{"id":"J_Muscle_Res_Cell_Motil-3-1-2045119","title":"Conduction velocities in amphibian skeletal muscle fibres exposed to hyperosmotic extracellular solutions\n","text":"Early quantitative analyses of conduction velocities in unmyelinated nerve studied in a constantly iso-osmotic volume conductor were extended to an analysis of the effects of varying extracellular osmolarities on conduction velocities of surface membrane action potentials in Rana esculenta skeletal muscle fibres. Previous papers had reported that skeletal muscle fibres exposed to a wide range of extracellular sucrose concentrations resemble perfect osmometers with increased extracellular osmolarity proportionally decreasing fibre volume and therefore diminishing fibre radius, a. However, classical electrolyte theory (Robinson and Stokes 1959, Electrolyte solutions 2nd edn. Butterworth & Co. pp 41\u201342) would then predict that the consequent increases in intracellular ionic strength would correspondingly decrease sarcoplasmic resistivity, Ri. An extension of the original cable analysis then demonstrated that the latter would precisely offset its expected effect of alterations in a on the fibre axial resistance, ri, and leave action potential conduction velocity constant. In contrast, other reports (Hodgkin and Nakajima J Physiol 221:105\u2013120, 1972) had suggested that Riincreased with extracellular osmolarity, owing to alterations in cytosolic viscosity. This led to a prediction of a decreased conduction velocity. These opposing hypotheses were then tested in muscle fibres subject to just-suprathreshold stimulation at a Vaseline seal at one end and measuring action potentials and their first order derivatives, dV\/dt, using 5\u201320 M\u03a9, 3 M KCl glass microelectrodes at defined distances away from the stimulus sites. Exposures to hyperosmotic, sucrose-containing, Ringer solutions then reversibly reduced both conduction velocity and maximum values of dV\/dt. This was compatible with an increase in Ri in the event that conduction depended upon a discharge of membrane capacitance by propagating local circuit currents through initially passive electrical elements. Conduction velocity then showed graded decreases with increasing extracellular osmolarity from 250\u2013750 mOsm. Action potential waveforms through these osmolarity changes remained similar, including both early surface and the late after-depolarisation events reflecting transverse tubular activation. Quantitative comparisons of reduced-\u03c7 2 values derived from a comparison of these results and the differing predictions from the two hypotheses strongly favoured the hypothesis in which Riincreased rather than decreased with hyperosmolarity.\nIntroduction\nA classic paper by Hodgkin (1954) (see also: Adrian 1975; Noble 1979) performed a quantitative cable analysis of the local circuit currents thought responsible for action potential propagation in unmyelinated nerve fibres studied in large volumes of constantly iso-osmotic extracellular fluid and demonstrated that their conduction velocities should vary as the square root of fibre radius, a. Action potential conduction velocity is also physiologically important in striated muscle: it ensures a rapid, sarcomeric activation leading to near-synchronous muscle contraction. The present paper accordingly extends these early analyses to effects of varying extracellular osmolarity on conduction velocities of surface membrane action potentials in Rana esculenta skeletal muscle. Skeletal muscle membrane differs from unmyelinated nerve in including an excitable transverse (T-) system whose activation initiates muscle contraction (Adrian et\u00a0al. 1969, 1970; Huang 1993; Nielsen et\u00a0al. 2004; Stephenson 2006). Nevertheless, it is likely that the initial sarcolemmal membrane activation precedes the subsequent inward tubular excitation and that the latter may take place partially independently of the propagation of the surface excitation wave. Sheikh et\u00a0al. (2001) suggested a partially separable T-tubule excitation initiated by Na+ channels selectively clustered around the mouths of the T-tubular lumina: detubulation produced by osmotic shock left surface membrane conduction velocities unchanged. Furthermore, increases in extracellular osmolarity did not alter the tubular diameters at the necks of the T-tubular system important in electrical connectivity between T-tubule network and remaining extracellular space (Launikonis and Stephenson 2002, 2004).\nHowever, variations in surface conduction velocities in skeletal muscle studied under varying osmolarities would be expected to differ from those of nerve fibres studied under constant, iso-osmotic conditions owing to the consequent changes in their cell volumes and fibre diameters. Nevertheless, skeletal muscle volume exhibits close to ideal osmotic behaviour, varying inversely with changes in extracellular osmolarity that would in turn predictably increase solute concentration. The Results section of this paper accordingly first develops the original treatment (Hodgkin 1954; Adrian 1975; Noble 1979) for situations in which fibre diameter is varied by alterations in extracellular osmolarity that would in turn alter intracellular ionic strength. This provided quantitative expectations for any resulting change in conduction velocity that corresponded to two contrasting situations. First, classical electrolyte theory (Robinson and Stokes 1959; Atkins 1998) would predict that increased intracellular ionic strength should proportionally decrease sarcoplasmic resistivity Ri. Our analysis then indicated that this would precisely offset any expected changes in fibre axial resistance, ri, produced by the osmotically induced alterations in fibre diameter and thereby leave conduction velocity unchanged. Secondly, Hodgkin and Nakajima (1972) reported that Ri increased with extracellular osmolarity, possibly due to increases in myoplasmic viscosity, although we certainly do not exclude possible factors arising from the more complex membrane structures found in muscle as opposed to nerve (see e.g. Sheikh et\u00a0al. 2001). This led to predictions that conduction velocity would decrease with increasing extracellular osmolarity.\nThese predictions were then investigated by experimental determinations of conduction velocity obtained from action potential records derived from microelectrode measurements made at known distances from defined stimulation sites in surface muscle fibres of Rana esculenta studied at a fixed (\u223c7\u00b0C) temperature at varied extracellular osmolarities. These findings demonstrated reversible changes in the conduction velocities of action potentials the nature of whose waveforms, including early surface and later tubular components, were otherwise unchanged, and distinguished between hypotheses through the observed dependence of conduction velocity on extracellular osmolarity.\nMaterials and methods\nCold-adapted frogs, Rana esculenta, were killed by concussion followed by pithing (Schedule I: Animal Procedures Act, Home Office, UK). The skin was removed and the tendon of insertion of the sartorius into the patella ligated, cut distally and dissected along its borders up to and including its origin at the pelvic girdle and acetabulum. This was performed at room temperature (21.7\u00a0\u00b1\u00a00.51\u00b0C; mean \u00b1 standard error of the mean; n\u00a0=\u00a015) in standard Ringer solution consisting of (mM): 115\u00a0NaCl, 2.5\u00a0KCl, 1.8 CaCl2, 3 Hepes, titrated to pH\u00a07, osmolarity 250\u00a0mOsm. The muscle was then stretched to 1.4 times its in situ length and secured in a transilluminated methacrylate polymer (Perspex) chamber by pinning the cleaned acetabulum and the ligature to minimize contractile artefact, which became less evident in solutions of higher osmolarities, and entirely absent with the higher (>350\u2212400\u00a0mM) sucrose concentrations. The isolated sartorius muscles had an in situ length of 30.6 \u00b1 0.94\u00a0mm and a length of 41.6\u00a0\u00b1\u00a01.15\u00a0mm (n\u00a0=\u00a015) when stretched. The muscle was laid over a supporting ramp with its dorsal surface uppermost.\nA watertight Perspex partition, coated with a layer of Vaseline, with the muscle running through a notch at its lower surface electrically isolated the recording chamber into two compartments. The only path conducting the brief voltage stimuli applied from two platinum electrodes at opposite ends of the chamber was therefore through the muscle across the partition. The platinum electrode on the side of the chamber containing the acetabulum was the cathode. The temperature of the bath solution was lowered by circulating distilled water cooled in a magnetically stirred ice bath. The water was circulated through a glass coil placed in the chamber in close proximity to the muscle, using a Minipuls 3 peristaltic pump (Gilson, France). A digital thermometer incorporating a remote thermistor (P. Frost, Department of Physiology, Development and Neuroscience, Cambridge), previously linearised and calibrated against a platinum film resistor was placed in the bathing solution near the muscle to allow the temperature to be continuously monitored and adjusted to remain constant throughout the recordings. The temperature was kept constant at 7.0 \u00b1 0.03\u00b0C (n\u00a0=\u00a015) (Sheikh et\u00a0al. 2001). Action potentials are temperature-sensitive so this allowed conditions to be optimised. Cooling also prolonged fibre viability especially in the hyperosmotic conditions.\nThe muscle was studied in the range of extracellular osmolarities from 250 to 750\u00a0mOsm. Solutions of different osmolarities were made by adding to the standard Ringer solution the membrane-impermeant solute sucrose at varying (150, 250, 350, 400, 450, 500\u00a0mM respectively) concentrations to yield solutions whose increased osmolarities (mOsm) were calculated from their total solute concentrations, and checked against measurements using a standard vapour pressure osmometer. Hepes was obtained from Sigma-Aldrich (Gillingham, Dorset, UK) and all other reagents from BDH Limited (UK). Solutions were changed, avoiding contact with the muscle, between tests using a vacuum pump (Edwards, UK) after which approximately 5\u201310\u00a0min elapsed before recording to permit time for both temperature (7\u00b0C) and osmotic equilibration; previous reports (Ferenczi et\u00a0al. 2004) have indicated that 200\u00a0s sufficed to permit complete volume changes in response to hyperosmotic solutions.\n3\u00a0M KCl-filled glass capillary microelectrodes (resistances 5\u201320\u00a0M\u03a9; tip potentials <5\u00a0mV: Adrian 1956) drawn from 1.2\u00a0mm (inner diameter) glass tubing were used to obtain membrane potentials. These were mounted via Ag\/AgCl junctions to a high impedance voltage follower balanced by matching Ag\/AgCl junctions earthing the bath. Only surface fibres showing clear-cut penetrations and stable resting membrane potentials were studied. Rates of rise and fall, dV\/dt, in the voltage traces, were obtained by electrical differentiation of the output, with voltage and dV\/dt channels filtered between cutoffs of 0\u00a0Hz\/5\u00a0kHz and 0\u00a0Hz\/20\u00a0kHz respectively. Action potentials were elicited by direct just-suprathreshold stimulation which varied between 1.5 and 5\u00a0V, to minimise electrotonic spread of the stimulus voltage and the number of fibres in which electrical activity was initiated, across the Perspex partition, through two platinum stimulating electrodes.\nConduction velocity was calculated by dividing the distance from the site of action potential initiation to the position of the recording microelectrode tip, ascertained by a Vernier scale, which gave measurements in cm to two decimal places, by the latency given by the time from a clear stimulus artefact generated to the peak of the dV\/dt input, which will be referred to as the maximum dV\/dt. The corresponding resting membrane potential, action potential overshoot and the value of maximum dV\/dt were also noted. These relatively early events in the timecourse of the regenerative response often could be measured even in records showing small contractile artefact, which often only became evident at later times in the recorded traces.\nResults\nThe experiments utilized an extension of early quantitative analyses (Hodgkin 1954; Adrian 1975) of conduction velocity in unmyelinated nerve studied in a constantly iso-osmotic volume conductor to changes that would result from varying the extracellular osmolarities in Rana esculenta skeletal muscle fibres. The initial analysis used the cable equation for the current density through any patch of flat membrane, expressed as current per unit membrane area,\nThis describes conduction in a cable whose own finite internal volume is small compared to that of the extracellular conducting fluid in which it is studied to give large ratios between longitudinal intracellular, ri, and extracellular, ro, resistances to current flow per unit length, i.e., ro\u00a0<<\u00a0ri, permitting extracellular terms relating to ro to be dropped.\nEquation (1) is combined with the expression for conduction velocity,  using the chain rule of differentiation, to give:where Vm is the internal potential across the membrane at distance x along the fibre. Conduction at constant velocity through local circuit spread of excitation then requires Im to be a single valued function of Vm. The term containing the intrinsic membrane parameters consisting of the area of membrane per unit length of fibre s, and the axial cytoplasmic resistance per unit length ri, is then constant, whence , and where the constant k only depends on passive local membrane electrical properties. Thus:\nFor unmyelinated cylindrical fibres for which the axial cytoplasmic resistance per unit length, ri, is related to fibre radius a and sarcoplasmic resistivity Ri by Ri\u00a0=\u00a0ri\u03c0 a2, the total fibre volume, vol,\u00a0=\u00a0\u03c0 a2L where L is the length of the fibre and for which the total surface area of the fibre, A\u00a0=\u00a0sL:\nIn the case of skeletal muscle fibres exposed to solutions of different osmolarities, osm, the volume, vol, behaves as a perfect osmometer following the relationship: and reductions in fibre volume due to increased extracellular osmolarity would correspondingly increase solute concentration. Classical electrolyte theory (Robinson and Stokes 1959) then predicts that each participating ion contributes a specific conductance Ksp determined by its solute concentration \u03b7C and a constant \u039b defined for any given ion in any specified solute and referred to the conductivity of the ion at a 1\u00a0M concentration:\nThe overall conductivity of such an ideal solution is then the algebraic sum of the conductivities of individual component ions. Decreases in fibre volume resulting from increased extracellular osmolarities then would increase intracellular ionic strength and in turn increase Ksp and proportionally decrease Ri: Equation (7) then gives: Equations (5) and (8) then give,thereby predicting a constant conduction velocity in view of the fact that the terms k\u2032, resulting from the additional proportionalities above, and A are both constants.\nIn contrast, Hodgkin and Nakajima (1972) suggested that sarcoplasmic resistivity, Ri, increased with increasing extracellular osmolarity, possibly owing to an increased myoplasmic viscosity, with reductions in fibre volume in hyperosmotic solutions, reporting a linear increase in Ri with increasing extracellular osmolarity:where D and E are constants.\nFrom (5) and (10):\nBecause k\u2032 and A are constants, we have:where D\u2032 and E\u2032 are also constants. This forms a contrasting expectation in which \u03b8 is expected to decrease with increasing extracellular osmolarity in a relationship modelled by Eq. (12) as a result of an increase in Ri. The alternative hypotheses could then be quantitatively tested by measurements of \u03b8 under different conditions of extracellular osmolarity.\nReversible effects of hyperosmotic extracellular solutions on action potential waveforms and latencies\nFigure\u00a01 shows typical action potential and dV\/dt waveforms obtained at a temperature of \u223c7\u00b0C before, during and after exposures to solutions with increased extracellular osmolarity with column A showing the action potential waveforms and column B their corresponding dV\/dt records. Panel a shows typical results from fibres in iso-osmotic Ringer at 7\u00b0C. The muscle was then exposed to a hyperosmotic (600\u00a0mOsm) Ringer solution and recordings resumed 5\u201310\u00a0min following this solution change (Panel b). Finally, the muscle was then returned to iso-osmotic Ringer and recordings again resumed 5\u201310\u00a0min after the solution was restored (Panel c). Latencies were measured from the clear stimulus artefact to the maximum point of the dV\/dt trace as seen in column B: this approach provided more consistent measurements of action potential latency than using arbitrarily chosen points on the action potential traces (see also Sheikh et\u00a0al. 2001).Fig.\u00a01Typical action potential (A) and dV\/dt traces (B) obtained from muscle fibres exposed successively to (a) iso-osmotic Ringer (b) hyperosmotic (600\u00a0mOsm) Ringer and (c) returned to iso-osmotic Ringer, in the same sartorius muscle\nThe action potential traces showed prolonged positive after-depolarisation phases lasting well beyond 20\u00a0ms following the surface action potential deflections (Fig.1Aa), consistent with persistent excitation of an intact transverse (T-) tubular system (Adrian and Peachey 1973). These persisted both in the hyperosmotic solution (Fig.\u00a01Ab) and the iso-osmotic solution to which the fibres were finally returned (Fig.\u00a01Ac), confirming a persistence in transverse tubular excitation and its excitation following initiation of the surface component of the action potential through these manipulations.\nUse of 600\u00a0mOsm as opposed to standard Ringer increased the action potential latencies and consequently the calculated values of conduction velocity. Thus, conduction velocities in the initial iso-osmotic Ringer solution, then at 600\u00a0mOsm-Ringer solution and finally, the returned iso-osmotic Ringer were 0.98\u00a0\u00b1\u00a00.092\u00a0m s\u22121 (n\u00a0=\u00a011 fibres), 0.58\u00a0\u00b1\u00a00.051\u00a0m s\u22121 (n\u00a0=\u00a010) and 0.72\u00a0\u00b1\u00a00.101\u00a0m s\u22121 (n\u00a0=\u00a06) respectively. The effect of hyperosmolarity on conduction velocity was thus at least partly reversible. However, this decrease in conduction velocity with increased osmolarity (from 250 to 600\u00a0mOsm) was not accompanied by qualitative changes in action potential or dV\/dt waveform. Likewise, the waveforms showed no qualitative changes following return to the iso-osmotic Ringer solution after hyperosmotic exposure.\nGrading of conduction velocity changes with extracellular osmolarity\nFigure\u00a02 summarises typical traces of action potentials (column A) and dV\/dt (column B) for experiments that systematically investigated the effects of graded changes in extracellular osmolarity reflecting different extracellular sucrose concentrations on conduction velocity, action potential waveform, and dV\/dt obtained at 7\u00b0C. Action potential waveforms again included both early rapid surface action potential deflections following the stimulus artefact and prolonged after-depolarisation phases that lasted well beyond 20\u00a0ms, reflecting T-tubular activation, at all the extracellular osmolarities studied (cf. Adrian and Peachey 1973). Measurements of latencies between the stimulus artefacts to the maximum value of the dV\/dt traces then suggested a noticeable overall decrease in the calculated conduction velocities with increasing osmolarity. All these changes occurred in the absence of any excessive depolarization in resting membrane potential over the range of explored osmolarities. Thus resting potential at an osmolarity of 250\u00a0mOsm was \u221285.27\u00a0\u00b1\u00a01.05\u00a0mV (n\u00a0=\u00a087); that at an osmolarity of 700\u00a0mOsm was \u221275.94\u00a0\u00b1\u00a02.20\u00a0mV (n\u00a0=\u00a033). However, resting potential at an osmolarity of 750\u00a0mOsm was \u221262.71\u00a0\u00b1\u00a02.82\u00a0mV (n\u00a0=\u00a017) (cf. Fraser et\u00a0al. 2006). At the latter extracellular osmolarity a small proportion of action potentials appeared to display markedly reduced amplitudes resulting in peak deflections that failed to show overshoots, and a firing of two consecutive action potentials after a single stimulation in the case of one fibre. Accordingly, the investigations were not performed at higher sucrose concentrations.Fig.\u00a02Typical action potential (A) and dV\/dt (B) traces recorded in the following extracellular osmolarities: (a) 250, (b) 400, (c) 500, (d) 600, (e) 650 and (f) 750 (all values given in mOsm). There is a notable overall increase in the latencies measured from (B)\nPlots of conduction velocity and maximum dV\/dt with increasing extracellular osmolarity\nFigure\u00a03 summarizes the results of systematic studies of the dependence of conduction velocity on extracellular osmolarity in a statistically larger number of muscle fibres. Action potentials and their corresponding dV\/dt were measured systematically in n\u00a0=\u00a087, 18, 27, 11, 10, 33 and 13 fibres at sucrose concentrations of 0, 150, 250, 350, 400, 450 and 500\u00a0mM corresponding in turn to osmolarities of 250, 400, 500, 600, 650, 700 and 750\u00a0mOsm respectively. Figure\u00a03 demonstrates that both conduction velocity (a) and maximum dV\/dt (means \u00b1 standard errors of the mean) (b) monotonically decreased with extracellular osmolarity. Figure\u00a03a then compares these experimental values of conduction velocity against predictions from the two hypotheses for the dependence of Ri on osmolarity. In both cases, \u03b8 was calculated from Ri and the extracellular osmolarity, osm, using the relationship derived from Eqs. (4) and (5) above that:Fig.\u00a03Experimentally measured conduction velocities (a) and maximum dV\/dt (mean \u00b1 standard error of the mean) plotted against extracellular osmolarity (\u25c6). Dashed lines in (a): velocities predicted for a situation in which Ri decreases proportionally as a result of increased intracellular ionic strength produced by changes in cell volume brought about by the osmolarity changes. Continuous line: predictions when Ri increases with increasing extracellular osmolarity suggested by Hodgkin and Nakajima (1972). The changes in (a) were accompanied by decreases in maximum dV\/dt (b) with increasing osmolarity, as expected for propagation brought about by local circuit currents\nThe dotted line denotes expectations from the first situation outlined above in which Ri decreases proportionally with increasing extracellular osmolarity: this predicts that conduction velocity does not alter with osmolarity from its control value in iso-osmotic solutions corresponding to a constant value of 0.85\u00a0m s\u22121 obtained in iso-osmotic solutions. The continuous line represents the second hypothesis in which Ri depends both on temperature and extracellular osmolarity as reported by Hodgkin and Nakajima (1972). The function displayed assumes the sarcoplasmic conductivity Gi(=\u00a01\/Ri) to have a Q10\u00a0=\u00a01.37, and a value of Ri at 2\u00b0C, of 298.51\u00a0\u03a9\u00a0cm in a muscle fibre within an extracellular solution of 250\u00a0mOsm osmolarity and of 390.63\u00a0\u03a9\u00a0cm in a muscle fibre within an extracellular solution of 600\u00a0mOsm osmolarity, as suggested by Hodgkin and Nakajima (1970, 1972). Then, let \u03b81, Ri1 and osm1 correspond to conditions in iso-osmotic Ringer and \u03b82, Ri2 and osm2 correspond to conditions in any hyperosmotic solution. Thus:Since \u03b81\u00a0=\u00a00.85\u00a0\u00b1\u00a00.029\u00a0m s\u22121 in iso-osmotic solution,where \nThe values generated by the above equations predicted a decline in conduction velocity with increasing extracellular osmolarity.\nTo test the statistical significance of the goodness-of-fit of the two contrasting predictions to the experimental data obtained, an Fx test that takes into account the difference of the two reduced-\u03c72 values in proportion to the first \u03c72 term was performed (Bevington 1969). This derived for each hypothesis a value of \u03c72 depicting the summed squared deviation of the original data, yi, to the predicted findings, y(xi), obtained at any extracellular osmolarity, xi, such that:\nValues for \u03c72 obtained in the two cases, \u03c712 where the predicted conduction velocity is a constant value and \u03c722 where there is an increase in Ri with extracellular osmolarity were 21.360 and 15.141 respectively. These values were then used to compute a F-statistic, given by:\nwhere n, the sample size = 200 and n\u22121 is the number of degrees of freedom.\nThe reduced-\u03c72 tests for goodness-of-fit yielded a F-statistic of 81.73 consistent with a significantly better fit (P\u00a0<<\u00a00.001) to predictions from a situation in which Ri increased as described by Hodgkin and Nakajima (1972) rather than a decrease in Ri with increasing extracellular osmolarity.\nDiscussion\nThis paper begins from classic analyses (Hodgkin 1954; Adrian 1975) of the dependence of action potential conduction velocities upon the diameter of nerve fibres studied in large volume conductors under iso-osmotic extracellular conditions. This had employed cable analysis that attributed these propagation processes to local circuit currents driven by Na+ currents, INa, generating the rising phase of the action potential that in turn discharged initially passive circuit components (Valdiosera et\u00a0al. 1974) equivalent to a membrane capacitance per unit area, Cm and resistance Rm of unit membrane area in series with an axial cytoplasmic resistance per unit length, ri. The latter is related to fibre radius a and sarcoplasmic resistivity Ri (k\u03a9\u00a0cm) by Ri\u00a0=\u00a0ri\u03c0a2 giving the original result that conduction velocity would vary as the square root of a.\nThe present paper extends this analysis to the effects of varying extracellular osmolarity on conduction velocities of surface membrane action potentials in Rana esculenta skeletal muscle, a situation that differed in a number of respects. First, skeletal muscle contains excitable transverse (T-) tubular membrane system responsible for initiating excitation-contraction coupling (Adrian et\u00a0al. 1969, 1970; Huang 1993; Nielsen et\u00a0al. 2004; Stephenson 2006). Nevertheless, the rapid initial sarcolemmal membrane activation that ensures rapid action potential propagation producing a synchronous sarcomeric activation likely largely precedes full tubular excitation (Adrian and Peachey 1973). Recent detubulation experiments left surface membrane conduction velocities unchanged suggesting a separation of surface and T-tubule excitation, the latter possibly initiated separately by Na+ channels localized around the T-tubular luminal mouths (Sheikh et\u00a0al. 2001). Increasing extracellular osmolarity did not increase the diameter of the necks of the T-tubules important in electrical connectivity between T-tubule network and the fibre membrane (Launikonis and Stephenson 2002, 2004). These findings would permit muscle fibre conduction velocities to be analysed in terms of surface cylinders. Second, skeletal muscle volume alters inversely with extracellular osmolarity (Blinks 1965; Ferenczi et\u00a0al. 2004) in turn correspondingly increasing solute concentration, in contrast to the situation represented by comparisons of nerve fibres of different diameters in similarly iso-osmotic extracellular solutions.\nThis paper then derived quantitative consequences from two possible hypotheses emerging from the above conditions. On the one hand, classical electrolyte theory (Robinson and Stokes 1959; Atkins 1998) predicts a specific conductance Ksp attributable to each intracellular ion species increasing proportionally with solute concentration following decreases in cell volume in hyperosmotic solution. This led to a prediction of a decrease in Ri precisely correcting out effects of any osmotically induced diameter change together leaving conduction velocity constant. On the other hand, empirical observations suggesting increases in Ri with extracellular osmolarity (Hodgkin and Nakajima 1972) permitted construction of a quantitative formula for the resulting variations in Ri with extracellular osmolarity as well as temperature. This led to predictions of a conduction velocity that decreased with increasing extracellular osmolarity.\nThe experiments described in this paper then sought to distinguish between the two hypotheses. It investigated the effects of extracellular osmolarity on conduction velocities of surface membrane action potentials in surface fibres, that would be maximally exposed to these solution changes, in frog skeletal muscle, following stimulation at a Vaseline seal at defined distances from the microelectrode recording site. Simultaneous records were made of the rate of change of membrane potential dV\/dt that would be provided a consistent time point at which there would be a maximal action potential slope as well as rate of discharge of the membrane capacitance, Cm, by local currents driven by the Na+ current INa.\nThese studies showed that conduction velocity declined with increasing extracellular osmolarity along with maximum dV\/dt as expected for a process dependent upon a local circuit current flow, despite relatively constant resting membrane potentials. These changes in conduction velocity in hyperosmotic solution were at least partially reversible. Furthermore, there were no changes in the nature of the action potential waveforms including early surface deflections and late after-depolarisation phases observed, consistent with a minimal change in the capacity for T-tubular excitation, through a still intact tubular system, that nevertheless followed generation of the initial surface component of the action potential wave.\nFurther systematic study in larger fibre numbers through a range of osmolarities (250\u2013750\u00a0mOsm) demonstrated corresponding declines in conduction velocity and maximum dV\/dt. At the highest osmolarity (750\u00a0mOsm), a small proportion of fibres variously showed low action potential overshoots which however did not correlate with the situations where there was a reduced conduction velocity, as well as multiple firing in a single muscle fibre. Nevertheless, further studies were not made at these and higher osmolarities; in any case, muscle fibres are thought only to act close to perfect osmometers at osmolarities up to around four times that of the iso-osmotic solution (Blinks 1965).\nThese findings are thus consistent with the second hypothesis in which sarcoplasmic resistivity, Ri, increases with the fibre shrinkage observed in hyperosmotic solutions, as suggested by Hodgkin and Nakajima (1972), as opposed to the first possibility outlined above in which conduction velocity should be constant. This was borne out by objective statistical analysis of the goodness-of-fit of the predictions derived from the two contrasting hypotheses as expressed in their resulting reduced-\u03c72 values, with the experimental values of conduction velocity generally assuming slightly higher values than predicted. The latter might reflect minor departures from a purely continuous conduction as postulated for unmyelinated cylindrical fibres, either due to contributions from peripheral regions of tubular membrane less isolated than the remaining tubular system (Hodgkin and Nakajima 1972), or the clustering of sodium channels around the tubular necks as reported by Sheikh et\u00a0al. (2001) that may contribute to a more saltatory-like conduction in the muscle that would be expected to generally speed up propagation of electrical activity.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\n49\u00a0kb doc","keyphrases":["conduction velocity","skeletal muscle","osmolarity"],"prmu":["P","P","P"]}
{"id":"Pediatr_Nephrol-3-1-1794138","title":"Pathophysiology of focal segmental glomerulosclerosis\n","text":"Focal segmental glomerulosclerosis (FSGS) is a major cause of idiopathic steroid-resistant nephrotic syndrome (SRNS) and end-stage kidney disease (ESKD). In recent years, animal models and studies of familial forms of nephrotic syndrome helped elucidate some mechanisms of podocyte injury and disease progression in FSGS. This article reviews some of the experimental and clinical data on the pathophysiology of FSGS.\nLearning objectives Discuss the experimental and clinical data on the pathophysiology of FSGSReview the alterations in glomerular structure and function associated with FSGSTo identify potential mechanisms responsible for disease progression in FSGSDistinguish some targets for the future therapy of FSGS\nFocal segmental glomerulosclerosis (FSGS) is a disease entity defined by findings on kidney biopsy [1, 2]. FSGS is the major cause of idiopathic steroid-resistant nephrotic syndrome (SRNS) in children and adults [3]. FSGS is the most common cause of acquired chronic renal insufficiency in children and frequently leads to progression to end-stage kidney disease (ESKD) [2]. FSGS may occur secondary to such disparate disease processes as HIV and obesity [1, 4]; this review focuses on the pathophysiology of primary FSGS (i.e., with no underlying illness).\nAlterations of normal glomerular structure and function have been found in FSGS [5]. Normal function requires that the three major components of the glomerular filter (the endothelial cells, podocytes, and glomerular basement membrane) are intact and are able to provide a permselective filtration barrier (Fig.\u00a01). Specialized tight junctions between podocyte foot processes create the slit diaphragm which is integral to preventing the loss of protein into the urinary space [6]. While the clinical presentation of FSGS is often heterogeneous, a characteristic feature of the disease is proteinuria, which implies the loss of this barrier [2, 7]. Indeed, electron microscopy has shown distortion of the normal architecture (or effacement) of the foot processes of podocytes in FSGS [1]. The connection between these projections of the epithelial cell and the underlying basement membrane can be disrupted, leading to the loss of nonspecific plasma proteins into the tubular filtrate [6].\nFig.\u00a01A Components of the normal glomerular filtration barrier: (1) glomerular basement membrane (GBM); (2) podocyte foot process; (3) endothelial cell; B Progressive changes seen in focal segmental glomerulosclerosis (FSGS) leading to sclerosis: (1) foot process effacement; (2) podocyte apoptosis\/loss and exposed glomerular basement membrane; (3) filtration of non-specific plasma proteins; (4) capillary expansion; (5) formation of synechiae; (6) misdirected filtration at points of synechiae; (7) mesangial matrix proliferation. Adapted from Kwoh et al. [9]\nHowever, unlike other causes of proteinuria and nephrotic syndrome, such as minimal change disease (MCD), FSGS often progresses to ESKD. While foot process effacement is seen in MCD as well as FSGS, histologically, FSGS is characterized by increased extracellular matrix within the glomerular tuft with obliteration of the glomerular capillary lumen. These sclerotic lesions occur focally and in only some segments of glomeruli, and are typically not associated with immune complex deposition [1]. The location of sclerotic lesions by light microscopy defines the variants of FSGS: perihilar variant (with sclerosis of the vascular pole), cellular variant (associated with hypercellularity of the capillary space), tip variant (involving the part of the glomerulus near the origin of the proximal tubule), and collapsing variant (with one or more glomeruli with global or segmental collapse) [1]. Clinically, the variants of FSGS differ; for example, the collapsing variant tends to progress more rapidly to ESKD and commonly occurs in the setting of HIV [1]. It is possible that different mechanisms may play a role in the pathogenesis of each variant of FSGS [7, 8].\nInsight into the pathogenesis of FSGS developed over the past decade from studies of genetic mutations in mice, models of progressive glomerulosclerosis (such as the rat remnant kidney model), and identification of gene mutations in some familial forms of nephrotic syndrome (including congenital nephrotic syndrome and familial and autosomal dominant FSGS) [7, 9, 10].\nKey in the pathogenesis of FSGS is podocyte damage and loss [5, 6]. Injury to podocytes occurs by four major mechanisms: alteration of the components of the slit diaphragm or interference with its structure, dysregulation of the actin cytoskeleton, alteration of the glomerular basement membrane or its interactions with the podocyte, or alteration of the negative surface charge of the podocyte [6, 9]. Damage to podocytes triggers apoptosis and their detachment of podocytes from the glomerular basement membrane [6, 9]. The ensuing reduction in podocyte number is felt to play an important role in the pathogenesis of FSGS [7]. The podocyte is normally a terminally differentiated cell with limited proliferative capacity in response to injury [7]. The initial insult to the podocyte leads to further damage mediated by cytokine release, mechanical stress, and further loss of polarity, resulting in sclerosis and scarring of the glomerulus [7, 9].\nGenetic mutations seen in congenital forms of nephrotic syndrome and FSGS enabled researchers to identify specific gene mutations involved in podocyte damage [10]. Mutations of the nephrin gene, a podocyte-specific transmembrane component of the slit diaphragm, are found in congenital Finnish-type nephrotic syndrome, and may lead to loss of normal caliber slit diaphragms [6, 9\u201311]. In mouse models, mutations of nephrin-like transmembrane genes (NEPH-1) which also localize to the slit diaphragm result in proteinuria and early death [6, 10].\nIt is unclear how alteration of the slit diaphragm results in podocyte loss. The slit diaphragm may be integral to maintaining cell polarity or its damage may alter the balance of cell signals, resulting in apoptosis. Mutations in a Fyn kinase (one of the src tyrosine kinases) that phosphorylates nephrin and may regulate cell cycle and apoptosis resulted in proteinuria and foot process effacement in a mouse model [9, 10].\nOther proteins which are part of the slit diaphragm complex include: podocin, CD2-associated protein (CD2AP), FAT, ZO-1, P-cadherin, an LAP (leucine rich repeat and PDZ domain) protein, and MAGI-1 [6, 10]. Mutations in podocin (a transmembrane protein that interacts with nephrin, NEPH-1 and CD2AP) have been identified in familial FSGS [9, 10, 12]. Recently, mutations in CD2AP, an immunoglobulin-like protein that is involved in nephrin integration with the podocyte cytoskeleton, have also been linked to genetic forms of FSGS [10, 13, 14]. In mouse models, the loss of FAT1 and FAT2 (transmembrane proteins with cadherin-like repeats) results in the absence of slit diaphragms, proteinuria, and early death [10]. The role of the other components of the slit diaphragm in the pathophysiology of FSGS is not yet clear.\nAlpha-actinin-4, an important structural component of the podocyte cytoskeleton, is mutated in some autosomal dominant forms of FSGS [10, 15\u201317]. Other mutations have been identified in association with FSGS in addition to abnormal structural proteins. For example, TRPC6 is a cation-selective ion-channel protein that mediates calcium signals and has also been associated with FSGS [18].\nCertain clinical variants of FSGS are suggestive of different mechanisms of injury to the podocyte. For example, a circulating factor which leads to glomerular basement membrane injury has been proposed in the pathogenesis of some types of FSGS [19, 20]. For example, there appears to be a role of a circulatory factor in the recurrence of FSGS in transplanted kidneys [20]. In some patients with recurrent FSGS, proteinuria remits in response to plasmapheresis and the removal of serum proteins. In addition, injections of serum from patients with recurrent FSGS were capable of inducing proteinuria in rats [20].\nAnother example of alternative mechanisms of injury is collapsing FSGS, which occurs in the setting of viruses such as HIV. In collapsing FSGS, dysregulation of the podocyte cell cycle appears to result in immature, proliferative podocytes [21, 22]. Finally, recent work has focused on the role of the parietal epithelial cell in the pathophysiology of FSGS [23]. Proliferation of parietal epithelial cells was identified in both a transgenic model of FSGS and a biopsy from a patient with collapsing FSGS [23].\nOf great clinical importance is the mechanism by which the initial podocyte injury progresses to the final sclerotic lesion (Fig.\u00a01). As podocyte numbers decline, there is a relative exposure of the glomerular basement membrane. Maladaptive interactions develop between the glomerular basement membrane and the parietal epithelial cells. Expansion of synechiae and\/or the leak of protein into Bowman\u2019s space results in the deposition of collagen. Ultimately, this results in the collapse of the capillary loop and the loss of endothelial cells [5].\nFactors resulting in the progression of FSGS to ESKD have also been the focus of recent research (Fig.\u00a02). Cytokines and vasoactive factors are believed to play a major role in the progression of FSGS. The overexpression of transforming growth factor \u03b2 (TGF\u03b2) or its effector proteins, the Smads, leads to glomerulosclerosis in animal models [8, 24]. Activation of the renin-angiotensin system upregulates TGF\u03b2 and is felt to further lead to the progression of disease [7, 24]. Other angiogenic factors, such as platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) may also play a role in disease progression [24]. The evidence for this is primarily based on animal models of progressive glomerulosclerosis, such as the rat remnant kidney model. In this model, PDGF and VEGF are upregulated and the later loss of VEGF expression correlates with progression of the glomerulosclerosis [24, 25].\nFig.\u00a02Factors involved in the progression of FSGS to end-stage kidney disease (ESKD): initial loss or injury to podocytes (related to defects in membrane proteins or cytoskeleton instability) leads to cytokine release, mechanical stress, hyperfiltration, and glomerular hypertrophy. These factors lead to upregulation of an inflammatory response mediated by monocytes, macrophages, and T-cells. The end result is collagen matrix deposition and fibrosis, and progression to ESKD\nMechanical stress is also believed to play a role in the progression of FSGS [9, 26]. Increased filtration due the defects of the filtration barrier results in increased single-nephron glomerular filtration rate (SNGFR). This hyperfiltration results in hypertrophy of glomeruli. The hypertrophy exacerbates the mismatch between the glomerular basement membrane and the decreased numbers of podocytes, resulting in further injury [9].\nAnother factor in the progression of FSGS is tubulointerstitial injury. Clinically, tubulointerstitial injury is a predictor of the loss of renal function in FSGS [1, 27]. The nonspecific entry of proteins into the tubular lumen is one potential source of damage to the interstitium. Indeed, persistence of nephrotic-range proteinuria is a negative prognostic factor for the progression of FSGS to ESKD [28]. While it is unclear if proteinuria itself is toxic to the tubulointerstitium, decreases in proteinuria achieved by angiotensin-converting enzyme (ACE) inhibitors and by angiotensin receptor blockers (ARB) appear to slow disease progression in some adults with FSGS [9, 29].\nThe presence of plasma proteins in the tubular filtrate may directly injure the tubulointerstitium. Cytokines (such as TGF\u03b2), when present in the tubules, will recruit monocytes, macrophage, and T-cells. This stimulates other cytokines, including interleukin-1, tumor necrosis factor alpha, and other chemokines [24]. The inflammatory infiltrate leads to mesangial matrix deposition, promoting the collapse of glomeruli. The cellular infiltrate and cytokines also damage tubular epithelial cells, and some tubular epithelial cells may undergo transformation to mesenchymal cells (an epithelial-mesenchymal transition or EMT) [24]. These mesenchymal cells, as well as recruited and stimulated fibroblasts, result in collagen matrix deposition and tubulointerstitial fibrosis [24].\nThe beneficial effects of blocking the renin-angiotensin system may not be limited to their antiproteinuric or antihypertensive effects. As noted earlier, angiotensin stimulates TGF\u03b2, contributing to fibrosis. It can also induce oxidative stress and it is stimulated by mechanical stress, such as hyperfiltration [24]. In addition, angiotensin affects intracellular calcium concentrations and the podocyte cytoskeleton [24]. Inhibition of angiotensin may slow progression by these local mechanisms [9, 29].\nWith the increasing incidence of FSGS in children [30], these pathways of podocyte injury and disease progression provide important targets for future intervention. Trials have already been initiated to antagonize cytokines, such as TGF\u03b2. Future therapeutic targets may include factors involved in podocyte protection or tubulointerstitial injury.\nQuestions (Answers appear following the reference list)\nWhich of the following statements is TRUE regarding the current understanding of the pathogenesis of focal segmental glomerulosclerosis (FSGS)? \nFSGS may result from immune-complex-mediated damage to endothelial cellsAlterations in components of the slit diaphragm may play a role in the pathogenesis of FSGSProliferation of podocytes leads to cytokine release and mechanical stress, resulting in scarring and sclerosis of the glomeruliMutations in a chloride channel have been associated with FSGS and may be pathogenicAll of the following are mutations of structural proteins that have been identified as pathogenic in FSGS EXCEPT: \nSodium channel mutationAlpha-actinin-4NephrinPodocinProgression of FSGS to end-stage kidney disease (ESKD) results from: \nDownregulation of transforming growth factor \u03b2 (TGF\u03b2)Decreased glomerular filtrationTubulointerstitial injuryBlockade of the renin-angiotensin systemProteinuria in the setting of FSGS: \nHas no effect on clinical courseMay be decreased by treatment with angiotensin-converting enzyme (ACE) inhibitorsResults from an increased number of glomerular foot processesLeads to the loss of mesangial matrixWhich of the following is FALSE: \nA circulating factor may play a role in the pathogenesis of FSGSProliferation of parietal epithelial cells has been identified in collapsing FSGSPodocyte loss due to necrosis appears to play a role in the pathogenesis of FSGSCD2-associated protein, FAT, nephrin, and podocin are examples of slit diaphragm proteins","keyphrases":["pathophysiology","focal segmental glomerulosclerosis (fsgs)","nephrotic syndrome","animal models","podocyte","injury","nephrin","podocin","tubulointerstitial","transforming growth factor (tgf\u03b2)"],"prmu":["P","P","P","P","P","P","P","P","P","R"]}
{"id":"Neurosurg_Rev-2-2-1564192","title":"Clinical and radiological features related to the growth potential of meningioma\n","text":"Clinical and radiological features that help predict the growth potential of meningioma would be beneficial. The purpose of this study is to clarify the characteristics related to proliferating potential using the MIB-1 staining index. We analyzed the relationship of MIB-1 staining indices to characteristics of 342 consecutive patients with meningioma surgically removed between 1995 and 2004 by logistic regression analysis. One hundred and forty-nine of the patients with meningioma were \u226560 in age; 89 male; 48 recurrent; 203 symptomatic; 157 at the skull base; 124 over 20 cm3; 24 multiple; 136 with edema; 117 with calcification. The MIB-1 staining index in 56 of 296 grade I meningiomas in WHO classification was \u2265 3.0; in 27 of 28 grade II; and in 17 of 18 grade III, respectively. Logistic regression analysis demonstrated that male (odds ratio [OR], 2.374, p=0.003), recurrence (OR, 7.574, p=0.0001), skull base (OR, 0.540, p=0.035), calcification (OR, 0.498, p=0.019) were independent risk factors for a high MIB-1 staining index (\u22653.0); age, symptomatic, volume, multiple, edema were not. Male, recurrence, non-skull base, absence of calcification are independent risk factors for a high proliferative potential. These should be taken into consideration when managing meningiomas.\nIntroduction\nAn estimated 2\u20133% of the population has an incidental asymptomatic meningioma in autopsy studies [14, 20]. With the wider use of CT and MRI, many meningiomas are discovered as incidental findings during investigation for unrelated symptoms [9, 13, 16, 17, 20, 21]. The growth potential of meningiomas varies. Some meningiomas remain unchanged in size for a long period, whereas others grow rapidly [15]. Sex, age, initial tumor size, and calcification were reported to be related to the tumor growth judging from follow-up scans [9, 13, 16, 17, 21].\nThe nuclear antigen Ki-67 expressed by proliferating cells has become available for routinely processed paraffin section. The MIB-1 antibody detects an epitope on the Ki-67 antigen, a nuclear protein present only during active phase of the cell cycle (G1, S, G2, and M) [2]. Several studies investigated how Ki-67 labelling indices could help to predict recurrence [1, 3, 4, 6\u20138, 11, 12, 18, 19]. An increased MIB-1 staining index was highly correlated with a shorter tumor volume doubling time [12]. In the cases that showed an MIB-1 of \u2265 3%, the tumor volume doubling time was <2 years. Nakaguchi et al. [12] found the formula which can calculate tumor doubling time (Td) from the MIB-1 staining index at surgery: log Td=31.4\u20130.14\u00d7MIB-1 SI (R2=0.556). The time interval to the next recurrence for recurrent meningiomas is associated with the MIB-1 staining index. Meningiomas with MIB-1 staining index of 3% or higher had a significantly higher tendency of recurrence [11]. Although these cell kinetics methods are valuable for growth potential, they can be applied only after the verification of pathology [15].\nThe purpose of this study was to clarify the clinical and radiological features related to meningioma proliferation using the MIB-1 staining index. Sex, age, calcification, edema, symptom, size, and shape of tumor were already reported to be related to the tumor growth and MIB-1 staining index [1, 3, 4\u20138, 11\u201313, 16\u201319, 21]. The results, however, were inconsistent because of a lack of sufficient case number and inadequate statistical analysis. Independent risk factors for high growth rate should be determined. We analyzed the relationship of MIB-1 staining indices to the characteristics of 342 consecutive patients with meningioma surgically removed between 1995 and 2004 by logistic regression analysis.\nMaterials and methods\nThree hundred and forty-two patients with meningiomas were surgically treated in our department of neurosurgery between 1995 and 2004. Radiological features were analyzed by CT scans and\/or MRI. Location of the tumor was classified as follows: convexity, falx, parasagittal, sylvian fissure, tentorial, ventricular, foramen magnum, olfactory groove, petroclival, petrous, sphenoid ridge, and tuberculum sellae. The latter six locations were considered as skull base. The tumor volume was calculated using the formula: length \u00d7 depth \u00d7 width \u00d7 0.5 [9]. When patients had multiple meningiomas, only the largest tumor was included. On the basis of conventional CT and bone window CT, patients were divided into two groups according to the low density area around the tumor and calcification in the tumor. A low density area due to surgical scar was not included in edema in patients with recurrent meningioma.\nThe tumors were histologically classified according to the World Health Organization classification of tumors [10]. An avidin-biotin immunoperoxidase or simple stain MAX-peroxidase (Nichirei, Tokyo) technique was used to perform MIB-1 monoclonal antibody (DAKO, Denmark) assay in selected sections of each case. All tissue sections were examined at high-power magnification (\u00d7400). The number of cells stained positively with MIB-1 and the total number of tumor cells were counted in several representative fields containing more than 1,000 cells. Their ratio was indicated as the MIB-1 staining index (%).\nStatistical analysis\nAll data were stored on a personal computer and analyzed using commercially available statistical software (SPSS version 12.0, SPSS Inc.). Chi-squired analysis was used to compare the MIB-1 staining index to characteristics of patients with meningioma. All variables were included in a logistic regression model to determine which variables were independently associated with a high MIB-1 staining index (\u22653.0). Significance was judged at a value of p<0.05 for all analyses.\nResults\nTable\u00a01 shows the characteristics and MIB-1 staining index of the 342 patients. One hundred and forty-nine of patients with meningioma were \u226560 in age; 89 male; 48 recurrent; 203 symptomatic; 157 at the skull base; 124\u226520\u00a0cm3 in volume; 24 multiple; 136 with edema; 117 with calcification. We compare these characteristics to the MIB-1 staining index. We divided them into two groups: <3.0 and \u22653.0 [11]. The MIB-1 staining index in 100 of 342 meningiomas was > 3.0. Sex (p=0.0001), recurrence (p=0.0001), symptomatic (p=0.013), volume (p=0.014), edema (p=0.001), and calcification (p=0.0001) were correlated with the MIB-1 staining index by chi-square test; age, skull base, and multiple were not. \nTable\u00a01Characteristics and MIB-1 staining index in 342 meningiomas\u00a0MIB-1 staining index (%)\u00a0Factor<3.0\u22653.0P ValueAge (years)0.937-49652450-723260-793470-2610Sex (male\/female)46\/19643\/570.0001Recurrence (yes\/no)14\/22834\/660.0001Symptomatic (yes\/no)133\/10970\/300.013Skull base (yes\/no)119\/12338\/620.059Volume (cm3)0.014-9.91133710-531520-7648Multiple (yes\/no)15\/2279\/910.244Edema (yes\/no)82\/16054\/460.001Calcification (yes\/no)97\/14520\/800.0001Total242100\nMeningothelial, transitional, and fibrous meningiomas were the three major subtypes, and they accounted for about three fourth of the total. Two hundred and ninety-six meningiomas belonged to grade I; 28 grade II; and 18 grade III. MIB-1 staining index in 56 of 296 grade I meningiomas was \u2265 3.0; that in 27 of 28 grade II; and that in 17 of 18 grade III, respectively (Table\u00a02).Table\u00a02Histological subtypes and MIB-1 staining index of 342 meningiomasSubtypeMIB-1 staining index (%)Total<3.0\u22653.0Grade IMeningothelial 10628134Fibrous671380Transitional431356Psammomatous606Angiomatous13215Microcystic101Secretory101Lymphoplasmacyte-rich101Metaplastic20224056296Grade IIAtypical12627Chordoid01112728Grade IIIRhabdoid011Papillary112Anaplastic0151511718Total242100342\nLogistic regression analysis demonstrated that male (odds ratio [OR], 2.374, p=0.003), recurrence (OR, 7.574, p=0.0001), skull base (OR, 0.540, p=0.035), calcification (OR, 0.498, p=0.019) were independent risk factors for a high MIB-1 staining index (\u2265 3.0); age, symptomatic, volume, multiple, and edema were not (Table\u00a03). \nTable\u00a03Logistic regression analysis for factors independently related to MIB-1 staining indexFactorOdds ratio95%CIP ValueAge1.1090.841\u20131.4610.464Sex2.3741.336\u20134.2190.003Recurrence7.5743.558\u201316.1240.0001Symptomatic1.4680.774\u20132.7840.240Skull base0.5400.305\u20130.9560.035Volume1.3320.944\u20131.8790.103Multiple1.0270.398\u20132.6510.957Edema1.5080.838\u20132.7110.170Calcification0.4980.278\u20130.8920.019\nDiscussion\nWe analyzed the relationship of the MIB-1 staining indices to the characteristics of 342 consecutive patients with meningioma surgically removed between 1995 and 2004 by logistic regression analysis. Logistic regression analysis demonstrated that male (odds ratio [OR], 2.374, p=0.003), recurrence (OR, 7.574, p=0.0001), skull base (OR, 0.540, p=0.035), calcification (OR, 0.498, p=0.019) were independent risk factors for a high MIB-1 staining index (\u2265 3.0); age, symptomatic, volume, multiple, and edema were not.\nThe relationship between the growth rate or MIB-1 staining index and age has been controversial: a higher MIB-1 staining index and higher growth rate were observed for younger patients [11, 13, 21]; but not in other reports [1, 12, 15, 19]. Our series of 342 patients with meningioma showed no relation.\nIt is well known that atypical and anaplastic meningiomas are predominant in males [10]. Matsuno et al. [11] reported that the mean MIB-1 staining index in 50 male patients was 5.5%, whereas that in 77 female patients was 2.7%. Our findings show that male (odds ratio [OR], 2.374, p=0.003) was an independent risk factor for a high MIB-1 staining index. We also found a higher MIB-1 staining index in males even in grade I meningioma (MIB-1 staining index in 32 of 226 females, and in 24 of 70 males was        \u2265 3.0, p=0.0001, chi-squire test).\nRecurrence (OR, 7.574, p=0.0001) was the most significant independent risk factor for a high MIB-1 staining index (\u2265 3.0). Therefore, we propose prompt management for recurrent meningiomas. In most of the recurrent meningiomas, the MIB-1 staining index was higher at the time of recurrence than at the time of initial surgery [1, 11, 19]. Changes in histological morphology and malignant transformation are also known in meningiomas.\nAlthough there is a significant difference in the MIB-1 staining index between symptomatic and non-symptomatic meningiomas by chi-square test, symptomatic is not an independent risk factor for a high MIB-1 staining index. Meningiomas commonly present with seizure disorders, and are associated with location, perilesional edema, and convexity location. Symptoms and signs of elevated intracranial pressure could be due to the large size of meningioma itself, or to the pronounced cerebral swelling resulting from reactive vasogenic edema. Focal neurological deficits caused by meningiomas are generally related to direct local brain, cranial-nerve compression, and can be predicted from the site of origin of the tumor [20]. Thus, symptomatic meningioma may not be related to a high MIB-1 staining index. The growth rate of incidental meningioma may be similar to that of symptomatic meningioma.\nOur results demonstrated that skull base (OR, 0.540, p=0.035) is an independent risk factor for a high MIB-1 staining index. No relationship has been reported between the MIB-1 staining index and the location of meningiomas [15, 19]. In general, the surgical risk for meningiomas is higher in skull base. A low proliferative potential in skull base meningiomas should be taken into consideration especially when treating elderly patients with asymptomatic meningiomas [9, 16].\nAlthough there was a significant difference of the MIB-1 staining index in tumor size by chi-square test [15], tumor size is not an independent risk factor for a high MIB-1 staining index. The tumor volume is associated with the annual growth rate but not with doubling time [13, 16, 21]. Assuming that a tumor shows a constant relative volume increase, larger tumors will show higher growth rates if the annual volume increase is expressed in absolute values. Large tumors should be carefully observed even though the initial volume is not a risk factor for a high MIB-1 index, and it is not correlated with doubling time.\nThe etiology of peritumoral brain edema associated with meningiomas is multifactorial. Factors that may influence the etiology of peritumoral edema include tumor size, histological subtypes, vascularity, venous stasis, and brain invasion [1, 4]. Ide et al. [4] found a significant correlation of both the MIB-1 staining index and tumor size with the extent of edema. A high MIB-1 staining index itself did not seem to be directly responsible for perifocal edema, since our logistic regression analysis demonstrated that edema is not an independent risk factor for a high MIB-1 index [15].\nTumors with calcification grew significantly less than those without calcification [9, 13, 16]. Absence of calcification on CT correlated strongly with doubling time [5]. Diffusely calcified meningiomas had a low mean MIB-1 staining index of 0.57%. Focally calcified tumors showed a relatively low proliferative potential (0.92%) compared with that of noncalcified tumors (1.75%) [15]. The results are always consistent when the relationship between calcification and proliferative potential or growth rate is compared. We also confirmed this characteristic in meningioma (OR, 0.498, p=0.019).\nIn conclusion, male, recurrence, non-skull base, absence of calcification are independent risk factors for a high proliferative potential. These should be taken into consideration when managing meningiomas.","keyphrases":["growth","meningioma","mib-1"],"prmu":["P","P","P"]}
{"id":"Mech_Dev-2-1-2428104","title":"Polydactyly in the mouse mutant Doublefoot involves altered Gli3 processing and is caused by a large deletion in cis to Indian hedgehog\n","text":"The mouse mutant Doublefoot (Dbf) shows preaxial polydactyly with 6\u20139 triphalangeal digits in all four limbs and additional abnormalities including a broadened skull, hydrocephalus, and a thickened, kinked tail. The autopod undergoes a characteristic expansion between late embryonic day (E) 10.5 and E11.5, following the onset of ectopic Indian hedgehog (Ihh) expression in the entire distal mesenchyme, except for the zone of polarising activity (ZPA), at E10.5. We show here that limb prepattern, as indicated by expression of Gli3 and Hand2 at E9.5 is unaffected by the mutation. As both Sonic hedgehog (Shh) and Ihh expression are present in Dbf limb buds at E10.5, we generated Dbf\/+;Shh\u2212\/\u2212 mutants to analyse the effects of different patterns of Hedgehog activity on the limb phenotype and molecular differentiation. Dbf\/+ embryos lacking Shh showed postaxial as well as preaxial polydactyly, and the Ihh expression domain extended posteriorly into the domain in which Shh is normally expressed, indicating loss of ZPA identity. Differences in gene expression patterns in wild type, single and compound mutants were associated with differences in Gli3 processing: an increased ratio of Gli3 activator to Gli3 repressor was observed in the anterior half of Dbf\/+ limb buds and in both anterior and posterior halves of compound mutant limb buds at E10.5. To identify the cause of Ihh misregulation in Dbf\/+ mutants, we sequenced \u223c20 kb of genomic DNA around Ihh but found no pathogenic changes. However, Southern blot analysis revealed a \u223c600 kb deletion disrupting or deleting 25 transcripts, starting 50 kb 5\u2032 of Ihh and extending away from the gene. The large deletion interval may explain the wide range of abnormalities in Dbf\/+ mutants. However, we did not detect anologous deletions in cases of Laurin\u2013Sandrow syndrome, a human disorder that shows phenotypic similarities to Dbf.\n1\nIntroduction\nThe Dbf mutant, which arose spontaneously in the 3H1 (C3H\/HeH\u00a0\u00d7\u00a0101\/H F1 hybrid) genetic background at Harwell (UK), is a polydactylous mouse that exhibits semidominant inheritance. Mice heterozygous or homozygous for Dbf have 6\u20139 digits in all four limbs; the extra digits are all triphalangeal and arise preaxially (Lyon et al., 1996; Hayes et al., 1998a). Dbf\/+ mice also show malformation of the tibia, a broadened skull, hydrocephalus, a thickened kinked tail, and reduced fertility and viability. Homozygotes additionally exhibit a midline facial cleft but cannot be recovered alive beyond embryonic day (E) 14.5.\nPolydactyly has been described in many mouse mutants, all except two of which show a discrete anterior domain of Sonic hedgehog (Shh) expression (Masuya et al., 1995; Hill et al., 2003). The Extra-toes (XtJ) mutant has an extended Shh domain due to functional inactivation of Gli3 (Hui and Joyner, 1993), whereas Dbf mice exhibit ectopic Indian hedgehog (Ihh) expression in the distal limb bud mesenchyme (Yang et al., 1998). Ectopic Ihh upregulation is first detectable at E10.5 (Crick et al., 2003), the stage at which hyperexpansion of the autopod begins; downstream targets of Shh signalling are ectopically up-regulated (Hayes et al., 1998b; Yang et al., 1998). However, the molecular mechanism by which the polydactyly arises from ectopic Ihh expression has not been investigated.\nThe polydactylous phenotype of the XtJ mutant was originally thought to result from the enlarged Shh expression domain (Hui and Joyner, 1993). However, Shh\u2212\/\u2212;Gli3\u2212\/\u2212 mutants exhibit polydactyly in a similar pattern to Gli3\u2212\/\u2212 mutants, suggesting that the polydactyly of Gli3-deficient mice is independent of Shh (te Welscher et al., 2002). In wild type (wt) limb buds, digital number and identity are regulated by interaction between Shh and Gli3 (Litingtung et al., 2002). In the presence of Shh, Gli3 remains as a 190\u00a0kDa activator species, Gli3A, that up-regulates Hedgehog (Hh)-responsive gene expression, while in the absence of Shh, Gli3A is processed to a smaller 83\u201386\u00a0kDa repressor form, Gli3R, which negatively regulates expression of Shh and its target genes (Dai et al., 1999; Shin et al., 1999; Sasaki et al., 1999). Litingtung et al. (2002) suggested that in wt limb buds the Gli3A:Gli3R ratio controlled by Shh limits the polydactylous potential of the autopod, imposing pentadactyl constraint. This is supported by the localization of Shh protein in wt limb buds, which extends anterior to the zone of polarising activity (ZPA) in a domain coincident with Patched1 (Ptc1) expression (Gritli-Linde et al., 2001), resulting in a posterior-to-anterior increase of the Gli3A:Gli3R ratio (Wang et al., 2000). Consistent with these observations, the Gli3 present throughout Shh\u2212\/\u2212 limb buds is mainly processed to Gli3R (Litingtung et al., 2002). Recently, the mutation underlying the polydactylous chicken talpid3 mutant has been reported to be in a novel gene and has also been shown to result in abnormal Gli3 processing (Davey et al., 2006). Given the evidence of involvement of abnormal Gli3 processing in the XtJ, Shh\u2212\/\u2212 and talpid3 mutants, it is possible that the polydactyly present in Dbf mice also results from aberrant Gli3 processing. This hypothesis is supported by evidence that Gli3 acts downstream of Ihh during endochondral skeletal development (Hilton et al., 2005; Koziel et al., 2005).\nTo investigate the mechanism by which polydactyly arises in Dbf we have analysed gene expression in Dbf\/+ limbs, where there is an excess of Hedgehog (Hh) signalling, and compared this to Shh\u2212\/\u2212 limbs, where there is none. Since Shh and Dbf are located on different chromosomes (5 and 1, respectively) (Blake et al., 2003; Hayes et al., 2001), we have been able to generate mutant mice that carry two copies of the disrupted Shh allele and are heterozygous for the Dbf mutation. To further dissect the mechanisms underlying the limb malformations in both Shh and Dbf mutants, we have analysed the effects of the ectopic Ihh expression associated with Dbf limb abnormalities in the Shh-null background by correlating altered patterns of gene expression with the phenotype of single and double mutants. Differences in Gli3 processing between each genotype suggest that Hh-Gli3 interactions govern the observed differences in digital number, and that postaxial polydactyly results from expression of Ihh, but not Shh, in the posterior ZPA mesenchyme.\nPrevious attempts to identify the Dbf mutation have been unsuccessful. Hayes et al. (2001) constructed a high resolution genetic map and localized the mutation to a 0.4\u00a0cM interval on mouse chromosome 1. This region contained 35 genes including several plausible candidates for the Dbf mutation. However, despite the sequencing of three of these genes, the Dbf mutation remained unidentified. Based on the misregulation of Ihh expression in Dbf, we sequenced \u223c20\u00a0kb of the surrounding genome but found no obvious pathogenic changes. To investigate whether a genomic rearrangement could be responsible, we used the mouse genome sequence to design a Southern blotting strategy to systematically screen the regions 5\u2032 and 3\u2032 of Ihh for copy number changes. We identified a \u223c600\u00a0kb deletion starting \u223c50\u00a0kb 5\u2032 of Ihh, which removes or interrupts 25 known and predicted transcripts. This raises the possibility that additional abnormalities seen in Dbf\/Dbf mice arise from loss of function of deleted genes, in addition to Ihh misregulation.\n2\nResults\n2.1\nThe prepattern of Dbf limb buds is unaffected\nExpression of Hand2 and Gli3 has been implicated in patterning the limb bud prior to Shh expression, and has been shown to be affected later by the absence of Shh (Chiang et al., 2001; te Welscher et al., 2002). We assayed expression of these two genes before (E9.5) and after (E11.5) the onset of ectopic Ihh at E10.5 in Dbf\/+ mutant embryos (Fig. 1). Gli3 expression is restricted to the anterior portion of the limb bud in wt embryos at E9.5 (Fig. 1A) and this expression pattern is not altered in the limb buds of Dbf\/+ mutants (Fig. 1B). Hand2 is expressed throughout the flank of wt embryos prior to formation of the limb bud, then becomes limited to the posterior region of the limb bud as it is initiated (Fig. 1C); this pattern is not altered in Dbf\/+ embryos at E9.5 (Fig. 1D). At E11.5, expression of Gli3 in Dbf\/+ limb buds differs from that in wt embryos in extending more distally; the domain is also broader although this probably simply reflects the greater breadth of the limb bud (Fig. 1F). Expression of Hand2 is limited to the proximal posterior margin in wt E11.5 limb buds (Fig. 1G); in contrast, the Hand2 domain in Dbf\/+ limb buds extends anteriorly and distally (Fig. 1H). Hence the limb prepattern as indicated by the expression of Hand2 and Gli3 at E9.5 is unaffected in Dbf\/+ limb buds, but the expression domains of both genes are altered in association with the presence of ectopic Ihh expression at E11.5 (Fig. 3H).\n2.2\nAltered limb phenotype of Dbf mutants in the absence of Shh\nAs Shh-null embryos die perinatally, gross morphological examination of wt, Shh\u2212\/\u2212, Dbf\/+ and Shh\u2212\/\u2212;Dbf\/+ embryos was conducted at E13.5 and alcian blue staining of the limb bones was carried out at E17.5 (Fig. 2). Both forelimb and hindlimb autopods of Shh\u2212\/\u2212;Dbf\/+ embryos resemble those of Dbf\/+ except that the broadened digital plate is more regular, shows fewer bifurcations, and is more extensive posteriorly (compare Fig. 2B, F and J with D, H and L). At E13.5 the autopod forms a 180\u00b0 fan, and the angle between the autopod and zeugopod on the postaxial side of the limb is decreased to 90\u00b0 (Fig. 2D, arrow).\n2.3\nIhh and Shh expression in compound mutant limbs is mutually exclusive\nThe expansion that characterizes the Dbf\/+ autopod takes place from late E10.5 to E11.5. We therefore analysed the expression domains of Shh and Ihh in limb buds immediately prior to (E10.5) and after (E11.5) the period of expansion. In both wt and Dbf\/+ limb buds at E10.5, Shh is expressed at the posterior margin (Fig. 3A and B), defining this region as the ZPA (Riddle et al., 1993). In wt mice Ihh is not expressed in limbs prior to E12.5 (St-Jacques et al., 1999) while in Dbf\/+ mutant mice, Ihh expression is present in the distal mesenchyme of the limb bud at E10.5 (Fig. 3C). This ectopic Ihh domain extends throughout the area anterior to the ZPA and may correspond to the progress zone. Its absence from the ZPA was confirmed by double in situ hybridization to show nonoverlapping juxtaposed Shh and Ihh expression (Fig. 3D). Expression of Ihh in E10.5 Shh\u2212\/\u2212;Dbf\/+ mutant limb buds extends throughout the distal mesenchyme including the posterior margin, i.e. the domain in which Shh is expressed in Dbf\/+ embryos (Fig. 3E).\nAt E11.5, Shh expression continues in the posterior margin of wt and Dbf\/+ limb buds (Fig. 3F and G). Expression of Ihh in Dbf\/+ mutant limb buds at E11.5 is progressively down-regulated from posterior to anterior, until it remains only in the anterior margin (Fig. 3H); in contrast, in Shh\u2212\/\u2212;Dbf\/+ mutant limbs, down-regulation of Ihh expression begins mid-distally, remaining strong in both the anterior and posterior mesenchyme (Fig. 3I).\n2.4\nGene expression is altered in Dbf limb buds lacking Shh\nTo gain insight into the mechanisms underlying the different patterns of polydactyly generated in the presence of different sources of Hh signalling in Dbf\/+ and Shh\u2212\/\u2212;Dbf\/+ limbs, we examined the expression of genes implicated in Shh signalling and limb patterning in wt, Dbf\/+, Shh\u2212\/\u2212 and Shh\u2212\/\u2212;Dbf\/+ limb buds at E10.5 (Fig. 4); as shown in Fig. 3, this is the stage at which Ihh expression is first detected. Expression of the transcriptional targets of Hh signalling, Ptc1 and Gli1, is expanded anteriorly in Dbf\/+ limbs; interestingly, expression of these genes is broader in the proximal mesenchyme of Dbf\/+limbs lacking Shh, suggesting expansion of the domain of Hh signalling in these limb buds. Conversely expression of Gli3, which is thought to be repressed by Hh signalling (Takahashi et al., 1998), shows a reduced expression domain in Dbf\/+ limbs. As expected, Gli3 is expressed throughout Shh\u2212\/\u2212 limbs at E10.5, but in the presence of Ihh in Shh\u2212\/\u2212;Dbf\/+ mutants it is dramatically down-regulated and required a prolonged colour development time for detection.\nIn wt and Dbf\/+ limbs at E10.5 there is a strong expression of Hand2 in the posterior mesenchyme, with a graded lower expression anteriorly, similar expression is seen in Shh\u2212\/\u2212 limbs. However, in Shh\u2212\/\u2212;Dbf\/+ limbs there appears to be a second strong anterior domain of Hand2 expression, consistent with the extended expression seen at E11.5 (Fig. 1H). As reported previously (Hayes et al., 1998b; Yang et al., 1998), the Hoxd13 domain is expanded anteriorly in Dbf\/+ limb buds; in Shh\u2212\/\u2212;Dbf\/+ limb buds, the domain shows even greater expansion, consistent with the more regular digital fan seen in these mutants. Expression of Fgf8 throughout the AER of expanded Dbf\/+ and Shh\u2212\/\u2212;Dbf\/+ limbs indicates that in both mutants Hh signalling between the mesenchyme and ectodermal AER is intact. Ectopic anterior expression of Fgf4 in the expanded limb buds of both mutants is consistent with their ectopic Ihh expression. Bmp4 expression in the progress zone was slightly down-regulated in Dbf\/+ limbs but up-regulated proximally; like wt limbs, it was absent from the AER. In contrast, Shh\u2212\/\u2212;Dbf\/+ limbs, which showed further down-regulation of Bmp4 in the mesenchyme of the progress zone, showed ectopic expression throughout the AER. Explanation for this pattern requires further investigation.\n2.5\nThe Dbf mutation affects the limb bud Gli3 ratio\nThe action of Gli3 protein as a transcriptional activator relies on its maintenance as Gli3A, which requires Hh signalling (Dai et al., 1999; Sasaki et al., 1999; Shin et al., 1999). To determine the effect of differential Hh signalling on Gli3 processing in mutant limb buds, we used a Gli3 antibody combined with Western blot analysis to assess the comparative levels of Gli3A and Gli3R in the anterior and posterior halves of E10.5 limb buds of all four genotypes (Fig. 5). As reported previously, wt limbs have a higher ratio of Gli3R to Gli3A anteriorly than posteriorly (Wang et al., 2000, and Fig. 5B and C). Dbf\/+ limb buds have a reduced level of the repressor relative to the activator, especially in the anterior half, where levels of the two forms of Gli3 are similar. In Shh\u2212\/\u2212 limb buds the difference between the anterior and posterior halves is greatly reduced with relatively high levels of Gli3R to Gli3A in both halves of the limb bud mesenchyme (Litingtung et al., 2002, and Fig. 5B and C). In Shh\u2212\/\u2212;Dbf\/+ mutants, both halves of the limb bud show a decreased ratio of Gli3R to Gli3A compared with the wt result; the ratio is similar in both halves of the limb bud, in contrast to the Dbf\/+ result which shows an A\u2013P asymmetry.\n2.6\nA \u223c600\u00a0kb deletion underlies Dbf\nTo determine the cause of the ectopic Ihh expression in Dbf limbs, we initially searched for the genetic lesion by sequencing 20\u00a0kb of the region around Ihh in Dbf heterozygotes and both parental strains, but found no pathogenic changes (data not shown). Subsequently we sought genomic rearrangements using a systematic Southern blotting strategy to interrogate the mouse genome sequence (http:\/\/genome.ucsc.edu\/), which initially identified the absence of a polymorphic 8\u00a0kb SpeI fragment in Dbf (see Section 4.5). Characterization of the breakpoint by Southern analysis and subsequently by inverse PCR led to identification of the centromeric breakpoint at position 75,098,488\u00a0bp on chromosome 1 (Fig. 6). Analysis of sequence 3\u2032 to this in Dbf\/+ DNA revealed the telomeric breakpoint to be at position 75,694,480\u00a0bp on chromosome 1. The deleted region therefore appears to be 595,992\u00a0bp; however this figure is not precise because the deletion encompasses a \u223c16\u00a0kb gap in the current mouse genome sequence (mm9 assembly) present between 75,102,130 and 75,118,131\u00a0bp. We confirmed the deletion by PCR using primers flanking the breakpoint and further demonstrated that three different loci distributed within the putatively deleted region were present only in a single copy in Dbf\/+ mutant DNA (see Section 4.5). Analysis of the wt sequences at the two breakpoints showed that the sequence at the centromeric breakpoint is unique, lying within the gene Non-homologous end joining factor 1 (Nhej1). However, a hexanucleotide motif CCAAAC present at the breakpoint is repeated 17 nucleotides upstream, separated by four copies of a trinucleotide CCT motif. The telomeric breakpoint resides within the 3\u2032 terminal region of a B1 repetitive element at the endpoint of a very T-rich motif (35 thymine residues in 47 bases) which is likely to represent the complement of an ancestral poly(A) tract related to the B1 element and does not disrupt any known gene. There is a three nucleotide ambiguity in the position of the breakpoint as the sequence ACA is present on both sides of the deletion (Fig. 6). In addition to disrupting Nhej1, the deletion completely removes 24 known and predicted genes (Fig. 6, Supplementary Table 2 and Section 3).\n2.7\nLaurin\u2013Sandrow syndrome does not result from large deletions 5\u2032 of IHH\nLaurin\u2013Sandrow syndrome (LSS) (MIM 135750) is rare human developmental disorder characterized by triphalangeal preaxial polydactyly of the hands and feet, with variable involvement of the proximal limb elements. It has been previously suggested that LSS shares many similarities with Dbf and may also arise from ectopic IHH expression (Innis and Hedera, 2004). To investigate the possibility that Dbf and LSS share a common etiology, we screened five patients diagnosed with LSS for copy number variation at 23 sites between IHH and EPHA4 using multiplex ligation-dependent probe amplification (see Supplementary Information). No copy number variation was detected (data not shown).\n3\nDiscussion\n3.1\nEctopic Ihh expression in Dbf\/+ is modified in the absence of Shh and is associated with loss of ZPA identity\nAlthough we have previously shown that expression of ectopic Ihh in Dbf\/+ limb buds coincides with the onset of limb bud expansion at E10.5 (Crick et al., 2003), it was not known whether the prepattern of Dbf limbs might be affected by the mutation prior to Ihh expression. However, no differences were detected in the expression of Gli3 or Hand2 in wt and Dbf\/+ embryos at E9.5 or E10.5, consistent with the hypothesis that ectopic Ihh expression represents the primary pathogenic event. By E11.5, expression domains of both Hand2 and Gli3 were more extensive in Dbf\/+ than wt limb buds, suggesting that Ihh signalling is able to modify their expression.\nIn Dbf\/+ limb buds, Ihh and Shh are expressed in discrete adjacent domains. Exclusion of Ihh from the Shh domain is reminiscent of the exclusion of the Hh-inducible gene Gremlin from this domain; Scherz et al. (2004) suggested that the effect may be due to high levels of intracellular autocrine Shh signalling. The loss of identity of the ZPA resulted in a abnormal expansion of the posterior limb bud mesenchyme in Shh\u2212\/\u2212;Dbf\/+ mice leading to the additional postaxial polydactyly seen in these mutants.\n3.2\nAbnormal gene expression leading to an aberrant Gli3 ratio underlies Dbf polydactyly\nTo elucidate the limb patterning underlying Dbf\/+ polydactyly and to investigate the generation of the broader, more regular fan of digits seen in Shh\u2212\/\u2212;Dbf\/+ mutants, we studied the expression of a range of limb patterning and development genes at E10.5. Dbf\/+ mutant limbs show an anterior expansion of the positive regulators of Hh signalling Ptc1, Gli1 and the downstream targets Hoxd13 at E10.5 and Hand2 by E11.5. Dbf\/+ limbs also show a reduction of Gli3 expression, which is thought to be negatively regulated by Hh signalling. Conversely, due to the complete lack of Hh activity in Shh\u2212\/\u2212;Dbf\/+ mutant limbs prior to E10.5, Gli3 is ubiquitously expressed in these limb buds until this stage, when it is down-regulated in Shh\u2212\/\u2212;Dbf\/+ but not Shh\u2212\/\u2212 mutants. Gli3R is thought to repress expression of Hoxd13 and Hand2 and Fgf4 in the anterior of wt limb buds while Gli3A induces the expression of Gli1 in the posterior region (reviewed in Tickle, 2006). Therefore, the postaxial polydactyly seen in Shh\u2212\/\u2212;Dbf\/+ mutants may be due to the loss of identity of the ZPA with concomitant posterior extension of the Ihh domain. In contrast, the preaxial polydactyly that is present in both Dbf\/+ and Shh\u2212\/\u2212;Dbf\/+ mutants is correlated with ectopic Gli3A-induced Hh signal transduction together with lack of repression of posterior patterning genes by Gli3R in the anterior of the limb bud. We suggest that the discrepancy between the very low level of Gli3 mRNA (Fig. 4) and the Gli3 protein detected in Shh\u2212\/\u2212;Dbf\/+ limb buds at E10.5 (Fig. 5) indicates the perdurance of protein after the gene has been down-regulated.\n3.3\nIdentification of the Dbf mutation\nThe interpretation of the mechanism of the Dbf mutation has been hampered previously by the failure of attempts to identify the underlying mutation. Using a Southern blotting and inverse PCR strategy we have demonstrated that a \u223c600\u00a0kb deletion underlies the Dbf phenotype. The presence of simple sequences at both breakpoints may have predisposed them to breakage; the lack of significant similarity between the breakpoints (except for a 3 nucleotide identity at the breakpoints themselves) suggests that the rearrangement is likely to have involved nonhomologous end joining (NHEJ). Further analysis of sequence at the breakpoints revealed that the distal breakpoint resides within the degenerate poly(A)n tract of a short retrotransposon (SINE) of the rodent B1 family, which, like human Alu repeats originate from 7Sl RNA (Vassetzky et al., 2003).\nThe deleted region in Dbf is relatively gene-dense and completely deletes 24 known and predicted transcripts as well as interrupting Nhej1 at the centromeric breakpoint. Several of these genes have previously been implicated in abnormal mouse phenotypes or human disease; information on the known expression patterns and functions of these genes is summarized in Supplementary Table 2. Abnormalities associated with genes in the deleted region may contribute to additional aspects of the heterozygous Dbf phenotype such as the broadened skull, hydrocephalus, reduced viability and fertility, thickened tail and supernumerary hair follicles. However, none of the homozygous null phenotypes resulting from specific targeting of the Ptprn, Des, Inha or Slc4a3 genes is lethal in late embryogenesis so the cause of death at E14.5 in Dbf homozygotes remains unclear. This could be attributable to loss of function of any of the genes within the interval for which homozygous mice have not yet been described, and\/or to homozygosity for the ectopic Ihh expression defect. Interestingly a recent study reported a human fetus with a balanced de novo translocation t(2;7)(q36;p22) with the chromosome 2 breakpoint interrupting the orthologue of Nhej1 at a position similar to the start of the Dbf deletion (Cantagrel et al., 2007). The consequence of this translocation, as in Dbf, would be to isolate the human IHH gene from possible regulatory sequences present on the opposite side of the NHEJ1 breakpoint. Although the terminated fetus exhibited syndactyly of all four limbs, polydactyly was not present, suggesting that the translocation did not result in ectopic IHH expression.\nWe have presented evidence that the prepattern of Dbf limb buds is unaffected and that the preaxial polydactyly is attributable to a reduction in Gli3R resulting from ectopic Ihh expression. It is interesting that preaxial polydactyly, the most striking aspect of the Dbf phenotype, is unlikely to result directly from haploinsufficiency of any of the genes in the deleted region. Rather, the deletion appears to affect a cis-acting regulatory element of Ihh, which could be a repressor located within the deletion, or an enhancer beyond the deleted region. Other examples of regulatory mutations acting at a distance have been reviewed by Kleinjan and van Heyningen (2005). Pinpointing the regulatory sequences involved remains a major challenge, one notable success being the identification of the ZPA sequence regulatory sequence (ZRS) which lies \u223c1.0\u00a0Mb upstream of Shh and regulates its expression in the ZPA; mutations in the ZRS lead to ectopic Shh expression resulting in preaxial polydactyly (Lettice et al., 2002). However, owing to the large size of the Dbf deletion and the large number of genes and highly conserved non-coding elements within it, it will be challenging to delineate the precise mechanism underlying ectopic Ihh expression in the Dbf mouse.\n4\nMaterials and methods\n4.1\nGeneration and identification of mutant mice\nMice heterozygous for the Shh null allele (Chiang et al., 1996) on the C57BL\/6J background were mated to Dbf\/+ mice on the 3H1 background. The Shh mutant allele was detected as previously described (Chiang et al., 1996). Homozygous Shh\u2212\/\u2212 embryos were identified by their phenotype.\nTo genotype Dbf\/+ embryos (prior to the identification of the causative deletion), Dbf\/+ mice were crossed with wt Mus musculus castaneus and the Dbf F1 progeny were bred with C3H wt mice. Embryos were genotyped using primers which amplify the marker D1Mit46 located \u223c2.3\u00a0cM from Ihh (P1 5\u2032-AGTCAGTCAGGGCTACATGATG-3\u2032, P2 5\u2032-CACGGGTGCTCTATTTGGAA-3\u2032). This produces amplification products of 276\u00a0bp and 320\u00a0bp on the C3H and Mus musculus castaneus backgrounds respectively.\n4.2\nWhole mount in situ hybridization\nDoubly heterozygous Shh+\/\u2212;Dbf\/+ were crossed with Shh+\/\u2212 mice and embryos of all six possible genotypes were collected for analysis of gene expression domains and morphology: wt, Shh+\/\u2212, Shh\u2212\/\u2212, Dbf\/+, Shh+\/\u2212;Dbf\/+ and Shh\u2212\/\u2212;Dbf\/+. To ensure consistency between developmental stages, only forelimb buds were analysed and a minimum of two samples were examined with each probe. Timing of embryos was by the vaginal plug method: 12.00 noon on the day on which the plug was observed was regarded as E0.5. Pregnant females were sacrificed on the appropriate day by cervical dislocation and the embryos were dissected from the uterus in ice cold phosphate buffered saline (PBS) (140\u00a0mM NaC1, 3\u00a0mM KCl, 10\u00a0mM Na2HPO4, 2\u00a0mM KH2PO4) followed by immersion in cold tissue fixative in accordance with the Animals (Scientific Procedures) Act, 1986. Where necessary, yolk sacs were removed for genotyping and embryos were fixed by immersion overnight at 4\u00a0\u00b0C in 4% paraformaldehyde in PBS. Embryos were dehydrated by sequential washing in 25%, 50%, 75% ethanol in PBT (PBS\u00a0+\u00a00.1% Tween 20) and finally by two washes in 100% ethanol. They were stored at \u221220\u00a0\u00b0C until required.\nSingle stranded digioxygenin-UTP labelled antisense riboprobes were generated from linearized plasmids containing cDNAs. Whole mount in situ hybridization was carried out essentially as described by Wilkinson (1992).\n4.3\nSkeletal preparations\nEmbryos for skeletal staining were dissected and fixed in 95% ethanol for 1\u20133 days. They were immersed in alcian blue stain (75% ethanol, 20% acetic acid, 3\u00a0mg\/ml alcian blue) for 21\u201328 days at 37\u00a0\u00b0C. They were cleared in 0.8% KOH, 20% glycerol. Following clearing, they were sequentially dehydrated and stored in 50% ethanol\/50% glycerol.\n4.4\nWestern Blotting\nThe polyclonal antibody specific for the amino terminus of Gli3 was a gift from Dr. Chin Chiang (Litingtung et al., 2002). Three \u03bcg of protein lysate derived from the anterior and posterior halves of \u223c6 E10.5 forelimb buds were resolved on 4\u201312% polyacrylamide gels. Gli3 protein was detected using anti-N-terminal Gli3 (1:300) primary antibody and biotinylated anti-rabbit immunoglobulin-\u03b3 secondary antibody (1:1000). Protein bands were visualized by incubation with a streptavidin-peroxidase conjugate followed by an enhanced chemiluminescence detection method (Amersham).\n4.5\nCharacterization of the Dbf deletion\nTo determine whether genomic rearrangements were associated with Dbf, single copy probes labelled with \u03b132P-dCTP were synthesized and used to hybridize Southern blots of DNA isolated from heterozygous 3H1 (C3H\u00a0\u00d7\u00a0H101 hybrid) Dbf mice and wt mice from both background strains. We used the mouse genome mm9 sequence release (July 2007) for all analyses presented in this paper. A probe corresponding to 75,099,314\u201375,099,651\u00a0bp revealed the absence of a polymorphic 8\u00a0kb SpeI fragment in Dbf, found in the C3H parental strain, suggesting the existence of a deletion. A further Southern blot using a probe corresponding to 75,097,987\u201375,098,255\u00a0bp revealed a 1.2\u00a0kb BspHI fragment present only in Dbf. This 1.2\u00a0kb breakpoint fragment was isolated by inverse PCR. Briefly, genomic DNA from Dbf was digested with BspHI, diluted to 10\u00a0ng\/\u03bcl and T4 DNA ligase was added to promote intramolecular ligation. Religated DNA was used directly in an inverse PCR using the primer pair: 5\u2032-GCATTTGAGATTGAGACAAGCACTCTCCACAC-3\u2032 and 5\u2032-ACAGCGCTAGACAGAAAGCCTGCTTGCT-3\u2032. DNA sequencing revealed 228\u00a0bp of unknown sequence that was shown by BLAST analysis to originate from a region of chromosome 1, \u223c596\u00a0kb telomeric from the breakpoint. PCR amplification with primers designed either side of the breakpoint (5\u2032-TGGTCTGGAGAGACAGCTCGTCCAGAG-3\u2032 and 5\u2032-GAGTTGAAGAGTTGGCATAGTGGTGCACAC-3\u2032) was employed to confirm the site of the deletion.\nTo confirm the Dbf lesion was a true deletion, primers were designed to amplify three regions within the deletion predicted to contain polymorphisms variable between the C3H and H101 background strains. These regions were located at \u223c150\u00a0kb intervals within the deleted region. The primer pairs used were site 1, 5\u2032-GCCCTCATGCTTGAGTACCTTGCCTGTGAT-3\u2032 and 5\u2032-GTCCTCCCAGGGGCTGAGCAGAGTG-3\u2032; site 2, 5\u2032-TAGACTGAGCACCCGGCCTAACATGCTC-3\u2032 and 5\u2032-TGTGTCATCCACCCGGTGCCTCTGACT-3\u2032; site 3, 5\u2032-TAGAATTCCCACTGGGTCCACCCACTC-3\u2032 and 5\u2032-CATACATCCGTGTACATGTACTGACTGTCACTG-3\u2032. Amplification products were digested with appropriate restriction endonucleases to discriminate between the alleles. Site 1 contained a novel TACC insertion polymorphism and was digested with HphI, site 2 contained a known C\/T polymorphism (rs31657679) and was digested with AvaI and site 3 contained a known polymorphism (rs3049959) and was digested with Hpy8I. In each case only the H101 allele was present indicating that the C3H chromosome carried the deletion. The presence of both background strains was confirmed on the centromeric side of the Dbf deletion by sequence polymorphisms observed during Southern blotting (data not shown). Both strain backgrounds were shown to be present on the telomeric side of the deletion by AseI restriction digest of a fragment containing a novel informative C\/T polymorphism which was amplified using the primer pair 5\u2032-CAACAAAGCCCACATCAATTCACTCAGGCCGTG-3\u2032 and 5\u2032-CACCCTGCCTCAACCTCTCACCTGCTAG-3\u2032.","keyphrases":["ihh","shh","limb patterning","digit formation"],"prmu":["P","P","P","R"]}
{"id":"Eur_Radiol-3-1-2077918","title":"Multi-detector row computed tomography angiography of peripheral arterial disease\n","text":"With the introduction of multi-detector row computed tomography (MDCT), scan speed and image quality has improved considerably. Since the longitudinal coverage is no longer a limitation, multi-detector row computed tomography angiography (MDCTA) is increasingly used to depict the peripheral arterial runoff. Hence, it is important to know the advantages and limitations of this new non-invasive alternative for the reference test, digital subtraction angiography. Optimization of the acquisition parameters and the contrast delivery is important to achieve a reliable enhancement of the entire arterial runoff in patients with peripheral arterial disease (PAD) using fast CT scanners. The purpose of this review is to discuss the different scanning and injection protocols using 4-, 16-, and 64-detector row CT scanners, to propose effective methods to evaluate and to present large data sets, to discuss its clinical value and major limitations, and to review the literature on the validity, reliability, and cost-effectiveness of multi-detector row CT in the evaluation of PAD.\nIntroduction\nBefore multi-detector row CT (MDCT) technology was available, the evaluation of peripheral arterial disease (PAD) using CT was restricted to imaging only a portion of the peripheral arterial tree [1\u20138]. With the introduction of four-detector row CT (4D-CT) in 1998, this major limitation was overcome. A complete coverage of the lower extremity inflow and runoff arteries was possible with one acquisition using a single-contrast bolus. With the launch of the 16-detector row CT (16D-CT), the spatial resolution increased to near isotropic voxels and the contrast medium efficiency improved [9\u201311]. True isotropic high spatial resolution of the entire volume was possible using the 64-detector row CT (64D-CT) scanner. In addition, improved X-ray tube capacity and scan speed allow submillimeter acquisition of a large coverage without limitations. These developments made multi-detector row CT angiography (MDCTA) an accurate alternative for the assessment of the peripheral arteries [12\u201325]. Using standardized scanning and reviewing protocols, peripheral CT angiography is a robust non-invasive technique for evaluating chronic and acute disease of the peripheral arteries. We present a review concerning our experience with 4-, 16-, and 64-detector row CT scanners in patients with PAD.\nTechnique\nPreparation\nThere are no specific prescanning preparations necessary for MDCTA of the peripheral arteries. The patient is placed comfortably to avoid movement, in the supine position with raised arms on the CT table. The legs are stabilized with cushions around the legs and slightly strapped with adhesive tape distally. It is important that the patient does not wear metal zippers or buttons on their clothing, since this can have a negative influence on the image quality, especially when using postprocessed images. Oral contrast should not be used, as this complicates postprocessing display (Table\u00a01). Contrast material needs to be administered at body temperature to decrease the viscosity. The protocol can be completely programmed into the scanner. \nTable\u00a01Prescanning preparationParameterDescriptionClothingNo metal parts on clothingOral contrastNoneI.V. cannula antecubitalMinimally 22 G (0.6\u00a0mm inner diameter, blue valve)PositioningSupine, stabilized and lightly strapped, feet-first and arms elevatedRespiratory phaseInspiration during abdominal-pelvic range\nTechnical parameters\nThe main challenge for peripheral CT angiography is the great range of the vascular system that needs to be depicted. Using a scanogram of approximately 1,500-mm length, the coverage of the acquisition is planned from the celiac trunk (T12 vertebral body) to the level of the talus using 4D-CT, or to the level of the feet using 16D-CT or higher (Fig.\u00a01).\nFig.\u00a01(a) Scout image with three planned reconstruction batches of the abdomen, the upper legs, and the lower legs to preserve postprocessed image resolution. The frames 3-1, 3-2, and 3-3 depict the field of view of the three data sets, which need to be as narrow as possible to optimize pixel size. Whole-body volume maximum intensity projection (MIP) images after semiautomated bone removal of the abdominal data set (b), the femoral data set (c), and the crural data set (d)\nScan duration\nThe optimal scan duration for peripheral CT angiography varies between approximately 20 to 40\u00a0s, depending on the number of detector rows and the collimation (Table\u00a02). The velocity of a contrast bolus to travel from the aorta to the popliteal arteries varies from 29 to 177\u00a0mm\/s in patients with PAD [26]. This large variability is unpredictable and does not correspond to the severity of PAD. Based on these bolus travel times, it is recommended to limit the maximum table speed on faster scanners to 30\u00a0mm\/s to avoid outrunning the bolus, leading to poor distal vessel opacification. This can be obtained, for example, by limiting the gantry rotation speed from 0.33 to 0.5 rotations per second or reducing the pitch (Table\u00a02). Moreover, it is advised to program a second acquisition protocol into the scanner to start immediately if delayed distal enhancement is detected (Fig.\u00a02). Because the time of the contrast bolus to travel from the aorta to the ankles varies from 7 to 40\u00a0s, a longer scan duration increases the risk of venous contamination, especially when there is critical ischemia and inflammation [26, 27]. Nevertheless, the discrimination of the arteries from the veins is often possible due to the stronger arterial enhancement and the anatomic 3D information [12]. \nTable\u00a02Acquisition parameters for various multi-detector row computed tomography (MDCT) configurations for the angiography of peripheral arteriesType of scannerSection collimation width (mm)bRotation time (s)PitchcTable feed (mm\/ rotation)Table speed (mm\/s)Scan duration (s) dCharacteristics4D-CTa4\u2009\u00d7\u20092.50.51.5153040Slow scan protocol, thick minimal slice width16D-CTa16\u2009\u00d7\u20090.750.51.3153040Slow scan protocol, high resolution16\u2009\u00d7\u20091.50.50.7173435Slow scan protocol, less resolution, better in obese patients16\u2009\u00d7\u20091.50.51.0244825Fast scan protocol, less resolution, reduction of contrast media64D-CTa2\u2009\u00d7\u200932\u2009\u00d7\u20090.60.50.8153040Slow scan protocol, high resolution, isotropic voxel, double z-sampling, scanning of obese patients possible2\u2009\u00d7\u200932\u2009\u00d7\u20090.60.330.8164825Fast scan protocol, reduction of contrast media, high resolution, isotropic voxel, double z-sampling, scanning of obese patients possible2\u2009\u00d7\u200932\u2009\u00d7\u20090.60.331.019.86020Fast scan protocol, reduction of contrast media, high resolution, isotropic voxel, double z-sampling, scanning of obese patients possible, risk of outrunning the bolusaProtocols designed for Siemens CT scanners (Siemens Medical Systems, Erlangen, Germany) and should be modified appropriately for other models and manufacturersbValues are number of sections times section widthcPitch as the ratio of the table feed per rotation over the total width of the collimated beamdScan times representing a scanned range of 120\u00a0cmFig.\u00a02Images from the first and second delayed acquisition of a 37-year old male with blue toe syndrome of the left hallux. (a and b) VRT images of the first acquisition show in the aneurysmatic abdominal aorta a short occlusion of the left femoral artery (white arrows) due to thrombo-embolism and an occlusion of the entire right superficial femoral artery. The anterior tibial arteries seem occluded in both legs. (c and d) VRT image of the feet with the first and the delayed second acquisition. The first acquisition (c) shows that the arteries of the feet are not enhanced yet due to slow flow (asterisk). The delayed acquisition (d) shows that both dorsal pedal arteries are patent and that the proximal arcuate artery and the first dorsal metatarsal artery (black arrows) of the left foot are occluded due to thrombo-embolism\nUsing recent MDCT scanners, fast scans (25\u00a0s or less) can be performed of the peripheral arteries to reduce the amount of contrast media. To allow fast scan speed using 16-detector row CT (16D-CT) scanners, a wider collimation must be used (Table\u00a02). The 64-detector row CT (64D-CT) even allows to perform fast scans while maintaining submillimeter collimation. However, to ensure distal opacification, the scanning delay must be increased appropriately in fast scans. Another difficulty of a fast scan is that there is a greater risk of asymmetric enhancement in patients with severe unilateral vascular disease. Therefore, it is safer to choose a slower scan speed.\nCollimation\nTo aim for maximal spatial resolution, a thin section collimation width allows a narrow effective slice width. Furthermore, the partial volume effect and blooming effect of calcium will be reduced (Fig.\u00a03) [10]. The collimation should be chosen as narrow as possible but still allowing for a table speed of 30\u00a0mm\/s and depends on the number of detector-rows and heat capacity. On a 4-detector row CT (4D-CT), the collimation is limited to 4\u2009\u00d7\u20092.5\u00a0mm, whereas the 16D-CT and 64-detector row CT (64D-CT) allow a submillimeter collimation of 16\u2009\u00d7\u20090.75\u00a0mm and 32\u2009\u00d7\u20092\u2009\u00d7\u20090.6\u00a0mm, respectively.\nFig.\u00a03a, bImages of 16-detector row CT (16D-CT) acquired with a collimation of 0.75\u00a0mm showing the effect of slice width (SW) on the blooming of the arterial wall calcifications. (a) Reconstructed axial image of the right external and internal iliac artery with SW of 3.0\u00a0mm using a B46 reconstruction kernel shows more blooming of the calcifications than (b). (b) Reconstructed axial image of the right external and internal iliac artery with SW of 0.75\u00a0mm using a B46 reconstruction kernel with less blooming of calcifications\nUsing 16D-CT in obese patients, the thin collimation protocol leads to unacceptable noise levels in the abdomen and pelvis because the tube is unable to deliver the necessary dose in this submillimeter configuration. In order to enable the tube to deliver a higher dose, a wider collimation (16\u2009\u00d7\u20091.5\u00a0mm) with a reduced pitch factor of 0.7 (Table\u00a02) is used to improve the image quality in obese patients. For the 64D-CT scanner, there is no longer a tradeoff between resolution and scan speed, and it allows, even in obese patients, a fast submillimeter scan protocol.\nContrast injection\nIt is important in peripheral CT angiography to obtain a high and homogenous enhancement of the arterial tree and to synchronize the acquisition with the enhancement. The optimization of acquisition timing and contrast medium delivery is essential for vascular assessment and image postprocessing. Normally, attenuation values higher than 200\u00a0HU in the arteries is considered suitable in MDCTA [12, 13]. For the intravenous injection of contrast medium in the antecubital vein, 22- and 20-gauge intravenous cannulas are needed for the maximal flow rates of 3.5 and 5.0\u00a0mL\/s, respectively.\nAcquisition timing\nDue to the interindividual hemodynamic variability in peripheral CT angiography, reliable timing techniques are preferred over using a fixed delay. The test-bolus technique relies on the dynamic monitoring of small contrast boluses to measure the contrast arrival and travel time at the proximal and distal arteries, respectively. The bolus-triggering technique is a commonly used timing technique that is based on repetitive low-dose sequential scans at the level of the abdominal aorta, to monitor the arrival time of the contrast media. The acquisition starts automatically when the preferred threshold is reached, approximately 100 to 150\u00a0HU above the baseline value. During a transition delay, which is the time needed for the table to move and start the scan, of approximately 4\u00a0s, breathing instructions can be given to the patient. During this delay, the enhancement of the aorta will further increase to an absolute value of more than 200\u00a0HU.\nFor a fast scan protocol, an extra delay must be added to the contrast arrival time to ensure distal arterial opacification [28]. This extra delay can be calculated as 35\u00a0s minus the scanning time. Thus, for a scan time of 25\u00a0s, a extra delay of 10\u00a0s. must be added. Another option is to monitor at the proximal level of the popliteal artery and to start the scan manually when enhancement is visualized. Consequently, the time of contrast arrival increases by approximately 8 s [26, 27] and the transition delay of the scanner increases to 11 s to travel from the knees to the diaphragm and then starting the acquisition.\nContrast injection\nThe volume of contrast material ranges from 120 to 160\u00a0ml for a typical scan duration of 40\u00a0s. The amount of contrast media depends on the scan duration and on the flow rate. Because the last volume of the bolus will not contribute to the enhancement when scanning below the knees, the injection duration can be shortened by 5\u00a0s, e.g., a 35-s injection time is used for an acquisition of 40\u00a0s. However, to ensure the enhancement of all arteries, the injection duration should not be shorter than 30 s and in fast scan protocols, a delay time needs to be added appropriately to prevent outrunning the contrast bolus. A flow rate of 3 to 4\u00a0ml\/s is necessary for adequate arterial enhancement [12]. This corresponds to an iodine administration rate of 1.0 to 1.4\u00a0g\/s using a contrast media concentration of approximately 320 to 350\u00a0mg I\/mL. Based on the reported literature the average values of contrast media volume, concentration, injection rate, and administration rate are 134\u00a0ml, 341\u00a0mg I\/mL, 3.5\u00a0ml\/s, and 1.2\u00a0g\/s, respectively [9\u201325, 29\u201336]. By increasing the iodine concentration to a concentration of 400\u00a0mg I \/mL, the iodine administration rate can be increased to 1.6\u00a0g\/s to increase the enhancement [37]. To optimize the enhancement, 20 to 60\u00a0mL of saline is injected immediately after the contrast media. A tighter bolus can be obtained to increase the attenuation.\nUsing a monophasic injection rate, the arterial enhancement increases over time to decrease at the end of the bolus. Consequently, the Hounsfield values of the enhanced arteries start lower at the level of the aorta and increase at the level of the popliteal artery to the highest attenuation value, and, subsequently, decrease distally in the runoff arteries, especially for longer scan durations [26]. A more homogenous enhancement can be achieved using a biphasic injection rate using a higher rate (5\u20136\u00a0ml\/s) at the beginning (during the first 5\u00a0s) of the injection and a lower rate (3\u00a0ml\/s) for the remaining volume. In clinical practice, a monophasic injection rate is often used because it is a simple method and has resulted in adequate image quality [37].\nPatient dose in MDCT\nA particular concern with MDCT scanners is delivering potentially higher radiation doses. To maintain the noise level in submillimeter slices, the dose needs to increase proportionally. On the other hand, with the increasing number of detector rows, the z-axis efficiency improves, since the overbeamed area decreases. Current MDCT scanners present an indication of patient dose on the scanner console for dose awareness and to help optimize the scan protocol. Useful in CT angiography is that, when reducing the X-ray energy, the contrast-to-noise ratio increases. Compared to a standard scan with 120\u00a0kVp, selecting 100\u00a0kVp, results in a dose saving of approximately 40% [38\u201340]. Furthermore, dose reduction can be achieved by decreasing the tube current using automatic tube current modulation. With angular tube current modulation, the tube current varies during the course of a rotation. The changing attenuation through different projections around the patient (e.g., at the level of the pelvis) can be used, to reduce unnecessary x-rays in the anterior\u2013posterior projection without any substantial effect on image quality [9, 41, 42]. With longitudinal tube current modulation, the tube current varies along the z-axis based on the size, shape, and attenuation to maintain a predefined noise ratio. Compared with constant tube current, this technique results in acceptable image noise and a dose reduction of 20% or more without compromising diagnostic image quality [9, 41, 42].\nThe average patient dose reported in the literature in the assessment of PAD with CT angiography is 7.47\u00a0mSv [9, 12, 24, 31, 43]. The radiation risk from these doses is not a major concern in patients with PAD. Their life expectancy is shorter than the latency period of a radiation-induced fatal malignancy [44\u201346].\nDisplay and evaluation\nImage reconstruction\nThe raw data set is reconstructed using an increment with 50% to 70% overlap. Peripheral CT angiography generates more than 1,500 axial images, depending on slice width and reconstruction increment. It is recommended to reconstruct separate data sets. Routinely, we calculate three separate data sets of the peripheral runoff (Fig.\u00a01). The first advantage is that it allows us to reconstruct thicker slices, e.g., of 1.5\u00a0mm for the abdominal and femoral data set, and thinner slices for the crural data set to optimize the resolution and to minimize the data load [9, 10, 16, 20, 23]. Secondly, longitudinal images that are calculated from the entire data set have a decreased resolution, due to the limited display matrix (e.g., 512\u2009\u00d7\u2009512) [32]. Images that are reconstructed from the separate data sets will preserve the initial longitudinal resolution.\nA smooth kernel (B20 for Siemens CT scanners) is generally used in CT angiography and leads to an accurate depiction of the diameter of the vessels and is very appropriate for postprocessing. A sharp kernel (B46) is used when stents or severe vessel wall calcifications are present, as it minimizes the blooming effect at the cost of some increase in the noise level [47].\nThe field of view (FOV) is selected as small as possible to optimize pixel size. A FOV of 380\u00a0mm, 350\u00a0mm, and 300\u00a0mm for the abdominal, femoral, and crural data sets, results in pixel sizes of approximately 0.74\u00a0mm, 0.68\u00a0mm, and 0.58\u00a0mm, respectively. Also, the FOV can be further decreased to 200\u00a0mm by including only one leg, leading to a pixel size of 0.4\u00a0mm.\nAdvanced postprocessing and image evaluation\nAdditional two-dimensional (2D) and three-dimensional (3D) postprocessing techniques are required to facilitate interpretation and presentation. Reviewing exclusively the transverse images is inefficient and less accurate than reviewing a combination of reformatted images.\nTo preserve the study quality for clinical decision making, a standard set of postprocessed images needs to be included in the protocol. These include thin-slab maximum-intensity projections (MIPs) through visceral and renal arteries and the abdominal aorta, through femoropopliteal arteries, and through crural arteries (Fig.\u00a04); whole-volume MIPs of the separate data sets after bone removal (Fig.\u00a01) and when necessary, after removal of vessel wall calcifications (Fig.\u00a05); and curved planar reformations (CPRs), e.g., through the iliac arteries. Volume-rendered (VR) images are fast and effortless created to present the pathology to clinicians, who normally do not have the possibility to review the data set interactively.\nFig.\u00a04a\u2013dStandard slab MIP images in the postprocessing protocol make MDCTA of peripheral arteries on a routine basis feasible. Slab MIPs are easy and fast to create and to evaluate. The images depict the vasculature without superimposing bones (a and b). From the abdominal data set, MIP images are created in coronal projection to depict the renal arteries (b) and in sagittal projection to depict the celiac trunk and mesenteric arteries (d). The aorta is also depicted for evaluation. (c) Standard coronal slab MIPs from the data set of the upper legs are created, which are parallel to the superficial femoral and popliteal artery. (d) Standard coronal slab MIPS from the data set of the lower legs display the crural arteriesFig.\u00a05a\u2013dVolume MIP images in anteroposterior projection show the result of three different threshold levels used for the segmentation of arterial wall calcifications. (a) Volume MIP before removal of the calcifications shows that the lumen is not visible. (b) Volume MIP after removal of the calcifications shows that, still, many voxels of calcification are present, hampering lumen assessment (arrows). (c) Volume MIP shows angiogram after calcium segmentation using a correct threshold level allowing lumen assessment. The rest of the voxels of the burden of calcifications are just visible as unesthetical noise, which is, however, preferable to introducing pseudo-stenoses (d) (arrows) by using a too low threshold level\nThe data sets are reviewed effectively by evaluating the standard set of postprocessed images and, interactively, exploring the data set using multiplanar reformations (MPRs) . The transverse images (or true cross-sectional images) need to be considered to verify diseased segments [4, 6, 19].\nWhen extensive calcifications or stents are present, the vessel lumen visibility and the clinicians\u2019 confidence in the CT images will decrease [25]. In whole-volume MIPs, superimposing calcifications can be selected to be removed digitally from the data set using thresholding and region-growing techniques (Fig.\u00a06). However, the removal of the numerous arterial wall calcifications can be very time consuming. Another limitation of these segmentation techniques is that readers should be aware of artificial stenoses and occlusions. These can be introduced when voxels that represent lumen are inadvertently removed when in close contact with the bones (Figs.\u00a07 and 8) or when a too low threshold value is used (Fig.\u00a05). In addition, in VR images, the lumen is also obscured by vessel wall calcifications and, as a result, should not be used for the lumen assessment (Fig.\u00a06). A more reliable technique for stenosis detection in extensive calcified arteries is CPR, which displays the lumen as a longitudinal cross-section (Fig.\u00a06). When using an application that semi-automatically traces the vessel lumen, the risk of an inaccurately positioned central lumen line is minimized. The CPR projection should include at least two perpendicular longitudinal projections and true cross-sectional images can be viewed for lumen assessment [19]. Software tools are available for automatic quantitative evaluation of the traced lumen and to generate a graphical presentation of luminal diameter (Fig.\u00a09). Multipath CPRs are under development and could enhance image evaluation.\nFig.\u00a06a\u2013dInfluence of vessel wall calcifications on postprocessed images and the ability for lumen assessment. (a) VRT image (medial view) of right femoropopliteal segment showing arterial wall calcification; does not allow luminal assessment. (b) CPR image (anteroposterior view) shows the interior of blood vessels as a longitudinal cross-section, even in the presence of the arterial wall calcifications. This is the preferred imaging technique when extensive calcifications of the vessel wall are present. Volume MIP (anteroposterior view) after bone removal using region-growing and threshold techniques (c) does not allow lumen evaluation. Volume MIP after additional calcification removal (d) removes superimposing calcification to enable lumen evaluationFig.\u00a07a\u2013cImages of segmentation artifacts due to bone removal in 16D-CTA. (a) Volume MIP after bone segmentation of the lower legs showing a pseudo-occlusion of both distal anterior crural arteries (arrows), which is caused by segmentation of the bones. (b) MIP of the lower legs showing the anterior tibial arteries in close proximity to the tibia (arrows), which is the cause of the false positive pseudo-occlusion. (c) Axial image of the lower legs just caudal from the pseudo-occlusion, showing the patency of both anterior tibial arteries of both legs in close proximity of the tibia (arrows)Fig.\u00a08a\u2013cApplying blue color to the voxels selected for removal helps to identify the sites of segmentation artifacts in VRT images. (a) VRT image before bone segmentation of the lower legs showing patent proximal anterior tibial arteries. (b) VRT with blue bones to indicate the voxels to be removed shows the voxels of the bone which are in contact with the proximal anterior tibial artery are not selected for removal and shows the voxels of the artery which are selected for removal. (c) Segmented VRT image showing the pseudo-occlusion of the anterior tibial arteryFig.\u00a09a, bResults of semiautomated quantitative lumen assessment in aortoiliac arteries of a patient with in stent thrombosis. (a) Graph (upper section) displaying the maximum and minimum diameters of the lumen to quantify stenosis. CPR (lower section) through the aortoiliac arteries, which can be rotated around its longitudinal axis, depicts the luminal obstruction (asterisk) due to a thrombus inside an iliac stent. (b) Corresponding transverse image confirms the occlusion of the iliac stent\nWall calcification problem\nThe depiction of vessel wall calcifications using MDCT can be valuable, since severely calcified arteries may have consequences for bypass surgery. On the other hand, these wall calcifications are known to hamper the assessment of the lumen [2, 10, 14, 19]. Approximately 20% to 50% of the vascular segments contain wall calcifications, of which, 10% severely calcified [11, 19]. Patients with a history of diabetes mellitus, cardiac disease, or elderly age are very likely to have extensive calcifications [48]. Furthermore, we found that patients with Fontaine stage III\/IV have more infrapopliteal arterial wall calcifications compared to stage IIb.\nHow can we deal with the vessel wall calcifications depicted with MDCTA? It is important to use a wider window width (WW) and higher window center (WC) level settings from the usual CT angiography level of around 150\u00a0WC\u2009\u00b1\u2009250\u00a0WW to 200\u00a0WC\u2009\u00b1\u20091000\u00a0WW for a better differentiation of calcifications and stents from the enhanced lumen and to minimize the effect of blooming. A further minimization of blooming is reached by using a sharper reconstruction kernel and higher spatial resolution.\nEspecially in MIP images, the lumen is hidden by the circumferential calcifications. In these circumstances, transverse images, CPR images, and the digital removal of the calcifications help to depict the lumen, at least for the larger arteries. Despite all of the available tools, in particular in the smaller crural arteries, the concentric calcifications still hamper lumen assessment [11]. Recent publications showed that a subtraction technique using two acquisitions is feasible in some patients with PAD using MDCTA [24, 49]. In the near future, automated 3D applications could help to minimize the impediment of the calcifications [50]. Whether dual-energy CT angiography can improve this limitation of CT needs to be evaluated.\nClinical value\nBecause MDCT angiography for the imaging of the peripheral arteries is a rather new non-invasive technique, there are a small number of studies published on its performance and reproducibility (Table\u00a03). The majority report on 4D-CT; two authors report on 16D-CT. There are no reports of the assessment of PAD using 64D-CTA. In our meta-analysis soon appearing in Radiology, which included 436 patients and 9,541 arterial segments, a pooled sensitivity and specificity for detecting a >50% stenosis of 92% and 93% was estimated, respectively. \nTable\u00a03Validity of CT angiography in peripheral arterial disease (PAD)AuthoraNo. of patientsNo. of analyzed segmentsNo. of detectorsReported sensitivity (%)eReported specificity (%)eAssessed segmentsStenosis category (%)hRichter et al. 199432ns184nsIliac>50Lawrence et al. 1995613419396Femorocrural>50Raptopoulos et al. 19963962419396Aortoiliac85\u201399Rieker et al. 199650400173\u201388b94\u2013100bFemorocrural75\u201399Rieker et al. 19973021019399Aortoiliac75\u201399Kramer et al. 199810ns294nsIliocrural>90\u201399Ishikawa et al. 199949ns19795Bypass graftsnsBourlet et al. 20002231819590Aortoiliac>50Puls et al. 20013118648986Total tree50\u201399Willman et al. 20034676949199Aortoiliac graftsnsOfer et al. 20031841049192Total tree>50Heuschmid et al. 200318568491c92cTotal tree>50Martin et al. 2003411,31249297Total tree75\u201399Catalano et al. 2004501,14849693Total tree>50Mesurolle et al. 20041616829193Total tree>50Ota et al. 20042447049999Total tree>50Poletti et al. 2004d12144482\/96gnsns>50Portugaller et al. 20045074049283Total treearea >70Romano et al. 2004423,40249395Total treensRomano et al. 2004221,78249294Total treensStueckle et al. 200452ns482100Total treensEdwards et al. 2005441,02447993Total tree50\u201399Fraioli et al. 2006751,425496\u201399h94\u201396hTotal tree50\u201399Schertler et al. 200517170169690Popliteocrural>50Willmann et al. 2005391,365169696Total tree>50Unpooled mean9194aBased on references [1\u20136, 8\u201310, 13\u201324, 31, 34, 35, 56, 57]bFor various anatomic levelscCalculated from the datadBased only on subtracted MDCTA images, the positive predictive value was 95%eSensitivity as published or calculated overall mean.fDiameter stenosis is mentioned unless specified (>50 means stenosis more than 50% including occlusion)gFor subtracted and nonsubtracted segments, respectivelyhDepending on the MDCTA protocol with varying mAs\nPublications on the reproducibility of CT angiography reported a good intertest agreement between MDCTA and DSA (Table\u00a04) and a good to excellent interobserver agreement for 4D-CTA [12, 21, 23, 35] and 16D-CTA (Table\u00a05) [9, 11]. A few studies provide stratified data on the aortoiliac, femoropopliteal, and crural tract and show that the accuracy and reproducibility of the crural tract is lower than for the aortoiliac and femoropopliteal tracts [9, 11, 20, 22, 23]. \nTable\u00a04Intertest agreement between CT angiography and digital subtraction angiography in PADAuthoraNo. of patientsNo. of assessed segmentsNo. of detectorsReported intertest agreementdAssessed segmentsRaptopoulos et al. 199639624190%AortoiliacBeregi et al. 199720521100%PoplitealTins et al. 200135219184%AortoiliacWalter et al. 2001224564\u03ba\u2009=\u20090.68 (0.50\u20130.97)cTotal treeRubin et al. 2001183514100%Total treeHeuschmid et al. 2003231,136486%Total treeOfer et al. 200318444478%Total treeRomano et al. 2004423,4024\u03ba\u2009=\u20090.68; 90%Total treeRomano et al. 2004221,7824\u03ba\u2009=\u20090.68; 90%Total treeaBased on references [3, 7, 12, 14, 16\u201318, 35, 55]bBased on 97% of the segmentscAverage of the reported kappa values (ranges) of the individual anatomical segmentsdAn unweighted kappa statistic (\u03ba) is reported for percentage agreementTable\u00a05Interobserver agreement of CT angiography in PADAuthoraNo. of patientsNo. of analyzed segmentsNo. of detectorsReported interobserver agreementbAssessed segmentsRieker et al. 1997302101\u03c1=0.95AortofemoralWalter et al. 2001224564\u03ba\u2009=\u20090.71\u20130.76cTotal treeTins et al. 200135219178%AortofemoralMartin et al. 2003411,3124\u03baw\u2009=\u20090.84Total treeRomano et al. 2004423,4024\u03ba\u2009=\u20090.84;0.86dTotal treeRomano et al. 2004421,7824\u03ba\u2009=\u20090.85, 0.88, \u03ba\u2009=\u20090.80eTotal treeCatalano et al. 2004501,1374\u03ba\u2009=\u20090.80Total treeOta et al. 2004244704\u03ba\u2009=\u20090.88IliacPortugaller et al. 2004507404\u03ba\u2009=\u20090.81Total treeKock et al.f732,2684\u03baw\u2009=\u20090.84Total treeOuwendijk et al. 2005792,41916\u03baw\u2009=\u20090.85Total treeWillmann et al. 2005391,36516\u03ba\u2009=\u20090.85\u20131Total treeaBased on references [4, 7, 9, 11, 15, 17\u201319, 21, 23, 35]bAn unweighted kappa statistic (\u03ba) is reported, unless indicated (\u03baw=weighted kappa statistic; \u03c1=intraclass agreement coefficients as a measure of agreement for ordinal or quantitative data). A linear weighting was used, except in one paper [15], where a quadratic weighting was usedcRange of kappa values of the individual anatomical segmentsdFor reader one and two, respectivelyeFor intraobserver (two readers) and interobserver agreement, respectivelyfBased on unpublished data\nMDCTA leads to adequate decision making for treatment recommendations concerning both the anatomical level and the technique of revascularization [51]. A cost-effectiveness study showed that MDCTA is a cost-effective diagnostic strategy in the work-up of PAD [52, 53]. Randomized controlled trials confirmed that MDCTA in PAD is the optimal diagnostic imaging technique [25, 54] and reduces the diagnostic costs when compared to DSA and CEMRA with comparable clinical utility and patient outcomes. Besides these evidence-based results, local expertise and availability also define which modality to use in clinical practice (Table\u00a06).\nTable\u00a06Advantages and limitations of multi-detector row CT angiography (MDCTA), contrast enhanced MR angiography (CEMRA), and digital subtraction angiography (DSA)\u00a0MDCTACEMRADSAIntermittent claudication (Fontaine II)+++Chronic critical ischemia (Fontaine III or IV)\u2212++Short examination time+\u2212\u2212Short postprocessing time\u2212++Outpatient setting++\u2212Availability+\u2212+Non-invasive technique\/patient comfortb++\u2212Low diagnostic imaging costs+\u2212\u2212Contrast media tolerance\u2212+\u2212Three-dimensional imaging++\u2212Non-interference of stentsc+\u2212+Radiation risk+(\u2212)d\u2212+(\u2212)dAcute clinical setting+\u2212+Hemodynamic assessment\u2212\u2212a+Extraluminal pathology visualization+\u2212a\u2212aIs only possible when using additional sequencesbFrom [58]cFrom [59]dNegligible risk in population with chronic obstructive PAD\nIt is reported that arterial wall calcifications lead to false-positive interpretations and a decreased reproducibility in reading MDCTA [2, 14, 11]. We have to acknowledge this limitation with current technology. A preferential indication for MDCTA in patients with intermittent claudication (Fontaine stage IIb) is clearly justified. However, patients with critical limb ischemia (Fontaine stage III\/IV), who are likely to have extensive calcifications of the smaller arteries, could be better off undergoing contrast-enhanced magnetic resonance angiography (CEMRA) or digital subtraction angiography (DSA).\nFinally, MDCTA is an accurate technique to evaluate the patency after revascularization procedures [43]. The technique can be used in the evaluation of acute ischemia, e.g., after a revascularization procedure or in thrombo-embolic disease (Fig.\u00a010). For aneurysmatic popliteal artery disease or entrapment syndromes of the popliteal artery, MDCTA is the preferred imaging modality (Fig.\u00a011) [55].\nFig.\u00a010a, bAcute thrombosis of the crural arteries in a 53-year-old woman with an acutely cold left leg after stopping anticoagulation therapy. The patient refused angiography. (a) VRT image (posteroanterior view) of MDCTA at the level of the crural arteries shows abrupt stoppage of arterial opacification in the left peroneal, anterior, and posterior tibial artery (arrows). The contralateral right crural arteries are patent. (b) Selective anterograde DSA image (posteroanterior view) confirms the occlusions of the three left crural arteries (arrows) due to thrombo-embolismsFig.\u00a011a\u2013dA 56-year old male patient who had a history of deep venous thrombosis with intermittent claudication of the right lower extremity. (a and b) Thin MIP image shows an aneurysmatic right popliteal artery with a tight stenosis distally. (c) VRT and volume MIP (d) confirm these findings and show patent proximal crural arteries\nConclusion\nMulti-detector row CT angiography (MDCTA) is an outstanding non-invasive imaging test in the evaluation of patients with peripheral arterial disease (PAD) and is currently the modality of choice in patients with intermittent claudication. The technique can be used in the evaluation of patency after revascularization procedures and in acute ischemia. MDCTA has been shown to have high diagnostic performance and reproducibility in evaluating peripheral arterial disease (PAD). MDCTA reduces diagnostic costs and provides adequate information for decision making. The most important drawback is the limited lumen evaluation of extensive calcified arteries. MDCTA appears to be clinically less valuable in critical limb ischemia because of extensive crural artery calcifications.","keyphrases":["tomography","sensitivity and specificity","human","peripheral vascular diseases","radiography","x-ray computed methods","reproducibility of results"],"prmu":["P","P","U","R","U","R","R"]}
{"id":"Graefes_Arch_Clin_Exp_Ophthalmol-4-1-2206250","title":"Ophthalmologists, suicide bombings and getting it right in the emergency department\n","text":"Background The number and extent of worldwide suicide attacks has risen sharply in recent years. The objectives of this retrospective study are: to determine the prevalence and outcome of the victims who sustained ocular injury, to describe the activities of ophthalmologists in the setting of an emergency department (ED) receiving mass casualties of a suicide bombing attack and to illustrate some of the treatment obstacles that they encountered and the protocol.\nThe number and extent of worldwide suicide attacks has risen sharply in recent years [1, 2]. The perpetrators typically mingle among crowds of civilians and detonate an explosive device that is usually strapped on their bodies with the intent of sacrificing their own lives in order to cause the death of as many others as possible. The injuries sustained by survivors of these well-planned attacks combine the lethal effects of penetrating trauma, blast injury, and burns [3]. Suicide attacks on civilians were historically confined to a limited number of countries, but the outrageous and devastating destruction of the Twin Towers in New York City on 11 September 2001 and the bombings in London and Madrid established the universality of such terrorism. With the rise in terror-related activities in urban settings, ophthalmologists worldwide may find themselves treating ocular trauma under conditions unlike any they had experienced before, and certainly remote from the relatively orderly setting of an emergency room in which traumatic ocular injuries can usually be counted on one hand at any given time.\nSince the beginning of the latest Israeli-Palestinian Intifada on 29 September 2000, more than 4,600 people\u2014mostly civilians\u2014have been killed or injured by suicide bombings in that area [4]. The Tel Aviv Sourasky Medical Center (TASMC) is the largest hospital in the Tel Aviv metropolitan area. As such, most of the victims of the suicide bombing attacks in its catchment area are evacuated to TASMC, and a special treatment protocol has evolved for coping with the unusual logistic needs of such events. The objectives of this retrospective study are: to describe the activities of ophthalmologists in the setting of an ED receiving mass casualties of a suicide bombing attack, to illustrate some of the treatment obstacles that they encountered and the protocol that evolved for overcoming them, and to determine the prevalence and outcome of the victims who sustained ocular injury.\nMethods\nIRB approval was obtained for this retrospective interventional case series study. Clinical data on all casualties evacuated to the TASMC due to suicide bombing-related injuries were collected from the trauma registry records and reviewed. Their demographic data were obtained from the main admitting office records.\nThe senior surgeon stationed at the ED entrance is rapidly provided essential information on the type\/location of injuries from the arriving ambulance\u2019s paramedical personnel. He\/she pages the designated on-site specialist according to prioritization for urgent management.\nOcular injuries are defined as any blunt, penetrating, or perforating trauma or blast-related damage to the eye, orbit, or ocular adnexa. The terms we used to describe ocular injuries conform to the recommendations the United States Eye Injury Registry and the International Society of Ocular Trauma [5]. Individuals with nonpenetrating or non-lacerating ocular injuries, non-penetrating debris in their eyelids, or superficial burns of the eyelids were excluded from this study, as were victims who died of their injuries before undergoing ophthalmologic treatment for whatever ocular damage had been suffered.\nThe ocular injuries for each eye were categorized according to type: it was possible to have multiple occurrences of the same type (i.e., multiple corneal lacerations) and of several types (i.e., corneal laceration and retinal detachment) in the same eye.\nAll records of ocular- and orbital-related trauma that were documented in the trauma registry were collected and analyzed together with hospital and outpatient clinic records. The analysis included age, gender, mechanism of injury, anatomic site of injury, Injury Severity Score (ISS) [6], length of stay, length of intensive care unit stay, and surgical procedures. Ophthalmic information included the initially diagnosed ocular condition, all surgeries performed during the ED stay and afterwards, and final ocular test findings. The data were entered using Excel spreadsheet (Microsoft Office) and a simple descriptive statistical analysis was performed.\nResults\nThere were 13 suicide bombing attacks in the Tel Aviv metropolitan area between October 2000 and October 2004. A total of 352 patients were evacuated to the TASMC ED, and 198 of them were hospitalized. The other 154 patients suffered from minor injury or shock for which they were given appropriate treatment and instruction and sent home. The overall severity of suicide bomb-related trauma was very high: the mortality rate was 8.4% when the attack occurred in open spaces, 15.5% in closed spaces and 20.3% when the bomb exploded inside a bus. The ISS was 1\u201314 for 74% of the patients (non-hospitalized) and \u226516 for the remaining 26% of the patients (admitted to hospital). One of the prominent hallmarks of suicide bombing injuries is the extremely high prevalence of head injuries: among our patients, 49% suffered from head and neck injuries, 9% head and extremity injuries, 4% head and torso injuries, and 38% torso and other injuries (all data are taken from the experience with the 13 bombings in Tel Aviv).\nSeventeen patients (4.8%) were listed in the trauma database as having any ocular or periocular trauma, and several had more than one type of injury. The types of recorded trauma were: open globe injuries (n=7), closed globe injury consisting of severe subconjunctival hemorrhage (n=2), partial thickness lamellar laceration of cornea (n=8) (lamellar flap) of which five were burn-related and three were due to small foreign bodies, and extraocular injuries (n=6), which included three orbital fractures due to primary blast injury and three eyelid lacerations. Primary repair of open globe was performed in six eyes that underwent primary closure of laceration. One patient who was diagnosed during the initial triage as suffering from open globe injury died during the initial trauma surgery, thus no ocular procedure was performed. Two other eyes underwent primary exploration of subconjunctival hemorrhage that was suspected as being open globe due to massive subconjunctival: no laceration was found intraoperatively, and contusion was diagnosed. The one eye that was found to be unsalvageable underwent primary enucleation.\nAfter the initial eye surgery, two patients died from their other injuries within 24 h of the explosions.\nOf the eight patients with partial thickness lamellar laceration of cornea, three were discharged and given instructions to return as outpatients on the following day. Five other patients who required and received medical treatment for non-ocular-related medical problems were hospitalized and continued eye treatment as inpatients. All patients in which superficial burns were found (n=5) were treated by manual removal of corneal foreign body and antimicrobial drops, and their recovery was uneventful.\nSecondary surgical intervention was performed on four patients (all surviving patients who initially underwent primary closure of open globe) within 1 week of the initial trauma: large intravitreal foreign bodies were extracted in three of them, and an intra-lenticular foreign body was extracted from the fourth (Table\u00a01). At final follow-up (\u22652 years post-trauma), the visual acuity of the three patients who suffered from large intravitreal foreign bodies were finger counting (FM)-hand movement (HM). The silicone oil was removed in two of these patients and retained in the third; two of them are wearing large cosmetic contact lenses due to corneal opacities that were cosmetically disturbing. The patient who underwent surgery due to intra-lenticular foreign body and penetrating keratoplasty had final visual acuity of 6\/12 (Table\u00a01).\nTable\u00a01Relevant data of suicide bombing survivors who sustained open globe injuryNo.Age, years\/sexPrimary surgical interventionSecondary surgical interventionForeign body extractedFurther surgeryFinal outcomeFinal visual acuity117\/fClosure of open globePatient died within 24 h of trauma216\/fNonePatient died within 24 h of trauma362\/mEnucleation417\/fClosure of open globeLensectomy, vitrectomy, removal of foreign body, endolaser silicone oil injectionMetal shrapnell (5\u00a0mm)Silicone oil removalPreserved globe flat retinaFC518\/fClosure of open globeLensectomy, vitrectomy, removal of foreign body, endolaser silicone oil injectionMetal ball (2\u00a0mm)Preserved globe flat retinaHM619\/fClosure of open globeLensectomy, vitrectomy, removal of foreign body, endolaser silicone oil injectionGlass fragment (3\u00a0mm)Silicone oil removalPreserved globe flat retinaHM727\/mClosure of corneal perforationPenetrating keratoplasty, lensectomy, vitrectomy endolaser, intraocular lens implantationGlass fragment (1\u00a0mm)6\/12FC, finger counting; HM, hand movement\nDiscussion\nThe dynamics of an emergency receiving center of victims of a suicide bombing attack are alien to most ophthalmologists. Today, the setting is characterized by large numbers of victims who sustain injuries that are more complex and more severe than those that had occurred during earlier periods of terror activity [7, 8]. Importantly, suicide bombings are more likely to occur in closed spaces, unlike other mass trauma scenarios such as car bombs, train wrecks, and other outdoor explosions: over 62% of injuries that occur in closed spaces are to the face, head, and neck, thus posing a far greater risk to the ocular structures.\nIn our experience, 17\/352 (4.8%) of the survivors of suicide bombing attacks sustained eye injuries, and 9\/17 (52%) required urgent attention. This rate is surprisingly lower when compared to previous reports, which documented that close to 10% of survivors of terrorist blasts have significant eye injuries [13]. We have no explanation for the low rate, but we can speculate that because of their close proximity to the hospital together with the efficiency of the Israeli Magen David (Red Cross), almost all survivors are speedily brought to the hospital for examination after terror-related episodes and are listed as admissions in the trauma records, even those with no complaints or only minor ones. Thus, the large number of admitted individuals artificially decreases the percentage of eye victims. Importantly, 41% of all of the reported ocular injuries [9\u201313] were severe in degree.\nThe final visual outcome of all the surgeries we performed was poor: globe preservation was successfully achieved in most cases (6\/7), but only one patient with an intra-lenticular foreign body had useful vision postoperatively.\nIn order to provide the best treatment in such a complicated setting, special adaptations must be made to the treatment algorithm of the ophthalmology team. In terms of individual trauma cases, the victim of a suicide bombing attack is no different from any other eye trauma patient. The sudden presentation of large numbers of injured patients, however, presents two types of challenges: the logistical one of rapidly processing masses of casualties through the system and the medical one of providing the best possible trauma care to severely wounded patients [14]. According to our protocol, all patients who complain of eye symptoms and all unconscious patients who sustain head or face injuries must be checked by an ophthalmologist. This requires special disaster on-call lists of ophthalmologists who are able to arrive to the hospital on extremely short notice since, thanks to the highly efficient organization of our Red Shield ambulance facilities, blast victims usually arrive at the hospital within minutes and are hurried to either diagnostic tests or directly to operating theaters. Upon arrival to the ED, every victim of a terrorist attack is triaged by a senior surgeon who synchronizes the activities of the multifaceted operation. The ophthalmologists already present in the hospital and the ones on-call who arrive to the ED are in contact with that surgeon in order to expeditiously locate the victims with ocular injuries, examine them, and send them to the operation theaters, intensive care units, imaging studies or home. There is a directive in our department that all available staff members must contact the hospital immediately upon learning about any suicide bombing attack to check whether their services are required. In the event of large-scale attacks, they are prepared to be recruited to assist in triaging and in treating all the victims, not just those with ocular injuries. The triage procedure is the key to the successful management of large trauma events: the most important rule is that all patients must be checked by the ophthalmologist wherever they are located on the hospital premises. Trauma patients invariably require urgent treatment and some are sent directly from the ER to either imaging units or immediately to the trauma surgical unit. The senior ophthalmologist on the premises must contact trauma registration services, get a list of all admitted patients (usually assigned numbers upon admission) and make sure that each and every one of them is examined, even during emergency surgery or during imaging interventions for non-ophthalmological injuries. When an open globe is suspected, the eye is immediately patched, and the finding is reported to the surgeon in charge of the patient: the staff is instructed not to intervene in the treatment of the eye. Further evaluation is done only when it is certain that there is no danger of expulsive hemorrhage.\nOnly patients suspected as suffering from open globe injury undergo urgent primary closure of the wound. Since that patient invariably presents with multiple injuries and may not be fit for transfer to the ophthalmology operating theater, however, special alterations to the surgical protocol may be required. For instance, no ophthalmological microscope is available in our trauma center because space is limited in the trauma room due to the concomitant performance of many surgeries and given the cumbersome structure of an ophthalmic microscope. There is, however, a high-quality neurosurgical microscope that has a long arm that can be placed at sufficient distance from the patient and the life-support machines so that it can be used without disturbing the anesthesiologists and other trauma teams as they work, and this microscope is used with great success during primary closure. Other eye surgery procedures are postponed, either until the patient has been stabilized or they are scheduled for a later date. This highlights the first critical responsibility of the ophthalmologist in the mass trauma setting, that of identifying which surgical procedures must be carried out immediately. The order of surgical intervention deserves special attention: due to the characteristic complexity of the injuries, most of the patients required multiple procedures immediately following the trauma. The established protocol adopted among our surgeons is (in descending order): trauma surgery (for life-threatening conditions, performed by either trauma surgeons or neurosurgeons), ophthalmologic interventions (immediate surgery or instructions for palliative care), and orthopedic and plastic surgery interventions.\nFinally, terrorist bombings present a danger to the ED staff members that is never associated with any other mass casualty situation: there is a very real chance of explosion by a second-hit, either by explosive material remaining on the perpetrator\u2019s body, or, even more threatening, a second suicide bomber who infiltrates the ED disguised as one of the victims and detonates the bomb inside the crowded ED. Thus, a unique caveat in the ED protocol for terrorist bombing attacks is heightened vigilance, starting from the chaotic first minutes after the arrival of the victims.\nProviding medical assistance in an ED to victims of suicide bombing attacks is a harrowing experience: physicians who work in an urban hospital are more and more likely to be exposed to such events [14]. In his excellent editorial, Hirshberg wrote \"Urban terrorism, the scourge of the 21st century, is already at our doorstep and surgeons are called upon to play leadership roles in shaping the emergency response in their hospitals. Learning from the experience of those for whom the unthinkable has become a daily reality can help us develop and implement more effective answers to the threats in our own communities\" [14].","keyphrases":["terror","ocular trauma","vitrectomy","intra ocular foreign body"],"prmu":["P","P","P","M"]}
{"id":"Int_J_Biochem_Cell_Biol-2-1-2267855","title":"Site-directed mutagenesis of Arginine282 suggests how protons and peptides are co-transported by rabbit PepT1\n","text":"The mammalian proton-coupled peptide transporter PepT1 is the major route of uptake for dietary nitrogen, as well as the oral absorption of a number of drugs, including \u03b2-lactam antibiotics and angiotensin-converting enzyme inhibitors. Here we have used site-directed mutagenesis to investigate further the role of conserved charged residues in transmembrane domains. Mutation of rabbit PepT1 arginine282 (R282, transmembrane domain 7) to a positive (R282K) or physiologically titratable residue (R282H), resulted in a transporter with wild-type characteristics when expressed in Xenopus laevis oocytes. Neutral (R282A, R282Q) or negatively charged (R282D, R282E) substitutions gave a transporter that was not stimulated by external acidification (reducing pHout from 7.4 to 5.5) but transported at the same rate as the wild-type maximal rate (pHout 5.5); however, only the R282E mutation was unable to concentrate substrate above the extracellular level. All of the R282 mutants showed trans-stimulation of efflux comparable to the wild-type, except R282E-PepT1 which was faster. A conserved negatively charged residue, aspartate341 (D341) in transmembrane domain 8 was implicated in forming a charge pair with R282, as R282E\/D341R- and R282D\/D341R-PepT1 had wild-type transporter characteristics. Despite their differences in ability to accumulate substrate, both R282E- and R282D-PepT1 showed an increased charge:peptide stoichiometry over the wild-type 1:1 ratio for the neutral dipeptide Gly-l-Gln, measured using two-electrode voltage clamp. This extra charge movement was linked to substrate transport, as 4-aminobenzoic acid, which binds but is not translocated, did not induce membrane potential depolarisation in R282E-expressing oocytes. A model is proposed for the substrate binding\/translocation process in PepT1.\n1\nIntroduction\nThe proton-coupled di- and tri-peptide transporter PepT1 (SLC15a1) is the major route of uptake of dietary nitrogen from the intestine, and is also important along with the higher affinity gene product PepT2 (SLC15a2) in the re-absorption of filtered peptides in the kidney (Daniel & Kottra, 2004; Meredith & Boyd, 2000). In addition, PepT1 is the route of entry of a wide class of orally bio-available pharmaceutically important compounds, including the \u03b2-lactam antibiotics, angiotensin-converting enzyme (ACE) inhibitors, antiviral and anticancer agents (Terada & Inui, 2004). Although these therapeutic compounds are not di- or tri-peptides, they are carried by virtue of their similar 3D shape to endogenous substrates, i.e. they are peptidomimetic, and modelling of the substrate-binding site from the features in common of this huge and diverse range of substrates has led to predictions concerning which parts of the PepT1 protein may be important. For example, a substrate template model has been developed by several groups (Bailey et al., 2000; Bailey et al., 2006; Biegel et al., 2005) which allows prediction of binding affinity for a potential substrate.\nThe rabbit PepT1 is a 707 amino acid protein, with twelve transmembrane spanning domains (TMDs) as confirmed by epitope mapping (Covitz, Amidon, & Sadee, 1998). In the absence of a crystal structure, attempts have been made to computer model the PepT1 transporter itself (Bolger et al., 1998), with site-directed mutagenesis used to test hypotheses generated by these models. One potential complication for these kind of studies is our recent report that PepT1 may form multimers in the plasma membrane (Panitsas, Boyd, & Meredith, 2006) although it is not clear how the subunits interact.\nSite-directed mutagenesis has been a useful tool to identify functionally important residues in PepT1. One such residue is arginine282 in the rabbit PepT1 sequence. The mutation of arginine282 to a glutamate produced a peptide transporter (R282E-PepT1) that was no longer driven by a proton-gradient but behaved more like a facilitated peptide transporter, whilst simultaneously exhibiting peptide-gated currents that were proposed to be through a non-specific cation channel activity (Meredith, 2004). Residue 282 is located approximately halfway down the predicted transmembrane domain 7 (TMD7), and is either an arginine or a lysine in all cloned mammalian PepT1 sequences to date. The presence of a charged amino acid residue in a TMD, along with its conservation, suggested a functional role. Here, we have systematically investigated the role of arginine282 in rabbit PepT1 by making further mutations to determine the requirement for the charge and have identified an interacting residue, aspartate341, located in putative TMD8. Some of these data have been previously published in abstract form (Pieri, Boyd, & Meredith, 2004).\n2\nMaterials and methods\n2.1\nSite-directed mutagenesis of the PepT1 gene\nOligonucleotides were custom synthesised (Sigma-Genosys, UK) for the following sequences (residues in bold are changed from wild-type PepT1):-R282-PepT1 mutants forward:where xxx was CAA for R282Q, AAG for R282K, GAT for R282D, CAT for R282H, and GCG for R282A.-D341-PepT1 mutants forward:where xxx was CGC for D341R.\nReverse primers for the PepT1 mutant PCR reactions were the reverse compliment of the forward primers. The site-directed PepT1 mutants were generated using the Quikchange protocol (Stratagene), and the resulting constructs confirmed by DNA sequencing (Department of Biochemistry, University of Oxford, UK).\n2.2\ncRNA synthesis and oocyte injection\nPepT1 constructs were linearised with XbaI (New England Biolabs, UK) and cRNA generated by in vitro transcription (T7 mMessage mMachine, Ambion, Cambridgeshire, UK). X. laevis oocytes were obtained under MS222 anaesthesia (0.2%, w\/v) in accordance with the UK Animals (Scientific Procedures) Act, 1986, and maintained at 18\u00a0\u00b0C in modified Barth's medium (88\u00a0mM NaCl, 1\u00a0mM KCl, 0.82\u00a0mM MgSO4, 2.4\u00a0mM NaHCO3, 0.42\u00a0mM CaCl2, 10\u00a0mM Hepes, 5\u00a0mM sodium pyruvate, 50\u00a0\u03bcg\u00a0ml\u22121 gentamicin (Fluka, Poole, UK), adjusted to pH 7.6 with 1\u00a0M NaOH). Transport measurements were performed at least 72\u00a0h after micro-injection of oocytes with 27nl cRNA (1\u00a0\u03bcg\/\u03bcl), with medium changed daily.\n2.3\nTransport experiments\nZero-trans uptake of [3H]-d-Phe-l-Gln (17.4\u00a0Ci\/mmole, custom synthesised, Cambridge Research Biochemicals, Stockton-on-Tees, UK) was performed as previously described (Meredith, 2004). Briefly, 5 oocytes were incubated in 100\u00a0\u03bcl of uptake medium (95\u00a0mM NaCl, 2\u00a0mM KCl, 1\u00a0mM CaCl2, 0.42\u00a0mM MgCl2, 10\u00a0mM Tris\/Hepes pH 7.4 or Tris\/Mes pH 5.5) with tracer (0.4\u00a0\u03bcM) [3H]-d-Phe-l-Gln. After incubation, the oocytes were washed sequentially five times in 1\u00a0ml of ice-cold 120\u00a0mM NaCl solution, lysed individually with 100\u00a0\u03bcl 2% (w\/v) SDS and liquid scintillation counted. As a control non-injected oocytes were also incubated in uptake medium with [3H]-d-Phe-l-Gln as above.\nThe affinity of wild-type and mutant PepT1 were assessed by competition studies with 0.4\u00a0\u03bcM [3H]-d-Phe-l-Gln and Gly-l-Gln present in the uptake medium in concentrations from 0 to 2\u00a0mM using the protocol above, and the Ki calculated using the method of Deves and Boyd (1989).\nEfflux studies were performed as previously described (Meredith, 2004), with the exception that the extracellular trans-stimulant Gly-l-Gln was used at 5\u00a0mM, and an efflux time-course was performed.\nDiethylpyrocarbonate (DEPC) inhibition of PepT1 was performed using a similar protocol to that of Terada, Saito, and Inui (1998). Briefly, PepT1 oocytes were preincubated with 1\u00a0mM DEPC for 15\u00a0min at pHout 5.5 in the absence or presence of the PepT1 substrates Gly-l-Gln, N-Acetyl-Phe (Meredith et al., 2000), and l-Ala-Tyramine (custom synthesised) and the non-substrate Tyr (all 5\u00a0mM). The oocytes were then washed in uptake medium before uptake assays were performed as detailed above.\n2.4\nElectrophysiology\nMeasurements of membrane potential were made by impaling oocytes with a single glass microelectrode (Intra 767 amplifier, WPI, Stevenage, Hertfordshire, UK) perfused in uptake medium (as above) with or without 0.4\u00a0\u03bcM d-Phe-l-Gln (synthesised in house), 0.6\u00a0mM Gly-l-Gln (Sigma, Poole, UK) or 10\u00a0mM 4-aminobenzoic acid (4-AMBA, Sigma). Two-electrode voltage clamp (TEVC) was performed by placing oocytes in a 0.1\u00a0ml recording chamber and perfusing with uptake solution (pH 5.5 or 7.4) at a rate of 15\u00a0ml\/min. Oocytes were impaled by two agarose-cushioned microelectrodes filled with 3\u00a0M KCl (0.5\u20132.0\u00a0M\u03a9) and voltage-clamped at \u221260\u00a0mV using a Geneclamp 500B amplifier and PCLAMP 8.1 software (Axon Instruments, CA, USA). The holding potential was stepped from \u221260\u00a0mV over the range of \u2212150 to +50\u00a0mV in 10\u00a0mV steps, each pulse lasting 100\u00a0ms, and returning to \u221260\u00a0mV in between test voltage pulses. Typically traces were filtered at 1\u00a0kHz during recording and digitized at 0.5\u20135\u00a0kHz using the DigiData 1200 interface (Axon Instruments, CA, USA). All experiments were carried out at room temperature.\n2.5\nData analysis\nAll data are expressed as mean\u00a0\u00b1\u00a0S.E.M., except for Fig. 6 where the error bars represent the maximum range of stoichiometry values when taking into account the errors for the uptake data and the currents.\nIn order to calculate the apparent charge:substrate stoichiometry, the peptide induced current that was measured by two-electrode voltage clamp at the oocyte resting membrane potential was divided by the radiolabelled dipeptide uptake in oocytes from the same preparation. This value was normalised to 1:1 for wild-type PepT1, the accepted stoichiometry for a neutral dipeptide (Fei et al., 1994; Steel et al., 1997).\n2.6\nStatistical analysis\nStatistical analyses were performed using one-way ANOVA with differences considered significant if p\u00a0<\u00a00.05 when data were compared to the wild-type control, as detailed in the text and\/or figure legend.\n3\nResults\n3.1\npH dependence of d-Phe-l-Gln uptake into R282 mutants\nFig. 1 shows the pH dependence of d-Phe-l-Gln uptake into oocytes expressing mutant PepT1 transporters where R282 has been changed into number of amino acid residues. Of the residues tested, R282K- and R282H-PepT1 behaved like the wild-type PepT1, in that the initial rate of uptake (1\u00a0h incubation time) of dipeptide was significantly faster (p\u00a0<\u00a00.05, one-way ANOVA) at an external pH of 5.5 than at 7.4. The other amino acid substitutions tested, R282E- (as previously reported, Meredith, 2004), R282D-, R282A- and R282Q-PepT1 all gave the same initial rate of uptake at pH 5.5 and 7.4, indicating that transport by these mutants is not stimulated by an inwardly directed proton gradient. These changes cannot be ascribed to changes in the affinity of the mutant PepT1 proteins for their substrate, as the Ki of Gly-l-Gln inhibiting 0.4\u00a0\u03bcM [3H]-d-Phe-l-Gln was unchanged in the mutants (Fig. 2).\n3.2\nCan the R282 mutant PepT1 transporters concentrate substrates?\nAn earlier finding was that, unlike the wild-type, the R282E-PepT1 mutant was unable to concentrate substrate even in the presence of an inwardly directed proton gradient (Meredith, 2004). The ability to concentrate substrate was therefore tested for the other R282 mutants (Fig. 3a and b shows representative time-course experiments at pHout 5.5 and 7.4, respectively), and the mean accumulation levels are shown for 8\u00a0h uptakes in Fig. 3c for pHout 5.5. In contrast to R282E-PepT1, all were found to be able to concentrate peptide well above the equilibrium level when the external pH was 5.5 (an oocyte was assumed to have a volume of 1\u00a0\u03bcl, Petersen & Berridge, 1996; Yao & Tsien, 1997). In the absence of the proton driving force (pHout 7.4, Fig. 3d) a similar level of intracellular d-Phe-l-Gln concentration was reached by all the mutant PepT1 transporters, including R282E-PepT1 if the incubation time was increased to 24\u00a0h (accumulation of 2.3\u00a0\u00b1\u00a00.4-fold compared to 3.1\u00a0\u00b1\u00a00.5-fold for wild-type PepT1). Although at 24\u00a0h incubation times we observed that cell survival can be a limiting factor, there was no statistical increase in the accumulation for the wild-type PepT1 at pHout 5.5 or 7.4 between 8 and 24\u00a0h incubations, nor between 8 and 24\u00a0h for the wild-type PepT1 at pHout 7.4 (8.0\u00a0\u00b1\u00a01.4 vs. 8.3\u00a0\u00b1\u00a01.0, 2.5\u00a0\u00b1\u00a00.5 vs. 3.1\u00a0\u00b1\u00a00.5 and 1.0\u00a0\u00b1\u00a00.4 vs. 1.5\u00a0\u00b1\u00a00.3 respectively, all p\u00a0>\u00a00.05, one-way ANOVA).\n3.3\nRates of d-Phe-l-Gln efflux from R282 mutants\nFig. 4 shows the rates of efflux of [3H]-d-Phe-l-Gln from oocytes expressing either wild-type PepT1, R282 PepT1 mutants or non-injected controls. All of the PepT1 constructs showed a significantly faster efflux than the non-injected oocytes (one-way ANOVA, p\u00a0<\u00a00.05), whilst R282E-PepT1 was significantly faster than the wild-type (one-way ANOVA, p\u00a0<\u00a00.05) as previously described (Meredith, 2004). Interestingly, R282E-PepT1 showed a faster efflux than all the other R282 mutants (one-way ANOVA, p\u00a0<\u00a00.05), which were not significantly different to the wild-type (p\u00a0=\u00a00.14).\n3.4\nApparent transport stoichiometry (charge to substrate) using two-electrode voltage clamp\nAs well as uptake being pH independent and non-concentrative, the electrophysiological characteristics of R282E-PepT1 were strikingly different to the wild-type (Meredith, 2004). Since R282D-PepT1 is similarly pH independent but does accumulate substrate, the apparent proton to peptide stoichiometry was examined, where the uptake of 0.4\u00a0\u03bcM d-Phe-l-Gln was compared to the peptide-induced current in the same batch of oocytes. The current at the oocyte resting membrane potential (\u221227.0\u00a0\u00b1\u00a01.1\u00a0mV at pHout 5.5, and \u221236.8\u00a0\u00b1\u00a02.2\u00a0mV at pHout 7.4, n\u00a0=\u00a012) was taken to represent the membrane potential under uptake conditions, as the addition of 0.4\u00a0\u03bcM substrate does not produce a detectable change in membrane potential (Fig. 5). The ratio of proton to neutral dipeptide co-transported through wild-type PepT1 is 1:1 (Fei et al., 1994; Steel et al., 1997), yet in R282E-, R282D- and R282A-PepT1 the apparent stoichiometry is substantially higher (4, 5 and 5 respectively at pHout 5.5, Fig. 6).\n3.5\nIs the extra current measured dependent on substrate transport?\nIn an attempt to see if the extra current carried by R282E-PepT1 was dependent on substrate translocation rather than simply substrate binding, the non-translocated PepT1 substrate 4-aminobenzoic acid (4-AMBA, Darcel, Liou, Tome, & Raybould, 2005; Meredith et al., 1998) was used. As found for wild-type PepT1, 4-AMBA failed to induce a depolarisation in R282E-PepT1 oocytes at 10\u00a0mM, three times its Ki (Meredith et al., 2000), in contrast to the known transported substrate Gly-l-Gln, also at three times Ki (Fig. 5).\n3.6\nIdentification of an interacting residue for R282\nThe conservation of a positively charged amino acid in a transmembrane domain (TMD7), and the results above, strongly suggested that the presence of a positive charged residue was necessary for wild-type-like PepT1 transport function. In TMD8, predicted to be at approximately the same level in the membrane, there is a conserved aspartate (D341), and it was an appealing hypothesis that the two oppositely charged side chains might be forming a charge pair. To test this, double mutants were made, R282E\/D341R- and R282D\/D341R-PepT1, to swap the charges over. As can be seen in Fig. 7, these double mutants showed the same pH dependence of influx as the wild-type transporter, providing strong evidence to support the hypothesis. Both R282E\/D341R- and R282D\/D341R-PepT1 were also able to concentrate substrate like the wild-type (data not shown).\n3.7\nDiethylpyrocarbonate inhibition of PepT1 function\nPreincubation of wild-type PepT1-expressing oocytes with 1\u00a0mM diethylpyrocarbonate (DEPC) for 15\u00a0min completely inhibited the PepT1 mediated dipeptide uptake measured over 1\u00a0h, as shown in Fig. 8. This inhibition by DEPC was largely prevented by the presence of the known PepT1 substrates Gly-l-Gln and N-Acetyl-Phe (Meredith et al., 2000), but not by the amino acid non-substrate Tyr. Interestingly, despite being a good PepT1 substrate (Ki approximately 0.1\u00a0mM, data not shown), a modified peptide lacking a carboxyl terminus (l-Ala-Tyramine) only partially prevented DEPC inhibition (Fig. 8).\n4\nDiscussion\nThe R282K mutation of rabbit PepT1 is not only the most conservative one regarding the charge, but in a number of species, including dog, rat and mouse, lysine is the naturally occurring residue at this position. Therefore it was not surprising to find that this mutant behaves like the wild-type rabbit PepT1 (pig, sheep, rhesus and crab-eating monkeys and human also have R282). The finding that R282H also behaved like the wild-type was interesting, as histidine has a side-chain that can be titrated over the range used in the experiments (pK of \u223c6 in free solution). Our findings could be interpreted in several ways, including the possibility that only at pHout 5.5 is there the formation of a positive charge by side-chain titration that gives a stimulation of uptake over that seen at pHout 7.4. A second possibility is that the protein environment surrounding H282 is such that its side chain pK is shifted away from 6 and it is therefore always protonated, and thus behaves more like an arginine or lysine. This effect has been shown for example in the enzyme protein tyrosine kinase (Tishmack, Bashford, Harms, & Van Etten, 1997), where histidine residues had a pK as high as 9.2 when analysed by NMR.\nIn the original study on R282E-PepT1, it was concluded that the uptake of peptide by the mutant transporter was uncoupled from the movement of protons, and that in addition to acting as a facilitated peptide transporter, R282E-PepT1 also displayed a peptide-gated non-specific cation conductance (Meredith, 2004). However, it is possible that this conclusion needs updating in the light of the current findings that there are R282 mutants that, like R282E-PepT1, are not pH stimulated, yet are still able to accumulate substrate above equilibrium when an inwardly directed proton gradient is imposed. For a transporter to be able to accumulate substrate above equilibrium, an energy source must be involved, in this case the proton electrochemical gradient. Therefore, the mutant PepT1 proteins that can accumulate substrate but do not show pH stimulation (R282A and R282D) must still be coupled to the movement of protons down their electrochemical gradient. The lack of pH stimulation could be attributed to the fact that during the transport cycle these specific mutants have a different rate limiting step to the wild-type, and that for these mutants that step is not pH dependent. It has already been reported that R282E-PepT1 has a faster rate of efflux than wild-type, consistent with an uncoupling of peptide uptake from the proton driving force (Meredith, 2004); the finding here that the rates of efflux for the other R282 mutants are not different from that of the wild-type is in agreement with the hypothesis that they are still proton-coupled, as shown by their ability to accumulate substrate above the extracellular concentration at pHout 5.5 but not 7.4.\nThe simplest hypothesis was that in R282E-PepT1, the extra inward charge movement associated with peptide uptake collapsed the membrane potential, which is known to be the major driving force for proton-coupled peptide uptake (Temple & Boyd, 1998), hence the apparent lack of substrate accumulation. Therefore by extension one would expect R282D- and R282A-PepT1 to have the same charge coupling as the wild-type, as they too accumulate substrate, but this was not the case: both R282E-, R282D- and R282A-PepT1 showed the same increased charge:peptide apparent stoichiometry, i.e. substantially larger than the wild-type. This stoichiometry itself showed pH dependence, with a lower value of around 2 for the mutants at pH 7.4, suggesting that the current is either carried by protons or is a pH-sensitive phenomenon. There was no difference in the uptake in the absence or presence of sodium, either for R282E-PepT1 (Meredith, 2004) or R282D-PepT1 (data not shown).\nThe finding that the R282E\/D341R- and R282D\/D341R-PepT1 double mutants (the latter being a charge pair reversal of the naturally occurring residues in rabbit PepT1) had the same characteristics as the wild-type protein strongly suggests that these two residues do interact in the 3D protein (Pieri et al., 2004) as previously proposed (Meredith, 2004). During the preparation of this manuscript, Kulkarni et al. (2007) reported findings consistent with R282 and D341 forming a charge pair in human PepT1. The initial hypothesis that in R282E-PepT1 repulsion between E282 and D341 allowed the movement of extra ions through the protein when a peptide is transported was not supported however by the finding that the single D341R mutant also behaved like the wild-type, as one might have thought that R282 and R341 would repel in much the same way as E282 and D341. That D341R-PepT1 behaved like wild-type suggests that although R282 and D341 seem to form a charge pair, in rabbit PepT1 residue 341 being negatively charged is not crucial to PepT1 function; interestingly, in human PepT1 the D341R single mutant had reduced function (Kulkarni et al., 2007). The reason for this difference between rabbit and human PepT1 is not clear. The observation that the non-transported PepT1 substrate 4-AMBA did not induce a depolarisation in R282E-PepT1-expressing oocytes clearly shows that the charge movement is linked to substrate binding and translocation and not to binding alone. One explanation for the increase in proton\u2013peptide stoichiometry is that in wild-type PepT1 the presence of a positively charged residue deep in the binding pocket at position 282 repels proton movement through the transporter protein during the translocation step. The data in Fig. 6 are consistent with this, as the apparent stoichiometry is lower at pHout 7.4 than it is at pHout 5.5, indicating that the proton electrochemical gradient is involved.\nIn the case of mutations where R282 was replaced with a non-positively charged amino acid, the rate-limiting step of the transport cycle must be insensitive to extracellular pH, hence the lack of stimulation when the pHout is dropped from 7.4 to 5.5. Kinetic analysis of peptide transport by PepT1 in rat renal brush border membrane vesicles (Temple & Boyd, 1998) showed that at pHout7.4\/pHin7.4, it was the protonation of the carrier protein that was the rate-limiting step (Temple, Bailey, Bronk, & Boyd, 1996), whereas at pHout5.5\/pHin7.4 it was the return of the unloaded carrier. The rate of peptide uptake by R282E-PepT1 (corrected for protein expression in the intact oocyte plasma membrane by luminometry, Panitsas et al., 2006) is the same as for the wild-type at pHout 5.5 (data not shown), but, unlike for the wild-type, is not slower at pHout 7.4. This indicates that the mutations to arginine282 that abolish pH sensitivity (R to E, D, A, or Q) are affecting the rate limiting step, so when pHout is 7.4 the rate limiting step has the same magnitude as that at pHout 5.5, hence the lack of sensitivity to changing pHout. The rate limiting protonation of the outward-facing carrier protein at pHout 7.4, the first step in the transport cycle (Temple et al., 1996), was proposed by us (Bailey et al., 2000; Meredith & Boyd, 1995) and others (e.g. Steel et al., 1997; Uchiyama, Kulkarni, Davies, & Lee, 2003) to be protonation of histidine57 (H57). In the R282-PepT1 mutants, except for R282K and R282H, the lack of a positively charged residue might result in a conformational change of the protein that changes the local environment and increases the pKa of H57, such that it is more easily protonated at a higher pHout. Thus at pHout 7.4 the rate limiting step is no longer the protonation of H57, but the return of the unloaded carrier, as it is at pHout 5.5, hence the similar transport rates at pHout 5.5 and 7.4.\nA model for how proton\u2013peptide transport might occur is shown in Fig. 9: the empty PepT1 is primed by protonation of H57, followed by the binding of a zwitterionic peptide, with the N-terminal co-ordinated at E595 and the C-terminal at H57-H+ (Meredith et al., 2000). Although the binding of the substrate C-terminal to His57-H+ is in disagreement with the conclusion of Terada et al. (1998), it is supported by the finding that N-Acetyl-Phe, a known PepT1 substrate which does not have a free amino terminal (Meredith et al., 2000), can protect PepT1 against inhibition by DEPC. Additionally, the finding that significant DEPC inhibition is still evident when oocytes were co-incubated with the PepT1 substrate l-Ala-Tyramine which lacks a carboxyl terminus further adds to the notion that H57-H+ binds the carboxyl terminus of the substrate (Fig. 8). H57-H+ then donates its proton to the C-terminal carboxyl group, and the transporter undergoes a conformational change that leads to the breaking of the salt bridge between R282 and D341, re-orientating the binding site to be inward facing as simultaneously the protonated N-terminal of the substrate binds to D341, neutralising the charge, and the R282 charge is stabilised by Y167 (the chemical properties of this tyrosine have been shown to be essential, Yeung et al., 1998). The peptide molecule is then released into the cytoplasm, whereby it returns to the zwitterionic state by releasing the proton from the carboxyl terminal. The transporter then undergoes the reverse conformational change to re-orientate the binding site to outward facing, and R282 reforms the salt bridge with D341.\nAs R282E-PepT1 cannot accumulate substrate, the implication must be that the movement of peptide is no longer coupled to the movement of the protons, whereas in all the other mutants coupling must be maintained. If for R282E-PepT1 the pKa of H57 was raised to the point that it was no longer favourable for it to donate its proton to the carboxyl terminus of the substrate, then peptide transport would no longer be proton-coupled, and this would explain the failure of R282E-PepT1 to accumulate substrate. Intriguingly, it can be seen in Fig. 6 that both R282D- and R282A-PepT1 appear to carry approximately one more charge per substrate peptide than R282E-PepT1, which is consistent with the hypothesis that one of the charges carried is coupled with the substrate.\nIn conclusion, the arginine at position 282 in rabbit PepT1 plays an intriguing role in the function of the transporter, with mutations to different residues revealing that a positive residue is required for pH dependence, whilst only R282E-PepT1 cannot concentrate substrate above equilibrium; this is despite other mutations, most notably R282D-PepT1, having a similarly increased charge:peptide stoichiometry. As previously proposed, R282 (TMD7) forms a charge pair with D341 (TMD8), with R282E\/D341R-PepT1 showing normal transport characteristics. Further biological testing or a crystal structure of PepT1 will be required to establish the validity of the model proposed.","keyphrases":["site-directed mutagenesis","slc15a1","epithelia","membrane transport","nutrient absorption","protein structure\u2013function"],"prmu":["P","P","U","R","M","M"]}
{"id":"Exp_Brain_Res-4-1-2373863","title":"Intramanual and intermanual transfer of the curvature aftereffect\n","text":"The existence and transfer of a haptic curvature aftereffect was investigated to obtain a greater insight into neural representation of shape. The haptic curvature aftereffect is the phenomenon whereby a flat surface is judged concave if the preceding touched stimulus was convex and vice versa. Single fingers were used to touch the subsequently presented stimuli. A substantial aftereffect was found when the adaptation surface and the test surface were touched by the same finger. Furthermore, a partial, but significant transfer of the aftereffect was demonstrated between fingers of the same hand and between fingers of both the hands. These results provide evidence that curvature information is not only represented at a level that is directly connected to the mechanoreceptors of individual fingers but is also represented at a stage in the somatosensory cortex shared by the fingers of both the hands.\nIntroduction\nThe neural representation of haptic information can be investigated using different approaches. The representation of object shape perceived with the fingers has mainly been studied using neurophysiological tools. It has been found that especially slowly adapting type I (SAI) mechanoreceptors in the finger but also fast-adapting type I (FAI) receptors are sensitive to curvature (Goodwin et al. 1997; Jenmalm et al. 2003). In order to perceive curvature, a combination of responses from a population of receptors is required (Goodwin and Wheat 2004). This processing occurs along several stages up to at least the somatosensory cortex (SI) (Gardner and Kandel 2000). Taking a neurophysiological approach is useful to uncover the pathways underlying curvature processing, but is less appropriate to establish the levels at which perceived curvature is essentially represented.\nA psychophysical approach that has been successful in providing greater insight into the neural representation of perceived properties is the study of the aftereffect, and especially, the transfer of the aftereffect. In vision, for example, the finding of partial, interocular transfer of the motion aftereffect has been explained by the involvement of both monocular and binocular cells in the processing of motion information from the stimulus (Moulden 1980; Wade et al. 1993; Tao et al. 2003). In a similar way, establishing the transfer characteristics of a haptic curvature aftereffect would provide insight into the representation of shape information. Finding aftereffect transfer between different fingers would indicate that curvature is represented at a level shared by these fingers, whereas no transfer would imply that each finger has a separate representation of curvature.\nA curvature aftereffect is the phenomenon whereby a flat test surface feels concave following prolonged contact with a convex adaptation surface (see Fig.\u00a01a). Curvature aftereffects have been found for different shapes and exploration modes. Gibson (1933) reported that a flat cardboard edge felt concave after the prolonged dynamic exploration of a convex cardboard edge. Vogels et al. (1996) demonstrated the existence of an aftereffect when the whole hand was placed on spherically curved shapes. They performed extensive experiments to examine the characteristics of this static curvature aftereffect. They found a linear relationship between the magnitude of the aftereffect and the curvature of the adaptation stimulus. Furthermore, they showed that the magnitude of the aftereffect increased with the adaptation time up to about 10\u00a0s. Finally, they found a decrease of the aftereffect with an increase of the interstimulus interval. In a follow-up study, they showed that the aftereffect also existed for alternative exploration modes, like touching a stimulus with only the five fingertips of the hand or performing small movements of the hand over the stimulus surface (Vogels et al. 1997). Given the strength and consistency of these findings, we supposed that curvature aftereffects should also occur for alternative ways of touching, such as the situation in which curved surfaces are statically being touched with only a single fingertip. However, this phenomenon has not yet been investigated, and consequently, any curvature aftereffect transfer between the fingers also remains unexplored.Fig.\u00a01a Schematic overview of a haptic curvature aftereffect: when you first touch a convex (concave) surface for some time, say 10\u00a0s, and subsequently touch a flat surface, this latter surface feels concave (convex). b Schematic drawings of the cross-sections of a convex and a concave stimulus. The stimuli had a cylindrical shape with a spherical top (see illustration a). The distance from the bottom to the centre of the top (h) was consistently 30\u00a0mm. The diameter of the cylinders (d) was also 30\u00a0mm. c Examples of two psychometric curves. The circular data points and the fit through these points results from adaptation to the convex adaptation stimulus. The PSE is represented by PV. The square data points and the fit through these points result from adaptation to the concave adaptation stimulus. In this case, the PSE is represented by PC. The magnitude of the aftereffect (AE) is defined as the difference between PV and PC\nThe purpose of the present study was to obtain a better understanding of the representation of haptically perceived shape information, by probing the transfer of the curvature aftereffect. In the first experiment, we established the existence of an aftereffect when a curved surface is touched by a single finger and measured whether this aftereffect transferred to other fingers of the same hand. The second experiment was set up to determine whether the aftereffect depended on the finger used. Finally, in the third and fourth experiments, we investigated the transfer of the aftereffect between fingers of both hands.\nMaterials and methods\nSubjects\nA total number of 40 subjects participated [n\u00a0=\u00a08 for experiments 1, 2 and 4, n\u00a0=\u00a016 for experiment 3; 18 were male and 22 were female; the mean age was 22\u00a0years; 37 were right-handed, 3 were left-handed, according to a standard questionnaire (Coren 1993)]. Subjects in experiments 1 and 2 received course credit for their participation. Subjects in the third and fourth experiments received monetary compensation.\nStimuli\nThe stimuli comprised of a compound of polyurethane foam and artificial resin (Cibatool BM 5460). A computer-controlled milling machine was used to produce cylinders with a flat bottom and a spherically curved top. The top was either pointing outward (convex) or inward (concave). Both convex and concave adaptation stimuli were used, with curvature values of +36 and \u221236\u00a0m\u22121, respectively; the curvature of the nine test stimuli ranged from \u221216 to +16\u00a0m\u22121, in steps of 4\u00a0m\u22121. Illustrations of the stimuli and their cross-sections are given in Fig.\u00a01a, b, respectively.\nProcedure\nA subject was seated behind a table. The preferred arm rested on a platform, which was 30\u00a0mm above the tabletop. In the third and fourth experiments, both arms rested on the platform. Only the fingertips projected over the platform. The experimenter placed the stimulus underneath a fingertip. A curtain prevented the subjects from seeing the stimulus. During a trial, the tip of one finger was placed on an adaptation stimulus for 10\u00a0s. Subsequently, the subject placed a finger on a test stimulus and had to judge whether this test stimulus felt convex or concave. Subjects were not allowed to move the finger over the stimulus surface, and the experimenter checked for this. No instructions were given on the force to contact the stimulus, nor was it measured. No feedback was provided on the response.\nThree conditions were measured in the first experiment. In all conditions, the adaptation stimulus was touched with the index finger. In one condition, the test stimulus was also touched with the index finger. In the other two conditions, the test stimulus was touched with the middle finger or the little finger of the same hand, respectively. Each condition consisted of 10 repetitions of a group of 18 trials (two adaptation stimuli\u00a0\u00d7\u00a0nine test stimuli) with trials randomized within a group. One complete condition was measured in a single session of about one and a half hours. The separate sessions were spread over different days. The order in which the conditions were conducted was counterbalanced for the first six subjects and randomly chosen for the last two subjects.\nIn the second experiment, both the adaptation and the test stimuli were touched by the middle finger. In the third and fourth experiments, the adaptation stimulus was contacted by the index finger of the preferred hand; the test stimuli were touched with the index finger (third experiment) or middle finger (fourth experiment) of the non-preferred hand.\nAnalysis\nThe data for each subject and each condition were analyzed separately for the convex and the concave adaptation stimuli. The percentage of \u201cconvex\u201d responses was plotted against the curvature of the test stimulus. The point of subjective equality (PSE) was determined by fitting a psychometric function (cumulative Gaussian) to the data. The PSE represents the curvature value that in 50% of the test cases was judged \u201cconvex\u201d and in 50% of the cases was judged \u201cconcave\u201d. The magnitude of the aftereffect is defined as the difference between the PSE resulting from the adaptation to a convex surface and the PSE resulting from the adaptation to a concave surface. Examples of psychometric curves for a convex and a concave adaptation are given in Fig.\u00a01c.\nResults\nThe mean results for the aftereffect values are shown in Fig.\u00a02. The error bars indicate the standard errors of the mean.Fig.\u00a02Mean results of the aftereffect. The indicated error bars are the standard error in the mean for each condition. Experiment 1: eight subjects participated. Adaptation was performed by the index finger of the preferred hand. Testing was done using the index finger, middle finger, or little finger of the same hand. Experiment 2: eight subjects participated. Adaptation and testing was performed by the middle finger of the preferred hand. Experiment 3: sixteen subjects participated. Adaptation was performed by the index finger of the preferred hand; testing was done by the opposite index finger. Experiment 4: eight subjects participated. Adaptation was performed by the index finger of the preferred hand; testing was done by the middle finger of the non-preferred hand\nExperiment 1\nWe tested the occurrence of an aftereffect in each condition by performing separate one-tailed t tests. A significant result was obtained in all conditions (t7\u00a0=\u00a06.3, P\u00a0<\u00a00.001 for the index finger; t7\u00a0=\u00a09.8, P\u00a0<\u00a00.001 for the middle finger; t7\u00a0=\u00a03.4, P\u00a0=\u00a00.006 for the little finger). Subsequently, an ANOVA with a repeated measures design was performed to determine any differences between conditions. A significant main effect was found (F2,14\u00a0=\u00a022.5, P\u00a0<\u00a00.001). Pairwise comparisons showed a significant difference between the index finger and the middle finger (P\u00a0=\u00a00.007) and between the index finger and the little finger (P\u00a0=\u00a00.004), but not between the middle finger and the little finger (P\u00a0=\u00a01.0). The P values were adjusted with a Bonferroni correction.\nExperiment 2\nA one-tailed t test showed that there was a significant aftereffect (t7\u00a0=\u00a08.0, P\u00a0<\u00a00.001). Inspection of Fig.\u00a02 shows that the aftereffect of the middle finger condition of the second experiment was comparable to the index finger condition of the first experiment and was much higher than the middle finger condition of the first experiment. Independent samples t test confirmed that there was no significant difference in the first case (t14\u00a0=\u00a00.6, P\u00a0=\u00a00.6), but that there was a significant difference in the second case (t7.4\u00a0=\u00a06.1, P\u00a0<\u00a00.001).\nExperiment 3\nA one-tailed t test highlighted a significant aftereffect (t15\u00a0=\u00a02.7, P\u00a0=\u00a00.009). The magnitude of this aftereffect was much lower than for the index finger condition of the first experiment. This was confirmed by an independent sample t test (t22\u00a0=\u00a05.0, P\u00a0<\u00a00.001).\nExperiment 4\nA significant aftereffect was obtained, as was confirmed by a one-tailed t test (t7\u00a0=\u00a07.4, P\u00a0<\u00a00.001).\nDiscussion\nThe first novel observation of this paper is that the perception of surface curvature by a single fingertip is influenced by preceding contact of this finger with another curved surface. The magnitude of this curvature aftereffect did not depend on the finger employed, as shown by a comparison between the results of the first and the second experiment. Previously, Vogels et al. (1996, 1997) reported the existence of a static curvature aftereffect, when the whole hand was used. We suppose that our finding of a one-finger aftereffect falls in the same class of phenomena. A quantitative comparison between the results of Vogels et al. (1996) and our finding can be made by calculating the relative magnitude of the aftereffect, i.e. the aftereffect divided by the difference between the adaptation stimuli. This value equals 0.17\u00a0\u00b1\u00a00.02 for the results of Vogels et al., whereas it was 0.15\u00a0\u00b1\u00a00.07 for the index finger condition of the first experiment and 0.17\u00a0\u00b1\u00a00.06 for the middle finger condition of the second experiment, respectively. These values are in the same order of magnitude, irrespective of the differences in manner of touching and curvature range of the stimuli.\nThe second important finding of our study is that the aftereffect partially transfers between fingers of the same hand. This means that the sensation of shape with a certain finger influences the perception of a shape touched by another finger. This suggests that the sensations obtained by the different fingers share a common representation. However, the transfer is far from complete, indicating that curvature perception by each finger also yields a substantial, individual part in the representation. Interestingly, the aftereffect does not only transfer from the index finger to the neighboring middle finger, but also to the distant little finger. This result is unlike recently performed localization (Schweizer et al. 2000) and learning studies (Sathian and Zangaladze 1997; Harris et al. 2001), in which the reported transfer effects were obtained in the neighboring finger, but not in the distant fingers. This indicates that the processes involved in detecting the finger that is stimulated or increasing the skills to discriminate punctate pressure or roughness are quite different from those concerned in shape perception of an object.\nThe third interesting result of this study is that there was a small, but significant transfer of the aftereffect between fingers of both hands, irrespective of whether opposite fingers (experiment 3) or different fingers (experiment 4) were employed. This result is different from the result reported by Vogels et al. 1997, who did not find intermanual transfer. However, in their experiments, whole hands were involved, whereas only single fingertips were used in our experiment. Moreover, their conclusion was based on the performance of only 2 subjects, whereas 24 participants provided the data for our study. The results of the third and fourth experiments suggest that the representation of shape information obtained with one hand is not completely distinct from the representation of shape information received by the other hand, but shares a common, bilateral component.\nHow can our findings be interpreted in the context of neurophysiological literature? Firstly, our finding that the aftereffect only transfers partially between fingers of the same hand shows that a substantial part of the processing occurs at a stage where each finger is individually represented. On this stage, which spreads from the mechanoreceptors in the fingers up to area 3b in SI, no overlap occurs in signals from the slowly adapting receptors and the fast-adapting receptors (Gardner and Kandel 2000). Slowly adapting receptors respond with a sustained discharge when the finger is in contact with a surface, whereas fast-adapting receptors only respond at the onset and removal phase of the finger (Johansson and Vallbo 1983). Vogels et al. (1996) showed that the magnitude of their curvature aftereffect increased with an increase in adaptation time. These findings point to an important role for the slowly adapting receptors in the curvature aftereffect. Therefore, we suggest that the aftereffect at the stage related to an individual finger mainly originates from the processing of the slowly adapting receptors. Secondly, the fact that we found a transfer between the fingers of the same hand implies that a significant part of the processing of curvature information occurs at a level shared by the different fingers. In physiological terms, this indicates that at least area 1 or 2 of SI are involved, as receptive fields in these areas cover several fingers of a single hand (Gardner and Kandel 2000), but processing may also occur at an even higher stage. Thirdly, our finding of an intermanual transfer shows that the processing of curvature information also takes place on a higher, bilateral level. We can only speculate on the neural correlates of this bilateral processing. Possible candidates include area 2 of SI, areas 5 and 7 of the posterior parietal cortex, and the secondary somatosensory cortex (Iwamura 2000; Gardner and Kandel 2000).\nIt is interesting to mention that the aftereffects in the intramanual transfer conditions (experiment 1) and the intermanual transfer conditions (experiment 3 and 4) are similar in magnitude. This suggests that no important curvature processing occurs at a level that is devoted to a single hand, but that all processing takes place at a higher stage. The similar results for experiments 3 and 4 provide further support that the hands and fingers are not somatotopically represented at this stage. From a previous study, it is known that subjects also performed similarly in intramanual and intermanual curvature discrimination tasks, but that higher performance was obtained when only a single finger was employed (Van der Horst and Kappers 2007). The analogy between that study and the current study is that curvature information is mainly represented at the level of the individual finger, but partly available at a higher, finger- and hand-independent level. We should be careful in ascribing a specific function to the involvement of the higher level areas in the processing of curvature information. The role of more cognitive aspects should not be excluded, since it is known that processes like tactile attention (Burton and Sinclair 2000; Spence and Gallace 2007), working memory (Burton and Sinclair 2000), and object recognition (Reed et al. 2004) also engage the somatosensory areas.\nThe aftereffect that we found in the present study is a similar phenomenon as the aftereffect that was previously reported by Vogels et al. (1996, 1997). However, this does not entail that the representation of curvature is identical for touching with a single finger or with the whole hand. Vogels et al. (1997) already showed that, although similar aftereffects were found when either the whole hand or only the five fingertips were used, only a small transfer between these exploration modes was obtained, which points to a limited overlap in representation. Similarly, we suppose that there is a difference in representation between curvature that is perceived by a single finger and curvature that is perceived by the whole hand. In the single finger case, the representation is mainly at the level of the individual finger, whereas in the whole hand case, the representation is spread over all fingers and the palm of the hand.\nThis study shows that establishing the intramanual and intermanual transfer of the aftereffect is a useful tool in obtaining more insight into the representation of object properties as perceived by the fingers. In general, studying aftereffect transfer is attractive, because it enables a connection between psychophysics and neurophysiology. The convergence of these approaches leads to a better understanding of human perception.","keyphrases":["transfer","curvature","aftereffect","touch","mechanoreceptor","somatosensory","tactile"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Eur_J_Appl_Physiol-3-1-1914221","title":"Physical fitness, fatigue, and quality of life after liver transplantation\n","text":"Fatigue is often experienced after liver transplantation. The aims of this cross-sectional study were to assess physical fitness (cardiorespiratory fitness, neuromuscular fitness, body composition) in liver transplant recipients and to explore whether physical fitness is related to severity of fatigue. In addition, we explored the relationship between physical fitness and health-related quality of life. Included were 18 patients 1\u20135 years after transplantation (aged 48.0 \u00b1 11.8 years) with varying severity of fatigue. Peak oxygen uptake during cycle ergometry, 6-min walk distance, isokinetic muscle strength of the knee extensors, body mass index, waist circumference, skinfold thickness, severity of fatigue, and health-related quality of life were measured. Cardiorespiratory fitness in the liver transplant recipients was on average 16\u201334% lower than normative values (P \u2264 0.05). Furthermore, the prevalence of obesity seemed to be higher than in the general population (17 vs. 10%). We found no deficit in neuromuscular fitness. Cardiorespiratory fitness was the only fitness component that was related with severity of fatigue (rs = \u22120.61 to rs = -0.50, P \u2264 0.05). Particularly cardiorespiratory fitness was related with several aspects of health-related quality of life (rs = 0.48 to rs = 0.70, P \u2264 0.05). Results of our study imply that cardiorespiratory fitness and body composition are impaired in liver transplant recipients and that fitness is related with severity of fatigue (only cardiorespiratory fitness) and quality of life (particularly cardiorespiratory fitness) in this group. These findings have implications for the development of rehabilitation programs for liver transplant recipients.\nIntroduction\nLiver transplantation (LTx) is the only definitive treatment for end-stage liver disease. Since 1988, patient survival and liver function after LTx have improved markedly, due to improved technical expertise, better selection of patients, improved post-LTx management of complications, and improved immunosuppressive therapy (Adam et al. 2003). In Europe, LTx has achieved a 1-year survival rate of 82% (Burroughs et al. 2006).\nAlthough studies have reported that quality of life improves after LTx (Gross et al. 1999; van der Plas et al. 2003), limitations in daily function still remain (Gross et al. 1999; van der Plas et al. 2003). Amongst these limitations, liver transplant recipients often experience fatigue (Aadahl et al. 2002; Belle et al. 1997; Leyendecker et al. 1993; van den Berg-Emons et al. 2006). Gross et al. (1999), and Belle et al. (1997) reported that, although the intensity of fatigue was reduced after LTx, fatigue remained the most distressing symptom 1\u00a0year after surgery. Leyendecker et al. (1993) found that 9\u00a0months after LTx, complaints of fatigue were more severe in the LTx group than in the general population. In a previous study, we found severe fatigue in 44% of patients up to 15\u00a0years after LTx, and these complaints did not decrease over time (van den Berg-Emons et al. 2006a).\nRehabilitation programs might be effective in reducing severity of fatigue after LTx, but, to develop appropriate programs, knowledge on the factors associated with fatigue after LTx is necessary. However, data on these factors are scarce. Aadahl et al. (2002) and van den Berg-Emons et al. (2006a) suggested that the fatigue experienced by liver transplant recipients is primarily physical, rather than psychological. Furthermore, van den Berg-Emons et al. (2006b) found that severe complaints of fatigue in liver transplant recipients are associated with low levels of everyday physical activity. A hypoactive lifestyle may lead to a negative spiral: hypoactivity leading to a reduction in physical fitness and deterioration of complaints of fatigue, leading to further hypoactivity.\nFew studies have investigated the physical fitness of liver transplant recipients, but the limited data available suggest a reduced physical fitness after LTx (Beyer et al. 1999; Stephenson et al. 2001; Unnithan et al. 2001). Stephenson et al. (2001) found a 40\u201360% lower maximal oxygen uptake than predicted in liver transplant recipients. Beyer et al. (1999) reported that, although the cardiovascular and neuromuscular fitness in liver transplant recipients improved after a supervised exercise program during the post-operative year, maximal oxygen uptake and muscle strength remained 10\u201320% lower compared to healthy gender and age-matched individuals. Also in pediatric liver transplant recipients, deficits in cardiovascular fitness, and abdominal muscle strength have been reported (Unnithan et al. 2001).\nIt may be hypothesized that deficits in physical fitness in liver transplant recipients are associated with complaints of fatigue. Furthermore, deficits in physical fitness may lead to impaired health-related quality of life (HRQoL). However, to our knowledge, no studies are available on the relationships between these parameters in liver transplant recipients.\nBecause of the scarcity of studies on physical fitness and related parameters in liver transplant recipients, the present study assessed physical fitness (cardiorespiratory fitness, neuromuscular fitness, and body composition) in liver transplant recipients and explored whether physical fitness is related to severity of fatigue in this group. The relationship between physical fitness and HRQoL was also explored.\nPatients and methods\nPatients\nTo obtain a representative sample of liver transplant recipients with respect to fatigue, we recruited liver transplant recipients with varying severity of fatigue, according to the distribution of severity of fatigue as found in our previous study in 96 liver transplant recipients (van den Berg-Emons et al. 2006a). Severity of fatigue in this previous study ranged from \u2018no signs of fatigue\u2019 to \u2018most disabling fatigue\u2019, and was assessed with the Fatigue Severity Scale of Krupp et al. (1989) (see below). Inclusion criteria for the present study were: LTx between 1 and 5\u00a0years ago, sufficient knowledge of the Dutch language, and age between 18 and 65\u00a0years. Exclusion criteria were: multiorgan transplant recipients, severe comorbidity, and contra-indication for a progressive maximal cycle ergometer test. Of the original sample of 96 patients in the previous study (van den Berg-Emons et al. 2006a), 4 patients had died, 1 patient had emigrated, 53 patients were transplanted more than 5\u00a0years previously, and 10 patients were not eligible because of contra-indications for a maximal cycle ergometer test. Of the 28 eligible patients, 18 patients (64%) agreed to participate. There were no significant differences in relevant characteristics between the patients who decided to participate and the non-participants. The study was approved by the Medical Ethics Committee of the Erasmus University Medical Center. Written informed consent was obtained from all subjects. Table\u00a01 shows the characteristics of the study group.\nTable\u00a01Characteristics of the study group (n\u00a0=\u00a018)Age (years)48.0\u00a0\u00b1\u00a011.8Gender\u00a0Male11\u00a0Female7Primary disease (n) a\u00a0Chronic17\u00a0Acute1Time since transplantation (years)3.3\u00a0\u00b1\u00a01.1Immunosuppressive agentsb\u00a0116\u00a021\u00a031Results are presented as mean\u00a0\u00b1\u00a0SD or numbersaChronic primary disease: cholestatic (n\u00a0=\u00a06), viral (n\u00a0=\u00a06), miscellaneous (n\u00a0=\u00a05); acute primary disease: intoxication (n\u00a0=\u00a01)bImmunosuppressive agents: 1 agent, cyclosporine or tacrolimus; 2 agents, prednisone with tacrolimus; 3 agents, prednisone with tacrolimus and azathioprine\nMeasurements\nPhysical fitness: cardiorespiratory\nCardiorespiratory fitness was measured with a progressive maximal aerobic test on a cycle ergometer (ER800, Jaeger Toennies, Breda, The Netherlands). The test was preceded by a 1-min warm-up period (20\u00a0W). The test started at 20\u00a0W, and resistance was increased every minute by 15 or 20\u00a0W, depending on the ability of the patients. Individual protocols were constructed such that the total exercise time ranged from 8 to 12\u00a0min. The pedal rate was 60\u00a0rpm and strong verbal encouragement was given during the test. The test was terminated when the subject voluntarily stopped due to exhaustion, or when the patient was unable to maintain the initial pedal rate. Gas exchange and heart rate (HR) were measured continuously using a breath-by-breath gas analysis system (K4b2, COSMED, Rome, Italy). Subjective strain was measured immediately after the final stage by the Borg Category Scale for Rating of Perceived Exertion (Borg 1982). Patients were asked to indicate how strenuous they had experienced the test by giving a number from 0 (no effort at all) to 10 (maximal effort). Cardiorespiratory fitness was defined as the mean oxygen uptake during the last 30\u00a0s of exercise [VO2peak, in ml\u00a0kg\u22121\u00a0min\u22121 and in ml\u00a0kg fat-free mass\u22121\u00a0min\u22121 (ml\u00a0kgFFM\u22121\u00a0min\u22121)]. In addition, the ventilatory anaerobic threshold (VAT, expressed as percentage of predicted VO2peak) was estimated by the ventilatory equivalent method, when VE\/VO2 and PetO2 increased while VE\/VCO2 and PetCO2 remained stable (Reinhard et al. 1979; Wasserman et al. 1999).\nFinally, patients performed the submaximal 6-min walk test (6MWT) (Guyatt et al. 1985). Patients were instructed to walk, not run, as far as they could along a 30-m marked tape in a hall during a 6-min period. Standardized encouragement was provided with the following phrases: \u201cYou are doing well\u201d and \u201cKeep up the good work\u201d. Patients were allowed to stop and rest during the test, but were instructed to resume walking as soon as they felt able to do so. The 6-min walk distance (6MWD) was registered.\nPhysical fitness: neuromuscular\nIsokinetic muscle strength of the knee extensors was assessed in both legs by a Biodex\u00ae dynamometer (Shirley, New York, USA), recording strength as torque in Nm. The patients were seated against a back-rest, firmly strapped at the hip and thigh. The rotational axis was aligned with the lateral femoral epicondyle. After five familiarization repetitions, isokinetic strength was measured at 60\u00b0\/s with 5 maximal contractions and at 180\u00b0\/s with 15 maximal contractions. Strong verbal encouragement was given during the test. Peak torque (PT) was defined as the maximum torque generated by the patients throughout one series of repetitions at each velocity.\nPhysical fitness: body composition\nHeight and body mass were measured without shoes. Body mass was measured using a Cormier Paribel\u00ae weighing chair (FH Balances Cormier, Romainville, France). Body mass index (BMI, kg\u00a0m\u22122) was calculated from height and body mass. Waist circumference (cm) was measured mid-way between the lowest rib and the iliac crest while standing. Thickness of four skinfolds (biceps, triceps, subscapular, suprailiaca region) was measured twice at the right side of the body with a Harpenden Skin-Fold Caliper (Burgess Hill, UK). The mean of the two measurements was used as representative for each site. Percentage body fat (BF) was predicted from skinfold thickness according to the method of Durnin and Womersley (1974).\nSeverity of fatigue\nSeverity of fatigue was assessed by the Dutch version of the Fatigue Severity Scale (FSS) (Krupp et al. 1989). The FSS is a self-administered questionnaire with answers ranging from 1 (\u2018strongly disagree\u2019) to 7 (\u2018strongly agree\u2019). The mean score of the nine inquiries ranges from 1 (\u2018no signs of fatigue\u2019) to 7 (\u2018most disabling fatigue\u2019). Internal consistency, reliability, validity, and sensitivity of the FSS have been established in several patient groups (Krupp et al. 1989; Merkies et al. 1999).\nIn addition to the FSS, severity of fatigue was assessed with a horizontal visual analogue scale (VAS). Patients were asked to mark the 100-mm line according to how intense they had experienced fatigue during the last month (0 denotes \u2018no fatigue experienced\u2019 and 100 denotes \u2018the most severe fatigue\u2019) (ter Borg et al. 2004). Visual analogue scales have been found to yield reliable and valid data (Huskisson 1982; Scott and Huskisson 1979).\nHealth-related quality of life\nHRQoL was assessed by the validated Dutch version (RAND-36) (van der Zee and Sanderman 1993) of the Medical Outcomes Study Short Form-36 (SF-36) (Ware and Sherbourne 1992). The SF-36 is a validated, self-administered questionnaire used internationally to measure health status with respect to different dimensions: physical functioning, social functioning, role limitations due to physical problems, role limitations due to emotional problems, pain, mental health, vitality, general health perception, and change in perceived health during the last 12\u00a0months. All raw scores were converted to a 0\u2013100 scale, with higher scores indicating higher levels of functioning or well-being.\nProcedure\nOn the day of the measurements, patients refrained from caffeine, nicotine, and heavy exercise. The order of the tests was standardized: patients started with the 6MWT, followed by the questionnaires (completed under supervision of the researcher), body composition measurements, strength test, and finally the progressive maximal aerobic test. Exercise tests were performed under supervision of a physician. There were sufficient rest periods between the tests.\nStatistics\nStatistical analysis was performed using SPSS 10.1 for Windows (SPSS Inc., Chicago, IL, USA). Results are presented as mean\u00a0\u00b1\u00a0standard deviation (SD), range or numbers. Results on cardiorespiratory fitness were compared to normative values for sedentary persons and to normative values for people who exercise no more than 1\u20132\u00a0h a week (recreational) (Vos et al. 2001). Results on neuromuscular fitness were compared with the normative values of Akima et al. (2001) and 6MWD was compared with the normative values of Enright and Sherill in healthy adults aged 40\u201380\u00a0years (1998). When patients were younger than 40\u00a0years, the normative values of Gibbons et al. (2001) were used. Obesity was defined as a BMI\u00a0\u2265\u00a030 (World Health Organization 1989), a waist circumference \u2265102\u00a0cm in men and \u226588\u00a0cm in women (Lean et al. 1995) or percentage BF \u226525% in men and \u226532% in women (Lohman 1992).\nDifferences in physical fitness between patients and normative values were tested with the non-parametric Mann-Whitney U Test. Relationships between physical fitness and severity of fatigue and between physical fitness and quality of life were explored using the non-parametric Spearman correlation coefficient (rs). A probability value \u22640.05 determined statistical significance. However, because of the relatively small study sample, also results on the \u03b1\u00a0=\u00a00.10 level are presented (indicating a trend).\nResults\nPhysical fitness\nMean score on the Borg Scale was 6.3\u00a0\u00b1\u00a02.2, indicating that the patients experienced the maximal ergometer test on average as heavy to very heavy. The mean HRmax was 90\u00a0\u00b1\u00a010% of what was predicted (predicted HRmax\u00a0=\u00a0220-age) (Fox et al. 1971).\nThe individual results of the patients are presented in Table\u00a02. Table\u00a03 shows the cardiorespiratory fitness and neuromuscular fitness of the patients compared with normative values. VO2peak in ml\u00a0kg\u22121\u00a0min\u22121 was 15\u00a0\u00b1\u00a022% (P\u00a0=\u00a00.07) and 34\u00a0\u00b1\u00a015% (P\u00a0=\u00a00.00) lower than normative values for, respectively, sedentary persons and those who exercise recreationally. VO2peak in ml\u00a0kgFFM\u22121\u00a0min\u22121 was 16\u00a0\u00b1\u00a019.2% (P\u00a0=\u00a00.02) and 33\u00a0\u00b1\u00a014.8% (P\u00a0=\u00a00.00) lower than normative values for, respectively, sedentary persons and those who exercise recreationally. 6MWD was 16\u00a0\u00b1\u00a014% (P\u00a0=\u00a00.01) lower than normative values. There was no significant deficit in neuromuscular fitness. Table\u00a04 shows the body composition of the LTx group. According to the cut-off points for obesity based on BMI, waist circumference and percentage BF, respectively 17, 36, and 41% of the patients were classified as obese.\nTable\u00a02Individual results on physical fitness in the 18 liver transplant recipientsAge (year\/Gender)BMI (kg\u00a0m\u22122)Waist (cm)aBody fat (%)bVO2peak (ml\u00a0kg\u22121\u00a0min\u22121)VO2peak (ml\u00a0kgFFM\u22121\u00a0min\u22121)bVAT (% predicted VO2peak sed) VAT (% predicted VO2peak recr)6MWD (m)PT extension 60\u00b0\/s (Nm)PT extension 180\u00b0\/s (Nm)56\/M28.3111.029.036.551.454.142.7652156.388.252\/M29.032.716.724.849.138.9428101.873.142\/F22.828.934.548.590.565.8530100.065.660\/F38.4118.542.013.122.543.632.5495101.978.664\/M29.7103.530.621.731.377.455.443099.962.624\/F21.072.025.929.940.438.831.1660159.9111.546\/M26.5100.025.828.238.048.638.7606151.688.563\/M24.420.520.926.469.449.741074.444.842\/M34.9132.022.830.925.8513145.9118.353\/F23.977.530.123.133.0108.664.846540.035.753\/M25.999.623.620.727.156.742.6510149.396.839\/M23.787.017.431.237.851.543.5547169.0111.833\/M24.088.019.835.143.767.959.9510155.9116.963\/F33.0103.034.815.824.257.943.230367.239.445\/F26.830.823.634.161.046.2514120.161.739\/F18.775.022.730.238.156.246.8600115.967.932\/M22.991.020.425.031.443.438.3576180.4124.658\/M25.199.025.018.524.648.637.9495128.691.9Abbreviations: BMI\u00a0(kg\u00a0m\u22122), body mass index; Waist (cm), waist circumference; VO2peak\u00a0(ml\u00a0kg\u22121\u00a0min\u22121), peak oxygen uptake per kg; VO2peak (ml\u00a0kgFFM\u22121\u00a0min\u22121), peak oxygen uptake per kg fat-free mass; VAT (% predicted VO2peak), ventilatory anaerobic threshold as percentage of predicted VO2peak; sed, sedentary normative values; recr, recreational normative values; 6MWD, 6-min walk distance; PT, peak torqueaWaist circumference: n\u00a0=\u00a014, because of thickness of the skin at the place of the cicatrice in four patientsbBody fat and VO2peak: n\u00a0=\u00a017, because the thickness of the subscapular skinfold could not be measured reliably in one patientTable\u00a03Cardiorespiratory fitness and neuromuscular fitness in the liver transplant groupLTx groupNorm valuesaPCardiorespiratory fitness\u00a0VO2peak (ml\u00a0kg\u22121\u00a0min\u22121)24.8\u00a0\u00b1\u00a06.929.4\u00a0\u00b1\u00a07.4 (sed)0.07\u202037.5\u00a0\u00b1\u00a06.7 (recr)0.00*\u00a0VO2peak (ml\u00a0kgFFM\u22121\u00a0min\u22121)b34.0\u00a0\u00b1\u00a08.741.0\u00a0\u00b1\u00a07.8 (sed)0.02*50.9\u00a0\u00b1\u00a05.9(recr)0.00*\u00a06MWD (m)513.6\u00a0\u00b1\u00a088.9609.6\u00a0\u00b1\u00a097.60.01*Neuromuscular fitness\u00a0PT extension at 60\u00b0\/s (Nm)123.2\u00a0\u00b1\u00a038.7138.5\u00a0\u00b1\u00a044.10.22\u00a0PT extension at 180\u00b0\/s (Nm) 82.1\u00a0\u00b1\u00a027.986.1\u00a0\u00b1\u00a030.10.59Results are presented as mean\u00a0\u00b1\u00a0SDAbbreviations: VO2peak\u00a0(ml\u00a0kg\u22121\u00a0min\u22121), peak oxygen uptake per kg; VO2peak (ml\u00a0kgFFM\u22121\u00a0min\u22121), peak oxygen uptake per kg fat-free mass; VAT (% predicted VO2peak), ventilatory anaerobic threshold as percentage of predicted VO2peak; sed, sedentary normative values; recr, recreational normative values (people who exercise no more than 1\u20132\u00a0h a week); 6MWD 6-min walk distance; PT, peak torqueaVos et al. (2001), Enright and Sherill (1998), Gibbons et al. (2001), Akima et al. (2001)bn\u00a0=\u00a017, because the thickness of the subscapular skinfold could not be measured reliably in one patient*\u00a0Significant (P\u00a0\u2264\u00a00.05) difference between patients and normative values\u2020\u00a0Difference between patients and normative values at the \u03b1\u00a0=\u00a00.10 level (trend)Table\u00a04Body composition in the liver transplant groupBody compositionMean\u00a0\u00b1\u00a0SDBody weight (kg)80.6\u00a0\u00b1\u00a018.3Height (m)1.74\u00a0\u00b1\u00a0.11Body mass index (kg\u00a0m\u22122)26.6\u00a0\u00b1\u00a05.0Waist circumference (cm)a96.6\u00a0\u00b1\u00a016.8Body fat (%)b26.9\u00a0\u00b1\u00a06.4Results are presented as mean\u00a0\u00b1\u00a0SDaWaist circumference: n\u00a0=\u00a014, because of thickness of the skin at the place of the cicatrice in four patientsbBody fat: n\u00a0=\u00a017, because the thickness of the subscapular skinfold could not be measured reliably in one patient\nRelationships\nTable\u00a05 shows the correlation coefficients between the physical fitness parameters and severity of fatigue as assessed with the FSS and VAS. None of the parameters of neuromuscular fitness or body composition were significantly related with severity of fatigue, assessed with either the FSS or the VAS.\nTable\u00a05Spearman correlation coefficients for the relationships between fitness parameters and severity of fatigue as measured with the Fatigue Severity Scale (FSS) and the Visual Analogue Scale (VAS) in 18 liver transplant recipientsPhysical fitnessFatigueFSSVASRsPRsPCardiorespiratory fitness\u00a0VO2peak (ml\u00a0kg\u22121\u00a0min\u22121)\u22120.400.10\u22120.520.03*\u00a0% of sedentary norm\u22120.170.50\u22120.42.0.08\u2020\u00a0% of recreational norm\u22120.320.20\u22120.530.03*VO2peak (ml\u00a0kgFFM\u22121\u00a0min\u22121)a\u22120.430.08\u2020\u22120.510.04*\u00a0% of sedentary norma\u22120.350.17\u22120.500.04*\u00a0% of recreational norma\u22120.450.07\u2020\u22120.610.01*\u00a0VAT (% predicted VO2peak sed)0.220.380.030.92\u00a0VAT (% predicted VO2peak recr)0.170.49\u22120.060.82\u00a06MWD (m)\u22120.440.07\u2020\u22120.530.03*\u00a0% of norm\u22120.250.32\u22120.520.03*Neuromuscular fitness\u00a0PT extension at 60\u00b0\/s (Nm)\u22120.390.11\u22120.310.22\u00a0% of norm\u22120.150.56\u22120.280.27\u00a0PT extension at 180\u00b0\/s (Nm)\u22120.300.22\u22120.120.64\u00a0% of norm0.020.930.040.87Body composition\u00a0Body mass index (kg\u00a0m\u22122)0.030.910.060.82\u00a0Waist circumference (cm)b\u22120.070.82\u22120.060.83Body fat (%)a0.320.210.150.58Abbreviations: VO2peak (ml\u00a0kg\u22121\u00a0min\u22121), peak oxygen uptake per kg; VO2peak (ml\u00a0kgFFM\u22121\u00a0min\u22121), peak oxygen uptake per kg fat-free mass; VAT (% predicted VO2peak), ventilatory anaerobic threshold as percentage of predicted VO2peak; sed, sedentary normative values; recr, recreational normative values (people who exercise no more than 1\u20132\u00a0h a week); 6MWD, 6-min walk distance; PT, peak torquean\u00a0=\u00a017, because the thickness of the subscapular skinfold could not be measured reliably in one patientbn\u00a0=\u00a014, because of thickness of the skin at the place of the cicatrice in four patients*\u00a0Significant (P\u00a0\u2264\u00a00.05) correlation\u2020\u00a0Correlation at the \u03b1\u00a0=\u00a00.10 level (trend)\nVO2peak in ml\u00a0kgFFM\u22121\u00a0min\u22121 (absolute and expressed as percentage of recreational normative values) and 6MWD (absolute) were related with severity of fatigue as assessed with the FSS at the \u03b1\u00a0=\u00a00.10 level (trend). With respect to the VAS, VO2peak in ml\u00a0kg\u22121\u00a0min\u22121 (absolute and expressed as percentage of recreational normative values), VO2peak in ml\u00a0kgFFM\u22121\u00a0min\u22121 (absolute and expressed as percentage of both sedentary and recreational normative values) and 6MWD (absolute and expressed as percentage of normative values) were significantly (P\u00a0\u2264\u00a00.05) correlated with severity of fatigue. VO2peak in ml\u00a0kg\u22121\u00a0min\u22121 as percentage of sedentary normative values was related with severity of fatigue as assessed with the VAS at the \u03b1\u00a0=\u00a00.10 level (trend). The VAT was not correlated with severity of fatigue, assessed with the FSS or the VAS.\nTable\u00a06 shows the correlation coefficients between physical fitness parameters and HRQoL. There were several significant correlations between physical fitness and HRQoL and particularly between cardiorespiratory fitness on the one hand and \u2018Physical functioning\u2019, \u2018Social functioning\u2019 and \u2018Vitality\u2019 on the other hand.\nTable\u00a06Spearman correlations coefficients for the relationships between physical fitness and health-related quality of life as assessed with the RAND\u221236 in 18 liver transplant recipientsPhysical fitnessRAND-36 domainPFSFRlpRleMHVTBPGHCHCardiorespiratory fitness\u00a0VO2peak (ml\u00a0kg\u22121\u00a0min\u22121)0.55\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u00a0% of sedentary norm\u20130.42\u2013\u2013\u20130.50\u2013\u2013\u2013\u00a0% of recreational norm0.48\u2013\u2013\u2013\u20130.51\u2013\u2013\u2013\u00a0VO2peak (ml\u00a0kgFFM\u22121\u00a0min\u22121)a0.570.510.41\u2013\u2013\u2013\u2013\u2013\u2013\u00a0% of sedentary norma\u20130.56\u2013\u2013\u20130.59\u20130.48\u2013\u00a0% of recreational norma0.460.58\u2013\u2013\u20130.58\u2013\u2013\u2013\u00a0VAT (% predicted VO2peak sed)\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u00a0VAT (% predicted VO2peak recr)\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u00a06MWD (m)0.670.57\u2013\u20130.53\u20130.54\u2013\u2013\u00a0% of norm\u20130.70\u2013\u2013\u20130.51\u2013\u20130.44Neuromuscular fitness\u00a0PT extension at 60\u00b0\/s (Nm)0.44\u2013\u2013\u2013.41\u2013\u2013\u2013\u2013\u00a0% of norm\u20130.68\u2013\u2013\u2013\u20130.56\u2013\u2013\u00a0PT extension at 180\u00b0\/s (Nm)\u2013\u2013\u2013\u2013\u2013\u20130.44\u2013\u2013\u00a0% of norm\u20130.51\u20130.54\u2013\u20130.50\u2013\u2013Body composition\u00a0Body mass index (kg\u00a0m\u22122)\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u00a0Waist circumference (cm)b\u2013\u2013\u20130.49\u20130.41\u2013\u2013\u2013\u2013\u2013\u00a0Body fat (%)a\u2013\u2013\u2013\u2013\u2013\u2013\u2013To enhance the clarity of the table, only significant correlation coefficients (P\u00a0\u2264\u00a00.05, bold) or trends (P\u00a0<\u00a00.10, not bold) are presentedAbbreviations: VO2peak (ml\u00a0kg\u22121\u00a0min\u22121), peak oxygen uptake per kg; VO2peak (ml\u00a0kgFFM\u22121\u00a0min\u22121), peak oxygen uptake per kg fat-free mass; VAT (% predicted VO2peak) ventilatory anaerobic threshold as percentage of predicted VO2peak; sed, sedentary normative values; recr, recreational normative values (people who exercise no more than 1\u20132\u00a0h a week); 6MWD, 6-min walk distance; PT, peak torque; PF, physical functioning; SF, social functioning; Rlp, Role limitations physical; Rle, Role limitations emotional; MH, mental health; VT, vitality; BP, bodily pain; GH, general health perception; CH, changes in healthan\u00a0=\u00a017, because the thickness of the subscapular skinfold could not be measured reliably in one patientbn\u00a0=\u00a014, because of thickness of the skin at the place of the cicatrice in four patients\nDiscussion\nThis is the first study in which relationships between several aspects of physical fitness and severity of fatigue are explored after LTx. The study is an initial step in identifying factors that are associated with fatigue in liver transplant recipients. A limitation of this study may be that the sample was relatively small. However, we believe that this sample is representative for patients after LTx and that the study provides important information for the development of rehabilitation programs for liver transplant recipients.\nPhysical fitness\nWe found average deficits in VO2peak of 16\u201334%, when we compared the results in the liver transplant recipients with normative values for, respectively, sedentary persons and those who exercise no more than 1\u20132\u00a0h a week. We believe that a value in between the sedentary and recreational normative values is representative for healthy Dutch persons of the same age as the liver transplant recipients that participated in our study. 6MWD was 16% lower than normative values. This subnormal level of cardiorespiratory fitness (both VO2peak and 6MWD) is in agreement with findings of previous studies (Beyer et al. 1999; Stephenson et al. 2001; Unnithan et al. 2001). Furthermore, the prevalence of obesity was higher in this study than in the general Dutch population; 17% of the patients had a BMI of more than 30 compared to 10% in the general Dutch population (Gezondheidsraad 2003).\nImpaired cardiorespiratory fitness in liver transplant recipients may be due to the use of immunosuppressive medication, e.g. glucocorticoids and calcineurin inhibitors, which may influence both the cardiovascular system and skeletal muscles (Hokanson et al. 1995; Mercier et al. 1996). Immunosuppressive medication can also induce appetite stimulation and diabetes mellitus; this in association with emotional conditions can cause a change in eating habits (Correia et al. 2003; Hussaini et al. 1998). Furthermore, patients with primary biliary cirrhosis and some patients with other chronic liver disease are hypermetabolic. LTx in these patients may lead to a reduction in resting metabolic rate and can also cause an increase in BF (Hussaini et al. 1998; Richardson et al. 2001). Besides immunosuppressive medication and changes in appetite and metabolism, also deconditioning (both before and after transplantation) may contribute to the impaired cardiorespiratory fitness and body composition (Epstein et al. 1998; Grose et al. 1995). In a previous study we found that severe complaints of fatigue were associated with low levels of everyday physical activity in liver transplant recipients (van den Berg-Emons et al. 2006b).\nBecause of the immunosuppressive medication and deconditioning, we also expected a deficit in neuromuscular fitness. However, this was not demonstrated in the present study, in contrast to Beyer et al. (1999), who found a 10\u201320% lower muscle strength in liver transplant recipients compared to age- and sex-matched sedentary individuals. They contributed this deficit to muscle weakness as a side-effect of glucocorticoids.\nThe discrepancy between the findings of our study and those of Beyer et al. (1999) may be explained by the use of Tacrolimus instead of cyclosporine in the majority of our subjects. Tacrolimus is known to have less side effects than cyclosporine (Maes and Vanrenterghem 2004).\nStephenson et al. (2001) reported an early VAT in liver transplant recipients (<45\u201350% of the predicted VO2max) compared to 50\u201360% of the VO2max in healthy persons found by Davis et al. (1997). They contributed this early anaerobic threshold in liver transplant recipients to the cyclosporine-induced decrease in mitochondrial oxygen consumption (2001). However, the European Respiratory Society (ERS Task Force 1997) reported that there is a wide range of normal predicted values (35\u201370%). Therefore, it is difficult to indicate whether the VAT in our patients was reduced compared to normal (58.6\u00a0\u00b1\u00a018.9 and 44.7\u00a0\u00b1\u00a011.1% of predicted VO2peak for, respectively, sedentary persons and those who exercise recreationally).\nPhysical fitness and severity of fatigue\nAlthough relationships between severity of fatigue (as measured with the FSS and VAS) and physical fitness were not univocal, this study demonstrates a relationship between cardiorespiratory fitness and severity of fatigue after LTx. Patients with more severe complaints of fatigue had larger deficits in cardiorespiratory fitness than patients with less severe complaints of fatigue. Although this cross-sectional study does not allow us to conclude that a reduced cardiorespiratory fitness results in fatigue (or vice versa), there may be an interaction between parameters: complaints of fatigue leading to decreased physical activity (van den Berg-Emons et al. 2006b) and decreased physical fitness, leading to further deterioration of complaints of fatigue. It may then be hypothesized that rehabilitation programs, aimed at enhancing cardiorespiratory fitness, can be effective in breaking through this negative spiral and (partly) reduce complaints of fatigue in this population. However, this hypothesis has to be confirmed in future randomized trials.\nIn contrast to our expectations, the present study indicates that other aspects of physical fitness, neuromuscular fitness and body composition, do not seem to be related with severity of fatigue after LTx. However, it should be realized that our study sample was relatively small and some of the studied relationships may have failed to show statistical significance.\nPhysical fitness and HRQoL\nPrevious studies on healthy persons indicate that physical activity, fitness, and body fatness are associated with HRQoL and mood (Han et al. 1998; Stewart et al. 2003). There are also indications that physical activity is related to HRQoL after LTx (Painter et al. 2001; van den Berg-Emons et al. 2006b). Therefore, we expected to find a relationship between physical fitness and HRQoL in our study group. In agreement with the study of Stewart et al. (2003) in healthy elderly subjects, we found several significant correlations between physical fitness (particularly cardiorespiratory fitness) and HRQoL in our liver transplant recipients. Patients with large deficits in physical fitness experienced worse HRQoL than patients with small deficits in physical fitness. However, in contrast with studies in healthy persons (Han et al. 1998; Stewart et al. 2003), we found only few relationships between body composition and HRQoL.\nOur results on the relationships between physical fitness and HRQoL imply that rehabilitation programs aimed at improving physical fitness (particularly cardiorespiratory fitness) may consequently result in improved HRQoL, particularly in improved physical and social functioning, vitality, and bodily pain. However, these implications have to be confirmed in future randomized trials on the effects of such rehabilitation programs in liver transplant recipients.\nConclusion\nCardiorespiratory fitness in the liver transplant recipients was distinctly impaired and the prevalence of obesity was higher than in the general population; there were no indications that neuromuscular fitness is impaired after LTx. Based on the relationships we found between cardiorespiratory fitness and severity of fatigue, a rehabilitation program aimed at enhancing cardiorespiratory fitness may help in reducing complaints of fatigue after LTx. Such rehabilitation programs may also result in improved HRQoL.","keyphrases":["fatigue","liver transplantation","body composition","peak oxygen uptake","isokinetic muscle strength"],"prmu":["P","P","P","P","P"]}
{"id":"Clin_Rev_Allergy_Immunol-4-1-2243253","title":"Anti-CCP2 Antibodies: An Overview and Perspective of the Diagnostic Abilities of this Serological Marker for Early Rheumatoid Arthritis\n","text":"The literature of the last 4 years confirms that the anti-CCP2 test is a very useful marker for the early and specific diagnosis of rheumatoid arthritis (RA). The anti-CCP2 test is very specific for RA (95\u201399%) and has sensitivity comparable to that of the rheumatoid factor (70\u201375%). The antibodies can be detected very early in the disease and can be used as an indicator for the progression and prognosis of RA. In this review, these interesting properties and some future possibilities of this diagnostic test are discussed.\nThe presence of autoantibodies in the serum of patients is a very typical phenomenon for autoimmune diseases. Most of these autoantibodies, however, can also be detected in patients with other conditions and are therefore not specific. A typical example is the rheumatoid factor (RF), which is present in most inflammatory conditions. However, in some cases, autoantibodies can give the clinician a more precise indication of the type of underlying disease because they occur specifically in a certain disease. For example, anti-Sm antibodies are linked almost exclusively to systemic lupus erythematosus (SLE); whereas anti-DNA topoisomerase-I antibodies are typically present in scleroderma patients. Among the most disease-specific autoantibodies described are the so-called ACPA (anti-citrullinated protein\/peptide antibodies). These antibodies occur specifically in RA and can be measured most conveniently via the anti-CCP (anti-cyclic citrullinated peptide) antibody test.\nThe first generation CCP test (CCP1) used in early diagnostic studies (2000\u20132001) contained a single cyclic citrullinated peptide derived from filaggrin as the substrate [1]. It could detect ACPA in 68% of patients with established RA with a very high specificity (98%). Because filaggrin is not expressed in the synovium, it is most likely not the natural citrullinated antigen for ACPA. Other peptides, not related to filaggrin, could therefore potentially provide better epitopes for detection of ACPA. Via screening of a number of peptide libraries, novel citrullinated peptides were obtained and incorporated into a second generation CCP test (CCP2). This test is commercially available, and as all companies use the same type of CCP2 peptides, standardization is achieved quite easily. The diagnostic properties of this test will be discussed below.\nThe CCP2 Test is Sensitive and Highly Specific for RA\nSince its appearance on the market in the second half of 2002, the diagnostic properties of the CCP2 test have been studied by many laboratories. This resulted in more than 120 publications dealing with this subject. The accumulated data, including only papers that appeared in PubMed till December 2006, are given in Table\u00a01. It is clear that the accumulated data confirm the earliest reports on specificity and sensitivity of the CCP2 test. The anti-CCP2 test demonstrates an RF-like sensitivity with a very high specificity for RA (see also recent reviews: [2, 3]). It is also commonly recognized that anti-CCP2 antibody may be present in up to 40% of RF-negative RA sera [4, 5].\nTable\u00a01Cumulative anti-CCP2 diagnostic data published between 2002 and 2006Patient groupNumberCCP2+Sensitivity (%)Specificity (%)RA total14,18310,13571.5\u00a0Early3,8762,36561.0\u00a0Established10,3077,77075.4Controls15,1566834.595.6\u00a0Non-RA11,5026475.694.4\u00a0Healthy3,654361.099.0In total, 122 independent studies were included. The selection for early RA has been adopted from the original reports.\nThe anti-CCP2 test enables clinicians to distinguish RA patients from other arthritic diseases, especially in cases where the RF test is not discriminative. This is, for example, the case with chronic hepatitis C virus (HCV) infection, a disease that can easily be misdiagnosed as it often reveals RA-like arthropathies and, in many cases, is accompanied by a positive RF. Several examples of such studies are given in recent reviews on this subject [6].\nRecently, there is also an interest to compare the diagnostic potential of anti-CCP2 with novel tests based on the use of a citrullinated antigen (for example, MCV\u2009=\u2009anti-mutated citrullinated vimentin [7], CPA\u2009=\u2009citrullinated protein antibodies, VCP\u2009=\u2009anti-viral citrullinated protein [8], antihuman fibrinogen alpha [9, 10], and CCP3 [11]). For a reliable comparison of these tests, it is essential that their performance is assessed under the same conditions, e.g., the sensitivities of the tests should be determined at the same level of specificity. Good examples of such stratified studies have been carried out recently by van der Cruijssen et al. [10] and Dejaco et al. [7]. For example, Dejaco et al. [7] showed, in a large cohort of patients (>600), that at a specificity of 98.7%, being the specificity of the anti-CCP2 test, the sensitivity of the anti-MCV test is 53.7% as opposed to 70.1% for the anti-CCP test. Coenen et al. [11] compared several commercial tests, including a very recent CCP3 test from Inova. At the cut-offs recommended by the various manufacturers, the positive predictive value of the three commercial CCP2 tests is about 90% with a specificity of around 96%. The specificity of the other tests (CCP3\u2009=\u200988%, MCV\u2009=\u200990%, CPA\u2009=\u200994%) is lower as are their positive predictive values [11]. These numbers may improve a little bit when the cut-off values are adjusted to more realistic data; nevertheless, the data allow the statement that, in absolute percentages, none of the tests performs better than the anti-CCP2 test. They also seem to indicate that some tests detect RA patient groups that are negative in the anti-CCP test, illustrating again that the autoantibody repertoire of RA patients is very heterogeneous.\nAnother risk for the specificity of a test that is based on a citrullinated antigen is the possibility that antibodies are not directed exclusively to the citrulline-containing epitope but also to other possibly overlapping epitopes present in the substrate antigen. This is particularly important when citrullinated versions of proteins like vimentin or fibrinogen are used. For example, it is known that antibodies to vimentin are present in several diseases different from RA [12, 13]. This particular problem has been addressed for CCP2 by Vannini et al. [14]. They used ELISA plates containing the control CCP2 antigens (Arg instead of Cit in the same peptide context), produced and made available by Euro-Diagnostica, Arnhem, The Netherlands, in parallel to the normal CCP2 test. The results of these comparative studies showed that in RA and most non-RA rheumatic disease sera, anti-CCP reactivity indeed is citrulline-dependent. However, in some patients, particularly autoimmune hepatitis patients, citrulline-independent reactivity with the antigen may occur. A positive CCP test in a rheumatic disease (almost always citrulline-specific) may thus suggest the future development of RA as has been suggested by several studies [15, 16]. A positive test in a nonrheumatic disease (very often not citrulline-specific), for example, liver disease, should be interpreted with care [14].\nAnti-CCP2 Antibodies are Present Early in Disease and have Predictive Potential\nBecause RA patients at first presentation often do not fulfill the criteria for the diagnosis\/classification of RA, an early, highly predictive marker would greatly assist the clinician in reaching an early diagnosis. There are several studies indicating that the anti-CCP2 test provides this help (reviewed by [2]).\nIn the recently published EULAR recommendations for the management of early arthritis [16], a list of factors has been proposed that predict persistent and erosive disease. These factors include: number of swollen and tender joints, ESR or CRP, level of RF and anti-CCP antibodies, and radiographic erosions. Most of these factors were also mentioned as being important in the prediction of early erosive RA (Visser et al. [17]). Subsequent studies by the same group gave an indication of the relative importance of these factors. When expressed as odds ratios (OR), the data was as follows: arthritis of three or more joints, 5.0; radiographic erosions, 8.7; positive IgM-RF, 1.7; and positive anti-CCP2, 38.6 [18]. These and other data (see also [19]) clearly show that the presence of anti-CCP antibodies is an important and independent prognostic factor for radiographic progression in not only early arthritis but also in early rheumatoid arthritis [16, 20].\nRecently, it has also been shown that IgM-CCP is present in early samples from both patients with undifferentiated arthritis (UA) and patients with RA as well as in follow-up samples from patients with RA. These data indicate the development of the anti-CCP isotype repertoire into full usage early in the course of arthritis and a continuous (re)activation of the RA-specific anti-CCP response during the further development of the disease [21].\nIt is, however, evident that besides the clinical and laboratory parameters mentioned above, some genetic factors are important as well. The effect of the HLA shared-epitope alleles on the development of ACPA has been firmly established. Citrullination is typically a process that occurs in apoptotic cells. Because such dying cells are generally removed from the environment via clearance by phagocytes, a process that is regulated by many genes, the immune system will normally not encounter citrullinated proteins. However, it has been shown that during inflammation, citrullinated proteins are detectable in the inflamed tissue, both in RA and non-RA patients [22]. This is probably caused by inefficient clearance of the massive numbers of dying cells, a process already described to occur in SLE [23]. This inefficient clearing could, in principal, also be the consequence of altered genes, i.e., the genetic background. The mere presence of citrullinated antigens in inflamed synovial tissue does not necessarily result in the occurrence of anti-CCP antibodies in serum or synovial fluid, neither in humans nor in mouse models of arthritis [24, 22]. Hill et al. [25] showed that the generation of anticitrulline antibody in mice actually is linked to the expression of the RA shared epitope (SE), and a similar link was also found in patients because the combination of SE, and anti-CCP has a very high predictive value for the future development of RA [26]. The specific structure of HLA molecules obviously plays an important role in the induction of autoimmunity to citrullinated proteins. These studies were extended by Huizinga et al. [27] who found that HLA-DRB1 alleles encoding the SE were only associated with anti-CCP positive RA and not with anti-CCP negative RA. In contrast, anti-CCP negative RA appeared to be associated with HLA-DR3 [28]. Therefore, the presence of citrullinated antigens, together with the appropriate genetic background (the SE and probably other types of HLA), appears to be the minimum requirement for an immune response to citrullinated polypeptides to be generated [29]. Next to that, other sensitizing genetic settings resulting from polymorphisms (e.g., PTPN22 [30]) might, given their association, aid in triggering of ACPA.\nConclusions and Future Perspectives\nThe present literature describes and confirms that the anti-CCP2 test is a very useful marker for the early and accurate diagnosis of RA. Anti-CCP antibody is very specific for RA and has a sensitivity comparable to that of RF. These antibodies can be detected very early in disease and may be used as an indicator for the progression and prognosis of RA. Initially, the test was available principally as a manual ELISA method through Euro-Diagnostica and Axis-Shield and their partners. More recently, fully automated anti-CCP assays have also been made available from Phadia (UniCap Elia CCP) and Abbott Diagnostics (AxSYM anti-CCP). A very novel format is the CCPoint, a Point-of-Care test for the detection of these antibodies in whole blood in 10\u00a0min [31]. A small drop of whole blood is applied onto the sample well followed by the addition of four drops of running buffer. After 10\u00a0min, the result is read by visual inspection of the detection zone for staining of the antigen line. As no special equipment is required to perform the assay, this test will bring the detection of anti-CCP antibodies into the office of the family doctor, facilitating a very fast referral to the rheumatologist when the test gives a positive answer. Such new applications will undoubtedly further enhance the utility of the anti-CCP autoantibody system in clinical practice.","keyphrases":["ccp2 test","rheumatoid factor","autoantibodies","anti-cyclic citrullinated peptide"],"prmu":["P","P","P","P"]}
{"id":"Histochem_Cell_Biol-3-1-2137947","title":"Nucleolus: the fascinating nuclear body\n","text":"Nucleoli are the prominent contrasted structures of the cell nucleus. In the nucleolus, ribosomal RNAs are synthesized, processed and assembled with ribosomal proteins. RNA polymerase I synthesizes the ribosomal RNAs and this activity is cell cycle regulated. The nucleolus reveals the functional organization of the nucleus in which the compartmentation of the different steps of ribosome biogenesis is observed whereas the nucleolar machineries are in permanent exchange with the nucleoplasm and other nuclear bodies. After mitosis, nucleolar assembly is a time and space regulated process controlled by the cell cycle. In addition, by generating a large volume in the nucleus with apparently no RNA polymerase II activity, the nucleolus creates a domain of retention\/sequestration of molecules normally active outside the nucleolus. Viruses interact with the nucleolus and recruit nucleolar proteins to facilitate virus replication. The nucleolus is also a sensor of stress due to the redistribution of the ribosomal proteins in the nucleoplasm by nucleolus disruption. The nucleolus plays several crucial functions in the nucleus: in addition to its function as ribosome factory of the cells it is a multifunctional nuclear domain, and nucleolar activity is linked with several pathologies. Perspectives on the evolution of this research area are proposed.\nIntroduction\nBrief history of the nucleolus\nAn ovoid body visible in the nucleus was probably the first observation of the nucleolus more than two centuries ago by F. Fontana. Since that time, the nucleolus has been the object of intense investigation and interestingly our vision of the nucleolus has evolved with technical progress. During the nineteenth century, using light microscopy, numerous cytologists described the variability of nucleolar morphology with great precision (Montgomery 1898). In 1934, McClintock proposed that the \u201cnucleolus is organized in the telophase through the activity of ... the nucleolar-organizing body\u201d (McClintock 1934). Since the nucleolar-organizing body corresponds to a specific region of chromosome 6 in Zea mays, this was the first time the nucleolus was related to gene activity. In the 1950\u2019s the presence of RNAs in the nucleolus was demonstrated, and in the 1960\u2019s in situ hybridization techniques made it possible to identify ribosomal genes (rDNAs) in the nucleolar organizer region (NOR) (Caspersson 1950; Perry 1962; Ritossa and Spiegelman 1965). During the same period, mass isolation of nucleoli became possible leading to the biochemical characterization of nucleolar components. Based on these results it was proposed that ribosome biogenesis occurs in nucleoli. Given that the nucleolus became a subject of great interest, the \u201cInternational symposium on the nucleolus\u2014its structure and function\u201d was organized in Montevideo in 1965 and the contributions published in Natl Cancer Inst Monogr no 23 (USA) in 1966. Since 1969, at the initiative of W. Bernhard and H. Busch, \u201cNucleolar Workshops\u201d on nucleolar organization, the biochemistry of nucleolar proteins, rRNA processing as well as variability in cancer cells were regularly organized. Several books on nucleoli were published; among them, the famous \u201cThe nucleolus and ribosome biogenesis\u201d is still a very useful source of information (Hadjiolov 1985). Between 1980 and 2000, the functional organization of the nucleolus was deciphered in large part due to the improvement of labeling by the electron microscopy (EM).\nRecently a new field of investigation was opened when molecules not involved in ribosome biogenesis were detected in the nucleolus (Carmo-Fonseca et al. 2000; Pederson 1998; Politz et al. 2002; Visintin and Amon 2000). In accordance with these nucleolar localizations, nucleolar mass spectrometry analyses identified \u223c700 nucleolar proteins, some of them not related to ribosome biogenesis (Andersen et al. 2005). The area of plurifunctional nucleolus was opened. Consequently \u201cThe nucleolus\u201d, a book that presents the state of the art was published (Olson 2004) as well as several reviews on the multiple functions of the nucleolus (Boisvert et al. 2007; Hernandez-Verdun 2006; Hiscox 2007; Raska et al. 2006).\nGeneral information\n\u201cThe nucleolus: an organelle formed by the act of building a ribosome\u201d (M\u00e9l\u00e8se and Xue 1995) reveals by its size and organization the efficiency of ribosome biogenesis. For example the nucleolus is a prominent nuclear structure in cycling cells but of limited size in the terminal stages of differentiation such as in lymphocytes or chick erythrocytes. If ribosome biogenesis is blocked, reorganization of the nucleolar components is visible in segregated nucleoli. In mammalian cells, the nucleolus is disorganized in prophase and reassembled at the end of mitosis using the nucleolar machineries from the previous cell cycle. On the contrary, in yeast the nucleolus is present and active throughout the cell cycle even though condensation of the rDNAs is necessary for transmission of the nucleolus in anaphase (D\u2019Amours et al. 2004; Sullivan et al. 2004; Torres-Rosell et al. 2004).\nThe nucleolus is the ribosome factory of the cell. In the nucleolus rDNAs are transcribed, the 47S precursor ribosomal RNAs (pre-rRNAs) are cleaved, processed and assembled with the 80 ribosomal proteins and the 5S RNA to form the 40S and 60S ribosomal subunits (selected reviews G\u00e9brane-Youn\u00e8s et al. 2005; Hernandez-Verdun and Jun\u00e9ra 1995; Scheer et al. 1993; Scheer and Hock 1999; Shaw and Jordan 1995; Thiry and Goessens 1996). This complex series of maturation and processing events, presently better characterized in yeast than in higher eukaryotes is under the control of about 150 small nucleolar RNAs (snoRNAs) and 2 large RNP complexes: (1) the small subunit (SSU) processome containing the U3 snoRNAs and 40 proteins or Utps (U three proteins) required for the 40S ribosomal subunit, and (2) the large subunit (LSU) processome required for the 60S ribosomal subunit (de la Cruz et al. 2004; Fatica and Tollervey 2002; Fromont-Racine et al. 2003; Sollner-Webb et al. 1996; Tollervey 1996). The snoRNAs associated with proteins, function in the maturation of rRNAs creating two types of modified nucleotides (2\u2032-O-methylation and pseudouridylation) and mediating endonucleolytic cleavages of pre-rRNAs (Gerbi and Borovjagin 2004).\nOur objective is to focus this review on the ribosome biogenesis processes occurring in the nucleoli that might help to decipher the global organization of nuclear functions. We describe nucleolar organization and dynamics, propose our view on nucleolar targeting, report the relationship between the nucleolus and the cell cycle, review particular relationships between nucleolus and virus, and nucleolus related to cancer.\nThe nucleolus is a model of compartmentation\nThree main components in the active nucleolus\nThe nucleolus has been proposed as the paradigm of nuclear functional compartmentalization (Strouboulis and Wolffe 1996). It is the site of ribosome biogenesis and in addition the nucleolar machineries are distributed in different compartments. When observed by EM, three main nucleolar components (compartments) can be discerned in mammalian cells: the fibrillar centers (FCs), the dense fibrillar component (DFC) and the granular component (GC) (Fig.\u00a01a). The FCs are clear areas, partly or entirely surrounded by a highly contrasted region (Goessens et al. 1987), the DFC. The FCs and the DFC are embedded in the GC, mainly composed of granules of 15\u201320\u00a0nm in diameter. The most contrasted structures in the EM sections stained with uranyl and lead correspond to high concentrations of nucleic acids. The condensed chromatin surrounding part of the nucleolus is visible using standard or preferential staining methods and also as a network within the nucleolus (Fig.\u00a01b). The global amount of intra-nucleolar chromatin is probably low since by light microscopy, DNA staining by DAPI excludes the nucleolus.\nFig.\u00a01The nucleolus of mammalian cells as seen by electron microscopy. a In the human HeLa cell, the three main nucleolar components are visible in a section of material fixed in glutaraldehyde and osmium tetroxyde, embedded in Epon and the section contrasted with uranyl acetate and lead citrate. FCs of different sizes are visible and the largest is indicated by an asterisk. The FCs are surrounded by the DFC and are embedded in the GC. b Preferential contrast of DNA using NAMA-Ur staining in a PtK1 cell (courtesy J. G\u00e9brane-Youn\u00e8s). The nucleolus is the gray structure surrounded by highly contrasted chromatin (arrow). Some chromatin filaments are also visible inside of the nucleolus (Nu). c, d Nucleolus of rat neurones (courtesy M. J. P\u00e9busque) in the day (c), and during the night (d) which is the active period for the nucleolus of the rat. In the nonactive period (c), the nucleolus is reticulated with small FCs (asterisk). In the active period, one giant FC is visible (d, asterisk). Bar in a: 0.5\u00a0\u03bcm and bars in b, c and d: 1\u00a0\u03bcm\nIt has become apparent that nucleoli of different cell types exhibit a variable number of FCs of different sizes, with an inverse proportion between size and number (Hozak et al. 1989; P\u00e9busque and Se\u00efte 1981). Generally cells with a high rate of ribosome biogenesis possess numerous small FCs. On the contrary, cells with greatly reduced metabolic and transcription activities, present small nucleoli with one large-sized FC such as in lymphocytes and in inactive mammalian neurons (Hoz\u00e0k et al. 1994; Lafarga et al. 1989). In the more active neurons, one giant FC (GFC) of 1\u20132\u00a0\u03bcm is observed together with small FCs (Fig.\u00a01c, d). It was demonstrated that the GFC is enriched in the upstream binding factor, the UBF transcription factor, in a small ubiquitin-like modifier (SUMO)-1 and Ubc9 but lack ubiquitin-proteasome and 20S proteasome (Casafont et al. 2007). However, the possibility that only one FC might play a role in storage and become a GFC during intense nucleolar activity is still an open question.\nIt is also remarkable that the tripartite nucleolar organization is not general since the nucleoli of Drosophila and insects lack FCs (Knibiehler et al. 1982; Knibiehler et al. 1984). It has been proposed that this difference in organization could be linked to the evolution of the rDNAs, in particular to the size of the intergenic sequences (Thiry and Lafontaine 2005).\nThe localization of the nucleolar machineries is related to their function in the production of the small and large ribosome subunits. These findings have led to assigning specific functions to specific compartments of the nucleolus. Nascent transcripts appear at the junction between the FCs and DFC and accumulate in the DFC (Cmarko et al. 2000; Guillot et al. 2005; Hoz\u00e0k et al. 1994; Puvion-Dutilleul et al. 1997; Shaw and Jordan 1995). This was recently confirmed in the GFC since no transcripts can be detected in these large structures (Casafont et al. 2007). Processing of the 47S pre-rRNA starts at the site of transcription in the DFC (Cmarko et al. 2000) and continues during the intra-nucleolar migration of the RNA towards the GC. The nucleolar proteins that participate in the early stages of rRNA processing, localize in the DFC, such as fibrillarin and nucleolin along with the U3 snoRNAs (Biggiogera et al. 1989; Ginisty et al. 1998; Ochs et al. 1985b; Puvion-Dutilleul et al. 1991), whereas proteins B23\/NPM (nucleophosmin) and PM-Scl 100 (rrp6 in yeast) that are involved in intermediate or later stages of processing have been localized to the GC (Biggiogera et al. 1989; Gautier et al. 1994). Recent advances in the isolation of large RNP complexes by tandem affinity purification and the characterization of their constituents demonstrated that two largely independent processing machineries exist in yeast nucleoli, the SSU processome (Dragon et al. 2002; Grandi et al. 2002) and the LSU processing\/assembly factors (Rau\u00e9 2004). The SSU\/90S processome is localized in the DFC and most of the 60S processing occurs in the GC. There is no particular domain characterized in the GC corresponding to the 43S subunit. This is most probably due to the limited events of 40S processing in the GC since the last step of processing occurs in the cytoplasm. In conclusion it seems that in the nucleoli, the vectorial distribution of the machineries successively involved in ribosome biogenesis correlates with the different processing steps of the biogenesis of the ribosome subunits.\nWhen ribosome biogenesis is active, the confinement of certain machineries in the FCs, DFC or GC makes it possible to reveal these subnucleolar constituents by immunofluorescence as illustrated for FCs (Fig.\u00a02A), DFC (Fig.\u00a02Ba, b), and GC (Fig.\u00a02Bc, d). The factors associated with the rDNA transcription machinery are distributed in several foci, most frequently inside the nucleolar volume as illustrated for UBF. These foci correspond to FCs. A distribution within the network inside the nucleolus is typical of the DFC as demonstrated for fibrillarin. Labeling of the nucleolar volume excluding small areas contained within the volume is typical of the GC as illustrated for B23\/NPM. These labeling patterns (FCs, DFC, GC) in the nucleoli provide a good indication of the step of ribosome biogenesis concerned and also reveal the blockage of ribosome biogenesis when this organization is disturbed (see below).\nFig.\u00a02The subnucleolar constituents revealed by fluorescence microscopy. (A) The rDNA transcription machinery, illustrated by UBF labeling, is localized in several foci corresponding to FCs in HeLa cells (a). rDNA transcription sites detected by in situ BrUTP incorporation (b), mainly colocalize with UBF as seen by the merge (c). The nucleus is visualized by Dapi staining (d). (B) HeLa cells expressing either fibrillarin-GFP fusion or DsRed-B23 fusion. Fibrillarin decorates the DFC (a, b) whereas B23 decorates the GC (c, d). In ActD-treated cells nucleoli are segregated, fibrillarin localizes in caps (e, f) contrary to B23 that localizes in the central body and outside the caps (g, h). Arrowheads point the caps. Bars: 10\u00a0\u03bcm\nSignature of impaired ribosome biogenesis\nTransient association of functionally related components appears necessary to generate a morphologically defined nucleolus with its three distinct components, thereby maintaining the nucleolus in its usual organization. This suggests that such an organization results from the activity of ribosome biogenesis. Indeed nucleolar reorganization is induced when ribosome biogenesis is impaired either by inhibiting rDNA transcription, or inhibiting rRNA processing and\/or transport.\nInhibition of transcription\nNucleolar segregation is observed upon rDNA transcriptional arrest either in physiological conditions or induced by low doses of actinomycin D (ActD). The segregation of nucleoli is characterized by the separation of the nucleolar components that remain close to each other but no longer intermingle (Fig.\u00a02Be\u2013h) (for reviews see Hadjiolov 1985; Hernandez-Verdun and Jun\u00e9ra 1995; Scheer and Benavente 1990). The effect of ActD on nucleolar organization follows sequential changes: first the fibrillar components (FCs and DFC) condense and migrate towards the periphery of the nucleolus, after which the nucleolar components segregate to finally form a central body associated with caps (Hadjiolova et al. 1995). In the caps are several proteins related to the RNA polymerase (pol) I transcription machinery such as UBF, close to fibrillarin-containing caps. In the central body are proteins derived from the GC, some of which are progressively released, such as PM-Scl 100. It was recently demonstrated that certain nucleolar caps of segregated nucleoli could recruit factors involved in mRNA splicing. In this case, localization is induced by inhibition of both RNA pol I (rRNA transcription arrest) and RNA pol II (mRNA transcription arrest) (Shav-Tal et al. 2005). This is not observed when only RNA pol I is inhibited indicating that the composition of a segregated nucleolus can be more complex when induced by general transcription inhibition.\nOne question that remains unanswered is how nucleolar components continue to be maintained in segregated nucleoli in spite of the absence of transcription or pre-rRNA processing. Nucleolar proteins may still be capable of forming complexes during inhibition of transcription, but why these complexes remain juxtaposed is presently unknown. Recently, it was reported that re-localization of proteins in specific caps of segregated nucleoli (after inhibition of RNA pol I and II transcription) is an energy-dependent repositioning process that requires active metabolism of the cells (Shav-Tal et al. 2005) most probably also ATP and GTP.\nDegradation of rRNAs\nA clue to the question of rRNA degradation in the nucleolus was recently proposed in yeast: a surveillance pathway that eliminates defective 60S pre-ribosomal subunits after addition of poly(A) tails was described (LaCava et al. 2005). RNA degradation appears to occur preferentially within a subnucleolar structure, the No-body, and is mediated by the exosome (Dez et al. 2006). Similarly, when the nuclear protein of the exosome rrp6 was deleted, poly(A) rRNAs and poly(A) U14 snoRNAs colocalized in one focus with Nop1 (fibrillarin in human), most probably the No-body (Carneiro et al. 2007). This body is distinct from the nucleolar body that functions in snoRNA maturation in yeast and could be a compartment where polyadenylation and degradation of nucleolar RNAs take place. This compartmentation would promote efficient recognition of rRNAs in view of degradation by the exosome (Carneiro et al. 2007). During nucleolar segregation induced by ActD in human cells, rRNAs are degraded. However, the formation of one focus containing PM-Scl 100 has not been described; it could be either an early event that was not carefully examined, or rRNA degradation could be different in yeast and mammalian cells.\nDisconnection of the nucleolar component\nDisconnection between rDNA transcription sites and the late rRNA processing proteins can be induced either by kinase inhibitors or by modifications of snoRNA domains (Chan et al. 1996; Colau et al. 2004; David-Pfeuty et al. 2001; Rubbi and Milner 2003; Sirri et al. 2002). The separation of the DFC and GC can be reversed by removal of a CK2 inhibitor, restoring nucleolar organization. The CK2 kinase is known to phosphorylate several nucleolar proteins (Meggio and Pinna 2003). We postulate that the connection between DFC and GC is controlled at least in part by phosphorylation of these proteins. This hypothesis was verified for B23 by mutation of the major site of CK2 phosphorylation (Louvet et al. 2006). In conclusion, the rRNA processing proteins can be disconnected from the rRNA transcription sites indicating that rRNA transcripts are not sufficient to attract the processing proteins in these conditions. The dynamics of nucleolar reformation and the connection between DFC and GC is ATP\/GTP dependent, sensitive to temperature, and is CK2-driven.\nTraffic and dynamics of nucleolar actors\nThe analysis in living cells of intranuclear dynamics has recently become possible using fluorescent fusion proteins. Time-lapse videomicroscopy can track the movement of large fluorescent complexes in the cell, and fluorescent recovery after photobleaching (FRAP) can measure the intracellular mobility or the residency time of fluorescent proteins (Lippincott-Schwartz et al. 2001). Inverse FRAP (iFRAP) quantifies the loss of fluorescence of the region of interest (ROI) after complete bleaching outside this region (Dundr et al. 2004). This constitutes a direct evaluation of the residency time of the proteins in the ROI. Another approach is the use of photoactivatable GFP (PA-GFP) to follow the traffic of the activated proteins (Patterson and Lippincott-Schwartz 2002). This process is similar to pulse-chase experiments since it makes it possible to follow a pool of labeled proteins starting at time zero. These technologies applied to nuclear dynamics have introduced new dimensions and unexpected concepts concerning nuclear functional compartmentation. The mobility of several GFP-tagged nuclear proteins (nucleolar proteins, histones, DNA binding proteins, transcription factors, splicing factors, nuclear receptors) has been estimated by FRAP and the recovery of fluorescence was slower than predicted for isolated diffusing proteins of similar size. FRAP recovery rates change with inhibition of transcription, decreased temperature and depletion of ATP indicating that recovery is correlated with nuclear activity.\nIt was demonstrated that nucleolar proteins rapidly associate with and dissociate from nucleolar components in continuous exchanges with the nucleoplasm (Phair and Misteli 2000). The recovery curve of GFP-fibrillarin (DFC marker) in the nucleolus reached a plateau, 60\u00a0s after bleaching and the plateau indicated an immobile fraction of \u223c15%. The diffusion coefficient of fibrillarin (estimated between 0.02 and 0.046\u00a0\u03bcm2\u00a0s\u22121) was 10\u00a0times lower in the nucleolus than in the nucleoplasm (Chen and Huang 2001; Phair and Misteli 2000; Snaar et al. 2000). This value is proposed to reflect the time of residency of fibrillarin engaged in nucleolar activity, and could explain the fact that the time of residency of fibrillarin is shorter in the Cajal body than in the nucleolus (Dundr et al. 2004). The nucleolar proteins engaged in rRNA transcription and processing (respectively UBF, B23, Nop52, nucleolin and Rpp29) also move with rapid recovery rates in the nucleolus as does fibrillarin (Chen and Huang 2001; Louvet et al. 2005). Conversely the recovery rates of ribosomal proteins are slow (\u223c3\u00a0times slower than nucleolar proteins). This could reflect a slower mechanism for ribosome protein assembly compared with transcription and processing (Chen and Huang 2001), or alternatively, more stable associations of ribosomal proteins with the pre-rRNAs.\nB23 (also designated NPM) is a multifunctional protein, abundant in the GC of the nucleolus that undergoes different phosphorylation events during the cell cycle. It was recently demonstrated by FRAP that the kinetics of B23 depends on its phosphorylation status (Negi and Olson 2006). During interphase, the half-time (t1\/2) recovery of B23 is 22\u00a0s in nucleoli but when the CK2 phosphorylation site is mutated (S125A) the t1\/2 increases to 44\u00a0s, and when a mutant mimicking phosphorylation charges of the four sites of mitotic CDK1 phosphorylation, the t1\/2 decreases to 12\u00a0s. This could indicate that the S125A-B23 protein has a higher affinity for the nucleolar components (Negi and Olson 2006). Alternatively this could correspond to a decreased turner-over in the nucleolar complexes in correlation with the disconnection of the DFC and GC occurring by overexpression of S125A-B23 (Louvet et al. 2006). Overexpression during interphase of B23 mimicking four sites of mitotic phosphorylation increased the mobility of B23. It is tempting to propose that this results from a defect of affinity for rRNAs of these B23s as demonstrated for mitotic phosphorylation of B23 (Okuwaki et al. 2002).\nInhibition of pol I transcription by ActD does not prevent traffic of nucleolar proteins. However, if the diffusion coefficient of nucleolar proteins in the nucleoplasm is similar for active and repressed pol I transcription, the traffic in segregated nucleoli changes differently for different nucleolar components. Traffic of UBF in the nucleolus is decreased by ActD, whereas it is similar for nucleolin or increased for ribosomal proteins (Chen and Huang 2001).\nIn contrast to the well-defined nucleolar structures visible by EM, all the nucleolar proteins involved in ribosome biogenesis that have been examined, cycle between the nucleolus and the nucleoplasm in interphase cells. To summarize, it is now established that rapid diffusion of nucleolar proteins occurs in the nucleoplasm and recruitment to the nucleolus is permanent. Moreover, the difference in kinetics of several proteins shared between the nucleolus and the Cajal body suggests the existence of compartment-specific retention (Dundr et al. 2004).\nTargeting to the nucleolus\nTo be localized or retained within the nucleolus\nIn eukaryotic cells, once imported or diffused into the nucleus, some proteins distribute throughout the nucleoplasm and others are targeted to specific nuclear compartments such as nucleoli. Proteomic analyses revealed that at least 700 proteins are localized in nucleoli (Andersen et al. 2002, 2005; Leung et al. 2003). Whereas the rules and signals that govern the nuclear localization and nuclear export of proteins are now well defined, those concerning nucleolar localization are still debated.\nContrary to the nuclear localization signals (NLSs), nucleolar localization signals or sequences (NoLSs) are not well characterized. Although several NoLSs have been described, no obvious consensus sequence has emerged. Nevertheless all NoLSs reported for nucleolus localizing virus proteins, such as HIV-1 Rev (Kubota et al. 1989), HIV-1 Tat (Dang and Lee 1989) and human T-cell leukaemia virus type 1 Rex (HTLV-1 Rex) (Siomi et al. 1988), and for cellular proteins such as the nucleolar protein p120 (Valdez et al. 1994), Survivin-deltaEx3 (Song and Wu 2005) and HSP70 (Dang and Lee 1989) are rich in basic residues. The capacity of numerous proteins to adopt nucleolar localization has been correlated with interaction of these proteins with B23. Owing to the ability of numerous nucleolar proteins to interact with B23 and because this major nucleolar protein shuttles constantly between the nucleus and the cytoplasm (Borer et al. 1989), it was frequently suggested that B23 might be a transporter for nucleolar proteins possessing a NoLS (Fankhauser et al. 1991; Li 1997; Valdez et al. 1994). Even if this tempting hypothesis has never been demonstrated, recent results obtained using stable U2OS-derived cell lines with reduced B23 expression levels showed that the nucleolar localization of ARF is linked to B23 (Korgaonkar et al. 2005). Indeed, reduced expression of B23 induced a partial delocalization of ARF from nucleoli to nucleoplasm. The authors therefore concluded that B23 targets ARF to nucleoli in a dose-dependent manner. Nevertheless, this result does not allow discriminating between a role for B23 in the transport of ARF from nucleoplasm to nucleoli and\/or in the retention of ARF in nucleoli.\nA NoLS, i.e. a sequence essential for nucleolar localization, is most probably a sequence involved in nucleolar retention by interacting with a nucleolar molecule such as B23 (Lechertier et al. 2007). Indeed, recent analyses of the intranuclear dynamics of proteins in living cells revealed that nuclear proteins could diffuse within the nucleoplasm (Phair and Misteli 2000; Sprague and McNally 2005). As for the nucleolus, it was demonstrated that nucleolar proteins rapidly associate with and dissociate from nucleolar components in a continuous exchange with the nucleoplasm (Chen and Huang 2001; Dundr et al. 2004; Phair and Misteli 2000; Snaar et al. 2000). There probably exist compartment-specific retention mechanisms for proteins in nuclear bodies, implying that the residency time of a particular molecule in a given nuclear body depends on its specific interactions (Misteli 2001). In support of this possibility, we have recently shown that the fusion of a B23-interacting sequence with fibrillarin makes it possible to re-localize fibrillarin from the DFC to the GC of nucleoli where B23 is mainly localized (Lechertier et al. 2007). Similarly, by fusing the B23-interacting sequence to MafG (part of the nuclear transcription factor NF-E2 composed of both MafG and p45), NF-E2 is redirected from the nucleoplasm to the GC. Therefore, interactions most probably govern the nuclear distribution of proteins and a NoLS is very likely a nucleolar molecule-interacting sequence.\nHowever, nucleolar localization of a protein is most probably governed by several factors and the presence of a NoLS in its sequence is not sufficient to predict nucleolar localization of the protein. In particular, a nucleolar protein must first be localized in the nucleus, and consequently all mechanisms that interfere with nuclear import and\/or nuclear export of a nucleolar protein will modify its localization at the steady state. A good illustration is provided by the major nucleolar protein, B23. This multifunctional protein is normally mainly located in the GC of nucleoli but exhibits an aberrant cytoplasmic localization in one-third of acute myeloid leukemias due to mutations in its C-terminal coding exon that causes a frameshift and the formation of an additional CRM1-dependent nuclear export signal (NES) (Mariano et al. 2006). Another example showing the difficulty encountered in predicting nucleolar localization is provided by the box C\/D snoRNPs: it seems clear that the nucleolar localization of box C\/D snoRNPs is linked to their biogenesis (Verheggen et al. 2001; Watkins et al. 2002). Indeed, by modifying the conserved stem II of the box C\/D motif present in the U14 snoRNA, both the specific assembly of the box C\/D snoRNP and nucleolar localization are lost (Watkins et al. 2002). Moreover, genetic depletion of one of the four core proteins, namely 15.5kD, Nop56, Nop58 and fibrillarin, also inhibits the nucleolar localization of box C\/D snoRNPs (Verheggen et al. 2001). However, targeting of box C\/D snoRNPs to nucleoli is not yet fully understood. Indeed, unexpectedly two nuclear export factors, PHAX and CRM1 appear to be stably associated with the U3 pre-snoRNPs (Boulon et al. 2004; Watkins et al. 2004). Boulon and coworkers proposed that U3 precursors bind PHAX, which targets the complex to the Cajal body, and that subsequently CRM1 further targets the U3 complexes to the nucleolus. Even if PHAX and CRM1 play an important role in the transport of box C\/D snoRNPs to the nucleus, the possibility that these proteins may also function in the nuclear export of snoRNPs cannot be excluded (Watkins et al. 2004). This possibility is reinforced by a recent study showing that in addition to nuclear export factors, the nuclear import factor Snurportin 1 is involved in U8 box C\/D snoRNP biogenesis (Watkins et al. 2007). Nucleolar localization of the components of the box C\/D snoRNPs would therefore depend on the biogenesis of the box C\/D snoRNP complexes, which would imply nuclear export.\nControl of rDNA transcription during cell cycle\nrDNA transcription machinery\nrDNAs are found in multiple, tandem, head-to-tail arrayed copies in the nucleoli of eukaryotic cells (Hadjiolov 1985). In mitotic human cells, rDNA clusters are localized on the short arm of the five pairs of chromosomes 13, 14, 15, 21 and 22 and are termed NORs. Each rDNA unit consists of a transcribed sequence and an external non-transcribed spacer (Hadjiolov 1985; Liau and Perry 1969) in which all the sequences necessary for proper RNA pol I transcription such as proximal promoters, spacer promoters and terminators are located (Hadjiolov 1985). In the rDNA promoter two important elements have been described, a CORE element and an upstream control element (UCE) (Haltiner et al. 1986; Windle and Sollner-Webb 1986; Xie et al. 1992) that function synergistically to recruit a transcriptionally competent RNA pol I complex. This complex contains in addition to RNA pol I, the upstream binding factor (UBF) (Pikaard et al. 1989; Voit et al. 1992), the selectivity factor protein complex SL1 (Learned et al. 1985) also called TIF-1B in mouse cells (Clos et al. 1986), consisting of the TATA-binding protein (TBP) and four transcription activating factors [TAFIs110, 63, 48 and 41 (Comai et al. 1994; Gorski et al. 2007; Zomerdijk et al. 1994)], the transcription initiation factor TIF-IA, the mouse homolog of Rrn3p (Bodem et al. 2000; Moorefield et al. 2000) and the transcription termination factor TTF-1 (Bartsch et al. 1988). The UBF containing HMG boxes (Bachvarov and Moss 1991; Jantzen et al. 1990) that confer a high affinity for DNA structures plays an architectural role on the rDNA promoter (Mais et al. 2005). It was proposed that UBF activates rDNA transcription because it stabilizes binding of SL1 required to recruit the initiation-competent subfraction of RNA pol I. This recruitment is achieved by interaction of UBF with the RNA pol I-associated factor PAF53 (Schnapp et al. 1994), and by interaction of SL1 with TIF-1A\/Rrn3p (Miller et al. 2001). TIF-1A\/Rrn3p interacts also with the RPA43 subunit of RNA pol I and thus facilitates linking between RNA pol I and SL1 complexes (Peyroche et al. 2000; Yuan et al. 2002). Following initiation, TIF-1A\/Rrn3p is released and can associate with another preinitiation complex. Recycling of TIF-1A\/Rrn3p requires a post-translational phosphorylation event that appears to play a role in its initiation activity (Cavanaugh et al. 2002; Zhao et al. 2003). Moreover, it was proposed that TTF-1 is not only involved in termination of transcription in cooperation with the release factor PTRF (Jansa and Grummt 1999), but also in the remodeling of ribosomal chromatin by recruiting ATP-dependent remodeling factors to the rDNA promoter (L\u00e4ngst et al. 1997). The nucleolar remodeling complex (NoRC) (Strohner et al. 2001), which acts in repression at the rDNA promoter level (Li et al. 2006; Santoro et al. 2002), and the transcription activator CSB (Cockayne syndrome group B protein), a DNA-dependent ATPase, interact with TTF-1 (Yuan et al. 2007). The finding that TTF-1 interacts with both CSB and NoRC suggests that competitive recruitment of CSB and NoRC may determine the epigenetic state of the rDNA.\nRegulation during the cell cycle\nIt is now established that the presence of a fully active nucleolus depends on cell cycle regulators. rDNA transcription is maximum in the S and G2 phases, silent in mitosis, and slowly recovers in G1. Post-translational modifications of the RNA pol I machinery are required for the formation of a productive preinitiation complex. The phosphorylation status of several components of the RNA pol I machinery can modify the activity and interactions of these proteins and thus can modulate rDNA transcription during the cell cycle. Concerning silencing of rDNA transcription during mitosis, it is well established that some components of the rDNA transcription machinery such as SL1 (Heix et al. 1998) and TTF-1 (Sirri et al. 1999), are mitotically phosphorylated by CDK1-cyclin B. As shown in vitro, CDK1-cyclin B-mediated phosphorylation of SL1 abrogates its transcriptional activity (Heix et al. 1998). Moreover CDK1-cyclin B is necessary not only to establish repression but also to maintain it from prophase to telophase. Indeed, in vivo inhibition of CDK1-cyclin B leads to dephosphorylation of the mitotically phosphorylated forms of components of the RNA pol I machinery and restores rDNA transcription in mitotic cells (Sirri et al. 2000). On the other hand, rDNA transcription also appears regulated by CDK(s) during interphase: the increase of rDNA transcription during G1 progression depends on phosphorylation of UBF by G1-specific CDK\u2013cyclin complexes (Voit et al. 1999), and CDK inhibitor treatments partially inhibit rDNA transcription in interphase cells (Sirri et al. 2000). Modifications of the phosphorylation status of UBF and\/or TAFI110 affect the interactions between UBF and SL1 necessary for recruitment of RNA pol I (Zhai and Comai 1999). In addition to phosphorylation, it has been speculated that acetyltransferases might also regulate the activity of RNA pol I transcription factors. Indeed, two studies have demonstrated that UBF and one of the SL1 subunits are acetylated in vivo (Muth et al. 2001; Pelletier et al. 2000). Functional studies indicated that acetylated UBF is transcriptionally more active than deacetylated UBF. However, acetylation of UBF does not affect its DNA binding activity as shown for other transcription factors, and it is unclear how this post-transcriptional modification modulates UBF activity. The TAFI68 subunit of SL1 is specifically acetylated by recruitment of PCAF (p300\/CBP associated factor) to the rDNA promoter. In vitro analyses indicate that acetylation of TAFI68 is likely to increase the activity of SL1 facilitating interaction of the complex with DNA. Sirtuins, the human homologues of the yeast Sir2 (silent information regulator) with NAD-dependent deacetylase and ADP-ribosyltranferase activity, have recently been implicated in the regulation of the RNA pol I machinery. In particular, nuclear sirtuin1 deacetylates TAFI68 and represses RNA pol I transcription in vitro (Muth et al. 2001). Conversely, the nucleolar sirtuin7 is described as activator of rDNA transcription by increasing RNA pol I recruitment to the rDNA, but no substrates of such activity have as yet been identified (Ford et al. 2006). Additional in vivo approaches are necessary to better understand the role of sirtuins in the regulation of rDNA transcription.\nSUMO modification is reported to influence the assembly of transcription factors on promoters and the recruitment of chromatin-modifying enzymes, and is often associated with transcriptional repression (Gill 2004). Recently, the colocalization of SUMO-1 and UBF in the GFC (Casafont et al. 2007) of neuronal cells and the nucleolar localization of the sentrin\/SUMO-specific proteases, SENP3 and SENP5 (Gong and Yeh 2006; Nishida et al. 2000) suggest a potential role of sumoylation on the regulation of rDNA transcription. Further studies of the identification of sumoylated nucleolar transcription factors will be necessary to verify this possibility.\nNucleolar assembly and disassembly\nIn higher eukaryotic cells at the beginning of mitosis when rDNA transcription is repressed, the nucleoli disassemble and are no longer observed throughout mitosis. Conversely nucleoli assemble at the exit from mitosis concomitantly with restoration of rDNA transcription and are functionally active throughout interphase.\nDisassembly in prophase\nIn late prophase when mitotic repression of rDNA transcription occurs, the rDNA transcription machinery remains associated with rDNAs in the NORs as revealed by the analysis of different components at the steady state (Roussel et al. 1993, 1996; Sirri et al. 1999). Nevertheless, more recent quantitative kinetic analyses have revealed that some RNA pol I subunits, including RPA39, RPA16 and RPA194, transiently dissociate from the NORs during metaphase and reappear in anaphase (Chen et al. 2005; Leung et al. 2004). As for the mechanism that governs disassembly of nucleoli in prophase, it may be assumed that it is linked to repression of rDNA transcription, most probably caused by CDK1-cyclin B-directed phosphorylation of components of the rDNA transcription machinery (Heix et al. 1998; Sirri et al. 1999).\nAt the beginning of prophase, the components of the pre-rRNA processing machinery do not remain in the vicinity of the rDNAs (Gautier et al. 1992) but become partially distributed over the surface of all the chromosomes (reviewed in Hernandez-Verdun et al. 1993). The nucleolar proteins that relocate to the chromosome periphery are components of the DFC and GC of the active nucleolus. In living cells, nucleolar proteins tagged with GFP are concentrated around the chromosomes during mitosis and migrate with the chromosomes (Savino et al. 2001). However, the mechanisms maintaining interactions of nucleolar processing proteins with chromosomes during mitosis have not been characterized. The colocalization of the different factors involved in pre-rRNA processing suggests that processing complexes are at least to some extent maintained during mitosis. It is as yet unknown whether migration of the nucleolar processing proteins occurring at the onset of mitosis (Fan and Penman 1971) takes place as a consequence of the arrest of pre-rRNA synthesis or whether it is also regulated. Indeed, it is noticeable that (1) during prophase, the components of the rRNA processing machinery appear to be delocalized before total repression of rDNA transcription occurs, and (2) the most recently synthesized pre-rRNAs accumulate as partially processed 45S pre-rRNAs (Dousset et al. 2000) suggesting that total repression of pre-rRNA processing could occur prior to total repression of rDNA transcription. These observations therefore raise the possibility that rDNA transcription and pre-rRNA processing are both repressed during prophase by distinct mechanisms.\nAssembly in telophase\nNucleoli assemble at the exit from mitosis concomitantly with restoration of rDNA transcription at the level of competent NORs (Roussel et al. 1996). Until recently it was admitted that transcriptionally active rDNAs, serving as nucleation sites, possessed by themselves the ability to organize the nucleoli (Scheer and Weisenberger 1994). Results obtained in the laboratory showed that (1) reactivation of rDNA transcription in mitotic cells does not lead to the formation of nucleoli (Sirri et al. 2000), (2) initiation of nucleolar assembly occurs independently of rDNA transcription (Dousset et al. 2000), and (3) at the exit from mitosis nucleologenesis is impaired in the presence of a CDK inhibitor even if rDNAs are actively transcribed (Sirri et al. 2002). Consequently, the formation of functional nucleoli at the exit from mitosis is not governed solely by the resumption of rDNA transcription. Based on previous studies (Sirri et al. 2000, 2002), we propose that the formation of nucleoli is a process regulated by CDK(s) at two levels: resumption of rDNA transcription but also restoration of rRNA processing.\nIn anaphase, early and late processing proteins (respectively fibrillarin, and Bop1, B23, Nop52) are homogeneously distributed around the chromosomes. During telophase and early G1, along the translocation pathway between chromosome periphery and transcription sites, processing proteins concentrate in foci designated prenucleolar bodies (PNBs), first described in plant cells (Stevens 1965). PNB formation is a general phenomenon occurring during the recruitment of the nucleolar processing proteins at exit from mitosis (Angelier et al. 2005; Azum-G\u00e9lade et al. 1994; Dundr et al. 2000; Jim\u00e9nez-Garcia et al. 1994; Ochs et al. 1985a; Savino et al. 2001). This appears to be a cell cycle regulated process since when the nucleolar function is established during interphase, recruitment of processing proteins is not associated with PNB formation. Inactivation of CDK1-cyclin B occurring at the end of mitosis induces the first events of nucleologenesis. Strikingly, fibrillarin concentrates in PNBs and rDNA clusters when decrease in CDK1-cyclin B activity overcomes the mitotic repression of RNA pol I transcription (Clute and Pines 1999), while Nop52 and other GC proteins are recruited later on transcription sites. This late recruitment is under the control of cyclin-dependent kinases since CDK inhibitors block this process (Sirri et al. 2002). Thus, it seems that recruitment of the processing machinery at the time of nucleolar assembly is a regulated process most probably dependent on cell cycle progression. This provides a physiological situation to investigate the formation, control and dynamics of nuclear bodies.\nThe dynamics of the processing nucleolar proteins was analyzed at the transition mitosis\/interphase using rapid time-lapse video microscopy (Fig.\u00a03). The first detectable assembly of proteins in foci occurred on the surface of the chromosome during telophase (Savino et al. 2001), followed by the progressive delivery of proteins to nucleoli ensured by progressive and sequential release of proteins from PNBs (Dundr et al. 2000). Based on the observations of different fixed cells, it was concluded that the early processing proteins are recruited first on transcription sites while the majority of the late processing proteins are still in PNBs (Fomproix et al. 1998; Savino et al. 1999). This sequence of events was confirmed in living HeLa cells. Fibrillarin resides briefly in PNBs (\u223c15\u00a0min) before recruitment to the nucleolus, while Nop52 is maintained longer in PNBs (\u223c80\u00a0min) (Savino et al. 2001). The relative dynamics of early and late rRNA processing proteins at the time of PNB formation was examined using co-expression of GFP-fibrillarin and DsRed-B23 (Angelier et al. 2005). Once near the poles, 1\u20132\u00a0min after the onset of telophase, numerous bright fluorescent foci containing both GFP-fibrillarin and DsRed-B23 appeared almost simultaneously. For about 10\u00a0min, the relative amount of B23 in foci was five to six times higher than that of the dispersed proteins whereas the amount of fibrillarin in the same foci was three to four times higher than that of dispersed proteins. Subsequently, fibrillarin was released while B23 was still present in the foci. This clearly illustrates the presence of the two types of nucleolar processing proteins in the same PNBs and suggests differential sorting of these proteins. Conversely in the same conditions, similar dynamics and flows of GFP-Nop52 and DsRed-B23 were observed. Thus the processing proteins passed through the same PNBs and were released simultaneously suggesting that these proteins could form complexes in PNBs.\nFig.\u00a03At the exit from mitosis, the dynamics of DsRed-B23 is followed in living cell. In telophase (0\u00a0min), the B23 signal is visible in small foci. These foci corresponding to PNBs are clearly visible 20\u00a0min later. The B23-containing PNBs are distributed in the nucleoplasm and B23 is progressively recruited in the incipient nucleolus (40\u00a0min). Nu nucleolus\nTime-lapse analysis of fluorescence resonance energy transfer (FRET) was chosen to determine whether nucleolar processing proteins interact along the recruitment pathway. The apparatus used to determine FRET performs tdFLIM (time domain fluorescence lifetime imaging microscopy) by the time and space-correlated single-photon counting method (Emiliani et al. 2003). This technique directly yields the picosecond time-resolved fluorescence decay for every pixel by counting and sampling single emitted photons. Positive FRET between GFP-Nop52 and DsRed-B23 in nucleoli indicates that the distance and most probably the interactions between the proteins can be evaluated by this approach (Angelier et al. 2005). Since it is possible to detect FRET between B23 and Nop52 in nucleoli, FRET was tracked during the recruitment of these proteins into nucleoli from anaphase to early G1. FRET was never detected during anaphase at the periphery of the chromosomes whereas it was registered in 20% of the PNBs at the beginning of telophase, in about 40% at the end of telophase, and in 55% in early G1. Thus, interaction between GFP-Nop52 and DsRed-B23 was established progressively in PNBs, as the number of PNBs exhibiting FRET increased. Such data indicate that Nop52 and B23 did not interact until they were recruited in PNBs. It is noteworthy that a given PNB can alternatively present or not present FRET. Based on the behavior of these two proteins, one possibility is that late rRNA processing proteins already interact in PNBs. Were this to be confirmed for other rRNA processing complexes, PNBs could be proposed as assembly platforms of processing complexes at this step of the cell cycle. It would be very interesting to establish whether this role can be extended to the early rRNA processing machinery (Angelier et al. 2005).\nIn conclusion, assembly of the nucleolus requires reactivation of the rDNA transcription machinery, and also recruitment and reactivation of the pre-rRNA processing machinery. Indeed cells exiting from mitosis in the presence of a CDK inhibitor exhibit neither relocalization of the late pre-rRNA processing components from PNBs to rDNA transcription sites, resumption of proper rRNA processing, nor formation of functional nucleoli.\nNucleolus and cancer\nThe link between cell proliferation, cancer and nucleolar activity has been well established during the past several decades (more than 5,000 references). Half of the studies related to the nucleolus and cancer are dedicated to the prognostic value of AgNOR staining, a technique revealing the amount of nucleolar proteins. The aim of this technique is to evaluate the proliferation potential of cancer cells by measuring nucleolar activity. B23, nucleolin, UBF and subunits of RNA pol I were found to be the argyrophilic proteins responsible for the silver-staining properties of nucleolar structures (Roussel et al. 1992; Roussel and Hernandez-Verdun 1994). In interphase cells, the amount of major AgNOR proteins, B23 and nucleolin, is high in S\u2013G2 and low in G1 phases and thus a higher value of AgNOR corresponds to actively cycling cells (Sirri et al. 1997). Standardization of the AgNOR staining method permits routine application of this technique for clinical purposes. The size of the nucleolus is generally enlarged in cancer cells, and this has been correlated with cell proliferation.\nA new field of research was recently opened by the discovery that several tumor suppressors and proto-oncogenes affect the production of ribosomes (Ruggero and Pandolfi 2003). rRNA synthesis is enhanced by c-Myc (Arabi et al. 2005) and it was proposed that this stimulation is a key pathway driving cell growth and tumorigenesis (Grandori et al. 2005). On the contrary, the decrease of ribosome production induces apoptosis in a p53-dependent or independent manner (David-Pfeuty et al. 2001; Pestov et al. 2001) and the disruption of the nucleolus mediates p53 stabilization (Rubbi and Milner 2003). The cross talk between the p53 pathway and the nucleolus is at least in part mediated by localization of Mdm2 in the nucleolus, an E3 ubiquitin ligase involved in p53 degradation. Nucleostemin, a nucleolar protein discovered in stem cells and in cancer cells interacts with p53 (Tsai and McKay 2005, 2002). It was proposed that nucleostemin might regulate p53 function through shuttling between the nucleolus and the nucleoplasm. The major nucleolar protein B23 is directly implicated in cancer pathogenesis as demonstrated by mutation of the gene in a number of hematological disorders (Grisendi et al. 2005). Importantly, in acute promyelocytic leukemia, the fusion protein NPM\/RAR\u03b1 localizes in the nucleolus indicating a role of this nucleolar protein in this disease (Rego et al. 2006).\nNucleolus and virus\nWithin the last few years, increasing evidence has revealed that viruses require the nucleus and in particular the nucleolus to target proteins indispensable for their replication. An increasing number of key proteins from both DNA- and RNA-containing viruses are localized in the nucleolus: viruses of the family Herpesviridae, Adenoviridae, Hepadnaviridae, Retroviridae, Rhabdoviridae, Orthomyxoviridae, Potyviridae, Coronaviridae and Flaviviridae, encode such proteins. Viruses have developed different strategies to facilitate targeting of their proteins to the nucleolus: (1) it was reported that the sequences of certain viral proteins harbor NoLS and NES (Harris and Hope 2000; Hiscox 2007; Kann et al. 2007). Recently (Reed et al. 2006) it was demonstrated by mutagenesis that the nucleocapsid (N) protein of infectious bronchitis virus (IBV), presents an 8 amino acid-long motif that functions as NoLS, and is necessary and sufficient for nucleolar retention of the N protein and colocalization with nucleolin and fibrillarin; the NoLS is required for interaction with cell factors. (2) Other viral proteins present sequences rich in arginine\u2013lysine (Ghorbel et al. 2006; Reed et al. 2006) known to be nucleolar retention signals; generally, these sequences overlap the NLS. (3) Some viral proteins that target the nucleolus present motifs with affinity for double-stranded RNA (dsRNA), for RNA binding or for DNA binding (Melen et al. 2007). (4) Other studies showed that nucleolar localization of viral proteins, is cell cycle-dependent (Cawood et al. 2007); using synchronization studies coupled to live cell confocal microscopy, the authors demonstrated that the concentration of N protein in the nucleolus was higher in the G2\/M phase than in other phases, and that in this phase the protein was more mobile in the nucleoplasm. In all the cases examined, the viral proteins depend on cell factors to successfully shuttle between the nucleolus and the cytoplasm.\nWhy must viral proteins target to the nucleolus?\nThe answer to this question is not clear; however, different authors had reported that such viral proteins are involved both in replication of the viral genome, and in transcriptional and post-transcriptional regulation of viral genome expression (Dang and Lee 1989; Pyper et al. 1998). For example, some plants viruses are known to encode a protein designated movement protein, responsible for long-distance movement of the viral RNA through the phloem (Ryabov et al. 1999). Movement strictly depends on the interaction of the viral movement protein with the nucleolus and the Cajal bodies, which contain snRNPs and snoRNPs (Kim et al. 2007a, b). The open reading frame (ORF) 3 of Groundnut rosette virus is one such protein; it is first localized in Cajal bodies and forms Cajal body-like structures, it is then localized in the nucleolus when the Cajal body-like structures fuse with the nucleolus, and finally it exits to the cytoplasm (Kim et al. 2007b). Another study showed that this shuttling is indispensable to form the RNPs essential for systemic virus infection (Kim et al. 2007a). In this process, the interaction of the viral ORF3 with fibrillarin is absolutely required. Interestingly, silencing of the fibrillarin gene blocks long-distance movement of the virus but does not affect virus replication and movement via plasmodesmata. Because the mobility of nucleolar components depends on the interactions and functions of the components (Olson and Dundr 2005), we suggest that targeting of viral proteins to the nucleolus could help viral protein traffic and diffusion of viral infection.\nThe activity of the human immunodeficiency virus (HIV)-1 Rev protein is essential for virus replication. Its subcellular localization is nucleolar, but it has the ability to shuttle continuously between the nucleus and the cytoplasm (Felber et al. 1989; Kalland et al. 1994). Rev possesses both an NES and an NLS; the NLS is associated with importin-\u03b2 as well as with B23 (Fankhauser et al. 1991; Henderson and Percipalle 1997). Rev-GFP movement in the nucleolus is very slow, implying that it is attached to affinity binding sites in this subcellular compartment (Daelemans et al. 2004). In addition, the transport of Rev from the nucleolus to the cytoplasm can be affected negatively by NF90, a cellular protein that colocalizes with Rev in the nucleolus (Urcuqui-Inchima et al. 2006) (Fig.\u00a04). This indicates that the transport of HIV transcripts by Rev to the cytoplasm is a regulated process. Because Rev is concentrated in the nucleolus, it was suggested that the passage of Rev to the nucleolus is an indispensable step for Rev function, and hence for HIV-1 replication. Indeed, based on HIV-1 RNA trafficking through the nucleolus, this organelle is an essential participant of HIV-1 RNA export (Michienzi et al. 2000).\nFig.\u00a04HIV Rev-GFP and NF90-RFP fusions were expressed in HeLa cells. Both proteins colocalize in nucleoli as seen by the merge. The nucleus is visualized by Dapi staining. Bars: 10\u00a0\u03bcm\nAs discussed above for Rev, it has been shown that the Herpes virus saimiri ORF57 protein is required for nuclear export of viral intronless mRNAs (Boyne and Whitehouse 2006). In addition, the expression of ORF57 induces nuclear trafficking, which is essential for nuclear export of such RNAs; the human transcription\/export protein involved in mRNA export, is redistributed to the nucleolus in the presence of the ORF57 protein. Based on these findings, the authors concluded that the nucleolus is required for nuclear export of the viral mRNAs.\nWhat are the consequences for the cells of the passage of viral proteins via the nucleolus?\nIt is known that all viruses whether with DNA or RNA genomes interfere with the cell cycle, affecting host-cell functions and increasing the efficiency of virus replication. The data obtained suggest that targeting of virus proteins to the nucleolus not only facilitates virus replication, but may also be required for pathogenic processes. Recent studies following infection by IBV, revealed a change in the morphology and protein content of the nucleolus (Dove et al. 2006). This included an enlarged FC and an increase in protein content; interestingly, the tumor suppressor protein p53, normally localized in the nucleus in virus infected cells, was redistributed mainly in the cytoplasm. The Hepatitis B virus (HBV) core antigen (HBcAg) is responsible for export of the virus with a mature genome (Yuan et al. 1999a, b). Indeed a change from isoleucine to leucine in position 97 (I97L) of HBcAg causes the cell to release virus particles with immature genomes. HBcAg with a mutation in position 97 (I97E or I97W) has been detected in the nucleolus colocalizing with nucleolin and B23, and this colocalization was often related with binucleated cells or apoptosis (Ning and Shih 2004), suggesting that the localization of HBcAg in the nucleolus could perturb cytokinesis. The authors propose that this event may be associated with liver pathogenesis.\nSome factors expressed by west nile virus (WNV) such as NS2B and NS3 and the WNV capsid (WNVCp) participate in WNV-mediated apoptosis (Oh et al. 2006; Ramanathan et al. 2006). It is well known that p53 is activated in response to oncogenic or DNA damaging stresses, inducing cell cycle arrest and apoptosis (Harris and Levine 2005). In normal conditions, HDM2 targets p53 and blocks abnormal accumulation of p53 by HDM2-mediated ubiquitinylation, followed by 26S proteasome-dependent degradation of p53 (Haupt et al. 1997; Kubbutat et al. 1997). Recently it was demonstrated that WNVCp could bind to and sequester HDM2 in the nucleolus, blocking p53-HDM2 complex formation (Yang et al. 2007). This phenomenon causes stabilization of p53 and Bax activation and thereafter apoptosis. In addition, the authors show that WNVCp is able to induce apoptosis-dependent processes, suggesting that the viral protein mediates apoptosis through p53-dependent mechanisms by retention of HDM2 in the nucleolus.\nRemarks and perspectives\nThe conclusions are based on the perspectives and the tendency that can be anticipated from the present research in the field of the nucleolus.\nWe propose that in the future, a better understanding of the complexity and variability of ribosome biogenesis will need to be established. For example, the difference between the information available in yeast and mammalian cells is of major importance. The different steps of ribosome biogenesis and protein complexes are well characterized in yeast due to easy access of mutants. Similarly, Miller chromatin spreading for electron microscopy in yeast strains carrying mutations reveals the coupling of RNA pol I transcription with rRNA processing (Schneider et al. 2007). Additionally, the compaction into SSU processomes of pre-18S ribosomal RNA before cleavage was observed on Miller spreads (Osheim et al. 2004). There is presently no comparable information for the mammalian genes. Yet the tendency is to generalize and suppose that the information is similar in the two models. In the future, differences will most probably be revealed as well as the complexity of the regulation in differentiated cells. Along this line, it was demonstrated that basonuclin, a cell-type-specific rDNA regulator transcribes only one subset of rDNAs of a cell (Zhang et al. 2007b). In such a differentiated cell, it remains to be established how the subset of rDNA repeats is selected.\nThe nucleolus was proposed to be a domain of the sequestration of molecules that normally operate outside this organelle, mainly in the nucleoplasm. Sequestration in the yeast nucleolus of the phosphatase cdc14 and its release into the cytoplasm at anaphase was demonstrated to be a key event in cell cycle progression (for a review see Cockell and Gasser 1999; Guarente 2000; Visintin and Amon 2000). However, it is important to recall that there is no nucleolus during mitosis in mammalian cells. In mammalian interphase cells, the nucleolus is a domain of retention of molecules related to cell cycle, life span, and apoptosis, and is in particular an actor of the p53-dependent pathway. Recently nucleolar retention of the Hand1 transcription factor was observed in trophoblast stem cells (Martindill et al. 2007). Phosphorylation of Hand1 induced nucleolar to nucleoplasm translocation of Hand1 and commitment of stem cells to differentiate into giant cells. Hand1 translocation to the nucleoplasm might regulate a crucial step of stem cell differentiation into polyploid giant cells but the targets of Hand1 in the nucleoplasm are still undefined.\nThe nucleolus is generally surrounded by highly condensed chromatin first described in rat hepatocytes and presently known as heterochromatin. By following the movements of chromosome sequences introduced in different sites in chromosomes of living cells, it was demonstrated that loci at nucleoli periphery and nuclear periphery are less mobile than in other sites. Disruption of the nucleoli by a CK2 inhibitor increases the mobility of the perinucleolar loci. It was proposed that the nucleolus and nuclear periphery could maintain the three-dimensional organization of chromatin in the nucleus (Chubb et al. 2002). Recently the perinucleolar ring of chromatin was brought to the fore when its role in the maintenance of inactive X (Xi) was demonstrated (Zhang et al. 2007a). During middle and late S phase, Xi contacts the nucleolar periphery when it is replicated during the cell cycle. It was discovered that the perinucleolar chromatin is enriched in Snf2h, the catalytic subunit of a remodeling complex required for replication of heterochromatin. These observations demonstrate the role of the perinucleolar compartment in maintaining the epigenetic state of Xi (Zhang et al. 2007a). The presence of inactive rDNA repeats in perinucleolar heterochromatin is known in many plant cells and in Drosophila. It was recently demonstrated that disruption in Drosophila of histone H3K9 methylation, a marker of heterochromatin, induced nucleolar disorganization and decondensation, and disorganization of rDNA repeats (Peng and Karpen 2007). The authors suggest that condensation of a part of the rDNA copies into heterochromatin could be a general strategy against recombination of these highly repeated genes.\nFor long, interest concerning the nucleolus was to establish how efficient ribosome biogenesis occurs and the link of this function with the cell cycle. More recently the effect of the disruption of ribosome biogenesis appeared very important when it was proposed that the nucleolus is a sensor of stress (Rubbi and Milner 2003). Indeed disruption of ribosome biogenesis releases ribosomal proteins from the nucleolus that bind to MDM2 and inhibit p53 degradation (Lindstrom et al. 2007). A connection between ribosomal stress and p53-dependent cell cycle arrest is now proposed (Gilkes et al. 2006).\nConsidering the diversity of the recent information gathered on the nucleolus, it is clear that this is a very dynamic and rapidly progressing research area. The most promising aspect is the contribution of new models (pseudo-NORs, Prieto and McStay 2007), new species (not only yeast), new approaches (Miller spreads using mutants, proteomics) and new questions (for instance the role of siRNAs or antisens RNAs in the activity of the nucleolus).","keyphrases":["nucleolus","assembly","virus","cancer","dynamics","nucleolar structure","pnb","cell cycle control"],"prmu":["P","P","P","P","P","P","P","R"]}
{"id":"Calcif_Tissue_Int-3-1-1914229","title":"Use of \u03b2-Blockers and the Risk of Hip\/Femur Fracture in the United Kingdom and The Netherlands\n","text":"Data from in vivo studies have indicated a role for \u03b2-blockers in the prevention of bone loss. Some epidemiological studies have found protective effects of \u03b2-blockers on fracture risk. However, there is limited information on the association with cumulative dose and type of \u03b2-blockers used. We conducted two case-control studies using data from the UK General Practice Research Database (GPRD) and the Dutch PHARMO Record Linkage System (RLS). Cases were patients with a first hip or femur fracture; controls were individually matched on practice\/region, gender, year of birth, and calendar time. Current use of \u03b2-blockers was defined as a prescription in 90 days before the index date. We adjusted for medical conditions and drugs associated with falling or bone mineral density. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using conditional logistic regression analysis. The study population included 22,247 cases and controls in the GPRD and 6,763 cases and 26,341 controls in the PHARMO RLS. Current use of \u03b2-blockers was associated with a reduced risk of hip\/femur fracture in both the GPRD (adjusted OR = 0.82, 95% CI 0.74\u20130.91) and PHARMO RLS (adjusted OR = 0.87, 95% CI 0.80\u20130.95) study populations. However, this reduction of risk was not associated with cumulative dose, lipophilicity, or receptor selectivity of \u03b2-blockers. The protective effect of \u03b2-blockers was only present among patients with a history of use of other antihypertensive agents (GPRD adjusted OR = 0.72, 95% CI 0.64\u20130.83; PHARMO RLS adjusted OR = 0.76, 95% CI 0.67\u20130.86) but not in patients using \u03b2-blockers only (GPRD adjusted OR = 0.97, 95% CI 0.82\u20131.14; PHARMO RLS adjusted OR = 1.01, 95% CI 0.90\u20131.14). Also, in patients with a history of use of other antihypertensive agents, no dose-response relationship with \u03b2-blocker use was found. The effect was constant with cumulative dose and the OR was below 1.0 even among patients who just started treatment with \u03b2-blockers. As the mechanism by which \u03b2-blockers could influence bone mineral density is likely to need some time to exert a clinically relevant effect, all these finding suggests that the association between \u03b2-blockers and fracture risk is not causal.\nBone remodeling comprises a phase of resorption by osteoclasts and a phase of formation by osteoblasts. Recent studies have shown bone metabolism to be mediated through the autonomic central nervous system. Leptin, a hormone produced in fat cells to signal energy insufficiency, regulates bone remodeling by modulating osteoblast proliferation and subsequent osteoclast activation via the osteoclast differentiation factor receptor activator of nuclear factor \u03baB ligand (RANKL). The antiosteogenic effect of leptin is not present in \u03b22-adrenergic receptor-deficient mice, which had actual increases in bone mineral density (BMD) [1\u20135]. Data from these in vivo studies indicate a role for \u03b2-blockers in the prevention of bone loss. In the early 1990s, propranolol was found to increase bone formation [6]. Some observational studies have reported that use of \u03b2-blockers was associated with a decreased risk of fractures [7\u20139], conflicting with other studies which found no association with fractures [10\u201312]. Studies on the effects of \u03b2-blockers on subclinical endpoints, like BMD or biochemical markers of bone resorption, have also yielded inconsistent results [7, 10, 12\u201314].\nA possible role for \u03b2-blockers in the prevention of fractures is of major clinical interest, given that fractures are a major source of morbidity, disability, hospitalization, and mortality. One of the most serious fractures resulting from accidental falls is hip fracture [15]. However, there is still a lack of knowledge with respect to the effects of cumulative dose and type of \u03b2-blockers used. Thus, the objective of this study was to assess the strength of the association between use of \u03b2-blockers and risk of hip\/femur fractures using data from two different large population-based databases in the United Kingdom and The Netherlands.\nMaterials and Methods\nSetting\nData for this study were obtained from the UK General Practice Research Database (GPRD) and the Dutch PHARMO Record Linkage System (RLS). The GPRD contains the computerized medical records of general practices across the United Kingdom (http:\/\/www.gprd.com). Approximately 6% of the total registered population of England and Wales is represented in the database, and it includes a cumulative total of over 5 million adult patients. The age and sex distribution of patients enrolled is representative of the general English and Welsh populations. Patient details accrued in the GPRD include demographic information, diagnoses, prescription details, preventive care provided, referrals to specialist care, hospital admissions, and related major outcomes [16]. Clinical data are stored and retrieved by means of Oxford Medical Information Systems and Read codes for diseases or causes of morbidity and mortality that are cross-referenced to the International Classification of Diseases (ICD-9). Several independent validation studies have shown that the GPRD has a high level of completeness and validity, including for hip fractures [17, 18].\nThe PHARMO RLS includes the demographic details and complete medication history of 950,000 community-dwelling residents of more than 25 population-defined areas in The Netherlands from 1985 onward. It is further linked to hospital admission records as well as several other health registries, including pathology, clinical laboratory findings, and general practitioner data (http:\/\/www.pharmo.nl). Since the majority of all patients in The Netherlands are registered only with one community pharmacy, independently of prescriber, pharmacy records are virtually complete with regard to prescription drugs. For this study, drug-dispensing and hospitalization data were used. The computerized histories record information on the type of drug dispensed, dispensing date, prescriber, amount dispensed, and prescribed dosage regimen. Hospital discharge records include detailed information on the primary and secondary discharge diagnoses; diagnostic, surgical, and treatment procedures; type and frequency of consultations with medical specialists; and dates of hospital admission and discharge. All diagnoses are coded according to the ICD-9-CM [19].\nDefinition of Cases and Controls\nGPRD\nA case-control study was conducted using GPRD data collected from January 1987 to July 1999. The details of this study have been described elsewhere [20, 21]. Briefly, cases were defined as patients aged 18 years and older with a first record of a hospital admission for a hip\/femur fracture (ICD-9 codes 820\u2013821) recorded in their medical records between the enrollment date of their practice in the GPRD and the end of data collection. The date of the occurrence of the hip\/femur fracture was the index date. Each case was matched by year of birth, sex, medical practice, and calendar time to one control patient without a history of a fracture. If no eligible control was available, the age criterion was expanded consecutively at 1-yearly intervals to a maximum of 10 years. If no eligible control patient could be found, then an age- and sex-matched control patient from another practice was selected.\nPHARMO RLS\nCases were patients aged 18 years and older with a first admission for a hip\/femur fracture between January 1, 1991, and December 31, 2002. The date of the hospital admission was the index date. Up to four control patients, who did not have a history of any type of fracture, were matched to each case by year of birth, gender, region, and calendar time.\nExposure Assessment\nFor each patient, we identified all prescriptions for (GPRD) and dispensing of (PHARMO RLS) \u03b2-blockers prior to the index date. Current users were defined as patients who had a prescription\/dispensing for \u03b2-blockers within 3 months prior to the index date. Recent users received a last prescription\/dispensing 3\u20136 months before the index date, past users 6\u201312 months before the index date, and distant past users more than 12 months before the index date. The last prescribed daily dose prior to the index date was obtained from the written dosage instructions. For each patient, cumulative exposure to \u03b2-blockers ever before the index date was calculated. The effect of cumulative dose was assessed both regardless of the timing of use and stratified to current, recent, past, and distant past use. Thus, current users could be classified to a low previous cumulative exposure (e.g., one or two 30-day prescriptions) or a high previous cumulative exposure (e.g., 100 30-day prescriptions). The estimated daily dose for each class of \u03b2-blocker was expressed as a fraction of the World Health Organization defined daily dose (DDD). A DDD is defined as the assumed average maintenance dose per day for a drug if used for its main indication in adults [22]. DDD equivalents can be used to compare drugs within a certain therapeutic group. In this study, we converted the DDD equivalents to milligrams of metoprolol, similar to the approach used in previous studies [21]. Furthermore, \u03b2-blockers were categorized according to receptor selectivity and lipophilicity based on data in handbooks on clinical pharmacology and therapeutics.\nStatistical Analysis\nThe strength of the association between use of \u03b2-blockers and risk of hip\/femur fractures was estimated using conditional logistic regression analysis (SAS version 9.1.3, PHREG procedure; SAS Institute, Cary, NC) and expressed as odds ratios (ORs) and 95% confidence intervals (CIs). Final regression models were determined by stepwise backward elimination using a significance level of 0.05. Smoothing spline regression plots were used to visualize the effect of cumulative \u03b2-blocker dose on risk of hip\/femur fractures [23].\nIn our analysis, we controlled for a wide range of clinical variables that have been associated with risk of falls or fractures. In the GPRD study, we included the following variables in the final model: history of diabetes mellitus, rheumatoid arthritis, hyperthyroidism, congestive heart failure, seizures, anemia, dementia, depression, psychotic disorder, cerebrovascular accident, chronic obstructive pulmonary disease, osteoporosis, and a record of back pain or falls in the year before the index date. Furthermore, prescriptions, in the 6 months prior to the index date, for anticonvulsants, nonsteroidal anti-inflammatory drugs (NSAIDs), methotrexate, hormone replacement therapy, other hypertensive drugs (low-ceiling diuretics, renin-angiotensin-aldosterone system [RAAS] inhibitors, calcium channel blockers), anxiolytics\/hypnotics, antipsychotics, antidepressants, anti-Parkinson drugs, oral and inhaled glucocorticoids, and bronchodilators as well as information on body mass index (BMI) were retained in the model. In the PHARMO RLS study, variables included in the final model were a dispensing of benzodiazepines in the 3 months prior to the index date, or, in the 6 months prior to the index date, a dispensing of oral glucocorticoids, inhaled glucocorticoids, bronchodilators, statins, BMD-modifying drugs, hormone replacement therapy, antipsychotics, antidepressants, opioids, antiepileptics, other hypertensive drugs (low-ceiling diuretics, RAAS inhibitors, calcium channel blockers), antidiabetics, laxants, disease-modifying antirheumatic drugs, NSAIDs, or metoclopramide. A history of hospital admission for cerebrovascular disease, cancer, endocrine disorders, inflammatory bowel disease and other bowel diseases, obstructive airway disease, musculoskeletal and connective tissue diseases, anemia, and skin diseases prior to the index date were also included in the final model.\nResults\nThe study population in the GPRD comprised 22,247 cases and 22,247 controls, whereas in the PHARMO RLS 6,763 cases and 26,341 controls were identified. The characteristics of both populations are displayed in Table\u00a01. The sex and age distributions of cases were similar in the two case-control sets, although more cases in the GPRD set were aged over 80 years.\nTable\u00a01.Characteristics of hip\/femur fracture cases and controls in the GPRD and PHARMO RLSCharacteristicGPRDPHARMO RLSCases (n = 22,247)Controls (n = 22,247)Cases (n = 6,763)Controls (n = 26,341)Gender\u00a0\u00a0Women75.8%75.8%72.9%72.7%Age (years)\u00a0\u00a0<6513.9%13.9%15.8%16.2%\u00a0\u00a065\u20137930.8%32.2%36.6%37.3%\u00a0\u00a0\u22658055.2%53.9%47.6%46.5%\u00a0\u00a0Smokinga22.1%20.6%No data availableBMIb\u00a0\u00a020\u20132546.6%42.4%\u00a0\u00a0<2018.0%9.7%No data available\u00a0\u00a0>2535.4%47.9%Antidepressants13.0%7.2%9.5%5.1%Oral glucocorticoids7.2%4.4%5.4%3.5%Thiazides11.9%12.9%12.1%11.4%Nitrates6.9%7.4%9.4%9.1%Hormone replacement0.6%1.2%1.1%1.3%a No data on smoking status for 45% of GPRD study populationb No data on BMI for 58.5% of GPRD study population\nCurrent use of \u03b2-blockers was associated with a significantly decreased risk of hip\/femur fracture in both databases, whereas recent and past use was not (Table\u00a02). Adjusted ORs for current \u03b2-blocker users were 0.83 (95% CI 0.75\u20130.92) in the GPRD and 0.87 (95% CI 0.80\u20130.95) in the PHARMO RLS. There was no strong effect of cumulative dose among current users of \u03b2-blockers in either data set (Fig.\u00a01), while assessing the effect cumulative dose irrespective of timing of use yielded similar results.\nTable\u00a02.Use of \u03b2-blockers and risk of hip\/femur fracture in the GPRD and PHARMO RLS\u03b2-blocker exposureGPRDPHARMO RLSCases (%)Controls (%)Crude OR (95% CI)Adjusted OR (95% CI)aCases (%)Controls (%)Crude OR (95% CI)Adjusted OR (95% CI)aTiming of use\u00a0\u00a0Current use4.55.90.70 (0.64\u20130.77)0.82 (0.74\u20130.91)12.413.70.91 (0.83\u20130.98)0.87 (0.80\u20130.95)\u00a0\u00a0Recent use0.60.60.89 (0.70\u20131.14)0.88 (0.68\u20131.15)1.71.71.01 (0.82\u20131.24)0.93 (0.75\u20131.15)\u00a0\u00a0Past use0.70.61.10 (0.86\u20131.40)0.99 (0.76\u20131.28)1.41.31.11 (0.88\u20131.40)0.99 (0.78\u20131.26)\u00a0\u00a0Distant past use3.72.81.10 (0.98\u20131.22)1.06 (0.94\u20131.20)6.86.31.07 (0.96\u20131.20)0.97 (0.87\u20131.09)Among current users of \u03b2-blockersSelectivity\u00a0\u00a0Low1.01.20.75 (0.63\u20130.90)0.86 (0.71\u20131.05)3.53.11.12 (0.97\u20131.30)1.04 (0.89\u20131.21)\u00a0\u00a0Medium0.10.20.79 (0.48\u20131.32)0.81 (0.47\u20131.41)0.30.50.40 (0.20\u20130.84)0.38 (0.18\u20130.79)\u00a0\u00a0High3.34.40.69 (0.62\u20130.76)0.77 (0.69\u20130.87)8.710.10.86 (0.79\u20130.95)0.84 (0.76\u20130.93)Lipophilicity\u00a0\u00a0Hydrophile3.24.20.69 (0.62\u20130.77)0.78 (0.70\u20130.88)5.15.20.98 (0.86\u20131.11)0.94 (0.83\u20131.06)\u00a0\u00a0Intermediate0.20.20.77 (0.50\u20131.20)0.88 (0.56\u20131.39)0.50.41.15 (0.77\u20131.73)1.04 (0.69\u20131.58)\u00a0\u00a0Lipophile1.11.40.72 (0.60\u20130.85)0.81 (0.68\u20130.98)6.98.00.85 (0.77\u20130.95)0.83 (0.74\u20130.92)First prescription\u00a0\u00a0Yes0.20.20.93 (0.58\u20131.48)1.18 (0.69\u20131.99)0.40.60.63 (0.42\u20130.95)0.62 (0.41\u20130.94)Last prescribed daily dose (DDD)b\u00a0\u00a0<0.670.91.20.72 (0.59\u20130.87)0.81 (0.65\u20131.00)8.79.80.89 (0.81\u20130.98)0.87 (0.79\u20130.96)\u00a0\u00a00.67\u20131.332.02.60.73 (0.64\u20130.83)0.85 (0.74\u20130.99)3.23.40.95 (0.82\u20131.11)0.90 (0.77\u20131.06)\u00a0\u00a0>1.331.42.00.64 (0.55\u20130.75)0.81 (0.69\u20130.97)0.40.40.95 (0.61\u20131.49)0.85 (0.54\u20131.35)a Adjusted for use of other antihypertensive drugs and general risk factors for falls and fractures (see Materials and Methods)b One DDD is equivalent to 150 mg metoprololFig.\u00a01.Spline visualization of cumulative dose among current \u00df-blocker users and risk of hip\/femur fractures (GPRD, dashed line, solid circles; PHARMO RLS, solid line, open circles). Cumulative dose is expressed in DDDs (1,000 DDDs are equivalent to 150 g of metoprolol). Odds ratios were adjusted for the same confounders as in Table 2.\nThe most frequently prescribed \u03b2-blocker in the GPRD was atenolol (3.0% among cases vs. 4.0% among controls, adjusted OR = 0.80, 95% CI 0.71\u20130.90). Current use of other \u03b2-blockers was infrequent (propranolol 0.4% vs. 0.5%, metoprolol 0.2% vs. 0.2%) and not associated with a decreased risk of hip\/femur fracture (adjusted OR = 0.90, 95% CI 0.76\u20131.06). In the PHARMO RLS, the most frequently used \u03b2-blockers at the index date were metoprolol (4.6% among cases vs. 5.6% among controls), atenolol (3.2% vs. 3.6%), sotalol (1.9% vs. 1.5%), and propranolol (0.9% vs. 0.9%). The adjusted ORs for current use of metoprolol and atenolol were 0.79 (95% CI 0.69\u20130.90) and 0.89 (95% CI 0.77\u20131.04), respectively. Current use of propranolol (adjusted OR = 0.98, 95 CI 0.74\u20131.21) and sotalol (adjusted OR = 1.15, 95% CI 0.93\u20131.42) were not associated with a protective effect of hip\/femur fracture.\nCategorizing \u03b2-blockers according to their lipophilicity, receptor selectivity, or last prescribed daily dose did not reveal major differences in effect (Table\u00a02), nor did stratification according to age and gender (Table\u00a03).\nTable\u00a03.Current use of \u03b2-blockers and risk of hip\/femur fractures in patient subgroupsCurrent use of \u03b2-blockersGPRDPHARMO RLSCases (%)Controls (%)Crude OR (95% CI)Adjusted OR (95% CI)aCases (%)Controls (%)Crude OR (95% CI)Adjusted OR (95% CI)aGender\u00a0\u00a0Men3.44.50.68 (0.55\u20130.84)0.77 (0.60\u20130.98)10.112.00.83 (0.70\u20130.98)0.77 (0.64\u20130.93)\u00a0\u00a0Women4.86.30.71 (0.64\u20130.78)0.83 (0.74\u20130.93)13.314.40.93 (0.95\u20131.02)0.90 (0.82\u20131.00)Age (years)\u00a0\u00a0<652.93.10.93 (0.68\u20131.26)0.91 (0.62\u20130.91)5.07.21.04 (0.80\u20131.35)0.94 (0.70\u20131.27)\u00a0\u00a065\u2013807.09.10.71 (0.62\u20130.80)0.84 (0.73\u20130.98)14.516.80.84 (0.58\u20131.12)0.80 (0.70\u20130.91)\u00a0\u00a080+3.54.60.65 (0.57\u20130.75)0.77 (0.66\u20130.91)12.413.50.94 (0.84\u20131.06)0.94 (0.83\u20131.07)History of any use of other antihypertensive drugs\u00a0\u00a0No2.22.40.89 (0.77\u20131.04)0.97 (0.82\u20131.14)8.38.11.02 (0.85\u20131.23)1.01 (0.90\u20131.14)\u00a0\u00a0Yes9.714.70.60 (0.53\u20130.67)0.73 (0.64\u20130.83)20.926.90.73 (0.65\u20130.83)0.76 (0.67\u20130.86)a Adjusted for use of other antihypertensive drugs and general risk factors for falls and fractures (see Materials and Methods) Percentages represent the proportion of current beta-blocker within each subcategory (e.g. male gender)\nThe protective effect of \u03b2-blockers was only present among patients who had been treated with other antihypertensive agents (e.g., low-ceiling diuretics, calcium antagonists, RAAS inhibitors), either concurrently or in the past (Table\u00a03). This finding was consistent in both the GPRD (adjusted OR = 0.73, 95% CI 0.64\u20130.83) and PHARMO RLS (adjusted OR = 0.76, 95% CI 0.67\u20130.86). Among patients using only \u03b2-blockers, the adjusted ORs were close to unity. In both data sets, the interaction term between current use of \u03b2-blockers and use of other antihypertensive drugs was statistically significant (P < 0.05). Furthermore, no effect of cumulative dose was found in either current users of \u03b2-blocker only or in current \u03b2-blocker users with a history of using other antihypertensive drugs (Fig.\u00a02). Further stratification according to high or low average daily dose during the study period indicated no effect of the intensity of \u03b2-blocker use on the risk estimates.\nFig.\u00a02.Spline visualization of cumulative dose among current \u00df -blocker users, stratified according to patients not having (A) or having a history of other antihypertensive drugs (B) (GPRD, dashed line, solid circles; PHARMO RLS, solid line, open circles). Cumulative dose is expressed in DDDs (1,000 DDDs are equivalent to 150 g of metoprolol). Odds ratios were adjusted for the same confounders as in Table\u00a02, except for the use of antihypertensive drugs.\nDiscussion\nIn both the GPRD and PHARMO RLS data sets, current use of \u03b2-blockers was associated with a decreased risk of hip\/femur fractures. However, there was no reduced risk of hip\/femur fractures among patients who did not have a history of using other antihypertensive drugs: a protective effect of \u03b2-blockers was only observed for patients with current or prior use of other antihypertensive agents. Even within this group of patients, no dose-response relationship with \u03b2-blocker use was found. The effect was constant with cumulative dose and the OR was below 1.0 even among patients who had just started treatment with \u03b2-blockers. As the mechanism by which \u03b2-blockers could influence BMD is likely to need some time to exert a clinically relevant effect, this finding suggests that the association between \u03b2-blockers and fracture risk is not causal.\nBased on in vivo and in vitro studies and the discovery that the central nervous system is involved in the regulation of bone, \u03b2-blockers have been implicated in a preventive role in patients with osteoporosis. Central effects of leptin have been found to be mediated by the sympathetic nervous system, acting via \u03b2 receptors on osteoblasts [3]. \u03b2 agonists stimulate bone-resorption activity in osteoclasts [24]. Previous data have shown that systemic application of \u03b2 agonists had a negative effect on bone mass in mice [25], whereas \u03b2 antagonists stimulated bone formation in rats [6].\nSeveral epidemiological studies have reported discrepant results on the association between use of \u03b2-blockers and risk of fracture [7\u201312], including a study that also used data from the GPRD [8]. The reason for the discrepancy is unclear. Emerging data from randomized controlled trials also support a lack of effect of \u03b2-blockers on the risk of fracture. Recently, a clinical trial among 41 normal postmenopausal women found no evidence that propranolol stimulates bone formation, as measured by bone turnover markers [26]. Furthermore, pooled data from nine clinical trials investigating the nonselective \u03b2-blocker carvedilol in the management of congestive heart failure did not provide evidence in support of an effect of \u03b2-blockers on fracture risk reduction [10]. The data from this meta-analysis are consistent with the results of our study as no effect on fracture was observed in patients treated with \u03b2-blockers without history of other antihypertensive drug use.\nTherapeutic uses of \u03b2-blockers include the treatment of hypertension as well as heart failure and secondary prevention post-myocardial infarction, -cardiac dysrhythmia, and -angina pectoris. Cardiovascular disease, heart failure, and hypertension have all been linked with decreased BMD [27\u201330]. Because thiazides, calcium channel blockers, and inhibitors of the renin-angiotensin system have also been associated with beneficial effects on bone [31], we stratified in our study on history of use of other antihypertensive drugs to separate the effect of current use of \u03b2-blockers from that of other antihypertensive agents. This method of stratification was not applied in earlier epidemiological studies on \u03b2-blockers and fractures.\nThe strength of our study is that it was population-based. Furthermore, we found the same results in both the UK and Dutch data sets. The prevalence of \u03b2-blocker use was nearly three times as high in the PHARMO RLS compared to the GPRD. In 2002, 5.1 million prescriptions for \u03b2-blockers were issued in The Netherlands (population 16 million) [32] compared to 22.4 million prescriptions in the United Kingdom in the same year (population 59 million) [33]. Taking into account that prescriptions in the United Kingdom are usually for 30 days and those in The Netherlands for 90 days, we can conclude that the observed difference in exposure prevalence is in line with prescribing data volumes in both countries.\nObservational studies like ours have potential for bias and confounding and can fuel debates on study interpretation and credibility [34\u201336]. Various drugs with effects on the central nervous system are known to increase the risk of falls and thereby fracture risk. Also, there are likely to be complex interactions between vascular disease and fracture risk, operating through falls risk, BMD, or common genetic or lifestyle factors. In this study, we had no information on BMD and we cannot exclude the possibility that cases and controls were different with respect to BMD. However, given that initiation of antihypertensive treatment in daily clinical practice will usually be independent of patient BMD, major confounding seems unlikely. Variables included in the final regression models were slightly different between the two data sets due to the nature of the data collections. However, multivariate adjustment had only modest effects on the OR of the exposure of interest. Information on smoking and BMI was not available in the PHARMO RLS database, but adjustment for these factors in the GPRD had no influence on risk estimates (smoking was not retained in the model). Data on BMI were missing for 58% of the GPRD study population, so we cannot exclude the concern that adjustment was suboptimal. We had no data on physical activity, diet, or socioeconomic status as such information is not available in the databases used in our studies [37]. Furthermore, we cannot exclude the possibility that residual confounding can explain part of our results.\nAn alternative explanation for our findings could be that the protective effect of \u03b2-blockers on fractures is an artifact caused by selective underuse in patients with an unmeasured comorbidity, a problem that has been described by Glynn et al. in a study on cardiovascular drug use and mortality [38]. In their study, among elderly subjects in the United States, they found that users of drugs from seven commonly prescribed therapeutic classes, including \u03b2-blockers, thiazide diuretics, and angiotensin-converting enzyme inhibitors, had reduced rates of death compared to nonusers, which was more likely to be explained by selective prescribing and nonadherence.\nA potential limitation is that we confined our study to hip\/femur fractures and did not evaluate other type of fractures. It is possible that potential beneficial effects of \u03b2-blockers are present only at sites other than the hip\/femur, but there is no evidence to support this.\nIn conclusion, the reduction in hip\/femur fracture risk was not related to cumulative dose of \u03b2-blockers and was only present in patients using \u03b2-blockers with a history of using other antihypertensive drugs as well. This suggests that the effect of \u03b2-blockers on hip\/femur fracture is not causal.","keyphrases":["\u03b2-blocker","fracture","epidemiology","case-control","osteoporosis"],"prmu":["P","P","P","P","P"]}
{"id":"Purinergic_Signal-4-2-2377322","title":"A case of serendipity*\n","text":"An account is given of how a sensitive bioassay system for measurement of the neurotransmitter acetylcholine serendipitously led to the identification of adenosine triphosphate (ATP) released in vitro from active skeletal muscle. Subsequent application of the identification procedures to exercising human muscle in vivo, cardiac muscle cells in vitro, and human erythrocytes exposed to hypoxia gave rise to the general concept of ATP as a molecule that could influence cell function from the extracellular direction. Mechanisms of ATP release from cells in terms of \u201ctrigger\u201d events such as mechanical distortion of the membrane, depolarization of the membrane, and exposure to hypoxia are discussed. Potential therapeutic uses of extracellular ATP in cancer therapy, radiation therapy, and a possible influence upon aging are discussed. Possible roles (distant and local) of extracellular ATP released from muscle during whole body exercise are discussed.\nIntroduction\nIn 1961 Boyd and Eadie [5] developed a highly sensitive bioassay for acetylcholine using an in situ perfusion technique of frog heart. Based on the great sensitivity of the frog heart to acetylcholine (Fig.\u00a01) a project was suggested by B. Katz to I. A. Boyd; they thought it possible that the amounts of acetylcholine released from stimulated motor nerve endings in frog skeletal muscle could be directly measured. The skeletal muscle to be used was the frog sartorius, about which a lot was known regarding the distribution and population of the motor nerve terminals. At that time the association of miniature end-plate potentials (MEPPs) with the release of acetylcholine in packets (\u201cquanta\u201d) together with the anatomical existence of synaptic vesicles at the motor nerve terminals was highly plausible. Any accurate measurement of acetylcholine release from motor nerve stimulation would provide valuable evidence for (or against) the association and the establishment of the \u201cquantal\u201d theory of neuromuscular transmission. In essence the experimental approach mimicked the classic experiment of Otto Loewi, where fluid perfusing a first frog heart was used to perfuse a second heart. When the vagus nerve supplying the first heart was stimulated cardiac arrest occurred, and shortly after this the second heart also stopped beating. Loewi termed the vagal inhibitory transmitter Vagusstoff. An isolated frog sartorius was to be stimulated via the motor nerve in a bathing solution and the solution then perfused through a frog heart. When a stimulated muscle solution was perfused a surprising stimulatory response was elicited, quite unlike the anticipated inhibitory effect of acetylcholine (Fig.\u00a01). Clearly this response would preclude any accurate assay for acetylcholine (as well as any successful conclusion to a thesis project aimed at substantiating the quantal theory of neuromuscular transmission!).\nFig.\u00a01The response of a very sensitive frog heart to graded concentrations of acetylcholine (g.ml\u22121). A concentration of 2.5\u2009\u00d7\u200910\u221213\u00a0g.ml\u22121 (1.54\u2009\u00d7\u200910\u221212\u00a0mmol.ml\u22121) produced a threshold response [19]\nIt was evident that for successful measurement of acetylcholine, the identification of the interfering substance, and its removal from the solution, had to be achieved. At this stage the basic question became: what substance(s) released from active skeletal muscle could possibly stimulate the frog heart? A \u201cblunderbuss\u201d approach was adopted, by perfusing through the heart as many available substances, known to exist in muscle, to see if any of them produced the stimulatory effect. The purine monophosphates and adenosine were eliminated since they did not stimulate the heart. Electrolyte changes, including an elevated potassium level, were also ruled out. Changes in calcium levels were hardly detectable in the bathing solution.\nOne obvious candidate as a stimulatory substance was catecholamine material. However, when the frog heart response to adrenaline was blocked by an ergot\/pronethalol perfusion, the stimulatory effect remained (Fig.\u00a03).\nFig.\u00a02Effect of graded concentrations of ATP (g.ml\u22121) perfused through a frog heart [6]. Note that low concentrations have a pure inotropic effect, while a triphasic response was produced by higher concentrations. Further analysis of this triphasic response has since been made [17]\nIndirect versus direct muscle stimulation The distribution of motor end plates in the sartorius muscle (there are no end plate regions at the pelvic end of the frog sartorius) allowed muscle fibers to be stimulated directly, without involvement of the neuromuscular junction in the activation process. Solutions bathing muscles stimulated directly produced the same stimulatory effect as the solutions from muscles stimulated via the motor nerve (indirect stimulation). This indicated that it was highly unlikely that the stimulatory substance had as its major source the activated motor nerve terminal.\nIdentification procedures\nIdentification of the stimulatory substance was established using three procedures: gel filtration, enzyme action of apyrase, and firefly tail luminescence.\nGel filtration chromatography The use of a gel filtration chromatography technique (Sephadex G-25, fine grade) enabled removal of protein from the stimulated muscle solution, allowing spectral absorbance analysis. An absorbance peak obtained at 265\u03bb was the first hint that a purine compound was present in the solution. The gel filtration technique also established a molecular weight of the stimulatory substance close to that of ATP.In the face of prevailing dogma that ATP could not cross cell membranes, perfusion with ATP was reluctantly performed. A stimulatory effect was obtained\u2014the author had become a citizen of the Republic of Serendip! A dose response of the frog heart to ATP is shown in Fig.\u00a02.\nFig.\u00a03Comparison of the stimulatory action of a stimulated muscle solution (\u2018X\u2019) on a frog heart before and after adrenergic blockade. Vertical interrupted line, 2-h period during which heart was perfused with pronethalol hydrochloride (10\u22126\u00a0g.ml\u22121) and ergotamine tartrate (10\u22126\u00a0g.ml\u22121). The stimulatory action of the stimulated muscle solution was unaffected by adrenergic blockade. Reproduced from Boyd and Forrester [6], with permission of the Physiological Society\nIncubation with apyrase In 1962 Traverso-Cori and Cori [61] demonstrated that an enzyme purified from potatoes (\u201capyrase\u201d) could selectively cleave the terminal phosphate of ATP. The stimulated muscle solution was incubated with apyrase, as were the stimulatory fractions from the gel filtration technique, and it was shown that the stimulatory effect was abolished, indicating that a phosphorylated compound was responsible for the stimulatory effect.\nThe luciferin\/luciferase test for ATP As long ago as 1947 McElroy [38] showed that when a crude extract of lampyrid beetles (Photinus pyralis) was exposed to ATP a light signal was generated. When the stimulated muscle solution was applied to luciferin extract a light signal was produced (see [18]).\nProcedures applied to perfused frog musculature\nAs this work developed the question \u201cdoes ATP appear extracellularly as a result of tissue damage?\u201d was frequently asked. Dissection damage to the sartorius muscle was avoided by the use of the perfused hindlimb preparation of the frog, where instrument trauma was limited to the insertion of cannulae. The identification procedures for ATP were applied to the perfusate from stimulated (indirect) hindlimb musculature. Table\u00a01 shows the relationship of ATP output to the frequency of motor nerve impulses [23]. When these amounts (0.24\u20131.8\u00a0nmol 100\u00a0g\u22121 min\u22121) are compared to the amounts of ATP released from isolated sartorius (50\u00a0nmol 100\u00a0g\u22121 min\u22121, [6]), it is seen that there is a great reduction detected in the perfusate sample, presumably as a result of exposure to greater ecto-ATPase activity that was not encountered in solutions bathing isolated muscles.\nTable\u00a01Relationship of ATP output to frequency of stimulation of perfused frog limbaHzpmol volley\u22121 100 g\u22121pmol 100 g\u22121 min\u2212114.024021.518051.4420103.01,800aData from Forrester and Hassan [23]\nProcedures applied to human muscle in vivo\nThe appearance of ATP in the perfusate from active frog hindlimb musculature raised the possibility that ATP might be released from active muscle into the circulating blood in vivo. The identification procedures were applied to human plasma obtained from resting and exercising human forearm muscle [20, 21]. Figure\u00a04 gives an example of the combined use of gel filtration chromatography, firefly luciferase, and apyrase for the identification and quantification of ATP in human plasma from exercising human forearm muscle [22]. Estimates of plasma ATP levels can only be approximate, since the accumulation in the blood is continuously offset by ATPase activity of both plasma and the surfaces of the erythrocyte and endothelial cells.\nFig.\u00a04Elution of ATP and plasma samples from exercising human forearm through a Sephadex column. Elution fractions were estimated with firefly extract. a ATP, 10\u22126\u00a0g.ml\u22121. b Post-occlusion human sample from a subject during forearm exercise. c Samples from another subject exercising forearm muscle, fraction at 9\u00a0ml was incubated with apyrase and retested on firefly extract. Fraction at 10\u00a0ml, incubation control. Reproduced from Forrester [22], with permission of the Physiological Society\nSequelae\nThe present-day knowledge and extensive classification of purine receptors (see review by [48]) has vindicated the concept of an extracellular system of ATP action influencing many tissues of the body. However, the mode of ATP release from cells remains a formidable challenge (see review by [56]). In the cases of skeletal muscle, cardiac muscle, and erythrocyte some specific circumstances leading to ATP release, e.g. membrane depolarization, hypoxia, and mechanical distortion [25], should be addressed further.\nAssociation of ATP release from depolarizing membrane  In 1962 Abood et al. [1] assessed outflux of various phosphates during membrane depolarization of excitable tissues. Prior to that Holton [32] detected ATP release from sensory nerves stimulated antidromically. An elegant experiment by Israel et al. [33] showed discrete release of ATP closely related to membrane depolarization in the electric organ of Torpedo. In 1973 Ohta et al. [40] studied and compared the mode of action of several veratrum alkaloid compounds on axonal membranes (squid giant axon). Some of them exerted a highly specific action on the resting sodium permeability, with veratridine having the most potent effect. Two results led them to conclude that the depolarization evoked by veratridine was caused by specifically increasing the membrane permeability to sodium ions: (1) application of tetrodotoxin (TTX), which specifically blocks sodium channels, restored the resting membrane potential after depolarization by veratridine and (2) removal of sodium ions from both external and internal phases of the axon, thus eliminating the sodium term from the Goldman-Hodgkin-Katz constant field equation describing the membrane potential [31], had the result that veratridine had no depolarizing action after sodium removal. In 1977 White [63] demonstrated ATP release from depolarizing isolated synaptosomes prepared from rat brain. The synaptosomes were suspended in a solution containing firefly extract. Depolarization was achieved by (1) elevation of extracellular potassium and (2) with application of veratridine. A light signal was evoked in both circumstances. Prior exposure of the synaptosomes to TTX, thus blocking the sodium channels, also blocked the light signal evoked by veratridine. This suggested that the release of ATP in response to veratridine in the absence of TTX was due to membrane depolarization following the opening of sodium channels. A record of one such experiment by Tom White is shown in a previous publication ([25] Fig. 16b).\nHypoxia The potency of hypoxia in evoking ATP release from heart cells and erythrocytes has been demonstrated [4, 24]. In the case of human erythrocyte, ATP release in response to hypoxia was very effectively blocked by the nucleoside transport blocker nitrobenzylthioinosine (NBTI). The nucleoside transporter has now been cloned [26] and is thought to be a member of a group of unclassified integral membrane proteins (see review by [8]). The connection between blockade of a nucleoside transporter and inhibition of nucleotide release (ATP) in response to hypoxia is unclear.Some headway has been made toward unraveling oxygen-sensing mechanisms in certain oxygen-sensitive cells. In 1988 Lopez-Barneo et al. [36], studying chemoreceptive carotid body cells, demonstrated that lowered oxygen tension inhibited potassium channels, resulting in cell depolarization. The presence of oxygen-sensing potassium channels has been reported in many different cell types, for example, in pulmonary vascular myocytes, which constrict due to membrane depolarization following hypoxia [44] and in rat pheochromocytoma cells [12]. Does ATP release associated with membrane depolarization, as reported above [63], occur in these cells? Perhaps release of ATP from heart cells and erythrocytes in response to hypoxia involves the presence of oxygen-sensitive potassium channels, the sequence of events being hypoxia, leading to inhibition of oxygen-sensitive channels, followed by membrane depolarization and ATP release. To date, these potassium channels have not been identified in heart cells or erythrocytes.\nMechanical distortion A broad spectrum of mechanical distortion exists, ranging from gross distortion of whole cells to the impact of suction electrodes in the patch-clamp techniques for recording of currents passing through single ion channels in the membrane. There is little doubt that mechanical distortion of the whole cell membrane can lead to the release of ATP into the immediate environment. Sprague et al. [59] showed a close relationship between the extent of erythrocyte deformation and ATP release. Increases in shear stress applied to endothelial cells have been shown to liberate \u201cvasoactive\u201d substances from vascular endothelial cells [10, 45, 47, 54]. ATP release into the pulmonary vascular bed occurs within seconds after an increase in flow rate [30]. Grygoczyk and Hanrahan [27] clearly showed that minimum mechanical disturbance, such as changing the solution bathing the preparation, enhanced ATP release within seconds. Schwiebert [55] emphasized that the degree of suction pressure in patch-clamp techniques may determine the rate of ATP appearance. A further factor complicating physical distortion of the cell membrane is the possible involvement of the underlying cytoskeletal system. Frequency of membrane channel opening is influenced by exposure to cytochalasin B, which disrupts the cytoskeleton [62]. A comprehensive review by Morris [39] outlining the widespread occurrence of mechanosensitive ion channels in a variety of tissues is highly recommended.\nPerspectives\nTowards the search for an ATP channel in the plasma membrane Braunstein et al. [7], investigating the mechanism of cell volume regulation, showed that the cystic fibrosis transmembrane conductance regulator (CFTR) restored cell volume (following hypotonic challenge) by a mechanism involving ATP release. This release could be blocked by DIDS and gadolinium, suggesting that ATP might be released through a channel separate from the CFTR. Their working hypothesis was that cell volume is sensed and transduced by an ABC transporter (ATP binding cassette) enhancing ATP release through activation of a separate but associated ATP channel. The earlier observation [4] that hypoxic-induced ATP release from erythrocyte was associated with \u201cband 3\u201d membrane prompted the speculation that band 3 could be equated with the CFTR protein moiety. However, these proteins have been cloned and sequenced and are quite dissimilar in character [34, 52].ATP passage across the inner mitochondrial membrane has long been known. Thinnes et al. [60] have identified a voltage\u2013dependent anion channel (VDAC, \u201cporin\u201d) which acts as a conduit for newly synthesized ATP in and out of the mitochondrion. VDAC is present in a multitude of tissue types, including human skeletal muscle [51] (Table\u00a01).\nExercise and circulating purines An early example of the appearance of purines in the circulating blood in response to graduated whole body exercise was obtained by Peter Parkinson [43]. It is known that a large proportion of circulating ATP is degraded in passage through the lung [15], perhaps, along with widespread ecto-ATPase activity, minimizing the effect of widespread vasodilatation and offsetting unwanted hypotension and collapse during exercise! The products of purine degradation are probably distributed generally, especially to tissues that cannot synthesize the purine ring moiety (see \u201cDiscussion,\u201d [11]). This phenomenon could appropriately be included in the concept of \u201cATP expansion\u201d proposed by Abraham et al. [2]. This begs the question: is one of the beneficial effects of an exercise regime due to the intermittent \u201cinternal transfusion\u201d of purines, including ATP, from exercising muscle to sustain \u201cATP expansion\u201d in the tissues?\nUse of ATP in cancer therapy Encouraging results have been obtained in the field of cancer therapy using extracellular ATP. A direct effect of ATP on tumor cells was demonstrated by Rapaport and coworkers [49, 50]. It was shown in various human cell lines that an increase in cellular ATP pools resulted in inhibition of DNA replication in the synthesis phase of the cell cycle, with subsequent arrest of cell growth. Inhibition of cancer growth in Ehrlich tumor cells by extracellular ATP has also been found [14, 35]. The dramatic weight loss seen in some forms of advanced cancer (cachexia) can be slowed with a regime of intravenous ATP [3]. As shown in Fig.\u00a05 maintenance of muscle strength was also a benefit.\nFig.\u00a05Changes in body weight and muscle strength in advanced non-small-cell lung cancer after ATP administration. a Weight change. b Elbow flexor performance (top); knee extensor performance (bottom). Reproduced from Agteresch et al. [3], with permission\nProtective effect of ATP against ischemia Improved recovery of ischemic liver, kidney, and myocardial tissues after perfusion of ATP-MgCl has been shown [9, 37, 41, 58].\nProtective effect of ATP in radiation therapy Impairment of wound healing following radiation therapy has been shown to be ameliorated with use of ATP-MgCl perfusion regimes [57].Although little understood, these protective effects may be associated with general elevation of tissue ATP levels, restoring the total \u201cenergy pool\u201d of the body (see \u201cATP expansion\u201d in review by [2]).\nAging and extracellular ATP levels The release of ATP from vascular endothelial cells in rat caudal artery is reduced with advancing age. Both spontaneous ATP release and release induced by methoxamine, an \u03b1-adrenoceptor agonist, were reduced in aged vascular endothelium [28]. It is interesting that enhanced release of adenyl purines from these cells was invoked by hypotension induced by exercise [29]. A clear example of the effect of age on the ATP content of human erythrocytes is shown in Table\u00a01 [46].Aging has also been shown to diminish phosphorus metabolites in left ventricular hypertrophy as measured by 31P magnetic resonance spectroscopy [42].If aging is associated with lower tissue levels of ATP, and applied exogenous ATP can replenish these levels (\u201cATP expansion\u201d see above), could aging reversal be brought about by increasing the exogenous ATP levels? At this juncture it should be noted that extracellular ATP can act as a powerful trigger of programmed cell death (apoptosis) in endothelial cells [13, 53]. Perhaps the phenomenon of apoptosis is designed to prevent the Malthusian nightmare of universal immortality!\nEpilogos\nAnd so what began as a project for the assessment of quantal release of acetylcholine from motor nerve terminals1 resulted in the finding that ATP was released from active skeletal muscle, leading to the establishment of a significant role for ATP in the hyperemia of exercising skeletal and heart muscle, as well as an appreciation of the widespread effects of extracellular ATP.\nIntrinsic to the concept of serendipity in science is the element of surprise, which, although conferring excitement to investigators, only reveals our frailty of prediction in scientific matters. Inspirata accident magis saepe quam quae speres\u2014What you do not expect happens more frequently than what you do expect (Plautus).","keyphrases":["serendipity","hypoxia","atp release from cells","extracellular atp","exercise hyperemia"],"prmu":["P","P","P","P","R"]}
{"id":"Biogerontology-4-1-2174522","title":"Energy metabolism, altered proteins, sirtuins and ageing: converging mechanisms?\n","text":"The predominant molecular symptom of ageing is the accumulation of altered gene products. Nutritional studies show that ageing in animals can be significantly influenced by dietary restriction. Genetics has revealed that ageing may be controlled by changes in intracellular NAD\/NADH ratio regulating sirtuin activity. Physiological and other approaches indicate that mitochondria may also regulate ageing. A mechanism is proposed which links diet, exercise and mitochondria-dependent changes in NAD\/NADH ratio to intracellular generation of altered proteins. It is suggested that ad libitum feeding conditions decrease NAD availability which also decreases metabolism of the triose phosphate glycolytic intermediates, glyceraldehyde-3-phosphate and dihydroxyacetone-phosphate, which can spontaneously decompose into methylglyoxal (MG). MG is a highly toxic glycating agent and a major source of protein advanced-glycosylation end-products (AGEs). MG and AGEs can induce mitochondrial dysfunction and formation of reactive oxygen species (ROS), as well as affect gene expression and intracellular signalling. In dietary restriction\u2013induced fasting, NADH would be oxidised and NAD regenerated via mitochondrial action. This would not only activate sirtuins and extend lifespan but also suppress MG formation. This proposal can also explain the apparent paradox whereby increased aerobic activity suppresses formation of glycoxidized proteins and extends lifespan. Variation in mitochondrial DNA composition and consequent mutation rate, arising from dietary-controlled differences in DNA precursor ratios, could also contribute to tissue differences in age-related mitochondrial dysfunction.\nNAD and life-span\nGenetic studies using a range of organisms have indicated that enzymes called sirtuins are linked to the control of ageing and life-span (Longo and Kennedy 2006; Leibiger and Berggren 2006; Lin and Guarente 2003). Sirtuins catalyse NAD-dependent deacetylation of histones (and other proteins) with the concomitant release of nicotimanide and O-acetyl-ADP-ribose (Howitz et\u00a0al. 2003). Other studies have suggested that metabolism of the redox couple NAD\/NADH provides a link between sirtuin activity and the control of cell senescence and organism life-span (Denu 2003, 2007; Belenky et\u00a0al. 2007; Bordone and Guarente 2005): NAD-dependent protein deacetylation helps maintain the juvenile phenotype, whereas inhibition of deacetylation activity by NADH or nicotinamide, or by NAD unavailability, promote the onset of cellular aging and decrease organism lifespan.\nAgeing, dietary restriction and NAD\nAgeing can be delayed in various organisms by dietary restriction (DR) induced by a permanent decrease in calorie intake (called caloric restriction\u2014CR). Recent observations have shown that an intermittent feeding (IF) protocol, which need not involve any overall reduction in calorie intake, can also delay ageing (Martin et\u00a0al. 2006; Masternak et\u00a0al. 2005; Mattson and Wan 2005). The mechanisms by which DR delays ageing and increases life-span are far from completely understood (Sinclair 2005). It is likely, however, that both CR and IF promote similar effects on the frequency of glycolysis and subsequent fasting periods (Hipkiss 2006a and 2007), i.e. glycolysis would be discontinuous, only operating post-prandialy. In contrast, glycolysis would be almost continuous under ad libitum (AL) feeding conditions. It is suggested that during the periods of fasting (induced by either CR or IF) the NAD\/NADH ratio would differ from that prevailing in the AL case where fasting would be unlikely or negligible. In the AL condition, continuous glycolytic throughput would tend to provoke an accumulation of NADH and lower NAD availability, whereas the CR- and IF-induced fasting would decrease glycolytic NAD demand and increase NADH oxidation and NAD regeneration.\nAgeing and accumulation of altered proteins\nAt the biochemical level ageing is characterized by the accumulation of altered protein molecules. The changes in protein structure result from intrinsic polypeptide instability as well as the actions of deleterious endogenous and exogenous agents (see Hipkiss 2006b; Schoneich 2006 and refs. therein). As yet it is unclear how changes in NAD metabolism might induce generation of altered proteins which characterise the aged phenotype.\nProtein glycation and ageing\nFormation of protein advanced glycation end-products (AGEs) is an important consequence of ageing and is increased particularly under conditions of uncontrolled glucose metabolism (e.g. hyperglycaemia) (see Ahmed and Thornally 2007; Thornalley 2007 and refs. therein for recent reviews). Protein AGEs can themselves induce inflammatory conditions and provoke production of reactive oxygen species (ROS) which can further compromise cell function. Indeed recent studies have shown that decreasing dietary AGE intake preserves defence functions against oxidative stress and decreases tissue damage in humans, and extends lifespan in mice, while increasing dietary AGE intake is correspondingly deleterious and accelerates ageing and decreases life-span (Cai et\u00a0al. 2007; Uribarri et\u00a0al. 2007a, b). Hence it is at least conceivable that decreasing metabolically-generated protein AGEs could help decrease the overall AGE load and could have beneficial effects by suppressing ageing and extending lifespan.\nNAD and accumulation of methylglyoxal, an endogenous glycating agent\nNAD is essential for the metabolism of the glycolytic intermediate glyceraldehyde-3-phosphate (G3P) via the action of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), generating 1,3-diphosphoglycerate (1,3DPG) and NADH as products. It is argued above that in the AL condition, glycolysis would be continuous, which would tend to lower NAD levels and raise NADH levels. This would occur especially should mitochondrial-mediated NADH re-oxidation to NAD be correspondingly lowered to compensate for the extra ATP synthesised via glycolysis, assuming cellular ATP demand remains unchanged. Limitation of NAD availability would lower GAPDH activity and promote an accumulation of G3P. The immediate precursor of G3P is dihydroxyacetone phosphate (DHAP); both of these trioses can glycate proteins. More importantly, however, is the fact that both G3P and DHAP can spontaneously decompose into methylglyoxal (MG), a highly toxic and very reactive glycating agent. It is likely therefore that changes in NAD availability could strongly influence MG production.\nIt has previously been proposed that differences in glycolytic frequency could help explain why dietary restriction delays cellular and organism aging, possibly due to decreased MG generation during DR conditions (Hipkiss 2006a). MG is a highly active glycating agent which is thought to be responsible for the increased protein\/lipid glycation detected during hyperglycaemic conditions and for much of the protein\/lipid glycation associated with diabetic complications (Ahmed and Thornalley 2007; Thornalley 2007). Although MG is a normal cellular constituent, its excessive production is deleterious (plasma MG levels are raised to around 800\u00a0nmol\/l in young diabetics compared to about 400\u00a0nmol\/l in young non-diabetic subjects, (Han et\u00a0al. 2007)). Importantly, MG can induce many of the deleterious physiological and biochemical changes characteristic of the aged phenotype, including increased ROS generation, mitochondrial dysfunction, apoptosis and inhibition of cell division (see Hipkiss 2006a and refs therein).\nA number of studies, some very recent, reinforce the notion that changes in cellular MG content are important determinants of the formation of altered protein that characterise senescence (Gomes et\u00a0al. 2006). Even at non-toxic concentrations, MG can influence cell proliferation by forming adducts with growth factor receptors (Cantero et\u00a0al. 2007). MG can also inhibit the activity of GAPDH (Lee et\u00a0al. 2005), causing triose phosphate accumulation and thereby increasing MG generation, and so inducing a highly deleterious cycle. MG can induce apoptosis (Nicolay et\u00a0al. 2006) and also affect gene expression and signal transduction, at least in cultured cells (Du et\u00a0al. 2003; Yao et\u00a0al. 2006; Ramasamy et\u00a0al. 2006). Two studies in Drosophila have shown that mutation in triosephosphate isomerase (the enzyme which converts DHAP into G3P, preceding GAPDH in the glycolytic pathway, and which is known to undergo age-related post-synthetic modification in\u00a0vivo (Gracy et\u00a0al. 1990)) is highly deleterious, causing paralysis, neurodegeneration and decreases life-span (Gnerer et\u00a0al. 2006; Celotto et\u00a0al. 2006), possibly because of MG accumulation. Human studies have shown that a deficiency in triosephosphate isomerase activity, causes increases in the levels of both DHAP (up to 20-fold (Schneider 2000)) and MG (Ahmed et\u00a0al. 2003), and induces neuromuscular degeneration and early death (Schneider et\u00a0al. 1965; Valentine 1966). Other studies have shown that MG induces apoptosis in neutrophils (Gawlowski et\u00a0al. 2007), inhibits extracellular matrix remodelling (Chong et\u00a0al. 2007) and can interfere with the stress response (Oya-Ito et al. 2006) by suppressing NF-kappaB-responsive gene activation (Laga et\u00a0al. 2007).\nHence it is reasonable to suggest that any increased MG generated in AL fed animals, compared to animals subjected to CR or IF, could make a significant contribution to cellular dysfunction. During the fasting periods in DR animals, NADH generated during glycolysis would be oxidized mitochondrially for ATP production, and NAD would be regenerated thereby allowing continued G3P metabolism, and preventing triose phosphate accumulation and consequently suppressing MG generation. This condition would decrease MG-induced macromolecular glycoxidation, mitochondrial damage, dysfunctional signalling and gene expression, as described above. Such a scenario is consistent with the findings that raising NAD levels, or lowering NADH levels by increasing its oxidation, also promote sirtuin activation, with concomitant beneficial effects on cell survival etc. Table\u00a01 illustrates the interrelationship and overlap between sirtuin regulation, generation of altered proteins and mitochondrial activity, exerted by metabolic effects on NAD and NADH levels.\nTable\u00a01Predicted effects of aerobic exercise and fasting induced by caloric restriction or intermittent feeding, on NAD and NADH levels, methylglyoxal (MG) levels, mitochondrial (mito) activity and sirtuin activityConditionsNADNADH MGMito activitySirtuin activityAd libitum fedLowHighHigh LoweredLoweredFastingHighLowLowIncreasedIncreasedAerobic exercise HighLowLowIncreasedIncreasedIncreased MG levels are partly responsible for the increased generation of altered proteins that accompanies ageing\nAny situation such as fasting which maintains NAD levels, either via regeneration from NADH, or by synthesis de novo or via a scavenging pathway, would facilitate metabolism of the MG precursors G3P and DHAP, and so decrease the incidence of MG-induced macromolecular damage. The increase in free-radical-mediated damage which occurs during AL feeding, compared to the CR and IF conditions, might occur as a result of not only MG-induced generation of ROS following its reaction with proteins etc., but also via plasma membrane-mediated NAD(P)H-oxidase activity. Furthermore, because less ATP is required from mitochondrial function due to continuous ATP synthesis via glycolysis in the AL-fed state, the decreased supply of electrons (as acetyl-CoA or from NADH) to the electron transport chain would tend to produce more incompletely reduced oxygen moieties i.e. oxygen free-radicals. Any increased intra-mitochondrial ROS production could also increase the probability of mitochondrial dysfunction.\nProtection against MG is afforded by the glyoxalase system which consists of two enzymes; glyoxalase I (GLX I), which uses glutathione to convert MG to a D-lactoyl-glutathione, and glyoxalase II (GLX II), which completes the detoxification by generating D-lactate and reduced glutathione. Over-expression of GLX I can inhibit formation of hyperglycaemia-induced AGEs (Shinohara et\u00a0al. 1998), while a deficiency in GLX I in humans is associated with increased protein glycation (Miyata et\u00a0al. 2001). GLX II activity may be rate-limiting in MG detoxification; GLX II over-expression is protective against MG-induced cell death, whilst its deficiency promotes MG-induced cell death (Xu and Chen 2006). It is also interesting that tumour necrosis factor can induce phosphorylation of GLX I which also results in substantial increase in cellular MG (van Herreweghe et\u00a0al. 2002).\nTissue differences in ageing susceptibility\nTissues appear to age at different rates as shown by the varied incidence of dysfunctional mitochondria between tissues in the same organism. Variation in tissue susceptibility to MG may reside partly in differing levels of the glyoxalase system together with those molecules (glutathione, polyamines, carnosine, creatine, pyridoxamine) which normally exert protective carbonyl scavenging activity towards glycating agents such as MG (Hipkiss 2005; de Arriba et\u00a0al. 2006).\nDietary-induced effects on metabolism could conceivably also affect mitochondrial DNA composition and hence mitochondrial protein structure and function. It has been found that mitochondrial DNA mutation rate may vary up to three-fold according to the relative concentrations of the four deoxyribonucleoside triphosphates in the nucleotide pool (Song et\u00a0al. 2005; Mathews and Song 2007). It is possible that dietary changes could affect the composition of the nucleotide pool and thereby affect mitochondrial DNA composition during its synthesis. Pool composition could vary between tissues, and any consequent differences in mitochondrial DNA mutation rate would contribute to tissue-specific age-related mitochondrial change. Thus mitochondrial dysfunction could be either a cause or a consequence of ageing (Hipkiss 2003), depending on the prevailing circumstances.\nThe beneficial effects of functional mitochondria on NAD regeneration\nThe recent observations (i) by Belenky et\u00a0al. (2007) showing that life-span extension in yeast is dependent upon NAD synthesis, (ii) that efficient mitochondrial function was necessary for maximal longevity in yeast (Piper et\u00a0al. 2006), and (iii) that mitochondrial uncoupling, which increases NADH oxidation, decreases telomere damage and delays senescence in cultured human fibroblasts (Passos et\u00a0al. 2007), are observations entirely consistent with the above proposal. The proposed beneficial effects of NADH oxidation to regenerate NAD via mitochondrial function would also help explain how aerobic exercise may delay development of the aged phenotype including production of altered proteins, as well as resolve the apparent paradox that increased oxygen utilization suppresses age-related change. The efficient regeneration of NAD via effective mitochondrial function is also consistent with mitochondrial ageing theories which postulate that mitochondrial dysfunction is key to the onset of ageing.\nAlso consistent with the present proposal are the very recent findings of Smith et\u00a0al. (2007) who concluded that, in the yeast Saccharomyces cerevisiae at least, elevated respiration is an important determinant of chronological longevity. They observed that growth on non-fermentable carbon sources, which forced the cells to employ respiration exclusively, extended lifespan, but which caloric restriction did not further enhance. This again illustrates, simplistically perhaps, the potential anti-ageing functions of aerobic respiration and the deleterious effects of glycolysis, both possibly mediated via changes NAD and NADH levels, which in turn regulate MG generation. Controversially, however, these authors also found that caloric restriction-mediated lifespan extension occurred independently of sirtuin activity in Saccharomyces cerevisiae.\nOther functions induced by DR\nAgeing is a complex phenomenon. It is likely that the rate-limiting event which increases cellular and hence organism vulnerability to death may vary according to circumstances. For example anti-oxidant functions may not be limiting in conditions where oxidative stress is not involved. There are an increasing number of findings suggesting that proteolytic dysfunction involving either proteasomes or autophagy cause altered protein to accumulate and compromise cell survival and which can be affected by dietary restriction (Bergamini et\u00a0al. 2003). Conversely activation of autophagy by inhibiting the target of rapamycin (TOR) signalling pathway can increase lifespan, at least in yeast (Bonawitz et\u00a0al. 2007) and a nematode worm (Henderson et\u00a0al. 2006). The recent observation that the sirtuin-like activity, histone deacetylase 6 (HDAC6), may provide a mechanistic link between the autophagic and ubiquitin-proteasome proteolytic systems in Drosophila (Pandey et\u00a0al. 2007), and the observation that up-regulation of neuronal sirtuin1 activity elevates the activity of the \u03b1-protease and prevents accumulation of the amyloid peptide (Qin et\u00a0al. 2006), support the idea that both formation and degradation of aberrant proteins are important for control of ageing and related disorders.\nConclusion\nIt is proposed that dietary-induced changes in NAD and NADH levels, as revealed by their regulation of sirtuin activity, may also control the concentration of deleterious glycolytic intermediates G3P and DHAP, and thereby also control formation of MG and generation of protein AGEs. The accumulation of MG and protein AGEs may compromise tissue function including mitochondrial activity and thereby contribute to organism ageing. Conversely, conditions that stimulate mitochondrial function will help regenerate NAD, maintain sirtuin activity and decrease formation of protein AGEs, intra- and extra-mitochondrial ROS can thereby delay ageing onset.","keyphrases":["altered proteins","ageing","dietary restriction","nad","nadh","methylglyoxal","glycolysis","deacetylases"],"prmu":["P","P","P","P","P","P","P","P"]}
{"id":"Behav_Genet-4-1-2257998","title":"Power Calculations Using Exact Data Simulation: A Useful Tool for Genetic Study Designs\n","text":"Statistical power calculations constitute an essential first step in the planning of scientific studies. If sufficient summary statistics are available, power calculations are in principle straightforward and computationally light. In designs, which comprise distinct groups (e.g., MZ & DZ twins), sufficient statistics can be calculated within each group, and analyzed in a multi-group model. However, when the number of possible groups is prohibitively large (say, in the hundreds), power calculations on the basis of the summary statistics become impractical. In that case, researchers may resort to Monte Carlo based power studies, which involve the simulation of hundreds or thousands of replicate samples for each specified set of population parameters. Here we present exact data simulation as a third method of power calculation. Exact data simulation involves a transformation of raw data so that the data fit the hypothesized model exactly. As in power calculation with summary statistics, exact data simulation is computationally light, while the number of groups in the analysis has little bearing on the practicality of the method. The method is applied to three genetic designs for illustrative purposes.\nIntroduction\nThe importance of statistical power in (behavior) genetic analyses is evident in the number of articles devoted to power calculations. Power has been studied in virtually all research designs, ranging from the classical twin design (Martin et al. 1978; Neale et al. 1994), to extended family designs (e.g., Heath et al. 1985; Heath and Eaves 1985; Posthuma and Boomsma 2000), to sibpair and family linkage and association designs, either in- or excluding gene by environment interaction (Abecasis et al. 2000a, b; Boomsma and Dolan 1998; Dolan et al. 1999; Fulker and Cherny 1996; Purcell 2002; Purcell and Sham 2002; Sham et al. 2000; Sham and Hewitt 1999; Sham et al. 2002; Van den Oord 1999). For a wide range of genetic designs, the Genetic Power Calculator1 (Purcell et al. 2003) can be used to calculate power. However, for customized designs and specific research questions, researchers may have to resort to their own procedure to carry out power calculations.\nPower calculation based on the likelihood with the general Pearson\u2013Nyman statistical decision theory takes two forms. First, the non-centrality parameter \u03bb of the non-null \u03c72-distribution can be calculated in the analysis of exact sufficient statistics (e.g., Dolan et al. 1999). If the distribution of the data is multivariate normal, the expected variance covariance matrix \u03a3 and the means vector \u03bc are sufficient statistics, as they define the likelihood of the data up to an arbitrary constant (Azzelini 1996). Second, when \u03a3 and \u03bc are not sufficient statistics, the non-centrality parameter \u03bb of the non-null \u03c72-distribution can be estimated on the basis of the analysis of simulated data using Monte Carlo simulation methods (e.g., Fulker and Cherny 1996; Abecasis et\u00a0al. 2002a, b; Purcell 2002; van den Oord 1999). The latter is computationally intensive, but does not require the presence of sufficient summary statistics, whereas the former is computationally light, but does require sufficient summary statistics.\nThe aim of the present note is to discuss a third method of power calculation, which we refer to as exact data simulation. This method is suitable when data are multivariate normal, and sufficient summary statistics are in principle available, but the number of possible groups is prohibitively large (say, in the hundreds). The large number of distinct groups renders power calculations on the basis of the summary statistics impractical. Usually, researchers resort to Monte Carlo based power studies under such circumstances. However, exact data simulation, in combination with the definition variable facilities in packages like Mplus (fourth edition, Muth\u00e9n and Muth\u00e9n 1998\u20131997) or the freely available Mx program2 (Neale et al. 2003), is also applicable, and is more efficient than raw data simulation. Exact data simulation was used by Dolan et al. (2005) to evaluate the effects of missing data on the power in structural equation modeling, and by Van der Sluis et al. (Under revision) to evaluate the power to detect gene by environment interaction in sib-pair association studies. Although the technique of exact simulation is in itself not new (Bollen and Stine provided the basics in 1993, and the technique of exact data simulation has recently been integrated as a distinct function in the freely available R-program3), we wish to bring it to the attention of geneticists since this method of calculating power has general value in the field of genetic modeling. Below, we shortly recapitulate the basics of power analysis, and then outline the procedure of exact simulation, which may be implemented readily (we use the freely available R program). The method is illustrated in three genetic designs. Although we confine ourselves in this paper to illustrations in the context of genetic designs, we stress that this form of simulation can be used for power calculations in a wide range of other designs such as random effects models, growth curve and simplex models, and structural models.\nPower calculation\nThe concept of power is closely related to the two types of statistical errors: the Type I error (i.e., the probability of rejecting a true hypothesis, \u03b1), and the Type II error (i.e., the probability of accepting a false hypothesis, \u03b2). Power is defined as 1\u00a0\u2212\u00a0\u03b2, i.e., the probability of rejecting a false hypothesis, or the probability of not making a Type II error. The basic aim of a power study is to determine the sample size N, which is required to achieve adequate power, given chosen \u03b1 and a particular effect size.\nFor example, suppose that we want to fit a classical univariate ACE twin-model (see Fig.\u00a01), and we expect that additive genetic effects (a) account for 40% of the phenotypic variance, besides effects of shared (c) and unique (e) environment. We denote this model 1, or hypothesis 1, H1. Under model 1, four parameters are estimated: the path coefficient for the additive genetic effects A (a), the path coefficient for the shared environment C (c), the path coefficient for the unique environment E (e, which includes measurement error), and the means of the twins (\u03bc), which are usually set to be equal within twin pairs and across MZ and DZ twins. The parameter vector \u03b8 for this model with df1\u00a0degrees of freedom, is \u03b81\u00a0=\u00a0{a, c, e, \u03bc}, from which the covariance matrices \u03a31mz and \u03a31dz and mean vector \u03bc1 can be derived. Now consider a second model, which we denote model 0, or H0, in which the additive genetic effects are assumed to account for only 5% of the phenotypic variance. This alternative model will be characterized by parameter vector \u03b80\u00a0=\u00a0{c, e, \u03bc}. Note that in model 0, parameter a is not estimated (and thus not part of \u03b8) but fixed at a value that corresponds exactly to 5% of the phenotypic variation being explained by additive genetic effects. This model has df0\u00a0degrees of freedom, covariance matrices \u03a30mz & \u03a30dz, and mean vector \u03bc0. This alternative model H0 is nested in the null-model H1, because the parameters in \u03b80 represent a subset of the parameters in \u03b81 (e.g., Bollen 1989; Satorra and Saris 1985). The study of power is subsequently concerned with the probability of rejecting the false model H0 in favor of the true model H1, given \u03b1, the true value of parameter a (the effect size), and sample size N.\nFig.\u00a01Classical univariate ACE-twin model\nTo calculate power, we adopt the method of Satorra and Saris (1985, see also Saris and Satorra 1993), which is based on the normal theory log-likelihood ratio test statistic T. In a single group, T is calculated as follows: where N is the sample size, p denotes the number of variables in the analysis, and \u03a3 and \u03bc, and S and m represent the theoretical and observed variance covariance matrix and the means vector, respectively. Given that the assumptions of normal theory maximum likelihood are met (e.g., multivariate normality, and a large sample of independently and identically distributed cases), and under the assumption that \u03a3 and \u03bc represent the true model (\u03a31 and \u03bc1), the test statistic T follows a \u03c72 distribution with df1\u00a0degrees of freedom, i.e., T\u00a0\u223c\u00a0\u03c72(df1) (Azzeline 1996; Bollen 1989). If \u03a3 and \u03bc do not represent the true model but the alternative model (\u03a30 and \u03bc0), and given regularity conditions are satisfied (practically amounting to multivariate normality, limited misfit and large sample size N), the test statistic T follows a non-central \u03c72 distribution with df0\u00a0degrees of freedom and non-centrality parameter \u03bb, i.e., T\u00a0\u223c\u00a0\u03c72(df0, \u03bb), where \u03bb\u00a0>\u00a00.\nGiven the significance level of the test \u03b1, and the difference in degrees of freedom between the true model and the alternative model, df1\u00a0\u2212\u00a0df0, the criterion level c\u03b1 can be obtained from a \u03c72 table. If the test statistic T exceeds this criterion level, i.e., T\u00a0>\u00a0c\u03b1, then the alternative model is rejected in favor of the true model (i.e., the fit of the alternative model to the data is significantly worse than the fit of the true model). The aim of power studies is to determine the probability of observing T\u00a0>\u00a0c\u03b1, i.e., P(\u03c72(df0, \u03bb)\u00a0>\u00a0c\u03b1), given \u03a30, \u03bc0, \u03a31, \u03bc1, N, and \u03b1, i.e., the probability of rejecting the alternative model in favor of the true model.\nThe non-centrality parameter \u03bb can be obtained by fitting the alternative model to the true \u03a31 and \u03bc1, whereby \u03bb equals the difference in the \u03c72 fit statistic of the model H1 and the \u03c72 fit statistic of the alternative model H0. That is (again in a single group), and the non-null distribution of this test statistic is \u03c72(df1\u00a0\u2212\u00a0df0,\u03bb). A variety of programs can subsequently be used to integrate the non-null distribution to obtain the power (e.g., R, Mx; see also Hewitt and Heath 1988). Note that some packages, such as the Mx program, also compute the total sample size that would be required (given the reported proportion of subjects in each group) to reject the hypothesis at various power levels.\nAs stated in the introduction, power calculations usually take on one of two forms. First, one may be in the position that all information present in the raw data can be summarized in the covariance matrix \u03a3 and the means vector \u03bc, in which case \u03a3 and \u03bc are sufficient statistics, because they define the likelihood of the data up to an arbitrary constant (Azzelini 1996). In that case, one can derive the expected population statistics \u03a3 and \u03bc for every group in the study design under H1 and H0, and base the power calculations on these summary statistics.\nThe second method of power calculation is applied when sufficient summary statistics are not available. For example, the population statistics \u03a3 and \u03bc do not summarize all information present in the raw data when the continuous data are a mixture (i.e., a convex combination of different distributions; McLachlan and Peel 2002), or when data are missing at random (MAR; Shafer and Graham 2002). In family studies, gene by environment interaction may render the summary statistics insufficient. For instance, Purcell (2002) showed how environmental moderation on the means and variances can be modeled (see Fig.\u00a02). For both MZ and DZ twins, the variance of twin i is calculated as: while the for MZ twins, covariance between twin i and twin j is calculated as: and for DZ twins as: The expected values of means for all twins are calculated as: Fig.\u00a02Univariate ACE-twin model including moderation on the variances and the means\nIf the environmental moderator is categorical or ordinal (e.g., gender or affection status), the sufficient statistics (\u03a3 and \u03bc) are available, assuming that the data are normally distributed conditional on the levels of the moderator. In that instance, the twins in a pair may be concordant with respect to the moderator (i.e., both twins score 0, or both twins score 1), or the twins may be discordant with respect to the moderator (i.e., scoring 0 and 1, respectively). For both MZ and DZ twins, \u03a3 and \u03bc can be formulated for all possible combinations, such that all information present in the raw data is summarized with 6 different variance covariance matrixes \u03a3 (3 for the MZ twins, and 3 for the DZ twins) and 3 different means vectors \u03bc (assuming no relation between zygosity and mean), as such distinguishing 6 different groups. By comparing the fit of the model including moderator effects on both the means and the variances, to a model in which the moderator only affects the means (say), one can obtain an estimate of non-centrality parameter \u03bb, from which the power to detect the effect of the moderator can be derived.\nHowever, if the moderator is continuously distributed, sufficient statistics cannot be calculated. In the absence of sufficient statistics, power calculation may be conducted by means of Monte Carlo simulation. This design implies determination of the values of the parameters of interest (e.g., based on previous studies or corresponding to realistic effect sizes), and subsequent (quasi-) random data generation according to the true model H1, with realistic sample size N. By fitting the false model H0 to the data simulated according to the true model H1, an indication of the power is obtained. However, in contrast to the situation in which sufficient summary statistics are available, parameter values are not recovered exactly when the H1 model is fitted, as the random data are the outcome of a stochastic sample process. Therefore, the difference in the \u03c72 statistic of the model H0 and the model H1 cannot be taken as an exact estimate of the non-centrality parameter \u03bb. To solve this, a large number of datasets are usually generated, and \u03bb is estimated as the mean of the difference in \u03c72 obtained in these data sets minus the number of degrees of freedom (df1\u00a0\u2212\u00a0df0). Since power studies often concern multiple parameters with multiple values, such Monte Carlo simulation studies can be prohibitively intensive. As an alternative, simulated sample sizes may be chosen very large to induce asymptotic behavior of the \u03c72 statistic. However, how large a sample size should be chosen depends on the study design in question, and very large sample sizes also render the analyses computationally intensive.\nExact data simulation\nPower calculations based on sufficient summary statistics are computationally relatively efficient to carry out. However, the actual feasibility of this type of power calculation depends on the number of distinct groups. If the number of groups is large (i.e.,\u00a0>100), it may be more convenient to carry out Monte Carlo based power calculations. We now introduce the concept of exact data simulation, which shares the virtues of the power studies based on summary statistics, but is more practicable given a large number of distinct groups.\nThe idea of exact data simulation is that data, which are randomly generated to begin with, can subsequently be transformed to fit the null-model H0 exactly. That is, first a data file is generated using a normal distribution quasi-random number generator. These data are then transformed, using a transformation proposed by Bollen and Stine (1993), so that the variance covariance matrix and means are exactly as specified under the model H0.\nAssume a total sample size of N, and k distinct groups with known probability pk. Let Y denote the Nk\u00a0\u00d7\u00a0q data matrix for group k, where Nk is N*pk (possibly rounded to the nearest integer) and q is the number of variables. Let m denote the q\u00a0\u00d7\u00a01 vector of observed means and S\u00a0=\u00a0YtY\/(N\u00a0\u2212\u00a01)\u2212mmt be the observed covariance matrix, \u03a3 the expected covariance matrix implied by model H1, and \u03bc the expected means vector implied by the model H1. Let S1\/2 and \u03a31\/2 then denote the square root factorization of the positive definite matrices S and \u03a3 such as given by a Cholesky factorization. It can then be shown that the covariance matrix and mean vector of data matrix Z, which is obtained through the following transformation of Yequals \u03a3 and \u03bc, exactly (Bollen and Stine 1993). In Eq. 7, J is a unit vector of length q, and \u2297 denotes the Kronecker product. This transformation allows one to create raw data for numerous groups that fit the null-model exactly. Consequently, when the null-model used for the generation of the data is fitted to these transformed data, all parameter values used for the simulation are recovered exactly. Let Log L0 and Log LA denote the maximum values of log-likelihood functions. The difference 2Log LA\u00a0\u2212\u00a02Log L0 equals the non-centrality parameter \u03bb.\nCompared to multi-group power calculation with summary statistics, which becomes unpractical when the number of groups is large, the practicality of exact data simulation is unaffected by the number of groups in the analysis. However, one issue does require attention. When the number of groups is large (e.g., 256, see Illustration 1 below), the probability pk that a subject belongs to a group k may be relatively small. In order to calculate the Cholesky decomposition of the observed variance covariance matrix S, the number of observations in a group Nk should equal at least q\u00a0+\u00a01 (i.e., to ensure that S is positive definite and the Cholesky decomposition is possible). There are two ways to handle this problem. First, one can choose N to be sufficiently large that all groups, even those with small probabilities, by choosing a very large overall sample size N for the simulation. Power analyses based on this very large sample size produce non-centrality parameters which can subsequently be used to calculate power for other, more realistic sample sizes. Second, one can choose a smaller overall sample size, and accept that not all possible groups will be represented in the power calculations. This choice is usually justified since very small groups (e.g., including 2 subjects out of a possible 10,000) do not contribute much to the power. However, power calculations are more precise when all groups are represented in the simulation, i.e., overall N is large. Furthermore, it is in principle possible that the presence of all groups is required for model identification.\nNote that packages like Mx and R will estimate the number of data vectors required for a power of e.g. 80%, given the proportion of subjects in each group. So while Nk\u00a0\u2265\u00a0q\u00a0+\u00a01 is required for exact simulation, Mx will return an overall sample size N, in which many groups may represented by fewer than q\u00a0+\u00a01 observations, which is in line with what one would expect to observe in research practice. Using the exact data simulation script subsequently to simulate data with the sample size advertised by Mx would not result in a power of 80% since the groups with Nk\u00a0<\u00a0q observations are not represented in the simulated data and thus do not contribute to the power.\nHaving discussed the concept of power, and the exact data simulation procedure, we will now illustrate the virtues of exact data simulation with three behavior genetics examples. We chose Mx to analyze the simulated data because of the program\u2019s inbuilt option to calculate the sample size required for different power levels given the non-centrality parameter. However, the non-centrality parameter can also be obtained through other software (e.g., LISREL, Mplus). The calculation of sample sizes required for different power levels can then be done using other programs like R. The R-scripts used to simulate the data, and the Mx scripts used to analyze the data are available in the Mx scripts library.4 A small R-script for power computations based on non-centrality parameters can be downloaded from the library as well.\nIllustration 1: multivariate ACE-model with data MCAR\nLet us consider a four-variate ACE-model with data obtained from MZ and DZ twins (no additional family members). We assume a model with one common genetic factor, one common shared environmental factor, and specifics for A, C and E for all four traits. The model is illustrated in Fig.\u00a03. Parameter values are chosen such that additive genetic influences, shared environmental influences, and non-shared environmental influences explain 50%, 30% and 20% of the total variance, respectively. Of the additive genetic variance, 60% is attributable to the common genetic factor (i.e., 30% of the total variance), and 40% to the specifics of A (i.e., 20% of the total variance). Of the shared environmental variance, 50% is attributable to the common shared environmental factor (i.e., 15% of the total variance), and 50% to the specifics of C (i.e., 15% of the total variance). Table\u00a01 contains the correlation matrices for the MZ and DZ twins in the case that data are not missing. Means for all traits are equal to zero. We consider the power to reject the alternative hypothesis that the genetic specifics for all four traits explain not 20% but only 5% of the total variance,5 i.e., one common genetic factor is (almost) sufficient to explain all genetic variance and covariance in the four traits. If there are no missing data (situation S1), then one could simply use summary statistics to obtain power information, as this is a 2-group analysis. However, suppose we want to study the influence of data missing completely at random (MCAR) on the power to reject the hypothesis that all genetic specifics are zero. Here we consider two scenarios. First, we study the case that the probability of data being MCAR is 20% for all variables (situation S2). Given that we have q\u00a0=\u00a02\u00a0\u00d7\u00a04 observations per family, this kind of missingness could yield 28\u00a0\u2212\u00a01\u00a0=\u00a0255 possible data patterns, i.e., 255 different groups (we discard the group in which all data are missing). In that case, power calculations using summary statistics are impractical, whereas exact data simulation is feasible. Note that some data patterns are rather unlikely, e.g., the probability of observing a valid observation for the first trait of the first twin only, while all other observations in the family are missing, is .8*(.27)\u00a0=\u00a01.024\u221205. Remember that the Cholesky decomposition cannot be calculated if the number of observations in a group Nk does not equal at least q\u00a0+\u00a01, so the simulated N needs to be very large if one wants all data patterns to be present in the simulated data set (about (q\u00a0+\u00a01)\/1.024\u221205\u00a0\u2248\u00a0900,000). Yet, as very rare observations will hardly contribute to the power (5 of the 900,000 cases in the present example), one can just as well adopt a smaller sample size, and accept the fact that some groups (i.e., patterns of observations) will not be represented, and calculate power given the most likely patterns of observations. \nFig.\u00a03Four-variate ACE-model with common factors for additive genetic and shared environmental effects, and specifics for A, C and ETable\u00a01Illustration 1: Four-variate cross-trait-cross-twin MZ correlations (below diagonal) and DZ correlation (above diagonal) for data without missingnessTwin 1Twin 2Trait1Trait2Trait3Trait4Trait1Trait2Trait3Trait4Twin 1Trait11.00.45.45.45.55.30.30.30Trait2.451.00.45.45.30.55.30.30Trait3.45.451.00.45.30.30.55.30Trait4.45.45.451.00.30.30.30.55Twin 2Trait1.80.45.45.451.00.45.45.45Trait2.45.80.45.45.451.00.45.45Trait3.45.45.80.45.45.451.00.45Trait4.45.45.45.80.45.45.451.00Note: Sample size N is not reported; as the simulations are exact, this correlation matrix should result independent of the sample size chosen for the simulations when data are not missing\nSecond, we study the case where the probability of data being MCAR is 40% for the first two variables, and zero for the second two (situation S3). This could be a realistic scenario in practice, for example, when a questionnaire study that measures two traits is extended during the data collection to include two additional traits, or when data from different studies are combined (one study in which all four traits were measured, while another study only included measurements of two traits for, e.g., economical reasons). Given that we have q\u00a0=\u00a02\u00a0\u00d7\u00a04 observations per family of which only four variables show missingness, this would yield 24\u00a0=\u00a016 possible data patterns, i.e., 16 different groups. With only 16 groups, power calculations using summary statistics would be feasible. However, one efficiently setup exact data simulation script can handle both this simple pattern of missingness, and more complex.\nFor the simulations we chose an overall sample size of 50,000 families (1\/3 MZ, 2\/3 DZ), which means that for situations S1 and S3, all groups (data patterns) are represented (50,000 and 49,999 cases simulated, respectively), while for situation S2, only the 163 most likely groups of the possible 255 are represented (49,999 cases simulated).\nThe three simulated data sets were subsequently analyzed in Mx. In the Mx-script, we specify different groups for the MZ and DZ twins. Because we use full information maximum likelihood to accommodate the missingness, we do not need to specify different groups for all possible missing data patterns. The Mx command \u2018option power\u2019 (\u03b1\u00a0=\u00a0.05, df\u00a0=\u00a04) was used to obtain an estimation of the total sample size that would be required for a power of 80%, given the current proportions of subjects in each group.\nWe find that for situation S1 (no missingness), 302 families are required for 80% power to reject the alternative hypothesis that the genetic specifics for all four traits explain 5% rather than 20% of the variance each, while for situations S2 (20% missingness for all variables) and S3 (40% missingness for only variable 1 and 2) the number of families required to obtain 80% power is estimated at 494 and 474, respectively. We hasten to note that these results are not informative for the case that data are missing at random (MAR), rather than MCAR (see Schafer and Graham 2002, for a comprehensive review on missingness and statistical procedures for handling missing data).\nThese power calculations took about 2\u00a0min for each situation S. Within the Monte Carlo framework, acquisition of similar power results would take at least T times as long for each situation S (where T is the number of replications one chooses to do). Given that the time required to write the data simulation script is equal for Monte Carlo simulation and exact simulation, it is clear that exact simulation saves a lot of time.\nIllustration 2: gene by environment interaction with latent G and measured, categorical E\nGene by Environment (G \u00d7 E) interaction is an important issue. From the perspective of the power study, a problem with the presence of G \u00d7 E when the E is continuously distributed is that it renders single summary statistics insufficient; in the presence of G \u00d7 E, (co)variances and means depend on the level of the environmental moderator, as we have seen in Eqs. 3\u20136. Purcell (2002) showed how G \u00d7 E on the means and variances can be modeled if G is latent, and E is measured.\nIn power calculations in the G \u00d7 E context, one can adopt a multi-group design, if the environmental moderator is categorical. For example, consider a classical ACE-twin design. If the environmental moderator is dichotomous (e.g., males versus females, young versus old, smoking versus non-smoking), the sample consisting of MZ and DZ twin pairs can be split up into twin pairs who are concordant with respect to the moderator (e.g., both twins do, or do not, smoke), and twin pairs who are discordant with respect to the moderator (only one of the twins in a pair smokes). With a dichotomous moderator and only two subjects per family, power calculations using summary statistics are feasible as there are only three distinguishable groups (not accounting for the distinction between MZ and DZ twins). However, suppose that you have measured an environmental moderator with 4 levels (coded 0, 1, 2, 3) in twin-pairs and their parents. With four persons per family and four possible moderator levels, there are 44\u00a0=\u00a0256 possible family configurations. In that case, multi-group analysis with summary statistics is impractical, and exact data simulation may be used instead.\nFor this illustration, parameters a, c, and e were all set to 1, such that the total variance equaled 3 (excluding moderating and main effects). The moderator, which was assumed independent of genotype in this illustration, was coded 0 to 3, such that the group with 0 on the moderator can be considered the baseline condition. The probability for all moderation levels was set to .25, and moderation levels were modeled as independent across family members (i.e., the probability for each family member\u2019s moderation level was independent of the moderation levels of the other family members). Moderation in C, E and the means was fixed to 0, but the regression weight of the moderator was set to .2 for the additive genetic effects, such that the moderator explained 20% of the variance in the total population (i.e., 0%, 13%, 24%, and 34% of the variance, respectively, depending on the level of the moderator). The model is illustrated in Fig.\u00a04. For the simulation, we chose an overall sample size N of 10,000 (1\/3 MZ and 2\/3 DZ twins). Fig.\u00a04Univariate ACE-model for parents and twin-offspring, including moderation on the variances and the means\nGiven these simulated data, we want to estimate the power to reject the alternative hypothesis that all moderator effects on the variances are zero (i.e., no G \u00d7 E, or C \u00d7 E, or E \u00d7 E). In practice, one would fix all regression weights concerning the moderating effects on the variances (\u03b2a, \u03b2c, and \u03b2e) to zero at once, resulting in a test with 3\u00a0degrees of freedom.\nThe data were analyzed in Mx: different groups were specified for the MZ and DZ twins, and the moderator featured as a so-called definition variable. The \u2018option power\u2019 command (\u03b1\u00a0=\u00a0.05, df\u00a0=\u00a03) was again used to obtain an estimation of the total sample size that would be required for a power of 80%, given the current proportions of subjects in each group.\nWith the probability for all moderation levels fixed to .25, all 256 groups were represented in both the MZ and the DZ twins in the simulated data file (9,984 cases simulated), and for the chosen values, the analysis shows that we would need 165 families for a power of 80%. If we were to change the moderator level probabilities from .25 for every level to .4, .3, .2, and .1 for levels 0, 1, 2 and 3 respectively, then 179 and 225 groups would be represented in MZ and DZ twins, respectively (9,705 cases simulated). In that case, 206 families would be required for a power of 80% even though the moderator effect (\u03b2a) is unchanged.\nThese power calculations took at most 1\u00a0minute in total. Again, acquisition of similar power results would take at least T times as long within the Monte Carlo framework (where T is the number of replications). Assembling the data simulation script takes equally long for both types of simulation, so overall, exact simulation saves time.\nIllustration 3: association for a tri-allelic locus with different allele frequencies \nThe aim of association studies is to determine whether genetic variation is associated with the risk for disease or the expression of a continuously distributed trait. Association studies may produce false positives, i.e., significant association in the absence of any true genetic effects. Population stratification is one source of false positives, i.e., the mixture of two populations with different allele frequencies and different phenotypic means. Fulker et al. \n(1999) showed that this type of spurious association can be avoided in a family-based study design. In this illustration, we focus on the situation in which data are available for pairs of siblings. Although this design allows for the simultaneous modeling of linkage and association, we limit the analysis to the association, but note that linkage information (i.e., IBD sharing estimation) could be included in exact data simulation scripts. For the present illustration, however, we assume that the locus under study is the QTL itself and not a marker in linkage disequilibrium with the QTL.\nIf a locus is diallelic, 22\u00a0=\u00a04 genotypes can be distinguished: AA, AB, BA and BB (of course, in practice, there are only 2\u00a0+\u00a01\u00a0=\u00a03 distinguishable groups as AB and BA are the same, but when simulating the data exactly, it is convenient simply to treat them as different groups). These 22 genotypes give rise to (22)2\u00a0=\u00a016 possible combinations of siblings (not accounting order), i.e., a 16 group analysis. Note that this is the simplest case: with 3 alleles, the number of possible sib-pairs is already (32)2\u00a0=\u00a081, and when the locus under study is a polymorphic marker, with, say, 15 possible alleles, the number of distinguishable sib-pairs is (152)2\u00a0=\u00a050,625. Clearly, multi-group analyses with sufficient summary statistics quickly become impractical as the number of alleles\u2013\u2013or loci\u2013\u2013increases.\nWe illustrate the use of exact data simulation in the context of the sib-pair association design, for a tri-allelic locus with alleles A, B, and C, with frequencies p, q, and r, respectively. The aim of this particular power calculation is to determine the influence of the allele frequencies on the power to detect a QTL. The biometrical model for a tri-allelic locus is summarized in Table\u00a02. As with the more familiar diallelic case, the expected genotypic value  is assumed zero, so that everything is scaled in terms of deviations. In the case of three alleles, 2 genotypic values are distinguished, which were both fixed to .206, so that AA was associated with an increase of .206, BB with an increase of .206, and CC with a decrease of\u2212.206 to .206\u00a0=\u00a0\u2212.412. Dominance was assumed to be absent, so the genotypic effect for the heterozygous genotypes AB, AC and BC was calculated as the mean of the effects of the homozygous groups. In the case of equal allele frequencies (p\u00a0=\u00a0q\u00a0=\u00a0r\u00a0=\u00a01\/3), this QTL explains 2.5% of the variance (as determined using regular regression with the phenotype as dependent variable and genotype as predictor). Note that the variance explained by the QTL depends on the allele frequencies, so even though the genotypic values remain the same across all simulations, varying the allele frequencies affects the effect size of the QTL effect. For all simulations, background variance was decomposed such that additive genetic effects explained 30%, and unique environmental influences (E) explained 70% of the variance that remained after the QTL-effects was taken into account. Overall sample size N was fixed to 10,000 (note that because of rounding, the actual N modeled will not be equal to the overall sample size N of 10,000; see Table\u00a03).Table\u00a02Expectations for a tri-allelic locus following the standard biometric model when dominance is assumed absentGenotypeAAABBBACBCCCGenotype frequency fijp22pqq22pr2qrr2Genotypic value gijx(x\u00a0+\u00a0y)\/2yx\u00a0+\u00a0z\/2\u00a0=\u00a0\u2212y\/2y\u00a0+\u00a0z\/2\u00a0=\u00a0\u2212x\/2zNote: p, q, and r denote the frequencies of alleles A, B and C, respectively; x is the genotypic value associated with genotype AA, y the genotypic value associated with genotype BB. As  the genotypic value for genotype CC is z\u00a0=\u00a0\u2212x \u2212 y (i.e., x\u00a0+\u00a0y\u00a0+\u00a0z\u00a0=\u00a00). \u03bcqtl denotes the expected contribution of the QTL to the population mean, and \u03c32qtl denotes the expected contribution of the QTL to the population variance (adapted from Falconer and Mackay 1996)Table\u00a03Results for illustration 3: Power calculations for sib-pair association with a tri-allelic locus with fixed genotypic valuesFrequencies alleles A, B, CEffect size (%)Actual NNr of groups represented \u03c72(6)Observed power N required for power of 80%.33\/.33\/.332.59,63981837.8241157.25\/.5\/.251.79,98581734.3571185.45\/.45\/.1.69,99381352.9631386.1\/.45\/.452.89,99280966.6981141Note: Effect size is defined as % of variance explained by QTL; Actual N refers to actual number of sib-pairs in the analysis; Nr of groups represented refers to the number of groups, of the possible 81, that were represented in the analysis; \u03c72(6): the \u03c72-value of the test for association when the genotypic effects for all 6 distinguishable genotypes are fixed to zero; Observed power refers to the power observed for the modeled sample size N\nNote that between and within effects were exactly equal (i.e., B\u00a0=\u00a0W) as we did not model population stratification; all between and within parameters can thus be fixed to be equal without loss of fit. The overall test for genetic association then involves fixing the genotypic effects for all 6 distinguishable genotypes (AA, AB, AC, BB, BC, and CC) to zero, i.e., 6\u00a0degrees of freedom.\nThe simulated sib-pair data were analyzed in Mx, using the \u2018option power\u2019 command (\u03b1\u00a0=\u00a0.05,df\u00a0=\u00a06) to obtain the sample size required for a power of 80%, given the current proportions of subjects in each group. The results presented in Table\u00a03 show that the power to detect a QTL with certain genotypic values depends on the allele frequencies. As expected, the power is greatest when the frequency for the allele with the largest genotypic value (allele C) is highest.\nWith exact data simulation, these power calculations took about 2\u00a0min for each choice of allele frequencies. Again, it would take at least T times as long to obtain similar power results within the Monte Carlo framework (where T is the number of replications), while the time required to write the data simulation script takes equally long for both types of simulation.\nConclusion\nIn this paper we discussed a third method of power calculation, which can be useful when sufficient summary statistics are available in principle, but the number of possible groups is so large to render a multi-group analysis impractical. The illustrations presented in this paper represent only a few of the possible (behavior genetics) designs in which exact data simulation may prove useful. Other models for which exact data simulation can be used include random-effects models, latent growth curve models, simplex models, and (hierarchical) structural models, either or not in the context of genetics, just to name a few. Exact data simulation does not require more programming skills, or programming time, than Monte Carlo simulation, but one may save a lot of time analyzing the simulated data and calculating power, especially when one wishes to construct graphs of power vs. effect size.\nIn this paper, we used the Mx program to analyze the simulated data because of its inbuilt power calculation function. Another useful option of Mx in this context is the possibility to output individual likelihood statistics for each raw data group. This information can be used to identify the groups that contribute most to the power to detect the effects of interest. Of course, various other statistical software packages (e.g., QTDT, LISREL, MPlus, R) can also be used in combination with exact data simulation to obtain the non-centrality parameters required for power calculations.\nWe emphasize that the power results obtained through exact data simulation are exactly similar to power results obtained through the analysis of summary statistics, and, just like power calculation using summary statistics, asymptotically similar to results obtained through Monte Carlo simulation (depending on the number of runs used in Monte Carlo). Differences between those two customary method of power calculation and exact data simulation only occur when subgroups have very low probabilities and the simulated overall sample size is not large enough to include all possible groups to sufficient extent; these small groups may then not be represented in the exact power simulation, while they may be (more or less) represented in other methods. However, as stated previously, the ensuing differences with respect to the power results, are very small as such small groups hardly contribute to the power anyway. Even so, to avoid the exclusion of small groups, one should choose a sufficiently large overall sample size in exact simulation, such that all groups are represented. This is perfectly doable, and does not alter the practicability of the method as it still involves analyzing a single (yet larger) dataset. The non-centrality parameter obtained in the analysis of the large simulated data set can subsequently be used to calculate the power for smaller, more realistic sample sizes. Alternatively, one may decide to accept the absence of certain groups, and the implied slight underestimation of power. Happily, the discrepancy between the intended N and the realized N is simple to calculate (as demonstrated in the R script available in the Mx scripts library), so that one can readily obtain an impression of the implications of this decision.\nThroughout the paper, we have assumed that the data, conditional on group, are normally distributed, so that sufficient statistics are in principle available. With respect to situations that preclude sufficient statistics, the present method may still have some use. For instance, a continuous moderator in a G \u00d7 E model, as discussed by Purcell (2002), might be approximated by a 5 point or 7 point Likert scale, which would render exact simulation possible in principle (see illustration 2).\nFinally we note that the extension of this method to discrete data would obviously be very useful, and does seem feasible.","keyphrases":["power","simulation"],"prmu":["P","P"]}
{"id":"Photosynth_Res-3-1-1779625","title":"A re-investigation of the path of carbon in photosynthesis utilizing GC\/MS methodology. Unequivocal verification of the participation of octulose phosphates in the pathway\n","text":"A GC\/EIMS\/SIM methodology has been developed to re-examine the path of carbon in photosynthesis. Exposing isolated spinach chloroplasts to 13CO2 on a solid support for a defined period followed by quenching and work-up provided a mixture of labelled sugar phosphates. After enzymatic dephosphorylation and derivatization, the Mox-TMS sugars were analysed using the above method. The purpose of the study was to try to calculate the atom% enrichment of 13C in as many of the individual carbons in each of the derivatized sugars as was practical using diagnostic fragment ions. In the event, only one 45 s experiment provided sufficient data to enable a range of enrichment values to be calculated. This confirmed that D-glycero-D-altro-octulose phosphate was present in the chloroplasts and was heavily labelled in the C4, C5 and C6 positions, in keeping with the hypothesis that it had an inclusive role and a labelling pattern consistent with a new modified pathway of carbon in photosynthesis.\nIntroduction\nIn two previous papers, we described a GC\/MS methodology, which is capable of providing a rapid, specific and quantitative means of analysis of 13C incorporation from 13CO2 into those sugar phosphates involved in the path of carbon in photosynthesis (Irvine et\u00a0al. 1992; MacLeod et\u00a0al. 2001). This provided a framework for a re-investigation of the photosynthetic carbon reduction (PCR) pathway in plants, commonly known as the Calvin Cycle (Calvin 1956). Although the PCR pathway as presented by Calvin (Fig.\u00a01) is widely accepted, its re-examination is warranted for the following reasons: (i) the evidence upon which Calvin based the pathway was incomplete, (ii) more recent investigations support the inclusion of other sugar phosphates, notably octulose phosphates, in the pathway (Williams and MacLeod preceding paper), and (iii) the substantial advances in analytical technologies since Calvin\u2019s data was collected in the 1950s, using 14C-labelling and paper chromatography, have the potential to provide more definitive and comprehensive results than were possible for Calvin to obtain.\nFig.\u00a01The path of carbon in photosynthesis (Calvin 1956). Each asterisk (*) represents a carbon atom labelled during one turn of the cycle. For explanation of abbreviations, see preceding paper\nThis paper describes the measurement, using GC\/EIMS\/SIM, of 13C incorporation into individual carbon atoms of C4 to C8 sugar phosphates following short-term exposure of isolated chloroplasts to 13CO2 during PS.\nMaterials and methods\nChemicals\nSorbitol was purchased from Koch-Light, UK, sodium pyrophosphate from Mallinckrodt, USA and Percoll from Pharmacia, Sweden. Enzymes and cofactors were obtained from either Sigma, USA or Boehringer-Mannheim, Germany. Ion exchange resins were obtained from BioRad, USA. Analytical grade chemicals were obtained from Ajax, Australia, BDH, UK or Merck, Germany. All solvents were distilled before use while pyridine was pre-dried and distilled from calcium hydride under argon. Water was obtained from a Millipore Milli-Q system. The 13CO2 gas (99.9% atom enriched) was purchased from Cambridge Isotopes, UK and prepared to 1% in nitrogen in a gas cylinder. A second cylinder was made up with 1% unenriched CO2 in nitrogen. Gas analysis of the cylinders was carried out on a Varian 6000 gas chromatograph using a 1.83\u00a0m\u00a0\u00d7\u00a00.32\u00a0cm glass column packed with Porapak N 100\u2013200 mesh stationary phase.\nPlant material\nSpinach (Spinacia oleracea, Yates Hybrid 102) seeds were obtained from Henderson Seed Co., Pty. Ltd., Lower Templestowe, Vic., Australia.\nGrowth of plant material; chloroplast isolation; chlorophyll assay; measurement of chloroplast intactness; polarographic measurement of chloroplast activity in suspension\nThe above methods have been described in detail in the accompanying paper (Williams and MacLeod preceding paper).\nMeasurement of chloroplast activity on filter membranes\nThe activity of the isolated chloroplasts mounted on a filter membrane was measured polarographically using a Clark oxygen electrode adapted for measurement of the oxygen evolution from leaf-discs (Delieu and Walker 1981). Methods for the preparation of chloroplasts for use in the leaf-disc oxygen electrode were based on those previously described (Cerovic et\u00a0al. 1987). Chloroplasts were mounted on a filter membrane by filtering a suspension of chloroplasts through a cellulose nitrate membrane (Sartorius, 50\u00a0mm diameter, pore size 8\u00a0\u03bcm). This was accomplished using a Millipore solvent clarification apparatus fitted with a 100\u00a0ml funnel. A suspension of chloroplasts equivalent to 250\u00a0\u03bcg chlorophyll in 40\u00a0ml resuspension medium at pH 7.6 containing 330\u00a0mM sorbitol, 50\u00a0mM HEPES-KOH, 2\u00a0mM EDTA, 1\u00a0mM MgCl2 and 1\u00a0mM MnCl2 was allowed to filter through the membrane under gravity or with a very light suction so that the chloroplasts became trapped on the surface of the membrane in a thin film of resuspension medium. The chloroplasts were washed with a further 10\u00a0ml of the resuspension medium and then given a final wash with 3\u00a0ml of assay medium containing 6\u00a0mM inorganic phosphate, 22.5\u00a0U\u00a0ml\u22121 alkaline phosphatase (Sigma, human placenta) and 1,000\u00a0U\u00a0ml\u22121 catalase in resuspension medium. A disc of 1,018\u00a0mm2 (representing 94% of the chloroplasts) was cut from the membrane and mounted in a leaf-disc oxygen electrode (Hansatech). Once assembled, the leaf-disc oxygen electrode was charged with an atmosphere of 1% CO2 in N2 at atmospheric pressure and sealed. The chloroplasts were exposed to incandescent light from a pair of bifurcated fibre optic light sources at an intensity of 1,200\u00a0\u03bcEm\u22122\u00a0s\u22121 measured at the underside of the leaf-disc electrode light window. The temperature of the water bath supplying the water jacket, which regulated the oxygen electrode temperature was maintained at 23\u00b0C. After a lag period of approximately 1.5\u00a0min, the evolution of O2 could be monitored as a digital voltage on the oxygen electrode control box and was also recorded on a Goerz Metrawatt SE 120 chart recorder. The rate of O2 evolution reached a linear and maximum rate at approximately 4\u00a0min after switching on the lights. The rate was calculated from the linear portion of the trace.\nExperiments in the leaf-disc oxygen electrode\nChloroplasts were prepared for experiments in the leaf disc oxygen electrode (as discussed for the assay of chloroplast activity on filter membranes in that equipment) and a linear rate of O2 evolution was established. At this time, a volume (60\u00a0ml) of 1% 13CO2 in N2 was flushed through the 6\u00a0ml chamber of the oxygen electrode that contained the membrane-mounted chloroplasts and after resealing the chamber, the chloroplasts were allowed to photosynthesize in the 13CO2-enriched atmosphere for a prescribed period of time before the reactions were terminated using the quenching methods described below. Experiments were conducted for 0, 30 and 45\u00a0s exposure to 1% 13CO2 in N2 and the \u201ccontrol\u201d experiment was conducted with chloroplasts exposed to 1% 13CO2 in N2 for 45\u00a0s in the dark. In addition, a \u201cblank\u201d experiment was conducted in which the full extraction procedure was followed in the absence of chloroplasts to measure the extent of any contaminants that may interfere with the analysis. Five repetitions of each experiment were carried out using the same chloroplast preparation.\nReactions were terminated by dissembling the apparatus as quickly as possible and plunging the chloroplast-containing membrane into a beaker of liquid nitrogen. It was estimated that this procedure took no more than 2.5\u00a0s. The beaker containing the membrane was then stored in a freezer at \u221220\u00b0C where the liquid nitrogen was allowed to evaporate.\nAfter overnight storage at \u221220\u00b0C the enzymes were denatured and the metabolites were extracted by removing the beaker containing the membranes from the freezer and immediately adding boiling 80% ethanol (75\u00a0ml) and boiling for a further 5\u00a0min. The membranes were then removed from the extract and thoroughly rinsed with water, adding the washings to the extract. After cooling, the pooled extracts were then evaporated to dryness at 37\u00b0C under a stream of dry nitrogen.\nDesign of a specific photosynthesis apparatus\nBecause of the limitations of the oxygen electrode for the type of experiments that were required, it was decided to design and construct an apparatus better suited for the purpose.\nThe basic requirements in the design and construction of the photosynthetic apparatus were (1) that the chloroplasts be mounted on a filter membrane inside a small chamber with a transparent window for the admission of light, (2) the filter membrane should be large enough to hold sufficient chloroplasts to provide enough extracted material for GC\/MS analysis, (3) the atmosphere above the membrane must allow the establishment of steady state photosynthesis with unenriched CO2 and the rapid replacement of this with the same concentration of 13CO2 whilst causing minimum disturbance to the steady state, (4) the volume of the chamber above and below the membrane should be small in order to minimize the time taken to flush out the 12CO2 with 13CO2 using a reasonable gas flow rate. Also the volume of the chamber below the membrane must be small in order to minimize the \u201cdead volume\u201d of residual 12CO2, and (5) there must be provision for an appliance to inject a liquid to quench the photosynthetic reactions after a given time and for the convenient removal of the chloroplast extract after the quench.\nFilter holders were available (Sartorius SM 165 08B) which, with minimal modification, provided a suitable basis on which to construct a prototype photosynthesis apparatus. The rapid replacement of unenriched CO2 with 13CO2 was provided for by the inclusion of two gas inlet ports each supplying an internal gas manifold around the outer perimeter of the chloroplast-supporting membrane. The manifolds directed 13CO2 across the entire membrane and drove excess gas out through a series of 6 gas outlet holes drilled in the housing above the centre of the membrane, which vented the expelled gases to the atmosphere. An electro-mechanical timer unit was constructed to permit the automatic and rapid switching of the gas supply from a gas cylinder containing 1% unenriched CO2 in nitrogen to another cylinder containing 1% 13CO2 in nitrogen via a pair of electrically operated gas valves, causing minimal disruption to the steady state of photosynthesis.\nThe inlet and outlet ports for the filter holder provided suitable ports for the entry and withdrawal of the quenching solution. A 10\u00a0ml syringe containing the quenching solution was attached to the filter inlet port via a tap, which could be opened to allow the injection of the quenching solution at the completion of the photosynthesis experiment. By applying a gentle suction via a tap at the filter outlet port, the extract could then be withdrawn into a flask containing boiling 80% ethanol located below the apparatus. The chloroplasts and apparatus were protected from the heat produced by the hotplate by the provision of a reflective heat shield. Light was provided via fibre optic cables, which greatly reduced the transmission of heat from the light source and reduced the need for cooling of the apparatus. The concentrations reported for chloroplast metabolites are variable but have usually been found to be in the range from 5 to 50\u00a0nmol per mg chlorophyll (Gerhardt et\u00a0al. 1987; Giersch et\u00a0al. 1980; Giersch 1979). A filter membrane loaded with chloroplasts equivalent to 250\u00a0\u03bcg of chlorophyll should therefore contain 1.25\u201312.5\u00a0nmol of most metabolites of interest. Assuming minimal losses during processing for GC\/MS analysis, this was considered to be adequate chloroplast material for each experiment.\nExperiments in the photosynthesis apparatus\nChloroplasts were prepared on a filter membrane as described for the experiments in the oxygen electrode. The loaded membrane was mounted intact in the photosynthesis apparatus. A gas mixture consisting of 1% unenriched CO2 in nitrogen was applied to the two gas inlet ports via an electrically operated valve connected to a specially made electro-mechanical timer unit. The gas stream (at a flow rate of 200\u00a0cm3\u00a0min\u22121) was humidified by bubbling it through a tube of water before entering the valve. The chloroplasts were illuminated through the transparent window in the apparatus by a pair of 150\u00a0W tungsten lamps, each directed through a bifurcated fibre optic light guide. The gas mixture was supplied for 4\u00a0min before the timer operated the valves supplying the gas and switched to the source supplying 1% 13CO2 in N2 for the prescribed period. Experiments were conducted for 0, 6, and 30\u00a0s exposure to 1% 13CO2 in N2 and the \u201ccontrol\u201d experiment was conducted with chloroplasts exposed to 1% 13CO2 in N2 in the dark for 30\u00a0s.\nThe reactions were terminated and the metabolites were extracted by injecting hot 80% ethanol (5\u00a0ml) into the apparatus and allowing the chloroplasts to stand in the solution for 10\u00a0s. The ethanolic chloroplast extract was then drawn through the membrane using gentle suction into a flask that contained boiling 80% ethanol (30\u00a0ml). The extraction process was repeated with a further volume of boiling 20% ethanol (5\u00a0ml) followed by boiling water (5\u00a0ml). The combined extract was then boiled for 5\u00a0min and allowed to cool. After cooling, the extract was evaporated to dryness at 37\u00b0C using a stream of dry N2 gas.\nPreparation of samples for GC\/MS analysis\nThe dried samples from the photosynthesis experiments described above were redissolved in water (2\u00a0ml) and then processed for GC\/MS analysis according to the following methods.\nThe chloroplast extract to be analyzed by GC\/MS was applied to an Elut Bond strong anion exchange (SAX) solid phase extraction column (500\u00a0mg, Varian Associates, USA) and washed through with deionized water (30\u00a0ml). The eluate was discarded and the fraction containing both mono- and bisphosphates of the sugars was eluted with 0.5\u00a0M NH4HCO3 (5\u00a0ml) and collected in a polypropylene centrifuge tube (50\u00a0ml). Most of the buffer was removed by blowing the sample dry with a stream of dry nitrogen gas, whilst warming the sample in a water bath at 37\u00b0C. The samples were then dephosphorylated using human prostatic acid phosphatase, which was chosen for its ability to catalyze dephosphorylation quantitatively without causing the transformations produced by other phosphatases (Irvine et\u00a0al. 1992). Dephosphorylation of up to 0.5\u00a0\u03bcmoles of sugar phosphates was carried out by the addition of 0.5 units of prostatic acid phosphatase to the sugar phosphate solution in 0.1\u00a0M ammonium acetate\u2013acetic acid buffer pH 4.6 (typically 200\u00a0\u03bcl) and incubated for 24\u00a0h at 30\u00b0C. The reaction was terminated by heating at 100\u00b0C for 2\u00a0min.\nFollowing dephosphorylation, the solutions containing the free sugars were deionized by stirring for 30\u00a0min with 0.5\u00a0g of mixed bed resin prepared using equal weights of anion (Bio-Rad AG 1\u00a0\u00d7\u00a08, 200\u2013400 mesh, HCO3\u2212 form) and cation (Bio-Rad AG 50\u00a0W\u00a0\u00d7\u00a08, 200\u2013400 mesh, H+ form) exchange resins together with deionized water. The resin was removed by filtration through a small column fashioned from a pipette tip (5\u00a0ml) plugged with cotton wool and containing a further 0.25\u00a0g of the mixed bed resin. The sample was washed through with water (15\u00a0ml), collected in a polypropylene centrifuge tube (50\u00a0ml) and lyophilized. The resulting residue was washed with deionized water (1\u00a0ml) into an Eppendorf tube (1.5\u00a0ml) and again lyophilized. The residue was redissolved in water (120\u00a0\u03bcl) and the entire sample was transferred to a Reacti-Vial (200\u00a0\u03bcl) and dried over P2O5. The sugars were subsequently dried under high vacuum for 1\u00a0h.\nDerivatization of dephosphorylated sugar phosphates\nSugars were prepared for GC\/MS analysis as the methoxime-TMS derivatives using methods similar to those first described by Sweeley (Sweeley et\u00a0al. 1963) for 1\u00a0mg scale derivatizations. In our investigation, smaller-scale derivatizations were required. Up to 350\u00a0nmol (approx. 50\u00a0\u03bcg) of the dried sugars in Reacti-Vials (0.2\u00a0ml) were derivatized by the addition, under dry nitrogen, of 1% methoxylamine in pyridine (40\u00a0\u03bcl) and refluxing at 80\u00b0C for 2\u00a0h in a Reacti-therm heater. The mixture was cooled to room temperature then silylation was accomplished by the addition under dry nitrogen, of 10\u00a0\u03bcl of Regisil (Pierce) followed by heating for 0.5\u00a0h at 80\u00b0C. The mixture was cooled to room temperature then diluted as appropriate with dry pyridine for GC\/MS analysis. Injections of 1\u00a0\u03bcl ideally contained no more than 125\u00a0pmol of the sugar under analysis in order to avoid saturation of the mass selective detector, as determined by sensitivity runs with derivatized glucose.\nGC\/MS analysis of derivatized sugars\nDerivatized sugars were analyzed by capillary GC\/MS on a Hewlett Packard 5890 gas chromatograph interfaced with a Hewlett Packard HP5970B MSD and controlled by a Hewlett Packard HP59970C ChemStation. Gas chromatography was performed on an HP-1 capillary column (12.5\u00a0m\u00a0\u00d7\u00a00.2\u00a0mm i.d., 0.33\u00a0\u03bcm cross-linked methyl silicone stationary phase) in the splitless mode with helium carrier gas (flow rate 1\u00a0ml\u00a0min\u22121). The column temperature was held at 100\u00b0C for 2\u00a0min with the filament off for the passage of the solvent and excess reagent through the column. The temperature was then increased to 250\u00b0C at 10\u00b0C\u00a0min\u22121, and held at 250\u00b0C for 3\u00a0min. Spectra were obtained by electron impact (EI) ionization at 70\u00a0eV. Full scan spectra of the derivatized sugars were usually obtained by scanning from m\/z 100 to 660 at a rate of 0.77 scans s\u22121 and represent an average of scans taken across the top of each relevant chromatographic peak. Selected ion monitoring (SIM) experiments on clusters of ions of interest in the spectra of the derivatized sugars were carried out in groups of 8\u201312 ions per peak with dwell times of 30\u00a0ms per ion. All results represent the mean of at least three separate injections unless otherwise stated. Corrections for natural abundance isotopes of C, H, N, O and Si and contributions from adjacent ions one mass unit lower of comparable intensity were made in calculating 13C enrichments in individual ions. Only fragment ions which had been found to have essentially a single origin as the result of the GC\/MS analysis of specifically 13C-labelled sugar standards were used in this study (Irvine et\u00a0al. 1992; MacLeod et\u00a0al. 2001).\nIdentification of sugar phosphates in chloroplast extracts\nThe individual unlabelled sugar phosphates present in the chloroplast extracts were identified by the retention times and full scan mass spectra of their dephosphorylated Mox-TMS derivatives on the above GC\/MS system when compared with those of authentic sugars run under identical conditions (Irvine et\u00a0al. 1992; MacLeod et\u00a0al. 2001).\nQuantification by GC\/MS of sugar phosphates in chloroplast extracts\nQuantification of analytes is best achieved by the inclusion in the sample mixture of a known amount of an internal standard having similar chemical and physical characteristics to those of the analytes.\nIdeally, for GC\/MS analysis the internal standard should be a stable isotope analogue of the analyte under investigation and when there are multiple analytes, a stable isotope of each analyte is preferred. In this investigation however, the analytes (derivatized sugars) are themselves expected to become multiply-labelled with the 13C isotope during photosynthesis and the stable isotope dilution method therefore could not be used. The alternative approach was used of adding a compound that is similar to the sugars under investigation but is unlikely to be encountered in chloroplasts and would be well separated from the analytes on GC\/MS.\nFor SIM, the mass spectrum of the derivatized standard chosen for this GC\/MS investigation needed to contain an ion which was common to all of the sugars being measured. In addition, this ion must not become labelled in a 13CO2 experiment. The sugar alcohols seemed to be an obvious choice. The mass spectra of the per-TMS derivative of these compounds show a prominent m\/z 147 ion representing the pentamethyldisiloxonium ion ([(CH3)3SiOSi(CH3)2]+), as do the mass spectra of all of the sugar methoxime TMS derivatives. Since this ion does not contain any carbons derived from the sugar, it cannot become labelled during a 13C isotope incorporation experiment. Thus, SIM of the m\/z 147 ion was the method of choice for quantitation studies.\nErythritol was chosen as the internal standard because (1) the m\/z 147 ion of its per-TMS derivative gave a linear and reproducible response when calibrated against the same ion in the mass spectra of Mox-TMS sugars at levels up to 120\u00a0pmol and (2) it was well separated on the HP-1 GC column from the sugars under consideration.\nSugars which were available in the free form with a high degree of purity were dried for 3\u00a0days over phosphorous pentoxide under a low vacuum. Approximately 0.02\u00a0g of the dried powder or syrup (ketopentoses) was weighed to five decimal places to prepare standard solutions of each sugar. Appropriate dilutions of these were taken to prepare a set of 0.5\u00a0mM standard solutions.\nSome sugars were only available as the phosphate esters and frequently in the presence of various amounts of buffer salts. In these cases, after weighing the dried powders and preparing standard solutions, they were assayed enzymically to establish their concentrations prior to use. Sedoheptulose, D-glycero D-ido- and D-glycero D-altro-octuloses were assayed as their bisphosphates using an aldolase-based assay (Bergmeyer and Bernt 1974). Erythrose was assayed as the monophosphate ester using a transketolase-based assay (Paoletti et\u00a0al. 1979). Sugar phosphates (3\u00a0\u03bcmol) were then evaporated to dryness in a rotary film evaporator and dephosphorylated in a solution (0.5\u00a0ml) which contained citrate buffer 50\u00a0\u03bcmol, pH 5.4) and human prostatic acid phosphatase (1.25 units). After allowing the dephosphorylation to proceed for 4\u00a0h at 37\u00b0C, the reaction was terminated by heating at 100\u00b0C for 2\u00a0min. The samples were then treated with mixed bed resin and lyophilized.\nRecovery from the dephosphorylation step was assessed by carrying out parallel dephosphorylations of glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-bisphosphate, all of which were assayed enzymically as the free sugars. The recovery of the monophosphates was 88% and of the bisphosphate was 78%. These figures provided a useful estimate of the likely recoveries of those sugars (above), which could not be assayed in the non-phosphorylated form.\nAppropriate dilutions of the lyophilized sugars were then prepared to produce a set of 0.5\u00a0mM solutions for use as standards. Groups of sugar mixtures were prepared, each containing 500\u00a0\u03bcl of the 0.5\u00a0mM standard solution. Each sugar was thus present in a volume of 2\u00a0ml and at a concentration of 0.125\u00a0mM. The first group contained erythrose, fructose and D-glycero D-ido-octulose. The second group contained glucose, sedoheptulose and D-glycero-D-altro-octulose. Pentoses were calibrated separately. A series of dilutions of the standard mixtures containing 0.025, 0.125, 1, 2, 3, 4 or 5\u00a0nmol of each sugar together with 5\u00a0nmol of the erythritol standard were added to separate ReactiVials.\nThe mixtures were then first dried over P2O5 then under high vacuum and derivatized (Mox-TMS). This gave solutions which contained 0.5, 2.5, 20, 40, 60, 80, or 100\u00a0pmol of each sugar together with 100\u00a0pmol of the erythritol standard in each \u03bcl of the derivatized solution.\nGC\/MS analysis was carried out by the measurement of the peak areas obtained for the m\/z 147 ion of each sugar using single ion monitoring. Three injections were made at each sugar level and the average ratio of sugar to erythritol peak area was determined. In those cases where the Mox-TMS derivative of the sugar showed peaks for both syn- and anti-forms, the area of the leading peak was used. A graph plotting the measured ratio of each sugar m\/z 147 ion area to the erythritol m\/z 147 ion area against the actual ratio of the sugar to erythritol for the series of sugar concentrations was then plotted. The slopes of these graphs then represent the calibration factors relating the sugars to the internal standard, erythritol, and were determined as the slope of the least squares line of regression of measured area ratio on actual ratio (Table\u00a01).\nTable\u00a01Sugar calibration factorsaSugarInterceptSlopeCoefficient of correlation (r)Erythrose\u22120.0171.0680.999Ribose\/Arabinose0.0010.6250.998Xylulose\/Ribulose\u22120.0101.0770.999Fructose\u22120.0080.3050.995Glucose\u22120.0110.5240.998Sedoheptulose\u22120.0130.6780.998D-g D-a-Octulose\u22120.0090.3180.996D-g D-i-Octulose\u22120.0050.1770.992aErythritol used as internal standard\nResults and discussion\nInitial experiments were carried out using the leaf-disc oxygen electrode during the time that the special photosynthesis apparatus was being constructed in-house. Obvious disadvantages inherent in this approach were (1) the difficulty in achieving complete, instantaneous displacement of 12CO2 with 13CO2 and (2) the time taken to dissemble the apparatus and quench the photosynthesis reaction after exposure of the chloroplasts to 13CO2, leaving open the possibility of partial back exchange of the 13C-labelled sugars. One advantage was that the photosynthetic activity of the chloroplast preparations could be measured in situ prior to carrying out the 13CO2 exchange experiments.\nThere were several pitfalls that had to be overcome before PS exchange experiments with 13CO2 could be performed. Foremost of these was the low concentrations of sugars of the PS pathway observed in the chloroplast extracts in initial experiments using unlabelled CO2, together with the presence of other sugar-like components in the GC\/MS analysis . The latter was eventually traced to adsorption and decomposition of the small quantities (pmol) of sugar phosphates present in the chloroplasts on the surfaces of glass vessels used during the workup procedure. To minimise decomposition, these were replaced where possible with non-glass vessels. Losses due to adsorption could not be completely avoided and necessitated combining extracts from five consecutive experiments (using the same chloroplast preparation) in order to try to obtain useful data on those sugars present in lowest concentrations. Contaminants introduced during the workup presented another problem in the GC\/MS analysis, which required extensive investigation of their sources. The only contaminant that could not be completely eliminated was sorbitol but this did not interfere with the sugar analysis as the retention time of its TMS derivative differed from those of the Mox-TMS sugars under investigation.\nThe experiment carried out in the oxygen electrode at zero time exposure of the chloroplast preparation to 1% 13CO2 in nitrogen showed, after work-up, no incorporation of label in the sugars. As well, the \u201ccontrol\u201d experiment carried out with chloroplasts exposed to 1% 13CO2 in the dark for 45\u00a0s produced no 13C enrichment in any of the sugars in the chloroplast extract.\nAfter analysis of the results of four experiments carried out in the oxygen electrode, involving exposure of the chloroplasts to 13CO2 for 30\u00a0s (twice) and for 45\u00a0s (twice), only one of the 45\u00a0s experiments produced a reasonably comprehensive set of results (Table\u00a02). The main factor contributing to the inadequacy of the remaining experiments was the lack of sufficient material to enable GC\/MS\/SIM runs to be performed on all of relevant ion clusters as set out in a previous paper (see Table\u00a02, MacLeod et\u00a0al. 2001).\nTable\u00a02Enrichment resultsa for 45\u00a0s experiment No. 2 in the leaf disc oxygen electrodeSugarm\/zCarbons13C013C113C213C313C413C5Xylose160C1-267.224.28.6205C4-580.319.7262C1-351.026.813.48.9307C3-561.221.512.54.7452C1-547.121.717.913.3Lyxose\/Arabinoseb160C1-266.724.88.5262C1-355.530.713.7307C3-568.518.912.6Xylulose205dC4-590.35.64.1263C1-354.626.513.55.4364C1-450.023.815.58.12.5452C1-546.522.116.99.93.90.8Ribose\/Ribulosec160C1-267.024.78.3205dC4-581.911.76.4262\/3eC1-359.123.112.75.1307C3-566.117.611.05.3319C2-550.424.114.77.92.8Fructose205C5-689.510.5263C1-365.917.811.64.8307C4-668.216.910.14.8319C3-658.620.311.86.42.9364C1-449.622.316.28.73.2Glucose160C1-273.619.27.2205dC5-678.014.67.4307fC4-660.921.511.66.1319C3-636.523.122.212.95.3364C1-433.227.322.212.54.9Unidentified heptulose307C5-757.422.612.97.1319C4-726.327.025.615.06.1421C3-724.324.524.116.38.02.8Sedoheptulose205dC6-772.419.48.3262C1-341.429.617.39.02.6307fC5-752.025.115.07.9319C4-725.526.025.616.26.7364C1-431.727.422.513.25.3466C1-517.021.526.220.010.74.5Octulose205dC7-878.614.17.4307fC6-858.222.912.76.3319C5-841.825.219.210.23.6331C4-831.221.521.314.57.83.8421C4-833.621.521.014.46.92.6466C1-534.622.321.513.36.02.3aAfter correction for natural abundance of 2H, 13C, 15N, 18O, 29, 30Si and contributions from some neighbouring ions. Results represent an average of at least three consecutive GC\/MS runs. Standard deviations for most sugars were <1% and for the less abundant sugars (xylose, arabinose\/lyxose, octulose and unidentified heptulose), standard deviations were <2%. When significantly different enrichments were measured for the syn and anti-methoxime isomers, it was assumed that some interference was present causing the higher result and the one showing the lower enrichment was used; otherwise both syn and anti-isomers where present were used in the calculationsbLyxose\/Arabinose refers to either one or both sugars which could not be separated on the capillary GC column used in these analysescRibose\/Ribulose refers to both sugars, which, although they could not be resolved were both shown to be present from their characteristic mass spectra (MacLeod et\u00a0al. 2001)dThe intensity of the m\/z 207 ion is enhanced due to column bleed, despite background correctioneThe MS of Ribose contains a small m\/z 262 ion while that of Ribulose has a large m\/z 263 ionfThe m\/z 305 ion contributes to m\/z 307 and 308. No correction has been made for this\nExperiments were then carried out using the custom-made photosynthesis apparatus, involving exposure of isolated chloroplasts to 1% 13CO2 in N2 for 30\u00a0s in the dark and for 0, 6 and 30\u00a0s under controlled light conditions. After work-up, both the \u201cdark\u201d experiment and zero time exposure showed no 13C incorporation in any of the sugars analysed. As was the case for all but one of the oxygen electrode experiments, the 6\u00a0s and 30\u00a0s experiments after work-up did not contain a sufficient concentration of the sugar Mox-TMS derivatives, even after combining five experiments, to enable selective ion profiles to be obtained on all of the relevant ions in all of the sugars. This was particularly disappointing as the specially designed apparatus performed well otherwise.\nIn all of the above experiments, the concentrations of the sugar phosphates present in the chloroplast extracts were determined by SIM of the m\/z 147 ion present in the mass spectra of the dephosphorylated Mox-TMS derivative, using erythritol as internal standard (see Materials and methods). A correction was made for losses incurred during the chloroplast work-up and dephosphorylation stages by adding 14C-labelled glucose 6-phosphate to a single membrane-mounted chloroplast preparation immediately prior to quenching in the special apparatus. The measured recovery figure of 73% of 14C-labeled glucose represents a minimum recovery value since the method eventually used involved the pooling of the extracts from five membranes, which would be expected to lead to reduced losses. Table\u00a03 lists the values obtained. As expected, the two \u201cblank\u201d runs showed only traces of glucose, fructose and pentoses while the 45\u00a0s \u201cdark\u201d experiment showed lower than normal levels of all sugars except glucose. The other six experiments, five using the oxygen electrode and one using the special apparatus, showed sugar concentrations which, although varying from experiment to experiment, were of the same order of magnitude to those previously reported (Lilley et\u00a0al. 1977).\nTable\u00a03Chloroplast metabolite concentrations (nmol (mg\u00a0chl)\u22121)Equipment for experimentApparatusLeaf disc oxygen electrodeExposure to 13CO2 (sec:)03004503045CompoundBlankaBlankaControlbNo 1No 2No 1No 2Erythrosec0.000.000.000.000.280.140.550.79 0.35Xylose0.000.390.000.000.000.000.861.100.61Lyxose\/Arabinose0.000.630.000.000.000.001.050.680.81Ribose\/Ribulose\/Xylulose0.205.740.211.864.702.549.7914.876.61Fructose0.2933.710.312.6729.6623.4842.06117.658.43Glucose0.3310.550.356.718.213.6015.6339.3816.18Unidentified heptulose0.000.000.000.000.290.000.851.040.84Sedoheptulose0.019.040.011.0612.971.1721.9538.9016.26D-g D-a-Octulose0.0000.170.000.280.400.001.533.53daExtract containing chloroplast medium without chloroplastsb45\u00a0s exposure to 13CO2 in the darkcTentatively identifieddm\/z 147 ion not monitored\nNo C3 sugars could be analysed under the GC conditions used since they eluted with the residual derivatizing agents. A tetrose sugar was present which had the same mass spectrum and elution time as erythrose (Table\u00a03) but subsequent analysis of label incorporation into selected ions showed no 13C enrichment. The unidentified heptulose listed in Table\u00a03 did show very significant 13C incorporation in the few ion clusters measured (Table\u00a02), comparable to that observed in sedoheptulose. D-g-D-a-Octulose was identified by the retention times of its syn- and anti-isomers but the earlier eluting D-g-D-i-octulose was not detectable.\nThe enrichment results obtained in Table\u00a02 were sufficient to calculate atom% excess in some but not all of the individual carbon atoms of the sugars listed (see Table\u00a02 in MacLeod et\u00a0al. 2001). For example, the enrichment in C-2 of ribose (23.8%) was obtained by subtraction of m\/z 307 (C3-5) from m\/z 319 (C2-5). Similarly, subtracting the 13C enrichments in the ions m\/z 307 and 205 (C4-5) gave the atom% excess in C3. C-1 (12.1%) was calculated by subtracting the above value for C2 from the enrichment in the m\/z 160 ion (C1-2). In some cases where it was not possible to obtain enrichment data for individual carbon atoms, values for consecutive two carbon units were calculated.\nWhile it is obvious that the results obtained fall short of the objective, for reasons given above, they nevertheless allow some pertinent observations to be made. The presence of D-g-D-a-octulose in isolated chloroplasts at concentrations between 0.2 and 3.5\u00a0nmol (mg\u00a0chl)\u22121 is unequivocally confirmed (Table\u00a03). In the 45\u00a0s experiment, the atom% 13C excess in carbons 4, 5 and 6 of the octulose is between 25% and 30%, comparable with the values calculated for C-2 and C-3 of ribose, C-3 and C-4 of fructose and glucose, and C-4 and C-5 of sedoheptulose (Table\u00a04). These sugars and their above carbons are implicated in CO2 fixation in Calvin\u2019s photosynthetic pathway (Fig.\u00a01) and it is therefore not unreasonable to speculate that D-g-D-a-octulose also has a role in this pathway, in support of the conclusions drawn in the preceding paper (Williams and MacLeod 2006). The pattern of 13C labelling of carbons 4, 5 and 6 of D-g-D-a-octulose presented here is precisely the same as that shown in the octulose 1,8-bisphosphate by NMR spectroscopy of a spinach leaf extract after photosynthesis in 13CO2 (Bartlett et\u00a0al. 1989).\nTable\u00a04Atom% excess 13C in individual carbons and 13C enrichments in C2 moietiesaCarbonXyloseLyxoseXyluloseRiboseFructoseGlucoseSedoheptuloseUnk. HeptuloseOctuloseC-1{24.2,{24.8,\u201312.1\u2013{19.2,\u2013\u2013\u2013C-28.68.5\u201323.8\u20137.2\u2013\u2013\u2013C-323.7\/24.1b16.7\u201324.5d14.140.1c\u20137.6\u2013C-4{19.7d,\u20138.4{12.5d,23.8\/24.7b27.523.6\/51b,c54.225.0C-50\u20137.10{10.5,{16d,46.3\/34.1b,c,d\u201328.2cC-600{21.1d\u201331.2dC-70{15.4,dC-80aA dash implies that the value could not be measured as a one- or two-carbon unitbCalculated from two separate sets of data, e.g. C1-3 minus C1-2 and C3-5 minus C4-5 both give a value for C3 of XylosecNot corrected for contribution of m\/z 305 to the m\/z 307 clusterdm\/z 207 from residual column bleed taken as zero in m\/z 205 cluster\nA further interesting observation was the presence of a second heptulose sugar, albeit in much lower concentrations than that of sedoheptulose (Table\u00a03). It had a substantial atom% excess at C-4 of 54.2%, comparable with that observed for the same carbon in sedoheptulose. A GC\/MS comparison of the retention time of its Mox-TMS derivative with that of mannoheptulose eliminated this as a possibility and so it remains unidentified.\nOne of the anomalies of the Calvin Pathway (Fig.\u00a01) has been the failure to identify erythrose phosphate in chloroplasts, even though it is proposed to play an integral role in CO2 fixation. In this study, although we were able to detect a four carbon sugar with the same retention time and mass spectrum as Mox-TMS-erythrose (Table\u00a03), GC\/MS\/SIM analysis showed no incorporation of 13C in any of its fragment ions. It could therefore be assumed that the level of erythrose phosphate participating in the pathway is below that detectable by GC\/MS\/SIM. However, the identification and evidence for the presence of Ery 4-P in the Calvin and Pentose pathways has always been weak and contentious (Williams et\u00a0al. 1980).\nUnfortunately, circumstances have not allowed us to repeat the above experiments on a scale which could provide sufficient amounts of sugar phosphates to allow more complete data on the time-course of 13C incorporation from 13CO2 during photosynthesis to be obtained. In support of an earlier study of gluconeogenesis in liver cells (Desage et\u00a0al. 1989) it has, however, been shown here that GC\/MS\/SIM offers a viable alternative methodology for the study of positional isotope analysis of intermediates formed from stable-isotope labelled precursors in complex biological systems.\nIn summary, the results reported here and in the preceding paper have established the formation and specific predictive labelling of octulose phosphate(s) in spinach chloroplasts by 13CO2 and 14CO2 during photosynthesis. The data do not unequivocally prove that octulose phosphates are direct and exclusive intermediates of a new set of partial reactions for the path of carbon that were inadvertently missed by Calvin and his colleagues. However we suggest that octulose phosphate formation may fulfil a role as mono- and bisphosphates in a shunt (as defined by Newsholme and Leech 1983) that is attached to the Calvin reaction scheme (Fig.\u00a01). Figure\u00a02 is a structural display of the new reaction scheme presented in the accompanying paper. The shunt shows that the octulose phosphate reactions intersect with and may in time and space compartmentalize Seh 1,7-P2 from SBPase and thereby limit its important role in the regulation of flux in the carbon path of PS.\nFig.\u00a02An extension of the Calvin pathway by the inclusion of an octulose phosphate shunt (vide infra). Each asterisk (*) represents a carbon atom labelled during one turn of the cycle. Sugar phosphates in red represent those participating in the octulose shunt. For explanation of abbreviations, see preceding paper","keyphrases":["chloroplasts","selected ion monitoring","mox-tms derivatives","octulose phosphate shunt","13c-enriched sugar phosphates","13co2 incorporation","gas chromatography\u2013mass spectrometry","modified calvin cycle"],"prmu":["P","P","P","P","M","R","M","R"]}
{"id":"Clin_Oral_Investig-4-1-2238781","title":"Is diagnosing exposed dentine a suitable tool for grading erosive loss?\n","text":"Quantifying tooth wear in general and erosion in particular mostly is made by distinguishing between lesions restricted to enamel and lesions reaching the underlying dentine. Various scores for grading have been used, but in all systems, higher scores are given in cases of exposed dentine, thus, indicating a more severe stage of the condition. Clinical diagnosis of exposed dentine is made by assessing changes in colour or optical properties of the hard tissues. This paper aims to review the literature and discuss critically problems arising form this approach. It appears that classifying the severity of erosion by the area or depth of exposed dentine is difficult and poorly reproducible, and taking into account the variation of enamel thickness, the amount of tissue lost often is not related simply to the area of exposed dentine. There has still been very little longitudinal investigation of the significance of exposed dentine as a prognostic indicator. Further work and discussion is needed to reevaluate the explanative power of current grading procedures.\nIntroduction\nThis review illustrates the problems that clinicians and researchers have experienced in quantifying tooth erosion. Assessing the degree of erosion in an individual may be difficult [32], but is important, as it reflects net exposure to the erosive forces and the opposing forces of protection, for example from saliva. Grading erosion present plays a part in the assessment of the problem, the need for treatment and, potentially, may indicate means for prevention of further tooth wear. If a reliable method for assessing the loss in vivo of tooth substance were available, then it might be possible to determine if erosion was continuing or had been halted, for example by preventive actions. Whilst some assessment of enamel erosion might be possible by assessing the progressive loss of anatomical contour, this is more difficult when erosion has reached the dentine. The diagnosis of exposed dentine is made by assessing changes in colour [43] or changes in the optical properties of the hard tissues, the validity of which, however, has not yet been investigated.\nThis paper will consider the following main points:\nDoes dentine exposure correlate with the severity of erosion?Can dentine exposure be reliably and reproducibly diagnosed?What is the interplay of factors causing tooth wear and how can other forms of tooth wear be distinguished from erosion when the wear has reached dentine?Are the parameters used for grading tooth erosion applicable to both the primary and secondary dentitions?Could symptoms of dentine hypersensitivity assist in the grading of erosive loss?\nDoes dentine exposure correlate with the severity of erosion?\nFor grading, the overwhelming majority of tooth wear and erosion indices use the differentiation between lesions restricted to enamel and lesions reaching the underlying dentine. The rationale for linking severity of erosion to extension into dentine probably stems from experience with dental caries and restorative procedures, but has not yet been critically discussed. It is an interesting question whether the progression of erosion from enamel into dentine has any significance in terms of disease, although it may well have a bearing in terms of treatment and, sometimes, the presence of symptoms of hypersensitivity felt by the patient.\nOne of the most widely used indices for assessing tooth erosion is that of Lussi et al. [30], albeit sometimes modified [6]. This index does indeed attempt to grade the severity of erosion by assessing the degree of dentine exposure in a simple and, presumably reproducible, way as shown in Table\u00a01. As can be seen, grades 2 and 3 record increased severity of erosion on facial surfaces by judging the area of exposed dentine, rather than loss of dentine. On other surfaces, grade 2 requires the involvement of dentine.\nTable\u00a01Use of the Lussi Index for grading the severity of dentine loss (in bold text)Index ScoreObservationFacial surfaces0No erosion, glazed appearance, absence of developmental ridges possible1Loss of surface enamel, dentine not involved2Erosion into dentine <50% of affected surface3Erosion into dentine >50% of affected surfaceOther surfaces0No erosion, glazed appearance, absence of developmental ridges possible1Slight erosion, rounded cusps, restorations stand proud of enamel, no dentine erosion2Severe erosion, more pronounced than score 1, dentine involved\nOther indices use thirds of the affected surface with exposed dentine as thresholds [13, 35, 41] or score the severity of lesions using a more detailed grading with respect to depth of the lesion, as enamel loss only as code 1 or 2, loss of enamel with exposure of dentine with the enamel dentine junction (EDJ) visible as code 3, loss of enamel and dentine beyond EDJ as code 4 and loss of enamel and dentine with exposure of pulp as code 5 [36]. Despite variations in the definition of scores, all indices give higher scores when dentine is exposed, thus, indicating a more severe stage of the condition.\nThis suggests that the amount of tissue loss must be higher when dentine is exposed than when enamel coverage is still present. When considering the thickness of enamel, however, it is obvious that this is not always the case. Enamel gradually thickens from the cervical region occlusally. At the coronal third of the crown, the enamel thickness is about 1.5\u00a0mm, in the cervical third, about 0.5\u00a0mm and becomes increasingly thinner towards the enamel cementum junction [26]. Thus, when the entire smooth surface of a tooth is exposed to acid, the dentine will be first exposed in the cervical region. At the occlusal area, enamel is thickest at the functional cusps, in upper molars. In lower molars, however, the enamel is thinnest at the mesiobuccal cusp, and is progressively thicker in order from the mesiolingual and distolingual to the distobuccal cusp tips [26]. This would explain the finding that lower first molars, particularly the mesio-buccal cusp tip, is the region where exposed dentine occurs most often. Taking into account the gross variation in enamel thickness, it is not surprising that the correlation between exposed dentine and the severity of wear is weak.\nA comparison of the amount of tissue loss assessed visually and histological findings revealed that the percentage of teeth with exposed dentine was high even in cases with minor substance loss and, in contrast, that enamel can be present in teeth with moderate to severe substance loss. Furthermore, dentine was exposed in all cases of cupping or grooving even if only minor substance loss occurred [17]. Similar results have been found in a study investigating primary teeth [5]. This showed that in 31 teeth with visually diagnosed wear, only three had enamel present histologically, and even in one of ten teeth in which no wear was diagnosed visually, the histological examination revealed exposed dentine.\nOnce dentine is exposed, it is more prone than enamel to abrasion [25, 21] since its microhardness is much lower than that of enamel [33]. When the severity of lesions is defined by the degree of exposure of dentine, the severity score could assume that tooth wear will progress more rapidly in the future. However, this has not been proven in incidence studies. The predictive value of a diagnosis of exposed dentine to future dental erosion or other forms of tooth wear is far from clear, and longitudinal studies are urgently needed to determine the progression, particularly of erosion, at different sites in the dentition.\nCan dentine exposure be reliably and reproducibly diagnosed?\nCupping of the cusp tips on molar teeth is one of the most common presentations of erosion, for example in teenagers (Fig.\u00a01). This has been found in a recent epidemiological study of erosion in Iceland [6] and was also reported in other studies [16, 30, 42].\nFig.\u00a01Cupping of the cusp tips on the molar teeth is an early sign of erosion where extension of erosion into dentine is difficult to determine\nOne major problem in assessing the degree of erosion in cupping is whether or not the dentine has been exposed at the base of the lesion. Because labial and palatal enamel surfaces have a greater surface area, the observer better appreciates their surface anatomy than with a narrow cup-like lesion formed into a tooth cusp tip. Thus, grading the severity of cuppings is more difficult than perhaps it is for labial and palatal surfaces.\nGanss et al. [17] demonstrated that when compared with histological examination, only 65% of areas with exposed dentine, 88% of areas with enamel present and 67% of all areas examined had been diagnosed correctly. Clinicians demonstrated relatively poor reliability and reproducibility in their diagnosis of dentine exposure regardless of their professional experience.\nAl Malik et al. [5] have also shown that while grading erosion from visual and photographic records gave comparable scores, the degree of erosion was underestimated compared with the degree of tooth wear determined histologically. One way to increase the reliability of indices is to keep the grading scheme simple because minor differences in severity are difficult to assess, at least with the naked eye. For example, in a study from van Rijkom et al. [43], two scores for enamel and dentine tissue loss each were used scoring slight enamel wear as 1, deep enamel wear as 2, wear into dentine as 3 and wear into dentine for more than one half of smooth surfaces as 4, and the Kappa values achieved were low. Larsen et al. [28], in contrast, attempted a prevalence study of tooth erosion using seven examiners and only three clear criteria defining grade 1 as erosion limited to the enamel, erosion exposing the dentine less than 50% of the area of the surface as 2 and erosion exposing dentine in more than 50% of the surface as 3. Despite a careful calibration, including a thorough discussion of the criteria and the experience gained during the study, substantial inter-examiner differences in diagnosis were found. On the other hand, a good strength of agreement was achieved with an index proposed by Larsen et al. [27] where five scores for incisal\/occlusal surfaces were used.\nFurther investigations specifically aimed at assessing the reliability and reproducibility of scoring the involvement of dentine in the grading of erosion is necessary, should this criterion be included a clinical index of erosion. One future perspective could be the development of instrument-supported approaches. A recent report of optical techniques involving nitrogen-laser-induced fluorescence [42] appeared to offer the possibility of differentiating accurately between erosion into enamel and into dentine. This method may, according to the authors, even be applicable in vivo, although the reported studies were in vitro.\nWhat is the interplay of factors causing tooth wear and how can other forms of tooth wear be distinguished from erosion when the wear has reached dentine?\nFactors leading to tooth wear, including erosive agents, such as dietary and gastric acid interact with protective factors from the diet, oral hygiene procedures and, particularly, saliva. Interestingly, one study of the relationship between erosion, soft drink consumption and reflux disease found the consumption of Coca-Cola\u00ae three times per week to be the dominant factor in the development of molar erosion [24], but the interplay of factors is often complex. Although the net outcome of erosive and reparative factors does not necessarily have to be tooth wear, it appears that once tooth wear has started, the interaction of these factors becomes ever more complex, not least by the time tooth wear has reached the dentine. Thus, the interplay of physical and chemical factors and the relevance of this to dentine wear will be considered together.\nRelevant physical forces are attrition and abrasion either from mastication or from oral hygiene procedures. Frequent exposure to dietary or gastric acid will lead to a softening of the dental hard tissues, making it easier for forces of attrition and abrasion to contribute significantly to the overall wear on the teeth. Whilst the effects of acids on enamel are relatively simple, leading to the dissolution of mineral and surface softening, the results on dentine are more complex. Chronic exposure to acids not only leads to an increasing loss of mineral but also to a progressive exposure of the organic dentine matrix, the effects of which, however, are not well known.\nThe most investigated interplay between erosion and physical forces is the abrasive effect of toothbrushing. An increased wear of eroded enamel and dentine was demonstrated in a number of studies [7\u20139, 14, 21, 38, 45], even if one study did not prove this effect for dentine [18]. Azzopardi et al. [11] have also shown that a combination of erosion and abrasion causes more tooth wear than either tooth wear force on its own. Results of epidemiological studies are ambiguous in demonstrating only weak [2, 29] or no [3, 37, 39] association between the occurrence of erosion and oral hygiene habits.\nWith respect to abrasion from mastication, the occlusal surfaces are at particular risk. When dentine is exposed, abrasive foods and oral hygiene products tend to hollow out softer surface regions [31]. This can take place when an abrasive diet is consumed or in cases of acid-weakened dental tissues where a less abrasive bolus could also have similar effects. Clinically, the shape of occlusal lesions can be remarkably similar, making the diagnosis of the predominant aetiological factor difficult to determine.\nPerhaps commonest among these non-erosive factors contributing to tooth wear is attrition [12]. There is little information about the relative prevalence of facets in relation to different aetiological factors for wear and their progression when dentine is reached. It appears, however, that attrition tends to be superimposed on abrasion in cases of abrasive diet, as facets seldom occur in ancient remains and are not more prevalent in subjects with acidic diet when compared to subjects with an average western diet [15].\nThe problems of measuring tooth erosion in an individual presenting with tooth wear as shown in Figs.\u00a02, 3 and 4 are considerable, as not only is it difficult to envisage how much dentine and enamel has been lost but it is also increasingly difficult to attribute the loss of tissue to one particular aetiological factor or another as the tooth wear progresses. Even in clear cases of acid erosion, there may be a combination of intrinsic and extrinsic acid contributing to the clinical appearance.\nFig.\u00a02Clinical case to illustrate the difficulties of attributing diagnosis of tooth wear. This example of severe tooth wear was thought to be attrition, superimposed on erosion, of the occlusal surfaces of posterior teeth and palatal surfaces of maxillary teethFig.\u00a03Erosion of the palatal cusps of maxillary molar and premolar teeth in a patient with gastro-oesophageal reflux disease. Note that dentine is visible on the mesio-palatal cusp of the first molar toothFig.\u00a04Typical appearance of erosion in a patient with gastro-oesophageal reflux disease showing palatal erosion of the maxillary tooth cusps and buccal erosion of the mandibular tooth cusps. The degree of tooth wear and the presence of restorations make grading difficult\nAre the parameters used for grading tooth erosion applicable to both the primary and secondary dentitions?\nOne particularly difficult problem is assessing tooth wear in the deciduous dentition where the tooth structure withstands the forces of tooth wear somewhat less than the permanent dentition. An example is shown in Fig.\u00a05 of a case where the aetiology of considerable erosion was never clear even when it continued into the permanent dentition. Indices used for the permanent dentition have also been used in the primary dentition [3, 35], but also, a special index for children has also been developed [36].\nFig.\u00a05Clinical appearance of severe erosion, probably combined with other forms of tooth wear, in the deciduous dentition of a patient in whom no pathological reflux disease was recorded. The reported diet was relatively normal but erosion later appeared in the first permanent molar teeth\nThe prevalence of erosive wear in the deciduous dentition varies between 6 and 100%, and the percentage of children with erosion into dentine varies between 1 and 50% [4, 10, 20, 23, 46]. The high prevalence of erosive wear in the deciduous dentition is remarkable when the short period in function is considered, but could be explained by the softer nature of the primary tooth substance leading to increased wear from physical impacts. The susceptibility to acids, in contrast, is probably not greater than for primary than for permanent dental tissues [22]. The enamel of deciduous teeth, however, is much thinner than that of permanent teeth [19], which will lead to the rapid exposure of dentine. These factors will tend to make scores greater for deciduous teeth, and the links with known aetiological factors seem less clear. Studies using the same criteria for the primary and permanent dentition revealed higher prevalence data for the primary than for the permanent dentition when lesions involving dentine are regarded [3, 35]. It is unclear if these data really reflect more severe tissue loss occurring in children or if the thin deciduous enamel leads to early exposure of dentine also in cases of minor wear, which would mean an overestimation of the condition.\nPerhaps the more relevant question is whether tooth erosion in the deciduous dentition is a prognostic indicator of likely erosion in the permanent dentition, as was the case in the subject illustrated in Fig.\u00a05. Meurman and ten Cate [34] have pointed out that there were no longitudinal clinical studies of progression of erosion in the deciduous teeth nor does the continuing of erosion into the permanent dentition appear to have received wide attention. In the last decade, only one study has demonstrated an increased risk of erosion in the permanent dentition when erosion had been present in the primary dentition. As study models were used, the relevance of exposed dentine was, however, not assessed [16].\nIdentifying the cause of erosion in the deciduous dentition may enable preventive measures, or treatment in the case of reflux disease, that could eliminate the risk of erosion in the permanent dentition. The UK National Guidelines in Paediatric Dentistry [40] notes the importance of recording the probable aetiological factors but notes specifically the unsuitability of the Smith and Knight Index [41] for measuring small longitudinal changes in tooth erosion and the problems of the complex interaction of causative and protective factors that make tooth erosion a more complex diagnostic and prognostic problem than would perhaps be thought initially to be the case.\nCould symptoms of dentine hypersensitivity assist in the grading of erosive loss?\nOnce tooth wear has extended into dentine, there is a possibility that the patient will experience symptoms of hypersensitivity [44]. There are numerous causes of dentine hypersensitivity and, therefore, great care is needed in diagnosing hypersensitivity as a result of tooth erosion reaching dentine. The mechanisms underlying hypersensitivity [44] strongly suggest that exposure of dentine tubules to the mouth will cause hypersensitivity in some, but not all, subjects. Consequently, the presence of symptoms of dentine hypersensitivity in a patient with signs of tooth erosion is strongly suggestive of extension of the erosion into dentine. While acidic drinks may expose the dentine tubules and remove early plaque formed on the dentine, thus leading to the symptoms of hypersensitivity, other factors such as toothbrushing may also contribute to this [1].\nIn conclusion, distinctions are commonly made between erosion into enamel and erosion into dentine. Methods of scoring are derived from parameters associated more with dental caries and restorative procedures for that disease rather than specifically for tooth wear. The interplay of tooth wear factors is complex and requires further research. Classifying the severity of erosion by the area or depth of exposed dentine is difficult and poorly reproducible and, particularly with respect to the variation of enamel thickness, the amount of tissue lost often is not related to dentine exposure. There has still been very little longitudinal investigation of the significance of exposed dentine as a prognostic indicator. Further work is needed to reevaluate the explanative power of current grading procedures.","keyphrases":["dentine","tooth wear","tooth erosion"],"prmu":["P","P","P"]}
{"id":"J_Abnorm_Child_Psychol-4-1-2268722","title":"Hot and Cool Forms of Inhibitory Control and Externalizing Behavior in Children of Mothers who Smoked during Pregnancy: An Exploratory Study\n","text":"This study examined whether children exposed to prenatal smoking show deficits in \u201chot\u201d and\/or \u201ccool\u201d executive functioning (EF). Hot EF is involved in regulation of affect and motivation, whereas cool EF is involved in handling abstract, decontextualized problems. Forty 7 to 9-year-old children (15 exposed to prenatal smoking, 25 non-exposed) performed two computerized tasks. The Sustained Attention Dots (SA-Dots) Task (as a measure of \u201ccool\u201d inhibitory control) requires 400 non-dominant hand and 200 dominant hand responses. Inhibitory control of the prepotent response is required for dominant hand responses. The Delay Frustration Task (DeFT) (as a measure of \u201chot\u201d inhibitory control) consists of 55 simple maths exercises. On a number of trials delays are introduced before the next question appears on the screen. The extent of response-button pressing during delays indicates frustration-induced inhibitory control. Prenatally exposed children showed poorer inhibitory control in the DeFT than non-exposed children. A dose\u2013response relationship was also observed. In addition, prenatally exposed children had significantly higher (dose-dependent) conduct problem- and hyperactivity-inattention scores. There were no significant group differences in inhibitory control scores from the SA-Dots. These results indicate that children exposed to prenatal smoking are at higher risk of hot but not cool executive function deficits.\nIntroduction\nPrenatal tobacco exposure has consistently been associated with children\u2019s externalizing behavior (e.g. Button et al. 2005; Huijbregts et al. 2007, in press; Kotimaa et al. 2003; Mick et al. 2002; Wakschlag et al. 2006). In contrast, studies into associations between prenatal tobacco exposure and children\u2019s cognitive functioning show very mixed results (e.g. Batstra et al. 2003; Fried et al. 1998, 2003 versus Breslau et al. 2005; Huijbregts et al. 2006; Trasti et al. 1999). This is surprising because externalizing behavior problems often co-occur with cognitive difficulties.\nCognition in ADHD, ODD\/CD, and ADHD+ODD\/CD\nCognition has been widely studied in ADHD (for reviews, see Barkley 1997; Castellanos et al. 2006; Sergeant et al. 2003; Wilcutt et al. 2005). This research has shown, among others, problems with inhibitory control, working memory, vigilance, reward sensitivity (delay aversion), and time processing and currently focuses on different cognitive endophenotypes of ADHD (i.e. biologically based phenotypes that carry genetic loadings and index liability to disease; see Castellanos and Tannock 2002). Oppositional Defiant Disorder (ODD), Conduct Disorder (CD) and high levels of physical aggression have also been associated with cognitive difficulties such as language-based verbal skills and executive function (EF) (Henry and Moffitt 1997; Hill 2002; S\u00e9guin et al. 1999). Controversy still exists about whether verbal and, particularly, EF deficits are present in ODD\/CD without comorbid ADHD (Pennington and Ozonoff 1996). Since Pennington and Ozonoff\u2019s review on EF in different forms of developmental psychopathology, an increasing number of studies have taken into account the high comorbidity of ODD\/CD and ADHD. This has resulted in reports of both qualitative and quantitative differences in EF-dysfunction between disorders. For example, Van Goozen and colleagues (2004) reported the absence of problems with Working Memory (WM) and planning in a group of children with Oppositional Defiant Disorder (ODD) and a group with combined ODD\/ADHD compared to healthy controls. Both are executive functions that have frequently been reported to be deficient in ADHD. Oosterlaan et al. (2005) compared ADHD, ODD\/CD, and ADHD+ODD\/CD groups and found that WM and planning deficits were specific to ADHD (and explained by ADHD in the combined disorder). Distinguishing ADHD and ODD\/CD is more difficult when it concerns inhibitory control (Oosterlaan et al. 1998). Inhibition has been defined and operationalized in a number of different ways, e.g. as stopping an ongoing response, interference control and inhibition of prepotent responses (Barkley 1997), and studies have generally not included tasks measuring such different forms of inhibition. Furthermore, inhibition has been listed as one of the executive functions (e.g. Pennington and Ozonoff 1996), but has also been described as an important cognitive ability underlying many different executive functions (Zelazo et al. 1997). Lack of inhibitory control has been proposed as the central deficit in both ADHD (Barkley 1997) and ODD\/CD\/physical aggression (e.g. Lau et al. 1995), but is not necessarily similarly dysfunctional in both types of disorders. Nigg (2003) has suggested that ADHD is predominantly associated with dysfunctional executive inhibition, whereas conduct problems are predominantly associated with dysfunctional motivational inhibition. Nonetheless, a \u2018motivational\u2019 pathway (or endophenotype) to cognitive deficits (e.g. altered reward sensitivity) in ADHD has also been identified (Solanto et al. 2001; Sonuga-Barke 2002). ODD\/CD and ADHD may be distinguished more easily on other aspects of emotional or social information processing. As a starting point it may therefore be good to use a theoretical framework that, in addition to motivation, accommodates more different aspects of emotional or social information processing. Such a model has been proposed by Zelazo and M\u00fcller (2002).\nHot and Cool Aspects of Executive Functioning\nZelazo and M\u00fcller (2002) made a distinction between the \u2018cool\u2019 aspects of EF more associated with dorsolateral regions of the prefrontal cortex (PFC) and the \u2018hot\u2019 aspects more associated with ventral and medial regions. Cool EF is elicited by abstract, decontextualized problems, whereas hot EF is elicited by problems that involve the regulation of affect and motivation (i.e., the regulation of basic limbic system functions). Neuro-anatomical evidence for a distinction between hot and cool executive functions stems from research into risky (\u2018hot\u2019) decision making versus ambiguous (\u2018cool\u2019) decision making (Krain et al. 2006). The authors performed a meta-analysis of studies measuring brain activity during risky decision making (involving low probabilities of high rewards and high probabilities of low rewards) and\/or ambiguous decision making (where the probability of a specific outcome is unknown or close to chance and choices do not differ in reward value), and found that there were significant differences between risky and ambiguous decision-making in regions of the orbitofrontal cortex (OFC: more activity in risky than in ambiguous decision-making), dorsolateral prefrontal cortex (DLPFC: more activity in ambiguous than in risky decision making) and anterior cingulate cortex (ACC: more activity in ambiguous decision-making than in risky decision-making in caudal regions, and more activity in risky than in ambiguous decision-making in rostral regions).\nZelazo and M\u00fcller use a problem-solving framework to discuss EF. Each stage of problem-solving (problem representation, planning, execution, and evaluation) is subserved by EF, which can take both hot and cool forms. They provide examples of each for every problem solving stage. For example, for the problem representation stage cool EF could be measured by asking participants to re-represent test items in different ways (but not act upon the re-representation). Hot measures of problem representation would include most tests of Theory of Mind, where participants are required to represent something (e.g. feelings, intentions) from multiple points of view. For the planning stage, tower tasks that require children to describe their plans without actually executing them are put forward as an example of \u2018cool\u2019 EF, whilst strategic deception, where participants are shown two pre-formulated plans, one deceptive and one not, and are asked to select the one that would trick the other person, was offered as an example of \u2018hot\u2019 EF. In the execution stage, card sorting (i.e. select according to two different rules\/principles) was given as an example of cool EF, and delay of gratification as an example of hot EF (e.g. choosing between a small reward immediately or a larger reward later (prudence) or between a reward for self now or a reward for self and other later (altruism)). Finally, in the evaluation phase error detection and error correction in the context of monitoring progress toward a goal are provided as examples of cool EF and error detection and correction in the context of extinction as examples of hot EF (i.e. after a certain amount of responding, it can be shown that the reward supply has been exhausted, a motivational input that might lead to response extinction). In the problem-solving framework inhibitory control is considered to underlie EF in all information processing stages and consequently has its hot and cool variants as well. Thus, when inhibitory control involves emotion, affect, and\/or motivation, it may be classified as a hot EF, whereas inhibitory control could be classified as cool EF when it is required in an \u201cabstract\u201d or \u201cneutral\u201d context.\nNeurobiology of Prenatal Tobacco Exposure and Externalizing Behavior\nThere is increasing evidence that maternal prenatal smoking is more strongly associated with ODD\/CD and with the co-occurrence of ODD\/CD and ADHD than with ADHD alone (Huijbregts et al. 2007; Nigg and Breslau 2007; Wakschlag et al. 2006). This evidence alone might be enough to suggest that children prenatally exposed to tobacco might specifically show cognitive dysfunction when emotion is involved, specifically when tasks involve inhibition. Further indirect support for such a hypothesis comes from neurobiological evidence showing similar or closely related effects on the central and peripheral nervous system associated with prenatal smoking and with antisocial\/disruptive behavior. Antisocial and disruptive behaviors have convincingly been associated with changes in the functioning of the autonomic nervous system (ANS) and the hypothalamic\u2013pituitary\u2013adrenal (HPA) axis. Both have very important roles in stress regulation. ANS-functioning is generally measured by (changes in) heart rate (HR), which reflects sympathetic and parasympathetic nervous system activity, and (changes in) skin conductance (SC), which reflects only sympathetic nervous system activity. HPA-functioning is generally measured by (changes in) the adrenal stress hormone cortisol. Resting HR, basal SC and basal cortisol levels were all shown to be lower in children with disruptive behavior disorders. In addition, reduced SC reactivity and blunted or absent cortisol responses to stress have consistently been reported (for reviews, see Ortiz and Raine 2004; Van Goozen et al. 2007). These findings have lead to the fearlessness theory of antisocial behavior (Raine 1993), which claims that low levels of arousal are markers of low levels of fear and the stimulation-seeking theory (Zuckerman 1979), which claims that low arousal represents an aversive physiological state and that individuals with tonically low arousal seek out stimulation to raise their arousal to optimal or normal level.\nAntisocial\/disruptive behavior has also been associated with neurotransmitter abnormalities, for example with impairments in the serotonergic (5-HT) system. The 5-HT system interacts with the HPA-system at several levels. For example, activation of (postsynaptic) 5-HT receptors in the hypothalamus stimulates HPA axis activity and causes elevation in cortisol, while a disruption of 5-HT neurotransmission by tryptophan depletion lowers HPA axis functioning (blunts cortisol-elevating effect of stressor). Glucocorticoids also modulate 5-HT receptor binding\/sensitivity (see Van Goozen et al. 2007). Importantly, the ANS\/HPA abnormalities in children with conduct problems have not been replicated in children with ADHD only (Herpertz et al. 2001; Snoek et al. 2004; Waschbusch 2002).\nAnimal studies investigating the consequences of prenatal nicotine exposure have shown an upregulation of nicotinic cholinergic receptor binding sites as well as persistent deficits in cerebrocortical choline acetyltransferase activity and hemicholium-3 binding to the presynaptic choline transporter, which is indicative of cholinergic hypoactivity (Seidler et al. 1992; Slotkin et al. 2006). These studies also show changes in noradrenergic responsiveness following prenatal nicotine exposure. The most important neurotransmitters of the ANS are acetylocholine (parasympathetic) and norepinephrine (sympathetic). In line with the chronic underarousal theories of antisocial\/disruptive behavior, it may be hypothesized that, without continued nicotine exposure, the increased number of cholinergic receptor binding sites will be understimulated, possibly resulting in stimulation-seeking and fearlessness. Furthermore, abnormalities in serotonergic neuromodulation following prenatal nicotine exposure have been indicated (suppression of the 5HT1A receptor subtype and upregulation of 5HT2 receptor subtype) (Seidler et al. 1992; Slotkin et al. 2006). We had already stressed the interactions between 5HT and HPA-functioning. The commonalities in the neurobiology of antisocial\/disruptive behavior and the neurobiology of prenatal smoking are striking. Moreover, factors affecting the same neurobiological system are more likely to reinforce each other\u2019s impact (Moffitt et al. 2005). A number of studies have already reported abnormalities in ANS\/HPA-functioning and levels of arousal in children of mothers who smoked during pregnancy (Browne et al. 2000; Horne et al. 2004; McDonald et al. 2006; Ramsay et al. 1996).\nThe Present Study\nTaken together, the strong associations between prenatal smoking and offspring conduct problems, the specific affect-mediated (\u2018hot\u2019) cognitive deficits supposedly associated with conduct problems, and the common neurobiology of prenatal nicotine exposure and conduct problems that particularly affects behavior and cognition under stressful conditions (i.e. behavior and cognition that is affect-mediated or \u2018hot\u2019), suggest that it would be useful to investigate aspects of hot cognition in children exposed to prenatal smoking (see also Raine 2002). Studies into associations between prenatal tobacco exposure and cognition have generally focused on academic achievement or rather broad cognitive constructs such as intelligence, verbal ability and executive functions (e.g. Batstra et al. 2003; Breslau et al. 2005; Huijbregts et al. 2006; Trasti et al. 1999), and have not yet used a distinction between hot and cool aspects of cognition. There are many possible causes for the inconsistent results which will be discussed later, but in the present study we will contrast hot and cool aspects of cognition, more specifically hot and cool forms of inhibitory control because a lack thereof has been hypothesized to be central to the behavior problems displayed by children of mothers who smoked during pregnancy. It is expected that children of mothers who smoked will particularly display problems with hot inhibitory control. Since a number of shared neural structures and neuromodulatory mechanisms are involved in cool and hot EF it cannot be ruled out that children prenatally exposed to nicotine will also have problems with cool inhibitory control but it may be hypothesized that these, if present, will be of a less serious nature.\nMethod\nParticipants\nForty children (16 boys, 24 girls; all of Caucasian ethnicity) attending two primary schools in Southampton (UK) participated in the study: 25 children from mothers who had not smoked during pregnancy (9 boys, 16 girls), 15 from mothers who had smoked during pregnancy (7 boys, 8 girls). Of these mothers, 6 smoked 1\u20139 cigarettes\/day and 9 smoked \u2265 10 cigarettes\/day (prenatal smoking was established through self-report by the mothers; although there is a risk for a social desirability bias, several studies have indicated a relatively strong association between retrospective self-report and blood\/urine cotinine-levels (i.e. the main nicotine metabolite), e.g. Law et al. 2003; Pickett et al. 2005). Mean age of the participants was 7.98 (range 7.0\u20139.0, SD\u2009=\u20090.68). Informed consent was obtained from all children and their parents\/caretakers and from the school boards. The study was approved by the University of Southampton\u2014School of Psychology Ethics Committee.\nTasks and Measures\nSustained Attention Dots Task\nThe Sustained Attention Dots task (SA-Dots, De Sonneville 1999) is a computerized task consisting of 600 presentations of 3-, 4-, and 5-dot patterns (random configurations). The 3, 4, or 5 dots are each presented 200 times. Participants use both hands for responding. They must press the mouse key of the non-dominant hand (the \u2018no-key\u2019) when 3 or 5 dots appear on the screen and the mouse key of the dominant hand (the \u2018yes-key\u2019) when 4 dots appear. A response has to be given between 250 and 6,000\u00a0ms after a signal. The fixed post-response interval is 250\u00a0ms. The program provides auditory feedback after an error. Since a response with the non-dominant hand is required twice as often as a response with the dominant hand, a response with the non-dominant hand becomes the prepotent response. Thus, inhibitory control is required when four dots appear. The less participants are capable of inhibiting biased response tendencies, the more misses (i.e. pressing the \u2018no-key\u2019 when four dots appear) they will produce relative to false alarms (i.e. pressing the \u2018yes-key\u2019 when three or five dots appear). The Bias score is the difference between number of misses and the mean of the number of false alarms when three dots appeared and when five dots appeared (see Huijbregts et al. 2002a). A second measure used to assess the quality of inhibitory control is post-error slowing. Post-error slowing is usually considered a strategic adjustment based on performance monitoring (Klein et al. 2007; Wiersema et al. 2007). In order to be able to slow down after an error inhibitory control is required. In order to control for general response speed, the difference between mean RT of correct responses after errors and overall mean RT of correct responses was selected as measure for post-error slowing (RTae).\nDelay Frustration Task\nThe Delay Frustration Task (DeFT, Bitsakou et al. 2006) is a task where participants are presented with a series of simple maths questions (only additions in the children\u2019s version) on a computer screen. Four possible solutions are displayed on the screen together with each question. Participants are asked to select the correct answer by pressing one of four buttons on a response box. The next question is presented as soon as a response has been recorded. However, on 16 out of 55 trials access to the next trial is delayed. On 8 out of the 16 trails in the post-response delay condition, a short delay (2\u201310\u00a0s) occurs, whilst on the remaining eight trials a delay of 20\u00a0s occurs. During the 20\u00a0s delay periods, the number (NP) and duration (TP) of responses made on a response key are recorded. These measures and their product (NP*TP) index delay frustration. In the instruction, participants are told that the computer shows signs of malfunctioning and that if the computer appears to not register their response, they should just wait until the next trial appears and do not have to respond again. It is considered to be indicative of inhibitory control if participants refrain from constantly pressing response box buttons during the delay periods. Practice sessions preceded both the SA-dots- and the DeFT test sessions, which were held in separate quiet rooms in both schools. Total test time per child was approximately 30\u00a0min (approximately 15\u00a0min for each task).\nStrengths and Difficulties Questionnaire (SDQ)\nThe parent\u2013teacher rated SDQ (Goodman 1997) is a short behavioral screening questionnaire of children aged 4 to 16\u00a0years, which can be completed within approximately 5\u00a0min. For the present study, teacher ratings were obtained, which correlate highly with parent ratings (Goodman 1997). The SDQ consists of 25 items comprising five different subscales (five items each): emotional problems, conduct problems, hyperactivity\/inattention, peer relationship problems, and prosocial behavior. The validity of this five-factor structure has been confirmed in large population samples (e.g. Goodman 2001). Each item is scored on a three-point scale: \u2018not true\u2019, \u2018somewhat true\u2019, or \u2018certainly true\u2019. \u2018Somewhat true\u2019 is always scored as 1, with the scoring of \u2018not true\u2019 and \u2018certainly true\u2019 varying with each item, being equal to either 0 or 2. A higher score indicates greater problems except for prosocial behavior, where a higher score indicates more positive behavior. A total difficulties score (range 0\u201340) is obtained by summing the scores of the emotional problems-, conduct problems-, hyperactivity\/inattention-, and peer relationship problems subscales. Of specific interest for the present study are the Conduct Problems (CP) scale, which has the following items: (1) often has temper tantrums or hot tempers; (2) generally obedient, usually does what adults request; (3) often fights with other children or bullies them; (4) often lies or cheats; and (5) steals from home, school, or elsewhere, and the Hyperactivity\/Inattention (HI) scale, containing the following items: (1) restless, overactive, cannot stay still for long; (2) constantly fidgeting or squirming; (3) easily distracted, concentration wanders; (4) thinks things out before acting; and (5) sees tasks through to the end, good attention span. Examples of items from the other scales are: \u201cPicked on or bullied by other children\u201d (peer relationship problems); \u201cMany worries, often seems worried\u201d (emotional problems); and \u201cHelpful if someone is hurt, upset or feeling ill\u201d (prosocial behavior). The SDQ has good reliability, whether it concerns internal consistency of the items (mean Cronbach\u2019s \u03b1\u2009=\u20090.73), cross-informant correlations or test\u2013retest reliability, and good criterion validity, whether it concerns its predictiveness of diagnosed psychiatric disorders, (mental health) service contact\/use, or the association of its scores with those on other behavioral screening questionnaires (Bourdon et al. 2005; Goodman 2001; Goodman and Scott 1999).\nStatistical Analysis\nMain analyses\nBecause the number of children participating in this study was not large and the scores on the SA-Dots Task, the Delay Frustration Task, and the behavior scales of the Strengths and Difficulties Questionnaire were not normally distributed, we used nonparametric tests. Spearman rank order correlations were used to test whether scores on the Delay Frustration Task were related to scores on the SA-Dots Task.\nMann\u2013Whitney U tests were employed to compare children of non-smokers and children of mothers who smoked during pregnancy on cognitive and behavioral (SDQ) measures. In order to test for dose-dependency children of smoking mothers were further subdivided into those whose mothers smoked between one and nine cigarettes\/day and those whose mothers smoked \u226510 cigarettes\/day. These two groups were compared to each other and to children of mothers who did not smoke using Kruskal\u2013Wallis H-tests.\nExploratory analyses\nOur final analyses were hierarchical linear regression analyses predicting those variables for which significant group differences were observed in Mann\u2013Whitney U tests from prenatal smoking and the sum of SDQ Hyperactivity-Inattention (HI) and Conduct Problems (CP) scores. Maternal prenatal smoking was introduced in a first block followed by CP+HI in a second block. These analyses should be considered exploratory because the sample is too small to reliably distinguish children with HI only or CP only. In order to determine whether HI or CP is more strongly associated with task performance than a combination of both of them, it would be necessary to have a HI only and CP only group as well (see Huijbregts et al. 2007; in press). Because the regression analyses are parametric tests, dependent variable scores were square root transformed in order to approach normal distributions. These exploratory analyses were performed because the relationship between prenatal smoking, externalizing behavior, and task performance can only be fully explored when all three variables are present in one analysis.\nResults\nMain analyses\nTable\u00a01 shows means (+SDs) of the smoking and non-smoking groups for the subscales of the SDQ and for the outcome measures of the SA-Dots and the DeFT. Scores on the SA-Dots Task and the Delay Frustration Task did not correlate significantly: Bias \u00d7 NP: r\u2009=\u20090.27, p\u2009=\u20090.143; Bias \u00d7 TP: r\u2009=\u20090.27, p\u2009=\u20090.139; Bias \u00d7 NP*TP: r\u2009=\u20090.26, p\u2009=\u20090.150; RTae \u00d7 NP: r\u2009=\u20090.16, p\u2009=\u20090.355; RTae \u00d7 TP: r\u2009=\u20090.13, p\u2009=\u20090.453; RTae \u00d7 NP*TP: r\u2009=\u20090.20, p\u2009=\u20090.267. Despite the fact that scores on all different subscales of the SDQ were related to each other, only the scores on the hyperactivity-inattention and conduct problems scales could be predicted by maternal prenatal smoking, as shown by the outcomes of a series of Mann\u2013Whitney tests. Thus, prenatal smoking did not predict the score on the emotional problems scale: Mann\u2013Whitney U\u2009=\u2009183.5, z\u2009=\u2009\u22120.46, p\u2009=\u20090.647, the peer relationships problem scale: Mann\u2013Whitney U\u2009=\u2009185.0, z\u2009=\u2009\u22120.44, p\u2009=\u20090.661, or the prosocial behavior scale: Mann\u2013Whitney U\u2009=\u2009194, z\u2009=\u2009\u22120.17 p\u2009=\u20090.865. In contrast, children of mothers who had smoked during pregnancy had more conduct problems (Mann\u2013Whitney U\u2009=\u2009134.5, z\u2009=\u2009\u22122.1, p\u2009=\u20090.038) and higher hyperactivity\/inattention (Mann\u2013Whitney U\u2009=\u2009124.5, z\u2009=\u2009\u22122.0, p\u2009=\u20090.041) than children of mothers who had not smoked (mean ranks for conduct problems: 25.7 for exposed children and 18.0 for non-exposed children; for hyperactivity-inattention: 25.1 for exposed children and 18.4 for non-exposed children) (see also Fig.\u00a01 for standardized CP and HI-scores for non-exposed versus exposed children). Dose\u2013response relationships were observed when behavior of non-exposed, moderately exposed and heavily exposed offspring was compared with Kruskal\u2013Wallis tests (conduct problems: mean ranks: 17.8, 22.4, and 26.8; \u03c72(2)\u2009=\u20095.7, p\u2009=\u20090.058; hyperactivity\/inattention: mean ranks 17.8, 19.1, and 29.1; \u03c72(2)\u2009=\u20096.5, p\u2009=\u20090.040) (Fig.\u00a02).\nTable\u00a01Mean scores (+SDs) for children with and without prenatal tobacco exposure on the strengths and difficulties questionnaire, the SA-dots task and the delay frustration taskGroup\u00a0Number (N)SDQSA-DotsDelay frustration taskCPHIRTaeBiasNPTPNP*TPOverall400.68 (1.2)2.8 (2.5)477.2 (507.1)9.0 (8.9)0.40 (.44)162.0 (145.5)123.2 (185.9)Not-Exposed250.48 (1.2)2.2 (2.2)473.6 (594.1)9.0 (10.3)0.29 (.40)116.9 (123.3)78.0 (139.5)Exposed151.0 (1.2)3.9 (2.6)483.6 (323.1)9.1 (6.5)0.57 (.46)234.1 (153.0)195.5 (229.6)SDQ: Strengths and Difficulties Questionnaire, CP: Conduct Problems, HI: Hyperactivity-Inattention, SA-Dots: Sustained Attention-Dots; Bias and RTae are the inhibitory control measures from the SA-Dots task: RTae: RT correct after error \u2013 RT correct general; Bias: Number of errors on 4-dot presentations \u2013 mean number of errors on 3 and 5 dot presentations. NP, TP and NP*TP are the inhibitory control measures from the Delay Frustration Task: NP: mean number of response button presses during 20-s delays. TP: time response buttons were pressed during 20-s delays.Fig.\u00a01Mean standardized scores on the SA-dots task, the Delay Frustration Task, and the Strengths and Difficulties Questionnaire for non-exposed vs exposed offspring. Bias and RTae are the inhibitory control measures from the SA-Dots task. DF-np (mean number of presses during 20\u00a0s intervals), DF-tp (mean duration of presses during 20\u00a0s intervals), and DF-np*tp (product of DF-np and DF-tp) are the inhibitory control measures from the Delay Frustration Task. SDQ-CP: Strengths and Difficulties Questionnaire\u2014Conduct Problems, SDQ-HI: Strengths and Difficulties Questionnaire\u2014Hyperactivity\/InattentionFig.\u00a02Dose-dependency in associations between maternal prenatal smoking and scores on the Delay Frustration Task and the Strengths and Difficulties Questionnaire. DF-np (mean number of presses during 20\u00a0s intervals), DF-tp (duration of presses during 20\u00a0s intervals), and DF-np*tp (product of DF-NP and DF-TP) are the inhibitory control measures from the delay frustration task. SDQ-CP: Strengths and Difficulties Questionnaire\u2014Conduct Problems, SDQ-HI: Strengths and Difficulties Questionnaire\u2014Hyperactivity\/Inattention\nWith respect to task performance, children of mothers who had smoked during pregnancy (mean rank\u2009=\u200925.5) showed a higher response frequency (number of button presses: NP) during intervals in the Delay Frustration Task than children of non-smokers (mean rank\u2009=\u200916.5) [Mann\u2013Whitney U\u2009=\u200997, z\u2009=\u2009\u22122.4, p\u2009=\u20090.016]. They also showed a longer response duration during intervals (time of button pressing: TP) (mean rank\u2009=\u200926.4) than children of non-smokers (mean rank\u2009=\u200916.0) [Mann\u2013Whitney U\u2009=\u200984, z\u2009=\u2009\u22122.8, p\u2009=\u20090.005] (Table\u00a01; Fig.\u00a01). A dose response relation was shown by Kruskal\u2013Wallis tests: NP: 0 cigs\/day: mean rank\u2009=\u200916.5; 1\u20139 cigs\/day: mean rank\u2009=\u200923.0; \u226510 cigs\/day: mean rank\u2009=\u200927.2; \u03c72(2)\u2009=\u20096.2, p\u2009=\u20090.044; TP: 0 cigs\/day: mean rank\u2009=\u200916.0; 1\u20139 cigs\/day: mean rank\u2009=\u200923.2; =10 cigs\/day: mean rank\u2009=\u200928.6, \u03c72(2)\u2009=\u20098.5, p\u2009=\u20090.014 (Fig.\u00a02). For frequency and duration combined (NP*TP), a Mann\u2013Whitney test also showed that children of smokers (mean rank\u2009=\u200926.0) showed less restraint during delays than children of non\u2013smokers (mean rank\u2009=\u200916.3): U\u2009=\u200990, z\u2009=\u2009\u22122.6, p\u2009=\u20090.009. A dose\u2013response relationship, indicating less inhibitory control during delays with an increase in prenatal smoking, was also observed (mean ranks for children of non-smokers, moderate smokers, and heavy smokers were 16.3, 22.8, and 28.1, respectively): \u03c72(2)\u2009=\u20097.5, p\u2009=\u20090.023 (Fig.\u00a02).\nThere were no significant differences between exposed and non-exposed offspring in inhibitory control scores from the Sustained Attention Task: Bias: U\u2009=\u2009130.5, z\u2009=\u2009\u22121.1, p\u2009=\u20090.255; RTae: z\u2009=\u2009\u22120.03, p\u2009=\u20090.988; Dose response: Bias: \u03c72(2)\u2009=\u20093.1, p\u2009=\u20090.209; RTae: \u03c72(2)\u2009=\u20091.4, p\u2009=\u20090.502.\nExploratory analyses\nAn important question that remains is whether the levels of CP and HI explain associations of prenatal smoking with DeFT-performance. As expected HI and CP were significantly related (Spearman\u2019s rho\u2009=\u20090.57, p\u2009<\u20090.001). Because not enough children with CP only or HI only could be distinguished in our sample, the sum of the SDQ CP- and HI-scores was used in subsequent regression analyses.\nHierarchical linear regression analyses introducing prenatal smoking in a first block and CP+HI in a second block to predict DeFT-scores NP, TP and NP*TP showed significant regression models for each of the DVs: NP: F(2,32)\u2009=\u20093.9, p\u2009=\u20090.029; TP: F(2,32)\u2009=\u20097.5, p\u2009=\u20090.002; and NP*TP: F(2,32)\u2009=\u20093.8, p\u2009=\u20090.039. In none of these models prenatal smoking remained a significant predictor when CP+HI was introduced: NP: \u03b2\u2009=\u20090.268 (p\u2009=\u20090.122), a reduction from \u03b2\u2009=\u20090.361 (p\u2009=\u20090.033) in the model without CP+HI; TP: \u03b2\u2009=\u20090.294 (p\u2009=\u20090.067), a reduction from \u03b2\u2009=\u20090.427 (p\u2009=\u20090.011); and NP*TP: \u03b2\u2009=\u20090.264, a reduction from \u03b2\u2009=\u20090.355 (p\u2009=\u20090.036). In models with maternal prenatal smoking, CP+HI most strongly predicted time\/duration of button pressing (TP): \u03b2\u2009=\u20090.393 (p\u2009=\u20090.016).\nDiscussion\nChildren of mothers who smoked during pregnancy exhibited problems with \u2018hot\u2019 inhibitory control, but not with \u2018cool\u2019 inhibitory control. Furthermore, children of mothers who smoked during pregnancy showed more conduct problems and hyperactivity-inattention than non-exposed children. The reliability of these effects was supported by the fact that dose\u2013response relationships were also observed. The finding that problems particularly appear when cognitive tasks involve the regulation of affect is in line with the well-replicated association between maternal prenatal smoking and behavior regulation problems. In addition, CP+HI could account for the effects of prenatal smoking on DeFT-performance. These findings further support the contention that children of mothers who smoked during pregnancy specifically have problems when cognitive task performance involves regulation of affect. Our results indicate the importance of employing a theoretical framework supported by neuro-anatomical and neurobiological data when studying associations between maternal prenatal smoking and children\u2019s cognitive and behavioral development.\nAlthough the pattern of results confirmed expectations, it was somewhat surprising that there were no group differences at all for the cool inhibitory control measures, nor were there significant correlations between SA-Dots inhibitory control scores and DeFT inhibitory control scores. Some associations were expected because hot and cool types of EF share common neural structures and neuromodulatory mechanisms, i.e. they are presumed to be part of an \u2018interactive functional system\u2019 (cf. Hongwanishkul et al. 2005), which is in line with the theory stating that cortical processes involving perception and cognition provide representations of the physical and social world to the sub-cortical motivational systems, whilst, in turn, these representations are influenced by appetitive and defensive needs (Derryberry and Rothbart 1997). We did not find evidence supporting such an overlap between hot and cool executive functions, but we cannot rule out that it would become apparent when other measures of cool inhibitory control are used.\nBecause cognition in children of mothers who smoked during pregnancy has always been studied without a clear theoretical framework, the employment of Zelazo and M\u00fcller\u2019s model is only a first step towards elucidating the cognitive profile of this population. Further explorations are warranted based on other cognitive models for which clear neuroanatomical evidence exists. It is possible that abnormalities in neuromodulation or neuroanatomical structures supporting hot EF only affect the most demanding cool EF-functions. The SA-Dots task in the present study had clear inhibitory control demands, but only minimal working memory or flexibility demands, which, particularly in combination with inhibitory control, might represent the most demanding forms of cool EF (e.g. Davidson et al. 2006; Huijbregts et al. 2002b).\nAlthough the present study was the first to use the hot versus cool distinction for examining EF in children whose mothers smoked during pregnancy, studies into ADHD and studies into differences in EF between children with ODD\/CD and ADHD have employed the same or similar distinctions earlier. In ADHD research significant empirical support has been found for a distinction between delay aversion (which can be considered a form of hot EF) and executive inhibitory control (which can be considered a form of cool EF) (e.g. Luman et al. 2005; Solanto et al. 2001, but see Geurts et al. 2006). Both characterize ADHD, but not necessarily simultaneously. Castellanos et al. (2006) formulated the interesting hypothesis that inattention symptoms may be associated with deficits in cool EF, whereas hyperactivity\/impulsivity symptoms reflect hot EF deficits. ADHD-research may however also have limited the conception of hot versus cool EF because hot EF has exclusively been studied in decision making paradigms, mostly (variants of) the Iowa Gambling Task (Bechara et al. 1994). Hot decision-making paradigms are indeed those that involve risks and rewards (Kerr and Zelazo 2004), but the definition of hot EF suggests that the distinction is applicable to most if not all executive functions.\nThis is important because, within the concept of hot EF, further distinctions may be justified based on which aspect of affect\/emotion is involved in performance of a task. For example, frustration and motivation (as measured by for example decision making or varying reward paradigms) are very different emotions with potentially different effects on task performance and different neurobiological correlates. Frustration is more closely related to stress, which, like nicotine, affects ANS\/HPA-functioning (stress: Dickerson and Kemeny 2004; Van Goozen et al. 2000; nicotine: Browne et al. 2000; Horne et al. 2004; McDonald et al. 2006; Ramsay et al. 1996). Despite some neuro-anatomical overlap between brain areas active during stress and during motivationally driven situations (e.g. orbitofrontal (OFC-) involvement: Hynes et al. 2006; Rolls 2004), there is no clear-cut evidence for strong ANS\/HPA-involvement during decision making tasks (i.e. tasks tapping into motivation). Thus, children of mothers who smoked during pregnancy might particularly demonstrate hot EF-deficits when stress or frustration are involved, but no hot EF deficits when motivation (or reward sensitivity) is involved.\nLimitations and recommendations for further studies\nLike prenatal smoking, deficient ANS\/HPA-functioning is also particularly associated with conduct problems and co-occurring conduct problems and hyperactivity (Herpertz et al. 2001; Waschbusch 2002). Unfortunately the number of participants in the present study was too small to reliably distinguish children with solely hyperactivity, solely conduct problems, and with co-occurring hyperactivity and conduct problems. Larger samples would also be required to investigate whether children with conduct problems present with different types of hot EF-deficits than children with ADHD. The main problem of a small sample size is that it limits the strength of confidence one can have in the generalizability of findings to other samples. That is why this study should be considered exploratory.\nFurthermore, it would be desirable for future studies to incorporate a number of control variables in the analysis of hot versus cool executive functions in children of mothers who smoked during pregnancy. There are many different types of variables that could account for associations between maternal prenatal smoking and hot EF-functioning. One important candidate is parental education or IQ, which unlike potential confounders such as alcohol or drug use during pregnancy, quality of parenting, family functioning, family status, mother\u2019s age when she had her first child and parental psychopathology, explained associations between maternal prenatal smoking and children\u2019s cognitive outcome measures in a number of studies (Breslau et al. 2005; Huijbregts et al. 2006; Trasti et al. 1999). Moreover, associations between maternal prenatal smoking and offspring cognition remained significant in studies where parental education or IQ were not introduced as potential confounders (e.g. Batstra et al. 2003). Despite the fact that these studies investigated associations of prenatal smoking with general cognitive outcome measures such as IQ and academic achievement, which have been reported to correlate significantly with measures of cool but not hot EF (Hongwanishkul et al. 2005), and despite the fact that participants in the present study were all from the same area of Southampton which can be considered relatively uniform regarding socio-economic status (family income, parental education etc.), we have not included a direct control measure for parental IQ or education and can therefore not rule it out as a potentially significant confounder. Moreover, confounders of the association between maternal prenatal smoking and hot EF may be different from those that explain associations between maternal prenatal smoking and cool EF. Another limitation is that, even though our study revealed a clear difference between results on the two inhibitory control tasks, we did not collect children\u2019s IQ\u2014or academic achievement measures ourselves. The interpretation of our results would have been stronger if, like Hongwanishkul and colleagues, we would have been able to show that such measures were associated with our cool EF-measure, whilst they would not have had a significant relation with our hot EF-measure.\nA final limitation we would like to address concerns the validity of our claim that the Delay Frustration Task measures hot inhibitory control whereas the SA-Dots task measures cool inhibitory control. Whereas the SA-Dots task has been used regularly before to measure inhibition of prepotent responding in different clinical populations characterized by deficient inhibitory control (e.g. Huijbregts et al. 2002a; Konrad et al. 2004; Slaats-Willemse et al. 2003), the Delay Frustration Task was originally developed to measure delay aversion (Bitsakou et al. 2006). In the dual pathway model of ADHD, delay aversion is considered a largely autonomous construct (Sonuga-Barke 2002). The advantage of considering this task a measure of delay aversion is that one could then make a stronger case for it being a measure of \u201chot EF\u201d, as such tasks have been used to measure this construct before. Still, the developers of this task recognize that it is likely that the Delay Frustration Task measures inhibitory control (i.e. participants have to stop a \u201cprepotent\u201d or \u201congoing\u201d response during the delays) (Bitsakou et al. 2006). Although based on Zelazo and M\u00fcller\u2019s theory DeFT should be regarded as a hot EF task regardless of whether it is considered a delay aversion or an inhibition task, it might be best for future studies to select tasks which are completely similar apart from an \u2018affect-\u2019 (e.g. frustration) component in one version.\nThe important message of this study is that two forms of EF can be distinguished which can be classified as hot and cool EF, and that in children of mothers who smoked during pregnancy a discrepancy can be observed between these two. Children whose mothers smoked during pregnancy are particularly at an increased risk of demonstrating (frustration-induced) hot EF-deficits. When these results are confirmed in larger samples that take into account the many potential confounders, intervention programs aimed at child factors could incorporate the training of specific emotion regulation skills in addition to the training of \u2018cool\u2019 EF-skills. Ideally, at risk families are identified before or early during pregnancy. Although prenatal smoking occurs in approximately 25% of pregnant women in Western countries (Breslau et al. 2005; Huijbregts et al. 2006), it has been shown that a family history of antisocial behavior in combination with prenatal smoking seriously compounds the risk of offspring disruptive behavior (Huijbregts et al. in press). Thus, like successful intervention programs aimed at increasing the knowledge about health (including smoking cessation) and care of mothers-to-be such as the Nurse Home Visitation Program (Olds et al. 1999), intervention programs aimed at improving child factors such as their emotion regulation skills should probably be provided to children with a combination of risk factors. When it is confirmed that ANS\/HPA abnormalities mediate associations between prenatal smoking and children\u2019s disruptive behavior and hot EF, clinical implications might also involve reinstating normal ANS\/HPA axis functioning pharmaceutically (Van Goozen et al. 2007), for example by temporarily saturating glucocorticoid receptors. By restoring stress response systems, emotion regulation and related cognitive functioning could be improved, perhaps even more strongly in disruptive children whose mothers smoked during pregnancy than in disruptive children whose mothers did not smoke during pregnancy.","keyphrases":["inhibitory control","prenatal smoking","conduct problems","hyperactivity","hot and cool executive functioning"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_J_Health_Econ-_-_-1388085","title":"The \u201cHealth Benefit Basket\u201d in The Netherlands\n","text":"This contribution describes the entitlements in Dutch health care and explores how these entitlements are determined and to whom they apply. The focus is on services of curative care. No comprehensive positive or negative list of individual services is included in formal laws. Instead, the legislation states only what general types of medical services are covered and generally the \u201cusual care\u201d criterion determines to which interventions patients are entitled. This criterion is not very restrictive and yields local variations in service provision, which are moderated by practice guidelines. It is conceivable, however, that the recent introduction of the DBC financing system will change the reimbursement and therefore benefit-setting policy.\nThis contribution gives a detailed account of entitlements in the Dutch health care system. Furthermore, it explains who decides about the services covered and the legal context in which this is done. It also describes which parts of the health benefit basket are defined more implicitly and which more explicitly, outlining the criteria used for the definition of the basket. We present the main characteristics of the Dutch health care system, highlighting those that impact on the entitlements of the insured. We then present a detailed account of health care entitlements in curative care. Finally, we conclude by identifying the aspects that determine the health care package and require special attention in a European context. We specifically ask what future developments are likely to impact on this system for definition of entitlements.\nThe main characteristics of the system\nThe Dutch Constitution states that the government should provide services to improve and protect public health. Therefore about 80% of health expenditures are paid from public funds (mainly social insurance), the rest being financed from private insurance and out-of-pocket payments [1]. The health insurance system broadly consists of three compartments (see Table\u00a01). First, every citizen is entitled to long-term care covered under the Exceptional Medical Expenses Act (Algemene Wet Bijzondere Ziektekosten, AWBZ). The AWBZ scheme pays for about 43% of Dutch health expenditure. Secondly, for acute care individuals are either publicly insured under the Sickness Fund Act (Ziekenfondswet, ZFW) or privately insured (37% and 15% of healthcare expenditure, respectively). The ZFW scheme covers among other things the costs of hospital admissions, treatment by physicians, pharmaceutical care, and medical aids.Table\u00a01 The three \u201cpillars\u201d of the Dutch health care systemFirst pillarSecond pillaraThird pillarType of careLong-term careBasic curative servicesAdditional servicesPrimary regulation (laws)AWBZb,cPrivate insurancec, WTZc, ZFWbComplementary insuranceb,cSecondary regulation (decrees)Decree on Entitlement to Exceptional Medical Expenses InsuranceHealth Insurance Treatment and Services Decree\u2013Tertiary regulation (discretion of Minister)Regulation on Subsidies in AWBZRegulations on Medical Specialist Care\u2013Regulations on Entitlements to AWBZ-careRegulations Governing the Provision of Paramedical Assistancea To be replaced in 2006 by a statutory health insurance scheme that covers the entire populationb Administered by sickness fundsc Administered by insurance companies\nPersons who do not qualify for compulsory health insurance but cannot satisfy private insurers\u2019 acceptance criteria (for example, if they represent a high risk) have access to a \u201cstandard insurance package\u201d which is similar to that of the ZFW. This entitlement is specified in the Medical Insurance Access Act 1998 (Wet op de toegang tot ziektekostenverzekeringen, WTZ). The third compartment consists of complementary insurance policies that may be bought to extend coverage.\nThe basic insurance package is defined mainly at the central government level, although only categories of services are identified. The AWBZ and ZFW (acts of Parliament) offer a list of the functional categories of health care services to which a patient is entitled (e.g., pharmaceutical care, hospital care, rehabilitation services). These formal laws indicate in general terms which areas of care are covered by the insurance scheme, but they do not specify the entitlements of the insured in detail. Usually further specification is provided in lower types of regulations by the government or the Ministry of Health, Welfare, and Sport and its advisory bodies. The lower forms of regulation specify the contents and the extent of the entitlements, specify conditions, and delegate responsibilities. Two important governmental decrees are associated with the AWBZ and the ZFW, respectively: the Decree on Entitlement to Exceptional Medical Expenses Insurance and the Health Insurance (Treatment and Services) Decree.\nThe social health insurance system ensures that patients receive the care to which they are entitled. Management of the two-tiered public\/private system, however, has proven difficult for the government, especially since the system lacks sufficient incentives for efficiency and has unfavorable equality characteristics. These conditions stimulated major reforms. Government involvement will be reduced significantly in 2006 when a new Health Insurance Act comes into force. This forms the final phase of the so-called Dekker plan (1987). The Dekker proposals set out conditions for the introduction of regulated competition in health care which should make the system more responsive to the preferences of consumers and less burdensome to manage. Health insurers have become main actors in promoting cost containment since they now hold full budget responsibility and may enter into contracting with providers. The government has promoted competition by enforcing open enrollment with health insurers and through establishing agencies that should regulate competition (e.g., an antitrust agency has been active in the health care sector since 1998). Nevertheless, the government will remain responsible for defining the health basket. A comprehensive description of the Dutch health insurance system is presented elsewhere [2].\nThe following section concentrates on the services made available in the health basket and the way in which these are decided. This description is limited to the curative sector. A complete account of health care services available in all health care sectors is offered by Stolk and Rutten [3]. Note that the integration of social and private insurance schemes into a national insurance implies that the coverage of the ZFW will be extended to the entire population. For this reason the present contribution describes only the ZFW benefit package.\nThe benefit package for curative care\nThe definition of entitlements for curative care is typical for the Dutch system: new interventions are usually implicitly introduced into a largely unspecified benefit package. A small negative list explicitly excludes services from reimbursement. This reflects the absence of a systematic procedure for the evaluation of curative services when defining the basket.\nMedical specialist care\nThe ZFW specifies a general entitlement to specialist care by hospitals (both inpatient and outpatient care): patients are entitled to medical, surgery, and obstetric care. Entitlement to hospital care also exists under the AWBZ if the patient must stay in hospital longer than 365\u00a0days. The provided care is limited merely by professional norms following the \u201cusual care\u201d principle (appropriateness according to professional standards). Also, a general statement is made that health care should be provided efficiently. Therefore the definition of entitlements for hospital care is rather implicit and the \u201cusual care\u201d criterion is not very restrictive. However, the entitlements are conditional upon the referral by a general practitioner, by another specialist to whom a general practitioner had referred the insured, or, where obstetric care is concerned, by a midwife.\nA ministerial decree, the Regulations on Medical Specialist Care, further specifies entitlements by excluding specific forms of transplantation and plastic surgery. It also determines that only certain types of transplantations are covered, and states that entitlement to some services (e.g., plastic surgery) only exist if specific conditions are met. Finally, this regulation explicitly excludes services such as eyelid, ear or body sculpturing, in vitro fertilization (IVF), uvoloplasty for persons who snore, sterilization or undoing sterilization, and circumcision. It should be noted that IVF is a special case and its reimbursement policy changed several times. Today, health insurers are subsidized to fund IVF treatment for the insured after the first attempt has been paid out of pocket.\nPharmaceuticals dispensed in hospitals are part of the entitlement to hospital care. They are generally financed out of the hospital budget, but the insurer may separately reimburse an additional percentage (up to 80%) of the cost for very expensive hospital drugs.\nThe law remains rather vague about entitlements to hospital care, but a recently introduced pricing system has led to more explicit benefit definition. In 2005 a new system for hospital financing was introduced by law: the Diagnose Behandeling Combinaties (DBCs), a DRG-like system describing all products that are provided in hospitals. Medical experts were involved in the determination of these DBCs, which are defined as the whole set of activities (diagnostic and therapeutic interventions) of the hospital and medical specialist starting from the first consultation and diagnosis of the medical specialist in the hospital until discharge. Each DBC is characterized by a code combining information on diagnosis (International Classification of Diseases, 10th edition) and treatment. This system facilitates negotiations between health insurers and hospitals on prices (on a bilateral level), at the same time providing a catalogue of medical services. Table\u00a02 shows how many different DBCs are defined within a medical specialty distinguishing between list A (prices fixed by the National Health Tariffs authority until further notice) and list B (prices negotiated by Sickness Funds and hospitals from January 2005 onwards). This catalogue of DBCs allows the specification of which DBCs are included in the basis package: the \u201cred\u201d list presents the number of DBCs not covered by compulsory insurance as determined by the Health Care Insurance Board on the basis of the \u201cusual care\u201d criterion and current explicit exclusions. Also an \u201corange\u201d list exists with DBCs for which reimbursement is limited to certain groups of patients. Finally, the table lists the number of product groups that have been defined to cluster DBCs into homogeneous price groups, as negotiation on the price of each single DBC would not be feasible.Table\u00a02 Overview of the DBC system (January 2005)SpecialtyNumber of DBCsProduct groupsaList AaList BaRed listbOrange listbOphthalmology1,7893181415Otorhinolaryngology6926\u20133514Surgery3,671182850553Plastic surgery13,7126320554433Orthopedics 3,0581412443Urology37,71795011617438Gynecology5362263434Neurosurgery1,25327151329Dermatology39610323311Internal medicine3,33519\u201361249Pediatrics3,4921091261Gastroenterology10,946190\u201334221Cardiology353\u2013\u20134439Respiratory medicine1,054145537143Rheumatology2,042129519617Allergology599\u2013\u20138Thorax surgery1,034\u2013105027Psychiatry648\u2013\u2013\u20133Neurology2,73918446143Geriatrics918\u2013\u2013\u201316Radiotherapy468\u2013\u201319514Radiology13,216\u2013\u20134728Anesthesiology582\u2013103819Clinical genetics100\u2013\u20133Total104,3501,3761,5113,649641a Published at the website of the National Health Tariffs authority (CTG\/ZAio): http:\/\/www.ctg-zaio.nl\/index.phpb Published at the website of the DBC maintenance organization: http:\/\/www.DBConderhoud.nl\nAn exception to the way in which medical specialist services are regulated is mental health care, which is currently covered under the AWBZ. Also the first year of treatment is covered by the AWBZ and not by the ZFW, as is the case for other medical specialist services. The reason is that psychotherapy is often a long and intensive treatment. Ambulatory psychotherapy is reimbursed (maximum of 25 sessions, 50 in the case of personality disorders) if referral is by a general practitioner or psychiatrist. Patients\u2019 entitlements to mental health care through the AWBZ include treatment (therapy sessions and medication), supportive guidance, and accommodation. Special regulations apply to mental health care in children and to treatment of addictions. The objective is to make mental health care a part of the basic benefit package under the new Health Insurance Act and therefore shift it to compartment 2 in a competitive environment. To permit this change DBCs in mental health care are now being defined.\nPrimary care, dental care, and paramedical services\nPrimary care, i.e., medical and surgical care provided by general practitioners, is covered under the ZFW, as specified in the Health Insurance (Treatment and Services) Decree. Care involves mainly consultations and visits, the prescription of pharmaceutical care, referral to medical specialists, and minor operations. Entitlements to these services are also defined implicitly: any type and quantity of care consistent with professional norms is covered by the scheme.\nEntitlements to dental care have been limited in the past and are defined explicitly. The ministerial regulation \u201cDental Care Health Insurance\u201d stipulates that persons up to the age of 18\u00a0years are entitled to dental care under the ZFW. Dental care includes 14 types of services, among which are periodic checkups, fluoride application treatment, sealing, and periodontal care. Adults are entitled to dental care only under special circumstances: special dental conditions and physical or mental disabilities.\nParamedical care\u2014consisting of physiotherapy, Mensendieck or Cesar remedial therapy, speech therapy and ergotherapy\u2014is partly covered. The first nine sessions are excluded from reimbursement, but thereafter some patients over the age of 18\u00a0years are entitled to physiotherapy, Mensendieck or Cesar remedial therapy for the treatment of chronic conditions that are defined in the Regulations Governing the Provision of Paramedical Assistance.\nCurative care at home\nUnder exceptional conditions and with authorization by the sickness fund, specialized services of curative care may be provided at home. First, persons are entitled to kidney dialysis at home or in a dialysis center under ZFW. If the dialysis takes place at home, the costs of training a person to carry out dialysis or providing assistance during the procedure are also covered. The costs of inspecting and maintaining equipment, modifying the home, providing special sanitary fittings, and heating are also reimbursable. Second, the ZFW regulates the use of treatment for chronic intermittent ventilation. Those covered by the ZFW are entitled to treatment at a ventilation center. A center may lend the patient the equipment for use at home or in a location where several persons can use the equipment. Specialist and pharmaceutical care provided by or on the advice of the ventilation center are also included.\nDiscussion\nA liberal benefit package?\nIn The Netherlands decisions about the medical services offered to a patient are often left to the physician (and\/or the health insurer). Negative lists are in certain categories used to exclude services (e.g., the new red and orange lists of DBCs). The extent to which new or old technologies (therapy, diagnostic services, and pharmaceuticals) are provided to the patient in the context of a DBC is left to the providers. However, some medical services were explicitly excluded for not being considered to belong to the domain of health care (e.g., plastic surgery). Additionally, limits were set to the provision of physiotherapy and psychotherapy for a lack of demonstrated effectiveness. Restrictive legislation only exists in the pharmaceutical sector where an itemized positive list of individual products is frequently updated [4]. For several other functional categories there are also positive lists, but these are not frequently updated (e.g., for medical devices and vaccines). Regarding pharmaceuticals it is interesting to observe that the reimbursement of extramural drugs (prescription filled by the community pharmacist) is centrally regulated, while for the use of drugs in the hospital the policy is liberal. The liberal policy has led to wide variations in the availability of certain expensive drugs across hospitals and to referral of \u201cexpensive\u201d patients to specialized centers. The new hospital financing system will change this and will probably force decision makers to regulate the provision of expensive drugs within a DBC.\nProcedures and criteria for limiting the health basket\nThe package can be limited by management of positive or negative lists, copayments, the definitions of conditions restricting eligibility, and promoting best practice, i.e., effective and efficient provision of care. Each instrument is applied within the Dutch system, although some sectors are more loosely managed than others. On the whole, emphasis is put on offering incentives for efficiency. Recently a no-claim system was introduced: if someone insured under ZFW has no expenses, he receives a bonus of \u20ac255 from his insurer or the difference if his expenses are less than \u20ac255. The liberal character of the basket is further illustrated by the fact that there is no systematic and central effort to use common criteria for defining the package as a whole. Nonetheless, decisions tend to be guided by a quite homogeneous set of considerations.\nThe respective Ministers of Health and more specifically the Health Care Insurance Board have been using health technology assessment (and cost-effectiveness information) since the middle 1980s as a tool for deciding on large public programs (e.g., national cancer screening programs) and on the addition of expensive curative programs (e.g., transplant programs) to the package [5]. Especially in the case of pharmaceuticals dispensed outside the hospitals the role of cost-effectiveness as criterion for reimbursement is quite prominent. If the manufacturer can demonstrate greater effectiveness than existing drugs and wants a premium price, he must submit a pharmacoeconomic dossier and figures allowing the Health Care Insurance Board to base its policy on information on (cost-)effectiveness.\nFurthermore, many technology assessment studies have been financed from a special fund managed by the Health Care Insurance Board and later by the National Medical Research Council that have produced valuable information on the cost-effectiveness of new diagnostic and therapeutic procedures. Although not legally required, this information has been used to define the benefit package or for the development of practice guidelines.\nFurther criteria have emerged as constant factors in decision making: severity of illness, individual vs. collective responsibility, affordability, and leakage (the probability of use outside a targeted patient group) [6]. Although initially consistent use of these criteria may have been doubted, gradually patterns emerged. Severity of illness, for example, played a role in deciding to finance the lung transplantation program although it was demonstrated to have a high cost-effectiveness ratio [7]. In relation to budget impact it also played a role in the decision not to fund PDE-5 inhibitors in the treatment of erectile dysfunction. Responsibility and affordability have played a role in deciding to restrict the reimbursement of pills for minor illnesses and dentistry. Leakage played a role in the decision to limit funding of some medicines (e.g., clopidogrel) to specific patient group. The increased understanding of the balance between the criteria is also recognized by policy makers.\nThe role of practice guidelines\nAn important way to influence the providing of service is through practice guidelines. There are many disciplinary and interdisciplinary practice guidelines for both primary and secondary care, some of which are also based on economic evidence. In The Netherlands a government program supported the development of practice guidelines in 1998\u20132002 which led to 31 consensus guidelines for medical practice addressing 23 disorders across seven disease groups as defined by the International Classification of Diseases. One example is the guideline for the use of cholesterol-lowering drugs in primary and secondary prevention which sets rules for the prescription of statins according to the patient\u2019s personal risk profile.\nFuture prospects\nAn important development is the change in hospital financing, involving description of the benefit package in terms of DBCs which are or are not reimbursed. Currently it is being discussed how the definition of DBCs can be more detailed to include specific regulations on the technologies to be used within a certain treatment. A so-called \u201cmaintenance organization for DBCs\u201d is set up to (re)define DBCs.\nThe reforms of 2006 are intended to reduce government control in the health care sector and provide incentives to patients and insurers for cost consciousness. Health care should follow the preferences of consumers. It follows that entitlements are formulated in functional form rather than as specific facilities or services. It is still not clear whether local actors will assume a role in defining the basket, and whether the government will centralize decisions.","keyphrases":["the netherlands","health benefit plans","health services","health priorities","national health programs"],"prmu":["P","R","R","M","R"]}
{"id":"Pediatr_Nephrol-4-1-2214827","title":"Hypertension in children with chronic kidney disease: pathophysiology and management\n","text":"Arterial hypertension is very common in children with all stages of chronic kidney disease (CKD). While fluid overload and activation of the renin\u2013angiotensin system have long been recognized as crucial pathophysiological pathways, sympathetic hyperactivation, endothelial dysfunction and chronic hyperparathyroidism have more recently been identified as important factors contributing to CKD-associated hypertension. Moreover, several drugs commonly administered in CKD, such as erythropoietin, glucocorticoids and cyclosporine A, independently raise blood pressure in a dose-dependent fashion. Because of the deleterious consequences of hypertension on the progression of renal disease and cardiovascular outcomes, an active screening approach should be adapted in patients with all stages of CKD. Before one starts antihypertensive treatment, non-pharmacological options should be explored. In hemodialysis patients a low salt diet, low dialysate sodium and stricter dialysis towards dry weight can often achieve adequate blood pressure control. Angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers are first-line therapy for patients with proteinuria, due to their additional anti-proteinuric properties. Diuretics are a useful alternative for non-proteinuric patients or as an add-on to renin\u2013angiotensin system blockade. Multiple drug therapy is often needed to maintain blood pressure below the 90th percentile target, but adequate blood pressure control is essential for better renal and cardiovascular long-term outcomes.\nIntroduction\nHypertension is one of the most common sequelae of chronic kidney disease (CKD) in children [1]. CKD-associated hypertension develops by a large variety of pathophysiological mechanisms. Blood pressure is one of the most critical determinants of the progression rate of renal failure in children [2, 3], and cardiovascular mortality in childhood onset renal failure [4]. Therefore, good antihypertensive management can substantially contribute to better renal and patient survival of adults with childhood-onset CKD.\nAs in many other complications of chronic renal failure, patients often do not report symptoms of hypertension, so an active screening approach is needed to prevent end-organ damage.\nThis article aims to give a short overview of the different pathophysiological pathways that lead to hypertension in CKD in order to explain how these can be targeted by different therapeutic approaches.\nPathophysiology of hypertension in CKD\nFundamentally, increased blood pressure is caused by an increase in cardiac output and\/or of total peripheral resistance. Both can be altered by a plethora of different mechanisms in uremia and renal failure. Additionally, children with certain underlying diseases, e.g. glomerulopathies and polycystic kidney disease, are especially susceptible to hypertension [3, 5]. Figure\u00a01 gives an overview of the most important pathways involved.\nFig.\u00a01Interplay of different factors in the generation of hypertension in chronic kidney disease (BP blood pressure, CO cardiac output, TPR total peripheral resistance, PTH parathyroid hormone, Na sodium)\nActivation of the renin\u2013angiotensin\u2013aldosterone system plays a pivotal role in renal hypertension. While plasma renin activity is typically found to be markedly elevated only in patients with renal artery stenosis, many patients with CKD have \u2018inappropriately normal\u2019 renin levels (i.e. lower levels would be expected, considering their degree of hypertension and fluid overload [6, 7]). Hyper-reninemia occurs probably due to renin secretion in poorly perfused areas such as cysts and scars or after microangiopathic damage or tubulo-interstitial inflammation [8, 9] and leads to angiotensin\u00a0II-mediated vasoconstriction as well as aldosterone-mediated salt retention, thus increasing both total peripheral resistance and blood volume. Additional delayed effects of a high angiotensin\u00a0II tone include inflammation, cardiac hypertrophy and endothelial cell damage, mesangial cell proliferation and fibrosis [10], which contribute further to hypertension and end-organ damage.\nSodium retention and consequent fluid overload have long been recognized as causes of hypertension in CKD. Hypertensive children on dialysis have lower residual urine output than their normotensive peers have [5]. While inter-dialytic weight gain is correlated with the inter-dialytic increase in ambulatory blood pressure, the correlation is rather weak (in children r\u2009=\u20090.41 [11]). This may be due to delayed effects [12] but also points to important volume-independent factors regulating blood pressure (BP) in patients on hemodialysis. This is also illustrated by the fact that nephrectomy in children on dialysis lowers mean blood pressure, despite causing anuria [13]. It has been proposed that fluid overload leads to hypertension only in those patients in whom peripheral resistance fails simultaneously to fall, i.e. when additional factors interfere with vascular autoregulation [14].\nA growing body of evidence suggests that increased activity of the sympathetic nervous system (SNS) is an important volume-independent cause of hypertension. Campese et al. demonstrated that renal denervation improves both hypertension and increased sympathetic activity caused by phenol injection into rat kidneys [15]. Muscle sympathetic nerve activity is also elevated in hypertensive patients with chronic renal failure [16]. The underlying mechanisms of this phenomenon are, as yet, unclear and may include afferent signals from the failing kidney as well as dopaminergic abnormalities and the accumulation of leptin in CKD [17, 18]. Interestingly, not only beta blockade but also angiotensin-converting enzyme (ACE) inhibition can reduce the sympathetic hyperactivation of CKD [16, 19]. However, as sympathetic hyperactivity is also a feature of renovascular hypertension [20], essential hypertension and hypertensive patients with polycystic kidney disease [13], it appears that sympathetic activation also occurs independently of renal function. The most established cause for sympathetic over-activation is renal ischemia caused by renal artery stenosis [20, 21], but renal cysts might also cause local renal ischemia.\nWhile children with end-stage renal disease (ESRD) usually have normal plasma noradrenaline and adrenalin concentrations, hemodialysis per se leads to substantial increase in both plasma renin activity and catecholamines, which can contribute to hypertension [22].\nRecent experimental evidence suggests that renalase\u2014an amine oxidase specifically expressed by the kidney\u2014lowers blood pressure and heart rate. Its activity is markedly reduced in patients with ESRD [23]. However, whether the cardiovascular effects of this enzyme are really due to its catecholamine-metabolizing activity is still controversial [24].\nThere has been debate about the role of nitric oxide (NO) in mediating endothelial cell damage and hypertension in CKD. Newer studies have demonstrated that, in uremic patients, reduced NO stimulation leads to reduced agonist-induced endothelium-dependent vasodilatation, whereas other vasodilatory pathways are not affected. Renal failure leads to the accumulation of endogenous NO synthase inhibitors such as asymmetric dimethyl-L-arginine (ADMA), which appears to be due to increased generation and decreased metabolism rather than decreased clearance [25]. ADMA independently predicts overall mortality and cardiovascular events in patients with ESRD [26], as well as progression of CKD [27]. While ADMA is related to blood pressure in animal models of CKD, clinical studies have not found differences in blood pressure [25].\nEndothelial NO synthase is also suppressed by hyperparathyroidism in rats with CKD [28]. In contrast to ADMA levels, those of serum parathyroid hormone (PTH) correlate highly with blood pressure in patients with CKD [29]. Whereas acute infusion of PTH has a hypotensive effect, chronic hyperparathyroidism leads to accumulation of calcium inside vascular smooth muscle cells, enhancing their sensitivity to calcium and norepinephrine [30, 31]. This effect can be blocked by calcium channel antagonists.\nA number of drugs commonly administered in CKD can cause iatrogenic hypertension. For example, erythropoietin (EPO) causes blood pressure elevation over several weeks. This may be via arterial wall remodeling, causing increased vascular resistance [32]. Vaziri et al. have proposed that EPO acts directly on voltage-independent calcium channels in smooth muscle cells, leading to a decreased sensitivity to the vasodilatory action of nitric oxide [33]. The resulting possibility of calcium channel antagonists as \u2018specific\u2019 therapy for EPO-induced hypertension has been successfully tested in rats [34].\nGlucocorticoids lead to fluid retention by their mineralocorticoid effect. Cyclosporine\u00a0A causes vasoconstriction of glomerular afferent arterioles and hyperplasia of the juxtaglomerular apparatus, with subsequent increased release of renin and angiotensin\u00a0II [35]. Increased circulating catecholamines and endothelin-1 precursors, and increased renal sodium absorption via the Na-K-2Cl co-transporter in the loop of Henle [36], have also been demonstrated after cyclosporine\u00a0A treatment. Tacrolimus appears to be less pro-hypertensive than cyclosporine in children after renal transplantation [37], but, for other reasons, it is less commonly used prior to transplantation [38].\nTreatment with growth hormone leads to water and sodium retention by the distal nephron [39], mediated by increased intra-renal insulin-like growth factor (IGF)-1. However, growth hormone (GH) does not appear to increase blood pressure in children with CKD or Turner syndrome, despite both groups being prone to hypertension [40, 41].\nManagement\nOwing to the lack of acute symptoms and to the serious long-term consequences of hypertension, an active screening approach is necessary to detect elevated blood pressures early and to prevent end-organ damage [42]. In contrast to other complications of CKD, which become prevalent only in later stages of CKD, hypertension is already very common in CKD stage\u00a01, with over 63% of children affected. In CKD stages\u00a04 and 5 the incidence increases further to 80% [1]. More than 50% of children with ESRD have uncontrolled hypertension, despite widespread use of antihypertensive drugs [5, 43]. The frequency of blood pressure measurement during screening and during therapy should be appropriate to the patient\u2019s risk of developing uncontrolled hypertension. A routine for this is suggested in the flow chart in Fig.\u00a02. In general, we would consider all children with CKD to be at least at intermediate risk, and, therefore, we recommend 3-monthly clinic blood pressure (CBP) measurements; children with ESRD, multiple pro-hypertensive medications or confirmed hypertension should be considered as at high risk. In our experience 24\u00a0h ambulatory blood pressure measurement (ABPM) is a very valuable tool and should be performed annually in high-risk populations such as renal transplant recipients or those with rapidly progressive renal disease. ABPM is also useful to exclude white-coat hypertension, which is a problem even in children under long-term medical care [44]. While there are equivocal results about the long-term consequences of white-coat hypertension, end-organ damage could be clearly demonstrated in children with masked hypertension, i.e. elevated ABPM but normal CBP [45], underlining the usefulness of this method.\nFig.\u00a02Flow diagram for the choice of method to measure blood pressure during screening, follow up and treatment (CBP clinic blood pressure ABPM 24\u00a0h ambulatory blood pressure monitoring, HBP home blood pressure measurement BP blood pressure, BMI body mass index)\nWhichever method is used, techniques should be in accordance with international consensus statements, and the appropriate pediatric reference ranges are very important. These are now available for clinic [46, 47], ambulatory [48] and home blood pressure measurements [49]. The diagnosis of hypertension should be based on at least three clinic blood pressure measurements above the 95th percentile [46]. As white-coat hypertension is very common, and the effects of thorough investigation and treatment can be far-reaching, we feel ABPM is advisable in nearly all newly diagnosed hypertensive children. In countries where health care providers do not cover the cost of ABPM, home blood pressure measurements may be helpful in confirming the diagnosis, but they do not pick up the nocturnal blood pressure dynamics [44].\nOnce hypertension has been confirmed, good management should not focus only on pharmacological therapy, but also on detection and treatment of end-organ damage and, where appropriate, improvement of the dialysis regime and consideration of therapeutic life-style changes.\nEchocardiography and ophthalmological examination are the most important assessments of end-organ damage in hypertensive children. Left ventricular hypertrophy (LVH) is common in children with CKD, even during antihypertensive therapy [50]. LVH in patients with normal clinic blood pressure may indicate masked hypertension in untreated children [45] or insufficient efficacy of antihypertensive drug therapy in treated children (e.g. insufficient dose, non-compliance, or short duration of action of selected antihypertensive drugs). Echocardiography and ophthalmological evaluation should be repeated at regular intervals in children with initial signs of end-organ damage [46]. From personal experience we would also recommend follow-up examination in children with persistent hypertension or in those at high risk (e.g. ESRD). Additional assessments of end-organ damage include measurement of carotid intima media thickness and of pulse wave velocity, which reflect functional alterations of arterial wall properties caused by hypertension and other factors such as hyperparathyroidism [51]. These investigations are still mainly used for research purposes, but clinical use has been facilitated by the recent provision of pediatric reference ranges [52].\nIn hypertensive patients who are undergoing renal replacement therapy, improvement of the dialysis prescription should be the primary therapeutic approach to hypertension, before pharmacological treatment is started.Extracellular volume overload can be efficiently reduced by adequately long dialysis times. Even though the reduction of fluid overload may take several weeks to translate into normalized blood pressure, drug-free control of hypertension is possible in many, if not most, hemodialysis patients [12]. A number of studies have shown improved BP control with short daily, long intermittent or nocturnal hemodialysis [53]. While conventional dialysis schedules can also improve control of hypertension by targeting dry weight more aggressively [54], this tends to be tolerated less well, with more intra-dialytic hypotensive episodes. Lower concentrations of dialysate sodium also produce a moderate fall in blood pressure on a population level, with best results in patients with previously high blood pressure [55].\nIn addition to intensified dialysis, dietary sodium restriction is a useful adjunct to intensified dialysis in avoiding sodium overload, and it reduces intra-dialytic hypotensive episodes. Indeed, some authors find dietary sodium restriction plus low sodium dialysate to be equally effective as time-intensified dialysis, and they maintain that with good dietary advice a low salt diet need not be unpalatable [56]. However, restriction of fluid and salt intake requires considerable patient motivation, which is often a problem in the adolescent population.\nTherapeutic life-style changes can be effective in lowering blood pressure if obesity contributes to the patient\u2019s hypertension. Obesity in children with CKD is uncommon and usually associated with steroid treatment; however, it is seen more commonly in populations with a higher background risk of obesity [3]. While weight loss can be effective in reducing blood pressure in overweight children with normal renal function [57], unfortunately, there are no controlled studies of life-style interventions in children with CKD. In our experience calorie-reduced diets are rarely effective in this population, probably due to the multifactorial etiology of hypertension in renal disease. Also, the recommended intake of fresh fruit and vegetables may be hard to achieve if a potassium- and phosphate-reduced diet is also necessary. Interestingly, weight loss improves salt-induced increases in blood pressure in obese children [58]; therefore, combined calorie- and sodium-reduced diets may be particularly effective in obese CKD children who are salt retainers.\nPharmacological treatment remains the mainstay of antihypertensive management in all stages of CKD. In approximately 75% of children with CKD stages\u00a02\u20134, blood pressure control below the 95th percentile can be achieved by antihypertensive monotherapy, but 50\u201360% of children need more than one drug if intensified BP control (<50th percentile) is targeted [Effect of Strict Blood Pressure Control and ACE Inhibition on Progression of CRF in Pediatric Patients (ESCAPE) trial, unpublished results]. In children with ESRD adequate control is much harder to achieve: a review of the North American Pediatric Renal Transplant Cooperative study (NAPRTCS) database also showed that over 50% of children on dialysis have uncontrolled hypertension [43], and a Polish nationwide survey found that in only 57% of children with ESRD was hypertension adequately controlled, despite the use of multiple drug therapy in 65% [5].\nEven though multiple drug therapy is often required, it is advisable to start with a single drug at a low dose and to titrate upward until blood pressure is controlled [46]. Exceptions are hypertensive emergencies, such as hypertensive encephalopathy, when intravenous (i.v.) treatment should be started promptly.\nACE inhibitors and angiotensin\u00a0II type\u00a01 receptor blockers (ARBs) are the most useful drugs, as they are not only antihypertensive but also slow down the progression of renal failure more efficiently than do other antihypertensive treatments [59]. Surveys among pediatric nephrologists from both sides of the Atlantic show that ACE inhibitors are increasingly popular and are now the most commonly used drugs for pediatric hypertension [60, 61]. The renoprotective effect of renin\u2013angiotensin system (RAS) blockade is due to a combination of reduced proteinuria, lower intra-glomerular pressure through selective dilatation of the glomerular efferent arteriole, and anti-inflammatory and anti-fibrotic effects [10]. Additionally, RAS inhibition reduces the sympathetic hyperactivity seen in CKD [16].\nThere is convincing evidence from studies of adults that all proteinuric patients should receive renin\u2013angiotensin system blockade, even if they do not have hypertension [42]. Major side effects are a moderate increase of potassium and creatinine; these should be monitored more closely with declining renal function. Sexually active adolescent girls must use contraception. ACE inhibitor-induced cough appears to be less common in children than in adults [62]. There is no clear evidence suggesting clinical superiority of ARBs over ACE inhibitors [63], and a number of drugs in both classes are labeled for children. Food and Drug Administration (FDA) approval for children under 6\u00a0years old is expected soon for a number of ARBs.\nCombination therapy with an ACE inhibitor and an ARB should be considered for patients who continue to show proteinuria while undergoing monotherapy, since it is effective in further reducing proteinuria (and progression of CKD) in adults [63, 64]. Experience in children is positive but very limited [65\u201368], and the increased risk of hyperkalemia and renal failure should be realized [69]. Therefore, for improved control of hypertension without proteinuria, the combination of single RAS blockade with a diuretic is preferable. The use of fixed-dose combinations of RAS antagonists with a thiazide diuretic may be an option in adolescents where compliance is an issue, but less so in younger children where frequent dose adjustments are required [46].\nFrom trials in hypertensive patients without renal disease it appears that the blood pressure lowering effect, per se, is relevant for cardiovascular risk protection, without any class-specific benefits. Therefore, in essential hypertension, the choice of agents should be guided by the matching of the side effects profile of the individual drugs to the patient-specific risk factor profile [47]. In patients with CKD, meta-analyses of adult trials demonstrated superior renoprotection by ACE inhibitors, even after adjustment for blood pressure and urine protein excretion [70]. There is no conclusive evidence as to whether the inhibition of the renin\u2013angiotensin system is superior to other antihypertensives in non-proteinuric CKD patients [59,71,72]. Recommendations for adults prefer to err on the side of caution and suggest a low threshold for considering CKD patients proteinuric (200 mg protein\/g creatinine in spot urine) [42].\nDiuretics are less commonly used in children with CKD than in adults with CKD, due to the preponderance of hypodysplastic kidney disorders, which frequently present as salt-losing nephropathies. In patients with evidence of hypervolemia, thiazides and loop diuretics have proven most useful for controlling volume overload, and they have a very good side-effects profile. It should be remembered that, while thiazides are a popular first-line therapy in mild-to-moderate CKD, they are less effective when glomerular filtration rate (GFR) falls below 60\u00a0ml\/min per 1.73\u00a0m2 body surface area, and they are ineffective below 30\u00a0ml\/min per 1.73\u00a0m2. Therefore, furosemide should be preferred and used in adequate doses for CKD stages\u00a04 and 5. Mineralocorticoid receptor antagonists (e.g. spironolactone) are theoretically attractive in CKD, due to their synergistic actions with RAS antagonists, and the new selective receptor blocker eplerenone is devoid of anti-androgenic side effects. However, monotherapy (and, even more, combination therapy with RAS antagonists) is limited by the potentiated risk of hyperkalemia. During diuretic therapy, patients should be monitored for volume depletion and electrolyte disturbances. Long-acting formulations help to increase patient compliance [42].\nCalcium channel blockers are very potent anti-hypertensive drugs and, therefore, useful as add-on therapy in children with resistant hypertension. Dihydropyridine (DHP) drugs (e.g. nifedipine, amlodipine) act mainly as vasodilators and do not have cardiac side effects. Amlodipine has pediatric labeling and is available as a suspension, and doses need not to be adjusted to renal function.\nHowever, DHP-type calcium channel blockers (CCBs) increase intra-glomerular pressure and proteinuria, while non-DHP-type calcium channel blockers (e.g. verapamil and diltiazem) have an additional anti-proteinuric effect. In a long-term clinical trial of elderly patients with type\u00a0II diabetic nephropathy, non-DHP calcium channel blockers showed as equally an effective slowing of CKD progression, reduction of proteinuria and antihypertensive efficacy as did the ACE inhibitor lisinopril (while both were superior to the beta blocker atenolol [73]). However, there are no published safety data on any of the non-DHPs in children with hypertension, so they should be used with caution due to their known prolongation of the PR interval in adults [74].\nIntravenous administration of nicardipine is an option for controlling hypertensive crises, especially when the level of renal function is unclear or changing rapidly. It has been used safely, even in very small children with hypertension, despite reports of hypotension in normotensive newborns with asphyxia [74].\nBeta blockers can be used as second-line therapy for renal hypertension in children. However, they are contraindicated in asthma and can cause fatigue. All beta blockers require dose reduction with progression of CKD. They should be used with caution in heart failure, and their adverse metabolic effects make them less suitable for diabetics. The largest clinical experience, especially for infants, is available for propranolol. A sustained-release form of this drug allows once daily administration in larger children. However, other agents, such as atenolol, which have the advantage of being both long acting and \u03b21-selective, may be preferred in clinical practice.\nOther drugs are used less commonly, mainly due to their more severe side-effects profiles. Alpha blockers (such as prazosin) can be used in patients who also require them for control of bladder emptying or Raynaud\u2019s phenomenon. Centrally acting alpha agonists (such as clonidine) act via reduction of sympathetic nervous outflow. Rebound hypertension after discontinuation is a major problem of this drug. The vasodilators hydralazine and minoxidil are less suitable in CKD as they are less effective and cause salt and water retention.\nThe aim of antihypertensive management is the regression of end-organ damage (especially LVH) and the lowering of blood pressure below target values while minimizing drug side effects. The currently recommended treatment goal in hypertensive patients with CKD is a blood pressure of < 130\/80\u00a0mmHg in adults [42, 47] and < 90th percentile in children [46]. However, a meta-analysis of 11 randomized trials in non-diabetic adults with CKD showed differing results for proteinuric and non-proteinuric patients: while there was no increase in adverse renal outcomes with higher blood pressure in non-proteinuric patients, ideal systolic blood pressure for proteinuric patients (> 1\u00a0g per day) was 110\u2013129\u00a0mmHg. In proteinuric patients, renal survival decreased, with systolic blood pressures below 110\u00a0mmHg and above 130\u00a0mmHg. These data are hard to interpret conclusively, as persistent hypertension during treatment may also be a reflection of more severe underlying disease [70]. In children, the pending results of the ESCAPE trial may help us to elucidate optimal blood pressure targets.\nConclusion\nAdequate management of hypertension in CKD requires an active screening approach in order to prevent the significant renal deterioration and cardiovascular morbidity and mortality associated with high blood pressure. Owing to the plethora of different pathophysiological mechanisms involved, a whole range of therapeutic options is available. Non-pharmacological options should not be disregarded for obese children or for children on hemodialysis. Inhibitors of the renin\u2013angiotensin system should be preferred for proteinuric patients and, probably, also for non-proteinuric patients. Multiple drug therapy is often necessary to reach target blood pressure below the 90th percentile. For this, diuretics and calcium channel blockers are the most suitable options.","keyphrases":["hypertension","children","chronic kidney disease","pathophysiology","life-style changes","antihypertension therapy"],"prmu":["P","P","P","P","P","P"]}
{"id":"Immunol_Lett-2-1-2430030","title":"Murine neutrophils present Class II restricted antigen\n","text":"Neutrophils were originally described as short lived, terminally differentiated phagocytes that contribute only to the innate immune response. Recent evidence of neutrophil cytokine production and expression of numerous cell surface proteins has suggested that neutrophils are likely to influence adaptive responses and may satisfy the criteria of antigen presenting cells. Under certain inflammatory conditions human neutrophils express major histocompatibilty complex (MHC) Class II and the costimulatory molecules CD80 and CD86. We have employed a murine T cell hybridoma with a transgenic T cell receptor specific for ovalbumin peptide 323\u2013339 (OVA323\u2013339), and a green fluorescent reporter of T cell receptor ligation, to directly investigate neutrophil-T cell interactions. These cells provide an ideal model system, allowing precise identification of antigen specificity and a clear readout of T cell activation. Additionally, whilst murine neutrophils have previously been shown to stimulate MHC Class I-dependent CD8+ T cell activation, CD4+ T cells stimulation via MHC Class II-expressing neutrophils has not been investigated. We addressed this by isolating murine neutrophils, loading with OVA323\u2013339 and co-culturing with T cells specific for the OVA323\u2013339\/MHC Class II complex, and this resulted in T cell activation, as determined by expression of the green-fluorescent protein reporter. Antigen-pulsed neutrophils were also able to prime na\u00efve OVA-specific CD4+ T cells in a contact-dependent manner, as shown by proliferation and cytokine production. Activation of lymphocytes was not due to contaminating macrophages. These studies demonstrate that murine neutrophils present MHC Class II-restricted peptides and induce T cell proliferation, confirming findings in human neutrophils, and demonstrate a novel pro inflammatory effect of murine neutrophils.\n1\nIntroduction\nThe range of functions ascribed to neutrophils has expanded significantly, from terminally differentiated, short-lived phagocytes that are solely involved in innate immune responses to cells that are now believed to influence the development of adaptive immune responses. Emerging evidence indicates that neutrophils may communicate with T cells through direct cell contact via major histocompatibility complex (MHC) Class I, Class II [1,2], and co-stimulatory molecules [3,4]. Additionally, a neutrophil-derived cytokine milieu [5] may have significant impact on the adaptive response to infection, for example via neutrophil-derived IL-12 [6,7].\nThere is considerable evidence to suggest that neutrophils satisfy the criteria of antigen presenting cells, with some capacity to process antigen. Human neutrophils express MHC Class II, either following in vitro activation via CD11b [8,9], with IFN\u03b3, and GM-CSF [10,11] or following in vivo activation in RA synovial fluid [4,12], in patients with active Wegener's Granulomatosis [13], in patients receiving GM-CSF [14], and in persistent localized Staphylococcus aureus infections [15]. In addition to MHC Class II expression, stimulation of normal human PMN with IFN\u03b3, or via CD11b induces CD80 and CD86 expression [8,11]. Human neutrophil presentation of superantigens is documented [2]. However, there are conflicting reports of neutrophils\u2019 capacity to process and present tetanus toxoid on MHC Class II and activate T cells [1,2]. A capacity for extracellular antigen processing had been suggested as neutrophil gelatinase may cleave collagen to a Class II restricted immunodominant epitope [16].\nHarding et al. demonstrated murine neutrophil Class I restricted antigen presentation [17] and additionally show that neutrophils processed phagocytosed bacteria via an alternate MHC Class I antigen-processing pathway, and such neutrophils may \u2018regurgitate\u2019 processed peptide into the extracellular space and this peptide may then bind MHC Class I on neighbouring cells (e.g. macrophages) for presentation to CD8 cells [17].\nHuman studies of antigen-specific neutrophil-T cell interactions are confounded by low frequency of antigen-specific T cells in peripheral blood, variation of MHC allele expression, which therefore impedes ideal peptide antigen identification. To overcome these complications, we have employed a murine model system. Moreover, despite the important role of the neutrophil in the pathogenesis of numerous inflammatory diseases, many of which are modelled in rodents, there is a relative paucity of information on murine Class II restricted antigen presentation. We, therefore, sought to investigate whether murine neutrophils present antigens via MHC Class II.\n2\nMaterials and methods\n2.1\nMurine peritoneal neutrophil and macrophage separation\nPeritoneal exudate cells (PEC) were obtained as previously described [18]. Briefly, 6\u20138-week-old BALB\/c mice (Harlan Olac, Bicester, UK) received 1.5\u00a0ml i.p. Brewers thioglycollate medium (Difco, East Molesey, UK) and 4\u00a0h later animals were sacrificed and cells harvested by peritoneal lavage. PEC were washed once then resuspended in 8\u00a0ml PBS, and layered over 3\u00a0ml Histopaque\u00ae 1083 (Sigma, Poole, UK), then centrifuged at 700\u00a0\u00d7\u00a0g for 30\u00a0min at 20\u00a0\u00b0C. The enriched population of neutrophils contained within the cell pellet was washed twice in RPMI (Invitrogen, Paisley, UK) then resuspended at 2\u00a0\u00d7\u00a0106\u00a0ml\u22121 in medium supplemented with 2\u00a0mM l-glutamine, 100\u00a0IU penicillin, 100\u00a0\u03bcg\/ml streptomycin (all Invitrogen) and 10% foetal calf serum (Harlan). Peritoneal macrophages were obtained by adhering PEC to a polystyrene flask for 3\u20134\u00a0h after which remaining adherent cells were incubated with ice cold PBS (without calcium or magnesium) at 4\u00a0\u00b0C for 10\u00a0min. Cells were harvested with a cell scraper, then washed in RPMI and resuspended at 2\u00a0\u00d7\u00a0106\u00a0ml\u22121.\n2.2\nMurine lymph node preparation\nDO11.10 mice, with CD4+ T cells specific for the OVA323\u2013339 peptide in the context of the MHC Class II molecule I-Ad\n[19] were obtained originally from N. Lycke, University of G\u00f6teborg, Sweden. This T cell receptor is recognized by the KJ1.26 clonotypic antibody. Single cell suspensions of lymph nodes from DO11.10 mice were obtained by passing tissues through 100\u00a0\u03bcm Nitex membrane (Cadish, London, UK) using a 5\u00a0ml syringe plunger. To enrich for lymphocytes in lymph node cell suspensions, cells were resuspended at 4\u00a0\u00d7\u00a0106\u00a0ml\u22121 in complete RPMI, and cultured in 150\u00a0cm3 polystyrene flasks (Iwaki, Japan), for 2\u00a0h after which non-adherent cells were removed. Cells were washed three times in RPMI and resuspended at the desired cell concentration in complete RPMI with 25\u00a0mM HEPES.\n2.3\nAssessment of cell proliferation and cytokine production\nCells were cultured under conditions indicated in figure legends. Where indicated, transwells (0.2\u00a0\u03bcm membrane, Fisher Scientific, UK) separated cell populations. Cell proliferation was assessed by incorporation of [3H]thymidine (0.5\u00a0\u03bcCi\/well) for the last 18\u00a0h of culture. Cells were harvested using a Betaplate 96-well harvester (Wallac Oy, Turku, Finland) and [3H]thymidine incorporation measured on a Betaplate liquid scintillation counter (Wallac). Concentration of cytokines in supernatants were estimated by ELISA. Paired antibodies (Pharmingen, Oxford, UK) were used in accordance with the manufacturer's recommendations. All assays were optimised and validated prior to use. Maxisorp 96-well plates (Nunc) were coated with capture antibody in 0.1\u00a0M NaHCO3 overnight at 4\u00a0\u00b0C. Plates were blocked for 2\u00a0h; samples and standards added for 2\u00a0h, followed by biotinylated secondary antibody for 1\u00a0h, then ExtrAvidin peroxidase (Sigma) conjugate for 45\u00a0min. Plates were developed with TMB (3,3\u2032,5,5\u2032-tetra-methylbenzidine, KPL, Gaithersburg, USA), and then read at 630\u00a0nm with a Dynex MRX II plate reader and analysed using Dynex Revelation 3.2 software (Dynex, Middlesex, UK). The levels of cytokine in supernatants were calculated by comparison with recombinant cytokine standards (R&D Systems, Abingdon, UK).\n2.4\nHybridoma cells\nThe DO11.10-GFP hybridoma (kind gift of David Underhill, Department of Immunology, University of Washington, Seattle, Washington [20]) cells were grown in medium containing a 0.5\u00a0mg\/ml geneticin. Co-cultures were established in which neutrophils or macrophages were incubated with 10\u00a0\u03bcg\/ml OVA323\u2013339 peptide and DO11.10-GFP hybridoma cells, in the ratio indicated in the figure legends. Cells were cultured for 14\u00a0h, washed, and then analysed by flow cytometry.\n2.5\nFlow cytometry\nAliquots of 1\u00a0\u00d7\u00a0106 cells in 12\u00a0mm\u00a0\u00d7\u00a075\u00a0mm polystyrene tubes (Falcon BD, Oxford, UK) were resuspended in 100\u00a0\u03bcl FACS buffer (PBS, 2% FCS and 0.05% NaN3) containing Fc Block (Becton Dickenson, Oxford, UK) as well as appropriate antibody or isotype control. Neutrophils were identified with anti GR-1 (RB6-8C5, Caltag), macrophages with anti F4\/80 (C1:A3-1, Caltag), and MHC Class II assessed (2G9, BD Biosciences), and T cells identified using anti CD4+ (RM4-5, BD) and a clonotypic antibody specific for the OVA323\u2013339 TCR (KJ1.26). After washing, samples were analyzed using a FACSCalibur flow cytometer equipped with a 488\u00a0nm argon laser and a 635\u00a0nm red diode laser and analyzed using CellQuest software (both BD).\n2.6\nStatistical analysis\nStatistical analysis was performed on data as indicated in the figure legends, using Minitab software.\n3\nResults\n3.1\nPurification of murine neutrophils\nNeutrophils were purified from thioglycollate-induced peritoneal exudate cells (PEC), as there is a relative paucity of neutrophils in murine peripheral blood (data not shown). PEC were harvested 4\u00a0h post-thioglycollate administration, previously shown to be the peak of neutrophil recruitment, and after which an increasing proportion of macrophages are recruited [18]. Approximately 65% of thioglycollate-induced PEC were identified as neutrophils on the basis of GR1 staining (data not shown). PEC were further enhanced for neutrophils with a Ficoll gradient purification as previously described [18]. Following purification, samples were routinely 85% GR1 positive (Fig. 1a). Importantly, samples contained few macrophages (<4%) as determined by F4\/80 staining (Fig. 1b). Cytospins of neutrophil preparations were prepared and demonstrated characteristic multilobed nuclei in >85% or cells (data not shown). Macrophage samples were 50\u201360% F4\/80 positive cells (data not shown). Murine neutrophils, expressing GR-1, were found to express MHC Class II (Fig. 1c), albeit at low levels relative to F4\/80 positive macrophages (Fig. 1d).\n3.2\nMurine neutrophils are able to present peptide antigen\nThe murine T cell hybridoma, DO11.10-GFP, is activated by OVA323\u2013339 peptide in the context of I-Ad, without any requirement for co-stimulation. The hybridoma does not respond to peptide alone (Fig. 2, \u2018background\u2019). Neutrophils or macrophages alone had no effect on GFP expression by DO11.10-GFP. However, neutrophils or macrophages loaded with OVA323\u2013339 stimulated significant GFP expression by hybridoma cells when cultured at a 1:1 ratio. A dilution of macrophages was performed to estimate the contribution of contaminating macrophages to the results observed with neutrophil populations, demonstrating that at a ratio of 1 macrophage to 10 DO11.10-GFP cells, there was significantly less expression of GFP protein compared to that induced by neutrophils at a 1:1 ratio (Fig. 2). At a ratio of 1:100, there was no consistent GFP expression detected (data not shown). As there were <5% F4\/80 positive macrophages contaminating the neutrophil samples, these titration experiments indicated that only a small proportion of the T cell hybridoma activation observed in the \u2018neutrophil presentation\u2019 assays could be attributable to macrophage contamination.\n3.3\nNeutrophils can drive na\u00efve T cell proliferation and cytokine production\nTo investigate neutrophils antigen presentation in a co-stimulation dependent system, and the ability of neutrophils not only to present but also to prime T cells, neutrophils were co-cultured with primary T cells. Lymph nodes were obtained from DO11.10 transgenic BALB\/c mice. Single cell suspensions of lymph nodes were allowed to adhere to plastic for 3\u00a0h to remove the majority of adherent antigen presenting cells, after which the population was routinely 70% CD3 positive (data not shown). The presence of remaining endogenous APCs in the lymph node facilitated a strong response to OVA323\u2013339 peptide alone (Fig. 3a, c and e). Therefore, neutrophils and macrophages were pulsed with OVA323\u2013339 for 2\u00a0h then washed three times to remove unbound peptide prior to the addition of transgenic T cells. Cells were co-cultured for 72\u00a0h at the ratios indicated in the figure legends. Neutrophils pulsed with OVA323\u2013339-induced proliferation (Fig. 3b), IL-2 (Fig. 3d) and IFN\u03b3 (Fig. 3f) production. Low levels of IL-5 were detected in some experiments (data not shown). The neutrophil-induced responses were less than those evoked by macrophages at the same cell ratio. A titration of macrophages was performed to give an estimate of the contribution of contaminating cells, demonstrating that only a small proportion of neutrophil-induced T cell responses could be attributable to contaminating macrophages.\n3.4\nCell contact dependence of neutrophil T cell interactions\nTo determine the relative contribution of cell contact dependent mechanisms and soluble factors in neutrophil-T cell interactions, cells were separated by 0.2\u00a0\u03bcm membranes placed between the cell populations, allowing diffusion of soluble proteins but preventing cell contact. The responding OVA323\u2013339-specific T cells were not a 100% pure T cell population and contained some APCs, demonstrated by the lymph node response to OVA323\u2013339 alone (Fig. 4a and b \u2018OVA\u2019). Thus, the transwells permitted investigation of whether lymph node-derived APCs were presenting soluble proteins derived from dead or degraded neutrophils. Bone marrow-derived dendritic cells (DC) were used as a positive control. Free OVA323\u2013339 or ConA crossed the transwell membrane and stimulated cell proliferation and cytokine production (Fig. 4a). However, when antigen pulsed neutrophils were separated from the lymph node cells by the transwell, antigen-specific proliferation and cytokine production was reduced (Fig. 4a and b). Importantly, antigen pulsed cell cultures did not release sufficient free antigen to stimulate a response from the OVA323\u2013339-specific T cells below (Fig. 4a). Thus, the antigen-specific T cell proliferation and cytokine production induced by neutrophil antigen presentation is cell contact dependent.\n4\nDiscussion\nData presented here demonstrate that murine neutrophils are indeed capable of directly presenting antigen on Class II MHC and activating T cells independently of other APCs. The DO11.10-GFP hybridoma cells respond exclusively in a Class II restricted manner, and our data demonstrate that neutrophils, rather than contaminating cells, were mediating this response.\nAt comparable cell ratios, neutrophil-mediated T cell activation was lower than that mediated by macrophages, commensurate with reduced expression of MHC Class II by neutrophils. Although neutrophils have been reported to express co-stimulatory molecules, we detected only low levels of CD40 and were unable to consistently detect CD80 or CD86 in our neutrophil preparations (in contrast to the F4\/80 positive macrophages which expressed CD40, CD80 and CD86; data not shown). Thus it is possible that: co-stimulatory molecule expression below the limits of detection of methods used here is sufficient for inducing a response, that T cells are responding in a co-stimulation independent manner, or that co-stimulation has occurred via alternative molecules.\nNeutrophil antigen processing capacity was investigated using whole ovalbumin protein compared to OVA323\u2013339. These studies indicated that these neutrophils did not process the ovalbumin effectively, demonstrated by a lack of response by DO11.10-GFP hybridoma (data not shown). An MHC Class I processing pathway within neutrophils was elegantly defined with E. coli transfected to express a MHC Class I restricted epitope of OVA. This demonstrated that neutrophils are capable of non-classical antigen processing for presentation on MHC Class I [17]. Although neutrophil enzyme mediated degradation of collagen generates a Class II epitopes (14), there is no current evidence of any Class II processing pathway. It has been proposed that as macrophage phagosomes are adequate for Class II antigen processing [21], therefore a parallel system may exist in neutrophils. A definitive investigation of murine neutrophil MHC Class II antigen processing awaits transfection of bacteria with OVA323\u2013339, thus elucidating the effects on antigen processing of concomitant neutrophil activation by bacteria.\nThere is increasing evidence documenting the potential for neutrophils to acquire macrophage [22] or dendritic cell (3) characteristics, often in response to in vitro conditions. We document in vitro antigen presentation by in vivo activated neutrophils. It will be of interest to delineate whether such neutrophil behaviour can be observed strictly in vivo. Physiologically, the location of such neutrophil-T cell interactions is likely to be restricted to sites of chronic or established inflammation, for example persistent bacterial infection [15]. Neutrophils have been shown to degrade proteins to release antigens recognized by auto reactive T cells [16], and such antigens may then be transported to the draining LN by migrating DCs. Due to the neutrophils\u2019 unique proteolytic activities, this may represent a mechanism of presentation of novel or cryptic antigens. Furthermore, there is evidence that neutrophils may passively acquire membrane proteins, including MHC, from other cells [23]. In our GFP-hybridoma studies (Fig. 2), this seems unlikely as the neutrophils were harvested from the peritoneum before there was significant in vivo macrophage migration, and immediately purified in vitro. However, this phenomenon may have contributed to the neutrophil mediated T cell activation in co-culture (Fig. 3).\nThere are overall fewer studies of murine neutrophils compared to human neutrophils, which is likely to be a reflection of the ease of acquiring human neutrophils that constitute 70% of circulating leucocytes, compared to limited quantity of murine peripheral blood available and the relative paucity of neutrophils, contributing only 20% of circulating leukocytes in rodents. These studies were only possible through use of rodent cells, as the TCR transgenic T cells absolutely control antigen specificity of response, which was central to this investigation of neutrophil-T cell interactions. However, the significantly different proportion of neutrophils in murine and human peripheral blood raises questions as to whether murine and human neutrophils perform parallel functions as is assumed. Our data document a novel, Class II restricted antigen presenting function of murine neutrophils, confirming findings in other human systems, and expanding our understanding of the role of the neutrophil in the adaptive immune response.","keyphrases":["murine","neutrophil","antigen presentation"],"prmu":["P","P","P"]}
{"id":"Eur_J_Clin_Pharmacol-3-1-2039832","title":"Evaluation of limited sampling strategies for tacrolimus\n","text":"Objective In literature, a great diversity of limited sampling strategies (LSS) have been recommended for tacrolimus monitoring, however proper validation of these strategies to accurately predict the area under the time concentration curve (AUC0\u201312) is limited. The aim of this study was to determine whether these LSS might be useful for AUC prediction of other patient populations.\nIntroduction\nThe calcineurin-inhibitor tacrolimus, used widely after organ transplantation, has a narrow therapeutic index and highly variable pharmacokinetic characteristics. Close monitoring of the drug concentration is required to achieve an optimum efficiency by minimizing the risk of subtherapeutic and toxic blood concentrations. Efficacy and side effects of tacrolimus are highly correlated with the area under the time concentration curve (AUC0\u201312) [1]. Elevated tacrolimus concentrations may lead to severe side effects such as nephrotoxicity, neurotoxicity and hyperglycaemia [2\u20134], while subtherapeutic tacrolimus concentrations increase the risk of transplant rejection enormously [5\u20137].\nThe most exact way to monitor the total tacrolimus exposure is by creating 12-h pharmacokinetic profiles, which implies that the tacrolimus concentration should be measured at at least six different time points. The AUC0\u201312 can then be calculated according to the trapezoidal rule using the tacrolimus concentrations measured at different time points. Since recording a complete 12-h pharmacokinetic profile for every patient is not feasible in clinical practice, traditionally many transplant centres have used tacrolimus trough (C0) concentrations to estimate the tacrolimus exposure. Although tacrolimus C0 concentrations are generally considered to be a good indication of the total systemic drug exposure [1, 8], its usefulness in differentiating graft rejection episodes from nephrotoxicity has been questioned [6, 9\u201311]. Recently, the correlation between individual tacrolimus concentrations and AUC0\u201312 has been studied in kidney [12\u201318], liver [19], heart [20, 21] and lung [22] transplant recipients. In these studies, a poor association was found between the tacrolimus C0 concentrations and the AUC0\u201312, while tacrolimus concentrations measured at other time points showed much better correlations with the AUC0\u201312. Additionally, strategies have been developed that included a limited number of sampling time points within a short time post-dose, the so-called limited sampling strategies (LSS). Several two- and three-time-point sampling strategies showed a high correlation with the AUC0\u201312 in published studies and were able to predict the AUC0\u201312 more accurately than the C0 concentration alone [12, 15\u201318, 20, 22].\nBased on the number of published studies regarding LSS for tacrolimus, there seems to be a growing interest for non-C0 concentration measurements as an indicator of between-patient variability and as a guide for dose adjustments. Most of these studies recommend different LSS, but these strategies have not been validated with an independent population. Ting et al. [23] recently reported that validation of the different LSS with an independent transplant population is an absolute prerequisite. The question was whether the LSS described in literature could be used in other centres with different populations. Predictive value of LSS from literature was evaluated using our own renal post-transplant group of 37 patients with known AUCs. Also the predictive value of trough levels (C0 and C12) determined in our own population was investigated.\nMaterials and methods\nPatient population\nIn total, 37 Caucasian renal transplant recipients for whom a complete 12-h time tacrolimus concentration curve had been determined in a past clinical trial were included in this study (Table\u00a01). The transplant recipients underwent a renal transplantation at least 1\u00a0year ago. Patients taking medication known to interact with tacrolimus, who suffered from gastro-intestinal or liver disease, pre-transplantation diabetes mellitus or other disorders that could have altered the absorption of tacrolimus were excluded from this study as illustrated in Table\u00a01.\nTable\u00a01Demographic characteristics of renal-transplant recipientsDemographic characteristicsPatients (n\u2009=\u200937)Gender (male\/female)24\/13Age (years, mean\u2009\u00b1\u2009SD)51.3\u2009\u00b1\u200910.9Length (cm, mean\u2009\u00b1\u2009SD)174\u2009\u00b1\u20098.4Weight (kg, mean\u2009\u00b1\u2009SD)77.4\u2009\u00b1\u200913.5Body mass index (kg\/m2, mean\u2009\u00b1\u2009SD)25.6\u2009\u00b1\u20093.42Primary kidney disease (n)\u00a0Glomerulonephritis1\u00a0Chronic pyelonephritis2\u00a0IgA nephropathy4\u00a0Hypertensive nephropathy7\u00a0Diabetes mellitus nephropathy0\u00a0Polycystic kidney disease8\u00a0Unknown4\u00a0Other11Transplantation number (n)\u00a0First30\u00a0Second6\u00a0Third or more1Tacrolimus mono therapy (n)29Tacrolimus dose (mg kg\u22121 day\u22121, mean\u2009\u00b1\u2009SD)0.054\u2009\u00b1\u20090.029C0 (ng\/mL, mean\u2009\u00b1\u2009SD)6.59\u2009\u00b1\u20091.39AUC0\u201312 (ng\u00d7h\/mL, mean\u2009\u00b1\u2009SD)122.5\u2009\u00b1\u200931.1Cmax (ng\/mL, mean\u2009\u00b1\u2009SD)20.9\u2009\u00b1\u20096.5Tmax (h, mean\u2009\u00b1\u2009SD)1.24\u2009\u00b1\u20090.43Use of azothioprine\/MMF\/rapamycine\/steroids3\/4\/0\/2Time since transplantation (days, mean and range)1,542 (453\u20134,128)Haemoglobin (mmol\/L, ref. M: 8.2\u201311.0, F: 7.3\u20139.7)8.52\u2009\u00b1\u20090.83Haematocrit fraction (ref. M: 0.41\u20130.52, F: 0.36\u20130.48)0.41\u2009\u00b1\u20090.04ALAT (units\/L, ref. M: <45, F: <35)24\u2009\u00b1\u200913ASAT (units\/L, ref. M: <35, F: <30)17\u2009\u00b1\u200910Serum albumin (g\/L, ref. 34\u201345)37.0\u2009\u00b1\u20093.84Serum creatinine (\u03bcmol\/L, ref. M: 71\u2013110, F: 53\u201397)128\u2009\u00b1\u200929Creatinine clearance (Cockcroft-Gault; mL\/min, ref. 90\u2013140)58.4\u2009\u00b1\u200926.6Ref. are the reference values applied in the Clinical Chemistry and Haematology Laboratory of the University Hospital in Maastricht. M Male, F female, MMF mycophenolate mofetil\nPrior to the blood sample collection, there was no tacrolimus dose change for at least 1\u00a0week. After overnight fasting, the blood samples were collected immediately before (C0) and 0.5 (C0.5), 1 (C1), 2 (C2), 3 (C3), 4 (C4), 5 (C5), 7.5 (C7.5) and 12 (C12) h after the morning tacrolimus administration. Patients were not allowed to take food until 1\u00a0h after ingesting the tacrolimus dose and were advised to avoid grapefruit intake to prevent alterations in the tacrolimus metabolism. Demographic as well as clinical data were determined at the time of recording the 12-h time tacrolimus concentration curve. The study was performed in accordance with the Declaration of Helsinki and its amendments. The protocol was approved by the local Medical Ethics Committee and written informed consent for participation in this study was obtained from all patients.\nDetermination of tacrolimus concentrations\nThe tacrolimus blood concentrations were determined in ethylene diamine tetra-acetic acid (EDTA) whole blood, using a method based on high-pressure liquid chromatography tandem mass spectrometry (HPLC-MS\/MS). The assay is linear from 1 to 300\u00a0\u03bcg\/l. Intra-assay precision and accuracy were 3.4, 2.2, and 3.0% and 102, 94 and 94% respectively at 3.04, 6.23 and 13.0\u00a0\u03bcg\/l (n\u2009=\u20096). Inter-assay precision and accuracy were 8.2, 5.2, and 4.6% and 102, 94 and 93% (n\u2009=\u20099) respectively. Lower limit of quantification was 1.0\u00a0\u03bcg\/l. The laboratory participates in the International Tacrolimus Proficiency Testing Scheme.\nLimited sampling strategies investigated\nIn our opinion, a suitable limited sampling strategy for tacrolimus should consist of two or three time concentration points within a short time post-dose (\u22644\u00a0h) including a trough level. We selected 24 LLS from the literature [12, 15, 16, 18, 20, 22]. Eighteen of these strategies were based on regression analysis [12, 16, 18, 20, 22], and six other strategies were based on Bayesian fitting [15]. Also strategies based on the tacrolimus C0 and C12 concentrations were developed for our own renal transplant patient population and compared with above-mentioned LSS.\nPharmacokinetics and statistical analysis\nThe area under the tacrolimus time concentration curve (AUC0\u201312) was calculated from the time versus tacrolimus concentration plot using the linear trapezoidal rule in MWPharm 3.50 (Mediware, Groningen, The Netherlands). The predicted AUC0\u201312 (AUCpred), calculated with the 24 different LSS, were validated by determining the predictive performance as described by Sheiner and Beal [24]. The percentage of the prediction error (PE) and the percentage of the absolute prediction error (APE) are parameters often used for validation in LSS [12, 14\u201316, 18, 20, 22]. Given the high pharmacokinetic variability, an APE of less than 15% was considered clinically acceptable [16, 25, 26].\nPrediction bias was measured as a percentage of the prediction error [PE (%)] using the following formula:\nPrediction precision was measured as a percentage of the APE using the following formula:\nThe variance in the strength of association between the AUCpred and the AUCactual was reflected by the linear regression coefficient of multiple determination (R2). All values are expressed as mean\u2009\u00b1\u2009SD. All statistical analyses were performed with use of SPSS 12.0 software for windows (Chicago, IL, USA).\nResults\nEvaluation of predictive performances of the limited sampling strategies\nTable\u00a02 shows an overview of the studies describing the LSS evaluated in the present study. The regression equations and the R2 found by the investigators for the evaluated LSS are summarised in Table\u00a03. Table\u00a04 describes the R2, which represents the association between AUCpred and AUCactual and the calculated PE and APE of the 24 evaluated LSS for our 37 pharmacokinetic profiles. Thirteen of the 18 LSS (72%) based on regression analysis had a predictivity of >90%. Additionally, all except three of the LSS examined gave a better prediction of the complete AUC0\u201312 in comparison with LSS based on a trough concentration C0 and C12 (mean 62%). Predictivity of all six LSS based on Bayesian fitting was <90% (mean 66.8%). Additionally, Fig.\u00a01 illustrates an overview of the performances of the 26 LSS evaluated in our well-characterized population of renal transplant recipients.\nTable\u00a02Overview of the characteristics of transplant patients included in the studies that described limited sampling strategiesStudyTransplanted organNumber of patientsaNumber of AUC0\u201312 curves for validation (Ib\/NIc)Analytical methoddTime since transplantationeInclusion criteriafWong et al. [16]Kidney180\/18Imx II2.5\u00a0years1,2Aumente Rubio et al. [20]Heart220\/25Imx<1\u00a0year\u2013Pisitkun et al. [18]Kidney150\/15Imx II8.7\u00a0months1,2,3Armendariz et al. [12]Kidney2213\/14ImxUnknown\u2013Scholten et al. [15]Kidney4364\/20ImxDiffersg2Ragette et al. [22]Lung150\/31Imx7.3\u00a0months\u2013aNumber of transplant patients used in the included study for both developing and validating the limited sampling strategies.bNumber of AUC0\u201312 used for developing the limited sampling strategies.cNumber of independent (I) and dependent (NI) AUC0\u201312 used in the study to validate the created limited sampling strategies.dThe analytical method used to determine the whole blood tacrolimus concentration.eThe mean time after transplantation.fThe inclusion criteria used for the transplant patients in the different studies. 1 Tacrolimus administrated when patients were in the fasting state, 2 patients selected for using no interfering medication with tacrolimus, 3 patients selected with a normal liver function test.gTwenty-two pharmacokinetic profiles were obtained within 2\u00a0weeks after transplantation, and 42 pharmacokinetic profiles were obtained between 6 and 52\u00a0weeks after transplantation.Table\u00a03Overview of limited sampling strategies and their reported coefficients of correlation (R2) with the complete tacrolimus AUC0\u201312EquationTime pointsRegression equationsR2Ref1.C00.542.C120.793.aC0, C2, C40.93[16]4.aC2, C40.93[16]5.aC0, C2, C40.97[20]6.C0, C40.95[20]7.C0, C1, C20.93[18]8.aC0, C1, C40.97[18]9.aC0, C2, C40.97[18]10.aC1, C2, C40.96[18]11.C0, C10.91[18]12.aC0, C40.95[18]13.a C1, C40.95[18]14.aC2, C40.96[18]15.a,bC0, C1, C40.97[18]16.a,bC0, C2, C40.96[18]17.C0, C1, C40.97[12]18.C0, C1, C3Bayesian estimation of the actual AUC0\u2013120.97[15]19.C0, C2, C3Bayesian estimation of the actual AUC0\u2013120.96[15]20.C0, C2, C4Bayesian estimation of the actual AUC0\u2013120.97[15]21.C0, C2Bayesian estimation of the actual AUC0\u2013120.94[15]22.C0, C3Bayesian estimation of the actual AUC0\u2013120.96[15]23.C0, C4Bayesian estimation of the actual AUC0\u2013120.95[15]24.aC0, C2, C40.98[22]25.C0, C40.96[22]26.aC2, C40.94[22]Limited sample strategies derived from the linear trapezoidal rule and the complete 12-h AUC.aLimited sampling strategies that are able to predict 90% of complete AUC0\u201312 of the renal transplant recipients within the absolute prediction error (APE) of 15%.bLimited sample strategies derived from the linear trapezoidal rule and the actual AUC0\u201312.Table\u00a04Evaluation of predictive performance of limited sampling strategies to estimate the complete AUC0\u201312 in the 37 renal transplant recipientsEquationTime pointsR2Mean PE (%)Mean APE (%)\u226415%a23.bC0, C40.760\u221214.9\u2009\u00b1\u200913.8 (\u221246.0\u201333.2)17.9\u2009\u00b1\u20099.43 (1.12\u201346.0)13 (35%)22.bC0, C30.779\u221211.5\u2009\u00b1\u200914.0 (\u221241.9 to 33.1)15.7\u2009\u00b1\u20098.83 (2.0\u201341.9)21 (57%)1.C00.5362.11\u2009\u00b1\u200914.8 (\u221227.1 to 24.4)12.3\u2009\u00b1\u20098.22 (0.7\u201327.1)22 (59%)11.C0, C10.7036.58\u2009\u00b1\u200914.8 (\u221226.5 to 43.7)12.6\u2009\u00b1\u200910.1 (0.1\u201343.7)24 (65%)2.C120.809.56\u2009\u00b1\u200911.6 (\u221212.7 to 29.9)12.0\u2009\u00b1\u20098.97 (0.3\u201329.9)24 (65%)19.bC0, C2, C30.502\u22124.44\u2009\u00b1\u200917.4 (\u221245.3 to 50.6)13.7\u2009\u00b1\u200911.4 (0.4\u201350.6)25 (68%)20.bC0, C2, C40.537\u22125.11\u2009\u00b1\u200916.3 (\u221243.1 to 50.3)12.9\u2009\u00b1\u200910.4 (0.2\u201350.3)28 (76%)18.bC0, C1, C30.5259.95\u2009\u00b1\u200919.4 (\u221229.7 to 88.8)13.1\u2009\u00b1\u200917.4 (0.4\u201388.8)30 (81%)25.C0, C40.911\u22127.83\u2009\u00b1\u20096.36 (\u221221.3 to 2.4)8.08\u2009\u00b1\u20096.02 (0.1\u201321.3)30 (81%)7.C0, C1, C20.8692.35\u2009\u00b1\u20099.96 (\u221217.2 to 27.3)8.03\u2009\u00b1\u20096.22 (0.0\u201327.3)31 (84%)6.C0, C40.896\u22125.97\u2009\u00b1\u20096.71 (\u221220.1 to 4.7)6.63\u2009\u00b1\u20096.04 (0.6\u201320.1)31 (84%)21.bC0, C20.802\u22123.69\u2009\u00b1\u200910.2 (\u221219.6 to 18.6)9.10\u2009\u00b1\u20095.67 (0.4\u201319.6)31 (84%)17.C0, C1, C40.9435.91\u2009\u00b1\u20097.06 (\u22128.8 to 26.3)7.02\u2009\u00b1\u20095.93 (0.2\u201326.3)33 (89%)15.cC0, C1, C40.9345.00\u2009\u00b1\u20097.28 (\u22129.8 to 25.8)6.81\u2009\u00b1\u20095.57 (0.2\u201325.8)34 (92%)14.C2, C40.9642.28\u2009\u00b1\u20096.58 (\u221217.1 to 16.1)5.45\u2009\u00b1\u20094.24 (0.7\u201317.1)35 (95%)24.C0, C2, C40.941\u22124.81\u2009\u00b1\u20095.26 (\u221217.3 to 2.8)5.32\u2009\u00b1\u20094.73 (0.1\u201317.3)35 (95%)13.C1, C40.9736.30\u2009\u00b1\u20094.84 (\u22125.9 to 17.8)6.68\u2009\u00b1\u20094.28 (0.3\u201317.8)36 (97%)8.C0, C1, C40.9673.37\u2009\u00b1\u20095.21 (\u22125.2 to 17.7)4.87\u2009\u00b1\u20093.80 (0.2\u201317.7)36 (97%)9.C0, C2, C40.9620.10\u2009\u00b1\u20096.37 (\u221216.7 to 14.7)4.71\u2009\u00b1\u20094.22 (0.3\u201316.7)36 (97%)26.C2, C40.9593.38\u2009\u00b1\u20095.24 (\u22127.6 to 15.5)5.20\u2009\u00b1\u20093.37 (0.0\u201315.5)36 (97%)10.C1, C2, C40.9763.07\u2009\u00b1\u20095.40 (\u221214.9 to 13.2)4.99\u2009\u00b1\u20093.64 (0.1\u201314.9)37 (100%)16.cC0, C2, C40.953\u22121.58\u2009\u00b1\u20095.29 (\u221214.9 to 10.1)4.00\u2009\u00b1\u20093.75 (0.0\u201314.9)37 (100%)12.C0, C40.9303.55\u2009\u00b1\u20096.30 (\u22129.8 to 14.3)6.29\u2009\u00b1\u20093.46 (0.1\u201314.3)37 (100%)4.C2, C40.963\u22121.66\u2009\u00b1\u20094.99 (\u221212.0 to 14.3)4.13\u2009\u00b1\u20093.20 (0.2\u201314.3)37 (100%)5.C0, C2, C40.9591.33\u2009\u00b1\u20095.24 (\u221211.8 to 14.0)4.22\u2009\u00b1\u20093.32 (0.5\u201314.0)37 (100%)3.C0, C2, C40.965\u22120.20\u2009\u00b1\u20094.79 (\u221210.4 to 13.7)3.64\u2009\u00b1\u20093.06 (0.2\u201313.7)37 (100%)aNumber and percentage of calculated AUC0\u201312 with a prediction error within 15%.bBayesian estimation of the actual AUC0\u201312.cLimited sample strategies derived from the linear trapezoidal rule and the actual AUC0\u201312.Fig.\u00a01An overview of the predictive performances of the limited sampling strategies published using 37 different pharmacokinetic profiles recorded in our renal transplant recipients. The number of limited sampling strategies categorized into the percentage of AUC0\u201312 within a prediction error of <15% is plotted on the x-axis, the number of limited sampling strategies is plotted on the y-axis\nDiscussion\nOur results confirm the results of several other studies [12, 14\u201316, 18, 20, 22] that trough concentrations C0 and C12 have a lower predictive value for the complete 12-h AUC than almost all other studied LSS. The predictivity of LSS based on Bayesian estimation of the AUCactual was lower than the LSS based on regression analysis. Therefore a trough level and one or two time points in the early phase (\u22644\u00a0h) post-dose seem not to be sufficient for a Bayesian estimation strategy to fit correctly most of the AUC0\u201312 and thus predict the complete AUC0\u201312 reliably. The differences in variability and shape between the curves of post-transplant recipients combined with just two or three sample points might have been caused by the large differences found between the AUCpred calculated according to the Bayesian estimation strategy and the complete AUCactual.\nIn contrast to most studies that describe LSS for tacrolimus in literature, we used an HPLC-MS\/MS assay to determine the tacrolimus concentration. Because there seems to be a fixed difference of about 15% between the immunoassay and the HPLC-MS\/MS, the prediction will change proportionally, and the predictivity of the LSS will be the same. Also potential interfering drug-drug interactions will have an equal influence on the different tacrolimus concentrations, which consequently has no effect on the predictivity of the different LSS.\nTing et al. [23] recently suggested that LSS should only be applied on transplant patient populations that are comparable with the transplant patient population that was used to develop the LSS. However, the renal transplant patient group examined in the present study was not exactly comparable with the transplant patient populations in which the equations for the LSS were developed. For example Aumente Rubio et al. [20] and Ragette et al. [22] used heart and lung transplant recipients respectively to develop and validate their LSS. Despite the fact that the LSS were developed with the pharmacokinetic profiles of patients who underwent a different kind of transplantation, Eqs. (5), (24) and (26) were able to predict at least 90% of the AUC0\u201312 within an APE of 15%, which suggests that these LSS are more robust than expected by Ting et al. [23]. Even though LSS gave a better reflection of the tacrolimus exposure, they are currently not often applied by clinical transplant practioners, possibly for logistical and financial reasons.\nIn conclusion, after validating several LSS from the literature, the present study indicates that all but three LSS gave a better prediction of the complete AUC0\u201312 than the trough concentrations C0 or C12. Moreover, LSS could produce satisfactory predictions for AUC0\u201312 recorded in an independent renal transplant patient population, although further evaluation of their reliability is necessary.","keyphrases":["limited sampling strategy","tacrolimus","auc0\u201312","trough level"],"prmu":["P","P","P","P"]}
{"id":"Appl_Microbiol_Biotechnol-3-1-1914294","title":"Functional analysis of genes involved in the biosynthesis of isoprene in Bacillus subtilis\n","text":"In comparison to other bacteria Bacillus subtilis emits the volatile compound isoprene in high concentrations. Isoprene is the smallest representative of the natural product group of terpenoids. A search in the genome of B. subtilis resulted in a set of genes with yet unknown function, but putatively involved in the methylerythritol phosphate (MEP) pathway to isoprene. Further identification of these genes would give the possibility to engineer B. subtilis as a host cell for the production of terpenoids like the valuable plant-produced drugs artemisinin and paclitaxel. Conditional knock-out strains of putative genes were analyzed for the amount of isoprene emitted. Differences in isoprene emission were used to identify the function of the enzymes and of the corresponding selected genes in the MEP pathway. We give proof on a biochemical level that several of these selected genes from this species are involved in isoprene biosynthesis. This opens the possibilities to investigate the physiological function of isoprene emission and to increase the endogenous flux to the terpenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate, for the heterologous production of more complex terpenoids in B. subtilis.\nIntroduction\nIsoprene (2-methyl-1,3-butadiene; I) or actually isopentenyl diphosphate (IDP; II) is the general precursor of all terpenoids, that represent a very diverse class of natural products. Two evolutionary distinct routes occur in nature for the biosynthesis of IDP. The spread of the two pathways is well investigated for organisms with a sequenced genome (Lange et al. 2000; Boucher and Doolittle 2000; Rohdich et al. 2001). In eukaryotes and archaea IDP and its isomer dimethylallyl diphosphate (DMADP; III) are formed via the mevalonate pathway (reviewed by Kuzuyama 2002). This pathway is well studied and for many organisms the enzymes are characterized and the encoding genes identified. More recently, another pathway to IDP was discovered in some eubacteria and in plastids of higher plants, that proceeds via the intermediate methylerythritol phosphate (MEP) (Rohmer et al. 1993; Rohmer 1999).\nMost Gram-negative bacteria, including Escherichia coli, use the MEP pathway and for E. coli this is now well documented. For Gram-positive bacteria the situation is less clear. Gram-positive cocci have been reported to only possess genes for the mevalonate pathway (Wilding et al. 2000). In Streptomyces aeriouvifer both pathways are found (Seto et al. 1996). In Bacillus subtilis, being regarded as the prototype for Gram-positives, only homologues of the genes for the MEP pathway are present (Wagner et al. 2000).\nFor E. coli the complete MEP pathway has been elucidated and the genes involved have been identified and the corresponding enzymes described (reviewed by Eisenreich et al. 2004). The pathway exists of seven subsequent enzymatic steps (Fig.\u00a01). The first reaction of the MEP pathway is catalyzed by a transketolase (DXS) and concerns the condensation of pyruvate (IV) and d-glyceraldehyde-3-phosphate (V) to 1-deoxy-d-xylulose 5-phosphate (VI) (Sprenger et al. 1997; Lois et al. 1998). 1-deoxy-d-xylulose is a branching intermediate in the biosynthesis of isoprenoids, thiamine (vitamin B1) (White 1978; David et al. 1981, 1982), and pyridoxol (vitamin B6), respectively (Hill et al. 1989). The second step is catalyzed by the IspC protein, initiating a rearrangement in the carbon skeleton followed by a reduction in the keto function of (VI), and finally delivering 2C-methyl-d-erythritol 4-phosphate (Takahashi et al. 1998). Five other subsequently acting enzymes are needed for the synthesis of IDP, including a phosphocytidyl transferase (IspD), a kinase (IspE), a cyclophosphate synthase (IspF), and two reductases (IspG, IspH) (Fig.\u00a01).\nFig.\u00a01Methylerythritol phosphate pathway of isoprenoids [Dxs (1), 1 deoxy-d-xylulose 5-phosphate synthase; IspC (2), 2C-methyl-d-erythritol 4-phosphate synthase; IspD (3), 4-diphosphocytidyl-2C-methyl-d-erythritol 4-phosphate synthase; IspE (4), 4-diphosphocytidyl-2C-methyl-d-erythritol kinase; IspF (5), 2C-methyl-d-erythritol 2,4-diphosphate synthase; IspG (6), 2C-methyl-d-erythritol 2,4-cyclodiphosphate; IspH (7), 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase; Idi (8), isopentyl diphosphate isomerase; IspA (9), farnesyl diphosphate synthase]. The putative genes are mentioned in italics for every biosynthetic step, including the essential gene YpgA, which is indicated with an *. Numbering of compounds and biosynthetic steps refers to the text\nBacteria use the precursors IDP and DMADP for the synthesis of several compounds including the side chains of ubiquinone or menaquinone. Next to that, several bacterial species use these precursors to synthesize isoprene, that is emitted to their environment (Kuzma et al. 1995). B. subtilis emits isoprene in high levels compared to other bacterial species as has been described for B. subtilis 6051, B. subtilis 23059, and B. subtilis 23856 (Kuzma et al. 1995). Using a bioreactor system, the emission of isoprene by B. subtilis 6051 was found to occur in three phases during the growth curve (Wagner et al. 1999). The phases correspond with respectively, glucose metabolism and secretion of acetoin, catabolism of acetoin, and the early stage of sporulation. The general laboratory strain B. subtilis 168 showed another pattern of isoprene emission, lacking phases 2 and 3 (Fall and Copley 2000). The function of the emitted isoprene has been postulated as being a signal molecule in the natural environment of the microorganism. Another possible explanation for the emitted isoprene is the efflux as an overflow metabolite in the bacterial pathway to isoprenoid structures (Fall and Copley 2000). The uptake of isoprene by microorganisms present in soil samples has been described as a sink of atmospheric isoprene (Cleveland and Yavitt 1998). However, there is no full evidence supporting one of the hypotheses mentioned. Isoprene originates from DMADP. The conversion of DMADP in isoprene is known to be an enzymatic process in the poplar tree from which a gene has been isolated and characterized (Miller et al. 2001; Schnitzler et al. 2005). Attempts to prove the enzymatic conversion to isoprene in B. subtilis suggested the involvement of an enzymatic step. Enzymatic activity has been partially purified, but the enzyme turned out to be very labile (Sivy et al. 2002). Until now the encoding gene could not be identified. A search for a homologue protein of the isoprene synthase from poplar tree in the genome of the B. subtilis did not yield a candidate. Based on homology with known genes, mostly from E. coli, candidate genes for the other enzymatic steps in the MEP pathway in B. subtilis have been suggested. Identification of the genes involved in this pathway should shed some light on the function of isoprene synthesis in Bacillus physiology. Due to the essentiality of the products downstream of the isoprenoid biosynthesis, like menaquinone, it is not remarkable that the candidate genes, except for the isomerase, have been shown to be essential genes for the survival of the bacterial cell (Kobayashi et al. 2003). In the construction of the BFAN collection these knock-out strains were not viable and therefore conditional mutants have been made using a pMUTIN3-vector. The expression in these strains can be regulated by the isopropyl-beta-d-thiogalactopyranoside (IPTG) inducible Pspac promoter (Yansura and Henner 1984; Vagner et al. 1998). Using this mutant system, expression levels can be regulated to a minimum for survival by decreasing the amounts of IPTG. In that way, the function of the genes can be investigated. Identification of the involvement of certain genes in the isoprenoid biosynthetic pathway, may create possibilities to redirect the metabolic flux toward IDP. The possibilities of pathway engineering have shown their strength already in other microorganisms, like E. coli and Saccharomyces cerevisiae. The metabolic flux was dependent mainly on the deoxyxylulose 5-phosphate synthase (DXS), the isopentenyl diphosphate isomerase (Idi), and the prenyltransferase delivering farnesyl diphosphate (IspA) (Harker and Bramley 1999; Kim and Keasling 2001; Kajiwara et al. 1997; Martin et al. 2003). By up-regulation of the MEP pathway, B. subtilis can be developed as an interesting host organism for the production of complex terpenoid compounds, such as the valuable drugs artemisinin and paclitaxel. In the present study we describe the functional analysis of genes in B. subtilis putatively encoding enzymes involved in the biosynthesis of the terpenoid precursor isopentenyl diphosphate. We used quantitative isoprene emission of different well controllable knock-out Bacillus strains in their environment to determine the function of the tested genes.\nMaterials and methods\nBacterial strains and media\nBacterial strains used are listed in Table\u00a01. B. subtilis strains 6051, 23059 and 23856 were obtained from the American Type Culture Collection (ATCC) (Rockville, USA). B. subtilis mutant strains were obtained through a chromosomal integration of pMUTIN3 derived plasmids (Vagner et al. 1998). Experiments were performed using Luria-Bertani (LB)-medium containing 1% bacto-tryptone, 0.5% yeast extract, and 0.5% NaCl (all purchased from Duchefa, Zwijndrecht, the Netherlands). If required, medium was supplemented with erythromycin (2\u00a0\u03bcg\/ml; Duchefa, the Netherlands) or the specified concentration of isopropyl-\u03b2-d-thiogalactopyranoside (IPTG; Duchefa, the Netherlands). For growing on plates the medium was solidified with 1.5% agar.\nTable\u00a01Putative genes for the MEP pathway in the genome of B. subtilis and the percentage of identities with known proteins from E. coli, the essentiality of each gene, the corresponding BFAN mutant strain (explained in detail in the text, with numbering of the respective enzymatic steps)EnzymeE. coliB. subtilisEssential geneMutant strainDxs (1)dxsdxs\/yqiE (43%)+168IyqiEIspC\/Dxr (2)dxryluB (43%)+168IyluBIspD (3)ygbPyacM (36%)+168IyacMIspE (4)ychByabH (27%)+168IyabHIspF (5)ygbByacN (56%)+168IyacNIspG (6)gcpEyqfY (46%)+168IyqfYIspH (7)lytByqfP (35%)+168IyqfPIdi Iidi\u2013\u2013Idi II (8)\u2013ypgA (39%)a\u2013168ypgAIpsA\/FPPS (9)ispAyqiD (43%)+168IyqiDaHomologue of Streptomyces protein (Kaneda et al. 2001)\nDetection of bacterial isoprene emission\nA single colony of the different B. subtilis strains was transferred from a plate to 10\u00a0ml Luria-Bertani (LB) medium (if required supplemented with 2\u00a0\u03bcg\/ml erythromycin and 100\u00a0\u03bcM IPTG) and grown over night (37\u00b0C; 300\u00a0rpm). Before the inoculation (1:100) of fresh LB medium containing different specified concentrations of IPTG, cells were gently washed three times with fresh LB-medium without IPTG by resuspending and centrifugating.\nIsoprene accumulation was measured on-line by sampling every 15\u00a0min, during a period of nine\u00a0hours, 15\u00a0ml of the air above 50\u00a0ml bacterial culture, growing (37\u00b0C; 300\u00a0rpm) in a 500\u00a0ml Erlenmeyer air tight flask (CBN, the Netherlands), and transferring into a gas-chromatography system suitable for the sensitive detection of isoprene (Syntech Spectras GC955 series 601, Synspec BV, the Netherlands) (Loreto and Delfine 2000). The air was pumped through a Tenax GA trap, desorbed at 180\u00b0C and transferred to an AT 5 column under a flow of 2.5\u00a0ml\/min nitrogen (3.7 bars; quality 5). The temperature program used was 3\u00a0min at 50\u00b0C followed by an increase in temperature to 70\u00b0C at 5\u00a0min; kept at this temperature until 12\u00a0min and than lowered to 50\u00b0C again. The isoprene present was detected by photo ionization at 10.6\u00a0eV. The gas chromatograph was calibrated using the dynamic gas dilution principle with several concentrations of gaseous isoprene using liquid isoprene (Sigma, USA) diluted in methanol and evaporated with a gas dilutor (MK5, MCZ Umwelttechnik, Germany). During the isoprene detection the growth of the bacterial culture was determined by measuring the optical density at 600\u00a0nm (OD600\u00a0nm) every hour.\nResults\nIsoprene emission in wild-type Bacillus strains\nThe emission of isoprene from B. subtilis wild type strains 168, 6051, 23059, and 23856 was investigated. For all four strains isoprene accumulated in the logarithmic phase of growth, leading to a high increase in isoprene in the flask. The absolute maximum of wild type strains varied between 400 and 700\u00a0\u03bcg\/m3. Starting from the late logarithmic phase to the beginning of the stationary phase the amount of isoprene in the flask decreased slowly.\nFigure\u00a02 shows the accumulation of isoprene during the growth of the wild type strains 168 and 6051. B. subtilis 6051 reached higher levels of isoprene, but this is probably not caused by a higher production rate rather than the amount of cells present, as reflected by the optical density of these strains. Corrected for the amount of cells, determined as the optical density at 600\u00a0nm, B. subtilis 168 accumulated at maximum 148\u00a0\u03bcg\/ m3 \/ OD600\u00a0nm compared to 166\u00a0\u03bcg\/ m3 \/ OD600\u00a0nm for B. subtilis 6051. The other two wild type strains tested, 23059 and 23856, showed a similar accumulation of isoprene as the strains described before. Corrected for the amount of cells, 23059 produced 192\u00a0\u03bcg\/ m3 \/ OD600\u00a0nm and 23856 produced 112\u00a0\u03bcg\/ m3 \/ OD600\u00a0nm isoprene at maximum accumulation (Table\u00a02).\nFig.\u00a02Isoprene emitted (solid lines) by wild type strains B. subtilis 168 (\u2022) and B. subtilis 6051 (\u25cb) during growth (dashed lines). Zero time (t\u2009=\u20090) indicates the transition point between the exponential and the post-exponential growth phasesTable\u00a02Maximum concentration of isoprene accumulated after emission by the different wild type Bacillus subtilis strains corrected for the amount of cells (OD600\u00a0nm)Bacillus subtilis strainMaximal isoprene accumulation (\u03bcg\/m3\/OD600\u00a0nm)16814960511662305919223856111\nMutant strains\nTo find the optimal conditions in which expression of the genes can be limited without killing the cells, each conditional mutant strain was subjected to IPTG depletion by growing the strains on agar plates containing different concentrations of IPTG varying from 0 to 1\u00a0mM. Decreasing the concentration of IPTG caused growth inhibition after overnight incubation resulting in smaller and finally no colonies (on plates without IPTG) at 37\u00b0C. Table\u00a03 shows the minimal concentration of IPTG at which still some growth was observed for the different conditional mutant strains. This concentration of IPTG was used in our experiments and defined as the condition causing limited induction. Most mutant strains indeed showed growth inhibition at lower concentration of IPTG. The mutant strain168IyqfY, the mutant for the putative gene encoding a homologue of the IspG protein (step 6) did not show any dependency on IPTG on plates (varying IPTG concentrations from 0 to 1\u00a0mM). Plates without IPTG incubated overnight resulted in the growth of normal colonies and the liquid cultures obtained growth curves comparable with wild type B. subtilis 168, independent of the concentration of IPTG. For some of the other mutant strains there was some growth of the bacterial cultures without IPTG as well, although severely impaired.\nTable\u00a03Maximum amount of isoprene accumulated after emission by the different mutant Bacillus strains, corrected for the amount of cells (OD600\u00a0nm)Bacillus strainLimited expression (\u03bcM IPTG)Maximal isoprene accumulation (\u03bcg\/m3\/OD600\u00a0nm)Relative decreaseLimited inductionFull inductionFull repression168IyqiE (1)1081192715.2168IyluB (2)131167135.4168IyacM (3)504113427.9168IyabH (4)252916075.6168IyacN (5)1014119208.8168IyqfY (6)\u2013n.d.194147\u2013168IyqfP (7)113130179.8168ypgA (8)\u2013\u2013\u2013103\u2013168IyqiD (9)101221661391.4The relative decrease is calculated as the maximal amount of isoprene at full induction devided by the maximal amount of isoprene at limited induction (dashes indicate that measurement did not apply here; explained in detail in the text, with numbering of the respective enzymatic steps).n.d., not determined\nFor all conditional knock out strains isoprene emission was measured at the concentration of IPTG with limited expression in comparison to 1\u00a0mM (full induction) and no IPTG (full repression).\nFigure\u00a03 represents the data for strain 168IyqiE, the conditional mutant strain for the first step in the biosynthetic pathway. Full induction of the dxs gene resulted in a normal growth curve of the bacterial culture and in an isoprene accumulation profile comparable to the profile of the wild type stain B. subtilis 168. Depletion of IPTG inhibited cell growth. The maximal concentration of isoprene accumulated in the flask and corrected for the amount of cells was 15 fold lower for the culture supplemented with 10\u00a0\u03bcM IPTG (8\u00a0\u03bcg\/ m3 \/ OD600\u00a0nm) than for the culture with 1\u00a0mM IPTG (119\u00a0\u03bcg\/ m3 \/ OD600\u00a0nm).\nFig.\u00a03Isoprene emitted (solid lines) by the B. subtilis mutant 168IyqiE during growth (dashed lines) in the presence of 1\u00a0mM IPTG (\u25be), 10\u00a0\u03bcM IPTG (\u25cb), and no IPTG (\u2022). Zero time (t\u2009=\u20090) indicates the transition point between the exponential and the post-exponential growth phases\nTable\u00a03 shows the maximum concentration of isoprene accumulated in the flask calculated on the amount of cells present at that time point for all tested mutant strains. Mutant strains for putative genes yluB, yacM, yabH, yacN, and yqfP encoding enzymes for respectively, steps 2, 3, 4, 5, and 7 showed significant differences in isoprene emission, varying from a 5\u201328 fold decrease at limited expression levels in comparison to full induction.\nThe knock-out strain of ypgA, putatively encoding an isopentenyl diphosphate isomerase (step 8), is the only knock-out of a nonessential gene. The mutant strain showed a normal growth curve and did not show a significant difference in the emission of isoprene in comparison to the wild type strain B. subtilis 168.\nWe also tested the function of yqiD, encoding a homologue for farnesyl diphosphate synthase (FDP; 7), synthase IspA (step 9) of E. coli. Depletion of IPTG to 10\u00a0\u03bcM did not result in a significant change in the isoprene emission by the mutant strain, while it did show to be dependent on IPTG for growth.\nDiscussion\nTo identify genes involved in the biosynthesis of the highly volatile compound isoprene, an efficient online detection system for this terpenoid (precursor) in the air above bacterial cultures was developed. Since the B. subtilis strains 6051, 23059, and 23856 were known to emit isoprene (Kuzma et al. 1995), we used these strains to set up the detection system for B. subtilis 168 for which the genome has been sequenced (Kunst et al. 1997) and functional knock outs have been made. All four strains emitted isoprene in our experiment. The differences in the amounts of isoprene emitted corresponded with the levels as described before, where B. subtilis 6051 and B. subtilis 23059 emitted higher amounts than B. subtilis 23856 (Kuzma et al. 1995). Since B. subtilis 168 showed emission of isoprene, the use of the experimental set up to investigate the isoprene synthesis by the mutant strains was justified. From the profile of isoprene emission with a maximum at the transition of the growth curve from the logarithmic phase to the stationary phase, it was concluded that from this time point on maximum isoprene concentrations had been reached and that amounts sampled from the culture every 15\u00a0min exceeded the production of new isoprene. We could not detect isoprene production in three phases using standard incubation in a shaking flask as has been described for B. subtilis 6051 grown in a bioreactor.\nThe conditional knock out strain of the yqiE encoding DXS, the transketolase responsible for the first step in the biosynthesis of isoprenoids, appeared to be highly dependent on IPTG. The dramatic decrease in isoprene production at low concentration of IPTG supported the role of this gene in the biosynthetic pathway of isoprene. yqiE has been identified as dxs encoding 1-d-deoxyxylulose-5-phosphate in B. subtilis before (Harker and Bramley 1999; Hecht et al. 2001). However, the confirming results for this mutant strain proved that the experimental set up of our work was valid to investigate the function of the other putative genes. Apparently, the isoprene emission is severely decreased when genes in the pathway are expressed at lower level than in the wild type strain.\nGrowth at the end of the incubation after several hours, as shown for many of the liquid cultures under IPTG depletion or even without suppletion of IPTG, may be explained by the occurrence of reversions in the mutant strain (Zanen et al. 2006). Another explanation could be the presence of a not fully repressed promoter in the complementation strain. It has been reported that in the used vector pMUTIN3 the Pspac promoter can give some low expression even in the absence of IPTG (Vagner et al. 1998; Petit et al. 1998). For genes showing low levels of expression in the wild type strain it is easy to get a pseudo wild type level of expression in the mutant strain. This may be in particular the case for highly efficient enzymes in the biosynthetic pathway.\nFor the putative genes yluB, yacM, yabH, yacN, and yqfP, encoding homologues of the enzymatic steps 2, 3, 4, 5 and 7, the isoprene accumulation of the conditional knock outs by depletion of IPTG clearly proved the involvement of these genes in the biosynthetic pathway isoprene. B. subtilis 168IyqfY (step 6) did not depend on IPTG and therefore nothing can be concluded from the data obtained with this mutant strain other than that this enzyme may be highly efficient leading to complementation already obtained at the low expression levels caused by leaking activity of the Pspac promoter (Zanen et al. 2006).\nThe nonessential gene ypgA encodes a homologue protein for an isopentenyl diphosphate isomerase (Idi) (step 8). The B. subtilis gene ypgA encodes for a so-called isopentenyl diphosphate isomerase type II protein (Takagi et al. 2004), while the genome of E. coli harbours a gene for a different Idi type I enzyme (Hahn et al. 1999). Interestingly, all archaea contain the Idi type II and all eukaryotes contain Idi type I. In genomes of eubacteria both are found, but there are also genomes without any of the described genes (Boucher and Doolittle 2000). In contrast to all other genes investigated in this study, the gene ypgA encoding for the IdiII was shown to be a nonessential gene in B. subtilis (Takagi et al. 2004). We also observed that the knock-out mutant of the bacterial strain is viable and produces isoprene. The idiI gene of E. coli has also been shown to be nonessential. Deletion mutants of this gene were viable on minimal medium (Hahn et al. 1999). Our findings, that the isoprene emission is not influenced by the knock out of the ypgA gene, can be explained by the enzymatic mechanism of the IspH protein, step 7, before the isomerase. IDP and DMADP can be synthesized independently by the catalytic action of IspH. A hypothetical mechanism for this reaction has been described (Eisenreich et al. 2004). This supports the nonessential character of the gene. The presence of isoprene proves that the isomer DMADP is still synthesized in the mutant and that the cells do not depend on the isomerase for the production of DMADP from IDP only. The isomerase functions in the balance of IDP and DMADP as a salvage protein (Eisenreich et al. 2004).\nThe first enzymatic biosynthetic step downstream the formation of the precursors IDP and DMADP is catalyzed by IspA, farnesyl diphosphate synthase (step 9). Therefore the putative gene is not involved in the biosynthesis of isoprene itself. The observation that isoprene is still emitted while the expression of the gene was depleted is easily explained by its function in the pathway downstream of IDP biosynthesis. Although some accumulation of the precursors IDP and DMADP could be expected by blocking yqiD, no significant differences in the flux toward the isoprene emission were detected. Apparently the efflux of isoprene is not influenced by the changes in FDP synthesis downstream in the isoprenoid pathway. This can be regarded as a contradiction to the hypothesis that isoprene emission functions as an efflux of an overflow metabolite.\nIn this study the involvement of several B. subtilis genes in the MEP pathway to isoprene was established by studying isoprene emission of mutants. Five genes, essential for viability, yluB, yacM, yabH, yacN, and yqfP, were shown to be essential for the isoprene production as well. Where these genes were at first instance depicted based on homology of the encoding proteins, the results of this study proved the functional involvement of the genes in the biosynthesis of isoprenoids on a biochemical level. A knockout of the sixth candidate gene, yqfY, did not yield a reduction in isoprene emission, nor a growth retardation, which might be explained by a not fully repressed Pspac promoter present in vector pMUTIN3. Knock-outs of the two other genes investigated in this study, showing no growth retardation as well as no reduction in isoprene emission, behaved in accordance with expectation.\nAlthough it remains unknown why isoprene is emitted by bacteria, the knowledge about the pathway to isoprene can be used for further investigations towards a better understanding of the metabolic flux to IDP and its physiological function. Next to that the IDP pool can be optimized by metabolic pathway engineering, creating a B. subtilis strain as an efficient Gram-positive host for the heterologous production of terpenoids, like the valuable plant-derived pharmaceuticals artemisinin and paclitaxel. The potential of using heterologous production organisms for the supply of terpenoids is already under investigation for E. coli and for several yeast strains (Martin et al. 2003; Lindahl et al. 2006; Ro et al. 2006; Dejong et al. 2006). For the purpose of metabolic pathway engineering it is interesting to search for a gene encoding isoprene synthase in B. subtilis as well. Evidence for an enzymatic bioconversion and the identification of this specific enzyme could give the possibility to block the efflux of isoprene thereby increasing the amount of isoprenoid available for the synthesis of terpenoids.","keyphrases":["isoprene","bacillus subtilis","terpenoid","mep pathway","methylerythritol phosphate pathway","gas chromatography"],"prmu":["P","P","P","P","R","M"]}
{"id":"Eur_J_Nucl_Med_Mol_Imaging-3-1-2121116","title":"Left ventricular dyssynchrony assessed by two three-dimensional imaging modalities: phase analysis of gated myocardial perfusion SPECT and tri-plane tissue Doppler imaging\n","text":"Purpose To compare left ventricular (LV) dyssynchrony assessment by phase analysis from gated myocardial perfusion SPECT (GMPS) with LV dyssynchrony assessment by tri-plane tissue Doppler imaging (TDI). Baseline LV dyssynchrony assessed with standard deviation (SD) of time-to-peak systolic velocity of 12 LV segments (Ts-SD) with TDI has proven to be a powerful predictor of response to CRT. Information on LV dyssynchrony can also be provided by GMPS with phase analysis of regional LV maximal count changes throughout the cardiac cycle.\nIntroduction\nCardiac resynchronization therapy (CRT) has become an established therapy for patients with end-stage heart failure [1]. However, up to 30% of the patients do not respond to CRT when selection is based on the traditional clinical and electrocardiographic criteria [New York Heart Association (NYHA) functional class III or IV, left ventricular ejection fraction (LVEF)\u2009<35%, and QRS duration\u2009>120\u00a0ms] [2]. In the search for better selection criteria for CRT, it has been shown that patients with LV dyssynchrony have a higher likelihood of a positive response to CRT [3\u20138]. Over the past few years, the assessment of LV dyssynchrony has been studied extensively with echocardiography.\nThe most frequently used technique is tissue Doppler imaging (TDI), which permits evaluation of timing of regional myocardial velocity and comparison of different regions yields information on LV dyssynchrony.\nBax et al. [3] have used a four-segment model to assess LV dyssynchrony (four basal LV segments: anterior, lateral, inferior and septal) and reported that a delay \u2265\u200965\u00a0ms among the four segments was a strong predictor of response to CRT. Other authors extended the analysis by evaluating the activation pattern of the entire LV. This method, originally described by Yu and co-workers [6\u20138], uses the standard deviation (SD) of electromechanical activation times based on a 12-segment model of LV (Ts-SD). The authors investigated 56 patients with heart failure who underwent CRT implantation and demonstrated that a cut-off value of 34.4\u00a0ms for Ts-SD could predict reverse LV remodeling after CRT with a sensitivity of 87% and a specificity of 81% [7]. However, to calculate this parameter of LV dyssynchrony, three apical views need to be acquired separately. A 3-dimensional (3D) TDI imaging modality (tri-plane TDI) has become available to overcome this limitation. Tri-plane TDI allows simultaneous acquisition of all LV segments during the same heartbeat rendering the technique more precise than the 2-dimensional (2D) TDI equivalent. Another recent development is the use of gated myocardial perfusion single photon emission computed tomography (GMPS) for assessment of LV dyssynchrony [9, 10]. A count-based method has been developed to extract phase information from the regional LV count changes throughout the cardiac cycle, and this phase information is related to the time interval when a region, in the 3D LV wall starts to contract (phase analysis). This technique has recently been implemented in the Emory Cardiac Toolbox as a diagnostic tool for assessment of LV mechanical dyssynchrony [9].\nTo further validate the use of GMPS with phase analysis for the assessment of LV dyssynchrony, a direct comparison with tri-plane TDI was performed in a cohort of heart failure patients.\nMaterials and methods\nPatients and study protocol\nForty consecutive patients with heart failure, referred for evaluation of potential eligibility for CRT, were included in this study. Traditional selection criteria for CRT were applied, including moderate-to-severe drug refractory heart failure (NYHA class III or IV), depressed LVEF (<35%) and prolonged QRS duration (>120\u00a0ms). Patients with a recent myocardial infarction (<3\u00a0months) or decompensated heart failure were excluded. Evaluation of the clinical status included assessment of NYHA functional class, quality-of-life score (using the Minnesota quality-of-life questionnaire) and exercise capacity (using the 6-min walk test). Extensive echocardiographic analysis, using 3D transthoracic echocardiography, was performed to assess LV dyssynchrony. Resting GMPS with technetium-99m tetrofosmin was clinically performed to exclude extensive ischemia and\/or viability [11, 12], in order to refer patients to revascularization if indicated. Thereafter, all scans were subjected to phase analysis to evaluate LV dyssynchrony. The results of the phase analysis with GMPS to assess LV dyssynchrony were subsequently compared with LV dyssynchrony derived from tri-plane TDI.\nEchocardiography\n3D data set acquisition\nAll patients underwent transthoracic echocardiography performed with a commercially available echocardiographic platform (VIVID 7, GE Vingmed Ultrasound, Horten, Norway) and equipped with a 3V-probe for 3D acquisition.\nPatients were scanned in left lateral decubitus position, from the apical window in tri-plane modus, acquiring simultaneously the apical four-, two- and three-chamber views. Care was taken to visualize the true LV apex. Color-coded TDI was applied to the tri-plane view to assess longitudinal myocardial regional function. Gain settings, filters and pulse repetition frequency were adjusted to optimize color saturation. Sector size and depth were optimized for the highest possible frame rate. At least two consecutive beats were recorded from each view, and the images were digitally stored for off-line analysis (EchoPac, GE Vingmed Ultrasound, Horten, Norway). The echocardiographic examination and the off-line analysis were performed by the same experienced echocardiographist, blinded to the GMPS and clinical data.\n3D analysis of LV dyssynchrony\nDuring post-processing, the tri-plane TDI dataset was used to analyze myocardial velocity curves as previously described with 2D TDI echocardiography [6\u20138]. Sample volumes were placed in the basal and mid segments of the septal, lateral, inferior, anterior, posterior and anteroseptal LV walls to calculate the time from the beginning of the QRS complex to peak systolic velocity (Ts), using the same heart beat (Fig.\u00a01). Subsequently, the standard deviation of Ts (Ts-SD) of the 12 myocardial segments was calculated and used as a parameter of LV dyssynchrony [6\u20138]. A cut-off value of Ts-SD \u2265\u200933\u00a0ms was used to define the presence of substantial LV dyssynchrony.\nFig.\u00a01Example of the myocardial velocity curves that can be derived by positioning the sample volume in any LV segment of the tri-plane dataset. This patient has substantial LV dyssynchrony: the postero-lateral and anterior walls (orange, light blue and red curves) are activated later than the septal and inferior walls (yellow and green curves); standard deviation of 12\u00a0LV segments' Ts is 57.5\u00a0ms\nGated myocardial perfusion SPECT\nResting GMPS with technetium-99m tetrofosmin (500\u00a0MBq, injected at rest) was performed using a triple head SPECT camera system (GCA 9300\/HG, Toshiba Corp.) equipped with low energy high-resolution collimators. Around the 140-KeV energy peak of technetium-99m tetrofosmin, a 20% window was used. A total of 90 projections (step and shoot mode, 35\u00a0s per projection, imaging time 23\u00a0min) were obtained over a 360-degrees circular orbit. GMPS acquisition involved 16 frames per cardiac cycle. Data were reconstructed by filtered backprojection and then re-oriented to yield gated short-axis images. Data reconstruction was performed over 360 degrees. These images were then submitted to the Emory Cardiac Toolbox for phase analysis [9]. GMPS images were analyzed at the department of radiology of Emory University School of Medicine by J.C. and E.V.G., who were blinded to the echocardiographic and clinical data. A phase distribution was extracted from a GMPS study, representing the regional onset of mechanical contraction of the LV. It can be displayed in a polar map or in 3D and used to generate a phase histogram. An example of a patient without LV dyssynchrony is shown in Fig.\u00a02a, whereas an example of a patient with substantial LV dyssynchrony as assessed with phase analysis by GMPS is shown in Fig.\u00a02b. The following four quantitative indices were obtained from the phase analysis of all patients: (1) histogram bandwidth, which includes 95% of the elements of the phase distribution; (2) phase SD, which is the SD of the phase distribution; (3) phase histogram skewness, which indicates the symmetry of the histogram; (4) histogram kurtosis, which indicates the degree to which the histogram is peaked [9]. In a healthy individual the LV contracts in a coordinated manner, and the phase distribution is nearly uniform with a highly peaked distribution of the histograms. As the LV contraction becomes dyssynchronous, the phase SD and histogram bandwidth are expected to increase (Fig.\u00a02b).\nFig.\u00a02a Example of a patient without LV dyssynchrony on GMPS. The non-normalized (upper panel) and normalized (lower panel) phase distributions are nearly uniform and the corresponding phase histograms are highly-peaked, narrow distributions. b Example of a patient (same as in Fig.\u00a01) with LV dyssynchrony on GMPS. The non-normalized (upper panel) and normalized (lower panel) phase distributions show significant non-uniformity and the corresponding phase histograms have widespread distributions\nStatistical analysis\nContinuous data are presented as mean\u2009\u00b1\u2009SD; dichotomous data are presented as numbers and percentages. Comparison of data was performed using the unpaired Student's t test or \u03c72 test when appropriate. Pearson\u2019s correlation analysis was performed to evaluate the relation between histogram bandwidth, phase SD, histogram skewness and histogram kurtosis by the phase analysis of GMPS and LV dyssynchrony (Ts-SD) by tri-plane TDI. A p-value\u2009<0.05 was considered to be statistically significant. A statistical software program SPSS 12.0 (SPSS Inc, Chicago, Il) was used for statistical analysis.\nResults\nStudy population\nA total of 40 consecutive patients with heart failure were included in this study [29 (72%) men; mean age 66\u00b110\u00a0years]. Baseline characteristics are summarized in Table\u00a01. Patients had severe LV dysfunction (mean LVEF 26\u2009\u00b17%), with extensive LV dilatation (mean LV end-diastolic volume 210\u00b148\u00a0ml). LV dyssynchrony assessment with tri-plane TDI demonstrated a mean value of Ts-SD of 34\u00b115\u00a0ms.\nTable\u00a01Baseline characteristics of the study population (n=40)Age (years)66\u00b110Gender (M\/F) 29\/11NYHA class 3.0\u00b10.46-MWT (m)282\u00b1126QoL score38\u00b117QRS duration (ms)147\u00b133Etiology, n (%)\u00a0Ischemic25 (62)\u00a0Idiopathic 15 (38)LVEF (%)26\u00b17LVEDV (ml)210\u00b148LVESV (ml)157\u00b143Medication, n (%)\u00a0ACE Inhibitors34 (85)\u00a0\u03b2-blockers28 (70)\u00a0Diuretics and\/or spironolactone36 (90)LVEDV = left ventricular end-diastolic volume; LVEF = left ventricular ejection fraction; LVESV = left ventricular end-systolic volume; 6-MWT = 6-min walk test; QoL = quality of life score; Ts-SD = standard deviation of time to peak systolic velocity of 12\u00a0LV segments\nHistogram bandwidth\nThe mean histogram bandwidth was 130\u00b0\u00b169\u00b0 (range 41\u00b0 to 260\u00b0); the normal value is 38.7\u00b0\u00b111.8\u00b0 for men and 30.6\u00b0\u00b19.6\u00b0 for women [9]. A good correlation between histogram bandwidth and LV dyssynchrony, assessed with tri-plane TDI, was observed (r=0.77, p<0.0001, Fig.\u00a03).\nFig.\u00a03Correlation between histogram bandwidth assessed with GMPS and LV dyssynchrony assessed with tri-plane TDI (Ts-SD)\nPhase SD\nThe mean phase SD was 40.2\u00b0\u00b118.8\u00b0 (range 14.5\u00b0 to 77.9\u00b0), whereas the normal value is 14.2\u00b0\u00b15.1\u00b0 for men and 11.8\u00b0\u00b15.2\u00b0 for women [9]. Pearson\u2019s correlation showed a good correlation between phase SD and Ts-SD (r=0.74, p<0.0001, Fig.\u00a04).\nFig.\u00a04Phase SD assessed with GMPS versus LV dyssynchrony assessed with tri-plane TDI (Ts-SD)\nHistogram skewness\nThe mean histogram skewness was 2.50\u00b10.81 (range 1.16 to 5.49), whereas the normal value is 4.19\u00b10.68 for men and 4.60\u00b10.72 for women [9]. A poor correlation with LV dyssynchrony assessed with tri-plane TDI was demonstrated by Pearson\u2019s correlation (r=-0.30, p=0.06).\nHistogram kurtosis\nThe mean histogram kurtosis was 7.38\u00b16.23 (range 1.08 to 37.42), whereas the normal value is 19.72\u00b17.68 for men and 23.21\u00b18.16 for women [9]. No correlation between histogram kurtosis and Ts-SD was shown by Pearson\u2019s correlation (r=\u22120.14, p=0.38).\nSubstantial versus no substantial LV dyssynchrony\nOf the 40 patients included, 20 (50%) had a substantial LV dyssynchrony (defined as Ts-SD\u2009\u2009\u2265\u200933\u00a0ms) [7, 8]. No significant differences in baseline characteristics were noted between the two patient groups (Table\u00a02). Histogram bandwidth and phase SD were significantly higher in patients with Ts-SD\u2009\u2009\u2265\u200933\u00a0ms compared to patients with Ts-SD\u2009<33\u00a0ms: 186\u00b0\u00b152\u00b0 versus 74\u00b0\u00b124\u00b0 (p<0.0001) and 55.3\u00b0\u00b113.6\u00b0 versus 25.1\u00b0\u00b17.6\u00b0 (p<0.0001), respectively (Fig.\u00a05a and b). In addition, histogram skewness was significantly lower in patients with Ts-SD\u2009\u2009\u2265\u200933\u00a0ms than in patients with Ts-SD\u2009<33\u00a0ms (2.23\u00b10.93 versus 2.78\u00b10.55, p=0.03). No significant difference between the two groups was observed for histogram kurtosis (6.36\u00b17.94 versus 8.4\u00b13.79, p=NS) (Fig.\u00a05c and d). An example of a patient with substantial LV dyssynchrony on both tri-plane TDI as well as on phase analysis with GMPS is provided in Figs.\u00a01 and 2b.\nTable\u00a02Baseline characteristics of patients with substantial (\u226533\u00a0ms) and no substantial LV dyssynchrony (<33\u00a0ms) assessed by SD of time-to-peak systolic velocity of 12\u00a0LV segments (Ts-SD)\u00a0Ts-SD\u2009\u226533\u00a0ms (n=20)Ts-SD\u2009<33\u00a0ms (n=20)p-valueAge (years)67\u00b11066\u00b19NSGender (M\/F)14\/615\/5NSQRS duration (ms)145\u00b131149\u00b136NSNYHA class 3.0\u00b10.43.0\u00b10.4NSEtiology, n (%)\u00a0Ischemic 14 (70)11 (55)NS\u00a0Idiopathic6 (30)9 (45)NSLVEF (%)25\u00b1726\u00b17NSLVEDV (ml)209\u00b147207\u00b150NSLVESV (ml)159\u00b146153\u00b142NSHistogram bandwidth (\u00b0)186\u00b15274\u00b124<0.0001Phase SD (\u00b0)55.3\u00b113.625.1\u00b17.6<0.0001Histogram skewness2.23\u00b10.932.78\u00b10.550.03Histogram kurtosis6.36\u00b17.948.40\u00b13.79NSFor abbreviations see Table\u00a01.Fig.\u00a05a Patients with substantial versus no substantial LV dyssynchrony (Ts-SD), using a cut-off value \u226533\u00a0ms. Histogram bandwidth is significantly higher in patients with substantial LV dyssynchrony. b Patients with substantial versus no substantial LV dyssynchrony (Ts-SD), using a cut-off value \u226533\u00a0ms. Phase SD is significantly higher in patients with substantial LV dyssynchrony. c Patients with substantial versus no substantial LV dyssynchrony (Ts-SD), using a cut-off value \u226533\u00a0ms. Histogram skewness is significantly lower in patients with substantial LV dyssynchrony. d Patients with substantial versus no substantial LV dyssynchrony (Ts-SD), using a cut-off value\u2009\u226533\u00a0ms. No significant difference in histogram kurtosis was demonstrated between patients without and patients with substantial LV dyssynchrony as assessed with tri-plane echocardiography\nIschemic versus idiopathic dilated cardiomyopathy\nIn total, 25 patients (62%) had an ischemic cardiomyopathy and 15 patients (38%) had an idiopathic dilated cardiomyopathy. No differences in echocardiographic variables, including LV dyssynchrony assessed by tri-plane TDI, or GMPS phase analysis indices were found between ischemic and non-ischemic patients. No significant differences in correlation coefficients were observed between patients with ischemic cardiomyopathy and patients with idiopathic dilated cardiomyopathy for the four different GMPS variables (Table\u00a03).\nTable\u00a03Correlation coefficients of LV dyssynchrony, assessed with tri-plane TDI, versus the different LV dyssynchrony parameters, derived from phase analysis of GMPS (idiopathic dilated cardiomyopathy versus ischemic cardiomyopathy)\u00a0Ischemic cardiomyopathy (n=25)Idiopathic dilated cardiomyopathy (n=15)p-valueHistogram bandwidthr=0.79r=0.78NSPhase SDr=0.77r=0.75NSHistogram skewnessr=\u22120.22r=\u22120.48NSHistogram kurtosisr=\u22120.07r=\u22120.35NS\nDiscussion\nThe main findings of the present study can be summarized as follows:\n(1) LV dyssynchrony assessment with phase analysis on GMPS is feasible in heart failure patients screened for eligibility for CRT implantation. The phase analysis variables histogram bandwidth and phase SD show good correlation with LV dyssynchrony as measured with tri-plane TDI (Ts-SD), suggesting that GMPS phase analysis can be used to quantify LV dyssynchrony. (2) Patients with substantial LV dyssynchrony on tri-plane TDI have significantly higher values of histogram bandwidth and phase SD derived from phase analysis with GMPS (histogram bandwidth: 186\u00b0\u00b152\u00b0 versus 74\u00b0\u00b124\u00b0, p<0.0001) and phase SD: 55.3\u00b0\u00b113.6\u00b0 versus 25.1\u00b0\u00b17.6\u00b0 (p<0.0001).\nCRT is now considered an important therapeutic option in the treatment of patients with end-stage heart failure [1]. Although encouraging beneficial effects have been reported, including improvement in LVEF and reduction in heart failure symptoms, it has also been demonstrated that up to 30% of patients do not have a favorable response to CRT. The presence of LV dyssynchrony is considered a key issue in the identification of potential responders to CRT [3\u20136]. Echocardiography and in particular TDI have been extensively used to assess LV dyssynchrony. Several parameters derived from myocardial velocity curves of different LV segments have been proposed to quantify LV dyssynchrony and predict response to CRT [3\u20138]. Yu and coworkers described a model (derived from 2D TDI data) to quantify global LV dyssynchrony using Ts-SD of 12 LV segments [6\u20138]. The authors reported a cut-off value\u2009>32.6\u00a0ms to distinguish between responders and non-responders [8] and a cut-off value\u2009>34.4\u00a0ms as the most powerful predictor of LV reverse remodeling [7]. In this study LV dyssynchrony was evaluated by Ts-SD from tri-plane TDI and compared with LV dyssynchrony assessment with phase analysis by GMPS. The tri-plane TDI technique avoids the problem of heart rate variability since simultaneous acquisition of the three apical views is possible; in the present study a cut-off value\u2009\u2009\u2265\u200933\u00a0ms was used to define substantial LV dyssynchrony on tri-plane TDI.\nInformation on cardiac dyssynchrony can also be derived from nuclear imaging. Kerwin et al. [13] evaluated interventricular dyssynchrony in 13 heart failure patients using multi-gated equilibrium blood pool scintigraphy and phase analysis. Patients were imaged during biventricular pacing and in sinus rhythm. During biventricular pacing, a significant improvement in interventricular synchrony was observed. Moreover, an improvement in LVEF was reported for all 13 patients during biventricular pacing (17.2\u00b17.9% to 22.5\u00b18.3%, p<0.0001), and the increase in LVEF correlated significantly with the improvement in interventricular synchrony (r=0.86; p<0.001). Fauchier et al. [14] used equilibrium radionuclide angiography and phase analysis to evaluate the prognostic value of interventricular and intraventricular dyssynchrony in 103 patients with idiopathic dilated cardiomyopathy. Phase images were generated from the scintigraphic data, and mean phase angles and SDs were calculated for the right and left ventricle. During a follow-up period of 27\u00b123\u00a0months, 18 major cardiac events occurred. Multivariate analysis revealed that LV intraventricular dyssynchrony and elevated pulmonary capillary wedge pressure were the only independent predictors of cardiac events.\nGMPS is currently used in patients with heart failure to exclude ischemia and\/or viability before referring for CRT implantation and to assess total scar burden in patients with ischemic cardiomyopathy [11, 12]. Indeed, various studies have shown that the presence of severe resting perfusion defects, as assessed with GMPS, limits the response to CRT [15\u201317]. Recently, Chen et al. [9] developed with GMPS a count-based method to extract phase information from the regional LV count changes throughout the cardiac cycle. The phase information is related to the regional onset of mechanical contraction of the LV (phase analysis) and provides information on the synchronicity of the LV contraction. The authors assessed in 90 normal individuals the normal range for four quantitative indices that can be used as markers of LV dyssynchrony (histogram bandwidth, phase SD, histogram skewness and histogram kurtosis) [9]. Recently, these four GMPS indices have been compared with LV dyssynchrony assessment by TDI in 75 patients with severe heart failure. It was shown that among the four quantitative indices of phase analysis, the variables histogram bandwidth and phase SD correlated best with LV dyssynchrony as assessed by TDI [10]. The current study involved a different patient subset and provides further support that phase analysis with GMPS can be useful in the evaluation of LV dyssynchrony.\nThe value of GMPS to assess LV dyssynchrony has been analyzed, and the indices derived from phase analysis were compared with Ts-SD measured by tri-plane TDI. It was shown that histogram bandwidth and phase SD correlated well with Ts-SD as evaluated by tri-plane TDI (histogram bandwidth: r=0.77 and phase SD: r=0.74). In addition, significantly higher values for histogram bandwidth and phase SD were observed in patients with substantial LV dyssynchrony on tri-plane TDI compared with patients without substantial LV dyssynchrony on tri-plane TDI.\nLimitations\nIn the present study, follow-up data after CRT implantation were not available. Therefore, the value of phase analysis to predict response to CRT could not be determined. Furthermore, GMPS may be less suitable for repeated analysis during the follow-up after CRT implantation, due to the radiation burden of the technique.\nConclusions\nThe results of this study confirm the feasibility to evaluate LV dyssynchrony with phase analysis by GMPS and its applicability in the clinical setting. In particular, histogram bandwidth and phase SD showed a good correlation with LV dyssynchrony measured with Ts-SD as assessed with tri-plane TDI. Future prospective studies in larger patient populations with follow-up after CRT implantation are needed to elucidate the potential role of GMPS with phase analysis for prediction of response to CRT.","keyphrases":["lv dyssynchrony","tdi","heart failure","nuclear imaging","gated spect"],"prmu":["P","P","P","P","R"]}
{"id":"Neuropsychol_Rev-3-1-2039835","title":"Motor-Skill Learning in Alzheimer\u2019s Disease: A Review with an Eye to the Clinical Practice\n","text":"Since elderly people suffering from dementia want to go on living independently for as long as possible, they need to be able to maintain familiar and learn new practical skills. Although explicit or declarative learning methods are mostly used to train new skills, it is hypothesized that implicit or procedural techniques may be more effective in this population. The present review discusses 23 experimental studies on implicit motor-skill learning in patients with Alzheimer\u2019s disease (AD). All studies found intact implicit motor-learning capacities. Subsequently, it is elaborated how these intact learning abilities can be exploited in the patients\u2019 rehabilitation with respect to the variables \u2018practice\u2019 and \u2018feedback.\u2019 Recommendations for future research are provided, and it is concluded that if training programs are adjusted to specific needs and abilities, older people with AD are well able to (re)learn practical motor skills, which may enhance their autonomy.\nIntroduction\nThe aging population is growing rapidly and by 2050 the number of elderly people aged 85\u00a0years old or older in Europe and North America is estimated to be approximately 19 million (Rom\u00e1n 2002). Since age is a high risk factor for dementia (Smith and Rush 2006), the expansion of the aging population and thus the number of people suffering from dementia has momentous consequences for national care systems as well as a large economic impact (Sch\u00f6lzer-Dorenbosch 2005). A way to contain these costly effects is by helping older people to stay independent for as long as possible, implying that the elderly must not stop learning. They apply old skills differently or acquire new skills, like learning how to use a walking aid, which gives rise to the following questions: Are demented elderly people able to learn such new skills? And are we, health professionals, able to train them?\nAs yet, psychopharmacological interventions, such as the use of cholinesterase inhibitors, may have some benefit in maintaining autonomy of elderly patients suffering from Alzheimer\u2019s disease (AD), as demonstrated by delayed nursing home placement (Becker et al. 2006). Recent pharmacological research also shows promising results for cognition: the treatments evaluated produced a moderate positive effect on global cognitive functioning (Grimley Evans et al. 2004; Sch\u00f6lzer-Dorenbosch 2005; Takeda et al. 2006). In their 2002 review on the placebo-controlled effects of rivastigmine on the cognition of AD patients, Birks et al. (2002) reported statistically-significant increases of 0.8 points on the Mini Mental State (MMS) Examination and 2.1 points on the Alzheimer\u2019s Disease Assessment Scale (ADAS-Cog). They also found benefits in the patients\u2019 activities of daily living, although the difference with placebo was not significant. Takeda et al. (2006) also reported reductions in the cognitive impairment of AD patients for donepezil and galantamine, but again, although showing potential, the improvements did not suggest a major difference in the daily lives of the patients.\nThese findings do not negate the importance of non-pharmacological approaches and it is possible that the interactions between medication and non-pharmacological approaches may be the most beneficial in maintaining patient\u2019s autonomy. In their review, Luijpen et al. (2003) conclude that the effect of non-pharmacological interventions with respect to cognition and affective behavior in dementia is similar to the effect of pharmacological regimens. Non-pharmacological treatments to improve autonomy in this patient population should hence be considered an additional option, especially since rehabilitation is increasingly being advocated as a means to optimize patients\u2019 overall functioning (De Vreese et al. 2001). Clare (2003) also concludes that neuropsychological rehabilitation applied in the context of progressive disorders like dementia do yield beneficial results.\nIn the present review we will focus on the ability of Alzheimer\u2019s patients to (re)learn practical motor skills. Currently, explicit or declarative learning methods are the starting points in most rehabilitation programs aimed at motor-skill learning in the cognitively unimpaired population (Van Cranenburg 2004). However, Zanetti et al. (2001) and R\u00f6sler et al. (2002) claimed that patients with dementia will profit more from implicit or procedural learning methods by showing that their AD cohorts were able to learn to waltz or to use a telephone when an implicit rather than an explicit learning approach was used. In implicit learning, skills are mastered without awareness, often simply by repeated exposure, and can be unconsciously revived from implicit memory (Buchner and Wippich 1998). The abovementioned studies were all focused on finding the best way to help older people with dementia learn or relearn practical (motor) skills, and although the results are encouraging, the patient samples were always small and it remains unclear how much was learned due to a lack of well-defined performance measures.\nIn the first part of our review we looked for corroborating evidence in experimental research for intact implicit motor-learning capacity in cohorts of elderly patients diagnosed with AD. In the second part we will elaborate on how these intact learning abilities can be utilized for their rehabilitation while taking the principles from theories of motor-learning into account. Two of the theories\u2019 core variables, i.e., practice and feedback (Schmidt and Wrisberg 2000) will be discussed more extensively, also in the light of research exploring these variables in AD. The results will be translated into practical instructions for more targeted rehabilitation training programs for this patient group.\nMaterials and Methods\nComputerized searches of the literature using the databases of PubMed and PsycLIT were conducted spanning a 20-year period, from 1985 up to and including 2005. The search terms (any field) used were procedural learning, sequence learning, motor-skill learning, or motor learning in combination with Alzheimer\u2019s disease. Only reports published in English were considered. For inclusion in this review the studies had to meet the following criteria: (a) a clinical diagnosis of Alzheimer\u2019s disease based on specified and generally accepted criteria; (b) a procedural task with motor responses; and (c) task performance expressed in time or error measures, and not only in fMRI or other imaging data. Ultimately, 23 studies were included in this review. Three studies will only be discussed in the second part of the review since they explicitly examined the role of feedback and type of practice.\nExperimental Research of Implicit Motor-Skill Learning in Alzheimer\u2019s Disease\nImplicit Learning Ability\nThe main results of the studies generated by our search of the literature are shown in Table\u00a01. Four studies using a Maze test in which blindfolded participants had to trace a complex pathway found that the AD patients were able to learn new motor-skills implicitly (Kuzis et al. 1999; Sabe et al. 1995; Starkstein et al. 1997; Taylor 1998). The nine studies that applied a Rotor-Pursuit task, in which participants had to maintain contact between a hand-held stylus and a rotating spot, also reported preserved learning abilities in their AD samples (Beatty et al. 1995; Deweer et al. 1994; Dick et al. 1995, 2001; Heindel et al. 1988; Heindel et al. 1989; Jacobs et al. 1999; Libon et al. 1998; Willingham et al. 1997). This is in agreement with the findings of Poe and Seifert (1997) based on a Puzzle-Assembly task and the results of Rouleau et al. (2002) involving a Mirror-Tracing task. Also, a Serial Reaction-Time Task (SRTT) was used in which participants needed to respond as fast as possible when a stimulus appeared in one of four places by pressing a corresponding response key (Grafman et al. 1990; Knopman and Nissen 1987; Knopman 1991; Willingham et al. 1997). Again, the AD patients showed implicit learning as reflected by the difference in reaction times (RTs) between blocks with a fixed sequence of stimuli presentation (decreasing RTs) and a random block (prolonged RTs). However, there are indications that the implicit learning ability in AD patients is affected because they generated inferior outcomes when accuracy was taken into account (Willingham et al. 1997) or when the data were log-transformed because of the unequal variance in RT (Knopman 1991). Ferraro et al. (1993) found preserved implicit SRTT learning only in the \u201cvery mildly demented\u201d group, and less in the \u201cmildly demented\u201d group although it is relevant to mention that none of the other studies used such a subtle severity classification.\nTable\u00a01Summary of results of experimental studies on motor-skill learning in Alzheimer\u2019s diseaseAuthorYearSample size and typesTask(s)Amount of learningaResults on learning capacitySabe, L., et al.199520\u00a0AD with co-morbid depression, 35\u00a0AD without co-morbid depression, 14 depressive, non-demented patients, 16 healthy controlsMaze testAD: 19%Co: 22%The AD patients showed significant deficits in declarative learning but only a minor (although statistically significantly) drop in procedural learning. The AD group with comorbid depression showed a similar learning pattern as the non-depressed AD group.GroupxTrial:p\u2009<\u20090.05Starkstein, S.E., et al.199755\u00a0AD (13 with mild, 12 with severe and 30 without anosognosia)Maze testAD no: 48%There was no group difference in declarative learning. As to procedural learning, the patients with severe anosognosia showed a significantly poorer performance whereas the patients with mild or no anosognosia showed no deficits.AD mild: 39%AD severe: \u221216%\u2013Taylor, R.199858\u00a0AD, 58 multi-infarct dementiaMaze test\u2013When age and overall neuropsychological functioning were taken into account, Maze performance was better in the AD patients than in the patients with multi-infarct dementia\u2013Kuzis, G., et al.199915\u00a0AD, 15\u00a0PD, 10\u00a0PD and dementia, 24 healthy controlsMaze testAD: 10%The AD group showed deficits on all measures of explicit memory. There were no significant between-group differences in the measures of implicit memory between the AD, control, and PD groups.Co: 39%\u2013Heidel, W.C., et al.198810\u00a0AD, 10\u00a0HD, 4 amnestic 20 healthy controlsRotor PursuitAD: 147%The AD patients showed preserved motor-skill learning while the patients with HD showed no motor learning.Co: 115%GroupxTrial: n.s.Heindel, W.C. et al.198916\u00a0AD, 13\u00a0HD, 17\u00a0PD, 22 healthy controlsRotor PursuitAD: 101%The AD patients showed preserved motor-skill learning while the patients with HD showed impaired motor learning.Co: 118%GroupxTrial: n.s.Beatty, W.W., et al.19954\u00a0AD, 1 corticbasal degenerationRotor Pursuit\u2013The AD patients showed preserved motor skill learning\u2013Deweer, B., et al.199413\u00a0AD institutionalized, 10 healthy controls, 17\u00a0AD out patients, 9 healthy controlsRotor PursuitAD in.:86%Explicit memory was severely impaired in the AD patients but they showed normal procedural learning.Co: 48%GroupxTrial: n.s.AD out: 161%Co: 139%GroupxTrial: n.s.Dick, M.B., et al.199512\u00a0AD, 12 healthy controlsRotor PursuitAD: 47%Performance significantly improved during the first 40 trials but additional practice provided no further beneficial effects. The AD patients showed minimal retention problems across four retention tests.Co: 81%GroupxTrial: n.s.Libon, D.J., et al.199816\u00a0AD, 14 vascular dementiaRotor PursuitAD: 60%The AD patients obtained a lower score on a verbal-learning task-recognition index and high scores on the Rotor Pursuit.\u2013Jacobs, D.H., et al.199912\u00a0AD, 12 healthy controlsRotor PursuitAD: 124%The AD patients and the controls were able to learn the motor task.Co: 106%GroupxTrial: p\u2009=\u20090.473Dick, M.B., et al.200118\u00a0AD, 18 healthy controlsRotor PursuitAD: 27%In normal-vision trials no differences in learning between the AD patients and the controls were found.Co: 36%GroupxTrial: n.s.Dick, M.B., et al.200399\u00a0AD, 100 healthy controlsRotor PursuitThe AD patients and controls receiving constant practice outperformed those in the blocked and random conditions. The AD patients only benefited from constant practice.Poe, M.K. et al.19979\u00a0AD, 14 healthy controlsPuzzle Assembly\u2013Even when the subjects had no explicit memory of practicing the task, they all demonstrated savings upon relearning.\u00a0GroupxTrial: n.s.Rouleau, I., et al.200212\u00a0AD, 12 healthy controlsMirror TracingAD: 44%Those AD patients that were able to perform the basic mirror-tracing task did not differ from the controls in level of performance, learning over trails, retention over a delay interval and generalization to other tasksCo: 49%GroupxTrial: n.s.Knopman, D.S., et al.198735\u00a0AD, 13 healthy controlsSRTTAD: 22%The AD patients showed learning of the repeated sequence, although they responded more slowly.Co: 38%GroupxTrial: n.s.Graftman, J., et al.199042\u00a0AD, 7\u00a0PSP, 44 healthy controlsSRTTAD: 36%The AD patients and controls showed motor-skill learning while the PSP patients did not.\u2013Knopman, D.199116\u00a0AD, 17 healthy controlsSRTTAD: 37%The AD patients showed learning of the sequence but they showed an inferior level of learning when the data were log-transformed.Co: 33%Ferraro, F.R., et al.199327 very mild AD, 15 mild AD, 17\u00a0PD, 26 healthy controlsSRTTAD mild: 11%The very mildly AD patients showed preserved learning comparable with the controls. The mildly AD patients and PD patients showed less implicit learning.AD very mild:22%Co: 20%Willingham, D.B., et al.199720\u00a0AD, 20 healthy controlsSRTT, Incompatible SRTT, Pursuit Tracking (randomized and repetitive pattern)SRTT:The dementia ratings predicted the ability to perform tasks but not the ability to learn them. AD patients can have a performance deficit but they have no general deficit in motor-skill learning.AD: 52%Co: 60%GroupXTrial: p\u2009>\u20090.2Pursuit:AD: 12%Co: 17%GroupxTrial: p\u2009>\u20090.2Hirono, N., et al.199736\u00a0AD, 19 healthy controlsBi-manual coordinated Tracing taskAD: 37%Skill learning in those AD patients that completed the tasks was as good as in the controls.\u00a0Co: 39%GroupxTrial: p\u2009=\u20090.193Dick, M.B., et al.199623\u00a0AD, 22 healthy controlsTossingThe AD patients given constant practice were able to learn and retain the tossing task similarly well as the controls. The AD patients showed less improvement when practicing at various distances.Dick, M.B., et al.200658\u00a0AD, 58\u00a0healthy controlsTossingThe AD patients showed significant improvements under constant practice only. None of the practice conditions facilitated intermediate transfer in the AD patients whereas constant practice did benefit them on tests assessing near transfer.AD= Alzheimer\u2019s disease; HD= Huntington\u2019s disease; PD= Parkinson\u2019s disease; PSP= \u2009Progressive supranuclear palsy.aexpressed as a percentage of the difference score between the last and first trial with respect to the score on the first trial. The GroupxTrial interaction for the AD and Co group is also reported when available.\nThus, irrespective of the task used, the studies assessing implicit motor-skill learning in AD we reviewed yielded positive outcomes. Indeed, in their 1997 study, Hirono and colleagues found that patients with mild AD were able to acquire motor and perceptual as well as cognitive skills in various procedural learning tasks.\nIt should be noted that in all studies the patients that could not perform the task were eliminated from the analyses. Yet, a failure to perform the prescribed task need not necessarily be related to learning problems. Willingham et al. (1997) attributed the phenomenon to other causes like the complexity of the instructions given or the type of skill to be performed. These factors may differ across tasks, which might explain their finding that the ability to complete one task did not predict the rate of improvement in another task. They conclude that AD patients have a performance deficit and not a generalized deficit in motor learning.\nPerformance and Amount of Learning\nFrom the above discussion of results we can presume that at least a subgroup of AD patients show preserved implicit learning abilities, but to what extent? Here, two aspects in motor learning should be differentiated, i.e., overall performance level and amount of learning (e.g., the increment in Total Time on Target in the Rotor-Pursuit task). All the studies found preserved motor-skill learning in AD patients although their overall performance levels in terms of reaction and movement time were always inferior to those of the controls. However, when we take the level of learning into account, the results are less consistent. Some of the results were not reported with enough detail to show unambiguously the amount of learning the AD patients showed compared to the controls (Poe and Seifert 1997). Some comparative studies did not include a healthy control group in addition to the patient groups (Beatty et al. 1995; Grafman et al. 1990; Libon et al. 1998; Starkstein et al. 1997; Taylor 1998), preventing patient-control comparisons from being made. The AD patients in the SRTT studies showed the same amount of learning (decrease in RT during the blocks with the fixed sequence) as the normal controls (Ferraro et al. 1993; Knopman and Nissen 1987; Knopman 1991; Willingham et al. 1997). In the nine studies that used a Rotor-Pursuit or Tracking task there were also no patient-control differences in amount of learning (Deweer et al. 1994; Dick et al. 1995, 2001; Heindel et al. 1988; Heindel et al. 1989; Hirono et al. 1997; Jacobs et al. 1999; Rouleau et al. 2002; Willingham et al. 1997). Two of the studies using a Maze test reported learning abilities in the AD group but less improvement across trials compared to the controls, (Kuzis et al. 1999; Sabe et al. 1995) findings which are perhaps explainable by the use of a task without visual feedback.\nTaken together, the reviewed studies all showed preserved implicit motor-skill learning in AD patients regardless of the task used. Their performance levels, however, never reached the levels of the healthy controls, demonstrated by their prolonged reaction and movement times. The AD patients\u2019 level of learning also differed depending on the task to be performed. Visual feedback appears to have a positive effect on their learning pace. They also seem to experience more problems with implicit learning when performing the SRTT, a task that involves two learning processes. The subjects have to master both spatial and motor regularities (Mayr 1996), and it is the learning of spatial regularities that may be compromised in AD patients, a process that is less implicated in the Rotor-Pursuit task in which normal implicit learning for the patients was found.\nTraining Patients with Alzheimer\u2019s Disease: Variables in Motor Learning\nThe studies discussed provide evidence that AD patients can learn new motor skills in an implicit way. It is therefore worthwhile to establish what would be the best way to train them. In the next section we will give a brief account of the two variables practice and feedback that play a role in (re)training motor skills. We will subsequently discuss the variables in relation to the findings reported in the relevant AD studies and conclude by making recommendations of how to enhance the acquisition of new motor skills in this population.\nWe will first, however, briefly address the existing views on the presence or absence of distinguishable learning stages in explicit and implicit learning. Generally, with explicit learning people tend to pass through three stages in the acquisition of motor skills (Fitts and Posner 1967). The first is the cognitive stage in which the focus is on understanding the task and developing strategies to perform it, requiring cognitive activity such as attention and executive functions. The second phase is the associative stage: the learner has selected the best strategy and now begins to refine the skill. Here, cognitive aspects are less important. And finally, there is the autonomous stage in which the skill becomes automatic, requiring a low degree of attention. Variables such as practice and feedback can be structured differently to enhance learning at each stage. Feedback in the cognitive stage, for example, may need to be more specific and applied more frequently to enhance learning, while feedback may be weaned toward the third stage of learning (Tse and Spaulding 1998).\nIn implicit learning, on the other hand, there is no clear distinction between these three stages. It has been proposed that in implicit learning the three stages might overlap or be ordered differently. There is support for a parallel development of implicit and explicit knowledge in learning (Willingham and Goedert-Eschmann 1999).\nPractice: Theory and Outcome Studies with AD Patients\nThe principle, \u201cThe more you practice, the more you learn,\u201d implies that the amount of practice should be maximized in therapy. But does more practice indeed improve the performance in AD patients? Dick et al. (1995) found that on the Rotor Pursuit both the AD and control group had reached their optimal performance after 40 trials because subsequent practice failed to yield any additional augmenting effect. It would be interesting to determine whether this also holds for other tasks like the Maze test in which, relative to the controls, an inferior amount of learning was observed for AD patients (Kuzis et al. 1999; Sabe et al. 1995).\nSince fatigue also plays a role in learning, the next question is how to alternate practice with rest to maximize learning in patients. Schmidt and Wrisberg (2000) distinguish two types of practice. In \u2018massed practice,\u2019 the greater proportion of the sessions is dedicated to training, while in \u2018distributed practice\u2019 the duration of rest equals or is greater than that of practice. To date, the effects of alternating these two training methods in the generally older AD patient group still requires further investigation.\nAnother factor that merits closer attention in the context of training programs for AD patients is whether the task should be learned as a whole or per constituent component. Training the components of a task separately before combining them into the whole pattern can be effective if the task itself can be naturally divided into components that reflect the inherent goal of the task (Schmidt 1988). For example, learning to drive a car can be easily divided into the components \u201clearning to shift gear\u201d and \u201clearning to steer,\u201d which can be trained individually. Learning to reach and grasp an item, on the other hand, does not lend itself well for phased training since reaching and grasping are integral components of a single, continuous movement.\nThe amount of variation in the practice session(s) is also a topic for further study. Task variables like the beanbag\u2019s weight and throwing distance in the Tossing task can be practiced in a random design so that the weight and distance can be varied systematically. Alternatively, they can be offered in a blocked design in which only one task variable per block is practiced repetitively. Another option is to use a constant design in which only one combination of task variables is trained. Note that over time, the connotation of the two terms has shifted: random and blocked practice now refer to the rehearsal of several distinct skills whereas varied and constant practice implies the rehearsal of different variations of the same skill (Schmidt and Wrisberg 2000). Nevertheless, in our report we will use the \u2018old\u2019 terms (random, blocked and constant) in their original meanings since these were terms and interpretations used in the reviewed literature. Early evidence suggests that random practice might be most effective for the acquisition and generalizability of a motor skill, whereas during the acquisition of a specific motor skill, performance benefits most from blocked practice (Schmidt 1988).\nAll available studies reviewed on this matter (Dick et al. 1996, 2000, 2003) show that AD patients learn best under constant practice conditions. According to Dick and his 1996 team, humans use their episodic memory of the training trials to accurately perform a task while learning a skill. They suggest that because AD patients experience problems with episodic memory, constant practice is more effective because repeated running of the same motor program does not require an intact episodic memory. The second reason why random practice may be less effective is that other cognitive functions that play a role in random practice, like the ability to switch tasks and divide attention, are affected in AD patients.\nDick et al. (1996, 2003) explained the AD patients\u2019 superior learning performance under constant practice conditions in terms of the schema theory originally developed by Schmidt (1975), and likewise propose a more open-loop account of motor control. Schmidt assumes the existence of generalized motor programs (GMPs) that are acquired through practice and that define the \u201cform\u201d of the action. These GMPs can be altered to meet environmental demands by a closed-loop system using sensory feedback. Schemata, e.g., for varying weight and distances in tossing, are learned and allow the action to be scaled to the environment (Schmidt 2003). When they considered their results in terms of this theory, Dick et al. (1996, 2003) concluded that AD patients can develop and access a GMP in training situations that emphasize movement consistency. However, they do not form the motor schemas needed to successfully achieve a movement when the environmental demands change because they are unable to encode and to store the different types of information about a motor pattern.\nThere are three other training approaches that can produce the desired learning effect: guidance, observation, and mental practice (Schmidt 1988). Guidance should only be used at the onset of training because experiments have shown that practice under unguided conditions seems to be more effective for retention and transfer (Shumway-Cook and Woollacott 1995). Observation conveys information about how a skill should be performed and seems to be especially beneficial for the acquisition of new movement patterns (Magill 1993). Our automated computer search and an extra search combining the three keywords with Alzheimer dementia both failed to generate any relevant studies that employed one of these training methods. The only study that provided some additional information on the topic is a report by Dick et al. (1988) which showed that AD patients could recall preselected (subject-defined) movements more accurately than constrained (experimenter-defined) movements on a linear positioning apparatus. This was explained by the patients\u2019 ability to profit from mental preparation of the movement prior to its execution. Without further systematic investigation, however, it cannot be inferred that the ability to profit from mental preparation also means AD patients will profit from mental practice. More research into the effects of all three practice types in AD is needed.\nFeedback: Theory and Outcome Studies with AD Patients\nA second crucial variable that influences motor learning is type of feedback. Intrinsic feedback encompasses the sensory information generated by motion, and extrinsic feedback entails information from an external source like a therapist (Schmidt and Wrisberg 2000). There are various ways to provide extrinsic feedback. It can be delivered during or after the movement, immediately following movement completion or delayed, and in a verbal or a non-verbal fashion. It can contain information on average performance (summary feedback) or it may reflect each movement or performance (constant feedback; Schmidt 1988). It is generally believed that constant feedback enhances only motor performance, not the level of learning (Shumway-Cook and Woollacott 1995). With less frequent feedback, learners have to rely more on other cues, which entails more elaborate encoding (Schmidt 1988). Extrinsic feedback can moreover be divided into \u2018knowledge of results,\u2019 in which the movement outcome is given in terms of the goal, and \u2018knowledge of performance,\u2019 so that the feedback concerns the movement pattern itself (e.g., in a Tossing task: increase the swing of your arm).\nIn almost all studies on motor-skill learning in AD, visual feedback was employed. Only the Maze tasks were administered under blindfolded conditions and the amount of learning in the AD patients proved inferior to the amount found for the controls (Sabe et al. 1995; Kuzis et al. 1999). In most Rotor-Pursuit tasks, the velocity of the target was individualized to equate initial performance. Controls generally tracked at a faster rate than the AD patients (Deweer et al. 1994; Dick et al. 1995; Jacobs et al. 1999; Libon et al. 1998). Possibly, AD patients can only perform this task at a slower rate because they rely more on visual feedback than controls.\nOnly one study using a Rotor-Pursuit task explicitly examined the role of visual feedback on performance in AD patients, showing a drop in performance when the visibility of the moving target was reduced during the learning phase (Dick et al. 2001). In contrast to that of the normal controls, the patients\u2019 performance did not improve across trials in the restricted-vision condition. In the full-vision condition the patients showed normal learning.\nIt can be tentatively concluded that for AD patients, constant visual feedback is important in learning motor skills, but more research is needed to confirm this hypothesis. We did not find any studies that were concerned with the frequency of external feedback, and whether knowledge of results and knowledge of performance makes a difference in this patient group. Based on the results cited above, it may be hypothesized that both forms of feedback knowledge probably place too much weight on the cognitive abilities in AD patients and therefore contribute little to successful performance.\nConclusions and Recommendations\nPeople with Alzheimer\u2019s disease are able to implicitly (re)learn motor skills to a certain extent and under specific conditions. The experimental research to date shows preserved implicit motor learning irrespective of the task used. Patients are capable of acquiring motor skills without awareness simply by repeated exposure, although their performances will not reach normal levels. This is expressed in their protracted performance relative to that of unimpaired controls. Moreover, extent of learning will differ depending on the task to be mastered.\nThe preserved implicit learning ability in AD can be of use for physical therapists working with this elderly patient group. Physical therapists can call upon neuropsychologists to provide information on their patients\u2019 learning capacities since they have quantitative measures at their disposal to assess a patient\u2019s level of functioning. However, the memory and learning tests currently available in the clinical practice evaluate explicit or declarative memory (Spaan et al. 2003). In order to get a satisfactory differential picture of the learning capacities in demented patients, implicit (motor) learning tasks need to be added to the neuropsychological assessment.\nThe evidence of intact implicit learning in AD further prompted the question how these intact learning abilities can best be translated into rehabilitation programs targeting this patient group. Learning is central in rehabilitation and knowledge of the system under treatment, like the motor system, must be combined with knowledge of how learning principles must be applied to achieve a successful training program (Baddeley 1993). With respect to patients with dementia, apart from the subtype of dementia and its specific neuropsychological syndrome, the training programs should apply the principles that emerge from theories of learning.\nThe studies we reviewed showed that in (re)learning motor skills, constant, or rather frequent and consistent practice is important in AD patients. This way of learning draws less on episodic memory and other cognitive functions compromised in AD patients. These data also suggest that practice under dual-task conditions should also be avoided.\nBecause AD patients have difficulty in generalizing the motor skills learned during the sessions, training has to take place in an environment that closely resembles the one in which the skill is going to be used and presumably with tools used by the AD patient in his or her daily life. If, for instance, an AD patient is trained in the use of a microwave, the device used during the training should be the same as the one available in the patient\u2019s household. The amount of training a patient needs will depend on the task being trained. The role of fatigue is also important in this respect. The effects of massed and distributed practice in this generally older patient group need to be addressed in future investigations.\nPatients with AD appear to remain dependent upon visual feedback throughout training and performance. Screening and subsequent correction of visual problems or the use of visual aids can be effective in the training process in this group for whom vision problems are very common (De Winter et al. 2004). The type and point in time when external feedback needs to be given and its effect on learning in AD also warrants attention in future research.\nIn the introduction of our review we asked whether patients with Alzheimer\u2019s disease might have intact motor-skill learning abilities. The answer is twofold. Clearly, AD patients show preserved implicit learning abilities that can be utilized in teaching (motor) skills, yet transfer to other skills is minimal. Accordingly, the professionals delivering the training programs should tailor the contents to the particular needs and abilities of this patient group or the individual patient. When the above guidelines are kept in mind and when our knowledge on this topic are widened, non-pharmacological interventions might contribute significantly in helping elderly people suffering from dementia to keep their autonomy. The extent to which pharmacological intervention may enhance these behavioral mechanisms and foster independent living in AD patients has yet to be determined.","keyphrases":["motor-skill learning","alzheimer\u2019s disease","rehabilitation","non-pharmacological interventions","procedural learning","implicit learning"],"prmu":["P","P","P","P","P","P"]}
{"id":"Mol_Immunol-1-5-2080686","title":"C1q deficiency promotes the production of transgenic-derived IgM and IgG3 autoantibodies in anti-DNA knock-in transgenic mice\n","text":"C1q-deficient mice have been shown to develop a lupus-like disease and to display an impaired clearance of apoptotic cells that are enriched in lupus autoantigens. However, the role of C1q in the regulation of autoreactive B cells remains debatable. To explore this we crossed MRL\/Mp C1q-deficient mice with knock-in transgenic (Tg) mice expressing an anti-ssDNA antibody (VH3H9R and VH3H9R\/VL\u03ba8R). Analysis of the VH3H9R mice showed that in the absence of C1q higher titres of Tg-derived IgM and IgG3 anti-ssDNA antibodies were detectable. In contrast, in the VH3H9R\/VL\u03ba8R C1q-deficient animals no increase in Tg antibody levels was observed. In both models the lack of C1q induced a marked reduction of marginal zone B cells and this was paralleled by a significant increase in the percentage of plasmocytes. Thus, one could postulate that in the absence of C1q the failure to clear efficiently dying cells provides an additional stimulus to the autoreactive Tg B cells resulting in their emigration from the marginal zone B cell compartment with subsequent increase in plasmocytes. However, the lack of C1q led to an increased production of Tg IgM and IgG3 antibodies only in VH3H9R mice indicating that additional genetic susceptibility factors are required to break self-tolerance.\n1\nIntroduction\nHereditary deficiencies of early components of the classical pathway of the complement system are known to predispose to systematic lupus erythematosus (SLE). Among these, C1q deficiency exhibits the strongest association with prevalence greater than 90% suggesting that a physiological activity of the early part of the classical pathway normally protects against the development of SLE (Pickering et al., 2000). Mice with targeted deletion of the C1q gene (C1qa\u2212\/\u2212) developed a spontaneous lupus-like disease characterised by the development of anti-nuclear autoimmunity and glomerulonephritis associated with the presence of multiple apoptotic bodies (Botto et al., 1998). Introgression of C1q deficiency onto different genetic backgrounds revealed that in mice C1q operates as a disease modifier. C57BL\/6.C1qa\u2212\/\u2212 mice displayed no increase of IgG autoantibodies (autoAbs) or glomerulonephritis (Mitchell et al., 2002), whilst C1q deficiency backcrossed onto the lupus-prone MRL\/Mp background accelerated both the onset and the severity of the autoimmune disease (Mitchell et al., 2002). Consistent with these observations, C1q reconstitution by bone marrow transplant attenuated the autoimmune disease present in MRL\/Mp.C1qa\u2212\/\u2212 mice (Cortes-Hernandez et al., 2004).\nCurrently, there are two main hypotheses to explain the role of complement in the development of SLE, neither of which is mutually exclusive. The first model, defined as the tolerance hypothesis, proposes a role for complement in determining the activation thresholds of lymphocytes, whereby complement enhances presentation of autoantigens to self-reactive immature B cells resulting in their elimination (Prodeus et al., 1998). The second one, known as the \u2018waste disposal\u2019 hypothesis, suggests that in addition to its role in the clearance of immune complexes, complement is involved in the physiological disposal of apoptotic cells (Botto et al., 1998), that have been shown to express lupus autoantigens on their surface (Casciola-Rosen et al., 1994). C1q plays a significant role in the clearance pathway of cellular debris by binding directly or indirectly to apoptotic blebs where it activates complement and mediates phagocytosis by professional and non-professional phagocytes (Korb and Ahearn, 1997; Mevorach et al., 1998; Nauta et al., 2004; Quartier et al., 2005; Taylor et al., 2000). Therefore, improper removal of dying cells in the setting of C1q deficiency could result in the stimulation of autoreactive cells leading to autoimmunity.\nImmunoglobulin Tg models have been instrumental in understanding B cell regulation revealing several key mechanisms, including receptor editing, deletion, anergy and ignorance. The hen egg lysozyme (HEL)\u2013anti-HEL (IgHEL) Tg model in particular has been widely used to demonstrate elimination of self-reactive clones by membrane-bound expressed antigen, anergy induction by soluble antigen (sHEL) or ignorance when the amount of antigen is so low that it does not reach the threshold to induce anergy. More recently, it has been shown that intracellular membrane-bound HEL failed to induce tolerance and was instead autoimmunogenic positively selecting IgHEL B1 cells and inducing large numbers of IgM autoantibody-secreting plasma cells (reviewed in Ferry et al., 2006). Taken together, these findings suggest that in this model the fate of self-reactive B cells is determined not only by the abundance, the avidity of the target self-antigen and the affinity of the B cell receptor but also by the location of the \u201cauto\u201d-antigen. The role of complement has been tested in the IgHEL Tg (MD4)\u2013sHEL (ML5) model by crossing the double Tg mice with mice deficient in C1q, C4, C3 or CD21\/CD35 (Cutler et al., 2001; Prodeus et al., 1998). IgG anti-HEL Abs remained undetectable in all complement deficient mice, but C4 and CD21\/CD35 deficient B cells displayed reduced surface IgM modulation, indicating a lower degree of anergy induction in these mice (Prodeus et al., 1998). However, this was not observed in the C1q deficient mice (Cutler et al., 2001). This discrepancy could either indicate that C4 operates independently from C1q or reflect differences in the genetic background of the mice used (Cutler et al., 2001). Nevertheless sHEL is not the ideal model to study the maintenance of tolerance in SLE as it is neither a natural autoantigen, nor are soluble plasma proteins typically targeted by the SLE autoAbs.\nMore recently, a model targeting an SLE antigen, DNA, was generated by Weigert and co-workers (Chen et al., 1995). In this model the rearranged variable heavy chain (VH) gene derived from a double stranded DNA-binding hybridoma developed in the autoimmune strain MRL\/Mp.lpr\/lpr, was inserted at the Igh locus and was referred to as VH3H9R. In contrast to conventional Tg mice, this knock-in model allows the Tg locus to undergo normal editing, isotype switching and somatic mutation. A variety of light chains can combine with the VH3H9 to yield anti-DNA Abs (Radic et al., 1991) but only few light chains are able to \u201csilence\u201d VH3H9R so that it no longer binds to DNA. By virtue of this characteristic the mice expressing only the VH3H9 chain (VH3H9R mice) can generate anti-DNA specificities. However, when the VH3H9R mice were crossed with a Tg knock-in light chain VL\u03ba8 (Prak and Weigert, 1995) to generate monospecific Tg mice (VH3H9R\/VL\u03ba8R mice) (Chen et al., 1997a), this combination of heavy and light chain V regions (VH3H9\/VLV\u03ba8) bound only ssDNA and not dsDNA (Prak and Weigert, 1995). Previous studies with the VH3H9R mice have shown that the autoreactive Tg B cells accumulated in the splenic marginal zone and were regulated by anergy on non-autoimmune backgrounds such as BALB\/c (Chen et al., 1995; Erikson et al., 1991) and C57BL\/6 (Fukuyama et al., 2005; Sekiguchi et al., 2002). However, tolerance could be broken in this model if T cell help was provided in the form of a chronic graft versus host disease (Sekiguchi et al., 2002). Consistent with these observations, the double VH3H9R\/VL\u03ba8R knock-in Tg B cells were regulated by anergy in non-lupus prone mice (BALB\/c), whilst in autoimmune prone MRL\/Mp.lpr\/lpr animals Tg B cells escaped tolerance induction and underwent class-switching and affinity maturation (Brard et al., 1999). These experiments suggested that the lpr mutation in the MRL background allowed the Tg autoreactive B cells to receive T cell help during a germinal center reaction.\nTo determine whether C1q is involved in selection of self-reactive B cells, we bred the C1q-deficient mice with the VH3H9R and the VH3H9R\/VL\u03ba8R mice and monitored the regulation and activation of anti-DNA Tg B cells over a period of 10 months. The mice in this study were on the autoimmune prone background MRL\/Mp expressing the CD95 (Fas) gene. The analysis of these mice revealed that the lack of C1q can influence the levels of IgM and IgG3 Tg-derived antibodies only in the VH3H9R model.\n2\nMaterials and methods\n2.1\nMice\nMRL\/Mp mice were obtained from Harlan Olac, Bichester, UK. MRL\/Mp.C1qa\u2212\/\u2212 deficient mice were generated as previously described (Mitchell et al., 2002). VH3H9R.MRL\/Mp (Chen et al., 1995), VL\u03ba8R.MRL\/Mp (Prak and Weigert, 1995) and VH3H9R\/VL\u03ba8R.MRL\/Mp mice were kindly provided by Prof. M. Weigert (Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, IL). MRL\/Mp.C1qa\u2212\/\u2212 mice were crossed with the VH3H9R\/V\u03ba8R.MRL\/Mp mice and the resulting VH3H9R\/VL\u03ba8R.MRL\/Mp.C1qa+\/\u2212 were then crossed either with MRL\/Mp or MRL\/Mp.C1qa\u2212\/\u2212 in order to generate littermate animals. Mice were bled every 3 months starting from 2 months of age and at 10 months they were sacrificed. The mice were genotyped by PCR using specific primers. PCR primers were as follow: mC1qA\/5\u2032 (5\u2032-GGGGCCTGTGATCCAGACAGG-3\u2032), mC1qA\/In2\u2212 (5\u2032-TAACCATTGCCTCCAGGATGG-3\u2032) and neo (5\u2032-GGGGATCGGCAATAAAAAGAC-3\u2032) for the C1q genotyping; MW114 (5\u2032-CTGTCAGGAACTGCAGGTAAGG-3\u2032) and MW162 (5\u2032-CATAACATAGGAATATTTACTCCTCGC-3\u2032) for the VH3H9R genotyping (Erikson et al., 1991); MW133 (5\u2032-GGTACCTGTGGGGACATTGTG-3\u2032) and MW157 (5\u2032-AGCACCGAACGTGAGAGG-3\u2032) for the VL\u03ba8R genotyping (Carmack et al., 1991). Animals were kept under specific pathogen-free conditions. All animal care and procedures were conducted according to institutional guidelines and approved by the local ethical committee.\n2.2\nFlow cytometry\nFlow cytometry was performed using a four-color staining of cells and analyzed with a FACSCalibur\u2122 (Becton Dickinson, Mountain View, CA). The following Abs were used: anti-B220 (RA3-6B2), anti-CD5 (53-7.3), anti-CD11b (M1-70), anti-CD19 (1D3), anti-CD23 (B3B4), anti-CD21\/CD35 (7G6), anti-CD90.2 (53-2.1), anti-CD138 (281-2), anti-IgM (II\/41). All Abs were purchased from BD Biosciences Pharmingen (San Diego, CA) with the exception of the anti-VH3H9 idiotype (1.209), a kind gift from Prof. M. Weigert (Gay et al., 1993). Biotinylated Abs were detected using an allophycocyanin-conjugated streptavidin Ab (BD Biosciences Pharmingen). Staining was performed in the presence of saturating concentration of 24G2 mAb (anti-FcRII\/III). Data were analyzed using WinMDI software (Version 2.8; Scripps Institute).\n2.3\nSerological analyses\nSerum levels of IgM and IgG were quantified by ELISA, as described previously (Cortes-Hernandez et al., 2004). Anti-ssDNA Abs, anti-chromatin Abs, anti-histone Abs and anti-dsDNA Abs were measured by ELISA as described previously (Burlingame and Rubin, 1990; Emlen et al., 1990). Microtiter plates were coated with ssDNA prepared from calf thymus DNA (Sigma), chromatin (Lorne Laboratories Ltd., Reading, UK) or histone (Calbiochem, Merck Biosciences, Darmstadt, Germany). For detecting anti-dsDNA Abs, plates were coated with streptavidin (Sigma). \u03a6X174 double-stranded plasmid DNA (Promega, Southampton, UK) was biotinylated with Photoprobe biotin (Vector Laboratories, Peterborough, UK) and added to the streptavidin. Serum samples were diluted appropriately in PBS 2%BSA, 0.05%Tween-20, 0.02% NaN3. Bound Abs were detected with alkaline phosphatase conjugated goat anti-mouse IgG (\u03b3-chain specific) (Sigma\u2013Aldrich, Dorset, UK) and anti-mouse IgM (Southern Biotechnology Associates, Inc., Birmingham, AL). All autoAb results are expressed in arbitrary ELISA units (AEU) in reference to a standard curve derived from serum pools containing high titers of autoAbs. The idiotype+ Abs were captured with the anti-VH3H9 idiotype (1.209) (Gay et al., 1993) and detected with alkaline phosphatase conjugated goat anti-mouse IgM or IgG subclasses specific Abs (Southern Biotechnology Associates). The standard curve was derived from an IgM VH3H9R\/V\u03ba8R mAb provided by Prof. Weigert or sera containing high IgG subclasses titers of the Tg.\n2.4\nRenal assessment\nProteinuria was assessed using Haema-combistix (Bayer Diagnostics, Newbury, UK). Kidneys were fixed in Bouin's solution and paraffin embedded, and sections were stained with periodic acid\u2013Schiff reagent. Glomerular histology was graded in a blind fashion scored on a scale of 0\u20134, as described before (Carlucci et al., 2007).\n2.5\nStatistics\nThe data are presented as median, with range of values in brackets, unless otherwise stated. The non-parametric Mann\u2013Whitney U-test was applied throughout with differences being considered significant for p-values\u00a0<\u00a00.05. Statistics were calculated using GraphPad Prism Version 3.0 (GraphPad Software, San Diego, CA).\n3\nResults\n3.1\nExpression of Tg autoantibodies\nC1q deficiency has been shown to accelerate the onset and progression of SLE in MRL\/Mp mice (Mitchell et al., 2002), but the mechanisms underlying these effects are still uncertain. The VH3H9R\/VL\u03ba8R Tg alleles were transferred onto C1q-deficient mice by crossing VH3H9R\/VL\u03ba8R.MRL\/Mp with MRL\/Mp.C1qa\u2212\/\u2212 and experimental cohorts of littermate mice were generated. Whilst in previous studies the expression of the Tg alleles was monitored by measuring the Tg-specific immunoglobulin allotype (Steeves and Marion, 2004), this analysis could not be applied in these mice as the Tg alleles and the endogenous immunoglobulin allotype were indistinguishable. Nevertheless, we were able to detect the combination of the VH3H9 with the VLV\u03ba8 using an idiotype specific monoclonal Ab named 1.209 (Gay et al., 1993). As the 1.209 mAb can also recognize the VH3H9 paired with other endogenous light chains such as VL\u03ba4 (Gay et al., 1993), this anti-idiotype Ab allowed us not only to assess the VH3H9R\/VL\u03ba8R Tg B cells but also some of the VH3H9R Tg B cells. C1q deficiency did not affect the proportion of peripheral blood B cells expressing the Tg in neither of the two models (VH3H9R\/VL\u03ba8R.MRL\/Mp.C1qa\u2212\/\u2212: 89.1\u00a0\u00b1\u00a01.1% versus VH3H9R\/VL\u03ba8R.MRL\/Mp: 90.7\u00a0\u00b1\u00a01.5%, p\u00a0=\u00a00.4118; VH3H9R.MRL\/Mp.C1qa\u2212\/\u2212: 25.3\u00a0\u00b1\u00a01.6% versus VH3H9R.MRL\/Mp: 22.3\u00a0\u00b1\u00a02.3% p\u00a0=\u00a00.1669). The low proportion of idiotype+ B cells in the VH3H9R.MRL\/Mp mice indicated that most of the B cells in these mice had either paired the VH3H9R Tg allele with a light chain that was not recognised by the anti-idiotype Ab, or edited the VH3H9R allele, or used the endogenous immunoglobulin allele. In the VH3H9R\/VL\u03ba8R.MRL\/Mp.C1qa\u2212\/\u2212 mice the B cell receptor was down-modulated to the same extent as in the VH3H9R\/VL\u03ba8R.MRL\/Mp mice (data not shown).\nSerological analyses were performed in order to establish whether the Tg autoAbs were expressed. The different cohorts of mice were bled at different time points, and the data presented here are those obtained at 10 month of age when the mice were sacrificed. Substantial levels of idiotype+ IgM, IgG3, IgG2a and IgG2b Abs were detected in the VH3H9R\/VL\u03ba8R.MRL\/Mp and in VH3H9R.MRL\/Mp mice indicating that the anti-DNA knock-in Tg alleles on this background were not regulated by anergy unlike when on the C57BL\/6 background (Fig. 1). In the VH3H9R\/VL\u03ba8R.MRL\/Mp.C1qa\u2212\/\u2212 mice the levels of idiotype+ Abs were similar to those detected in the strain-matched C1q-sufficient animals. In contrast, the VH3H9R.MRL\/Mp.C1qa\u2212\/\u2212 mice had significantly increased levels of idiotype+ IgM and IgG3 Abs compared to the VH3H9R.MRL\/Mp mice (Fig. 1A and B), but similar levels of idiotype+ IgG2a and IgG2b Abs (Fig. 1C and D).\nWe then measured the levels of IgM and IgG autoAbs. A significant increase in the titre of IgM anti-ssDNA Abs was observed in the VH3H9R\/VL\u03ba8R.MRL\/Mp.C1qa\u2212\/\u2212 mice compared to the VH3H9R\/VL\u03ba8R.MRL\/Mp mice (Fig. 2A). Of note, the VH3H9R\/VL\u03ba8R.MRL\/Mp.C1qa\u2212\/\u2212 mice displayed only markedly increased levels of IgM autoAbs directed against ssDNA (the specificity encoded by the Tg alleles) but not against other lupus autoantigens such as dsDNA, histone or chromatin (Fig. 2B\u2013D). In the VH3H9R.MRL\/Mp mice the pairing of the VH3H9 with endogenous L chains could yield to more specificities. Consistent with this, VH3H9R.MRL\/Mp.C1q\u2212\/\u2212 mice had significantly increased levels of IgM anti-ssDNA, anti-dsDNA, anti-histone and anti-chromatin Abs (Fig. 2A\u2013D). We then analysed the levels of IgG autoAbs and found that these were similar between the C1q-sufficient and -deficient cohorts (Supporting information Table S1).\n3.2\nRenal assessment\nAt 10 month of age all the animals were sacrificed. VH3H9R\/VL\u03ba8R.MRL\/Mp and VH3H9R.MRL\/Mp animals showed histological evidence of a mild glomerulonephritis (median score 1.0, range: 0.0\u20133.0 and 1.5, range: 1.0\u20133.0, respectively) reminiscent of the disease observed in MRL\/Mp non-Tg mice (Mitchell et al., 2002). C1q-deficiency in MRL\/Mp mice has previously been shown to worsen the kidney pathology (Mitchell et al., 2002). However, in the Tg mice the absence of C1q did not exacerbate the renal disease compared to the strain-matched C1q-sufficient mice (VH3H9R\/VL\u03ba8R.MRL\/Mp.C1qa\u2212\/\u2212 mice: median score 1.0, range: 0.0\u20133.0, p\u00a0=\u00a00.4287; VH3H9R.MRL\/Mp.C1qa\u2212\/\u2212 mice: median range 2.0, range: 1.0\u20132.0, p\u00a0=\u00a00.4564).\n3.3\nFlow cytometric analysis of splenic and peritoneal B cells\nIn order to determine if the serological data were accompanied by phenotypic changes in T and B lymphocytes, we performed a comprehensive analysis of the various splenic and peritoneal subpopulations. Cells of at least 7 mice from each cohort were analysed by FACS at the time of the sacrifice using the combinations of markers shown in Table 1. In agreement with the findings in other genetic backgrounds (Li et al., 2002), the VH3H9R.MRL\/Mp mice had a larger marginal zone (MZ) B cell population compared to VH3H9R\/VL\u03ba8R.MRL\/Mp (Fig. 3A) and an increased proportion of idiotype+ B cells in the MZ compartment compared to follicular (FO) compartment (5.9\u00a0\u00b1\u00a01.0 versus 4.2\u00a0\u00b1\u00a00.6, paired t-test p\u00a0=\u00a00.0157) (Table 1). More interestingly, in the VH3H9R.MRL\/Mp.C1qa\u2212\/\u2212 mice, which produced higher levels of IgM autoAbs, the MZ B cell population was significantly decreased compared to the VH3H9R.MRL\/Mp animals (Fig. 3A) and less idiotype+ B cells were found in the MZ compared to the FO compartment (3.4\u00a0\u00b1\u00a00.7 versus 4.8\u00a0\u00b1\u00a01.1, paired t-test p\u00a0=\u00a00.0042) (Table 1). In the VH3H9R\/VL\u03ba8R.MRL\/Mp mice there was no preferential localization of idiotype+ B cells in the MZ zone (paired t-test p\u00a0=\u00a00.056) (Table 1). C1q deficiency was associated with a significant reduction in the percentage of MZ B cells (Fig. 3A) and a decrease of idiotype+ B cells in the MZ compared to the FO area (4.2\u00a0\u00b1\u00a00.9 versus 17.5\u00a0\u00b1\u00a03.2, paired t-test p\u00a0=\u00a00.0013). These findings indicated that MZ B cells might have been activated in the absence of C1q. Activated MZ B cells have been shown to migrate into the T cell zone and differentiate into plasma cells. Indeed, the percentage of plasma cells was increased in the two C1q-deficient cohorts compared to the respective MRL\/Mp controls (Fig. 3B).\nThe peritoneal cavity in mice has been shown to be a site where some self-reactive B cells can escape tolerance (Fagarasan et al., 2000) and B-1 cells, one of the major sources of circulating IgM, accumulate. More cells were recovered from the peritoneum of VH3H9R.MRL\/Mp.C1qa-\/-mice compared to VH3H9R.MRL.Mp mice (p\u00a0=\u00a00.0051) but not in the VH3H9R\/VL\u03ba8R.MRL\/Mp.C1qa\u2212\/\u2212 versus VH3H9R\/VL\u03ba8R.MRL\/Mp animals (p\u00a0=\u00a00.1923) (Table 1). When the peritoneal B cell subpopulations were analyzed in more detail, all subsets were increased in the VH3H9R.MRL\/Mp.C1qa-\/mice compared to the VH3H9R.MRL.Mp mice (Table 1). On the contrary, in the VH3H9R\/VL\u03ba8R C1q-deficient animals only B-1a cells were slightly increased compared to their wild type strain-matched controls (Table 1), but this did not reach statistical significance (p\u00a0=\u00a00.0518). We then analysed the VH3H9\/VL\u03ba8 idiotype expression on the different peritoneal B cells. Surprisingly in the peritoneum of the VH3H9R\/VL\u03ba8R.MRL.Mp and VH3H9R.MRL.Mp mice the majority of the B cells were not expressing the VH3H9\/VL\u03ba8 idiotype (Table 1). Of note the C1q-deficient mice had similar percentage of idiotype+ B cells in the different peritoneal B cell subpopulations compared to C1q-sufficient mice. The reduced proportion of idiotype+ B cells in the peritoneum compared to the peripheral blood and the spleen (data not shown) suggests that in this organ the B cells either preferentially used the endogenous alleles or had been activated and undergone editing.\n4\nDiscussion\nC1q deficiency in humans and in mice has been associated with the development of a lupus-like illness. However, the role of C1q in the regulation of autoreactive B cells remains debatable. Here, we explored the regulation of autoreactive B cells by C1q using anti-DNA knock-in Tg models (VH3H9R\/VL\u03ba8R and VH3H9R) on the lupus prone MRL\/Mp genetic background. The analysis of these mice revealed that the MRL\/Mp background was in itself sufficient to allow the expression of anti-DNA Tg autoAbs and that the lack of C1q modified this effect only in the single Tg model (VH3H9R). However, in the absence of C1q the MZ B cell compartment was significantly reduced in both models and this was accompanied by an increase in plasmocytes.\nRecently the VH3H9R\/VL\u03ba8R anti-DNA knock-in Tg model has been widely used for investigating the mechanisms of regulation of autoAb production in murine models of SLE. There is an increasing evidence that the VH3H9R\/VL\u03ba8R and VH3H9R anti-DNA Tg B cells, which have a relatively weak affinity to ssDNA, are regulated by anergy on normal genetic backgrounds such as BALB\/c and C57BL\/6, but could be induced to lose tolerance when transferred onto lupus models such as (NZB\u00a0\u00d7\u00a0NZW)F1 or MRL\/Mp.lpr\/lpr (Brard et al., 1999; Chen et al., 1995; Erikson et al., 1991; Fukuyama et al., 2005; Sekiguchi et al., 2002). In light of these observations it was important for our study to establish whether the autoimmune prone MRL\/Mp background was in itself capable of breaking tolerance. MRL\/Mp mice are known to develop a mild autoimmune disease which can be accelerated with different disease-modifying genes such as Yaa (Merino et al., 1989), lpr and gld (Cohen and Eisenberg, 1991). The analysis of the idiotype+ (VH3H9R\/VL\u03ba8R) Abs revealed that the Tg MRL\/Mp mice indeed had in circulation these idiotype+ Abs (IgM, IgG2a, IgG2b, IgG3) indicating that a break of tolerance had spontaneously occurred in these mice. One explanation for this is an intrinsic defect in MRL\/Mp B cells and there is some evidence in support of this. MRL\/Mp mice have been reported to exhibit a defect in maintaining the developmental arrest of VH3H9\/VL\u03bb anti-dsDNA conventional Tg B cells (Mandik-Nayak et al., 1999, 2000) and to have a spontaneous B cell hyperactivity in the absence of Ag in the IgHEL experimental model (Nijnik et al., 2006). However, the VH3H9\/VL\u03bb anti-dsDNA autoreactive B cells, despite not being any longer developmentally arrested as in a BALB\/c mice, exhibited follicular exclusion and failed to differentiate into plasma cells (Mandik-Nayak et al., 1999). Furthermore Tg MRL\/Mp mice expressing IgHEL have been shown to be able to down-regulate their B cell receptor and to be unable to secrete detectable levels of anti-HEL Abs in the presence of sufficient amount of sHEL (Nijnik et al., 2006). Similarly anti-laminin Tg MRL\/Mp mice were found to be tolerant (Rudolph et al., 2002). Another potential explanation for the break of the B cell tolerance in the VH3H9R\/VL\u03ba8R anti-DNA Tg mice, is that in this model the MRL\/Mp background was able to provide sufficient T cell help and the presence of idiotype+ IgG subclasses favour this hypothesis.\nWe next examined whether C1q could modulate the phenotype of the anti-ssDNA Tg B cells. In the VH3H9R\/VL\u03ba8R.MRL\/Mp mice the absence of C1q increased significantly the circulating levels of IgM against ssDNA but not against other autoantigens including dsDNA. As the pairing of VH3H9 with VL\u03ba8 prevents the binding of VH3H9 to dsDNA, the elevated amount of IgM anti-ssDNA observed could have been the result of an increased Tg expression. However, the idiotype analysis failed to demonstrate a difference in the levels of IgM idiotype+ Abs between VH3H9R\/VL\u03ba8R.MRL\/Mp.C1qa\u2212\/\u2212 and VH3H9R\/VL\u03ba8R.MRL\/Mp mice, questioning whether the source of the increased levels of IgM anti-ssDNA was indeed the Tg B cells. On the other hand, the VH3H9R.MRL\/Mp.C1qa\u2212\/\u2212 mice displayed significantly higher levels of IgM and IgG3 idiotype+ Abs. As the VH3H9R heavy chain can pair with different endogenous light chains generating a wider range of autoAbs, other specificities were tested. Indeed the absence of C1q increased significantly IgM levels against all the lupus autoantigens analysed.\nTo gain insight into the mechanisms regulating the autoAb production in the C1q-deficient mice we then carried a detailed analysis of the different B cell populations. Several studies have proposed a possible role for MZ B cells in the development of lupus in mouse models. Although studies in immunoglobulin Tg mice have shown that autoreactive B cells can accumulate in the marginal zone under various experimental situations (Li et al., 2002; Mandik-Nayak et al., 1997, 1999, 2006; Qian et al., 2004; Wen et al., 2005), it remains to be established whether MZ B cells secrete pathogenic autoAbs in any model of lupus. B cells producing potentially pathogenic autoAbs are thought to home to the MZ (Chen et al., 1997b; Li et al., 2002) and sequestration to this site is believed to prevent them from entering into the germinal centres and developing the properties of pathogenic B cells. However, recent studies in lupus-prone mice have reported both enlargements (Grimaldi et al., 2001; Mackay et al., 1999; Wither et al., 2000) and impaired development of the MZ B cell compartment (Amano et al., 2003; Samardzic et al., 2002). Consistent with previous observations in the VH3H9R\/VL\u03ba8R model (Wen et al., 2005), the MZ was found to be enlarged in the VH3H9R.MRL\/Mp mice but no accumulation of MZ B cells was observed in VH3H9R\/VL\u03ba8R.MRL\/Mp mice. Importantly C1q deficiency decreased dramatically the proportion of MZ B cells in these mice and this was accompanied by an increase in the percentage of plasma cells. More interestingly, in the C1q-deficient Tg mice we observed a significant disappearance of idiotype+ B cells from the MZ suggesting that these cells had been activated. Marginal zone B cells bearing low affinity self-reactive BCR can react to repetitive Ag and produce natural autoAbs of the IgM isotype upon contact with blood-borne pathogens or self Ag (Oliver et al., 1999). Moreover, MZ B cells can undergo T cell independent switching to IgG, IgA and IgE in response to pathogen associated molecular patterns (PAMP). As C1q has been shown to be involved in the clearance of apoptotic cells, which are enriched in the typical lupus autoantigens, one could postulate that the increased IgM Tg secretion and the reduction of the MZ B cells in the VH3H9R.MRL\/Mp.C1qa\u2212\/\u2212 mice might be related to a failure to clear antigens associated with dying cells. In the absence of C1q the ineffectively cleared autoAgs could stimulate the autoreactive MZ Tg B cells to differentiate into plasmocyte resulting into the decrease of MZ B cells and increase of circulating IgM autoAbs. Similarly, in the spleens of VH3H9R\/VL\u03ba8R.MRL\/Mp.C1qa\u2212\/\u2212 mice significantly less idiotype+ B cells were present in the MZ compartment and more plasmocytes were found. However, these cellular changes were not paralleled by an increase of idiotype+ IgM Abs in circulation indicating in this model they were not sufficient to induce autoantibody production.\nAnother potential source of IgM autoAbs are the B1 cells. B1 cells have long been associated with the secretion of natural Abs against self and foreign pathogens, which can occur without obvious inflammatory response. The importance of B1 cells in the pathogenesis of lupus continues to be debated. A major argument for a role of B1a cells in mouse lupus relates to the expansion of this subset in NZB and (NZB\u00a0\u00d7\u00a0NZW)F1 mice. Reduction of B1 cells for instance via intraperitoneal injection of H2O delayed disease onset and reduced disease severity in (NZB\u00a0\u00d7\u00a0NZW)F1 mice (Murakami et al., 1995). However, the expansion of B1a cells was shown not to be critical for the production of IgM or IgG autoAbs in this murine model of SLE (Atencio et al., 2004). The Abs secreted by B1 cells tend to be polyreactive with low-affinity cross-reactivity to a variety of self Ags and these characteristics are very different from the pathogenic IgG Abs produced by lupus mice. In our experimental model, we found an increase in the total number of peritoneal and B1 cells in the VH3H9R.MRL\/Mp.C1qa\u2212\/\u2212 mice compared to their wild type counterparts (Table 1). A similar trend was observed for the B1a cells in the VH3H9R\/VL\u03ba8R.MRL\/Mp.C1qa\u2212\/\u2212 mice but this did not reach statistical significance. However, in both models the percentages of idiotype+ B1 cells between C1q-deficient and -sufficient mice were similar. These findings would indicate the lack of C1q may favour the expansion of peritoneal B1 cells and that these cells might have contributed to the increase of serum IgM autoAb levels observed in the VH3H9R.MRL\/Mp C1q-deficient mice. Supporting this hypothesis, a recent study showed that C1q deficiency increases the positive selection of B-1 cells and IgM autoAb production by a membrane-bound intracellular auto-Ag (Ferry et al., 2007).\nIn conclusion, using mice expressing site-directed transgenes for anti-DNA autoAbs we have shown that: (i) the MRL\/Mp background was in itself capable of inducing the expression of anti-DNA Tg autoAbs, and (ii) VH3H9R.MRL\/Mp.C1qa\u2212\/\u2212 could influence the production of Tg-derived IgM and IgG3 autoAbs possibly as a result of an impaired disposal of cellular debris. However, we found no evidence of a direct role of C1q in the regulation of self-reactive conventional B cells. Further studies on non-lupus prone genetic backgrounds such as C57BL\/6 will be necessary to determine if C1q deficiency can play a more substantial role in shaping the repertoire of the autoreactive B cells.","keyphrases":["transgenic","b cells","complement","autoimmunity","ab, antibody","aeu, arbitrary elisa units","fo, follicular","hel, hen egg lysozyme","mz, marginal zone","tg, transgenic","sle, systematic lupus erythematosus","rodent"],"prmu":["P","P","P","P","R","R","R","R","R","R","R","U"]}
{"id":"Ulster_Med_J-76-3-2075581","title":"The use of mechanical bowel preparation in elective colorectal surgery\n","text":"Background Mechanical bowel preparation (MBP) prior to elective colorectal surgery has been in use for many years. It is considered important in preventing post-operative infectious complications after colorectal surgery. The evidence to support these claims is lacking within the medical literature and yet this still remains standard practice in many hospitals. A literature search was undertaken to ascertain the evidence available regarding the use of MBP in elective colorectal surgery.\nBACKGROUND\nMechanical bowel preparation (MBP) prior to elective colorectal surgery has been in use for many years. Early observational studies and long-standing clinical experience have shown that removal of faecal matter from the bowel lumen prior to surgery has been associated with decreased patient morbidity and mortality1. It is still commonly used in routine practice today2. In fact, in a recent survey of members of the American Society of Colon & Rectum Surgeons, 99% of respondents routinely use MBP although 10% question its use3. This is in keeping with common belief that clinical practice often is not evidence based but is based on tradition, previous teaching and anecdote.\nMBP is considered important in preventing post-operative infectious complications after colorectal surgery2,4\u201310. Important infectious complications include wound infection, intra-abdominal abscess formation and anastomotic leakage. There are a number of ways in which MBP is thought to act. It may decrease intraoperative contamination with faecal material thereby reducing the incidence of post-operative wound infection and residual intra-abdominal infection6,7,9,10. It may prevent mechanical disruption of the anastomosis by the passage of hard faeces9 and improves the handling of the bowel intra-operatively2,7. It may reduce the bacterial count within the colon7,10. Conversely, it may also be associated with bacterial translocation through the bowel wall hence possibly contributing to post-operative infectious complications5,11. The evidence to support these claims is lacking within the medical literature and yet this still remains standard practice in many hospitals4,12.\nPrimary colonic anastomosis is considered unsafe in unprepared bowel but there is little data to suggest that infectious complications are decreased by MBP10. Bowel preparation is unpleasant for patients and can be associated with complications such as dehydration, nausea, vomiting, mucosal lesions, hypokalaemia and other electrolyte disturbances1,9,11. The omission of this practice from pre-operative preparation would be welcomed by nursing staff and patients alike.?\nMETHODS\nA literature search was undertaken to ascertain the evidence available regarding the use of MBP in elective colorectal surgery. This included a search of PubMed, Medline and Embase using the keywords \u201cmechanical bowel preparation\u201d, \u201cbowel cleansing\u201d and \u201celective colorectal surgery\u201d, a search of recent relevant journals including Diseases of the Colon and Rectum and British Journal of Surgery and backward chaining from articles obtained. The search was restricted to English language articles and a timescale of 10 years was chosen to give a balanced view of this topic.\nIn this review, mechanical bowel preparation will be defined as an oral preparation given prior to surgery to clear faecal material from the bowel lumen. There are a number of different preparations available including polyethylene glycol, mannitol and sodium picosulphate. Rectal enemas may also be administered before low anterior resections to ensure that the rectum is empty.\nElective colorectal surgery is defined as any surgery undertaken on a planned basis for any condition of the colon or rectum requiring bowel resection and primary anastomosis. This will include colorectal carcinoma and inflammatory bowel disease.\nThere are a number of recent randomised controlled trials (RCTs) to evaluate the use of MBP prior to elective colorectal surgery,10,11,13,14 and specifically for left-sided resections5. Many of these studies are underpowered therefore introducing the possibility of a Type II error and limiting the use of these results in clinical decision-making (Table I).\nTable I\nRCTs examining MBP\nZmora 2003\nFa-Si-Oen 2005\nRam 2005\nBucher 2005\nMiettinen 2000\nNo. of patients included\n415\n250\n329\n153\n267\nNo. of patients excluded\n35\n0\nNot given\n0\n12\nNo. of pt (MBP\/no MBP)\n187\/193\n125\/125\n164\/165\n78\/75\n138\/129\nMean age (MBP\/no MBP)\n68\/68\n68\/70 (median)\n68\/68\n63\/63\n61\/64\nCancer % (MBP\/no MBP)\n78\/78\n90\/92\n75\/88\n32\/28\n46\/55\nL colon surgery % (MBP\/no MBP)\n68\/72\n48\/58\n89\/85\n100\/100\n45\/47\nType of prep\nPolyethylene glycol\nPolyethylene glycol\nSodium phosphate\nPolyethylene glycol\nPolyethylene glycol\nAntibiotic\nYes\nYes\nYes\nYes\nYes\nSame length of prophylaxis\nNo\nYes\nYes\nNo\nYes\nRectal enema\nYes\nNo\nNo\nYes\nNo\nAnastomosis % (stapled \/ handsewn)\nNot given\n7\/93 (MBP) 8\/92 (no MBP)\n94\/6 (MBP) 98\/2 (no MBP)\nNot given\n60\/30 (MBP) 62\/28 (no MBP)\nSurgeon\/trainee %\nNot given\n42\/59 (MBP) 50\/50 (no MBP)\n37\/63 (MBP) 32\/68 (no MBP)\nNot given\nNot given\nAnastomotic leak % (MBP \/ no MBP)\n3.7\/2.1 (NS)\n5.6\/4.8 (NS)\n0.6\/1.2 (NS)\n6\/1 (NS)\n4\/2 (NS)\nWound infection % (MBP \/ no MBP)\n6.4\/5.7 (NS)\n7.2\/5.6 (NS)\n9.8\/6.1 (NS)\n13\/4 (NS)\n4\/2 (NS)\nIntra-abdominal abscess % (MBP \/ no MBP)\n1.1\/1 (NS)\nNot given\n0.6\/0.6 (NS)\n1\/3 (NS)\n2\/3 (NS)\nNS = not significant\nThis lack of power in studies is somewhat overcome by the use of meta-analyses and systematic reviews of the literature but the reader must be aware that these methods also have their limitations. A number of meta-analyses and systematic reviews were used in this review1,2,7\u20139,15.\nDISCUSSION\nSix systematic reviews were identified in the literature assessing the role of MBP in preventing infectious complications following colorectal surgery1,2,7\u20139,15.\nThe meta-analysis carried out by Platell & Hall7 found a statistically significant increase in the incidence of wound infection in those patients receiving MBP when considering the three included RCTs. This may have been influenced by the rate of wound infection seen in one trial that used a five day regime of MBP. The anastomotic leakage rate was also higher in the MBP group but not significantly so. Each RCT used a different type of MBP and this lack of standardisation affects the validity of the results. The included studies were also underpowered thereby introducing a high possibility that they failed to detect a significant difference in the results (type II error). Evidence from the prospective and retrospective studies was in favour of no MBP in pre-operative period.\nA subsequent review of the literature by Zmora et al9 appraised four RCTs. One of the studies found an increased risk of anastomotic leakage and intra-abdominal infection but no increased risk of wound infection in the group of patients receiving MBP. The remaining RCTs found no significant difference in intra-abdominal infection rate but a slight increase in wound infection rate in the MBP group.\nThere were conflicting results in the non-randomised studies with some showing an increased rate of infection and others reporting no difference in infection rates between the groups.\nThe remaining four meta-analyses only included RCTs1,2,8,15. All authors agreed that MBP was of no benefit in preparation for colorectal surgery and it may be detrimental to the patients' outcome1,2,8. Wille \u2013Jorgensen et al15found that although initial analysis showed a significantly higher rate of anastomotic leakage in the MBP group, this significance disappears when sensitivity analyses are applied thereby weakening the conclusion that MBP leads to an increased rate of anastomotic leakage.\nSlim et al8 found there was significantly more anastomotic leakage in the group of patients receiving MBP and a tendency to a higher rate of wound infection but this was not statistically significant. This group repeated the analysis excluding the poor quality trials and the results still favoured a no MBP regime although this was not statistically significant.\nOnly two meta-analyses looked at MBP in rectal surgery specifically1,15. Willie-Jorgensen et al15 found that when results were stratified for colonic and rectal surgery there was no trend in either direction. Guenaga et al1 found that the results of stratification favoured no MBP but this was not statistically significant. This is of more clinical importance as it may be difficult to perform a low anterior resection and anastomosis with a loaded rectum15. Both authors suggest that further trials evaluating the use of rectal preparation with enemas may be useful. Guenaga et al1also mention that the use of pre-operative radiotherapy would be an important consideration in assessment of bowel preparation for rectal surgery as many patients with rectal cancer undergo pre-operative radiotherapy.\nAll but one of the five RCTs examining MBP5,10,11,13,14 found no significant difference in the rate of anastomotic leakage and wound infection between patients receiving MBP or not10,11,13,14. The largest trial was undertaken by Zmora et al10 with 415 patients recruited. There are several flaws in the methodology of this trial introducing bias and compromising the validity of the results.\nThere was no difference found in the rate of post-operative infectious complications between the two groups. The rate of diarrhoea post-operatively was significantly more common in the group receiving MBP but this is of little clinical significance, as many patients will experience an increased stool frequency once the bowels become active. The authors acknowledge that separating the role of MBP in post-operative infection rate is difficult and ideally all other measures should be constant. They also note that the study is underpowered to detect a 5% difference in infection rate.\nThe RCT conducted by Bucher et al5 comparing MBP with no MBP in patients undergoing elective left-sided colorectal surgery found an increase in the total incidence of infectious abdominal complications in the group receiving MBP (22% v 8%; p=0.028). This led the authors to conclude that there was good evidence to suggest that the practice of MBP should be re-evaluated. They gave an enema pre-operatively to all patients undergoing an anterior resection regardless of whether they had been randomised to MBP or not, decreasing the internal validity of the results. If anastomotic leak rate (a more clinically important outcome than wound infection) were to be used as the primary end-point then the study would need 514 patients in each group.\nThe trial conducted by Ram et al14 was not properly randomised, introducing methodological bias and limiting the value of the results of this study. There was no definition of sample size and patients with low rectal anastomosis were excluded. Again, the assessor of outcome was not blinded to the intervention, introducing another source of bias. No statistically significant difference in the frequency of infectious complications was observed between the groups yet the authors concluded that \u201cmechanical bowel preparation is unnecessary for safe elective colonic and colorectal surgery\". But they recommend MBP in selected cases including the resection of small tumours when palpation of the colon may be necessary or when intra-operative colonoscopy may be performed.\nFa-Si-Oen et al13 conducted a well-designed multi-centre RCT, reported in 2005. Approximately half the resections carried out in this study were left-sided. This is important as it is now generally accepted that right-sided anastomosis is safe without MBP. This study excluded patients undergoing rectal surgery. There was no significant difference in wound infection or anastomotic leak rate but the bacterial swab results used to define wound infection in this study were only correctly obtained in 185 out of 250 patients therefore this may not be an accurate reflection of the true rate of wound infections. This study could not demonstrate an additional protective effect for MBP but it was an interim analysis and was underpowered. As a result, conclusions for clinical practice cannot be drawn from these results.\nMiettinen et al11 reported the results of a prospective, randomised study including patients undergoing rectal surgery. There was no significant difference in infectious complications found between the two groups but it is difficult to conclude on the influence on anastomotic leakage from these results as the study included patients who did not undergo an anastomosis.\nA number of these reported trials are underpowered thereby limiting their ability to detect a clinically significant difference in outcome between the two study groups10,11,13. One way of overcoming the problem of small sample sizes is to carry out a multi-centre trial where a larger number of patients are easier to recruit. A limitation is that they introduce heterogeneity in operative and peri-operative techniques. This is important, as surgical technique may be the single most important factor in influencing the surgical outcome9. All these studies agree that elective colorectal surgery may be safely performed without MBP and that there is no evidence to continue this invasive practice with potentially negative side effects.\nMemon et al16 carried out a retrospective non-randomised trial based on operating surgeon preferences using a questionnaire. The validity of this questionnaire is unclear as no pilot study was carried out prior to the collection of definitive data. Follow-up of the patients was obtained using the hospital records therefore relying on accurate clinical notes, which are not always available.\nOne hundred and thirty six patients who underwent elective left-sided colorectal procedures for non-obstructive large bowel pathologies were identified using the hospital computer system. Coding errors may mean that some eligible patients were excluded from the analysis. This, along with the lack of randomisation, would introduce significant bias.\nNo statistical difference was found between the two groups for all infectious complications and mortality. The authors recognise the limitations of their results and do not recommend any changes in practice but do suggest that a prospective randomised trial should be performed to demonstrate the impact of MBP on morbidity and mortality in patients undergoing elective colorectal surgery.\nA prospective, observational trial performed by van Geldere et al17 assessed the outcome of 250 consecutive patients who underwent resection and primary anastomosis of the colon and upper rectum under the care of a single surgeon. None of these patients received MBP pre-operatively. Both emergency and elective procedures were included in analysis. Results were favourable with an overall wound infection rate of 3.3% and an anastomotic failure rate for left-sided resections of 1.2%. The authors recommend that more powerful randomised trials are needed but in the hands of a single surgeon, primary anastomosis of unprepared bowel is safe with relatively few complications.\nA small observational study conducted by Ahmad et al4 found an anastomotic leak rate of 4.2% and a wound infection rate of 8.5%. The average age of the sample was lower than that of the typical population undergoing colorectal surgery. This fact, plus the small sample size, compromises the external validity or the extent to which the results can be generalised to other samples or situations.\nCONCLUSION\nThere are a number of meta-analyses, systematic reviews and RCTs looking at the efficacy of MBP in preventing post-operative infectious complications following elective colorectal surgery. Unfortunately many of these trials are underpowered and have a high chance of a type II error10,11,13. Most authors recommend that colorectal surgery is safe without pre-operative MBP but that there may some situations in which it may be beneficial (e.g. if there is a small tumour or the possible need for intra-operative colonoscopy)14.\nThe implication for clinical practice in this situation is that there is not enough strength of evidence at present to recommend a change in practice. There is a need for further higher powered trials to try to answer this question definitively. The only way that this may be achieved is by multi-centre trials where it is easier to recruit a large number of patients but it must be taken into consideration that this will introduce heterogeneity in the operative and peri-operative techniques which may have an influence on overall outcome9. There is a need for larger clinical trials in this area to address whether MBP, with its potential side effects, is truly necessary prior to elective colorectal surgery.\nFurther studies are required to assess the use of rectal preparation alone prior to rectal surgery1,15 and also to include patients who have undergone pre-operative radiotherapy1 as this is a common occurrence in patients who have rectal carcinoma and these patients may subsequently undergo resection with primary anastomosis. It is clear that further research is needed to clarify the role of MBP in elective colorectal surgery to ensure that the patients are receiving the most appropriate treatment with the least adverse effects.","keyphrases":["mechanical bowel preparation","colorectal surgery"],"prmu":["P","P"]}
{"id":"Qual_Life_Res-3-1-2039860","title":"Demographic characteristics and quality of life of patients with unexplained complaints: a descriptive study in general practice\n","text":"Objective About 13% of GPs\u2019 consultations involve unexplained complaints (UCs). These complaints can progress to chronic conditions like medically unexplained symptoms, chronic functional symptoms or somatoform disorders. Little is known about the demographic characteristics and quality of life of patients with early stage UCs. Our study objective was to describe these characteristics. Additionally we compared them with other patient groups to serve as a frame of reference.\nIntroduction\nA patient\u2019s complaints are said to be unexplained if the general practitioner (GP) cannot decide on a specific diagnosis after adequate history taking, physical examination and careful consideration of the patient\u2019s psychosocial context [1]. On average, 13% of consultations involve complaints considered unexplained by Dutch GPs [2]. These unexplained complaints often concern fatigue, abdominal and musculoskeletal complaints.\nIt is often assumed that unexplained complaints are mild and self-limiting, because patients do often not revisit their GP for them after a first consultation [3, 4]. However, an unexplained complaint can be a first sign of somatic or psychosocial pathology or a precursor of more chronic unexplained complaints such as chronic medically unexplained symptoms (MUS), chronic functional symptoms or somatoform disorders [5].\nPatients with chronic unexplained conditions often have high levels of medication and other healthcare use and frequently show significant psychological distress [6]. Patients with multiple medically unexplained symptoms (MMUS) have impaired quality of life [7]. Furthermore, patients often do not feel taken seriously, do not feel helped and feel treated as malingerers. They, therefore, tend to lose trust in their GPs [8]. In brief, the long-term consequences of many unexplained complaints are unfavourable.\nGPs too consider unexplained complaints problematic. One study found that in consultations with such patients GPs feel frustrated and helpless though devoted to help [9].\nResearch on unexplained complaints is increasing. So far, most research has focused on patients with chronic consequences of unexplained complaints. This research varies from discussions on nomenclature and taxonomy [10\u201312], to research on the characterization of these patient groups [13, 14] and possible treatment options [15\u201317]. Little is known about patients with unexplained complaints in their early stages. We performed this study to describe the demographic characteristics and quality of life of patients with early stage unexplained complaints. To provide some frame of reference, we compared these descriptive measures for the unexplained complaints to those of an unselected group of patients visiting their GP, and a group of depressed patients. For practitioners, better delineation, in terms of quality of life, of a group of patients that frequently seeks their help may sensitize them to consider more structured monitoring in order to achieve a firmer diagnosis be it somatic, psychological, or psychosocial.\nMethods\nGPs and patients\nGPs were recruited from the southern and the western part of the Netherlands. GPs were given the Dutch College of General Practitioners\u2019 definition of unexplained complaints: those complaints that remain of unclear origin for the GP after adequate history taking, physical examination and careful consideration of the patient\u2019s psychosocial context [1]. This definition supposes that complaints are labelled \u201cunexplained\u201d early on in the clinical episode before elaborate investigations were performed.\nBetween February 2002 and December 2003, participating GPs included adult patients, presenting with a complaint that the GP designated as \u201cunexplained\u201d at the end of the first consultation. Patients who had presented with the same complaint in the previous 6\u00a0months were not included. Patients were eligible if they presented with fatigue, abdominal complaints, musculoskeletal complaints, weight changes or itch as their main complaint.\nIndividual GPs decided whether a complaint was \u201cunexplained\u201d. There was no standardization of this process other than that implied by the Dutch College of General Practitioners\u2019 definition. GPs invited patients to participate in the study at the end of the consultation.\nInformed consent procedure and medical ethics committees\nAll participating patients gave written informed consent after having read information provided by the GP directly after the consultation. The medical ethics committees of the University of Amsterdam and Maastricht University approved the study protocol.\nData collection: study population\nThe data for this study were collected as part of baseline measurements for a randomized diagnostic trial on unexplained complaints in general practice [18]. After the entry consultation, patients filled out a questionnaire on demographic characteristics. The RAND 36-item Health Survey (RAND-36) was used to measure generic health-related quality of life. The RAND-36 [19] is a Dutch version of the Medical Outcome Study 36-item Short Form Health Survey (SF-36) [20] and is composed of 36 questions and standardized response choices, organized into eight multi-item scales (domains). These eight domains are: physical functioning (PF), role limitations due to physical health problems (RP), social functioning (SF), general mental health (MH), role limitations due to emotional problems (RE), vitality (VT), bodily pain (BP) and general health perception (GH). Raw RAND-36 scores on the eight domains are linearly converted to 0\u2013100 scales with higher scores indicating better quality of life.\nData collection: reference populations\nReference population for comparison of demographic characteristics\nThe demographic characteristics of a Dutch general practice population were taken from the second Dutch National Survey of General Practice, which is a large representative population-based survey that takes place every 10\u00a0years. Methods of this study have been published elsewhere [21]. Briefly, this national survey contains data about health and healthcare-related behaviour of 375,899 persons, registered in 104 practices with 195 GPs in 2001.\nReference populations for comparison of quality of life\nThe results section of this manuscript shows that the quality of life of patients with early stage unexplained complaints is poor. Our initial objective was purely descriptive. However, when such low quality of life figures were found, we felt the need to compare these to other patient groups. First, we wondered whether maybe all patients consulting their GP would have such low quality of life figures and therefore compared the unexplained complaints group with a Dutch general practice population. We also thought that maybe our patient population included a lot of depressed patients which could explain the poor quality of life and therefore also compared our quality of life figures with those of a population of depressed patients.\nFigures of the Dutch general practice population came from a study on functional status, health problems, age and comorbidity in primary care patients [22]. In this study 60 GPs from 43 general practices handed out a written questionnaire to 100 patients of 18\u00a0years and older, consecutively visiting their practice. In total 4,024 patients responded.\nFigures of the depressed patients came from the Netherlands Mental Health Survey and Incidence Study (NEMESIS). This NEMESIS survey, was based on a random sample drawn from the Dutch general adult population aged 18\u201364 (n\u00a0=\u00a07,076) of whom 204 had been diagnosed with a major depression within the past month [23].\nStatistical analysis\nFirst, we compared the demographic characteristics of the five unexplained complaint categories to each other. Those of the total UC study population were compared to those of a Dutch general practice population. Differences were tested using the Fisher\u2019s exact test.\nSecond, mean domain scores on the RAND-36 were compared for each of the five unexplained complaint groups using linear regression with the \u201cfatigue\u201d complaint group as the reference category. We calculated robust variance estimates (Huber-White sandwich estimator) [24] to allow for potential dependence of quality of life scores within a single GP.\nLastly, we compared mean domain scores of the total UC study population to those of a Dutch general practice population sample and to a random sample of depressed patients. Differences were statistically tested with t-tests in which equal variances were not assumed. Analyses were performed using STATA, version 9.2.\nResults\nGeneral characteristics of the study group\nOf the 91 GPs who intended to participate, 18 dropped out before including any patients. They predominantly reported lack of time as the reason for drop out. Ten GPs did not include any patients during the inclusion period, although they did not formally drop out. Thus, 63 GPs (69%) included 513 patients with unexplained complaints (range: 1\u201336 patients per GP). Questionnaires from 466 (91%) patients were available for analysis. Forty-seven patients stopped their participation to the study or did not fill out this particular questionnaire.\nDemographic characteristics of the study group\nThe demographic characteristics are summarized in Table\u00a01, for the total UC study population, per complaint group and for the Dutch general practice population. The mean age of the total UC study population was 44\u00a0years (interquartile range 31\u201355) and 74% were women. Most patients had completed secondary or higher level education (91%) and were married or living together with a partner (71.5%).\nTable\u00a01Demographic characteristics of the total UC study population, per complaint group and of a Dutch general practice populationDemographic characteristicCategoriesTotal UC study population % (n\u00a0=\u00a0466)Fatigue % (n\u00a0=\u00a0295)Abdominal complaints % (n\u00a0=\u00a060)Musculoskeletal complaints % (n\u00a0=\u00a069)Other complaint groups % (n\u00a0=\u00a042)Dutch GP population % (n\u00a0=\u00a03,85,461)SexMale26.426.12524.633.349.5Female73.6a73.97575.466.750.5AgeMean (years)44.241.842.352.2 b50.3Unknown0\u2013194.96.16.71.1023.420\u20133937.142451330.930.440\u20136445.9a41.74066.7503365+1210.28.318.819.113.2Educational levelNone1004.3 b2.416Elementary97.53.317.4 b14.318.9Secondary68.9a68.18059.5b64.348.1Higher22.1a24.416.718.81917Marital stateSingle28.529.82030.428.6UnknownMarried\/cohabiting71.570.28069.571.4UnknownInsurance TypePublic65.265.868.369.65067.4Private34.834.231.730.45032.6NationalityDutch95.396.396.791.39386.2Not Dutch4.73.73.38.7713.8Region of the NetherlandsWest56.457.646.762.352.4UnknownSouth43.642.453.337.747.6Unknowna P\u00a0<\u00a00.05 (Fisher\u2019s exact test, reference group is GP population)b P\u00a0<\u00a00.05 (Fisher\u2019s exact test, reference groups are fatigue and abdominal complaints)\nThe main reason for encounter was fatigue (63.3%) and only few patients with weight changes or itch were included. In all further analyses, these latter two groups were combined into an \u201cother complaints\u201d group.\nPatients with musculoskeletal complaints were significantly older and less educated than patients with fatigue or abdominal complaints (P\u00a0<\u00a00.05).\nCompared to patients from the Dutch general practice population (second Dutch National Survey of General Practice), unexplained complaint patients were more likely to be women (74% vs. 51%), older (46% vs. 33% in the age group 40\u201364) and more highly educated (91% vs. 65% secondary or higher level education). These differences were statistically significant (P\u00a0<\u00a00.05).\nQuality of life of the total UC study population and per unexplained complaint group\nThe overall quality of life for the total UC study population was poor (Table\u00a02). All domains showed a mean score of less than 70 (on a scale from 0 to 100), except Physical Functioning (mean 73.4). The lowest score was in the Role Functioning Physical domain (mean 37.2). Also the Vitality domain scored low (mean 40.5) both in the total UC study population and in the separate complaint groups.\nTable\u00a02Quality of life of the total UC study population and per complaint groupAllFatigueAbdominalMusculoskeletalOtherN466295606942Domains RAND-36MeanMeanMean\u0394 (95% CI)Mean\u0394 (95% CI)Mean\u0394 (95% CI)Physical functioning73.473.381.38.1 (2.2 to 14.0)a62.2\u22128.0 (\u221214.8 to \u22121.3)a76.33.0 (\u22124.3 to 10.4)Social functioning66.562.570.88.3 (\u22120.2 to 16.9)76.413.9 (6.9 to 21.0)a729.5 (\u22120.7to 19.7)Role functioning physical37.231.556.324.7 (11.9 to 37.5)a397.5 (\u22123.5 to 18.5)4715.4 (\u22126 to 31.4)Role functioning emotional60.254.174.420.3 (9.0 to 31.6)a74.220.1 (6.6 to 33.6)a59.35.2 (\u221210.4 to 20.8)Mental health63.861.869.47.6 (2.3 to 12.9)a67.96.1 (1.1 to 11.1)a62.91.0 (\u22126.8 to 8.8)Vitality40.533.653.720.1 (15.1 to 25.1)a54.120.5 (13.8 to 27.2)a48.514.9 (8.2 to 21.5)aBodily pain67.772.761.7\u221210.0 (\u221215.9 to \u22126)a55.5\u221217.2 (\u221225.2 to \u22129.2)a61.3\u221211.4 (\u221221.5 to \u22121.2)aGeneral health55.755.261.15.9 (3.4 to 8.4)a53.8\u22121.4 (\u22123.6 to 0.8)54.3\u22120.9 (\u22125 to 3.3)Differences (\u0394) indicate differences with the fatigue subgroup, where the mean of the fatigue subgroup was subtracted from the other subgroup\u2019s meanConfidence intervals (95% CI) were calculated using the Huber-White sandwich variance estimator which accounts for within physician correlation (for details, see main text)aP\u00a0<\u00a00.05 (linear regression, reference group is fatigue)\nExcept for the Physical Functioning and Bodily Pain scales, patients with unexplained fatigue significantly scored the worst (P\u00a0<\u00a00.05) in every domain, especially on the Role Functioning Physical domain (mean 31.5). Patients with musculoskeletal complaints showed the significantly lowest score on Physical Functioning (mean 62.2) and Bodily Pain (mean 55.5) (P\u00a0<\u00a00.05).\nComparison of quality of life with other patient groups\nTable\u00a03 shows that the total UC study population scored lower on all domains than the Dutch general practice population. These differences were statistically significant (P\u00a0<\u00a00.05) for all domains except Bodily Pain.\nTable\u00a03Comparison of RAND-36 scores of different patient groupsDomains RAND-36Total UC study populationDutch GP populationDepressionAge18\u20138718\u201380+18\u201364N4664,024204MeanMean\u0394 (95% CI)Mean\u0394 (95% CI)Physical functioning73.478.55.1 (2.9 to 7.3)a81.27.8 (3.9 to 11.7)aSocial functioning66.574.58 (5.4 to 10,6)a62.5\u22124 (\u22128.4 to 0.4)aRole functioning physical37.262.425.2 (21.5 to 28.9)a63.426.2 (19.3 to 33.1)aRole functioning emotional60.27514.8 (10.6 to 19.0)a42.6\u221217.6 (\u221224.6 to \u221210.6)aMental health63.869.65.8 (4.0 to 7.6)a46.6\u221217.2 (\u221220.4 to \u221214.0)aVitality40.558.518 (16.1 to 19.9)a40.3\u22120.2 (\u22123.6 to 3.2)Bodily pain67.768.40.7 (\u22121.7 to 3.1)680.3 (\u22124.3 to 4.9)General health55.765.710 (8.9 to 11.1)a55.70 (\u22123.2 to 3.2)Differences (\u0394) indicate differences with the total UC study population, where the mean of the total UC study population was subtracted from the other patient group\u2019s mean95% CI: 95% confidence intervalaP\u00a0<\u00a00.05 (t-test with equal variances not assumed, reference group is total UC study population)\nPatients from the depression group scored significantly lower on the Role Functioning Emotional and Mental Health domains. On all the other domains the total UC study population scored equally high or significantly lower (Physical Functioning and Role Functioning Physical, P\u00a0<\u00a00.05) than patients from the depression group. In other words, unexplained complaint patients scored lowest on predominantly physically oriented domains, whereas depressed patients scored lowest on predominantly mentally\/emotionally oriented domains.\nDiscussion\nOur findings indicate that patients with early stage unexplained complaints are mainly women in their forties, with secondary or higher education levels and with an overall remarkably poor quality of life. Their quality of life in all but one domain of the RAND-36 is significantly worse than that of patients from a general practice population, even taking into account that such a population also includes (around 13% of) unexplained complaint patients. Patients with unexplained complaints predominantly score badly on physically oriented domains, compared to depressed patients, who predominantly score badly on mental\/emotional oriented domains. For the remaining domains they score on a comparably low level. Therefore, practitioners may consider to pay attention to quality of life aspects of patients with early stage unexplained complaints, even though this may not always lead to a firmer diagnosis or instant improvement in treatment options for most patients. A more intense monitoring of these patients could, however, be advised.\nThe total UC study population scored very low on the Vitality domain of the RAND-36 (mean 40.5). This domain consists of questions regarding spirit and energy. The Vitality domain of the SF\/RAND-36 is known to be correlated with subjective feelings of fatigue [25] and we therefore interpret this finding as a logical consequence of the high prevalence of patients with fatigue in the study population. Patients with unexplained complaints do not seem to have predominantly psychosocial problems. The domains Social Functioning and Mental Health were not the worst scoring domains in the study population (mean 66.5 and 63.8 respectively). The Role Functioning Physical domain scored much lower (mean 37.2), suggesting that patients with unexplained complaints feel that their physical complaints hinder them in their daily functioning. Of the five categories of unexplained complaints, patients with unexplained fatigue have the poorest quality of life.\nA first potential limitation of our study can be that different definitions and conceptualizations of unexplained complaints limit the generalizability of our findings. In our study, unexplained complaints were labelled \u201cunexplained\u201d by the GPs in the first consultation after onset, before more elaborate diagnostics. This is in contrast to medically unexplained symptoms (MUS), which refer to symptoms that have been ruled out to have an explainable cause (diagnosis by exclusion). Our concept of early stage unexplained complaints does not allow one to draw firm inferences as to whether the lower quality of life levels can be attributed to the unexplained complaints or should be attributed to underlying, though not yet diagnosed conditions, or concurrent comorbidity.\nSecond, we cannot fully exclude the possibility that some degree of selection bias is present. Participating GPs may have selectively included older patients or those with poorer quality of life. However, in a non-inclusion study in the participating general practices, we searched the electronic medical files by means of text words for eligible but not included patients with unexplained complaints. This non-inclusion study did not show major sex and age differences between included and not-included patients. However, differences may exist on other characteristics.\nA third limitation of our study is that no specific depression or anxiety questionnaire was used. At the start of the study, such a poor quality of life was not anticipated and, therefore, only a more general questionnaire was considered sufficient. The RAND-36 mentally\/emotionally oriented domain figures and the differences we found when comparing the quality of life profile of unexplained complaint patients with depressed patients however, are not pointing towards the presence of depression or anxiety. Furthermore, the GPs did not consider the included patients to be depressed or suffering from an anxiety disorder, otherwise they would not have labelled the patient as having unexplained complaints by definition. It is not impossible however, that depression or anxiety disorders might play a role in the poor quality of life of patients with unexplained complaints. Maybe these diagnoses are established only by GPs over time, and do not become clear in (one of) the first consultations.\nFinally, the patient groups used for comparison of quality of life are perhaps not totally comparable to our study population. For example, there are older patients included in our study population than in the depressed patient groups. Since age has its influence on quality of life this can have influenced the contrast. Also, a depression is a treatable condition, whereas early stage unexplained complaints are not treated yet. The better quality of life in this patient group on some domains can therefore be a treatment effect. We did, however, not intend to study a fully comparable contrast in this sense beforehand, it was a result driven comparison.\nAlthough much research has been performed in patients with more chronic consequences of unexplained complaints, to our knowledge, no other study on demographic characteristics and quality of life of patients with early stage unexplained complaints in general practice has been published.\nPatients with unexplained complaints appear to be mainly highly educated women in their forties. They report remarkably poor levels of quality of life. Future research should explore whether and how quality of life scores and other characteristics could help in early identification of patients at risk of developing chronicity. Until then practitioners should at least be aware that early stage unexplained complaints may not always be as mild as is frequently assumed. Early stage unexplained complaints may be associated with considerable suffering on a daily basis. Awareness of potential poor quality of life may help physicians realise that they are dealing with a patient, at least, in need of more intense monitoring but maybe also of more intense treatment approaches.","keyphrases":["quality of life","unexplained complaints","descriptive study","fatigue","family practice"],"prmu":["P","P","P","P","M"]}
{"id":"Sleep_Breath-4-1-2276561","title":"Low intense physical exercise in normobaric hypoxia leads to more weight loss in obese people than low intense physical exercise in normobaric sham hypoxia\n","text":"Training in mild to moderate hypoxia (14\u201317% O2 in breathing air) and extended resting in moderate hypoxia (9\u201313% O2) have been shown to have effects in animals and humans on lipid and glucose metabolism, appetite loss, and, in part, on body weight. The causality for these effects is not yet known in detail, and the available data in humans from high-altitude and low-pressure chamber studies are scarce. New technical developments by German companies in the production of artificial climates with normobaric hypoxic conditions in larger rooms at reasonable energy costs allow now to perform hypoxia weight loss studies in obese humans with stable experimental conditions and protocols with a sham hypoxia control. Thirty-two obese people were recruited for a mild intense training study in normobaric hypoxia (15 vol.% O2) and normoxia\/sham hypoxia (20.1 vol.% O2). Twenty of these [mean age 47.6 years, mean body mass index (BMI) 33.1, 16 m, 4 f) were willing to follow up on an 8-week, three times per week, 90-min low intense physical exercise in their individual fat burning mode, which has been determined by an exercise testing with spiro-ergometry upfront. The subjects were evenly randomized into a hypoxia and sham hypoxia group. The difference of the two groups in weight loss and changes in HBa1C values were analyzed before and after the training period. No nutritional diet was applied. Subjects in the hypoxia group in mean lost significantly more weight than in the sham hypoxia group (\u03941.14 kg vs \u03940.03 kg; p = 0.026). This resulted in a tendency to reduce the BMI more in the hypoxia group (p = 0.326). In the mean, there was no HbA1C exceeding normal values (mean 5.67 and 5.47%), and the HbA1C stayed basically unchanged after the 8-week training. Mild physical exercise three times per week for 90 min in normobaric hypoxia for 8 weeks led to significantly greater weight loss in obese persons than the exercise in sham hypoxia in this, to our knowledge, first sham hypoxia controlled study.\nIntroduction\nHigh altitude exposure which cause hypoxaemia has various serious effects on the human body. Those effects are studied by physiologists since the nineteenth century, and science has shed light on a lot of physiological and pathophysiological body reactions [1].\nWeight loss in high altitude under hypoxic climate conditions is one of those effects in which the causes remain still unclear, and it is not known if it is dose\/altitude-dependent and affects everybody or only certain individuals [3\u20135].\nThe detection of the hypoxia-induced factor-alpha 1 (HIF-\u03b11) and the chain reaction on mediators that this factor starts have prompted some speculations and investigations on various nutritional signals. One of the nutritional signals that seem to be triggered by HIF is the peptide hormone leptin [6\u20139]. This could lead to the direction that the human lipid metabolism is affected by hypoxia and that hypoxia causes signals to reduce food intake and also changes in fat mobilization and fat uptake to the body [10].\nBecause the societies in first and second world countries struggle with an increased body weight, pathological fat levels, and the increasing number of individuals with metabolic syndrome in their populations, the urge for various inexpensive treatment forms including a lifestyle change has risen. The idea is coming up whether training or stay at moderate altitude or simulated altitude could enhance weight loss and improvement of fat and sugar metabolism because of the known effects of hypoxia on weight loss. Several open trial field studies with low numbers of individuals have been carried out so far, but they do not have controls [11\u201313]. In the AMAS 2000 study, patients with metabolic syndrome have trained and stayed for 3\u00a0weeks at 1,700\u00a0m altitude or within a control group at sea level. In this randomized controlled study, significant improvements in fat metabolism and regarding weight loss have been found for the patients at altitude compared to patients at sea level [14].\nThe construction design of our facility with normobaric hypoxic climate rooms allows pretending hypoxic conditions without actually running the rooms in hypoxic climate (sham hypoxia) and to perform a single blind randomized controlled weight loss study in simulated altitude.\nOur aim was to perform a physical exercise weight loss study in obese subjects under moderate hypoxia (15\u00a0vol.% O2) and to compare the effects on body weight and fat metabolism to a control group training on the same level in sham hypoxic conditions.\nMaterials and methods\nSite Our facility for simulated altitude in the renovated Kurmittelhaus der Moderne in Bad Reichenhall in southeast Bavaria has three rooms (90, 37, and 25\u00a0m2) in which a hypoxic climate at normobaric conditions can be produced up to an simulated altitude of 6,000\u00a0m (approximately 7\u00a0vol.% O2; see Fig.\u00a01).\nFig.\u00a01The 90\u00a0m2 hypoxia training room with exercise devicesThe rooms are two well-equipped training facilities and one sleeping and rest room with a full bath. To reach a hypoxic climate, the air in the room is flushed with nitrogen, and the volume percent of oxygen is reduced to the determined level. To control the climate, oxygen and carbon dioxide are constantly controlled. The data are transmitted to a computer, and the computer controls via specialized software the electronic climate unit and the inflow of nitrogen and fresh air through the air condition (software by Low Oxygen Systems, Berlin-Buch, Germany; hardware by Atlas Copco, Walter Bau and Siemens and Fujitsu-Siemens, all Germany). The nitrogen is produced via a filtration system that eliminates oxygen from pressurized air. Because of the constant air flow, there is a low noise level from the air condition in the room. The level of simulated altitude is usually displaced in the rooms. To keep subjects uninformed about the oxygen level in the room while the air condition was running at all times, this display was covered during the study times and made invisible to the subjects.\nSubjects Thirty-two healthy subjects (22 men, 10 women) with a minimum age of 16\u00a0years and a minimum body mass index (BMI) of 27 were asked to participate in an 8-week trial with three times 90-min moderate physical exercise. Estimation of sample size was calculated to reach a statistical significance for a change in BMI of one point. Acquisition of subjects was done by local radio announcement and personal contact. Subjects did not receive any kind of reimbursement for participation. All subjects were asked to continue with their normal diet\/nutrition which they had before the study.Twenty subjects were finally willing to perform the whole 8-week training period and gave written consent. Subjects were randomly allocated to the hypoxia training and the sham hypoxia training group. Subjects were not told to which group they were allocated. Antropometric data of the subjects in the two groups are shown in Figs.\u00a02 and 3. Both groups were comparable in terms of age and sex.\nFig.\u00a02Mean age (SD), weight, and body mass index (BMI) of the subjects who finished the trial in the hypoxia groupFig.\u00a03Mean Age, weight, and BMI of the subjects who finished the trial in the sham hypoxia groupIn all 20 subjects, spiro-ergometry was performed to determine the max O2 consumption (ml kg\u22121 min\u22121) at exercise and the training level for the study set at the heart frequency value at 60% of the max O2 (ml kg\u22121 min\u22121). In all subjects, an echocardiography was performed for safety reasons to exclude subjects with heart insufficiency levels NYHA 2\u20134.\nStudy parameters Primary study parameter was bodyweight (kg) before and after the trial. Secondary study parameters were: BMI and the laboratory parameters (serum and EDTA blood sample); HbAC1, cholesterol, high- and low-density lipoproteins and triglycerides. Other exercise and blood parameters were not part of this trial but are available on request for reviewers from the laboratory and exercise test data sheets.Blood samples were drawn at the beginning and end of the 8-week trial; bodyweight and height were measured via a regular medical weight scale.\nTraining period Subjects performed exercise at a heart rate level which corresponded to 60% of the heart rate level at their individual max O2 (ml kg\u22121 min\u22121) on three different training (stepper, treadmill, and bicycle ergometer) devices for 90\u00a0min on 3\u00a0days per week, 8\u00a0weeks, either in normobaric hypoxia (15\u00a0vol.% O2, equivalent to 2,500-m altitude) or in normobaric normoxia (20\u00a0vol.% O2, equivalent to 450-m altitude). For safety reasons, SaO2 was controlled regularly during the training via pulseoximeter (Konica Minolta, Japan) in all subjects, and the subjects were asked for the main symptoms of acute altitude sickness (headache, nausea, and training untypical weakness in the legs). Pulse oximetry was blinded to subjects. Training was under constant control of medical personnel.\nStatistical analysis The differences between the two groups were analyzed via Student\u2019s t test, statistical significance determined as p\u2009<\u20090.05 (statistical software used: Excel, Microsoft, Redwood, WA, USA). Data are presented as descriptive data, and values are given as mean values.\nEthics and informed consent The study protocol was approved by the ethical committee of the Paris Lodron University Salzburg. All subjects gave written informed consent to participate in the study. Subjects under 18 were finally excluded; therefore, written consent by the parents was not necessary.\nResults\nAll 20 subjects who were included in the training sessions finished the study without problems. No adverse side effects were reported by the subjects, including no signs of acute mountain sickness in those who exercised in 15.2\u00a0vol.% oxygen level.\nThe average heart rate at 60% O2max for all subjects was 118\/min. Subjects kept this heart frequency level stable during exercise (\u00b13\/min). This meant that because of the slightly increased heart frequency at rest in hypoxia, the hypoxia group exercised at a lower wattage.\nBecause of the neutral smell of the air-conditioned air, subjects could not tell if they are in hypoxia or sham hypoxia. More than 50% of the subjects guessed wrongly as to which group they belong.\nWeight loss: The hypoxia group lost 1.14\u00a0kg in mean; the sham hypoxia group lost no bodyweight at all (p\u2009=\u20090.02, see Figs.\u00a04 and 5). This led to a non-statistical significant tendency in the hypoxia group to reduce the BMI more than the sham hypoxia group (p\u2009=\u20090.33). The number of subjects had been too low to reach statistical significance.\nFig.\u00a04Mean (SD) weight loss after 7\u00a0weeks in the hypoxia group vs the sham hypoxia groupFig.\u00a05Weight reduction or increase in the single subjects (matched pairs by age) of the hypoxia vs sham hypoxia group (y-axis = kg, x-axis = number of subject). Weight loss occurred in six subjects of the hypoxia group and in four of the sham hypoxia group, weight increase in two of the hypoxia group and four of the sham hypoxia group, weight stayed unchanged in two subjects of hypoxia (four and eight) and two of the sham hypoxia group (eight and nine)\nLipid levels and HbA1C: None of the differences in lipid levels reached statistical significance. However, the hypoxia group showed little non-statistical significant tendencies to lose more cholesterol (\u221210.7 mg\/ml vs 6.0 mg\/ml; p\u2009=\u20090.68), triglycerides, and low-density lipoproteins than the sham hypoxia group, but less high-density lipoproteins (Figs.\u00a06 and 7).\nFig.\u00a06Mean (SD) reduction and increase in triglycerides values of the hypoxia vs sham hypoxia groupFig.\u00a07Mean (SD) reduction in high-density lipoproteins (HDL) in the hypoxia vs sham hypoxia group\nThe healthy subjects of both groups were not diabetics. They had HbA1C levels on the edge but still in normal values (mean 5.67 and 5.47%), and those levels did not change after the training period of 8\u00a0weeks.\nDiscussion\nThe here presented study on fat metabolism and weight loss in mild to moderate hypoxia is, to our knowledge, the first one with a sham hypoxia control protocol.\nWe could show that obese subjects did lose significantly more weight during an 8-week training period with three times per week 90-min low intense exercise when this exercise took place in 15\u00a0vol.% O2 hypoxia than in normal air at 450-m altitude (20\u00a0vol.% O2). We could also show that there have been non-statistical differences in fat metabolism or at least in serum levels of lipoproteins and triglycerides between the hypoxia training and the sham hypoxia training group.\nThere are, due to the nature of the study and above that, several limitations, which have to be addressed. The number of subjects is not big enough to show a reduction in BMI, and power analysis shows that we would need about 32 subjects to have a significant BMI reduction. We wanted to get more subjects into the study, but the reduction of subjects to a limited number due to time restrictions and willingness is a common problem in weight loss protocols with obese people. The difference in weight loss in mean with 1.14\u00a0kg seems, for 8\u00a0weeks of training, not really a lot. However, several subjects did significantly lose more weight in the hypoxia group, and the mean bodyweight loss is not due to natural swings in weight change.\nThis also happened although the hypoxia group trained at a slightly lower wattage due to a slightly increased heart frequency in hypoxia. We cannot fully exclude that the normoxia group decreased body fat and increased muscle mass due to the slightly higher level of muscle load, but it seems unlikely with this very low intense training.\nBecause we did not put our subjects on a stringent diet, there might have been the chance that some individuals did eat more than normally and some less. Keeping the subjects on their routine food seemed to us the natural choice of being uninfluenced in the weight change by a special nutritional diet. The other point is that weight loss in hypoxia might be caused by reduced food intake, and that was a part of the study. To let the subjects reduce their food intake, you have to have them on a longer leash. This is especially true if leptin and hypothalamic signals come into play and might let subjects be less hungry, respectively, less hungry for fat. To achieve more accuracy, the only chance would be to exactly control the nutrition of all subjects already before the study and during the training period. To do this, subjects have to be under full supervision all the time. This is an almost impossible achievement in ambulatory care or with independent volunteers.\nOur findings are consistent with previous studies which have shown that low intense training in moderate hypoxia leads to weight loss [11\u201314]. The protocol with a sham hypoxia control group, which did not lose weight with the same amount of exercise, strengthens this hypothesis. A definite reason why this weight loss occurs is still not known. Several investigations could prove that the body reduces fat as a substrate for energy production due to the higher use of oxygen necessary in fat burning [2, 13], but this is still controversially discussed [14]. One of the reasons for a controversial discussion is that so far, nobody did observe subjects on a longer term [14]. Most studies have dealt with exercise in hypoxia for hours or a maximum of a few days, not for 2\u00a0months as we did.\nIn our investigation, the subjects in hypoxia had reduced their triglycerides, their cholesterol, and their low-density lipoproteins and kept their high-density lipoproteins stable, whereas the subjects in normoxia did not do so. However, none of the effects reached statistical significance. Due to the actual literature, this can be explained by several possible causes: Either the fat mobilization is reduced due to hypoxia over time and so the serum levels of lipids are lower at the point of measurement or the increased catecholamine levels during hypoxia lead to reduced lipolysis and a depot of intracellular fat [16, 17].\nMost likely, it seems that increased leptin levels lead to a reduced fat intake via food, and that is the reason why triglyceride levels decrease [6, 10]. The answer is still open, and more research is needed to clarify this. Other mediators besides catecholamines, like insulin like growth factor, might influence both fat and carbohydrate metabolism in hypoxia [15].\nIn conclusion, there is more and more evidence that even mild moderate hypoxia in comfortable simulated altitude, if combined with exercise, leads to weight loss.\nWeight loss regimes with exercise and diets or just exercise could be combined with normobaric hypoxia in the future. Future investigations should include whether the stay in moderate hypoxia alone without exercise increases the weight loss and if such a regimen would still be safe.","keyphrases":["physical exercise","normobaric hypoxia","weight loss"],"prmu":["P","P","P"]}
{"id":"Biotechnol_Lett-3-1-2151841","title":"Engineering eukaryotic protein factories\n","text":"The biopharmaceuticals market is currently outperforming the pharmaceuticals market and is now valued at US$ 48 billion with an average annual growth of 19%. Behind this success is a 100-fold increase in productivities of eukaryotic expression systems. However, the productivity per cell has remained unchanged for more than 10 years. The engineering of the ER-resident protein folding machinery is discussed together with an overview of signal transduction pathways activated by heterologous protein overexpression to increase cell specific productivities.\nIntroduction\nThe demand for therapeutic proteins, i.e. monoclonal antibodies, has been forecast to exceed production capacities (Werner 2004). Moderate increases in product titers and yields are expected to result in significant savings in capital investment and consumer costs (Werner 2004). Over the last decade product titers have increased from \u223c20\u00a0mg\u00a0l\u22121 to 2\u00a0g\u00a0l\u22121 (Werner 2004; Wurm 2004). These improvements are largely attributable to increases in viable cell densities. However, cell specific productivities remained nearly constant during this time (Wurm 2004).\nTo improve cell specific productivities the bottleneck for heterologous protein secretion has to be identified and resolved. In expression systems exploiting multiple gene copies or strong promoters the amount of secreted heterologous protein does not increase proportionally with gene copy number (Schr\u00f6der 2007), messenger ribonucleic acid (mRNA) (Schr\u00f6der et\u00a0al. 1999), or even the intracellular amount of the heterologous protein (Schr\u00f6der and Friedl 1997). In these cells, the protein accumulates in intracellular aggregates (Schr\u00f6der and Kaufman 2005), associates with the molecular chaperone heavy chain-binding protein (BiP)\/glucose-regulated protein of 78\u00a0kDa (GRP78)\/karyogamy 2 protein (Kar2p) (Schr\u00f6der and Kaufman 2005), and induces dilation of the endoplasmic reticulum (ER) (Dorner et\u00a0al. 1989; Gennaro et\u00a0al. 1991). Therefore, exit of the correctly folded polypeptide chain from the ER is the rate-limiting step for heterologous protein secretion. I will discuss strategies to improve cell specific productivities by engineering protein folding in the ER and signal transduction pathways activated by overexpression of heterologous proteins.\nProtein folding in the ER\nThe ER-resident protein folding machinery has four components: (1) Molecular chaperones assisting other proteins to fold, (2) enzymes such as protein disulfide isomerases (PDIs) and cis-trans peptidyl prolyl isomerases (PPIs), (3) a degradation machinery, and (4) signal transduction pathways that co-ordinate protein folding demand with capacity. At least three hierarchically organized chaperone systems operate in the ER, the heat shock protein of 70\u00a0kDa (HSP70) chaperones BiP and luminal HSP seventy 1 protein (Lhs1p)\/chaperone in the ER 1 protein\/Ssi1p\/GRP170\/oxygen-regulated protein of 150\u00a0kDa (ORP150), the HSP90 chaperone GRP94\/adenotin\/endoplasmin\/tumor rejection antigen glycoprotein of 96\u00a0kDa (gp96)\/ER protein of 99\u00a0kDa\/HSP108\/Ca2+-binding protein 4\/protein kinase of 80\u00a0kDa, and the lectin chaperones calnexin\/p88, calreticulin\/Ca2+-binding protein of 63\u00a0kDa (CAB-63)\/calregulin\/Ca2+-regulated protein of 55\u00a0kDa (CRP55)\/high affinity Ca2+-binding protein (HACBP), and calmegin\/calnexin-t (Fig.\u00a01). BiP works on completely unfolded polypeptide chains and translocates nascent polypeptide chains into the ER, whereas GRP94 and the lectin chaperones work on partially folded substrates.\nFig.\u00a01Hierarchy of chaperone systems in the ER. Abbreviations: Glc\u00a0=\u00a0d-glucose, Pi\u00a0=\u00a0inorganic phosphate ()\nBiP consists of an N-terminal ATPase domain and a C-terminal substrate binding domain. BiP cycles through rounds of adenosine triphosphate (ATP) hydrolysis and adenosine diphosphate (ADP) ATP exchange (Fig.\u00a01), which makes BiP a chaperone foldase (Winter and Jakob 2004). At least six DnaJ or HSP40 co-chaperones stimulate the ATPase activity of BiP and two growth after phage induction E (GrpE) co-chaperones the ADP ATP exchange reaction. In the ADP-bound form BiP has high affinity for unfolded substrates. Substrates bound to BiP are conformationally locked. ADP ATP exchange decreases the affinity of BiP for unfolded substrates. Substrate binding stimulates the ATPase activity of BiP. Thus, protein folding requires ATP (Dorner and Kaufman 1990). BiP and Lhs1p coordinate their activities. Lhs1p is a nucleotide exchange factor for BiP, and BiP stimulates the ATPase activity of Lhs1p (Steel et\u00a0al. 2004).\nCytosolic HSP90 chaperones are ATP-consuming chaperone foldases (Fig.\u00a01) regulated by several co-chaperones that affect its ATPase activity, load substrates onto HSP90 chaperones, assist in protein folding, and target substrates to degradation pathways (Strudwick and Schr\u00f6der 2007). GRP94 co-chaperones have not been identified, but other ER luminal chaperones found in complexes with GRP94 may be GRP94 co-chaperones. The KM value of GRP94 for ATP is \u223c100\u00a0\u03bcM and its intrinsic ATPase activity is barely detectable. These data indicate that GRP94 may be a chaperone holdase, a chaperone that binds to unfolded proteins, but does not cycle through ATP hydrolysis cycles. A drop of adenine nucleoside concentrations in stress situations may activate GRP94 (Rosser et\u00a0al. 2004).\nThe lectin chaperones share an N-terminal globular oligosaccharide-binding domain and a C-terminal extended hairpin loop, the P domain, which provides the chaperone function (Strudwick and Schr\u00f6der 2007). Calnexin and calmegin are transmembrane proteins. Calreticulin is a soluble protein. Most proteins entering the ER are glycosylated by addition of the oligosaccharide Glc3Man9GlcNAc2 (Glc\u00a0=\u00a0d-glucose, Man\u00a0=\u00a0d-mannose, GlcNAc\u00a0=\u00a0N-Acetyl-2-d-glucosamine). \u03b1-Glucosidases I and II rapidly remove the two terminal d-glucose residues. Calnexin and calreticulin bind to this monoglucosylated oligosaccharide. Removal of the third d-glucose moiety by \u03b1-glucosidase II releases the protein from calnexin\/calreticulin. Reglucosylation of unfolded proteins by uridine diphosphate (UDP)-glucose:glycoprotein glucosyl transferase (UGGT) triggers repeated interactions with calnexin\/calreticulin (Fig.\u00a01). This reglucosylation reaction consumes UDP-glucose. Therefore, the calnexin chaperones are chaperone foldases. The Saccharomyces cerevisiae genome encodes a calnexin homolog, but no homolog for UGGT, suggesting that the lectin chaperones may also function as holdases. Demannosylation of N-linked oligosaccharides trigger export of proteins to the Golgi complex or their targeting for retrotranslocation into the cytosol and proteasomal degradation (Lederkremer and Glickman 2005; Ruddock and Molinari 2006).\nAn expanding family of PDIs catalyzes the formation and isomerization of disulfide bonds. Disulfide bond formation requires the regeneration of oxidized PDI catalyzed by the flavin adenine dinucleotide (FAD)-dependent oxidases ER oxidation 1 protein (Ero1p)\/ERO1-L\u03b1, ERO1-L\u03b2 and essential for respiration and viability 2 protein (Erv2p) (Fig.\u00a02). The final electron acceptor is molecular oxygen. Oxidative protein folding accounts for \u223c25% of all reactive oxygen species (ROS) formed in a cell (Tu and Weissman 2004) and is increased by ER stress (Haynes et\u00a0al. 2004). Disulfide bond isomerization is independent of the Ero1p oxidases. Reduced glutathione provides reducing equivalents to remove incorrectly formed disulfide bonds. PDI has chaperone holdase activity that is independent of its protein disulfide isomerase activity (Wilkinson and Gilbert 2004) and redox-regulated foldase activity (Tsai et\u00a0al. 2001).\nFig.\u00a02Disulfide bond formation and isomerization reactions catalyzed by PDI\nSlowly folding proteins or folded incompetent proteins have to be removed from chaperone cycles to prevent them from poisoning the protein folding machinery. This is achieved by targeting these proteins for retrotranslocation into the cytosol and degradation by the proteasome in a process called ER associated protein degradation (ERAD) (Yoshida 2007) (Fig.\u00a03). Proteins destined for ERAD are recognized by BiP, PDI, and lectin sensors such as ER-degradation enhancing \u03b1-mannosidase-like proteins 1\u20133 (EDEM1\u20133)\/homologous to mannosidase I 1 protein (Htm1p)\/mannosidase-like 1 protein (Mnl1p) and osteosarcoma-9 (OS-9)\/yeast OS-9 homolog protein (Yos9p) (Gauss et\u00a0al. 2006a). The lectin sensor EDEM extracts unfolded proteins from the calnexin cycle. ERAD substrates are then retrotranslocated into the cytosol via the secretory 61 (SEC61) channel or a channel formed by derlin proteins, ubiquitinated by action of ubiquitin-conjugating enzymes (E2 enzymes) and ubiquitin ligases (E3 enzymes), deglycosylated, and degraded by the proteasome. Redundancy exists in the ubiquitination machinery involved in ERAD. In yeast and mammalian cells at least three distinct E2\u00b7E3 complexes are present in the ER membrane. The substrate specificities of these complexes, as well as their substrate selection rules are poorly understood. In yeast, the E3 ligase Hrd1p co-operates with Hrd3p and Yos9p to select its substrates (Gauss et\u00a0al. 2006b). Another selection criterion is the time an unfolded protein spends in foldase cycles. In the calnexin cycle, demannosylation of N-linked oligosaccharides generates glycostructures recognized by lectins such as EDEM1\u20133. Association of cytosolic HSP70s with the nucleotide exchange factor B cell leukemia\/lymphoma 2 (Bcl-2) associated athanogene protein 1 (BAG-1) (Alberti et\u00a0al. 2003) and of cytosolic HSP90s with the ubiquitin ligase carboxyl terminus of heat shock cognate protein of 70\u00a0kDa (HSC70) interacting protein (CHIP) (H\u00f6hfeld et\u00a0al. 2001) targets proteins for proteasomal degradation. Association of the DnaJ co-chaperone 58\u00a0kDa inhibitor of double-stranded RNA activated protein kinase (PKR, p58IPK) with HSP70 chaperones at the translocation pore targets proteins, whose translocation into the ER is stalled, for proteasomal degradation (Oyadomari et\u00a0al. 2006). If similar targeting mechanisms for proteins folding slowly in the BiP foldase cycle exist, is currently unknown.\nFig.\u00a03ER associated protein degradation. The exact nature of the retrotranslocation channel is unknown. The list of E2 and E3 enzymes catalyzing ubiquitination of ERAD substrates is not exhaustive. Abbreviations: NEF\u00a0=\u00a0HSP70 co-chaperone, Ub\u00a0=\u00a0ubiquitin\nEngineering of chaperone machineries\nEngineering of chaperone holdases should be straightforward. Holdases hold onto an unfolded protein and act in an equimolar ratio to the unfolded substrate, but are unable to support folding reactions. Holdases may provide a buffer capacity to prevent aggregation of unfolded proteins. Co-overexpression of chaperone foldases may be beneficial to overexpression of holdases (Smith et\u00a0al. 2004; Zhang et\u00a0al. 2006b). Overexpression data for calnexin\/calreticulin and PDI are most easily explained by assuming that this buffer capacity is increased. Calnexin or calreticulin overexpression generally improved heterologous protein secretion (Chung et\u00a0al. 2004; Conesa et\u00a0al. 2002; Kato et\u00a0al. 2005). Overexpression of one cycle constituent should not increase cycle capacity, if this constituent is not limiting, and if constituent levels are not co-regulated. The hypothesis that calnexin or calreticulin are limiting for the calnexin cycle seems unlikely, because of the high abundance of these proteins. Alternatively, overexpression of calnexin or calreticulin may inactivate lectins such as EDEM (Molinari et\u00a0al. 2003; Oda et\u00a0al. 2003), which target unfolded proteins for ERAD. Further, elevated calnexin or calreticulin levels not supported by concomitant increases in activities of other calnexin cycle components, may convert these surplus amounts of calnexin and calnexin into holdases or inactive chaperones.\nPDI overexpression increased secretion of some, but not all, heterologous proteins (Butz et\u00a0al. 2003; Damasceno et\u00a0al. 2006; Inan et\u00a0al. 2006; Kato et\u00a0al. 2005; Mohan et\u00a0al. 2007; Schr\u00f6der 2007; Zhang et\u00a0al. 2006b). PDI overexpression provides increased holdase activity in the ER. For example, overexpression of catalytically inactive PDI improved heterologous protein secretion (Hayano et\u00a0al. 1995). PDI overexpression also improved secretion of heterologous proteins containing no disulfide bonds (Powers and Robinson 2007; Smith et\u00a0al. 2004; Vad et\u00a0al. 2005). Substrate selectivity of PDI may explain why PDI overexpression failed to increase heterologous protein secretion in some studies (Butz et\u00a0al. 2003; Damasceno et\u00a0al. 2006; Mohan et\u00a0al. 2007; Schr\u00f6der 2007). If this is the case, co-expression of an alternative holdase, i.e. GRP94, may yield more consistent results for different heterologous proteins. Alternatively, increased rates of disulfide bond formation or isomerization in PDI overexpressing cells may explain increased heterologous protein secretion. As an enzyme, PDI should function at substochiometric ratios to its substrates. In disulfide bond formation, regeneration of oxidized PDI may become rate-limiting in PDI overexpressing cells. PDI is an abundant ER protein, making it unlikely that its overexpression can significantly augment its catalytic activities.\nEngineering of foldases seems more difficult, because of the complex regulation of these chaperones (see above). BiP overexpression increased heterologous protein secretion for about half of the heterologous proteins studied (Chung et\u00a0al. 2004; Damasceno et\u00a0al. 2006; Dorner et\u00a0al. 1992; Hsu and Betenbaugh 1997; Kim et\u00a0al. 2003; Schr\u00f6der 2007; Smith and Robinson 2002; Zhang et\u00a0al. 2006b), but can also have negative effects (Dorner et\u00a0al. 1992; Schr\u00f6der 2007). In cells overexpressing BiP one of its co-chaperones, Lhs1p, or ER luminal ATP may become limiting, converting BiP into a holdase or inactive chaperone. This may target proteins towards ERAD, stall translocation, convert Lhs1p into a holdase or inhibit Lhs1p. Increased BiP activity may stall the GRP94 and\/or calnexin\u2013calreticulin chaperone machineries, because of the hierarchy of ER luminal chaperone systems. This again may target heterologous proteins for ERAD. BiP overexpression attenuates the unfolded protein response (UPR, see below). Thus, BiP levels in WT and BiP overexpressing cells expressing a heterologous protein may not be dramatically different. Further, BiP overexpressing cells may experience an imbalance in their chaperone machineries, because the UPR co-ordinates expression of several chaperones. Moreover, if overexpression of BiP shifts the folding bottleneck to the GRP94 and calnexin chaperone systems, upregulation of these chaperone systems by the UPR may be blunted, because of suppression of UPR activation by elevated BiP levels.\nUnfolded protein response (UPR)\nER stress is the perturbation of the balance between the folding capacity and the folding demand imposed on the ER. To restore ER homeostasis, the UPR activates expression of chaperone, PDI, PPI, and ERAD genes, attenuates transcription of genes encoding secretory proteins and general translation, induces phospholipid synthesis, and induces an inflammatory response. Apoptosis is induced if these actions fail to restore ER homeostasis or in response to prolonged ER stress (Schr\u00f6der and Kaufman 2005).\nThe ER membrane of higher eukaryotes harbors at least three classes of transmembrane proteins activated by ER stress, basic leucine zipper (bZIP) transcription factors synthesized as type II transmembrane proteins, of which the best characterized are activating transcription factor 6\u03b1 (ATF6\u03b1) and ATF6\u03b2\/cyclic adenosine monophosphate response element binding protein (CREB)-related protein (CREB-RP)\/G13, the protein kinase eukaryotic translation initiation factor 2\u03b1 (eIF2\u03b1) kinase 3 (EIF2AK3)\/pancreatic eIF2\u03b1 kinase (PEK)\/PKR-like ER kinase (PERK), and the protein kinase-endoribonucleases ER to nucleus signaling 1\u03b1 (ERN1\u03b1)\/inositol-requiring 1\u03b1 (IRE1\u03b1) and IRE1\u03b2 (Fig.\u00a04). Several ATF6 paralogs, CREB3, CREB4, CREB-H, box B binding factor 2 (BBF2), and old astrocyte specifically induced substance (OASIS) have recently been described. In unstressed cells, these stress sensors are bound to BiP and kept in an inactive state. Upon ER stress, BiP is released coinciding with their activation. Whether BiP release is triggered by sequestration by unfolded proteins, conformational changes in the ER luminal domains, or reactivation of the BiP ATPase cycle is currently being debated (Kimata et\u00a0al. 2004; Shen et\u00a0al. 2005). ER stress also alters interactions of pro- and antiapoptotic Bcl-2 proteins at the ER membrane. These proteins regulate ER luminal Ca2+ homeostasis, ER morphology, ER stress signaling, and apoptosis induced in response to ER stress (Hetz 2007).\nFig.\u00a04Signal transduction pathway in the UPR. In the human UPR caspase 4 substitutes for caspase 12. In yeast and filamentous fungi, the IRE1-XBP-1 (Hac1p\/HACA) pathway is the only known UPR signal transduction pathway. [Reprinted in modified form with permission from Bentham Science Publishers from Schr\u00f6der M, Kaufman, RJ (2006) Divergent roles of IRE1\u03b1 and PERK in the unfolded protein response. Curr Mol Med 6:5\u201336]\nBiP release from ATF6 induces translocation of ATF6 to the Golgi complex where its bZIP transcription factor domain is proteolytically released from the Golgi membrane by site 1 and 2 proteases (S1P and S2P) (Fig.\u00a04). This cytosolic fragment translocates to the nucleus and induces expression of chaperone genes (Strudwick and Schr\u00f6der 2007). The functions of the ATF6 paralogs in the ER stress response is less well understood. OASIS contributes to activation of the BiP gene (Kondo et\u00a0al. 2005), and ATF6\u00b7CREB-H heterodimers activate inflammatory genes (Zhang et\u00a0al. 2006a).\nBiP release from PERK triggers oligomerization and activation of the protein kinase domain of PERK. PERK phosphorylates the bZIP transcription factor nuclear factor erythroid 2 (NF-E2) related factor 2 (NRF2) and eIF2\u03b1 (Strudwick and Schr\u00f6der 2007) (Fig.\u00a04). NRF2 phosphorylation induces its translocation to the nucleus, where a NRF2\u00b7ATF4 heterodimer activates transcription of antioxidant response genes (Harding et\u00a0al. 2003) to counteract ROS formation by repeated folding attempts of nascent polypeptide chains in ER stressed cells. eIF2\u03b1 phosphorylation attenuates general translation and decreases the influx of nascent unfolded polypeptide chains into the ER. It also clears short-lived proteins from the cell, i.e. D-type cyclins, resulting in cell cycle arrest in G1 phase, and of inhibitors of nuclear factor \u03baB (NF-\u03baB) (I\u03baB), activating the pro-inflammatory transcription factor NF-\u03baB. eIF2\u03b1 phosphorylation also induces preferential translation of capped mRNAs containing several short upstream open reading frames (uORFs) and cap-independent translation of mRNA via internal ribosomal entry sites (IRES). Currently, the only known mRNA whose translation is stimulated by ER stress is the mRNA encoding the bZIP transcription factor ATF4. ATF4 induces expression of CCAAT enhancer binding protein (C\/EBP) homologous protein (CHOP)\/CHOP-10\/growth arrest and DNA damage-inducible gene 153 (GADD153). CHOP represses transcription of anti-apoptotic BCL-2 (McCullough et\u00a0al. 2001) and induces expression of pro-apoptotic tribbles-related protein 3 (TRB3) (Ohoka et\u00a0al. 2005). Translational attenuation by PERK is transient and countered by several eIF2\u03b1 phosphatases. The regulatory subunit of protein phosphatase 1 (PP1), GADD34, is induced late in ER stress by ATF4, and targets PP1 to eIF2\u03b1.\nUpon BiP release, IRE1 autophosphorylates in trans and activates its endoribonuclease domain (Strudwick and Schr\u00f6der 2007). Activated IRE1 cleaves exon-intron junctions in the mRNAs encoding the bZIP transcription factors homologous to ATF\/CREB1 protein (Hac1p) in yeast, HACA in filamentous fungi, and X box-binding protein 1 (XBP-1)\/hepatocarcinogenesis-related transcription factor (HTF)\/tax-responsive element binding protein 5 (TREB5) in metazoans (Fig.\u00a04). In yeast, the HAC1 exons are ligated by transfer RNA ligase. Both transcription factors activate expression of genes encoding ER-resident molecular chaperones, protein foldases, and genes encoding proteins involved in ERAD. Hac1p and XBP-1 also stimulate phospholipid biosynthesis. Spliced Hac1p represses genes under control of the transcriptional regulator unscheduled meiosis 6 protein (Ume6p), including the early meiotic genes and a large array of genes involved in carbon and nitrogen metabolism. This function of spliced Hac1p may explain the slow growth phenotype of yeast cells overexpressing Ire1p or spliced Hac1p (Mori et\u00a0al. 2000). Attenuation of metabolism by Hac1p in yeast may substitute for attenuation of general translation by PERK. XBP-1 activation is delayed compared to ATF6 and PERK activation (Yoshida et\u00a0al. 2003), allowing for an early folding only phase in the UPR followed by a folding and degradation phase. In mammals, phosphorylated IRE1 sequesters tumor necrosis factor receptor associated factor 2 (TRAF2) from pro-caspase 12, resulting in clustering and activation of this caspase and initiation of apoptosis (Strudwick and Schr\u00f6der 2007) (Fig.\u00a04). TRAF2 recruits apoptosis signal-regulating kinase 1 (ASK1) and I\u03baB kinase to IRE1. ASK1 activates the mitogen-activated protein (MAP) kinases p38 and ju-nana (jun) N-terminal kinases (JNKs). p38 and JNKs phosphorylate and potentiate pro-apoptotic transcription factors, i.e. CHOP and c-Jun, and phosphorylate and inhibit anti-apoptotic Bcl-2 and Bcl-xL to induce apoptosis (Szegezdi et\u00a0al. 2006). Inhibition of JNK phosphatases by ROS formed in oxidative protein folding also contributes to JNK activation (Kamata et\u00a0al. 2005). Association of the pro-apoptotic Bcl-2 family proteins Bax and Bak with the cytosolic portion of IRE1 is required for efferent IRE1 signaling, i.e. XBP-1 mRNA splicing and JNK1 activation (Hetz et\u00a0al. 2006).\nEngineering of the UPR\nEngineering of the UPR holds promise to increase concentrations of several chaperones in a functionally meaningful ratio, thus preventing inadvertent conversion of chaperone foldases into holdases, or even inactivating chaperones because of lack of concomitant increases in co-chaperones and co-factors. The UPR is as much a survival response as it is a response to boost protein secretion. Clearly, some aspects of the physiological UPR are not desirable in an expression system, for example inhibition of general translation by PERK, stimulation of ERAD and apoptosis. Overexpression of spliced Hac1p or its filamentous fungi homolog HACA increased heterologous protein secretion in Saccharomyces cerevisiae and Aspergillus niger (Valkonen et\u00a0al. 2003a, b). In Chinese hamster ovary (CHO)-K1 cells XBP-1 overexpression improved secretion of several heterologous proteins (Ku et\u00a0al. 2007; Tigges and Fussenegger 2006). In yeast and filamentous fungi, secretion of several heterologous proteins was not improved or even decreased by overexpression of Hac1p (Gasser et\u00a0al. 2006; Rakestraw and Wittrup 2005; Valkonen et\u00a0al. 2003a). One drawback of some of these studies is that wild-type (WT) cells with a functional UPR are compared to WT cells overexpressing spliced Hac1p, which may explain why in some cases only marginal differences between the two cell types have been observed. The more consistent effect of spliced XBP-1 overexpression on protein secretion (Ku et\u00a0al. 2007; Tigges and Fussenegger 2006) may be caused by a preferential activation of UPR signaling pathways that stimulate cellular functions beneficial for heterologous protein secretion, i.e. increased chaperone gene expression. This increased ER-resident chaperone machinery attenuates all UPR signaling, and most importantly, those UPR signaling pathways likely to decrease protein production, for example general translation inhibition by PERK, stimulation of ERAD and of apoptosis (\u00d6zcan et\u00a0al. 2004).\nConclusions\nEngineering of chaperone systems by overexpressing a single component of the ER-resident protein folding machinery has overall yielded mixed results. Our basic understanding of protein folding in the ER is still incomplete. Addressing these open questions should underpin engineering approaches to improve the performance of chaperone systems. A more detailed understanding of chaperone function and regulation should inform future work to improve chaperone systems. Co-expression of different holdases or targeting of heterologous or cytosolic holdases to the ER may yield more consistent improvements for different heterologous proteins. The function of overexpressed chaperones may not be the same as at their normal physiological concentrations, because of the lack of a corresponding increase in co-chaperones and co-factors. To improve foldase function, concomitant elevation of BiP, its co-chaperones, and ATP levels should be attempted.\nEngineering of the UPR suggests that mimicking an UPR by expression of its activated signaling molecules does not consistently improve productivities. Dissection of UPR signaling activities may be necessary to improve heterologous protein secretion. To place engineering of the UPR on an informed basis, we need to understand the UPR in more detail. It is still not clear what the most upstream events in activation of the UPR are, how UPR signaling integrates into cellular signaling, and how the UPR decides between a prosurvival and an apoptotic response to ER stress. Cell engineering also needs to address the potential problem that increased oxidative protein folding may be inherently toxic to cells, because of elevated cellular ROS levels.","keyphrases":["protein folding","molecular chaperone","unfolded protein response","endoplasmic reticulum associated protein degradation","heterologous protein production"],"prmu":["P","P","P","R","R"]}
{"id":"Pharm_World_Sci-4-1-2253652","title":"Minimising treatment-associated risks in systemic cancer therapy\n","text":"Aim of the review To review the consequences of drug-related problems (DRP) in systemic cancer therapy and identify specific contributions of the pharmacist to minimise treatment-associated risks. Method Searches in PubMed, Embase and the Cochrane Library were conducted. Bibliographies of retrieved articles were examined for additional references. Only papers in English between 1980 and 2007 were included. Results In systemic cancer therapy there is an enormous potential for DRP due to the high toxicity and the complexity of most therapeutic regimens. The most frequently reported DRP can be classified into adverse effects, drug\u2013drug interactions, medication errors, and non-adherence. Pharmacists have enhanced efforts to assure quality and safety in systemic cancer therapy together with other health care providers. In consequence, oncology pharmacy has evolved as a novel specialist discipline. The endeavour to merge and co-ordinate individual activities and services of the pharmacist has led to pharmaceutical care concepts which aim at offering novel solutions to the various DRP. Conclusion Pharmaceutical care for cancer patients should be developed within research projects and integrated into disease management programs in order to ensure broad implementation.\nImpact of findings on practice\nThere is an enormous potential for drug-related problems in cancer therapy\nThe most frequently reported drug-related problems in systemic cancer therapy are adverse effects, drug\u2013drug interactions, medication errors, and non-adherence\nPharmacists can contribute substantially to risk minimisation in systemic cancer therapy by adding specific drug-related knowledge to the treatment team and offering patient-related services\nPharmaceutical care for cancer patients offers novel solutions to the various drug-related problems and should be further developed in research projects\nIntroduction\nIn systemic cancer therapy, drug regimens are administered following established protocols which have been carefully evaluated in clinical trials. The administration of supportive medication is not as standardised as with antineoplastic therapy. Major components of the supportive therapy are selected by the general practitioners or the patients themselves rather than by the oncologist. Furthermore, patients tend to see more than one physician involved in the cancer care process as well as alternative practitioners. Patients are also exposed to a tremendous variety of drugs which are available to the customer without prescription (over-the-counter, OTC).\nThe more complex drug therapy is, the higher the risk of experiencing drug-related problems (DRP) such as adverse effects, interactions, medication errors, and non-adherence. Drug-related problems in cancer chemotherapy can have severe consequences originating from the high toxicity and narrow therapeutic range of anticancer drugs. The tragic case of Betsy Lehman who died from an overdose of cyclophosphamide at the Dana Faber Cancer Center in Boston demonstrated this risk painfully [1]. It must be the goal of all health care providers to minimise treatment-associated risks as much as possible.\nOver the last few decades the pharmacy profession has experienced a change from traditional drug-oriented toward patient-oriented services. In oncology, pharmacists have established central services for compounding cytotoxic drugs and started to offer industry-independent drug information for physicians and patients as well as therapeutic drug monitoring for critical dose drugs, e.g. aminoglycosides in neutropenic patients. As the specialist knowledge of pharmacists in this field has increased continuously \u201concology pharmacy\u201d has evolved into a new pharmaceutical discipline with its own curriculum. In 1995 the International Society for Oncology Pharmacy Practitioners (ISOPP) was founded. The aim of the society is \u201cto determine the optimal medical treatment for cancer patients, thereby improving their quality of life\u201d. Currently, the concept of pharmaceutical care is being adapted to the needs of the cancer patient as a further step to optimising individual drug therapy [2].\nThe intention of this review is to summarise possible solutions for DRP in oncology focussing on the specific tasks of the pharmacist.\nAim of the review\nIt was the aim of this article to review and analyse the most frequent DRP and their consequences in systemic cancer therapy. Moreover, specific contributions of the pharmacist to minimise treatment-associated risks should be identified.\nMethod\nSearches in PubMed, Embase and the Cochrane Library were conducted. Database terms included DRP, adverse effects, drug\u2013drug interactions, medication errors, adherence, compliance, pharmaceutical care in connection with oncology or cancer. Bibliographies of retrieved articles were examined for additional references. Only papers in English between 1980 and 2007 were included.\nResults\nVarious DRP can occur in the treatment path of systemic cancer therapy (Table\u00a01). In a recently published study conducted on an oncology ward of a Swedish hospital 114 DRP in 58 patients were identified indicating their high incidence in cancer patients [3]. In this section, major problems related to adverse effects, drug\u2013drug interactions, medication errors, and non-adherence are reviewed separately.\nTable\u00a01Drug-related problems can originate from several steps of the treatment path\nAdverse effects\nAccording to the World Health Organization (WHO) an adverse drug reaction (ADR) is defined as a response to a drug which is noxious and unintended, and which occurs at doses normally used or tested in man for prophylaxis, diagnosis or therapy of disease. In cancer chemotherapy such ADRs are strongly connected to the treatment itself. Because of the fact that most cytotoxic agents cannot distinguish between normal and neoplastic cells, most ADRs seem to be unavoidable. They are often accepted not only by patients but also by health care providers.\nIn order to illustrate the most common ADRs in oncology patients in a cancer centre in Australia were observed and the incidence as well as the predictability, preventability, and severity of the occurred ADRs were assessed. Among the ten most common ADRs constipation ranked first but was connected directly to the use of opioids rather than to cytotoxic chemotherapy. Nausea and vomiting, fatigue, alopecia, drowsiness and myelosuppression ranked second to sixth. Of these ADRs 88% were predictable and about 50% even probably preventable, because of inadequate use of preventative measures [4].\nAs well as the incidence of ADRs in modern chemotherapy the patients\u2019 perceptions of the impact of these ADRs on well being and quality of life are increasingly taken into account. Patient perceptions have changed markedly over the last two decades. Whereas in 1983 physical symptoms such as nausea, vomiting and hair loss were most troubling from the patients\u2019 point of view, in 2002 psychosocial complaints ranked among the top ten symptoms, with the complaint \u201caffects my family or partner\u201d rated as the most severe ADR. Alopecia and fatigue ranked second and third place [5, 6].\nAs a consequence, the management of ADRs has become a focus of clinical research and new drugs such as aprepitant, a neurokinin-1-receptor antagonist for the prevention of nausea and vomiting, were developed [7]. Despite these new therapeutic options the minimisation of toxic effects of chemotherapy still seems to be a challenging task.\nThe first and most important step to a better management of ADRs is the formulation and implementation of evidence-based clinical practice guidelines for the different symptoms in a multidisciplinary approach. Several studies have shown a positive effect of guidelines for antiemetic prophylaxis and therapy on both clinical and economic outcomes [8\u201311]. The most important influencing factors seem to be the appropriate use of 5-HT3 receptor antagonists and the application of oral drug formulations. Nevertheless, altering the usage patterns of physicians to comply with evidence-based guidelines seems to be difficult. Methods such as the use of computerised decision support systems and educational outreach mechanisms appear to be most effective particularly when used in combination [12]. The pharmacist can support and promote the adherence to guidelines [13].\nA second step to a better management of ADRs is the implementation of standardised chemotherapy order forms that also contain supportive care medication. By using standardised order forms a more appropriate prescribing of antiemetics based on the level of emetogenicity of administered chemotherapy and thus a reduction in drug expenditure can be achieved [14]. Such order forms should be elaborated following a multidisciplinary approach including physicians, pharmacists and nurses.\nDrug\u2013drug interactions\nThe large number of prescribed drugs administered to cancer patients leads to a high potential for drug\u2013drug interactions. As indicated previously many cancer patients also use over-the-counter medication as well as alternative and complementary treatment options. Frequently, cancer patients suffer from concomitant chronic diseases that require the intake of other drugs, further increasing the risk of interactions. This problem is often underestimated in oncology as highlighted in several review articles [15\u201318]. Moreover, drug\u2013food, drug\u2013disease and drug\u2013diagnostics interactions have to be considered that are, however, beyond the scope of this article.\nDrug\u2013drug interactions must be avoided as they can lead to overdosing or underdosing of anticancer drugs with the consequence of toxicity or a loss of effectiveness, respectively. However, not all published interactions are also clinically relevant. A rational and safe drug therapy includes a check for potential drug\u2013drug interactions based on the individual medication and a decision whether and how identified drug\u2013drug interactions have to be considered or not.\nKnowledge of the mechanisms of interactions is crucial to assess the clinical relevance of an interaction. Drug\u2013drug interactions are usually classified as pharmaceutical, pharmacokinetic or pharmacodynamic interactions (Table\u00a02). The risk of interactions can differ substantially between individual drugs, even within the same class of drugs. This can be considered when selecting a particular drug for an individual patient.\nTable\u00a02Classification of drug\u2013drug interactionsPharmaceutical interactions Mostly physical or chemical incompatibilities, e.g. chemical reactions or precipitations due to drug admixtures.Pharmacokinetic interactions Occur at the level of drug absorption, distribution, excretion, and metabolism. Frequently, the cytochrome P450 metabolising enzyme system and drug transporters such as P-glycoprotein are involved in such interactions.Pharmacodynamic interactions Directly related to the desired or undesired drug effects, e.g. the antitumoral effect or drug-associated toxicity.\nFor some drugs with a high potential for interactions, therapeutic drug monitoring can be useful to detect and control interactions. The advantage of this approach is that the dosage of the drug of risk can be adapted to the measured individual plasma concentrations. Thus the treatment of both interacting drugs may be continued. Examples are anticonvulsant, antidepressant and antifungal drugs which are often administered to cancer patients as supportive medication [19].\nIn general, it is almost impossible for the physician to keep in mind all of the interaction mechanisms and potential consequences for the individual patient. Regular interaction checks by the pharmacist may solve this problem as he is the only health care professional who may have an overview of the drugs prescribed by various physicians and the medication taken by the patient on his own initiative. The avoidance of drug\u2013drug interactions should be regarded as a multidisciplinary task [20].\nThe basis for each interaction check is to take the medication history of each patient and to update it regularly. Nowadays software tools are available facilitating rapid interaction checks and providing information on the mechanism and clinical relevance of an interaction. Each hospital and community pharmacy should have access to at least one of these tools.\nMedication errors\nIn addition to the DRP described above, there are numerous risks for the occurrence of medication errors along the therapeutic path in oncology. A medication error is defined as any preventable event that may cause or lead to inappropriate medication use or patient harm, while the medication is in the control of the health care professional, patient, or consumer [21]. In 2000 the American Institute of Medicine published their report \u201cTo err is human: Building a Safer Health System\u201d which analysed flaws in the health system and offered suggestions for its improvement [22]. Based on the results of a study conducted in Utah and Colorado in 1992 an incidence of about 3% adverse events in hospitalised patients was found, of which the majority of the non-operative events were adverse drug events (ADE) [23]. The authors concluded that iatrogenic injury continues to be a significant public health problem. Due to the narrow therapeutic range of many anticancer drugs, the impact of an ADE is more serious (and at worst lethal) compared to other systemically administered drugs. Antineoplastic agents were found to be the second most common group of drugs which caused lethal medication errors [24]. As well as quality and safety issues, the reduction of medication errors has a financial effect.\nThe first source of medication errors is the prescription and ordering process: the wrong protocol may be chosen, the cumulative dose may be mixed up with the single dose, the route of administration may not be clearly indicated or misinterpretation of the physicians\u2019 handwriting may lead to errors. A study carried out in a cytotoxic preparation unit in a French hospital revealed in only a 6\u00a0month period more than 300 medication errors out of 1,262 prescriptions for 285 patients. Most errors (>70%) were found to be simple physicochemical incompatibilities, e.g. incompatibility of the drug with the used matrix. However, also over- and underdosage as well as wrong medications were identified [25]. The authors conclude that most of these errors could possibly be avoided by a computerised prescription network. Computer-assisted solutions enable physicians to order the medication electronically which is nowadays one way to reduce the incidence of those errors. Mekhjian et\u00a0al. evaluated the benefits of physician order entry systems and found that transcription errors could be reduced and speed in the ordering process could be improved. Even the duration of the stay in hospital could be reduced by the system [26]. Particularly in oncology, the computerised physician order entry (CPOE) has obvious advantages. A recent study has shown that CPOE in electronic medical records improves completeness of the medical record compared to paper charts independent of regimen complexity [27]. Prepared protocols facilitate correct ordering and double checking by the programme itself and by the pharmacist responsible. In addition, supportive medication, such as hydration or the antiemetic or antiallergic medication can be automatically proposed depending on the selected regimen. Moreover, the risk of misinterpretation of handwriting is eliminated. Recently, a CPOE system with integrated clinical decision support has been implemented in Ontario (Canada). This system, for example, alerts the clinician when maximum cumulative doses are reached, calculates body surface area, and estimates the creatinine clearance of the patient [28]. With all the advantages of these systems it has to be taken into account that new risks may arise from those new processes such as information errors due to the lack of clearness on the monitor [29].\nThe next step after ordering, the compounding of cytotoxic drugs also holds various risks, especially when performed on the ward. The implementation of central cytotoxic preparation units in pharmacy departments in the late 1980s has been one of the first measures to standardise the process in order to increase safety. Nevertheless, of 30,819 preparations surveyed in a study on incidence and risk factors of preparation errors 140 were found to be defective (0.45%) [30]. Less than half of those cases were classified as major errors including wrong dosage, wrong labelling or the use of incompatible diluents. Although the incidence is fairly low it must still be the aim to reduce this further by analysing the risk factors such as workload. Centralisation itself offers many more options for safety improvement. Standardised pharmaceutical validation leads to an increased interception of medication errors in antineoplastic treatment. This can be explained by the improved knowledge of the pharmacists involved in the validation process. In consequence of a standardised pharmaceutical validation process in the hematology\u2013oncology department of a 550-bed university hospital the number of detected medication errors that did not reach the patients could be increased by 41% (from 14.08\u00a0medication errors\/1,000\u00a0patient days to 19.83\u00a0medication errors\/1,000\u00a0patient days) in the second year after the introduction of pharmacists in the multidisciplinary oncology team [31]. Using their expertise, pharmacists can facilitate protocol development and adherence, dose verifications and education of other health care practitioners [32].\nFinally, storage and administration errors of cytotoxic drugs form a group of major medication errors with serious consequences for the patient. Therefore, it is mandatory that the assigned staff are well trained in all aspects of chemotherapy storage and administration. For many drugs, special storage conditions, e.g. refrigeration or protection from light, have to be considered. Detailed knowledge of the administered drugs is also very important in order to educate the patient and to react appropriately when incidents such as extravasations occur. In case of extravasation the necessary measures have to be initiated. Different drugs require different measures which the oncologically trained staff needs to know. An extravasation kit including essential items for first aid must be made available on site [33]. Incorrect or delayed treatment can cause serious damage to the patient, resulting in even the loss of a limb.\nVarious surveys have been undertaken in order to find solutions for the prevention of medication errors. The non-punitive reporting of errors is a necessary requirement to be able to detect, analyse and consequently minimise the incidence and severity of medication errors in oncology. Safety measures with error reporting and analysis have been developed and implemented in order to improve the entire system [34]. The most effective improvements of treatment performance have been achieved by multidisciplinary system approaches integrating physicians, pharmacists and nurses [2, 32, 34\u201336].\nNon-adherence\nAdherence (or compliance) is generally defined as the extent to which a person\u2019s behaviour, taking medication, following a diet, and\/or executing lifestyle changes, corresponds with agreed recommendations from a health care provider [37]. Non-adherence strongly compromises the success of a patient\u2019s therapy, results in additional, potentially unnecessary, diagnostic and therapeutic procedures and thus generates further costs and possibly health problems as a consequence of the treatments themselves. It is important to note that the traditional concept that patients are solely responsible for taking their medication (and therefore non-adherence is a patient-driven problem) is misleading. The WHO understands adherence as a phenomenon consisting of five dimensions (Table\u00a03). The magnitude of this problem is further emphasised by the fact that adherence rates for many long-term drug therapies range from only 40 to 78% [38, 39].\nTable\u00a03The five dimensions of adherence and examples of associated factors (adapted from [34]) Social\/economic factorsHealth care team\/system-related factorsCondition-related factorsTherapy-related factorsPatient-related factorsEconomic statusPatient-provider relationshipSeverity of symptomsComplexity of regimenAnxiety about side effectsCultural beliefsEducation of providersLevel of disabilityTreatment durationPatients\u2019 motivationIlliteracyCapacity of systemRate of progressionChanges in treatmentPatients\u2019 expectationsAgeDuration of consultationsCo-morbiditiesSide effectsForgetfulnessDistance from treatment centerMedication distribution systemAvailability of effective treatmentsPrevious treatment failuresPatients\u2019 knowledge about illness\nIt is a difficult task to assess patient adherence reliably. Patient self-reports, rates of prescription refills, patient diaries or pill counts as the sole basis for the measurement of adherence have been shown to be inadequate [38\u201340]. It has been demonstrated that these methods overestimate the degree to which patients adhere to their tamoxifen regimen [40]. The use of microelectronic adherence monitoring (Medication Event Monitoring System, MEMSTM, Fig.\u00a01) provides a valuable estimate of the timing of events and insights into patients\u2019 behaviour in taking medication. However, to prove actual intake of the medication, additional plasma concentration monitoring would be necessary. To obtain an optimal measurement of patient adherence, a combination of several methods is suggested [38].\nFig.\u00a01Medication event monitoring system, MEMSTM: The pill bottle cap contains a micro-electronic circuit that registers when the bottle is opened. This data may then be transferred to a computer via a reading device\nStudies conducted in various disease areas have shown that a wide variety of methods to improve adherence is available. A recently published review classifies these methods into four general categories: patient education, improved dosage, increased hours when clinics are open, and improved communication between health care providers and patients [38]. All four categories require a collaborative approach including all members of the multidisciplinary health care team. Due to their position in the chain of health care providers, this requires the contribution of pharmacists, not only in the hospital setting but also in community pharmacies [41]. It has been shown that no single intervention strategy appears consistently more effective than another. Successful methods are complex and comprehensive as well as labour-intensive [38, 42]. The Cochrane Review on Interventions to Enhance Medication Adherence concludes that there is no evidence that low adherence can be \u201ccured\u201d and hence efforts to improve adherence must be maintained for as long as the treatment is needed [43].\nCancer patients seem to benefit especially from interventions towards an optimised adherence, resulting in improved outcomes [42]. Research investigating adherence in oncology settings has been mostly focussed on palliative care and supportive medication because chemotherapy has mainly been administered intravenously in hospitals or clinics. However, with the increasing availability and importance of oral anticancer drugs such as capecitabine, etoposide, vinorelbine, erlotinib and sorafenib, patients increasingly take over the responsibility for the correct administration of their prescribed therapy. Although cancer patients might be more adherent than other patient groups due to a high level of motivation, the slightest non-adherence can endanger the therapeutic goals [39].\nOnly few studies have been published investigating the level of adherence of cancer patients. In a cohort of outpatients receiving chemotherapy for haematologic malignancies adherence to the allopurinol and prednisolone prescription was measured based on plasma concentrations. It was shown that control patients without intervention were adherent only 17% of the time with allopurinol and 27% of the time with prednisone. Three specially developed intervention packages including education and home visits resulted in increased adherence rates of 44\u201348% and 33\u201338% of the time for allopurinol and prednisone, respectively [44]. Another study assessed adherence in patients with breast cancer receiving oral cyclophosphamide and found that 43% of the patient population met criteria for non-adherence according to both behavioural and dosage definitions [45]. In a recently published study in a cohort of 2816 women adherence to tamoxifen treatment was investigated using prescription refill data: 22.1% of the patients exhibited a discontinuation of treatment (non-persistence) within 1\u00a0year [46]. An open controlled trial is currently undertaken at the University of Bonn investigating the impact of intensified pharmaceutical care provision on the adherence of patients receiving oral capecitabine using the MEMS\u2122-technology. Pharmacists\u2019 interventions include the extensive provision of drug information to the patients and health care professionals, routine checks for drug interactions, preparation of written drug intake plans for patients, regular pharmacist consultations and structured documentation of the pharmaceutical care process [47].\nDiscussion\nThe recognition of the described risks of the individual patient associated with complex drug therapies has led to the development of a conceptual framework for an advanced pharmacy practice philosophy. The concept of pharmaceutical care was introduced as a further development of the pharmaceutical profession gaining acceptance in Europe and worldwide [48, 49]. The American Society of Health System Pharmacists set up guidelines for standardised pharmaceutical care to ensure that pharmacists practicing pharmaceutical care work to the same standard [50].\nPatients with complex drug regimens and\/or chronic diseases and those who frequently need to be hospitalised might benefit from pharmaceutical care in particular. These criteria apply to many cancer patients. Within the pharmaceutical care process the application of agreed therapeutic algorithms can be assured on the individual basis. The adherence of the patient can be improved by patient education before and during the treatment cycles combined with patient counselling regarding drug therapy, adverse effects and complementary treatment options.\nThe London oncology pharmacy group introduced guidelines for pharmaceutical care of cancer patients which not only include the actual \u2018pharmaceutical care\u2019 as such, but standardise the clinical pharmacy activities, dispensing, updating therapeutic policies, cytotoxic reconstitution, drug information, clinical trials, and the oncology training of pharmacists [51]. A group of British experts has drawn up a policy framework for commissioning cancer services. They suggest the establishment of structures which support the seamless care of cancer patients in the community setting in a network of all parties in order to make use of the respective specialist knowledge [52]. Accordingly, information flow at discharge from hospital to the community should be optimised utilising pharmaceutical care plans to ensure the efficient distribution of medication to the patient is not interrupted.\nAlthough there are some reports on the implementation of pharmaceutical care for cancer patients there is still little scientific evidence on the feasibility of pharmaceutical care and its actual benefit to the patient. In Canada, projects have been carried out which suggest to implement suitable outcome parameters to evaluate the impact of pharmaceutical services in oncology [53]. These have stimulated a wide discussion in Canadian health care politics regarding the necessity of the services offered. In Scotland, research is concentrating on the documentation and thereby standardisation of the provision of pharmaceutical care to cancer patients [54]. In Germany, various research projects are being carried out which investigate the feasibility and benefit of pharmaceutical care for patients with different indications. Patients with breast and colorectal cancer as well as patients receiving oral chemotherapy are the focus of projects at the University of Bonn. Another project is being carried out in Hamburg developing the provision of pharmaceutical care to lung cancer patients [2].\nIn terms of cancer many disciplines contribute to the care process. Thus, cross-profession and cross-sector cooperation is crucial in order to improve information flow. Over the last few years disease management programs (DMPs) have increasingly been introduced to cover the whole care process which begins with the early diagnosis of the disease. They aim at providing the optimal medical care by the implementation of evidence-based guidelines. Pharmaceutical care concepts seem to have the potential to support the goals of DMPs and could be easily integrated in programmes for cancer patients. However, it is necessary to document the impact of pharmaceutical care on patient outcomes in order to comply with the demand for transparency.\nConclusions\nSystemic cancer therapy is particularly complex and hence associated with multiple risks for the patient as described above. Many of these risks are preventable by specific measures which can be taken by a particular health care provider or the patient. The pharmacist with his central position relating to drug dispensing and utilisation can contribute substantially by adding specific drug-related knowledge and offering patient-related services.\nPharmaceutical care is designed as a framework that integrates individual contributions of the pharmacist into the entire therapeutic path. Model projects are urgently needed to assess the clinical, humanistic and economic outcome of patient-oriented pharmaceutical services. The integration of pharmaceutical care into disease management programmes might facilitate efficient collaboration between all health care professionals and hence improve effectiveness and safety of systemic cancer therapy.","keyphrases":["cancer","drug-related problems","medication errors","oncology","pharmaceutical care","chemotherapy"],"prmu":["P","P","P","P","P","P"]}
{"id":"Arch_Dermatol_Res-3-1-1910890","title":"German evidence-based guidelines for the treatment of Psoriasis vulgaris (short version)\n","text":"Psoriasis vulgaris is a common and chronic inflammatory skin disease which has the potential to significantly reduce the quality of life in severely affected patients. The incidence of psoriasis in Western industrialized countries ranges from 1.5 to 2%. Despite the large variety of treatment options available, patient surveys have revealed insufficient satisfaction with the efficacy of available treatments and a high rate of medication non-compliance. To optimize the treatment of psoriasis in Germany, the Deutsche Dermatologische Gesellschaft and the Berufsverband Deutscher Dermatologen (BVDD) have initiated a project to develop evidence-based guidelines for the management of psoriasis. The guidelines focus on induction therapy in cases of mild, moderate, and severe plaque-type psoriasis in adults. The short version of the guidelines reported here consist of a series of therapeutic recommendations that are based on a systematic literature search and subsequent discussion with experts in the field; they have been approved by a team of dermatology experts. In addition to the therapeutic recommendations provided in this short version, the full version of the guidelines includes information on contraindications, adverse events, drug interactions, practicality, and costs as well as detailed information on how best to apply the treatments described (for full version, please see Nast et al., JDDG, Suppl 2:S1\u2013S126, 2006; or http:\/\/www.psoriasis-leitlinie.de).\nIntroduction\nThe Deutsche Dermatologische Gesellschaft (DDG) and the Berufsverband Deutscher Dermatologen (BVDD) have initiated a project to develop evidence-based guidelines for the treatment of plaque psoriasis. The full version of the Guidelines has been published in the Journal der Deutschen Dermatologischen Gesellschaft (JDDG 2006 Suppl 2) and is available at http:\/\/www.psoriasis-leitlinie.de [149]. This article summarizes the key points of the Guidelines.\nBackground\nPsoriasis vulgaris is a common and chronic inflammatory skin disease which has the potential to significantly reduce the quality of life in severely affected patients. The incidence of psoriasis in Western industrialized countries is 1.5\u20132% [2]. Studies on the impairment of life quality have shown that, depending on the severity of the disease, a significant burden may exist in the form of a disability or psychosocial stigmatization [3]. Patient surveys have shown that the mental and physical impairment associated with psoriasis is comparable to that of other significant chronic conditions, such as type 2 diabetes or chronic respiratory diseases [4].\nPatient surveys have shown that only 25% of psoriasis patients are highly satisfied with the outcome of their treatment, another 50% indicate moderate satisfaction, and approximately 20% report low treatment satisfaction [5]. In addition, there is a high non-compliance rate in the intake of medication of up to 40% [1].\nExperience has shown that treatment selection for patients with psoriasis vulgaris is more commonly based on traditional concepts than on evidence-based data on the efficacy of various therapeutic options. In addition, it appears that systemic therapies are occasionally not applied in situations where they are needed due to the increased effort involved in monitoring the patients for unwanted side effects and possible interactions with other drugs.\nGoal of the Guidelines\nThe overall objective of the Guidelines is to provide dermatologists in clinics and private practice with an accepted and evidence-based decision-making tool for the selection and implementation of a suitable and efficacious treatment for patients with psoriasis vulgaris. The focus of the Guidelines is on the induction therapy of mild, moderate, and severe plaque-type psoriasis in adults.Physicians\u2019 personal experiences and traditional therapeutic concepts should be supplemented and, if necessary, replaced by an evidence-based assessment of the efficacy of individual therapies in psoriasis vulgaris.The guidelines provide in-depth explanations of the available systemic and topical treatments for psoriasis, including the different photo- and photochemical therapies, and provide detailed descriptions of the administration and safety aspects.By providing background information on the profile of the available drugs, including efficacy, safety, and aspects of practicality and costs, the Guidelines should facilitate the process of selecting an appropriate treatment for each individual patient. This should help increase compliance and optimize the benefit\/risk ratio of psoriasis therapies.\nMethods\nA detailed description of the methodology employed in developing the Guidelines can be found in the Method Report on the Guidelines (http:\/\/www.psoriasis-leitlinie.de).\nBasis of data\nA systematic search of the literature was carried out in May 2005 with the objective of assessing the effectiveness of individual therapeutic options. This search yielded 6,224 publications of which 142 studies fulfilled the inclusion criteria for the Guidelines (see Box\u00a01) and were therefore included in the assessment of the effectiveness of the relevant treatment. Various other aspects were evaluated on the basis of information presented in available literature, without a systematic analysis, and the years of personal experience of the experts.\nEvidence assessment\nThe efficacy and effectiveness of each intervention was evaluated using evidence-based criteria.\nThe methodological quality of each individual study was assessed using the following grades of evidence (GE):A1Meta-analysis that includes at least one randomized study with grade A2 evidence. In addition, the results of the different studies included in the meta-analysis must be consistent with one another.A2A high-quality (e.g. sample-size calculation, flow chart, intention-to-treat (ITT) analysis, sufficient size) randomized, double-blind comparative clinical study.BRandomized clinical study of lesser quality or other comparative study (non-randomized, cohort, or case-control study).CNon-comparative study.DExpert opinion.Individual interventions (i.e., as monotherapy) were rated according to the following levels of evidence (LE):Intervention is supported by studies with grade A1 evidence or studies with grade A2 evidence whose results are predominantly consistent with one another.Intervention is supported by studies with grade A2 evidence or studies with grade B evidence whose results are predominantly consistent with one another.Intervention is supported by studies with grade B evidence or studies with grade C evidence whose results are predominantly consistent with one another.Little or no systematic empirical evidence\nTherapeutic recommendation\nA distinct rating of the therapeutic options or a strict clinical algorithm cannot be defined for the treatment of psoriasis vulgaris. The criteria for selecting a particular therapy are complex. The decision for a specific treatment should be based on the profile of the available drugs and the characteristics of a given patient. The decision for or against a therapy remains a case-by-case decision. These Guidelines provide a reasonable form of assistance in deciding on a suitable therapy and are an instrument for optimizing the required therapeutic process.\nThe recommendations formulated in the text are supported graphically in selected key recommendations by the following indications of the strength of the therapeutic recommendation:\u2191\u2191Measure is highly recommended\u2191Measure is recommended\u2192Neutral\u2193Measure is not recommended\u2193\u2193Measure is highly inadvisableThe strength of the recommendation reflects both a treatment\u2019s efficacy and the level of evidence supporting it as well as aspects of safety, practicality, and the cost\/benefit ratio. A consensus on the strength of the recommendation was reached during the Consensus Conference.\nResults\nTherapeutic options are named and discussed in alphabetical order.\nTherapeutic strategies\nFig.\u00a01\nOverview of the therapeutic options evaluated for chronic plaque psoriasis (the therapeutic options are listed alphabetically and do not represent a ranking)\nEvaluation of topical and systemic therapies in tabular form\nThese tables are intended to provide a rough orientation for evaluating the therapeutic options. Cumulative calculations of the individual aspects for the overall evaluation of the therapeutic options are not possible and cannot be drawn upon for the final analysis of a therapeutic option. The product assessment for each individual patient may deviate greatly. A direct comparison between systemic and topical therapies is not possible because of the different severity of the psoriasis lesions of patients treated with topical or systemic treatments. The evaluations reported here were made on the basis of data extracted from the literature and expert opinions.\nFor further details refer to the Methods Report at www.psoriasis-leitlinie.de\nTopical monotherapy\nPhototherapy and systemic monotherapy\n(a) Efficacy The evaluation of the efficacy column reflects the percentage of patients who achieved a reduction in the baseline Psoriasis Area and Severity Index (PASI) of \u226575%. \nScaleSystemic therapyTopical therapy++++Approx. 90%Approx. 60%+++Approx. 70%Approx. 45%++Approx. 50%Approx. 30%+Approx. 30%Approx. 15%+\/\u2212Approx. 10%Approx. 5%\u2013Not definedNot defined\nThe evidence level applies only to the estimate of efficacy.\n(b) Safety\/tolerance in induction therapy or maintenance therapy This refers to the risk of occurrence of severe adverse drug reactions or the probability of adverse drug reactions that would result in the discontinuation of therapy.\n(c) Practicality (Patient) This evaluation analyzes the effort involved in handling and administrating the treatment regimen by the patient.\n(d) Practicality (Physician) This aspect considers the amount of work (documentation, explanation, monitoring), personnel and equipment needs, time for physician\/patient interaction, remuneration of therapeutic measures, invoicing difficulties\/risk of recourse claims from the health insurance companies.\n(e) Cost\/benefit Consideration of the costs of an induction therapy or a maintenance therapy.\nThe evaluations of safety\/tolerance in induction therapy or maintenance therapy as well as practicality for the physician or patient and the cost\/benefit were performed using a scale ranging from poor (\u2013) to good (++++). The gradation between these two extremes was made based on expert opinion and unsystematic literature search. A level of evidence was not given for these evaluations since no systematic literature review was performed.\nEvaluation of topical therapies\nCalcineurin inhibitors\nTable\u00a01\nTabular summary\nCalcineurin inhibitors\nFirst approved in Germany\n\u00a0Pimecrolimus (Elidel\u00ae)\n2002 (Atopic dermatitis, not approved for psoriasis vulgaris)\n\u00a0Tacrolimus (Protopic\u00ae)\n2002 (Atopic dermatitis, not approved for psoriasis vulgaris)\nRecommended initial dosage\nPimecrolimus cream: 1\u00d7 to 2\u00d7 dailyTacrolimus: 1\u00d7 to 2\u00d7 daily (application on the face: Begin with 0.03% salve; increase later dosage to 0.1% salve)\nRecommended maintenance dosage\nIndividual therapeutic adjustment\nExpected beginning of clinical effect\nAfter approximately 2\u00a0weeks\nResponse rate\nNo data available\nImportant contraindications\nPregnancy and nursing (due to lack of experience) skin infections, immune suppression\nImportant adverse drug reactions (ADRs)\nBurning sensation on skin, skin infections\nImportant drug interactions\nNone known\nOther\nCave: Do not combine with phototherapy! Photoprotection!\nCorticosteroids\nTable\u00a02\nTabular summary\nCorticosteroids\nFirst approved in Germany\n1956 (Psoriasis vulgaris)\nRecommended control parameters\nNone\nRecommended initial dosage\nOne to two times daily\nRecommended maintenance dosage\nGradual reduction following onset of effect\nExpected beginning of clinical effect\nAfter 1\u20132\u00a0weeks\nResponse rate\nBetamethasone dipropionate, two times daily: marked improvement or clearance of the skin lesions in 46\u201356% patients after 4\u00a0weeks (LE\u00a01)\nImportant contraindications\nSkin infections, rosacea, perioral dermatitis\nImportant ADRs\nSkin infections, perioral dermatitis, skin atrophy, hypertrichosis, striae\nImportant drug interactions\nNone\nOther\n\u2013\nCoal tar\nTable\u00a03\nTabular summary\nCoal tar\nFirst approved in Germany\nListed active ingredient since 2000 (DAC on page 170), historical application, various tar-containing externals are licensed as drugs, application of tar as anti-psoriatic following publication by Goeckermann in 1925\nRecommended control parameters\nAfter long-term application\/application on large areas: if needed clinical controls for potential development of skin carcinoma\nRecommended initial dosage\n5\u201320% salve preparations or gels for local therapy, 1\u00d7 daily\nRecommended maintenance dosage\nNo long-term application (max. 4\u00a0weeks, DAC 2000)\nExpected beginning of clinical effect\nAfter 4\u20138\u00a0weeks, efficacy improves in combination with UV application\nResponse rate\nThere is insufficient data available on the response rate as a monotherapy (LE 4)\nImportant contraindications\nPregnancy and nursing\nImportant ADRs\nColor, odor, carcinogenic risk, phototoxicity\u2014which is part of the desired effect\nImportant drug interactions\nNot known with topical use\nOther\nDAC 2000 (on page 170), Hazardous Goods Directive Appendix 4 No. 13\nDithranol\nTable\u00a04\nTabular summary\nDithranol\nFirst approved in Germany\nPsoralon\n1983 (Psoriasis vulgaris)\nPsoradexan\n1994 (Psoriasis vulgaris)\nMicanol\n1997 (Psoriasis vulgaris)\nRecommended control parameters\nIntensity of irritation\nRecommended initial dosage\nBegin with 0.5% preparation for long-term therapy or 1% for short-contact therapy, then increase if tolerated\nRecommended maintenance dosage\nNot recommended for maintenance therapy \nExpected beginning of clinical effect\nAfter 2\u20133\u00a0weeks\nResponse rate \nMarked improvement or clearance of skin lesions in 30\u201350% of the patients (LE 2)\nImportant contraindications\nAcute, erythrodermic forms of psoriasis vulgaris; pustular psoriasis\nImportant ADRs\nBurning and reddening of the skin in >10%\nImportant drug interactions\n\u2013\nOther\n\u2013\nTazarotene\nTable\u00a05\nTabular summary\nTazarotene\nFirst approved in Germany\n1997 (psoriasis vulgaris)\nRecommended control parameters\nCheck development of skin irritations\nRecommended initial dosage\nBegin with one treatment daily of tazarotene gel 0.05% in the evening for approximately 1\u20132\u00a0weeks\nRecommended maintenance dosage\nIf necessary continue for 1\u20132\u00a0weeks with tazarotene gel 0.1%\nExpected beginning of clinical effect\nAfter 1\u20132\u00a0weeks\nResponse rate\nAfter 12\u00a0weeks therapy with 0.1% tazarotene gel improved findings of at least 50% in approximately 50% of the patients (LE 2)\nImportant contraindications\nPregnancy, nursing\nImportant ADRs\nPruritus, burning sensation of skin, erythema, irritation\nImportant drug interactions\nAvoid concomitant use of preparations with irritating and drying properties \nOther\n\u2013\nVitamin D3 and analogues\nTable\u00a06\nTabular summary\nVitamin D3 and analogues\nFirst approved in Germany\n\u00a0Calcipotriol\n1992 (Psoriasis vulgaris)\n\u00a0Tacalcitol\n1994 (Psoriasis vulgaris)\n\u00a0Calcitriol\n1999 (Psoriasis vulgaris)\n\u00a0Calcipotriol\/Betamethasone \n2002 (Psoriasis vulgaris)\nRecommended control parameters\nMonitor for skin irritations \nRecommended initial dosage\nCalcipotriol: 1\u00d7 to 2\u00d7 daily to affected locations, up to a maximum of 30% of the body surface\nTacalcitol: 1\u00d7 daily to affected locations, up to a maximum of 20% of the body surface\nCalcitriol: 2\u00d7 daily to affected locations, up to a maximum of 35% of the body surface\nRecommended maintenance dosage\nCalcipotriol: 1\u00d7 to 2\u00d7 daily, up to 100\u00a0g\/week for up to 1\u00a0year\nTacalcitol: 1\u00d7 daily for 8\u00a0weeks, for up to 18\u00a0months, on a maximum of 15% of the body surface with up to 3.5\u00a0g daily\nCalcitriol: insufficient experience with the application for more than 6\u00a0weeks \nExpected beginning of clinical effect\nAfter 1\u20132\u00a0weeks\nResponse rate \nBetween 30 and 50% of the patients demonstrated a marked improvement or clearance of the lesions after 4\u20136\u00a0weeks (LE 1)\nImportant contraindications\nDiseases with abnormal calcium metabolism, severe liver and renal diseases\nImportant ADRs\nSkin irritation (reddening, itching, burning)\nImportant drug interactions\nDrugs which elevate the calcium levels, (e.g. thiazide diuretics), no concomitant application of topical salicylic acid preparations (inactivation)\nOther\nExposure to UV light results in inactivation of the vitamin D3-analogues\nPhototherapy\nTable\u00a07\nTabular summary\nPhototherapy\nFirst approved in Germany\nClinical experience, depending on the modality for >50\u00a0years\nRecommended control parameters\nRegular skin inspection (UV erythema)\nRecommended initial dosage\nIndividual dose depends on skin type; options:\n\u2022 UVB: 70% of minimum erythema dose (MED)\n\u2022 Oral PUVA (photochemotherapy): 75% of the minimum phototoxic dose (MPD)\n\u2022 Bath\/cream PUVA: 20\u201330% of MPD\nRecommended maintenance dosage\nIncrease according to degree of UV erythema\nExpected beginning of clinical effect\nAfter 1\u20132\u00a0weeks \nResponse rate\nIn >75% of the patients PASI, 75 after 4\u20136\u00a0weeks (LE 2)\nImportant contraindications\nPhoto-dermatoses\/photosensitive diseases, skin malignancies, immunosuppressionOnly PUVA: pregnancy and nursing\nImportant ADRs\nErythema, itching, blistering, malignanciesOnly oral PUVA: nausea \nImportant drug interactions\nDrugs causing phototoxicity or photoallergy\nOther\nCombination with topical preparations acts synergistically, PUVA may not be combined with cyclosporine\nEvaluation of systemic therapies\nEfalizumab\nTable\u00a08\nTabular summary\nEfalizumab\nFirst approved in Germany\nSeptember 2004 (psoriasis vulgaris)\nRecommended control parameters\nPrior to therapy exclusion of significant infections, complete blood count, liver values \nRecommended initial dosage\n0.7\u00a0mg\/kg body weight (BW) per week\nRecommended maintenance dosage\n1\u00a0mg\/kg BW per week\nExpected beginning of clinical effect \nAfter 4\u20138\u00a0weeks\nResponse rate\nPASI 75 for approximately 30% of the patients after 12\u00a0weeks (LE 1)\nImportant contraindications (limited selection)\nChronic or acute infections, pregnancy, malignant diseases, immune deficiencies, no vaccinations before or during treatment\nImportant ADRs (limited selection)\nFlu-like injection reactions, leukocytosis and lymphocytosis, rebound, exacerbation and arthralgia, thrombocytopenia\nImportant drug interactions\nNot known \nOther\nStop therapy due to the risk of exacerbation and rebound if a PASI reduction of 50% is not achieved after 12\u00a0weeks\nTable\u00a09\nLaboratory controls during treatment with efalizumab\naHb (hemoglobin), HCT (hematocrit), erythrocytes, leukocytes, differential blood count, platelets\nbALT alanine aminotransferase, AST aspartate aminotransferase\nEtanercept\nTable\u00a010\nTabular summary\nEtanercept\nFirst approved in Germany\n2002 (Psoriasis arthritis )\/2004 (psoriasis vulgaris)\nRecommended control parameters\nPrior to therapy exclusion of tuberculosis, complete blood count, liver and renal values, urinanalysis \nRecommended initial dosage\nTwice 25\u00a0mg per week or 2\u00d7 50\u00a0mg\/week\nRecommended maintenance dosage\nTwice 25\u00a0mg per week\nExpected beginning of clinical effect \nAfter 4\u20138\u00a0weeks, at the latest after 12\u00a0weeks\nResponse rate\nPASI 75 in 34% (2\u00a0\u00d7\u00a025\u00a0mg) or 49% (2\u00a0\u00d7\u00a050\u00a0mg) of the patients at the end of the induction phase (12\u00a0weeks) (LE 1)\nImportant contraindications (limited selection)\nInfections, pregnancy, nursing, heart failure NYHA III\u2013IV\nImportant ADRs (limited selection)\nLocal reactions, infections \nImportant drug interactions (limited selection)\nAnakinra (IL-1 receptor antagonist)\nOther\n\u2013\nTable\u00a011\nLaboratory controls during treatment with etanercept\nInfliximab\nTable\u00a012\nTabular summary\nInfliximab\nFirst approved in Germany\n2004 (Psoriasis arthritis)\/2005 (psoriasis vulgaris)\nRecommended control parameters\nPrior to therapy exclusion of tuberculosis, during therapy: leukocyte and platelet counts, liver value controls, signs of clinical infection\nRecommended initial dosage\n5\u00a0mg\/kg BW at week 0, 2, 6\nRecommended maintenance dosage\n5\u00a0mg\/kg BW in dosage intervals of 8\u00a0weeks\nExpected beginning of clinical effect \nAfter 1\u20132\u00a0weeks\nResponse rate\nPASI 75 in \u226580% of the patients with moderate to severe psoriasis vulgaris (LE 1)\nImportant contraindications (limited selection)\nAcute or chronic infections, tuberculosis, cardiac failure NYHA III\u2013IV, pregnancy and nursing\nImportant ADRs (limited selection)\nInfusion reactions, severe infections, progression of heart failure NYHA III\u2013IV, very rare liver failure, autoimmune phenomena\nImportant drug interactions (limited selection)\nAnakinra (IL-1 receptor antagonist)\nOther\n\u2013\nTable\u00a013\nLaboratory controls during treatment with infliximab\naHb, HCT, erythrocytes, leukocytes, differential blood count, platelets\nCyclosporine\nTable\u00a014\nTabular summary\nCyclosporine\nFirst approved in Germany\n1983 (Transplantation medicine)\n1993 (Psoriasis vulgaris)\nRecommended control parameters\nInterview\/examination: \n\u2022 Status of skin and mucous membranes\n\u2022 Signs of infection\n\u2022 Neurological, gastrointestinal symptoms\n\u2022 Blood pressure\nLaboratory controls: see Table\u00a014\nRecommended initial dosage\n2.5\u20133 (max. 5) mg\/kg BW\nRecommended maintenance dosage\nInterval therapy (between 8 and 16 weeks) with dosage reduction at the end of the induction therapy (e.g., 0.5 mg\/kg BW every 14\u00a0days) or\nContinuous long-term therapy with dosage reduction (e.g. by 50\u00a0mg every 4\u00a0weeks after week 12) and a dosage increase by 50\u00a0mg with relapse\nMaximum total duration of therapy: 2\u00a0years\nExpected beginning of clinical effect\nAfter approximately 4\u00a0weeks\nResponse rate\nDose-dependent: after 8\u201316\u00a0weeks with 3\u00a0mg\/kg BW; PASI 90 in approximately 30\u201350% of patient and PASI 75 in approximately 50\u201370% of patients (LE 1)\nImportant contraindications (limited selection)\nAbsolute:\nReduced renal function, insufficiently controlled arterial hypertension, uncontrolled infections, relevant malignancies (current or previous, in particular hematologic diseases and cutaneous malignancies with the exception of basal cell carcinoma)\nRelative:\nRelevant hepatic dysfunction, pregnancy and nursing, concomitant use of substance which interacts with cyclosporine, concomitant UV-therapy or prior PUVA-pre-therapy with cumulative dosage >1000 J\/cm\u00b2, concomitant application of other immunosuppressives, retinoids or long-term prior-therapy with methotrexate (MTX)\nImportant ADRs (limited selection)\nRenal failure, increase of blood pressure, liver failure, nausea, anorexia, vomiting, diarrhea, hypertrichosis, gingival hyperplasia, tremor, weariness, parasthesia, hyperlipidemia\nImportant drug interactions (limited selection)\nIncrease of the cyclosporine level (CYP3A inhibition) through:\nAllopurinol, calcium antagonists, amiodarone, antibiotics (macrolides, clarithromycin, josamycin, ponsinomycin, pristinamycin, doxycycline, gentamicin, tobramycin, ticarcillin, quinolones), ketoconazole, oral contraceptives, methylprednisolone (high dosages), ranitidine, cimetidine, grapefruit juice\nDecrease of the cyclosporine level (CYP3A induction) through: Carbamazepine, phenytoin, barbiturates, metamizole, St. John\u2019s wort \nPossible reinforcement of nephrotoxic adverse drug reactions through: Aminoglycosides, amphotericin B, ciprofloxacin, acyclovir, non-steroidal antiphlogistics\nSpecific interactions:\nPotassium-saving substances: increased risk of hyperpotassemia\nReduced clearance of: Digoxin, colchicine, prednisolone, HMG-CoA reductase inhibitors (e.g. lovastatin), diclofenac\nOther\nIncreased risk of lympho-proliferative diseases in transplant patients. Increased risk of squamous cell carcinoma in psoriasis patients following excessive phototherapy.\nOnly moderately effective in and not approved for psoriatic arthritis \nAlso used successfully in the therapy of chronic-inflammatory diseases in children\nTable\u00a015\nLaboratory controls during treatment with cyclosporine\naErythrocytes, leukocytes, platelets\nbALT, AST, AP (alkaline phosphatase), \u03b3GT (gamma glutamyl transpeptidase), bilirubin\ncSodium, potassium\ndRecommended twice (fasting) and week-2 and 0\neOnly with indication (e.g. muscle cramps)\nFumaric acid esters\nTable\u00a016\nTabular summary\nFumaric acid esters\nFirst approved in Germany\n1995 (Psoriasis vulgaris) \nRecommended control parameters\nSerum creatinine, transaminases\/\u03b3GT, complete blood count including differential blood count, urinanalysis\nRecommended initial dosage\nAccording to recommended dosage regimen see Table\u00a017\nRecommended maintenance dosage\nIndividually adapted dosage\nExpected beginning of clinical effect\nAfter approximately 6 weeks\nResponse rate\nPASI 75 in 50\u201370% of the patients at the end of the induction phase after 16 weeks (LE 2)\nImportant contraindications (limited selection)\nChronic diseases of the gastrointestinal tract and\/or the kidneys and chronic diseases, which are accompanied by an impairment of the leukocyte count or functions, malignant diseases, pregnancy and nursing \nImportant ADRs (limited selection)\nGastrointestinal complaints, flush, lymphopenia, eosinophilia\nImportant drug interactions\nNone known\nOther\n\u2013\nTable\u00a017\nDosage regimen for Fumaderm therapy\nFumaderm initial\nFumaderm\nWeek 1\n1-0-0\nWeek 2\n1-0-1\nWeek 3\n1-1-1\nWeek 4\n1-0-0\nWeek 5\n1-0-1\nWeek 6\n1-1-1\nWeek 7\n2-1-1\nWeek 8\n2-1-2\nWeek 9\n2-2-2\nTable\u00a018\nLaboratory controls during treatment with fumaric acid esters\naLeukocytes, platelets, erythrocytes, differential blood count\nMethotrexate\nTable\u00a019\nTabular summary\nMethotrexate\nFirst approved in Germany\n\u00a0Lantarel\u00ae\n1991 (Psoriasis vulgaris)\n\u00a0Metex\u00ae 7.5\/10\u00a0mg\n1992 (Psoriasis vulgaris)\n\u00a0Metex\u00ae 2.5\u00a0mg\n2004 (Psoriasis vulgaris)\nRecommended control parameters\nComplete blood count (Hb, HCT, differential blood count, platelets), renal function (serum creatinine, urea, urine sediment), liver values (serum transaminases), III-procollagen amino terminal propeptides \nRecommended initial dosage\n5\u201315\u00a0mg per week\nRecommended maintenance dosage\n5\u201322.5\u00a0mg per week depending on effect\nExpected beginning of clinical effect\nAfter 4\u20138\u00a0weeks\nResponse rate\nPASI 75 in approximately 60% of the patients at the end of the induction phase of 16\u00a0weeks (LE 3)\nImportant contraindications (limited selection)\nAbsolute contraindications:\nDesire to have children (for both men and women), pregnancy and nursing, inadequate contraception, drug consumption, alcoholism, known sensitivity to active ingredient methotrexate (e.g. pulmonary toxicity), bone marrow dysfunction, severe liver disease, severe infections, immunodeficiency, active peptic ulcers, hematologic changes (leucopenia, thrombocytopenia, anemia), renal failure\nRelative contraindications:\nKidney disorders, liver disorders, history of arsenic consumption , chronic congestive cardio-myopathy, adiposity, old age, diabetes mellitus, history of hepatitis, lack of patient compliance, ulcerative colitis, diarrhea, NSAID use, gastritis\nImportant ADRs (limited selection)\nLiver fibrosis\/cirrhosis, pneumonia\/alveolitis, bone marrow depression, renal damage, alopecia (reversible), nausea, weariness, vomiting, elevated transaminases, infection, gastrointestinal ulcerations, nephrotoxicity\nImportant drug interactions (limited selection)\nCyclosporine, salicylates, sulfonamides, probenecide, penicillin, colchicin, NSAIDs (naproxene, ibuprofene, etc.), ethanol, co-trimoxazole, pyrimethamine, chloramphenicol, sulfonamides, prostaglandin synthesis inhibitors, cytostatics, probenecide, barbiturates, phenytoin, retinoids, sulfonamides, sulfonylurea, tetracyclines, co-trimoxazol, chloramphenicol, dipyridamole, retinoids, ethanol, leflunomide\nOther\nConsistent avoidance of alcohol, X-ray of the lungs prior to beginning therapy \nTable\u00a020\nLaboratory controls during treatment with MTX [137]\naHb, HCT, erythrocytes, leukocytes, differential blood count, platelets\nbALT, AST, AP, \u03b3GT, albumin, bilirubin, lactate dehydrogenase (LDH)\ncLiver biopsy instead of a liver sonography in risk groups\nRetinoids\nTable\u00a021\nTabular summary\nAcitretin\nFirst approved in Germany\n1992 (Psoriasis vulgaris)\nRecommended control parameters\nErythrocyte sedimentation rate (ESR), complete blood count, liver values, renal values, blood lipid values, pregnancy test, x-ray control of the bones in case of long-term therapy\nRecommended initial dosage\n0.3\u20130.5\u00a0mg\/kg BW per day for approximately 4\u00a0weeks, then 0.5\u20130.8\u00a0mg\/kg BW\nRecommended maintenance dosage\nIndividual dosaging dependent on the results and tolerance \nExpected beginning of clinical effect\nAfter 4\u20138\u00a0weeks\nResponse rate\nWidely variable and dose-dependent, no definite statement possible, partial remission (PASI 75) in 25\u201375% of the patients (30\u201340\u00a0mg per day) (LE 3) in studies\nImportant contraindications (limited selection)\nRenal and liver damage, desire to have children in female patients of child-bearing age, pregnancy, nursing, alcohol abuse, manifest diabetes mellitus, wearing of contact lenses, history of pancreatitis, hyperlipidemia requiring drug treatment\nImportant ADRs (limited selection)\nHypervitaminosis A (e.g., cheilitis, xerosis, nose-bleeding, alopecia, increased skin fragility) \nImportant drug interactions (limited selection)\nPhenytoin, tetracyclines, methotrexate, alcohol, mini-pill\nOther\nContraception up to 2\u00a0years after discontinuation in female patients of child-bearing age\naDouble contraception is recommended (e.g., condom\u00a0+\u00a0pill; IUD\/Nuva-Ring\u00a0+\u00a0pill; cave: no low-dosed progesterone preparations\/mini-pills) during and up to 2\u00a0years after the end of therapy; effectiveness is reduced by acitretin\nTable\u00a022\nLaboratory controls during treatment with Acitretin\naHb, HCT, leukocytes, platelets\nbPreferably assayed twice (week-2 and 0); HDL, high-density lipidoprotein\nOther therapies\nClimate\/balneotherapy\nTable\u00a023\nTabular summary\nClimate\/balneotherapy\nFirst approved in Germany\nClinical experience with balneotherapy has existed for more than 200\u00a0years \nRecommended control parameters\nRegular skin inspection\nRecommended initial dosage\nTherapy regimens vary depending on the institution\/location\nRecommended maintenance dosage\nTherapy regimens vary depending on the institution\/location\nExpected beginning of clinical effect\nVaries greatly\nResponse rate\nVaries greatly (LE 4)\nImportant contraindications\nDependent on modality selected\nImportant ADRs\nDependent on modality selected\nImportant drug interactions\nNot applicable\nOther\nBalneotherapy and climate therapy are frequently combined\nPsychosocial therapy\nNotes on the use of the Guidelines\nThe presentation of the therapies deliberately focused on those aspects deemed particularly relevant by a panel of experts. Aspects which are not of specific importance for a certain intervention, but which are part of the physician\u2019s general obligations to the patient, such as the investigation of intolerance and allergies toward certain drugs or the exclusion of contraindications, are not individually listed, but it is nevertheless taken for granted that these are part of the physician\u2019s duty to provide care.\nIt is recommended that each and every user carefully reads and follows the product information and compare it with the recommendations in the Guidelines on dosaging, contraindications, and drug interactions for completeness and currentness. Every dose or application is administered at the user\u2019s own risk. The authors and the publishers kindly request that users inform them of any inaccuracies that they might notice. The users are requested to keep themselves constantly informed of any new findings subsequent to the publication of the Guidelines.","keyphrases":["evidence-based guidelines","treatment","psoriasis vulgaris"],"prmu":["P","P","P"]}
{"id":"Support_Care_Cancer-3-1-2092408","title":"Testicular cancer: a longitudinal pilot study on stress response symptoms and quality of life in couples before and after chemotherapy\n","text":"Goals of work The current study was designed to longitudinally examine stress response symptoms (SRS) and quality of life (QoL) in couples confronted with disseminated testicular cancer. The objectives were to examine couples\u2019 patterns of adjustment over time and possible differences in adjustment between the patient and his partner.\nIntroduction\nTesticular cancer is a rare disease, although it is the most common tumor in men aged 20\u201335\u00a0years. About 500 new cases are diagnosed each year in The Netherlands [1]. Testicular cancer is distinguished into seminomas and nonseminomas, and each type accounts for about half of the total. Half of the men with nonseminoma are diagnosed with disseminated disease that is treated with chemotherapy [2]. Since the introduction of cisplatin-based chemotherapy in the late 1970s, up to 80% of the patients with disseminated disease can be cured [3\u20135].\nChemotherapy for testicular cancer has several acute physical side effects like nausea, fatigue, and neuropathy [6]. On a psychological level, patients receiving chemotherapy have been found to report anxiety, depression, and distress [7\u201310]. The partners of testicular cancer patients receiving chemotherapy may encounter adverse sequelae as well. Partners have to struggle with the fear of potentially losing their significant other. Partners are the primary source of information for family members and friends while their husbands are admitted to the hospital. In addition to this social task, partners often take on the caregiver role between chemotherapy cycles. Caregiving for cancer patients was found to negatively impact a caregiver\u2019s physical and mental health [11\u201313].\nThe impact of testicular cancer on younger couples might differ from the impact on couples who face cancer at an older age. The young couples confronted with testicular cancer might not have been together for a substantial period of time. This can make the relationship more vulnerable to the stressors induced by major life events. Earlier studies have shown that relationships of shorter duration are more sensitive to disruption after a testicular cancer diagnosis [14, 15], although another study did not find such a connection [16]. Also, couples are confronted with possible treatment-related infertility and sexual difficulties at a time in life where partners are often focused on starting a family. Couples may face several individual and dyadic stressors after the diagnosis of testicular cancer, leading to the experience of distress and lowered quality of life (QoL). This is particularly true when chemotherapy is part of the treatment protocol, as it is a more demanding treatment modality than surgery alone in the case of testicular cancer.\nSeveral prospective studies focused on dyadic adjustment and functioning from cancer diagnosis in a variety of sites, up to 1 1\/2\u00a0years later [17\u201320]. Although both patients and partners were reported to suffer from distress, levels of distress did not necessarily correspond within couples. Different patterns of distress and adjustment were found to be associated with gender and health status. Some studies reported that female partners were most vulnerable because they reported higher levels of distress than female and male patients and male partners [20\u201322].\nOne pilot study examined the course of distress and QoL in testicular cancer patients before, during, and after chemotherapy (complete data on ten patients). Patients reported highest levels of distress shortly before the start of chemotherapy. Distress and anxiety decreased over time [23]. Another recent prospective study on a large group of testicular cancer patients receiving chemotherapy focused on global QoL during the first 2\u00a0years after diagnosis. A considerable impairment in global QoL was found after 3\u00a0months. However, after 2\u00a0years 36% of patients reported improved functioning compared to baseline [24]. At the University Medical Center Groningen (UMCG) in The Netherlands, a tertiairy referral center for patients with testicular cancer, and at the University of Texas M. D. Anderson Cancer Center, USA considerable institutional research was done into epidemiology, short- and long-term medical, and sexual outcomes of testicular cancer [25\u201328]. These cross-sectional and retrospective studies were done on QoL of testicular survivors [29], their partners [30], and marital and sexual satisfaction [16, 31\u201333]. We chose to expand these findings by performing a prospective study, and the findings of Trask and Fossa by including partners in our prospective study. Patterns of stress response symptoms (SRS) and QoL of patients and partners were examined at three time points during the first year. SRS can occur after a range of traumatic events, including cancer. They involve intrusive thoughts and avoidance of thoughts and situations that remind them of the event, and are often studied in cancer patients [34]. Goals of the present study were: (1) to explore differences and relationships between patients and partners\u2019 SRS and QoL and to relate those to relationship aspects (duration and presence of children); (2) to examine change over time in SRS and QoL in patients and partners; (3) to relate earlier levels of SRS and QoL in patients and partners to later levels; and (4) to examine differences in QoL of patients and partners with that of a reference group of men and women.\nMaterials and methods\nProcedure and participants\nAll patients diagnosed with a disseminated nonseminomatous testicular tumor who consecutively visited the UMCG in The Netherlands for treatment between April 2001 and March 2004 and who were married or cohabiting were approached for this study. Only patients who received chemotherapy and had a steady relationship (n=30) could be included in this study. Other inclusion criteria were age over 18\u00a0years at study entry, sufficient command of the Dutch language, no previous treatment for cancer, and absence of a psychiatric history. The study was introduced to the patients and their partners after orchiectomy (removal of the affected testicle) was performed. Couples received a questionnaire at the following three time points: after orchiectomy but before the start of chemotherapy (T1), immediately after completion of chemotherapy, which is approximately 3\u00a0months after T1 (T2), and 1\u00a0year after T1 (T3). The patients received four cycles of cisplatin, etoposide, and bleomycin with a 3-week interval between each cycle. Couples received a letter with information about the objectives of the study, an informed consent form, the questionnaires, and a prepaid return envelope. Thirty of the 70 patients diagnosed during the above-mentioned time period appeared to meet all of the inclusion criteria. They and their partners were approached to participate in the study. Of the eligible 30 couples, 21 participated (70%). Two couples did not complete all three measurement times, therefore, only 19 couples provided complete data. The study was approved by the Medical Ethics Committee of the UMCG.\nMeasurements\nSociodemographics: Data on the following sociodemographic variables were collected at T1: age, educational level, employment status, presence of children, and duration of the relationship. Highest educational level completed was measured on a seven-point scale: primary school [1], and lower vocational [2], lower secondary [3], middle secondary [4], high secondary [5], higher vocational degrees [6], and advanced university [7]. Employment status could be indicated as employed for wages, housekeeping, student, unemployed, unable to work, or retired.\nSRS were measured with the Impact of Event Scale (IES) [35, 36]. This scale (15 items) makes an inventory of the extent to which a subject is currently occupied with an event by measuring intrusion (intrusively experienced ideas, images, feelings, or bad dreams about the event; 7 items) and avoidance of unpleasant feelings or memories of the event (8 items), resulting in a total score of SRS. Patients and partners rated the frequency of SRS with respect to his cancer during the preceding 7\u00a0days. Examples of items include: \u201cAny reminder brings back feelings about it\u201d (intrusion) and \u201cI try to banish it from my memory.\u201d The IES is a valid instrument for measuring cancer-related SRS [37, 38]. With this questionnaire, information was obtained about the degree to which confrontation with testicular cancer was influencing the current daily life of the respondent. Higher scores indicate more SRS. The Dutch version of the IES indicates a total score of more than 26 as severe SRS, for which psychological help is recommended. Reliability of this scale was good for patients (Cronbach\u2019s alpha for the different measurement times ranged from 0.82 to 0.89) and for partners (Cronbach\u2019s alpha for the different measurement times ranged from 0.78 to 0.93).\nQoL was measured with three subscales of the RAND-36 [39]: physical functioning (ten items), social functioning (two items) and mental health (five items). To avoid statistical problems due to multiple comparisons, we chose these three subscales as a representation of overall QoL. The RAND-36 measures generic QoL. After recoding and transformation, scores on the subscales could range from 0 to 100. Higher scores indicate a better QoL. Reliability of these scales was good to very good for patients (Cronbach\u2019s alpha for the different measurement times ranged from 0.67 to 0.90) and for partners (Cronbach\u2019s alpha for the different measurement times ranged from 0.75 to 0.92). The Dutch manual for the RAND-36 provides reference scores. These comprised the mean scores of a group of 691 nonselected men and 372 nonselected women from a random representative sample of persons aged 18\u00a0years and older from the population register of a municipality in the north of The Netherlands (number of inhabitants=108,000). The mean age of the persons in the total random sample was 44.1\u00a0years (range 18\u201389\u00a0years) [39].\nStatistical analyses\nThe database consisted of matched pairs of patients and partners, making analyses on pair level possible. Paired t tests and chi-square test were performed to examine differences in sociodemographics between patients and partners. Wilcoxon signed rank test (because of small sample size) and correlations were computed to examine differences and relationships in SRS and QoL between patients and partners. Independent t tests were performed for having children (yes or no) with SRS and QoL. Pearson\u2019s product moment correlations were calculated to examine the relationship between duration of the relationship and patients and partners\u2019 SRS and QoL. Repeated measures analysis of variance (ANOVA) was used to examine change over time in functioning. Pearson\u2019s product moment correlations were calculated to examine relationships between the functioning of patient and that of the partner and between measurement times. Strong correlation coefficients (>0.50) indicate consistency between measurement times and stable responses between earlier and later levels of functioning. Independent t tests were performed to compare QoL of patients and partners with those of a reference group of men and women.\nResults\nSociodemographic and treatment-related variables\nSociodemographic and treatment related variables are reported in Table\u00a01. Patients were older than partners (t=\u22123.1, p<0.01). The mean time couples had been together was 5.7\u00a0years. Eighteen patients were employed for wages and 1 was a student, whereas 11 partners were employed for wages, 4 were home-keepers and the remaining 4 partners were students (chi-square=19.0, p=0.04). Of the 21 couples, 5 had children and 14 did not. After chemotherapy was completed (at T2), all patients were restaged. Patients without biochemical or radiological abnormalities were considered to have reached complete remission (n=8). In case of proven residual disease, a resection of residual retroperitoneal tumor mass (RRRTM) was performed. Eleven patients underwent this surgery and were considered to be in complete remission afterward. None of the 19 patients experienced a relapse during follow-up (up to T3). \nTable\u00a01Descriptives of sociodemographic and treatment-related variables\u00a0PatientPartnerMeanSDMeanSDAge (years)b31.66.628.97.6\u00a0Range19.9\u201343.519.8\u201344.7Duration relationship (years)5.76.3\u00a0Range1\u201322Education level3.61.44.01.6N%N%Employment statusb\u00a0Employed for wages18951158\u00a0Student15421\u00a0Housewife421Children\u00a0Yes526\u00a0No1474RRRTM +\u00a0Yes1158\u00a0No842RRRTM Resection of residual retroperitoneal tumor massap<0.01bp<0.05\nPatients\u2019 and partners\u2019 SRS and QoL\nAt T1, SRS of patients were moderately strongly and negatively related to those of partners (r=\u22120.48, p<0.05). At T2, social functioning of patients was moderately strongly and positively related to that of partners (r=0.53, p<0.05). At the three measurement times all other correlations between patients\u2019 SRS and QoL and those of partners were low to moderate, ranging from r=0.04 to 0.34, and not statistically significant. At T1, in two couples, both patient and partner experienced SRS above the cut-off point. At T2 and T3 there were no couples in which both patient and partner experienced SRS above the cut-off point. Wilcoxon tests showed only one significant difference between patients and partners\u2019 functioning. At T2, patients reported a lower level of physical functioning than did partners (Z=\u22122.6, p<0.01).\nIndependent t tests did not show a significant relationship between having children and SRS for patients at all measurement times, and for partners at T1 and T2. At T3 partners with children reported higher levels of SRS than partners who did not have children (t=2.6, p=0.018). The presence of children was unrelated to patients\u2019 and partners\u2019 QoL at all measurement times. The duration of the relationship was not significantly related to the patients\u2019 or partner\u2019s SRS or QoL at all measurement times and relationships were absent to very weak.\nThe course of SRS and QoL over time in patients and partners\nRepeated measures ANOVA showed that the SRS of patients fluctuated according to a quadratic trend: The highest level was reported at T1 and after a decrease at T2, the level was increased again somewhat at T3. Earlier levels of SRS in patients were highly positively related to later levels (Table\u00a02). At T1, five of the patients (26%) reported stress response levels above the cut-off point; at T2, two patients (11%); and at T3, three patients (16%). Two patients reported clinically elevated levels of stress response levels at all measurement times. For partners the level of SRS declined, via a linear trend. Partners\u2019 reports of stress response levels were moderately strongly related between T1 and T2, strongly between T2 and T3, and the relationship between T1 and T3 was weak (Table\u00a02). At T1, ten of the partners (53%) reported stress response levels above the cut-off point, at T2, six (32%), and at T3, two (10.5%). One partner reported clinically elevated levels of stress response levels at all measurement times. \nTable\u00a02SRS and QoL of patients and partners\u00a0T1T2T3Reference groupRepeated measures ANOVAT1\u2013T2T2\u2013T3T1\u2013T3Mean (SD)Mean (SD)Mean (SD)Mean (SD)FprprprpStress response symptomsPatient18.1 (13.0)10.2 (10.7)12.3 (9.9)7.90.0120.650.0020.710.0010.700.001Partner25.8 (9.3)15.2 (12.6)12.1 (12.4)17.70.0010.41ns0.640.0030.16nsPhysical functioningPatient93.4 (10.8)70.5 (23.4)92.6 (13.3)84.5 (22.3)a,b,c21.40.0010.28ns0.38ns0.20nsPartner90.2 (19.0)90.8 (19.0)89.7 (19.9)80.7 (23.6)b,d0.19ns0.790.0010.800.0010.970.001Social functioningPatient77.6 (19.7)71.1 (22.8)90.8 (13.1)88.4 (19.6)d,e9.00.0080.610.0090.19ns0.17nsPartner72.1 (20.2)77.6 (20.7)f84.5 (16.5)86.1 (20.9)a12.90.0020.600.0070.37ns0.680.001Mental healthPatient68.2 (22.3)77.5 (11.9)81.9 (13.4)79.4 (17.3)d6.70.0190.550.0170.33ns0.23nsPartner69.1 (13.6)71.2 (15.6)74.3 (15.1)75.5 (18.9)2.6ns0.720.0010.640.0040.520.023ap<0.01 (independent t test T1 and reference group)bp<0.05 (independent t test T2 and reference group)cp<0.05 (independent t test T3 and reference group)dp<0.05 (independent t test T1 and reference group)ep<0.01 (independent t test T2 and reference group)fp<0.01 (Wilcoxon test patients and spouses)\nLower physical functioning was reported by patients at T2 compared to T1, but physical functioning returned to baseline level at T3. Earlier levels of physical functioning in patients were not significantly related to later levels, and correlations were low. A decline in social functioning of patients was also found at T2 compared to T1, but higher social functioning than at baseline was found at T3. Social functioning of patients at T1 was positively and strongly related to functioning at T2, but the relationships between T2\u2013T3 and T1\u2013T3 were low to moderate. Mental health of patients improved over time. Mental health at T1 was positively and strongly related to levels at T2, and weakly to levels at T3. The relationship between mental health at T2 and T3 was moderately strong.\nFor partners, no significant time effects were found for physical functioning and mental health. Earlier levels of physical functioning and mental health of partners were highly and positively related to later levels. Social functioning of partners improved over the year and was highly positively related between T1 and T2 and between T1 and T3. The relationship between T2 and T3 was moderately strong. (Table\u00a02).\nQoL of patients and partners compared to that of a reference group of men and women\nAt T1, patients (t=\u22123.4, p<0.01) and partners (t=\u22122.1, p<0.05) reported better physical functioning than the reference groups. Patients (t=2.3, p<0.05) and partners (t=2.9, p<0.01) reported worse social functioning than the reference groups at T1, and patients (t=2.2, p<0.05) reported worse mental health at T1. At T2, patients reported worse physical functioning than the reference group of men (t=2.6, p<0.02) but partners reported better physical functioning than the reference group of women (t=\u22122.2, p<0.05). Patients also reported worse social functioning than the reference men at T2 (t=3.3, p<0.01). No differences were found between the patients\u2019 and partners\u2019 mental health and the norm groups at T2. Only one significant difference was found at T3: Patients reported better physical functioning than men in the reference group (t=\u22122.6, p<0.02) (Table\u00a02).\nDiscussion\nThe present study was the first to prospectively and longitudinally examine psychosocial functioning in both testicular cancer patients and their partners. We focused on SRS and QoL (physical functioning, social functioning, and mental health) after orchiectomy but before the start of chemotherapy, immediately after completion of chemotherapy, and 9\u00a0months later (1\u00a0year follow-up).\nSRS in couples were most salient before the start of chemotherapy. Twenty-six percent of patients reported clinically elevated SRS at this time, a number comparable to the 30% of patients with clinically elevated distress found in a recent study on testicular cancer patients [23]. A review of stress response syndromes in adult cancer populations showed that the incidence of clinically elevated SRS ranged from 3 to 4% in patients recently diagnosed with early stage cancer [34]. The majority of these studies used the same questionnaire for measuring SRS as we did. The same review identified younger age, greater proximity to diagnosis, more advanced disease, and greater treatment intensity as risk factors for a higher level of SRS. The much higher percentage found in our study at the first measurement time may be explained by the prevalence of these four risk factors in our patient group.\nNot only did patients appear distressed, but also almost twice as many of the partners compared to patients reported clinically elevated levels of SRS. Female sex was found to correlate with greater stress response symptomatology in cancer populations [34]. Of course, these spouses are not patients themselves, but they obviously face cancer-related fears and worries as well. Our results affirm earlier findings that female spouses of cancer patients are vulnerable to distress, and often report higher levels of distress compared to their male counterparts [20, 21, 40\u201342]. It was also found that partners who had children experienced more SRS a year after diagnosis compared to partners without children. This higher level of distress might also be a result of caregiving tasks that are often the domain of female partners.\nSRS in couples decreased after completion of chemotherapy. One year after diagnosis, stress response levels in patients rose slightly, but they continued to decrease in partners. These patterns suggest that the period before chemotherapy commences is most stressful, and that recovery seems to occur within a year. At the time of the first measurement the responses of the couples seem to be colored by the fact that they had recently learned the diagnosis, and the outcome of treatment remained uncertain. Concerns of couples about side effects of chemotherapy such as nausea, the possibility of immunodeficiency, and hair loss may contribute to distress. Partners also have caregiving tasks that can exacerbate their distress [22]. All couples in this study received positive news about the results of treatment, and 1\u00a0year after diagnosis none of the patients had experienced a relapse of disease. Couples seemed to have recovered from this major life event over the year, possibly as a consequence of the success of treatment reducing insecurity about outcome.\nAn interesting finding was that before commencement of chemotherapy, the level of SRS of patient and partner was inversely related. When one spouse was reporting more SRS, the other reported less. This might be a psychological mechanism through which spouses want to protect one another from their own distress, a finding reported before in studies on couples facing cancer [40]. Another possible interpretation might be that patients who expressed very low levels of distress were using denial, avoidance, or other repressive psychological mechanisms while their partners carried the psychological burden of stress. A patient who expresses high distress may also allow his spouse to assume a stronger supportive role, and thus reduce her expression of stress.\nIn line with the medical trajectory, physical functioning of patients was worst after completion of chemotherapy. As a consequence of chemotherapy, many patients still experience adverse side effects, including fatigue and a sense of physical exhaustion. It is surprising to note that physical functioning of patients was better than that of the reference group before start of chemotherapy and 1\u00a0year later. The first measurement occurred shortly after removal of the affected testicle, and patients may have experienced physical relief after the initiation of treatment. It may also be that age contributed to the difference found between the patient group and the reference group. Younger age is associated with better physical functioning [39], and the mean age of the patients in this study was somewhat more than 10\u00a0years lower than that of the reference group. There may have been other differences from the reference group as well, such as physical activity and SES. Partners\u2019 physical functioning did not change over the year but they also experienced better physical functioning than the reference group before and after chemotherapy. In an earlier cross-sectional study we found that partners of men who survived testicular cancer between 1\u201320\u00a0years reported better physical functioning than a reference group of women, even years after diagnosis [30]. Partners of testicular cancer patients may have changed the evaluation of their own health in a positive way after witnessing the diagnosis and treatment of a life-threatening illness.\nIn patients, social functioning was worst immediately after completion of chemotherapy, and in spouses before start of chemotherapy. Before start of chemotherapy, both partners reported worse social functioning compared to the reference groups. Before chemotherapy starts, couples are probably overwhelmed by the implications of a cancer diagnosis and focus more on the treatment to come rather than on being socially active as usual. As a consequence of chemotherapy, patients may still suffer from negative side effects like fatigue and impaired physical functioning, which in turn may affect their social functioning. Social functioning in patients and partners was positively related after completion of chemotherapy, meaning that when one partner experienced better social functioning the other also did. Couples had comparable social functioning to that of the reference groups 1\u00a0year after diagnosis; they seem to have returned to their usual social activities. This finding is in line with studies on testicular cancer survivors that show little or no change in social contacts and work activities [6, 43].\nMental health of patients improved over the year. It was poorer than that of men in the reference group but only before start of chemotherapy. Mental health of partners was comparable throughout the year and to that of a reference group of women, despite the high level of SRS they reported before start of chemotherapy. SRS apparently are a different expression of mental functioning and may be encapsulated or separated out.\nIt appeared that functioning in couples facing testicular cancer was not similar in patients and partners. Testicular cancer patients showed u-shaped trajectories of SRS, physical functioning, and social functioning. This pattern seemingly followed the medical trajectory they had undergone and was found before in a large group of testicular cancer patients [24]. Mental health of patients improved over the year. Spouses reported a decline in SRS and an improvement in social functioning, but no change in physical functioning or mental health over the year. Differences between patients and partners were also noticeable in stability of functioning. In patients, earlier levels of SRS were strongly predictive of later levels while less individual stability was found with regard to physical and social functioning and to mental health. Partners reported less individual stability in SRS over time, although individual stability was found between T2 and T3. However, partners were individually highly consistent in their reports of physical functioning and somewhat less strong in mental health and social functioning. In addition, correlations between the functioning of patients and partners were moderate to very low. We also found that at the second and third measurement time, there were no couples in which both the patient and the partner reported clinically elevated levels of SRS. This finding confirms the lack of correspondence in functioning between patient and partner.\nThese findings support recent studies that found different adjustment patterns for patients and partners, and a lack of correspondence in functioning [19, 20]. Research is needed to examine if these different reaction patterns to a cancer diagnosis affect the marital relationship.\nThis study has some limitations. First, the possibility of including couples was limited because testicular cancer has a low incidence, and because of the young age of patients at diagnosis, part of this group will not yet have established a steady relationship. Second, no information was available on the functioning of couples who declined to participate. They may have been those who were functioning best or worst, thus biasing the results in either direction. Third, because of the limited sample size, variables that measure more in depth relationship aspects like marital satisfaction were not included in this study. In a previous retrospective study on testicular cancer, factors identified as important for couple adjustment like good communication, spousal support, and marital satisfaction all appeared to facilitate better functioning [32]. These moderating factors in adjustment of both patient and spouse deserve to be studied prospectively in the future.\nIn summary, this study was the first longitudinal prospective exploration of functioning in couples facing testicular cancer during the first year after the diagnosis. Patients confronted with disseminated testicular cancer and their partners reacted differently to this stressor. Clinically elevated stress response levels were present in one third of patients and half of the partners before start of chemotherapy. Patients reported worst mental health before start of chemotherapy, and worst physical and social functioning immediately after completion of chemotherapy. Partners reported an improvement in social functioning, and no change in physical functioning and mental health over the year. QoL of patients and partners was comparable to that of the reference groups a year after start of chemotherapy, patients even reported better physical functioning at that time. These findings support earlier retrospective studies in testicular cancer survivors and their spouses that also reported few long-term effects in psychosocial functioning in the group overall, but that identified a small group that remains distressed [16, 31, 32]. Little correspondence was found in the functioning of the partners. Findings should be regarded as preliminary, as the study sample was limited in size. However, clinicians can be reassured that the effect of disseminated testicular cancer on the QoL of patients and their partners seems to be temporary, although a minority does seem to need clinical attention for SRS.","keyphrases":["testicular cancer","stress response symptoms","quality of life","chemotherapy","partners"],"prmu":["P","P","P","P","P"]}
{"id":"Knee_Surg_Sports_Traumatol_Arthrosc-4-1-2413121","title":"Effects of biophysical stimulation in patients undergoing arthroscopic reconstruction of anterior cruciate ligament: prospective, randomized and double blind study\n","text":"Pre-clinical studies have shown that treatment by pulsed electromagnetic fields (PEMFs) can limit the catabolic effects of pro-inflammatory cytokines on articular cartilage and favour the anabolic activity of the chondrocytes. Anterior cruciate ligament (ACL) reconstruction is usually performed by arthroscopic procedure that, even if minimally invasive, may elicit an inflammatory joint reaction detrimental to articular cartilage. In this study the effect of I-ONE PEMFs treatment in patients undergoing ACL reconstruction was investigated. The study end-points were (1) evaluation of patients\u2019 functional recovery by International Knee Documentation Committee (IKDC) Form; (2) use of non-steroidal anti-inflammatory drugs (NSAIDs), necessary to control joint pain and inflammation. The study design was prospective, randomized and double blind. Sixty-nine patients were included in the study at baseline. Follow-up visits were scheduled at 30, 60 and 180 days, followed by 2-year follow-up interview. Patients were evaluated by IKDC Form and were asked to report on the use of NSAIDs. Patients were randomized to active or placebo treatments; active device generated a magnetic field of 1.5 mT at 75 Hz. Patients were instructed to use the stimulator (I-ONE) for 4 h per day for 60 days. All patients underwent ACL reconstruction with use of quadruple hamstrings semitendinosus and gracilis technique. At baseline there were no differences in the IKDC scores between the two groups. At follow-up visits the SF-36 Health Survey score showed a statistically significant faster recovery in the group of patients treated with I-ONE stimulator (P < 0.05). NSAIDs use was less frequent among active patients than controls (P < 0.05). Joint swelling resolution and return to normal range of motion occurred faster in the active treated group (P < 0.05) too. The 2-year follow-up did not shown statistically significant difference between the two groups. Furthermore for longitudinal analysis the generalized linear mixed effects model was applied to calculate the group \u00d7 time interaction coefficient; this interaction showed a significant difference (P < 0.0001) between the active and placebo groups for all investigated variables: SF-36 Health Survey, IKDC Subjective Knee Evaluation and VAS. Twenty-nine patients (15 in the active group; 14 in the placebo group) underwent both ACL reconstruction and meniscectomy; when they were analysed separately the differences in SF-36 Health Survey scores between the two groups were larger then what observed in the whole study group (P < 0.05). The results of this study show that patient\u2019s functional recovery occurs earlier in the active group. No side effects were observed and the treatment was well tolerated. The use of I-ONE should always be considered after ACL reconstruction, particularly in professional athletes, to shorten the recovery time, to limit joint inflammatory reaction and its catabolic effects on articular cartilage and ultimately for joint preservation.\nIntroduction\nArticular cartilage performs mechanical functions absorbing the different loads applied to a joint in the course of daily activity [19]. Homeostasis and mechanical competence of cartilage are regulated by the activity of the chondrocytes that maintain the function and the integrity of the extracellular matrix, proteoglycans and collagen.\nIn consideration of the scant repairability of the cartilage, even modest damages resulting from trauma or inflammation may be the starting point for cartilage degeneration leading over time to extensive lesions that deepen into the thickness of the cartilage itself, ultimately exposing the subchondral bone tissue [3, 17].\nJoint injury may involve synovial tissue, cartilage and subchondral bone leading to joint inflammation, swelling and pain. Surgical interventions must certainly be included among the triggers of inflammatory reaction in a joint [12]. The development of arthroscopic procedures has undoubtedly limited joint damage associated to surgery for reconstruction of ligaments; nevertheless, it does not avoid the inflammatory response. Thus, while arthroscopic procedures make surgery less invasive, the inflammatory response at the joint remains and the release of pro-inflammatory cytokines in the synovial fluid is associated with an increase in the aggrecanase activities that lead to a degradation of the cartilage matrix, and also inhibit proteoglycan synthesis [11, 15, 18]. To prevent cartilage damage, current pharmacological therapies aim to control the catabolic effects of the pro-inflammatory cytokines and enhance anabolic activity, proteoglycan synthesis and proliferation of chondrocytes. Drugs that combine the above effects are called chondroprotectors; in this category should be included drugs with A2A adenosine receptor agonist activity, able to stimulate the physiological pathways that control inflammation and promote chondrocyte anabolic activities. Nevertheless, these drugs are in early stages of clinical testing [5].\nPre-clinical studies have shown that pulsed electromagnetic fields (PEMFs) in vitro favour the proliferation of chondrocytes [6, 16], stimulate proteoglycan synthesis [7] and demonstrate an A2A adenosine receptor agonist activity [20, 21]. Electromagnetic fields in vivo prevent degeneration of articular cartilage and down-regulate the synthesis and release of pro-inflammatory cytokines in the synovial fluid [2, 4, 8, 9]. These findings suggest that electromagnetic fields may be used to control joint inflammation and to stimulate cartilage anabolic activities, finally resulting in chondroprotection.\nA clinical study performed in patients undergoing arthroscopic treatment for cartilage lesions showed that biophysical treatment with PEMFs was well tolerated by the patients and led to a decrease in the use of non-steroidal anti-inflammatory drugs (NSAIDs) and to an early functional recovery; the positive effect of the treatment was maintained at a 3-year follow-up [22].\nArthroscopic reconstruction is the treatment of choice following anterior cruciate ligament (ACL) rupture; although minimally invasive, the procedure is associated with joint reaction involving the synovia and it is expected to lead to an increase of pro-inflammatory cytokines in the synovial fluid with catabolic effect on articular cartilage. In this study, we evaluated whether the treatment with PEMFs could be used to control joint inflammatory response in patients undergoing ACL reconstruction. The end points of the study were: (1) patients\u2019 functional recovery evaluated by International Knee Documentation Committee (IKDC) form; (2) use of NSAIDs, necessary to control joint pain and inflammation.\nMaterials and methods\nPatients and study design\nIn 2004\u20132005, 84 patients undergoing ACL reconstruction were evaluated for inclusion in the study at five clinical centres. Of these, 69 gave their informed consent to participate in the study. The prospective randomized and double-blind study was approved by the local ethical committees. Inclusion criteria were the following: age between 18 and 45\u00a0years, ACL complete lesion following acute trauma or consequence of ligament chronic degeneration. All lesions were documented by MRI and confirmed during the intervention. The following were the exclusion criteria: osteonecrosis of the femoral condyle, rheumatoid arthritis, autoimmune disease, systemic disease and patients requiring meniscus repair.\nThe patients were assigned to the active or placebo group according to the following randomization criteria: age (18\u201330 or 31\u201345), sex, smoking status, origin of ACL rupture (traumatic or degenerative). For randomization of patients, a computer-generated schedule was prepared by a biostatistician. In this process, a random number seed was entered into the computer to generate a list that assigned equal numbers of active and placebo stimulators. The minimum number of patients per group required was calculated by power analysis taking into account the results of a previous study [22].\nOf the 69 patients included, two never started the therapy, two dropped out within 2\u00a0weeks of therapy, and five did not return at follow-up visits; a total of 60 patients were therefore available for subsequent analysis. The ACL rupture occurred during sports activity in 49 patients (24 active and 25 placebo), daily activity in eight patients (four active and four placebo) and traffic accident in three patients (three active). At the time of ACL reconstruction 29 patients underwent also meniscectomy: 15 in the active group and 14 in the placebo.\nClinical evaluation\nThe patients were evaluated by IKDC Form before the intervention and at 30, 60 and 180\u00a0days afterwards. The different parts of the questionnaire, IKDC Current Health Assessment Form (SF-36 Health Survey), IKDC Subjective Knee Evaluation Form and IKDC Knee Examination Form were analysed separately. As regards the scores of the questionnaires, for each subject we considered the changes at follow-up visits with respect to the values recorded at baseline, before surgery.\nPain intensity was evaluated by visual analogue scale (VAS) of 10-cm length: 0\u00a0cm no pain, 10\u00a0cm maximum pain. The patients were allowed to use NSAIDs to control knee pain when present and had to report doing so.\nA 2-year follow-up telephone interview was conducted and the patients were asked: (a) if they had undergone further surgery at the knee, (b) if they had pain at the knee, (c) if they had functional limitation in daily activity, (d) if they returned to previous sport activity level.\nSurgical technique\nACL arthroscopic repair was performed by quadruple hamstrings semitendinosus and gracilis technique. Tendons were harvested with the tendons stripper through a 2\u20133\u00a0cm vertical incision on the antero-medial tibial area. Diameter of the quadruple hamstrings semitendinosus and gracilis tendons was measured, while the tibial tunnel and same size femoral tunnel (30\u00a0mm length) were prepared. The graft was pulled up through the tibial tunnel with the knee at 90\u00b0 of flexion and suspended on the external femoral cortex (Endobutton, Smith and Nephew, London, UK). Distally, the graft was fixed with an interference absorbable screw at the tibia at 10\u00b0 of flexion.\nRehabilitation\nAll the patients underwent standard rehabilitation using passive knee flexion daily. Exercises started within the third post-operative day with isometric quadriceps contractions and then progressed to active closed-chain exercises by 4\u20136\u00a0weeks postoperatively. During the first 20\u00a0days patients were instructed to use two crutches and then progressive weight bearing until the end of the second month.\nBiophysical stimulation\nThe patients were treated with active or placebo devices. The active stimulators (I-ONE; IGEA, Carpi, Italy) generated a magnetic field of peak intensity of 1.5\u00a0mT at a frequency of 75\u00a0Hz; no heat or vibration was felt by the patient during treatment (Fig.\u00a01).\nFig.\u00a01Left I-ONE PEMFs generator. Right wave form of magnetic field, 1.5\u00a0mT peak value (top); electric field induced in a standard coil probe made of 50 turns (0.5\u00a0cm \u2205) of copper wire (0.2\u00a0mm \u2205), peak value 3\u00a0mV\/cm (bottom)\nThe patients were instructed to use the stimulator for 4\u00a0h per day, not necessarily consecutively, for 60\u00a0days. Treatment started within 7\u00a0days from the surgery. Each device contained a clock to monitor the hours of use.\nStatistical analysis\nThe results were analysed with SPSS 13.0 (Statistical Packages for Social Sciences Inc, Chicago, IL, USA). Comparison among the continuous variables in the two groups was performed with Student\u2019s heteroschedastic t test; comparison of continuous variables within each group during follow-up was performed with Student\u2019s paired t test.\nBinomial and categorical variables were compared by contingency tables applying the chi-square test for 2\u00a0\u00d7\u00a02 tables and the Cochran Mantel Haenszel test for larger size tables.\nGeneralized linear mixed effects model was applied to the SF-36 Health Survey, IKDC Subjective Knee Evaluation and VAS data to test if a different trend between the two groups was present during follow-up by correcting for the following covariates: sex, age, weight, height, hours of treatment, smoking status, use of NSAIDs. In this analysis, a mathematical model is built which takes into account the trend over time of individual patients belonging to each group (Group\u00a0\u00d7\u00a0Time interaction) and determines if a statistical difference exists between the groups during the follow-up [10].\nThe minimum significance level for all the statistical tests was set at P\u00a0<\u00a00.05.\nResults\nAt baseline, the two groups of study were homogeneous for age, weight, height, VAS, SF-36 Health Survey and IKDC Subjective Knee Evaluation score (Table\u00a01).\nTable\u00a01Characteristics of the groups at baselinePlacebo #29I-ONE #31PMeanSEMeanSEAge29.61.632.51.40.17Weight7227330.73Height175117420.59VAS2.40.33.20.50.27SF-36 Health Survey3723720.95IKDC Subjective Knee Evaluation Form4834730.90\nAverage daily treatment was the same in both groups: 3.92\u00a0\u00b1\u00a00.5\u00a0h\/die versus 3.13\u00a0\u00b1\u00a00.3\u00a0h\/die in the I-ONE group and the placebo group, respectively (P\u00a0=\u00a0n.s.).\nThe average pain was modest and almost absent at 6\u00a0months\u2019 follow-up: 0.7\u00a0\u00b1\u00a00.2\u00a0cm among placebo and 0.9\u00a0\u00b1\u00a00.2\u00a0cm among active. At 30\u00a0days, less patients in the active group used NSAIDs: 8% in the I-ONE group versus 27% in the placebo group (P\u00a0<\u00a00.05).\nThe SF-36 Health Survey score decreased significantly at 30\u00a0days, in both groups (P\u00a0<\u00a00.0005). At 60\u00a0days the mean SF-36 Health Survey score in the I-ONE patients already exceeded the initial value (by 3.2 points), whereas in the patients of the placebo group SF-36 Health Survey score was slightly below the initial mean value (by \u22120.7 units). At 6\u00a0months a significant (P\u00a0<\u00a00.005) increase was observed for SF-36 Health Survey average values in both groups; the patients of the I-ONE group were above the initial values by 10.1 units, while the placebo group exceeds the baseline value by 7.2 units. The mean changes of SF-36 Health Survey score in the I-ONE group are systematically higher with respect to placebo during follow-up, P\u00a0<\u00a00.05 (Fig.\u00a02).\nFig.\u00a02Mean changes of SF-36 Health Survey (\u00b1SE) versus baseline in the two groups (P\u00a0<\u00a00.05)\nThe IKDC Subjective Knee Evaluation score increased over 6\u00a0months and did not show significant differences between the two groups at any follow-up visit.\nThe IKDC Knee Examination Form outlined both groups including subjects with joint swelling before surgery (one in placebo and two in I-ONE group, P\u00a0=\u00a0n.s.) and at 30\u00a0days\u2019 follow-up (five in placebo and six in I-ONE group, P\u00a0=\u00a0n.s.). On day 60, joint swelling was observed in the placebo group (two patients) only. Joint swelling was not observed any more at 6\u00a0months\u2019 follow-up. Limitation in the passive range of motion of the knee was more frequent in the placebo group than in the I-ONE group (P\u00a0<\u00a00.05) (Fig.\u00a03).\nFig.\u00a03Patients with limitation in passive range of motion in the two groups, P\u00a0<\u00a00.05\nFinally, the generalized linear mixed effects analysis revealed a significantly different trend (group\u00a0\u00d7\u00a0time interaction, P\u00a0<\u00a00.0001) between the two groups for SF-36 Health Survey score, IKDC Subjective Knee Evaluation score and for VAS, showing a positive effect of I-ONE treatment. The estimate coefficients and significance of independent variables for three models are displayed in Table\u00a02.\nTable\u00a02Generalized linear mixed effects models in which the dependent variables considered are: SF-36 Health Survey score, IKDC Subjective Knee Evaluation score and VAS, respectivelyCoefficientStd. err.z testP\u00a0<\u00a0SF-36 Health Survey \u00a0Group0.3612.6900.130.893\u00a0Hours of treatment\u22120.0140.008\u22121.790.073\u00a0Sex\u22128.3484.810\u22121.740.083\u00a0Weight\u22120.4230.171\u22122.480.013\u00a0Height0.2200.2540.870.386\u00a0Age\u22120.2120.137\u22121.550.120\u00a0Smoking status0.7682.0750.370.711\u00a0Use of NSAIDs\u22124.6672.726\u22121.710.087\u00a0Time0.1250.0344.580.000\u00a0Group\u00a0\u00d7\u00a0Time0.0510.0143.670.0001\u00a0Constant37.94040.3020.940.347IKDC Subjective Knee Evaluation\u00a0Group\u22121.4993.417\u22120.440.661\u00a0Hours of treatment\u22120.0070.011\u22120.620.533\u00a0Sex\u221219.3906.172\u22123.140.002\u00a0Weight\u22120.5780.216\u22122.680.007\u00a0Height0.1500.3220.470.642\u00a0Age\u22120.7540.185\u22124.080.000\u00a0Smoking status\u22122.2142.783\u22120.800.426\u00a0Use of NSAIDs\u22122.9413.805\u22120.770.440\u00a0Time0.1450.0512.890.000\u00a0Group\u00a0\u00d7\u00a0Time0.1670.0315.370.000\u00a0Constant94.56151.3821.840.066VAS \u00a0Group0.7990.4551.760.079\u00a0Hours of treatment\u22120.0000.001\u22120.220.824\u00a0Sex1.5830.8061.960.050\u00a0Weight0.0200.0290.670.505\u00a0Height0.0270.0430.640.524\u00a0Age0.0330.0221.50.133\u00a0Smoking status0.2460.3370.730.464\u00a0Use of NSAIDs\u22120.8250.434\u22121.900.058\u00a0Time\u22120.4410.2443.890.000\u00a0Group\u00a0\u00d7\u00a0Time\u22120.0090.002\u22124.000.000\u00a0Constant\u22125.5936.721\u22120.830.405The Group\u00a0\u00d7\u00a0Time interaction term describes the different trend between the groups\nAt the 2-year follow-up interview 86% of the patients in the I-ONE group and 75% in the placebo group reported complete functional recovery, no knee pain and return to sport activity.\nACL reconstruction and meniscectomy\nWhen the cohort of patients, undergoing both ACL reconstruction and meniscectomy, was analysed separately, the SF-36 Health Survey score confirmed the faster recovery trend among I-ONE treated patients compared to placebo, P\u00a0<\u00a00.05 (Fig.\u00a04). At 6\u00a0months, SF-36 Health Survey average score increase was 11.4 in the I-ONE group (P\u00a0<\u00a00.005 vs. baseline) and 7.1 in placebo group (P\u00a0=\u00a0ns vs. baseline). Further, the average values of SF-36 Health Survey were significantly higher in the I-ONE group compared to the placebo (45.2\u00a0\u00b1\u00a01.5 vs. 37\u00a0\u00b1\u00a02.7, P\u00a0<\u00a00.05).\nFig.\u00a04Patients undergoing ACL and meniscectomy: mean changes of SF-36 Health Survey (\u00b1SE) versus baseline in the two groups (P\u00a0<\u00a00.05)\nThe percent of patients with limitation in the passive range of motion was lower in the I-ONE group compared to the placebo one (34% I-ONE vs. 50% placebo at day 30 and 4% I-ONE vs. 17% placebo at day 60, P\u00a0<\u00a00.05).\nDiscussion\nArthroscopic surgery has gained a large success and led to a significant increase in its use: about 650,000 procedures are performed in the USA each year [14]. However, the access into the joint space is always associated to an inflammatory reaction that may jeopardize the benefits expected from surgery. Joint inflammation has a catabolic effect on extracellular matrix and inhibits chondrocyte activity; thus, all means capable of locally controlling the inflammation should be adopted to prevent the onset and limit the progression of cartilage damage. Furthermore, unlike bone tissue after damage, the cartilage will not completely recover its competence: once lost, the articular cartilage does not reform [13].\nMany efforts are made to develop strategies able to control joint inflammation and to favour the anabolic activities of chondrocytes; these are challenging objectives, and up to now the pharmacological approaches based on the use of drugs, whether by systemic or by local route, have not yet been able to demonstrate a genuine chondroprotective effect in humans [19].\nPre-clinical studies have shown PEMFs to have a chondroprotective effect, mediated by the control of inflammation and by the stimulation of chondrocyte activity; thus, we hypothesized that after arthroscopic surgery PEMFs treatment can be used for articular cartilage protection and ultimately joint preservation.\nThis prospective, randomized and double-blind study investigated whether and to what extent the employment of I-ONE, by controlling joint reaction to arthroscopy, could accelerate functional recovery in patients undergoing ACL reconstruction. The I-ONE treatment was well tolerated by the patients and no adverse side effects were observed. The results show that, at 30\u00a0days after surgery, in I-ONE group significantly fewer patients used NSAIDs to control pain, compared to patients in the placebo group; afterwards, the use of NSAIDs was not necessary in either group.\nWhen IKDC Subjective Knee Evaluation average scores were analysed, we found no statistically significant difference between the I-ONE and placebo group; this is in agreement with the findings of other authors who reported that this parameter does not correlate with the other clinical information collected using the SF-36 Health Survey form [1]. However, when the results of the two groups were analysed by generalized linear mixed effects model, which takes into account the trend of each patient in both groups and the effect of confounding factors, we could evidence a positive significant effect of I-ONE treatment also in the Subjective Knee Evaluation (Table\u00a02).\nThe SF-36 Health Survey average scores at baseline were the same in the I-ONE and placebo groups; however, the high standard deviation testify the large distribution of initial score values. To monitor patient\u2019s recovery after ACL reconstruction, we considered the SF-36 Health Survey score changes with respect to baseline for each individual subject. At 2 and 6\u00a0months SF-36 Health Survey increase is undoubtedly higher in I-ONE group than in the placebo group. This result indicates a faster recovery in the treated patients. This positive effect of I-ONE treatment is confirmed by the generalized linear mixed effects analysis. Further, when the cohort of patients who underwent both ACL reconstruction and meniscectomy was analysed, we observed that the average increase of SF-36 Health Survey at 60\u00a0days in the I-ONE group was the same as that of placebo group at 6\u00a0months (6.0 vs. 7.1, P\u00a0=\u00a0n.s.).\nThe IKDC Knee Examination Form showed how in the placebo group the resolution of joint swelling and the recovery of complete range of motion occur later compared to the I-ONE group; no significant difference in scoring was observed among centres.\nThe study end-points were thus demonstrated: fewer patients in the I-ONE group required the use of NSAIDs and their functional recovery was faster.\nAt 2-year follow-up no statistically significant difference was observed between two groups, although the percent of patients with complete recovery was slightly higher in the I-ONE group.\nIn this study we applied a statistical analysis specifically developed for longitudinal studies that allows to calculate the group\u00a0\u00d7\u00a0time interaction. This test, that considers individual patient\u2019s score at different time points and the possible influence of confounding factors, supports the positive effect of I-ONE treatment on the recovery of patients undergoing ACL reconstruction.\nOur data confirm the results reported by Zorzi et al. [22] in a group of patients treated with I-ONE following an arthroscopic treatment for cartilage lesions. To the authors\u2019 knowledge, there are no other reports of use of biophysical stimulation after surgical procedures of the knee.\nBiophysical stimulation allows treating individual joints, permeating the whole cartilage surface and thickness, the synovia and the subchondral bone. The effectiveness of biophysical stimulation is not limited by considerations such as diffusion ability and concentration gradient, which are present and important in the dynamic of a pharmacological intervention; joint tissues are paramagnetic, they do not attenuate the biophysical signal and thus are all homogenously exposed to the treatment efficacy. Biophysical stimulation is an effective therapeutic intervention to control the detrimental consequences of the inflammation over articular cartilage in the absence of negative side effects.\nI-ONE should always be considered after ACL reconstruction, particularly in professional athletes, to shorten the recovery time, to limit joint inflammatory reaction and ultimately for joint preservation.","keyphrases":["anterior cruciate ligament","pulsed electromagnetic fields","joint preservation","chondroprotection","biophysical stimuli"],"prmu":["P","P","P","P","M"]}
{"id":"Int_J_Hematol-4-1-2330061","title":"Identification of genes potentially involved in supporting hematopoietic stem cell activity of stromal cell line MC3T3-G2\/PA6\n","text":"Although coculture of hematopoietic stem cells (HSCs) with stromal cells is a useful system to study hematopoiesis in the niche, little is known regarding the precise cellular and molecular mechanisms of maintaining HSCs through cell\u2013cell interactions. The murine preadipose stromal cell line MC3T3-G2\/PA6 (PA6) has been demonstrated to support HSCs in vitro. In this study, microarray analysis was performed on PA6 cells and HSC-nonsupporting PA6 subclone cells to identify genes responsible for supporting HSC activity. Comparison of gene expression profiles revealed that only 144 genes were down-regulated by more than twofold in PA6 subclone cells. Of these down-regulated genes, we selected 11 candidate genes and evaluated for the maintenance of HSC function by overexpressing these genes in PA6 subclone cells. One unknown gene, 1110007F12Rik (also named as Tmem140), which is predicted to encode an integral membrane protein, demonstrated a partial restoration of the defect in HSC-supporting activity.\nIntroduction\nHematopoietic stem cells (HSCs) are capable of self-renewal and multilineage differentiation and generate all types of blood cells throughout the lifetime. A major challenge for a long time has been the establishment of culture conditions that can facilitate ex vivo expansion of HSCs. To date, a large number of attempts using combinations of multiple cytokines have resulted in limited success, with a several-fold increase in HSC numbers under the optimal culture conditions [1, 2]. A major problem is that culture conditions established thus far have induced HSC proliferation coupled with differentiation, leading to a progressive loss of in vivo long-term repopulating potential [3\u20138]. Therefore, a better understanding of mechanisms that regulate HSC self-renewal and differentiation is required to achieve ex vivo expansion of HSCs.\nIn the adult bone marrow, HSCs are thought to reside in a specific microenvironment, referred to as the niche, which is composed of stromal cells that play a vital role in determining stem cell fate. Indeed, several stromal cell lines have been established not only from bone marrow but also from fetal liver and the aorta-gonad-mesonephros region and have been shown to maintain HSCs in vitro [9\u201316]. Coculture of HSCs with stromal cell lines is a useful system to study hematopoiesis in the niche. Although physical contact between HSCs and stromal cell lines has been demonstrated to be essential, little is known regarding the precise cellular and molecular mechanisms of maintaining HSCs by stromal cell lines.\nMC3T3-G2\/PA6 (PA6) is a preadipose stromal cell line derived from newborn mouse calvaria that can support long-term hematopoiesis in vitro [17]. In addition, PA6 subclones incapable of supporting the proliferation of HSCs have been isolated [18]. It has also been shown that the hematopoiesis-supporting ability of PA6 cells is not solely conferred by their expression of c-kit ligand, stem cell factor (SCF). In this study, microarray analysis was performed to identify genes responsible for supporting HSC activity of PA6 cells. Eleven genes specifically down-regulated in the subclone cells were selected and characterized by overexpressing the corresponding cDNAs in the subclone cells.\nMaterials and methods\nMice\nC57BL\/6 (B6-Ly5.2) mice were purchased from Charles River Laboratories Japan (Yokohama, Japan). C57BL\/6 mice congenic for the Ly5 locus (B6-Ly5.1) were obtained from RIKEN BRC. B6-Ly5.1\/Ly5.2 F1 mice were obtained from mating pairs of B6-Ly5.1 and B6-Ly5.2 mice. All animal experiments were approved by the Animal Experiment Committee of the RIKEN Tsukuba Institute.\nStromal cell culture and lentiviral transduction\nThe OP9 stromal cell line was obtained from RIKEN BRC Cell Bank. PA6 and OP9 cells were maintained in Minimum Essential Medium Eagle-\u03b1 (MEM-\u03b1) (Sigma-Aldrich, St Louis, MO) containing 20% fetal bovine serum (FBS) (Sigma-Aldrich) at 37\u00b0C in a 5% CO2 atmosphere. FANTOM cDNA clones (kindly provided by Dr. Y. Hayashizaki and Dr. J. Kawai, RIKEN GSC) corresponding to the genes used in this study were subcloned into the lentiviral vector plasmid pCSII-EF-MCS-IRES2-Venus. Recombinant lentiviral vectors were produced as described previously [19]. PA6 subclone cells were transduced with lentiviral vectors expressing cDNAs at a multiplicity of infection of 200 and >90% of transduction efficiency was confirmed by fluorescence-activated cell sorting (FACS) analysis for Venus expression.\nPurification of CD34\u2212KSL cells and coculture with stromal cells\nCD34\u2212\/lowc-Kit+Sca-1+lineage marker\u2212 (CD34\u2212KSL) cells were purified as described previously with minor modifications [20]. Briefly, bone marrow cells isolated from 10- to 16-week-old B6-Ly5.2 mice were stained with a lineage marker antibody cocktail consisting of biotinylated anti-Gr-1, anti-Mac-1, anti-B220, anti-IgM, anti-CD4, anti-CD8, and anti-Ter119 antibodies (eBioscience, San Diego, CA). Lineage marker+ cells were depleted using streptavidin-coupled Dynabeads M-280 (Invitrogen, Carlsbad, CA). The remaining cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD34, phycoerythrin (PE)-conjugated anti-Sca-1, and allophycocyanin (APC)-conjugated anti-c-Kit antibodies (all from BD Biosciences, San Jose, CA). The biotinylated antibodies were developed with streptavidin-APC-Cy7 (BD Biosciences). FACS was performed with a FACSVantage SE (BD Biosciences). CD34\u2212KSL cells were sorted into individual wells of a 96-well plate containing stromal cells (1\u00a0\u00d7\u00a0104 cells\/well) irradiated with 1.5\u00a0Gy and were cocultured in 150 \u03bcL of MEM-\u03b1 containing 20% FBS.\nColony-forming cell assay\nAfter 10\u00a0days of coculture, CD34\u2212KSL HSCs were collected and plated in a 12-well plate containing 0.6\u00a0mL of methylcellulose medium (MethoCult GF M3434) (StemCell Technologies, Vancouver, Canada) containing 50\u00a0ng\/mL rmSCF, 10\u00a0ng\/mL rmIL-3, 10\u00a0ng\/mL rhIL-6, and 3\u00a0units\/mL rhEPO. After 12\u00a0days of incubation, colonies were recovered, cytospun onto glass slides, and then subjected to Hemacolor (Merck KGaA, Darmstadt, Germany) staining for morphological examination. Colony-forming unit-granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM), CFU-granulocyte, erythrocyte, monocyte (CFU-GEM), CFU-granulocyte, monocyte (CFU-GM), CFU-granulocyte (CFU-G), CFU-monocyte (CFU-M), and burst forming unit-erythrocyte (BFU-E) were scored using standard scoring criteria. The total colony number was expressed as colony-forming cell (CFC).\nCompetitive repopulation assay\nThe competitive repopulation assay was performed by using the congenic Ly5 mouse system as described previously [8]. CD34\u2212KSL HSCs after coculture were mixed with 2\u00a0\u00d7\u00a0105 total bone marrow competitor cells from B6-Ly5.1 mice and transplanted into lethally (9.5\u00a0Gy) irradiated B6-Ly5.1 mice. At 12 to 16\u00a0weeks after transplantation, peripheral blood cells of the recipient mice were collected by retro-orbital bleeding. After lysis of red blood cells with ammonium chloride buffer, the remaining nucleated cells were stained with FITC-conjugated anti-Ly5.2, PE-conjugated anti-Ly5.1, biotinylated anti-Mac1, and biotinylated anti-Gr1 antibodies, followed by addition of streptavidin-PerCP. The cells were stained simultaneously with APC-conjugated anti-B220 antibody or a mixture of APC-conjugated anti-CD4 and anti-CD8 antibodies. FACS analysis was performed with a FACSCalibur. Donor chimerism was determined as the percentage of Ly5.2+ cells. When the percent chimerism was >1.0% for all myeloid, B-lymphoid, and T-lymphoid lineages, recipient mice were considered to be multilineage reconstituted.\nMicroarray analysis\nTotal RNA was isolated from stromal cells using the ISOGEN reagent (Nippon Gene, Tokyo, Japan) and purified with the RNeasy MinElute cleanup kit (Qiagen, Hilden, Germany). Biotin-labeled cRNA was prepared from 1\u00a0\u03bcg of the purified total RNA with a one-cycle cDNA synthesis kit and 3\u2032-amplification reagents for IVT labeling (Affymetrix, Santa Clara, CA) and was hybridized to an Affymetrix Gene Chip Mouse Genome 430 2.0 array (Affymetrix), which contains approximately 45,000 probe sets for analyzing the expression levels of more than 34,000 mouse genes. After washing and staining with the antibody amplification procedure, the microarrays were scanned with an Affymetrix GeneChip Scanner 3000 7G. All these procedures were carried out according to the manufacturer\u2019s instructions. The expression value (Signal) and detection call (Present (P), Absent (A), or Marginal (M)) for each probe set were calculated using GeneChip Operating Software version 1.4 (Affymetrix). The Signal values were normalized so that their mean in each experiment was 100 in order to adjust for minor differences between the experiments. The change value (Signal Log Ratio) and change call (Increase, Marginal Increase, No Change, Marginal Decrease, or Decrease) for each probe set were calculated by Comparison Analysis of the software. All experiments were performed in duplicate using two independent cell samples. To identify differentially expressed genes, we selected probe sets that showed a change call of Decrease and a Signal Log Ratio value of \u2264\u20131 (more than twofold down-regulation) or a change call of Increase and a Signal Log Ratio value of \u22651 (more than twofold up-regulation) in each of the two independent experiments.\nQuantitative real-time polymerase chain reaction\nTotal RNA was isolated from stromal cells using the ISOGEN reagent, and cDNA was synthesized from 1\u00a0\u03bcg of total RNA using SuperScript II reverse transcriptase (Invitrogen). Real-time polymerase chain reaction (PCR) amplification with two independent cell samples was carried out using the LightCycler FastStart DNA Master SYBR Green I kit (Roche, Penzberg, Germany) according to the manufacturer\u2019s instructions. The primer sets used in this study are listed in Table\u00a01. Data were normalized GAPDH mRNA levels. Gene-specific amplification was confirmed by determining the melting curves of the PCR products and by a single band of the expected size in agarose gel electrophoresis.\nTable\u00a01Primers used in quantitative real-time PCRGenePrimer sequenceProduct size (bp)1110007F12RikForward5\u2032-GCCCTGTGCCTGATGTTCTAC-3\u2032111\u00a0Reverse5\u2032-GCCCATGTCCTCCTTCCAC-3\u20322900064A13RikForward5\u2032-GTTTGACCCTGTCCGAGTCG-3\u2032205\u00a0Reverse5\u2032-CGGGAGAACCATCATCATAACC-3\u2032Ccl2Forward5\u2032-TTAAAAACCTGGATCGGAACCAA-3\u2032121\u00a0Reverse5\u2032-GCATTAGCTTCAGATTTACGGGT-3\u2032Ccl9Forward5\u2032-TCAGATTGCTGCCTGTCCTAT-3\u2032117\u00a0Reverse5\u2032-GAACCCCCTCTTGCTGATAAAG-3\u2032Cxcl5Forward5\u2032-TGCGTTGTGTTTGCTTAACCG-3\u2032107\u00a0Reverse5\u2032-AGCTATGACTTCCACCGTAGG-3\u2032IL-1rnForward5\u2032-GCTCATTGCTGGGTACTTACAA-3\u2032132\u00a0Reverse5\u2032-CCAGACTTGGCACAAGACAGG-3\u2032IL-6Forward5\u2032-TAGTCCTTCCTACCCCAATTTCC-3\u203276\u00a0Reverse5\u2032-TTGGTCCTTAGCCACTCCTTC-3\u2032\nResults and discussion\nSubclones of the PA6 cell line are defective in supporting HSCs\nAlthough PA6 subclones 2, 12, and 14 have been isolated in terms of inability to support long-term hematopoiesis in vitro, their HSC-supporting capacity has not been precisely assessed [18]. Therefore, we evaluated the maintenance of HSC function after coculture with PA6 subclones 2 and 12 (hereinafter referred to as S-2 and S-12, respectively) by in vitro CFC and in vivo competitive repopulation assays using CD34\u2212KSL cells as highly purified HSCs [20]. Murine bone marrow-derived OP9 stromal cell line that can support HSCs was also used as a control [13]. As shown in Table\u00a02, the CFC frequency significantly decreased after 10\u00a0days of coculture with PA6 S-2 and S-12 cells compared with PA6 cells, and lower levels of engraftment were observed in transplanted mice. These results indicate that PA6 subclone cells have a substantial defect in supporting HSCs even after short-term coculture.\nTable\u00a02CFC frequency and competitive repopulation capacityStromal cellsCFC frequencyNo. of reconstituted mice (%)% ChimerismPA610.8\u00a0\u00b1\u00a07.516\/9 (66.7)13.2\u00a0\u00b1\u00a019.9PA6 S-20.60\u00a0\u00b1\u00a00.97*4\/10 (40.0)4.5\u00a0\u00b1\u00a011.2**PA6 S-123.2\u00a0\u00b1\u00a03.05*3\/8 (37.5)3.4\u00a0\u00b1\u00a05.9**OP928.2\u00a0\u00b1\u00a06.615\/9 (55.6)8.6\u00a0\u00b1\u00a011.5A total of 80 CD34\u2212KSL HSCs (10 cells\/well) were cocultured with stromal cells for 10\u00a0days and were then subjected to the CFC assay. The CFC frequency represents the colony number per ten input CD34\u2212KSL HSCs. For the competitive repopulation assay, 30 CD34\u2212KSL HSCs were cocultured with stromal cells for 10\u00a0days and then transplanted into lethally irradiated mice. At 12\u00a0weeks after transplantation, peripheral blood cells of the recipient mice were analyzed. Data represent the mean \u00b1 SD of two independent experiments*P\u00a0<\u00a00.01, **P\u00a0>\u00a00.2 versus PA6, Student\u2019s t-test\nIdentification of genes that were specifically down-regulated in PA6 subclone cells\nWe hypothesized that genes responsible for supporting HSCs would be down-regulated in PA6 subclone cells. To identify genes specifically down-regulated in PA6 subclone cells, microarray analysis was performed on PA6 cells, PA6 S-2 and S-12 cells, and OP9 cells using an Affymetrix GeneChip array containing approximately 45,000 probe sets representing over 34,000 mouse genes. As expected, gene expression profiling showed a few differences in gene expression between PA6 cells and PA6 subclone cells. Compared to PA6 cells, only 144 genes were down-regulated by more than twofold in both PA6 subclone cells. Of these down-regulated genes, 41 genes were of unknown function, and 67 of 103 (65%) known genes encoded membrane and extracellular space proteins according to the Gene Ontology cellular component classification. On the other hand, 121 genes were up-regulated by more than twofold and 30 of 67 (45%) known genes encoded membrane and extracellular space proteins. The signal values and detection calls for this analysis are provided in supplemental data. Among the down-regulated genes, eight genes of known\/predicted function including chemokine- and cytokine-related genes, which were expected to effect on the HSC-supporting activity, and three genes of unknown function, which were predicted to encode membrane proteins, were selected for further analysis (Table\u00a03).\nTable\u00a03List of candidate genes down-regulated in PA6 subclone cells compared with PA6 cellsGene symbolGene titleRefseq IDMicroarrayqPCRvs. PA6vs. OP9vs. PA6vs. OP9PA6 S-2PA6 S-12PA6 S-2PA6 S-12PA6 S-2PA6 S-12PA6 S-2PA6 S-121110007F12RikRIKEN cDNA 1110007F12 geneNM_1979860.300.460.110.190.290.230.160.131200009O22RikRIKEN cDNA 1200009O22 geneNM_0258170.300.212.791.48NDNDNDND2900064A13RikRIKEN cDNA 2900064A13 geneNM_1337490.410.371.231.160.290.330.480.55Ccl2Chemokine (C-C motif) ligand 2NM_0113330.090.130.040.060.160.070.090.04Ccl9Chemokine (C-C motif) ligand 9NM_0113380.140.190.030.040.100.090.040.03Cd53CD53 antigenNM_0076510.230.150.280.14NDNDNDNDCxcl5Chemokine (C-X-C motif) ligand 5NM_0091410.330.230.430.380.200.130.900.60HgfHepatocyte growth factorNM_0104270.410.410.850.88NDNDNDNDIl1rn(IL-1rn)Interleukin 1 receptor antagonistNM_0311670.090.120.260.31<0.030.04<1.251.45Il6(IL-6)Interleukin 6NM_0311680.380.442.072.620.300.162.421.31Ppap2bPhosphatidic acid phosphatase type 2BNM_0805550.220.220.090.09NDNDNDNDRelative expression levels in PA6 subclone cells (S-2 and S-14) compared with PA6 and OP9 cells were calculated using data from microarray and quantitative real-time PCR (qPCR) analyses. Data represent the average of fold changes from two independent experimentsND not determined\nCharacterization of candidate genes\nTo determine the requirement of candidate genes for the supportive activity of PA6 cells, PA6 S-2 cells were transduced with lentiviral vectors containing the corresponding cDNAs. In all experiments, the transduction efficiency was more than 90% as determined by FACS analysis for Venus-positive cells. CD34\u2212KSL HSCs were cocultured with PA6 S-2 cells expressing candidate genes for 10\u00a0days and then subjected to the CFC assay. As shown in Fig.\u00a01, the CFC frequency increased when cocultured with PA6 S-2 cells overexpressing the candidate genes except Hgf compared with PA6 S-2 cells. Morphological evaluation of the colonies revealed that the number of CFU-GEMM, which is the most primitive colony type observed in the CFC assay, increased except Ccl2 and Ccl9. Of note, although the overexpression of IL-6 increased the number of colonies to the same level as PA6 cells, a large proportion (\u223c80%) of colonies were CFU-M.\nFig.\u00a01Effect of over expression of candidate genes on the CFC frequency. A total of 80 CD34\u2212KSL HSCs (10 cells\/well) were cocultured with PA6 cells, PA6 S-2 cells, or PA6 S-2 cells expressing the indicated genes for 10\u00a0days and then subjected to the CFC assay. Individual colonies were scored according to their morphology. Data represent the average of two independent experiments\nWe next performed the competitive repopulation assay to determine the effect of overexpressing seven candidate genes including Ccl9, IL-6, Ppap2b, and Cxcl5, and three unknown genes. As shown in Table\u00a04, successful multilineage reconstitution was observed in all mice transplanted with HSCs cocultured with PA6 S-2 cells overexpressing 1200009O22Rik or IL-6 as well as with PA6 cells. However, comparison of the chimerism showed no significant differences from that of PA6 S-2 cells, suggesting that the overexpression of neither 1200009O22Rik nor IL-6 completely restored the defective HSC-supporting activity of PA6 S-2 cells. Microarray analysis showed that the expression of IL-6 was approximately twofold up-regulated in PA6 S-2 and S-12 cells when compared with OP9 cells; this finding was confirmed by quantitative real-time PCR analysis (Table\u00a03). It has also been demonstrated that IL-6 alone or in combination with SCF is incapable of maintaining CD34\u2212KSL HSCs [8]. Although IL-6 is involved in various steps of hematopoiesis and has been used for the ex vivo expansion of HSCs [21], IL-6 may not be crucial for the maintenance of HSCs governed by stromal cells.\nTable\u00a04Effect of overexpression of candidate genes on the competitive repopulation capacityNo. of reconstituted mice (%)% ChimerismTotalMyeloidB-lymphoidT-lymphoidPA69\/9 (100)22.1\u00a0\u00b1\u00a025.130.9\u00a0\u00b1\u00a030.925.1\u00a0\u00b1\u00a026.517.4\u00a0\u00b1\u00a023.0PA6 S-24\/10 (40)7.3\u00a0\u00b1\u00a014.04.0\u00a0\u00b1\u00a07.410.8\u00a0\u00b1\u00a018.89.2\u00a0\u00b1\u00a017.81110007F12Rik7\/10 (70)16.2\u00a0\u00b1\u00a014.8*21.8\u00a0\u00b1\u00a026.318.7\u00a0\u00b1\u00a016.915.5\u00a0\u00b1\u00a016.71200009O22Rik8\/8 (100)8.2\u00a0\u00b1\u00a010.214.5\u00a0\u00b1\u00a012.29.6\u00a0\u00b1\u00a013.48.3\u00a0\u00b1\u00a011.32900064A13Rik2\/5 (40)3.3\u00a0\u00b1\u00a04.610.5\u00a0\u00b1\u00a020.63.6\u00a0\u00b1\u00a05.41.0\u00a0\u00b1\u00a02.2Ccl92\/5 (40)11.1\u00a0\u00b1\u00a015.014.2\u00a0\u00b1\u00a019.512.7\u00a0\u00b1\u00a017.611.4\u00a0\u00b1\u00a015.7IL-69\/9 (100)8.5\u00a0\u00b1\u00a013.58.1\u00a0\u00b1\u00a07.111.6\u00a0\u00b1\u00a016.99.2\u00a0\u00b1\u00a016.3Ppap2b8\/10 (80)9.7\u00a0\u00b1\u00a010.420.5\u00a0\u00b1\u00a026.79.2\u00a0\u00b1\u00a09.79.6\u00a0\u00b1\u00a014.6Cxcl53\/5 (60)3.0\u00a0\u00b1\u00a04.012.8\u00a0\u00b1\u00a023.62.5\u00a0\u00b1\u00a03.51.9\u00a0\u00b1\u00a01.8Thirty CD34\u2212KSL HSCs were cocultured with PA6 cells, PA6 S-2 cells, or PA6 S-2 cells expressing the indicated genes for 10\u00a0days and then transplanted into lethally irradiated mice. At 16\u00a0weeks after transplantation, peripheral blood cells of the recipient mice were analyzed. Data represent the mean\u00a0\u00b1\u00a0SD*P\u00a0=\u00a00.18 versus PA6 S-2, Student\u2019s t-test\nPA6 S-2 cells overexpressing 2900064A13Rik or CCl9 had no apparent effect on the HSC-supporting activity. In contrast, the overexpression of 1110007F12Rik, Ppap2b, or Cxcl5 resulted in a certain increase in the frequency of reconstituted mice. Although these genes as well as 1200009O22Rik and IL-6 could significantly increase the CFC frequency, the effect on the HSC-supporting activity assessed by the competitive repopulation assay was limited. This may be because 10\u00a0days of coculture was not long enough to detect a significant difference in HSC-supporting activity. It is of course well known that many growth factors and cytokines that can increase the CFC frequency have little influence on the maintenance and proliferation of HSCs. Of note, the overexpression of 1110007F12Rik showed a substantial increase in the chimerism, although the difference did not reach statistical significance (Table\u00a04). To confirm the effect of 1110007F12Rik expression on the supportive activity of PA6 cells, we performed the following experiments. First, down-regulation of 1110007F12Rik expression in PA6 S-2 and S-12 cells compared to that in PA6 and OP9 cells was confirmed by quantitative real-time PCR analysis (Table\u00a03). Next, PA6 S-2 cells were transduced with a myc-tagged 1110007F12Rik expression lentiviral vector and its expression was detected by Western blotting and immunofluorescence staining with anti-myc antibody. Coculture with PA6 S-2 cells overexpressing myc-tagged 1110007F12Rik resulted in an increase in the CFC frequency at a level similar to that shown in Fig.\u00a01 (data not shown). The 1110007F12Rik cDNA encodes a predicted protein of 185 amino acids, which is rich in leucine (23%) and has no significant homology with any known protein sequences. 1110007F12Rik is also named as Tmem140 (transmembrane protein 140), which is predicted to be an integral membrane protein gene according to the Gene Ontology classification. Further studies are required to determine whether 1110007F12Rik is involved in the HSC-supporting activity of PA6 cells.\nThus far, a large number of microarray analyses of stromal cell lines derived from a variety of tissue types have been reported. Comparison of gene expression profiles between HSC-supporting and HSC-nonsupporting stromal cell lines has resulted in the identification of a number of differentially expressed genes [22]. In the present study, we expected a few differences in gene expression by comparing HSC-supporting PA6 cells with HSC-nonsupporting PA6 subclone cells and indeed, we could narrow down the candidate genes involved in maintaining HSCs. Although one of the analyzed candidate genes, 1110007F12Rik, demonstrated a partial restoration of the defect in HSC-supporting activity of PA6 S-2 cells, it is possible that other down-regulated genes in PA6 subclone cells may also be required for the maintenance of HSCs. Continuing studies to identify genes responsible for HSC-supporting activity of PA6 cells would facilitate the understanding of the mechanisms that control stem cell fate through interaction with stromal cells.\nElectronic supplementary material\nBelow is the link to the Supplemental data.\nElectronic supplementary material (XLS 388 kb)","keyphrases":["hematopoietic stem cells","stromal cells","mc3t3-g2\/pa6","coculture","microarray"],"prmu":["P","P","P","P","P"]}
{"id":"Breast_Cancer_Res_Treat-3-1-2137942","title":"Stromal mast cells in invasive breast cancer are a marker of favourable prognosis: a study of 4,444 cases\n","text":"Purpose We have previously demonstrated in a pilot study of 348 invasive breast cancers that mast cell (MC) infiltrates within primary breast cancers are associated with a good prognosis. Our aim was to verify this finding in a larger cohort of invasive breast cancer patients and examine the relationship between the presence of MCs and other clinical and pathological features.\nIntroduction\nMast cells (MCs) are part of the innate immune system and are recruited to and activated in the microenvironment of a developing tumor. MCs originate from multipotential hemopoietic stem cells in the bone marrow, and express the proto-oncogene c-kit, a transmembrane type III tyrosine kinase receptor protein [1]. After leaving the vascular bed they differentiate and acquire functional maturity.\nMC infiltrates have been described in a variety of human cancers, including non-small-cell lung cancer [2\u20134], breast cancer [5], colorectal cancer [6], basal cell carcinoma [7] and pulmonary adenocarcinoma [8]. MCs are attracted to the tumor by tumor-derived chemo attractants where they either degranulate to release potential tumor cytotoxic compounds or become innocent bystanders depending on local tumor conditions [9]. There is controversy about the pro- and anti-tumorigenic effects of MCs in different cancers. The accumulation of MCs has been associated with enhanced growth and invasion of several human cancers [10]. On the other hand, MC infiltration has been associated with good prognosis in breast [11, 12] ovarian [13], lung [14] and colorectal [15] carcinomas.\nMurine models have shown that tumor incidence and growth are inversely correlated with the MC density in MC-deficient mice compared to normal [16].\nOur previously published pilot report of 348 case series of breast carcinomas showed that the presence of stromal MCs correlated with a good prognosis (P\u00a0=\u00a00.0036) in invasive breast cancer [11].We present here a large tissue microarray (TMA) study of 4,444 cases of invasive breast carcinomas with clinical outcome data. This study validates the independent prognostic significance of stromal MCs in breast cancer.\nMaterials and methods\nPatient selection\nA total of 4,620 archival samples from patients with invasive breast carcinoma referred to the British Columbia Cancer Agency between January 1986 and September 1992 were used for TMA construction. The Clinical Research Ethics Board of the University of British Columbia approved the study. Patients with in-situ disease, metastatic disease at presentation, and male breast cancer were excluded from analysis, thus bringing the final tally to 4,444 cases. This represents 34% of all patients diagnosed with breast cancer in the province of British Columbia during this time period. This large, well characterized cohort is derived from a consecutive series of patients who were referred to the BC Cancer Agency for consultation and had tumor samples sent to a central laboratory at the Vancouver General Hospital for estrogen receptor (ER) status. Consequently, for all of these patients we have available detailed demographic and outcome data, as well as formalin fixed paraffin embedded (FFPE) primary tumor samples for immunohistochemical analysis. Available clinical information includes age, histology, tumor grade, tumor size, lymph node status, type of local and adjuvant systemic therapy, and dates of first recurrence and death. Clinical and pathological variables were determined following well-established criteria. A portion of this cohort of patients was recently used in a population study validating the on-line breast cancer prognostic calculator ADJUVANT! Online [17].\nTMA construction\nThe Vancouver Hospital ER laboratory retained single archival tumor blocks from each case in this patient cohort. The material had been frozen prior to neutral buffered formalin fixation. All the paraffin sections were first stained with H&E and reviewed by a pathologist. Representative tumor areas were circled and matched with the donor blocks. 0.6-mm cores were punched out from the donor blocks and embedded 1\u00a0mm apart in 17 recipient blocks using Tissue Microarrayer (Beecher instruments Silver Springs, MD). These tissue arrays have been used to test the new ER SP1 antibody that improves the sensitivity for detecting ER by IHC [18].\nImmunohistochemistry (IHC)\nSections from TMA were cut at 4\u00a0\u03bcm and immunostained with antibody to KIT (CD117), a transmembrane tyrosine kinase acting as a type III receptor for MC growth factor. Slides were also concurrently stained for ER and Her2 using standard immunoperoxidase techniques. The antibodies and antigen retrieval methods are summarized in Table\u00a01.\nTable\u00a01Details of antibodies used for immunohistochemistryAntibodyIsotype CompanyAntigen retrievalConcentrationc-kitRabbit polyclonalDakoVentana1:100Her2 SP3Rabbit monoclonalLab visionSteam 30\u00a0min, 0.05\u00a0M TRIS buffer (pH 10)1:100ER SP1Rabbit monoclonalLab visionCitrate buffer (pH 6)1:250\nImmunohistochemical image processing and scoring\nThe stained slides were digitally scanned with a BLISS automated digital imaging microscope (Bacus Laboratories, Lombard, IL) which consists of a microscope with a scanning stage, video camera and software designed for scanning TMAs. A relational database was constructed using identification information and immunohistochemistry scores for each tissue core in the microarrays. An internet website was then constructed using this database and a WebSlideViewer Java applet provided by the manufacturer to view the microarray images and allow for an image zooming functionality. This website is publicly accessible through http:\/\/www.gpecimage.ubc.ca\/tma\/web\/viewer.php.\nThe slides were scored manually by two independent pathologists, blinded to the clinical outcome, as previously described [11]. Total number of stained MCs in each core was recorded. ER status was assessed using the rabbit monoclonal antibody (SP1) antibody [18]. The fractions of ER positive tumor nuclei were scored as 0 (<1%), 1 (1\u201325%), 2 (25\u201375%), and 3 (>75%). Her2 SP3 rabbit monoclonal from LabVision (NeoMarkers) was used to stain the TMA slides and were scored using Hercept test\u00ae (Dako Corporation, Carpinteria, CA) scoring system. Final Her2 score was derived using both IHC and Fluorescent in situ hybridization (FISH) assays. Cases with Her2 IHC Herceptest score\u00a0=\u00a03, were scored as positive. Those cases with Her2\u00a0IHC Herceptest score\u00a0=\u00a02 were re-evaluated using FISH assay, and only those cases with Her2 FISH amplification ratio \u22652.0 were scored as Her2 positive. C-Kit and ER scores were binarized for statistical analysis as follows: CD-117: 0\u00a0=\u00a0no MCs; 1\u00a0=\u00a0any MCs; ER: 0\u00a0\u2264\u00a01% nuclei stained; 1\u00a0\u2265\u00a01% nuclei stained. We excluded cases for which it was not possible to assign a score to the immunostaining (insufficient invasive tumor in the core, or missing core).\nStatistical analysis\nThe raw scores were entered into an Excel database and each TMA core was assigned a unique core ID number. The spreadsheet was then processed utilizing TMA-Deconvoluter 1.06 software that had been adapted for TMA analysis [19]. A database was created by incorporation of all deconvoluted marker data into the clinicopathologic patient database.\nWe applied a split-sample validation technique for our statistical analysis. Our 4,444 patient cohort was stratified into eight subgroups (Table\u00a02) based on adjuvant treatment received and then randomized into equal sized training and validation sets. The two groups were balanced with respect to treatment received; but there are also no significant differences in clinical or pathological variables, including age, tumor size and grade, nodal status, and ER status. For this study, and future studies using this TMA, the primary investigator is given access to all clinical, outcome, and TMA data from the training set only. The training set is used to generate and refine hypotheses regarding the biomarker under study. Significant findings are then formally presented at a bimonthly joint scientific group meeting of the Genetic Pathology Evaluation Centre (GPEC, a collaborative group of scientists and pathologists) and Breast Cancer Outcomes Unit (BCOU, a group of oncologists and epidemiologists based at the BC Cancer Agency). Those findings considered to be of clinical and scientific interest are then re-tested on the validation set. A separate researcher who did not participate in the training set analysis performs the re-testing on the validation set. Our statistical approach is intended to minimize false positive results, particularly with subgroup analysis.\nTable\u00a02Summary of clinical-pathological characteristics of the 4,444 breast cancer patientsTest setValidation setn% within group% within known valuesn% within group% within known valuesTotal2,2222,222Age at diagnosis (years)Median (range)60 (25\u201395)60 (23\u201391)<401707.77.71577.17.140\u20135445720.620.646821.121.155\u20136970231.631.680936.436.4\u22657089340.240.278835.535.5GenderFemale2,21199.599.52,21099.599.5Male110.50.5100.50.5Nodal statusNegative1,27257.257.41,25656.556.8Positive94342.442.695743.143.2Unknown70.390.4Number of positive nodesMedian (range)2 (1\u201324)2 (1\u201328)1\u2013358662.164.361464.266.24\u2013924325.826.722923.924.7\u226510828.79.0848.89.1Unknown323.4303.1ER Status at diagnosisNegative47221.221.847221.221.9Positive1,68976.078.21,68775.978.1Unknown612.7632.8Tumor size (cm)Median (range)2.0 (0.1\u20139.9)2.0 (0.1\u20139.9)0.1\u20131.026511.912.128512.813.01.1\u20132.086438.939.486438.939.52.1\u20135.094942.743.291541.241.8>5.01175.35.31265.75.8Unknown271.2321.4Tumor grade11255.66.01034.64.9287639.442.084438.040.231,08348.752.01,15351.954.9Unknown1386.21225.5\nStatistical analysis was performed using SPSS 14.0. In univariate analysis, breast-cancer specific survival (BCSS) was estimated using Kaplan\u2013Meier (KM) curves, and significant differences determined by Log rank tests. For BCSS, survival time was censored at the date of death if the cause was not breast cancer or at the date of last follow-up if the patient was still alive at the end of the study period. 6 patients with unknown cause of death were excluded from BCSS analysis. Cox proportional hazards models were used to calculate adjusted hazard ratios accounting for covariates. Kendall\u2019s tau-b and the Mann-Whitney tests were used to measure the correlation of c-kit status to pathological variables. All statistical tests were two-sided. The Bonferroni correction for multiple comparisons was applied during validation set analysis, and the alpha value for each comparison is 0.05\/n, where n is the total number of comparisons.\nX-tile analysis\nWe also used X-tile software [20] to find the optimal cut-off point for the total number of MCs that will predict prognosis in breast cancer patients. X-tile program split the cohort randomly into a matched training and validation set as a method for selecting optimal cut-points. It than calculated a P value for every possible division of the cohort expression data. A two-dimensional graph with its corresponding survival curves was plotted where each colored pixel was proportional to is \u03c72 Value. The program automatically calculated the maximum \u03c72 value which served as a cut-point to separate the number of MCs that predicted prognosis.\nResults\nOut of 4,620 cases on the TMAs, we selected 4,444 breast cancers that showed invasive tumor in the cores. The clinico-pathologic characteristics of patients included in the study are depicted in Table\u00a02. The total number of stained MCs was recorded as a continuous variable with counts ranging from 0\u00a0to 24\u00a0MCs per core. They were seen as 4\u201320\u00a0\u03bcm round to oval mononuclear cells with granular cytoplasm and single oval nucleus. The cytoplasmic granules were ganglion-, net-, or crystal-shaped (Fig.\u00a01).\nFig.\u00a01TMA core showing stromal mast cells stained with c-kit (CD-117). Magnification, 20\u00d7. MCs are seen as brown, granular stained oval, spindle or polygonal cells\nTraining set results\nSurvival analysis\nA total of 2,222 patients were included in the training set analysis. After excluding cases which had insufficient invasive tumor, missing core or un-interpretable staining pattern, 1,801 cases were carried forward for the analysis. Out of these, MCs were present in 508 (28.2%) cases. The mean survival time of patients with presence of stromal MCs was 15.0\u00a0years (95% CI, 14.5\u201315.5) compared to 13.9\u00a0years (95% CI, 13.5\u201314.2) for those who did not have positively stained MCs in their tumor stroma. KM survival analysis (Fig.\u00a02a) showed that the presence of stromal MCs was a favourable prognostic marker in the entire training set (BCSS @ 18.4\u00a0years, Log rank [Mantel Cox], P\u00a0=\u00a00.001).\nFig.\u00a02KM survival curve for all patients in training set (a) and validation set (b) with presence of stromal mast cells\nCorrelation with other biomarkers\nThere was positive correlation between MCs and ER (Kendall\u2019s tau-b [\u03c4b], 0.034, P\u00a0=\u00a00.148), Bcl2 (\u03c4b\u00a0=\u00a00.077, P\u00a0=\u00a00.002), and Her2 (\u03c4b\u00a0=\u00a00.049, P\u00a0=\u00a00.052), and negative correlation between MCs and EGFR (\u03c4b\u00a0=\u00a0\u22120.029, P\u00a0=\u00a00.228) and CK5\/6 (\u03c4b\u00a0=\u00a0\u22120.003, P\u00a0=\u00a00.906) in the training set analysis (Table\u00a03). As these correlations were either not significant or extremely weak, they were not carried forward to the validation set for further analysis.\nTable\u00a03Correlations between mast cells and other biomarkersTraining setMast cellsERKendall\u2019s tau-b0.034Significance (2-tailed)0.148N1,788EGFRKendall\u2019s tau-b\u22120.029Significance (2-tailed)0.228N1,646Her2Kendall\u2019s tau-b0.049Significance (2-tailed)0.052N1,746CK5\/6Kendall\u2019s tau-b\u22120.003Significance (2-tailed)0.906N1,624Bcl2Kendall\u2019s tau-b0.077Significance (2-tailed)0.002N1,616N\u2014scorable for both markersAll scores binarized as detailed in the text\nNodal status \nKM survival analysis showed no statistically significant difference in the survival between tumors with and without MCs in node-negative (BCSS @ 18.1\u00a0years, Log rank [Mantel Cox], P\u00a0=\u00a00.1199) and a significant difference in the node-positive group (BCSS @ 18.3\u00a0years, Log rank [Mantel Cox], P\u00a0=\u00a00.0140). Hence, this result was also not carried forward to the validation set.\nMultivariate analysis\nCox proportional hazard model was used to carry out the multivariate analysis and included age, tumor grade, tumor size, nodal status, ER and Her2 as independent predictors of BCSS. All the above variables achieved statistical significance as shown in Table\u00a04(a). Presence of MCs achieved statistical significance (P\u00a0=\u00a00.041) with a HR\u00a0=\u00a00.804, 95% CI 0.653\u20130.991.\nTable\u00a04Cox proportional hazard regression analysis showing hazard ratios and P-values in patients with invasive breast carcinomaSignificanceHRBCSS 95% CI for HRLowerUpper(a) Training setMast cells0.0410.8040.6530.991ER0.0180.7770.6310.957Her20.0031.4391.1291.834Size of the lesion\u00a0\u00a0\u00a0\u00a02\u20135\u00a0cm vs. \u22642\u00a0cm0.0001.8841.5472.295\u00a0\u00a0\u00a0\u00a0>5\u00a0cm vs. \u22642\u00a0cm0.0011.9311.3302.803Grade*\u00a0\u00a0\u00a0\u00a0Grade 1, 2 vs. Grade 3 0.0011.3901.1391.696Nodal status0.0002.3801.9652.882Age\u00a0\u00a0\u00a0\u00a040\u201349 vs. <400.0000.5670.4200.765\u00a0\u00a0\u00a0\u00a050\u201365 vs. <400.0130.6890.5130.924\u00a0\u00a0\u00a0\u00a0>65 vs. <400.0190.6770.4890.938(b) Validation setMast cells0.1280.8460.6831.049ER0.02890.7930 .6440 .976Her20.00181.4631.1521.859Size of the lesion\u00a0\u00a0\u00a0\u00a02\u22125\u00a0cm vs. \u22642\u00a0cm3.07\u00a0\u00d7\u00a010\u221241.4391.1811.753\u00a0\u00a0\u00a0\u00a0>5\u00a0cm vs. \u22642\u00a0cm1.66\u00a0\u00d7\u00a010\u221262.2581.6183.151Grade*\u00a0\u00a0\u00a0\u00a0Grade 1, 2 vs. Grade 3 1.96\u00a0\u00d7\u00a010\u221261.6581.3462.042Nodal status7.06\u00a0\u00d7\u00a010\u2212192.4101.9842.927Age\u00a0\u00a0\u00a0\u00a040\u201349 vs. <400.4321.1580.8031.671\u00a0\u00a0\u00a0\u00a050\u201365 vs. <400.6341.0900.7651.552\u00a0\u00a0\u00a0\u00a0>65 vs. <400.071.3940.9731.998*\u00a0Grade 1\u2014well differentiatedGrade 2\u2014moderately differentiatedGrade 3\u2014poorly differentiatedHR\u00a0=\u00a0adjusted hazard ratio, CI\u00a0=\u00a0confidence interval and BCSS\u00a0=\u00a0Breast Cancer Specific Survival\nValidation set results\nSurvival analysis\nThis group included the remaining 2,222 patients from the whole cohort. The mean age at diagnosis was 60\u00a0years and the median follow-up was 12.4\u00a0years. The median tumor size was 2.0\u00a0cm. 50% of patients had Grade 3 tumors, 43% were node positive, and 76% were ER positive. After excluding cases that had insufficient invasive tumor, missing core or un-interpretable staining pattern, 1,796 cases were carried forward for the analysis. Out of these, MCs were present in 494 (27.5%) cases. KM survival analysis (Fig.\u00a02b) showed that the presence of stromal MCs was a favourable prognostic marker in the validation set (P\u00a0=\u00a00.006).\nMultivariate analysis\nCox proportional hazard model was used to carry out the multivariate analysis and included age, tumor grade, tumor size, nodal status, ER and Her2 as independent predictors of BCSS. All the above variables achieved statistical significance as shown in Table\u00a04(b). Presence of MCs did not achieve statistical significance (P\u00a0=\u00a00.128) but retained a similar HR\u00a0=\u00a00.846, 95% CI 0.683\u20131.049 as in the training set analysis.\nX-tile analysis results\nWe assessed the association between patient outcome and number of positively stained MCs in the stroma of tumors using X-tile software. This software allowed us to define an optimal cut-off point that defined the number of MCs needed to predict good prognosis in the cancer patients. The program divided the population into low and high-risk groups based on the number of MCs detected. It converted continuous data into ordinal classes for statistical analysis. The X-tile plot showed that breast cancer patients with any number of stromal MCs had better prognosis compared to those who have no MC infiltration in their stroma. MCs ranging between 1 and 22 in the tumor stroma were present in 507 (27.61%) patients and were grouped together in the low population group of the training set. The high population group comprised of 1,329 (72.39%) patients in the training set. It reiterated the findings obtained by KM survival analysis that the presence of any number of stromal MCs predicts good prognosis in invasive breast cancer patients.\nDiscussion\nThe importance of the reciprocal relationship between tumor and stroma is being increasingly recognized [21], and is the role of stromal inflammatory cells like MCs, macrophages, fibroblasts and T cell subtypes in cancer initiation and progression. cDNA microarray analysis has shown that genes expressed by stromal cells correlate with differences in the biology of the tumors and are prognostic predictors in breast cancer [22].\nThere is an ongoing debate about possible detrimental or beneficial effects of MC accumulation in the stroma of solid tumors. In-vitro studies have shown that MC inhibits tumor growth [23]. This is in agreement with other studies showing increase in MC count in early stage of non-small cell lung cancer and thereby supporting their anti-tumor role. [3]. Increased islet\/stromal MC-ratio and presence of tumor islet MCs were shown to be independent good prognostic indicators in non-small-cell lung cancer [24]. Recent studies in ovarian cancer [13] and colorectal cancer [15] have also confirmed their correlation with prognosis.\nMCs secrete factors like heparin, interleukin-8 (IL-8) and vascular endothelial growth factor that promote neovascularization; histamine which suppresses immune response; platelet-derived growth factor, nerve growth factor and stem-cell factors which are mitogenic; and proteases that promote metastasis [9]. On the other hand, MCs can inhibit tumor growth by releasing endogenous peroxidase that is cytotoxic to mammalian tumor cells [25], by natural cytotoxicity [21] and by recruiting neutrophils, eosinophils, lymphocytes and macrophages [26]. MCs can secrete protective substances without degranulation in the presence of tumor-derived blockers like oxidized polyamines and thus be detrimental to the tumor [27]. They can destroy tumor cell surface structures directly and indirectly in a fashion similar to the effect of arginase [28]. MCs can also inhibit tumor growth by secreting beneficial cytokines like IL-1, IL-4, IL-6, and tumor necrosis factor-\u03b1 that induce apoptosis of endothelial cells [29]; and chondroitin sulphate which inhibits metastases [30]. Cathepsin G secreted by MC activates platelets, lymphocytes and macrophages, and is known to be cytotoxic to some mammalian cells [31]. Its inhibition by a tumor cell product was implicated in the progression of advanced squamous cell tumors [32] suggesting its cytotoxic capability against target cells. MCs secrete chymase, which stimulates apoptosis in different target cells [33] and inhibits angiogenesis by exerting cytotoxic effect on vascular smooth muscles [34]. MC tryptase stimulates inflammation and recruits fibroblasts leading to tumor fibrosis, and thereby limiting tumor growth and metastasis [35]. Tryptase, however, is also implicated in angiogenesis and its effect correlated with a better response to chemotherapy in ovarian cancer. This suggests that MC tryptase-mediated fibrosis and angiogenesis may be responsible for the good prognostic effect seen in advanced ovarian cancer [13].\nIn this TMA study, we showed that the presence of even one or more MCs in the tumor microenvironment was sufficient to exert a positive prognostic effect. Our finding supports the idea of assessing inflammatory cell infiltrates as prognostic markers in cancer. In our previously published pilot report, we had observed no correlation between the presence of stromal MCs with either B-cells (CD20-positive) or T-cells (CD3-positive) [11], and hence we did not stain for these inflammatory cells in this study. We also did not find a statistically significant prognostic effects of MCs in the node-negative group in the training set analysis. This differs from our earlier published report [11] on a 348 case series which concluded that the presence of MCs was a favourable prognostic factor in the node-negative patients (P\u00a0=\u00a00.018) but not the node-positive group (P\u00a0=\u00a00.384). This finding underscores the importance of validating clinically relevant findings on a larger series of patients and using the test-validation approach to arrive at a meaningful conclusion.\nA number of studies have elaborated on the role of immune cells in cancer etiopathogenesis. T regulatory (Treg) cells are known to be associated with poor outcome in ovarian cancer [36] and the initial results of a clinical trial aimed at specifically eliminating Treg cells has shown promising results [37]. Lymphoma-associated macrophages have been used to predict the outcome of follicular lymphoma [38] and reactive macrophages are used to predict breast, prostate, ovarian, and cervical cancer outcome [39]. Tumor-infiltrating neutrophils are used to predict outcome in patients with adenocarcinoma of the bronchioloalveolar carcinoma subtype [40] and gastric carcinoma [41]. In a recent report, it was concluded that the type, density, and location of immune cells within the tumor samples are a better predictor of patient survival than the histopathological methods currently used to stage colorectal cancer [42].\nIn conclusion, we confirm our earlier pilot study findings and confirm that stromal MCs correlate with a good prognosis in a large cohort of 4,444 invasive breast cancer patients with long-term follow-up. It highlights the critical role that the host stromal reaction, in particular the inflammatory cell infiltrate, plays in modulating cancer progression. MCs can be used as markers for risk stratification in invasive breast cancers.","keyphrases":["stroma","mast cells","breast cancer","inflammatory infiltrate"],"prmu":["P","P","P","R"]}
{"id":"Immunogenetics-3-1-1914236","title":"The SPINK gene family and celiac disease susceptibility\n","text":"The gene family of serine protease inhibitors of the Kazal type (SPINK) are functional and positional candidate genes for celiac disease (CD). Our aim was to assess the gut mucosal gene expression and genetic association of SPINK1, -2, -4, and -5 in the Dutch CD population. Gene expression was determined for all four SPINK genes by quantitative reverse-transcription polymerase chain reaction in duodenal biopsy samples from untreated (n = 15) and diet-treated patients (n = 31) and controls (n = 16). Genetic association of the four SPINK genes was tested within a total of 18 haplotype tagging SNPs, one coding SNP, 310 patients, and 180 controls. The SPINK4 study cohort was further expanded to include 479 CD cases and 540 controls. SPINK4 DNA sequence analysis was performed on six members of a multigeneration CD family to detect possible point mutations or deletions. SPINK4 showed differential gene expression, which was at its highest in untreated patients and dropped sharply upon commencement of a gluten-free diet. Genetic association tests for all four SPINK genes were negative, including SPINK4 in the extended case\/control cohort. No SPINK4 mutations or deletions were observed in the multigeneration CD family with linkage to chromosome 9p21-13 nor was the coding SNP disease-specific. SPINK4 exhibits CD pathology-related differential gene expression, likely derived from altered goblet cell activity. All of the four SPINK genes tested do not contribute to the genetic risk for CD in the Dutch population.\nIntroduction\nCeliac disease (CD) is a chronic inflammatory condition of the small intestine because of an immunological intolerance for the food protein gluten. Patients have to adhere to a life-long diet devoid of gluten to prevent the detrimental effects of a prolonged nutrient and mineral deficiency (Green and Jabri 2003). Susceptibility for CD is predominantly determined by genetic factors, and the complex inheritance patterns suggest the interaction of multiple genes (van Heel et al. 2005). It is well established that the adaptive immune response to gluten plays a pivotal role in the pathogenesis of CD. Th1 activation of CD4+ T cells follows gluten-peptide presentation by DQ2 or DQ8 molecules expressed on antigen-presenting cells (Sollid 2002). The HLA-DQA and -DQB gene variants coding for these molecules are the major genetic determinants for CD susceptibility (Koning et al. 2005). Recently, the importance of innate immunity in CD pathogenesis was also underscored by the observation of induced IL15 expression and NKT cell chemotaxis through the MICA and NKG2D molecules (Hue et al. 2004; Meresse et al. 2004). However, no genetic contribution of the cognate genes has been demonstrated. The notion of crosstalk between the adaptive and innate immune systems is not limited to CD and gets much attention in studies of the inflammatory process (Hoebe et al. 2004). This raises the question whether some aspects of innate immunity may contribute to the genetic susceptibility for CD. The innate immune system uses a wide array of defense mechanisms against the invasion of pathogens. These encompass the expression of pattern recognition receptors, release of antimicrobial molecules, and preservation of epithelial barrier and tissue integrity by, e.g., serine protease inhibitors (Kimbrell and Beutler 2001).\nOne branch of the family of serine protease inhibitors is that of the Kazal type (SPINK) that originally consisted of four members in humans (SPINK1, SPINK2, SPINK4, and SPINK5). Recently, as part of a cluster of SPINK genes on chromosome 5q32 that already included SPINK1 and SPINK5, five new SPINK(-like) members were identified that were located more distally: SPINK5L2, SPINK6, SPINK5L3, SPINK7, and SPINK9, respectively (NCBI Map Viewer, build 36.1). However, these new members lack functional annotation and were therefore not included in this study. SPINK family members 1, 2, and 4 have a comparable size and structure coded for by 4 exons with a single Kazal type serine protease inhibitor domain. SPINK5, in contrast, contains 33 exons that encode 15 inhibitory domains. All four SPINK members are thought to be involved in the protection against proteolytic degradation of epithelial and mucosal tissues, although their major site of expression may differ. SPINK1 is expressed in the pancreas and the gastrointestinal tract, and mutations in this gene are reported in various forms of pancreatitis (Pfutzer and Whitcomb 2001). SPINK2 (located on 4q12) is expressed in the testis, epididymis, and seminal vesicle, where its antimicrobial function may be involved in protection of fertility (Rockett et al. 2004). SPINK4 was originally isolated from pig intestine (Agerberth et al. 1989) and is abundantly expressed in human and porcine goblet cells in the crypts of Lieberk\u00fchn but was also found in monocytes and in the central nervous system (Metsis et al. 1992; Norberg et al. 2003). SPINK5 is expressed in the thymus, vaginal epithelium, Bartolin\u2019s glands, oral mucosa, tonsils, and the parathyroid glands (Magert et al. 1999). Mutations in SPINK5 are responsible for the Netherton syndrome, a lethal skin disorder characterized by ichthyosis, hair shaft defects, atopy, skin barrier defects, and recurrent bacterial infections (Bitoun et al. 2002). Mouse models of the Netherton syndrome have shown enhanced proteolysis of desmoglein 1 and filaggrin in SPINK5 mutants (Descargues et al. 2005; Hewett et al. 2005). Moreover, SPINK5 has also been associated with asthma and atopic dermatitis (Blumenthal 2005).\nInterestingly, both SPINK1 and SPINK5 are located on chromosome 5q32. This region contains the CELIAC2 susceptibility locus that emerged repeatedly in linkage studies (Babron et al. 2003). Despite the fact that this region is rich in candidate cytokine genes and intense mapping efforts were made, no closely associated genes were identified (Ryan et al. 2005). Likewise, SPINK4 is located on chromosome 9p13.3 and resides within a linkage region (9p21-13) where we previously identified a novel CD locus that segregated within a four-generation Dutch family (van Belzen et al. 2004). Taken together, the role of SPINK genes in epithelial and mucosal protection and the important genetic locations of SPINK1, SPINK4, and SPINK5 prompted us to subject the four conventional members of the SPINK family to gene expression and genetic association analyses to ascertain their possible role in CD pathogenesis.\nMaterials and methods\nPatient material\nDuodenal biopsy samples were collected by endoscopy as part of a routine CD diagnostic procedure or to monitor the response to a gluten-free diet in previously diagnosed patients. All patients were classified using the Marsh nomenclature according to the UEGW criteria (Report of a working group of the United European Gastroenterology Week in Amsterdam 2001). Two biopsy samples taken in parallel to those used for histological examination were pooled and used for determination of gene expression. In total, 62 individuals were examined with quantitative reverse transcription polymerase chain reaction (qRT-PCR), of which 16 were normal controls, and 46 were CD patients (Fig.\u00a01). The patient group consisted of 15 untreated cases with villous atrophy (MIII) and 31 patients treated with a gluten-free diet who were in various stages of mucosal recovery: MII (crypt hyperplasia; n\u2009=\u200911), MI (lymphocyte infiltration; n\u2009=\u20098), and M0 (complete remission; n\u2009=\u200912). The biopsies of all participating patients were reevaluated by an experienced pathologist, and only CD patients with a proven original Marsh III lesion were included in this study. The genetic association study on all four SPINK genes was initially conducted on a cohort of 310 independent CD patients and 180 independent age- and sex-matched controls, all of which were from Dutch Caucasian decent. In a second stage, exclusively focused on all SPINK4 SNPs, we added 360 controls to a total of 540. In case of three SPINK4 variants with suggestive P values, the power of the study was further enhanced by adding 169 extra CD cases to a total of 479. In parallel, we also examined the SPINK4 gene in a previously described four-generation Dutch CD family (van Belzen et al. 2004). Six family members and a CEPH control were subjected to DNA sequence analysis of the SPINK4 coding regions and splice sites boundaries. Additionally, we performed SPINK4 SNP haplotype analysis in this family. All patients and family members that volunteered for this study signed an informed consent. The study was approved by the Medical Ethics Committee of the University Medical Center Utrecht.\nFig.\u00a01Results of qRT-PCR of SPINK genes in normal controls (NC) and CD patients, either untreated (MIII) or on a gluten-free diet (MII\u2013M0). The Marsh stages refer to the pathological conditions of the mucosa, characterized by atrophy of the villi (MIII); hyperplastic crypts between the villi (MIII\u2013MII); and enhanced lymphocyte infiltration (MIII\u2013MII\u2013MI). Stage M0 indicates complete remission comparable to controls. The genes tested were as follows: SPINK1 (a); SPINK2 (b); SPINK4 (c); and SPINK5 (d). Measurements were made in triplicate, on pools of separately prepared cDNA samples. Expression data were normalized to the normal control pool.(e) Relative expression of all four SPINK genes with respect to SPINK2 in the healthy duodenal mucosa. Note the logarithmic scale here. The GUSB gene was used as an endogenous control in all tests. Errorbars indicate standard deviations\nExpression study\nThe isolation of total RNA from biopsy samples and the analysis of gene expression by real-time qRT-PCR on an ABI Prism 7900HT was performed as described before (Wapenaar et al. 2004). We used the commercially available Assay-on-Demand test for SPINK1 (Hs00162154_m1), SPINK2 (Hs00221653_m1), SPINK4 (Hs00205508_m1), SPINK5 (Hs00199260), and the endogenous control gene GUSB (PDAR 4326320E; Applied Biosystems, Foster City, CA). All samples were tested in triplicate on pooled cDNA samples representing each Marsh class. The results were confirmed with cDNA from individual samples tested in duplicate. Relative levels of gene expression were obtained using the SDS2.1 software (Applied Biosystems).\nGenetic association and data analysis\nHaplotype tagging SNPs were selected for SPINK1, SPINK2, SPINK4, and SPINK5 based on HapMap (Phase I) data using Haploview (Barrett et al. 2005). For each haploblock containing SNPs in high linkage disequilibrium, one or more representative SNPs were selected that should capture the genetic variation within that block. For the four SPINK genes tested, this resulted in a set of 18 haplotype tagging SNPs and one coding SNP (see Table\u00a01 and Fig.\u00a02). SNP assays were obtained from Applied Biosystems and analyzed on an ABI Prism 7900HT. Hardy\u2013Weinberg Equilibrium (HWE) was evaluated separately in cases and controls for all SNPs tested. Allele frequencies were compared between cases and controls, and P values were obtained by \u03c72 analysis. \nTable\u00a01Allelic distribution of SPINK haplotype tagging SNPs in a Dutch CD case-control cohort\u00a0Cases (n\u2009=\u2009310)Controls (n\u2009=\u2009180)\u00a0\u00a0AlleleAllele countsAllele frequency (%)Allele countsAllele frequency (%)Gene nameSNP i.d.Position*Minor\/majorMinorMajorMinorMajorMinorMajorMinorMajor\u03c72P valueSPINK1rs10515593147,178,993A\/G11944921797626822.177.90.1660.6834SPINK1rs3777125147,184,010C\/G22833640.459.615020042.957.10.5270.4681SPINK1rs4705204147,195,313C\/A12542922.677.46027617.982.12.8130.0935SPINK1rs891992147,205,707G\/A7748313.886.24730313.486.60.0190.8906SPINK2rs1001563057,520,070A\/G21632839.760.313621638.661.40.1020.7489SPINK2rs78154257,528,232G\/A22039036.163.912322135.864.20.0090.9237SPINK4rs56335333,206,428G\/C12047220.379.76727919.480.60.1120.7375SPINK4rs56351233,206,484G\/A505568.391.73631010.489.61.2430.2648SPINK4rs89167133,222,271G\/T7054011.588.55230414.685.41.9980.1575SPINK4rs706107**33,230,225A\/G11749919818126923.176.92.3580.1246SPINK4rs70610933,230,668A\/G11848619.580.58227622.977.11.5490.2133SPINK4rs70611533,243,605G\/C7753712.587.55230814.485.60.7160.3975SPINK5rs3756688147,422,972G\/A23238037.962.113522137.962.10.0000.9968SPINK5rs4472254147,433,830A\/C240376396114221439.960.10.0810.7757SPINK5rs4519913147,452,004G\/A27933145.754.316819246.753.30.0790.7792SPINK5rs1422987147,466,552T\/C495598.191.9273297.692.40.0700.7917SPINK5rs3815740147,471,386G\/A385746.293.8213375.994.10.0470.8291SPINK5rs2052532147,476,500G\/A20041432.667.411724132.767.30.0010.9723SPINK5rs3764930147,485,309G\/A19941332.567.511724332.567.50.0000.9958*Basepair position according to NCBI build 35.1**Coding SNP, nonsynonymous change (Val7Ile)Fig.\u00a02Genomic organization of the four SPINK genes. The upperhorizontalline indicates exon locations (verticalbars) and SNP positions (numberedasterisks). The SNPs are numbered for each gene consecutively as they appear in Table\u00a01. SPINK4 SNP no. 4 represents the nonsynonymous (Val7Ile) coding SNP rs706107. The arrowpoints indicate the orientation of transcription. The lowerportion of the figure shows the pairwise linkage-disequilibrium structure between indicated SNPs given by D\u2032 statistics based on the European population in the HapMap database (Phase II). Darkerred intensities indicate higher D\u2032 values (numbers indicate D\u2019 value, whereas SNP pairs without number have a D\u2032\u2009=\u20091)\nDNA sequence analysis\nDNA sequence analysis was performed on SPINK4 in six members of a four-generation Dutch CD family and one CEPH control (family, 1,331; individual, 2). Of these six family members, four were affected (index 02, 08, 32, and 41) and carried the disease-linked haplotype, and two were nonaffected (index 21 and 31) without this haplotype (Fig.\u00a03). All coding sequences of the SPINK4 gene were PCR-amplified, including the intron\u2013exon boundaries (for primers and protocols, see supplementary data Table\u00a01). PCR products were examined on a 2% agarose gel and purified with a Millipore Vacuum Manifold (Billerica, MA). Samples were prepared with the BigDye terminator cycle sequencing ready kit (Applied Biosystems) according to the manufacturer\u2019s protocol. PCR and sequencing amplification were performed on a GeneAmp PCR system 9700 (Perkin Elmer, Foster City, CA). Sequences were run on an ABI Prism 3730 analyzer (Applied Biosystems). Analysis and sequence alignment was carried out with Sequence Navigator (Applied Biosystems) and Vector NTI (InforMax, Massachusetts) software packages.\nFig.\u00a03Pedigree of the Dutch multigeneration CD family. Only affected descendents are depicted (10 out of 13 siblings in the second generation were affected). The grandparental SPINK4 haplotypes that are boxed and shaded are identical to the grandmaternal at-risk haplotype (noninformative). The SNPs are ordered (top-to-bottom) as they appear in Table\u00a01. Genotype numbers 1, 2, 3, and 4 refer to A, C, G, and T alleles, respectively. Sequence analysis was performed on family members 02, 08, 21, 31, 32, and 41. Family member index numbers are indicated in bold\nResults\nSPINK gene expression in the CD mucosa\nThe expression of all four conventional members of the human SPINK family was determined by real-time qRT-PCR on duodenal biopsy-derived cDNA pools from normal controls and CD patients, either untreated or in various stages of remission on a gluten-free diet. The results shown in Fig.\u00a01 indicate that only SPINK4 (Fig.\u00a01c) is differentially expressed, and that its transcriptional activity, which is at its highest in Marsh III (20-fold compared to controls), decreases sharply (fourfold) when patients improve and make a transition to Marsh II. To preclude that the results for SPINK4 might be biased by fortuitous differences in individual expression levels within the generated pools, we also examined the control and case samples each separately. This did not change the observed drop in SPINK4 expression during tissue recovery (see supplementary Fig.\u00a01). Likewise, we performed the same analysis for the other three SPINK genes without affecting the profile already observed in the pools (results not shown). We also examined the relative expression of the four SPINK genes with respect to each other in the normal intestinal mucosa. This showed that both SPINK1 and SPINK4 have the highest expression, which is respectively 480-fold and 240-fold higher compared to SPINK2, whereas SPINK5 is in the same order of magnitude (fivefold) as SPINK2 (Fig.\u00a01e). In conclusion, only the SPINK4 gene appears to be differentially regulated in the intestinal mucosa during recovery from the gluten-evoked CD lesion. This observation prompted us to examine whether SPINK4, or any of the other SPINK genes, could also be causally related to the CD pathogenesis.\nGenetic association analysis of SPINK genes\nWe designed a haplotype tagging SNP strategy to capture all genetic variation in SPINK1, -2, -4, and -5. An overview of these four SPINK genes with their genomic organization, linkage-disequilibrium structure, and the position of the haplotype tagging SNPs used is depicted in Fig.\u00a02. Initially, these haplotype tagging SNPs were tested in 310\u00a0CD cases and 180 controls (Table\u00a01) and showed no significant association for any of the haplotype tagging SNPs in the four SPINK genes. Despite the initial negative result, we decided to pursue SPINK4 further because it is expressed in goblet cells (Metsis et al. 1992), displayed a CD pathology-related differential expression in the intestinal mucosa, and mapped within a CD linkage region (van Belzen et al. 2004). Initially, we expanded the control group with 360 samples to a total of 540 for all SPINK4 SNPs tested. As a result, the Val7Ile coding variant rs706107 and its flanking haplotype tagging SNPs rs891671 and rs706109 yielded suggestive but nonsignificant P values of 0.0595, 0.0510, and 0.1122, respectively (data not shown). To increase the power of the study even further, we subsequently added 169\u00a0CD cases to a total of 479. The effect on the P values of the three SNPs tested was such that they dropped below the significance threshold (see Table\u00a02). From this, we conclude that the four SPINK genes tested do not contribute to the genetic susceptibility in the Dutch CD population. \nTable\u00a02Allelic distribution of three selected SPINK4 SNPs in the extended Dutch CD case-control cohort\u00a0Cases (n\u2009=\u2009479)Controls (n\u2009=\u2009540)\u00a0\u00a0AlleleAllele countsAllele frequency (%)Allele countsAllele frequency (%)Gene nameSNP i.d.Position*Minor\/majorMinorMajorMinorMajorMinorMajorMinorMajor\u03c72P valueSPINK4rs89167133,222,271G\/T12681413.486.615890414.985.10.8890.3457SPINK4rs706107**33,230,225A\/G20573721.878.224281422.977.10.3820.5365SPINK4rs70610933,230,668A\/G21272422.677.424281622.977.10.0140.9053*Basepair position according to NCBI build 35.1**Coding SNP, nonsynonymous change (Val7Ile)\nSPINK4 sequence analysis in a multigeneration family\nWe have previously described a four-generation CD family with an extraordinary high incidence of affected individuals (see Fig.\u00a03). The disease segregated with a grandmaternal haplotype on chromosome 9p21-13 (van Belzen et al. 2004), a region that encompasses SPINK4. The apparent dominant inheritance pattern could be caused by a mutation that is rare in the general CD population but present with a high phenotypic penetration in this specific family. To assess if any functional variants of the SPINK4 gene were present in this family, we sequenced all its exons and intron\u2013exon boundaries in six family members. However, we did not observe mutations in any of the samples tested (results not shown). Neither was the exon 1 coding SNP rs706107 specific for affected individuals as all seven individuals tested (including the CEPH control) carried the most frequent GG genotype (Fig.\u00a03). To exclude the possibility of deletions in SPINK4 to be misinterpreted from the sequence data as homozygous genotypes, we also performed segregation analysis of the grandparental SPINK4 haplotypes within the entire family but observed no suspect inheritance pattern (Fig.\u00a03). In conclusion, we have found no evidence that SPINK4 is a candidate gene for the chromosome 9p21-13 CD locus in the Dutch population in general or in the multigeneration Dutch CD family specifically.\nDiscussion\nChronic inflammatory conditions and autoimmune disorders are typically characterized by a deregulated adaptive and innate immune system. The innate defense consists of multiple components that include physical barriers, antimicrobial molecules, pattern recognition receptors, circulating phagocytes, and the complement system (Hoebe et al. 2004). A breach of the epithelial barrier and loss of microbial containment is often the first of a series of events that trigger or sustain chronic inflammatory diseases (Tlaskalova-Hogenova et al. 2004) as described, e.g., in Crohn\u2019s disease, atopic eczema, asthma, and psoriasis (Schreiber et al. 2005). In CD, the gut\u2013lumen separation is undermined by dietary gluten that evokes a combined innate and adaptive immune response (Londei et al. 2005). It is the joined action of gluten peptides, environmental factors, and genetic determinants that precipitates this enteropathy. The human leukocyte antigen locus is the major genetic contribution to the adaptive Th1 reaction (Koning et al. 2005). Recently, we identified MYO9B as a susceptibility gene in the Dutch population that possibly has an effect on epithelial barrier integrity (Monsuur et al. 2005). Several other studies have underscored the involvement of innate immunity in CD, however, without identification of underlying causative gene variants (Londei et al. 2005). Interestingly, it was also reported that the epithelial glycocalyx and the bacterial composition in the CD gut is distinct (Forsberg et al. 2004; Tjellstrom et al. 2005).\nIn search of genes that may have a primary contribution to CD pathogenesis, we focused our attention to the SPINK family of serine protease inhibitors that play an important role in tissue preservation through the containment of uncontrolled proteolysis and bacterial growth. In this study, we demonstrated differential gene expression of mucosal SPINK4 in CD. Crypt hyperplasia is a feature of the Marsh III and Marsh II stages of CD, and the concomitant increase in the number of goblet cells may contribute to the increased SPINK4 expression. However, the observed sharp decrease in gene expression sets in during the MIII\/MII transition, whereas crypt normalization is observed only later at the MII\/MI recovery phase. This suggests that SPINK4 downregulation sets in soon after commencement of the gluten-free diet. This SPINK4 differential expression probably reflects altered goblet cell activity, but its functional significance and regulatory mechanism in CD pathology remains to be established.\nThe combination of functional relevance and mapping to CD linkage intervals pointed to the SPINK family members as attractive functional and positional candidate genes. We have chosen a robust strategy for genetic association testing based on haplotype tagging SNPs and linkage-disequilibrium structure of the SPINK loci applied to a considerably sized Dutch case-control cohort. With our study design, we had 75% power to confirm association with SPINK1, -2, and -5 (relative risk 2.0; allele frequency 0.1\u20130.45; 95% confidence interval), whereas this was even 95% (RR 2.0) and 80% (RR 1.6) for SPINK4. These power estimates reflect a Type I error rate of 0.05, which is appropriate for testing a previously reported result. Initial detection of a new genetic association would require much more stringent criteria to assure reproducibility, and power would be correspondingly less.\nIn parallel, we examined the extended Dutch CD family for variants and deletions in SPINK4. We hypothesized that a specific SPINK4 mutation, although rare in the general population, could have a dramatic impact on mucus composition, bacterial containment, and gluten sensitivity, thereby explaining the apparent dominant and high penetration inheritance pattern in our extended CD family. With both approaches, we were not able to establish a genetic involvement of the SPINK genes tested. However, we cannot completely rule out the possibility of a rare noncoding mutation in SPINK4 (outside the splice donor and acceptor regions) that might specifically segregate in this atypical CD family, characterized by an exceptional high prevalence of affected members.\nDespite this negative result in the Dutch CD population, we cannot formally rule out the possibility of genetic contribution of SPINK genes to CD in other European populations like the Italian in whom, unlike the Dutch (van Belzen et al. 2003), chromosome 5q linkage was established (Greco et al. 1998; Percopo et al. 2003). Genuine population heterogeneity has been reported before, e.g., between CARD15\/NOD2 and Crohn\u2019s disease (Lesage et al. 2002; Croucher et al. 2003) and between SPINK5 and asthma (Blumenthal 2005; Jongepier et al. 2005). The new SPINK members on chromosome 5q (SPINK5L2, SPINK6, SPINK5L3, SPINK7, and SPINK9) were not part of this study. Currently, no functional annotation is available for these genes that are located near SPINK1 and SPINK5 in a chromosomal region that appears to have been subjected to gene duplication during evolution. Therefore, we cannot exclude their possible involvement in CD or any other inflammatory disorder.\nElectronic Supplementary Material\nBelow is the link to the electronic supplementary material.\nSupplementary Fig.\u00a0S1\n(GIF 14.0\u00a0kb)\nSupplementary Table\u00a01\nPrimers and reaction conditions used for SPINK4 DNA sequence analysis. (PDF 80.0\u00a0KB)","keyphrases":["spink genes","celiac disease","genetic association","quantitative reverse-transcription polymerase chain reaction"],"prmu":["P","P","P","P"]}
{"id":"Appl_Psychophysiol_Biofeedback-4-1-2259255","title":"EEG Biofeedback as a Treatment for Substance Use Disorders: Review, Rating of Efficacy, and Recommendations for Further Research\n","text":"Electroencephalographic (EEG) biofeedback has been employed in substance use disorder (SUD) over the last three decades. The SUD is a complex series of disorders with frequent comorbidities and EEG abnormalities of several types. EEG biofeedback has been employed in conjunction with other therapies and may be useful in enhancing certain outcomes of therapy. Based on published clinical studies and employing efficacy criteria adapted by the Association for Applied Psychophysiology and Biofeedback and the International Society for Neurofeedback and Research, alpha theta training\u2014either alone for alcoholism or in combination with beta training for stimulant and mixed substance abuse and combined with residential treatment programs, is probably efficacious. Considerations of further research design taking these factors into account are discussed and descriptions of contemporary research are given.\nIntroduction\nSubstance use disorders (SUD) include disorders related to the taking of a drug of abuse (including alcohol), and represent the most common psychiatric conditions (APA 2000) resulting in serious impairments in cognition and behavior. Acute and chronic drug abuse results in significant alteration of the brain activity detectable with quantitative electroencephalography (qEEG) methods. The treatment of addictive disorders by electroencephalographic (EEG) biofeedback (or neurofeedback, as it is often called) was first popularized by the work of Eugene Peniston (Peniston and Kulkosky 1989, 1990, 1991) and became popularly known as the Peniston Protocol. This approach employed independent auditory feedback of two slow brain wave frequencies, alpha (8\u201313\u00a0Hz) and theta (4\u20138\u00a0Hz) in an eyes closed condition to produce a hypnagogic state. The patient was taught prior to neurofeedback to use what amounts to success imagery (beingsober, refusing offers of alcohol, living confidently, and happy) as they drifted down into an alpha-theta state. Repeated sessions reportedly resulted in long-term abstinence and changes in personality testing. Because the method seemed to work well for alcoholics, it has been tried in subjects with cannabis dependence and stimulant dependence\u2014but with limited success until the work of Scott and Kaiser (Scott and Kaiser 1998; Scott et\u00a0al. 2002, 2005). They described treating stimulant abusing subjects with attention-deficit type EEG biofeedback protocols, followed by the Peniston Protocol, with substantial improvement in program retention and long-term abstinence rates. This approach has become known widely as the Scott\u2013Kaiser modification (of the Peniston Protocol).\nThis \u201cwhite paper\u201d on EEG biofeedback for SUD will offer an assessment of efficacy according to the guidelines jointly established by the Association for Applied Psychophysiology and Biofeedback (AAPB) and the International Society for Neurofeedback and Research (ISNR). Assessing the efficacy of neurofeedback for SUD involves several considerations. The first of these involves difficulties assessing the efficacy of any treatment method for SUD. Outcome benchmarks (i.e., total abstinence, improved function and quality of life) and time points of outcome (i.e., one year, two years post treatment) are not clearly established.\nOutcome assessment for treatment of SUD in itself is a complex topic well beyond the scope of this article. Because different drugs of abuse are associated with different patterns of EEG abnormality, as will be discussed in detail in this article, it is difficult to assign broad-brush EEG biofeedback solutions to SUD as a whole. Any statements of efficacy will need to describe specific EEG biofeedback protocols for specific substances of abuse. Furthermore substance abuse is often mixed substance type and comorbid conditions are common and vary from subject to subject, as will also be borne out in this article. As of yet there are no gold standard medication or other treatments for the various types of SUD and efficacy of any SUD treatment method likely falls into the \u201cpossibly effective\u201d to \u201cprobably effective\u201d range according to the efficacy guidelines jointly established by the AAPB and ISNR. Finally, all of the studies of EEG biofeedback in SUD to date employ EEG biofeedback as an add on to cognitive behavioral or twelve step treatment regimes, so any statements of efficacy would have to acknowledge that EEG biofeedback is not a stand alone treatment for SUD.\nThis article is divided into several sections. In the first section after \u201cIntroduction,\u201d we review SUD prevalence and describe qEEG changes typical for the most widespread drugs of abuse (alcohol, marijuana, heroin, cocaine, and methamphetamine). The second section describes treatment studies employing EEG biofeedback in SUD. Studies that have used the Peniston Protocol are described first, along with critical commentaries of these studies. In the second part of this section, a description of the Scott\u2013Kaiser modification is given, along with some discussion of a rationale for why this approach may be more successful with stimulant abusers. This section also describes some current research. The third section assesses efficacy of the Peniston Protocol and the Scott\u2013Kaiser modification. The fourth section takes a look at the clinical implications of comorbidities in neurobiofeedback treatment of alcohol and drug abuse. The fifth section discusses the clinical implications of standard cognitive-behavioral therapies in SUD treatment and reviews the rationale for the application of qEEG-guided neurofeedback intervention in SUD in conjunction with these therapies. The final section summarizes findings in qEEG and neurofeedback in SUD and additionally proposes further directions for clinical research in this area.\nThis article represents an update of earlier reviews (Trudeau 2000, 2005a, b) of EEG biofeedback for addictive disorders extended with a review on qEEG in SUD. This review is presented as one of a series of papers in both The Journal of Neurotherapy and The Journal of Applied Psychophysiology & Biofeedback describing and reviewing biofeedback applications for adult populations. No attempt will be made to review the fields of qEEG and neurobiofeedback generally (see current reviews by Hammond 2006; Kaiser 2006), or the field of addictive disorders generally, although some references will be made to specifics the authors feel are pertinent to a discussion of emerging concepts of qEEG as a sensitive tool for the brain function assessment in SUD, and EEG biofeedback as a treatment approach for SUD.\nSUD Prevalence and qEEG Changes\nDrug addiction can be described as a mental disorder with idiosyncratic behavioral, cognitive, and psychosocial features. The SUD commonly referred to as \u201cdrug addiction\u201d is characterized by physiological dependence accompanied by the withdrawal syndrome on discontinuance of the drug use, psychological dependence with craving, the pathological motivational state that leads to the active drug-seeking behavior, and tolerance, expressed in the escalation of the dose needed to achieve a desired euphoric state. Drug addiction is a chronic, relapsing mental disease that results from the prolonged effects of drugs on the brain (Dackis and O\u2019Brain 2001; Volkow et\u00a0al. 2003, 2004). Drug addiction can take control of the brain and behavior by activating and reinforcing behavioral patterns that are excessively directed to compulsive drug use (Di\u00a0Chiara 1999; Gerdeman et\u00a0al. 2003).\nFrom the 11 classes of substances listed in the DSM-IV we will discuss in our review only alcohol, cannabis (marijuana), heroin, and such psychostimulants as cocaine and methamphetamine. Addiction leads to behavioral, cognitive, and social adverse outcomes that incur substantial costs to society. In 2002, it was estimated from the Substance Abuse and Mental Health Service Administration (SAMHSA 2004) that 22\u00a0million Americans have a substance abuse or dependence disorder, and 2\u00a0million of them were current cocaine users (Vocci and Ling 2005). In 2005, there were 2.4\u00a0million persons who were current cocaine users, which is more than in 2004 (SAMHSA 2006). The number of current crack users increased from 467,000 in 2004 to 682,000 in 2005. According to the 2004 revised National Survey on Drug Use and Health, nearly 12\u00a0million Americans have tried methamphetamine, and 583,000 of them are chronic methamphetamine users (SAMHSA 2004). In 2005, an estimated 22.2\u00a0million persons aged 12 or older were classified with substance dependence or abuse in the past year (9.1% of the population aged 12 or older). Of these, 3.3\u00a0million were classified with dependence on or abuse of both alcohol and illicit drugs, 3.6\u00a0million were dependent on or abused illicit drugs but not alcohol, and 15.4\u00a0million were dependent on or abused alcohol but not illicit drugs. There were 18.7\u00a0million persons classified with dependence on or abuse of alcohol in 2005 (7.7%). The specific illicit drugs that had the highest levels of past year dependence or abuse in 2005 were marijuana, followed by cocaine and pain relievers. Of the 6.8\u00a0million persons aged 12 or older classified with dependence on or abuse of illicit drugs, 4.1\u00a0million were dependent on or abused marijuana in 2005. This number represents 1.7% of the total population aged 12 or older and 59.9% of all those classified with illicit drug dependence or abuse. Marijuana was the most commonly used illicit drug (14.6\u00a0million past month users). In 2005, it was used by 74.2% of current illicit drug users. Among current illicit drug users, 54.5% used only marijuana, 19.6% used marijuana and another illicit drug, and the remaining 25.8% used only an illicit drug other than marijuana in the past month (SAMHSA 2006).\nFatal poisoning, which include overdoses (ODs) on illicit drugs, alcohol, and medications, is the leading cause of injury death for individuals age 35\u201344 and the third leading cause of injury death overall, trailing motor vehicle accidents and firearm-related deaths (CDC 2004). Heroin-related ODs have increased at an alarming rate in portions of the US and other countries (Darke and Hall 2003; Landen et\u00a0al. 2003), and OD has surpassed HIV infection as the primary cause of death for heroin users. Not surprisingly, heroin is frequently associated with opioid-related ODs, both as a single drug and in combination with other substances (CDC 2004).\nMany patients seeking treatment for addiction have multiple drug dependencies and psychiatric comorbidities (Volkow and Li 2005). Information from epidemiological surveys indicates that drug addiction is a common phenomenon and is associated with significant effects on both morbidity and mortality. Large individual and societal costs of drug abuse make research and treatment of drug addiction imperative (French et\u00a0al. 2000; Mark et\u00a0al. 2001). Recently through intensive clinical neurophysiological research and biological psychiatric studies many specific components of cognitive, emotional, and behavioral deficits typical for SUD have been identified and investigated. However, the practical values of these cognitive neuroscience and applied psychophysiology-based treatment (e.g., neurofeedback) findings depend on a further integration of these methodological approaches.\nqEEG in Substance Use Disorders\nEEG in Alcoholism\nEEG alterations have been described extensively in alcoholic patients (Porjesz and Begleiter 1998), but any attempt at drawing a common picture from qEEG data is difficult due to significant methodological differences, such as different definitions of frequency bands, different filtering methodology, number of channels, reference choice, etc. However, most reports of alcoholic patients agree in describing alterations mainly within the beta (Bauer 1997, 2001a; Costa and Bauer 1997; Rangaswamy et\u00a0al. 2002, 2004) and\/or alpha bands (Finn and Justus 1999).\nThe qEEG and LORETA mapping studies of detoxified alcohol-dependent patients, as compared with normal controls, showed an increase in absolute and relative beta power and a decrease in alpha and delta\/theta power (Saletu et\u00a0al. 2002), which is in agreement with earlier reports of low-voltage fast EEG patterns, as often encountered by visual EEG inspection (Niedermeyer and Lopes da Silva 1982). As slow activities are considered to be inhibitory, alpha activity may be viewed as an expression of normal brain functioning and fast beta activities as excitatory, the low-voltage fast desynchronized patterns may be interpreted as hyperarousal of the central nervous system (CNS) (Saletu-Zyhlarz et\u00a0al. 2004). The investigations by Bauer (2001a) and Winterer et\u00a0al. (1998) showed a worse prognosis for the patient group with a more pronounced frontal CNS hyperarousal. It may be hypothesized that these hyperaroused relapsing patients require more CNS sedation than abstaining ones.\nThe EEG maps of alcohol-dependent patients differ significantly from those of normal controls and patients suffering from other mental disorders and might be useful for diagnostic purposes (Pollock et\u00a0al. 1992; Saletu et\u00a0al. 2002; Saletu-Zyhlarz et\u00a0al. 2004). Decreased power in slow bands in alcoholic patients may be an indicator of brain atrophy and chronic brain damage, while an increase in the beta band may be related to various factors such as medication use, family history of alcoholism, and\/or hallucinations, suggesting a state of cortical hyperexcitability (Coutin-Churchman et\u00a0al. 2006).\nAbnormalities in resting EEG are often associated with a predisposition to development of alcoholism. Subjects with a family history of alcoholism were found to have reduced relative and absolute alpha power in occipital and frontal regions and increased relative beta in both regions compared with subjects with a negative family history of alcoholism. These results suggest that resting EEG alpha abnormalities are associated with risk for alcoholism, although their etiological significance is unclear (Finn and Justus 1999).\nAlcohol-dependent individuals have different synchronization of brain activity than light drinkers as reflected by differences in resting EEG coherence (Kaplan et\u00a0al. 1985, 1988; Michael et\u00a0al. 1993; Winterer et\u00a0al. 2003a) and power (e.g., Bauer 2001ab; Enoch et\u00a0al. 2002; Rangaswamy et\u00a0al. 2002; Saletu-Zyhlarz et\u00a0al. 2004). Most differences in EEG coherence and power are found in the alpha and beta bands. Non-alcohol-dependent relatives of alcohol-dependent individuals also have EEG differences in alpha and beta coherence (Michael et\u00a0al. 1993) and power (Bauer and Hesselbrock 2002; Finn and Justus 1999; Rangaswamy et\u00a0al. 2002, 2004) as compared to subjects without alcohol-dependent relatives. This indicates that differences in functional brain activity as measured with qEEG in alcohol-dependent patients not only relate to the impact of long-term alcohol intake, but possibly also to genetic factors related to alcohol dependence.\nBoth alcohol dependence (Schuckit and Smith 1996) and EEG patterns (Van Beijsterveldt and Van Baal 2002) are highly heritable. In addition, some genes coding for GABA receptors in the brain, which mediate the effects of alcohol, are related to certain EEG patterns (Porjesz et\u00a0al. 2005; Winterer et\u00a0al. 2003b). Moreover, some GABA-receptor genes that are related to EEG patterns are also associated with the risk to develop alcohol dependence. These associations again suggest that genetic factors play a major role in the EEG differences associated with alcohol dependence.\nThe EEG coherence analysis is a technique that investigates the pairwise correlations of power spectra obtained from different electrodes. It measures the functional interaction between cortical areas in different frequency bands. A high level of coherence between two EEG signals indicates a co-activation of neuronal populations and provides information on functional coupling between these areas (Franken et\u00a0al. 2004). De Bruin et\u00a0al. (2004, 2006) investigated the pure effects of alcohol intake on synchronization of brain activity, while minimizing the confounding influence of genetic factors related to alcohol dependence. They showed that heavily drinking students with a negative family history had stronger EEG synchronization at theta and gamma frequencies than lightly drinking students with a negative family history. This study suggests that, in students, heavy alcohol intake has an impact on functional brain activity, even in the absence of genetic factors related to alcohol dependence.\nThe findings of studies on the effects of alcohol dependence on EEG coherence can be summarized as follows: Kaplan et\u00a0al. (1985) reported lower frontal alpha and slow-beta coherence in alcohol-dependent males and females. Michael et\u00a0al. (1993) found higher central alpha and slow-beta coherence, but lower parietal alpha and slow-beta coherence in males with alcohol dependence. Winterer et\u00a0al. (2003a, b) described higher left-temporal alpha and slow-beta coherence and higher slow-beta coherence at right-temporal and frontal electrode pairs in alcohol-dependent males and females. De Bruin et\u00a0al (2006) showed that moderate-to-heavy alcohol consumption is associated with differences in synchronization of brain activity during rest and mental rehearsal. Heavy drinkers displayed a loss of hemispheric asymmetry of EEG synchronization in the alpha and slow-beta band. Moderately and heavily drinking males additionally showed lower fast-beta band synchronization.\nTherefore, qEEG alterations have been described extensively in alcoholics. Most EEG reports in alcoholic patients agree in describing alterations mainly within the beta and alpha bands. Patients with a more pronounced frontal hyperarousal have worse prognosis. Decreased power in slow bands in alcoholic patients may be an indicator of chronic brain damage, while increase in beta band may be related to various factors suggesting cortical hyperexcitability. Abnormalities in resting EEG are highly heritable traits and are often associated with a predisposition to alcoholism development. The studies on the effects of alcohol dependence on EEG coherence can be summarized as lower frontal alpha and slow-beta coherence in alcohol-dependent patients with some topographical coherence abnormality differences between alcohol-dependent males and females.\nEEG in Marijuana Abuse\nSeveral lines of evidence suggest that cannabis (marijuana, tetrahydrocannabinol\u2014THC) may alter functionality of the prefrontal cortex and thereby elicit impairments across several domains of complex cognitive function (Egerton et\u00a0al. 2006). Several studies in both humans and animals have shown that cannabinoid exposure results in alterations in prefrontal cortical activity (Block et\u00a0al. 2002; O\u2019Leary et\u00a0al. 2002; Whitlow et\u00a0al. 2002), providing evidence that cannabinoid administration may affect the functionality of this brain area. Despite the fact that a number of transient physiological, perceptual and cognitive effects are known to accompany acute chronic marijuana (THC) exposure in humans, persistent qEEG effects in humans resulting from continuing exposure to this drug have been difficult to demonstrate (Wert and Raulin 1986). In early reviews of EEG and ERP studies of acute and chronic THC exposure in humans (Struve et\u00a0al. 1989, 1994), it was reported that significant associations between chronic exposure and clinically abnormal EEG patterns had not been demonstrated and that attempts to use visual EEG analyses to detect transient acute THC exposure induced EEG alterations failed to demonstrate consistent THC\u2013EEG effects across studies.\nQuantitative methods of analyzing EEG spectra from single posterior scalp derivations began to be applied to studies of acute THC exposure. These early studies reported that acute THC exposure produced transient increases in either posterior alpha power, decreases in mean alpha frequency or increases in alpha synchrony (Fink et\u00a0al. 1976; Struve et\u00a0al. 1989; Tassinari et\u00a0al. 1976; Volavka et\u00a0al. 1971, 1973). These studies found that THC produced a transient dose-dependent rapid onset: (1) increase in relative power (amount, abundance) of alpha; (2) decrease in alpha frequency; and (3) decrease in relative power of beta as measured from posterior scalp electrodes.\nLater studies of Struve et\u00a0al. (1998, 1999, 2003) demonstrated and replicated a significant association between chronic marijuana use and topographic qEEG patterns of persistent \u201calpha hyperfrontality\u201d (i.e., elevations of alpha absolute power, relative power, and interhemispheric coherence over frontal cortex) as well as reductions of alpha mean frequency. These findings from chronic users are consistent with both non-topographic (Hockman et\u00a0al. 1971; Tassinari et\u00a0al. 1976; Volavka et\u00a0al. 1973) and topographic (Lukas et\u00a0al. 1995; Struve et\u00a0al. 1994) transient EEG effects of acute THC administration. Therefore, chronic daily THC use was found to be associated with distinct topographic qEEG features. Compared with non-users, THC users had significant elevations of absolute and relative power, and interhemispheric coherence of alpha activity over the bilateral frontal cortex (referred to as \u201calpha hyperfrontality\u201d). A second finding was that the voltage (not relative power or coherence) of all non-alpha frequency bands was significantly elevated in THC users, although the voltage increase was generalized and not frontally dominant. A third finding involved a widespread decrease in the relative power of delta and beta activity for cannabis users, particularly over the frontal cortical regions. A fourth finding was that interhemispheric coherence of theta and possibly delta activity was also significantly elevated over frontal cortex for marijuana users. Because most studies included daily THC users and non-users drawn from an inpatient psychiatric population, the effects of psychiatric diagnoses or medication were not controlled.\nThus, qEEG studies on acute THC exposure reported a transient dose-dependent increase in relative power of alpha, decrease in alpha frequency, and decrease in relative power of beta at posterior EEG recording sites. Chronic marijuana abuse is known to result in a number of physiological, perceptual and cognitive effects, but persistent qEEG effects from continuing exposure to THC have been difficult to demonstrate. However, recent studies of Struve and his colleagues have demonstrated a significant association between chronic marijuana use and topographic qEEG patterns of persistent elevations of alpha absolute power, relative power, and interhemispheric coherence over frontal cortex, as well as reductions of alpha mean frequency. Another important qEEG finding was the elevated voltage of all non-alpha bands in THC users. A third qEEG finding involved a widespread decrease in the relative power of delta and beta activity over the frontal cortical regions in marijuana users.\nEEG in Heroin Addiction\nOnly a few studies have investigated qEEG changes in heroin addicts. Qualitative changes were observed in more than 70% of heroin addicts in the early abstinence (acute withdrawal) period, and these included low-voltage background activity with diminution of alpha rhythm, an increase in beta activity, and a large amount of low-amplitude delta and theta waves in central regions (Olivennes et\u00a0al. 1983; Polunina and Davydov 2004). Franken et\u00a0al. (2004) found that abstinent heroin-dependent subjects have an enhanced fast beta power compared with healthy controls, and this finding is concordant with other EEG studies on alcohol and cocaine abusing subjects (Costa and Bauer 1997; Herning et\u00a0al. 1994b; Rangaswamy et\u00a0al. 2004; Roemer et\u00a0al. 1995). Spectral power and event-related potentials (ERP) in heroin addicts strongly relate to abstinence length (Shufman et\u00a0al. 1996, Bauer 2001a; Polunina and Davydov 2004). Most studies showed considerable or even complete normalization of EEG spectral power or magnitude of ERP components in heroin ex-addicts who maintained abstinence for at least 3\u00a0months (Bauer 2001b, 2002; Costa and Bauer 1997; Papageorgiou et\u00a0al. 2001; Polunina and Davidov 2004; Shufman et\u00a0al. 1996).\nSome quantitative changes were also reported in methadone-maintenance heroin addicts (Gritz et\u00a0al. 1975), current heroin addicts, and subjects in heroin abstinence less than 80\u00a0days (Shufman et\u00a0al. 1996). Gritz et\u00a0al. (1975) demonstrated a significant slowing of occipital alpha rhythm peak frequency in 10 methadone-maintained patients and the same trend in 10 abstinent heroin-addicted subjects. In one study (Polunina and Davydov 2004), slowing of slow alpha (8\u201310\u00a0Hz) mean frequency was significantly related to the amount of heroin taken by these patients daily before withdrawal. The prolongation of ERP component latencies in heroin addicts was also reported (Papageorgiou et\u00a0al. 2001), and these delays significantly correlated with years of heroin use, rather than with abstinence length in the study of Bauer (1997). Polunina and Davydov (2004) demonstrated frequency shifts in the fast alpha range at the frontal and central recording sites and a slowing of slow alpha mean frequency at the central, temporal, and occipital sites of recording in heroin abusers who used heroin for at least 18\u00a0months.\nIn general, pronounced desynchronization is characteristic for acute heroin withdrawal, but as it was mentioned above, several studies (Bauer 2001a, 2002; Costa and Bauer 1997; Papageorgiou et\u00a0al. 2001; Polunina and Davydov 2004; Shufman et\u00a0al. 1996) showed that spectral power of EEG tends to normalize almost completely after several weeks of abstinence. The most consistent changes in EEG of heroin addicts were reported in alpha and beta frequencies, and included a deficit in alpha activity and an excess of fast beta activity in early heroin abstinence. The latter abnormality appears to reverse considerably when heroin intake is stopped for several months, and therefore it may be viewed as an acute withdrawal effect. The dynamics and characteristics of spectral power changes within the early opiate withdrawal suggest the participation of catecholamine imbalances, especially noradrenaline and perhaps to a lesser degree dopamine, which are widely recognized as a main cause of opiate physical dependency symptoms (Devoto et\u00a0al. 2002; Maldonado 1997). Acute opiate administration has been shown to increase, while abstinence from chronic opiate use has been shown to decrease extracellular dopamine (DA) in the nucleus accumbens. In contrast, extracellular DA in the prefrontal cortex is not modified by acute opiate use, but is markedly increased during morphine and heroin abstinence syndrome (Devoto et\u00a0al. 2002). Relationships between theta and beta frequencies shifts and neurotransmitter imbalances characteristic for heroin withdrawal remain unclear.\nWithdrawal state in heroin addicts is known to elicit a strong craving for drug, anxiety, nervousness, deficits in inhibitory control, dysphoric motivational state, and intrusive thoughts related to drugs (Franken 2003; Franken et\u00a0al. 1999, 2004; Stormark et\u00a0al. 2000). Research on functional connectivity in drug withdrawal states is restricted to a few studies on coherence of the EEG signal in abstinent heroin users (Franken et\u00a0al. 2004; Fingelkurts et\u00a0al. (2006a), active heroin abusers (Fingelkurts et\u00a0al. 2006b), and in abstinent polysubstance abusers (Roemer et\u00a0al. 1995). In a study on 22 opioid-dependent patients under acute opioid influence, Fingelkurts et\u00a0al. (2006b) showed that longitudinal opioid exposure impairs cortical local and remote functional connectivity, and found that local connectivity increased, whereas the remote one decreased. These findings were interpreted as specific signs of independent processing in the cortex of chronic heroin addicts. It has been suggested that such independent processes may constitute the candidate mechanism for a well-documented pattern of impairment in addicts that expresses the lack of integration of different cognitive functions for effective problem solving and helps to explain the observed deficits in abstract concept formation, behavioral control, and problems in the regulation of affect and behavior.\nSpecifically, Fingelkurts et\u00a0al. (2006b) found that the number and strength of remote functional connections among different cortical areas estimated by the index of EEG synchrony was significantly higher in patients in acute heroin withdrawal than in healthy controls for most categories of functional connections. Although this result was observed in the alpha as well as in the beta frequency bands, it was most prominent for the beta range. In the same patient sub-sample under acute opioid influence the authors (Fingelkurts et\u00a0al. 2006a) observed the opposite: a significant decrease in the number and strength of remote functional connections, when compared with healthy controls. Thus, the increase of remote synchronicity among cortical areas during the short-term withdrawal period may indicate the selective attentional focus on cues and memories related to drugs while ignoring neutral cues (Franken et\u00a0al. 2000; Sokhadze et\u00a0al. 2007). Generally this can explain a narrowing of the behavioral repertoire and compulsive drug seeking in abstinent addicted subjects (Vanderschuren and Everitt 2004). Therefore, the elevated synchrony within the beta frequency band in these studies (Fingelkurts et\u00a0al. 2006a, b) may reflect a state of CNS activation toward reward-seeking behavior, with this being a prerequisite of relapse among opiate drug dependent patients (Bauer 2001a).\nqEEG changes in heroin addicts in the acute withdrawal period have been described as low-voltage background activity with a diminution of alpha rhythm, an increase in beta activity, and a large amount of low-amplitude delta and theta waves in central regions. In general, pronounced desynchronization is characteristic for acute heroin withdrawal, but the spectral power of EEG tends to normalize almost completely after several weeks of abstinence. The most consistent changes in EEG of heroin addicts were reported in the alpha and beta frequencies, and included a deficit in alpha activity and an excess of fast beta activity in early heroin abstinence. The excess of beta appears to reverse considerably when heroin intake is stopped for several months, and therefore it may be viewed as an acute withdrawal effect. Recent studies found that the number and strength of remote functional connections among different cortical areas estimated by the index of EEG synchrony for the beta range was significantly higher in patients in acute heroin withdrawal than in healthy controls for most categories of functional connections.\nEEG in Cocaine Addiction\nQualitative and quantitative EEG measures are highly sensitive to the acute and chronic effects of neurointoxication produced by such psychostimulants as cocaine, as well as effects from withdrawal and long-term abstinence from cocaine use (Ehlers et\u00a0al. 1989). However, some EEG characteristics observed in cocaine addicts are considered to be due to the toxic effects of this drug on the brain, whereas some EEG characteristics in cocaine addicts may also indicate a predisposition toward the development of SUD (Porjesz et\u00a0al. 2005).\nHans Berger (1937, cited by Gloor 1969; Herning et\u00a0al. 1985) was the first to study the effects of cocaine on human EEG, reporting an increase in activity in the beta bandwidth. This was replicated in subsequent studies with a larger number of subjects (Alper 1999; Alper et\u00a0al. 1990, 1998; Costa and Bauer 1997; Herning et\u00a0al. 1985; Noldy et\u00a0al. 1994; Prichep et\u00a0al. 1996, 1999, 2002; Roemer et\u00a0al. 1995). Beside beta effects, studies have reported an increase in delta activity (Herning et\u00a0al. 1985) and frontal alpha activity (Herning et\u00a0al. 1994b), while others have reported an increase in alpha wave EEG associated with bursts of cocaine-induced euphoria (Lukas 1991). More recently, researchers have begun analyzing qEEG profiles of cocaine-dependent patients using the spectral power of each primary bandwidth over the different topographic cortical areas. Excess alpha activity (Alper et\u00a0al. 1990; Herning et\u00a0al. 1994b; Lukas 1991; Prichep et\u00a0al. 1996) and decreased delta activity (Alper et\u00a0al. 1990; Noldy et\u00a0al. 1994; Prichep et\u00a0al. 1996; Roemer et\u00a0al. 1995) have been reported, while others have reported increased beta power (Herning et\u00a0al. 1985, 1994b; Noldy et\u00a0al. 1994) in cocaine-dependent patients, recorded in eyes closed, resting conditions. The qEEG abnormalities, primarily found in anterior cortical regions, were shown to correlate with the amount of prior cocaine use (Herning et\u00a0al. 1996a; Prichep et\u00a0al. 1996; Roemer et\u00a0al. 1995; Venneman et\u00a0al. 2006). The qEEG has been used more often to characterize the effects of withdrawal in cocaine-dependent patients. Several studies reported that during protracted abstinence from cocaine qEEG effects are featured by long-lasting increases in alpha and beta bands together with reduced activity in delta and theta bands (Alper et\u00a0al. 1990; Prichep et\u00a0al. 1996; Roemer et\u00a0al. 1995).\nRecently Reid et\u00a0al. (2006) investigated qEEG profiles in cocaine-dependent patients in response to an acute, single-blind, self-administered dose of smoked cocaine base (50\u00a0mg) versus placebo. Cocaine produced a rapid increase in absolute theta, alpha, and beta power over the prefrontal cortex, lasting up to 25\u00a0min after administration of the drug. The increase in theta power was correlated with a positive subjective drug effect (\u201chigh\u201d), and the increase in alpha power was correlated with nervousness. Cocaine also produced a similar increase in delta coherence over the prefrontal cortex, which was correlated with nervousness. Placebo resulted only in a slight increase in alpha power over the prefrontal cortex. These data demonstrate the involvement of the prefrontal cortex in the qEEG response to acute cocaine, and indicate that slow wave qEEG, delta and theta activity are involved in the processes related to experiencing rewarding properties of cocaine.\nPrichep et\u00a0al. (1999, 2002) extended the idea of relating baseline EEG activity to outcome in cocaine-dependent patients in treatment programs. Subjects with cocaine dependence have persistent changes in brain function assessed with qEEG methods, present when evaluated at baseline, 5\u201314\u00a0days after last reported crack cocaine use, and persistent at one and six month follow-up evaluations (Alper 1999; Alper et\u00a0al. 1990, 1998; Prichep et\u00a0al. 1996, 2002; Venneman et\u00a0al. 2006). Several recent studies employing qEEG techniques have already demonstrated an association between the amount of beta activity in the spontaneous EEG and relapse in cocaine abuse (Bauer 1997, 2001a). A decrease in the delta and theta bands of the EEG can be regarded as a specific sign of brain dysfunction.\nHowever, this sign, as well as other qEEG abnormal patterns, can be found in many different psychiatric disorders and none of them can be considered as pathognomonic of any specific mental or neurological disorder. EEG coherence in cocaine addiction was investigated in only one study (Roemer et\u00a0al. 1995). The authors reported globally reduced interhemispheric coherence in the delta and theta bands, and frontally in the beta band. It should be noted that subjects in this study were cocaine-preferring polysubstance abusers during abstinence and these results can hardly be generalized to crack cocaine-only users or other categories of cocaine-dependent subjects not enrolled in any treatment.\nTherefore, acute effects of smoked crack cocaine have been shown to produce a rapid increase in absolute theta, alpha, and beta power over the prefrontal cortex, lasting up to half-an-hour after administration of the drug. The increase in theta power was reported to correlate with a positive subjective drug effect, while the increase in alpha power was reported to correlate with nervousness. qEEG measures are also sensitive to the acute and chronic effects of cocaine, as well as the effects from withdrawal and long-term abstinence from cocaine use. Some EEG characteristics observed in cocaine addicts are considered to be due to the neurotoxic effects, whereas some EEG characteristics in cocaine addicts may also indicate a predisposition toward the development of cocaine addiction. qEEG has been used more often to characterize the effects of withdrawal in cocaine-dependent patients. During protracted abstinence from cocaine qEEG effects are featured by long-lasting increases in alpha and beta bands together with reduced activity in delta and theta bands. Several recent studies employing qEEG techniques have demonstrated an association between the amount of beta activity in the spontaneous EEG and relapse in cocaine abuse.\nEEG in Methamphetamine Addiction\nSeveral studies have examined the neurobiological consequences of methamphetamine dependence using qEEG methods (e.g., Newton et\u00a0al. 2003, 2004). It was found that methamphetamine dependent patients exhibited a significant power increase in the delta and theta bands as compared to non-drug-using controls (Newton et\u00a0al. 2003). These results are in accordance with other neurocognitive studies (Kalechstein et\u00a0al. 2003) suggesting that methamphetamine abuse is associated with psychomotor slowing and frontal executive deficits. Within the methamphetamine-dependent subjects, increased theta qEEG power was found to correlate with response time and was accompanied with reduced accuracy (Newton et\u00a0al. 2004). To our knowledge, qEEG patterns associated with acute withdrawal and recent abstinence in methamphetamine dependence have not yet been sufficiently described. One study reported (Newton et\u00a0al. 2003) that methamphetamine dependent volunteers with 4\u00a0days of abstinence had increased EEG power in the delta and theta but not in the alpha and beta bands. Within the methamphetamine dependent group, a majority of the conventional EEGs were abnormal (64%), compared to 18% in the non-methamphetamine using group.\nThe qEEG may provide a sensitive neurophysiological outcome measure of methamphetamine abuse-related persistent alterations in neurocognitive functions (Newton et\u00a0al. 2004). In a study by Simon et\u00a0al. (2002), when performance of patients with SUD was compared to their matched non-using control groups, both methamphetamine and cocaine abusers were impaired on cognitive measures, but the type and degree of impairments were somewhat different. Some of these differences between methamphetamine and cocaine effects on cognitive functions and electrophysiological alterations can be explained by differential pharmacokinetics of these two drugs, as cocaine is rapidly metabolized with an elimination half-life of several hours, whereas methamphetamine is eliminated more slowly, with an elimination half-life averaging 12\u00a0h (Cook et\u00a0al. 1993; Jeffcoat et\u00a0al. 1989). Moreover, cocaine differs from methamphetamine in that cocaine inhibits the reuptake of dopamine, serotonin, and norepinephrine, whereas methamphetamine mobilizes and releases these monoamines from storage granules, thus producing rapid and large increases in synaptic concentrations (Simon et\u00a0al. 2002, 2004). This might be responsible for the discrepancies in observed qEEG manifestations associated with chronic methamphetamine and cocaine abuse.\nOnly a few studies have examined the qEEG consequences of methamphetamine dependence. They report that methamphetamine dependent patients exhibited a significant power increase in the delta and theta bands as compared to non-drug-using control. The qEEG patterns associated with acute withdrawal and recent abstinence in methamphetamine dependence have not yet been sufficiently described. One study reported that abstinent methamphetamine dependent patients had increased EEG power in the delta and theta but not in the alpha and beta bands. In general, qEEG studies of methamphetamine addiction are in accordance with other neurocognitive studies suggesting that methamphetamine abuse is associated with psychomotor slowing and frontal executive deficits.\nP300 Abnormalities in Cocaine, Methamphetamine, Heroin Addiction, and Alcoholism\nThe P300 component of the ERP, occurring 300\u2013600\u00a0ms post-stimulus, is the most widely used ERP in psychiatry and other clinical applications (Polich et\u00a0al. 1994; Polich and Herbst 2000; Pritchard 1981, 1986; Pritchard et\u00a0al. 2004). The amplitude of the P300 reflects the allocation of attentional resources, while the latency is considered to reflect stimulus evaluation and classification time (Katayama and Polich 1998; Polich and Herbst 2000). The P300 is usually obtained in an oddball paradigm, wherein two stimuli are presented in a random order, one of them frequent (standard) and another one rare (target) (Polich 1990). A modification of the oddball task has been used where a third, also rare stimulus (distracter), is presented along with standard and target stimuli. It was reported that these infrequent distracters elicit a frontocentral P300, so called P3a, whereas the rare targets elicit a parietal P300, so called P3b (Katayama and Polich 1996, 1998). The P3a is recorded at the anterior scalp locations and has been interpreted as reflecting frontal lobe activity (Gaeta et\u00a0al. 2003; Knight 1984). Though the P300 response in general is thought to represent \u201ccontext updating\/closure,\u201d in a three-stimuli oddball task the P3a is interpreted as \u201corienting,\u201d and the P3b is viewed as an index of the ability to maintain sustained attention to target (N\u00e4\u00e4t\u00e4nen 1990). The anterior P3a indexes the contextual salience of the rare stimuli, whereas the posterior P3b is indexing task-relevance of the stimuli (Gaeta et\u00a0al. 2003).\nA robust finding in ERP studies on alcoholism is that alcoholics as well as individuals at high risk to develop alcoholism have been shown to have a low P300 amplitude in various task paradigms (Cohen et\u00a0al. 2002; Hada et\u00a0al. 2000; Porjesz et\u00a0al. 2005; Porjesz and Begleiter 1998). Kouri et\u00a0al. (1996) examined the P300 component in patients who were dually dependent on cocaine and heroin. The results showed no P300 amplitude differences between the patients and healthy non-drug-dependent volunteers when patients presented for detoxification. However, after the course of detoxification, the P300 amplitude was significantly smaller in the cocaine- and heroin-dependent group than in the non-dependent control group. In a study by Bauer (2001b) the P300 did not differentiate among patients characterized by histories of either cocaine, or cocaine and alcohol, or heroin dependence. Across all the patient groups, the P300 was significantly reduced in amplitude relative to the P300 ERPs recorded from individuals with no history of alcohol or drug dependence. This study also demonstrated that continued abstinence from heroin and from cocaine and alcohol is also associated with a trend toward normalization of the P300. In a recent study of Papageorgiou et\u00a0al. (2004) the P300 component was evaluated during the anticipatory period of a short memory task in 20 patients characterized by a past history of heroin dependence (6\u00a0months abstinence), in 18 current heroin users and in 20 matched healthy subjects. Abstinent heroin addicts exhibited a significant reduction of the P300 amplitude at the central frontal region, relative to the other two groups.\nThe results of early work examining the effect of cannabis use and THC administration on visual and auditory ERPs have been inconclusive (Rodin et\u00a0al. 1970; Roth et\u00a0al. 1973). Later studies of Patrick et\u00a0al. (1995, 1997) could not find P300 latency differences in audio and visual oddball tasks between THC users without psychiatric problems and controls. Although THC users displayed reduced auditory and visual P300 amplitudes in this study, when age differences between THC users and controls were removed, all significant P300 amplitude differences were removed as well.\nAcute and chronic use of cocaine exerts neuropharmacological effects on amplitude and latency of both anterior and posterior P300 ERP components (Biggins et\u00a0al. 1997; Fein et\u00a0al. 1996; Herning et\u00a0al. 1994a; Kouri et\u00a0al. 1996; Polich 1990). Longer P300 (P3b) latency without abnormalities in amplitude was reported in several studies on cocaine withdrawal (Herning et\u00a0al. 1994a; Lukas 1993). Noldy and Carlen (1997) demonstrated effects of cocaine withdrawal on the latency of the P300 in an auditory oddball task. In cocaine-dependent patients, P3a amplitude decrements over frontal areas are persistent even after long periods of abstinence (Bauer 1997). The latency of the P3a was delayed and the amplitude was reduced to novel non-targets in cocaine and alcohol-dependent subjects compared to controls (Biggins et\u00a0al. 1997; Hada et\u00a0al. 2000) in auditory and visual three-stimuli oddball tasks.\nSeveral studies have investigated ERP changes associated with methamphetamine abuse and dependence. The P300 component of the auditory ERP was reported to show a prolonged latency in the oddball task in methamphetamine dependent subjects with a history of psychosis, compared to normal controls (Iwanami et\u00a0al. 1994, 1998). In particular, the patients with methamphetamine dependence showed reduced P3a amplitude in the reading task and delayed P3b latency with normal P3b amplitude in the auditory oddball task. This was interpreted as indicating a prolonged central noradrenergic dysfunction due to earlier methamphetamine use.\nIn most ERP studies the P300 did not differentiate among patients characterized by histories of either cocaine, or cocaine and alcohol, or heroin dependence. Across all the patient groups, the P300 was significantly reduced in amplitude relative to P300 ERPs recorded from individuals with no history of alcohol or drug dependence. The latency of the frontal and parietal P300 was reported to be delayed, and the amplitude was reduced to novel non-targets in cocaine and alcohol-dependent subjects compared to controls in auditory and visual three-stimuli oddball tasks. Continued abstinence from heroin, cocaine, and alcohol was shown to be associated with a trend toward P300 normalization. Several studies have investigated ERP changes associated with methamphetamine abuse and dependence. In general, chronic psychoactive substance abuse and drug dependence are associated with delayed and attenuated cognitive ERP in auditory and visual oddball tasks.\nqEEG and ERP Abnormalities in Addiction: Psychopharmacological Effects or Trait Markers?\nWhether qEEG alterations and P300 decrements found in most of SUD are only a coincident \u201cmarker\u201d of vulnerability or make a direct etiologic contribution to risk for substance dependence is still unknown (Bauer and Hesselbrok 2001; Carlson et\u00a0al. 2002; O\u2019Connor et\u00a0al. 1994; Polich et\u00a0al. 1994; Porjesz and Begleiter 1998). The P300 reduction and abnormal qEEG patterns are seen in mental disorders that often are comorbid with substance abuse, such as conduct disorder (Bauer and Hesselbrock 1999, 2001), ADHD (Bauer 1997; O\u2019Connor et\u00a0al. 1994), and bipolar or major affective disorder (Friedman and Squires-Wheeler 1994). Reduced P300 amplitude related to prefrontal brain dysfunction may suggest that a deficit in inhibitory control is an underlying mechanism shared by different psychopathologies (Bauer and Hesselbrock 1999; Clark et\u00a0al. 1999; Tarter et\u00a0al. 2003). According to Bauer (2002), certain ERP and qEEG abnormalities and impaired functioning on complex cognitive tests in patients formerly dependent on cocaine might not be proximately caused by drug use per se but be more related to comorbid alcohol use or another psychiatric condition. Taken together, the findings converge on the conclusion that there exists an inherited predisposition for an externalizing psychopathology that includes ADHD, conduct disorder, and substance abuse. PTSD seems to heighten the risk for addiction as well. Thus, the reviewed findings support the hypothesis that addicted subjects may manifest a P300 amplitude reduction and qEEG abnormalities as a trait reflecting the CNS disinhibition, which may be a predisposing factor for addiction liability, resistance to drug habit extinction, and relapse vulnerability.\nHeritability and Neurotransmitter Considerations in Substance Use Disorders\nThere has been a consistent drift in addiction research between the psychosocial, cognitive and behavioral aspects of addiction and the biological and genetic emphasis. In much of the present data relating to genetics and animal models (Blum et\u00a0al. 2006; Porjesz et\u00a0al. 2005; Ryabinin and Weitemier 2006; Samochowiec et\u00a0al. 2006), studies suggest that a genetic predisposition for SUD is an accepted concept. Much of the genetic research addresses the influence of alleles thought responsible in coding for genes that express phenotypic neurotransmitter production and distribution; mainly involving endorphins, dopamine and serotonin. These neurotransmitters, dopamine in particular, are also suspect in other appetitive and mood disorders and psychopathologies, of particular note, Reward Deprivation Syndrome (RDS). RDS is described as a dysfunction in the Brain Reward Cascade and proposes that abnormal craving behavior is a consequence of defects in the DRD2 and D1, D3, D4 and D5 dopaminergic receptor genes (Blum et\u00a0al. 2006).\nBlum and colleagues (1990, 1993, 1996) described this syndrome and identified the D2 dopamine receptor gene as a possible candidate for susceptibility to alcoholism in severe alcoholics (Blum et\u00a0al. 1993) and proposed this gene\u2019s association with dopamine production and distribution may produce a sevenfold increase in the likelihood of developing alcohol use problems (Uhl et\u00a0al. 1993). This DRD2 dopamine receptor gene and polymorphisms within its genetic coding specific to addiction remain unclear due to its involvement in other disorders; including, obesity (Blum et\u00a0al. 2006), Tourette\u2019s syndrome (Comings et\u00a0al. 1991) pathological aggression and violence, PTSD (Comings et\u00a0al. 1996) and schizoid\u2014avoidant disorder (Chen et\u00a0al. 2005). SUD were classified as a subtype of RDS and treatment regimens for these disorders have been classified as inadequate (Blum et\u00a0al. 2007) and research continues in developing possible genetic interventions that may produce dopamine and other neurotransmitter regulation in substance-induced rapid dopamine increase in limbic regions (Blum et\u00a0al. 2007).\nIt is clear that heritability plays an important role in addictive disorders, however, to what extent environment, perception and synaptic permanency and plasticity influence the course of genetic adaptation or maladaptive traits requires further investigation. Suggested neuroanatomical substrates involved in SUD implicate mesolimbic and diencephalon regions; including the substantia nigra, reticular formation, medial forebrain bundle, nucleus accumbens, septum pediculum, olfactory tubercule and hippocampus and suggest that any concentration of alcohol exposure to these regions would make alcohol use virtually unavoidable (Myers and Privette 1989).\nStudies of EEG Biofeedback in Substance Abuse Treatment\nThe Peniston Protocol (Alpha-Theta Feedback)\nThe early studies of Kamiya (e.g., Nowlis and Kamiya 1970) on self-regulation of alpha rhythm elicited substantial interest in the potential clinical applications of alpha biofeedback for SUD treatment. There were reported several uncontrolled case studies and conceptual reviews on alpha EEG training for alcohol (DeGood and Valle 1978; Denney et\u00a0al. 1991; Jones and Holmes 1976; Passini et\u00a0al. 1977; Tarbox 1983; Watson et\u00a0al. 1978) and drug abuse treatment (Brinkman 1978; Goldberg et\u00a0al. 1976, 1977; Lamontagne et\u00a0al. 1977; Sim 1976), but the impact of alpha biofeedback training as a SUD therapy was not significant.\nThe bulk of the literature to date regarding EEG biofeedback of addictive disorders is focused on alpha-theta biofeedback. The technique involves the simultaneous measurement of occipital alpha (8\u201313\u00a0Hz) and theta (4\u20138\u00a0Hz) and feedback by separate auditory tones for each frequency representing amplitudes greater than pre set thresholds. The subject is encouraged to relax and to increase the amount of time the signal is heard, that is to say, to increase the amount of time that the amplitude of each defined bandwidth exceeds the threshold. A variety of equipment and software has been used to acquire, process, and filter these signals, and there are differences in technique inherent with equipment and software.\nAlpha-theta feedback training was first employed and described by Elmer Green and colleagues (Green et\u00a0al. 1974) at the Menninger Clinic. This method was based on Green\u2019s observations of single lead EEG during meditative states in practiced meditators, during which increased theta amplitude was observed following an initial increased alpha amplitude, then a drop off of alpha amplitude (theta\/alpha crossover). When the feedback of the alpha and theta signal was applied to subjects, states of profound relaxation and reverie were reported to occur. The method was seen as useful in augmenting psychotherapy and promoting individual insight. It could be seen as a use of brain wave signal feedback to enable a subject to maintain a particular state of consciousness similar to a meditative or hypnotic relaxed state over a 30- or 40-min feedback session.\nGoslinga (1975) gave the first description of the use of alpha-theta feedback in a SUD treatment program. This integrated program started in 1973 at the Topeka VA, and included group and individual therapies. Daily 20-min EEG biofeedback sessions (integrated with EMG biofeedback and temperature control biofeedback) were conducted over 6\u00a0weeks, resulting in free, loose associations, heightened sensitivity, and increased suggestibility. Patients discussed their insights and experiences associated with biofeedback in therapy groups several times a week, augmenting expressive psychotherapy. The first published clinical reports of efficacy of alpha-theta training at the Topeka VA were by Twemlow and Bowen (1976), who explored the impact of alpha-theta training on psychodynamic issues in 67 non-psychotic chronic male alcoholics in an inpatient treatment program. In this non-controlled study, they found that \u201creligiousness\u201d as a predictor of \u201cself-actualization\u201d may have increased as a result of imagery experienced in theta states. This was seen as positive to the program goal of augmenting Alcoholics Anonymous as a recovery philosophy. The high suggestibility of the method was acknowledged; \u201ctreatments such as brainwave training, which utilize abstract, ill understood techniques are potential repositories of magical projection and fantasy and would logically be more acceptable to alcoholics who are able to have \u2018faith\u2019 (devoutly or moderately religious)\u201d (Twemlow and Bowen 1977). In another uncontrolled study at the Topeka VA, 21 alcoholics were reported to exhibit within and across session increases in raw theta amplitudes at occipital areas bilaterally measured by single lead EEG during the course of alpha-theta training, becoming more able to achieve deep states as manifested by EEG (Twemlow et\u00a0al. 1977). These initial studies advanced the utility of biofeedback induced theta states in promoting insight and attitude change in alcoholics, with the assumptions that biofeedback-induced theta states are associated with heightened awareness and suggestibility, and that this heightened awareness and suggestibility would enhance recovery. Outcome data regarding abstinence were not reported.\nIn the first reported randomized and controlled study of alcoholics treated with alpha-theta EEG biofeedback, Peniston and Kulkosky (1989) described positive outcome results. Their subjects were inpatients in a VA hospital treatment program, all males with established chronic alcoholism and multiple past failed treatments. Following a temperature biofeedback pre-training phase, Peniston\u2019s experimental subjects (n\u00a0=\u00a010) completed 15 30-min sessions of eyes closed occipital alpha-theta biofeedback. Compared to a traditionally treated alcoholic control group (n\u00a0=\u00a010), and nonalcoholic controls (n\u00a0=\u00a010), alcoholics receiving brainwave biofeedback showed significant increases in percentages of EEG recorded in the alpha and theta rhythms, and increased alpha rhythm amplitudes (single lead measurements at international site O1). The experimentally treated subjects showed reductions in Beck Depression Inventory scores compared to the control groups. Control subjects who received standard treatment alone showed increased levels of circulating beta-endorphin, an index of stress, whereas the EEG biofeedback group did not. Thirteen-month follow-up data indicated significantly more sustained prevention of relapse in alcoholics who completed alpha-theta brainwave training as compared to the control alcoholics, defining successful relapse prevention as \u201cnot using alcohol for more than six contiguous days\u201d during the follow-up period. In a further report on the same control and experimental subjects, Peniston and Kulkosky (1990) described substantial changes in personality test results in the experimental group as compared to the controls. The experimental group showed improvement in psychological adjustment on 13 scales of the Millon Clinical Multiaxial Inventory compared to the traditionally treated alcoholics who improved on only two scales and became worse on one scale. On the 16-PF personality inventory, the neurofeedback training group demonstrated improvement on seven scales, compared to only one scale among the traditional treatment group. This small n study employed controls and blind outcome evaluation, with actual outcome figures of 80% positive outcome versus 20% in the traditional treatment control condition at 4-year follow up.\nThe protocol described by Peniston at the Fort Lyons VA cited above is similar to that initially employed by Twemlow and colleagues at the Topeka VA and Elmer Green at the Menninger Clinic, with two additions, i.e., (1) temperature training and (2) script. Peniston introduced temperature biofeedback training as a preconditioning relaxation exercise, along with an induction script to be read at the start of each session. This protocol (described as follows) has become known as the \u201cPeniston Protocol\u201d and has become the focus of research in subsequent studies. Subjects are first taught deep relaxation by skin temperature biofeedback for a minimum of five sessions that additionally incorporates autogenic phrases. Peniston also used the criteria of obtaining a temperature of 94\u00b0 before moving on to EEG biofeedback. Participants then are instructed in EEG biofeedback and in an eyes closed and relaxed condition, receive auditory signals from an EEG apparatus using an international site O1 single electrode. A standard induction script employing suggestions to relax and \u201csink down\u201d into reverie is read. When alpha (8\u201312\u00a0Hz) brainwaves exceed a preset threshold, a pleasant tone is heard, and by learning to voluntarily produce this tone, the subject becomes progressively relaxed. When theta brainwaves (4\u20138\u00a0Hz) are produced at a sufficiently high amplitude, a second tone is heard, and the subject becomes more relaxed and according to Peniston, enters a hypnagogic state of free reverie and high suggestibility. (Although theta increase and alpha decrease are thought by Peniston to be associated with a deeply relaxed state where hypnagogic reverie is present, this may simply represent drowsiness) (Niedermeyer 1999). Following the session, with the subject in a relaxed and suggestible state, a therapy session is conducted between the subject and therapist where the contents of the imagery experienced is explored and \u201cabreactive\u201d experiences are explored (Peniston and Kulkosky 1989, 1990, 1991).\nSaxby and Peniston (1995) reported on 14 chronically alcohol dependent and depressed outpatients using this same protocol of alpha-theta brainwave biofeedback. Following treatment, subjects showed substantial decreases in depression and psychopathology as measured by standard instruments. Twenty-one month follow-up data indicated sustained abstinence from alcohol confirmed by collateral report. These male and female outpatients received 20 40-min sessions of feedback.\nBodenhamer-Davis and Calloway (2004) reported a clinical trial with 16 chemically dependent outpatients, 10 of whom were probationers classified as high risk for re-arrest. Subjects completed an average of 31 alpha-theta biofeedback sessions. Psychometrics demonstrated improvements in personality and mood. Follow-up at 74\u201398\u00a0months indicated 81.3% of the treatment subjects were abstinent. Re-arrest rates and probation revocations for the probation treatment group were lower than those for a probation comparison group (40% vs. 79%).\nFahrion (1995) gave a preliminary report (n\u00a0=\u00a0119) on a large randomized study of alpha-theta training for addiction in the Kansas Prison System using group-training equipment. A report of the completed study (n\u00a0=\u00a0520) (Fahrion 2002) showed little difference between the two groups overall at 2-year outcome. But, when results were analyzed for age, race and drug of choice, neurofeedback emerged as a more efficacious treatment for younger and non-white and non-stimulant abusing participants. Interestingly, this protocol was not effective for cocaine abusers. (Stimulant abusers will be discussed later in this article under the Scott\u2013Kaiser modification of the Peniston protocol.)\nThe issue of alpha-theta biofeedback in culturally sensitive groups that have not responded to traditional modes of addiction treatment (such as confrontational group therapies) has been considered in an open case series reported by Kelly (1997). This three year follow-up study presented the treatment outcomes of 19 Dine\u2019 (Navajo) clients. Four (21%) participants achieved \u201csustained full remission,\u201d 12 (63%) achieved \u201csustained partial remission,\u201d and 3 (16%) remained \u201cdependent.\u201d The majority of participants also showed a significant increase in \u201clevel of functioning\u201d.\nSchneider et\u00a0al. (1993) used slow cortical potential biofeedback to treat 10 unmedicated alcoholic patients in four neurofeedback sessions after hospitalization. Seven patients participated in a fifth session an average of 4\u00a0months later. Six out of these seven patients had not had a relapse at the follow-up. These results are similar to those reported for alpha theta training.\nSeveral other studies using the Peniston protocol and its modifications reported cases with positive clinical effects (Burkett et\u00a0al. 2003, DeBeus et\u00a0al. 2002; Fahrion et\u00a0al. 1992; Finkelberg et\u00a0al. 1996; Skok et\u00a0al. 1997). These studies suggest that an applied psychophysiological approach based on an alpha-theta biofeedback protocol is a valuable alternative to conventional substance abuse treatment (Walters 1998). Nevertheless, most of these results were reported at the society meetings, and only few of these studies were published in mainstream peer-reviewed journals other than The Journal of Neurotherapy.\nA critical analysis of the Peniston Protocol is discussed at length in the previous reviews (Trudeau 2000, 2005a, b). Several controlled studies of the Peniston protocol for addictions, completed by Lowe (1999), Moore and Trudeau (1998), and Taub and Rosenfeld (1994), suggest that alpha-theta training for addictions may be non-specific in terms of effect when compared to suggestion, sham or controlled treatment, or meditational techniques. By contrast, Egner et\u00a0al. (2002) showed that alpha-theta training results in an increase of theta\/alpha ratios, as compared to a control condition. In an in depth critical analysis that examines inconsistencies reported in the original Peniston papers, Graap and Freides (1998) raise serious issues about the reporting of original samples and procedures in these studies. In their analyses, the results may have been due as much to the intense therapies accompanying the biofeedback as due to the biofeedback itself. The subjects may have been comorbid for a number of conditions, which were not clearly reported, particularly PTSD, which may have been the focus of the treatment. In his reply to these criticisms, Peniston (1998) acknowledges that it \u201cremains unknown whether the temperature training, the visualizations, the ATBWNT (alpha-theta brain wave neurotherapy), the therapist, the placebo, or the Hawthorne effects are responsible for the beneficial results.\u201d The criticism raised above by Graap and Friedes (1998) regarding Peniston\u2019s papers could also be applied to earlier replication studies. Neither Peniston\u2019s studies nor the replication studies provide sufficient detail regarding the specifics of the types of equipment used for alpha-theta feedback, including filtering methods for the EEG signal or other technical information, to permit exact reproduction of the feedback protocols with other equipment. Outcome criteria also vary in the replication studies, with varying measures of abstinence and improvement. An exception to these concerns is the report of Scott et\u00a0al. (2005), which will be discussed later in greater detail.\nIt should be noted that psychostimulant (cocaine, methamphetamine) addictions may require approaches and neurofeedback protocols other than alpha\/theta training. Persons who are cocaine-dependent are cortically under-aroused during protracted abstinence (Roemer et\u00a0al. 1995). qEEG changes, such as a decrease in high beta (18\u201326\u00a0Hz) power are typical for withdrawal from cocaine (Noldy et\u00a0al. 1994). Cocaine abusers who are still taking this drug often show low amounts of delta and excess amounts of alpha and beta activity (Alper 1999; Prichep et\u00a0al. 1999), whereas chronic methamphetamine abusers usually exhibit excessive delta and theta activity (Newton et\u00a0al. 2003). Thus, cocaine and methamphetamine users may warrant a different EEG biofeedback protocol, at least at the beginning stages of neurofeedback therapy.\nThe Scott\u2013Kaiser Modification of the Peniston Protocol\nScott and Kaiser (1998) describe combining a protocol for attentional training (beta and\/or SMR augmentation with theta suppression) with the Peniston protocol (alpha-theta training) in a population of subjects with mixed substance abuse, rich in stimulant abusers. The beta protocol is similar to that used in ADHD (Kaiser and Othmer 2000) and was used until measures of attention normalized, and then the standard Peniston protocol without temperature training was applied (Scott et\u00a0al. 2002). The study group is substantially different than that reported in either the Peniston or replication studies. The rationale is based in part on reports of substantial alteration of qEEG seen in stimulant abusers associated with early treatment failure (Prichep et\u00a0al. 1996, 2002) likely associated with marked frontal neurotoxicity and alterations in dopamine receptor mechanisms (Alper 1999). Additionally, preexisting ADHD is associated with stimulant preference in adult substance abusers, and is independent of stimulant associated qEEG changes. These findings of chronic EEG abnormality and high incidence of preexisting ADHD in stimulant abusers suggest they may be less able to engage in the hypnagogic and auto-suggestive Peniston protocol (Trudeau et\u00a0al. 1999). Furthermore, eyes-closed alpha feedback as a starting protocol may be deleterious in stimulant abusers because the most common EEG abnormality in crack cocaine addicts is excess frontal alpha (Prichep et\u00a0al. 2002).\nIn their initial report, Scott and Kaiser (1998) described substantial improvement in measures of attention and also of personality (similar to those reported by Peniston and Kulkosky 1990). Their experimental subjects underwent an average of 13 SMR-beta (12\u201318\u00a0Hz) neurofeedback training sessions followed by 30 alpha-theta sessions during the first 45\u00a0days of treatment. Treatment retention was significantly better in the EEG biofeedback group and was associated with the initial SMR-beta training. A subsequent published paper (Scott et\u00a0al. 2005) reported on an expanded series of 121 inpatient drug program subjects randomized to condition, followed up at 1\u00a0year. Subjects were tested and controlled for the presence of attentional and cognitive deficits, personality states and traits. The experimental group showed normalization of attentional variables following the SMR-Beta portion of the neurofeedback, while the control group showed no improvement. Experimental subjects demonstrated significant changes (p\u00a0<\u00a0.05) beyond the control subjects on 5 of the 10 scales of the MMPI-2. Subjects in the experimental group were also more likely to stay in treatment longer and more likely to complete treatment as compared to the control group. Finally, the one-year sustained abstinence levels were significantly higher for the experimental group as compared to the control group.\nThe approach of beta training in conjunction with alpha-theta training has been applied successfully in a treatment program aimed at homeless crack cocaine abusers in Houston, as reported by Burkett et\u00a0al. (2003), with impressive results. Two hundred and seventy (270) male addicts received 30 sessions of a protocol similar to the Scott Kaiser modification. One-year follow-up evaluations of 94 treatment completers indicated that 95.7% of subjects were maintaining a regular residence; 93.6% were employed\/in school or training, and 88.3% had no subsequent arrests. Self-report depression scores dropped by 50% and self-report anxiety scores by 66%. Furthermore, 53.2% reported no alcohol or drug use 12\u00a0months after biofeedback, and 23.4% used drugs or alcohol only one to three times after their stay. This was a substantial improvement from the expected 30% or less expected recovery in this group. The remaining 23.4% reported using drugs or alcohol more than 20\u00a0times over the year. Urinalysis results corroborated self-reports of drug use. The treatment program saw substantial changes in length of stay and completion. After the introduction of the neurofeedback to the mission regimen, length of stay tripled, beginning at 30\u00a0days on average and culminating at 100\u00a0days after the addition of neurotherapy. In a later study the authors reported follow-up results on 87 subjects after completion of neurofeedback training (Burkett et\u00a0al. 2005). The follow-up measures of drug screens, length of residence, and self-reported depression scores showed significant improvement. It should be noted that this study had limitations, because neurofeedback was positioned only as an adjunct therapy to all other faith-based treatments for crack cocaine abusing homeless persons enrolled in this residential shelter mission and was an uncontrolled study. Yet the improvement in program retention is impressive and may well be related to the improved outcome.\nContinuing Research\nSelf-Perception and Experimental Schemata in the Addicted Brain\nRex Cannon, Joel Lubar, and Deborah Baldwin of the Brain Research and Neuropsychology Laboratory at University of Tennessee at Knoxville are performing research with three goals in mind: First, to attempt to reconcile and integrate data from all disciplines involved in addiction research in order to develop a novel approach for neurophysiological study pertaining to SUD and conceivably determine and describe EEG source generators that are instrumental in the processes of self-perception and experiential schemata utilizing a recently developed assessment instrument. Second, to utilize this information to develop an integrative treatment model for addictive disorders based on this research, involving novel group processing methods and spatial specific neurophysiological operant learning (LORETA Neurofeedback) (Cannon et\u00a0al. 2006, 2007; Congedo 2003; Congedo et\u00a0al. 2004), and finally, third, to utilize both the assessment and neurophysiological data for development of statistical models for possible diagnostic and predictive purposes and to provide a means for a neurophysiological measure of treatment efficacy.\nResearch indicates that substance abusers have elevated beta activity in an EEG resting state as compared with normative groups (Rangaswany et\u00a0al. 2002) and elevated alpha activity after administering a mood altering substance (Cohen et\u00a0al. 1993; Kaplan et\u00a0al. 1985). It is suggested that many of the neurophysiological markers may provide information about the state of the individual prior to the development of an addictive disorder and that these brain functions are under genetic control (Porjesz et\u00a0al. 2002, 2005; Tapert 2004). Kaplan et\u00a0al. (1985) reported lower frontal alpha and slow-beta coherence in alcohol-dependent males and females. Michael et\u00a0al. (1993) found higher central alpha and slow-beta coherence, but lower parietal alpha and slow-beta coherence in males with alcohol dependence; contrarily, other findings suggest that morphine, alcohol and marijuana show increased alpha 2 power in the spectral EEG and relate this to the euphoric state produced by the drug (Lukas 1989, 1995). Winterer et\u00a0al. (2003a, b) described higher left-temporal alpha and slow-beta coherence and higher slow-beta coherence at right-temporal and frontal electrode pairs in alcohol-dependent males and females. De Bruin et\u00a0al. (2004) showed that moderate-to-heavy alcohol consumption is associated with differences in synchronization of brain activity during rest and mental rehearsal. Heavy drinkers displayed a loss of hemispheric asymmetry of EEG synchronization in the alpha and low-beta band. Moderately and heavily drinking males additionally showed lower fast-beta band synchronization. Decision-making processes and the ability to form a resistance to drugs, i.e., the ability to say no, involve numerous brain regions; including, the insular, somatosensory, orbitofrontal, anterior cingulate and dorsolateral prefrontal cortices, as well as the amygdala, hippocampus and thalamic nuclei (Bechara 2005).\nThis research considers the integration of the features of addicted persons as reported in earlier studies, case reports and theoretical concepts as vital in understanding behavioral manifestations of the suspected neural pathways that are premised to be involved in the development of SUD. Some of the fundamental descriptions of addicted individuals portray them as passive with dependent strivings, emotionally immature, abounding with fears of responsibility or independent action and ultimately, infantile inadequate personalities (Coodley 1961), as well as emotionally, socially, and educationally underdeveloped (Meyerstein 1964), and immature and regressive (Dorsey 1961; Gerard and Kornetsky 1955; Hill 1962). These individuals are reported to struggle with affirming positive thoughts of self-esteem, tendencies to undervalue themselves and be self deprecating, and exhibit difficulty adjusting to others and these tendencies are veiled by overt behavioral patterns, including, physical or verbal abuse.\nIndividuals with SUD present with a vast number of paradoxical characteristics; including an overwhelming sense of inadequacy disguised by an apparent overwhelming sense of confidence. Similarly, an apparent abundance of anger and aggression utilized as a disguise for a paralyzing sense of fear, more specifically, fear of people, economic insecurity, rejection, and alienation, which paradoxically are exacerbated by the continued use of the substance. One of the more profound idiosyncratic characteristics of this population is the tendency to ruminate and associate past events, perceptions and the associated emotions with both present and future. The perception of experience is often clouded by the personalization of events (real or imagined) and reinforced with a deliberate, ambiguous effort to avoid reconciling this confound, which reinforces an uninhibited association of all current interactions and situations with past events. Opposite to what often is implied, these features may not originate from the consequences of substance abuse, but from earlier periods in development (Vos 1989), and in the perspective of this research these features and others have an etiology in specific neurophysiological regions that are the direct result of dendritic pruning that occurs in early development that continues on into adolescence and, unless intervention or awareness of these schemata are achieved, they remain problematic into adulthood.\nTo date, studies identifying such schematic source generators and their relationship with SUD using qEEG and standardized low-resolution electromagnetic tomography (sLORETA) are scant. This research is designed to assess the neural activation patterns relative to schemata regarding the self in recovering addicts and identify possible generators in the cortex as compared to controls. In this research, it is hypothesized that there is dendritic pruning early in developmental phases that contribute to frequency specific activity in neuronal populations in the ventromedial portions of the prefrontal cortex and limbic regions. Furthermore, it is proposed that these neural pathways hinder the integration of affect, cognition, reward and decision-making processes and adversely influence the perception of self and self in relation to experience and the development of adaptive schemata and personality characteristics.\nIntegration of Cognitive Neuroscience Approaches in Assessment of Functional Outcomes of Neurofeedback and Behavioral Therapy Based Interventions in SUD\nSokhadze et\u00a0al. (2007a) in their conceptual review proposed an integrated approach to assessment and treatment utilizing cognitive neuroscience methods (e.g., qEEG, ERP), conventional psychotherapeutic treatment, and neurofeedback therapy to assess the recovery of cognitive and emotional functions affected by chronic psychostimulant drug abuse co-occurring with PTSD. Cognitive neuroscience methodologies used for assessment of the outcome effects of psychotherapy and neurofeedback interventions for comorbid disorders have significant potential for additionally identifying neurophysiological and clinical markers of treatment progress (Sokhadze 2005). These outcome markers may provide useful information for planning bio-behavioral interventions in this form of dual diagnosis.\nStotts and colleagues (2006) at the University of Texas at Houston, in collaboration with researchers at Rice University, used motivational interviewing (MI) with personalized feedback, particularly employing the ERP markers of deficiencies in selective attention task produced by cocaine abuse in crack addicts. In a randomized, controlled pilot study these authors (Stotts et\u00a0al. 2006; Sokhadze et\u00a0al. 2004, 2005) evaluated the feasibility and preliminary efficacy of a brief MI intervention using EEG\/ERP graphical feedback for crack cocaine abusers. Treatment-seeking cocaine abusers (N\u00a0=\u00a031) were randomly assigned to a two-session MI intervention or a general educational drug conseiling (control) condition. All participants received EEG assessments based on dense-array ERP tests in a selective attention task at intake and post-treatment. Results indicated that the MI intervention was feasible and the subjective impact of the EEG\/ERP feedback was positive. Significant group differences in percentage of cocaine positive urine screens across the study were found, favoring the MI group; 84.9% for the control group and 62.6% in the MI group.\nIn a current study at the University of Louisville, Tato Sokhadze and his colleagues are utilizing dense-array qEEG\/ERP variables and measures of behavioral performance on mental tasks (reaction time, accuracy) to explore the cognitive functions in patients with cocaine abuse\/dependence diagnosis, and the recovery of these functions during bio-behavioral intervention based on an integrated neurofeedback approach (NFB, Scott\u2013Kaiser protocol) and motivational enhancement therapy (MET) in an outpatient population. The purpose of this research is also to characterize changes in cognitive functioning associated with the success rate of three arms for cocaine addiction treatment (MET, NFB, combined MET\u00a0+\u00a0NFB). Prior, during, and subsequent to the above bio-behavioral therapies, individual differences in qEEG and dense-array ERP are being assessed during cognitive tasks containing drug-related and generally affective cues, and during cognitive tasks aimed to test cortical inhibitory capacity, selective attention, response error processing, and cortical functional connectivity. Preliminary data from this study were presented at the 2007 annual meeting of ISNR (Sokhadze et\u00a0al. 2007b) and are being prepared for the publication.\nEfficacy of Alpha Theta Training\nThe Guidelines for Evaluation of Clinical Efficacy of Psychophysiological Interventions (LaVaque et\u00a0al. 2002), which have been accepted by AAPB and ISNR, specify five types of classification for the effectiveness of biofeedback procedures, ranging from \u201cNot empirically supported\u201d to \u201cEfficacious and Specific.\u201d The requirements for each classification level are summarized in brief below. A more complete description may be found in LaVaque et\u00a0al. (2002).\nCriteria for Levels of Evidence of Efficacy\nLevel 1:Not empirically supported. This classification is assigned to those treatments that have only been described and supported by anecdotal reports and\/or case studies in non-peer reviewed journals.\nLevel 2:Possibly efficacious. This classification is considered appropriate for those treatments that have been investigated in at least one study that had sufficient statistical power, well-identified outcome measures, but lacked randomized assignment to a control condition internal to the study.\nLevel 3:Probably efficacious. Treatment approaches that have been evaluated and shown to produce beneficial effects in multiple observational studies, clinical studies, wait list control studies, and within-subject and between-subject replication studies merit this classification.\nLevel 4:Efficacious. In order to be considered \u201cefficacious,\u201d a treatment must meet the following criteria:In a comparison with a no-treatment control group, alternative treatment group, or sham (placebo) control utilizing randomized assignment, the investigational treatment is shown to be statistically significantly superior to the control condition or the investigational treatment is equivalent to a treatment of established efficacy in a study with sufficient power to detect moderate differences;The studies have been conducted with a population treated for a specific problem, from whom inclusion criteria are delineated in a reliable, operationally defined manner;The study used valid and clearly specified outcome measures related to the problem being treated;The data are subjected to appropriate data analysis;The diagnostic and treatment variables and procedures are clearly defined in a manner that permits replication of the study by independent researchers, andThe superiority or equivalence of the investigational treatment have been shown in at least two independent studies\u201d (LaVaque et\u00a0al. 2002, p. 280).\nLevel 5:Efficacious and Specific. To meet the criteria for this classification, the treatment needs to be demonstrated to be statistically superior to a credible sham therapy, pill, or bona fide treatment in at least two independent studies.\nUsing these criteria and based on the studies reported to date alpha-theta training can be classified as Level 3\u2014probably efficacious\u2014when combined with an inpatient rehabilitative treatment modality in subjects with long standing alcohol dependency. This classification is based on the original randomized and controlled study of the Peniston Protocol (Peniston and Kulkosky 1989, 1990, 1991) and multiple observational and uncontrolled studies that preceeded (Twemlow and Bowen 1977, Twemlow et\u00a0al. 1977) and followed these studies (DeBeus et\u00a0al. 2002; Burkett et\u00a0al. 2003; Fahrion et\u00a0al. 1992; Finkelberg et\u00a0al. 1996; Skok et\u00a0al. 1997; Bodenhamer-Davis and Calloway 2004; Saxby and Peniston 1995; Fahrion 1995).\nUsing these criteria and based on reported studies to date the Scott\u2013Kaiser modification of the Peniston Protocol can also be classified as probably efficacious (Level 3) when combined with residential rehabilitation modalities in stimulant abusers. This rating is based on one controlled study of 121 subjects in which Peniston\u2019s outcomes of both psychometric improvement and abstinence improvement were replicated (Scott et\u00a0al. 2005) and one observational study of 71 subjects (Burkett et\u00a0al. 2003).\nAlpha-theta training protocols do not completely meet the criteria for the Level 4, \u201cefficacious\u201d classification. Although there are sufficient studies that show statistically significant superiority of randomly assigned treatment groups to no-treatment control groups, and studies have been conducted with populations treated for a specific problem, from whom inclusion criteria are delineated in a reliable, operationally defined manner, and the studies cited use valid and clearly specified outcome measures related to the problem being treated with data subjected to appropriate data analysis, there remains the shortcoming cited by Graap and Freides (1998) for the initial reports of Peniston and Kulkosky (1989, 1990, 1991). We recall the qualifying limitations of LaVaque et\u00a0al. (2002), who stated that \u201cthe diagnostic and treatment variables and procedures are not clearly defined in a manner that permits replication of the study by independent researchers\u201d (p. 280). However, the Scott et\u00a0al. (2005) report does appear to clearly delineate treatment variables and procedures. One other independent study showing the superiority of modified alpha-theta training to control condition would meet the stated criteria for a Level 4 \u201cefficaceous\u201d classification.\nTo be considered Level 5 (\u201cefficacious and specific\u201d) modified alpha-theta training would need to be shown to be superior to sham or bona fide treatment. It has not been demonstrated that the Peniston type alpha-theta feedback is more efficacious than sham treatment (Trudeau 2000, 2005a, b; Lowe 1999; Moore and Trudeau 1998) or alternative treatment that involves meditation (Taub and Rosenfeld 1994).\nClinical Considerations: Comorbidities of SUD and Implications for Individualized (qEEG-Guided) Neurofeedback\nThere are several conditions commonly associated with addictive disorders that have known neurophysiological aberrations. The co-occurrence of alcohol and other SUD with other psychiatric disorders has been widely recognized. Co-occurrence of SUD and other psychiatric diagnosis (e.g., PTSD, antisocial personality disorder, ADHD, unipolar depression etc.) is highly prevalent (Drake and Walach 2000; Evans and Sullivan 1995; Grant et\u00a0al. 2004; Jacobsen et\u00a0al. 2001). Persons with co-occurring other mental disorders and SUD have a more persistent illness course and are more refractive to treatment than those without dual diagnoses (Brown et\u00a0al. 1995; O\u2019Brien et\u00a0al. 2004; Schubiner et\u00a0al. 2000; Swartz and Lurigio 1999). Depression occurs in approximately 30% of chronic alcoholics (Regier et\u00a0al. 1990). In treatment settings, these depressed patients can present particular challenges to the clinician, as they may not respond as well to treatment as other patients, may have greater relapse, attrition, and readmission rates, and may manifest symptoms that are more severe, chronic, and refractory in nature (Sheehan 1993). Independent of other psychiatric comorbidity, ADHD alone significantly increases the risk for SUD (Biederman et\u00a0al. 1995). Associated social and behavioral problems may make individuals with comorbid SUD and ADHD treatment resistant (Wilens et\u00a0al. 1998). In males ages 16\u201323, the presence of childhood ADHD and conduct disorder is associated with non-alcohol SUD (Gittleman et\u00a0al. 1985; Manuzza et\u00a0al. 1989). In summary childhood ADHD associated with conduct disorder in males is an antecedent for adult non-alcohol SUD and anti-social personality disorder (Wender 1995). The incidence of ADHD in clinical SUD populations has been studied and may be as high as 50% for adults (Downey et\u00a0al. 1997) and adolescents (Horner and Scheibe 1997). Adult residual ADHD is especially associated with cocaine abuse and other stimulant abuse (Levin and Kleber 1995). Monastra et\u00a0al. (2005) in a white paper review of ADHD, cite positive treatment outcomes of just under 80% in treatment of ADHD with neurofeedback.\nRates of PTSD occurring in persons primarily identified with or in treatment for substance abuse vary from 43% (Breslau et\u00a0al. 1991) up to 59% (Triffleman et\u00a0al. 1999). In a general population study, Cottler et\u00a0al. (1992) reported that cocaine abusers were three times more likely to meet diagnostic criteria for PTSD compared to individuals without a SUD. Kalechstein et\u00a0al. (2000) found that methamphetamine-dependent individuals are at greater risk to experience particular psychiatric symptoms. There was reported a significant dependence-by-gender effect, with methamphetamine-dependent females reporting significantly more overall posttraumatic stress symptomatology compared to females reporting no dependence, whereas males significantly differed only with respect to depression. Peniston and Kulkosky (1991) reported effective treatment of PTSD using a protocol similar to the one they employed for alcoholics.\nHughes and John (1999) review the applicability of qEEG findings in SUD. They note that in numerous qEEG studies there is a consensus of increased beta relative power in alcoholism and increased alpha in cannabis and crack cocaine users. They conclude that the evidence provided by studies to date is insufficient to recommend qEEG as a routine clinical assessment tool in SUD, although it may be useful in differential diagnosis in difficult cases. A number of specific qEEG abnormalities have been described as specific to suspected neurotoxicities associated with chronic stimulant abuse. These studies (Alper et\u00a0al. 1990; Noldy et\u00a0al. 1994; Prichep et\u00a0al. 1996; Roemer et\u00a0al. 1995; Trudeau et\u00a0al. 1999) based on reasonably uniform abstinence times and employing different EEG technology and analytical approaches, have produced remarkably similar findings of alpha relative amplitude excess with delta relative amplitude deficit that is striking. Excess alpha amplitude with slowing of alpha frequency associated with chronic cannabis abuse has been reported (Struve et\u00a0al. 1998). As noted, Scott and Kaiser (1998) describe combining a protocol for attentional training (beta reward) with alpha-theta training in a population of subjects whose primary drugs of abuse were stimulants and who had features of ADHD.\nIt may make good sense clinically to consider specific neurotherapy treatment of these disorders either in place of or preceding alpha-theta therapy, similar to the Scott\u2013Kaiser approach. Second, applicable neurotherapy approaches are attractive alternative therapies for coexisting or underlying conditions in SUD clients who have high-risk behaviors for medication treatment, such as overdosing, abuse, or poor compliance. While there are no published systematic studies of neurotherapy treatment of co-occurring depression, TBI, ADHD, PTSD, or drug neurotoxicity on the course and outcome of addictive disorders, several recent reports of neurotherapy for addictions based on qEEG findings, which in turn may be related to comorbidities, have been presented. Basically, this technique involves the use of qEEG to identify patterns of EEG that deviate from standardized norms, and individualized EEG biofeedback protocols to correct them (Romano-Micha 2003). DeBeus et\u00a0al. (2002) are presently conducting a randomized controlled study of neurotherapy for SUD that examines the difference between a qEEG-based treatment, a research-based (Scott\u2013Peniston) treatment, and a wait-list control for chemically dependent outpatients. Preliminary results are promising. While historically, alpha-theta training has been the accepted approach in treating chemical dependency, this study suggests qEEG-based training is a viable alternative, demonstrating similar outcomes for personality change and abstinence rates. Future directions include determination of those likely to benefit from one of the particular treatments or a combination of the two and analysis of long-term abstinence rates. Gurnee (2004) has presented data on a series of 100 sequential participants with SUD who were treated by qEEG-based neurotherapy, with marked heterogeneity of qEEG subtypes and corresponding symptom complexes. In this clinically derived scheme, qEEGs that deviate from normative databases, mainly with excess alpha amplitude, are associated more often with depression and ADD. Those with deficient alpha amplitude are associated with anxiety, insomnia, and alcohol\/drug abuse. Beta excess amplitude is associated with anxiety, insomnia, and alcohol\/drug abuse. Central abnormalities are interpreted as mesial frontal dysfunction and are associated with anxiety, rumination, and obsessive compulsive symptoms. The therapeutic approach is to base neurotherapy on correcting identified qEEG abnormalities, i.e., train beta excess amplitude down when present, while monitoring symptoms.\nTentative findings suggest that qEEG variables may be used to predict those alcoholics and drug abusers most at risk for relapse. Winterer et\u00a0al. (1998) were able to predict relapse among chronic alcoholics with 83\u201385% success, significantly outperforming prediction from clinical variables. Although they found more desynchronized (less alpha and theta and more beta activity) over frontal areas in alcoholics in general, those individuals who relapsed displayed even more of this activity. Bauer (2001) obtained EEG data on alcohol, cocaine or opioid dependent patients after 1\u20135\u00a0months of sobriety. Those who had relapsed by 6\u00a0months later were also characterized by increased beta (19.5\u201339.8\u00a0Hz) activity relative to those maintaining abstinence. Relative beta power was superior to severity of the alcoholism, depression level, antisocial personality disorder, childhood conduct problems, family history, or age as predictors, and was unaffected by the substance of abuse. The EEG differences between relapse-prone and abstinence-prone groups were found to be related to the interaction of two premorbid factors: childhood conduct disorder and paternal alcoholism. These findings receive further support from Bauer (1993) and from Prichep et\u00a0al. (1996) who also found that beta activity was predictive of treatment failure. They found two clusters among cocaine addicts: One had more severe damage (alpha) and tended to remain in treatment. Those with less severe alpha excess and more beta activity tended to leave treatment. They also discovered that dropouts could not be determined from the presence of anxiety or depression or demographic variables.\nTreatment of patients with substance abuse disorder by neurofeedback may become more complicated when patients present various psychiatric conditions. When addiction is comorbid with ADHD it is suggested that SMR (or beta increase, theta decrease) training should be conducted to address the related ADHD disorder first (Biederman et\u00a0al. 1997). Applicability of neurofeedback methods to treat anxiety and affective disorders is reviewed by Hammond (2006). Peniston and Kulkosky (1990) describe personality normalization in alcoholics treated with EEG biofeedback. Alpha-theta feedback has also been reported as efficacious in alcoholics with depressive symptoms (Saxby and Peniston 1995). There are only a few case studies on the efficacy of neurofeedback for treating generalized anxiety disorder (Vanathy et\u00a0al. 1998) and PTSD (Huang-Storms et\u00a0al. 2006; Graap et\u00a0al. 1997). Alpha-theta feedback has been described as efficacious in post-combat PTSD (Peniston and Kulkosky 1991; Peniston et\u00a0al. 1993). However, additional research needs to be completed to determine the clinical outcome and efficacy of bio-behavioral treatment based on brain wave self-regulation in addiction disorders that are comorbid with various anxiety disorders and PTSD.\nClinical Considerations: Cognitive-Behavioral and Neurofeedback Treatment in Substance Use Disorders\nBecause of its chronic nature, long-term treatment for SUD is usually necessary (Crits-Christoph et\u00a0al. 1997, 1999). Effective agonist and antagonist pharmacotherapies as well as symptomatic treatments exist for opioid dependence, but neither agonists nor antagonists have been approved as uniquely effective for treatment of stimulant abuse or dependence (Grabowski et\u00a0al. 2004). There is no current evidence supporting the clinical use of carbamazepine (Tegretol), antidepressants, dopamine agonists (drugs commonly used to treat Parkinson\u2019s and Restless Leg Syndrome), disulfiram (Antabuse), mazindol (an experimental anorectic), phenytoin (Dilantin), nimodipine (Nimotop), lithium and other pharmacological agents in the treatment of cocaine dependence (de Lima et\u00a0al. 2002; Venneman et\u00a0al. 2006). Because no proven effective pharmacological interventions are available for cocaine addiction or for methamphetamine addiction, treatment of stimulant addiction has to rely on existing cognitive-behavioral therapies (CBT) or CBT combined with other biobehavioral approaches (Van den Brink and van Ree 2003).\nAccording to Volkow et\u00a0al. (2004) successful strategies for behavioral treatment in drug addiction may include (1) interventions aimed to decrease the reward value of the drug and simultaneously increase values of natural reinforcement; (2) approaches aimed to change stereotype conditioned drug-seeking behaviors; and (3) methods to train and strengthen frontal inhibitory control. Because stressful events can result in relapse to drug taking behavior (Koob and Le Moal 2001), an adjunct treatment strategy is to interfere with the neurobiological responses to stress (Goeders 2003; Koob and Le Moal 2001). Treatment of comorbid mental conditions may also require the concurrent treatment of drug addiction. In some cases, however, comorbid drug addiction may result from attempts to alleviate the psychiatric disorder through self-medication (i.e., co-occurring cocaine use and ADHD and\/or heroin addiction co-occurring with PTSD). In other cases, severity of a psychiatric disorder symptom may increase as a result of drug abuse (Volkow et\u00a0al. 2003, 2004).\nIn patients with drug abuse arising from an attempt to self-medicate (Khantzian 1985, 1997), treatment of the comorbid mental disorder may help prevent abuse. For instance, treatment of the preexisting condition of ADHD may prevent cocaine abuse (Biederman et\u00a0al. 1995, 1997). In some cases though the persistent qEEG abnormalities associated with chronic SUD may happen to be independent from ADHD clinical status (Trudeau et\u00a0al. 1999). The co-occurrence of ADHD and SUD has received considerable attention in the recent clinical and scientific literature (Davids et\u00a0al. 2005). These two disorders are often linked to one another. Because the core symptoms of ADHD may be mimicked by the effects of psychoactive drugs, it is difficult to diagnose one disorder in the presence of the other (Davids et\u00a0al. 2005). ADHD has been found to be associated with an earlier onset of SUD (Horner and Scheibe 1997). It is generally assumed that untreated ADHD is a risk factor for SUD development (Biederman et\u00a0al. 1997, 1998; Manuzza et\u00a0al. 1998; Trudeau 2005a, b).\nIn a case of comorbidity in which the use of drugs antecedes a mental disease (e.g., substance-induced anxiety disorder, DSM-IV-TR, APA 2000) or is not driven by self-medication strategies, the simultaneous treatment of both psychiatric conditions may be required. In this situation, treatment could be guided by the two following concepts: (1) Behavioral interventions to activate and strengthen circuits involved in inhibitory control, such as bio-behavioral self-regulation training, may increase successful abstinence from drug taking. (2) Considering the important role of cognitive and emotional processes involved in the predisposition for drug abuse, the development of non-pharmacological interventions (e.g., CBT, stress management, neurofeedback) is a feasible strategy.\nDirections for Further Research \nSpecific patterns of qEEG abnormality associated with specific substance use toxicity such as those found in stimulant abuse or alcohol abuse or with comorbidities such as ADHD (Chabot and Serfontein 1996), PTSD (Huang-Storms et\u00a0al. 2006) or TBI (Thatcher et\u00a0al. 1989) suggest underlying brain pathologies that might be amenable to EEG biofeedback that is tailored to the person. These approaches would likely be individualized rather than protocol based, and would be used independently or in conjunction with classic alpha-theta training. By way of example, these could include protocols specific to the qEEG abnormality, such as frontal delta reward to correct the frontal delta deficit in cocaine abuse that Alper (1999) hypothesizes may be related to cocaine sensitization and changes in dopamine transmission. To our knowledge this has never been studied and is clearly a research (not a clinical) recommendation. The qEEG patterns and abnormalities depend significantly on whether the subject is still currently using, the chronicity of use, and the current stage of withdrawal or protracted abstinence. A neurofeedback protocol selected for an individual client with SUD should be directly related to the level of current substance use or abstinence, especially in such classes of drugs as heroin, where the withdrawal syndrome results in substantial physiological manifestations including transient qEEG changes.\nEven though there are no reported systematic studies of EEG biofeedback treatment of commonly occurring comorbidities of SUD, it makes sense that clinical EEG biofeedback treatment study protocols consider the presence of ADHD, TBI, depression, and drug-associated neurotoxicity. This approach may improve outcome, especially in conventional treatment resistant participants.\nUnfortunately, only a few large-scale studies of neurofeedback in addictive disorders have been reported in the literature. Most, if not all of the recommendations previously made regarding further research (Trudeau 2000, 2005b) have yet to be implemented. These recommendations are summarized as follows.Studies require external, systematic replicability of brain wave feedback methods and results in diverse populations that include various control and alternative treatment conditions wherein the groups are matched on key dimensions.Details need to be given regarding the equipment that was used and the associated technical specifications (e.g., details about amplification, filtering, spectral extraction, windowing, and other pertinent information) needed by neurofeedback specialists for replication and comparison.The essential components and durations for brain wave feedback required for therapeutic advantage need to be stated, including double-blinded studies that control for all other possible therapeutic effects.Open clinical trials that investigate efficacy of the types of protocols used for ADHD, PTSD, depression, and TBI remediation with SUD subjects comorbid for those conditions need to be reported.Open clinical trials that assess the efficacy of EEG biofeedback in addressing the specific qEEG changes of chronic alcohol, heroin, cannabis and stimulant abuse need to be reported.The physiological and psychological processes of the therapeutic effects of EEG biofeedback, including studies of qEEG and ERP changes, need to be investigated and reported.Studies need to adhere to clearly defined outcome measures that have established reliability and validity.\nOther important recommendations for future development of the field are listed below:The availability of an increased number of channels for EEG and ERP recording (e.g., higher spatial sampling rate) makes it possible to better localize the source of brain activity. More focused research of this type seems warranted.There are several specific functional diagnostic tools from the cognitive neuroscience arsenal that are very specific for testing addictive disorders. Those that may be especially valuable include cue reactivity tests using qEEG and ERP measures. Cue reactivity is a very sensate test of motivational relevance of drug-related items (Carter and Tiffany 1999) that can be detected using EEG methods.In addition to using more traditional neurocognitive tests (TOVA, IVA+, etc.) that are commonly included in neurofeedback research (e.g., in particular in studies on effectiveness of neurotherapy in ADHD treatment) there may be value in incorporating standardized tests with EEG\/ERP recording to assess executive functions in addicts. Tests that warrant mention are the Continuous Performance Test (Go-NoGo task), Stroop test, Eriksen flanker test, etc. Some of these tests are sufficiently sensitive for assessing recovery of cortical inhibition function commonly known to be impaired in patients with SUD.Testing emotional reactivity and responsiveness in addiction is another important domain where qEEG and ERP methods may help to obtain more effective evaluation of the affective state of recovering addicts.\nIn future neurofeedback treatment for SUD attempts should be made to integrate neurotherapy with other well known behavioral interventions for drug abuse, such as cognitive-behavioral therapy (CBT) and motivation enhancement therapy (MET, Miller and Rollnick 2002). As a population, drug addicts are very difficult to treat, characterized by a low motivation to change their drug habit and a reluctance to enter inpatient treatment. CBT and MET are powerful psychotherapeutic interventions that can help to bring about rapid commitment to change addictive behaviors. These behavioral therapies are especially useful for enhancing compliance with drug-dependent individuals and facilitating their neurofeedback treatment engagement.\nNeurofeedback may be among the most promising biofeedback modalities for the treatment of adolescents with addictive disorders because of the neuroplasticity potential of the adolescent brain. While there is little work available on the prevention and treatment of SUD in adolescents utilizing neurotherapy, there is no reason to suspect that the approaches used in adults would not be applicable in SUD adolescents (Trudeau 2005b).\nThe EEG biofeedback treatment of ADHD may be important in prevention for children and adolescents at risk for developing SUD. It may be possible that EEG biofeedback therapy of childhood ADHD may result in a decrease in later life SUD (Wilens et\u00a0al. 1998). This remains speculative, as there have been no reported studies of the effects of neurofeedback treatment on prevention of SUD to date.\nThere are several important applications of the neurofeedback protocols for enhancement of cognitive performance in healthy subjects (reviewed in Vernon 2005). This promising new line of neurofeedback-based cognitive neuroscience research (Barnea et\u00a0al. 2005; Egner and Gruzelier 2001, 2003, 2004a, b; Egner et\u00a0al. 2004; Vernon et\u00a0al. 2003) has significant potential to elucidate neurobiological mechanisms explaining how neurofeedback training may alter and enhance cognition and behavioral performance in patients with SUD as well.\nDrugs of abuse can impair cognitive, emotional and motivational processes. More qEEG and cognitive ERP research is needed to characterize the chronic and residual effects of drugs on attention, emotion, memory, and overall behavioral performance. More research is needed also to relate cognitive functionality measures to clinical outcome (e.g., relapse rate, drug screens, psychiatric status, etc.). Such qEEG\/ERP studies may facilitate the translation of clinical neurophysiology research data into routine practical tools for assessment of functional recovery both in alcoholism and addiction treatment clinics. We believe that administration of some of above described qEEG assessments at the pre-treatment baseline might provide useful predictors of clinical outcome and relapse risk. Incorporation of cognitive tests with EEG and ERP (e.g., P300) measures into cognitive-behavioral and neurofeedback based interventions may have significant potential for identifying whether certain qEEG\/ERP measures can be used as psychophysiological markers of treatment progress (and\/or relapse vulnerability), and also may provide useful information in planning cognitive-behavioral and neurotherapy treatment when substance abuse is comorbid with a mental disorder.\nWith the advances made in the last several years, it is hoped that continued interest will be generated to further study brainwave biofeedback treatment of addictive disorders. Effectiveness in certain \u201chard to treat\u201d populations (conventional treatment resistant alcoholics, crack cocaine addicts, cognitively impaired substance abusers) is promising. The prospect of an effective medication free, neurophysiologic, and self-actualizing treatment for a substance based, brain impaired, and self-defeating disorder such as SUD is attractive.","keyphrases":["eeg biofeedback","substance use disorder","neurofeedback","alcoholism","neurotherapy","erp","quantitative eeg","cognitive-behavioral treatment"],"prmu":["P","P","P","P","P","P","P","R"]}
{"id":"Psychopharmacologia-4-1-2362139","title":"Expression of amphetamine sensitization is associated with recruitment of a reactive neuronal population in the nucleus accumbens core\n","text":"Rationale Repeated exposure to psychostimulant drugs causes a long-lasting increase in the psychomotor and reinforcing effects of these drugs and an array of neuroadaptations. One such alteration is a hypersensitivity of striatal activity such that a low dose of amphetamine in sensitized animals produces dorsal striatal activation patterns similar to acute treatment with a high dose of amphetamine.\nIntroduction\nRepeated exposure to psychostimulant drugs causes a long-lasting enhancement of certain behavioral responses to the drug, such as psychomotor activity and stereotypy, and behaviors related to incentive motivation, a process termed behavioral sensitization (Stewart and Badiani 1993). Behavioral sensitization is known to be associated with long-lasting functional changes within limbic corticostriatal systems (Pierce and Kalivas 1997; Robinson and Kolb 2004; Vanderschuren and Kalivas 2000). These systems comprise functionally and anatomically heterogeneous areas with a fine-grained specificity of anatomical projections connecting the divisions within the dorsal striatum, ventral striatum, and prefrontal cortex (Groenewegen et al. 1997; Voorn et al. 2004). This anatomical and functional heterogeneity is of potential importance to the roles of these areas in sensitization.\nWithin the dorsal striatum, subareas termed patches (or striosomes) show more reactivity to amphetamine than the surrounding matrix areas in sensitized animals (Canales and Graybiel 2000; Vanderschuren et al. 2002). Our previous studies demonstrated that this pattern of neuronal reactivity is also seen in acutely challenged animals with the important difference that sensitized animals show preferential activation in patches at much lower doses of amphetamine than those required to produce this type of differentiation in activation in drug-naive animals (Vanderschuren et al. 2002). An imbalance in patch\u2013matrix activation has been suggested to underlie stereotyped behavior (Canales and Graybiel 2000). Because our drug treatment regimen caused robust locomotor sensitization, which is incompatible with profound stereotypy, we hypothesized that hyperreactivity of patch compartments is not sufficient to produce stereotypy (Vanderschuren et al. 2002). To extend our previous findings, we ran new experiments using the same doses and regimen of amphetamine administration previously used and measured locomotor activity, stereotypy, grooming, and rearing to establish whether our drug treatment regimen, which causes hyperreactivity of dorsal striatal patches, produces stereotypy.\nThe ventral striatum, specifically the nucleus accumbens (Acb), and the prefrontal cortex (PFC) are both involved in behavioral sensitization (Pierce and Kalivas 1997; Vanderschuren and Kalivas 2000), an involvement which has received particular attention because of the important role that both areas play in appetitive and consummatory properties of both natural and drug rewards (Everitt and Wolf 2002; Robbins and Everitt 2002; Salamone et al. 2003; Volkow and Li 2004). There are functional differences between subregions within both the Acb, i.e., core and shell, and within subregions of the PFC (Cardinal et al. 2002; Robbins and Everitt 2002). However, there is presently inconclusive evidence on the respective roles that the subregions play during the expression of behavioral sensitization to psychostimulants. Studies using neurochemistry, lesions, cellular activity markers, and study of morphological changes have suggested exclusive roles for the core (Cadoni et al. 2000; Li et al. 2004; Phillips et al. 2003) or the shell (Filip and Siwanowicz 2001; Hsieh et al. 2002; Pierce and Kalivas 1995; Todtenkopf et al. 2002a). Other studies, including previous work from our own laboratory, suggest a lack of sensitization of accumbens activity all together (Ostrander et al. 2003; Vanderschuren et al. 2002). The medial PFC, and particularly the prelimbic area, has been implicated in induction of psychostimulant sensitization (Tzschentke and Schmidt 1998, 2000), although conflicting results have been found for cocaine- vs. amphetamine-induced sensitization (Tzschentke and Schmidt 2000). The dorsomedial prefrontal cortex has been shown to be involved in the expression of sensitization (Pierce et al. 1998). However, the roles of the orbital and lateral areas in psychostimulant sensitization remain to be investigated. Thus, clarification of the specific roles of the Acb and PFC subregions is needed. To study the activation of the Acb and PFC in detail during the expression of behavioral sensitization, we examined levels of c-fos-like proteins (henceforth c-fos) in detail in the subregions of the Acb and PFC of the rat after an amphetamine challenge in behaviorally sensitized rats.\nMaterials and methods\nAnimals and drug treatments\nAll experiments were approved by the Animal Ethics Committee of the Vrije Universiteit and were conducted in agreement with Dutch laws (Wet op de Dierproeven 1996) and European regulations (Guideline 86\/609\/EEC).\nA total of 48 male Wistar rats weighing 180\u2013200\u00a0g upon arrival in the laboratory (as in, e.g., De Vries et al. 1996; Vanderschuren et al. 1999a, b, 2002) were housed in Macrolon cages in groups of two animals per cage under controlled laboratory conditions (lights on 0700 to 1900 hours). Food and water were available ad libitum. Drug treatment started after an acclimatization period of at least 1\u00a0week. Animals were briefly handled during the 2\u00a0days before all injections. In the acute amphetamine experiments, animals were injected with either saline or 1, 2.5, or 5\u00a0mg\/kg d-amphetamine sulfate (O.P.G., Utrecht, The Netherlands; n\u2009=\u20094 per dose). Sensitization regimens were according to a protocol previously established to produce locomotor sensitization in our laboratory (De Vries et al. 1996; Vanderschuren et al. 1999a, b), and doses were the same as used previously in a c-fos study in our laboratory (Vanderschuren et al. 2002). Animals received once daily injections for five consecutive days of 2.5\u00a0mg\/kg d-amphetamine sulfate or saline in the home cage (pretreatment phase). Two weeks post treatment, half of the animals from each pretreatment group were given challenge injections of 1\u00a0mg\/kg d-amphetamine sulfate while the other half was injected with saline. This gave a total of four experimental groups, n\u2009=\u20098 per group: amphetamine-pretreated, amphetamine-challenged (AA); amphetamine-pretreated, saline-challenged (AS); saline-pretreated, amphetamine-challenged (SA); and saline-pretreated, saline-challenged (SS).\nLocomotor activity quantification and behavioral scoring\nAll injections for the acute experiments and challenge injections for the sensitization experiments took place in our locomotor activity setup. On the challenge injection day, animals were first placed in the Perspex cages (length\u2009\u00d7\u2009width\u2009\u00d7\u2009height\u2009=\u200940\u2009\u00d7\u200940\u2009\u00d7\u200935\u00a0cm) in which locomotor activity was measured and allowed to acclimate for 2\u00a0h. After that period, challenge injections were administered and horizontal activity was measured in 10-min blocks for 90\u00a0min using a video tracking system (EthoVision, Noldus Information Technology B.V., Wageningen, The Netherlands), which determined the position of the animal five times per second. Behavior of the animals was also videotaped and scored afterwards for three mutually exclusive categories: grooming (rubbing two paws over head and\/or body), rearing (both front paws off of the ground but not grooming), or stereotypical behavior (repeated movements without horizontal movement, e.g., head shaking). Behavior was scored for 5\u00a0min of every 15\u00a0min, giving a total of seven measurements per animal, by an observer unaware of the treatment of the animals using a time-sampling program written in PC Basic. Time spent performing each behavior was expressed as the percentage of total time for each 5-min block. Replication scoring several months after initial scoring produced results identical to initial observations, demonstrating the reliability of our scoring procedures.\nc-fos Immunocytochemistry\nAt 90\u00a0min after the challenge injection, animals were decapitated, the brains were snap-frozen in isopentane and stored at \u221280\u00b0C until use. Sections of 20\u00a0\u03bcm were cut on a cryostat and mounted onto coated slides (SuperFrost Plus) which were dried and stored at \u221280\u00b0C until use. For visualization of c-fos, sections were defrosted and fixed in a 4% paraformaldehyde solution in phosphate-buffered saline (PBS; 0.1\u00a0M, pH\u00a07.4). Sections were washed with Tris-buffered saline (TBS, 0.1\u00a0M, pH\u00a07.4) then incubated with primary antibody against c-fos (1:1,800, Oncogene Research, Burlington, MA, USA) in TBS with 0.5% Triton-X and 0.5% bovine serum albumin (TBS-TX-BSA) overnight at 4\u00b0C. After rinsing with TBS, endogenous peroxidase activity was removed by incubation of sections in a 1% hydrogen peroxide solution for 15\u00a0min. Sections were rinsed with TBS, then incubated in biotinylated goat antirabbit antibody (1:100, Dako, Denmark) in TBS-TX-BSA for 1\u00a0h, washed in TBS and incubated in avidin\u2013biotin complex with horseradish peroxidase (HRP) (1:100, Vector Laboratories, Burlingame, CA, USA) for 1\u00a0h. Sections were rinsed in Tris\u2013HCl then incubated in 3\u20323-diaminobenzidine (DAB; Sigma Chemical, 0.05% DAB in Tris\u2013HCl) and rinsed in Tris\u2013HCl. Sections containing the prefrontal cortex were incubated with Hoechst 33258 (1:2,000; Molecular Probes, Eugene, OR, USA), a fluorescent nuclear stain used to visualize cytoarchitecture. Sections were dried and finally coverslipped with Merckoglas (Merck, Darmstadt, Germany).\nHistological quantification\nQuantification of c-fos immunopositive nuclei was performed using an MCID Elite imaging system (Imaging Research, Ontario, Canada). Images of the nucleus accumbens in the c-fos DAB immunostained sections were digitized using an objective magnification of \u00d710 on a Leica DM\/RBE photomicroscope with a Xillix MicroImager digital camera (1,280\u2009\u00d7\u20091,024\u00a0pixels). Digitized images were combined so that the core and the shell areas were included, using the MCID tiling tool. Three (in some cases, two) sections per rat were chosen for quantification at the rostral\u2013caudal levels in which inputs from the prefrontal cortex, thalamus, and amygdala have been particularly well characterized (Wright and Groenewegen 1995). The prefrontal cortex was digitized in the same fashion with the exception that color digital images were acquired using a Sony HAD camera (Sony DXC 950v, 640\u2009\u00d7\u2009512\u00a0pixels) of both the DAB staining and the epifluorescence of the Hoechst 33258 staining. The core and shell areas of the nucleus accumbens were delineated on the basis of atlas drawings from sections stained for calbindin (Jongen-Relo et al. 1993). The prefrontal cortex was delineated into prelimbic, infralimbic, orbital, and lateral areas on the basis of cytoarchitectonic criteria visible in the Hoechst 33258 staining.\nThe c-fos immunopositive nuclei in the nucleus accumbens were segregated from background staining levels using several point operators and spatial filters combined in an algorithm designed to detect local changes in the relative optical density (ROD). Briefly, images underwent histogram equalization and smoothing (low-pass filter, kernel size 7\u2009\u00d7\u20097). The unfiltered image was subtracted from the smoothed image, followed by a series of steps to optimize the processed image and make it a suitable measuring template for detecting objects the size and shape of c-fos immunopositive nuclei. This algorithm was preferred over ROD thresholding because it does not involve an observer-dependent operation. The number of nuclei counted was corrected with a factor indicating approximate size of a c-fos immunopositive nucleus, thus preventing two groups of stained pixels touching one another in the image being mistakenly counted as one nucleus. The results of all counting were expressed as the number of nuclei per surface area (mm2). Integrated ROD for each segmented immunopositive nucleus was determined. Segregation of c-fos positive nuclei in the color-digitized images was performed in a similar fashion.\nSubsequently, we set out to compare c-fos positive density (i.e., the number of cells per surface area) in a manner that accounts for labeling intensity: dark, light, or midrange. First, histograms of c-fos nuclei ROD values (for black and white images) or intensity values (for color images) were constructed for each brain area for each treatment group and qualitatively compared. These histograms were used to determine the value of the 33rd and 66th percentile optical density within the SS group. Based on these values, all nuclei from all animals for each area were binned as \u201clight\u201d (ROD values under the 33rd percentile of the SS group), \u201cmidrange\u201d (ROD values between the 33rd and 66th percentile of the SS group) or \u201cdark\u201d (ROD values above the 66th percentile of the SS group). The number of immunopositive nuclei per bin was counted per rat, and the group averages were determined from the rat averages. For the prefrontal cortex, the same technique was used, but as these areas were digitized in color, intensity was used to bin rather than optical density. An increase in the number of nuclei in the \u201cdark\u201d bin would signify a rightward shift in the histograms, indicating that the increased cellular activity measured was primarily the result of more c-fos expression in the same group of neurons. An increase in the number of nuclei in the \u201cmidrange\u201d bin would signify an upward shift in the histograms, indicating that the increase in the total number of nuclei measured was the result of the addition of a new group of nuclei to the cellular response (Fig.\u00a01).\nFig.\u00a01Theoretical conceptualization of shifts in the ROD histograms. The total number of c-fos positive nuclei per mm2 counted is represented as the area under the curves to the right of the detection level, indicated as an arrow on the x-axis. Curve 1 indicates the control group; vertical lines indicate the ROD used to separate the neurons into light, midrange, and dark. An increase in the treatment group compared to the control group in the total number of c-fos positive nuclei per mm2 could be the result of an increase in the frequency of c-fos positive nuclei, indicated by an increase in frequency in curve 2, causing an increase in the number of c-fos positive nuclei in the midrange. This would indicate that a new group of neurons is being recruited in the c-fos response, represented by the difference between curves 1 and 2 in frequency. Alternatively, the same number of neurons could be active, but expressing more c-fos protein. This would cause a rightward shift in the curve (curve 3) and allowing more c-fos positive nuclei to come above the detection level and causing more c-fos positive nuclei to be measured in the dark range. A similar line of reasoning can be followed for other options, for instance a leftward shift in the case of reduced levels of c-fos protein (not illustrated)\nThe dorsal striatum of the AA group was qualitatively inspected by two observers, both blind to the experimental conditions. The distribution pattern of c-fos positive nuclei was described and compared with that in a series of closely adjacent sections from the same animals stained immunocytochemically for the \u03bc-opioid receptor to visualize striatal patches (Vanderschuren et al. 2002).\nStatistics\nFor the quantification of c-fos immunoreactivity, the experimental groups were compared for effects of pretreatment and challenge (saline vs. amphetamine) and for interactions between these effects using a two-way ANOVA test followed by a Tukey post hoc test. For locomotor activity and behavioral scores, a repeated-measures ANOVA was conducted using time as within-subjects factor and followed by a Tukey post hoc test.\nResults\nBehavioral results\nFor all behavioral measures, time was included as a within-subjects measure in a repeated-measures ANOVA followed by a post hoc test when significant time\u2009\u00d7\u2009pretreatment or time\u2009\u00d7\u2009challenge interactions were found. For brevity, only the most relevant of the results of these post hoc test results are described in this section and other results are presented in the figures.\nBehavioral measures: responses to acute amphetamine\nLocomotor activity was dose-dependently altered in animals treated acutely with amphetamine (Fig.\u00a02a; main effect dose F(3,10)\u2009=\u200943.914; p\u2009<\u20090.001). Saline-treated animals showed generally low activity levels, averaging a total of 1,335\u2009\u00b1\u2009514\u00a0cm traveled during the 90-min period. The groups treated with 1\u00a0mg\/kg (14,045\u2009\u00b1\u20091,836\u00a0cm) and 5\u00a0mg\/kg amphetamine (14,906\u2009\u00b1\u20092,321\u00a0cm) showed comparable levels of locomotor activity, whereas the highest locomotor response to amphetamine was observed in the 2.5\u00a0mg\/kg group (25,543\u2009\u00b1\u20091,704\u00a0cm). All amphetamine-treated groups differed from saline in the post hoc test. Acute amphetamine caused a significant increase in rearing (F(3,12)\u2009=\u20097.971; p\u2009<\u20090.005; Fig.\u00a02b), although not dose-dependently, as all doses differed significantly from the saline-treated group in post hoc testing, but there were no significant differences among the amphetamine-treated groups (average percent time spent rearing, saline\u2009=\u20092.1\u2009\u00b1\u20090.75; 1\u00a0mg\/kg\u2009=\u200915.2\u2009\u00b1\u20092.7; 2.5\u00a0mg\/kg\u2009=\u200920.4\u2009\u00b1\u20093.5; 5\u00a0mg\/kg\u2009=\u200916.8\u2009\u00b1\u20097.7). No significant effect of amphetamine on grooming was seen (main effect of dose: F(3,12)\u2009=\u20093.098, n.s.; Fig.\u00a02b). Stereotypy was exclusively observed in the group treated with 5\u00a0mg\/kg of amphetamine (average percent time spent in stereotypical behavior, 5\u00a0mg\/kg\u2009=\u200928.4\u2009\u00b1\u200916.5; all other groups average\u2009=\u20090\u2009\u00b1\u20090; Fig.\u00a02b). A clear main effect of dose was seen (F(3,12)\u2009=\u200911.724; p\u2009<\u20090.005). The 5-mg\/kg group differed significantly from all other groups in the post hoc test.\nFig.\u00a02Locomotor activity (a) and behavioral observations (b) for animals treated acutely with saline or 1, 2.5, or 5\u00a0mg\/kg amphetamine. The highest levels of locomotor activity were seen in the 2.5-mg\/kg treated group, which differed significantly from all other groups in the post hoc tests. Stereotypical behavior was observed in the animals treated with 5\u00a0mg\/kg amphetamine, which differed significantly from all other groups, but no stereotypy was seen in any of the other treatment groups. No significant differences were seen in grooming behavior (b). All amphetamine-treated groups showed significantly more rearing than the saline-treated group (b), but the amphetamine treatment groups did not differ from one another. Bars indicate group averages, error bars represent SEM, n\u2009=\u20094 per group. Asterisks indicate significant difference in post hoc testing (p\u2009<\u20090.05)\nBehavioral measures: responses to challenge after repeated amphetamine\nPretreatment with amphetamine caused a clear-cut augmentation of the locomotor response to amphetamine, as illustrated in Fig.\u00a03a. Overall across the 90-min period, the AA group of amphetamine-pretreated rats that were challenged with amphetamine on the test day showed a 70% increase in activity compared to the SA group, which was pretreated with saline and challenged with amphetamine (total traveled distance during the 90-min test period\u2009=\u200920,432\u2009\u00b1\u20093,403\u00a0cm by AA compared to 11,924\u2009\u00b1\u20092,149\u00a0cm by SA). Both groups that were challenged with saline on the test day (AS and SS, pretreated with amphetamine and saline, respectively) showed considerably less locomotor activity, showing approximately 10% of the activity displayed by the AA group (total traveled distance during 90\u00a0min, SS\u2009=\u20091,770\u2009\u00b1\u2009493\u00a0cm; AS\u2009=\u20092,196\u2009\u00b1\u2009623\u00a0cm). These differences were reflected in significant main effects of pretreatment (F(1,20)\u2009=\u20096.15; p\u2009<\u20090.05) and challenge (F(1,20)\u2009=\u200962.07; p\u2009<\u20090.001), and a significant pretreatment\u2009\u00d7\u2009challenge interaction (F(1,20)\u2009=\u20095.029; p\u2009<\u20090.05).\nFig.\u00a03Locomotor activity (a) and behavioral observations (b) in amphetamine-pretreated and amphetamine-challenged animals and control groups. Bars represent group average of locomotor activity (a) or percentage of time spent displaying each mutually exclusive category of behavior (grooming, rearing or stereotypical behavior; b). No bars are present representing stereotypical behavior because this behavior was not observed in any animals included in this experiment. Error bars indicate SEM, n\u2009=\u20096 per group. AA amphetamine-pretreated, amphetamine-challenged; SA saline-pretreated, amphetamine-challenged; AS amphetamine-pretreated, saline-challenged; SS saline-pretreated, saline-challenged. Asterisks indicate significant difference in post hoc testing (p\u2009<\u20090.05)\nAs shown in Fig. 3b, the evaluation of the percentage of time spent rearing revealed an increase in rearing in the AA group compared to all other groups (average over 90\u00a0min, AA\u2009=\u200914.3\u2009\u00b1\u20092.9%, AS\u2009=\u20091.84\u2009\u00b1\u20090.64%, SA\u2009=\u20097.09\u2009\u00b1\u20091.2%, SS\u2009=\u20093.34\u2009\u00b1\u20090.92%). This was confirmed by a significant main effect of challenge (F(1,20)\u2009=\u200927.52; p\u2009<\u20090.001) and pretreatment\u2009\u00d7\u2009challenge interaction (F(1,20)\u2009=\u20097.96; p\u2009<\u20090.05). No main effect of pretreatment was observed for this parameter (F(1,20)\u2009=\u20093.41; n.s.).\nPercentages of time spent grooming were generally low with average scores under 4% in all groups (average over 90\u00a0min, AA\u2009=\u20091.66\u2009\u00b1\u20090.64, AS\u2009=\u20093.54\u2009\u00b1\u20091.97, SA\u2009=\u20091.78\u2009\u00b1\u20090.86, SS\u2009=\u20093.97\u2009\u00b1\u20090.87; Fig.\u00a03b). Despite the somewhat higher averages in the saline-challenged groups compared to the amphetamine-challenged groups, no significant effects of pretreatment, challenge, or interaction between the two were seen on grooming (pretreatment: F(1,20)\u2009=\u20090.64, n.s.; challenge: F(1,20)\u2009=\u20093.42, n.s.; pretreatment\u2009\u00d7\u2009challenge: F(1,20)\u2009=\u20090.21, n.s.).\nNo stereotyped behavior was observed in any animal in any of the groups included in the repeated amphetamine experiments during any of the time periods scored (Fig.\u00a03b).\nCellular reactivity results\nIn the nucleus accumbens and prefrontal cortex, the quantification of c-fos positive nuclei was tested for statistical significance by two-way ANOVAs using pretreatment and challenge as factors, followed by a Tukey HSD post hoc. For brevity, results of these post hoc tests are presented in the figures and in Table\u00a01.\nTable\u00a01Mean\u00b1SEM density (number of cells per mm2) of c-fos immunoreactive nuclei in light, dark, and midranges of cellular staining intensity\u00a0Treatment groupAAASSASSNucleus accumbensCoreLight21.4\u2009\u00b1\u20096.917.6\u2009\u00b1\u20094.217.9\u2009\u00b1\u20093.221.2\u2009\u00b1\u20094.2Midrange\u00a734.2\u2009\u00b1\u20092.8&19.0\u2009\u00b1\u20093.2#20.5\u2009\u00b1\u20093.722.1\u2009\u00b1\u20095.1Dark\u00b6\/****94.5\u2009\u00b1\u200914.1&\/+35.6\u2009\u00b1\u20094.6#61.2\u2009\u00b1\u200912.424.1\u2009\u00b1\u20093.6#ShellLight28.8\u2009\u00b1\u20098.726.9\u2009\u00b1\u20097.328.8\u2009\u00b1\u20095.938.3\u2009\u00b1\u20096.4Midrange42.3\u2009\u00b1\u20093.241.7\u2009\u00b1\u20099.234.5\u2009\u00b1\u20096.640.3\u2009\u00b1\u20096.6Dark*96.1\u2009\u00b1\u200917.163.6\u2009\u00b1\u20099.286.5\u2009\u00b1\u200920.744.5\u2009\u00b1\u20097.6Prefrontal cortexPrelimbicLight48.2\u2009\u00b1\u20095.034.9\u2009\u00b1\u20096.739.3\u2009\u00b1\u20095.432.2\u2009\u00b1\u20093.8Midrange**50.0\u2009\u00b1\u20096.931.0\u2009\u00b1\u20096.046.4\u2009\u00b1\u20097.027.7\u2009\u00b1\u20096.3Dark***74.3\u2009\u00b1\u200912.432.0\u2009\u00b1\u20096.7%82.28\u2009\u00b1\u200918.8+\/&28.7\u2009\u00b1\u20097.9%InfralimbicLight37.6\u2009\u00b1\u20093.833.1\u2009\u00b1\u20095.830.4\u2009\u00b1\u20095.629.9\u2009\u00b1\u20095.1Midrange46.7\u2009\u00b1\u20098.334.9\u2009\u00b1\u20097.835.0\u2009\u00b1\u20097.928.3\u2009\u00b1\u20095.4Dark***78.1\u2009\u00b1\u200913.7+40.7\u2009\u00b1\u20097.170.3\u2009\u00b1\u200914.530.2\u2009\u00b1\u20096.9#OrbitalLight27.8\u2009\u00b1\u20093.624.7\u2009\u00b1\u20096.123.0\u2009\u00b1\u20094.321.0\u2009\u00b1\u20094.4Midrange**61.6\u2009\u00b1\u20098.5+32.6\u2009\u00b1\u20097.745.0\u2009\u00b1\u20098.430.7\u2009\u00b1\u20095.9#Dark****129.7\u2009\u00b1\u200921.7&\/+32.6\u2009\u00b1\u20098.6#120.4\u2009\u00b1\u200924.2+26.3\u2009\u00b1\u20095.7#\/%LateralLight*22.8\u2009\u00b1\u20092.314.8\u2009\u00b1\u200910.018.1\u2009\u00b1\u20092.814.4\u2009\u00b1\u20093.3Midrange*39.9\u2009\u00b1\u20095.219.3\u2009\u00b1\u20095.127.4\u2009\u00b1\u20097.618.4\u2009\u00b1\u20093.7Dark****69.5\u2009\u00b1\u200911.6&\/+18.3\u2009\u00b1\u20094.4#65.1\u2009\u00b1\u200915.6+16.7\u2009\u00b1\u20094.6#\/%Results of ANOVA testing are shown in the column indicating ROD range as follows: paragraph mark significant main effect of pretreatment, asterisk significant main effect of challenge, section mark significant pretreatment\u2009\u00d7\u2009challenge interaction; one symbol: p\u2009<\u20090.05, two symbols: p\u2009<\u20090.01, three symbols: p\u2009<\u20090.005, four symbols: p\u2009<\u20090.001. Results of post hoc testing are indicated in the cells containing the value of the average number of nuclei as follows: plus sign significantly different from SS, percent sign significantly different from SA, ampersand significantly different from AS, number sign significantly different from AA.\nSensitization alters distribution of reactivity in dorsal striatum\nIn the dorsal striatum, visual inspection of the sections from animals both pretreated and challenged with amphetamine (AA group) showed overall more c-fos positive nuclei compared to the AS group, and a differential distribution throughout the area (Fig.\u00a04). c-fos positive nuclei were more abundant medially than laterally. Within the general distribution, heterogeneity of reactivity was seen conforming to patterns previously observed in patches in sensitized animals. Comparison of c-fos staining pattern with \u03bc-opioid stained patches in sections from another series from the same animals confirmed the location of high concentrations of nuclei in \u03bc-opioid stained patches.\nFig.\u00a04Digital micrographs of c-fos immunopositive nuclei in the dorsal striatum of representative animals from groups pretreated with amphetamine and challenged with either saline (a, group AS) or amphetamine (b, group AA). Scale bars indicate 100\u00a0\u03bcm, arrows point to c-fos immunopositive nuclei. Note the higher number of c-fos positive nuclei in the AA animal compared to the SA animal, and the inhomogeneous distribution of nuclei in the AA animal\nCellular reactivity sensitizes in nucleus accumbens core, but not accumbens shell or prefrontal cortex\nWithin the nucleus accumbens, the core and shell subdivisions showed different c-fos immunoreactivity response patterns to an amphetamine challenge after the sensitizing regimen (Fig.\u00a05a\u2013d and Fig.\u00a06). The core showed a clear effect of sensitization with a number of c-fos positive nuclei in the AA group 48% higher than that of the SA group and more than 100% compared to both saline-challenged groups (Fig.\u00a06). Significant main effects of pretreatment (F(1,28)\u2009=\u200910.59; p\u2009<\u20090.005) and challenge (F(1,28)\u2009=\u200944.75; p\u2009<\u20090.001) and a significant pretreatment\u2009\u00d7\u2009challenge interaction (F(1,28)\u2009=\u20097.55; p\u2009<\u20090.05) confirmed this observation. In the nucleus accumbens shell, more c-fos positive nuclei were counted in the amphetamine than saline-challenged groups, but no sensitization effect was seen (Fig.\u00a06). A significant main effect of challenge (F(1,28)\u2009=\u20094.41; p\u2009<\u20090.05), but not of pretreatment (F(1,28)\u2009=\u20090.57; n.s.), was observed, and no pretreatment\u2009\u00d7\u2009challenge interaction (F(1,28)\u2009=\u20090.04; n.s.) was present in the ANOVA of the nucleus accumbens shell data.\nFig.\u00a05Digital micrographs of c-fos immunopositive nuclei and representations of nuclei quantified in the nucleus accumbens from representative animals treated acutely with amphetamine (SA group; a, c, and e) and challenged with amphetamine after amphetamine pretreatment (AA group; b, d, and f). The low magnification overviews of nucleus accumbens in a (SA animal) and b (AA animal) show differences in the number of c-fos immunopositive nuclei between the two experimental groups. Corresponding detail images of the nucleus accumbens core, shown in c (detail from b) and d (detail from b), are medial to anterior commissure. Nuclei counted for quantification in c and d are illustrated in e and f, respectively. Red squares indicate immunoreactive nuclei classified as \u201cdark,\u201d green squares as \u201cmidrange,\u201d and blue squares as \u201clight\u201d (see text for classification procedures). Broad white arrows in d and f indicate a \u201cdark\u201d immunopositive nucleus and its representation in the counted nuclei; gray arrows represent \u201cmidrange\u201d; narrow white arrows indicate \u201clight.\u201d Scale bar in a indicates 300\u00a0\u03bcm for a and b; scale bar in c represents 100\u00a0\u03bcm for c and d. cp caudate putamen, ac anterior commissure, core nucleus accumbens core, shell nucleus accumbens shellFig.\u00a06Mean number of c-fos immunopositive nuclei in nucleus accumbens core and shell and prelimbic, infralimbic, orbital, and lateral prefrontal cortex. Two-way ANOVA analysis of accumbens data showed significant main effects of pretreatment and challenge and a pretreatment\u2009\u00d7\u2009challenge interaction in the nucleus accumbens core. A significant effect of challenge was observed in the nucleus accumbens shell and for all PFC areas measured. Bars represent the number of c-fos immunopositive nuclei per mm2, error bars indicate SEM, n\u2009=\u20098 per group. Asterisk indicates significant differences in post hoc testing. See Fig.\u00a03 for abbreviations of treatment groups\nIn the prefrontal cortex, an increase in c-fos positive nuclei in amphetamine-challenged groups compared to saline-challenged groups was observed in all four subregions measured. No differences were seen between amphetamine- and saline-pretreated animals in the number of c-fos positive nuclei after an amphetamine challenge (Fig.\u00a06). Significant main effects of challenge were observed in all areas (PL: F(1,28)\u2009=\u200913.87, p\u2009<\u20090.001; IL: F(1,28)\u2009=\u20096.94, p\u2009<\u20090.05; orbital PFC: F(1,28)\u2009=\u200925.96, p\u2009<\u20090.001; lateral PFC: F(1,28)\u2009=\u200920.55, p\u2009<\u20090.001). No significant effects of pretreatment (PL: F(1,28)\u2009=\u20090.14, n.s.; IL: F(1,28)\u2009=\u20091.20, n.s.; orbital PFC: F(1,28)\u2009=\u20090.945, n.s.; lateral PFC: 0.79, n.s.) or pretreatment\u2009\u00d7\u2009challenge interactions (PL: F(1,28)\u2009=\u20090.08, n.s.; IL: F(1,28)\u2009=\u20090.037, n.s.; orbital PFC: F(1,28)\u2009=\u20090.039; lateral PFC: F(1,28)\u2009=\u20090.67) were observed.\nFrequency distributions of relative optical densities across groups show sensitization of midrange in accumbens core\nWhen comparing the ROD of the c-fos positive nuclei of each area across groups, significantly higher (=darker) averages were seen in amphetamine- compared to saline-challenged groups in the core (optical density 0.1216\u2009\u00b1\u20090.0026 in saline-challenged, 0.1372\u2009\u00b1\u20090.0040 in amphetamine-challenged; F(1,28)\u2009=\u200910.815, p\u2009<\u20090.005), the shell (optical density 0.1208\u2009\u00b1\u20090.0023 in saline-challenged, 0.1307\u2009\u00b1\u20090.0035 in amphetamine-challenged; F(1,28)\u2009=\u20095.513, p\u2009<\u20090.05), the infralimbic area (intensity 0.7173\u2009\u00b1\u20090.0057 in saline-challenged, 0.6985\u2009\u00b1\u20090.0660 in amphetamine-challenged; F(1,28)\u2009=\u20094.368, p\u2009<\u20090.05), the orbital prefrontal area (intensity 0.7181\u2009\u00b1\u20090.0048 in saline-challenged, 0.6921\u2009\u00b1\u20090.0073 in amphetamine-challenged; F(1,28)\u2009=\u20098.258, p\u2009<\u20090.001), and the lateral prefrontal area (intensity 0.7208\u2009\u00b1\u20090.0045 in saline-challenged, 0.6992\u2009\u00b1\u20090.0067 in amphetamine-challenged; F(1,28)\u2009=\u20096.753, p\u2009<\u20090.05). Significant differences were seen between the AA and SS groups and between the SA and SS group in the core in post hoc testing. No significant differences were seen in the shell in post hoc testing.\nThe distributional patterns of the cellular ROD suggested differences in the distributions of ROD across the treatment groups. To further analyze the optical densities and compare the frequencies of the various optical densities between groups, histograms of the optical densities of all cells per area, per group were constructed (Figs.\u00a05e\u2013f and 7a and b) and used to divide immunostained nuclei into light, midrange, and dark optical density (OD) ranges based on the 33rd and 66th percentile values in the SS group (Table\u00a01), as described in the \u201cMaterials and methods\u201d section. Two-way ANOVAs for pretreatment\u2009\u00d7\u2009challenge were conducted, followed by Tukey HSD post hoc tests. For brevity, the results of the post hoc tests are indicated in Table\u00a01.\nFig.\u00a07Representations of distributions of optical densities in the nucleus accumbens. In a and b, histograms of relative optical densities of individual c-fos immunopositive nuclei in nucleus accumbens core (a) and shell (b) are depicted. T1 and T2 lines represent the 33rd and 66th percentile values, respectively, for the SS group. In the nucleus accumbens core, the AA group shows more neurons in the midrange segment (between T1 and T2), as confirmed by the analysis of number of cells per segment (c). A significant pretreatment\u2009\u00d7\u2009challenge interaction is present in the midrange of the nucleus accumbens core. The amphetamine-challenged groups both contain more neurons in the dark segment of both core (c) and shell (d) compared to saline-challenged groups, as evidenced by a significant effect of challenge in the ANOVA test. No significant differences were observed in post hoc testing in the shell; asterisks indicate significant differences in post hoc testing for core in c (p\u2009<\u20090.05). Line colors and patterns or bar fillings indicating groups are as shown in a and c. For c and d, bars represent the number of c-fos positive nuclei in each OD segment per mm2, error bars indicate SEM, n\u2009=\u20098 per group. See Fig. 2 for abbreviations of treatment groups\nIn both the core and the shell, amphetamine-challenged animals showed higher numbers of c-fos positive nuclei than saline-pretreated animals in the dark range (core challenge: F(1,28)\u2009=\u200923.928, p\u2009<\u20090.001; shell challenge: F(1,28)\u2009=\u20096.421, p\u2009<\u20090.05). A significant effect of pretreatment was seen in the core in this range (F(1,28)\u2009=\u20095.202, p\u2009<\u20090.05), and no effect was seen in the shell (F(1,28)\u2009=\u20090.949, n.s.). No pretreatment\u2009\u00d7\u2009challenge interactions were observed in the core (pretreatment\u2009\u00d7\u2009challenge: F(1,28)\u2009=\u20091.227, n.s.; Fig.\u00a07c and Table\u00a01) or shell (pretreatment\u2009\u00d7\u2009challenge: F(1,28)\u2009=\u20090.103, n.s.; Fig.\u00a07d and Table\u00a01) in this range. In the midrange nuclei, the core area showed a distinct pattern of distribution compared to all other areas measured as a sensitization effect was confirmed by a significant pretreatment\u2009\u00d7\u2009challenge interaction (F(1,28)\u2009=\u20094.901, p\u2009<\u20090.05; Fig.\u00a07c), while no significant main effects were observed (pretreatment: F(1,28)\u2009=\u20091.985, n.s.; challenge: F(1,28)\u2009=\u20093.195, n.s.). No differences between groups were observed in the midrange nuclei in the shell (pretreatment: F(1,28)\u2009=\u20090.474, n.s.; challenge: F(1,28)\u2009=\u20090.146, n.s.; pretreatment\u2009\u00d7\u2009challenge: F(1,28)\u2009=\u20090.229, n.s.; Fig.\u00a07d). No differences between groups were seen in the light range nuclei in the core (Fig.\u00a07c; pretreatment: F(1,28)\u2009=\u20090.000, n.s.; challenge: F(1,28)\u2009=\u20090.002, n.s.; pretreatment\u2009\u00d7\u2009challenge: F(1,28)\u2009=\u20090.532, n.s.) or the shell (Fig.\u00a07d; pretreatment: F(1,28)\u2009=\u20090.624, n.s.; challenge: F(1,28)\u2009=\u20090.280, n.s.; pretreatment\u2009\u00d7\u2009challenge: F(1,28)\u2009=\u20090.632, n.s.).\nWithin the prefrontal cortex, the areas measured showed a relatively homogenous pattern of distribution of c-fos immunoreactivity across the OD ranges (Table\u00a01). In the dark range, all areas showed significantly more c-fos positive nuclei in the amphetamine-treated groups compared to the saline-treated groups (main effect challenge: PL: F(1,28)\u2009=\u200914.974, p\u2009<\u20090.005; IL: F(1,28)\u2009=\u200911.563, p\u2009<\u20090.005; orbital: F(1,28)\u2009=\u200931.370, p\u2009<\u20090.001; lateral: F(1,28)\u2009=\u200923.751, p\u2009<\u20090.001) but no effects of pretreatment (main effect pretreatment: PL: F(1,28)\u2009=\u20090.035, n.s.; IL: F(1,28)\u2009=\u20090.645, n.s.; orbital: F(1,28)\u2009=\u20090.209, n.s.; lateral: F(1,28)\u2009=\u20090.092, n.s.) or interaction between pretreatment and challenge (pretreatment\u2009\u00d7\u2009challenge: PL: F(1,28)\u2009=\u20090.207, n.s.; IL: F(1,28)\u2009=\u20090.014, n.s.; orbital: F(1,28)\u2009=\u20090.008, n.s.; lateral: F(1,28)\u2009=\u20090.015, n.s.). In the midrange, increased numbers of c-fos positive nuclei in amphetamine-challenged animals were seen in the prelimbic (F(1,28)\u2009=\u20098.081, p\u2009<\u20090.01), orbital (F(1,28)\u2009=\u20097.941, p\u2009<\u20090.01), and lateral (7.029, p\u2009<\u20090.05) areas, but not the infralimbic area (F(1,28)\u2009=\u20091.545, n.s.). No effects were seen in the midrange segment of pretreatment (main effect pretreatment: PL: F(1,28)\u2009=\u20090.265, n.s.; IL: F(1,28)\u2009=\u20091.511, n.s.; orbital: F(1,28)\u2009=\u20091.453, n.s.; lateral: F(1,28)\u2009=\u20091.461, n.s.), and no pretreatment\u2009\u00d7\u2009challenge interactions were seen (pretreatment\u2009\u00d7\u2009challenge: PL: F(1,28)\u2009=\u20090.001, n.s.; IL: F(1,28)\u2009=\u20090.113, n.s.; orbital: F(1,28)\u2009=\u20090.928, n.s.; lateral: F(1,28)\u2009=\u20091.076, n.s.). In the light range, only the lateral prefrontal cortex showed a significant increase in c-fos positive nuclei in the amphetamine-challenged groups (main effect challenge: F(1,28)\u2009=\u20094.228, p\u2009<\u20090.05). No other challenge effects were observed (main effect challenge: PL: F(1,28)\u2009=\u20093.751, n.s.; IL: F(1,28)\u2009=\u20090.243, n.s.; orbital: F(1,28)\u2009=\u20090.302, n.s.). No effects of pretreatment (main effect pretreatment: PL: F(1,28)\u2009=\u20091.225, n.s.; IL: F(1,28)\u2009=\u20091.060, n.s.; orbital: F(1,28)\u2009=\u20090.863, n.s.; lateral: F(1,28)\u2009=\u20090.793, n.s.) or of sensitization (pretreatment\u2009\u00d7\u2009challenge interaction: PL: F(1,28)\u2009=\u20090.356, n.s.; IL: F(1,28)\u2009=\u20090.156, n.s.; orbital: F(1,28)\u2009=\u20090.015, n.s.; lateral: F(1,28)\u2009=\u20090.573, n.s.) were seen in the light range of the PFC c-fos immunopositive nuclei.\nDiscussion\nIn the present study, we set out to characterize the behavioral response to amphetamine in drug-naive and amphetamine-pretreated animals, as we had previously observed that treatment with a high dose (5\u00a0mg\/kg) of amphetamine in drug-naive animals resulted in a similar pattern of cellular reactivity in the dorsal striatum as a challenge with an intermediate dose (1\u00a0mg\/kg) in amphetamine-pretreated, behaviorally sensitized rats (Vanderschuren et al. 2002). Furthermore, we aimed to characterize the reactivity of subregions within the nucleus accumbens and prefrontal cortex during the expression of psychostimulant sensitization. Our results showed that previously demonstrated similarity of cellular activation patterns in the dorsal striatum after amphetamine in sensitized or drug-naive rats was not accompanied by similar behavioral responses. Thus, animals given acute amphetamine at a dose that produces heightened patch activation (5\u00a0mg\/kg) displayed intense stereotypy, which was not seen during the expression of sensitization at a challenge dose (1\u00a0mg\/kg) that also produces higher activation of patches than surrounding matrix. Cellular activation was sensitized in response to a drug challenge in the nucleus accumbens core, but not the shell or the prefrontal cortex. Examination of the distribution patterns of the optical densities of the individual immunopositive nuclei revealed an upward shift in the histograms of the core in the AA group, resulting in a significant increase in the midrange stained nuclei in the accumbens core in sensitized animals.\nSensitization of locomotor activity and rearing without stereotypy\nAfter amphetamine pretreatment, an augmented locomotor and rearing response to amphetamine was seen compared to saline-pretreated, amphetamine-challenged animals, thus supporting the notion that stereotypy and patch hyperreactivity could take place independently. The behavioral pattern of the amphetamine-pretreated, amphetamine-challenged group most resembled the animals treated acutely with 1.0 or 2.5\u00a0mg\/kg of amphetamine. No stereotyped behavior was observed in the sensitized animals after an amphetamine challenge, while intense stereotypy was seen in animals treated acutely with 5\u00a0mg\/kg of amphetamine. In our previous paper (Vanderschuren et al. 2002), we noted that the decrease in locomotion normally observed in animals treated acutely with a high dose of amphetamine was likely a result of stereotypy, an idea which is strengthened by the present observations.\nIn previous studies by our laboratory and others, animals treated acutely with the relatively high dose of 5\u00a0mg\/kg of amphetamine (Graybiel et al. 1990; Vanderschuren et al. 2002) or with the same amphetamine regimen for sensitization followed by a challenge with 1\u00a0mg\/kg amphetamine used in the present study (Vanderschuren et al. 2002) show enhanced ratios of response in patches compared to the matrix in the dorsal striatum, findings visually confirmed in the present set of experiments. We concluded on the basis of our previous results that the changes seen in striatal reactivity represent a shift in sensitivity to amphetamine rather than long-term adaptations in circuitry. If the increased sensitivity to amphetamine underlying behavioral sensitization paralleled the hyperreactivity of patches, the same pattern of behavior would be expected in animals showing similar immediate-early gene expression patterns, namely, the 5-mg\/kg acutely challenged group and the amphetamine-sensitized, amphetamine-challenged group. However, the expression of stereotypy in the 5-mg\/kg group, not seen in the sensitized animals, and the erratic locomotor activity seen in the 5-mg\/kg group, clearly indicate behavioral differences after the two treatments. It is interesting to note that the dissociation of hyperreactivity of patches and sensitized behavioral responses manifests in a lack of stereotypical behavior in sensitized animals, as increased reactivity of patches compared to the matrix has been suggested to underlie stereotypical behavior (Canales and Graybiel 2000). Given the fact that the sensitization regimen and challenge dose used in the present study causes heightened patch to matrix ratios of c-fos expression, but does not cause stereotyped behavior, changes in striatal patterns of activity are apparently not causally related to stereotypy.\nSensitization of immediate-early gene activation in a specific neural population of the nucleus accumbens core\nThe sensitized immediate-early gene expression we observed in the nucleus accumbens core during the expression of psychostimulant sensitization after a withdrawal period of 14\u00a0days contradicts a number of previous immediate-early gene studies where no sensitization effect was observed in the core (Ostrander et al. 2003; Todtenkopf et al. 2002a), including one study from our own laboratory (Vanderschuren et al. 2002). There are two main differences between the present study and the previous study from our group. First, in our previous study, a 3-week abstinence period was observed, while in the present study, animals were tested after 14\u00a0days of abstinence. It is interesting to note that studies examining immediate-early gene expression after shorter abstinence periods (2\u00a0days) do show sensitization of reactivity within the core (Hedou et al. 2002; Todtenkopf et al. 2002a), indicating that abstinence time is of potential importance for sensitization of c-fos in the nucleus accumbens core. However, this explanation seems unsatisfactory as the study by Totenkopf et al. (2002a) also tested c-fos expression after 2\u00a0weeks of abstinence and found no sensitization of c-fos expression in the nucleus accumbens core. This discrepancy might be explained by a second difference between the studies where no effect was observed and the present study: in studies finding no effect of sensitization on the nucleus accumbens core, animals were at least partially pretreated and challenged in the same environment. Exposure to amphetamine in a relatively novel environment has been shown to potentiate the c-fos response to the drug in the nucleus accumbens core (Ostrander et al. 2003).The importance of the testing environment was recently underscored by a study showing that sensitized c-fos responses to cocaine in the nucleus accumbens only occurred when cocaine was always administered in a discrete environment outside of the home cage (Hope et al. 2006).\nExpression of c-fos in the striatum can be elicited by stimulation of dopamine receptors (Berretta et al. 1992), which have been demonstrated to be crucially involved in the expression of amphetamine sensitization (Vanderschuren and Kalivas 2000). As mentioned in the \u201cIntroduction\u201d, a number of studies point to the sensitization of dopamine transmission in the accumbens core during the expression of sensitization (Cadoni et al. 2000; Phillips et al. 2003), and increased dopamine is also seen in the core compared to the shell in yoked controls in cocaine and heroin self-administration studies (Lecca et al. 2007a; Lecca et al. 2007b). It is thus possible that dopamine may play a role in eliciting the increased c-fos response seen in the accumbens core in the present study.\nIn the accumbens core, more cells in the midrange of optical densities were present in the amphetamine-pretreated, amphetamine-challenged group than in all other groups. This \u201cupward\u201d shift in sensitized animals, visible in the histogram of optical density frequencies, indicates that the increase in c-fos positive nuclei is not due to nuclei that were under our detection threshold for c-fos increasing their reactivity enough to be measured. In the case of increased c-fos expression within the same population of nuclei that normally respond to amphetamine, a rightward shift in the histogram of optical densities would be expected, which would manifest in a sensitization effect in the dark range and increase of the average optical density. No increase was seen in the overall average optical density, no significant interaction observed between pretreatment and challenge in the dark group, and there is no significant difference between the AA and SA groups in the dark group. However, an obvious trend to sensitization in the darkly stained group is visible in Fig.\u00a06. Although the lack of significance precludes firm conclusions, it might be that some neurons which normally respond to amphetamine by producing c-fos react by producing more c-fos after sensitization.\nThe increase of frequency of neurons stained in the midrange without a significant shift in average optical density suggests the recruitment of a new population of nuclei becoming responsive to amphetamine in the sensitized animals. The population most likely to be involved in that sensitized response is the dopamine D2 receptor-containing, enkephalin-positive population that project via the subthalamic nucleus to the substantia nigra pars reticulata and entopenduncular nucleus. Indeed, sensitization of cocaine-induced c-fos expression in the nucleus accumbens in animals treated with the drug in a discrete environment was only found in enkephalin-positive cells, but not in dynorphin-positive, dopamine D1 receptor-expressing neurons that directly project to the substantia nigra (Hope et al. 2006). Differences in responsivity to amphetamine in these two cellular populations have also been demonstrated in a study where dopamine D2 receptor-containing neurons became activated when amphetamine is administered in a novel environment (Badiani et al. 1999). Remarkably, c-fos expression in dopamine D1 receptor-containing accumbens neurons was shown to be important for the induction of cocaine sensitization (Zhang et al. 2006), suggesting that these two different cell populations play distinct roles in the induction and expression of behavioral sensitization.\nImmediate-early gene responses to acute amphetamine in the nucleus accumbens shell and prefrontal cortex\nThe cellular response of the nucleus accumbens shell to acute amphetamine, which did not sensitize after repeated amphetamine, corresponds well with previous results from our laboratory demonstrating the same effect (Vanderschuren et al. 2002). The role of the shell in the expression of psychostimulant sensitization is not clear-cut. On the one hand, psychostimulant sensitization-induced long-term changes in cellular reactivity or cellular morphology are not generally found in the nucleus accumbens shell after psychostimulant administration (Cadoni et al. 2000; Li et al. 2004; Todtenkopf et al. 2002a). Moreover, post induction lesions of the shell leave the expression of cocaine sensitization intact (Todtenkopf et al. 2002b), although preinduction lesions of a subarea of the shell result in reduced sensitized, but not acute responses to cocaine (Brenhouse and Stellar 2006). On the other hand, microinjection of amphetamine or cocaine into the shell (but not core) produces sensitized psychomotor responses and augmented dopamine levels in animals pretreated with cocaine (Filip and Siwanowicz 2001; Pierce and Kalivas 1995), and sensitized cellular reactivity has been observed in specific subareas within the accumbens shell after repeated cocaine administration (Brenhouse et al. 2006; Todtenkopf et al. 2002a). Discrepancies between studies finding the effects of sensitization in the shell and the present study could be due to the use of cocaine pretreatment and\/or challenges in other studies and amphetamine pretreatment and challenge in the present study. Cocaine and amphetamine have been observed to produce different activation patterns in the dorsal striatum with acute amphetamine producing activation of patches and acute cocaine producing a more homogenous staining pattern (Graybiel et al. 1990); it is possible that cocaine and amphetamine pretreatment produce different patterns of reactivity in the ventral striatum as well.\nWithin the prefrontal cortex, all areas showed c-fos expression in response to amphetamine, but there was no effect of amphetamine preexposure. To our knowledge, this is the first examination of c-fos expression in orbital and lateral prefrontal areas. However, the lack of sensitization of c-fos expression in the medial prefrontal cortex corresponds well with results from other groups showing similar results after 2\u00a0weeks of abstinence (Todtenkopf et al. 2002a). Sensitization of c-fos immunoreactivity does occur when animals are challenged after 2\u00a0days of abstinence (Hedou et al. 2002; Todtenkopf et al. 2002a, b), suggesting a role in the induction or early phases of sensitization. In support of a role for the prefrontal cortex in the induction and early phases of expression, lesion studies have demonstrated region-specific effects in the involvement of prefrontal areas in the induction of cocaine sensitization (Tzschentke and Schmidt 2000), and lesions of the entire medial prefrontal cortex have been shown to prevent the induction of amphetamine sensitization (Wolf et al. 1995; Cador et al. 1999, but see Tzschentke and Schmidt 2000). Studies examining expression of psychostimulant sensitization suggest that the dorsal areas of the medial prefrontal cortex that project to the nucleus accumbens core are involved in the expression of psychostimulant sensitization (Steketee 2003). Although no sensitization of immediate-early gene expression was observed in the present study, a dorsal\u2013ventral gradient was seen in acute response to amphetamine with the prelimbic area showing a 74% increase in the SA group over the SS group and the infralimbic area showing a smaller increase at 45%. The high responsivity of lateral prefrontal neurons to amphetamine (114% increase in SA over SS) is also interesting in this respect, as the projections from this area to the nucleus accumbens are nearly exclusive to the nucleus accumbens core (Berendse et al. 1992). The highly active prefrontal cortical inputs projecting to the core may play a role in enhancing the activation of the core during the expression of sensitization.\nRemarkably, the highest responsivity to acute amphetamine was found in the orbital frontal area, a prefrontal cortex area that projects primarily to the dorsal striatum and only very sparsely to the nucleus accumbens (Berendse et al. 1992). Metabolic and structural changes within the orbital prefrontal cortex have been demonstrated in human drug addicts and nonhuman primates and rodents with a history of drug self-administration (Crombag et al. 2005; Porrino and Lyons 2000; Volkow and Li 2004). A recent study showed that rats sensitized to cocaine are impaired on a task that is sensitive to orbital prefrontal lesions (Schoenbaum et al. 2004). The high responsivity of this area to acute amphetamine indicates that corticostriatal circuits not involving the nucleus accumbens also play an important role in the acute response to amphetamine.\nIn conclusion, challenging animals sensitized to amphetamine with a dose of the drug that produces preferential activation of dorsal striatal patches did not produce stereotyped behavior, unlike the behavioral pattern observed in animals treated acutely with a dose of amphetamine that causes the same pattern of neural activation. Sensitized immediate-early gene activity was found in the nucleus accumbens core, but not the accumbens shell or the prefrontal cortex. The gradients observed in the present study in cellular responses to amphetamine in subareas of the prefrontal cortex with specific afferents to the nucleus accumbens suggest a specific role for various corticostriatal loops in the behavioral responses to psychostimulant drugs, further supporting the importance of accumbens\u2013prefrontal cortex interactions in drug addiction (Volkow and Li 2004; Robbins and Everitt 2002).","keyphrases":["stereotypy","behavioral sensitization","striatum","prefrontal cortex","locomotion","immediate-early gene"],"prmu":["P","P","P","P","P","P"]}
{"id":"Eur_J_Epidemiol-4-1-2226058","title":"Apolipoprotein E gene is related to mortality only in normal weight individuals: The Rotterdam study\n","text":"Objective To investigate the relationship between the apolipoprotein E (APOE) gene and the risk of mortality in normal weight, overweight and obese individuals. Methods and Results In a population-based study of 7,983 individuals aged 55 years and older, we compared the risks of all-cause and coronary heart disease (CHD) mortality by APOE genotype, both overall and in subgroups defined by body mass index (BMI). We found significant evidence for interaction between APOE and BMI in relation to total cholesterol (p = 0.04) and HDL cholesterol (p < 0.001). Overall, APOE*2 carriers showed a decreased risk of all-cause mortality. Analyses within BMI strata showed a beneficial effect of APOE*2 only in normal weight persons (adjusted hazard ratio (HR) 0.7[95% CI 0.5\u20130.9]). APOE*2 was not associated with a lower risk of all-cause mortality in overweight or obese persons. The effect of APOE*2 in normal weight individuals tended to be due to the risk of CHD mortality (adjusted HR 0.5 [95% CI 0.2\u20131.2]). Conclusion The APOE*2 allele confers a lower risk of all-cause mortality only to normal weight individuals.\nApolipoprotein E is a plasma protein involved in the metabolism of cholesterol. The apolipoprotein E isoforms \u03b52, \u03b53 and \u03b54, determined by the three alleles of the apolipoprotein E gene (APOE*2, APOE*3 and APOE*4), differ in their binding affinity to serum cholesterol and hence in their ability to clear dietary fat from the blood [1]. Accordingly, total serum cholesterol levels differ between APOE genotypes, with APOE*2 carriers having lower total serum cholesterol levels and APOE*4 carriers having higher levels than APOE*3 homozygotes [1\u20134].\nIn line with the clear differences in total serum cholesterol levels associated with the APOE genotype, one would expect that APOE*2 carriers have lower risks of cardiovascular morbidity and mortality and APOE*4 carriers have higher risks. Yet, the evidence for this association is inconsistent. A recent meta-analysis showed that APOE*4 was associated with a higher risk of coronary heart disease (CHD), but found no association with APOE*2 [5]. APOE*2 has been associated with a lower risk of mortality [6] and APOE*4 with a higher risk [7\u201311], but also these relationships were not found by others [12\u201319].\nA possible explanation for these inconsistencies may be found in the role of body weight. Several studies have demonstrated that the relationship between the APOE gene and lipids differs between normal weight, overweight and obese individuals [20\u201322]. Total serum cholesterol levels were higher among obese than among non-obese APOE*4 carriers [20], and obese APOE*2 carriers had higher serum triglycerides and higher LDL-cholesterol levels than non-obese APOE*2 carriers [20, 21]. While APOE*2 carriers generally have the lowest cholesterol levels, those with high BMI were found to have similar total serum cholesterol levels compared to APOE*3 homozygotes and APOE*4 carriers [20]. These findings suggest that the adverse effects of high body weight may outweigh the beneficial effect of APOE*2 status. If so, a protective effect of APOE*2 on mortality may be largest among normal weight individuals.\nDespite this evidence for a moderating effect of body weight on the relation between the APOE gene and total serum cholesterol, no studies so far have performed a stratified analysis on the relationship between the APOE gene and the risk of mortality. The aim of the present study was to investigate the extent to which the relationship between APOE genotypes and the risk of mortality differs between normal weight, overweight and obese individuals. We examined this relationship for both all-cause and CHD mortality.\nMethods\nStudy population and procedures\nThe present analyses were performed in the Rotterdam Study, an ongoing population-based study on the determinants of disease and disability among persons 55\u00a0years and older. Details of this study have been described elsewhere [23]. Baseline data were collected between 1990 and 1993. During home visits, a trained investigator obtained information on health status, medical history, medication use and lifestyle. Subsequently, participants were invited to the study center where they underwent an extensive clinical examination. The Medical Ethics Committee of the Erasmus Medical Center approved the study protocol and all participants provided written informed consent. From 10,275 eligible subjects, 7,983 (78%) individuals agreed to participate in the study and were examined at baseline.\nData collection\nClinical and laboratory assessments\nThe clinical examination included weight, height, systolic and diastolic blood pressure, serum glucose, total serum cholesterol and high-density lipoprotein (HDL) cholesterol levels. BMI was computed as weight (kg) divided by height squared (m2). Participants were classified as underweight (BMI\u00a0<\u00a018.5\u00a0kg\/m2), normal weight (18.5\u00a0kg\/m2\u00a0\u2264\u00a0BMI\u00a0<\u00a025\u00a0kg\/m2), overweight (25\u00a0kg\/m2\u00a0\u2264\u00a0BMI\u00a0<\u00a030\u00a0kg\/m2) or obese (BMI\u00a0\u2265\u00a030\u00a0kg\/m2) [24]. The waist and hip circumferences were measured, and the waist-hip-ratio (WHR) was calculated as indirect assessment of abdominal fat. Systolic and diastolic blood pressures were measured twice in a sitting position after 5\u00a0min rest using a random-zero sphygmomanometer. The mean of the two measurements was used for the analyses. Hypertension was defined as systolic blood pressure higher than 160\u00a0mmHg, diastolic blood pressure higher than 100\u00a0mmHg, or the use of antihypertensive medication indicated to treat high blood pressure (hypertension grades 2 and 3) [25]. Diabetes was diagnosed based on a random or post-load glucose level higher than 11.0\u00a0mmol\/l and\/or the use of anti-diabetic medication [26]. Total serum cholesterol and HDL-cholesterol were measured using an automated enzymatic procedure [27]. Hypercholesterolemia was defined as a total serum cholesterol level above 6.2\u00a0mmol\/l [28]. The APOE genotype was determined on DNA samples using a polymerase chain reaction followed by enzymatic digestion using methods previously described [29]. The frequencies of APOE genotypes were APOE 2\/2 0.8%, APOE 2\/3 12.8%, APOE 2\/4 2.7%, APOE 3\/3 58.4%, APOE 3\/4 22.9% and APOE 4\/4 2.4%. The proportions of the APOE alleles and genotypes were in Hardy-Weinberg equilibrium (p\u00a0=\u00a00.71).\nMortality data\nInformation on the vital status of the participants was obtained at regular intervals from the municipal population registry. Causes of death were obtained from the general practitioners by means of a standardized questionnaire relating to the circumstances of death, most likely cause of death and time and place of death. Two independent research physicians coded all events according to the International Classification of Diseases, 10th edition (ICD-10) [30]. CHD mortality was defined as death from diseases coded I20\u2013I25, I46, I50 or R96. Mortality data were available up to December 2003.\nStatistical analyses\nOf the 7,983 individuals who participated at baseline, 5,817 (73%) had APOE genotyped successfully and had complete information on BMI, WHR and cholesterol levels. Because persons with an extremely low BMI may suffer from life-threatening diseases such as cancer, individuals with a BMI below 18.5\u00a0kg\/m2 (n\u00a0=\u00a051) were excluded from the analyses. Also, APOE 2\/4 individuals were excluded from the analyses (n\u00a0=\u00a0158) to distinguish the effect of the APOE alleles unambiguously. Hence, data from 5,608 participants were available for the analyses.\nDifferences in baseline characteristics by APOE genotypes were tested using the chi-squared statistic (categorical variables) or ANOVA (continuous variables). P for trend was obtained by testing the linearity of the sum of squares from the ANOVA analyses. Multiple linear regression analysis was used to assess the interaction between APOE genotype and BMI on total serum cholesterol and HDL-cholesterol levels. Differences in survival probabilities were examined by comparing Kaplan-Meier survival curves and tested using the log rank test. Kaplan-Meier plots were constructed using age as the time scale to take proper account of the effect of age [31, 32]. Risks of mortality were quantified as hazard ratios (HRs) using Cox proportional hazards analyses with age as the time scale. The most common genotype (APOE 3\/3) was used as the reference category. The proportionality assumption of all models was verified by testing the Schoenfeld residuals [33]. Because the proportionality assumption was not met, HRs were calculated for early and late mortality using 80\u00a0years as the cut-off age. This cut-off age was selected because it led to proportionality of the models below and above the cut-off age. HRs were calculated adjusted for gender, smoking status, education level, total serum cholesterol, HDL-cholesterol, waist to hip ratio hypertension and diabetes mellitus.\nResults\nThe mean age at entry was 68.7\u00a0years (SD\u00a0=\u00a08.7\u00a0years) and 41.8% of the participants were men. The mean follow up time was 11.1\u00a0years (SD\u00a0=\u00a03.8\u00a0years). Baseline characteristics of APOE*2 carriers, APOE*3 homozygotes and APOE*4 carriers are presented in Table\u00a01. As expected, cholesterol levels differed significantly between the genotype groups. The prevalence of hypercholesterolaemia was lowest among APOE*2 carriers (49%) and highest among APOE*4 carriers (66%). Furthermore, there were statistically significant differences in BMI scores between the genotype groups, with APOE*2 carriers having the highest mean BMI (26.7\u00a0\u00b1\u00a03.6\u00a0kg\/m2) and APOE*4 carriers the lowest (26.1\u00a0\u00b1\u00a03.4\u00a0kg\/m2). There were no statistically significant differences in WHR between the genotype groups.\nTable\u00a01Baseline characteristics by APOE genotype groupsAPOE*2 carriersAPOE 3\/3APOE*4 carrierspn7823,3691,457Sex (men)3842420.05Age at entry (years)68.8 (8.8)68.9 (8.7)68.3 (8.5)0.14Body mass index (kg\/m2)26.7 (3.6)26.4 (3.6)26.1 (3.4)<0.01Normal weight343940Overweight494747<0.05Obese171413Waist-to-hip ratio0.9 (0.1)0.9 (0.1)0.9 (0.1)0.18Systolic blood pressure (mmHg)140 (22)140 (23)138 (2)0.04Diastolic blood pressure (mmHg)74 (11)74 (12)73 (11)0.06Hypertension3634330.25Total serum cholesterol (mmol\/l)6.4 (1.3)6.6 (1.2)6.8 (1.2)<0.001HDL-cholesterol (mmol\/l)1.4 (0.4)1.3 (0.3)1.3 (0.4)<0.001Cholesterol lowering medication3230.71Hypercholesterolemia496066<0.001Smoking status (current)3935330.02Education level\u00a0\u00a0\u00a0\u00a0Lower434345\u00a0\u00a0\u00a0\u00a0Intermediate4342400.21\u00a0\u00a0\u00a0\u00a0Higher141515Diabetes mellitus101090.75Values are means (standard deviations) for continuous variables and percentages for categorical variables. p values are obtained by ANOVA for continuous variables and by \u03c72 for categorical variables\nFigure\u00a01 presents mean total serum cholesterol and HDL-cholesterol levels by APOE genotype in normal weight, overweight and obese persons. In all BMI strata, APOE*2 carriers had the lowest and APOE*4 carriers the highest total serum cholesterol (pfor trend\u00a0<\u00a00.01). The association between HDL-cholesterol and APOE genotype was in the opposite direction (pfor trend\u00a0<\u00a00.01). Note that even though APOE*2 carriers had the lowest total serum cholesterol levels in each BMI group, still 50% of the overweight and 57% of the obese APOE*2 carriers had hypercholesterolaemia compared to 41% of the normal weight APOE*2 carriers (p\u00a0=\u00a00.003). There was no significant evidence for interaction between APOE genotype and BMI in relation to total serum cholesterol (p\u00a0=\u00a00.26) and HDL-cholesterol (p\u00a0=\u00a00.25). However, when BMI was analyzed as a continuous trait, then the p value for interaction was 0.04 for total serum cholesterol and less than 0.001 for HDL-cholesterol.\nFig.\u00a01Mean total serum cholesterol and HDL-cholesterol levels by APOE genotypes for normal weight, overweight and obese individuals. HDL: high-density lipoproteins\nA total of 1,918 deaths occurred during follow-up. Of those, 257 occurred in 782 APOE*2 carriers (33%), 1,160 in 3,369 APOE*3 homozygotes (33%) and 501 in 1,457 APOE*4 carriers (34%). Differences between the Kaplan-Meier survival curves of the APOE*2 carriers, APOE*3 homozygotes and APOE*4 carriers were small but statistically significant (p\u00a0=\u00a00.03) with APOE*2 carriers having slightly lower mortality risks (Fig.\u00a02). After adjustment for other cardiovascular risk factors, APOE genotype was not associated with all-cause mortality before or after 80\u00a0years of age in the overall population (Table\u00a02). The differences between the curves were only statistically significant in normal weight individuals (p\u00a0=\u00a00.04; Fig.\u00a03). Normal weight carriers of the APOE*2 allele had a significantly lower risk of mortality before age 80\u00a0years (HR [95% CI]\u00a0=\u00a00.7 [0.5\u20130.9]), but not after age 80\u00a0years (crude HR [95% CI]\u00a0=\u00a01.0 [0.8\u20131.4]). There were no significant differences in the risk of mortality between genotype groups among overweight or obese persons (Table\u00a02).\nFig.\u00a02Kaplan Meier survival curves for all-cause mortality by APOE genotypesTable\u00a02Incidence rates and hazard ratios for all-cause mortality by APOE genotypes and BMI categoriesFollow-upIncidence rateHazard ratioFollow-upIncidence rateHazard ratioDeaths(py)Deaths\/1,000 py (95% CI)(95% CI)Deaths(py)Deaths\/1,000 py (95% CI)(95% CI)Before 80\u00a0years of ageAfter 80\u00a0years of ageAllAPOE*2 carriers1016,78214.9 (12.3\u201318.1)0.9 (0.7\u20131.1)15621,7647.2 (6.1\u20138.4)0.9 (0.8\u20131.1)APOE 3\/348128,70316.8 (15.3\u201318.3)67995,8087.1 (6.6\u20137.6)APOE*4 carriers19812,69315.6 (13.6\u201317.9)1.0 (0.8\u20131.2)30341,6377.3 (6.5\u20138.1)1.2 (1.0\u20131.4)Total78048,17816.2 (15.1\u201317.4)1,138159,2097.1 (6.7\u20137.6)Normal weightAPOE*2 carriers282,37011.8 (8.2\u201317.1)0.7 (0.5\u20130.9)577,2897.8 (6.0\u201310.1)1.0 (0.8\u20131.4)APOE 3\/320611,31418.2 (15.9\u201320.9)25638,0456.7 (6.0\u20137.6)APOE*4 carriers785,08015.4 (12.3\u201319.2)1.0 (0.7\u20131.3)12717,3057.3 (6.2\u20138.7)1.3 (0.9\u20131.7)Total31218,76416.6 (14.9\u201318.6)44062,6397.0 (6.4\u20137.7)OverweightAPOE*2 carriers593,30517.9 (13.8\u201323.0)1.1 (0.9\u20131.5)6511,0645.9 (4.6\u20137.5)0.9 (0.7\u20131.1)APOE 3\/321713,57316.0 (14.0\u201318.3)31045,3666.8 (6.1\u20137.6)APOE*4 carriers906,04714.9 (12.1\u201318.3)0.9 (0.7\u20131.2)13719,3987.1 (6.0\u20138.4)1.2 (1.0\u20131.5)Total36622,92416.0 (14.4\u201317.7)51275,8276.8 (6.2\u20137.4)ObeseAPOE*2 carriers141,10712.6 (7.5\u201321.4)1.0 (0.5\u20131.8)343,41110.0 (7.1\u201314.0)1.0 (0.6\u20131.5)APOE 3\/3583,81615.2 (11.7\u201319.7)11312,3989.1 (7.6\u201311.0)APOE*4 carriers301,56619.2 (13.4\u201327.4)1.3 (0.9\u20132.0)394,9347.9 (5.8\u201310.8)1.1 (0.8\u20131.6)Total1026,48915.7 (12.9\u201319.1)18620,7429.0 (7.8\u201310.4)Hazard ratios adjusted for sex, waist-hip-ratio, smoking, education level, total serum cholesterol, HDL cholesterol hypertension and diabetes; py: person-years; CI: Confidence intervalFig.\u00a03Kaplan Meier survival curves for all-cause mortality by APOE genotypes for normal weight, overweight and obese individuals\nTo investigate whether the lower risk of mortality observed among normal weight APOE*2 carriers was due to a lower risk of mortality from CHD, we calculated the HRs separately for CHD mortality. APOE genotype was not significantly associated with CHD mortality. Analysis within BMI strata showed that normal weight APOE*2 carriers tended to have lower risks of CHD mortality (adjusted HR [95% CI]\u00a0=\u00a00.5 [0.2\u20131.2]). No differences in the risk of CHD mortality between genotype groups were found in overweight or obese participants (Table\u00a03).\nTable\u00a03Incidence rates and hazard ratios for CHD mortality by APOE genotypes and BMI categoriesFollow-upIncidence rateHazard ratioFollow-upIncidence rateHazard ratioDeaths(py)Deaths\/1,000 py (95% CI)(95% CI)Deaths(py)Deaths\/1,000 py (95% CI)(95% CI)Before 80\u00a0years of ageAfter 80\u00a0years of ageAllAPOE*2 carriers266,7823.8 (2.6\u20135.6)1.0 (0.6\u20131.5)412,10319.5 (14.4\u201326.5)1.1 (0.8\u20131.5)APOE 3\/312628,7034.4 (3.7\u20135.2)1718,74419.6 (16.8\u201322.7)APOE*4 carriers5912,6934.6 (3.6\u20136.0)1.1 (0.8\u20131.4)663,47219.0 (14.9\u201324.2)1.1 (0.8\u20131.4)Total21148,1784.4 (3.8\u20135.0)27814,31919.4 (17.3\u201321.8)Normal weightAPOE*2 carriers52,3702.1 (0.9\u20135.1)0.5 (0.2\u20131.2)1566322.6 (13.6\u201337.5)1.6 (0.9\u20132.9)APOE 3\/35611,3144.9 (3.8\u20136.4)483,07515.6 (11.8\u201320.7)APOE*4 carriers265,0805.1 (3.5\u20137.5)1.2 (0.7\u20131.9)211,28616.3 (10.6\u201325.1)1.1 (0.6\u20131.8)Total8718,7644.6 (3.8\u20135.7)845,02416.7 (13.5\u201320.7)OverweightAPOE*2 carriers163,3054.8 (3.0\u20137.9)1.4 (0.8\u20132.4)1598915.2 (9.1\u201325.2)0.7 (0.4\u20131.3)APOE 3\/35113,5733.8 (2.9\u20134.9)924,03522.8 (18.6\u201328.0)APOE*4 carriers216,0473.5 (2.3\u20135.3)0.8 (0.5\u20131.4)351,61921.6 (15.5\u201330.1)1.0 (0.7\u20131.5)Total8822,9243.8 (3.1\u20134.7)1426,64321.4 (18.1\u201325.2)ObeseAPOE*2 carriers51,1074.5 (1.9\u201310.8)1.1 (0.4\u20133.4)1145024.4 (13.5\u201344.1)1.4 (0.7\u20133.1)APOE 3\/3193,8165.0 (3.2\u20137.8)311,63419.0 (13.3\u201327.0)APOE*4 carriers121,5667.7 (4.4\u201313.5)1.6 (0.8\u20133.5)1056817.6 (9.5\u201332.7)1.1 (0.5\u20132.2)Total366,4895.5 (4.0\u20137.7)522,65119.6 (14.9\u201325.7)Hazard ratios adjusted for sex, waist-hip-ratio, smoking, education level, total serum cholesterol, HDL cholesterol hypertension and diabetes; py: person-years; CI: Confidence interval\nDiscussion\nOur study shows that the APOE*2 allele has a protective effect on overall mortality. This protective effect seems to be limited to mortality from CHD. Analyses within BMI strata demonstrated that this genetic advantage of APOE*2 was observed only in normal weight individuals, but not in overweight and obese APOE*2 carriers.\nThe results of the present analyses may seem to contradict our earlier work in this population. We previously reported that the APOE gene was not significantly related to mortality [12]. While our findings come from the same population, the mean follow-up time of the participants has increased from 5.4\u00a0years to 11.1\u00a0years and the number of deaths increased by 53% from 18% to 34%. This extended follow-up has improved the statistical power of our study to show a modest effect of APOE genotype on overall mortality. Research on the relationship between APOE and mortality has merely provided conflicting results [6\u201319, 34]. Three out of eleven studies showed a protective effect of APOE*2 [6, 14, 18]. and three a deleterious effect of APOE*4 [8\u201310]. The latter effect may partly mediated by the increased risk of Alzheimer disease in association with APOE*4 and mortality [10, 11].\nNote that our analysis showed small significant difference in the Kaplan-Meier survival curves, demonstrating a protective effect of APOE*2 before age 80. This may be due to the effect of age that becomes apparent when performing survival analysis with age as time scale, as we did in the present paper. Such finding is compatible with previous reports suggesting that genes affecting human lifespan might be age-specific [35]. Genetic and environmental interactions at older ages vary from those at early ages and therefore explain the differences in the association between APOE and mortality before and after 80\u00a0years of age. A previous report showed that among nonagenarians, APOE genotype has no effect on cognitive function, cognitive decline or survival [36].\nIn line with previous studies [20\u201322], we found that the total serum cholesterol levels associated with APOE genotype differed between normal weight, overweight and obese individuals. As expected, mean levels of total serum cholesterol increased with BMI and APOE*2 carriers had the lowest mean total serum cholesterol in each BMI group. APOE*4 carriers had higher cholesterol levels and lower HDL-cholesterol concentrations than APOE*2 carriers despite the presence of a lower BMI and equal WHR. Previous reports showed that APOE genotype, BMI and WHR determine together the lipid levels [20\u201322, 37]. However, in our study abdominal fat, as measured by WHR, did not have any effect on the lipid levels by APOE genotype, while BMI was an important determinant. The significant interaction between APOE genotype and BMI in relation to total serum cholesterol found previously [20], was also supported by this study. Although the mechanism of such interaction has not been yet elucidated, evidence suggests that obesity and abdominal fat increase low density lipoprotein cholesterol and therefore total cholesterol at a higher degree among APOE*4 carriers [20\u201322, 37]. This is in line with our observation that the deleterious effect of increased BMI prevails over the beneficial effects of the APOE*2 allele. Furthermore, previous research in the Rotterdam Study and the Dutch population indicated that among APOE*2 homozygous carriers, the expression of Hyperlipoproteinemia type III is determined to a great extent by hyperinsulinemia and the presence of insulin resistance syndrome [38], conferring and increased risk of CHD through accelerated atherosclerosis [39].\nAlthough the association between high BMI and high cholesterol levels is well known and the APOE gene is associated with cholesterol levels as previously described, this is the first study that examined the effect of APOE genotype on mortality in BMI strata. We found that presence of the APOE*2 allele was associated with a decreased risk of mortality only in normal weight individuals and that this lower risk was partly explained by lower risk of CHD mortality. These results were in line with the lower total cholesterol and higher HDL levels among normal weight APOE*2 carriers. Our finding that the protective effect of APOE*2 was only found in normal weight individuals suggests that the negative influences of increasing BMI outweigh the positive effects of genetic predisposition. When these results are confirmed by other population-based studies, a challenging question remains whether weight loss in overweight and obese APOE*2 carriers restores the genetic advantage of their APOE status.","keyphrases":["apoe","mortality","bmi","cholesterol","chd mortality"],"prmu":["P","P","P","P","P"]}
{"id":"Dev_Genes_Evol-3-1-1784541","title":"Free-living flatworms under the knife: past and present\n","text":"Traditionally, regeneration research has been closely tied to flatworm research, as flatworms (Plathelminthes) were among the first animals where the phenomenon of regeneration was discovered. Since then, the main focus of flatworm regeneration research was on triclads, for which various phenomena were observed and a number of theories developed. However, free-living flatworms encompass a number of other taxa where regeneration was found to be possible. This review aims to display and to compare regeneration in all major free-living flatworm taxa, with special focus on a new player in the field of regeneration, Macrostomum lignano (Macrostomorpha). Findings on the regeneration capacity of this organism provide clues for links between regeneration and (post-)embryonic development, starvation, and asexual reproduction. The role of the nervous system and especially the brain for regeneration is discussed, and similarities as well as particularities in regeneration among free-living flatworms are pointed out.\nIntroduction\nRegeneration research in flatworms has a long standing history, but was focussed on triclads since its beginning in 1774, when Pallas (Pallas 1774) described the regeneration of the triclads Dendrocoelum lacteum and Bdellocephala punctata (from Br\u00f8ndsted 1969). Since then, a vast amount of studies about regeneration in flatworms has appeared (for recent reviews, see Agata and Watanabe 1999; S\u00e1nchez Alvarado 2000, 2003, 2004, 2006; Sal\u00f3 and Bagu\u00f1\u00e0 2002; Newmark and S\u00e1nchez Alvarado 2002; Agata 2003; Agata et al. 2003; Reddien and S\u00e1nchez Alvarado 2004; Reuter and Kreshchenko 2004; S\u00e1nchez Alvarado and Kang 2005; Sal\u00f3 2006), but many of the old questions and many of the most intriguing phenomena that have been discovered cannot be explained today. Why can some animals regenerate, while others cannot? How is the duplication of heads or tails (heteromorphoses) possible in adult organisms? What conditions are sufficient and what are necessary for successful regeneration? Is regeneration a side-product of asexual reproduction, or the other way round? Is regeneration recapitulating pathways used in embryonic and postembryonic development? How are stem cells controlled and directed in regeneration? Are stem cells in adult flatworms totipotent, and can they be likened to embryonic blastomeres? This review provides a brief synopsis on what is known about the regeneration capacity in free-living flatworms, and addresses open questions about regeneration, with special emphasis on an emerging model organism, Macrostomum lignano (Egger et al. 2006a,b).\nA comparison of the regeneration capacity of the macrostomorph flatworm M. lignano with other free-living flatworms necessarily involves a look at their phylogenetic relationship. The Macrostomorpha belong to the largest taxon Rhabditophora, which also encompasses the Polycladida, Lecithoepitheliata, Prolecithophora, Proseriata, Bothrioplanida, Tricladida, Rhabdocoela, and the parasitic Neodermata (Ehlers 1985; Rieger 1996; Tyler et al. 2006). Macrostomorpha are considered to be the most basal taxon within the Rhabditophora due to a simple pharynx, entolecithal eggs, and the lack of vitellaria (Ehlers 1985). Referring to their small size (millimeter range), macrostomorphans are members of the \u201cmicroturbellarians\u201d, a nonsystematic denomination. Polyclads and triclads are the only taxa belonging to the \u201cmacroturbellaria\u201d, with forms generally in the centimeter range. Triclads, as more derived rhabditophorans, have vitellaria, a rather complicated embryonic development (e.g., Cardona et al. 2005) and an often very pronounced regeneration capacity (see Br\u00f8ndsted 1969).\nThe status of the acoels\nWhile many molecular (e.g., Bagu\u00f1\u00e0 and Riutort 2004a,b) and also some morphological studies (e.g., Reuter et al. 2001, summed up in Egger and Ishida 2005) have placed the acoels outside the Plathelminthes, a number of characteristics (apomorphies) speak in favor of acoels being flatworms (Ehlers 1985), most notably the stem cell system and the special mode of epidermal replacement (Smith et al. 1986; Tyler and Hooge 2004). In this review, acoels and nemertodermatids are considered members of the Plathelminthes.\nWhat is a planarian?\nIn flatworm regeneration research, other groups than triclads are often neglected and can easily be overlooked. This may in part be due to an ambiguous use of the term \u201cplanarian\u201d, which is most often referring merely to triclads (more specifically, to paludicolans, e.g., Agata 2003; Reddien and S\u00e1nchez Alvarado 2004), but is also commonly used as a synonym for all free-living flatworms (e.g., Bagu\u00f1\u00e0 1998; Sal\u00f3 and Bagu\u00f1\u00e0 2002) or even used interchangeably for free-living flatworms and triclads alike (e.g., Newmark and S\u00e1nchez Alvarado 2001). The term \u201cfreshwater planarian\u201d, while somewhat more specific, is still ambiguous, as a number of nontriclad flatworms, e.g., Stenostomum and Microstomum, are freshwater dwellers as well. In publications dealing with several flatworm taxa, it is often unclear what the term planarian signifies.\nIt seems therefore desirable to agree on using less ambiguous denominations in scientific contexts, such as unequivocal scientific names, and to get rid of the term planarian.\nOverview of regeneration capacity in free-living flatworm groups\nAll major free-living flatworm taxa with their tentative general regeneration capacities are given in Fig.\u00a01. Asexually reproducing forms are only known for acoels, catenulids, macrostomorphans, and triclads. Within free-living flatworms, either catenulids (Ehlers 1985) or acoelomorphs (acoels and nemertodermatids, Rieger 1996) are considered as the basal-most taxon. All other flatworms are encompassed by the rhabditophorans, with macrostomorphans and polyclads at the base (Ehlers 1985; Rieger 1996).\nFig.\u00a01The regeneration capacity of the major free-living flatworm taxa. In all groups with asexual reproduction, species with a pronounced regeneration capacity can be found (green shades). Not all species in the listed taxa are necessarily showing the same regeneration capacity; only a broad classification of regeneration capacities is given. The major taxa were compiled after Ehlers (1985), Rieger (1996), and Tyler et al. (2006)\nCatenulida\nThe regeneration capacity of two catenulid genera, namely, Stenostomum and Catenula, has been extensively investigated. Probably linked with asexual reproduction (paratomy), excellent regeneration capacity has been reported after artificial amputation (Ritter and Congdon 1900; Child 1903a,b; Ruhl 1927a; van Cleave 1929; and Palmberg 1990 for Stenostomum; Moraczewski 1977 for Catenula). Regeneration from the so-called organ primordia was found to be possible for all organ systems.\nAcoela\nBased on experiments mainly with Polychoerus caudatus (Stevens and Boring 1905; Child 1907; Keil 1929) and Paramecynostomum diversicolor (Peebles 1913), Hyman notes that acoels \u201chave limited powers of regeneration\u201d (Hyman 1951, p 182). The studied species were not able to regenerate ganglia (brain) or the statocyst. Later studies with Amphiscolops langerhansi (Steinb\u00f6ck 1954, 1955, 1963a,b) and Hofstenia giselae (Steinb\u00f6ck 1966, 1967) illustrated a substantial regeneration capacity in these acoels, where H. giselae was found to even regenerate the statocyst after amputation (Steinb\u00f6ck 1966). Several species can regenerate their eyes (Hanson 1967 for A. langerhansi and Pseudohaplogonaria sutcliffei, Yamasu 1991 for Praesagittifera naikaiensis, and \u00c5kesson et al. 2001 for Convolutriloba longifissura).\nAcoels are now known to show all main modes of asexual reproduction, that is architomy (e.g., C. longifissura, \u00c5kesson et al. 2001), paratomy (e.g., Paratomella unichaeta, Ax and Schulz 1959), and budding (e.g., C. retrogemma, Hendelberg and \u00c5kesson 1988). Architomy is characterized by fission before formation of new organs, paratomy by formation of new organs before fission. Budding refers to a special case of paratomy, where the axis of the new piece is not corresponding to the axis of the old piece\u2014in C. retrogemma, the axis of the buds is tilted by 180\u00b0 in relation to the main animal (Hendelberg and \u00c5kesson 1988, 1991).\nNemertodermatida\nNo account of regeneration potential or asexual reproduction has been given for animals from this taxon.\nMacrostomorpha\nAs already observed in catenulids and acoels, the regeneration capacity of macrostomorphans is closely associated with the mode of reproduction. Thus, in the asexually reproducing taxa Microstomum lineare and Alaurina (paratomy), the regeneration capacity is most pronounced and all organs can be regenerated (von Graff 1908; Ruhl 1927a; Palmberg 1986, 1990, 1991). Regeneration research with the exclusive sexually reproducing genus Macrostomum started with Ruhl (1927a), who found that Macrostomum tuba cannot regenerate the head (brain, eyes, and pharynx) and requires at least a quarter of the gut in the anterior piece to be able to regenerate posterior parts, including copulatory organs. More recently, Ladurner et al. (1997) noted that in Macrostomum hystricinum marinum and Macrostomum pusillum the anterior part can fully regenerate the posterior part, while the posterior part cannot regenerate the anterior part. Similar findings were reported for M. lignano: cut in midbody, only the anterior part was found to regenerate the missing part (Rieger et al. 1999). Salvenmoser et al. (2001) described the regeneration of the musculature in M. lignano after the removal of the posterior end. Using the same species, Egger et al. (2006a,b) characterized the regeneration capacity at various cutting levels. Anterior regeneration is possible if the animals are cut in front of the brain; posterior regeneration can be successful from cutting levels starting with the posterior end of the pharynx. A distinct regeneration blastema occurs in M. lignano, but not in M. lineare, where organ primordia are formed without conspicuous accumulation of neoblasts at the wound site (Palmberg 1986).\nPolycladida\nThe best-studied polyclad genus regarding regeneration is Leptoplana. Leptoplana alcynoe (now Letoplana alcinoi) and Leptoplana velutinus, as well as Thysanozoon brocchi and Cryptocelis alba, were shown to regenerate posterior parts by Monti (1900a). Using Leptoplana atomata, Schultz (1901, 1902) observed the regeneration of the posterior part, including gonads and copulatory organs, whereas head and pharynx were not regenerated. Similar results were obtained with Leptoplana tremellaris and L. alcinoi, although the pharynx was found to regenerate here (Child 1904a\u2013c, 1905a; von Levetzow 1939). L. tremellaris can regenerate both anterior and posterior parts if only about half the brain is removed by transversal amputation. At amputation levels posterior of the brain, no complete anterior regeneration is possible (Child 1904a\u2013c). Notoplana humilis shows a comparable regeneration capacity (Ishida 1998). Besides more posterior parts, T. brocchi is also able to regenerate the main tentacles, gut, and pharynx (Monti 1900a; von Levetzow 1939). Studying regeneration in Leptoplana saxicola, Pucelis litoricola, and Planocera californica, Olmsted (1922a) reports that these species follow the \u201cpolyclad rule for regeneration\u201d, that is, regeneration of all amputated parts (including eyes) is possible if the brain remains intact. Several studies were dedicated to find out whether polyclads can regenerate the brain (Morgan 1905 for Leptoplana littoralis and von Levetzow 1939 for T. brocchi), and how the behavior changes after excision of the brain (Morgan 1905; Olmsted 1922b; von Levetzow 1939; Koopowitz et al. 1975, 1976) or after severance of the main lateral nerve cords (Olmsted 1922b; Koopowitz et al. 1975, 1976). Severed nerve cords and brain halves can fuse and resume their functions (Olmsted 1922b; Faisst et al. 1980), but the brain is not regenerated. Full anterior regeneration, including the brain, after complete removal of the brain was only observed in Cestoplana, provided the amputation level is a short distance behind the brain (Child 1905b). All mentioned species, except T. brocchi, are members of the taxon Acotylea (Tyler et al. 2006). From what is known, both Cotylea and Acotylea seem to share a comparable regeneration capacity.\nLecithoepitheliata\nOnly scarce reports about regeneration are available for this neoophoran taxon. For Prorhynchus stagnalis, Ruhl (1927a) remarks that no regeneration capacity whatsoever could be detected at anterior, posterior, and midbody cutting levels. The posterior pieces survived for 30\u00a0days. When cutting Geocentrophora baltica a short distance behind the pharynx, no regeneration could be observed (Steinb\u00f6ck 1927). All pieces died at the latest after 17\u00a0days. A closer examination of the regeneration capacity of Geocentrophora sphyrocephala revealed that these animals can approximately rebuild the mouth opening anteriorly in 3\u20135\u00a0weeks but cannot regenerate the brain. Both parts die after 40\u00a0days if the animals are cut just behind the pharynx. Cut in midbody or more posteriorly, the anterior part can regenerate the tail with adhesive organs in 2\u20133\u00a0weeks, although the animals were never observed to reach the original body length. The posterior part dies without regeneration after 1\u00a0day (Hagleithner 1946).\nProlecithophora\nAccording to Pechlaner (1957), Plagiostomum lemani is reported to have a low regeneration capacity (Keller 1894), while Plagiostomum girardi can regenerate all amputated parts (Monti 1900b).\nProseriata\nvon Graff (1882) describes amputation experiments with Monocelis fusca, where posterior pieces resumed movement after 3\u20135\u00a0days. In M. fusca, Monocelis lineata and other monocelidids (Coelogynopora, Archilopsis), and also in otoplanids (Itaspiella, Bothriomolus), Giesa (1966) observed posterior regeneration of parts containing the brain in 3\u20134\u00a0days. Even pieces amputated just behind the brain can regenerate. The brain does not regenerate, though. In Otomesostoma auditivum, Pechlaner (1957) found promising regeneration potential, severely limited by a short life span of 9\u201310\u00a0months and low living temperatures (about 4\u00b0C). Gut, part of the gonads, and sensory organs can regenerate. First signs of pharynx, eye, and brain regeneration were observed after 9\u201315\u00a0weeks, but the animals died before regeneration was complete. The statocyst was not regenerated.\nBothrioplanida\nWhile no extensive regeneration studies have been undertaken, Sekera (1911) found that Bothrioplana semperi is able to regenerate the pharynx posteriorly.\nTricladida\nDalyell (1814) noted that Planaria nigra (today: Polycelis nigra) could \u201calmost be called immortal under the edge of the knife\u201d (in Br\u00f8ndsted 1969). However, not all triclad species are equally powerful regarding regeneration. \u015civickis (1930) distinguished five groups of triclads according to their ability to regenerate a head. This classification was subsequently refined into eight types by Teshirogi et al. (1977). For the Phagocata velata type, the Dugesia dorotocephala type, the Polycelis auriculata type, and the Dendrocoelopsis lactea type 2 (Sapporo population), regeneration of a head is possible from any part of the body, but with varying probability of success. Animals of the Dendrocoelopsis lactea type 1 (Kuroishi population) fail to regenerate a head from tail pieces up to the male gonopore. The Dendrocoelopsis ezensis type and the Dendrocoelum lacteum type 1 are very similar in that they are generally only able to regenerate a head if amputated anterior to the pharynx, whereas the Bdelloura type (e.g., Bdelloura candida) completely lacks the ability to regenerate a head. While a vast amount of literature on regeneration in triclads is available, recent publications are not so much concerned with studying the regeneration capacities of different triclads, but more with unraveling cellular and molecular mechanisms of regeneration in only a few model triclads, such as Dugesia japonica and Schmidtea mediterranea.\nRhabdocoela\nThe typhloplanoid flatworm Mesostoma ehrenbergi cannot regenerate any organs, only approximately restore the original shape posteriorly (Steinmann and Bresslau 1913). Regeneration experiments with Mesostoma productum, Mesostoma lingua, and Mesostoma punctatum revealed that anterior regeneration of parts in front of the brain is possible, although often done imperfectly. The tail can regenerate; the more posterior the cut is made, the better is the regeneration. Brain and pharynx cannot be regenerated (Fulinski 1922). In Phaenocora unipunctata and Phaenocora megalops (also typhloplanoids), no regeneration capacity was found (Fulinski 1922). For the the dalyelliid Dalyellia millportiana and the kalyptorhynch Gyratrix hermaphroditus, Ruhl (1927a) remarks curtly that all animals died within 1\u00a0day after amputation and no regeneration being observable. Dalyellia viridis and Typhloplana viridata survive the amputation of the caudal-most part of the tail, but show no signs of regeneration, not even of the adhesive organs. In Rhynchomesostoma rostratum, another typhloplanoid, the amputated tail tip is regenerated, or at least a tail-like shape is restored (Hein 1928).\nComparison of the regeneration capacity between free-living flatworm taxa\nWhile the experimental evidence for some groups is still patchy, we find three main types of regeneration capacity in free-living flatworms. Some taxa cannot regenerate at all, such as most rhabdocoels and some lecithoepitheliates. On the other end of the scope are species that can regenerate all organs, such as the acoel Convolutriloba, the catenulid Stenostomum, the macrostomorphan Microstomum, the polyclad Cestoplana, and many triclads. Most taxa however lie in between these extremes, lacking the ability to regenerate brain, eyes, pharynx, or statocyst (many acoels), but are able to regenerate gonads, copulatory organs, sucker, duo-gland adhesive systems, gut, and the anterior-most part in front of the brain.\nThe regeneration capacity of M. lignano can be likened most closely to triclads of the Bdelloura group, which cannot regenerate a head (brain or eyes). Most polyclads, although often able to regenerate a pharynx, are also roughly comparable to Macrostomum regarding their regeneration capacity. Members of other diverse taxa like lecithoepitheliates and rhabdocoels are bearing resemblance with regeneration in Macrostomum as well. Such similarities can be found crossing the boundaries of the major flatworm taxa, making the regeneration capacity a feature of no immediate phylogenetic value. Close relatives like Microstomum and Alaurina (both macrostomids) surpass Macrostomum\u2019s regeneration capacity by far.\nAsexual reproduction and regeneration\nNot surprisingly, the ability to regenerate is often linked to asexual reproduction (Fig.\u00a01, Br\u00f8ndsted 1969; S\u00e1nchez Alvarado 2000). In taxa lacking asexual reproduction, the regeneration capacity is generally less pronounced than in taxa with asexual reproduction, where regeneration is necessarily involved (Br\u00f8ndsted 1969; Reuter and Kreshchenko 2004). As described in the section \u201cAcoela\u201d, in architomy, regeneration events follow fission, while in paratomy and budding, the term \u201cpregeneration\u201d seems to be more accurate, as organs are duplicated in the presence of the old organs in the new animal (zooid) before fission occurs. The notable difference between regeneration and pregeneration is the presence of a brain in the latter, which is regarded as a decisive organ for regeneration in many species (Henley 1974; Kreshchenko et al. 2001). It is interesting to note that the catenulid Stenostomum leucops, reproducing asexually by paratomy, often fails to regenerate a head if amputated artificially (van Cleave 1929). This finding hints at an actual difference between paratomy and architomy regarding the influence on regeneration capacity.\nAsexual reproduction in flatworms occurs in more basal taxa (Acoela, Catenulida, and Macrostomorpha) as well as in more derived taxa (Tricladida and Neodermata). Triclads, like acoels, show paratomy (Dugesia fissipara and Dugesia paramensis, Hyman 1951) as well as architomy (e.g., Dugesia tahitiensis, Peter et al. 2001). Just like regeneration capacity, asexual reproduction is too dispersed among diverse taxa to provide insight into phylogenetic relationships.\nWhy can some flatworm species regenerate while others cannot?\nIf a profound regeneration capacity is a plesiomorphic feature, there might have been a selection pressure against regeneration during evolution (Goss 1991; in S\u00e1nchez Alvarado 2000). Why would such a useful feature be reduced or abolished? Because regeneration is dangerous, as a plethora of evidence of double-headed or double-tailed flatworms testify. Such aberrant forms are easily produced in species that can regenerate (see section \u201cHeteromorphoses\u201d), but are not known from flatworms lacking regeneration capabilities. Other than as a side effect of asexual reproduction, the ability to regenerate is seemingly not useful enough to outweigh the inherent dangers. What are the chances that a predator spares a tail fragment that can regenerate to a complete animal in a couple of days if left alone? What are the chances that a disease only affects a part of the animal, but spares the rest to readily regenerate the damaged organs? A totipotent stem cell system out of control might be too high a risk for comparably little reward. This could be the reason why regeneration capabilities decreased in species that no longer propagate through asexual reproduction.\nIn this light, it seems more likely that in evolutionary terms, asexual reproduction is a primitive and not a derived feature (Rieger 1986; Gremigni 1992; Reuter and Kreshchenko 2004), and that regeneration capability results from asexual reproduction as an added bonus. Regeneration can be seen as a form of asexual reproduction, only triggered by different stimuli.\nWhat is the decisive factor that determines the regeneration capacity of a species? Citing regeneration experiments with Dendrocoelum and Bdellocephala, Br\u00f8ndsted (1969) comes to the conclusion \u201cthat the posterior part of the body in these two species simply lacks the potential to respond to head inducing substances\u201d. This may also hold true for M. lignano, considering that in transversal amputations a head was never regenerated, but in longitudinal incisions a second head\u2014in the presence of the original head\u2014could be formed (Fig.\u00a02c,d, Egger et al. 2006a,b). This experiment suggests that the ability to induce head formation, although present in adult M. lignano, is restricted to the head region only. Serotonin and possibly other substances related to the brain or nervous system might be key factors deciding on the regeneration capacity of flatworms.\nFig.\u00a02Heteromorphoses resulting from embryogenesis and regeneration. a Twin embryo of M. lignano, 8-cell stage. The two embryos are not separated by a dividing wall. In some cases, such embryos may grow to siamese twins. Diameter of individual embryos is about 150\u00a0\u03bcm. b Different animal than a found in culture dishes. The juvenile animals are connected at the level of the pharynx, possibly the result of two embryos growing together. c Adult M. lignano, 1\u00a0week after longitudinal incision (arrowhead). Only a small indentation is visible. d Same specimen as in c 29\u00a0days after longitudinal incision. A second head with brain, a single eye, and pharynx has emerged. Scale bars are 100\u00a0\u03bcm. c, d Same scale bar\nIt is interesting to note that double-headed specimens of M. lignano can sometimes be observed in untreated culture dishes. These aberrant forms might rather be the result of twin embryos (Fig.\u00a02a) that have grown together during embryonic development, than adults that were accidentally incised longitudinally (Sekera 1911; Steinmann and Bresslau 1913; Seilern-Aspang 1958).\nOvaries restricting regeneration?\nThe possible evolutionary origin and function of regeneration aside, what genetical, physiological, developmental, or environmental factors are responsible to determine to what extent an organism can regenerate? Even close relatives may differ vastly in regeneration capacity (e.g., Microstomum\u2013Macrostomum), and also the same species in different developmental stages. Stenostomum alternates seasonally between asexual and sexual reproduction (protandric hermaphroditism). Asexual and male specimens of Stenostomum grande can regenerate all organs, but lose the ability to regenerate a head after the appearance of ovaries (van Cleave 1929). While the presence of ovaries and the loss of regeneration capacity might be coincidental, it appears possible that in S. grande ovaries are producing substances that inhibit regeneration. Could sexualization be directly affecting a general decrease of regeneration potential? At present, the molecular nature of such an inhibitory substance remains unclear. Vasa-related genes are expressed strongly both in gonads and in the early blastema in D. japonica and in M. lignano (Shibata et al. 1999; Pfister and Ladurner 2005), making an inhibitory function of vasa during regeneration improbable, at least in these two species. Also, contrary to the findings in Stenostomum, several triclads are known to regenerate all organs in the presence of gonads (e.g., S. mediterranea, S\u00e1nchez Alvarado 2003). Two possibilities may explain differences in the regeneration capacity in the presence of ovaries: either the ovaries in Stenostomum are producing a unique inhibitory substance that is not synthesized in Schmidtea, or Schmidtea is able to counteract the effects of such an inhibitory agent.\nRegeneration in juveniles\nIf gonads, specifically ovaries, are indeed hindering regeneration in some species, then sexually immature juveniles could possibly be more potent regenerates than adults. In the rhabdocoel Mesostoma, young animals (the author does not specify their age) are reported to regenerate better than adults (Fulinski 1922). Contrastingly, in the acoel species H. giselae, no regeneration even of large juveniles (2.5\u00a0mm long) takes place (Steinb\u00f6ck 1967).\nIn most cases, however, juveniles regenerate as well (or bad) as adults. An explanation is that gene expression for the building of gonads is already occurring in immature animals, thus diminishing possible differences in the regeneration capacity between juveniles and adults. Regeneration in juvenile triclads has been studied in B. punctata by Br\u00f8ndsted, who found that animals recently hatched from cocoons were able to regenerate the head at the same rate as mature adults (Br\u00f8ndsted 1942, 1969). Pechlaner (1957) studied the regeneration of freshly hatched animals, juveniles and mature adults in the proseriate O. auditivum. He did not observe significant differences in the regeneration capacity between these age groups. Neither juvenile nor adult dalyellids were found to regenerate (Hein 1928). In the lecithoepitheliate G. sphyrocephala, the regeneration capacity of juveniles corresponds to that of adults (Hagleithner 1946). The results for M. lignano support these findings and show that even very young animals (1\u00a0day after hatching) are capable of regeneration, indicating that the neoblast stem cell system is already present and functional as found in adults. Accordingly, BrdU labeling of 1-day-old hatchlings revealed a similar pattern of S-phase cell distribution as in adults, with the majority of labeled cells along the sides of the animal, and no labeled cells in the rostrum. Just like adults, juveniles were not able to regenerate a head (Egger et al. 2006a,b).\nSimilarities between postembryonic development and regeneration\nHowever similar, regeneration has some characteristics not shared with postembryonic development. Unlike postembryonic development, regeneration in flatworms is triggered by a disruptive event like fission or amputation (but not by chemical destruction of tissue, burning, or high voltage; see Br\u00f8ndsted 1969). As in other flatworms, in M. lignano, the wound is soon closed after amputation by contraction of the ring musculature minimizing the wound surface, and by flattening of the surrounding epidermal cells (Salvenmoser et al. 2001). In the next days, undifferentiated cells (neoblasts) accumulate at the wound site, forming a distinct blastema, covered by new and old epidermal cells. Subsequently, differentiation of microorgans and organs becomes prevalent within the blastema, superseding the blastema proper (Egger et al. 2005, 2006a,b). Epidermal cells are not undergoing mitoses, as new epidermal cells and blastema cells are derived from neoblasts located in the mesodermal space. Different to triclads, neoblasts are proliferating also within the blastema, not only at the blastema border (Egger et al. 2005). While wound closure and blastema formation are regeneration-specific events, similarities between regeneration and postembryonic development become prevalent starting with differentiation in the (former) blastema. In extreme cases, where only a small piece remains for regeneration (e.g., in pharynx-level regenerates, Egger et al. 2006a,b), resemblance with postembryonic development is particularly striking (Fig.\u00a03). The remaining organs in these regenerates undergo extensive morphallactic changes to accommodate to the new body proportions (Fig. 3n\u2013o in Egger et al. 2006a).\nFig.\u00a03Comparison of M. lignano hatchlings with posterior regenerates amputated at the pharynx level. b and d are depicting the same individual. a Hatchling up to 24\u00a0h old. b Pharynx level regenerate 7\u00a0days after amputation. c 7-day-old hatchling. d Pharynx level regenerate 14\u00a0days after amputation. Scale bar for all subpanels is 100\u00a0\u03bcm\nOne week after amputation, pharynx-level regenerates of M. lignano are confronted with similar challenges as freshly hatched juveniles: both have to build gonads and the male and female genital organs, grow a full set of duo-gland adhesive glands, and increase their size by five or six times (Figs.\u00a03a,b, and 4). It seems plausible, that after wound healing and restoration of the basic functionality of the gut, pharynx-level regenerates are making use of similar or same developmental pathways that are used in hatchlings (Fig.\u00a04) (cooption, S\u00e1nchez Alvarado 2000). This view is supported by the ability of hatchlings and juveniles to regenerate just like adults, and by the similarity of the stem cell system in hatchlings, juveniles, and adults (Ladurner et al. 2000; Egger et al. 2006a,b). Vasa gene expression is similar in hatchlings and in gonad-recovering regenerates as well (Pfister and Ladurner 2005). Also, the time needed for developing to a full-grown adult in M. lignano is about the same for hatchlings (Morris et al. 2004) and 1-week-old pharynx-level regenerates, that is about 2\u00a0weeks (Fig.\u00a03 and Egger et al. 2006a,b). In the proseriate M. fusca, the regeneration of small anterior pieces also goes through stages similar to postembryonic development (Giesa 1966), which is observed in triclads as well (Bagu\u00f1\u00e0 1998). Starved adults, reducing their size and breaking down the gonads (Nimeth et al. 2004), are also likely recapitulating their postembryonic development while growing back to full size (Fig.\u00a04). Not only is regeneration akin to postembryonic development, amputated animals can even be rejuvenated by regeneration (Fig.\u00a04 and Haranghy and Bal\u00e1zs 1964 for triclads, Egger et al. 2006a,b for M. lignano). While untreated animals were found to live for about 10\u00a0months in culture conditions, they were outlived by animals amputated 45 times over a period of 20\u00a0months, effectively doubling their life span so far (Egger et al. 2006a,b; Egger, unpublished observations). At the time of writing, many of these repeatedly amputated animals are still alive.\nFig.\u00a04Similarities between (post-)embryonic development, regeneration, and starvation considering as example M. lignano. Successive developmental stages from left to right, from embryonic to postembryonic development (formation of gonads and genital organs) until adulthood. Regeneration If an adult animal is amputated just posterior of the pharynx, the anterior piece will pass stages similar to early juveniles and subadults before regeneration to a normal adult is completed. The same animal can be amputated repeatedly, each time seemingly repeating parts of its postembryonic development. Repeated regeneration was also shown to have a rejuvenation effect. A connection between the formation of organ primordia during embryonic development and the organ differentiation in regenerating animals is possible, but has not been shown yet for M. lignano. Some developmental pathways are likely shared between late embryos and regenerates as well, e.g., the initial building of microorgans like the duo-gland adhesive systems. Both in embryos and posterior regenerates, these organs are built from scratch. Starvation Animals starved for 30\u00a0days or longer break down their gonads and genital organs shrink, but can recover and regain sexual prowess after feeding. Starvation leading to an extended life span was shown for triclads and is also likely to occur in M. lignano\nA fountain-of-youth effect through starvation was shown to occur in triclads (Haranghy and Bal\u00e1zs 1964), but was not yet proven for M. lignano (Fig.\u00a04).\nEmbryonic development and regeneration\nEarly embryogenesis is vastly different between diverse free-living flatworm taxa (for recent publications, see Jondelius 2004 for nemertodermatids; Ramachandra et al. 2002 for acoels; Morris et al. 2004 for macrostomorphans, Younossi-Hartenstein and Hartenstein 2000 for polyclads, Cardona et al. 2005 for triclads, and Younossi-Hartenstein and Hartenstein 2001 for rhabdocoels). Even comparably close relatives show significant differences in their early embryonic development. Lacking vitellaria and therefore producing entolecithal eggs, Macrostomorpha and Polycladida are the only archoophoran Rhabditophora, placing them at the basal-most positions in this largest flatworm taxon (Ehlers 1985; Rieger 1996). Nonetheless, their early embryonic development starts to deviate after about the third cleavage (Fig.\u00a05). If developmental pathways from the embryonic development are reused in regeneration, it is most likely from the formation of organ primordia and later stages (Fig.\u00a04). Cardona et al. (2005) point out the similarities between embryogenesis and early regeneration in triclads concerning the building of a provisional epidermis and the development of muscles and nerves, suggesting that not only postembryonic, but also embryonic developmental pathways are utilized during regeneration, a viewpoint shared with Vannini (1966). A comparison of gene expression patterns between regenerates (see Reddien et al. 2005 for triclads), late embryos and hatchlings seem to be a worthwhile task.\nFig.\u00a05Early embryonic development of two basal rhabditophorans. a The 12-cell stage of the polyclad Pseudostylochus intermedius. Egg without eggshell. b 12-cell stage of the macrostomorphan M. lignano. Different to the clear spiral cleavage of polyclads, M. lignano forms the so-called hull cells after the third cleavage to enclose the rest of the embryo. The diameter of the embryos is about 150\u00a0\u03bcm\nEarly blastomere ablation in polyclads has revealed a determinative development as in other spiralians (Boyer 1986), the fate of blastomeres depending on micromere\u2013macromere interaction (Boyer 1989). Polyclads do not have an invariant cell lineage, however (Boyer 1992). Polyclad embryos can be obtained devoid of an eggshell (Fig.\u00a05a) and still develop normally in a culture dish treated with antibiotics. Regeneration experiments with polyclad embryos may thus be relatively easy to carry out and could provide additional clues about the requirements for regeneration in flatworm embryos. Also, the regeneration capacity of polyclad larvae, as compared to hatchlings of directly developing polyclads, would be interesting to know. A connection between regulation in early embryonic development and regeneration capacity does not seem to exist. This notion is supported by regeneration experiments with embryos of the triclad P. nigra. Regeneration does not take place before organ systems, such as the nervous system, reach a certain degree of differentiation in the embryo, so that stage five embryos are unable to regenerate, while stage six embryos with a more differentiated nervous system readily regenerate lost parts (Le Moigne 1966). This author held that the nervous system plays a critical role in the formation of a blastema during regeneration.\nNervous system and role of the brain for regeneration\nThe nervous system, especially the brain, has long been considered to play a key role in regeneration (Kreshchenko et al. 2001). In many free-living flatworms, posterior parts can be regenerated, while the brain cannot. In anterior regenerates of M. lignano missing the brain, a blastema-like structure was observed, which was unable to give rise to all missing organs. The more posterior the animals were amputated, the less pronounced was this abortive anterior blastema (Egger et al. 2006a,b). In Macrostomum, are the brain and the nerve cell clusters associated with the pharynx (Fig.\u00a06) required to release factors needed for the regeneration of brain, eyes, and pharynx? The study of gene expression in head-amputated regenerates of the triclads D. japonica and S. mediterranea revealed necessary genes for regeneration of the brain (Cebri\u00e0 et al. 2002a; Koinuma et al. 2003; Cebri\u00e0 and Newmark 2005). Also, with nou-darake a factor was identified that restricts brain tissue to the head region of D. japonica. If nou-darake is interrupted by RNA interference, brains appear in all body regions (Cebri\u00e0 et al. 2002b). In species that are not able to regenerate a brain, nou-darake or a similar factor might be expressed not only in the brain region but also throughout the body, thus preventing the formation of a new brain even if the old brain was lost.\nFig.\u00a06Immunocytochemical stainings of the nervous system of M. lignano. Confocal images. a GYIRFamidergic immunoreactivity. Clusters of immunoreactive cells are found lateral to the pharynx and in front of the eyes. b, c Serotonergic immunoreactivity in the head region. b Full stack of confocal images. Note the stained cell clusters lateral to the pharynx and in front of the eyes. c Subset of image stack in b. In this focal plane, stained cells anterior to the eyes become apparent. Arrowheads denote the level of the eyes\nIn M. hystricinum marinum and M. pusillum, large clusters of serotonergic cells have been found besides the posterior part of the pharynx (Ladurner et al. 1997). These results were corroborated for M. lignano by staining of the serotonergic and the GYIRFamidergic nervous system (Fig.\u00a06). Amputees cut in the middle of the pharynx lose this cluster of nerve cells, as well as a large part of the pharynx, which is known to be a critical structure for regeneration in some triclads (Br\u00f8ndsted 1969) and polyclads (Ishida 1998). Ladurner et al. (1997) held that the distribution of serotonergic nerve cells is correlated with the regeneration capacity. In species where serotonergic neurons are restricted to the head area, the posterior part of the animals cannot regenerate the head. In the asexually reproducing species M. lineare and S. leucops, serotonergic nerve cells are found over the whole length of the main nerve cords.\nThe inability of M. lignano to regenerate eyes after transversal amputation or oblique amputation between the eyes, is possibly linked with a severe brain damage caused by these amputations, and the loss of strongly immunoreactive cells in front of the eyes (Fig.\u00a06c). In two cases of longitudinal incisions, lost eyes were regenerated or supernumerary eyes were built\u2014but in these cases, the brain was only slightly injured. In a series of experiments, Lender (1950, 1951a\u2013c) and Wolff and Lender (1950a,b) stress the importance of the head ganglia for the regeneration of eyes in P. nigra. If the brain was repeatedly excised or X-irradiated, no regeneration of eyes took place: an intact brain was obviously required to induce eye regeneration. These results were corroborated for Dugesia (now Schmidtea) lugubris by T\u00f6r\u00f6k (1958). However, L. littoralis, L. saxicola, and P. litoricola can regenerate eyes even if the brain was excised (Morgan 1905; Olmsted 1922a).\nVannini (1965) observed in S. lugubris and P. nigra that the testes do not regenerate in the absence of a brain. Ghirardelli (1965) found that if a two-headed Dugesia was decapitated on one side, the testes on the same side degenerated. Contrastingly, the gonads in M. lignano are viable for an extended period of time after decapitation or oblique amputation between the eyes, and are only reduced if the pharynx is lost as well, causing malnourishment and starvation (Egger et al. 2006a,b). This may be an indication that the building of the gonads is not under the primary control of the brain in M. lignano, just like the tail plate can be built without the presence of a head. The regeneration of a tail in headless regenerates is possible in M. lignano as well as in triclads of the Dendrocoelum group (Br\u00f8ndsted 1939, 1969), in the polyclads L. littoralis, L. saxicola, and P. litoricola (Morgan 1905; Olmsted 1922a), and also in the rhabdocoel Mesostoma (Fulinski 1922). Therefore, the head in all of these organisms is not required to control the regeneration of the tail. In Cestoplana and T. brocchi, the pharynx can regenerate in the absence of the brain (Child 1905c; von Levetzow 1939); in Cestoplana often a secondary pharynx is built in the presence of the old pharynx (Child 1905c). Also, the regulation of tissue breakdown (e.g., gonads) and body size reduction during starvation is possible in decapitated pieces, so the brain is not responsible for this regulation as well. Headless M. lignano with intact pharynx are able to feed, in accordance with the polyclads Notoplana acticola and Planocera gilchristi (Koopowitz et al. 1976). These animals are able to identify and devour food in the absence of a brain, while Enchiridium punctatum, a polyclad possessing a tubular instead of a plicate pharynx, cannot feed without the brain (Koopowitz et al. 1976). Similarly, some triclads, also equipped with a tubular pharynx, are not able to feed if the brain was amputated (Hyman 1951). Brainless M. ehrenbergi cannot feed but can move normally (Steinmann and Bresslau 1913), whereas brainless Phaenocera is reported to behave similarly to normal animals (Fulinski 1922). G. sphyrocephala, different to M. lignano, cannot adhere to the substrate after decerebration (Hagleithner 1946).\nThe main function of the brain in M. lignano is probably to be found in directed motion (e.g., light avoidance, predator avoidance; see also Koopowitz et al. 1976 and literature therein), satiation reflex (Koopowitz et al. 1976), mating behavior (Scharer et al. 2004), and chemical analyzation of the medium.\nThe totipotency of neoblasts\nPharynx-level regenerates of M. lignano have shown that the regeneration of gonads is possible after removal of all gonadal tissue. The viability of the regenerated gonads has been demonstrated by breeding experiments with fully regenerated animals. Besides the gonads, also the gut and the copulatory organs have been rebuilt de novo in pharynx-level regenerates (Egger et al. 2006a,b). In similar experiments, Morgan (1902) removed all gonads in Schmidtea lugubris, and the animals regenerated to fully functional sexual organisms. It is still possible that the remaining neoblasts in the amputee are only pluripotent and not totipotent, different types of neoblasts being competent for different (groups of) tissues. A convincing experiment to prove the totipotency of neoblasts would be the injection of a single neoblast into a lethally X-irradiated animal that is to be amputated some weeks after the injection. In case the animal survives, and is able to restore all removed organs, then either redifferentiation of cells would have taken place, or (at least some) adult neoblasts would be totipotent. A similar experiment, with the injection of 20,000\u201324,000 cells enriched in neoblasts (30\u201388% neoblasts) into X-irradiated S. mediterranea, was performed by Bagu\u00f1\u00e0 et al. (1989). This experiment was not replicated in the literature, but the results made highly probable that neoblasts as a whole are totipotent in routine cell turnover, leaving open the questions if this holds also true for regeneration and if this totipotency applies to one single cell type or to several different types of stem cells.\nAre neoblasts in species with limited regeneration potential not totipotent, or are they just not receiving the right stimulus or guidance for regeneration? Supporting the latter notion is the ability of Macrostomum to duplicate heads after longitudinal incision (Fig.\u00a02d and Egger et al. 2006a,b), while decapitated animals were never observed to regenerate a head (Egger et al. 2006a,b). In this species, it seems that stimuli from the brain are required to duplicate or regenerate the brain. Is there a specific subset of predetermined \u201cbrain\u201d neoblasts in species that cannot regenerate a brain? These neoblasts could be located in the vicinity of the brain and therefore lost by amputation of the brain. Brain excision experiments in polyclads, where only the encapsuled brain was removed (von Levetzow 1939; Koopowitz et al. 1976), do not support the existence of such brain-specific neoblasts, as these animals were not able to regenerate the brain. Also, amputation experiments in M. lignano, where only brain and the neoblast-free rostrum were amputated, showed no brain regeneration (Egger et al. 2006a,b). While neoblasts are required for regeneration in general, they do not determine whether a species is able to regenerate a specific organ such as the head, as was found with grafting experiments in triclads (Br\u00f8ndsted 1969).\nCan a single neoblast from an adult develop to a whole organism in an adequate environment, just like a zygote? For the parasitic flatworm Taenia crassiceps, it was shown that single cells of trypsinated cysticerci are able to produce complete cysticerci in the mouse host (Toledo et al. 1997). So in these cestodes, a single totipotent cell, other than a zygote, is able to recover a whole animal. No such experiment is known for free-living flatworms. Besides the difficulty in providing a suitable culture medium, distinct differences between a single neoblast and a zygote are yolk content or surrounding yolk cells and maternal determinants that are present in eggs, but not in neoblasts. Do (all or some) neoblasts have embryonic characteristics, as some authors held (e.g., Keller 1894)? When and where do neoblasts origin in the embryo? Polyclads with their clear and traceable embryonic development appear to be suitable flatworms for investigating these questions.\nHeteromorphoses\nHetermorphoses, i.e., the occurrence of supernumerary heads or tails, in species that cannot regenerate an amputated head, have shown that the regeneration potential of neoblasts is not to be equated with the regeneration capacity of the species. Heteromorphoses are also a placative warning sign for the dangers coming with the ability to regenerate (all) organs. Last but not the least, hetermorphoses in free-living flatworms show that the duplication of axes is not only possible during embryonic development (Fig.\u00a02b, siamese twins), but also in adults, even after comparably minor surgery. The M. lignano specimen depicted in Fig.\u00a02c,d shows that a longitudinal incision in the anterior part of the animal can establish a second anteroposterior body axis parallel to the original axis. The new anteroposterior axis reaches from the rostrum tip to the caudal tip and duplicates all organs including head, pharynx, female genital opening, and male copulatory organ. Even the breadth of the animal approximates that of two single specimens. Over time, however, the animal degenerated in both halves losing all eyes, and was not able to recover to a viable state (Egger et al. 2006a,b). A decreased viability in animals with heteromorphoses was also observed in Macrostomumappendiculatum and Bothrioplana bohemica (Sekera 1911), as well as in S. leucops (Ruhl 1927b).\nIn triclads, duplicated or even multiple heads (e.g., 10-headed D. lacteum, Lus 1924) and tails have been generated and studied by a great number of researchers (see Br\u00f8ndsted 1969). Randolph (1897) made longitudinal and lateral incisions in Planaria maculata [today: Dugesia (Girardia) tigrina] leading to additional eyes and pharynges. It was found that when a duplicated head or tail was amputated close enough to the second main axis, regeneration was inhibited by the presence of the remaining head or tail (Rand and Mildred 1926). In the polyclad Leptoplana, double-heads and double-tails were experimentally produced by longitudinal incision (Child 1905a). With the same method, tails, but not heads, could be duplicated in the rhabdocoel Mesostoma (Fulinski 1922) and in the acoel P. caudatus (Keil 1929). Constrastingly, heads, but not tails could be duplicated by incision in the catenulid S. leucops (Ruhl 1927b).\nAnother type of heteromorphoses is created by provoking a body axis reversed to the original body axis. This was found in M. lignano after longitudinal incision in the head, that induced the creation of a tail plate appendage in the anterior body region (Egger et al. 2006a,b). Agata et al. (2003) suggested that one of the main functions of a blastema in triclads is producing positional signals and inducing intercalary regeneration between stump and blastema. An incision may cause the building of a very small blastema at the wound site, which provides anterior or posterior positional signals. Depending on the signal the blastema produces, either a head or a tail plate is subsequently formed.\nIncisions may also have a long-range effect, as was observed by Randolph (1897) in triclads, where outgrowths appeared at body regions not affected by incision. A similar phenomenon as in M. lignano was observed by Steinb\u00f6ck (1967) in H. giselae, where an animal was divided into 10 pieces, one of which developed a tail-like structure at the anterior end besides the head. Just as was observed in M. lignano (Egger et al. 2006a,b), the anterior tail in H. giselae was gradually moving along the side of the animal towards the posterior end, when after 36\u00a0days the additional tail was located at the posterior tip of the animal.\nConclusion\nA pronounced regeneration capacity and the existence of possibly totipotent stem cells in adults are two key features of many flatworms not found in this combination in any other bilaterian taxa. It could be shown that regeneration follows similar principles in many different taxa of free-living flatworms, even if the regeneration capacity can vastly differ. Such comparisons make it possible to recognize unique patterns, and also to form general ideas how, why, and when regeneration works. For this reason, it appears to be valuable to establish several flatworm model organisms from various taxa, to address old and upcoming questions regarding regeneration and the neoblast stem cell system. One of these comparably new model flatworms is M. lignano, a small but versatile worm that facilitates regeneration studies on the cellular and molecular level.","keyphrases":["rhabditophora","planarian","catenulida","acoela","platyhelminthes"],"prmu":["P","P","P","P","U"]}
{"id":"Eur_J_Nucl_Med_Mol_Imaging-3-1-1998890","title":"Treatment of patients with gastro-entero-pancreatic (GEP) tumours with the novel radiolabelled somatostatin analogue [177Lu-DOTA0,Tyr3]octreotate\n","text":"Medical treatment and chemotherapy are seldom successful in achieving objective tumour reduction in patients with metastatic neuroendocrine tumours. Treatment with the radiolabelled somatostatin analogue [90Y-DOTA0,Tyr3]octreotide may result in partial remissions in 10\u201325% of patients. The newer analogue [DOTA0,Tyr3]octreotate (octreotate) has a ninefold higher affinity for the somatostatin receptor subtype 2 as compared with [DOTA0,Tyr3]octreotide. Also, labelled with the beta- and gamma-emitting radionuclide 177Lu, it has proved very successful in achieving tumour regression in animal models. The effects of 177Lu-octreotate therapy were studied in 35 patients with neuroendocrine gastro-entero-pancreatic (GEP) tumours who underwent follow-up for 3\u20136 months after receiving their final dose. Patients were treated with doses of 100, 150 or 200 mCi 177Lu-octreotate, to a final cumulative dose of 600\u2013800 mCi, with treatment intervals of 6\u20139 weeks. Nausea and vomiting within the first 24 h after administration were present in 30% and 14% of the administrations, respectively. WHO toxicity grade 3 anaemia, leucocytopenia and thrombocytopenia occurred after 0%, 1% and 1% of the administrations, respectively. Serum creatinine and creatinine clearance did not change significantly. The effects of the therapy on tumour size were evaluable in 34 patients. Three months after the final administration, complete remission was found in one patient (3%), partial remission in 12 (35%), stable disease in 14 (41%) and progressive disease in seven (21%), including three patients who died during the treatment period. Tumour response was positively correlated with a high uptake on the octreoscan, limited hepatic tumour mass and a high Karnofsky Performance Score. Because of the limited efficacy of alternative therapies, many physicians currently adopt an expectant attitude when dealing with patients with metastatic GEP tumours. However, in view of the high success rate of therapy with 177Lu-octreotate and the absence of serious side-effects, we advocate its use in patients with GEP tumours without waiting for tumour progression.\nIntroduction\nNeuroendocrine gastro-entero-pancreatic (GEP) tumours, which comprise pancreatic islet cell tumours, non-functioning neuroendocrine pancreatic tumours and carcinoids, are usually slow growing. If the tumour is localized, the therapy of choice is surgery. When a metastatic tumour causes a syndrome by hormonal overproduction (i.e. carcinoid syndrome, hypergastrinaemia), treatment with somatostatin analogues results in symptomatic relief in most cases. In terms of objective tumour reduction (complete and partial remission), however, treatment with somatostatin analogues is seldom successful, whether or not it is given in combination with interferon-\u03b1 [1, 2, 3].\nA new development in cytoreductive therapy for GEP tumours is the use of radiolabelled somatostatin analogues. Initial studies with high doses of [111In-DTPA0]octreotide (111In-octreotide; Octreoscan) in patients with metastatic neuroendocrine tumours were encouraging, although partial remissions were exceptional [4, 5]. However, 111In-coupled peptides are not ideal for peptide receptor radionuclide radiotherapy (PRRT) because of the small particle range and therefore short tissue penetration. Therefore, another radiolabelled somatostatin analogue, [90Y-DOTA0,Tyr3]octreotide, was developed. Using this compound, partial remissions have been reported in 10\u201325% of patients with neuroendocrine tumours [6, 7, 8].\nRecently, it was reported that the somatostatin analogue [DOTA0,Tyr3]octreotate has a ninefold higher affinity for the somatostatin receptor subtype 2 as compared with [DOTA0,Tyr3]octreotide [9]. Also, labelled with the beta- and gamma-emitting radionuclide 177Lu, this compound was shown to be very successful in achieving tumour regression and animal survival in a rat model [10]. In a comparison in patients, we found that the uptake of radioactivity, expressed as a percentage of the injected dose of [177Lu-DOTA0,Tyr3]octreotate (177Lu-octreotate), was comparable to that after 111In-octreotide for kidneys, spleen and liver, but was three- to fourfold higher for four of five tumours [11].We concluded that 177Lu-octreotate potentially represents an important improvement because of (a) the higher absorbed doses that can be delivered to most tumours with about equal doses to potentially dose-limiting organs and (b) the lower tissue penetration range of 177Lu as compared with 90Y, which may be especially important for small tumours.\nIn this study we present the first data on the side-effects as well as the antitumoral effects of 177Lu-octreotate therapy in 35 patients with GEP tumours, who had a follow-up of 3\u20136 months after receiving their final dose.\nMaterials and methods\nPatients Thirty-five patients with GEP tumours were studied. All patients had tumour uptake on the octreoscan preceding the therapy that was at least as high as the uptake in the normal liver tissue. None of the patients had received prior treatment with other radiolabelled somatostatin analogues. Eight patients used Sandostatin s.c.; this medication was discontinued from 1 day before to 1 day after the treatment. Prerequisites for treatment were: Hb \u22656\u00a0mmol\/l, WBC \u22652\u00d7109\/l, platelets \u226580\u00d7109\/l, creatinine \u2264150\u00a0\u03bcmol\/l and Karnofsky Performance Score \u226550.\nAll patients gave written informed consent to participate in the study, which was approved by the medical ethical committee of the hospital.\nMethods [DOTA0,Tyr3]octreotate was obtained from Mallinckrodt, St Louis, Mo., USA. 177LuCl3 was obtained from NRG, Petten, the Netherlands and Missouri University Research Reactor (MURR), Missouri, Mo., USA, and was distributed by IDB, Baarle-Nassau, the Netherlands. 177Lu-octreotate was prepared as described previously [11].\nGranisetron 3\u00a0mg was injected i.v. and an infusion of amino acids (lysine 2.5%, arginine 2.5% in 1\u00a0l 0.9% NaCl; 250\u00a0ml\/h) was started 30\u00a0min before the administration of the radiopharmaceutical and lasted up to 3.5\u00a0h afterwards. The radiopharmaceutical was co-administered via a second pump system. The treatment doses of 100\u00a0mCi were injected in 20\u00a0min and those of 150\u00a0and 200\u00a0mCi were injected in 30\u00a0min. The interval between treatments was 6\u20139 weeks. Patients were treated up to a cumulative dose of 750\u2013800\u00a0mCi (27.8\u201329.6\u00a0GBq) (corresponding to a radiation dose to the bone marrow of 2\u00a0Gy) [11], unless dosimetric calculations indicated that the radiation dose to the kidneys would then exceed 23\u00a0Gy; in these cases the cumulative dose was reduced to 600\u2013700\u00a0mCi.\nRoutine haematology, liver and kidney function tests, hormone measurements and serum tumour markers were measured 1 week prior to each therapy, as well as at follow-up visits. EORTC quality of life forms (QLQ-C30) [12] were filled out by the patients at each visit.\nCT or MRI scanning was done within 3 months before the first therapy, and 6\u20138 weeks, 3 months and 6 months after the last treatment.\nIn vivo measurements. The tumours on the CT or MRI scans were measured and scored according to the WHO solid tumour response criteria. The uptake on the pretreatment octreoscans was scored visually on planar images on a 4-point scale: grade 1, lower than normal liver tissue uptake; grade 2, equal to normal liver tissue uptake; grade 3, greater than normal liver tissue uptake; grade 4, higher than normal spleen\/kidney uptake.\nStatistics Analysis of variance (ANOVA), paired t tests and chi-square tests were used. P values <0.05 were considered significant.\nResults\nThe study population comprised 14 men and 21 women with a mean age of 54 years(range 19\u201378 years). Twelve had carcinoid tumour, 12 neuroendocrine pancreatic tumour, 8 neuroendocrine tumour of unknown origin and 3 gastrinoma. Twelve patients had been operated in the past, 1 had had radiotherapy, 3 had had chemotherapy and 14 had been treated with octreotide (Sandostatin). Sixteen of the 35 (46%) patients had documented progressive disease within 1 year before the start of the therapy. Cycle doses were 100\u00a0mCi in seven patients, 150\u00a0mCi in 14 and 200\u00a0mCi in the remaining 14. Higher dosages were not administered, since 200\u00a0mCi 177LuCl3 is typically bound to 180\u2013300\u00a0\u03bcg [DOTA0,Tyr3]octreotate, and higher doses would result in a lower percentage tumour uptake owing to receptor saturation. In 30 patients, the final intended cumulative dose of 600\u2013800\u00a0mCi was administered. Three of the five remaining patients had progressive disease and died before completing their treatment; the other two patients, who were both elderly, stopped their treatment after reaching a cumulative dose of 600\u00a0mCi because of the burden they felt the treatment to be.\nNausea and vomiting within the first 24\u00a0h after the administration were present in 30% and 14% of the administrations, respectively, and were independent of the administered dose. Some abdominal pain was noticed during 11% of the administrations, especially in patients with liver metastases. Increased, mild hair loss was present in 17 of the 35 patients; hair regrowth had occurred at follow-up 3 and 6 months after the final administration.\nWHO toxicity grade 2 or 3 anaemia (Hb 4.95\u20136.2 or 4.0\u20134.9\u00a0mmol\/l, respectively), leucocytopenia (WBC 2.0\u20132.9 or 1.0\u20131.9\u00d7109\/l, respectively) and thrombocytopenia (platelets 50\u201374.9 or 25.0\u201349.9\u00d7109\/l, respectively) occurred after 8% and 0%, 5% and 1%, and 3% and 1% of the administrations, respectively. Toxicity grade 2 or 3 leucocytopenia or thrombocytopenia occurred in two out of three (67%) patients who had had previous chemotherapy, as against seven out of 32 (22%) patients who had not. Mean haematological parameters rose again during the follow-up after the final administration. Serum creatinine, creatinine clearance and serum HbA1c did not change significantly (data not shown). In patients without thyroid hormone medication, serum TSH and fT4 levels did not change. In women, serum LH and FSH concentrations did not change significantly; in men, serum testosterone decreased and serum LH concentrations increased significantly. Also inhibin-B concentrations decreased and serum FSH levels increased significantly.\nThe effects of the therapy on tumour size were evaluable in 34 patients. Three months after the final administration (on average 9 months from the start of the treatment), a complete remission (CR), evaluated with CT scanning, MRI and somatostatin receptor imaging, was found in one patient (3%), partial remission (PR) in 12 (35%), stable disease (SD) in 14 (41%) and progressive disease (PD) in 7 (21%), including the three patients who died during the treatment period (Table\u00a01) (Figs.\u00a01, 2). Follow-up evaluation 6 months after the final therapy was available for 19 of the 34 patients. All seven patients who had PR after 3 months still had PR after 6 months; in 10 of the 12 patients with SD, the evaluation was unchanged, whereas one had a minimal response (MR) and one had PD.Table\u00a01. Tumour responses\nin 34 patients, 3 months after the final administration of 177Lu-octreotate. Three\npatients with PD died before reaching their final doseTumour typeResponseTotalCRPRSDPDCarcinoid4 (33%)6 (50%)2 (17%)12NE pancreas1 (8%)1 (8%)7 (58%)3 (25%)12NE unknown origin4 (57%)1 (14%)2 (29%)7Gastrinoma3 (100%)3Total1 (3%)12 (35%)14 (41%)7 (21%)34Fig.\u00a01.A\u2013C  Planar scans of the abdomen, 3 days after the injection of 200\u00a0mCi 177Lu-octreotate in a patient with liver metastases of an operated neuroendocrine pancreatic tumour. A After the first treatment; B after the second treatment; C after the fourth treatment. Note the loss of intensity of uptake in the liver lesions (arrows in A). This sign virtually always indicates a tumour volume response. D, E CT scans of the same patient: before treatment (D) and 3 months after the last treatment (E). Tumour (arrows in D) is not demonstrated on E. Neither MRI nor octreoscan could demonstrate definite tumour deposits at that timeFig.\u00a02. A, B MRI scans in a woman with hepatic metastases of a gastrinoma. Before treatment (A) she was inoperable because of the size and localization of the metastases. Three months after completion of her treatment with 800\u00a0mCi 177Lu-octreotate she had a PR (B). Note also that the hypertrophy of the gastric wall (arrow in A) had diminished. Concomitantly, serum concentrations of gastrin (closed dots) and chromogranin-A (open dots) had decreased markedly (C). At 6 months the MRI showed no further regression. The patient underwent left partial hepatectomy and the two right-sided lesions were drained and injected with alcohol. MRI 3 months after surgery showed further regression of the right-sided lesions\nTumour response (CR or PR) was significantly more frequent in patients whose tumours had a high uptake on the octreoscan [6\/7 (86%) with grade 4 uptake vs. 7\/27 (26%) with grade 2 or 3 uptake; chi-square test: P<0.05]. Five out of the seven patients (71%) with PD had hepatomegaly and diffuse liver metastases vs 6 out of 27 (22%) with CR, PR or SD (chi-square test: P<0.05). Tumour response was more frequent in patients with documented PD within 1 year before the start of the treatment (8\/15; 53%) than in those without (5\/19; 26%), although this difference was not statistically significant (P=0.11; chi-square test). Of the 15 patients with documented PD before the start of the treatment, 3 (20%) had SD at follow-up. Patients who had a Karnofsky Performance Score of less than 80 before the treatment more frequently had PD (5\/8; 63%) than those who had higher scores (2\/26; 8%) (P<0.01; chi-square test).\nThe serum tumour marker chromogranin-A was elevated in 29 patients. During the treatment and follow-up, there was a clear decrease in serum chromogranin-A concentrations in patients with PR or CR, whereas these concentrations were virtually unchanged in patients with SD, and initially showed an increase in patients with PD (Fig.\u00a03).Fig.\u00a03. Serum chromogranin-A concentrations during and after therapy in patients with PR, SD or PD. The reduction in the number of patients during the course of the follow-up was due to death or missing values. Note the logarithmic y-axis\nThe patient-assessed quality of life, according to the EORTC-QLQ30 questionnaire, was evaluated in 25 patients. Ten patients were excluded because of progressive disease\/death (n=5) or missing forms during the follow-up period (n=5, foreign patients). Scores before the start of the treatment, after receiving 400\u2013600\u00a0mCi 177Lu-octreotate and at follow-up 3 months after the final treatment were evaluated. There were no significant differences for functional scales or single symptom scales. The global health scale, on which patients were asked to assign marks regarding both their general health and their quality of life, and which ranged from 0 to 100, was judged as higher than 70 by nine patients (36%) before the start of the treatment, by 17 (68%) patients during the treatment and by 16 (64%) patients after the treatment (P<0.05; chi-square test).\nDiscussion\nThere are few treatment options for metastatic GEP tumours. The use of radiolabelled somatostatin analogues for tumour regression is a promising new development. With [90Y-DOTA0,Tyr3]octreotide, PR (or CR) has been reported in 10\u201325% of patients with neuroendocrine tumours [6, 7, 8]. In the present study we found objective tumour shrinkage in 38% of the patients, but PD before the start of the treatment was documented in only 46% of the patients. This last fact is important, because documented PD was present in more than 80% of patients in two of the reported series treated with [90Y-DOTA0,Tyr3]octreotide [6, 8]. As with chemotherapy, an objective tumour response after PRRT is more likely in patients with (fast) growing tumours. Indeed, in our series we also found a trend towards a more favourable treatment outcome in patients with documented PD before the start of the treatment. It may therefore be concluded that the percentage of patients with significant tumour shrinkage in our study might have been even higher had the percentage of patients with PD prior to treatment been comparable to that in studies with [90Y-DOTA0,Tyr3]octreotide. The side-effects of treatment with 177Lu-octreotate are few and mostly transient, with mild bone marrow depression as the most common finding. Neither renal nor pituitary function deteriorated in any of our patients. Other side-effects can be ascribed to the radiation dose to the testes in men. This dose leads to significantly lower serum testosterone and inhibin-B levels which in turn give rise to higher serum LH and FSH concentrations, thereby substantiating that the pituitary function is unimpaired.\nThe patient-assessed global health score improved in 30% of patients during the treatment and the follow-up period. This is an important finding which reflects the improvement in patient well-being and stresses the scarcity of side-effects as perceived by patients. The fact that other scores mainly addressing symptoms did not change significantly is likely due to the diversity of symptoms between patients, the use of Sandostatin by symptomatic patients and the small size of the patient group.\nMany physicians adopt an expectant attitude when dealing with patients with metastatic GEP tumours owing to the low success rates of chemotherapy protocols. However, given that treatment with 177Lu-octreotate resulted in PR (or CR) in 38% of our patients, this attitude may be questioned. Although there was a (statistically non-significant) tendency towards more frequent tumour regression in patients who had documented PD before the start of the treatment, those of our patients who had an objective tumour response more frequently had a limited tumour load. This implies that waiting for tumour progression might place patients in a worse position, as PD during or after treatment was more frequent in patients with an extensive tumour load, especially in the liver. We therefore advocate treatment with177Lu-octreotate at an earlier stage of metastatic disease, when even CR may be possible. Another argument in favour of early treatment is that neuroendocrine tumours can dedifferentiate in the course of the disease, and lose their somatostatin receptors, making treatment with radiolabelled somatostatin analogues impossible. If serious side-effects of treatment with 177Lu-octreotate remain absent during longer patient follow-up, such treatment could also be considered in an adjuvant setting in patients with neuroendocrine tumours who are operated on with curative intent.\nLastly, as it has been shown in animal experiments that 90Y-labelled somatostatin analogues are more effective for larger tumours and 177Lu-labelled somatostatin analogues are more effective for smaller tumours [13, 14], combination therapy with 90Y-labelled and 177Lu-labelled octreotate may be tried in the near future. Such therapy might yield even better results than treatment with either of the radionuclides alone [15].","keyphrases":["octreotate","somatostatin receptor","gep tumours","radionuclide therapy","lutetium-177"],"prmu":["P","P","P","R","U"]}
{"id":"Glycoconj_J-4-1-2234449","title":"Characterization of anticoagulant heparinoids by immunoprofiling\n","text":"Heparinoids are used in the clinic as anticoagulants. A specific pentasaccharide in heparinoids activates antithrombin III, resulting in inactivation of factor Xa and\u2013when additional saccharides are present\u2013inactivation of factor IIa. Structural and functional analysis of the heterogeneous heparinoids generally requires advanced equipment, is time consuming, and needs (extensive) sample preparation. In this study, a novel and fast method for the characterization of heparinoids is introduced based on reactivity with nine unique anti-heparin antibodies. Eight heparinoids were biochemically analyzed by electrophoresis and their reactivity with domain-specific anti-heparin antibodies was established by ELISA. Each heparinoid displayed a distinct immunoprofile matching its structural characteristics. The immunoprofile could also be linked to biological characteristics, such as the anti-Xa\/anti-IIa ratio, which was reflected by reactivity of the heparinoids with antibodies HS4C3 (indicative for 3-O-sulfates) and HS4E4 (indicative for domains allowing anti-factor IIa activity). In addition, the immunoprofile could be indicative for heparinoid-induced side-effects, such as heparin-induced thrombocytopenia, as illustrated by reactivity with antibody NS4F5, which defines a very high sulfated domain. In conclusion, immunoprofiling provides a novel, fast, and simple methodology for the characterization of heparinoids, and allows high-throughput screening of (new) heparinoids for defined structural and biological characteristics.\nIntroduction\nGlycosaminoglycans (GAGs) are long, unbranched, negatively charged polysaccharide chains. The precursor of heparin and heparan sulfate (HS) is composed of alternating N-acetylglucosamine (GlcNAc) and d-glucuronic acid (GlcA) residues. In chondroitin sulfate (CS) and dermatan sulfate (DS), the aminosugar is a N-acetylgalactosamine (GalNAc) instead of GlcNAc. The polysaccharide chain precursors are modified by a sequential series of enzymatic reactions, including N-deacetylation\/N-sulfation, C5-epimerization of GlcA to l-iduronic acid (IdoA), and O-sulfation at various positions [27, 32].\nHeparin is widely used as an anticoagulant. A specific pentasaccharide sequence (GlcNAc\/NS(6OS)\u2013GlcA\u2013GlcNS(3,6OS)\u2013IdoA(2OS)\u2013GlcNS(6OS)) [5, 23, 37] in heparin binds with high affinity to the enzyme inhibitor antithrombin III, causing a conformational change, resulting in the exposition of its active sites and in inactivation of factor Xa. For the inactivation of factor IIa, an additional site in heparin proximal to the pentasaccharide is necessary. The formation of a ternary complex between antithrombin III, factor IIa and heparin, which requires at least 18 saccharide residues for efficient formation, subsequently results in the inactivation of factor IIa [23]. Next to heparin, other heparinoids are used, including preparations containing other GAGs such as HS and DS, low molecular weight heparins (LMWHs), and fondaparinux, a synthetic pentasaccharide identical in structure to the antithrombin III-binding pentasaccharide. Heparinoids are used to treat patients with venous thrombosis and pulmonary embolism [2, 5, 10, 22]. In addition, they are used to prevent thrombosis in patients that undergo surgery and to prevent clotting in the extracorporeal circulation during hemodialysis [2, 5, 10, 22, 24, 28]. Due to their lower molecular weight, LMWHs have a high anti-factor Xa activity compared to their anti-factor IIa activity resulting in a more refined regulation of coagulation [4, 24]. In addition, the biological half-life of LMWHs\/fondaparinux is longer in comparison to heparin, and the incidence of heparin-induced thrombocytopenia (HIT) is substantially reduced [6, 7, 23].\nLMWHs are synthesized from heparin by controlled chemical or enzymatic depolymerization processes, resulting in the production of oligosaccharides of various lengths (Table\u00a01). Dalteparin and nadroparin are produced by deamination using nitrous acid, resulting in the formation of an unnatural 2,5-anhydromannitol residue at the reducing end of the resulting oligosaccharides. Enoxaparin is produced by chemical \u03b2-elimination at alkaline pH, whereas tinzaparin is produced by enzymatic \u03b2-elimination by heparinase. Both processes result in the formation of an unsaturated uronic acid residue at the non-reducing end of the GAG fragments [12, 17].\nTable\u00a01Characteristics of therapeutic heparinoidsHeparinoid: generic name (brand name)Composition (and method of heparin modification)Molecular weight, kDaHeparinHeparin5.0\u201330.0Dalteparin (Fragmin)LMWH (deaminative cleavage with nitrous acid)4.0\u20136.0Nadroparin (Fraxiparine)LMWH (deaminative cleavage with nitrous acid)2.4\u20137.2Enoxaparin (Clexane, Lovenox)LMWH (\u03b2-eliminative cleavage by alkaline treatment)3.5\u20135.5Tinzaparin (Innohep, Logiparin)LMWH (\u03b2-eliminative cleavage by heparinase)3.4\u20135.6Sulodexide (Vessel Due F)80% \u2018fast-moving\u2019 heparina, 20% DS<8.0Danaparoid (Orgaran)84% HS, 12% DS, 4% CS4.0\u201310.0Fondaparinux (Arixtra)Pentasaccharide: C31H43N3Na10S81,728All drugs are derived from porcine intestinal mucosa, with the exception of the chemically synthesized pentasaccharide fondaparinux.LMWH Low-molecular weight heparin, HS heparan sulfate, DS dermatan sulfate, CS chondroitin sulfateaHeparin that is less sulfated and has a lower molecular weight [42].\nAll heparinoids, with the exception of fondaparinux, are heterogeneous mixtures of different GAG chains. Structural analysis of heparinoids is generally done by nuclear magnetic resonance spectroscopy, high pressure liquid chromatography, mass spectrometry, capillary electrophoresis, or polyacrylamide gel electrophoresis [17]. In general, these techniques require sophisticated instrumentation and sometimes extensive sample preparation. Alternative methodologies that are fast and simple are needed. Immunoprofiling of heparinoids could be a fast and simple method to characterize the complex heparinoids. Recently, a number of phage\u2013display-derived antibodies were generated, reactive with various domain structures within heparin. In this study nine antibodies\u2013all defining different heparin epitopes\u2013were applied to study their reactivity with heparinoids.\nMaterials and methods\nHeparinoids\nThe following heparinoids were included in the study: heparin (Sigma, St. Louis, MO), dalteparin (Pharmacia, Woerden, The Netherlands), nadroparin (Sanofi-Synth\u00e9labo, Maassluis, The Netherlands), enoxaparin (Aventis Pharma, Hoevelaken, The Netherlands), tinzaparin (LEO Pharma, Breda, The Netherlands), sulodexide (Alfa Wassermann S.p.A., Bologna, Italy), danaparoid (Organon, Oss, The Netherlands), and fondaparinux (Sanofi-Synth\u00e9labo, Maassluis, The Netherlands). Characteristics are given in Table\u00a01.\nQuantification of glycosaminoglycans by Farndale assay\nA Farndale assay was done to determine the concentration of GAGs in the preparations [13]. To 50\u00a0\u03bcl of the standard (heparin, 0\u2013100\u00a0\u03bcg\/ml) and heparinoids 1\u00a0ml Farndale reagent (40.5\u00a0mmol\/l glycine, 40.6\u00a0mmol\/l NaCl, and 0.05\u00a0mmol\/l 1,9-dimethylmethylene blue, pH\u00a03.0) was added and absorbance was measured directly at 525\u00a0nm.\nDetermination of glycosaminoglycan classes by agarose gel electrophoresis\nThe different classes of GAGs in the heparinoids were separated by agarose gel electrophoresis, fixed in 0.1% (w\/v) azure A in 10\u00a0mmol\/l MgCl2\u00b76H2O, 50\u00a0mmol\/l Na\u2013formiate and 0.001% (w\/v) Na\u2013azide (pH\u00a03.5), and stained by combined azure A\u2013silver treatment [38].\nEnzymatic digestion of heparinoids\nHeparinoids were diluted in heparinase I digestion buffer (50\u00a0mmol\/l NaAc and 50\u00a0mmol\/l Ca(Ac)2, pH\u00a07.0) and\/or chondroitinase ABC digestion buffer (25\u00a0mmol\/l Tris and 2\u00a0mmol\/l Mg(Ac)2, pH\u00a08.0) to a final content of 4\u00a0mg\/ml. The enzymes were added to a final concentration of 0.02\u00a0IU\/ml for heparinase I (E.C. 4.2.2.7; from Flavobacterium heparinum; Seikagaku, Tokyo, Japan) and 0.04\u00a0IU\/ml for chondroitinase ABC (E.C. 4.2.2.4; from Proteus vulgaris; Sigma). Heparinase I cleaves the linkages GlcNS\u00b16S\u2013IdoA2S predominantly present in heparin and to a minor extent in HS [11]. Chondroitinase ABC cleaves CS and DS [11]. The solutions were incubated for 16\u00a0h at 37\u00b0C.\nDetermination of glycosaminoglycan size by polyacrylamide gel electrophoresis\nHeparinoids and their enzymatic digestion products were separated by polyacrylamide gel electrophoresis using a 33% gel [36], fixed in 0.8% (w\/v) alcian blue in 2% (v\/v) HAc, and stained by combined azure A\u2013silver treatment [38].\nProduction of anti-heparin antibodies\nAntibodies (Table\u00a02) were obtained by biopanning against HS or heparin, using bovine kidney HS (antibodies HS4C3 [41], HS3A8, HS4E4 [8], and LKIV69 [43]), porcine intestinal mucosa heparin (antibodies EW3D10 and EW4G2 [39]), human lung HS (antibody NS4F5; Smits et al., unpublished results), and mouse and human skeletal muscle GAG (antibodies AO4B08 and RB4EA12 [15], respectively). Large scale production of soluble antibodies was performed as described before [8, 41].\nTable\u00a02Characteristics of epitope-specific anti-heparin antibodiesAntibodyVH CDR3 sequenceVH familyDP geneGAG used for selectionEssential modificationsInhibitory modificationsHS4C3GRRLKD338Bovine kidney HS [41]N-sulfation\u00a02-O-sulfation (minor importance)6-O-sulfation3-O-sulfationHS3A8GMRPRL338Bovine kidney HS [8]N-sulfation\u00a0C5-epimerization2-O-sulfation6-O-sulfation (likely)HS4E4HAPLRNTRTNT338Bovine kidney HS [8]N-acetylation2-O-sulfationa 6-O-sulfationN-sulfationC5-epimerizationLKIV69GSRSSR338Bovine kidney HS [43]N-sulfation6-O-sulfationC5-epimerization (likely)2-O-sulfationEW3D10GRTVGRN338Porcine intestinal mucosa heparin [39]Sulfation required, position(s) unknownbEW4G2GKVKLPN338Porcine intestinal mucosa heparin [39]Sulfation required, position(s) unknownbNS4F5SGRKGRMR353Human lung HScN-sulfation\u00a0C5-epimerization2-O-sulfation (high)6-O-sulfation (high)AO4B08SLRMNGWRAHQ347Mouse skeletal muscle GAG [15]N-sulfation\u00a0C5-epimerization2-O-sulfationd6-O-sulfation (high)RB4EA12RRYALDY332Human skeletal muscle GAG [15]N-acetylation\u00a0N-sulfation6-O-sulfationMPB49 (control)WRNDRQ338\u2013\u2013Given are the antibody name, amino acid sequence of the VH complementary determining region 3 (CDR3), VH germ line gene family, DP gene number, selection moiety, and relevant modification reactions required for antibody binding. Table derived from Wijnhoven et al. unpublished results.GAG Glycosaminoglycan, HS heparan sulfateaAntibody HS4E4 possibly requires as yet unspecified O-sulfated residues [16].bAlthough the chemical nature of the specific heparin\/HS structure recognized is not exactly known, the antibody defines a unique sulfated heparin\/HS structure as demonstrated by its specific staining pattern on renal cryosections [8, 39].cSmits et al., unpublished resultsdAntibody AO4B08 requires an internal 2-O-sulfated iduronic acid residue [16].\nEnzyme-linked immunosorbent assay\nReactivity of the anti-heparin antibodies with the heparinoids was evaluated by enzyme-linked immunosorbent assay (ELISA). Wells of microtiter plates were coated overnight by incubation with the heparinoids that bound by passive adsorption [41]. After blocking with PBS containing 3% (w\/v) BSA and 1% (v\/v) Tween-20 for 1\u00a0h, anti-heparin antibodies [in PBS containing 1% (w\/v) BSA and 0.1% (v\/v) Tween-20] were added for 1.5\u00a0h. As a negative control, antibody MPB49 was used, which is >95% identical to most antibodies used, but does not bind any GAG. Bound antibodies were detected using mouse IgG anti-VSV tag antibody P5D4 (1:10; Boehringer Mannheim, Mannheim, Germany), followed by incubation with alkaline phosphatase-conjugated rabbit anti-mouse IgG (1:2,000; Dako, Glostrup, Denmark), both for 1\u00a0h. Enzyme activity was detected using 100\u00a0\u03bcl 1\u00a0mg p-nitrophenyl phosphate (ICN, Aurora, OH)\/ml 1\u00a0M diethanolamine\/0.5\u00a0mM MgCl2 (pH\u00a09.8) as a substrate. Absorbance was measured at 405\u00a0nm [43]. Background reactivity, measured with the control antibody MPB49, was substracted. The absorbance with control antibody MPB49 was comparable to the absorbance measured when primary antibody was omitted.\nImmunofluorescence staining\nHuman kidney cryosections (2\u00a0\u03bcm) were incubated with anti-heparin antibodies in the absence or presence of 100\u00a0\u03bcg\/ml of the heparinoids. Bound antibodies were detected as described [43].\nResults\nGeneral biochemical analysis of heparinoids\nThe different classes of GAGs in the heparinoids were separated by agarose gel electrophoresis (Fig.\u00a01). Heparin showed a band at about the same position as the HS standard, whereas the LMWHs dalteparin, nadroparin, enoxaparin, and tinzaparin migrated somewhat further into the gel. Danaparoid displayed bands at the level of HS and DS, whereas the CS band, which only represents 4% of the prepration (Table\u00a01), could not be detected. Sulodexide clearly showed two bands, representing the \u2018fast-moving\u2019 heparin (heparin that is less sulfated and has a lower molecular weight [42]) and DS fractions. The chemically synthesized pentasaccharide fondaparinux, which has a much lower molecular weight compared to the other heparinoids, migrated further into the gel.\nFig. 1Agarose gel electrophoresis of heparinoids. The different classes of GAGs in the drugs were visualized by a combined azure A\u2013silver staining procedure. The standard contains 40\u00a0ng HS, 20\u00a0ng DS, and 20\u00a0ng CS\nTo analyze the molecular range of the heparinoids, gel electrophoresis using 33% polyacrylamide was applied. The LMWHs clearly displayed distinct fragments with a lower molecular weight compared to heparin (Fig.\u00a02a). The smaller fragments of enoxaparin compared to tinzaparin (both prepared by \u03b2-elimination) can be explained by a preference of heparinase for 2-O-sulfated iduronic acid in contrast to chemical alkaline treatment, which results in cleavage at both sulfated and non-sulfated iduronic acid residues. Sulodexide and danaparoid showed a broad smear with less distinct fragments, in line with their methods of preparation, which does not include a specific depolymerization step. The pentasaccharide fondaparinux demonstrated primarily one individual band. To further establish the nature of the heparinoids, they were subjected to enzymatic digestion using heparinase I and\/or chondroitinase ABC (Fig.\u00a02b for dalteparin, enoxaparin, and sulodexide). Heparin, dalteparin, nadroparin, enoxaparin, tinzaparin, and fondaparinux were only cleaved by heparinase I, in accordance with their derivation from heparin. Sulodexide was cleaved by heparinase I as well as chondroitinase ABC, which is in line with the heparin\/DS nature of the preparation. Danaparoid was primarily cleaved by chondroitinase ABC, reflecting the presence of DS\/CS, whereas heparinase I was less effective.\nFig. 2Thirty-three percent polyacrylamide gel electrophoresis of undigested (a) and enzymatically digested (b) heparinoids. Heparinoids were visualized by a combined alcian blue-silver staining procedure. Treatment with heparinase I resulted in cleavage of dalteparin and enoxaparin, whereas treatment with chondroitinase ABC did not. Sulodexide was cleaved by heparinase I as well as chondroitinase ABC\nCharacterization of heparinoids using anti-heparin antibodies\nReactivity of anti-heparin antibodies with immobilized heparinoids was analyzed by ELISA (Table\u00a03). For reference, reactivity of antibodies with heparin was set at 100%. A distinct antibody-binding profile could be observed for all antibodies, although also similar reactivity with a number of antibodies was noticed. For instance, enoxaparin and tinzaparin, both prepared by \u03b2-elimination, reacted differently with antibodies LKIV69 and EW4G2, whereas they reacted similarly with the other antibodies. Nadroparin reacted very poorly with all the antibodies tested, which may be in line with its overall low amount of sulfated disaccharides in comparison with other LMWHs [17]. In contrast, sulodexide and danaparoid reacted strongly with antibody HS4E4, which defines a low-sulfated and N-acetylated epitope, in comparison to the LMWHs. This is in line with the presence of low-sulfated heparin (HS) and HS in sulodexide and danaparoid, respectively, and which contain a substantial amount of N-acetylated glucosamine. Compared to heparin, the other heparinoids reacted poorly with antibody NS4F5, which defines a very highly sulfated oligosaccharide.\nTable\u00a03Reactivity of anti-heparin antibodies with heparinoids (ELISA)\u00a0HS4C3HS3A8HS4E4LKIV69EW3D10EW4G2NS4F5AO4B08RB4EA12Heparin100100100100100100100100100Dalteparin75\u2009\u00b1\u2009644\u2009\u00b1\u200948\u2009\u00b1\u200953\u2009\u00b1\u2009133\u2009\u00b1\u2009649\u2009\u00b1\u200980\u2009\u00b1\u2009016\u2009\u00b1\u2009413\u2009\u00b1\u20092Nadroparin29\u2009\u00b1\u200935\u2009\u00b1\u200924\u2009\u00b1\u200930\u2009\u00b1\u200903\u2009\u00b1\u200917\u2009\u00b1\u200940\u2009\u00b1\u200901\u2009\u00b1\u200911\u2009\u00b1\u20091Enoxaparin80\u2009\u00b1\u2009956\u2009\u00b1\u200987\u2009\u00b1\u2009310\u2009\u00b1\u2009236\u2009\u00b1\u2009826\u2009\u00b1\u200971\u2009\u00b1\u2009128\u2009\u00b1\u2009721\u2009\u00b1\u20096Tinzaparin84\u2009\u00b1\u2009155\u2009\u00b1\u200928\u2009\u00b1\u2009344\u2009\u00b1\u20091045\u2009\u00b1\u20091274\u2009\u00b1\u200919\u2009\u00b1\u2009220\u2009\u00b1\u2009325\u2009\u00b1\u20098Sulodexide102\u2009\u00b1\u2009571\u2009\u00b1\u200911283\u2009\u00b1\u20092151\u2009\u00b1\u2009743\u2009\u00b1\u200910100\u2009\u00b1\u200930\u2009\u00b1\u20090102\u2009\u00b1\u20091526\u2009\u00b1\u20098Danaparoid69\u2009\u00b1\u20091122\u2009\u00b1\u2009358\u2009\u00b1\u2009172\u2009\u00b1\u200922\u2009\u00b1\u200911\u2009\u00b1\u200910\u2009\u00b1\u2009063\u2009\u00b1\u200963\u2009\u00b1\u20091Anti-heparin antibodies were applied to heparinoids immobilized on microtiter plates. Given is the reactivity of the antibodies with the heparinoids relative to unmodified heparin. Values are given as mean \u00b1 SEM (n\u2009=\u20094). Each hepainoid shows a distinct immunoprofile.\nHeparinoids could be ranked according to their reactivity with antibodies. For instance, reactivity of antibody EW4G2 was highest with heparin and sulodexide, followed by tinzaparin, dalteparin, and enoxaparin. Reactivity with the other heparinoids was very low or absent. Antibody LKIV69 reacted strongly with heparin, followed by sulodexide and tinzaparin, whereas other heparinoids reacted poorly. Reactivity of antibody AO4B08 was highest with heparin and sulodexide, followed by danaparoid and enoxaparin\/tinzaparin\/dalteparin, whereas reactivity with nadroparin was absent. These data indicate that distinct heparin domain structures are present in different commercially available heparinoids.\nThe only chemically defined heparinoid studied was the pentasaccharide fondaparinux (GlcNS(6OS)\u2013GlcA\u2013GlcNS(3,6OS)\u2013IdoA(2OS)\u2013GlcNS(6OS)). Due to its small size (1,728\u00a0kDa), it was assayed using a competition ELISA (Table\u00a04). Heparin was included as a reference and values obtained for fondaparinux were related to those obtained for heparin. Antibodies HS4E4 and RB4EA12, both requiring N-acetylation in their epitopes, did not react with fondaparinux. Antibody NS4F5, which reacts with an extremely high sulfated oligosaccharide, was also not reactive. These results are in line with the chemical composition of fondaparinux, which contains no N-acetylated glucosamine and a non-sulfated glucuronic acid residue. Reactivity with antibody LKIV69, which reacts poorly with 6-O-sulfated epitopes, was very weak and reflects the high amount of 6-O-sulfation in fondaparinux. Reactivity with antibody AO4B08, which needs C5-epimerization, N-sulfation, high 6-O-sulfation and an internal 2-O-sulfated iduronic acid residue, was only moderate\/poor, reflecting the lack of an internal 2-O-sulfated iduronic acid (i.e. flanked by other iduronic acid residues). Reactivity with antibody HS4C3, which requires N-, 3-O-, and 6-O-sulfation, and antibody HS3A8, which requires at least C5-epimerization and N- and 2-O\u2013sulfation, was high, which corresponds with the chemical structure of fondaparinux. Reactivity was also high with antibodies EW3D10 and EW4G2, both defining an as yet undefined, but sulfated oligosaccharide. These data indicate that reactivity with antibodies may provide some chemical information of heparinoids.\nTable\u00a04Reactivity of anti-heparin antibodies with heparin and fondaparinux (competition ELISA)\u00a0HS4C3HS3A8HS4E4LKIV69EW3D10EW4G2NS4F5AO4B08RB4EA12Heparin0.3\u2009\u00b1\u20090.10.4\u2009\u00b1\u20090.12.7\u2009\u00b1\u20090.1 0.2\u2009\u00b1\u20090.00.1\u2009\u00b1\u20090.00.1\u2009\u00b1\u20090.00.1\u2009\u00b1\u20090.01.2\u2009\u00b1\u20090.2<0.01Fondaparinux1.7\u2009\u00b1\u20090.21.3\u2009\u00b1\u20090.1 >20010.3\u2009\u00b1\u20090.80.2\u2009\u00b1\u20090.01.5\u2009\u00b1\u20090.5 11.7\u2009\u00b1\u20090.8 12.3\u2009\u00b1\u20093.821.5\u2009\u00b1\u20090.5Ratio fondaparinux\/heparin5.73.3>7451.521511710.3>2150Given are the amounts of heparinoids (micrograms per milliliter), which result in a 50% inhibition of the binding of the anti-heparin antibodies to immobilized HS\/heparin (IC50 values), and the ratio between antibody reactivity with fondaparinux and heparin. Values are given as mean \u00b1 SEM (n\u2009=\u20093). Antibody EW3D10 showed a strong reactivity with fondaparinux, followed by antibodies HS4C3, HS3A8, and EW4G2. The other antibodies showed a relatively weak or no reactivity.\nReactivity of the anti-heparin antibodies with immobilized heparinoids was supported by immunofluorescence analysis (Fig.\u00a03). When human renal cryosections were simultaneously incubated with anti-heparin antibodies and heparinoids, staining was completely abolished with those heparinoids, which reacted strongly with the antibodies in ELISA (e.g. heparin and sulodexide for antibodies HS4C3 and HS4E4). An obvious decreased staining was noticed for those heparinoids, which reacted moderately with the antibodies (e.g. nadroparin for antibody HS4C3), whereas no major differences in staining were observed for those heparinoids, which did not bind to antibodies (e.g. nadroparin for antibody HS4E4).\nFig. 3Immunofluorescence staining of human renal cryosections with anti-heparin antibodies HS4C3 and HS4E4 in the absence and presence of heparinoids. Staining was abolished when renal cryosections were incubated with HS4C3 or HS4E4 in the presence of heparin or sulodexide. HS4C3 staining was decreased in the presence of nadroparin, whereas HS4E4 staining was unaffected. Bar represents 50\u00a0\u03bcm; magnification is identical for each photograph. G Glomerulus, BC Bowman\u2019s capsule, T renal tubule\nDiscussion\nIn this study eight heparinoids were characterized by their reactivity with nine different anti-heparin antibodies. All heparinoids showed a distinct antibody-binding profile in line with differences in GAG composition (heparin, HS, CS, DS), molecular weight, and method of preparation. A number of antibodies reacted strongly with certain heparinoids, but not with others. It may therefore be possible to deduce chemical and biological information of a specific heparinoid by virtue of its antibody profile. For instance, antibody NS4F5 showed reactivity with only heparin. This antibody defines a stretch of trisulfated (N-, 2-O-, and 6-O-sulfated) and epimerized disaccharides and therefore defines a highly sulfated heparin domain (Smits et al., unpublished results). The lack of reactivity of the other heparinoids for NS4F5 indicates the absence or the destruction of the domain during their preparation. Absence may hold for sulodexide and danaparoid, which contain HS rather than heparin. Destruction by the depolymerization processes may hold for the LMWHs. In this respect, reactivity with antibody NS4F5 may be indicative for the degree of depolymerization. The antibody profile of the chemically synthesized pentasaccharide fondaparinux is compatible with its chemical structure GlcNS(6OS)\u2013GlcA\u2013GlcNS(3,6OS)\u2013IdoA(2OS)\u2013GlcNS(6OS). Reactivity with antibodies HS4C3 and HS3A8 indicates it to contain iduronic acid and N-, 2-O-, 3-O-, and 6-O-sulfate residues, whereas poor\/no reactivity with antibodies HS4E4, LKIV69, AO4B08, and RB4EA12 indicates the lack of internal 2-O-sulfated iduronic acid residues (i.e. flanked by other iduronic acids) and N-acetylated glucosamine residues. Lack of reactivity with antibody NS4F5 indicates that fondaparinux does not contain a stretch of tri-sulfated disaccharides.\nNext to information on the chemical properties of the heparinoids, the antibody profile may be indicative for their biological activity, e.g. the anti-Xa\/anti-IIa ratio. This ratio is of essential importance since it is associated with a more predictable anticoagulant effect. The anti-Xa\/anti-IIa ratio is 1 for unfractionated heparin, 2\u20134 for LMWHs, 0.5 for sulodexide, and infinite for fondaparinux [2, 23]. Antibodies such as HS4E4 and AO4B08 (Tables\u00a03 and 4), which are highly reactive with heparin and sulodexide, but poorly\/not with LMWHs and fondaparinux, likely bind to a heparin domain structure at a site proximal to the pentasaccharide sequence and may be indicative for a high anti-factor IIa activity. These results indicate that when a heparinoid is recognized by antibodies HS4C3, HS3A8, EW3D10, and EW4G2 (which showed a high reactivity with fondaparinux; Table\u00a04), as well as by antibodies HS4E4 and AO4B08, the heparinoid is able to form a ternary complex with antithrombin III and factor IIa, resulting in inactivation of both factors Xa and IIa [23]. On the other hand, when a heparinoid is recognized by antibodies HS4C3, HS3A8, EW3D10, and EW4G2, but not by antibodies HS4E4 and AO4B08, it is does not contain the heparin domain structure at the site proximal to the pentasaccharide required for factor IIa binding. In this case only factor Xa will be inactivated. When a ratio is calculated from reactivity of heparinoids with antibodies HS4C3 and HS4E4 (indicative for 3-O-sulfates and for the presence of domains allowing IIa activity, respectively), and heparin is assigned a ratio of 1, the following numbers are found: dalteparin, 9; nadroparin, 7; enoxaparin, 11; tinzaparin, 11; sulodexide, 0.4; and fondaparinux, >>13 (no antibody HS4E4 reactivity observed). These numbers are in line with the above mentioned anti-Xa\/anti-IIa ratios, which were determined by enzymatic assays [2, 23], and indicate that biological characteristics are associated with antibody profiles.\nHeparin-induced thrombocytopenia (HIT) is a major complication in patients administered heparin. Heparin binding to platelet factor 4 (PF4) causes a conformational change in the protein, rendering it antigenic. Susceptible patients develop antibodies to the heparin-PF4 complex, and binding of the antibody-PF4-heparin complex to its receptor on platelets results in platelet activation and aggregation. In addition, platelets are removed by activated immune cells, resulting in thrombocytopenia [6]. Highly sulfated domains within heparin\/HS are involved in the binding to PF4 [31], and an increase in sulfation and size is associated with an increased risk of HIT [6, 19]. This may explain why the incidence of HIT is lower in patients treated with LMWHs, danaparoid, or fondaparinux compared to unfractionated heparin [7, 23]. Antibody NS4F5, which defines a highly sulfated domain, reacted strongly with heparin, but not with the other heparinoids. Reactivity with this antibody may thus be used to obtain information about the risk of a heparinoid to induce HIT.\nAlthough we have focussed here on the immunoprofiling of heparinoids in the context of anticoagulation, this technique may also be useful to evaluate heparinoids in other settings. Heparin and LMWHs have been used to treat patients with kidney disease since an ameliorating effect on a.o. proteinuria has been found [1, 14, 21, 25, 30, 33, 34, 40]. Likewise, heparinoids are reported to have anti-tumor and anti-metastatic effects [3, 20, 29], the degree of sulfation being of crucial importance. Heparinase III-digested heparin (highly sulfated fragments), inhibited tumor growth, whereas heparinase I-digested heparin (low sulfated fragments) promoted tumor growth [18], underscoring the necessity to characterize the preparation used. In addition to the implementation of these antibodies to characterize the therapeutic potential of heparinoids, they may be used to stage a disease (e.g. kidney disease, cancer) characterized by a specific HS profile in tissue, blood, and\/or urine.\nSince the antibody profile of the heparinoids used in these studies may be correlated to a specific biological effect, antibodies may be used for the identification and subsequent characterization of the active structures within the heparinoids, which may result in the development of more specific drugs. The antibodies described here may also be used to counteract bleeding in patients administered an overdose of heparinoids. This is generally accomplished by treatment with protamine sulfate [9, 26], but this rather unspecific polycation may cause hypotension, bradycardia, and thrombocytopenia. In addition it may cause other severe side effects including anaphylactic and anaphylactoid reactions, resulting in respiratory distress, circulatory collapse, capillary leak, and pulmonary hypertension [26]. There is a need for safer agents that inhibit the anticoagulant activity of heparinoids, and some antibodies described here may be useful, especially those that also react with fondaparinux, which is difficult to inhibit in vivo. It has already been described that antibodies HS4C3 and EW3D10 strongly inhibited heparin-induced anticoagulation (APTT clotting assay) [35, 39]. Antibody HS4C3 also blocked the anticoagulant activities of heparin and fondaparinux in an anti-factor Xa assay [35]. These studies indicate that anti-heparin antibodies may react with heparinoids thereby neutralizing their anticoagulant effect.\nFinally, our antibodies may be used for fast industrial screening of heparinoids. A quality check of heparinoid batches may be performed using the antibodies since each heparinoid shows a specific immunoprofile.\nIn conclusion, this study presents a novel way of characterizing heparinoids using immunoprofiling. Reactivity with antibodies was found indicative for chemical and biological aspects of the heparinoids, and may therefore be used for a fast and simple screening of industrial batches for defined characteristics.","keyphrases":["anticoagulation","heparinoid","immunoprofiling","antibody","heparin-induced thrombocytopenia"],"prmu":["P","P","P","P","P"]}
{"id":"Ann_Hematol-4-1-2413090","title":"Cytogenetic features in myelodysplastic syndromes\n","text":"Myelodysplastic syndromes (MDS) comprise a group of bone marrow diseases characterized by profound heterogeneity in morphologic presentation, clinical course, and cytogenetic features. Roughly 50% of patients display clonal chromosome abnormalities. In several multicentric studies, the karyotype turned out to be one of the most important prognostic parameters and was incorporated into statistical models aiming for a better prediction of the individual prognosis like the International Prognostic Scoring System. However, due to the profound cytogenetic heterogeneity, the impact of many rare abnormalities as well as combinations of anomalies occurring in a substantial portion of patients with MDS is still unknown and can only be delineated on the basis of large international multicentric cooperations. Recently, the German\u2013Austrian MDS Study Group presented cytogenetic findings in 2,072 patients with MDS, which serve as a basis for the characterization of the cytogenetic subgroups discussed in this article. The availability of new therapeutic options for low- and high-risk MDS targeted against distinct entities characterized by specific chromosome abnormalities, like 5q-deletions, monosomy 7, and complex abnormalities underlines the important role of cytogenetics for the clinical management of MDS. This article thus focuses on the clinical and prognostic relevance, the molecular background, and therapeutic perspectives in these three cytogenetic subgroups.\nIntroduction\nThe profound heterogenetity of myelodysplastic syndromes (MDS) is well-known from morphological and clinical studies, which finally lead to the establishment of classification, and prognostic scoring systems. It is becoming increasingly obvious that this heterogeneity also manifests itself against the background of genetic heterogeneity of MDS. In sharp contrast to the situation in chronic myeloid leukemia (CML) where one single cytogenetic abnormality, the translocation t(9;22)(q34;q11), is the genetic hallmark of the disease, there is an enormous variability of cytogenetic abnormalities in MDS hampering not only the prognostic classification but also the delineation of the molecular background of cytogenetic aberrations in MDS. As an example in our recent multicentric cytogenetic analysis of patients with MDS, we observed 684 different types of chromosome abnormalities in a cohort of 1,080 patients with MDS and an abnormal karyotype [1].\nThe cytogenetic profile of MDS\nIn general, MDS show a characteristic genetic profile with an overweighing of unbalanced abnormalities. Most frequently, a loss of genetic material in the form of deletions and monosomies can be observed. A gain of genetic material with the appearance of total or partial trisomies is less frequent. Loss or gain of genetic material can also be the result of unbalanced translocations, which are frequently observed in MDS with multiple abnormalities. Taken together, it is thus obvious to assume that a prime molecular mechanism in MDS is the loss or inactivation of tumor suppressor genes, while the activation of oncogenes seems to be less relevant in myelodysplasia.\nIn contrast to AML, balanced structural abnormalities like translocations and inversions are rare in MDS. Due to the profound genetic heterogeneity, the knowledge about distinct cytogenetic alterations was mainly restricted to the most frequent abnormalities (\u22125\/5q\u2212, \u22127\/7q\u2212, +8, 20q\u2212, and \u2212Y), although, in MDS, rare chromosome abnormalities are present in a substantial portion of patients. In a recent analysis of the German\u2013Austrian MDS Study Group, 59% of all 2,370 abnormalities observed in 1,080 patients with MDS where rare; that is, they occurred with a frequency of less than 2% [1]. In this situation, prognostic knowledge can only be deepened by large-scale multicentric studies with a high enough number of abnormal cases with follow-up data. This development was initiated by the International MDS Risk Assessment Working Group (IMRAW) ending up with the establishment of the International Prognostic Scoring System (IPSS), which was based upon the analysis of 816 patients with de novo MDS of whom 327 had abnormal karyotypes [2]. A next step forward was the study of the Spanish cooperative group with 500 abnormal cases [3] followed by our German\u2013Austrian dataset with 1,080 patients with chromosome abnormalities [1]. The situation is furthermore complicated by the fact that chromosome abnormalities in principle can occur in three different conditions: as isolated abnormality, together with one additional change, and as part of complex abnormalities with at least two additional cytogenetic alterations. Table\u00a01 shows the incidence of the 21 most frequent abnormalities according to the number of accompanying alterations occurring in our German\u2013Austrian patient cohort.\nTable\u00a01Incidence of chromosome abnormalities in MDS related to 2,072 patients examined successfullyAnomalytotal, n (% of all cases)Isolated, n (%a)With one additional abnormality, n (%a)As part of complex abnormalities, n (%a)5q\u2212312 (15.1)146 (47)52 (17)114 (36)\u22127\/7q\u2212230 (11.1)86 (37.5)31 (13.5)113 (49)+8173 (8.4)81 (46.8)37 (21.4)55 (31.8)\u221218\/18q\u221278 (3.8)3 (3.8)2 (2.6)73 (93.6)20q\u221274 (3.6)36 (48.6)10 (13.5)28 (37.8)\u2212569 (3.3)1 (1.4)4 (5.8)64 (92.8)\u2212Y58 (2.8)41 (70.7)5 (8.6)12 (20.7)+2145 (2.2)5 (11.1)18 (40)22 (48.9)\u221217\/17p\u221242 (2.0)1 (2.4)1 (2.4)40 (95.2)inv\/t(3q)41 (2.0)16 (39)8 (19.5)17 (41.5)\u221213\/13q\u221240 (1.9)5 (12.5)6 (15)29 (72.5)+1\/+1q37 (1.8)3 (8.1)6 (16.2)28 (75.7)\u22122133 (1.6)3 (9.1)4 (12.1)26 (78.8)+1128 (1.4)6 (21.4)4 (14.3)18 (64.3)\u22121226 (1.3)02 (7.7)24 (92.3)12p\u221225 (1.2)7 (28)6 (24)12 (48)t(5q)24 (1.2)6 (25)3 (12.5)15 (62.5)11q\u221223 (1.1)8 (34.8)4 (17.4)11 (47.8)9q\u221223 (1.1)8 (34.8)3 (13)12 (52.2)t(7q)22 (1.1)6 (27.3)6 (27.3)10 (45.5)\u22122022 (1.1)0022 (100)aOf cases with the respective abnormality\nCytogenetic prognosis\nThe first large-scale cytogenetic studies in patients with MDS encompassing more than 100 patients where published more than 20\u00a0years ago [4\u20137]. In the following years, the patients cohorts increased step by step from nearly 250 patients [8] to some 400 patients [9, 10]. In 1997, Greenberg published a collaborative multicentric international data set of more than 800 patients with de novo MDS, which was the basis for the establishment of the IPSS [2]. This database was further surmounted by the analyses of the Spanish cooperative group with 968 patients examined (Table\u00a02), which represented the largest number of patients with MDS karyotyped so far [3]. Recently, the German\u2013Austrian Study Group published their multicentric analyses of more than 2,100 patients with MDS [1].\nTable\u00a02Cytogenetic prognostic findings in publications with greater than 100 patients examinedAuthor, yearNumber of patientsAbnormal (%)FavorableIntermediateUnfavorableKnapp 198517466 (38)Normal (NN)ComplexNowell 1986\u2013198914463 (44)5q\u2212, 20q\u2212+8\u22127\/7q\u2212Billstr\u00f6m 198816974 (44)NN, 5q\u2212+8\u22127\/7q\u2212Pierre 1989247106 (43)NNComplexMorel 1993408151 (37)NN, 5q\u2212, \u2212Y, \u22127\/7q\u2212, 20q\u2212+8ComplexToyama 1993401200 (50)+8\u22127\/7q\u2212ComplexWhite 199419875 (38)NN, 5q\u221212p\u2212, +21Complex, +8, 20q\u2212Greenberg 1997816327 (40)NN, 5q\u2212, 20q\u2212, \u2212YAll othersComplex, abnormal #7Sol\u00e9 2005968500 (51)NN, 5q\u2212, 20q\u2212, \u2212Y, 11q\u2212, 12p\u2212rea 3q, + 8, +9, t11q, 17p\u2212Complex, \u22127\/7q\u2212, i17qHaase 20072,0721,080 (51)NN, +1\/+1q, t(1q), 5q\u2212, t(7q), 9q\u2212, 12p\u2212, abnormal #15, t(17q), 20q\u2212, \u221221, + 21, \u2212X, \u2212Yrea 3q, \u22127, 7q\u2212, +8, 11q\u2212, t(11q23), +19, complex (=3)Complex (>3), t(5q)rea Rearranged\nWhat has changed and what has been achieved during these last 25\u00a0years of cytogenetic analyses in MDS? If the aberration rate is considered, there is a gradual increase in the portion of abnormal cases from under 40% in the most earlier studies to 50% in the more recent analyses of Toyama, Sol\u00e9, and Haase [1, 3, 10]. Although the composition of the patient cohorts may play a role for the portion of clonal abnormalities in a given collective, other factors like the improvement of culture conditions possibly due to the use of recombinant myeloid growth factors [11] might have contributed to the increase in the aberration rate in cytogenetic studies. To date, an aberration rate of 50% can be regarded as an international standard.\nPrognostic relevance of chromosome abnormalities\nThe prognostication of patients with MDS has become an important means for the development of therapeutic strategies based on more individualized risk assessment. Even in the most recent prognostic scoring system, the World Health Organization classification-based Prognostic Scoring System, cytogenetics play a decisive role [12]. In general, three to four prognostic cytogenetic categories can be distinguished. In several multicentric investigations, cytogenetics have been proven to be highly relevant independent prognostic parameters ([2, 3, 13], see also Table\u00a02).\nGood prognosis\nEven in the earliest study considered here, Knapp et al. [4] described the prognostic relevance of cytogenetic findings with a normal karyotype as favorable and complex abnormalities as being associated with bad prognosis. The good prognosis of a normal karyotype was confirmed by nearly all other groups. Thus, in contrary to the situation in AML where patients with a normal karyotype have an intermediate outcome and prognosis is significantly influenced by additional molecular alterations, in MDS, a normal karyotype is undisputedly associated with good prognosis. Further, well-established abnormalities defining good-risk subgroups are deletions of 5q and 20q as well as loss of the Y-chromosome, although the question whether this latter abnormality is age related, or a real clonal marker is unsolved as yet. The Spanish group identified further new cytogenetic abnormalities with a good prognosis (12p\u2212 and 11q\u2212) [3].\nIn the German\u2013Austrian multicentric study, the following abnormalities were associated with a favorable clinical course with a median survival between more than 9\u00a0years and 32\u00a0months: normal karyotype, t(1q), 5q\u2212, t(7q), 9p\u2212, 12q\u2212, t(15q), t(17q), 20q\u2212, +21, \u221221, \u2212X, \u2212Y. However, prognosis was only favorable when not more than one additional abnormality was present [1].\nIntermediate prognosis\nIn most studies, patients with trisomy 8 displayed an intermediate clinical course. Until the Spanish group published their results on more than 980 patients, the knowledge of cytogenetic findings with an intermediate prognosis was very limited. In the IPSS, all abnormalities neither belonging to the good-risk group (isolated 5q\u2212, 20q\u2212, and loss of Y-chromosome) nor to the bad risk cohort (complex [greater than or equal to three abnormalities] or any chromosome 7 abnormality) were designated to be of intermediate prognosis not by availability of survival data but by definition [2]. In their large Spanish dataset, Sol\u00e9 et al. delineated new abnormalities associated with an intermediate clinical course (3q abnormalities, trisomy 9, 11q translocations, and 17p deletions) [3]. The results of White et al. [14] and Toyama et al. [10] who identified \u22127\/7q\u2212 and 12p\u2212 and trisomy 21, respectively, as abnormalities with an intermediate prognosis were not confirmed by other groups. Recently, the multicentric database of the German\u2013Austrian MDS Study Group revealed several infrequent abnormalities with an intermediate prognosis. A median survival time of 23\u201326\u00a0months was observed in patients with trisomy 8 and 11q\u2212 (called Intermediate-I). A more worse prognosis with a median survival time between 20 and 14\u00a0months was found for 11q23 translocations, chromosome 3q aberrations, trisomy 19, 7q deletions, and complex abnormalities with three different chromosomal alterations and monosomy 7 (called Intermediate-II) [15].\nPoor prognosis\nThere is a great consensus in all publications on cytogenetic prognosis in MDS that complex abnormalities characterize a MDS subgroup with bad prognosis and a median survival time significantly below 1\u00a0year, although the threshold at which the number of abnormalities confers bad prognosis is a matter of debate. While in most publications, the term \u201ccomplex\u201d is used for three or more abnormalities, in the Medical Research Council AML trials, bad prognosis was assigned to cases with five or more abnormalities [16]. Recently, we clearly could show that, in patients with MDS, median survival is significantly reduced only when more than three abnormalities are present, reducing the median survival from 17\u00a0months in cases with three abnormalities to less than 9\u00a0months in cases with four or more anomalies [1]. Besides complex changes, partial and total monosomy 7 was ranked to the cytogenetic findings with an unfavorable clinical course [3, 5\u20137]. Within the IPSS, any chromosome 7 abnormality was counted to the bad prognostic findings [2]. As mentioned above, in the German\u2013Austrian dataset, \u22127\/7q\u2212 showed a significant better prognosis (14 and 19\u00a0months median survival time, respectively) as compared to the complex abnormality group defined by four or more chromosomal changes (8.7\u00a0months) and was thus attributed to the intermediate-II group [15]. The findings of Toyama et al. [10] who observed an unfavorable course in patients with trisomy 8 or 20q deletions was not confirmed by any other group. Sol\u00e9 et al. described a median survival of under 12\u00a0months in ten patients with an isochromosome of 17q [3]. Since, in the German\u2013Austrian dataset, this abnormality was too infrequent for a statistically meaningful analysis, the Spanish findings are convincing but still need further confirmation.\nPrognostic scoring\nIn the IPSS, three different cytogenetic subgroups were established and weighted against bone marrow blast counts and cytopenias by multivariate analysis [2]. Applying the cytogenetic part of the IPSS to our German\u2013Austrian dataset, 59% of the patients had a good risk karyotype (normal, isolated 5q\u2212, 20q\u2212, or loss of Y-chromosome) with a median survival of 54\u00a0months, 19% of the collective had an intermediate prognosis (neither good risk nor bad risk cytogenetics) with a median survival of 31\u00a0months, and 22% displayed a dismal prognosis (any chromosome 7 anomaly, complex [\u22653] changes) with a median survival of 11\u00a0months [1].\nThe application of the new cytogenetic findings derived from the German\u2013Austrian dataset allows the definition of four different cytogenetic prognostic subgroups (Table\u00a03 and Fig.\u00a01). The low-risk group covers 73% of patients with 14 cytogenetic categories and a median survival of 55\u00a0months, the intermediate groups I and II (15.5% of patients) with eight cytogenetic categories and a median survival of 29 (intermediate-I) and 15\u00a0months (intermediate-II), and the high-risk group (11.5% of patients) with a median survival of 8\u00a0months [15]. In comparison to the IPSS, our new cytogenetic prognostication splits the intermediate group into two distinctly separate subgroups with significantly different median survival. The bad-risk group is more strictly defined with a lower median survival time (8 vs. 11\u00a0months). While the IPSS assigned an intermediate risk by exclusion and not by knowledge, our intermediate cytogenetic subgroup was based exclusively on available survival data of patients treated with supportive care only thus reflecting the natural course of the disease. Taking into account only those patients with a known prognosis (in IPSS only good- and bad-risk patients), 81% of our patients could be categorized by the IPSS in comparison to 93% by the German\u2013Austrian prognostic system.\nFig.\u00a01Kaplan\u2013Meier survival curves according to the cytogenetic prognostic classification of the German\u2013Austrian MDS Study Group. Log-rank test: p\u2009<\u20090.0001 (3 degrees of freedom) [15]Table\u00a03New cytogenetic prognostic subgroups of the German\u2013Austrian MDS Study Group in 1,202 patients treated with supportive care onlyCytogenetic riskCytogenetic findingNumber (%)Median survival (months)Good12p\u22127 (0.6)n.r.9q\u22126 (0.5)n.r.t(15q)6 (0.5)n.r.15q\u22125 (0.4)n.r.+2113 (1.1)100.85q\u2212132 (11)77.220q\u221224 (2)71.0\u2212X6 (0.5)56.4normal karyotype622 (51.7)53.4\u2212Y33 (2.8)39.4t(1q)7 (0.6)34.7t(7q)7 (0.6)34.7t(17q)6 (0.5)32.1\u2212216 (0.5)32.0Intermediate-I11q\u221211 (0.9)26.1+864 (5.3)23.0Intermediate-IIt(11q23)6 (0.5)20.0Any 3q abnormality16 (1.3)19.9+195 (0.4)19.87q\u221211 (0.9)19.0Complex (=3 anomalies)32 (2.7)17.0\u2212742 (3.5)14.0PoorComplex (>3 anomalies)134 (11.1)8.7t(5q)7 (0.6)4.4n.r. Median survival not reached\nA further element of the IPSS, the weighting of cytogenetics in comparison to other relevant parameters like the bone marrow blast counts, has become questionable. According to the IPSS, 0 scoring points are attributed to good-risk cytogenetics and blasts less than 5%, intermediate cytogenetics as well as 5\u201310% blasts get 0.5 scoring points, and bad-risk cytogenetic findings are scored with 1.0. Eleven to 20% blasts are scored with 1.5 and 21\u201330% blasts with 2.0 points. Thus, bad-risk cytogenetics with a median survival of 11\u00a0months get less scoring points than patients with 11\u201320% blasts and a median survival in our cohort of 16.5\u00a0months. According to our observations, only patients with 21\u201330% blasts (11.7\u00a0months median survival) had a prognosis comparable to patients with bad-risk cytogenetics. Thus in a revision of the IPSS the weight of bad-risk cytogenetics has to be readjusted [17].\nDelineation of the most relevant cytogenetic subgroups 5q\u2212, monosomy 7, and complex abnormalities\n5q deletions\nDeletions within the long arm of chromosome 5 are the most frequent cytogenetic changes in MDS accounting for roughly 30% of abnormal cases [1, 2, 3]. The deletions can have variable size; however, the common deleted region always spans the chromosome band 5q31. Due to intensive research during the last decade, our knowledge of the molecular background of these abnormalities is increasing. It is generally accepted that not the illegitimate fusion of deoxyribonucleic acid (DNA) sequences at the variable breakpoints of the deletions but loss of genetic information represents the relevant pathomechanism. There could be two different deleted regions in 5q31. One that is located more centromeric is possibly associated with bad prognosis, complex abnormalities, and high-risk, as well as therapy-related, MDS. A second area is located more telomeric in the vicinity of band 5q32 and is supposed to be related to the good-risk 5q\u2212 syndrome [18\u201321. Recently, it has been convincingly shown by the means of sophisticated systematic knockout experiments on 41 candidate genes in the critical 5q\u2212 region that the gene for a ribosomal subunit protein, RPS41, seems to be a relevant gene in patients with 5q\u2212 syndrome on the basis of haploinsufficiency [22]. The 5q\u2212 syndrome has been first described by Van den Berghe in 1974 [23]. It is cytogenetically characterized by an isolated deletion of chromosome 5q. A female preponderance is well documented. The clinical appearance is characterized by a refractory macrocytic anemia, normal or elevated platelets, and mild leukocytopenia. The clinical course is mild and long lasting with a very low risk for leukemic transformation. The bone marrow smears reveal less than 5% blasts, while dysplasia of the erythroid and granulocytic lineages usually are discrete or even absent. Megakaryopoesis shows profound and characteristic dysplasias with either separated multiple nuclei, hypolobulation, microkaryocytes, and most characteristically monolobulated megakaryocytes with a round nucleus.\nThe prognosis of 5q deletions in MDS is generally favorable if they are not part of complex abnormalities; however, it is significantly modified by single additional cytogenetic changes [1, 24]. Figure 2 shows the Kaplan\u2013Meier survival curves for patients treated with supportive care only with isolated 5q\u2212, 5q\u2212 plus one additional abnormality, and 5q\u2212 as part of complex abnormalities derived from the German\u2013Austrian database [1].\nFig.\u00a02Median survival according to accompanying abnormalities in patients with 5q deletions. Log-rank test: p\u2009\u2264\u20090.0001 (2 degrees of freedom), p\u2009=\u20090.30 (isolated vs. +1), p\u2009\u2264\u20090.0001 (isolated vs. complex), p\u2009=\u20090.0001 (+1 vs. complex)\nIt is evident that additional abnormalities negatively influence survival in patients with 5q deletions. However, due to cytogenetic heterogeneity, the additive prognostic impact of distinct single additional abnormalities remains obscure as yet (Table\u00a04).\nTable\u00a04Frequencies (in percent of all cases with the respective primary abnormality) of accompanying abnormalities5q\u2212 (n\u2009=\u200959)\u22127\/7q\u2212 (n\u2009=\u200938)trisomy 8 (n\u2009=\u200944)Additional anomalyPercentAdditional anomalyPercentAdditional anomalyPercent+817+21105q\u221223+21135q\u221210+2111\u221220\/20q\u22128+810der(3q21\/q26)7\u221277inv(3q)5+117der\/del(12p)7del(12p)5\u221275der\/del(3p)5t(11q23)5del(12p)5der\/t(21q)5iso(17)(q10)5+135t(11q23)3Others50+145Others35+8*5del(1p)5Others22\nOver a long period, the therapeutic standard in patients with noncomplex 5q deletions was supportive care only. In clinical trials, retinoic acid turned out to be inefficient, and low-dose cytarabine was more effective but led to a pronounced increase in severe neutropenic infections [25]. A preferential response to other modern therapeutic strategies like immunosuppression and suppression of DNA methyltransferase or histone deacetylase was not reported as yet. However, recently, List et al. reported a remarkably high response rate to the immunomodulating agent lenalidomide, which was especially pronounced in MDS patients with 5q deletions. Nine out of 12 patients with a 5q deletion displayed complete cytogenetic remissions, and 10 out of 12 experienced an erythroid response [26]. Thus, a new promising agent may target cell clones in MDS bearing 5q deletions. These results could recently be confirmed on the basis of a large muliticentric trial [27].\nMonosomy 7\nMonosomy 7 is the second most frequent distinct chromosome abnormality in MDS occurring in some 25% of abnormal cases. It can present as total or partial monosomy. In the latter case, variable deletions of parts of the long arm lead to loss of genetic material of different size. As yet, no significant differences concerning the prognostic relevance have been observed between total and partial monosomy 7. In the German\u2013Austrian dataset, 36% of monosomy 7 were isolated ones, 14% displayed one additional abnormality, and 50% occurred as part of complex abnormalities [1]. Comparable to the situation in 5q, also in 7q, at least two and maybe more distinct regions of common deletions have been identified: the band 7q22 and the more telomeric regions 7q31\u201332 and 7q36. It is interesting to note that interstitial as well as terminal deletions might be due to cryptic unbalanced translocations. In single individuals, more than one different deletion has been found in one and the same patient, either within the same copy of chromosome 7 with retention of sequences between the deleted regions or within different cell clones [28, 29]. These observations clearly underline the regional genetic instability of the long arm of chromosome 7, which makes this region especially prone to deletions of different size and localization as well as to structural abnormalities. On the other hand, it can be assumed that there must be a cluster of genes with tumor-suppressive features distributed over several chromosome bands in 7q with the same or very similar consequences when being inactivated by chromosomal abnormalities.\nHowever, in contrast to some presumptions in the 5q deletions, as yet, no prognostic differences have been elaborated between different regions of deletions (reviewed in [20]). Investigations of the molecular background of monosomy 7 MDS are on the way. An association of this cytogentic subgroup with RAS mutations, mutations of AML1, and hypermethylation of p15INK4B have been reported [30, 31]. In an analysis of gene expression profiles in CD34+ cells from MDS patients with monosomy 7, a malignant phenotype with highly proliferative potential was found with an overexpression of HOX9A, PRAME, BMI-1, PLAB, and the DNA repair gene BRCA2. Parallelly, downregulation of the tumor suppressor gene p21, GATA2, and MAP was observed [32]. Clinically, monosomy 7 is characterized by a lower median age of the affected patients as compared to 5q deletions, severe refractory cytopenias, and a proneness to life-threatening infections. Predisposing conditions recurrently observed in cases with monosomy 7 are antecedent aplastic anemia, Fanconi\u2019s anemia, neurofibromatosis type I, cyclic neutropenia, and long-term treatment with granulocyte colony-stimulating factor (G-CSF), as well as Schwachman\u2019s syndrome. It is of special interest that myeloid cells harboring monosomy 7 seem to be preferentially responsive to a stimulation by myeloid growth factors like G-CSF and granulocyte\u2013macrophage CSF in vitro [33, 34] as well as in vivo [35]. Although being obvious, this phenomenon never was exploited therapeutically for a targeted priming strategy for patients with monosomy 7.\nIn extremely rare cases, for the so-called monosomy 7 syndromes, the abnormality has a familiar background. In a substantial portion of patients, a mutagen exposition (benzene, solvents, irradiation, or radio- and\/or chemotherapy) was documented.\nIn contrast to the situation in 5q deletions, in monosomy 7, additional abnormalities do not have such a profound impact on outcome as seen in 5q deletions, since monosomy 7 even as an isolated abnormality confers a significantly bad prognosis. In our Austrian\u2013German dataset, median survival times were 14\u00a0months for the isolated abnormality, 11\u00a0months for cases with one additional change, and 8\u00a0months for monosomy 7 as part of complex abnormalities (Fig.\u00a03) [1].\nFig.\u00a03Median survival according to accompanying abnormalities in patients with \u22127\/7q\u2212. Log-rank test: p\u2009=\u20090.03 (2 degrees of freedom), p\u2009=\u20090.94 (isolated vs. +1), p\u2009=\u20090.07 (isolated vs. complex), p\u2009=\u20090.43 (+1 vs. complex)\nTherapeutic options in the monosomy 7 subgroup are unsatisfying as yet. If age and clinical condition are adequate, patients should be treated with allogeneic stem cell transplantation whenever possible. Conventional intensive chemotherapy bears a high risk of early death and nonresponse. Even if a complete remission can be achieved, this frequently is of only short duration with a high risk of early relapse. In a recent report on 34 patients with MDS or AML treated with 5-azacytidine, the group of Mufti observed a preferentially good response of MDS patients with monosomy 7 to the demethylating agent 5-azacytidine. In this trial, five of the seven (71%) complete responders had isolated chromosome 7 abnormalities and achieved a continuous complete remission (10\u00a0months follow-up) in contrast to patients with other karyotype abnormalities like trisomy 8 who relapsed within the first 6\u00a0months [36]. Supporting results have been recently published for the use of decitabine by L\u00fcbbert et al. [37]. Future multicentric therapy trials are needed to verify this observation in a higher number of patients.\nComplex abnormalities\nAccording to the International System for Human Cytogenic Nomenclature criteria, complex chromomosome abnormalities are defined by the simultaneous occurrence of at least three independent abnormalities within one cell clone [38]. This cytogenetic subtype is present in some 15% of all patients with MDS (roughly 30% of all abnormal cases) and thus occurs in a frequency comparable to 5q deletions [1\u20133]. Complex abnormalities may be the result of a multistep process with sequential accumulation of abnormalities, called karyotype evolution. In some cases, karyotype evolution can be observed if repeated cytogenetic analyses are performed, unraveling the stepwise accumulation of secondary abnormalities with the starting point of a primary abnormality. In a few patients, cytogenetic analysis detects cell clones with primary and others with primary and secondary and possibly further abnormalities at one and the same time point. However, frequently, this process cannot be proven in the individual patient who presents with multiple chromosomal changes at first diagnosis. In most of these cases, complexity is profound with aberration numbers exceeding the threshold of three by far. Frequently, in these cases, cell-to-cell variations occur, and the chromosomal complement presents with a chaotic, mutator-like shape resembling cytogenetics of solid tumors and malignancies in chromosomal breakage syndromes. Thus, it is conceivable that, in these cases, the process leading to complex abnormalities and genetic instability must proceed rapidly on the one hand and must involve mechanisms like DNA repair and cell cycle control comparable to the situation in DNA repair deficiency syndromes. Recent results of gene expression analyses in patients with MDS and complex chromosome abnormalities support these assumptions and are considered below in more detail [39]. In a substantial portion of patients with complex abnormalities, an exposition to therapeutically applied mutagens, like anthracyclines, topoisomerase II inhibitors, alkylating agents, and\/or irradiation, can be traced in the medical history [40]. Within the subgroup of patients with complex changes, a broad range of the number of abnormalities with the majority of patients displaying greater than or equal to five chromosomal changes can be observed [39].\nThere is a strong association with unbalanced structural abnormalities affecting most frequently chromosomes 5q and 7q. Chromosomes less frequently involved in loss of genetic material were chromosomes 3 (p- and q-arms), 12p, 13q, 16q, 17p, 18q, and 20q. Genomic gains were observed for chromosome 8\/8q, 11q, and 21q [41]. Mutations of p53 have been observed in up to 90% of patients with AML and complex abnormalities [39]. Matrix comparative genomic hybridization (CGH) analyses in patients with complex abnormalities revealed an association of complex karyotype changes with amplifications in the chromosomal regions 8q24, 9p24,,11q23, 12p13, 13q12, 20q11, and 2q1q22 [42].\nRecently, characteristic profiles of complex abnormalities in AML were delineated by gene expression analyses. In this respect, a significant overexpression of RAD21, a double-strand-break DNA repair enzyme, as well as overexpression of other genes related to DNA-repair, apoptotic mechanisms, and cell-cycle control (RAD1, RAD 9A, RAD23B, RAD51AP1, NBS1, MSH6, SUMO1, and PARP2) was observed [39].\nTaken together, these data might help to understand complex abnormalities as a maximum manifestation of genetic instability by allowing cells with significant DNA damages to circumvent physiologically protective apoptosis and to escape cytotoxicity of chemotherapy by intensified DNA repair. This model is also compatible with the high extent of chemo-resistance of hematopoietic cells with complex chromosome abnormalities, which is a well-known and severe problem in the clinical management of high-risk MDS. Further clinical associations are the high median age of patients within this cytogenetic subgroup [43] and, related to conventional intensive chemotherapy, short remission durations and a high risk for early relapse [44]. Allogeneic stem cell transplantation, the only curative option for these patients, will only be feasible for a minority of individuals with complex chromosomal changes due to age-related multimorbidity and organ dysfunctions. New therapeutic strategies targeting hypermethylation, deacetylation, and immunomodulation, which have proven their tolerability also in aged patient populations, are now available for the treatment of patients with high-risk MDS. In this respect, two studies have to be mentioned. Cytogenetic responses in a substantial portion of patients with high-risk MDS have been observed in a multicentric study of decitabine, a potent demethylating agent. Major cytogenetic responses (MajCR) were observed in 38% of 50 patients. Remarkably, 10 out of 26 patients (38%) with high-risk cytogenetics displayed a MajCR; five of them had complex chromosome abnormalities. In the patients\u2019 group with MajCR, median survival time was 24\u00a0months as compared to the significantly shorter median survival time of 11\u00a0months in patients with persisting cytogenetic aberrations [45]. These observations are further supported by observations of our group. Within a cohort of 14 patients with high-risk MDS, we recently observed hematologic and cytogenetic responses to 5-azacytidine according to the modified International Working Group criteria [46] in three out of four patients with complex abnormalities [47].\nAnother new therapeutic mechanism, immunomodulation, might be effective in cases with complex karyotypic changes too. In this connection, a complete cytogenetic response to the immunomodulatory drug lenalidomide has been reported in patients with high-risk MDS with complex chromosome abnormalities harboring 5q deletions [48]. Further multicentric clinical trials are needed to confirm these observations.\nConclusions\nIn contrast to CML where the bcr\u2013abl fusion forms the homogenous genetic basis of the disease [49], MDS show a profound heterogeneity, not only on the morphologic and clinical level but also on the genetic presentation. Three cytogenetic subgroups with prognostic relevance have been identified and were incorporated into prognostic scoring by the IPSS. However, in the great majority of cytogenetic changes, the prognostic relevance is still obscure. Genetic heterogeneity in MDS leads to the paradox situation that rare abnormalities can be observed frequently [1] with the consequence that, for a relevant portion of abnormalities, their prognostic impact is still unknown. The only chance to overcome these shortcomings is to establish multicentric cooperations, which are the goals of the German\u2013Austrian MDS Study Group, which is now cooperating with the MD Anderson Cancer Centre as well as the IMRAW group [50\u201352]. New therapeutic strategies targeting immunomodulation and epigenetic changes proved to be of outstanding effectiveness and tolerability in comparison to established therapies not only for the low-risk 5q syndrome but also for high-risk MDS with as yet deleterious cytogenetic alterations like monosomy 7 and complex abnormalities.\nHowever, what about the subgroup of nearly 50% of patients presenting with a normal karyotype? New techniques will help to further delineate the genetic background in these cases. Thus, very recently, two groups published their results of single nucleotide polymorphism analyses in MDS [53, 54]. The group of Mufti performed high-resolution single-nucleotide polymorphism (SNP) array analyses in 119 low-risk MDS patients of whom 32% displayed clonal chromosome abnormalities to search for cryptic chromosomal aberrations not detectable by chromosomal banding analyses. They found uniparental disomy (UPD), a copy number neutral loss of heterozygosity, in 46% of patients, small deletions in 10%, and amplifications in 8% of the cohort. The changes of copy numbers were acquired while UPDs were constitutional. The former aberrations were associated with a worsened outcome of the patients. The authors speculated that the high frequency of UPD may be indicative for a constitutional genomic instability in these patients [53]. This could be a predisposing factor for MDS. Maciejewski et al. applied 250 K SNP arrays to 94 patients with MDS and observed SNP aberrations in 75% of patients as compared to 59% clonal chromosomal abnormalities detected by metaphase analyses. Previously unknown lesions were found in patients with a normal\u2014as well as in others with an abnormal\u2014karyotype. In this study, UPD occurred in 20% of the MDS patients [54].\nNew analytical tools like matrix CGH, microarray gene expression analyses, proteomics, and methylation profiling will add substantially to the understanding of pathogenesis, delineation of therapeutic targets, and individualization of therapy in MDS.\nTaken together cytogenetics is still the gold standard of genetic diagnosis in MDS providing \u201clabels\u201d like 5q\u2212, monosomy 7, or complex abnormalities for disease entities with a common biological behavior and clinical as well as prognostic impact. In the future, scientific progress will depend on the ability to combine established and newly developed methods to gain a comprehensive understanding of MDS, which will translate into an individualized and highly effective and well-tolerable treatment for all patients with MDS.","keyphrases":["cytogenetics","mds","karyotype","prognosis","monosomy 7","complex abnormalities","5q\u2212"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Ann_Surg_Oncol-4-1-2234441","title":"A Novel Function of the Receptor for Advanced Glycation End-Products (RAGE) in Association with Tumorigenesis and Tumor Differentiation of HCC\n","text":"Background The expression of the receptor for advanced glycation end products (RAGE) has an impact on the mechanisms giving rise to characteristic features of various cancer cells. The purpose of this study was to elucidate the clinicopathological relevance of the level of RAGE expression in patients with hepatocellular carcinoma (HCC) and to explore the effect of RAGE expression on the characteristic features of HCC.\nAlthough many cancers arise from chronic inflammation, the relationships between carcinogenesis, cancer promotion, and its molecular characteristics remain poorly understood. Hepatocellular carcinoma (HCC), which typifies an inflammation-related tumor, is one of the most common malignancies in the world, especially in Asia and Africa. Japan has a high incidence of chronic viral hepatitis, cirrhosis, and HCC. The resolution of inflammatory activity at the molecular level may correlate with prevention of hepatocarcinogenesis and cancer promotion.\nThe receptor for advanced glycation end-products (RAGE) is a multiligand receptor classified as an immunoglobulin superfamily cell surface molecule and acts as a counter-receptor for high-mobility group box 1 (HMGB1),1 advanced glycation end-products (AGEs), S100\/calgranulins, and amyloid-\u03b2 peptides. These interactions trigger the activation of key cell signaling pathways (e.g., p38 and p44\/42 MAP kinase,2 NF-\u03baB, cdc42\/rac,3 and the generation of reactive oxygen species, and result in the production of proinflammatory cytokines.5 RAGE-mediated proinflammatory processes are now considered to contribute to the progression of many chronic diseases, such as neuropathy, nephropathy,6 macrovascular disease, amyloidoses, inflammatory conditions (e.g., rheumatoid arthritis and inflammatory bowel disease) and sepsis.5,7 In addition to RAGE-mediated proinflammatory events, recent studies have revealed that the interaction of RAGE and its ligands and the resultant signaling play a causative role in the characteristic modulation of cancer cell functions, i.e., increasing tumor invasion and metastasis.8 Furthermore, several clinical studies have demonstrated the (strong) association of RAGE expression with the malignant potential of various cancers such as gastric cancer,9 colon cancer,10,11 common bile duct cancer,12 pancreatic cancer,13 and prostate cancer,14 although one report showed a reverse correlation between RAGE expression and tumor progression.15 Thus, RAGE expression may be expected play have a significant role in the development of HCC, although no data have been reported for this tumor. In terms of HCC, it is known that the pathophysiological conditions or circumstances surrounding tumorigenesis are quite different from those reported for other cancers. For example, small HCC tumors at an early stage are continuously exposed to low oxygen and high glucose as well as the noncancerous hepatic tissues with cirrhosis in which tissue derangement occurs.15 In addition, RAGE is the receptor for AGEs produced from excessive glucose metabolism,16 extracellular HMGB1 released from necrotic cells which could be induced by hypoxia and inflammation, and serum amyloid A (SAA) produced in response to the proinflammatory cytokine IL-6,17 and these RAGE ligands are possibly generated and\/or released from inflamed hepatic tissues. Our central hypothesis is thus that RAGE expression may play a particular role in tumorigenesis of HCC, in addition to the role in the invasive and\/or metastatic potential of cancer cells. With this background, the objectives of this study were: (1) to clarify the relationship between RAGE expression and the clinico-pathological features of HCC and (2) to investigate the functional role of RAGE expression in HCC development.\nMATERIAL AND METHODS\nHuman samples\nFrom March 2000 to September 2005, 65 patients with primary HCC were treated surgically in the Department of Surgical Oncology and Digestive Surgery, Kagoshima University School of Medicine. Of these 65 patients, 12 who had diabetes mellitus and 6 who underwent preoperative therapy were excluded from the study. RAGE expression for Diabetes patients upregulates at various tissues.18,19,20 A further 11 patients were excluded because their RNA samples were degraded. Samples from the remaining 36 patients (30 men and 6 women with a mean age of 67.1 years) were included in the study. Seven patients (19.4%) were positive for hepatitis B surface antigen and 21 (58.3%) were positive for the antibody to hepatitis C virus. Eight patients (22.2%) were negative for both of these viruses. Twenty-four patients had chronic hepatitis and four had liver cirrhosis. The mean tumor size was 49.7 mm (range, 16\u2013150 mm). The histological grade of each tumor was determined according to the general rules for the clinical and pathological study of primary liver cancer (The Liver Cancer Study Group of Japan, 2000).21 Four tumors (11.1%) were well-differentiated HCC, 28 (77.8%) moderately differentiated HCC, and 4 (11.1%) poorly differentiated HCC. Postoperative tumor recurrence was observed for 11 patients (30.6%) (Table\u00a01). For immunohistochemical study, a further 12 HCC samples (six well- and six poorly differentiated HCC), obtained surgically during the same period at JA Kagoshima Kouseiren Hospital, were added and used to verify the correlation of RAGE expression with tumor differentiation. Finally, a total of 48 HCC nodules (10 well-differentiated HCC, 28 moderately differentiated HCC, and 10 poorly differentiated HCC) were studied. As a control study, six normal liver samples were collected from patients with benign or metastatic liver tumors. Written informed consent, recognized by the ethical committees of Kagoshima University School of Medicine and JA Kagoshima Kouseiren Hospital, was obtained from each patient before tissue acquisition.\nTABLE\u00a01.Background of patientsGender\u00a0\u00a0Male30 (83%)\u00a0\u00a0Female6 (17%)Mean age67.2 yearsVirus type\u00a0\u00a0B7 (19.4%)\u00a0\u00a0C21 (58.3%)\u00a0\u00a0None8 (22.2%)Background of livers\u00a0\u00a0Chronic hepatitis24 (60.7%)\u00a0\u00a0Liver cirrhosis4 (11.1%)\u00a0\u00a0Normal liver8 (22.2%)\u00a0\u00a0Mean tumor size 49.7mmHistological grade\u00a0\u00a0Well differentiated4 (11.1%)\u00a0\u00a0Moderately differentiated28 (77.8%)\u00a0\u00a0Poorly differentiated4 (11.1%)Postoperative tumor recurrence 11 (30.6%).\nImmunohistochemistry\nConsecutive 4-\u03bcm sections were cut from each paraffin-embedding block. Sections were immunostained by anti-RAGE antibody (Santa-Cruz, CA, USA) according to the conventional immunoperoxidase technique. Briefly, after peroxidase blocking with 3% H2O2\/methanol for 10 min, specimens were blocked with phosphate buffered saline (PBS) containing 5% normal horse serum (Vector Laboratories, Inc., Burlingame, CA, USA). Anti-RAGE antibody was used at 1\/200. After overnight incubation at 4\u00b0C with the primary antibody, specimens were briefly washed in PBS and incubated at room temperature with the secondary antibody conjugated with peroxidase. The specimens were then washed in PBS and color-developed by diaminobenzidine solution (DAKO). After washing with water, specimens were counterstained with Meyer\u2019s hematoxylin (Sigma Chemical Co., St Louis, MO, USA). Immunostaining of all cases was performed at one time to ensure the same conditions of antibody reaction and DAB exposure. A total of 48 HCC nodules (10 well-differentiated HCC, 28 moderately differentiated HCC, and 10 poorly differentiated HCC) were studied. To evaluate the immunohistochemical staining, ten fields were selected and expression in 1000 tumor cells (100 cell\/fields) was evaluated with high-power (\u00d7200) microscopy. The immunohistochemical expression of RAGE was defined as positive if distinct staining of the cell membrane was observed in at least 10% of tumor cells.\nCell and cell culture\nHepatoma cell lines HepG2, HuH7, HT17, and Li7 were kindly provided by the Cell Resource Center for Biomedical Research Institute of Development, Aging, and Cancer, Tohoku University and Hep3B was obtained from the European Collection of Cell Cultures. HepG2, Hep3B, and HT17 were cultured with Dulbecco\u2019s Modified Eagle\u2019s Medium (DMEM) (1000 mg\/l) and HuH7 and Li7 were cultured with RPMI. RAGE-transfected Cos7 and its mock-transfectant were kindly provided by Drs. Yamamoto, Department of Biochemistry and Molecular Vascular Biology, Kanazawa University. These transfectants were maintained with DMEM supplemented with 10% Fetal Calf Serum (FCS) in the presence of 650\u00a0\u03bcg\/ml G418. A hypoxic environment was created by placing a cell culture dish with one pouch of Anaero Pack into an airtight jar. (Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan). This created nearly 0% O2 conditions.\nGene silencing of RAGE with specific siRNA\nCells were seeded in 12-well plates at a density of 1\u00a0\u00d7\u00a0105 cells per well and allowed to adhere overnight. Then siRNAs for the target gene or its control oligoribonucleotide mixed with Dharma FECT2TM transfection reagent (Dharmacon Inc. Chicago, USA) was added to the cells and incubated for 48h at 37\u00b0C The efficacy of gene silencing was evaluated using immunoblot analysis.\nMTT assay\nCell viability was monitored after incubation for 24, 36, and 48 hours by MTT assay. Briefly, 0.5\u00a0mg\/mL 3-[4,5]-2,5-diphenyltetrazolium bromide (MTT) in fresh medium was added to each well and the cells were incubated for an additional 3\u00a0hours. Afterwards, the blue formazan crystals were dissolved in 1\u00a0mL isopropanol and measured spectrophotometrically at 570\u00a0nm.\nImmunoblot analysis\nWhole cell lysates were prepared as per the Santa Cruz protocol. One milliliter of Radio-Immunoprecipitation Assay (RIPA) buffer was added to a 100 mm cell culture plate. The plates were gently rocked for 15 min at 4\u00b0C. Adherent cells were scrpaed with a cell scraper, followed by incubation for 30\u201360 min on ice. The cell lysate was microcentrifuged at 10,000g for 10 min at 4\u00b0C. The supernatant fluid was the total cell lysate. The supernatant was transferred to a new microfuge tube and the pellet was discarded. Twelve microgram lysates were subjected to immunoblot analysis using a 12.5% sodium dodecyl sulfate (SDS) -polyacrylamide gel followed by electrotransfer onto nitrocellulose filters. The filters were immunoreacted with anti-RAGE antibody (a gift from TORAY Research Institute, Sagamihara, Japan) or with anti-HMGB1 antibody (BD Biosciences, Tokyo, Japan) and then incubated with peroxidase-conjugated anti-goat IgG (Medical and Biological Laboratories, Nagoya, Japan). The immune complex was visualized using the Enhanced Chemiluminescence (ECL) Western blot detection system (PIERCE, Rockford, IL, USA). The amount of B-actin as an internal control was also examined using a specific antibody (Cytoskelton Inc., Denver, CO, USA and Santa Cruz, CA, USA). At least three independent experiments were performed.\nQuantitative RT-PCR\nFor reverse-transcription PCR (RT-PCR) and real-time quantitative PCR, total RNA was extracted from 30 mg frozen tissue using Total RNA Mini (VIOGENE, CA, USA). For cDNA synthesis, the RNA samples (1 \u03bcg) were converted into cDNA by reverse transcription (RT) using random primers (TAKARA, Siga, Japan) according to the manufacturer\u2019s instructions. To estimate the mRNA expression levels of several genes quantitatively, polymerase chain reaction (PCR) amplification was performed using a Light-Cycler system (Roche, Mannheim, Germany) and the Light-Cycler Fast Start DNA Master SYBER green I kit (Roche). Primers were as follows: RAGE: 5\u2032-AAA CAT CAC AGC CCG GAT TG-3\u2032 and 5\u2032-TCC GGC CTG TGT TCA GTT TC-3\u2032, HMGB1: 5\u2032-GCT CAG AGA GGT GGA AGA CCA-3\u2032 and 5\u2032-GGT GCA TTG GGA TCC TTG AA-3\u2032 (14) , GAPDH: 5\u2032-TTG GTA TCG TGG AAG GAC TCA-3\u2032 and 5\u2032-TGT CAT CAT ATT TGG CAG GTT T-3\u2032. Amplification was carried out in 20 \u03bcL reactions containing 4 mM MgCl2, 2 \u03bcL of primer, 2 \u03bcL of Light-Cycler-FastStart DNA Master SYBR green I reagent, and 2 \u03bcL of cDNA. Reaction conditions were an initial incubation at 95\u00b0C for 10 min, followed by 50 cycles at 95\u00b0C for 10 s for denaturation, 64\u00b0C for 10 s for annealing of the RAGE primers, 54\u00b0C for 10 s for annealing of the HMGB1 primers and 60\u00b0C for 10 s for annealing of the GAPDH primers and 72\u00b0C for 10 s for extension. Melting curves were obtained according to the protocol under the following conditions: 0 s denaturation period at 95\u00b0C, starting temperature of 65\u00b0C, end temperatures of 95\u00b0C, and rate of temperatures increase of 0.1\u00b0C\/s. The quantitative value of the target gene (RAGE mRNA) in each sample was normalized using GAPDH expression as an internal control. The quantitative RT-PCR assay was carried out twice and the mean value was calculated. Finally, the mRNA expression ratio of cancerous (C) to noncancerous (N) tissues was calculated using the following formula: R=log{target gene (C)\/GAPDH (C)}, R=log{target gene (N)\/GAPDH (N)}. These experiments were carried out twice to confirm reproducibility.\nStatistical analysis\nStatistical analysis was performed using the JMP IN version 5.1.2 software system (SAS institute Inc., Cary, NC, USA). Each value of mRNA expression was log transformed before statistical analysis. Gene expression was compared among normal liver, hepatitis, and HCC using Student\u2019s t-test. The relationships between RAGE-, HMGB1-mRNA expression levels and clinicopathological features were evaluated using Student\u2019s t-test and the Mann\u2013Whitney U test, as appropriate. Immunohistochemical study of RAGE in HCC tissues was evaluated using the \u03c72 test. A p value of less than 0.05 was considered to be statistically significant.\nRESULTS\nRAGE and HMGB1 expression in normal liver, hepatitis, and HCC\nRAGE antibody yielded a strong band compared to control mouse lung extracts. Using Western blotting and RT-PCR, protein and mRNA expression of RAGE and HMGB1 were examined in both cancer and noncancerous tissues from three cases (Fig.\u00a01a, b). All 3 cases showed co-expression of RAGE and HMGB1 protein and mRNA in these tissues.\nFIG.\u00a01.(a) RAGE expression by Western blotting (A) and RT-PCR (B). (b) HMGB1 expression by Western blotting (A) and RT-PCR (B). Abbreviations: normal, normal liver; CH, chronic hepatitis.\nQuantitative RAGE mRNA expression in HCC and noncancerous lesions\nComparing the quantitative expression of RAGE mRNA in paired cancer and noncancerous tissues (i.e., chronic hepatitis or liver cirrhosis) of 36 cases, HCC tissues showed significantly higher expression than noncancerous tissues (p\u00a0<\u00a00.01, Fig.\u00a02a). Moreover, the mean values of RAGE mRNA expression in cancer and noncancerous tissues were higher (p\u00a0<\u00a00.01 and p\u00a0=\u00a00.08) than that in normal liver tissues (i.e., non-inflamed liver from benign or metastatic liver tumor patients) (Fig.\u00a02b).\nFIG.\u00a02.(a) Quantitative RAGE mRNA expression in paired cancerous (HCC) and noncancerous tissues from 36 cases. (b) Quantitative RAGE mRNA expression in normal liver (n\u00a0=\u00a06) and noncancerous and cancerous tissues (n\u00a0=\u00a036).\nRelationship between RAGE mRNA expression and clinicopathological features\nTo elucidate the biological significance of RAGE expression in HCC, we compared the levels of RAGE mRNA expression with the clinico-pathological features of 36 patients. As shown in Table\u00a02, we noted significant differences in RAGE mRNA expression in association with gender, age, the levels of protein induced by vitamin K absence or antagonist (PIVKA-II), and postoperative recurrence. In terms of gender and age, the results are in accordance with a previous report.23 The expression levels of RAGE mRNA tended to be lower in poorly differentiated tumors compared to well- or moderately differentiated tumors (p\u00a0=\u00a00.06). Moreover, the levels of RAGE mRNA showed a negative correlation with PIVKA-II levels and the presence or absence of recurrence. There were no significant differences regarding tumor size, intrahepatic metastasis, and vascular invasion. On the other hand, it should be noted that a significant difference in the levels of RAGE expression was observed between hepatitis virus-positive and virus--egative tissues (p\u00a0<\u00a00.01) in the study of noncancerous inflammatory liver tissues (i.e., chronic hepatitis or liver cirrhosis) (data not shown).\nTABLE\u00a02.Relationship between tumor RAGE expression and clinicopathological featuresFactorsTumor RAGE mRNA expressionp valuenMean\u00a0\u00b1\u00a0SDGender\u00a0\u00a0Male302.267\u00a0\u00b1\u00a00.3410.03\u00a0\u00a0female62.777\u00a0\u00b1\u00a00.619Age\u00a0\u00a0\u226565 years262.468\u00a0\u00b1\u00a00.5680.03\u00a0\u00a0<65 years102.052\u00a0\u00b1\u00a00.638Tumor size (mm)\u00a0\u00a0\u226530112.395\u00a0\u00b1\u00a00.6130.39\u00a0\u00a0<30252.333\u00a0\u00b1\u00a00.620Portal invasion\u00a0\u00a0Absent222.358\u00a0\u00b1\u00a00.6550.52\u00a0\u00a0Present142.348\u00a0\u00b1\u00a00.594Venous invasion\u00a0\u00a0Absent262.339\u00a0\u00b1\u00a00.5770.47\u00a0\u00a0Present102.357\u00a0\u00b1\u00a00.633Vascular invasion\u00a0\u00a0Absent192.362\u00a0\u00b1\u00a00.6330.46\u00a0\u00a0Present172.340\u00a0\u00b1\u00a00.601Intrahepatic metastasis\u00a0\u00a0Absent272.378\u00a0\u00b1\u00a00.6620.33\u00a0\u00a0Present92.273\u00a0\u00b1\u00a00.435Gross classification\u00a0\u00a0Localized type212.448\u00a0\u00b1\u00a00.6360.13\u00a0\u00a0Invasive type152.217\u00a0\u00b1\u00a00.564Differentiation\u00a0\u00a0Well42.365\u00a0\u00b1\u00a00.5660.43\u00a0\u00a0Moderately282.420\u00a0\u00b1\u00a00.5800.08\u00a0\u00a0Poorly41.860\u00a0\u00b1\u00a00.783Stage\u00a0\u00a0I,II162.367\u00a0\u00b1\u00a00.6430.55\u00a0\u00a0III,IV202.340\u00a0\u00b1\u00a00.597PIVKA\u2021U\u00a0\u00a0Normal92.704\u00a0\u00b1\u00a00.385\u00a0\u00a0High252.186\u00a0\u00b1\u00a00.6300.03AFP\u00a0\u00a0Normal122.557\u00a0\u00b1\u00a00.5860.16\u00a0\u00a0High242.250\u00a0\u00b1\u00a00.607Virus\u00a0\u00a0B72.237\u00a0\u00b1\u00a00.606\u00a0\u00a0C212.382\u00a0\u00b1\u00a00.569\u00a0\u00a0None82.374\u00a0\u00b1\u00a00.779Recurrence\u00a0\u00a0Absent252.485\u00a0\u00b1\u00a00.6280.04\u00a0\u00a0Present112.050\u00a0\u00b1\u00a00.456\nRAGE expression and tumor differentiation in HCC\nTo verify the correlation of RAGE expression with HCC differentiation, we carried out an immunohistochemical examination. (Fig.\u00a03a\u2013c) Well- and moderately differentiated HCC showed a high percentage of RAGE positivity (70% and 64%, respectively); only 2 of 10 (20%) were positive in poorly differentiated HCC (Fig.\u00a03d). Statistically, there were significant differences in the rates of positivity between well- and moderately differentiated HCC and poorly differentiated HCC.\nFIG.\u00a03.RAGE expression by immunohistochemical staining: (a) well-differentiated HCC, (b) moderately (mod.) differentiated HCC, (c) poorly differentiated HCC, (e) the number of RAGE positive or negative cases according to tumor differentiation.\nRAGE expression in hepatoma cell lines\nRAGE antibody yielded a strong band on Western blot analysis of the control RAGE transfected Cos7 cells (Fig.\u00a04a, lane 1). In this experimental setting, the well-differentiated HCC cell line HuH7 (lane 6) expressed RAGE protein at a modest level. The poorly differentiated HCC cell line HT17 (lane 7) expressed RAGE protein at a low level. Hepatoma cell lines Hep3B and Li7 (lane 4 and 5), derived from tumors of unknown state of differentiation, expressed RAGE protein at a modest level. HepG2 (lane 3) did not express RAGE protein at all. These results were very similar to RAGE mRNA expression and depend on the pathological differentiation of HCC.\nFIG.\u00a04.(a) RAGE expression in hepatoma cell lines by Western blotting. (b) RAGE expression in hepatoma cell lines under hypoxic conditions by Western blotting. N, normoxic conditions; A, anaerobic conditions.\nEnhanced RAGE expression under hypoxic conditions\nEarly carcinogenesis occurs in an avascular environment and under conditions of hypoxia.15,23,24 In order to clarify the role of RAGE expression under hypoxic conditions, all hepatoma cell lines were cultured in an anaerobic environment. Li7 could survive for 48 h, Hep3B for 12 h, and HuH7 for 24 h under such conditions, up to the appearance of signs of cell death. HepG2 and HT17 rapidly died under anaerobic conditions. The RAGE-positive cell lines (Li7, Hep3B and HuH7) were more tolerant of anaerobic conditions than RAGE-negative or weakly expressing cell lines (HepG2 and HT17). Moreover, RAGE expression in the cell lines that survived was clearly upregulated in response to the hypoxic conditions (Fig.\u00a04b).\nEnhancement of cell survival in anaerobic conditions following RAGE transfection\nThese findings led to the hypothesis that RAGE expression could confer tolerance to hypoxia in HCC. Therefore, we investigated the survival of RAGE-transfected and mock-transfected cells under hypoxic conditions. In continuous incubation under hypoxic conditions, as expected, RAGE-transfected Cos7 cells clearly survived longer than mock-transfected Cos7 cells (observed at 36 h and 48 h) (Fig.\u00a05).\nFIG.\u00a05.Comparison of cell survival of RAGE-transfected Cos7 and mock-transfected Cos7 cells under anaerobic conditions. Cell survival of both groups was estimated from six dishes at each time point using the MTT assay. *p\u00a0<\u00a00.01 by Student\u2019s t-test.\nDecline of cell survival in hypoxic conditions following RAGE reduced by siRNA\nConsistent with the previous result, reduction of RAGE expression with anti-RAGE siRNA re-increased susceptibility to hypoxia-induced injury in RAGE-overexpressed Cos7 transfectant (Fig.\u00a06a,b), suggesting that RAGE expression might play an important role in the acquirement of hypoxia-resistant cellular phenotype.\nFIG.\u00a06.RAGE level in RAGE-transfected Cos7 cells were reduced by siRNA RAGE-transfected Cos7 cells reduced RAGE level by siRNA decreased more than the cells mixed with only transfection reagent. *p\u00a0<\u00a00.01 by Student\u2019s t-test.\nDISCUSSION\nHepatocellular carcinogenesis and associated tumorigenesis occur in a stringent and restrictive environment, such as the cirrhotic liver in which tissue oxygen supply is insufficient. Moreover, in the liver, with a dual blood supply from the hepatic artery and portal vein, liver-specific oxygen and glucose supply are available dependent upon the degree of liver disorder. The multiligand receptor RAGE is particularly relevant in this context. The ligands for RAGE may be produced abundantly during HCC development. For example, HMGB1 released from necrotic cells and AGE generated in the process of higher hepatic glucose metabolism are considered to be major candidates (i.e., the presence of HMGB1 and carboxymethyl lysine in HCC; data not shown). Hepatic SAA production in response to IL-6 may also be included.19 In addition, RAGE expression and its downstream signaling are now considered to play a significant role in the progression of various cancers as well as the development of inflammatory lesions.\nAnother aspect of the biology of RAGE was suggested by the features of retinoic acid-induced neuroblastoma differentiation, in which RAGE expression played a more important role in cellular survival than in neurite outgrowth.25 In this model, inhibition of RAGE function partially blocked the increase in levels of the anti-apoptotic protein Bcl-2 in the process of neuronal differentiation,25 indicating that RAGE and its signaling also might contribute to the survival of certain cancer cell types undergoing \u201cdifferentiation\u201d. Furthermore, although indirectly, our previous study demonstrated that NF-\u03baB, currently considered as a causative transcriptional factor for various inflammatory events and also known as a major RAGE-mediated signal, played an important role in the survival of neuronal tumor cells26,27 and in experimentally developed HCC.28\nThe present study demonstrated that increased RAGE expression was highly associated with the status of pathological \u201cdifferentiation\u201d in HCC, which played a significant role in acquisition of the hypoxia-resistant phenotype of tumor cells. This conclusion is supported by several lines of experimental evidence. First, the level of RAGE expression was higher in well- and moderately differentiated HCC, while it diminished as the tumors dedifferentiated to poorly differentiated HCC. This was consistent with the evidence that a negative correlation was observed between the level of RAGE mRNA expression and either the level of PIVKA II or the incidence of postoperative recurrence. Second, the analysis of five HCC lines revealed that three of these (Li7, Hep3B, and HuH7) that are resistant to hypoxic stress characteristically showed higher levels of RAGE expression compared to the two hypoxia-intolerant cell lines HepG2 and HT17. Third, sublethal hypoxia exposure induced significantly increased RAGE expression in hypoxia-resistant HCC lines. In the analysis of the association between the level of RAGE expression and the \u201cdifferentiation status\u201d of HCC lines, the level of RAGE expression was higher in \u201cpossibly differentiated\u201d lines (i.e., HCC with low Alpha-Fet protein (AFP) production), consistent with the results from clinical samples. Finally, cells overexpressing RAGE exogenously showed prolonged survival under hypoxic conditions compared to control mock-transfected cells, and siRNA experiments demonstrated similar results.\nOur conclusion also provides a new hypothetical concept that hepatic RAGE expression may be relevant to the stage or severity of inflammation and the incidence of carcinogenesis and early tumorigenesis of HCC (Fig.\u00a07). In the process of the development of an HCC lesion, increased RAGE expression by inflamed hepatocytes may confer adaptation to an advanced hypoxic environment during remodeling and carcinogenesis with accelerated cell proliferation. In this context, an HCC lesion which is highly associated with inflammation caused by either hepatitis viruses or drugs is developed through a process of multistage carcinogenesis, i.e., from inflammatory lesions (e.g., hepatitis, cirrhosis and precancerous lesion) to adenomatous hyperplasia, and eventually into HCC. Once cancer is established, HCC dedifferentiates step-by-step to a more malignant histology, from well- and moderately to poorly differentiated HCC. It has also been pointed out that the vascular supply changes significantly between each stage of tumorigenesis. According to the evidence from several clinical studies,15,29,30 both adenomatous hyperplasia and well-differentiated HCC are hypovascular tumors primarily fed by the portal vein system, while moderately and poorly differentiated HCC are hypervascular tumors primarily fed by arterial blood. In addition, early carcinogenesis and the development of the malignant phenotype generally occur in an avascular environment.31\u201333 Increased RAGE expression, at least in part, may thus play an important role in the mechanism of early HCC development from precancerous inflammatory lesions. In addition, about the expression of RAGE being lower in poorly differentiated HCC when compared to well and moderately differentiated HCC, gastric and colon cancers have been showed that RAGE and other ligands interacts act on tumor promotion in clinical materials.9,11 However, prostate cancer was demonstrated to show no correlation between RAGE expression and tumor differentiation and depth.14 Moreover, non-small-cell lung cancers have been showed a reverse correlation between RAGE expression and tumor stage.34 This result is partially similar to our results. RAGE expression of each clinical samples is various.\nFIG.\u00a07Scheme of the change of RAGE expression according to the sequential change of liver tissue: normal \u2192 chronic hepatitis (CH) \u2192 cirrhosis \u2192 HCC. The value indicates the quantitative RAGE mRNA expression.\nSome reports have shown that RAGE-expressed cells have invasion and migration potential. Our data from clinical samples did no\u2019 relate to the potential. In vivo, invasive and metastatic potential are reflected by many factors, which may have caused our results.\nOur results raise many questions concerning mechanistic and practical processes. It is important to know whether RAGE can bind HMGB1 secreted from activated macrophages in hepatic inflammation, or if occupancy of RAGE by inhibitors would obviate binding of the stimulatory ligands. The functional role of downstream signaling of the RAGE would also be important to determine the cytoprotective mechanism under hypoxia.\nOur findings have provided the first evidence of the clinical relevance and function of RAGE in HCC, namely differentiation-associated RAGE expression that confers a hypoxia-resistant phenotype. Although other mechanisms may also be important, our data also introduce the concept that RAGE and its functions may be possible candidates for therapeutic targets in the treatment of HCC.","keyphrases":["receptor for advanced glycation end-products ( rage )","hepatocellular carcinoma ( hcc )","immunohistochemistry","hypoxia-reverse-transcription polymerase chain reaction ( rt-pcr )"],"prmu":["P","P","P","M"]}
{"id":"Qual_Life_Res-3-1-2039846","title":"Choosing between measures: comparison of EQ-5D, HUI2 and HUI3 in persons with hearing complaints\n","text":"Objectives To generate insight into the differences between utility measures EuroQol 5D (EQ-5D), Health Utilities Index Mark II (HUI2) and Mark III (HUI3) and their impact on the incremental cost-effectiveness ratio (ICER) for hearing aid fitting\nIntroduction\nHearing loss affects the ability to exchange information and therefore affects a person\u2019s quality of life [1]. Of the western adult population, approximately 15% are hearing impaired [2], and for these persons hearing aid use has proven to be effective [3, 4]. The growth of the elderly population has far-reaching implications for auditory health service delivery and expenditure since the prevalence increases heavily with age. As a result, increasingly more economic evaluations are undertaken on interventions such as hearing aid fitting. In economic evaluations health related quality of life (HRQoL) should be measured with a preference-based utility measure [5]. Until recently, the benefits of hearing aid fitting had not translated into a statistically significant improvement in health state utility [6\u20138]. In 2004 Barton et\u00a0al [9] confirmed this for the EuroQol 5D (EQ-5D), but did find statistically significant utility gain after hearing aid fitting with the Health Utilities Index Mark III (HUI3). This difference may result from differences in the descriptive system, and the way the scoring function is derived.\nAlso with regard to other conditions, a number of studies have found that different utility measures tend to lead to different utility scores [10\u201322]. In general there is need for head-to-head comparisons of utility measures, in order to assess the implications for the interpretation and comparability of economic analyses, especially in conditions where only subtle changes after treatment are expected [23, 24]. In these comparisons the comparison of different tariffs should also be incorporated. But most importantly, the impact of the differences between measures on incremental cost-effectiveness ratios (ICERs) should be made apparent. Surprisingly, the latter has not yet received much attention.\nThe objective of this article is to compare the two most frequently used utility measures in economic analyses [25] in a Dutch population with hearing complaints. Utility scores derived with the EQ-5D UK tariff [26], the EQ-5D Dutch tariff [27], the Health Utilities Index Mark 2 (HUI2) and HUI3 [28] are compared to generate further insight into the differences between the measures, and the impact on the ICER for hearing aid fitting. In particular, this article considers:practicality of using the EuroQol and HUI in a population with hearing complaints;construct validity of the EQ-5D UK tariff, EQ-5D Dutch tariff, HUI2 and HUI3;agreement between the EQ-5D UK tariff, EQ-5D Dutch tariff, HUI2 and HUI3;responsiveness of the EQ-5D UK tariff, EQ-5D Dutch tariff, HUI2 and HUI3 after hearing aid fitting;and the impact of on the ICER for hearing aid fitting.\nMethods\nThe EQ-5D, HUI2 and HUI3\nThe five questions of the EQ-5D descriptive system each represent one dimension of health-related quality of life (mobility, self-care, usual activities, pain\/discomfort and anxiety\/depression) [29]. Each question has three levels and the questions together classify persons into one of 243 health states. The commonly used scoring function is based on a British study (EQ-5D UK tariff) [26], with preferences derived with the time trade-off (TTO) method, in a representative sample of the UK population of 2,997 respondents. The scoring function is additive and the possible range of utility scores is \u22120.59 to 1.00. Recently, Lamers et\u00a0al [27] developed a Dutch scoring function for the EQ-5D (EQ-5D Dutch tariff), based on TTO in a sample of 298 respondents, with utility scores ranging from \u22120.33 to 1.00.\nThe 15 questions of the HUI descriptive system classify respondents into either HUI2 or HUI3 health states. The HUI system focuses on health \u2018within the skin\u2019, meaning that they purely focus on impairment and not on the social context of the impairment [28]. HUI2 consists of seven attributes (sensation, mobility, emotion, cognition, self-care, pain and fertility), with three to five levels, leading to 24,000 possible health states. The optional fertility attribute was not used in the present study. The multiplicative scoring function was derived using standard gamble (SG) and Visual Analogue Scale (VAS) in a random sample of 293 Canadian respondents and utility scores range from \u22120.03 to 1.00 [30]. HUI3 consists of eight attributes (vision, hearing, speech, ambulation, dexterity, emotion, cognition and pain), with five to six levels and leads to 972,000 possible health states. The multiplicative scoring function was derived from SG and VAS, in a random sample of the Canadian general population (n\u00a0=\u00a0504), resulting in possible utility scores varying from \u22120.36 to 1.00 [31].\nStudy population and data collection\nData were collected as part of a before-after study examining direct hearing aid provision versus provision by referral [32]. The study was carried out in 2004\u20132005 in three regions (Maastricht, Rotterdam and Amsterdam) in the Netherlands. Persons with hearing complaints were recruited from the participating Ear Nose and Throat (ENT) departments, audiological centres and hearing aid dispensers. Both EQ-5D and HUI were administered at the first visit to the ENT department. Questionnaires were completed at the department, and respondents were helped by a trained interviewer if requested. Patients who were fitted with a hearing aid were asked to attend the ENT department for a follow-up visit to evaluate the hearing aid fitting. During the evaluation at the ENT department this subset of patients completed both questionnaires for a second time. Again, questionnaires were completed at the department, and respondents were helped by a trained interviewer if requested.\nPracticality of the questionnaires\nEspecially in an elderly population, an important aspect of a utility measure is the ease of completion. The practicality of using the EQ-5D and the HUI in a population with hearing complaints was therefore assessed by the completion rate, using a Wilcoxon Signed Ranks Test to test whether the completion rates were significantly different. Additionally we examined the item non-response.\nConstruct validity\nIn absence of a gold standard to measure health state utility, there is no clear technique to determine the construct validity of utility measures. A way to examine the construct validity is to examine whether utility scores are different for distinctive groups [33, 34]. Other studies have demonstrated differences of quality of life scores by sex [33, 35, 36] and age [35\u201338]. Comparisons were made between EQ-5D UK and Dutch tariff, HUI2 and HUI3 scores by age (above versus below median) and sex. It was expected that persons of a higher age and females have lower utility scores. In addition, the respondents were divided into five clinically distinctive groups, based on their hearing loss and hearing aid use. Hearing loss was defined as the better ear pure tone average (BEPTA) hearing loss for the frequencies 1000, 2000 and 4000\u00a0Hz. The groups were: persons who were not entitled to reimbursement of a hearing aid (BEPTA\u00a0<\u00a035\u00a0dB); persons who were entitled to reimbursement (BEPTA\u00a0\u2265\u00a035 dB) but did not apply for a hearing aid (non-applicants); first time hearing aid applicants; experienced hearing aid users who were about to have a new hearing aid fitted (re-applicants); and experienced hearing aid users who did not have a new hearing aid fitted.\nIt was expected that persons with a BEPTA smaller than 35\u00a0dB would have a higher quality of life score than persons in the other four groups, because they are likely to experience less problems with hearing. It was also expected that non-applicants had a higher utility score than first time applicants, since the latter group is expected to experience more hearing complaints, resulting in fitting a hearing aid as a solution for their hearing problems.\nDescriptive summary statistics were provided and normality was tested for all data using the Kolmogorov\u2013Smirnov test. Kruskal\u2013Wallis one way ANOVA and pairwise comparison tests (Mann\u2013Whitney U) were used to explore the differences between the groups.\nAgreement\nTo assess agreement between the measures, a Wilcoxon Signed-Rank test and a Paired t-test were used to test whether the scores of the EQ-5D (UK and Dutch tariff), HUI2 and HUI3 had the same distribution and mean. Correlations (Kendall\u2019s Tau) and the Intra-class Correlation Coefficient (ICC) were computed. The ICC was based on a two-way mixed effect model, such that the subject effect was random and the instrument effect was fixed, and computed at the individual patient level. An ICC below 0.75 implies poor to moderate agreement; above 0.75 implies good agreement [34].\nResponsiveness\nResponsiveness was tested in the subpopulation with a valid score both before and after hearing aid fitting. Effect size and standardized response mean were calculated. Effect size is the change in score divided by the standard deviation of scores at baseline. Standardized response mean is the change in score divided by the standard deviation of the change in score. Both were interpreted using benchmarks for effect size: 0.20 through 0.49 is interpreted as small, 0.50 through 0.79 as moderate and \u22650.80 as large [39]. Also, a Wilcoxon Signed Ranks Test and a Paired t-test were conducted on the before and after scores. The change in score after hearing aid fitting was tested for differences between first time hearing aid applicants and re-applicants. It was expected that re-applicants had a smaller change in utility, since they already had a hearing aid and therefore expectedly less hearing problems at baseline.\nImpact on the ICER\nTo illustrate the impact on the ICER, straightforward one year ICERs of hearing aid fitting versus no hearing aid fitting (doing nothing) were calculated. Quality Adjusted Life Years (QALYs) were calculated using the area under the curve method, for the EQ-5D (UK and Dutch tariff), HUI2 and HUI3 [5]. Baseline utility scores were used to estimate the effects of \u2018no hearing aid fitting\u2019. Costs of no hearing aid fitting were assumed to be zero. This was compared to the costs and effects after hearing aid fitting. The costs of hearing aid fitting were calculated prospectively for each respondent, including General Practitioner (GP) visit(s), ENT visit(s) and hearing aid(s). We used standard costs for medical consumption [40], and the actual price of the hearing aid. As costs and utility scores are generally not normally distributed, a non-parametric bootstrap sampling method was used to calculate the 95% confidence interval around the ICERs [41]. Cost-effectiveness acceptability curves for all measures were created to characterize the likelihood that hearing aid fitting will be deemed cost-effective based on the incremental costs and outcomes, for a range of ceiling ratios. Ceiling ratios reflect societies\u2019 maximum willingness to pay for a unit of outcome.\nResults\nPracticality\nOf the 337 persons with hearing complaints included in the study, in total 315 (93%) fully completed both the EQ-5D and the HUI descriptive system at baseline. Each item on the EQ-5D had six missing values, except for mobility (n\u00a0=\u00a05). Regarding the HUI, all questions had six or seven missing values, except for the questions on hearing in a group conversation (n\u00a0=\u00a012), pain and discomfort (n\u00a0=\u00a010), and hearing in a conversation with one other person (n\u00a0=\u00a08). Although completion rates were high for both questionnaires, EQ-5D was fully completed by 328 persons (97%), which is significantly more than the 318 persons (94%) who completed the HUI (P-value 0.012).\nOf the 315 persons who completed both EQ-5D and HUI at baseline, 173 persons (55%) had a hearing aid fitted. Of them, 82 (47%) attended the ENT department for the follow-up visit after hearing aid fitting. Ninety-one respondents (53%) who had a hearing aid fitted did not show at the follow-up visit because they had not finished their hearing aid fitting before the end of the study (n\u00a0=\u00a037), or because they had a hearing aid fitted at a dispenser not participating in the study (n\u00a0=\u00a054), and were therefore lost to follow up. Of the 82 respondents who did attend the follow-up visit, 70 (85%) fully completed both the EQ-5D and HUI descriptive system. Each item on the EQ-5D had five missing values, except for pain\/discomfort (n\u00a0=\u00a06). Regarding the HUI, the hearing questions both had four missing values, and the other questions had four to seven missing values. Seventy-six persons (93%) fully completed the EQ-5D, while 71 persons (87%) fully completed the HUI. This difference is not statistically significant (P-value 0.059).\nConstruct validity\nMean age of the respondents was 69.6\u00a0years (sd 8.9; median 70), and BEPTA was on average 42 dB. The respondents were divided into groups below 70\u00a0years old (n\u00a0=\u00a0156) versus 70\u00a0years and older (n\u00a0=\u00a0159) and male (n\u00a0=\u00a0189) versus female (n\u00a0=\u00a0126), see Table\u00a01. The scores on the EQ-5D, HUI2 and HUI3 were not normally distributed (Kolmogorov\u2013Smirnov test, P-value 0.000). Only the EQ-5D detected differences in reported health by both age and sex. HUI3 detected a difference in utility between the age groups, HUI2 did not detect any differences.\nTable\u00a01Comparison of mean scores, median scores and interquartile range (IR) at baseline according to different characteristics of the respondentsAgeSexBEPTAEQ-5D UK tariffEQ-5D Dutch tariffHUI2HUI3NMean % MaleMean MeanMedian (IR) MeanMedian (IR) MeanMedian (IR) MeanMedian (IR)Age<7015661.964%37.40.860.94 (0.24) 0.880.95 (0.20)0.780.79 (0.15)0.640.65 (0.36)\u22657015977.057%47.10.810.80 (0.27)0.840.84 (0.23)0.760.80 (0.17)0.580.59 (0.38) P-value*0.0100.0250.1340.034SexMale18969.3100%43.40.850.88 (0.26)0.870.90 (0.19)0.770.80 (0.13)0.610.62 (0.37)Female12669.90%40.50.810.80 (0.27)0.830.84 (0.23)0.760.78 (0.18)0.610.63 (0.39)P-value*0.0190.0120.5750.965Clinically distinctive groupsBEPTA\u00a0<35\u00a0dB6961.954%21.30.840.81 (0.27)0.860.84 (0.20)0.800.80 (0.20)0.700.66 (0.37)Non-applicants4674.259%42.10.800.87 (0.27)0.830.90 (0.23)0.770.78 (0.15)0.620.60 (0.41)First time applicants10871.262%46.90.840.85 (0.27)0.860.87 (0.23)0.760.78 (0.16)0.580.61 (0.28)Re-applicants6569.666%53.60.850.85 (0.27)0.880.86 (0.20)0.770.79 (0.17)0.560.61 (0.41)P-value**0.9960.9930.2640.004* Mann\u2013Whitney U test** Kruskal\u2013Wallis test\nFurthermore, the respondents were divided into five clinically distinctive groups: BEPTA\u00a0<\u00a035\u00a0dB (n\u00a0=\u00a069), non-applicants (n\u00a0=\u00a046), first time hearing aid applicants (n\u00a0=\u00a0108), re-applicants (n\u00a0=\u00a065), and experienced hearing aid users not applying for a new hearing aid (n\u00a0=\u00a012). The latter group was excluded from the analysis because of the small sample size. Fifteen persons could not be classified into a clinical group because they were lost to follow-up after the first visit.\nBased on the EQ-5D and HUI2, no distinction could be made between any of the clinically distinctive groups. A logarithmic transformation was performed on the EQ-5D and HUI2 data to compensate for skewness. Even after transformation, and also when correcting for age, sex and BEPTA, no differences were found between the groups. Only HUI3 scores demonstrated a significant difference between the clinically distinctive groups (Kruskall\u2013Wallis; P-value 0.004). More specifically, HUI3 found significant differences between persons with a BEPTA\u00a0<\u00a035 dB and first time applicants (Mann\u2013Whitney U; P-value 0.002), and between persons with a BEPTA\u00a0<\u00a035 dB and re-applicants (P-value 0.001). HUI3 did not confirm our expectation that non-applicants had significantly higher utility scores than first time applicants. As expected, non-applicants stated less problems on the hearing attribute than first time applicants, but they also stated more problems on the ambulation and pain attributes.\nAgreement\nA summary of the utility scores is presented in Table\u00a02. Mean utility scores for the population with hearing complaints were higher for the EQ-5D Dutch tariff (mean 0.86; standard deviation 0.18) and EQ-5D UK tariff (0.83; sd 0.21) than for the HUI2 (0.77; sd 0.14) and HUI3 (0.61; sd 0.24). The differences in mean scores and distributions are statistically significant. The UK and Dutch tariff of the EQ-5D and HUI2 and HUI3 were found to have good agreement, since they were strongly correlated (0.90; 0.71) and had an ICC of 0.98 and 0.74, respectively. The scores on all other measures showed statistically significant, but low correlations and their agreement was moderate to poor (Table\u00a03).\nTable\u00a02Utility scores in baseline population with hearing complaints (n\u00a0=\u00a0315)MeasureMinimumMaximumMedian*Interquartile rangeMean**Standard deviationEQ-5D UK tariff\u22120.251.000.850.270.830.21EQ-5D Dutch tariff\u22120.031.000.860.190.860.18HUI20.231.000.790.150.770.14HUI3\u22120.071.000.620.380.610.24* All statistically significantly different: Wilcoxon Signed-Rank test; P\u00a0<\u00a00.01** All statistically significantly different: Paired t-test; P\u00a0<\u00a00.01Table\u00a03Agreement in the baseline population with hearing complaints (n\u00a0=\u00a0315)Pairs of utility functionsKendall\u2019s Tau*ICC(95% Confidence interval)EQ-5D UK tariff versus HUI2r\u00a0=\u00a00.410.51(0.42\u20130.59)EQ-5D UK tariff versus HUI3r\u00a0=\u00a00.370.47(0.38\u20130.55)EQ-5D UK versus Dutch tariffr\u00a0=\u00a00.900.98(0.97\u20130.98)HUI2 versus HUI3r\u00a0=\u00a00.710.74(0.68\u20130.78)HUI2 versus EQ-5D Dutch tariffr\u00a0=\u00a00.400.51(0.42\u20130.59)HUI3 versus EQ-5D Dutch tariffr\u00a0=\u00a00.360.44(0.35\u20130.53)*\u00a0All statistically significant, P\u00a0<\u00a00.01\nCeiling effects were observed in the EQ-5D UK tariff (Figs.\u00a01 and 2), results were similar for EQ-5D Dutch tariff. As measured with the EQ-5D (both UK and Dutch tariff), 44% of the respondents reported perfect health, despite their hearing complaints. Measured with the HUI2 or HUI3, less than 1% of the respondents reported perfect health. For respondents reporting perfect health on the EQ-5D, mean utility scores were 0.83 on the HUI2 (range 0.35\u20131.00) and 0.71 on the HUI3 (range 0.06\u20131.00).\nFig.\u00a01Scatterplot of utility scores derived with EQ-5D UK tariff and HUI2Fig.\u00a02Scatterplot of utility scores derived with EQ-5D UK tariff and HUI3\nResponsiveness\nResponsiveness was tested in the subpopulation with a valid score both before and after hearing aid fitting (n\u00a0=\u00a070). The effect size and standardized response mean of the EQ-5D UK (0.05; 0.05) and Dutch tariff (0.03; 0.02) were less than small. HUI2 and HUI3 were more responsive to change after hearing aid fitting, since both had a moderate effect size (0.64; 0.55) and standardized response mean (0.57; 0.66).\nMean change in utility after hearing aid fitting (Table\u00a04) was highest when measured with the HUI3 (mean 0.12; sd 0.18) and HUI2 (0.07; sd 0.13), while almost no change was measured with the EQ-5D (UK tariff 0.01, sd 0.13; Dutch tariff 0.00, sd 0.12). The change in utility measured with HUI2 and HUI3 is statistically significant (Paired t-test, P-values 0.005 and 0.000). No change was observed in any attribute of the EQ-5D (Fig.\u00a03a). The change in HUI2 utility score after hearing aid fitting occurred in the sensation attribute (Fig.\u00a03b) and in the HUI3 score in the hearing attribute (Fig.\u00a03c). Almost no change was observed in any of the other attributes of the HUI2 and HUI3.\nTable\u00a04Change in health state utility after hearing aid fitting and ICER with 95% Confidence Interval (CI) (n\u00a0=\u00a070)MeasureMeanStandard deviationMedianInterquartile rangeMinimumMaximumICERa \u20ac\/QALY(95% CI) \u20ac\/QALYEQ-5D UK tariff0.010.130.000.04\u22120.600.27286,866(inferiorb\u201347,082)EQ-5D Dutch tariff0.000.120.000.04\u22120.600.28647,209(inferiorb\u201361,934)HUI20.07*0.130.08**0.12\u22120.500.4025,337(19,356\u201338,012)HUI30.12*0.180.13**0.22\u22120.220.6015,811(11,664\u201324,654)*\u00a0Statistically significant; Paired t-test; P\u00a0<\u00a00.01**\u00a0Statistically significant: Wilcoxon Signed-Rank test; P\u00a0<\u00a00.01a\u00a0ICER based on mean scoresb\u00a0Inferior means higher costs and lower utilityFig.\u00a03Percentage of responses for each level of each attribute before (b) and after (a) hearingaid fitting for EQ-5D, HUI2 and HUI3\nThe mean change in utility score after hearing aid fitting, when measured with HUI2 and HUI3, was higher for first-time hearing aid applicants (0.08; 0.13) than for re-applicants (0.06; 0.10). This outcome was in line with our expectations, but is not significantly different.\nImpact on the ICER\nMean costs of doing nothing were zero. The mean costs of hearing aid fitting were \u20ac1,877. The latter consisted of GP visits (\u20ac37), ENT visits (\u20ac295) and hearing aid(s) (\u20ac1545). All hearing aids were digital, and hearing aids were bilaterally fitted in 83% of the respondents.\nThis resulted in mean one-year incremental costs of hearing aid fitting versus doing nothing of \u20ac1,877. The mean utility gain of 0.01 (sd 0.13), measured with the EQ-5D UK tariff, resulted in a ratio of \u20ac286,866 per QALY, with a 95% confidence interval of inferior (higher costs, lower utility) to \u20ac47,082\/QALY. There was a 36% probability that hearing aid fitting was both more costly and less effective (inferior). The mean utility gain of 0.003, measured with the EQ-5D Dutch tariff, resulted in an ICER of \u20ac647,209 per QALY (95% confidence interval: inferior to \u20ac61,934\/QALY). There was a 42% probability that hearing aid fitting was inferior. Applying the HUI2 and HUI3, the ICER was \u20ac25,337 per QALY (95% confidence interval: \u20ac38,012\/QALY to \u20ac19,356\/QALY) and \u20ac15,811 per QALY (95% confidence interval: \u20ac24,654\/QALY to \u20ac11,664\/QALY) respectively. For both measures there was no probability that hearing aid fitting was inferior.\nThe informal Dutch ceiling ratio of \u20ac20,000\/QALY [42] implied that hearing aid fitting was only cost-effective when utility was measured with the HUI3. A cost-effectiveness plane with incremental cost and effect pairs for 1,000 bootstrap replications, for all measures, is shown in Fig.\u00a04. Figure\u00a05 shows the cost-effectiveness acceptability curves for all measures.\nFig.\u00a04Incremental cost-effectiveness ratio for hearing aid fitting. Incremental costs (euro) are displayed at the vertical line, incremental effects (QALYs) on the horizontal lineFig.\u00a05Cost-effectiveness acceptability curves of EQ-5D UK and Dutch tariff, HUI2 and HUI3\nConclusion and discussion\nHearing loss affects the exchange of information and therefore affects a person\u2019s quality of life [1]. Hearing impaired persons can benefit from using a hearing aid, since hearing aid use has proven to be effective, it improves social, emotional and communication functions and reduces depression [3, 4].\nThe results of this study in a relatively healthy population with hearing complaints provide insight in the differences between two widely used utility measures, the EQ-5D and the HUI system.\nRegarding practicality, both questionnaires had high completion rates, with the EQ-5D having a higher completion rate than the HUI.\nWith the EQ-5D, differences were detected in utility by age and sex, indicating construct validity. The HUI3 detected differences by age, but not by sex. Differences between clinically distinctive groups were only detected by HUI3. However, the HUI3 did not confirm our expectation that non-applicants would have higher utility values than first-time applicants. An explanation for this may be that non-applicants had more health problems other than their hearing, as reflected in the ambulation and pain dimensions of the HUI3.\nOverall, HUI2 and HUI3 scores were lower than EQ-5D scores and agreement was moderate to poor. Although these measures intend to assess the same construct, namely health state utility, this result was expected as the instruments differ in their underlying assumptions about what constitutes health state utility.\nAs to responsiveness, only HUI2 and HUI3 measured statistically significant improvement after hearing aid fitting, the EQ-5D UK and Dutch tariff both were not able to capture this effect. Half of the patients (53%) who were fitted with a hearing aid were lost to follow up, either because they had not finished their hearing aid fitting before the end of the study, or because they had their hearing aid fitted at a dispenser not participating in our study. As these patients did not differ from the follow-up group in baseline utility, hearing loss and age, we did not expect this low response rate to influence the results.\nThe HUI2 and HUI3 change scores resulted in smaller ICERs for hearing aid fitting. Although they were only illustrative, the different ICERs found in the present study clearly show that the choice of a utility instrument in the economic evaluation of hearing aid fitting may heavily influence the cost-effectiveness outcome.\nTo calculate the ICER of hearing aid fitting we included the total population of respondents considered for hearing aid fitting, regardless of the type of hearing aid fitting. This makes the ICERs found in the present study representative for hearing aid fitting in general in the Netherlands. However, the one year ICERs calculated in the present study were merely illustrative of the impact of different utility scores on the ICER of hearing aid fitting, as the cost-effectiveness of hearing aid fitting has thoroughly been examined by Joore et\u00a0al [8].\nOur results confirm Barton et\u00a0al [9, 43], who compared the EQ-5D UK tariff and HUI3 before and after hearing aid fitting in a UK sample. However, the change in utility after hearing aid fitting derived with the HUI3 in the present study was twice the change in utility derived with the HUI3 in the study of Barton et\u00a0al [9]. Respondents in the latter study had a lower baseline utility score and a higher range in utility change after hearing aid fitting, which suggests a somewhat different study population. Our results confirm previous studies that concluded that the EQ-5D lacks sufficient sensitivity to measure the benefit of hearing aid fitting [6, 7, 44]. The lower utility scores and higher responsiveness of the HUI3 in relation to the EQ-5D were also found in a population with visual impairment [22]. Joore et\u00a0al [8] calculated that hearing aid fitting costs \u20ac15,807 per QALY, using the EQ-5D as utility measure. The results of the present study indicate that using the HUI3 as utility measure probably had resulted in a more favourable ICER for hearing aid fitting. Vuorialho et\u00a0al [45] recently concluded that counseling of hearing aid users to reduce the number of non-users is highly cost-effective, although they were unable to measure any change in utility. As they used the EQ-5D, it is possible that they would have been able to demonstrate favourable costs per QALY when they would have used the HUI3 to measure change in utility.\nThree questions arise from the results of the present study: can differences be explained by differences in the measures, are the differences observed between the measures important, and what are the implications of the findings for utility measurement and cost-utility analysis in populations with hearing complaints?\nFirst, differences in utility scores can be explained by differences in the descriptive system and the way the utility scoring function is derived. Regarding the descriptive system, the focus of the EQ-5D on physical, mental and social functioning [46] differs from the \u2018within-the-skin\u2019 perspective of HUI, which focuses on the underlying level of impairment. However, this does not explain why the EQ-5D does not measure change after hearing aid fitting, as previous studies have found that hearing aid fitting improves social and emotional functioning [3, 4]. Also, the measures differ in the content and number of attributes, items and levels used, and therefore differ both in the number and in the content of possible health states. It has already been suggested not to use the EQ-5D in relatively healthy populations, given the presence of a ceiling effect [15]. The ceiling effect of the EQ-5D found in the present study is likely to contribute to the differences in responsiveness. When, as found in our study, 44% of the respondents report perfect health at baseline, it is unlikely to find a considerable utility gain from any intervention. Furthermore, since the HUI descriptive system pays explicit attention to hearing abilities, it is to be expected that in a relatively healthy population with hearing complaints HUI and EQ-5D utility scores differ, and HUI is more responsive. As the HUI3 also pays explicit attention to visual abilities, this may explain why comparisons of EQ-5D and HUI3 in hearing and vision show similar results [22].\nThere are also differences in the utility scoring functions. Although in general SG (used for HUI) leads to higher scores than TTO (used for EQ-5D) [5], in the present study the EQ-5D scores were considerably higher than the HUI scores. Although different populations do not necessarily yield different results [5], the population sample in which the preferences are measured may also have impact on the differences. In the present study, differences between utilities derived with the UK and Dutch tariff were observed. This difference may be the result of differences in health valuation between people from the Netherlands and the UK, but may also result from the somewhat questionable representativeness of the population sample used to develop the Dutch tariff [27].\nDifferences also exist in the type of scoring function. EQ-5D uses an additive system, assuming no interaction for preferences among attributes at all. The HUI uses a multiplicative scoring function, with the effect that the loss of utility associated with a particular dimension is dependent on the level of impairment on other dimensions [5]. For example, Barton et\u00a0al [43] illustrated that hearing impairment (\u2018unable to hear at all\u2019) has a greater impact on HRQoL as measured with the HUI3 when one has no other health problems (\u22120.53), than when one also has moderate to severe pain and is unable to see at all (\u22120.05). It seems rational that persons find their hearing loss a less important aspect of their health state utility when they experience more comorbidity. The multiplicative scoring function of HUI takes this influence of comorbidity into account and seems to be more suitable for modeling utility scores.\nAre the differences observed between the measures important? The answer to this question is a clear \u2018yes\u2019. The impact of different utility measures on the ICER for hearing aid fitting is of a magnitude that can alter policy decisions and emphasizes that comparisons of QALYs across studies and interventions should be interpreted with caution [12]. The general purpose of a utility measure is to capture the health effects in terms of HRQoL of a policy or program for use in economic analyses. Health economic analyses are a tool to allocate resources in a way that maximizes health (or welfare). In order to use the outcomes of economic analyses for policy decisions, there should however be a clear notion of what should be maximized. The results of the present study show that the potential benefit of an intervention heavily depends on the assumptions of what constitutes health underlying the utility measure that is used. Beyond the apparent necessity of psychometric quality of a utility measure, an important area for future research is whether societies wish, or should, maximize life expectancy corrected for HRQoL from a more functional perspective as in the EQ-5D, or from a \u2018within-the-skin\u2019 perspective as in the HUI. Other studies that have thoroughly examined the psychometric differences between utility measures seem to pass over this important question [11, 13, 19, 21, 24].\nWhat are the implications of the findings for utility measurement and cost-utility analysis in populations with hearing complaints? Generally, it has been recommended that the instrument that is most sensitive to the health states in which one is interested should be selected [5, 12, 47]. From clinical experience it is plain that hearing aid use is effective in alleviating hearing loss and does improve health-related quality of life, but the EQ-5D lacks the sensitivity to capture this improvement. In an otherwise healthy population, HUI3 has proven to be more responsive and therefore more appropriate for evaluating HRQoL in a population with hearing complaints, and is therefore the instrument of first choice in this population.","keyphrases":["hearing loss","questionnaires","costs and cost analysis","quality-adjusted life years","outcome assessment (health care)"],"prmu":["P","P","R","M","M"]}
{"id":"Eur_J_Health_Econ-_-_-1388079","title":"The \u201cHealth Benefit Basket\u201d in Denmark\n","text":"Until 2007, when the new legislation on health care becomes effective, the right to receive free health care services in Denmark, or \u201chealth benefits,\u201d are described in a comprehensive set of legislation, including laws, executive orders and legal guidelines. This contribution provides an overview of the current main legislation regulating the Danish \u201chealth benefit basket\u201d and describes the regulatory mechanisms for the provision of curative care at Danish hospitals and primary health care offices. Although the services are both financed and planned by the counties, they differ substantially in the way that benefits are regulated.\nThe Danish health care system offers a vast amount of benefits and entitlements to persons living in Denmark. The benefits are more or less specified health services provided at hospitals or in primary and tertiary health care institutions. Entitlements are rights to receive these services free of charge or against a fee covering a part of the costs. The benefits and entitlements altogether form the so-called \u201chealth benefit basket\u201d or \u201cbasic package of health services\u201d [1]. Although the notion of a benefit basket is not commonly used in Denmark, health benefits are indeed described in the legislation. The following pages provide an overview of the laws regulating health benefits and a description of the regulatory mechanism for the provision of curative care in hospitals and in primary care offices. Curative care services are financed and planned by the 14 counties and the municipal authorities of Copenhagen and Frederiksberg, and there are considerable differences between in- and outpatient services in the way in which benefits are regulated and services are provided and reimbursed.\nTable\u00a01 provides an overview of the four most important laws regulating benefits in the Danish health sector:Table\u00a01 The Danish \u201chealth benefit basket\u201d: overview of main legislationHospital Act (and ancillary legislation)Public Health Insurance Act (and ancillary legislation)Medicines Act (and ancillary legislation)Social Services Act (and ancillary legislation)Other (specific) legislationHC.1.1 Inpatient curative care+\u2013(+)\u2013\u2013HC.1.2 Day-cases of curative care+\u2013(+)\u2013\u2013HC.1.3 Outpatient care(+)+(+)\u2013\u2013HC 1.4 Curative home care++(+)\u2013\u2013HC.2.1 Inpatient rehabilitative care+\u2013\u2013(+)\u2013HC.2.2 Day cases of rehabilitative care+(+)\u2013(+)\u2013HC 2.3 Outpatient rehabilitative care(+)+\u2013+\u2013HC 2.4 Services of rehabilitative home care(+)(+)\u2013++HC.3.1 Inpatient long-term care+\u2013\u2013+\u2013HC.3.2 Day cases of long-term care+\u2013\u2013+\u2013HC 4.1 Clinical laboratory +++\u2013\u2013HC.4.2 Diagnostic imaging+++\u2013\u2013HC.4.3 Patient transport ++\u2013+\u2013HC.4.9 All other miscellaneous services++\u2013(+)\u2013HC.5.1 Pharmaceuticals, etc.\u2013++\u2013\u2013HC 5.2 Therapeutic devices etc.(+)(+)\u2013+\u2013HC.6.1 Maternal and child care++\u2013(+)+HC 6.2 School health services\u2013\u2013\u2013\u2013+HC.6.3 Prevention of communicable diseases\u2013(+)\u2013\u2013+HC.6.4 Prevention of noncommunicable diseases\u2013(+)\u2013\u2013+HC.6.5 Occupational health services\u2013\u2013\u2013\u2013+HC.6.9 All other services\u2013\u2013\u2013\u2013+Legal statusLawLawLawLawLawsDecision makerCounty + physicianCounty + GPCounty, GP (physician)Municipality + countyCounty + municipality + GPPurposeEntitlements, delegation and defining responsibility of countiesEntitlements, reimbursement, copaymentRole of Danish Medicines Agency, process of approval of medicinesEntitlements, delegation and defining responsibility of counties and municipalitiesEntitlements, defining roles and responsibilities of actors etcPositive\/negative definition of benefitsPositivePositivePositivePositivePositive Degree of explicitnessa11 (2 and 3)2 (and 3)1 (2 and 3)1, 2, 3If itemized: goods, procedures, indicationsNot itemizedProcedures (indications)(Goods)(Goods, indications)IndicationsUpdatingNoNoNoNoNoCriteria used for defining benefits\u00a0\u00a0Need+++++\u00a0\u00a0Costs\u2013\u2013\u2013\u2013\u2013\u00a0\u00a0Effectiveness\u2013\u2013+\u2013\u2013\u00a0\u00a0Cost-effectiveness\u2013\u2013\u2013\u2013\u2013\u00a0\u00a0Budget+\u2013\u2013\u2013\u2013\u00a0\u00a0Other\u2013+\u2013\u2013\u2013a 1, \u201cAll necessary\u201d; 2, areas of care are described; 3, services are linked to specific itemsThe Hospital Act establishes the regulatory framework for all types of inpatient care and day care, patient transport, maternity care, ambulatory and rehabilitative care provided at hospitals (Bekendtg\u00f8relse af lov om sygehusvaesenet, LBK 766, 28 March 2003). The law delegates the responsibility for the planning, providing, and financing of hospital services to the county council. A number of executive orders and directives further specify this law.The Public Health Insurance Act (PHIA) and its supplementary legislation provides the regulatory frame for entitlements to outpatient care and to some extent to rehabilitative care, ancillary services for outpatients, maternity and child care, and reimbursement of pharmaceuticals (Lov om offentlig sygesikring, LOV 509, 1 July 1998; og DSK 9926, 20 January 1999; og LOV 467 og 469, 31 May 2000; og LOV 495, 7 June 2001; og LOV 1031, 23 November 2000; og LOV 1045, 23 December 1998; og LOV 1118, 29 December 1999; og LOV 1207, 27 December 2003; og LOV 1431, 22 December 2004). Benefits and services are further specified in a number of executive orders as well as in the used fee schedule.The Medicines Act regulates the population\u2019s access to pharmaceuticals and specifies the process of approval of medicines by the Danish Medicines Agency, marketing authorizations, etc. (Bekendtg\u00f8relse af lov om laegemidler, LBK 656, 28 July 1995).The Consolidated Social Services Act provides the regulatory frame for social services (Bekendtg\u00f8relse af lov om social service, LBK 708, 29 June 1998). The law delegates the planning, financing and provision responsibility to the counties for the specialized care and to municipalities for the less specialized care. This law covers the long-term inpatient, outpatient and home care. It also describes the provision responsibility for rehabilitative care not following a hospital treatment and also regulates the entitlement to therapeutic devices, nonacute patient transport, and other services.\nDanish legislation on health care furthermore contains a number of specific laws dealing with preventive services, for example, for children (Forebyggende sundhedsordninger for b\u00f8rn og unge, LOV 438, 14 June 1995), with explicit and specific definitions of benefits. The main decision makers in the Danish health sector at political and administrative levels are the local governing bodies responsible for the service, hence the elected politicians for the county and the municipal councils. At clinical level especially the general practitioner plays a very important gate-keeper role, but hospital physicians are also important in defining medical needs. Most benefits are explicitly but vaguely defined by the law, leaving it to decision makers to specify which services to offer to patients. The Hospital Act states that \u201cthe county shall provide hospital treatment free of charge for its citizens\u201d (Sect.\u00a05). The degree of explicitness is described as \u201call necessary\u201d (1) by all laws; some laws further describe areas of care (2) and some are linked to items (3). The PHIA and the Social Services Act are to some extent more explicit, by linking benefits to items.\nThis contribution is confined to curative health services, and the description of benefits in curative health services in Denmark is divided into three sections. First, the main actors along with their roles and responsibilities are outlined and differences between in- and outpatient services are emphasized. Second, benefits in the hospital sector are described and third, an outline of benefits under the Health Care Reimbursement Scheme (HCRS) for outpatient services is provided. Benefits are described with regard to the legislation and other regulatory mechanisms defining them, how they are classified, and how the fee schedule under the HCRS, a benefit \u201ccatalogue,\u201d is structured.\nMain actors in defining benefits: their roles and responsibilities\nThe Danish Parliament (Folketing) legislates on health care. The Minister for the Interior and Health is responsible for further specifying the law, setting rules in certain areas and initiating reforms and bills. Unless it is explicitly mentioned that the Minister is empowered to specify the law, it can only be changed by the Folketing. Regarding outpatient care the Minister must approve all negotiated agreements between the Health Care Reimbursement Negotiation Committee (HCRNC) and the health personnel trade unions.\nThe National Board of Health (NBH) is a subdivision of the Ministry of the Interior and Health. The NBH is responsible for supervising and providing advice to \u201chealth care persons\u201d (the Danish word sundhedspersoner used in the legislation refers to all types of health care professionals) and health administrative authorities, for elaborating and issuing guidelines and reference programs as well as for monitoring and documenting health services. Guidelines from the NBH define the current best practice within specific health care programs or interpret the law. The \u201chealth care persons\u201d are for their part obliged to show clinical diligence and act according to \u201cbest practice.\u201d\nAccording to the Hospital Act, the counties and the Copenhagen Hospital Corporation are responsible for the planning and financing of hospital services (Sect.\u00a01) and for providing free hospital treatment for their citizens (Sect.\u00a05). The providers of hospital services may be county-owned hospitals, private hospitals, or foreign hospitals (Sect.\u00a03). Regarding outpatient care the county reimburses the services provided by private specialists and general practitioners (GPs). County representatives in a subcommittee under the HCRNC decide on the actual supply of services, for example, who is allowed to establish a private practice in which county.\nA county health plan coordinating health services in the county is to be elaborated every 4\u00a0years (Hospital Act, Sect.\u00a011; PHIA Sect.\u00a027e). The plan includes a description of the county\u2019s population health status (premise of the plan), the physical capacity of the county hospitals, including how and where specialties are represented, and the number of beds and employees etc. in each specialty. Furthermore, preventive services, outpatient services, and cooperation between GPs, specialists, dentists, and the hospital sector, cooperation with municipalities, ambulance services, and local emergency services are coordinated and described in the plan. The NBH supervises and comments on the plan. However, the counties are not, in any legal sense, obliged to follow the advice provided by the NBH.\nHospital and clinical department managers have considerable freedom within the legal, clinical, and economic framework set by their superiors at national and county level. The heads of department may therefore establish treatment protocols and indications for treatment for different patient categories as well as set up guidelines for prioritizing between patients on waiting lists.\nIn general the Danish legislation is rather vague with regard to the specification of services and very often leaving decisions about benefits to medical judgement. Thus physicians play a certain role in the definition of benefits. Especially, the GP is important, as she\/he is responsible for referring patients from general practice to practically all parts of the health sector.\nDefinition of benefits\nHospital care\nBenefits provided in the Danish hospital sector are defined at four levels of decision making: at national level by means of legislation, at county level by planning and supply control, and at hospital and clinical level by local guidelines and clinical decision making. According to the Hospital Act, citizens have the right to free hospital treatment (Sect.\u00a05) at any hospital in the country (Sect.\u00a05b) which they choose. If the waiting time for treatment exceeds 2\u00a0months, they have the right to be treated at a private hospital in Denmark or abroad at their county\u2019s expense (Sect.\u00a05\u00a0g). The Act includes inpatient care, hospital ambulatory (day) care and in some cases home care for somatic and mental patients. Benefits are explicitly but vaguely formulated by the law. In principle, patients are entitled to any treatment that is clinically indicated, but in practice limitations apply. At national level the Hospital Act and its amendments directly or indirectly specify which services to offer (positive definition of benefits) or not to offer (negative definitions of benefits) to whom and under which circumstances:\nPositive definition of benefits (examples):For persons suffering from a \u201clife-threatening\u201d disease, defined as patients with certain heart conditions or cancer patients, a maximum national waiting time is defined.The county offers a breast examination (mammography) to women aged 50\u201369\u00a0years every second year (Hospital Act; Sect.\u00a05e). However, this benefit is accompanied by a clause stating that the Minister will decide when this service is to be implemented in the counties. The service is expected to be implemented by 2008. Today it is only offered in three counties.Patients with a clinically defined need for rehabilitation have the right to receive a plan from the hospital describing when and where rehabilitation is to take place at discharge, and who will coordinate between the county and municipal providers (Hospital Act; Bekendtg\u00f8relse om udarbejdelse af genoptraeningsplaner ved udskrivning fra sygehus, BEK 1009, 9 December 2003).\nNegative definition of benefits (examples):Induced abortions after week 12 of pregnancy are only allowed after permission from the Minister of Justice and upon medical or social indication (Bekendtg\u00f8relse af lov om svangerskabsafbrydelse og fosterreduktion, LBK 541, 16 June 2004, og BEK 540, 16 June 2004, og VEJ 57, 16 June 2004). Sterilization cannot be provided to persons below the age of 25\u00a0years (Bekendtg\u00f8relse af Lov om sterilisation og kastration, LBK 661, 12 July 1994, og BEK 1131, 13 December 1996).Unmarried women and women over the age of 45\u00a0years are exempt from in vitro fertilization treatment by a physician (specialist; Lov om Kunstig befrugtning i forbindelse med laegelig behandling, diagnostik, forskning mv, LOV 460, 10 June 1997; Bekendtg\u00f8relse om kunstig befrugtning, BEK 728, 17 September 1997).Free choice of private hospitals (in the case the waiting time for treatment at county hospitals exceeds the limit of 2\u00a0months) does not include transplantation, sterilization, reproductive health services, hearing aid, cosmetic surgery, sex change, psychiatric treatment, alternative treatment, or treatment characterized as research (Bekendtg\u00f8relse om amtskommuners betaling for sygehusbehandling ved en anden amtskommunes sygehusvaesen, BEK 594, 23 June 2003).\nIn general, conventional treatments not included in the benefit package are not offered. For patients suffering from life-threatening cancer a referral needs to be approved by a board of specialists, a \u201cSecond Opinion Panel\u201d established under the NBH (information available at: http:\/\/www.sst.dk\/planlaegning_og_behandling\/second opinion.aspx, accessed 4 August 2005). If the alternative such as conventional treatment is approved by the board, the expenses are covered by a special government grant. However, nonconventional treatment, such as a spa therapy, zone therapy, and homeopathy are not included in the benefit package [2].\nFinancial instruments\nHospitals are financed by the counties through tax revenue and block grants from the state. Following the annual negotiations between the counties and the government on the level of taxation and size of block grant the government has introduced new financial measures as a means to influence the development of the health sector. Special block grants are earmarked to high priority areas such as heart surgery and cancer treatment. These grants accompany recommendations from the NBH on treatment improvements and expansion of the capacity, as described in the \u201cHeart Plan\u201d and the \u201cCancer Plan.\u201d Furthermore, with the aim of shortening waiting lists, a provision of activity-based funding for additional production has been made available from 2002 for counties which can document an increase in their activity (Hospital Act; (Cirkulaer om udbetaling af statstilskud i 2004 til \u00f8get aktivitet p\u00e5 sygehusomr\u00e5det CIR 6, 5 January 2004. Bekendtg\u00f8relse om \u00f8konomiske rammer for frit valg til private specialsygehuse, BEK 627, 20 June 2004.). Financial restrictions are imposed on the counties as their obligation to finance care at certain private hospitals including hospices, sclerosis hospitals, arthritis sanatoria, and brain rehabilitation hospitals is limited to a certain annual amount. Thus patients are either referred to a waiting list or must pay for the treatment themselves at a private clinic. At county level the counties determine the content and costs of hospital activity through the use of detailed budgets, enabling them to specify treatments offered, technologies used, service standards, and capacity available. There is an indirect regulation of benefits available to county inhabitants through the control of the supply. In principle, the county can decide not to offer a certain treatment, for example, in vitro fertilization treatment to women who have already had one child by this means, or an expensive cancer treatment. However, the patient can choose to be treated in another county due to the right of free choice of hospital with the home county having to pay for it, which actually displays certain limitations to the county\u2019s planning freedom. In the hospital sector there are no benefit \u201ccatalogues\u201d as such. However, a wide number of classifications are used to document the activity and prices based on diagnosis-related groups (DRG). DRGs are used for the remuneration of hospital services provided in other counties or at private hospitals.\nOutpatient services: the Health Care Reimbursement Scheme\nUnlike hospital services, benefits regarding outpatient services are under the HCRS explicitly and specifically detailed by the law and in a fee schedule negotiated on services and prices between the private providers and the HCRNC. The PHIA states that primary health care is available to (almost) everyone with a permanent address in Denmark. All citizens must choose between insurance groups I and II with a trade-off in terms of free choice of the provider and the size of reimbursement. Persons in group I, more than 98% of the population, are entitled to free services from GPs and to partial reimbursement of dentist services, physiotherapy treatment, etc. With a few exceptions public reimbursement of specialist and paramedic services are subject to GP referral. Persons in group II, fewer than 2% of the population, have a wider choice of providers, but are less entitled to reimbursement. Everyone receives a personal card clearly stating the entitlements to the services (PHIA; Bekendtg\u00f8relse om valgfri indplacering i sygesikringsgrupper, udstedelse af sygesikringsbevis mv, BEK 198, 21 March 2003; Bekendtg\u00f8relse om begraensning i adgang til ydelser efter sygesikringsloven for visse persongrupper, BEK 119, 17 March 1976, og BEK 115 af 21 February 1990). A large number of executive orders and the fee schedule for reimbursement of private providers further specify entitlements and benefits laid down by the PHIA (PHIA; Bekendtg\u00f8relse om befordringsgodtg\u00f8relse efter sygesikringsloven, BEK 3, 3 January 2001; Bekendtg\u00f8relse om tilskud efter sygesikringsloven til fodbehandling, BEK 129, 18 March 2003; Bekendtg\u00f8relse om tilskud efter sygesikringsloven til kiropraktisk behandling, BEK 181, 22 March 2004; Bekendtg\u00f8relse om tilskud efter sygesikringsloven til fysioterapeutisk behandling, BEK 405, 18 May 2001; Bekendtg\u00f8relse om tilskud efter sygesikringsloven til psykologbehandling for saerligt udsatte grupper, BEK 472, 18 June 2002; Bekendtg\u00f8relse om tilskud efter sygesikringsloven til sondeernaering og andre ernaeringspraeparater, BEK 531, 18 June 2003; Bekendtg\u00f8relse om tilskud efter den offentlige sygesikring til betaling af briller til b\u00f8rn under 16 \u00e5r, BEK 543, 8 December 1980; Bekendtg\u00f8relse af lov om tandpleje, LBK 1261, 15 December 2003, og BEK 1073, 11 December 2003; Bekendtg\u00f8relse om tilskud efter sygesikringsloven til tandpleje, BEK 147, 6 March 2004; Bekendtg\u00f8relse om adgang til laegehjaelp efter sygesikringsloven, BEK 180, 18 March 2003).\nSome benefits are defined very explicitly, e.g., a specific maximum amount in Danish crowns to be covered for spectacle lenses and spectacle frames; (BEK 543, 8 Decemberr 1980, see above), while others are defined in more general or vague terms, e.g., proportional reimbursement of negotiated fee for physiotherapy treatment (PHIA; BEK 405, 18 May 2001, see above), or proportional reimbursement of negotiated rates for dental treatment (LBK 1261, 15 December 2003; og BEK 1073, 11 December 2003, see above; BEK 147, 6 March 2004, see above). There are positively defined benefits (e.g., the right to spectacle lenses and frames) and exclusions through negatively defined benefits, e.g., a service excluded from reimbursement from the public travel insurance benefits (Bekendtg\u00f8relse om befordringsgodtg\u00f8relse efter sygesikringsloven, BEK 3, 3 January 2001). While, for example, GP services, ear nose and throat medicine, psychiatric, and oculist services are free of charge for patients, dentist services are only partly covered by public funds. Some services are regulated by legislation but not included in the benefit basket, unless certain specific criteria are fulfilled (e.g., psychologist counseling, chiropody, and chiropractic services (BEK 129, af 18 March 2003, see above; BEK 181, af 22 March 2004, see above; BEK 472, 18 June 2002, see above). Table\u00a02 provides an overview of the main services under the HCRS, showing the extent and criteria for coverage and whether the services are subject to a referral from the GP.Table\u00a02 Benefits regarding outpatient services according to the Health Care Reimbursement Scheme. Services fully, partially or not covered by public fundsType of service\/providerCoverage by public fundsCoverage for special groupsReferral from GP required (Group I insured)General practiceFull\u2013\u2013Dentist servicesPartialFull coverage for children at school age, disabled, low income elderlyNoTube feeding\/nutritional preparationsFull\u2013YesPhysiotherapyPartialFull coverage for patients with specific diagnoses (muscular-skeletal) YesPsychiatristFull\u2013YesPsychologistNone\/(partial)Partial coverage for persons exposed to traumatic incidentsYesChiropodistPartialPartial coverage for diabetics and low-income elderlyYesChiropractorPartial\u2013YesOpthalmologistsFull\u2013NoEar-nose-throat physiciansFull\u2013NoDieticiansNone\u2013NoOther specialistsFull\u2013Yes\n\u201cOther specialists\u201d whose services are fully part of the benefit basket upon GP referral include: anesthetists, specialists in dermatology and venereal diseases, specialists in diagnostic radiology, gynecologists, obstetricians, internists, surgeons, biochemists, neurologists, orthopedics, specialists in pathological anatomy or plastic surgery, pediatricians, rheumatologists, and specialists in tropical medicine or laboratory examinations.\nThe fee schedule\nWhereas the laws describe mainly the extent to which outpatient services are included in the benefit basket, for example, the proportion of public funding and the criteria for coverage, the fee schedule negotiated between the HCRNC and the health personnel trade unions specifies the services for which the providers are paid. Since the schedule lists services which are reimbursed from public funds and are therefore provided free of charge to the patients, the fee schedule may\u2014in an indirect way\u2014be regarded as a benefit catalogue (Sygesikringens Forhandlingsudvalg, takstkatalog, April 2005).\nItems in the fee schedule are negotiated every 3\u00a0years while the tariffs are revised twice a year. Health technology assessments, cost-effectiveness analyses, and clinical studies are adduced by the professional committees and included in the negotiation but they are not mandatory. Criteria for the decisions concerning the inclusion or the exclusion of an item are neither systematic nor public. An example of an argument over the inclusion of services regards progress in treatment methods (substitution of outdated services with more up-to-date procedures). There is usually a reimbursement rate when benefits such as those for contraceptive advice from the GP are specifically stated by the law (A. Bonne, HCRNC, personal communication).\nThe catalogue is divided into specialties with a subdivision into type of services offered (items). Items may be directly linked to the legislation that specifies the benefits, for example, antenatal care consultation and first child health check-up at the age of 5\u00a0weeks (general practice). They are also directly linked to the logic applied by the legislation, for example, following certain age groups, certain timing (week 12, first and second consultations, etc.), or specific agreement, such as on working hours or transport fees for the professional group involved. When the legislation does not specify the benefits, items are either linked to consultation type (visits), goods (e.g., laboratory tests) procedures (e.g., spirometry) or even indications (e.g., podiatry for diabetics) in some rare cases. There is generally no specification of the technology to be used with a few exceptions.\nConcluding remarks\nThe Danish benefit basket for curative services is regulated directly by law and influenced indirectly by more \u201csoft\u201d regulatory mechanisms. In the hospital sector which is the main part of the health sector in terms of expenditure, the law delegates the responsibility for providing, financing, organizing, and planning services to the 14 counties. Thus there may be regional variations in the services offered. Benefits are explicitly defined but not specified by the law. In principle, all clinically indicated hospital services are included in the benefit basket. However, central initiatives, such as legislation on the patient\u2019s free choice of hospital, waiting time guarantees for some patient groups and financial constraints influence the counties\u2019 decisions and narrow the room for regional variation. Regarding outpatient services the picture differs slightly as benefits in the HCRS are regulated at central level through explicit and specific legislation while being further specified in agreements about items and tariffs between county and provider representatives.\nIn 2007 the Danish health sector will be subject to a structural reform which will significantly influence the way in which health services are financed and organized. The current political layer of 14 counties will be abolished and be replaced by five regions responsible for providing hospital services but without the taxation rights that they have today. The municipalities will merge into fewer units, assume financing responsibilities for more health services than today and will purchase hospital services from the regions.\nThe reform\u2019s immediate impact on health benefits is hard to predict, but there will be some modification as to legislation at least. A law, \u201cthe Health Act\u201d (Sundhedsloven, LOV nr. 546, 24 June 2005), recently passed by the Folketing, collects the previous vast amount of legislation on health care into a single document. Among other things the goals of Danish health care services, for example, that citizens should have easy and equal access to health care, have now been made explicit. The rights to health care services do not change fundamentally, nor do decision makers and their roles and responsibilities. The Act contains many clauses empowering the Minister for the Interior and Health to set specific rules, thus giving the Minister much more decision-making power than today. However, the Health Act provides a regulatory framework with explicitly mentioned rights to health services not being different from what is outlined here. The specification of benefits will still take place at the lower levels of decision making, by specifications of the law by the Minister, by the National Board of Health, in agreements between regions and municipalities, through a payment schedule, or at clinical level.","keyphrases":["denmark","health services","health benefit plans","health priorities","national health programs"],"prmu":["P","P","R","R","R"]}
{"id":"J_Comput_Aided_Mol_Des-4-1-2270916","title":"How to do an evaluation: pitfalls and traps\n","text":"The recent literature is replete with papers evaluating computational tools (often those operating on 3D structures) for their performance in a certain set of tasks. Most commonly these papers compare a number of docking tools for their performance in cognate re-docking (pose prediction) and\/or virtual screening. Related papers have been published on ligand-based tools: pose prediction by conformer generators and virtual screening using a variety of ligand-based approaches. The reliability of these comparisons is critically affected by a number of factors usually ignored by the authors, including bias in the datasets used in virtual screening, the metrics used to assess performance in virtual screening and pose prediction and errors in crystal structures used.\nIntroduction\nBased on the large number of papers recently published, it has become obvious that a large proportion of the computational chemistry community, both in academia and in industry, is very interested in evaluating and comparing software for a number of different purposes. A large number of publications have appeared over the last 5\u00a0years or so that are focused on the evaluation of docking tools for pose prediction [1], virtual screening [2] and affinity prediction [3]. There have also been a number of recent publications examining the performance of ligand-based tools in similar tasks. The ligand-based tools have also been evaluated in the areas of pose reproduction (by conformer generators [4\u20136]), virtual screening [7] and affinity prediction [8]. In the following sections some issues with studies on pose prediction and virtual screening will be discussed.\nPose prediction\nA common method of evaluating a docking program is to gauge its performance in cognate re-docking or self-docking. In this process a ligand is extracted from a co-crystal structure with its target protein and the program is challenged to pose the ligand as closely as possible to its experimentally identified structure. It may be argued that cognate re-docking is not a task commonly faced in the normal use of docking tools, since cross-docking (docking of a ligand into a structure with which it was not crystallised) is the actual application of a docking tool [9]. However, the exercise remains popular, doubtless in part due to the relative ease of execution of a self-docking study and partly for comparison with previous studies. As has been pointed out previously [10], comparing docking programs for their ability to predict the bioactive pose of a ligand is difficult for a number of reasons, some of which are obvious while others are subtler. However, the operational difficulties in comparing programs in a robust way should not cause other sources of error to be ignored. It is a truism that it is meaningless to compute a property with greater precision than the accuracy of the experiment that measures that property. Unfortunately, as will be seen in subsequent sections, this is often ignored by authors of papers in the area of pose prediction.\nWhen comparing tools for pose prediction, the heavy atom root mean square deviation (RMSD) between the computed and experimental poses is the de\u00a0facto standard. A regrettably common method to compare docking tools for pose prediction success\u2013\u2013illustrated, for example, in the papers presenting results from the MolDock program [11] or the Glide XP evaluation [8]\u2013\u2013is to compare the average RMSDs across a set of structures. The use of the average RMSD admits of many possible problems of interpretation, not least of which is the biasing of the average by a few very large or very small numbers. In the case of the MolDock results, the mean RMSD across a set of structures was used to suggest that the performance of MolDock was comparable to that of GLIDE and superior to that of Surflex (see Table\u00a01). However, the use of the median RMSD, which is far less biased by a few extrema, suggests a different conclusion\u2014that MolDock is in fact somewhat superior to Surflex, but not as good as GLIDE. For a similar analysis on the drawbacks of using average RMSD as a comparator, see Cole et\u00a0al. [10]. A number that is noticeably absent from this, and all other comparisons of docking tools using RMSD, is an error bar on the average RMSD (which can be calculated by bootstrapping). Without such an error bar it is impossible to assert that any tool compared in this study is actually better than any other.\nTable\u00a01Comparison of RMSD results from a set of docking enginesMolDockGLIDESurflexMean1.381.381.86SD1.491.742.02Median0.920.691.10\nA separate issue with these two experiments involves contamination of the dataset used to evaluate the performance of the tools. In the case of the Glide results the RMSD\u2019s given are not to the deposited crystallographic pose but rather to one that results from a pre-processing step, as noted by the authors of the MolDock paper [11]. As such this is not an \u201capples to apples\u201d comparison, since the Glide pre-processing step optimizes protein and ligand coordinates using the force-field component of the Glide scoring function, which necessarily introduces bias in the structure. In the MolDock case the authors have essentially trained the MolDock fitness function on the 77 complexes that they use to evaluate its performance. As such the reported results give no indication of the likelihood of success in predicting a pose for a system upon which MolDock was not trained.\nThe RMSD between two poses is a geometric measure, comparing the atomic positions between the experimental structure and the docked or predicted structure. Other metrics based on comparing the geometry of the experimental and the computed pose have been developed, such as relative displacement error (RDE) [12]. These, and all other atom-based metrics, suffer from the drawbacks pointed out by Cole et\u00a0al. [10]. A more serious problem for metrics like RMSD and RDE is that they attempt to indicate the quality of reproduction of a model for the data, not the crystallographic data itself, i.e., the electron density. This disconnection between RMSD as a metric of quality for pose prediction and the original crystallographic data has been a cause for concern. Some attempts have been made to arrive at metrics that better reflect the reproduction of the actual crystallographic data, in particular real-space refinement (RSR) [13]. Unfortunately, RSR has not been widely used, possibly because it is more difficult to calculate than atom-based metrics like RMSD or RDE. Other metrics that are not based purely on atom position, such as interaction-based accuracy classification (IBAC) [14], try to reflect the ultimate use of the predicted pose, i.e., determining the nature of the interactions that the ligand makes with the protein. While the IBAC approach has value in that it assesses a computed pose by its interactions with the protein, it is not amenable to automation; it is therefore tedious to assemble sufficient data to make statistically robust comparisons between tools based on IBAC.\nHowever, the problem of choosing which metric to use to compare pose prediction studies is dwarfed by the difficulty in choosing a dataset of protein\u2013ligand co-complexes upon which to perform the comparison. A widespread tendency in conformer reproduction and pose prediction studies is to ignore even the possibility of error in the crystal structures that are being reproduced. Crystal structures are often treated as perfect, infinitely precise and accurate representations of the atomic details of a protein\u2013ligand complex. There are a number of reasons why this is not so; a few will be discussed in the following paragraphs.\nCrystal structures are models\nThis statement makes up the warp and weft of crystallography, yet in the transition of crystallographic data from crystallographer to computational chemist the distinction between the actual data and a model for that data is often lost. The actual data in crystallography is, of course, diffraction data leading to electron density. The atom positions that make up a crystal structure are a model that attempts to explain this data in as complete a fashion as possible. This is illustrated in Fig.\u00a01.\nFig.\u00a01Fitting atoms into electron density produces a crystallographic model\nThe process of fitting or modeling the atoms (of a ligand or of a protein) into the electron density is not always straightforward, and the problems are often more serious when the fitting of a ligand into density is being carried out. The sources of these problems include:Incomplete or fragmentary density;The electron density not defining the positions of all atoms unambiguously;Poor structural parameters are used for the fitting process, which can give inappropriate conformations (particularly of ligands);Errors by the users, arising from careless treatment of the data or lack of expertise with small molecules.\nA variety of metrics can be calculated for a given crystal structure that attempts to give an indication of the quality of the model. While no single number can encompass the quality of a structure, some of the metrics of quality are more useful than others. The most commonly used is the nominal resolution. This is a measure only of the quantity (of data collected) and not of the quality of the data nor, most especially, of the quality of the model fitted to that data. (For a discussion on the problems with resolution, see Ref. [15]) It is therefore unfortunate that nominal resolution is often the only criterion used to select a protein structure, and that its meaning is often misconstrued. For example, in a paper [16] by Nissink et\u00a0al. the following incorrect statement is made: \u201cThe resolution of a protein structure is directly related to the accuracy of the data.\u201d Not only is this statement not entirely true, it also confuses what the nominal resolution means (quantity of data collected) with the accuracy of a model, which are two very different things. A metric that does attempt to assess the quality of the fit of the crystallographic model to the source data is the so-called Rfree, introduced by Brunger [17]. Rfree is an indication of how well an atomic model explains a small percentage of the density data that was omitted during the fitting process, and is thus an unbiased metric that can be reliably used to distinguish a well fitted model from a poorly fitted model. Unfortunately, Rfree is infrequently used as a metric of quality when selecting crystal structures for docking studies or other purposes (see Ref. [18] for the use of Rfree as a criterion for selection of structures). More frequent use of Rfree as one of a set of metrics for selection of crystal structures could help to avoid the selection of poorly fitted models for pose reproduction.\nCrystal structures have unavoidable imprecision\nAs already discussed, nominal resolution is not a metric of quality for a structure and although Rfree indicates the quality of the fit of the model to the original data it can provide no estimate of the uncertainty in the atomic positions within that model. It is often assumed that the experimental uncertainty in the atomic positions in a crystallographic model can be estimated by use of the isotropic B-factor, which is supposedly a measure of the thermally driven fluctuation in atomic positions. However B-factors are a refined parameter and so they cannot be compared between structures without detailed knowledge of the restraints used [19]. It is therefore impermissible to use low B-factors as a sure indication of low positional uncertainty. Nor is it possible that there exists a uniform cut-off for B-factors that indicates low positional uncertainty in all structures, though such an assumption is frequently made [20]. Another potential problem with B-factors is that they can be over-refined in the pursuit of better quality metrics for a structure. An example is given in Fig.\u00a02, which shows the ligand (leucinol) from the structure 5ER1 from the RCSB [21] annotated with its B-factors. The B-factors for every atom in the ligand are <1. In contrast, B-factors even for well-located atoms in high quality models, are generally not <5 [22]. In this instance the crystallographer has over-refined the B-factors of the ligand atoms in order to improve the quality of the overall model. In this model the B-factors are unphysically low only as a consequence of a pathology of the refinement process. Accordingly, B-factors as an indication of local mobility in a protein structure should be treated with caution, as they are not simply an experimentally derived quantity but are free parameters in the refinement process.\nFig.\u00a02B-factors for the ligand in the 5ER1 crystal structure\nAn alternative to using the average B-factor for estimating the uncertainty of the position of atoms an atom in a structure is the average coordinate precision (or diffraction-component precision index, DPI). The DPI expresses the average precision for the atomic coordinates in a protein structure [23] and as such can be used as a measure of the experimental precision of the atomic positions in that structure. The original formulation of DPI was very complex and has recently been recast in a more easily calculable way by Blow [24]. The use of DPI as a metric of quality for crystal structures used for docking studies was introduced to the computational community by Goto et\u00a0al. [18], and their formula is given in Eq.\u00a011.\nIn this treatment the standard error of position, \u03c3(r), is related to the number of atoms in the unit cell, Natoms, the volume of the unit cell, Va, the number of crystallographic observations, nobs and the Rfree. It should be noted that the formula presented by Goto et\u00a0al. is not precisely the same formula that Blow derives in his paper. In Eq.\u00a01 the prefactor is given as 2.2, while in the original work by Blow the prefactor is given as 1.28. This is because Blow is calculating coordinate error for a particular axis, \u03c3(x, y, or z), while Goto et\u00a0al. are calculating the error in the distance, \u03c3(r), giving rise to the \u221a3 difference between the two prefactors. As such it is appropriate to consider the error in the coordinates, \u03c3(x, y, z) as a measure of the uncertainty in the atomic positions, and the error in inter-atomic distances, \u03c3(r), when comparing a computed and an experimental structure.\nThe resolution of a crystallographic model, as has been mentioned, is often used to select protein structures for pose prediction by docking or conformer generation studies, on the assumption that resolution imparts some information on the quality and precision of the model. The DPI is a much more direct estimate of the reliability of crystallographic models when it comes to comparing experimental and computed atom positions (as is done in conformer reproduction or pose prediction). It is therefore of interest to compare the nominal resolution for a large number of \u201cgood quality\u201d crystal structures with the DPI (\u03c3(r)) for the same structures. A good dataset for this comparison is provided by the extensive investigations performed by Kirchmair et\u00a0al. [4]. Here 776 co-crystal structures were used to provide experimental ligand structures that were then compared to sets of computed conformations from conformer generation applications. For 556 of these crystal structures there exists sufficient data to allow the DPI to be calculated and the relationship between the nominal resolution for these structures and their DPI is shown in Fig.\u00a03. It is obvious from Fig.\u00a03 that the statement by Kirchmair that \u201c0.5\u00a0\u00c5 approximately represents the accuracy of protein X-ray crystallography\u201d is not supported by the actual properties of the crystal structures they studied. In fact, in those cases where the DPI can be calculated, almost 56% of the structures from their paper have DPIs\u00a0>\u00a00.5\u00a0\u00c5.Fig.\u00a03Coordinate error for 556 structures from the paper by Kirchmair et\u00a0al. [4]\nFigure\u00a03 illustrates a number of other interesting points. While the expectation that greater coordinate precision will arise from structures with better nominal resolution is generally borne out by the data, there are many exceptions. Table\u00a02 shows some examples of structures where the nominal resolution gives an unexpected estimation of coordinate precision. In the top half of the table are structures with good nominal resolution but unexpectedly high DPI, while the lower half of the table shows some structures with low nominal resolution and either unexpectedly low or unexpectedly high DPIs. Accordingly, simply using nominal resolution as a metric of quality for structures to be used in a pose prediction or conformer generation study is insufficient to guarantee that structures of appropriate quality will be used.Table\u00a02Resolution and DPI for selected structures from the Kirchmair datasetPDB codeResolution (\u00c5)DPI (\u00c5)1FC71.380.691FDO1.380.601JJT1.81.371JJE1.81.251CIB2.55.541ILH2.760.141C8M2.80.181QJX2.80.25\nWith the DPI for a structure in hand one can set a lower limit on the precision with which a computed conformation can reproduce an experimental one\u2014the RMSD between the two conformations cannot be less than the DPI for the experimental structure. It can be seen that over half (55.6%) of the structures in this set has DPIs\u00a0>\u00a00.5\u00a0\u00c5, while Kirchmair et\u00a0al. report pose reproduction statistics both at <0.1\u00a0\u00c5 and at <0.5\u00a0\u00c5 RMSD. Since in over half of the structures in this dataset the DPI is >0.5\u00a0\u00c5, Kirchmair et\u00a0al. report pose reproductions at <0.5\u00a0\u00c5 RMSD that are more precise than the accuracy of the source data allows. This analysis is made on the conservative assumption that the error in the atomic positions in the computed pose is zero. In the Goto et\u00a0al. paper [18] the assumption is made that the errors in the computed pose are the same as for the experimental pose. In this analysis a computed and experimental pose must be different by an RMSD of \u221a2\u00a0\u00d7\u00a0DPI for the difference to be significant, which would mean that an even higher proportion of the poses in the Kirchmair set have been reproduced with a greater precision than the experimental accuracy.\nThe same tendency to reproduce experimental data with a precision greater than the experimental accuracy is seen frequently in pose prediction experiments with docking engines. In Fig.\u00a04 nominal resolution is plotted against the DPI for crystal structures from two well known docking validation sets, those for GOLD [25] and GLIDE [3]. The two graphs are plotted on the same scale to allow direct comparison. On each graph is also plotted an estimate of the theoretical lower limit for the atomic precision (the pink line). A formula describing the relationship between nominal resolution and DPI is given in Eq.\u00a022 below, based on a derivation by Blow [24].Fig.\u00a04The nominal resolution versus the coordinate error for a subset of the Gold (structures with resolution <2.5\u00a0\u00c5) and the Glide data sets\nThe variables found in Eq.\u00a02 are as follows: s is the percent solvent present in the crystal, Vm is the asymmetric unit volume to molecular weight ratio, C is the completeness of the data, and dmin is the nominal resolution of the structure. The ideal lines shown in Fig.\u00a04 were calculated using Eq.\u00a02 and assuming an s of 0.0, a Vm of 2.5, a C of 100%, and that Rfree is equal to the resolution\/10.\nInspection of Fig.\u00a04 shows that the GOLD set contains one structure (1YEE) whose calculated DPI lies below the theoretical lower limit. This is probably due to a mistranscription error in the PDB file. The PDB file for the 1YEE structure gives the number of reflections as 77209, while the number of unique reflections is 21342\u2014a disparity that cannot be reconciled by reference to the data redundancy for this structure. Accordingly the calculated DPI for 1YEE is too low because the reported number of reflections is erroneously high. In the GLIDE set all structures have DPIs higher than our predicted theoretical minimum. In fact, in this set 52% of the structures have a DPI\u00a0>\u00a00.5\u00a0\u00c5, and in 31% of the cases the reported RMSD for redocking is less than the DPI (so that the prediction is more precise than experimental accuracy allows). This is only one example of a publication in which protein structures are assumed to be free of uncertainty in their atomic positions; the literature abounds with others.\nThe combination of Rfree and the DPI for a structure can give a good overall picture of the quality of a model and the reliability of the atom positions within that model. In spite of the availability of these and other measures of quality, there are a number of test sets of protein co-crystal structures used for evaluating docking engines that appear to have been selected by other criteria that do not relate in any way to their quality [26]. Accordingly, the results from these studies should be treated with some caution.\nCrystal structures have avoidable errors\nThe Rfree and the DPI are global measures of quality; other, local, measures of quality are also useful. For example, if a small portion of the atoms in a structure have been poorly fitted to the density, this will not be revealed by any global measure of fit. Only a local measure would reveal the error. A relatively common problem in co-crystal structures in the PDB is poor fitting of small molecules to the density, giving unrealistic ligand structures. These poorly fitted ligand structures are then used as a \u201cstandard\u201d to judge the quality of a docking program\u2019s or conformer generator\u2019s performance. While the poor quality of some ligand structures in PDB models has been known for some time [27], these reports have been anecdotal and few systematic attempts to avoid such poorly solved structures (other than visual inspection) appear to have been undertaken. If appropriate attention is not paid to selecting good quality ligand structures, then a dataset could be constructed that contains ligand structures that have significant errors. For pose prediction studies, poorly solved ligand structures in the dataset must be avoided. It is senseless to try to computationally reproduce an \u201cexperimental\u201d ligand structure that has been incorrectly fitted to the electron density. Examples of some possible errors in ligand structures that should result in structures that are not computationally reproducible are shown in Figs.\u00a05 and 6.Fig.\u00a05Ligand conformation from 1A8T structure. The conformation has two serious atom\u2013atom clashesFig.\u00a06Ligand conformation from the 1A4K structure. The cis-amide group is an error of fitting\nIn the case of the 1A8T structure (Fig.\u00a05), the deposited ligand coordinates contain two serious atom\u2013atom clashes that give the resulting conformation very high energy (28.4\u00a0kcal\/mol above a refitted structure by the MMFF94 forcefield [28]). The deposited coordinates are clearly in error in this case and no docking engine or conformer generator should be expected to reproduce such a structure.\nThe error shown in Fig.\u00a06 for the 1A4k structure is of a more subtle nature. Here the crystallographer has fitted a highly strained cis-amide into the electron density with no compelling reason from the electron density to do so. The amide group is packing against a tyrosine residue from a symmetry mate in the unit cell, and makes no polar interaction with it. The corresponding trans-amide, which fits the experimental density just as well, is 15.5\u00a0kcal\/mol lower in energy (using the MMFF94 force-field). Also this trans amide is able to make a hydrogen bond (with a backbone carbonyl group) that is not available to the cis conformation. Once again, this model should not be reproduced by a docking engine or conformer generator, as it is obviously not the correct solution.\nOne of the only studies that bears on the issue of ligand strain was published by Perola and Charifson in 2004 [29]. The authors examined ligand strain in a number of public structures from the RCSB database and proprietary structures from Vertex internal collection and found that 10% of the ligand structures examined had high strain (10\u00a0kcal\/mol or greater above the global minimum). The two examples discussed above, 1A8T and 1A4K, are both part of the Perola dataset. Clearly these two ligand structures have high strain energies due to errors in fitting and not due to a fundamental property of the ligand\u2019s conformation in complex with the protein. Close examination of the rest of the models for the ligands (where structure factors are available) showed that a number of them are incorrect, and re-solving them provided lower energy structures in about 85% of the cases [30]. Recent re-examination of the Perola dataset by the Snyder group has provided further insights into the large strain energies originally reported [31]. It is therefore most likely that the vast majority of ligand conformations in complex with a protein show strain energies less than 6\u00a0kcal\/mol, unless the ligand is rather large [32]. Those ligand conformations with strain energies higher than 10\u00a0kcal\/mol are almost certainly incorrect. As such low ligand strain should be among the criteria for selection of structures for pose prediction, as structures with high strain are very likely to arise from errors in the fitting process.\nIt is clear from the foregoing discussion that, while crystal structures are an invaluable source of information on protein\u2013ligand binding, these structures are not without many sources of confounding errors. These errors, those inherent to the data and the process of fitting, as well as those introduced by human error or the insufficiencies of the fitting program, should be borne in mind before using crystal structure data. The selection of a reliable set of structures for pose prediction is a therefore not a trivial task. An excellent study considering these issues can be found in Hartshorn et\u00a0al. [33].\nVirtual screening\nVirtual screening can be defined as any method that ranks a set of compounds by some score. Successful virtual screening relies on having a scoring method that assigns good scores to interesting molecules (usually defined as active against a target protein of interest) and worse scores to uninteresting (inactive) molecules. Accordingly a successful virtual screen will provide, from the top of this ranked list, a set of compounds for experimental screening that is highly enriched in active molecules. This topic has been of great interest both in academia and in the pharmaceutical industry in recent years, and a large number of publications have appeared on the subject. While a few of the publications have investigated virtual screening conducted prospectively [34\u201336], the vast majority have been concentrated in the area of retrospective virtual screening. In the rest of this article we shall concern ourselves solely with the retrospective experiments. The goal of such experiments is often to identify an application that performs well on a given target, or across a wide range of targets, with a view to utilizing this application in prospective virtual screens.\nThere are a number of approaches to quantitating the success of a particular tool for virtual screening. The most often used, and simplest to calculate, is enrichment at a given percentage of the database screened. Enrichment (EF) is defined according to Eq.\u00a033 (Hitssampledx%\u00a0=\u00a0number of hits found at x% of the database screened, Nsampledx%\u00a0=\u00a0number of compounds screened at x% of the database, Hitstotal\u00a0=\u00a0number of actives in entire database, Ntotal\u00a0=\u00a0number of compounds in entire database).\nEnrichment appears to measure the quantity of most interest to those performing virtual screening: the ability of a tool to place a large proportion of the active compounds at the top of the ranked list. Enrichment is also simple to calculate and understand, so it seems the ideal metric with which to compare tools for their virtual screening performance. However, enrichment suffers from a number of significant drawbacks, especially when comparing results between studies or using enrichment to predict future performance:It is dependent on the structure of the dataset, in that datasets with larger proportions of actives will have a narrower range of possible enrichments.It penalizes ranking one active compound above another.It exhibits pernicious behaviour at the cut-off at which the enrichment is calculated.It gives no weight to where in the ranked list a known active compound appears. Thus to calculate enrichment at 1% in a virtual screen of 10,000 compounds, the number of actives (N) in the top ranked 100 compounds is needed. However the enrichment at 1% is the same whether the N active compounds are ranked at the very top of the list or at the very bottom of the top ranked 100.It is difficult to calculate analytically errors in enrichment, and there is no available literature for such a calculation.\nWith regard to point (i), experiments performed on different datasets cannot be compared when using enrichment, as the dynamic range of enrichment will be different for different datasets, but there are several cases where this has been done [37]. Early enrichment can also suggest overly impressive performance for tools that rank a small fraction of the active molecules very early in the list, but fail to give good ranks to the majority of the actives [37]. It has been shown by Seifert [38] that enrichment does not detect significant pathologies in the ranking function. Other metrics have been developed by some groups specifically to address some of these problems: RIE by Merck [39], cumulative probability by Molsoft [40] and average number of outranking decoys by Schrodinger [3]. These metrics have, historically, only been used by the groups that invented them and are thus not useful for comparison to studies conducted by other groups. This lack of direct comparability across studies is compounded by the fact that these metrics cannot be converted into a more commonly used metric that could be used to compare results. Also, as with enrichment, it is unknown how to estimate analytically errors in any of these metrics.\nA metric used to determine success in detecting a signal in a background of noise is the receiver operator characteristic (ROC). The ROC curve is derived by plotting noise (fraction of false positives) on the x-axis versus signal (fraction of true positives) on the y-axis. The area under the ROC curve (AUC) is a widely used measure in a variety of fields including medical statistics, criminology and bioinformatics [41, 42]. When applied to virtual screening the ROC illustrates success in ranking actives (signal) above decoys (noise). The AUC for the ROC curve shows performance of a given tool when screening across the entire database is examined, not just at fixed, early points in the screen as enrichment does. The theoretically perfect performance of a virtual screening application gives the maximum area under a ROC curve (1.0), while random performance of a tool gives an AUC of 0.5. Areas under the curve of less than 0.5 imply a systematic ranking of decoys higher than known actives. For recent applications of the ROC curve in virtual screening, see [2, 43]. The AUC for the ROC curve is also known as the \u2018discrimination\u2019. Discrimination is defined as the fraction of occurrences that a randomly chosen true positive (active) is given a better score than a randomly chosen true negative (decoy). This number then allows prediction of the likely effectiveness of a tool in experiments that have not yet been conducted. This predictive ability is not provided by metrics such as enrichment, cumulative probability and average number of outranking decoys, because while the ROC describes a property of the application studied, the other metrics essentially describe a property of the experiment. The AUC assesses virtual screening performance across the entire database and many practitioners of virtual screening are, rightly, most concerned about early performance of the tools they use. This is one reason why enrichment is so commonly used to measure success. The metric of early performance based on the ROC curve is the true positive rate at fixed false positive rates. The true positive rate at a false positive rate of, for example, 1% is a much more robust measure than the enrichment at 1% and provides similar information about the early performance of a tool.\nThe AUC also offers the advantage that a statistically robust estimate of its errors can be estimated analytically from the AUC itself, using a method developed by Hanley [41]. This is not a property possessed by enrichment, average number of outranking decoys etc. For these metrics errors can only be estimated (by bootstrapping or other approaches) from the raw data, which is rarely provided. The error in the AUC, as with other metrics, is reduced by increasing the number of positives (active compounds) and by increasing the number of negatives (inactives). The Hanley treatment shows that the error in the AUC is most significantly reduced by increasing the number of actives, while increasing the number of decoys has a much smaller impact on the error. Therefore virtual screening datasets with high proportions of active compounds will provide results with lower error bars for the AUC. With the error for an AUC available, meaningful comparisons can be made between two or more different tools. However the other metrics used for virtual screening mentioned above do not allow an analytical estimation of their errors. Comparisons designed to determine which tool is superior for a given purpose, that are based on metrics assumed to be free of errors, are fraught with difficulty.\nWhile the choice of metric may affect the relative ranking of tools compared on the same datasets, the composition of those datasets has a very profound effect on the results generated. Until very recently it has been common practice to assemble a dataset for virtual screening by seeding a set of active molecules against a target of interest into a background of compounds (decoys) chosen essentially at random. These decoys compounds were often drawn from public sources such as vendor catalogues. An example of this approach is the seminal paper on virtual screening by docking from the Rognan lab where decoy compounds were selected at random from the publicly available compounds [44]. This set of decoys, or a subset thereof, has been used extensively since its publication in 2000 [2 and references therein] so that this same set of decoy compounds has been used in more than 45 different published virtual screening experiments on a wide variety of target systems. Given the huge variety in the types of active molecules camouflaged in this same set of decoys it seems intuitively obvious that in some cases the active compounds for a given target will be very easily discriminated from these decoys. As such a number of the virtual screening experiments performed using this set of decoy compounds have given good results purely due to differences in simple properties between the actives and the decoys (vide infra). In the Rognan dataset there were 10 active compounds for each of the targets studied and 990 randomly selected decoys. There are two problems arising from constructing a dataset in this way. The first is that the small number of active compounds means that the errors can be very high (vide supra). The second is that trivial property differences between the active compounds and the decoys can result in undeservedly good performance. The first issue, low prevalence, is still widespread in retrospective virtual screening studies. Although low prevalence reduces the reliability of the results, many virtual screening experiments are still conducted using very low numbers of active compounds. Reasons for this could include a desire to mimic \u201creal\u201d HTS experiments, where hit rates are often on the order of 0.01\u20130.1% [45] or to allow enrichment, the most commonly used metric for success in these studies, the maximal dynamic range. Tribelleau et\u00a0al. [43] show that the dynamic range of enrichment, the difference between random and maximal performance, decreases as the proportion of active molecules in a dataset rises. For a recent example of a retrospective virtual screening study with deliberately large numbers of actives, providing high statistical power and small error bars, see Ref. [37].\nThe second issue, systematic differences in simple properties between decoys and actives in retrospective virtual screening experiments, is much more serious. As has been pointed out in a number of publications, scoring functions in docking programs, which are almost always additive, are sensitive to molecular size or heavy atom count, the number of hydrogen bonds that the molecule can make etc. Accordingly, systematic differences in these simple molecular properties between actives and decoys will cause systematic differences in ranking. For example, active compounds with higher average heavy atom counts will tend to rank better than the decoys when scored by a function that is sensitive to heavy atom count. For a fuller discussion of this issue see Verdonk et\u00a0al. [46]. An example of the influence of the selection methods for decoys on virtual screening performance is shown in Fig.\u00a07. In this figure, two retrospective virtual screening studies against CDK-2 using the docking tool FRED [47] are compared using ROC curves. In both cases the same actives were docked against the same co-crystal structure, while different decoy compounds were used. In one case (the green line), the decoys were chosen at random from the Maybridge compound collection [48], in the other (the red line), decoys were chosen to match the properties of the actives based on simple 1D properties. The striking difference, especially in early performance, is obvious. Clearly the performance of FRED is heavily affected by the nature of the decoy compounds, and to obtain a predictive indication of the utility of FRED in virtual screening decoy sets similar to those giving the red line should be used. It is worthy of note that the AUC\u2019s for the two experiments shown are different by more than their respective 95% confidence intervals, so that this difference is indeed statistically significant.\nFig.\u00a07Effect of decoy selection method on virtual screening by docking\nAnother example of how much of the signal that separates active from inactive compounds arises from systematic differences between their properties is shown in Fig.\u00a08. In these eight examples, drawn from the Surflex-Dock dataset [2], the performance of three 3D virtual screening methods\u2013\u2013Surflex-Dock, ROCS [49] and FRED\u2013\u2013is compared to a simple 1D method. In this 1D approach [46, 50], compounds are ranked by distance in an Euclidean property space to the centre of the space defined by the active compounds. The Euclidean space is defined by five simple molecular properties: number of donors, number of acceptors, number of rotatable bonds, XlogP and 0.01\u00a0\u00d7\u00a0molecular weight. This concept of ranking compounds by distance in a high dimensional Euclidean property space has recently been published as a virtual screening method, known as DACCS [51].\nFig.\u00a08AUCs for various virtual screening methods on part of the Surflex-Dock validation set\nIt is clear from Fig.\u00a08 that in four of the eight cases (OPPA, HIV-PR, TK and PARP) the active compounds are very dissimilar from the background set, as the 1D ranking method gives very good virtual screening performance. In a fifth case (TS) the performance of 1D method is as good as any of the 3D methods, although none of the tools perform particularly well. Accordingly, judging virtual screening performance for any tool using such datasets is unlikely to be productive as most of the \u201csignal\u201d separating actives from decoys lies purely in differences in simple molecular properties. A further confounding issue with these datasets is that a large number of the active compounds in these sets are close structural analogues of one another. For ligand-based methods this high structural similarity amongst the actives can cause the actives to be very easily discriminated from the decoys, while a structure-based method may have more difficulty. Accordingly, while property bias is an important consideration in constructing decoy sets, analogue bias should be carefully considered when selecting sets of active compounds.\nA recent effort to avoid some pathologies arising from poorly selected decoy sets has come from the work of Huang et\u00a0al. [52] with the Database of Useful Decoys, or DUD. In this work decoys were selected to match the same simple molecular properties of the active compounds using a similar approach to that mentioned earlier, so that the decoys are not trivially separable from the actives. DUD represents a very wide-ranging dataset (actives against 40 target proteins) that has been designed to evaluate the underlying performance of docking tools, and not the sensitivity of that tool\u2019s scoring metric to differences in simple molecular properties. Note that the DUD decoy selection approach uses a discontinuous representation of the molecular properties, while the approach mentioned above uses a continuous representation. The similarity between these two approaches for decoy selection and the DACCS approach for active selection is striking. It is a topic of further investigation whether much of the reported success of the DACCS method is due to over-training\/poor active compound selection.\nThe work of Huang et\u00a0al. with the DUD dataset showed that, at least for DOCK, this approach of specifically matching the properties of the decoys to those of the actives can produce more difficult decoy sets than those chosen purely for drug-likeness in a number of cases. It should be noted that the differences in performance reported in the DUD paper are essentially anecdotal, since no error bars are reported. With that caveat in mind inspection of Fig.\u00a04 of the DUD paper, in which comparisons of performance of DOCK between the DUD \u201cown\u201d decoy sets and some commonly available agnostic decoys are shown, is instructive. In 10 of the 12 cases presented the DUD \u201cown\u201d decoy set is the most challenging of the four decoy sets compared, implying that a property-matched decoy set can provide a more difficult background set than an \u201cagnostic\u201d or general decoy set. For a drug-like decoy set chosen without specific reference to the active compounds being screened for, which is therefore to be expected to be less challenging than decoy sets designed with specific reference to the actives being searched for, see Ref [53]. The DUD datasets also have relatively high prevalence (the design goal was to achieve a prevalence close to 3%, though this varies slightly from target to target), giving the results generated with DUD reasonably low errors (except for those cases where the number of actives is small). This is not to say that DUD is perfectly constructed\u2013\u2013there are still some large differences between the properties of the actives and the decoys in some cases and in some cases there are so few active molecules that statistically robust results cannot be generated. An important property not considered in the selection of the DUD decoys is formal charge. As such, there are some sets of the DUD actives that are easily discriminated from the decoys based on formal charge. For example, the mean formal charge on the neuraminidase ligands is +1.76, while the mean charge on the decoys is +0.76. For acetylcholinesterase, the mean charge on the actives is \u22121.68, while the mean charge on the decoys is \u22120.76. As with the datasets illustrated in Fig.\u00a08, the DUD active sets were not chosen with a view to structural diversity and some of the active sets consist entirely of closely analogous compounds. Very recently the original DUD dataset has been extended by adding more active compounds and by clustering the actives to remove trivially graph similar actives from the set [54]. This makes \u201cDUD 2.0\u201d a suitable dataset not only for docking approaches but also ligand-based techniques. It should be noted that there is a limit to the acceptable level of similarity between actives and presumptive decoys. When the decoys are too similar to the actives the assumption that the decoys are inactive becomes increasingly untenable, giving rise to large numbers of \u201cfalse false positives\u201d. Accordingly the problem of decoy selection is not yet completely solved, and may not admit of a single solution for all problems or tools. However, since in retrospective work the point is purely to gain a measure of the expectation of performance in as yet unperformed studies, the use of carefully designed decoy sets is mandatory.\nIt is unfortunate that the docking targets in DUD (39 crystal structures and 1 homology model) were not selected with as much care as the small molecule datasets. In 6 of the 38 co-crystal structures in DUD (there is one apo structure in the set), the DPIs are 1.5\u00a0\u00c5 or more, resulting in significant uncertainty in the positioning of any atom in these structures. These structures are ALR2 (1AH3), COX-2 (1CX2), EGFR (1M17), GR (1M2XZ), InhA (1P44) and p38 (1KV2). Accordingly docking results from these structures should be interpreted with great care.\nConclusions\nLarge numbers of evaluations and comparisons of tools for pose prediction and virtual screening have been published in recent years, an indication of significant interest in identifying tools that will have robust performance in one or both of these areas. Unfortunately the vast majority of these studies have been invalidated by poor choice of datasets, lack of consideration of error in source data and use of metrics that do not permit robust comparisons. For those papers using crystal structure data, too little account is taken both of the unavoidable imprecision in these structures and of the errors of fitting that are regrettably frequently seen in structures in the RCSB. In many cases nominal resolution, a measure of the quantity of data gathered, is confused with a measure of quality for the structure and other metrics indicating quality and reliability (DPI and Rfree) are ignored. When performing pose prediction geometric measures such as RMSD are almost always used to compare the experimental and predicted pose. These measures are uniformly used without taking into account either the inevitable imprecision in the atomic positions in crystal structures or the fact that using geometric measures necessarily implies comparing a model for the source data with a computed pose. In almost no cases are crystal structures inspected for errors in fitting. Without taking all these sources of error into account the results of any publication that uses crystal structure data will be suspect and of little use in deciding what tools are the most suitable for the task at hand. In papers concerned with virtual screening there has been, until recently, too little focus on eliminating trivial reasons for good performance from a given tool. The DUD dataset [52] has illustrated ways in which challenging virtual screening datasets can be constructed and, since it is publicly available, DUD offers the opportunity for a common benchmark upon which a wide variety of tools can be compared. The plethora of metrics used to judge and compare virtual screening performance serves merely to confuse the field rather then to clarify it. The lack of confidence intervals on metrics for success makes meaningful comparisons between tools almost impossible to interpret. The AUC for ROC offers great promise as a metric for virtual screening, as it offers the possibility of predictive value along with robust errors. For an exemplary use of ROC in virtual screening tests see a recent paper by Jain [55]. It is hoped that the field will soon converge to a single metric of virtual screening performance, such as the ROC, that will allow robust and direct comparisons between tools and between studies.","keyphrases":["pose prediction","virtual screening","coordinate error","property bias","software evaluation"],"prmu":["P","P","P","P","R"]}
{"id":"Childs_Nerv_Syst-4-1-2413122","title":"Secondary meningioma in a long-term survivor of atypical teratoid\/rhabdoid tumour with a germline INI1 mutation\n","text":"Objective We report on a patient who developed a meningioma more than two decades after removal at a young age of an atypical teratoid\/rhabdoid tumour (AT\/RT), which was due to a germline INI1 mutation, and radio- and chemotherapy.\nIntroduction\nWe previously reported on a family in which four cousins developed an atypical teratoid\/rhabdoid tumour (AT\/RT) at young age, due to inheritance of a germline G>A mutation in the donor splice site of exon 4 of the tumour suppressor gene INI1 [2]. More than 20\u00a0years after resection of the AT\/RT, the oldest of the four cousins developed an intracranial meningioma and, after an additional year, a myoepithelioma of the lip. In addition to the constitutional INI1 mutation and loss of the normal INI1 allele, we detected an identical rearrangement in the NF2 gene, not affecting its coding regions, in the AT\/RT and myoepithelioma of the patient and concluded that they both originated from a common precursor cell. However, the rearrangement in NF2 and loss of the normal INI1 allele could not be demonstrated in the meningioma. Despite this, the development of the meningioma may be the consequence of the presence of the constitutional INI1 mutation, because mutations in this gene have been identified in meningiomas [8]. Alternatively, the meningioma could be induced by the radiation therapy, which our patient received after removal of the AT\/RT and which is a well-known consequence of this treatment [1]. We investigated the meningioma for the presence of genetic changes reported in radiation-induced meningiomas and performed a comparative loss of heterozygosity (LOH) analysis to demonstrate that such radiation-induced genetic changes are absent in the AT\/RT and myoepithelioma of our patient.\nMaterials and methods\nTumour samples and DNA isolation\nDNA from the AT\/RT and the myoepithelioma was isolated from formalin-fixed and paraffin-embedded tumour material, and DNA from the meningioma was isolated from a fresh frozen tumour sample using commercially available kits (Qiagen, Venlo, The Netherlands).\nSNP analysis\nA genome-wide single nucleotide polymorphism (SNP) analysis of normal and meningioma DNA was conducted with the Affymetrix GeneChip Human Mapping 10K Array Xba 142 2.0. The array contains 10,204 SNPs distributed across the genome. The GeneChips were processed by an authorised Affymetrix Service Provider using standard Affymetrix protocols (Service XS, Leiden, The Netherlands). LOH regions were identified by comparing the SNP profiles of normal and meningioma DNA and scoring heterozygous SNP markers (AB) in the normal DNA that were reduced to homozygosity (AA or BB) in the meningioma DNA. Intervals were determined on the basis of locations of SNP markers in the UCSC Genome Browser, March 2006 Build, at http:\/\/genome.ucsc.edu\/.\nLOH analysis\nLoss of heterozygosity (LOH) analysis was performed with microsatellite markers as described previously [3] using primer sequences and conditions for polymerase chain reaction given by The Genome Database at http:\/\/www.gdb.org\/. Because of rather extensive degradation of DNA extracted from the AT\/RT, only markers generating PCR fragments smaller than 150\u00a0bp were used.\nCase report\nHistory\nOur patient was diagnosed with a tumour in the fourth ventricle and right cerebellar hemisphere at the age of 4.5\u00a0years. Resection of the tumour was incomplete. The tumour was originally classified as anaplastic ependymoma WHO grade III, but upon extensive genetic analysis and histopathological re-evaluation, reclassified as AT\/RT [2]. He received adjuvant chemotherapy (methotrexate, vincristine and prednisolone) followed by craniospinal radiotherapy (3,300\u00a0cGy in 22 doses) with a boost of 2,100\u00a0cGy on the location of the tumour. Fifteen years later, a brain CT without contrast showed no signs of recurrent disease or of other new pathology. Almost 6\u00a0years later, 20.5\u00a0years after initial diagnosis, he presented again with bifrontal headaches, increasing ataxic gait disturbances and fatigue. CT examination showed a large isodens space-occupying lesion in the left temporal region with oedema, midline shift and contrast enhancement. One year after resection of this tumour, he developed a myoepithelioma of the upper lip. The patient is alive at 29\u00a0years of age.\nHistological findings\nHistopathological examination of the left temporal tumour revealed an atypical meningothelial meningioma WHO grade II with many vessels and up to six mitoses per 2\u00a0mm2.\nSNP and LOH analysis Radiation-induced meningiomas usually display a complex karyotype [1]. To determine the genetic changes, we performed a genome-wide scan by SNP analysis of the meningioma and corresponding normal DNA. The identified chromosomal regions displaying LOH are listed in Table\u00a01. Next, we tested several markers from within each of the five largest deleted regions by conventional microsatellite analysis to determine their LOH status in the meningioma and in the AT\/RT and myoepithelioma. Presence of LOH in the meningioma, but absence of LOH in AT\/RT and myoepithelioma was found for D1S199 in 1pter-p34.3 (Fig.\u00a01a), D2S327 in 2pter-p12, D3S3727 in 3p26.1-cen (Fig.\u00a01b), D16S3020 in 16pter-p12.1 and D17S1872 in 17qcen-q21.31 (Fig.\u00a01c). In contrast, as determined previously [2] and exemplified by marker D22S430 in Fig.\u00a01d, there was LOH for chromosome 22 markers in the AT\/RT and myoepithelioma, but not in the meningioma.\nFig.\u00a01LOH analysis using markers from chromosome regions a 1pter-p34.3 (D1S199), b 3p26.1-cen (D3S3727), c 17qcen-q21.31 (D17S1872) and d 22q (D22S430) of AT\/RT (A), meningioma (M) and myoepithelioma (My) DNAs of the patient. N denotes normal DNA derived from the patient\u2019s blood leucocytes. Arrowhead indicates position of lost alleleTable\u00a01LOH regions in the meningioma, as determined by SNP analysisChromosome regionExact interval (Mb)1pter-p34.30\u201338.901p33-p3346.99\u201351.091p32.2-1p31.358.19\u201365.502pter-p120\u201379.283p26.1-cen6.11\u201388.847q36.1-qter150.04\u2013158.6616pter-p12.10\u201322.7516q23.1-q23.373.46\u201381.3317cen-q21.3123.09\u201340.3519pter-p13.20\u20138.83\nDiscussion\nIn a previous study, we provided evidence for the involvement of INI1, by constitutional mutation and independent somatic loss, in the development of both the AT\/RT and the myoepithelioma of our patient. Moreover, we could demonstrate that these tumours originated from an identical precursor cell [2]. Although involvement of INI1 has been described in sporadic meningiomas [8], until now meningiomas have not been reported to occur as a consequence of the inheritance of an INI1 mutation. However, this might be due to the extreme rarity of families with an inherited INI1 mutation, with only four published cases so far [2, 5, 6, 11]. In this context, it is interesting to note that (as demonstrated before and exemplified by marker D22S430 in Fig.\u00a01d) the meningioma of our patient has no LOH 22 and the retained INI1 allele carries no additional inactivating mutation [2], indicating that this tumour is not the result of inheritance of the mutated INI1 gene. In contrast, the meningioma displayed genetic changes that are characteristic for radiation-induced meningiomas [1, 7, 9], such as a complex karyotype, as evidenced by the SNP analysis (Table\u00a01), including extensive losses on chromosome arm 1p, but no loss of chromosome 22, and no mutation in the NF2 gene [2]. The genetic changes found in the meningioma proved not to be present in the AT\/RT or myoepithelioma (Fig.\u00a01a\u2013c, and data not shown), demonstrating that the meningioma is genetically not related to these tumours and should not be considered as a late and more differentiated recurrence or metastasis of the AT\/RT.\nIn conclusion, the clinical and genetic characteristics of the meningioma clearly indicate that the tumour developed as a consequence of the radiation therapy. Recent evidence suggests that long-term survival of patients with a childhood AT\/RT can occur especially after aggressive treatment including radiotherapy [10, 12]. The possible late effects of radiation therapy have been considered as serious drawbacks of this treatment. This is the first case in literature in which such a possible consequence in the form of a radiation-induced meningioma is actually documented in a long-term survivor of an AT\/RT. Compared to sporadic meningiomas, radiation-induced meningiomas are more aggressive and have a higher chance to recur [1]. Because of this unfavourable prognosis, continuous medical surveillance of our patient is warranted.","keyphrases":["meningioma","atypical teratoid\/rhabdoid tumour","ini1","radiation therapy","genetic predisposition"],"prmu":["P","P","P","P","M"]}
{"id":"Diabetologia-4-1-2362134","title":"Increased osteoclastic activity in acute Charcot\u2019s osteoarthopathy: the role of receptor activator of nuclear factor-kappaB ligand\n","text":"Aims\/hypothesis Our aims were to compare osteoclastic activity between patients with acute Charcot\u2019s osteoarthropathy and diabetic and healthy controls, and to determine the effect of the receptor activator of nuclear factor-kappaB ligand (RANKL) and its decoy receptor osteoprotegerin (OPG).\nIntroduction\nAlthough Charcot\u2019s osteoarthropathy is characterised by increased local bone resorption [1], the exact cellular mechanisms contributing to the pathogenesis of this condition remain unresolved. Osteoclasts have been shown to be the principal cell type responsible for bone resorption [2]. These cells originate from the haemopoietic lineage and are known to undergo various stages of proliferation, fusion and differentiation before they are fully functionally active, mature osteoclasts. Recently, receptor activator of nuclear factor-kappaB (RANK) ligand (RANKL) has been identified as an essential mediator of osteoclast formation and activation [3]. RANKL is expressed on a variety of cell types such as bone forming osteoblasts, T lymphocytes, dendritic cells, endothelial cells and fibroblasts. RANKL mediates the process of osteoclastogenesis by binding to its RANK, which is expressed on mononuclear osteoclast precursors. The effects of RANKL\u2013RANK interaction are physiologically counterbalanced by osteoprotegerin (OPG), which acts as a soluble receptor decoy for RANKL and blocks the interaction of RANKL with RANK. The ratio of RANKL to OPG has been suggested to regulate the extent of osteoclast formation and resorption. Therefore, any alteration in the RANKL\/OPG ratio could be critical in the pathogenesis of osteolytic bone disorders [4].\nRecently, Jeffcoate hypothesised that the RANK\/RANKL\/OPG pathway may play an important role in the osteolysis seen in acute Charcot\u2019s osteoarthropathy [5]. Using an in vitro technique to generate functional human osteoclasts from peripheral blood monocytes (PBMCs) [6] in the presence of macrophage-colony stimulating factor (M-CSF) [7] and soluble RANKL, it is possible to determine the cellular mechanisms involved in the process of osteoclast formation and resorption in physiological and pathological conditions. To our knowledge, this technique has not yet been studied in patients with Charcot\u2019s osteoarthropathy.\nThe aims of this study were: (1) to generate functional human osteoclasts in vitro from diabetic patients with acute Charcot\u2019s osteoarthropathy and from healthy and diabetic control participants; (2) to compare the extent of osteoclast formation and resorption; and (3) to determine the role of the RANK\/RANKL\/OPG pathway in osteoclastic activity in Charcot\u2019s osteoarthropathy.\nMethods\nPatients\nWe studied nine consecutive diabetic patients with recent onset of acute Charcot\u2019s osteoarthropathy (five men, four women; five type 1, four type 2 diabetes), eight diabetic patients with no previous history of Charcot\u2019s osteoarthropathy (five men, three women; four type 1, four type 2) and eight healthy control participants (five men, three women). Patients with acute Charcot\u2019s osteoarthropathy were matched for age and duration of diabetes with the diabetic control patients and for age with the healthy control participants. The mean age was similar between patients with Charcot\u2019s osteoarthropathy and diabetic control patients (53\u2009\u00b1\u20092.8 versus 59\u2009\u00b1\u20092.9\u00a0years [mean\u2009\u00b1\u2009SEM], p\u2009=\u20090.167) as was the mean age between the former and healthy control participants (53\u2009\u00b1\u20092.8 versus 47\u2009\u00b1\u20092.7\u00a0years, p\u2009=\u20090.114). The mean duration of diabetes was similar in both groups with diabetes (31\u2009\u00b1\u20095.1 [Charcot patients] versus 27\u2009\u00b1\u20094.6\u00a0years, p\u2009=\u20090.606). Diabetes control as indicated by glycated Hb was also similar in the two diabetes groups (7.7\u2009\u00b1\u20090.6 [Charcot\u2019s] versus 7.8\u2009\u00b1\u20090.4%, p\u2009=\u20090.743).\nDiagnosis of Charcot\u2019s osteoarthropathy was made on the presentation of a hot swollen foot, with skin foot temperature 2\u00b0C greater than the corresponding site on the contralateral foot and with typical radiological changes of subluxation, dislocation or fragmentation of bone on standard foot radiographs [8]. All patients had intact feet and no evidence of foot infection or ulceration.\nEthical permission for this study was obtained from the King\u2019s College Hospital Research Ethics Committee and all participants gave written informed consent.\nIsolation and culture of monocytes\nPeripheral blood mononuclear cells were isolated as previously described [6]. Briefly, blood was diluted 1:1 in \u03b1-minimum essential medium (MEM; Invitrogen, Paisley, UK), layered over Histopaque and centrifuged (693\u00a0g) for 20\u00a0min. The interface layer was resuspended in MEM, then centrifuged (600\u00a0g) for a further 10\u00a0min. The resultant cells were resuspended in MEM with 10% heat-inactivated FCS and counted in a haemocytometer following lysis of erythrocytes by a 5% (vol.\/vol.) acetic acid solution.\nTo assess the extent of osteoclast formation and resorption, PBMCs were cultured on glass coverslips and dentine slices. Initially, 5\u2009\u00d7\u2009105 PBMCs were added to 6-mm diameter glass coverslips and 4-mm diameter dentine slices in MEM containing 100\u00a0UI\/ml penicillin, 100\u00a0\u03bcg\/ml streptomycin and 10% FCS (Gibco, Paisley, UK). After 2\u00a0h incubation, coverslips and dentine slices were vigorously rinsed in medium to remove non-adherent cells. The cultures were maintained in MEM\/FCS under three different culture conditions: (1) human M-CSF (R&D Systems Europe, Abingdon, UK) alone at 25\u00a0ng\/ml; (2) M-CSF plus 100\u00a0ng\/ml human soluble RANKL (sRANKL; Peprotech, London, UK) (a concentration known to facilitate differentiation of osteoclast precursors to active bone-resorbing osteoclasts in vitro); and (3) M-CSF plus sRANKL plus 250\u00a0ng\/ml human OPG (R&D Systems Europe).\nCoverslips and dentine slices were cultured at 37\u00b0C in 5% CO2 for 14 and 21\u00a0days respectively.\nOsteoclast formation\nAfter 14\u00a0days, the coverslips were examined histochemically for the expression of tartrate-resistant acid phosphatase (TRAcP), an osteoclast marker. Coverslips with newly formed osteoclasts were collected and rinsed in PBS buffer, fixed with formalin (10% [vol.\/vol.] in PBS buffer) for 10\u00a0min and rinsed in distilled water. TRAcP was histochemically revealed by a simultaneous coupling reaction using Naphtol AS-BI-phosphate as substrate and Fast violet B as the diazonium salt. The coverslips were incubated for 90\u00a0min at 37\u00b0C in a dark room, rinsed three times in distilled water and the residual activity was inhibited by 4% NaF (wt\/wt) for 30\u00a0min. Coverslips were then rinsed in distilled water, counterstained with DAPI for 20\u00a0min and allowed to dry before mounting, using an aqueous medium. TRAcP-positive cells with more than three nuclei were identified as osteoclasts. The number of newly generated osteoclasts was assessed using a light microscope examination.\nOsteoclast resorption\nAfter 21\u00a0days, the dentine slices were removed from the culture wells, placed in NH4OH (1\u00a0mol\/l) for 30\u00a0min and sonicated for 5\u00a0min to remove any adherent cells. They were then rinsed in distilled water and stained with 0.5% (vol.\/vol.) toluidine blue prior to examination by light microscopy. The surface of each dentine slice was examined for evidence of lacunar resorption and the extent of eroded surface on each dentine slice was determined using image analysis and expressed as the percentage of surface area resorbed.\nStatistical analyses\nData were expressed as a mean\u2009\u00b1\u2009SEM. Initially the difference within the three study groups (Charcot patients, healthy and diabetic controls) was assessed with the non-parametric Kruskall\u2013Wallis test. Then the differences between Charcot and diabetic patients, and Charcot patients and healthy controls were assessed by the non-parametric Mann\u2013Whitney U test. In each patient group, the differences between the various culture conditions were also assessed using the Mann\u2013Whitney U test. Differences were considered significant at p\u2009<\u20090.05.\nResults\nOsteoclast cultures in the presence of M-CSF\nOsteoclast formation The mean number of newly formed TRAcP-positive multinucleated osteoclasts in the presence of M-CSF alone was significantly greater in the patients with acute Charcot\u2019s osteoarthropathy (48.6\u2009\u00b1\u200918.2) than in diabetic (6.8\u2009\u00b1\u20092.7) and healthy control participants (5.0\u2009\u00b1\u20090.7) (p\u2009=\u20090.008). The number of TRAcP-positive multinucleated osteoclasts formed in acute Charcot\u2019s osteoarthropathy was 7.2 and 9.7 times greater than those formed in diabetic (p\u2009=\u20090.010) and healthy control groups (p\u2009=\u20090.003), respectively.\nOsteoclast resorption The newly formed osteoclasts exhibited increased functional activity as demonstrated by the extent of resorption on dentine slices, with percentage area resorption significantly elevated in the patients with acute Charcot\u2019s osteoarthropathy (0.264\u2009\u00b1\u20090.06%) compared with diabetic (0.000\u2009\u00b1\u20090.00%) and healthy control groups (0.004\u2009\u00b1\u20090.01) (p\u2009<\u20090.0001). The percentage of resorption was significantly greater in the Charcot patients than in the diabetic (p\u2009=\u20090.001) and healthy control groups (p\u2009=\u20090.001).\nOsteoclast cultures in the presence of M-CSF and sRANKL\nOsteoclast formation The addition of sRANKL led to an increase in the number of TRAcP-positive multinucleated osteoclasts in all three groups of patients. The mean number of these osteoclasts in patients with acute Charcot\u2019s osteoarthropathy was 96.0\u2009\u00b1\u200921.6, which was significantly greater than that in the diabetic (56.5\u2009\u00b1\u200911.5) and healthy (29.0\u2009\u00b1\u20095.1) control groups (p\u2009=\u20090.010; Fig.\u00a01a,c,e). The number of TRAcP-positive multinucleated osteoclasts in the patients with acute Charcot\u2019s osteoarthropathy was 1.7 times higher than in diabetic control patients, but this finding did not reach significance (p\u2009=\u20090.105). However, the number of these osteoclasts in the acute Charcot group was 3.3 times (and significantly) higher than in the healthy control group (p\u2009=\u20090.005). When the number of cells in the cultures with M-CSF alone was compared with that after the addition of sRANKL, there was a significant increase in the diabetic control patients (from 6.8\u2009\u00b1\u20092.7 to 56.5\u2009\u00b1\u200911.5, p\u2009=\u20090.003) and in the healthy participants (from 5.0\u2009\u00b1\u20090.7 to 29.0\u2009\u00b1\u20095.1, p\u2009=\u20090.002), while the increase in the number of TRAcP-positive multinucleated osteoclasts in the acute Charcot group failed to reach significance (increase from 48.6\u2009\u00b1\u200918.2 to 96.0\u2009\u00b1\u200921.6, p\u2009=\u20090.059; Fig.\u00a02a).\nFig.\u00a01Multinucleated TRAcP-positive cells were formed on glass coverslips (a, c, e) capable of lacunar resorption (b, d, f) after 14 and 21\u00a0days incubation, respectively, in the presence of 25\u00a0ng\/ml human M-CSF and 100\u00a0ng\/ml sRANKL. Newly formed osteoclasts were numerous and highly active in Charcot\u2019s patients (a, b) compared with diabetic (c, d) and healthy control (e, f) participants. Scale bars, 10\u00a0\u03bcmFig.\u00a02a Quantitative comparison between the number (n) of TRAcP-positive cells formed in cultures with M-CSF alone (white bars) or with M-CSF and sRANKL (black bars) in patients with Charcot\u2019s osteoarthropathy and diabetic and healthy control participants. b Quantitative comparison between the percentage area resorption in the same cultures and patient groups. Statistical differences between the groups were determined using the Mann\u2013Whitney U test, with significance as follows: a Charcot\u2019s p\u2009=\u20090.059, diabetic control p\u2009=\u20090.003, healthy control p\u2009=\u20090.002; b Charcot\u2019s p\u2009<\u20090.0001, diabetic control p\u2009<\u20090.0001, healthy control p\u2009<\u20090.0001\nOsteoclast resorption The percentage area resorption on dentine slices with M-CSF and sRANKL was significantly increased in the acute Charcot group (41.6\u2009\u00b1\u20098.1%) compared with that in the diabetic (14.2\u2009\u00b1\u200916.5%) and healthy control groups (10.5\u2009\u00b1\u20091.9%; p\u2009=\u20090.005). Resorption in the Charcot patients was 2.9 times higher than in diabetic control patients (p\u2009=\u20090.008) and four times higher than in healthy participants (p\u2009=\u20090.005; Fig.\u00a01b,d,f). The addition of sRANKL to the cultures with M-CSF led to the following rises in the percentage area resorption when compared with M-CSF alone: Charcot\u2019s 0.264\u2009\u00b1\u20090.06% to 41.6\u2009\u00b1\u20098.1%, p\u2009<\u20090.0001; diabetic control 0.000\u2009\u00b1\u20090.00% to 14.2\u2009\u00b1\u200916.5%, p\u2009<\u20090.0001; healthy control 0.004\u2009\u00b1\u20090.01% to 10.5\u2009\u00b1\u20091.9%, p\u2009<\u20090.0001 (Fig.\u00a02b).\nOsteoclast cultures in the presence of M-CSF, sRANKL and excess concentrations of OPG\nOsteoclast formation The addition of excess concentrations of OPG led to a reduction in the number of TRAcP-positive multinucleated osteoclasts in the cultures with M-CSF, sRANKL and OPG in all the three groups of patients. However, after the addition of OPG, the number of TRAcP-positive multinucleated osteoclasts was still significantly increased in the Charcot group (54.4\u2009\u00b1\u200917.6), as compared with diabetic (8.8\u2009\u00b1\u20095.3) and healthy control participants (4.4\u2009\u00b1\u20091.2; p\u2009=\u20090.003). In the cultures with M-CSF, sRANKL and OPG, the number of TRAcP-positive multinucleated osteoclasts was greater in the Charcot patients than in the diabetic (p\u2009=\u20090.005) and healthy control groups (p\u2009=\u20090.001).\nWhen OPG was added to the cultures with M-CSF and sRANKL, the reduction in the number of TRAcP-positive cells in Charcot patients was not significant (96.0\u2009\u00b1\u200921.6 versus 54.4\u2009\u00b1\u200917.6, p\u2009=\u20090.189). OPG on the other hand significantly inhibited the number of TRAcP-positive cells in M-CSF and RANKL-mediated cultures from diabetic (reduced from 56.5\u2009\u00b1\u200911.5 to 8.8\u2009\u00b1\u20095.3, p\u2009=\u20090.005) and healthy control participants (29.0\u2009\u00b1\u20095.1 to 4.4\u2009\u00b1\u20091.2, p\u2009=\u20090.003; Fig.\u00a03a).\nFig.\u00a03a Comparison between the number (n) of TRAcP-positive cells formed in cultures with M-CSF and sRANKL (black bars) or with M-CSF, sRANKL and excess concentrations of OPG (250\u00a0ng\/ml) (grey bars) in patients with Charcot\u2019s osteoarthropathy and diabetic and healthy control participants. b Comparison between the percentage area resorption in the same cultures and patient groups. Statistical differences between the groups were determined using the Mann\u2013Whitney U test, with significance as follows: a Charcot\u2019s p\u2009=\u20090.189, diabetic control p\u2009=\u20090.005, healthy control p\u2009=\u20090.003; b Charcot's p\u2009=\u20090.001, diabetic control p\u2009=\u20090.001, healthy control p\u2009<\u20090.0001\nOsteoclast resorption The addition of OPG led to a marked reduction of the percentage area resorption on dentine slices in Charcot patients (from 41.6\u2009\u00b1\u20098.1% to 5.9\u2009\u00b1\u20092.4%, p\u2009=\u20090.001) and also in diabetic (14.2\u2009\u00b1\u200916.5% to 0.45\u2009\u00b1\u20090.31%, p\u2009=\u20090.001) and healthy control (from 10.5\u2009\u00b1\u20091.9% to 0.00\u2009\u00b1\u20090.00%, p\u2009<\u20090.0001) participants (Fig.\u00a03b).\nHowever, the percentage area resorption on the dentine slices was still greater in the cultures with M-CSF, RANKL and OPG from the patients with acute Charcot\u2019s osteoarthropathy (5.9\u2009\u00b1\u20092.4%) than in those from diabetic (0.45\u2009\u00b1\u20090.31%) and healthy control (0.00\u2009\u00b1\u20090.00%) participants (p\u2009=\u20090.003). Resorption on the dentine slices was greater in the Charcot patients than in diabetic (p\u2009=\u20090.005) and healthy control (p\u2009=\u20090.003) groups.\nDiscussion\nThis study shows that monocytes from patients with acute Charcot\u2019s osteoarthropathy cultured in the presence of M-CSF alone were capable of differentiating into mature osteoclasts that exhibited increased resorption compared with diabetic and healthy control participants. Furthermore, osteoclasts generated after the addition of sRANKL were functionally more aggressive, exhibiting a considerable increase in the extent of resorbing activity in patients with acute Charcot\u2019s osteoarthropathy. This resorption was partially blocked by the addition of excess concentrations of OPG, a soluble receptor decoy for RANKL. This suggests that the increased osteoclastic activity in patients with acute Charcot\u2019s osteoarthropathy is mediated through both a RANKL-dependent and a RANKL-independent pathway.\nCultures from the patients with Charcot\u2019s osteoarthropathy showed increased osteoclast formation and resorption when cultured with M-CSF alone. Although M-CSF is an essential factor for proliferation, differentiation and survival of the monocyte-macrophage lineage [9, 10], it is not an osteoclastogenic factor and it is unusual to detect osteoclast formation and resorption in cultures with M-CSF, as was seen in the diabetic and healthy controls. This observation suggests that in acute Charcot\u2019s osteoarthropathy there may be increased levels of other circulating pro-inflammatory factors such as TNF-\u03b1 [11, 12], IL-6 [13], IL-8 [14] and LIGHT (homologous to lymphotoxins exhibiting inducible expression and competing with herpes simplex virus glycoprotein D for herpes virus entry mediator [HVEM], a receptor expressed by T lymphocytes) [15], which have been previously shown to stimulate osteoclastogenesis independently of RANK\/RANKL mechanisms. The concentrations of these circulating factors in diabetic and healthy control participants may not be sufficient to induce the formation and differentiation of active osteoclasts in the presence of M-CSF alone.\nAfter the addition of sRANKL to M-CSF cultures, the newly formed osteoclasts exhibited markedly increased resorption in the patients with Charcot\u2019s osteoarthropathy, although the number of osteoclasts did not significantly increase in these patients compared with cultures with M-CSF alone. These observations may not be unique to Charcot\u2019s osteoarthropathy, and indeed similar observations have been reported in other conditions associated with increased bone resorption, such as rheumatoid arthritis where the addition of sRANKL resulted in a significant increase in lacunar resorption, but did not lead to a significant increase in the number of TRAcP-positive cells [16]. Overall, the observed extensive resorption in acute Charcot patients, in the presence of M-CSF and sRANKL, as compared with the diabetic or healthy control groups, may suggest that the osteoclast precursors circulating in acute Charcot patients are in a higher activated state and as such are more primed to becoming osteoclasts (mediated through RANKL) than those in the control groups.\nIn order to ascertain that RANKL was a major osteoclastic activator in patients with Charcot\u2019s osteoarthropathy, excess concentrations of OPG, the soluble receptor decoy to RANKL, were added to the cultures with M-CSF and RANKL. The rationale for this approach was that if osteoclastogenesis is mediated solely through RANK\u2013RANKL interaction, addition of excess concentrations of OPG (as had been previously determined to be sufficient to block osteoclastogenesis through RANKL [15]) would completely abolish the process of osteoclast differentiation and activation. In the current study, although osteoclast formation and resorption in the diabetic and healthy control groups was completely blocked by the addition of OPG, the latter did not achieve total inhibition of osteoclast formation and resorption in patients with acute Charcot\u2019s osteoarthropathy. These results suggest that although RANKL-dependent pathways do play a significant role in the osteoclastic activity of Charcot\u2019s osteoarthropathy, an alternative pathway (other than RANK\/RANKL) may also be involved. Osteoclastogenic mediators other than RANKL that have been reported to stimulate osteoclast differentiation independently of the RANKL pathway include TNF-\u03b1 [11, 12], IL-6 [13], IL-8 [14] and LIGHT [15]. In acute Charcot\u2019s osteoarthropathy, it is possible that one or a combination of these factors may have initiated the circulating osteoclast precursors to be in a more \u2018primed\u2019 condition, a situation which as such could explain the observed resorption in Charcot monocyte cultures supplemented with M-CSF alone, without the exogenous addition of any osteoclastogenic mediators.\nThe osteolysis of Charcot\u2019s osteoarthropathy may be explained by our observation that osteoclast precursors from Charcot patients develop into mature osteoclasts that exhibit increased resorptive activity, especially in response to RANKL, unlike the increased resorption in response to bacterial infection, which is not mediated by RANKL [17]. Increased expression of RANKL has been previously demonstrated in pathological osteolysis associated with the development of various bone diseases [18] and a similar mechanism may contribute to osteolysis of Charcot\u2019s osteoarthropathy [5]. Furthermore, patients with Charcot\u2019s osteoarthropathy have severe neuropathy, which itself can also lead to increased expression of RANKL as a result of the loss of nerve-derived peptides known to antagonise its effect such as calcitonin gene-related peptide [5]. In addition to the RANKL-dependent pathway, our results suggest that a RANKL-independent pathway, mediated by pro-inflammatory cytokines, may also be important. Indeed, Charcot\u2019s foot is characterised by excessive inflammation and proinflammatory cytokines have been implicated in its pathogenesis [19]. In support of this, a recent immunohistochemical analysis of bone samples isolated from Charcot\u2019s osteoarthropathy patients showed excessive osteoclastic activity in a microenvironment enriched with mediators of bone resorption (IL-1, IL-6 and TNF-\u03b1) [20]. Thus a RANKL-independent pathway, which is also known to play a role in other osteolytic disorders such as rheumatoid arthritis [21] and aseptic loosening [22], could contribute also to the pathogenesis of the Charcot\u2019s osteoarthropathy.\nThis study has indicated, for the first time that the RANKL-dependent pathway is important in the pathogenesis of Charcot\u2019s osteoarthropathy, thereby raising the possibility of the use of RANKL inhibition in the management of Charcot\u2019s foot. However, our observations also suggest that a RANKL-independent pathway may play a role, but further investigation is required to fully clarify the mechanism involved. If confirmed, specific pharmacological agents that counteract the RANKL-independent pathway, such as anti-TNF strategies, may be useful in the treatment of Charcot\u2019s osteoarthropathy. Whatever the relative importance of either pathway, this in vitro technique of generating human osteoclasts from PBMCs may allow specific characterisation of osteoclastic activity in each patient and could, in the future, lead to individually tailored anti-osteoclastic treatment for the patient with acute Charcot\u2019s osteoarthropathy.","keyphrases":["osteoclasts","charcot\u2019s osteoarthropathy","rankl","opg","resorption","osteolysis"],"prmu":["P","P","P","P","P","P"]}
{"id":"J_Chem_Ecol-3-1-1915630","title":"Jasmonic Acid-Induced Changes in Brassica oleracea Affect Oviposition Preference of Two Specialist Herbivores\n","text":"Jasmonic acid (JA) is a key hormone involved in plant defense responses. The effect of JA treatment of cabbage plants on their acceptability for oviposition by two species of cabbage white butterflies, Pieris rapae and P. brassicae, was investigated. Both butterfly species laid fewer eggs on leaves of JA-treated plants compared to control plants. We show that this is due to processes in the plant after JA treatment rather than an effect of JA itself. The oviposition preference for control plants is adaptive, as development time from larval hatch until pupation of P. rapae caterpillars was longer on JA-treated plants. Total glucosinolate content in leaf surface extracts was similar for control and treated plants; however, two of the five glucosinolates were present in lower amounts in leaf surface extracts of JA-treated plants. When the butterflies were offered a choice between the purified glucosinolate fraction isolated from leaf surface extracts of JA-treated plants and that from control plants, they did not discriminate. Changes in leaf surface glucosinolate profile, therefore, do not seem to explain the change in oviposition preference of the butterflies after JA treatment, suggesting that as yet unknown infochemicals are involved.\nIntroduction\nPlants can be attacked by many herbivorous insects and have evolved a variety of defense strategies, including morphological barriers, synthesis of toxic or repellent secondary metabolites, and the release of synomones that attract natural enemies of the herbivores. These defenses can be constitutive, i.e., expressed independent of the presence of an attacker, or inducible, in which case defense compounds accumulate in response to attack (Karban and Baldwin, 1997). Herbivores can detect induced defensive compounds and respond by avoiding these plants, which signal lower suitability as a host plant (Landolt, 1993; De Moraes et al., 2001; Kessler and Baldwin, 2001; Meiners et al., 2005). Induced plant defense can affect herbivorous insects directly through the production of toxic compounds or indirectly through the production of cues that indicate intra- or interspecific competition for the herbivores (Schoonhoven et al., 2005). Moreover, induced plant defense signals can reduce the enemy-free space for the herbivores. For parasitoids and predators, induced infochemicals may indicate the presence of their host or prey on the plant (Turlings et al., 1990; Dicke and Vet, 1999). Phenotypic changes in individual plants may therefore affect insects at different trophic levels, and thus, the composition of the insect community and food web associated with the plant (Price et al., 1980; Van Zandt and Agrawal, 2004; Takabayashi et al., 2006).\nAlready in the 19th century, Kirby and Spence (1863) observed that Pieris brassicae females preferred to lay their eggs on plants devoid of eggs. Later, this was confirmed under more controlled conditions by Rothschild and Schoonhoven (1977) for both P. brassicae and Pieris rapae. This avoidance of infested plants is caused by a physiological response of the plant to oviposition, rather than by compounds excreted by the butterflies themselves (Blaakmeer et al., 1994). The butterflies also avoid egg deposition on leaves with feeding larvae (Rothschild and Schoonhoven, 1977). It was postulated that butterflies avoid laying eggs on herbivore-infested plants because herbivore attack induces defense compounds in plants that can influence the performance of their offspring and to reduce the risk of inter- or intraspecific competition and parasitism (Thompson and Pellmyr, 1991; Shiojiri et al., 2002). Egg-induced chemical changes in Brassica plants are also known to arrest Trichogramma parasitoids that parasitize Pieris eggs (Fatouros et al., 2005).\nOviposition-site selection involves an important behavioral decision in the life cycle of an herbivorous insect because hatching larvae have limited dispersal capacity (Renwick and Chew, 1994). P. rapae is a solitary butterfly that lays one egg at a time, whereas P. brassicae is gregarious and lays batches of about 20\u2013100 eggs. P. rapae appears to spread the risk of larval mortality, laying few eggs within any patch. This has the advantage of being able to exploit isolated plants (Davies and Gilbert, 1985). P. brassicae, however, needs patches of plants because one large egg batch will require more than one plant for all caterpillars to develop into adults. Oviposition-site selection is performed in consecutive phases of searching and contact evaluation. P. rapae butterflies use visual, olfactory and tactile cues during these phases (Rothschild and Schoonhoven, 1977; Renwick and Radke, 1988). Acceptance of a site may be determined by the balance of positive and negative factors (Renwick and Radke, 1988). Renwick and Radke (1988) suggest that olfaction does not play a role in attraction to a host plant, but may be involved in avoidance of non-host plants. P. rapae and P. brassicae are crucifer specialists and are known to use glucosinolates, toxic secondary metabolites characteristic for Brassicaceae, as oviposition stimulants. Glucobrassicin and sinigrin are effective oviposition stimulants for P. brassicae and P. rapae (Renwick et al., 1992; Van Loon et al., 1992a).\nA major signal-transduction pathway involved in induced plant defense is the octadecanoid pathway (Arimura et al., 2005). A central compound in the pathway is jasmonic acid (JA), which has an important role in direct and indirect defense in many plant species. In response to JA or methyl jasmonate (MeJA) treatment, increased concentrations of several compounds have been documented in a range of plant species, e.g., proteinase inhibitors (Moura and Ryan, 2001), polyphenol oxidases (Thaler et al., 1996), nicotine (Baldwin et al., 1996), trypsin inhibitors (Cipollini and Sipe, 2001), glucosinolates (Cipollini and Sipe, 2001; Van Dam et al., 2004; Mewis et al., 2005), and increased volatile emission (Boland et al., 1995; Dicke et al., 1999; Koch et al., 1999).\nHerbivores are reported to be affected by JA treatment of plants. Several studies have focused on the larval stage of the herbivores and have shown reduced relative growth rates and leaf consumption (Van Dam et al., 2000; Gols et al., 2003; Van Dam et al., 2004). In field experiments, spraying of JA decreased the abundance of caterpillars, flea beetles, aphids, and thrips (Thaler et al., 2001). Other studies addressed the influence of JA application to plants on oviposition-site selection behavior of adult herbivores. These studies showed that JA application can result in induced resistance as well as induced susceptibility (Stanjek et al., 1997; Kessler and Baldwin, 2001; Lu et al., 2004).\nHere, we studied how JA application affects oviposition of two specialist herbivores on cabbage, Pieris rapae L. and P. brassicae L. (Lepidoptera: Pieridae) that are closely related, yet differ drastically in the amount of eggs they put on one plant. Our study is the first to compare closely related herbivores with a different oviposition strategy, which might affect the consequences of JA-induced responses. JA is known to mediate the induction of chemical defense responses in plants to feeding damage and deposition of eggs (Dicke and Van Poecke, 2002; Hilker and Meiners, 2006; Mumm and Hilker, 2006). By using JA, we were able to examine the effects of induced defense responses in cabbage. Moreover, JA application has the advantage that visually detectable damage and the presence of herbivores or eggs are avoided. Finally, JA allows control over the strength of induction through controlled dosages. We hypothesized that JA treatment would inhibit the oviposition of the butterflies. We made solvent extracts to address the identity of the active plant compounds that influenced butterfly behavior. Rather than testing whole-leaf extracts, however, we extracted the glucosinolates from the surface of both control and JA-treated plants and tested the oviposition preference of the butterflies for these glucosinolate fractions on a neutral substrate. Furthermore, we included a control experiment to exclude a potential direct effect of JA on oviposition behavior. We addressed the following questions: (1) does JA treatment of cabbage plants affect host plant selection of the two Pieris butterfly species; (2) are there differences between solitary and gregarious butterflies; (3) does JA treatment affect glucosinolate levels in leaf surface extracts; and (4) do changes in glucosinolate levels determine the changes in oviposition preference?\nMethods and Materials\nPlants and Insects Brussels sprouts plants, Brassica oleracea var. gemmifera L. (Brassicaceae) cultivar Cyrus were grown from seed in a greenhouse in plastic pots (11\u2009\u00d7\u200911\u2009\u00d7\u200911\u00a0cm) at 20\u201328\u00b0C, 40\u201380% RH and a 16-hr light\/8-hr dark photoperiod. All experiments were conducted with 6- to 7-wk-old plants. Stock colonies of the large cabbage white P. brassicae and the small cabbage white P. rapae were maintained on Brussels sprouts in a climatized room at 20\u201322\u00b0C, 50\u201370% RH and a 16-hr light\/8-hr dark photoperiod.\nChemical Analysis Brussels sprouts plants were sprayed with 0.1\u00a0mM JA or control solution. JA ((\u00b1) jasmonic acid, purity >97%; Sigma-Aldrich, St Louis, MO, USA) was applied to the surface of the leaves, i.e., plants were sprayed with a JA solution with 0.1% Tween 20 until run-off or just with 0.1% Tween 20 for the control. The next day, glucosinolates (GLS) were extracted from the surface of the intact Brussels sprouts leaves. Each sample consisted of four leaves (between the third to sixth leaf from the base of a plant) that were cut at the base of the petiole. Directly after cutting, the lamina was dipped for 5\u00a0sec in 300\u00a0ml of dichloromethane, and after a 5-sec interval, it was dipped for 5\u00a0sec in 150\u00a0ml of methanol (St\u00e4dler and Roessingh, 1990; Van Loon et al., 1992a; Griffiths et al., 2001). The methanol was evaporated from the crude methanol dip-volume with a rotary evaporator (IKA-Werke GmbH, Staufen, Germany). For each treatment, 11 plants were sampled. The extract was redissolved in methanol, desulphatased on a DEAE-Sephadex A25 column, and separated on a reverse phase C-18 column by using high performance liquid chromatography (HPLC) as described in Van Dam et al. (2004). Glucosinolate detection was performed with a photodiode array (PDA) detector (200\u2013350\u00a0nm) with 229\u00a0nm as the integration wavelength. Sinigrin (sinigrin monohydrate, ACROS, NJ, USA) was used as an external standard. We used the correction factors at 229\u00a0nm from Buchner (1987) and the EC (EC, 1990) to calculate the concentrations of the glucosinolates. Desulfoglucosinolate peaks were identified by comparison of HPLC retention times and UV spectra with standards kindly provided by M. Reichelt, MPI Chemical Ecology and a certified rape seed standard (Community Bureau of Reference, Brussels, code BCR-367R). The surface area was measured directly after dipping, and the dry mass of the leaves was measured after drying at 50\u00b0C for 72\u00a0hr. The GLS content was calculated in pmol per cm2 leaf material.\nHerbivore Oviposition Preference Test Pieris adults emerged from pupae in a large oviposition cage (67\u2009\u00d7\u2009100\u2009\u00d7\u200975\u00a0cm) in a greenhouse compartment at 22\u201324\u00b0C and 50\u201370% RH. Apart from natural daylight, cages were illuminated by sodium vapor lamps (type SON-T, 500\u00a0W, Philips, The Netherlands) from 8:00\u00a0a.m. until 2:00\u00a0p.m. In this cage, they were provided with a 10% sucrose solution and an oviposition substrate; depending on the experiment, a plant or an artificial leaf made of green cardboard paper sprayed with sinigrin. For the experiments, one male and one female butterfly were introduced per oviposition cage (67\u2009\u00d7\u200950\u2009\u00d7\u200975\u00a0cm) in the same greenhouse compartment, on the day before the experiment. In these cages, the butterflies were also provided with sucrose solution. At 8:30\u00a0a.m., the treated leaves or papers and respective controls were introduced into the cages, and the butterflies were allowed to oviposit until the beginning of the afternoon. At 2:00\u00a0p.m., the leaves were removed, and the number of eggs was counted. The experiments were carried out in several cages per day and 3\u20134\u00a0d per treatment with new pairs of butterflies each day, adding up to a total of 24\u201336 independent replicates.\nSurface Application of JA The effect of JA-induced changes in Brussels sprouts plants on butterfly behavior was tested in oviposition experiments with P. brassicae and P. rapae. Three concentrations of JA solution, 0.01, 0.1, and 1\u00a0mM, corresponding to approximately 1.25, 12.5, and 125\u00a0\u03bcg JA\/g fresh weight (or 0.25, 2.5, and 25\u00a0nmol JA\/cm2) respectively, were sprayed on the plants and tested against a control (plants treated with 0.1% Tween 20). The next morning, just before the start of the experiment, the fourth, fifth, and sixth leaves from the base of the plants were cut, and their petioles were placed directly into a vial with tap water and introduced into the cages with butterflies.\nSystemic Uptake of JA For P. rapae, two application methods were used to assess the effect of JA-induced changes in Brussels sprouts on oviposition preference. For the second application method, the fourth, fifth, and sixth leaves were cut from untreated plants and placed in a 0.1-mM aqueous JA solution 22\u00a0hr before the start of the experiment. Total uptake of the solution was on average 6.3\u2009\u00b1\u20091.5\u00a0ml per control leaf and 6.0\u2009\u00b1\u20091.6\u00a0ml for JA-treated leaves (corresponding to approximately 20\u00a0\u03bcg JA\/g fresh weight or 5\u00a0nmol JA\/cm2, assuming homogeneous distribution over the leaf tissue after uptake).\nEffect of Pure JA on Oviposition Preference In the next experiment, green cardboard paper sprayed with an oviposition stimulant was used to test the effect of pure JA on the oviposition behavior of P. rapae on an inert substrate. Sinigrin has been shown to be a suitable oviposition stimulant for Pieris butterflies (Van Loon et al., 1992a) and was therefore used to stimulate oviposition on the artificial substrate in this experiment. The paper (8\u2009\u00d7\u200911.5\u00a0cm) was treated with 1\u00a0ml of a 5-mM sinigrin solution (Janssen Pharmaceutica, Tilburg, The Netherlands) by spraying it with a Desaga chromatographic sprayer (Heidelberg, Germany). Subsequently, after drying, papers were sprayed with either 1\u00a0ml of a 1-mM JA solution or water (control substrates) just before the test (210\u00a0\u03bcg JA\/carton or 11\u00a0nmol JA\/cm2).\nBioassays with Purified Glucosinolate (GLS) Fractions GLS were extracted from the leaf surface as described for the chemical analysis. For each treatment, control and 0.1\u00a0mM JA, 60 plants were used for the extraction, of which four to five leaves per plant were dipped. Subsequently, the extracts were fractionated following the protocol of S\u00f8rensen (S\u00f8rensen, 1990). The GLS fractions were stored in the freezer until analysis. The GLS were dissolved in methanol to make two concentrations, one corresponding to the amount of GLS extracted from the material of two plants in 0.8\u00a0ml and one concentration corresponding to the amount of GLS from one leaf in 0.8\u00a0ml. Hereafter, we will express these concentrations in gram leaf equivalents (gle). One gle corresponds to the amount of GLS extracted from 1\u00a0g fresh and intact leaf. With an average weight of 6\u00a0g per leaf, the highest concentration corresponds to 48\u00a0gle and the lower concentration to 6\u00a0gle. With a sprayer, a volume of 0.8\u00a0ml of one of the solutions was sprayed on green paper following the same method as described above for the test of pure JA. P. rapae butterflies were offered a two-choice situation, with one paper sprayed with GLS extracted from control plants and one paper with GLS from JA-treated plants.\nPerformance of P. rapae Caterpillars The development of first instar caterpillars to pupae was observed on control and JA-treated plants. Control plants were sprayed with a 0.1% Tween 20 solution and JA-treated plants with a solution of 0.5\u00a0mM JA with 0.1% Tween 20. Thirty newly hatched P. rapae caterpillars were evenly distributed over two plants per treatment, 24\u00a0hr after treatment, and were placed in cages (67\u2009\u00d7\u200950\u2009\u00d7\u200975\u00a0cm) in a greenhouse compartment at 22\u201324\u00b0C and 50\u201370% RH. The plants were replaced with new plants twice a week, so that the maximum time between induction and larval feeding never exceeded 5\u00a0d. The number of days until pupation and pupal weight were recorded.\nStatistical Analyses Each individual butterfly female was subjected to a two-choice situation, in which most individuals oviposited on both control and JA-treated leaves. As the egg load differed between individuals, the number of eggs on each treatment per individual was treated as a paired sample. The oviposition data for P. rapae were normally distributed; therefore, they were analyzed with a paired t-test. The oviposition data for P. brassicae were not normally distributed, and therefore, analyzed with the nonparametric equivalent of the paired t-test, the Wilcoxon matched-pair signed-ranks test. The data on the developmental time of the caterpillars in the performance test were not normally distributed and were analyzed with a Mann\u2013Whitney U test for differences between the treatments. Pupal weight was normally distributed and analyzed with an analysis of variance (ANOVA). Changes in GLS content were analyzed with a Mann\u2013Whitney U test. Statistical analyses were performed with SPSS 11.0.\nResults\nChemical Analysis Five GLS were detected in B. oleracea leaf surface samples: glucoiberin, sinigrin, 4-hydroxyglucobrassicin, glucobrassicin, and 4-methoxyglucobrassicin (Table\u00a01). No significant difference was detected between JA-treated and control leaves for the total amount of GLS per cm2. The amounts of glucobrassicin, the most abundant glucosinolate in these samples, 4-methoxyglucobrassicin, and sinigrin, did not significantly differ between control and JA-treated leaves. The amounts of glucoiberin and 4-hydroxyglucobrassicin collected in the leaf surface extracts were significantly lower for JA-treated leaves compared to control leaves (Table\u00a01). The same results were obtained when calculated for the GLS content expressed as nmol per mg dry weight (not shown). \nTable\u00a01Glucosinolate content in surface extracts of Brassica oleracea leaves in pmol\/cm2 for control and JA-treated plantsCompoundControl Treatment Median (range)a,bJA Treatment Median (range)a,cZPGlucoiberin10.7 (6.3\u201321.7)0 (0\u20136.0)\u22122.7130.007Sinigrin9.3 (0\u201323.5)4.2 (0\u20136.8)\u22121.7740.0764-Hydroxyglucobrassicin0.7 (0\u20136.1)0 (0\u20130)\u22122.2070.027Glucobrassicin34.9 (18.4\u201398.2)88.6 (38.6\u2013132.5)\u22121.4790.1394-Methoxyglucobrassicin0.5 (0\u20132.5)0 (0\u20131.3)\u22121.3700.171Total amount of glucosinolates64.1 (27.0\u2013155.7)90.0 (42.9\u2013138.8)\u22120.5630.573aInterquartile range from first to third quartilebN\u2009=\u200910cN\u2009=\u200911\nHerbivore Oviposition Preference: Surface Application of JA As P. brassicae lays its eggs in batches, both the number of egg batches and the number of eggs per leaf were counted. For the 1\u00a0mM JA treatment, the number of batches was significantly lower on JA-treated leaves than on control leaves (N\u2009=\u200936, Z\u2009=\u2009\u22122.628, P\u2009=\u20090.009, Wilcoxon matched-pair signed-ranks test), and the total number of eggs was significantly lower as well (N\u2009=\u200936, Z\u2009=\u2009\u22122.035, P\u2009=\u20090.042, Wilcoxon matched-pair signed-ranks test). For the 0.1-mM JA treatment, the result was similar (N\u2009=\u200927, batches: Z\u2009=\u2009\u22122.223, P\u2009=\u20090.026; eggs: Z\u2009=\u2009\u22122.138, P\u2009=\u20090.032, Wilcoxon matched-pair signed-ranks test) (Fig.\u00a01). Experiments with 0.01\u00a0mM JA application did not show discrimination by the butterflies between the treated and control leaves (results not shown). Also, P. rapae butterflies significantly preferred to oviposit on control leaves compared to JA-treated leaves (Fig.\u00a02). The leaves treated with the two highest concentrations of JA tested, 1 and 0.1\u00a0mM, were avoided in favor of the control leaves (paired t-test, respectively, t\u2009=\u20093.805, df\u2009=\u200923, P\u2009=\u20090.001 and t\u2009=\u20093.681, df\u2009=\u200923, P\u2009=\u20090.001). The lowest concentration of JA tested, i.e., 0.01\u00a0mM, did not affect the distribution of eggs over the leaves (t\u2009=\u2009\u22120.662, df\u2009=\u200923, P\u2009=\u20090.52, paired t-test).\nFig.\u00a01Pieris brassicae oviposition on control and JA-treated plants. Two concentrations of JA were tested against a control. The box represents the interquartile range from first to third quartile, the line across the box indicates the median; asterisks indicate statistical differences between the preference for control and JA-treated plants (*P\u2009<\u20090.05, **P\u2009<\u20090.01, Wilcoxon matched-pair signed-ranks test). a Egg batches per female per leaf. b Eggs per female per leafFig.\u00a02Pieris rapae oviposition preference (measured as the number of eggs per female per leaf) between control and JA-treated B. oleracea. Three concentrations of JA were tested against a control in 24 replicated experiments for each concentration. Mean numbers of eggs per female + SEM are given; asterisks indicate statistical differences between the preference for control and JA-treated plants (n.s. P\u2009>\u20090.05, **P\u2009<\u20090.01, paired t-test)\nSystemic Uptake of JA In the experiment with P. rapae that employed systemic uptake of JA through the petiole, the same result was obtained: the number of eggs on the JA-treated leaves (10.0\u2009\u00b1\u20091.57) was lower than on the control (19.12\u2009\u00b1\u20092.82) leaves (t\u2009=\u20093.976, df\u2009=\u200931, P\u2009<\u20090.001, paired t-test).\nEffect of Pure JA on Oviposition Preference When paper treated with sinigrin and JA was compared to paper with only sinigrin, there was no difference in the number of eggs the butterflies deposited on the two substrates (t\u2009=\u2009\u22120.438, df\u2009=\u200926, P\u2009=\u20090.67, paired t-test) (Fig.\u00a03). These results show that the observed effect of the JA treatment on herbivore oviposition behavior was due to induced changes in leaf tissue rather than to a direct repellent or deterrent effect of JA itself.\nFig.\u00a03Oviposition of P. rapae on green paper sprayed with sinigrin plus JA and green paper with sinigrin only. Mean number of eggs per female per paper leaf + SEM (n.s. P\u2009>\u20090.05, paired t-test, N\u2009=\u200927)\nBioassays with Purified Glucosinolate Fractions The butterflies did not discriminate between the two GLS fractions (Table\u00a02), the number of eggs on paper with the GLS from control plants, and the number of eggs on paper with GLS from JA-treated plants was not different for either concentration (concentration 6\u00a0gle: Z\u2009=\u2009\u22121.514, N\u2009=\u200920, P\u2009=\u20090.130, Wilcoxon matched-pair signed-ranks test; 48\u00a0gle: t\u2009=\u2009\u22120.523, df\u2009=\u200919, P\u2009=\u20090.607, paired t-test) (Fig.\u00a04). \nTable\u00a02Glucosinolate (GLS) content in fractions from leaf surface extracts from control and JA-treated plants in pmol\/cm2, Z and P-values of Mann\u2013Whitney U testGlucosinolateGLS from control plantGLS from JA-treated plantGlucoiberin4.93.9Sinigrin3.15.14-HydroxyglucobrassicinNot detectedNot detectedGlucobrassicin16.081.04-MethoxyglucobrassicinNot detected0.8Fig.\u00a04Oviposition of P. rapae on green paper sprayed with purified GLS-fractions from leaf surface extracts of control and JA-treated plants. Mean number of eggs per female per paper leaf + SEM (n.s.\u2009P\u2009>\u20090.05, paired t-test, N\u2009=\u200920)\nPerformance of P. rapae Caterpillars About two-thirds of the caterpillars survived until pupation, and a similar number of caterpillars reached the pupal stage on both treatments, 19 on control and 18 on JA-treated plants (Fig.\u00a05). The caterpillars on JA-treated plants pupated on average after 15\u00a0d, while the caterpillars on the control plants pupated significantly sooner, on average after 13\u00a0d (Mann\u2013Whitney U, Z\u2009=\u2009\u22124.071, P\u2009<\u20090.001). The average pupal weight on the control plants, 165\u2009\u00b1\u20093.4\u00a0mg, was similar to that on the JA-treated plants, 158\u2009\u00b1\u20093.3\u00a0mg (ANOVA, F\u2009=\u20092.665, df\u2009=\u20091, P\u2009=\u20090.112).\nFig.\u00a05Development time of P. rapae caterpillars from hatching until pupation on control [open squares (\u25a1)] and JA-treated [closed triangles (\u25b4)] plants. Cumulative number of pupae per treatment\nDiscussion\nOur data show that JA treatment of Brussels sprouts leaves reduces the acceptance of leaves for oviposition by P. rapae and P. brassicae in a similar way. Treatment of leaves with 0.1 or 1\u00a0mM JA reduced the proportion of eggs the butterflies laid on these leaves. A concentration of 0.01\u00a0mM JA did not change oviposition preference. The former concentrations are comparable to the concentrations of JA or MeJA that were applied to several plant species in other studies and that reduced development of Spodoptera exigua, Trichoplusia ni, Manduca sexta, thrips, and aphids (Thaler et al., 1996; Avdiushko et al., 1997; Van Dam et al., 2000; Omer et al., 2001), and abundance of Manduca quinquemaculata, S. exigua, thrips, and flea beetles in the field (Kessler and Baldwin, 2001; Thaler et al., 2001). For cabbage plants, Lu et al. (2004) found inducible resistance in a susceptible Brassica species (Chinese cabbage, Brassica campestris L.) and induced susceptibility in a resistant Brassica species (common cabbage, B. oleracea) for Plutella xylostella L.\nTo exclude that JA itself caused the above effect, we tested the phytohormone on an inert substrate and studied two different application methods to the leaf material. We considered this an essential control that is lacking in other studies. The results of these experiments show that it was not JA itself that caused the difference in oviposition preference between control and JA-treated leaves, thus providing proof that processes in the plant induced by the JA treatment changed the acceptability of the leaves. It has also been reported previously that development of cabbage looper or tobacco hornworm larvae is not affected when MeJA is added to an artificial diet, but it is retarded when MeJA was applied to cabbage or tobacco plants (Avdiushko et al., 1997).\nIn leaf surface extracts of JA-treated and untreated Brussels sprouts, we found five glucosinolates. After JA application, glucobrassicin, the major glucosinolate in the B. oleracea cultivar we used, occurred at a level twice as high as in control plants, and glucoiberin and 4-hydroxyglucobrassicin concentrations decreased. The total glucosinolate content did not change significantly. However, most other studies on glucosinolate content in Brassicaceous plants after induction by JA- or MeJA-treatment or insect attack have reported an increase in glucosinolates, although there is substantial variation among different species, or even genotypes, and type of induction (Bodnaryk, 1994; Cipollini and Sipe, 2001; Mikkelsen et al., 2003; Mewis et al., 2005). Moreover, glucosinolates may not be evenly distributed throughout the leaf. We measured glucosinolate content in a surface extract after 24\u00a0hr, whereas most studies have measured glucosinolate content in whole leaf extracts and after a longer induction time. Recently, Reifenrath et al. (2005) postulated that the wax layer does not contain glucosinolates, and the polar glucosinolates that are found by using the solvent extraction method are washed from the inner leaf to the outside through the stomata. Nevertheless, we chose a surface extraction method because the butterflies retrieve chemosensory information from the leaf surface, as they do not damage the leaf before ovipositing. Surface extracts are thus likely to give a better reflection of the chemosensory information used than whole leaf extracts.\nBoth butterfly species distinguish between induced and non-induced leaves, most likely based on chemical differences, as JA-induced leaves do not display herbivore presence or damage. The different levels of two out of five glucosinolates in the surface extracts may provide a chemosensory basis for the oviposition preference observed, although the isolated GLS from the two treatments yielded no differences in acceptance of the paper for oviposition. While the isolated GLS on paper stimulated oviposition behavior, they appear not to be the main cue to discriminate between the JA-induced and non-induced cabbage plants.\nWe did not quantify other chemicals, stimulants, deterrents, or precursors that might mediate preference behavior, such as isothiocyanates, terpenoids, other glycosides, or amino acids (Huang et al., 1993; Renwick and Chew, 1994; Soldaat et al., 1996; Agrawal and Kurashige, 2003). Both P. rapae and P. brassicae can perceive a broad range of chemicals (Van Loon et al., 1992b; Hern et al., 1996). Electroantennogram responses to a range of plant volatiles are similar for both species (Van Loon et al., 1992b), although host plant selection by Pieris butterflies appears largely based on contact chemoreception rather than olfaction (Renwick and Chew, 1994). Host plant selection is suggested to depend on a balance of stimulants and deterrents and not just on the detection of presence or absence of particular compounds (Huang et al., 1993; Bruce et al., 2005). Therefore, glucosinolates, in combination with other stimulants or deterrents, may determine the acceptance of a host plant by the butterflies.\nThe octadecanoid pathway, in which JA is a key molecule, is involved in induction of synomones in response to oviposition and to herbivore damage (Meiners and Hilker, 2000; Dicke and Van Poecke, 2002; Hilker and Meiners, 2006; Mumm and Hilker, 2006). JA treatment of plants results in emission of synomones that attract natural enemies like predatory mites and parasitoids (Dicke et al., 1999; Hilker and Meiners, 2002; Van Poecke and Dicke, 2002; Hilker and Meiners, 2006). This attraction results in a higher natural enemy density around damaged plants, and therefore, it is advantageous for the herbivores to avoid oviposition on induced plants. Moreover, intact plants lack competitors, either intra- or interspecific. Herbivores may use induced plant cues to detect the presence or absence of other herbivores, especially because plant cues are, although less reliable, often easier to detect than cues from the herbivores themselves (Vet and Dicke, 1992).\nFurthermore, induced plants may affect herbivores directly by influencing the performance of their offspring. For P. rapae, the development time differed between caterpillars feeding on JA-induced and caterpillars feeding on non-induced plants. Development of the caterpillars to pupae took longer on the induced plants, which exposes them to natural enemies for a longer time and gives them a disadvantage in the competition for resources with other herbivores. These results comply with those of Agrawal and Kurashige (2003), who showed that growth of P. rapae larvae was reduced on herbivore-induced Brassicaceae.\nIn summary: (1) JA treatment of B. oleracea results in avoidance of host plants by the two Pieris butterflies; (2) the related gregarious and solitary butterfly species tested responded in a similar fashion to JA-treated plants; (3) JA treatment reduced the contents of two out of five glucosinolates in leaf surface extracts of Brussels sprouts; and (4) the purified GLS fractions could not explain the observed avoidance behavior. The results indicate that JA-induced infochemicals play an important role in host plant selection behavior of these butterflies; however, the phytochemicals involved still have to be elucidated.","keyphrases":["cabbage","pieris rapae","glucosinolates","pieris brassicae","performance","octadecanoid pathway","host plant selection"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Pediatr_Nephrol-4-1-2335295","title":"Improved renal survival in Japanese children with IgA nephropathy\n","text":"Since the beginning of the 1990s, Japanese medical practitioners have extensively prescribed angiotensin-converting enzyme (ACE) inhibitors for children with mild IgA nephropathy (IgA-N) and steriods for those with severe IgA-N. We have performed a retrospective cohort study to clarify whether the long-term outcome has improved in Japanese children with IgA-N. Renal survival was defined as the time from onset to end-stage renal disease (ESRD). We divided the study period into two time periods based on the occurrence of the initial renal biopsy:1976\u20131989 and 1990\u20132004. Actuarial survivals were calculated by Kaplan\u2013Meier method, and comparisons were made with the logrank test. The Cox proportional hazard model was used for multivariate analysis. Between 1976 and 2004, 500 children were diagnosed as having IgA-N in our hospitals. The actuarial renal survival from the time of apparent disease onset was 96.4% at 10 years, 84.5% at 15 years and 73.9% at 20 years. Renal survival in the 1990\u20132004 period was significantly better than that in 1976\u20131989 (p = 0.008), and a marked improvement in renal survival in patients with severe IgA-N was also observed (p = 0.0003). Multivariate analysis indicated that diagnosis year was a significant factor for ESRD-free survival independently of baseline characteristics. The results of this study show that there has been an improvement in terms of renal survival in Japanese children with IgA-N.\nIntroduction\nIgA nephropathy (IgA-N) is the most common primary glomerulonephritis worldwide. It was initially considered to be a benign disease with a favorable prognosis, but data from long-term follow-up studies subsequently revealed that the disease progressed to renal failure in 20\u201350% of adult patients [1, 2]. Although there has been a prevailing belief that the prognosis of IgA-N is more benign in children, the results of more recent studies do not support this [3]. At the beginning of the 1990s, a strict policy of treatment for IgA-N was adopted by the majority of physicians [4]; this consisted of the extensive use of angiotensin-converting enzyme inhibitors (ACEIs) as an almost universally used therapy for IgA-N, and other therapies, including corticosteroids, for more severe IgA-N [4]. This approach has also been adopted in Japan since the beginning of the 1990s, with ACEIs generally prescribed for children with mild IgA-N showing focal mesangial proliferation and steroids prescribed for children with severe IgA-N showing diffuse mesangial proliferation. Combined therapy with prednisolone, azathioprine, heparin\u2013warfarin and dipyridamole for 2\u00a0years in patients with severe IgA-N showing diffuse mesangial proliferation was started at the Kobe University and Wakayama Medical University hospitals in 1990 [5, 6]. At present, the effect of this therapy on long-term renal survival in children with IgA-N remains unknown as there have been few studies on outcome in sufficiently large cohorts of pediatric patients [4].\nIn the study reported here, we investigated data from 500 children with IgA-N to determine whether the long-term outcome has improved in Japanese children with IgA-N.\nMethods\nPatients\nThe medical history of children under the age of 20\u00a0years who underwent routine renal biopsies at Kobe University and Wakayama Medical University hospitals between January 1976 and December 2004 were analyzed retrospectively. Clinical data and follow-up information were obtained from the medical records, and if needed, further complementary information was obtained by telephone contact with the patients, family members and or physicians.\nA diagnosis of IgA-N was based on the presence of IgA as the sole or predominant immunoglobulin in the glomerular mesangium in the absence of systemic disease [7]. Diffuse or focal mesangial proliferation was defined on the basis of World Health Organization criteria [8]: diffuse mesangial proliferation was defined as more than 80% of glomeruli showing moderate or severe mesangial cell proliferation, i.e. more than three cells per peripheral mesangial area; focal mesangial proliferation was defined as less than 80% of glomeruli showing moderate or severe mesangial cell proliferation. All children with IgA-N were diagnosed by one investigator (N.Y.) using the same criteria during the entire study period. Renal biopsies were performed in children with persistent proteinuria (early morning urine protein\/creatinine ratio \u22650.2g\/g) with or without hematuria. Renal biopsy criteria were not changed during the entire study period.\nRenal survival was defined as the period from the time of apparent disease onset to end-stage renal disease (ESRD) requiring renal replacement therapy.\nGiven the change of policy for treating IgA-N patients in the early 1990s, we divided the study period when the initial renal biopsy had been performed into two time periods: 1976\u20131989 (early period) and 1990\u20132004 (late period).\nTreatment\nSince we started a strict policy of treatment for IgA-N at the beginning of the 1990s, we investigated the initial treatment profile of children with IgA-N. After a pilot study period, we started the first randomized controlled trial (RCT) by the Japanese Pediatric IgA Nephropathy Treatment Study Group (JPIGANTS) for treatment of children with severe IgA-N showing diffuse mesangial proliferation in 1990 [5]. We started the second RCT by the JPIGANTS for treatment of children with severe IgA-N in 1994 [6]. We started the third RCT by the JPIGANTS for treatment of children with severe IgA-N in 2001. The details of each treatment are shown in Table\u00a01.\nTable\u00a01Treatment in three randomized controlled trials for childhood IgA nephropathy with diffuse mesangial proliferationPeriod1990\u20131993 (first RCT)1994\u20131998 (second RCT)2001\u2013present (third RCT)Group 1PrednisolonePrednisolone (as in group 1 in the first period)Prednisolone\u00a0\u00a02\u00a0mg\/kg\/day, 4\u00a0weeks\u00a0\u00a02\u00a0mg\/kg\/day, 4\u00a0weeks\u00a0\u00a02\u00a0mg\/kg\/2\u00a0days, 4\u00a0weeks\u00a0\u00a02\u00a0mg\/kg\/2\u00a0days, 4\u00a0weeks\u00a0\u00a01.5\u00a0mg\/kg\/2\u00a0days, 4\u00a0weeks\u00a0\u00a01.5\u00a0mg\/kg\/2\u00a0days, 4\u00a0weeks\u00a0\u00a01\u00a0mg\/kg\/2\u00a0days, 21\u00a0months\u00a0\u00a01\u00a0mg\/kg\/2\u00a0days, 9\u00a0months\u00a0\u00a00.5\u00a0mg\/kg\/2\u00a0days, 12\u00a0monthsAzathioprineAzathioprine (as in group 1 in the first period)No azathioprine\u00a0\u00a02\u00a0mg\/kg\/day, 24\u00a0monthsDipyridamoleDipyridamole (as in group 1 in the first period)Dipyridamole\u00a0\u00a05\u00a0mg\/kg\/day, 24\u00a0months\u00a0\u00a06\u20137\u00a0mg\/kg\/day, 24\u00a0monthsHeparinNo heparin\u00a0\u00a0APTT 60\u00a0s, 28\u00a0daysWarfarinWarfarinWarfarin\u00a0\u00a0TT 30\u201350%, 23\u00a0months\u00a0\u00a0TT 30\u201350%, 24\u00a0months\u00a0\u00a0TT 20\u201350%, 24\u00a0monthsMizoribine\u00a0\u00a04\u00a0mg\/kg\/day, 24\u00a0monthsGroup 2Heparin-WarfarinDipyridamole (as in group 1)Prednisolone (as in group 1)Prednisolone (as in group 1)Mizoribine (as in group 1)TT, Thrombotest; APTT, activated partial thromboplastin time; RCT, randomized controlled trial\nFor the treatment of children with mild IgA-N showing focal mesangial proliferation we started a RCT by the JPIGANTS in 1990. During this trial (1990\u20131993) about half of the children with mild IgA-N in our hospitals received Sairei-to, a Chinese herb, for 24\u00a0months, and the other half received no medication. We began to use ACEIs for treatment of mild IgA-N with focal mesangial proliferation as the first choice from the beginning of 2000.\nStatistical analyses\nThe results were analyzed with SAS ver. 9.1.2 (SAS Institute Japan Ltd., Tokyo, Japan). The associations between categorical variables were examined using the Fisher\u2019s exact test. Continuous characteristics of the groups were compared with the Mann-Whitney U-test. Actuarial survival curves were calculated according to the Kaplan-Meier method [9], and comparisons were made with the logrank test [10]. Univariate and multivariate analyses were performed to assess the difference between the two periods in terms of renal survival. For multivariate analysis, we used the Cox proportional hazard model [11]. A two-tailed p value of less than 0.05 was taken as the level of significance.\nResults\nPatients\nBetween 1976 and 2004, 1759 children underwent a first renal biopsy examination at the Kobe University and Wakayama Medical University hospitals. Among these, 500 Japanese children (28.4%; 279 boys and 221 girls) were diagnosed as having IgA-N: 219 in 1976\u20131989 and 281 in 1990\u20132004. There was no evident change in the number of patients per year between the early (1976\u20131989) and late (1990\u20132004) periods. The median patient age at diagnosis was 10.9\u00a0years (range 2.5\u201319.6\u00a0years), and the median follow-up period for the patients overall was 5.9\u00a0years (range 1.3\u201320.5\u00a0years).\nThe baseline characteristics of children with IgA-N are shown in Table\u00a02. There were significant differences in some characteristics between the two periods. Age at diagnosis had a tendency to be higher in the late group, while the ratio of asymptomatic proteinuria and hematuria at initial presentation was significantly higher in the late group. The ratio of heavy proteinuria (\u2265 1\u00a0g\/m2 per day) at diagnosis was significantly higher in the early period, but the ratio of patients showing diffuse mesangial proliferation was higher in the late period. Based on these data, we concluded that there was no evident difference in disease severity between the two periods.\nTable\u00a02Baseline characteristicsCharacteristicNumber of patients (%)pTotal (n\u2009=\u2009500)1976\u20131989 (n\u2009=\u2009219)1990\u20132004 (n\u2009=\u2009281)Sex (M\/F)279\/221132\/87147\/1340.08Age at diagnosis, year, median [range]10.9 [2.5\u201319.6]10.1 [3.4\u201316.8]11.6 [2.5\u201319.6]< 0.001Initial presentationAsymptomatic proteinuria and hematuria384 (76.8%)150 (68.5%)234 (83.3%)< 0.001Macroscopic hematuria93 (18.6%)60 (27.4%)33 (11.7%)< 0.001Edema23 (4.6%)9 (4.1%)14 (5.0%)0.67Proteinuria at diagnosis (g\/m2\/day)\u00a0\u00a0<1361 (72.2%)146 (66.7%)215 (76.5%)0.008\u00a0\u00a0\u22651139 (27.8%)73 (33.3%)66 (23 .5%)Estimate creatinine clearance at diagnosis (ml\/min per 1.73\u00a0m2)\u00a0\u00a0<6013 (2.6%)3 (1.4%)10 (3.6%)0.16\u00a0\u00a0\u226560487 (97.4%)216 (98.6%)271 (96.4%)Renal biopsy at diagnosis\u00a0\u00a0 Diffuse mesangial proliferation171 (34.2%)63 (28.8%)108 (38.4%)0.03\u00a0\u00a0 Focal mesangial proliferation329 (65.8%)156 (71.2%)173 (61.6%)\nTreatment\nThe initial treatment for IgA-N showing focal mesangial proliferation or diffuse mesangial proliferation is given in detail in Table\u00a03. There was a clear change of treatment for IgA-N between the early and late periods. This is particularly true in terms of ACEI use for focal mesangial proliferation and combined therapies for diffuse mesangial proliferation, both of which increased dramatically during the late period.\nTable\u00a03Change of initial treatment for IgA nephropathy in Japanese childrenTreatmentFocal mesangial proliferationDiffuse mesangial proliferation1976\u20131989 (n\u2009=\u2009156)1990\u20132004 (n\u2009=\u2009173)1976\u20131989 (n\u2009=\u200963)1990\u20132004 (n\u2009=\u2009108)No treatment96 (61.6%)23 (13.2%)19 (30.2%)1 (0.9%)Antiplatelet and\/or anticoagulant35 (22.4%)2 (1.2%)14 (22.2%)7 (6.5%)Prednisolone, (\u00b1 antiplatelet and\/or anticoagulant)4 (2.6%)6 (3.5%)7 (11.1%)25 (23.1%)Prednisolone + immunosuppressant, (\u00b1 antiplatelet and\/or anticoagulant)7 (4.5%)8 (4.6%)19 (30.2%)74 (68.5%)Chinese herb (Sairei-to)14 (9.0%)46 (26.5%)4 (6.3%)0 (0.0%)ACEI and\/or ARB0 (0.0%)88 (50.9%)0 (0.0%)1 (0.9%)ACEI, Angiotensin-converting enzyme inhibitor; ARB, angiotensin II receptor blocker\nRenal survival\nAmong a total of 500 patients who had IgA-N therapy, the actuarial renal survival from the time of apparent disease onset was 96.4% at 10\u00a0years, 84.5% at 15\u00a0years and 73.9% at 20\u00a0years (Table\u00a04). As shown in Fig.\u00a01 and Table\u00a04, in children diagnosed as having IgA-N in 1976\u20131989, the actuarial renal survival from the time of apparent disease onset was 94.0% at 10\u00a0years, 80.1% at 15\u00a0years and 70.1% at 20\u00a0years. In children diagnosed as having IgA-N in 1990\u20132004, the actuarial renal survival from the time of apparent disease onset was 98.8% at 10\u00a0years, 98.8% at 15\u00a0years (p\u2009=\u20090.008; Fig.\u00a01, Table\u00a04).\nTable\u00a04Actuarial renal survival analysis of 500 children with IgA nephropathy\u00a0Initial renal biopsy yearNumber of patients10-year renal survival15-year renal survival20-year renal survivalpaTotal1976\u2013200450096.4%84.5%73.9%Total1976\u2013198921994.0%80.1%70.1%0.0081990\u2013200428198.8%98.8%\u2013Diffuse mesangial proliferation1976\u201319896378.5%68.6%b\u20130.00031990\u2013200410897.8%97.8%b\u2013Focal mesangial proliferation1976\u20131989156100.0%97.7%\u20130.51990\u20132004173100.0%100.0%\u2013ap values on the logrank testbThe 13-year survivalFig.\u00a01Kaplan\u2013Meier plot of renal survival stratified by the initial biopsy year for children with IgA nephropathy. 95% CI 95% Confidence interval\nFigures\u00a02 and 3 show the long-term outcome for children in the two different mesangial proliferation groups. For children with severe IgA-N showing diffuse mesangial proliferation, both the 10- and 13-year renal survivals were 97.8% when the diagnosis was made in the period 1990\u20132004; when the diagnosis was made in the period 1976\u20131989, renal survivals were 78.5% and 68.6%, respectively (p\u2009=\u20090.0003; Fig.\u00a02, Table\u00a04). For children with mild IgA-N showing focal mesangial proliferation, both the 10- and 15-year renal survivals were 100.0% when diagnosis was made in 1990\u20132004, compared with 100.0% and 97.7%, respectively, in 1976\u20131989 (p\u2009=\u20090.5; Fig.\u00a03, Table\u00a04). Although we observed better renal survival in patients diagnosed in the period 1990\u20132004 than in the period 1976\u20131989 for children with IgA-N showing focal mesangial proliferation, the difference did not reach statistical significance. The children with IgA-N showing diffuse mesangial proliferation in 1990\u20132004 had excellent long-term renal survival.\nFig.\u00a02Kaplan-Meier plot of renal survival stratified by the initial biopsy year for children with severe IgA nephropathy showing diffuse mesangial proliferationFig.\u00a03Kaplan-Meier plot of renal survival stratified by the initial biopsy year for children with mild IgA nephropathy showing focal mesangial proliferation\nTable\u00a05 shows the results of the univariate analysis using the logrank test and the multivariate analysis using the Cox proportional hazard model of prognostic factors for ESRD-free survival. Mesangial proliferation degree (focal or diffuse), proteinuria at diagnosis (<1 or \u22651\u00a0g\/m2 per day), estimate of creatinine clearance at diagnosis (\u226560 or <60\u00a0ml\/min per 1.73\u00a0m2) and initial renal biopsy (diagnosis) year (1976\u20131989 or 1990\u20132004) were included as factors for analyses. Mesangial proliferation degree and initial renal biopsy year were significant in both the univariate and the multivariate analysis. For children with IgA-N, the most influential prognostic variable was mesangial proliferation degree. Proteinuria at diagnosis was significant in the univariate but not in the multivariate analysis. These results from the multivariate analysis showed that initial renal biopsy year was a significant factor for ESRD-free survival independently of mesangial proliferation degree, proteinuria at diagnosis and estimate of creatinine clearance at diagnosis (hazard ratio = 0.08, 95% CI 0.004\u20130.43; Table\u00a05).\nTable\u00a05Univariate and multivariate analysis of the prognostic value of factors for end-stage renal disease-free survivalFactorUnivariateMultivariateHR95% CIpHR95% CIpMesangial proliferation focal; diffuse10.922.80\u201372.46<0.00110.272.42\u201370.750.001Proteinuria at diagnosis <1; \u22651 (g\/m2\/day)5.271.65\u201319.770.012.140.57\u20138.780.26CCl at diagnosis \u2265 60; < 60 (ml\/min per m2)14.122.11\u201357.410.015.580.74\u201330.220.09Initial renal biopsy year 1976\u20131989; 1990\u201320040.140.01\u20130.740.020.080.004\u20130.430.002CI, Confidence interval; HR, hazard ratio; CCl, creatinine clearance\nDiscussion\nAlthough this was a retrospective study, the data seem to provide unique and valuable information about IgA-N in children for several reasons. First, the study subjects were a complete non-selected cohort of patients with IgA-N from among all children who underwent first renal biopsy examinations at Kobe University and Wakayama Medical University hospitals between 1976 and 2004. Five hundred children with IgA-N participated in our study, which to our knowledge is the largest series used to investigate the outcome of IgA-N in children to date. Furthermore, these patients were diagnosed histologically by a single investigator (N.Y.) based on the same criteria during the whole study period. Renal biopsy criteria were also unchanged during the whole study period. Therefore, the cohort in the present study is considered to have been rigidly homogeneous in terms of the analysis of the disease outcome.\nOne of the conditions that facilitated our study was the school screening program, which was started by the Japanese government in 1974. In Japan, all children between the ages of 6 and 18\u00a0years are screened annually, and those found to have urinary abnormalities are referred for further investigation. Thus, in general, treatment for IgA-N is started early in the course of disease because the duration of disease before treatment is short as a result of this school screening program [12]. Therefore, in this study we were able to observe the disease courses of patients during the initial renal biopsy period, 1976\u20132004, who underwent initial treatments for IgA-N at a relatively homogeneous stage of the disease. Accumulated experience indicates that long-term corticosteroid and\/or immunosuppressive treatment during the severely progressive stage of the disease does not confer any benefit in adult patients [2]. In contrast, it is known that centers in countries with active urine screening programs are more likely to diagnose mild disease with a good prognosis, thus favorably influencing the overall outcome of the cohort [13].\nAnother favorable parameter of this study was the enforcement of consecutive systemic nationwide clinical trials of IgA-N. After a pilot study period, the first RCT for treatment of children with severe IgA-N showing diffuse mesangial proliferation was started by the JPIGANTS in 1990 [5]. To date, two RCTs of treatment for children with severe IgA-N showing diffuse mesangial proliferation by the JPIGANTS have been completed [5, 6], and the third RCT of treatment for children with severe IgA-N showing diffuse mesangial proliferation is currently being conducted by the same group. For the treatment of children with mild IgA-N and focal mesangial proliferation, a RCT was also started by the JPIGANTS in 1990. After completion of the RCT, we have conducted prospective clinical trials of treatment for children with mild IgA-N and focal mesangial proliferation. These prospective trials have enabled for us to clarify the optimum treatment for IgA-N and to collect precise information on patients with IgA-N.\nBecause of the variable rate of progression to renal failure and the probable multifactorial pathogenesis of IgA-N, it is desirable to evaluate the effectiveness of any treatment by a prospective controlled trial. However, although the ultimate endpoint in any clinical trial of progressive IgA-N is the development of chronic renal insufficiency, most pediatric patients do not develop it during the study period [14]. Therefore, data from a properly analyzed long-term retrospective study may also be important to evaluate disease outcome and treatment effectiveness [4, 9\u201311]. Retrospective studies with accurate statistical evaluation using life-time analysis and multivariate survivorship analysis according to the Cox regression model are thought to work effectively together with well-designed RCTs.\nThe primary purpose of this study was to clarify whether long-term outcome was improved in Japanese children with IgA-N retrospectively using adequate statistical methods. Although it seemed that there were no clinically significant differences in the baseline characteristics of the groups, there were some statistically significant differences in the baseline characteristics of the groups between the two periods when initial renal biopsies were performed. These different baseline characteristics may influence the final outcomes reported in this study. To compensate for this limitation, we used a multivariate survivorship analysis according to the Cox regression model. Since we have to adjust for differences of baseline characteristics, which were thought to be important prognostic factors [15], mesangial proliferation degree, proteinuria at diagnosis, estimate of creatinine clearance at diagnosis and the initial renal biopsy (diagnosis) year were included as factors for analyses. This multivariate analysis indicated that the initial renal biopsy year was a significant factor for ESRD-free survival after adjustment of these baseline characteristics. These data support an improvement of renal survival in Japanese children with IgA-N.\nThere was a notable lack of the usual dominance of males over females in our study and, in addition, there was a difference between the 1976\u20131989 and the 1990\u20132004 periods, with more females being recruited in the second period. Although this difference was not statistically significant (p\u2009=\u20090.08), we decided that it be worthwhile discussing possible reasons for the differences in gender representation between the two periods. Therefore, we analyzed males and females separately. However, there was no gender-based factor in the results (data not shown).\nAt the beginning of the 1990s, a strict policy of treatment was adopted by the majority of physicians in Japan. It is likely that therapeutic intervention early after the onset of clinically apparent disease will provide the best opportunity for improving the outcome of patients with IgA-N, thus reducing the number of patients who develop ESRD [5, 6]. This study showed a clear change in the treatment of IgA-N between the early and late groups (Table\u00a03). In particular, the use of ACEIs for focal mesangial proliferation and combination therapies for diffuse mesangial proliferation increased dramatically in the late group.\nCorticosteroids have been widely used to treat moderate to severe IgA-N, particularly in pediatric patients. Some evidence has been obtained recently pertaining to the role of corticosteroids in the treatment of IgA-N [2, 13, 14, 16\u201318]. With regard to children and based on the results of two multicenter RCTs, we reported previously that the treatment of childhood IgA-N with diffuse mesangial proliferation using prednisolone, azathioprine, warfarin and dipyridamole for 2\u00a0years early in the course of disease reduced the severity of immunologic renal injury and prevented any increase in the percentage of sclerosed glomeruli [5, 6]. In contrast, in the first RCT, heparin\u2013warfarin and dipyridamole treatments for 2 years did not reduce urinary protein excretion, serum IgA concentration and mesangial IgA deposition, and they did not prevent any increase of sclerosed glomeruli [5]. The results of the second RCT showed that the treatment of children with severe IgA-N using prednisolone alone for 2\u00a0years reduced the severity of immunologic renal injury, but did not prevent any further increase of glomerular sclerosis [6]. Therefore, treatment with the combination therapy using prednisolone and immunosuppressant, such as azathioprine, may be better than the prednisolone monotherapy for patients with severe IgA-N.\nWe found an improvement of long-term renal survival in Japanese children with IgA-N (Table\u00a04, Figs.\u00a01 and 2). Renal survival in 1990\u20132004 was significantly better than in 1976\u20131989 (Fig.\u00a01), and a marked improvement of renal survival in IgA-N showing diffuse mesangial proliferation was observed over time (Fig.\u00a02). This improvement may be related to the 2-year therapy, including corticosteroids, for all patients with IgA-N showing diffuse mesangial proliferation as a treatment policy, although in principle the effectiveness of any treatment can only be evaluated properly by a controlled trial. It is conceivable that the outcome in the period 1976\u20131989 may have reflected the natural course of disease, whereas that in 1990\u20132004 may have reflected modification of the disease course by the treatments.\nThe median follow-up period of the patients overall was 5.9\u00a0years (range 1.3\u201320.5\u00a0years). Although this is a considerably long period, it may not be sufficiently long for the follow-up of IgA-N, which has a slow progressive course. Since the elapsed time from apparent disease onset to ESRD was used to evaluate the disease outcome, chronic renal insufficiency before ESRD was not considered in our study. Thus, we do not know whether the observed improvement of outcome means a complete cure of the disease or merely a delay of the disease course. Further follow-up of the cohort for a long period is therefore important.\nOne aspect which we should consider in our study is the possibility that at least some of the differences in outcome between the early and late groups may be due to general changes in care that have occurred over time. Such changes are frequently not recognized, such as a gradually greater awareness of managing marginally raised blood pressure, among others.\nIn conclusion, this study has demonstrated a substantial improvement of long-term renal survival in Japanese children with IgA-N. On the basis of the results of this study we were unable to provide the reason for this improved outcome directly; however, it is likely that adequate management using a strict policy of treatment may have been responsible.","keyphrases":["corticosteroid","mesangial proliferation","proteinuria","ace inhibitors","immunosuppressive drug"],"prmu":["P","P","P","R","M"]}
{"id":"Bioinformation-2-2-2174421","title":"DoD2007: 1082 molecular biology databases\n","text":"Molecular biology databases are an integral part of biological research. To date, many databases were established with varied options to access associated biological data. Depending on the data being annotated, some are architecturally similar while others are specialized. In order to provide a partial solution to data integration, we report Database of Databases (DoD2007), constructed using html and javascript. The database has a web-based user interface with simple global search, specific database search, keyword help as well as links to abstracts, full-text and database home pages. Majority of data were derived form Nucleic Acids Research database issue and other published resources. The current release includes 15 categories with updated descriptions and links to 1082 databases, of which, 209 are new entries. New databases included in this issue are represented with \u2018+\u2019 sign before the name and a \u2018*\u2019 symbol provided for those that remained silent.\nBackground\nMolecular Biology databases have become an integral part of scientific research. They are widely used to understand the underlying mechanism of genomes, expression patterns, bimolecular \ninteractions, metabolism, understanding evolutionary relationships etc. as well as providing knowledge that helps to examine specific state of a disease or condition and assist in drug discovery \nand development. This sort of biological knowledge is disseminated to a variety of scientific researchers through specialized databases made possible through internet technologies, software's and \ntools. As more and more genomes are being sequenced and annotated, huge amount of data are accumulating. [1] Biological databases designed would \ncater to meet the needs of the scientific community. But depending on the data being annotated, some are architecturally similar while the others are specialized. The challenges to develop an integrated \nsystem are due to several factors such as variety and amount of data available and data heterogeneity in different sources. [2] Therefore, \ndata integration has proved problematic and to provide a partial solution, we report an update on collection of molecular biology databases with a search interface, Database of Databases (DoD2007). \nThe latest edition, DoD2007 was compiled with similar capabilities [3,4] but with additional features such as a global search for all databases \nand links to database entries from category list, added patents database as a new category and one sub-category each in nucleotide, Genomics database and two in Protein Sequence database, respectively.\nMethodology\nDoD2007 was updated from the reported database issue of Nucleic Acids Research [5] and various journal sources. The 14 categories reported in \nour previous update [4] remain unchanged except a new category; \u2018patents db\u2019 has been incorporated in DoD2007 update, keeping in view the importance \nof patent rights in scientific discipline. Currently, this category includes two databases, patome [6] and DNA patents database. [7] \nDoD2007 was constructed using html and javascript. The modes of access of databases listed in DoD2007 vary depending on the content, format and access methods. Majority of the databases are provided with \ndirect access to search data from DoD2007. Some databases require multiple search items while some others require sequence or annotation to be pasted in the search box and therefore they are provided with a \ndirect link to the database home page. Over time, DoD2007 has come to enhance the listed molecular biology databases as its use by researchers, educators and students in diverse disciplines has expanded. \nThe recent release DoD2007 contains three distinct newly developed functionalities, which are outlined below. \nFirst, we have developed a user interface to search databases globally. In other words, a global search can be utilized to search the categories reported in DoD2007, database names and other pages located in \nthe database. Keywords used to search the database can be general or specific and depends on the terms indexed for search. \nSecond, an organized category list was created in a separate page. The availability of these databases as a list is intended to promote better understanding of database segregation used to generate \ncategories and to quickly recognize the new ones with those that are inaccessible. These databases have links to the database entries because a link back to the main page makes it easy for both new and \nproficient users to conduct a variety of searches. However, some users may not realize the keywords that need to be used to search a database and even some users may show interest in the published resource. \nHence, a keyword help and links to abstract or full-text articles were created in respective main pages for intuitive and efficient presentation. Finally, a number of sub-categories are created such \nas \u2018Operons\u2019 and \u2018Comparative genomics\u2019 in \u2018nucleotide db\u2019 and \u2018Genomics db\u2019 respectively along with two sub-categories viz. \u2018Protein Interactions\u2019 and \u2018Amino Acid Repeats\u2019 in \u2018Protein Sequence db\u2019.\nFeatures of DoD2007\nDoD2007 is a unique, categorized and easy-to-use interface for all molecular biology databases. New databases in various categories were appended and the number of databases in DOD2007 reached up to 1082 that are 209 databases ahead \nfrom the previous issue. Major additions, 49 new entries, were reported in Genomics database. However, the list is not complete. A brief description of databases under all categories was given as a pop-up window with a provision to view \nfull-text of the published article and a direct link to the database home page. The search option under \u2018link to database\u2019 leads to the respective home pages of the database. Boolean search items are not allowed in the DoD2007 but spaces \nbetween search strings enables the search. During the search, results are displayed in a separate window as this helps in further database scan without losing the home page.\nDoD2007 facilitates integrated database search to scan a number of relevant databases from respective categories. A few databases incorporated in DoD2007 require user log-in or restricted access and a few are inaccessible or \ndiscontinued. To distinguish such databases, new entries in DoD2007 are provided with a \u2018+\u2019 sign and inaccessible ones are recognized by a \u2018*\u2019 sign before the name. The current list of database categories and the number of entries in \neach category are given in table 1 (supplementary material). An image of the database is given in figure 1. Since its first appearance in the year 2005, DoD constituted 719 molecular biology databases and the year 2005 resulted in an \naddition of 154 entries making up to 873 databases (DoD2006) and the year 2006 has 209 new entries being appended in DoD2007. On the other hand, nearly 4 percent of databases are inaccessible such as HLA Ligand\/Motif database \n[8] of Immunological database or discontinued while few others remained silent such as European rRNA Database [9] \nand Small RNA Database [10] of RNA sequence database etc.\nOn an average, there is a continuous increase in the number of databases which specifies the importance of such databases to biological community. Therefore, in order to account for the rise in number of databases, a graph as shown in \nfigure 2 was plotted for each category since year 2004. The displayed graph showed a substantial increase in Nucleotide, Protein and Genomics databases respectively.\nUtility\nDoD2007 provides the updated descriptions and links to existing and new databases that serves as an interface and user-friendly access to molecular biology scientific community. Number of inaccessible databases reported in the \nyears 2005 and 2006 remain unchanged in DoD2007 so as to enable the users to know the type of database that once existed.\nConclusion\nDoD2007, a freely available web-based database resource enabling ease of access, serves as a general reference source both for community of researchers working in molecular biology and educators who deal with particular databases \nof their interest. Brief description about the databases and keyword help makes the users familiar with the contents of the database, respective keywords and database links. The database shall be updated on a yearly basis.\nSupplementary material\nData 1","keyphrases":["molecular biology","database","data integration","javascript"],"prmu":["P","P","P","P"]}
{"id":"Eur_J_Epidemiol-3-1-2071967","title":"The Rotterdam Study: objectives and design update\n","text":"The Rotterdam Study is a prospective cohort study ongoing since 1990 in the city of Rotterdam in the Netherlands. The study targets cardiovascular, neurological, ophthalmological and endocrine diseases. As of 2008 about 15,000 subjects aged 45 years or over comprise the Rotterdam Study cohort. The findings of the Rotterdam Study have been presented in some 600 research articles and reports (see http:\/\/www.epib.nl\/rotterdamstudy). This article gives the reasons for the study and its design. It also presents a summary of the major findings and an update of the objectives and methods.\nIntroduction\nThe Rotterdam Study was designed in the mid-1980s as a response to the demographic changes that were leading to an increase of the proportion of elderly people in most populations [1]. It was clear that this would produce a strong rise in elderly people living with diseases, as most diseases cluster at the end of life, and that to discover the causes of diseases in the elderly one would have to study risk factors of those diseases [2]. A major approach to finding causes is the prospective follow-up study, which has proven quite effective in finding causes of heart disease and cancer. Remarkably, in the 1980s there were hardly any follow-up studies focussing on the elderly.\nThe design of the Rotterdam Study\nThe basic design of the study was straight-forward: a prospective cohort study among 7,983 persons living in the well-defined Ommoord district in the city of Rotterdam in the Netherlands (78% of 10,215 invitees). They were all 55\u00a0years of age or over and the oldest participant at the start was 106\u00a0years [3]. The study started with a pilot phase in the second half of 1989. From January 1990 onwards participants were recruited for the Rotterdam Study. Figure\u00a01 gives a diagram of the various cycles of the study.\nFig.\u00a01Diagram of examination cycles of the Rotterdam Study (RS). RS1 refers to the baseline examination of the original cohort (pilot phase 07\/1989-12\/1989; cohort recruitment 01\/1990-09\/1993). RS2, RS3 and RS4 refer to re-examination of the original cohort members. RSPlus1 refers to the extension of the cohort with persons in the study district that became 55\u00a0years since the start of the study or those of 55\u00a0years or over that migrated into the study district. RSPlus2 refers to the re-examination of the extension cohort. RSYoung1 refers to the baseline examination of all persons aged 45 and over living in the study district that had not been examined (i.e. mainly comprising those aged 45\u201355\u00a0years)\nIn 1999, 3,011 participants (out of 4,472 invitees) who had become 55\u00a0years of age or moved into the study district since the start of the study were added to the cohort.\nIn 2006, a further extension of the cohort was initiated in which about 6,000 subjects aged 45\u201354\u00a0years, living in the Ommoord district, were invited (expected number of participants about 4,000). By the end of 2008, the Rotterdam Study is therefore expected to comprise about 15,000 subjects aged 45\u00a0years or over.\nThe participants were all examined in some detail at baseline. They were interviewed at home (2\u00a0h) and then had an extensive set of examinations (a total of 5\u00a0h) in a specially built research facility in the centre of their district. These examinations focussed on possible causes of invalidating diseases in the elderly in a clinically state-of-the-art manner, as far as the circumstances allowed. The emphasis was put on imaging (of heart, blood vessels, eyes, skeleton and later brain) and on collecting bodily fluids that enabled further in-depth molecular and genetic analyses. These examinations were repeated every 3\u20134\u00a0years in characteristics that could change over time. And so we had examination cycles from 1990 to 1993, from 1993 to 1995, from 1997 to 1999, from 2000 to 2001, from 2002 to 2004, from 2004 to 2005 and from 2006 to 2008 (Fig.\u00a01).\nThe participants in the Rotterdam Study are followed for a variety of diseases that are frequent in the elderly (and many are also in the not so elderly): coronary heart disease, heart failure and stroke, Parkinson disease, Alzheimer disease and other dementias, depression and anxiety disorders, macular degeneration and glaucoma, diabetes mellitus and osteoporosis.\nThe Rotterdam Study has been approved by the institutional review board (Medical Ethics Committee) of the Erasmus Medical Center and by the review board of the Netherlands Ministry of Health, Welfare and Sports. The approval has been renewed every 5\u00a0years. Separate approval has been obtained for the introduction of major new elements in the study (e.g. MRI investigations).\nIn the remainder of this article the objectives and major findings will be presented with an update of the methods for cardiovascular diseases, neurological diseases, ophthalmologic diseases, psychiatric diseases, endocrine diseases, as well as for genetic and for pharmaco-epidemiologic studies.\nCardiovascular diseases\nObjectives\nResearch on the epidemiology of cardiovascular diseases focuses on three primary areas of interest: studies on risk factors for atherosclerosis and coronary heart disease, studies on the detection of subjects at high risk of coronary heart disease, and studies on cardiovascular conditions in older age.\nTwo groups of putative risk factors for atherosclerosis and coronary heart disease are included. The first are endocrine factors, including estrogens and androgens, insulin and insulin-like growth hormone I, and thyroid gland and adrenal gland hormones. The second group contains factors involved in haemostasis, inflammation and endothelial function. Research also focuses on genetic factors in these areas in relation to risk of coronary heart disease.\nThe ability of classical cardiovascular risk factors to identify subjects at high risk of coronary heart disease is limited. Risk stratification may be improved when based on the presence of atherosclerosis. To this end, repeated measurements of non-coronary atherosclerosis and measurements of coronary, carotid and aortic arch calcification have been included in the study.\nAnother line of research focuses on cardiovascular diseases in the elderly that are in large part the consequence of ischemic heart disease, like heart failure and atrial fibrillation. An important topic in this area is the early diagnosis of heart failure using echocardiographic assessment of asymptomatic systolic and diastolic dysfunction of the left ventricle. Atrial fibrillation is another major chronic condition frequent at older age. Examination of the determinants and prognosis of atrial fibrillation is part of this research line.\nMajor findings\nFindings on determinants of atherosclerosis and coronary heart disease include the relation between hypothyroidism and myocardial infarction [4] and the association of genetic variation in the estrogen receptor alpha gene with risk of myocardial infarction in women [5]. The study demonstrated that high levels of CRP [6] and lipoprotein-associated phospholipase A2 activity [7] were associated with risk of coronary heart disease and stroke. Genetic findings in this area include a positive association of coronary heart disease with genetic variation in the complement factor H gene [8] but not with variation in the CRP gene [6].\nThe study showed that carotid intima-media thickness and plaques measured by ultrasound, the ankle-arm index, and aortic calcification detected by X-ray are independent predictors of risk of coronary heart disease [9]. The study also showed that vessel wall stiffness, as measured by aortic pulse wave velocity (PWV), improved prediction of cardiovascular disease beyond the classical risk factors [10]. The Rotterdam Study was the first to publish results from population-based research on the predictive value for coronary heart disease of coronary calcification assessed by electron-beam CT [11].\nThe study enabled accurate assessment of the incidence and lifetime risk of heart failure and atrial fibrillation in an elderly population [12, 13]. It was shown that atherosclerosis and inflammation are also involved in these conditions [14, 15].\nMethods update\nRepeated measures of non-coronary atherosclerosis included carotid intima-media thickness and plaques by ultrasound, the ankle-arm index and aortic calcification by X-ray [9]. Electron-beam CT and multi-detector CT were used to accurately quantify calcification in the coronary, aortic arch and carotid arteries [11]. In the second additional cohort of the Rotterdam Study (RS Young1, see Fig.\u00a01), the measurement of plaque vulnerability with high-resolution MRI of the carotid arteries will be added. Other outcome measures include electrocardiography, echocardiography and measures of vessel wall stiffness (PWV and carotid distensibility) [10].\nDeterminants were assessed by physical examinations, collection of blood samples, and by questionnaires and interview. The role of genetic factors is studied using the candidate gene approach and more recently the genome wide association study.\nClinical cardiovascular outcomes are collected during our continuous follow-up and include non-fatal myocardial infarction and cardiac death, revascularizations, heart failure and atrial fibrillation. The methods of diagnosis of these outcomes have been described in previous papers [6, 12, 13].\nNeurological diseases\nObjectives\nNeuroepidemiologic research in the Rotterdam Study focuses on the frequency, etiology and early recognition of the most frequent neurologic diseases in the elderly, including dementia (in particular Alzheimer disease), Parkinson disease and stroke. In neurodegenerative and cerebrovascular disorders clinical symptoms typically become manifest late in the disease course, the occurrence of clinical disease does not reflect the underlying spectrum of disease-related pathology, and most of the clinical syndromes are etiologically heterogeneous. Therefore, an additional research focus is on the causes and consequences of pre-symptomatic brain pathology that can be assessed with non-invasive imaging modalities.\nMajor findings\nNeurodegenerative and cerebrovascular diseases are highly frequent in the elderly. The prevalence increases from age 55 to 65\u00a0years to age 90\u00a0years and above from less than 1% to over 40% for dementia [16], from less than 0.5% to more than 4% for Parkinson disease [17], and from approximately 1% to nearly 10% for stroke. The incidence figures follow this pattern of a strong increase with age over the entire age range, with the age-specific incidence of dementia being identical for men and women at least until the age of 85 [18] but with men having a higher age-specific incidence of both stroke and Parkinson disease than women throughout the age range [19, 20].\nVascular pathology and vascular risk factors are associated with worse cognitive performance [21], which also translates in people with vascular pathology or risk factors for vascular disease having an increased risk of dementia, including Alzheimer disease [22]. Moreover, several life style factors are associated with the risk of dementia and Alzheimer disease [23\u201325], suggesting that onset of dementia may at least partly be delayed or prevented. Commonly used drugs may have a role in this [26].\nThe classical risk factors for stroke also predict risk of stroke in the Rotterdam Study [27]. More recently identified risk factors, including inflammatory markers, may be etiologically relevant but thus far add little to the identification of people at risk [28]. Possibly underlying this is that a large amount of stroke goes clinically undetected [29]. Nearly 20% of elderly people have at least one silent brain infarct, and thereby a nearly fourfold increased risk of clinical stroke, a more than doubled risk of dementia including Alzheimer disease, and an increased risk of depression [29].\nNeuroimaging reveals that brain pathology is widespread [30] and can go clinically undetected for a long time. In addition to the silent infarcts, many apparently healthy elderly have ischemic changes in their cerebral white matter that are associated with an increased risk of dementia, stroke and depression [31]. Also brain atrophy, especially of the hippocampus, is already present years before onset of even the earliest sign of cognitive impairment or subjective complaints [32]. This emphasizes the need to shift the attention in etiologic research of neurodegenerative and cerebrovascular disease to the causes of pre-symptomatic and underlying brain changes.\nMethods update\nAssessment of dementia and Alzheimer disease\nIn the baseline and follow-up examinations participants undergo an initial screen for dementia with the Mini Mental State Examination (MMSE) and the Geriatric Mental Schedule (GMS), followed by an examination and informant interview with the Cambridge Examination for Mental Disorders of the Elderly (CAMDEX) in screenpositives (MMSE <26 or GMS >0), and subsequent neurological, neuropsychological and neuroimaging examinations [16, 18]. Of subjects who cannot be reexamined in person, information is obtained from the GPs and the regional institute for outpatient mental health care. A consensus panel makes the final diagnoses in accordance with standard criteria (DSM-III-R criteria; NINCDS-ADRDA; NINDS-AIREN).\nAssessment of parkinsonism and Parkinson disease\nParticipants are screened in the baseline and follow-up examinations for cardinal signs of parkinsonism (resting tremor, rigidity, bradykinesia or impaired postural reflexes). Persons with at least one sign present are examined with the Unified Parkinson Disease Rating Scale and a further neurologic exam. Parkinson disease is diagnosed if two or more cardinal signs are present in a subject not taking antiparkinsonian drugs, or if at least one sign has improved through medication, and when all causes of secondary parkinsonism (dementia, use of neuroleptics, cerebrovascular disease, multiple system atrophy or progressive supranuclear palsy) can be excluded [17, 19].\nAssessment of stroke and stroke subtypes\nHistory of stroke at baseline was assessed through interview and verified in medical records. Putative incident strokes get identified through the linkage of the study database with files from general practitioners, the municipality, and nursing home physicians\u2019 files, after which additional information (including brain imaging) is collected from hospital records. A panel discusses all potential strokes and subclassifies strokes into ischemic, hemorrhagic or unspecified [20, 28].\nAssessment of cognitive function\nGlobal cognitive function is measured through the Mini Mental State Examination (MMSE) in all surveys. From the third survey onwards we added a 30\u00a0min test battery that was designed to assess executive function and memory function, and which includes a Stroop test, a Letter Digit Substitution Task, a Word Fluency Test, and a 15 words Word List Learning test.\nRotterdam Scan Study: brain imaging within the Rotterdam Study\nIn 1991, a random sample of 111 participants underwent axial T2-weighted magnetic resonance (MR) imaging to assess presence and severity of white matter lesions [33]. In 1995, a random sample of 563 non-demented participants underwent brain MR imaging in the context of the Rotterdam Scan Study. The scanning protocol included series of axial proton-density, T2-weighted and T1-weighted images, as well as a high-resolution 3D-HASTE sequence [31]. From August 2005 onwards, a dedicated 1.5 Tesla scanner is operational in the research centre of the Rotterdam Study, and brain imaging is performed in all study participants without contra-indications. The scanning protocol includes 4 high-resolution axial sequences (3D T1-weighted; 2D PD-weighted; 2D FLAIR; and 3D T2* GRE), 2D phase-contrast imaging, and diffusion tensor imaging (DTI).\nPsychiatric diseases\nObjectives\nThe aim of the psychiatric research in the Rotterdam Study is to investigate the determinants, correlates and consequences of common psychiatric problems in the elderly. The focus has been on depressive disorders but anxiety disorders, sleep disturbances and complicated grief are also being studied.\nMajor findings\nThe one-month prevalence of both major and minor depression at baseline was 1.5%, the prevalence of anxiety disorder was 8.2%.\nWe found evidence for the vascular depression hypothesis. More severe coronary and extra-coronary atherosclerosis were associated with a higher prevalence of depression, as were cerebral haemodynamic changes [34, 35]. However, we could not rule out that earlier depressive episodes may have contributed to the development of atherosclerosis. Moreover, our data did not support a specific symptom profile of vascular depression as previously defined [36].\nMost of our studies of nutritional and metabolic factors provided no support for an etiological role in depression. Folate deficiency, homocysteine, fatty acid ratio (omega-6\/omega-3) and vitamin E had little or no effect on the risk of depression after control for health and health related behaviour [37\u201339]. In contrast, low levels of vitamin B12 and high levels of inflammatory protein IL-6 convey a strong risk for depression, although the latter effect may be confined to high-risk groups [40].\nSeveral SNPs have been investigated, mostly with negative results. However, we found that an ER-alpha polymorphism had a substantial effect on anxiety but not on depression in women [41].\nMethods update\nIn the first years of the Rotterdam Study psychiatric data collection was very limited. However, from the third examination onwards, which began in 1997 (see Fig.\u00a01), depressive symptoms and disorders are have been ascertained in all participants. The 4,603 participants with valid depression scores in this examination thus constitute the baseline population for longitudinal studies of depression. Because assessment of anxiety disorders, sleeping disturbances and complicated grief were added in the fourth examination, they can currently be studied only cross-sectionally.\nAssessment of determinants\nPsychiatric research in the Rotterdam Study focuses on biological risk factors. The vascular depression hypothesis was tested with different measures of atherosclerosis, arterial stiffness and cerebral blood flow [34, 35]. We also examined whether blood levels of vitamins and fatty acids, immune parameters, and markers of folate metabolism increased the likelihood of depression [37\u201340]. In one ongoing project, diurnal patterns of cortisol secretion are related to psychiatric and other disorders. Studies of genetic polymorphisms and brain morphology are underway. Current data collection includes a dexamethasone suppression test to measure hypothalamic-pituitary-adrenal axis activity in all participants, which is unique in a population-based study.\nAssessment of outcomes\nDepression is assessed using a two step procedure. First, participants completed the Center for Epidemiologic Studies Depression scale (CES-D) during the home interview to measure self-reported depressive symptoms [42]. As a second step, screen positive subjects, who scored above a well-established CES-D cut-off of 20 points, have a psychiatric interview using the Present State Examination (PSE\/SCAN) [43]. This semi-structured interview is conducted by a clinician and yields DSM-IV diagnoses including major and minor depression and dysthymia.\nThe following anxiety disorders are assessed with a slightly adapted Munich version of the Composite International Diagnostic Interview: generalized anxiety disorder, specific and social phobia, agoraphobia without panic disorder, and panic disorder [44]. Sleep quality and disturbance is measured with the Pittsburgh Sleep Quality Index. In addition, sleep duration and fragmentation are assessed with actigraphy, a method that infers wakefulness and sleep from the presence or absence of limb movement [45]. The Inventory of Complicated Grief is used to identify traumatic grief [46]. This is a condition distinct from normal grief and bereavement-related depression, characterized by symptoms like disbelief about the death and searching for the deceased.\nOphthalmologic diseases\nObjectives\nThe primary objectives of the ophthalmological part of the Rotterdam Study are to study frequency and risk factors of common and invalidating eye diseases with emphasis on age-related macular degeneration (AMD) and primary open angle glaucoma (POAG). Another line of research focuses on the associations between retinal vessel abnormalities and cardiovascular and neurologic diseases.\nMajor findings\nAMD was the main cause of blindness in the high age-group [47]. Major risk factors that were found for AMD are smoking, atherosclerosis, hyperopia [48\u201350]. The APO-E \u03b5-4 allele showed an inverse association with AMD [51]. First-degree relatives of patients with late ARM developed ARM at an increased rate at a relatively young age [52]. The heterogeneity of genetic risk among AMD families is considerable, and the proportion of high-risk families is relatively small [53]. The anticipated protective effect of cholesterol-lowering drugs on AMD could not be substantiated [54]. A protective effect on AMD was demonstrated in participants using a diet rich of antioxidants and zinc intake in our study population. Support for a multifactorial origin of AMD was found in the cumulative effects of aberrant CFH and CRP genes, inflammations and smoking as genetic and environmental stimulators of the complement cascade [55].\nThe prevalence estimates of POAG in Rotterdam was highly dependent on the applied criteria [56]. That was a major reason for proposing a classification system without final, subjective adjudication [57]. Systemic blood pressure and hypertension were associated with elevated intraocular pressure but not with prevalent POAG [58]. Relatives of patients with POAG have a strongly increased risk of glaucoma. Enlarged cup-disc ratio was the earliest and most prominent feature of familial aggregation [59]. The incidence of OAG rises significantly with age. Most of the patients with incident OAG were unaware of having OAG [60]. The incidence of visual field loss rises fivefold between 55 and 80\u00a0years to 20\/1,000 person years in the general population [61]. Glaucoma is the main cause for visual field loss, followed by stroke [62].\nLarger retinal venular diameters are associated with generalized atherosclerosis, inflammation and cholesterol levels. Retinal venular diameters are variable and may play their own independent role in predicting cardiovascular disorders [62]. Dilated retinal venules at baseline were predictive for stroke, cerebral infarction, dementia, white brain matter lesions, impaired glucose tolerance, diabetes mellitus and mortality [63, 64]. In systemic hypertension, the arteriolar diameters are narrow and arteriolar and venular narrowing precedes the onset of hypertension [65].\nMethods update\nRepeated ophthalmic measurements include best corrected ETDRS visual acuity, refractive error, Goldmann applanation tonometry, keratometry, slitlamp examination of the anterior segment and visual field testing. In pharmacological mydriasis we make colour photographs of the lens, 35 degree photography of the macular area, and simultaneous stereoscopic imaging of the optic disc and macular area. Digital photography of the macular area and optic disc are added since the fourth follow-up examination. Scanning laser ophthalmoscopy (HRT II) measurements of the optic disc, macular pigment density measurements and optical coherence tomography of the macular area and optic disc were added during follow-up.\nFor the assessment of AMD we use stereoscopic 35\u00b0 macular images centred on the fovea. The images are graded according to the International Classification and Grading System for AMD [66]. \nThe procedure for assessing POAG includes Goldmann applanation tonometry, visual field screening, ophthalmoscopy and stereoscopic fundus photography [57].\nAssessment of retinal vascular diameters is done by analyses of digitized fundus transparencies with a semi automated system [58, 67].\nGenetic studies\nObjectives\nThe first objective of the laboratory team is to collect, store and manage the biological tissues sampled in the Rotterdam Study. The second objective of the group concerns genotyping and assessment of biomarkers.\nMajor findings\nBoth from biomarker studies as well as molecular genetic studies important findings have emerged. For example, among the biomarker analyses the study documenting the relationship between homocysteine and osteoporosis has been novel [68] and since widely replicated. Several candidate gene studies have also yielded major new insights coming from both exploratory studies as well as from collaborative replication efforts across all disease-oriented research lines in the Rotterdam Study. A unique feature of the Rotterdam Study is exploited by studying the relationship between pleiotropic gene variants and not one but multiple diseases and disease-related endpoints. For example, the studies on the promoter region of the IGF-1 gene revealed a series of consistent associations ranging from birth weight to diabetes [69], while other consistent associations involve the estrogen receptor alpha (ESR1) gene in relation to osteoporosis [70], osteoarthritis, height, myocardial infarction [5], age-at-menopause, and depression.\nThe Rotterdam Study is involved as a major collaborative centre for studies for novel genes coming from genome-wide analysis (GWA) studies, including CFH in age-related macula degeneration [71], NOS1AP in QT interval [72], and several SNPs involved in height, type 2 diabetes, and breast cancer. Finally, Rotterdam Study investigators play a leading role in the emerging large global consortia focussing on the contribution of complex disease gene variants by prospective meta-analyses across many epidemiological cohorts, such as for osteoporosis in the GENOMOS Study and EUROSPAN.\nMethods update\nData collection, storage and management\nAt each examination, blood, serum, plasma (citrate, heparine and EDTA based), sputum, and urine are collected. Fasting blood samples are collected along with challenged samples as part of a glucose tolerance test. Sputum is collected before and after a dexamethasone-suppression test. Sputum is frozen at \u2212196\u00b0C before and after the challenge and stored at \u221280\u00b0C. To obtain serum and plasma, tubes are centrifuged according to a protocol standardising time and conditions from the drawing of blood to centrifugation. All samples are snap frozen at \u2212196\u00b0C using liquid nitrogen and stored at \u221280\u00b0C. RNA is isolated from blood within 5\u00a0h after sampling and stored at \u221220\u00b0C. DNA is isolated from blood and extraction has been recently automated using a Hamilton STAR pipetting platform and AGOWA magnetic bead technology. DNA sample storage is in Matrix 2D-barcode tubes in 96-well format. Overnight urine samples are collected, frozen at \u2212196\u00b0C and stored at \u221280\u00b0C. For data management, an in-house customized laboratory management system has been developed. Sample retrieval will be automated with an in-house customized laboratory track and trace system.\nBlood assessments\nFor all participants, serum cholesterol, HDL, LDL, triglycerides, glucose and glucose levels are assessed. In urine, micro albumin and creatinine are determined in all participants. There have been a large number of specific blood\/serum\/plasma-based biomarker assessments including steroids (e.g. estrogens, androgens, vitamin D, cortisol), interleukins, CRP, IGF1, insulin, iron-parameters (iron, ferritin and transferrin saturation), fibrinogen, homocysteine, folic acid, riboflavine, pyridoxine, SAM\/SAH ratio, cobalamine, Lp-PLA2, Fas\/Fas-L, vitamins, a-beta42\/40 and thyroid hormones (TSH).\nGenotyping facilities\nAffiliated laboratory facilities include a medium\/high-throughput platform for candidate gene studies and genome wide association (GWA) analyses. The facilities use high-end automated machinery including a Caliper\/Zymark ALH 3000 pipetting robot (including a TwisterII, and integrated plate sealer, plate reader (OD 260\/280), a Tecan EVO 150 Freedom pipetting robot, a Deerac Equator NS808 nanoliter liquid dispenser, 15 electronic PCR machines (ABI 9700, 2 \u00d7 384), an ABI7900HT Taqman machine (running 1\u00a0ng gDNA in 2\u00a0\u03bcl reactions), a WAVE 3500HT dHPLC, and two ABI3100 sequencing machines. DNA sample handling is centred on 384-well plates. Candidate gene studies are done mostly using Taqman genotyping with throughputs at 30,000 genotypes per day. Continuous efforts are focussed on reducing the required amount of genomic DNA, which is now down to 1\u00a0ng per genotype. Genome-wide genotyping studies are based on 500\/1,000\u00a0K Affymetrix arrays and 317, 550 and 1,000\u00a0K Illumina arrays with throughputs at 100\u2013300 arrays per week.\nThe genotyping facility has been partly sponsored by NWO investment grants (911-03-012; 175.010.2005.011), is part of the ErasmusMC Biomics core facility, and serves as knowledge centre for polymorphism analysis attracting national and international interested parties, both academic and industrial.\nCandidate gene studies\nWe have genotyped >200 polymorphisms across the complete cohort and conducted a large number of candidate gene studies in the Rotterdam Study. These mostly concern individual potentially functional single nucleotide polymorphisms (SNPs) per gene, but sometimes also haplotype tagging SNPs (e.g. ESR1, ESR2, HSD11B1, fibrinogen), and also high-density SNP screening (e.g. the vitamin D receptor gene [73]). The candidate genes studied include the apolipoprotein E gene (APOE), the angiotensin-converting enzyme (ACE), the gene encoding angiotensinogen (AGT), angiotensin II type 1 receptor (AT1R) gene, G protein beta3 (GNB3), adducine gene, Cholesteryl Ester Transfer Protein (CETP), Hepatic Lipase, Phosphodiesterase 4D (PDE4D), ALOX5AP encoding 5-lipoxygenase activating protein, a polymorphism in the regulatory region of the Insulin-like Growth Factor 1 (IGF-1) gene, the hemochromatosis (HFE) gene, Complement factor H gene (CFH), and several polymorphisms in genes from the estrogen-, thyroid-, cortisol-, vitamin D-, IGF-, and Wnt-signalling pathways, the homocysteine pathway, and several matrix molecules.\nGenome wide association (GWA) studies\nA recently developed approach to identify robust genetic factors for complex disease is the Genome Wide Association (GWA) analysis. This is based on genotyping epidemiological cohorts with ultra-high density SNP arrays with up to 1\u00a0million SNPs. The method has already been shown to successfully identify genetic factors for several traits and diseases including age-related macula degeneration, inflammatory bowel disease, body mass index, type 2 diabetes and breast cancer. Through a large grant from the Dutch research organization NWO one of the world\u2019s largest GWA studies is conducted involving 10,000 DNA samples from the Rotterdam Study. This GWA is based on the Illumina 550\u00a0K arrays and finished by the end of 2007. The GWA data will be useful for all research lines within the Rotterdam Study, and will also serve as a control GWA dataset for other research centres in and outside the Netherlands for both SNP frequencies as well as copy number variations (CNVs). In addition to this, a pilot study has been performed on 500 women from the Rotterdam Study using the Affymetrix 500\u00a0K platform allowing direct platform comparisons and merging of data to further increase genome coverage. In addition, our group has been active in developing new software for GWA analyses [74].\nPharmaco-epidemiologic studies\nObjectives\nA major objective of the pharmaco-epidemiologic studies is to investigate the role of drugs as determinants of disease in the Rotterdam Study. This includes studying efficacy and effectiveness of drugs, as well as adverse reactions to drugs.\nMajor findings\nImportant findings have been published on pharmaco-epidemiological topics concerning the main outcomes in the Rotterdam Study. Studies about the association between dementia and antihypertensive drugs [75] and NSAIDs [26] have strongly suggested a protective effect of both groups of drugs. Several studies have been performed on cardiovascular topics [76\u201378]. In one of these studies, NSAIDs were associated with an increased risk of heart failure [79]. In line with the suspicion that QTc-prolonging drugs may cause sudden cardiac death, it was demonstrated in the Rotterdam Study that a prolonged QTc is indeed an important risk factor [77]. Furthermore, in one study it was demonstrated that high-dose corticosteroids increase the risk of atrial fibrillation [78]. In the important area of locomotor diseases, studies have demonstrated that thiazide diuretics protect against hip fracture [79] and that statins reduce the risk of vertebral fracture [80]. On the other hand, the risk that long-term use of certain NSAIDs may aggravate signs of osteoarthritis has been emphasized [81]. In the area of ophthalmological diseases, a protective effect of cholesterol-lowering agents on macular degeneration has been suggested [82]. In other areas, such as pharmacogenetics and other causes of interactions between drugs, several important findings have been published [83\u201391].\nMethods update\nFor several reasons, a drug is a highly attractive determinant in clinical epidemiology. First, drugs are probably the most important therapeutic intervention in health care. Despite rigorous clinical research before registration, many important effects of drugs are discovered after marketing. Second, all marketed drugs have proven biological activity, meaning that it concerns a determinant which really matters. Third, and as a consequence of the availability of complete medication histories in Dutch health care, the role of drug exposure can be assessed in a detailed way.\nIn the Rotterdam Study, there is an almost complete coverage of the population as of 1 January, 1991 thanks to the fact that all pharmacies which serve the Ommoord region are on one computer network. To date, almost 3\u00a0million prescriptions have been delivered to the population of the Rotterdam Study and of each prescription, details are available about the product name and contents, ATC-code, dosage and duration of drug therapy.\nDrugs are a group of determinants, which can be studied in association with a large variety of diseases. In the Rotterdam Study, there is a strong interest in the association between drugs and the cardiovascular, neurological, endocrinological and ophthalmological diseases, which have been the main topics since starting. However, there is also important information about the association with psychiatric diseases, cancer, and chronic obstructive pulmonary disease. Moreover, important information about secondary outcomes, such as drug blood levels, other laboratory information, and information about hospital discharge diagnoses, is gathered on a continuous basis to facilitate pharmaco-epidemiological studies.\nManagement of the Rotterdam Study\nThe Rotterdam Study is directed by a Management Team comprising Jan Heeringa, MD, study coordinator, Eric Neeleman, head IT, Frank van Rooij, head data-management, and the scientific principal investigators Albert Hofman (PI Rotterdam Study, chairman), Monique Breteler (PI Neurological diseases), Cornelia van Duijn (PI Genetic studies), Gabriel Krestin (PI Radiology), Huibert Pols (PI Endocrinology), Bruno Stricker (PI Pharmaco-epidemiology), Henning Tiemeier (PI Psychiatric diseases), Andr\u00e9 Uitterlinden (PI Genome wide analysis), Johannes Vingerling (PI Ophthalmologic diseases) and Jacqueline Witteman (PI Cardiovascular diseases).","keyphrases":["cohort study","heart failure","stroke","parkinson disease","alzheimer disease","dementia","macular degeneration","glaucoma","diabetes","osteoporosis","pharmaco-epidemiology","myocardial infarction","genome wide analysis"],"prmu":["P","P","P","P","P","P","P","P","P","P","P","P","P"]}
{"id":"Intensive_Care_Med-2-2-1315315","title":"SAPS 3\u2014From evaluation of the patient to evaluation of the intensive care unit. Part 2: Development of a prognostic model for hospital mortality at ICU admission\n","text":"Objective To develop a model to assess severity of illness and predict vital status at hospital discharge based on ICU admission data.\nIntroduction\nOne of the crucial steps in the evaluation of risk-adjusted outcomes is the choice of the reference database for estimating adequate reference lines for the analyzed variables. For the SAPS\u00a03 to reflect the standard of practices and outcome in intensive care at the beginning of the 21st century, we decided to collect data from a large sample of intensive care units (ICUs) worldwide. Other models have restricted data collection to large ICUs in Europe or North America\u2014SAPS\u00a0II [1], MPM\u00a0II [2], APACHE II [3] and APACHE\u00a0III [4], a strategy that minimizes the heterogeneity of the sample but restricts the generalization of the results.\nAt the statistical level, there is also a need for change, in order to take into account the hierarchic nature of our data [5, 6]. Current general outcome prediction models do not consider the existence of clinical and nonclinical factors, aggregated at the ICU level, that can have an important impact on prognosis. Instead, they assume that these factors are either not important or are randomly distributed throughout large samples and that the variation between ICUs is small. This assumption is not likely to be borne out at the ICU level for either nonclinical factors (e.g. organization and management, organizational culture) or clinical factors (e.g. clinical management, diagnostic and therapeutic strategies). If the variation between ICUs is not negligible, it will compromise the stability of the equations used to compute predicted mortality. Furthermore, the published models consider the relation between performance and severity of illness to be constant, and that may not be the case, since performance can vary within ICUs according to the severity of illness of the patients [7, 8]. To overcome this problem, we chose to adopt a new strategy for the development of the SAPS\u00a03 score and to apply statistical modelling techniques that control for the clustering of patients within ICUs instead of assuming the independence of observations. Conceptually, the SAPS\u00a03 admission core comprises the following parts:\nFirst, the SAPS\u00a03 ADMISSION SCORE, represented by the arithmetic sum of three subscores, or boxes:Box I: What we know about the patient characteristics before ICU admission: age, previous health status, co-morbidities, location before ICU admission, length of stay in the hospital before ICU admission, and use of major therapeutic options before ICU admission.Box II: What we know about the circumstances of ICU admission: reason(s) for ICU admission, anatomic site of surgery (if applicable), planned or unplanned ICU admission, surgical status and infection at ICU admission.Box III: What we know about the presence and degree of physiologic derangement at ICU admission (within 1\u00a0h before or after admission). Second, the SAPS\u00a03 PROBABILITY OF DEATH during a certain period of time (in the case of the main model, the probability of death at hospital discharge).\nGiven our objective of evaluating not only individual patient outcome but also the effectiveness of ICU practices, we focused the model on data available at ICU admission or shortly thereafter. This model will be completely open and available free of any direct or indirect charges to the scientific community.\nMethods and statistical analysis\nPrimary variable selection\nBased on the SAPS\u00a03 Hospital Outcome Cohort as described in Part 1 of this report, continuous predictive variables were categorized in mutually exclusive categories based on smoothed curves such as LOWESS [9], showing the univariate dependence of hospital mortality on the predictive variables. Classes of categorical variables were also collapsed according to their univariate hospital mortality levels using multidimensional tables and clinical judgment as appropriate, depending on the nature of the data. Additively, regression trees (MART) [10] were applied to check the cutoffs.\nMissing values were coded as the reference or \u201cnormal\u201d category for each variable. When dual data collection was used\u2014maximum and minimum values recorded during a certain time period\u2014missing maximum values of a variable were replaced by the minimum, if documented, and vice versa. Some regression imputations were performed if noticeable correlations to available values could be exploited. For a detailed description of data collection and handling, see Part 1 of this report.\nSelection of variables was done according to their association with hospital mortality, together with expert knowledge and definitions used in other severity of illness scoring systems. The objective of using this combination of techniques rather than regression-based criteria alone was to reach a compromise between over-sophistication of the model and knowledge from sources beyond the sample with its specific case mix and ICU characteristics.\nCross validation\nFor being able to cross-validate the model, we randomly extracted five roughly equal-sized parts based on number of patients from the database, as suggested previously [11]. In a second approach, partitioning was based on ICUs and not on patients. It was thus possible to run the model-building procedure five times in each of the two approaches, each time taking four parts of the sample as a development set and the remaining one as the validation set. This allowed to estimate the variability of prediction resulting from the construction process of the prognostic score. A further check of the stability of the predictions was made by partitioning the sample according to major patient characteristics, such as surgical status and infection status.\nThe quality of predictions in the validation sets was assessed by looking at the goodness-of-fit in terms of the p values for the Hosmer-Lemeshow tests \u0108 and \u0124 [13] and the discriminative capability of the models by the use of the area under the receiver operating characteristic (aROC) curve [14, 15]. Another criterion to judge the appropriateness of the model was the fit in certain subsamples, defined according to major patient typologies [16]. \nReducing model complexity\nTo reduce the complexity of the model classes, we concentrated on logistic regression. In the first step a stepwise logistic regression was used to identify the significant predictors in each of the five subsamples. A threshold of 0.01 for the p value was generally applied for inclusion in the model to separate irrelevant predictors [12]. At this stage we also evaluated if interactions among these predictors would influence results. Interactions, however, did not make a valuable contribution for the prediction.\nSignificant predictors (n=70) were in a second step entered into a logistic regression model. The criterion for a predictor to enter the model was homogeneity across the five model-building processes: in principle, predictors should enter the model in all five development sets, but depending on the frequency of the predictor in the samples, the magnitude of the effect, and medical reasoning, some predictors were included if they appeared in the model in at least three subsamples. An example is the presence of Acquired Immunodeficiency Syndrome (AIDS): it was selected as a comorbidity in only 81 patients (0.48%), but the mortality\u2014without controlling for other variables\u2014in these patients was 42%. By taking all the above steps to identify the set of predictors, although deliberately not using any formal numeric criterion, we reduced the complexity of the model to minimize the amount of overfitting: This process resulted in 61 item classes (representing 20 variables) remaining in the final model.\nUsing the parameter estimates from the logistic regression as starting values, a multilevel model was applied in the next step, using patient characteristics as fixed effects and ICUs as a random effect. Estimates were again calculated for the five development sets (for both, patient and ICU -based development subsamples).\nAt this stage it was checked if rounding of coefficients (which allows for an easier manual computation of the score) would influence results, which was found not to be the case. Consequently, this was the approach chosen for the final construction of the SAPS\u00a03 admission score sheet.\nThe stability of the processes of variable selection and reducing complexity was further checked by bootstraping with replacement the total sample 100 times, both at patient level and at ICU level.\nPredicting hospital mortality\nAfter this step was completed, a shrinking power transformation was applied. This procedure uses log-transformation of the score to reduce the influence of extreme score values (outliers) on the mortality prediction. For this purpose, the SAPS\u00a03 score and the transformed log (SAPS\u00a03 + g) scores were used to predict hospital mortality. Conventional logistic regression was used in the evaluation of this step because of convergence problems for the corresponding multilevel model in a few subsamples. The best shrinkage model then was selected (excluding the trivial model with the SAPS\u00a03 score as the single predictor) by checking which of the terms in the model contributed best to the prediction and was moreover stable over the respective validation sets and specific subsamples. This procedure was applied on both, patient and ICU -based subsamples.\nAfter finishing these steps of cross-validation, the final estimates for the selected predictors of the SAPS\u00a03 score as well as the selected shrinkage procedure were then calculated from the total sample of patients.\nTo arrive at the customised models for each major geographic region, specific customised equations were calculated, relating, by logistic regression, the transformed log (SAPS\u00a03 + g) admission scores computed as described above to the vital status at hospital discharge. This process allows both the intercept and the slope of the curve relating the SAPS\u00a03 admission score to change across different regions. The goodness-of-fit of these equations was evaluated by means of the same methodology used for the global sample.\nSAS for Windows, version 8.02 (SAS Institute Inc., Cary, NC, USA) and MLwiN version 1.10.0007 (Centre for Multilevel Modelling, Institute of Education, London, UK) and the R Software Package (http:\/\/www.r-project.org) were used for the development of the model.\nResults\nBased on the methodology described, 20 variables were selected for the SAPS\u00a03 admission score (Tables\u00a01 and\u00a02):Five variables for evaluating Box I: age, co-morbidities, use of vasoactive drugs before ICU admission, intrahospital location before ICU admission, and length of stay in the hospital before ICU admission;Five variables for evaluating Box II: reason(s) for ICU admission, planned\/unplanned ICU admission, surgical status at ICU admission, anatomic site of surgery, and presence of infection at ICU admission and place acquired;Ten variables for evaluating Box III: lowest estimated Glasgow coma scale, highest heart rate, lowest systolic blood pressure, highest bilirubine, highest body temperature, highest creatinine, highest leukocytes, lowest platelets, lowest hydrogen ion concentration (pH), and ventilatory support and oxygenation.Table\u00a01 SAPS\u00a03 admission scoresheet\u2014Part 1Box I035678911131518Age, years<40>=40<60>60<70>=70<75>=75<80>=80Co-MorbiditiesCancer therapy 2)Chron, HF (NYHA IV),Haematological cancer 3),4)Cirrhosis, AIDS 3)Cancer 5)Length of stay before ICU admission, days 1)<14>=14<28>=28Intra-hospital location before ICU admissionEmergency roomOther ICUOther 6)Use of major therapeutic options before ICU admissionVasoactive drugsBox II034569ICU admission: Planned or UnplannedUnplannedReason(s) for ICU admissionplease see Part 2 of the scoresheetSurgical status at ICU admissionScheduled surgeryNo surgery 7)Emergency surgeryAnatomical site of surgeryplease see Part 2 of the scoresheetAcute infection at ICU admissionNosocomial 8)Respiratory 9)Box III1513111087532024578Estimated Glasgow Coma Scale (lowest), points3\u20134567\u201312>=13Total bilirubine (highest), mg\/dL<2>=2<6>=6Total bilirubine (highest), \u00b5mol\/L<34.2>=34.2<102.6>=102.6Body temperature (highest), Degrees Celsius<35>=35Creatinine (highest), mg\/dL<1.2>=1.2<2>=2<3.5>=3.5Creatinine (highest), \u00b5mol\/L3\u2013456<106.1>=106.1<176.8>=176.8<309.4>=309.4Heart rate (highest), beats\/minute<120>=120<160>=160Leukocytes (highest), G\/L<15>=15Hydrogen ion concentration (lowest), pH<=7.25>7.25Plateletes (lowest), G\/L<20>=20<50>=50<100>=100Systolic blood pressure (lowest), mm\u00a0Hg<40>=40<70>=70<120>=120Oxygenation 10), 11)PaO2\/FiO2<100 and MVPaO2\/FiO2>=100 and MVPaO2<60 and no MVPaO2>=60 and no MVThe definition for all variables can be found in detail in Appendix C of the ESM. For names and abbreviations which are differing from those in the ESM, explanations are given below. Generally, it should be noted that no mutually exclusive conditions exist for the following fields: Comorbidities, Reasons for ICU admission, and Acute infection at ICU admission. Thus, if a patient has more than one condition listed for a specific variable, points are assigned for all applicable combinations.1 This variable is calculated from the two data fields: ICU Admission date and time\u2014Hospital admission date and time (see Appendix C of the ESM) 2 Cancer Therapy refers to the data definitions in Appendix C of the ESM: Co-Morbidities: Chemotherapy, Immunosupression other, Radiotherapy, Steroid treatment3 If a patient has both conditions he\/she gets double points.4 Chronic HF (NYHA IV)\/Haematological cancer refer both to the data definitions in Appendix C of the ESM: Co-Morbidities: Chronic heart failure class IV NYHA, Haematological cancer. 5 Cancer refers to the data definitions in Appendix C of the ESM: Co-Morbidities: Metastatic cancer.6 Other refers to the data definitions in Appendix C of the ESM: Intra-hospital location before ICU admission: Ward, Other.7 No surgery refers to the data definitions in Appendix C of the ESM: Surgical Status at ICU Admission: Patient not submitted to surgery.8 Nosocomial refers to the data definitions in Appendix C of the ESM: Acute infection at ICU admission\u2014Acquisition: Hospital-acquired.9 Respiratory refers to the data definition in Appendix C of the ESM: Acute infection at ICU admission\u2014Site: Lower respiratory tract: Pneumonia, Lung asbcess, other. 10 PaO2, FIO2 refer to the data definitions in Appendix C of the ESM: Arterial oxygen partial pressure (lowest), Inspiratory oxygen concentration.11 MV refers to the data definition in Appendix C of the ESM: Ventilatory support and mechanical ventilation.Table\u00a02SAPS 3 admission scoresheet \u2013 Part 2Box II \u2013 continuedICU admission 12)16Reason(s) for ICU admission\u00a0\u00a0\u00a0Cardiovascular: Rhythm disturbances 13)\u20135\u00a0\u00a0\u00a0Neurologic: Seizures 13)\u20134\u00a0\u00a0\u00a0Cardiovascular: Hypovolemic hemorrhagic shock, Hypovolemic non hemorrhagic shock. \/ Digestive: Acute abdomen, Other 3)3\u00a0\u00a0\u00a0Neurologic: Coma, Stupor, Obtuned patient, Vigilance disturbances, Confusion, Agitation, Delirium4\u00a0\u00a0\u00a0Cardiovascular: Septic shock. \/ Cardiovascular: Anaphylactic shock, mixed and undefined shock 3)5\u00a0\u00a0\u00a0Hepatic: Liver failure6\u00a0\u00a0\u00a0Neurologic: Focal neurologic deficit7\u00a0\u00a0\u00a0Digestive: Severe pancreatitis9\u00a0\u00a0\u00a0Neurologic: Intracranial mass effect10\u00a0\u00a0\u00a0All others0Anatomical site of surgery\u00a0\u00a0\u00a0Transplantation surgery: Liver, Kidney, Pancreas, Kidney and pancreas, Transplantation other\u201311\u00a0\u00a0\u00a0Trauma \u2013 Other, isolated: (includes Thorax, Abdomen, limb); Trauma \u2013 Multiple\u20138\u00a0\u00a0\u00a0Cardiac surgery: CABG without valvular repair\u20136\u00a0\u00a0\u00a0Neurosurgery: Cerebrovascular accident5\u00a0\u00a0\u00a0All others012)Every patient gets an offset of 16 points for being admitted (to avoid negative SAPS 3 Scores).13) If both reasons for admission are present, only the worse valve (\u20134) is scored.\nAn estimation of the variability of the coefficients in the overall sample and in the five disjoint subsamples is given in Table E8 of the Electronic Supplementary Material (ESM), together with their respective coefficients (unrounded and rounded) and p values. The SAPS\u00a03 admission score can thus, in theory, vary from a minimum of 0 points to a maximum of 217 points. The distribution of the SAPS\u00a03 admission score in our sample is presented in Fig.\u00a01. The minimum value observed was 5, and the maximum value was 124, with a mean of 49.9\u00b116.6 (mean \u00b1 SD) and a median of 48 (38\u201360). The highest explanatory power came from Box I, with Box II and Box III being less important for the outcome; the three boxes represent 50%, 22.5% and 27.5%, respectively, of the total Nagelkerke\u2019s R-Square. The relationship between the SAPS\u00a03 and vital status at hospital discharge is given by the equation: \nLogit = \u221232.6659 +ln(SAPS\u00a03 score +20.5958) \u00d77.3068\nand the probability of mortality by the equation: \nProbability of death = elogit\/(1+elogit).Fig.\u00a01 Distribution of the SAPS\u00a03 admission score in the SAPS\u00a03 database\nThe relationship between the SAPS\u00a03 admission score and the respective probability of death in the hospital is described in Fig.\u00a02. Overall, no combined discrepancy between observed and expected outcomes across all of the strata was outside sampling variability as demonstrated a Hosmer-Lemeshow goodness-of-fit test \u0124 of 10.56 (p=0.39) and a Hosmer-Lemeshow goodness-of-fit test \u0108 of 14.29 (p=0.16) (Figs.\u00a03, 4 and Table E9, ESM). The overall discriminatory capability of the model, as measured by aROC curve, was 0.848. The goodness-of-fit according to major patient typologies (surgical status, trauma, and infection) can be found in Table\u00a03. Calibration and discrimination presented differences across different geographic areas: the best predictive results were achieved in patients from Northern Europe (observed-to-expected [O\/E] mortality ratio 0.96 [0.83\u20131.09]) and the worst predictive results were obtained in patients from Central and South America (O\/E mortality ratio, 1.30 [1.23\u20131.37]); see also Table\u00a04 and Fig.\u00a05 and Appendix B in the ESM.Fig.\u00a02 Relationship between the SAPS\u00a03 admission score and the respective probabilities of hospital mortalityFig.\u00a03 Hosmer-Lemeshow goodness-of-fit test \u0108 in the overall sample. Predicted risk of hospital death, observed hospital mortality rate, and the corresponding number of patients per decile are shown. Columns: Number of patients; squares: mean SAPS\u00a03-predicted mortality per decile; circles: mean observed mortality per decileFig.\u00a04Hosmer-Lemeshow goodness-of-fit test \u0124 in the overall sample. Predicted risk of hospital death, observed hospital mortaliy rate, and the corresponding number of patients per decile are shown. Columns: Number of patients; squares: mean SAPS\u00a03-predicted mortality per decile; circles: mean observed mortality per decileTable\u00a03 Performance of the model across major patient typologiesPatient characteristics GOF test \u0124pGOF test \u0108pO\/E ratio95% CIaROCTrauma patients19.920.039.030.531.030.93\u20131.120.854Non-operative admissionsa14.860.1417.80.061.010.98\u20131.040.825Scheduled surgerya11.50.3227.39<0.010.970.90\u20131.030.825Emergency surgerya4.970.8912.880.231.000.95\u20131.050.809No infectionb8.570.5714.770.141.000.97\u20131.020.846Community-acquired infectionc8.40.5911.760.31.000.96\u20131.050.786Hospital-acquired infectiond15.210.127.110.721.020.97\u20131.070.77GOF: Hosmer-Lemeshow goodness-of-fit; O\/E: observed-to-expected mortality; CI: 95% confidence interval; aROC: area under receiver operating characteristic (curve)aNon-operative admissions, scheduled surgery emergency surgery: see data definitions appendix C, ESMbNo infection: Patients not infected at ICU admissioncCommunity-acquired infection: Patients with community-acquired infection at ICU admissiondHospital-acquired infection: Patients with hospital-acquired infection at ICU admissionTable\u00a04 Performance of the model in the global sample and in different geographic areasRegionsGOF test \u0124pGOF test \u0108pO\/E ratio95% CIaROCAustralasia15.250.128.090.620.920.85\u20130.990.839Central, South America78.01<0.0180.82<0.011.301.23\u20131.370.855Central, Western Europe56.45<0.0147.89<0.010.840.79\u20130.900.861Eastern Europe19.450.0318.690.041.091.00\u20131.190.903North Europe2.440.992.340.990.960.83\u20131.090.814Southern Europe, Mediterranean countries14.180.1620.780.021.020.98\u20131.050.834North America10.570.399.630.470.910.78\u20131.040.812Global database10.560.3914.290.1610.98\u20131.020.848GOF: Hosmer-Lemeshow goodness-of-fit; O\/E: observed-to-expected mortality; CI: 95% confidence interval; aROC: area under the receiver operating characteristic (curve).Fig.\u00a05 Observed-to-expected (O\/E) mortality ratios by region. Observed-to-expected (O\/E) mortality ratios are shown by region. Bars indicate 95% confidence intervals\nFor a more precise estimation of the probability of death in the hospital across the different geographic regions, specific customised equations were calculated (Table\u00a05). This customised approach allows each ICU to choose its own reference line for the prediction of hospital mortality: either the overall SAPS\u00a03 hospital mortality sample or its own regional subsample. This approach can be supplemented in the future by customised equations at the country level if data are available and if a more precise estimation of outcome in a specific setting is needed. The overall goodness-of-fit of these customised equations for each region is presented in Table\u00a05. A complete list of the number of patients and the respective O\/E mortality ratios by country, according to the global equation and the regional equations, are presented in Tables E10 and E11 of the ESM, with point estimates varying at the global level from 0.68 (0.56\u20130.80) to 2.05 (1.27\u20132.82). Most O\/E ratios are close to the identity line, as expected for a stable model.Table\u00a05 Customized SAPS\u00a03 admission equations for the different geographic areasAreaEquationGOF \u0124pGOF \u0108pO\/ECIAustralasiaLogit=\u221222.5717 + ln (SAPS\u00a03 score + 1) \u00d75.316310.430.402.200.991.000.93\u20131.07Central, South AmericaLogit=\u221264.5990 + ln (SAPS\u00a03 score + 71.0599) \u00d713.23228.940.547.030.721.000.94\u20131.06Central, Western EuropeLogit=\u221236.0877 + ln (SAPS\u00a03 score + 22.2655) \u00d77.986715.130.1312.150.271.000.94\u20131.06Eastern EuropeLogit=\u221260.1771 + ln (SAPS\u00a03 score + 51.4043) \u00d712.684710.130.437.120.711.000.92\u20131.08North EuropeLogit=\u221226.9065 + ln (SAPS\u00a03 score + 5.5077) \u00d76.27463.450.972.220.991.000.86\u20131.14Southern Europe, Mediterranean countriesLogit=\u221223.8501 + ln (SAPS\u00a03 score + 5.5708) \u00d75.57095.280.8713.120.221.000.97\u20131.03North AmericaLogit=\u221218.8839 + ln (SAPS\u00a03 score + 1) \u00d74.39794.220.934.470.921.000.86\u20131.14GOF \u0124: Hosmer-Lemeshow goodness-of-fit \u0124 test; GOF \u0108: Hosmer-Lemeshow goodness-of-fit \u0108 test; p: respective p-values;  O\/E: observed-to-expected mortality ratio; CI: 95% confidence interval\nDiscussion\nWe have presented the results of a large multicentric, multinational study aimed at updating the SAPS\u00a0II model. This study was necessary for several reasons. First, the reference line used by SAPS\u00a0II was derived from a database collected in the early 1990s; since that time, there have been changes in the prevalence of major diseases and in the availability and use of major diagnostic and therapeutic methods that are associated with a shift toward poor calibration of older models such as SAPS\u00a0II and APACHE III [17, 18]. Second, SAPS\u00a0II was developed from a database built exclusively from patients in Europe and North America. This sample may not be representative of the case mix and medical practices that constitute the reality of intensive care medicine in the rest of the world (e.g. Australasia or South America), where variability in structures and organization is probably related to outcome [19].\nThird, since computation of predicted mortality is based on a reference database, the user should be able to choose between them, i.e., a global database, which provides a broader comparison at the potential cost of less relevance to local conditions, and a regional database, which provides a better comparison with ICUs in geographic proximity but at the cost of losing comparability with ICUs in other parts of the world. A third possibility could be added\u2014a country-representative database\u2014but such a database would raise the problem of whether the ICUs selected were representative of a certain country.\nFourth, the development of computers in recent years has created easy access to strong computational power. One of the implications of this is that it is now possible to develop a new outcome prediction model, based on digital data acquisition and analysis, with minimal differences in definitions and application criteria. These advances were coupled with extensive automatic logical and error-checking capabilities and the availability of data collection manuals online. Moreover, developers of the SAPS\u00a03 model could take advantage of computer-intensive methods of data selection and analysis, such as the use of additive partition trees and logistic regression with random effects. Several new statistical techniques have been used in recent years to allow a more stable prediction of outcome, such as genetic algorithms and artificial neural networks [20, 21], dynamic microsimulation techniques [22], and first- and second-level customization strategies [23\u201325]. However, the value of these techniques is for the moment limited, usually because they are based on regional databases [24\u201326] that prevent extrapolation to other settings; moreover, their superiority in even the regional setting still needs to be established.\nFinally, the SAPS\u00a03 conceptually dissociates evaluation of the individual patient from evaluation of the ICU. Thus, for individual patient assessment, the system separates the relative contributions to prognosis of (i) chronic health status and previous therapy, (ii) the circumstances related to ICU admission, and (iii) the presence and degree of physiologic dysfunction. It is interesting to note that one half of the predictive power of the model is achieved with Box I, i.e., with the information that is available before ICU admission. The prognostic capabilities of the model can be further improved by 22.5% by using data related to the circumstances of the ICU admission (Box II), and by another 27.5% by the incorporation of physiologic data (Box III). These numbers are different from those published by Knaus et al. [4] but are based on what we have learned in the last years about prognostic determinants in the critically ill patient.\nFor performance evaluation, several reference lines should be used, with risk-adjusted mortality in different patient typologies and not only O\/E mortality ratios at hospital discharge in the overall ICU population [27]. The results of the SAPS\u00a03 study showing that different O\/E ratios were observed in different regions of the world should be explored further, since, apart from regional differences in case mix (not taken into account by the model), they can also be related to regional variations in structures and organization of acute medical care, to different lifestyles (e.g., prevalence of obesity, or alcohol and tobacco use) and\/or\u2014though less likely\u2014to genetic differences among populations.\nWe would like to re-emphasize that the model presented here is based exclusively on data (including physiologic data) available within 1\u00a0h of ICU admission and calibrated for manual data acquisition; consequently, it should be expected to overestimate mortality when an automatic patient data management system with a high sampling rate is used [28, 29]. Limiting acquisition of physiologic data to the hour of ICU admission should minimise the impact of this factor when compared with models based on the most deranged data from the first 24\u00a0h after ICU admission, probably at the expense of a small decrease in the ROC curve, a greater sensitivity to the exact time point at which admission to ICU occurs, and therefore more reliant on the assumption that measured physiology alone (as opposed to changes in physiology) predict outcome. It also allows the prediction of mortality to be done before ICU interventions take place. This gives the SAPS\u00a03 admission model a major advantage over existing systems, such as the SAPS\u00a0II or the APACHE II and III, since all these systems can be affected by the so-called Boyd and Grounds effect: the occurrence of more abnormal physiologic values during the first 24\u00a0h in the ICU, leading to an increase in computed severity of illness and a corresponding increase in predicted mortality. These increases may, however, be due not to a greater intrinsic severity of illness of the patient but to the provision of suboptimal care in the first 24\u00a0h of ICU admission, when a stable patient may be allowed to deteriorate [30].\nFurther studies should be done of factors occurring after ICU admission that influence risk-adjusted mortality. We should keep however in mind that this approach comes with one potential pitfall: a possible decrease in the amount of data available for the computation of the model; also, the shorter time period for data collection can eventually increase the likelihood of missing physiological data and the reliance on the assumption that missing physiological data are normal. This effect should be small, considering the widespread availability of monitoring and point-of-case analysers.\nHaving demonstrated the internal validity of the SAPS\u00a03 admission model by the extensive use of cross-validation techniques, we should stress that external validation is also necessary. The fact that the overall database was not collected to be representative of the global case-mix (and especially the case-mix of specific regional areas or patient typologies such as specific diseases) should be empirically tested. Furthermore, the rate of deterioration of our estimates over time should be followed by the appropriate use of temporal validation, especially to avoid what Popovich called grade inflation [18].\nThe SAPS\u00a03 system was developed to be used free of charge by the scientific community; no proprietary information regarding the scientific content is retained. All the coefficients needed for the computation of outcome probabilities are available in the published material. The SAPS\u00a03 can even be computed manually, using a simple scoresheet, although it was designed to be integrated into computerised data acquisition and storage systems that allow the automatic check of the quality of the registered data.\nIn conclusion, we can say that at the end of this stage of the project, we have been able to overcome some of the problems inherent in current risk-adjustment systems. We have minimized user-dependent problems through the publication of careful, detailed definitions and criteria for data collection [31]. We have also addressed the patient-dependent problems by expanding the reference database and making it more representative of reality, in order to include the maximum possible range of variations for patient-centred variables and resulting patient-centred outcomes. This approach was complemented by the development of specific customised equations for major areas of the world, allowing ICUs to choose a reference line for outcome prediction\u2014the global database or the regional database for their own area.\nUsers of these models should keep in mind that benchmarking is a process of comparing an ICU with a reference population. The appropriate choice of reference population is difficult, and we cannot simply change it because the observed-to-predicted mortality rate is not the one we want. For this reason, the choice should depend on the objective of the benchmark: more precise estimation will need local or regional equations, developed from a more homogeneous case mix. A generalisable estimation will, on the other hand, need more global equations developed from a more representative case mix.\nLast but not least, we have successfully addressed some of the problems of prognostic model development, especially those related to the underlying statistical assumptions for the use of specific methods for selection and weighting of variables and the conceptual development of outcome prediction models. In the future, multi-level modelling with varying slopes (and not just random intercepts) might be able to give a better answer to researchers but for the moment they would make the models to complex to be managed outside a research environment.\nElectronic Supplementary Material\n(PDF 794 KB)","keyphrases":["intensive care unit","hospital mortality","severity of illness","icu mortality","risk adjustment"],"prmu":["P","P","P","R","M"]}
{"id":"Matern_Child_Health_J-2-2-1592155","title":"Preconception Care in International Settings\n","text":"Objectives: This literature review briefly describes international programs, policies, and activities related to preconception care and resulting pregnancy outcomes. Methods: Electronic databases were searched and findings supplemented with secondary references cited in the original articles as well as textbook chapters, declarations, reports, and recommendations. Results: Forty-two articles, book chapters, declarations, and other published materials were reviewed. Policies, programs, and recommendations related to preconceptional health promotion exist worldwide and comprise a readily identifiable component of historic and modern initiatives pertaining to women's health, reproductive freedom, and child survival. Conclusions: The integration of preconception care services within a larger maternal and child health continuum of care is well aligned with a prevention-based approach to enhancing global health.\nIntroduction\nPreconception health is widely recognized as a critical component of domestic and international maternal and child health promotion. Broadly defined as the provision of biomedical and behavioral interventions prior to conception in order to optimize women's wellness and subsequent pregnancy outcomes [1], the notion of preconception care can be found in various global policy and practice recommendations concerning women's health and child survival. Although programs and guidelines may vary in response to local needs, the overarching concept of preconception care is present among developing and industrialized countries, within socialized or market-based health care systems, and independent of political, cultural, or religious beliefs. Furthermore, the incorporation of various preconception care strategies and ideologies within international maternal and child health programs and policies spans nearly 30 years, indicating a long-term recognition of the relative importance of such interventions as a means of optimizing pregnancy outcomes.\nThis article describes international efforts to reduce adverse maternal and infant outcomes through programs, policies, and activities related to preconception care. Because widespread support for preconception health promotion is comparatively recent, explicit mention of preconception care programs and policies per se is often difficult to identify within the international literature. Furthermore, because many developing countries adopt strategies guided by the declarations of international agencies or other coalitions [2], the development of preconception health initiatives must be traced through the history of various international health movements, starting with the promotion of global primary health care, followed by Safe Motherhood initiatives, and culminating with the women's rights and health movements. Thus, whereas the activities described herein may not be immediately identifiable as preconception care, they represent essential pieces of the preconception health puzzle.\nMaterials and methods\nA MEDLINE search with English language and human subject restriction was conducted for the years 1980 through May 9, 2005 using the reference terms \u201cpreconception (pre-conception) care,\u201d \u201cpreconception counseling,\u201d \u201cpreconceptional care,\u201d \u201cpericonception care,\u201d and \u201cpericonceptional care.\u201d A total of 756 citations were identified using the initial search parameters. References for which preconception care programs or activities outside of the United States comprised the primary topic of interest were included. Articles detailing clinical or scientific evidence of specific periconceptional interventions were excluded. Also reviewed were other published articles as well as textbook chapters, declarations, reports, and recommendations not retrievable from these databases. The search terms were also entered in various internet search engines and relevant web-based information was used to supplement the findings. Pertinent abstracts from the National Summit on Preconception Care (CDC, June 2005) were also reviewed.\nResults\nA total of 42 articles, book chapters, declarations, and other published materials were reviewed. The findings were categorized into three main areas in order to present the material in a coherent fashion: international conferences and accords, professional organizations, and international preconception care programs.\nInternational conferences and accords\nAs preconception health is closely linked to women's health, language in support of preconception care is found in declarations and agreements derived at international conventions on the topic of women's wellness and reproductive health. For example, although the primary goal of the 1978 International Conference on Primary Health Care was to promote the health of all by outlining essential primary health care strategies to be implemented throughout the world, one important provision of the declaration outlined the importance of maternal and child health care and family planning as integral components of primary health care [2\u20134].\nThe Safe Motherhood Initiative, initially drafted in 1987 at the first international Safe Motherhood Conference in Nairobi, Kenya, focused on reducing morbidity and mortality associated with pregnancy and childbirth among developing countries [3]. The Safe Motherhood Inter-Agency Group (IAG) cosponsored the event and is now comprised of various international and national agencies whose mission is to enhance maternal and neonatal survival through the development and promotion of cost-effective interventions. In a global technical consultation held 10 years after the launch of this initiative, the IAG assessed relevant health indicators and developed a package of services to be offered to all women as a means of ensuring safe motherhood. Various preconception care services were outlined, including family planning, adolescent reproductive health education services, and community-based education pertaining to sexual and reproductive health as well as safe motherhood [5].\nCovenants related to preconception care were adopted by the 1988 World Congress of Obstetrics and Gynaecology and its predecessor, the General Assembly of the International Federation of Gynaecology and Obstetrics. The 1988 World Congress heralded the promotion of women's health with the first step being prevention as a social responsibility; the General Assembly of the International Federation advocated governments undertake concrete measures to improve women's health and social status [6]. The second International Congress for Maternal and Neonatal Health, held in Monastir, Tunisia, in November 1984, included in its recommendations the need to promote the expansion of health services including family planning services and other preconception interventions such as smoking cessation, increased birth spacing, and tetanus vaccination [7].\nThe 1993 Report of the World Conference on Human Rights reaffirmed the need for equal access to health care for women, including a wide range of family planning services and called for reductions in infant and maternal mortality rates and overall improvements in women's health and nutrition [5, 8]. Similarly, the International Day of Action for Women's Health, first held on May 28, 1990, through the collaborative efforts of the Women's Global Network for Reproductive Rights and the Latin American and Caribbean Women's Health Network\/ISIS International, reiterated the need for significant reductions in maternal mortality, particularly in Latin American countries [3].\nInternational conferences held in subsequent years reflected an important paradigm shift in which pregnancy outcomes and maternal health were considered within a broader context encompassing reproductive health and women's health as well as socio-cultural factors [3]. Held in Cairo in 1994, the third decennial International Conference on Population and Development (ICPD) emphasized the importance of reproductive freedom and developed a definition of reproductive health that included \u201caccess to appropriate health care services that enable women to go safely through pregnancy and childbirth and provide couples with the best chance of having a healthy infant\u201d [9]. In 1995, two additional international meetings took place\u2014the World Summit for Social Development and the Fourth World Conference on Women\u2014in which the tenets set forth by the ICPD with regard to maternal mortality and morbidity were reaffirmed and new goals for improving access to maternal health services were also derived [3, 5, 9, 10].\nMore recent international activities related to preconception health include the 1998 World Health Day and the Millennium Declaration. World Health Day 1998 focused on safe motherhood with the assertion that every pregnancy faces risks that may be ameliorated by enhancing access to quality maternal health services, delayed childbearing, and preventing unwanted pregnancies [11]. The Millennium Declaration, adopted by all member states of the United Nations in September 2000, included eight millennium goals to be achieved on a global level. The goals were designed as a measure of development against which countries may compare health indicators, programs, and policies. Maternal health and child mortality are explicitly detailed in two main goals and are closely related to other key areas including poverty, gender equality, nutrition, education, and infectious diseases [3, 12].\nReferences to preconception health can also be found in the declarations of conferences addressing the issues of child health and survival. For example, the 1989 Convention on the Rights of the Child recognized the right of children to attain the highest possible standard of health and contained provisions for reductions in infant mortality, prenatal and postnatal care for mothers, family planning education, and services and health education for children and parents regarding nutrition, clean drinking water, and environmental contaminants [13]. In addition, the World Declaration on the Survival, Protection, and Development of Children, developed at the 1990 World Summit for Children, included tasks pertaining to the reduction of maternal mortality with a specific emphasis on family planning and child spacing as well as efforts to reduce infant and child mortality [14].\nProfessional organizations and associations\nInternational health care professionals also have long recognized the utility of preconception care, as evidenced by directives and recommendations supported by various professional organizations. In 1952, after significant changes to the system of reporting maternal deaths sparked concern within the medical community about maternal mortality rates in the United Kingdom, a series of inquiries were initiated that ultimately resulted in the ongoing Confidential Enquiries into Maternal and Child Health [3, 15]. In its current manifestation, the program seeks to improve the health of mothers, infants, and children through careful investigation of all maternal and perinatal deaths as well as issues associated with diabetes during pregnancy and child health.\nIn 1982, the International Federation of Gynaecologists and Obstetricians (FIGO) joined the World Health Organization (WHO) in creating a task force to assess safe motherhood within regional and international communities. Since then, FIGO has conducted needs assessments and funded demonstration projects to ascertain cost-effective methods of identifying and managing pregnancy complications [3, 16, 17]. One such project, the Averting Maternal Death and Disability Program (AMDD), was established at the Columbia University Mailman School of Public Health and has partnered with numerous international agencies and communities to improve access to quality emergency obstetric care [17].\nNurses and midwives also have played an important role in the promotion of preconception care among the medical community. Themes associated with International Nurses Day have incorporated various components of the preconception care paradigm, including safe motherhood, school health, healthy families, women's health, community health, domestic violence, and the AIDS stigma. The International Confederation of Midwives, a member of the FIGO working group on Safe Motherhood and Newborn Health, has hosted a number of workshops related to safe motherhood and midwifery. Topics addressed at the workshops have included HIV\/AIDS, gender-based violence, and most recently, maternal and newborn health during birth and the postnatal period [3, 18].\nInternational preconception care guidelines and programs\nThe structure of preconception care programs in the international community differs considerably according to the type of health system employed and the level of economic support. Wealthier countries tend to have better organized health systems [19] and therefore use broader policies and guidelines to direct the provision of preconception services within the health care sector. Thus, countries such Canada [1], the United Kingdom [20, 21], Spain [22], Australia, Hungary [23], and the Netherlands [24] are more likely to adopt various recommendations related to preconception health, whereas less-developed countries in the regions of Latin America [25], Africa, India, and the Middle East tend to use more targeted interventions [26]. For example, many African countries provide continuing education to community health care workers to help them deal with issues in preconception counseling and reproductive choices for patients with HIV.\nWithin the developing countries of Latin America, preconception care has afforded such nations as Ecuador, Honduras, Nicaragua, Paraguay, and Peru an opportunity to address inequitable (as compared with more developed countries) maternal and perinatal morbidity and mortality rates [25]. Maternal mortality in these countries is cited as one of the primary causes of death among women 15 to 39 years of age. Using a $250,000 grant provided by WHO, these countries have undertaken a demonstration project to deliver preconception health education via community programs. The specific aims of the project are to develop and deliver a comprehensive preconception care package that includes (1) risk assessment (identify individual, family, and social risks and barriers to prenatal care); (2) health promotion (ensure proper nutrition; avoid substance, tobacco, and alcohol use; provide family planning; perform PAP smear screening, and provide ongoing care); and (3) treatment delivery (treat medical conditions and infections such as malaria and sexually transmitted diseases, update immunizations, provide nutritional supplementation such as folic acid, and conduct home visits).\nIn China before 2003, a premarital health check was required of all couples planning to marry and consisted of medical examinations and testing as well as health education. The requirement has since been abolished and this change has been followed by concomitant declines in the rates of premarital examinations [27]. As a result, pilot studies have been designed to test the feasibility and content of a social marketing campaign for preconception care in China. The findings indicate that although women are interested in information about preconception health, numerous barriers to implementing a national program exist, including vertical health systems, a lack of coordinated efforts among governmental organizations providing family planning and primary health care, and an abundance of potentially confusing media health messages [27]. Preconception care services are sometimes provided in private clinics such as the Pre-Pregnancy Preparation Service (PPPS) in Hong Kong which provides pre-pregnancy counseling, medical testing, health assessments, and educational services to approximately 4,000 couples each year [28].\nTo reach women and children residing in remote regions of the Philippines, a mobile health services program has been developed whereby a multidisciplinary team of physicians and nurse midwives travel to specified areas and provide services for 1 to 3 days per month. The team screens pregnant women for various risk factors and nonpregnant women of reproductive age for chronic medical conditions. Family planning services are also provided [29].\nThe Russian Association of Gynecologists and Obstetricians has developed and implemented the People's Health Movement with a primary objective of promoting health and preventing disease throughout the stages of a woman's reproductive life. Based on policy recommendations arising from an alert that the state of maternal and child health was one of crisis proportion, preconception care services inclusive of family planning are now being initiated [30].\nThe Office de la Naissance et de l\u2019Enfance in Belgium is currently establishing a national preconception care campaign to educate all women of reproductive age and all health professionals engaged in the care of women and children regarding the importance of preconception health care. The marketing tools and plans for disseminating the information are now being developed [31].\nIn France, preconception care has focused on more targeted recommendations, such as good glycemic control prior to conception. It had previously been established that preterm delivery among diabetic mothers was much higher than among the general population and that fetal loss and congenital malformations were assoicated with poor glycemic control [32]. Using one of the goals of the 1989 International Diabetes Federation meeting in St. Vincent\u2019s, Italy, tertiary perinatal centers began offering preconception care in an attempt to reduce the level of adverse pregnancy outcomes of diabetic mothers [33]. Services included risk assessment of potential diabetic complications, education on nutritional and glycemic self-monitoring, and optimization of insulin treatment regimens as directed by treatment guidelines. Data from a cross-sectional study of 12 perinatal centers in France indicate that nearly half of all women with Type 1 diabetes received preconception care during 2000\u20132001 as compared to 24% of women with Type 2 diabetes. Because rates of adverse outcomes among infants of diabetic mothers were similar to those during 1986\u20131988, it was concluded that more effort was needed to achieve the targets set forth in the St. Vincent's declaration [33].\nCanada's National Guidelines on Family-Centred Maternity and Newborn Care devotes an entire chapter to preconception care and describes the multitude of intrinsic and extrinsic factors that influence preconception health. Accordingly, various settings appropriate for the administration of preconception care interventions are discussed as well as various social and medical issues, including stress, social support, abuse and violence, healthy lifestyle practices, and nutrition [1].\nAlthough information about the development of international preconception care clinics is scant, such programs have been noted in Hungary, the United Kingdom, and the Netherlands. In 1989, Hungary established the Optimal Family Planning Service (OFPS) under the direction of the WHO Collaborating Centre for the Community Control of Hereditary Diseases. The OFPS was comprised of 32 regional health care centers providing periconceptional care free of charge. The aim of this program was to reduce adverse perinatal outcomes and protect maternal health among all women. In 1996, the Hungarian government increased the number and scope of the centers to incorporate these services within primary health care [23]. Assessments of various indicators in Hungary 10 years after the creation of the OFPS indicate that the rates of major congenital anomalies decreased and that the use of protective factors such as folic acid supplementation, rubella vaccination, and infection screening increased. Barriers to the use of preconception care services included unnecessary medical examinations, an inability to effect change among unintended pregnancies, and additional costs [24].\nDespite the existence of numerous clinical genetic centers in the Netherlands, only two preconception clinics have been described in the literature. The first, an outpatient preconception care clinic established by the University Medical Center of Nijmegen, provides thorough evaluations of potential pregnancy risk and preconceptional interventions for women referred by health care practitioners [34]. Treatment for hyperhomocysteinemia and changes in drug therapies for women taking potentially teratogenic medications were the most common preconception interventions at the clinic. A pilot preconception clinic in Maastricht was developed as a forum in which women planning a pregnancy could address questions and concerns. Couples attending the clinic responded favorably to the program and indicated that they would not have asked their general practitioner such questions [35]. These findings are supported by the results of recent surveys of Dutch women in which more than 70% of the respondents reported an interest in preconception counseling when offered by their own general practitioner [36]. However, although the majority of Dutch health care providers are in favor of establishing preconception care clinics [37, 38], lack of specific knowledge and time constraints have been cited as barriers to the provision of adequate preconception counseling [38].\nPreconception clinics have also been described in London and Glasgow. The London clinic opened in 1978 and consisted of one obstetrician who advised women with previous pregnancy complications about the management of subsequent pregnancies [39, 40]. During the first 18 months of the clinic's operation, 56 women were seen. Opened in 1982, the Glasgow clinic also served women affected by prior adverse pregnancy outcomes. The clinic remained operational for 9 years and relied on a research nurse for initial evaluation and screening followed by a physician consultation 4 weeks later [40, 41]. The most common reasons for referral to the clinic were previous miscarriage, previous fetal abnormality, and chronic maternal disease. Improvements in pregnancy outcome were noted only among the women with chronic conditions [41].\nConclusions\nGlobal recognition of the potential benefits associated with preconception health promotion is not new. International policy directives and practice recommendations related to women's health, reproductive freedom, and child survival almost always include provisions for the enhancement of women's wellness and social status as a means of reducing adverse pregnancy outcomes. However, the relative success of preconception care programs in both developed and developing countries is directly related to the availability and accessibility of health care for women. Accordingly, WHO's World Health Report 2005: Make Every Mother and Child Count details the inherent interrelation of the needs of the mother and child, indicates that reproductive health comprises an essential element of the continuum of maternal and child health, and calls for a reformulation of interventions from vertical programs to those offering a wider range of services [42]. A similar longitudinal approach to women's wellness and reproductive health has been promoted in the U.S. in light of increasing rates of low birth weight and persistent racial disparities in maternal and infant outcomes [43\u201345].\nThe present description of international programs and perspectives on preconception care is limited in scope by numerous factors. The literature search was as comprehensive as possible but not exhaustive because of language restrictions, difficulty in retrieving documentation of international preconception care programs and policies, the relative scarcity of published information about international preconception care practices, and the myriad of maternal and child health outcomes that fall under the umbrella of preconception health and for which both population-based and targeted interventions exist. Finally, data pertaining to maternal and infant outcomes after the introduction of preconception care activities is often unavailable. Thus, this review provides only a broad overview of international activities related to preconception health. However, the findings are instructive as a preliminary step in the compilation and dissemination of information related to international preconception care recommendations and practices. Future research should include a regionalized inspection and comparison of preconception care programs, policies, and recommendations within and among various countries. The impact of these programs on community-specific indicators of maternal and perinatal health should also be evaluated.\nIrrespective of setting, the wider application of the core components of preconception care (i.e., risk assessment, health promotion, and intervention) has the potential to enhance the health and overall well-being of women, infants, and children around the globe. In light of the numerous factors influencing pregnancy outcome, an integrated approach to preconception health promotion has been proposed as perhaps the most effective and efficient means of implementing preconception health care [43\u201347]. The benefits of service integration are apparent in even the most resource-poor settings, as evidenced by the introduction and expansion of the WHO's strategy, the Integrated Management of Childhood Illness, in various countries [48]. Indeed, the perspective that preconception care is part of a larger continuum of care is well aligned with a prevention-focused public health paradigm. As such, the widespread promotion of preconception health may be the most timely and logical step toward ensuring global health.","keyphrases":["preconception care","international health","reproductive health"],"prmu":["P","P","P"]}
{"id":"Clin_Exp_Metastasis-4-1-2413104","title":"Serum proteome analysis for profiling protein markers associated with carcinogenesis and lymph node metastasis in nasopharyngeal carcinoma\n","text":"Nasopharyngeal carcinoma (NPC), one of the most common cancers in population with Chinese or Asian progeny, poses a serious health problem for southern China. It is unfortunate that most NPC victims have had lymph node metastasis (LNM) when first diagnosed. We believe that the 2D based serum proteome analysis can be useful in discovering new biomarkers that may aid in the diagnosis and therapy of NPC patients. To filter the tumor specific antigen markers of NPC, sera from 42 healthy volunteers, 27 non-LNM NPC patients and 37 LNM NPC patients were selected for screening study using 2D combined with MS. Pretreatment strategy, including sonication, albumin and immunoglobulin G (IgG) depletion, was adopted for screening differentially expressed proteins of low abundance in serum. By 2D image analysis and MALDI-TOF-MS identification, twenty-three protein spots were differentially expressed. Three of them were further validated in the sera using enzyme-linked immunosorbent assay (ELISA). Our research demonstrates that HSP70, sICAM-1 and SAA, confirmed with ELISA at sera and immunohistochemistry, are potential NPC metastasis-specific serum biomarkers which may be of great underlying significance in clinical detection and management of NPC.\nIntroduction\nNasopharyngeal carcinoma (NPC) is a rare malignancy in most parts of the world, though it is highly prevalent in Southern Asia, where the incidence is about a 100-fold higher than in other populations. It is one of the most confusing, commonly misdiagnosed and poorly understood diseases. Previous studies show that the cancer is an Epstein-Barr virus-associated malignancy with a remarkable racial and geographical distribution. The etiology of NPC is thought to be associated with a complex interaction of genetic, viral, environmental and dietary factors. Thanks to the advancements in genomics, proteomics and bioinformatics in recent decades, the etiology, carcinogenesis and progression of the disease is better understood. Research into these components may unravel the pathways in NPC development and potentially decipher the molecular characteristics of the malignancy [1, 2]. NPC is an insidious tumor and is usually at the stage of metastasis involving lymph nodes or other organs before it can be found. Thus early and accurate diagnosis is very important for therapy and prognosis of NPC patients. Unfortunately, no effective method of accurately diagnosing new-onset NPC is available now. We presume if specific serum biomarkers associated with NPC metastasis can be identified on the basis of advancements in genomics, proteomics and bioinformatics, an approach to the early detection and monitoring of NPC may be found.\n2D comparative proteome analysis is a new technology for the separation and identification of disease-specific proteins, and it has been applied successfully to screen potential biomarkers for NPC in cell lines and tumor tissues [3\u20138]. In our previous studies, we employed proteomic techniques to study protein changes of CNE2, a poorly differentiated squamous carcinoma cell line of human NPC cells, induced by 12-O-Tetradecanoyl-phorbol-13-acetate (TPA). It is likely that TPA promotes NPC in necessary cooperation with EBV, or that it may function as an antiproliferative or differentiative revulsant in noninitiated cells [4]. However, if a marker can be detected only in surgical specimens, its clinical significance is limited, especially for early screening or diagnosis. Recently, Saeid R. et\u00a0al. reported their pioneering work on NPC serum analysis by 2D without any pretreatment, revealing the enhanced expression of relatively abundant proteins as ceruloplasmin (CPL) [9]. In order to find relatively low-abundant serum proteins which may be more valuable in predicting NPC progression, we pretreated serum with sonication, albumin and IgG depletion before 2D analysis. By comparing 2D image analyses for healthy volunteers, non-LNM NPC and LNM NPC patients, we may identify the differentially expressed proteins so that specific serum biomarkers associated with NPC metastasis can be found.\nMaterials and methods\nSubjects\nSerum samples from 42 healthy volunteers and 64 NPC patients were available for comparative proteomic analysis. Samples from NPC patients were obtained before any clinical treatment. All the patients had a nasopharyngeal biopsy and NPC was confirmed by two independent pathologists unaware of the patient\u2019s condition. Careful medical examinations and CT scan determined non-LNM cases and LNM cases in the NPC patients. Clinical data of all the subjects for 2D and ELISA are shown in Table\u00a01. In the screening study, serum samples from the same group were pooled to smooth intrinsic individual differences and enhance common characteristic traits only related to disease status [10], aliquoted, and stored at \u221280\u00b0C before analysis. In the validation study, 26 of the patients received a mean of 17.8\u00a0months of follow-up (range, 6\u201322\u00a0months) after radiotherapy and\/or chemotherapy. Six of the followed-up patients died of NPC, and the other survived the follow-up period.\nTable\u00a01Serum samples in the studySamples for 2-DESamples for ELISAHealthyNPCHealthyNPCLymph node status\/NegativePositive\/NegativePositiveNumber422737302730Gender (Male\/Female)22\/2021\/623\/1415\/1521\/620\/10Age (\u00b1SD)42\u00a0\u00b1\u00a07.943\u00a0\u00b1\u00a010.950.5\u00a0\u00b1\u00a011.742.5\u00a0\u00b1\u00a08.943\u00a0\u00b1\u00a010.947.6\u00a0\u00b1\u00a010.2\nDepletion of high-abundance proteins\nSigma-Aldrich ProteoPrep\u00ae Immunoaffinity Albumin and IgG Depletion Kit was used for depletion of proteins, albumin and IgGs, allowing visualization of co-migrating proteins with albumin and IgG on a 2D gel and also higher sample loads for improved visualization of lower abundance proteins. A total of 30\u00a0\u03bcl of serum was diluted with 100\u00a0\u03bcl of equilibration buffer from the depletion kit (Sigma) and sonicated using four ultrasonic bursts of 15\u00a0s each. Between sonications, the samples were chilled on ice. Then serum albumin and IgG was depleted according to the manufacturer\u2019s instructions. After collection of the bound proteins, the majority (>95%) of the unbound proteins were left in the depleted serum sample. Modified Bradford assay was used to measure the protein concentrations of all serum samples [11].\n2D analysis\nThe Immobiline Dry strip (pH 4\u20137, length 17\u00a0cm, Bio-Rad) was rehydrated with 600\u00a0mg protein in 350\u00a0ml rehydration buffer containing 7\u00a0M urea, 2\u00a0M thiourea, 4% CHAPS, 65\u00a0mM DTT, 5\u00a0mM tributylphosphine, 1% IPG buffer and 0.002% bromophenol blue for 14\u00a0h at room temperature. IEF was performed using Protean IEF cell (Bio-Rad, Hercules, CA, USA) with a total of 70\u00a0kVh. Then the strip was subjected to two-step equilibration in equilibration buffer containing 6\u00a0M urea, 30% glycerol, 2% SDS and 50\u00a0mM Tris\u2013HCl (pH 6.8) with 1%\u00a0w\/v DTT for the first step, and 2.5%\u00a0w\/v iodoacetamide for the second step. The second-dimensional SDS-PAGE gel (13% T, 2.7% C, and 20\u00a0\u00d7\u00a020\u00a0\u00d7\u00a01\u00a0mm3) was carried out using a Protean II xi 2D cell (Bio-Rad) according to the following procedure: 30\u00a0min at a constant current of 12\u00a0mA followed by 24\u00a0mA per gel until the bromophenol blue front reached the bottom of the gel. Subsequently the gels were stained with MS accommodated silver staining [12]. 2D of each sample was run in triplicate to minimize run-to-run variation. The silver-stained 2D gel were scanned using a Power-Look 1100 imaging scanner (Umax, Dallas, TX, USA) and analyzed using PDQuest 7.1 software package (Bio-Rad).\nIn-gel enzymatic digestion\nProtein spots were excised from gel with an operating knife blade, destained twice with 30\u00a0mM potassium ferricyanide and 100\u00a0mM sodium thiosulfate (1:1\u00a0v\/v) and then equilibrated in 50\u00a0mM NH4HCO3 to pH 8.0. After dehydrating with ACN and drying in N2 at 37\u00b0C for 20\u00a0min, the gel pieces were rehydrated in 15\u00a0ml trypsin solution (10\u00a0\u03bcg\/ml in 25\u00a0mM NH4HCO3) at 4\u00b0C for 30\u00a0min and incubated at 37\u00b0C overnight. Peptides were extracted twice using 0.1% TFA in 50% CAN and dried with N2.\nMALDI-TOF-MS identification\nThe peptide mixtures were solubilized with 0.5% TFA, with saturated \u03b1-cyano-4-hydroxy-trans-cinnamic (CHCA) solution in 0.1% TFA\/50% acetonitrile as the matrix and analyzed using M@LDI R (Micromass, Manchester, UK). Mass spectra were externally calibrated with lock mass 2,465.199\u00a0Da and internally calibrated with autodigested peaks of trypsin (MH+: 2,211.105\u00a0Da).\nProtein identification and database searching\nProtein identification using peptide mass fingerprinting (PMF) was performed by the MASCOT search engine (http:\/\/www.matrixscience.com\/, MatrixSicence Ltd., London, UK) against the MSDB protein database. The errors in peptide masses were in the range of 25\u00a0ppm. One missed tryptic cleavage site per peptide was allowed during the search. Proteins matching more than four peptides and with a MASCOT score higher than 64 were considered significant (P\u00a0<\u00a00.05). Carboamidomethylation of cysteine was selected as the static modification and oxidation of methionine as the differential modification. Protein identification results were filtered with peakErazor software.\nEnzyme-linked immunosorbent assay (ELISA) confirmation\nELISAs were conducted to confirm the protein identification and differential expression of sICAM-1, SAA and HSP70 in sera from the three groups. Measurements were done by commercially available ELISA kits (StressXpress Hsp70 ELISA Kit: EKS-700B, Stressgen Biotechnologies, Victoria; High sensitivity sICAM-1 ELISA Kit: BMS241, Bender Medsystems Company, Australian; Human SAA ELISA Kit: EL10015, YES Biotech Laboratories Ltd, Canada), in accordance with the manufacturer\u2019s instructions. All sera were stored at \u221280\u00b0C before they were measured. Both standards and samples were run in duplicate. The main protocols were as follows: monoclonal coating antibody was adsorbed onto microwells; target protein present in the sample or standard bound to antibodies was adsorbed to the microwells; second antibody was added and bound to target protein captured by the first antibody; following incubation unbound enzyme was removed during a wash step; a color reaction was formed and absorbance was measured at 450\u00a0nm. The standard curve was used to determine the concentration of target protein in an unknown sample.\nImmunohistochemistry (IHC) assay\nThe previous studies showed that sICAM-1 in serum could be released by membrane-associated ICAM-1 of cancer cell [13, 14]. However, SAA is synthesized and secreted into serum by hepatocytes in the liver during the acute phase [15, 16]. In the present study, therefore, expressions of only HSP70 and ICAM-1 were detected in tissue by IHC assay. Three-micrometer sections of paraffin-embedded tissues were cut for a prior antigen retrieval step in boiling 10\u00a0mM citrate buffer pH 6 for 20\u00a0min. The fixed tissues were subjected to immunostaining using an ultrasensitive S-P technique (MaiXin, China). Briefly, the slides were incubated overnight at 4\u00b0C with goat anti-ICAM and anti-HSP70 polyclonal antibody (1:200) (Wuhan Boster Biological Technology Co., Ltd., China). The concentrations of the primary antibodies used have been optimized previously. After washing with PBS three times, they were treated with biotin-conjugated second antibody before adding streptavidin-peroxidase. For color reaction, diaminobenzidine (DAB) was used. All series included positive controls. For negative controls, the antibody was replaced by PBS. To evaluate the expression, the cellular localization of immunoreactivity was determined and scored for both intensity (negative, weak, moderate and strong) and proportion (5\u201325, 26\u201350, 51\u201375 and 76\u2013100%) of stained cells. To allow comparison of immunostaining with pathological stage and metastasis for each cancer, integer values were assigned to the scores of intensity (0\u20133) and proportion of tumor cells stained (0\u20134). If the evaluations did not agree, the specimens were reevaluated and then classified according to the assessments given, most frequently, by the observers. The proportion and intensity scores were then added to obtain a total score, which was labeled as one of the four following categories: (a) high positive (+++), total score of 6\u20137; (b) moderate positive (++), total score of 4\u20135; (c) low positive (+), total score of 2\u20133; (d) negative (\u2212), total score of 0\u20132.\nStatistical analysis\nAll the analyses performed were descriptive. The serum levels of protein expressions were compared using one-way ANOVA test. Associations between immunohistochemical scores and clinicopathological variables of tissue specimens were evaluated by Fisher\u2019s exact test or \u03c72 test. PDQuest software was used to establish a master gel representing each group [17]. For differential expression of matched gels, protein spots whose intensities were either increased or decreased two-fold or greater were marked and then confirmed by manual inspection of all relevant 2D gel, not only those included in the matchsets, to ensure consistency. Quantitative analysis was performed using the Student\u2019s t-test among three groups of gels. All statistical analysis was performed with SPSS 10.0 software and P value <0.05 were considered statistically significant.\nResults\nQuantitative comparison and identification of protein spots on 2D gels\nAmong the three groups, we found twenty-three spots significantly altered (P\u00a0<\u00a00.05) or absent\/emergent, 13 of which were identified successfully (Fig.\u00a01). The corresponding detailed differential expression patterns are shown in Fig.\u00a02a. Quantitative comparison and MS identification of the 13 differentially expressed proteins are shown in Fig.\u00a02b and summarized in Table\u00a02, respectively. Overall, transferrin (TRF) and transthyretin (TTR) were down-regulated and 12-lipoxygenase (12-LOX), serum amyloid A1 protein precursor (SAA), soluble intercellular adhesion molecule-1 (sICAM-1) and lysine-specific histone demethylase1 (LSD1) were up-regulated in two NPC groups compared with the healthy group. Furthermore, increased expression levels of 12-LOX, sICAM-1 and SAA were observed in LNM NPC as compared with non-LNM NPC. However, heat shock protein 70 (HSP70) and cytochrome P450 (CYP450) were expressed significantly and constantly only in LNM NPC patients. The MALDI-TOF-MS mass spectra are shown in Fig.\u00a03.\nFig.\u00a01Representative 2D patterns of albumin and IgG depleted serum from healthy (a), non-LNM NPC (b) and LNM NPC groups (c). In the 2D images, spots \u20181\u201310\u2019 indicate up-regulation and \u201811\u201313\u2019 down-regulation in NPC group. Black arrows indicate protein spots that were identified successfully by MSFig.\u00a02Detailed 2D patterns of the thirteen differentially expressed proteins among healthy, non-LNM and LNM NPC groups (a). Corresponding gel positions are shown in Fig. 1. Magnified 2D maps demonstrating the different expressions of selected proteins are listed in Table 2. The densitometric analysis of each protein was calculated from nine different gels using PDQuest 7.1 software package (b). Each bar represents the mean\u00a0\u00b1\u00a0S.D. of intensity, with significant differences between groups found by two-tailed student\u2019s-test. Note that this panel has a different vertical (y) axis and a break in the axis to accommodate the poor performance of the subject spot No.6. The break region is from 5000 to 6000. *P\u00a0<\u00a00.05, compared with healthy group; **P\u00a0<\u00a00.05, compared with non-LNM NPC groupTable\u00a02Identification results of differentially expressed proteins among healthy volunteers, non-LNM NPC and LNM NPC patientsNo.aProtein descriptionMSDB IDProtein levelbMASCOT scorecSequence coveraged (%)Theoretical Mr\/pIe1Ovochymase precursorQ7RTY7\u219111652124,947\/8.672Arachidonate 12-lipoxygenaseAAA51587\u21911936875,694\/5.823sICAM-1Q99930\u2191112544270\/6.404Cytochrome P450Q53EX9\u21912075755,635\/6.835Serum amyloid A1 protein precursorYLHUS\u21911708513,524\/6.286Hemoglobin beta subunits are s-nitrosylated1BUWB\u21912075515,565\/6.767E1A 10S proteinQ9YLA2\u21911436928,493\/4.098Lysine-specific histone demethylase 1O60341\u21911725893,358\/6.119KIAA1622 proteinQ9HCF0\u21917861102,351\/8.4210Heat shock 70\u00a0kDa protein (HSP70)Q6G1F9\u21911575368,161\/4.8811TransferrinQ9NQB8\u21931676777,050\/6.8112UPF0366 protein C11orf67AAD40378\u21931045812,798\/8.5813TransthyretinAAA61181\u2193876912,836\/5.35aThe numbers indicate the spot positions in 2D gel as shown in Figs.\u00a01 and 2b\u2191, up-regulation in NPC patients; \u2193, down-regulation in NPC patientscBy MALDI-TOF MS analysisdCalculated by amino acid counteCalculated from the database entry without any processingFig.\u00a03PMF of protein spots No. 3, 5 and 10, respectively, representing sICAM-1 (panel A), SAA (panel B), and HSP70 (panel C), which were expressed differentially between the groups of non-LNM and LNM NPC (MASCOT scores are 65, 85 and 78, respectively, P\u00a0<\u00a00.05)\nELISA confirmation\nFigures\u00a04 and 5 show the ELISA results of sICAM-1, SAA and HSP70 performed in the three groups. There were statistically significant differences between health and NPC groups (P\u00a0<\u00a00.01) as well as between non-LNM and LNM NPC groups (P\u00a0<\u00a00.05) (Fig.\u00a04). And with the TNM stage advanced, the serum levels of sICAM-1 and HSP70, were detected significantly elevated, but that of SAA not (Fig.\u00a05). Furthermore, bivariate correlation analysis showed a significant relationship between the serum levels of the three proteins (P\u00a0<\u00a00.01). The follow-up showed that the serum levels of sICAM-1 and HSP70, but not of SAA, were strongly associated with NPC mortality (Student\u2019s t-test, P\u00a0=\u00a00.001, 0.001 and 0.132, respectively). Our ELISA findings were consistent with results of 2D and silver-staining.\nFig.\u00a04Quantitative comparison of sICAM-1, HSP70 and SAA in sera among healthy, non-LNM NPC and LNM NPC groups. Each bar represents the mean\u00a0\u00b1\u00a0S.D. of the concentration, with significant differences between groups found by one-way ANOVA test. Note that this panel has a different vertical (y) axis and a break in the axis to accommodate the poor performance of the subject SAA. The break region is from 500 to 4500\u00a0\u03bcg\/l. *P\u00a0<\u00a00.05, compared with healthy group; **P\u00a0<\u00a00.05, compared with non-LNM NPC groupFig.\u00a05Quantitative comparison of sICAM-1, HSP70 and SAA in sera among NPC patients at different TNM stage. Each bar represents the mean\u00a0\u00b1\u00a0S.D. of the concentration, with significant differences between groups found by one-way ANOVA test. Note that this panel has a different vertical (y) axis and a break in the axis to accommodate the poor performance of the subject SAA. The break region is from 600 to 2000\u00a0\u03bcg\/l. *P\u00a0<\u00a00.05, compared with NPC patients at TNM stage I; **P\u00a0<\u00a00.05, compared with NPC patients at TNM stage II; ***P\u00a0<\u00a00.05, compared with NPC patients at TNM stage III\nIHC assay\nIn NPC patients, HSP70 and ICAM-1 were observed in tumor cells but not in stromal, endothelial or inflammatory cells. The immunoreactivity was located in both membrane and cytoplasm for ICAM-1 and only in cytoplasm for HSP70 (Fig.\u00a06). The immunoreactivities of HSP70 and ICAM-1 were observed in 37 (64.9%) and 43 (75.4 %) of the 57 cases of NPC, respectively. The correlation of positive rates showing HSP70 and ICAM-1 immunoreactivity with clinicpathological characteristics of the cases is summarized in Table\u00a03. Statistically, positive expression rates of HSP70 and ICAM-1 were significantly correlated with lymph node metastasis (LNM) of NPC. Furthermore, we found that positive expression rates of ICAM-1 (6\/6, 100%) and HSP70 (5\/6, 83.3%) were high in the cases that died of NPC during the follow-up period (P\u00a0>\u00a00.05). The results manifested that ICAM-1 and HSP70 expressions were strongly associated with NPC metastasis and mortality, indicating poor prognosis.\nFig.\u00a06Immunohistochemical detection of HSP70 and ICAM-1 in NPC (Magnification \u00d7200). The immunoreactivity was located in both membrane and cytoplasmic stainings for ICAM-1 but only in cytoplasmic staining for HSP70. The black arrows indicate the target proteins stained in brown-yellow in the NPC nest. The red arrow in E shows negative immunoreactivity of normal nasopharyngeal epithelia. a, negative expression of HSP70; b and c, positive expressions of HSP70; d, negative expression of ICAM-1; e and f, positive expressions of ICAM-1; a, b, d and e, non-LNM NPC; C and F, LNM NPCTable\u00a03Characteristics of NPC patients dependent on ICAM-1 and HSP70 expressionsCharacteristicsNumberICAM-1 expressionHSP70 expressionNegativePositive (%)P valueNegativePositive (%)P valueGender\u00a0\u00a0\u00a0\u00a0Male41932 (78.0) 0.4641526 (63.4) 0.704\u00a0\u00a0\u00a0\u00a0Female16511 (68.8) 511 (68.8) AgeMean (45.4) \u00a0\u00a0\u00a0\u00a0<45\u00a0years26719 (73.1) 0.704917 (65.4) 0.945\u00a0\u00a0\u00a0\u00a0\u226545\u00a0years31724 (77.4) 1120 (64.5) Lymph node status\u00a0\u00a0\u00a0\u00a0Positive30327 (90.0) 0.012525 (83.3) 0.002\u00a0\u00a0\u00a0\u00a0Negative271116 (59.3) 1512 (44.4) TNM stage\u00a0\u00a0\u00a0\u00a0I\u00a0+\u00a0II241113 (54.2) 0.0021212 (50.0) 0.044\u00a0\u00a0\u00a0\u00a0III\u00a0+\u00a0IV33330 (90.9) 825 (75.8) Survival rate\u00a0\u00a0\u00a0\u00a0Survival20614 (70)0.280614 (70)1.000\u00a0\u00a0\u00a0\u00a0Mortality606 (100)15 (83.3)Note: Values in parentheses are percentages\nDiscussion\nSerum is a complex body fluid, containing a large diversity of proteins. More than 10,000 different proteins are present in the human serum and many of them are secreted or shed by cells during different physiology or pathology processes [18]. Consequently, proteomics has raised great expectations for the discovery of biomarkers to improve diagnosis or classification of a wide range of diseases, including cancers [19]. Serum is expected to be an excellent source of protein biomarkers because it circulates through, or comes in contact with, all tissues. During this contact it is likely to pick up proteins secreted or shed by tissues, which has recently been tested and confirmed [20]. However, serum has been termed as the most complex human proteome [21] with considerable differences in the concentrations of individual proteins, ranging from several milligrams to less than one pictogram per milliliter [22]. Another analytical challenge for biomarker discovery arises from the high variability in the concentration and state of modification of some human plasma proteins between different individuals [23]. Despite these limitations, human serum holds immense diagnostic potential. In the last decade, several large-scale projects have been initiated, aimed at characterizing the human plasma\/serum proteome.\nIn the mean time, several serum proteomic studies on NPC have been reported. Cho et\u00a0al. performed protein chip profiling analysis with surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) technology on sera from NPC patients and demonstrated that SAA may be a potentially usefully biomarker for NPC [24]. However, the technology for discovery of new cancer biomarkers has recently been questioned for its flaws, such as its qualitative nature, high identification error rate, poor reproducibility and nonspecific absorption matrices [25\u201327]. 2D-based comparative proteome analysis, though a new technology for the separation and identification of disease-specific proteins, has been applied successfully to screen potential biomarkers for NPC in cell lines and tumor tissues [3\u20138].\nRecently, Saeid et\u00a0al. reported their pioneering work on NPC serum analysis by 2D, revealing the enhanced expression of such relatively abundant proteins as ceruloplasmin (CPL) [9]. However, in 2D study, abundant proteins, such as albumin and IgG, that account for approximately 60\u201397% of the total serum proteins [28], mask other proteins that migrate to the surrounding areas and limit the loading amount of serum. As albumin and IgG are known to function as carriers and transporters of important proteins such as hormones, cytokines, and lipoproteins within the blood [29\u201331], the depletion of these two highly abundant proteins may result in the loss of potentially important proteins bound to them at the same time. In order to release those adsorbed or bound proteins, we sonicated the diluted sera before the depletion and desalting steps as suggested by Quero et\u00a0al. [32, 33]. The improvements we made in sample preparation enabled us to find some valuable low abundant proteins.\nOf the 13 successfully identified protein spots, we focused on 10 up-regulated proteins in NPC for further validation. We also accumulated certain knowledge about three proteins by literature profiling. Finally, we decided to further investigate sICAM-1, HSP70 and SAA, which seemed more associated with our research interest, using both ELISA and IHC to validate their differential expressions. Intriguingly, most of these identified proteins have been reported to be associated with carcinogenesis and tumor metastasis. Our research is herein chiefly concerned with the functional implications of the three proteins to NPC at the serum level.\nAlthough the source of sICAM-1 has not been fully elucidated, researches show it can be released by cancer cells [13, 14] as well as by peripheral blood mononuclear, endothelial, and fibroblastic cells [34]. Proteolytic cleavage of membrane-bound ICAM-1 may be the most likely mechanism for the generation of sICAM-1 [35]. In patients with certain malignancies, the serum sICAM-1 titers have been found elevated in association with tumor growth and distant metastasis of malignant melanoma [36], lung [37], breast [38], gastric [39], hepatocellular [40], and colorectal cancers [41]. Poor survival of cancer patients correlated with a high level of serum sICAM-1 has also been demonstrated [36, 38, 39]. In our study, the positive expression of tissue ICAM-1 and levels of serum sICAM-1 were significantly correlated to the presence, progression, metastasis and mortality of NPC. sICAM-1 possesses most of the necessary extracellular structures to retain the functional activities of ICAM-1 [13, 35]. It has been reported that ICAM-1 on the surface of cancer cells or antigen presenting cells (i.e., macrophages) is a costimulatory factor that stabilizes T-cell receptor-mediated binding between these cells and T lymphocytes [42]. sICAM-1 would work as an immunosuppressive agent by blocking LFA-1 on T lymphocytes, thus rendering it less available for binding with ICAM-1 on the surface of cancer cells [43]. In this manner, the shedding of sICAM-1 may speed up the metastatic process by escaping host immune surveillance. This, probably, presents an additional potential mechanism accounting for high serum levels of sICAM-1 in NPC patients who have metastasized via hematogenous and lymphatic routes. As serum sICAM-1 may be useful for monitoring hematogenous metastasis, measuring the serum sICAM-1 level might be potentially significant in clinic.\nSAA is an acute-phase protein with various isoforms in a molecular mass range of 11\u201314. In normal individuals, SAA is produced by hepatocytes in the liver [16]. After secreted into serum, it rapidly binds to high-density lipoprotein, with 90% of the protein particles bound [15]. A review of the literature shows that only a low level of SAA can be found in the sera of healthy individuals, despite the ubiquitous nature of SAA [44]. This is in sharp contrast to the patients with neoplastic diseases, such as those with renal [45] and colorectal [46] cancers, who showed dramatic elevation of serum SAA. The prognostic significance of SAA for other cancers has also been found by conventional radioimmunoassay, in line with the findings of this study [47]. Cho et\u00a0al. demonstrated that SAA may be a potentially usefully biomarker for NPC [24]. The report fully confirmed relatively huge concentrations of SAA in serum (0.2\u20132\u00a0g\/l) that are thousands of times higher than classical cancer biomarkers (such as CEA, PSA, CA125, etc.) originating from tumor cells. In a similar way, we further confirmed that serum SAA was much elevated in NPC patients, particularly at the process of lymph node metastases when compared with non-LNM patients. However, such biomarkers (acute-phase reactants) are not commonly considered as cancer-specific ones and expected to be elevated in other malignant diseases or inflammatory diseases as well [26, 27]. In previous reports, SAA was found elevated in different malignancies, such as cancers of kidney, colon and prostate, as well as in leukemias and lymphomas [24, 48]. Therefore, SAA may represent a cancer epiphenomenon, unlikely to be of much clinical use in diagnosing and monitoring cancer [49], but it will be interesting to explore whether the rapid production of SAA in the liver or the epithelia of different organs [50] may be related to the stimulation by cytokines abundantly present in the NPC cells. It is also meaningful to investigate how SAA is produced at different stages of clinical manifestation in NPC patients.\nHSP70 is the main protein produced during cellular response to varied stresses, such as heat shock, ischemia\/reperfusion, and oxidative changes [51, 52]. Because HSP70 confers cell protection against different stresses, it has been hypothesized that it plays a protective role in tumor growth in\u00a0vivo [53, 54]. Indeed, HSP70 is overexpressed in human tumors of varied origins [55], such as colorectal [56], breast [57, 58], prostate [59], liver [60] cancers and melanoma [61]. LNM and poor survival of cancer patients correlated with HSP70 overexpression has also been documented [57, 58, 61\u201363]. It is suggested that HSP70 is needed for in\u00a0vivo tumor progression. In addition, HSP70 may be released from tumor cells involving involves both active secretion and passive release from necrotic cells [64]. Physiological mechanisms include co-secretion in exosomes of HSC70 with TRF and the active HSP70 secretion by the nonclassical pathway employed by cytokines [65]. Therefore, HSP70 is detectable in serum so that it could potentially be used as a biomarker for diagnosis or disease classification. Abe et\u00a0al. reported that HSP70 is a marker of prostate cancer, and may be used in conjunction with PSA to identify patients with early-stage prostate cancer [59]. However, to the best of our knowledge, the association between serum level of HSP70 and NPC status has not been reported. Since our findings demonstrated that there was a modest association between serum HSP70 level and NPC staging, the serum HSP70 level should not be considered as an independent prognostic factor in NPC patients, although it might be a prognostic predictor by univariate analysis. In short, serum HSP70 may have an adjunctive clinical value in monitoring tumor progression and evaluating prognosis in NPC patients, though its clinical application as a major tumor marker is limited.\nIn conclusion, our serum proteomic analysis, using a comprehensive pretreatment strategy, provides a practical and exemplary tool of screening progression-associated serum proteins in NPC research. After comparing 2D image analyses for healthy volunteers, non-LNM NPC and LNM NPC patients, we successfully identified 13 differentially expressed protein spots. We further explored the differential expressions of three of the proteins, namely, sICAM-1, HSP70 and SAA, by ELISA at serum and IHC at tissue, though more work should be completed to pinpoint the direct correlation between serum level of sICAM and HSP70 and their expression level in the tissues. We suggest that the three proteins may be potential serum biomarkers which can serve as effective target points for early diagnosis and therapy of NPC patients, though further clinical research should be done before the potential come true.","keyphrases":["serum proteome","carcinogenesis","lymph node metastasis","nasopharyngeal carcinoma"],"prmu":["P","P","P","P"]}
{"id":"Arch_Orthop_Trauma_Surg-3-1-2111040","title":"Trochanteric fractures in the elderly: the influence of primary hip arthroplasty on 1-year mortality\n","text":"Introduction The aim of the study was to compare the mortality risk and complication rate after operative treatment of pertrochanteric fractures with primary arthroplasty, dynamic hip screw (DHS) or proximal femoral nail (PFN).\nIntroduction\nWhile relative consensus exists about the treatment of femoral neck fractures for elderly patients, the optimal treatment for per- and intertrochanteric fractures is still under debate [1, 7]. In a previous study of 1,173 patients with proximal femoral fractures it appeared that trochanteric fractures are still a challenge, as 1-year mortality risk and complication rate were considerably higher compared with femoral neck fractures [7]. As restoration of the preoperative ambulatory level correlated with survival rate after 1\u00a0year [7] and elderly patients are often unable to cooperate with partial weight bearing, the primary stability of the device is crucial to allow early mobilization to prevent cardio-pulmonal complications and thrombosis. Excessive collapse of the fracture site and varus displacement is a common problem of sliding hip screws combined with plates or femoral nails especially in elderly women who often suffer from osteoporosis and poor bone quality [16]. Therefore, primary arthroplasty was proposed by some authors [4, 10, 28] but their patient collectives ranging from 37 to 54 were to small to allow general recommendations. Because we used hip arthroplasty even for trochanteric fractures for a long time as a standard procedure [7, 26], we are now able to report on our experiences in a large number of patients.\nThis study compares the mortality risks and 1-year postoperative complications between arthroplasty and osteosynthetic fixation (DHS or PFN) for intertrochanteric fractures. A 1-year mortality was chosen as a main indicator as it depends on the surgical trauma as well as the rapid return to preinjury activity and further complications. A 1-year period was chosen as the mortality risk approaches that of an age-matched reference population after this interval [32, 35].\nPatients and methods\nA total of 308 patients who were treated for trochanteric femoral fractures between 1992 and 2005 with a minimum age of 60\u00a0years were enrolled in this study. Seventeen patients with pathologic fractures due to metastases were excluded. Information about survival after 1\u00a0year could be achieved from 283 of the 291 patients.\nTwo hundred and seventeen patients (76.7%) were female. The mean age at the time of surgery was 80.5 (\u00b19)\u00a0years ranging from 60 to 98\u00a0years. Women (82\u00a0\u00b1\u00a08\u00a0years) of our cohort were significantly older than men (75.5\u00a0\u00b1\u00a011\u00a0years; P\u00a0<\u00a00.01).\nThe fracture type was classified according to the System of the Orthopedic Trauma Association (AO\/OTA) [24]. Pertrochanteric two part fractures are classified as A1-type, multi-part fractures as A2-type and reversed oblique intertrochanteric fractures as A3-type fractures. While all A1-type fractures are considered to be stable, most of the A2 and all A3 type fractures are unstable [19]. The distribution of the fracture types within the treatment groups is displayed in Fig.\u00a01.\nFig.\u00a01Distribution of the surgical treatment depending on the fracture type. The dynamic hip screw (DHS) was mostly used in more stable fractures, while the proximal femoral nail (PFN) was mainly used in A3-type fractures (X\u00b2\u00a0=\u00a022.4, P\u00a0<\u00a00.001)\nA total of 109 patients (38.5%) were treated with a \u201cdynamic hip screw\u201d (DHS by Synthes\u00ae). Within this group there were mainly stable fractures type A1 (52%) or A2 (44%; Table 1); 132 patients (46.6%) were treated with primary arthroplasty, 117 with a total hip replacement (THR) and 15 with a bipolar hemiarthroplasty (HA). Since 2000 PFN (Synthes\u00ae) was used in 42 of the 100 cases, mainly for unstable fractures in patients without advanced osteoarthritis. As demonstrated in Table\u00a01, these patients were younger and more frequently male (P\u00a0<\u00a00.05).\nTable\u00a01Description of the baseline variables within the treatment groupsArthroplastyDHSPFNAge (years)83\u00a0\u00b1\u00a0779\u00a0\u00b1\u00a0975\u00a0\u00b1\u00a012Number of patients13210942Gender\u00a0Female 115 (87%)80 (73%)26 (62%)\u00a0Male17 (13%)29 (27%)16 (38%)\u22654 comorbidities63 (48%)56 (51%)17 (41%)Fracture type\u00a0A137 (28%)57 (52%)7 (17%)\u00a0A282 (63%)48 (44%)29 (69%)\u00a0A312 (9%)5 (4%)6 (14%)Year of injury\u00a01992\u20131999111 (84%)72 (66%)0 (0%)\u00a02000\u2013200521 (16%)37 (34%)42 (100%)\nOutcome variables\nPrimary outcome variable was the 1-year mortality risk (Table\u00a02). Furthermore, the occurrence of complications which led to further interventions was analyzed in multiple regression analysis. These were dislocation of the hip, non-union of the fracture, cutting out of the hip screw, infections, thrombosis and hematoma.\nThe Merle d'Aubigne test (0 to 18 points) was used to assess the patients [10]. We further questioned whether the patients returned to their preoperative status (e.g. living at home).Table\u00a02The distribution of fracture type and treatment and its specific mortality1992\u201319992000\u201320051992\u20132005nDeathMortality (%)nDeathMortality (%)nDeathMortality (%)A1711926.829413.81002323.0\u00a0THR34926.50034926.5\u00a0HA003133.33133.3\u00a0DHS371027.019315.8561323.2\u00a0PFN000.0700.0700.0A2952829.5661319.71614125.5\u00a0THR652436.96233.3712636.6\u00a0HA001218.31218.3\u00a0DHS30413.318211.148612.5\u00a0PFN0030826.730826.7A317635.35120.022731.8\u00a0THR12541.60012541.6\u00a0HA000000\u00a0DHS5120.0005120.0\u00a0PFN005120.05120.0All fractures1835329.01001818.02837125.1\u00a0THR1113834.26233.31174034.2\u00a0HA0015213.315213.3\u00a0DHS721520.837513.51092018.4\u00a0PFN0042921.442921.4THR total hip replacement, HA hemiarthroplasty, PFN proximal femoral nail, DHS dynamic hip screw\nSurgical technique\nThe proximal femoral nail (PFN by Synthes\u00ae-Switzerland) or the dynamic hip screw (DHS by Synthes\u00ae-Switzerland) was used according to the manufacturer\u2019s instructions. For closed reduction the patients were positioned supine on a fracture table. Only 135\u00b0 four hole-DHS plates without additional trochanteric stabilizing plates or tension band wires were used in this series.\nFor hemiarthroplasty a standard cemented stem (Weller, Aesculap, Germany) and a bipolar head (DePuy, Leeds, England) were used. All femoral stems were cemented using the modern 3D generation cementing technique [2]. In cases of total hip replacement, a polyethylene cup (Aesculap\u00ae, Germany) was cemented in the same manner (Fig.\u00a03).\nStatistics\nFirst we performed univariate analysis of independent variables (age, sex, treatment, preoperative risk factors, etc.) and mortality using Fischer\u2019s exact test for qualitative variables and ANOVA for quantitative variables. The homogeneity of variance was checked by Levene test. All tests were two-sided and a P\u00a0\u2264\u00a00.05 was considered to be significant.\nThe influence of treatment on mortality was checked by a multiple logistic regression model adjusted for the presence of preoperative comorbidities (four or more vs. up to three), fracture classification, age, gender, complications requiring revision surgery and year of surgery.\nWe used RR additional to OR because the more frequent the outcomes becomes, the more the odds ratio will overestimate the risk ratio when it is more than 1 or underestimate the risk ratio when it is less than 1 [34]. Odds ratios (OR) were transferred to relative risks (RR) according to the method of Zhang et al. [34] with the formula: RR\u00a0=\u00a0OR\/[(1\u00a0\u2212\u00a0P0)\u00a0+\u00a0(P0\u00a0\u00d7\u00a0OR)], where P0 is the incidence of the outcome in the nonexposed group (reference category), and OR the odds ratio given by the multiple analysis. A RR\u00a0<\u00a01 refers to a factor category that is mitigating (decreasing) the risk of the outcome (e.g. dying) when present, an RR\u00a0>\u00a01 refers to a factor category that is increasing that risk when present compared with the reference category.\nAll analyses were conducted with the SPSS statistic software for Windows 12.0 (SPSS, Chicago, IL, USA).\nResults\nSurgery\nEighty-five percent of our patients were operated within 1 day after admission. In the other cases, operation was delayed to improve the preoperative condition of the patient, e.g. by transfusion or change of medication. This was independent of the treatment groups (P\u00a0=\u00a00.307). Mean blood loss and operating time differed significantly depending on the type of operation. The mean blood loss for arthroplasty (1,050\u00a0\u00b1\u00a0700\u00a0ml) was significantly higher than for fixation with a DHS (409\u00a0\u00b1\u00a0360\u00a0ml) or PFN (332\u00a0\u00b1\u00a0277\u00a0ml; P\u00a0<\u00a00.001). Implantation of a hip replacement took a mean of 115\u00a0\u00b1\u00a036\u00a0min, that of a DHS 73\u00a0\u00b1\u00a038\u00a0min and of a PFN 84\u00a0\u00b1\u00a032\u00a0min (P\u00a0<\u00a00.001) including time for closed reduction on the extension rag. Fracture classification significantly influenced operating time if an arthroplasty was performed. It was 100\u00a0\u00b1\u00a024\u00a0min for A.1, 121\u00a0\u00b1\u00a039\u00a0min for A.2 and 127\u00a0\u00b1\u00a026\u00a0min for A.3-fractures (P\u00a0=\u00a00.012). The blood loss was similarly influenced (800\u00a0\u00b1\u00a0460\u00a0ml for A.1, 1,200\u00a0\u00b1\u00a0800\u00a0ml for A.2, 900\u00a0\u00b1\u00a0500\u00a0ml for A.3 fractures; P\u00a0=\u00a00.019)\nMortality\nSix patients (2.1%) died during hospitalization, 25 (8.9%) within 90\u00a0days. These were patients with considerable preoperative risk factors; 71 patients (25.2%) died within 1\u00a0year (Table\u00a02).\nInfluence of gender and age\nAs demonstrated in Fig.\u00a02, survival was correlated to age and gender; 34.8% of the male and 24.9% of our female patients dies within the first year (relative risk 0.34, P\u00a0=\u00a00.004; Table\u00a04). Not considering the different treatment groups patients over the age of 80\u00a0years had an increased mortality risk (P\u00a0=\u00a00.004).\nFig.\u00a02Influence of age and gender. Independent of the treatment and fracture type the mortality risk was significantly influenced by age for both genders (P\u00a0=\u00a00.018 for men, P\u00a0=\u00a00.002 for women). After multivariate analysis, the risk to die was significantly lower for woman (relative risk 0.34, P\u00a0=\u00a00.004) and higher for patients over the age of 90\u00a0years (relative risk 3.61; P\u00a0=\u00a00.009)\nInfluence of comorbidities\nThe comorbidities and their prevalence are shown in Table\u00a03. Of the single comorbidities only cardiac arrhythmia and cerebral diseases (P\u00a0=\u00a00.04) showed a significant influence on mortality. Logistic regression analysis demonstrated that four or more comorbidities increased the risk to die by 78% (relative risk 1.78, P\u00a0=\u00a00.009; Table\u00a04). Forty-eight percent of our patients had four or more comorbidities and showed a mortality risk of 33.3% compared with 17.4% if they had less than four (P\u00a0=\u00a00.032 in \u03c72-test).\nTable\u00a03The prevalence and relative risks of the most common comorbiditiesComorbiditynPoP (Fisher\u2019s exact)RRORCI 95%P (log. reg.)Heart attack1240.3400.431.121.190.56\u20132.140.69Cardiac arrhythmia470.3540.01**1.101.161.02\u20132.430.04*Cardiac insufficiency1110.3040.821.441.790.67\u20133.180.55Hypertonus1540.2681.000.980.970.55\u20131.730.92Pulmonary diseases660.2880.731.071.10.57\u20132.160.77Anemia470.3190.351.261.430.69\u20132.960.33Renal insufficiency470.2611.000.850.810.38\u20131.750.60Cerebral diseases1270.3360.301.421.811.02\u20133.220.04*Diabetes mellitus920.3260.281.241.410.79\u20132.520.25Metabolic diseases300.2330.820.750.70.27\u20131.800.46Alcohol\/nicotine\/other drugs290.3210.351.511.980.79\u20134.990.14Gastrointestinal disease420.2440.310.760.710.32\u20131.640.43Others570.3040.851.011.010.51\u20132.030.96Relative risks (RR) are computed from odds ratios (OR) by the following formula: RR\u00a0=\u00a0OR\/[(1\u00a0\u2013\u00a0P0)\u00a0+\u00a0(P0\u00a0\u00d7\u00a0OR)]P0 is the proportion of patients with the respective disease dying within 1-year after proximal femoral fractureRR\u00a0<\u00a01.0 represents a minor risk of mortality; RR\u00a0>\u00a01.0 represents a major risk of mortality compared with patients without this criteria; *\u00a0P\u00a0\u2264\u00a00.05; **\u00a0P\u00a0\u2264\u00a00.01Table\u00a04Multiple logistic regressions analysis of a 1-year mortality after proximal femoral fractures of elderly patientsDescription of the analysisInfluence of interaction of kind of treatment and fracture classification after adjusting age, gender, comorbidities, revision surgery and date of surgery Odds ratio95% CIPRelative riskInfluence: interaction of kind of treatment and fracture classificatione\u00a0A1*PFN0.0000.0000.9990.00\u00a0A1*DHS0.6380.228\u20131.7870.3920.71\u00a0A2*TEP1.3910.566\u20133.4200.4721.25\u00a0A2*PFN1.1240.272\u20134.6470.8721.08\u00a0A2*DHS0.3720.112\u20131.2370.1070.46\u00a0A3*TEP2.1200.502\u20138.9540.3071.59\u00a0A3*PFN1.0170.087\u201311.8330.9891.01\u00a0A3*DHS1.0930.096\u201312.3870.9431.06Influence: age categoryc\u00a070\u201379\u00a0years1.2310.334\u20134.5320.7551.19\u00a080\u201389\u00a0years2.7980.785\u20139.9700.1122.23\u00a0>90\u00a0years6.3811.603\u201325.4030.0093.61Influence: genderd\u00a0Female gender0.3380.161\u20130.7080.0040.44Influence: comorbidities complications and year of surgery \u00a0\u22654 comorbidities2.2381.219\u20134.1090.0091.78\u00a0Revision surgery1.1950.448\u20133.1840.7221.13\u00a0Surgery after 20000.7750.343\u20131.7520.5410.83Relative risks (RR) are computed from OR (odds ratio) by the following formula:RR\u00a0=\u00a0OR\/[(1\u00a0\u2212\u00a0P0)\u00a0+\u00a0(P0\u00a0\u00d7\u00a0OR)]P0 is the proportion of patients in the respective reference category dying within 1-year after proximal femoral fractureRR\u00a0<\u00a01.0 represents a minor risk of mortality; RR\u00a0>\u00a01.0 represents a major risk of mortality compared with patients without this criteriaNumber of cases in all models: 282aReference: TEPbReference: A1cReference: <70\u00a0yearsdReference: male gendereReference: A1*TEP\nInfluence of fracture-classification and treatment\nOnly pertrochanteric fractures were enrolled in this study and sorted according to the AO\/OTA classification (Fig.\u00a01). While the mortality did not differ between A1- (23.0%) and A2-type fractures (25.5%), it was higher in A3-type fractures (31.8%; Table\u00a02). As this fracture type affected only 7% of our patients, it did not significantly influence the mortality risk (P\u00a0=\u00a00.42).\nAbout 18.4% of the patients who received a dynamic hip screw, 21.4% of those who received a proximal femoral nail and 33% in the arthroplasty groups (THR and HA) died within 1\u00a0year (P\u00a0=\u00a00.011). As the indication for surgery was not randomized but decided on fracture classification and clinical status we attempted to reduce these influences in a multiple logistic regression analysis (Table\u00a04). All potential influencing factors as gender, age, fracture type and amount of comorbidities were taken into account. If categorical variables were used to evaluate the interaction between fracture classification and treatment, we could not find a statistical influence of surgical treatment on mortality (Table\u00a04). The relative risk to die was decreased for A.1- and A.2-type fractures, treated with a DHS, but this did not reach significance levels. As only seven patients with a A.1-type fracture were treated with a PFN (and all of them survived), no sensible statistical analysis can be done for this category. As all devices showed good results for this kind of fracture, the decision should be based on other parameters for A.1-type fractures.\nBecause our treatment regime was changed in the beginning of 2000 (introduction of the PFN, new bipolar hemiarthroplasty instead of total hip arthroplasty; Table\u00a02), we implicated the time of surgery in our analysis. Since then the 1-year mortality after arthroplasty decreased from 34.2 to 19% (P\u00a0=\u00a00.26, Fisher\u2019s exact test). Although over all mortality decreased from 29 to 18%, no influence of time of surgery on the mortality risk could be found using multivariate regression analysis (P\u00a0=\u00a00.54; Table\u00a04).\nInfluence of complications\nThe only postoperative complication which influenced the survival rate was postoperative pneumonia. It occurred in 13 cases independent of the treatment groups (P\u00a0=\u00a00.26). Six of the patients died within 1\u00a0year (P\u00a0=\u00a00.048).\nThe DHS had a revision rate of 8.1%, while the PFN needed reoperations in nine cases (22.5%). The main reason was cutting out of the screw in the osteoporotic bone and postoperative hematoma. Due to cutting out and fixation failure secondary hip arthroplasty was performed after failed internal fixation ten times. It happened six times (5.4%) after a DHS and four times (9.8%) after a PFN.\nThe main complication after arthroplasty was dislocation, which occurred in 16 patients (12%). It did not occur with bipolar hemiarthroplasty. While dislocation was only a minor problem in A1-type fractures (7%), it occurred in ten A2-type (12%) and two of the nine A3-type fractures (22%). Patients were usually treated by closed reduction and physiotherapy and it did not influence mortality (P\u00a0=\u00a00.78).\nRehabilitation\nThe mean hospital stay was 18\u00a0days and did not differ significantly between the three treatment groups (P\u00a0=\u00a00.7). It prolonged to a mean of 30\u00a0days if a reoperation became necessary (P\u00a0=\u00a00.003). Patients who were ambulatory before the injury could walk with a walker or sticks at time of discharge; 80% of the surviving patients who were independent before the injury regained their preoperative status after 1\u00a0year. Since the year 2000 patients were encountered to a prospective registry. The mean Merle d\u2019Aubign\u00e9 score (0\u201318 points) for the surviving 78 patients was 13.8\u00a0\u00b1\u00a02.5. No significant differences could be found between the three treatment groups (P\u00a0=\u00a00.122).\nDiscussion\nWhile primary arthroplasty is a standard procedure for femoral neck fractures, little experience exists for trochanteric fractures [4, 5, 9, 28]. They were primarily used as a salvage procedure after failed internal fixation [12, 15, 21, 23, 31]. Most authors implanted special calcar replacement devices, which are expensive and require the removal of large portions of the proximal femur [3, 8, 13, 28]. We used a standard cemented stem and either a standard cemented cup or a bipolar head since the 1970s in our hospital. As it proved superior to former methods of internal fixation even for pertrochanteric fractures it was the treatment of choice since 1992 [26]. From the year 2000 on hemiarthroplasty was routinely used instead of total hip arthroplasty and the proximal femoral nail was introduced for patients without advanced osteoarthritis.\nThe ideal treatment for intertrochanteric fractures is still under debate as none of the existing osteosynthetic devices could prove its superiority in former studies [16, 25, 29, 30]. The most frequent problem in elderly patients who usually are not able to walk without weight bearing was a cutting out of the hip screw which occurred in 4 to 20% of the reported cases [4, 6]. In our series this complication was only seen in comminuted fractures (A2 and A3 type). Thus, 10 times (6.2% of the internal fixation devices) a secondary hemiarthroplasty was performed. Immediate full weight bearing regardless of bone quality is an advantage of primary cemented arthroplasty. In our hospital, all patients are encouraged to stand up the day after surgery. Dislocation was the major complication in patients treated with a total hip replacement after comminuted pertrochanteric fractures. This high dislocation rate is rarely seen after primary hip arthroplasty but well known even for femoral neck fractures [22] or revision surgery [33] and can be attributed to insufficient muscular stability. Fractures of the greater or lesser trochanter might exaggerate this problem. The use of bipolar arthroplasty instead of total hip replacements can reduce this complication to an acceptable rate [11, 28]. Since we use total hip arthroplasty only in patients of good physical shape with severe osteoarthritis we did not see any dislocations.\nAmbulatory levels and clinical scores are considerable affected by the preoperative status. Since clinical examination usually cannot be obtained for all patients, reliable comparison is difficult. Some authors use telephone interviews [4] or report about less than 50% of their patients for a 1-year follow-up [10]. In both cases it can be assumed that patients with bad functional results are not regularly included. We could also examine only 78% of the last 100 patients clinically and therefore do not want to overemphasize these findings. Instead we chose the 1-year mortality risk as the primary outcome parameter as it could be obtained for all patients and is related to the postoperative ambulatory status as well as to the operative trauma [14]. In a previous study with more than 500 fractures, we could demonstrate that patients who regained their independency and were able to attend a rehabilitation institution had a significantly lower mortality risk [7], which was further supported in this study.\nMortality was significantly influenced by patient related factors such as gender, age and comorbidities but not by the fracture type. In accordance with our former studies and other authors [7, 17, 20, 26] few single comorbidity influenced the mortality risk whereas the sum of four or more comorbidities increased the risk to die by approximately 78% (Tables\u00a03 and 4). Patients who received a PFN or DHS were significantly younger and healthier than those in the arthroplasty group. These influences were reduced by multivariate regression analysis (Table\u00a04). Furthermore, the interaction of fracture type and treatment was taken into account. Considering these influences, no significant influence of one of the three treatment groups on the mortality risk could be found. In studies that only use internal fixation devices mortality risks comparable with our over all mortality were found [27], thus\u2014in accordance with other authors [3, 13]\u2014we think that cemented hemiarthroplasty is an alternative to internal fixation in elder patients especially as our revision rate was significantly lower for arthroplasty compared with internal fixation.\nOnly Chan et al. [4] published a study about the use of a standard hemiarthoplasty for the treatment of displaced intertrochanteric fractures. In their small group of 54 patients they found a mortality rate of 31.5% within 1\u00a0year which did not differ from comparable age-matched studies. The walking ability of their patients was favorable compared with other studies with internal fixation. We agree that immediate full weight bearing, which can be achieved after cemented arthroplasty, is crucial for these patients.\nHaentjens et al. compared 37 patients who received a calcar replacement device with 42 patients of a retrospective group, who were treated by internal fixation and found an advantage in functional outcome [10]. Although the mortality rate could not be decreased, early walking with full weight bearing reduced the incidence of pressure sores, pulmonary infection and atelectasis. Kim et al. [18] who compared a calcar replacement prosthesis with intramedullary fixation in a prospective study in two small groups of 29 patients could not find a significant difference concerning the functional outcomes, but the cut-out rate of the hip screw was 7% in their patients.\nUp to now a clear indication of arthroplasty in the treatment of pertrochanteric fractures does not exist. Although operation time and blood loss are higher no significant difference concerning the mortality rate between primary arthroplasty and internal fixation could be found, but the revision rate in the arthroplasty group was significantly smaller especially since we use bipolar hemiarthroplasty instead of a total hip replacement. If the cemented arthroplasty has been performed accurately, there is very little concern about weight bearing. Furthermore, primary arthroplasty eliminates the possibility for malunion, cut-out of the hip screw and avascular necrosis of the femoral head. But primary arthroplasty is a technically challenging procedure. All lose fragments including the greater and the lesser trochanter have to be attached with cerclage wires before cementing the stem to prevent extrusion (Fig.\u00a03) and especially in utterly comminuted fractures orientation of the rotation and leg length can be demanding.\nFig.\u00a03Examples of treatment of A2-type fractures with a hemiendoprosthesis and tension band wires. Depending on the fracture type different ways of fixating the trochanteric region with wires were used. In the case of a calcar fragment reaching distal to the lesser trochanter a temporary reduction with a cerclage wire or reduction bone clamp was used before reaming. Final fixation of the greater trochanter was performed after reduction of the hip with cerclages or a tension band\nThe DHS is not suitable for reverse oblique and comminuted fractures [19, 30] but for stable fractures it seems to be advantageous to short femoral nails regarding reoperation rate and fracture fixation failure in the literature [16, 29]. We further support the recommendation that the DHS should be used for stable A1- and A2.1-type fractures while an intramedullary device is advantageous for unstable fractures. Primary cemented arthroplasty is a viable option for the treatment of trochanteric fractures in a selected group of previously independent mobile patients especially if osteoporosis would prevent full weight bearing or osteoarthritis would make further operations necessary. The routine use of total hip arthroplasty was abandoned in our hospital as the luxation rate was higher than after hemiarthroplasty. It is still used as salvage procedure after failed internal fixation in elder patients with bad bone quality.","keyphrases":["mortality risk","trochanteric femoral fractures","total hip replacement"],"prmu":["P","P","P"]}
{"id":"Breast_Cancer_Res_Treat-3-1-2045691","title":"Differential effects of tamoxifen and anastrozole on optic cup size in breast cancer survivors\n","text":"Introduction The main purpose of this study was to determine whether the optic cups of tamoxifen users and anastrozole users differ in size, with the cups of the tamoxifen users being smaller.\nIntroduction\nSelective estrogen receptor modulators (SERMS) and aromatase inhibitors are the two major classes of medications used in the United States as adjuvant endocrine therapy for early-stage, hormone-receptor-positive breast cancer. Of the SERMS that are FDA-approved for this purpose [1], tamoxifen has been the most widely used, and for several decades it has been prescribed extensively for women of all ages [2]. The first aromatase inhibitor to be FDA-approved for early-stage breast cancer was anastrozole (Arimidex\u00ae), in 2002 [2]. However, because aromatase inhibitors do not block estrogen production adequately for pre-menopausal women [3], they are indicated for use by post-menopausal women only [4], for whom estrogen production is entirely non-ovarian [5]. Initial reports indicate that anastrozole is more effective than tamoxifen at preventing cancer recurrence [6, 7]. Thus, anastrozole now is being recommended for many women who would have been prescribed tamoxifen several years ago [8]. Because the use of aromatase inhibitors is likely to continue to increase and because the use of tamoxifen may become progressively more restricted to younger women, the potential side effects of anastrozole need to be evaluated, and the influence of age on tamoxifen side-effects needs to be closely examined.\nOf all the documented or putative effects of tamoxifen on the visual system [9], tamoxifen retinopathy may be the most widely known. However, tamoxifen retinopathy is uncommon [10] and possibly cumulative-dose-dependent [11]. In contrast, Eisner et al. [12] found that short-term tamoxifen users (i.e., \u22642\u00a0years of use) often have smaller optic cups than age-matched female control subjects, in a study of women who had been selected for the absence of any overt eye disease. This result was consistent with the possibility that tamoxifen often causes a subclinical degree of swelling at the optic cup, particularly since tamoxifen is a potent blocker (at least in culture) of swelling-activated chloride channels [13, 14], such as those in astrocytes [15, 16], and astrocytes are the predominant glial cell in the cup [17, 18]. If tamoxifen indeed causes optic-cup swelling via its ability to block swelling-activated chloride channels, the cup sizes of anastrozole users would be expected to more closely resemble those of female control subjects than those of tamoxifen users. The present study tested this prediction.\nBecause of changing trends in adjuvant breast cancer treatment, the present study also assessed the effect of age on optic nerve head (ONH) parameters. The results support the inference that tamoxifen usage can lead to small cups, although apparently mainly for women older than about 50\u00a0years. All measurements for this study were obtained using confocal scanning laser ophthalmoscopy with the Heidelberg Retina Tomograph II (HRT). This commercial instrument was introduced in 1999 for routine clinical use, but until now, it has been used mainly to assess glaucoma and related conditions.\nMethods\nSubjects\nThree groups of amenorrheic women ages 40\u201369\u00a0years old were recruited for this study: (1) women using the standard dose of 20\u00a0mg tamoxifen daily as adjuvant therapy for breast cancer for at least 4\u00a0months but no longer than 2\u00a0years, (2) women using the standard dose of 1\u00a0mg anastrozole daily as adjuvant therapy for breast cancer for at least 4\u00a0months but no longer than 2\u00a0years, and (3) women not using any hormonally acting medications. This third group served as an approximately age-matched control group. The 2-year duration-of-use requirement was based on results from several studies of vision [19, 20] and the eye [12] indicating that short- versus long-term tamoxifen use can be distinguished operationally using a 2-year cutoff. The 4-month duration-of-use requirement was included to help ensure that the medications had time to act and that the body\u2019s response had a chance to stabilize [21, 22].\nAll tamoxifen and anastrozole users had completed primary treatment for breast cancer, and all were fully active and able to perform their daily pre-cancer activities without restriction. None of the control subjects had positive breast cancer histories or had previously used breast cancer medication for any purpose. Similarly, none of the tamoxifen users had previously used a hormonally acting breast cancer medication other than tamoxifen, and none of the anastrozole users had previously used a hormonally acting breast cancer medication other than anastrozole. In addition, none of the subjects in any group had ever used the SERM raloxifene.\nWith one exception, all analyses for this report are based on data from subjects who had been amenorrheic for at least 6\u00a0months at the time of testing. The one exception is an analysis comparing data from control subjects with data from an additional group of women (the fourth subject group overall) who met the same eligibility criteria as the control group, except for the absence of menses.\nAll subjects, regardless of group, met a rigorous set of eligibility criteria for excellent ocular health in order to reduce the roles of confounding factors that could complicate the data. These are the same criteria we have used previously for several types of studies [12, 19, 23]. These criteria are: (1) 20\/20 or better visual acuity in one eye and 20\/25 or better visual acuity in the other eye, (2) no evidence or suspicion of eye disease on undilated direct ophthalmoscopic examination and on subsequent evaluation of individuals\u2019 stereoscopic color fundus and ONH photographs by a glaucoma specialist (author JRS) who was masked from identifying subject information, (3) no history of eye disease or ocular hypertension, (4) no diabetes, (5) intraocular pressure (IOP) \u226422\u00a0mmHg on Goldmann applanation and no between-eye IOP difference >2\u00a0mm Hg, (6) no myopia >5 diopters, (7) no use of any medication (other than tamoxifen) known to affect vision, (8) no history of ocular surgery, and (9) normal color vision (i.e., no worse than a single minor transposition error on the D-15 test conducted under Macbeth illumination). There was no suggestion of papilledema for any subject (as for criterion #2).\nDemographic data for eligible subjects with usable HRT data (see second paragraph of Procedures) are given in Table\u00a01 for each of the three amenorrheic subject groups plus the additional group comprised of women without breast cancer histories and still experiencing menses. The mean ages of the tamoxifen users were slightly lower than the mean ages of the anastrozole users and amenorrheic control subjects possibly because some tamoxifen users were not menopausal despite being amenorrheic [24]. For reference, the median age of natural menopause in American society is 51\u00a0years [25].\nTable\u00a01Demographic data for each of the subject groupsAmenorrheic subjects 40\u201369\u00a0years oldAmenorrheic subjects 51\u201369\u00a0years oldNot amenorrheic, no breast cancer history n\u00a0=\u00a019Anastrozole n\u00a0=\u00a020Tamoxifen n\u00a0=\u00a034Control, no breast cancer history n\u00a0=\u00a032Anastrozole n\u00a0=\u00a018Tamoxifen n\u00a0=\u00a025Control, no breast cancer history n\u00a0=\u00a029Age (years)58.2 (SD\u00a0=\u00a06.8)54.5 (SD\u00a0=\u00a05.2)57.2 (SD\u00a0=\u00a06.0)59.6 (SD\u00a0=\u00a05.4)56.5 (SD\u00a0=\u00a04.7)58.2 (SD\u00a0=\u00a05.1)48.0 (SD\u00a0=\u00a03.3)IOP (mmHg)15.7 (SD\u00a0=\u00a02.9)15.5 (SD\u00a0=\u00a02.3)15.6 (SD\u00a0=\u00a02.9)15.7 (SD\u00a0=\u00a03.0)15.5 (SD\u00a0=\u00a02.5)15.7 (SD\u00a0=\u00a03.0)15.5 (SD\u00a0=\u00a02.3)Duration of medication use (years)1.07 (SD\u00a0=\u00a00.45)1.31 (SD\u00a0=\u00a00.49)01.09 (SD\u00a0=\u00a00.471.36 (SD\u00a0=\u00a00.48)00IOPs are from subjects\u2019 test eyes only\nAll subjects were unpaid volunteers. Recruitment methods have been described previously [19]. All subjects were Caucasian, except for one Asian subject still experiencing menses. After being informed of the nature and possible consequences of the study, all subjects gave written informed consent to participate in this study. The study protocol adhered to the tenets of the Declaration of Helsinki and was approved by the OHSU Institutional Review Board and the OHSU Cancer Institute. For each subject, HRT data and IOP measurements were obtained at the same testing session. Subject recruitment and assessment lasted several years, until usable HRT data had been collected for 20 eligible anastrozole users.\nProcedures\nONH scans were obtained using the HRT II according to standard techniques, after which a contour line is drawn on the average of three rapid-succession scans to define the margin of the ONH (i.e., of the optic disk) [26]. Stereo color photographs of the ONH were used to assist with contour-line placement, and extensive use was made of the ability of the HRT II software to rotate images in three dimensions. Contour lines were drawn with the grader masked from all other subject information, including subject group and age. Additional details have been presented previously [12]. However, we had previously placed the contour line as close as possible to the dark-to-light color change in the HRT reflectance image in order to define the margin of the ONH [12], but for the present study we ensured that the contour line was placed on stable rim tissue on top of the ring of Elschnig [27], which is often most discernable from the underside of the 3D image, as a ridge circumscribing the ONH. With these 3D-based contour lines, the mean measured disk areas for each of our subject groups were very nearly centered within the norms provided by Heidelberg Engineering on the HRT II printout. Thus, with one exception, all analyses of HRT values are based on the contour lines drawn according to the more recent set of criteria, which are intended to place the contour line exactly at the disk margin, rather than slightly within it. The one exception was made to assess the importance (or lack of importance) of the precise contour line placement for documenting between-group differences. Thus, we conclude the Results section by briefly reporting the results of analyses based on data obtained using the earlier contour-line placement criteria.\nEyes were considered to have usable HRT data only if the average standard deviation for the height of each pixel in the 3 HRT scans did not exceed 50\u00a0\u03bcm, since average topographic standard deviation values above 50\u00a0\u03bcm are considered by Heidelberg Engineering to signify \u201clow image quality\u201d [28]. Image quality was equally good for each subject group. The mean average-topographic-standard-deviation for each of the three amenorrheic subject groups ranged from 20 to 22 (SD\u00a0=\u00a07\u20138) \u03bcm for all eligible subjects and also for subjects older than 50\u00a0years. Among women still experiencing menses, the mean was 23 (SD\u00a0=\u00a09) \u03bcm.\nAlthough HRT scans were obtained for each subject\u2019s 2 eyes, only one eye per subject was used for data analysis for this paper. In this way, analyses could be limited to eyes with 20\/20 or better acuity for a maximal number of subjects. Thus, test eyes were defined initially using the following steps applied in order as necessary: (a) the eye with the better acuity, even by 1 letter, than the fellow eye, (b) the eye with a lesser degree of spherical equivalent refractive error than the fellow eye, and (c) subject preference. In cases where the initially designated test eye had an HRT average topographic standard deviation value exceeding 50\u00a0\u03bcm, the fellow eye became the test eye for analysis purposes, but only if the fellow eye had 20\/20 or better acuity and an HRT average topographic standard deviation value \u226450\u00a0\u03bcm. These are the same methods we used previously to designate test eyes [12].\nData analyses\nComparisons of central tendency across more than two subject groups were made using Kruskal\u2013Wallis non-parametric analyses of variance (ANOVAS) because the distributions of some of the HRT indices were significantly non-Gaussian (1-sample Kolmogorov-Smirnov test) in ways consistent with the literature [29]. Similarly, post hoc comparisons between pairs of groups were made using Mann\u2013Whitney U tests. Relations of HRT indices to age were evaluated for significance using Spearman rank-order coefficients because the strongest age trends were not linear. None of the reported P-values have been adjusted for multiple comparisons, but the P-values required for statistical significance of post-hoc tests as determined using a step-down Bonferroni procedure [30] are provided in the legend to Table\u00a02. All analyses were conducted using SYSTAT 10.2 (Richmond, CA). All P-values are for 2-sided tests.\nTable\u00a02ONH indices: between-group comparisons (subjects 40\u201369 years old)ANOVA (Kruskal\u2013Wallis)Anastrozole versus tamoxifen Tamoxifen versus control Anastrozole versus control Cup volumeP\u00a0=\u00a00.021P\u00a0=\u00a00.007P\u00a0=\u00a00.052P\u00a0=\u00a00.560 Maximum cup depthP\u00a0=\u00a00.016 P\u00a0=\u00a00.008P\u00a0=\u00a00.036P\u00a0=\u00a00.430Mean cup depthP\u00a0=\u00a00.028P\u00a0=\u00a00.011P\u00a0=\u00a00.050P\u00a0=\u00a00.645Cup areaP\u00a0=\u00a00.074P\u00a0=\u00a00.028P\u00a0=\u00a00.106P\u00a0=\u00a00.612Cup\/disk area ratioP\u00a0=\u00a00.030P\u00a0=\u00a00.013P\u00a0=\u00a00.045P\u00a0=\u00a00.763Cup shapeP\u00a0=\u00a00.593P\u00a0=\u00a00.325P\u00a0=\u00a00.797P\u00a0=\u00a00.429Rim areaP\u00a0=\u00a00.115P\u00a0=\u00a00.173P\u00a0=\u00a00.053P\u00a0=\u00a00.492Rim volumeP\u00a0=\u00a00.084P\u00a0=\u00a00.059P\u00a0=\u00a00.061P\u00a0=\u00a00.940Disk areaP\u00a0=\u00a00.510P\u00a0=\u00a00.474P\u00a0=\u00a00.653P\u00a0=\u00a00.232Mean RNFL thicknessP\u00a0=\u00a00.718P\u00a0=\u00a00.654P\u00a0=\u00a00.438P\u00a0=\u00a00.735RNFL cross sectional areaP\u00a0=\u00a00.562P\u00a0=\u00a00.573P\u00a0=\u00a00.287P\u00a0=\u00a00.721Height variation contourP\u00a0=\u00a00.617P\u00a0=\u00a00.375P\u00a0=\u00a00.893P\u00a0=\u00a00.387Left-most data column\u2013\u2013P-values for the non-parametric ANOVAS (Kruskal\u2013Wallis) across all three amenorrheic subject-groups simultaneously. Comparisons based on the results for the larger contour circles (see Methods). Right 3 columns\u2013\u2013unadjusted P-values for the post hoc comparisons (Mann\u2013Whitney U tests) between pairs of subject-groups. The top 6 rows of variables are for cup data, the next 2 rows are for rim data, and the bottom 4 rows are for disk-margin data. The bold entries signify P-values that are considered statistically significant. For the ANOVAS, statistical significance was considered to be p\u00a0\u2264\u00a00.05. For the post-hoc comparisons, a step-down Bonferroni approach was used, so that the first (i.e., the most significant) comparison required a significant ANOVA plus P\u00a0\u2264\u00a00.05\/3, the second most significant comparison required a significant first comparison plus P\u00a0\u2264\u00a00.05\/2, and the third most significant comparison required a significant second comparison plus P\u00a0\u2264\u00a00.05. \u201cRNFL\u201d signifies retinal nerve fiber layer thickness. The height variation contour signifies the maximal minus the minimal retinal nerve fiber layer thickness, as defined in the text\nResults\nThe optic cup volumes, maximum optic cup depths, and cup\/disk area ratios were each significantly smaller for the tamoxifen users than for the anastrozole users. In contrast, none of the comparisons between anastrozole users and control subjects approached significance for any cup dimension. Table\u00a02 provides P-values for the between-group comparisons for every summary ONH index. The cup-volume results are represented graphically as a function of age in Fig.\u00a01, which presents the data from the three groups of amenorrheic subjects: tamoxifen users (filled symbols), anastrozole users, (shaded symbols), and control subjects (unfilled symbols).\nFig.\u00a01Graph of cup volume versus age for all amenorrheic subjects. Filled circles represent tamoxifen users, shaded circles represent anastrozole users, and unfilled circles represent control subjects\nThe data in Fig.\u00a01 indicate that among tamoxifen users, cup volumes tended to be relatively small mainly for those subjects older than about 50\u00a0years. Indeed, cup volumes decreased significantly with age (Spearman r\u00a0=\u00a0\u22120.41, P\u00a0=\u00a00.018) among tamoxifen users. However, a correlational analysis would necessarily reduce or obscure the significance of any age-related change if such change occurred predominantly within a narrow age range. Thus, although the corresponding results for cup area (Spearman r\u00a0=\u00a0\u22120.33), maximum cup depth (Spearman r\u00a0=\u00a0\u22120.30), and cup\/disk area ratio (Spearman r\u00a0=\u00a0\u22120.34) were each non-significant when assessed using correlations, the cup volumes, cup areas, maximum cup depths and cup\/disk area ratios were each significantly lower (P\u00a0<\u00a0.05) for subjects older than 50\u00a0years compared to subjects 50\u00a0years and younger when based on between-group comparisons of central tendencies. An age cutoff at 51 rather than 50\u00a0years yielded the same result. For each of these 2 age cutoffs, the difference in cup volumes between the older and younger tamoxifen users was highly significant (p\u00a0\u2264\u00a0.005).\nAmong control subjects and anastrozole users, there were not enough younger subjects to make meaningful comparisons between these same 2 age classes. However, we also tested an additional group of women without breast cancer histories, comprised of subjects who met all the same eligibility criteria as the control group, except for the absence of menses. None of the ONH indices differed significantly between the amenorrheic control group (32 women with an average age of 57.2\u00a0years) and this additional group (19 women with an average age of 48.0\u00a0years), and there was no suggestion of any differences in the cup indices specifically. For example, the median cup volume equaled 0.077\u00a0mm3 for the 32 control subjects and 0.076\u00a0mm3 for the 19 subjects still experiencing menses. Similarly, the median cup\/disk area ratio equaled 0.20 for the 32 control subjects and 0.21 for these 19 additional subjects. Among the 32 control subjects themselves, cup volume was not correlated with age (Spearman r\u00a0=\u00a0\u22120.08). Among anastrozole users, Spearman r\u00a0=\u00a00.27 (P\u00a0=\u00a00.255), but the regression line relating cup volume to age accounted for only 5% of the total variance, and for only 2% when the calculation was restricted to subjects older than 50\u00a0years.\nBecause cup dimensions varied appreciably with age only for tamoxifen users, and because this variation appeared to be quite marked at about age 50\u00a0years, we recomputed the between-group comparisons of all the ONH indices for subjects older than 50\u00a0years. (None of the 19 women still experiencing menses were included in any of these comparisons). The results are shown in Table\u00a03. As expected, all of the between-group differences that were significant when the data were compared for all subjects remained significant when the data were compared for only the older subjects, and many of the comparisons between tamoxifen users and control subjects became significant (e.g., for cup volume). In addition, rim volumes (i.e., the volume above and outside the cup but within the disk) now differed significantly between groups when compared for all three groups simultaneously, but not when compared for any two groups on subsequent post hoc analysis. Had we used a less conservative means of comparison (a parametric ANOVA followed by Tukey\u2019s HSD test), the rim volumes of the tamoxifen users would have been considered to be significantly larger than the rim volumes of the anastrozole users and also than the rim volumes of the control users. This is probably the appropriate interpretation, since rim volumes, unlike cup volumes, were fairly normally distributed, allowing the use of parametric statistics.\nTable\u00a03ONH indices: between-group comparisons (subjects 51\u201369 years old)ANOVA (Kruskal\u2013Wallis)Anastrozole versus tamoxifen Tamoxifen versus control Anastrozole versus control Cup volumeP\u00a0=\u00a00.006P\u00a0=\u00a00.003P\u00a0=\u00a00.019P\u00a0=\u00a00.330Maximum cup depthP\u00a0=\u00a00.007P\u00a0=\u00a00.005P\u00a0=\u00a00.014P\u00a0=\u00a00.309Mean cup depthP\u00a0=\u00a00.016P\u00a0=\u00a00.007P\u00a0=\u00a00.038P\u00a0=\u00a00.463Cup areaP\u00a0=\u00a00.025P\u00a0=\u00a00.009P\u00a0=\u00a00.051P\u00a0=\u00a00.431Cup\/disk area ratioP\u00a0=\u00a00.011P\u00a0=\u00a00.005P\u00a0=\u00a00.020P\u00a0=\u00a00.540Cup shapeP\u00a0=\u00a00.340P\u00a0=\u00a00.146P\u00a0=\u00a00.400P\u00a0=\u00a00.463Rim areaP\u00a0=\u00a00.113P\u00a0=\u00a00.161P\u00a0=\u00a00.049P\u00a0=\u00a00.511Rim volumeP\u00a0=\u00a00.030P\u00a0=\u00a00.018P\u00a0=\u00a00.035P\u00a0=\u00a00.678Disk areaP\u00a0=\u00a00.435P\u00a0=\u00a00.301P\u00a0=\u00a00.883P\u00a0=\u00a00.217Mean RNFL thicknessP\u00a0=\u00a00.542P\u00a0=\u00a00.350P\u00a0=\u00a00.336P\u00a0=\u00a01.000RNFL cross sectional areaP\u00a0=\u00a00.482P\u00a0=\u00a00.369P\u00a0=\u00a00.259P\u00a0=\u00a00.878Height variation contourP\u00a0=\u00a00.341P\u00a0=\u00a00.160P\u00a0=\u00a00.775P\u00a0=\u00a00.242Same as Table\u00a02, except based on data from subjects older than 50\u00a0years)\nTable\u00a04 presents the means and standard errors of the mean, along with the corresponding medians, for every ONH index for each of the three groups of subjects restricted to women older than 50\u00a0years. Note that the means and medians for many ONH indices (e.g., rim volume) were similar within groups, but that for some ONH indices (e.g., cup volume), means and medians were dissimilar, signifying a skewed distribution. Thus, Figs.\u00a02\u20136 present between-group comparisons of medians with interquartile intervals plus overall ranges for cup volume (Fig.\u00a02), cup area (Fig.\u00a03), maximum cup depth (Fig.\u00a04), cup\/disk area ratio (Fig.\u00a05), and rim volume (Fig.\u00a06), in all cases for subjects older than 50\u00a0years. Although the cup areas of the tamoxifen users were not significantly smaller than those of the control subjects, their cup\/disk ratios were significantly lower, probably because a major source of anatomic variability unrelated to tamoxifen use now was factored out.\nTable\u00a04ONH indices: means and standard errors of the mean, and medians (subjects 51\u201369 years old)Anastrozole n\u00a0=\u00a018Tamoxifen n\u00a0=\u00a025Control n\u00a0=\u00a029Cup volume (mm3)0.107\u00a0\u00b1\u00a00.0170.043\u00a0\u00b1\u00a00.0150.090\u00a0\u00b1\u00a00.0170.1040.0120.073Maximum cup depth (mm)0.611\u00a0\u00b1\u00a00.0470.423\u00a0\u00b1\u00a00.0400.559\u00a0\u00b1\u00a00.0400.5910.3780.594Mean cup depth (mm)0.207\u00a0\u00b1\u00a00.0170.145\u00a0\u00b1\u00a00.0140.189\u00a0\u00b1\u00a00.0140.2060.1300.198Cup area (mm2)0.476\u00a0\u00b1\u00a00.0590.275\u00a0\u00b1\u00a00.0500.423\u00a0\u00b1\u00a00.0570.5230.1390.385Cup\/disk area ratio0.207\u00a0\u00b1\u00a00.0220.123\u00a0\u00b1\u00a00.0210.193\u00a0\u00b1\u00a00.0220.2290.0790.192Cup shape\u22120.229\u00a0\u00b1\u00a00.015\u22120.199\u00a0\u00b1\u00a00.012\u22120.219\u00a0\u00b1\u00a00.013\u22120.229\u22120.205\u22120.210Rim area (mm2)1.724\u00a0\u00b1\u00a00.0661.895\u00a0\u00b1\u00a00.0711.697\u00a0\u00b1\u00a00.0611.7971.8671.732Rim volume (mm3)0.453\u00a0\u00b1\u00a00.0250.593\u00a0\u00b1\u00a00.0420.475\u00a0\u00b1\u00a00.0280.4610.5800.506Disk area (mm2)2.200\u00a0\u00b1\u00a00.0942.169\u00a0\u00b1\u00a00.0742.120\u00a0\u00b1\u00a00.0672.3162.1562.154Mean RNFL thickness (mm)0.245\u00a0\u00b1\u00a00.0100.264\u00a0\u00b1\u00a00.0140.246\u00a0\u00b1\u00a00.0080.2460.2620.242RNFL cross sectional area (mm2)1.281\u00a0\u00b1\u00a00.0591.366\u00a0\u00b1\u00a00.0141.266\u00a0\u00b1\u00a00.0491.2821.3350.242Height variation contour (mm)0.359\u00a0\u00b1\u00a00.0190.401\u00a0\u00b1\u00a00.0190.383\u00a0\u00b1\u00a00.0150.3750.3880.391Fig.\u00a02Box plot of cup volume for women older than 50\u00a0years for each of the three amenorrheic subject groups. For each group, the horizontal line inside the box represents that group\u2019s median value. The bottom and top of the box represent the upper and lower hinges, respectively, i.e., the first and third quartiles between which lie 50% of all the values. The whiskers represent the range of values that fall within a distance D from the hinges, where D equals 1.5 times the inter-quartile range. Asterisks represent individual points that fall between a distance of D and 2D from the hinges. Open circles represent individual points that are more distant outliersFig.\u00a03Same as Fig.\u00a02, except that box plots are for cup areasFig.\u00a04Same as Fig.\u00a02, except that box plots are for maximum cup depthsFig.\u00a05Same as Fig.\u00a02, except that box plots are for cup\/disk area ratiosFig.\u00a06Same as Fig.\u00a02, except that box plots are for rim volumes\nWhen the between-group comparisons of the ONH indices were further restricted by excluding tamoxifen and anastrozole users using these medications for less than 1\u00a0year, every comparison that was significant (bold numerical entries in Table\u00a03) remained significant. In addition, the duration of medication use was observed to matter little or not at all for the anastrozole users. For instance, the regression line relating cup volume to the duration of anastrozole use accounted for only 5% of the total cup-volume variance among all 20 anastrozole users, and for only 4% when the calculation was restricted to the 18 anastrozole users older than 50\u00a0years. These several sets of results indicate that the cup differences between the tamoxifen and anastrozole groups did not occur because the anastrozole users had experienced too short a period of medication use.\nThe bottom 4 indices listed in Tables\u00a02\u20134 represent biomorphometric assessments at the disk margin alone. These are: (1) disk area, (2) mean retinal nerve fiber layer (RNFL) thickness, (3) RNFL cross-sectional area, and (4) the height variation contour, calculated as the maximal minus the minimal RNFL thickness along the contour line. None of these indices differed significantly between groups, although there was some suggestion of greater RNFL thickness for the tamoxifen users (see Table\u00a04).\nAs stated in the Methods section, we also conducted analyses using the ONH indices derived from contour lines placed slightly interior to the anatomic disk margin rather than exactly at the disk margin. Every statistically significant result concerning the cup remained significant when based on the smaller contours. Conversely, whereas some of the cup-area comparisons involving tamoxifen users were not statistically significant when based on the larger contours (see Tables\u00a02, 3), every corresponding comparison became significant when based on the smaller contours. In particular, the cup-area values for the older tamoxifen users (>50\u00a0years) became significantly lower than the cup-area values for the older control subjects (>50\u00a0years), presumably because using the smaller contours enhanced the importance of effects occurring predominantly within the interior of the cup.\nDiscussion\nBecause tamoxifen users and anastrozole users share similar medical histories, the results indicate that tamoxifen often leads to reductions of optic-cup size among middle-aged women older than about 50\u00a0years. Any effects of anastrozole at the ONH remain unproven, and if they exist at all, they are probably minor and opposite to the effects of tamoxifen. In contrast to tamoxifen [9, 10], there seem to be no reports in the peer-reviewed literature concerning any visual or ocular side effects of anastrozole. One study has found that anastrozole can cause regression of breast cancer metastases within the choroid [31].\nBecause all of the women in this study met a rigorous set of criteria for excellent ocular health, any cup-size changes presumably would be designated as subclinical. Data from several reports suggest that the color vision of tamoxifen users is often altered [10, 19, 20], although such alterations either are subtle or can be revealed only under specialized laboratory conditions, and there is no evidence that color vision changes are caused by effects occurring at the ONH. A recent survey found that about 13% of tamoxifen users reported experiencing vision changes that they attributed to tamoxifen [32], but the nature of these purported changes was not specified, nor were the subjects\u2019 reports related to the results of any eye examination. Nevertheless, the subjects reporting vision changes had significantly higher serum levels of tamoxifen and a potent tamoxifen metabolite (N-DMT) than did the women not reporting these changes [32]. In contrast, reports of well-known side effects associated with tamoxifen use, such as hot flashes and vaginal dryness, were not related significantly to these same serum levels [32]. Cases of tamoxifen-induced optic neuropathy have been described in the literature [12, 33\u201338], but such cases usually are considered rare [9, 10, 38]. Perhaps tamoxifen users sometimes experience a degree of optic neuropathy that would not be detected or confirmed on a presenting eye examination owing to the wide range of normal cup appearances. If so, the results of this study point to a potential new application for intraocular imaging devices such as the HRT, which provides spatial resolution at the ONH on the order of tens of microns and is designed to detect longitudinal change [39]. Several types of high-resolution intraocular imaging device are now available clinically [39], and they allow high-resolution biomorphometric data to be collected in ways that are non-invasive and not intimidating to patients. The ease of data collection with these devices may allow patients who report tamoxifen-related vision change to be evaluated before and after cessation of tamoxifen use, and also after any rechallenge. Until this is done, and then only if changes in vision are shown to correspond to anatomical changes, might it be justified for the type of results described in this study to direct therapy. Presently, the only assured clinical application is to alert ophthalmologists and neurologists that assessment of the ONH for conditions such as glaucoma or optic neuritis may be complicated by the use of tamoxifen.\nA human-subjects study such as ours cannot definitively identify the mechanism(s) by which tamoxifen may reduce optic-cup size in vivo. Nevertheless, there is evidence to suggest that tamoxifen might reduce cup size via astrocytic swelling. First, tamoxifen is a potent blocker of swelling- (or \u201cvolume-\u201d) activated chloride channels for astrocytes assessed in culture [16, 40], and astrocytes are abundant in the optic cup [18, 41]. Second, although astrocytes are present in the RNFL [17, 41], they comprise proportionally more of the neural volume as the cup is descended [41], and astrocytes along with ganglion cell axons are abundant at the base of the cup and also within the sides [42]. However, if the ganglion cell axons increased in volume, one would expect more visual dysfunction than is observed. In addition, there is evidence that tamoxifen-induced development of posterior subcapsular cataracts [10] may involve the blockage of volume-activated chloride channels [43], and that the utility of high-dose tamoxifen as salvage therapy for astrocytomas [44] may depend partly on the ability of tamoxifen to cause astrocytic swelling [45]. Based on photographic assessment of the ONH and retina by a neuro-ophthalmologist (author JF), there did not appear to be any difference in the vasculature of the three subject groups that would have led to the between-group cup differences quantified in this study and also observed in the ONH photographs when examined for groups of subjects rather than for isolated subjects.\nThe presence of a steep reduction of cup size with age at about 50\u201351\u00a0years among the tamoxifen users suggests that any ability of tamoxifen to reduce cup size, by whatever means, depends on the absence or near absence of natural female hormones, given that the median age of natural menopause is 51\u00a0years [25] and given that tamoxifen elevates the estrogen levels of pre-menopausal women [46]. Hormone replacement therapy might, in principle, prevent or alleviate tamoxifen-induced reductions in cup size, but hormone replacement is generally contraindicated for breast cancer survivors, especially for women who have had hormone-receptor-positive tumors [47] (although diverse opinions exist [48]). Hormonal reduction by itself, whether occurring naturally or induced by anastrozole, does not appear to result in cup-size reductions of nearly the magnitude or the frequency inferred for the older tamoxifen users, at least not for the age range that we investigated. The presence of small cups among the tamoxifen users probably did not result from selectively low IOP levels, since the measured IOPs for each subject group were quite similar, differing by no more than 0.2\u00a0mmHg on average. Because the presence of small cups among tamoxifen users appeared to be unaccompanied by corresponding significant effects at the disk margin, the effects of tamoxifen on the ONH may be regarded as predominantly localized to the cup.\nFor this study, we found no evidence of any age-related change of ONH indices for healthy women not using hormonally acting medications. However, a recent study of hundreds of subjects has documented the presence of ONH differences between healthy men and women older than 65\u00a0years [49]. In that study, several ONH indices were found to differ significantly between men and women, but the greatest percentage difference was for cup area, which averaged about 14% smaller for women than for men [49]. There is separate evidence that cup areas can depend slightly on the phase of the natural menstrual cycle [50]. These 2 sets of results along with our own results suggest that further studies be conducted to elucidate the effects of hormonally acting medications on the ONH. Such studies should include elderly women using tamoxifen, if possible, since elderly women have higher circulating levels of tamoxifen than do younger women [51].","keyphrases":["tamoxifen","anastrozole","optic cup","breast cancer","adjuvant endocrine therapy","eye","optic nerve head"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"J_Gastrointest_Surg-3-1-1915638","title":"Long-term Results of a Primary End-to-end Anastomosis in Peroperative Detected Bile Duct Injury\n","text":"The management of a bile duct injury detected during laparoscopic cholecystectomy is still under discussion. An end-to-end anastomosis (with or without T-tube drainage) in peroperative detected bile duct injury has been reported to be associated with stricture formation of the anastomosis area and recurrent jaundice. Between 1991 and 2005, 56 of a total of 500 bile duct injury patients were referred for treating complications after a primary end-to-end anastomosis. After referral, 43 (77%) patients were initially treated endoscopically or by percutaneous transhepatic stent placement (n = 3; 5%). After a mean follow-up of 7 \u00b1 3.3 years, 37 patients (66%) were successfully treated with dilatation and endoscopically placed stents. One patient died due to a treatment-related complication. A total of 18 patients (32%) underwent a hepaticojejunostomy. Postoperative complications occurred in three patients (5%) without hospital mortality. These data confirm that end-to-end anastomosis might be considered as a primary treatment for peroperative detected transection of the bile duct without extensive tissue loss. Complications (stricture or leakage) can be adequately managed by endoscopic or percutaneous drainage the majority of patients (66%) and reconstructive surgery after complicated end-to-end anastomosis is a procedure with relative low morbidity and no mortality.\nIntroduction\nBile duct injury (BDI) after laparoscopic cholecystectomy (LC) is still a major problem in current surgical practice. BDI is associated with reduced survival, increased morbidity, and poor long-term quality of life (QoL).1,2 The incidence of BDI at laparoscopic cholecystectomy has been reported between 0.3 to 1.4%,3\u20135 depending on the criteria used to define the injury as well as the study population. Of these injuries, one-third is detected during the procedure.6 Measures to prevent and recognize BDI are outlined in many publications.6\u20138,9\u201311 The optimal treatment strategy and short- and long-term outcome has been published extensively.12\u201314 Controversy exists however about the management of peroperative detected BDI. The most important factor is the extent of tissue loss of the common bile duct, but also severity of inflammation and the size and diameter of the proximal duct. The peroperative management range from simple drainage and referral to a tertiary center to an end-to-end anastomosis (EEA) (duct to duct, with or without T-tube drainage) or a hepaticojejunostomy (HJ).\nIt has been suggested that EEA is associated with a relative high stricture rate up to 70\u201380% and consequently a high incidence of secondary repair.15 Therefore, many tertiary centers prefer to perform a HJ instantly. A secondary repair after EEA should be associated with an increased risk of postoperative complications as the formation for strictures and stenosis.16,17 Others, however, consider EEA as a relative simple definitive repair, and also an optimal initial drainage procedure before reconstructive surgery in a secondary setting.18 Reports on large consecutive series to analyze the outcome of EEA are scary because this procedure is generally not performed in referral centers. One should realize that patients referred to such a center after previous EEA elsewhere are a negative selection of the EEA population. So far, a systematic analysis of a large group of patients with an EEA has not been performed and therefore this study was conducted.\nThe aim of the present study was to analyze short- and long-term outcome in patients who are referred after failure of a primary EEA.\nPatients and Methods\nPatients Cohort and Data Collection\nBetween January 1991 and January 2006, 500 consecutive patients were referred to the Academic Medical Center (AMC) in Amsterdam for the management of a BDI after cholecystectomy. Patient data was induced in a prospective database. All types of BDI were included, also minor injuries such as leakage from the cystic duct or ducts of Luschka. To define the location of BDI, the Bismuth classification was used.18 For the present study, the medical charts of all patients who underwent a primary EEA were retrospectively reviewed to analyze the initial operation reports and clinical data.\nData from the referring hospital included: indication for cholecystectomy, type of initial procedure, location of injury, type of repair including the use of a T-tube, the postoperative diagnostic interventions, and the therapeutic interventions before referral. Data from the present center included: symptoms at referral, diagnostic work-up, type of treatment, short-term, and long-term complications.\nEndoscopic, Radiological, and Surgical Treatment for complicated EEA\nEndoscopic treatment was performed by balloon dilatation or catheter dilatation before stent placement. The biliary stent is placed over the guide wire bridging the stenosis. Two or more stents were inserted if possible. For multiple stent insertion, an endoscopic sphincterotomy was performed to facilitate stent placement. Stents were replaced after 6\u00a0weeks and subsequently exchanged every 3\u00a0months to avoid cholangitis.\nPercutaneous transhepatic catheterization was performed by injecting the contrast medium from the right intercostal approach. A right or left approach for the percutaneous transhepatic biliary drainage was chosen depending on ultrasound images illustrating the biliary anatomy, and the possibility of puncturing a dilated intrahepatic bile duct. Catheterization of intrahepatic bile ducts was performed in standard fashion. A guide wire was advanced through the biliary stricture into the duodenum. When this was achieved, a biliary drainage catheter was inserted. All drainage procedures were performed with the administration of broad-spectrum antibiotics.\nIn case of a surgical reconstruction, the procedure was performed via a Roux-en-Y hepaticojejunostomy. The stricture in the CBD is transected and the hilar plate is opened. The hepatic ducts of different segmental bile ducts are mobilized and from there opened over the left hepatic duct. Intrahepatic segmental ducts are mobilized and if possible sutured together before one or two jejunal anastomosis are made. A closed suction drain is placed during operation and removed 24\u201348\u00a0hours after surgery. Percutaneous transhepatic drains, when inserted before surgery are left in place and removed after 10\u00a0days till 6\u00a0weeks, depending on the clinical course, the level of anastomosis and the surgeons\u2019 preference.\nOutcome\nFollow-up data was obtained through outpatient records and the records of the general practitioner. The outcome of treatment was analyzed by the number complications and late restenosis during follow-up. Failure of treatment was defined as recurrent stenosis after stent therapy followed by surgery or recurrent stenosis after surgical reconstruction followed by additional therapy.\nStatistical Analysis\nData from patient characteristics, management, and outcome show descriptive statistics in number of patients and percentages. Mean and median values are given with a minimum and maximum. Long-term stricture-free survival was analyzed by Kaplan Meier Survival Analysis. Data analyses were performed using SPSS\u00ae software (SPSS, Chicago, Illinois, USA).\nResults\nPatients\u2019 Characteristics at Referral\nThe referral pattern of BDI patients (n\u2009=\u2009500) throughout the last 15\u00a0years are summarized in Fig.\u00a01. From the total of 500 patients, 56 (11.5%) underwent a primary EEA. Patient characteristics are listed in Table\u00a01. The laparoscopic cholecystectomy (n\u2009=\u200948, 86%) was converted in all patients. An open cholecystectomy was performed in eight patients (14%). In 49 patients (88%), the anastomosis was made over a T-tube. The tube was removed at the referring hospital or at the AMC after a mean of 52\u00a0days (range 2\u2013145\u00a0days).\nFigure\u00a01Referred patients for treatment of bile duct injury. Total number of referred patients (red), patients referred after a primary end to end anastomosis (blue), and patients referred after a primary biliodigestive reconstruction (green).Table\u00a01Patient Characteristics\u00a0Primary EEAn\u2009=\u200956%Age at cholecystectomyMean (years)52GenderFemale4377Indication for cholecystectomySymptomatic cholelithiasis4580Cholecystitis59Cholecystitis a froid61Type of initial operationOpen procedure814Laparoscopic to open procedure4886Anastomosis over T-tube4988Duration of T-tube in situDays, median (range)42(2\u2013145)\nAfter the primary EEA, 19 patients (34%) underwent other therapeutical interventions before referral (Table\u00a02) (Fig.\u00a02). These patients underwent a range of one to three procedures before referral (median 2). The interventions included a relaparotomy in two patients (4%), percutaneous drainage of fluid collections in five patients (9%), endoscopicially placed stents in 12 patients (21%), a papillotomy in nine patients (16%), and percutaneous transhepatic drainage in two patients (4%). The median interval from the primary EEA to referral was 16\u00a0weeks (range 0\u2013141\u00a0weeks). At referral, a biliary stricture was diagnosed in 38 patients (68%); in 10 patients (18%), bile leakage was diagnosed and combination of both in eight patients (14%). Symptoms at referral were cholestasis (n\u2009=\u200914, 25%), cholangitis (n\u2009=\u200910, 18%), and abdominal pain (n\u2009=\u200915, 27%). Three patients were referred because of uncontrolled sepsis (n\u2009=\u20092) and peritonitis (n\u2009=\u20091). According to the Bismuth classification, the majority of injuries (leakage of stricture) (n\u2009=\u200947, 84%) was located below the bifurcation. In nine patients (16%), the injury (mostly strictures) involved the bifurcation or the right or left hepatic duct (i.e., Bismuth classification grades IV and V). \nTable\u00a02Referral Pattern\u00a0Primary EEAn\u2009=\u200956%Time interval between injury and referral\u00a0Weeks, median (range)16 (0\u2013141)Intervention after EEA and before referral\u00a0Explorative relaparotomy24\u00a0Percutaneous drainage59\u00a0Endoscopic stenting1221\u00a0Endoscopic papillotomy916\u00a0PTDa24Symptoms at referral\u00a0Cholestasis1425\u00a0Cholangitis\/fever1018\u00a0Abdominal pain1527\u00a0Abces\/biloma47\u00a0Uncontrolled sepsis\/peritonitis35Diagnosis at referralStenosis3868Leakage1018Combination of stenosis and leakage814Location of injury at referralb\u00a0\u00a0I916\u00a0\u00a0II2138\u00a0\u00a0III1730\u00a0\u00a0IV712\u00a0\u00a0V24aPercutaneous transhepatic drainagebAccording to Bismuth classificationFigure\u00a02ERCP showing successful (aggressive) stent therapy after primary EEA. a Stenosis of the common bile duct. b Stents in situ. c After stent removal within a year.\nManagement after Referral\nDiagnostic work-up was performed by CT-scan (n\u2009=\u20099; 16%), endoscopic cholangiography (n\u2009=\u200938; 68%), and transhepatic cholangiography (n\u2009=\u20099; 16%). The definitive treatment of BDI patients after EEA is shown in the flow diagram (Fig.\u00a03). After work-up, three patients (5.3%) were treated with percutaneous transhepatic cholangiographic drainage (PTCD) and 40 patients (71.4%) were treated endoscopically. Thirteen patients (23%) underwent reconstructive surgery after work-up; eight patients because of a complete stenosis of the CBD, in three patients reconstructive surgery was performed after failure of stent therapy at the referring hospital and in two patients because of a percutaneous fistula and persistent bile leakage.\nFigure\u00a03Flow diagram of the success and failure rates after a multidisciplinary treatment of patients who underwent a peroperative end to end anastomosis for bile duct injury. Given percentages are calculated from the number of patients in the previous flow box. PTCD Percutaneous transhepatic catheter dilatation.\nRadiological and Endoscopic Treatment\nThree patients were successfully treated by PTCD. In two patients, a stenosis was treated by transhepatic dilatation and in one patient, bile leakage was treated by external transhepatic stent insertion.\nForty patients (71.4%) were treated endoscopically (Fig.\u00a02). In 37 patients (66%), stent insertion was successful and in three patients (5%), adequate drainage succeeded by papillotomy. The median number of stent replacements was five (range 1\u201315) with a median duration of treatment of 359\u00a0days (range 39\u20131,355). Complications occurred in nine patients (24%). Stent dislodgment (n\u2009=\u20093), clogging (n\u2009=\u20092), and cholangitis (n\u2009=\u20095) were mild complications and were successfully treated by stent exchange or administration of antibiotics. One severe complication occurred in a 75-year-old patient. After 4\u00a0years of stent therapy, the stent migrated and perforated the duodenum. Finally, the patient died due to multiple organ failure and sepsis.\nSurgical Treatment\nAfter referral and during the follow-up period, a new hepatobiliary anastomosis was performed by hepaticojejunostomy in 13 patients (23.2%). Mean duration of hospital stay was 9.1\u2009\u00b1\u20093.1\u00a0days. Postoperative complications occurred in one patient (7.6%) who underwent a PTC procedure after leakage of the anastomosis. No hospital mortality occurred in patients who underwent a reconstructive procedure after a previous EEA.\nLong-term Follow-up\nAfter a mean follow-up of 7.1\u2009\u00b1\u20093.3\u00a0years, seven patients (13%) have died. One endoscopically treated patient died due to a complication of treatment as described above. The other patients died due to malignancy (n\u2009=\u20094) and myocardial infarct (n\u2009=\u20092).\nThe long-term results in patients treated with endoscopic and radiological treatment are as follows: from a total of 43 patients treated with endoscopic or PTCD procedures, 86% (n\u2009=\u200937) was successful. In three patients (7%), signs of restenosis occurred after stent removal after 2, 3.5, and 4\u00a0months. Continued stent therapy was successful in all three patients. Five patients underwent reconstructive surgery after prolonged endoscopic stenting. Postoperative complications occurred in two of the five patients and these patients received additional therapy for wound infection (n\u2009=\u20091) and postoperative cholangitis (n\u2009=\u20091).\nThe long-term results of surgical treatment after EEA are as follows; from 13 patients who underwent a HJ after work-up, a stenosis of the anastomosis occurred in two patients (15%). Both patients underwent successful percutaneous transhepatic dilatation, respectively 9 and 35\u00a0months after surgery.\nThe overall 5\u00a0years stricture free survival in the total cohort (n\u2009=\u200956) is 91%, shown by a Kaplan Meier curve in Fig.\u00a04.\nFigure\u00a04Kaplan\u2013Meier plot showing proportion of patients without restenosis among 56 bile duct injury patients treated for complications after EEA.\nDiscussion\nThe present study describes a selected group of BDI patients, who were referred for treatment after a complicated EEA. This group of patients is a negative selection, representing the worst complications of EEA; otherwise, patients were not referred for additional treatment. So, this study does not provide any information about the success rate of EEA. The present study shows a long-term stricture free survival of 91% in EEA patients after treatment in a tertiary center. The analysis showed that even the majority of complications after primary AEE in a general hospital can successfully be treated by endoscopic and radiological interventions. In only one-third of the patients, a secondary surgical repair is necessary. The surgical reconstruction after EEA was associated with acceptable morbidity and without mortality.\nAround 40 to 45 patients are referred annually without any sign of decrease over the last years. Considering 15.000 LC\u2019s per year in the Netherlands, we still consider 0.4\u20130.5% mentioned in the reviews as an underestimation of the real incidence of BDI, at least in the Netherlands.19 In 20% of the patients referred to the AMC, the injury was detected during the initial surgical procedure. This finding is similar to reports in literature.6,20  From the total of 500 referred BDI patients, 11.2% was referred for the treatment of a complication after peroperative EEA. Because referred patients only represent the complications after EEA, we do not know the real incidence of EEA procedures in BDI.\nPeroperative repair in BDI detected during surgery can be performed by EEA (with or without the use of a T-tube) or by a primary HJ. A HJ is a more complex procedure and one should be very careful not to further extend the injury into the intrahepatic ducts or subsequently damage the arterial supply (bleeding and clipping or ligation of right hepatic artery).21,22 The present study shows that if complications occur after EEA, these can successfully be treated by percutaneous or endoscopic balloon dilatation and\/or stenting in the majority of patients. A HJ in the acute setting without dilated bile ducts is even more difficult and therefore consulting a surgeon with experience in reconstructive hepatobiliary surgery is recommended. In contrast with a primary HJ an EEA is a relatively simple procedure and can also be performed in less experienced hands. The risk to increase damage is smaller in an EEA procedure and with the use of a T tube instant bile drainage is realized. If indicated, reconstructive surgery by means of an elective HJ can be performed. It is strongly advised to perform a HJ after classification the injury and analyzing the biliary anatomy. Preoperative cholangiography (with the use of the T tube) will illustrate the location of the stenosis and the extension of dilation of the proximal bile ducts. A reconstructive procedure for stenosis of EEA has a satisfying outcome, as peroperative conditions are good after the inflammation has subsided and the bile ducts are dilated due to stenosis.\nIn a situation in which peroperative bile leakage is due to (extensive) tissue loss, in particular, in patients with more proximal lesions at the bifurcation or intrahepatically, no primary repair should be performed. In this situation, adequate drainage of the upper right abdomen is strongly advised and the patient should be referred for elective reconstruction. Referral to tertiary center in this situation has a positive effect on outcome.2\nEnd-to-end anastomosis is reported to be associated with a high incidence of recurrent jaundice due to stricture formation of the anastomotic area.15 Therefore, some authors suggest that EEA is almost never appropriate if the bile duct has been completely transected,15,23 while others favor this strategy when there is no extensive tissue loss.18 Stent therapy for iatrogenic bile duct strictures has changed during the last decade and therefore the long-term outcome after stenting has improved.24 A more aggressive approach with more stents and smaller time intervals between stent changes is favored. With this new approach, 80% of the patients who undergo an ERCP for postoperative bile duct stenosis, have a 10-year stricture-free survival.25 Although complications occur at a significant rate, these are usually mild. The only severe complication occurred in the present series, due to a migrated stent, was not reported in previous series.24,25 After stent removal, recurrent stenosis develops in 20% of patients within 2\u00a0years of stent removal.25 Therefore, endoscopic treatment should be the initial management of choice for postoperative bile duct strictures. Without signs of improvement after endoscopic stenting, reconstructive surgery is indicated in otherwise fit patients.\nOf interest is the evaluation of the long-term stricture-free survival after treatment for complications after EEA. After a mean follow-up of 7.1\u00a0years, restenosis after treatment developed in 9% of the patients. In all patients who underwent initial endoscopic therapy, restenosis occurred a relatively short time after stent removal, diagnosed within 2 to 8\u00a0months follow-up. Therefore, endoscopic treatment is not associated with a high rate of long-term restenosis after stent removal. In two patients, a restenosis occurred within 3\u00a0years after a hepaticojejunostomy. Symptoms were cholestasis and cholangitis. In both patients, transhepatic dilatation was successful to resolve the stenosis. The long-term stricture-free survival of 91% in the present series provides evidence for a good outcome after treating complicated EEA patients. If BDI is detected during surgery, in particular if there is no extensive tissue loss, the local anatomy is clear and there is no inflammation, EEA could be considered as a sufficient treatment strategy. Patients with postoperative complications (stricture or leakage) should be treated by a multidisciplinary team of gastroenterologists, radiologists, and surgeons. Postoperative complications can adequately be managed by endoscopic or percutaneous drainage in two-third of the patients. Reconstructive surgery after a complicated EEA is associated with low morbidity and no mortality.","keyphrases":["end-to-end anastomosis","bile duct injury","cholecystectomy"],"prmu":["P","P","P"]}
{"id":"Osteoporos_Int-4-1-2267483","title":"Loss of treatment benefit due to low compliance with bisphosphonate therapy\n","text":"Summary Among 8,822 new female bisphosphonate users, non-compliant bisphosphonate use was associated with a 45% increased risk of osteoporotic fracture compared to compliant use (MPR \u226580%). Classifying compliance into five categories, fracture risk gradually increased with poorer compliance. These results emphasize the importance of treatment compliance in obtaining maximal treatment benefit.\nIntroduction\nOsteoporosis is a systemic condition characterized by low bone mass and microarchitectural deterioration of bone tissue, leading to enhanced bone fragility and, consequently, an increased fracture risk [1]. Osteoporosis has clinical and public health importance because osteoporotic fractures are one of the most common causes of disability and a major contributor to medical costs in many regions of the world [2]. Bisphosphonates are potent inhibitors of the osteoclast-mediated resorption of bone and widely used to treat osteoporosis and reduce the risk of osteoporotic fractures [3].\nLong-term therapy with bisphosphonates is required to realise the full benefits of this treatment [3, 4]. This is supported by a recent PHARMO study that showed that the risk of fractures was reduced by 26% after one year persistent bisphosphonate use and up to 32% after two year persistent use [5]. However, in daily clinical practice, 50% to 75% of patients discontinue bisphosphonate use already in the first year of therapy [5\u20138]. Moreover, non-compliance with bisphosphonates has also been reported to be a frequent issue, with rates varying from 35% to 65% [6, 9]. Reasons for low compliance with bisphosphonate treatment are the stringent regimen to minimize the risk of oesophageal irritation, the existence of drug-related gastrointestinal side effects, and the fact that osteoporosis is often asymptomatic in early stages [10]. Caro et al. showed that patients who were compliant with their osteoporosis medication, including bisphosphonates, estrogens and calcitonin, experienced a 16% lower fracture rate compared to non-compliant users [11]. The objective of this study was to investigate the risk of osteoporotic fractures associated with low compliance with bisphosphonates in more detail. In addition to dichotomizing compliance, we also classified compliance into five categories.\nPatients and methods\nSetting\nData were obtained from the PHARMO Record Linkage System (PHARMO RLS) which includes several databases, among which drug dispensing and hospitalization data of more than two million residents of the Netherlands. The drug dispensing histories contain data on the dispensed drug, the type of prescriber, the dispensing date, the amount dispensed, the prescribed dose regimens, and the duration of use. All drugs are coded according to the Anatomical Therapeutic Chemical (ATC) Classification. The hospital records include detailed information concerning the primary and secondary diagnoses, procedures, and dates of hospital admission and discharge. All diagnoses are coded according to the International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM).\nStudy cohort\nThe source population included all new users of bisphosphonates in the period from January 1999 until July 2004. These patients were not dispensed any anti-osteoporotic drugs (bisphosphonates, raloxifene, tibolone, estrogens, hormone replacement therapy, calcitonin or teriparatide) for at least one year before their first bisphosphonate dispensing. All female new users of alendronate (10\u00a0mg daily or 70\u00a0mg weekly) or risedronate (5\u00a0mg daily or 35\u00a0mg weekly) (i.e. the dosages indicated for the prevention or treatment of postmenopausal osteoporosis), aged \u226545\u00a0years or with diagnosed postmenopausal osteoporosis (ICD-9-CM code 733.01), without gaps in registration in PHARMO RLS, and with a registration of at least one year before and one year after starting bisphosphonate treatment were included in the study cohort.\nAll study patients were followed from the first bisphosphonate dispensing until the occurrence of the outcome of interest, death, end of registration in PHARMO RLS or end of the study period, whichever event was earliest.\nCompliance\nCompliance with bisphosphonates during follow-up may change and was measured over 90-day intervals using the Medication Possession Ratio (MPR). MPR was defined as the sum of days\u2019 supply of all alendronate and risedronate dispensings during or overlapping the 90-day interval divided by 90 potential days of bisphosphonate therapy. For each dispensing only the days\u2019 supply covering a specific interval were counted. E.g. of a 100-days supply dispensed at follow-up day 60, only 30\u00a0days were counted when calculating the MPR over the first interval (day 0\u201390) and 70\u00a0days for the MPR over the second interval (day 91\u2013180). The maximum value of a MPR was set at 1. Subdividing follow-up in larger intervals was assumed too rough and subdividing in smaller intervals was not feasible regarding the usual prescription length of 90\u00a0days.\nOsteoporotic fractures\nThe outcome variable osteoporotic fracture was defined as hospitalisation for an osteoporotic fracture during follow-up. Hospitalisations were selected based on primary discharge diagnosis for probable (ICD-9-CM codes 805.2 (vertebral thoracic), 805.4 (vertebral lumbar), 820 (proximal femur), 812 (proximal humerus), 813.4 (distal radius\/ulna), 814 (carpus)) or possible (ICD-9-CM codes 823.0 (proximal tibia\/fibula), 807.0 (rib), 807.2 (sternum), 808.0 (pelvis)) osteoporotic fractures.\nCovariates\nFor all patients, a history of osteoporotic fracture was assessed. Furthermore, we determined use of non-steroidal anti-inflammatory drugs, analgesics, benzodiazepines and antidepressants in the year after starting bisphosphonate treatment as these drugs are associated with an increased risk of fractures [12\u201314]. Similarly, we assessed use of thiazide diuretics, \u00df-blockers and statins in the year after start as these drugs are associated with a reduced risk of fractures [15\u201317].\nStatistical analyses\nThe association between low compliance with bisphosphonates and the risk of fractures was analysed univariately and multivariately using time-dependent Cox regression analysis to account for changing compliance over time. In this analysis, at the time of each fracture, the cumulative compliance up to and including the corresponding 90-day interval of women who had experienced a fracture was compared to the cumulative compliance of those women who remained fracture-free at this time. The cumulative compliance of the fracture-free patients was measured up to the interval of corresponding fracture.\nIn a first analysis, compliance was dichotomized and a MPR <80%, i.e. non-compliant bisphosphonate use was compared to a MPR \u226580%, i.e. compliant use. In a second analysis, compliance was classified into five categories: <20%, 20\u201349%, 50\u201369%, 70\u201389% and \u226590% (reference). A Wald test for trend was performed. Assuming that the effect of bisphosphonates is not immediate, and in line with Caro et al. [11], fractures occurring in the first 182\u00a0days of follow-up were excluded from the analyses. In a sub-analysis, this exclusion period was varied. Furthermore, an additional analysis was performed excluding women with only one bisphoshonate dispensing during follow-up; in these patients it is less sure that they actually used the drug.\nThe multiple regression models included age, history of fracture and all covariates that were univariately associated with the risk of fractures and significantly contributed to the multivariate model, i.e. inclusion of the covariate resulted in a change of the compliance hazard ratio (HR) of 5% or over, starting with the most potent covariate. HRs and 95% confidence intervals (CI) were estimated using SAS V8.2 UNIX (Cary, NC, USA).\nResults\nThe study cohort included 8,822 new female users of daily or weekly alendronate or risedronate, who contributed a total of 22,484 person-years of follow-up during the study period. More than half of the patients were older than 70\u00a0years of age (n\u2009=\u20094,708, 53%) and about one quarter of the patients used corticosteroids (irrespective of quantity) (Table\u00a01). The first bisphosphonate was mainly prescribed by the general practitioner (64%). Only 197 patients (2%) were hospitalized for an osteoporotic fracture in the year before starting treatment.\nTable\u00a01General characteristics of new bisphosphonate users between 1 January 1999 and 30 June 2004 (N\u2009=\u20098,822)CharacteristicN%Age class (years)45\u20135488910.155\u2013693,22536.5\u2265704,70853.4Mean age (years) \u00b1 sd 69.4\u2009\u00b1\u200910.3Year of start1999 \/ 20002,25025.52001 \/ 20022,76931.42003 \/ 20043,80343.1Initial bisphosphonateDaily bisphosphonate4,22247.9Weekly bisphosphonate4,60052.1First prescriberGeneral practitioner5,70464.7Internist, rheumatologist, orthopedist2,31326.2Other prescribers8059.1Hospitalization for an osteoporotic fracture in the year before start 1972.2Use of corticosteroids in the year before start (irrespective of quantity)2,38927.1Follow-up time (months)12\u2013232,93933.324\u2013352,26825.7\u2265363,61541.0\nDuring follow-up, 216 patients (2%) experienced an osteoporotic fracture, of which 40 patients during the first six months and 78 during the first year. About two third of all osteoporotic fractures were located at the proximal femur.\nThe percentage of patients with a MPR < 80% (i.e. non-compliant patients) increased from 34% (3,018\/8,822) after six months of follow-up to 42% (3,743\/8,822) after one year, 51% (3,014\/5,883) after two years and 60% (2,149\/3,615) after three years of follow-up. Subdividing MPR in classes (Fig.\u00a01), the majority of patients either had a MPR \u226590% or a MPR <20%, with the first group decreasing and the latter group increasing over time.\nFig.\u00a01Compliance with bisphosphonates, classified in five MPR categories, after six months, one year, two years and three years of follow-up. MPR: medication possession ratio\nDichotomizing compliance, a MPR <80%, i.e. non-compliant bisphosphonate use was associated with a 40% increased risk of osteoporotic fracture more than six months after starting treatment (HR 1.41 95% CI 1.04\u20131.91, adjusted for age and history of fracture (see Table\u00a02 for association between co-variates and fracture risk)) compared to a MPR \u226580%, i.e. compliant use. Varying the exclusion period, i.e. excluding fractures occurring in the first year of follow-up or including all fractures during follow-up, yielded similar adjusted increased risks (HR 1.50; 95%CI 1.06\u20132.13 and HR 1.45; 95%CI 1.10\u20131.91, respectively). Excluding women who received only one bisphosphonate dispensing during follow-up also did not change the results (adjusted HR 1.45; 95% CI 1.05\u20131.99, based on 156 fracture patients and 7,758 fracture-free patients).\nTable\u00a02Association between co-variates and fracture risk more than six months after starting treatment\u00a0Fracture patients (N)Fracture-free patients (N)N%N%HRcrude95% CITotal1761008,606100Age (years) at start\u00a045\u20135474.087910.21.00reference\u00a055\u2013692916.53,18937.11.160.51\u20132.66\u00a0\u22657014079.54,53852.74.282.00\u20139.15Year of start\u00a01999\/20008648.92,15825.11.681.04\u20132.71\u00a02001\/20026335.82,69131.31.550.97\u20132.49\u00a02003\/20042715.33,75743.61.00referenceHistory of osteoporotic fracture52.81902.21.520.63\u20133.70Comedication in the year after start1\u00a0Analgesics6838.62,13624.81.821.34\u20132.46\u00a0Benzodiazepines8045.53,32438.61.300.96\u20131.74\u00a0Antidepressants2916.51,04612.21.521.02\u20132.26\u00a0\u00df-blockers4726.71,92922.41.350.97\u20131.89HR: hazard ratio, CI: confidence interval1 Univariate non-significant comedications (i.e. non-steroidal anti-inflammatory drugs, thiazides and statins) are not shown.\nClassifying compliance into five categories, fracture risk gradually increased with poorer compliance (p-value <0.05 for trend). Compared to a MPR \u226590%, the adjusted risk of fracture increased from 1.2 times for a MPR between 50% and 90% to 1.8 times for a MPR less than 20% (Fig.\u00a02).\nFig.\u00a02The relationship between compliance with bisphosphonates, classified in five MPR categories, and fracture risk more than half a year after starting treatment. HRs are adjusted for age and history of fracture and are compared to a MPR \u226590%. MPR: medication possession ratio, HR: hazard ratio, CI: confidence interval\nDiscussion\nThis study indicates that non-compliant bisphosphonate users had an approximately 50% higher likelihood of osteoporotic fracture compared to compliant users. Classifying compliance into five categories, fracture risk gradually increased with poorer compliance to an 80% risk increase with very low compliance compared to very high compliance. These results emphasize the importance of treatment compliance in obtaining maximal treatment benefit.\nTo quantify the loss of treatment benefit with low compliance, information on fracture risk in women not treated with bisphosphonates is necessary. The above mentioned percentage risk increases are compared to compliant use and cannot be translated into percentage loss of treatment benefit. We chose not to use untreated patients as reference because of the heterogeneity of patterns of treatment use that exist within this group. In a meta-analysis of 11 trials of alendronate for the treatment of postmenopausal osteoporosis, a consistent 50% reduction in fracture risk was seen compared to placebo [18]. For risedronate, a meta-analysis of 8 trials revealed a 25 to 35% reduction in fracture risk compared to placebo [19]. In other words, untreated women are 1.5 to 2 times as likely to experience a fracture compared to women using bisphosphonates. Assuming that the patients with a MPR \u226590% in our study are comparable to the treated patients in trials, the 1.6 to 1.8 times increased fracture risk we observed with a MPR <50% can be translated into a 80 to 100% loss of treatment benefit. Apparently, patients with the lowest compliance are essentially comparable to non-treated patients.\nAt present, in addition to Caro et al., others have reported on the relationship between compliance with bisphosphonates and fracture risk [11, 20, 21]. Siris et al. [20] found a relative fracture risk reduction of 25% with compliant use of alendronate or risedronate, i.e. a 33% risk increase with non-compliant use. There was a progressive relationship between compliance and fracture risk reduction, commencing at a MPR around 50% and becoming more pronounced at a MPR \u226575%. This finding, like our results, suggests that low-compliant patients gain only marginal benefit from bisphosphonate therapy. Among women starting drug therapy for osteoporosis (of which two-third bisphosphonates) Weycker et al. [21] found that compared to women with a MPR <30%, the odds of fracture was similar (1.02) for women with a MPR between 30 and 69% and women with a MPR between 70 and 89%, but lower (0.70) for women with a MPR \u226590%. The latter corresponds to a 1.4 times increased risk of fracture with a MPR <30% compared to a MPR \u226590%. Caro et al. [11] reported that compared to a MPR >90%, fracture risk was significantly increased at lower compliance levels, i.e. up to 1.4 times with a MPR \u226450%. However, it is not clear whether this effect was gradual. In a study population of mainly users of hormone replacement therapy, Huybrechts et al. [22] found that compared to women with a MPR \u226590%, fracture risk was similar (HR 1.09) for women with a MPR between 80 and 90%, but higher for women with a MPR between 50 and 80% and women with a MPR <50% (HR 1.18 and 1.21, respectively). Our associations are about similar than reported by Siris et al., but stronger than found by Caro et al., Weycker et al. and Huybrechts et al. The most likely explanation for this is that our and Siris\u2019 study population was restricted to bisphosphonate users, while the others also included users of other, less potent anti-osteoporotic drugs (30% to 65% of the study population).\nThere is a previous PHARMO study on bisphosphonate use and risk of fractures, using persistence [5]. Besides a different measure of drug exposure, the current study differs from this previous study in that the study period was extended, different inclusion criteria were applied and new patients were included besides previously studied patients. Although persistence and compliance are measured differently, with persistence referring to the duration of uninterrupted drug use, they are closely related. Patients classified as non-compliant would also have been classified as non-persistent and vice versa. The results of our current study are in line with our previous study in which the risk of fractures was reduced by 26% after one year persistent bisphosphonate use and up to 32% after two years of persistent use [5]. Similarly, Gold et al [23] reported that persistent bisphosphonate use for at least 6\u00a0months was associated with a 26% lower fracture risk. Expressing these results the other way around, non-persistent bisphosphonate use is associated with an about 40% fracture risk increase. Among a population of mainly users of hormone replacement therapy, one year of uninterrupted therapy reduced the likelihood of fractures with significant odds ratios of 0.38 for hip fractures and 0.60 for vertebral fractures [7].\nOur analyses may be limited by some issues common to observational database research in daily clinical practice. Most importantly, although we considered a large number of known confounders in our analyses, including history of fractures, there may be other confounding factors for which information was not available, such as bone mineral density (BMD). BMD is an important determinant of fracture risk [24] and BMD testing has been shown to be positively associated with persistence and compliance [25, 26]. Consequently, the results of our analyses are likely to be conservative in the sense that compliant patients may have had an increased fracture risk because of low BMD, and history of fractures only partially controlled for this. Furthermore, it is possible that patients have started using other anti-osteoporotic drugs, e.g. raloxifene, during follow-up and stopped bisphosphonate use. This means that part of the patients who were non-compliant with bisphosphonates may have been protected against fractures by another anti-osteoporotic drug and the observed increased fracture risk with non-compliant bisphosphonate use therefore may be underestimated. However, we know from our database that this concerns only about 7% of patients in the first year of bisphosphonate treatment. In addition, compliance with treatment was based on dispensing data. As it is unknown whether a patient actually used the drug, compliance may have been overestimated. However, it seems likely that patients who obtain prescription refills do take their medication. Excluding women who filled only one bisphosphonate dispensing during follow-up from the analyses did not change the results. Another limitation relates to the classified analysis. With the use of five compliance categories studying a trend is possible but it does not give information on the point at which there is a significant shift in fracture risk. Ideally, 10% MPR categories should be used. However, patient numbers per 10% MPR categories (data not shown) did not allow for this detailed analysis.\nTo increase the treatment benefit of bisphosphonates, compliance should be improved. Related to the inconvenient, stringent intake regimen, dosing frequency is an important determinant of compliance with bisphosphonates. In recent earlier studies, once-weekly dosing was associated with better compliance and persistence compared to daily dosing. However, even with the weekly regimen, compliance and persistence were suboptimal in about half of the patients [6, 8, 9]. Bisphosphonates with less frequent dosing regimes than weekly, e.g. monthly or three-monthly ibandronate or annual zolendronate, are either available yet or upcoming and may further improve compliance and therefore treatment benefit. In a cross-over trial, women with postmenopausal osteoporosis preferred once-monthly ibandronate therapy and found it more convenient than once-weekly alendronate therapy [27]. There are no data yet on the actual impact of this once-monthly, and other formulations on compliance with bisphosphonate therapy and reduction of fracture risk in daily practice. Other keys to improve compliance are e.g. healthcare provider - patient communication and continuous reinforcement of the importance of treatment. Reminder systems may help the patient comply with therapy, especially with infrequent dosing regimes. However, results from a review show that current methods of improving patient compliance for several chronic health problems are mostly not very successful [28]. Furthermore, although a number of demographic and clinical variables are associated with compliance with bisphosphonates these predictors, either individually or together, have been shown to have low predictive value for identifying patients who will become non-compliant [25].\nIn conclusion, the results of this study show a statistically significant association between level of compliance with bisphosphonates and level of fracture risk, emphasizing the importance of treatment compliance in obtaining maximal bone protection.","keyphrases":["benefit","bisphosphonates","non-compliance","fracture","osteoporosis"],"prmu":["P","P","P","P","P"]}
{"id":"Skeletal_Radiol-3-1-2042033","title":"Whole-body imaging of the musculoskeletal system: the value of MR imaging\n","text":"In clinical practice various modalities are used for whole-body imaging of the musculoskeletal system, including radiography, bone scintigraphy, computed tomography, magnetic resonance imaging (MRI), and positron emission tomography-computed tomography (PET-CT). Multislice CT is far more sensitive than radiographs in the assessment of trabecular and cortical bone destruction and allows for evaluation of fracture risk. The introduction of combined PET-CT scanners has markedly increased diagnostic accuracy for the detection of skeletal metastases compared with PET alone. The unique soft-tissue contrast of MRI enables for precise assessment of bone marrow infiltration and adjacent soft tissue structures so that alterations within the bone marrow may be detected before osseous destruction becomes apparent in CT or metabolic changes occur on bone scintigraphy or PET scan. Improvements in hard- and software, including parallel image acquisition acceleration, have made high resolution whole-body MRI clinically feasible. Whole-body MRI has successfully been applied for bone marrow screening of metastasis and systemic primary bone malignancies, like multiple myeloma. Furthermore, it has recently been proposed for the assessment of systemic bone diseases predisposing for malignancy (e.g., multiple cartilaginous exostoses) and muscle disease (e.g., muscle dystrophy). The following article gives an overview on state-of-the-art whole-body imaging of the musculoskeletal system and highlights present and potential future applications, especially in the field of whole-body MRI.\nIntroduction\nThe skeletal system is a frequent target of metastatic spread from various primary tumors like carcinoma of the breast, lung and prostate cancer. Moreover, primary malignancies may also originate from the bone marrow, such as lymphoma and multiple myeloma [1]. Therefore, it is highly important to accurately assess manifestations of malignant diseases within the bone marrow in order to facilitate adequate therapy and predict prognosis.\nOnly pronounced destruction of bone with loss of bone mineral content exceeding 50% is readily visible in radiographic examinations [2]. Computed tomography (CT) is definitely more sensitive than radiography and it is the image modality of choice to evaluate the extent of destruction of trabecular and cortical bone and to assess stability and fracture risk. Magnetic resonance imaging (MRI), on the other hand, allows bone marrow components, such as hematopoietic and fat cells, to be visualized. Moreover, tumor infiltration into the spinal canal and paravertebral soft tissues is clearly depicted. Compared with other imaging modalities like radiography, CT or bone scintigraphy, it is the most sensitive technique for the detection of pathologies restricted to the bone marrow, even if trabecular bone is not destroyed [3, 4]. It has been reported that up to 40% of skeletal metastases occur outside the field of view covered by a routine assessment of the axial skeleton, underlining the importance of whole-body bone marrow imaging [5]. In the past, different requirements for patient positioning and coil set-up complicated the introduction of MRI as a practicable whole-body application. With multi-channel whole-body MRI (WB-MRI) scanners, however, head-to-toe assessment of the whole skeletal system has become a realistic option without compromises in image quality compared with dedicated examinations of limited anatomical areas. Beyond the assessment of malignant bone neoplasms, WB-MRI has recently been proposed for the whole-body imaging of systemic muscle disease and may prove useful for an evidence-based screening of patients suffering from diseases that predispose to bone malignancy (e.g., multiple cartilaginous exostoses).\nTechnical aspects of whole-body MRI\nDue to its lack of ionizing radiation MRI seems suitable for whole-body imaging, but for a long time its primary application has been the assessment of focal pathologies within particular organs and body parts. The most severe challenges of WB-MRI in the past have been long examination times, mainly caused by time-consuming patient repositioning and changing of the array configuration. Initially, the sequential scanning approach for WB-MRI of the skeletal system consisted of separate scanning steps of T1-weighted and STIR (short tau inversion recovery) imaging at five body levels with at least one patient repositioning process using conventional head, neck, body, and spine array coils. For a complete whole-body examination, including dedicated imaging of the spine in sagittal orientation, a total room time of at least 60\u00a0min had to be taken in account. Steinborn et al. introduced for the first time this whole-body bone marrow scanning concept for the screening of bone metastases and, despite the considerable complexity of the examination, reported advantages in diagnostic accuracy for MRI compared with conventional WB imaging techniques, like skeletal scintigraphy [6]. Later, attempts to overcome FOV restrictions and increase patient comfort were based on a rolling platform concept mounted on top of the scanner table, making the patient glide in between a \u201ccoil sandwich\u201d comprised of the body coil and the integrated spine coil (AngioSURF\u2122\/BodySURF\u2122; MR-Innovation, Essen, Germany). However, with this approach considerable compromises in spatial resolution, especially in peripheral body regions like the head\/neck and lower extremities, had to be tolerated [7]. With the introduction of multi-channel MR scanners, using a system of multiple phased array coils covering the whole body like a matrix, imaging of the total skeletal system without compromises in spatial resolution became possible. In particular, the combination of free table movement with parallel imaging acquisition techniques (PAT) resulted in substantially shorter room time and allowed to integrate otherwise time-consuming, but indispensable sequence types for bone marrow imaging (e.g., STIR sequences). The proposed imaging protocol for high resolution T1-weighted turbo spin echo (TSE) and STIR imaging from head to toe is performed with 1.3\u2009\u00d7\u20091.1-mm and 1.8\u2009\u00d7\u20091.3-mm plane resolution respectively (5-mm slices, matrix 384, PAT 2\u20133). Additionally, dedicated T1-weighted TSE and STIR imaging of the complete spine is performed (1.0\u2009\u00d7\u20091.0-mm plane, 3-mm slices, matrix 384), which results in a total scan time of 43\u00a0min.\nA promising new concept for WB-MRI is the continuously moving table technique with the use of PAT. Recently, a SENSE reconstruction algorithm has been successfully applied on stationary receiver coils with arbitrary coil dimensions for continuously 3D gradient echo imaging of the complete body without significant constraints in image quality [8]. Zenge et al. have reported promising initial results for 3D whole-body continuous data acquisition using the rolling table concept as a new potential strategy for WB-MRI metastases screening, especially for large field-of-view imaging in short bore systems [9].\nTechnical aspects of whole-body CT\nMultislice CT (MS-CT) is frequently used in oncologic imaging, and in the detection of bone destruction CT is far more sensitive than radiography [10]. Moreover, it is unique in its ability to evaluate the extent of osseous destruction and to assess stability and fracture risk [11]. Whole-body imaging usually implies CT of the neck, thorax, abdomen\/pelvis in axial orientation with multi-planar reconstructions and recalculation of bone window setting. Continuous hardware improvements for CT imaging from single-slice scanners to 64-slice scanners have resulted in larger FOV and faster acquisition times with high resolution. Examination time now is reduced to 1\u20132\u00a0min and improvement in resolution with the resulting formation of isotropic voxels allows high quality multiplanar reconstructions. In particular, the use of ultrathin collimation (0,5\u00a0mm) can provide excellent image quality for the neck and peripheral skeleton. Furthermore, with automated tube current dose modulation systems exposure of the patient to ionizing radiation could be reduced by 10\u201368% on average, depending on the anatomical region, without substantially sacrificing image quality [12]. Absolute dose reduction (described as volume CT dose index) ranges from 11.97\/8.18\u00a0mGy for a pelvic examination to 23.28\/7.45\u00a0mGy when a combined angular and z-axis modulation system is used.\nMultislice CT enables bone destruction to be detected as well as osteoblastic and mixed patterns with osteosclerosis and destruction. In cases of diffuse bone marrow infiltration inhomogeneous osteoporosis may be detected. However, as in radiography, this may mimic osteoporosis, unless areas of bony destruction are present. 3D reconstruction algorithms, like VRT (volume rendering technique), can be used to display complex fractures (e.g., pathological fracture) and further increases the reliability to detect occult vertebral fractures compared with plain radiographs. Furthermore, the 3D morphology of vertebral fractures may give indications concerning the nature of the fracture (e.g., tumorous vs. osteoporotic fracture).\nClinical application of whole-body MRI\nWhole-body MRI is increasingly used in the field of oncologic imaging as an adjunct or alternative to established multi-modality approaches (e.g., radiographs, MS-CT, ultrasound, scintigraphy) for initial tumor staging or screening for tumor recurrence after curative therapy. Promising results have been reported for the detection of distant metastatic disease, especially in tumors that frequently metastasize to the bone, liver, and brain (Fig.\u00a01) [13, 31]. Recently, WB-MRI has been proposed as a sensible application for a more integrated assessment of multiple myeloma and systemic muscle diseases [16, 17, 18, 19].\nFig.\u00a01A 60-year-old patient with a malignant melanoma. a Coronal T1-weighted turbo spin echo (TSE) whole-body (WB) magnetic resonance imaging (MRI) indicates multifocal metastatic disease of the liver (arrow). b Whole-body positron emission tomography-computed tomography (PET-CT) shows multiple areas of pathologic [18F]-fluorodeoxyglucose (FDG)-uptake in the right atrium (arrow), the liver (arrowheads), and in the right iliac bone (arrowhead). c Contrast-enhanced CT reveals a metastasis within the right atrium. d T1-weighted TSE WB-MRI confirms a hypointense lesion in the right iliac bone indicating bone metastasis. e Axial fat-saturated contrast-enhanced MRI of the pelvis shows another bone metastasis in the right sacral bone (arrow). f, g The corresponding CT images in the bone window setting show extensive osteolysis within the right iliac bone; however, no morphologic changes are found in the right sacral bone\nMetastasis\nBased on morphological criteria in radiography, CT, and MRI, skeletal metastases are classified as osteolytic (approximately 50%), osteoblastic (35%) ,and mixed type (15%). For MRI bone screening, the combination of unenhanced T1-weighted spin echo and turbo-STIR sequences proved to be highly sensitive in discriminating benign from malignant marrow disorders [20]. On T1-weighted sequences tumor spread is identified by replacement of normal fat containing marrow, resulting in a hypointense signal . Fat-suppressed sequences, such as STIR, depict neoplastic lesions by virtue of the hyperintense signal due to increased content of water within the tumor cells (Fig.\u00a02). However, osteoblastic metastases may be depicted in STIR sequences with variable signal intensities from hypointense in dense sclerotic lesions to hyperintense when more cellular components are present [21]. The unique soft-tissue contrast of MRI allows precise assessment of tumor infiltration within the bone marrow and even diffuse infiltration of the bone marrow with neoplastic cells, not associated with focal bone destructions or formation of new bone, is detected [22]. In some cases additional sequences, such as dynamic studies of signal enhancement after gadolinium injection, may be performed for accurate differentiation from benign bone marrow changes, such as hyperplastic bone marrow formation [23]. Finally, MRI allows for precise assessment of adjacent paraosseous structures, such as the spinal canal.\nWhole-body MRI compared with bone scintigraphy\nIn clinical practice multi-modality algorithms are most commonly used when metastatic bone disease is suspected. They include radiography, bone scintigraphy, PET, CT, and MRI. In many institutions 99mTc-phosphonate-based bone scanning is performed as the method of initial bone marrow screening. However, scintigraphy provides only limited spatial resolution and at an early stage of disease lesions may remain invisible in the absence of an osteoblastic response [24]. Furthermore, misinterpretation of tracer uptake in healing fractures or degenerative disease may lead to false-positive findings. The diagnostic performance of MRI compared with bone scintigraphy for the detection of skeletal metastases has been examined in various studies and higher specificity and sensitivity in the early detection of skeletal metastases have been reported [6, 24, 25]. Steinborn et al. compared combined T1-weighted and STIR WB-MRI with bone scintigraphy using the sequential scanning approach in a lesion-by-lesion analysis [6]. WB-MRI reliably detected more confirmed skeletal metastases (91%) than bone scintigraphy (85%). Another study group reported higher sensitivity and specificity of WB-MRI (92%\/90%) compared with scintigraphy (83%\/80%) on a patient-by-patient basis [20]. Daldrup-Link et al. analyzed both methods for the detection of bone metastases in 39 children and young adults and observed a higher sensitivity of 82% for WB-MRI compared with 71% for scintigraphy, with this difference increasing, especially in medium-sized lesions between 1 and 5\u00a0cm [3].\nInterestingly, both methods showed differences in performance according to lesion location. Most false-negatives in scintigraphy were found in the spine, while diagnostic problems for WB-MRI occur in the thoracic cage and skull, especially when coronal imaging orientation is used, a problem that is certainly increased by motion artefacts. These problems might be overcome when using fast turbo spin echo sequences for thoracic imaging in combination with axial slice orientation.\nWhole-body MRI compared with MS-CT\nMultislice CT is far more sensitive than radiography in the assessment of focal bone destructions. Krahe et al. compared radiography and CT examinations in 112 patients with metastases of the spine [10]. MS-CT identified 268 involved vertebrae while radiography depicted 88% of these lesions when the vertebral body was involved and only 66% when other parts of the vertebrae were affected. Intraspinal and paravertebral tumor extension was correctly assessed by plain radiography in only 23% and 33% of cases respectively. Recently, multidetector MS-CT has been proposed for whole-body screening of the skeletal system as an alternative to bone scintigraphy [26]. Groves et al. investigated 43 patients with suspected bone metastases using bone scintigraphy and 16-detector MS-CT and detected metastatic disease in 14 out of 43 and 13 out of 43 patients respectively, with an agreement of 84%, suggesting a similar diagnostic performance of both modalities. The authors concluded that in case of suspected skeletal metastasis, scintigraphic studies may not be needed, when an adequate whole-body MS-CT tumor staging with recalculation of bone window setting has been performed. Consecutively, this might shorten diagnostic pathways and save resources. However, it is not yet clear whether MS-CT is equal to MRI for the assessment of bony metastases. First results have demonstrated the superior detection rate of MRI (Fig.\u00a01) [27].\nWhole-body MRI compared with PET-CT\nIn contrast to MRI and MS-CT, positron emission tomography (PET) using [18F]-fluorodeoxyglucose (FDG) provides functional information by tracing increased FDG uptake directly into the tumor cells. Recent studies indicate that whole-body FDG-PET increases specificity of bone screening compared with bone scintigraphy, although there is conflicting evidence whether there is a significant gain in sensitivity [28]. Still, FDG is a tracer that is not tumor-specific and may also accumulate in the presence of inflammation and thus lead to false-positive findings. Also, FDG is not suitable for several tumor entities due to poor tracer uptake, e.g., prostate cancer, myxoid tumors of the gastrointestinal tract, low-grade sarcomas or renal cell carcinomas [29]. A clear technical disadvantage of PET, similar to scintigraphy, is its poor anatomical resolution, which often makes the exact localization of a lesion difficult. Fused PET-CT scanners combine the functional data of PET with the detailed anatomical information of MS-CT scanners in a single examination and have further improved diagnostic accuracy. Various authors have reported a significant decrease in ambiguous lesions and an improvement in the specificity of PET-CT compared to that of PET alone in the detection of malignant disease [14, 30].\nOnly a few study groups have directly compared the performance of WB-MRI with FDG-PET-CT in the detection of skeletal metastases [15, 31]. Antoch et al. analyzed the accuracy of both modalities in 98 patients in terms of TNM-based tumor staging. Both imaging procedures revealed a similar diagnostic sensitivity to the detection of distant metastases (WB-MRI\u2009\u200993%, PET-CT 94%). Regarding bone metastases, the sensitivity was significantly higher when using WB-MRI (85%) instead of PET-CT (62%). Our own observations confirm these data. We examined 41 patients with suspected skeletal metastases with both FDG-PET-CT and WB-MRI using a multi-channel scanner with PAT [31]. In a lesion-by-lesion analysis 102 malignant and 25 benign bone lesions were confirmed by histology or follow-up. WB-MRI showed a significantly higher diagnostic accuracy than PET-CT (91% vs. 78%, Figs.\u00a01, 2). In particular, lesions smaller than 5\u00a0mm were visualized by WB-MRI with a cut-off size of 2\u00a0mm for WB-MRI compared with 5\u00a0mm for PET-CT. Lesions less than double the size of the spatial resolution of the PET scanner (usually 6\u00a0mm) can especially lead to false-negative results. Additionally, 10 bone metastases in distal parts of the body were revealed by WB-MRI due to the larger field of view (the FOV of a routine PET-CT is restricted to the diagnostic spiral CT, usually ranging from the skull base to the proximal femora). However, specificity was higher in PET-CT (PET-CT 80% vs. WB-MRI 76%). Here, certainly, the additional metabolic information of PET plays the most important role in reliably discriminating between malignant and benign lesions (e.g., atypical haemangioma) [32].\nFig.\u00a02A 28-year-old man with non-Hodgkin\u2019s lymphoma. a Coronal whole-body STIR imaging shows suspect areas with hyperintense signal in the right clavicular region and both iliac bones. b Magnification shows a mass extending from the right clavicular fossa and infiltrating the right apical thoracic cage. c The lesion shows a pathological FDG uptake in PET-CT, indicating malignancy. d The T1-weighted sagittal image of the spine shows extensive multifocal lymphoma manifestations. e PET-CT underestimates the degree of tumor involvement by showing tracer uptake only in the thoraco-lumbar region\nOn the other hand, at least in breast cancer, different patterns of FDG uptake have been reported in osteoblastic, osteolytic or mixed lesions, indicating that sclerotic lesions may be less FDG-avid [33]. The additional morphologic information of PET\/CT compared with PET alone is certainly of great value in increasing diagnostic sensitivity. Also, significant improvement in diagnostic accuracy has been reported when 18F-fluoride is used for PET or PET-CT for the assessment of malignant skeletal disease. 18F-fluoride is a tracer that, similar to 99mTC-diphosphonate, specifically adsorbs onto bony surfaces with a predilection for sites of active bone formation [30].\nAn important indication in bone imaging is lesion monitoring after chemotherapy or radiation therapy. It has to be taken into account that on MRI necrotic bone metastases may remain virtually unchanged in morphology or signal characteristics, which may make evaluation of therapy response difficult. Compared with viable tumors, the contrast enhancement is frequently less pronounced and slower in tumors that have responded to a particular therapy. Reduction in tumor size may be delayed and is not a sensitive sign in the assessment of response. Tumor metabolism, and consequently FDG uptake, is highly susceptible to chemotherapy. On the CT image, osteolytic metastases often demonstrate typical sclerotic transformation. In this setting, future studies are needed to evaluate the sensitivity of FDG-PET-CT in assessing the response to chemotherapy and whether sensitivity is reduced in the first days after initiation of therapy. After radiation therapy MRI is particularly helpful, because irradiated lesions are easily distinguishable from new lesions because of the high signal of normal bone marrow on T1-weighted SE images, which is due to conversion of hematopoietic into fatty bone marrow.\nMultiple myeloma\nMultiple myeloma is a frequent neoplastic disease affecting the bone marrow. It is characterized by expansive growth of malignant plasma cell clones with consecutive destruction of the bony architecture. Predilection sites are the axial skeleton (spine and pelvis), but also the ribs, the shoulder region, skull, and proximal femurs, stressing the need for total body imaging for adequate assessment of the extent of disease. The bone marrow may either be diffusely infiltrated or there may be focal accumulations of atypical plasma cells (Fig.\u00a03).\nFig.\u00a03A 70-year-old man with multiple myeloma. a The radiograph of the pelvis is inconspicuous. b Coronal MS-CT reconstruction of the pelvis in a bone window setting reveals a large area of destruction within the left iliac bone (arrow). c) STIR-WB-MRI confirms focal tumor manifestation within the iliac bone (arrow) and reveals multiple small nodular infiltrations within the sacral bone and pelvis. d MS-CT of the spine shows a compression fracture of Th9. e, f T1-weighted SE- and STIR imaging of the spine reveals diffuse myeloma infiltration of the spine. g Coronal STIR sequences of the pelvis show additional focal infiltration of the left femoral head missed on radiography and MS-CT\nIn patients with myeloma the basic diagnostic work-up in many institutions includes radiographic examinations of the skull (two planes), the rib cage, the upper arms, the spine (two planes), the pelvis, and the upper legs. This diagnostic approach is still represented in the classic Salmon and Durie staging system of the disease, which includes radiographic, immunohistochemical, and serological factors of the disease, and defines the selection of adequate therapy [34]. However, diagnostic sensitivity of radiography in the detection of myeloma manifestations is rather low and thus allows diagnosis only at advanced stages of the disease when at least 50% of the bone mineral content has been lost [2]. In particular, a diffuse bone marrow infiltration pattern may cause diagnostic problems as it can easily be misdiagnosed as senile osteoporosis. In a study conducted by Baur et al. 55% of focal and 59% of diffuse infiltrations were missed by radiography [35]. With the use of contrast-enhanced sequences and calculation of percentage signal increases the sensitivity of MRI for diffuse myeloma infiltration, especially at earlier stages, can be further increased [36].\nSchreiman et al. examined 32 patients with multiple myeloma using single-row CT and radiography [37]. Twelve patients showed osseous affections in both modalities. CT, however, usually demonstrated a more extensive involvement and in 6 out of 13 patients only CT detected myeloma involvement despite normal radiographs. Another study group recently proposed FDG-PET-CT as an alternative to radiographic imaging of the whole body [38]. PET-CT detected more lesions in 16 out of 28 patients (57%) and 9 of these patients had completely negative radiography.\nGhanem et al. compared WB-STIR-MRI using the conventional radiographic skeletal survey in 54 patients with plasma cell neoplasms [16]. Whole-body MRI correctly revealed bone marrow infiltration in 74% of patients, while radiography depicted pathological changes in 55% of patients. Moreover, WB-MRI showed a larger extent of infiltration in 90% of concordant findings.\nIn two studies, the diagnostic accuracy of MS-CT and MRI was analyzed. Mahnken et al. compared results of four-detector MS-CT and MRI examination of the spine and pelvis in 18 patients with stage III multiple myeloma [39]. A total number of 325 vertebral bodies were evaluated. In MS-CT 231 vertebral bodies were classified as \u201caffected\u201d, compared with only 224 vertebral bodies in MRI. These \u201cfalse-negative\u201d findings might have been a consequence of response to previous treatment. However, no information about previous therapy was provided in this study. On the other hand, MRI showed 5 affected vertebrae that were considered normal on MS-CT. The false-negatives on MS-CT might be due to early bone marrow infiltration without signs of osseous destruction. Furthermore, the surprisingly high sensitivity of MS-CT in this study may also be caused by pre-selection of a patient group with advanced disease.\nIn our own study, 30 patients with multiple myeloma were examined with a 1.5-Tesla multi-channel WB-MRI scanner and a 16- and 64-detector MS-CT scanner [17]. MRI showed superior diagnostic accuracy to MS-CT. In particular, the diagnostic sensitivity of MS-CT was inferior to that of WB-MRI (Fig.\u00a03). The high amount of false-negatives on MS-CT may be explained by the fact that early stages of the disease can be visualized on MRI, displaying bone marrow replacement before any destruction of trabecular and cortical bone occurs. False-positive results on MS-CT, on the other hand, may be due to misinterpretation of inhomogeneous osteoporosis as diffuse myeloma infiltration.\nBaur et al. were able to show that an extended staging system, including MRI of the spine, has a significant influence on the assessment of prognosis. Using the classic staging system of Durie and Salmon without MRI, 25 out of 77 patients would have been understaged, thus underlining the importance of incorporating the MRI bone marrow status into staging and therapy planning of this disease [40].\nMuscle disease\nDiagnostic imaging of muscle disease (e.g., polymyositis or muscle dystrophy) represents a challenge as it requires high-resolution whole-body coverage of soft tissue structures to adequately assess the pattern, distribution, and extent of the disease. CT, scintigraphy, and ultrasound are not suitable for this purpose as all these modalities lack sufficient soft tissue contrast, spatial resolution or large FOV imaging options. Other diagnostic tests frequently used, like electromyography, are unspecific and muscle biopsies used as the gold standard have been reported to be false-negative in 10\u201325% of cases due to sampling errors [41]. MRI, with its precise delineation of fat, muscle, and bone is an ideal candidate for imaging of systemic muscle disease. Moreover, substantial dose exposure in a predominantly younger patient cohort commonly affected by muscle diseases can be avoided. Previously, MRI has been used for a targeted assessment of muscle disease [41, 42]. A combination of T1-weighted SE and T2-weighted pulse sequences with fat saturation, such as STIR, are indispensable for assessing increased fat and water content within the muscle tissue respectively (Fig.\u00a04). Contrast enhancement with Gadolinium chelates also adds to diagnostic accuracy, especially when combined with fat suppression. Since various muscle groups and body parts may be involved in an unpredictable distribution, coverage of the whole body is important.\nFig.\u00a04a Whole-body MRI of a 60-year-old man suffering from severe progressive muscle dystrophy, symmetrically affecting the muscles of the rib cage, abdomen, pelvis, and lower extremities. b WB-STIR shows signs of diffuse reactive edematous processes in the left upper thighs and muscles of the distal lower limbs. c, d T1-weighted imaging displays fatty tissue replacement affecting most of the muscles of the pelvis. Residual adductor muscle tissue is shown with high contrast due to its hypointense signal. e Progressive muscle dystrophy has also occurred in the lower extremities, affecting all compartments\nO\u2019Connell et al. previously described WB-STIR imaging for the diagnosis of patients suffering from polymyositis [18]. With the use of parallel imaging techniques WB-STIR imaging can be performed in under 13\u00a0min at an inplane resolution of 1.3\u2009\u00d7\u20091.1\u00a0mm. With this method, symmetry of muscle involvement and the extent and severity of inflammation can be analyzed, so that an adequate target for biopsy can be identified. During therapy, WB-MRI is particularly helpful in monitoring the disease outcome by visualizing the muscle-to-fat tissue relation or to verify a decrease in inflammation. Also, various drugs, such as steroids, may have side effects that involve the muscles and result in various types of myopathy. Lenk et al. proposed coronal and axial WB-STIR imaging in combination with coronal T1-weighted SE imaging and sagittal T1-\/T2-weighted imaging of the spine, as a sensible protocol for systemic muscle disease, resulting in an approximate total scan time of 45\u00a0min [19].\nHowever, assessment of distal parts of the upper extremity may be limited. This is due to positioning of the arms on the pelvis and thighs. If the muscles of the forearm are in the focus of interest, additional examination with dedicated coil systems may be required.\nPatients with muscular dystrophy, an X-chromosome recessive disease, suffer from progressive destruction of muscle tissue with subsequent replacement by fatty and fibrous tissue. In addition to the morphological information concerning the distribution and extent of replacement of muscle by fatty tissue as provided by WB-MRI, exact quantification of the muscle-to-fat relation is of substantial diagnostic and prognostic importance. In particular, T1-weighted axial WB-MRI in combination with CAD (computer aided diagnostics) applications may be useful for the quantification of total body fat distribution. Interesting results have been reported by Brennan et al. who introduced a fast, automated approach to body fat measurement in healthy individuals as a useful alternative to the body mass index [43].\nFuture applications\nEustace et al. have proposed WB-MRI for trauma assessment in patients referred for suspected child abuse [44]. For a rapid assessment of acute trauma, especially when multiple organs are affected, MS-CT provides fast and detailed systemic information on potentially life-threatening organ, vessel or bone injury and is the imaging technique of choice. Recently, an accelerated triage MS-CT protocol has been proposed by Koerner et al. for time-effective handling of mass casualty incidents using MS-CT. For a scenario of 15 multiple trauma patients admitted over a period of 2\u00a0h within a mass casualty incident a mean total time in the scanner room of 8.9\u00a0min was calculated, including patient preparation and image reconstruction [45].\nFinally, there may be potential for WB-MRI in the secondary screening of congenital skeletal diseases predisposing to malignancies, like multiple cartilaginous exostoses or histiocytosis X (Fig.\u00a05). However, only case reports have been published on the potential benefits or therapeutic impact in these specific patient groups.\nFig.\u00a05A 35-year-old man with multiple cartilaginous exostoses. a, b Whole-body MRI shows typical manifestations in the metaphyseal parts of the long bones of the upper and lower extremities. c Enlargement of the left knee joint shows an exostosis at the medial side of the femur and at the proximal tibia. d Deformation of the radius. e Exostoses and deformation of both femoral necks. In summary, no indications of malignant transformation (e.g., widening of the cartilaginous cap) were found in this patient\nConclusion\nWhole-body imaging is increasingly successfully applied in musculoskeletal imaging, especially in the field of systemic malignant diseases affecting the bone and in diseases predisposing to malignant transformation. Whole-body MRI is able to depict bone marrow pathologies with high resolution and excellent soft tissue contrast by demonstrating signal alterations due to changes in its fat, water, and hematopoietic cell components with high resolution and excellent soft tissue contrast. Together with CT or PET-CT and its valuable additional metabolic information, it has great potential in the more comprehensive, more accurate, and earlier diagnosis of musculoskeletal diseases. Although further evaluation of the true potential of whole-body applications is awaited, they are promising tools aiding the more efficient management of patients suffering from systemic malignant or benign diseases of the soft tissue and bone.","keyphrases":["whole-body","imaging","musculoskeletal","magnetic resonance imaging"],"prmu":["P","P","P","P"]}
{"id":"Cell_Tissue_Res-3-1-2039796","title":"Effect of thrombin peptide 508 (TP508) on bone healing during distraction osteogenesis in rabbit tibia\n","text":"Thrombin-related peptide 508 (TP508) accelerates bone regeneration during distraction osteogenesis (DO). We have examined the effect of TP508 on bone regeneration during DO by immunolocalization of Runx2 protein, a marker of osteoblast differentiation, and of osteopontin (OPN) and bone sialoprotein (BSP), two late markers of the osteoblast lineage. Distraction was performed in tibiae of rabbits over a period of 6 days. TP508 (30 or 300 \u03bcg) or vehicle was injected into the distraction gap at the beginning and end of the distraction period. Two weeks after active distraction, tissue samples were harvested and processed for immunohistochemical analysis. We also tested the in vitro effect of TP508 on Runx2 mRNA expression in osteoblast-like (MC3T3-E1) cells by polymerase chain reaction analysis. Runx2 and OPN protein were observed in preosteoblasts, osteoblasts, osteocytes of newly formed bone, blood vessel cells and many fibroblast-like cells of the soft connective tissue. Immunostaining for BSP was more restricted to osteoblasts and osteocytes. Significantly more Runx2- and OPN-expressing cells were seen in the group treated with 300 \u03bcg TP508 than in the control group injected with saline or with 30 \u03bcg TP508. However, TP508 failed to increase Runx2 mRNA levels significantly in MC3T3-E1 cells after 2\u20133 days of exposure. Our data suggest that TP508 enhances bone regeneration during DO by increasing the proportion of cells of the osteoblastic lineage. Clinically, TP508 may shorten the healing time during DO; this might be of benefit when bone regeneration is slow.\nIntroduction\nDistraction osteogenesis (DO) is a well-established technique, originally developed in orthopedic surgery for bone lengthening (Kojimoto et al. 1988) and later also used to treat hereditary malformations in the craniomaxillofacial region (Amir et al. 2006). DO is characterized by the formation of new bone between two osteotomized bone segments, which are separated by gradual traction. One of the clinical phases of the distraction technique is the bone consolidation period, defined as the time between the end of active distraction and the removal of the distraction device. It represents the time needed for a complete bridging of the distraction gap by bone and a further maturation of this bone. Typically, the bone consolidation phase takes approximately 6\u201312\u00a0weeks in the craniomaxillofacial region and 3\u20136\u00a0months in long bones (Fischgrund et al. 1994; Smith et al. 1999; Felemovicius et al. 2000; Amir et al. 2006). Although DO is believed to be superior to other bone augmentation techniques, many attempts have been made to improve the technique to enhance the bone regeneration process in the distraction gap (Hagiwara and Bell 2000; Li et al. 2002; Mofid et al. 2002; Takamine et al. 2002; Schortinghuis et al. 2005). Such improvements would provide the opportunity to shorten the bone consolidation period and hence minimize complications such as the development of non-union, infection or fracture. Various methods have been tested to promote bone formation in the distraction gap, e.g. electrical and mechanical stimulation (Hagiwara and Bell 2000; Mofid et al. 2002), transplantation of osteoblast-like cells (Takamine et al. 2002) or administration of growth factors such as bone morphogenetic proteins (Li et al. 2002) or fibroblast growth factor 2 (FGF-2; Okazaki et al. 1999). Another procedure to accelerate bone regeneration involves the application of thrombin-related peptide 508 (TP508; Ryaby et al. 2000; Sheller et al. 2004; Li et al. 2005a,b; Wang et al. 2005).\nTP508 is a synthetic peptide consisting of 23 amino acids, which represent the natural amino acid sequence of the receptor-binding domain of human thrombin (pro-thrombin amino acids 508\u2013530; Sower et al. 1999; Stiernberg et al. 2000). Thrombin is an important factor in blood homeostasis, inflammation and wound healing (Coughlin et al. 1992). It plays a role in the formation of fibrin clots and platelet activation. Thrombin also stimulates the cell proliferation, migration and\/or differentiation of various cell types, such as fibroblasts, endothelial cells and lymphocytes. All these cell types contain one or more thrombin receptors (Chen and Buchanan 1975; Belloni et al. 1992). TP508 appears to mimic and accelerate many of the effects of native thrombin in initiating wound healing through a non-proteolytic pathway (Sower et al. 1999). Histological studies have indicated that TP508 is chemotactic for neutrophils, lymphocytes and monocytic cells; it enhances the formation of granulation tissue, neovascularization and other wound-healing events (Sower et al. 1999; Ryaby et al. 2000; Naldini et al. 2004).\nTP508 has also been tested for its potential to enhance bone formation (Ryaby et al. 2000; Sheller et al. 2004; Li et al. 2005a,b; Wang et al. 2005). A single injection of 1\u00a0\u03bcg TP508 accelerates fracture healing in rat long bone fracture. The breaking strength of bones of young and aged rats injected with TP508 has been enhanced over that of controls (Ryaby et al. 2000). One recent study of TP508 treatment during DO has revealed a significantly greater bone mineral density in TP508-treated groups than in saline-treated controls (Li et al. 2005a,b). Histological analysis has also shown advanced bone consolidation and remodeling after TP508 treatment.\nIn the present study, we report the effect of TP508 in the regulation of bone regeneration during DO in more detail. We have examined Runx2\/Cbfa1, osteopontin (OPN) and bone sialoprotein (BSP) protein expression in tissue sections of rabbit long bones undergoing DO. Runx2 is an essential transcription factor for osteoblast differentiation and for extracellular matrix gene expression (Komori et al. 1997; Karsenty et al. 1999). In Runx2 knockout mice, both intramembranous and endochondral ossification are blocked because of the maturational arrest of osteoblasts (Komori et al. 1997). Runx2 can directly stimulate the transcription of the collagen type I and osteoblast-related genes, such as those for OPN and BSP, both major non-collagenous proteins found in the extracellular matrix of bone (Karsenty et al. 1999). We have hypothesized that TP508 treatment during DO enhances the expression of Runx2, OPN and BSP.\nMaterials and methods\nAnimal model of DO\nThe paraffin blocks containing distracted rabbit long bones were those prepared previously (Li et al. 2005a,b) from 30 adult male New Zealand White rabbits (age 24\u00a0weeks, body weight 2.6\u20133.5\u00a0kg). External fixators were applied to stabilize the osteotomized mid-tibia for 7 days of the latency period and were activated for 6\u00a0days, once a day at a rate of 1.4\u00a0mm. This distraction rate was intentionally chosen in order to copy the poor bone healing condition as previously described (Li et al. 1997).\nDuring the experiment, seven animals were excluded in the study because of soft tissue complications (n\u2009=\u20092), pinhole fracture (n\u2009=\u20093) and anaesthetic death (n\u2009=\u20092). The remaining 23 rabbits were randomly divided into three experimental groups, each group consisting of minimally seven rabbits. TP508 (OrthoLogic, Tempe, USA) at two different concentrations (30 and 300\u00a0\u03bcg TP508) or saline were injected percutanously at the beginning and at the end of the lengthening phase. The first group (n\u2009=\u20098) received injections of 300\u00a0\u03bcl saline containing 300\u00a0\u03bcg TP508, the second group (n\u2009=\u20098) received injections of 300\u00a0\u03bcl saline containing 30\u00a0\u03bcg TP508 and the third group (n\u2009=\u20097) received injections of 300\u00a0\u03bcl saline alone and served as a control group. The injections were given in three different areas (100\u00a0\u03bcl in each area): the proximal, central and distal area of the distraction gap. Two weeks after the lengthening phase, all animals were killed. Tissue samples consisting of the distraction gap and 5\u00a0mm proximal and distal to the pre-existing bone were then harvested and immediately fixed in 95% ethanol for further examination. Tissues were decalcified with EDTA and embedded in paraffin. Serial sections (5\u00a0\u03bcm thick) were cut and mounted on poly-lysine-coated glass slides. The peripheral quantitative computed tomography and histological examination of these bones has been published previously (Li et al. 2005a,b).\nAll animal experimental procedures were approved and performed following the guidelines for animal scientific procedures (Animal Scientific Procedures Act 1986, British Home Office).\nAntibodies\nThe mouse recombinant monoclonal antibody Pebp2alphaA was kindly donated by Dr. K. Sasaguri, Kanawaga Dental School, Japan. This antibody has been used and characterized previously and reacts with all isotypes of Runx2 (Bronckers et al. 2001, 2005). The mouse anti-rat OPN monoclonal antibody (MPIIIB101) was obtained from the Developmental Studies Hybridoma Bank (developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences Iowa City, IA 52242, USA). The mouse anti-human BSP (monoclonal antibody A4232.1) was purchased from Immunodiagnostik, Bensheim, Germany. Both anti-OPN and anti-BSP antibodies have been used and specified previously (Ibrahim et al. 2000; Bronckers et al. 2005; Zerbo et al. 2005).\nImmunohistochemical procedures\nTissue sections were de-paraffinized in xylene and rehydrated through a graded series of aqueous ethanol solutions. Endogenous peroxidase was quenched by incubation in 3% H2O2 in 100% ethanol (5\u00a0min). A pre-incubation in hot citrate buffer (0.01\u00a0M, pH 6.0, 95\u00b0C, 20\u00a0min) was performed for antigen retrieval prior to incubation with 30% normal horse serum (30\u00a0min). Sections were then incubated overnight with primary monoclonal anti-Runx2 antibody (1:200), anti-BSP antibody (1:400) or anti-OPN antibody (1:600) at 4\u00b0C in a humidified chamber. After washes with phosphate-buffered saline (PBS), biotinylated horse anti-mouse IgG (ABC-peroxidase Elite kit; Vector Laboratories, Burlingame, Calif., USA) was added at a dilution of 1:200 and the sections were incubated at room temperature for a minimum of 60\u00a0min. The sections were washed again with PBS, incubated with ABC peroxidase complex for another 60\u00a0min followed by a maximum of 20\u00a0min in 3,3\u2032 diaminobenzidine substrate solution (Vectastain kit) to visualize the immunoreaction, counterstained with methyl green for 2\u20133\u00a0min, mounted in 50% glycerol and covered with a glass coverslip. As negative controls, sections were incubated with non-immune mouse IgG in place of the primary antibody.\nScoring\nSections were examined by using a Leica DM RA microscope equipped with a Leica DC 200 digital camera. The central areas of the gap were selected as the region of interest (ROI). First, the boundaries of the distraction gap (indicated by the osteotomy lines) were determined and subsequently a grid was positioned over the ROI halfway between the two osteotomy lines. The grid was 609\u00a0\u03bcm in width and 479\u00a0\u03bcm in length. The ROI consisted of some newly formed bone (mostly in the group treated with 300\u00a0\u03bcg TP508) and fibrous tissue (Fig.\u00a01a). Counting was performed by using the grid at a magnification of 200\u00d7, with a minimum of three sections, at least 30\u00a0\u03bcm apart, per animal. Some tissue blocks were of poor quality or no longer contained a complete intact gap area with an ROI; this reduced the number of animals per group to 5\u20136 animals. The total number of cells was counted (immunopositive and immunonegative cells). The sections were coded and randomized. Quantification was performed by one investigator (L.A.) who was unaware of the origin of the sections. Numbers of immunopositive cells were noted in absolute terms and were expressed as the percentage of all (immunopositive and negative) cells present in the ROI.\nFig.\u00a01Immunolocalization of Runx2 and osteopontin proteins in TP508-treated groups (a-c, e 300\u00a0\u03bcg TP508 group, d 30\u00a0\u03bcg TP508 group). a Overview of immunoreactions in a biopsy taken from rabbit long bone after distraction osteogenesis Runx2 staining (FT fibrous tissue, NB new bone, P periosteum, PC periosteal callus). A white grid was used to count the number of immunopositive and negative cells in the region of interest. \u00d750. b Runx2 staining in the nuclei and cytoplasm of the cells. Runx2 was strongly expressed in osteoblasts (Ob). Note that young osteocytes (Ocy), which had recently embedded in the bone matrix, expressed Runx2 protein. Cells adjacent to the osteoblast layer, presumably pre-osteoblasts (Pre-Ob), also expressed Runx2, but with lower intensity (B bone). \u00d7400. c Immunostaining for osteopontin. Positive staining was found in the cytoplasm of the cells (white arrows). Note that expression was stronger towards the newly formed bone (NB). \u00d7200. d Runx2 immunostaining in tissue of a rabbit injected with 30\u00a0\u03bcg TP508. Expression was much lower than that after injection with 300\u00a0\u03bcg TP508 and was similar to saline-injected control tissue (not shown). Fewer fibrocartilage-like cells (FCC) were stained than after injection with 300\u00a0\u03bcg TP508 (HC hypertrophic cartilage-like cells). \u00d7200. e Negative control stained with non-immune IgG instead of primary antibodies to Runx2. Note the lack of positive staining. Counter-staining with methyl green. \u00d7200. Bars 20\u00a0\u03bcm (b, d), 50\u00a0\u03bcm (c, e), 300\u00a0\u03bcm (a)\nCell cultures\nThe mouse osteoblast-like MC3T3-E1 cell line, derived from newborn mouse calvaria, was routinely maintained in \u03b1-MEM (Gibco, Paisley, UK) supplemented with 10% fetal bovine serum (HyClone), 120 \u03bcg\/ml penicillin (Sigma, St Louis, Mo., USA), 100\u00a0\u03bcg\/ml streptomycin sulphate (Sigma), 1.25\u00a0\u03bcg\/ml fungizone (Gibco), 50\u00a0\u03bcg\/ml sodium ascorbate (Merck, Darmstadt, Germany), 10\u00a0mM \u03b2-glycerophosphate (Sigma) and 300\u00a0\u03bcg\/ml glutamine (Sigma) at 37\u00b0C in a humidified atmosphere of 5% CO2 in air. This cell line is capable of expressing Runx2 mRNA (Tsuji et al. 1998). To stimulate cells to express osteoblastic markers, they were first exposed to 10\u00a0nM dexamethasone in 25\u00a0cm2 flasks (Greiner BioOne, Solingen, Germany) for 1\u00a0week. Upon confluency (typically after 5\u00a0days), cells were harvested by using 0.25% trypsin and 0.1% EDTA in PBS, centrifuged at 600g for 10\u00a0min, washed and plated in 24-well culture dishes (Greiner BioOne) at three different cell densities (3\u00d7104, 1.5\u00d7104 and 7.5\u00d7103 cells\/well) in order to obtain a comparable number of cells at the end of each experiment. After 1\u00a0day of culture, the medium was replaced by media containing various concentrations of TP508 (0, 10 or 100\u00a0\u03bcg\/ml). Throughout the experiment, all media were supplemented with 10\u00a0nM dexamethasone. Cells were exposed to TP508 for 24, 48 and 72 h, after which times, the cells were collected. The experiment was carried out in quadruplicate and repeated once.\nRNA analysis and quantitative real-time polymerase chain reaction\nTotal RNA from cultured cells was isolated by using TRIZOL reagent (Gibco) according to the manufacturer\u2019s instructions. The RNA content was determined by measuring the absorbance in water at 260\u00a0nm by means of an Ultrospec III spectrophotometer (Amersham, Buckinghamshire, England). cDNA synthesis was performed by using 750\u00a0ng total RNA in a final reaction volume of 20\u00a0\u03bcl containing 5\u00a0U transcriptor (Roche Applied Science), 5\u00d7 polymerase chain reaction (PCR) buffer, 4\u00a0U random primers (Roche), 20\u00a0U protector RNase inhibitor (Roche), 1\u00a0mmol each dNTP and 10\u00a0\u03bcl template. The reverse transcription step was performed on a Gene Amp 9700 Thermocycler (Applied Biosystems, Foster City, Calif., USA) at 55\u00b0C for 30 min followed by 85\u00b0C for 5\u00a0min.\nReal-time PCR was performed on the ABI PRISM 7700 sequence detection system (Applied Biosystems). The phosphobilinogen deaminase gene (PBGD) served as the endogenous reference (de Vries et al. 1999) to normalize Runx2 expression. The primers for the amplification of Runx2 mRNA were 5\u2032-ATGCTTCATTCGCCTCAC-3\u2032 and 5\u2032-ACTGCTTGCAGCCTTAAAT-3\u2032 (GenBank database, accession no.\u00a0NM\u00a0001024630). The PCR primers for PBGD, amplified as the internal reference, were 5\u2032-AGTGATGAAAGATGGGCAACT-3\u2032 and 5\u2032-TCTGGACCATCTTCTTGCTGA-3\u2032 (accession no.\u00a0BC 003861). For the amplification of the Runx2 and PBGD products, 37.5\u00a0ng cDNA was added to the PCR mixture containing SYBR Green PCR Master Mix consisting of SYBR Green I Dye, AmpliTaq Gold DNA polymerase, dNTPs with dUTP, a passive reference and buffer (Applied Biosystems) and 300\u00a0nM of each primer, in a final volume of 25\u00a0\u03bcl. The enzyme was activated by preheating the samples for 10 min at 95\u00b0C, followed by a two-step PCR procedure consisting of a denaturation step at 95\u00b0C for 15 s and an annealing and extension step at 60\u00b0C for 1 min for 45 cycles. Relative expression was calculated by using the comparative Ct method. Samples were normalized for the expression of PBGD by calculating \u0394Ct (CtRunx2\u2212CtPBGD); subsequently, the \u0394\u0394Ct values were calculated as \u0394Ctsample\u2212\u0394Ctcalibrator , where the calibrator was the control sample (without TP508 incubation). Relative expression of the Runx2 gene was expressed as 2-(\u0394\u0394 Ct) (Livak and Schmittgen 2001).\nStatistical analysis\nValues obtained from the scoring of tissue sections and normalized mRNA levels determined by PCR analysis (presented as means and standard deviations) were analysed by using GraphPad Prism 4 for Windows. For the analysis of the immunostaining, an average value was calculated per animal for each protein and treatment; these average values were used to determine the mean and standard deviation for each treatment group (5\u20136 animals\/group). Analysis of variance (ANOVA) was used to test for statistical significance, which was accepted when P<0.05 (two-tailed). Tukey\u2019s post test was performed to compare individual pairs of groups if P<0.05. Linear regression analysis was used to examine the correlation between Runx2 mRNA expression and culture time.\nResults\nImmunohistochemistry demonstrated different localization patterns for Runx2, OPN and BSP proteins. Cells expressing these proteins were detected throughout the distraction gap and in the periosteum, the periosteal callus and the old (predistraction) bone (Fig.\u00a01a). Runx2 staining was located in nuclei and cytoplasm, whereas strong staining for OPN and BSP was localized in the cytoplasm (Figs.\u00a01b,c,2i,3c); weak staining for BSP and OPN was also seen in the extracellular bone matrix. Figures\u00a01, 2, 3 present the different staining patterns and Table\u00a01 summarizes the average intensity of immunostaining in the ROI.\nFig.\u00a02Runx2 (a, d, g), OPN (b, e, h) and BSP (c, f, i) expression in the distraction gap (B bone, FT fibrous tissue, FC fibrous cartilage-like tissue, asterisks central area of distraction gap, hatch layers of cells adjacent to the newly formed bone, arrows osteoblasts, arrowheads osteocytes) of the various groups (a-c 0\u00a0\u03bcg TP508 group, d-f 30\u00a0\u03bcg TP508 group, g-i 300\u00a0\u03bcg TP508 group). Inset (i): Newly formed bone in the gap. Original magnifications: a 50\u00d7; d, g 100\u00d7; b, e, h 100\u00d7; c, f, i, inset 200\u00d7. Bars 20\u00a0\u03bcm (c, f, i), 50\u00a0\u03bcm (b, d, e, g, h), 100\u00a0\u03bcm (a)Fig.\u00a03Immunolocalization of Runx2, OPN and BSP in the 300\u00a0\u03bcg TP508 group. a Runx2 staining of a vessel wall (EC endothelial cells, SMC smooth muscle cells). Note that some cells near the blood vessel weakly express Runx2 (white arrows). \u00d7400. b Osteopontin staining of a vessel wall. Note that the cells surrounding the blood vessel are immunonegative and only stain weakly for methyl green (white arrows). \u00d7400. c BSP staining is restricted to the layer of osteoblasts (Ob) and hardly occurs in fibrous tissue \u00d7 400. d OPN staining is expressed in osteoblasts (Ob) and osteocytes of new bone (NB) and some fibrous tissue cells close to osteoblasts, presumably pre-osteoblasts (Pre-Ob). \u00d7200. Bars 20\u00a0\u03bcm (b), 50\u00a0\u03bcm (c, d), 100\u00a0\u03bcm (a)Table\u00a01Intensity scores of immunostaining in region of interest (\u00b1 weak, + moderate, ++ strong, +++ very strong). The intensity of immunostaining was scored in the central area of the gap (region of interest) at 40\u00d7 final magnification. Variation exists between sections in each animal and between animals within groupsMarkerGroup with 0\u00a0\u03bcg TP508Group with 30\u00a0\u03bcg TP508Group with 300\u00a0\u03bcg TP508RUNX2+ to +++ to ++++ to +++OPN\u00b1 to ++\u00b1 to ++++BSP\u00b1 to +++\u00b1+ to +++\nExpression of Runx2, OPN and BSP in the group injected with saline (control group) was similar to that in the group treated with 30\u00a0\u03bcg TP508 (Figs.\u00a01d, 2a\u2013f). The intensity of the staining in both groups was however much lower than that in the 300 \u03bcg TP508 group (Figs.\u00a01c,2g,h,3c,d). Runx2 and OPN were detected in active osteoblasts, in fibroblasts next to the osteoblastic layer (presumably pre-osteoblasts), in osteocytes in the old (predistraction) and newly formed bone, in fibroblasts of the periosteum, in the central area of the gap, in endothelial and smooth muscle cells of the vessels and in chondrocyte-like cells next to new bone. We found two types of cartilage-like tissue in the group treated with 30\u00a0\u03bcg TP508 and in control group, i.e. fibrocartilage-like (collagen fibres between lacunae) and hypertrophic-like (evenly dispersed chondrocytes, without clear fibres; Fig.\u00a01d). Higher numbers of cells associated with these two tissues were found in the group treated with 30\u00a0\u03bcg TP508 and in the control group than in the group treated with 300\u00a0\u03bcg TP508 (Fig.\u00a02a,d,g). More fibrocartilage-like cells than hypertrophic cartilage-like cells were positive for Runx2 (Fig.\u00a01d). Sections in which primary antibodies had been replaced with non-immune antibodies were negative (Fig.\u00a01e).\nThe Runx2- and OPN-positive blood vessel cells (both the endothelial and smooth muscle cells) were more evident in the group with 300\u00a0\u03bcg TP508 (Fig.\u00a03a,b). They appeared to occur at locations with high bone formation activity. Connective tissue cells in close vicinity to Runx2-positive blood vessels also stained faintly for Runx2 but not for OPN. Immunostaining for BSP was more restricted than that for Runx2 and OPN and was seen in osteoblasts and young osteocytes (Fig.\u00a03c,d). Weak expression was found in some of the cells considered to be pre-osteoblasts. Fibroblast-like cells in the central fibrous area of the distraction gap expressed Runx2 (Fig.\u00a02a,d,g) and OPN (Fig.\u00a02b,e,h) but were negative for BSP. The number and the intensity of Runx2 and OPN in the gap increased from fibrous tissue towards the newly formed bone (Fig.\u00a01c). Weak BSP staining was seen in the area within the gap in which bone trabecules started to be formed (Fig.\u00a02c,f,i).\nIn the group injected with 300\u00a0\u03bcg TP508, immunostaining for Runx2, OPN and BSP was distributed throughout the regenerating areas. The immunostaining for Runx2, OPN and BSP in the connective tissue in the gap (endosteal area) was relatively high and as intense as in the periosteal callus tissue. In the 30\u00a0\u03bcg TP508 group and in the control group, staining was less intense in the central gap area than in the periosteal callus area.\nQuantitative immunohistochemistry was performed to investigate the effect of TP508 injection on the total number of cells and on the number of differentiated bone cells in the central area of the gap. The total number of cells (immunostained and unstained) in the ROI was not significantly different between the three groups. Mean values and the standard deviation of the total number of cells in the tissues treated with 0, 30\u00a0\u03bcg TP508 and 300\u00a0\u03bcg TP508 were 958\u00b1217, 1140\u00b1137 and 1161\u00b1282, respectively (ANOVA, P=0.5). This indicated that TP508 injection had no overall effect on total number of cells in the distraction gap. Next, we counted the number of cells, in the ROI, expressing each of the three antibodies tested. Absolute values of immunopositive cells in the 0, 30\u00a0\u03bcg TP508 and 300\u00a0\u03bcg TP508 groups were as follows: for Runx2, 263\u00b161, 288\u00b1171, 560\u00b1115, respectively; for OPN, 299\u00b1201, 244\u00b140, 677\u00b1216, respectively; for BSP, 255\u00b1111, 252\u00b173, 480\u00b131, respectively. We also expressed the number of immunopositive cells as a percentage of the total number of cells (Fig.\u00a04). The number of Runx2- and OPN-expressing cells was significantly higher in the group treated with 300\u00a0\u03bcg TP508 than in the control group that received only saline (P<0.05; Fig.\u00a04). The number of BSP-expressing cells was also higher in the 300\u00a0\u03bcg TP508 group than in the 30\u00a0\u03bcg TP508 group (P<0.05). No significant differences were found between the 30\u00a0\u03bcg TP508 group and the control group receiving saline (P>0.05).\nFig.\u00a04Effect of TP508 injection on the number of immunopositive cells in the central portion of the distraction gap (black bars Runx2-positive cells, grey bars OPN-positive cells, white bars BSP-positive cells). Data are presented as percentage cells immunopositive for each of the three proteins examined (means\u00b1SD). *The percentages of Runx2-, OPN- and BSP-positive cells in the 300\u00a0\u03bcg TP508 group were significantly higher than for cells with the corresponding proteins in the saline-treated control group; the percentage of BSP-positive cells in the 300\u00a0\u03bcg group was also significantly higher than that in the 30\u00a0\u03bcg group (ANOVA, P<0.05)\nThe previous data suggested that TP508 increased the differentiation of osteogenic cells into osteoblasts in situ. To determine whether TP508 had any direct effect on osteogenic cells that were in the process of turning into osteoblasts, we performed in vitro experiments with the (mouse) osteoblast-cell line MC3T3-E1 after pre-culture in the presence of dexamethasone. After a 24-h stimulation with TP508, we found a significant (but slight) downregulation of Runx2 mRNA at 10\u00a0\u03bcg\/ml TP508 (P=0.008) but no change at 100\u00a0\u03bcg\/ml TP508 (Fig.\u00a05).\nFig.\u00a05Quantitative reverse transcription\/polymerase chain reaction analysis of Runx2 mRNA expression in mouse osteoblast-like (MC3T3-E1) cells cultured for 24, 48 and 72\u00a0h in the presence of TP508 (black bars control group with 0\u00a0\u03bcg\/ml TP508, hatched bars 10\u00a0\u03bcg\/ml TP508, white bars 100\u00a0\u03bcg\/ml TP508). Data are presented as means\u00b1SD (n=8) and are representative of two experiments. *The 10\u00a0\u03bcg\/ml TP508 group at 24\u00a0h of incubation was significantly lower than the control group (one-way ANOVA; Tukey\u2019s multiple comparison test, P<0.01). Correlation analysis between mRNA and culture time: r2=0.94, P=0.06 for 10\u00a0\u03bcg\/ml TP508; r2=0.99, P=0.06 for 100\u00a0\u03bcg\/ml TP508\nDiscussion\nThis study demonstrates a positive effect of TP508 on the synthetic activity of bone-forming cells in the distraction gap in rabbit long bones. After injection of a high dose of TP508, significantly more cells expressing Runx2, OPN and BSP are located in the central part of the distraction gap, predominantly consisting of soft connective tissue. We have also found that these cells stain more intensely for Runx2, OPN and BSP suggesting that TP508 not only proportionally increases the number of osteogenic cells in the distraction gap but also their expression level. The finding that TP508 enhances bone-forming activity in the soft tissue gap area is in agreement with previous morphometric data indicating that TP508 stimulates bone regeneration in the distraction gap (Li et al. 2005a,b).\nThe underlying molecular mechanism of the stimulation of Runx2 expression in the distraction gap after TP508 treatment is not clear. Our in vitro experiment with osteoblast-like MC3T3-cells suggests that TP508 does not have a significant positive effect on Runx2 mRNA. However, we cannot rule out that the MCT3C3 cells are too far advanced along the osteoblast differentiation pathway to respond to TP508 for they had been pre-cultured in dexamethasone-containing medium before they were exposed to TP508. No definitive conclusions can therefore be drawn from these in vitro data. In vivo, TP508 may have stimulated the differentiation of osteogenic cells though the activity of other non-osteogenic cell types. Injection of TP508 into a bone fracture model upregulates FGF-2 mRNA in (not further specified) tissues at the fracture site (Wang et al. 2005). Since FGF-2 is known to be an upstream regulator of Runx2 (Franceschi and Xiao 2003), one explanation for the increased Runx2 expression in the gap is via an enhanced cytokine release by inflammatory cells, which in turn stimulates osteoblast differentiation (Okazaki et al. 1999; Stiernberg et al. 2000; Franceschi and Xiao 2003; Naldini et al. 2004). Alternatively, TP508 stimulates blood vessel growth in vivo (Norfleet et al. 2000; Li et al. 2005a,b; Vartanian et al. 2005; Wang et al. 2005) and this subsequently enhances bone formation. TP508 increases recruitment of human aortic and microvascular endothelial cells through chemotaxis (Norfleet et al. 2000; Li et al. 2002), upregulates angiogenesis-related genes (Wang et al. 2005) and acts directly on microvascular cells to accelerate sprouting (Vartanian et al. 2005). Vascularization provides the oxygen that is required for the metabolic activity of the osteoblasts (Tuncay et al. 1994; Salim et al. 2004); it provides homes for circulating stem cells that, under proper stimulation, differentiate into osteoblastic cells (Kuznetsov et al. 2001). In addition, vessel cells secrete paracrine factors that can regulate bone cell metabolism (Villars et al. 2000; Street et al. 2002). Thus, the angiogenic properties of TP508 might help to enhance bone growth.\nIn the present study, we have also found Runx2 protein expression in endothelial cells, vascular smooth muscle cells and loose connective cells surrounding developing blood vessels at sites of active bone and tissue formation. Recent studies have presented evidence for transient Runx2 mRNA and protein expression during angiogenesis in embryonic human orofacial tissues (Bronckers et al. 2005), in human primary bone marrow endothelial cells and in endothelial cell lines (Namba et al. 2000; Sun et al. 2001). Thus, our data suggest that, with respect to Runx2 expression, the formation of blood vessels in regenerating adult tissues during DO resembles vessel formation in embryonic tissues. Of note, under certain conditions such as inflammation, cells associated with vessels (e.g. pericytes, endothelial cells and smooth muscle cells) or their precursors are capable of differentiating into osteoblast-like cells (Schor et al. 1995; Reilly et al. 1998; Kinner and Spector 2002) suggesting that the forming microvasculature is a potential source for osteogenic cells.\nThe distribution and staining intensity of Runx2, OPN and BSP varies between the different groups. In the control group and in the group treated with a low dose of TP508, staining is more pronounced in the periosteal callus than in the tissue in the distraction gap. However, in the group treated with a high dose of TP508, we have found the same high staining in both periosteal callus and gap tissue. Periosteal callus formation is a common finding during fracture healing (Gerstenfeld et al. 2003). New woven bone is formed rapidly around the fractured segments, because of stimulation of the periosteum that contains many osteoprogenitor cells. In the same section, a high staining intensity in the periosteal callus can therefore be considered as an internal reference. Hence, as the staining for Runx2, OPN and BSP in the distraction gap is as intense as that in the periosteal callus at the high dose of TP508, this is a clear indication that the high dose of TP508 stimulates bone formation in the distraction gap. This observation is in accordance to a previous histological study that has demonstrated advanced bone consolidation in a group treated with a high dose of TP508 (Li et al. 2005b). TP508 might stimulate the migration of osteoblast precursors to the distraction gap (Li et al. 2005a). Li and colleagues have shown that human periosteal-derived osteoblasts and human microvascular endothelial cells are chemotactic to TP508 (Li et al. 2005a). Therefore, the presence of TP508 in the gap might attract osteoprogenitor cells in the periosteum to migrate into the distraction gap.\nRunx2 is a critical gene for osteoblast differentiation and for the function of the fully differentiated osteoblast (Komori et al. 1997; Karsenty et al. 1999). The Runx2 protein binds to the osteoblast-specific element 2 that is present in the promoter regions of all major extracellular matrix genes expressed in osteoblasts, including the genes for osteocalcin (OC), OPN and BSP and it regulates the expression of these proteins (Karsenty et al. 1999). OPN and BSP belong to the Small Integrin-Binding LIgand, N-linked Glycoprotein (or SIBLING) family (Fisher et al. 2001). Among other functions, members of this family are known to have a role in mineralization, as they bind strongly to hydroxyapatite (Fisher et al. 2001).\nIn conclusion, we have demonstrated that the increase of bone regeneration by thrombin-related peptide TP508 is associated with an increase in the immunostaining for Runx2, an essential transcription factor of the osteoblastic lineage, and for the bone matrix proteins BSP and OPN. TP508 may thus be a candidate for enhancing bone regeneration when bone regeneration is slow, as occurs in elderly patients.","keyphrases":["thrombin peptide","distraction osteogenesis","bone regeneration","runx2","osteopontin","bone sialoprotein","immunohistochemistry","rabbit (new zealand white, adult, male)"],"prmu":["P","P","P","P","P","P","P","R"]}
{"id":"J_Med_Internet_Res-7-2-1550646","title":"A Case Study of a Retracted Systematic Review on Interactive Health Communication Applications: Impact on Media, Scientists, and Patients\n","text":"Background In October 2004, a flawed systematic review entitled \u201cInteractive Health Communication Applications for People with Chronic Disease\u201d was published in the Cochrane Library, accompanied by several press releases in which authors warned the public of the negative health consequences of interactive health communication applications, including the Internet. Within days of the review's publication, scientists identified major coding errors and other methodological problems that invalidated the principal conclusions of the study and led to a retraction. While the original study results and their negative conclusions were widely publicized in the media, the retraction seemed to go unnoticed.\nIntroduction\nPublication of the Review\nOn October 18, 2004, the Cochrane Collaboration, a organization which produces and disseminates systematic reviews of health care interventions [1], published a review entitled \u201cInteractive Health Communication Applications for People with Chronic Disease\u201d [2], which from this point on will be referred to as the \u201cIHCA review.\u201d The IHCA review was edited by the Cochrane Consumers and Communication Review Group [3]. Those who prepare reviews volunteer to work in one of many Collaborative Review Groups, with editorial teams overseeing the preparation and maintenance of the reviews.\nInteractive health communication applications (IHCAs) were defined in the IHCA review as \u201ccomputer-based, usually Web-based, health information packages for patients that combine information with social-, decision-, or \u2018behavior change'-support\u201d [2]. The results of the IHCA review showed that IHCAs had a positive effect on knowledge and on social support, no effect on behavioral outcomes, and a negative effect on clinical outcomes.\nThe principal conclusion of the review was \u201cconsumers whose primary aim is to achieve optimal clinical outcomes should not use an IHCA\u201d [2]. This conclusion was the focus of a press release which the mass media widely circulated (as will be documented later). However, only days later, the IHCA review was found to be flawed and was retracted.\nRetractions\nThe National Library of Medicine (NLM) is a leader in the bibliographic handling of retractions. The Medical Subject Headings (MeSH) contain the concept \u201cretracted publication,\u201d which identifies a citation previously published and now retracted through a formal issuance from the author, publisher, or other authorized agent. In January 2005, the PubMed query \u201cRetracted Publication[Publication Type] AND 1971:2004[edat]\u201d retrieved 619 retracted citations that entered PubMed between 1971 and 2004. Since the query \u201c1971:2004[edat]\u201d retrieves approximately 12.5 million citations, less than 1 in 10000 publications have been retracted.\nFriedman [4] studied 60 fraudulent articles by one scientist. Journals in which the scientist had published were notified of the fraud. Only 7 articles were subsequently tagged in PubMed with \u201cRetracted Publication.\u201d The delay between publication of a paper and its retraction often has deleterious effects [5]. Furthermore, journals and institutions are hesitant to issue a statement of errors in published work unless the author of the work confesses to the error, which authors may resist doing because such an admission can be career-damaging.\nWhile very few publications are officially retracted, the concern about factors related to retractions is substantial. The study of retractions itself might be indexed with MeSH concepts such as \u201cscientific misconduct,\u201d although the fraction of retractions that stem from error as opposed to scientific misconduct is not known. The query \u201cScientific Misconduct[majr] AND 1971:2004[edat]\u201d in PubMed retrieved 1840 citations. This body of literature recommends that medical researchers constructively criticize the research practices of others in their institution to reduce the likelihood of misconduct [6].\nThe objective of this paper is to document the IHCA review as an event in the history of medical publishing, to identify the factors leading to the publicizing of a retracted publication, and to assess the implications.\nMethods\nThe objectives of this research called for various study methods. The author employed the following three methods: (1) historical processes of collecting documents about a contemporary event and organizing them thematically; (2) ethnographic processes of author participation in the event, personal communication with other participants in the event, interpretation of communications, and construction of models; (3) content analyses based on bibliographic database and Internet searches, coding of the retrieved documents, and tallying of the code frequencies.\nThe ethnographic method employs the extended case method, and the extended case method applies reflexive science to ethnography. Buroway describes reflexive science as follows: \u201cReflexive science starts out from dialogue, virtual or real, between observer and participants, embeds such dialogue within a second dialogue between local processes and extralocal forces that in turn can only be comprehended through a third, expanding dialogue of theory with itself\u201d [7].\nVarious database and Internet searches were employed to study the impact of the review and to quantify the difference between mass media coverage of the original publication and its retraction. LexisNexis Academic databases of health news and general news were searched, as was Google. The queries were designed in an iterative process that began with keywords from the question to be addressed but refined the query based on study of the query retrieval results. The retrieved results were coded, and the coding language was also developed in an iterative process. First, the obvious codes \u201cabout the review\u201d and \u201cabout the retraction\u201d were introduced. Each retrieved document was classified into a single code by the author. If the retrieved document was not appropriately described by an existing code, then the coding language was augmented. The Web of Science was also queried to identify academic citations, but no citations were identified (data not shown). Most database and Internet searches were conducted in May 2005.\nTo better understand how special the publicity accorded the IHCA review was, this study was extended to three other publications: 2 of these were retracted publications tagged as \u201cRetracted Publication\u201d (1 Cochrane review, but not eHealth related, and 1 non-Cochrane review, but eHealth related), and 1 was a meta-analysis with a scope similar to that of the IHCA review. These 3 reports were identified through PubMed searches.\nResults\nThe following qualitative results on the impact of the IHCA review are organized into three main sections: scientist reaction, mass media reaction, and patient reaction.\nThe section on scientist reaction considers Cochrane reviewers' reactions and how eHealth scientists responded to the IHCA review in the comment section of the Cochrane database. The mass media section provides the Cochrane retraction and then explores, via LexisNexis and Google results, the reaction of the mass media to the IHCA review. The patient reaction section shares dialogue from patient-patient online discussions that reveals the reactions of patients to the IHCA review.\nScientist Reaction\nThe Cochrane Collaboration allows anyone to submit comments to the published reviews. Two scientists' comments on the IHCA review appeared independently on October 28, 2004. Kummervold and Eysenbach criticized the IHCA review for both its protocol and its coding.\nKummervold explained in detail how the coding of the meta-analysis was incorrect: \u201cWe can't get the numbers to add up, it looks like they are reversed in 8 of the 11 studies...\u201d [8]. He delineated the facts and the interpretation for each of the 8 studies at issue; for example, regarding the HbA1c measurement in the Lehmann 2003 paper, he stated that Lehmann reported a reduction in HbA1c of 0.8 for the intervention group, and 0.1 for the control group, which should be interpreted as a positive result for the intervention group. Kummervold added: \u201cWe also find it strange that you focus so much on the overall estimates when there is so much heterogeneity in the material. The conclusion seems to be overstated\u201d [8].\nEysenbach had similar comments, stressing that a formal meta-analysis of these heterogeneous studies was problematic, and that the three studies which contributed most to the \u201cnegative\u201d result were in fact positive: \u201cApart from the fact that I do not think that it is legitimate to do a formal meta-analysis using papers measuring totally heterogeneous outcomes with different types of interventions, I also notice that the overall effect estimate is \u2018negative' (eg, \u2018favoring control') because of three studies\u2026. However, when I read these three studies I cannot find that their result[s] are negative\u2026. If my suspicion is correct, then this is quite a catastrophic error, and quite an embarrassment for Cochrane to let such an error slip through peer-review\u201d [9].\nOn November 10, 2004, the Cochrane Consumers and Communication Review Group reacted to the discovered errors [10] with a notice that included the following: \u201cThe review will be withdrawn as soon as possible\u2026. As the corrections to the review have not been completed yet, it would be premature to announce any reversal of the review's findings at this stage.\u2026 The original press releases regarding this review were made not by the Cochrane Collaboration itself but by University College London\u2026.\u201d\nJohn Wiley & Sons (the publisher of the Cochrane Database) released to EurekAlert a retraction on December 6, 2004: \u201cThe review originally determined that\u2026chronically ill people using interactive programmes had worse clinical outcomes than those who did not. Regrettably, errors in data analysis meant that these outcomes were reported incorrectly.... It is expected that the revised results will be published in April 2005\u201d [11].\nThe April 2005 edition of the Cochrane Systematic Reviews did not mention the IHCA review. Royle, the chief executive officer of the Cochrane Collaboration, said that further review of the revised report was ongoing and no date could be given as to when the review might be published (personal communication, April 25, 2005).\nMass Media Reaction\nThe Cochrane Database of Systematic Reviews is not read by the typical consumer. However, Murray's employer, the University College London (UCL), worked with Murray to widely publicize the result. UCL posted a news bulletin on its website on October 18, 2004 that remained there as of May 25, 2005. The bulletin was titled \u201cKnowledge may be hazardous to web consumers' health\u201d and stated the following: \u201cPeople who use their computers to find information about their chronic disease often wind up in worse condition than if they had listened to their doctor, according to a UCL review of studies on internet health.\u2026 One reason\u2026might be because knowledge-seekers become so steeped in information from the Internet they make treatment choices on their own, contradicting advice from their doctors\u201d [12].\nMost significantly, the UCL bulletin was circulated to information intermediaries that are considered the main entrance to the world's mass media, including AlphaGalileo and EurekAlert.\nA search on LexisNexis Academic with the query \u201cElizabeth Murray AND health\u201d for the period June 2004 to May 2005 revealed a total of 15 relevant press reports, in the following categories:\nMedical and Health News: There were 9 publications with titles such as UCL's press release title of \u201cKnowledge may be hazardous to web consumers' health.\u201d The publications appeared in places like Life Science Weekly, Law and Health Weekly, and Health and Medicine Week.\nGeneral News\u2013Major Papers: There were 5 relevant articles, such as one entitled \u201cWhy medical advice from the internet can be bad for your health\u201d in the British The Daily Telegraph and another entitled \u201cMedical Web sites may be unhealthy places to learn about ills\u201d in the Omaha World Herald. Only 1 article was about the retraction, published in the Ottawa Citizen on October 18, 2004.\nTime Incorporated Publications: There was 1 article in the November 1, 2004 issue of Time entitled \u201cClick to Get Sick?\u201d [13].\nAmong the 15 results from the LexisNexis Academic database, only 1 newspaper report, authored by Tom Spears, dealt specifically with the retraction [14]. Spears, in personal communication with this author (November 18, 2005), said, \u201cI was fairly stunned today to learn that it [IHCA review] has been withdrawn; I found out only because I was looking up the study for my daughter, a science student. Now I'm covering the sequel for tomorrow's paper.\u2026 I scan EurekAlert faithfully, as many reporters do, and never saw a hint of anything there.\u201d\nTo further test whether the media emphasized the false negative result but minimally covered the retraction, a content analysis on Google was performed on May 24, 2005. The query was \u201chealth AND Cochrane AND Murray AND (interactive OR web OR internet)\u201d for English pages, within the past year. Of the first 200 retrieved hits, 170 pages were not related to the IHCA review. Of the remaining 30 pages, 23 (77%) were reports about the original publication that did not mention the retraction, and an additional page was a bibliography (at a UCL site) that included a citation to the IHCA review, again without mentioning the retraction. All reports (except the bibliography) used a title such as \u201cClick to Get Sick?\u201d and emphasized the negative impact on clinical outcomes of using the Web. The reports came from such reputable sources as the British Broadcasting Corporation and US News and World Report. In contrast, only 6 pages (20%) addressed the retraction: 2 were the original press releases now marked with \u201cretraction\u201d but still emphasizing in their particulars the negative health impact, 3 were Web pages at Cochrane sites, and 1 was an announcement from MedicalNews entitled \u201cUpdated press release to October 2004 Cochrane Review.\u201d The latter was the only non-Cochrane\u2013related page primarily addressing the retraction.\nThe grey literature reported on the mass media. For example, The Neuroscience for Kids Newsletter summarized [15] the \u201cClick to Get Sick?\u201d Time article by Sanjay Gupta, and a Web archive for patient education at the Samaritan Health Center pointed patients to Gupta's article. This author wrote to Gupta and asked him to write about the retraction, but Gupta did not reply.\nNLM indexed the IHCA review and entered the citation for it (including its abstract) in PubMed on October 21, 2004. The \u201cRetracted Publication\u201d tag did not, however, appear in PubMed until March 24, 2005.\nPatient Reaction\nSome patients reported the news about the IHCA review to their patient-patient online discussion groups. In a neurology patient discussion group [16], a patient posted the entire BBC news story. Patients responded in two ways. Some rejected the IHCA review result and added strong comments, such as \u201cI have gotten more help and answers for problems from knowledgeable people on this Internet Forum than I have from any of the multitude of doctors I have seen over the last 12 years.\u201d Others accepted the conclusion but insisted that patients could filter bad information from good and benefit in the end from the web. These patients were not aware of the retraction of the IHCA review.\nThis author reported the Time \u201cClick to Get Sick?\u201d article to two head-and-neck cancer patient discussion groups to which he belongs. A day later he reported the retraction from the Cochrane Database. One member of the discussion group replied: \u201cThanks for the update\u2013the negative findings seemed odd to me when I read it, so I'm glad it's being revised.\u201d This author, in his role as a cancer patient, also formally commented on the IHCA review at the Cochrane Database site [17].\nThe typical patient with a chronic disease has no formal medical training and is ill prepared to critique a meta-analysis of clinical trials. However, the typical patient is vulnerable to cultural pressures, as they are partially shaped by and reflected in the mass media.\nComparison With Another Cochrane Retraction\nFor comparison, a search for further retracted Cochrane reviews using the PubMed query \u201cCochrane Database Syst Rev[TA] AND Retracted Publication[PT] AND 1971:2005\/5\/25[edat]\u201d was conducted. One reference, in addition to the IHCA review already discussed, was identified, which was a retracted review by Brewster et al [18] about antihypertensives. The retraction for the Brewster et al review is explained on the Cochrane website as follows: \u201cThis systematic review has been withdrawn temporarily because its contents are potentially misleading.\u201d\nA search on LexisNexis with the query \u201cBrewster AND antihypertensive\u201d for the period November 2004 to May 2005 retrieved no articles in either the \u201cGeneral News\u2013Major Papers\u201d category or the \u201cMedical and Health News\u201d category.\nA search on Google for \u201cBrewster antihypertensive\u201d followed by an examination of the first 100 retrieved pages identified 23 relevant pages, which had a very different content pattern than the hits for the IHCA review. They all contained citations of papers from Brewster et al, who have published elsewhere on the same subject as in their review. The Brewster et al publication attracting the most attention was an article [19] in the Annals of Internal Medicine that was not retracted but has the same title as the Cochrane review. Thus, the only other retracted Cochrane review had a very different mass media, scientific, and web impact than the IHCA review.\nComparison With Other Retracted Articles Related to eHealth\nTo determine whether other articles on a similar topic to the IHCA review have been retracted, a search was first made for articles on a similar subject that had been MeSH indexed in PubMed. The article by Demiris [20] seemed relevant, and its two MeSH index terms were \u201cDisease Management\u201d and \u201cInternet.\u201d A search on PubMed for \u201cRetracted Publication[PT] AND Disease Management[majr] AND Internet[majr] AND 1995:2005\/5\/25[edat]\u201d returned no citations. When the search was broadened by removing the term \u201cDisease Management,\u201d 1 retracted reference was retrieved, entitled \u201cThe quality of surgical information on the Internet\u201d [21]. As previously described in the Journal of Medical Internet Research, this article was retracted due to a case of cyberplagiarism, with large sections of the paper having been lifted from different websites [22].\nA search on LexisNexis Academic with the query \u201cMcKinley and surgical and Internet\u201d for the period 1995 to May 2005 revealed no relevant press reports, neither in the \u201cGeneral News\u2013Major Papers\u201d category (three hits were all not relevant to the McKinley article) or in the \u201cMedical and Health News\u201d category.\nA search on Google for English pages with the query \u201cMcKinley surgical Internet\u201d revealed 96 irrelevant pointers in the first 100 results. Of the remaining 4 relevant hits, 1 was the article about the plagiarism [22], which precipitated the retraction of the McKinley et al manuscript, and 3 were academic references to the McKinley et al article, which did not note it being retracted.\nThus, the only other retraction of a published article appearing in PubMed similar in topic (the Internet) to the IHCA review had a very different pattern of reactions than the IHCA review.\nA Similar Meta-Analysis on eHealth\nThe IHCA review addressed a topic that the mass media found interesting. Have any other recent publications also been a meta-analysis on the impact of interactive applications on health, and, if yes, what was the mass media reaction? Using the query \u201cMeta-analysis AND Web AND Chronic Illness\u201d in PubMed, we found only 1 citation: Wantland et al [23] did a meta-analysis on Web-based health interventions that was published (in the Journal of Medical Internet Research) about the same time as the IHCA review. The paper concluded that \u201cthe effect size comparisons in the use of Web-based interventions compared to non-Web-based interventions showed an improvement in outcomes for individuals using Web-based interventions to achieve the specified knowledge and\/or behavior change for the studied outcome variables.\u201d\nWhat has been the impact of the Wantland et al paper and how does that compare to the impact of the IHCA review? The Wantland et al paper was not announced with a press release in EurekAlert. A search on LexisNexis Academic for newspaper articles about the Wantland et al paper retrieves no articles. The queries performed were similar to those performed for the IHCA review and included \u201cWantland AND health\u201d for 2004 through 2005 in General News\/Major Papers.\nA search was done on Google for \u201cWantland health Web\u201d on May 24, 2005. Of the first 200 returns, 182 were not relevant. Of the remaining 18 hits, 15 pages contained academic citations to Wantland et al, 2 announced the appearance of the article, and 1 was a personal blog that commented on the article.\nThus, most of the Google returns that gave Wantland et al citations are academic in character and very different from the mass media coverage afforded the IHCA review.\nDiscussion\nAs shown, the IHCA review provides a perhaps unprecedented case from which lessons should be drawn. Only one other Cochrane review (about antihypertensives) has been retracted, and that one received negligible mass media attention. The only retracted publication in PubMed that is indexed under the MeSH concept of \u201cInternet\u201d (the IHCA review did not have time to get indexed before it was withdrawn) received no newspaper coverage. The paper most similar to the IHCA review in topic and method (the Wantland et al report [23]) received considerable academic attention but no newspaper coverage. In other words, special circumstances must have come together for the IHCA review situation.\nThis section next presents a framework based on tiers of response. The first tier is medical scientists. The second tier is the mass media spreading medical press releases. The third tier is the patient community reacting to the mass media and the scientists.\nFirst-Order Problem\nIn an effort to critique the problem that occurred, one might build on the analysis of misconduct in toxicology by Purchase. Purchase [24] identified four roots of misconduct:\nIntention of the work\nConduct of the studies\nDesign and interpretation of studies\nBias from conflict of interest\nIn the case of the IHCA review, the intention was scientifically appropriate, namely to gain further insight about IHCAs through a systematic review. In the other three categories, fault can be found:\nThe errors in the coding of data should not have been made. The coauthors Nazareth and Tai, who are credited with doing the coding, have good enough credentials to not lay the blame on lack of experience: Nazareth is a Professor at UCL and is Scientific Director of the British Medical Research Council's General Practice and Research Framework, and Tai has coauthored several articles over the past two decades that appeared in refereed medical journals. An explanation for the miscoding in terms of experience of the coders is not apparent.\nThe design of the study has been criticized as lumping together studies which are too heterogeneous in their design, interventions, and outcomes [8,9]. The protocol might have been more rigorously vetted by the Cochrane Review Group, and the authors should have been more cautious in their interpretation of results and emphasized the weakness of the design in their publicity.\nThe reporting of the work suggests possible bias. The authors and their employers have sensationalized a result that catches the media's attention. For some observers, the review appeared biased in that the authors, who are affiliated with medical institutions, concluded that patients should listen to their doctor, instead of seeking help on the Internet.\nPurchase [24] claims that a partial solution to this first-order problem is the institutionalization of quality controls. In the 1970s, good laboratory practice regulations were introduced, but comparable regulations do not exist for meta-analyses. For quality control of a meta-analysis the scientific community relies on the research team, the researchers' institution, and the referees. A medical research institution, such as the UCL Medical School, presumably embraces results from its researchers that can earn mass media coverage and is not the appropriate institution to prevent sensationalizing. Referees can not be expected to detect when laboratory data are intentionally modified [25]; however, in this case they could have been expected to detect when data available to them are miscoded. Problems with refereeing have been frequently noted and in particular for the Cochrane Database [26].\nOpen commentary, as exists for the Cochrane Database after a publication, is one way to identify flaws. Extending the open commentary to the refereeing phase might reduce the likelihood of something going to press with errors. A submitted article might be available to the public and a community of hundreds of registered scientists could be invited to make anonymous comment. Submissions online would require extensive online commenting that reached a consensus before a submission could be considered \u201cpublished.\u201d Other approaches to increase the commentary on the research process include refereeing the protocol phase [27], which is done by the Cochrane Collaboration but apparently not with the necessary rigor or topic expertise.\nSecond-Order Problem\nThe second-order problem is a press release and subsequent mass media coverage of the release. Winsten's classic study of science and the media shows how the truth is repeatedly misrepresented by journalists and researchers: \u201cThe most striking finding which emerged from the interviews [of medical journalists] is the dominant distorting influence of the competitive force in journalism.\u2026 As economic competition among hospitals has intensified, they have begun to compete aggressively for publicity.\u2026 With increasing frequency\u2026scientists\u2026are using the media to attach their names to important findings before their competitors do.\u2026 The result has been a spiraling competition, sometimes characterized by exaggerated claims\u201d [28].\nOnline media have stimulated further competition [29]. The case of the IHCA review reflects these pressures. The UCL press release contained inaccuracies, even if the review would have been scientifically sound, in order to gain mass media attention. For instance, the subtitle of the press release was \u201cKnowledge may be hazardous to web consumers' health.\u201d In truth, the IHCA review was not about Web applications, per se, but about IHCAs, which are defined more broadly than \u201cWeb applications.\u201d However, writing a news article about IHCAs is less likely to catch attention than an article about the Web. The UCL press release did not introduce and define the term IHCA, and Murray issued statements that implied the Web was the issue. By the time the information from the press release made it into the mass media, the material had been modified enough to lose any mention of IHCAs. For instance, the Time article said, \u201cPeople who use the Web to get information about their chronic diseases often wind up in worse shape than before they logged on.\u201d\nOne way for researchers to prevent the mass media from misrepresenting the truth is for researchers to understand how the media work and to interact with the media accordingly [28]. Murray should have known that her words might be twisted to emphasize what would sell newspaper space and should not have wildly speculated. The reputations of the Cochrane Collaboration and UCL partially account for the wide dissemination of the original press release. Yet, neither organization has taken adequate steps to undo the impact of the media reporting on the IHCA review.\nThe honesty of the press could be improved with the Internet [30]. Online health care mass media publications could allow the public to make comments on news articles. Rating techniques, such as employed at eBay and Slashdot, might be used to give prominence to quality feedback [31].\nThird-Order Problem\nThe third-order problem concerns the long-term impact of the mass media. While electronic publications might be erased from a computer or marked as retracted, this does not consistently happen. Furthermore, some of the mass media coverage of the IHCA review is on paper and sits on people's bedside tables with no practical way to be retracted [32].\nAlthough this author did not (yet) find any citations to the IHCA review in Web of Science, previous studies have confirmed that a retracted scientific publication may continue to have impact without readers recognizing its retracted status. For instance, one study [33] tracking the citation pattern of 82 retracted articles revealed that, together, they were cited 733 times after their retraction, but only a small fraction of the citations referred to the retraction. In the case of the mass media, retracted publications might be read by people without them seeing the separate retraction notice.\nIf and when the revised IHCA review is published, what could it say that would undo the effect of the original publication? If the conclusion is that IHCAs result in improved clinical outcomes, then the medical profession will want to closely study the protocol and might have grounds to discredit the conclusion. The media trumpeted the IHCA review conclusion partly because it was counterintuitive but was backed by top-notch institutions. If the conclusion becomes intuitive, then the media are unlikely to be interested in it.\nThe reactions to the IHCA review in patient online discussions highlight the importance of virtual communities in helping patients deal with published information. Simple extensions to Web-based, patient, discussion systems could help patients connect to Web-based publications. For instance, when a patient posts a message to a Web-based discussion board, the Web system could parse the message and provide links from the message to relevant articles on the Web. Patients might follow the links and engage in discourse about the validity and implications of the literature. This might lessen the potential ill effects of publications that are wrong or misleading.\nConclusions\nThis special medical publishing event was marked by incorrect coding and a desire for maximum publicity. The IHCA review authors, their employers, and the Cochrane Collaboration were responsible for quality control, and failed. The mass media played their part by widely publicizing a sensational message but not reacting to the notice that that sensational message was false. The false result that patients are clinically harmed by interactive applications was very strongly delivered to patients worldwide. The broad lesson to be re-learned is that potentially sensational results should be carefully scrutinized before being sensationalized.","keyphrases":["patients","mass media","retraction of publication","online systems","medical errors","editorial policies"],"prmu":["P","P","R","R","R","M"]}
{"id":"Virchows_Arch-4-1-2329729","title":"The significance of lobular neoplasia on needle core biopsy of the breast\n","text":"The management of a core biopsy diagnosis of lobular neoplasia is controversial. Detailed radiological\u2013pathological review of 47 patients with cores showing classical lobular neoplasia was performed (patients with pleomorphic lobular carcinoma in situ (LCIS) or associated risk lesions were considered separately). Immediate surgical excision in 25 patients showed invasive carcinoma in 7, ductal carcinoma in situ (DCIS) in 1 and pleomorphic LCIS in 1; radiological\u2013pathological review showed that the core biopsy missed a mass in 5, missed calcification in 2 and that calcification appeared adequately sampled in 2. Nineteen patients had follow-up of at least 2 years. Four patients developed malignancy at the site of the core biopsy (invasive carcinoma in three, DCIS in one); one carcinoma was mammographically occult, one patient had dense original mammograms and two had calcifications apparently adequately sampled by the core. In conclusion, most carcinomas identified at the site of core biopsy showing lobular neoplasia were the result of the core missing the radiological lesion, emphasising the importance of multidisciplinary review and investigation of any discordance. Some carcinomas were found after apparently adequate core biopsy, raising the question of whether excision biopsy should be considered after all core biopsy diagnoses of lobular neoplasia.\nIntroduction\nLobular carcinoma in situ (LCIS) was described by Foot and Stewart [25]. The term atypical lobular hyperplasia (ALH) was later introduced to describe a similar lesion, but with less marked changes. We prefer the term lobular neoplasia [30], which includes both LCIS and ALH, particularly for core biopsies in which there is limited tissue, making distinction between ALH and LCIS difficult.\nThe relationship between lobular neoplasia and invasive carcinoma of the breast is controversial [28]. Lobular neoplasia is often multifocal and bilateral [4]. There is evidence that it is a risk factor for invasive carcinoma in both breasts: after a diagnosis of lobular neoplasia, there is an increased risk of subsequent invasive carcinoma in both breasts [30], and the majority are ductal in type. Recent evidence suggests that lobular neoplasia may, like ductal carcinoma in situ (DCIS), be a non-obligate precursor for invasive carcinoma at the same site. There is a higher risk of invasive carcinoma in the ipsilateral than in the contralateral breast. The proportion of subsequent carcinomas that are of lobular type is higher than in unselected series of carcinomas [45]. The morphology of the cells in lobular neoplasia and invasive lobular carcinoma is similar. The same truncating mutation in the E-cadherin gene has been found in invasive lobular carcinoma and adjacent LCIS [57].\nThe significance of lobular neoplasia in breast needle core biopsies is uncertain. Invasive carcinomas are sometimes identified after a core biopsy diagnosis of lobular neoplasia. Some studies suggest this occurs because the core biopsy missed the lesion and so does not explain the clinical or radiological abnormality [41]. Other studies suggest that carcinomas may be identified after the diagnosis of lobular neoplasia on core biopsy even if the core biopsy explains the clinical or radiological abnormality [20]. An important consideration in radiological\u2013pathological correlation is whether classical lobular neoplasia is associated with calcification. The traditional view is that lobular neoplasia does not have a radiological correlate [4], but recent reports have described an association between classical lobular neoplasia and calcification. We also wished to investigate our impression that, in core biopsies containing classical lobular neoplasia, calcification is often associated with columnar cell change.\nPleomorphic LCIS is a recently described variant of lobular neoplasia. The growth pattern and absence of E-cadherin expression resemble classical LCIS. However, the marked nuclear pleomorphism, frequent presence of central necrosis and calcification and expression of c-erbB-2 are similar to high-grade DCIS [50]. In the past, pleomorphic LCIS would usually have been diagnosed as high-grade DCIS.\nThe main aim of this study was to assess the risk of invasive carcinoma and DCIS at the site of classical lobular neoplasia diagnosed on breast needle core biopsies. In view of the clear morphological differences, pleomorphic LCIS was considered separately. A second aim was to investigate the localisation of calcification in core biopsies containing lobular neoplasia.\nMaterials and methods\nThis project was discussed with the chair of the Nottingham University Hospitals Research Ethics Committee who considered that it was a service evaluation and, therefore, did not require formal ethical approval. A multidisciplinary review of the pathology and radiology of patients with a core biopsy diagnosis of lobular neoplasia from July 1998 to June 2006 was performed. The biopsies from July 1998 to June 2000 were included in a previous study [35]. The diagnosis of lobular neoplasia was made on haematoxylin and eosin sections. Immunohistochemistry for E-cadherin was performed for lesions with a differential diagnosis including DCIS or pleomorphic LCIS. Lobular neoplasia was subdivided into ALH and LCIS using the criteria described by Page et al. [44]. Pleomorphic LCIS was defined as a high-grade dyscohesive intra-acinar proliferation that was E-cadherin negative.\nPatients were excluded if they had synchronous or previous invasive carcinoma or DCIS in the same breast. Two patients with contralateral invasive carcinoma were included. Core biopsies containing invasive carcinoma, DCIS or an area suspicious of either diagnosis were excluded. Cores containing pleomorphic LCIS, an atypical intraductal epithelial proliferation (including atypical ductal hyperplasia), radial scar or a papillary lesion were considered separately. Thus, the main group of core biopsies studied contained classical lobular neoplasia with no other risk lesions: we termed this group \u201csimple\u201d classical lobular neoplasia.\nThe core biopsies were reviewed for the following features: location of any calcification and presence of columnar cell change. Six cores were not available, so could not be reviewed. The frequency of columnar cell change was compared with 87 consecutive core biopsies reported by one observer (AHSL) as normal or benign.\nResults\nSimple classical lobular neoplasia on core biopsy\nForty-nine core biopsies with lobular neoplasia from 47 women satisfied the entry criteria (0.3% of the 14,597 diagnostic core biopsies performed during this period). Two patients had two core biopsies from the same area of the breast containing lobular neoplasia: both are only counted once in the following results. The median age was 52\u00a0years (range 33 to 81).\nTwenty-six patients presented with mammographic screening abnormalities: 20 with calcification, 3 with a mass, 2 with calcification and distortion and 1 with a mass and calcification. Nineteen patients presented symptomatically: ten with a mass, seven with a thickening and two with a thickening and calcification. Two women had calcification identified by mammography performed in one patient as part of the investigation of a contralateral carcinoma and, in the other, as follow-up of a contralateral carcinoma. Eight core biopsies were freehand, 14 were ultrasound guided and 25 were performed under stereotactic guidance.\nThere was an association between lobular neoplasia and columnar cell change (see Table\u00a01). Calcification was seen in 25 of 41 cores with slides available for review. It was seen in lobular neoplasia in six biopsies (15%); in one the calcification was only seen in the lobular neoplasia, but in the other five, it was also present in other changes (columnar cell change in three, sclerosing adenosis in one, fibroadenoma in one). Of the other 19 biopsies with calcification, columnar cell change was the most commonly associated pathology (10 biopsies).\nTable\u00a01Extent of columnar cell change in cores with lobular neoplasia and controlsColumnar cell changeCore biopsy with lobular neoplasiaControls (normal or benign core biopsy)None12 (29%)58 (67%)One lobule or duct9 (22%)14 (16%)At least two lobules or ducts20 (49%)15 (17%)\u03c72\u2009=\u200918, P\u2009=\u20090.0001\nSurgical excision within 2\u00a0months of core biopsy was performed in 25 patients. Excision showed malignancy in nine patients (see Table\u00a02). In seven patients, pathology\u2013radiology correlation showed that the core biopsy did not explain the radiological abnormality (a mass in five and calcification in two). Five of these seven patients were seen in the first 2\u00a0years of this study. Excision showed benign changes in 16, although one woman had invasive carcinoma of mixed ductal and lobular type diagnosed 33\u00a0months after the core biopsy in a separate quadrant of the same breast.\nTable\u00a02Details of patients with invasive carcinoma, DCIS or pleomorphic LCIS at the site of the core biopsy showing simple lobular neoplasiaAgePresentationCore pathologyTime to excision\/monthsFinal diagnosis (surgical procedure)Pathology\u2013radiology correlation60Screen-detected massALH, CCCh, calcs113 mm IDC, G3, LN 0\/4 (Mx)Core missed mass75Symptomatic massLCIS, UEH, CCCh26 mm IDC, G2, LN not examined (Mx)Core missed mass63Screen-detected distortion and calcification. Mass on USALH114 mm ILC, G2, LN 0\/5 (WLE)Core missed mass54Screen-detected calcificationALH14 mm tubulobular carcinoma G1 + 50\u00a0mm DCIS, LN 0\/6 (Mx)Core missed calcs43Symptomatic massLCIS19 mm IDLC, G2, LN 0\/7 (Mx)Core missed mass68Screen-detected massALH112 mm ILC, G2, LN 0\/6 (Mx)Core missed mass64Symptomatic mass, no mass on US. Mammographic calcificationALH, cyst, calcs17 mm DCIS (WLE)Calcs sampled by core54Screen-detected calcificationALH112 mm tubular carcinoma + 35 mm DCIS, LN 0\/4 (WLE)Core missed calcs45Symptomatic cysts. Mammographic calcificationLCIS, calcs15 mm pleomorphic LCIS (WLE)Calcs sampled by core52Screen-detected calcificationALH, UEH, CCCh, calcs319 mm ILC, G2, LN 1\/6 (Mx)Mammographically occult mass70Screen-detected calcificationALH, calcs2936 mm IDC, G3, LN 6\/16 (Mx)Dense original mammogram51Screen-detected calcificationALH, CCCh, calcs2630 mm DCIS + extensive LCIS (Mx)Calcs sampled by core55Screen-detected calcificationALH, CCCh, calcs29IDC 8\u00a0mm, G3, 45 mm DCIS, LN 1\/4 (Mx)Calcs sampled by coreUS ultrasound, ALH atypical lobular hyperplasia, LCIS lobular carcinoma in situ, UEH epithelial hyperplasia of usual type, CCCh columnar cell change, calcs calcification, IDC invasive ductal carcinoma, ILC invasive lobular carcinoma, IDLC invasive ductal and lobular carcinoma, DCIS ductal carcinoma in situ, G histological grade, LN axillary lymph nodes, Mx mastectomy, WLE wide local excision\nNineteen patients who had not had a diagnostic surgical excision had follow-up of at least 2\u00a0years (range 25 to 105\u00a0months). None of these patients received radiotherapy to the breast. Malignancy was identified in seven women. In four, the malignancy was at the same site as the core biopsy (see Table\u00a02), in one DCIS was found in a different quadrant of the same breast and two developed contralateral invasive carcinoma. Three women had no surgical excision and follow-up of less than 2\u00a0years. Neither of the two women with a previous history of contralateral invasive carcinoma developed carcinoma at the site of the core biopsy showing lobular neoplasia.\nNo significant relationships were found between the diagnosis of malignancy (DCIS, pleomorphic LCIS or invasive carcinoma) at the site of the core biopsy and the following features: age of the woman, diagnosis of ALH or LCIS on the core biopsy, whether core biopsy or vacuum-assisted biopsy was performed and the method of guidance (freehand, ultrasound or stereotactic).\nPleomorphic LCIS on core biopsy\nBoth patients with pleomorphic LCIS on core biopsy presented with calcification detected by mammographic screening. In both, calcification was seen histologically in association with the pleomorphic LCIS. Both had a diagnostic surgical excision, which showed cribriform DCIS in one and further LCIS in the other.\nAtypical intraductal epithelial proliferation\/radial scar\/papillary lesion\nEight patients had atypical intraductal epithelial proliferation, radial scar or a papillary lesion in addition to lobular neoplasia on the core biopsy. All eight presented with abnormalities detected by mammographic screening: calcification in five, calcification and distortion in one, mass in one and distortion in one. Seven patients had a diagnostic surgical excision, and one patient had her lesion removed with vacuum-assisted mammotomy. DCIS was found in one of three women with core diagnosis of atypical intraductal epithelial atypia and in one of two with core diagnosis of radial scar. Papillary DCIS with a 3-mm focus of invasion was found after a core diagnosis of papillary lesion with atypical intraductal epithelial proliferation. One patient with a core diagnosis of radial scar with atypical intraductal epithelial proliferation and one with a papillary lesion had benign findings at excision.\nDiscussion\nRelation between lobular neoplasia and calcification\nAn important question in the management of lobular neoplasia on core biopsy is the relationship between lobular neoplasia and calcification. Lobular neoplasia is not typically associated with any specific clinical abnormality, and there are no characteristic macroscopic features. Early papers suggested that classical lobular neoplasia does not have any diagnostic mammographic features [4]. In the past, surgical biopsies containing lobular neoplasia were frequently performed for mammographic calcification, but pathologically, the calcification was mostly associated with fibrocystic change and identified within the lobular neoplasia in about 20% [4, 11, 24, 55]. Calcification is associated with the lobular neoplasia in between 8% and 53% of core biopsies containing classical lobular neoplasia in different studies [1, 3, 14, 20, 41]. Benign calcification is frequently seen close to invasive carcinomas in surgical specimens [52]. It is well recognised that malignancy may be an incidental discovery on a biopsy for calcification that is associated only with benign disease [49].\nThe present study confirms the association between lobular neoplasia and columnar cell change [8, 41]. In older studies, the majority of surgical biopsies containing lobular neoplasia were performed for fibrocystic change [30]. In the present study, calcification was associated with lobular neoplasia in 15% of core biopsies, but commonly was also seen in fibrocystic change as well. Calcification was associated with columnar cell change in 32% of cores with lobular neoplasia. The association of calcification with columnar cell change is well recognised [27]. Our results support the view that, in biopsies containing lobular neoplasia, most of the calcification is associated with fibrocystic change, particularly columnar cell change. Importantly, the lobular neoplasia is incidental to the calcification in the majority of biopsies.\nA recent study of collagenous spherulosis found that 15 of 59 patients had associated LCIS [48]. In contrast, only one of the patients in the present study with lobular neoplasia had associated collagenous spherulosis.\nRelationship between simple classical lobular neoplasia and carcinoma\nIn this study, 44 women with simple classical lobular neoplasia on core biopsy had surgical excision or follow-up of at least 2\u00a0years. Twenty-five women had immediate surgical excision, which showed invasive carcinoma, DCIS or pleomorphic LCIS in nine (36%). In the majority, the core biopsy was judged to have missed the clinical\/radiological lesion. This emphasises the importance of clinico-pathological review and further investigation of any discordance. Most of the missed lesions occurred in the early part of the study, suggesting that the accuracy of radiological localisation of core biopsies may have improved later in the series. A smaller number of carcinomas occurred after lobular neoplasia on core biopsy apparently incidental to the clinical or radiological lesion. This raises the question of whether excision biopsy should be considered after all core biopsy diagnoses of lobular neoplasia. The frequency of malignancy if the patients with radiological\u2013pathological discordance are excluded is 11% (2\/18).\nFour of 19 patients with at least 2\u00a0years follow-up developed carcinoma at the site of the core biopsy. In one, the carcinoma was mammographically occult, although it was clinically palpable, in one the original mammogram was dense and two women had calcification apparently adequately sampled by the core biopsy. Definite comment cannot be made, but the clinical histories suggest that it is possible that the carcinoma may have been identified earlier in some of these patients if they had had an immediate surgical biopsy. If these women are added to those without radiological\u2013pathological discordance in the above paragraph, the frequency of malignancy is 16% (6\/37).\nPrevious published studies of simple lobular neoplasia on core biopsy were reviewed [1\u20133, 6, 9, 10, 12, 14, 16\u201318, 20, 21, 23, 25, 26, 31, 33, 36, 37, 39, 41\u201343, 46, 47, 51, 54, 58, 59]. The data in most studies are not representative. Probably the major bias is that only about half of the lesions had a surgical biopsy and it is not clear how patients were selected for excision biopsy. Furthermore, most studies only report image-guided or stereotactic biopsies. A surgical biopsy was performed in 561 patients with a core biopsy diagnosis of lobular neoplasia with invasive carcinoma found in 50 or 49 and DCIS in 32 or 33 (82 [15%] in total). It is \u2018impossible to know if some of the reported cases are those in which the targeted lesion has been missed and re-excision was done for failure to successfully sample the target\u2019 [15]. In reviewing the previous studies, we have assumed that sampling was adequate for core biopsy performed for calcification if calcification was present in the core biopsy. Of those 52 malignancies with details available, 25 (48%) had a radiological\u2013pathological discordance (missed mass in 22 and missed calcification in 3), and 27 had apparently adequately sampled calcification.\nOne hundred four patients who did not have immediate surgical excision had reported follow-up. The only four patients (4%) with invasive carcinoma or DCIS on follow-up at the site of core biopsy are the four patients described in the present study. The absence of subsequent malignancy in other studies is very surprising given that lobular neoplasia is a well-established risk factor for later carcinoma. Nevertheless, the low rate suggests that the chance of invasive carcinoma or DCIS is lower in those not having immediate surgical excision.\nIt is difficult to estimate the chance of finding invasive carcinoma or DCIS if all patients had an excision biopsy. The 15% rate found in the subset of patients who had immediate excision biopsy is probably an overestimate. If the cases with radiological\u2013pathological discordance are excluded, the risk is probably about half of this, approximately 8%. If one assumes that half of the patients who did not have an immediate excision did not have invasive carcinoma or DCIS at the core site, this gives an estimate of about 4%. This represents an estimate of the lower limit of risk.\nSome studies suggest that the risk of finding invasive carcinoma or DCIS is a little higher if the core biopsy shows LCIS rather than ALH [13]. In view of the subjectivity of this distinction on core biopsy, it is probably of limited use in routine practice.\nIt is clearly appropriate to investigate patients with simple classical lobular neoplasia on core biopsy that does not explain the clinical or radiological abnormality. If a definitive diagnosis cannot be made with further core biopsy, then an excision biopsy is indicated.\nThe management of simple classical lobular neoplasia on core biopsy with no radiological\u2013pathological discordance is less straightforward. As discussed above, the literature suggests that the risk of malignancy is in the range of 4% to 8%. This risk is comparable to that associated with radial scar or papillary lesion with no epithelial atypia [7, 34, 35, 40, 53]. Both radial scars and papillary lesions are excised in many centres according to current guidance [19]. The major difference with lobular neoplasia, however, is that both are usually clearly defined lesions on radiological examination and therefore easy to excise.\nThere is a need for prospective studies with surgical excision of all lesions so that an unbiased assessment of the risk of simple classical lobular neoplasia on core biopsy can be made. Careful radiology\u2013pathology correlation is essential. It may be possible to stratify the risk within this group using clinical, radiological or pathological features.\nLobular neoplasia associated with radial scars, papillary lesions and atypical intraductal epithelial proliferations\nMost centres excise radial scars, papillary lesions and atypical intraductal epithelial proliferations diagnosed on core biopsy. In the present study, carcinoma was found on excision of three of the eight lesions with these lesions in combination with lobular neoplasia. This supports the practice of excising such lesions.\nPleomorphic LCIS\nIn the present study, both cores with pleomorphic LCIS had associated calcification. In contrast to the controversial nature of the association between calcification and classical LCIS, the literature suggests that there is a clear relationship between calcification and pleomorphic LCIS [29, 50]. There are limited data on the significance of pleomorphic LCIS on core biopsy. One of the two patients in the present study had DCIS in the excision specimen. Three of the six patients described in the literature [5, 20, 37] had invasive carcinoma on excision, but radiology showed a density in one and an architectural distortion in another. Although the follow-up data are limited, we believe that, until there is further evidence, it is prudent to recommend that pleomorphic LCIS is managed as DCIS because of the morphological similarity of pleomorphic LCIS to high-grade DCIS. The present study may underestimate the frequency of pleomorphic LCIS, as E-cadherin immunohistochemistry was infrequently performed early in the study period and some cases may have been diagnosed as high-grade DCIS.\nSome lobular neoplasia shows features overlapping with DCIS and is difficult to classify even with E-cadherin immunohistochemistry [32, 38]. Lobular neoplasia with comedo necrosis, but without marked nuclear pleomorphism, has recently been described [22]. The clinical significance of these rare clinico-pathological scenarios is uncertain. Until there is more evidence, it would be prudent to recommend surgical biopsy of such lesions because of the overlap of features with DCIS. Mass forming lobular neoplasia can occur rarely [56], but is an example of clinico-pathological discordance, so surgical biopsy is prudent.\nIn conclusion, we recommend excision after a core biopsy diagnosis of lobular neoplasia in the following circumstances:\nDiscordance of clinical or radiological findings with the pathological changes (this includes mass-forming lobular neoplasia)Lobular neoplasia with atypical histological features including pleomorphic LCIS, lobular neoplasia with necrosis, and when it is not possible to exclude DCIS despite E-cadherin immunohistochemistryIf there is an associated risk lesion, such as atypical intraductal epithelial proliferation, radial scar or papillary lesion.\nWe suggest that further studies are needed to guide the management of simple classical lobular neoplasia, as the data on the risk of associated carcinoma remain unclear in this group of patients.","keyphrases":["lobular neoplasia","breast","pleomorphic lobular carcinoma in situ","lobular carcinoma in situ","atypical lobular neoplasia","needle biopsy"],"prmu":["P","P","P","P","R","R"]}
{"id":"Anal_Bioanal_Chem-3-1-1592252","title":"Identification of sulfation sites of metabolites and prediction of the compounds\u2019 biological effects\n","text":"Characterizing the biological effects of metabolic transformations (or biotransformation) is one of the key steps in developing safe and effective pharmaceuticals. Sulfate conjugation, one of the major phase II biotransformations, is the focus of this study. While this biotransformation typically facilitates excretion of metabolites by making the compounds more water soluble, sulfation may also lead to bioactivation, producing carcinogenic products. The end result, excretion or bioactivation, depends on the structural features of the sulfation sites, so obtaining the structure of the sulfated metabolites is critically important. We describe herein a very simple, high-throughput procedure for using mass spectrometry to identify the structure\u2014and thus the biological fate\u2014of sulfated metabolites. We have chemically synthesized and analyzed libraries of compounds representing all the biologically relevant types of sulfation products, and using the mass spectral data, the structural features present in these analytes can be reliably determined, with a 97% success rate. This work represents the first example of a high-throughput analysis that can identify the structure of sulfated metabolites and predict their biological effects.\nIntroduction\nThe study of drug metabolism is one of the critical steps in drug development [1\u20134]. At physiological conditions, drugs undergo a variety of biotransformations, which produce metabolites with different chemical structures. The resulting metabolites differ in both pharmacological and toxicological properties compared to their parent drugs [1]. Therefore, to identify and characterize the structures and properties of drug metabolites formed in vivo, an extensive investigation of the structures generated by biotransformation is required.\nSulfate conjugation (sulfation) is one of the most important phase II reactions that occurs during the biotransformation of a variety of structurally diverse endogenous compounds, xenobiotics, and drugs [5\u20139]. During this process, a sulfonate moiety (SO3) is transferred from the donor 3\u2032-phosphoadenosine-5\u2032-phosphosulfate (PAPS) to the substrates through the catalysis of sulfotransferase enzymes, resulting in sulfated products [10]. Sulfation is generally considered as a detoxification pathway, because the sulfated products are more water-soluble; this facilitates their elimination from the body [11\u201313]. However, sulfation can also lead to the bioactivation of certain type of compounds which include benzylic, allylic alcohols and aromatic hydroxylamines. Sulfated products of these compounds can undergo loss of , resulting in reactive electrophilic carbocation or nitrenium ion intermediates, which covalently bind to cellular macromolecules, leading to mutagenicity and carcinogenicity [6, 14, 15]. The production of the electrophilic intermediates relies on the structural feature of sulfation sites, as shown in Fig.\u00a01. Under physiological conditions, the sulfate anions in substrates a\u2013c in Fig.\u00a01 act as good leaving groups to form resonance-stabilized intermediates [6, 14]. For the other types of sulfated products in Fig.\u00a01, this process is not favorable due to instability of cation products.\nFig.\u00a01Mechanism of forming bioactive carbocation or nitrenium ion intermediates from sulfated products. Substrates a, b and c undergo the bioactivation process while d\u2013g do not. a sulfated benzylic alcohol; b sulfated allylic alcohol; c sulfated aromatic hydroxylamine; d) sulfated aliphatic alcohol; e sulfated phenol; f sulfated aliphatic amine; g sulfated aromatic amine\nThe biological sulfation of tamoxifen is one example that demonstrates how the sulfation site dictates the biological fate of the molecule. Tamoxifen is a pharmaceutical that is widely used in the treatment and prevention of breast cancer. It is hydroxylated during phase I metabolism to generate two products: 4-hydroxytamoxifen (4-OH-TAM) and alpha-hydroxytamoxifen (\u03b1-OH-TAM). The sulfation of 4-OH-TAM and that of \u03b1-OH-TAM have completely different biological effects. The sulfation of 4-OH-TAM, which occurs at a phenol site (as in Fig.\u00a01), leads to detoxification, whereas the sulfation of \u03b1-OH-TAM, which involves sulfation of a benzylic alcohol (as in Fig.\u00a01a), leads to bioactivation [16, 17]. This example (along with many others) demonstrates the fact that the biological effects of sulfation can be determined by characterizing the sulfation site of the substrate.\nIt is widely known that aromatic or aliphatic hydroxyl and amine groups are the major sites in molecules that can be sulfated during metabolism [18]. However, identifying the sulfation sites can be difficult because many potential sulfation sites can be produced or modified during phase-I biotransformations [5]. For example, hydroxyl groups can be added to a benzene ring or carbon chain through aromatic or aliphatic hydroxylation, generating new potential sulfation sites as in the tamoxifen example. An amine group can be changed to a hydroxylamine group through N-oxidation [1], which modifies the structural feature of the potential sulfation site. Since these new sites are formed in vivo, characterizing the biological effect of their sulfation can be problematic. It is thus essential to develop a method that can characterize the sulfation sites of unknown sulfated metabolites. With this information, the biological effect of sulfation can be estimated, and the properties of sulfated metabolites can be characterized.\nAnalytical techniques such as NMR spectroscopy and mass spectrometry have been utilized to identify sulfated metabolites [19\u201331]. NMR is a powerful technique that can not only detect sulfated metabolites [20, 21, 24], but also help to determine the position of sulfation in the substrate molecules [19, 22, 23]. However, due to the relatively high sample requirements, the complexity of data analysis, and the need to characterize purified compounds, the application of NMR in identification and characterization of drug metabolites is limited in high-throughput metabolite profiling. Compared to NMR, mass spectrometry is more widely used in drug metabolite identification, due to its high selectivity and sensitivity, low detection limit, and ability to analyze mixtures [32]. It is known that mass spectrometry can identify sulfation by detecting the 80\u00a0Da mass increase in MS mode, or the characteristic ions in MS\/MS mode [21, 25\u201331]. However, limited information about structural features of sulfation sites currently can be obtained from MS\/MS analysis, with the well-known exception that the product ion m\/z 97 () can be used to differentiate alicyclic sulfates from aromatic sulfates [25, 26].\nIn order to facilitate the identification of sulfation sites in pharmaceuticals, we developed a mass spectrometry-based protocol that differentiates between the biologically relevant sulfation sites. After completing the studies on several classes of sulfated products, a set of rules was developed to predict the sulfation sites. With these prediction rules, structural feature of sulfation sites can be determined by detecting MS\/MS fragmentation pathways of their corresponding sulfated products. With the structural information of sulfation sites, the two key different biological effects of sulfation, \u201cdetoxification\u201d and \u201cbioactivation\u201d, can be differentiated, based on literature precedence that links the type of sulfation to its biological fate. Based on that information, relevant properties of sulfated products can be estimated.\nExperimental\nReagents Sulfates and sulfamates which include 4-nitrocatechol sulfate dipotassium salt, L-ascorbic acid 2-sulfate dipotassium salt, indoxyl sulfate potassium salt, \u03b2-estradiol 3-sulfate sodium salt, potassium 4-nitrophenyl sulfate, \u03b2-Estradiol 3-sulfate sodium salt, 5-Br-4-Cl-3-indolyl sulfate potassium salt, 4-methylumbelliferyl sulfate potassium salt, 2-aminoethyl hydrogen sulfate, poly (vinyl sulfate) potassium salt, D-glucose 6-sulfate potassium salt, chondroitin disaccharide \u0394di-6S sodium salt, N-acetylglucosamine 6-sulfate sodium salt, N-cyclohexylsulfamic acid, 3-hydroxypropyl-sulfamic acid monopotassium salt, D-glucosamine 2-sulfate sodium salt, and butyl-sulfamic acid were purchased from Sigma-Aldrich (St. Louis, MO). One sulfamate, 4-methylphenyl- sulfamic acid, was purchased from Scientific Exchange, Inc. (Center Ossipee, NH). (R)(+)-\u03b1-phenethylsulfamic acid was purchased from Norse Laboratories (Newbury Park, CA).The benzylic alcohols, allylic alcohols, hydroxylamines which include (R)-(+)-\u03b1-methyl-2-naphthalenemethanol, benzyl alcohol, (R)-1-phenyl-2-propen-1-ol, 4-chloro-2-methylbenzyl alcohol, 3-ethoxybenzyl alcohol, 2-ethoxybenzyl alcohol, 4-ethoxybenzyl alcohol, crotyl alcohol, furfuryl alcohol, N-methylhydroxylamine hydrochloride, N-isopropylhydroxylamine hydrochloride, N-cyclohexylhydroxylamine hydrochloride, N, N-diethylhydroxylamine and N-benzoyl-N-phenylhydroxylamine were purchased from Sigma-Aldrich (St. Louis, MO). These benzylic alcohols, allylic alcohols, and hydroxylamines were utilized to synthesize their corresponding sulfated products. The other reagents used in the sulfation of alcohols and hydroxylamines were also purchased from Sigma-Aldrich (St. Louis, MO).\nSulfation of benzylic, allylic alcohols and tertiary hydroxylamines Sulfated products were prepared based on slight modifications to a previous protocol [33] by dissolving 1.0 equivalent (5.0\u00a0mmol) of the substrate in 5.0\u00a0ml dimethylformamide (DMF) and adding sulfur trioxide-dimethylformamide complex (DMF-SO3 5.5\u00a0mmol) and pyridine (5.5\u00a0mmol). The reaction mixture was stirred at 40 \u00b0C for 1\u00a0h. H2O (35\u00a0ml) was added to the product solution, and the product was extracted with 3\u00d715\u00a0mL ethyl acetate (EtOAc). Evaporation of the solvent afforded the corresponding sulfated product.\nSulfation of secondary hydroxylamines To a dry THF (10\u00a0ml) solution of NaH (10.0\u00a0mmol) was added secondary hydroxylamines (5\u00a0mmol) at 0\u00a0\u00b0C. Then the solution was stirred at room temperature for 2\u00a0h, followed by reaction with DMF-SO3 (5.0\u00a0mmol) overnight. The corresponding sulfated products were obtained by evaporation of THF.\nSample pretreatment Sulfated compounds that were purchased were dissolved to a final concentration of 1.0\u00d710\u22124\u00a0M with 50% MeOH\/ 50% H2O, and directly injected into the mass spectrometer. Sulfated benzylic and allylic alcohols and tertiary hydroxylamines were prepared by diluting 10\u00a0\u03bcl of the liquid product by 100 fold with 50% MeOH\/50% H2O, and then injected to the mass spectrometer. Sulfated secondary hydroxylamines were prepared by dissolving 10\u00a0mg solid product in 1.0\u00a0ml 50% MeOH\/ 50% H2O, followed by injection into the mass spectrometer.\nMass spectrometry The mass spectra were recorded using a Quattro Ultima (Waters Corp., Milford, MA) triple quadrupole mass spectrometer equipped with an electrospray ionization source. Data was acquired in the negative ion mode using a capillary voltage of 2.80\u00a0kV and a cone voltage was 45\u00a0V. The source temperature and desolvation gas temperature were 80 and 150 \u00b0C, respectively. Argon is used as the collision gas, and the pressure in collision cell is 1.7E(\u22123) mbar. The collision energy applied in MS\/MS for all the samples was 35\u00a0eV.\nResults and discussion\nThe structures of the sulfated products are in Fig.\u00a02. These compounds are representative of different types of sulfated products that are produced by sulfation at different sites. Mass spectrometric studies were conducted in the negative ion mode since sulfated products are deprotonated at physiological pH [11, 15]. The common product ions from the MS\/MS experiments are summarized in Table\u00a01. As the table indicates, common product ions include m\/z [M-H-80], m\/z 80, m\/z 96 and m\/z 97. The presence of m\/z [M-H-80], m\/z 96 or m\/z 97 ions are quite dependent on the site of sulfation. The presence of the negative ion with m\/z 80, however, is common in the fragmentation of almost every type of sulfated products, so it is less helpful in identification of sulfation sites. Thus this ion, m\/z 80, is used to identify sulfation sites when no other characteristic ions can be detected. Based on the MS\/MS data, each type of sulfated product possesses a characteristic fragmentation pathway, which is described below. An example of MS\/MS data for each type of sulfated product is provided in Fig.\u00a03 with the characteristic ion labeled by an asterisk.\nFig.\u00a02Structures of selected sulfated compounds a sulfated aromatic alcohols or enols; b sulfated aliphatic alcohols with \u03b2 hydrogens on sp3carbons; c sulfated benzylic or allylic alcohols; d sulfated hydroxylamines; e sulfated aminesFig.\u00a03Characteristic (-) ESI-MS\/MS data for compounds in this study: a sulfated aromatic alcohol, from group a; b sulfated aliphatic alcohol with \u03b2 hydrogen on sp3carbon, from group b; c sulfated benzylic alcohol, from group c; d sulfated aliphatic hydroxylamine, from group d; e sulfated aromatic hydroxylamine, from group d; f sulfated aliphatic amine, from group e; g sulfated aromatic amine, from group eTable\u00a01Characteristic fragmentation of selected sulfated productsGroupNo.Chemical nameAbundance of characteristic ions in MS\/MS (%)m\/z [M-80]-m\/z 80m\/z 96m\/z 97a14-nitrocatechol sulfate100a\u2013\u2013\u20132L-ascorbic acid 2-sulfate31a\u2013\u2013\u20133Indoxyl sulfate24a52\u2013\u201344-nitrophenyl sulfate100a\u2013\u2013\u20135\u03b2-estradiol 3-sulfate100a\u2013\u2013\u201365-Br-4-Cl-3-indolyl sulfate47a59\u2013\u201374-methylumbelliferyl sulfate100a\u2013\u2013\u2013b82-aminoethyl hydrogen sulfate \u20131001867a9Poly (vinyl sulfate)\u2013\u2013\u201397a10D-glucose 6-sulfate\u2013\u2013\u2013100a11Chondroitin disaccharide \u0394di-6S \u2013\u2013\u201331a12N-acetylglucosamine 6-sulfate\u2013\u2013\u201393ac132-naphthalenemethanol, \u03b1-methyl- sulfate\u20136.77.3a10014Benzyl sulfate\u201316100a\u201315(R)-1-phenyl-2-propen-1-sulfate\u201326100a12164-Cl-2-methylbenzyl sulfate\u2013\u2013100a95173-ethoxybenzyl sulfate\u201310013a6.4182-ethoxybenzyl sulfate\u201310017a8.5194-ethoxybenzyl sulfate\u201310020a8.720crotyl sulfate\u20135591a5021furfuryl sulfate\u2013\u201353a39d22N-methyl-hydroxylamine-O-sulfonic acid\u201310073a9.223N-isopropyl- hydroxylamine-O-sulfonic acid\u201368100a5.124N-cyclohexyl-hydroxylamine-O-Sulfonic acid\u201327100a1125N,N-diethyl-hydroxylamine-O-sulfonic acid\u201392100a1926N-benzoyl-N-phenyl-hydroxylamine-O-sulfonic acid\u2013\u2013100a\u2013e27N-cyclohexylsulfamic acid\u2013100a\u2013\u2013284-methylphenyl-Sulfamic acid\u2013100a\u2013\u2013293-hydroxypropyl-sulfamic acid\u2013100a\u2013\u201330D-glucosamine 2-sulfate\u2013100a\u2013\u201331butyl-sulfamic acid\u2013100a\u2013\u201332(R)(+)-\u03b1-phenethylsulfamic acid\u2013100a\u2013\u2013*A threshold of 5.0% is used for the relative abundance of characteristic ions. The long dash (\u2013) means that the characteristic ion can not be detected or the relative abundance is below 5.0%aRepresents the specific characteristic ion of each group that can help to identify the sulfation site\nGroup a: sulfated aromatic alcohols or enols\nCompounds 1\u20137 are sulfated aromatic alcohols or enols. For these compounds, the sulfate group is attached to an sp2 carbon (benzene ring or double bond). These sulfated products share the same fragmentation pathway by undergoing the neutral loss of 80\u00a0Da, and forming a characteristic ion at m\/z [M-H-80], as seen in Fig.\u00a03a. After undergoing the neutral loss of SO3, phenoxide or enolic anions are formed, and the negative charge is resonance stabilized, as shown in Scheme\u00a01 [33].\nScheme\u00a01Sulfated aromatic alcohols or enols (group a) dissociate to produce a characteristic ion with neutral loss of 80 Da. Group b: sulfated aliphatic alcohols with \u03b2 hydrogens on sp3carbons\nThe second group of sulfated products originates from the sulfation of aliphatic alcohols: compounds 8\u201312. For these species, the sulfate group is attached to an sp3 carbon. All sulfated products of this type can produce the characteristic ion m\/z 97 in MS\/MS, and one example is shown in Fig.\u00a03b. The ion m\/z 97 is generated when the proton from the \u03b2 carbon is transferred to the sulfate moiety, and the C-O bond is broken to form a bisulfate anion [33]. See Scheme\u00a02.\nScheme\u00a02Sulfated aliphatic alcohols (group b) produce the characteristic ion m\/z 97\nThis rearrangement occurs via an energetically accessible six-membered ring transition state, and a stable neutral product is formed. As a result, this fragmentation is predominant in the MS\/MS data of this group of sulfated products. The mechanism shows that the availability of a \u03b2 hydrogen on an sp3 carbon is required for this fragmentation. Unlike compounds in group a, neutral loss of 80\u00a0Da is not observed from this group of compounds. This is likely due to the fact that the product ion that would be generated from loss of 80\u00a0Da (SO3) is not resonance stabilized, so the loss is not favorable. Compound 8 is a special case because it gives a characteristic ion with m\/z 96, in addition to m\/z 97. The relevant mechanism for this loss is addressed in the discussion of compounds in group d.\nGroup c: sulfated benzylic or allylic alcohols\nIn the third group of sulfated compounds (13\u201321), each contains a sulfate group attached to benzylic or allylic carbon. All the compounds in this group produce a characteristic ion with m\/z 96. An example of an MS\/MS data for this type is shown in Fig.\u00a03c. The characteristic ion is produced by homolytic cleavage, producing benzylic or allylic radicals. The radical is resonance stabilized, which is demonstrated in Scheme\u00a03 [33].\nScheme\u00a03Sulfated benzylic and allylic alcohols (group c) produce the characteristic ion m\/z 96\nIn addition to m\/z 96, the negative ion m\/z 97 can also be obtained for some of the compounds in this category. For example, compounds 13 and 16 both have an abundant peak at m\/z 97, due to the availability of a hydrogen on the sp3 carbon in close proximity to the sulfate group. Even though compound 13 is a benzylic sulfate, a hydrogen attached to the \u03b2 sp3 carbon is present. Therefore, just like compounds in group b, it undergoes a rearrangement to form the ion, m\/z 97. Compound 16 possesses a methyl group in the ortho position, and the availability of hydrogen attached to the sp3 carbon in methyl group enables a similar rearrangement (eight-membered ring) to form the product ion, m\/z 97, as illustrated in Scheme\u00a04. Even though \u03b2 hydrogens are available in other benzylic and allylic sulfates in this group, the fragmentation to produce m\/z 97 is not as favorable, since the hydrogens are on the sp2 hybridized carbons. The neutral loss of 80\u00a0Da is not favorable either, because the product ion that would be generated from such a loss is not resonance stabilized.\nScheme\u00a04Fragmentation mechanism producing m\/z 97 from compound 16\nGroup d: sulfated hydroxylamines\nThe fourth group of sulfated products originates from sulfation of hydroxylamines, and as a result, the sulfate group is directly attached to the amine: See compounds 22\u201326. All the sulfated products of this group can produce the characteristic ion m\/z 96, and examples of MS\/MS data are shown in Fig.\u00a03. A characteristic ion with m\/z 96 can be obtained for sulfated products originating from both aliphatic (Fig.\u00a03d) and aromatic (Fig.\u00a03e) hydroxylamines. For these sulfated compounds, homolytic cleavage is favorable. This is due to the small electronegativity difference (about 0.5) between N and O, compared to electronegativity difference of around 1.0 for C \u2013N and O-S [34]. The small electronegativity difference causes the electrons to be split equally (homolytic cleavage) between the oxygen and nitrogen when the bond breaks, instead of both electrons moving onto the oxygen or nitrogen, which would be the case during heterolytic cleavage. In Scheme\u00a05, the mechanism of this homolytic cleavage is depicted. An amine radical is formed as the product.\nScheme\u00a05Sulfated hydroxylamines (group d) produce the characteristic ion m\/z 96\nThis mechanism can also be used to explain the presence of the ion with m\/z 96 in the MS\/MS spectrum of compound 8. Scheme\u00a06 shows how this ion is produced from compound 8. In this case, homolytic cleavage can be used to distribute a single electron onto the nitrogen. Compounds that contain a nitrogen two carbons away from the sulfation site, like this one, could also produce m\/z 96 as a product ion.\nScheme\u00a06Fragmentation mechanism producing m\/z 96 from compound 8\nGroup e: sulfated amines\nThe last group of sulfated products, sulfamates, originates from the sulfation of amines. Compounds 27\u201332 belong to this type. The only characteristic ion for this type of sulfated products is the negative ion, m\/z 80. This characteristic ion can be obtained from fragmentation of sulfated products originating from both aliphatic amines (Fig.\u00a03f) and aromatic amines (Fig.\u00a03g). This group of sulfated products has another type of bond, a N-S bond, that has a small electronegativity difference between the two atoms (0.5) [34], making the homolytic cleavage between the nitrogen and sulfur favorable. The mechanism that shows production of m\/z 80 is illustrated in Scheme\u00a07. No other fragmentation pathway is as favorable as this homolytic cleavage pathway, making m\/z 80 the predominant ion in all of the MS\/MS data for these compounds.\nScheme\u00a07Sulfated amines (group e) produce the characteristic ion m\/z 80\nPrediction rules\nFor each type of sulfated product, there is a predominant fragmentation pathway that results in a characteristic product ion during MS\/MS fragmentation. The specific characteristic ion for each group is obtained from every sulfated product in that group, as shown in Table\u00a01: the ion with the neutral loss of 80\u00a0Da can be seen in MS\/MS spectrum of every sulfated product in group a; an ion with m\/z 97 can be detected in all the MS\/MS data of group b compounds; an ion with m\/z 96 can be obtained from MS\/MS data of all the sulfated products of both group c and group d. A negative ion with m\/z 80 can always be found in MS\/MS data of sulfated products in group e.\nWe have shown that characteristic fragmentation pathways of sulfated products are dependent upon the structural features of the sulfation sites. Conversely, relevant structural information of sulfation sites can be obtained by determining characteristic fragmentation pathways or characteristic ions. To facilitate this information transformation, a set of prediction rules is described as follows: \nIf the characteristic ion with a neutral loss of 80\u00a0Da can be detected, this indicates that the sulfate group is attached to an sp2 carbon, the sulfated product is therefore produced by the sulfation of a phenol or an enol.If the characteristic ion with m\/z 96 can be detected, the sulfated product is produced by sulfation of a benzylic or allylic alcohol, or a hydroxylamine.If the characteristic ion with m\/z 97 can be obtained, and it is more abundant than the ion, m\/z 96 (if it is detected), the sulfate group is attached to an sp3 carbon. This is the sulfated product produced by sulfation of aliphatic alcohols with an available \u03b2 hydrogen attached to an sp3 carbon;If none of the ions, m\/z [M-H-80]\u2212, m\/z 96, or m\/z 97 can be detected, and only the ion m\/z 80 is observed, then the sulfated product is produced by sulfation of an amine.\nThere are occasions when the conditions in rule 2 and 3 might both be observed. For example, both of these apply to compound 13, which suggests that the sulfation site is not only an alcohol with available \u03b2 hydrogen (on an sp3 carbon), but also a benzylic or allylic alcohol; and this estimation is consistent with the actual structure of compound 13. Among the selected 32 sulfated products, 31 of them follow the proposed prediction rules. The exception, compound 8, possesses an amine group in the \u03b2 position. The amine at this position enables the compound undergo the fragmentation pathway to produce ion with m\/z 96 (see Scheme\u00a06). In this case, rule 2 incorrectly predicts the sulfation site for compound 8. As a result, the obtained data in Table\u00a01 demonstrates a 97% (31\/32=0.97) success rate of these prediction rules.\nPotential application of the prediction rules in characterizing unknown sulfated metabolites\nAs one of the major phase II biotransformations, sulfate conjugation is involved in the metabolism of an enormous range of substrates [5]. In general, sulfation is a detoxification or deactivation pathway, but it can also bioactivate the substrate molecules when sulfation sites are benzylic, allylic alcohols (group c) or aromatic hydroxylamines (group d) [6, 8, 14]. According to the prediction rules developed herein, sulfation at any of these sites will generate products that produce the ion m\/z 96 in MS\/MS, so the structural feature of the sulfation sites can be determined by rule 2. Sulfation of other sites, like phenols, aliphatic alcohols or amines, will cause other characteristic ions to appear in MS\/MS data. Therefore, the sulfation sites can be determined by rule 1, 3 or 4. In these cases, sulfation facilitates detoxification.\nWith the proposed prediction rules, biological effects of sulfation can be determined, and the properties of sulfated metabolites can be estimated. Figure\u00a04 demonstrates how the proposed method works to characterize unknown sulfated metabolites. Since high-quality MS\/MS data is a necessary prerequisite of using this method, if the metabolite is present in a complex biological matrix, some mass spectral optimization may be necessary. After obtaining reliable (-)ESI-MS\/MS data on the unknown sulfated metabolite, the prediction rules are used to determine sulfation site by identifying the characteristic ions present in the MS\/MS data. If the MS\/MS data matches rule 1, 3 or 4 (instead of rule 2), the sulfation sites can be phenols (or enols), aliphatic alcohols (with \u03b2 hydrogen on sp3 carbon) or amines. In these cases, the sulfation undergoes a detoxification pathway and the sulfated metabolite is more easily eliminated, compared to the parent drug. If the MS\/MS data is consistent with rule 2, the sulfation site should be benzylic, allylic alcohols or hydroxylamines. This would indicate that the sulfation might follow a bioactivation pathway, and lead to carcinogenic sulfated products. One potential caveat to this second condition is that a few nontoxic sulfated metabolites that have structures similar to compound 8, might also produce the characteristic ion with m\/z 96, following the mechanism in Scheme\u00a06. Another possible drawback of this characterization is that the diagnostic ion m\/z 96 does not discriminate between sulfated products of aliphatic and aromatic hydroxylamines; and thus far only aromatic hydroxylamines have been proven to be toxic. However, distinguishing between aromatic and aliphatic hydroxylamines is not critically important because sulfated aliphatic hydroxylamines are rather uncommon metabolites. While it is possible that a few compounds could be misassigned as \u201ctoxic\u201d, none of the sulfated metabolites that are known to be bioactivated would be misdiagnosed as \u201cnontoxic\u201d, as demonstrated herein.\nFig.\u00a04The method for characterizing unknown sulfated metabolites\nConclusion\nA method was developed to determine the structural features of sulfation sites, by detecting the characteristic fragmentation pathway of the corresponding sulfated products in (-) ESI-MS\/MS. By summarizing MS\/MS data from five different types of sulfated products originating from different sulfation sites, their characteristic fragmentation pathways and characteristic ions were determined. Based on this information, a set of prediction rules was developed to transfer information about the fragmentation pathway of sulfated products to the structural features of the sulfation site. As a result, the proposed prediction rules can be applied in drug metabolite profiling to characterize sulfation sites, to further estimate the biological effect of sulfation, and to evaluate relevant properties of sulfated metabolites.","keyphrases":["sulfation","metabolites","pharmaceuticals","mass spectrometry","icp-ms","bioanalytical methods","biological samples"],"prmu":["P","P","P","P","U","M","R"]}
{"id":"Anal_Bioanal_Chem-4-1-2270354","title":"Neutron activation analysis and X-ray Rayleigh and Raman scattering of hair and nail clippings as noninvasive bioindicators for Cu liver status in Labrador Retrievers\n","text":"The heritability of chronic hepatitis in the Labrador Retriever is studied with the aim of identifying the related gene mutation. Identification of cases and controls is largely based on instrumental neutron activation analysis (INAA) Cu determination in liver biopsies. The burden for these companion animals may be reduced if nail clippings and hair (fur) could serve as a noninvasive indicator for the hepatic Cu concentrations. No correlation was found between hepatic Cu concentrations and Cu concentrations in hair and nail samples. However, hair and nail samples were also analyzed by X-ray tube excitation, taking advantage of the X-ray Compton, Rayleigh, and Raman scattering which reflects the organic components such as the type of melanin. Principal component analysis provided first indications that some differentiation between healthy and sick dogs could indeed be obtained from hair and nail analysis.\nIntroduction\nChronic hepatitis in dogs is a histological diagnosis, characterized by the presence of fibrosis, inflammation, and necrosis of the liver cells. Symptoms of chronic hepatitis are quite variable but the most common signs are lethargy, loss of appetite, and diarrhea. Pets may also drink and urinate more. As the disease progresses, many dogs develop yellowish gums, eyes, and skin (icterus\/jaundice), and a swollen abdomen which is filled with fluid (ascites). In some cases toxins affect the nervous system and the dogs become blind and obtuse. This can progress to seizures, coma, and death. The disease is characterized, amongst others, by copper accumulation in the liver. Previous studies have shown elevated hepatic Cu concentrations in the order of 1,000\u20132,000\u00a0mg\u00a0kg\u22121 compared with normal Cu levels of <\u2009200\u2013500\u00a0mg\u00a0kg\u22121. Such hepatic copper accumulation can result from increased uptake of copper, a primary metabolic defect in hepatic copper metabolism, or from altered biliary excretion of copper. Several studies have already been conducted at Utrecht University on inherited copper toxicosis and hepatic copper storage in companion dogs such as Bedlington Terriers [1] and Dobermanns [2]. Currently, one of the programs is investigating the genetic basis of the disease by examination of family members from Labrador Retrievers with copper-associated chronic hepatitis.\nThe selected dogs undergo blood sampling, and retrieval of a liver biopsy (by use of the Menghini technique). The liver biopsy is examined histologically and stained for copper granules. As this project deals with companion animals from the Labrador population in The Netherlands and not with test animals, cooperation of the dog owners is crucial. Following the Delft group\u2019s involvement and over 20\u00a0years\u2019 experience investigating nail clippings as a bioindicator for the trace element status in man [3, 4], a feasibility study was undertaken to assess the usefulness of nail clippings and hair (fur) as a noninvasive indicator for the Cu liver levels with the aim of replacing the transcuteaneous full-needle biopsy.\nTwo approaches were tested. The most straightforward was to verify if Cu levels in nail clippings and hair would reflect the Cu liver levels. The other approach was initiated by the observation that a copper deficiency may result in hair\/coat color changes. This is ascribed to the activity of cuproenzymes, which catalyze the biosynthesis of melanin from L-tyrosine. A copper deficiency causes, among others, pigmented hair on the head and face to lose its normal color and get a \u201cwashed out\u201d appearance and become gray [5].\nIn 2005, Bueno et al. demonstrated that X-ray Rayleigh peaks and especially the peakless part in the X-ray Raman scattering continuum in X-ray spectra obtained by tube excitation carry information on the organic constituents of biological materials [6, 7]. A study of Poodles in which all spectral information was processed by dedicated principal component analysis (PCA) resulted in a clear differentiation of all dogs examined on the basis of their hair color and, moreover, the separation of healthy and sick dogs on the basis of the hair analysis. Hair pigmentation is due to two major types of melanin, viz., eumelanin and phaeomelanin (Fig.\u00a01). The type of melanin manifesting in the hair can be attributed to the hair color genotype [8, 9], whereas the intensity and distribution of the granules may also be affected by diseases in which sulfur metabolism is involved.\nFig.\u00a01Chemical structures of eumelanin and phaeomelanin\nBoth approaches were tested here, i.e., (i) Cu determination via instrumental neutron activation analysis (INAA) in liver biopsies, nail clippings, and hair samples; and (ii) X-ray spectrum acquisition after X-ray tube excitation, followed by full spectrum data (i.e. all channel contents) processing by PCA, including the often neglected peakless part of the X-ray scattering region resulting from Compton, Rayleigh and Raman effects.\nExperimental\nLiver biopsy samples, nail clippings, and hair were collected from approximately 100 dogs with chronic hepatitis as well as from healthy animals. Nail clippings were taken from phalanx I (the thumb nail), since this nail is not in contact with the ground, thus minimizing contamination problems. Nail clippings could not be collected from all animals as in some cases breeders had removed the nail permanently shortly after birth, since sometimes such a thumb nail hinders the dog in working trials. Hair was sampled from the median abdominal wall, just cranial to the belly button.\nThe liver biopsy samples were freeze-dried at the veterinary clinic. The nail clippings and hair samples were analyzed without cleaning to prevent changes in the nail and hair morphology that might affect the scattering information from the X-ray fluorescence (XRF) analysis. It should be noted that a Cu contamination of the nail clipping and hair is highly unlikely; the nail of the thumb nail is never in contact with, e.g., soil, and external contamination of the hair was also unlikely.\nINAA\nThe needle biopsies were transferred into polyethylene capsules and lyophilized, resulting in sample masses varying typically between 1.5 and 50\u00a0mg dry weight. Nail clippings had masses of 5\u201350\u00a0mg, and the typical mass of the hair samples was approximately 100\u2013200\u00a0mg.\nThe Cu levels were established by INAA via the determination of 66Cu. This is the radionuclide of choice although 64Cu is also produced; however, the associated 511-keV peak is insufficient for reliable Cu determination, whereas the 1,345-keV peak provides insufficient sensitivity. Quantification of all element mass fractions was based on the single comparator method [10]. Samples were irradiated one-by-one for 3\u00a0min in the Hoger Onderwijs Reactor in a thermal neutron fluence rate of 1.7\u2009\u00d7\u20091017\u00a0m\u22122\u00a0s\u22121. A Zn flux monitor was included in each irradiation; the neutron fluence rate was determined by measurement of the induced 69mZn activity. The activated biopsy, nail, and hair samples were measured 30\u00a0s after irradiation for 3\u00a0min at a 1-cm distance from the end-cap of a side-looking 12% Ge detector. The samples were rotated during counting. The Zn monitors were measured in the same geometry for 60\u00a0s after a 4-min decay time. The samples were processed in batches of 14 samples, one internal quality control sample, and a blank. A sample of NBS 1577b Bovine Liver (certified Cu content 160\u2009\u00b1\u20098\u00a0mg\u00a0kg\u22121) was used for internal quality control. Details of the irradiation facility, gamma-ray spectrometer, and associated quality assurance have been published elsewhere [11]. The spectrometer is equipped with ORTEC DSPEC-PLUS electronics, which allows for adequate dead-time correction [12]. It is obvious that, particularly in the nail and hair samples, several other elements such as Ca, Mg, and S were also quantified on the basis of their short halflife radionuclides, measured simultaneously with 66Cu.\nX-ray tube excitation\nThe measuring procedure consisted of weighing approximately 100\u00a0mg of each hair sample and 10\u00a0mg of each nail sample directly into appropriate cells and submitting them to blank rhodium X-ray tube radiation, in triplicate. A common laboratory energy dispersive XRF instrument (EDX 700, Shimadzu) was used under the same irradiation conditions: 50-kV applied voltage in the tube, 25% dead time, 10-mm beam collimation, and 100-s irradiation time. It should be noted that the methodology of using the region in the X-ray spectrum resulting from scattering implies that only the spectrum\u2019s shape is needed for the data processing by PCA. There is no need for quantification towards Cu mass fractions, and hence no reference materials were used for quality control as was the case in the hair, nail, and liver biopsy analysis by INAA.\nThe chemometric method [13] used for data analysis was PCA, processed via the software The Unscrambler, version 9.2, from Camo.\nResults and discussion\nCu could not be determined in all available nail samples because of the small sample masses and the detection limit of the analytical protocol selected. Detection limits on the basis of 66Cu were typically in the order of 25\u00a0ng, corresponding to a minimum detectable Cu mass fraction of 5\u00a0mg\u00a0kg\u22121 in a 5-mg nail clipping sample. Cu levels in hair and nail are given in Fig.\u00a02, together with the associated Cu liver levels. Uncertainties are not depicted for clarity reasons; typical uncertainties (combined standard uncertainty) were approximately 5\u201320% for the liver analyses, 15\u201330% for the nail, and 5\u201310% for the hair analyses. It is clear from Fig.\u00a02 that the Cu levels in hair and nail do not reflect the Cu liver levels. This indicates that the gene(s) responsible for the Cu accumulation in the liver is(are) only active in the liver cells and not in the cells at the nail base or in the hair pocket.\nFig.\u00a02Cu levels in nail clippings and hair of Labrador Retrievers as a function of the Cu levels in liver biopsies taken from the same dogs: \u25cb hair, \u25cf nail; uncertainties are omitted for clarity (see text)\nPrincipal component analysis (PCA) results of the processing of the channel contents of the X-ray spectra of all hair samples are given in Fig.\u00a03, and the related factor loadings of PC1 and PC2 are shown in Fig.\u00a04 (similar results were found for the processing of the channel contents of the X-ray spectra of the nail samples, although these are not shown here). The PC1 loading graph indicates the dependent variables that explain most of the data variance, and the PC2 loading graph indicates variables that explain the second data variance. PC1 and PC2 are orthogonals and therefore independent of each other.\nFig.\u00a03PCA analysis of all hair samples, classified here by the hair color: \u25a1 yellow, * chocolate, \u25a0 blackFig.\u00a04PC1 loadings for hair samples (top). PC2 loadings for hair samples (bottom)\nThe variables included in the PCA analysis were from the channel contents between 0 and 40\u00a0keV, i.e., covering the entire spectum recorded.The symbols in Fig.\u00a03 reflect the hair color. The differentiation with respect to coat color results most likely from differences in type of melanin in Labradors with a black, liver (chocolate), or yellow-colored coat. Table\u00a01 shows the genotypes of the hair color inheritance of Labrador Retrievers [8], in which each character represents a specific gene (denoted in pairs, of course). An uppercase character represents a dominant gene, a lowercase character represents a recessive gene; no character in a gene pair implies that it is not relevant for the genotype if the locus is occupied by a dominant or recessive gene. As produces black without any tan on the dog and there are other genes that can modify the black to liver (chocolate Labrador). If As is present, in most cases the dog will be able to produce only eumelanin pigment. Some shades of liver (chocolate), though a eumelanin pigment, overlap some shades of tan, a phaeomelanin pigment. The e, recessive red, overrides whatever gene is present at the A locus to produce a dog which shows only phaeomelanin pigment in the coat. The related melanin types are also included in Table\u00a01 and the color differentiation by the principal component analysis is most probably based on the differences in melanin corresponding to the different genotypes. The wide spread of the groups might reflect homozygotes and heterozygotes for the genes involved.\nTable\u00a01Labrador genotypes [8] for hair color inheritance and corresponding types of melaninColorGenotypeMelanin typeBlackAs\u2013\u00a0\u00a0B\u2013\u00a0\u00a0E\u2013EumelaninLiver (chocolate)As\u2013\u00a0\u00a0bb\u00a0\u00a0E\u2013EumelaninYellow, liver nose\u2013 \u2013\u00a0\u00a0B\u2013\u00a0\u00a0eePhaeomelaninYellow, black nose\u2013 \u2013\u00a0\u00a0bb\u00a0\u00a0eePhaeomelanin\nThe PCA also revealed a differentiation to gender, as is further exemplified in Fig.\u00a05 for nails. This is in agreement with earlier work for Poodles in which a similar gender separation was observed [6].\nFig.\u00a05PCA analyses of all nail samples, classified by gender\nThese results (Figs.\u00a03, 4, 5) indicate that PC1 allows for a separation of dogs by gender and PC2 for separation by color, particularly when using hair samples. The loading graphs of the PCs (Fig.\u00a04) show that sulfur and Rh scatter (Compton, Rayleigh, and Raman) explain the variance in PC1. This is most likely attributed to variations in (sulfur bondings in) keratin structures. This effect contributes to almost 100% of the data variance. The rest of it, PC2, is not very large (approximately 1\u201310%) and allows for separations due to differences in sulfur, and to some extent with melanin.\nThe physiological cause of the variation of the keratin structures in hair and nail by gender is not yet fully clear, but there are several indications that the structure of hair is different for males and females. It is well known that there is a relationship between testosterone and hair formation (andropogenic hair). Andropogenic hair has a different growth rate and a higher weight than other hair. It has also been observed that the type of sulfur is different for females and males: rhombic (alpha) sulfur predominates in female hairs, monoclinical (beta) sulfur in male hairs [14]. Less information is available about nails. Genetic variation in the proteins of the human nail has been observed [15], as well as a sex variation in lipid composition of human fingernail plates [16].\nAnother possible explanation for the separation of dogs in terms of genders in the hair analysis might be found in the results of Scott et al. [17] who concluded that the human melanocortin 1 receptor (MC1R) is regulated by, amongst others, specific endocrine sex hormones and by UV radiation. In confirmation of the latter study, Broekmans et al. [18] found that male subjects were more sensitive to UV irradiation than female subjects. It was already known that eumelanin is photoprotective, whereas phaeomelanin may contribute to UV-induced skin damage due to its potential to generate free radicals in response to UV radiation [19]. Skin melanin content, which was positively associated with hair color in men, was the main phenotypical determinant of sensitivity to UV irradiation.\nThe PCA results could also be plotted as a function of the INAA Cu liver biopsy values. Results for hair and nail are given in Fig.\u00a06 for a differentiation towards dogs with Cu liver levels of <\u2009600\u00a0mg\u00a0kg\u22121 or >\u2009600\u00a0mg\u00a0kg\u22121. A Cu biopsy level of >\u2009600\u00a0mg\u00a0kg\u22121 is considered to be positively indicative for liver Cu accumulation. In addition, plots were also made in which the hair and nail samples are identified by correlating with Cu biopsy values > and <\u2009200\u00a0mg\u00a0kg\u22121 as well as at > and <\u2009400\u00a0mg\u00a0kg\u22121. At these lower Cu levels no clear differentiation is obtained, as could be expected, which confirms that the PCA is not producing an artifact, accidentally coinciding at the 600\u00a0mg\u00a0kg\u22121 Cu liver biopsy level.\nFig.\u00a06PCA results, nail X-ray spectral data vs. INAA Cu liver biopsy values (top): \u25a0 dogs with Cu liver levels 0\u2013600\u00a0mg\u00a0kg\u22121, \u25a1 dogs with Cu liver levels >\u2009600\u00a0mg\u00a0kg\u22121 , N\u2009=\u200960. PCA results, hair X-ray spectral data vs. INAA Cu liver biopsy values (bottom): \u25a0 dogs with Cu liver levels 0\u2013600\u00a0mg\u00a0kg\u22121, \u25a1 dogs with Cu liver levels >\u2009600\u00a0mg\u00a0kg\u22121, N\u2009=\u200943\nIn all PCA, it could be justified to remove some outliers on the basis of high leverage values and high energy variance residuals in PCs. High values of leverage and residuals are caused, for instance, by imperfect quality and positioning of the sample during the X-ray irradiation. In addition, outliers may sometimes result from the presence of dogs with strong parental relationships [6].\nThe strength of using the information of the scattered radiation is emphasized by the role of sulfur in the separation towards gender and Cu accumulation. Total sulfur, as determined by INAA via 37S, hardly shows any correlation with INAA Cu liver values (correlation coefficients 0.03 and 0.32 for nail and hair, respectively). However, the INAA results provide only the total sulfur content, i.e., sulfur present in keratin, melanin, and any other compound, whereas the PCs of the channel contents of the X-ray spectra, especially due to the peakless scattered region, apply to sulfur directly related to keratin and\/or melanin. This explains the much better correlation of \u201ckeratin\/melanin\u201d sulfur with Cu liver values compared with total S with Cu.\nThe results of our study show that a fairly sharp differentiation can be obtained between dogs with low and high Cu liver levels if PCA is performed on a dataset obtained from all channel contents of X-ray spectra obtained by X-ray tube excitation of nail or hair samples together with the spectral information of often neglected X-ray (Rayleigh, Raman) scattered radiation.\nA practical application of this technique would start with construction of a dataset using samples of known origin, encompassing all varieties expected. New and unknown samples can then be projected on this model, and subsequently classified. This approach will be further validated with new samples to be collected from Labrador Retrievers still being observed and treated along medicinal and dietary approaches.\nConclusions\nHair and nail clippings are widely used as bioindicators for the trace element status of man and, to a much lesser extent, also for animals. The results of this study emphasize that it is quite risky to assume a priori that the trace element levels in hair and nail reflect differences between classes of individuals, e.g., \u201chealthy\u201d and \u201csick\u201d people or animals. In this study related to chronic hepatitis, the Cu levels in liver biopsies are not reflected by Cu levels in hair and nail.\nThe X-ray Rayleigh scattered and X-ray Raman continuum contain valuable information about the organic, low-Z elemental matrix composition of an object. In this study, first indications were obtained that the Cu accumulation possibly may have an effect on the nail and hair melanin, thus offering a potentially favorable outlook for noninvasive monitoring of the Cu liver status. An additional advantage of the approach is that a measurement by X-ray tube excitation takes only about 2\u00a0min per sample so that, once a database has been built up, the technique might be valuable for large-scale screening purposes.","keyphrases":["neutron activation analysis","trace elements","chemometrics","x-ray spectroscopy","biological samples"],"prmu":["P","P","P","M","R"]}
{"id":"J_Gastrointest_Surg-3-1-1852374","title":"Risk of Fecal Diversion in Complicated Perianal Crohn\u2019s Disease\n","text":"The purpose of the study was to determine the overall risk of a permanent stoma in patients with complicated perianal Crohn\u2019s disease, and to identify risk factors predicting stoma carriage. A total of 102 consecutive patients presented with the first manifestation of complicated perianal Crohn\u2019s disease in our outpatient department between 1992 and 1995. Ninety-seven patients (95%) could be followed up at a median of 16 years after first diagnosis of Crohn\u2019s disease. Patients were sent a standardized questionnaire and patient charts were reviewed with respect to the recurrence of perianal abscesses or fistulas and surgical treatment, including fecal diversion. Factors predictive of permanent stoma carriage were determined by univariate and multivariate analysis. Thirty of 97 patients (31%) with complicated perianal Crohn\u2019s disease eventually required a permanent stoma. The median time from first diagnosis of Crohn\u2019s disease to permanent fecal diversion was 8.5 years (range 0\u201323 years). Temporary fecal diversion became necessary in 51 of 97 patients (53%), but could be successfully removed in 24 of 51 patients (47%). Increased rates of permanent fecal diversion were observed in 54% of patients with complex perianal fistulas and in 54% of patients with rectovaginal fistulas, as well as in patients that had undergone subtotal colon resection (60%), left-sided colon resection (83%), or rectal resection (92%). An increased risk for permanent stoma carriage was identified by multivariate analysis for complex perianal fistulas (odds ratio [OR] 5; 95% confidence interval [CI] 2\u201318), temporary fecal diversion (OR 8; 95% CI 2\u201335), fecal incontinence (OR 21, 95% CI 3\u2013165), or rectal resection (OR 30; 95% CI 3\u2013179). Local drainage, setons, and temporary stoma for deep and complicated fistulas in Crohn\u2019s disease, followed by a rectal advancement flap, may result in closing of the stoma in 47% of the time. The risk of permanent fecal diversion was substantial in patients with complicated perianal Crohn\u2019s disease, with patients requiring a colorectal resection or suffering from fecal incontinence carrying a particularly high risk for permanent fecal diversion. In contrast, patients with perianal Crohn\u2019s disease who required surgery for small bowel disease or a segmental colon resection carried no risk of a permanent stoma.\nIntroduction\nCrohn\u2019s disease was initially described as a nonspecific inflammatory bowel disease, affecting mainly the terminal ileum and characterized by a subacute or chronic necrotizing and cicatrizing inflammation.1 Eventually, gastrointestinal Crohn\u2019s disease became recognized as a full-thickness disease of the gastrointestinal wall that may affect the entire gastrointestinal tract, including the perianal region.2\u20136\nPerianal lesions are common in patients with Crohn\u2019s disease.7\u201313 Clinical manifestations vary from asymptomatic skin tags to severe, debilitating perianal destruction and sepsis. Asymptomatic perianal lesions require no treatment, but because they become painful and disabling, they may require surgical treatment. Surgical management needs to be conservative and should focus on the drainage of septic sites, preserving sphincter function and palliating symptoms.10\u201313 Medical management has had some success in improving symptoms, but as yet, it has not been able to eliminate most perianal complaints permanently.14,15\nFecal diversion was successfully used to achieve remission in colonic Crohn\u2019s disease. Moreover, it was utilized to allow severe perianal disease to settle, thereby avoiding proctectomy.16\u201319 However, restoring the intestinal passage carries the risk of recurrent perianal disease activity, possibly resulting in a decreased quality of life compared to the situation with fecal diversion.\nWe investigated the overall risk of a permanent stoma in patients with severe perianal Crohn\u2019s disease and tried to identify risk factors predicting permanent stoma carriage.\nMethods\nPatients\nA total of 102 consecutive patients with Crohn\u2019s disease presented with the first manifestation of a perianal fistula or a perianal abscess in our outpatient department between 1992 and 1995. Patients were investigated in Trendelenburg\u2019s position by perianal inspection, proctoscopy, rectoscopy, and rigid sigmoidoscopy. Endoanal ultrasound was performed in case of suspected perianal abscess formation, and MRI was conducted of the pelvic floor in case of complicated fistulizing disease or intrapelvic abscess formation. All patients were documented prospectively. Follow-up data of 97 patients (95%) were available by a standardized questionnaire mailed to the patients and by a standardized chart review. There were 50 female and 47 male patients (ratio 1.06:1) with a median age of 23\u00a0years (range 8\u201351\u00a0years). Patients were evaluated with respect to the recurrence of perianal abscesses, fistulas, or surgical treatment of Crohn\u2019s disease over the years. The median interval between the first diagnosis of Crohn\u2019s disease and last follow-up was 16\u00a0years (range 8\u201337\u00a0years). Four patients had isolated small intestinal disease, 11 patients had isolated colonic disease, and 82 patients had small intestinal and colonic disease.\nThe abscess location was categorized as subcutaneous, intersphincteric, deep perianal, ischiorectal, and above the pelvic floor. Abscess formations were categorized into simple (subcutaneous, intersphincteric, deep perianal, and ischiorectal, circular extension less than 90\u00b0, pelvic floor not involved) and complicated (circular extension more than 90\u00b0 [horse shoe abscess] or pelvic floor involved). Fistulas were classified according to Parks et al.20 into subcutaneous, intersphincteric, extrasphincteric, transsphincteric, rectovaginal, and suprasphincteric, as described previously.21 We divided fistulas into simple fistulas (no more than two perianal openings) and complex fistulas (rectovaginal, three or more perianal openings).\nA variety of factors, such as sex of the patient, perianal fistula, rectovaginal fistula, abscess formation, anal stricture, fecal incontinence, or abdominal surgery were evaluated with regard to their predictive character for permanent stoma carriage by univariate and multivariate analysis.\nStatistical Analysis\nKaplan\u2013Meier analysis using a log-rank test was used for comparing risk rates over time. Factors that might influence permanent fecal diversion were analyzed using the chi-square test. Fisher\u2019s exact test was used for univariate analysis and multiple logistic regression and Wald\u2019s test for multivariate analysis. Subgroup analysis was performed for rectovaginal fistulas, as present in female patients only. Data are given as numbers of cases and percentages or median and interquartile ranges. A P\u2009<\u20090.05 was considered as significant.\nResults\nOverall Risk of Permanent Fecal Diversion\nThirty of 97 patients (31%) with perianal Crohn\u2019s disease eventually required a permanent stoma. Nineteen patients were female and 11 male (ratio 1.73:1). The median time from first diagnosis of Crohn\u2019s disease to permanent fecal diversion was 8.5\u00a0years (range 0\u201323\u00a0years; Fig.\u00a01). Increased rates of permanent fecal diversion were observed in 53% of patients with previous temporary fecal diversion, in 54% of patients with complex perianal fistulas, in 54% of female patients with rectovaginal fistulas, and in 83% of patients with fecal incontinence (Table\u00a01). Patients who required subtotal colon resection (60%), left-sided colon resection (83%), or rectal resection (93%) needed a permanent stoma at high rates (Table\u00a02).\nFigure\u00a01Relative proportion of patients without permanent stoma in complicated perianal Crohn\u2019s disease (n\u2009=\u200997) during follow-up.Table\u00a01Patients with First Manifestation of Complicated Perianal Crohn\u2019s Disease (n\u2009=\u200997) and the Rate of Fecal Diversion During Follow-up\u00a0PatientsTemporary StomaPermanent StomaOverall9751 (53%)30 (31%)Abscess  formation7532 (43%)21 (28%)Simple abscess formation33 (44%)13 (39%)7 (21%)Complex abscess formation42 (56%)23 (55%)15 (36%)Fistulas8851 (58%)26 (30%)Simple perianal fistulas42 (48%)17 (40%)8 (19%)Complex perianal fistulas46 (52%)34 (74%)23 (50%)Rectovaginal fistulas26 (54% of \u2640)18 (69% of 26)14 (54% of 26)Overlap between abscess formation and presence of fistulas exists, and abscesses led to fecal diversion in combination with fistulas only.Table\u00a02Abdominal Procedures and the Rate of Fecal Diversion During Follow-up in Patients with Complicated Perianal Crohn\u2019s Disease (n\u2009=\u200997)\u00a0Patient NumberPermanent Stoma (% of Patients)Small Bowel ProceduresStricturplasty70Small bowel resection because of stenosis230Small bowel resection because of enteroenteric fistula70Anastomosis resection because of inflammatorystenosis300Stoma revision40Large bowel proceduresSegmental colon resection140Right-sided colon resection204 (20)Ileocecal resection6824 (35)Subtotal colon resection3521 (60)Left-sided colon resection65 (83)Rectal resection1312 (92)Patients that required small bowel resections carried no risk of fecal diversion, whereas patients with colon resections carried an increased risk of fecal diversion.\nAbscess Formation\nSeventy-five of 97 patients (77%) had at least one perianal abscess at first presentation or during follow-up. Surgical therapy for abscesses consisted of seton drainage, mushroom catheter drainage, or incision and drainage, as described previously.21 A permanent fecal diversion because of recurrent abscess formations, always in combination with fistula problems and perianal sepsis, became necessary in 21 of 75 cases (28%). Simple abscess formations occurred in 33 patients, and 7 of those patients (21%) required permanent fecal diversion (Fig.\u00a02). Complex abscess formations were present in 42 patients, with permanent fecal diversion being necessary in 14 cases (33%; Table\u00a01).\nFigure\u00a02Relative proportion of patients without permanent stoma in complicated perianal Crohn\u2019s disease with simple (n\u2009=\u200942) and complex (n\u2009=\u200946) fistulas during follow-up. The presence of a complex fistula significantly increased the risk of permanent fecal diversion (P\u2009<\u20090.001).\nPerianal and Rectovaginal Fistulas\nPerianal fistulas affected 88 of 97 patients (91%), including 26 female patients with rectovaginal fistulas. Symptomatic fistulas with abscess formation were treated by simple drainage procedures and provided with a seton. Azathioprine therapy was tried if purulent secretion persisted. Infliximab was rarely used in recent years at the discretion of the referring gastroenterologist. Overall, permanent fecal diversion became necessary in 26 of 88 patients (30%). Forty-two patients (48%) had simple fistulas, whereas 46 patients (52%) had complex fistulas. Of these, permanent fecal diversion was documented in 14 patients (54%), whereas simple fistulas eventually required a permanent stoma in only 8 cases (16%). Twenty-six of 48 female patients had rectovaginal fistulas, of which 14 (54%) eventually required permanent fecal diversion (Table\u00a01).\nThirty-four of 97 patients (35%) developed anal strictures, whereas fecal incontinence was documented in 12 patients (12%) during the follow-up. In 14 of 34 patients (41%) with anal stricture, a permanent fecal diversion became necessary, whereas 10 of 12 patients (83%) suffering from fecal incontinence required a permanent stoma.\nAbdominal Surgery\nEighty-three of 97 patients with perianal Crohn\u2019s disease (86%) underwent abdominal surgery at least once during follow-up because of Crohn\u2019s disease activity, with a total of 227 abdominal procedures being performed. In 68 of 227 operations (29%), intestinal stenosis required surgery. Segmental colon resection was performed 108 times (46%), whereas subtotal colectomy for fulminant colitis was performed 35 times (15%). One patient required surgery because of a fistula carcinoma. Patients with complicated perianal Crohn\u2019s disease who required a small bowel resection carried no risk of permanent fecal diversion, whereas the majority of patients with left-sided colon resection, subtotal colon resection, or rectal resection needed permanent fecal diversion (Table\u00a02). Patients that needed abdominal surgery repeatedly carried an increased risk for a permanent stoma (permanent stoma rate: three or more abdominal operations, n\u2009=\u200920, 50%; less than two abdominal operations, n\u2009=\u200920, 15%; P\u2009<\u20090.043). Patients with permanent fecal diversion had undergone previous abdominal surgery three times as often as those patients without (P\u2009<\u20090.05; Table\u00a03). Eventually, 17 patients (18%) needed proctectomy, of which 13 were female patients with rectovaginal fistulas. In 14 patients with a permanent stoma, a rectal stump remained in place and was controlled at yearly intervals. Patients with complicated perianal Crohn\u2019s disease and large bowel resection carried a significantly increased risk of permanent fecal diversion compared to patients with complicated perianal Crohn\u2019s disease and small bowel resection (48 vs 0%, P\u2009<\u20090.001) (Fig.\u00a03). \nTable\u00a03Abdominal Procedures per Patient and Percentage of Patients with Abdominal SurgeryAbdominal ProceduresMedian(25\u201375%)Patients with Abdominal Surgery (%)P ValuePermanent fecal diversion3(2\u20134)100P\u2009<\u20090.05Without permanent fecal diversion1(1\u20133)Temporary fecal diversion3(2\u20134.25)80P\u2009<\u20090.05Without temporary fecal diversion1(0\u20132.5)Anal stricture2(1\u20134)60n.s.Without anal stricture2(1\u20133)Incontinence2(2\u20134.5)10n.s.Without incontinence2(1\u20133)Patients with Crohn\u2019s disease and complicated perianal fistulas who required temporary or permanent stoma had more abdominal procedures than patients who did not need a stoma, whereas patients with anal stricture or fecal incontinence had no increased abdominal procedure rate (the number of abdominal procedures per patient is given as median with interquartile ranges). Abdominal surgery and fecal diversion correlated on univariate analysis (P\u2009<\u20090.05).Figure\u00a03Treatment algorithm for patients with complicated perianal Crohn\u2019s disease.\nTemporary Fecal Diversion\nTemporary fecal diversion, done mostly by loop ileostomy, became necessary in 51 of 97 patients (53%). Increased rates of temporary fecal diversion were observed in 55% of patients with complex abscess formations, including those with severe perianal sepsis, in 77% of patients with complex perianal fistulas, and in 69% of female patients with rectovaginal fistulas. In 24 of 51 patients (47%) the temporary stoma could be removed after perianal disease had subsided. Fistulas were closed in these patients by rectal advancement flaps,23 whereas cutting setons, fistulectomy, or infliximab infusions were not employed in these patients.\nRisk Factors Predictive of a Permanent Fecal Diversion\nUnivariate Analysis\nComplex perianal fistulas (P\u2009<\u20090.04), fecal incontinence (P\u2009<\u20090.001), and rectovaginal fistulas in female patients (P\u2009<\u20090.001) carried an increased risk for a permanent stoma. Patients with high rates of abdominal surgery had a significantly increased risk for fecal diversion, whereas the frequency of abdominal surgery was not influenced by the presence of anal stricture or fecal incontinence (Table\u00a03). The need for subtotal colon resection (P\u2009<\u20090.001), rectal resection (P\u2009<\u20090.001), or temporary fecal diversion (P\u2009=\u20090.001) also resulted in an increased risk of permanent fecal diversion. A variety of factors did not increase the risk of a permanent stoma, such as sex of the patient, anal stenosis, perianal abscesses, and abdominal surgery, excluding subtotal colon and rectal resection (Table\u00a04). \nTable\u00a04Risk Factors for Permanent Fecal Diversion by Univariate and Multivariate Analysis in Patients with Complicated Perianal Crohn\u2019s DiseaseRisk FactorsUnivariate AnalysisMultivariate AnalysisPermanent Fecal Diversion (%)P ValueOR95% CIP ValueRectal resection92P\u2009<\u20090.001305\u2013179P\u2009<\u20090.002Fecal incontinence83P\u2009<\u20090.001213\u2013165P\u2009<\u20090.02Subtotal colectomy60P\u2009<\u20090.001Rectovaginal fistulas54P\u2009<\u20090.001Temporary fecal diversion51p\u2009=\u20090.00182\u201335P\u2009<\u20090.02Complex perianal fistulas54P\u2009<\u20090.0452\u201318P\u2009<\u20090.03\nMultivariate Analysis\nAccording to multivariate analysis, complex perianal fistulas (P\u2009<\u20090.03), fecal incontinence (P\u2009<\u20090.02), temporary fecal diversion (P\u2009<\u20090.02), or rectal resection (P\u2009<\u20090.002) still carried a significantly increased risk for permanent fecal diversion (Table\u00a04).\nDiscussion\nPerianal disease is a feature of Crohn\u2019s disease that afflicts approximately one third of patients, but prevalence rates vary between 8 and 90%.19 Patients with colonic involvement will have perianal disease in more than 50% of cases, whereas patients with small bowel disease are affected in less than 20%.24 Perianal Crohn\u2019s disease can present as minor lesions, such as skin tags, skin excoriations, and fissures, which rarely need surgical treatment. In contrast, perianal fistulas often result in abscesses or perianal sepsis, requiring urgent surgery, and fistulas and abscesses are considered as suppurative complications of perianal Crohn\u2019s disease.25 We conducted surgery as conservatively as possible because sphincter function should be preserved as much as possible.19,22,23 Throughout the study period, 77% of patients developed abscess formations, which is considerably more than the 48% rate reported previously with a shorter mean follow-up of 32\u00a0months.22\nThe incidence of perianal fistulas in Crohn\u2019s disease is somewhat lower, affecting 10\u201334% of patients.26 Healing rates of 60\u201370% in patients with Crohn\u2019s disease and perianal fistulas were reported in earlier series.21,26\u201328 However, these series contained a large proportion of simple fistulas, whereas most of our patients had complex transsphincteric or rectovaginal fistulas and fistulas that did not heal through conservative treatment. Perianal fistulas that resulted in abscess formation were drained surgically, and in case of massive purulent secretion or perianal sepsis, oral antibiotics were used additionally. Setons were put into place to prevent recurrent abscess formation, a treatment that is well known as a possible means for controlling perianal sepsis and preventing recurrent abscess formation.19,21\u201323,29 We did not use cutting setons nor fistulotomy for transsphincteric fistulas, as fecal incontinence may succeed.19,22,23 Infliximab was successfully shown to reduce fistula secretion, and outer fistula openings may heal.30 However, fistula tracks persist with varying degrees of residual inflammation, which may cause recurrent fistulas and perianal abscesses.31\nRectal advancement flaps can be used to close the inner opening of transsphincteric or rectovaginal fistulas when perianal disease has abated and the rectal mucosa is not inflamed. However, fistulas eventually recurred in about one third of patients after a mean follow-up of 19\u00a0months, and some patients developed new fistulas.23 Hyman32 reported an initial 79% healing rate in a series of 14 patients with Crohn\u2019s disease and perianal fistulas, but observed longer term success in only 50% of patients. Our current approach is to use rectal advancement flaps for transsphincteric fistulas while delaying surgery if there is an acute flare of Crohn\u2019s disease, in particular, if proctitis is present.23,33 Perianal fistulas in Crohn\u2019s disease closed by a rectal advancement flap have a recurrence rate of 50\u201360%, but can be attempted repeatedly.23,34 If a second rectal advancement flap fails, the failure rate increases up to 75%,34 and a temporary stoma is suggested to the patient before a third attempt is made. By this approach, we were able to heal perianal fistulas in 24 out of 51 patients (47%) who required a temporary stoma. In a previous study, diversion was the most powerful factor influencing healing according to multiple regression analysis.23 The chances of a symptomatic fistula recurrence were increased without a stoma (52% vs 14%, 21), and patients with Crohn\u2019s colitis carried an increased risk of fistula recurrence.23\nA particular problem is rectovaginal fistulas, which almost always open at the dentate line.19 They occur in 3\u201310% of patients with Crohn\u2019s disease.19 In our series, 54% of female patients had rectovaginal fistulas, indicating that a large patient proportion had complex perianal fistulas; referral bias might have contributed to this high proportion of rectovaginal fistulas, as our outpatient clinic for Crohn\u2019s disease is well known regionally. Rectovaginal fistulas carry a poor prognosis,18 and a 70% recurrence rate was observed 24\u00a0months after a rectal advancement flap was performed.23 In the experience of Keighley et al.35, 11 of 13 patients with rectovaginal fistulas required proctectomy with a permanent stoma. In our series, 8 out of 26 rectovaginal fistulas healed by a rectal advancement flap. Eighteen patients required a temporary stoma, of which four could be removed after a rectal advancement flap was tried again successfully (overall healing rate 12 of 26, 46%), resulting in a permanent stoma rate of 14 of 26 patients (54%). Healing rates of rectovaginal fistulas associated with Crohn\u2019s disease vary widely, and low patient numbers are usually reported.18 In the Cleveland clinic, 16 of 37 rectovaginal fistulas (43%) healed using an endorectal advancement flap, but patients not having Crohn\u2019s disease were included in the study.36 In low anovaginal fistulas, higher healing rates of up to 68% were reported in patients with Crohn\u2019s disease.37 Recurrent rectovaginal fistulas were shown to heal after repeated rectal advancement flap procedures were conducted at about the same rate even in Crohn\u2019s disease, but surgery should be delayed for at least 3\u00a0months after a previous repair,38 which is standard policy at our clinic. It is interesting to note that Halverson et al.38 reported that the presence of a diverting stoma significantly increased the fistula recurrence rate (67 vs 50%).\nAfter years of perianal or transvaginal pus secretion and recurrent abscess formations, patients are often tired of the suffering involved. Antibiotics, azathioprine, or 6-mercaptopurine may be used successfully in suppurative perianal Crohn\u2019s disease.15,18,38,39 In our department, this is routinely tried if perianal disease does not settle after surgical drainage is provided. However, azathioprine or 6-mercaptopurine are not tolerated by all patients and may also fail to relieve perianal Crohn\u2019s disease. In these cases, fecal diversion can be offered as a relatively minor procedure with low morbidity,18 and might be performed laparoscopically in selected cases.40 However, overall healing rates of perianal disease are only around 40%, with 87% of those being diverted eventually retaining their stoma.18 We diverted 51 patients, of which 27 retained there stoma (53%), which compares favorably, but indicates a considerable risk for a permanent stoma if diversion is tried, and being a significant risk factor according to multivariate analysis in our series.\nPerianal disease in combination with fecal incontinence might necessitate fecal diversion as well. Factors that may contribute to fecal incontinence in patients with Crohn\u2019s disease are reduced stool consistency, sphincter injuries after abscess incisions, and keyhole deformities after laying open perianal fistulas. The latter was reported to result in fecal incontinence in 5 of 27 patients with perianal fistulas in Crohn\u2019s disease,41 which is why we did not use this technique or cutting setons in transsphincteric fistulas. We always incise perianal abscesses along a circular perianal line, meticulously preserving sphincter integrity. Nevertheless, fecal incontinence became prevalent in 12% of our patients during follow-up and was a significant predictor of permanent fecal diversion by multivariate analysis. Rectal advancement flap procedures may influence continence,22 and a 9% rate of worsened continence was reported thereafter.33 In particular, the risk of fecal incontinence was increased after flap repairs in patients with previous surgical fistula repairs,33 but only 2 out of 12 patients with fecal incontinence had a previous rectal advancement flap repair in our study. A high rate of abdominal procedures was associated with an increased risk for a temporary or a permanent stoma, possibly reflecting high intestinal disease activity of Crohn\u2019s disease in these patients. All 30 patients that ended with a permanent stoma had frequently undergone abdominal procedures (median of three procedures).\nBecause of the suffering involved and unsuccessful previous attempts to heal perianal fistulas, about half of our patients eventually opted for temporary ileostomy, being brought into contact with a stoma nurse and stoma carriers with Crohn\u2019s disease before, and 31% of all patients remained with a permanent stoma. In general, the need for a stoma was reduced during recent decades in Crohn\u2019s disease,43 but the overall long-term risk for a stoma in patients with Crohn\u2019s disease who require abdominal surgery was reported between 30 and 40%.44,45 Fecal diversion remains an option to subside perianal disease activity, with an early response rate of about 70\u201380%.46\u201348 Unfortunately, 75% of those patients eventually experienced a relapse, and the restoration of intestinal continuity was achieved in 10% only.48 In 130 patients with surgically treated perianal Crohn\u2019s disease, a permanent stoma became necessary in 24%, and this rate increased to 53% in patients with Crohn\u2019s colitis.45 In this light, considering that all of our patients had complicated perianal disease and almost all (93 of 97 patients, 96%) had colonic involvement, we judge a 31% permanent stoma rate as a success in a subset of patients carrying a particularly high risk for a stoma. Local drainage, setons, and, if not successful, a temporary stoma after a rectal advancement flap, resulted in closure of the stoma in 47% of the time, which is an improvement compared to previous studies where restoration of intestinal continuity was reported in only 10\u201340% of patients, with most studies providing the lower end percentages.44,46\u201348 If perianal Crohn\u2019s disease was the indication for creating a stoma, stoma closure was successful in 2 out of 15 patients only (13%).47\nEventually, proctectomy became necessary in 18% of patients, which is in the range others have reported.24,44,49,50 However, if the presence of colonic disease was considered separately, seven out of eight patients with complicated perianal Crohn\u2019s disease needed proctectomy, again indicating that an 18% proctectomy rate in our high-risk population is rather low. All of our patients who needed proctectomy had colonic disease, and 13 of 17 had a rectovaginal fistula.\nIn a recent study investigating the quality of life in patients with Crohn\u2019s proctocolitis, patients in remission had a health-related quality of life similar to controls. Patients with active disease had a reduced quality of life, and only the symptom index negatively predicted a reduced quality of life, whereas neither previous colonic surgery nor the presence of a stoma did.51 Accordingly, carrying a stoma does not necessarily mean a reduced quality of life, although a slight reduction in two out of eight domains (physical and emotional role) was detected.51 However, in patients with perianal disease, the cumulative abscess rate during 3\u00a0years of follow-up and the symptomatic fistula recurrence rate with a mean follow-up of 22\u00a0months were both greatly reduced with a stoma (76 vs 13% and 52 vs 14%), indicating reduced suffering and need for surgery.21,22\nIn summary, patients with complicated perianal Crohn\u2019s disease, colonic involvement, and a high rate of abdominal procedures carried a significant risk for a permanent stoma. However, if the risk factors identified were taken into account, the rate of patients eventually requiring a permanent stoma seemed low and showed a decrease compared to previous studies, indicating that multiple treatment episodes and complex surgery, including temporary fecal diversion, might eventually heal at least some patients in this high-risk population, as was also observed by others.50","keyphrases":["fecal diversion","crohn\u2019s disease","perianal abscesses","fistulas"],"prmu":["P","P","P","P"]}
{"id":"Plant_Mol_Biol-3-1-1805040","title":"Genome-wide transcriptional analysis of salinity stressed japonica and indica rice genotypes during panicle initiation stage\n","text":"Rice yield is most sensitive to salinity stress imposed during the panicle initiation (PI) stage. In this study, we have focused on physiological and transcriptional responses of four rice genotypes exposed to salinity stress during PI. The genotypes selected included a pair of indicas (IR63731 and IR29) and a pair of japonica (Agami and M103) rice subspecies with contrasting salt tolerance. Physiological characterization showed that tolerant genotypes maintained a much lower shoot Na+ concentration relative to sensitive genotypes under salinity stress. Global gene expression analysis revealed a strikingly large number of genes which are induced by salinity stress in sensitive genotypes, IR29 and M103 relative to tolerant lines. We found 19 probe sets to be commonly induced in all four genotypes. We found several salinity modulated, ion homeostasis related genes from our analysis. We also studied the expression of SKC1, a cation transporter reported by others as a major source of variation in salt tolerance in rice. The transcript abundance of SKC1 did not change in response to salinity stress at PI stage in the shoot tissue of all four genotypes. However, we found the transcript abundance of SKC1 to be significantly higher in tolerant japonica Agami relative to sensitive japonica M103 under control and stressed conditions during PI stage.\nIntroduction\nRice is one of the most economically important crops of the world. Rice yields can be reduced by up to 50% when grown under moderate (6\u00a0dS\u00a0m\u22121) salinity levels (Zeng et al. 2002). Soil salinity is a serious constraint to rice cultivation under irrigated agriculture in countries like Pakistan and elsewhere due to poor quality of water (Abdullah and Ahmad 1982). Rice is the major crop of many coastal regions which are prone to sea water ingress during high tide and of rainfed systems which experience increasing salinity in the root zone due to a rising water table. Despite significant yield losses under saline conditions, rice is potentially a good candidate for cultivation in areas prone to coastal flooding due to its ability to tolerate standing water in the field for long periods. In California, salinity is an increasing concern for rice cultivation due to irrigation practices that involve use of recirculating water systems and the requirement of water holding after pesticide application during early growth stages (Scardaci et al. 1996).\nThe susceptibility of rice to salinity stress varies with growth stage. Rice is relatively salt-tolerant at germination, and in some cases is not affected significantly by up to 16.3\u00a0dS\u00a0m\u22121 of salinity (Heenan et al. 1988; Khan et al. 1997). Rice becomes very sensitive at the young seedling stage, which impacts the stand density in salt-affected fields (Lutts et al. 1995). It is relatively less sensitive during late reproductive stage (grain ripening). From an agronomic perspective, sensitivity of yield components is of primary importance. Salinity significantly reduces the tiller number per plant, spikelet number per panicle, fertility, panicle length and primary branches per panicle (Heenan et al. 1988; Cui et al. 1995; Khatun et al. 1995; Zeng et al. 2002). Reductions in tiller number per plant and spikelet number per panicle were reported to be the major causes of yield loss in one cultivar of rice under salinity stress (Zeng and Shannon 2000). The number of spikelets per panicle was determined to be the most sensitive yield component. This component is determined at early reproductive stage, around panicle initiation (PI) (Hoshikawa 1989; Counce et al. 2000). The loss in spikelet number per plant was most significant when the stress was imposed before PI (3-leaf stage) or between PI and booting stage (\u223c16\u00a0days after PI) (Zeng et al. 2001). This loss of potential spikelets is attributed to degeneration of primary and secondary branches and flower primordia (Zeng et al. 2001). Considerable genetic variability for this yield parameter was reported based on evaluation of several diverse genotypes for salinity stress response (Zeng et al. 2002). Considerable variation for salt tolerance at critical stages in the cultivated gene pool has also been reported by other workers (Yeo and Flowers 1982; Moradi et al. 2003). This genetic variability can be utilized for improvement of salt tolerance by focusing on specific yield components. However, the underlying genes conferring tolerance during PI are at present unknown.\nIn the current study, we have focused on the salinity stress imposed during the PI stage considering the significant impact it has on rice yields and a lack of prior information on molecular components of the response to salinity stress at this critical stage. The genotypes selected for this study have contrasting physiological responses to salinity stress at PI based on several seasons of experimentation at the U.S. Salinity Laboratory, Riverside (Zeng et al. 2002). One of the most salt-tolerant genotypes identified by Zeng et al. (2002) was IR63731, an indica line developed from a cross between tolerant land race Nona Bokra and sensitive cultivar IR28. This genotype had the most favorable combination of salt tolerance at early vegetative and PI stages. This genotype possesses good yield potential under salinity stress due to high tillering ability and panicle weight (Zeng et al. 2002). Another genotype, Agami, was ranked high for its ability to yield high under salinity stress. Agami is a salt-tolerant japonica subspecies cultivar grown in Egypt. Several studies have reported the popular California japonica cultivar M103, to be very sensitive at all stages (Zeng et al. 2002, 2003). Therefore, for the current work we have used IR63731 and Agami as the tolerant genotypes representing indica and japonica subspecies and IR29 and M103 as the sensitive genotypes for each of the subspecies.\nPreviously, we focused on transcript accumulation responses of rice to salinity stress imposed during the early vegetative stage in two indica subspecies genotypes which differed in salt tolerance as manifested by higher tillering capacity and maintenance of favorable ion homeostasis (Walia et al. 2005). The sensitive genotype used for that study was IR29, a widely used sensitive standard which is susceptible at the vegetative and reproductive stages. IR29 was the sensitive parent of a recombinant inbred population developed at the International Rice Research Institute (IRRI) for mapping salt tolerance quantitative trait loci (QTL) in rice using Pokkali as the tolerant parent. A salt-tolerant recombinant inbred line (RIL) FL478 from this population was used as the tolerant genotype in our prior study (Walia et al. 2005).\nThe transcriptome of rice under salinity stress at PI has been largely uncharacterized. Considering the sensitivity of rice yield to salinity stress when imposed around PI, it is important to identify specific genes that are involved in the response to stress at this stage. Responsive genes are expected to include those with a role in salt adaptation and tolerance, and which respond to salt injury or constitute non-adaptive responses. The use of genotypes with well-characterized phenotypic responses to salinity stress at a particular growth stage can help in identifying potential candidate genes which may be important for heritable salt tolerance. Here we perform whole genome transcript profiling of genotypes from two major subspecies of cultivated rice chosen for their representative tolerance or sensitivity to salinity stress during the early reproductive stage, specifically at PI. By including indica genotype IR29 in the present study on PI, we now have mRNA expression level responses at both of the two most salt sensitive, yield-determining stages for this genotype (early vegetative as per Walia et al. 2005, and early reproductive in this study). Physiological characterization of the lines was also performed as part of the present study, and gene expression results were interpreted in the physiological context. We also incorporated information from known salt tolerance QTL in rice with the objective of correlating gene expression with gene location on the rice genome.\nMaterial and methods\nPlant materials for expression studies\nSeeds of rice (Oryza sativa L.) genotypes IR29, IR63731-1-1-4-3-2 and Agami were obtained from G. B. Gregorio at IRRI of the Philippines and then propagated through quarantine at the George E. Brown, Jr. Salinity Laboratory, Riverside, CA, USA. Seed for M103 was supplied by California Cooperative Rice Research Foundation Inc., at Biggs, California. These genotypes were selected from the germplasm collections at the two sites based on their differences in salt tolerance in terms of agronomic and physiological performance. Extensive characterization of these lines was performed by Zeng et al. (2002, 2003) at George E. Brown, Jr. Salinity Laboratory, Riverside, CA, USA.\nPlant culture and salinity treatments\nThe experiment was conducted in the greenhouse at Riverside, California [33\u00b058\u203224\u2033\u00a0N latitude, 117\u00b019\u203212\u2033\u00a0W longitude] between May and July 2005. Plants were cultured in tanks (122\u00a0\u00d7\u00a061\u00a0\u00d7\u00a046\u00a0cm) filled with sand and irrigated with nutrient solution (Yoshida et al. 1976). The nutrient solution consisted of NH4NO3 (1.43\u00a0mM), NaH2PO4\u00a0\u00b7\u00a02H2O (0.37\u00a0mM), K2SO4 (0.5\u00a0mM), CaCl2 (1.00\u00a0mM), and MgSO4\u00a0\u00b7\u00a07H2O (1.6 mM). Nutrient solution pH was maintained between 5.0 and 6.5 by adding sulfuric acid as needed. Irrigation solutions were prepared in 1600\u00a0L reservoirs and pumped to provide irrigation to the sand tanks. Overflow irrigation was returned to the reservoirs through drainage by gravity. Each reservoir provided irrigation water to three sand tanks (replicates) three times daily for 30\u00a0min per irrigation cycle. Four rows of each genotype were planted per tank for control tanks. An extra row at each end of the tank was used as the border row. Each genotype was planted in separate tanks which were marked for salinization later in the experiment. The water level was maintained at the sand surface until emergence of the seedlings from the sand and then increased to 6\u20138\u00a0cm above the sand surface. Air temperature ranged from 30\u00b0C to 42\u00b0C during the day and 20\u201324\u00b0C during night. Relative humidity ranged from 40% to 80%. Light averaged 1104\u00a0\u03bcmol\u00a0m\u22122\u00a0s\u22121 at noon. Weak plants were removed from the tanks. Plant growth stages were closely monitored. Rough estimates of the days to PI stage for each genotype were available from growing the genotypes for several years in the same greenhouse conditions and time of the year during previous experiments (Zeng et al. 2002). Salinity treatment was applied by adding NaCl and CaCl2 (5:1 molar concentration) in two steps over a period of 3\u00a0days to each genotype about 9\u201310\u00a0days before PI (Fig.\u00a01). Electrical conductivities (ECw) of nutrient solutions were monitored using an EC meter daily (Hanna Instruments, Woonsockett, RI). The salinity in the treatment reservoir was allowed to stabilize for 5\u00a0days to a final electrical conductivity of 7.0\u00a0dS\u00a0m\u22121. The non-saline control reservoir with Yoshida solution had an ECw of 1.1\u00a0dS\u00a0m\u22121. Two plants were randomly selected each day for each genotype when the plants were approaching PI. The main culms were dissected under a microscope to observe the development of young panicles. The day when we observed any plant of a genotype with panicle about 0.5\u00a0mm long was defined as the PI date for that genotype under the treatment in which it was growing (control or salt stressed). Salinity delayed PI in the sensitive genotype M103, but not in other three lines.\nFig.\u00a01Experimental design of salinity stress treatment. Salinity stress was applied 9\u201310\u00a0days before panicle initiation (PI). A final salinity level of 7\u00a0dS\u00a0m\u22121 was reached by a two-step addition of NaCl and CaCl2. Conductivity in the control tanks is represented by a dotted line and the treated tank by a solid line. Diamonds represent the PI and ovals represent the harvest time points. The crown and growing point of the main shoot were harvested 4\u20135\u00a0days after reaching PI\nPhenotypic characterization and gene expression studies\nPlants were characterized for phenotypic responses to salinity stress on the day of harvest for RNA extraction. For phenotypic characterization the shoot tissue from six plants was harvested for ion analysis. Plants were washed with deionized water, dried in a forced air oven (70\u00b0C), and then ground into fine powder. Shoot Na+ and K+ concentrations were determined on nitric-perchloric acid digests by inductively coupled plasma optical emission spectrometry (ICP, Perkin-Elmer Co., Norwalk, CT, USA). Transpiration rate of the youngest fully expanded leaf was measured between 10.00\u00a0h and 12.00\u00a0h one day before harvesting the tissue for RNA using a LI-COR 6400 Photosynthesis System (LI-COR Biosciences, Lincoln, NE). The following conditions for leaf gas measurements were used: photosynthetic photon flux density, 1200\u00a0\u03bcmol\u00a0m\u22122; chamber CO2 concentration, 380\u00a0\u03bcmol\u00a0CO2\u00a0mol\u22121; leaf temperature, 27\u00b0C; and chamber vapor concentration, 20\u00a0mmol\u00a0H2O\u00a0mol\u22121.\nThe plants were harvested 4 or 5\u00a0days after reaching PI. The main shoot was dissected for RNA extraction to obtain the growing point and crown tissue which was snap frozen. Approximately 12 plants were harvested per genotype per tank and tissue pooled to make each biological replicate for RNA extraction. Three biological replicates (one per array) were used for each treatment. Two plants from each tank were allowed to grow to maturity to verify that plants survived the imposed salinity stress.\nRNA extraction and processing for GeneChip analysis\nRNA samples were processed as recommended by Affymetrix, Inc. (Affymetrix GeneChip Expression Analysis Technical Manual, Affymetrix, Inc., Santa Clara, CA) at the Core Instrumentation Facility at the University of California, Riverside by Barbara Walter. Total RNA was initially isolated from frozen shoot tissue using TRIzol Reagent. The RNA was purified using an RNeasy spin column (Qiagen, Chatsworth, CA) and an on-column DNase treatment. Eluted total RNAs were quantified with a portion of the recovered total RNA and adjusted to a final concentration of 1\u00a0\u03bcg\/\u03bcl. All RNA samples were quality assessed prior to beginning target preparation\/processing steps by running out a small amount of each sample (typically 25\u2013250\u00a0ng\/well) onto a RNA Lab-On-A-Chip (Caliper Technologies Corp., Mountain View, CA) that was evaluated on an Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). Single-stranded, then double-stranded cDNA was synthesized from the poly(A)+ mRNA present in the isolated total RNA (10\u00a0\u03bcg total RNA starting material each sample reaction) using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen Corp., Carlsbad, CA) and poly(T)-nucleotide primers that contained a sequence recognized by T7 RNA polymerase. A portion of the resulting ds cDNA was used as a template to generate biotin-tagged cRNA from an in vitro transcription reaction (IVT), using the Affymetrix GeneChip IVT Labeling Kit. Fifteen micrograms of the resulting biotin-tagged cRNA was fragmented to strands of 35\u2013200 bases in length following prescribed protocols (Affymetrix GeneChip Expression Analysis Technical Manual). Subsequently, 10\u00a0\u03bcg of this fragmented target cRNA was hybridized at 45\u00b0C with rotation for 16\u00a0h (Affymetrix GeneChip Hybridization Oven 320) to probe sets present on an Affymetrix Rice Genome array. The GeneChip arrays were washed and then stained (SAPE, streptavidin-phycoerythrin) on an Affymetrix Fluidics Station 450 followed by scanning on a GeneChip Scanner 3000.\nRice Genome array\nThe Rice Genome array (Affymetrix, Santa Clara, CA) contains probe sets designed from \u223c48,564 japonica and 1,260 indica sequences. The sequence information for this array was derived from NCBI UniGene Build #52 (http:\/\/www.ncbi.nlm.nih.gov\/UniGene), GenBank mRNAs and 59,712 gene predictions from TIGR\u2019s osa1 version 2.0. Gene models which had any indication of transposable elements were removed from the list of TIGR predicted genes. The array is believed to represent about 46,000 distinct rice genes. About 26,000 of these are 3\u2032 anchored Unigene EST and mRNA clusters, including known rice full length cDNA clones and 19,431 are solely from TIGR gene predictions. To obtain annotations for the salt-regulated probe sets, we extracted the target sequence of identified probe sets from the sequence information file (.sif) for the rice genome array. The target sequence extends from the 5\u2032 end of the 5\u2032-most probe to the 3\u2032 end of the 3\u2032-most probe. The target sequences were then searched using BLASTn against the TIGR rice pseudomolecules, release 3 (http:\/\/www.tigr.org\/tdb\/e2k1\/osa1) and the TIGR Arabidopsis database, version 5.\nStatistical analysis of array data\nThe triplicated array data set was analyzed using GeneChip Operating Software (GCOS 1.2) and DChip (Li and Wong 2001; http:\/\/www.DChip.org) software. The scanned images were examined for any visible defects. Satisfactory image files were analyzed to generate raw data files saved as CEL files using the default settings of GCOS 1.2 from Affymetrix. We used a default target intensity value (TGT) setting of 500. The normalization factor value was set to 1 to extract data without normalization. Default parameter settings for the Rice Genome array were used. The scaling factor for the arrays ranged from 3.1 to 8.5. The detection calls (present, absent or marginal) for the probe sets were made by GCOS. Further analysis was done using DChip which incorporates a statistical model for expression array data at the probe level. The DChip program was set to import GCOS signals. The normalization of all arrays was performed using an invariant set approach. For calculating the expression index of probe sets we used the PM model and opted for truncating the low expression values to 10th percentile of the probe set signals called absent. The expression values were log2 transformed after calculating the expression index. DChip was used for comparative analysis of samples from salt stress and control treated plants for both genotypes. We identified the differentially expressed genes using the empirical criterion of more than 10-fold change (E-B\u00a0>\u00a01 or B-E\u00a0>\u00a01) and significant t-tests of P\u00a0<\u00a00.05 based on three biological replicates. The baseline array, which is a control sample, is denoted by B and salinity-treated experimental sample is denoted by E. The P-values assigned to differentially expressed genes were used as a ranking criterion for ordering of up-regulated and down-regulated genes. We used the DChip feature to assess the false discovery rate (FDR) in our data set using 250 permutations in most comparisons. Empirical FDR was below 15% when identifying differentially expressed genes. All microarray data from this work is available from NCBI GEO (http:\/\/www.ncbi.nlm.nih.gov\/geo) under the series entry GSE4438.\nExpression validation by semi-quantitative RT-PCR\nSeveral key expression profiles obtained from chip hybridizations were further validated by semi-quantitative RT-PCR using first-strand cDNA. The japonica sequence of each gene was obtained from the TIGR rice database. Exonic sequences were used for the design of primers using Primer Express (Perkin-Elmer Applied Biosystems, Foster City, CA). The primer sequences for 18S rRNA (F-ATGATAACTCGACGGATCGC, R-CTTGGATGTGGTAGCCGTTT) were used as an internal control. The primer sequences for SKC1 were obtained from Walia et al. (2005). A cDNA first strand was synthesized using Taq-Man Reverse Transcription Reagents (Applied Biosystems, Forster City, CA; Ref: N808-0234) following the manufacturer\u2019s instructions. Two micrograms of total RNA was converted into cDNA. Each cDNA was diluted 40 times and 5\u00a0\u03bcL of cDNA was used for three-step PCR. The number of PCR cycles was optimized (37\u00a0cycles) for the 5 genes selected for expression validated and 34\u00a0cycles for SKC1.\nResults\nA moderate level of salinity stress was imposed gradually nine or ten days before PI stage and plants were sampled for phenotypic characterization and expression profiling five days after PI (Fig.\u00a01). All four genotypes used in this experiment were known to reach PI at different time points based on prior characterization of these genotypes over several growing seasons under comparable growing conditions and time of the year.\nOne confounding factor in experiment involving salinity stress is that salinity is known to delay PI in all genotypes. With this in mind we designed our experiment to harvest the control and salinity stressed tissue of each of the genotype at similar developmental stage i.e. 4\u20135\u00a0days after the plants reached PI. This was done because developmental equivalence when comparing control plants with stressed plants within each genotype is more relevant when the focus is on a specific sensitive stage such as PI.\nShoot ion uptake of salt-tolerant and sensitive lines\nShoot sodium ion analysis when assayed under salinity stress clearly distinguished tolerant from sensitive genotypes. Salinity stress increased the Na+ concentration in all genotypes significantly (Table\u00a01). The increase was nearly 10-fold in all genotypes except IR63731. Sodium ion accumulation in the tolerant genotypes, IR63731 and Agami, was much lower than in the sensitive genotypes. Agami, the salt-tolerant japonica, maintained a lower Na+ concentration even under control conditions and had the lowest Na+ concentration under stressed conditions. Shoot K+ concentration decreased in Agami, while the concentration in other three genotypes did not change significantly under salinity stress. Since K+\/Na+ ratio under salinity is considered a more important index of salinity tolerance than ion concentrations separately, we ranked the four genotypes by K+\/Na+ ratio in descending order: Agami (4.9)\u00a0\u2248\u00a0IR63731 (4.7)\u00a0>\u00a0M103 (3.7)\u00a0>\u00a0IR29 (2.9). Under control conditions, indicas had a lower K+\/Na+ ratio than japonica varieties. In addition to measuring Na+ and K+ levels in the shoot tissue, we also analyzed the chloride levels. Shoot chloride concentrations increased in all genotypes under salinity stress. Sensitive japonica, M103 however, maintained a much lower Cl\u2212 level (433\u00a0mmol\u00a0kg\u22121 compared to 535\u00a0mmol\u00a0kg\u22121 in Agami) than other genotypes.\nTable\u00a01Physiological responses of salt-sensitive and salt-tolerant genotypesTreatmentsNa+K+Cl\u2212TrM103 control23.1\u00a0\u00b1\u00a01.1872\u00a0\u00b1\u00a052.1355\u00a0\u00b1\u00a030.412.1\u00a0\u00b1\u00a01.6M103 salt236\u00a0\u00b1\u00a016.5881\u00a0\u00b1\u00a040.2433\u00a0\u00b1\u00a032.58.9\u00a0\u00b1\u00a00.7IR29 control24\u00a0\u00b1\u00a03.0799\u00a0\u00b1\u00a023.2351\u00a0\u00b1\u00a024.310.8\u00a0\u00b1\u00a01.3IR29 salt279\u00a0\u00b1\u00a033.2808\u00a0\u00b1\u00a049.5525\u00a0\u00b1\u00a012.67.6\u00a0\u00b1\u00a00.3Agami control16.5\u00a0\u00b1\u00a0.96772\u00a0\u00b1\u00a023350\u00a0\u00b1\u00a030.57.0\u00a0\u00b1\u00a00.6Agami salt140\u00a0\u00b1\u00a020.1688\u00a0\u00b1\u00a025.8535\u00a0\u00b1\u00a012.14.2\u00a0\u00b1\u00a00.6IR63731 control25.7\u00a0\u00b1\u00a02.9740\u00a0\u00b1\u00a030.5279\u00a0\u00b1\u00a01111.0\u00a0\u00b1\u00a00.8IR63731 salt166\u00a0\u00b1\u00a042.7791\u00a0\u00b1\u00a033.2545\u00a0\u00b1\u00a035.57.2\u00a0\u00b1\u00a00.8The shoot Na+, K+ and Cl\u2212 concentrations were measured in mmol\u00a0kg\u22121 dry weight. Transpiration rate (Tr) is expressed as m\u22122\u00a0s\u22121. Ion concentration values were obtained from six replicates. Tr values are mean\u00a0\u00b1\u00a01\u00a0S.E. from 6 to 7 replicate measurements\nTo further characterize the physiological status of the four genotypes, we measured the transpiration rate of the youngest fully expanded leaf of the main shoot (Table\u00a01). The transpiration rate decreased in all genotypes under stress. A noteworthy observation was the significantly lower rate of transpiration maintained by tolerant japonica Agami under control and stressed conditions relative to all other genotypes. Tolerance in Agami therefore seems to depend on an avoidance mechanism. We also observed the plants for visual damage symptoms of salinity stress. The sensitive genotypes, especially IR29, had necrosis on some leaves about 1\/3 the length of the leaf from the tip roughly one week after the initiation of stress.\nExpression responses of tolerant and sensitive genotypes to salinity stress\nWe used two japonica and indica rice genotypes with contrasting salt tolerance for global gene expression under salinity stress. The developmental stage targeted in this study was PI. Salinity stress was applied nine or 10\u00a0days before PI and the tissue (crown and growing point) was harvested 5\u00a0days after the genotypes reached PI stage (Fig.\u00a01; see \u201cMaterials and methods\u201d). To define differentially expressed genes we used the following criteria: (1) t-test P-value\u00a0<\u00a00.05, (2)\u00a0\u2265\u00a02-fold change, and (3) FDR below 15% (see Materials and methods). Using these criteria we identified 292 and 346 probe sets which were up- and down-regulated in salt-sensitive japonica M103 respectively (Fig.\u00a02). The same selection criteria identified 54 up- and 54 down-regulated probe sets in the salt-tolerant japonica, Agami. A comparable number of probe sets (55) were differentially up-regulated in salt-tolerant indica IR63731 under salinity stress. A total of 35 probe sets were down-regulated in IR63731. The expression response of sensitive indica, IR29 was characterized by up-regulation of 589 probe sets. In contrast, only 57 probe sets were significantly down-regulated in response to salinity stress. Complete lists of probe sets identified as differentially expressed in each of the genotypes are provided as supplemental data (Datasheet 9\u201311). Five probe sets identified as differentially expressed from the microarray datasets were checked using semi-quantitative RT-PCR (Supplemental Fig. 1). The results agreed for four of the genes examined. The primers for the fifth gene, Os02g52390, did not amplify from the indica subspecies, since they were derived from japonica exonic sequence. Nineteen probe sets were significantly up-regulated in all genotypes in response to salinity stress. A selected list of probe sets which were commonly induced is presented in Table\u00a02. Only one probe set, Os.17057.1.S1_at, with no hits to the database was down-regulated by salinity stress in all genotypes.\nFig.\u00a02Overlap of up-regulated probe sets between different genotypes. (A) Overlap between the sensitive genotypes M103 and IR29. (B) Overlap between the tolerant genotypes. (C) Overlap among the japonica subspecies. (D) overlap among the indica subspecies. The up-regulated probe sets were identified using the combined criteria of a 2 or more fold change, P-value threshold of 0.05 and false discovery rate below 15%Table\u00a02Selected genes commonly induced in all four genotypes and their P-values during the salinity stress imposed during reproductive stagePutative functionProbe setRice locusP-valueIR29M103IR63731AgamiWSI76 protein\u2013\u2013riceOs.2677.1.S1_atOs07g488300.0020.010.0180.038Low-temperature-induced protein LTI30Os.12633.1.S1_s_atOs11g267900.0010.0090.0020.0005Myb-like DNA-binding domainOs.10333.1.S1_atOs07g028000.00010.0010.0010.001RNase S-like protein (drought induced)Os.12922.1.S1_atOs09g367000.0020.00040.000060.004MtN3Os.16044.1.S1_atOs02g309100.0360.0260.00030.006Low affinity nitrate transporterOs.32686.1.S1_atOs01g651100.00030.00090.01680.007Putative Myb-like DNA-binding ProteinOs.25453.1.A1_atOs03g555900.00020.00270.00360.015Bowman\u2013Birk serine protease inhibitorOs.7612.1.S1_atOs03g608400.0140.0120.0240.008Nitrate transporter (NTP2)Os.45923.1.S1_atOs01g375900.00050.00030.00430.0013Anthocyanin 5-O-glucosyltransferaseOsAffx.4277.1.S1_atOs05g087500.0070.0230.0120.006Putative function is based on BLAST hits against TIGR rice, TIGR Arabidopsis and NCBI nr database\nWe made several comparisons to identify genes which characterize the response of japonica and indica to salinity as well as genes which are expressed in tolerant and sensitive genotypes (Fig.\u00a02). We identified 42 probe sets which were induced commonly between the two japonicas, Agami and M103. Similar comparison between the indica genotypes identified 41 probes sets as commonly induced. In an effort to search for transcripts which may be associated with salt tolerance of Agami, we identified 12 probe sets which were induced in Agami but not in M103. Two of these probe sets (Os.11975.2.A1_at and OsAffx.25546.1.S1_s_at) were induced in Agami but down-regulated in M103. Probe set Os.11975.2.A1_at had no sequence match to known genes. Probe set OsAffx.25546.1.S1_s_at represented a germin-like gene (GPL4), Os03g44880.. We also identified several probe sets which were induced in IR63731 but not in sensitive indica, IR29. These included RD22, a jacalin lectin protein and nitrate reductase (NR1). The expression of RD22 is known to be induced in response to salt stress and dehydration (Yamaguchi-Shinozaki and Shinozaki 1993).\nBesides focusing on the genes which were differentially expressed in response to salinity stress within each of the genotypes, we also performed genotype\u2013genotype comparisons under control conditions. This analysis was performed to identify genes associated with salt tolerance that may be inherently transcribed at a higher level in one genotype than in another. Interpretation of such genotypic comparisons using plants profiled under control conditions is done in light of the fact that the compared genotypes have similar developmental stage but not same chronological age. We found 539 probe sets which were significantly up-regulated in Agami relative to M103 under control conditions (Supplemental Data Sheet 13). A similar comparison yielded 686 probe sets expressed at a higher level in IR63731 than IR29 (Supplemental Data Sheet 14). We BLAST searched the target sequence of all the probe sets against the rice genomic sequence assembled at TIGR (http:\/\/www.tigr.org) to obtain the genomic position of the best BLAST hit gene model. We found several probe sets which were elevated in Agami relative to M103 to be tightly clustered. Clusters on chromosome 1, 5, 10 and 12 were particularly large (Fig.\u00a03A). A dense 19-probe set cluster occurred on the long arm of chromosome 10. This cluster localized to a segment of 1.1 Mb and represented 15 rice gene loci. Another 11-probe set cluster on chromosome 1 extends from 11.07\u00a0Mb to 12.61\u00a0Mb (1.54\u00a0Mb in length). This region is of particular interest because two QTL for salt tolerance have been mapped to this region (Bonilla et al. 2002; Lin et al. 2004). The number of gene clusters obtained from the comparison of indica genotypes was relatively small and most were found on chromosome 1 (Fig.\u00a03B). Two of the clusters on chromosome 1 were located in the region of a salt tolerance QTL Saltol. The first was an 8-probe set (six gene loci) cluster (9.03\u20139.91\u00a0Mb) and the second was a 14-probe set (eight gene loci) cluster extending from 11.19\u00a0Mb to 12.62\u00a0Mb. The positions of these two clusters may coincide with two separate QTL in the Saltol region reported by Lin et al. (2004) and Bonilla et al. (2002). Additionally, a dense 16-probe set, 537\u00a0kb long cluster was located on the distal end of chromosome 1.\nFig.\u00a03Rice chromosomes with gene clusters based on genotypic comparisons. (A) Rice gene model clusters on chromosomes 1, 5, 10 and 12 obtained from genotypic comparison of M103 with Agami under control conditions. (B) Rice gene model clusters on chromosome 1, obtained by genotypic comparison of IR29 with IR63731 using unstressed samples. The chromosome displays only up-regulated rice genes in the tolerant lines under control conditions. The gene clusters with shaded area co-localize to previously identified as a salt tolerance QTL on chromosome 1\nGenes involved in ion homeostasis\nMaintenance of low Na+ and a favorable K+\/Na+ ratio are desirable traits for rice growing under saline conditions and ultimately manifest as salt tolerance. Considering the importance of ion transporters in exclusion, redistribution, and compartmentalization of ions we searched for and identified several cation transport-related genes which significantly responded to the salinity treatment (Table\u00a03). One such gene was a sodium transport protein (Os02g07830) induced (P\u00a0<\u00a00.02) in IR29 only. In contrast a sodium\/calcium exchanger was down-regulated in M103. Another gene (Os12g25200), encoding for CLC-d chloride channel was induced in M103 and IR29 by 17-fold (P\u00a0<\u00a00.0001) and 7.3-fold (p\u00a0<\u00a00.00008), respectively. Several genes associated with transport of K+ were differentially regulated in response to salinity stress. Three of these genes belong to the KT-HAK-KUP family of transporters associated with high affinity K+ uptake from the roots (Santa-Maria et al. 1997). In M103, a transporter (Os08g36340) similar to HAK4 was up-regulated by stress, while HAK5 (Os07g01220) was down-regulated. Another cation channel gene (Os01g52070), with sequence similarity to KAT3 was down-regulated in M103. A potassium transporter, KUP1 (Os09g27580), was induced in IR29 by more than 5-fold (P\u00a0<\u00a00.002) under salinity stress. We did not detect any cation transporters significantly responding to salinity stress in the tolerant genotypes.\nTable\u00a03Genes from selected categories which were differentially expressed under saline conditions in one or more rice genotypesPutative functionProbe setLocusM103IR29IR63731AgamiIon homeostasisK+ transporter (HAK4)Os.2358.1.S1_atOs08g363401.20.63\u2013\u2013CLC-d chloride channelOs.27207.1.S1_atOs12g252004.162.880.78\u2013Cation channel protein (KAT3)OsAffx.21536.1.S1Os01g52070\u22121.05\u2013\u2013\u2013Na+\/Ca2+ exchanger proteinOs.20354.1.S1_atOs03g45370\u22121.11\u2013\u2013\u2013K+ transporter (HAK5)Os.27454.1.S1_xOs07g01220\u22121.54\u2013\u2013\u2013Cation (Na+) transport proteinOs.50590.2.A1_atOs02g07830\u20131.0\u2013\u2013K+ transporter (KUP1)Os.6037.2.S1_x_atOs09g27580\u20132.51\u2013\u2013Flowering and inflorescenceCentroradialis protein (CEN)Os.57548.1.S1_atAt2g275501.551.44\u2013\u2013Zinc finger protein (ID1)Os.4766.1.S1_at1.470.62\u2013\u2013Constan-like protein (CO6)Os.7920.1.S1_atOs06g444501.391.00.68CCT motifOs.16422.1.S1_s_atOs08g150501.04\u2013\u2013\u2013CLAVATA1 receptor kinaseOs.7123.2.S1_x_at0.821.05\u2013\u2013AP1-like MADS-box proteinOs.2348.1.S1_a_at\u22120.91\u22121.09\u2013\u2013Gigantea-like proteinOs.7987.1.S1_atOs01g08700\u22121.04\u2013\u2013\u22121.74Floral homeotic protein (AP1)Os.12750.2.S1_s_atAt1g69120\u22121.12\u2013\u2013\u2013Flowering locus TOsAffx.15765.1.S1Os06g35940\u22121.131.7\u2013\u2013Homeodomain (KNAT7)Os.4164.2.S1_a_atAt1g62990\u22121.66\u2013\u2013\u2013Sex determination tasselseed 2Os.15281.1.S1_at\u20133.93\u2013\u2013No apical meristem (NAM)Os.37548.1.S1_at\u20131.81\u2013\u2013AuxinCYP83B1Os.11110.1.S1_atAt4g31500\u22121.71\u2013\u2013\u2013Auxin-repressed protein ARP1Os.12735.1.S1_at1.21\u2013\u2013\u2013Auxin-responsive proteinOs.20151.1.S1_atAt3g25290\u20131.27\u2013\u2013Auxin transporter PIN1Os.37330.1.S1_at\u22120.94\u2013\u2013\u22121.1Axi1Os.52961.1.S1_at\u22121.0\u2013\u2013\u2013Phototropic-responsive NPH3Os.56849.1.S1_atAt1g030101.03\u2013\u2013\u2013NPK1-related protein kinaseOs.5940.1.S1_at\u20131.46\u2013\u2013Auxin response factor 10Os.8374.1.S1_atOs04g43910\u22121.17\u2013\u2013\u2013Anthocyanin pathwayChalcone, stilbene synthasesOs.11154.1.S1_aOs03g067001.111.460.921.3Phenylalanine ammonia-lyaseOs.25687.1.S1_xOs02g41680\u20131.25\u2013\u2013Isoflavone reductaseOs.32454.1.S1_atAt4g39230\u22121.96\u2013\u2013\u20134-coumaroyl-CoA synthase 3Os.4377.1.S1_atAt1g65060\u22121.4\u2013\u2013\u2013Flavonoid 3\u2032-hydroxylase Os.46551.1.S1_atAt5g07990\u2013\u22121.09\u2013\u2013Dihydroflavonol 4-reductaseOs.48545.1.S1_atOs04g53810\u20131.94\u2013\u2013Ferulate-5-hydroxylaseOs.9727.1.S1_atAt4g36220\u22121.64\u2013\u2013\u2013Chalcone isomerase (CHI)Os.9929.1.S1_atAt3g55120\u22121.31\u2013\u2013\u20135-hydroxyferulic acidOsAffx.13783.1.S1At5g54160\u20131.02\u2013\u2013Fold change in each genotype is expressed as log2 (log21.2 is equal to 2.2-fold change numerically). The P-value threshold of 0.05 was used for significant differential expression. A (\u2013) indicates no change detected\nSince the shoot ion analysis indicated a strong Na+ exclusion phenotype in the tolerant lines, we further explored our data set for any association of cation transporters. On comparing the tolerant Agami with M103 under control and salinity stress conditions, we found a cation transporter (Os01g20160) to be expressing at a higher level in Agami by 2.6-fold (P\u00a0<\u00a00.01) and 2.8-fold (P\u00a0<\u00a00.003) compared to M103 under control and salt stressed conditions, respectively. On extending the genotypic comparison to indica genotypes, we found Os01g20160 transcript to be present in IR63731 at a higher level than in IR29 (1.98-fold, P\u00a0<\u00a00.01) under control conditions. The cation transporter gene 01g20160 is located in the vicinity of the Saltol region (Bonilla et al. 2002) and has a perfect sequence match to the SKC1 gene reported to be the tolerance conferring gene in the salt tolerance QTL studied by Ren et al. (2005). We pursued the expression of this gene using semi-quantitative RT-PCR in roots and shoot tissue of several rice genotypes which were part of the salinity stress experiment conducted during the vegetative stage study (Walia et al. 2005). The expression of 01g20160 in genotypes IR63731, Pokkali, FL478 and IR29 was analyzed in the root and shoot tissues for vegetative stage (V), and shoot tissue of IR63731 and IR29 at the PI stage (R) under control and stressed conditions (Fig.\u00a04). The expression of Os01g20160 increased in response to salinity in the roots of tolerant lines IR63731, Pokkali and FL478 but not in salt-sensitive IR29. The mRNA accumulation of Os01g20160 in shoot tissue increased in response to salinity in all four genotypes analyzed during the vegetative stage.\nFig.\u00a04Expression of SKC1 in different indica genotypes. Semi-quantitative RT-PCR was performed to study the expression of SCK1 in the root and shoot tissue of IR63731 and IR29 under control and stressed conditions during vegetative (V) and reproductive (R) stages. Expression was analyzed for roots and shoot tissue of tolerant lines Pokkali and FL478 during vegetative stage of growth. The curly bracket encloses a control and the corresponding salt stressed sample\nDifferential expression of auxin associated genes in rice under salinity stress\nPhytohormones including auxin indole 3-acetic acid (IAA) play a central role in plant growth, and development. Auxin is also known to mediate responses to light and gravity. In our expression analysis, several auxin-related genes were differentially regulated by salinity stress, indicating that salinity stress is associated with auxin response in rice. Almost all of these auxin related genes were differentially expressed in sensitive genotypes M103 and IR29. Most striking was the response of M103 to salinity in this context. The expression of cytochrome P450 83B1 (CYP83B1) was significantly (P\u00a0<\u00a00.04) down-regulated under salt stress. The expression level of CYP83B1 is known to determine auxin levels in plants (Barlier et al. 2000). Other auxin related genes down-regulated in response to salinity include a putative axi1 and two auxin response factors. In contrast, a mitogen activated protein kinase cascade gene, NPK1 which is known to suppress early auxin response gene transcription is induced in response to salinity in IR29. An increased level of nonphototropic hypocotyl 3 (NPH3) transcript was observed in M103 under salt stress. A rice ortholog of NPH3 is involved in phototropic response of coleoptiles through lateral auxin translocation (Haga et al. 2004). Another gene involved in auxin transport, PIN1 was found to be down-regulated in Agami. Several of the auxin related genes and their response to salinity are listed in Table\u00a03.\nEffect of salinity stress on floral transition and development in rice\nThe transition from vegetative to reproductive stage, termed floral transition, is a dramatic phase change in plant development. The transition controlled by a complex genetic network that integrates environmental signals such as day length and temperature (Simpson et al. 1999). We found several rice genes which are associated with floral transition and morphological development to be responsive to salinity stress imposed at the PI stage. Centroradialis (CEN), a gene which plays a role in phase transition and panicle morphology, was induced in both the sensitive genotypes, M103 and IR29 in response to salinity. A zinc-finger protein indeterminant1 (ID1) linked to phase transition signaling in maize, was induced at a higher level (2.7-fold, P\u00a0<\u00a00.0001) in M103 compared to IR29 (1.5-fold, P\u00a0<\u00a00.02). In contrast, the flowering locus T (FT) (Os06g35940) gene was differentially regulated in M103 (down-regulated) compared to IR29 (up-regulated) under salinity stress. FT is known to be downstream of gigantea, which represses flowering in rice. A gigantea-like gene (Os01g08700) was down-regulated in both japonica genotypes M103 and Agami. Several other genes involved in phase transition and organ identity and size determination were differentially regulated in M103, IR29 and Agami but not in IR63731. On comparing the expression patterns of IR63731 with IR29, we found several genes including gigantea, FT, terminal flower 1 (TFL1), and embryonic flower 2 (EMF2) to be expressing at a higher level in IR63731 under control conditions. Some of these differentially regulated genes and their expression levels in different genotypes are detailed in Table\u00a03.\nDiscussion\nIn this study, we applied global gene expression profiling analysis to study the transcriptional responses of four rice genotypes comprised of tolerant and sensitive indicas and japonicas. For these genotypes, each with well-characterized salinity tolerance from prior studies, we measured gene expression during PI, the most salt-sensitive stage of development, considered to be the major factor in yield loss in rice. The results from this study, which used a moderate, agronomically relevant salinity stress, demonstrate striking differences in expression responses between tolerant and sensitive genotypes. The genotypes were also characterized for their physiological responses to obtain insights which enabled us to correlate some of the phenotypic observations with expression results.\nUnder salinity stress two key features, one physiological and the other within the transcriptomes, clearly distinguished the tolerant genotypes from the sensitive genotypes. First is the ability of the tolerant lines IR63731 and Agami to exclude Na+ ion from the shoot tissue compared to sensitive lines. The second feature, observed at the gene expression level, is the strikingly small number of genes which responded to salinity stress in the tolerant genotypes. This is likely a manifestation of the low shoot Na+ trait in tolerant lines which experience significantly lower toxicity at the cellular level and therefore requires fewer adjustments of cell metabolism and architecture. Among the genes which were commonly induced by salinity in all genotypes were abiotic stress response related genes such as water stress induced (WSI76), low temperature induced protein (LTI30) and a drought-induced RNase S-like gene. The WSI76 encodes for galactinol synthase (Takahashi et al. 1994) and was responsive to low temperature and osmotic stress. Galactinol synthase is required for the committed step in raffinose family oligonucleotides (RFOs). It was also induced in the tolerant line FL478 when salinity was imposed at early vegetative stage, but not in IR29 (Walia et al. 2005). Besides the genes involved in response to dehydration, we also detected at least two genes involved in nitrate transport, which may be compensating for salinity induced nutritional deficiency. A nitrate transporter, NRT1 was induced in Mesembryanthemum crystallinum in leaf and root tissue under salt stress (Popova et al. 2003). A low-affinity nitrogen transporter and NRT2, both localizing to chromosome 1 of rice were induced in all rice genotypes in response to salinity stress (Table\u00a02).\nIon homeostasis in rice under salinity stress\nOur data indicated that the tolerant genotypes IR63731 and Agami maintained significantly lower Na+ concentration in the shoot tissue compared to sensitive lines under salinity stress. Sodium ion concentration in the plant is considered to be a good indicator of the ion toxicity levels to which the cells are exposed. The lower ion specific stress in tolerant genotypes is reflected in the relatively small number of genes which respond to salinity in tolerant lines when compared to sensitive IR29 and M103. We found several ion homeostasis related genes such as ion transporters which were responsive to salinity stress in the sensitive genotypes. One such gene encoding for Na+ transporter (Os02g07830) was induced by salinity stress in IR29. This gene has high sequence similarity to the ArabidopsisHKT1. The barley ortholog of Os02g07830 was up-regulated under salt stress in the shoot tissue of a barley genotype, Morex during early seedling stage (Walia et al. 2006, submitted). This barley genotype particularly and barley in general, is known to accumulate a high level of Na+ in the shoot tissue during salinity stress. Salt tolerance in barley is believed to be mainly achieved through efficient compartmentalization of toxic ions besides other mechanisms conferring tissue tolerance. High Na+ in the shoot tissue was also documented for rice genotype IR29 at vegetative and reproductive stage under salinity stress (Walia et al. 2005; Table\u00a01). The expression of Os02g07830 did not change significantly in the other three genotypes examined. Based on these pieces of information, we can speculate that Os02g07830 may be involved in redistribution or compartmentalization of Na+ within the plant in genotypes which are relatively inefficient in Na+ exclusion from the plant.\nAnother rice gene recently proposed to confer salt tolerance through recirculation of Na+ ions is SKC1 (Ren et al. 2005). SKC1 was identified as gene underlying a rice QTL for maintaining high shoot K+ in the shoot under salt stress (Lin et al. 2004). The SKC1 gene also encodes a HKT-type transporter and functions as a Na+-selective transporter in rice. Rice locus, Os01g20160 previously annotated as a cation transporter corresponds to SKC1 based on sequence similarity. This locus has been reported to be induced in shoot tissue by salt stress in both the sensitive IR29 and tolerant FL478 genotypes under salinity stress imposed during early vegetative stage (Walia et al. 2005). However, the induction in salt-tolerant FL478 was significantly higher than in IR29 under stress. In contrast to these prior results with vegetative stage stress, we did not detect a significant up-regulation of SKC1 in the shoot tissue of any of the four rice genotypes at PI profiled under salt stress in this study. However, on making genotype\u2013genotype comparisons between Agami-M103 and IR63731-IR29, we found SKC1 to be expressing at a higher level in Agami compared to M103 under both control and saline conditions. Interestingly, the shoot Na+ ion analysis indicated that Agami maintains lower Na+ concentration than M103 under both control and saline conditions (Table\u00a01).\nThe expression of SKC1 was reported to be up-regulated by salt stress in root but not shoot tissue (Ren et al. 2005). In contrast, our results with indica genotypes at vegetative stage indicated an up-regulation of SKC1 by salt stress in the shoot (crown plus growing point tissues), an observational discrepancy that may be attributed to the very different methods of applying salt stress between the two studies. We pursued the expression analysis of SKC1 further by analyzing the shoot and root tissues of tolerant indicas Pokkali, IR63731 and FL478 and sensitive line IR29 (Fig.\u00a04). Our results indicate that SKC1 is salt up-regulated in the shoots of all four indica genotypes under the conditions that we used. Under the more gradual and moderate stress conditions that we applied, SKC1 was up-regulated in the root tissues only in salt-tolerant lines, not in salt-sensitive IR29.\nAuxin and response of rice to salinity stress\nSalinity modulates the expression of several auxin-associated genes in the sensitive japonica genotype, M103. The expression level of the gene encoding CYP83B1 was down-regulated in M103 but not in other genotypes. A loss-of-function mutation in CYP83B1 resulted in accumulation of indole 3-actaldoxime (IAOx) which is redirected to auxin biosynthesis (Bak et al. 2001). Taken together, it is plausible that auxin homeostasis in M103 is disturbed in response to salinity by virtue of a reduction of CYP83b1 gene expression. Another gene involved in auxin signal transduction, axi1 was also down-regulated in M103 (Hayashi et al. 1992). Although the mechanisms of auxin signal transduction in plants are still largely obscure, there is considerable evidence linking auxin perception and various ion transporters located in plant cell membranes (Blatt and Thiel, 1993; Barbier-Brygoo, 1995). Anion channels have been indicated to be involved in auxin signaling (Thomine et al. 1997). More specifically, auxin stimulated Cl\u2212 efflux\/influx has been of interest for several decades (Bentrup et al. 1973; Rubinstein, 1974; Babourina et al. 1998). Bentrup et al. (1973) reported that IAA significantly reduced the uptake of Cl\u2212 by cells of Petroselinum sativum, while Babourina et al. (1998) reported a stimulatory effect of auxin on Cl\u2212 uptake by oat coleoptiles. The modulation of auxin related genes in M103 during salt stress is attractive in context of the shoot Cl\u2212 concentration in M103. The sensitive genotype maintained a significantly lower Cl\u2212 concentration under salt stress compared to other genotypes (Table\u00a01). Interestingly, a CLC-d chloride channel (Os12g25200) was induced in M103 by 17-fold (P\u00a0<\u00a00.0001) in response to salinity stress.\nThree of the genes induced in IR29 in response to salinity were associated with auxin. The chloride channel (Os12g25200) was induced in IR29 by 7-fold (P\u00a0<\u00a00.00008). Also induced was a gene for an NPK1 kinase-like protein under salinity stress. Constitutive expression of a tobacco MAPKK NPK1 is reported to dampen auxin responsiveness of a reporter gene (Kovtun et al. 1998) and confer salt tolerance (Kovtun et al. 2000). Additionally, expression profiling of the salt-overly sensitive (sos2-1) mutant in comparison to wild type Arabidopsis revealed differential induction of auxin-related genes in the mutant under salt stress (Kamei et al. 2005). These reports and our data indicate a significant role of auxin in response of some rice genotypes to salinity stress.\nSalinity stress and floral transition\nIn the current work we focused on shoot meristem tissue under salinity stress imposed during PI. We detected several genes associated with developmental transitions and flowering to be modulated by salinity stress. Salinity delays PI in rice (Zeng et al. 2002), and in our study this delay was generally greater for sensitive than tolerant genotypes. In this context we found the up-regulation of rice genes with sequence match to terminal flower 1 (TFL1)\/centroradialis (CEN) in the sensitive genotypes to be interesting. Over expression of TFL1\/CEN homologs of rice delayed phase transition and altered panicle morphology (Nakagawa et al. 2002). The delay in initiation of panicle in sensitive genotypes, especially M103 under salinity stress, was longer than tolerant genotypes. The expression of TFL1\/CEN was not significantly changed in the tolerant lines under stress. Both TFL1 and CEN belong to the family of phosphatidyl-ethanolamine-binding proteins (PEBPs) (Bradley et al. 1996). Another gene which is involved in flowering transition is FT, which is also a member of the PEBP family. The role of FT is however antagonistic to TFL1, as FT induces floral transition. The expression level of FT was down-regulated in M103 as expected due to a delayed floral transition under salinity stress. Surprisingly, FT was up-regulated in IR29. Up-regulation of FT in IR29 was also observed during the vegetative stage (Walia et al. 2005). The expression of FT was not modulated by salinity stress in tolerant lines IR63731 and Agami during PI and in FL478 during vegetative stage. An up-stream member of the floral induction pathway to which FT belongs is gigantea (GI). Suppressed expression of the rice ortholog of gigantea (OsGI) through RNA-mediated interference (RNAi) resulted in a late flowering phenotype under short day conditions relative to wild-type plants (Hayama et al. 2003). The expression level of gigantea-like gene was significantly reduced in M103 and Agami under salinity stress (Table\u00a03). None of these genes were differentially modulated in tolerant indica IR63731. In brief, the gene expression analysis identified genes which explain the observed delay in PI of M103 under salinity stress.\nHigh salt tolerance has often been linked to genotypes which take relatively long to reach maturity. The genotypes selected for this experiment also differ in their time to reach maturity. Therefore it can be argued that the late maturing genotypes IR63731 and Agami are likely to be more salt tolerant merely as a consequence of higher biomass translating into higher yield. However, Zeng et al. (2002) reported a lack of correlation between the duration of reproductive growth period and salt tolerance based on seed yield. This study also included three of the four genotypes used in the current study, namely IR63731, Agami and M103 and employed a comparable level of salinity stress. Since difference in time to maturity for different genotypes can potentially lead to confounding effects on gene expression measured by microarrays, we decided to focus on the developmental stage just after PI was observed for each treatment regardless of the growth conditions (control or stressed) and genotype. This involved staggered harvesting of control and stressed plants for each of the genotype, hence normalizing for delay in PI resulting from salt stress. The effect of difference in development among genotypes used for transcriptome analysis can be avoided by using nearly isogenic lines which differ in salinity tolerance at PI but have similar developmental behavior. However such optimal lines focusing on tolerance at PI are presently not available.\nCommonalties in the salinity stress response of IR29 at early vegetative versus reproductive stage\nSensitive genotype IR29 responded to salinity stress during PI by inducing a large number of genes. Up-regulation of large number of genes was also reported for IR29 under salinity stress during early vegetative stage relative to tolerant genotype FL478 (Walia et al. 2005). We explored the extent of commonalities in salinity induced responses of IR29 by comparing the induced genes during vegetative and reproductive stage. One of the most striking stress responses of IR29 during the vegetative stage experiment was the induction of genes involved in the flavonoid pathway. In the present study, we again observed that several of the flavonoid pathway related genes were induced in IR29 under salt stress, in this case at PI. Among the genes induced include those encoding for phenylalanine ammonia lyase, chalcone synthase, dihydroflavonol 4-reductase, and flavonone 3-hydroxylase. Therefore, up-regulation of the flavonoid pathway as a response to salinity stress appears to be a general characteristic of IR29. Interestingly, some of the genes involved in the flavonoid pathway were down-regulated in the sensitive japonica, M103. So the induction of flavonoid pathway in response to salinity stress is not a universal response of sensitive rice genotypes.\nWe further compared the IR29 data from the vegetative stage study with the current IR29 PI data. In general, we found that the genes responding to salinity stress at vegetative stage were strikingly different from those which responded when the stress was imposed during PI. A total of 524 probe sets were differentially expressed in response to salinity at reproductive stage but not during vegetative stage. We were especially interested in genes which mapped to regions of the rice genome associated with salt tolerance based on QTL studies. One such gene (Os01g24710) encoding for SALT protein was induced during the reproductive stage. This gene has a jacalin-like lectin domain and physically maps to the Saltol region on chromosome 1 (13.88\u00a0Mb). Interestingly, another gene (Os01g25280) also encoding a SALT-related protein was induced during the vegetative stage in IR29 (Walia et al. 2005). This gene also maps to the Saltol region (14.26\u00a0Mb) on chromosome 1. The salT gene in rice is responsive to salt stress and the phytohormones abscisic acid and jasmonic acid (JA) (Claes et al. 1990; Moons et al. 1997). We found some genes involved in JA biosynthesis, and JA responsive genes, to be induced by salinity stress in IR29 during reproductive stage but not during vegetative stage. These include two lipoxygenases (Os03g52860 and Os12g37260), a gene encoding for 12-oxo-phytodienoic acid reductase (OPR2) (Os06g11240) and a jasmonate induced protein (JIP). The induction of JA biosynthesis and responsive genes during salinity stress is interesting in context of the report that post-treatment of salt-stressed rice with JA dramatically improves the physiological activities otherwise inhibited by stress (Kang et al. 2005). Results from our laboratory have also indicated an ameliorating role of JA pretreatment in salt-stressed barley on shoot Na+ concentrations and photosynthetic performance (Walia et al. 2007).\nBesides salT, we found several other key transcripts related to salinity stress response as differentially expressed during reproductive stage. A potassium uptake protein (Os09g27580) induced by salinity stress during the reproductive stage in IR29 was one such transcript. This gene localizes to a region on chromosome 9 (16.7\u00a0Mb) which is associated with a major QTL for K+ uptake in rice identified by Koyama et al. (2001). This region on chromosome 9 was more recently linked to a QTL controlling root Na+ concentration (Lin et al. 2004). Another gene Os12g39400 (Os.16453.1.S1_at) encoding for a salt-tolerance zinc finger protein (STZ\/ZAT10) was up-regulated during reproductive stage under salinity stress. The STZ\/ZAT10 is a transcriptional repressor which is believed to regulate the COR\/RD gene expression (Lee et al. 2002). The expression of STZ\/ZAT10 was not up-regulated in any other genotype under salt stress. Based on this comparison, we can state that very few genes are commonly induced in response to salinity stress in a given genotype when sampled at two different developmental stages.\nElectronic supplementary materials\nBelow are the links to the electronic supplementary materials.\nESM 1 (XLS 64 kb)\nESM 2 (XLS 74 kb)\nESM 3 (XLS 21 kb)\nESM 4 (XLS 21 kb)\nESM 5 (XLS 111 kb)\nESM 6 (XLS 23 kb)\nESM 7 (XLS 21 kb)\nESM 8 (XLS 18 kb)\nESM 9 (XLS 126 kb)\nESM 10 (XLS 32 kb)\nESM 11 (XLS 128 kb)\nESM 12 (XLS 29 kb)\nESM 13 (XLS 16 kb)","keyphrases":["rice","panicle initiation","salt stress","microarray"],"prmu":["P","P","P","P"]}
{"id":"Skeletal_Radiol-4-1-2335296","title":"Plantar fasciitis and calcaneal spur formation are associated with abductor digiti minimi atrophy on MRI of the foot\n","text":"Objective To determine the association of atrophy of the abductor digiti minimi muscle (ADMA), an MRI manifestation of chronic compression of the inferior calcaneal nerve suggesting the clinical diagnosis of Baxter\u2019s neuropathy, with MRI markers of potential etiologies, including calcaneal spur formation, plantar fasciitis, calcaneal edema, Achilles tendinosis and posterior tibial tendon dysfunction (PTTD).\nIntroduction\nEntrapment of the first branch of the lateral plantar nerve (inferior calcaneal nerve), or Baxter\u2019s neuropathy, produces medial heel pain which may be indistinguishable from heel pain due to plantar fasciitis [1\u20133]. Baxter\u2019s neuropathy accounts for up to 20% of cases of heel pain [4] but is frequently overlooked as a potential cause of pain, even when pain persists after surgical intervention for other potential causes. On MRI, the presence of atrophy of the abductor digiti minimi muscle (ADMA) reflects chronic compression of the inferior calcaneal nerve and suggests the clinical diagnosis of Baxter\u2019s neuropathy [1, 5]. Treatment for Baxter\u2019s neuropathy includes conservative therapy and, in cases in which non-interventional management is ineffective, surgical release [6].\nEntrapment of the inferior calcaneal nerve may result from altered biomechanics, reflected by posterior tibial tendon dysfunction (PTTD) or Achilles tendinosis, or may result from direct mechanical compression of the nerve due to plantar fasciitis and\/or plantar calcaneal enthesophytes [1, 7]. Our study was performed to assess the frequency of MRI manifestations of these potential etiologic factors in patients with and without ADMA to test the hypothesis that MRI findings reflecting altered biomechanics or direct compression of the inferior calcaneal nerve occur more frequently in patients with ADMA.\nMaterials and methods\nPatient population\nThis retrospective study included 200 individuals who were referred for MRI of the hindfoot due to the presence of foot pain. These were individuals seen in a subspecialty teleradiology group practice with participant referrals from 40 states. The study was conducted with data from August 2006 to January 2007 and included patients of all ages. The study group was composed of 100 consecutive individuals with abductor digiti minimi atrophy and 100 consecutive individuals without abductor digiti minimi atrophy. No eligible participants were excluded. The subspecialty teleradiology group practice does not have an Institutional Review Board (IRB), and the study was conducted in compliance with the Declaration of Helsinki principles and the Health Insurance Portability and Accountability Act. Owing to the retrospective nature of the study, informed consent was not obtained.\nParticipants\u2019 ages ranged from 10\u201392\u00a0years [mean\u2009=\u200949.0\u00a0years, standard deviation (SD)\u2009=\u200916.9\u00a0years] and included 139 women and 61 men.\nMRI technique\nPatients were scanned in magnets ranging from 0.2\u00a0T to 1.5\u00a0T. Long- and short-axis fat and fluid sensitive sequences were obtained without the administration of gadolinium. The technical parameters for all magnet strengths were as follows: sagittal T1-weighted, sagittal short-tau inversion recovery (STIR), oblique axial STIR, coronal T1-weighted, coronal STIR, plantar flexed axial proton density, axial T2-weighted, and axial T2-weighted with fat saturation or STIR.\nData acquisition\nIn our retrospective analysis, potential patients were identified through review of reports of consecutive MRI studies obtained over a 6-month period. One hundred patients with ADMA and 100 patients without ADMA were selected for inclusion through review of reports, with subsequent confirmation of the presence or absence of ADMA through review of images. Patients were identified by review of MRI reports, with subsequent review of MR images. We reviewed 1,780 MRI reports in order to identify 100 patients with ADMA. Two radiologists (U.C., and A.L.), each with 4.5\u00a0years of experience of MRI interpretation and who were blinded to the initial interpretation, by consensus interpreted the MR images of both groups. Each MR image was evaluated for presence and absence of plantar fasciitis, calcaneal spur formation, calcaneal edema, PTTD, and Achilles tendon dysfunction. Atrophy was defined by the presence of any amount of fatty replacement of muscle mass on T1-weighted images (Figs.\u00a01 and 2). Plantar fasciitis was defined by thickening, partial or full thickness tear of the plantar fascia. Calcaneal spur formation was defined by the presence of a bony outgrowth at the medial calcaneal tuberosity. Calcaneal edema was defined by increased signal intensity within the calcaneus adjacent to the insertion of the plantar fascia on fluid-sensitive sequences. PTTD was defined by tendon thickening, tenosynovitis or partial or full thickness tear. Achilles tendon dysfunction was defined by tendon thickening, peritendonitis or partial or full thickness tear.\nFig.\u00a01Coronal T1-weighted image demonstrating normal abductor digiti minimi (ADM) muscleFig.\u00a02Coronal T1-weighted image demonstrating fatty replacement of the abductor digiti minimi muscle (ADM), indicating atrophy\nStatistical analysis\nAge and gender distribution, as well as the frequency of plantar fasciitis, calcaneal spur formation, calcaneal edema, PTTD, and Achilles tendon dysfunction, were compared in patients with and without ADMA. Age was compared with analysis of variance (ANOVA). For the categorical variables we used, as appropriate, Pearson chi-square analyses or Fisher\u2019s exact test to compare the proportion of those with atrophy to those without. We used logistic regression to determine odds ratios and 95% confidence intervals for each of the variables to atrophy. First, we included each variable in a univariate model to determine its relationship to atrophy. We then simultaneously included all the variables in a multivariate analysis to determine the relationship of the variable to atrophy, while simultaneously adjusting for all the other variables. Alpha level was at 0.05, and all tests were two-sided. SPSS version\u00a015.0 (2006) (SPSS, Chicago, USA) was used for the analyses.\nResults\nOverall, of 200 patients in the study (100 with ADMA and 100 without ADMA), 25 (12.5%) had Achilles tendinosis, 18 (9%) had calcaneal edema (n\u2009=\u200918), 55 (27.5%) had calcaneal spurs, 41 had other tendon abnormalities, 63 (31.5%) had plantar fasciitis, and 43 (21.5%) had PTTD. Fourteen (7%) patients had isolated ADMA in the absence of any other identified pathological condition.\nComparison of the frequency of the above findings in patients with and without ADMA is shown in Table\u00a01. Those with ADMA were significantly older than those without atrophy (P\u2009<\u20090.001). There were no gender differences.\nTable 1Comparisons of demographic and clinical variables for those with and without atrophy of the abductor digiti minimi muscleVariableNo atrophy (n\u2009=100) % (no.)Atrophy (n=100) % (no.)PAge (years)a<0.001[Mean (SD)]40.8 (15.8)57.2 (13.7)Gender0.28Women66.0% (66)73.0% (73)Men34.0% (34)27.0% (27)Achilles tendinosis<0.001No97.0% (97)78.0% (78)Yes3.0% (3)22.0% (22)Calcaneal edema0.005No97.0% (97)85.0% (85)Yes3.0% (3)15.0% (15)Calcaneal spur<0.001No93.0% (93)52.0% (52)Yes7.0% (7)48.0% (48)Other tendon abnormality0.60No81.0% (81)78.0% (78)Yes19.0% (19)22.0% (22)Plantar fasciitisb<0.001No89.0% (89)47.5% (47)Yes11.0% (11)52.5% (52)PTTDNo89.0% (89)68.0% (68)Yes11.0% (11)32.0% (32)<0.001aMean and SDs compared with ANOVA, since continuous variable. All other analyses with Pearson\u2019s chi-square, except for Achilles tendinosis and calcaneal edema, which were with Fisher\u2019s exact test due to small subgroupsbData missing for one individual with atrophy\nPatients with atrophy compared to those without atrophy had significantly greater proportions of Achilles tendinosis, calcaneal edema, calcaneal spur, plantar fasciitis, and PTT dysfunction.\nTable\u00a02 shows logistic regression analyses for the variables. In univariate analyses, increased age, presence of Achilles tendinosis, calcaneal edema, calcaneal spur, plantar fasciitis, and PTT dysfunction were all significantly associated with increased odds ratios for atrophy. In the multivariate analysis, only increased age, presence of calcaneal spur, and presence of plantar fasciitis were significantly associated with atrophy. Presence of Achilles tendinosis only had a P value of 0.09, and calcaneal edema and PTT dysfunction had non-significant P values. The multivariate analysis was also repeated, using data only from the individuals older than 21\u00a0years, in case inclusion of data from patients younger than 21\u00a0years (n\u2009=\u200915) had affected the results; the odds ratios, P values and significance levels were almost exactly the same.\nTable\u00a02Logistic regression analyses for atrophy of the abductor digiti minimi muscle (OR odds ratio, CI confidence interval)VariableUnivariate OR (95% CI)PMultivariate OR (95% CI) PAge (years)1.08 (1.05, 1.11)<\u20090.0011.06 (1.03, 1.09)<\u20090.001Gender0.280.51\u00a0Women1.001.00\u00a0Men0.72 (0.39, 1.32)0.75 (0.33, 1.73)Achilles tendinosis<\u20090.0010.09\u00a0No1.001.00\u00a0Yes9.12 (2.63, 31.60)3.69 (0.81, 16.88)Calcaneal edema0.0070.47\u00a0No1.001.00\u00a0Yes5.71 (1.60, 20.39)1.89 (0.35, 10.19)Calcaneal spur<\u20090.0010.02\u00a0No1.001.00\u00a0Yes12.26 (5.18, 29.10)3.60 (1.28, 10.17)Other tendon abnormality0.600.71\u00a0No1.001.00\u00a0Yes1.20 (0.60, 2.39)1.19 (0.48, 2.97)Plantar fasciitis<\u20090.0010.01\u00a0No1.001.00\u00a0Yes8.95 (4.27, 18.77)3.35 (1.31, 8.56)PTTD0.0010.31\u00a0No1.001.00\u00a0Yes3.81 (1.79, 8.10)1.72 (0.60, 4.88)\nOf the 200 patients in the study, the consensus reviewers (U.C. and A.L) disagreed with findings in the initial report of eight , three with ADMA and five without ADMA. In two of the cases of ADMA, the presence of a calcaneal spur was not mentioned in the initial report. In the third case of ADMA, the presence of posterior tibial tendinosis was not mentioned in the initial report. For three of the five patients without ADMA, the presence of a calcaneal spur was not mentioned in the initial report. For one of the patients without ADMA, the presence of Achilles tendinosis was not mentioned in the initial report. For one patient without ADMA, the presence of plantar fasciitis was not mentioned.\nDiscussion\nPatients with ADMA had a significantly greater frequency of Achilles tendinosis, calcaneal edema, calcaneal spur, plantar fasciitis, and posterior tibialis tendon dysfunction than those without ADMA. After logistic regression analysis, only increased age, calcaneal spur, and plantar fasciitis remained significantly associated with ADMA.\nThe inferior calcaneal nerve supplies motor branches to the flexor digitorum brevis, quadratus plantae and abductor digiti minimi muscles and sensory fibers to the long plantar ligament and calcaneal periosteum [1]. The inferior calcaneal nerve originates from the lateral plantar nerve near the trifurcation of the posterior tibial nerve at the level of the medial malleolus and courses between the abductor hallucis and quadratus plantae muscles [8] (Figs.\u00a03 and 4). When it reaches the inferior border of the abductor hallucis muscle, it turns laterally, passing anterior to the medial calcaneal tuberosity (or spur, if present), between the quadratus and underlying flexor digitorum brevis muscles until it reaches the abductor digiti minimi muscle [7, 9\u201311] (Fig.\u00a03). The orthopedics and podiatrics literature suggests two possible sites of entrapment of the inferior calcaneal nerve which might result in Baxter\u2019s neuropathy: first, in patients with altered biomechanics, such as excessive pronation (flatfoot deformity), the nerve may be compressed as it turns laterally between the quadratus plantae and abductor hallucis muscles [12], and second, the nerve may be compressed as it passes anterior to the medial calcaneal tuberosity, or, if present, plantar calcaneal spur [7, 8, 13]. Our study evaluated potential markers of inferior calcaneal nerve compression due to either etiology. PTTD and Achilles tendinopathy were assessed as markers of altered biomechanics which might predispose the individual to inferior calcaneal nerve compression. Calcaneal spur formation and plantar fasciitis were assessed as potential causes of inferior calcaneal nerve compression as the nerve passes anterior to the medial calcaneal tuberosity. The persistence of an association between calcaneal spur formation and plantar fasciitis after logistic regression analysis supports the importance of these factors in the etiology of Baxter\u2019s neuropathy. The presence of 14 patients with isolated ADMA without other abnormalities suggests that Baxter\u2019s neuropathy may also occur in the absence of mechanical entrapment secondary to altered biomechanics.\nFig.\u00a03An anatomic drawing of the sole of the foot after removal of the layers containing the skin, plantar fascia, and part of the flexor digitorum brevis muscle and abductor hallucis muscle cut away. Posterior tibial nerve trifurcation. PTN posterior tibial nerve, ICN inferior calcaneal nerve, LPN lateral plantar nerve, MPN medial plantar nerveFig.\u00a04An anatomic drawing of a coronal section through the right talocrural and talocalcaneal joints (ADM abductor digiti minimi muscle, FDB flexor digitorum brevis muscle, AH abductor hallucis muscle)\nBaxter\u2019s neuropathy can produce chronic heel pain. Clinically, the symptoms of Baxter\u2019s neuropathy may be indistinguishable from those of plantar fasciitis, and the two conditions are often superimposed [5, 7, 14, 15]. Initial treatment for either condition is the same (taping to prevent abnormal motion, Achilles\/plantar fascia stretching exercises, and medications such as steroids or non-steroidal anti-inflammatory agents [7]. Subsequent treatment includes injections and physical therapy, with surgical intervention reserved for patients who fail to respond to conservative measures.\nFor patients in whom heel pain persists despite conservative therapy, MRI evaluation is indicated prior to any surgical intervention. When a plantar fasciotomy is performed for clinically presumed plantar fasciitis in patients with occult Baxter\u2019s neuropathy, symptoms often persist, because at least a portion of the underlying cause of pain has not been addressed [14]. When pre-operative MRI demonstrates abductor digiti minimi atrophy, the surgeon is afforded the opportunity to advise the patient of the underlying abnormality and its prognosis, and, when indicated, to alter the surgical approach to include surgical release of the inferior calcaneal nerve.\nThe radiology literature rarely mentions ADMA as an MRI manifestation of Baxter\u2019s neuropathy. One study identified ten subjects with MRI evidence of ADMA out of 476 subjects who underwent MRI evaluation of the foot or ankle and found that ADMA was associated with obesity and the presence of a heel spur [5]. Another study evaluated patterns of muscle atrophy on MRI of the foot and found that, of 29 patients with focal denervation atrophy involving the muscles of the foot, 19 had ADMA. Of patients with denervation in the lateral plantar nerve distribution, 18 had atrophy involving the ADM and one had atrophy of the adductor hallucis muscle [16]. The largest study to date was a prospective evaluation of the prevalence of ADMA on MRI, which found ADMA in 6.3% of 602 consecutive patients who underwent MRI of the foot and ankle [17]. The prevalence of ADMA in that study is corroborated by the findings in our study, in which it was necessary for us to review 1,780 MRI studies in order to identify 100 patients with ADMA, corresponding to a prevalence of 5.6%. The study also found that the most common associated findings in patients with ADMA were plantar enthesophytes and plantar fasciitis, but it did not include a statistical analysis of the prevalence of associated findings in patients with and without ADMA. Our study includes the largest number of patients with ADMA on MRI and is the first study to perform logistic regression analysis to determine independent variables associated with ADMA.\nLimitations of the study include retrospective data collection, which may lead to selection bias; however, we attempted to minimize selection bias by including 100 consecutive patients in each study group. In addition, the inclusion of 15 pediatric patients might have affected the results, as younger patients are less likely to have degenerative conditions of the foot or ankle and could potentially have falsely decreased the frequency of findings in the group of patients without abductor digiti minimi atrophy. However, a repeat statistical analysis, from which the pediatric patients had been excluded, demonstrated no significant difference in results.\nIn summary, our study showed a significant association between atrophy of the abductor digit minimi muscle, an MRI manifestation of Baxter\u2019s neuropathy, with age, plantar calcaneal spur formation, and plantar fasciitis. These findings support the notion of an etiologic role for compression of the inferior calcaneal nerve as it passes anterior to the medial calcaneal tuberosity in the development of Baxter\u2019s neuropathy.","keyphrases":["plantar fasciitis","mri","foot","muscle atrophy","muscle denervation"],"prmu":["P","P","P","P","R"]}
{"id":"Eur_Radiol-2-2-1705470","title":"New coil concept for endoluminal MR imaging\n","text":"Our aim was to conduct a prospective study to evaluate staging accuracy of a new coil concept for endoluminal magnetic resonance imaging (MRI) on ex vivo gastric carcinomas. Twenty-eight consecutive patients referred to surgery with a clinically proven primary gastric malignancy were included. Surgical specimens were examined with a foldable and self-expanding loop coil (8-cm diameter) at 1.5 Tesla immediately after total gastrectomy. T1- and T2-weighted and opposed-phase sequences (axial, frontal sections; 3- to 4-mm slice thickness) were acquired. Investigators blinded to any patient information analyzed signal intensity of normal gastric wall, gastric tumor, and lymph nodes. Findings were compared with histopathological staging. On surgical specimens, 2\u20135 gastric wall layers could be visualized. All gastric tumors (26 carcinomas, two lymphomas) were identified on endoluminal MR data (100%). Overall accuracy for T staging was 75% (18\/24); sensitivity to detect serosal involvement was 80% and specificity 89%. N staging correlated in 58% (14\/24) with histopathology (N+ versus N\u2212). The endoluminal coil concept is feasible and applicable for an ex vivo setting. Endoluminal MR data provided sufficient detail for gastric wall layer differentiation, and therefore, identification of T stages in gastric carcinoma is possible. Further investigations in in vivo settings should explore the potential of our coil concept for endoluminal MR imaging.\nIntroduction\nDespite the fact that the incidence of gastric cancer is declining in most Western countries, it remains the second leading cause of cancer mortality worldwide [1, 2]. Simultaneously, the incidence and prevalence of cancer arising from the gastric cardia has been increasing since the 1970s [3]. Adenocarcinoma of the esophagogastric junction can be classified according to Siewert et al. based on morphology and anatomical location [4]. Adequate surgical resection (R0) is the only potentially curative therapy for eligible patients with gastric carcinoma [4]. In the West, gastric cancer is often diagnosed at an advanced stage of disease not eligible for surgery. Fewer than 50% of patients undergo R0 resection [5, 6]. Recently, new concepts of multimodal treatment strategies for locally advanced gastric carcinoma have been investigated [7, 8]. Local treatment by endoscopic mucosal resection for early gastric carcinomas is currently being evaluated [9, 10]. Several phase II and III clinical trials for neoadjuvant chemotherapy in gastric carcinoma showed its feasibility and safety [11, 12]. Its purpose is to eliminate or delay systemic metastasis and reduce micrometastatic spread of disease. Another benefit is potential reduction of tumor volume in initially unresectable advanced tumor stages (downstaging), therefore increasing resectability rate. An adequate treatment strategy, especially in regard to the concept of neoadjuvant chemotherapy, requires precise clinical staging to depict relevant prognostic factors and identify resectable tumor stages. This also accounts for endoscopically treated early gastric cancers as well as for adenocarcinomas of the gastroesophageal (GE) junction since each type of tumor requires a different surgical approach. Thus, the exact knowledge of tumor morphology and tumor invasion into gastric wall is a crucial information for clinical staging.\nUp to now, computed tomography (CT) and magnetic resonance imaging (MRI) have not provided the spatial resolution to fulfill this requirement with sufficient accuracy [1, 13\u201316]. Results for staging accuracy of endoscopic ultrasound (EUS) also seem to be improvable [17\u201319]. The main aspect of gastric wall differentiation is spatial resolution. By using endoluminal radiofrequency (RF) coils for MRI, image quality and spatial resolution can be enhanced. MRI provides superior soft tissue contrast, which makes it useful for tumor invasion detection; furthermore, information not obtainable by other imaging modalities is acquired. Various authors reported the use of endoluminal RF coils as a diagnostic and staging tool in gastrointestinal diseases and rectal and prostate cancer [20\u201327]. A major problem with endoluminal RF coils is placement close to the region of interest and depth of visualization. Our approach uses a foldable and afterward self-expanding loop coil design (8-cm diameter), which enhances spatial resolution and depth of visualization The aim of our study was to assess normal gastric wall architecture and signal intensity (SI) on endoluminal MRI as well as signal intensity and appearance of gastric carcinomas and their related lymph nodes. Subsequently, we used endoluminal MRI as staging modality on ex vivo gastric carcinomas. Findings were compared with histopathological staging.\nMaterials and methods\nPatients\nA prospective study was conducted on patients with gastric malignancies referred for total gastrectomy to the surgery department of our university hospital. They were included in the study if the lesion was identified by EUS or CT suggesting a gastric carcinoma or if histological workup of biopsies confirmed the diagnosis. The study protocol was approved by the institutional review board, and informed consent was acquired from each patient prior to surgery. We investigated 28 consecutive patients, 11 women and 17 men (age range 46\u201387, median 67 years). Tumors were located at the cardiac region in 11 cases, the fundal area in two, the corpus in six, and the antropyloric area in nine. Immediately after surgery, gastrectomy specimens were taken to the MR suite to conduct the examination within a time frame of 2\u20133\u00a0h (Fig.\u00a01). Specimens where then sent to histopathology unchanged from the procedure.\nFig.\u00a01Flowchart of study design\nEndoluminal RF coil\nThe endoluminal RF coil (patent issued 10\/2005, German Patent and Trade Mark Office, No: 10127850.0\u201335, Grenacher et al.), developed in collaboration with Fraunhofer Institute for Biomedical Engineering (IBMT, St. Ingbert, Germany) consists of a foldable and self-expanding receiver loop (8-cm diameter). It is coated with a biocompatible material (silicone) to prevent direct contact of the wire with stomach tissue [29]. The flexible characteristics of the shape memory metal (nitinol) used allow passage through the instrument channel (13-mm diameter) of a specially designed MR-compatible endoscope. The MR-compatible endoscope was not used in this study and is described elsewhere, but it is mentioned in this context because RF coil design is reflected by the specifications of the endoscope. A nonferromagnetic tuning box connects the RF coil via a standard interface with a 1.5-Tesla MRI scanner (Symphony, Siemens, Erlangen, Germany), which was used in this study. Gastrectomy specimens were placed in a container with 3\u20134\u00a0l of 0.9% sodium chloride solution to ensure good contrast with gastric tissue and adequate expansion of gastric folds. The loop of the RF coil was placed on the mucosa side of the tumor. After the tumor was located with a scout sequence, images where acquired in axial and frontal sections of 3- to 4-mm thickness. Scanning was performed with T1- and T2-weighted and opposed-phase sequences (Table\u00a01). \nTable\u00a01Imaging sequence parametersSequenceRepetition time (TR)Echo time (TE)AcquisitionsSlice thickness (mm)MatrixField of view (FOV) (mm)Pixel size (mm)T1-weighted TSE5511424512x5122000.39x0.39T2-weighted TSE3,52070231024x10242200.21x0.21T2-weighted GRE1,0503513512x5122200.43x0.43T1-weighted GRE opposed phase187743256x2652200.86x0.86TSE turbo spin echo, GRE gradient recalled echo\nImage evaluation\nImage data were read on certified diagnostic work stations independently by two board-certified radiologists with 10 and 12\u00a0years of experience, respectively, and one board-certified surgeon with 8\u00a0years of experience in endoscopic ultrasound on gastric carcinomas. Investigators were blinded to any clinical information as well as patient identification. After separate reading, a discussion was held about the quality of data and unique features of the case. Additionally, final agreement was achieved if disagreement on tumor stage had initially occurred. With respect to the unique imaging technique, the resolution and detail of image data a consensus staging was reasonable. Radiological and histopathological classification of the tumor was done according to the Tumor Node Metastisis (TNM) staging system of the Union Internationale Contre le Cancer (UICC) [30]. Determination of tumor stage was done as follows: \nT1: tumor invasion of the lamina propria of mucosa or invasion of submucosa, wall thickening, and signal intensity changes confined to these layers.T2: tumor invasion extended to the muscularis propria, and additional thickening through gastric layers with signal intensity changes showing either a homogeneous or inhomogeneous lesion without serosal abnormalities. If invasion extended beyond muscularis propria into an adjacent fat tissue plane without serosal infiltration, it was considered as T2.T3: tumor invasion of mucosa, submucosa, and muscularis propria, with infiltration of the serosa or changes in signal intensity presenting with micronodular strands as growth into extraserosal fat tissue.T4: tumor invasion into adjacent organs or structures clearly presenting as contiguous tumor extension or a mass with similar signal intensity as the gastric tumor.\nN staging was done counting the lymph nodes detectable on MRI, regarding them as pathologic due to their signal intensity. Since gastrectomy samples varied in lymph node content due to surgical technique and extension of the gastrectomy not comparable to an in vivo situation, N factor was graded into N+ for positive findings of lymph nodes and into N\u2212 for absence of lymph nodes. In each case, normal gastric wall features were assessed. Signal intensities of the mucosa, submucosa, muscularis propria, and serosa or subserosa were recorded, as well as the amount of layers in which the normal gastric wall could be differentiated into. A score system from 1 to 5 (1=poor; 5=very good) was used for several study items to weigh the power of their findings. Each sequence, for example, was rated for image quality on a range from 1 to 5, representing insufficient to very good. Signal intensity and morphology of the gastric tumors and demarcation to normal gastric wall were used to describe characteristics of tumor presentation. The lesion was identified by irregular architecture of gastric wall, abnormal thickening, and change in signal intensity of gastric wall layers, which usually present with sharp and clear demarcation into three to five different layers with distinct signal intensities and thickness. Tumor size was not measured, as it does not influence T staging or provide further information. Histopathology was considered the gold standard, and radiological data where compared with its results.\nAll data are presented as absolute numbers and relatively as percentages. Concordance of histopathological results and radiological findings are reported as overall accuracy. Sensitivity, specificity, and accuracy for T factor, N+\/\u2212 factor are provided. Detection rate of serous membrane invasion or the differentiation between T2 and T3 stages was assessed, as it defines advanced stages of disease. Ninety-five percent confidence intervals (CI) are given if statistically applicable.\nResults\nThe setup of our study proved to be feasible, as shown elsewhere [29]. Two cases were not included because of insufficient image quality. Histopathology found 26 carcinomas (15 adenocarcinomas, nine signet cell carcinomas, two mixed type carcinomas) and two gastric lymphomas. Four tumors were classified as pT1, 15 as pT2, 3 as pT3, and 2 as pT4. Gastric lymphomas were excluded from the study although they where detected as such in both cases (100%).\nGastric tumor staging\nIn 24 (100%) cases, the tumor was identified. Initially 46% (11\/24) of T stages were staged correctly; 1\/4 of pT1 tumors, 7\/15 of pT2 tumors, 2\/3 of pT3 tumors, and 1\/2 of pT4 tumors were classified correctly (Table\u00a02). Interpretation of image data was done without knowledge of tumor location in respect to the gastric region, such as cardia, fundus, corpus, or antrum, as it would be possible on an in vivo setting such as endoscopy or CT. In a second reading session, the interpreters were informed about the gastric region of the primary tumor for each case. In knowledge of gastric region, six, first as T3 classified carcinomas of the cardia, were correctly staged as pT2 and one, first as T3 classified tumor (cardia), was correctly identified as pT4 (Table\u00a03). This gives an overall accuracy of 75% (18\/24)\u00b1 \u201317.32 (CI) (Table\u00a04). The mean score for T-factor staging was 3.54 (range 2\u20135). Sensitivity, specificity, and accuracy for detecting serosal involvement was 80%\u00b116.00, 89%\u00b112.27, and 88%\u00b113.23, with a mean score of 4.5 (range 3\u20135; Table\u00a05). Overstaging occurred in 4\/24, three pT1 tumors were classified as T2 or T3 stages, and one pT2 tumor was identified as T3 stage (Table\u00a06). In this case, the tumor replaced all gastric wall layers and invaded perigastric fat tissue but did not infiltrate the serous membrane. Insufficient separation of gastric wall layers and the presence of abnormal signal intensities most likely desmoplastic reactions or peritumor inflammation were identified retrospectively as reasons for overstaging of pT1 tumors. Two tumors (pT2, pT3) were understaged as T1 and T2 stage. One case was a diffuse type pT2 adenocarcinoma with disseminated infiltration into the muscularis propria, which is not visualized and thus presented as T1 stage. \nTable\u00a02Comparison of histologic with endoluminal findings without knowledge of tumor region\u00a0Endoluminal magnetic resonance imaging (MRI) findingsHistologic findingsT1T2T3T4Total (n=24)T1121\u20134T2177 \u201315T3\u201312 \u20133T4\u2013\u201311 2Table\u00a03Comparison of histologic findings with endoluminal findings acquired with knowledge of tumor region\u00a0Endoluminal magnetic resonance imaging (MRI) findingsHistologic findingsT1T2T3T4Total (n=24)T1121 \u20144T21131\u201415T3\u201312 \u20133T4\u2013\u2013\u201322Table\u00a04Sensitivity, specificity, and accuracy for each T stage; overstaging and understaging rates\u00a0T1T2T3T4OverallSensitivity1\/413\/15 2\/3 2\/2 \u2013Specificity19\/20 6\/9 19\/21 22\/22 \u2013Accuracy20\/24 19\/24 21\/ 24 24\/24 18\/24 Overstaging3\/4 1\/15 \u2013\u20134\/24 Understaging\u20131\/15 1\/3 \u20132\/24 Table\u00a05Results for serosal invasion detectionEvaluation of serosal invasionSensitivity4\/5Specificity17\/19Accuracy21\/24False positive2Table\u00a06Signal intensities of gastric wall layers on T1-weighted, T2-weighted, and opposed-phase imaging from inside to outside\u00a0T1 imaging (n=21)aT2 imaging (n=27)aOpposed phase imaging (n=3)aTwo layers (n=7)bHigh\u2013\u2013IntermediateThree layers (n=33)bHighIntermediateHighLowHighLowIntermediateIntermediateHighFour layers (n=10)bHighIntermediate\u2013LowLowIntermediateIntermediateLowLowfive layers (n=1)*\u2013Intermediate\u2013HighIntermediateLowIntermediateaNumber of sequences analyzedbNumber of sequences with n layers\nNodal involvement\nLymph node detection resulted in N+ for detected lymph nodes in 17 cases with N\u2212 in 7 cases. Sensitivity for detecting N+ was 71%\u00b118.22, specificity 29%\u00b118.07, and accuracy 58%\u00b119.72. This reflects the limitation of the study design. Ex vivo gastrectomy specimens do not provide the same quantity of lymph nodes as pathological workup. The mean score rating N factor staging was 4.4 (range 2\u20135).\nImage analysis\nSeparation of gastric wall layers ranged from a minimum of two visible layers to five distinct visible layers (mean 3.10) in 51 analyzed sequences: 21 T1-weighted and 27 T2-weighted sequences and three opposed-phase sequences (Table\u00a06). T2-weighted sequences depicted an average of 3.37 gastric wall layers; T1-weighted sequences 2.76. Mean score for image quality of the sequences used was 3.88 (range 2\u20135), with 4.11 for T2- and 3.62 for T1-weighted sequences. Image analysis revealed that in four out of six incorrectly staged T factors, tumors were described as inhomogeneous or diffuse concerning demarcation to normal gastric wall. This may be one reason for misinterpretation of T factor. Signal intensity of the mucosa for T1-weighted sequences was mostly classified as hyperintense; for T2-weighted, it was intermediate. Submucosa, muscularis propria, and serosa or subserosa presented with hypointense, intermediate, hypointense SI for T1-weighted; hyperintense, intermediate, hypointense for T2-weighted sequences (Table\u00a06). Lymph node and tumor appearance correlated in 86%\u00b19.9 (44\/51 sequences), with intermediate SI for T1- and T2-weighted sequences. Out of eight sequences without correlation in lymph node and tumor SI, two sequences showed a homogeneous tumor in T1-weighted and an inhomogeneous lesion in T2-weighted images (Fig.\u00a03). In three opposed-phase sequences, lymph nodes (hypointense) showed different signal intensity than the primary tumor (intermediate). T2-weighted sequences (Table\u00a04) visualized more gastric wall layers and provided the interpreter with a better demarcation of tumor volume from normal gastric wall than did T1. T1-weighted sequences showed a better delineation and contrast of lymph nodes in adjacent structures (Fig.\u00a02). \nFig.\u00a02a, bTwo consecutive T2-weighted images (a, b) of a T1 early gastric carcinoma (white arrowheads), well differentiated (intestinal type of Lauren classification), located at the subcardial region. Lymph nodes in adjacent fat tissue with a high signal intensity (white arrows) are visualized. Morphology of normal gastric wall is pointed out by open arrows. (*) marks the position of the receiver coilFig.\u00a03a, bT2 signet cell tumor of the cardia region (diffuse type of Lauren classification, white arrowhead) on T1-weighted (a) and T2-weighted imaging (b). The extent of tumor mass and the diffuse infiltration into gastric wall is better visualized on T2-weighted images due to the mucinous character of the tumor (black arrowheads). The tumor appears more homogeneous on T1-weigthed images (a). (*) marks the position of the receiver coil\nDiscussion\nEndoluminal MRI has been used by various groups to asses its ability for locoregional imaging of esophageal and gastrointestinal disease. The first group [20, 31] to introduce an MR endoscope reported promising results for the diagnosis of gastrointestinal diseases and pointed out limitations of their design [20, 31]. Image quality of endoluminal MRI was not as clear as those acquired by EUS, and their success rate of obtaining clear images of the stomach was low (58%) [20]. Although their study achieved 89% for T-staging accuracy, its patient collective consisted mainly of advanced gastric carcinomas (T3, T4). Other authors reported the use of an MR endoscope with a similar design [21, 28, 32], sharing the incorporation of the RF coil with 10\u00d730 and 10\u00d747\u00a0mm into the tip of the MR endoscope. This is a useful design for imaging at the esophagus, where both groups showed good results in staging esophageal cancer [20, 28]. The small cylindrical RF coil design implemented in the tip of the MR endoscope may be a reason for problems in obtaining sufficient images in the stomach. Our approach using an insertable and self-expandable RF coil (8-cm diameter), independent from the MR endoscope, seems to be more suitable for imaging of the stomach by providing enhanced depth of visualization. In 93%, sufficient image data was obtained. Detection of resectable lesions can only be accomplished by exact knowledge of tumor invasion in relation to gastric wall layers. New multimodal treatment strategies require the information of tumor stage, as it defines enrollment into neoadjuvant therapy approach. Clinical staging based on imaging studies can provide important information for adequate therapy decision if they fulfill this requirement. Gastric wall differentiation and identification is essential for accurate staging of gastric malignancies.\nOur approach can depict between two to five gastric wall layers and therefore deliver sufficient detail as basis for tumor invasion detection. Signal intensities of gastric layers assessed in our study correspond mainly to those reported previously by other authors [20, 33, 34]. Visualization of the serosa layer remains a problem in all published studies with MRI or endoluminal MRI on gastric wall. Some authors investigated ex vivo gastrectomy specimen with a 1.0-Tesla MRI device and were able to differentiate up to five different gastric layers, but visualization of the serosa or subserosa was not achieved [35]. One reported study used a 2.4-Tesla MRI system and failed, as did another study with a 4.7-Tesla MRI system, to visualize serosa or subserosa as a distinct layer [34, 36]. One work group reported possible identification of serosa or subserosa on gastrectomy specimens after fixation with formalin [33]. With the use of a 1.5-Tesla MRI device and a 4-cm-diameter loop coil, muscularis propria and serosa or subserosa showed isointense signal intensities but were outlined by subserosal fat tissue with contrasting signal intensity. The results were confirmed by another work group using a similar setup [37]. Identification of the serosa is necessary to securely differentiate between T2- and T3-stage gastric carcinoma. Although a detection rate of 88% for serosal involvement was achieved in this study, serosa as a single gastric wall layer was not clearly visualized. In seven cases with four to five visible gastric layers, a thin outer layer on T2-weighted sequences was detected and could possibly be identified as serosa or subserosa with subserosal fat tissue. This finding was inconsistent and varied in different T-stage carcinomas. This may be because of isointense signal intensity of muscularis mucosae and serosa or subserosa or the fact that the thickness of the serosa layer is not sufficient for secure identification [38]. It is not certain whether tumor invasion into or close to the serosa or subserosa layer changes its signal intensity presentation. Indirect detection by subserosal fat tissue seems possible, but in our experience, there is some variance in the thickness of subserosal fat tissue in gastric carcinoma specimens. One reason for this could be tumor cachexia.\nAn interesting learning point is reflected by the results of T staging. Knowledge of the gastric region where the tumor is located is important since T staging of the stomach depends on the gastric region. Tumors of the cardia and part of the fundus region where a serosal layer is missing are considered T2 whereas they would be T3 in other gastric regions. The study setup was intended to be as close as possible to a future in vivo setting with an MR endoscope, but tumor location and gastric region cannot be read on image data from gastrectomy specimens. An in vivo setting would enable investigators to acquire this information. After providing the tumors' gastric region, T staging accuracy improved to 75%. Thus, the gastric region of tumor location is an important fact for adequate staging of tumor invasion. While this study used an ex vivo model of gastric carcinomas and the amount of patients included could be higher, the entire range of T factors for gastric carcinomas is covered. Another limitation of this study is the missing congruence of lymph nodes present in the gastrectomy specimens due to surgical technique to those available for histopathology. This is reflected by N-factor staging results. An in vivo setting should be more accurate but poses new problems, such as motion artefacts due to patient or endoscope movement. Modern rapid MRI sequences and spasmolytic agents may be able to overcome these obstacles in an in vivo setting, but this is a goal for future studies. It is important to point out pitfalls of this imaging technique, as overstaging is a problem in many imaging modalities. Peritumor inflammation, micronodular affection of the serosal membrane, and diffuse-type gastric carcinoma were retrospectively identified as reasons for misinterpretation of tumor invasion. Overstaging occurred in 17%, with three T1 tumors not correctly classified. This is encouraging since all T1 tumors were detected as tumors but in two cases were classified as T2 tumors and one as T3. Only one incorrectly classified T2 tumor contributed to overstaging. This indicates that overstaging was not a problem of differentiation between T2 and T3 stages of disease.\nIt is necessary to focus on T1 carcinomas, as experience with T1 carcinomas in imaging modalities in Western countries is low due to the fact that most gastric cancers are diagnosed at an advanced stage. It is important to learn more about signal intensity and morphologic presentation of early gastric carcinomas on endoluminal MRI to improve detection rate. Endoluminal MR imaging provides enough detail to visualize a T1 tumor (Fig.\u00a02). Endoscopic mucosal resection as one treatment option for early gastric carcinoma requires an imaging method that is able to visualize and identify these stages securely and provide information that is observer independent. Further studies should include a larger quantity of early stages of gastric carcinomas to analyze accuracy potential of endoluminal MRI. MR images provide information not obtainable by other imaging modalities. A gastric tumor can present with different appearances on T1-weighted imaging than on a T2-weighted imaging (Fig.\u00a03). Depth of visualization of a method is essential for staging a gastric tumor, assessing its depth of invasion into gastric wall and into adjacent organs and structures, as well as depicting involved lymph nodes. Our image data can provide detailed information about gastric wall invasion as well as organ invasion (Figs.\u00a02 and 4).\nFig.\u00a04a, bT4 gastric tumor (intestinal type of Lauren classification, open arrows) on T2-weighted images of the same plane. Transition of normal gastric wall into tumor mass (white arrows). Pancreas (white arrowheads) adjacent to the gastric wall demonstrates depth of visualization (a). Tumor invasion into pancreas (black arrowheads) (b)\nIn this study, we have shown that our concept is functional and feasible for MRI of the stomach, and staging results comparable with other imaging modalities were achieved. Endoluminal MRI in combination with conventional MRI could assess local and regional imaging in one session, giving accurate information about local staging and metastatic spread of disease. Secondary contrast media should be evaluated for its use in improving gastric wall layer discrimination and identification as well as tumor demarcation. Additional studies are needed to assess the clinical feasibility of this coil concept in vivo for imaging of the upper gastrointestinal tract. High-resolution imaging of the pancreas could be another possible application. Today, most tumors of the stomach and esophagus are staged by conventional methods, such as endoscopy, EUS, or CT. Future prospects of endoscopic MRI may be limited to certain tumor entities, such as early or advanced carcinomas, but it could be able to answer additional specific clinical questions for relevant therapy decisions.\nSummary Endoluminal MRI with the coil concept is feasible and applicable. T staging of ex vivo gastric carcinomas is possible. Results for T-factor staging are preliminary but promising, as they reflect the first experience with this technique. Image quality and resolution obtained with the endoluminal RF coil are convincing for future use in gastrointestinal imaging. Limitations of this study include missing congruence in quantity of lymph nodes for N staging, a limited amount of some T-stage gastric carcinomas, and the ex vivo setting. Taking the limitations into account, the coil design should be investigated in an in vivo setting after assessing safety aspects to evaluate the prospects given by the ex vivo results. The clinical potential may be limited by providing a staging tool for certain tumor entities. With more experience in endoluminal MRI and interpretation of endoluminal image data, its use could be extended toward other fields in gastrointestinal imaging.","keyphrases":["coils","magnetic resonance imaging","stomach neoplasms","neoplasm staging","experimental study"],"prmu":["P","P","M","M","M"]}
{"id":"Clin_Oral_Investig-4-1-2238791","title":"How valid are current diagnostic criteria for dental erosion?\n","text":"In principle, there is agreement about the clinical diagnostic criteria for dental erosion, basically defined as cupping and grooving of the occlusal\/incisal surfaces, shallow defects on smooth surfaces located coronal from the enamel\u2013cementum junction with an intact cervical enamel rim and restorations rising above the adjacent tooth surface. This lesion characteristic was established from clinical experience and from observations in a small group of subjects with known exposure to acids rather than from systematic research. Their prevalence is higher in risk groups for dental erosion compared to subjects not particularly exposed to acids, but analytical epidemiological studies on random or cluster samples often fail to find a relation between occurrence or severity of lesions and any aetiological factor. Besides other aspects, this finding might be due to lack of validity with respect to diagnostic criteria. In particular, cupping and grooving might be an effect of abrasion as well as of erosion and their value for the specific diagnosis of erosion must be doubted. Knowledge about the validity of current diagnostic criteria of different forms of tooth wear is incomplete, therefore further research is needed.\nThe process of diagnosis and current criteria for dental erosion\nFrom the chemical view, the aetiology of dental erosion can be defined as the chronic exposure of the teeth to extrinsic or intrinsic acids under the condition that the oral fluids are undersaturated with respect to tooth mineral [23, 26]. Under in vitro conditions without physical impact, teeth demineralise centripetally (Fig. 1), a feature of substance loss which is normally not observed in the mouth. In fact, the multitudes of physical and chemical assaults occurring during a lifetime result in a more or less characteristic pattern of tooth wear. The classification of wear is therefore made from morphological features which are frequently seen clinically. The tooth morphology as apparent after eruption is the idealised status, deviations of which, if not caries or trauma, are diagnosed as (erosive) tooth wear. Various forms of wear including dental erosion are listed in the International Classification of Diseases [52] thus defining them as a disease (for critical discussion of this notion see [12]).\nFig.\u00a01Effect of the continuous exposure of a human third molar to 10% citric acid. The amorphous, centripetal tissue loss is obvious (a unaffected tooth, b tissue loss after 4, c 8, and d 12\u00a0h immersion time)\nLooking at what \u201cdiagnosis\u201d is, one will find mostly definitions like \u201cidentification of disease from signs or symptoms\u201d, implicating that the physician or scientist \u201creads off\u201d from the patient thus detecting the disease. Health and disease, however, is not a given condition, but is constituted by the theoretical concepts and the discursive practice the physician or scientist is subjected to [12]. That is to say, rather than reading off the disease from the patient, a pattern of criteria is projected onto the diagnosed subject which determines the diagnostic procedure and outcome. Following this approach, the diagnostic process, in a first step, is a theoretical concept ordering signs and symptoms to diseases which takes place within the configurative power of the discourses. In whatever discourse the physician acts, the act of clinical diagnosis, in a second step, is classification. The conclusion reached through this process is called \u201ca diagnosis\u201d. From this background, it becomes obvious that the diagnostic process must be accompanied by a recurrent reconsideration of its theoretical concepts and the reevaluation of the criteria used. The latter is what the paper aims to achieve.\nThe diagnosis of tooth wear in general and erosion in particular is made from its lesion characteristics, from the results of nutritional, medical and occupational analysis, and from dietary records. The diagnostic process can be more differentiated with the individual patient, whereas in field trials it is restricted to the classification of lesion shape.\nThroughout the literature, there is more or less consensus about the aetiology of the various forms of tooth wear, the clinical criteria for dental erosion and its differential diagnosis [12, 14].\nThe early signs of erosive tooth wear appear as changes of the optical properties of enamel resulting in a smooth silky\u2013shining glazed surface. When the tissue loss continues, changes in the original morphology occur. On smooth surfaces, convex areas flatten or concavities develop, the width of which clearly exceeds the depth. Lesions are located coronal from the enamel\u2013cementum junction (CEJ) with an intact enamel rim along the gingival margin. On occlusal and incisal surfaces, rounding and cupping of the cusps and grooving of the incisal edges occur, and restorations may rise above the level of the adjacent tooth surfaces. In advanced cases, the whole occlusal morphology disappears.\nThe validity of these clinical criteria for erosion, however, has not been under critical consideration. Validity, in general terms, means the degree, to which a measurement measures what it purports to measure. For the issue addressed here, criterion validity is the relevant term, meaning the extent to which the measurement correlates with an external criterion under study. A further aspect of validity is concurrent validity. Concurrent validity means to which extent the measurement and the criterion refer to the same time [24]. In the case of erosion this would address the question if (a) the diagnostic criteria reflect lesions being an effect of an exposure to acids and (b) if the presence of characteristic signs is concurrent with an acid exposure.\nThe implementation of current diagnostic criteria\nConsidering how the diagnostic criteria for erosion were established, the literature reveals that it was individual clinical experience and case reports rather than systematic research. One of the first publications on the characteristics of acid induced tissue loss was in 1946 from Robinson [43] and in 1947 from Stafne and Lovestedt [47]. Robinson, whilst being in doubt about the aetiology of dental erosion, described the lesions as located on smooth surfaces and as wedge shaped, cup shaped, disk shaped, irregular in form, L- or U-shaped, or simply as small round depressions or larger surface lesions. It is not clear how these descriptions were established, but Robinson referred to a work from McClure and Ruzicka [31] describing the morphology of lesions of rats teeth after being fed with lactate and citrate drinking fluid.\nStafne and Lovestedt presented their observations in subjects with known acid exposure, amongst them 50 patients with frequent consumption of lemon juice. They did not give a concrete description of lesion shape, but attributed hypersensitivity, absence of stain and defects with rounded margins as effect of the action of the acids. Their most important sign of diagnostic value was the presence of fillings projecting above the surface of the tooth. In their publication, a number of clinical images were included, presenting lesions clearly matching current erosion criteria.\nMore than twenty years later, in 1970, it was Pindborg [40] who gave the often cited definition of erosion as being superficial loss of dental hard tissue by a chemical process which does not involve bacteria. He described the clinical signs of chemically-induced tissue loss as usually located to the gingival third of the facial surfaces, possibly also located at proximal surfaces, lesions to appear shallow, disc-shaped, smooth, polished, or scooped out. In contrast to abrasion, he attributed erosive lesions to be located evenly on the left and right side.\nIt appears noteworthy, that Pindborg, as well as Robinson, ascribed cupping of the cusps, loss of the occlusal morphology or loss of crown height, and incisal grooving to attrition which in their publications was defined as result of mastication.\nIt is probably Eccles and Jenkins [8, 10] who were the first to give a detailed and systematic description of the lesion characteristics and also suggested a system for classification. The basis of the development of the clinical criteria was a sample of 72 patients seen in the dental hospital over a period of 9\u00a0years [8]. All cases were thoroughly documented with respect to their medical and dietary history, that is, it was a group of subjects with known exposure to intrinsic or dietary acids. The findings derived from this group of subjects (Table\u00a01) have been retained nearly unchanged until now. The main aspects are in general terms loss of surface contour, shallow concavities on smooth surfaces, cupping and grooving on occlusal\/incisal surfaces and restorations rising above the level of the adjacent tooth surface.\nTable\u00a01Diagnostic criteria for dental erosion as outlined by Eccles and Jenkins [10] and Eccles [8]Diagnostic criteria for dental erosionInitialAbsence of developmental ridges of the enamel, smooth glazed surfaceAdvanced\u00a0Facial\/oral surfacesConcavities whose breadth greatly exceeds their depthLesion ovoid or crescentic in outline, concave in cross section orLesion entirely in the crown, irregular in outline, punched out appearance\u00a0Occlusal\/incisal surfacesSurfaces appear flattened, depression of the cusps (cupping) and on the incisal edges (grooving), edges of restorations raising above the level of the adjacent tooth surface\nConclusions from epidemiological studies using current diagnostic criteria\nCurrent criteria, derived from a relatively small sample, have been applied in case reports [8, 9, 16, 32, 39, 50] and studies with risk groups [15, 17, 21, 25, 34, 38, 44, 48, 51]. Indeed, when used in subjects with known or strongly assumed exposure to acids, most studies revealed a higher prevalence of lesions in the exposed groups compared to the control groups (Table\u00a02).\nTable\u00a02Prevalence of lesions in risk groups deriving from the use of current diagnostic criteria for dental erosion\u00a0IndexGroup sizePrevalence risk groupPrevalence control groupIntrinsic acid exposureMeurman et al. [34] reflux diseaseEccles and Jenkins indexn\u2009=\u200911728\/117\u2009=\u200924%No control groupRyt\u00f6maa et al. [44] eating disordersEccles and Jenkins indexn\u2009=\u200914022\/35\u2009=\u200963%12\/105\u2009=\u200911%\u00d6hrn et al. [38] eating disordersLussi indexn\u2009=\u200913379\/81\u2009=\u200998%Minor, less severeIncisal\/occlusalIncisal\/occlusalGrade 1: 93%Grade 1: 73%Grade 2: 52%Grade 2: 23%BuccalBuccalGrade 1: 30%Grade 1: 19%Grade 2: 9%Grade 2: 6%PalatalPalatalGrade 1: 21%Grade 1: 10%Grade 2: 5%Grade 2: 0%Extrinsic acid exposureLinkosalo and Markkanen [25] vegetariansEccles and Jenkins indexn\u2009=\u20095216\/26\u2009=\u200960%0\/26\u2009=\u20090%Wiktorsson et al. [51] wine tastersEccles and Jenkins indexn\u2009=\u20091914\/19\u2009=\u200974%No control groupGanss et al. [15] raw food dietLussi indexn\u2009=\u2009206127\/130\u2009=\u200998%66\/76\u2009=\u200987%\nThese observations support the finding that subjects with continuous exposure to acids have a higher rate of lesions with a specific characterisation. This is, however, not enough support for the assumption that, vice versa, subjects presenting with such defects must be exposed to acids.\nAnalytical epidemiological studies on random or cluster samples attempted to relate the occurrence of lesions with any of the known aetiological factors for erosion, but only few studies of this type have been published. Most of them include children or adolescents, but there is lack of studies on older population groups. Furthermore, off the few studies, some used the Tooth Wear Index (TWI [46]) which is not designed to assess dental erosion specifically. The overall findings (Table\u00a03) are controversial since some authors found no or only a partial relation between aetiological factors and the occurrence or severity of lesions [2, 3, 5, 7, 20, 30, 35, 37, 49], whereas others revealed strong relationships [22]. In addition, a relation to the intake of yoghurt or other foodstuff which certainly has no erosive potential was mentioned [1, 27, 35].\nTable\u00a03Analytical epidemiological studies attempting to relate the occurrence of (erosive) wear to aetiological factors\u00a0Index, group size, age and prevalenceConclusionJ\u00e4rvinen et al. [22]Eccles & Jenkins indexCitrus fruits: odds ratio (OR) 2Case-control, n\u2009=\u2009100 eachSoft drinks: OR 413\u201383-year-oldsLussi et al. [27]Lussi indexSignificant relation to the consumption of fruit, acidic drinks, yoghurt, vomitingn\u2009=\u200941726\u201330- and 46\u201350-year-olds at least 36 and 43% resp. with any erosionBartlett et al. [5]TWI (Smith and Knight)No significant relation to drinks or other acidic foodn\u2009=\u2009210Significant relation to heart burn11\u201314-year-olds57% had wear in enamel on more than 10 teethJaeggi et al. [20]Lussi indexNo relation to any aetiological factorn\u2009=\u200941719\u201325-year-olds at least 82% with erosionAl-Dlaigan et al. [1]TWI (Smith and Knight)Significant relation to drinks and fruit, but also to milk, yoghurt and beern\u2009=\u200941814-year-olds48% low, 51% moderate 1% severe lesionsAl-Majed at al. [2]TWI (Smith and Knight) modified for erosionNo association to erosive drinks for the total samplen\u2009=\u2009862Significant association to frequency of drinks at night and duration of drinks retained in the mouth only in advanced cases (n\u2009=\u200995)12\u201314-year-olds95% with erosionMathew et al. [30]Lussi indexNo relation to the intake of sport drinksn\u2009=\u200930418\u201328-year-olds37% with erosionVan Rijkom et al. [49]Modified Lussi indexNo relation to acidic drinks and fruitsn\u2009=\u200940015\u201316-year-olds30% with visible smooth wearArnadottir et al. [3]Modified Lussi indexNo significant association to risk factorsn\u2009=\u200927815-year-olds72% grade 124% grade 25% grade 3Nunn et al. [37]TWI (Smith and Knight) modified for erosionNo significant association with dietary factorsn\u2009=\u20091726Significant relationship with gastro-oesophageal symptoms4\u201318-year-olds36, 56 and 34% with any erosion on buccal and palatal surfaces of the incisors, and first permanent molars resp.Dugmore and Rock [7]TWI (Smith and Knight) modified for erosionDrinking fizzy pop: odds ratio 1.59\u20132.52 depending on amount and frequencyn\u2009=\u2009114912-year-oldsNo relation to eating apples, citrus fruit56% with erosionMilosevic et al. [35]TWI (Smith and Knight) on labial and lingual surfaces in front teeth, occlusal surfaces of first molarsNo association to apples, fresh orangesn\u2009=\u20092385Weak association (OR 1\u20131.4) to yoghurt, grapefruit, salad dressing, vinegar, fruit juice, fizzy drinks14-year-oldsStrong association to herbal\/lemon tea (OR 3.97)53.5% with exposed dentine\nThere are many points of discussion for explanation. At first, (erosive) wear is the effect of various concurrent or past chemical exposures from different sources, the variety of which can hardly be covered by simple questionnaires, and especially in younger people the dietary intake and lifestyle might be open to variations. Furthermore, questionnaires may reflect only in part the dietary habits due to interpretation by the responder or seasonal changes in daily life habits. When the young age of the groups studied is considered, the determinants for dental erosion may not have acted for long enough, and, in addition, there might be considerable differences in the individual susceptibility to erosive demineralisation [15].\nIt must, however, also be taken into consideration that current diagnostic criteria might not be valid enough to really reflect the effect of a chronic acid exposure.\nTaking into account that tooth wear is the result of various factors, the occlusal and incisal surfaces of teeth are not only exposed to acids but are particularly prone to physical impacts from mastication. From this background, the finding that many studies using erosion indices reveal that occlusal lesions are the most prevalent and that it is the first lower molar which is most affected [19], is of particular importance. Occlusal surfaces might be the only location exhibiting a relation between lesion severity and prevalence, and age [28] thus indicating a significant contribution of abrasion from mastication. Furthermore, particularly the teeth from ancient remains exhibit defects being strikingly of the same shape as those attributed to erosion today (Fig. 2).\nFig.\u00a02a Occlusal aspect of a subject living on a raw food diet with multiple acid impacts, and a medieval subject b with an assumed abrasive diet (images a, b, and c samples from [13, 15]). Occlusal\/incisal defects in a subject with chronic vomiting d and in a medieval subject c. The shape of lesions from predominantly erosive and predominantly abrasive aetiology is strikingly similar\nComparative studies on lesion characteristics of wear\nAttempts have been made to characterise the shape of wear resulting from abrasion and erosion [6, 13].\nA comparison of subjects with known exposure to a non-acidic but coarse diet and subjects with an acidic, but refined diet using silicon impressions revealed that in the former, the scooped out dentine was significantly shallower than in the latter. Furthermore, the deepest lesion region in teeth from subjects with abrasive diet was located at the functional cusps, whereas in teeth from subjects with an erosive diet, the deepest region of the defects was located more centrally. The authors concluded that in cases when the dentine is severely scooped out, the causative agent is most likely of erosive origin whereas scooping tends to be shallower when wear is caused by abrasion. The conclusion of the study was to suggest a quantitative diagnostic procedure by calculating the depth\/breadth ratio for clinical differential diagnosis [6].\nAnother approach was to compare the shape of defects occurring in subjects with substantially different nutrition patterns [13]. The study included three groups of age matched individuals \u2014 randomly selected contemporary subjects, medieval remains and a group of subjects living on raw food diet. From the latter group, extensive information was available on their intake of acid food qualifying their nutrition as rather erosive. For the medieval group, an abrasive nutrition was assumed from knowledge about the general nature of the early medieval diet.\nAs to the smooth surfaces, the most interesting finding was that in the medieval group, no lesions were observed (Table\u00a04). Even in cases with severe wear, the lingual and buccal aspects of the teeth appeared undisturbed and, in many cases, even developmental ridges were present. The lack of cervical defects is also reported by Aubry et al. [4] and Kaidonis (in this issue) and also inherently supported by the fact that the overwhelming number of anthropological studies on tooth wear only deal with occlusal and proximal wear and that the indices used there do not include criteria for cervical wear [11, 36]. There are, however, few studies reporting wear on lingual surfaces in archaeological remains [18, 41] and there is one publication discussing regurgitation as a possible aetiological factor [42]. In this study, 151 adult pre-Conquest British skulls were examined using the TWI. Indeed, buccal and lingual wear was observed to the same degree as in a contemporary comparison group, but cervical wear did not develop beyond criterion 0 in most individuals. In 20% of the skulls, however, a cervical wear to TWI score of 2\u20133 occurred. No information was given about the shape of the lesions, and from the images included, it appears that their characteristics would not match the current criteria for dental erosion.\nTable\u00a04Prevalence of lesions of defined shape in three groups (n\u2009=\u2009100 each) of subjects with substantially different nutrition patterns [13]\u00a0Abrasive diet (medieval group)Acidic diet (raw food group)Average western diet\u00a0Incisal\/occlusal surfacesIncisors\/caninesGrooving93%96%90%n.s.\u00a0Molars\/premolarsShallow cupping (<0.5\u00a0mm)87%59%47%p\u2009<\u20090.001Deep cupping (>0.5\u00a0mm)78%45%4%p\u2009<\u20090.0001\u00a0Smooth surfaces (all teeth)Concavity coronal to the CEJ0%63%8%p\u2009<\u20090.0001V-shaped defects0%38%10%p\u2009<\u20090.0001\nIn contrast to the findings in the medieval group, shallow defects located coronal from the CEJ were considerably prevalent in the raw food group (Table\u00a04, Fig. 3b), whereas in the western diet group, the prevalence of this kind of lesions was much lower and corresponds to its general prevalence in Germany [45].\nFig.\u00a03a Buccal aspect of teeth 44\u201347 with significant loss of crown height, but without any lesion in a medieval remain [13] with severe generalised occlusal wear c. b Occlusal defects in a subject living on a raw food diet with a high intake of acidic food [15]. The shape of the occlusal lesions is similar to c, but combined with shallow lesions with intact cervical rim lesions. d Same subject with Fig. 4b with an initial buccal lesion\nAs to the occlusal\/incisal surfaces, grooving and cupping was common in all groups even though most often in the medieval group, followed by the raw food group, and the western diet group.\nThe conclusion from this study was that shallow defects on smooth surfaces might be a valid criterion for dental erosion, whereas cupping, and especially incisal grooving, was common in all groups and therefore not valid for a differential diagnosis.\nThe similarity of occlusal\/incisal defects in all groups might be explained from a tribological view [29]. Wear, as a result of abrasion, occurs as three body wear that means the intervention of an abrasive slurry or bolus. During mastication, the abrasive tends to hollow out softer surface regions. Cupping or grooving can occur when deeper enamel regions with lower microhardness [33] are exposed or when the dentine is reached. In the case of erosion, tissue loss is on one hand caused by direct dissolution of mineral but also due to an increased susceptibility of acid softened surfaces to physical wear. In these cases, also a non- or less abrasive bolus could cause similar defects when acting on acid weakened surfaces.\nHence, the occlusal\/incisal substance loss observed in individuals prone to dietary acids may be explained as pronounced abrasion\/demastication of acid softened surfaces. Therefore, it is questionable if the occlusal morphological criteria used for the diagnosis of occlusal erosion per se are valid.\nEven though there are often striking similarities between lesions of predominantly abrasive or predominantly erosive origin, there is, on the other hand, often also considerable variation in lesion shape in cases of erosion. The morphology of occlusal lesions in subjects with known exposure to acids varies from deep hollowing out of the cusps to totally amorphous loss of the occlusal structure even in subjects with a similar dietary history (Fig. 4).\nFig.\u00a04Occlusal tissue loss from erosive aetiology can also be of strikingly different shape either presenting as deeply hollowed out lesions (a subject with raw food diet [15], b subject with excessive consumption of orange juice) or as amorphous generalised tissue loss affecting the entire surface (c, d subjects with excessive consumption of erosive drinks). An interesting feature is seen in an adolescent with a history of severe anterior open bite with only the molars being in function e Substance loss occurred from excessive consumption of a cola type drink. In the premolars, dentine is proud of the surface. f Hollowing out the entire occlusal surface with enamel remnants in the centre, aetiology is the excessive consumption of sport drinks\nConclusion\nThe validity of current diagnostic criteria for dental erosion has not been systematically studied, even though there is consensus about their definition.\nThe following conclusions can be drawn:\nGrooving of incisal surfaces is a common phenomenon and possibly the effect of any physical or chemical impact. It should be considered to abandon grooving of anterior teeth and canines as a clinical criterion for dental erosion.Shallow defects located coronal from the CEJ may predominantly occur as effect of chronic acid exposure and might be pathogonomic for dental erosion. This assumption is supported by the finding that these types of lesions are not present in ancient remains even in cases of severe wear.Cupping of cusps is the most uncertain criterion because it can be an effect of abrasion as well as of erosion. In industrialised countries, abrasion is not expected to be a significant factor in young people. Cupping occurring at younger ages can therefore be an effect of erosion. At older ages, however, physical and chemical impacts add up increasingly and cupping will therefore be of little diagnostic value in adults.\nThese conclusions are drawn from very few studies; therefore systematic research on this issue is needed. Nevertheless, there is enough support for a criticism of current diagnostic criteria particularly in the light of the development of a new index.","keyphrases":["validity","criteria","erosion","epidemiology","diagnosis"],"prmu":["P","P","P","P","P"]}
{"id":"J_Comput_Aided_Mol_Des-4-1-2270923","title":"What do we know and when do we know it?\n","text":"Two essential aspects of virtual screening are considered: experimental design and performance metrics. In the design of any retrospective virtual screen, choices have to be made as to the purpose of the exercise. Is the goal to compare methods? Is the interest in a particular type of target or all targets? Are we simulating a \u2018real-world\u2019 setting, or teasing out distinguishing features of a method? What are the confidence limits for the results? What should be reported in a publication? In particular, what criteria should be used to decide between different performance metrics? Comparing the field of molecular modeling to other endeavors, such as medical statistics, criminology, or computer hardware evaluation indicates some clear directions. Taken together these suggest the modeling field has a long way to go to provide effective assessment of its approaches, either to itself or to a broader audience, but that there are no technical reasons why progress cannot be made.\nIntroduction\nVirtual screening in the pharmaceutical industry is an essential part of molecular modeling\u2019s contribution to lead discovery and, to a lesser extent, lead optimization. This has led to considerable research into what method or approach works best, typically by means of \u2018retrospective\u2019 evaluations, i.e. attempting to predict future, i.e. prospective, behavior by appraising techniques on known systems. Despite this there is no agreed upon theory as to how to conduct a retrospective evaluation. As a consequence, it is very difficult for an outsider to assess if methods are getting better, have stayed the same, or even worsened over time. In a practical enterprise, such as drug discovery, the proposed benefits of virtual screening, i.e. avoiding the cost and time of a real screen, have to be weighed against one simple question: does it actually work? Without proper metrics of success, i.e. ones that go beyond the anecdotal, molecular modeling is not guaranteed a vibrant future.\nObserved as a general exercise, there are four elements that ought to be standard for any prediction study, whether of a virtual screen, or any general pattern recognition method. The first is whether the study is well designed. The second is what metrics are used to evaluate the outcome. The third is a consideration of significance, i.e. error analysis. And the fourth is a reliable assessment of whether the results are particular or general. All four aspects are important and yet it is rare for a study in any field to meet\u00a0all these criteria. Even in the most critical part of drug discovery, i.e. clinical trials, there is considerable room for improvement, as several recent retrospective studies of the medical literature have demonstrated [1, 2]. In reports on virtual screening, in fact in molecular modeling in general, it is rare to find an adequate consideration of any of these issues.\nWhy is this? Why is the modeling field so poor at the most basic elements of evaluation? A charitable view would be that, as with communication skills, most modelers receive little appropriate formal training. Certainly there is no central resource, whether scholastic review, book or paper. A slightly less charitable view is that journals have not developed standards for publication and as such there is little Darwinian pressure to improve what the community sees as acceptable. It is to be hoped that this is a learning curve, i.e. that editors will eventually appreciate what is required in a study. An extreme view is that we are poor at evaluations because we simply do not matter very much. If large fortunes were won or lost on the results from computational techniques there would be immense debate as to how to analyze and compare methods, on what we know and exactly when we know it. There would be double blind, prospective and rigorously reviewed studies of a scale and depth unknown in our field but common in, for instance, clinical trials. In short, there would be standards.\nIn the hope that virtual screening is, in fact, worthwhile we provide comment, suggestions and research on two important aspects, namely experimental design and performance metrics. Although the two are intimately linked, i.e. an experiment should be designed with a mind to what is being measured, there are distinguishable aspects. On experimental design, extensive properties, such as number of targets, actives and inactives, need to reflect a statistical understanding of the current unreliability, or high variance, of methods [3\u20135]. So dominant is this variance that it almost renders moot any discussion of other matters, such as decoy design. However, ultimately all aspects of design are important. On decoy selection we suggest the necessity of clarifying design intent and suggest four broad categorizations. In analyzing results, the issue of correlation is considered. This often arises in the context of the 2D similarity of actives from congeneric series, but the general issue also concerns decoys, targets and methods. Research is proposed that would clarify essential and poorly understood issues, such as the transference of predictability between closely related systems. On evaluation metrics we examine the AUC (Area Under the Curve) of ROC (Receiver Operator Characteristic) curves [6\u20139]. Consideration of why the AUC is a popular measure in many disciplines suggests standards by which virtual screening metrics ought to be judged. Finally, by evaluating average properties of large numbers of systems, and by considering simple cost\/benefit examples, we bring into question the validity and utility of metrics proposed to capture \u2018early\u2019 behavior.\nExperimental design\nIn what follows we consider the importance of both intensive and extensive properties of an experiment. An intensive property is something intrinsic to a design, whereas extensive properties change with the size of the system. For example, the type of decoys used in a retrospective study is an intensive property; the number of such is an extensive property. We believe the most overlooked intensive characteristic is the design goal, i.e. what is trying to be proved. This typically falls into a few discrete classes and appropriate labeling would help combine lessons from different studies. For extensive quantities we consider how common statistical approaches can aid the choice of numbers of actives, decoys and targets. Finally, actives, decoys, targets or methods are not always independent and this has to be quantified even in as simple a matter as comparing two programs. Techniques for accounting for correlation within an experimental design are known but rarely applied.\nIntensive properties\nOne of the most basic issues in designing a retrospective screen is how to chose decoys. Typically there are a certain number of active compounds and one wishes to see if a method can distinguish these from a second set, presumed inactive. This is the most basic of classification problems. Is X of type A or type B? The legal system often has the same dilemma, e.g. was X at the scene of a crime or not? A police line-up has all the components of a virtual screen. Usually the number of actives (suspects) is small, usually one. The number of decoys (called \u2018fillers\u2019) has to be sufficient that random selection does not compete with real recognition; a minimum of four is usual. But it cannot be so large that guilt is hidden within the statistical variance of the innocent. The fillers need to be convincing, i.e. not outlandishly dissimilar to the guilty party, but not too similar or even potentially also at the scene (i.e. false false positives). As courtroom verdicts can depend on the appropriateness of a line-up, standard procedures are well known.\nWe make the argument for four types of virtual screening experiments; each with its own intent. Each of the four designs ultimately consists of a random selection of decoys but after the application of different filters. Universal. Any compound available to be physically screened, typically either from vendors or corporate collections.Drug-like. Available and drug-like, typically by applying simple chemical filters.Mimetics. Available, drug-like and matched to known ligands by simple physical properties.Modeled. Available, drug-like and derived using 3D modeling on known ligands or the intended targets.Although no classification scheme could be perfect, fair comparison of studies requires an alignment of intent. In general, decoys get \u2018harder\u2019 from A to D, although this is not necessarily true on a case-by-case basis and is itself an interesting area of research.\nThe first, and perhaps least in favor, is the universal selection of decoys. A catalogue of compounds from a vendor or set of vendors is treated as a general population from which to draw. An example of a virtual study with universal decoys can be found in Rognan et\u00a0al. [10]. Although this method is now uncommon, it has an interesting intent. Faced with all compounds available for testing, does a method distinguish known actives without using prior knowledge of what makes a compound active? Drug discovery has a long and successful history of grinding up exotic plants and animals and screening for activity and so this is a reasonable, if old-fashioned, approach. In the Rognan set, for example, we find I3, not likely to be a drug but none-the-less an interesting molecule. The problem with universal decoys is two fold. First, is it random enough? The space of all possible chemistry is exceedingly vast [11, 12]. The concept that a few thousand compounds, in particular from a vendor database, could act as a thorough sampling is implausible. In fact, there is now evidence suggesting known chemistry is very restrictive [12]. Because of \u2018inductive bias\u2019, a concept frequently highlighted by Jain [13, 14], we tend to make what we know might work, instead of sampling of what can be made. Second, is it possible a universal decoy such as I3\u00a0might stand out pretty much the same way a shady character would stand out against a selection of school children, shop clerks and nuns? Paul Hawkins has described this as the \u2018dog\u2019 test [15], i.e. if your dog could tell the difference between the actives and inactives what have you really proved? Actually, potentially a lot but only if the rest of the experiment is designed with this choice of decoys in mind. The problem here is one of dynamic range. If it is too easy to distinguish an active then the only way to distinguish between the methods is to test many, many times, i.e. to improve the statistical power necessary to rank one method above another. As is well known, and discussed below, the error in any metric depends on both the number of actives and the number of inactives. While it is trivial to increase the number of (presumed) inactives almost without bound, the number of actives is normally very finite. Only in some of the more impressive published studies does the number of actives exceed a hundred [3, 4] and it is this limitation that really hinders random decoys being an effective experimental design. We note that this is only a presumed inadequacy of universal decoys; in fact such decoys may prove difficult for some computation methods, the Hawkins dog test not with-standing. The point is that a presumed limitation can be overcome by applying basic error analysis.\nA more typical selection procedure is to choose from a decoy set that is \u2018drug-like\u2019. Of course, there is no rigorous definition of \u2018drug-like\u2019, but this does not stop it being widely used. The intent is to mimic modern physical screens and not test everything but instead be guided by current dogma as to what a drug might look like. The most prevalent of these descriptions is the famous Lipinski Rule-of-Five, but there are many variants [16]. This is not dissimilar to how police line-ups are actually constructed; \u2018fillers\u2019 are normally acquired from local jails. In theory, this should also be a harder test of methods because there are less easily discernable inactives, although this is not proven. Examples of this approach are the studies of McGaughey et\u00a0al. and Warren et\u00a0al. One potential advantage of this approach is that because decoys are derived from characterized collections they are more likely to be known to be inactives. This is typically only an assumption for universal decoy sets. It is debatable as to how big a problem false decoys are, but clearly they do not help. There are also issues with drug-like decoys. Some companies\u2019 collections are heavily biased towards certain targets that may or may not be related to the retrospective study at hand. The study by McGaughey et\u00a0al. reported significant differences in the efficiency of decoys chosen from the MMDR, a kind of \u2018consensus\u2019 drug-like collection, compared to ones from their internal Merck database. This trade of generality for local applicability is a characteristic of many aspects of evaluations. For instance, should targets be chosen to represent all possible systems, a subset of pharmaceutical interest or a class within that subset? What is gained in local applicability is often lost to generality and prospective predictability.\nThe third approach is to find mimetic or modeled decoys. These are meant to stress-test methods and should be used to compare approaches, rather than necessarily evaluate real-world performance. Mimetic decoys are constructed so that \u2018simple\u2019 methods cannot tell known ligands from decoys. The rationale is utilitarian; why should one chose to use sophisticated methods when simple, ligand-based, ones can do just as well? Approaches include matching physical properties, for instance size, number of hydrogen bond donors and acceptors, lipophilicity, charge or flexibility [17]. An example of this approach is the DUD dataset [18] of Irwin et\u00a0al. Here, for each target thirty-six decoys are found for each active by matching physical properties, forming a mimetic set referred to as DUD-self. The combined set, i.e. across all targets is drug-like and is referred to as DUD-all. Mimetic decoys can sometimes be depressingly effective, as illustrated by Irwin et\u00a0al. However, in not all cases were DUD-self decoys harder to distinguish than decoys from DUD-all. This at least suggests physical property mimetics are not guaranteed to provide a reality check for methods claiming to capture the physics of drug-target interaction.\nModeled decoys go one step further than mimetics by eschewing the concept of comparison to simple, practical, methods and instead designing directly against the method under study. As an example, suppose decoys for a docking study were chosen such that every decoy had good shape complementarity with some part of the active site, i.e. it fit well. It is widely known that basic shape complementarity is usually necessary for binding and forms a major component of most scoring functions. Such a set of decoys would them make for a challenging test of scoring functions. But is this a good test of docking? If essentially random performance is seen an observer might decide docking is without merit, whereas an appropriate conclusion would be that complex scoring functions are useless. The inevitable desire of methods to be seen to be useful often prevents modeled decoys being chosen, even though they potentially address the most interesting scientific questions. Irwin et\u00a0al. had intended their mimetic decoys to act more as modeled decoys, i.e. the aim was to make things harder for docking programs, but, as mentioned, this was not always achieved.\nAlthough mimetic and modeled decoy selections have virtues, they can also hamper comparison between different studies. In the case of the mimetic approach, the definition of \u2018simple\u2019 evolves over time, especially as property calculations improve or change. To arrive at a set of modeled decoys the procedure applied must be scrupulously described, e.g. how is the protein prepared, how is the ligand protonated etc, and complete and accurate descriptions of published virtual screening procedures are rare. However, there seems no reason a consensus could not be reached by interested parties. Standard protocols could be developed, shared and used to verify results. The problems are more of will than ingenuity.\nGiven the above discussion, what is the appropriate decoy set to use? A universal set with sufficient actives to enable discrimination between methods? A drug-like set built from one group\u2019s definition of a corporate collection, but perhaps without general applicability? A mimetic set to produce physically similar decoys? Or a modeled set defined so as to tease out specific differences between methods, even at perception of poor performance? A suggestion by Geoff Skillman [19] provides a novel framework. Given the speed of modern computers and the cost of storage and transmittal of information, there seems no reason a retrospective study could not contain all decoy types, with careful labeling of individual intent. The authors can make of their data what they will, for instance by reporting performance against a subset of decoys. However, if a broader set is included in the supplementary material, others can make use of the data for potentially different purposes. One of the proposals of this paper is for modeling to move beyond the anecdotal towards the systematic. Full reporting of data is essential but a further step would be to include alternate data so that others can construct purposes beyond the original intent.\nExtensive properties\nIn addition to intensive properties, there are the extensive properties such as how many actives, decoys and targets are used. Once again the important consideration is knowing what we want to know. If the purpose is to evaluate a single method on a single target the necessary extensive properties are quite different than for a broad study on the efficacy of several methods on many targets. We illustrate this with some basic error analysis.\nThe Central Limit Theory (CLT) states that the average of M measurements tends towards the true mean with an error proportional to \u221a(V\/M), where V is the average squared difference of a quantity from its estimated mean. Thus, the error is an intrinsic quantity, the square root of V, divided by an extrinsic quantity, the square root of the number of measurements. Famously, we have to take four times the number of measurements to reduce the error by a factor of two. What does this say as to the number of actives, decoys or targets that should be used to accurately measure the performance of a method? If the performance is similar no matter what actives, decoys or targets are used then the variance is small and M can be small. However, this is not the situation for modeling techniques applied to real systems. Instead, it is the ruling zeitgeist that \u2018performance may vary\u2019 [3\u20135].\nJust how variable are virtual screening methods? Figure\u00a01a and b illustrate the extent of the problem by presenting a reanalysis of the Warren et\u00a0al. study from GSK, with eight different docking methods and our own work on the DUD dataset (DUD-self decoys) using four different virtual screening techniques. The performance metric is the AUC averaged over each dataset. The number of targets for Warren et\u00a0al. is eight and for DUD forty, i.e. a five fold difference. As expected, the error bars, i.e. the confidence limits at 95%, are slightly more than twice as big for a method assessed against the GSK set than against DUD. In addition it is clear that although methods in the GSK test could be broadly classified as better or worse, this is subject to considerable statistical dispute. The resolving power of DUD begins to be apparent in Fig.\u00a01b, where one can begin to put some significance to the generally held belief that ligand-based methods perform better than docking and about as equivalently as 2D methods [4, 21]. The average AUC and error bars for GOLD from the Warren study are included in for comparison purposes only. A more quantitative analysis of this data will be presented below in the section on correlation between methods.\nFig.\u00a01(a) Average AUC values across docking programs in the Warren study, with 95% confidence intervals. Where programs were run in multiple modes the best average AUC was retained. (b) Average AUC values plus error bars across the DUD dataset for four in-house methods. Docking: FRED, Ligand-based: ROCS, 2D: Lingos and MACCS Keys [20]. Also included for comparison purposes is the average AUC for GOLD against the Warren set with associated error bars\nWhat is the source of so much variation such that even forty targets are barely able to provide statistically supportable conclusions? In general, given a property measurement that has N independent sources of error, the expected error is formed from the root mean square of the individual sources of error, thus:\nFor our case we can write:\nThe variances are intrinsic properties to \u2018targets\u2019, \u2018actives\u2019 and \u2018inactives\u2019. How do we know what these variances are? One way is to boot-strap, i.e. leave out a randomly chosen fraction of the targets, or subset of actives or inactives, and measure changes in performance. Repeating this procedure many times gives a statistical sampling of the sensitivity to outliers and the number of measurements. Alternatively, in some cases the variance can be established more precisely. In the case of AUC, for example, it can be shown that for a particular target the variance for both actives and inactives can be approximated by:\nwhere the sums are over all actives or inactives, pi is the probability this active i has a higher score than an inactive, qj is the probability an inactive j has a higher score than an active and \u2329\u232a represents the average of a quantity [7]. Typically, the variances of both actives and inactives are roughly equal. This leads to useful insights as to the required ratio of decoys to actives. When this ratio is 100:1, the net error is only larger by 0.5% than if we were to use an infinite number of decoys. A ratio of 40:1, roughly that of the DUD-self set, yields an impact about 1%. At 10:1, this impact is about 5% and at 4:1 about 11%. Note that these effects on the error estimates, not on the actual average. What does this look like in practice? Figure\u00a02 shows the AUC values for FRED applied to DUD (self-decoys), along with associated 95% confidence intervals for each system. Given these AUCs and contributions to variance from actives and inactives, we can directly address whether the source of the variance across targets is due to insufficient sampling of actives and decoys, or an intrinsic property of methods. By the CLT,Fig.\u00a02AUC values ordered from left to right by number of actives for each target in the DUD set. Program used: FRED with Chemscore as the posing and scoring function. Error bars are 95% confidence intervals for each virtual screen\nwhere\nTherefore\nTable\u00a01 shows contributions to the square of the average error in the mean AUC across DUD for our four methods calculated in this manner. First, as expected the contribution from the inactives is about forty times less than that of the actives (because the intrinsic variances are similar and there are thirty-six times more decoys than actives in DUD-self). Secondly, it is clear that the errors due to target variability is roughly ten times higher than that due to actives. As independent errors add as squares, this implies only about 5% of the observed confidence limit on the target-averaged AUC is due to the finite number of actives. This leaves 95% of the 95% confidence limit due entirely to the considerable variation from target to target. Comparing DUD-self to the careful evaluation of McGaughey et\u00a0al. from Merck, and Warren et\u00a0al. from GSK, the latter have roughly four times more actives and four times less systems, i.e. they are more careful studies of particular systems (error bars are 50% smaller per system) but substantially less useful for general conclusions (error bars are twice as big per method). Remarkably, even if the number of actives in each DUD-self set were reduced by a factor of ten, causing a threefold increase in the error estimation per target, the net error of averages over all systems is only increased by about 30%.Table\u00a01The contribution to observed variance from actives, decoys and targets over the DUD dataset (DUD-self decoys)Method\u2329Err2\u232a\u00a0\u2212\u00a0Decoys\u2329Err2\u232a\u00a0\u2212\u00a0Actives\u2329Err2\u232a\u00a0\u2212\u00a0ObservedEst. \u2329Err2\u232a\u00a0\u2212\u00a0TargetsFRED0.0000480.00200.0230.021ROCS0.0000250.00220.0410.039MACCS 0.000040.00170.0300.028LINGOS0.0000390.00170.0350.033The estimated error (squared) from the variation between targets is estimated from the observed variance and corresponds to that which would be obtained if the number of actives and inactives were infinite\nThe conclusions from this analysis of extensive properties are straightforward. When calculating the properties of a single system the number of actives is fairly important, but the number of inactives does not have to be substantially larger. A ratio of decoys to actives of 4:1 only has an error 11% higher than the limiting value from an infinite number of inactives. It would be more useful to include sets of inactives designed for different purposes than to attempt to \u2018overwhelm\u2019 the actives with decoys.If the purpose is to test a method against other methods with 95% confidence then the number of systems required is very large, much larger than even DUD. In our analysis the contributions to the variance from a limited numbers of actives is almost insignificant compared to the target-to-target variation. For example, it would take over 100 test systems to tease apart the difference between the ligand-based method ROCS and the docking program FRED with 95% confidence. (See below.)The variance between systems is such that the number of actives per target does not need to be very large, perhaps even as low as ten. As such, suggestions to only include representatives of chemical classes, e.g. see Good and Oprea [22], may be statistically quite valid.\nCorrelations\nA key assumption underlying much statistical analysis is the independence of samples, for instance that any two measurements are uncorrelated. This is often a good assumption but it is not hard to find counterexamples. Consider the case where a decoy is included twice. We have gained no new information. Yet Ni, the number of decoys, has increased and so the error goes down. Clearly the error has not really been reduced. Instead, the decoys are no longer independent. In the line-up analogy, this would correspond to including identical twins as fillers. While this is an unlikely mistake, what about two individuals that look very similar? How independent are two molecules and what does this even mean? The temptation is to reach for the familiar chemical definition, i.e. 2D similarity. Even though there is no rigorous definition of chemical similarity, it is a major concern in selecting active populations from chemically related (congeneric) series. Methods that either rely on chemical similarity, or are heavily influenced by it, may not be making independent assessments. Clearly 2D methods fall into this category, and sometimes ligand-based 3D methods. Ideally, methods that use protein structure, such as docking, ought to be less affected, but this is far from proven. Suggestions as to how to improve matters include reducing the set of active to a smaller set of representative structures [22], or giving more weight to the first compound discovered in a series [23]. (Application of similar protocols to decoys is seldom discussed, perhaps because they are less likely to be congeneric). These are practical suggestions derived from knowing the nature of drug discovery. There is also a general approach that eschews the particulars. Two compounds are considered operationally dependent if their rankings under different tests are correlated. For instance, a method that had a size-bias would tend to rank a pair of molecules of comparable extent similarly, no matter what the target. Even without 2D similarity, this implies a less than perfect independence. Imagine a method where all the decoys are of one size and all the actives another. No matter what the actual number of actives and inactives, there are essentially only two molecules, an active and an inactive, and our ability to extract meaningful statistics is severely compromised. Note that the operational part of this definition depends on the nature of the method, i.e. dependence is conditional on the nature of the procedure investigated.\nSimilar situations occur in assessments of genetic linkage. The degree of dependence amongst a set of markers is evaluated by constructing a matrix where the entries are the correlation of phenotypic scores between any two markers. The eigenvalue spectrum of this matrix is then used to assess the actual number of degrees of freedom [24]. Crucially, though, correlation can only be estimated by knowing the behavior of a pair of samples\/compounds over many tests\/targets. At first glance this suggests that the same set of decoys should be used across all targets in a study. If decoy selection is universal or drug-like this is typically a part of the design, i.e. the decoy set is reused. However, mimetic and modeled decoys rely on the nature of the actives, which will vary from target to target. This might seem a dilemma, i.e. we want to reuse decoys so we know if they are correlated but we cannot reuse them because one set of decoys may be completely inappropriate for another target. Here Skillman\u2019s suggestion is again useful, i.e. there is nothing to stop us including the decoy set for one target in the virtual screen of a second target not as decoys but rather to gain information on operational independence. To distinguish the role of decoys from one system applied to a second system for purposes other than assessing performance, we suggest the term \u2018latent\u2019 since these secondary decoys should be hidden from the calculation of performance metrics. In addition to the concept of latent decoys, latent actives can be used to measure operational independence independent of 2D similarity. Another possibility is to use the actives from one system as explicit decoys in other systems. For instance, in the Warren et\u00a0al. study each set of actives also formed the decoy set for the other targets. The intent was just to produce a set of drug-like decoys, but it fortuitously provides the most compact form for a rigorous estimation of decoy\/active independence. This work will be presented elsewhere, along with an elaboration of the techniques for assessing operational correlation.\nWe turn now to the question of target independence. As we have seen, and is widely appreciated, the variation of performance of methods from target to target is considerable. But do certain targets, or classes of targets, behave similarly for certain methods? For instance, one would expect that a docking program parameterized against certain binding motifs would perform similarly across all targets with this motif, if only because of inductive bias. Or one might assume that isoforms of a target are sufficiently akin that docking methods would perform similarly on each. Fortuitously, the Warren study provides one such example in the inclusion of PDFE and PDFS. Figure\u00a03 shows the difference in performance of methods used on isoforms versus the average difference between all other pairs of targets. It is clear there is less variance between the isoforms than unrelated targets. If this were a generalizable result it would have two consequences. On the positive it would mean that methods could be quantified for certain types of problems without requiring large numbers of targets, i.e. because the variance is smaller. On the negative, it would mean that just as considerations need to be made for the true statistical power of closely related actives, or inactives, similar considerations need to be made for targets, increasing the number of targets required to either discern general differences between methods or to reliably gauge progress of a single approach. And, as we have seen, to measure global performance on independent systems already requires sampling beyond common practice.Fig.\u00a03Docking performance against the two isoforms in the Warren study (PDFS and PDFE), compared to the averaged difference over all other pairs of targets\nThe PDFE\/S example is a single data point. It is entirely possible that the variation between targets of similar class, or highly conserved isoforms, or even different forms of the same protein structure is not small and that calculating mean properties is still formidable. One might imagine this is a well-researched area, but this appears not to be the case. Retrospective experiments are designed, however poorly, to give an estimate of to what to expect for the next new target and so targets are chosen to be diverse. Software would seldom be used in default, out-of-the-box, mode when there is considerable domain knowledge, i.e. within a set of closely related targets. Hence, the question of method variance over similar systems appears to have been over-looked.\nThe final aspect of independence is correlation between methods. Suppose we have method A and method B, each tested on the same set of targets with the same set of actives and decoys and the results show A is consistently slightly better than B. How can we prove this difference is statistically significant? At first glance this would seem difficult. As illustrated in Fig.\u00a01a and b, the variance of any one computational method over a set of targets in invariably large. As such, the error bars on an average property, such as an AUC or enrichment, are big. So although the average behavior of method A is slightly better than B, this difference would appear statistically insignificant. However, if the test systems are indeed identical this is not the correct assessment. Instead, the CLT is applied to the set of measured differences between methods, e.g. for an AUC example the variance becomes:\nThe formula for Vardiff can be rewritten as:\nHere Corr(A,B) is a measure of the correlation between methods A and B and is related to the Pearson correlation coefficient, thus:\nIf the tests of methods A and B are independent then the correlation is typically assumed zero and the variance is just the sum of the variances of both methods. If variances were roughly equal, this would typically be a joint error bar \u221a2 larger than the individual error bars. (This also means the common practice of evaluating whether two methods are statistically different by whether their individual error bars overlap is generally incorrect.)\nHowever, if the tests applied to methods A and B are identical, correlation needs to be explicitly included. In the case of A always slightly better than B, we need to assess whether the mean difference is larger than the joint confidence limits generated from the variance of the difference between A and B. In fact, if A is better than B by a constant amount we are guaranteed statistical significance because the variance of the difference is zero. In general, methods tend to be positively correlated so that the joint confidence limits are lower than from independent measurements.\nConfidence limits are convenient because they give a visual estimate of the possible range of true values, typically at a 95% level of confidence. However, joint confidence limits are less graphical as they pertain to pairs of methods. In addition, there is considerable concern in other fields as to the arbitrariness of the 95% value. The origin of this number is R. A. Fisher, whose work in the 1920s still dominates much of the field of practical statistics. Fisher, primarily an agriculturalist, observed a 10% increase in cabbage yield when manure was used. He also observed that only one in twenty plots without manure showed a yield greater than 10%, and so the 95% cut-off was born! From this, and because of the general utility of Fisher\u2019s work on experimental design, a p-value of 0.05 dominates many fields, in particular clinical trials. Essentially, a p-value is the probability a null hypothesis can be rejected. In our case the null hypothesis would be that method B is in fact better than method A, despite average values suggesting the reverse. Under the assumption the difference in performance between two methods is as predicted by the CLT (i.e. Gaussian), we can assign a (p-)value to the probability one method is better only because of random chance. We do this by calculating the area under the normal form for which one appears better than the other. The mathematics of this is shown below:\nwhere \u2329A\u2013B\u232a is the average difference between the methods, the other variables are as defined as above and erf is the inverse cumulative Gaussian, or error, function. If Nt is small, i.e. less than twenty, then a slightly different functional form is more accurate (i.e. from the Student t-test) because the CLT only applies in the limit of large N. For practical purposes the difference can be ignored. The smaller p, the stronger the case for A being better than B. Note, we are not proving how much better A is than B. The best estimate of A\u2019s superiority is still the mean difference of whatever property we are measuring. Rather the p-value refers to the dichotomous question, is A better than B?\nTable\u00a02 illustrates the above concepts for the four methods listed in Fig.\u00a01b applied to DUD (DUD-self decoys). The diagonal entries are the mean values of the AUC of each method, followed by the associated 95% confidence limits. The upper triangle of the table contains the na\u00efve joint confidence limits, i.e. by summing individual confidence limits, the joint confidence limits assuming independent tests and the joint confidence limits properly calculated with correlation. The lower diagonal contains the Pearson correlation coefficient for each pair of methods, followed by the p-value for the hypothesis that the better method is so only by chance. So, for example, the probability that the 2D and ligand based methods are better than the docking program FRED by chance are around the 10% level, whereas the differences between these three methods themselves is close to 50:50, i.e. in this example we can distinguish ligand-based methods from a docking protocol but not one ligand-based method from another. This is most likely because DUD is not designed to test ligand-based retrieval containing, as it does, many 2D similar actives. Several others have made this point, including the curators themselves [14, 18, 22]. Examples from other fields of how to apply these procedures to differences in AUC can be found in Hanley and McNeil [9].Table\u00a02Statistical measures necessary to accurately assess the relative performance of methods, here applied to the DUD data set (DUD-self decoys)MethodFREDROCSMACCSLINGOSFRED0.684\/0.0430.11\/0.08\/0.070.1\/0.07\/0.060.1\/0.07\/0.065ROCS0.17\/0.090.732\/0.0650.12\/0.085\/0.050.125\/0.09\/0.05MACCS 0.03\/0.050.70\/0.470.734\/0.0550.115\/0.08\/0.055LINGOS0.19\/0.140.65\/0.360.54\/0.310.72\/0.061Diagonal terms: average AUC\/95% confidence limits. Upper triangle terms: na\u00efve joint confidence limits\/joint confidence limits assuming different tests\/joint confidence limits assuming same tests and accounting for correlation. Lower triangle terms: Pearson correlation coefficients\/p-values that a method has a higher mean AUC by random chance\nOne of the advantages of p-values is the statistical machinery, again developed by Fisher [25], for combining values from different studies. A classic example is the effects of tobacco. It was not one study that convinced the medical profession, but a series of studies and the facility to combine the results that lead to the overwhelming conclusions as to the health risks of smoking. In statistics this is referred to as \u201cMeta-Analysis\u201d. Despite this, and the wide application of p-values in other fields, they are largely absent from modeling, with a few exceptions [5].\nIn conclusion, correlation is important in all aspects of virtual screening, but perhaps most important and most easily corrected for in the comparison of pairs of methods. Neglecting the effects of correlation between tests is a frequent problem even in clinical studies [2] and to our knowledge has not been properly applied to comparing methods in virtual screening.\nMetrics\nGiven an experimental design, what quantities should be measured to assess performance? The question suggests a sequential process, i.e. design the experiment and then measure something, whereas good design takes into account what is going to be measured, in particular to what accuracy. However, assuming a given design, how do we extract useful information? In this section we consider what should be measured and why. This is not a quandary specific to virtual screening, in fact is it universal to all prediction exercises. This very commonality can help suggest worthwhile approaches. It also suggests that measures constructed specifically, even uniquely, for chemical virtual screening should be held to a similar standard to those prevalent in the wider world. Is virtual screening really so different from, say, Internet page ranking? In particular, we will consider the issue of \u2018early\u2019 behavior, i.e. measures that reward ranking some active compounds near the top of a list. By considering real-world financial parameters we ask whether \u2018early\u2019 behavior is even necessarily to be prized. By looking at a large number of virtual screens, we will ask whether such \u2018early\u2019 measures are necessary and whether they can be predicted from more fundamental and well-understood properties. Finally, the application of accurate error bounds will be shown to suggest at least one way of quantifying the advantage an expert brings to well-studied systems.\nProperties of virtual screening metrics\nA long list of metrics has been applied to virtual screening. What makes for a good metric? The unfortunate answer with some papers is \u201cany metric that will make my method look good\u201d. And if no known metric will suffice, then simply make a one up. This is a typical indicator of an under-regulated and under-developed field. Computer manufacturers used to habitually make up their own measures for the latest processor or operating system, leading to much confusion and annoyance. As a consequence, in 1988 SPEC (Standard Performance Evaluation Corporation) was formed and SPEC Marks became the standard benchmark of anything worth measuring. SPEC had a simple philosophy: \u201cThe key realization was that an ounce of honest data was worth more than a pound of marketing hype\u201d [26]. SPEC Marks have evolved over time to now cover CPU, graphics, Java, mail servers, file servers, parallel performance, high performance computing and other aspects. In other words, SPEC is not a single measure because not all users want the same thing, but this does not mean manufacturers can create their own metrics. Rather SPEC is an umbrella organization for a set of open and diverse groups that consider, ratify and develop benchmarks. In this spirit, this section will concentrate on what ought to be general characteristics of a good metric rather than all prevalent quantities.\nIn a somewhat circular manner, one of the first characteristics of a good measure is that everyone uses it. Clearly one of the problems with a field with diverse measures is incomparability, the \u201capples and oranges\u201d problem. The most straightforward solution is not imposition of a particular standard but full disclosure of all data. The authors of a study may want to present enrichment at 5%, but if the data is freely available others may calculate the enrichment at 1% or 13% or whatever they wish. This would inevitably lead to standardization as independent parties harvest data from many sources, publishing larger and larger studies on the advantages and disadvantages of different methods and measures. This would provide another example of meta-analysis described above. Sometimes a valid excuse against disclosure is that compounds or targets are proprietary. However, just providing lists of actives and inactives in rank order with unique, but not necessarily identifying, tags is enough to calculate most of the metrics for a particular virtual screen. Currently the field of modeling lacks even an agreed upon format for the exchange of such rarely available information.\nHowever, if we are going to report a statistic what properties should it have? From considering measures that have become standard in other fields, what characteristics define a good measure? We suggest the following short list:Independence to extensive variablesRobustnessStraightforward assessment of error boundsNo free parametersEasily understood and interpretable\nTake for example the very popular \u201cenrichment\u201d measure. Everyone understands the concept of enrichment: swirl a pan of water and gravel from the Klondike river in 1896 in just the right way and you ended up with mostly gold. In virtual screening you look at the top few percent and see whether there are more actives than you would expect by chance. As a mathematical formula this is typically presented as:\nThe problem with this measure is that the enrichment becomes smaller if fewer inactives are initially present. Imagine panning for gold with all the sand removed. There would still be the same gold in the pan, along with maybe some pebbles and small rocks, but the eventual relative improvement after \u2018panning\u2019, i.e. \u2018enrichment\u2019, is reduced. The problem is that the (Fraction of Actives Found) contradicts requirement (i), i.e. is a function of extensive quantities, the number of actives and inactives. This means that enrichment is not actually a measure of a method; it is a measure of a method and a particular experiment. If the ratio of inactives to actives becomes very large it is assumed this problem disappears, i.e. that the limiting behavior obeys (i). This is not true if the enrichment at a given percent is large, i.e. at precisely the points of most interest. Also, enrichment does not meet requirements (ii). At a small enough percentage the enrichment becomes an unstable function of the exact positions of actives in a list. There is also no agreed upon percentage, making this an adjustable parameter (often abused). Finally, other than by bootstrapping, the author knows of no simple assessment of error bounds. However, it is an intuitive measure, easily understood, passing rule (v), and so almost uniquely to this field is the most common metric reported.\nSome have been aware of the lack of robustness of enrichment and proposed metrics that average over all percentages with weighting schemes. Before we consider these measures we point out that a simple fix to the common variant of enrichment is to make the enrichment refer to the fraction of inactives, not to the fraction of all compounds. This simple change makes the enrichment independent of extensive quantities, more robust, accessible to analytic error approximation [27] and yet suffers only a slight reduction in interpretability. Only a few, such as Jain [14] have used this alternate form. Perhaps the only important failing of this measure is that it lacks a specific name. For the purpose of this paper it will be referred to as the ROC enrichment to distinguish it from the widely abused variety.\nROC enrichment has better properties because is related to an even better metric, the AUC, defined as the area under a ROC curve. A ROC curve is simply a plot of the discovered active fraction versus the discovered inactive fraction. (Each point on the ROC curve can be translated to a ROC enrichment by dividing by the fraction of inactives). The AUC is the average of this property over all inactive fractions. Many excellent treatises can be found [6\u20139] and it has become a standard for classification performance in many disciplines (medical diagnostics, radiology, clinical testing, criminology, machine learning, data mining, psychology and economics to name a few). It satisfies all of the criteria listed above as a metric, including (v), ease of interpretation. The AUC is simply the probability that a randomly chosen active has a higher score than a randomly chosen inactive. The main complaint against the AUC is that is does not directly answer the questions some want posed, i.e. the performance of a method in the top few percent. This is akin to complaining that SPEC Marks do not do a good job of evaluating mobile phone processors; a fair complaint perhaps but hardly justifying creating a new benchmark without the strengths of existing standards. The AUC ought to at least be held as such a standard against which new measures are judged.\nEarly performance in virtual screening\nFigure\u00a04 illustrates the supposed limitations of the AUC as a measure of performance. The graph shows two ROC curves, each with an AUC of exactly 0.5. Overall this means that an active is as equally likely to out-rank an inactive than the other way around. However, clearly in the case of the solid line a certain fraction of actives is being scored significantly higher than most inactives, while another fraction is being scored worse, i.e. it is only the average behavior that appears even-handed. Similarly, the dashed curve illustrates the case where the actives are all scored better then a certain fraction of the inactives but worse than another fraction. The solid and dashed curves are instances of bimodal score distributions for the actives and inactives respectively. Since the goal of a virtual screen is to save us the trouble of actually screening all the compounds it is entirely reasonable to prefer good \u2018early\u2019 behavior. And yet the AUC does not distinguish between such curves and so, it is claimed, is not appropriate.Fig.\u00a04Example ROC plots for \u201cearly\u201d and \u201clate\u201d methods\nIt is against this backdrop that metrics such as RIE [28] and BedROC [29] were developed. In both cases the essential idea is to give early rankings of actives more weight than late rankings. In RIE\/BedROC actives are given a weight depending on their position in the list using an exponential function running from 1.0 for the top ranked compound to a number typically close to zero for the lowest ranked. The exponential factor, beta, determines how fast this exponential dies away from the top rank and controls how much the RIE\/BedROC parameter focuses on the top of the list. The larger beta the greater the early focus. In RIE the sum of active weights is normalized by the RIE of a random distribution of actives. In BedROC normalization is by the maximum dynamic range, i.e. the score with all the actives ranked at the top minus the score with all the actives ranked at the bottom. In addition, by first subtracting the score of the worst-case scenario, BedROC has the elegant property of running from 0.0 to 1.0. The rational behind using these approaches is to give precedence to actives ranking early but not to fall into the trap of choosing a single enrichment value, i.e. be more robust to perturbations in the rank ordering. Impressively, Truchon and Bayly also derive analytic estimations of the error bounds for BedROC and give some suggested values for the beta parameter. Some incorrect statements regarding the AUC mar their work, for instance that random scores do not give an AUC of 0.5, and that the AUC is dependent on the number of actives and inactives, but overall the work is an interesting attempt to answer a perceived need. Applied to the examples in Fig.\u00a04, the ratio of the BedROC score of the solid line to the dashed is about two for a beta of ten and about ten for a beta of twenty.\nSo does BedROC or RIE qualify as a good metric for virtual screening? Comparing against the five criteria listed above, both are more robust than enrichment, and the error protocols for BedROC satisfies criteria (iii). RIE suffers from having an ill-defined numerical interpretation (i.e. how good is an RIE of 5.34?). BedROC attempts to overcome this by scaling between 0.0 and 1.0, but does this qualify as being understandable? There is no absolute, interpretable meaning to a BedROC (or RIE) number, only a relative meaning when ranking methods.\nUnfortunately, neither BedROC nor RIE satisfy criteria (i) or (iv), i.e. both are dependent on extrinsic variables and have an adjustable parameter, the exponential factor beta. The former, as we have seen, means that scores can only be compared in the limiting case of an excess of inactives and, as in the case of enrichment, this excess has to persist even when the enrichment of actives is very high, i.e. it is exactly when the actives are predominantly at the top of the list that both BedROC and RIE (and enrichment) are most sensitive to the total number of inactives. Interestingly, it would be possible to reformulate both metrics to avoid this problem. Just as ROC enrichment is a better metric than enrichment, an exponential weighting across the ROC curve, rather than to the individual rankings of actives amongst inactives, would remove the sensitivity of these measures to extensive properties. However, there would still remains the issue of the arbitrariness of the exponential factor beta. Just as with enrichment thresholds there is nothing intrinsically wrong with the freedom to select a threshold that is of interest to the particular research group. Some companies might have the facility to physically screen ten percent of their database, another only one percent. However, as a characteristic of a method, or a class of methods, it is a disadvantage. A proponent of a method has a free parameter with which to make their method look favorable or even just less unfavorable. In the example in Fig.\u00a04, a factor of two in BedROC between the methods (beta\u00a0=\u00a05.0) does not sound anywhere near as bad as a factor of ten (beta\u00a0=\u00a020.0).\nCost structures of virtual screening\nThere is no fundamental meaning to BedROC or RIE. Neither gets to the real heart of why the solid curve in Fig.\u00a04 represents a better method than the dashed curve. In what follows we will argue that this can only be stated with respect to a set of assigned costs, assumed but never stated. We start by noting that the current focus on early enrichment is actually a change in values for the industry. This author recalls conversations in the mid-1990s wherein the concept of missing any potential lead compound was deemed unacceptable. By contrast, a preference for early behavior implies it is acceptable to miss a significant fraction of potential actives in favor of finding a few good leads. There is merit in this approach. Often a chemistry team can only follow up on a small number of leads. High throughput screens can take several months to design and bring on-line, time chemists could use to explore initial leads from a smaller focused set designed by a virtual screen [30]. What are not made explicit in this shift are the costs of the four components of any virtual screen: true positives (TP), false negatives (FN), false positives (FP) and true negatives (TN). Not wanting to miss anything is equivalent to assigning an infinite cost to a false negative. This was never sensible, but reflected a \u2018lottery\u2019 mentality prevalent at the time. The reality is that virtual screening never finds drugs; at best it can find things that might, after considerable effort, become drugs. In addition, the attrition rate at many stages in the drug design process means any lead-like compound is at best a bet that will often fail, costing many millions of dollars. A lottery ticket is potentially worth millions; the expected value, i.e. averaged over all contingencies, is usually less than the cost of the ticket. The assumption behind virtual screening is that the value of a true positive similarly averaged is worth the cost of computers and modelers. This is an unproven conjecture.\nThe assignment of a cost structure to the components of a screen is common in the field of medical diagnostics. Here the costs can be estimated with some reliability. A true positive represents the successful diagnosis of a condition that will save money when treated. A false positive means further, costly, tests will need to be performed. A false negative might cost a lot if a more severe condition develops. Finally, a true negative can be set to the cost of the test or a small saving if compared to a more expensive test. If these values are assigned to each \u201ctruth table\u201d component (TP, FP, TN, FN), a ROC curve can be transformed into a cost curve. A small caveat is that the ROC curve deals with true and false positive rates and so to transform to real costs the expected number of actives and inactives is required, or at least the ratio of the two. Suppose we apply a cost structure to Fig.\u00a04 as follows:TP\u00a0=\u00a08.0FN\u00a0=\u00a0\u22122.0FP\u00a0=\u00a0\u22120.16TN\u00a0=\u00a00.02\nPositive numbers are favorable, for instance the cost assigned to a true negative is the saving from not physically screening a compound. At any point in the curve the cost of progressing with all compounds higher than a given threshold t depends on the False Positive Rate (FPR) and True Positive Rare (TPR):\nLet us assume Na\/Ni\u00a0=\u00a01\/100, then:\nThis is a simple linear scaling of the graphs in Fig.\u00a04, as shown in Fig.\u00a05a. As expected, the best approach is to take the method with early performance over the later performance. Notice that the late performing method is never cost effective and even the early method is only cost effective for a narrow range of rankings.Fig.\u00a05(a) Cost weighted versions of the curves in Figure 4 as per the first description in the text. (b) Cost weighted versions of the curves in Fig. 4 as per the second description in the text\nNow consider a slightly difference weighting:TP\u00a0=\u00a08.0FN\u00a0=\u00a0\u22122.0FP\u00a0=\u00a0\u22120.04TN\u00a0=\u00a00.03\nFigure\u00a05b illustrates the effect of these new weightings. By reducing the cost of a false positive by 75%, i.e. to around the savings of a true negative, both methods are always cost effective. Furthermore, although the early method has a clear maximum at around 20% of the database, it is actually worth physically screening about 75% of the database.\nThese examples are obviously only illustrative, but the point they make is real. Early enrichment is important only because of an assumed cost structure. Clearly much more complicated models could be constructed, possibly with real data, as with medical tests. However, to the author\u2019s knowledge this has never been published, presented or even discussed within the industry. It is an assumption that early enrichment is better. Likewise, it is also an assumption that virtual screening itself is a productive exercise compared to physical screening.\nAveraged properties of virtual screening\nSuppose the cost structure of virtual screening does favors early enrichment. Can we at least say metrics such as RIE and BedROC, perhaps reformulated to be independent of extensive variables, are superior to AUC? If the early behavior shown in Fig.\u00a04 were indeed repeated from system to system then clearly this would be the case. In Fig.\u00a06 we show data from Warren et\u00a0al. for twenty docking procedures averaged over all eight targets in the study. Examination of these curves reveals nothing that resembles the biphasic nature anticipated from Fig.\u00a04. Individual curves might occasionally suggest biphasic behavior but there is little evidence for this in targetaveraged ROC curves. Figure\u00a07 shows similar curves for the four methods in Fig.\u00a01b averaged across the DUD set. The curves in Fig.\u00a07 are smoother because the averaging across forty targets in DUD is more extensive than the eight from GSK and show even less evidence of biphasic behavior. There are two possibilities for these observations. Either the individual curves are not biphasic or the averaging dilutes this characteristic. It is possible to imagine a technique that would rank one type of actives well, perhaps hydrophobic moieties, but ranks others badly, e.g. hydrophilic ones, but that the proportions of each set differ target to target such that the total behavior appears monophasic. To see if this might be the case we examined two hundred and seventy virtual screens from the Warren study, looking for a divergence between BedROC, with exponential parameter 5.0, and AUC, i.e. an abnormally low AUC and a high BedROC, although possibly the reverse. The results are shown in Fig.\u00a08. Clearly there is a strong correlation between BedROC and AUC. Similar correlations were also seen when higher exponential factors were employed and suggest no evidence for biphasic behavior. A better AUC naturally leads to a better BedROC value and does so with surprisingly little variation. It might still be possible that one of the methods has average biphasic behavior, for instance if all the BedROC\/AUC points for a method trended higher. Figure\u00a09 shows this is not the case. Here, the average AUC per method is compared to the average BedROC value for that method. In addition, the correlation is stronger the better the BedROC value, so that methods that have a good AUC will also have a (strongly correlated) good BedROC score.\nFig.\u00a06Averaged ROC curves for twenty methods in the Warren study for which scores for all eight targets where available. Programs and scoring functions listed to the right of the graphFig.\u00a07Average ROC curves for FRED, ROCS, MACCS keys and LINGOS over DUD, with DUD-self decoys. FRED was run with the ChemGauss3 scoring functionFig.\u00a08BedROC scores with an exponential factor of 5.0 versus the AUC for 270 virtual screens from the Warren studyFig.\u00a09The average AUC for each method run against all eight targets in the Warren study versus the averaged BedROC score for each such method\nThere is one exception to monophasicity, shown circled in Fig.\u00a09. This point lies outside the 95% confidence limits of both AUC and BedROC. Its BedROC score is significantly higher than expected and its AUC is around 0.5, i.e. random ranking. The target protein represented by this point is PPAR-\u03b4 and the method is MVP, a program developed in-house at GSK by Mill Lambert. In conversation Lambert freely admitted that not only did he have extensive knowledge of this target, he used all of this information to tune MVP. Unfortunately, because of certain aspects of the target he could only select one of three chemical classes for this \u2018hands-on\u2019 treatment at a cost to the two other classes. Hence, MVP had to be biphasic. It seems interesting that out of two hundred and seventy virtual screens the only outlier from the BedROC-AUC correspondence is an example of expert intervention. An unintended consequence of this study might be a method to spot and quantify expert contributions to virtual screening, i.e. by comparing early behavior, either with BedROC or other metrics, to that predicted from the fundamental measure of AUC.\nConclusions\nIn this study we have considered several aspects of experimental design and performance metrics for virtual screening. There is clearly interest in doing things the right way, not least because of a popular, if unproven, belief that virtual screening saves the pharmaceutical industry money. As with many relatively young endeavors, molecular modeling has been long on promises and short on standards, and it is standards that ultimately deliver the proof that our field is useful. For many years the computer industry suffered from similar growing pains. Not only were there few, if any, reliable comparison metrics for different processors, operating systems, compilers and so forth, the proposed benefits of computers were more assumed than quantified. These days no one doubts the impact of the computing revolution. It is to be hoped that a similar statement can one day be made for molecule modeling. It is with this in mind that the following observations and recommendations are made.\nOn the issue of experimental design we propose:Decoy selection needs to be properly labeled as to intent to facilitate inter-study comparison. We have suggested four classifications, universal, drug-like, mimetic and modeled based on examples from the literature and on typical use-case analysis.Providing access to primary data would allow the field to gain cumulative knowledge. The field of modeling has almost no \u201cmeta-analysis\u201d, i.e. research combining the results from studies, largely because of a lack of standards as to procedures and measures, but also due to the lack of primary data. A comprehensive format for virtual screening information would be useful.The inclusion of multiple decoy sets of different design and intent for each target in an evaluation would, in combination with (i) and (ii) above, greatly increase the cumulative value of published studies.The number of targets, actives and inactives need to be carefully considered with respect to the purpose of the experiment and the required accuracy of the results. These can be derived from simple statistical methods that are almost never applied.The effects of correlation between actives or inactives can be generally defined as an operational quantity. This could be investigated if actives and inactives for one target were included as explicit or latent decoys for all other targets. Warren et\u00a0al. provides an example of the first, i.e. decoy sets were made from the actives of other targets. The second is an extension of point (iii), i.e. include multiple sets of decoys in a study but for different purposes. In conjunction with (ii) above, this would provide material for a rigorous analysis of operational correlation in virtual screening.Correlation between targets needs further research, in particular the question of the variance of computational methods on closely related systems.Differences between methods, especially within a single study over multiple targets, should only be reported if the effects of correlation are included. Editors of journals should never publish papers that suggest one method is better than another if these basic statistics are not employed. At a minimum it is recommended that the method variances along with correlation-corrected joint confidence limits be reported. This would allow the estimation of p-values for any assessment of method superiority.On the issue of performance metrics we propose:Deciding on the metrics to be reported should be a community effort, although access to primary data to encourage \u201cmeta-analysis\u201d would aid the autonomous adoption of metrics.There are good reasons metrics such as the AUC are popular in other fields and any new or additional measures for virtual screening need to be assessed against the characteristics that have made such metrics successful. Five characteristics required for a metric to be of similar heft to the AUC are proposed: independence to extensive variables, robustness, error bounds, no adjustable parameters and ease of interpretation. As an illustration, an improvement to the common enrichment measure is described. We propose the term \u201cROC enrichment\u201d for this new measure. Similar improvements to early measures are proposed.Currently, it would seem that providing AUCs and a few ROC enrichment values for the early part of a screen, e.g. 1% and 2%, would capture most average behavior of interest.The assumption that \u2018early\u2019 behavior is necessarily a benefit is based on an assumed cost structure that may or may not hold. Similar statements are true for virtual screening in general. A rigorous attempt to assign real-word costs would be of use to the field.We have found very little evidence that suggests average behaviors cannot be accurately predicted by AUC or obvious extensions there of. Those suggesting otherwise need to provide clear-cut, statistically valid, evidence.Divergence from (v) may be an indicator of local or domain knowledge, i.e. knowing the right answer and\/or extensive knowledge of the system under study. A potential future area of research is whether this is also an indicator of over-parameterization, posterior system preparation or other reliance on retrospective knowledge. Interestingly, 2D methods applied to DUD, showed no evidence of such a divergence.In conclusion, there is no reason it is not possible to establish standards in the field of molecular modeling necessary to enhance the quality of publications and allow a reliable assessment of methods and progress. However, there are also powerful incentives not to be rigorous. As one invested scientist was heard to pronounce, \u201clivelihoods are at stake\u201d. This is true; we suggest the livelihood of the entire field. Whether the modeling community has the will to enact such measures may well determine whether future generations of scientists look back and see a field that became essential to drug discovery or one that became a mere footnote.","keyphrases":["virtual screening","metrics","statistics","auc","roc curves"],"prmu":["P","P","P","P","P"]}
{"id":"Ann_Hematol-2-2-1705486","title":"Lenalidomide in the context of complex karyotype or interrupted treatment: case reviews of del(5q)MDS patients with unexpected responses\n","text":"Lenalidomide has particular activity in patients with transfusion-dependent del(5q) myelodysplastic syndromes (MDS), but mechanistic information is limited regarding the relationship between erythroid and cytogenetic responses. We reviewed medical records from three distinct subgroups of del(5q) MDS patients who had unexpected effects with lenalidomide treatment: 1. two patients with complex karyotypes who achieved both cytogenetic remissions and transfusion independence; 2. two patients with 5q- syndrome who took lenalidomide for less than 12 weeks but remained transfusion independent for 15+ months still displaying del(5q) metaphases after 6 and 12 months; and 3. one patient who was a non-responder on lenalidomide during treatment but became transfusion independent for 13+ months after discontinuation. All but the latter patient in this series had reduction of affected metaphases, suggesting that erythroid responses might be mediated by result from partial or complete suppression of the malignant clone, either directly or indirectly through modulation of the bone marrow microenvironment. These clinical observations illustrate the heterogeneity of del(5q)MDS pathogenesis and the diversity of lenalidomide responses within this patient subset.\nIntroduction\nMyelodysplastic syndromes (MDS) with chromosomal deletion of 5q [del(5q) MDS] are heterogeneous diseases [3, 5]. Apart from the 5q- syndrome [7, 16], patients with del(5q) MDS may present with one additional chromosomal abnormality, with multiple additional chromosomal abnormalities leading to a complex karyotype, or with an increase of bone marrow and\/or peripheral blasts irrespective of chromosomal complexity. These distinct disease subgroups have dramatically different prognostic features [3, 5, 6, 12].\nLenalidomide is a thalidomide analog with a distinct clinical profile that has demonstrated erythroid responses leading to RBC transfusion independence, particularly in del(5q) MDS [10, 11] and in some patients with Low or Int-1 MDS lacking the del(5q) chromosomal abnormality [14]. Although the exact mechanism of action has not been defined, lenalidomide is known to have multiple biological activities including anti-angiogenesis, immunomodulation, anti-cytokine, and direct toxic effects on malignant bone marrow cells [1, 4].\nWe experienced unexpected effects of lenalidomide in del(5q) MDS patients that are suggestive of the mode of action in this patient subgroup and may have implications for future use of the substance in this patient population. To our knowledge, these types of responses to lenalidomide have not been previously described.\nStudy design\nBetween November 2003 and May 2006, 43 patients with del(5q) MDS with or without additional chromosomal abnormalities were treated with lenalidomide at St. Johannes Hospital, Medizinsche Klinik II, Duisburg, Germany. As of December 27, 2005, lenalidomide has been approved for clinical use in the United States, but not in Europe. These patients received lenalidomide as participants of clinical trials or through an expanded access program. Of the cases reported in this paper, only one patient took part in a lenalidomide clinical trial, and that patient was a non-responder. Patients were informed of inclusion of their information in the present report and consent was given.\nResults and discussion\nPatients with complex karyotypes\nPatient 1\nA 59-year-old man diagnosed in October 2005 with International Prognostic Scoring System (IPSS) [9] Int-2 risk MDS, >5% bone marrow blasts (RAEB-I) [2, 17], one cytopenia, and complex karyotype: 44, XY, del (5)(q13q33), -7, -15, -18, -19, -19, +mar (15)\/46, XY(7) was transfusion-dependent and received two units of packed RBC every 2 to 3\u00a0weeks since diagnosis. He had a previous history of two myocardial infarctions, insulin-dependent diabetes mellitus, arterial hypertension, and deep venous thrombosis. Treatment with lenalidomide (10\u00a0mg p.o., once daily) commenced in December 2005 and this patient has remained on treatment as of May 2006. He experienced grade 1 neutropenia and grade 2 thrombocytopenia, which required no treatment. The time to transfusion independence was immediate; no further transfusions were required after the initiation of lenalidomide treatment. Hemoglobin was >13\u00a0g\/dl after 8\u00a0weeks, although the time to normalization of hemoglobin (normal level at local laboratory=14\u00a0g\/dl) has not been reached. Repeat cytogenetic analyses conducted in January and March 2006 showed consistent reductions in the number of abnormal metaphases (Table\u00a01). \nTable\u00a01Sequential bone marrow cytogenetic analysesPatient numberAnalysis dateKaryotype110\/200544, XY, del(5)(q13q33), \u22127, \u221215, \u221218, \u221219, \u221219, +3mar [15]\/46, XY [7]1\/200644, XY, del(5)(q13q33), \u22127, \u221215, add(18)(p11), \u221219, add(19)(q12)[8]\/46, XY [7]3\/200645, XY, del(5)(q13q33), \u22127, der(12;14)(q10;10), \u221214, \u221215, add(18)(p11), \u221219, add(19)(q12), +3mar [3]\/46; XY [17]23\/200446, XX, der(1)t(1;2)(p13;?), der(2)t(1;2)(p13;q31)del(2)(p23), del(5)(q15q31) [19]\/ 46, XX [1]12\/2004 (FISH)No evidence of 5q31 deletion6\/2005 (FISH)No evidence of 5q31 deletion1\/2006No evidence of 5q31 deletion32\/200446, XX, del(5)(q13q33) [20]5\/200546, XX, del(5)(q13q33) [12]\/ 46, XX [13]45\/200446, XY, del(5)(q13q33) [20]12\/200546, XY, del(5)(q13q33) [13]\/ 46, XY [8]51\/199646, XX, del(5)(q13q33) [15]10\/2002No change, non-mosaic pattern of del(5q)3\/200312\/20036\/200411\/200406\/2006(FISH) indicates that additional fluorescence in situ hybridization was performed.\nPatient 2\nA 78-year-old woman with IPSS Int-1 MDS [no blasts, one cytopenia, complex karyotype: 46, XX, der(1)t(1:2)(p13;?), der(2)t(1;2)(p13;q31)del(2)(p23), del(5)(q15q31)(19)\/46, XX (1)], who was diagnosed in March 2004, had an RBC transfusion requirement of two packed RBC every 8\u00a0weeks. This lady\u2019s case report has been published in an earlier issue of this journal, but with a shorter follow-up [6]. She had a previous medical history of grade 1 renal insufficiency. Treatment with lenalidomide (10\u00a0mg p.o. once daily) commenced in July 2004. The dosage was reduced to 5\u00a0mg p.o. once daily from November 2004 to November 2005, and was subsequently reduced to 5\u00a0mg p.o. every other day from November 2005 to present. The actual treatment duration was 22\u00a0months.\nAdverse events included grade 2 neutropenia, grade 2 thrombocytopenia, and grade 3 scalp itching. Whereas neutropenia and thrombocytopenia required no additional medication, scalp itching was treated with antihistamines and low doses of prednisone (10\u00a0mg) for 2\u00a0weeks. Transfusion independence was reached after 1\u00a0month and normalization of hemoglobin was reached after 4\u00a0months of treatment. Repeat cytogenetic analyses showed complete cytogenetic remission with no evidence of 5(q31) deletion in December 2004 (4\u00a0months after start of treatment), in June 2005 and in January 2006 (Table\u00a01). This del(5q) patient with a complex karyotype achieved both durable complete cytogenetic remission and long-term RBC transfusion independence.\nPatients with transfusion independence despite long-term interruption of medication\nPatient 3\nA 33-year-old woman was diagnosed in February 2004 with IPSS Low-risk MDS (no blasts, one cytopenia) and del(5)(q13q33) as the sole karyotypic abnormality, disease characteristics that are consistent with 5q- syndrome. She had a transfusion requirement of two packed RBC units every 3 to 4\u00a0weeks. Lenalidomide treatment was started in November 2004, but therapy was interrupted after 28\u00a0days for erythematous lesions of the skin and grade 4 neutropenia. Transfusion independence was reached after 8\u00a0weeks without further medication and normalization of hemoglobin was achieved after 20\u00a0weeks. Grade 1 neutropenia persisted. Repeat bone marrow cytogenetics in May 2005 showed a reduction in del(5q) metaphases (Table\u00a01). Although treatment was interrupted due to toxicity, this patient continued to improve with respect to both erythroid response and the number of del(5q) metaphases. This patient remains transfusion independent at 21\u00a0months after commencement of lenalidomide therapy and 20\u00a0months after discontinuation of therapy.\nPatient 4\nA 49-year-old man with IPSS Low-risk MDS typical of 5q- syndrome [no blasts, one cytopenia, isolated del(5)(q13q33)] was diagnosed May 2004. He was transfusion-dependent, requiring two packed RBC units every 2\u00a0weeks before the start of lenalidomide therapy in November 2004. However, treatment was stopped after 12\u00a0weeks (February 2005) because of grade 3 skin itching. Transfusion independence had been reached after 8\u00a0weeks of treatment. Normalization of hemoglobin was achieved after 16\u00a0weeks and was ongoing at last visit (21\u00a0months) despite receiving no additional treatment. Mild leukopenia has persisted. A repeat karyotype analysis in December 2005 (13\u00a0months after onset of therapy) showed a reduction in del(5q) metaphases and no evidence of cytogenetic evolution (Table\u00a01). The lengthy duration of response in this patient was notable and unexpected considering the relatively short treatment period and the persistence of the del(5q) clone.\nPatient with no response during treatment but transfusion independence after discontinuation\nPatient 5\nA 58-year-old woman with no relevant previous history was diagnosed January 1996 with IPSS Low-risk MDS (no blasts, one cytopenia) with del(5)(q13q33) as the sole karyotypic abnormality. She was transfusion-dependent, receiving two packed RBC units every 4\u00a0weeks. Low-dose cytarabine was given in December 1996, resulting in 10\u00a0months of transfusion independence, but was again given in January 1998 without effect. All-trans-retinoic acid was administered from July 2002 to January 2003 without improvement of transfusion burden. She subsequently received lenalidomide from January 2004 to November 2004 without reduction in transfusions.\nThis prolonged treatment of more than 10\u00a0months was administered in the hope of induction of a late response to the drug. Her hemoglobin value at commencement of lenalidomide therapy was 7.1\u00a0g\/dl, and she needed two red blood cell transfusions every 3\u00a0weeks to maintain these levels throughout drug intake. However, 16\u00a0weeks after lenalidomide discontinuation, transfusion independence was achieved in this patient (March 2005) and without additional measures. The response had been ongoing for 16\u00a0months, without normalization of hemoglobin (10.9\u00a0g\/dl at last visit in June 2006). Six repeat karyotype analyses from Oct 2002 to June 2006 all showed the initial finding of a non-mosaic pattern of del(5q) (Table\u00a01).\nDiscussion\nClinical trials of lenalidomide have demonstrated cytogenetic and erythroid responses in patients with del(5q) MDS [10, 11] that have not been achieved with other non-cytotoxic agents [8]. The activity of lenalidomide in patients with complex karyotypes including del(5q) represents a major advance, as those patients have an ominous prognosis and are expected to transform rapidly to acute myeloid leukemia [6]. Patient 1, who had Int-2 MDS with del(5q) and a complex karyotype, is of special interest because repetitive karyotyping showed the number of affected metaphases to be gradually reduced during lenalidomide treatment. Patient 2, who also had del(5q) with a complex karyotype, experienced both complete cytogenetic response and normalization of hemoglobin. Two other patients with complex cytogenetic abnormalities are being treated at our institution, but the duration of treatment is too short for final evaluation.\nWhereas cytogenetic remission with lenalidomide treatment correlates closely with erythroid response [10], complete suppression of the malignant clone was not required for transfusion independence in four of the five cases (patients 1, 3, 4, and 5) reported here. Hematologic remissions occurred in three patients despite treatment interruption and persistence of the del(5q) karyotype (patients 3, 4, and 5). Patient 5 had a late effect and reached transfusion independence after discontinuation of lenalidomide. The long-term hematologic responses seen with patient 3\/4 patients may be an exception, as another patient at our department had worsening of anemia 3\u00a0months after stopping lenalidomide after a short period of administration (5\u00a0weeks).\nTwo other patients at our institution had interrupted lenalidomide treatment after prolonged intake (at months\u00a024 and 13, respectively) in complete hematologic and cytogenetic remission for reasons other than an adverse event. Both patients remain in complete hematologic remission, ongoing for more than 6 and 21\u00a0months. Thus, altogether six out of 43 patients at our site have interrupted lenalidomide treatment for non-toxicity reasons. Five experienced an ongoing erythroid response and one patient relapsed after 3\u00a0months.\nAlthough all of the patients reported here have achieved transfusion independence, lenalidomide treatment does not uniformly lead to transfusion independence in del(5q) MDS. Of note, the lenalidomide MDS 003 study in del(5q) MDS patients shows that recurrence of the cytogenetic aberration or cytogenetic evolution is not an uncommon feature in this patient population during lenalidomide treatment: New chromosomal abnormalities occurred in 24 out of 119 patients, and no single type was seen in more than one patient [10]. Interestingly, chromosome 7 abnormalities occurred in only one patient, in contrast with the higher incidence in the MDS 001 study [11].\nList et al. suggested that lenalidomide may act by suppression of the del(5q) clone [11]. All but one patient in our series reported here had reduction of affected metaphases, which supports this hypothesis. However, this may not be a result of an immediate cytotoxic effect, as evidenced by the slow decrease in malignant metaphases as seen with patient 1. The other responding patients, particularly patient 5, showed a slow erythroid response more consistent with a sustained effect on the bone marrow microenvironment. Effective bone marrow modulation might, in some instances, lead to (partial) suppression of del(5q) clones. However, even if clonal suppression did not occur, normal hematopoiesis may be sufficiently strengthened to support a partial erythroid response in some patients.\nExamples of such erythroid response without alteration of the underlying cytogenetic abnormality have occurred in studies with other immunomodulating drugs like antithymocyte globulin and cyclosporine A [15], differentiation-inducing agents like all-trans-retinoic acid [8], and with cytokine treatment like darbepoietin [13]. Thus, among individual patients within the del(5q) MDS subset, lenalidomide may affect the del(5q) clone and the bone marrow microenvironment to different degrees to result in hematopoietic normalization. Further study is needed to better understand the relationship between erythroid response and cytogenetic remission with lenalidomide treatment.\nIn conclusion, although the above case reports obviously represent only selected cases out of a larger number of lenalidomide treated del(5q) MDS patients, these unusual and unexpected cytogenetic and erythroid responses suggest that some patients with complex karyotypes including del(5q) or who discontinue therapy may still benefit from lenalidomide treatment.","keyphrases":["lenalidomide","transfusion-dependent","myelodysplastic syndromes","del(5q) mds","case studies"],"prmu":["P","P","P","P","R"]}
{"id":"Psychopharmacologia-3-1-2080347","title":"Lack of evidence for reduced prefrontal cortical serotonin and dopamine efflux after acute tryptophan depletion\n","text":"Rationale Acute tryptophan depletion (ATD) is a widely used method to study the role of serotonin (5-HT) in affect and cognition. ATD results in a strong but transient decrease in plasma tryptophan and central 5-HT synthesis and availability. Although its use is widespread, the evidence that the numerous functional effects of ATD are caused by actual changes in 5-HT neuronal release is not very strong. Thus far, decreases in 5-HT efflux (thought to reflect synaptic release) were only reported after chronic tryptophan depletion or when ATD was combined with blockade of 5-HT reuptake.\nIntroduction\nA widely applied method for the study of the role of serotonin (5-HT) in cognition and affective processing is acute tryptophan depletion (ATD; Biggio et al. 1974; Fadda et al. 2000b; Fadda 2000). Through dietary depletion of the essential amino acid tryptophan, peripheral and central availability of this precursor of serotonin can be rapidly lowered (Biggio et al. 1974; Moja et al. 1989). This, in turn, leads to reduced synthesis of the monoamine 5-HT (Gessa et al. 1974) and reduced central availability of this neurotransmitter (Fadda et al. 2000b; Lieben et al. 2004a).\nSince the introduction of this method, ATD has been successfully applied in both human and rodent research and has implicated the serotonergic system in cognition (e.g. memory impairments, reduced attention and increased impulsivity) and affective processes (e.g. relapse of depressed patients, mild depressive state, aggression and irritability; Fadda 2000; Riedel et al. 2002). Various studies have, furthermore, reported ATD-induced effects on typical \u2018prefrontal\u2019 tasks, like reversal learning (Rogers et al. 1999; Finger et al. 2007) and intra- and extradimensional set shifting (Park et al. 1994), but also ATD-induced changes in prefrontal cortex (PFC) activity as measured with functional magnetic resonance imaging (Rubia et al. 2005; Allen et al. 2006; Evers et al. 2005).\nRecent literature, however, shows that the reported cognitive and behavioural effects of ATD are not always reproducible and suggests that ATD as a method for studying 5-HT depletion is particularly suitable for subpopulations that show a vulnerability to 5-HT dysregulation (Fadda 2000; Booij et al. 2003; Fusar-Poli et al. 2006; Jans et al. 2007). These subgroups include remitted depressed patients (Bremner et al. 1997; Young and Leyton 2002; Neumeister et al. 2004), females (Nishizawa et al. 1997; Schmitt et al. 2000; Booij et al. 2002; Ellenbogen et al. 1996) and subjects with certain genetic variations in the 5-HT transporter gene (Roiser et al. 2006; Finger et al. 2007). Fusar-Poli et al. (2006) furthermore suggest that the effects observed after ATD might not be mediated solely through a 5-HT-related mechanism, but rather indirectly via compensatory mechanisms of other transmitter systems (see also Praschak-Rieder et al. 2004). One candidate for this is dopamine (DA). Although direct evidence for ATD-induced effects on other transmitter systems, including DA, is lacking, there is compelling evidence that 5-HT can directly regulate dopaminergic activity and, consequently, function (for a current review, see Alex and Pehek 2007).\nOn the other hand, evidence for the effect of ATD on the actual neuronal release of 5-HT in the brain is sparse and, in most cases, indirect. Human studies are limited in their measurement to imaging studies of central 5-HT synthesis (Nishizawa et al. 1997), 5-HT receptor occupation (Udo de Haes et al. 2002; Praschak-Rieder et al. 2004) and cerebrovascular fluid measurement of tryptophan and 5-HT metabolism (Carpenter et al. 1998). Although these studies partially confirm reduced synthesis of 5-HT and do report lowered levels of the metabolite of 5-HT in cerebrospinal fluid, evidence for reduced release of 5-HT is absent.\nA number of rodent studies, in general employing the same method of ATD, suggest that in addition to reduced tryptophan availability and reduced 5-HT synthesis, the actual synaptic release of 5-HT is lowered. Evidence for the assumption that ATD does in fact lower 5-HT release is, however, inconclusive. Microdialysis studies have been able to show reduced extracellular 5-HT after ATD only in the presence of a selective serotonin reuptake inhibitor (SSRI; Fadda et al. 2000b; Bel and Artigas 1996; Stancampiano et al. 1997), while there are no reports that show decreased or unaffected 5-HT efflux in animals that are not treated with SSRIs. This is an important observation, as non-physiological blockade of serotonin reuptake can interfere with normal transmitter release, making a clear-cut interpretation of the data difficult. Furthermore, Marco and Meek (1979) show that SSRI treatment in rats can reduce synthesis, which, in turn, could augment the depletion of 5-HT pools induced by ATD (see also Carlsson and Lindqvist 1978; Moret and Briley 1997; Fuxe et al. 1982 and Yamane et al. 2001).\nIn line with the evidence for prefrontal mediated effects of ATD mentioned earlier, we examined the effect of ATD on extracellular concentrations of both 5-HT and DA in the PFC in spontaneously active, and behaviourally stimulated, animals. We aimed to investigate whether successful lowering of blood plasma tryptophan concentration with ATD leads to a reduction of medial prefrontal 5-HT efflux in the absence of a serotonergic reuptake blockade.\nMaterials and methods\nAll experiments were approved by the Animal Experimentation Committee of the Royal Netherlands Academy of Arts and Sciences and were carried out in agreement with Dutch Laws (Wet op de Dierproeven, 1996) and European regulations (Guideline 86\/609\/EEC).\nSubjects\nSubjects were 20 male outbred Wistar rats (Harlan\/CPB, Horst, The Netherlands) weighing 175\u2013200\u00a0g at arrival. Upon arrival, the animals were socially housed in groups of four in standard type IV macrolon cages where they were kept under a reversed day\/night cycle (dimmed red light from 7\u00a0a.m. until 7\u00a0p.m., white light from 7\u00a0p.m. until 7\u00a0a.m.) for the duration of the experiment. After surgery and during experimental procedures, the animals were kept in individual cages (25\u2009\u00d7\u200925\u2009\u00d7\u200935\u00a0cm).\nSurgery\nTwo weeks after arrival, the animals were subjected to surgery for placement of a jugular vein catheter and implantation of bilateral microdialysis probes. The weight of the animals at time of the surgery was 297\u2013332\u00a0g. Rats were anesthetised with intramuscular Hypnorm (0.24\u00a0mg\/kg fentanyl citrate and 7.5\u00a0mg\/kg fluanisone, Janssen); subcutaneous Dormicum (0.75\u00a0mg\/kg midazolam, Roche) was given for muscle relaxation. A silicon catheter was placed in the right jugular vein to allow for blood sampling, as described by Steffens (1969). For the subsequent implantation of the microdialysis probes, the animals were mounted in a stereotactic frame with the toothbar set at \u22122.5\u00a0mm. Bilateral probes were then placed in the medial PFC at an angle of 12\u00b0 (A\u2009+\u200930\u00a0L\u2009\u00b1\u200918; V\u2009\u2212\u200955). Both microdialysis probes and jugular vein cannula were secured to the scull with dental cement and two cranial screws. Postoperative pain reduction was achieved with Temgesic (0.10\u00a0mg\/kg buprenorphine, Schering-Plough), 0.03\u00a0ml\/100\u00a0g b.w. s.c approximately 2\u00a0h after surgery. After surgery, the animals returned to individual cages for the remainder of the experiments.\nExperimental procedure\nApproximately 1\u00a0week after surgery, the animals were assigned to either the experimental or control condition, with the body weight of the animals matched between groups. Fourteen hours before the experiment, food was removed from the home cages. On the day of the measurement, the animals were transferred to the experimentation room where they were kept in cages that contained their own bedding material. For the duration of the experiment, the animals were connected to the microdialysis tubing and blood sampling lines, which were attached to a swivel and counterbalanced beam out of reach of the animals. In this way, the animals were unrestricted in their movement, and the collection of blood samples could be achieved without handling the animals.\nThe collection of hourly blood samples started as soon as stable efflux of DA and 5-HT was observed (see below), which was approximately 2\u00a0h after connection. A first administration of the diet (see below) followed the initial blood sampling; a second administration was given 90\u00a0min after the first. Two and a half hours after the first diet administration, the rats were subjected to 20\u00a0min of \u2018novelty\u2019 stimulation.\nDuring this period, the animals were given access to an identically shaped cage, filled with new bedding material, by means of an opaque sliding door connecting the measurement cage with the \u2018novelty\u2019 cage.\nOptical movement detectors, connected to a PC running locally written software, were placed on top of the cages to detect possible changes in locomotor behaviour due to diet administration.\nAll animals were tested individually on separate days. A radio played to mask background noise and minimise interference from experimental procedures.\nTreatment\nThe experimental rats (TRP minus group) were treated orally with a protein\u2013carbohydrate mixture lacking tryptophan (TRP; 4.0\u00a0g\/kg Solugel P\u2122 and 2.0\u00a0g\/kg Maltodextrine) in a volume of 10\u00a0ml\/kg (see Table\u00a01). Two diet administrations were given, spaced 90\u00a0min apart. Control animals received the same mixture with added TRP (0.28% TRP of total protein). The exact composition of the mixture and the procedure for administration is identical to that described in Lieben et al. (2004a). To minimise stress, the rats were daily handled and habituated to oral injections with normal tap water (10\u00a0ml\/kg). To minimise TRP intake through the normal rat chow, food was removed from the home cages 14\u00a0h before the start of the experiment. Water, however, remained freely available during this period.\nTable\u00a01Composition of the nutritional mixture and determination of the amino acids content of the gelatin-based proteinMixtureValuesProtein (Solugel P\u00ae) in 100\u00a0ml tap water100Aspartic acid + asparagine5.2Glutamic acid + glutamine9.3Hydroxyproline12.1Serine3.1Glycine22.5Histidine0.5Arginine8.8Threonine1.1Alanine9.3Proline13.3Tyrosine0.4Valine2.1Methionine0.6Cysteine0.2Isoleucine1.4Leucine3.0Hydroxylysine1.4Phenylalanine1.9Tryptophan0.1Lysine3.6Carbohydrate (Malthodextrine) in 80\u00a0ml tap water50KCl0.094CaCl2\u00b72H2O2.32l-Tryptophan (Tryp minus group)0l-Tryptophan (control group)0.28The composition of the administered mixture used in the current experiment is depicted in bold. The amino acid content (%) of the protein was obtained from PB Gelatins, Tessenderlo, Belgium.\nBlood sampling and quantification of TRP content\nOn the experimental day, hourly blood samples were taken via the jugular vein catheter and analysed for TRP content. We collected six consecutive blood samples during the experiment. The first sample was taken after a stable microdialysis baseline was established and just before the first diet administration was given. Before the start of the experiment, the rats were habituated to blood sampling to minimise stress (i.e. a syringe was attached to the catheter and blood was withdrawn and immediately returned).\nBlood samples (200\u00a0\u03bcl) were collected in heparin-filled tubes and deproteinised with 5-sulfosalicylic acid (5%). After centrifuging (15\u00a0min at 3,220\u00d7g), the samples were stored at \u221280\u00b0C until analysis.\nAfter completion of the experiment, the plasma samples were thawed at 4\u00b0C, and 20\u00a0\u03bcl supernatants were transferred to a high-performance liquid chromatograph (HPLC) for analysis (Waters 600E pump and Waters 717plus autosampler, Waters Chromatography b.v., Netherlands; Jasco FP-920 intelligent fluorescence detector, Jasco, Benelux B.V., Netherlands and Shimadzu Class-vp\u2122 software v.5.03 Shimadzu Duisburg, Germany).\nThe mobile phase consisted of 0.06\u00a0mol\/l sodium acetate, 9\u00a0mmol\/l citric acid, 0.37\u00a0mmol\/l sodium 1-heptane sulfonate (HSA) and 12.5% methanol in milliQ water. The flow rate was kept constant at 1.0\u00a0ml\/min. Separation of TRP from other components was achieved with a Supelcosil column (LC-18-DB 25\u00a0cm\u2009\u00d7\u20094.6\u00a0mm\u2009\u00d7\u20095\u00a0\u03bcm), with a 2-cm guard-column of the same material (Supelco Superguard\u2122, Supelco USA), at a constant temperature of 40\u00b0C. Quantification was achieved by means of fluorescence detection, with the excitation wavelength set at 335\u00a0nm and an emission wavelength of 440\u00a0nm. TRP content was measured against a calibration curve of an external TRP standard.\nMicrodialysis probes and measurements\nConcentric dialysis probes, manufactured and routinely used in this laboratory (Feenstra and Botterblom 1996), were constructed from fused silica (i.d. 0.075\u00a0mm) protected by 25-G needles as inlet and outlet. A Hospal membrane (o.d. 0.32\u00a0mm, i.d. 0.24\u00a0mm) with 3-mm exposed length was fitted for these experiments.\nA week after surgery (Feenstra et al. 2000), the animals were transferred to the test room where the dialysis probes were connected to PEEK (polyetheretherketone)-tubing (o.d. 0.51\u00a0mm; i.d. 0.13\u00a0mm; Aurora Borealis, Netherlands) attached to a dual-channel swivel (Pronexus, Sk\u00e4rholmen, Sweden). A Univentor 801 microinfusion syringe pump delivered a ringer solution (145\u00a0mmol\/l NaCl, 1.2\u00a0mmol\/l CaCl2, 2.7\u00a0mmol\/l KCl, 1.0\u00a0mmol\/l MgCl2) at a flow rate of 1.3\u00a0\u03bcl\/min. Dialysate was directly introduced into the electrically actuated HPLC injector (Valco, VICI, C6W), and 50\u00a0\u03bcl was injected every 15\u00a0min. DA and 5-HT were separated on a Supelcosil column (LC-18-DB 25\u00a0cm\u2009\u00d7\u20094.6\u00a0mm\u2009\u00d7\u20095\u00a0\u03bcm) and detected by means of an ANTEC VT-03 electrochemical flowcell (50-\u03bcm spacer) set at +0.65\u00a0V against a Ag\/AgCl reference electrode.\nThe mobile phase consisted of 0.06\u00a0mol\/l sodium acetate, 9\u00a0mmol\/l citric acid, 0.37\u00a0mmol\/l HSA and 12.5% methanol in milliQ water. The flow rate was kept constant at 1.0\u00a0ml\/min; a pulse damper (ANTEC, Leyden) was placed with the column inside the DECADE detector unit (ANTEC) and kept at a constant temperature of 40\u00b0C.\nThe DECADE unit was programmed to automatically make an injection every 15\u00a0min and simultaneously start registration of the detector output. 5-HT and DA content was measured against a calibration curve of an external standard using Shimadzu Class-VP\u2122 software (Shimadzu, v.5.03). The detection limit (ratio signal to noise\u2009=\u20092) was 0.15\u00a0pg of 5-HT and DA injected onto the column.\nDrugs and chemicals\nThe Gelatine hydrolysate (Solugel P\u2122) was obtained from PB Gelatins (Tessenderlo, Belgium). Glucodry 200 was obtained from the Amylumgroup (Koog aan de Zaan, the Netherlands). Potassium chloride (KCl), calciumchloride\u2013dihydrate (CaCl2\u00b72H2O), sodium chloride (NaCl), sodium 1-heptane sulfonate (HSA), methanol and tryptophan were obtained from Sigma-Aldrich Chemie, Germany. 5-Sulfosalicyclic acid dehydrate and magnesium chloride hexahydrate (MgCl2\u00b76H2O) were obtained from Merck Schuchhardt. Sodium acetate and citric acid were purchased at Janssen Chimica, Geel, Belgium\nHistological examination\nAfter completion of the experiments, the rats were humanely killed by inhalation of a mixture of CO2\/O2 (70\/30) followed by 100% CO2. The brains were rapidly taken out of the skull and frozen at \u221220\u00b0C. Coronal (20\u00a0\u03bcm) sections were cut on a cryostat and stained with thionine for examination with the microscope for the precise location of the microdialysis probes.\nData analysis\nChanges in extracellular concentration of DA and 5-HT were calculated as deviations from the baseline defined as the last two samples preceding the first diet administration. Changes in plasma levels of TRP were compared to the first sample preceding diet administration. \u2018Movement-counts\u2019, in arbitrary units, registered by the optical movement detector were taken as measure for possible changes in activity. All data were analysed using a repeated measures analysis of variance (ANOVA) with time as within-factor and group\/treatment as between-factor. If indicated by the Mauchly\u2019s test of sphericity, the number of degrees of freedom was adjusted by a Huynh\u2013Feldt correction.\nIf indicated by an ANOVA group or interaction effect, subsequent evaluation of the effects of the treatment on individual samples was done by the Student Newman\u2013Keuls test with repeated measures in which all samples are mutually compared. The level of significance was p\u2009<\u20090.05.\nThe effect of novelty exposure on 5-HT and DA release and general activity during the measurement was assessed by recalculation of a baseline from the two samples before novelty exposure. The data were then analysed as described above. All data are presented as means\u00b1SEM.\nResults\nHistological examination and animal exclusion Histological sections were examined for placement of the microdialysis probes. Data from rats with incorrectly placed probes were not included in the analysis. After exclusion, both the control group and the TRP minus group included eight animals.\nPlasma concentrations Figure\u00a01 shows the change of plasma tryptophan concentrations for both the control and treated group. Due to blockade of several jugular vein cannulas, the control group included seven and the TRP minus group, five animals. Average plasma concentrations (\u03bcg\/ml) of tryptophan for the initial baseline samples were 18.80\u2009\u00b1\u200911.92 for the control group and 21.51\u2009\u00b1\u200915.64 for the TRP minus group.\nFig.\u00a01Plasma tryptophan concentration. Relative concentration (mean\u00b1SEM) of tryptophan in plasma compared to baseline. Open triangles correspond to the TRP minus group; closed squares correspond to the control group. The vertical grey bars correspond to, respectively, first diet administration, second diet administration and novelty exposure. Asterisk indicates a significant difference between the two groups, p\u2009<\u20090.05; sigma indicates a significant difference compared to the baseline value, p\u2009<\u20090.05A repeated measures ANOVA with group (TRP minus\/control) as between-subjects factor and time as within-subjects factor resulted in a main effect of time [F(4.45,44.50)\u2009=\u20093.07 p\u2009<\u20090.03] and group [F(1,10)\u2009=\u200938.76, p\u2009<\u20090.0005] as well as a group\/time interaction [F(4.45,10)\u2009=\u20098.09, p\u2009<\u20090.0005]. An additional t test revealed a significant reduction of the TRP minus group compared to the control group (time point 3: t\u2009=\u20096.00, p\u2009<\u20090.0005; time point 4: t\u2009=\u20097.66, p\u2009<\u20090.0005; time point 5: t\u2009=\u20095.76, p\u2009<\u20090.0005; time point 6: t\u2009=\u20093.15, p\u2009=\u20090.01; Fig.\u00a01). In contrast to the control group that showed an initial increase in plasma tryptophan (up to 167%) and returned to baseline values [F(5,30)\u2009=\u20093.50, p\u2009<\u20090.02], the TRP minus group showed a persistent decrease in plasma tryptophan after the second administration, with a minimum of 29% of baseline 2\u00a0h after the first diet administration [F(2.51,10.06)\u2009=\u200915.26, p\u2009=\u20090.001]. A simple contrast ANOVA for the control group revealed that time points 3 and 4 were significantly different from baseline; for the TRP minus, time points 3, 4, 5 and 6 were different from baseline.\nMicrodialysis\nMicrodialysis of 5-HT Figure\u00a02a shows the relative change, compared to two baseline samples, in 5-HT efflux over the entire day. An ANOVA with repeated measures revealed no significant group [F(1,14)\u2009=\u20090.57, p\u2009>\u20090.4], time [F(13.49,188.78)\u2009=\u20091.19, p\u2009>\u20090.2] or interaction effect [F(13.49,14)\u2009=\u20090.97, p\u2009>\u20090.4]. Average transmitter concentrations (pg\/50\u00a0\u03bcl) of serotonin for the initial baseline samples were 1.41\u2009\u00b1\u20090.27 (mean\u2009\u00b1\u2009SEM) for the control group and 1.48\u2009\u00b1\u20090.23 (mean\u2009\u00b1\u2009SEM) for the TRP minus group.\nFig.\u00a02Microdialysis of 5-HT and DA and general activity counts. Relative change in extracellular 5-HT and DA (mean\u00b1SEM) and movement compared to baseline. Open triangles correspond to the TRP minus group; closed squares correspond to the control group. a 5-HT, b DA and c movement counts. The vertical grey bars correspond to, respectively, first diet administration, second diet administration and novelty exposure. No significant changes were observedNo significant differences between treated and control group were observed during novelty exposure when values were expressed relative to the original baseline or to a recalculated baseline just before the exposure.\nMicrodialysis of DA Figure\u00a02b shows the relative change, compared to two baseline samples, in DA efflux over the entire day. An ANOVA with repeated measures revealed a significant effect of time [F(6.95,60.58)\u2009=\u20092.76, p\u2009<\u20090.02], but not of group [F(1,14)\u2009=\u20091.62, p\u2009>\u20090.2] or of interaction [F(8.14,14)\u2009=\u20090.98, p\u2009>\u20090.4]. Subsequent analysis of individual groups showed no time effect for either group.Average extracellular transmitter concentrations (pg\/50\u00a0\u03bcl) of dopamine for the initial baseline samples were 0.64\u2009\u00b1\u20090.15 (mean\u00b1SEM) for the control group and 0.72\u2009\u00b1\u20090.16 (mean\u00b1SEM) for the TRP minus group.No significant differences between treated and control group were observed during novelty exposure when values were expressed relative to the original baseline or to a recalculated baseline just before the exposure.\nMovement detection\nFigure\u00a02c shows the average number of activity counts for both the control group and the TRP minus group. Average movement counts for the initial baseline samples were 123.59\u2009\u00b1\u200917.71 (mean\u00b1SEM) for the control group and 110.38\u2009\u00b1\u200914.22 (mean\u00b1SEM) for the TRP minus group. These data support the behavioural observation of sustained general activity throughout the measurements (e.g. grooming and explorative movement).\nA repeated measures ANOVA revealed a significant effect of time over the entire day [F(10.27,143.72)\u2009=\u20092.69, p\u2009<\u20090.005]. No group [F(1,14)\u2009=\u20090.42, p\u2009>\u20090.5] or interaction [F(10.27,14)\u2009=\u20090.55, p\u2009>\u20090.8] effect was observed.\nThe novelty stimulation did not cause any differential effect on overall activity.\nDiscussion\nTo gain insight into the relation between ATD and the release of serotonin (5-HT) in the prefrontal cortex, we measured plasma levels of tryptophan and, simultaneously, PFC efflux of 5-HT and DA in animals that underwent ATD. In line with current literature (Lieben et al. 2004a), ATD induced a rapid decline of free plasma tryptophan within 2\u00a0h of the initial administration that lasted throughout the experimental procedure. However, despite this reduction, efflux of prefrontal serotonin (5-HT), as measured in the medial PFC with microdialysis, was unaffected in these animals. In contrast to previous studies, these measurements, in behaviourally active animals, were performed in the absence of a serotonergic reuptake blocker. These results indicate that successful lowering of plasma tryptophan, to levels associated with behavioural effects, does not necessarily induce lowering of 5-HT efflux. Considering the widespread use of ATD as a method for studying the role of serotonin in behavioural and cognitive research, these findings are especially relevant.\nThe current experiment combined blood sampling through a chronic jugular vein catheter with online microdialysis measurements of 5-HT and DA. This combination of techniques was used to ensure that blood sampling did not hinder the animals in their behaviour or cause unwanted effects on transmitter release.\nIn line with current literature, we measured a rapid decrease of plasma tryptophan of more than 70%. Although the possibility exists that a greater reduction of plasma tryptophan, which can be achieved with chronic tryptophan depletion (Fadda et al. 2000a), could have induced a reduction of 5-HT efflux, it would not explain the effects on behaviour and affect observed after tryptophan depletions of a magnitude similar to those observed in the current experiment (e.g. Riedel 2004; Lieben et al. 2004b; Delgado 2006). The 4.5-h time-window in which we measured is well within the period for which behavioural effects have been reported, reducing the likelihood that a decrease in 5-HT efflux would have been observed had we measured for a longer period. Lack of sensitivity of the measurement equipment does not seem likely either; with the current detection limit, reductions of either transmitter would have been measurable.\nThe absence of a clear effect of novelty stimulation on either general activity or transmitter efflux might suggest that the animals were inactive. However, behavioural observation, as well as quantitative movement detection, indicates otherwise, showing that the animals were in fact active throughout the measurements (e.g. grooming, and explorative behaviour), although the active novelty exposure did not induce the expected increase in locomotor activity.\nLiterature data from both human and rodent studies show that ATD leads to lowering of plasma tryptophan (Biggio et al. 1974; Moja et al. 1989) and decreased synthesis (Gessa et al. 1974) and availability of central 5-HT (Ashley and Curzon 1981; Fadda et al. 2000b; Lieben et al. 2004a), but, to date, it has not been convincingly shown that the ATD lowers efflux of 5-HT in the brain. The few rodent studies that do show reduced efflux after ATD either used chronic treatment with a tryptophan-deficient diet (Fadda et al. 2000b; Van der Stelt et al. 2004) or combined the measurements with drugs that prevent reuptake of 5-HT (Fadda et al. 2000b; Bel and Artigas 1996; Stancampiano et al. 1997; but see Bel and Artigas (1996) who observe an increase, rather than a decrease, of 5-HT efflux in the PFC after ATD in rats on free food). To the best of our knowledge, the current experiment is the first to report on the effect of ATD on prefrontal 5-HT efflux after overnight fasting in the absence of a reuptake blocker, an experimental design that parallels the behavioural studies in both rodents and human subjects. As pointed out in the \u201cIntroduction\u201d, this is especially important, as the addition of reuptake inhibition can lead to decreased 5-HT synthesis, and thus, augment the effect of ATD. In light of the behavioural effects observed after acute tryptophan depletion and the general notion that a reduction of synaptic 5-HT release underlies these effects, understanding the effect of ATD on 5-HT efflux, which is thought to reflect release, is essential.\nOur data seem to suggest that successful tryptophan depletion does not alter 5-HT release. ATD, however, has convincingly shown to be an effective method to manipulate mood and induce cognitive\/behavioural effects in both humans (Fusar-Poli et al. 2006; Riedel et al. 2002) and rodents (Fadda 2000; Riedel 2004). This apparent discrepancy will be discussed in the next section.\nAlthough mood effects after ATD have been reported across populations of subjects, groups with vulnerability for 5-HT dysregulation (e.g. remitted depressed patients, women and people that show a genetic variation in the 5-HT transporter gene) seem particularly sensitive to ATD (Delgado 2006; Booij et al. 2002; Moore et al. 2000; Neumeister 2003; Riedel et al. 2002; Roiser et al. 2006; Jans et al., 2007). It is argued that especially these groups are sensitive to ATD because they already show a reduced availability of central 5-HT that is further reduced by tryptophan depletion. In contrast to non-vulnerable subjects, these subpopulations will be less able to compensate for a temporary reduction in 5-HT synthesis and will experience a greater impact of ATD. Likewise, SSRI-treated animals will experience depletion of 5-HT stores (Marco and Meek 1979) and more readily show decreased 5-HT efflux after ATD than unmedicated rats. Measurements of indices of 5-HT release in response to ATD in \u2018vulnerable\u2019 rodents, like serotonin transporter knockout animals (Holmes et al. 2003), or females have not been performed.\nIn contrast to effects on mood, behavioural and cognitive effects are generally observed in a broader population that also includes healthy subjects (for a review, see Riedel et al. 2002) that do not show a particular sensitivity to 5-HT dysregulation.\nThe most parsimonious explanation that can unify those observations with the current data is that brain areas outside the medial PFC, which have not been measured in the present experiment, might mediate the behavioural effects. As the innervation of 5-HT in the brain is not uniform (McQuade and Sharp 1997; see for an overview Frazer and Hensler 1999) and some areas appear to be more sensitive to ATD than others (Lieben et al. 2004a; Fusar-Poli et al. 2006), the absence of 5-HT efflux reductions in the medial PFC might not reflect ATD-induced changes in other (prefrontal) areas.\nHowever, this explanation seems to hold only partially at best. As mentioned in the \u201cIntroduction\u201d, ATD studies have implicated prefrontal 5-HT in various \u2018prefrontal\u2019 tasks, like reversal learning (Rogers et al. 1999; Finger et al. 2007) and intra-, extradimensional set shifting (Park et al. 1994), and shown ATD-induced changes in PFC activity (Rubia et al. 2005; Allen et al. 2006; Evers et al. 2005).\nThe possibility that other transmitter systems might mediate ATD effects (Fusar-Poli et al. 2006) was investigated by simultaneous measurements of DA. As we reported that DA efflux did not respond to the ATD, the current data cannot confirm this hypothesis, although other possible transmitters remain to be investigated.\nWhile it is not possible to extrapolate these findings to brain areas outside the medial PFC, our present data show that an effective reduction of plasma tryptophan with ATD does not necessarily imply a similar reduction in 5-HT efflux. As ATD continues to be an important and widely used paradigm to study specific roles of 5-HT, it is important to clarify through which mechanism ATD exerts its effects and to what extent changes in 5-HT contribute to the functional effects of ATD.","keyphrases":["tryptophan","serotonin (5-ht)","dopamine (da)","microdialysis","medial prefrontal cortex"],"prmu":["P","P","P","P","R"]}
{"id":"Ann_Biomed_Eng-2-2-1705491","title":"Bioactive Hydrogel Substrates: Probing Leukocyte Receptor\u2013Ligand Interactions in Parallel Plate Flow Chamber Studies\n","text":"The binding of activated integrins on the surface of leukocytes facilitates the adhesion of leukocytes to vascular endothelium during inflammation. Interactions between selectins and their ligands mediate rolling, and are believed to play an important role in leukocyte adhesion, though the minimal recognition motif required for physiologic interactions is not known. We have developed a novel system using poly(ethylene glycol) (PEG) hydrogels modified with either integrin-binding peptide sequences or the selectin ligand sialyl Lewis X (SLeX) within a parallel plate flow chamber to examine the dynamics of leukocyte adhesion to specific ligands. The adhesive peptide sequences arginine\u2013glycine\u2013aspartic acid\u2013serine (RGDS) and leucine\u2013aspartic acid\u2013valine (LDV) as well as sialyl Lewis X were bound to the surface of photopolymerized PEG diacrylate hydrogels. Leukocytes perfused over these gels in a parallel plate flow chamber at physiological shear rates demonstrate both rolling and firm adhesion, depending on the identity and concentration of ligand bound to the hydrogel substrate. This new system provides a unique polymer-based model for the study of interactions between leukocytes and endothelium as well as a platform to develop improved scaffolds for cardiovascular tissue engineering.\nIntroduction\nLeukocyte adhesion to sites of inflammation is crucial to eliminate the cause of irritation and repair the surrounding tissue. After the initiation of inflammation by cytokines such as interleukin-1\u03b2 (IL-1\u03b2), tumor necrosis factor-\u03b1 (TNF-\u03b1), lipopolysaccharide, and interleukin-3, (IL-3), leukocyte velocity slows dramatically due to leukocyte contact with the vascular wall.9,27,34  Rolling, the initial transient interaction, is mediated by selectin molecules present on the activated endothelial cell surface. E-selectin is an inducible surface glycoprotein that is known to bind the carbohydrate ligand sialyl Lewis X (SLeX), which is present on various leukocytes10,11,13,35,41,43 and mediates stronger adhesions and slower rolling than P- or L-selectin.27 Following this primary contact, firm adhesion takes place via activated \u03b22-integrins binding intracellular adhesion molecule-1 (ICAM-1) expressed on the endothelium, and through the most important member of the \u03b21 integrin subfamily, very late antigen-4 (VLA-4; \u03b14\u03b21). VLA-4 binds vascular cell adhesion molecule-1 (VCAM-1), and is responsible for lymphocyte adhesion to vascular endothelium and leukocyte recruitment to the inflamed area.1,10,11,27 Though well studied, the exact mechanism of adhesion to the vascular endothelium is not known. There are two suggested mechanisms of integrin activation; one proposing that activation is a result of chemokine stimulation of leukocytes2,43 and another suggesting that selectin binding leads to integrin activation.12,42\nSeveral systems have been developed to examine the dynamics of rolling and firm adhesion and to elucidate the exact mechanism of the binding cascade. Leukocytes isolated from human blood have been studied under static and flow conditions on activated endothelial cell monolayers as well as on cells engineered to express endothelial cell adhesion molecules.3,28,37,40 Polystyrene microspheres coated with adhesion ligands have also been shown to interact with stimulated cell monolayers17 and on substrates coated with selectins and other cell adhesion molecules.11 Yeast engineered to display E-selectin has been observed rolling on substrates coated with SLeX. 2 Recently, poly(ethylene glycol) (PEG) has been tethered to gold surfaces to create cell resistant surfaces and spatial gradients of PEG on gold surfaces have been used to study the kinetics of static cell adhesion.32,33 While each of these systems has revealed new insight into the dynamics of leukocyte interactions with endothelial cell adhesion molecules, the ultimate goal must be to establish a simple, cell-free system that more closely mimics the in vivo environment.\nPEG hydrogels are crosslinked hydrophilic networks that demonstrate excellent biocompatibility, being highly resistant to protein adsorption and cell adhesion, and causing minimal inflammatory responses.20,21,22,36 These highly swollen networks have similar water content and mechanical properties to soft tissues and may be engineered to contain cell adhesion peptides, growth factors, and therapeutics for localized drug delivery.5,6,26,29,30,39,44 PEG diacrylate hydrogels may be covalently modified with cell adhesive peptide sequences to encourage cell adhesion, spreading, and migration and the interactions of cells with these hydrogels has been extensively studied under static conditions.15,16,24,25,29,31,38 A key benefit of such materials is that the base material, PEG, is intrinsically resistant to protein adsorption, so the adhesive interactions with cells are limited to the factors that are specifically engineered into the hydrogel network. In this work we propose a novel system to study leukocyte adhesion under shear using photopolymerized PEG copolymer hydrogels. After forming thin flat PEG diacrylate base hydrogels, we are able to polymerize a layer of monoacrylate PEG-peptide (or SLeX) to the surface, and using a parallel plate flow chamber, we can observe cell adhesion using video microscopy.\nMaterials And Methods\nAll chemicals were purchased from Sigma\u2013Aldrich (St. Louis, MO) unless otherwise stated.\nSynthesis of Polyethylene Glycol Diacrylate\nPolyethylene glycol diacrylate (PEG-DA) was synthesized by dissolving 12\u00a0g dry PEG (MW: 6000; Fluka, Milwaukee, WI) in 16\u00a0ml anhydrous dichloromethane (DCM) with an equimolar amount of triethylamine and 0.72\u00a0g acryloyl chloride (Lancaster Synthesis, Windham, NH) added dropwise. The mixture was stirred under argon for 24\u00a0h, washed with 2\u00a0M K2CO3, and separated into aqueous and DCM phases to remove HCl. The DCM phase was dried with anhydrous MgSO4 (Fisher Scientific, Pittsburgh, PA), and the PEG diacrylate was then precipitated in diethyl ether, filtered, and dried under vacuum at room temperature overnight. The resulting polymer was dissolved in N,N-dimethylformamide-d7 and characterized via proton NMR (Avance 400\u00a0MHz; Bruker, Billerica, MA) to determine the extent of acrylation.\nSynthesis of PEG Derivatives Containing Cell Adhesion Molecules\nThe cell adhesive peptide sequence used in this study include arginine\u2013glycine\u2013asparagines\u2013serine (RGDS; American Peptide Company, Inc., Sunnyvale, CA), and a peptide containing the cell adhesive leucine\u2013asparagines\u2013valine sequence (glycine\u2013proline\u2013glutamic acid\u2013isoleucine\u2013leucine\u2013asparagines\u2013valine\u2013serine\u2013threonine, GPEILDVST), which was synthesized using standard fluorenylmethoxycarbonyl (Fmoc) chemistry on an Applied Biosystems 431A peptide synthesizer (Foster, CA). The high affinity 4-((N\u2032-2-methylphenyl)ureido)-phenylacetyl-leucine-aspartic acid-valine-proline (Bio1211; Commonwealth Biotechnologies, Inc., Richmond, VA)5,6 was also used as an alternate LDV-containing compound. The non-adhesive sequences used as negative controls were arginine\u2013glycine\u2013glutamic acid\u2013serine (RGES; American Peptide Company, Inc., Sunnyvale, CA) and glycine\u2013proline\u2013glutamic acid\u2013isoleucine\u2013leucine\u2013glutamic acid\u2013valine\u2013serine\u2013threonine (GPEILEVST), also synthesized using Fmoc chemistry on a peptide synthesizer.\nPeptides were conjugated to PEG monoacrylate by reaction with acryloyl-PEG-N-hydroxysuccinimide (PEG-NHS; MW 3400; Nektar Therapeutics, Huntsville, AL) in 50\u00a0mM sodium bicarbonate (pH 8.5) at a 1:1 molar ratio for 2\u00a0h. The mixture was then dialyzed (MWCO 1000), lyophilized, and stored at \u221220\u00b0C. Gel permeation chromatography with UV and evaporative light scattering detectors (Polymer Laboratories, Amherst, MA) was used to determine the coupling efficiency.\nThe selectin ligand sialyl Lewis X (SLeX) was conjugated to PEG using an avidin\u2013biotin bridge. PEG-NHS (25\u00a0mg) was reacted with a lysine\u2013biotin conjugate (biocytin) at a 1:2 molar ratio in 50\u00a0mM sodium bicarbonate (pH 8.5) for 2\u00a0h. SLeX-biotin (500\u00a0\u03bcg; Glycotech, Gaithersburg, Maryland) was reacted with avidin (10\u201315\u00a0units\u00a0mg\u22121) at a ratio of 2\u00a0units avidin per mole of SLeX-biotin in 50\u00a0mM sodium bicarbonate (pH 8.5) for 2\u00a0h. The reaction mixtures were then combined to allow further conjugation of avidin and biotin, thereby linking acryloyl\u2013PEG\u2013NHS\u2013lysine\u2013biotin to avidin\u2013biotin\u2013SLeX.\nSynthesis of Bilayered PEG Copolymer Hydrogels\nHydrogels were formed by first dissolving 0.2\u00a0g\u00a0ml\u22121 PEG diacrylate in 10\u00a0mM HEPES buffered saline (HBS, pH 7.4); the polymer solution was then filter sterilized using a 0.22\u00a0\u03bcm filter (Gelman Sciences, Ann Arbor, MI). The photoinitiator 2,2-dimethoxy-2-phenyl acetophenone in N-vinylpyrrolidinone (300\u00a0mg\u00a0ml\u22121) was added at 10\u00a0\u03bcl\u00a0ml\u22121 polymer solution. This mixture was injected between rectangular glass plates separated by 0.5\u00a0mm spacers and polymerized under UV light (365\u00a0nm, 10\u00a0mW\u00a0cm\u22122) for 30\u00a0s. The top plate was removed and the hydrogel surface rinsed with sterile PBS. A second layer, consisting of 5\u00a0\u03bcmol\u00a0ml\u22121 of either acryloyl-PEG-peptide or acryloyl-PEG-SLeX in HBS and 10\u00a0\u03bcl\u00a0ml\u22121 2,2-dimethoxy-2-phenyl acetophenone in N-vinylpyrrolidinone was then layered on top of the PEG diacrylate base gel, the upper glass plate replaced, and the second layer photopolymerized by exposure to UV light (365\u00a0nm, 10\u00a0mW\u00a0cm\u22122) for 1\u00a0min.\nCell Maintenance\nJURKAT cells (human T-lymphocytes; ATCC, Manassas, VA) were maintained in RPMI-1640 prepared with 10% fetal bovine serum (FBS; BioWhitaker, Walkersville, MD), 2\u00a0mM l-glutamine, 1\u00a0unit\u00a0ml\u22121 penicillin, and 100\u00a0mg\u00a0l\u22121 streptomycin (GPS). 300.19\/E cells (mouse pre-B lymphoblast; ATCC, Manassas, VA) were sustained in RPMI-1640 prepared with 10% FBS, 1% GPS, and 0.1\u00a0mM 2-mercaptoethanol. Cells were maintained at 37\u00b0C in a 5% CO2 environment.\nCell Adhesion and Rolling on Adhesive PEG Gels\nFlow assays were performed using a circular parallel plate flow chamber (Glycotech, Gaithersburg, MD) mounted on the stage of a Zeiss Axiovert 135 microscope (Carl Zeiss Inc., Thornwood, NY). The chamber was placed on top of photopolymerized PEG copolymer hydrogels and vacuum sealed to the surface using a portable vacuum pump (Fisher Scientific, Pittsburgh, PA) as shown in Fig.\u00a01. Cell suspensions were drawn through the flow field (1\u00a0cm path width, 0.01 in thickness) using a programmable syringe pump (BS-8000 Multi-Phaser\u2122 Programmable Syringe Pump, Braintree Scientific Inc., Braintree, MA) at varying flow rates corresponding to a shear stress range of 3.5\u201335\u00a0dynes\u00a0cm\u22122, which is comparable to in vivo shear rates. Cellular interactions with the hydrogels were monitored using a Nikon CoolPix 5000 camera (Nikon Inc., Melville, NY) and transferred to videotape for further analysis.\nFigure\u00a01.Schematic of parallel plate flow chamber system. A small pump is used to create a vacuum, sealing the chamber on the surface of the gel. Cells are then perfused over the gel surface using a programmable syringe pump.\nCation Dependent Binding\nJURKAT cells were treated with 2\u00a0mM magnesium (Mg2+), calcium (Ca2+), or manganese (Mn2+) or with 10\u00a0mM EDTA and then perfused through the flow chamber and allowed to settle on the gel for 5\u00a0min. Controls were exposed to standard formulations of RPMI-1640 containing 0.4\u00a0mM Ca2+ and 0.4\u00a0mM Mg2+. Ten fields of view were scanned to get an average number of cells per field of view. Flow rates corresponding to shear stresses of 0.5, 1.0, and 10\u00a0dynes\u00a0cm\u22122 were used to wash away unbound cells. The number of cells remaining for each shear stress was counted and averaged over several fields of view.\nLDV Specificity\nJURKAT cells treated with 2\u00a0mM Mg2+ were allowed to settle on the LDV gel for 5\u00a0min and an average number of cells per field of view was determined. Specificity was demonstrated by the addition 7\u00a0\u03bcg\u00a0ml\u22121 of either a mouse anti-human monoclonal antibody that blocks VLA-4 binding to VCAM-1 (anti-CD49d, clone BU49; Ancell Corporation, Bayport, MN) or an IgG1 isotype control (purified mouse myeloma IgG1; Invitrogen Corporation, Carlsbad, CA) at a shear stress of 0.5\u00a0dynes\u00a0cm\u22122. The average number of cells bound per field of view was again counted to determine the amount of cells remaining bound to the surface.\nSpecificity was also demonstrated by the addition of a solution of either 10\u00a0mM EDTA or 150\u00a0\u03bcM Bio1211 introduced into the flow chamber under a shear stress of 0.5\u00a0dynes\u00a0cm\u22122. An average number of cells bound to the gel under flow was determined every minute for 13\u00a0min.\nVideo Analysis\nCells were allowed to settle on each gel for 5\u00a0min. An average of 10 fields of view was scanned and the number of cells settled on the peptide gel was counted. After flow began, fields of view were scanned again and the number of cells remaining (bound to the gel) was counted. After flow was established on the SLeX gels, video was paused and the number of interacting cells was counted. The numbers were averaged over 10 fields of view for each shear stress.\nStatistical Analysis\nData were compared with two-tailed, unpaired t-tests; p-values less than 0.05 were considered to be significant.\nResults\nSynthesis of PEG Hydrogels\nPEG hydrogels were formed under UV light in the presence of a photoinitiator between two glass plates. Hydrogels 0.5\u00a0mm thick were formed after 30\u00a0s of exposure. The addition of an acryloyl-PEG-peptide derivative mixed with the photoinitiator to the surface of the hydrogels resulted in a covalently bound layer of cell adhesive moiety on the surface (Fig.\u00a01).\nQuantification of Cell Adhesion and Rolling\nCell rolling on SLeX hydrogels was quantified over a range of shear stresses, and 86.8\u00a0\u00b1\u00a011.6 cells per field of view rolled on the gel surface at 3.5\u00a0dynes\u00a0cm\u22122 with an average rolling velocity of 141.3\u00a0\u03bcm\u00a0s\u22121 (Fig.\u00a02). Rolling decreased with increasing shear stress (7.0\u00a0dynes\u00a0cm\u22122: 15.2\u00a0\u00b1\u00a03.8 cells, average rolling velocity 232.7\u00a0\u03bcm\u00a0s\u22121; 14\u00a0dynes\u00a0cm\u22122: 8.2\u00a0\u00b1\u00a01.9 cells, average rolling velocity 486.8\u00a0\u03bcm\u00a0s\u22121; 21\u00a0dynes\u00a0cm\u22122: no rolling observed).\nFigure\u00a02.300.19\/E cells rolling on SLeX modified gel. 300.19 cells transfected with human E-selectin was perfused over a SLeX modified gel. The number of rolling cells were counted per field of view. SLeX ability to support rolling decreased as shear stress increased (*p\u00a0<\u00a00.02 as compared to shear rate of 3.5\u00a0dyne\u00a0cm\u22122).\nApproximately 98.5\u00a0\u00b1\u00a018.6% of cells contacting hydrogel surfaces modified with Bio1211 adhered to the gel surface. In comparison, 41.5\u00a0\u00b1\u00a013.4% of cells in contact with the surface of PEG-LDV, an interaction specific for the \u03b14\u03b21 integrin expressed on JURKAT cells, adhered to the hydrogels and 23\u00a0\u00b1\u00a08.5% of cells contacting PEG-RGDS hydrogels adhered to the gel surface (Fig.\u00a03). No cells adhesion to PEG-DA or PEG-RGES hydrogels was observed; however the control LEV peptide-modified gels had 1.6\u00a0\u00b1\u00a01.2% of cells contacting the surface adhere.\nFigure\u00a03.Peptide ability to bind VLA-4. JURKAT cells were allowed to settle on PEG-DA gels conjugated with different peptides at similar densities. After 5\u00a0min, cells were subjected to a shear stress of 0.5\u00a0dynes\u00a0cm\u22122 to remove unbound cells and the remaining cells were counted (*p\u00a0<\u00a00.02 compared to Bio1211).\nCation Dependant Binding\nCation sensitivity was assessed by the addition of cations or EDTA to cell cultures prior to their exposure to PEG-LDV hydrogels. The presence of 10\u00a0mM EDTA had the greatest inhibitory effect on binding, followed by 2\u00a0mM Ca2+. Mg2+ and Mn2+ had little effect on the ability of JURKAT cells to bind LDV, and a higher number of cells exposed to both calcium and magnesium were able to bind to the gel surface than those exposed to calcium alone (Fig.\u00a04).\nFigure\u00a04.Cation dependent binding of VLA-4 to LDV peptide. JURKAT cells were incubated with 2\u00a0mM cations or 10\u00a0mM EDTA prior to settling on the LDV gel; controls were exposed to standard formulations of RPMI-1640 containing 0.4\u00a0mM Ca2+ and 0.4\u00a0mM Mg2+. The percentage of cells on the gel was determined based on the total number of cells before initiating a shear stress of 0.5\u00a0dynes\u00a0cm\u22122 (*p\u00a0<\u00a00.05 compared to Control).\nLDV Specificity\nJURKAT cell binding to PEG-LDV hydrogels was reversed upon the addition of a monoclonal anti-VLA-4 (3.4\u00a0\u00b1\u00a02.1% bound). Cells exposed to an isotype-matched control antibody were still able to adhere to the hydrogel surface (88.8\u00a0\u00b1\u00a033.4% bound), demonstrating specificity of LDV for the VLA-4 integrin. The higher affinity of Bio1211 compared to the LDV peptide was also observed by the addition of unbound Bio1211 after cells were allowed to settle on PEG-LDV hydrogels (Fig.\u00a05). Bio1211 was able to remove cells from the gel surface better than EDTA through competitive binding of VLA-4.\nFigure\u00a05.LDV Specificity for VLA-4. 10\u00a0mM EDTA or 1.5\u00a0uM Bio1211 was introduced into the flow chamber at a wall shear stress of 0.5\u00a0dynes\u00a0cm\u22122 after JURKAT cells were allowed to settle on the LDV gel for 5\u00a0min. Bio1211, which has a higher affinity for VLA-4 was able to compete the cells off the LDV modified gel surface. EDTA was not as effective as Bio1211 as cells detached more slowly and not completely. The dashed line represents the uninhibited controls, in which 40\u00a0\u00b1\u00a015.2% of the cells initially exposed to the hydrogel remained bound through the duration of the experiment. Each data point represents the average percent of bound cells per 10 fields of view and error bars represent the standard deviation of the percent of bound cells within those 10 fields of view.\nThe influence of peptide concentration on cell adhesion was determined by varying the amounts of LDV bound to the hydrogel surface (Fig.\u00a06). The number of cells bound to the gel increased with LDV concentration, and LDV was better able to support adhesion at lower shear stresses for all concentrations when compared to higher rates of shear.\nFigure\u00a06.Dilution effect of LDV. The number of JURKAT cells bound to the gel decreased as the LDV peptide concentration was lowered. LDV was better able to support adhesion at lower shear stresses for all concentrations (*p\u00a0<\u00a00.05 comparing shear rates at each concentration).\nDiscussion\nThe system of hydrophilic hydrogels modified with cell adhesion peptides implemented in this study exhibits the ability to mimic the cell adhesion cascade that occurs during the onset of inflammation in the vascular system. The capacity to modify hydrogels with cell adhesion molecules for the study of cellular interactions with biomaterials for drug delivery and tissue engineering is well documented.4,7,15,16,18,24\u201326,29 \u201331,38,39,44 This study implements the use of these materials under physiological flow conditions to examine the mechanisms of leukocyte rolling and firm adhesion.\nThe formation of thin flat PEG diacrylate hydrogels for use with a parallel plate flow chamber improves upon earlier systems that utilize hydrophobic surfaces with adsorbed cell adhesion molecules. The highly crosslinked structure of swollen PEG networks have water content similar to native vessels, and covalent incorporation of cell adhesive peptides guarantees control of concentration and allows for patterning of one or more adhesion sequences on the gel surface.23 This system is based on a flexible substrate with tunable stiffness, the properties of which can be exploited to examine the responses of different cell types in microenvironments that mimic native tissues in various states of development, remodeling, regeneration, and disease. It has been suggested that the differentiation of cellular function and response could depend significantly on matrix elasticity,8,14,45 and varying the polymer composition or concentration in the hydrogel can alter the permeability and mechanical properties,36 simulating a range of biologically relevant conditions. In addition, the local mobility of adhesive ligands in the solvated hydrogel system should be considerably better than on solid substrates, allowing more variable orientations which could contribute to a greater fraction of accessible ligands available to receptors on the cell surface.\nLeukocyte cell adhesion to the surfaces of modified PEG-DA hydrogels was highly specific, reversible, and sensitive to ligand site density and affinity, demonstrating the efficacy of the system to mimic the events leading to the firm adhesion of immune cells on activated endothelium. The slow rolling of 300.19\/E cells on the surfaces of PEG-SLeX hydrogels confirms the integration of SLeX into the system through the use of an avidin\u2013biotin linkage, which did not disrupt the active binding site of the carbohydrate. Incorporation of the RGD and LDV peptides encouraged firm cell adhesion to the materials surface under shear in a concentration dependant manner, and cells remained adherent throughout the duration of the flow experiment. These results illustrate that this method of studying leukocyte adhesion succeeds in mimicking the interactions seen on the in vivo vascular wall under shear. With further optimization, such as incorporation of signaling molecules, this system using of PEG gels can improve insight into the mechanisms of rolling and firm cell adhesion at sites of active vascular disease.\nConclusions\nThe covalent modification of PEG hydrogel surfaces with cell adhesive peptides was accomplished in continuous layers of adhesive regions. These materials encouraged vascular cell adhesion through both transient interactions with selectin molecules and firm binding via integrins. The system presented here to expose adhesive hydrogels to cells under flow conditions represents a method to study cell\u2013material interactions in environments that closely mimic in vivo environments.","keyphrases":["hydrogel","flow chamber","leukocyte adhesion","poly(ethylene glycol)"],"prmu":["P","P","P","P"]}
{"id":"Pflugers_Arch-3-1-2137943","title":"A refined radio-telemetry technique to monitor right ventricle or pulmonary artery pressures in rats: a useful tool in pulmonary hypertension research\n","text":"Implantable radio-telemetry methodology, allowing for continuous recording of pulmonary haemodynamics, has previously been used to assess effects of therapy on development and treatment of pulmonary hypertension. In the original procedure, rats were subjected to invasive thoracic surgery, which imposes significant stress that may disturb critical aspects of the cardiovascular system and delay recovery. In the present study, we describe and compare the original trans-thoracic approach with a new, simpler trans-diaphragm approach for catheter placement, which avoids the need for surgical invasion of the thorax. Satisfactory overall success rates up to 75% were achieved in both approaches, and right ventricular pressures and heart and respiratory rates normalised within 2 weeks. However, recovery was significantly faster in trans-diaphragm than in trans-thoracic operated animals (6.4 \u00b1 0.5 vs 9.5 \u00b1 1.1 days, respectively; p < 0.05). Stable right ventricular pressures were recorded for more than 4 months, and pressure changes, induced by monocrotaline or pulmonary embolisms, were readily detected. The data demonstrate that right ventricular telemetry is a practicable procedure and a useful tool in pulmonary hypertension research in rats, especially when used in combination with echocardiography. We conclude that the described trans-diaphragm approach should be considered as the method of choice, for it is less invasive and simpler to perform.\nIntroduction\nOver the last decade, new medical treatments became available for the treatment of pulmonary hypertension (PH) patients, and this development set off a renewed interest in the pathophysiology and haemodynamics of the pulmonary circulation [6, 12, 22]. Experimental PH studies are still largely confined to single measurements of pulmonary arterial (PA) or right ventricular (RV) pressure in anaesthetised, open-chest animals undergoing artificial respiration, which does not allow determining changes of pressures in time. Measuring pulmonary haemodynamics have improved considerably since RV telemetry was firstly introduced by Hess et al. [4], as it allows for continuous monitoring of RV or PA pressures, without introducing artefacts caused by stress or anaesthetics [9]. Moreover, animals fitted with telemetry can serve as their own controls, thus permitting the use of smaller groups of animals than is possible with the single measurement approach in anaesthetised animals [9]. Although many investigators have embraced this new technology with enthusiasm and despite its proven potential, studies actually using this methodology in rats are scarce [4, 13, 16, 18, 19].\nOne possible explanation for this discrepancy is that the original procedure was only described briefly before [4], complicating its implementation for potential new users. Secondly, the described trans-thoracic approach involves RV catheterisation through complex and highly invasive thoracotomy [4]. In the present study, we hypothesised that RV catheterisation performed via an opening in the diaphragm, thereby omitting a thoracotomy, may be a simpler, less invasive alternative.\nIn the present report, we provide a detailed description of the alternative trans-diaphragm approach together with a comparison with the original trans-thoracic approach. Furthermore, we illustrate its usefulness when studying the pulmonary circulation, by presenting novel comprehensive pulmonary and cardiac haemodynamic data during the development of monocrotaline-induced pulmonary hypertension.\nMaterials and methods\nAll experiments were approved by the Institutional Animal Care and Use Committee of the VU University and were conducted in accordance with the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes, and the Dutch Animal Experimentation Act.\nAnimals\nThirty-eight male Wistar rats were used (250\u2013300\u00a0g; Harlan, Horst, The Netherlands), of which 33 were operated and 5 served as controls (no surgery). The animals were allowed to adapt to their new environment for at least 1\u00a0week before surgery. The animals were conventionally housed in pairs under controlled conditions (temperature 21\u201322\u00b0C; humidity 60\u201365%; 12:12\u00a0h light\u2013dark cycle) and had free access to filtered water and standard rat chow (Global 2016, Harlan Teklad, Blackthorn-Bicester, England).\nTelemetry system\nAn implantable radio-telemetry system for blood pressure measurements for small laboratory animals was used, comprising a radio-transmitter (TA11PA-C40) fitted with a 10-cm-long catheter (Data Science International [DSI], St. Paul, MN). Transmitters were turned on a day before implantation and stored in sterilised saline, following the manufacturer\u2019s instructions (http:\/\/www.datasci.com\/information\/index.asp).\nThe pressure was monitored continuously during surgery to ensure proper position of the tip of the pressure catheter. After successful implantation of the transmitter, RV or PA pressures and locomotor activity were recorded for 10\u00a0s every 5\u00a0min. From the pressure recordings mean, systolic and diastolic pressure, heart rate, respiratory rate and circadian rhythm were subsequently derived (Dataquest A.R.T. software 4.0, DSI).\nPre-operative care and anaesthesia\nAll animals received pre-surgical intramuscular buprenorphine analgesia (0.10\u00a0mg\/kg; Schering-Plough, Maarssen, The Netherlands). For general anaesthesia, isoflurane (2.0% in 1:1 O2\/air mix; Pharmachemie, Haarlem, The Netherlands) was used via induction chamber and tracheal intubation (16\u00a0G\u2009\u00d7\u200951-mm Teflon tube; ventilator settings: breathing frequency 80\/min, pressures: 9\/0\u00a0cmH2O, inspiratory\/expiratory ratio 1:1). For extra local analgesia, lidocaine spray (100\u00a0mg\/ml; AstraZeneca, Zoetermeer, The Netherlands) was applied at the surgical region. Animals were maintained under anaesthesia for an average time of 80\u00a0min.\nAnimals were placed on a heating pad to maintain body temperature and positioned in dorsal recumbency. Hydromellose drops (0.3%; Ratiopharm, Zaandam, The Netherlands) were applied to prevent drying of the eyes. After shaving and disinfection with 70% ethanol of the chest and abdomen, animals were covered with sterile incision foil and the animals were then covered with sterile incision foil (Opraflex, Lohmann & Rauscher, Almere, The Netherlands). A surgical microscope was used (magnification \u00d716\u201364; Carl Zeiss, Sliedrecht, The Netherlands) for optimal view.\nSurgery: trans-thoracic approach\nImplantation of the telemetry transmitter in the abdomen\nThe abdominal cavity was accessed via a 5-cm midline laparotomy, starting just below the xiphoid process. The transmitter was placed in the peritoneal cavity, with the catheter pointing caudally to prevent liver injury and tissue reaction. The abdominal cavity was covered with gauzes soaked in warm saline and left open until the end of the procedure.\nRouting of the pressure catheter to the right ventricle\nThe heart was exposed by means of a left thoracotomy performed at the sixth intercostal space and mid-clavicular line. The thorax was opened with blunt scissors, respecting the anatomy of the overlying muscle layers and cautiously avoiding injury to the lungs. The pressure catheter was then tunnelled subcutaneously from the peritoneal cavity to the opening in the thorax and temporarily laid aside. Four individual small hooks were used to retract the ribs and expose the heart through a wider, 2\u2009\u00d7\u20092-cm window. The pericardium was then opened with two dressing forceps.\nCatheterisation of the right ventricle\nA superficial purse string (6\u20130 Prolene, Ethicon, St-Stevens-Woluwe, Belgium) was placed on the right ventricle free wall, near the apex. Through the purse string, the right ventricle was punctured with a 19\u00a0G syringe needle in the direction of the RV outflow tract, avoiding any coronary vessel. After removing the needle, the site was wiped with a sterile cotton stick. The tip of the catheter was then carefully inserted into the right ventricle, using a vessel canulation forceps (0.5\u20131.0\u00a0mm outer diameter, Fine Science Tools, Heidelberg, Germany), avoiding loss of gel from the tip at all times. Live pressure waveform trace confirmed proper RV catheterisation (RV systolic\/diastolic pressures are typically \u223c25\/1\u00a0mmHg). Depending on the protocol, the pressure catheter was subsequently advanced for about 2\u00a0cm more, positioning the tip of the catheter beyond the pulmonary valves. Again, live pressure waveform trace confirmed correct positioning of the pressure catheter in the pulmonary artery (PA systolic\/diastolic pressures are typically \u223c25\/10\u00a0mmHg; Fig. 1, left panel). Finally, the catheter was fixed by the purse string and a small drop of tissue adhesive at the site of insertion (10\u00a0\u03bcl dispensed by a pipette; Vetbond, 3M, St. Paul MN; Electronic Supplementary Material (ESM) Fig. E-1a).\nFig.\u00a01left panel: Live pressure recordings while advancing the pressurecatheter into the pulmonary artery. Notice the stepwise increase in diastolic pressures when manoeuvring the catheter beyond the pulmonary valves (arrow), whereas the systolic pressures remained unchanged. right panel: Live pressure recordings while injecting a bolus injection of microspheres (arrow; 19\u00a0\u03bcm, 1.5 million\/100\u00a0g i.v.). Upon embolisation, a significant increase in systolic, diastolic and developed RV pressures was observed. Furthermore, in both recordings, the effect of breathing is noticeable\nClosing of the chest and abdomen\nAfter the catheterisation, blood clots if any were removed from the pleural cavity with a moist cotton stick. The ribs were approximated with two individual sutures (5\u20130 Vicryl, Ethicon). To promote full expansion of the lungs, a small burst of positive pressure was applied (maximally 10\u00a0cmH2O for 1\u00a0s). A chest tube (18\u00a0G), used as air outlet, was removed after proper thorax excursions were observed. The chest muscles were laid back in layers (no suturing necessary). Then, the transmitter was sutured to the abdominal muscle (5\u20130 Perma-Hand, Ethicon) incorporating the suture rib of the device. The abdominal wall was sutured in a running subcuticular pattern (5\u20130 Vicryl). Finally, the skin of the chest and abdomen was closed with a running suture (5\u20130 Vicryl).\nSurgery: trans-diaphragm approach\nOpening of abdomen and the diaphragm\nThe abdominal cavity was accessed via a 5-cm midline laparotomy, with the abdominal wall retracted by two individual hooks. To expose the diaphragm, a retraction suture was used to lift the xiphoid process during the whole procedure. The visceral organs were covered temporarily with wet gazes, and the liver was gently pushed down by the weight of a blade holder. A small midline incision in the diaphragm was then made, with small sharp scissors, from the xiphoid process until the tendinous part of the diaphragmatic membrane. The two sides of the diaphragm were held aside with two retraction sutures, providing optimal exposure of the heart (ESM Fig. E-1b).\nCatheterisation of the right ventricle\nRV catheterisation was performed similarly, as described above. In short, the pericardium was opened by two dressing forceps, and a purse string was placed just right of the apex. The pressure catheter was then inserted into the right ventricle through a small puncture made with a syringe needle and fixed in place.\nClosing the diaphragm and abdomen\nAfter catheterisation, blood clots in the thorax, if any, were removed. The diaphragm was closed with a running suture (5\u20130 Vicryl), starting from the ventral side, with the pressure catheter sticking out the diaphragm at the dorsal end of the incision. After that, the retraction suture through the xiphoid process was removed. To promote full expansion of the lungs, a small burst of positive pressure was applied, as described above, with the chest tube between the sutures of the diaphragm, until proper thorax excursions were visually confirmed. To further secure closure of the diaphragm, a small amount of tissue adhesive (Vetbond, 3M) was applied. Finally, the transmitter was implanted in the peritoneal cavity, fixed to the abdominal wall with the catheter facing caudally, and the abdomen was closed, as described above.\nPost-operative care\nTo compensate for loss of fluids, all animals received warm sterile saline at the end of the procedure (5\u00a0ml intraperitoneal). After final skin closure, the animals were allowed to regain consciousness.\nIn the first 24\u00a0h of recovery, the animals were housed in individual cages. Each cage was placed halfway on a heating pad, in such a way that half of the cage was maintained above it and the other at room temperature. During this period, the animals were monitored three times, received post-surgical analgesia when clinically indicated (buprenorphine 0.10\u00a0mg\/kg subcutaneous) and were provided with drinking gel pads and softened rat chow. After 24\u00a0h, the animals were conventionally housed in pairs for a full recovery, and they were inspected and weighted daily. Animals were considered fully recovered from surgery when their appearance and behaviour were normal, their surgical wounds healed, their pre-surgical body weight regained, when the pressures normalised and circadian rhythm was restored. The study period was ended after 4\u00a0months.\nExperimental protocol\nTo induce acute and chronic pulmonary hypertension, four animal received an intravenous bolus injection of microspheres [25] (tail; 1.5 million \/100\u00a0g body mass in sterile saline; polystyrene, mean diameter 19\u00a0\u03bcm (#7520), Duke Scientific, Fremont, CA) and three animals monocrotaline subcutaneous [24] (MCT; 80\u00a0mg\/kg in sterile saline; Sigma-Aldrich, Zwijndrecht, The Netherlands), respectively. Experimental interventions were performed no earlier then 2\u00a0weeks after the surgery.\nRV echocardiography\nAfter a 2-week recovery, trans-thoracic echocardiographic measurements (ProSound SSD-4000 system equipped with a 13-Mhz linear transducer [UST-5542], Aloka, Tokyo, Japan) were performed on anaesthetised but spontaneously breathing rats (isoflurane 2.0% in 1:1 O2\/air mix), according to the standards of the American Society of Echocardiography [10, 15]. To minimise the effects of isoflurane on cardiac function, the time under anaesthesia was set to a maximum of 15\u00a0min [14].\nThe measurements were compared with untreated rats to check for possible adverse effects related to the procedure. Cardiac output, tricuspid annular plane systolic excursion (TAPSE), RV wall thickness (RVWT) and RV end diastolic diameter (RVEDD) were measured, as described before by Hardziyenka et al. [3]. TAPSE is a parameter for RV systolic function, which is measured in the apical four-chamber view of the heart and expresses the displacement of the lateral tricuspid annulus towards the apex during systole [10].\nEstimation of pulmonary vascular resistance and RV power output\nAdditional echocardiographic measurements were performed in MCT-treated rats and untreated but operated controls, just before the injection, 2\u00a0weeks after injection and when the first clinical signs of RV heart failure developed (as indicated by weight loss, dyspnoea and lethargy, after which the rats were euthanised). Pulmonary vascular resistance (PVR) and RV power output (RV-power) were estimated, by combining telemetric pressure data and echocardiographic flow data.\nPVR was estimated by Poiseuille\u2019s law: [PVR]\u2009\u2248\u2009[mean PAP]\/[cardiac output]\u2009\u2248\u2009(0.61\u2009\u00d7\u2009[systolic RV pressure]\u2009+\u20092\u00a0mmHg)\/[cardiac output] [1, 23].\nRV-power was estimated using a simplified pressure\u2013volume analysis, assuming rectangular pressure\u2013volume loops: [RV-power]\u2009\u2248\u2009[systolic RV pressure]\u2009\u00d7\u2009[stroke volume]\u2009\u00d7\u2009[heart rate]\u2009=\u2009[systolic RV pressure]\u2009\u00d7\u2009[cardiac output] [21, 23].\nAutopsy\nTo investigate potential effects of chronic implantation of the telemetry transmitters, the animals were euthanised at the end of the study protocol by exsanguination under isoflurane (4.0% in 1:1 O2\/air mix). Macroscopic examination of right ventricle, lungs, diaphragm and intercostal muscles were performed, and organ weights (heart, lungs, liver, spleen, kidneys) were measured and compared with controls. Animals treated with MCT or microspheres were excluded from these analyses.\nStatistical analyses\nAll measurements, unless otherwise stated, are presented as mean \u00b1\u2009standard error of the mean. Normal distribution was verified, and Student\u2019s t test, analysis of variance or Fisher Exact test were used, when appropriate (SPSS 13.0, SPSS, Chicago IL). A p value less than 0.05 was considered statistically significant.\nResults\nSuccess rate\nSeventeen animals were operated using the trans-thoracic approach, and 16 animals were operated using the trans-diaphragm approach.\nIn the trans-thoracic group, 8 animals showed stable pressure signals for the whole study period of 4\u00a0months. Five animals did not recover form surgery, and 4 animals developed instable pressure signals, caused by clot formation inside the pressure catheter within 2\u00a0weeks of recovery. Of the 9 failed procedures, 4 occured in the first four attempts.\nIn the trans-diaphragm group, 9 animals showed stable pressure signal for the whole study period of 4\u00a0months. Six animals did not recover form surgery, and one animal developed instable pressure signals, caused by clot formation inside the pressure catheter within 2\u00a0weeks of recovery. As in the trans-thoracic group, 4 of the failures occurred in the first four attempts.\nBy discarding the first 4 animals in each procedure, as we consider this the learning curve, the overall success rate was 8 of 13 (62%) for the trans-thoracic approach and 9 of 12 (75%) for the trans-diaphragm approach.\nRecovery from surgery\nWithin a week post-surgery, heart and respiratory rates and circadian rhythm normalised in all rats, and no differences were observed between the two approaches (Table\u00a01). Although all animals regained their pre-surgical body weight within 2\u00a0weeks, full recovery was significantly faster in the trans-diaphragm- than the trans-thoracic-operated animals (6.4\u2009\u00b1\u20090.5 vs 9.5\u2009\u00b1\u20091.1\u00a0days, respectively; p\u2009<\u20090.05).\nTable\u00a01Main results of this study, all values in mean\u2009\u00b1\u2009SEM\u00a0Trans-thoracic approach (n\u2009=\u200917)Trans-diaphragm approach (n\u2009=\u200916)Controls (n\u2009=\u20095)Success ratea8\/13 (62%)7\/9 (79%)n.r.Recovery\u00a0BW to pre-surgical level (days)d9.5\u2009\u00b1\u20091.16.4\u2009\u00b1\u20090.5*n.r.\u00a0Heart rate (bpm)e362\u2009\u00b1\u20094.4359\u2009\u00b1\u20095.7350\u2013400b [8, 17]\u00a0Respiratory rate (rpm)e90\u2009\u00b1\u20092.491\u2009\u00b1\u20093.880\u2013100b [11, 17, 20]\u00a0Circadian rhythm (days)e3.4\u2009\u00b1\u20090.43.7\u2009\u00b1\u20090.6n.r.Pressurese\u00a0RVSP (mmHg)25\u2009\u00b1\u20091.124\u2009\u00b1\u20090.921\u201326b [16]\u00a0RVDP (mmHg)1.7\u2009\u00b1\u20090.32.2\u2009\u00b1\u20090.21\u20135c [5]RV echocardiographyd\u00a0Cardiac output (ml\/min)107\u2009\u00b1\u20094.9116\u2009\u00b1\u20096.9110\u2009\u00b1\u20094.9\u00a0TAPSE (mm)3.6\u2009\u00b1\u20090.13.7\u2009\u00b1\u20090.23.4\u2009\u00b1\u20090.1\u00a0RVWT (mm)1.0\u2009\u00b1\u20090.11.0\u2009\u00b1\u20090.10.9\u2009\u00b1\u20090.1\u00a0RVEDD (mm)3.6\u2009\u00b1\u20090.13.7\u2009\u00b1\u20090.13.6\u2009\u00b1\u20090.1Autopsyf, organ weights (g)\u00a0Heart1.4\u2009\u00b1\u20090.11.4\u2009\u00b1\u20090.21.3\u2009\u00b1\u20090.1\u00a0Lungs1.5\u2009\u00b1\u20090.11.5\u2009\u00b1\u20090.21.5\u2009\u00b1\u20090.2\u00a0Liver15.4\u2009\u00b1\u20090.515.0\u2009\u00b1\u20090.615.1\u2009\u00b1\u20090.5\u00a0Spleen0.65\u2009\u00b1\u20090.030.68\u2009\u00b1\u20090.040.64\u2009\u00b1\u20090.04\u00a0Kidneys2.4\u2009\u00b1\u20090.22.3\u2009\u00b1\u20090.22.3\u2009\u00b1\u20090.1n Number of animals, n.r. not relevant, BM body mass, RVSP RV systolic pressure, RVDP RV diastolic pressure, TAPSE tricuspid annular plane systolic excursion, RVWT RV wall thickness, RVEDD RV end diastolic diameter*p\u2009<\u20090.05aFirst four attempts were discarded.bNormal values for Wistar rats, awake and at rest, measured by radio-telemetrycNormal values for Wistar rats, measured during acute pressure measurements under anaesthesiadAnalyses on surviving animals onlyeAnalyses on successfully operated animals only (stable signal)fAnalyses on surviving and untreated animals only (no microspheres or monocrotaline)\nRV pressures normalised in all animals within 2\u00a0weeks after surgery (RV systolic\/diastolic pressures \u223c25\/1\u00a0mmHg), irrespective of the method used (Table\u00a01). PA systolic\/diastolic pressures were estimated to be \u223c25\/10\u00a0mmHg (Fig.\u00a01, left panel). Activity had no significant effect on the stability of the pressure signals.\nEchocardiography revealed normal cardiac output (\u223c110\u00a0ml\/min), TAPSE (\u223c3.5\u00a0mm), RVWT (\u223c1.0\u00a0mm) and RVEDD (\u223c3.6\u00a0mm) in all rats, and no differences were seen between the differently operated animals and controls (Table\u00a01). Furthermore, other signs of RV dysfunction, like pericardial effusion and tricuspid regurgitation, were not present in any animal.\nAcute pressure effects of pulmonary embolisation by microspheres\nRV telemetry could readily detect acute changes in RV pressure, induced by a bolus injection of microspheres (Fig.\u00a01, right panel). Upon embolisation, systolic RV pressures increased from 21\u2009\u00b1\u20091 to 38\u2009\u00b1\u20093\u00a0mmHg (p\u2009<\u20090.01) and diastolic RV pressures increased from 2\u2009\u00b1\u20091 to 7\u2009\u00b1\u20092\u00a0mmHg (p\u2009<\u20090.05).\nHaemodynamic changes during the development of MCT-induced pulmonary hypertension\nChanges in haemodynamic parameters during the development of MCT-induced PH are shown in Fig.\u00a02 (also in ESM Fig. E-2). Four weeks after MCT injection, the first clinical signs of RV heart failure developed. During this period, there was a gradual rise in RV systolic pressure, from 22\u2009\u00b1\u20092\u00a0mmHg at baseline to 38\u2009\u00b1\u20094\u00a0mmHg at 2\u00a0weeks and 72\u2009\u00b1\u20095\u00a0mmHg at 4\u00a0weeks (p\u2009<\u20090.01 vs control). At 4\u00a0weeks, cardiac output dropped dramatically, from 100\u2009\u00b1\u20097\u00a0ml\/min at baseline to 107\u2009\u00b1\u20098 at 2\u00a0weeks and 52\u2009\u00b1\u20094\u00a0ml\/min at 4\u00a0weeks (p\u2009<\u20090.01 vs control). In addition, the poor haemodynamic condition of the MCT-treated rats at 4\u00a0weeks was confirmed by a simultaneous decline in TAPSE from 3.6\u2009\u00b1\u20090.1\u00a0mm during the first 2\u00a0weeks to 1.8\u2009\u00b1\u20090.2\u00a0mm at 4\u00a0weeks (p\u2009<\u20090.01 vs control). During the development of PH, the right ventricle remodelled initially by increasing RVWT (1.0\u2009\u00b1\u20090.1\u00a0mm at baseline, 1.1\u2009\u00b1\u20090.1\u00a0mm at 2\u00a0weeks and 1.3\u2009\u00b1\u20090.1\u00a0mm at 4\u00a0weeks, respectively; p\u2009<\u20090.01 vs control) but subsequently by increasing RVEDDs as well (3.5\u2009\u00b1\u20090.1\u00a0mm at baseline, 3.6\u2009\u00b1\u20090.1\u00a0mm at 2\u00a0weeks and 7.0\u2009\u00b1\u20090.5\u00a0mm at 4\u00a0weeks, respectively; p\u2009<\u20090.01 vs control).\nFig.\u00a02Haemodynamic changes during the development of MCT-induced pulmonary hypertension. Four weeks after the rats received MCT, clinical signs of RV heart failure developed. In these 4\u00a0weeks, systolic pressures continued to rise, while cardiac output could not be maintained. The presence of RV remodelling was indicated by increased wall thickness and diameter. The pulmonary vascular resistance continued to rise as well, while at 4\u00a0weeks, the increase in the RV-power was inadequate to compensate for the dramatically increased afterload. Triangles connected by dashed lines: MCT-treated; squares connected by uninterrupted lines: control group; n\u2009=\u20093 for both groups. All values are in mean\u2009\u00b1\u2009SEM, Asterisk, p\u2009<\u20090.05; double asterisk, p\u2009<\u20090.01 compared to control (post-hoc analyses). Please note that sometimes the icons conceal the error bars. For explanation of the abbreviations, see corresponding text\nDuring the development of PH, PVR increased continuously, from 1.2\u2009\u00b1\u20090.2\u2009\u00d7\u2009109\u00a0N m\u22125 s at baseline, to 1.9\u2009\u00b1\u20090.2\u2009\u00d7\u2009109\u00a0N m\u22125 s at 2\u00a0weeks and 7.9\u2009\u00b1\u20090.9\u2009\u00d7\u2009109\u00a0N m\u22125 s at 4\u00a0weeks (p\u2009<\u20090.01 vs control). RV-power was increased at 2\u00a0weeks, but no further increase was seen at 4\u00a0weeks (RV-power\u2009=\u20094.9\u2009\u00b1\u20090.7\u00a0mW at baseline, 9.1\u2009\u00b1\u20090.9\u00a0mW at 2\u00a0weeks, 8.3\u2009\u00b1\u20090.7\u00a0mW at 4\u00a0weeks, respectively; p\u2009<\u20090.01 vs controls).\nAutopsy\nChronic implantation of the telemetry transmitter in both approaches did not result in any differences in organ weight as compared to control (Table\u00a01), and no apparent pulmonary embolisms were seen.\nAutopsy revealed only local fibrosis of the right ventricle near the insertion point of the catheter (ESM Fig. E-3a). In the trans-thoracic approach, animals showed normal healing of the intercostal muscles. In the trans-diaphragm approach, no herniation or dehiscence of the diaphragm were seen in any animal, and often, the liver was partially accreted to the healed wound of the diaphragm (ESM Fig. E-3b).\nDiscussion\nThe present study describes in detail two surgical techniques to monitor RV or PA pressures over time by radio-telemetry in rats. To our best knowledge, we are the first research group to describe the trans-diaphragm approach and its comparison with the previously used trans-thoracic approach. We have demonstrated that: (1) in our hands, both the trans-diaphragm as well as the trans-thoracic approach have satisfactory success rates, especially when considering the complexity of the procedures, (2) the time-to-recovery was significantly shorter with the trans-diaphragm approach than the trans-thoracic approach, (3) measured physiological parameters recovered fully in both methods, and there were no permanent detrimental effects on RV, intercostal muscles or diaphragm, (4) using RV-telemetry, acute and chronic pressure changes in the pulmonary circulation can be readily detected and (5) RV-telemetry, in combination with echocardiography, allow thorough monitoring of pulmonary and cardiac haemodynamic changes over time.\nFully recovered animals showed normal RV structure, as expressed by RVWT and RVEDD, and normal function, as expressed by cardiac output and TAPSE. In addition, in both approaches, RV pressure normalised shortly after surgery, and autopsy revealed only minor fibrosis in the direct proximity of the catheter. Therefore, permanent detrimental effects of RV-telemetry on the RV can be excluded for both techniques. In addition, examination of other organs revealed no abnormalities, as was described by others [4]. In animals operated using the trans-diaphragm approach, macroscopic examination revealed normal wound healing of the diaphragm. Although we have not tested the strength of the healed diaphragm, others did not find significant differences between a normal (untouched) and a sutured diaphragm [7]. The natural history of small defects in the diaphragm is relatively benign, as they almost always heal spontaneously [2], especially when the diaphragm is fixated by other organs [26]. Furthermore, the observation of a normal respiratory rate seen in the recovered animals confirms that the trans-diaphragm approach is safe and a good alternative for the trans-thoracic approach.\nIn our experience, insertion of the pressure catheter into the RV and its advance further into the pulmonary artery when desired is comparatively easy to perform in the trans-diaphragm approach. This relative ease of RV catheterisation is reflected in the higher success rate compared to the trans-thoracic approach and might be well explained by the more caudal and inferior direction by which the catheter enters the heart. Approaching the heart in this manner entails minimal manipulation and manoeuvring of the catheter during the process of RV catheterisation, resulting in a lower incidence of clot formation inside the catheter. This complication occurred more frequently in trans-thoracic-operated animals as a direct consequence of excessive manoeuvring and accidental squeezing of the catheter causing it to lose or displace some of its anti-thrombolitic gel. The strongest advocate of the trans-diaphragm approach over the trans-thoracic approach, however, is the significantly faster recovery of the animals. Prompt recovery from surgery is particularly important in rats with MCT-induced PH because of low survival rates beyond 4\u00a0weeks after MCT injection.\nIn this study, we used echocardiography to validate RV-telemetry in vivo. Additionally, through the combination of pressure data, obtained by RV-telemetry, with echo-Doppler flow data, we were able to study the haemodynamics of the pulmonary circulation in greater detail [23]. MCT induced pulmonary and cardiac remodelling, at the same time as PVR, RVWT and RVEDD increased. Cardiac adaptation to overcome the increasing afterload became inadequate by the fourth week of MCT injection, in such that there was no further increase in RV-power to maintain adequate cardiac output while RV systolic pressure continued to rise. Detailed studies of these observations are planned for the near future.\nIn the present study, we have focussed on the refinement of the RV-telemetry technique in rats, but this does not exclude its applicability in mice and other rodents. Indeed, in their recent study, Schwenke et al. [18] reported long-term monitoring of PA pressure in mice using radio-telemetry.\nIn conclusion, we described a new, easier-to-perform, mildly invasive trans-diaphragm-based RV-telemetry approach for long-term monitoring of PA and RV pressures in the rat model of MCT-induced PH. Our findings may be applied to improve our understanding of the disease processes involved in PH and develop better treatment strategies for the disease.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nElectronic Supplementary Material\nM.L. Handoko et al. A refined radio-telemetry technique to monitor right ventricle or pulmonary artery pressures in rats. Pflugers Arcg \u2013 Eur J Physiol 2007. Fig. E-1, E-2, E-3. (PDF 292\u00a0kb)","keyphrases":["pulmonary artery","diaphragm","heart","respiration","circulation","blood pressure","cardiac output","power output","hypertensive rats","vascular remodelling"],"prmu":["P","P","P","P","P","P","P","P","R","R"]}
{"id":"J_Comp_Physiol_A_Neuroethol_Sens_Neural_Behav_Physiol-4-1-2248214","title":"Far field scattering pattern of differently structured butterfly scales\n","text":"The angular and spectral reflectance of single scales of five different butterfly species was measured and related to the scale anatomy. The scales of the pierids Pieris rapae and Delias nigrina scatter white light randomly, in close agreement with Lambert\u2019s cosine law, which can be well understood from the randomly organized beads on the scale crossribs. The reflectance of the iridescent blue scales of Morpho aega is determined by multilayer structures in the scale ridges, causing diffraction in approximately a plane. The purple scales in the dorsal wing tips of the male Colotis regina act similarly as the Morpho scale in the blue, due to multilayers in the ridges, but the scattering in the red occurs as in the Pieris scale, because the scales contain beads with pigment that does not absorb in the red wavelength range. The green\u2013yellow scales of Urania fulgens backscatter light in a narrow spatial angle, because of a multilayer structure in the scale body.\nIntroduction\nMany butterflies have vivid, colourful wing patterns, created by rows of partly overlapping scales with intricate spatial structures that scatter incident light. The scattered light can interfere coherently or incoherently. When the scale structures have spatial periodicity, the interference of coherent light waves often results in striking iridescences. The displayed colours are then called structural or physical. Without periodicity, light scattering is incoherent or random, resulting in white scales, unless they contain pigment that selectively absorbs in a certain wavelength range. In the latter case a pigmentary or chemical colour results (Fox and Vevers 1960; Vukusic and Sambles 2003; Kinoshita and Yoshioka 2005). Many butterfly species feature structural as well as pigmentary colours (Rutowski et al. 2005; Yoshioka and Kinoshita 2006a).\nEach butterfly wing scale is the cuticular product of a single cell, with a rather flat, unstructured lower scale lamina and a highly structured upper lamina, typically consisting of longitudinal ridges connected by crossribs (Ghiradella 1998). The fine structure of butterfly scales is highly variable (Vukusic et al. 2000). For instance, the crossribs of the white scales of pierids are adorned with granules (Yagi 1954; Giraldo and Stavenga 2007; Morehouse et al. 2007), the ridges of Morpho scales and many male Pieridae are elaborated into multilayer structures (Ghiradella et al. 1972; Vukusic et al. 1999; Kinoshita et al. 2002), and many papilionids and lycaenids have scales with photonic crystal properties (Vukusic et al. 2002; Vukusic and Sambles 2003; Kertesz et al. 2006).\nThe scales reflect only part of the incident light flux, and the remaining part is transmitted unless it is absorbed by pigment. Because the scales are arranged in layers on both sides of the wing, incident light suffers reflection and transmission in each layer of the scale stacks, and therefore the wing reflectance is not solely due to backscattering by the top layer, the cover scales, but it is the cumulative effect of the scale stacks on both wing sides. Yoshioka and Kinoshita (2006b) investigated this phenomenon in Morpho wings, and to explain the reflectance spectra of intact wings from the spectra of individual scales they used a simplified scale stack model, assuming that normally incident light rays are also reflected and transmitted normally. Stavenga et al. (2006) developed a more general model for the reflectance of butterfly wings to interpret reflectance measurements performed on intact as well as partly or completely denuded wings of the small white butterfly, Pieris rapae. The basic assumption of the latter model, that the scales scatter randomly, was however not specifically demonstrated.\nKnowledge of the spatial and spectral reflectance characteristics of single scales is essential to further develop our understanding of the coloration principles applied by butterflies. In the present study we investigate the scattering properties of wing scales of a variety of butterflies, and we correlate the scattering diagrams with the electron micrographs of the scale structure. We show that white scales of pierid butterflies are approximately random scatterers. Iridescent scales, with multilayer structures, exhibit directional reflection. Whereas some butterfly species appear to have scales with dominant iridescence, other species combine iridescence and scattering properties.\nMaterials and methods \nAnimals \nWe investigated the scales of a variety of butterflies. The small white, P. rapae, was obtained from a continuous culture maintained by Dr J. J. van Loon, Entomology Department, University of Wageningen (the Netherlands). The black jezebel, Delias nigrina, was captured near Bateman\u2019s Bay, Australia. The Morpho aega was purchased. The purple tip, Colotis regina, was received from the butterfly collection of the Royal Museum for Central Africa, Brussels (Belgium; curator Dr U. Dall\u2019Asta). The moth Urania fulgens was a gift from Dr Marta Wolff, Entomology group, University of Antioquia (Medell\u00edn, Colombia).\nAngular distribution of scattering by single scales\nSingle scales were isolated by gently pressing the wing of a butterfly on a glass plate. Subsequently, an isolated scale was glued to the tip of a glass micropipette (tip diameter ca. 5\u00a0\u03bcm). The micropipette was then mounted on a micromanipulator in the optical setup shown in Fig.\u00a01a. The scale was always hanging with the longitudinal ridges of the scale oriented vertically. Light from a xenon lamp was focused on a pinhole with diameter 50\u00a0\u03bcm, which was subsequently imaged on the scale. The light beam, which had an aperture of <10\u00b0, passed a small hole in a screen before it reached the scale. The scale reflected (that is, back-scattered) part of the incident light. The angular spread of the reflected light was documented by photographing the light distribution at the white backside of the screen (Fig.\u00a01a). We used a Nikon Coolpix 990 (Fig.\u00a02) and an Olympus DP70 (Fig.\u00a06). The digital images were processed with MatLab (Fig.\u00a06).\nFig.\u00a01Diagram of the optical system used for measuring the angular distribution of the scale reflectance. Light from a light source was focused on a pinhole, which was imaged on the scale. a A white screen with a small hole was placed in between the imaging lens and the scale. The light reflected by the scale caused a light pattern on the screen, which was photographed. b The light reflected by the scale was collected by a lightguide, which relayed the captured light to a spectrometer. The lightguide was mounted on a motorized stage rotating in the horizontal plane (top view drawing) Fig.\u00a02Angular and spectral reflectance of isolated butterfly scales. a\u2013d White scale of the dorsal forewing of a male Pieris rapae. e\u2013h White scale from the dorsal forewing of a male black jezebel, Delias nigrina. i\u2013l Blue scale of a Morpho aega. m\u2013p Purple scale from the tip of the dorsal forewing of a male purple tip, Colotis regina. q\u2013t Orange\u2013white scale of Urania fulgens. First column: scanning electron microscopy of scale cross-sections made by cutting pieces of wing with a razor blade. Bars 1\u00a0\u03bcm. Second column: photographs of the angular distribution of light reflected by a scale and subsequently reflected by a white screen, where the scale was illuminated with a narrow beam of white light (see Fig.\u00a01a). The scales were glued at the tip of a glass micropipette, which was suspended from above on a micromanipulator; see the black shadow in the upper part of b and f. The bright spot at the right of the central black hole is due to light forward scattered by the scale. The ellipses in b (due to the slightly oblique position of the camera) represent directional angles in steps of 10\u00b0, from 10\u00b0 to 60\u00b0, as well as that for 65\u00b0. The hole in the screen is ca. 8\u00b0. Third column: angular dependence of the reflectance in the horizontal plane, measured with the setup of Fig.\u00a01b. Fourth column: reflectance spectra of single scales measured with a microspectrophotometer \nSpectrophotometry \nThe angular dependence of the light scattering by the scale was measured with a lightguide, which was mounted on a rotating motor and connected to a spectrometer (Yoshioka and Kinoshita 2006b). The scales were adjusted so that the scale plane was perpendicular to the light beam, as judged by the symmetrical reflection pattern with respect to the axial direction. The experiments were run using a Labview interface, which allowed the measurement of reflectance spectra in angular steps of 1\u00b0 over a 180\u00b0 angle (Fig.\u00a01b). Angular reflectance curves were calculated for a series of wavelengths with 10\u00a0nm interval by sequentially averaging the reflectance spectra over 10\u00a0nm wavelength ranges. In addition, the spectral reflectances of single scales were measured with a microspectrophotometer, consisting of a xenon light source, a Leitz Ortholux microscope, and a fiber optic spectrometer. The microscope objective was an Olympus 20\u00d7, NA 0.46. A white reflectance standard (Spectralon, Labsphere, North Sutton, NH, USA) served as the reference in all cases.\nElectron microscopy \nSubsequent to the microspectrophotometry, the single scales were prepared for scanning electron microscopy (SEM). Additionally, pieces of wing were cut and put on the SEM-holder in different positions to visualize the upper surface as well as cross-section of the scales. Samples were sputtered with palladium for 5\u00a0min with 800\u00a0V and 200\u00a0mTorr (Hummer, Alexandria, VA, USA). A Philips XL-30 scanning electron microscope with a voltage of 3\u00a0kV was used to investigate the scale anatomy. For transmission electron microscopy, wing pieces were prepared as usual. In brief, samples were embedded in agar for better handling, prefixed in 2% glutaraldehyde in 0.1\u00a0M sodium cacodylate buffer, and postfixed in 1% OsO4\/1.5% K4 Fe(CN)6 in 0.1\u00a0M cacodylate. Subsequent washing with double-distilled water and dehydration with an alcohol series that ended with 100%, were followed by propylene oxide during 30\u00a0min and embedding in Epon. Post-microtomed samples were contrasted with uranyl acetate in methanol for 2\u00a0min and lead in water for 1\u00a0min, and were then examined with a Philips 201 transmission electron microscope.\nResults\nThe white scales of the small white, P. rapae, are marked by ovoid beads that adorn the crossribs (Fig.\u00a02a). The angular light scattering of a single scale can be readily visualized with the setup of Fig.\u00a01a, where a beam of white light is focused on a scale via a small hole in a screen. Figure\u00a02b is a photograph of the screen, showing the angular distribution of the light reflected by a white scale taken from the dorsal wing of a male small white, P. rapae. The scale scatters light approximately circular-symmetrically, suggesting that the scale acts as a diffuse scatterer.\nThe angular distribution of the scattering was quantitatively investigated with the setup of Fig.\u00a01b, where the light reflected by the scale is measured as a function of angle in the horizontal plane. Figure\u00a02c presents the reflectance of the scale of Fig.\u00a02b as a function of angle for a number of wavelengths, normalized to the maximal reflectance value; the angle is 0\u00b0 for the normal to the scale. The scale\u2019s reflectance spectrum for normally incident light is given in Fig.\u00a02d. The reflectance is high in the visible wavelength range, but it is low in the ultraviolet, because of an ultraviolet-absorbing pigment, presumably leucopterin (Wijnen et al. 2007), which is concentrated in pigment granules, the ovoid beads (Fig.\u00a02a). The beads act as strong scatterers at wavelengths where the pigment absorption is negligible (Stavenga et al. 2004; Giraldo and Stavenga 2007; Morehouse et al. 2007). In addition to the beads, the other structures of the scale, that is the ridges and crossribs of the upper lamina of the scale as well as the lower lamina, contribute to the scattering (Fig.\u00a02c).\nFigure\u00a02e\u2013h presents a similar set of data for a white scale of the dorsal forewing of a male black jezebel, D. nigrina. This case is obviously very similar to that of the white P. rapae scale. The scale anatomy with crossribs studded with beads is very similar (Fig.\u00a02e), the scattering is again approximately random (Fig.\u00a02f, g), and the reflectance spectrum is also high in the visible and low in the UV (Fig.\u00a02h).\nThe strikingly blue M. aega has scales where the lamellae of the ridges form multilayers (Fig.\u00a02i). Different from Morpho didius, for example, which has glass cover scales and strongly pigmented ground scales (Vukusic et al. 1999), cover and ground scales of M. aega cannot be distinguished. The scattering diagram of a scale of a M. aega is a horizontal stripe (Fig.\u00a02j), perpendicular to the vertically oriented scale ridges. The angular dependence of the reflectance varies strongly with the wavelength (Fig.\u00a02k), and the reflectance measured with normally incident light features a distinct peak in the blue (Fig.\u00a02l), which is due to the multilayered structure of the scale ridges (Fig.\u00a02i; see also Vukusic et al. 1999; Kinoshita et al. 2002; Yoshioka and Kinoshita 2004). A reflectance peak value of more than two results, because the scale\u2019s scattering is highly directional and the reflectance was measured relative to the diffusely scattering white standard (Fig.\u00a02l).\nThe purple scales at the dorsal wing tips of the male purple tip, C. regina, have ridges that are fine-structured similarly as in the case of M. aega (Fig.\u00a02m). The purple scale features a scattering diagram with a blue stripe and a red diffuse pattern (Fig.\u00a02n), which is a mixture of the stripe phenomenon of Fig.\u00a02j and the diffuse patterns of Fig.\u00a02b, f. The blue stripe is due to light backscattered by the fine-structured ridges, and the red diffuse pattern results from randomly scattered light, filtered by a pigment contained in granules below the multilayered ridges (Fig.\u00a02m). The blue peaking reflectance spectrum, shown in Fig.\u00a02p, is mainly due to reflection by the ridges, and the red band, above 600\u00a0nm, is mainly caused by the light scattering pigment granules (Fig.\u00a02p).\nA green\u2013yellow reflecting scale of the moth U. fulgens has between the upper and lower scale laminae (Fig.\u00a02q) an elaborate multilayer system, which yields a spatially restricted, directional scattering diagram (Fig.\u00a02r, s). The high amplitude of three of its reflectance spectrum is again due to the directionality of the scale reflectance (Fig.\u00a02t). The reflectance spectrum features a distinct band, peaking at 590\u00a0nm, with halfwidth about 120\u00a0nm, indicating that an interference reflector is involved.\nTo evaluate whether the beaded scales of P. rapae, D. nigrina and C. regina act as diffuse scatterers, the angular reflectances of these species were normalized and compared with a Lambertian reflector (Fig.\u00a03). The wavelengths selected are outside the absorption bands of the pigments, which means that the wavelengths chosen for P. rapae and D. nigrina are in the visible spectrum, but for C. regina only in the red wavelength range. The white scale of D. nigrina (Fig.\u00a03b) well approximates a Lambertian reflector. The angular distribution patterns of the white scale of P. rapae slightly deviate from the ideal curve (Fig.\u00a03a), but notably the purple scale of C. regina is not a perfectly diffused red scatterer. Presumably this is due to the highly structured, multilayered ridges.\nFig.\u00a03Normalized angular reflectances measured from the scales of the three pierid butterflies, P. rapae, D. nigrina, and C. regina, compared to a Lambertian reflector (bold line). Angular reflectance curves are shown in the wavelength range where the reflectance is high, that is, where pigment absorption is low. Four wavelengths covering the visible range are shown for P. rapae (a) and D. nigrina (b). The scattering diagrams of D. nigrina well approximate a Lambertian reflector, but the angular dependence of the scattering by C. regina shows regular peaks, which indicates that the light scattered by the pigment granules is affected by the multilayered ridges\nDiscussion\nThe relationship between the optical properties of butterfly scales and their structure has been the topic of several studies (e.g., Vukusic et al. 1999, 2002; Kinoshita et al. 2002). Most of the previous investigations have focused on iridescent scales. Here, we have compared the reflection pattern of five differently structured single butterfly scales that scatter light coherently or incoherently, or both.\nWe started with a simple white scale common to many species of the pierid subfamily Pierinae. Due to the characteristic beaded structure light scattering is strong, thus causing the intense white colour. We find that the white pierid scales approximate the properties of a Lambertian diffuser, at least in the wavelength range where pigment absorption is negligible (Fig.\u00a03).\nThe ridges of the scales of Morpho butterflies cause a blue colour. Melanin pigment below the multilayered ridges absorbs stray light over the whole visible wavelength range, including the ultraviolet, thus supporting the strikingly blue wing colour (Yoshioka and Kinoshita 2006a). The pigment of the scales in the dorsal wing tips of C. regina also absorbs stray light, but not in the long-wavelength range. The remaining red light together with the blue iridescence causes the purple colour.\nA similar, however, short-wavelength-shifted case is formed by the yellow scales of the dorsal wings of many male butterflies of the pierid subfamily Coliadinae, where a UV and blue absorbing pigment is combined with UV iridescence (Ghiradella et al. 1972; Rutowski et al. 2005). Figure\u00a04a is a transmission electron micrograph of a yellow cover scale from the dorsal wing of a male brimstone, Gonepteryx rhamni. The UV iridescent, yellow scales of the male brimstone have numerous beads, which contain the yellow pigment xanthopterin (Wijnen et al. 2007). The beads are seen in Fig.\u00a04a as empty ovoids, but this is presumably due to the procedures for transmission electron microscopy (Morehouse et al. 2007).\nFig.\u00a04Transmission electron microscopy images of a cover scale of the dorsal wing of a male brimstone, Gonopteryx rhamni (a), and of a scale of Urania fulgens (b). Ridges are indicated by closed arrows. The brimstone scale has multilayers restricted to the ridges. The space between ridges and crossribs is studded with pigment granules (open arrow). U. fulgens has continuous multilayers connected by numerous pillars that result in spreading of the scattering. Bar: 1\u00a0\u03bcm\nThe scattering diagram of U. fulgens is not perfectly directional (Figs.\u00a02r, s), which should have been the case when the scales consisted of an ideal multilayer. Transmission electron microscopy shows that between the multilayers exist pillars (Fig.\u00a04b), which presumably cause the spread in the scattering diagram. Of course, the scales\u2014and therefore the multilayers\u2014are also not perfectly flat. The ridges (see Figs.\u00a02q, 4b) will further contribute to some diffuse scattering.\nKnowing how light is scattered by individual scales is necessary for a proper understanding of butterfly wing coloration. As has been previously demonstrated, wing reflectance is a cumulative effect due to multiple reflection and transmission by the layers of scales on the butterfly wing (Yoshioka and Kinoshita 2006b; Stavenga et al. 2006). The scales are often arranged in quite regular rows and have approximately the same orientation with respect to the wing surface, but of course there are deviations from perfect order. This will be rather irrelevant in the case of the small white, P. rapae, where scattering is approximately diffuse, but in cases where scattering is highly directional, as in Morpho butterflies, disordered scale arrangement will result in broadening of the scattering diagrams. This is illustrated in Fig.\u00a05, where scattering by a single scale of M. aega (Fig.\u00a05a) is compared with scattering by an array of scales on the wing (Fig.\u00a05c). The far-field scattering diagram of a single scale is a narrow stripe (Fig.\u00a05b; see also Fig.\u00a02j), but that of a set of scales is a distinctly broadened stripe (Fig.\u00a05d). The angular extent of the scattering by the single scale in the vertical plane is <10\u00b0, but that of the scale set is about 30\u00b0 (Y, Fig.\u00a06). The broadening of about 20\u00b0 will be due to a rotational variation of the scale plane around the long axis of the scales. The tilt angle, that is the angle of the scale plane with the wing, will have a similar variation, as the scattering diagram of the scale set is about 20\u00b0 wider than that of the single scale (X, Fig.\u00a06). Further broadening of the scattering diagram will occur when reflection by the complete wings of a Morpho butterfly is considered. The dynamic changes of light scattering by a Morpho flying in a natural environment, and how the butterflies will be perceived by conspecifics and predators will be interesting themes for future research.\nFig.\u00a05Scattering by scales of Morpho aega. a Single scale about normally illuminated by a beam with about 50\u00a0\u03bcm diameter; the outline of the scale is visible because of an additional, obliquely illuminating beam (bar: 50\u00a0\u03bcm). b Far field scattering pattern of the single scale photographed at a white screen (see Fig.\u00a02j). The bright white spot is due to light scattered at the hole in the white screen and direct scattering by the scale. The interrupted circles indicate the angular directions of the scattering in steps of 10\u00b0. c A wing piece about normally illuminated by a beam with about 900\u00a0\u03bcm diameter; cover and ground scales cannot be distinguished (bar: 200\u00a0\u03bcm). d Far field scattering pattern of the wing piece. The white spot is due to light scattered at the hole in the white screen and to direct scattering by the scaleFig.\u00a06Spatial profiles of the scattering diagrams of Fig.\u00a05b, d. The extent of the scattering in the horizontal plane (X) is much broader than the scattering in the vertical plane (Y) for both the single scale as for the intact wing piece. The angular width in the vertical plane of the scattering by the single scale is <10\u00b0, but the width in the horizontal plane is about 100\u00b0. The vertical angular extent of the scattering by the wing piece is about 30\u00b0, meaning a broadening of about 20\u00b0 with respect to the single scale, and the extent in the horizontal plane is similarly broadened","keyphrases":["scattering","angular reflectance","lambertian reflector","single butterfly scale","structural colour"],"prmu":["P","P","P","P","R"]}
{"id":"BMC_Bioinformatics-4-_-521197","title":"EasyGene \u2013 a prokaryotic gene finder that ranks ORFs by statistical significance\n","text":"Background Contrary to other areas of sequence analysis, a measure of statistical significance of a putative gene has not been devised to help in discriminating real genes from the masses of random Open Reading Frames (ORFs) in prokaryotic genomes. Therefore, many genomes have too many short ORFs annotated as genes.\nBackground\nAs of February 2003, 106 microbial genomes have been sequenced and made publicly available and the race is now on to mine genomes such as these for interesting and\/or valuable genes and motifs. It has been estimated [1] that 60\u201380% of the genes in newly sequenced organisms have known homologues in other species. This percentage will grow as genomic annotations progress and perhaps there will be a time when virtually all genes can be found by homology matches to known proteins. That day, however, is not around the corner and even if it were, the occasional odd genes which would nevertheless escape detection by homology may very well be the truely novel and most wanted ones.\nIt is a common misconception that identification of genes in prokaryotes is almost trivial. Any random sequence, as well as non-coding regions in real genomes, contain a large number of open reading frames (ORFs). Most of these are too short to be possible protein coding genes, but in many genomes there are many 'random' ORFs longer than e.g. 100 amino acids, a cut-off that is often used for considering an ORF a real gene. The large number of short 'random' ORFs makes is difficult to discriminate real genes from random ORFs below a certain length, which depends on the genome and in particular its GC content. Therefore many genomes are over-annotated [2]. In one genome, that of A. pernix [3], all ORFs longer than 100 amino acids are annotated as genes, but probably only around half the annotated genes are real [2]. The most severe problem today is to disciminate between short genes and random ORFs, and here the meaning of 'short' is quite organism dependent. One of the most important contributions of this paper is a way to deal with this problem by introducing a statistical significance for an ORF being a gene.\nComputational gene finding exploits the statistical differences in codon usage between coding and non-coding regions of DNA [4-6]. The search for a mathematical frame work to efficiently capture these differences in codon usage led to Markov chain models and the GeneMark algorithm [7]. In order to facilitate the combination of various Markov chain scores, the application of Hidden Markov Models (HMMs) to gene finding was introduced in a gene finder for E. coli, Ecoparse [8]. These methods relied on a set of known genes for estimating parameters. More recently methods have been developed which start from a raw genome and automatically extract data for estimation. One of these, Glimmer [9], employs interpolated Markov models in order to use the maximum Markov chain order which can reliably be estimated for every oligomer. Another one, Orpheus [1], appeared the same year and calculates coding potentials of ORFs based on codon frequency of similarity-derived genes. Most of these gene finders also extract Shine-Dalgarno sequences in order to improve prediction of start codons.\nDue to their modular structure, HMMs are a suitable frame work for gene finding, and they are now used in GeneMark.hmm [10], GeneMarkS [11,12] and Frame-by-Frame [13]. GeneMarkS uses a mixture of Markov chains of order 0, 1 and 2 in combination with features of already annotated genomes to bootstrap an initial estimation of coding statistics, which is then iteratively improved by the GeneMark.hmm2.1 algorithm. Gibbs sampling is also used to detect Ribosome Binding Sites (RBS). The Frame-by-Frame method was conceived to improve the accuracy of GeneMark.hmm and it employs standard Viterbi parsing of all six reading frames independently and subsequently combines these into a global parse.\nIn this paper, we describe a fully automated method for making an organism specific gene finder. It starts from a raw genome and searches for protein matches to get a good training set. Then an HMM with states for coding regions as well as RBS is estimated from the data set. This HMM is used to score all the ORFs in the genome and finally the score is converted to a measure of significance \u2013 R \u2013 which is the expected number of ORFs that would be predicted in one megabase of random DNA. The main advantage of this significance measure is that it takes the length distribution of random ORFs properly into account. The method is shown to match or exceed other gene finders currently available.\nMethods\nAutomatic extraction of training set\nIn order to fully automate the construction of the gene finder, a data set of reliable genes was obtained through the following procedure, which is essentially the same as that used in [1]. All ORFs with a significant match to a protein from a different organism are assumed to be real genes. A subset of those have only one possible start codon, because there is only one start between the most upstream protein match and the nearest upstream in-frame stop codon. The details of the method are:\n1. Extract the maximal ORFs longer than 120 bases from the query-genome. For every stop codon, extract the region from the first downstream (in frame) start codon to the next downstream (in frame) stop codon.\n2. Translate the ORFs to proteins and search for significant protein matches in Swiss-Prot [14] using BLASTP without gaps and a threshold of 10-5 [15]excluding proteins from the query-genome and proteins listed with one or more of the keywords putative, unknown, possible, hypothetical, probable, bacteriophage, transposon, insertion, reverse transcriptase.\n3. For each ORF with at least one significant protein match, identify the most upstream position of these matches (to compensate for random matches, we actually remove 6 bases from the most upstream match before proceeding). If there is no alternative start codon between this position and the start of the ORF, put it in set A' of genes with certain starts. The remaining ORFs are put in set B' of certain genes with uncertain starts.\n4. Reduce similarity in set A' by removing genes with similarity within the set. All pairs of genes are compared using BLASTN with a significance cut-off of 0.001. Two genes that match are called neighbours. Genes are removed starting with the one having the largest number of neighbours. This continues until no gene has neighbours left in the set [16]. The reduced set is henceforth referred to as A.\n5. Unite set A' and B' into set T' and reduce similarity of T' to obtain set T .\n6. Add 50 bases of upstream flank to genes of all sets and 10 bases downstream flank.\nThis procedure is a means to identify ORFs in a genome which are almost certain to be (protein coding) genes. The ORFs in set A and T make out reliable and balanced sets of positive examples which may be used to estimate the model parameters.\nHMM architecture\nWe use a hidden Markov model (HMM) to model the gene structure. An HMM is a probabilistic model in which it is possible to model the various types of signals in a gene in one integrated model. For introductions to HMMs, see e.g. [17-19]. We model standard 'text book genes' with an unbroken open reading frame, i.e. genes with no programmed frame shifts, no sequencing errors nor any other special feature obstructing the reading frame.\nThe general architecture of the EasyGene HMM is shown in figure 1. There is a begin and end state marked B and E respectively. Then, at each side of the gene model, there are null models to model everything that is not part of a gene nor lies in the immediate vicinity of a gene. The next submodel is the RBS model which models the RBS as well as the nucleotides between the RBS and the start codon. After the start codon we model 3 bases explicitly since it appears that the codon usage immediately downstream of start codons differs from the rest of the gene [13]. Similarly we model the last codon before the stop codon explicitly and 6 bases after the stop in order to capture information present around the stop codon.\nFigure 1\nThe overall HMM architecture. Each box corresponds to a submodel with more than one state. The number above the boxes indicates the number of bases modelled by the submodel. An 'X' indicates a variable number.\nWe employ 3 looped codon submodels of the interior of the gene as depicted on the right hand side of figure 2. The reason for using several codon models is to embed a realistic length distribution in the HMM instead of the geometric distribution which would be implicit in having only one looped codon submodel [[17], Section 3.4]. We chose 3 because it results in a good fit to the empirical length distribution (see Results). The states in the codon model are of 4th order [20] in order to capture the inter-codon dependencies. The three looped codon models are identical, i.e. they have the same emission and transition probabilities (the states are tied).\nFigure 2\nEnlargement of null model and internal looped codons. LEFT: The state structure of the NULL model. The background state is of third order and models the general composition of the genome. The three shadow states model coding regions on the complementary strand. There are transitions from the background state to the first RBS state and to the first state modelling the start codon. RIGHT: Details of model of internal codons. A codon is modelled by three states with a transition from the last state back to the first and one out of the codon model. By putting several codon models in series, the length distribution of coding regions can be captured. From the last state there is a transition to the first state of the 'BSTOP' model, which models the last codon before the stop codon.\nThe RBS model in figure 3 models the ribosome binding site as well as the nucleotides between the RBS and the start codon. It has 7 states to capture any ribosome binding patterns and 12 tied spacer states for modelling the region between the RBS and the start codon. From the first spacer state there are transitions to all but the last of the following states, so the length distribution (with a minimum of 3 and maximum of 12) can be modelled exactly without imposing Gaussian assumptions as is done in e.g. [21]. These spacer states are of order zero.\nFigure 3\nThe state structure of the RBS model. The RBS model consists of seven states for modelling the ribosome binding site followed by a set of tied states for the variable region between the RBS and the start codon. From the last state there is a transition to the first of the three states modelling the start codon.\nThe null model depicted on the left side of figure 2 has a third order state for capturing intergenic regions and a reverse codon model for modelling reverse genes ('shadows') with states of second order. Note that transitions are allowed directly from the null model to a start codon; this facilitates detection of genes inside operons which may not have a clear RBS.\nWe found that the inclusion of two more branches of internal codons improved performance. This is presumably because it allows the HMM to keep separate statistics for atypical genes, some of which may be horizontally transfered. Adding a fourth branch did not improve performance further, so we stopped at three lending some support to the hypothesis that there are essentially three classes of genes in prokaryotic genomes [22].\nModel estimation\nThe HMM parameters (transition and emission probabilities) are estimated with the Baum-Welch algorithm, which is a maximum likelihood approach that finds the parameters maximizing the probability of the training set, see e.g. [17]. The estimation is done in these stages:\n1. The emission probabilities of the background state are estimated from both strands of the complete genome.\n2. The genes from start to stop codons are extracted from the training set and reverse complemented. The shadow model (consisting of three states) is estimated from these sequences. The parameters of this model are fixed.\n3. The RBS, start and astart submodels (cf. figure 1) are estimated using set A exclusively since this set has reliable gene starts and therefore aligned upstream regions. Regions of 50 bases upstream from the start codon are extracted from all genes in set A. A null model (with fixed parameters) is included before the RBS model. The RBS model is initially primed with a high probability for the consensus GG (a dinucleotide common to most RBS), but the precise pattern is found by the estimation method. During estimation, a type of simulated annealing is used, where noise of a decreasing amount is added to the parameters in each iteration of the Baum-Welch algorithm [23]. After training on set A, the parameters of the RBS, start and astart models are fixed.\n4. The null model, RBS, start and astart models are now combined with the internal codons, bstop, stop and astop models to make up the complete model. The non-fixed parameters of this model are then trained on the (larger) set T .\nThe whole procedure can be completely automated. Note that no experimentally mapped RBSs are used for estimating the RBS model, the RBS is discovered during the estimation procedure.\nWhen estimating the complete model (stage 4 above) we use labelled estimation [17,20], where each base of the sequence is labelled as coding or non-coding. For the part of set T where the exact start is not known, we leave the part of the sequence from the most upstream start codon until the first significant protein match unlabelled. The weight with which each base in the unlabelled region contributes to the estimation of the parameters in the coding states is automatically determined during the iterative estimation procedure.\nDecoding\nBy decoding we mean the process of finding an optimal parse of the DNA string into coding and non-coding regions.\nThe commonly used Viterbi decoding returns the most probable path of the sequence given the model [17], but this is not appropriate when the length is modelled by duplicating codon states, since this length modelling is realized only as a sum over many HMM paths. Therefore we use posterior decoding where one calculates, for each nucleotide i, the probability that it was emitted by a given state S. The calculation is done by adding the probabilities of all paths compatible with having state S emit nucleotide i [24,17]. We use this to calculate the posterior probability of the first state of the start codon model, and thus obtain the probability that a gene starts at any position in the sequence. Given our assumption of perfect 'textbook-genes' with no errors or frame shifts, there is exactly one stop codon for each start, and thus the probability of a gene start is equal to the probability of the whole gene. The independent scoring of start codons makes it trivial to report several possible start codons for a gene in cases where there is no clear \"winner\".\nNote that the HMM architecture in figure 1 is non-looped \u2013 ie. it would find only one gene if we were using Viterbi decoding, which only gives the single most likely parse. This architecture however, is the correct one for scoring ORFs with posterior decoding. It has the further advantage that overlapping genes are easily handled since each gene start will be scored independently whether or not it overlaps other genes. In contrast, using a looped model and Viterbi decoding would not facilitate detection of overlapping genes unless the model contains explicit states for overlapping genes as described in e.g. [8].\nThe state posterior probability itself is not a useful score, because it is a probability of the whole sequence, not just a single gene, and it therefore depends on the length of the sequence it is part of. By dividing the posterior probability by the probability of the whole sequence (the genome) according to the null model, the contribution to the state posterior probability of the sequences flanking a gene will cancel and effectively make the ratio independent of the flanking sequences (except the parts very close to the gene), see the Appendix. The log of this ratio is called the log-odds score, and that is our basic score for a gene.\nSignificance\nAs mentioned above, it is important to take into account the chance that a random ORF of the same length scores as high as a given gene. This is implicitly taken into account by our HMM because it models the length distribution of genes, but it turns out that one can calculate a significance score, which works slightly better (se results) and has a more intuitive interpretation.\nThe probability of finding high-scoring ORFs in a random sequence is highly length dependent; the number of ORFs decays exponentially with the length so there will be a lot more short ORFs than long ones. For a random sequence of given length (e.g. 1 Mb) the expected number of ORFs of length l' can be written as\nN(l') = exp (A - Bl'), \u00a0\u00a0\u00a0 (1)\nwhere A and B are constants that can be found from linear regression of the log of the number of ORFs against the length. All lengths are measured in codons and we count both start and stop codon. For convenience we introduce the variable length l', which is the length of the ORF modelled by the looped codon submodels. A number of codons l0 are modelled explicitly in the beginning and end of the ORF (l0 = 4 in our model), so l' = l - l0.\nIf the log-odds score is denoted \u03b2, we show in the appendix that \u03b2' = \u03b2 - (n - 1) log(l' - n\/2) is approximately normally distributed with a mean \u03b1\u03bc + \u03b3\u03bc l' and variance \u03b1\u03c3 + \u03b3\u03c3 l', which are both linear in l' (the \u03b1's and \u03b3's are constants). Here n is the number of looped codon submodels (3 in our model). The coefficients of the linear mean and variance are estimated by linear regression on ORFs from random sequences. Then we define the standard score\nwhich is normally distributed with mean 0 and variance 1.\nFor any given length l' and standard score \u0393, one can now estimate the expected number C(l', \u0393) of ORFs of the same length scoring higher than \u0393 in a fixed (long) random sequence using equation (1),\nC(l', \u0393) = exp(A - Bl') [1 - \u03a6 (\u0393)], \u00a0\u00a0\u00a0 (3)\nwhere \u03a6 is the cumulative normal distribution. This number can be used directly to judge the significance of a gene predicted with length l and a standard score \u0393. However, it would be preferable to know the total number of expected genes (of all lengths) predicted in a random sequence, rather than the expected number with a certain length. Therefore, instead of using C(l', \u0393) to judge significance, we use the total number of expected predictions in a random sequence.\nSuppose a gene of length l1 is predicted with a standard score \u03931. Then the expected number of genes of that length predicted in a random sequence is C = C(, \u03931). Now we want to calculate the total number of genes of all lengths predicted in a random sequence at the threshold C. For any length above a certain lC, the expected number of ORFs will fall below C due to the exponential decay of the length distribution. Therefore the total number of predicted genes (regardless of length) is roughly lC C + the sum of predicted genes above lC (a sum of a geometric series). We end up with the following expression for the total number of predicted genes in a random sequence:\nThe number 16 arises from the fact that the minimum ORF length considered is 20 codons corresponding to a variable length of 20 - 4 = 16. See Appendix for details. This is the expected number of genes predicted in a random sequence for a given value of C, and that number is the one we quote for each predicted gene. The constants A and B characterize the random sequence.\nR depends on the standard score \u0393 through C. This dependence is illustated in Figure 4. It clearly shows that a short ORF needs to have a much higher standard score than a long ORF in order to be significant (in the sense of having a low R-value).\nFigure 4\nRelationship between R, \u0393 and variable length in codons l'. The numbers are taken from the E. coli runs described in Results and Discussion, but the qualitative behavior is independent of the genome\nWe have chosen to normalize R to a random sequence of 1 Mb rather than a random sequence the length of the genome, because then significance can be compared across genomes. The precise recipe we use for calculation of statistical significance is:\n\u2022 For the genome in question, generate 40 Mb of random sequence using the 3rd order background model (estimated from the genome). Extract all ORFs and estimate the parameters A and B by linear regression of the log of the number of ORFs against the length and normalize to 1 Mb. To avoid distortion of regression lines due to noisy statistics of long ORFs, restrict the variable ORF length used to lie below 70 codons. This makes the range of variable length 16\u201370 codons, which corresponds to a total length range of 20\u201374 codons.\n\u2022 Score all the ORFs in the random sequence with the model and calculate \u03b2' for all ORFs. For each length, calculate the average and variance and estimate the parameters \u03b1\u03bc, \u03b3\u03bc, \u03b1\u03c3, and \u03b3\u03c3 by linear regression, again using ORFs in the range 20\u201374 codons.\n\u2022 To calculate the significance of an ORF in the genome, first calculate the standard score \u0393 from equation 2, then C from equation 3, and finally the significance value R using equation 4.\nThere are of course other possible choices of significance measure, but we believe that this is a simple and intuitively clear one and we have prefered it to the more traditional significance measure. By reporting the number R of expected false positives in one megabase of random sequence, it is easy for the end-user to estimate the number of false positives in a random sequence the length of the entire query genome \u2013 one simply has to multiply R with the size of the genome measured in megabases.\nUsing other gene finders\nIn order to benchmark EasyGene we compare it with some of the existing gene finders.\nGeneMark 2.4, Frame-by-Frame, GeneMark.hmm 2.1 and GeneMark.hmm\/S all belong to the GeneMark suit of programs and are accessible via the web interfaces listed in the references.\nFor GeneMark.hmm2.1, GeneMark.hmm\/S and Frame-by-Frame the output is a coordinate listing (start and stop positions) of all predicted genes.\nGeneMark2.4 outputs a list of stop codons and corresponding high-scoring start codons. Each start\/stop is listed with scores for coding potential and RBS. We collect all starts for a given stop and output the \"Avg Prob\" of the start with the highest RBS score. Whenever a threshold was needed for comparison purposes, we used 0.5 which is the default set on the web page.\nGlimmer2.02 and Orpheus2 were installed locally. We changed the minimum ORF length predicted by these gene finders to 60 bp which seems to be the minimum used by the other gene finders. Orpheus and Glimmer provide two kinds of output: a verbose coordinate list of starts, stops and ORF scores and a simpler, post-processed list of coordinates for ORFs regarded as genes.\nIn order to test their ORF scoring we had to parse the scored output. We had some difficulties interpreting the scored Orpheus output since some ORFs were scored several times with identical results (several identical \"Start chosen\"). In cases where multiple copies were found, we simply chose one of them and used the corresponding \"Coding potential\" (with the recommended threshold of -1) for further analysis.\nFor Glimmer2.02 the scored output was parsed simply by selecting the Gene Score attributed to every scored ORF and using the recommended threshold of 90.\nFinally, we used RBSfinder [25] for an alternative post-processing of Glimmer2.02 output. RBSfinder is designed to look for RBS sequences upstream of genes predicted by Glimmer2.02. If there are no RBS patterns in this region, RBSfinder searches for a start codon having a RBS pattern in the same reading frame upstream or downstream and relocates the start codon accordingly. The program may be iterated several times using revised predictions as new inputs. We found that running it twice was better than once but running it three times did not improve things further, so we chose to run it twice (with default options).\nResults and Discussion\nA number of tests were conducted in order to optimize the model architecture. We tested the number of codon models in series and found that three models yield a very good fit to the observed length distribution, see figure 5 for an example.\nFigure 5\nGene length distribution imposed by HMM architecture. The model length distribution given by a negative binomial (equation 8 with n = 3) compared to the length histogram of set A genes for H. pylori J99.\nThe results of our experiments with the number of branches in the coding model and the order of the coding states are shown in figure 6 and 7 respectively, which show (cross-validated) performance curves (ROC curves) for varying numbers of branches and various orders. The performance curves are made by plotting the average true positive rates for a range of average false positive rates (the fraction of false positives made on average by the 10 different cross-validation models in 1 Mb of random sequence).\nFigure 6\nAssessing the optimal number of HMM coding branches. Performance curves for 1,2,3 and 4 Markov branches of looped codon submodels for E. coli. The performance curves are made by the following procedure: First we sort the positive R-values in ascending order for each of the 10 subsets of set T (test sets). Then for each ascending R-value we calculate the fraction of genes in set T scoring below R (true positive rate) and the fraction of ORFs (with lengths greater than or equal to 20 codons) in one megabase double-stranded sequence scoring below R (false positive rate). The resulting 10 files with true and false positive rates are concatenated and 30 false positive cutoffs are selected (from 0 to 0.15 with steps of 0.005). The false positive entries in the 10 files which fall between these cutoffs are found and the corresponding true positive entries are averaged. Hence for each average false positive rate (halfway between two consecutive false positive cutoffs) we associate an average true positive rate and these tuples are then plotted.\nFigure 7\nAssessing the optimal order of looped codon states. Performance curves for 3rd, 4th and 5th order Markov states of looped codon submodels for E. coli. For explanation of the construction of performance curves please confer the caption of figure 6.\nFigure 6 indicates that 3 branches is the best choice, while figure 7 suggests that the optimal order for these branches is 4. Note that the Y-axes of these figures have been zoomed in order to allow visual inspection of the performance differences. Note also that all architectures and orders in fact yield a relatively high true positive rate even at false positive rates below 0.02. Similar figures were also made for other organisms (not shown) and although the results were not always as clear as for E. coli, a choice of 3 branches of 4th order states as default models of the internal parts of genes works well for the organisms we tested.\nSimilarly, figure 8 shows performance curves to compare log-odds and significance scores and the significance scoring is seen to be slightly better in that it allows detection of more true positives for a given false positive rate. The significance scoring has the additional advantage of being genome independent and has an intuitively appealing interpretation.\nFigure 8\nComparing significance and log-odds. Performance curves comparing significance and log-odds scores for E. coli. For explanation of the construction of performance curves please confer the caption of figure 6.\nTable 1 shows the average true and false positive rates for selected R-values in the case of E. coli models with three branches and fourth order branch states. This table gives an impression of the approximate R values corresponding to the graphs in figures 6, 7 and 8.\nTable 1\nTrue and false positive rates for selected R-values.\nR-value\nTP rate\nFP rate\n0.1\n0.971\n0\n2\n0.980\n2.3e-6\n10\n0.984\n3.0e-5\n50\n0.987\n3.1e-4\n150\n0.991\n8.7e-4\n500\n0.995\n2.8e-3\n10000\n0.999\n0.059\nTrue and false positive rates averaged over 10 cross-validations for selected R-values in the case of the three branches, fourth order E. coli model. The FP rate is measured as the average fraction of ORFs (with lengths greater than or equal to 20 codons) in one megabase of double-stranded random sequence scoring lower than the given R-value. The TP rate is measured as the average fraction of ORFs in data set T scoring lower than the given R-value\nIn order to test the validity of the approximations used in the derivation of the significance measure, we generated 1 Mb of random sequence from the Markov chain corresponding to the background state of the E. coli model. The plots in Figure 9 show the mean and variance of the length-corrected log-odds score, \u03b2', for each length. They are very close to being linear as we assumed. The length distribution of the ORFs in the same random sequence is also shown, which confirm the geometric length distribution. The predicted number of significant ORFs in 1 Mb random sequence is compared with the theoretical significance value R in Figure 10 and the agreement is seen to be rather good. We also compared the distribution of standard (3) scores for ORFs in the same random sequences to a normal distribution of unit variance and zero mean and the agreement also turned out to be good in this case as seen in Figure 11. We conclude that the assumptions and approximations used in the calculation of R are quite accurate.\nFigure 9\nStatistical characteristics of random sequences. The top two panels show the mean and variance of log-odds scores versus variable ORF length in random sequences (E. coli model). Lowest subplot shows a logarithmic plot of the length distribution of random ORFs. The linear regression lines are shown in all three plots.\nFigure 10\nComparing predicted and found number of false positives. Empirical and theoretical number of false positives per Mb double-stranded random sequence according to the E. coli model.\nFigure 11\nProbability density functions for the standard score. Empirical (dots) and theoretical (line) probability density functions for the standard scores (\u0393) in random sequences (E. coli model). The lower plot is an enlargement of the distribution tail.\nTable 2 shows the percentage of genes found for eight different gene finders and some sets of high-confidence genes from E. coli as well as the number of genes found in the whole genome and in random sequences. The eight gene finders are: EasyGene, Glimmer2.02, Glimmer2.02 with RBSfinder post-processing, Orpheus, GeneMark2.4, GeneMark.hmm2.1\/GeneMarkS hybrid, \"pure\" GeneMark.hmm2.1 and Frame-by-Frame.\nTable 2\nSpecificity, sensitivity and precision estimates for different gene finders in E. coli.\nData set\nEasyGene\nGlim\nrbs-Glim\nOrpheus\nGm24\nGmS\nGmhmm\nFrame\nA'-% found\n98.4\n98.9\/98.9\n98.9\n98.0\/95.3\n91.5\n97.2\n98.1\n97.0\nA'-% exact\n93.8\n98.9\/95.3\n84.1\n95.1\/92.4\n41.6\n88.0\n85.7\n93.2\nB'-% found\n98.4\n98.5\/98.6\n98.6\n95.9\/96.5\n90.2\n96.6\n97.2\n96.4\nT-% found\n98.1(98.0)\n98.3\/98.4\n98.4\n96.5\/95.6\n89.8\n96.3\n97.1\n96.1\nGenome\n4145\n6827\/5756\n5756\n9333\/7543\n3552\n4064\n4230\n4064\nzero order\n7\n169\/211\n211\n6761\/5430\n6\n153\n1459\n0\nfirst order\n7\n545\/723\n723\n6836\/4804\n13\n241\n830\n0\nthird order\n1\n2423\/2694\n2694\n6582\/4817\n43\n659\n866\n1\nshadows\n0\n19\/21\n21\n22\/9\n1\n0\n2\n0\nUpper part shows the percentage of genes found exactly (both 5' and 3' end) and partially (only 3' end exact) for different gene finders and sets of high confidence genes in E. coli. For Glimmer and Orpheus, the numbers before the \"\/\" are based exclusively on their ORF scores and recommended threshold whereas the numbers after the \"\/\" are based on their post-processing procedures. The number of genes predicted in the whole genome is also shown. This should be compared to the 4288 annotated genes in E. coli. The lower part of the table shows the number of false positives predicted in random sequences generated by Markov chains of order 0, 1 and 3 and the very last row shows the number of false predictions in the shadows of the high-confidence genes in data set A. All values listed for EasyGene are based on an R-value threshold of R = 2.\nSequence set A' consist of the 1136 genes with high-confidence starts extracted from the E. coli genome as described in Methods. The \"% exact\" row indicates the percentage of predictions where both start and stop codon are correctly predicted whereas the \"% found\" row indicates that only the stop codon is correct. Note that all genes in set A have the most upstream start (they are Longest Possible Open Reading Frames \u2013 LPORFs) by construction, and hence performance on this set favours gene finders which are biased towards LPORFs (such as Glimmer). Set B' consists of 1690 high-confidence genes with uncertain starts extracted as described in Methods. Due to the uncertainty of start codon placement, one cannot evaluate the exact start prediction performance for this set. The same is true for set T which is a similarity reduced union of A' and B' (2042 sequences).\nThe Genome row shows the number of genes predicted in the E. coli genome using default parameters and thresholds for the various gene finders. For Glimmer and Orpheus the minimum length of predicted genes was lowered to 60 in order to make their performance comparable to the others'. The next three rows show the number of false positives found in both strands of 1 Mb random sequences generated by zero, first and third order Markov chains estimated from the entire E. coli genome. The last row of the table shows the number of predictions wholly within the shadows of set A' \u2013 i.e. wholly within regions complementary to the genes in set A' where, ideally, no genes (or at least very few) should be predicted.\nThe test sets overlap with the training sets for EasyGene. Therefore, for set T the 10-fold cross validation sensitivity is shown in parenthesis for EasyGene and it is seen to be reassuringly close to the non-crossvalidated sensitivity suggesting that EasyGene employs an appropriate model complexity and steers free of overfitting. Note also that the other gene finders have also been estimated from sets that overlap (or even contain) set T .\nFor Orpheus and Glimmer we show two numbers N1\/N2 for each entry corresponding to before and after post-processing. For Orpheus, N1 is the number of unique ORFs having a Coding Potential above the recommended minimum of -1 and N2 is the number of entries in the post-processed orfnuc file. The post-processing removes some overlaps but also appears to employ a less restrictive cutoff than the recommended -1. For Glimmer, N1 is the number of ORFs with Gene Scores greater than or equal to the recommended threshold of 90 and N2 is the number of entries in the post-processed list of putative genes. The post-processing elects (\"votes\") some ORFs as gene candidates despite a low score. On the other hand the post-procesing removes some same-strand overlaps in different reading frames so the N2 may be greater or less than N1 depending on the relative extent of overlaps and \"voting\".\nIt is always difficult to asses the specificity of a gene finder, because it is difficult to find genomic regions that are certain to contain no genes. We have therefore assessed specificity in three different ways. First, by counting the number of predicted genes in a genome. If this number is much higher than the number of annotated genes, it is likely that there are many false positive predictions, i.e. poor specificity. Our second test is based on random sequence. Clearly, a high number of predicted genes in a random sequence of bases indicates a poor specificity. However, it is probably not possible to find an exact quantitative correspondence between predictions in random sequences and real genomes. Also, it is not clear what sort of random sequence to use for such a test. By 0'th order we mean a sequence with bases generated randomly and independently with the base frequencies of the genome. Bases are quite correlated in DNA sequences, so we have also tested on sequences that are generated by Markov chains of orders 1 and 3. These Markov chains are estimated from the genome, so the sequences will have the same distribution of dinucleotides and 4-nucleotides, respectively, as the genome. Finally, the third test is the number of genes predicted on the opposite strand of genes (shadows); these shadow regions should contain very few genes if any.\nAll gene finders except EasyGene, Frame and GeneMark2.4 predict a rather large number of false positives in random sequences, but for GeneMark.hmm and GeneMarkS we do not see large over-prediction in the genome or in shadows. Evidently, Glimmer and Orpheus predict a lot more genes in the genome than the other gene finders, suggesting that these gene finders have very high false positive levels. This is supported by the high numbers of genes predicted in random sequences, and (to a much lesser extent) in shadows. Orpheus and Glimmer actually predict more genes pr. nucleotide in the third order random sequence than they do in the genome, suggesting that the coding potential calculated in these gene finders is far from optimal.\nThe HMM used by Frame assumes a minimum gene length of 69 bases which could make its false positive level seem somewhat better (lower) than it is, but there was no convenient way to lower the minimum length so we simply left it. It should also be noted that Frame relies on pre-existing annotations for training and is therefore not a self-training gene finder like Glimmer, Orpheus, GeneMarkS and Easygene.\nThe sensitivity of the gene finders is tested on sets of high-confidence genes. Glimmer has the highest sensitivity for all sets, but this is largely due to heavy over-prediction. One ought always to bear in mind that it is not difficult to achieve high sensitivity if high levels of false predictions are tolerated at the same time \u2013 sensitivity is 100% if all ORFs are predicted as genes! Although there are some very close competitors, EasyGene comes out as the second best in sensitivity for all sets.\nThe exact prediction of start codons is tested on set A' and on an experimentally ver-ified set. As mentioned above, set A is biased, because all genes of this set are LPORFs. Glimmer always predicts the most upstream start and consequently achieves a high performance on this set. When Glimmer's output is post-processed by RBSfinder the performance drops considerably.\nThe prediction of start codons was evaluated further on a set of 195 E. coli genes with experimentally verified starts [26] with results shown in table 3. Set LiC is the subset of 133 genes which coincide with the longest possible open reading frame (LPORF) while set LiD is the remaining 62 genes whose starts are downstream of the LPORF start. The goal is to find the starts of the challenging LiD set without loosing too many of the more trivial starts of set LiC. Table 3 shows that while most gene finders partially locate all genes in set LiC and LiD, there are large variations in their exact localization ability. Selecting for the highest combined performance on set LiC and LiD, one sees that EasyGene, GeneMarkS and Frame-by-Frame are best. Their performances also exceed that of [27] in which a cross-validated performance of 84.9% +\/- 4% is reported on a subset of 184 genes out of the 195. In the low end we have Glimmer finding 0% of set LiD exact (1.6% with post-processing) and GeneMark2.4 finding 49.6% of set LiC exact. Using the RBSfinder post-processing [25] on the Glimmer predictions improves performance on set LiD to 75.8%, but at the cost of a substantial dip in set LiC performance to 88.7%.\nTable 3\nSensitivity and precision estimates for experimentally verified E. coli genes.\nData set\nEasyGene\nGlim\nrbs-Glim\nOrpheus\nGm24\nGmS\nGmhmm\nFrame\nLiC-% found\n100\n100\/100\n100\n97.7\/91.7\n97.7\n100\n100\n100\nLiC-% exact\n94.0\n100\/97.0\n88.7\n96.2\/90.2\n49.6\n94.0\n90.2\n98.5\nLiD-% found\n100\n100\/100\n100\n96.8\/98.4\n100\n100\n100\n100\nLiD-% exact\n96.8\n0\/1.6\n75.8\n51.5\/51.6\n67.7\n95.2\n80.6\n87.0\nPercentage of genes found exactly and partially in two subsets of the 195 experimentally verified genes published by [26]. All values listed for EasyGene are based on an R-value threshold of R = 2.\nMany gene finders are first developed for E. coli and then later adapted to other organisms. It is therefore important also to test gene finders on other organisms. Based on table 2 and 3 we chose EasyGene, GeneMarkS and Frame-by-Frame as the gene finders with the best overall performances and then conducted further comparisons between these for M. tuberculosis [28], H. pylori [29] and B. subtilis [30]. M. tuberculosis presents a challenge due to GC richness, H. pylori due to small genome size and B. subtilis was chosen on account of its reputation of being well annotated [11]. The results are presented in table 4 with the same rows as table 2.\nTable 4\nSpecificity, sensitivity and precision estimates for M. tuberculosis, H. pylori J99 and B. subtilis.\nM. tuberculosis\nData set\nEasyGene\nGmS\nFrame\nA'-% found\n96.7\n97.2\n96.0\nA'-% exact\n89.1\n80.9\n87.9\nB'-% found\n96.8\n97.1\n96.3\nT-% found\n96.9(96.6)\n97.3\n96.4\nGenome\n3749\n3983\n4341\nzero order\n0\n-\n8\nfirst order\n3\n-\n2\nthird order\n2\n-\n12\nshadows\n1\n0\n1\nH. pylori J99\nData set\nEasyGene\nGmS\nFrame\nA'-% found\n99.2\n99.2\n99.2\nA'-% exact\n97.5\n95.7\n96.7\nB'-% found\n100\n99.6\n98.9\nT-% found\n99.7(98.8)\n99.5\n99.1\nGenome\n1491\n1518\n1479\nzero order\n2\n1479\n2\nfirst order\n1\n336\n2\nthird order\n0\n403\n0\nshadows\n2\n0\n0\nB. subtilis\nData set\nEasyGene\nGmS\nFrame\nA'-% found\n99.3\n98.1\n98.8\nA'-% exacts\n94.8\n94.1\n93.3\nB'-% found\n99.2\n99.0\n98.2\nT-% found\n99.3(99.2)\n99.0\n98.4\nGenome\n4083\n4221\n4006\nzero order\n1\n675\n0\nfirst order\n2\n457\n0\nthird order\n1\n813\n2\nshadows\n0\n0\n0\nGenes found exactly and partially for different gene finders and sets of high confidence genes in M. tuberculosis, H. pylori J99 and B. subtilis, where the number of annotated genes is 3918, 1491 and 4100 respectively. There are no pre-trained GeneMarkS models for M. tuberculosis, so it was not possible to obtain a false positive estimate for this organism. All values listed for EasyGene are based on an R-value threshold of R = 2.\nFor M. tuberculosis GeneMarkS and EasyGene are comparable, although GeneMarkS seems to over-predict slightly (assuming that the 3918 annotated genes are close to being correct), and Easygene might under-predict. We believe that Frame predicts too many genes in this organism and at the same time it has lower sensitivity than the two others, suggesting a worse performance overall. This indicates that Frame is not very robust with respect to high GC content.\nThe small dataset from H. pylori might give a slight over-fitting in EasyGene, where there is one percent difference between cross-validated results and non-crossvalidated. For this organism the three gene finders seem to have very similar performances. Finally, for B. subtilis EasyGene comes closest to the number of annotated genes and have higher sensitivity than the other methods.\nConclusions\nThe emerging overall picture is that the sensitivity of EasyGene tends to be comparable to GeneMarkS and higher than Frame. With regards to specificity, EasyGene and Frame tend to be comparable and both higher than GeneMarkS. Hence, EasyGene comes out with the best combined sensitivity\/specificity performance. When it comes to exact starts, EasyGene also generally performs best.\nGlimmer and Orpheus have good sensitivities but at the cost of very low specificities in this comparison. We have lowered the ORF length cutoff from their default values in these methods to make the results comparable. This may be unfair, but since the challenge is to find the short genes, we believe that any gene finder should be able to score them.\nAt present it is not possible to automatically find all genes in a prokaryotic genome. We believe the aim of a gene finding system is to help expert annotators as much as possible, and we consider the statistical significance of a gene an important help in classifying the predictions into almost certain genes and border-line genes needing more attention. Contrary to most other gene finders discussed here, it is up to the user which significance cut-off to use. EasyGene also predicts sub-optimal start codons if need be, so it will be easy to see if e.g. two alternative starts have almost the same score.\nA shortcoming of the significance value as calculated here is that long ORFs score well simply on account of their length, since very long ORFs occur rarely in random sequences. For this reason, EasyGene also provides a log-odds score in the output which may be held up against the R-value for ORFs longer than say 500 bp. Genes of this length ought to have high log-odds values. If they do not \u2013 i.e. if they score below 0 \u2013 then they are probably not real genes despite their length (sometimes very long non-coding ORFs occur in regions of repetitive DNA).\nIf the amount of available genomic DNA is very small (as it may be in partially sequenced genomes) the 3 branches of 4th order coding models may have too many parameters to be reliably estimated. In such cases, one could reduce the parameter space simply by using fewer branches and\/or lower orders. More generally, one could develop a method for automatic fine-tuning of HMM architecture for every new organism. Other lines of future research could focus on modelling of genes with errors and frame shifts.\nFinally, it may be noted that a prototype of EasyGene has already been used in the annotation of S. typhi [31].\nAppendix: The length dependent score distribution\nThe log-odds score distribution has several components. The probability of a sequence z containing and ORF given a model M may be written as\nwhere c1, . . . , cl denotes the codons in the ORF and flanks denotes all the rest of the sequence. P (l|M) is the length distribution of the HMM. In logarithmic form we have\nSimilarly, the denominator of the log-odds score, log P (z|N ), may be rewritten in exactly the same way, so the entire log-odds score becomes\nwhere\nWe will now look at the distribution of each of these terms.\nThe null model consists of a state with a loop and three reverse codon states with a loop. For a long sequence one of the loops will usually dominate the probability, so the length distribution is well approximated by a geometric distribution\nP(l|N) k1 exp (-k1l)\nwhere k1 is a constant greater than zero.\nThe length distribution from the looped codon states is a negative binomial [17],\nwhere p is loop probability and n is the number of looped codon submodels (in casu, n = 3). Some of the ORF (start and end) is modelled by non-looped states. The number of nucleotides modelled in the beginning and end of the ORF is denoted l0 and the variable length is denoted l', so the total ORF length is l = l0 + l'. Using that log \u00a0 (n - 1) log(l' - n\/2) - log(n-1)! for l' >>n, we obtain\nThe next term, Q, in equation 6 is a sum of random variables since we are considering random sequences. The sum Q will therefore (according to the central limit theorem) converge to a normal distribution N (\u03bcl, \u03c3), for large l.\nThe submodels flanking the gene model are identical to the null model. Therefore p(flanks|M) and p(flanks|N) will almost cancel in the last term of equation 6, except from the contribution from the RBS model and the states after the stop codon. This contribution will be independent of the ORF length. Since it is again a sum of random terms it is well approximated by a normal distribution.\nApart from the non-linear term from the negative binomial we have now shown that all terms in equation 6 are constant or scale linearly with l. Therefore\nis normally distributed for fixed length with an average and variance that are linear functions of l:\naverage = \u03b1\u03bc + \u03b3\u03bcl'\u00a0\u00a0\u00a0\u00a0\u00a0(11)\nThe parameters of these linear functions can readily be estimated from random sequences by doing linear regression between the variable length and the mean and variance of log-odds scores as shown in the two upper plots of figure 9. (Note that one can switch from the variable length l' to the total length l in the formulas by changing the constants.)\nFinally it is convenient to introduce the standard score \u0393, defined in equation 2, which is normally distributed with average 0 and variation 1.\nSince random ORF lengths are geometrically distributed (cf. equation 1), the expected number of ORFs of length l scoring more than \u0393 in a sequence is\nC(l', \u0393) = exp (A - Bl') [1 - \u03a6(\u0393)], \u00a0\u00a0\u00a0 (13)\nwhere the exponential term is the expected number of ORFs of variable length l' and \u03a6 is the cumulative normal distribution. Thus, for a given score we can calculate the number of expected predictions in a random sequence.\nFor lengths l \u2265 lC this expectation is always less than C (due to the exponential factor). So if we require that no more than C predictions be made at any length, the total number of predictions in a random genome is\nwhere ls is the shortest ORF length considered (we use 20). lC is given by\nStrictly speaking, we should take the smallest integer larger than , but using the real expression introduces only an insignificant error. Inserting into equation 14 finally yields equation 4,\nR = {(A - log C)\/B - (ls - l0)}C + C\/(1 - exp(-B)). \u00a0\u00a0\u00a0 (16)\nWeb sites used\nFrame-by-Frame:\nGeneMark.hmm 2.1:\nGeneMark.hmm 2.1 using GeneMarkS models:\nGeneMark 2.4:","keyphrases":["statistical significance","computational gene finding","hidden markov model","short open reading frames","automated genome annotation"],"prmu":["P","P","P","R","R"]}
{"id":"Hernia-4-1-2231409","title":"Impact of the mesh fixation technique on operation time in laparoscopic repair of ventral hernias\n","text":"Background Fixation of the prosthesis is one of the critical components of laparoscopic repair of ventral and incisional hernia (LRVIH). The impact of the fixation technique used on operative time has never been analyzed. We compared the duration of the operation according to the fixation technique used in a series of 138 patients with primary umbilical hernia.\nIntroduction\nLaparoscopic repair of ventral and incisional hernia (LRVIH) is gaining increasing popularity due to its low recurrence rate, short hospital stay, and low complication rate. Fixation of the prosthesis is one of the critical components of LRVIH. Currently, the two most popular methods of mesh fixation are the use of helical tackers either with or without transabdominal sutures (TAS). Overall it appears that the two laparoscopic mesh fixation techniques are similar in outcomes [1]. However, the impact of the applied fixation technique on operative time has never been analyzed. We compared the duration of the operation according to the fixation technique used in a series of patients with primary umbilical hernia (PUH) who underwent a straightforward repair by using completely standardized techniques.\nPatients and methods\nData were collected from operative reports of all 167 patients who underwent laparoscopic repair of PUH up to January 2007. To make the data more homogenous, we excluded patients who required adhesiolysis (n\u00a0=\u00a013) or simultaneously underwent another procedure (n\u00a0=\u00a012), or with whom a minor complication occurred intraoperatively, i.e., bleeding or equipment problem (n\u00a0=\u00a04). The remaining 138 patients underwent straightforward repair of PUH by using a completely standardized technique, and they represented the study group used to compare operative times between the two mesh fixation techniques.\nPneumoperitoneum was established by using a Veress needle. Three trocars (10, 5, and 5\u00a0mm) were inserted left laterally. A 1-mm-thick expanded polytetrafluoroethylene mesh (DualMesh\u00ae, WL Gore, Flagstaff, AZ) of 15 by 10\u00a0cm was used to overlap the hernia opening by at least 3\u20134\u00a0cm. The mesh was fixed using one of the following two techniques: either (1) with tackers (ProTack\u00ae, TycoUSS, Norwalk, CT) along the periphery of the mesh at intervals of 1\u20131.5\u00a0cm and eight TAS placed equidistant also along the periphery (n\u00a0=\u00a0107, further called the TAS technique) or (2) with a double ring of tackers alone without the use of TAS (n\u00a0=\u00a031, further called the DC technique). With this technique, also known as a \u201cdouble crown technique\u201d [2], the outer ring of tackers is the same as in the TAS technique. The inner ring of tackers is placed around the hernia opening about 1\u00a0cm apart. Transabdominal sutures were pulled through the abdominal wall with a suture passer (Gore Suture Passer Instrument\u00ae, WLGore, Flagstaff, AZ). In the first 84 patients, the method of mesh fixation was based on the surgeon\u2019s preference. For the last 54 patients, the mesh fixation technique performed was randomly chosen in conjunction with another study being done at our hospital that compares postoperative pain following these two methods of mesh fixation. Once the fixation was completed, the abdomen was desufflated and the trocar site of 10\u00a0mm and skin were closed. The time taken from the stab wound for insertion of a Veress needle to completion of skin closure was recorded to the nearest minute.\nStatistical analysis was done using the t test. Significance was set at a P\u00a0<\u00a00.05.\nResults\nOf the 138 patients in our study group, 106 were males and 36 females. Mean age was 55.0\u00a0\u00b1\u00a012.3\u00a0years. The TAS and DC groups were found to be well matched for sex and age. Mean operating time for the TAS technique was 50.6\u00a0min compared to 41.4\u00a0min for the DC technique. Mean difference in operating time was 9.2\u00a0min. This difference was significant (P\u00a0=\u00a00.002).\nMean postoperative hospital stay was identical for both groups and equaled 1\u00a0day. Postoperative complications included seroma lasting longer than 6\u00a0weeks in seven patients and chronic pain at TAS sites in one patient. All seromas resolved without intervention. The patient with persisting pain underwent relaparoscopy and removal of all eight TAS that provided complete pain relief. There were no significant differences in morbidity between the two groups. No recurrences were detected during a mean follow-up of 26.4\u00a0\u00b1\u00a025.6\u00a0months.\nDiscussion\nBesides prosthetic material that must sufficiently overlap the hernia defect, a reliable fixation of the prosthesis against the abdominal wall is a crucial component for success of LRVIH. Since the early years of LRVIH, there has been a strong belief among pioneers of this technique that reliable fixation of the mesh can only be achieved with the TAS technique [3, 4] and, in addition, that sutured mesh fixation is an imperative in LRVIH [5]. Another technique of fixation introduced later and consisting of a double crown of tackers only has been gaining increasing popularity due to a few specified advantages: technical simplicity, fewer incisions in the skin and possibly shorter operative time [2, 6]. One of the most interesting issues on LRVIH currently being debated is which of these two techniques is better. Prospective randomized studies comparing the two techniques are missing. Meta-analysis of published literature indicates that the two laparoscopic mesh fixation techniques are similar in main outcome parameters such as recurrence and complication rate [1]. In the same analysis, the use of tackers alone resulted in a slightly shorter operative time than when the TAS technique was used. However, in most of the studies that were included in this meta-analysis, fixation with tackers was performed by applying only a single row of tackers or technical details of the tacker fixation technique were not explained. Reliable data comparing TAS technique and DC technique are definitely missing.\nWe decided to compare the two techniques in a maximally homogenous model of the procedure: same site of hernia, same logistics of the operation, same prosthetic material, same fixation device, and same operation technique until the moment that the outer ring of tackers is completed. The only difference between the two techniques was the completion of the mesh fixation. In one technique eight TAS had to be inserted and in the other technique usually 6\u20138 tackers of the inner crown. Once the fixation had been completed, whatever the method was, the rest of the procedure was again identical. Obviously, the difference in operative times between the two operative techniques can be entirely accounted to the difference in time needed for insertion of eight TAS compared to the time needed for application of an inner crown of tackers. Since tacking of the inner crown takes definitely not more than 1\u00a0min in laparoscopic repair of PUH, insertion of eight TAS required at least 8\u00a0min. This strongly indicates that insertion of every single TAS prolongs LRVIH by approximately 1\u00a0min.\nA laparoscopic repair of PUH is definitely the least complex procedure among all LRVIHs. Insertion of TAS is probably easier than in other LRIVHs due to a central location of the hernia, a general absence of adhesions, maximal space between the distended abdominal wall and the bowel underneath, and an excellent view. It may be anticipated that insertion of TAS during more complex laparoscopic repairs of incisional hernias at less suitable sites, in the presence of adhesions and proximity of the bowel can be much more challenging and as a consequence will require more time than during repair of a PUH.\nOur results indicate that LRVIH by using the DC technique indeed requires less operative time than when the TAS technique is used. As long as no significant differences between the two fixation techniques are demonstrated on issues of recurrence, complications, and postoperative pain, the time difference we have measured might be an argument in favor of the DC technique, especially when mesh fixation would require a large number of TAS.","keyphrases":["hernia","umbilical","laparoscopy","study, time","surgical mesh","surgical fixation devices"],"prmu":["P","P","U","R","M","M"]}
{"id":"Ann_Surg_Oncol-4-1-2266786","title":"Liver Transplantation for Hepatocellular Carcinoma\n","text":"Background Orthotopic liver transplantation (OLT) is the best available option for early hepatocellular carcinoma (HCC), although its application is limited by stringent selection criteria, costs, and deceased donor graft shortage, particularly in Asia, where living donor liver transplant (LDLT) has been developed.\nHepatocellular carcinoma (HCC) is the third most common cause of cancer-related death worldwide.1 Orthotopic liver transplantation (OLT) is the best therapeutic option for early, unresectable HCC, although it is limited by graft shortage and the need for appropriate patient selection. In the late 1980s, the results after OLT for HCC were disappointing, with high early recurrence and 5-year survival rates ranging between 18 and 40%.2 This discouraging experience and shortage of deceased donor grafts compelled the transplant community to establish stringent selection criteria to predict posttransplant survival of HCC patients. Recognizing that patients with small, incidentally found tumors had survival rates after liver transplantation equivalent to those after transplantation for benign disease, Mazzaferro et al. in Milan established criteria for OLT in a landmark study published in 1996.3 They showed that a subgroup of patients with radiologic evidence of a single tumor \u22645 cm in diameter, or two to three tumors \u22643 cm in diameter had 5-year and recurrence-free survival rates of 75 and 83%, respectively. The Milan criteria were subsequently adopted by the United Network for Organ Sharing (UNOS) staging system for allocating organs for OLT in the United States.\nOLT is limited by a shortage of deceased donor liver grafts, particularly in Asia, where the rate of deceased donors is negligible. To overcome this shortage, living donor liver transplantation (LDLT) has been developed with favorable preliminary results. Other areas of study include expanding the criteria for liver transplantation to include larger tumors, as shown by the group from the University of California, San Francisco (UCSF) and molecular profiling of HCC to improve prognostication and patient selection.\nMILAN CRITERIA FOR LIVER TRANSPLANTATION IN HCC\nIn the past 10 years, results of OLT have improved steadily because of careful patient selection pioneered by the introduction of the Conventional Milan Criteria (CMC) in 1996.3 The aim of these criteria was to achieve a good outcome in patients who fulfilled the criteria and avoid a poor prognosis in patients who exceeded them. This aim was achieved by the Milan group, who showed that the 10-year overall survival surpassed 70% in 300 liver transplants for HCC that fulfilled the CMC. Such good results have been confirmed worldwide.4,5 The elements of the CMC, namely size and number of tumors, have been shown in multivariate analysis to be the only independent variables predicting patient survival and tumor recurrence, with other biological prognostic factors playing a role only within the \u201csize and number\u201d limits (Table\u00a01).\nTABLE\u00a01.Prognostic factors affecting survival of 250 patients transplanted for HCC according to Conventional Milan Criteria at the National Cancer Institute of Milan10-year survival: multivariate analysisVariableHRCI (95%)P valueOverall patient survivalConventional Milan criteria (in vs out)3.11.35\u20136.93.007Tumor-free survivalConventional Milan criteria (in vs out)5.51.39\u201321.27.01Microsatellites (yes vs no)3.61.5\u20138.71.004Microvascular invasion (yes vs no)3.41.36\u20138.76.009Tumor grading (G3 vs G1-2)3.41.04\u201311.14.04\nThe successful outcome of OLT based on the CMC has led to more patients with HCC being routed to transplantation and an increasing number of proposals for expansion of the CMC.6,7 None of these expanded criteria are, however, supported by a robust statistical sample size or prospective validation. In order to investigate proposals to expand the CMC, an unprecedented multicenter study has been conducted in 24 European centers, collecting data from 466 patients transplanted for HCC whose tumors exceeded the CMC at posttransplant pathologic assessment (http:\/\/www.hcc-olt-metroticket.org).5,7 The results have been plotted in a tumor size-and-number Cartesian contour plot showing the 5-year survival probability according to the size and number of HCC nodules detected in the explanted liver. Based on a preliminary analysis, a \u201cHCC forecast chart\u201d has been developed, which can predict 5-year posttransplant survival rates on the basis of morphological tumor characteristics (Fig.\u00a01). It is conceivable that similar chart models obtained from large sample sizes could replace the CMC for OLT in patients with HCC. Unlike the CMC or expanded proposals, which are based on tumor morphology, these chart models can incorporate important variables for OLT in HCC, including priority score for HCC and dropout rate based on donor availability at different centers. Preliminary ad interim analysis shows a significant shift of the isograms of the proposed chart depending on the presence of vascular invasion in explant specimens, confirming the need for reliable biological markers of important tumor characteristics, such as vascular invasion.\nFIG.\u00a01.HCC forecast chart showing probability of 5-year survival based on tumor size and number in explanted liver.\nMILAN VERSUS EXPANDED CRITERIA FOR LIVER TRANSPLANTATION\nFollowing the 1996 publication by the group from Milan, using a restrictive set of criteria for OLT in patients with HCC, excellent 5-year posttransplant patient survival of at least 70% has been reported from many centers.4,5 The growing experience and success of OLT for HCC have fueled controversies related to expansion of the Milan criteria for OLT, since many studies have suggested that tumor stage beyond the Milan criteria does not necessarily predict worse survival after OLT.8,9 Among the proposed expanded criteria, the UCSF criteria (single tumor nodule up to 6.5 cm; or three or fewer tumors, the largest of which is \u22644.5 cm with the sum of the tumor diameters \u22648 cm) reflect a modest expansion of tumor size limits beyond the Milan criteria.6 At least three transplant experiences from Europe and the United States, however, underlined the limitations of applicability of the UCSF criteria in the pretransplant setting, considering that most of the patients adhering to the UCSF were also within the Milan criteria.10\u201312 Even though the UCSF criteria have been independently validated in several studies, the overlapping population of patients adhering to the UCSF but not the Milan criteria is often negligible and estimated to be <10% of the total transplanted population.12,13 Such a limitation was evidenced by a multicenter study from France, in which 39 of 461 patients (8.7%) had explanted tumors beyond the Milan but within the UCSF criteria.10 Although the 5-year survival of 67% for patients within the UCSF criteria was equivalent to that of the 183 patients meeting the Milan criteria (and significantly better than the 34% 5-year survival rate among 238 patients exceeding both criteria), the 44 patients meeting UCSF but exceeding Milan criteria at pretransplant staging had a 5-year survival rate of only 48%, compared with 60% observed in the 272 patients within Milan criteria and with 37% in the 121 patients beyond both criteria.\nThe limitations of pretransplant imaging studies, exemplified by tumor understaging in 20% of patients, have been a major concern for liberalizing the existing criteria for OLT.5 The UCSF group applied their proposed criteria according to preoperative imaging in 138 patients over a 5-year period; the 1- and 5-year recurrence-free probabilities were 95 and 91%, and the respective probabilities for recurrence-free survival were 91 and 80%.14 The 106 patients with HCC meeting the Milan criteria (T1\/2) had 1- and 5-year recurrence-free probabilities of 96 and 90%, respectively, compared with 93% at both 1 and 5 years for patients with HCC exceeding Milan but meeting UCSF criteria (T3A). Understaging by preoperative imaging was observed in 21% of T1\/2 and 28% of T3A tumors. When explant tumor stage exceeded the UCSF criteria, the 1- and 5-year recurrence-free probabilities were 79 and 61%, compared with 98 and 97% within the UCSF criteria. Patients meeting T3A criteria did not have a significantly higher incidence of poorly differentiated tumor grade or vascular invasion.\nIn the current era of increasing demand and unrelenting organ shortage, the foundation of the debate regarding expansion of the Milan criteria for HCC may ultimately rest on what the transplant community would consider an acceptable survival after OLT for HCC. Some groups have proposed a 50% 5-year patient survival to be the minimum acceptable cutoff.5 This mark may have been surpassed by the UCSF group, who have applied expanded criteria to benefit an additional 10% of patients with HCC with respect to posttransplant survival and tumor recurrence.\nLIVING DONOR LIVER TRANSPLANTATION FOR HCC: EASTERN EXPERIENCE\nLiver transplantation offers the best chance of cure for early unresectable HCC. However, its role has been limited by the shortage of deceased donor liver grafts, which is particularly severe in Asia, where the deceased donor organ rates are fewer than 5 donors per million population, compared with 10\u201335 donors per million population in Western countries.15 In most Asian countries, HCC is the most common cancer and the most frequent indication for OLT, aggravating the unmet demand for liver grafts. The development of living donor liver transplantation (LDLT), especially adult-to-adult right lobe liver transplantation, has allowed more patients with HCC to benefit from OLT.16 LDLT can theoretically provide an unlimited source of liver grafts and eliminate the uncertainty of prolonged waiting times and the risk of dropout due to tumor progression. However, LDLT is a novel treatment for HCC with unresolved issues regarding indications and results.\nRecent studies on LDLT for HCC suggest favorable long-term survival results.17,18 However, it remains unclear whether the outcome after LDLT for HCC is equivalent to that of deceased donor liver transplantation (DDLT). A study from a Korean group reported similar 3-year survival rates after LDLT and DDLT (91.4 vs 89.9%) in patients with tumors fulfilling the Milan criteria, after excluding perioperative mortality.13 However, others have found significantly higher rates of tumor recurrence after LDLT compared with DDLT for HCC.19 This may be related to selection bias, as LDLT eliminates the waiting period for grafts. In DDLT the waiting period provides time for a natural selection process in which patients with biologically more aggressive tumors drop out due to tumor progression. In addition, the ischemic-reperfusion injury associated with small-for-size grafts in LDLT and angiogenesis associated with liver regeneration may theoretically promote growth of tumors in the transplanted liver after LDLT, although the actual clinical impact of such biological processes remains unclear.20,21\nLiver transplantation is conventionally offered to Child-Pugh class C patients with unresectable early HCC. Recently, there have been heated debates on whether liver transplantation should be used as first-line therapy for Child-Pugh class A patients with early HCC.22 While some previous studies showed that liver transplantation for early HCC may achieve better survival compared with resection, this may be partly related to selection bias in favor of transplantation because patients with more aggressive tumors drop off the waiting list for DDLT.19 Recent studies suggest that for patients with preserved liver function and early stage HCC, hepatic resection can achieve a 5-year survival rate of 70%, comparable to that after OLT.13,23\nThe availability of grafts from dedicated live donors has been considered one of the main arguments favoring LDLT as primary therapy for patients with early HCC and preserved liver function. However, even in Asian countries where LDLT is commonly performed, up to half of the patients with early HCC may not have suitable living donors for various reasons, including ABO-incompatibility, hepatitis serology, and patient refusal to accept living donation.18 Furthermore, the risks of donor hepatectomy, with morbidity and mortality rates of 14\u201321% and 0.25\u20131%, respectively, should be carefully balanced against the benefit of LDLT.19 Risking the life of a donor for HCC patients who have an alternative option of hepatic resection, which achieves long-term survival equivalent to LDLT, is ethically not acceptable to many Asian surgeons. Most Asian centers still consider resection as first-line treatment for HCC patients with preserved liver function and reserve LDLT as an option for salvage transplantation in patients with recurrent tumors.13,23,24\nAnother matter of debate in LDLT involves expanding the indications beyond the CMC or UCSF criteria. LDLT is currently being performed in some Asian centers for patients with HCC beyond the Milan criteria, with results that are expectedly worse than those for patients within the Milan criteria.13,17 Some transplant surgeons argue that despite the poorer results, LDLT for advanced HCC may be justified, since donors voluntarily accept the risks of donor hepatectomy to dedicate a graft for HCC patients, who may otherwise have no effective treatment options. However, others argue that the medical profession should not relegate the issue to individual donor autonomy.25 With the lack of clear data showing benefits of LDLT for advanced HCC, the medical community should take a conservative moral stand and limit the use of LDLT for HCC that meets the same criteria as DDLT.\nLIVING DONOR LIVER TRANSPLANTATION FOR HCC: WESTERN EXPERIENCE\nOver the past 10 years, LDLT has developed as an alternative to DDLT because of the scarcity of deceased donor livers. Patient survival after LDLT is similar to that after DDLT, despite data from the UNOS database showing lower post-LDLT graft survival, possibly due to the learning curve for LDLT and lower graft-to-recipient body weight ratio.26 In the United States, enthusiasm for LDLT peaked in 2001, when more than 500 LDLTs were performed; the aftermath of a widely-publicized donor death in 2002 led to a nationwide retrenchment, with no more than 325 procedures per year since then. Nevertheless, LDLT has an important role in the treatment of HCC. While previously there was concern over possible tumor stimulation due to regeneration after LDLT, it appears that the type of graft (living vs deceased donor) has little, if any, impact on post-OLT tumor progression.\nTypically, LDLT results in a liver graft that is smaller than the expected liver volume of the recipient. The outcome of a relatively small donor organ depends not only on graft size but also on the recipient\u2019s preoperative degree of liver failure and portal hypertension. Compared with patients awaiting transplant for end-stage cirrhosis, candidates with HCC generally have better-preserved liver function and less portal hypertension, and are thus better able to tolerate a small graft.\nReady availability is the most important advantage of LDLT for HCC. Posttransplant tumor recurrence is tied to pretransplant tumor stage; progression of tumor while awaiting transplant can only worsen the prognosis. This fact is reflected in the United States allocation scheme for deceased donor livers, which accords priority to patients with HCC meeting the Milan criteria. Since adoption of the Model of End-Stage Liver Disease organ allocation policy in 2002, candidates with T2 HCC (1 nodule between 2 and 5 cm; or 2\u20133 nodules, all \u22643 cm) have enjoyed priority such that in many regions of the United States, waiting times are short, obviating the need for LDLT.27 In some regions, however, longer waiting times and attendant higher dropout rates support the use of LDLT in cases of HCC within the Milan criteria.\nThe Milan criteria were adopted because they identify a subgroup of candidates with HCC for whom transplant results are similar to those in patients transplanted for end-stage cirrhosis without HCC. It is widely recognized, however, that many patients with HCC beyond the Milan criteria can be cured by OLT.8,9 In the United States, adoption of expanded priority criteria for DDLT, as proposed by the UCSF group, is currently under consideration. LDLT can be undertaken in candidates who do not meet criteria for waiting list priority. Broadening criteria based on tumor size and number will inevitably lead to more recurrence; nevertheless, Roayaie et al. demonstrated 55% freedom from recurrence at 5 years in patients transplanted with HCC between 5 and 7 cm and no macroscopic vascular invasion.28 Based on data such as these, many centers offer LDLT to patients with HCC meeting expanded criteria that are estimated to yield a 5-year survival of approximately 50%.5\nSELECTION IN HCC: BIOLOGY OR MORPHOLOGY\nThe distinction between biology and morphology in simple terms can be described as behavior versus appearance. Ultimately, the behavior of HCC is the final deciding factor on patient outcome. The ability to predict the biology of HCC is desperately needed for patient selection in OLT. The current staging criteria for HCC takes into account tumor morphology but not tumor biology, with transplantable stages stratified according to tumor size and number. For treatment options other than liver transplantation (i.e., liver resection, chemotherapy, and cytoablative therapies), tumor morphology as described by the pathological TNM staging system is reasonably adequate. For liver transplantation, however, gross and microscopic morphology are inadequate for predicting outcomes because of the scarcity of the donor organs that must be judiciously allocated. The issue would be less significant if resources were unlimited.\nIdeally, the TNM or morphology-based staging system would be able to stratify patients into homogeneous groups with the same outcomes. However, the current staging systems are not precise enough to segregate patients with HCC and cirrhosis into such homogeneous groups. As almost all patients with gross invasion of the hepatic venous system, positive lymph nodes, or metastatic disease experience recurrence and are therefore not candidates for transplantation, the N and M components of the TNM classification can immediately be eliminated from the organ allocation scheme. This leaves only the T component upon which we can base our biological prediction, namely tumor size and number.\nIn our efforts to refine our current staging systems, morphological or chemical factors have been studied, including microscopic vascular invasion, encapsulation, plasma albumin mRNA, and serum alpha-fetoprotein.5 To date, however, none of these have improved upon the current systems. Part of the problem has been radiologic techniques are not yet sufficient to perfectly define tumor number and size and will never be able to determine microscopic vascular invasion. Moreover, many new staging systems require pretreatment biopsy, which poses a small but real risk of tumor seeding.\nTo understand the biological behavior and identify genes associated with survival after OLT, Marsh and colleagues at the University of Pittsburgh performed microdissection on explanted tissue and studied DNA mutations near 9 tumor-associated gene loci to create an index of cumulative mutational damage, termed the fractional allelic imbalance (FAI).29,30 They found that FAI and vascular invasion were the strongest independent predictors of tumor-free survival. Thus, incorporation of gene mutational data allows desegregation of HCC patients from imprecise, morphology-based staging systems and allows improved prognostication.\nCONCLUSIONS\nWhile patients with early resectable HCC and preserved hepatic function should undergo surgical resection, in those with unresectable disease due to underlying liver dysfunction, orthotopic liver transplantation (OLT) offers the best chance for cure. In the past 10 years, results of OLT have steadily improved because of careful patient selection pioneered by the introduction of the Milan criteria in 1996. Supported by studies showing that many patients with tumor stage beyond the Milan criteria can be cured by OLT, a number of expanded criteria have been proposed. While expanding the criteria for OLT allows more patients to be eligible for transplantation, arguments against expanding the criteria include the increased risk of vascular invasion and tumor recurrence at higher stages of HCC. The principal limitation of OLT for HCC is the shortage of deceased donor living grafts, especially in Asia. The development of LDLT has allowed more patients to benefit from OLT with favorable preliminary survival results. Given the shortage of organs available for OLT and lack of predictive power of currently used staging systems, improved prognostic tools are needed to predict outcomes after OLT. Molecular markers of cancer progression may add significant discriminatory power to the current staging systems and may improve organ allocation schemes for patients with HCC.","keyphrases":["transplantation","hepatocellular","hepatoma"],"prmu":["P","P","U"]}
{"id":"Pediatr_Radiol-4-1-2292499","title":"Sonographic findings in bacterial meningitis in neonates and young infants\n","text":"Cranial sonography plays an important role in the initial evaluation of infants with suspected bacterial meningitis and in monitoring for complications of the disease. Echogenic widening of the brain sulci, meningeal thickening and hyperemia suggest the diagnosis in an at-risk population. Sonography can identify the presence of extra-axial fluid collections, and color Doppler sonography can be very helpful in differentiating benign enlargement of subarachnoid spaces from subdural effusions. Intraventricular debris and stranding, and an irregular and echogenic ependyma are highly suggestive findings associated with ventriculitis. Sonography can play an important role in the detection of postinfectious hydrocephalus, in the determination of the level of obstruction, and in the evaluation of intracranial compliance. Focal or diffuse parenchymal involvement can represent parenchymal involvement by cerebritis, infarction, secondary hemorrhage or early abscess.\nIntroduction\nBacterial meningitis is an uncommon but serious condition in the newborn and infant child. The incidence of bacterial meningitis has shown a dramatic decline in the last 25\u00a0years in the United States [1]. But despite a number of recent advances in neonatal intensive care, rapid bacteriological assays, and potent antibiotics, neonatal bacterial meningitis remains an important cause of neurological disability worldwide [2\u20134]. Early diagnosis of the primary disease and its complications is essential for the prevention of neurodevelopmental sequelae. Cranial sonography is often the first imaging modality used in the evaluation of critically ill infants with suspected meningitis. Thus, familiarity with the pathophysiology and sonographic appearance of bacterial meningitis is important for the radiologist and neonatologist taking care of these patients. In this article we review and update the US features of bacterial meningitis and its complications with emphasis on the relationship between imaging findings and underlying pathological changes.\nPathophysiology of meningitis\nBacterial meningitis usually occurs as a result of bacteremia and sepsis, with initial seeding of the central nervous system via the choroid plexus. The infection then spreads into the cerebrospinal fluid (CSF) and can cause inflammation of the ventricular system (ventriculitis). Inflammation of the meninges typically follows [2, 5]. A key feature of arachnoiditis is the presence of vasculitis, predominantly in small and medium-size veins that traverse the subarachnoid space [5]. Meningeal inflammation extends to the walls of the bridging and cortical veins to cause thrombophlebitis and vascular occlusion, which is associated with cortical infarcts [5, 6]. A number of bacterial virulence factors and inflammatory cytokines contribute to the inflammatory process, resulting in diffuse cerebral edema, increased intracranial pressure, and alterations of cerebral blood flow [7\u20139]. Lipopolysaccharides found in the bacterial cell wall can lead to a disruption in the blood\u2013brain barrier and invasion of microorganisms into deeper cerebral structures [10, 11]. Arachnoiditis and ventriculitis might also contribute to a more generalized encephalopathy characterized by neuronal loss, gliosis and periventricular leukomalacia [2]. Later pathological findings include diffuse cerebral cortical and white matter atrophy, hydrocephalus, which can also be seen in the early phase, multicystic encephalomalacia, porencephaly and ventricular septations [2, 3, 5, 12, 13].\nIn the acute phase of meningitis, marked vasodilatation of cortical vessels is often present as the result of potent vasogenic effects of bacterial toxins. In later stages of the disease, there is a global reduction in cerebral blood flow attributed to impairment of cerebral autoregulation and brain edema [14].\nIn the newborn infant, streptococcus group B, Escherichia coli, and Listeria monocytogenes are the most commonly isolated organisms in CSF. Although other gram-negative enteric organisms such as Klebsiella species and Citrobacter species are less common, they can have far more devastating consequences [15].\nSonographic features of meningitis\nSonographic abnormalities are present in approximately 65% of infants with acute bacterial meningitis [3, 16\u201322]. However, the frequency of imaging abnormalities in patients with a clinical presentation complicated by persistent seizures, abnormal neurological findings and a deterioration of CSF examination within the first 48\u00a0h can be as high as 100% [17]. The high incidence of positive sonograms in infants with suspected complications resulted in the recommendation by some authors to use sonography only when there is a clinical suspicion of a complication [3]. Han et al. [3] found that no infant without a clinical indication of complications of meningitis had clinically significant findings on sonography. However, other authors suggest a sonographic examination should be used on every infant with or without evidence of complications on the basis of reports of a high incidence of positive findings in infants without any clinical indication of complications [17, 23, 24]. Rosenberg et al. [23] suggest an initial examination at the time of diagnosis of bacterial meningitis and a follow-up study within 1\u00a0week if the initial study demonstrates any parenchymal or ventricular abnormality.\nWe recommend cranial sonography as a baseline study in every infant who has an adequate size fontanel if the diagnosis of bacterial meningitis is suspected clinically. Because the major advantage of sonography is its ability to be safely repeated, a second study should be performed if any clinical deterioration occurs, such as increasing head circumference, occurrence of new neurological findings, and lack of response to therapy. However, in patients with complicated bacterial meningitis whose clinical situation is sufficiently stable to leave the nursery, MRI should be the next study of choice. In children, MRI is widely accepted for its safety and high soft-tissue resolution. It is superior to sonography in showing the existence and extension of the complications, especially in the posterior fossa. It has the further advantage of not using ionizing radiation.\nIt is essential to use state-of-the-art equipment with optimized settings including linear, curved and vector probes with variable megahertz in order to perform a complete evaluation of the meninges because of the often subtle sonographic changes of bacterial meningitis [25, 26].\nMeningeal and extra-axial findings\nIn healthy individuals, the pia-arachnoidal membrane is seen as an echogenic line on the surface of the brain. Normal thickness of the membrane measured from the surface of a frontal gyrus (single layer) and within a sulcus (double layer) should not exceed 1.3\u00a0mm and 2\u00a0mm, respectively [27]. Echogenic widening of brain sulci, or meningeal thickening, is the most common and earliest sonographic sign of meningitis, seen in 26\u201383% of affected patients (Fig.\u00a01) [3, 16\u201320, 28]. Prominent cortical vessels can be seen within the pia-arachnoid on color Doppler sonography (Fig.\u00a02). These findings reflect the intense inflammatory exudate that accumulates in the depths of the fissures and sulci, especially around the pial and subarachnoid vessels [5]. Despite their common occurrence, signs of meningeal inflammation are temporary and unrelated to any significant clinical or neurological outcome [3, 16, 20].\nFig.\u00a01Thickened meninges. a Coronal sonogram through frontal lobes in an 11-week-old infant with group streptococcus group B meningitis demonstrates diffuse echogenic thickening of the leptomeninges (arrows) and prominent extra-axial fluid spaces (asterisk). b, c Coronal sonogram on a different infant with group streptococcus group B meningitis (b) shows marked thickening of the suprasellar cisterns (arrows), compared to a normal infant (c)Fig.\u00a02Vasodilatation of pial vessels. Angled sagittal midline color Doppler sonogram in a 3-week-old infant with streptococcus group B meningitis shows prominent vascularity along the surface of the brain parenchyma and a patent superior sagittal sinus (arrowheads)\nAccumulation of extra-axial fluid might be present in 8\u201333% of infants with bacterial meningitis. Most commonly, these represent sterile, reactive subdural effusions that have no prognostic significance [3, 16, 19, 20, 28]. On sonography, they appear as hypoechoic concave fluid spaces, sometimes containing mobile echogenic debris (Fig.\u00a03). Empyema is a very rare complication of meningitis, present in up to 1% of affected patients [3, 5, 17, 24]. Although it is often difficult to differentiate a sterile effusion from an early empyema [28], an enlarging complex extra-axial fluid space is a concerning finding. Such spaces can become quite large and cause a mass effect on the adjacent brain parenchyma [29] (Fig.\u00a04). Clinical findings including persistent fever, new focal neurological signs or seizures are not sufficient for suspicion of subdural empyema because they are not specific for the diagnosis of empyema [30]. Hence, a combination of imaging modalities and clinical evaluation is necessary for the early diagnosis of subdural empyema. The final diagnosis is based on the analysis of the subdural effusion [31].\nFig.\u00a03Subdural effusion. a Coronal midline sonogram in a 3-week-old infant with streptococcus group B meningitis shows echogenic debris within subdural fluid. b Axial diffusion-weighted MR image shows markedly hyperintense signal in the extra-axial fluid consistent with restricted diffusion caused by meningeal edema or pusFig.\u00a04Subdural empyema. a Angled sagittal sonogram in a 1-week-old infant with E. coli meningitis shows thickened septations in the subdural space (arrows). b Repeated sonogram at 2\u00a0weeks of age shows a complex heterogeneous collection consistent with focal empyema (arrows). Needle aspiration confirmed the presence of pus\nThe distinction between subarachnoid and subdural location of extra-axial fluid is important in the management of infants with meningitis. Isolated subarachnoid fluid spaces up to 3.3\u00a0mm are considered incidental findings of no clinical importance in meningitic infants, whereas neurosurgical evacuation is the therapy of choice for infected subdural effusions (empyema) [25, 32]. Early detection with frequent sonographic monitoring of subdural effusions might lessen the neurological sequelae in infantile and neonatal purulent meningitis [33]. Color Doppler sonography can be very helpful in differentiating benign enlargement of subarachnoid spaces from subdural effusions [34, 35]. When subarachnoid fluid is present cortical vessels on the brain surface are surrounded by fluid, whereas fluid in the subdural space compresses the cortical vessels along the surface of the brain (Fig.\u00a05).\nFig.\u00a05Subdural fluid. Midline coronal color Doppler sonogram in an 11-week-old infant with group streptococcus group B meningitis shows cortical vessels along the surface of the left hemisphere (blue vessels). Vasculature is located in the pia-arachnoid space and is not surrounded by fluid. This finding strongly suggests that fluid is in the subdural space. Note the superior sagittal sinus (asterisk)\nIntraventricular findings\nThe most common sonographic signs of ventriculitis include an irregular and echogenic ependyma, the presence of intraventricular debris and stranding, often associated with ventricular dilatation. Debris in the ventricles can be caused by any infecting organism; however, it is most frequently seen with E. coli meningitis [3, 36]. The amount and coarseness of intraventricular debris can vary considerably (Fig.\u00a06) and reflects the extent of ventriculitis. Sonographic resolution of intraventricular debris reflects the efficacy of antibiotic treatment [37]. Inflammation within the ventricles can cause intraventricular adhesions and formation of septae as a more chronic complication of meningitis in about 10% of patients [21, 28, 38]. These adhesions can cause compartmentalization in the ventricles, leading to intraventricular cyst formation (Fig.\u00a07). The identification of the ventricular septations is very important in planning for appropriate shunt placement. Sonography is the study of choice for the demonstration of these septations, often demonstrating them better than CT [38].\nFig.\u00a06Ventriculitis. a\u2013c Coronal (a), left sagittal (b), and mastoid sonograms (c) obtained in a 12-day-old premature infant with E. coli meningitis show extensive septations and echogenic debris clots filling the lateral, third and fourth ventricles. Note the echogenic thickening of edematous ependyma (arrows). d Repeat coronal sonogram at 19\u00a0days of age shows a decrease in size and heterogeneity of the intraventricular debris. Note progression of ventricular dilatationFig.\u00a07Cyst formation. Coronal image obtained in a 5-week-old infant with E. coli meningitis shows a midline cyst displacing the third ventricle (arrow) superiorly and to the right\nAlthough histopathological studies have shown exudative inflammation of the choroid plexus (plexitis) and of the ependymal lining (ventriculitis) in up to 93% of those autopsied [5, 13], only those with more severe involvement exhibit abnormal findings on cranial sonography [3, 16, 20, 28]. By US examination, the choroid plexus can appear more echogenic and its contours irregular and poorly defined [16, 28, 38, 39]. The ependyma can have a thick, irregular and echogenic appearance, as well [20, 28, 37\u201339].\nParenchymal findings\nAbnormal parenchymal echogenicity\nAreas of abnormal brain echogenicity have been reported in 12% to 65% of infants with bacterial meningitis [3, 20, 28]. Lesions can be focal or diffuse and can represent parenchymal involvement by cerebritis, infarction, secondary hemorrhage or early abscess [3, 17, 40]. The presence and size of parenchymal lesions are associated with significant neurological sequelae and are indicators of a poor prognosis [3].\nOne of the most devastating complications of bacterial meningitis is abscess formation. It has been reported with a frequency of between 1% and 18% in newborns with meningitis [2, 3, 16, 21, 28]. Mortality rates are significant (15\u201375%), and up to 66% of survivors develop neurological sequelae [41, 42].\nMost cerebral abscesses are caused by a member of the Enterobacteriaceae family (Citrobacter and Enterobacter species) [43, 44]. Citrobacter koseri (formerly C. diversus) is the main causative agent leading to abscess formation in the brain [43, 45]. Up to 77% of infected patients develop an intracerebral abscess. The major pathological feature of Citrobacter meningitis is vasculitis followed by infarction, with necrosis and liquefaction of large portions of the white matter of the hemispheres [45, 46]. Hemorrhagic necrosis and liquefaction can also occur [43, 46]. Surgical drainage of these lesions is not recommended to avoid further damage to surrounding brain parenchyma [46].\nThe sonographic appearance of abscess varies with the stage of the infection. During the initial stage of cerebritis, sonography might only show a poorly marginated area of increased echogenicity with increased vascularity using color or power Doppler [2] (Fig.\u00a08). As the abscess matures the lesion becomes a well-circumscribed, complex solid mass with highly echogenic walls [3, 21]. After progression with\/or without antibiotic therapy the lesions progress to cavitation with marked peripheral hyperemia [16, 21, 47, 48]. Sonographic differentiation between hemorrhagic necrosis and abscess is not always possible, and percutaneous sampling of the intracavitary fluid might be necessary. Sonographic guidance can be useful for puncture, aspiration, and irrigation of the abscess cavity [49, 50].\nFig.\u00a08Liquefactive cerebritis. a, b Coronal (a) and sagittal right (b) sonograms through the frontal lobes in a full-term infant with Citrobacter meningitis show extensive echogenic cerebritis involving the right frontal lobe and basal ganglia. c Repeated sonogram 5\u00a0days later shows early liquefaction of the frontal lobe (arrow). d, e Coronal sonogram obtained after 7\u00a0days (d) demonstrates complete liquefaction of involved brain and surrounding hyperemia on color Doppler sonography (e). Needle aspiration revealed necrotic tissue with no organism\nBrain edema\nIn the acute stage of meningitis, both intra- and extracellular edema can occur. As a result, the lateral ventricles as well as the other cisternal structures can be compressed or obliterated [5]. The range of ventricular size is quite wide, from mild compression to complete sonographic obliteration, the latter being a poor prognostic factor [28, 51]. On sonography, there might be a diffuse or heterogeneous increase in the echogenicity of the brain with effacement of the sulci and gyri [20, 23, 28, 50, 52] (Fig.\u00a09). Increased intracranial pressure is associated with increased pulsatility of arterial flow, reflected in elevated resistive index (RI) values on pulsed Doppler sonography. Other causes of elevated pulsatility of flow include venous thrombosis (Fig.\u00a010), large extra-axial fluid collections, and postinfectious hydrocephalus. Retrograde blood flow during diastole is an indication of markedly altered intracranial compliance [53\u201357]. When equivalent antegrade and retrograde blood flow is present during numerous cardiac cycles, this might be an indication of nonviable brain blood flow [35, 58].\nFig.\u00a09Brain edema. Coronal sonogram in a 7-day-old infant with group B streptococcus meningitis shows generalized edema with compression of lateral ventricles and sulci and diffuse increased heterogeneity of the cerebral hemispheresFig.\u00a010Sagittal sinus thrombosis, nonviable blood flow. a Coronal color Doppler image an 18-day-old infant with group B streptococcus meningitis shows flow limited to internal carotid arteries at the base of the brain. b On the magnified coronal color Doppler image no flow is identified in the superficial sagittal sinus (arrows), and the thrombosis of the sinus was also confirmed on sagittal scans using high-sensitivity Doppler settings (not shown). c Duplex Doppler waveform of the anterior cerebral artery without fontanel compression shows reversal of flow during the entire diastolic phase\nDiffuse cerebral atrophy, multicystic encephalopathy, and porencephaly might be seen as the end-stage of complications of bacterial meningitis.\nHydrocephalus\nVentricular dilatation is present in 14\u201365% of infants with bacterial meningitis and can occur in either the acute or the chronic phase of the disease [16\u201321, 28]. The level of obstruction to CSF flow is usually outside the ventricular system but can occur within the narrowest portions of the ventricles as a result of ependymitis and secondary adhesion. Common locations for intraventricular obstruction include the aqueduct of Sylvius and the exit foramina of the fourth ventricle [16].\nSonography can play an important role in the detection of hydrocephalus, in the determination of the level of obstruction, and in the evaluation of intracranial compliance. In aqueduct stenosis the lateral and third ventricles are disproportionately dilated, the aqueduct is thickened and echogenic and the fourth ventricle is of normal size (Fig.\u00a011). Detection of CSF flow through the aqueduct of Sylvius is possible by imaging the aqueduct using the mastoid fontanel view. Using power Doppler sonography, the anterior fontanel is compressed and quickly released. Particulate debris within the CSF allows power Doppler signal to be detected when CSF flows in a retrograde fashion through a patent aqueduct into the third ventricle [59].\nFig.\u00a011Postinfectious hydrocephalus. a Midline sagittal sonogram in an infant with aqueductal stenosis following group B streptococcus meningitis shows dilatation of the lateral and third ventricles; the fourth ventricle is normal. b Mastoid fontanel view of another infant with group B streptococcus meningitis shows markedly dilated lateral, third, and fourth ventricles secondary to obstruction of the fourth ventricle outlet foramina. Note the echogenic particle in the third ventricle, which could be debris or a clot\nPulsed Doppler interrogation of the anterior cerebral artery before and after compression of the anterior fontanel can be a useful adjunct in the serial monitoring of infants with postinfectious hydrocephalus [53, 54, 57]. Increasing response to fontanel compression as manifested by increasing changes in arterial RI can be an indication for shunt placement in infants with rapidly progressive hydrocephalus.\nSonographic evaluation of the thoracolumbar spine in infants with meningitis might reveal the presence of echogenic debris within the spinal subarachnoid space. This finding has been associated with a higher risk of progressive hydrocephalus [60].\nConclusion\nCranial sonography continues to be an excellent front-line imaging modality in the initial diagnosis and monitoring of infants with acute bacterial meningitis and its potential complications. Its utility is enhanced by the use of alternative scanning approaches and Doppler techniques for identification of ventriculitis, extra-axial fluid collections, and hemodynamic alterations. Spinal sonography might also play a role in assessing the risk of progressive hydrocephalus.","keyphrases":["meningitis","doppler","children","ultrasonography","bacterial infections","central nervous system infections"],"prmu":["P","P","P","U","R","R"]}
{"id":"Vision_Res-2-1-2386851","title":"Contrast discrimination: Second responses reveal the relationship between the mean and variance of visual signals\n","text":"To explain the relationship between first- and second-response accuracies in a detection experiment, Swets, Tanner, and Birdsall [Swets, J., Tanner, W. P., Jr., & Birdsall, T. G. (1961). Decision processes in perception. Psychological Review, 68, 301\u2013340] proposed that the variance of visual signals increased with their means. However, both a low threshold and intrinsic uncertainty produce similar relationships. I measured the relationship between first- and second-response accuracies for suprathreshold contrast discrimination, which is thought to be unaffected by sensory thresholds and intrinsic uncertainty. The results are consistent with a slowly increasing variance.\n1\nIntroduction\nFirst applied to psychophysical data by Tanner\nand Swets (1954), signal-detection theory (SDT) posits that\nall stimuli elicit some sensation. However, due to noise, sensations experienced\nin the absence of stimulus can sometimes be more intense than sensations\nactually elicited by a stimulus. Crucial evidence for these faint hallucinations\ncomes from Swets, Tanner, and Birdsall\u2019s\n(1961) two-response, four-alternative forced-choice (2R4AFC)\ndetection experiment, in which observers reported both their first and second\nchoices for the temporal position of a visual target.1 SDT\npredicts that second-guessing should be better than chance, and this is what\nSwets et al. found.\n1.1\nSignal-detection theory\nAccording to SDT (Green & Swets,\n1966), each stimulus X, gives rise\nto a Gaussian probability-density function (PDF)of sensory intensity x. In its simplest\nform, all PDF\u2019s have the same standard deviation, i.e.,\n\u03c3X\u00a0=\u00a0\u03c3,\u00a0 \u2200X.\n1.2\nIncreasing variance\nSimple SDT proved incapable of explaining Swets et al.\u2019s (1961) 2R4AFC detection experiment.\nInstead, they proposed that sensation variance increased with sensation mean:Increases in the \u201csigma-to-mean ratio\u201d2\nr\u00a0=\u00a0d\u03c3X\/d\u03bcX,\nproduce decreases in both first- and second-response accuracies, but the\nsecond-response accuracies to decrease faster. Swets et al. tried several\nvalues for this ratio and obtained as good fit to their data when\nr\u00a0=\u00a00.25.\n1.3\nIntrinsic uncertainty\nElsewhere (Solomon, 2007),\nSwets et al.\u2019s (1961)\n2R4AFC data have been successfully fit with an alternative model,\nincorporating intrinsic uncertainty. Intrinsic-uncertainty models posit that\nperceived intensity depends on the maximum activity in several independent\nsensory mechanisms, only one of which is actually sensitive to the stimulus.\nGiven a sufficient number M, of these mechanisms, the\nvariance of their maximum activity will decrease as the intensity of the\nstimulus decreases (see Pelli,\n1985, for a graphical demonstration of this). Thus, to\nsome degree, intrinsic uncertainty mimics Swets et al.\u2019s proposal of\nincreasing variance. With suprathreshold stimuli, the maximum activity\nalways occurs in the appropriately tuned mechanism, and the others have no\ninfluence on perception.\n1.4\nLow-threshold theory\nAt odds with SDT is the idea of a sensory threshold, which weak stimuli\nmust exceed to be detected. In a detection task, stimuli that do not exceed\nthe threshold can be selected only when no other stimulus exceeds it, and\nthe observer is forced to make a choice. Swets\net al. (1961) developed this idea into a\n\u201clow-threshold\u201d hybrid of signal-detection and threshold\ntheories. Unlike other models with this name, Swets et al. claimed theirs\ncould fit the 2R4AFC results. Elsewhere (Solomon, 2007), I have corroborated this claim, and\nshown that the fit is not quite as good as those produced by models\nincluding either intrinsic uncertainty or increasing variance.\n1.5\nThis study\nNeither intrinsic uncertainty (Pelli, 1985; Tanner, 1961) nor Swets et al.\u2019s (1961) low-threshold\ntheory requires sensation variance to increase with sensation mean. These\ntheories are somewhat special because they are thought to affect the\nvisibility of only very faint stimuli. Increasing variance, on the other\nhand, has implications for suprathreshold contrast discrimination. For this\nreason, I decided to conduct a 2R4AFC contrast-discrimination experiment.\nThe goal was to obtain an estimate of the sigma-to-mean ratio, which would\nnot be contaminated by intrinsic uncertainty or a low threshold.\n2\nMethods\nThere were five observers: the author (JAS), another psychophysicist who\nunderstood the purposes of the experiment (MJM), two experienced psychophysical\nobservers who were na\u00efve to the purposes of this experiment (FV and MT) and\none further observer who had no previous laboratory experience (NN). As\ndescribed below, NN produced a very high proportion of \u201cfinger\nerrors.\u201d This suggested to us a general unreliability, and no further\nanalyses were performed on his data.\nThe Psychophysica (Watson & Solomon,\n1997a) software used in these experiments is available at\nhttp:\/\/vision.arc.nasa.gov\/mathematica\/psychophysica.html.\nThe 23.5-cd\/m2 display (a Sony GDM F-520 CRT) was viewed in a\ndark room from 1.15\u00a0m. Luminances of vertically adjacent\npixels were effectively independent, and could obtain any value between 1.06 and\n46\u00a0cd\/m2. Stimuli were horizontal,\ncosine-phase Gabor patterns whose wavelength and spatial spread were\n\u03bb\u00a0=\u00a00.25\u00b0 and \u03c3\u00a0=\u00a00.18\u00b0, respectively. Stimuli were flashed simultaneously, in\nfour positions, each marked by four dark spots. The centers of these positions\nformed a 5.6\u00b0\u00a0\u00d7\u00a05.6\u00b0 square\ncentered on fixation (see Fig.\n1).\nOn each trial, three stimuli appeared with a pedestal contrast, which\nvaried between blocks of 90 trials each. The contrast of the fourth stimulus was\nsomewhat greater. After each 0.18-s stimulus exposure, observers gave two\nresponses. The first response indicated which of the four positions the observer\nthought most likely to have contained the high-contrast target. The second\nresponses from JAS and MJM indicated their second choices for the target\nposition. Following their second responses, JAS and MJM received auditory\nfeedback indicating which\u2014if either\u2014of their responses was\ncorrect.\nThe na\u00efve observers were not told that three of the four stimuli would\nhave the same contrast. They were instructed merely to indicate their choices\nfor the positions containing the two highest contrasts, in order. This\nencouraged them to fully consider their second responses, even when they felt\nconfident about their first. The na\u00efve observers received no\nfeedback.\nAlthough I was primarily concerned with suprathreshold contrast\ndiscrimination, I was also eager to replicate Swets\net al.\u2019s (1961) findings at detection threshold. Since\nI was therefore committed to measuring both the left and right ends of the\nthreshold-vs.-contrast function (Nachmias &\nSainsbury, 1974; Fig.\n2), I decided to devote a few\nextra trials to get the middle as well. (Note: due to limited availability, FV\nperformed only the critical conditions, i.e., those with supra-threshold\npedestals.)\nTable 1 shows the number of trials each observer performed with each\npedestal contrast. In it, and in the discussion below, I use the conventional\ndecibel scale of contrast energy: if m is the maximum\navailable contrast, then an x dB stimulus is one that has\na contrast of\nm10x\/20.\nPrior to each trial, the quest procedure (Watson & Pelli, 1983) estimated the performance\nthreshold ct, i.e., the contrast\nincrement required for 62%-accurate first responses. This is halfway between\nchance performance (25%) and a hypothetical ceiling of 99%.\nAs a means of more accurately estimating the ceiling, or equivalently, the\nfrequency of finger errors, a \u221210\u00a0dB increment was used\non one-ninth of the trials. For JAS, the target was given an increment of either\nct\u00a0\u2212\u00a02\u00a0dB or\nct\u00a0+\u00a02\u00a0dB, with equal probability, on the remaining\ntrials. For the other observers, target increments were either\nct\u00a0\u2212\u00a02\u00a0dB or\nct . This modification allowed\nbetter sampling of their psychometric functions. Finally, to further encourage\nthe na\u00efve observers to fully consider their second responses, one of the\nthree alternatives to each of the \u201cobvious\u201d (\u221210\u00a0dB) targets was fixed at \u221216\u00a0dB.\n3\nResults\n3.1\nPsychometric functions\nFor each observer and each pedestal, first-response accuracies were\nmaximum-likelihood3\nfit with a modified Gaussian distribution.In the preceding expression, c is the\nincrement (in dB), \u03a81 is the\nprobability of a correct first response and f is the\nPDF defined in Eq. (1). Threshold\nct, and\n\u03c3 were free parameters, but the frequency\nof finger errors \u03b4, was not allowed to vary\nwith pedestal contrast. These psychometric fits were obtained for purely\ndescriptive purposes. Unlike some of the fits described below, these were\nnot driven by any particular model of performance. Best-fitting values for\n\u03b4 were 0.006, 0.002, 0.048, 0.018 and 0.095\nfor JAS, MJM, FV, MT and NN, respectively. When debriefed, NN reported a\ntendency to respond before the end of a trial.\n3.2\nThreshold-vs.-contrast functions (first response\nonly)\nFig. 2 shows how threshold\nvaries with pedestal contrast. JAS\u2019s and MJM\u2019s thresholds were\nsimilar, and formed the classic \u201cdipper\u201d shaped function. MT did\nnot suffer as much masking. That is, his thresholds with high-contrast\npedestals were lower than the JAS\u2019s and MJM\u2019s. However, his\ndetection threshold\u2014obtained with pedestals having zero contrast (or\n\u2212\u221e dB)\u2014was similar: between \u221224 and \u221223\u00a0dB. FV\u2019s thresholds with high-contrast pedestals fall\nwithin the range spanned by the other observers\u2019, thus we can be\nreasonably confident that these pedestals exceeded her detection threshold,\nas they did for the other observers.\n3.3\nSecond-vs.-first-response-accuracy functions:\nDetection\nFig. 3 shows how first- and second-response accuracies co-varied\nwhen, as in Swets et al.\u2019s\n(1961) experiment, there was no pedestal. Appendix\nA contains a full description\nof the raw data. Several features of Fig.\n3 deserve a detailed description.\nThe axes differ from those used by Swets et\nal. (1961). For the horizontal axes, instead of signal\nstrength, which may not be a linear function of contrast, I prefer\nfirst-response accuracy \u03a81. For\nthe vertical axes, Swets et al. used second-response accuracy, divided by\nthe proportion of first-response errors. However, both first- and\nsecond-response accuracies are subject to measurement error. When one\nuncertain statistic is divided by another, the confidence intervals for the\nquotient are necessarily very large. To avoid this problem, I get rid of the\ndenominator and plot simply second-response accuracy\n\u03a82.\nOne argument against this way of plotting the results is that a large\nportion of the graph will be wasted because\n\u03a82\u00a0\u2a7d\u00a01\u00a0\u2212\u00a0\u03a81. A dotted line has been\nadded to each graph to indicate this upper limit for second-response\naccuracy. Similarly, \u03a82\u00a0\u2a7d\u00a0\u03a81. Of course, given a\nfinite number of trials, we may find that the frequency of correct second\nresponses P2 exceeds the frequency\nof correct first responses P1, but\nonly a perverse observer would have a greater\nprobability of being correct in his or her second\nresponse. Therefore I have added another dotted line to each graph to\nindicate this other upper limit for second-response accuracy.\nEach point in Fig. 3\nrepresents data collected with a unique target contrast. Several of these\npoints reflect only a few responses made at the beginning of the experiment,\nbefore the adaptive staircase had converged. We can thus have little\nconfidence in the likelihood of a correct first or second response with\nthese targets. To convey this confidence (or lack thereof), I have plotted\n95% confidence intervals, both horizontally and vertically, about each\npoint. These intervals are based on binomial probabilities, calculated from\nthe range defined by the limits described in the preceding paragraph (see\nAppendix B for details).\n3.4\nModeling finger errors\nSDT can be modified to accommodate finger errors. Let\n\u03c81 denote the first-response\naccuracy without errors. For those trials containing a first-response finger\nerror, the probability of a correct first response is (1\u00a0\u2212\u00a0\u03c81)\/3. Thus, if the\nfinger-error rate is \u03b4, the overall probability\nof a correct first response is (1\u00a0\u2212\u00a0\u03b4)\u03c81\u00a0+\u00a0\u03b4(1\u00a0\u2212\u00a0\u03c81)\/3.\nTo derive the formula for second-response accuracy, it helps to\nunderstand that first-response finger-errors will be incorrect with\nprobability 1\u00a0\u2212\u00a0[(1\u00a0\u2212\u00a0\u03c81)\/3]\u00a0=\u00a0(2\u00a0+\u00a0\u03c81)\/3. Without loss of\ngenerality, we may assume that observers correct some proportion\n\u03b5 of first-response finger errors with\ntheir second response. Thus on these trials, the probability of a correct\nsecond response is \u03c81. When\nfirst-response finger-errors are not explicitly corrected, I will assume\nthat the second response is completely random. On these latter trials, the\nsecond response will be correct with probability [(2\u00a0+\u00a0\u03c81)\/3]\/3\u00a0=\u00a0(2\u00a0+\u00a0\u03c81)\/9. Thus, on those\ntrials in which a first-response finger error occurred, the second-response\naccuracy should beSecond-response accuracy overall will be (1\u00a0\u2212\u00a0\u03b4)\u03c82\u00a0+\u00a0\u03b4\n[\u03b5\u03c81\u00a0+\u00a0(1\u00a0\u2212\u00a0\u03b5)(2\u00a0+\u00a0\u03c81)\/9], where\n\u03c82 would have been the\nsecond-response accuracy, had there been no first-response finger\nerrors.\nTo estimate \u03b5, trials containing an\n\u201cobvious\u201d (\u221210\u00a0dB) increment and an\nincorrect first response were examined. (Because the highest pedestals had\nthe potential for masking even these large increments, they were excluded\nfrom this analysis.) On these trials, we may assert that\n\u03c81\u00a0=\u00a01, and we can then solve Eq. (4) for \u03b5.\nSolutions were 0.70, 0, 0.58 and 0 for JAS, MJM, FV and MT,\nrespectively.\n3.5\nMaximum-likelihood fits\nThese values of \u03b5 were assumed when\ncalculating the curves in Figs.\n3\u20136. In Fig. 3, the solid black curves represent the\nprediction of simple SDT, that is, when r\u00a0=\u00a00 in Eq. (2). The dashed lines represent the prediction of\nhigh-threshold theory, which ascribes all errors to unlucky guesses rather\nthan faint hallucinations. (See Swets et al.,\n1961 or Solomon,\n2007, for derivations of these predictions.)\nMaximum-likelihood fits were also obtained for three further\nmodifications of SDT. They were: (i) increasing variance with power-law\ntransduction, (ii) intrinsic uncertainty and (iii) low-threshold theory.\nDetails of these three models can be found in Appendix C, and receiver-operating characteristics\nfor all three models applied to a yes\/no-detection task appear in\nNachmias (1972). The fits\nappear as solid green, blue and red curves, respectively, in Fig. 3.\nGoodness-of-fit is indicated by the generalized (log) likelihood-ratios\nin Table 2. Specifically, these values reflect the maximum log\nlikelihoods, minus the conventional upper-bound on log likelihood described\nin Footnote 3. Note that unlike\nsome generalized likelihood-ratios (Mood,\nGraybill, & Boes, 1974), these cannot be expected to\nfollow the chi-square distribution because there are so many conditions\n(i.e., specific increment contrasts) containing only one or two trials\n(Wichmann & Hill,\n2001).\nOnly for JAS, low-threshold theory produced, by far, the best fits of\nthe three SDT models. In fact, the best-fitting \u201clow\u201d threshold\nfor JAS was effectively a high threshold, never exceeded by zero-contrast\npedestals. That is why JAS\u2019s red curve in Fig. 3 is visually indistinguishable from the dashed\nblack line. Since JAS\u2019s results are consistent with a high threshold,\nthey are inconsistent with the findings and conclusions of Swets et al. (1961).\nFor the other two observers, the sigma-to-mean ratios of the\nbest-fitting increasing-variance models were between 0.26 and 0.28; similar\nto Swets et al.\u2019s (1961)\nestimate of 0.25. Thus, these results can be considered a successful\nreplication of Swets et al.\u2019s. At the end of this section, I speculate\non why JAS\u2019s results differ.\n3.6\nTransducer-independent estimates of\nr\nAt detection threshold, as described above, the raw data were simply\nfit with a multiplicative-noise\/power-law-transducer version of SDT.\nHowever, with suprathreshold pedestals, we cannot be certain what shape the\ntransducer takes. Increasing-variance models (e.g., Kontsevich, Chen, & Tyler, 2002) use a\nsimple power-law transducer, but constant-noise models (e.g., Legge & Foley\u2019s, 1980), use a\ntransducer that is initially expansive, then compressive, as the pedestal\nincreases. The compressive non-linearity is required to produce masking;\ni.e., threshold elevation from high-contrast pedestals.\nWe do not really have to worry about the form of transducer, because\nSDT\u2019s predictions for the relationship between first- and\nsecond-response accuracies are independent of signal transduction\n(Solomon, 2007; Swets et al., 1961). We merely need to\nquantify how these predictions change with the sigma-to-mean ratio, and find\nthe values most consistent with the data. This examination of a\ntransducer-independent facet of contrast-discrimination data is\ncomplementary to attempts at modeling contrast-discrimination data without\nputting any constraint on the form of the transducer (Katkov, Tsodyks, & Sagi, 2006a; Katkov, Tsodyks,\n& Sagi, 2006b; Klein,\n2006).\nTransducer-independent estimates of the sigma-to-mean ratio\nr were obtained by maximizing the likelihood of\nobserving second-response accuracies\nP2, given the first-response\naccuracies P1. A complete\ndescription of this process appears in Appendix D. The best fits were 0.56 for JAS, 0.32 for MJM and\n0.31 for MT. These values are illustrated in Fig.\n4. All of these fits are comparable to the\nmaximum-likelihood fits described above.\n3.7\nBinning accuracy\nFig. 4 is less cluttered than\nFig. 3. It has fewer data\npoints and no horizontal confidence intervals. Nonetheless, the same data\nappear in both figures. For legibility, I have decided to combine data from\nincrements producing similar first-response accuracies, as determined by the\npsychometric functions described above. I have adopted the relatively\narbitrary decision to use 5\u00a0dB bin-widths. There is one\nvisible consequence of this manipulation: a rightward shift for one data\npoint in JAS\u2019s panel. However, without binning, the suprathreshold\ndata presented below would be impossible to read.\nI have also decided to forgo plotting data from increments producing\nboth floor (i.e., 0.25) and ceiling (i.e., 1\u00a0\u2212\u00a0\u03b4, see Eq.\n(3)) accuracies. Such data are\nworthless at discriminating between candidate models. Finally, also for the\nsake of legibility, I have also decided to cull data points representing\nfewer than 10 trials. Of course, these latter data are not completely\nworthless; they have not been excluded from any model fits. A complete\ndescription of the binned data appears in Appendix A.\n3.8\nSecond-vs.-first-response-accuracy functions: Suprathreshold\ndiscrimination\nFig. 5 shows how first- and\nsecond-response accuracies co-varied for the four highest pedestals. (The\nbinned data are tabulated in Appendix A.) Plotting conventions have been inherited from\nFig. 4.\nIt should be apparent that the second responses with these\nsuprathreshold pedestals tend to be more accurate than the second responses\nat detection threshold. Using the same procedure as was described for the\ndetection data (above and in Appendix D), values for the sigma-to-mean ratio\nr were found that maximized the likelihood of\nobserving second-response accuracies\nP2, given the first-response\naccuracies P1. The best fits were\n0.11 for JAS, 0.16 for MJM, 0.21 for FV and 0.09 for MT. These values are\nillustrated in Fig. 5.\nThese values of r are smaller than those\nrequired to fit the detection data. FV\u2019s relatively high value may\nhave something to do with her relatively high finger-error rate. No\ndetection data from FV are available for comparison.\nPedestal-by-pedestal estimates of r appear in\nFig. 6. Most of these\nestimates remain near or below the value of 0.25, selected by Swets et al. (1961), when neither the\neffect of a low threshold nor that of intrinsic uncertainty is considered.\nThe results from JAS are different; best-fitting values of\nr start at 0.56, and decrease to 0 as pedestal\nintensity increases.\nJAS\u2019s small-pedestal estimates of r are\nstrangely high. As noted above, they are not incompatible with the notion\nthat visual noise never exceeds the threshold of visibility in the absence\nof a stimulus. Previous attempts to replicate Swets et al.\u2019s (1961) 2R4AFC results have also met\nwith mixed success. Despite their chronological precedence, Kincaid and Hamilton\u2019s (1959) results\nhave been described as both successful and unsuccessful replications of\nSwets et al.\u2019s (Green & Swets,\n1966 and Blackwell,\n1963, respectively). As yet, I have not been able to\ntrack down a copy of Kincaid and Hamilton\u2019s publication. One further\nattempt to replicate Swets et al. was described by Eijkman and Vendrik (1963). They reported\nthat second responses for light detection were greater than chance in just\none of three observers.\nAn alternative interpretation for JAS\u2019s high-threshold-like\nperformance is that he simply ignored sensory information and selected his\nsecond responses more-or-less randomly. This explanation would be easier to\nswallow if all his estimates of r were high. However,\nwith suprathreshold pedestals, his r is no higher\nthan that of the other observers. Expectation may have caused JAS to change\nhis strategy with pedestal intensity, but I have no definitive\nanswer.\n4\nDiscussion\nThe most equitable summary of these results is that they are consistent\nwith a performance-limiting source of noise, which increases slightly with\nsuprathreshold contrast.\nBest estimates for the rate varied from 0.09 to 0.21. I wondered whether\nsuch small sigma-to-mean ratios would be sufficient to model the high\ncontrast-discrimination thresholds obtained with high-contrast pedestals, or\nwhether a saturating transducer function for stimulus contrast would also be\nnecessary. Previous attempts (Kontsevich et al.,\n2002; Solomon,\n2007) to fit contrast-discrimination data without compressive\ntransduction have not focused on the minimum necessary sigma-to-mean ratio, but\nthat ratio can be inferred from the published parameter values.\n4.1\nFitting contrast discrimination\nIn those previous attempts, the standard deviation of visual signals\nwas allowed to increase as a decelerating power function of the mean. Specifically,(Kontsevich et al. considered only suprathreshold contrast and\nthus could set \u03c30\u00a0=\u00a00; Solomon used\n\u03c30\u00a0=\u00a01.) Therefore, the sigma-to-mean ratios decreased\nas the means increased. From the best-fitting parameter values, I have used\na contrast of 100% to infer the minimum sigma-to-mean ratios required to\nexplain contrast discrimination without compressive transduction. The\nsmallest of these ratios was 0.13 (obtained using the parameter values fit\nto observer SV with \u201csustained\u201d stimuli in Kontsevich et al., 2002). Thus it seems\nthat the sigma-to-mean ratios estimated in the present study may in fact be\nable to produce appreciable masking.\nFour different models were maximum-likelihood fit to all of\nJAS\u2019s, MJM\u2019s and MT\u2019s 2R4AFC data. Details of all four\nmodels appear in Solomon (2007).\nOne of these models was a four-parameter, non-linear transducer model with\nconstant Gaussian noise (i.e., where r\u00a0=\u00a00). This transducer is initially\nexpansive, then compressive, as the input increases. Foley (1994) obtained good fits to 2AFC\ncontrast-discrimination thresholds with this model, but we already know this\nmodel will over-estimate second-response accuracies, particularly in a\n(zero-pedestal) detection experiment (Solomon,\n2007; Swets et al.,\n1961).\nIn the other three models that were fit to JAS\u2019s, MJM\u2019s and\nMT\u2019s data, the variance of visual signals was allowed to increase with\nthe mean. Two of these models have already proven capable of producing\nacceptable fits to some of Foley\u2019s\n(1994) 2AFC thresholds (see Fig. 13 of Solomon, 2007). In one of these, a\npower-law transducer is responsible for the \u2018dip\u2019 in the\ncontrast-discrimination function. In the other, intrinsic uncertainty\nproduces the dip. The remaining increasing-variance model explored in this\npaper uses a low threshold to produce the dip.\nFit details appear in Table\n3. Initially, four\nparameters were allowed to vary freely in each fit. For JAS and MJM the\nbest-fitting model was the one with increasing variance model and intrinsic\nuncertainty. In general, the fits improved as uncertainty\nM, increased. For JAS and MJM, the fits of the\nincreasing-variance\/intrinsic-uncertainty model remained superior when\nM was fixed at a value of 10,000. When the\nrelationship between signal mean and standard deviation was forced to be\nlinear (i.e., q\u00a0=\u00a01 in Eq. (5)), the fits of the\nincreasing-variance\/intrinsic-uncertainty model remained superior. They even\nremained superior when M was fixed at a value of\n1000. Thus, all of JAS and MJM\u2019s data can be satisfyingly summarized\nby a relatively simple model, combining increasing variance with intrinsic\nuncertainty. Within the context of this model the best-fitting values of\nr (regardless of constraints on\nM or q) were 0.16 for JAS\nand 0.14 for MJM. These values are nearly identical to the\ntransducer-independent estimates (0.14 for JAS and 0.16 for MJM), described\nabove.\nWhen MT\u2019s data were fit with a 3-parameter\nincreasing-variance\/intrinsic uncertainty model, the best-fitting values for\nthe sigma-to-mean ratio and intrinsic uncertainty were\nr\u00a0=\u00a00.11 and\nM\u00a0=\u00a0440,\nrespectively. However, his data are best-fit by the constant-variance,\n4-parameter, nonlinear-transducer model (Foley,\n1994). Thus, in two out of three cases, the\ncontrast-discrimination data can be satisfyingly summarized by a 3-parameter\nmodel of intrinsic uncertainty and increasing variance. Compressive\ntransduction is not required.\n4.2\nSensory thresholds for contrast\ndiscrimination\nAlthough \u201csensory threshold\u201d means different things to\ndifferent people (Swets et al.,\n1961), it is usually understood to be some sort of\nbarrier weak stimuli must overcome to be perceived (Swets, 1961). However, it is no less valid\nto apply the concept to the task of contrast discrimination, even with large\npedestals. That is, forced-choice errors may occur simply because all the\nalternatives appear identical and the observer simply guesses\nincorrectly.\nInspection of Fig. 5 should\nbe sufficient to rule out any \u201chigh\u201d sensory threshold for\ncontrast discrimination. The data shown there are even less similar to the\n(dashed) high-threshold prediction than Swets et\nal.\u2019s (1961) detection data (not shown). However,\nsome proportion of correct second responses may indeed have been just lucky\nguesses, which is to say my data cannot rule out a \u201clow\nthreshold\u201d for \u201csuprathreshold\u201d contrast\ndiscrimination.\n4.3\nOther models\nThe logic of this study hinges on the assumption that unmasked,\nsuprathreshold contrast discrimination can be modeled with a single sensory\nmechanism. Although this assumption is popular, it is not completely\nuncontroversial. Before I describe other possible models, I should first\nstress the importance of the word \u201cunmasked.\u201d In a highly\ninfluential paper, Foley (1994)\nargued that the mechanism responsible for contrast discrimination was not\nimmune to the activity in differently tuned mechanisms. By manipulating the\nspatial phase, orientation and temporal frequency of masking stimuli,\nFoley and Boynton (1994) were\nable to probe the interactions between mechanisms responsible for contrast\ndiscrimination. However, when no masks are present, most models (including\nall of Foley\u2019s) consider contrast discrimination to be mediated by a\nsingle mechanism or channel; the one best tuned to the target.\nThere are three notable exceptions. Teo and\nHeeger (1994) and Yu, Klein,\nand Levi (2004) have developed models with greater\nphysiological plausibility, in which individual mechanisms have very limited\ndynamic ranges. Elsewhere (Watson & Solomon,\n1997b) I have argued this type of model is well\napproximated by the more popular, single-mechanism model for contrast\ndiscrimination. The two types of model can be considered equivalent when\nperformance-limiting noise is added after the outputs of multiple mechanisms\nare combined.\nThe third exception was recently proposed by Henning and Wichmann (2007) to account for their finding\nthat the low-contrast \u201cdip\u201d of threshold-vs.-contrast functions\n(e.g., Fig. 2) disappears in the\npresence of a notched-noise mask. This result suggests the dip is due to\noff-frequency looking. That interpretation may be correct, but I am\nobligated to note their results are also consistent with intrinsic\nuncertainty, which would attribute a less-pronounced dip to uncertainty\nreduction. Indeed, Blackwell\n(1998) argued that noise, both within the\ndetector\u2019s pass band and outside it, could reduce intrinsic\nuncertainty and facilitate detection. She also provided psychophysical\nevidence for this facilitation.","keyphrases":["contrast","discrimination","detection","threshold","uncertainty","psychophysics","noise"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Histochem_Cell_Biol-4-1-2228381","title":"Protein quality control: the who\u2019s who, the where\u2019s and therapeutic escapes\n","text":"In cells the quality of newly synthesized proteins is monitored in regard to proper folding and correct assembly in the early secretory pathway, the cytosol and the nucleoplasm. Proteins recognized as non-native in the ER will be removed and degraded by a process termed ERAD. ERAD of aberrant proteins is accompanied by various changes of cellular organelles and results in protein folding diseases. This review focuses on how the immunocytochemical labeling and electron microscopic analyses have helped to disclose the in situ subcellular distribution pattern of some of the key machinery proteins of the cellular protein quality control, the organelle changes due to the presence of misfolded proteins, and the efficiency of synthetic chaperones to rescue disease-causing trafficking defects of aberrant proteins.\nIntroduction\nFolding and assembly of proteins and their function depend on each other. Like in industrial production lines, in cells the quality of newly synthesized proteins is monitored in regard to proper folding and correct assembly in the early secretory pathway, the cytosol and the nucleoplasm (Bennett et al. 2005; Bukau et al. 2006; Dobson 2003; Ellgaard and Helenius 2003; Park et al. 2007; Ravid et al. 2006; Roth 2002; Sitia and Braakman 2003; Zhang and Kaufman 2006). Protein quality control is a basic cellular phenomenon through which aberrant proteins become eliminated. Aberrant proteins can occur as waste products at a certain rate during de novo synthesis, or are caused by cellular stress, or due to disease-causing mutations (Aridor and Hannan 2002; Gregersen et al. 2006; Kim and Arvan 1998; Kopito 2000; Lukacs et al. 1994; Pet\u00e4j\u00e4-Repo et al. 2000; Schubert et al. 2000; Turner and Varshavsky 2000; Ward and Kopito 1994). Once recognized as non-native or incompletely assembled, those proteins will be removed and degraded by a process generally termed ERAD, for ER-associated degradation (Hirsch et al. 2004; Meusser et al. 2005).\nFor secretory and membrane proteins, the molecular machinery involved in the recognition, retention and dislocation of aberrant proteins has been identified to a certain detail (Carvalho et al. 2006; Denic et al. 2006; Hirsch et al. 2003; Ismail and Ng 2006; Katiyar et al. 2005; Li et al. 2006; Lilley and Ploegh 2004; Schuberth and Buchberger 2005; Tsai et al. 2002; Ye et al. 2003, 2004). The ensuing final step consists of polyubiquitination of aberrant proteins condemning them for degradation by proteasomes (Eisele et al. 2006; Hochstrasser 1996; Jarosch et al. 2002; McCracken and Brodsky 2005; Wolf and Hilt 2004; Zwickl et al. 2002). In addition to various chaperones aiding proteins to achieve their proper conformation, various machinery proteins are involved in the recognition and retention of aberrant proteins. For glycoproteins, the importance of specific oligosaccharidic structures generated initially by trimming glucosidase II and UDP-glucose:glycoprotein glucosyltransferase and later on by ER-mannosidase I has been recognized (Helenius and Aebi 2004; Parodi 2000; Roth 2002; Roth et al. 2002). Glycoproteins bearing monoglucosylated oligosaccharides will be bound by calnexin or calreticulin. If the aberrant glycoproteins are considered, binding to calnexin or calreticulin will protect them temporarily from degradation. The complete deglucosylation by glucosidase II will result in their exit from the calnexin\/calreticulin cycle. Subsequent trimming of mannose residue(s) of the oligosaccharide B branch by ER-mannosidase I opens the gate to dislocation and degradation of aberrant proteins. The link between the calnexin\/calreticulin cycle and the dislocation process is apparently provided by two lectin-like proteins: EDEM1 (yeast ortholog Htm1p\/Mnl1p) (Hosokawa et al. 2001; Jakob et al. 2001; Kanehara et al. 2007; Nakatsukasa et al. 2001; Oda et al. 2003) and Yos9p (mammalian orthologues OS-9 and XTP3-B) (Bhamidipati et al. 2005; Buschhorn et al. 2004; Gauss et al. 2006; Kanehara et al. 2007; Kostova and Wolf 2005; Szathmary et al. 2005).\nDepending on the type of protein and the location of the lesion, different ERAD dislocation pathways have been identified (Carvalho et al. 2006; Denic et al. 2006; Ismail and Ng 2006; Schuberth and Buchberger 2005). Aberrant luminal proteins and membrane proteins with a defect in their luminal domain undergo the ERAD-L pathway, which is defined by the E3 ubiquitin ligase Hrd1p complex. The Hrd1p complex consists of several proteins including Hrd3p, an E2 complex (Ubc7p and its membrane-anchoring factor Cue1p), the Cdc48p complex (AAA-ATPase Cdc48p or p97, the Ufd1 and Npl4 cofactors, and the Ubx2p membrane anchor), Der1p, Yos9p, Kar2p (BiP) and Usa1p. It should be noted that the actual function of some of those proteins in the complex remains to be established. Aberrant membrane proteins with lesions in their cytosolic domain enter the ERAD-C pathway organized by the E3 ubiquitin ligase Doa10p complex. This complex is comparably simple and consists in addition to Doa10p only of the E2 complex and the Cdc48p complex. The ERAD-M pathway is followed by membrane proteins with a lesion in their transmembrane domain and involves only Hrd1p and Hrd3p. These dislocation pathways were established for yeast cells but most probably will apply to higher eukaryotes as well because of the evolutionary conservation of the ERAD pathways. The Doa10p complex also operates in the polyubiquitination of aberrant cytosolic and nuclear proteins, in addition to the ERAD-C pathway (Neuber et al. 2005; Ravid et al. 2006; Swanson et al. 2001).\nThis review will focus on how immunocytochemical labeling and electron microscopic analysis have helped to disclose the in situ subcellular distribution pattern of some of the key machinery proteins of the protein quality control, the organelle changes due to the presence of misfolded proteins, and the efficiency of synthetic chaperones to rescue disease-causing trafficking defects of aberrant proteins.\nMachinery proteins of the protein quality control reside beyond the ER\nFor the quality control of glycoprotein folding, glucosidase II (Gls II) and UDP-glucose:glycoprotein glucosyltransferase (GT) in connection with the calnexin\/calreticulin cycle are of eminent importance (Helenius and Aebi 2004; Parodi 2000; Roth 2002). Gls II is a luminal glycoprotein, which exists in two isoforms (Pelletier et al. 2000; Ziak et al. 2001) and does not contain known ER retention signals of the C-terminal KDEL type, nor any hydrophobic region characteristic of transmembrane proteins (Flura et al. 1997; Trombetta et al. 1996). As depicted in Fig. 1a, Gls II acts second to glucosidase I by removing the two inner \u03b11,3-linked glucose residues (Brada and Dubach 1984; Burns and Touster 1982). The presence of three or two glucose residues on oligosaccharides can be considered to represent a trimming glyco-code whereas one glucose residue represents a trimming as well as folding glyco-code (Fig.\u00a01b) (Jakob et al. 1998b). The involvement of Gls II and of mono-glucosylated oligosaccharides generated by the enzyme in the protein quality control is well documented (Hammond et al. 1994; Hebert et al. 1995; Jakob et al. 1998a, b). By confocal immunofluorescence, Gls II not unexpectedly exhibited a pattern typically observed for the ER as shown in Fig. 2b (Roth et al. 2003; Zuber et al. 2001). By high-resolution immunoelectron microscopy, ER localization of Gls II could be definitely established (Lucocq et al. 1986; Zuber et al. 2000, 2001). In addition to the rough ER including the nuclear envelope and the transitional ER, the smooth ER was also positive for Gls II. However, with the superior resolution of electron microscopic immunogold labeling, Gls II was additionally found in tubulovesicular clusters between transitional ER and the cis Golgi apparatus. They represent pre-Golgi intermediates involved in antero- and retrograde transport of cargo (Appenzeller-Herzog and Hauri 2006; Bannykh and Balch 1997, 1998; Hammond and Glick 2000; Palade 1975; Saraste et al. 1987; Schweizer et al. 1988).\nFig.\u00a01Schematic presentation of the oligosaccharide trimming pathway by glucosidase I (Gls I), glucosidase II (Gls II), UDP-glucose:glycoprotein glucosyltransferase (GT) and ER-mannosidase I (ER-Man I)Fig.\u00a02Double confocal immunofluorescence for UDP-glucose:glycoprotein glucosyltransferase (a) and glucosidase II (b) demonstrates co-distribution (c) of the two protein quality control machinery proteins in cultured clone 9 hepatocytes. Immunogold localization of UDP-glucose:glycoprotein glucosyltransferase in an ultrathin frozen section of clone 9 hepatocytes reveals immunoreactivity in the rough ER (ER) including nuclear envelope and a pre-Golgi intermediate (pGI). The cisternal stack of the Golgi apparatus (GA) is not labeled\nMono-glucosylated oligosaccharides of glycoproteins are targeted by the calnexin\/calreticulin cycle and after being deglucosylated by Gls II, will be targeted by GT if not correctly folded (Fig.\u00a01b). GT apparently senses exposed patches of charged amino acids and reglucosylates the aberrant glycoproteins, which is followed by their re-entry in the calnexin\/calreticulin cycle (Parodi et al. 1983; Sousa and Parodi 1995; Trombetta and Parodi 2003). When the subcellular distribution of GT was studied by confocal immunofluorescence, its labeling pattern (Fig.\u00a02a) was alike that observed for Gls II (Fig.\u00a02b; Zuber et al. 2001). By immunoelectron microscopy, GT was detectable in the rough ER including the nuclear envelope and the transitional ER as well as the smooth ER (Fig.\u00a02d). Unlike Gls II, for which the labeling intensity over rough and smooth ER was equal, labeling intensity for GT over smooth ER was only 11% that of the rough ER. GT immunolabeling was also discovered in the pre-Golgi intermediates (Fig.\u00a02d). Notably, the pre-Golgi intermediate immunolabeling for GT was approximately twice that of rough ER (Zuber et al. 2001). Double immunogold labeling for GT combined with the pre-Golgi intermediate marker ERGIC-53 and the COPII component sec23p (Hughes and Stephens 2008) proved the identity of the GT-labeled structures. Interestingly, specific immunogold labeling for calreticulin was also observed in the pre-Golgi intermediates (Zuber et al. 2000, 2001).\nTogether, these results provided new insight into the in situ subcellular organization of some key elements of the protein quality control machinery. Gls II, GT and calreticulin were not only present in the rough ER, as expected, but also in the smooth ER and unequivocally present in pre-Golgi intermediates. This pattern was found in different rat cell lines and tissues as well as Drosophila tissue and cell lines. The presence of three functionally closely associated proteins is a strong evidence for the involvement of pre-Golgi intermediates in protein quality control. Of course, immunolocalization provides no direct evidence for the functionality of the detected protein at a certain location. However, there is no reason to assume that Gls II, GT and calreticulin would be only functional in the ER. Studies in yeast have provided strong evidence that multiple, sequentially acting quality control checkpoints exist along the secretory pathway extending as far as to the Golgi apparatus (Arvan et al. 2002; Caldwell et al. 2001; Sayeed and Ng 2005; Taxis et al. 2002; Vashist et al. 2001; Vashist and Ng 2004; Younger and Chen 2006). In mammalian and insect cells, protein quality control is apparently not restricted to the ER, and the pre-Golgi intermediates appear to be involved in this fundamental cellular process as well. As will be discussed later, pre-Golgi intermediates represent not only a quality control checkpoint, but are also sites of accumulation of aberrant proteins.\nThe ERAD factor EDEM1 defines a novel vesicular ER exit pathway\nAs mentioned in the \u201cIntroduction\u201d, an impressive body of molecular and functional data exists concerning the macromolecular assemblies involved in the various ERAD pathways. The current well-founded basic conception of ERAD in yeast and higher eukaryotes is that the aberrant proteins after being removed from folding cycles are dislocated to the cytosol and eventually degraded by the 26S proteasome, and that this occurs in the ER. It is not clear whether this is a randomly occurring event or a more structured affair. Recent studies on EDEM1 in mammalian cells have provided preliminary evidence for a high level of subcellular organization.\nThe discovery that the Man8 B isomer oligosaccharide was actively involved in ERAD-L (Fig.\u00a01c) in yeast (Jakob et al. 1998a) and mammalian cells (Liu et al. 1999) paved the way to the identification of a lectin-like protein with sequence similarity to class I \u03b11,2-mannosidases in yeast -Htm1p\/Mnl1p- and mammalian cells -EDEM1- (Hosokawa et al. 2001; Jakob et al. 2001; Nakatsukasa et al. 2001). EDEM1 of mammalian cells is a soluble glycoprotein (Olivari et al. 2005; Zuber et al. 2007), which is regulated by the unfolded protein response (Hosokawa et al. 2001) and seems to connect the calnexin\/calreticulin cycle to the dislocation process (Molinari et al. 2003; Oda et al. 2003). EDEM1 appears to exist in complex with the dislocation proteins Derlin-2 and -3, and the AAA ATPase p97 (Oda et al. 2006). It is not fully understood how EDEM1 interacts with aberrant proteins. However, there is evidence for interaction with ER-mannosidase I-trimmed oligosaccharides such as depicted in Fig. 1c (Hosokawa et al. 2003). Overexpression of EDEM1 has been shown to prevent formation of dimers of misfolded Null Hong Kong variant of alpha1-antitrypsin (Hosokawa et al. 2006). Notably, EDEM1 and ER-mannosidase I do not exist in complexes, which can be immunoprecipitated (Hosokawa et al. 2003).\nRecently, the subcellular distribution of endogenous EDEM1 in various mammalian cell types was established with a specific anti-peptide antibody (Zuber et al. 2007). Unexpectedly, its immunofluorescence pattern did not correlate with that of calnexin and other ER marker proteins. Rather, an unusual pattern of well distributed punctate structures along with some localized finger-like structures was revealed (Fig.\u00a03a\u2013c). The distribution patterns of endogenous EDEM1 and that of overexpressed tagged EDEM1 were dramatically different: instead of a punctate, non-ER pattern, a typical reticular ER pattern plus punctate staining was observed (Zuber et al. 2007). This striking difference in subcellular distribution between endogenous EDEM1 and overexpressed tagged EDEM1 was confirmed by Optiprep density gradients. Endogenous EDEM1 was restricted to the densest fractions, whereas tagged EDEM1 showed the same broad distribution as observed for calnexin, sec61\u03b2, and Derlin-1 and -2 (Zuber et al. 2007). In this context, it needs to be emphasized that previous biochemical analyses of EDEM1 interaction with quality control machinery proteins and ERAD substrates were performed with cells transiently overexpressing tagged EDEM1 (Hosokawa et al. 2003; Molinari et al. 2003; Oda et al. 2003). The nature of the EDEM1 immunofluorescence pattern was clarified by immunogold labeling and serial section analysis (Fig.\u00a03d\u2013h). It revealed the presence of EDEM1-reactive buds along rough ER cisternae which apparently gave raise to \u223c150\u00a0nm vesicles. These buds and vesicles were devoid of a COPII coat, formed outside the canonical ER exit sites of the transitional ER and were not found in the tubulovesicular clusters of pre-Golgi intermediates (Fig.\u00a03i). Occasionally, EDEM1 luminal immunolabeling in limited parts of distended ER cisternae was observed, which accounted for approximately 11% of the immunogold labeling for GT. Double confocal immunofluorescence for endogenous EDEM1 in rat hepatoma clone 9 cells stably expressing the Null Hong Kong variant alpha 1-antitrypsin showed co-distribution of the two proteins (Zuber et al. 2007). Together, these data revealed the existence of a vesicular transport pathway out of the rough ER through which the ERAD factor EDEM1 and an ERAD substrate became sequestered from the early secretory pathway. Through this pathway potentially harmful aberrant luminal proteins can be removed. These findings also indicate that the Gls II and GT containing pre-Golgi intermediates appear to be not involved in the dislocation of an ERAD-L substrate.\nFig.\u00a03Double confocal immunofluorescence for endogenous EDEM1 (a) and calnexin (b) reveals different distribution patterns for the two proteins (c) in human HepG2 cells. Detection of endogenous EDEM1 by immunogold labeling of ultrathin frozen sections (e) or pre-embedding immunoperoxidase labeling (d, f\u2013h) reveals sparse labeling in the lumen of ER cisternae (arrowheads in e and f) and intense labeling over ER buds and vesicles pinching-off the ER (from Zuber et al. 2007). The subcellular distribution pattern of endogenous EDEM1 is schematically depicted in (i). In contrast to COPII-coated buds, which are formed at the transitional ER (TE) and give raise to COPII-coated vesicles present in pre-Goli intermediates (pre GI), EDEM1-positive buds occur outside the transitional ER and EDEM1-positive COPII-unreactive vesicles form clusters in the cytoplasm\nEndomannosidase assigns glucose trimming function to the Golgi apparatus\nIt is generally assumed the glucose trimming occurs exclusively by Gls I and II and, therefore, is limited to the ER and pre-Golgi intermediates. However, under conditions of inhibition of trimming glucosidases, formation of mature oligosaccharides has been observed. This apparent paradox could be explained by the existence of an alternate glucose-trimming pathway by neutral endo-alpha-mannosidase (Lubas and Spiro 1987, 1988; Moore and Spiro 1990, 1992; Spiro 2000). Endomannosidase is currently the only known endoglycosidase. In contrast to the trimming Gls I and II, it cleaves internally between the glucose-substituted mannose and the remaining oligosaccharide (Fig.\u00a04a). Its substrate specificity (Fig.\u00a04a) is basically that of Gls I and II (Moore and Spiro 1990, 1992; Rabouille and Spiro 1992). However, unlike Gls I and II, ER-mannosidase I trimmed mono-glucosylated oligosaccharides are a substrate of endomannosidase. The resulting Man8\u20135 GlcNAc isomer A is the specific product of endomannosidase. It should be noted that this oligosaccharide is no more a substrate for reglucosylation by GT. Biochemically, activity for endomannosidase was found to be enriched in Golgi membranes (Lubas and Spiro 1987). By immunofluorescence (Dong et al. 2000; Zuber et al. 2000), endomannosidase exhibited a crescent-shaped perinuclear staining and fine punctate staining throughout the cytoplasm which partially overlapped with immunofluorescence for Gls Golgi mannosidase II (Fig.\u00a04b\u2013d). High-resolution immunoelectron microscopy demonstrated endomannosidase in the peripheral and Golgi-associated pre-Golgi intermediates as well as cis and medial cisternae of the Golgi apparatus (Fig.\u00a04e) (Zuber et al. 2000). Trans cisternae of the Golgi apparatus and the trans Golgi network were unreactive. Quantification revealed \u223c85% of the immunogold labeling for endomannosidase in the Golgi apparatus and \u223c15% in pre-Golgi intermediates. Although, both endomannosidase and Gls II could be detected in pre-Golgi intermediates by double immunogold labeling, they labeled different elements of the vesiculotubular clusters (Fig.\u00a04e). Thus, endomannosidase and Gls I and II exhibited non-overlapping subcellular distributions (Roth et al. 2003; Zuber et al. 2000). Functionally, the presence of endomannosidase in the ER would interfere with the action of glucosyltransferase by preventing the reglucosylation of misfolded glycoproteins. Together, these findings demonstrating a predominantly Golgi apparatus localization of endomannosidase strongly indicated that glucose trimming of N-linked oligosaccharides is not limited to the ER.\nFig.\u00a04The various oligosaccharidic substrates of endomannosidase are depicted in (a). Like trimming glucosidases, endomannosidase trimms Gls1\u20133Man9GlcNAc2, and unlike trimming glucosidase II, monoglucosylated oligosaccharides with mannose-trimmed B and C branches. Double confocal immunofluorescence for endomannosidase (b) and Golgi mannosidase II (c) reveals co-distribution of the two enzymes (d) in clone 9 hepatocytes. Double immunogold labeling for endomannosidase (small gold particles, arrowheads) and glucosidase II (large gold particles, arrows) reveals endomannosidase localization in cis and middle Golgi apparatus cisternae (g), whereas glucosidase II is observd in rough ER including nuclear envelope. Non-overlapping immunogold labeling for both enzymes exists in pre-Golgi intermediates. N: nucleus, PM: plasma membrane. Micrographs b\u2013e are from Zuber et al. (2000)\nSince glucose trimming is indispensable for the synthesis of mature oligosaccharide side chains, deglucosylation by endomannosidase in the Golgi apparatus ensures that this important process is not blocked. Further biochemical and morphological analyses demonstrated that Golgi apparatus localized endomannosidase-processed oligosaccharides of alpha 1-antitrypsin irrespective of their folding state (Torossi et al. 2006). From the literature, it is well known that disease-causing misfolded glycoproteins to a certain extent might escape the protein quality control and become secreted (Cox 2001; Desnick et al. 2001). As a case in point, in humans suffering from alpha1-antitrypsin deficiency, the Z-variant of alpha1-antitrypsin not only becomes partially secreted, but also is active as serine protease inhibitor (Cabral et al. 2002; Teckman and Perlmutter 1996). As experimentally shown for the Z-variant of alpha1-antitrypsin (Torossi et al. 2006), endomannosidase provided a back-up mechanism for its de-glucosylation en route through the Golgi apparatus. Processing of its oligosaccharides to mature ones is apparently important for their proper trafficking and correct functioning.\nOrganelle changes due to intracellular accumulation of misfolded proteins\nAs a general rule, misfolded proteins become targeted by the protein quality control and following polyubiquitination will be degraded by proteasomes (Eisele et al. 2006; Hochstrasser 1996; Jarosch et al. 2002; McCracken and Brodsky 2005; Wolf and Hilt 2004; Zwickl et al. 2002). Depending on various factors such as the efficiency of the ubiquitin\u2013proteasome system, the intracellular amounts of misfolded glycoproteins and their biophysical properties as well as interactions with other proteins, a whole spectrum of organelle changes can be observed in protein folding diseases.\nFor some protein folding diseases, no significant structural aberrations of the early secretory pathways could be observed. An example is Fabry\u2019s disease, an inherited deficiency of lysosomal alpha-galactosidase A (alpha-Gal A), which causes progressive lysosomal glycosphingolipid accumulation (mainly globotriosylceramide Gb3) (Desnick et al. 2001). Disease-causing mutant alpha-Gal A could be shown by immunofluorescence to be retained in the ER where it existed in complexes with the chaperone BiP (Yam et al. 2005, 2006). From this, we concluded that recognition and ER-retention of the mutant alpha-Gal A by the protein quality control machinery constituted the mechanism leading to lysosomal deficiency in alpha-Gal A. Electron microscopic analysis of cultured fibroblast from Fabry patients harboring different mutations did not reveal significant changes of the morphology of the ER and the pre-Golgi intermediates. As expected, the fibroblasts contained numerous large lysosomes with characteristic multilamellar inclusions. Thus, the intracellularly retained mutant alpha-Gal A apparently became dislocated and was efficiently degraded by the ubiquitin\u2013proteasome system. A similar situation was observed for a polytope membrane protein, aquaporin-2, whose folding mutants can cause renal diabetes insipidus (Canfield et al. 1997; Morello and Bichet 2001). The T126M mutant aquaporin-2 was found to be retained in the ER and efficiently degraded by proteasomes without causing ER dilatation (Hirano et al. 2003). ER retention and rapid proteasomal degradation are also hallmarks of the pulmonary form of alpha 1-antitrypsin deficiency (Lomas and Parfrey 2004; Sifers et al. 1988). However, other types of protein folding diseases have been shown to result in the distention of the ER cisternae. One example is the congenital hypothyroid goiter in which the mutant thyroglobulin is misfolded (Kim et al. 1996; Kim and Arvan 1998; Kim et al. 1998, 2000; Medeiros Neto et al. 1996). In disorders of procollagen biosynthesis, distended ER cisternae were also observed (Bogaert et al. 1992). Other examples are represented by LDL receptor class 2 mutants (Lehrman et al. 1987; Pathak et al. 1988).\nThere are protein folding diseases associated with both distended ER cisternae and enlarged pre-Golgi intermediates. A misssense mutation of the insulin 2 gene (Cys96Tyr) in Akita mice disrupting one of the two interchain disulfide bonds is associated with intracellular accumulation of misfolded proinsulin (Wang et al. 1999). This resulted in a significant increase of the volume density of dilated ER profiles and of the pre-Golgi intermediates (Fig.\u00a05a, b) (Fan et al. 2007; Zuber et al. 2004). For the latter, a significant increase of the tubular elements was observed. Although the mutant proinsulin was degraded through proteasomes (Wang et al. 1999), its accumulation in the early secretory pathway caused an activation of the unfolded protein response and induced apoptosis (Oyadomari et al. 2002a; Oyadomari et al. 2002b). Other mutant proteins such as the cystic fibrosis (Kopito 1999; Riordan 1999) causing delta F508 variant of the chloride channel (Gilbert et al. 1998) and misfolded major histocompatibility complex class I protein (Hsu et al. 1991; Raposo et al. 1995), have been shown to accumulate in the expanded pre-Golgi intermediates.\nFig.\u00a05Details of an insulin-producing pancreatic beta cell from Akita mice. The presence of misfolded proinsulin results in the local distention of rough ER cisternae (RER*). Arrows point to regions of transition of non-distended ER (RER) to distended ER (RER*). In addition, the pre-Golgi intermediates (pGI) are greatly enlarged. G: Golgi apparatus, TE: transitional ER. In (B), the organelle changes in terms of differences of their volume density (Vv) and differences in proinsulin distribution pattern (LI) are schematically shown (from (Zuber et al. (2004)\nCertain other misfolded proteins are accompanied by the formation of insoluble aggregates in the lumen of the ER, which physically precludes dislocation to the cytosol and exposure to proteasomes. The stress-induced so-called intracisternal granules in the pancreas of starved guinea pigs (Palade 1956), which are composed of aggregated proenzymes (Fig.\u00a06a) (Geuze and Slot 1980; Pavelka and Roth 2005), form a classical example. For the liver-disease-causing alpha 1-antitrypsin Z variant, about 15% of the non-secreted mutant protein is polymerogenic and thus forms insoluble aggregates in the ER lumen, which cannot be degraded (Lomas et al. 1992, 2004) The Glu342Lys substitution of the Z-variant results in a spontaneous loop-sheet polymerization of the protein. In contrast to the above-mentioned protein folding diseases, which all have in common a loss-of-function pathogenesis, the Z-variant-caused alpha 1-antitrypsin deficiency seems to involve a pathologic gain-of-function pathogenesis (Hidvegi et al. 2005). The ER inclusions described above belong to the category of Russell bodies. Russell bodies represent subregions of the rough ER in which insoluble proteins accumulate (Fig.\u00a06b) (Alanen et al. 1985; Kopito and Sitia 2000; Valetti et al. 1991). They are typically found in cells synthesizing mutant immunoglobulins (Alanen et al. 1985; Kopito and Sitia 2000; Mattioli et al. 2006; Valetti et al. 1991) but also in cells synthesizing, for instance, mutant myocilin. Mutations of the myocilin gene are associated with primary open-angle glaucoma (Tamm 2002). Mutant myocilins are secretion-incompetent and have been shown biochemically to form intracellular detergent-insoluble complexes (Gobeil et al. 2004; Jacobson et al. 2001; Sohn et al. 2002). In cultured cells transfected to express both mutant and wild-type myocilin, heteromeric, detergent\u2013insoluble protein complexes were formed which were segregated into typical Russell bodies (Yam et al. 2007c). Thus, myocilin-caused open-angle glaucoma represents a protein folding disease. Its pathogenesis involves a pathological gain-of-function mechanism because of the interaction and complex formation of mutant with wild-type myocilin (Gobeil et al. 2004; Joe et al. 2003; Sohn et al. 2002; Yam et al. 2007c). As a consequence, unfolded protein response factors and pro-apoptotic factors were up-regulated and cells underwent apoptosis (Yam et al. 2007c) as detected by the appearance of lobulated nuclei and the TUNEL assay (Taatjes et al. 2008).\nFig.\u00a06a Intracisternal granules (asterisks) in the rough ER of exocrine rat pancreatic cells induced by puromycin treatment. These granules correspond to mini Russell bodies and are composed of aggregated proenzymes. b Russell bodies (RB) induced by heat shock in CHO cells. They represent distended parts of rough ER cisternae filled with protein aggregates. Note the structurally normal appearing rough ER cisternae in their neighborhood\nAll the mutant proteins discussed above are luminal or membrane proteins. What happens to aberrant cytosolic and nuclear proteins? Same like ER proteins, one extreme situation is that they become efficiently degraded by cytosolic and nuclear proteasomes subsequent to polyubiquitination (Schubert et al. 2000; Turner and Varshavsky 2000). Thus, cytosolic and nuclear quality control in normal cells suppresses the formation of aggregates of aberrant proteins by degrading them. The other extreme is represented by the formation of cytosolic and nuclear inclusion bodies due to inefficient degradation of aberrant proteins by the ubiquitin\u2013proteasome system. Cytosolic, non-membrane bounded inclusion bodies are generally called aggresomes (Corboy et al. 2005; Kopito and Sitia 2000). They consist of pericentriolar protein aggregates surrounded by a cage of intermediate (vimentin) filaments that are the most consistent component of aggresomes in addition to ubiquitin, proteasomes and molecular chaperones. Aggresomes can be induced experimentally by forced overexpression of aggregation-prone mutant proteins or by experimentally inhibiting proteasomes (Fig.\u00a07) (Anton et al. 1999; Fan et al. 2007; Johnston et al. 1998; Wigley et al. 1999). On the other hand, it has been shown that protein aggregates can directly impair the function of the ubiquitin\u2013proteasome system (Bence et al. 2001). The formation of aggresomes is a multi-step process, which depends on the intact microtubules. Aggresomes are formed by the coalescence of small protein aggregates transported from the cell\u2019s periphery along microtubules to centrioles (Garcia-Mata et al. 1999; Johnston et al. 1998; Kawaguchi et al. 2003; Vidair et al. 1966; Wigley et al. 1999; Wojcik et al. 1996). In the nucleus, the inclusion bodies can be found in association with the promyelocytic leukemia oncogenic domains (Anton et al. 1999).\nFig.\u00a07Formation of pericentriolar aggresomes following proteasome inhibition by lactacystin in CHO cells stably expressing misfolded proinsulin. Irregularly shaped, electron dense flocculent material is present in the cytoplasm and surrounded by intermediate filaments (a). At higher magnification, the spatial relationship between the protein aggregates and a centriole can be seen (b). Micrographs from Fan et al. (2007)\nInclusion bodies have been observed in association with a number of chronic neurodegenerative diseases such as Parkinson\u2019s disease, Huntington\u2019s disease, Alzheimer\u2019s disease and amyotrophic lateral sclerosis (Johnston et al. 2000; Kabashi and Durham 2006; Rubinsztein 2006; Selkoe 2003; Shults 2006; Soto 2003). Inclusion bodies named Lewy bodies are a morphological hallmark of Parkinson\u2019s disease and other neurodegenerative disorders (McNaught et al. 2002b; Olanow et al. 2004; Shults 2006). Lewy bodies in the dopaminergic neurons resemble aggresomes and represent spherical bodies commonly composed of a core of granular material and peripheral radiating filaments. They contain a variety of proteins such as alpha-synuclein, the alpha-synuclein-binding protein synphilin-1 torsin A, neurofilaments, ubiquitin, proteasomal subunits and various heat shock proteins as well as ubiquitin-activating enzyme, ubiquitin-conjugating enzyme, ubiquitin ligase enzymes and proteasome activators. Furthermore, they contain centrosome-related gamma-tubulin and pericentrin. Thus, it has been proposed that the formation of Lewy bodies represents an aggresome-like response in dopaminergic neurons (McNaught et al. 2002c). Considering the observed impairment of the ubiquitin\u2013proteasome system in patients with Parkinson\u2019s disease (McNaught et al., 2001, 2002a, 2006), it is assumed that Lewy bodies, by segregating increasing levels of aberrant and potentially cytotoxic proteins, might protect the neurons (Olanow et al. 2004). It should be stressed, however, that the mechanism leading to selective neuronal death in Parkinson\u2019s disease is not fully understood and the role Lewy bodies might be playing needs to be studied further studies (Lindholm et al. 2006).\nSynthetic chaperones for treatment of protein folding disease\nThe various protein folding diseases mentioned above can be classified based on the pathogenetic mechanism. Efficient proteasomal degradation of the misfolded protein is characteristic of the loss-of-function pathogenesis. This is the case in protein folding diseases such as cystic fibrosis, the lung form of alpha 1-antitrypsin deficiency, aquaporin 2-caused renal diabetes insipidus, Gaucher\u2019s disease and Fabry\u2019s disease. Here, the missing function of the degraded protein alone can be the cause of the clinical symptoms, or secondary effects due to substrate accumulation like in lysosomal storage diseases. Intracellular accumulation due to inefficient proteasomal degradation of misfolded proteins is representative of a pathological gain-of-function mechanism, which is combined with a loss of function. Intracellular accumulation of misfolded proteins associated or not with protein aggregation can result in the activation of the unfolded protein response leading to ER stress and apoptosis. A pathological gain-of-function mechanism can be also the cause of a dominant clinical course when the wild-type protein in complexes with the mutant protein is retained inside the cells. Examples for pathological gain-of-function pathogenesis-associated protein folding diseases are myocilin-caused open-angle glaucoma, familial hypophyseal diabetes insipidus, Parkinson\u2019s disease and Huntington\u2019s disease.\nMany attempts have been made to at least partially correct the protein misfolding in order to overcome their trafficking defect and to alleviate ER stress. Among other approaches, small molecule synthetic chaperones have been used in order to shift the folding equilibrium of mutant proteins towards a more native state (Arakawa et al. 2006; Chaudhuri and Paul 2006; Cohen and Kelly 2003; Papp and Csermely 2006; Perlmutter 2002). Chemical chaperones include osmotically active substances such as DMSO, glycerol, polyols or deuterated water, and other compounds such as 4-phenylbutyric acid (Burrows et al. 2000; Lim et al. 2004; Liu et al. 2004; Pedemonte et al. 2005; Rubenstein and Zeitlin 2000; Tamarappoo and Verkman 1998; Tveten et al. 2007; Welch and Brown 1996). Other substances such as enzyme inhibitors (Fan et al. 1999; Matsuda et al. 2003; Sawkar et al. 2002) and receptor ligands or antagonists {Pet\u00e4j\u00e4-Repo, 2002 #16211;Egan, 2002 #12283} have been shown to function as pharmacological chaperones.\nHere, we have chosen two examples from our work to demonstrate how immunocytochemistry and microscopy in combination with biochemical analyses can be applied to demonstrate the functionality of a chemical and a pharmacological chaperone in rescuing the consequence of disease-causing protein misfolding. It has been mentioned above that open-angle glaucoma-causing mutant myocilin forms insoluble protein aggregates in the ER lumen (Russel bodies), which result in ER stress and apoptotic cell death (Yam et al. 2007b). Among the other tested chemical chaperones, treatment with sodium 4-phenylbutyrate significantly reduced the amount of intracellular detergent\u2013insoluble myocilin aggregates and thereby the number of Russel bodies in the cells (Fig.\u00a08a\u2013c), diminished mutant myocilin interaction with calreticulin and restored the secretion of mutant myocilin. As a consequence, the ER stress was released and most interesting, the apoptosis rate was reduced close to levels observed in control cells expressing wild-type myocilin (Fig.\u00a08d). Thus, sodium 4-phenylbutyrate exerts a beneficial effect by protecting the cells from the deleterious effects of mutant myocilin. Since sodium 4-phenylbutyrate is a tissue and cell-permeable molecule, it holds the potential for topical administration in the treatment of myocilin-caused primary open-angle glaucoma.\nFig.\u00a08Confocal double fluorescence of HEK 293 cells stably expressing GFP-wt and FLAG-wt myocilin reveals an ER and fine punctate pattern (a). HEK 293 cells coexpressing GFP-mutant myocilin and FLAG-wt myocilin exhibit distinct cytoplasmic aggregates, which correspond to Russell bodies (b). Treatment with the chemical chaperone sodium 4-phenylbutyrate results in reduction of the percentage of cells with myocilin-containing Russell bodies (c) and a drastic reduction of the apoptosis rate (d). Empty columns in d show values for untreated cells and filled columns for sodium 4-phenylbutyrate treated cells. From Yam et al. (2007a)\nThe second example concerns Fabry\u2019s disease, a lysosomal storage disorder caused by a deficiency of alpha-Gal A activity in lysosomes that results in the accumulation of glycosphingolipid globotriosylceramide (Gb3). The lysosomal trafficking of mutant alpha Gal A is impaired because the enzyme is retained in the ER by the protein quality control (Yam et al. 2005). Others had demonstrated that the activity of mutant alpha-Gal A in vitro at neutral pH could be stabilized with the competitive enzyme inhibitor 1-deoxygalactonorijimycin (DGJ) (Fan et al. 1999). Treatment of cells expressing mutant alpha-Gal A with a non-inhibitory dose of DGJ enhanced the intracellular enzyme activity (Yam et al. 2005, 2006). In addition, we could demonstrate by immunofluorescence and quantitative immunogold labeling that the mutant enzyme was redistributed from the ER to lysosomes and that this trafficking was mannose 6-phosphate-dependent. The DGJ treatment resulted in release of mutant alpha-Gal A from the chaperone BiP and in its conversion in the mature lysosomal form. Double confocal immunofluorescence and immunogold labeling demonstrated that the lysosomal Gb3 storage was cleared and that the size of the lysosomes became normalized (Yam et al. 2005, 2006). Together, this demonstrated that DGJ exhibited a chaperone-like effect and induced the trafficking of ER-retained mutant alpha Gal A to lysosomes where the enzyme was catalytically active. Therefore, the pharmacological chaperone DGJ potentially offers a convenient and cost-efficient therapeutic alternative to enzyme replacement therapy.","keyphrases":["erad","protein folding disease","glucosidase ii","glucosyltransferase","edem1","endomannosidase","chemical chaperones"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Pflugers_Arch-3-1-1950587","title":"Transmural variations in gene expression of stretch-modulated proteins in the rat left ventricle\n","text":"The properties of left ventricular cardiac myocytes vary transmurally. This may be related to the gradients of stress and strain experienced in vivo across the ventricular wall. We tested the hypothesis that within the rat left ventricle there are transmural differences in the expression of genes for proteins that are involved in mechanosensitive pathways and in associated physiological responses. Real time reverse transcription polymerase chain reaction was used to measure messenger RNA (mRNA) levels of selected targets in sub-epicardial (EPI) and sub-endocardial (ENDO) myocardium. Carbon fibres were attached to single myocytes to stretch them and to record contractility. We observed that the slow positive inotropic response to stretch was not different between EPI and ENDO myocytes and consistent with this, that the mRNA expression of two proteins implicated in the slow response, non-specific cationic mechanosensitive channels (TRPC-1) and Na\/H exchanger, were not different. However, mRNA levels of other targets, e.g. the mechanosensitive K+ channel TREK-1, Brain Natriuretic Peptide and Endothelin-1 receptor B, were significantly greater in ENDO than EPI. No targets had significantly greater mRNA levels in EPI than ENDO. On the basis of these findings, we suggest that the response of the ventricle to stretch will depend upon both the regional differences in stimuli and the relative expression of the mechanosensitive targets and that generally, stretch sensitivity is predicted to be greater in ENDO.\nIntroduction\nIt is known that mechanical stimulation of the myocardium (stress and strain) can lead to both acute and chronic changes in the electrical and mechanical activity of the heart and contribute to the development of cardiac hypertrophy [7, 11]. It is acknowledged that the ventricular myocardium is not homogenous and that there are regional differences in the basic properties of ventricular myocytes, e.g. [1, 10]. In the whole heart, transmural gradients in ventricular wall stress and strain exist [6], which may influence the expression of genes for proteins that are sensitive to mechanical stimuli. This may contribute to regional variations in physiological characteristics and thereby to transmural differences in the response to stimuli such as stretch.\nTherefore, regional variations in wall stress and strain, in conjunction with regional differences in gene expression, may regulate the response of the heart. To date, relatively little is known about these possibilities, although it has been shown that the messenger RNA (mRNA) expression and current density of the mechanosensitive twin-pore K+ channel TREK-1 is greater in the rat sub-endocardium (ENDO) than the sub-epicardium (EPI) [12, 8, 6]. The purpose of this study was to test the hypothesis that the mRNA expression levels of several proteins, which are thought to modulate the mechanical and electrical response to stretch, vary across the rat left ventricular wall. Additionally, we wished to test whether the slow contractile response to axial stretch [2], which is thought to be modulated by some of the chosen targets, differs between myocytes from different regions of the left ventricular free wall, thus potentially linking gene expression to function.\nMaterials and methods\nAll animal experimentation was carried out in accordance with the Animals (Scientific Procedures) Act 1986 and the local Institutional ethical review panel.\nIsolation of RNA\nTen Sprague\u2013Dawley rats (194\u2013240\u00a0g) were killed and the hearts removed. The left ventricular free wall was excised and snap frozen in liquid nitrogen between the large flat ends of a pair of tongs. The thickness of the left ventricular wall was measured with pre-cooled callipers, and 25-\u03bcm sections, equivalent to one third of the wall thickness, were collected on a cryostat from the EPI and ENDO surfaces, leaving a distinct mid-myocardial region. All samples were stored at \u221280\u00b0C until RNA extraction. Total RNA extraction was performed following the Qiagen mini-kit protocol for striated muscle, with the exception that an additional volume of RLT was added to the Proteinase K solution before binding of the RNA onto the mini-columns. Integrity and concentration of the isolated RNA were assessed by electrophoresis through 0.6% formaldehyde\/1% agarose gels with ethidium bromide and the concentration of RNA adjusted to 1\u00a0g\/l. Complementary DNA (cDNA) was prepared from 4\u00a0\u03bcg of total RNA with random priming using Superscript III first-strand synthesis system (Invitrogen, Life Technologies, Rockville, MD) and diluted 1:10 in TE (10\u00a0mM Tris-HCl [pH\u00a07.5], 1\u00a0mM ethylenediamine tetraacetic acid) before use in real-time polymerase chain reaction (PCR).\nReal-time RT-PCR\nReal-time reverse transcription PCR (RT-PCR) was performed using TaqMan low-density arrays (Micro Fluidic Cards, Applied Biosystems, Foster City, CA). Each card consisted of 384 wells, preloaded with pre-designed fluorogenic TaqMan probes and primers, configured to allow detection of 48 transcripts for 7 experimental and 1 calibrator samples. Our calibrator sample consisted of a mixture of cDNA from EPI and ENDO regions. Each of the eight sample lanes in a card was loaded with 100\u00a0\u03bcl of a 1:1 mixture of cDNA (equivalent to 77\u00a0ng input RNA) and TaqMan Universial PCR master mix (Applied Biosystems). PCR was done according to the recommended protocol (50\u00b0C for 2\u00a0min and 94.5\u00b0C for 10\u00a0min, followed by 40 cycles at 97\u00b0C for 30\u00a0s and 59.7\u00b0C for 1\u00a0min) on an ABI RISM 7900HT Sequence detection system (Applied Biosystems). Data were collected with instrument spectral compensations by the Applied Biosystems SDS 2.2 software and analysed using the threshold cycle relative quantification method. Relative transcript expression was normalised to the housekeeper gene GAPDH.\nMechanosensitive targets\nmRNA targets chosen for this investigation were: TREK-1 (a two-pore-domain K+-selective channel) and TRPC-1 (transient receptor potential canonical, a cationic non-selective mechanosensitive channel [MSC]); the Na\/H exchanger, thought to be central to the slow increase in force that occurs upon axial stretch; endothelin-1 and its receptor types A and B and the angiotensin II receptor A, known to regulate the hypertrophic response to mechanical stimulation and possibly the slow inotropic response; caveolin-3 a critical component of caveolae, which are potential mechanosensors; caveolin-1, a major component of caveolae in non-cardiac muscle and recently identified in cardiac myocytes; brain natruiretic peptide (BNP) and the \u03b1 and \u03b2 sub-types of myosin heavy chain (MHC), the expression of both BNP and \u03b2-MHC are known to be increased by stretch [11].\nIsolation of left ventricular myocytes\nThe isolation of single rat left ventricular myocytes and the measurement of the rapid and slow responses to axial stretch by attached carbon fibres was performed as previously described [2]. In brief, excised hearts were perfused on a Langendorff apparatus with a collagenase\/protease containing physiological solution. EPI and ENDO strips, each one third of the wall thickness, were manually cut from the left ventricular free wall with scissors leaving a distinct and intact mid-myocardial region. Further incubation with enzyme solution resulted in the isolation of separate populations of EPI and ENDO myocytes.\nMyocytes were attached to carbon fibres to record auxotonic force development in response to external stimulation and to axially stretch myocytes. Force was calculated from fibre motion, and stretch was calculated from the increase in sarcomere length. The rapid response to stretch was measured within 20\u00a0s of an increase in sacromere length, whereas the slow response was calculated as the further increase in force 5\u00a0min after the stretch. These experiments were carried out at a stimulation frequency of 1\u00a0Hz at a temperature of 22\u201324\u00b0C (to improve the experimental success rate) using a bicarbonate-based buffer containing (in mM): NaCl 118.5; NaHCO3 14.5; KCl 4.2; KH2PO4 1.2; MgSO4 1.2; glucose 11.1; CaCl2 1, equilibrated with 95% O2\/5% CO2 (pH\u00a07.4).\nStatistics\nData were analysed using paired t tests to compare relative mRNA expression from the EPI vs ENDO region of each heart and unpaired t tests to compare the magnitude of the rapid and slow inotropic responses. Statistical significance was assumed when P\u2009<\u20090.05.\nResults\nThere was some consistency in the pattern of regional expression of mechanosensitive genes (Table\u00a01). Although the expression levels of some targets were unchanged, others were significantly greater in ENDO but never so in EPI. This observation was not due to a systematic error of the technique as levels of mRNA of targets known to be greater in EPI, e.g. the potassium channel component, Kv4.2, were significantly greater in EPI than ENDO (results not presented). Indeed, if \u03b1-MHC is excluded (being chosen as a counterpoint to \u03b2-MHC), 9 of the 11 targets had a larger mean mRNA expression in ENDO than EPI; this trend is statistically significant (P\u2009<\u20090.05, sum of ranks test). \nTable\u00a01Comparison of mRNA expression of mechanosensitive proteins from the EPI and ENDO region of rat left ventricleAssay ID referenceCommon nameEPIENDOPaired t testMean\u2009\u00b1\u2009SEMMean\u2009\u00b1\u2009SEMSlow response and\/or hypertrophic Agtr1a-Rn00578456_m1Angiotensin II receptor 1A1.64\u2009\u00b1\u20090.161.30\u2009\u00b1\u20090.22NS Edn1-Rn00561129_m1Endothelin-11.17\u2009\u00b1\u20090.141.58\u2009\u00b1\u20090.24NS Ednra-Rn00561137_m1Endothelin-1 receptor type A1.19\u2009\u00b1\u20090.141.38\u2009\u00b1\u20090.12NS  Ednrb-Rn00569139_m1Endothelin-1 receptor type B1.27\u2009\u00b1\u20090.171.93\u2009\u00b1\u20090.220.001 ENDO\u2009>\u2009EPI Nppb-Rn00580641_m1Brain natriuretic peptide1.42\u2009\u00b1\u20090.152.77\u2009\u00b1\u20090.420.002 ENDO\u2009>\u2009EPI Slc9a1-Rn00561924_m1Sodium\u2013hydrogen exchanger1.33\u2009\u00b1\u20090.091.51\u2009\u00b1\u20090.17NSMyofilaments Myh6-Rn00568304_m1\u03b1-myosin heavy chain1.56\u2009\u00b1\u20090.121.37\u2009\u00b1\u20090.17NS Myh7-Rn00568328_m1\u03b2-myosin heavy chain1.20\u2009\u00b1\u20090.232.19\u2009\u00b1\u20090.360.006 ENDO\u2009>\u2009EPIMechanosensitive channels Kcnk2-Rn00597042_m1Kcnk2 (TREK-1)0.84\u2009\u00b1\u20090.041.04\u2009\u00b1\u20090.040.001 ENDO\u2009>\u2009EPI Trpc1-Rn00585625_m1Transient receptor potential canonical-10.72\u2009\u00b1\u20090.040.72\u2009\u00b1\u20090.05NSMechanotransducers (caveolae) Cav-Rn00755834_m1Caveolin-10.90\u2009\u00b1\u20090.030.96\u2009\u00b1\u20090.030.015 ENDO\u2009>\u2009EPI Cav3-Rn0055343_m1Caveolin-30.81\u2009\u00b1\u20090.030.85\u2009\u00b1\u20090.05NSData shown are mean\u2009\u00b1\u2009SEM, expression is relative to GAPDH. The probability of paired t tests is shown in the table. P\u2009>\u20090.05 (n\u2009=\u200910 pairs).NS Not significantly different\nWhen single myocytes were stretched from their resting sarcomere length (ENDO 1.83\u2009\u00b1\u20090.09\u00a0\u03bcm; EPI 1.82\u2009\u00b1\u20090.07\u00a0\u03bcm) by an equivalent amount (ENDO 7.8\u2009\u00b1\u20090.9 %; EPI 7.5\u2009\u00b1\u20090.5%, n\u2009=\u200911 ENDO, 18 EPI myocytes) as illustrated in Fig.\u00a01a, we observed that the magnitude of neither the rapid nor slow inotropic response was dependent upon regional origin (Fig.\u00a01b).\nFig.\u00a01The inotropic response of left ventricular myocytes to axial stretch. a Representative trace showing the typical biphasic inotropic response of a myocyte to a stretch of 8% from a resting sacromere length (SL) of 1.85\u00a0\u03bcm. The trace shows changes in active force, resting force has been subtracted with a sample and hold device. The rapid increase in force is seen immediately upon an increase in SL, the slow response develops over the following minutes. b Mean data showing the rapid and slow response to stretch of \u22458% from a resting SL of \u22451.82\u00a0\u03bcm in sub-epicardial (EPI) and sub-endocardial (ENDO) left ventricular myocytes. The responses from EPI and ENDO cells were not significantly different from each other. P\u2009>\u20090.05, n\u2009=\u200918 EPI , n\u2009=\u200911 ENDO\nDiscussion\nThis investigation of transcripts for proteins sensitive to stretch, or involved in mechanosensitive pathways, has allowed us to detect a number of small but nevertheless significant differences in mRNA levels between ENDO and EPI samples.\nRapid and slow inotropic responses to axial stretch\nFollowing equivalent amounts of axial stretch, the rapid inotropic response was not significantly different in EPI and ENDO rat myocytes. This observation is consistent with findings in untrained intact myocytes [10]. However, a greater stretch-induced increase in myofilament Ca2+ sensitivity (\u03b4pCa50) has been reported in untrained skinned ENDO myocytes than EPI myocytes when larger stretches (from sarcomere lengths of 1.9 to 2.3\u00a0\u03bcm) are applied [3].\nWe observed that the slow response did not vary with myocyte regional origin. This is an important and novel observation because the multicellular cardiac preparations typically used to study the slow response (trabeculae and papillary muscles) are ENDO in nature. Additionally, previous whole heart studies recorded the aggregate response from the whole left ventricle while our own previous single cell experiments did not distinguish between myocyte origin. Consistent with the functional data, we observed that the mRNA expression levels for proteins implicated in the development of the slow inotropic response in rat (non-specific cationic MSCs, possibly TRPC-1) and the Na\/H exchanger, see [2], are not regionally different. It should be noted however, that although TRPC-1 has been proposed as a non-specific cationic MSC [9], such a role has not yet been established in the myocardium.\nMSCs and mechanotransducers\nAlthough levels of TRPC-1 mRNA were not regionally different, we found that TREK-1 mRNA expression was greater in ENDO than EPI. This latter finding agrees with the observations of Tan et al. [12] and Kelly et al. [6], although the regional difference in our study was smaller than previously reported. We would therefore hypothesise that an equivalent mechanical stimulus that activated TRPC-1 and TREK-1 would produce a larger electrical response in ENDO tissue (see [6]). Whether increased TREK-1 in ENDO would stabilise ENDO membrane potential during the diastolic period and be potentially anti-arrhythmic or reduce the transmural gradient in action potential duration and be potentially pro-arrhythmic [1] remains to be resolved.\nAlthough we found no evidence for a regionally different expression of caveolin-3, an essential component of cardiac caveolae, the role of caveolae may also be influenced by regional expression of the receptors and other signalling molecules that they aggregate. The observation that mRNA for caveolin-1 is greater in ENDO is intriguing as this isoform has only recently been identified in cardiac myocytes, and its role in myocyte caveolae is unknown.\nIndicators of cardiac hypertrophy\nThe ATII\/ET-1 signalling pathways are known to play a role in the hypertrophic response to mechanical stimulation and, in some species, the slow inotropic response, see [4] for review. We only observed significantly greater levels of mRNA for the ET-1B receptor whereas it is the ET-1 A receptor that Cingolani et al. report to be important for the slow response. We also saw increased mRNA levels of BNP and of \u03b2-MHC in ENDO. Previous studies have variously reported increased [5] or similar [3] levels of \u03b2-MHC protein in normal rat ENDO vs EPI myocardium. Both BNP and \u03b2-MHC are indicators of hypertrophy provoked by mechanical stimuli. It is therefore interesting that wall stress and, in some studies, wall strain, is thought to be greater in ENDO [6].\nAssumptions and limitations\nIn this study, we have made the common, implicit assumption that there is a link between levels of mRNA expression and protein activity. This assumption can only be validated by measuring in situ protein activity, a huge task given the diverse nature of our targets. However, in measuring the slow inotropic response to stretch and the mRNA expression of the targets thought to underlie this effect, we have attempted to address this problem. We chose the rat for consistency with our previous work on regional differences and on the slow response to stretch [10, 2] and because of the ready availability of mRNA sequences for this species. Given that regional variations exist in other species [1], it seems reasonable to expect that our observations on mechanosensitive targets are relevant to other mammalian species, although investigation of other species is required to confirm this.\nConclusions\nThe level of mRNA expression of some proteins that are modulated by mechanical stimuli vary across the rat left ventricular wall. In vivo, the response to mechanical stimulation is likely to be complex. Targets (e.g. in the present study caveolin-3 and TRPC-1) whose mRNA expression are not regionally different might still be differentially regulated by transmural variation in the mechanical stimuli, whereas other permutations of increased\/decreased mRNA expression and protein activity with increased\/decreased stimuli are possible. Based upon our present observations and those from the literature reporting increased stretch sensitivity of various responses in ENDO, e.g. [5, 10, 3, 6], it seems that in general, ENDO tissue is likely to be more influenced by, and be responsive to, mechanical stimulation than EPI tissue.","keyphrases":["gene expression","stretch","cardiac myocytes","trp channels","stretch-activated channel"],"prmu":["P","P","P","M","M"]}
{"id":"Histochem_Cell_Biol-4-1-2413110","title":"The desmosome and pemphigus\n","text":"Desmosomes are patch-like intercellular adhering junctions (\u201cmaculae adherentes\u201d), which, in concert with the related adherens junctions, provide the mechanical strength to intercellular adhesion. Therefore, it is not surprising that desmosomes are abundant in tissues subjected to significant mechanical stress such as stratified epithelia and myocardium. Desmosomal adhesion is based on the Ca2+-dependent, homo- and heterophilic transinteraction of cadherin-type adhesion molecules. Desmosomal cadherins are anchored to the intermediate filament cytoskeleton by adaptor proteins of the armadillo and plakin families. Desmosomes are dynamic structures subjected to regulation and are therefore targets of signalling pathways, which control their molecular composition and adhesive properties. Moreover, evidence is emerging that desmosomal components themselves take part in outside-in signalling under physiologic and pathologic conditions. Disturbed desmosomal adhesion contributes to the pathogenesis of a number of diseases such as pemphigus, which is caused by autoantibodies against desmosomal cadherins. Beside pemphigus, desmosome-associated diseases are caused by other mechanisms such as genetic defects or bacterial toxins. Because most of these diseases affect the skin, desmosomes are interesting not only for cell biologists who are inspired by their complex structure and molecular composition, but also for clinical physicians who are confronted with patients suffering from severe blistering skin diseases such as pemphigus. To develop disease-specific therapeutic approaches, more insights into the molecular composition and regulation of desmosomes are required.\nIntroduction\nDesmosomes are intercellular adhering junctions serving to attach neighbouring cells to each other. They are most numerous in tissues subjected to significant mechanical stress such as the stratified squamous epithelia of the skin (Bizzozero 1864) and of mucous membranes (Farquhar and Palade 1963) as well as the myocardium (Fawcett and Selby 1958). Moreover, desmosomes are found in simple epithelia and in non-epithelial cells such as the meningeal cells of the arachnoidea (Gusek 1962) and the follicular dendritic cells of lymph follicles (Swartzendruber 1965).\nDesmosomes were discovered as cell contacts in the middle of the nineteenth century (Calkins and Setzer 2007). By the means of light microscopy, desmosomes were first described in the epidermis by the Italian pathologist Bizzozero in (1864). In his histology text book, the anatomist Josef Schaffer from Vienna introduced the term \u201cdesmosome\u201d, by combining the greek words \u201cdesmos\u201d (bond) and \u201csoma\u201d (body) although, to that time, he, like most others in the field believed that desmosomes were cytoplasm-filled intercellular bridges (Schaffer 1920). It took almost another century until Keith Porter, using electron microscopy, was able to confirm the basic observation of Bizzozero that desmosomes rather are contacts between adjacent cells and to allow the first description on desmosome ultrastructure (Porter 1956). With these new technical advances at hand, several studies were performed in the following years on the distribution and organization of desmosomes in various tissues. In addition, starting in the 1970s, biochemical approaches and molecular cloning techniques were applied to identify the desmosomal components and to characterize their interactions (Drochmans et al. 1978; Moll et al. 1986; Moll and Franke 1982; Schwarz et al. 1990; Skerrow and Matoltsy 1974).\nSignificant insights into the regulation of desmosomal adhesion also came from the field of dermatology since it was demonstrated that autoantibodies in patients suffering from the autoimmune blistering skin diseases pemphigus vulgaris (PV), and pemphigus foliaceus (PF), are directed to Ca2+-sensitive cell surface proteins within desmosomes (Eyre and Stanley 1987, 1988; Jones et al. 1986b; Karpati et al. 1993), which were identified as the desmosomal cadherins desmoglein 1 (Dsg 1) and Dsg 3 (Amagai et al. 1991; Koulu et al. 1984). The term \u201cpemphigus\u201d comes from the greek word \u201cpemphix\u201d (blister) and is being used in dermatology since 1791 (Schmidt et al. 2000), long before it was found that pemphigus is associated with autoantibodies against keratinocyte surface antigens (Beutner and Jordon 1964) and that these antibodies are sufficient to cause acantholysis, i.e. loss of cell\u2013cell adhesion, in human skin in vivo and in vitro (Anhalt et al. 1982; Schiltz and Michel 1976). The final break-through was the finding that autoantibodies against the extracellular domains of Dsg 3 and Dsg 1 in PV and in PF are pathogenic (Amagai et al. 1995, 1994a, 1992). Therefore, autoantibodies from pemphigus patients have been used to characterize the mechanisms involved in the regulation of desmosomal adhesion. Except from pemphigus, other diseases in which desmosomal adhesion is altered by mutations or bacterial toxins helped to elucidate the functional role of the different desmosomal components.\nDuring the last past several years, a number of comprehensive reviews have been published on both desmosome structure and function (Dusek et al. 2007b; Garrod et al. 2002; Getsios et al. 2004b; Green and Simpson 2007; Holthofer et al. 2007; Kitajima 2002; Kottke et al. 2006; Muller et al. 2008a; Yin and Green 2004) and\/or on the mechanisms involved in pemphigus pathogenesis (Amagai 2003; Hashimoto 2003; Lanza et al. 2006; Payne et al. 2004; Sharma et al. 2007; Sitaru and Zillikens 2005; Stanley and Amagai 2006), which indicates that the perspective of the existing model of the desmosome and its role in pemphigus pathogenesis are constantly reshaped. Moreover, because even textbook knowledge such as on the molecular composition of myocardial intercalated discs needs revision (Borrmann et al. 2006; Franke et al. 2006), it becomes obvious that after almost 150\u00a0years of desmosome research, our knowledge is still far from complete. This article focuses on the mechanisms regulating desmosomal adhesion, which are compromised in diseases such as pemphigus.\nThe ultrastructure and composition of desmosomes\nThe first detailed analysis of desmosome ultrastructure was provided by Odland (1958). Desmosomes are discoid junctions with a diameter of about 0.2\u20130.5\u00a0\u03bcm and are composed of two electron-dense plaques in each of the two cells which are separated by an intercellular cleft of 24\u201330\u00a0nm (Figs.\u00a01, 2) (Farquhar and Palade 1963; Odland 1958). Within the plaques, an outer dense plaque can be separated from a less dense inner plaque, the latter of which is linked to loops of intermediate filament bundles (Kelly 1966). Desmosomes contain members of at least three protein families. Desmosomal cadherins form the intercellular adhesive interface, whereas armadillo and plakin family proteins built up the plaques. It is believed that the cytoplasmic tail of Dsgs and Dscs interact with plakoglobin which in turn binds to desmoplakin (Fig.\u00a01). Desmoplakin finally is anchored to the intermediate filament cytoskeleton (Green and Simpson 2007). These interactions seem to be stabilized laterally by plakophilin (Hatzfeld 2007).\nFig.\u00a01Molecular model of the desmosome. The desmosomal cadherins desmoglein and desmocollin undergo homophilic and heterophilic binding via interaction with the amino-terminal extracellular (EC) 1 domain of partner molecules on the same (cis) as well as on the neighbouring cell (trans). The cytoplasmic domains are largely embedded in the outer dense plaque (ODP) where they are associated with plakoglobin and plakophilin. In the inner dense plaque (IDP), desmoplakin links these adaptor molecules to the intermediate filament cytoskeletonFig.\u00a02Ultrastructure of the desmosome. The electron micrograph of a keratinocyte desmosome shows the desmosomal plaque with inserting cytokeratin intermediate filaments as well as some fuzzy material within the extracellular space likely reflecting the extracellular domains of desmosomal cadherins\nDesmosomes and desmosome-like junctions\nAdhering junctions are divided into two main forms: (1) desmosomes, which serve as anchoring structures for intermediate filaments to desmosomal cadherins, and (2) adherens junctions, which contain cell-type specific adhesion molecules from the cadherin super-family that are linked to the actin cytoskeleton. Both, desmosomes and adherens junctions can be found as constituents of more elaborated cell contact complexes. Moreover, chimeric cell contacts exist which share features of both adherens junctions and desmosomes.\nJunctional complexes\nPolarized epithelial cells display junctional complexes located at the uppermost section of the baso-lateral membrane. In apico-basal direction, the complex is composed of the zonula occludens (tight junction), the zonula adherens and a desmosome (macula adherens) (Farquhar and Palade 1963). Accompanied by a line of separated desmosomes, the zonula occludens and the zonula adherens span the entire cell by forming continuous junction belts. These junctional complexes are regarded as hallmarks of polarized epithelial cells but differ in terms of size and ultrastructure in cell type-specific manner.\nArea composita\nThe intercalated discs of the myocardium also consist of three types of cell junctions, i.e. adherens junctions, desmosomes and gap junctions (Fig.\u00a03). Although \u201ctransitional forms\u201d between adherens junctions and desmosomes were described in the very beginning (Fawcett and Selby 1958), most morphological studies regarded the intercalated discs to be composed of separated desmosomes and adherens junctions, the former linked to the desmin type intermediate filament cytoskeleton and the latter to actin filaments of the myofibrills (Fig.\u00a03) (Fawcett and McNutt 1969; McNutt and Fawcett 1969; Shimada et al. 2004). This view seemed to be supported by immuno-localization studies, which showed that desmoplakin as a desmosomal marker and the myocardial adherens junction protein N-cadherin displayed mutually exclusive spatial distribution patterns (Angst et al. 1997; Gutstein et al. 2003). However, a recent comprehensive set of studies unequivocally demonstrated that the desmosomal components desmoglein 2 (Dsg 2), desmocollin 2 (Dsc 2), desmoplakin and plakophilin-2 as well as the adherens junction components N-cadherin, cadherin-11, \u03b1-catenin and \u03b2-catenin, afadin, vinculin and ZO-1 are present in all parts of intercalated discs irrespective of whether their ultrastructure resembles more closely  typical desmosomes or adherens junctions (Borrmann et al. 2006; Franke et al. 2006). It seems that the two types of junctions coalesce within the first-year postpartum and that plakophilin 2 is of special importance for junction integrity (Pieperhoff and Franke 2007; Pieperhoff et al. 2008). Therefore, the intercalated discs were now reclassified as \u201carea composita\u201d, a mixed type of adhering junctions.\nFig.\u00a03Ultrastructure of the area composita of a myocardial intercalated disc. The electron micrograph shows an intercalated disc containing a gap junction (GJ) in its longitudinal section as well as an adhering junction with an extensive electron-dense plaque in the section perpendicular to the cellular axis. Note that insertion of actin filaments, which is typical for adherens junctions, is present in some parts of the junction (asterisk) but not in others (hash key). Based on the recent finding that all parts of these adhering junctions contain the same set of desmosomal components, they are now defined as area composita\nComplexus adhaerentes and meningeal junctions\nLymphatic endothelial cells in certain lymphatic vessels and in the sinus of lymph nodes form a kind of chimeric cell contact, which contains components from desmosomes (desmoplakin, plakoglobin), adherens junctions (VE-cadherin, \u03b1-catenin, \u03b2-catenin, afadin) as well as from tight junctions (claudin-5 and ZO-1) (Hammerling et al. 2006; Schmelz and Franke 1993; Schmelz et al. 1994). The ultrastructure of complexus adhaerentes shares features of both adherens junctions and desmosomes. Complexus adhearentes form continuous belt-like junctions similar to adherens junctions of vascular endothelial cells, whereas their junctional plaques are more similar to desmosomes. It was proposed that plakoglobin in these contacts is responsible for recruitment of desmoplakin (Kowalczyk et al. 1998). Recently, in meningeoma cells, a new type of adhering junctions was discovered in which adherens junctions also contained the desmosomal plaque protein plakophilin 2 (Akat et al. 2008).\nThe desmosomal components\nDesmosomal cadherins\nThe desmosomal members of the cadherin superfamily, desmogleins (Dsg 1\u20134) and desmocollins (Dsc 1\u20133), are single-pass transmembrane glycoproteins, which mediate adhesion in Ca2+-dependent manner (Buxton et al. 1993; Garrod et al. 2002; Getsios et al. 2004b; Nollet et al. 2000). Desmosomal adhesion molecules have been first isolated from desmosomal intercellular regions (Gorbsky and Steinberg 1981). Using specific antibodies to localize proteins at the cell surface and to inhibit desmosome formation, Dsc 1 (130\u00a0kDa) and Dsc 2 (115\u00a0kDa) were shown to be directly involved in cell\u2013cell adhesion (Cowin et al. 1984) and later on were identified to be cadherin family proteins (Collins et al. 1991; Koch et al. 1991b). Similarly, Dsg 1 (165\u00a0kDa), Dsg 2 (116\u00a0kDa), Dsg 3 (130\u00a0kDa) and Dsc 3 (110\u00a0kDa) were characterized (Amagai et al. 1991; Arnemann et al. 1991; Jones et al. 1986a; King et al. 1995; Koch et al. 1991a, 1990; Schafer et al. 1994; Schmelz et al. 1986a, b). More recently, Dsg 4 (108\u00a0kDa) was found to be the principal Dsg expressed in hair follicles (Kljuic et al. 2003; Whittock and Bower 2003). The genes encoding desmosomal cadherins, which share an amino acid identity of approximately 30\u201350%, both with each other and with classical cadherins, are all located on chromosome 18 in humans (Cowley et al. 1997). Mouse models revealed that Dsg 2 and Dsc 3 are the most important desmosomal cadherin members because deficiency caused embryonic lethality (Table\u00a01) (Den et al. 2006; Eshkind et al. 2002). Because lethality induced by Dsc 3 deficiency occurred before mature desmosmes were formed, and because Dsg 2 was observed to be also localized outside of desmosomes in embryonal stem cells, non-desmosmal functions of Dsc 3 and Dsg 2 seemed to be responsible in this context. In contrast, ablation of cadherins with more restrictive expression patterns such as Dsc 1 led to localized superficial epidermal acantholysis or mucosal and deep epidermal splitting in traumatized skin accompanied by hair loss in the case of Dsg 3 (Chidgey et al. 2001; Koch et al. 1998). Dsg 4 mutations are followed primarily by defective hair formation (Kljuic et al. 2003).\nTable\u00a01Desmosome-associated diseases in humans and in transgenic mouse modelsMouse model Desmosomal componentHuman diseaseInactivationGenetic alteration\u2013Dsg 1Striate palmoplantar keratoderma (SPPK) (Rickman et al. 1999)Embryonic lethalityDefective stem cell proliferation(Eshkind et al. 2002)Dsg 2Arrhythmogenic right ventricular cardiomyopathy (ARVC) (Pilichou et al. 2006)Suprabasal epidermal acantholysisOral erosionsHair loss(Koch et al. 1998)Dsg 3\u2013Impaired hair keratinisationHyperproliferation (Kljuic et al. 2003)Dsg 4Hypotrichosis (Kljuic et al. 2003)Epidermal granular layer acantholysisImpaired barrier functionHyperproliferation(Chidgey et al. 2001)Dsc 1\u2013\u2013Dsc 2Arrhythmogenic right ventricular cardiomyopathy (ARVC)(Syrris et al. 2006; Heuser et al. 2006)Embryonic lethality(Den et al. 2006)Dsc 3\u2013Embryonic lethalityMyocardial fragilityEpidermal subcorneal acantholysis(Bierkamp et al. 1996, 1999; Ruiz et al. 1996)PlakoglobinNaxos disease:Arrhythmogenic right ventricular cardiomyopathy (ARVC)Palmoplantar keratodermaWoolly hair(McKoy et al. 2000)Embryonic lethalityDefects in heart, skin, blood vesselsNeuroepithelium(Gallicano et al. 1998, 2001)DesmoplakinArrhythmogenic right ventricular cardiomyopathy (ARVC)(Rampazzo et al. 2002)Striate palmoplantar keratoderma (SPPK)(Armstrong et al. 1999; Whittock et al. 1999)Carvajal syndrome:Dilated left ventricular cardiomyopathyStriate palmoplantar keratoderma (SPPK)Woolly hair(Norgett et al. 2000)Lethal acantholytic epidermolysis bullosa:-suprabasal epidermal blistering-universal alopecia-nail loss(Jonkman et al. 2005)\u2013Plakophilin 1Ectodermal dysplasia and skin fragility syndrome:Skin blistering around mouth, on soles and palms(McGrath 1997)Embryonic lethalityHeart defects(Grossmann et al. 2004)Plakophilin 2Arrhythmogenic right ventricular cardiomyopathy (ARVC)(Gerull et al. 2004)Bacterial toxinsSuperficial epidermal acantholysis(Melish and Glasgow 1970; Amagai et al. 2000a, b)Dsg 1Staphylococcus scalded skin syndrome:Superficial skin splittingFever, erythemaSkin tenderness(first described by Ritter von Rittershain 1878)AutoantibodiesSuperficial epidermal acantholysis(Amagai et al. 1995)Dsg 1Pemphigus foliaceus (PF)Superficial epidermal acantholysisPemphigus vulgaris (PV, mucocutaneous)Suprabasal epidermal acantholysisMucosal erosions (mouth, larynx, nasal cavity, vagina)(Lever 1953; Koulu et al. 1984; Amagai et al. 1991)Suprabasal epidermal acantholysisMucosal erosions(Anhalt et al. 1982; Amagai et al. 1992; Mahoney et al. 1999)Dsg 3Pemphigus vulgaris (PV, mucosal dominant)Suprabasal epidermal acantholysis(Lever 1953; Amagai et al. 1991)\nThe structure of desmosomal cadherins\nDesmosomal cadherins are type I integral membrane proteins. The amino-terminal extracellular domain of desmosomal cadherins consists of four cadherin repeats (EC1\u20134) of about 110 amino acids followed by a less related membrane-proximal domain (EC 5) (Dusek et al. 2007b). Based on the crystal structure of classical cadherins (Boggon et al. 2002; Overduin et al. 1995; Shapiro et al. 1995), the EC 1\u20134 domains are thought to be connected via flexible linkers which are rigidified by binding of up to three Ca2+ ions each (Pertz et al. 1999). In the cytoplasmic domain, a juxtamembranuous anchor (IA) region is located which, at least in the case of Dsc 1a, contains a desmoplakin-binding element (Troyanovsky et al. 1994b) and maybe be involved in binding and trafficking of p120catenin similar to its role in E-cadherin (Miranda et al. 2003). The following cadherin-typical sequence (ICS) is required for binding to plakoglobin (Mathur et al. 1994; Troyanovsky et al. 1994a). For desmocollins, in which a long and short \u201ca\u201d and \u201cb\u201d isoform is generated by alternative splicing (Collins et al. 1991), it has been shown that the ICS domain is lacking in the b isoform which therefore is unable to bind plakoglobin but instead associates with plakophilin 3 (Bonne et al. 2003; Troyanovsky et al. 1993). The functions of the C-terminal proline-rich linker (L), the Dsg-specific repeated unit domains (RUDs) and the desmoglein terminal domain (DTD), which are all only present in desmogleins, are not clear (Dusek et al. 2007b).\nThe Ca2+-dependency of desmosomal cadherin-mediated binding\nIt is well established that binding of desmosomal and classical cadherins is strictly Ca2+-dependent (Chitaev and Troyanovsky 1997; Heupel et al. 2007; Pertz et al. 1999; Waschke et al. 2007). For Dsg 1, Ca2+-dependency of homophilic binding has been characterized in more detail. It was found that the Ca2+ concentration for half-maximal binding activity of Dsg 1 is 0.8\u00a0mM Ca2+ and that binding is highly cooperative with the Hill coefficient being \u22655 (Waschke et al. 2007). This indicates that Dsg 1 binding is strong only at extracellular Ca2+concentrations higher than 0.8\u00a0mM. Although the exact extracellular Ca2+concentration within the epidermis is unknown, it has been shown that a gradient exists with low Ca2+concentrations in the basal layers and high concentrations in the superficial epidermis (Elias et al. 2002; Menon and Elias 1991). Therefore, if homophilic binding of Dsg 1 occurs in vivo, it has to be considered that it may contribute to effective intercellular adhesion only in the superficial epidermis.\nTransinteraction mechanisms of desmosmal cadherins\nAt present, it is still a matter of debate how desmosmal cadherins interact with each other in vivo. However, several lines of evidence indicate that the N-terminal EC 1 domain is important. Similar to classical cadherins, desmosomal cadherins contain a cell adhesion recognition (CAR) site containing a central alanine residue (Blaschuk et al. 1990). However, instead of the conserved tripeptide HAV sequence of classical cadherins, the sequence is YAT for Dsc 1 and RAL for Dsg 1, respectively (Tselepis et al. 1998). Peptides derived from these sequences were able to block homophilic adhesion mediated by Dsg 1 and Dsc 1 and to inhibit desmosomal adhesion in epithelial cells when the peptides for Dsg 1 and Dsc 1 were applied together, indicating that the CAR site in the EC 1 domain is critical for maintenance of desmosmal adhesion (Runswick et al. 2001; Tselepis et al. 1998). This hypothesis is supported by studies in which mechanisms underlying the loss of keratinocyte cohesion in pemphigus were investigated. AK23, a monoclonal Dsg 3 antibody from a PV mouse model directed against the predicted binding motif of the EC 1, has been shown to be pathogenic in vivo whereas antibodies targeting other parts of the Dsg 3 extracellular domain were not (Tsunoda et al. 2003). Together with the recent finding that AK 23 similar to Dsg 3 antibodies from PV patients, which are known to be also primarily directed against the N-terminal part of EC 1 (Sekiguchi et al. 2001), is able to directly interfere with Dsg 3 binding (Heupel et al. 2007), these data demonstrate that interaction of EC 1 is crucial for Dsg 3 binding. In addition to these functional studies, morphologic studies sought to address the mode of desmosomal cadherin interaction within desmosomes by using electron tomography imaging of epidermal tissue (Al-Amoudi et al. 2007; He et al. 2003). Based on predictions from the C-cadherin crystal structure, He and colleagues reported that in mouse epidermis several desmosomal cadherins form knots in which cadherins display stochastic arrangement. In these knots, desmosomal cadherins seemed to interact via their EC 1 domains with both molecules on the same cell in cis as well as with molecules from opposing cells in trans (He et al. 2003). Al-Amoudi and co-workers refined the technique by employing cryo-electron microscopy in human epidermis. They confirmed cis- and trans-interactions of the EC 1 domains, possibly via insertion of the tryptophane 2 into the hydrophobic pocket of the CAR site (Al-Amoudi et al. 2007). However, they found that cis-interactions of the EC 4\u20135 regions may also occur and that desmosomal cadherins rather show periodically zipper-like arrangements similar to classical cadherins (Boggon et al. 2002).\nHomophilic and heterophilic binding of desmosomal cadherins\nIn contrast to classical cadherins from adherens junctions which primarily bind in homophilic manner, data indicate that desmosomal cadherins undergo both homophilic and heterophilic transinteraction. Using EC 1\u20132 fragments of Dsg 2 and Dsc 2, it was shown that homophilic binding occurs in vitro (Syed et al. 2002). Similarly, homophilic binding of Dsg 3 was found (Amagai et al. 1994b). When recombinant proteins consisting of the complete extracellular domain were used for atomic force microscopy (AFM) measurements, it was demonstrated that the unbinding force of single homophilically transinteracting molecules was about 37\u201368\u00a0pN (depending on the retrace velocity 300\u20136,000\u00a0nm\/s) for Dsg 1 with a lifetime of 0.17\u00a0s and about 50\u00a0pN for Dsg 3 (Heupel et al. 2007; Waschke et al. 2005, 2007), which is in the same range as observed for other types of cadherins characterized by the same method such as VE-cadherin, N-cadherin or LI-cadherin (Baumgartner et al. 2003, 2000; Wendeler et al. 2007). These data indicate that the molecular binding properties of homophilic adhesion of desmosomal cadherins may be comparable to other cadherins.\nHeterophilic binding of Dsg 2 to Dsc 1 or Dsc 2 was also found on the molecular level (Chitaev and Troyanovsky 1997; Syed et al. 2002) but no interaction of Dsg 1 with Dsg 3 (Heupel et al. 2007). Aggregation assays of transfected cells indicated that in cells, heterophilic binding of Dsgs and Dscs might be of even greater importance than homophilic binding to induce strong intercellular adhesion (Kowalczyk et al. 1996; Marcozzi et al. 1998; Runswick et al. 2001) and that adhesion is strictly dependent on the ratio of the respective Dsgs and Dscs (Getsios et al. 2004a). This view is supported by the recent finding that a conditional Dsc 3-deficiency in mice induced a severe pemphigus-like phenotype with epidermal blistering (Chen et al. 2007). Because antibodies in typical pemphigus are usually not directed against Dsc 3 but against Dsg 1 and Dsg 3, it has to be considered that heterophilic binding of these three molecules is important for epidermal cohesion in vivo.\nArmadillo family proteins\nFrom the Armadillo family, plakoglobin and plakophilins 1\u20133 are important components of desmosomes.\nPlakoglobin\nPlakoglobin (82\u00a0kDa), also termed \u03b3-catenin, is the only essential desmosomal component which is also found in typical adherens junctions (Cowin et al. 1986; Franke et al. 1989, 1983). The gene encoding for plakoglobin was mapped to chromosome 17 (Aberle et al. 1995). Plakoglobin binds to the cytoplasmic cadherin-typical sequence of Dsgs and Dscs via its first three armadillo repeat domains (Chitaev et al. 1998). Because the same binding site is required for interaction of plakoglobin to \u03b1-catenin, the latter is excluded from desmosomes. Similarly, although the armadillo repeat domain of \u03b2-catenin can also bind to Dsg 2, its flanking domains inhibit this interaction, which may explain the absence of \u03b2-catenin from desmosomes (Troyanovsky et al. 1996; Wahl et al. 1996). Plakoglobin has been demonstrated to interact with other desmosomal plaque components such as desmoplakin, plakophilins and also with cytokeratin filaments (Bonne et al. 2003; Chen et al. 2002; Kowalczyk et al. 1997; Smith and Fuchs 1998). The importance of this linker function can be concluded from studies in which inactivation of plakoglobin led to embryonic lethality due to mechanical fragility of the myocardium and, when mice are viable, to subcorneal skin blistering indicating that plakoglobin is essential for desmosomal stability (Table\u00a01) (Bierkamp et al. 1996; Ruiz et al. 1996).\nBesides its function as a desmosomal adaptor protein, plakoglobin seems to be involved in nuclear signalling. It has been shown that plakoglobin, comparable to \u03b2-catenin in the canonical wnt signalling pathway, confers transcriptional activity together with TCF-4\/LEF transcription factors (Maeda et al. 2004). This mechanism seems to interfere with \u03b2-catenin-mediated transcription (Hu et al. 2003). Because plakoglobin like \u03b2-catenin is a target of glycogen synthase kinase-3 \u03b2, which drives proteosomal degradation of both proteins (Kodama et al. 1999; Muller et al. 2008a; Williamson et al. 2006), a complex pattern of direct and indirect transcriptional regulation seems likely. A target gene of Lef-1\/plakoglobin signalling is c-Myc, the expression of which was inhibited in keratinocytes but enhanced in transformed rat kidney epithelial cells (Kolligs et al. 2000; Kolly et al. 2007; Williamson et al. 2006). This indicates that the role of plakoglobin transcriptional regulation is strictly cell type-dependent. In keratinoytes, c-Myc repression by plakoglobin is required to stop proliferation and to allow terminal differentiation (Williamson et al. 2006). Another potential target gene is the anti-apoptotic molecule Bcl\u2013XL, which was found to be upregulated in plakoglobin-deficient cells leading to reduced apoptosis and thus might also be repressed by plakoglobin (Dusek et al. 2007a). Taken together, plakoglobin serves as functional linker between intercellular adhesion and regulation of the cell cycle. This might also be important for cancer progression because many tumors are characterized by loss of plakoglobin expression.\nPlakophilins\nPlakophilin 1 (80\u00a0kDa) was first identified as an \u201caccessory\u201d plaque protein because, in contrast to plakoglobin and desmoplakin, it was found in cells from certain stratified and complex epithelia only (Franke et al. 1983; Hatzfeld et al. 1994; Heid et al. 1994). Afterwards, it became clear that plakophilin 2 (100\u00a0kDa) is ubiquitously present in all desmosomes and also plakophilin 3 (87\u00a0kDa) is present in most simple and stratified epithelia (Mertens et al. 1999; Schmidt et al. 1999). The genes encoding plakophilin 1, 2 and 3 are located on chromosomes 1, 12, and 11, respectively (Bonne et al. 1998). Plakophilin 1 and 2 exist in two splice variants with a shorter \u201ca\u201d and a longer \u201cb\u201d form (Mertens et al. 1996; Schmidt et al. 1997). In addition to their localization in desmosomes, plakophilins 1 and 2 are also found in the karyoplasm in a variety of cells and plakophilin 1 b is exclusively located in the nucleus. Plakophilin 2 deficiency leads to embryonic death due to heart defects indicating that the presence of at least one member of the plakophilin family is required (Table\u00a01) (Grossmann et al. 2004). Under these conditions, cytokeratin filaments were retracted from cell borders and desmoplakin was localized in the cytoplasm rather than at desmosomes in cardiomyocytes, demonstrating the relevance of plakophilin 2 to desmosplakin recruitment. Because cardiomyocytes in contrast to epithelial cells express only plakophilin 2 but not plakophilin 1 and 3, defects were present in the heart only, whereas epithelia were not affected.\nPlakophilins can directly interact with all other desmosomal components including cytokeratins via the aminoterminal head domain (Bonne et al. 2003; Hatzfeld 2007; Hatzfeld et al. 2000). Plakophilin 1 recruits desmosomal components to the cell membrane, increases size and number of desmosomes and therefore seems to be a scaffolding protein, which induces desmosome assembly (Hatzfeld et al. 2000; Kowalczyk et al. 1999; Wahl 2005). On the other hand, because plakophilin 1 is located in the dense inner desmosomal plaque whereas cytokeratin filaments only loop into the outer plaque, it is believed that plakophilin 1 enhances desmoplakin lateral interactions but does not directly associate with cytokeratin filaments in vivo (Hatzfeld 2007; North et al. 1999). It has been demonstrated that desmosome formation is mediated by the aminoterminal domain, whereas recruitment of plakophilin 1 itself to the plasma membrane is dependent on the carboxyterminal region (Sobolik-Delmaire et al. 2006).\nIn addition to its function in the regulation of desmosome assembly, plakophilins may also regulate signalling mechanisms, both at cell borders as well as in the nucleus. Plakophilin 1 associates with actin filaments at cell borders and has been reported to interact with a GTP exchange factor (GEF) for Rho and thereby could regulate activity of Rho GTPases similar to the closely related p120-catenin, which is known to inhibit Rho A and to activate Rac 1 and Cdc42 (Anastasiadis and Reynolds 2001; Hatzfeld 2007). In addition to their localization within the desmosomal plaque, plakophilin 1 and 2 are also present inside nucleus and plakophilin 2 has been found to be part of the polymerase III complex which is responsible for generation of tRNA and rRNA (Mertens et al. 2001). By these two mechanisms, it is possible that plakophilins may regulate cell adhesion and cell growth (Hatzfeld 2007).\nPlakin family proteins\nPlakin family proteins are linkers between the cytoskeleton and cell\u2013cell or cell\u2013matrix contacts (Jefferson et al. 2007). Desmoplakin, which exists in two spice variants of a protein encoded by a single gene on chromosome 6 (desmoplakin I: 322\u00a0kDa; desmoplakin II: 259\u00a0kDa), is an essential component of the desmosomal plaque and therefore is regarded as the prototype of this family (Armstrong et al. 1999; Hatsell and Cowin 2001; Mueller and Franke 1983; Sonnenberg and Liem 2007). Other members such as plectin, envoplakin and periplakin were also found in desmosomes, but their significance for the structure and function of desmosomes is less clear. Especially, plectin is primarily important in hemidesmosmes, which anchor epithelia to the extracellular matrix.\nDesmoplakin consists of an aminoterminal plakin domain, which can interact with all other desmosomal plaque proteins such as plakoglobin and plakophilins but also with Dsc 1a (Kowalczyk et al. 1997; Smith and Fuchs 1998; Troyanovsky et al. 1994b). The central coiled-coil rod domain, which is important for dimerization, is followed by the carboxyterminal tail consisting of three globular subdomains with several plakin-repeats, which serve as linkers for different intermediate filament types (Choi et al. 2002; Green et al. 1990; Stappenbeck and Green 1992). It is well established that desmoplakin is the main linker protein between the desmosomal cadherin\u2013plakoglobin complex and the intermediate filament cytoskeleton. This has been shown in vitro and was ultimately demonstrated in desmoplakin-deficient mice, which had a reduced number of desmosomes and died at embryonic stage just after implantation (Table\u00a01) (Bornslaeger et al. 1996; Gallicano et al. 1998). Similar to the findings in epidermal-specific desmoplakin-deficient mice, which suffered from skin blistering, desmosomes were not anchored to intermediate filaments (Vasioukhin et al. 2000). Moreover, when desmoplakin was rescued in extraembryonic tissues so that embryos further developed, defects were present not only in the myocardium and epidermis but also in the vasculature and in the neuroepithelium, underlining the importance of desmoplakin for tissue differentiation (Gallicano et al. 2001).\nDiversity of desmosomes in different tissues and specific epithelial layers\nAlthough it was discovered about 25\u00a0years ago that the structure of desmosomes is not identical in all types of cells and tissues (Giudice et al. 1984; Jones et al. 1986b), the knowledge on the diversity of desmosomes is still uncomplete and matter of discussion (Garrod et al. 2002; Getsios et al. 2004b; Green and Simpson 2007; Hatzfeld 2007; Holthofer et al. 2007; Kottke et al. 2006; Yin and Green 2004). The diversity of desmosomes has implications for tissue differentiation and also is of high-medical relevance because diseases caused by genetic alteration of or by an autoimmune reponse against a specific desmosomal component may affect only certain but not all desmosome-containing tissues.\nSome desmosomal components such as Dsg 2, Dsc 2 and the plaque proteins desmoplakin, plakoglobin and plakophilin 2 are ubiquitously expressed in all cells and tissues in which desmosomes are found. Plakophilin 3 is present in most simple epithelia except hepatocytes as well as in stratified epithelia, whereas plakophilin 1 is restricted to stratified and complex epithelia. In epithelia, the desmosomal cadherins show typical expression patterns. Simple epithelia and urothelium usually express Dsg 2 and Dsc 2 only. Apparently, exceptions are the additional presence of Dsg 1 in the mucosa of uterus, stomach, intestine and in epithelia of liver and pancreas as well as the expression of Dsc 1 in intestine and liver or Dsc 3 in stomach, prostate, salivary gland and urothelium. Dsg 4 has a unique tissue distribution in skin and several simple epithelia such as those present in pancreas, salivary glands, testis, prostate and hepatic epithelium.\nThe Dsg 1\/Dsc 1 and Dsg 3\/Dsc 3 pairs are largely confined to stratified epithelia where the expression patterns of the Dsg and Dsc isoforms usually conform. Interestingly, in the stratified corneal epithelium, only Dsg 1 and Dsc1 are present indicating that these desmosomal cadherins in the absence of Dsg 1 and Dsg 3 are sufficient to maintain cohesion in stratified epithelia also. In the epidermis, the plaque proteins plakoglobin, desmoplakin and plakophilin 3 are expressed in all layers (Fig.\u00a04). In contrast, plakophilins 1 and 2 display inverse distribution with plakophilin 1 being more abundant in the superficial epidermis. These inverse expression patterns are also typical for the Dsg 1\/Dsc 1 and Dsg 3\/Dsc 3 pairs. Dsg 1\/Dsc 1 are the predominant desmosomal cadherins in the superficial epidermis, whereas Dsg 3\/Dsc 3 are primarily expressed in the lower epidermis. In contrast to Dsg 1, which can be detected in some desmosomes in keratinocytes of the basal layer also, Dsc 1 and Dsg 4 are absent in the basal layer (Dusek et al. 2007b; Mahoney et al. 2006; Spindler et al. 2007). Because Dsg 3 is expressed throughout the spinous layers (Fig.\u00a05), the expression patterns of Dsg 1 and Dsg 3 largely overlap in human adult epidermis (Mahoney et al. 2006; Spindler et al. 2007). Dsc 2 is enriched in the deep epidermis with lower levels in the superficial epidermis, whereas Dsg 2 is restricted to basal and suprabasal cells but is present in very faint amounts only (Mahoney et al. 2006) indicating that this pair of proteins may be primarily important for cell cohesion in simple epithelia and myocardium rather than in complex epithelia. However, it is unclear at present which desmosomal cadherin isoforms are capable to heretophilically bind to each other and thus interpretation of these distribution patterns with respect to their relevance for mechanical adhesion is preliminary. In multilayered squamous epithelium of mucous membranes, for instance of the oral cavity, Dsg 1 and Dsg 3 are strongly expressed throughout all layers, whereas Dsg 4 shows strong expression in superficial layers but is missing in the basal layer (Mahoney et al. 2006).\nFig.\u00a04Expression patterns of desmosomal components in the epidermis. The schematic drawing of the epidermis (left) indicates the basal (BL), spinous (SL), granular (GL) and corneal (CL) layer of the epidermis. On the right, the expression patterns of desmosomal components in the specific epidermal layers are illustrated. For instance, Dsg 1 and Pkp 1 are most prominent in the superficial layers, whereas expression of Dsg 3 and Dsc 3 is strongest in the deep epidermis. Dsg desmoglein, Dsc desmocollin, Pkp plakophilin, PG plakoglobin, DP desmoplakinFig.\u00a05Immunostaining of Dsg 1 and Dsg 3 in human epidermis. Intact human epidermis was immunostained using monoclonal antibodies against Dsg 1 (a) and Dsg 3 (b). A merge of both panels is shown in c. Dsg 1 is most abundant in the superficial epidermis but is also present in the basal layer. Dsg 3 is expressed in the basal layer as well as throughout the spinous layer indicating that in human epidermis the expression patterns of these two proteins broadly overlap. Scale bar is 20\u00a0\u03bcm\nIt is important to note that the specific distribution patterns of desmosomal components in stratified epithelia are important for epithelial differentiation and function (Green and Simpson 2007). It was shown that forced overexpression of Dsg 3 in the suprabasal epidermis led to abnormal differentiation and hyperproliferation as well as perinatal lethality due to transepidermal water loss (Elias et al. 2001; Merritt et al. 2002). Similarly, forced suprabasal Dsg 2 and Dsc 3 overexpression resulted in hyperproliferation and formation of papillomas, possibly via altered \u03b2-catenin\/wnt signalling (Brennan et al. 2007; Hardman et al. 2005).\nDesmosome assembly and disassembly\nThe mechanisms participating in desmosome assembly and disassembly have been reviewed in detail elsewhere (Getsios et al. 2004b; Green and Simpson 2007; Kitajima 2002; Yin and Green 2004). For instance, extracellular Ca2+ and protein kinase C (PKC) signalling are well known to be involved in desmosome assembly. Ca2+ concentrations >0.1\u00a0mM allow formation of adherens junctions and desmosomes (Hennings and Holbrook 1983). Desmosomal plaques with inserted cytokeratin filaments became visible as early as after 5\u00a0min after the Ca2+ switch followed by appearance of assymetrical desmosomes after 10\u00a0min and of symmetric desmosomes after 1\u00a0h. Increased extracellular Ca2+ induced incorporation of desmosomal components such as Dsgs, plakoglobin and desmoplakin into the desmosomal plaque (Hennings and Holbrook 1983; Pasdar et al. 1995; Pasdar and Nelson 1988, 1989). Activation of PKC is required for translocation of desmosomal components to the cell membrane and for desmosome assembly (Sheu et al. 1989), but also was found to reduce desmosomal adhesion and to increase Ca2+-dependence of desmosomes (Kimura et al. 2007) indicating that regulation of desmosomal adhesion by PKC is complex.\nBefore desmosome assembly, adhesion zippers of E-cadherin-containing puncta form on filopodial processes of neighbouring cells, an event that requires both \u03b1-catenin and VASP-driven actin reorganization (Vasioukhin et al. 2000). Afterwards, these intermediate junctions mature to adherens junctions and desmosomes are assembled at regions where membranes are brought together. It appears that Dscs initiate the formation of desmosomes. This is based on the observations that Dsc 2 is the first desmosomal component at the cell surface followed by Dsg 2 in MDCK cells (Burdett and Sullivan 2002) and that, in keratinocytes, N-terminally deleted Dsc 3, which compromised desmosome formation was still able to bind to \u03b2-catenin. Therefore, it can be speculated that Dsc 3 could localize to pre-existing adherens junctions to induce desmosome formation (Hanakawa et al. 2000). Desmosomal cadherins seem to be transported in vesicles from the Golgi along microtubules whereas non-membranous cytoplasmic particles containing desmoplakin and plakophilin are associated with intermediate filaments and move towards cell-junctions by actin-based motility (Godsel et al. 2005; Green and Simpson 2007). Desmoplakin trafficking seems to be dependent on intracellular Ca2+ levels because patients with Darier\u2019s disease, which results from mutations in a sarcoplasmic reticulum Ca2+ pump show desmoplakin retention in the cytoplasm and altered desmosome structure (Dhitavat et al. 2003; Dhitavat et al. 2004; Sakuntabhai et al. 1999).\nOn the cell surface, the desmosomal cadherins together with plakoglobin and desmoplakin are sufficient to nucleate a desmosomal plaque (Kowalczyk et al. 1997). Further plaque enlargement and desmoglein clustering are dependent on plakoglobin together with plakophilin (Bornslaeger et al. 2001; Koeser et al. 2003). Therefore, keratinocytes deficient for either plakoglobin or desmoplakin display reduced numbers of desmosomes, disturbed plaque formation and reduced anchorage of cytokeratin filaments (Bierkamp et al. 1999; Vasioukhin et al. 2001). It appears that during desmosome assembly, Dsg3-containing clusters are formed in the beginning, which, upon attachment to cytokeratin filaments, become integrated in desmosomes (Sato et al. 2000). Once they are formed, desmosomes are stable throughout the cell cycle and are not disrupted during mitosis, although the desmosomal components are subjected to a significant turnover with a half-life of about 30\u00a0min like it was shown for Dsc 2a (Windoffer et al. 2002). Finally, it has to be emphasized that a reciprocal dependence of desmosomes and adherens junctions seems to exist. This can be concluded from experiments in which expression of N-terminally deleted Dsc 3 or desmoplakin deficiency resulted in impaired formation of both desmosomes and adherens junctions (Hanakawa et al. 2000; Vasioukhin et al. 2001).\nRegulation of keratinocyte proliferation by desmosomal cadherins\nEvidence is accumulating that desmosomal cadherins such as Dsg 3 regulate keratinocyte proliferation (Muller et al. 2008a). It has been shown that autoantibodies from pemphigus vulgaris patients induce continuing keratinocyte proliferation by impaired Dsg 3\/plakoglobin signalling, which finally leads to c-Myc overexpression (Muller et al. 2008a; Williamson et al. 2007, 2006). According to this concept, in healthy epidermis Dsg 3 binding results in inhibition of glycogen synthase kinase 3 (GSK3) via activation of phosphatidylinositol trisphosphate kinase (PI3K) and Akt. In consequence, GSK3 phosphorylation-dependent degradation of plakoglobin is abolished which allows plakoglobin to translocate into the nucleus and to induce growth arrest via suppression of c-Myc (Muller et al. 2008a; Williamson et al. 2006).\nDesmosome-associated diseases\nSeveral diseases have been found in which impaired desmosomal adhesion contributes to pathogenesis. Inactivation of desmosomal function may be reduced by completely different mechanisms including genetic defects of desmosomal components, cleavage of desmosomal cadherins by bacterial toxins and binding of autoantibodies to desmogleins 1, the latter of which is the cause of the autoimmune disease pemphigus. Although altered expression of desmosomal cadherins such as Dsg 2\/Dsc 2 and Dsg 3\/Dsc 3 have been observed in human carcinomas such as squamous cell carcinoma as well as gastric, colorectal and breast carcinomas, mutations are usually absent (Bazzi and Christiano 2007). Therefore, the role of desmosomal cadherins in cancer is unclear at present.\nGenetic diseases\nMutations in desmosomal plaque components in humans affect the myocardium as well as the epidermis with its appendages (Table\u00a01). Mutations in genes for the essential desmosomal plaque components desmoplakin and plakoglobin result in heart, skin and hair defects (Bazzi and Christiano 2007; Green and Simpson 2007; McGrath 2005). In contrast, genetic alterations of Dsg 2, Dsc 2 and plakophilin 2 selectively lead to heart defects because these are the only isoforms of their protein families in the myocardium. On the other hand, mutations in Dsg 1 and plakophilin 1, which are primarily expressed in the epidermis, cause skin defects whereas loss of Dsg 4 in hair follicles results in hair loss.\nGenetic heart defects\nInterestingly, all defects of desmosomal components causing heart defects such as desmoplakin, plakoglobin, plakophilin 2, Dsg 2 and Dsc 2 lead to the phenotype of arrhythmogenic right ventricular cardiomyopathy (ARVC) which is clinically characterized by right bundle block and arrhythmia and histologically by fibrofatty replacement of cardiomyocytes, possibly due to impaired cell adhesion caused by loss and alterations of desmosomes (Asimaki et al. 2007; Gerull et al. 2004; McKoy et al. 2000; Pilichou et al. 2006; Rampazzo et al. 2002; Syrris et al. 2006; Heuser et al. 2006). This is in line with embryonic lethality due to myocardial rupture in mice models deficient in these proteins. Therefore, the thinnest parts of the right ventricle are the most severely affected, but left ventricle involvement also occurs (van Tintelen et al. 2007). However, fibrofatty transdifferentiation of cardiomyocytes cannot be simply explained by impaired desmosomal adhesion, but rather seems to be caused by altered wnt\/ \u03b2-catenin signalling in response to nuclear translocation of plakoglobin (Garcia-Gras et al. 2006).\nGenetic defects of skin and its appendages\nHaploinsufficiency of the gene encoding Dsg 1 results in the autosomal dominant skin disease striate palmoplantar keratoderma (SPPK), which is characterized by linearly arranged thickening of the stratum corneum on the palms, soles, knees, ankles and finger knuckles (Milingou et al. 2006; Rickman et al. 1999). However, blisters are absent indicating that disturbed differentiation is the primary mechanism underyling this entity rather than a loss of keratinocyte adhesive function. Similarly, mutated Dsg 4 leads to autosomal recessive inherited hypotrichosis due to defective hair follicle differentiation, a phenotype related to the lanceolate hair mouse (Kljuic et al. 2003). In contrast, ablation of plakophilin 1 results in the recessive skin-fragility ectodermal dysplasia syndrome, which in 1997 was the first genetic desmosome-associated disease to be described (McGrath 2005). Here, both loss and alterations of desmosomes and lacking insertion of cytokeratin filaments due to inability to recruit desmoplakin cause skin blistering around the mouth as well as on palms and soles accompanied by dystrophic hair and nails (McGrath et al. 1997; McMillan and Shimizu 2001).\nMutations in plaque proteins with involvement of various tissues\nMutations in plakoglobin are the cause of Naxos disease in which ARVC and palmoplantar keratoderma are associated with woolly hair (McKoy et al. 2000). Interestingly, in contrast to plakoglobin-deficient mice (Bierkamp et al. 1996), acantholysis is absent indicating that some mechanical functions of plakoglobin are maintained in these patients.\nThe most variable phenotypes are the consequence of desmoplakin alterations. An autosomal recessive disorder with dilated cardiomyopathy, keratoderma and woolly hair called Carvajal syndrome is comparable to Naxos disease (Norgett et al. 2000). Haploinsufficiency leads to SPPK, whereas non-sense mutations are accompanied by skin fragility leading to blisters in the face as well as on extremities and trunk and also with wolly hair (Armstrong et al. 1999; Whittock et al. 1999, 2002). However, the most severe disorder is lethal acantholytic epidermolyis bullosa, which is caused by C-terminally truncated desmoplakin and was fatal in a 10-day-old hair and nailless newborn due to extensive blistering leading to transcutaneous fluid loss (Jonkman et al. 2005). On the ultra-structural level, desmosomes were reduced in desmoplakin-related SPPK similar to SPPK caused by mutations in Dsg 1 (Wan et al. 2004), but not in lethal acantholytic epidermolysis bullosa. However, desmosome shedding, alterations of desmosomal plaques and impaired cytokeratin insertion were typically associated with desmoplakin mutations (Jonkman et al. 2005; Norgett et al. 2000; Wan et al. 2004). At present, it is unclear why different mutations affect different tissues.\nInfectious diseases\nStaphylococcal scalded skin syndrome (SSSS), which was first described by Ritter von Rittershain in 1878, is the systemic variant of epidemic pemphigus neonatorum or sporadic bullous impetigo and is characterized by superficial epidermal splitting accompanied by fever, erythema and skin tenderness (Farrell 1999; Lyell 1983; Stanley and Amagai 2006). Most cases are caused by staphylococcal exfoliative toxin (ET), a serine protease, which has been shown to selectively cleave Dsg 1 between EC 3 and 4 in conformation-dependent manner, but not Dsg 3 or E-cadherin (Table\u00a01) (Amagai et al. 2000a, 2002; Hanakawa et al. 2002; Melish and Glasgow 1970). Assuming that ET does not cleave other superficially expressed desmosomal cadherins such as Dsg 4 or Dsc 1, SSSS is a good example that extensive epidermal blistering can be induced by proteolytic cleavage of a single adhesion molecule. According to its bacterial pathogenesis, SSSS can be effectively treated with antibiotics (Stanley and Amagai 2006).\nPemphigus\nPemphigus is an autoimmune blistering skin disease, which is characterized by intraepidermal blistering (Lever 1953). The two major types of pemphigus are the more severe pemphigus vulgaris (PV), which accounts for 80\u201390% of cases, and pemphigus foliaceus (PF) (Bystryn and Rudolph 2005; Schmidt et al. 2000; Stanley and Amagai 2006). Pemphigus is a rare disease with a yearly incidence of 0.75\u20135 cases per million and apart from being present in humans, is also found in horses, dogs and cats. In contrast to other autoimmune diseases, which primarily affect women, pemphigus is equally distributed between both genders, and is diagnosed mostly between the fourth and sixth decades. In PV, there are two main forms, the mucosal-dominant and the mucocutaneous type. In both cases, the disease most commonly begins with painful non-healing ulcerations not only in the mucous membranes of the mouth, but also in the larynx, nose and vagina. Later on, flaccid blisters may occur on the scalp, trunk, groin and axillae, which easily rupture, leaving sharply outlined erosions and heal without scarring (Fig.\u00a06). In contrast to PV, PF only affects the epidermis and because epidermal splitting is restricted to the superficial epidermis, lesions appear as crusted erosions on the upper torso, face and scalp. It has to be mentioned that pemphigus can be induced by drugs such as penicillamine, penicillin, captopril and \u03b2-blockers and also can occur as a paraneoplastic entity accompanying or preceding lymphoma and lung carcinoma (Yeh et al. 2003). Moreover, in South America an endemic form of PF, called Fogo selvagem, exists, which is thought to be transmitted by insect vectors (Aoki et al. 2004; Diaz et al. 1989). Currently, the therapy of pemphigus is based on immunosupression and reduction of autoantibody load. Conventional therapy includes high-dose corticosteroids, intravenous immune globulin and cytotoxic agents. Before systemic corticosteroids were available, 75% of PV patients died within a year. Second-line therapies for refractory PV include rituximab, an antibody directed against B cell CD20, which reduces autoantibody-producing B cells as well as plasmapheresis to physically remove autoantibodies (Ahmed et al. 2006; Shimanovich et al. 2008).\nFig.\u00a06Clinical phenotype of Pemphigus vulgaris and Pemphigus foliaceus. Patients suffering from the mucocutaneous form of pemphigus vulgaris (PV) usually have flaccid blisters and erosions on the trunk (a) accompanied by mucosal ulcerations in the mouth (b). In contrast, Pemphigus foliaceus patients are characterized by crusted epidermal erosions (c) whereas involvement of mucous membranes is absent\nHistology and autoantibody profile in pemphigus\nBesides the clinical phenotype, diagnosis of pemphigus is based on histology and the patients\u2019 autoantibody profile (Bystryn and Rudolph 2005). The histologic hallmark of pemphigus is acantholysis, i.e. loss of cell\u2013cell adhesion between keratinocytes. In PV, the epidermal cleavage plane is located in the deep epidermis, usually right above the basal layer (Fig.\u00a07). In contrast, in PF, epidermal splitting occurs between granular layers. Skin blisters can be induced by rubbing on healthy-appearing epidermis, a phenomenon referred to as Nikolsky sign.\nFig.\u00a07Typical histology of epidermal lesions from pemphigus patients. Hematoxylin eosin-stained paraffin sections from PV (a) and PF (b) patients showed suprabasal epidermal cleavage in the PV and superficial granular blistering in PF. Scale bar is 50\u00a0\u03bcm\nAutoantibodies in pemphigus are sufficient to cause blistering in human skin in vivo and in vitro (Anhalt et al. 1982; Schiltz and Michel 1976). In contrast to other autoimmune blistering skin diseases such as bullous pemphigoid or epidermolysis bullosa acquisita (Sitaru and Zillikens 2005; Yancey 2005), pemphigus antibodies do not require the complement system or leukocytes to induce blisters in vivo (Anhalt et al. 1986). It is generally accepted that PV and PF are characterized by different autoantibody profiles, which generally correlate with disease activity (Bystryn and Rudolph 2005; Harman et al. 2001; Ishii et al. 1997; Stanley and Amagai 2006; Stanley et al. 1984; Yeh et al. 2003). Patients suffering from mucosal-dominant PV usually have antibodies directed against Dsg 3 but not Dsg 1, whereas mucocutaneous PV is characterized by both Dsg 3 and Dsg 1 autoantibodies (Amagai et al. 1999; Ding et al. 1997; Jamora et al. 2003; Miyagawa et al. 1999). In contrast, in PF patients usually antibodies against Dsg 1 but not Dsg 3 are detected (Amagai et al. 1999). However, it is also known that in some cases this correlation between the clinical phenotype and the autoantibody profile was not found (Baykal et al. 2002; Jamora et al. 2003; Yoshida et al. 2005; Zagorodniuk et al. 2005).\nOver the last decade, there is a debate whether these autoantibodies against desmosomal cadherins are pathogenic (Amagai et al. 2006). It has been shown by passive transfer of affinity-purified Dsg antibody fractions as well as by depletion of pathogenic activity by absorption against desmoglein extracellular domains that Dsg 1 antibodies in PF and the combination of Dsg 1 and Dsg 3 autoantibodies in PV as well as in paraneoplastic pemphigus are sufficient to induce skin blistering (Amagai et al. 1995, 1994a, 1992, 1991, 1998; Koulu et al. 1984; Mahoney et al. 1999). An active PV mouse model in which Dsg 3-deficient mice were immunized with Dsg 3 before splenocytes from these animals were transferred to lymphopenic Rag-2-deficient mice supported the notion that Dsg 3 antibodies alone can cause mucosal erosions (Amagai et al. 2000b). Similar in keratinocyte cultures, depletion of Dsg 1-specific antibodies from PF-IgG by preincubation with recombinant Dsg 1 but not after preincubation with VE-cadherin completely abolished keratinocyte dissociation (Waschke et al. 2005).\nPemphigus IgG were found to include a plethora of more than 20 different autoantibodies against keratinocyte antigens such as antibodies against Dsg 1, Dsg 4, Dsc 1-3, desmoplakin, plakoglobin and E-cadherin and several other proteins not associated with cell junctions (Amagai et al. 2006; Evangelista et al. 2008; Kljuic et al. 2003; Korman et al. 1989; Nguyen et al. 2000c). For instance, in all PF sera as well as in 79% of mucocutaneous PV sera, autoantibody activities against E-cadherin were detected, most of which were due to Dsg 1 autoantibodies cross-reacting with E-cadherin (Evangelista et al. 2008). Some of the different autoantibodies have clearly been shown not to be pathogenic such as the Dsg 4-cross-reacting Dsg 1 antibodies in PF (Nagasaka et al. 2004). Therefore, similar to other autoimmune diseases, the pathogenetic relevance of autoantibodies against a specific protein in pemphigus has to be challenged until it has been convincingly demonstrated (Amagai et al. 2006). However, it has been reported that antibodies others than those directed to desmogleins also contribute to epidermal blistering because PV-IgG not containing Dsg 1 antibodies were effective to cause blistering in Dsg 3-deficient mice (Nguyen et al. 2000c). It is possible that these antibodies include antibodies to cholinergic receptors and to pemphaxin, which have both been detected in 85% of PV and PF sera (Grando 2006a; Nguyen et al. 2000b). The pathogenic relevance of antibodies against cholinergic receptors was concluded from experiments where preincubation of monkey oesophagus with PV-IgG blocked staining by a rabbit acetylcholine receptor antibody and the fact that this antibody induced keratinocyte dissociation in culture (Nguyen et al. 2000a). However, antibodies against pemphaxin alone were not sufficient to induce skin blistering (Nguyen et al. 2000b). Moreover, it has not been demonstrated so far that autoantibodies from pemphigus patients, which target cholinergic receptors are capable to induce acantholysis. Therefore, at present it is safe to believe that epidermal blistering in pemphigus is primarily caused by antibodies against Dsg 1 and Dsg 3. These pathogenic antibodies in PV and PF mainly belong to the IgG 4 and IgG 1 subclasses (Bhol et al. 1995; Rock et al. 1989; Spaeth et al. 2001).\nThe knowledge that autoantibodies against desmosomal cadherins are sufficient to induce acantholysis in complement- and leukocyte-independent manner makes pemphigus one of the best-characterized models to study the direct mechanisms underlying autoimmune diseases. Besides the role of autoantibodies, the contribution of Dsg 3 autoreactive T helper (Th) cells has also been characterized for PV and endemic PF (Hertl et al. 2006). Th1 and Th2 cells in PV recognize the extracellular domain of Dsg 3 when presented on the HLA class II alleles HLA-DR\u03b21*0402 and HLA-DQ\u03b21*0503, whereas in Fogo selvagem patients HLA-DR\u03b21*0402 and HLA-DR\u03b21*0101 were most common. In PV patients as well as in healthy carriers of the PV-associated HLA II alleles, Dsg 3 and Dsg 1-autoreactive T cells were found. In healthy individuals, Th1 cells with characteristics of regulatory T (Tr1) cells which inhibit T cell activation were most prevalent. In contrast, in PV patients the levels of Tr1 cells were reduced while Th2 cells were increased (Veldman et al. 2004). Therefore, it is possible that an imbalance of autoreactive Tr1 and Th 2 cells plays a role in the induction of PV by promoting the proliferation of anti-Dsg 3 producing B cells.\nThe mechanisms underlying pemphigus skin blistering\nAs a first concept it was proposed that proteolytic cleavage of molecules responsible for intercellular adhesion was the mechanism underlying pemphigus skin blistering. Later on, with the identification of desmosomal cadherins as the target antigens of pemphigus autoantibodies and with more sophisticated cell biologic tools at hand, the ideas of direct antibody-mediated inhibition and of indirect signalling-mediated reduction of desmoglein binding were developed (Fig.\u00a08).\nFig.\u00a08The two principal mechanisms underlying pemphigus skin blistering. Two principal mechanisms have been proposed by which autoantibodies specific for Dsg 1 and Dsg 3 could impair desmosomal adhesion. First, antibodies could directly interfere with desmoglein transinteraction (a). Second, antibody binding has been shown to trigger intracellular signalling pathways, which indirectly results in loss of desmoglein-mediated binding (b)\nProteolytic cleavage of desmosomal cadherins\nProteolytic cleavage of cell adhesion molecules has first been suggested to be involved in pemphigus acantholysis because protease inhibitors blocked pemphigus IgG-induced cell detachment in culture (Farb et al. 1978). Moreover, plasminogen activator activity and expression of the urokinase-type plasminogen activator receptor (uPAR) system were found to be increased following treatment with PV- and PF-IgG\/serum in keratinocytes in vitro as well as in PV patients\u2019 skin (Feliciani et al. 2003; Hashimoto et al. 1983; Lo Muzio et al. 2002; Schaefer et al. 1996; Seishima et al. 1997; Yamamoto et al. 2007b), possibly via phospholipase C (PLC)-mediated signalling (Esaki et al. 1995). Anti-uPA antibodies and a PA inhibitor were sufficient to block acantholysis induced by PV- or PF-IgG in several studies (Feliciani et al. 2003; Hashimoto et al. 1983; Morioka et al. 1987) but not in that by Schuh et al. (2003). However, a definitive evaluation of the uPAR system for pemphigus acantholysis became possible since a study using uPA- and tissue PA-deficient mice showed extensive skin blistering in response to PV- and PF-IgG (Mahoney et al. 1999). Thus, the plasminogen activator system does not appear to be essential for pemphigus skin blistering but may aggravate the phenotype, especially when secondary inflammatory mediators such as IL-1\u03b1 and TNF-\u03b1 are released (Feliciani et al. 2000, 2003).\nThe same may hold true for other proteases such as matrix metalloproteinases (MMP) or proteases of the ADAM (a disintegrin and metalloproteinase) family. MMP-9, which was overexpressed but not activated following treatment with PV serum was reported to specifically cleave Dsg 3 during apoptosis (Cirillo et al. 2007a, d). ADAM17 on the other hand, was upregulated by activation of the epidermal growth factor receptor (EGFR) and caused shedding of Dsg 2 (Bech-Serra et al. 2006; Santiago-Josefat et al. 2007). These results may be important for PV because EGFR activation was observed following treatment with PV-IgG (Chernyavsky et al. 2007a; Frusic-Zlotkin et al. 2006). The presence of proteolytic enzymes in PV sera may also explain why IgG-depleted PV sera were found to be pathogenic in culture (Cirillo et al. 2007c). However, because no direct evidence was provided that MMP-9 or ADAM17 or any other proteinase cleaves members of the Dsg family in pemphigus, the significance of these findings for acantholysis in pemphigus is unclear and the specific proteolysis hypothesis proposed for pemphigus requires further experimental substantiation (Cirillo et al. 2008). Nevertheless, the fact that specific cleavage of Dsg 1 by staphylococcal exfoliative toxin in bullous impetigo is sufficient to cause a histologic phenotype comparable to PF (Amagai et al. 2000a; Hanakawa et al. 2002) indicates that, in principle, specific proteolysis could be an effective mechanism in pemphigus.\nDirect inhibition of desmoglein binding\nSince it was discovered that autoantibodies in pemphigus are directed to desmosomal adhesion molecules, it was  believed that these autoantibodies might directly interfere with desmoglein binding (Fig.\u00a08) (Amagai et al. 1991; Jones et al. 1986a; Koulu et al. 1984), a mechanism also refered to as \u201csteric hindrance\u201d(Sharma et al. 2007). This model is attractive because it has been shown that autoantibodies against Dsg 3 and Dsg 1 in PV and PF patients primarily target the aminoterminal part of the EC 1 domain (Futei et al. 2000; Hacker-Foegen et al. 2003; Ishii et al. 2008; Muller et al. 2008b; Sekiguchi et al. 2001). The EC 1 domain, according to morphologic studies on desmoglein transinteraction in desmosomes, is increasingly recognized as the part of the desmosomal cadherin ectodomain, responsible for trans-interaction (Al-Amoudi et al. 2007; He et al. 2003) and may harbour the putative transadhesive interface, based on data from the crystal structure of classical cadherins (Boggon et al. 2002; Overduin et al. 1995; Shapiro et al. 1995). Moreover, it seems that autoantibody reactivity to the aminoterminal parts (EC 1) of the Dsg 3 ectodomain correlates with high disease activity as well as epidermal or mucosal involvement in PV although the titers of these antibodies do not show this correlation (Amagai et al. 1992; Muller et al. 2006, 2008b; Salato et al. 2005).\nFirst functional data that anti-Dsg 3 antibodies in PV may directly interfere with Dsg 3 binding were provided using monoclonal antibodies derived from the active PV mouse model (Amagai et al. 2000b). AK 23, which was directed against the aminoterminal part of EC 1 was found to be pathogenic and capable to induce epidermal blistering in vivo, at least when PF-IgG or exfoliative toxin A was added to inactivate Dsg 1 (Shimizu et al. 2005; Tsunoda et al. 2003). Antibodies to other parts of the Dsg 3 extracellular domain such as AK 9 and AK 18 were ineffective to induce blisters. Recently, by using single-molecule atomic force microscopy (AFM), it was shown that PV-IgG as well as AK 23 directly interfered with homophilic Dsg 3 binding under cell free conditions (Heupel et al. 2007) which supports the hypothesis of direct inhibition of Dsg 3 binding in PV (Stanley and Amagai 2006). However, no direct inhibition of Dsg 1 binding by PV-IgG and PF-IgG was detected by AFM. These autoantibodies induced keratinocyte dissociation and reduced binding of both Dsg 3- and Dsg 1-coated microbeads to the surface of cultured keratinocytes, as revealed by laser trapping (Heupel et al. 2007; Waschke et al. 2005). These data suggest that autoantobodies interfere with Dsg 1 binding rather by indirect, cell-dependent mechanisms.\nFinally, it has to be noted that, if direct inhibition occurs, it is not possible to discriminate at present whether interference with Dsg 3 binding in PV was mediated by steric hindrance, i.e. by blocking trans-interaction of desmoglein molecules by the bound autoantibody, or rather by allosteric effects, i.e. autoantibody-induced conformational changes of the Dsg 3 ectodomain, which in turn interfere with Dsg 3 transinteraction. An antibody directed against the putative transadhesive interface may directly induce steric hindrance, whereas antibodies directed against other parts of the desmoglein ectodomain could indirectly inhibit desmoglein binding by allosteric mechanisms. The fact that AK 18 and AK 9, which were directed to the middle and the carboxyterminal parts of the Dsg 3 ectodomain, were not pathogenic and did not directly interfere with Dsg 3 binding suggests that these specific antibodies were not capable of causing allosteric hindrance (Heupel et al. 2007; Tsunoda et al. 2003). On the other hand, an antibody directed against the EC2 domain, although this part of the molecule may not be involved in transinteraction, might be large enough to cause steric hindrance of Dsg 3 transinteraction. Therefore, \u201cdirect inhibition\u201d, instead of \u201csteric hindrance\u201d of desmoglein binding should be used until discrimination between steric and allosteric effects is possible.\nDesmoglein compensation in pemphigus\nThe desmoglein compensation hypothesis was proposed to explain the different clinical phenotypes of PV and PF on the basis of their different autoantibody profiles (Amagai 2003; Payne et al. 2004; Sharma et al. 2007; Shirakata et al. 1998; Stanley and Amagai 2006; Udey and Stanley 1999). According to this concept, in the deep epidermis which contains both Dsg 1 and Dsg 3, Dsg 3 compensates for the functional loss of Dsg 1 induced by Dsg 1-specific autoantibodies, resulting in more superficial blistering in PF (Fig.\u00a09). In PV, when only Dsg 3 antibodies are present, no epidermal blistering would occur because Dsg 1 is considered to compensate for autoantibody-induced loss of Dsg 3 binding. However, acantholysis occurs in mucous membranes where Dsg 3 is assumed to be the predominantly expressed Dsg isoform, leading to the phenotype of mucosal-dominant PV. When autoantibodies to Dsg 1 are also produced in PV, epidermal blistering occurs. However, it is unclear why the cleavage plane is restricted to the deep epidermis in PV since in PF Dsg 1 autoantibodies cause superficial blistering (Fig.\u00a010). For this reason and other reasons such as the cases of pemphigus in which the autoantibody profiles do not correlate with the clinical phenotype or the presence of other desmosomal cadherin isoforms in the epidermis, this concept has been challenged (Amagai et al. 2006; Bystryn and Rudolph 2005; Muller et al. 2002; Spindler et al. 2007).\nFig.\u00a09The desmoglein compensation hypothesis. Based on the different autoantibody profiles in PV and PF together with the findings that Dsg 3 is present in the deep epidermis only whereas Dsg 1 is primarily expressed in the superficial epidermis, the desmoglein compensation hypothesis has been proposed to explain the epidermal cleavage planes typical for PV and PF. According to this model, blistering in PF affects the superficial epidermis because Dsg 3 is present in the deep epidermis to compensate for the autoantibody-induced loss of Dsg 1 binding. In PV, epidermal involvement would occur only when autoantibodies against both Dsg 1 and Dsg 3 are present because Dsg 1 is found in all epidermal layers and could compensate for loss of Dsg 3 binding when antibodies to Dsg 3 are solely presentFig.\u00a010Immunostaining of Dsg 1 and Dsg 3 in PV lesional epidermis. Epidermis from a patient with mucocutaneous PV was stained for Dsg 1 (a) and Dsg 3 (b). A merge of both panels is shown in c. Both Dsg 1 and Dsg 3 are expressed in the basal layer underneath the blister as well as in keratinocytes in the blister roof. However, Dsg 3 staining appears to be fragmented throughout the epidermis whereas Dsg 1 staining is more continuous. Note that in the level of the cleavage plane the apical membrane of basal cells shows strong immunostaining for Dsg 1 and Dsg 3 (arrows). Therefore, based on the desmoglein compensation hypothesis the expression patterns of Dsg 1 and Dsg 3 cannot explain why the cleavage plane is located suprabasally in PV but not in other epidermal layers. Scale bar is 20\u00a0\u03bcm\nExperimental support for the desmoglein compensation hypothesis in vivo was obtained in mice. It was shown that PF-IgG were sufficient to cause skin blistering in Dsg 3-deficient mice but not in normal mice (Mahoney et al. 1999). In skin layers where Dsg 1 and Dsg 3 were found, autoantibodies against both desmogleins were required for blistering. In line with these findings, forced expression of Dsg 3 in the superficial epidermis abolished the ability of PF-IgG to induce acantholysis in mice (Wu et al. 2000). In contrast, in human skin and in cultured human keratinocytes in vitro, PF-IgG were effective to induce acantholysis despite of the presence of both Dsg 1 and Dsg 3 (Spindler et al. 2007). The discrepancy between these conflicting findings may be explained in part by the notion that the desmoglein compensation hypothesis is based on the following two assumptions: (1) the expression pattern of Dsg 3 and Dsg 1 do not substantially overlap in epidermal and mucosal layers where the cleavage plane in PV and PF is located. (2) Dsg 1- and Dsg 3-specific autoantibodies only lead to inactivation of either Dsg 1 or Dsg 3, respectively. Because of the latter, the desmoglein compensation hypothesis has been used to promote the idea that autoantibodies reduce Dsg binding by direct inhibition rather than by unspecific proteolysis (Mahoney et al. 1999).\nRegarding the distribution of Dsg 1 and Dsg 3, it is important to note that Dsg 3 expression patterns in specific epidermal layers are different in mice and men. In mice, expression of Dsg 3 is restricted to the basal and immediately suprabasal epidermal layer (Mahoney et al. 2006, 1999). In human skin, when PV and PF were used for staining, a similar staining pattern was revealed (Amagai et al. 1996; Shimizu et al. 1995). In contrast, when specific antibodies or in situ hybridisation were used for Dsg 3 mapping in human epidermis, it was demonstrated that Dsg 3 is present throughout the spinous layers and thus Dsg 3 distribution showed substantial overlap with expression of Dsg 1 (Arnemann et al. 1993; Mahoney et al. 2006; Spindler et al. 2007). However, immunostaining of human epidermis using another monoclonal antibody detected expression of Dsg 3 in the lower epidermis only (Wu et al. 2000). In oral mucosa, equally strong expression of Dsg 1 and Dsg 3 was found throughout the epithelium when specific antibodies were used (Mahoney et al. 2006), whereas Dsg 1 staining intensity was found to be much lower when PV-IgG were used for immunstaining (Shirakata et al. 1998). Taken together, the expression patterns of Dsg 1 and Dsg 3 broadly overlap in human epidermis and appear to be identical in oral mucosa.\nWith respect to the second assumption the desmoglein compensation is based on, i.e. selective inactivation of Dsg 1 but not of Dsg 3 by Dsg 1-specific antibodies, it was shown recently that both PF-IgG (only containing Dsg 1-specific antibodies) and PV-IgG from patients with only Dsg 3-specific antibodies were equally effective to reduce binding of Dsg 1- and Dsg 3-coated beads to the surface of cultured keratinocytes (Heupel et al. 2007; Spindler et al. 2007). These data indicate that PV-IgG and PF-IgG can reduce binding of Dsg 1 and Dsg 3, at least on the keratinocyte cell surface. Taken together, the relevance of desmoglein compensation for pemphigus pathogenesis in humans cannot be concluded from experiments in mice, especially because distribution patterns of Dsg 1 and Dsg 3 substantially differ in the two species. Therefore, alternative models have to be worked out to explain the different epidermal cleavage planes in PV and PF. These may involve different signalling pathways required for maintenance of desmosomal adhesion in the specific epidermal layers as outlined below.\nSignalling pathways in pemphigus and in desmosome disassembly\nSince it has been shown that PV-IgG upon binding to keratinocytes induced a rapid transient increase of intracellular Ca2+ (Seishima et al. 1995), several signalling pathways have been shown to be involved in pemphigus pathogenesis (Fig.\u00a08) (Kitajima 2002; Lanza et al. 2006; Sharma et al. 2007; Sitaru and Zillikens 2005). Interestingly, evidence has been provided that transadhering non-desmosomal cadherins, for instance Dsg 3, are involved in \u201coutside-in\u201d signalling and that binding of pemphigus IgG interferes with this function (Muller et al. 2008a). This can be concluded from experiments which showed that autoantibody binding as well as keratinocyte separation started between desmosomes (Sato et al. 2000; Takahashi et al. 1985) and that non-junctional Dsg 3 and plakoglobin were depleted first before changes in the desmosomal fractions were present (Aoyama and Kitajima 1999; Williamson et al. 2006; Yamamoto et al. 2007a). Interestingly, to trigger Dsg-induced signalling, autoantibody-mediated cross-linking of Dsg 3 and Dsg 1 seems not to be required because monovalent Fab fragments and single-chain variants of PV- and PF-IgG were effective to cause skin blistering in vivo and to disrupt the desmosomal plaque in vitro (de Bruin et al. 2007; Ishii et al. 2008; Payne et al. 2005; Rock et al. 1990).\nCa2+, PLC and PKC\nIt was shown that PV-IgG caused a rapid, transient phospholipase C (PLC)-dependent increase of inositol 1,4,5 trisphosphate and of intracellular Ca2+ leading to activation of both PKC and plasminogen activator (PA) (Esaki et al. 1995; Kitajima et al. 1999; Memar et al. 1996; Osada et al. 1997; Seishima et al. 1995, 1999). Because a chelator of intracellular free Ca2+ blocked keratinocyte dissociation in vitro and inhibitors of calmodulin, PLC and PKC were effective to block PV-IgG-induced acantholysis in vivo (Arredondo et al. 2005; Sanchez-Carpintero et al. 2004), it is possible that this signalling pathway may be involved in PV acantholysis. However, because the PA system is not believed to be crucial in this process, PKC signalling may contribute to PV acantholysis by other pathways such as phosphorylation of \u03b2-catenin in adherens junctions (Chernyavsky et al. 2007b). This hypothesis is supported by experiments, which showed that keratinocyte adhesion was negatively regulated by pharmacologic PKC activation (Kimura et al. 2007).\np38MAPK\nActivation of p38MAPK at present is the most promising signalling mechanism to be responsible for acantholysis in pemphigus. It has been demonstrated in vivo that p38MAPK and one of its downstream targets, heat shock protein (HSP) 25, were phosphorylated in response to PV-IgG and PF-IgG and that pharmacologic inhibition of p38MAPK abolished blister formation (Berkowitz et al. 2006, 2007b). Similarly, p38MAPK and the human homolog HSP 27 were found to be phosphorylated in skin lesions of PV and PF patients (Berkowitz et al. 2007a). In cultured human keratinocytes, phosphorylation of p38MAPK and HSP27 occured after 30\u00a0min of exposure to PV-IgG (Berkowitz et al. 2005; Kawasaki et al. 2006). However, another study found that activity of p38MAPK was not increased before 120\u00a0min and that activity peaked after 240\u00a0min of PV-IgG treatment (Chernyavsky et al. 2007a). In the latter study it was shown that activation was mediated, at least in part, by Dsg 1- and\/or Dsg 3-specific antibodies because Dsg depletion by siRNA reduced p38MAPK activation by 50%. Inhibition of p38MAPK blocked autoantibody-induced keratinocyte dissociation, Rho A inactivation, cytokeratin retraction and reorganization of the actin cytoskeleton (Berkowitz et al. 2005; Chernyavsky et al. 2007a; Waschke et al. 2006). These results demonstrate that p38MAPK is involved in the mechanisms leading to acantholysis and that inhibition of p38MAPK could be a beneficial approach to treat pemphigus patients. Nevertheless, based on the finding that activation of p38MAPK can also be a consequence of cell detachment in rat intestinal epithelium (Rosen et al. 2002), it was proposed that p38MAPK activation is a consequence of cell dissociation (Sharma et al. 2007). However, this is unlikely because significant pemphigus-IgG-induced acantholysis usually takes 12\u201324\u00a0h to occur (Berkowitz et al. 2006; Caldelari et al. 2001; Lanza et al. 2008; Mahoney et al. 1999; Nagasaka et al. 2004; Nguyen et al. 2000c; Shu et al. 2007; Spindler et al. 2007; Takahashi et al. 1985; Tsunoda et al. 2003; Waschke et al. 2005; Waschke et al. 2006; Williamson et al. 2006).\nThe mechanisms by which p38MAPK and HSP27 lead to keratinocyte dissociation are largely unclear but may involve reorganization of the keratinocyte cytoskeleton. It has been shown recently that serine phosphorylation of cytokeratin 8 by p38MAPK induced cytokeratin network disassembly (Woll et al. 2007). Similarly, HSP27 was found to associate with cytokeratin filaments and to inhibit assembly of glial fibrillary acidic protein (GFAP) (Perng et al. 1999). Similarly, HSP25 was found to interact with actin filaments and to prevent formation of aggregates of thermally denatured actin (Panasenko et al. 2003). However, because p38MAPK-mediated phosphorylation of HSP27 or HSP 25 promoted actin assembly and stabilized F-actin against depolymerization in response to cytochalasin D or heat shock (Benndorf et al. 1994; Geum et al. 2002; Guay et al. 1997), one would expect that PV-IgG-induced HSP27 phosphorylation also would stabilize actin filaments. Therefore, it is more likely that HSP27 is part of a salvage pathway in response to PV-IgG and does not directly contribute to PV-IgG-induced actin reorganization.\nRho A GTPase\nRho GTPases are important regulators of the cytoskeleton and of cell adhesion (Braga and Yap 2005; Bustelo et al. 2007; Fukata et al. 1999; Jaffe and Hall 2005). In the epidermis, Rac 1 was found not to be crucial for maintaining epithelial integrity but for stem cell differentiation, presumably via negative regulation of c-Myc expression (Benitah et al. 2005; Chrostek et al. 2006). Recently, it has been shown that PV- and PF-IgG-induced epidermal splitting, keratinocyte dissociation, as well as loss of Dsg 1 and Dsg 3 binding in vitro were accompanied by p38MAPK-dependent inactivation of Rho A and that specific activation of Rho A by the bacterial toxin cytotoxic necrotizing factor y (CNFy) abolished these effects (Spindler et al. 2007; Waschke et al. 2006). Moreover, toxin-mediated inactivation of Rho A and Rac 1 resulted in epidermal splitting, keratinocyte dissociation and actin reorganization similar to treatment with pemphigus IgG. Especially, inactivation of Rho GTPases by toxin B caused deep epidermal acantholysis comparable to the effects of PV-IgG. Based on this observation, it is tempting to speculate that the cleavage plane in pemphigus may in part be explained by different, layer-specific signalling pathways important for keratinocyte cohesion.\nBecause cytokeratin retraction and actin reorganization in response to pemphigus IgG were also abrogated by activation of Rho A, these results suggested that Rho A could be involved in the regulation of desmoglein cytoskeletal anchorage (Waschke et al. 2006). This idea was supported by Triton extraction experiments which showed that Rho A activation reduced Dsg 3 in the non-cytoskeleton bound fraction. Alternatively, Rho GTPases may regulate endocytosis of desmosomal cadherins similar to their role in E-cadherin turnover (Akhtar and Hotchin 2001; Izumi et al. 2004; Kamei et al. 1999). Interestingly, endocytosis and depletion of Dsg3 are increasingly recognized to contribute to pemphigus acantholysis (Calkins et al. 2006; Yamamoto et al. 2007a). Taken together, Rho GTPases seem to be important for maintenance of desmosomes and activation of Rho A could be used to develop new strategies in pemphigus treatment.\nThese data are in contrast to previous findings, which suggested that Rho GTPases are not involved in the regulation of desmosomal adhesion. This was concluded from experiments in keratinocytes which showed that Rac 1 inactivation by transfection with a dominant-inactive mutant as well as inactivation of Rho A by C3 toxin for 25 min was sufficient to displace E-cadherin but not desmoplakin from cell junctions (Braga et al. 1997). These studies led to the hypothesis that Rho GTPases are important regulators of keratinocyte adherens junctions but not desmosomes (Braga and Yap 2005). However, because inactivation of Rho A by a cell-permeable C3 fusion toxin was sufficient to displace Dsg 3 from cell borders after 180\u00a0min, the negative results from the early study might be caused by the shorter incubation period (Waschke et al. 2006).\nPlakoglobin\nBesides its involvement in PV pathogenesis, plakoglobin is important for several aspects of desmosomal adhesion. Plakoglobin deficiency resulted in subcorneal acantholysis, loss of desmosomes, and impaired cytoskeletal anchorage of desmoplakin and desmogleins in vivo and in vitro, supporting the notion that plakoglobin is an important cytoskeletal linker and is crucial for desmosome assembly (Bierkamp et al. 1999; Yin et al. 2005). However, at least in part, plakoglobin functions seem to be compensated by other desmosomal plaque proteins such as plakophilin 1 because a plakoglobin-deficient keratinocyte cell line displayed Dsg 3 cytoskeletal anchorage and keratinocyte aggregation similar to wild-type cells (Caldelari et al. 2001). The linker function of plakoglobin seems to be regulated by tyrosine phosphorylation of plakoglobin because following EGFR activation, phosphorylated plakoglobin remained associated with Dsg 2 but not with desmoplakin (Gaudry et al. 2001). Moreover, plakoglobin phosphorylation was shown to be required for EGFR-induced loss of cell adhesion and to regulate binding of plakoglobin to desmoplakin (Miravet et al. 2003; Yin et al. 2005). These data indicate that plakoglobin is required to integrate the effects from extracellular cues and from different signalling pathways in order to allow coordinated modulation of desmosomal adhesion. In addition, plakoglobin regulates the  turn over of desmosomal components because the protein levels of desmogleins as well as of plakoglobin and desmoplakin were decreased in plakoglobin-deficient cells (Yin et al. 2005).\nWith respect to pemphigus, it has been shown that plakoglobin is critical for PV pathogenesis because keratinocytes from plakoglobin-deficient mice were resistant to PV-IgG-induced keratinocyte dissociation, cytokeratin retraction and disruption of the desmosomal plaque (Caldelari et al. 2001; de Bruin et al. 2007). These studies provided first evidence that direct inhibition of Dsg binding alone cannot account for acantholysis in PV and raised the hypothesis that plakoglobin could be part of a receptor complex required to transfer the signal from autoantibody-bound Dsg 3 into the cell, a phenomenon referred to as \u201coutside-in\u201d signalling (Muller et al. 2008a). As outlined above, it has been shown that plakoglobin is involved in c-Myc repression, which is required for keratinocyte differentiation and that PV-IgG by depleting noncytokeratin-anchored plakoglobin on the cell surface also led to c-Myc overexpression (Williamson et al. 2006). However, as discussed below, the role of c-Myc signalling in acantholysis is unclear at present.\nEGFR\nThe involvement of the EGFR in PV pathogenesis has been reported by showing that EGFR activity increased after 30\u00a0min and peaked after 60\u00a0min of PV-IgG incubation. This was followed by activation of ERK1\/2, c-Jun phosphorylation and finally by keratinocyte apoptosis (Chernyavsky et al. 2007a; Frusic-Zlotkin et al. 2006). Inhibition of EGFR reduced EGFR signalling as well as apoptosis and acantholysis (Frusic-Zlotkin et al. 2006). This finding was surprising because EGFR signalling usually is considered to promote cell survival and proliferation (Bazley and Gullick 2005; Muller et al. 2008a). However, because 60\u00a0h were required to induce acantholysis, very high concentrations of IgG (5\u00a0mg\/ml) were used and activation of p38MAPK was not detected in this study, the significance of these results is unclear.\nEGFR could contribute to PV pathogenesis by stimulating desmosome disassembly. It has been shown that EGF-induced loss of keratinocyte aggregation was mediated by plakoglobin phosphorylation, which led to uncoupling of the desmoglein 2-plakoglobin complex from desmoplakin and thus from the cytoskeleton (Gaudry et al. 2001; Yin et al. 2005). Moreover, EGFR seems to negatively regulate the protein levels of desmosomal cadherins, in part by promoting their metalloproteinase-mediated degradation, as well as the incorporation of desmosomal cadherins into the desmosomal plaque (Bech-Serra et al. 2006; Lorch et al. 2004; Santiago-Josefat et al. 2007).\nSrc\nThe tyrosine kinase Src is activated by PV-IgG within 30\u00a0min and seems to contribute to EGFR and p38MAPK activation because inhibition of Src reduced EGFR and p38MAPK phosphorylation by 45 and 30%, respectively (Chernyavsky et al. 2007a). Because Src inhibition reduced PV-IgG-induced loss of keratinocyte cohesion, cytokeratin retraction and apoptosis in vitro and inhition of tyrosin kinases blocked PV-IgG-induced acantholysis in vivo (Chernyavsky et al. 2007a; Sanchez-Carpintero et al. 2004), these results indicate that Src is significantly involved in PV acantholysis. Besides activation of p38MAPK and EGFR, a recent study provided another possible underlying mechanism: Src was shown to directly phosphorylate p120catenin in response to PV-IgG, which correlated with the degree of acantholysis (Chernyavsky et al. 2007b). Because p120catenin is involved in stabilization of classical cadherins such as E-cadherin on the cell surface as well as in cadherin-dependent regulation of Rho A and Rac 1 activity (Alema and Salvatore 2007; Kowalczyk and Reynolds 2004), Src-mediated phosphorylation of p120catenin may be important for PV pathogenesis.\nCholinergic receptors\nThe cholinergic system of the human epidermis involves two classes of cholinergig receptors, the nicotinic and the muscarinic acetylcholine receptors (nAChR and mAChR), which in keratinocytes are involved in the regulation of cell\u2013cell and cell\u2013matrix adhesion as well as in cell migration (Grando 2006a). As outlined above, it is unclear whether autoantibodies against cholinergic receptors in pemphigus directly contribute to acantholysis. However, it was shown that cholinergic agonists can ameliorate PV acantholysis in vivo and in vitro (Nguyen et al. 2004b) and that local application of cholinergic agonists such as pilocarpine had therapeutic effect on oral and skin lesions in PV patients (Iraji and Yoosefi 2006; Namazi 2004). Cholinergic agonists increased protein levels of Dsg 1, Dsg 3 and E-cadherin and antagonists to cholinergic receptors resulted in keratinocyte dissociation, which was paralleled by phosphorylation of these molecules (Grando 2006a). Recently, a mechanism by which signalling from cholinergic receptors could directly interfere with PV-IgG-induced effects was demonstrated. The M1 mAChR agonist pilocarpine inhibited PV-IgG-induced acantholysis by reducing serine phosphorylation of \u03b2-catenin and Src-mediated tyrosine phosphorylation of p120catenin by activation of the specific protein phosphatases (Chernyavsky et al. 2007b). Similarly, an agonist of the \u03b1 7 nAChR reduced p120catenin phosphorylation both on the level of Src activation as well as by activation of the tyrosine phosphatase. Moreover, it was shown that M3 and M4 receptors can lead to activation of Rho A and that carbachol, which activates \u03b1 3 and \u03b1 7 nAChR, activates Rho A, Rac 1 and Cdc42 (Chernyavsky et al. 2004a, b; Ruiz-Velasco et al. 2002), which also might be involved in the inhibition of PV-IgG-induced acantholysis. Taken together, these data indicate that activation of cholinergic receptors could provide a promising strategy to treat PV patients.\nRegulation of cell cycle and gene expression\nRecent data support the hypothesis that PV-IgG change expression patterns of molecules involved in cell cycle regulation and that these molecules might be involved in acantholysis. As outlined above, PV-IgG-induced plakoglobin depletion resulted in c-Myc overexpression in cultured keratinocytes (Muller et al. 2008a; Williamson et al. 2006). Accordingly, c-Myc overexpression was found in the epidermis of PV patients, but interestingly not in PF skin, indicating that this mechanism may only be important for PV (Williamson et al. 2007, 2006). Because pharmacologic inhibition of c-Myc as well as of plakoglobin degradation by blocking GSK 3 abrogated pemphigus-IgG-induced skin blistering, these results indicate that this mechanism could also be important to induce acantholysis. However, the findings that c-Myc overexpression was found after 24\u00a0h, whereas PV-IgG-induced loss of cell aggregation was observed as early as after 12\u00a0h and that c-Myc overexpression was absent in PF skin indicate that c-Myc overexpression likely contributes to ongoing proliferation of keratinocytes in PV, whereas it seems not to be essential for acantholysis. Therefore, other mechanisms must exist in addition to induce keratinocyte dissociation. These mechanisms may, amongst others, involve expression of genes different from c-Myc, which is suggested by the fact that plakoglobin deficiency resulted in protection of keratinocytes from apoptosis, possibly via overexpression of the anti-apoptotic molecule Bcl-XL (Dusek et al. 2007a).\nSimilar to c-Myc, cyclin-dependent kinase 2 (cdk2), another kinase involved in the regulation of cell cycle progression, seems to be involved in keratinocyte proliferation and acantholysis in pemphigus (Lanza et al. 2008). PV serum increased protein levels of cdk2 and PV serum-induced acantholysis in vivo was abolished by pharmacologic inhibition of cdk2. Moreover, siRNA-mediated down-regulation of cdk2 blocked cell dissociation in cultured keratinocytes indicating that cdk2 may be important for PV acantholysis in vivo and in vitro. However, at present, it is unclear how continuing keratinocyte proliferation in consequence to increased c-Myc and cdk2 signalling contributes to acantholysis in PV.\nOne explanation is that reduced expression of desmosomal components in hyperproliferating cells may foster the loss of keratinocyte cohesion in pemphigus. PV-IgG do not only induce direct signalling effects on cell junctions and the cytoskeleton but also change gene expression patterns, which may also contribute to pemphigus pathogenesis. It has been shown by DNA microarray that PV-IgG within 8\u00a0h down-regulated transcription of 198 genes including genes encoding for adhesion molecules including Dsg 3 and desmoplakin, cytoskeletal proteins such as different cytokeratins as well as molecules involved in cell cycle regulation such as p53 and cyclin D2 in cultured keratinocytes and that expression of some of these genes was antagonistically regulated by methylprednisolone (Nguyen et al. 2004a). When PV serum was used for 24\u00a0h, the expression of even more genes was altered. In cultured human keratinocytes, transcription of 231 genes was decreased including Dsc 2 and plakophilin 3 whereas transcription of 329 genes was increased. In vivo, expression of 1114 genes was reduced whereas transcription of 349 other genes was inceased (Lanza et al. 2008). Pharmacologic inhibition of cdk2 blunted the effect of PV-IgG on the expression of most of the genes including desmoplakin and also blocked PV-IgG-induced acantholysis. Thus, it is conceivable that altered desmosome formation may be involved in the mechanisms underlying keratinocyte dissociation in pemphigus.\nApoptosis\nApoptosis has been detected in skin lesions and in perilesional skin of PV and PF patients (Gniadecki et al. 1998; Puviani et al. 2003; Wang et al. 2004a) and hallmarks of apoptosis such as DNA fragmentation, increased expression of pro-apoptotic molecules Fas, FasL, Bax, p53, depetion of anti-apoptotic Bcl-2 and FLIPL as well as activation of caspases 1, 3 and 8 have been observed following treatment of cultured keratinocytes with PV-IgG or PV serum (Arredondo et al. 2005; Baroni et al. 2004; Chernyavsky et al. 2007a; Frusic-Zlotkin et al. 2005, 2006; Pelacho et al. 2004; Puviani et al. 2003; Wang et al. 2004a, b). Hence, compelling evidence has been provided for the presence of programmed cell death in PV although the phenotype of acantholytic cells is different from cells undergoing apoptosis (Arredondo et al. 2005). However, for several experiments prolonged incubation times of 48\u201372\u00a0h (Arredondo et al. 2005; Baroni et al. 2004; Frusic-Zlotkin et al. 2005, 2006; Wang et al. 2004a, b) were required whereas in most studies from the literature, acantholysis was clearly present after 18\u201324\u00a0h (Berkowitz et al. 2006; Caldelari et al. 2001; Lanza et al. 2008; Mahoney et al. 1999; Nagasaka et al. 2004; Nguyen et al. 2000c; Shu et al. 2007; Spindler et al. 2007; Takahashi et al. 1985; Tsunoda et al. 2003; Waschke et al. 2005; Waschke et al. 2006; Williamson et al. 2006). In another study, apoptosis was detected by TUNEL reactivity starting after 6\u00a0h of PV-IgG treatment (Chernyavsky et al. 2007a). However, in this study, loss of intercellular adhesion was present after 120\u00a0min and thus before onset of apoptosis. Therefore, it is conceivable that apoptosis may be an event parallel to or in consequence of acantholysis. On the other hand, it was reported that activated caspase 3 cleaves Dsg 3 (Weiske et al. 2001) and that caspase and calpain inhibitors can block PV-IgG-induced acantholysis in keratinocyte monolayers and in skin organ culture (Arredondo et al. 2005; Wang et al. 2004a, b; Weiske et al. 2001). Taken together, it is unclear at present to which extent PV-IgG-induced acantholysis is caused by apoptosis.\nTargets of signalling pathways in pemphigus\nPemphigus is a desmosomal disease because pathogenic autoantibodies are directed against Dsg 1 and Dsg 3. Moreover, these antibodies result in the depletion of desmosomal components from the cell surface, alterations of desmosomal plaques and a loss of desmosomes (Aoyama and Kitajima 1999; Aoyama et al. 1999; Calkins et al. 2006; de Bruin et al. 2007; Sato et al. 2000; Shu et al. 2007; Waschke et al. 2006; Williamson et al. 2006; Yamamoto et al. 2007a). However, it is unclear at present whether desmosomes are the primary targets of autoantibodies or whether components outside of desmosomes cause disassembly of desmosomes via mechanisms involving adherens junctions or the cytoskeleton, which finally results in acantholysis. The latter hypothesis is supported by the observation that intercellular spaces between desmosomes widen before desmosomes separate (Takahashi et al. 1985).\nEffects on desmosomes and adherens junctions\nIt is well established that pemphigus autoantibodies lead to depletion of desmosomal components from desmosomes as well as from the cell surface. Within 30\u201360\u00a0min, PV-IgG induced internalization and lysosomal degradation of cytoskeleton-unbound Dsg 3 together with plakoglobin (Aoyama and Kitajima 1999; Aoyama et al. 1999; Calkins et al. 2006; Sato et al. 2000; Williamson et al. 2006; Yamamoto et al. 2007a). After 24\u00a0h, Dsg 3 but not Dsg 2, plakoglobin or desmoplakin was also depleted from the cytoskeletal fractions leading to reduced total cellular Dsg 3 levels (Calkins et al. 2006; Yamamoto et al. 2007a). This effect was mediated by Dsg 3-specific antibodies because monoclonal Dsg 3 antibodies such as AK23 were effective to induce Dsg 3 depletion and this effect correlated with the pathogenic activity of these autoantibodies in vivo and in vitro (Shu et al. 2007; Yamamoto et al. 2007a). However, no strong correlation between Dsg 3 depletion and keratinocyte dissociation was observed. Dsg 3 depletion was observed in DJM-1 cells at 40% confluence as well as in normal human keratinocytes. In contrast, in confluent HaCaT monolayers, a reduction of Dsg 3 half-life but no depletion of total cellular Dsg 3 was detected (Cirillo et al. 2006; Waschke et al. 2006). Because depletion of Dsg 3 was first found in parallel with serine phosphorylation of Dsg 3, it was suggested that phosphorylation might be required for Dsg 3 degradation (Aoyama and Kitajima 1999; Aoyama et al. 1999; Bystryn and Rodriguez 1978; Rodriguez and Bystryn 1977). However, phosphorylation of Dsg 3 was observed in some studies but not in others, which might be due to different cell culture models indicating that Dsg phosphorylation is not necessary for acantholysis (Chernyavsky et al. 2007b; Nguyen et al. 2004a).\nSome evidence exists that depletion of Dsg 1 may also occur in pemphigus. However, studies to systematically investigate Dsg 1 turnover in pemphigus are lacking. Dsg 1 internalization and decreased immunostaining of Dsg 1 have been observed following treatment with PF-IgG\/-serum and PV serum (Cirillo et al. 2007b; Waschke et al. 2005). This is in line with previous findings from the literature, which demonstrated that PF-IgG did not bind to keratinocytes in the lower epidermis of PF patients but strongly labelled basal keratinocytes in the epidermis of healthy subjects suggesting that in PF the lower epidermis was depleted from Dsg 1 (Bystryn and Rodriguez 1978; Rodriguez and Bystryn 1977). Taken together, it is unclear at present to which extent Dsg depletion from desmosomes causes  antibody-mediated acantholysis. However, even if depletion of desmosomal components occured secondary to keratinocyte dissociation, it likely would further aggravate the loss of keratinocyte cohesion.\nThe involvement of adherens junctions in pemphigus pathogenesis is unclear at present. It has been shown that formation of adherens junctions is a prerequisite for desmosome assembly and \u03b1-catenin-deficient keratinocytes had reduced numbers of desmosmes (Vasioukhin et al. 2000). Because Rho GTPases are known to primarily regulate adhesion mediated by classical cadherins in adherens junctions whereas the requirement of Rho A and Rac 1 activity for maintenance of desmosomal adhesion was reported just recently (Braga and Yap 2005; Fukata et al. 1999; Spindler et al. 2007; Waschke et al. 2006), it was suggested that pemphigus IgG-induced inactivation of Rho A causes disassembly of desmosomes via destabilization of adherens junctions (Sharma et al. 2007). Although alterations of adherens junctions in response to PV-IgG were negligible compared to the effects on desmosomes (Calkins et al. 2006; de Bruin et al. 2007; Muller et al. 2008a; Waschke et al. 2006), this scenario cannot be completely ruled out at present. In fact it was reported recently that PV-IgG lead to phosphorylation of \u03b2-catenin and p120catenin by PKC and Src, respectively (Chernyavsky et al. 2007b). However, more studies are required to address the question how catenin phosphorylation in adherens junctions is associated with keratinocyte dissociation in pemphigus.\nEffects on the cytoskeleton\nAlterations of the cytoskeleton have been described both in pemphigus skin lesions as well as in keratinocytes exposed to pemphigus autoantibodies,. These effects include dysorganization of cytokeratin filaments, the actin cytoskeleton as well as of microtubules.\nFirst, it has been described that cytokeratin filaments become detached from cell junctions and accumulate in the perinuclear region, a phenomenon commonly referred to as \u201ckeratin retraction\u201d (Berkowitz et al. 2005; Caldelari et al. 2001; Calkins et al. 2006; Chernyavsky et al. 2007a; Jinbu et al. 1992; Kitajima et al. 1987; Waschke et al. 2006; Wilgram et al. 1961, 1964; Williamson et al. 2006). It was shown that cytokeratin retraction could be detected as early as 4\u20136\u00a0h after addition of PV-IgG, i.e. when first alterations of desmosomes became visible, and was fully established after 24\u00a0h (Berkowitz et al. 2005; Calkins et al. 2006; Chernyavsky et al. 2007a). However, because no cytokeratin filament reorganization was observed before cell detachment and the desmosomes of acantholytic cells remained anchored to cytokeratin filament bundles (Kitajima et al. 1986; Shimizu et al. 2004; Spindler et al. 2007), it appears that cytokeratin retraction is not required for acantholysis but may be a secondary event.\nRecent studies demonstrated that acantholysis in pemphigus was paralleled by alterations of actin filaments including increased stress fiber formation as well as fragmentation of actin filament bundles. Moreover, pharmacological inhibition of pemphigus IgG-induced acantholysis also was effective to reverse the effects on the cytoskeleton (Berkowitz et al. 2005; Spindler et al. 2007; Waschke et al. 2005; Waschke et al. 2006). Similarly, microtubule distribution was found to be altered by pemphigus IgG (Kitajima et al. 1986). Taken together, pemphigus IgG cause profound cytoskeletal reorganization but it is unclear at present whether these effects contribute to acantholysis or are triggered consequent to keratinocyte dissociation. Nevertheless, it is likely that alterations in the cytoskeleton may account for the changes in cell shape during keratinocyte dissociation, which seem to start between existing desmosomes (Bystryn and Grando 2006; Takahashi et al. 1985).\nThe role of the different mechanisms in blister formation\nAs outlined above, both direct and indirect mechanisms finally leading to a loss of desmoglein-mediated adhesion have been found to be involved in pemphigus acantholysis. At present, compelling evidence indicates that acantholysis in PV and in PF is initiated by cellular signalling pathways rather than by direct inhibition of Dsg binding (Fig.\u00a011). This was first shown using plakoglobin-deficient keratinocytes, which were resistant against PV-IgG-induced acantholysis and was further supported by the findings that inhibition of p38MAPK and activation of Rho A is sufficient to abolish PV-IgG and PF-IgG-induced skin blistering (Berkowitz et al. 2005, 2006, 2007b; Caldelari et al. 2001; Waschke et al. 2006). Moreover, low temperature-abrogated keratinocyte dissociation but not antibody binding to the keratinocyte cell surface indicating that direct inhibition of cadherin transinteraction by autoantibody binding is not sufficient to cause acantholysis (Calkins et al. 2006). If extracellular autoantibody-mediated direct inhibition of Dsg binding would be the primary cause of acantholysis, it is hard to imagine how this process should be blocked by modified intracellular signalling or low temperature. It is possible that antibody-mediated direct inhibition of Dsg binding may contribute to trigger cellular signalling events (Muller et al. 2008a; Sharma et al. 2007; Tsunoda et al. 2003). However, because PF-IgG were shown to induce cellular signalling events but not to directly reduce Dsg 1 binding, it seems that direct inhibition of Dsg transinteraction is not essential to alter Dsg-mediated signalling (Heupel et al. 2007; Waschke et al. 2005).\nFig.\u00a011The mechanisms involved in pemphigus acantholysis. Accumulating evidence indicates that PV-IgG and PF-IgG initiate keratinocyte dissociation via intracellular signalling pathways including p38 MAPK, Rho A and plakoglobin (PV only). In addition, other mechanisms such as direct inhibition of Dsg 3 binding and Dsg 3 depletion from desmosomes as well as other signalling events seem to contribute to PV pathogenesis, whereas their role for acantholysis in PF is unclear. These mechanisms may account for the more severe clinical phenotype of PV compared to PF. PLC phospholipase C, PKC protein kinase C, cdk 2 cyclin-dependent kinase 2, EGFR epidermal growth factor receptor\nIt is tempting to speculate that the clinical phenotype of PV may be more severe compared to PF because additional mechanisms could be involved in PV (Fig.\u00a011). Indeed, some mechanisms have only been found to contribute to acantholysis in PV but not in PF. For instance, direct inhibition of Dsg 3 transinteraction was found in PV but not in PF under conditions where antibodies caused acantholysis (Heupel et al. 2007). Therefore, it is possible that direct inhibition as a second pathway to reduce Dsg 3 binding in PV may explain the more severe phenotype of PV. Similarly, depletion of Dsg 3 has been convincingly shown in PV whereas depletion of Dsg 1 in both PV and PF is less clear (Yamamoto et al. 2007a). Moreover, altered plakoglobin signalling leading to c-Myc overexpression was found in PV patients\u2019s skin but not in PF (Williamson et al. 2007, 2006). Other pathways have been investigated for PV only whereas data for PF are lacking. As outlined above, inhibitors of calmodulin, PKC, EGFR, cdk2 as well as of tyrosin kinases have been shown to inhibit PV-IgG-induced acantholysis.\nFinally, it has to be emphasized that desmoglein proteolysis, mechanical stress as well as secondary changes for example in the extracellular Ca2+ concentration might contribute to skin blistering. For instance, acantholysis may foster skin blistering by derangement of the epidermal Ca2+ gradient, which in turn would result in loss of desmosomal cadherin binding, especially when the tight junctions of the granular layer are affected.\nConcluding remarks\nThe major goal for the future is to elucidate the primary signalling pathways responsible for the diverse effects of pemphigus IgG such as inhibition of desmoglein binding, depletion of desmosomal components, loss of desmosomes, reorganization of the cytoskeleton and finally the induction of acantholysis. Other pathways will be identified to represent salvage mechanisms to rescue keratinocyte cohesion (Grando 2006b). Moreover, future experiments may reveal how the different signalling pathways are involved in the regulation of desmosomal adhesion in the specific epidermal layers. This knowledge may elucidate why the cleavage planes in PV and PF usually are different. The second goal is to characterize whether acantholysis in pemphigus is caused by mechanisms directly targeting desmosomes or whether cellular signalling pathways regulate desmosomal adhesion via reorganisation of the cytoskeleton or via other types of cell contacts such as adherens junctions.","keyphrases":["desmosomes","pemphigus","autoantibodies","desmogleins","steric hindrance","desmoglein compensation"],"prmu":["P","P","P","P","P","P"]}
{"id":"Mol_Immunol-1-5-2075531","title":"The different effector function capabilities of the seven equine IgG subclasses have implications for vaccine strategies\n","text":"Recombinant versions of the seven equine IgG subclasses were expressed in CHO cells. All assembled into intact immunoglobulins stabilised by disulphide bridges, although, reminiscent of human IgG4, a small proportion of equine IgG4 and IgG7 were held together by non-covalent bonds alone. All seven IgGs were N-glycosylated. In addition IgG3 appeared to be O-glycosylated and could bind the lectin jacalin. Staphylococcal protein A displayed weak binding for the equine IgGs in the order: IgG1 > IgG3 > IgG4 > IgG7 > IgG2 = IgG5 > IgG6. Streptococcal protein G bound strongly to IgG1, IgG4 and IgG7, moderately to IgG3, weakly to IgG2 and IgG6, and not at all to IgG5. Analysis of antibody effector functions revealed that IgG1, IgG3, IgG4, IgG5 and IgG7, but not IgG2 and IgG6, were able to elicit a strong respiratory burst from equine peripheral blood leukocytes, predicting that the former five IgG subclasses are able to interact with Fc receptors on effector cells. IgG1, IgG3, IgG4 and IgG7, but not IgG2, IgG5 and IgG6, were able to bind complement C1q and activate complement via the classical pathway. The differential effector function capabilities of the subclasses suggest that, for maximum efficacy, equine vaccine strategies should seek to elicit antibody responses of the IgG1, IgG3, IgG4, and IgG7 subclasses.\n1\nIntroduction\nOnly recently has the full complement of horse Ig heavy (H) chain constant region genes (one \u03b4, one \u03bc, seven \u03b3, one \u03b1 and one \u025b) been described (Wagner et al., 2004). The horse has the highest number of IgG constant region (\u03b3 or IGHG) genes of any mammalian species examined to date and all the seven IgG subclasses they encode appear to be expressed in vivo (Wagner et al., 2004). Early studies described five equine IgG subclasses named IgGa, IgGb, IgGc, IgG(T), and IgG(B) or \u2018aggregating immunoglobulin\u2019. Following identification of the seven horse heavy chain constant region genes, the IgG subclasses have been reassigned as IgG1 to IgG7 (Wagner, 2006). Of the originally described IgG subclasses, IgGa corresponds to IgG1, IgGb to IgG4 and IgG7, IgGc to IgG6, and IgG(T) to both IgG3 and IgG5 (Wagner et al., 2002, 2004).\nIgG is the predominant antibody class in equine serum and colostrum (Sheoran et al., 2000) and is present at the mucosal surfaces, where it is the most abundant isotype in the equine urinary tract, lower respiratory tract and lung (Butler, 1998). In equine serum, IgGb (IgG4 and IgG7) is the most prevalent isotype followed by IgG(T) (IgG3 and IgG5), IgGa (IgG1) and IgGc (IgG6). In colostrum, IgGb is predominant, followed by IgGa and IgG(T), while IgGc is barely detectable. In nasal wash samples from adult horses only the IgGa and IgGb subclasses have been detected (Sheoran et al., 2000). Systemic and\/or mucosal IgG antibody responses are known to play an important role in protection against several equine pathogens including equine influenza virus (Nelson et al., 1998; Breathnach et al., 2006) and Streptococcus equi (Galan and Timoney, 1985; Galan et al., 1986; Sheoran et al., 1997), and may limit the severity and spread of equine herpes virus (EHV)-1 (Kydd et al., 2006).\nAlthough a role for equine IgG antibodies in protection against disease has long been recognised, the structures and functions of the individual IgG subclasses are not well characterised. Identification and cloning of the full complement of IgG H chain genes has provided a fresh resource for the study of equine IgG proteins. Here, we describe the first expression of recombinant versions of all seven equine IgG subclasses and present an analysis of their individual physical and biological properties.\n2\nMaterials and methods\n2.1\nConstruction of equine \u03b3 H chain expression vectors\nThe mouse VH gene specific for NIP was subcloned as a HindIII-BamHI fragment from a previously described human IgA1 expression vector (Morton et al., 1993) into pcDNA3.1\/Hygro (+) (Invitrogen, Paisley, UK) to produce pcDNA3.1Vnip. Isolation of genomic DNA for equine IGHG3, IGHG4 and IGHG7, and cDNA for equine IGHG1, IGHG2, IGHG5 and IGHG6 has been previously described (Wagner et al., 2002, 2004). For construction of the equine IgG3 H chain vector, a 2.5\u00a0kb BamHI fragment containing the IGHG3 gene along with 630\u00a0bp of 5\u2032 UTR and 577\u00a0bp of 3\u2032 UTR was placed downstream of the mouse VH gene in pcDNA3.1Vnip. To construct mammalian expression vectors for the remaining six horse IgGs, each \u03b3 constant region was amplified by PCR and placed downstream of the 630\u00a0bp horse IGHG3 5\u2032 UTR (also amplified by PCR) within the cloning vector pBluescript II (Stratagene, Amsterdam, The Netherlands). For each subclass, the 5\u2032 UTR and \u03b3 constant region cassette was then subcloned into pcDNA3.1VNip downstream of the mouse VH gene.\n2.2\nExpression of reqIgGs in CHO-K1 cells\nCHO-K1 cells, which had previously been stably transfected with the mouse \u03bb L chain specific for NIP, were transfected with one of the \u03b31\u2013\u03b37 H chain expression vectors, as previously described (Morton et al., 1993). Supernatant from individual resistant CHO clones was screened for Ig production by antigen-capture ELISA as previously described (Morton et al., 1993) except that the detection antibodies used were either rabbit anti-mouse \u03bb L chain\u2013HRP conjugate (0.25\u00a0\u03bcg\/ml in PBS-0.5% Tween [PBS-T]) or goat anti-horse IgG Fc-HRP conjugate (0.4\u00a0\u03bcg\/ml in PBS-T) (both Rockland, Gilbertsville, PA, USA).\n2.3\nPurification and analysis of reqIgGs\nReqIgGs were purified using NIP-affinity chromatography as previously described (Morton et al., 1993), then subjected to gel filtration on a Superose6 column using an \u00c4KTA FPLC system (GE Healthcare, Little Chalfont, UK). Only fractions corresponding to intact antibody (H2L2) were pooled for further analysis. The antibodies were analysed by SDS-PAGE and Western blotting on 8.4% acrylamide gels under reducing and non-reducing conditions. Gels were stained with Coomassie brilliant blue and Western blots were probed with HRP-conjugated rabbit anti-mouse \u03bb L chain (0.2\u00a0\u03bcg\/ml) or anti-horse IgG antibodies. Horse serum IgG (Sigma\u2013Aldrich, Poole, UK) and recombinant human IgG1 (Pleass et al., 1999) were used as controls.\n2.4\nReactivity of commercially available anti-horse IgG antibodies with the reqIgGs\nThe following antibodies were tested for reactivity against the reqIgGs in ELISA: goat HRP-conjugated polyclonal antibodies specific for horse IgGa, IgGb, IgGc or IgG(T), (kindly provided by Serotec, Oxford, UK and Bethyl Laboratories, Montgomery, TX, USA), and mouse monoclonal antibodies (mAb) against horse IgGa (CVS48), IgGb (CVS39), IgGc (CVS53) and IgG(T) (CVS38) (kindly provided by Serotec). Secondary antibody used to detect binding of the mAb was a goat anti-mouse IgG1-HRP conjugate diluted 1\/10,000 (kindly provided by Serotec). In addition, HRP-conjugated goat polyclonal anti-horse IgG H\u00a0+\u00a0L chain (kindly provided by Bethyl Laboratories) and goat anti-horse IgG Fc (Rockland) were tested in ELISA. Antigen-capture ELISAs were performed as above using 250\u00a0ng\/ml of each purified reqIgG subclass. Detection antibodies were diluted 1\/10,000 (goat polyclonals) or 1\/100 (anti-IgGa mAb), 1\/200 (anti-IgGb mAb), 1\/10 (anti-IgGc mAb) and 1\/500 (anti-IgG(T) mAb). The goat polyclonal anti-horse IgGa, IgGb, IgGc and IgG(T) antibodies were also tested in immunoblotting. Purified reqIgGs (1\u00a0\u03bcg) were electrophoresed under reducing and non-reducing conditions and the detection antibodies were diluted as for ELISA.\n2.5\nAnalysis of reqIgG glycosylation\nReqIgGs (1\u00a0\u03bcg) were electrophoresed on 8.4% acrylamide gels under reducing conditions alone or following reduction and de-glycosylation. Removal of N-linked sugars was carried out using a GlycoPro enzymatic deglycosylation kit (Prozyme, Inc., Leandro, CA, USA) according to the manufacturer's instructions. Following transfer to nitrocellulose, membranes were probed with the appropriate HRP-conjugated goat anti-horse IgG subclass antibody or either Con A-biotin (diluted 1\/10,000) or jacalin-biotin (diluted 1\/5000) (both Vector Laboratories, Inc., Burlingame, CA, USA) followed by streptavidin-AP (Dako, Ely, UK). Recombinant human IgG1 and IgA1 (Pleass et al., 1999) were used as positive controls.\n2.6\nInteraction with staphylococcal protein A and streptococcal protein G\nInteraction of reqIgGs with protein A and protein G was analysed by ELISA. NIP-BSA coated wells were incubated with 100\u00a0\u03bcl (0.25\u00a0\u03bcg\/ml) of one of the purified reqIgGs or recombinant human IgG1 for comparison. Binding to protein A or protein G was detected by incubation with 100\u00a0\u03bcl of serial dilutions of either HRP-conjugated protein A (0\u201350\u00a0\u03bcg\/ml in PBS-T) or HRP-conjugated protein G (0\u20135\u00a0\u03bcg\/ml in PBS-T) (both Sigma, Poole, UK). Plates were developed with ABTS and absorbance read at 405\u00a0nm.\n2.7\nFunctional assays\nChemiluminescence assays were carried out as previously described (Pleass et al., 1999) using 10\u00a0\u03bcg\/ml of each reqIgG and equine PBLs, isolated from blood drawn under appropriate licence from the UK Home Office. Complement binding and activation was assessed by ELISA on NIP-BSA coated wells incubated with recombinant human IgG3 (Bruggemann et al., 1987) or each of the reqIgGs (0\u201310\u00a0\u03bcg\/ml in PBS-T, 100\u00a0\u03bcl\/well) for 1\u00a0h at room temperature. Following washing, plates were incubated with human serum diluted 1\/100 in veronal buffered saline containing 0.5\u00a0mM MgCl2, 2\u00a0mM CaCl2, 0.05% Tween-20, 0.1% gelatin and 0.5% BSA for 1\u00a0h at room temperature. After washing, plates were incubated with either a 1\/800 dilution of sheep anti-C1q-HRP (Serotec) or a 1\/500 dilution of biotin-conjugated anti-C5b-9 (Quidel, Santa Clara, CA, USA), followed by streptavidin-HRP (Dako) diluted 1\/1000 in PBS-T, 0.5% BSA for 1\u00a0h at room temperature. Plates were developed as above.\n3\nResults\n3.1\nAnalysis of purified antibody by SDS-PAGE and Western blotting\nTransfection of each of the equine \u03b31\u2013\u03b37 H chain expression vectors into CHO cells stably expressing a compatible mouse \u03bb light (L) chain allowed expression of all seven subclasses. Expressed Ig was purified by antigen affinity chromatography followed by FPLC. All seven IgGs eluted from FPLC as a single major peak representing monomer. Minor peaks eluting earlier were attributed to antibody that had aggregated during purification. For each subclass, fractions corresponding to the major peak were pooled and used for all subsequent experiments.\nSDS-PAGE analysis of FPLC purified reqIgGs confirmed that the reqIgs assembled into intact Igs stabilised by interchain disulphide bridges (Fig. 1A). Under reducing conditions, analysis of the seven IgGs revealed that the IgG2 H chain migrated the most quickly, followed closely by those of IgG4 and IgG7, then IgG6, IgG1, IgG5 and IgG3 (Fig. 1B). Under the more sensitive conditions of Western blotting using an anti-mouse \u03bb L chain antibody to detect the horse IgGs (Fig. 1C), some lower molecular weight species were noted for IgG4 and IgG7 under non-reducing conditions. These may represent species such as half molecules (HL) and L chain dimers (L2), suggesting that a small proportion of the IgG4 and IgG7 molecules lack inter-H and\/or H\u2013L disulphide bonds and are stabilized by non-covalent interactions alone. Hence, under the native conditions of FPLC only a single major peak of fully assembled IgG was seen.\n3.2\nReactivity of anti-horse IgG antibodies with the reqIgG subclasses\nCurrently available anti-horse IgG antibodies are categorised on the basis of reactivity against horse IgG subclasses defined under the previous nomenclature of IgGa, IgGb, IgGc, and IgG(T). We sought to analyse the reactivity of these antibodies with the seven reqIgG subclasses. The results, obtained by ELISA, are summarised in Table 1. The polyclonal antibody directed against horse IgGa was specific for a single subclass, namely IgG1. Similarly a polyclonal antibody preparation directed against IgGc was found to recognise just one subclass, in this case IgG6. Goat anti-horse IgGb showed strong reactivity with IgG4 and IgG7 but also weakly recognised IgG1. The goat anti-horse IgG(T) polyclonal antibody reacted most strongly with IgG5, but also recognised IgG2 and IgG3. The pattern of recognition of the IgG subclasses by the mAbs was similar to that of the polyclonal antibodies except that anti-horse IgGb mAb recognised IgG4 and IgG7 but not IgG1, and anti-horse IgG(T) mAb strongly recognised IgG5, weakly recognised IgG3 but showed no reactivity with IgG2. Reactivity of both the polyclonal and monoclonal anti-IgGb antibodies with IgG4 and IgG7 is not surprising as these two subclasses show 97% amino acid identity.\nThe polyclonal anti-horse IgG (H\u00a0+\u00a0L) and anti-horse IgG Fc antibodies recognised all seven IgG subclasses but gave varying signal strengths in the ELISA (see Table 1). Anti-horse IgG (H\u00a0+\u00a0L) gave a strong ELISA signal with IgG1, IgG4, IgG5 and IgG7, an intermediate signal with IgG2 and IgG3, and only a weak signal with IgG6. The observed reactivity is directed towards the heavy chain of the recombinant IgGs only, because no binding of the anti-horse IgG (H\u00a0+\u00a0L) was seen with recombinant forms of either equine IgA or human IgA (Morton et al., 1993), both of which carry identical light chains to those of the eqIgGs (data not shown). Anti-horse IgG Fc gave strong ELISA signals with IgG1, IgG4 and IgG7, intermediate signals with IgG3 and IgG5, and only weak signals with IgG2 and IgG6.\nThe reactivities of the anti-horse polyclonal Abs with the horse IgG subclasses when assessed by immunoblotting under non-reducing conditions (Fig. 2A) mirrored those of ELISA. However, under reducing conditions (Fig. 2B) goat anti-horse IgGb recognised IgG4 and 7 but no longer recognised IgG1, suggesting that the IgG1 epitope(s) recognised by this antibody may be discontinuous and depend on the intact disulphide-stabilised structure of the antibody. Several previously described anti-equine IgG monoclonal antibodies were thought to recognise conformational epitopes on equine IgG as they functioned under native conditions but not in immunoblotting (Sheoran et al., 1998). Under reducing conditions, goat anti-horse IgG(T) used at a 1\/10,000 dilution recognised IgG3 and 5 but in contrast to non-reducing SDS-PAGE and ELISA no longer reacted with IgG2 but with IgG1 instead. However, recognition of IgG2 under reducing conditions could be achieved with a higher concentration (1\/1000 dilution) of detection antibody (data not shown).\nUnder non-reducing conditions, immunoblotting with goat anti-IgGb (Fig. 2A) revealed additional molecular weight species for IgG4 and IgG7 consistent with those observed with anti-L chain blotting. These additional bands may represent species such as free H-chain, H-chain dimers (H2) and half molecules (HL), suggesting, again, an absence of inter-H and\/or H\u2013L disulphides in small fractions of these two subclasses.\n3.3\nAnalysis of the glycosylation of reqIgGs\nAll seven reqIgGs contain a conserved potential N-glycosylation site within the CH2 domain. Additional potential sites are found in the CH1 domain of IgG3 and IgG5 and in the CH2 domain of IgG2. To determine whether N-glycans were attached to any of these sites, the reqIgGs were probed under reducing conditions with concanavalin A (Con A), which is specific for N-linked glycans. Con A bound to all reqIgG subclasses confirming that the IgGs are indeed N-glycosylated (Fig. 3A). There was, however, some difference in the intensity of the bands, with IgG2 and IgG3 blotting more weakly than the other subclasses, most likely a reflection of the number, type or accessibility of N-glycans present.\nBy contrast, jacalin, which is specific for O-linked glycans, bound only to reqIgG3 and to the O-glycosylated human IgA1 used as a positive control (Fig. 3B).\nTreatment of reqIgGs with N-glycanase resulted in a noticeable reduction in the molecular weights of their H chains (Fig. 4), confirming their N-glycosylation status. For the IgG2 subclass, reactivity of the detecting antibody (goat anti-horse IgG(T)) was lost following the removal of N-glycans, suggesting that the epitopes recognized by this anti-horse reagent are in some way glycan-dependent. The IgG5 H chain migrated as two discrete bands following treatment with N-glycanase, possibly reflecting partial removal of the two potential N-linked glycans in a proportion of the molecules treated.\n3.4\nInteraction with staphylococcal protein A and streptococcal protein G\nBinding of protein A to reqIgGs was generally much weaker than the binding of protein G (Fig. 5), in keeping with earlier reports (Sheoran and Holmes, 1996; Burton and Woof, 1992). A 10-fold higher concentration range of protein A than protein G was required to see significant binding to equine and human IgG.\nProtein A showed the highest level of binding to IgG1 and relatively low levels of binding to the other subclasses in the order: IgG3\u00a0>\u00a0IgG4\u00a0>\u00a0IgG7\u00a0>\u00a0IgG5\u00a0=\u00a0IgG2\u00a0>\u00a0IgG6. Sheoran and Holmes (1996) reported moderate adherence of protein A to serum IgGa (i.e., IgG1) but saw no binding of IgGb (IgG4 and 7) or IgG(T) (IgG3 and IgG5). The fact that slight binding of protein A to IgG3, IgG4, IgG5 and IgG7 was observed in our study may be explained by the use of high concentrations of protein A (up to 50\u00a0\u03bcg\/ml) and a sensitive method of assessing the interaction. Sugiura et al. (2000) also reported some binding of equine IgGb and IgG(T) to protein A. In agreement with previous work (Sheoran and Holmes, 1996; Sugiura et al., 2000), binding of IgG6 to protein A was barely detectable above background levels.\nProtein G showed strong binding to IgG4 and IgG7, closely followed by IgG1. IgG3 displayed intermediate binding, while binding to IgG6 and IgG2 was low, and IgG5 did not bind. Sheoran and Holmes (1996) also showed that the binding affinity of IgGb (IgG4 and IgG7) to protein G was higher than that of IgGa (IgG1). Furthermore, they identified two fractions of IgG(T), one which bound strongly to protein G and one that bound weakly, which most likely correspond to IgG3 and IgG5, respectively.\n3.5\nFunctional assays\nWe studied Fc\u03b3R-mediated cellular responses by assessing the capacity of the antibodies to trigger a respiratory burst in equine peripheral blood leukocytes (PBL). Recombinant IgG1, IgG4, IgG5 and IgG7, and to a lesser extent IgG3, all triggered strong respiratory bursts, whereas IgG2 and IgG6 elicited only very weak responses (Fig. 6).\nBinding of C1q and activation of the classical complement pathway by the reqIgGs was assessed using ELISA. Human serum was used as a source of complement due to the ready availability of antibodies to detect human C1q and human C5-9 complex. The C1q\u2013IgG interaction is highly conserved throughout evolution and C1q is known to react with IgG from different species (Burton and Woof, 1992). The ability of the reqIgGs to bind C1q (Fig. 7A) was reflected in their ability to activate the classical complement pathway to its terminal components (Fig. 7B). In control experiments, both C1q binding and C5-9 deposition were reduced to zero for all IgGs following heat inactivation of serum (data not shown). IgG3 was the most potent activator of complement, followed by IgG1, IgG4 and IgG7, which all bound C1q and activated complement to the same extent. Recombinant IgG2, IgG5 and IgG6 did not bind C1q or activate complement.\n4\nDiscussion\nOur studies show that the CHO expression system previously described for human antibodies (Morton et al., 1993) can be used to generate a stable supply of equine IgG antibodies. These recombinant IgGs retain affinity for their antigen, are recognised by anti-mouse \u03bb L-chain and anti-horse IgG antibodies, are glycosylated, and assemble as covalently stabilised four chain (H2L2) molecules with the expected relative molecular weight of approximately 150\u00a0kDa. Hence, in terms of structure, they appear to be representative of natural horse IgGs.\nInterestingly, horse IgG3 appears to have O-linked as well as N-linked glycans attached to the H chain. O-glycosylated forms of rabbit IgG and mouse IgG2b in which the O-glycans are attached to the hinge region have been described (Fanger and Smyth, 1972a, 1972b; Kim et al., 1994; Kabir et al., 1995). Serine and threonine residues, often in clusters, are known sites for O-glycan attachment. A prominent feature of O-glycosylation sites is an increased frequency of proline residues, especially at positions \u22121 or +3 relative to the glycosylated residue (Wilson et al., 1991). The horse IgG3 hinge is rich in proline and threonine and contains two Thr residues with a Pro at position \u22121 and a number of Thr residues with a Pro at position +3. Hence, it is probable that O-glycans are attached to the hinge region of horse IgG3. O-glycosylation of the hinge region of IgG confers an increased resistance to proteolysis by various proteases including pepsin, papain and elastase (Fanger and Smyth, 1972b; Kabir et al., 1995; Parham, 1983) and therefore may play a role in protection of eqIgG3 from degradation.\nA minor feature of both equine IgG4 and IgG7 was the presence of species such as free H chain, H chain dimers (H2), half molecules (HL) and L chain dimers (L2) under denaturing conditions, suggesting that a small proportion of the molecules lack H\u2013L or H\u2013H disulphide bonds, akin to human IgA2m(1) and human IgG4, respectively. The human IgG2, IgG3 and IgG4 subclasses are disulphide bonded to the L chain through Cys131 in the CH1 domain. The equivalent Cys is missing in equine IgG4 and IgG7, and may account for the tendency for a minor proportion of these subclasses to exist in forms lacking H\u2013L bonds. The majority of equine IgG4 and IgG7 molecules are present at molecular weights corresponding to intact (H2L2) antibodies, suggesting that H\u2013L disulphide bonds can exist. Human IgG1, which lacks Cys131 in the CH1 domain, instead forms a disulphide bond to the L chain through Cys220 in the upper hinge (Burton and Woof, 1992). Hence, it is feasible that hinge cysteines in equine IgG4 and IgG7 may participate in H\u2013L disulphide bond formation. Likewise, this could be the case for equine IgG6, which also lacks the corresponding CH1 Cys residue. The appearance of half molecules of equine IgG4 and IgG7 would suggest that a fraction of both these subclasses lack inter-H chain disulphide bonds, with the molecules being stabilised by non-covalent interactions instead. This arrangement has already been noted in a fraction of human IgG4 molecules which are seen as HL half molecules under denaturing, non-reducing conditions (Aalberse and Schuurman, 2002). The deficiency in inter-H-chain bonds in human IgG4 has been attributed to a Ser within the core hinge sequence (CPSC), which replaces the Pro (CPPC) found in isotypes such as human IgG1 and IgG2 that do not have this deficiency. This Ser seems to increase the likelihood that the two core hinge Cys residues form intra- rather than interchain disulphide bonds possibly due to greater hinge flexibility (Angal et al., 1993; Bloom et al., 1997). In equine IgG1, IgG3, IgG5 and IgG6, which all form disulphide bonded H2L2 molecules, the corresponding core hinge sequence is CPKC. In contrast, the core hinge sequences of eqIgG7 and IgG4 are CPTC and CPAEC respectively, suggesting that deviations from central Lys to either Thr or Ala-Glu may decrease the efficiency of interchain disulphide bridge formation in some way.\nOur results for the binding of the reqIgGs to protein A and protein G agree well with those for the binding of horse IgG subclasses which had been purified from serum and classified under the previous nomenclature (Sheoran and Holmes, 1996; Sugiura et al., 2000). The binding sites for protein A and G on the human IgG Fc lie at the CH2-CH3 domain interface, with considerable but incomplete overlap (Deisenhofer, 1981; Sauer-Eriksson et al., 1995) (Fig. 8). The amino acid sequences of equine IgG2, IgG3, IgG4, IgG5, IgG6 and IgG7 all contain at least one amino acid difference from human IgG1 within the regions contributing to protein A binding (Fig. 8). These differences may be sufficient to explain the low affinity of these IgGs for protein A because a single amino acid substitution of His to Arg at position 435 in a human IgG3 allotype is known to prevent binding to protein A (Burton and Woof, 1992). Similarly the differential reactivity of the subclasses with protein G can be rationalized on the basis of amino acid substitutions at key positions in the protein G interaction site (Fig. 8). The fact that the highest level of binding is seen with eqIgG1, IgG4 and IgG7, which like human IgG1 all possess His433 and Tyr436, may suggest that these two residues play key discriminatory roles in the binding of horse IgGs to protein G.\nIgGs mediate effector function through interaction with leukocyte Fc\u03b3Rs and activation of the classical pathway of complement. These effector functions differ with IgG subclass (Burton and Woof, 1992). There is little available information with regard to comparative binding of the horse IgG subclasses to Fc\u03b3Rs. An early study found that eqIgG (subclass unspecified) but not eqIgG(T) (i.e., eqIgG3 and\/or IgG5) was able to interact via the Fc region with equine monocytes and neutrophils (Banks and McGuire, 1975). In the present study, we have demonstrated that equine IgG1, IgG3, IgG4, IgG5 and IgG7 are able to elicit a strong respiratory burst from equine PBL, predicting that these five subclasses can interact with Fc\u03b3R on the surface of these cells. IgG2 and IgG6 stimulated little or no response suggesting that these two subclasses are unable to interact efficiently with Fc\u03b3R. Several amino acids in the lower hinge region of human IgG1 at the N-terminus of the CH2 domain (Leu234-Leu235-Gly236-Gly237-Pro238-Ser239) (Fig. 8) play a central role in binding to human Fc\u03b3R and deviations from this motif help to explain human IgG subclass specificity for Fc\u03b3R (Burton and Woof, 1992; Woof and Burton, 2004). In the horse IgGs, IgG1 and IgG3, which elicit bursts, retain this motif while IgG2 and IgG6, which do not elicit bursts, display significant differences from the motif (Fig. 8). Thus, amino acid differences in this region may account, at least in part, for the observed differences in ability to trigger a respiratory burst. However, despite IgG4, IgG5 and IgG7 lacking the full motif, these subclasses are still capable of eliciting strong respiratory bursts, suggesting that regions other than the lower hinge region may influence interaction with at least some classes of equine Fc\u03b3Rs.\nActivation of the classical complement pathway by IgG may be initiated through the binding of the first complement component C1q, to two or more adjacent IgG molecules. Human IgG subclasses bind C1q in the rank order IgG3\u00a0>\u00a0IgG1\u00a0>\u00a0IgG2 while IgG4 shows no significant binding (Burton and Woof, 1992). Early studies of equine anti-Lac antibodies (Klinman et al., 1966) using guinea pig complement showed that a preparation comprising IgGa and IgGb (i.e., IgG1, IgG4 and IgG7) was able to fix complement whilst IgGc (i.e., IgG6) was not. The current study confirmed and extended these results. We found that eqIgG3 was the most potent activator of complement, followed by eqIgG1, IgG4 and IgG7, while eqIgG2, IgG5 and IgG6 failed to bind C1q or activate complement. While our results were obtained using human serum as a source of complement, and we cannot rule out the possibility of subtle differences of reactivity with horse complement, we feel that our findings provide a good reflection of the likely relative complement-activating capabilities of the different eqIgG subclasses.\nThe C1q binding site lies within the CH2 domain of IgG, but appears not to be conserved precisely across species. Glu318, Lys320 and Lys322 are necessary for C1q binding by mouse IgG2b, whilst in human IgG1 or IgG3 Asp270, Lys322, Pro329 and Pro331 appear important with minimal contribution from Glu318 and Lys320 (Duncan and Winter, 1988; Idusogie et al., 2000; Thomessen et al., 2000). A comparison of the equine IgG CH2 amino acid sequences does not reveal a common difference between the complement activating and non-activating subclasses at the residues corresponding to Asp270, Glu318, Lys320, Pro329 or Pro331 (Fig. 8). However, all of the complement-activating subclasses have a Lys corresponding to Lys322. The fact that this residue is a serine in eqIgG2 and IgG5 may account, at least in part, for the inability of these subclasses to activate complement. Curiously, IgG6, which possesses a lysine at position 322, does not activate complement, underlining the subtle species differences that are apparent in complement activation.\nOur data on the effector function capabilities of the IgG subclasses have important new implications for the design of effective horse vaccines. It is clear that to achieve maximal protection via Fc\u03b3R- and complement-mediated elimination mechanisms, vaccines should seek to elicit IgG antibodies of the IgG1, IgG3, IgG4 and IgG7 subclasses. Vaccines that trigger only IgG2, IgG5 or IgG6 antibodies are predicted to offer less effective protection. Since IgG plays key roles in both serum and mucosal compartments in the horse, these considerations are applicable to both systemic and mucosal vaccination strategies.\nFurther, our findings on the relative effector function capabilities of the different IgG subclasses lend themselves to a rationalization of the protection offered by natural IgG immune responses against various infectious agents. Thus, investigations into the role of individual subclasses in protective immunity have demonstrated that IgGa and IgGb (i.e., the Fc\u03b3R- and complement-engaging isotypes IgG1, IgG4 and IgG7) contribute to protection against equine influenza virus (Nelson et al., 1998; Breathnach et al., 2006; Soboll et al., 2003), Streptococcus equi (Sheoran et al., 1997) and Rhodococcus equi (Lopez et al., 2002). Indeed, IgGb (IgG4 and IgG7) has been suggested to be most important in equine protective antibody-mediated immune responses to intracellular pathogens (Nelson et al., 1998; Lopez et al., 2002; Goodman et al., 2006), whereas IgGc (IgG6) is suggested to be least important (Sheoran et al., 2000). The current study helps rationalize this observation as both IgG4 and IgG7 are able to stimulate a potent respiratory burst and activate complement via the classical pathway. By contrast, these effector functions are absent in the IgGc (IgG6) subclass. The requirement of equine IgGs to recruit effector molecules for effective immunity is illustrated by several studies. For example, IgG-mediated neutralisation of equine arteritis virus (EAV) (Balasuriya and MacLachlan, 2004) and acute phase EHV-1 (Snyder et al., 1981) is complement dependent, and opsonisation by specific IgG is necessary for efficient phagocytosis of Rhodococcus equi, Escherichia coli and Actinobacillus equuli by equine neutrophils or alveolar macrophages (Hietala and Ardans, 1987; Grondahl et al., 2001; Cauchard et al., 2004). Drawing all this information together, it is clear that the enhanced effector function capabilities of equine IgG1, IgG3, IgG4 and IgG7 equip these subclasses for key protective roles.\nInternational meetings have highlighted the need for further research into the functional roles of equine IgG subclasses and for the development of tools to study equine Igs (Lunn et al., 1998). The reqIgGs described here are a reliable source of pure and homogeneous equine IgG subclasses and will serve as useful reference proteins for the production and screening of equine specific reagents. In particular, the generation of mAb able to discriminate between the seven subclasses is an ongoing project. Moreover, they will provide a valuable resource for future research, in particular in delineation of the function of individual IgG subclasses in antibody-mediated immunity of the horse.","keyphrases":["igg","protein a","respiratory burst","complement","horse","reqigg, recombinant equine igg","ehv-1, equine herpes virus-1","nip, 3-nitro-4-hydroxy-5-iodophenylacetate","pbs-t, pbs-tween","fc\u03b3r, receptor specific for the fc region of igg","pbl, peripheral blood leukocyte","recombinant antibodies"],"prmu":["P","P","P","P","P","R","M","M","M","R","R","R"]}
{"id":"Cogn_Process-4-1-2254470","title":"Coherence and recurrency: maintenance, control and integration in working memory\n","text":"Working memory (WM), including a \u2018central executive\u2019, is used to guide behavior by internal goals or intentions. We suggest that WM is best described as a set of three interdependent functions which are implemented in the prefrontal cortex (PFC). These functions are maintenance, control of attention and integration. A model for the maintenance function is presented, and we will argue that this model can be extended to incorporate the other functions as well. Maintenance is the capacity to briefly maintain information in the absence of corresponding input, and even in the face of distracting information. We will argue that maintenance is based on recurrent loops between PFC and posterior parts of the brain, and probably within PFC as well. In these loops information can be held temporarily in an active form. We show that a model based on these structural ideas is capable of maintaining a limited number of neural patterns. Not the size, but the coherence of patterns (i.e., a chunking principle based on synchronous firing of interconnected cell assemblies) determines the maintenance capacity. A mechanism that optimizes coherent pattern segregation, also poses a limit to the number of assemblies (about four) that can concurrently reverberate. Top-down attentional control (in perception, action and memory retrieval) can be modelled by the modulation and re-entry of top-down information to posterior parts of the brain. Hierarchically organized modules in PFC create the possibility for information integration. We argue that large-scale multimodal integration of information creates an \u2018episodic buffer\u2019, and may even suffice for implementing a central executive.\nIntroduction\nWhen we return home from work we sometimes find ourselves deeply engaged in thinking about an unsolved problem. Yet, at the same time we manage to find our way home and avoid accidents. This indicates that apparently we are able to automatically control our behavior without needing conscious attention. If suddenly an obstacle appears, however, we are also able to switch attention in a split second. Given the right cues, we may also remember that in the morning we planned to do some shopping on the way home. That too would put solving the problem to a halt and bring us to figure out the best route to the shop. This shows that control may be taken over by retrieving a plan, and that in order to carry it out we need access to a large database, including a route planner. If later on we resume thinking about the problem, we may figure out a solution by combining known elements in a novel configuration, and then start thinking how to use it in the future.\nThe description given above is an example of the coordinated and goal-directed thoughts and actions that are generated in our brains all the time. Clearly, such coordinated behavior requires a versatile system of cognitive control for which as yet there does not seem to exist an adequate concept. Some of the contours of such a control system, however, are discernable in the example given above, like the ability to maintain representations, to direct attention and to combine previously unrelated information.\nAn early and influential framework for the study of control and coordination of cognitive behavior is the multi-component model of working memory (Baddeley and Hitch 1974). Working memory (WM) refers to a limited capacity system for temporary holding and manipulating information, that is required for performing a wide range of cognitive tasks such as comprehension, learning and reasoning. Originally the model distinguished three-components. A general control system, or \u2018central executive\u2019, and two subsidiary slave systems, a phonological loop and a visuo-spatial sketch pad, each capable of maintaining a limited amount of information. More recently (Baddeley 2003), an episodic buffer was added to connect working memory with long-term memory and to allow binding together information from different sources into integrated episodes. The central executive is the most important but least understood component of WM, and it looks conspicuously like a homunculus. Baddeley is well aware of this criticism, but he defends the central executive concept by pointing out that it defines a problem area for which the processes have to be specified.\nAn important conceptual model was suggested by Norman and Shallice (1986). They proposed a \u2018data base system\u2019 for automatic habitual action routines, containing well-learned stimulus-response associations and cognitive and behavioral skills, and a \u2018supervisory attentional system\u2019 capable of controlling behavior by selectively biasing and reconfiguring the available skills and schemas. Automatic control develops gradually through practice as learning processes create associative pathways between perception and action. The conscious form of control is applied when we are confronted by novel and unexpected stimuli, and when we have the intention to attain specific goals. In this case, automatic actions to stimulus patterns in the environment have to be suppressed and replaced by novel task- and goal-directed actions.\nThe model of Norman and Shallice aimed to explain deficits of executive control that are often observed in patients with damage in prefrontal cortical areas. It is now generally accepted that the prefrontal cortex (PFC) is of crucial importance when behavior must be guided and controlled by internal states and intentions, when automatic responses have to be suppressed, and when tasks require the establishment of new or rapidly changing mappings between perception and action (e.g., Goldman-Rakic 1996; Miller and Cohen 2001; Smith and Jonides 1999; Wood and Grafman 2003). Anatomically, the PFC is well positioned to coordinate processing in the rest of the brain. It consists of a number of strongly interconnected areas that collectively have reciprocal connections with virtually all other neocortical and subcortical structures. It is also an area that shows late development, both phylogenetically and ontogenetically, reaching maturity only in adolescence (Fuster 2001).\nBased on these anatomical considerations, we have suggested (Phaf and Wolters 1997) that working memory or, more general, executive functions, may have emerged as a consequence of an evolutionary development of the PFC. This development may have created the possibility for neural processes that could run (partly) independent of present input and output, but that also could modulate, and thus control, perception-action relations in the rest of the cortex. In this view, the role of PFC in controlling behavior is modulatory rather than transmissive, which is similar to the role assigned to PFC in several other models (e.g., Norman and Shallice 1986; Miller and Cohen 2001; O\u2019Reilly et al. 1999). Whereas simple adaptive behavior rests on a cycle of perception, action, and perception-of-action results, the added PFC would allow an internalization of this loop, freeing the organism of the restriction of being aware of, or acting upon, physically present objects or situations only. In fact, it would implement a medium for creating a virtual world, i.e., manipulating internal representations that are independent of the present environment (for a similar idea, describing thinking as simulated behavior and perception, see Hesslow 2002).\nMore specifically, we suggested that the development of PFC created the possibility to maintain physically absent information in an active state by recurrent connections (loops) between PFC and the rest of the cortex. The recurrent activity in these loops may affect subsequent perceptual and motor processing, i.e., it can redirect attention and control actions by activating or inhibiting particular motor programs. By assuming interactions and integration between loops, more complex forms of representation and control may develop. For instance, recurrent connections with memory systems would allow access to all stored information, and mechanisms for combining information in the loops would allow the formation and updating of future goal states, and ways to achieve them.\nA taxonomy of working memory functions\nAlthough there is no generally accepted taxonomy of executive functions, we may speculate which functions would be required. It can be argued, for instance, that to be able to exert top-down control over cognitive processes, patterns of activity representing task relevant information must be actively maintained in the PFC. Moreover, PFC must be able to generate biasing signals to guide the flow of information by selectively inhibiting or activating particular representations and pathways in other parts of the brain. This interaction between PFC and posterior parts of the brain would also be needed to retrieve any relevant information stored in memory. Finally, PFC must be able to integrate information from different sources to implement goal directed behavior over time. We suggest, therefore, that three main executive functions have to be distinguished in PFC. The first function is maintenance, i.e., holding a limited amount of currently needed information, i.e., all task-relevant information supplied by preceding events, in an active form. The second function is attentional control, i.e., top-down controlled selective activation of task-relevant stimulus representations and responses. Only selective activation is required, because in a competitive system this automatically induces selective inhibition of task-irrelevant stimuli and responses. The third function is integration. This function consists of the ability to flexibly combine and reorganize information from different sources in the service of controlling task execution. This also includes control over search in LTM, monitoring and evaluating results of actual or imagined actions, and sequencing operations that are needed for planning, decision making and problem solving. In our view, the central executive operations in PFC are strongly interdependent and continuously interacting with processing in other cortical areas (see also Duncan 2001, for emphasis on interdependence of control, working memory and attention in PFC). A somewhat similar view of PFC functions was suggested by Smith and Jonides (1999). They proposed a distinction between short-term storage and two executive processes, selective attention and task management.\nIn this paper we will first discuss the neuroanatomical correlates of these hypothesized interdependent working memory functions. Then we will present a neural model for binding and segregation in working memory to simulate the maintenance function. This model is based on recent ideas about synchronization of activation patterns in networks and it is able to explain capacity limitations of maintenance in WM. Next, extensions of this model will be discussed to realize the attentional control mechanism. It is suggested that modulatory effects within PFC may selectively enhance or suppress representations in posterior cortex by recurrent connections. Finally, we will present some ideas on how to realize integration and manipulation functions, given that large-scale multi-modal integration is necessary to explain coherence and coordination of behavior.\nNeuroanatomical correlates of working memory in PFC\nGenerally, the brain shows a remarkable specialization of anatomically distinctive areas performing specific cognitive functions. Also the PFC comprises a number of cyto-architectural distinctive areas or modules that differ in patterns of connectivity with other brain regions (e.g., Miller and Cohen 2001; Wood and Grafman 2003). Much effort has been invested in finding functional specializations within the PFC, i.e., the extent that different areas or modules of PFC are selectively involved in encoding particular types of information (e.g., verbal versus visual), or in implementing various processing functions (e.g., maintenance versus attentional control).\nAlthough this debate is still ongoing, there seems to be a growing consensus that broadly speaking, three major areas in PFC seem to be distinguishable in terms of processing specific types of information (Fuster 2001; Wood and Grafman 2003), probably with additional sub-specializations. First, ventromedial and orbitofrontal regions (BA 11\/12\/47) seem specifically involved in representing and processing reward and affective information, and thus participate in emotionally and motivationally driven behavior (e.g., Fuster 2001; Rolls 2000). These regions have major reciprocal connections with the temporal lobe and the amygdale complex. Second, medial regions of the PFC (in particular the anterior cingulate cortex, BA 24\/32) are suggested to be involved in error monitoring, and in detecting conflicts between competing stimuli and responses (e.g., Ridderinkhof et al. 2004). Third, lateral and anterior PFC areas (BA 9\/10\/44\/45\/46; see Petrides 2005, for a review of the architectonic organization of this area) are supposedly involved in all executive functions that are needed for organized goal-directed behavior, such as the selection of goal-relevant information, the manipulation and maintenance of information, and monitoring multiple-events (e.g., Miller and Cohen 2001; Petrides 2000). This area has strong reciprocal connections with parietal and temporal association areas, the hippocampal formation and with all other PFC regions. Damage to lateral PFC typically impairs the ability to formulate and carry out plans and sequences of actions (Fuster 2001) and to control long-term memory encoding and retrieval (Blumenfeldt and Ranganath 2006; Tomita et al. 1999).\nThere is also increasing evidence, however, that localization of informational content is not precise, but rather a matter of degree. A review of imaging results showed that quite different cognitive demands (i.e., response conflict, task novelty, WM delay and load, and perceptual difficulty) induced very similar patterns of prefrontal recruitment, mainly involving lateral and medial PFC areas (Duncan and Owen 2000). This finding is corroborated by single cell studies that have shown a substantial adaptability of function at the level of individual neurons. Many lateral PFC neurons show highly specific activity patterns depending upon the current task and task conditions (e.g., Asaad et al. 1998, 2000; White and Wise 1999; Quintana and Fuster 1999; Wallis et al. 2001).\nThese results led Duncan (2001) to postulate the \u2018adaptive coding\u2019 model. According to this model, neurons in PFC adjust their function to match the requirements of particular tasks that are carried out. Apparently any given cell can be driven by many different kinds of input, both from posterior parts of the brain and from other PFC areas. The collective responsiveness pattern of all PFC cells thus reflects the particular task relevant conditions. Duncan also noted, however, that the potential of cells to process different types of information, does not exclude regional specializations. This would reconcile results indicating regional specialization, with results from single cell studies showing large scale integration and adaptation. For example, a statistical rather than an absolute specialization seems to apply for left-hemisphere PFC recruitment for verbal material and right-hemisphere PFC recruitment for non-verbal stimuli (see, e.g., Passingham et al. 2002).\nA slightly different perspective on functional specialization of PFC areas is to assume that some areas are relatively specialized, whereas others serve more general integrative purposes and come into play in almost any task. Gruber and von Cramon (2003), for instance, found both modality specific and amodal areas in PFC in verbal and visuo-spatial working memory tasks. Other findings suggest that many cells in ventrolateral areas of PFC are specifically sensitive to maintaining representations of single stimuli (e.g., objects or locations), whereas cells sensitive to maintaining complex integrated stimuli and elaborative rehearsal are found more often in dorsolateral areas (Prabhakaran et al. 2000; Owen 2000; Wagner et al. 2001). Wallis and Miller (2003) presented evidence suggesting that orbitofrontal cortex, which is specifically involved in reward and affect processing, passes on this information to dorsolateral areas of PFC where it is combined with other information to control behavior. Similarly, Ridderinkhof et al. (2004) concluded from a review that detection of response conflicts and response errors elicits overlapping foci of activation in medial PFC areas, and that this activation serves as a signal that engages regulatory processes in lateral PFC areas.\nLearning and working memory\nOur view of working memory is that its informational content consists of the active part of long-term memory representations that is available at any point in time for controlled processing (e.g., Cowan 1999; O\u2019Reilly et al. 1999). These representations presumably are stored in the temporal, parietal and occipital cortex, so the functions of working memory (maintenance, selection and integration) that apply to these representations are realized by an interaction between PFC and posterior cortical areas. In this view, PFC is not itself involved in storing long-term memory information. Its representational role would be limited to temporarily maintaining and integrating information stored elsewhere. This view seems to be endorsed by the fact that the neural circuits and pathways assumed to be involved in the two main learning processes (Eichenbaum and Cohen 2001), and the corresponding procedural and declarative memory systems (Squire and Zola-Morgan 1991), do not include the PFC as a representational medium.\nThe suggestion that PFC implements WM functions, but is not itself involved in associative learning, would fit the requirement that it is maximally flexible in the service of generating novel combinations of representations that are stored somewhere else. There are no indications, however, that the neural architecture and processes of the PFC differ fundamentally from those in posterior cortex which intrinsically generate a learning capacity. Therefore, some learning (and representational) capacity in PFC cannot be ruled out a priori.\nSome evidence suggests that the information used in working memory tasks does not have to be stored in PFC. First, prefrontal patients generally perform quite well in tasks requiring the retrieval of previously learned procedural and declarative knowledge. However, they are typically impaired in episodic memory tasks requiring for instance elaboration of stimuli, ordered recall and source monitoring. So the problem with prefrontal damage does not seem an inability to store information in memory, but an inability to control encoding and retrieval processes (e.g., Shimamura 1995). Second, several recent studies have linked activity in PFC areas with promoting effective LTM formation (e.g., Blumenfeld and Ranganath 2006; Buckner 2003). They suggest, however, that the information itself is not stored in PFC. Instead, the interaction between PFC and posterior cortex controls what is selectively activated, maintained and elaborated, and what is subsequently stored as a novel representation via the medial temporal lobe and the hippocampus (see, e.g., Ranganath et al. 2005). Third, an increasing number of studies showed that the activation patterns of many single neurons or neuron ensembles in PFC quickly and flexibly adapt to represent completely arbitrary rules (Asaad et al. 2000; Wallis et al. 2001; White and Wise 1999), or to arbitrary action sequences (Averbeck et al. 2006). Although in all of these studies a number of learning trials precedes the development of task specific activation patterns, the speed and flexibility of the adaptation process seems more suggestive of an adaptive coding than of an associative learning process.\nOther authors have suggested that learning in PFC does occur. O\u2019Reilly et al. (1999), for example, suggested that the information that is maintained in WM consists of activation patterns in PFC that are sustained by strong mutual excitation of the neurons involved. To explain the flexibility in combining these representations without losing their specificity, they assumed that PFC representations had to be relatively isolated from each other. Therefore, learning in PFC was supposed to be slow, taking place over many years, and eventually producing a rich palette of independent PFC representations that enable flexible problem-solving skills. Such representations might have a hierarchical structure with an increasing level of abstraction along a posterior-anterior PFC axis, probably with the most posterior PFC representations being closely connected to detailed memory representations in posterior cortex.\nA worked out version of this model by Rougier et al. (2005) showed that extensive training of their model with various tasks resulted in the development of abstract rule-like PFC representations that supported flexible generalization in novel tasks. The PFC representations developed slowly, but once learned adaptive behavior was mediated by a search for a task appropriate pattern of activity (cf. the adaptive coding mechanism of Duncan 2001), rather than the need to update connection weights. Although the simple tasks simulated in this model may also apply to more realistic and complex rules, the authors point out that it still leaves unexplained how PFC representations can be dynamically recombined and can interact with other systems (such as episodic memory, language and affect).\nIt remains to be seen whether the supposed PFC learning mechanism is viable. Over brief periods of time, learning probably does not play a major role, but a role of learning over extensive periods cannot be ruled out.\nEvidence for three interdependent working memory functions in PFC\nMaintenance\nNeurophysiological studies have established persistent activity during delays (i.e., after stimulus offset) as the main candidate for a neural substrate of the maintenance function of WM. Single cells showing persistent firing during a delayed-matching task were first discovered in PFC, and later in other neocortical areas like inferotemporal (IT) cortex (Fuster and Alexander 1971; Goldman-Rakic 1996; Nakamura and Kubota 1995).\nDifferent areas, especially in posterior parts of PFC, may be involved in maintaining modality specific information. Smith and Jonides (1999), for instance, suggested a relatively specialized role of left and right PFC hemispheres in maintaining verbal and visual information, respectively. Moreover, they proposed that maintenance of spatial information may involve more dorsally located regions than maintenance of object information (see also Baddeley 2003).\nIn principle, there are two ways to maintain activity in neurons during delays. One is to assume that input activation creates an activation pattern in a network of cells that is maintained within the network by auto-association (Amit 1995; Deco and Rolls 2003), and possibly augmented by a dopamine gating mechanism (O\u2019Reilly et al. 1999). The other possibility is that the activation pattern oscillates in a recurrent loop between different networks of cells.\nThere is much evidence that supports an important role of recurrent loops in maintaining information during working memory tasks. Strongest evidence is that during delays continued activity is not only found in PFC, but also in modality specific areas in IT cortex when coding for objects (Fuster et al. 1985; Miller et al. 1993; Tomita et al. 1999; Ungerleider et al. 1998), and in parietal cortex when coding for locations (Curtis and d\u2019Esposito 2003; Rowe et al. 2000; Sakai et al. 2002). Moreover, localized cooling of either PFC or IT cortex interferes with activity in the other area and causes behavioral deficits in a working memory task (see Fuster 2001). From a review of the literature, Ranganath (2006) concluded that information in visual WM tasks is maintained through persistent activity in visual cortical areas (e.g., inferotemporal and parietal areas) that is promoted by top-down input from PFC. So according to Ranganath, maintenance would be an interaction between PFC and modality or object specific posterior areas. An additional loop involving the medial temporal lobe (i.e., the hippocampus and related areas) may be required to maintain complex novel stimuli, and to quickly create new long-term representations (e.g., Ranganath et al. 2005).\nRanganath (2006) is somewhat unclear about the nature of the feedback from PFC in maintenance. As a further specification we would suggest a fixed connection scheme (probably shaped by long-term learning) between posterior brain areas, containing long-term memory or newly created representations of stimuli, and adjacent areas in PFC. These pathways convey to PFC what information is being maintained. This may also explain the finding that during maintenance, activation in posterior areas is more vulnerable to distraction than activation in PFC (Miller et al. 1993; O\u2019Connor et al. 2002). Maintenance may resist such distraction because PFC activation can reinstate activation of corresponding lower level representations. Such reinstatement probably also underlies memory retrieval and mental imagery. It has been shown, for example, that in tasks requiring participants to imagine faces or buildings, category specific regions in the IT cortex became activated (e.g., Ishai et al. 2002; O\u2019Craven and Kanwisher 2000).\nAs was argued by O\u2019Reilly et al. (1999), there are two seemingly incompatible core requirements of working memory. The first is to robustly maintain goals and task instructions over time even when faced with strong interference. The second is to rapidly and flexibly update goals and tasks if circumstances change. Their solution to combine robust maintenance with flexible updating has been to assume a dopamine gating mechanism. Under tonic dopamine levels a strong mutual excitation within a cell assembly would lead to sustained activity and resistance to interference by irrelevant input. Phasic shifts of dopamine levels induced by significant events (e.g., success or failure in a task or novel stimuli predicting reward) would enhance the strength of afferent input and cause updating of the activation state, which subsequently may lead to representing novel strategies, rules, goals or task states.\nAlthough a dopamine-gating mechanism is an interesting possibility, we believe that re-entry of activation through recurrent neuronal circuits is the main mechanism for the maintenance function of WM. It is likely that there are many of such recurrent loops for different types of information. These loops link the perceptual, memory and motor representational areas in posterior cortex to PFC. They feed information into PFC and, in turn, are activated by the recurrent activation from PFC. Modulating activity in these loops by other PFC sources would modulate the top-down recurrency. We also suggest and that these loops form a hierarchy, at the lowest level maintaining simple stimuli or features and at higher levels maintaining increasingly complex stimulus relations and rules. We will show how this idea can be implemented and explore the feasibility of such an implementation. We agree, however, that especially for the highest and most global or integrative levels of maintaining information, a dopamine gating mechanism for auto-associative maintenance cannot be ruled out.\nAttentional control\nAttentional control by executive functions in PFC requires top-down effects on local information processing in posterior brain areas (e.g., Desimone and Duncan 1995; Miller and Cohen 2001; Shimamura 2000). Evidence for such a role comes, for instance, from findings showing that an important characteristic of behavioral deficits following damage to the PFC is extreme distractability (i.e., an inability to suppress interfering information) and disinhibition (i.e., an inability to suppress inappropriate responses).\nA mechanism for top-down attentional control is suggested by the \u2018biased competition model\u2019 (Chelazzi et al. 1993; Desimone and Duncan 1995; Downing 2000; Reynolds et al. 2000; Gazzaley et al. 2005). According to such a model, top-down control activates corresponding representations that are then in a better position to compete with irrelevant information for perceptual awareness and control of motor behavior. Such top-down attentional modulation of neural responses, i.e., relative enhancement of neuronal responses to task-relevant stimuli and relative suppression of neuronal responses to task-irrelevant stimuli, has been shown throughout the visual system as early as the primary visual cortex (e.g., Desimone and Duncan 1995; Reynolds et al. 1999), and the exact areas that are biased depend on the task that is performed (e.g., Kastner et al. 1999).\nTop-down attentional bias not only pre-activates specific target representations but also spreads activation to related representations. Distractors with visual similarities to targets also attract attention (are positively biased), as well as distractors with semantic or associative links to the target (Moores et al. 2003). Task specific activity in PFC also generates top-down signals involved in the selection of actions, and in long-tem memory retrieval (Hasegawa et al. 1998) and storage (Brewer et al. 1998; Kyd and Bilkey 2003; Blumenfeld and Ranganath 2006; Wagner et al. 1998). This perspective thus suggests a single underlying mechanism of cognitive control by the PFC, namely a top-down biasing effect on processing in specialized sensory, motor and memory systems responsible for task performance (see, e.g., Miller and Cohen 2001).\nWe will explore the feasibility of a mechanism for attentional control based on modulating feedback signals from PFC in the recurrent circuits that are used for maintenance. We suggest that attentional control is mediated by biasing the recurrent loops between PFC and posterior cortex. In this view, the biasing signals stemming from higher integrative areas in PFC would modulate the recurrent feedback to posterior cortical areas (see also Deco and Rolls 2003).\nIntegration\nThe importance of a large-scale integrative system for the coordination of behavior has been widely acknowledged. Such a system would provide the combination and integration of all information necessary for controlling task-relevant behavior, i.e., it would comprise all task-relevant information supplied by the present context and goals, and by knowledge retrieved from memory. Baddeley (2000), for instance, suggested an episodic buffer as an additional subsystem in his WM model which is capable of integrating and maintaining information from different sources. In a similar vein, Miller and Cohen (2001) argued for a cognitive control system in the PFC that represents goals and the means to achieve them. They suggest that this system integrates converging inputs from many sources. It exerts control by divergent feedback signals to sensory, memory and motor areas in posterior cortex which mediate directed attention, response selection, and guide retrieval from LTM.\nA high level integrative control system was also proposed by Koechlin et al. (2003). They suggested a cascade model, consisting of three nested levels of cognitive control. At the top of this cascade, located in rostral (anterior) parts of the PFC, they assume an episodic control system involved in selecting task-set representations \u2018according to events that previously occurred or to ongoing internal goals\u2019 (p. 1181). The view that progressively \u2018higher\u2019 neural areas support functions that are increasingly more integrative also has been endorsed also by Fuster (2001). He argued that a cascaded control model, assuming several nested levels of control, with a highly integrative system at the top, may explain neuropsychological results showing that damage to the top system only affects tasks that require a high degree of information integration (e.g., planning and problem solving), but does not interfere with tasks that can be executed at a lower level of control.\nPotentially the highest level of integration is attained in the anterior PFC (aPFC), in humans corresponding to BA10. In a recent discussion, Ramani and Owen (2004) pointed out this area as a likely candidate for being the apex of a hierarchical system of PFC modules, because it is predominantly (or even exclusively) reciprocally connected to other supramodal PFC areas. Therefore, it is in the position to integrate everything the brain is capable of representing. Moreover, the aPFC has anatomical characteristics (a high dendritic spine density combined with a low density of cell bodies) that make it likely to be involved in integration. The aPFC has been suggested to be specialized for processing internal mental states and introspective evaluation (Christoff and Gabrieli 2000), monitoring successful retrieval (Ranganath and Paller 2000), management and monitoring of goals and sub-goals (Koechlin et al. 2000; Braver and Bongliatti 2002), integration of information over time (Braver et al. 2001; Koechlin et al. 2003; Sakai et al. 2002), and manipulation of relational knowledge (Kroger et al. 2002).\nA hierarchical model of integration based on the convergence of input from lower level specialized PFC modules on higher integrative levels, is consistent with claims that a distinction can be made between PFC areas involved in simple maintenance, and others involved in maintenance of complex information and executive processes (D\u2019Esposito et al. 1999; Sakai et al. 2002). For example, several findings suggest a distinction between ventrolateral (vlPFC) and dorsolateral (dlPFC) areas in terms of the level of abstractness of information processed (e.g., Koechlin et al. 2003; O\u2019Reilly et al. 2002). Tasks requiring processing of single words or concepts mainly seem to involve vlPFC (e.g., Wagner et al. 2001), whereas in more complex tasks, like sentence processing, enhanced involvement of dlPFC has been found (e.g., Hashimoto and Sakai 2002; Kerns et al. 2004b). A relatively high integrative role for the dlPFC (as compared to vlPFC) is also suggested by findings showing this area to be involved in tasks that go beyond simple maintenance, such as elaborative rehearsal (Blumenfeldt and Ranganath 2006), processing relations between stimuli (Kroger et al. 2002), and solving conflicts that are signalled by medial areas of PFC (Egner and Hirsch 2005; Kerns et al. 2004a).\nWe endorse the idea that the PFC is hierarchically organized, with subordinate modules being (relatively) specialized in processing simple aspects of tasks, and super-ordinate modules, located in dlPFC and frontopolar cortex, being specialized in large scale integration (i.e., binding) of inputs from different sources. At the apex of the supposed hierarchy, the aPFC might integrate all goal related information over space and time (see Fig.\u00a01). Here, emotional and motivational evaluations (from orbitofrontal areas), results of memory retrieval, language and rule-like representations (from dlPFC), and anticipated action effects, response conflicts and bodily states (from medial PFC areas) converge. So the aPFC would be crucial for integrating multiple forms of information in the pursuit of general goals (e.g., Ramnani and Owen 2004), or when information from temporally dispersed events has to be integrated (e.g., Koechlin et al. 2003, 2006). Conversely, the ensuing activation patterns in aPFC set the stage for task and goal directed behavior, with control being exerted by top-down modulation of subordinate modules. In this way a hierarchy of top-down coordinated control is implemented by entraining successively lower order areas, eventually biasing perceptual processing, memory search, and action selection.\nFig.\u00a01A schematic view of the hierarchical structure of PFC. Many details regarding sensory input and motor output processing in subcortical centres and cerebellum are not shown. Double arrowheads indicate recurrent connections. Connections and modules shown are not suggested to be anatomically fully complete and accurate. aPFC anterior PFC; dlPFC dorsolateral PFC; vlPFC ventrolateral PFC; oPFC orbital and ventromedial PFC; mPFC medial PFC (anterior cingulate cortex); pm cortex premotor cortex\nA neurocomputational model of maintenance, control and integration\nElsewhere (Raffone and Wolters 2001), we have presented a model for the temporary holding in (visual) working memory of a limited number of neural patterns, simulating either single features or integrated objects. The model implemented a cortical mechanism of maintenance in a network of model neurons with biologically plausible parameters. Although the model implemented a visual working memory system, the principles may be applicable to any form of information or type of working memory.\nIn the model WM was assumed to be based on recurrent connections between IT cortex containing representations of objects or features, and corresponding neurons in PFC. The IT representations were modelled as strongly associated neural assemblies that generate synchronized firing patterns when activated by external input. The simultaneous activation of independent assemblies in IT causes competition via inhibitory interneurons. Due to the neuron characteristics, this leads to desynchronization among the activation patterns of competing assemblies resulting in a sustained phase-locked activation of multiple assemblies over time.\nMaintenance in cortical circuits of visual working memory was shown to be possible in terms of oscillatory reverberations between PFC and IT modules. Firing rate oscillations induced during stimulus presentation were maintained after stimulus offset by active feedback from prefrontal areas. Neurophysiological plausible model parameters enforced a limitation of about three to four independent assemblies that could be maintained in this way. This number closely coincides with recent estimates of the maintenance capacity of WM (e.g., Cowan 2001). The same mechanism that optimizes coherent pattern segregation, also poses a limit to the number of assemblies (about four) that can concurrently reverberate. The model thus indicated that selective synchronization and desynchronization of feedback-based oscillatory reverberations creates a suitable medium for a visual working memory. Simulations showed that the model was able to explain both the existence of severe limits in the number of assemblies (stimuli) that can be held (e.g., Luck and Vogel 1997; Luck and Beach 1998), and the absence of a limit on the size of assemblies, i.e., representing either simple stimuli or complex chunks (e.g., Ericsson and Delaney 1999). We introduced the concept of \u2018chunking fields\u2019 to account for the creation of more complex neural assemblies (e.g., higher order information units or chunks) through previous Hebbian learning (e.g., Hummel and Biederman 1992; Singer 1995). The model can potentially account for different degrees of within-object feature integration (Olson and Jiang 2002) in terms of graded synchrony between neurons coding for features of the same object.\nHere, we will explore an extension of the model of Raffone and Wolters (2001), simulating not only maintenance, but also a selective attention mechanism and a particular characteristic of an integration mechanism. The network architecture presented here to model these functions, is composed of three modules, which we assume to correspond to an IT module, a ventrolateral prefrontal module (vlPFC), and a dorsolateral prefrontal module (dlPFC), respectively (see Fig.\u00a02). We assume that visual features are coded by individual assemblies of neurons in IT, which are \u201cmatched\u201d to one assembly in vlPFC in a recurrent circuit. Moreover, we assume that different subsets of four vlPFC assemblies coding for given visual chunks, are bi-directionally connected to dlPFC assemblies (one for each set of four vlPFC assemblies).\nFig.\u00a02Scheme of the cortical network architecture. In the IT module, 20 neural assemblies code for 20 hypothetical visual features or separate representational elements. The figure shows the case with five four-feature chunks, with synchronizing connections between the assemblies coding the features of the same object (depicted as diamond-like configurations). The IT module also comprises an assembly of globally inhibitory neurons, which are implicitly modeled through inhibitory postsynaptic potentials (IPSPs). The vlPFC module is a set of 20 assemblies of neurons, with a coding structure \u201cmatching\u201d the IT module structure. For simplicity, each IT assembly is recurrently connected with one vlPFC assembly, with a signaling delay of 15\u00a0ms in both directions. The vlPFC assemblies coding for a given chunk are reciprocally connected with a dlPFC assembly, which propagates synchronous firing before Hebbian learning takes place in the IT module\nIn the IT module, strong connections are implemented within and weak connections between assemblies coding different \u2018stimuli\u2019. We also implemented a global inhibition (competition) mechanism between IT assemblies. This circuitry, in which a given assembly is inhibited by the firing of the other assemblies in the IT module, mediates an active desynchronization mechanism.\nStimulus input to the IT module is given by stochastic spike trains from (not explicitly modeled) lower visual areas, during a limited onset-offset period. During stimulus presentation all stimulus specific IT neurons received a high frequent train of spikes as input, which was added to a continuous stochastic low frequency spike input to all IT neurons. The vlPFC module has a coding structure \u201cmatching\u201d the structure of the IT module (see Fig.\u00a02). Each IT assembly is recurrently connected with one vlPFC assembly, with a signaling delay of 15\u00a0ms in both directions. Although we implemented a monosynaptic feedback circuit, in real cortical networks this delayed feedback is likely to be mediated by multisynaptic circuitries of the \u2018synfire\u2019 type, with a stable transmission along multiple diverging\/converging synaptic links (Abeles et al. 1993a, b; see also Villa and Fuster 1992). In vlPFC and dlPFC there are no inhibitory neurons and no inter-assembly connections.\nAlthough stimuli are coded by large assemblies of neurons in real cortical networks, in the present model individual stimuli are coded by single neurons. Our simulations will show that the functional processes we have investigated previously at the level of assemblies of neurons (Raffone and Wolters 2001) also hold at the level of single neurons, thus pointing out the robustness of the observed effects. The use of single neurons instead of assemblies of tens of neurons, is also motivated by the sake of running relatively fast simulations with a smaller time-step in numerical integration of neuronal equations (higher computational accuracy) than in our previous investigations.\nThe model, of course, is very simplified with respect to the complexity of the real cortical networks. Feedback from the vlPFC module does no more than maintaining the oscillatory state of IT assemblies after the stimulus offset. More realistic network versions might include \u201cclosed\u201d reverberatory circuits within prefrontal areas, making prefrontal assemblies independent from the IT assemblies in maintaining the delay activity. This would allow modality shifts by activating other prefrontal assemblies, which in turn would trigger reverberatory circuitries in lower cortical areas, in the absence of actual physical stimulation. Moreover, we used a simplified one-to-one matching between IT and vlPFC assemblies, whereas it seems more realistic to assume that prefrontal assemblies are relatively non-specific, thus being connected to multiple sets of neurons in posterior areas. Instead of the one-to-one matching of assemblies, dlPFC neurons might simply send back activation to all vlPFC neurons, and these in turn to all IT neurons from which they receive activation. However, the present simple architecture is sufficient for demonstrating the functional principles we have specified earlier.\nSimulations\nSimulating maintenance in working memory\nWe first replicated our earlier results (Raffone and Wolters 2001) with stimulus features coded by individual neurons, rather than by sets of neurons, as well as with a higher temporal resolution of the simulations (computational accuracy) and a more realistic global inhibition mechanism. In this simulation the weights of the synchronizing inter-neuron connections in IT were set to zero, so all neurons code for different independent features. As shown in Fig.\u00a03, about four elements are retained in the network after offset of the stimuli, which is in accordance with actual capacity limitations (e.g., Cowan 2001; Luck and Vogel 1997). The frequency of the reverberatory oscillations is approximately 30\u00a0Hz. Figure\u00a03a and b show the retention of three out of four retained items, Fig.\u00a03c and d the retention of four out of four, and Fig.\u00a03e and f the retention of four out of eight items. Note the automatic phase-segregation due to the mutual inhibition desynchronizing effects (see Raffone and Wolters 2001, for a systematic investigation of retention capacity with different network parameters). The missed retention of some items (Figs.\u00a03a, b, e, f) is due to the transient inhibition propagated by the firing of other competing neurons in IT. This inhibition \u201ccounteracts\u201d the feedback input from the matched vlPFC neuron, thus interrupting the reverberatory cycle after stimulus offset. Such an interruption is more likely to occur with multiple reverberatory activities and stronger inhibition, as in that case the probability of the simultaneous arrival of strong inhibition and feedback signals increases.\nFig.\u00a03Limited retention capacity related to between-item segregation. Due to mutual inhibitory activity, the neuronal action potentials (spikes) become spaced in the oscillatory phase, thus allowing a markedly discriminative oscillatory reverberation and retention of the coded items. Often not all reverberations survived the stimulus offset. The panels show the fluctuating membrane potentials of neurons, as well as their spikes by short vertical bars above the membrane potentials. The panels a, c, and e shows the dynamic behavior of IT neurons, and the panels b, d and f the responses of respectively matched vlPFC neurons. In (a, b), three out of the four neurons remained active. In (c, d), all the reverberations remained active, with all the four items being retained. In (e, f), four out of eight neurons maintained their sustained oscillation. Note that the oscillatory reverberations tend to be optimally spaced in the phase-lag, depending on the number of reverberating neurons\nIn this simulation, we did not replicate our previous demonstration (Raffone and Wolters 2001) that the capacity of the reverberatory circuit is independent of the size of the IT representations. As we showed there, larger cell assemblies of connected neurons with fast-signaling positive weights, quickly synchronize and behave as single units. We demonstrated that the maintenance capacity of our model is not a function of the size of a cell assembly, but of the presence and strength of associations between the units of an assembly. This probably mimics the fact that working memory capacity is not a function of the amount of information per se, but of the level of organization or chunking of the material to be maintained (Miller 1956).\nSimulating attentional control in working memory\nGiven the severe limitation of the capacity of working memory, neural mechanisms are necessary to restrict access to it, depending on the behavioral relevance of the information. As shown by Asaad et al. (1998, 2000), prefrontal neurons of monkeys performing a \u2018delayed-matching-to-sample\u2019 task exhibited a higher firing rate when they coded for a rule-defined target-object. Such neurons were suggested to be involved in both the selection and maintenance of behaviorally relevant information.\nTo model a selective attention mechanism by which selection and retention is controlled by working memory cortical circuits, a top-down input to IT neurons has to be supplied by a prefrontal source involved in supervisory control rather than in mere information maintenance. A similar assumption was made in a model for attentional control by Deco and Rolls (2003). Such a mechanism implements the top-down biasing of the competition between neural assemblies in posterior cortex as suggested by Desimone and Duncan (1995). In our model this supervisory control signal could be supplied through the dlPFC and vlPFC modules.\nWe first considered an additive (voltage-independent) input to a subset of four (the bottom four in Fig.\u00a04a) out of eight independent IT neurons all receiving external input. This additional input was modeled in terms of additional spikes with an excitatory postsynaptic potential (EPSP) amplitude equal to the external input signals and a spiking probability equal to 0.04. As can be seen in Fig.\u00a04a, the four items receiving top-down input exhibited a higher (subthreshold) membrane potential before the stimulus onset. This top-down bias was crucial in selecting the items to be retained in terms of reverberatory oscillations. All four biased (and one unbiased) item were maintained after stimulus offset.\nFig.\u00a04Attentional modulation of reverberatory maintenance. Four out of the five maintained activities are of neurons (the first four neurons from below in the panels) the activities of which are selectively enhanced either by additive (voltage-independent) modulation (a), or by voltage-dependent (multiplicative) signals (b). Note that with additive modulation the membrane potentials of the four \u2018focused\u2019 neurons are higher before the stimulus onset (a), whereas the amplification mostly takes place after stimulus onset in the voltage-dependent modulation case (b)\nWe also implemented a voltage-dependent modulatory input to IT neurons. A voltage-dependent gain effect simulates the possible role of NMDA receptors (i.e., receptors that activate a neuron only if a signal arrives at an already depolarized synapse). NMDA-based gain effects are suggested to play a crucial role in cognitive coordination and control (Phillips and Silverstein 2003, see also Raffone et al. 2003), and such gain effects are often modelled as a multiplication of input signals. This multiplicative effect was modeled in terms of the product of the voltage-independent external input (VI) and voltage-dependent top-down signals (VD) with amplitude equal to 0.5\/5, and spiking probability equal to 0.25, according to the following equations Following suggestions of Tononi et al. (1992), the product of equation (2) was set equal to 0 when a membrane potential (ui) was less than a voltage threshold VT (set equal to 0.5). In that case, only a voltage-independent stimulus-related input determines the net input to the neuron, without any amplification.\nAs can be inferred from Fig.\u00a04b, the attentional bias (attentional input for four out of eight items) in the voltage dependent condition is expressed after the stimulus-input onset, in terms of an amplification effect. Also in this simulation all four biased (and one unbiased) items were maintained after stimulus offset. We believe that voltage-dependent input to high-level areas of the visual system may enable an effective top-down control mechanism, since it produces strong amplification effects, but only in the presence of relevant stimulus-input (see also Hirsch and Gilbert 1991; Tononi et al. 1992, about voltage-dependent signaling in the visual system). This multiplicative signaling effect would prevent spurious activation of representations in the visual system in the absence of any bottom-up sensory evidence.\nA series of follow-up simulations showed that the probability of item retention depends on the relative biasing top-down input for a given item. A strong attentional input to one or two items may \u2018narrow\u2019 visual working memory capacity to one or two highly focused elements, due to the higher firing rate (e.g. bursting) of the neurons coding for the focused items.\nMechanisms for integration in working memory\nAny domain-specific account of functions or representations in the brain ultimately implies a binding problem, since the specialized neural representations need to be dynamically bound to enable the creation (and a conscious readout) of coherent and integrated representations of complex events. These binding processes may occur in terms of the selective synchronization of reverberating cell assemblies, as shown in our simulations. In this framework, segregated neural representations may be bound via reciprocal synchronizing connections that originate within the prefrontal cortex (for a discussion of this and other forms of binding, see Murre et al. 2006; see also Engel et al. 2001; Varela et al. 2001).\nIn our model, dlPFC can play a crucial role in what we call \u201cassociative control\u201d, i.e. in selecting meaningful or currently salient conjunctions of otherwise separated representational elements held in a temporarily active state within vlPFC-IT circuits. We have shown that reciprocal fast signaling between a dlPF neuron and its corresponding vlPFC neurons may \u201centrain\u201d all these vlPFC neurons coding for different features or units to fire in a nearly synchronous manner. This synchronous firing is propagated to IT neurons, which in turn fire almost coincidently before the action of the mutual inhibitory signals within the IT module (Fig.\u00a05a, b). We suggest that such a top-down controlled selective synchronization of independent representations is a likely candidate of the process underlying integration in PFC. This mechanism ensures the simultaneous and coherent activation of independent representations that may be widely dispersed over various brain areas. It may cause, for instance, the large-scale task-dependent synchronization in the gamma band, as observed by Rodriguez et al. (1999).\nFig.\u00a05Associative control on vlPFC neuronal activities by dlPFC neurons. The panels a, c, and e shows the dynamic behavior of IT neurons, and the panels b, d and f the responses of respectively matched vlPFC neurons. As shown in panels a, b, inter-neuron synchronization of the four vlPFC neurons is induced by a dlPFC neuron (activity not shown), and is then propagated to the matched IT neurons. As shown in panels c, d, the joint effect of dlPFC feedback and Hebbian associative signals in IT may induce a higher firing rate of neurons in IT and vlPFC. Panels e, f show that a stable synchronization of IT and vlPFC neurons is observed after Hebbian strengthening of IT chunking synapses, with feedback from the dlPFC neuron being switched-off\nThe same mechanism may allow Hebbian learning to occur between meaningful or behaviorally-salient conjunctions of independent elements (see Miltner et al. 1999). In order to account for the retention of integrated units in visual working memory, in previous simulations we defined units by assuming already established chunking fields, i.e. pre-existing associative connections between IT neurons, coding different features of the same unit. A major problem, however, is how \u2018chunking fields\u2019 or integrated representations in long-term memory are formed, i.e. how associative Hebbian learning may take place in the simulated cortical circuits of (visual) working memory. This learning process should operate against the phase-segregation tendency due to mutual desynchronizing inhibition. In the present model, the dlPFC module may perform this temporary synchronization control processes.\nIn a series of simulations, we used the entrainment of vlPFC and IT neurons by a top-down dlPFC signal and combined it with a timing-dependent Hebbian learning rule in IT (simulating timing-dependent plasticity of synapses, see e.g., K\u00f6rding and K\u00f6nig 2000; Song et al. 2000, see Fig.\u00a05c, d). These simulations showed that the synchronous firing in IT, induced by a top-down recurrent signal, causes the weights between the IT neurons to increase, resulting in a \u2018chunked\u2019 representation of initially independent features. As synaptic weights increased according to the timing-dependent learning rule, the synchronization among related IT neurons becomes gradually less dependent on the feedback action of dlPFC neurons on related vlPFC neurons, ultimately leading to an \u201cautomatic\u201d within-chunk integration within the IT module, in the absence of any control feedback from the dlPFC module (Fig.\u00a05e, f).\nControl simulations with the same model and learning parameters, showed that a relatively high number of simultaneous presentations of initially independent elements, may slowly give rise to novel chunks with a variable degree of stability, even in the absence of feedback from dlPFC to vlPFC. This slow chunk-learning process may be related to an implicit learning process, rather than the more rapid process of controlled chunk formation by feedback from dlPFC.\nDiscussion\nMiller and Cohen (2001) concluded that PFC is critical when top-down control of processing is needed for the guidance of behavior by internal states or intentions. In this paper we conjecture that such control is possible by assuming that the PFC is a brain system that is reciprocally connected to posterior parts of the brain and therefore capable of modulating direct perception-action relations. We argued that three interdependent functions are required for such control: maintenance of activation patterns even in the face of distraction, large scale integration of information from different sources, and top-down selective attention. We also showed that these functions can be simulated, at least in principle, with a biologically plausible model assuming recurrent connections and a hierarchical structure of PFC. Importantly, the functions were simulated using a single framework consisting of a hierarchy of recurrently connected modules. This framework proved capable of maintaining a limited number of mutually desynchronized patterns, of controlling selective attention by top-down modulatory signals, and of high level integration by top-down synchronization of the activity of independent patterns.\nOf course, the model suggested here is extremely simplified and it does not capture many of the complexities and intricate details of the real system. For instance, we did not try to incorporate modulatory effects of neurotransmitters and we have not discussed the many possible interactions of the PFC with subcortical systems, like the basal ganglia and amygdala, and the cerebellum. It may also be noted that we discussed, but did not try to incorporate, the possibly important role of a dopamine gating system for maintaining high level integrative activation states (O\u2019Reilly et al. 1999, 2002).\nOne of our aims to develop the model was to try to find out how the structural and functional characteristics of the PFC may explain the central executive component of working memory assumed by Baddeley (2003). In the latest version of this model an additional component, an episodic buffer, was proposed. This component was thought necessary to explain interactions between working memory and LTM, for example to explain how chunking may supplement the capacity for immediate serial recall. A continuous interaction between working memory and LTM is a basic assumption in our model, and we have shown that chunking is a natural consequence of this interaction.\nAccording to Baddeley (2003), the episodic buffer allows information from different systems to be integrated, and it may be regarded as the \u2018storage component of the executive\u2019. Moreover, Baddeley suggested that the buffer is a separate temporary store in which long-term memory information is downloaded in order to be manipulated and used for creating new representations. In our view, the structure of such an episodic buffer would actually be a hierarchy of PFC modules maintaining and integrating information at successively higher levels. Information is not copied and downloaded in PFC, but selected task-relevant perceptual and memory information in posterior cortical areas is kept in an active state by recurrent loops. We assume that at the highest integration levels, presumably located in anterior and dorsolateral areas of PFC, information can be integrated over space and time. At this level, general goals, and plans to achieve them, would be generated. We suggest that this may be the implementation of an episodic buffer and even of a central executive. The creation of short-term and long-term goals at high levels of integration, allows to control behavior by top-down regulation of activation in subordinate modules and eventually by modulating perception-action systems. Current goals and tasks would have to be maintained here in order to control ongoing behavior. More distant goals, however, may be stored in LTM to be retrieved later on.\nWe do not imply that control is always hierarchical. Because the PFC is assumed to be a modulatory system, simple tasks may be executed automatically without PFC involvement. Only controlled processing requires modulation of automatic processes by PFC feedback. Also here, however, control does not need to be completely hierarchical. Depending on the type and complexity of a task, control may be executed by specialized modules at subordinate levels. Automatic processing and controlled processing under the guidance of subordinate levels in PFC, is a requirement to free higher integrative areas to engage in the kind of simulated actions and perceptions, and using anticipated outcomes, that are the contents of conscious thought and planning.\nSo there are things we can do automatically, using previously established associations between perceptual events and effective responses, and there are things we can do in a controlled way, using the possibility of PFC to modulate and thus control perception and action. The possibility of PFC to work off-line, to use and integrate all present and past knowledge for creating virtual worlds and for making plans and carry them out if conditions are appropriate, has generated a tremendously flexible potential for control. The model we propose is vastly insufficient to simulate such cognitive feats that we engage in daily. We believe, however, that the principles for a controlling system we have suggested may be a first step towards a better understanding of what is until now a cognitive Terra Incognita.","keyphrases":["maintenance","integration","working memory","prefrontal cortex","control of attention","synchronization","recurrent networks"],"prmu":["P","P","P","P","P","P","R"]}
{"id":"Eur_Spine_J-2-2-1602195","title":"Preadolescent presentation of a lumbar chordoma: results of vertebrectomy and fibula strut graft reconstruction at 8 years\n","text":"Chordoma is a tumour of notochordal origin which usually involves the sacrum or skull base presenting in adulthood. Chordoma in a mobile spinal segment is infrequent and the authors report an extremely rare presentation of L3 chordoma in a child aged 7 years. Although a benign tumour, mobile segment chordoma is more locally aggressive, more likely to metastasise and has a poorer 5 year survival than sacral and clival lesions. Wide surgical excision and reconstruction is the treatment of choice in vertebral chordoma. This case was treated with staged vertebrectomy and fibular strut graft reconstruction and the results of clinical and radiological follow up at 8 years are presented.\nCase report\nA previously well 7-year old child presented with a 6\u00a0month history of right anterior thigh and knee pain. Examination revealed a marked list to the right, painful restriction of spine movements in all directions, an absent right knee jerk with reduced sensation in the right L3 distribution. There was no motor weakness or sphincter disturbance. Back pain restricted straight leg raise bilaterally to 30\u00b0. No history of weight loss, systemic symptoms and no past medical and family history of relevance. Blood tests were within normal limits. Inflammatory markers were not elevated. Plain radiographs revealed an osteolytic lesion in the posterior body of L3. MRI of the whole spine identified an isolated lesion at L3 with right pedicle involvement and tumour extension anterior to the dural sac at L4 (Fig.\u00a01). Isotope bone scan showed no increased uptake. A staging CT of the chest showed no evidence of metastases.\nFig.\u00a01Pre-operative MRI scan of lumbar spine showing the lesion involving L3 vertebra\nTranspedicular biopsy at L3 was performed and histology revealed fragments of remodelling bone, haemorrhage and a myxoid tissue containing strips and ribbons of cohesive, vacuolated cells. These cells had the appearance of notochordal-type tissue. Immunohistochemistry was positive for Cytokeratin S-100.\nIn view of the histological findings and atypical young age of presentation a radical 360\u00b0 L3 vertebrectomy with pedicle screw stabilization and fusion was planned.\nAt operation the L3 tumour had extended through the lamina and transverse process on the right compromising the right L3 nerve root. There was both cranial and caudal extradural extension of the tumour anterior to the dural sac with tenting from L2 to L4. A wide decompression was performed with resection of the posterior elements and partial posterior vertebrectomy at L3. To ensure that the tumour within the spinal canal was completely excised it was necessary to resect the inferior lamina of L2 and most of the lamina at L4 bilaterally. Hence it was felt appropriate to extend the instrumentation inferiorly to L5.\nPedicle screw instrumentation was performed at L2 and L5 bilaterally. Free fat graft was placed over the dura and nerve roots. Bone graft was placed posterolaterally from L2 to L5 (Fig.\u00a02). An epidural infusion was used postoperatively for pain relief. Initial post operative neurological assessment was normal.\nFig.\u00a02AP and lateral X-rays following first stage of the procedure\nThe following day deterioration in her lower limb neurological status was noted with no active movement or sensation in her lower limbs. That this was not recognised immediately was in part attributed to her epidural and to some difficulty with neurological assessment in a young child with systemic analgesia after major surgery. She was returned to theatre where a large haematoma was found and drained. Although subsequent sensory recovery was good, her motor recovery was slow with grade 1 power in her lower limbs.\nTwo weeks later, once progressive neurological recovery was established, she underwent second stage anterior vertebrectomy of L3 through a retroperitoneal approach. Unilateral L2, L3 and L4 segmental vessels were ligated to expose L3. A complete excision of the remaining L3 and posterior longitudinal ligament was performed in order to adequately decompress the dural sac anteriorly. Three fibular strut autografts from L2 to L4 were used to reconstruct the anterior column. Posterior iliac crest graft was used for further augmentation (Fig.\u00a03).\nFig.\u00a03AP and lateral X-rays following second stage of the procedure showing the fibular strut grafts used\nNeurological recovery continued post-operatively and she had regained normal sensation and grade 5 motor power in both lower limbs by 6\u00a0months. Bladder sphincter dysfunction has persisted, requiring intermittent self catheterisation and anti-cholinergic medication. She was nocturnally continent. Bowel function remained normal throughout.\nRadiographic fusion was evident at 6\u00a0months. Metal work was removed at 12\u00a0months and intra-operative biopsy showed no tumour recurrence (Fig.\u00a04).\nFig.\u00a04AP and lateral X-rays following removal of metal work showing consolidation of the strut grafts\nShe remained under annual follow-up with MRI and plain radiographs. At 8\u00a0years she has no evidence of tumour recurrence. Her neurological status is unchanged. Imaging has shown excellent remodelling of the graft (Figs.\u00a05, 6). She is skeletally matured with no lower limb malalignment and experienced no fibular donor graft site morbidity.\nFig.\u00a05Chronological X-rays showing graft incorporationFig.\u00a06Eight year follow-up MRI Scan\nDiscussion\nChordoma is a primary bone tumour arising from notochord remnants. It is a rare tumour accounting for only 1\u20134% of primary bone tumours [1]. Chordoma is usually a slow growing tumour, often with local recurrence and it may metastasise late in its course [1]. Typically it presents in late middle age and may occur anywhere along the spinal column. Chordoma is found most commonly in the clivus, sacrum and infrequently in the mobile vertebrae [2].\nThe optimum treatment for vertebral chordoma is wide surgical excision. Radiotherapy may provide short term benefit when adequate excision is not possible and in cases of local recurrence [3]. Surgical treatment has become more aggressive in recent years evolving from intralesional debulking to en bloc resection, as documented by Boriani et al., over a 45-year period in their treatment of 21 cases of chordoma located in the spine above the sacrum [4].\nChordomas are considered to be low grade tumours and metastases are infrequent at presentation [1, 5]. The prognostic value of staging is therefore diminished, compared to the value of staging in other bone tumours, because all these tumours are stage IA or IB according to the Musculoskeletal Tumor Society Staging System [6].\nMobile segment chordomas have a 50% survival rate at 5\u00a0years and a 28% survival rate at 10\u00a0years [4, 7]. Sacral chordomas on the other hand are reported as having 86% 5\u00a0year survival [3]. Treatment outcome is significantly influenced by the size and site of chordoma [1, 4]. Chordomas found in the vertebral bodies appear to be more aggressive than those arising in the clivus or the sacrum [3]. Metastases have been reported in 80% of the vertebral body chordomas, as compared to a rate of 43% of all chordomas [3]. However, the survival rate appears to be affected more by local tumour progression than by metastases [1].\nConclusion\nThis case is unusual given the young age at presentation. She is the youngest patient reported in world literature. Despite the poorer published results of mobile segment chordomas, radical surgical excision and reconstruction has resulted in an 8\u00a0year disease free survival. Fibular strut graft reconstruction proved an excellent reconstructive option in the growing skeleton with potential for remodelling. It is likely that the young age has contributed to the near complete neurological recovery.","keyphrases":["chordoma","vertebrectomy","mobile spinal segment","benign tumour","fibular strut graft reconstruction"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_J_Clin_Pharmacol-4-1-2254473","title":"Prior outpatient antibiotic use as predictor for microbial aetiology of community-acquired pneumonia: hospital-based study\n","text":"Objective The causative micro-organism in community-acquired pneumonia (CAP) is often difficult to predict. Different studies have examined chronic morbidity and clinical symptoms as predictors for microbial aetiology of pneumonia. The aim of our study was to assess whether prior outpatient antimicrobial treatment is predictive for determining the microbial aetiology of CAP.\nIntroduction\nCommunity-acquired pneumonia (CAP) remains a major reason for hospital admission and a common cause of death in developed countries [1, 2]. The initial management of patients hospitalised with pneumonia consists mostly of empirical antimicrobial treatment [3, 4]. Determination of the appropriate antimicrobial treatment is essential as inadequate antimicrobial treatment, generally defined as microbial ineffective therapy against the causative pathogen, can negatively influence patient outcome [5]. Because it is not always possible to identify the causative pathogen, especially during the first days following hospitalisation, many studies have focussed on other parameters suggestive of the causative pathogen. The most frequently studied parameters for this purpose are patient characteristics (age and co-morbidities) and clinical signs [6, 7], although nonresponsiveness to prior outpatient antimicrobial treatment has also been suggested as a predictor of the aetiology of pneumonia. However, to date, this latter parameter has not been comprehensively quantified and documented. The aim of the study reported here was to assess whether prior outpatient antimicrobial treatment is a predictor of microbial aetiology in patients admitted to hospital for CAP.\nMethods\nThe study was conducted in the St. Antonius Hospital, a 600-bed teaching hospital in Nieuwegein, The Netherlands.\nPatient population\nThis was a prospective observational study of patients with confirmed pneumonia admitted between October 1, 2004 and August 1, 2006. Pneumonia was defined as a new or progressive infiltrate on a chest X-ray plus at least two of the following criteria: cough, sputum production, temperature >38\u00b0C or <35\u00b0C, ausculatory findings consistent with pneumonia, leucocytosis or leucopenia (>10\u00a0g\/l, <4\u00a0g\/l or >10% rods in leucocyte differentiation), C-reactive protein >3 times the upper reference value for normal. Patients who were immune compromised (systemic steroid use at admission (prednisone equivalent >20\u00a0mg\/daily for more than 3\u00a0days), haematological malignancies and other immunosuppressive therapy) were excluded. The study was approved by the local Medical Ethics Committee, and informed consent was obtained from each patient.\nMicrobial aetiology workup\nAt least two blood cultures were performed, and sputum was taken for Gram-staining and culture and subsequently analysed by Taqman real-time PCR for Mycoplasma pneumoniae, Legionella pneumophila and Chlamydophyla psittaci [8]. Pharyngeal samples were taken for viral culture. Urine was sampled for antigen testing on Streptococcus pneumoniae and L.pneumophila (Binax NOW; Binax, Scarborough, ME) [9, 10]. In addition, serum samples taken on the day of admission and on day 10 were analysed in pairs for detection of a fourfold rise of antibodies to respiratory viruses, Coxiella burnetii, M. pneumoniae, and C. psittaci by complement fixation assay [11]. For each patient, the total workup was completed, and the microbiology department was blinded to data on outpatient antibacterial drug use. When both viruses and bacteria were identified in a patient, the search prevailed for a definite aetiology for the bacteria.\nExposure to antimicrobial therapy\nData on outpatient antimicrobial drug use were acquired through community pharmacy dispensing records that captured all drug exposures the year prior to hospital admission. A patient was considered to be exposed to an antimicrobial drug when a prescription was filled within 14\u00a0days prior to hospitalisation. The name, dosage and amount of antimicrobial drug dispensed were also ascertained. The prescribed antimicrobial drug was classified as appropriate or inappropriate in accordance with current Dutch guidelines on the initial treatment of patients with suspected pneumonia [12, 13].\nCo-morbidity assessment\nIn addition to outpatient antimicrobial drug use, co-morbidities and other relevant patient characteristics were identified to address factors related with the aetiology of CAP. Co-morbidities were defined based on the presence of conditions for which the patient was under active medical supervision or was receiving treatment at the time of hospital admission. The co-morbidities evaluated were pulmonary diseases (chronic obstructive pulmonary disease or treated asthma), congestive heart failure, diabetes (both type I and type II), history of stroke and end-stage renal disease (serum creatinine >150\u00a0\u03bcmol\/l). The patients were also classified according to the Pneumonia Severity Index (PSI) developed by Fine et al. [14]. This index classifies patients into five categories of predicted mortality risk (with the fifth category being that of highest mortality risk). The outpatient use of oral corticosteroids and gastric acid-suppressing drugs was also ascertained.\nStatistical analysis\nThe SPSS statistical package (ver. 12.0.1 for Windows; SPSS, Chicago, IL) was used for the statistical analyses. Continuous data were expressed as the mean \u00b1 SD or as the median (interquartile range) where appropriate. To study the association between prior outpatient antimicrobial treatment and the aetiology of pneumonia, we applied multivariate logistic regression analyses. The analyses were conducted for overall aetiology and relevant pathogens separately. All baseline characteristics were considered potential predictors. Potential predictors were included in the multivariate model when they were retained after backward stepwise elimination. Significance was set at a p value < 0.05. The model\u2019s performance (goodness-of-fit and discriminative ability) was tested by performing the Hosmer and Lemeshow test and calculating the area under the receiver operator characteristic (ROC) curve.\nResults\nIn total, 201 patients with pneumonia were included in the study. The mean age of the patients was 63\u2009\u00b117\u00a0years, and 124 patients were male. Three patients (1%) were admitted from a nursing home. Pneumonia severity as well as co-morbid illnesses are summarised in Table\u00a01. The overall median duration of the hospital stay was 10\u00a0days [7 \u201314], and 21 patients were admitted to the intensive care ward. During the hospital stay, ten patients died, all due to pneumonia. The overall 28-day mortality rate was 5%. Forty-seven patients (23%) had received antimicrobial treatment in the 14-day time-window prior to hospital admission. The antimicrobial drugs dispensed to these patients are summarised in Table\u00a02.\nTable\u00a01Demographics, co-morbidities and pneumonia severity index of 201 patients with community-acquired pneumonia (CAP)Characteristicn (%)Age (Years)\u00a0\u00a0<6074 (37)\u00a0\u00a060\u20136939 (19)\u00a0\u00a070\u20137950 (25)\u00a0\u00a0>8038 (19)Gender\u00a0\u00a0Male124 (62)\u00a0\u00a0Female77 (38)Co-morbidities\u00a0\u00a0Pulmonary diseases71 (35)\u00a0\u00a0Heart failure18 (9)\u00a0\u00a0Diabetes35 (17)\u00a0\u00a0History of stroke17 (9)\u00a0\u00a0End-stage renal disease10 (5)\u00a0\u00a0Nursing home resident3 (1)Co-medication\u00a0\u00a0Oral corticosteroids58 (29)\u00a0\u00a0Gastric acid suppressing drugs61 (30)Fine score at admissiona\u00a0\u00a0I30 (15)\u00a0\u00a0II34 (17)\u00a0\u00a0III53 (26)\u00a0\u00a0IV56 (28)\u00a0\u00a0V28 (14)aFine et al. [14]Table\u00a02Outpatient antibiotics utilization profile prior to hospitalisation for CAPType of antimicrobial drugNumber of users (%)aAppropriatebClavulanic acid18 (38)YesAmoxycillin12 (26)YesDoxycycline7 (15)YesClarithromycin5 (11)YesCo-trimoxazole4 (8)NoCiprofloxacin2 (4)NoNorfloxacin1 (2)NoAzithromycin1 (2)YesaTotal percentage exceeds 100% because some patients (n\u2009=\u20093) had two prescriptionsbBased on current Dutch guidelines NVALT and SWAB [12, 13]\nThe majority of the patients (79%) had their prescription filled within 4\u00a0days prior to hospital admission, and 85% of the prescribed antimicrobial drugs complied with current Dutch guidelines [12, 13]. A microbial aetiology could be determined in 128 (64%) of the patients. Table\u00a03 shows the results of different tests used to determine the aetiological diagnosis of CAP.\nTable\u00a03Results of of different tests used to determine the aetiology of CAP\u00a0Sputum cultureSputum PCRAntigen testingBlood cultureSerologyViral cultureNumber of samples1487818318213088Number of positive samples781436193814Percentage of positive samples531820102916Streptococcus pneumoniae33\u20133017\u2013\u2013Gram-negative strain\u00a0\u00a0Heamophilus influenzae19\u2013\u20130\u2013\u2013\u00a0\u00a0Other10\u2013\u20131\u2013\u2013Atypical\u00a0\u00a0Mycoplasma pneumoniae\u20137\u2013\u20138\u2013\u00a0\u00a0Legionella spp.156\u20137\u2013\u00a0\u00a0Other\u20132\u2013\u20131\u2013Viral\u2013\u2013\u2013\u20132214Other\u00a0\u00a0S. aureus6\u2013\u20131\u2013\u2013\u00a0\u00a0Gram-positive other2\u2013\u20130\u2013\u2013\nFewer causative pathogens were found in the population hospitalised following prior outpatient antimicrobial treatment than in patients without prior antimicrobial treatment [57 vs. 66%; crude odds ratio (OR) 0.71, 95% confidence interval (CI) 0.36\u20131.38]. In patients with prior beta-lactam treatment, aetiology in the group comprising atypical bacterial pathogens was more probable (eight of 29 cases, 28%; crude OR 4.66, 95% CI 1.73\u201312.56). The aetiology of S. pneumoniae was less prevalent in patients with prior beta-lactam antimicrobial treatment (four of 29 cases, 14%; crude OR 0.33, 95% CI 0.11\u20130.99). In multivariate analyses, these associations remained significant (OR 3.51, 95% CI 1.25\u20139.99 and OR 0.30, 95% CI 0.10\u20130.90, respectively) (Table\u00a04). Additional significant predictors included in these models were heart failure, pulmonary comorbidity, pneumonia severity index and the use of gastric acid suppressing drugs. The goodness-of-fit of both multivariate models was good, with a p value of 0.899 (Hosmer and Lemeshow test) for the model predicting pneumococcal pneumonia and a p value of 0.995 for the model predicting pneumonia of an atypical aetiology. The corresponding areas under the ROC curve were 0.62 and 0.67, respectively.\nTable\u00a04Odds ratios (OR) for aetiology and prior outpatient beta-lactam treatment in patients admitted to hospital for CAPAetiologyPrior outpatient beta-lactam treatment (n = no. of patients)OR (95% CI)Yes (%)No (%)Total no. of samples29 (100)172 (100)Univariate\u00a0\u00a0Pneumococcal4 (14)56 (33)0.33 (0.10\u20130.99)\u00a0\u00a0Atypical8 (28)13 (8)4.66 (1.72\u201312.56)\u00a0\u00a0Viral2 (7)14 (8)0.84 (0.18\u20133.89)\u00a0\u00a0Gram-negative strains2 (7)21 (12)0.53 (0.12\u20132.40)\u00a0\u00a0Other1 (3)7 (4)0.84 (0.10\u20137.11)\u00a0\u00a0Unidentified12 (41)61 (36)1.28 (0.58\u20132.87)Multivariate\u00a0\u00a0Pneumococcal\u2013\u20130.30 (0.10\u20130.90)a\u00a0\u00a0Atypical\u2013\u20133.51 (1.25\u20139.99)bOR, Odds ratio; CI, confidence IntervalaAdjusted for heart failure and use of gastric acid suppressing drugsbAdjusted for pulmonary diseases and pneumonia severity index\nIn patients aged <60\u00a0years without co-morbidities, the aetiology of atypical bacterial pathogens was more prevalent (OR 4.64, 95% CI 1.72\u201312.56). Pulmonary co-morbidity was associated with the finding of S. pneumoniae and H. influenzae as causative pathogens (OR 1.87, 95% CI 1.00\u20133.47; OR 3.72, 95% CI 1.20\u201311.57, respectively). Patients who received macrolides prior to hospitalisation had an increased probability of viral pneumonia (two of five cases, 40%) (crude OR 8.67, 95% CI 1.34\u201356.23).\nDiscussion\nThe results of our study show that among patients admitted for CAP, a threefold decreased chance of having a S. pneumoniae infection and a threefold increased probability of having pneumonia of atypical aetiology were associated with the patient having received an initial beta-lactam treatment as an outpatient. These findings indicate that information on prior outpatient antimicrobial therapy has a predictive value in the diagnostic workup aimed at identifying the causative pathogen and planning the corresponding treatment in patients with pneumonia.\nThe initial management of patients hospitalised with pneumonia has been under constant study in different settings during the past decades. The choice of antimicrobial treatment, time to first antimicrobial drug administration and route of administration have all appeared to be relevant factors associated with the outcome of pneumonia [15\u201317]. A knowledge of the predominant microbial patterns in CAP is therefore essential when choosing an essential empirical antimicrobial treatment. Previous studies have found that S. pneumoniae, H. influenzae, Influenza virus A and B, Legionella spp. and C. pneumoniae are the most frequent pathogens in CAP [13], which is in accordance with our results. Because S. pneumoniae is the most frequently appearing pathogen, the administration of beta-lactam antibiotics is the initial empirical antimicrobial treatment of choice in the treatment guidelines on CAP [3, 12, 13]. Beta-lactam antibiotics, however, do not cover Legionella spp., C. pneumoniae and M. pneumoniae, the so-called atypical pathogens. Therefore, patients with pneumonia of atypical aetiology who are treated with beta-lactam antibiotics as an outpatient will probably not respond to treatment, with the possible consequence being a deterioration of the situation and subsequent hospital admission. Our finding of an increased prevalence of atypical pathogens in patients with prior outpatient beta-lactam treatment supports such an explanation, but also confirms what has already been suggested in the different guidelines for the management of community-acquired pneumonia in adults [13, 18]. These guidelines state that following the failure of the initial empirical treatment with beta-lactam antibiotics, the microbiological examination should be reassessed with a view to excluding the less common pathogens, such as atypical pathogens, and that antimicrobial treatment covering atypical pathogens should be considered. Our study supports the choice of antimicrobial treatment covering atypical pathogens (e.g. macrolides) for all patients with CAP who are admitted to hospital after prior treatment with beta-lactam antibiotics. However, whether this protocol will result in improved clinical outcome should be subject to additional study.\nThe observed reduction in the frequency of S. pneumoniae in patients who received prior outpatient antimicrobial treatment could also be due to a failure to detect the organisms in cultures. This could mask S. pneumoniae as the causative pathogen. However, we believe that this explanation is less plausible, especially since we also used antigen testing to identify the causative pathogen [19]. In addition, such a mechanism can not explain the finding of an increased probability of pneumonia caused by atypical pathogens. To the best of our knowledge, our study is the first to specifically document and quantify the failure of initial outpatient antibiotic treatment as a predictor of the microbial aetiology of CAP.\nThis study was conducted in a single teaching hospital in The Netherlands, but we believe that the conclusions drawn will apply to other settings. First, the percentage of identified aetiology (64% in this study) is in agreement with that of other studies using similar microbiological techniques [20\u201322]. Second, our patient characteristics comply to a great extent with a previous nationwide study on prior outpatient antibacterial therapy as a prognostic factor for mortality in patients hospitalised for pneumonia [23]. In that large database study, the percentage of patients hospitalised after initial outpatient antimicrobial treatment was almost identical to that observed in our study (27 vs. 23%, respectively). In addition, age distribution, co-morbidities and the antibiotic utilisation profile of the outpatients were very similar as were the median duration of hospital stay and in-hospital mortality. Unfortunately, due to limited numbers, we were unable to study an association between prior outpatient antimicrobial treatment and mortality in our study. A very reassuring finding was that 85% of all outpatient antibiotic prescriptions complied with national guidelines on the initial treatment of adults with suspected pneumonia. This reduces the possibility that the findings, rather than being associated with antibiotics, might reflect the diagnostic acumen of the physicians who saw the patients in primary care. However, we cannot rule out the possibility that typical signs of infection with atypical pathogens may have been missed by primary care physicians.\nAs well as finding a relation between prior antimicrobial treatment and aetiology, we also found an association between aetiology and age and pulmonary co-morbidity. Patients aged <60\u00a0years without co-morbidities were more likely to have an aetiology comprising viral or atypical bacterial pathogens, and pulmonary co-morbidity was independently associated with S. pneumoniae and H. influenzae as causative pathogens. These findings confirm the results of previous studies on the impact of age and co-morbidity on the microbial aetiology of CAP [6]. A limitation of the present study, however, is that we were not able to adjust for smoking habits and alcohol intake of the patients. Previous studies on determinants for pneumonia aetiology found that these factors are significant predictors of pneumococcal infection [6, 7]. On the other hand, we do not expect prior antimicrobial therapy and smoking and alcohol intake to coincide in such a way that this would result in a null effect when all the information is available.\nIn conclusion, among patients admitted for pneumonia, whether or not a patient has received prior antimicrobial therapy as an outpatient provides relevant information in the diagnostic workup, in particular in terms of identifying the causative pathogen and planning the initial treatment at the time of hospital admission. This finding supports a further strengthening of the continuity of care at the interface between the extramural and hospitalised settings.","keyphrases":["antibiotics","aetiology","pneumonia","diagnosis","outcome assessment"],"prmu":["P","P","P","P","R"]}
{"id":"Histochem_Cell_Biol-4-1-2248610","title":"Retrograde traffic in the biosynthetic-secretory route\n","text":"In the biosynthetic-secretory route from the rough endoplasmic reticulum, across the pre-Golgi intermediate compartments, the Golgi apparatus stacks, trans Golgi network, and post-Golgi organelles, anterograde transport is accompanied and counterbalanced by retrograde traffic of both membranes and contents. In the physiologic dynamics of cells, retrograde flow is necessary for retrieval of molecules that escaped from their compartments of function, for keeping the compartments\u2019 balances, and maintenance of the functional integrities of organelles and compartments along the secretory route, for repeated use of molecules, and molecule repair. Internalized molecules may be transported in retrograde direction along certain sections of the secretory route, and compartments and machineries of the secretory pathway may be misused by toxins. An important example is the toxin of Shigella dysenteriae, which has been shown to travel from the cell surface across endosomes, and the Golgi apparatus en route to the endoplasmic reticulum, and the cytosol, where it exerts its deleterious effects. Most importantly in medical research, knowledge about the retrograde cellular pathways is increasingly being utilized for the development of strategies for targeted delivery of drugs to the interior of cells. Multiple details about the molecular transport machineries involved in retrograde traffic are known; a high number of the molecular constituents have been characterized, and the complicated fine structural architectures of the compartments involved become more and more visible. However, multiple contradictions exist, and already established traffic models again are in question by contradictory results obtained with diverse cell systems, and\/or different techniques. Additional problems arise by the fact that the conditions used in the experimental protocols frequently do not reflect the physiologic situations of the cells. Regular and pathologic situations often are intermingled, and experimental treatments by themselves change cell organizations. This review addresses physiologic and pathologic situations, tries to correlate results obtained by different cell biologic techniques, and asks questions, which may be the basis and starting point for further investigations.\nIntroduction\nThe biosynthetic-secretory route is traveled by newly synthesized luminal and membrane proteins and glycoproteins from their sites of synthesis at bound ribosomes of the rough endoplasmic reticulum (RER) to their final destinations inside and outside the cells (for review e.g. Farquhar and Hauri 1997; Mellman and Warren 2000). It involves complex and highly dynamic organelles, such as the Golgi apparatus, pre-Golgi intermediates, and post-Golgi organelles, which all have central roles in transport regulation, sorting, and targeting. The route that leads out of the RER at special ER-export sites, involves complicated ER-Golgi intermediate compartments, enters the Golgi apparatus stacks at their cis side, leads across the stacks of Golgi cisternae, where the traversing molecules are subjected to major modifications, and continues at the trans Golgi side. The trans Golgi network (TGN) is a central place of molecule sorting, and packaging of cargo molecules into vehicles for transport to the final intra or extracellular destinations, where the molecules eventually exert their specific functions, such as organelles of the lysosomal system, diverse domains of the plasma membrane, and the extracellular space. At all levels of the biosynthetic-secretory route, anterograde transport of membranes and cargo is counterbalanced by retrograde traffic (for review Sannerud et al. 2003). This is necessary for several reasons, which include the retrieval of molecules that have escaped from the sites of their specific functions, for membrane balance and the maintenance of the functional integrities of organelles and compartments along the secretory route and repeated use of molecules, and molecule repair. Major antero-retrograde traffic cycles are located at the pre-Golgi junction between the ER, the intermediate compartment, and the Golgi apparatus, as well as at the post-Golgi junctions between the TGN, early and late endosomes, and plasma membrane. Within the Golgi apparatus stacks, anterograde transport is assumed to be counterbalanced by retrograde traffic as well. Internalized molecules have been shown to travel along retrograde biosynthetic pathways (e.g. Gonatas et al. 1983; Pavelka et al. 1998; Vetterlein et al. 2002; Volz et al. 1995), and physiologic retrograde routes are misused by harmful substances to reach the sites of their specific toxic effects (e.g. Pelham et al. 1992; Rapak et al. 1997; Sandvig et al. 1991, 1992, 2002; for review Sandvig and van Deurs 2002, 2005). Since the early report in 1992 by Kirsten Sandvig and colleagues, who for the first time showed that along such pathways toxins could be transported en route from the plasma membrane into early compartments of the biosynthetic-secretory system, such retrograde trails of toxins are at the center of cell biologic and medical research.\nFor both the cell physiologic retrograde routes, and misused pathways, knowledge about molecular machineries, and insights into regulatory mechanisms are increasing rapidly (e.g. Gokool et al. 2007; Mari et al. 2008; Popoff et al. 2007; W\u00e4lchli et al. 2008; Yamane et al. 2007; for review Bonifacino and Rojas 2006; Sannerud et al. 2003). In immuno and fine structural analyses, multiple fine details of the compartments, structures and complex architectures involved have been visualized (e.g. Mari et al. 2008; Marsh 2005; Mogelsvang and Howell 2006; Mogelsvang et al. 2004; Pavelka 2007; Pavelka et al. 1998; Vetterlein et al. 2002, 2003). However, there still exists a considerable lack in understanding the mechanisms involved and uncertainties in the interpretations of individual results obtained by molecular biologic and genetic investigations on one hand, and morphologic findings on the other hand. Well known functional processes yet cannot be exactly attributed to concrete defined compartments, and vice versa, the functional implications of multiple structures and architectures need to be clarified. There is a strong need to bring together the biochemical, molecular biological, genetic, morphologic, immuno-cytochemical, and fine structural results.\nRetrograde traffic in cell physiology\nAnterograde transport in the secretory route is accompanied and counterbalanced by retrograde traffic at each section of the secretory route, including pre- and post-Golgi areas, and presumably at the level of the stacks of Golgi cisternae as well. The diverse retrograde routes and cycles cannot be seen separately. There exist similarities in the mechanisms and machineries, close relationships, and mutual influences.\nRetrograde Golgi-to-ER traffic and pre-Golgi circuits\nBy retrograde Golgi apparatus-to-ER traffic, ER-resident luminal and membrane proteins are retrieved (Munro and Pelham 1987; Semenza et al. 1990), and membrane proteins, including the cargo receptor ERGIC-53 (Appenzeller et al. 1999; Schweizer et al. 1988), and Golgi-resident proteins, such as glycosyltransferases, recycle (Lee et al. 2004; Lippincott-Schwartz et al. 1990; Storrie et al. 1998; Storrie 2005). It is evident that multiple retrograde traffic routes work in parallel in order to retrieve molecules to the ER, and cycle proteins and lipids between ER and Golgi apparatus. The first retrograde Golgi-to-ER pathway described was the KDEL-receptor mediated transport of luminal ER proteins (Lewis and Pelham 1990; Munro and Pelham 1987; Semenza et al. 1990), which following the export out of the ER together with the flow of multiple other proteins destined for transport to other destinations have to be retrieved to their sites of functions in the ER. This traffic, as well as the transport of type-I transmembrane proteins bearing the double-lysin motif signal (Letourneur et al. 1994) is mediated by COPI-coated vesicles (Nickel and Wieland 2002). The retrieval of escaped ER-proteins takes place most efficiently from the ER-Golgi intermediate compartment (ERGIC) but can occur from successive Golgi apparatus compartments up to the TGN as well. Luminal acidification, and calcium concentration in the lumina of the respective compartments have been suggested to be regulatory factors (reviewed in Sannerud et al. 2003).\nFurthermore, COPI-independent transport machineries act in Golgi-to-ER retrograde traffic (Johannes and Goud 2000). Such routes are known to involve Rab6 GTPases (Girod et al. 1999; Storrie 2005; White et al. 1999), key regulators of intracellular membrane traffic (Hammer and Wu 2002; Stenmark and Olkkonen 2001; Zerial and McBride 2001). Three different isoforms are known, Rab6A, Rab6A\u2032, an alternatively spliced variant of Rab6A (Echard et al. 2000), and the brain-specific Rab6B (Wanschers et al. 2007). Recently, it has been shown that Rab6A and Rab6A\u2032 perform different non-overlapping functions in cells, and the Rab6A\u2032 isoform is shown mainly regulating the COPI-independent retrograde pathway to the ER (Del Nery et al. 2006). Rab6A GTPases interact with a subunit of the dynein\u2013dynactin complex, and transport compartments move between Golgi apparatus and ER bidirectionally along the microtubules (Matanis et al. 2002; Short et al. 2002). COPI-independent Rab6A-dependent retrograde pathways are used by Golgi apparatus-resident proteins, such as glycosyltransferases, which cycle continuously between the Golgi apparatus and ER (Martinez et al. 1997; Rhee et al. 2005; Storrie 2005; Storrie et al. 1998).\nRetrograde traffic within the Golgi apparatus\nIn the anterograde biosynthetic-secretory traffic, newly synthesized molecules arriving from the ER and ERGIC are taken up into the Golgi apparatus, visit cisternae of the stacks to be modified in well known subsequent steps, and are sorted to different further destinations at the trans side and TGN. Since decades, it is well established that the stacks of Golgi cisternae are subcompartmentalized into separate functional spaces, in each of which defined sets of enzymes are active, and collaborate in performing specific modifications of the molecules that visit the respective cisternae, e.g. perform changes of the sugar chains of the glycoconjugates, such as secretory and plasma membrane glycoproteins, enzymes of the lysosomal system, and lysosomal membrane constituents (Berger 1985; Farquhar and Palade 1998; Glick 2000; Pavelka 1987; Puthenveedu and Linstedt 2005; Rambourg and Clermont 1997; Roth 1997; Storrie 2005). It is assumed that the cisternae of the Golgi apparatus stacks are visited by the molecules to be modified, and there occurs a flow of membranes and contents across the stacks. However, at present, it is not clear, how in the dynamics of the flow the Golgi apparatus subcompartmentalization, which ensures effective glycosylation and other processing in the secretory pathway, is maintained. Conflicting results make interpretations complicated but there exist indications that retrograde transport of membrane constituents, such as the subcompartment-specific glycosyltransferases, is involved. The question how, and even whether, newly synthesized membrane and cargo proteins traverse the stacked Golgi cisternae, is a major point of debate. Several models are discussed, suggesting either an anterograde transport via vesicles, or anterograde flow by progression of the cisternae themselves, or by tubular connections between the cisternae, or by a signal-mediated temporary opening of channels connecting adjacent cisternae (Kartberg et al. 2005; Malhotra and Mayor 2006; Marsh and Howell 2002; Marsh et al. 2004; Mironov et al. 2005; Pelham and Rothman 2000; Puthenveedu and Linstedt 2005; Rodriguez-Boulan and M\u00fcsch 2005; Sallese et al. 2006; Storrie 2005, Trucco et al. 2004). Contradictory results also concern the retrograde traffic, which at least in the \u201ccisternae progression model\u201d is required for maintenance of the specific subcompartments. COPI-coated vesicles, also being involved in the retrograde Golgi-to-ER traffic (see the previous chapter), have been proposed as the candidates for retrograde intra-Golgi traffic but the results are conflicting (summerized by Rabouille and Klumperman 2005), and alternative mechanisms are considered as well, such as traffic across tubular connections between the Golgi cisternae (Marsh et al. 2004; Trucco et al. 2004). The retrograde transport and the cycling of Golgi-resident glycosyltransferases between Golgi apparatus and ER have been shown to be functionally connected with the maintenance of the structure of the Golgi apparatus (Starr et al. 2007; Storrie 2005). Recently, it has been shown that the Rab6-binding protein TMF (TATA element modulatory factor), which is involved in the Rab6-dependent retrograde transport processes both from endosomes to the Golgi apparatus, and from the Golgi apparatus to the ER, via its cytoplasmic region is implicated in the retention of N-acetylgalactosaminyltransferase-2 (Yamane et al. 2007).\nRetrograde plasma membrane-to-Golgi traffic and post-Golgi circuits\nIn part comparable with the pre-Golgi retrograde routes, post-Golgi retrograde traffic on one hand represents a backward flow that counterbalances forward flow by anterograde traffic to the final destinations of newly synthesized molecules to the plasma membrane, extracellular space, secretory granules, endosomes, or lysosomes. Post-Golgi retrograde trafficking concerns TGN-resident proteins, such as TGN38, and receptors involved in the sorting of newly synthesized molecules of the trans Golgi apparatus and TGN to their specific sites of action. This transport is bidirectional; the receptors recycle to the TGN to be reused in further rounds, possibly also being modified or repaired (Bonifacino and Rojas 2006; Rohn et al. 2000; Snyder and Rogers 1985; Volz et al. 1995). Best-studied examples are the cation-independent and the cation-dependent mannose-6-phosphate receptors (CI- and CD-MPR; Ghosh et al. 2003). Other recycling proteins are the multi-ligand receptor sortilin (Mari et al. 2008), TGN38 (Banting and Ponnambalam 1997; Ghosh et al. 1998; Stanley and Howell 1993), GPP130 and GP73 (Puri et al. 2002), processing enzymes, such as the transmembrane endoproteases furin and carboxypeptidase D (Molloy et al. 1999; Varlamov and Fricker 1998), and SNAREs (soluble N-maleimide-sensitive fusion protein \/NFS\/ attachment protein receptor; Hettema et al. 2003; Hong 2005). At least, two independent retrograde routes from early endosomes to the TGN exist involving specific components of the Rab and SNARE machineries. One leads to the TGN via late endosomes (Barbero et al. 2002; Carrol et al. 2001; Lombardi et al. 1993; Mallet and Maxfiled 1999); the other one is a direct route from early and\/or recycling endosomes to the TGN, thus bypassing late endosomes (Mallard et al. 1998, 2002). During the past years, insights into the regulatory mechanisms of retrograde traffic from endosomes to the TGN considerably increased. Multiple constituents of the molecular transport machineries have been characterized and their roles specified; these include Rab9 and TIP47 (tail-interacting protein 47 kDa; Diaz et al. 1997), PACS1 (phosphofurin acidic cluster sorting protein 1) and AP-1 (Crump et al. 2001; Meyer et al. 2000), EpsinR (Saint-Pol et al. 2004), the t-SNAREs syntaxin 16 and 5 (Amessou et al. 2007), and constituents of the retromer complex with the two subcomplexes, a membrane-bound coat that consists of the phosphinositide binding proteins Sorting nexin 1 and possibly Sorting nexin 2, and the cargo-binding proteins Vps26, Vps29, and Vps35 (Arighi et al. 2004; Gokool et al. 2007; Popoff et al. 2007; Restrepo et al. 2007; Rojas et al 2007; Seaman et al. 1998; Seaman 2004, 2005; for review Bonifacino and Rojas 2006). Clathrin and the retromer complex are suggested to function in consecutive retrograde sorting steps on early endosomes (Popoff et al. 2007).\nThere exist several different carrier compartments for retrograde endosome-to-TGN traffic that can form at the same vacuolar early endosome (Mari et al. 2008): tubular sorting endosomes (Peden et al. 2004), and tubular endosomal networks (Bonifacino and Rojas 2006), and the endosome-to-TGN carriers (Mari et al. 2008). The tubular endosomal network (TEN) is connected to vacuolar early endosomes, which exhibit bilayered coats composed of clathrin and Hrs (hepatocyte-growth-factor-regulated tyrosine kinase substrate) thought to contain the ESCRT machinery that targets proteins to the intraluminal vesicles, thus being sorted to the multivesicular-late endosomal-lysosomal route (Hurley and Emr 2006; Sachse et al. 2002). The vacuolar endosomal part is suggested to receive endocytic cargo from the plasma membrane on one hand, and biosynthetic cargo, e.g. lysosomal enzymes, from the TGN on the other hand; it progressively becomes acidic, leading to the release of the cargo from the receptors. It is proposed that the vacuolar endosomal region matures into late endosomes, whereas from the TEN-region, cargos are sorted to different destination, such as to the TGN and Golgi apparatus along the retrograde route but also to different regions of the plasma membrane for recycling and transcytosis, and to specialized storage compartments, such as melanosomes. The TEN is equipped with different transport machineries including the retromer. It is considered that the pericentriolar endocytic recycling compartment (Ghosh et al. 1998; Maxfield and McGraw 2004; Ullrich et al. 1996) could be a specialized subdomain of TEN. With respect to its central sorting role, TEN is proposed to represent a \u201cmirror image\u201d of the TGN (Bonifacino and Rojas 2006). A possibly comparable endocytic trans-Golgi network has been shown to develop after internalization of wheat germ agglutinin (WGA; Pavelka et al. 1998; Vetterlein et al. 2002; Figs.\u00a01, 2). Recently, a novel class of carriers, the endosome-to-TGN carriers (ETCs), has been characterized (Mari et al. 2008). These carriers transport mannose-6-phosphate receptors and sortilin; they are dependent on the presence of sorting nexin 1 (SNX1), and show unique structures appearing as non-branched tubules and vesicles, clearly different from the tubular sorting endosomes (Peden et al. 2004) and tubular networks (Bonifacino and Rojas 2006).\nFig.\u00a01The Golgi apparatus reorganizes during endocytosis of WGA within a period of 30\u00a0min. ainsert: The insert shows WGA-HRP reaction products concentrated in a clathrin-coated pit, and a coated vesicle (arrows), as well as lining the membrane of a small uncoated vesicle (arrowhead). \u00d735,000. a Small endosomes (arrowheads), and a large vacuolar endosome (arrow) are apparent in the cytocentre (c), and close to a small Golgi apparatus stack (Golgi). A distinct TGN is not visible. \u00d730,000. b Globular early endosomes are accumulated close to the trans side of Golgi apparatus stacks (Golgi); some of the endosomes are covered with clathrin coats (arrowheads); partly, they exhibit WGA reaction products attached to the membranes, partly contained within the lumina. Some of endosomes appear to contact each other, and fine bridges are visible (short arrows). WGA reactions are apparent within the cisternae of a small Golgi apparatus stack (large arrow). \u00d731.500. c A network, an endocytic trans Golgi network (endocytic TGN), is apparent consisting of interconnected globular pieces (arrowheads) that resemble the earlier globular endosomes. Again here, the WGA reaction products are either attached to the membranes, or fill the lumina. Parts of this endocytic TGN are attached to stacks of Golgi cisternae at their trans sides (arrows), and are associated with trans Golgi ER. At the cis side, ER-Golgi-intermediate compartments (ERGIC) are visible. \u00d728,000. d Endocytic TGN consisting of interconnected globular pieces (arrowheads) and filled with WGA reaction products are partly attached to Golgi apparatus stacks (arrows), and associated with trans Golgi ER. \u00d732,000Fig.\u00a02Portions of the endocytic TGN are integrated in the Golgi apparatus stacks a The WGA-labeled endocytic TGN (long arrows) represents an integrated part of this Golgi apparatus stack, and is closely associated to the transmost Golgi cisterna at one side, and with trans Golgi ER at the opposite side. One large vacuolar part (V) of the endocytic TGN is visible; some of the globular pieces of the endocytic TGN are covered with clathrin coats (short arrow). \u00d735,000. b The endocytic TGN (arrows) consists of globular pieces, and a large vacuole part (V), which here is attached to the Golgi apparatus stack. \u00d744,000Fig.\u00a03a\u2013d This 3D-model of a Golgi apparatus stack containing integrated endocytic TGN, has been formed according to the data of an electron tomography tilting series. The model is shown from different sides. The endocytic TGN (green) consists of a free portion (long arrows), and another portion integrated in the stack (short arrows), and is closely associated with trans Golgi ER (red). In both the free and the Golgi-associated portions, globular elements are visible. Stacked Golgi cisternae are shown in yellow. In c, the stacked Golgi cisternae are removed. Spikes mark clathrin coats\nRetrograde traffic of internalized molecules\nThe plasma membrane-endosome-TGN pathway used by plasma membrane proteins for reentry into the biosynthetic system for reuse, or modification and repair (Snyder and Rogers 1985; Volz et al. 1995; reviewed in Bonifacino and Rojas 2006) also opens the secretory pathway for entry of extracellular ligands, including harmful substances, such as bacterial and plant toxins (for review Sandvig and van Deurs 2005), and provides tracks for drug delivery to the interior of cells (for review Tarrago-Trani and Storrie 2007).\nRetrograde routes of lectins and toxins\nSince more than two decades, it is known that lectins, and bacterial and plant toxins, such as the Shiga and Cholera toxins, the Pseudomonas exotoxin A, and ricin, utilize the retrograde routes within the cells to travel to the TGN and Golgi apparatus, ER, and cytosol (e.g. Gonatas et al. 1983, Sandvig and Brown 1987; van Deurs et al. 1986, 1987). Sandvig et al. (1992) were the first, who showed that Shiga toxin is transported from the cell surface en route to the endoplasmic reticulum. Shiga toxin consists of an enzymatically active A-subunit that is noncovalently linked to a pentamer of B-chains. The latter binds to the glycosphingolipid Gb3, and mediates the toxin\u2019s transport. Following internalization, the toxin is transported to early endosomes, sorted to the TGN and Golgi apparatus, and further transported to the ER, from where after cleavage the A-subunit is retrotranslocated to the cytosol, the site where it exerts its toxic action by inactivation of ribosomes, and inhibition of protein synthesis (Johannes and Goud 1998, 2000; Sandvig and van Deurs 1994, 1996, 2002, 2005). It is well established that different uptake mechanisms are used, and there exists not only one route to the endoplasmic reticulum but different retrograde pathways may be traveled by different toxins, and trafficking of one class of toxin is not limited to one route, e.g. Pseudomonas exotoxin A has been shown to travel to the endoplasmic reticulum along multiple pathways (Smith et al. 2006). There also appears to be fundamental differences between the endosomal sorting into the retrograde pathway to the TGN of Shiga and Cholera toxins (Bujny et al. 2007; Chinnapen et al. 2007; Feng et al. 2004; Massol et al. 2004; Torgersen et al. 2001). Multiple details concerning different uptake mechanisms, and retrograde traffic of Shiga toxin and ricin have been elucidated during the past years (e.g. Amessou et al. 2007; Garred et al. 1995; Grimmer et al. 2005, 2006; Johannes et al. 1997; Lauvrak et al. 2004, 2006; Mallard et al. 1998; Rapak et al. 1997; R\u00f6mer et al. 2007; Skanland et al. 2007; Slominska-Wojewodzka et al. 2006; Tai et al. 2004; Torgersen et al. 2007; Utskarpen et al. 2006, 2007; Yoshino et al. 2005). A key compartment is the early endosome (see previous chapter), from where internalized molecules, are either recycled to the plasma membrane to be reused, or sorted to the late endosomal pathway and to the lysosomes to be degraded, or transferred to a direct pathway to the TGN, travelled by endogenous proteins, like the mannose-6-phosphate receptors (for review Bonifacino and Rojas 2006). It is the latter route that is travelled by Shiga toxin and ricin. For efficient transport of Shiga toxin from early endosomes to the TGN and Golgi apparatus, the retromer complex is required (Bujny et al. 2007; Popoff et al. 2007; Utskarpen et al. 2007). The ricin endosome-to-TGN and Golgi traffic has been shown to be facilitated by depletion of sphingolipid (Grimmer et al. 2006), and to be dependent on Rab6A and Rab6A\u2032 (Utskarpen et al. 2006), which are also regulators in pre-Golgi circuits (see previous chapter). The syntaxins 5 and 16 being involved in retrograde transport of mannose-6-phosphate receptors, also are necessary for efficient retrograde traffic of Shiga toxin, as well as for trafficking into the cells of ricin and cholera toxin (Amessou et al. 2007). In the center of interest are the regulatory roles of phosphoinositides (for review De Matteis and Godi 2004), and the importance of signaling (Pelkmans et al. 2005; Perret et al. 2005; von Zastrow and Sorkin 2007). Recently, it has been demonstrated that retrograde traffic of Shiga toxin and ricin are phosphoinositide-regulated (Skanland et al. 2007; Utskarpen et al. 2007). The phosphoinositide-binding proteins sorting nexins 1 and 2, being part of the retromer complex (Seaman 2004, 2005), are necessary for efficient transport of Shiga toxin to the Golgi apparatus (Bujny et al. 2007; Popoff et al. 2007; Utskarpen et al. 2007). Sorting nexins have been shown to be in crosstalk with the phosphatidylinositol (PI) 3-kinase hVps34; it is proposed that hVps34 produces a specific PI(3)P pool needed for the localization of sorting nexins on endosome vesicles, which in turn is required for retrograde endosome-to-Golgi traffic of ricin (Skanland et al. 2007).\nShiga toxin is an active player in its own transport mediating both internalization, and intracellular transport. Upon binding to the plasma membrane or entry into the cells, it is able to trigger signaling cascades (Ikeda et al. 2000; Lauvrak et al. 2006; W\u00e4lchli et al. 2008). Shiga toxin activates the tyrosine kinase Syk, by which clathrin phosphorylation and uptake of Shiga toxin is induced (Lauvrak et al. 2006). Protein kinase C\u03b4 is specifically activated by Shiga toxin regulating endosome-to-Golgi transport (Torgersen et al. 2007). Evidence is provided for activation of a signaling cascade that involves a crosstalk between Ca2+ and the MAP kinase p38; it is suggested that Shiga toxin, by modifying Ca2+ homeostasis, recruits p38 to endosomes for regulation of transport to the Golgi apparatus (W\u00e4lchli et al. 2008).\nThe entry of internalized molecules into the secretory pathway at the TGN and Golgi apparatus level is a major event influencing further retrograde traffic to the ER and cytosol. Although it is well established, and has been shown ultrastructurally very early for several toxins, that transport to the TGN and Golgi apparatus is followed by uptake into cisternae of the Golgi apparatus stacks (e.g. Sandvig et al. 1992; van Deurs et al. 1987), the involved mechanisms are still poorly understood. A detailed ultrastructural analysis has been undertaken using a HepG2 hepatoma cell model, and uptake of wheat germ agglutinin labeled with horseradish peroxidase (WGA-HRP; Pavelka et al. 1998; Pavelka 2007; Vetterlein et al. 2002).\nWheat germ agglutinin is an N-acetyl-glucosamine and sialic acid-specific lectin, which is known for many years to be transported to the Golgi apparatus in retrograde direction (e.g. Gonatas et al. 1977; Stieber et al. 1984); it is also used in connection with the developments of drug delivery systems (Lochner et al. 2003; Weissenb\u00f6ck et al. 2004). WGA reacts with numerous binding sites at the cell surface, and is taken up in large amounts, thus mimicking normal situations in cells, and here particularly reflecting the functions of hepatocytes in the liver tissue. HepG2 hepatoma cells are especially well suited for these studies, since prior to, and in the initial phases of WGA endocytosis, the Golgi apparatus mainly consists of small inconspicuous stacks of cisternae (Fig.\u00a01a), and the changes and reorganizations of the Golgi apparatus during WGA-endocytosis are clearly visible (Figs.\u00a01, 2, 4). Briefly, the results show that internalized WGA, following internalization via clathrin-coated vesicles (Fig.\u00a01a insert) and possibly other mechanisms as well, is rapidly transported to the Golgi apparatus, and induces dramatic Golgi reorganizations. Early globular endosomes accumulate at the trans Golgi side (Fig.\u00a01b) and a network is formed, an endocytic trans-Golgi network (endocytic TGN) that consists of interconnected globular pieces (Fig.\u00a01c), which in dimensions and shapes resemble the earlier globular endosomes (Fig.\u00a01b). In these compartments, the WGA reaction products in part detach from the limiting membranes, and fill the lumina (Fig.\u00a01b, c, d) indicating that the luminal milieu is changing. Portions of the endocytic TGN in close association with trans-Golgi ER, attach to trans Golgi cisternae (Fig.\u00a01c, d), thus becoming integrated parts of the Golgi apparatus stacks (Figs.\u00a02a, b, 4a; various views of a 3D-model are presented in Fig.\u00a03a\u2013d). The trans-Golgi attachment of the endocytic TGN leads to interconnections of the small Golgi stacks (Figs.\u00a01c, d, 4b), and causes the formation of Golgi apparatus ribbons. Concomitantly, and in part prior to the formation of an endocytic TGN, internalized WGA appears within cisternae of Golgi apparatus stacks (Figs.\u00a01b, d, 4a). Stacks that contain high amounts of WGA within the lumina of all the cisternae are mainly found at later periods of WGA endocytosis, e.g. after 45\u201360\u00a0min (Fig.\u00a04b). Very seldom, internalized WGA can be localized within the ER cisternae. At all periods of WGA endocytosis, internalized WGA appears within multivesiculated bodies (Fig.\u00a04a, b), indicating that internalized WGA not only trafficks into the Golgi apparatus but is sorted to the late endosomal\u2013lysosomal pathway as well.\nFig.\u00a04WGA is taken up into the stacked Golgi cisternae a Free portions (long arrows), and Golgi-integrated portions (short arrows) of the WGA-reactive endocytic TGN are visible. Fine WGA reactions also are apparent at confined regions of some of the stacked Golgi cisternae (double arrow). Several multivesiculated bodies (MVB) are densely filled with WGA-reaction products, and show domains, where tubular and vesicular transport carriers are formed. \u00d720,000. b A Golgi ribbon is visible, in which the individual stacks of cisternae are interconnected at the cis, as well as at the trans side. Most of the stacked Golgi cisternae are densely filled with WGA reaction products; others show reactions at limited regions (arrow). At the right lower corner, connections of medial cisternae with the cis-most cisterna of the same stack, and of other stacks are shown. In a vacuolar endosome (V), WGA reaction products are attached to the limiting membrane, and to an intra-vacuolar vesicle (arrowhead). \u00d723,000\nThe detailed knowledge of these endocytosis-induced Golgi reorganizations has been used to develop a precise time schedule for regulated retrograde transport of WGA into the endoplasmic reticulum by treatment with Brefeldin A (Vetterlein et al. 2003). However, multiple questions are open, which concern the mechanisms of formation of the endocytic TGN, and its attachment to the Golgi stacks, the delivery of cargo, and uptake of membranes, interactions of transport carriers with Golgi subcompartments, mechanisms of retrograde transport within the Golgi apparatus stacks, signaling and the role of contact points, the importance of lipid transfer (De Matteis et al. 2007; Hanada et al. 2007), and possible direct endocytic TGN-to-ER traffic via trans-Golgi associated ER. Of particular interest are questions, as to whether secretory anterograde flow influences retrograde traffic, and vice versa, as to whether retrograde flow influences Golgi apparatus size and localization, and whether the formation of Golgi ribbons by retrograde flow is comparable with the formation of Golgi ribbons by input of membrane derived from the ER (Marra et al. 2007).\nConcluding remarks\nIn future studies, it will be important to correlate the detailed fine structural findings with the molecular biologic and genetic results on the machineries and regulatory mechanisms of retrograde traffic (e.g. Amessou et al. 2007; Bonifacino and Rojas 2006; De Matteis et al. 2007; Hanada et al. 2007; Levine and Loewen 2006; Marra et al. 2007; Missiaen et al. 2007; von Zastrow and Sorkin. 2007; W\u00e4lchli et al. 2008). The crucial roles of retrograde traffic in the biosynthetic-secretory route for cellular homeostasis and intoxication of cells, the importance for assessment of effects and side effects of drugs (e.g. Sandoval and Molitoris 2004), and the development of strategies for targeted drug delivery to the interior of cells (El Alaoui et al. 2007; Johannes and Decaudin 2005; Kreitman 2006; Smith et al. 2002; Tarrago-Trani and Storrie 2007; Weissenb\u00f6ck et al. 2004) will be the driving forces.","keyphrases":["retrograde traffic","er","golgi apparatus","endocytosis","biosynthetic secretory route"],"prmu":["P","P","P","P","R"]}
{"id":"J_Mol_Evol-3-1-1894752","title":"The Mechanisms of Codon Reassignments in Mitochondrial Genetic Codes\n","text":"Many cases of nonstandard genetic codes are known in mitochondrial genomes. We carry out analysis of phylogeny and codon usage of organisms for which the complete mitochondrial genome is available, and we determine the most likely mechanism for codon reassignment in each case. Reassignment events can be classified according to the gain-loss framework. The \u201cgain\u201d represents the appearance of a new tRNA for the reassigned codon or the change of an existing tRNA such that it gains the ability to pair with the codon. The \u201closs\u201d represents the deletion of a tRNA or the change in a tRNA so that it no longer translates the codon. One possible mechanism is codon disappearance (CD), where the codon disappears from the genome prior to the gain and loss events. In the alternative mechanisms the codon does not disappear. In the unassigned codon mechanism, the loss occurs first, whereas in the ambiguous intermediate mechanism, the gain occurs first. Codon usage analysis gives clear evidence of cases where the codon disappeared at the point of the reassignment and also cases where it did not disappear. CD is the probable explanation for stop to sense reassignments and a small number of reassignments of sense codons. However, the majority of sense-to-sense reassignments cannot be explained by CD. In the latter cases, by analysis of the presence or absence of tRNAs in the genome and of the changes in tRNA sequences, it is sometimes possible to distinguish between the unassigned codon and the ambiguous intermediate mechanisms. We emphasize that not all reassignments follow the same scenario and that it is necessary to consider the details of each case carefully.\nIntroduction: Distinguishing Possible Mechanisms of Codon Reassignment\nNow that many complete genomes of organisms and organelles are available, there is ample evidence that the genetic code is not as universal (Knight et al. 2001a; Yokobori et al. 2001; Santos et al. 2004) as previously believed (Crick 1968). Many cases are now known where a codon (or related group of codons) has been reassigned from one amino acid to another, from a stop to an amino acid, or from an amino acid to a stop. If a change in the translation system occurs in an organism such that a codon is reassigned, most of the occurrences of this codon will still be at places where the old amino acid was preferred. We would expect the changes causing the codon reassignment to be strongly disadvantageous and to be eliminated by selection. It is possible for mutations to cause disappearance of the codon in its original positions and reappearance in positions where the new amino acid is preferred. Mutations throughout the genome are required for it to readjust to the change in the genetic code. The problem is therefore to understand how codon reassignments can become fixed in a population despite being apparently deleterious in the intermediate stage before the genome has time to readjust.\nVarious mechanisms have been proposed to explain the process of codon reassignment. We have recently shown (Sengupta and Higgs 2005) that these mechanisms can be described within a framework that we call the gain-loss framework. \u201cGain\u201d refers to the gain of a new tRNA gene that is able to translate the reassigned codon as a different amino acid, or the gain of function of an old tRNA gene (e.g., by base modification in the anticodon) so that it translates the reassigned codon in addition to the codons it previously interacted with. \u201cLoss\u201d refers to the deletion of an existing tRNA for the reassigned codon or the loss of function of the tRNA so that it can no longer translate this codon. We identified four mechanisms in the gain-loss framework, as described below.\nCodon disappearance (CD) mechanism This was originally proposed by Osawa and Jukes (1989, 1995). For an amino acid (or stop) with more than one codon, it is possible for all occurrences of a codon to be replaced by synonymous codons, so that the first codon disappears entirely from the genome. After this, the gain and loss in the translation system are neutral changes that do not affect the organism. After the gain and loss occur, the codon may reappear in the genome by mutations at sites where the new amino acid is preferred. The distinguishing element of this mechanism is that the codon disappears first, and the gain and loss occur during a period in which the codon is absent. For the other three mechanisms described here, the codon does not need to disappear before the change.\nAmbiguous intermediate (AI) mechanism This was proposed by Schultz and Yarus (1994, 1996). They argued that a codon does not need to disappear in order to be reassigned and proposed that there is a transient period when the codon is ambiguously translated as two distinct amino acids. In terms of our gain-loss framework, this corresponds to the case where the gain occurs before the loss, i.e., there are two different tRNAs specific to the amibiguous codon during the intermediate period, and the new code becomes established when the old tRNA is lost.\nUnassigned codon (UC) mechanism This mechanism arises as a natural possibility in our gain-loss framework (Sengupta and Higgs 2005). It corresponds to the case where the loss occurs before the gain. There is an intermediate period where there is no tRNA available that can efficiently translate the codon; hence we say the codon is unassigned. The new code becomes established when the gain in function of the new tRNA occurs and the codon is reassigned to the new amino acid. If a codon were truly unassigned, and no tRNA could translate it at all, then the loss of the original tRNA would be lethal if the codon had not previously disappeared. However, several cases are known where an alternative tRNA is able to translate a codon (albeit less efficiently) after the tRNA that was specific to that codon has been deleted (Yokobori et al. 2001). Deletion of tRNAs appears to be frequent in mitochondrial genomes and we argue below that deletion of a tRNA is the primary event that instigated several of the mitochondrial codon reassignments.\nCompensatory change mechanism The final mechanism that occurs in the gain-loss framework is referred to as compensatory change because of its analogy with compensatory mutations in molecular evolution. Kimura (1985) considered a pair of mutations such that each is deleterious when it occurs alone, but when both occur together they are neutral, e.g., in the paired regions of RNA secondary structures (Higgs 1998, 2000; Savill et al. 2001). The gain and the loss in codon reassignment are changes in two different parts of a genome that form a compensatory pair. It is possible that one of these changes occurs but remains infrequent in the population until the second change occurs in an individual that already has the first change. Once the gain and loss are present in the same individual, they can spread simultaneously through the population, although they did not occur at the same time. In the compensatory change case there is no point at which individuals with ambiguous codons or UCs are frequent in the population.We showed using a population genetics simulation (Sengupta and Higgs 2005) that all four mechanisms can occur within the same model depending on the parameter values. Here, we consider which mechanisms occur in real cases. We limit ourselves to mitochondrial changes because most of the observed changes are in mitochondria and because the availability of substantial numbers of complete mitochondrial genomes makes it possible to pinpoint the changes and to study codon usage and tRNA gene content in the genomes before and after the reassignment. Studies of this type were carried out several years ago by Knight et al. (2001a, 2001b) and more recently by Swire et al. (2005). We compare our results with these previous surveys in the discussion section.In order to interpret the changes in the genetic code, phylogenetic trees are required. We split our set of species into groups that are consistent with previous studies. Figure\u00a01 shows fungi and related species, and Fig.\u00a02 shows plants\/algae and related species. These were obtained by our own phylogenetic analysis of mitochondrial genes. Figure\u00a03 shows alveolates\/stramenopiles and related species, and Fig.\u00a04 shows metazoa. These were obtained using combined information from other sources. Details of phylogenetic methods are given in the Supplementary Information.\nFig.\u00a01.Phylogeny of fungi and related species derived from mitochondrial proteins.Fig.\u00a02.Phylogeny of plants and algae derived from mitochondrial proteins.Fig.\u00a03.Phylogeny of alveolates, stramenopiles, and haptophytes according to published sources.Fig.\u00a04.Phylogeny of metazoa according to published sources.\nReassignments That Can Be Explained by Codon Disappearance\nReassignments of UGA from Stop to Trp\nThe UGA Stop-to-Trp change is the most frequently occurring reassignment known. The review by Knight et al. (2001a) lists six of these in mitochondria (and four in nuclear genomes). Our updated analysis of the mitochondrial data identifies the following 12 cases in mitochondria.Metazoa, Monosiga, and Amoebidium: This change is shared by all Metazoa and by their two closest known relatives (Fig.\u00a01 and Lang et al. 2002).Acanthamoeba: Fig\u00a01. and Burger et al. (1995).Basidiomycota (Crinipellis and Schizophyllum): Fig.\u00a01.Ascomycota (group containing Penicillium and relatives): Fig.\u00a01.Ascomycota (group containing Yarrowia and relatives): Knight et al. (2001a) list a single change in the ancestor of Metazoa, Acanthamoeba, and Fungi. However, Fig.\u00a01 shows that UGA remains a stop codon in Chytridiomycota, Zygomycota, and Dictyostelium. Therefore, it is likely that cases i, ii, and iii are separate reassignments. The reassignment has also occurred in almost all the Ascomycota, with the exception of the Schizosaccharomyces group. This implies that cases iv and v in the Ascomycota are also separate reassignments. These conclusions depend on the argument that a reversal of this change (from Trp to Stop) is very unlikely, which we discuss below.Rhodophyta (Chondrus, Porphyra): Fig.\u00a02 and Burger et al. (1999).Pedinomonas: Fig.\u00a02 and Turmel et al. (1999).Haptophytes: This change was reported in a subgroup of haptophytes including Phaecocystis and Isochrisis by Hayashi-Ishimaru et al. (1997). The complete genome of Emiliana (Sanchez-Puerta et al. 2004) shows that it also possesses the reassignment. This is consistent with our phylogeny (Fig.\u00a03).Ciliates (Paramecium, Tetrahymena): Knight et al. (2001) place this change at the base of the alveolates. However, our analysis of codon usage and sequences from Plasmodium species discussed below shows that UGA remains a Stop codon in Plasmodium. Thus the reassignment is not shared by all alveolates.Cafeteria: Fig.\u00a03.Bacillariophyta (Skeletonema, Thalassiosira): Fig\u00a03 and Ehara et al. (2000)Kinetoplastida (Trypanosoma, Leishmania): Inagaki et al. (1998) (not shown in figures).\nIn species in which only the UGG codon codes for Trp, the tRNA-Trp has a CCU anticodon. Mutation of the wobble position C to a U creates a UCU anticodon that can pair with both UGA and UGG: hence the reassignment of UGA to Trp. We have analyzed the tRNA-Trp sequences of all available mitochondrial genomes to determine in which species this mutation has occurred. We find that all species in which UGA is Stop have a CCU anticodon, as expected. Almost all species in which UGA is Trp have a UCU anticodon, but we find some exceptions below. Table\u00a01 shows the number of occurrences of each of the standard Stop codons together with the tRNA-Trp anticodon. When UGA is Stop it is usually used less frequently than the preferred stop codon UAA. When the reassignment occurs, many UGG Trp codons mutate synonymously to UGA; hence UGA becomes frequent. This is seen in all species having a UCA anticodon. However, UGA is also frequent in Amoebidium, Crinipellis, and Schizophyllum, which have a CCA anticodon. Therefore there must be a posttranslational modification of the C base in the anticodon that permits translation of UGA, rather than a mutation in the gene. Another special case is the kinetoplastids. These have no mitochondrial tRNAs, and import all the required tRNAs from the nucleus. The tRNA-Trp from the nucleus has CCA anticodon because the canonical code is used in the nucleus. The same tRNA is imported to the mitochondrion and then undergoes a base modification so that it can translate both UGA and UGG (Alfonzo et al. 1999).\nTable\u00a01.Codon usage data relevant to reassignments of Stop codons UGA and UAGUGA istRNA-Trp anticodonCodon usageUGAUAAUAGSpecies in Fig.\u00a01\u00a0\u00a0Amoebidium castellaniiTrpCCAa782812\u00a0\u00a0Dictyostelium discoideumStopCCA2308\u00a0\u00a0Monosiga brevicolisTrpTCA124284\u00a0\u00a0Homo sapiensbTrpTCA9283\u00a0\u00a0Rhizophidium sp. 136StopCCA532117c\u00a0\u00a0Spizellomyces punctatusStopCCA1220140c\u00a0\u00a0Hyaloraphidium curvatumNot usedCCA0162\u00a0\u00a0Monoblepharella sp. JEL 15StopCCA1205\u00a0\u00a0Harpochytrium sp. JEL105Not usedCCA0122\u00a0\u00a0Harpochytrium sp. JEL94Not usedCCA0131\u00a0\u00a0Allomyces macrogynusNot usedCCA01614\u00a0\u00a0Mortierella verticillataRareCCA2e214\u00a0\u00a0Rhizopus oryzaeNot usedCCA0204\u00a0\u00a0Crinipellis perniciosaTrpCCAa1247316\u00a0\u00a0Schizophyllum communeTrpCCAa25191\u00a0\u00a0Penicillium marneffeiTrpTCA61161\u00a0\u00a0Hypocrea jecorinaTrpTCA89145\u00a0\u00a0Schizosaccharomyces japonicusNot usedCCA061\u00a0\u00a0Schizosaccharomyces octosporusNot usedCCA080\u00a0\u00a0Schizosaccharomyces pombeRareCCA1e70\u00a0\u00a0Yarrowia lipolyticaTrpTCA57122\u00a0\u00a0Candida stellataTrpTCA3480\u00a0\u00a0Candida albicansTrpTCA4958\u00a0\u00a0Saccharomyces cerevisiaeTrpTCA124190Species in Fig.\u00a02\u00a0\u00a0Malawimonas jakobiformisStopCCA1462\u00a0\u00a0Cyanidioschyzon merolaeStopCCA2293\u00a0\u00a0Chondrus crispusTrpTCA101223\u00a0\u00a0Porphyra purpureaTrpTCA117274\u00a0\u00a0Chaetosphaeridium globosumStopCCA7309\u00a0\u00a0Chara vulgarisStopCCA8308\u00a0\u00a0Prototheca wickerhamiiNot usedCCA0351\u00a0\u00a0Pseudoendoclonium akinetumStopCCA114021\u00a0\u00a0Pedinomonas minorTrpTCA62110\u00a0\u00a0Scenedesmus obliquuseStopCCA12141b\u00a0\u00a0Chlamydomonas eugametosNot usedCCA0122\u00a0\u00a0Chlamydomonas reinhardtiiNot usedCCA062Species in Fig.\u00a03\u00a0\u00a0Emiliana huxleyiTrpUCA73192\u00a0\u00a0Rhodomonas salinaStopCCA1349\u00a0\u00a0Naegleria gruberiNot usedCCA0379\u00a0\u00a0Plasmodium reichenowiNot usedNone030\u00a0\u00a0Plasmodium falciparumNot usedNone030\u00a0\u00a0Paramecium aureliaTrpTCA832917\u00a0\u00a0Tetrahymena pyriformisTrpTCA228440\u00a0\u00a0Tetrahymena thermophilaTrpTCA228450\u00a0\u00a0Caferteria roenbergensisTrpTCA190322\u00a0\u00a0Phytophthora infestansStopCCA1390\u00a0\u00a0Saprolegnia feraxNot usedCCA0421\u00a0\u00a0Chrysodidymus synuroidesNot usedCCA0343\u00a0\u00a0Ochramonas danicaNot usedCCA03014\u00a0\u00a0Laminaria digitataStopCCA4296\u00a0\u00a0Pylaiella littoralisStopCCA7387aThese species have a CCA anticodon but UGA is Trp. This suggests that the C base is modified in the tRNA.bIn vertebrates AGA and AGG are also Stop codons. Each is used once in Homo sapiens.cUAG is reassigned to Leu in these species.dThese codons are presumed to be inefficiently translated as Trp by the tRNA-Trp(CCA).eIn S. obliquus, UCA is a novel Stop codon, occurring 17 times.\nThe predictions from our theory and simulations (Sengupta and Higgs 2005) were that Stop codons are most likely to be reassigned via the CD mechanism because they are rare in the first place (and chance disappearance is therefore relatively likely) and, also, because if the codons do not disappear, the penalty for read-through of a stop codon is likely to be larger than the penalty for mistranslation of an amino acid. (However, read-through of a stop codon is not necessarily lethal, especially if the number of additional codons translated until the next random occurrence of a stop codon is not too large.) The figures in Table\u00a01 make a strong case that the CD mechanism is responsible. UGA is rare in almost all species where it is used as a Stop codon. Many of these genomes have high AU content, which is probably the reason UAA is preferred over UGA as a Stop codon. Many of the closest relatives to the species where UGA is reassigned have particularly low usage of UGA, e.g., Allomyces and Rhizopus, close relatives of the Basidiomycota, have zero; Cyanidioschyzon, a close relative of Chondrus and Porphyra, has only two; Scenedesmus and Chlamydomonas, close relatives of Pedinomonas, have one and zero respectively; and Plasmodium, a close relative of Paramecium and Tetrahymena, has zero. This last example shows that UGA is not reassigned in Plasmodium, as mentioned in case ix above. These examples clearly show that disappearance of UGA is possible and that, in many of the cases, there is good evidence that UGA was absent or almost absent at the time it was reassigned.\nA mutation pressure from GC to AU, which is implicated in the disappearance of UGA, will also tend to cause rapid mutations from UGG Trp codons to UGA after the Trp tRNA gains the ability to decode UGA. This is one reason that reversal of the change is unlikely. There are often around 100 UGAs in genomes where the reassignment has occurred, and it would be very difficult for this large number to disappear by chance because this would act against the mutation pressure. A second reason is that the reassignment to Trp would be associated with the loss of function of the release factor that originally interacted with the UGA. A reversal would also require regaining of the function of the release factor.\nThe unlikeliness of the reversal of the reassignment is important in our interpretation of cases i to v above. The codon usage and tRNA-Trp anticodon data (Table\u00a01) show that UGA is not established as a Trp codon in Dictyostelium, Chytridiomycota, Zygomycota, or Schizosaccharomyces. This means that multiple reassignment events are required to explain the observed pattern. It appears that UGA was rare and prone to disappearance right from the base of the Metazoa\/Fungi tree in Fig.\u00a01, and the release factor may already have lost its function. Therefore all that is required for the reassignment to occur is the simple mutation of the tRNA anticodon. In groups where the release factor was lost, but the tRNA mutation did not occur, the UGA codon would be effectively unassigned and would be selected against. Seif et al. (2005) argue that this has occurred in Mortierella and Schizosaccharomyces, where there is evidence of a small number of UGA acting as Trp codons that are translated very inefficiently by the standard Trp tRNA with CCA anticodon. A genetic code change might easily become established in these species in the future if the mutation occurred in the tRNA.\nThe Probability of Disappearance of UGA Codons\nIt is possible to calculate the probability Pdis that a codon will disappear using a simple model of the mutation process. Swire et al. (2005) have used this method to show that in most cases of stop codon reassignment, the probability of disappearance of the codon was relatively large, whereas in many cases of sense codon reassignments, the probability was extremely small. Hence they argue that stop codons were reassigned via CD but sense codons were not. We agree with this conclusion in almost all cases, but there are a few cases where we argue for CD in sense codon reassignments as well.\nIn this section, we consider only UGA stop codons. The probability of UGA CD can be calculated as follows. Let the equilibrium frequencies of the bases under the mutational process be \u03c0A, \u03c0C, \u03c0G, and \u03c0U. Let fUGA, fUAA, and fUAG be the relative frequencies of the three stop codons (fUGA + fUAA, + fUAG = 1). If there is no selective preference of one stop codon over another, we expect these frequencies to be in equilibrium under mutation. Therefore \nHence . In mitochondrial genomes, the two strands of the genome are not equivalent, the four base frequencies are all different, and it is not true that \u03c0C = \u03c0G and \u03c0A = \u03c0U (see Urbina et al. 2006). The values of the frequencies can be estimated from the frequencies of the bases at fourfold degenerate (FFD) sites. These are calculated by summing over all third-position sites that are FFD. If the total number of stop codons in the genome is Nstop, then the probability of disappearance of UGA is .\nWe do not know the values of Nstop or fUGA at the point where the codon reassignment occurred, but we can use the species that are close to the reassignment point on the tree as an estimate, as shown in Table\u00a02. Nstop is the the sum of UAA and UAG from Table\u00a01. UGA codons do not contribute to Nstop because these are now Trp codons in these species. We used Monosiga as a proxy for case i. The resulting probability is high (Pdis = 0.17) and is consistent with the CD mechanism. Amoebidium was not used in this case because it has a derived multichromosome structure of the mitochondrial genome, and metazoa were not used because they have a much reduced genome size. Both of these would be poor estimators of what the genome was like at the reassignment point. In all these species, \u03c0G is much less than \u03c0A, but the bias is more extreme in some species than others. In case ix, if either of the Tetrahymena species is used as proxy, a fairly high Pdis is obtained, but if Paramecium is used, Pdis is much lower. Similarly in case iii, Pdis is high if we use Schizophyllum, but low if we use Crinipellis. These two species differ in base frequencies and also in the number of genes on the genome (i.e., Nstop). Although Nstop = 89 in Crinipellis (also known as Moniliophthora perniciosa), this genome has many genes labeled as \u201chypothetical protein\u201d that do not have homologues in related species. These genes may be recent insertions or may not even be expressed sequences. Thus, Nstop was probably much less than 89 at the time of the reassignment, and Pdis would be correspondingly higher. In both these cases, it is reasonable to conclude that the properties of the genome at the point of reassignment were such that CD was not too unlikely.\nTable\u00a02.Estimates of stop codon disappearance probabilitiesCaseSpeciesNstop%G%Afcodon (%)PdisUGA CDiMonosiga brevicolis322.8547.525.360.17iiAcanthamoeba castellanii409.8926.3521.446.4E-05iiiCrinipellis perniciosa899.1932.8417.942.3E-08iiiSchizophyllum commune203.4842.876.990.23ivPenicillium marneffei172.3835.645.890.36ivHypocrea jecorina194.6840.149.460.15vYarrowia lipolytica141.5549.012.970.65vCandida stellata80.9441.572.170.84viChondrus crispus256.0836.6112.463.6E-02viPorphyra purpurea3110.9835.8019.011.4E-03viiPedinomonas minor111.6021.316.520.48viiiEmiliana huxleyi219.2636.5016.832.1E-02ixParamecium aurelia4614.8719.6030.146.8E-08ixTetrahymena pyriformis443.2242.276.624.9E-02ixTetrahymena thermophila452.6340.465.757.0E-02xCafeteria roenbergensis326.1732.5613.746.6E-03UAG CDiRhizophidium373.3334.058.184.3E-02iSpizellomyces punctatus326.0330.6914.107.7E-03iiScenedesmus obliquus36.6435.7313.550.65\nThe only other case where Pdis is low is case ii, where Pdis = 6.4 \u00d7 10\u22125 if Acathamoeba is used as proxy. There is no other related species for which data are available. We have already seen that fluctuations in base frequencies can change Pdis by several orders of magnitude. Therefore a value of order 10\u22125 does not appear ridiculously small, and the true value could well have been much higher at the point the reassignment actually occurred. Finally, we comment on case 17 of Swire et al., Table 2a, where they estimate the low probability of Pdis = 2.7 \u00d7 10\u221211. This corresponds to our case vi, where we estimate Pdis = 3.6 \u00d7 10\u22122 if Chondrus is used as proxy and Pdis = 1.4 \u00d7 10\u22123 if Porphyra is used. Both of these are much higher than the estimate of Swire et al. We also note that, according to our phylogeny, these two species are related and we assume a single reassignment in their ancestor, whereas Swire et al. treat them as independent (cases 16 and 17). The change in the phylogeny would not affect our estimate of Pdis. Thus, we conclude that all these examples of UGA codon reassignment are consistent with the CD mechanism. (Cases xi and xii are not included in Table\u00a02 because there are insufficient data to perform the calculation.)\nReassignments of UAG Stop Codons\nTwo cases of reassignment of UAG from Stop to Leu are known: one in the two chytrids, Rhyzophidium and Spizellomyces (Fig.\u00a01; Laforest et al. 1997) and the other in Scenedesmus (Fig.\u00a02; Hayashi-Ishimaru et al. 1996; K\u00fcck et al. 2000). In Table\u00a01, UAG is also seen to be rare in general and rare particularly in the close relatives of the species reassigned: only two in the other chytrid, Hyaloraphidium; and zero in Pedinomonas, relative of Scenedesmus. These reassignments show the same pattern as the UGA Stop-to-Trp examples and can also be attributed to the CD mechanism. Disappearance of UAG would also be favored by mutation pressure increasing the AU content.\nThe reason UAG is reassigned less frequently than UGA may be because of the relative difficulty of the required change in the tRNA. In the case of UGA, the existing tRNA-Trp can simply mutate its anticodon. In the UAG case it is necessary to create a new tRNA-Leu with a CUA anticodon to pair with UAG. This can be done by making a mutation in the second position of a tRNA-Leu with a CAA anticodon. Such a mutation in the anticodon would not change the amino acid specificity of the tRNA because the anticodon of the tRNA-Leu does not act as an identity element for recognition by the leucyl-tRNA synthetase (Asahara et al. 1993), in contrast to most other amino acyl-tRNA synthetases, which recognize the anticodon. However, this can only be done after duplication of the tRNA-Leu, because one copy of this gene is still required to translate UUG Leu codons. Thus, this reassignment is relatively rare because it requires a prior tRNA duplication. It is interesting to note that in most genomes, both UUA and UUG would be Leu, and there would be a single tRNA with UAA anticodon to translate both these. Another peculiarity of Scenedesmus is that UUA codons are not used (K\u00fcck et al. 2000), but UUG codons are frequent. This is clearly related to the fact that the tRNA-Leu has CAA anticodon in this species.\nUAG has been reassigned to Ala in a group of green algae that are closely related to Scenedesmus (Hayashi-Ishimaru et al. 1996). Complete genomes are not available for this group, therefore they are not included in our Fig.\u00a02. It seems likely that UAG disappeared in the ancestor of Scenedesmus and the other algae, and that the codon was captured by Leu in Scenedesmus and by Ala in the other species.\nTable\u00a02 also shows the probability of disappearance of the UAG codon for these two cases of UAG reassignment. This is calculated in the same way as for UGA above. These values do not appear unreasonably small in view of the uncertainty in the genome properties at the point of reassignment. Therefore, we conclude that these changes are consistent with the CD mechanism.\nSense Codon Reassignments Linked to Codon Disappearance\nIt is not only Stop codons that can be reassigned by the CD mechanism. The following examples show sense codon changes occurring via this mechanism. In the canonical code, both CUN and UUR code for Leu and these two codon blocks are accessible to one another by a synonymous mutation at the first position. Similarly CGN and AGR both code for Arg and are also accessible to one another via a synonymous mutation at first position. In AU-rich genomes, CUN and CGN codon families are subject to disappearance and replacement by UUR and AGR. Table\u00a03 gives the number of occurrences of the codon groups for Leu and Arg in several Fungi. Base frequencies at FFD sites are also shown. C and G frequencies are very low in all the species in Table\u00a03, indicating a strong mutation pressure toward A and U.\nTable\u00a03.Codon usage in some Fungi lineages: intronic ORFs excluded (E) or included (I)Leu codonsArg codonsIle and Met CodonsFrequency at FFD sitesCUNUURCGNAGRAUUAUCAUAAUG%U%C%A%GS. japonicus (E)7919873213340324876.03.419.21.3S. octosporus (E)68236234161340d5761.11.934.82.2S. pombe (E)5319273311339495156.41.340.51.8Y. lipolytica (E)446180c751748727711948.31.149.01.5C. stellata (E)3279122912381565457.30.241.60.9C. albicans13239747261198122910055.44.837.72.1C. parapsilosis (E)6654739453033219311768.60.729.80.9C. parapsilosis (I)137728601024104929914365.33.029.02.7P. canadensis (E)2571418672741856210549.20.749.40.7P. canadensis (I)2774620742982058610950.00.948.40.6A. gossypii80a2910c40215795d,e3457.20.042.80.0K. lactis (E)0b2860c48213167d6344.01.653.31.1K. lactis (I)0b3120c552561627d6543.42.652.11.9K. thermotolerans (E)16a304244204172d5647.70.951.00.4K. thermotolerans (I)42a44010722982330d7848.32.147.62.0C. glabrata (E)11a2941c452072116d,e7346.70.652.00.7C. glabrata (I)28a4151c603182585d,e7848.60.949.80.7S. cerevisiae(E)33a3337492393160d,e7348.62.347.12.0S. castellii (E)19a2740c402037101d,e5647.51.649.91.0S. servazzii (E)22a3000c462181195d,e7037.20.959.52.4aCUN is reassigned to Thr and an unusual tRNA-Thr gene is present in these species.bThere is no tRNA for the CUN block in this species.cThe tRNA-Arg is deleted in these species.dThe tRNA-Ile(CAU) is deleted in these species.eAUA is reassigned to Met in these species.\nThe species in the top half of Table\u00a03 (P. canadensis and above) use CUN for Leu, as in the canonical code. These species possess a tRNA-Leu with anticodon UAG for this family. In all these cases the CUN family is less frequent than the UUR family. The six species followed by a superscript a in Table\u00a03 have undergone a reassignment of the CUN family to Thr. These species possess an unusual tRNA-Thr with anticodon UAG (Sibler et al. 1981; Osawa et al. 1990) and the usual tRNA-Leu(UAG) is not found in the genome. In K. lactis, the codon family is not used at all. Significantly, this is the only species in which no tRNA exists that pairs with this codon family. The fact that the tRNA-Thr(UAG) appears only in species where the tRNA-Leu(UAG) is absent suggests that the new gene evolved directly from the old tRNA-Leu(UAG) gene. This requires no change in the anticodon, but changes elsewhere are required in order that the tRNA is recognized by the threonyl-tRNA synthetase and not the leucyl-tRNA synthase. Although many amino acyl-tRNA synthetases recognize the anticodon, recognition of tRNA-Leu by leucyl tRNA-synthetase is exclusively determined by the bases in the large variable arm (Asahara et.al. 1993). Changes in the variable arm could have prevented the tRNA acting as a tRNA-Leu and would have left it open to evolving a new function as a tRNA-Thr.\nThe codon usage figures in Table\u00a03 indicate that the change is attributable to the CD mechanism. The number of occurrences of CUN was most likely driven to zero prior to the branching of A. gossypii (see Fig.\u00a01). A major change in the tRNA-Leu(UAG) gene was then possible, allowing it to be charged with Thr. CUN codons later reappeared with a new meaning. In the context of our gain-loss framework (Sengupta and Higgs 2005), this example is interesting in that the gain and loss of function occur in the same tRNA.\nAlthough it is likely that the unusual tRNA-Thr in these species evolved directly from the old tRNA-Leu, the alternative scenario is that there was a duplication of the tRNA-Thr(UGU) gene that decodes the normal ACN Thr codons. One of these genes might then have changed its anticodon to UAG (two mutations required). In order to determine the origin of the tRNA-Thr(UAG) gene, we constructed a phylogeny of all tRNAs from P. canadensis, K. thermotolerans and S. castellii, i.e., from representative species before and after the codon reassignment. The tRNAs of each type formed monophyletic triplets, with the exception of the genes with UAG anticodons. The tRNA-Thr(UAG) genes in K. thermotolerans and S. castellii showed no close relationship to either the tRNA-Thr(UGU) genes or the tRNA-Leu(UAG) gene in P. canadensis. Thus, neither of the two possible scenarios was supported. Despite this, it stills seems most likely that the new tRNA-Thr gene evolved from the old tRNA-Leu, a conclusion also reached by Sibler et al. (1981) and Osawa et al. (1990).\nOne possibility that cannot entirely be ruled out from the codon usage data is that the changes in the tRNA-Leu(UAG) occurred when the CUN codons were very rare but not entirely absent. The changes might have been such as to immediately cause the tRNA to be charged by Thr, or might have happened more gradually, so that the same tRNA could be ambiguously charged by Leu and Thr during the changeover period. We would then have to count this as an example of the AI mechanism. Nevertheless, the low CUN number resulting from mutation pressure is clearly a major factor in this case, and we therefore feel comfortable in classifying it under the CD mechanism. The tRNA gene has undergone considerable modification, including an unusual insertion that makes the anticodon loop larger than the standard seven bases. In our opinion, it is unlikely that such a large change could have happened while the tRNA remained simultaneously functional for both amino acids.\nTable\u00a03 also shows disappearances in the Arg codons that happen in parallel with those in the Leu codons. Mutation pressure away from C causes replacement of CGN codons by AGR. Once CGN codons have disappeared, there is no penalty to the deletion of the tRNA-Arg(ACG) gene. This gene is absent in the species followed by a superscript c in Table\u00a03, and CGN is absent in these species (or apparently there is just one in C. glabrata). On the other hand, the gene is still present in S. cerevisiae and K. thermotolerans, and a handful of CGN codons still remain in these species. The tRNA-Arg(ACG) genes in S. cerevisiae and K. thermotolerans show clear sequence homology to that in P. canadensis. From the phylogeny of these species in Fig.\u00a01, we see that it requires four independent deletions of the tRNA-Arg(ACG) to explain the observed pattern of absences of the gene, indicating that deletion of redundant genes is a rapid phenomenon.\nFigure\u00a01 shows several separate reassignments of CGN from Arg to unassigned and only one reassignment of CUN from Leu to Thr. This is because the symbols label points where the tRNAs were deleted or mutated, not points where the codon disappeared. Our interpretation is that both CUN and CGN codons were absent in the ancestor of the group of yeast species prior to the branching of A. gossypii, and that change in the tRNA-Leu occurred at this point, whereas the deletions of the tRNA-Arg genes did not occur till after the split between the species. This would permit small numbers of CGN codons to reappear in some lineages where the gene was not deleted (e.g., S. cerevisiae and K. thermotolerans). The final twist in this story is that the CUN codons are absent in K. lactis. This means that there must have been a further reassignment of CUN from Thr to unassigned in this species only. Clearly the codons have disappeared in this case, which could be due to drift, or because of poor functioning of the tRNA-Thr(UAG) gene in this species, which would have caused selection against this codon block. We have little evidence in this case, but for the purposes of our summary table in the Discussion below (Table\u00a06), we classified it as CD.\nTable\u00a03 shows another example of the disappearance of the CGN block in Y. lipolytica. The corresponding tRNA-Arg is also deleted in this species (Kerscher et al. 2001). In S. pombe and C. stellata, the species that branch before and after Y. lipolytica, the tRNA-Arg is still present and the CGN codons are still used. CGN has thus become unassigned in this single species. This also appears to be a result of mutation pressure causing disappearance of CGN before deletion of the tRNA-Arg.\nThe Probability of Disappearance of CUN and CGN Codons\nThe number of Leu and Arg codons in these genomes is much larger than the number of stops. Therefore, it is more difficult for these sense codons to disappear than it is for stops. Nevertheless, the probability of CD is not unreasonably low in these cases. If we assume that all six Leu codons are in equilibrium, we find \nfrom which the sum of the relative frequencies of all the CUN block codons is \nThe probability of disappearance of the CUN block is , where NLeu is the number of Leu codons in the genome.\nHowever, Table\u00a03 shows that this calculation is hardly necessary. In A. gossypii, the species branching immediately after the proposed point of the codon reassignment, %C = 0 at FFD sites, i.e., the mutational bias against C is so strong that there are no C bases at all in any FFD site. Thus fCUN = 0, and Pdis = 1. Presumably the chance of mutating to a C is not precisely zero in A. gossypii, but it is small enough that all the FFD C-ending codons disappear. Therefore it is not unreasonable that the CUN block should have disappeared in the ancestor of this group.\nAs we do not know that %C was exactly zero in the ancestor, we would like to know how small it must have been in order for there to be a significant probability of disappearance of CUN. It can be seen that %C is low in all species in Table\u00a03, but variations of \u00b11% make a big difference in estimation of Pdis. Estimation of base frequencies is further complicated by the presence of intronic ORFs in many of these species. It has previously been shown that codon usage patterns differ between the intronic ORFs and the other genes (Bullerwell et al. 2003; Talla et al. 2005). For several species in Table\u00a03, we have listed separate codon information for the genes excluding the intronic ORFs (E) and for the full set of genes including the intronic ORFs (I). In every case considered, %C is slightly higher if the intronic ORFs are included. These ORFs vary in number between species, and are not present in some species, including A. gossypii. This makes it difficult to know the number of genes in the ancestral genome at the point of CUN reassignment. It also suggests that these sequences may be recent insertions and that the base composition may not have adjusted to the equilibrium base composition of the rest of the genome. Furthermore, the presence of rare codons in the intronic ORFs suggests that these sequences may not be expressed at a high level or that their expression may not be important for the organism. For example, in K. lactis and K. thermotolerans, AUA is avoided because of deletion of the corresponding tRNA (to be discussed below), but there are substantially more AUA codons in the intronic ORFs than the regular genes. For these reasons, it is likely that the base frequencies in the genes excluding the intronic ORFs are better indicators of the equilibrium base frequencies of the mutational process affecting the most important genes in the genome. We also note that the presence of the intronic ORFs is sometimes associated with alternative splicing, so that the same exon is used in several proteins (e.g., S. cerevisiae and C. stellata). Clearly we do not want to count the same exon twice when counting codons. A full investigation of all these effects on intronic ORFs is beyond the scope of this paper.\nGiven all the caveats above, it is clear that any estimate of Pdis is subject to a very large uncertainty. However, we wish to give a fair estimate for a case where %C is not exactly zero. We use the figures for C. glabrata because the base frequencies with and without the intronic ORFs differ less than in most species. To be conservative, we use the figures including the ORFs. From Table\u00a03, we obtain fCUN = 0.0354, and Pdis = (0.9646)415 = 3.2 \u00d7 10\u22127. Slight reductions in %C would increase this figure by orders of magnitude. We conclude the bias against C must have been at least as strong as it is in C. glabrata (i.e., %C < 0.9) in order for the CUN codons to have a reasonable chance of disappearing. The fact that %C becomes exactly zero in A. gossypii and is less than 0.9% in several other cases in Table\u00a03 shows that these extreme biases do occur. Swire et al. (2005) have calculated many examples of CD probabilities. The CUN reassignment corresponds to case 3 of Swire et al. (2005, Table 2b). For some reason, they estimated a vanishingly small probability of disappearance of the CUN codons (Pdis = 6.8 \u00d7 10\u2212427), which is the lowest of any of the cases they list. They thus argued that CD was impossible in this case. We disagree, since we have shown that Pdis is high due to the extreme bias against C bases. This case is exceptional, however. In all the other examples of sense-to-sense codon reassignments discussed by Swire et al., we agree that the probability of CD is indeed unrealistically low and that the reassignment must have occurred without CD.\nUsing an argument similar to that above, the relative frequency of CGN codons is , and the probability of disappearance of the CGN block is . We have already seen that %C = 0 in A. gossypii and is very low in related species. NArg is much lower than NLeu, so disappearance of CGN in the ancestor of this group is therefore quite likely, and is easier than disappearance of CUN. For the case of Y. lipolytica, if we use the figures from this species as a proxy for the ancestor, we obtain Pdis = (1 \u2013 0.0426)75 = 0.038, which is not unreasonably small.\nClearly it is much more difficult for sense codons to disappear than stop codons, as the number of codons involved is much larger. For this reason, we need an extremely strong base frequency bias in order to cause disappearance. However, in the above cases we have shown that the bias is indeed extreme and that the CD mechanism seems likely. A reviewer of this paper pointed out that Osawa and Jukes (1989), the original proponents of the CD model, insisted that, according to their theory, every single occurrence of the codon must disappear from the genome before the reassignment occurred. From current genome data it will never be possible to tell whether every occurrence of the codon disappeared at the time the reassignment occurred. Therefore, if we insist on this strict definition, it is impossible to assign a mechanism to any of the reassignment events. However, to be more constructive and more practical, we wish to make a clear distinction between the examples discussed above and those that we discuss in the remainder of the paper. In the above examples, there is a clear case that CD is likely due to the direction of the mutation pressure and the best estimates of the CD probabilities are not too low. The extreme rarity (and probably absence) of these codons was clearly a major factor in allowing the codon reassignment to occur. In contrast, in the examples below, there is clear evidence that the codon was not rare at the time of the reassignment and that the codon reassignment was initiated by some other event not related to CD.\nReassignments That Cannot Be Explained by Codon Disappearance\nReassignment of AUA from Ile to Met Is Initiated by Loss of a tRNA-Ile Gene\nIn the canonical code, the AUN codons are unusual in that AUU, AUC, and AUA code for Ile, and only AUG codes for Met. The tRNAs need to distinguish between codons ending in A and those ending in G, which is not usually necessary (except for the UGG Trp codon). The translation system in bacteria uses two tRNAs for Ile: one with anticodon GAU, which translates AUY codons, and one with anticodon CAU, which translates AUA only. It is known from studies on E. coli (Muramatsu et al. 1988) that the C at the wobble position undergoes a posttranscriptional base modification to K2C (lysidine), which allows this tRNA-Ile to pair with the AUA codon. The same gene also is present in many mitochondrial genomes in which AUA is translated as Ile, and it is presumed that the same base modification occurs. However, the tRNA-Ile(CAU) gene is absent in mitochondrial genomes in which AUA is reassigned to Met. Before codon reassignment, tRNA-Met also has a CAU anticodon, which pairs with AUG only, but modification of the wobble-position C allows it to pair with both AUA and AUG (Tomita et al. 1999b).\nIn order to locate and understand the cases of AUA codon reassignment in mitochondria, it is crucial to establish which genomes contain the tRNA-Ile(CAU) gene. This is complicated by the fact that some genomes contain three tRNAs with CAU anticodons (a Met initiator, a Met elongator. and an Ile), and the Ile tRNA is often mistakenly annotated as Met. To uncover misannotations, we constructed a phylogeny of all the tRNAs with CAU anticodon from all the fungi genomes in our data set. The genes fell into three groups that could be reliably identified. Supplementary Table S3 lists the position of the three tRNAs in each genome and, hence, shows in which genomes there have been gene deletions.\nFrom this, we note that the tRNA-Ile is missing from the seven species from A. gossypii downward in Table\u00a03. Hence, this gene must have been deleted prior to the branching of A. gossypii, as shown in Fig.\u00a01. Before the deletion of the tRNA-Ile(CAU) gene, AUA and AUU are frequent codons for Ile but AUC is rare (e.g., C. albicans, C. parapsiplopsis, and P. canadensis). This is to be expected from the FFD base frequencies. Mutation pressure is therefore in the wrong direction to cause disappearance of AUA, and in fact AUA is the most frequent Ile codon in P. canadensis, the closest relative to the species concerned. We therefore argue that this reassignment was initiated by loss of the tRNA-Ile while the AUA codon was frequent in the genome. Hence, it is an example of the UC mechanism, according to our classification.\nImmediately after the loss of this tRNA, the AUA codon had no specific tRNA to pair with it, and it may be said to have been unassigned. The tRNA loss could not have been lethal, however, so there must have been another way of decoding AUAs. In Metazoa, some genomes are known where the tRNA-Ile(CAU) gene has been deleted, but the AUA codon has not been reassigned. Yokobori et al. (2001) have shown that in such cases, AUA is translated as Ile by the tRNA-Ile(GAU), even though this requires GA mispairing at the wobble position. It is presumed that this is tolerated if there is no better alternative available, i.e., if the tRNA-Ile(CAU) has been deleted but no modification to the tRNA-Met has occurred. This may be what is happening in the two Kluyveromyces species, where usage of AUA is very low, despite the fact that A is also frequent at FFD sites in these species. This demonstrates a definite avoidance of AUA, probably due to the fact that there is no tRNA that translates the AUA codon efficiently. Talla et al. (2005) argue that AUA is assigned to Met in K. thermotolerans based on only two occurrences of this codon in the VAR1 gene. It may be more reasonable to say that AUA is unassigned in both K. lactis and K. thermotolerans. In C. glabrata, Koszul et al. (2003) conclude that AUA is associated with Met based on comparison of C. glabrata sequences with S. cerevisiae sequences from which they find that 8 of 16 AUA codons in C. glabrata appear at conserved Met sites encoded by AUA in S. cerevisiae. There is still some avoidance of AUA codons in C. glabrata nevertheless. In A. gossypii and the Saccharomyces species, AUA is frequent, and we presume that it is efficiently translated as Met. This may imply that there have been two independent adaptations of the tRNA-Met to deal with AUA in these two lineages.\nWe wish to underline the difference between the AUA reassignment and the reassignments of CUN and CGN in the same species (see previous section). For CUN and CGN, mutation pressure drives the disappearance. In both these cases there is only one tRNA for the block of four codons, and if this were deleted (in the CGN case) or drastically mutated (as probably happened in the CUN case), there would be no plausible alternative tRNA that could step in. Hence these changes can only occur after the codon disappears. In contrast, mutation pressure leads to frequent usage of AUA for Ile. Loss of the tRNA-Ile(CAU) is possible without CD because the tRNA-Ile(GAU), and possibly also the tRNA-Met(CAU), has some ability to pair with this codon. A calculation of the AUA CD probability also serves to highlight the difference with the previous section. If the three Ile codons are in equilibrium, , and . Using C. glabrata (I) as an example, as we did for the CUN disappearance, we obtain Pdis = (1\u22120.502)343 = 1.4 \u00d7 10\u2212104, which is indeed vanishingly low. As described in the previous paragraph, the adaptation to using AUA for Met seems to have been gradual. A possible reason for this could be that the change in the tRNA-Met is due to a base modification of the wobble position C, not a mutation (as in the tRNA-Trp, where the wobble position C mutates to U when the UGA codon is reassigned to Trp). The difference between the avoidance of AUA seen in Kluyveromyces and the fairly frequent use of AUA as Met in Saccharomyces may lie in the fraction of tRNAs in which this base gets modified.\nThe other species of fungus in which tRNA-Ile(CAU) is missing is Schizosaccharomyces octosporus, although the gene is still present in S. pombe and S. japonicus. Table\u00a03 shows that AUA is absent altogether in S. octosporus (excluding intronic ORFs), whereas comparison with the other two species suggests that there were 30 or 40 AUA codons present before the loss of the tRNA. Once again, it is unlikely that these codons would have disappeared by chance, and A is a frequent base at FFD sites in all three Schizosaccharomyces species. Hence the disappearance of AUA in S. octosporus is due to selection against this codon after the loss of the tRNA. Bullerwell et al. (2003) show that AUA has not disappeared entirely in the intronic ORFs of S. octosporus. This suggests that there is weaker selection on codon usage in the intronic ORFs because these genes are less strongly expressed than the standard genes. It also confirms that selection must have driven the disappearance of AUA in the standard genes, because if mutation pressure were responsible, it would have affected the intronic ORFs equally. Thus, we conclude that the initial event must have been the deletion of the tRNA-Ile(CAU), and hence this corresponds to the UC mechanism. In this case, the codon has not been reassigned to Met, but there is the potential for this to occur in the future if there is a gain of function in the tRNA-Met.\nFrom Supplementary Table S3, we see that C. albicans, C. parapsilosis, and Y. lipolytica have only one tRNA-Met. Possibly this plays the role of both initiator and elongator. We do not consider it further because this deletion is not associated with any codon reassignment.\nAUA is also reassigned to Met in some Metazoa. The tRNA-Ile(CAU) gene is present in close relatives of the Metazoa, Amoebidium, and Monosiga (Burger et al. 2003) and, also, in Porifera (Lavrov et al. 2005). AUA is Ile in these species. The gene is deleted in all remaining Metazoa (see Fig.\u00a04), which suggests that it was lost after the divergence of the phylum Porifera from other metazoan phyla. Cnidaria are unusual in that they have lost almost all tRNAs from the mitochondrial genome and require import of tRNAs from the nucleus. AUA remains Ile in this group, presumably because the tRNA-Ile(CAU) is imported. There are also two other groups in which AUA remains as Ile: Platyhelminthes, or more specifically Rhabditophora (Telford et al. 2000), and Echinoderms\/Hemichordates. These phyla retain sufficient tRNAs to translate the full code and there is no suggestion of tRNA import. It therefore appears that AUA is translated as Ile by the tRNA-Ile(GAU). This is consistent with the argument of Yokobori et al. (2001) that GA pairing at the anticodon wobble position is tolerated if there is no better alternative anticodon-codon pairing solution available.\nIn the remaining metazoan phyla, AUA is reassigned to Met. The usual tRNA-Met anticodon is CAU. This can be modified in several different ways to pair with AUA in addition to AUG. In Drosophila, modification from C to 5-formylcytidine (f5C) at the wobble position occurs (Tomita et al. 1999b). These authors also show that the unmodified CAU anticodon can translate both codons if the base in the 37th position of the tRNA is t6A (N6-threonylcarbamoyladenosine) instead of A (see also Kuchino et al. 1987). Note that the 37th position is the nucleotide immediately 3\u2019 of the anticodon. In urochordates, the tRNA-Met has a U*AU anticodon (where U* is an unknown derivative of U). This is a third means by which the tRNA-Met has gained the ability to translate AUA. There is insufficient evidence to say exactly where these modifications to the tRNA-Met evolved, and there are many species for which the details of the tRNA-Met are not known. Nevertheless, the existence of these different modifications to the tRNA-Met suggests that the reassignment of AUA to Met has occurred independently at least three times in different phyla after the loss of the original tRNA-Ile(CAU) gene (as shown in Fig.\u00a04). Note that the AUA codon is common in Porifera and Cnidaria, and we did not find any example of disappearance of this codon in any of the Metazoa (see Table\u00a04). Hence, there is no suggestion that the codon disappeared before the loss of the tRNA-Ile(CAU). As the tRNA loss occurred first, this is an example of the UC mechanism, very similar to the case of AUA reassignment in Fungi.\nTable\u00a04.Codon usage in some metazoan lineagesIle and Met codonsSer and Arg codonsAsn and Lys codonsFrequency at FFD sitesAUUAUCAUAAUGAGUAGCAGAAGGAAUAACAAAAAG%U%C%A%GAxinella corrugate222452051307423 35b1710714 862835.911.730.621.8Geodia neptuni207182181187915 55b1510418 892440.77.135.716.4Metridium senile190591101385821 43b128431 792242.517.525.015.0Acropora tenuis182441151136811 34b169015 723143.511.517.627.4Limulus polyphemus241107171a43228 661210344 661935.717.138.88.4Daphnia pulex18793 90a524920 68 1e7543 484136.220.224.918.7Drosophila melanogaster35516216a13300 74 0e19310 81550.52.443.63.6Caenorhabditis elegans25723134a446161263913912 951450.24.137.58.2Trichinella spriralis136114193a964620 71346880 653429.615.242.213.0Katharina tunicate22649125a454636 883513447 793043.112.332.012.5Lumbricus terrestris194105194a642014 63137066 702228.224.336.910.5Terebratulina retusa137147152a34623 78184667 801922.635.334.47.7Fasciola hepatica1198 3996928 741502 18f4468.25.46.919.4Schistosoma mansoni1341515512310210 6250544 39f6052.84.323.319.5Taenia crassiceps17421261011041 4329895 59f4964.31.419.914.5Paracentrotus lividus146531651021114 66154450 88f5425.022.942.69.6Asterina pectinifera12966178782223 54194058110f4828.825.135.410.6Balanoglossus carnosus97148 69771835 19 0e2098 04522.437.829.110.6Saccoglossus kowalewskii18572 96703422 4 0e6068 471240.723.031.25.1Halocynthia roretzi17012112a601254 68c88785 294253.03.626.117.2Cionia intestinalis29719237a34737221c37143161032256.54.833.35.4Branchiostoma lanceolatum18647139a637830 12 0e8626 403437.79.132.820.5Branchiostoma floridae18746138a637333 12 0e8724 403338.58.533.020.0Epigonichthys maldivensis15970122a594935 31 2e7040 373534.713.629.422.3Myxine glutinosa210154182a442328 2d 169771021529.227.434.98.5Homo sapiens124196167a401439 1d 132132 851014.540.438.76.4aAUA is Met in these species.bAGR remains Arg.cAGR is Gly.dAGR is Stop.eAGG is avoided, probably because there is an unmodified G in the wobble position of tRNA-Ser(GCU).fAAA is reassigned to Asn.\nWe interpret the decoding of AUA as Ile in Platyhelminths and Echinoderms\/Hemichordates as indicating that an appropriate change in the tRNA-Met did not occur, and that AUA has always been Ile in these lineages. An alternative is that AUA was assigned to Met once at the point of the loss of the tRNA-Ile(CAU) and that there were reversals of the change in these two groups (as shown in Fig.\u00a02 of Knight et al. [2001a] and Fig.\u00a01 of Yokobori et al. [2001]). However, this scenario does not explain why more than one type of modification of the tRNA-Met should exist. Also, once the AUA codon is captured by Met and mutations have occurred throughout the genome (so that AUA appears in positions where a Met rather than an Ile is required), then the organism is better adapted than it would be if AUA were inefficiently translated by the tRNA-Ile(GAU). Thus we see no reason why a reversal of this change should occur. Telford et al. (2000) have also discussed these two alternative scenarios in a parsimony treatment, however, they did not consider the position of the tRNA-Ile(CAU) gene loss or the fact that there are several independent modifications to the tRNA-Met, both of which are essential points in our interpretation.\nReassignment of AUA from Ile to Met has also been observed (Ehara et al. 1997) in some species of yellow-green algae (Xanthophytes). The entire genome of these species is not yet available and the lack of information about the tRNA genes and codon usage makes it impossible to deduce the mechanism of change. However, it is clear that the change occurred independently of similar changes observed in Metazoa and Fungi.\nReassignments Involving the AGR Block Are Initiated by Loss of a tRNA-Arg Gene\nThe AGR block, assigned to Arg in the canonical code, has undergone multiple reassignments in Metazoa. The tRNA-Arg(UCU) is present in Porifera but is absent in all other Metazoa. In Cnidaria, almost all tRNAs are missing, and tRNA import from the cytoplasm is required. This is not associated with codon reassignment. In the remaining Metazoa (i.e., Bilateria) the loss of the tRNA-Arg(UCU) gives rise to subsequent changes in the genetic code. Loss of this gene leaves the tRNA-Ser(GCU) as the only candidate for translating these codons. Hence, AGR is reassigned to Ser at the base of the Bilateria (Fig.\u00a04). This is classed as the UC mechanism, as it is initiated by a tRNA loss. Note that AGR codons are frequent in Porifera and Cnidaria; therefore there is no suggestion of CD (Table\u00a04).\nThe GCU anticodon of tRNA-Ser usually interacts with only AGY codons prior to the loss of the tRNA-Arg. However, pairing of the tRNA-Ser with AGR codons is also possible to some extent, once there is no longer any competition from the tRNA-Arg (Yokobori et al. 2001; Yokoyama et al. 1995). In Drosophila it is known that the tRNA-Ser(GCU) translates AGA (Tomita et al. 1999), i.e., there is a GA mispairing at the wobble position in the same way as in the mispairing of AUA with tRNA-Ile(GAU) after the loss of the tRNA-Ile(CAU). In Drosophila, AGG is absent, which may indicate that this codon is avoided because of selection against an unfavorable GG mismatch that would occur at the wobble position. It should also be noted that %G and %C are very low in Drosophila (Table\u00a04), so the absence of AGG may simply be a result of mutation pressure. In Daphnia there is only one AGG codon, even though %G is high. This suggests definite avoidance of this codon. However, not all arthropods show this pattern. As an example, there are 12 AGG codons remaining in Limulus. Two other groups in which AGG is absent or very rare are the hemichordates and cephalochordates. Here the tRNA also has a G at the wobble position, which we presume is unmodified.\nIn contrast, Table\u00a04 shows many other cases where AGA and AGG are both frequent. In several invertebrate groups, the G wobble position is mutated to a U, which allows it to pair with all the AGN codons. This is the case with C. elegans and T. spiralis in Table\u00a04, where we see relatively high usage of AGG. Alternatively, in some echinoderms (Matsuyama et al. 1998) and mollusks (Tomita et al. 1998) a base modification from G to m7G also permits it to decode the entire AGN block as Ser.\nThe AGR block has undergone further changes in urochordates, where it is reassigned to Gly, and craniates, where it is reassigned to Stop. The most likely state prior to these changes would be that AGA was moderately frequent and AGG was rare or absent (as with the current hemichordates and cephalochordates). Urochordates contain an additional tRNA-Gly with anticodon UCU (Gissi et al. 2004). This has arisen by duplication of the standard tRNA-Gly(UCC), followed by anticodon mutation. This new tRNA pairs efficiently with AGR codons and outcompetes the tRNA-Ser(GCU). AGR codons become very frequent after the reassignment (Table\u00a04) because mutation from GGR glycine codons occurs. Note that %A is greater than %G in all these genomes, so mutation pressure favors synonymous mutations from GGR to AGR. Since AGA was a moderately frequent codon for Ser prior to the reassignment, and since %A is high, we cannot argue that mutation pressure caused the disappearance of AGA and replacement by AGR prior to the change. Thus, it seems unlikely that CD is responsible for this change. This argument also applies for the reassignment of AGR to Stop in the craniates. AGA is rare in craniates after the change because Stop codons are always rare. These genomes have very high %A, so unless there was a short period when mutation pressure was in the opposite direction to that seen in all the current genomes, then we must suppose that this reassignment occurred without disappearance of the codon. This is puzzling since it implies premature termination of translation of genes containing AGA Ser codons.\nSpruyt et al. (1998) argued that AGA is also read as Gly in Branchiostoma lanceolatum. They found a putative tRNA with anticodon UCU that may have arisen by duplication and mutation of the tRNA-Gly(UCC), as occurred in the urochordates. However, the codon usage numbers suggest that this is not the case and that the sequence identified does not function as a tRNA-Gly. The pattern seen for AGA and AGG in B. lanceolatum resembles that in the hemichordates and some of the arthropods, where there is a single tRNA-Ser(GCU). Also, other cephalochordate genomes are now available, including B. floridae (Boore et al. 1999) and Epigonichthys maldivensis (Nohara et al. 2005), which also show no evidence of reassignment of AGA to Gly. The distinction between B. lanceolatum and B. floridae (e.g., Knight et al. 2001a) therefore seems unfounded.\nReassignment of AAA from Lys to Asn May Proceed Via an Ambiguous Intermediate\nThe reassignment of AAA from Lys to Asn occurred independently in echinoderms and platyhelminthes. Furthermore, the AAA codon is absent in the hemichordate Balanoglossus (see Table\u00a04) but present in the related species, Saccoglossus, where it is translated as Lys, as in the canonical code (M.J. Smith, personal communication).\nMost metazoans in which AAA retains its standard assignment of Lys have one tRNA-Lys(UUU), which pairs with AAR codons, and one tRNA-Asn(GUU), which pairs with AAY codons. Mispairing between a G in the anticodon wobble position and an A-ending codon is implicated in both the reassignment of AUA from Ile to Met and the reassignment of AGR from Arg to Ser, as discussed above. In the case of the tRNA-Asn(GUU), mispairing with AAA is prevented by posttranscriptional modification of the wobble position G to queuosine (Q), which allows pairing with U and C but inhibits pairing with A (Morris et al. 1999).\nIn echinoderms and platyhelminthes, the tRNA-Lys anticodon is mutated to CUU (Tomita et al. 1999a), which pairs only with AAG. Several changes to the tRNA-Asn occur that increase its ability to pair with AAA. The modification of G to Q does not take place (Yokobori et al. 2001), which may allow some degree of GA mispairing. In echinoderms, modification of the second anticodon position from U to Psi (pseudouridine) enhances the ability of the GPsiU-Asn anticodon to recognize the AAA codon. Also in some echinoderms, the mutation of the base immediately adjacent to the 5\u2019 end of the anticodon from a U to a C seems to help the GUU anticodon to recognize AAA (Castrasena et al. 1998). In Balanoglossus the tRNA-Lys has also changed to CUU, but there seem to be no modifications in the tRNA-Asn that would allow AAA to be reassigned to Asn. AAA is therefore unassigned.\nIn all groups in which AAA is still Lys, it is a relatively frequent codon and it is usually more frequent than AAG. %A is usually quite high at FFD sites. Thus, even though Castrasena et al. 1998 argue for the CD mechanism, it seems unlikely to us that AAA disappeared in either the ancestor of the platyhelminthes or the echinoderms. Note that the absence of AAA in Balanoglossus is not shared with Saccoglossus, so we cannot use this to argue that AAA disappeared in the common ancestor of echinoderms and hemichordates. The AI mechanism seems the most likely mechanism for the reassignments in platyhelminthes and echinoderms. The process would have begun by a gain in function of the tRNA-Asn that gave it some ability to pair with AAA, and hence made AAA ambiguous, e.g., the Q modification ceased to occur. Then further changes to the tRNA-Asn would have increased the fraction of AAA codons translated as Asn, and a straightforward mutation of the tRNA-Lys could then have removed this ambiguity.\nAlternatively, it is possible that the changes occurred via the UC mechanism. If the tRNA-Lys mutation happened first, this would leave the tRNA-Asn able to pair inefficiently with the AAA, and subsequent changes to the tRNA-Asn would allow it to recognize AAA more easily. This argument is analogous to the case of the AUA:Ile to Met change or the AGR:Arg to Ser. However, in those two cases the loss of function is the deletion of the original tRNA for the codon in question, which is irreversible. In the AAA case, the loss of function is just the mutation of U to C in the anticodon. This would be a deleterious mutation that could easily reverse, so it is difficult to see why the change would go to fixation. This makes the UC mechanism seem less plausible for AAA. We conclude, therefore, that of all the reassignments considered in this paper, these two examples of AAA reassignment are the best candidates for the AI mechanism.\nIn the case of Balanoglossus, the AAA codon cannot disappear by mutation pressure, and therefore we require a selective reason why this codon should be absent. This can be attributed to either UC or AI mechanisms. If a gain of function of the tRNA-Asn(QUU) brought about by the loss of Q modification happened first, the resulting tRNA-Asn(GUU) would have acquired some ability to decode AAA as Asn and consequently AAA would be ambiguously translated. The negative selection against such ambiguous translation may have driven the AAA codon to disappear. Subsequently, the loss of function of tRNA-Lys(UUU) occurred, brought about by a mutation from U to C at the first anticodon position, and this removed the ambiguity in AAA decoding. Further experimental information on the state of modification of the tRNA-Asn in Balanoglossus would be useful to confirm this.\nAlternatively, the loss of function of tRNA-Lys that prevents it from translating AAA may have occurred first. In the gain-loss framework, this would be a loss without a gain. AAA would be unassigned, and selection against this codon would drive its usage down to zero. This is analogous to the situation in Schizosaccharomyces octosporus, where we argued that the AUA codon was driven to zero after the loss of the tRNA-Ile(CAU) gene. However, this case is again made less plausible by the fact that the change in the tRNA-Lys would be reversible, whereas the loss of the gene from S. octosporus is irreversible. Indeed, the current situation in Balanoglossus seems unstable. A mutation of the tRNA-Lys(CUU) back to UUU would be neutral in the absence of AAA codons. If this occurred, then synonymous mutations of AAG codons to AAA would rapidly occur (since %A is greater than %G). This would drive the system back to the canonical code. The fact that AAA remains absent in Balanoglossus suggests that there is some reason why the reversal of the tRNA-Lys back to UUU would not be neutral. One possibility may be that CUU pairs more efficiently than UUU with the AAG codons, so translational efficiency would favor retention of the CUU anticodon once AAG became the dominant codon for Lys. Another possibility is that unmodified wobble position U bases can often pair with all four bases in four codon families. For this reason, wobble position U\u2019s are often modified when they occur in tRNAs for two-codon families only (Yokobori et al. 2001). Thus prior to the codon reassignment, the tRNA-Lys anticodon would be U*UU, where U* is a posttranscriptional modification of U that permits it to pair with A and G only. If the ability to modify the U were lost by Balanoglossus when the anticodon mutated to CUU, then a reversal of this mutation back to UUU would leave an unmodified U in the wobble position that could pair with both Asn and Lys codons. This would be selected against due to the ambiguity created in the Asn codons. Both these explanations are rather speculative.\nIntroduction of New Stop Codons\nIn addition to the AGR block, which becomes Stop in vertebrates, there are two further cases where a sense codon has been reassigned to Stop. According to the GenBank entry AF288091 (G. Burger, unpublished), UUA has been reassigned from Leu to Stop in the stramenopile Thraustochytrium aureum (Fig.\u00a03). One tRNA-Leu gene in T. aureum has a CAA anticodon instead of the usual UAA anticodon, hence UUA is no longer translated as Leu. %A is high in this and related species, and UUA is frequently used as Leu in related species. This makes it unlikely that UUA disappeared due to mutation pressure. The mechanism cannot be assigned with certainty, but the most likely explanation is that UUA was driven to become rare after the mutation in the tRNA-Leu occurred because of inefficient pairing with the CAA anticodon. Gain of function of the release factor could then have occurred. This scenario corresponds to the UC mechanism (loss before gain). An alternative is that changes in the release factor began to occur in such a way that UUA became ambiguously translated as Leu and Stop. In this case, UUA would be selected against as a Leu codon and would be driven to low frequency, and selection would favor the mutation to C in the anticodon, so that UUA was no longer translated as Leu. This would correspond to the AI mechanism (gain before loss). It is difficult to decide between these scenarios.\nA second similar case is the reassignment of UCA from Ser to Stop in Scenedesmus obliquus (K\u00fcck et al. 2000). In most species, a tRNA-Ser(UGA) would decode UCN codons, but in S. obliquus, the tRNA-Ser has a GGA anticodon that can only pair with the UCY codons. UCA has become the standard Stop codon, used in the 13 standard mitochondrial protein genes. Alongside this, UCG is absent, presumably because it no longer pairs with the tRNA-Ser. The Stop codons UAA and UGA are not used, while UAG has been reassigned to Leu (as discussed above under \u2018reassignments of UAG stop codons\u2019).\nTable\u00a05 shows that %A is fairly high in S. obliquus and related species, and that UCA is a fairly common codon in both P. minor and C. eugametos, where it retains its assignment to Ser (we consider C. reinhardtii separately in the following section). It seems unlikely that UCA would disappear due to mutational pressure. Here again, it is possible to explain the result in terms of a UC or an AI scenario. In the UC scenario, the tRNA-Ser anticodon would mutate to GGA, which would drive UCA and UCG to very low values and permit the release factor to change so that it interacts with UCA. In the AI scenario, the release factor would change first, making UCA ambiguous and causing it to be selected against and driven to low frequency, and allowing the mutation in the tRNA-Ser to occur.\nTable\u00a05.Codon usage in some green algaeLeu codonsIleSer codonsThr codonsGlu codonsArg codonsFFD sitesUUAUUGAUAUCAUCGACAACGGAAGAGAGAAGG%A%GPedinomonas minor41184115538270581135221.31.6Scenedesmus obliquus038401301115731409335.76.6Chlamydomonas eugametos3533817680398653423048.511.7Chlamydomonas reinhardtii0251000000550026.13.7Note. Intronic and other nonstandard ORFs are excluded. Unusual selective effects influence S. obliquus and C. reinhardtii.\nThe full codon usage table of S. obliquus is given by K\u00fcck et al. (2000) and Nedelcu et al. (2000). In Table\u00a05 we have chosen selected codons in order to illustrate that S. obliquus is unusual in other ways. The usual Leu UUA codon is absent, and the tRNA-Leu has a CAA anticodon, which translates UUG only (the same as in T. aureum except that UUA is not used as a Stop codon). AGA is also absent, but there is a tRNA-Arg with CCU anticodon that translates AGG. Nedelcu et al. (2000) identified a tRNA-Arg(UCU) that should interact with AGA and a tRNA-Ile(UAU) that should interact with AUA. However, AGA and AUA are both absent. They state that these two tRNAs are redundant because their corresponding codons are not used. However, we argue that these two tRNAs must be nonfunctional or not expressed, otherwise the corresponding codons would be used. Note that %A > %G in S. obliquus, so if there were functional tRNAs with U at the wobble position, the A-ending codons would be frequent. As an illustration, the Glu codons in S. obliquus do follow the expected trend from the mutational frequencies: GAA is more frequent than GAG because these are both translated by a tRNA-Glu(UUC) with U at the wobble position. The Thr codons shown in Table\u00a05 also have ACA more frequent than ACG, but one final peculiarity of the S. obliquus genome is that it possesses no tRNA-Thr. It is presumed that a tRNA-Thr(UGU) is imported from the cytoplasm.\nImportation of tRNAs from the Cytoplasm to the Mitochondria\nAlthough there are many mitochondrial genomes that possess sufficient tRNAs to translate the full genetic code, there are also many genomes in which this is not the case, and where import of nuclear-encoded tRNAs from the cytoplasm to the mitochondrion must be occurring. Supplementary Table S1 lists the amino acids for which tRNAs are present\/absent in the mitochondrial genome of each species. In the figures, we have used a white arrow symbol to indicate positions where import of multiple tRNA genes must have evolved. We estimate at least nine independent origins of tRNA import: Acanthamoeba, Dictyostelium, Cnidaria, Chytridiomycota, land plants, Pedinomonas, Chlamydomonas, Naegleria+Alveolata, and kinetoplastids (the latter are not shown but are known to use tRNA import). This count depends on the details of the phylogeny: it is possible that Acanthamoeba and Dictyostelium have a common ancestor, which would decrease the count by one, and it is possible that Naegleria is not a sister group to the alveolates, which would increase the count by one.\nIn all the above groups, at least four tRNAs are absent, and it is clear that the remaining tRNAs are insufficient, thus there is no doubt that import must occur. However, there are also many other groups where only a small number of tRNAs are missing. We have already discussed several cases above where the loss of a tRNA leads to the corresponding codon becoming unassigned or reassigned to a new amino acid. These cases leave clear signals in the codon usage patterns. This means that there is no import of a replacement tRNA from the cytoplasm in the cases discussed in previous sections. In contrast, we now discuss several cases where a small number of tRNAs are absent from the mitochondrial genomes but there is no change in the genetic code or unusual codon usage pattern. In these cases it appears that import of one or a few specific tRNAs is occurring.\nThere is no tRNA for Thr in any of the stramenopile genomes. In addition, tRNA-Thr is absent from Reclinomonas, the Rhodophyta, Mesostigma, Scenedesmus, and all the groups where multiple tRNAs are missing. This makes tRNA-Thr the most frequently deleted tRNA. We consider this significant, but we have no plausible explanation why. The tRNA-Thr attracts attention because there are many genomes in which this is the only tRNA missing. Thr codons are nevertheless frequent in these genomes, so there must be a way of translating them. Import of tRNA-Thr from the cytoplasm is quite possible, but we do not know why this specific tRNA should be imported. Alternatively, in the genomes where tRNA-Thr is one of the only ones missing, it is possible that another tRNA is post-transcriptionally edited to become a tRNA-Thr. Two types of tRNA would thus arise from the same gene. This point has been suggested previously (Saks et al. 1998), but as far as we are aware, there is no direct evidence that this occurs.\nThere is no tRNA for the Arg CGN block in several of the stramenopiles (Cafeteria, Thraustochytrium, Chrysodidymus, Ochromonas, Laminaria, Pylaiella) but it is present in Phytophthora and Saprolegnia. Complete genomes are not available for the remainder of the stramenopiles shown in Fig.\u00a03, hence we cannot tell if the gene is present. If the phylogeny is correct, this implies three independent losses of the tRNA-Arg. Either tRNA import or some other tRNA change must be compensating. Recall that this same tRNA-Arg is also absent in several fungi and that this leads to the CGN block becoming unassigned (as shown in Fig.\u00a01). So tRNA-Arg is not imported in the fungi.\nSupplementary Table S1 lists several other genomes where just one or two tRNAs are missing. It is possible that tRNA import occurs or that there are unknown tRNA modifications that compensate for these individual losses. However, we consider this as somewhat uncertain in cases where only one genome is known with the missing gene. This could simply be a failure to locate the gene on the genome or a problem of misannotation.\nThe codon usage of Chlamydomonas reinhardtii is markedly different from C. eugametos (Table\u00a05 and Denovan-Wright et al. 1998). Both of these species have only three tRNAs (Supplementary Table S1), hence tRNA import is required. C. eugametos uses almost all codons, and the codon usage is in line with what we would expect from the FFD site frequencies. However, there are many codons that are absent entirely from the C. reinhardtii genome. This suggests that a restricted set of tRNAs is being imported in C. reinhardtii, which causes selection against codons that cannot be translated, whereas a much broader set is being imported in C. eugametos, which allows a more standard pattern of codon usage. There are several parallels between the codon usage in C. reinhardtii and Scenedesmus obliquus (a sister species; see Fig.\u00a02). UUA (Leu), AUA (Ile), UCG (Ser), and AGA (Arg) are all absent from both. In S. obliquus, this is due to peculiarities in the tRNAs encoded by the mitochondrial genome (see previous section). The correspondence between the codon usage and tRNA set in S. obliquus shows that there is no tRNA import in this species. It therefore appears that the tRNAs that were first imported in C. reinhardtii correspond to those that were needed according to the codon usage pattern of the ancestor of C. reinhardtii and S. obliquus. In C. eugametos, the evolution of tRNA import has proceeded further. A more complete set is imported and the codon usage has relaxed back to a more standard pattern due to mutation.\nDiscussion\nDistinguishing the Mechanisms\nThe gain-loss framework that we use to classify mechanisms of codon reassignment was introduced in discussion of our computer simulations (Sengupta and Higgs 2005). Here we wish consider to what extent this classification is applicable to the real events. The CD mechanism is defined by the fact that the codon disappears from the genome and is replaced by synonymous codons prior to any changes in the tRNAs and release factors. The reasons for the disappearance are random drift and directional mutation pressure. In simulations it is obvious whether a codon has disappeared. In real examples, there is clear evidence if the codon usage is zero or close to zero in the species close to the reassignment point, and there is also clear evidence if mutation pressure is in the right direction to cause CD. It is, of course, never possible to say that the codon usage was exactly zero in some genome in the past. A practical definition of the CD mechanism for real cases is that absence or extreme rarity of the codon is the major factor that initiates the codon reassignment. There should be evidence that the codon was rare and that the probability of CD was not too low. We have shown that this is true in the cases where we argue that the CD mechanism occurred. Therefore, the distinction of CD from the alternatives is one that we feel can be reliably made in real examples.\nIn cases where we have determined that the codon did not disappear, the central question is to distinguish between UC and AI mechanisms. The UC mechanism is defined by the fact that the loss occurs before the gain, whereas in the AI mechanism, the gain occurs before the loss. Although this distinction is obvious in simulations, it can be more difficult in real cases, as we did not observe the order of events. Nevertheless, we gave several examples where we feel a reliable classification of UC can be made. In particular, the reassignments of AUA from Ile to Met and AGR from Arg to Ser are both associated with the deletion of a tRNA from the genome. The loss event is an irreversible gene deletion that leaves the organism in a deleterious state. The gain event is then positively selected in response to this loss. In contrast, in the case of the reassignment of AAA from Lys to Asn, the loss event is a mutation in the anticodon of the tRNA-Lys. This is reversible, so selection would favor mutation back to the original state, which seems more likely to occur than making a codon reassignment. The gain in this case is probably due to the cessation of the Q modification process, which could occur due to deleterious mutations in the modifying enzyme or disruption of the transport of the enzyme to the mitochondria. These changes seem less easily reversible than a single mutation in the anticodon. Therefore we argue that the gain occurred first and that the loss occurred in response. This makes the AAA reassignment the most likely example of the AI mechanism. The cases where both loss and gain seem easily reversible are the most difficult to classify, and both AI and UC scenarios can be proposed. (These arguments are based on the assumption that the ambiguous and unassigned states are deleterious with respect to the original code. We deal with the alternative suggestion that ambiguous translation can be positively selected later in the Discussion.)\nWe just argued that the AUA Ile-to-Met case is initiated by deletion of the tRNA-Ile(CAU) and that this change can be reliably classed as UC. Nevertheless, it is possible that ambiguous translation plays a role in this reassignment in the following sense. After the deletion, the AUA codon would be translated inefficiently by the tRNA-Ile(GAU), but it is possible that there might also be some interaction with the tRNA-Met(CAU). When the tRNA-Met becomes modified, it definitely gains the ability to translate AUA. So, ambiguous translation of AUA as both Ile and Met at some points in this process is not unlikely. Before the original tRNA-Ile(CAU) is deleted, any slight ability of the other two tRNAs to translate AUA would be irrelevant. Also, after the modification of the tRNA-Met, any slight ability of the tRNA-Ile(GAU) to translate AUA would be irrelevant. The ambiguity, if it existed, would only be relevant when there is a competition between two poorly adapted tRNAs. Despite all this, the gain in function of the tRNA-Met only occurs after the loss of the tRNA-Ile(CAU), so this reassignment counts as UC not AI. We recommend the use of the term AI only when ambiguous translation occurs as a result of a gain occurring before a loss, and where competition occurs between two well-adapted tRNAs.\nThe reassignment of AGR from Ser to Gly in urochordates involves a gain due to the duplication and anticodon mutation of the tRNA-Gly. In this case, too, it is plausible that an AI stage for AGR decoding may have resulted after the gain of the new tRNA-Gly. The ambiguity would eventually be removed due to the selection of the superior recognition of AGR by the new tRNA-Gly. However, this only happens after the deletion of the original tRNA-Arg(UCU). We have counted the changes from Arg to Ser and Ser to Gly as separate events because the amino acid changes twice. However, we may think of the duplication of the tRNA-Gly as the gain that finally compensates for the loss of the tRNA-Arg considerably earlier in evolution. The AGR:Ser to Gly case is one of the examples where the number of tRNAs increases (see Table\u00a06 and discussion below), but this only returns the number back to its value in the canonical code; it does not represent an increase with respect to the canonical code.\nTable\u00a06.Summary of mechanisms of codon reassignment in mitochondriaCodon reassignmentNo. of timesCan this be explained by GC\u2192AU mutation pressure?Change in no. of tRNAsIs mispairing important?Reassignment mechanismUAG: Stop \u2192 Leu2Yes. G \u2192 A at 3rd position.+1NoCDUAG: Stop \u2192 Ala1Yes. G \u2192 A at 3rd position+1NoCDUGA: Stop \u2192 Trp12Yes. G \u2192 A at 2nd position.0Possibly. CA at 3rd position.CDCGU\/CGC\/CGA\/CGC: Arg \u2192 unassigned5Yes. C \u2192 A at 1st position.\u20131NoCDCUU\/CUC\/CUA\/CUG: Leu \u2192 Thr1Yes. C \u2192 U at 1st position.0NoCDCUU\/CUC\/CUA\/CUG: Thr \u2192 unassigned1No\u20131NoCDAUA: Ile \u2192 Met or unassigned3\/5aNo\u20131Yes. GA at 3rd position.UCAAA: Lys \u2192 Asn2No0Yes. GA at 3rd position.AIAAA: Lys \u2192 unassigned1No0Possibly. GA at 3rd position.UC or AIAGA\/AGG: Arg \u2192 Ser1No\u20131Yes. GA at 3rd position.UCAGA\/AGG: Ser \u2192 Stop1No0NoAIbAGA\/AGG: Ser \u2192 Gly1No+1NoAIbUUA: Leu \u2192 Stop1No0NoUC or AIUCA: Ser \u2192 Stop1No0NoUC or AINote. CD, codon disappearance; UC, unassigned codon; AI, ambiguous intermediate.aThe loss of the tRNA-Ile has occurred three times (once in metazoa, once in yeasts, and once in S. octosporus) but the reassignment of the codon to Met has occurred at least five times (three times in metazoa and twice in yeasts).bIf AGR was fully reassigned to Ser prior to these changes, then they are probably AI. However, they may also be considered as a final response to the loss of the original tRNA-Arg, in which case we might count them as UC.\nA slow response to a tRNA deletion occurs for both the tRNA-Ile(CAU) and the tRNA-Arg (UCU), which are deleted at roughly the same time in the ancestral Bilateria. This would leave AUA inefficiently translated as Ile and AGR inefficiently translated as Ser. Both these situations remain in some phyla today. However, in other groups, AUA has been captured by Met, and AGR either has become a useful Ser codon (due to mutation of the tRNA-Ser) or has been reassigned to Gly or Stop. These secondary changes may have occurred considerably later than the deletions of the original two tRNAs.\nThe fourth mechanism occurring in the gain-loss framework is the compensatory change mechanism. This would correspond to a case where the loss and gain become fixed in the population at the same time. Organisms with either the gain or the loss but not both would only occur as rare variants, and would never be frequent in the population. This mechanism is expected in the theory due to the analogy of the problem with compensatory mutation theory (Kimura 1985; Higgs 19982000). In the simulations of codon reassignment (Sengupta and Higgs 2005) it was possible to distinguish a few cases where this occurred. It may well be that the compensatory change mechanism occurs in real cases, and it could be an explanation in cases where both AI and UC seem equally plausible. However, it would be very difficult to distinguish this from either the UC or the AI mechanism after the event.\nComparison with Previous Surveys\nRecent papers by Knight et al. (2001b) and Swire et al. (2005) have also carried out surveys of changes in the mitochondrial genetic code. Knight et al. (2001b) highlighted the fact that variation in GC content plays a major role in the CD mechanism. Most mitochondrial genomes are subject to mutation pressure from GC to AU. Since third codon positions are often synonymous, they argued that the CD mechanism would predict that the codons that are reassigned should have C or G at the third position. As this is not always the case, they argued against the CD mechanism. However, it is not the third codon position per se that matters; it is the position at which the synonymous mutation occurs. Table\u00a06 gives examples where synonymous mutations driven by GC-to-AU mutation pressure have occurred at all three positions. Knight et al. (2001b) came down strongly in favor of the AI mechanism. We wish to redress the balance here. Our data show the CD mechanism is the best explanation in over half the cases. We agree with Swire et al. (2005) that CD applies well to stop codon reassignments, but does not usually apply to sense codon reassignments. The cases of CUN and CGN reassignments are exceptions to this, where we argue for CD with sense codons.\nIn their proposal of the AI mechanism, Schultz and Yarus (1994) identified three specific types of codon-anticodon mispairing that might be important in allowing ambiguous translation of a codon: GA at the third codon position, CA at the third codon position, and GU at the first codon position. Knight et al. (2001b) considered whether these mispairing events were implicated in the reassignments in mitochondrial genomes. In Table\u00a06, we also do this (cf. Table\u00a03 of Knight et al.). We agree that GA mispairing at the third codon position is important in the reassignments of AAA and AGR. For the AUA Ile-to-Met transition, we argue that GA mispairing between tRNA-Ile(GAU) and the AUA codon is important, otherwise deletion of the tRNA-Ile(CAU) would not be possible. Mispairing of the unmodified tRNA-Met(CAU) with AUA (as in the Knight et al. table) might also be relevant, but we do not think this is significant prior to the deletion of the tRNA-Ile(CAU). The reverse transition of AUA from Met back to Ile listed by Knight et al. does not occur in our interpretation (Fig.\u00a04). Also, the reassignment of UAA from Stop to Tyr listed by Knight et al. is no longer thought to occur. Knight et al. argue that CA mispairing is important for the UGA Stop to Trp transition. There is evidence for this in Bacillus subtilis (Lovett et al. 1991, Matsugi et al. 1998), where UGA is ambiguous. It is not clear whether this is a key feature in the UGA reassignments in mitochondria. There are many species where the canonical system works fine (UGA is Stop, UGG is Trp, and the tRNA-Trp has CCA anticodon). It is possible that if CA mispairing started to occur due to some change in the tRNA-Trp outside the anticodon, UGA would become ambiguous, and this would provide a selective pressure against UGA codons that would help to drive them to disappear. However, we are not aware of any evidence for this in mitochondria. The disappearance of UGA is likely to occur in any case due to mutation pressure and drift, and this seems to be a more important factor. The other cases listed by Knight et al. where mispairing is implicated do not occur in mitochondria, and we did not consider them. From Table\u00a06, we would argue that mispairing is important in some but not all reassignments. However, the existence of mispairing does not necessarily imply the AI mechanism occurred, and mispairing is not a diagnostic feature of AI, as previously envisioned.\nIf translation of a codon becomes inefficient due to tRNA loss, then selection will act against it and it will tend to be replaced by synonymous codons. If this situation remains for some time without any compensating gain of function in another tRNA, then the codon may in fact disappear. We have argued that this is the case in S. octosporus, where the tRNA-Ile(CAU) has been deleted and AUA has disappeared, and also in the two Kluyveromyces species, in which AUA is very rare (Table\u00a03). A similar thing happens with the AGR reassignment: there are several invertebrate groups in which AGG becomes rare after the deletion of the tRNA-Arg because of inefficient pairing with the tRNA-Ser when it retains its original GCU anticodon (Table\u00a04). In cases where there is a full gain in function of the new tRNA, the codon in no longer selected against. This is seen when modifications occur in both the tRNA-Met and the tRNA-Ser.\nGiven that the loss of the tRNA in the first step of the UC mechanism is likely to be deleterious, anything that offsets the deleterious effect to some extent will increase the likelihood of the UC mechanism occurring. It has been suggested that deletion of a tRNA gene might be favored since it reduces the total length of the genome and hence allows more rapid replication (Andersson and Kurland 1991, 1995). We considered this effect in our simulations (Sengupta and Higgs 2005) and showed that selection for reduction in genome length does indeed increase the likelihood of the UC mechanism. This factor will be more important in smaller genomes because the relative change in length for deleting a tRNA will be larger. For the cases of deletion of the tRNA-Ile and tRNA-Arg that we are considering, we do not have an experimental measurement of the selective disadvantage to the translation system of loss of the gene, so it is not possible to say whether the potential advantage of shortening the genome is large or small compared to this. We do not wish to argue that these reassignments are \u201ccaused\u201d by selection to reduce the genome size, but we do wish to say that it is the chance deletion of these tRNAs that initiated the reassignment in these cases, and that the fixation in the population of the genome with the deletion could well be aided by selection for more rapid genome replication.\nHowever, there is no general trend for tRNA deletion at codon reassignment. We have given the changes in tRNA number associated with our interpretation of the reassignment events in Table\u00a06. There are three cases of tRNA gain, each of which occurs once. These are due to a tRNA duplication followed by an anticodon mutation. There are four cases of tRNA deletion, which together occur 10 times. Changes in release factors are not included in this table because these genes are not coded by the mitochondrial genome. In Table\u00a06 in the supplementary information of Swire et al. (2005), a distinction is made between the theoretical and the observed changes in tRNA number. Given the genome information now available, this distinction is no longer necessary. The AGR Arg to Ser reassignment in Bilateria is classed as an observed gain by Swire et al. (their case 9) because they are comparing with Cnidaria. However, it is clear that tRNA import has evolved separately in Cnidaria and that it does not occur in either Porifera or the ancestral Bilaterian. The proper comparison for Bilateria is with Porifera, where the tRNA-Arg is present; therefore it is evident that the gene has been deleted in Bilateria at the point of the codon reassignment. Case 6 of Swire et al. is the AUA Ile to Met that occurs in yeasts. We have shown in this paper that the tRNA-Ile(CAU) gene is present in the fungi prior to this reassignment and is deleted at the point of the reassignment. Once again, this is a clear deletion and there is no difference between the observed and the expected change.\nAn important issue in the AI mechanism is the nature of the selective effect on an ambiguous codon. In our model (Sengupta and Higgs 2005), we assumed that ambiguous translation would be unfavorable because the wrong amino acid would be inserted part of the time. However, it has been suggested that ambiguous translation may be favorable (Santos et al. 1999). Swire et al. (2005) also gave an argument related to the cost of amino acid biosynthesis, whereby ambiguous translation might be favorable because the savings in the cost of using an energetically cheaper amino acid might outweigh the penalty of the ambiguity. The ambiguity penalty may be very small at particular sites where the amino acid substitution is neutral or almost neutral; hence, the cost savings would make this change advantageous. This mechanism is possible and we are currently in the process of studying it using simulations of the same type as those used by Sengupta and Higgs (2005). A problem we see with this argument is that, if an amino acid substitution were favored by cost saving, it would be easier to achieve this by a straightforward mutation in the gene without changing the genetic code. Such mutations could occur in places where it was favorable and not in places where it was not. There must be some sites in the protein where the substitution is deleterious, even if there are also some neutral sites. On the other hand, if the codon were reassigned, this would force changes in all sites in the protein, whether favorable or not, which would act against the postulated benefit of the change.\nWe do not think that positive selection for ambiguous translation can be a general explanation of sense-to-sense reassignments. We have already given one case where a sense-to-sense reassignment is better explained by the CD mechanism and several where it is better explained by UC. Swire et al. (2005) did not distinguish between AI and UC. However, the cost-savings argument is less relevant for UC than AI. In AI, if the cost-savings scenario occurred, it would be central to the argument because it would provide a reason why the gain occurred first, i.e., it would be the driving force for the reassignment. In the UC mechanism, the cause of the transition is the chance loss event, which is presumed to be deleterious and is not driven by selection. The gain event would then be positively selected because it would get rid of the penalty from the slow translation that occurs when there is no well-adapted tRNA. The cost-savings argument might also favor the gain event in this case. This would change the details of the selective forces favoring the gain, but this would only be relevant after the initial deletion. However, there is no need to postulate the existence of the neutral sites and cost savings in the UC case, because the gain would be driven by selection to improve translational efficiency anyway. Thus, if the cost-savings scenario occurred in a UC reassignment, it would be a minor detail, not a driving force.\nIn any case, the best-documented example of ambiguous translation of a codon is the CUG codon in the Candida nuclear genome (Santos et al. 1999, 2004), and this example now provides evidence that ambiguous translation is deleterious. It has been shown that the reassignment is driven by the gain of a new tRNA-Ser with anticodon CAG that translates the CUG codon, which is normally Leu. This case was formerly cited as an example of positive selection for ambiguous translation. However, Massey et al. (2006) have now shown that there is selection against the ambiguous CUG codons that causes them to become very rare in their original Leu positions. Most occurrences of CUG in Candida are newly evolved in Ser positions. This shows that there is negative selection on the ambiguous codon. (Note that this is the third reason for a codon to become rare: in the CD mechanism, the codon disappears due to mutation pressure; in the UC mechanism, the codon is inefficiently translated and selected against and therefore might become rare; in the AI mechanism, the codon might be selected against because it is ambiguous, and therefore might become rare.) Another study suggesting that ambiguous translation is deleterious is that by Kim et al. (2000), who studied many examples of anticodon mutations in tRNA-Phe from E. coli. In cases where the cells expressing the mutant tRNA were viable, it was found that the mutant tRNA was charged with the amino acid corresponding to the new anticodon, i.e., there was no ambiguous translation. However, in other cases, expression of the mutant tRNA was lethal. It was presumed that the mutant tRNA was still charged with Phe, although its anticodon corresponded to another amino acid. This would cause ambiguous translation, which was apparently deleterious in the experiment.\nThe arguments in this paper apply only to codon reassignments that have occurred since the establishment of the canonical code. They do not apply to the origin and buildup of the canonical code. We believe that there is a good case that positive selection was important in the establishment of the canonical code (Freeland et al. 2003; Ardell and Sella 2002) and that this accounts for the nonrandomness and apparent optimization of the code. Adding a new amino acid to the code opens up a whole new realm of protein sequences that can be formed using the new amino acid. Therefore, there is ample scope for positive selection to act in determining the codons to which each amino acid is initially assigned. Translational error rates and mutation rates may have been large in early organisms, so minimizing the effects of these errors may have been important. However, we do not think that positive selection guides the codon reassignments that have occurred after the establishment of the canonical code. There is little to be gained from a codon reassignment in modern organisms because no new amino acid is being added, and the effects of minimizing error will be very small when both translational error and mutation rates are as small as they are today.\ntRNA Evolution\nAlthough Table\u00a06 shows no strong trend to reduce the number of tRNAs at codon reassignment events, we feel that this obscures the very large role that tRNA deletion has had in the evolution of the mitochondrial translation system. A major difference between bacterial and mitochondrial systems is that four-codon families in bacteria require at least two tRNAs\u2014one with wobble position G to translate U- and C-ending codons and one with wobble position U to translate A- and G-ending codons. Most mitochondria require only one tRNA with wobble position U for all four codons. In metazoan mitochondria the second tRNA has been deleted in all eight four-codon families. The same genes have been deleted in other groups too, and we suspect that there have been independent deletions of the same gene, although demonstrating this would be difficult because we do not have a fully resolved tree for the earliest branches of the eukaryotes. Nevertheless, the same process has occurred at least once in all eight four-codon families, which is relevant to the discussion on genome streamlining, even though there is no change in the code. Loss of a tRNA with wobble position G may have been slightly deleterious originally when it occurred in the early stages of mitochondrial evolution, but it is possible that the ribosome has become sufficiently flexible to tolerate this. It could also be that the loss still comes with a price of decreased accuracy and\/or speed of translation. Selection for reduction in genome length might well play a role in these deletions also. The 22 tRNAs in animal mitochondria represent the minimal possible set. It is only possible to reduce the number further by making codons unassigned or by evolving a mechanism of tRNA import from the cytoplasm. We have seen that tRNA import has evolved multiple times. If import is possible, then there will be no deleterious effect of further tRNA deletions from the mitochondrial genome, and selection for shorter genome length may favor the fixation of variants in which tRNAs have been deleted. Thus rapid tRNA deletion might be expected in lineages in which a general import process is operating.\nIt is known that many protein genes formerly present on the mitochondrial genome have been transferred to the nuclear genome (Blanchard and Lynch 2000). Many of these code for proteins that are required in the mitochondrion and are targeted back to the organelle after synthesis. In cases where tRNAs are imported, it is interesting to ask whether these tRNAs are in fact formerly mitochondrial tRNA genes that have been transferred to the nucleus, or whether the existing nuclear tRNAs have become functional in the mitochondria and the mitochondrial genes have simply been deleted. We attempted to answer this question with the kinetoplastids, Trypanosoma brucei and Leishmania tarentolae, which possess no mitochondrial tRNAs and where import is known to occur (Simpson et al. 1989; Hancock and Hajduk 1990; Schneider and Marechal-Drouard 2000). The full set of tRNAs in the nuclear genome of these species is known. We carried out phylogenetic analysis comparing these genes with tRNAs from other eukaryotic nuclei, bacteria, and mitochondria. For each separate tRNA, the kinetoplastid genes clustered with eukaryotic nuclear genes and not with mitochondria. We do not show these results because the trees have very low statistical support due to the short sequence length of the tRNAs. Nevertheless, we found no evidence that any of the kinetoplastid tRNAs had been transferred from the mitochondrial genome.\nThe identity elements and antideterminants of tRNAs are key aspects of the molecules that play a crucial role in the proper aminoacylation and prevention of misacylation of a tRNA (Gieg\u00e9 et al. 1998). These are important factors in understanding changes in the genetic code. Most aminoacyl tRNA-synthetases use the anticodon as a key identity element for proper recognition of the tRNA. Two exceptions are Leu and Ala, where the anticodon has no role to play in the proper charging of the cognate tRNA with the amino acid. In such cases, mutations in the anticodon do not affect the aminoacylation of the tRNA. Such mutations can result in codon reassignment if the resulting anticodon acquires the ability to pair with a codon originally associated with a different amino acid. The reassignment of UAG from Stop to Leu in the mitochondrial genome of some species of green plants provides such an example (Laforest et al. 1997). Another possibility is that misacylation of a tRNA due to a change in an aminoacyl tRNA-synthetase could be a cause of a codon becoming ambiguous. The tRNA would be ambiguously charged, but there would only be one type of tRNA for the codon. However, this does not seem to occur in the mitochondrial reassignments we considered here.\nConclusions\nWe have given arguments above as to which mechanisms seem most likely in each of the codon reassignments in mitochondrial genomes. We have shown that the many reassignments of stop codons to sense codons are readily explained by CD, given the biased base composition of most mitochondrial genomes and the small total number of occurrences of stop codons in these genomes. Disappearance of sense codons is more difficult because the total number of codons for the corresponding amino acid is large. A very strong mutational bias is required for sense codons to disappear. However, in one group of yeast species, an extreme bias against C does exist, and we argue that the reassignment of CUN and CGN codons in these species is attributable to CD. In the other examples of sense codon reassignments, the mutational bias is in the wrong direction for causing CD, and the probability of disappearance is negligible.\nWhere the codon does not disappear, we have emphasized the important distinction between the UC and AI mechanisms. The case for the UC mechanism is most clear when the reassignment is associated with a tRNA deletion. We then argue that the deletion initiated the process and the codon reassignment occurred as a response to this. The UC mechanism does not rely on selection for reducing genome length, but if such selection were significant, this would increase the likelihood of this mechanism. Many nonessential tRNAs have been deleted during mitochondrial genome evolution and these did not initiate codon reassignments because the original code was still functional after the deletion. However, this makes it clear that chance tRNA deletion is a relatively common event. We also observed several cases where a good argument can be made for the AI mechanism, i.e., where the reassignment arose because the codon first became ambiguous. In other cases, scenarios for both AI and UC seemed equally plausible, and it is difficult to distinguish them after the event.\nWe see these genetic code changes as chance events, rather than as changes governed by positive selection. Disappearance is a chance event that occurs under drift when there is strong mutational bias in base frequencies. If a change in tRNAs or release factors happens to occur while a codon is absent, then a codon reassignment can occur. However, this is a chance event: the codon frequency could drift back to a higher level without any reassignment occurring. Our interpretation of reassignments via UC and AI mechanisms is that they too are initiated by chance events, such as the deletion of a tRNA gene or a change in the process of base modification in an anticodon. These changes are probably slightly deleterious, but efficient functioning of the translation system can be restored by making the codon reassignment. The view that AI states are driven by positive selection seems unlikely to us at present. The origin of the canonical code is outside the scope of the present paper. However, we emphasize that the situation in codon reassignments in modern organisms is different from that during the early evolution of the canonical code, where positive selection probably had an important role.\nWe conclude that our gain-loss framework is suitable as a description of the real codon reassignment events. It emphasizes that there are several mechanisms that are alternatives within a larger picture, and that it is not always profitable to discuss these mechanisms as though they were mutually exclusive. These mechanisms can and do occur in nature, and one mechanism is not sufficient to explain all cases.\nElectronic Supplementary Material\nSupplementary material","keyphrases":["codon reassignment","mitochondrial genetic codes","unassigned codon mechanism","ambiguous intermediate mechanism","codon disappearance mechanism"],"prmu":["P","P","P","P","R"]}
{"id":"Transgenic_Res-3-1-1829418","title":"Effects of FVB\/NJ and C57Bl\/6J strain backgrounds on mammary tumor phenotype in inducible nitric oxide synthase deficient mice\n","text":"The ability to genetically manipulate mice has led to rapid progress in our understanding of the roles of different gene products in human disease. Transgenic mice have often been created in the FVB\/NJ (FVB) strain due to its high fecundity, while gene-targeted mice have been developed in the 129\/SvJ-C57Bl\/6J strains due to the capacity of 129\/SvJ embryonic stem cells to facilitate germline transmission. Gene-targeted mice are commonly backcrossed into the C57Bl\/6J (B6) background for comparison with existing data. Genetic modifiers have been shown to modulate mammary tumor latency in mouse models of breast cancer and it is commonly known that the FVB strain is susceptible to mammary tumors while the B6 strain is more resistant. Since gene-targeted mice in the B6 background are frequently bred into the polyomavirus middle T (PyMT) mouse model of breast cancer in the FVB strain, we have sought to understand the impact of the different genetic backgrounds on the resulting phenotype. We bred mice deficient in the inducible nitric oxide synthase (iNOS) until they were congenic in the PyMT model in the FVB and B6 strains. Our results reveal that the large difference in mean tumor latencies in the two backgrounds of 53 and 92 days respectively affect the ability to discern smaller differences in latency due to the Nos2 genetic mutation. Furthermore, the longer latency in the B6 strain enables a more detailed analysis of tumor formation indicating that individual tumor development is not stoichastic, but is initiated in the #1 glands and proceeds in early and late phases. NO production affects tumors that develop early suggesting an association of iNOS-induced NO with a more aggressive tumor phenotype, consistent with human clinical data positively correlating iNOS expression with breast cancer progression. An examination of lung metastases, which are significantly reduced in PyMT\/iNOS\u2212\/\u2212 mice compared with PyMT\/iNOS+\/+ mice only in the B6 background, is concordant with these findings. Our data suggest that PyMT in the B6 background provides a useful model for the study of inflammation-induced breast cancer.\nIntroduction\nTransgenic mouse models that develop spontaneous mammary adenocarcinomas have proven valuable in revealing molecular mechanisms underlying tumorigenesis and metastasis (Cardiff 2003; Fargiano et\u00a0al. 2003). Models target specific pathways depending on the transgene being expressed under the control of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) or whey acid protein (WAP) mammary gland promoters and thereby replicate genetic defects in subsets of human tumors (Cardiff and Wellings 1999; Rosner et\u00a0al. 2002). Molecules from pathways known to be important in the pathogenesis of human breast cancer such as the Wnt, Ras and ErbB2\/Neu pathways have been utilized, however few spontaneous mammary tumor model systems develop metastasis, a crucial aspect of cancer progression.\nAs demonstrated by gene expression analysis (Ma et\u00a0al. 2003), the polyomavirus middle T (PyMT) model in the FVB\/N (FVB) background activates the same signaling pathways as erbB2, an oncogene amplified or overexpressed in approximately 30% of human breast cancers. The model has been particularly useful for genetic studies due to its short tumor latency and high incidence of pulmonary metastasis (Guy et\u00a0al. 1992). These features have enabled global expression profiling of PyMT tumors from different genetic backgrounds to identify signatures of tumor virulence (Qiu et\u00a0al. 2004) that are consistent with a gene set predictive of metastasis in human tumors (Ramaswamy et\u00a0al. 2003). Hence, the model has been used effectively to dissect early and late stages of mammary tumor development (Guy et\u00a0al. 1994; Lin et\u00a0al. 2001; Webster et\u00a0al. 1998; Williams et\u00a0al. 2004). In women with breast cancers involving this pathway, the course of the disease varies widely. One powerful means to understand that variability is to identify useful animal models. The PyMT breast cancer model can be used in genetic crossing experiments to identify loci having modifier effects on either tumorigenesis or metastasis (Le Voyer et\u00a0al. 2000). An understanding of the genetic polymorphisms underlying these effects is important in the study of human cancer as genetic variability affects host-tumor interactions in numerous ways and is an important factor in the response to therapy.\nWidely varied tumor responses to polyoma virus infection among different inbred strains of mice have been well documented (Freund et\u00a0al. 1992; Gross 1983). Much of this difference can be attributed to the major histocompatibility complex (MHC) which results in immunologically mediated resistance associated with an H-2b haplotype such as in C57Bl strains (Law et\u00a0al. 1967; Ting and Law 1965). Other inbred strains, notably those with the H-2d and H-2k haplotypes have moderate and weak tumor resistance respectively (Freund et\u00a0al. 1992). The FVB strain has an H-2q haplotype, which may be an additional MHC locus that confers susceptibility to polyoma virus induced tumorigenesis. Alternatively, non-MHC genes can also result in a dominant pattern of tumor susceptibility as in the case of the C3H\/BiDa strain (Freund et\u00a0al. 1992).\nStrain variations are also relevant to the study of genetic mutations in mouse models of disease. An analysis of targeted disruptions on mammary tumorigenesis in the PyMT model requires breeding of knockout models usually developed in the 129\/SvJ-C57Bl\/6J background into the FVB background. It is commonly recognized that the FVB strain is susceptible while the C57Bl\/6J (B6) strain is more resistant to PyMT mammary tumorigenesis and F1 crosses of these strains increase tumor latency by approximately 6\u00a0days (Lifsted et\u00a0al. 1998). However, it is not clear what effects continued backcrossing has on the analysis of targeted mutations in the PyMT model.\nPreviously we reported that inducible nitric oxide synthase (iNOS) deficiency in the PyMT model in the B6 background resulted in an increase in tumor latency that was not observed in the FVB background (Ellies et\u00a0al. 2003). Since the issue of strain background is an important one for analysis of mouse models we have made iNOS knockout mice congenic in the FVB and B6 backgrounds and carried out a comparison of tumorigenesis and metastasis in both strains.\nMaterials and methods\nAnimals\nPyMT (FVB\/N-Tg(MMTV-PyVT)634Mul\/J) mice (Guy et\u00a0al. 1992) were obtained from W. Muller (McMaster University, Ontario, Canada). We acquired PyMT mice that had been backcrossed 5 generations into the B6 background from A. Varki (UCSD, La Jolla, CA). Control (C57Bl\/6J) and iNOS deficient mice (B6.129P2-Nos2tm1Lau\/J; Laubach et\u00a0al. 1995) were purchased from The Jackson Laboratory, Bar Harbor, ME. The PyMT B6 mice were further backcrossed until congenic in the B6 background and then bred with iNOS\u2212\/\u2212 mice. iNOS\u2212\/\u2212 mice were bred into the FVB background until congenic (>10 generations) and then crossed with PyMT FVB mice. Female mice heterozygous for the PyMT transgene and homozygous for the wild type or mutated iNOS gene were used in these studies. For clarity and brevity, we have designated FVB\/N-Tg(MMTV-PyVT)634Mul\/JxB6.129P2-Nos2tm1Lau\/J+\/+ mice PyMT\/iNOS+\/+ and FVB\/N-Tg(MMTV-PyVT)634Mul\/JxB6.129P2-Nos2tm1Lau\/J\u2212\/\u2212 mice PyMT\/iNOS\u2212\/\u2212. FVB mice were palpated twice weekly from 4\u00a0weeks of age and B6 mice once weekly from 8\u00a0weeks of age to monitor mammary tumor development. Tumors were measured in 2 dimensions using calipers and tumor volume estimated using the standard calculation for a sphere 4\/3\u00a0\u00d7\u00a03.14\u00a0\u00d7\u00a0a\u00a0\u00d7\u00a0b2 where a is the smaller diameter and b is the larger diameter. After euthanizing the mice, mammary tumors were dissected and weighed and the total tumor burden calculated (tumor weight\/body weight). All studies followed the NIH guidelines for the care and treatment of experimental laboratory rodents.\nMammary gland whole mounts\nPairs of inguinal mammary glands were fixed in formalin or Carnoy\u2019s fixative, dehydrated through a graded series of ethanol solutions and defatted in xylene. Following rehydration, the mammary epithelium was stained with carmine stain (Sigma Chemical, St Louis, MO) for 30\u00a0min. After removing excess stain by washing in water, samples were dehydrated and stored in methyl salicylate (Sigma Chemical, St Louis, MO).\nTumor microvascular density\nCryosections of OCT embedded tumor samples were cut at 7\u00a0\u03bcm and stained with anti-CD31 clone 13.3 (1:200, BD Pharmingen, San Diego, CA) as previously described (Ellies et\u00a0al. 2003). Five random fields per tumor were captured on the Spot RT digital camera system (Diagnostic Instruments, Sterling Heights, MI) at 200\u00d7 magnification and analyzed using the public domain NIH Image program (http:\/\/rsb.info.nih.gov\/nih-image\/). Microvessel areas were normalized to 1\u00a0mm2 of epithelium.\nLung metastases\nParaffin embedded lungs were serial sectioned and stained with hematoxylin and eosin. Both the numbers and areas of lung metastases were measured by light microscopy using images and software from the Spot RT digital camera system. Lung area per section was measured using a dissecting scope and metastatic tumor burden calculated (mm2 of lung metastases\/cm2 of lung tissue) for each animal using data from 3 sections at least 100\u00a0\u03bcm apart. At least 13 PyMT\/iNOS+\/+ or PyMT\/iNOS\u2212\/\u2212 mice were used in this analysis.\nStatistical analysis\nThe percentage of mice tumor free in each group was analyzed using a Kaplan-Meier survival analysis, and the log-rank statistic was used for comparison of the curves between groups. The t-test was used for comparison of 2 groups and the analysis of variance (ANOVA) followed by the Bonferroni post-test was applied to multiple comparisons. The Mann\u2013Whitney U test was used to compare metastastic burden in the PyMT\/iNOS+\/+ and PyMT\/iNOS\u2212\/\u2212 groups.\nResults\nPyMT tumorigenesis is delayed in the C57Bl\/6 strain\nThe PyMT model was originally developed in the FVB strain due to its high fecundity. Tumor growth occurs rapidly and metastatic tumors form in the lungs (Guy et\u00a0al. 1992). To compare tumorigenesis in the FVB strain with that in the C57Bl\/6 strain onto which many genetically modified strains are bred, we made PyMT mice congenic in the B6 background. Examination of tumor latency showed a dramatic difference with a mean latency of 53\u00a0days for PyMT tumor development in FVB mice and 92\u00a0days in B6 mice, a delay of approximately 6\u00a0weeks (Fig.\u00a01A). Whole mounts of mammary glands from the two strains also showed that even at weaning, hyperplasias were more advanced in FVB than B6 PyMT mice (Fig.\u00a01B).\nFig. 1PyMT tumorigenesis in the FVB and B6 strains. (A) The day at which the first mammary tumor was detected by palpation in each mouse was recorded and a Kaplan-Meier survival plot generated. Log rank analysis indicated a highly significant difference, P\u00a0<\u00a00.001 between the curves.  FVB mice N\u00a0=\u00a044, B6 mice N\u00a0=\u00a046. (B) Whole mounts of mammary glands from 3\u00a0week old PyMT\/iNOS+\/+ mice of the FVB (left panel) and B6 (right panel) strains\nEffects of iNOS deficiency are more apparent in the C57Bl\/6 background \nTo determine how this difference in tumor latency may affect the phenotype observed following crossbreeding with genetically altered mice, we bred in iNOS\u2212\/\u2212 mice congenic in the two backgrounds. As previously reported, we observed a 3\u20134\u00a0week delay in palpable tumors in B6 PyMT mice lacking iNOS activity (Ellies et\u00a0al. 2003). However, this difference was difficult to discern in the FVB background due to the rapid tumor onset (Fig.\u00a02A). Mammary gland whole mounts reflected the latency data with a clear difference in tumor growth with iNOS deficiency being observed in the #4 glands of B6 PyMT mice (Fig.\u00a02A).\nFig.\u00a02Effects of iNOS deficiency on mammary tumor development. (A) Whole mounts of PyMT\/iNOS+\/+ and PyMT\/iNOS\u2212\/\u2212 #4 mammary glands from 11\u00a0week FVB mice or 20\u00a0week B6 mice. LN = lymph node. (B) Kaplan Meier plots of the tumor latency of PyMT\/iNOS+\/+ annd PyMT\/iNOS\u2212\/\u2212 mice of the 2 strains. Data were analyzed by the log rank test. PyMT\/iNOS+\/+ FVB N\u00a0=\u00a044, PyMT\/iNOS\u2212\/\u2212 FVB N\u00a0=\u00a057, PyMT\/iNOS+\/+ B6 N\u00a0=\u00a046, PyMT\/iNOS\u2212\/\u2212 B6 N\u00a0=\u00a026\nPyMT tumor growth kinetics vary in FVB\/N and C57Bl\/6 strains \nThe pattern of tumor growth differed between strains and may in part account for the greater disparity in tumor development observed in the absence of iNOS in PyMT B6 mice. Once the mean tumor volume reached 1\u00a0cm3, tumor growth proceeded exponentially in the PyMT FVB mice leading to sacrifice within 3\u00a0weeks (Fig.\u00a03A). To achieve the same final tumor volume, the PyMT B6 mice required approximately 7\u00a0weeks and growth followed a more linear pattern, suggesting more variable rates of growth of individual tumors. Indeed, when we analyzed the tumor latency of all tumors, we were able to define two distinct phases of tumor growth in the B6 strain: early, from 12\u201317\u00a0weeks and late, from 19\u201323\u00a0weeks (Fig.\u00a03B). The major effect of iNOS deficiency appears to be a delay in the early phase of tumor development.\nFig.\u00a03Mammary tumor growth kinetics. (A) Mean tumor volume per mouse. FVB N\u00a0=\u00a032, B6 N\u00a0=\u00a044. Data are means\u00a0\u00b1\u00a0SEM. (B) Tumor latency for individual B6 PyMT\/iNOS+\/+ and PyMT\/iNOS\u2212\/\u2212 tumors. Data were derived from examining 460 B6 PyMT\/iNOS+\/+and 260 PyMT\/iNOS\u2212\/\u2212tumors\nDeficiency in iNOS-modulated tumor growth is site specific\nIn concordance with increased tumor latency and decreased tumor growth (Ellies et\u00a0al. 2003) tumor burden was significantly lower in PyMT\/iNOS\u2212\/\u2212 mice (Fig.\u00a04A). An examination of tumor burden in individual mammary glands revealed a reduction in all PyMT\/iNOS\u2212\/\u2212 glands compared to controls, although the numbers were only significant in the #3 and #4 mammary glands (Fig.\u00a04A). It is interesting to note that these are the glands with the largest fat pads and may indicate a role for the stroma in the iNOS effect. To assess a role for tumor vascularization in this effect we examined tumor microvascular density (MVD) in #1and #2\u20135 tumors of both genotypes. Although there was a slight reduction in the mean MVD in #1 versus #2\u20135 tumors in the PyMT\/iNOS+\/+ and PyMT\/iNOS\u2212\/\u2212 genotypes, it was not statistically significant, suggesting this is not the mechanism for the difference observed in tumor growth at different locations (Table 1).\nFig.\u00a04Distribution of B6 mammary gland tumor burden. (A) Total tumor burden was significantly lower in PyMT\/iNOS\u2212\/\u2212 mice, ***P\u00a0<\u00a00.001. (B) Examination of individual tumors indicated that a significant difference in genotypes was only found in the #3 and #4 mammary glands, although in each gland the mean tumor burden was lower in PyMT\/iNOS\u2212\/\u2212 mice. *P\u00a0<\u00a00.05 ANOVA followed by Bonferroni post tests. PyMT\/iNOS+\/+ B6 N\u00a0=\u00a024 (black bars), PyMT\/iNOS\u2212\/\u2212 B6 N\u00a0=\u00a025 (grey bars)Table\u00a01Quantitative analysis of tumor microvascular densityGenotypeLocationNMVD (\u03bcm2\/mm2)PyMT\/iNOS+\/+#19216\u00a0\u00b1\u00a020PyMT\/iNOS+\/+#2\u201359176\u00a0\u00b1\u00a012PyMT\/iNOS\u2212\/\u2212#16201\u00a0\u00b1\u00a011PyMT\/iNOS\u2212\/\u2212#2\u2013511189\u00a0\u00b1\u00a011Data are means\u00a0\u00b1\u00a0SEM\nThis site-specific effect prompted us to examine tumor latency in individual glands. We had observed during palpations that the #1 mammary gland was usually the first to develop tumors, however we were surprised to find a clear difference between the latency of the #1 tumors compared with all other tumors (Fig.\u00a05). This difference was more noticeable in the absence of iNOS. The greater difference in mean latency in the #4 glands compared with the #1 glands was consistent with the tumor burden results being more significant in the #4 glands.\nFig.\u00a05Tumor latency of individual B6 mammary glands. Tumor latency was lower in the #1 mammary gland in PyMT\/iNOS+\/+ mice compared with all other mammary glands (upper panel). A similar result was observed in PyMT\/iNOS+\/+ mice, with an increase in disparity between the latency of the #1 gland and all other glands (middle panel). When the #1 and #4 latencies for both genotypes were overlayed, a greater difference in mean tumor latency was observed between the #4 glands when compared with the #1 glands (lower panel) \niNOS effects on tumor metastasis \nThe PyMT model has been useful for the study of tumor metastasis due to the high penetrance of this clinically important characteristic of tumor progression. However, we found that in the FVB strain, tumors were so aggressive that no significant difference could be observed in the metastatic burden occurring in PyMT\/iNOS+\/+ and PyMT\/iNOS\u2212\/\u2212 mice Fig\u00a06. Although the incidence of tumor metastasis was less in the B6 strain, a statistically significant difference between the genotypes was found. We have maintained PyMT\/iNOS\u2212\/\u2212 mice for up to 32\u00a0weeks and found no increase in metastatic burden (data not shown), suggesting that the decrease in metastasis is not due simply to the delayed tumor latency in these mice.\nFig.\u00a06Tumor metastasis. (A) HE stained paraffin embedded sections of PyMT\/iNOS+\/+ (left panel) and PyMT\/iNOS\u2212\/\u2212 (right panel) lungs. (B) Comparison of metastatic tumor burden in FVB and B6 mice *P\u00a0<\u00a00.05 Mann-Whitney U test. PyMT\/iNOS+\/+ FVB N\u00a0=\u00a014, PyMT\/iNOS\u2212\/\u2212 FVB N\u00a0=\u00a013, PyMT\/iNOS+\/+ B6 N\u00a0=\u00a017, PyMT\/iNOS\u2212\/\u2212 B6 N\u00a0=\u00a021\nDiscussion\nMouse models of mammary tumorigenesis have provided important insights into the molecular basis of human breast cancer and are essential for gathering preclinical data regarding therapeutic drug efficacy. The PyMT model in the FVB strain has a mean tumor latency of 53\u00a0days and has been used extensively in studies of mammary tumorigenesis (Cardiff 2003; Muller and Neville 2001). The model is frequently crossed with gene-targeted strains that have been generated in the B6\/129 mixed background or backcrossed onto the B6 background. We had highly variable results examining iNOS effects in a mixed B6\/FVB PyMT background and sought to characterize the phenotype of this mutation in congenic FVB and B6 strains. Our study demonstrates that the genetic background of the PyMT model can affect the ability to discern differences in phenotype due to genetic alterations that require time to influence tumor progression. Differences observed in the B6 background were obscured in the FVB background due to the signficantly shorter tumor latency and more rapid tumor growth in this strain.\nFurther analysis of mammary tumorigenesis in the B6 background indicated that tumor formation occurred in 2 phases: early, from 12\u201317\u00a0weeks and late, from 19\u201323\u00a0weeks. It is the early phase of tumor formation that is most affected in the absence of iNOS, consistent with recent data linking iNOS expression with transcriptional upregulation of signal transducers and activators of transcription 3 (STAT3) by nuclear epidermal growth factor receptor (EGFR) (Lo et\u00a0al. 2005). EGFR\/ErbB1, a member of the ErbB family of receptor tyrosine kinases that signal via the same pathways as PyMT (Webster et\u00a0al. 1998), is also found in the nucleus of highly proliferative cells where is functions as a transcription factor (Marti et\u00a0al. 1991; Wells and Marti 2002). Structural and functional characterization of the iNOS promoter has identified binding elements for both EGFR and STAT3 (Lo et\u00a0al. 2005). Immunohistochemical studies of breast carcinomas show that nuclear EGFR positively correlates with iNOS and survival analysis demonstrates that high levels of iNOS are a prognostic indicator of poor survival (Lo et\u00a0al. 2005). Thus the B6 PyMT mice in the presence or absence of iNOS deficiency are likely to provide an important model for further analysis of these novel molecular interactions.\nMammary tumor latency for each of the ten murine mammary glands has generally been assumed to be stoichiometric. However our data demonstrate that the #1 mammary glands have tumor initiating properties that are distinct from the other 4 pairs of mammary glands. The #1 mammary glands have the smallest fat pads and are located in the head and neck region, which is highly vascular, so we hypothesized that an increase in vascular density in the #1 tumors could be responsible for this difference. An examination of the microvacular density (MVD) of #1 tumors versus all others showed a trend in which #1 tumors had a higher mean MVD than #2\u20135 tumors as determined by quantitative analysis of CD31 staining. However, these values were not statistically significant, suggesting a different mechanism is responsible for the observed site-specific differences in tumor burden and tumor latency. Other factors in the stromal microevironment may also play a role in mediating this effect. The release of epidermal growth factor (EGF) from the submandibular salivary gland has been shown to promote mouse mammary tumorigenesis in virus mediated and chemical carcinogen induced models (Kurachi et\u00a0al. 1985; Molinolo et\u00a0al. 1998). The proximity of the #1 glands to the submandibular gland make EGF induced proliferation a plausible explanation, however further investigations will be required to determine whether the release of this growth factor primarily affects the #1 glands.\nAnother possible explanation for this intriguing difference is that the #1 mammary gland is developmentally distinct from the other mammary glands. The #1 gland is not the first to develop since placode 3 develops first at E11.0\u201311.5 on the ectodermal streak between the forelimb and hindlimb (Eblaghie et\u00a0al. 2004; Mailleux et\u00a0al. 2002). Placodes 1 and 5 arise from the axillary and inguinal streaks respectively and placodes 4 and 2 form where the mammary line abuts these streaks (Veltmaat et\u00a0al. 2004). Recent studies indicate that signaling molecules essential for mammogenesis are placode dependent. Placodes 2 and 3 do not form in the absence of Lef1 mediated canonical Wnt signaling (Boras-Granic et\u00a0al. 2006) and placodes 1, 2, 3 and 5 require Fgf10 signaling from somites underlying the ectoderm (Mailleux et\u00a0al. 2002; Veltmaat et\u00a0al. 2006). It is clear there are developmental differences between mammary glands and it is possible that early developmental factors result in an increased propensity for malignant transformation in the #1 mammary glands, though the identity of these factors remains to be determined.\nSince metastasis is an aspect of tumor progression that is critical to patient survival, we examined the effect of iNOS on tumor metastasis in both the FVB and B6 backgrounds. No significant difference in metastatic burden was found in PyMT\/iNOS\u2212\/\u2212 mice in the FVB background, however the tumor burden was reduced in lungs of PyMT\/iNOS\u2212\/\u2212 mice in the B6 background compared with controls. Furthermore, the reduction in metastatic burden was not due simply to the delayed tumor latency as we maintained PyMT\/iNOS\u2212\/\u2212 mice longer to compensate for the difference and found that this did not alter our previous findings (Ellies et\u00a0al. 2003).\nHuman clinical studies have associated increased iNOS expression with poor prognosis in breast cancer patients (Loibl et\u00a0al. 2005), and the use of non-steroidal anti-inflammatory drugs (NSAIDs) with a 22\u201339% reduction in the risk of breast cancer (Harris et\u00a0al. 2005; Khuder and Mutgi 2001). To increase our understanding of the molecular pathways involved in these effects, it is essential to have animal models that recapitulate the clinical findings. We have shown that in the PyMT B6 background loss of iNOS, an important inflammatory mediator, results in increased tumor latency and decreased tumor metastasis that is not due simply to the difference in latency. This model will be useful in determining molecular pathways underlying the role of iNOS in breast cancer.","keyphrases":["fvb\/nj","c57bl\/6j","strain","mouse","breast","cancer","inos","polyomavirus middle t antigen"],"prmu":["P","P","P","P","P","P","P","M"]}
{"id":"Eur_J_Pediatr-4-1-2234440","title":"Moebius-Poland syndrome and hypogonadotropic hypogonadism\n","text":"Case report\nA male infant was born at 39\u00a0weeks of gestation, with birth weight (3.1\u00a0kg) and body length (49\u00a0cm) (both 50th percentile). The occipitofrontal circumference was 33\u00a0cm (25th percentile). The newborn\u2019s face was inexpressive due to complete facial diplegia, bilateral ophthalmoplegia with impairment of the vertical gaze of the right eye, palpebral ptosis, a carp-shaped mouth, and a high-arched palate. Muscle and skeletal abnormalities were also apparent, including the absence of the pectoralis major and trapezius muscles, as well as left cubitus valgus; left hand was hypoplastic and showed 5th finger clinodactyly. These features prompted the diagnosis of Moebius-Poland syndrome. No previous cases were known in the patient\u2019s family. Clinical examination revealed no other dysmorphic features except for micropenis. The karyotype was normal (46 XY). During follow-up, a mild psychomotor delay was confirmed by Denver Developmental Screening Test-II. Cortical-subcortical atrophy with no other structural abnormalities was observed in magnetic resonance imaging (MRI) of the brain. Delayed puberty was recorded when the patient was 15\u00a0years of age (Tanner stage I was recorded after physical examination). The testicular volume (measured using a Prader orchidometer) was 2\u00a0ml. Anosmia-tested by a standard olfactory test (CCCRC)- was not present. An endocrinological investigation revealed low baseline serum FSH (0.5\u00a0mU\/ml), LH (0.1\u00a0mU\/ml) and testosterone (2\u00a0ng\/dl) levels. To confirm the suspected hypogonadotropic hypogonadism, a gonadotrophin releasing hormone stimulation test was performed and a very subnormal response obtained (after 180\u00a0min and 24\u00a0h FSH concentrations were 3.5\u00a0mU\/ml and 0.7\u00a0mU\/ml, respectively, and LH concentrations 0.7\u00a0mU\/ml and 0.2\u00a0mU\/ml, respectively). GH, TSH, and ACTH levels were normal. A further MRI scan of the brain showed no pathological lesions in the hypothalamus or pituitary gland. With a diagnosis of hypogonadotropic hypogonadism established, the child was treated with gonadotrophic hormone followed by depot preparations of testosterone. The patient eventually reached a normal weight and height and attained full sexual development (final testicular volume 6\u20138\u00a0ml).\nDiscussion\nThe usual diagnostic criteria for Moebius syndrome include congenital facial palsy with impaired ocular abduction [1, 2, 4, 6]. Nevertheless, in a thorough review, Verzijl et al. reported that abducens nerve palsy is not always present [6]. Vertical gaze palsy was recorded in the right eye of the present patient, suggesting involvement of the 3rd cranial nerve (Fig.\u00a01). MRI scans often demonstrate anomalies of the posterior fossa in patients with Moebius syndrome, and it has been proposed that the latter be understood as a complex congenital anomaly affecting the rhombencephalon [6]. The present patient, however, showed no such anomalies. In addition to the main signs, more than half of all patients with Moebius syndrome have other anomalies, usually limb defects [1, 3]. The specific combination of both Moebius syndrome and Poland syndrome\u2014a rare congenital anomaly characterised by pectoralis muscle defect and ipsilateral hand abnormalities\u2014has been reported before, and is then referred to as Moebius-Poland syndrome [5]. Some authors suggest it may represent a single developmental condition that may be due to the variable expression of a single gene or involve contiguous genes [6].\nFig.\u00a01Patient photo\nHypogonadotropic hypogonadism is unusual in Moebius syndrome [1\u20133]. The present patient is only the 7th case reported to date, and the first with Moebius-Poland syndrome.","keyphrases":["hypogonadotropic","hypogonadism","moebius syndrome","poland syndrome"],"prmu":["P","P","P","R"]}
{"id":"Breast_Cancer_Res_Treat-4-1-2190785","title":"Impact of a programme of mass mammography screening for breast cancer on socio-economic variation in survival: a population-based study\n","text":"Background After a systematic mass mammography breast cancer screening programme was implemented between 1991 and 1996 (attendance 80%), we evaluated its impact on survival according to socioeconomic status (SES).\nIntroduction\nMammography screening aims at early detection of breast cancer so that adequate treatment will eventually lower breast cancer mortality. In a mass screening programme, it is therefore especially important to reach women who have the highest chance of being diagnosed with advanced stage or have the lowest survival rates.\nWomen from lower socio-economic strata are less likely to attend population screening programmes [1\u20134] and are also more likely to present with unfavourable stage at diagnosis, [1, 5, 6] although not all studies confirm this [7\u20139]. Lower breast cancer survival rates among the disadvantaged are usually attributed to advanced stage at presentation, but also to suboptimal access to adequate treatment. A recent population-based study in Switzerland found social class to be an independent prognostic factor [10].\nThe mass breast cancer screening programme was introduced in 1991 for women of 50\u201369\u00a0years and became fully implemented in 1996 in the south of the Netherlands covered by the population-based Eindhoven Cancer Registry, with a continuous high participation rate. Based on previous work [11] and a new postcode-based indicator of socio-economic status (SES) introduced by Statistics Netherlands [12] we were able to investigate survival according to SES for a sufficient period of time after introduction.\nWe studied whether survival according to SES was affected differentially by the implementation of the screening programme.\nMethods\nThe Eindhoven Cancer Registry records data on all patients newly diagnosed with cancer in the south\u2013eastern part of the Netherlands, an area with now 2.4\u00a0million inhabitants (about 15% of the Dutch population) and only general hospitals. Trained registry personnel actively collect data on diagnosis, staging, and treatment from the medical records after notification by pathologists and medical registration offices.\nIn the area of the Eindhoven Cancer Registry, a biennial breast cancer screening programme for women aged 50\u201369\u00a0years was started in 1991 and fully implemented in 1996. The attendance rate was more than 80% [13].\nFor our analyses we included all patients age 50\u201369\u00a0years diagnosed in 1983\u20132002 with invasive breast cancer in the eastern part of the registration area (about 1\u00a0million inhabitants). This population has been followed-up for vital status up to 1-1-2005. Information on the vital status of all patients was obtained initially from the municipal registries and since 1998 from the Central Bureau for Genealogy. These registers provide virtually complete coverage of all deceased Dutch citizens.\nAn indicator of socioeconomic status was developed by Statistics Netherlands [12] being based on individual fiscal data from the year 2000 on the economic value of the home and household income and provided at aggregated level for each postal code (average of 17 households). Socioeconomic status was categorized according to quintiles ranging from 1 (low) to 5 (high), with a separate class for postal codes with a care-providing institution (such as a nursing home). This measure is assumed to be valid ten years before and after the basic year (2000), so for patients diagnosed before 1990 we used a measure which was also based on postal code of residence, but socio-economic status (five categories) was based on data from a marketing agency (self-reported occupation and education define 45 social classes, collapsed into a 5-level indicator based on average number of years of education), as described before [11]. We also used both SES indicators for the whole study period (1983\u20132002) to make sure any effect of diagnostic period was not attributable to the indicator we used.\nWe calculated distribution of age and stage of disease according to period of diagnosis.\nStage was categorized according to the TNM classification [14]. Patients with either positive lymph nodes or metastases were considered to have advanced disease.\nChi-square test was performed of changes in the distribution across the three diagnostic periods. T-tests were performed of differences between two groups.\nCrude survival analyses were performed. The log-rank test was used to evaluate significant differences between survival curves in univariate analyses. We used Cox regression models to compute multivariate rates. The proportional hazard assumption of the predictor was evaluated by applying Kaplan\u2013Meier Curves. The predictor satisfied the assumption of proportionality as the graphs of the survival function versus the survival time resulted in graphs with parallel curves as did the graphs of the log(\u2212log(survival)) versus log of survival time. The independent prognostic effect of SES was investigated, adjusting for age and stage of disease, and stratified according to period of diagnosis (1983\u20131990, 1991\u20131996, 1997\u20132002). We also calculated the age and stage-adjusted effect of period of diagnosis stratified according to SES.\nResults\nMedian age was similar for all three periods of diagnosis (59, 60, and 59\u00a0years, respectively).\nPatients diagnosed between 1997 and 2002 had a significantly more favourable stage at diagnosis than patients diagnosed in earlier periods (P\u00a0<\u00a00.0001): the proportion diagnosed with stage I (tumour smaller than 2\u00a0cm, no axillary lymph nodes involved) increased from 30% in 1983\u20131990 to 41% in 1991\u20131996 and 45% in more recent years (Table\u00a01). The proportion with advanced disease, i.e. stage III or IV, was significantly lower in the most recent period (9.7%) compared to 1991\u20131996 (14%) and 1983\u20131989 (26%, P\u00a0<\u00a00.0001). Treatment varied over time, with a large proportion receiving systemic therapy in recent years (50%, vs. 36% and 29%).\nTable\u00a01Characteristics of all women age 50\u201369\u00a0years diagnosed with invasive breast cancer between 1983\u20132002 in Southeastern Netherlands1983\u201319901991\u201319961997\u20132002Totaln%n%n%n%TNMI4653064241838451,94539II6384266543805442,10843III27818152101156.254511IV1157.5684.4653.52485.0unknown382.5312.0241.3931.9Treatment*S alone27118341222491386117S\u00a0+\u00a0RT7665062840658362,05242S\u00a0+\u00a0RT\u00a0+\u00a0ST3052042227673361,40028S\u00a0+\u00a0ST10871238231134629.4ST alone261.7231.5231.3721.5Other583.8211.4130.7921.9Socio-economic status1 (low)336222851826214883182325213192034219986203308202791835519942194154102741835819786165 (high)3022031520414221,03121institution#00.0231.5331.8561.1unknown1097.1634.0834.52555.2Total1,5341,5581,8474,939100*\u00a0S\u00a0=\u00a0Surgery, RT\u00a0=\u00a0Radiotherapy, ST\u00a0=\u00a0Systemic therapy#institution\u00a0=\u00a0care-providing institution such as a nursing home\nThe proportion of patients from the lowest socio-economic class decreased from 22% in 1983\u20131990 to 18% in 1991\u20131996 and 14% in 1997\u20132002 (P\u00a0<\u00a00.0001), whereas the proportion from in the higher social classes increased.\nStage distribution improved significantly over time for each social class (P\u00a0<\u00a00.01). It was similar for all SES groups in 1983\u20131990 (P\u00a0=\u00a00.7, Fig.\u00a01), although the proportion with stage IV was somewhat lower in the highest classes. The stage distribution was marginally more favourable for high SES compared to the lowest SES group in both 1991\u20131996 and 1997\u20132002 (P\u00a0=\u00a00.06\u00a0both periods), although the overall effect of SES on stage was not significant in the last period of time (P\u00a0=\u00a00.4).\nFig.\u00a01Stage distribution according to socio-economic status and period of diagnosis of patients age 50\u201369\u00a0years with invasive breast cancer in Southeastern Netherlands\nSurvival improved for all socio-economic strata over time (Fig.\u00a02). Survival rates did not differ among patients from each of the socio-economic classes diagnosed 1983\u20131990 in the period (P\u00a0=\u00a00.9), 5-year survival rates being 70%, 70%, 70%, 68% and 69% for patients from the lowest to the highest social class, respectively. For patients diagnosed in 1991\u20131996, survival of patients with a high SES was better than that of all other socio-economic strata (P\u00a0=\u00a00.01), 5-year survival rates being 76%, 76%, 80%, 78%, and 87%, respectively. For patients diagnosed in 1997\u20132002 an increasing gradient in survival was observed (P\u00a0=\u00a00.002) ranging from the lowest rates for the lowest SES group to the highest for the higher classes (80%, 84%, 83%, 85%, and 89%, respectively).\nFig.\u00a02Trend in survival according to socio-economic status for all women age 50\u201369\u00a0years diagnosed with invasive breast cancer in Southeastern Netherlands\nMultivariate analysis (Table\u00a02) showed that patients diagnosed between 1991 and 1996 from the lower social classes had a 29% higher risk of death compared to the highest socio-economic group, after adjusting for age and stage at diagnosis (HR for the lowest versus the highest SES group: 1.29, 95%CI: 1.0\u20131.7). The risk of death for low SES patients diagnosed since 1997 was twice as high as that for the highest SES group (HR: 2.01, 95%CI: 1.3\u20133.0). The overall effect of socio-economic status was significant in the last period (P\u00a0=\u00a00.02). Additional adjustment for treatment did not change risk estimates more than 5% (data not shown).\nTable\u00a02Multivariate regression analysis of survival of breast cancer patients age 50-69\u00a0years according to period of diagnosis, Southeastern Netherlands1983\u201319901991\u201319961997\u20132002HR*a95% CIHR*a95% CIHR*a95% CIAge (continuous)1.031.0\u20131.01.041.0\u20131.11.011.0\u20131.0Socio-economic status1 (low)1.010.8\u20131.21.291.0\u20131.72.011.3\u20133.021.030.8\u20131.31.281.0\u20131.71.541.0\u20132.330.950.8\u20131.21.180.9\u20131.61.531.0\u20132.340.990.8\u20131.31.391.0\u20131.81.330.9\u20132.05 (high)b1.001.001.00\u03a72 trend0.58 (P\u00a0=\u00a00.97)5.9 (P\u00a0=\u00a00.21)11.4 (P\u00a0=\u00a00.02)TNM stageIb1.001.001.00II1.751.5\u20132.12.331.9\u20132.92.001.4\u20132.8III3.112.5\u20133.84.673.5\u20136.25.393.6\u20138.1IV9.857.6\u20131316.012\u20132216.511\u201324unknown2.251.4\u20133.52.561.4\u20134.64.812.3\u201310* HR\u00a0=\u00a0Hazard Ratio, CI\u00a0=\u00a0Confidence IntervalaAdjusted for all variables listedbReference\nAge and stage-adjusted survival improved over time for all socioeconomic strata (Table\u00a03), the largest improvements were found for the highest social classes.\nTable\u00a03Multivariate regression analysis of survival according to socio-economic status (SES) of breast cancer patients age 50\u201369 years in Southeastern NetherlandsSES1 (low)2345 (high)HR*a95% CIHR*a95% CIHR*a95% CIHR*a95% CIHR*a95% CIPeriod of diagnosis1983\u20131990b1.001.001.001.001.001991\u201319960.870.7\u20131.10.720.6\u20130.90.800.6\u20131.00.840.6\u20131.10.600.5\u20130.81997\u201320020.770.6\u20131.10.490.4\u20130.70.610.4\u20130.80.490.3\u20130.70.360.2\u20130.5X2 trend3.21 (P\u00a0=\u00a00.21)20.9 (P\u00a0<\u00a00.001)9.2 (P\u00a0=\u00a00.01)14.1 (P\u00a0<\u00a00.001)35.6 (P\u00a0<\u00a00.001)HR\u00a0=\u00a0Hazard Ratio, CI\u00a0=\u00a0Confidence IntervalaAdjusted for age at diagnosis and stage of diseasebReference\nDiscussion\nWe found that the proportion of breast cancer patients with a low SES has decreased since the introduction of a mass biennial mammography screening programme with high response rates. Although stage distribution improved for all socio-economic groups, the proportion with advanced disease decreased the most in the highest socio-economic group. In the 1980s survival was similar for all socio-economic groups, but since the introduction of screening the survival of women with a high SES has improved more than that for low socio-economic classes, also after adjustment for age and stage.\nWe used an indicator of socio-economic status based on the postal code of a residential area. This aggregate covers a relatively small geographical area, and thus represents a reliable approximation of individual socio-economic status. Furthermore, routinely collected income tax data (no questionnaires or interviews) have been found to provide reliable estimates of household income. Previous studies have proven that socio-economic differences based on neighbourhood data tend to reflect socio-economic differences well at the individual level [15\u201317]. Furthermore, this objective measure of SES is also applicable for older women (born before 1955), whose occupation or education does not always properly reflect their social class [18]. We also repeated the analyses comparing both SES indicators if they were applied for the whole study period (1983\u20132002) to ensure any that effect of diagnostic period was not attributable to the indicator we used, and it was not.\nThe lower proportion of patients with a low SES since the introduction of screening is not likely to reflect the higher attendance rate of women from a higher social class because of the very high participation rate, although this is not known according to social class. Studies from other countries have shown that SES does play a role in participation in the screening programme, [2] sometimes [3] but not always [4] due to the costs of a screening mammogram. However, the costs for the mass screening programme in the Netherlands are completely covered by public funds. Furthermore, the mean attendance rate in the Netherlands has always been rather high (about 80%), [13] and in our study area even higher than the national mean (85% in 2005) [19].\nForeign-born women are more likely to be non-attenders in the Netherlands, [20] as well as in Sweden, [21] Australia, [3] and the US, [22] for a variety of reasons. However the incidence of breast cancer among these groups of migrants is relatively low in the Netherlands and the stage distribution is comparable to that of women born in the Netherlands [20]. So this is unlikely to have affected survival rates in our study.\nA lower attendance rate of low social classes will result in more advanced disease stages at presentation [23]. Before the start of the mammography screening programme, we found that the stage distribution for breast cancers diagnosed in 1980\u20131989 was slightly more favourable for the highest socio-economic group [24]. We have now shown that this was also true after the introduction of screening, although the differences were small. In fact, we found that, although stage distribution became more favourable for all socio-economic groups, the proportion with advanced disease decreased less in the lower socio-economic group. This differential stage distribution was also described in a recent Danish study, [6] although our differences were smaller.\nThe variation in survival according to SES may also be related to differences in treatment, which depends on the disease stage and varies over time. The use of surgery and radiotherapy was similar across SES groups. However, we found that the administration of adjuvant chemotherapy varied across the social strata among stage II patients (8% of the lowest SES group versus 17% of the highest SES group, P\u00a0<\u00a00.001). Patients with a higher SES seem to have benefited more from the general trend towards more adjuvant chemotherapy independent of the disease stage. This may explain, at least in part, the diverging trend in survival rates.\nAnother explanation for differential survival could be socio-economic variations in lifestyle. Smoking has become relatively more prevalent among low SES groups [25, 26]. This may have had an adverse effect on survival due to a poor general health while undergoing breast cancer treatment or to smoking related diseases (such as chronic obstructive pulmonary diseases (COPD) or cardiovascular disease).\nAlso related to an unhealthy lifestyle is obesity, which has become an increasingly important problem in the last decade, [27, 28] especially among women from the lower social classes [29].\nSerious concomitant diseases besides breast cancer also affect survival rates, [30] which may explain differences in survival if comorbidity occurs more frequently in low SES groups.\nSince the Eindhoven Cancer Registry has recorded comorbidity for all newly diagnosed patients since 1993, we checked whether the prevalence varied across socioeconomic strata. Indeed, the proportion of patients with comorbidity was higher among those with a lower SES (70% of patients in the lowest SES group had one or more concomitant conditions compared to 60% of the high SES group). In particular, the prevalence of diabetes and cardiovascular disease was highest in the low SES groups (diabetes in 10% with low SES and 4% with high SES, cardiovascular disease 7% and 4%, respectively).\nSeveral studies have reported increased survival rates after the introduction of breast cancer screening 31-35. As far as we know, no studies describe a differential effect of the introduction of screening on survival rates for socio-economic strata. However, socioeconomic inequalities in mortality have been widening in recent decades in western European countries [36]. In fact, socio-economic differences in breast cancer mortality increased between 1983 and 1993 among women in Finland and Italy (Turin), but remained stable in Denmark and decreased somewhat in Norway where a mass screening programme was only introduced later [36, 37].\nIn conclusion, despite a very high participation rate women from lower socio-economic strata clearly benefited less from the introduction of the breast cancer screening programme than those with a lower SES, probably due to a higher prevalence of comorbidity and suboptimal treatment (for both the cancer and the concomitant disease).","keyphrases":["breast cancer","survival","mass screening","socio-economic status"],"prmu":["P","P","P","P"]}
{"id":"Eur_J_Clin_Pharmacol-3-1-1914266","title":"Coumarin anticoagulants and co-trimoxazole: avoid the combination rather than manage the interaction\n","text":"Objective The objective of our study was to examine the management of the interaction between acenocoumarol or phenprocoumon and several antibiotics by anticoagulation clinics and to compare the consequences of this interaction on users of co-trimoxazole with those for users of other antibiotics.\nIntroduction\nCoumarin-type anticoagulants have a narrow therapeutic range. One important aspect of their safety is their sensitivity to drug interactions, many of which have been described [9, 11].\nThere are several reasons why antibiotic use can be considered to be indicative of a change in anticoagulation status in users of coumarin-type anticoagulants. When the antibiotic is used for febrile illness, it may be associated with overanticoagulation [5, 12]. In two studies on the interaction between coumarin anticoagulants and antibiotics, the risk of severe overanticoagulation, defined as an International Normalised Ratio (INR) \u22656.0, was increased more in users of sulfamethoxazole-trimethoprim (co-trimoxazole) than in users of other antibiotics [12, 21]. Sulfamethoxazole is a strong inhibitor of CYP2C9 [24], the main liver enzyme involved in the metabolism of warfarin [13], acenocoumarol [19] and probably phenprocoumon [20], which could explain this stronger association with overanticoagulation. Current clinical guidelines in The Netherlands for the management of coumarin drug interactions advise healthcare givers to avoid prescribing the concurrent use of co-trimoxazole and coumarins [1]. Nevertheless, in daily practice co-trimoxazole is frequently prescribed to users of coumarins, since physicians in anticoagulation clinics assume that an interaction with co-trimoxazole can be managed in a manner similar to those used to manage interactions that arise with the concurrent use of coumarins with other antibiotics. An anticoagulation clinic will initiate one of the following procedures once it has been notified of the initiation of the use of an antibiotic: (1) measurement of the INR during the antibiotic course and adjustment of the coumarin dose depending on the INR value (a reactive dose-adjustment); (2) a preventive (coumarin) dose reduction (PDR) preceding an INR measurement during or after the antibiotic course, assuming that use of an antibiotic or the intercurrent infection itself increases the risk of overanticoagulation. The PDR approach seems even more relevant to co-trimoxazole than to other antibiotics because the CYP2C9-inhibiting effect of the former might increase the risk of overanticoagulation more than the infectious state alone. However, PDR could also lead to temporary undertreatment, and evidence for the effectiveness of this approach is currently lacking. There are no official guidelines for such dose adjustments, and the application of PDR strongly depends on the personal view of the responsible physician.\nThe aim of the present study was to examine the management of the interaction between coumarin anticoagulants and antibiotics by anticoagulation clinics and its consequences for users of co-trimoxazole and other antibiotics. To this end, we conducted a prospective follow-up study at four anticoagulation clinics in The Netherlands.\nMaterials and methods\nStudy design\nThis was a follow-up study conducted at four anticoagulation clinics in The Netherlands. We included patients who were stabilised on one of the coumarin anticoagulants acenocoumarol or phenprocoumon and who had started using one of the following antibiotics between January 2001 and October 2003: co-trimoxazole, amoxicillin, amoxicillin-clavulanic acid, clarithromycin, doxycyclin, nitrofurantoin, norfloxacin or trimethoprim. In addition to co-trimoxazole, we chose the other antibiotics based on their use for the same kind of infections, mainly those of the urinary and respiratory tract.\nThe subjects included in our study were prospectively followed during the antibiotic course until the last INR measurement, which occurred within 6\u00a0weeks following the starting date of the antibiotic (follow-up time). We did not intervene in the daily routine of the participating anticoagulation clinics and, in particular, we made no agreements on checking the INR of patients during the antibiotic course, on making additional INR measurements, on the time intervals between INR measurements after the antibiotic courses or on dose adjustments when antibiotics were prescribed. To assess the consequences of interaction management reliably and to avoid confounding by an unstable anticoagulation status preceding the antibiotic course, we only included stabilised patients in our study. Criteria for the assessment of stability were: (1) use of the coumarin anticoagulant for at least 50\u00a0days before initiation of the antibiotic; (2) availability of at least four INR measurements before the initiation of the antibiotic; (3) the last two INR measurements before initiation of the antibiotic were within the therapeutic range; (4) a maxim of one out of the last four INR measurements or a maxim of 30% of the INR measurements during the 50\u00a0days immediately preceding initiation of the antibiotic were outside of the therapeutic range, with no INR being above 5.5. Similar criteria for stability have been used in other studies [12, 21].\nWe excluded subjects from our analyses in whom the INR was not measured during the course of the antibiotic and who used the antibiotic for a period shorter than 3\u00a0days and longer than 14\u00a0days. If the INR was not measured during the course, an interaction effect of the antibiotic could be missed. Antibiotics used for less than 3\u00a0days or more than 14\u00a0days are usually prescribed for prophylaxis not for acute infections.\nAll patients were informed of the aims of the study and were asked for their written consent to participate in the study.\nSetting and attitudes of anticoagulation clinics on antibiotic use\nAll anticoagulation clinics in The Netherlands monitor the INR in outpatients at a frequency varying from a few days to maximally 6\u00a0weeks. The two target therapeutic ranges are the normal therapeutic range (INR: 2.0\u20133.5) and the high therapeutic range (INR: 2.5\u20134.0).\nThe initiation of the use of an antibiotic is usually reported to the anticoagulation clinics by the patients, their pharmacists and\/or the prescribing physicians.\nThe four anticoagulation clinics participating in this study had different attitudes on the management of the interaction between coumarins and antibiotics. The approach of three of the anticoagulation clinics was to decrease the coumarin dose preventively if co-trimoxazole was prescribed; one of the anticoagulation clinics applied a PDR of 20\u201325% in the case of co-trimoxazole use. If one of the other antibiotics examined in this study was prescribed, the application of a PDR would depend on the seriousness of the disease and on the occurrence of fever. The fourth anticoagulation clinic had no established protocols for dose reduction but indicated that it would monitor the INR of every user of co-trimoxazole within 3\u20135\u00a0days after initiation of the course.\nData collection\nWe collected relevant data on the participating patients and recorded these in a database: sex and age of patient; dosage and indication of the coumarin; prescribed antibiotics (indication, dosage and duration of use); results of INR measurements before, during and after the antibiotic course; co-medication; relevant co-morbidities (malignancies, thyroid diseases, heart failure). These data were retrieved from the medical files of the anticoagulation clinics. Patients were asked to indicate on a questionnaire for which infection the antibiotic was prescribed and whether they had suffered from fever during the antibiotic course. We recorded this as fever yes\/no in our database. If the coumarin-dose was reduced as soon as the antibiotic was started in the absence of an actual INR, we recorded this as a preventive dose-reduction and calculated the percentage of the dose reduction from the data on dosage in the file of the anticoagulation clinic.\nIn order to assess the anticoagulation status shortly after the antibiotic course, we recorded the time spent within, above and under the therapeutic range from the starting date of the antibiotic until the last INR measurement within 6\u00a0weeks following the starting date of the antibiotic. Six weeks is the maximal period between two INR measurements if a patient is well stabilised. Furthermore, after a longer follow-up period, differences between patients could be more attributable to other factors than to the infection or antibiotic use. If after the first INR during the antibiotic course no second INR measurement was available within the 6-week period after the starting date of the antibiotic, we recorded no follow-up time and no time spent within, above or under the therapeutic range.\nOutcomes\nThe end points of our study were chosen to assess the effectiveness of the management of the interaction between coumarin anticoagulants and co-trimoxazole and other antibiotics.\nWe examined the following parameters in users of co-trimoxazole with and without PDR as well as in users of other antibiotics with and without PDR: \noccurrence of moderate overanticoagulation (INR >4.5) and severe overanticoagulation (INR >6.0);time spent within, above and under the therapeutic INR range from the starting date of the antibiotic until the last INR measurement within 6\u00a0weeks following the starting date of the antibiotic.\nCalculations and statistical analysis\nWe assessed the effects of the PDR within the group of users of co-trimoxazole and within the group of users of other antibiotics by comparing the occurrence of overanticoagulation in patients for whom a PDR had been applied with the occurrence of overanticoagulation in patients for whom PDR had not been applied (logistic regression models). We also compared the occurrence of overanticoagulation and time spent within, under and above the therapeutic range of co-trimoxazole users with users of other antibiotics (reference group). These comparisons were made for patients with PDR and for patients without PDR. Finally, we compared the time spent within, under and above the therapeutic range in patients for whom a PDR had been applied with those for whom a PDR had not been applied (reference) within the groups of co-trimoxazole users and users of other antibiotics (linear regression models). In all models we adjusted for the potential confounding covariates sex, age, target therapeutic range and fever, as indicated by the patient. Covariates were added to the statistical models one at a time. We adjusted for a covariate if it changed the point estimation of the outcome of interest by 5% or more upon inclusion in the model.\nTime spent within, above and under the therapeutic INR range was calculated by the step-up method described by Rosendaal et al. [15].\nAlthough all patients were stable when they were included in our study, we re-analysed our statistically significant outcomes after excluding patients in whom destabilisation could be due to factors other than those of infection and\/or fever (presence of thyroid disease, malignancy or use of other enzyme-inhibiting or-inducing drugs).\nAll statistical analyses were performed with the statistical software package SPSS ver. 12 (SPSS, Chicago, Ill.).\nResults\nA total of 424 patients who met the inclusion criteria gave their informed consent to participate in our study. Of these patients, 81 did not have assesment of the INR during the antibiotic course, 14 used the antibiotic for less than 3 days, and 3 used the antibiotic for more than 14 days.\nA PDR was applied more frequently for users of co-trimoxazole (28\/43; 65%) than for users of other antibiotics (60\/283; 21.2%) (Table\u00a01). \nTable\u00a01Characteristics of patients (n = 326) using antibiotics, treated by four anticoagulation clinicsCharacteristicCo-trimoxazole (n\u2009=\u200943)Other antibioticsa (n\u2009=\u2009283)PDR+b (n\u2009=\u200928)PDR-c (n\u2009=\u200915)PDR+b (n\u2009=\u200960)PDR-c (n\u2009=\u2009223)Men, no. (%)22 (78.6)10 (66.7)30 (50.0)114 (51.1)Age in years, mean (SD)75.4 (10.9)75.1 (8.2)72.6 (10.9)71.4 (11.2)Users of acenocoumarol, no. (%)24 (85.7)10 (66.7)52 (86.7)169 (75.8)Follow-up time, mean (SD) 33.2 (5.6)28.9 (8.0)30.4 (7.2)30.2 (7.2)Fever, no. (%)18 (64.3)6 (40.0)27 (45.0)120 (53.8)Normal target therapeutic range, no. (%)d19 (67.9)5 (33.3)30 (50.0)112 (50.2)Respiratory infections, no. (%)8 (28.6)3 (20.0)33 (55.0)116 (52.0)Urinary tract infections, no. (%)13 (46.4)8 (53.3)11 (18.3)53 (23.8)Malignancies, no. (%)1 (3.6)3 (20.0)3 (5.0)9 (4.0)Thyroid diseases, no. (%)001 (1.7)11 (4.9)Users of inhibiting drugs, no. (%)01 (6.7)5 (8.3)17 (7.6)Users of inducing drugs, no. (%)1 (3.6)01 (1.7)4 (1.8)INR measurements, mean no. (SD)3.5 (0.9)3.9 (1.5)3.5 (1.1)3.1 (1.1)Acenocoumarol, mean dose, mg\/day (SD)2.42 (1.26)2.41 (1.41)2.61 (1.06)2.60 (1.12)Percentage PDR applied, mean (SD) in acenocoumarol users15.0 (7.6)10.3 (11.1)Phenprocoumon, mean dose, mg\/day (SD)2.81 (0.86)2.53 (1.02)2.99 (1.31)2.36 (1.00)Percentage PDR applied, mean (SD) in phenprocoumon users17.9 (15.8)11.4 (7.0)Percentage PDR applied, all coumarins, mean (SD)15.4 (8.8)10.5 (10.6)aOther antibiotics: Trimethoprim (n\u2009=\u20093), doxycyclin (n\u2009=\u2009104), amoxicillin (n\u2009=\u200977), amoxicillin-clavulanic acid (n\u2009=\u200936), clarithromycin (n\u2009=\u200914), norfloxacin (n\u2009=\u200933), nitrofurantoin (n\u2009\u2009=\u2009\u200916)bPDR+, Preventive dose reduction appliedcPDR-, Preventive dose reduction not applieddNormal target therapeutic range: INR 2.0\u20133.5\nThe PDR applied was significantly greater in users of co-trimoxazole than in users of other antibiotics (15.0 and 10.3%, respectively; P value for difference: 0.036; two-sided t-test). The number of INR measurements during follow-up was significantly higher in both users of co-trimoxazole (PDR applied and PDR not applied) and users of other antibiotics (PDR applied) than in users of other antibiotics in whom a PDR was not applied. (P values of 0.028, 0.006 and 0.007, respectively; two-sided t-test). Mean daily dosages for acenocoumarol were lower in users of co-trimoxazole than in users of other antibiotics, but this difference was not statistically significant and even smaller (0.14\u00a0mg) after adjustment for differences in age (Table\u00a01).\nIn co-trimoxazole users, the PDR protected strongly against both moderate and severe overanticoagulation [adjusted odds ratio (OR): 0.06, 95% confidence interval (CI): 0.01\u20130.51 for INR >4.5; adjusted OR: 0.09, 95% CI: 0.01\u20130.92 for INR >6]. For other antibiotics, the effect of the PDR on overanticoagulation was not as strong and not statistically significant (Tables\u00a02 and 3). \nTable\u00a02Occurrence of overanticoagulation and time spent within, above and under the therapeutic range by patients using co-trimoxazole and other antibioticsaOutcomeCo-trimoxazoleOther antibioticsPDR+b (n\u2009=\u200928)PDR-b (n\u2009=\u200915)PDR+b (n\u2009=\u200960)PDR-b (n\u2009=\u2009223)INR >4.5, no. (%)3 (10.7)25 (89.3)9 (15.0)45 (20.2)dINR > 6.0, no. (%)1 (3.6)4 (26.7)5 (8.3)14 (6.3)eTime within therapeutic range, mean % (95%CI)c71.1 (60.4\u201381.8)51.8 (34.6\u201369.0)76.2 (69.5\u201382.9)75.7 (72.2\u201379.3)fTime above therapeutic range, mean % (95%CI)15.0 (5.7\u201324.3)20.3 (10.7\u201329.8)12.3 (6.8\u201317.7)18.9 (15.5\u201322.2)gTime under therapeutic range, mean % (95%CI)14.0 (5.6\u201322.2)27.9 (7.7\u201348.1)11.5 (6.9\u201316.1)5.4 (3.7\u20137.2)ha Calculated for the time from the date the antibiotic was first taken until the last INR measurement within 6\u00a0weeks following the starting date of the antibiotic. Time within, above and under therapeutic range was calculated for antibiotic users in whom at least one INR measurement had been performed within 6\u00a0weeks after the INR measurement during the antibiotic course. This resulted in the exclusion from the analysis of the following number of subjects: co-trimoxazole (1 1); other antibiotics 14 (1 13). The numbers in parenthesis indicate the number of subjects with a PDR and those without a PDR, respectively.bPDR+, Preventive dose reduction applied; PDR-, preventive dose reduction not applied.c95% CI, 95% Confidence interval for the reported mean valuedRange: 7.1% for nitrofurantoin to 27.6% for norfloxacineRange: 0% for nitrofurantoin and trimethoprim to 8.6% for doxycyclinfRange: 71.8% for amoxicillin to 84.8% for norfloxacingRange: 8.6% for norfloxacin to 22.0% for amoxicillinhRange: 3.3% for norfloxacin to 11.1% for clarithromycinTable\u00a03Odds ratios for effect of preventive dose reduction (PDR) and for (severe) overanticoagulation in users of co-trimoxazole compared with users of other antibiotics\u00a0Odds ratios (95%CI)PAdjusted Odds ratios (95% CI)aPEffect of PDR on overanticoagulationb\u00a0Co-trimoxazole\u00a0\u00a0\u00a0PDR applied, INR >4.50.10 (0.02\u20130.50)0.005*0.06 (0.01\u20130.51)0.010*\u00a0\u00a0\u00a0PDR applied, INR >6.00.10 (0.01\u20131.02)0.0510.09 (0.01\u20130.92)c0.042*\u00a0\u00a0\u00a0PDR not appliedReferenceReference\u00a0Other antibiotics\u00a0\u00a0\u00a0PDR applied, INR >4.50.70 (0.32\u20131.52)0.37N.A.d\u00a0\u00a0\u00a0PDR applied, INR >6.01.36 (0.47\u20133.93)0.57N.A\u00a0\u00a0\u00a0PDR not appliedReferenceRisk of overanticoagulation\u00a0PDR not applied\u00a0\u00a0\u00a0Co-trimoxazole, INR >4.54.52 (1.56\u201313.1)0.006*3.96 (1.33\u201311.8)0.013*\u00a0\u00a0\u00a0Co-trimoxazole, INR >6.05.43 (1.53\u201319.2)0.009*3.86 (1.03\u201314.6)0.046*\u00a0\u00a0\u00a0Other antibioticsReferenceReference\u00a0PDR applied\u00a0\u00a0\u00a0Co-trimoxazole, INR >4.50.68 (0.17\u20132.73)0.59N.A.\u00a0\u00a0\u00a0Co-trimoxazole, INR >6.00.41 (0.04\u20133.66)0.420.30 (0.03\u20133.05)e0.30\u00a0\u00a0\u00a0Other antibioticsReferenceReference*Statistically significant difference at P\u2009\u2264\u20090.05aAdjusted for differences in fever as indicated by patient, age, sex, target therapeutic range, unless otherwise indicatedbPDR, Preventive dose reductioncAdjusted for differences in age and sexdN.A., Adjustment not applied because the inclusion of covariates in our model did not result in a change of at least 5% in the odds ratios (see text)eAdjusted for differences in age, sex and fever as indicated by patient\nIf PDR was applied, the risk of overanticoagulation was not increased in users of co-trimoxazole compared with users of other antibiotics. However, if PDR was not applied, there was a strongly increased risk of moderate as well as severe overanticoagulation in co-trimoxazole users compared with users of other antibiotics (adjusted OR: 3.96, 95% CI: 1.33\u201311.8 for INR >4.5; adjusted OR: 3.86, 95%CI: 1.03\u201314.6 for INR >6.0) (Tables\u00a02 and 3).\nDuring the 6-week follow-up, co-trimoxazole users with a PDR spent more time within and less time under the therapeutic range than co-trimoxazole users without a PDR, but these differences were not statistically significant. Users of co-trimoxazole without a PDR spent significantly less time within the therapeutic range than users of other antibiotics with a PDR, whereas significantly more time was spent under the therapeutic range. Moreover, co-trimixazole users with a PDR also spent significantly more time under the therapeutic range than did all users of other antibiotics (adjusted mean difference: 6.9%; 95%CI: 1.0\u201312.9) (Tables\u00a02 and 4). \nTable\u00a04Comparisons of time spent within, under and above the therapeutic range by users of co-trimoxazole and other antibioticsaMean differencePAdjusted mean differencebPPDR applied\u00a0\u00a0Co-trimoxazole, % time within TRc\u22125.1 (\u221217.2 to 6.9)0.40\u22124.2 (\u221217.1 to 8.6)0.51\u00a0\u00a0Co-trimoxazole, % time above TR2.7 (\u22127.3 to 12.7)0.601.9 (\u22128.9 to 12.7)0.73\u00a0\u00a0Co-trimoxazole, % time under TR2.4 (\u22126.3 to 11.1)0.58N.A.d\u00a0\u00a0Other antibioticsReferenceReferencePDR not applied\u00a0\u00a0Co-trimoxazole, % time within TR\u221223.8 (\u221238.2 to \u20139.6)< 0.001*\u221222.3 (\u221236.6 to \u20138.0)e0.002*\u00a0\u00a0Co-trimoxazole, % time above TR1.4 (\u221211.7 to 14.5)0.83N.A.\u00a0\u00a0Co-trimoxazole, % time under TR22.5 (14.4 to 30.6)< 0.001*20.4 (12.4 to 28.5)f< 0.001*\u00a0\u00a0Other antibioticsReferenceReferenceCo-trimoxazole\u00a0\u00a0PDR applied, % time within TR19.3 (0.7 to 37.9)0.042*14.6 (\u22125.8 to 35.1)f0.16\u00a0\u00a0PDR applied, % time above TR\u22125.3 (\u221219.6 to 9.0)0.46\u22124.4 (\u221219.1 to 10.3)g0.55\u00a0\u00a0PDR applied, % time under TR\u221214.0 (\u221231.6 to 3.6)0.16\u221210.7 (\u221229.0 to 7.5)g0.24\u00a0\u00a0PDR not appliedReferenceReferenceOther antibiotics\u00a0\u00a0PDR applied, % time within TR0.5 (\u22127.0 to 8.1)0.890.6 (\u22126.8 to 8.2)0.87\u00a0\u00a0PDR applied, % time above TR\u22126.6 (\u221213.5 to 0.3)0.061N.A.\u00a0\u00a0PDR applied, % time under TR6.1 (2.0 to 10.1)0.003*N.A.\u00a0\u00a0PDR not appliedReferenceReferenceCo-trimoxazole, PDR applied\u00a0\u00a0% time within TR\u22124.7 (\u221215.1 to 5.6)0.37\u22123.0 (\u221213.5 to 7.5)f0.58\u00a0\u00a0% time above TR\u22122.4 (\u221211.9 to 7.1)0.61\u22123.7 (\u221213.3 to 6.0)h0.46\u00a0\u00a0% time under TR7.2 (1.2 to 13.1)0.018*6.9 (1.0 to 12.9)h0.022*\u00a0\u00a0Other antibiotics\u00a0\u00a0\u00a0\u00a0\u00a0PDR applied + PDR not appliedReferenceReference*Statistically significant difference at P\u2009\u2264\u20090.05aCalculated for the time from the starting date of the antibiotic until the last INR measurement within 6\u00a0weeks following the starting date of the antibiotic.bAdjusted for differences in fever as indicated by patient, age, sex, target therapeutic range, unless otherwise indicated.cTR, Therapeutic range;dN.A., Adjustment not applied because including covariates in our model did not result in a change of at least 5 % of mean difference (see text).eAdjusted for differences in sex.fAdjusted for differences in sex and target therapeutic range.gAdjusted for differences in age, sex and target therapeutic range.hAdjusted for differences in sex, target therapeutic range and fever as indicated by patients.\nCo-trimoxazole users with more than a 20% PDR spent significantly more time under the therapeutic range than users of other antibiotics (adjusted mean difference: 7.4\u00a0mg; 95%CI: 0.9\u201314.0; P\u2009=\u20090.027). If less than a 20% PDR was applied, the difference between the users of co-trimoxazole and those of other antibiotics shrunk and was no longer significant.\nThe application of a PDR differed between anticoagulation clinics. Three of the four anticoagulation clinics participating in this study applied PDR as a rule in co-trimoxazole users (83.3\u201385.7%). In terms of users of other antibiotics, the application of a PDR was more varied: in three of the anticoagulation clinics PDR was sometimes applied (in 17.6\u201350.8% of all cases), whereas one anticoagulation clinic did not apply the PDR approach at all. The overall percentage of time spent within the therapeutic range during the first 6\u00a0weeks after initiation of an antibiotic ranged from 73.7 to 78.0% at all four anticoagulation clinics. In the anticoagulation clinic that did not apply a PDR, overanticoagulation (INR>4.5) occurred most frequently for the all antibiotics class (26.9 vs.10.8\u201322.7% in the other clinics), with the difference being most marked for co-trimoxazole (54.4 vs. 14.3\u201316.7% in the other clinics).\nWe also analysed our data separately for users of acenocoumarol and phenprocoumon. There were no differences in the point estimates of most of our main outcomes between users of either of these coumarins, with the exception of percentage of time spent under the therapeutic range in phenprocoumon users for whom PDR was applied. However, most of the results that were statistically significant for all coumarin users were also significant for users of acenocoumarol (n\u2009=\u2009252; 78.2 %), whereas they were in most cases not significant for the smaller group of users of phenprocoumon (n\u2009=\u200971; 21.8%) (Table\u00a05). \nTable\u00a05Main outcomes stratified for users of acenocoumarol and phenprocoumonOutcomeAcenocoumarol (n\u2009=\u2009254)Phenprocoumon (n\u2009=\u200971)Protective effect of PDRAdjusted OR (95%CI)aPAdjusted OR (95%CI)aPCo-trimoxazole\u00a0\u00a0\u00a0PDR applied, INR >4.50.08 (0.01\u20130.70)0.022*0.16 (0.01\u20134.48)>0.3b\u00a0\u00a0\u00a0PDR not appliedReferenceReferenceRisk of overanticoagulation\u00a0\u00a0\u00a0PDR not applied\u00a0\u00a0\u00a0Co-trimoxazole, INR >4.54.40 (1.15\u201316.8)0.030*3.83 (0.55\u201326.7)0.18\u00a0\u00a0\u00a0Other antibioticsReferenceReference% Time within or under TRMean difference (95%CI)Mean difference (95%CI)\u00a0\u00a0\u00a0Co-trimoxazole, % time within TR\u221222.1 (\u221239.1 to \u20135.0)0.011*\u221221.4 (\u221249.4 to 6.6)0.13\u00a0\u00a0\u00a0Co-trimoxazole, % time under TR20.3 (10.9\u201329.7)<0.001*22.6 (5.7\u201339.5)0.010*\u00a0\u00a0\u00a0Other antibioticsReferenceReference\u00a0\u00a0\u00a0Co-trimoxazole, PDR applied\u00a0\u00a0\u00a0% time under TR9.1 (3.0\u201315.1)0.004*\u22126.7 (\u221223.0 to 9.6)0.42\u00a0\u00a0\u00a0Other antibiotics\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0PDR applied + PDR not appliedReferenceReference*Statistically significant difference at P\u2009\u2264\u20090.05aAdjusted for differences in fever as indicated by patient, age, sex and target therapeutic rangebAdjustment not applied because the number of patients with INR >4.5 was zero; OR was calculated by increasing the values of each of the cells of the crosstable by 0.5\nRe-analysis of our results after excluding patients with thyroid diseases and malignancy or those using enzyme-inhibiting or -inducing drugs gave similar point estimates or trends, significance for severe overanticoagulation in users of co-trimoxazole compared to other antibiotics and for time spent within the therapeutic range for users of co-trimoxazole in whom PDR was not applied (data not shown).\nDiscussion\nThe results of the present study, in which we evaluated the management of the interaction between antibiotics and coumarin anticoagulants by anticoagulation clinics, demonstrated that a PDR reduces the risk of overanticoagulation in co-trimoxazole users to the level of other antibiotic users, but also that management of the interaction between coumarins and co-trimoxazole results in a significantly longer period of undertreatment during the first 6\u00a0weeks after initiation of the antibiotic.\nIn three of the four anticoagulation clinics PDR was applied more frequently and was significantly higher in users of co-trimoxazole than in users of other antibiotics, indicating that anticoagulation clinics are aware of the seriousness of the interaction between coumarins and co-trimoxazole. In the cases and case series that have reported on overanticoagulation and bleeding with the concurrent use of antibiotics and co-trimoxazole [2, 3, 6\u20138, 10] an effect of the intercurrent infection on the anticoagulation status could not be ruled out. However, Penning-van Beest et al. (case control study) and Visser et al. (follow-up study) both demonstrated that an increased risk of severe overanticoagulation (INR >6.0) was particularly associated with co-trimoxazole [12, 21]. A plausible explanation is the strong inhibition of the main metabolising enzyme, CYP2C9, of the coumarins by sulfamethoxazole, the sulphonamide component of co-trimoxazole [24].\nAlthough PDRs as applied in clinical practice are effective in reducing the overanticoagulation risk in co-trimoxazole users, the price that has to be paid for the concurrent use of co-trimoxazole is a significantly prolonged period of underanticoagulation compared with the use of other antibiotics during the first 6\u00a0weeks after the antibiotic course. This difference was more marked in the subgroup of subjects in whom PDR was not applied. Possible explanations for this result are (1) the usually shorter time span between PDR and the first INR measurement (always within the course) compared to the time span between a reactive dose reduction following supratherapeutic INR and subsequent INR measurement (usually after the course) and (2) the higher reactive dose reduction which is applied in the case of severe overanticoagulation (INR >6.0). However, even co-trimoxazole users for whom the PDR had been applied had a significantly prolonged period of underanticoagulation compared with all of the users of other antibiotics (PDR applied and PDR not applied taken together). This last comparison is totally logical because our results strongly suggest that a PDR should always be applied in co-trimoxazole users, whereas this is as a rule not required in users of other antibiotics. The adjusted difference in time spent under the therapeutic range \u2013 ranging from 6.9 (PDR applied) to 22.5% (PDR not applied) \u2013 corresponds to about 2\u20137\u00a0days of the mean follow-up time of 30\u00a0days in otherwise stabilised patients; this time interval is clinically relevant and can be avoided by substituting co-trimoxazole.\nIt is not difficult to explain the prolonged period of underanticoagulation in co-trimoxazole users. The application of a PDR, which was in this study higher in co-trimoxazole users, might overcompensate for overanticoagulation, whereas the reactive dose reduction following overanticoagulation carries the same risk of overcompensation and undertreatment as PDR. Consequently, the inhibition of CYP2C9 by co-trimoxazole superimposes an additional problem upon the already potentially destabilising effects of the infection and fever. Because our results for acenocoumarol in the separate analyses were predominantly in agreement with the overall results, our findings primarily apply to acenocoumarol users. It is possible that users of phenprocoumon are less sensitive to interactions with CYP2C9 inhibitors such as co-trimoxazole [20, 22]. We do expect that our results also apply to users of warfarin, which seems to be even more CYP2C9 sensitive than acenocoumarol [18].\nOur study has several limitations. Because we retrieved medical data from anticoagulation clinics, it is possible that not all of the relevant data on potentially destabilising factors, such as malignancies, thyroid diseases and the use of other inhibitors of coumarin metabolism, were available. However, by only including patients who were obviously stable at the moment of initiation of the antibiotic, we decreased the chance that such factors changed the anticoagulant status during the antibiotic course. A second limitation is the absence of data on the presence of polymorphisms of the genes encoding the coumarin-metabolising enzyme CYP2C9 or the pharmacodynamic target of coumarins, VKORC1. The genotypes of both CYP2C9 [16, 22] and VKORC1 are strongly associated with interindividual variability in coumarin dose requirements [4, 14, 17, 23]. Further studies would be needed to assess whether the risk of overanticoagulation in co-trimoxazole users differs between carriers of a CYP2C9 or VKORC1 polymorphism and wild-type patients. It should be clear that our results only apply to patients with a stabilised anticoagulation state at the initiation of the antibiotic course.\nIn conclusion, if co-trimoxazole is prescribed to users of coumarin anticoagulants, the interaction can be managed by applying PDR, which adequately decreases the risk of overanticoagulation, but this successful management comes at the cost of a prolonged period of underanticoagulation after the course. Consequently, rather than managing the interaction it is better to avoid prescribing co-trimoxazole as a therapeutically equivalent alternative is always available.","keyphrases":["coumarins","co-trimoxazole","acenocoumarol","phenprocoumon","antibiotics","anticoagulation clinic","drug interaction"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Appl_Microbiol_Biotechnol-3-1-2039825","title":"Properties, production, and applications of camelid single-domain antibody fragments\n","text":"Camelids produce functional antibodies devoid of light chains of which the single N-terminal domain is fully capable of antigen binding. These single-domain antibody fragments (VHHs or Nanobodies\u00ae) have several advantages for biotechnological applications. They are well expressed in microorganisms and have a high stability and solubility. Furthermore, they are well suited for construction of larger molecules and selection systems such as phage, yeast, or ribosome display. This minireview offers an overview of (1) their properties as compared to conventional antibodies, (2) their production in microorganisms, with a focus on yeasts, and (3) their therapeutic applications.\nIntroduction\nThe field of recombinant antibody technology has rapidly progressed during the last two decades, mainly because of the interest in their human therapeutic use. The ability to select specific human antibodies by display technologies and to improve their affinity, stability, and expression level by molecular evolution has further boosted the field. Whole antibodies are complex molecules that consist of heavy and light chains (Fig.\u00a01a). They contain an N-linked oligosaccharide attached to the second heavy-chain constant domain (CH2) that is essential for antibody effector functions such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytolysis (CDC), and for retaining a long serum half-life.\nFig.\u00a01Schematic diagram of conventional (a) and heavy-chain (b) antibodies and fragments thereof. Variable domains derived from the antibody heavy (VH) and light (VL) chains are shaded dark gray and light gray, respectively, whereas constant domains (CH and CL) are not shaded. Note the absence of the light chain and CH1 domain in heavy-chain antibodies. Antibody domains that pair by noncovalent interactions are indicated by overlaying them. The B-subunits of naturally pentamerizing toxins that are used to generate pentabodies are indicated as hatched spheres\nAlthough isolated antibody heavy (Utsumi and Karush 1964) and light chains (Yoo et al. 1967) can retain antigen-binding specificity, their affinity and solubility is often reduced (Ward et al. 1989). However, the paired N-terminal variable domains of heavy (VH) and light (VL) chains are sufficient for antigen binding (Sundberg and Mariuzza 2002). Such antibody fragments can be produced as monovalent antibody fragment (Fab) or as single-chain Fv (scFv) where the VH and VL domains are joined by a polypeptide linker (Fig.\u00a01a). Their production in microbial cells is often cumbersome, especially when producing multivalent formats, because of the requirement for domain association.\nThe discovery that camelids (bactrian camels, dromedaries, and llamas) produce functional antibodies devoid of light chains (Hamers-Casterman et al. 1993) formed a further breakthrough because their single N-terminal domain (VHH, also referred to as Nanobody\u00ae) binds antigen without requiring domain pairing. These heavy-chain antibodies also lack the CH1 domain, which in a conventional antibody associates with the light chain and to a lesser degree interacts with the VH domain (Fig.\u00a01b). Although single-domain antibodies later were also identified in particular cartilaginous fish (Greenberg et al. 1995), most research on the biotechnological application of single-domain antibodies was done using camelids because of their ease of handling, including immunization. Methods to isolate antigen-specific VHHs from immune (Arbabi-Ghahroudi et al. 1997; Van der Linden et al. 2000a), nonimmune (Tanha et al. 2002; Yau et al. 2003; Verheesen et al. 2006), or semisynthetic (Goldman et al. 2006) libraries using phage, yeast, or ribosome display are now well established. For further reading on these topics, we refer to recently published reviews (Muyldermans 2001; Dufner et al. 2006).\nProperties\nSequence analysis (Hamers-Casterman et al. 1993; Muyldermans et al. 1994; Vu et al. 1997; Harmsen et al. 2000) and elucidation of the crystal structure (Desmyter et al. 1996, 2001; Spinelli et al. 1996) has revealed several structural features of VHH domains. Similar to conventional VH domains, VHHs contain four framework regions (FRs) that form the core structure of the immunoglobulin domain and three complementarity-determining regions (CDRs) that are involved in antigen binding. This contrasts with shark single-domain antibodies that have a vestigial CDR2 that does not contribute to antigen binding (Streltsov et al. 2004). As compared to human VH domains, the VHH FRs show a high sequence homology of more than 80%, and their 3D structures can be superimposed (Muyldermans et al. 2001; Holliger and Hudson 2005).\nThe most characteristic feature of VHHs is the presence of amino acid substitutions at four FR2 positions (positions 37, 44, 45, and 47; Kabat numbering) that are conserved in conventional VH domains and that are involved in forming the hydrophobic interface with VL domains. Occasionally, antigen-binding single-domain antibody fragments that lack these characteristic FR2 substitutions are isolated from camelids. These fall into two groups. The low-affinity binders isolated from a nonimmune library originated from conventional antibodies, presumably because of the polymerase chain reaction crossover cloning artifact, as they were linked to the CH1 domain (Tanha et al. 2002). We refer to these as VHH-like conventional VHs. However, such single-domain antibody fragments with conventional-like FR2 sequences that bind antigen with high affinity are isolated from immune libraries with the high efficiency of about 10% (Conrath et al. 2001a; Saerens et al. 2004; Harmsen et al. 2005a, 2007), which equals their presence in unselected libraries (Harmsen et al. 2000). This is not expected when such clones originate from a cloning artifact. Unlike the clones isolated by Tanha et al. (2002), these clones often contain a hydrophilic residue (mostly arginine) at position 103. This substitution is probably important for their single-domain nature (Desmyter et al. 2001) because conventional antibodies contain a highly conserved hydrophobic residue (tryptophan) at this position that contacts VL. This suggests that these represent functional VHH domains derived from recombination of conventional VH gene segments with heavy-chain constant gene segments during B cell maturation. This was confirmed by the absence of the CH1 domain when such VHH domains were reisolated from the original immune repertoire using a CDR3-specific primer (De Haard, unpublished observation). Therefore, we refer to these as conventional-like VHH domains. Although the increased hydrophilicity of VHHs predominantly relies on the aforementioned changes in the former VL interface, some amino acids at positions that form a slightly hydrophobic patch on conventional VH domains that contacts CH1 (Lesk and Chothia 1988) are also changed into hydrophilic residues in VHHs (Muyldermans et al. 1994; Harmsen et al. 2000).\nFurthermore, the CDRs of VHHs contain some characteristic features. Firstly, the N-terminal part of CDR1 is more variable (Vu et al. 1997; Harmsen et al. 2000; Nguyen et al. 2000). Secondly, many dromedary VHHs have an extended CDR3 that is often stabilized by an additional disulfide bond with a cysteine in CDR1 or FR2 (Muyldermans et al. 1994) resulting in the folding of the CDR3 loop across the former VL interface (Desmyter et al. 1996). A particular subfamily of llama VHHs (VHH3) also contains an extended CDR3 that is stabilized by an additional disulfide bond with a cysteine at position 50 in FR2. However, VHHs of this subfamily are rarely isolated, and most llama VHHs have CDR3 loops similar in length to those found in human VHs.\nVHHs have many advantages for biotechnological applications, which are summarized in Table\u00a01. An important advantage is their high microbial production level (see next section).\nTable\u00a01Advantages of camelid single-domain antibody fragments as compared to conventional antibody fragmentsAdvantageMolecular basisFacile genetic manipulationSingle-domain natureIncreased functional size of immune librariesNo decrease in library size because of reshuffling of VL and VH domainsFacile production of multivalent formatsMore flexible linker design and no mispairing of VL and VH domainsFacile production of oligoclonal preparations from single cellsNo mispairing of VL and VH domainsHigh physicochemical stabilityEfficient refolding due to increased hydrophilicity and single-domain natureHigh solubilityIncreased hydrophilicityRecognition of hidden antigenic sitesSmall size and extended flexible CDR3Rapid tissue penetration, fast clearanceSmall sizeWell expressedEfficient folding due to increased hydrophilicity and single-domain natureSee text for references\nSeveral advantages result from their single domain nature. Thus, VHH libraries generated from immunized camelids retain full functional diversity. This contrasts with the diminished diversity of conventional antibody libraries because of reshuffling of VL and VH domains during library construction. As a result, high-affinity antigen-binding VHHs can be isolated by directly screening a limited number of clones from immune libraries without prior selection using display technologies (Frenken et al. 2000; Harmsen et al. 2005b). Furthermore, the single-domain nature facilitates subsequent molecular manipulation. For example, for many applications, it is advantageous to engineer monovalent antibody fragments into multivalent formats to increase functional affinity (termed avidity) or to produce bispecific antibody fragments that can simultaneously bind to different antigens. Such molecules (diabodies, Fig.\u00a01a) can be produced using conventional recombinant antibodies using linkers between the VH and VL domains of a specific length, although this often results in aggregation and reduced affinity because of mispairing of VH and VL domains (Glockshuber et al. 1990; Whitlow et al. 1993). VHHs are more suitable for production of such formats because they allow more flexible linker design, which is important for simultaneous binding of multivalent antigens, without the problems posed by domain mispairing. Thus, several functional trivalent-bispecific VHHs have been successfully produced (Coppieters et al. 2006; Roovers et al. 2007).\nThe use of mixtures of a limited number of monoclonal antibodies (oligoclonal antibodies) is advantageous over single monoclonal antibodies for particular applications, such as toxin neutralization (Nowakowski et al. 2002). Because of regulatory requirements, such oligoclonals are preferentially produced from single cells. Again, VHHs are predicted to be more suitable for single-cell production of oligoclonals because of the absence of domain mispairing, although this is yet to be demonstrated experimentally.\nContrary to conventional antibodies, VHHs have been shown to remain functional at 90\u00b0C (Van der Linden et al. 1999) or after incubation at high temperatures (Van der Linden et al. 1999; Perez et al. 2001). This high apparent stability is mainly attributed to their efficient refolding after chemical or thermal denaturation and to a lesser extent because of an increased resistance against denaturation (Perez et al. 2001; Dumoulin et al. 2002; Ewert et al. 2002). The increased apparent stability is probably due to an increased hydrophilicity of the former VL interface region because a \u201ccamelized\u201d human VH fragment that contains several of the hallmark hydrophilic amino acid residues of VHHs was more stable than the original VH fragment (Davies and Riechmann 1995, 1996), whereas \u201cdecamelization\u201d of a VHH to mimic a VH domain reduces its thermodynamic stability (Conrath et al. 2005). In addition to these specific mutations, the packing of extended CDR3 loops against this former VL interface contributes to domain stability (Bond et al. 2003). Furthermore, refolding of VHHs only requires domain refolding, whereas conventional antibodies also require association of VH and VL domains.\nVHHs can also recognize antigenic sites that are normally not recognized by conventional antibodies such as enzyme active sites (Lauwereys et al. 1998; De Genst et al. 2006) and conserved cryptic epitopes (Stijlemans et al. 2004). This facilitates their use as enzyme inhibitors or in diagnosis of trypanosome infections. The ability to recognize these recessed antigenic sites has been attributed to their smaller size and the ability of the extended CDR3 loop to penetrate into such sites (Desmyter et al. 1996; De Genst et al. 2006). It is interesting to note that this structure\u2013function relation is also observed in a rare example of a broadly reactive human mAb that recognizes the recessed and conserved CD4-binding cavity of human immunodeficiency virus type 1 gp120 by virtue of an extended CDR3 (Zwick et al. 2003). With respect to antigen binding, the single-domain nature could be a disadvantage for binding to small antigens such as haptens and peptides because these normally bind in a groove or cavity at the VH\u2013VL interface (Sundberg and Mariuzza 2002). Indeed, llamas immunized with clenbuterol developed conventional but not heavy-chain antibodies against this hapten (Lange et al. 2001). However, hapten- and peptide-binding VHHs have been successfully isolated using strong selection systems (Spinelli et al. 2000; Yau et al. 2003; Alvarez-Rueda et al. 2007; Harmsen et al. 2007). The affinities of VHHs are generally comparable to those of conventional antibody fragments (Muyldermans et al. 2001). Occasionally, VHHs with affinity constants (KD) as low as 100\u00a0pM are isolated (Saerens et al. 2004; De Genst et al. 2006; Harmsen et al. 2006), which equals the affinity ceiling proposed for natural antibodies (Sundberg and Mariuzza 2002).\nBecause of their small size of about 15\u00a0kDa, VHHs rapidly pass the renal filter, which has a cutoff of about 60\u00a0kDa, resulting in their rapid blood clearance. In addition, the small size results in a fast tissue penetration. This is advantageous for targeting of VHHs coupled to toxic substances to tumors (Cortez-Retamozo et al. 2004), in vivo diagnosis using imaging, and treatment of snake bites (Harrison et al. 2006). However, for other therapeutic applications, such as treatment of infectious or inflammatory diseases, the short serum half-life of about 2\u00a0h (Cortez-Retamozo et al. 2002; Harmsen et al. 2005a) is a disadvantage.\nProduction in microorganisms\nAlthough a fully active nonglycosylated IgG was recently produced at high level in Escherichia coli, most functional complete antibodies can only be efficiently produced using mammalian cells, especially when their appropriate glycosylation is required for therapeutic applications. However, antibody fragments that lack the Fc with its N-linked oligosaccharide are preferably produced in microbial systems (Arbabi-Ghahroudi et al. 2005). These have a shorter development time from gene to product and require simple well-established fermentation conditions that can be performed on large-scale resulting in costs of goods that can be as low as $1 per gram heterologous protein (Estell 2006). Most large-scale microbial production systems are based on E. coli, yeasts, or filamentous fungi. Production in E. coli can be done by secretion into the oxidizing periplasmic space or expression in the reducing cytosol. The latter requires the often cumbersome refolding of antibody fragments (Arbabi-Ghahroudi et al. 2005). Using eukaryotic microbial hosts, antibody fragments are generally produced by targeting to the secretory pathway. This enables efficient disulfide bond formation, addition of N-linked oligosaccharide, and secretion of soluble, correctly folded product to the culture medium.\nVHHs have often been produced in E. coli (Arbabi-Ghahroudi et al. 1997; Rahbarizadeh et al. 2005). There is only one example of VHH production in filamentous fungi, which resulted in limited proteolytic degradation of the secreted product (Joosten et al. 2005) because of the high levels of proteases secreted by filamentous fungi (Gerngross 2004). VHHs have also often been produced in the yeast Saccharomyces cerevisiae (Frenken et al. 2000; Thomassen et al. 2002; Van der Vaart 2002). VHH production by the favored yeast expression host Pichia pastoris was only recently described (Rahbarizadeh et al. 2006). Occasionally, yeast-produced VHHs are N-glycosylated (Frenken et al. 2000; Harmsen et al. 2005a). This can affect antigen binding (Van der Vaart et al. 2006). Furthermore, it could complicate their therapeutic use because the addition of yeast-specific high-mannose oligosaccharides results in a high immunogenicity and decreased serum half-life because of binding to specific mannose receptors on cells of the reticulo-endothelial system (Sethuraman and Stadheim 2006).\nAlthough VHHs are generally well produced in microorganisms, the production level of different clones can vary by a factor of 100 (Frenken et al. 2000; Harmsen et al. 2005b; Van de Laar et al. 2007). Several VHH sequence patterns can be associated with their production level. First, the presence of a potential N-linked glycosylation site often increases production levels in yeast (Sagt et al. 2000). Second, in our experience (Harmsen, unpublished observations), conventional-like VHHs are generally produced at reduced levels in yeast. This contrasts with the reported efficient production in E. coli of VHH-like VHs (Tanha et al. 2002) but is consistent with the increased production level of \u201ccamelized\u201d conventional VH domains in E. coli (Davies and Riechmann 1995). Third, the presence of unpaired C-terminal cysteines reduces expression levels (Simmons et al. 2006). Fourth, replacement of hydrophobic residues of conventional VH domains normally interacting with CH1 increased scFv production in E. coli (Nieba et al. 1997), suggesting that the hydrophilic mutations that naturally occur at these positions in VHHs also contributes to their high expression level. However, there are many examples of VHHs that differ by only a few amino acids and are produced at highly variable levels where the exact amino acid change responsible for the difference in production level is difficult to predict (Frenken et al. 2000; Harmsen et al. 2005b). Furthermore, without such knowledge, VHH production can be improved by random molecular evolution using deoxyribonucleic acid shuffling (Van der Linden et al. 2000b), as has often been done for conventional antibody fragments (Dufner et al. 2006). The high refolding capability of VHHs, which is a consequence of their sequence, has also been correlated with a high production level in E. coli (Jespers et al. 2004; Olichon et al. 2007).\nIn addition to the nature of the VHH, host factors affecting VHH production have been identified. In baker\u2019s yeast, the specific VHH production rate is correlated with growth rate (Thomassen et al. 2005) and can be up to fivefold increased by growing on ethanol as the carbon source (Van de Laar et al. 2007). Supplementation of the medium with sorbitol, casamino acids, or ethylenediamine tetraacetic acid improves VHH production by P. pastoris (Rahbarizadeh et al. 2006).\nIn addition to monovalent VHHs, several expression formats for the production of VHH multimers have been described (Fig.\u00a01b). These include genetic fusions of two (Conrath et al. 2001b; Harmsen et al. 2005a) or three VHHs (Coppieters et al. 2006; Roovers et al. 2007) that either recognize different antigens or the same repeating antigen to increase functional affinity. Although such VHH fusions are less efficiently produced than their monovalent versions, their production level exceeds that of their conventional-antibody-based fusion counterparts without aggregation or low solubility. However, antigen binding by the C-terminal VHH in such fusions can be compromised (Conrath et al. 2001b) presumably because of steric hindrance by the N-terminal VHH. The avidity of VHHs has also been strongly increased using genetic fusions to the B-subunits of an E. coli toxin that self-assembles into a homopentamer (Zhang et al. 2004), resulting in pentameric recombinant antibodies (\u201cpentabodies,\u201d Fig.\u00a01b).\nVHHs on their own cannot recruit effector functions such as ADCC and CDC. This limits their therapeutic application. Although such effector functions can be indirectly recruited using bispecific (conventional) antibody fragments binding to host immunoglobulin (Holliger et al. 1997), it may be more efficient to recruit these functions by fusing VHHs to host Fc domains. Production of such functional antibodies requires the correct glycosylation of the CH2 domain, which until recently could only be accomplished using higher eukaryotic cells (Nguyen et al. 2003) but not by microbial production. However, this may now be feasible using P. pastoris strains with an engineered glycosylation machinery that are able to produce proteins with a specific human glycoform (Hamilton et al. 2006). Furthermore, transgenic mice containing hybrid llama\/human antibody loci that contain llama V regions and human D, J, and C regions have recently been used to generate human heavy-chain antibodies in mice (Janssens et al. 2006).\nTherapeutic applications\nAlthough VHHs are highly suited for applications that require a high stability, such as use in shampoo for the prevention of dandruff (Dolk et al. 2005), as capturing reagents in immunoaffinity purification (Verheesen et al. 2003), or use in biosensors (Pleschberger et al. 2004), we would like to focus on their therapeutic applications, which are more challenging. Several VHHs are now being studied for use in various disease areas, including oncology (Revets et al. 2005) and in infectious, inflammatory, and neurodegenerative diseases (Table\u00a02).\nTable\u00a02Examples of therapeutic applications of camelid VHHsDiseasePathogenTarget antigenVHH valency for disease targetAdditional fusion partnerReferenceSleeping sicknessTrypanosomesVSG oligomannoseMonovalentApolipoprotein L-IBaral et al. 2006Infant diarrheaRotavirusUnknownMonovalentNoneVan der Vaart et al. 2006Infant diarrheaRotavirusUnknownMonovalentLactobacillus cell-surface anchorPant et al. 2006Piglet diarrheaE. coliF4 fimbriaeMonovalentNoneHarmsen et al. 2006CariesS. mutansI\/II adhesionMonovalentNoneKruger et al. 2006FMDFMD virusVP1MonovalentPEGHarmsen et al. 2007SepsisN. meningitidisLPSMonovalentNoneEl Khattabi et al. 2006Cancer\u2013CEAMonovalent\u03b2-LactamaseCortez-Retamozo et al. 2004Cancer\u2013EGF receptorBivalentAnti-albumin VHHRoovers et al. 2007Rheumatoid arthritis\u2013TNF\u03b1BivalentAnti-albumin VHHCoppieters et al. 2006Brain disorders\u2013\u03b1 (2,3)-SialoglycoproteinMonovalentNoneMuruganandam et al. 2002Neurodegenerative diseases\u2013BaxMonovalentNoneGueorguieva et al. 2006\nVHHs are especially suited for oral immunotherapy because of their resistance against extremes of pH and the capacity to bind to the target at high concentrations of chaotropic agents (Dumoulin et al. 2002, 2003). Administration to piglets of a VHH that effectively prevents intestinal attachment of E. coli bacteria that cause diarrhea resulted in poor in vivo protection (Harmsen et al. 2005b) because of degradation by gastrointestinal proteases (Harmsen et al. 2006). However, by selection for proteolytic stability, a VHH could be isolated from the original library that was not degraded in vivo (Harmsen et al. 2006). VHHs that successfully prevented diarrhea caused by rotavirus in a mouse model were similarly selected for resistance against the acidic environment of the stomach (Van der Vaart et al. 2006). Alternatively, VHH proteolysis can be prevented by local VHH production using natural gut commensal bacteria. Thus, diarrhea could also be prevented by lactobacilli that produce rotavirus-neutralizing VHHs fused to a cell surface anchor (Pant et al. 2006). Treatment of caries, caused by Streptococcus mutans, with VHHs conferred only limited protection (Kruger et al. 2006). Because these VHHs should function in the oral cavity, the low level of protection cannot be due to proteolytic VHH degradation within the gastrointestinal tract.\nThe short serum half-life because of a rapid renal clearance limits the efficacy of VHHs in many parenteral applications. Therefore, VHHs have been targeted to normally long-lived serum proteins such as albumin (Coppieters et al. 2006; Roovers et al. 2007) or immunoglobulin (Harmsen et al. 2005a) using bispecific VHHs recognizing these serum proteins in addition to the therapeutic target, resulting in half-lives that equal the half-life of albumin (2\u00a0days in mice) and immunoglobulin (9\u00a0days). An alternative well-known approach to increase serum half-life of proteins is the chemical addition of polyethylene glycol (PEG). Such PEGylation of foot-and-mouth disease (FMD) virus-neutralizing VHHs not only increased serum half-life but also increased in vitro neutralizing potency to levels above that of the hyperimmune serum (Harmsen et al. 2007). However, in contrast to the full protection afforded by the hyperimmune serum, these VHHs poorly protected guinea pigs from FMD viral challenge infection, suggesting that Fc-mediated effector functions are required for efficient in vivo protection (Harmsen et al. 2007).\nNevertheless, many diseases were successfully treated with VHHs in the absence of Fc-mediated effector functions. These VHHs either are used as targeting devices for toxic enzymes or block a specific molecular interaction. For example, sleeping sickness was successfully treated with VHHs that bind to a trypanosome coat protein and were fused to the apolipoprotein L-1 enzyme, resulting in trypanosome lysis (Baral et al. 2006). In oncology, a VHH directed against carcinoembryonic antigen was used for targeting the genetically fused \u03b2-lactamase to tumor cells. This enzyme then converts an injected nontoxic prodrug into a toxic drug in the vicinity of the targeted tumor cells, leading to their killing (Cortez-Retamozo et al. 2004). Several VHH therapies are also being developed for treatment of oncology or inflammatory diseases based on blocking molecular interactions. VHHs binding to epidermal growth factor receptor (EGFR) can block epidermal growth factor (EGF) binding to its receptor, which can be used to treat solid tumors (Roovers et al. 2007). Tenfold more potent EGFR-binding VHHs could be obtained by construction of bivalent formats. It is interesting to note that the recently approved conventional antibody Panitumumab directed against EGFR also blocks EGF binding and is expected to give poor ADCC and CDC (Reichert and Valge-Archer 2007). Furthermore, by blocking receptor interaction, VHHs binding to tumor necrosis factor-\u03b1 can be used for treatment of rheumatoid arthritis (Coppieters et al. 2006). The potency of bivalent formats was 500-fold increased as compared to monovalent VHHs and even exceeded the potency of clinically used conventional antibodies both in vitro and in a murine arthritis model. Similarly, lipopolysaccharide (LPS)-binding VHHs were isolated that block LPS binding and signaling to host cells for treatment of sepsis (El Khattabi et al. 2006).\nThe potential immunogenicity of VHHs could compromise their parenteral therapeutic use, especially in treatments that require repeated injections. Until now, multiple injections of VHHs have not shown any immunogenicity in mice, as assessed by the presence of specific antibodies, T cell proliferation, or cytokine levels (Cortez-Retamozo et al. 2002; Coppieters et al. 2006). This could rely on their high sequence homology to conventional VH domains and on their high stability because aggregation of proteins is known to increase immunogenicity (Hermeling et al. 2004). If necessary, technologies developed to decrease immunogenicity of mouse monoclonal antibodies (Presta 2006) could also be applied to VHHs. Alternatively, immunogenicity could be reduced by the use of conventional-like VHHs, which have an even higher structural homology to conventional VH domains.\nFor their use in targeting drugs across the blood\u2013brain barrier (BBB) into the brain, VHHs were selected that transmigrate the human BBB in an in vitro model and accumulate in the brain after intravenous injection into mice (Muruganandam et al. 2002). These could be used for treatment of neurological disorders. Finally, Bax-specific VHHs have been expressed in the cytoplasm, resulting in so-called intrabodies, to prevent oxidative-stress-induced apoptosis that is implicated in several neurodegenerative diseases (Gueorguieva et al. 2006). Because of their stability, VHHs are especially suited for intrabody production because this requires expression in the reducing environment of the cytoplasm (Gueorguieva et al. 2006; Rothbauer et al. 2006).\nConclusions\nSince the discovery of heavy-chain antibodies in 1993, the field of single-domain antibody fragments has been rapidly growing. VHHs have many advantages for biotechnological applications. They can be economically produced in microorganisms and have a high stability. Furthermore, they are highly suited for expression as multivalent, including bispecific, formats or as enzyme fusions. This permits a plug-and-play approach, where, depending on the target, biology potency can be increased by multivalent constructs or bispecific VHH recognizing two different targets can be made. This also enables the tailor-made design of serum half-life using site-directed PEGylation or by targeting to long-lived serum proteins using bispecific VHHs. Although fusions of targeting VHHs to Ig-binding VHHs or Fc can be used to recruit effector functions most current research on VHHs focuses on therapeutic applications where such effector functions are not required. Finally, conventional whole antibodies occasionally give side effects because of their bivalent nature, which can result in target cross-linking, or the presence of the Fc region. Evidently, such side effects are not expected to occur using monovalent VHHs. This, however, is yet to be confirmed as the first VHH has entered phase I clinical trials in 2007 (http:\/\/www.ablynx.com).","keyphrases":["single-domain","yeast","microbial production","glycosylation"],"prmu":["P","P","P","P"]}
{"id":"J_Headache_Pain-4-1-2245992","title":"Performances in cerebellar and neuromuscular transmission tests are correlated in migraine with aura\n","text":"In previous studies, we described subclinical abnormalities of neuromuscular transmission and cerebellar functions in migraineurs. The aim of this study was to search if these two functions are correlated in the same patient. Thirteen migraineurs [five without aura (MO) and eight with aura (MA)] underwent both stimulation-SFEMG and 3D-movement analysis. Single fiber EMG (SFEMG) results were expressed as the \u201cmean value of consecutive differences\u201d (mean MCD). Precision of arm-reaching movements (measured with an infrared optoelectronic tracking system) was expressed as the average deviation in the horizontal plane. Median values of mean MCD and mean horizontal deviation were not different between MO and MA. However, in MA, but not in MO, both variables were positively correlated. Thus, we conclude that neuromuscular transmission and cerebellar functions are correlated in the same patient when affected by migraine with aura. We suggest that this correlation might be due to a common molecular abnormality.\nIntroduction\nMigraine is a paroxysmal neurological disorder with a high prevalence in the general population. There is strong epidemiological and genetic evidence that genetic factors play a major, though variable role, in its pathogenesis. Migraine patients are characterized between attacks on various CNS-evoked responses by a deficit of habituation [1, 2], which may have a familial character [3\u20135]. The lack of habituation may play a role in migraine pathogenesis [6]. By contrast, other mild abnormalities have been identified that are unlikely to play a pathogenic role: a decreased safety factor at the neuromuscular junction on single fiber EMG (SFEMG) [7\u20139] and subclinical cerebellar hypermetria in the horizontal plane on opto-electronic analysis of upper arm-reaching movements [10]. It was suggested that these two abnormalities might reflect the same underlying genetic abnormality. If this is correct, one may expect that in the same individual they be of a similar degree. We have therefore compared in the same migraine patient the SFEMG and 3D-movement analysis.\nMaterials and methods\nPatients\nThirteen migraine patients (ICHD-II) [11] were recruited from the Headache Clinic of the Headache Research Unit in Li\u00e8ge, Belgium. Eight patients suffering from typical aura with migraine headache (MTA\u2013ICHD-II code 1.2.1; three women and five men; median age 28.5\u00a0years; range 13\u201367) and five patients with migraine without aura (MO\u2013ICHD-II code 1.1; three women and two men; median age 40.0\u00a0years; range 31\u201351) underwent both stimulation-SFEMG and 3D-movement analysis. All patients were right-handed. None of them had any other medical condition detectable by history and clinical examination; none was taking drugs on a regular basis, nor had taken any drug within 3\u00a0days before the recordings. The recordings took place at least 3\u00a0days after and before an attack (checked by telephone-interview).\nThe study was conducted after approval of our Institution\u2019s ethics committee and performed in accordance with the ethical standards of the 1964 Declaration of Helsinki, with the understanding and consent of each involved subject.\nSFEMG recordings\nA Nicolet Viking IV device (Nicolet\u00ae Biomedical, Madison, Wisconsin, USA) was used for stimulation single fiber electromyography [12]. Single muscle fiber activity was recorded with 25-mm-long single fiber needles (Medelec Neurodiagnostic Accessories, ref: 16829, Witney, Oxfordshire, UK), and the motor nerve was stimulated with Nicolet teflon-insulated monopolar needles. We stimulated suprathreshold the motor branch of the radial nerve and assessed the variability in latency, i.e. the jitter (Fig.\u00a01), of single fiber action potentials in m. extensor digitorum communis (EDC) of the right arm. Stimulations\u2013acquisitions were 100 per muscle fiber, and stimulation rate was 10\u00a0Hz. We recorded 25 EDC muscle fibers per patient. Off-line analyses of recordings were performed, and on average 18 artifact-free EDC muscle fibers per patient were selected to assess their mean MCD. Results were expressed as the \u201cmean value of consecutive differences\u201d (MCD) of successive interpotential intervals, as usual in SFEMG studies.\nFig.\u00a01Figures representing a normally (on the top) and an abnormally (on the bottom) jittered fibers\n3D-movement analysis\nMovements were recorded at 100\u00a0Hz in three dimensions (3D) using an infrared optoelectronic-tracking-system (ELITE\u2122, Milan, Italy) with a reflective marker attached to the tip of the index finger and another marker to the movement target.\nParticipants were seated with the target in the medio-sagittal plane on eye level. They were instructed to start with the right arm extended to the right, to touch the target with high precision (but fast) without any trunk movement, to go back to the starting position and to repeat above movements, in a given pace over 15\u00a0s (one trial) to result in 8\u201310 movements (Fig.\u00a02). The used Cartesian coordinate system is head-fixed with a nasooccipital, a horizontal and a vertical axis and the origin in the target. Since in our previous study [10] abnormalities in migraineurs were most pronounced in the horizontal plane, we limited ourselves to an analysis of the mean deviation in the horizontal plane (in millimeters), measured over four trials, each consisting of 8\u201310 arm movements.\nFig.\u00a02Experimental setup for the movement task\nQuantitative variables in each group of migraine patients (MO and MTA) were expressed as medians. Differences between groups were analysed with the Mann\u2013Whitney U test. The Spearman rank order correlation test was used to compare the values of the mean MCD on SFEMG and the mean horizontal deviation on 3D-movement analysis.\nResults\nOn SFEMG, the median value for mean MCD was not significantly different between MO (16.05\u00a0\u03bcs; range 9.50\u201322.93) and MTA (18.91\u00a0\u03bcs; range 11.50\u201324.55). The median value of mean horizontal deviation was 4.01\u00a0mm (range \u22123.25 to 18.12) in MO and 10.74\u00a0mm (range 1.60\u201317.03) in MTA, a nonsignificant difference.\nIn MO, there was no significant correlation between mean MCD and mean horizontal deviation. By contrast, in MTA, both variables were positively correlated (R\u00a0=\u00a00.71, P\u00a0=\u00a00.046) (Fig.\u00a03).\nFig.\u00a03Scatter plot relating mean MCD on SFEMG (X axis, \u03bcs) and mean horizontal deviation on 3D analysis of a reaching arm movement (Y axis, mm), and linear regression lines. Migraine without aura patients (MO): squares, dashed line. Migraine with aura patients (MTA): triangles, continuous line\u00a0\nDiscussion\nOur within-patient analysis shows that, in migraine with aura, the mean MCD on SFEMG increases significantly with the degree of horizontal deviation in a visually guided reaching movement. This suggests that, in subgroups of migraine patients, neuromuscular transmission (NMT) performance and control of ballistic movements by the lateral cerebellum are similarly influenced by a common biochemical and\/or neural mechanism.\nAlthough no gene mutations have been identified till now, the genetic load is thought to be higher in the pathogenesis of migraine with aura than that without aura [13]. In familial hemiplegic migraine 1 (FHM1) [14], mutations have been found in the CACNA1A gene, which codifies for the main subunit of P\/Q-type Ca2+ channels, heavily concentrated at the neuromuscular junction and in the cerebellum [15\u201318]. Thus, we initially hypothesized that the NMJ and cerebellar abnormalities found in some migraine patients might be due to dysfunctioning Ca++ channels. However, SFEMG studies were normal in FHM1 [19], and mutations in the CACNA1A gene are not found in patients suffering from the common forms of migraine, with or without aura [20]. Some evidence that the CACNA1A gene may be involved in migraine with typical aura [21], and the facts that NMT impairment can be found in episodic ataxia type 2, an allelic disorder of FHM1 [22], and normalizes after treatment with acetazolamide [23] are still in favor of a possible involvement of Ca++ channels, particularly in migraine with aura.\nOther proteins implicated in migraine pathophysiology may constitute a common link between neuromuscular junctions (NMJ) and the cerebellum. This is unlikely for the alpha-2 subunit of the Na-K ATPase, of which the gene ATP1A2 is mutated in FHM2 [24], because the alpha-2 isoform is found at intracellular membranes [25] and not at the NMJ, where the major role is played by the alpha-3 isoform [26]. Moreover, SFEMG results were found normal in FHM2 patients [19].\nBy contrast, a different group of calcium channels, the R-type, influence neuromuscular transmission and can compensate for dysfunctioning P\/Q channels [27]. Moreover, they seem to play a role in cerebellar functions [28]; thus, their activity may influence in parallel NMJ and cerebellar performances, similarly to what we found in the subgroup of migraineurs with aura. The CACNA1E gene, which codes for the alpha-1E subunit of R-type channels, is precisely located on locus 1q31, for which significant linkage was found in the common forms of migraine [29], and a single nucleotide polymorphism was more frequent in subgroups of patients affected by migraine with aura [30].\nIn conclusion, the correlation between NMJ and cerebellar performances that we have found in migraine with aura patients might be due to a common genetically determined molecular mechanism, possibly influencing ion channels functions, but this has still to be proven by appropriate genetic studies.","keyphrases":["migraine","genetics","neuromuscular junction","cerebellum"],"prmu":["P","P","P","P"]}
{"id":"J_Chem_Ecol-4-1-2266969","title":"Prey and Non-prey Arthropods Sharing a Host Plant: Effects on Induced Volatile Emission and Predator Attraction\n","text":"It is well established that plants infested with a single herbivore species can attract specific natural enemies through the emission of herbivore-induced volatiles. However, it is less clear what happens when plants are simultaneously attacked by more than one species. We analyzed volatile emissions of lima bean and cucumber plants upon multi-species herbivory by spider mites (Tetranychus urticae) and caterpillars (Spodoptera exigua) in comparison to single-species herbivory. Upon herbivory by single or multiple species, lima bean and cucumber plants emitted volatile blends that comprised mostly the same compounds. To detect additive, synergistic, or antagonistic effects, we compared the multi-species herbivory volatile blend with the sum of the volatile blends induced by each of the herbivore species feeding alone. In lima bean, the majority of compounds were more strongly induced by multi-species herbivory than expected based on the sum of volatile emissions by each of the herbivores separately, potentially caused by synergistic effects. In contrast, in cucumber, two compounds were suppressed by multi-species herbivory, suggesting the potential for antagonistic effects. We also studied the behavioral responses of the predatory mite Phytoseiulus persimilis, a specialized natural enemy of spider mites. Olfactometer experiments showed that P. persimilis preferred volatiles induced by multi-species herbivory to volatiles induced by S. exigua alone or by prey mites alone. We conclude that both lima bean and cucumber plants effectively attract predatory mites upon multi-species herbivory, but the underlying mechanisms appear different between these species.\nIntroduction\nPlants can employ arthropod natural enemies and defend themselves against herbivorous insects and mites. Such arthropod natural enemies can have an impact on local herbivore populations and thereby also on plant fitness (Sabelis and van der Meer 1986; Dicke and Sabelis 1989; Pels and Sabelis 1999; van Loon et al. 2000; Fritzsche Hoballah and Turlings 2001; Kessler and Baldwin 2001). Plants can promote the effectiveness of natural enemies by providing alternative food, shelter, or volatiles (Price et al. 1980; Dicke and Sabelis 1988). The emission of herbivore-induced plant volatiles (HIPV) has been demonstrated in several families (e.g., Dicke et al. 1990a; Turlings et al. 1990; van den Boom et al. 2004; van Poecke and Dicke 2004). Many parasitoid wasps, predatory mites, and predatory insects use HIPV to locate their prey or hosts (reviewed in Dicke 1999; Dicke and Vet 1999; Sabelis et al. 1999). HIPV are thus thought to have an important influence on the interactions between plants, herbivores, and natural enemies in food webs (Turlings et al. 1995; Dicke and Vet 1999).\nTo date, studies on the production of HIPV by plants and the responses of natural enemies to these have been carried out mostly for systems with one species of plant, herbivore, and natural enemy. Yet, in the field, most plants are likely to be attacked by several herbivore species, or by herbivores and pathogens, at the same time. It is difficult to predict whether or not changes in HIPV blends upon multi-species herbivory affect attraction of natural enemies, and in what direction. Several studies have attempted to fill this gap by analyzing the chemical composition of volatile blends upon multi-species infestation of plants (Shiojiri et al. 2001; Cardoza et al. 2002; Rodriguez-Saona et al. 2003; Rost\u00e1s et al. 2006; Moayeri et al. 2007; Rasmann and Turlings 2007; Soler et al. 2007). For instance, Shiojiri et al. (2000) showed that the parasitoid wasp Cotesia plutellae prefers the HIPV blend induced by its host Plutella xylostella to the blend induced by multi-species herbivory by its host and the non-host caterpillar Pieris rapae although volatile blends are similar (Shiojiri et al. 2001). Cotesia glomerata, on the other hand, prefers HIPV blends induced by its host P. rapae and non-host caterpillars to blends induced by one caterpillar species feeding alone (Shiojiri et al. 2000; Vos et al. 2001). Cotesia marginiventris and Microplitis rufiventris do not discriminate between the multi-species-induced HIPV blend of plants infested with their host (beet armyworm caterpillars) and a plant pathogen or plants infested with their host alone, although the pathogen significantly suppresses volatile induction (Rost\u00e1s et al. 2006). Similarly, C. glomerata does not discriminate between mustard plants infested with a root herbivore and its host, or plants infested with its host alone (Soler et al. 2007). The parasitoid C. marginiventris and the entomopathogenic nematode Heterorhabditis megidis exhibited a reduced attraction to volatiles from maize plants that were infested by a non-host in addition to their host, although the non-host was feeding on different tissues from the host, i.e., leaves vs. roots (Rasmann and Turlings 2007).\nIn this study, we investigated the effects of multi-species herbivory by spider mites (Tetranychus urticae) and beet armyworm caterpillars (Spodoptera exigua) on volatile emissions by two different plant species: lima bean (Phaseolus lunatus) and cucumber (Cucumis sativus), and evaluated the responses of Phytoseiulus persimilis, a specialist natural enemy of spider mites. Both herbivore species are known to induce volatile emission in several plant species, and remarkable differences in HIPV blend composition have been found in lima bean infested by either of these herbivores (e.g., Ozawa et al. 2000; Schmelz et al. 2003; de Boer et al. 2004). We statistically compared volatile emission by multi-species-infested plants with the sum of volatile emissions of plants infested with either herbivore species alone to evaluate the presence of potentially antagonistic or synergistic effects among signal transduction pathways (e.g., Engelberth et al. 2001). Plant responses to multi-species herbivory may depend on feeding modes of the herbivore species involved (Walling 2000), but whether or not plant species differ in these responses is unknown. Therefore, we compared two plant species in their relative responses to multi-species vs. single-species herbivory by using the same two herbivore species to attack both plant species. We also studied the olfactory responses of predatory mites (P. persimilis). In nature, plants are commonly attacked by more than one herbivore. Knowledge on natural enemy responses to volatile blends induced by multi-species vs. single-species herbivory is essential to understand whether differences in volatile composition are relevant in an ecological context.\nMethods and Materials\nPlants, Herbivores, and Predators Lima bean plants (Phaseolus lunatus L. cv Sieva) and cucumber plants (C. sativus L. cv Lange Groene Giganten) were grown in a greenhouse at 20\u201330\u00b0C, 50\u201370% R.H. and 16\u00a0hr of light. Bean plants were used when their primary leaves had unfolded (and were about 8\u201312\u00a0cm wide), which was 10\u201315\u00a0d after sowing. Cucumber plants were used when their first two leaves were about 8\u201312\u00a0cm wide, which was 15\u201335\u00a0d after sowing.Two-spotted spider mites, Tetranychus urticae Koch (Acari: Tetranychidae), were reared on lima bean plants in a greenhouse under the same conditions as uninfested plants. Eggs of the beet armyworm Spodoptera exigua H\u00fcbner (Lepidoptera: Noctuidae) were obtained from a colony reared on artificial diet in the laboratory of Virology, Wageningen University, The Netherlands (Smits et al. 1986). Two to three batches of eggs were placed in a Petri dish together with a lima bean or cucumber leaf in a growth chamber at 23\u2009\u00b1\u20091\u00b0C. Larvae were used in experiments within 24\u00a0hr of hatching.A colony of predatory mites, Phytoseiulus persimilis Athias-Henriot (Acari: Phytoseiidae), was kept on spider mite-infested lima bean leaves in a growth chamber at 23\u2009\u00b1\u20091\u00b0C, 50\u201370% R.H. and continuous light. To obtain females of the same age for olfactometer experiments, gravid females were allowed to oviposit on a spider mite-infested bean leaf for 2\u00a0d. The female offspring were used in experiments 9\u00a0d after initiation of the colonies, i.e., 1\u20132\u00a0d after their final molt. Before the experiments, females were kept individually in 1.5-ml microcentrifuge tubes with a small droplet of water, for 24\u2009\u00b1\u20092\u00a0hr at 23\u2009\u00b1\u20091\u00b0C.\nPlant Treatments Treatments were applied to the two oldest leaves of lima bean or cucumber plants 3\u00a0d before an experiment. Plants were incubated in plastic cages in a climate-controlled chamber at 24\u2009\u00b1\u20091\u00b0C, 50\u201370% R.H. and 16L:8D, each treatment in a separate cage. We used the following four treatments:\nT. urticae: Adult female spider mites were transferred to experimental plants. Twenty mites per leaf were used on lima bean, and 100 mites per leaf were used on cucumber because the same number of spider mites leads to lower attraction of P. persimilis to cucumber than to lima bean plants (Dicke et al. 1990b).S. exigua: Newly hatched S. exigua larvae were placed in clip cages to keep caterpillars on the leaves (clip cages were made of two plastic cylinders, 2.5\u00a0cm diam., and a hairpin; the upper side was covered with gauze and the underside with Parafilm). Two larvae in one clip cage were used per leaf. Clip cages were moved daily to a new position on the same leaf, and any missing larvae were replaced. The clip cages were supported by sticks to prevent the leaves from bending and incurring damage due to the weight of the cages.Multi-species herbivory: Two newly hatched S. exigua larvae in a clip cage and 20 (lima bean) or 100 (cucumber) T. urticae females were placed on each leaf.Uninfested: Plants without herbivores were treated in the same way as plants of the other treatments. This treatment was only used for volatile collections of cucumber. We did not analyze volatile emission from uninfested lima bean plants because it is documented that they emit low amounts of volatiles (e.g., de Boer et al. 2004).When clip cages were used to keep caterpillars in place, T. urticae-infested or uninfested plants were treated in the same way with empty clip cages to avoid any cage effects.\nVolatile Collections and Analyses Just before volatile collections, plants were cut above the soil line. Stems of individual plants were wrapped in moist cotton and aluminum foil to prevent them from wilting during volatile collection, which lasted at most 4\u00a0hr. All clip cages, caterpillars, and their products were removed. Five plants of one treatment were transferred to a 5-l glass vessel. A viton O-ring and a metal clamp were used to attach the glass lid to the vessel to make it airtight. Purified air (filtered through silica, a molecular sieve, activated charcoal, and 90\u00a0mg Tenax) was split into two air streams of about 100\u00a0ml\/min each and led into the vessels through teflon tubing. Volatiles from two treatments were collected simultaneously in parallel. The system was purged for 30\u00a0min before attaching a collection tube with Tenax (90\u00a0mg for bean samples, 200\u00a0mg for cucumber samples) to the air outlet in the lid of the vessel. Bean volatiles were collected for 25\u00a0min, cucumber volatiles were collected for 3\u00a0hr. For lima bean, volatile collection was repeated four times for each treatment, except for uninfested from which volatiles were not sampled. For cucumber, volatile collections were replicated seven times for the uninfested, six times for T. urticae, and five times for S. exigua and multi-species herbivory each. One sample of the T. urticae and one of the S. exigua treatments of cucumber were lost during the analysis.Volatiles from lima bean and cucumber were analyzed on two different gas chromatography-mass spectrometry (GC-MS) systems.\nLima bean Volatiles were released from Tenax traps with a thermodesorption cold trap setup (Chrompack, Middelburg, The Netherlands) by heating at 250\u00b0C for 10\u00a0min, with a He-flow of 12\u00a0ml\/min. Desorbed compounds were collected in the cold trap at \u221290\u00b0C. Volatiles were injected in splitless mode into the DB5 column (60\u00a0m\u2009\u00d7\u20090.25\u00a0mm ID, 0.25\u00a0\u03bcm film thickness) by heating the cold trap to 220\u00b0C. After an initial column temperature of 40\u00b0C for 4\u00a0min, the temperature was raised to 280\u00b0C at 4\u00b0C\/min. The column was directly coupled to the ion source of a Finnigan MAT 95 mass spectrometer, which was operating in the 70-eV electron impact (EI) ionization mode with scanning from mass 24 to 300 at 0.5 scan\/sec. Compounds were identified by comparison of mass spectra with those in the NIST 98 library and in the Wageningen Mass Spectral Database of Natural Products, and by checking the retention indices.\nCucumber Volatiles were released from Tenax traps with a thermodesorption cold trap setup (Markes, UK) by heating at 200\u00b0C for 10\u00a0min, with a He-flow of 30\u00a0ml\/min. The desorbed volatiles were collected in the cold trap at \u221210\u00b0C. Volatiles were injected in splitless mode into the RTX-5Silms column (Restec, 30\u00a0m\u2009\u00d7\u20090.32\u00a0mm ID, 0.33\u00a0\u03bcm film thickness) by heating of the coldtrap to 270\u00b0C. After an initial column temperature of 40\u00b0C for 2\u00a0min, the temperature was raised to 95\u00b0C at 3\u00b0C\/min, then to 165\u00b0C at 2\u00b0C\/min, and subsequently to 250\u00b0C at 15\u00b0C\/min. The column was directly coupled to the ion source of a Finnigan quadrupole mass spectrometer, which was operated in the 70-eV EI ionization mode and scanned from mass 33 to 300 at three scans\/sec. Compounds were identified by comparison of mass spectra with those in the NIST 98 and Wiley 7th edition spectral libraries, and by checking the retention indices.\nAnalyses Not all compounds were detected in every replicate of every treatment. We therefore added one peak area unit to all measurements for lima bean and 10 units to all measurements for cucumber. These were the lowest recorded non-zero values for bean and cucumber, respectively. This enabled us to plot volatile emission on a log scale (Figs.\u00a01a and 2a), and to determine ratios among treatments (see below).\nFig.\u00a01Effect of multi-species herbivory on volatile emission by lima bean plants. (a) Volatile emission (mean + SE peak area units) upon single herbivory by Tetranychus urticae (20 mites per leaf) (open bars), single herbivory by Spodoptera exigua (two larvae per leaf) (filled bars), or multi-species herbivory (hatched bars). Asterisks indicate compounds that were significantly affected by herbivore treatment (P\u2009<\u20090.05, Kruskal\u2013Wallis tests). (b) Ratio of emission rates upon multi-species herbivory to the sum per volatile emitted by the two single herbivore treatments (see \u201cMethods and Materials\u201d for detailed explanation). Symbols indicate mean ratio, and error bars indicate upper and lower 95% confidence limits. Asterisks indicate compounds that were significantly more strongly induced by multi-species herbivory than by the sum of T. urticae and S. exigua (lower 95% confidence limit larger than 0). N\u2009=\u20094 for all treatments. Compound numbers: (1) (Z)-3-hexen-1-ol, (2) methyl salicylate, (3) (E)-\u03b2-ocimene, (4) (E)-4,8-dimethyl-1,3,7-nonatriene, (5) (3E,7E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene, (6) p-mentha-1,3,8-triene, (7) (Z)-3-hexen-1-ol acetate, (8) 2-methylbutanal-O-methyl oxime, (9) 3-methylbutanal-O-methyl oxime, (10) linalool, (11) 1-octen-3-ol, (12) hexyl acetate, (13) limonene, (14) \u03b2-caryophyllene, (15) nonanal, (16) indole, (17) (E)-2-hexen-1-ol acetate, (18) 3-pentanone, (19) 3-octanone, (20) 2-methylbutanal nitrile, (21) (Z)-\u03b2-ocimene, (22) rose furan, (23) unknown 95B, 150, (24) 3-methylbutanal nitrile, (25) unknown 91B, 148, (26) unknown 41, 69B, 164, (27) 2-methylpropanal-O-methyl oximeFig.\u00a02Effect of multi-species herbivory on volatile emission by cucumber plants. (a) Volatile emission (mean \u00b1 SE peak area units) upon single herbivory by Tetranychus urticae (100 mites per leaf, N\u2009=\u20095, open bars), single herbivory by Spodoptera exigua (2 larvae per leaf, N\u2009=\u20094, filled bars), or multi-species herbivory (N\u2009=\u20094, hatched bars). Asterisks indicate compounds that are significantly affected by herbivore treatment (P\u2009<\u20090.05, Kruskal\u2013Wallis tests). (b) Ratio of emission rates upon multi-species herbivory to the sum per volatile emitted by the two single herbivore treatments (see \u201cMethods and Materials\u201d and for detailed explanation). Symbols indicate mean ratio, and error bars indicate upper and lower 95% confidence limits. Asterisks indicate compounds that were significantly less induced by multi-species herbivory than by the sum of T. urticae and S. exigua (upper 95% confidence limit smaller than 0) (N\u2009=\u20094) Compound numbers: (1) (E)-2-hexenal + (Z)-3-hexanal, (2) (Z)-3-hexen-1-ol acetate, (3) indole, (4) (E,E)-\u03b1-farnesene, (5) butyl aldoxime, (6) 3-methylbutanal-O-methyl oxime, (7) (E)-4,8-dimethyl-1,3,7-nonatriene, (8) (3E,7E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene, (9) (E)-\u03b2-ocimene. Note that peak area units in Fig.\u00a02a cannot be compared with Fig.\u00a01a because measurements were done on a different GC-MS systemFor cucumber, we first determined which volatile compounds were induced by herbivory by using Kruskal\u2013Wallis tests per compound. Variation in volatile emission levels of uninfested plants was low, indicating that other types of inducing factors (e.g., general stress) were absent or had only minor effects. Only herbivore-induced volatiles were used in subsequent analyses. We assumed that all compounds emitted by infested lima bean plants were induced by herbivores based on previous measurements of volatile emissions by uninfested lima bean plants (e.g., de Boer et al. 2004). Per plant species, we then used Kruskal\u2013Wallis tests to analyze which compounds were differentially induced by the three herbivore treatments (T. urticae, S. exigua, or multi-species herbivory) (\u03b1\u2009=\u20090.05). Note that we did not statistically compare absolute levels of volatile emission across plant species because volatiles were collected for different periods of time, and analyses were run on two different GC-MS systems.We also tested whether the effect on HIPV of multi-species herbivory was the same as the combined effect of the two herbivores feeding alone, i.e., T. urticae + S. exigua. Per replicate, the amount of each volatile compound of the multi-species herbivory treatment was divided by the sum of the amounts emitted by T. urticae and S. exigua. The ratios were log-transformed before taking their averages. An average ratio of 0 means the effect of multi-species herbivory and the sum of T. urticae and S. exigua is equal. We concluded that a compound was significantly more strongly induced by multi-species herbivory than by the sum of T. urticae and S. exigua if the lower 95% confidence limit was larger than 0. Similarly, we concluded a significantly weaker effect of multi-species herbivory if the upper 95% confidence limit was smaller than 0 (see Figs.\u00a01 and 2). We also used a \u03c72 test to test whether the number of compounds with a ratio >0 and a ratio <0 differed from a 50:50 distribution. A 50:50 distribution would be expected if the effect of multi-species herbivory and the sum of T. urticae and S. exigua is equal. One replicate of the T. urticae treatment in cucumber was excluded from this analysis because we had no corresponding replicates of the S. exigua and multi-species herbivory treatments. Note that these tests reflect volatile responses to multi-species herbivory relative to responses to single-species herbivory. This allows us to compare these data between lima bean and cucumber because the data no longer depend on the absolute levels of volatiles detected by the different GC-MS systems.\nY-tube Olfactometer Experiments A closed system Y-tube olfactometer setup was used to test the responses of predatory mites to volatiles induced by multi-species herbivory (Takabayashi and Dicke 1992; de Boer et al. 2004). Odor sources consisted of four leaves of the same treatment that were cut from the plant just before an experiment; the petioles were wrapped in wet cotton wool and aluminum foil. Individual female predators were observed for a maximum of 5\u00a0min. A choice was recorded when the finish line, halfway one of the olfactometer arms, was reached within this period. Otherwise, it was recorded as \u201cno-choice\u201d. Sixteen to 23 predators were tested per experimental day, and each experiment was repeated on 4 different days. Per replicate, new groups of predatory mites and new sets of leaves were used. We tested the responses of predatory mites to volatiles from bean or cucumber plants of the multi-species herbivory treatment vs. volatiles from plants of the T. urticae or S. exigua treatments.The choices of predatory mites between two odor sources in the Y-tube olfactometer were analyzed with two-sided binomial tests to investigate whether the distribution of the predators differed from 50:50. Predators that did not make a choice were excluded from statistical analyses.\nResults\nVolatile Emission by Lima Bean Plants The main compounds emitted by T. urticae-infested bean plants were methyl salicylate (MeSA; compound number 2 in Fig.\u00a01a), (3E)-4,8-dimethyl-1,3,7-nonatriene [(E)-DMNT] (4), and (3E,7E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene [(E,E)-TMTT] (5). Herbivory by S. exigua alone resulted in the emission of similar amounts of (E)-DMNT (4), but amounts of MeSA (2) and (E,E)-TMTT (5) were small. Instead, S. exigua-infested bean leaves emitted large amounts of (E)-\u03b2-ocimene (3) and (Z)-3-hexen-1-ol acetate (7). Multi-species herbivory on bean plants induced the same volatile compounds as S. exigua feeding alone. Multi-species-infested plants emitted larger amounts of all volatile compounds than plants infested with a single herbivore species, except for (Z)-3-hexen-1-ol (1) that was emitted in larger amounts by S. exigua-infested plants, and MeSA (2) that was emitted in somewhat larger absolute amounts by T. urticae-infested plants.In lima bean, herbivore treatment had a significant effect on the emission of (Z)-3-hexen-1-ol (1), (E)-\u03b2-ocimene (3), (Z)-3-hexen-1-ol acetate (7), 2-methylbutanal-O-methyl oxime (8), linalool (10), 1-octen-3-ol (11), hexyl acetate (12), \u03b2-caryophyllene (14), indole (16), (E)-2-hexen-1-ol acetate (17), (Z)-\u03b2-ocimene (21), and two unknown compounds (23 and 26) (all P\u2009<\u20090.05, Kruskal\u2013Wallis tests, Fig.\u00a01a). These compounds were all induced in larger amounts by S. exigua and\/or multi-species herbivory than by T. urticae. Total volatile emission was also significantly affected by treatment (P\u2009<\u20090.006, Kruskal\u2013Wallis test, Fig.\u00a01a).We compared the ratio of volatiles emitted in the multi-species herbivory treatment to the sum of volatiles emitted by the T. urticae and S. exigua treatments to determine whether infestation by two herbivore species simultaneously had additive, synergistic or antagonistic effects. Figure\u00a01b shows the average ratio of multi-species herbivory to the sum of T. urticae and S. exigua for lima bean. Eight of 27 compounds were significantly more strongly induced by multi-species herbivory than by the sum of the two herbivore species feeding alone, suggesting synergistic effects (Fig.\u00a01b). These compounds included members of various groups of chemicals, for example, (E)-2-hexen-1-ol acetate (17) and (Z)-\u03b2-ocimene (21). In addition, more compounds had a ratio >0 than expected (\u03c72\u2009=\u20099.80, P\u2009=\u20090.002). None of the compounds was significantly less induced by multi-species herbivory, suggesting the absence of antagonistic effects of T. urticae and S. exigua feeding simultaneously on lima bean.\nVolatile Emission by Cucumber Plants Nine compounds were induced by herbivory on cucumber (all P\u2009<\u20090.10, Kruskal\u2013Wallis tests; data not shown). The main compounds emitted by T. urticae-infested cucumber plants were (Z)-3-hexen-1-ol acetate (compound number 2 in Fig.\u00a02a), (E,E)-\u03b1-farnesene (4), 3-methylbutanal-O-methyl oxime (6), (E)-DMNT (7), and (E)-\u03b2-ocimene (9) (Fig.\u00a02a). Similar compounds were emitted in the largest amounts by S. exigua-infested cucumber plants. Multi-species herbivory resulted in a volatile blend that consisted of the same compounds as the blends induced upon herbivory by each of the herbivore species alone. The multi-species herbivory treatment resulted in the strongest induction of 3-methylbutanal-O-methyloxime (6) and (E)-\u03b2-ocimene (9), while T. urticae-infested plants emitted the largest amounts of (Z)-3-hexen-1-ol acetate (2). Herbivory by T. urticae alone and multi-species herbivory induced larger amounts of (E,E)-TMTT (8) than single herbivory by S. exigua alone. No MeSA was detected in any of the cucumber samples.In cucumber, herbivore treatment had an effect on the emission of 3-methylbutanal-O-methyloxime (6) and (E)-DMNT (9) (P\u2009<\u20090.05, Kruskal\u2013Wallis tests, Fig.\u00a02a). Both compounds were induced in larger amounts by T. urticae or multi-species herbivory than by S. exigua. Total volatile emission was not affected by herbivore treatment in cucumber (P\u2009>\u20090.10, Kruskal\u2013Wallis test, Fig.\u00a02a).Two volatile compounds [(E)-2-hexanal\/(Z)-3-hexenal (1) and (Z)-3-hexen-1-ol acetate (2)] were induced significantly less by multi-species herbivory than by the sum of T. urticae and S. exigua (Fig.\u00a02b), suggesting the presence of antagonistic effects of the two herbivore species feeding on cucumber. The seven other compounds were equally induced by multispecies herbivory and the sum of the two herbivores feeding alone, indicating an additive effect of T. urticae and S. exigua. The distribution of compounds with a ratio >0 or <0 did not differ from 50:50 (\u03c72\u2009=\u20090.06, P\u2009=\u20090.81).\nResponses of P. persimilis We tested the preferences of the predatory mite P. persimilis for the volatile blends analyzed above in a Y-tube olfactometer. On bean and cucumber plants, predatory mites preferred HIPV induced by multi-species herbivory to blends induced by either T. urticae or S. exigua alone (Fig.\u00a03, P\u2009\u2264\u20090.02, binomial tests).\nFig.\u00a03Responses of P. persimilis to volatile blends in the Y-tube olfactometer. Odor sources consisted of four leaves from lima bean (a and b) or cucumber plant (c and d) simultaneously infested by Tetranychus urticae and Spodoptera exigua or infested by one of the herbivore species alone (S. exigua: a and c, T. urticae: b and d). Bars present the overall percentages of predators choosing for each odor source. Numbers in bars are the total numbers of predators responding to each odor source. Choices between odor sources were analyzed with a two-sided binomial test (*P\u2009<\u20090.05; ***P\u2009<\u20090.001)\nDiscussion\nWe investigated volatile emissions by lima bean and cucumber plants upon single and multi-species herbivory by T. urticae and S. exigua. In lima bean, we found that most compounds were more strongly induced by multi-species herbivory than by the sum of each of the herbivores feeding separately (Fig.\u00a01b). This suggests that the two herbivores feeding together on the same plant have more than an additive effect on volatile emission, potentially indicating that biosynthetic pathways may act synergistically. It is also possible that one or both herbivore species inflicted more damage on multi-species-infested plants than on plants with conspecifics only. However, it is unlikely that spider mite feeding was affected by the presence of S. exigua because in a preliminary experiment oviposition rates of mites, which are correlated with food intake rates, were similar on leaf discs with or without S. exigua damage (de Boer et al., unpublished). Some studies have observed that feeding by S. exigua is enhanced by the presence of aphids or a fungal pathogen (Rodriguez-Saona et al. 2005; Cardoza and Tumlinson 2006), but other studies have found no effect of the presence of whiteflies or a fungus (Rodriguez-Saona et al. 2003; Rost\u00e1s et al. 2006).\nIn contrast to bean, we found that in cucumber plants, two volatile compounds were induced less by multi-species herbivory than by the sum of the two herbivores feeding separately, suggesting that their induction was suppressed by simultaneous feeding of T. urticae and S. exigua. Whether this reduction is caused by a negative interaction between the signal transduction pathways induced by spider mites and beet armyworms, or to reduced feeding of one or both herbivore species as a result of feeding on the same plant, remains to be investigated. From our data, it appears that plant species is an important determinant in the way in which herbivore-induced defense pathways interact upon multispecies herbivory.\nThe plant hormones salicylic acid (SA) and jasmonic acid (JA) play important roles in plant defense pathways and the production of HIPV (e.g., Kessler and Baldwin 2002; van Poecke and Dicke 2004). Interaction between these pathways may occur, for example, when SA inhibits JA-dependent defense pathways (e.g., Doares et al. 1995; Niki et al. 1998; Engelberth et al. 2001). To determine whether synergistic (lima bean) and antagonistic (cucumber) interactions between biosynthetic defense pathways caused the changes in HIPV blends that we observed, endogenous levels of JA and SA should be determined in future studies. Arimura et al. (2002) showed that both JA and SA levels are significantly elevated in lima bean after 3\u00a0d of spider mite feeding, whereas several Spodoptera species are known to increase JA levels in corn and broad bean (Blechert et al. 1995; Schmelz et al. 2003). We are not aware of analyses of SA levels in response to Spodoptera feeding. No studies of JA and SA levels in plants upon multi-species herbivory are known to us either.\nAnother important question that needs to be addressed is the effect of herbivore sequence on volatile emission patterns, since in nature two herbivore species may infest an individual plant sequentially. In fact, some herbivore species are known to be attracted to HIPV themselves (e.g., Bolter et al. 1997; Kalberer et al. 2001), but spider mites are not attracted to S. exigua-infested lima bean plants (Horiuchi et al. 2003).\nTo determine whether the differences in volatile profiles upon multi-species herbivory and single-species herbivory are relevant in an ecological context, we investigated the behavior of the predatory mite P. persimilis, a specialist natural enemy of spider mites. Our results show that P. persimilis prefers volatiles induced by multi-species herbivory by spider mites and the non-prey caterpillar S. exigua to volatiles induced by one of the herbivore species feeding alone (Fig.\u00a03). The strong preference of P. persimilis for the volatiles induced by multi-species herbivory may be explained by the relatively large amount of some specific compounds. Of the three major compounds emitted by spider mite-infested lima bean, two, namely (E,E)-TMTT and especially (E)-DMNT, were induced more strongly by multi-species herbivory, whereas induction of the third, MeSA, was somewhat reduced (Fig.\u00a01a). Of the five major compounds emitted by spider mite-infested cucumber, four were induced more strongly by multi-species herbivory: (E)-DMNT, (E)-\u03b2-ocimene, (E,E)-\u03b1-farnesene, and 3-methylbutanal-O-methyl oxime, while (Z)-3-hexen-1-ol acetate emission was reduced (Fig.\u00a02a). (E)-DMNT, (E,E)-TMTT, and (E)-\u03b2-ocimene are known attractants for P. persimilis (Dicke et al. 1990a, de Boer et al. 2004), and (E,E)-TMTT can be used by P. persimilis to discriminate between complex blends of HIPV (de Boer et al. 2004). Similarly, when S. exigua-induced volatiles are compared to the blend induced by multi-species herbivory, all of these compounds are produced in larger amounts in the latter. This suggests that a greater emission of (the sum of) attractive compounds may be the main determinant in attracting the predatory mite P. persimilis.\nPrevious studies on the responses of parasitoid wasps to HIPV also demonstrated a preference for HIPV induced by multi-species herbivory compared to single-species herbivory (Shiojiri et al. 2000; Vos et al. 2001; Cardoza et al. 2003; Moayeri et al. 2007). Rodriguez-Saona et al. (2005) even showed that na\u00efve Cotesia marginiventris females were only attracted to tomato plants infested with their host S. exigua when the same plant was also infested with aphids. The presence of aphids thus made the host-infested plants more detectable. The opposite, i.e., multi-species herbivory-induced volatiles being less attractive than volatiles induced by only the host has been found in two cases: C. plutellae wasps preferred volatiles induced by their host, P. xylostella, to volatiles induced by their host and the non-host caterpillar P. rapae (Shiojiri et al. 2000), and C. marginiventris as well as the entomopathogenic nematode H. megidis were less attracted to volatiles from maize plants that were infested by a non-host in addition to their host (Rasmann and Turlings 2007). In such cases, the presence of non-host or non-prey herbivores may decrease the detectability or reliability of HIPV cues for natural enemies. For example, feeding by a non-prey or non-host herbivore may suppress the induction of volatile compounds used by natural enemies. Multi-species herbivory thus adds a new dimension to the trade-off between reliability and detectability of searching cues used by natural enemies of herbivorous arthropods (Vet and Dicke 1992).","keyphrases":["food webs","signal transduction","gc-ms","methyl salicylate","indirect defense","terpenes","tritrophic interactions"],"prmu":["P","P","P","P","M","U","M"]}
{"id":"Exp_Brain_Res-4-1-2248215","title":"Attenuation of N2 amplitude of laser-evoked potentials by theta burst stimulation of primary somatosensory cortex\n","text":"Theta burst stimulation (TBS) is a special repetitive transcranial magnetic stimulation (rTMS) paradigm, where bursts of low-intensity stimuli are applied in the theta frequency. The aim of this study was to investigate the effect of neuronavigated TBS over primary somatosensory cortex (SI) on laser-evoked potentials (LEPs) and acute pain perception induced with Tm : YAG laser stimulation. The amplitude changes of the N1, N2, and P2 components of LEPs and related subjective pain rating scores of 12 healthy subjects were analyzed prior to and following continuous TBS (cTBS), intermittent TBS (iTBS), intermediate TBS (imTBS), and sham stimulation. Our results demonstrate that all active TBS paradigms significantly diminished the amplitude of the N2 component, when the hand contralateral to the site of TBS was laser-stimulated. Sham stimulation condition had no significant effect. The subjective pain perception also decreased during the experimental sessions, but did not differ significantly from the sham stimulation condition. The main finding of our study is that TBS over SI diminished the amplitude of the N2 component evoked from the contralateral side without any significant analgesic effects. Furthermore, imTBS produced responses similar to those observed by other forms of TBS induced excitability changes in the SI.\nIntroduction\nFunctional neuroimaging studies have provided unequivocal evidence of the participation of the primary somatosensory cortex (SI), secondary somatosensory cortex (SII), and insula in pain processing (Talbot et al. 1991; Casey et al. 1994; Apkarian et al. 1999; Gelnar et al. 1999). Evidence suggests that the nociceptive input into these regions at least partially underlies the perception of sensory features of pain (Bushnell et al. 1999; Coghill et al. 1999; Peyron et al. 1999; Chen et al. 2002; for a reviews see: Peyron et al. 2000; Apkarian et al. 2005).\nElectrophysiological studies have also confirmed the participation of the SI in pain processing, contralateral to the stimulated side. Tarkka and Treede (1993) first reported pain induced-activity in the SI using electroencephalogram (EEG) and applying brain electric source analysis (BESA). In their model, the peak latency measured at the SI was very similar to that of SII. In a combined magnetoencephalogramm (MEG) and laser-evoked potential (LEP) study Ploner et al. (1999) reported SI activity also contralateral to the side of stimulation and further to this proposed a parallel pain processing in SI and SII. This parallel activation of SI and SII was confirmed by other studies (Ploner et al. 2000, 2002) and other groups (Timmermann et al. 2001; Kanda et al. 2000; Inui et al. 2003; Nakata et al. 2004). However, some previous LEP dipole modelling studies showed that a dipole source in SI area was necessary to explain the scalp LEP topography, none of them reported a clear correspondence between the SI activity and a definite LEP component (Tarkka and Treede 1993; Ploner et al. 2002; Kanda et al. 2003). Kanda et al. (2000) detected SI activity following painful stimulation by recording intracranial EEG, but recently, intracerebral depth recordings in an epileptic patient have shown no reliable LEP response from the area 3b of the SI after painful laser stimulation, although a reliable N2\u2013P2 response could be recorded at Cz (Valeriani et al. 2004). Inui et al. (2003) reported the absence of activation from area 3b of the SI after noxious electrical stimulation as well, however they found activity in the area 1 of SI. Other studies found no pain-related activation of the SI at all (for a review see: Garcia-Larrea et al. 2003; Kakigi et al. 2005).\nRecent studies showed, that several kinds of external stimulation methods such as single-pulse transcranial magnetic stimulation (TMS) (Kujirai et al. 1993), 1\u00a0Hz repetitive TMS (rTMS) (Enomoto et al. 2001), paired associative stimulation (PAS) (Tsuji and Rothwell 2002; Wolters et al. 2005), transcranial direct current stimulation (tDCS) (Matsunaga et al. 2004; Dieckhofer et al. 2006) or theta burst stimulation (TBS) (Ishikawa et al. 2007) modulate the amplitude of cortical components of median nerve somatosensory evoked potentials (SEPs). It was recently reported that, cathodal tDCS over the SI (Dieckhofer et al. 2006) significantly reduced the N20 amplitude of median nerve SEPs. Furthermore, cathodal polarization over the SI induced a prolonged decrease of tactile discrimination (Rogalewski et al. 2004) and diminished acute pain perception and the amplitude of the N2 component of LEPs (Antal et al. 2007).\nRecently Huang et al. (2005) developed a special \u201ctheta burst\u201d paradigm to modulate human motor cortex (M1) excitability using low intensity, repetitive bursts of magnetic stimuli. The authors distinguished three stimulation patterns, which were proved to have different effects over M1 activity, when it was monitored by the amplitude of transcranial motor evoked potentials (MEPs). Continuous TBS (cTBS) caused a significant reduction in MEP amplitudes, which was probably due to the inhibition of specific excitatory circuits (I1-wave inputs to corticospinal neurons), as later confirmed by another study (Di Lazzaro et al. 2005). In contrast, intermittent TBS (iTBS) facilitated M1 activity and produced increase in MEP amplitudes. Interestingly, intermediate TBS (imTBS) had no effect at all. In addition, TBS has also been shown to have an effect on the human premotor (Mochizuki et al. 2005) and visual cortex (Franca et al. 2006).\nNon-invasive cortical stimulation of M1 for the treatment of certain kinds of chronic and experimentally induced pain has recently attracted much interest. Both low and high frequency rTMS was reported to reduce subjective pain perception and has been used experimentally to reduce chronic pain (for reviews see: Leo and Latif 2007; Fregni et al. 2007). However, although the involvement of SI in pain perception and processing is reported by several imaging studies (for reviews see: Peyron et al. 2000; Apkarian et al. 2005), the magnetic stimulation of this cortical area in order to modify pain perception is neglected. Therefore, the aim of our study was to investigate the effects of cTBS, iTBS, and imTBS, on the early (N1) and late (N2, P2) components of LEPs and related subjective pain perception when applied over the left SI. We hypothesized that the three TBS types, which have short durations (maximum 190\u00a0s), would affect LEP components in a specific, paradigm-dependent manner similarly to the effect over M1 as revealed by Huang and et al. (2005).\nMethods\nSubjects\nNineteen healthy volunteers between 18 and 35\u00a0years were informed about all aspects of the experiments and signed an informed consent. Three subjects chose not to continue the experiment after the first or second session and during off-line EEG analysis four further subjects were dropped out because of their hardly detectable LEP components. Hence, twelve of the subjects (six male, six female; mean age\u00a0=\u00a026.33\u00a0\u00b1\u00a03.17\u00a0years) were included in the final analysis. We conformed with the Declaration of Helsinki and the experimental protocol was approved by the Ethics Committee of the University of G\u00f6ttingen. None of the subjects suffered from chronic pain syndromes, nor took any medication regularly. None had a history of neurological or psychiatric illness. All of them participated in all four sessions, the three TBS and sham stimulations.\nTBS stimulation\nTheta burst stimulation was applied over the hand area of the left SI using a standard, figure-of-eight-coil (MCF-B65 Butterfly Coil) and MagPro stimulator (Medtronic, Denmark) with an outer half-coil radius of 75\u00a0mm, with a posterior\u2013anterior\u2013posterior current flow in the coil. Stimulus intensity was 80% of active motor threshold (AMT) (Huang et al. 2005).\nFor AMT determination, the coil was placed tangentially to the scalp, with the handle pointing backwards and laterally 45\u00b0 from mid-line. MEPs of the right abductor digiti minimi muscle (ADM) were recorded by Ag-AgCl-electrodes in a belly tendon-montage before each stimulation. The signals were amplified and filtered (1.59\u00a0Hz-1\u00a0kHz, sampling rate of 5\u00a0kHz), digitalized with a micro 1401 AD converter (Cambridge Electronic Design, Cambridge, UK), recorded by a computer using SIGNAL software (Cambridge Electronic Design, Version 2.13). Complete muscle relaxation was controlled though auditory and visual feedback of EMG activity. AMT was defined as the minimum intensity eliciting a MEP of a superior size when compared to spontaneous moderate muscular activity in at least three of six pulses.\nThe pattern of TBS consisted of bursts containing three pulses at 50\u00a0Hz which were repeated at 200\u00a0ms intervals (i.e., 5\u00a0Hz) for up to 600 pulses for 40\u00a0s continuously (cTBS), or triads repeated at 200\u00a0ms intervals for 2\u00a0s intermittently with 8\u00a0s breaks for up to 600 pulses (iTBS). In the case of imTBS the triads (three pulses at 50\u00a0Hz) were repeated at 200\u00a0ms intervals for 5\u00a0s, in every 15\u00a0s up to 600 pulses (Huang et al. 2005). In separate experimental sessions, sham stimulation was applied with the cTBS protocol using the same coil held over the same position but tilted to a 90\u00b0 angle (one-wing 90\u00b0) (Lisanby et al. 2001) with only the margin of the coil in contact with the scalp.\nDetermination of the primary somatosensory cortex (SI)\nAnatomical magnetic resonance imaging (MRI) (Siemens 3 T, T1 weighted) dataset was used to determinate the coil localization for the stimulation of SI for all subjects using the Brainsight neuronavigation system (Rogue Research Inc., Montreal Quebec, Canada). The hand area was determined by previous fMRI studies (Bushnell et al. 1999; Blankenburg et al. 2003) and was located and marked in the MRI dataset as the target for TMS application (Fig.\u00a01).\nFig.\u00a01Three dimensional anatomical MRI of a single subject. Anatomical magnetic resonance imaging (MRI) (Siemens 3 T, T1 weighted) dataset was used to determinate the coil localization for the stimulation of SI by all subjects using the Brainsight neuronavigation system. The hand area was located and marked in the MRI dataset as target for TMS application. The black point indicates the hot-spot of the stimulation coil\nLaser stimulation\nA Tm\u00a0:\u00a0YAG laser system (WaveLight Laser Technologie AG, Erlangen, Germany) was used to induce painful stimulation. The thulium laser emits near-infrared radiation (wavelength 2,000\u00a0nm, pulse duration 1\u00a0ms, laser beam diameter 7\u00a0mm) with a penetration depth of 360\u00a0\u03bcm into the human skin and allows a precise restriction of the emitted heat energy to the termination area of primary nociceptive afferents without affecting the subcutaneous tissue (Treede et al. 2003). The distal handpiece of the laser was positioned 30\u00a0cm from the radial part of the dorsal surface of the hand. The pain threshold was determined on both hands at the beginning each session before baseline EEG recording by applying laser stimuli from 200 mJ in 50\u00a0mJ steps. During EEG recordings, each laser stimulus was delivered with an intensity of 1.4\u20131.6 times the threshold intensity to a slightly different spot in a 5\u00a0\u00d7\u00a05\u00a0cm square on the dorsum of the hand in order to reduce receptor fatigue or sensitization by skin overheating (Treede et al. 2003). Skin temperature of the stimulated area was checked prior to every switch between hands, and corrected with a heating lamp if it fell below 35\u00b0C.\nPsychophysical evaluation\nWe used the verbal analogue score (VAS) to assess the subjective intensity of pain. The subjects were instructed to pay attention to the laser stimuli and to rate the perceived pain verbally (1-warm, 1.1 smallest pain, and 1.9 most intense pain) about 2\u20133\u00a0s after each laser-impulse. The values were individually averaged separately for both hands in each session and conditions. The ears of the subjects were plugged during the measurements to avoid auditory artifacts accompanying laser stimulation.\nElectrophysiological recordings\nThe EEG was recorded using a 64-channel montage applying 64 ring electrodes (inner diameter: 6\u00a0mm, outer diameter: 12\u00a0mm) (EasyCap; Falk and Minow GmbH, M\u00fcnich, Germany). The electrodes were placed in accordance with the extended international 10\u201320 system. The impedance was kept <5\u00a0k\u03a9. The Fz was used as reference, the ground was placed 2\u00a0cm anterior to the tragus of the right ear. Data were collected at a sampling rate of 1,000\u00a0Hz with the BrainAmp system (Brain Products GmbH, Munich, Germany) and were analyzed offline. The obtained data were re-referenced to the connected mastoids (TP9-TP10). A 0.5\u00a0Hz low-cutoff as well as a 30\u00a0Hz high-cutoff filter was used. In addition to automatic artifact detection (200\u00a0\u03bcV amplitude criterion) all epochs were visually inspected, and those containing eye blinks or muscle movement artifacts were excluded. Baseline correction was performed on the basis of the 100\u00a0ms prestimulus interval. The amplitudes of N1 (referring to Fz) and N2\u2013P2 (referring to TP9-TP10) components were measured.\nAlthough we recorded data on 64 channels, we assessed LEPs according to the scalp distribution on the analyzed peaks. The N2 is a negative component (referring to TP9-TP10) was peaking around 160\u2013240\u00a0ms. The amplitude of the early N1 negative peak which came before N2 on T7 and T8 channels (referring to Fz) was analyzed. The P2 positive component after N2 was peaking around 300\u2013360\u00a0ms. The N2 component is largest over the lateral temporal and fronto-central areas on electrodes Fz, Cz, CPz, F1\u2013F4, FC1\u2013FC6, C1\u2013C6, T7, T8, and CP1\u2013CP6. In contrast, the P2 peak has its maximum amplitude over the vertex on electrodes FCz, Cz, CPz, Pz, F1\u2013F4, FC1\u2013FC4, C1\u2013C4, CP1\u2013CP4, and P1\u2013P4. For the analysis of LEPs according to regional distribution, we defined three distinct areas with pooling the data: central (with all the mid-line electrodes such as Fz, FCz, Cz, CPz, and Pz), left (F3, FC3, C3, CP3, P3, FC5, C5, CP5, and T7) and right (electrodes according to the left side) instead of separate electrodes.\nExperimental design\nThe subjects were sitting in a reclining chair. In case of all sessions first the EEG cap was placed on the head. After pain threshold determination the baseline LEP measurements were performed. Every run for the LEP recording consisted of 40\u00a0epochs of laser stimulation on each hand. The interstimulus interval of the stimulation ranged from 8 to 15\u00a0s (Raij et al. 2003). Thus the LEP recording lasted for 8\u201310\u00a0min for the first hand and also for 8\u201310\u00a0min for the second hand laser-stimulation. In all three TBS and sham TBS conditions, the right hand was stimulated first in half of the cases and the left hand was stimulated first in the other half. This order was kept for the subjects for all conditions. After baseline LEP recording the AMT was measured (\u223c15\u201320\u00a0min) and the TBS were applied for 40\u2013190\u00a0s through the cap. After TBS the impedance of the EEG electrodes were retested and corrected below 5\u00a0k\u03a9 if it was necessary. The TBS was followed by a post-stimulation LEP recording in \u223c5\u00a0min after TBS. Thus, the interval between the two LEP recordings with regard to the same hand was about 30\u201335\u00a0min including all subjects and conditions.\nThe experimental sessions were separated from each other by at least 5\u00a0days. The subjects were blinded as to the type of magnetic stimulation. The order of the sessions was randomized across subjects.\nData analysis\nThe individually averaged VAS values and LEP amplitudes were entered into a repeated-measures-ANOVA for both hands and LEP components separately [four TBS CONDITION (cTBS, iTBS, imTBS, and sham)\u00a0\u00d7\u00a02 TIME (before, after TBS)]. We considered a psychophysical or an electrophysiological change only if the CONDITION\u00a0\u00d7\u00a0TIME interaction was significant. In case of the LEP components we investigated if this effect was dependent on the defined areas by calculating the CONDITION\u00a0\u00d7\u00a0TIME\u00a0\u00d7\u00a0REGION interaction. Post hoc analysis was done using Student\u2019s t tests (paired samples, two-tailed, level of significance P\u00a0<\u00a00.05).\nResults\nPsychophysics\nThe intensity of the laser stimulation (1.4\u20131.6\u00a0\u00d7\u00a0of the pain threshold) was 19.88\u00a0mJ\/mm2 for cTBS, 20.53\u00a0mJ\/mm2 for iTBS, 20.52\u00a0mJ\/mm2 for imTBS, and 20.33\u00a0mJ\/mm2 for sham stimulation. None of the subjects reported any side-effect after the stimulation.\nIn case of the contralateral hand (right) stimulation, repeated-measures-ANOVA revealed no main effect of CONDITION [F(3,33)\u00a0=\u00a00.828, P\u00a0=\u00a00.488] but the TIME was significant [F(1,11)\u00a0=\u00a027.270, P\u00a0<\u00a00.001]. The CONDITION\u00a0\u00d7\u00a0TIME interaction was also not significant [F(3,33)\u00a0=\u00a00.080, P\u00a0=\u00a00.97]. In case of the ipsilateral hand (left) stimulation, there was no significant main effect of CONDITION [F(3,33)\u00a0=\u00a01.329, P\u00a0=\u00a00.282] but the TIME was significant [F(1,11)\u00a0=\u00a015.395, P\u00a0<\u00a00.005]. The CONDITION\u00a0\u00d7\u00a0TIME interaction was also not significant [F(3,33)\u00a0=\u00a00.716, P\u00a0=\u00a00.55] (Fig.\u00a02).\nFig.\u00a02The effects of the TBS on subjective pain perception. The VAS-values were standardized for each subject, for each condition by calculating the after\/before ratio. The independent variables were the CONDITION and HAND in order to determine if there is any difference between stimulation of the two hands. There was no main effect of the stimulation CONDITION and the HAND (left or right). The interaction CONDITION\u00a0\u00d7\u00a0TIME was also not significant\nElectrophysiology\nThe N1, N2, and P2 components could clearly be identified in all subjects. The LEPs are presented in Figs.\u00a03 and 4.\nFig.\u00a03The effects of the TBS on the N1 component. The figure shows N1 amplitude changes during the experimental sessions on the grand averages of LEPs. The N1 was analyzed over the electrodes T7 and T8 referred to Fz (international 10\u201320 electrode system) in case of both hands. The solid line shows LEPs before and the intermittent line after TBS interventions. There was no significant difference between stimulation conditionsFig.\u00a04Grand averages of LEPs obtained by contralateral (right) hand laser stimulation for three scalp regions (central, left and right) re-referenced to the connected mastoids (TP9-TP10), before and after the three TBS conditions and sham stimulation. The solid line shows LEPs before and the intermittent line after TBS interventions\nThe N1 component\nWe analyzed the amplitudes of the early N1 components on channels T7 and T8 (referring to Fz). There was no significant main effect of CONDITION [contralateral hand: F(3,33)\u00a0=\u00a02.216, P\u00a0=\u00a00.105; ipsilateral hand: F(3,33)\u00a0=\u00a02.865, P\u00a0=\u00a00.052]. The TIME was significant if the contralateral hand was stimulated [F(1,11)\u00a0=\u00a06.186, P\u00a0=\u00a00.030] but not for the ipsilateral hand [F(1,11)\u00a0=\u00a03.305, P\u00a0=\u00a00.096]. The interaction of the CONDITION\u00a0\u00d7\u00a0TIME resulted in no significant interaction neither after the contraleteral [F(3,33)\u00a0=\u00a00.727, P\u00a0=\u00a00.543] or ipsilateral [F(3,33)\u00a0=\u00a01.694, P\u00a0=\u00a00.187] hand stimulation (Fig.\u00a03).\nThe N2 component\nThe repeated-measures-ANOVA (ipsilateral hand) showed no significant main effect of CONDITION [F(3,33)\u00a0=\u00a00.555, P\u00a0=\u00a00.65], but the effect of TIME was significant [F(1,11)\u00a0=\u00a011.769, P\u00a0=\u00a00.006]. The CONDITION\u00a0\u00d7\u00a0TIME interaction resulted in no significance [F(3,33)\u00a0=\u00a00.149, P\u00a0=\u00a00.93] (Fig.\u00a05a). In case of the (contralateral hand) the main effect of CONDITION [F(3,33)\u00a0=\u00a00.250, P\u00a0=\u00a00.86] was not significant, but the TIME was significant [F(1,11)\u00a0=\u00a032.034, P\u00a0<\u00a00.001]. The CONDITION\u00a0\u00d7\u00a0TIME interaction was also significant [F(3,33)\u00a0=\u00a04.058, P\u00a0=\u00a00.015] (Fig.\u00a05b). The interaction with electrode position was not significant [F(6,66)\u00a0=\u00a01.068, P\u00a0=\u00a00.39]. The post hoc t test showed that all active TBS stimulation significantly decreased the amplitudes of the N2 component at all defined regions for the contralateral hand stimulation. Table\u00a01 summarizes the results of t tests.\nFig.\u00a05The mean N2 amplitude changes during the four TBS conditions at the ipsilateral (left) (Fig.\u00a05a) and contralateral (right) (Fig.\u00a05b) hand laser-stimulation for the three calculated regions (central, left and right). The stars mark significant differences between before-after TBS conditions (post hoc t-tests, paired samples, two-tailed, P\u00a0<\u00a00.05)Table\u00a01Post hoc analysis of the N2 componentcTBSiTBSimTBSshamBefore versus afterBefore versus afterBefore versus afterBefore versus afterCentralP-levels0.00330.00020.00210.3942t-values\u22123.730\u22125.499\u22124.005\u22120.887LeftP-levels0.00540.00040.00310.5921t-values\u22123.451\u22125.041\u22123.775\u22120.552RightP-levels0.03940.00850.00390.8118t-values\u22122.337\u22123.196\u22123.6450.244Shows the results of the Student\u2019s t-tests (paired samples, two-tailed) in case of the right hand stimulation. The level of the significance was P\u00a0<\u00a00.05\nThe P2 component\nThe repeated-measures-ANOVA showed no significant main effect of CONDITION [contralateral: F(3,33)\u00a0=\u00a01.571, P\u00a0=\u00a00.22; ipsilateral: F(3,33)\u00a0=\u00a01.054, P\u00a0=\u00a00.38], but the TIME was significant [contralateral: F(1,11)\u00a0=\u00a017.038, P\u00a0=\u00a00.002; ipsilateral: F(1,11)\u00a0=\u00a015.362, P\u00a0=\u00a00.002]. The CONDITION\u00a0\u00d7\u00a0TIME interaction was also not significant [contralateral: F(3,33)\u00a0=\u00a02.669, P\u00a0=\u00a00.064; ipsilateral: F(3,33)\u00a0=\u00a00.418, P\u00a0=\u00a00.74].\nThe means of the different LEP components from all 12 subjects are presented in Table\u00a02.\nTable\u00a02The mean amplitudes of the LEP componentsPeakcTBSiTBSimTBSshamBeforeAfterBeforeAfterBeforeAfterBeforeAfterLeft handN1T7\u22124.42\u00a0\u00b1\u00a02.75\u22124.60\u00a0\u00b1\u00a03.13\u22124.68\u00a0\u00b1\u00a02.52\u22123.71\u00a0\u00b1\u00a02.65\u22123.33\u00a0\u00b1\u00a03.22\u22123.50\u00a0\u00b1\u00a03.15\u22124.98\u00a0\u00b1\u00a02.70\u22125.20\u00a0\u00b1\u00a03.08T8\u22126.13\u00a0\u00b1\u00a02.17\u22124.78\u00a0\u00b1\u00a03.60\u22126.73\u00a0\u00b1\u00a03.44\u22125.13\u00a0\u00b1\u00a02.89 \u22124.99\u00a0\u00b1\u00a04.27\u22124.64\u00a0\u00b1\u00a03.62\u22126.56\u00a0\u00b1\u00a03.47\u22126.33\u00a0\u00b1\u00a03.11N2Central\u22129.77\u00a0\u00b1\u00a05.48\u22127.92\u00a0\u00b1\u00a04.91\u221210.29\u00a0\u00b1\u00a05.01\u22128.88\u00a0\u00b1\u00a03.98\u22129.04\u00a0\u00b1\u00a05.43\u22127.25\u00a0\u00b1\u00a05.52\u221210.02\u00a0\u00b1\u00a04.43\u22128.11\u00a0\u00b1\u00a06.16Left\u22127.13\u00a0\u00b1\u00a03.60\u22126.05\u00a0\u00b1\u00a03.44\u22127.44\u00a0\u00b1\u00a03.49\u22126.69\u00a0\u00b1\u00a02.91\u22126.81\u00a0\u00b1\u00a03.69\u22125.55\u00a0\u00b1\u00a03.88\u22127.13\u00a0\u00b1\u00a03.55\u22125.98\u00a0\u00b1\u00a04.28Right\u22126.70\u00a0\u00b1\u00a03.05\u22124.59\u00a0\u00b1\u00a03.25\u22126.42\u00a0\u00b1\u00a03.54\u22125.41\u00a0\u00b1\u00a02.56\u22125.88\u00a0\u00b1\u00a03.05\u22124.70\u00a0\u00b1\u00a03.19\u22126.11\u00a0\u00b1\u00a03.24\u22124.82\u00a0\u00b1\u00a03.85P2Central14.79\u00a0\u00b1\u00a08.0211.74\u00a0\u00b1\u00a07.0314.86\u00a0\u00b1\u00a07.6912.43\u00a0\u00b1\u00a07.0013.19\u00a0\u00b1\u00a07.7411.26\u00a0\u00b1\u00a08.2313.83\u00a0\u00b1\u00a06.7512.03\u00a0\u00b1\u00a06.31Left8.40\u00a0\u00b1\u00a03.897.18\u00a0\u00b1\u00a03.669.19\u00a0\u00b1\u00a04.74 7.74\u00a0\u00b1\u00a03.597.36\u00a0\u00b1\u00a04.806.63\u00a0\u00b1\u00a05.167.90\u00a0\u00b1\u00a03.646.84\u00a0\u00b1\u00a03.67Right8.80\u00a0\u00b1\u00a03.806.78\u00a0\u00b1\u00a03.038.68\u00a0\u00b1\u00a04.657.59\u00a0\u00b1\u00a03.687.91\u00a0\u00b1\u00a03.706.90\u00a0\u00b1\u00a03.988.28\u00a0\u00b1\u00a04.277.76\u00a0\u00b1\u00a04.17Right handN1T7\u22128.18\u00a0\u00b1\u00a04.79\u22125.98\u00a0\u00b1\u00a03.39\u22126.38\u00a0\u00b1\u00a04.67\u22125.12\u00a0\u00b1\u00a03.62\u22127.34\u00a0\u00b1\u00a04.70\u22125.88\u00a0\u00b1\u00a03.73\u22127.43\u00a0\u00b1\u00a03.30\u22126.38\u00a0\u00b1\u00a03.21T8\u22124.92\u00a0\u00b1\u00a04.43\u22123.96\u00a0\u00b1\u00a03.24\u22123.25\u00a0\u00b1\u00a03.29\u22122.98\u00a0\u00b1\u00a02.35\u22124.56\u00a0\u00b1\u00a04.61\u22123.03\u00a0\u00b1\u00a02.39\u22124.37\u00a0\u00b1\u00a03.53\u22124.15\u00a0\u00b1\u00a02.62N2Central\u221210.11\u00a0\u00b1\u00a03.83\u22127.44\u00a0\u00b1\u00a04.52\u221211.05\u00a0\u00b1\u00a04.87\u22126.69\u00a0\u00b1\u00a04.23\u22129.99\u00a0\u00b1\u00a05.06\u22126.40\u00a0\u00b1\u00a04.27\u22129.57 3.97\u22128.94\u00a0\u00b1\u00a05.40Left\u22127.74\u00a0\u00b1\u00a02.70\u22125.67\u00a0\u00b1\u00a02.77\u22128.62\u00a0\u00b1\u00a03.65\u22125.71\u00a0\u00b1\u00a03.43\u22127.77\u00a0\u00b1\u00a03.80\u22125.09\u00a0\u00b1\u00a02.64\u22126.79\u00a0\u00b1\u00a03.29\u22126.42\u00a0\u00b1\u00a03.78Right\u22126.58\u00a0\u00b1\u00a03.37\u2212 5.12\u00a0\u00b1\u00a03.34\u22126.76\u00a0\u00b1\u00a03.00\u22124.80\u00a0\u00b1\u00a03.26\u22126.52\u00a0\u00b1\u00a03.59\u22124.24\u00a0\u00b1\u00a02.72\u22125.82\u00a0\u00b1\u00a02.74\u22126.00\u00a0\u00b1\u00a03.99 P2Central14.30\u00a0\u00b1\u00a06.5611.62\u00a0\u00b1\u00a05.9314.52\u00a0\u00b1\u00a07.1410.90\u00a0\u00b1\u00a05.4914.04\u00a0\u00b1\u00a07.7910.74\u00a0\u00b1\u00a07.1311.37\u00a0\u00b1\u00a06.3810.75\u00a0\u00b1\u00a05.73Left8.47\u00a0\u00b1\u00a03.876.95\u00a0\u00b1\u00a03.038.90\u00a0\u00b1\u00a04.147.00\u00a0\u00b1\u00a02.918.65\u00a0\u00b1\u00a04.796.64\u00a0\u00b1\u00a03.376.21\u00a0\u00b1\u00a03.456.62\u00a0\u00b1\u00a03.22Right8.52\u00a0\u00b1\u00a03.836.73\u00a0\u00b1\u00a02.758.29\u00a0\u00b1\u00a04.176.41\u00a0\u00b1\u00a03.328.75\u00a0\u00b1\u00a04.296.90\u00a0\u00b1\u00a03.766.84\u00a0\u00b1\u00a04.446.67\u00a0\u00b1\u00a04.04The mean amplitudes of the LEP components before and after stimulation in all four conditions. (mean\u00a0\u00b1\u00a0standard deviation)\nDiscussion\nThe main finding of our study is that all theta burst paradigms over the SI were able to diminish the amplitude of the N2 component of LEPs significantly when compared to sham stimulation. Surprisingly, the imTBS condition, that is suggested to be used as a placebo condition, when it applied over the M1 (Huang et al. 2005), also caused a strong amplitude decrease. The N1 and P2 components and the subjective pain rating scores were not significantly influenced by any type of TBS.\nRecent studies using the theta burst paradigm have concentrated on the effects of continuous (cTBS) and intermittent theta burst stimulation (iTBS) (Franca et al. 2006; Andoh et al. 2007; Ishikawa et al. 2007; Koch et al. 2007; Mochizuki et al. 2007). None of them investigated the intermediate (imTBS) pattern since Huang and et al. published that it has no effect over the M1 as revealed by MEPs and could thus be used as a sham condition (Huang et al. 2005).\nAccording to our knowledge, only five studies applied TBS over non-motor cortical areas. First Franca et al. (2006) used the theta burst pattern of rTMS over the visual cortex. They found that cTBS increased phosphene thresholds whilst iTBS was found to be ineffective. In another study, both cTBS and iTBS over the left dorsal premotor cortex decreased the transcallosal inhibition revealed by pairs of transcranial magnetic stimuli (Koch et al. 2007). In a recent study, Wernicke\u2019s area was stimulated with iTBS while the reaction time of auditory word detection was measured (Andoh et al. 2007). In this work, iTBS facilitated the detection of foreign words when compared with native words.\nConcerning the SI, a shorter form of the iTBS (300 pulses) over the left SI resulted in a significant oxy-hemoglobin decrease at the contralateral SI and M1, detected by near infrared spectroscopy (Mochizuki et al. 2007). In another study cTBS of the SI resulted in a temporary decrease (13\u00a0min), whereas stimulation of the M1 caused a long-lasting increase (up to 53\u00a0min) of the amplitudes of cortical components of the median nerve SEPs (Ishikawa et al. 2007). In summary, these results suggest that cTBS has an inhibitory effect on non-motor areas; whereas the effect of iTBS is more facilitatory, but clearer results have still to emerge. Our results are the first demonstrating that all three TBS paradigms, but not by sham stimulation over the SI resulted in similar after-effects regarding the amplitude of the N2 component of LEPs evoked by the laser stimulation of the contralateral hand.\nThe N2 component (peaking around 160\u2013220\u00a0ms), is generated bilaterally in the operculoinsular region and in the anterior cingulate cortex (ACC) (Garcia-Larrea et al. 2003) and reflects sensory, discriminatory processes (Garcia-Larrea et al. 1997; Iannetti et al. 2005); whilst the P2 component (peaking around 300\u2013360\u00a0ms) arose mostly from the ACC and represents attentional, cognitive and affective factors of pain perception and processing (Treede 2003). However, other studies did not find significantly different brain sources for N2 and P2, revealing both parasylvian and ACC contributions for the N2\u2013P2 components (Raij et al. 2003; Ohara et al. 2004). Thus, LEP changes in N2 or P2 component might result from changes in either sensory-discriminative or cognitive aspects of pain. Studies using subdural recordings (Kanda et al. 2000; Ohara et al. 2004) or MEG (Kanda et al. 2000) demonstrated that the LEP components can be recorded over the SI and SII simultaneously, and the N2 peak may indicate the arrival of input originating from nociceptors.\nWith regard to the N1 component which is an early LEP potential (peaking around 140\u2013170 ms) and reflecting the early sensory-discriminative processing of pain perception (Iannetti et al. 2005), we did not find any significant change in amplitude. According to scalp topography (maximum near T3 and T4), the N1 is generated near to the SII in the fronto-parietal operculum (Treede et al. 2003). The participation of operculoinsular cortex in coding the pain intensity was recently suggested by using LEP measurements (Ianetti et al. 2005). However, another study (Gracia-Larrea et al. 1997) did not find any significant correlation between the amplitude of the N1 component and subjective pain rating.\nThe fact that we found a decrement of both the N2 and P2 amplitudes in the sham condition as well, should be discussed. This phenomenon is known as habituation and has been described in previous LEP studies (Spiegel et al. 2000; Tamura et al. 2004a). Still, the effects of real TBS conditions on N2 amplitudes were greater than that of the sham condition and it was significant above the stimulated left, the neighboring central and the contralateral area as well when the right hand was laser stimulated. Attention can also directly influence the N2\u2013P2 components of LEPs as well as subjective pain rating as it was suggested by previous experimental results (Gracia-Larrea et al. 1997; Ohara et al. 2006). In our experiment the subjects were asked to pay attention to each laser stimuli and since the TBS and sham condition were applied in a randomized order and the subjects were blinded as to the type of magnetic stimulation, the significant difference between sham and the other three TBS on the N2 amplitude is more than simply habituation or the effect of the different attentional states.\nThe possible origin of the N2 component is mainly the bilateral operculoinsular region and the ACC (Garcia-Larrea et al. 2003). Therefore, when we inhibited the left SI, the activity of the pain related cortical network decreased due to the widespread cortical connections between SI and other cortical areas. However, the involvement of the contralateral SI and bilateral SII\u2014parasylvian region in the N2 component generation was also reported (Kakigi et al. 2005; Kanda et al. 2000; Ohara et al. 2004). According to this, it is also possible that the inhibition of the SI itself may cause direct effects on LEP components. In this study we found no significant difference between the stimulation conditions with regard to subjective pain perception (Fig.\u00a02), however, the subjective pain rating decreased during the experimental sessions after every type of TBS including the sham condition. It is important to mention that the placebo effect is high in almost every pain perception study, regardless of the paradigm used (Khedr et al. 2005; Lefaucheur et al. 2004). The explanation of this discrepancy between the electrophysiological and psychophysical changes is rather speculative, because the relationship between the anatomical origin of the N2\u2013P2 components and their psychophysical correlates controversially discussed in the literature. In a PET study different cortical activations for pain threshold, intensity and unpleasantness have been found (T\u00f6lle et al. 1999). One possibility is that modulation the excitability of the left SI by TBS activated some elements of the pain related network that caused a decrease in the N2 amplitude. However, this stimulation intensity or duration was not strong or adequate enough to modify the subjective pain rating.\nIn our previous study (Antal et al. 2007), we found that cathodal tDCS over the SI, similarly to the present findings, significantly decreased the N2 component of LEPs. In contrast to the present findings the subjective pain perception in healthy subjects was also diminished. TDCS is a method that modifies the resting membrane potentials of cortical neurons intracortically (Nitsche and Paulus 2000, 2001). Cathodal stimulation decreases, whilst anodal stimulation increases cortical excitability (Nitsche and Paulus 2000, 2001). However, in that study we have used a large electrode size (5\u00a0\u00d7\u00a07\u00a0cm) in order to optimize stimulation\u2019s parameters (Nitsche and Paulus 2000) and therefore we might have covered a large part of the SI. It is possible that we have stimulated one part of the somatosensory association cortex (BA 5\/7) that is posterior to the SI. Activation of human BA5\/7 has also been linked to pain perception (Apkarian et al. 1999; Forss et al. 2005). BA 5\/7 is anatomically connected to other nociceptive brain areas such as the ACC, insula, thalamus and primary motor cortices (Friedman et al. 1986). In order to increase the focality of the transcranial stimulation in the present study we used a neuronavigation system to determine the hand area over the SI.\nMany of the previous pain-related studies stimulated the M1. The effects of low and high frequency rTMS of M1 on experimentally induced acute pain perception seem to depend on the type of noxious stimulation. C-fiber mediated acute pain as induced by intradermal capsaicin administration could be attenuated by 1\u00a0Hz rTMS over the M1 (Tamura et al. 2004b), whereas it increased A\u03b4-fiber mediated laser-induced pain in another study (Tamura et al. 2004a). Similarly, controversial effects were observed after 20\u00a0Hz rTMS (Johnson et al. 2006; Summers et al. 2004). In contrast 10\u00a0Hz rTMS over M1 increased electrically induced A\u03b4-fiber mediated pain threshold (Yoo et al. 2006), but others found that 10\u00a0Hz rTMS has only an effect on the unpleasantness of the pain without any effect on pain threshold (Mylius et al. 2007). In case of chronic pain the high frequency rTMS seems to more effective than the application of low frequencies (Leo and Latif 2007).\nThe neuronal mechanism of the theta burst paradigm is highly speculative. The results of the experiments with single trains of TBS suggest that in the human M1 TBS produces a mixture of facilitatory and inhibitory effects on synaptic transmission (Huang and Rothwell 2004). Huang and Rothwell proposed, that facilitation develops faster than inhibition, thus in case of the inhibitory cTBS, several seconds after an initial facilitation the inhibition overrides this effect. ITBS uses only the early excitatory effect in the initial 2\u00a0s and after this the stimulation is interrupted for 8\u00a0s. Most likely, the underlying mechanisms will involve many of the basic elementary mechanisms described previously in the LTP\/LTD literature (Paulus 2005). Recently Huang et al. (2007) have demonstrated that the after-effects produced by both iTBS or cTBS are NMDA-receptor dependent and hence they are more likely to involve plasticity-like changes at the synapse in the M1. More recently, it was found that the excitatory effects of iTBS were reversed after NMDA receptor activation by D-cycloserin (Teo et al. 2007). This is in contrast with the findings of tDCS induced LTP where the excitatory effects are prolonged by D-cycloserin (Nitsche et al. 2004). However, it is important to note that these observations were done on the M1. It is possible that the different effectiveness observed between TBS protocols on motor and sensory cortices could be due to differences in the physiological and functional states of the stimulated cortex.\nIn summary, in our study we found a significant decrease of the N2 component of the LEPs after cTBS, iTBS, and imTBS when compared to sham stimulation over the SI. In addition we found, that the subjective pain perception did not show significant differences among the stimulation conditions suggesting that this method is probably not the most effective in decreasing subjective pain perception. Further findings show that imTBS resulted in more impressive modification of the LEPs, than were found in previous TMS-studies over the M1 using MEP measures (Huang et al. 2005), implying that it should not be used in further studies as \u2018sham\u2019 condition, at least with regard to LEPs and when it is applied over the SI. However these results are not directly comparable with the effects of TBS on M1, thus further studies are needed to clarify the effects of imTBS on different cortical areas. Future studies should also clarify the effectiveness of the different TBS paradigms applied over the M1 and non-motor cortical areas, such as the SII on acute pain perception and in chronic pain.","keyphrases":["laser-evoked potentials","primary somatosensory cortex","neuronavigation","pain","theta burst transcranial magnetic stimulation"],"prmu":["P","P","P","P","R"]}
{"id":"Plant_Mol_Biol-4-1-2295252","title":"The microRNA regulated SBP-box genes SPL9 and SPL15 control shoot maturation in Arabidopsis\n","text":"Throughout development the Arabidopsis shoot apical meristem successively undergoes several major phase transitions such as the juvenile-to-adult and floral transitions until, finally, it will produce flowers instead of leaves and shoots. Members of the Arabidopsis SBP-box gene family of transcription factors have been implicated in promoting the floral transition in dependence of miR156 and, accordingly, transgenics constitutively over-expressing this microRNA are delayed in flowering. To elaborate their roles in Arabidopsis shoot development, we analysed two of the 11 miR156 regulated Arabidopsis SBP-box genes, i.e. the likely paralogous genes SPL9 and SPL15. Single and double mutant phenotype analysis showed these genes to act redundantly in controlling the juvenile-to-adult phase transition. In addition, their loss-of-function results in a shortened plastochron during vegetative growth, altered inflorescence architecture and enhanced branching. In these aspects, the double mutant partly phenocopies constitutive MIR156b over-expressing transgenic plants and thus a major contribution to the phenotype of these transgenics as a result of the repression of SPL9 and SPL15 is strongly suggested.\nIntroduction\nDuring maturation, plants pass through several developmentally distinct growth phases in which the shoot gradually gains reproductive competence (Poethig 1990). After the transition from embryonic to postembryonic growth, plants undergo at least two further phase transitions, the vegetative as well as the reproductive phase change. During vegetative growth, rosette leaves are initiated at the flanks of the shoot apical meristem (SAM) with a certain frequency, referred to as plastochron (Erickson and Michelini 1957). After going through the reproductive phase transition, also known as the floral transition, the SAM starts to initiate floral buds instead of leaves. In Arabidopsis, as in many other plants showing day length dependent flowering, the floral transition is preceded by a transition from juvenile to adult growth. This switch, known as the vegetative phase change, is physiologically defined as achieving competence to respond to photoperiodic induction of flowering (Poethig 1990).\nThe transition from juvenile to adult growth is gradual and rather subtle but generally can be followed by several morphological markers. In Arabidopsis, for example, leaves produced in the juvenile phase have long petioles, are small, round and lack abaxial trichomes. In contrast, short petioles, elliptical anatomy and the development of trichomes on the abaxial side represent adult traits (Telfer et\u00a0al. 1997).\nRegulation of these developmental transitions is largely dependent on (changes in) environmental cues such as day length, light intensity and temperature, as well as on endogenous factors such as the plant hormone gibberellin (Telfer et\u00a0al. 1997). Whereas the molecular genetic mechanisms underlying the floral transition are already worked out in increasing detail (Komeda 2004), it has only been recently that we begin to understand the molecular genetic basis of the vegetative phase change. Most genes suggested to play a role in promoting the latter phase change have been identified by the analysis of mutants showing a precocious onset of adult traits and intriguingly, link vegetative phase change to RNA silencing pathways. These genes include the Arabidopsis ortholog of exportin 5\/MSN5, HASTY (HST; Telfer and Poethig 1998; Bollman et\u00a0al. 2003), the zinc-finger-domain protein encoding locus SERRATE (SE; Clarke et\u00a0al. 1999) and ZIPPY (ZIP), an AGO-family member (Hunter et\u00a0al. 2003). More recently, screens for mutations with zip-like phenotypes resulted in alleles of SUPRESSOR OF GENE SILENCING3 (SGS3) and RNA-DEPENDENT POLYMERASE6 (RDR6), both genes required for posttranscriptional gene silencing (PTGS) and acting in the same pathways as ZIP and HST (Peragine et\u00a0al. 2004). Furthermore, a precocious vegetative phase change has also been found in dicer-like 4 (dcl4) mutants (Gasciolli et\u00a0al. 2005; Xie et\u00a0al. 2005; Yoshikawa et\u00a0al. 2005).\nOne explanation for the observed effects could be that target genes of this silencing pathway play a positive role on the vegetative phase change and their down-regulation consequently promotes juvenility. Hence, mutations in genes involved in this silencing pathway, as the ones described above, cause an accelerated vegetative development.\nIn line with this idea, members of the plant specific SBP-box gene transcription factor family have been implicated in promoting vegetative and floral phase transitions. In particular, overexpression of the Arabidopsis SBP-box gene SPL3 leads to early flowering and a significant earlier appearance of abaxial trichomes on the rosette leaves (Cardon et\u00a0al. 1997; Wu and Poethig 2006). Interestingly, together with 10 of 16 other family members, SPL3 expression is post-transcriptionally controlled by miR156 and probably also by the very closely related miR157 (Rhoades et\u00a0al. 2002; Schwab et\u00a0al. 2005; Wu and Poethig 2006; Gandikota et\u00a0al. 2007). Consistent with its role of down-regulating SPL3 and related SPL target-genes, constitutive overexpression of miR156 encoding loci has been shown to cause the production of a significantly larger number of leaves with juvenile characteristics and a delay in flowering (Schwab et\u00a0al. 2005; Wu and Poethig 2006).\nAlthough the available data clearly point to a regulatory role for the miRNA regulated SPL genes in the temporal development of the Arabidopsis shoot, the contribution of the single genes to the described phenotypes remains to be determined. Therefore, we identified and isolated mutant alleles for single SPL genes. In comparison to other miR156 targeted SPL genes, available expression data (AtGenExpress; Schmid et\u00a0al. 2005) show SPL9 and SPL15 to be already quite active in the vegetative shoot apex. Accordingly, their mutant phenotypes were found to affect vegetative development. Here we report the mutant analysis of SPL9 and SPL15, two likely paralogous members of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor family (Cardon et\u00a0al. 1999), and discuss their redundant regulatory role on the vegetative phase change and the temporal initiation of rosette leaves.\nMaterials and methods\nPlant material and plant growth conditions\nAll of the genetic stocks described in the paper were in Columbia background. The T-DNA insertion lines SALK_006573 (spl9-2), SALK_074426 (spl15-1) and SALK_138712 (spl15-2) were obtained at the Nottingham Arabidopsis Stock Centre (NASC). The T-DNA insertion lines GABI-Kat 544F04 (spl9-3) and WiscDsLox 457 (spl15-3) were obtained from GABI-Kat and the Arabidopsis Biological Research Centre (ABRC), respectively. Insertion mutant information for NASC- or ARBC-lines was obtained from the SIGNAL website at http:\/\/signal.salk.edu. Plants homozygous for the T-DNA insertions were identified by PCR using T-DNA left border- and gene-specific primers. T-DNA specific left border primers for SALK, GABI-Kat and WiscDsLox T-DNAs were 5\u2032-GCGTGGACCGCTTGCTGCAACT-3\u2032, 5\u2032-ATATTGACCATCATACTCATTGC-3\u2032 and 5\u2032-TGGCAGGATATATTGTGGTGTAAACA-3\u2032, respectively. In combination with the respective left border primer we used the following gene-specific primers: 5\u2032-GCTATGGCTTAAGCCTTAAGTTAAAAGG-3\u2032 for SALK_006573, 5\u2032-CGTAGCTGTCGTGGACTAGTGTCAATC-3\u2032 for SALK_074426 and SALK_138712, 5\u2032-AACCTCTGTTCGATACCAGCCACAG-3\u2032 for GABI-Kat 544F04 and 5\u2032-AGCCATTGTAACCTTATCGGAGAATGAG-3\u2032 for WiscDsLox 457. The stable En-1 insertion mutant 5ABA33-H1 (spl9-1) was obtained from the ZIGIA-population (Unte 2001). Plants homozygous for a four base pair insertion in the first exon of SPL9 caused by the excision of the En-1 transposon were backcrossed with wild type twice to obtain plants exclusively containing the four base-pair insertion without any further transposon contamination. In order to identify plants containing the mutation we used the following primer combination: 5\u2032-AGTAAGAGGAAACCACCATGGAGATGG-3\u2032 (forward) and 5\u2032-AACCTTCCACTTGGCACCTTGGTATA-3\u2032 (reverse, recognises the insertion).\nAll plants were grown in plastic trays or pots filled with ready-to-use commercial, pre-fertilized soil mixture (Type ED73, Werkverband eV, Sinntal-Jossa, Germany). For stratification, seeds were kept on moist paper at 4\u00b0C in the dark for 4\u20135\u00a0days before transferring to soil (i.e. \u201csowing\u201d) in growth chambers at 22\u00b0C, 50% relative humidity. Germination and cultivation of the plants in long-day conditions (16\u00a0h light, 8\u00a0h dark) were either under approx. 70\u00a0\u03bcE\/cm2\/s (LD1) or 175\u00a0\u03bcE\/cm2\/s (LD2) light provided by fluorescent tubes (L58W\/840 and L58W\/25 Osram, Munich, Germany). Plants in short-day (SD) conditions (8\u00a0h light, 16\u00a0h dark) were cultivated under approx. 450\u00a0\u03bcE\/cm2\/s light. To determine sensitivity to photoperiodic induction of flowering, stratified seeds were germinated in a modified SD with a 9\u00a0h light period. The developing plants were kept in these conditions for 21\u00a0days before they were transferred to similar growth chambers with continuous light provided by Osram HQIT 400\u00a0W lamps. Batches of plants were returned to the modified SD conditions after 1, 3 and 5\u00a0days.\nPhenotypic analysis\nFlowering time was measured as the time between sowing and anthesis (opening of first flower). Bolting time was recorded when the main inflorescence had reached a height of 0.5\u00a0cm. Inflorescence height was measured between the rosette and the first flower of the main inflorescence of plants with their first siliques fully ripened. In order to count the number of site shoots, all site shoots longer than 0.5\u00a0cm were scored. Abaxial trichomes were scored using a Leica MZFLIII stereomicroscope (Wetzlar, Germany). An estimation of the rosette leaf initiation rate (L\/D day\u22121) was obtained by dividing the number (L) of rosette leaves having reached at least 0.5\u00a0cm in length through the number of days (D) between sowing and determination. Note that this value reflects but does not equalize (average) plastochron as it should be corrected for the true start of initiation of the first leaf as well as the time needed for the first adult leaf to reach a size of 0.5\u00a0cm.\nHistological analysis\nApical regions were isolated from plants grown in SD for 41\u00a0days by trimming with a razor blade. The tissue was fixed in 4% formaldehyde\/0.1\u00a0M PO4, pH 7.0 for 48\u00a0h and embedded in paraffin using a Leica ASP300 tissue processor (Wetzlar, Germany). Embedded apices were cut into 8\u00a0\u03bcm thin cross sections using a Jung Autocut 2055 and photographed using a Zeiss Axiophot microscope (G\u00f6ttingen, Germany) equipped with a KY-F5U 28CCD camera (JVC, Yokohama, Japan). The first cross section in which the apex was visible plus two successive sections were used to determine the diameter of the apical region. From these three measured values the average was taken for comparison. In addition, cross sectional area and circularity factor (=4\u03c0\u00a0A\u00a0P\u22122, A is area, P is perimeter) of the leaf primordia, outlined by hand on the photographs, were determined as well. The measurements were performed with help of the program ImageJ 1.35s (Wayne Rasband, National Institutes of Health, USA).\nGA3 treatments\nCol-0, the spl9 spl15 double mutant and the 35S::MIR156b overexpressor were grown in LD1 conditions. Immediately after germination, half of the plants were treated by spraying 100\u00a0\u03bcM GA3, 0.02% Tween 20 and this was repeated twice per week until they started flowering. The other half of the plants was similarly treated with 0.02% Tween 20.\nPhylogenetic comparison\nMultiple alignments of amino acid sequences were generated by the program ClustalW of the MacVector 7.2.2 software package (Accelrys Ltd., Cambridge, UK) using the BLOSUM 30 matrix with an open gap penalty of 10 and an extend gap penalty of 0.05. Only the SBP-domain was used for the phylogenetic reconstruction. The tree was constructed using the neighbour-joining algorithm of the MacVector 7.2.2 software package.\nQuantitative real-time PCR analysis\nTo perform quantitative RT-PCR (using the iQ5 real-time PCR detection system, Bio-Rad, Munich, Germany) apical regions were collected (roots and as much of the leaves as possible were removed using tweezers) of plants cultivated 5, 9, 13, 27 and 32\u00a0days after sowing in LD1 conditions. Total RNA was extracted using the RNeasy plant mini kit (Qiagen, Hilden, Germany), including an on-column DNase digestion. First-strand cDNA was synthesized using SuperScript III RNase H reverse transcriptase (Invitrogen) starting with 2\u00a0\u03bcg of total RNA primed with an oligo(T)12\u201318 primer (Gibco BRL, Karlsruhe, Germany). SPL9-specific primers, 5\u2032-AGAACATTGGATACAACAGTGATGAGG-3\u2032 (forward) and 5\u2032-GTTTGAGTCGCCAATTCCCTTGTAGC-3\u2032 (reverse) as well as SPL15-specific primers, 5\u2032-TTGGGAGATCCTACTGCGTGGTCAACC-3\u2032 (forward) and 5\u2032-AGCCATTGTAACCTTATCGGAGAATGAG-3\u2032 (reverse), were designed to generate a PCR product of 171 and 300\u00a0bp, respectively. Based on the analysis of Czechowski et\u00a0al. (2005) PP2A expression was used as reference for transcript normalization with the primer pair 5\u2032-TAACGTGGCCAAAATGATGC-3\u2032 (forward) and 5\u2032-GTTCTCCACAACCGCTTGGT-3\u2032 (reverse). The PCR efficiencies for SPL9, SPL15 and PP2A primers were determined to be 96, 95.5 and 96%, respectively. Quantifications, in triplicate, were performed using the Brilliant SYBRGreen QPCR kit (Stratagene, La Jolla, CA, USA), according to the manufacturer\u2019s protocol, in a final volume of 25\u00a0\u03bcl. PCR was carried out in 250\u00a0\u03bcl optical reaction vials (Stratagene) heated for 10\u00a0min at 95\u00b0C to hot-start the Taq polymerase, followed by 40 cycles of denaturation (30\u00a0s at 95\u00b0C), annealing (30\u00a0s at 58\u00b0C) and extension (30\u00a0s at 72\u00b0C).\nSemi-quantitative RT-PCR analysis\nTotal RNA was extracted from seedlings of the Col-0 wild-type, mutant- and transgenic lines using the RNeasy plant mini kits (Qiagen). RT-PCR with equal amounts of RNA was performed using the one-step RT-PCR kit (Qiagen). SPL9 knockout lines were identified using the following primer pair: 5\u2032-GGTCGGGTCAGTCGGGTCAGATACC-3\u2032 (forward) and 5\u2032-ACTGGCCGCCTCATCACTCTTGTATCC-3\u2032 (reverse). SPL9 mRNA is expected to yield a 415\u00a0bp fragment whereas genomic SPL9 DNA is expected to yield a 1,138\u00a0bp fragment. SPL15 knockout lines were identified using the following primer pair: 5\u2032-AGAAGCAAGAACCGGGTCAATACC-3\u2032 (forward) and 5\u2032-AGCCATTGTAACCTTATCGGAGAATGAG-3\u2032 (reverse). SPL15 mRNA is expected to yield a 666\u00a0bp fragment whereas genomic SPL15 DNA is expected to yield a 1,004\u00a0bp fragment. RT-PCR of the loading control (RAN3; At5g55190) was performed with the primer pair 5\u2032-ACCAGCAAACCGTGGATTACCCTAGC-3\u2032 (forward) and 5\u2032-ATTCCACAAAGTGAAGATTAGCGTCC-3\u2032 (reverse) to yield a fragment of 531\u00a0bp when derived from RAN3 mRNA (genomic RAN3 is expected to yield a 1,314\u00a0bp fragment).\nStatistics\nGraphical representations of numerical data were generated with the Microsoft Excel program (Microsoft Germany, Munich) and statistical tests were performed using the Student\u2019s t-Test within this program. P-values lower than 0.05 were considered to be statistically relevant and the data involved to represent significant differences.\nRemaining techniques and methods\nStandard molecular biology techniques were performed as described by Sambrook et\u00a0al. (1989). Graphical plots and digital photographic images were cropped and assembled using Adobe Photoshop (Adobe Systems, San Jose, CA, USA).\nResults\nMolecular characterization of the Arabidopsis SBP-box genes SPL9 and SPL15\nMutant alleles for the Arabidopsis SBP-box genes SPL9 (At2g42200) and SPL15 (At3g57920) were obtained from screening publicly available electronic databases and seed stock centres for transposon or T-DNA tagged SPL genes. For SPL9, we identified three insertion alleles designated as spl9-1 to -3 and confirmed the nature and position of their mutations (see \u201cMaterials and methods\u201d; Fig.\u00a01a). The first allele, spl9-1, was identified in the En-transposon mutagenised ZIGIA population (Baumann et\u00a0al. 1998; Unte 2001) and most likely resulted from the excision of an inserted En-1 transposon leaving behind a 4-bp insertion footprint in the first exon. The result of this is a frame shift in the coding sequence and the generation of a stop-codon 86 base pairs after the insertion site. Both spl9-2 and spl9-3 represent T-DNA insertion mutant alleles identified within, respectively, the SALK collection (Alonso et\u00a0al. 2003) and the GABI-Kat collection (Li et\u00a0al. 2007).\nFig.\u00a01Molecular characterization of SPL9 and SPL15. (a) Schematic representation of the genomic loci of SPL9 and SPL15. The positions of the mutations identified are indicated by open triangles, numbered according to the respective alleles. Boxes represent exons. The SBP-box sequences are depicted in black, the remaining coding sequences in grey and the untranslated 5\u2032 and 3\u2032 regions are left blank. (b) Changes in transcript levels of SPL9 and SPL15 in the shoot apical region during plant development in LD1 as determined with qRT-PCR and normalized against PP2A. For comparison, relative transcript levels were arbitrarily set to one for SPL9 5\u00a0days after sowing. Error bars indicate standard deviation. (c) Absence of SPL9 and SPL15 transcripts in seedlings of the respective mutants as validated by RT-PCR. Presence of the respective transcripts in Col-0 wild type seedlings is shown for comparison and the amplification of RAN3 transcript as quality control and reference for quantification. Fragment lengths are indicated on the left in base pairs (bp)\nAlso three independent T-DNA insertion lines for SPL15 could be obtained and confirmed (see \u201cMaterials and methods\u201d; Fig.\u00a01a). Two alleles designated as spl15-1 and spl15-2 were identified within the SALK collection and one, spl15-3, within the WiscDsLox T-DNA collection.\nAccording to data available from the AtGenExpress micro-array database (Schmid et\u00a0al. 2005), both SPL9 and SPL15 transcript levels increase during development and are preferentially found in the shoot apical region and in young flowers. We confirmed this temporal expression pattern with the help of qRT-PCR (Fig.\u00a01b). In LD1 growing conditions (see \u201cMaterials and methods\u201d), SPL9 and SPL15 transcript levels remain comparable during the first 2\u20133\u00a0weeks. Thereafter, the expression level of SPL9 starts to increase followed by that of SPL15. Around 32\u00a0days after sowing (DAS), at about the time Col-0 plants have undergone their reproductive phase transition, SPL9 transcript levels have become approximately two and a half times higher in comparison to SPL15 and six times in comparison to day 5. Arabidopsis lines carrying as a transgene a genomic fragment encompassing the locus for SPL15 and with a GUS reporter gene inserted downstream of the ATG start codon, confirmed the predominantly apical expression of SPL15 (Supplementary Fig.\u00a01).\nRT-PCR performed on mRNA isolated from whole seedling plants homozygous for any of the three SPL9 or SPL15 mutant alleles (see \u201cMaterials and methods\u201d) did not result in the detection of RNA derived of the respective genes (Fig.\u00a01c). This strongly suggests that all mutant alleles isolated represent functional null-alleles. Accordingly, plants homozygous for any of the three spl9 mutant alleles showed highly identical phenotypes, as did all three homozygous spl15 mutants (see phenotypic analysis below; Supplementary Fig.\u00a02). Allelic tests confirmed that the observed phenotypes are indeed due to mutation in either SPL9 or SPL15, respectively (data not shown).\nWith over 75% of their amino acid residues identical, SPL9 and SPL15 show high similarity on the level of their proteins. Also a phylogenetic comparison based on the SBP-box of all 17 SPL genes in Arabidopsis revealed SPL9 and SPL15 as most closely related and most likely forming a pair of paralogous genes (Fig.\u00a02). Based on this close relationship some degree of functional redundancy could be expected and, therefore, we created double mutant lines to uncover such redundancy. To ascertain that phenotypic changes in the mutant plants are solely due to the loss-of-function of SPL9 and SPL15 we generated two different homozygous double mutant lines with the allelic combinations spl9-1 spl15-1 and spl9-2 spl15-2, respectively. Both lines exhibit the same phenotype as described in the next section. For further detailed analysis the spl9-1 spl15-1 line was chosen and in the following referred to as spl9 spl15 for simplicity.\nFig.\u00a02Phylogenetic relationship of the Arabidopsis SBP-box genes as based on the conserved SBP-domain. The orthologous sequence of Chlamydomonas CRR1 has been used as outgroup. The likely paralogous pair SPL9 and SPL15 is boxed in grey. MiR156\/157 targeted SPL genes are marked with an asterisk. Only bootstrap values over 50% are shown\nPhenotypic analysis of spl9 and spl15 mutants\nFor a phenotypic analysis, we compared spl9 and spl15 single mutants, spl9spl15 double mutants to Col-0 wild type as well as to an 35S::MIR156b transgenic line (kindly provided by D. Weigel and R. Schwab).\nAn interesting aspect of the MIR156b over-expressing plants, as already noticed by Schwab and co-workers (2005) is an increased rate of rosette leaf initiation which, in combination with a modest delayed flowering, results in the obvious denser rosettes of fully developed plants (Fig.\u00a03a). In addition, advanced 35S::MIR156b plants became very bushy (Fig.\u00a03c). We found these phenotypic aspects also displayed by the spl9 spl15 double mutant, albeit less pronounced (Fig.\u00a03a, b). To quantify the contribution of SPL9 and SPL15 to these phenomena, we compared the number of rosette and cauline leaves of the respective single and double mutants and of the MIR156b overexpressor to wild type (Table\u00a01). Whereas in LD2 growing conditions, the 35S::MIR156b line produced ca. eleven more rosette leaves in comparison to wild type, the single mutant lines produced, on average, only 1\u20132 rosette leaves more. Again, with ca. six extra rosette leaves, the spl9spl15 double mutant differed more from wild type than the single mutants and showed a stronger tendency towards the phenotype of the MIR156b overexpressor. The number of cauline leaves remained very comparable among all mutants and wild type, although some reduction may be observed particularly in the spl9 mutants.\nFig.\u00a03Phenotypic analysis of spl9 and spl15 mutants. (a) Flowering spl9, spl15 and spl9spl15 double mutant plants shown next to Col-0 wild type and the MIR156b overexpressor. Plants shown next to each other are of the same age and grown in parallel under LD2 conditions. (b, c) Col-0 wild type, spl9spl15 double mutant (b) and MIR156b overexpressor (c) at a more advanced stage of development in comparison to the plants shown in aTable\u00a01Phenotypic evaluation of spl9 and spl15 mutant alleles in comparison to Col wt and a 35S::MIR156b transgene under LD conditionsRosette leavesCauline leavesBolting (DAS)Anthesis (DAS)Juvenile leavesaInfloresc. heightb (cm)MeanSDMeanSDMeanSDMeanSDMeanSDMeanSDCol-0\u00a0wt13.11.13.90.516.31.220.91.55.51.212.11.3spl9-114.31.13.4c0.715.91.419.6c1.78.30.88.01.0spl9-215.61.23.5c0.516.91.220.81.49.20.98.20.8spl9-315.71.33.3c0.617.5c1.521.31.59.60.78.60.8spl15-115.61.13.3c0.416.60.820.91.07.10.710.31.2spl15-214.91.03.50.517.11.920.91.97.30.811.4c1.1spl15-316.11.03.70.717.2c0.821.81.17.60.711.1c1.3spl9-1 spl15-119.5d1.43.40.918.5d1.422.3c1.810.9d0.86.9d1.4spl9-2 spl15-218.9d1.33.3c0.619.0d1.522.8c1.810.8d0.46.8d0.835S::MIR156b24.4e2.43.2c0.819.31.822.4c2.114.8e1.12.3e0.716 plants per genotype were used for determinationDAS, days after sowing; SD, standard deviationValues significantly different from Col-0\u00a0wt at 0.001 confidence level are shown in italicsaNumber of rosette leaves formed before the first leaf with abaxial trichomesbMeasured from rosette to first flowercValues significantly different from Col-0\u00a0wt at 0.05 but not at 0.001 confidence leveldValues significantly different from single mutants at 0.05 confidence leveleValues significantly different from double and single mutants at 0.05 confidence level\nAlso with respect to the development of side shoots, the spl9spl15 double mutant differed more from wild type than the single mutants. In fact, spl9-1 and spl15-1 single mutants were found not to differ significantly from Col-0 plants that had formed, on average, 0.9\u00a0\u00b1\u00a00.6 side shoots of at least 0.5\u00a0cm in length by the time that the first siliques ripened. With an average of 2.1\u00a0\u00b1\u00a01.1 side shoots, the spl9spl15 double mutant did significantly differ from wild type as did the 35S::MIR156b transgenic line with, on average, 4.1\u00a0\u00b1\u00a00.8 side shoots. Taken together, the phenotypic data of the spl9spl15 double mutant clearly suggests a redundant function of SPL9 and SPL15 in shoot development and in the maintenance of apical dominance.\nMiR156 is assumed to target, besides SPL9 and SPL15, exclusively other SPL genes (Rhoades et\u00a0al. 2002). These too were shown to be down regulated in MIR156b over-expressing plants (Schwab et\u00a0al. 2005). As in comparison to the spl9 spl15 double mutant the MIR156b over-expressor displays an even more severe aberrant phenotype, it can also be deduced that in addition to SPL9 and SPL15, other miR156-controlled SPL genes act redundantly to control shoot development and apical dominance.\nIn addition to the number of leaves formed before the appearance of the first flowers, we also determined for the same plants the time they needed to bolt as well as to anthesis (Table\u00a01). On average, the spl9 and spl15 single mutants behaved similar to wild type but, as expected based on the data of Schwab et\u00a0al. (2005), the 35S::MIR156b line bolted and flowered somewhat later. The spl9 spl15 double mutants showed an intermediate behaviour. Whereas for the single mutants the few more leaves formed may be accounted for by the slight delay in the transition to flowering, this delay is unlikely to explain the increased rosette leaf number of the spl9 spl15 double mutant. In line with the observation of Schwab and co-workers (2005) who reported a leaf-initiation rate per day in SD of 2.2 vs. 1.4 for the MIR156b overexpressor and the wild type, respectively, this is probably best explained by assuming a shortened plastochron during vegetative growth.\nTo uncover a possible cause or consequence for this increased rate of leaf initiation, we microscopically examined cross sections of the vegetative shoot apex to determine size and phyllotaxy of the spl9spl5 double mutant and the MIR156b overexpressor and compared these to wild type. To this purpose, plants were grown for 41\u00a0days in SD conditions, whereafter the number of rosette leaves having reached at least 0.5\u00a0cm in length were recorded and their apices dissected, fixed and embedded in paraffin (see \u201cMaterials and methods\u201d). At this age, Col-0 plants were found to have formed on average 24.5 leaves of 0.5\u00a0cm or more, the double mutant 33.8 and the MIR156b overexpressor plants already 43.4 (Fig.\u00a04a). As the plants were of the same age, these differences most likely reflect differences in plastochron. Alternatively, one may assume large temporal differences per genotype concerning initiation of the first leaf and\/or development of the last leaf recorded to have reached 0.5\u00a0cm in length. However, we obtained no indications for such discrepancies and noted increasing differences in rosette density during the entire vegetative growth phase of wild type and mutants. From these data, a relative 1.8-fold (43.4\/24.5) increase in leaf initiation rate of the 35S::MIR156b transgenics over wild type can be deduced, a value that quite well matches the observation of Schwab et\u00a0al. (2005). Leaf initiation rate of the double mutant seem to be increased by a factor of 1.4 (33.8\/24.5) in comparison to wild type.\nFig.\u00a04Leaf formation of spl9spl15 double mutant in comparison to wild type and MIR156b overexpressor. (a) Determination of the average number of rosette leaves of at least 5\u00a0mm in length formed by the primary shoot and (b) of the average diameter of the primary shoot apex of spl9spl15 double mutants, Col-0 wild type and MIR156b overexpressor plants after having grown for 41\u00a0days in SD. (c, d) Average circularity (c) and cross sectional area (d) of leaf primordia as determined from cross section through primary shoot as shown in e\u2013g. Values represent averages of 10 subsequent primordia as indicted in different shades of grey according to the legend shown in d. (e\u2013g) Cross sections through primary shoot apices of a Col-0 wild type (e), a spl9spl15 double mutant (f) and a MIR156b overexpressor plant (g) after having grown for 41\u00a0days in SD. Error bars in a\u2013d indicate standard deviation (n\u00a0=\u00a06). Successive leaf primordia in e\u2013g are sequentially numbered starting with the youngest (P1). Marginal meristem on one side of leaf number 17 (L17; counted from the centre outwards) is encircled in e and g. Scale bar in e\u2013g represents 200\u00a0\u03bcm\nAfter sectioning the paraffin embedded material, a small but not significant difference in average SAM-diameter of the spl9spl15 double mutant and Col-0 wild type could be observed (Fig.\u00a04b, e\u2013f). However, with an average diameter of 104\u00a0\u03bcm, the MIR156b overexpressor showed also a slight but yet significant (P\u00a0<\u00a00.05) decrease in its SAM size compared to Col-0 (Fig.\u00a04b, e, g). Furthermore, both the spl9spl15 double mutant and the MIR156b overexpressor exhibited the same phyllotaxy as wild type with rosette leaves initiated either clock- or anticlockwise with an angle of divergence of about 137.5\u00b0 between successive leaves and forming a spiral lattice with a parastichy pair (3,5) (Fig.\u00a04e\u2013g). From these observations, it is concluded that the observed shorter plastochron is neither the result nor the cause of an altered phyllotaxy in the spl9spl15 double mutant or the MIR156b overexpressor. The shortened plastochron, however, seems to correlate with a reduced SAM size.\nAs obvious from cross sections shown in Fig.\u00a04e, g, the young leaves of the MIR156b overexpressor appear more roundish in shape in comparison to wild type leaves at similar positions. In particular, the vacuolated cells surrounding their midveins seem larger and the developing laminas reduced, i.e. represented by less small cytoplasm rich cells along their lateral margins. In addition, the stipules of the MIR156b overexpressor seem to be more prominent. In these aspects of leaf development, the spl9spl15 double mutant seems to behave intermediate (Fig.\u00a04e).\nTo quantify the difference in shape and size of the leaf primordia, we determined their circularity and cross sectional area (see \u201cMaterials and methods\u201d) starting from the first leaf cross section found to be separated from the apical meristem. To reduce effects due to imperfect cross sectioning, i.e. not absolute perpendicular to the longitudinal axes, as well as in correlating sequentially numbered primordia of different sections, we averaged the values obtained over 10 successive primordia. As shown in Fig.\u00a04c, circularity of the youngest 10 leaf primordia was highly similar between the different genotypes. Circularity of subsequent older primordia decreased in all, however, more rapidly in wild type such that, on average, leaf 10\u201320 did differ significantly between the genotypes. Interestingly, it cannot be excluded that this difference is a direct consequence of a shortened plastochron in the mutant lines. In particular as the leaf initiation rate of the MIR156b overexpressor line lies roughly one and a half times above that of wild type. Accordingly, and with respect to absolute age, leaf 11\u201320 of the MIR156b overexpressor may be more comparable to leaves 6\u201315 of wild type and to which indeed no significant difference in circularity was found. However, in cross sectional area these young leaves differed significantly. On average, 6.5\u00a0\u00b1\u00a02.4\u00a0\u00d7\u00a0103\u00a0\u03bcm2 for wild-type leaves 6\u201315 and 26.7\u00a0\u00b1\u00a016.2\u00a0\u00d7\u00a0103\u00a0\u03bcm2 for leaf 11\u201320 of the MIR156b overexpressor.\nIt is known that the shape and other characteristics of newly formed leaves progressively change in correlation with the vegetative phase transition (Telfer et\u00a0al. 1997). Furthermore, likely due to a changed plastochron, the correlation between leaf number and flowering seems to differ for the mutants and wild type. Therefore, we further investigated the possibility that the observed differences correlate with relative altered timing of the vegetative phase transition.\nFunctional analysis of SPL9 and SPL15 during the vegetative phase transition\nIn order to determine the timing of the vegetative phase change we used the absence or presence of abaxial trichomes on rosette leaves as a morphological marker for leaves formed during the juvenile or adult growth phase, respectively (Telfer et\u00a0al. 1997). On average, in our LD2 growing conditions, the first abaxial trichomes developed on rosette leaf number six of Col-0 wild-type plants (Table\u00a01). The spl9spl15 double mutants displayed its first abaxial trichomes on leaf number twelve and the 35S::MIR156b overexpressor on leaf number 16. Although less than the spl9spl15 double mutant, the respective single mutants also developed significantly more juvenile leaves than wild type (Table\u00a01).\nWe distinguished juvenile and adult growth phase based on a phase dimorphism, i.e. in abaxial trichomes. However, on a plant-physiological level the juvenile phase in Arabidopsis is characterized as being incompetent to respond to photoperiodic induction of flowering (Poethig 1990). To determine if this competence was indeed affected, small populations of 20\u201322 plants of wild type, the spl9, spl15 single and double mutants and the MIR156b overexpressor were germinated and cultivated for 3\u00a0weeks in non-inductive SD conditions (see \u201cMaterials and methods\u201d). The plants were then brought into continuous light and batchwise shifted back to SD after either 1, 3 or 5\u00a0days. Their flowering response was recorded within a 3-week period after this inductive treatment. Plants not flowering within this period also did not flower after 2\u00a0months like plants of a control group representing all genotypes that were kept continuously in SD. As shown in Table\u00a02, the 5-day inductive treatment caused a flowering response in 100% of the plants of all genotypes. Three days also sufficed to induce all or almost all of the wild-type and single mutant plants, whereas the response of the double mutant and particular of the MIR156b overexpressor already declined. One day of continuous light, still enough to induce half or more of the wild-type and single mutant plants, did not induce flowering in any of the MIR156b overexpressor plants and only in one-tenth of the double mutants. These results thus demonstrate that also according to physiological criteria, SPL9 and SPL15 redundantly promote the juvenile-to-adult phase transition. In addition, other miR156-regulated SPL genes are expected to contribute as well based on the behaviour of the MIR156b overexpressor.\nTable\u00a02Photoperiodic floral induction in wild type and spl mutantsPercentage of plantsa induced after treatment with continuous light for1 D3 D5 DCol-0\u00a0wt60100100spl9-159100100spl15-15095100spl9 spl1598610035S::MIR156b027100D, daysaTwo populations of 10\u201311 plants per genotype were evaluated within a 3\u00a0week period following inductive treatment\nThe role of gibberellin in the function of SPL9 and SPL15\nThe plant hormone gibberellin (GA) is known to promote flowering in many plants and in Arabidopsis it is particularly required for flowering in SD (Wilson et\u00a0al. 1992). Exogenous application of GA will induce abaxial trichomes on leaves where these are normally not present although they will not appear earlier than leaf three (Telfer et\u00a0al. 1997). In order to test whether the spl9spl15 double mutant and the MIR156b overexpressor are defective in gibberellin sensitivity or biosynthesis, we exogenously applied GA3 and compared the onset of abaxial trichome production to mock treated and wild-type plants grown in LD1 (Fig.\u00a05). Like in wild type, the GA3 treatment strongly reduced the number of rosette leaves without abaxial trichomes, i.e. juvenile leaves, with about a factor of three. This result shows that the spl9spl15 double mutant and the MIR156b overexpressor remained sensitive to GA3. However, in both the spl9spl15 double mutant and the MIR156b overexpressor the amount of GA3 applied could not reduce the number of juvenile leaves to that of obtained in GA3 treated wild type.\nFig.\u00a05Effect of GA on spl9spl15 double mutant and MIR156b overexpressor plants in comparison to wild type. The number of juvenile rosette leaves formed is shown for plants that were either regularly sprayed with GA3 (100\u00a0\u03bcM GA3) or mock treated. Error bars indicate standard deviation (n\u00a0=\u00a08)\nDiscussion\nTransgenic plants constitutively over-expressing the plant specific miRNA156 have been described for Arabidopsis (Schwab et\u00a0al. 2005; Wu and Poethig 2006) and rice (Xie et\u00a0al. 2006). Recently, overexpression of a miR156 encoding locus has also been shown to be the cause of the natural maize mutant Corngrass1 phenotype (Chuck et\u00a0al. 2007). Interestingly, in all three different species, overexpression of this well conserved miRNA (Axtell and Bartel 2005; Arazi et\u00a0al. 2005) causes a similar phenotype suggesting an evolutionary conserved role for the function of miR156 and its SPL target genes. Generally, in comparison to the respective wild type, miR156 over-expressing plants are smaller, flower later, tend to lose apical dominance and initiate more leaves with a shorter plastochron. MiR156 targets eleven SBP-box genes in Arabidopsis but the results presented here clearly show that already simultaneous silencing of the two likely paralogous target genes, SPL9 and SPL15, well approximate the miR156 over-expressing phenotype regarding the traits mentioned above. SPL9 and SPL15 thus act as important and functionally redundant transcription factors regulating diverse processes in shoot maturation and most likely in combination with other miR156 regulated SPL genes. In agreement with this latter statement is our observation that in addition to spl9-1 and spl15-1, mutation of a third miR156 controlled gene, SPL2 (At5g43270; T-DNA insertion line SALK_022235), results in triple mutant plants showing an even better approximation to the MIR156b overexpressor phenotype (Supplementary Fig.\u00a02; despite the absence of detectable SPL2 transcript, single homozygous spl2-1 mutant plants lack an obvious mutant phenotype, data not shown). In greenhouse LD conditions we found the triple mutant to have produced on average 17.3\u00a0\u00b1\u00a02.1 (n\u00a0=\u00a016) rosette leaves in comparison to 15.6\u00a0\u00b1\u00a02.6 for the spl9 spl15 double mutant. Col-0 wild-type and the MIR156b overexpressor plants grown in parallel produced 12.9\u00a0\u00b1\u00a01.7 and 22.5\u00a0\u00b1\u00a03.5 rosette leaves before flowering, respectively.\nSPL9 and SPL15 positively regulate the juvenile-to-adult growth phase transition\nIt became clear with the detailed analysis of Arabidopsis MIR156b overexpressors by Wu and Poethig (2006) as well as with the description of the Corngrass1 mutant in maize by Chuck et\u00a0al. (2007), that one of the major phenotypic alterations in miR156 over-expressing plants is an extended juvenile growth phase. This suggests that one of the important functions of miR156 targeted SBP-box genes is to promote the vegetative phase change. In agreement with this observation, Wu and Poethig (2006) showed that overexpression of the miRNA156 regulated gene SPL3 and its likely paralogs leads to a greatly shortened juvenile phase in Arabidopsis. Based on morphological markers (abaxial trichomes) and physiological parameters (response to photo-inductive stimulus) we found that the spl9spl15 double mutant exhibit a delayed vegetative phase transition and, therefore, conclude that both genes are very likely involved in the positive regulation of this developmental process in a redundant fashion. Most likely, because of this redundancy, photoperiodic induction of the single spl9 and spl15 mutants is not much affected. However, the effect on the appearance of abaxial trichomes as a marker for the juvenile-to-adult phase transition appears to be stronger in the spl9 mutant in comparison to spl15. This may be due to the fact that in shoot apical development expression of SPL9 starts to increase before that of SPL15 (Fig.\u00a01b).\nAs SPL9 and SPL15 promote the juvenile-to-adult growth phase transition and thus competence to respond to photoperiodic induction of flowering, it is interesting to note that both SPL9 and SPL15 themselves are strongly upregulated in the shoot apex upon such induction (Schmid et\u00a0al. 2003). An additional role for these genes in establishing inflorescence or floral meristem identity may thus be suggested.\nSPL9 and SPL15 negatively regulate leaf initiation rate\nOur data on the leaf initiation rate suggest that SPL9 and SPL15 act negatively on leaf initiation. Silencing of both genes leads to a shorter plastochron. Other miR156 regulated SPL genes may act similarly as the plastochron is even further shortened in the MIR156b overexpressor plants. A few mutants are known to cause a shortened plastochron and most of them simultaneously affect phyllotaxy. However, we found that shortening of the plastochron due to loss of SPL gene function is neither the cause nor the result of a changed spatial distribution of leaf primordia at the shoot apex. A shorter plastochron without an altered phyllotaxy has also been reported for two rice mutants, plastochron1 and -2 (pla1, -2; Itoh et\u00a0al. 1998; Miyoshi et\u00a0al. 2004; Kawakatsu et\u00a0al. 2006). PLA1 encodes a cytochrome P450 protein, whereas PLA2 encodes a MEI2-like RNA binding protein. In both mutants the reduction in plastochron is accompanied by an increase in the size of the SAM and a higher rate of cell division. However, although the SAM of pla2 is actually smaller than that of pla1, it has a shorter plastochron. Furthermore, higher cell division activity associated with constitutive overexpression of CyclinD shortened the plastochron in tobacco without altering SAM size (Cockcroft et\u00a0al. 2000). These observations suggest, as already noticed by Kawakatsu et\u00a0al. (2006), that not SAM size but rather cell division rate is decisive in plastochron duration. Also our results may lend support to this hypothesis as both the spl9spl15 double mutant and the MIR156b overexpressor exhibit a clearly shorter plastochron than wild-type plants but their SAM sizes differ only marginally. Therefore, it will be of interest to determine if SPL9, SPL15 and other miR156 regulated SPL genes control cell division rate in the SAM and, if so, in particular if their role is mediated through the phytohormone cytokinin. Not only is cytokinin a major positive regulator of cell proliferation and division in plants (Werner et\u00a0al. 2001) it is also, in mutual dependence of auxin, a major determinant in the outgrowth of lateral shoots (Sachs and Thimann 1967; Chatfield et\u00a0al. 2000). This latter aspect may explain the reduced apical dominance observed for the spl9spl15 double mutant and the MIR156b overexpressor. Finally, mutants disrupting cytokinin signalling are known to result in reduced leaf initiation rates in addition to a smaller SAM and other effects (Nishimura et\u00a0al. 2004; Higuchi et\u00a0al. 2004).\nDo SPL9 and SPL15 negatively regulate leaf maturation rate?\nBased on their observations of the pla mutants in correlation to the expression of the respective genes in leaf primordia but not in the SAM, Kawakatsu et\u00a0al. (2006) proposed that the rate of leaf maturation plays a significant role in regulating the rate of leaf initiation. In addition, these authors postulated a model in which the inhibitory effect of pre-existing leaf primordia on the initiation of the next leaf is lost as they mature. Similarly, a shortened plastochron in the spl9, spl15 mutants and the MIR156b overexpressor may also be due to precocious maturation of their leaves as suggested by our comparison of cross sections through successive leaf primordia of wild type and the MIR156b overexpressor. Even if the shape, i.e. circularity, of the primordia may not significantly differ after correction for an altered plastochron by comparing primordia based on age and not on serial sequence number, their cross sectional area seems to increase more rapidly in the MIR156b overexpressor. In particular, the cells surrounding the midvein in MIR156b overexpressor leaves appear to enlarge more rapidly.\nSPL9 and SPL15 do not modulate the role of GA in the vegetative phase change\nExogenous application of GA3 has been found to accelerate abaxial trichome production in Arabidopsis suggesting that gibberellins function to regulate vegetative phase change (Telfer et\u00a0al. 1997). These findings are also supported by mutant analysis. For example in spindly (spy) mutants, which undergo constitutive GA response, abaxial trichomes occur on leaves initiated significantly earlier than in wild type (Jakobsen and Olszewski 1993; Telfer et\u00a0al. 1997). On the other hand, Telfer et\u00a0al. (1997) found that mutants blocked in GA biosynthesis as well as GA insensitive mutants are significantly delayed in the appearance of abaxial trichomes. Loss-of-function mutants for the here examined SPL9 and SPL15 genes clearly delay the appearance of abaxial trichomes.\nOur treatment of spl9spl15 double mutant and 35S::MIR156b transgenic plants with high doses of GA3 showed that, like in wild type, their number of juvenile leaves can be reduced but not to numbers equal to those found for similarly treated wild type. In fact the ratios of juvenile to adult leaves of wild type and mutant phenotypes remain highly comparable to untreated plants. From this we conclude that the role SPL9, SPL15 and other miR156 controlled SPL genes play in the vegetative phase change, is unlikely to be GA mediated although a minor contribution to GA sensitivity can not be excluded.\nOutlook\nMiR156 targeted members of the SBP-box family of transcription factors in both mono- and dicots appear to play an important role as positive regulators of shoot maturation and of the vegetative to reproductive phase transition in particular. Both genetic factors, i.e. miR156 and SBP-box genes, have also been suggested to be major determinants in the transition from undifferentiated to differentiated embryogenic calli of rice (Luo et\u00a0al. 2006). As the interaction between SBP-box genes and miR156 is of ancient origin in land plants (Arazi et\u00a0al. 2005; Riese et\u00a0al. 2007) it will be interesting to learn to what extent their molecular interplay is of importance to developmental phase transitions in plants in general.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nSupplementary Fig. 1\nExpression of SPL15 as detected with a GUS reporter gene construct. A pSPL15::GUS:SPL15 reporter was constructed by subcloning a 2,793\u00a0bp genomic fragment of SPL15 beginning 1,260\u00a0bp upstream of the SPL15-ATG start codon and extending to 132\u00a0bp downstream of the SPL15-stop codon into the binary vector pGJ2148 (kindly provided by Guido Jach, MPIZ Cologne). A \u00df-glucuronidase (GUS) reporter was subsequently cloned in frame shortly downstream of the SPL15-ATG start codon and the whole construct stably transformed into Arabidopsis Col-0 background. (A\u2013B) GUS staining of mature embryo gently squeezed out of the imbibed seed (A) and of a ca. 10 day old seedling grown in LD (B). Bars in A and B represent respectively 200\u00a0\u03bcm and 2\u00a0mm. (TIF 26,178 KB)\nSupplementary Fig. 2\nPhenotypes of spl9 and spl15 related mutants. (A) Flowering plants homozygous for spl9-1, -2 and -3 alleles in Col-0 background next to wild type. (B) Flowering plants homozygous for spl15-1, -2 and -3 alleles in Col-0 background next to wild type. (C) Flowering plants, from left to right, of Col-0 wild type, the double mutants spl9-1 spl15-1 and spl9-2 spl15-2, the triple mutant spl2-1 spl9-1 spl15-1 and the MIR156b overexpressor. (TIF 32,914 KB)","keyphrases":["sbp-box genes","shoot maturation","arabidopsis","phase change","juvenile phase","mirna"],"prmu":["P","P","P","P","P","P"]}
{"id":"Virus_Res-2-1-2194287","title":"Novel vectors expressing anti-apoptotic protein Bcl-2 to study cell death in Semliki Forest virus-infected cells\n","text":"Semliki Forest virus (SFV, Alphavirus) induce rapid shut down of host cell protein synthesis and apoptotic death of infected vertebrate cells. Data on alphavirus-induced apoptosis are controversial. In this study, the anti-apoptotic bcl-2 gene was placed under the control of duplicated subgenomic promoter or different internal ribosome entry sites (IRES) and expressed using a novel bicistronic SFV vector. The use of IRES containing vectors resulted in high-level Bcl-2 synthesis during the early stages of infection. Nevertheless, in infected BHK-21 cells translational shutdown was almost complete by 6 h post-infection, which was similar to infection with appropriate control vectors. These results indicate that very early and high-level bcl-2 expression did not have a protective effect against SFV induced shutdown of host cell translation. No apoptotic cells were detected at those time points for any SFV vectors. Furthermore, Bcl-2 expression did not protect BHK-21 or AT3-neo cells at later time points, and infection of BHK-21 or AT3-neo cells with SFV replicon vectors or with wild-type SFV4 did not lead to release of cytochrome c from mitochondria. Taken together, our data suggest that SFV induced death in BHK-21 or AT3-neo cells is not triggered by the intrinsic pathway of apoptosis.\n1\nIntroduction\nSemliki Forest virus (SFV) is a positive-stranded RNA virus in Alphavirus genus (family Togaviridae), a widely distributed group of human and animal pathogens (Strauss and Strauss, 1994). SFV genomic RNA (so-called 42S RNA) is approximately 11.5\u00a0kb long and encodes four non-structural proteins designated nsP1\u20134; these are involved in viral RNA synthesis. The remaining proteins form the virus capsid and envelope and are not essential for virus replication.\nAfter virus entry, the 42S RNA is translated into a large non-structural polyprotein, which is processed to form an early and subsequently a late replicase complex (Strauss and Strauss, 1994). The early replicase mediates synthesis of the negative-stranded RNA complementary to the genomic 42S RNA. Minus strands are used by the late replicase as templates for the synthesis of new positive strand 42S RNA, and for transcription of subgenomic mRNAs encoding the structural proteins.\nThe structural genes of SFV are not required for replication and can be removed or replaced with a polylinker and\/or with foreign gene sequences. This property forms the basis of the SFV-based replicon vector systems (Liljestrom and Garoff, 1991; Smerdou and Liljestrom, 1999). SFV-based replicon vectors mediate high-level expression of heterologous proteins. However, as with virus, vectors cause shutdown of cellular biosynthesis and induce apoptotic death (Glasgow et al., 1997, 1998; Scallan et al., 1997). This precludes long-term foreign gene expression, and several attempts to reduce the cytotoxicity of alphavirus vectors have been made (Fazakerley et al., 2002; Lundstrom et al., 2003, 2001; Perri et al., 2000).\nThe anti-apoptotic gene bcl-2 is an antagonist of the intrinsic mitochondrial pathway of apoptosis (for reviews see Ashe and Berry, 2003; Cory and Adams, 2002; Tsujimoto and Shimizu, 2000). Bcl-2 can prevent release of cytochrome c from mitochondria, thus, precluding the apoptotic cascade (Kluck et al., 1997; Yang et al., 1997). Bcl-2 can block apoptosis induced by several viruses, including influenza virus and reovirus (Nencioni et al., 2003; Rodgers et al., 1997). Existing data on Bcl-2 in SFV- or Sindbis virus-induced apoptosis are contradictory. On one hand it has been shown that alphavirus-induced apoptosis of baby hamster kidney (BHK) cells, Chinese hamster ovary cells, rat insulinoma cells and rat prostatic adenocarcinoma (AT3) cells can be prevented by over-expression of Bcl-2 (Levine et al., 1993; Lundstrom et al., 1997; Mastrangelo et al., 2000; Scallan et al., 1997). Similarly, a Sindbis virus expressing Bcl-2 produces reduced encephalitis in infected mice (Levine et al., 1996). That Bcl-2 expression can block apoptosis, suggests involvement of intrinsic pathway of apoptosis. In contrast, other studies using rat embryo fibroblasts and monocyte cell lines overexpressing Bcl-2 failed to detect a protective effect against alphavirus-induced apoptosis (Grandgirard et al., 1998; Murphy et al., 2001).\nThe aim of this study was to determine whether expression of anti-apoptotic Bcl-2 directly from SFV-based replicon vectors in BHK-21 cells could be used to prolong co-expression of marker proteins from a bicistronic SFV replicon. Using the SFV1 vector system (Liljestrom and Garoff, 1991), the bcl-2 gene was placed either under the control of a duplicated SFV subgenomic promoter or an internal ribosome entry site (IRES). It is possible that expression of Bcl-2 from the subgenomic promoter occurs too late to prevent cell death. Expression from an IRES element within the genomic RNA should be more rapid. We tested two different IRES elements, the Encephalomyocarditis virus IRES (EMCV-IRES) and the crucifer-infecting tobamovirus IRES (CR-IRES). The latter is a 148-nt element, which precedes the CR coat protein gene and displays IRES activity across all kingdoms (Dorokhov et al., 2002). Using this novel approach we demonstrate that early Bcl-2 expression does not protect SFV-infected BHK-21 cells from alphavirus-induced translational shutdown or cell death. Moreover, our results indicate that SFV-induced cell death in BHK-21 cells does not involve the release of cytochrome c from mitochondria, and most likely does not occur by the apoptotic intrinsic pathway.\n2\nMaterials and methods\n2.1\nPlasmid construction\nThe BamHI-XmaI multicloning site of the pSFV1 replicon (Liljestrom and Garoff, 1991) was replaced with a BamHI, ApaI, ClaI, AvrII, NruI, NsiI and XmaI multicloning site; the resulting construct was designated as pSFV-PL. The spliced sequences encoding the mouse Bcl-2 alpha protein (locus AAA37282), the EMCV-IRES (pIRES2-EGFP; BD Clontech) and the 148\u00a0bp CR-IRES (Ivanov et al., 1997) were amplified by PCR, cloned and verified by sequence analysis. Each IRES was fused to the Bcl-2 coding sequence and cloned into NsiI-XmaI digested pSFV-PL vector; obtained constructs were designated as pSFV-EMCV-bcl2 and pSFV-CR-bcl2. To create constructs expressing Bcl-2 protein from the duplicated subgenomic promoter, the IRES from pSFV-EMCV-bcl2 was replaced by an oligonucleotide duplex representing the minimal SFV subgenomic promoter (Hertz and Huang, 1992); the resulting construct was designated pSFV-PR-bcl2. The d1EGFP reporter gene (BD Clontech) was amplified by PCR, sequenced and cloned into pSFV-PL, pSFV-EMCV-bcl2, pSFV-CR-bcl2 and pSFV-PR-bcl2 vectors treated with ClaI-NsiI. Resulting constructs were designated as pSFV-PL-d1EGFP, pSFV-d1EGFP-EMCV-bcl2, pSFV-d1EGFP-CR-bcl2 and pSFV-d1EGFP-PR-bcl2, respectively (Fig. 1). Sequences and primers are available upon request.\nTo construct SFV replicons expressing mutated chromoprotein HcRed (from the reef coral Heteractis crispa), HcRed was PCR amplified (from pHcRed1-N1; BD Clontech), cloned and sequenced. The sequence encoding Bcl-2 from pSFV-d1EGFP-EMCV-bcl2, pSFV-d1EGFP-CR-bcl2 and pSFV-d1EGFP-PR-bcl2 was replaced with HcRed to give constructs pSFV-d1EGFP-EMCV-HcRed, pSFV-d1EGFP-CR-HcRed and pSFV-d1EGFP-PR-HcRed (Fig. 1).\nTo obtain constructs used for viability analysis under puromycin selection, the sequence encoding d1EGFP from pSFV-PL-d1EGFP, pSFV-d1EGFP-EMCV-bcl2, pSFV-d1EGFP-CR-bcl2 and pSFV-d1EGFP-PR-bcl2 was replaced by that of puromycin acetyltransferase (Pac), and constructs were designated pSFV-PL-Pac, pSFV-Pac-EMCV-bcl2, pSFV-Pac-CR-bcl2 and pSFV-Pac-PR-bcl2.\nTo generate infectious RNA, constructs were linearised by SpeI digestion and in vitro transcription was carried out as previously described (Karlsson and Liljestrom, 2003).\n2.2\nCells and viruses\nBHK-21 cells were grown in Glasgow's Minimal Essential Medium containing 5% foetal calf serum, 0.3% tryptose phosphate broth, 0.1\u00a0U\/ml penicillin and 0.1\u00a0\u03bcg\/ml streptomycin. AT3-neo and AT3-bcl2 cells were grown in Roswell Park Memorial Institute-1640 medium containing 10% foetal calf serum, 0.1\u00a0U\/ml penicillin and 0.1\u00a0\u03bcg\/ml streptomycin. All cells were grown at 37\u00a0\u00b0C in a 5% CO2 atmosphere. SFV4 was derived from the infectious cDNA clone pSP6-SFV4 (Liljestrom et al., 1991).\n2.3\nTransfection and collection of virus-like particles (VLPs)\nBHK-21 cells were co-transfected with equal amounts of vector and helper RNA (Liljestrom and Garoff, 1991). Helper RNA encodes the structural proteins under subgenomic promoter. Transfected cells were grown at 28\u00a0\u00b0C for 72\u00a0h and the VLPs collected, concentrated, purified and titrated as described by Karlsson and Liljestrom (2003). Although the replicase encoded by the replicon vector will amplify both RNAs, helper RNA is not packed into VLPs due to a missing packaging signal. To determine if any replication-proficient viruses were formed due to the recombination between replicon vector RNA and helper RNA, batches of VLPs were tested as described by Smerdou and Liljestrom (1999). All infections with VLPs were carried out in BHK-21 cells at a multiplicity of infections 10 (moi\u00a0=\u00a010) for 1\u00a0h at 37\u00a0\u00b0C.\n2.4\nMetabolic labelling\nBHK-21 cells in 35\u00a0mm diameter plates were infected with SFV VLPs as described above. Infected cells were washed twice with PBS, once with methionine and cysteine free Dulbecco's Modified Eagle Medium followed by 30\u00a0min labelling with 50\u00a0\u03bcCi\/ml of [35S]methionine and [35S]cysteine (RedivuePRO-MIX, Amersham Biosciences). After labelling, cells were washed with PBS, lysed in Laemmli buffer and analyzed by SDS-PAGE. Gels were dried under vacuum and exposed to film.\n2.5\nImmunoblot analysis\nBHK-21 cells were infected and samples collected as described above. After SDS-PAGE, proteins were transferred to a nitrocellulose membrane, probed with rabbit polyclonal antisera against SFV nsP1, EGFP (in-house), HcRed (Clontech), with a mouse monoclonal antibody against Bcl-2 (Santa Cruz Biotechnology, Inc.) or with a mouse monoclonal antibody against beta-actin (C4) (Cruz Biotechnology, Inc.) and visualized by ECL immunoblot detection kit (Amersham Life Science).\n2.6\nAnalysis of bicistronic SFV vector cytotoxicity in BHK-21 cells\nCytotoxicity of SFV vectors was analyzed as described by Garmashova et al., 2006. Replicon RNA was obtained from pSFV-PL-Pac, pSFV-Pac-EMCV-bcl2, pSFV-Pac-CR-bcl2 and pSFV-Pac-PR-bcl2. 106 BHK-21 cells were electroporated with 5\u00a0\u03bcg of RNA. Cells were seeded into wells (growth area 2.0\u00a0cm2 per well; Cellstar, Greiner bio-one plates) and selected with puromycin (10\u00a0\u03bcg\/ml) from 6\u00a0h post-transfection. Viable adherent cells were determined at 6, 24, 48 and 72\u00a0h post-transfection using Trypan blue (Flow Laboratories).\nThe viability of infected cells was also analyzed by WST-1 assay (Roche). The assay is based on the reduction of WST-1 to a water-soluble formazan dye by viable cells. BHK-21 cells were seeded in 96-well plates (7\u00a0\u00d7\u00a0103\u00a0cells\/well), grown for 18\u00a0h and infected with VLPs containing recombinant replicons at moi\u00a0=\u00a010. Control cells were mock-infected. Infected cells were analyzed 6, 24 or 48\u00a0h post-infection (p.i.) by adding 10\u00a0\u03bcl of WST-1 to each well, followed by incubating the plate for 1\u00a0h and measuring the change in color intensity at 450\u00a0nm in a microplate reader.\n2.7\nImmunofluorescence microscopy\nCells were grown on cover slips and infected for selected times, mock-infected cells were used as controls. Cells were washed with PBS, fixed with 4% paraformaldehyde for 10\u00a0min at room temperature and permeabilized with cold methanol for 7\u00a0min at \u221220\u00a0\u00b0C. Cells were then washed with PBS, blocked in the 3% BSA-PBS and incubated for 1\u00a0h with primary antibody (mouse anti-cytochrome c monoclonal antibody (BD Pharmingen) or rabbit polyclonal antibody against SFV nsP1). Then the cells were washed again with PBS and incubated with a AlexaFluor 568 (Invitrogen) or Cy3 conjugated secondary antibody for 1\u00a0h, washed three times with PBS and air-dried. Staurosporine (final concentration 0.5\u00a0\u03bcM) was added to mock-infected cells 1\u20132\u00a0h before fixing to induce release of cytochrome c from mitochondria. Samples were analyzed on an Olympus U-RFL-TX microscope or a Bio-Rad MRC-1024 confocal microscope.\n2.8\nAnalysis of the viability of AT3 cells infected with SFV VLPs\n3\u00a0\u00d7\u00a0106 AT3-neo or AT3-bcl2 cells were infected with VLPs in serum-free RPMI1640 media supplemented with 0.2% BSA. The amount of VLPs used for infections corresponded to a moi\u00a0=\u00a020 for BHK-21 cells. At 12\u00a0h p.i., EGFP positive cells were separated using a BD FACSAria cell sorter; EGFP-positive cells were seeded in 24-well plates. Viable cells were determined at 24 and 48\u00a0h p.i. using Trypan blue (Flow Laboratories).\n3\nResults\n3.1\nExpression of d1EGFP and Bcl-2 using bicistronic SFV vectors\nTo study the expression of foreign proteins using the bicistronic replicons, BHK-21 cells were infected with SFV-d1EGFP-CR-bcl2, SFV-d1EGFP-EMCV-bcl2 and SFV-d1EGFP-PR-bcl2 VLPs and in addition, with monocistronic SFV-PL-d1EGFP VLPs. Samples were collected at 2, 4, 6, 8, 12 and 24\u00a0h p.i. and analyzed by immunobloting. Expression of SFV nsP1 was generally detectable by 2\u00a0h p.i. and increased up to 8\u201312\u00a0h p.i. (Fig. 2a\u2013e). The amount of nsP1 was approximately equal for all bicistronic vectors (Fig. 2b\u2013d). Expression of d1EGFP was also detectable by 2-4\u00a0h post-infection (Fig. 2a\u2013d) and was highest in SFV-PL-d1EGFP infected cells (Fig. 2a). Major differences were observed with Bcl-2 expression, which was detected on immunoblots as two bands (Fig. 2b\u2013d). Bcl-2 expression was strongest and earliest (2\u00a0h p.i.) in SFV-d1EGFP-EMCV-bcl2 infected cells (Fig. 2c). In cells infected with SFV-d1EGFP-CR-bcl2 (Fig. 2b), expression of Bcl-2 was detected 4\u00a0h p.i. Expression of Bcl-2 was also found in cells infected with SFV-d1EGFP-PR-bcl2 (Fig. 2d) although levels were lower compared to IRES containing constructs.\nTo assess the ability of the bicistronic vectors to express proteins in general, Bcl-2 sequences were replaced with non-cytotoxic HcRed. The resulting replicons SFV-d1EGFP-EMCV-HcRed, SFV-d1EGFP-CR-HcRed and pSFV-d1EGFP-PR-HcRed were used to infect BHK-21 cells. There was no significant difference in the expression of nsP1 and d1EGFP proteins between bicistronic vectors also expressing HcRed or Bcl-2 (Fig. 2e for SFV-d1EGFP-EMCV-HcRed, data not shown for SFV-d1EGFP-CR-HcRed and pSFV-d1EGFP-PR-HcRed). Expression of HcRed was strongest for SFV-d1EGFP-EMCV-HcRed (detectable by Western blotting by 4\u00a0h p.i.; Fig. 2e). This is in agreement with previous observations suggesting that the highest expression levels of the second target proteins are achieved with SFV-d1EGFP-EMCV vectors. The apparent delay in detection of protein expression (2\u00a0h p.i. for Bcl-2 versus 4\u00a0h p.i. for HcRed) is most likely due to the quality of the HcRed antibody (Clontech).\nThus, vectors containing IRES elements expressed Bcl-2 earlier and to higher levels. Presence of a duplicated subgenomic promoter or IRES element following the Bcl-2 encoding sequence did not have any major effect on the time-course of infection, however, differences in expression levels of the first marker protein, d1EGFP, were observed.\n3.2\nEffects of infections by recombinant SFV VLPs on host cell protein synthesis\nMetabolic labelling was used to study the effects of infection by SFV VLPs on host cell protein synthesis. Infected BHK-21 cells were pulse labelled at 2, 4, 6, 8, 12 and 24\u00a0h p.i. In contrast to mock-infected cells, where protein synthesis is ongoing (Fig. 3A), translation in infected cells was rapidly inhibited (Fig. 3B\u2013E). Shutdown of host protein synthesis was detected as early as 4\u00a0h p.i. (Fig. 3B\u2013E). High-level and continuous expression of Bcl-2 was visible in cells infected with SFV-d1EGFP-EMCV-bcl2 (Fig. 3D). In cells infected with other bicistronic vectors, expression of Bcl-2 could only be detected by Western blot (Fig. 2b\u2013d). These results indicate that high-level, continuous expression of Bcl-2 from a bicistronic replicon vector does not protect against shut-off of host cell protein synthesis.\n3.3\nSurvival of cells infected with Bcl-2 expressing bicistronic replicons\nIt has been shown that similar to wild-type virus, alphavirus based replicon vectors induce apoptotic death of infected vertebrate cells (Glasgow et al., 1997, 1998; Scallan et al., 1997) and that over-expression of Bcl-2 can protect cells against alphavirus-induced apoptosis (Lundstrom et al., 1997; Scallan et al., 1997).\nTo study the effect of Bcl-2 expression from bicistronic vectors on cell death, BHK-21 cells were transfected with SFV-PL-Pac, SFV-Pac-EMCV-bcl2, SFV-Pac-CR-bcl2 and SFV-Pac-PR-bcl2 replicons or mock-transfected, and puromycin selection was applied 6\u00a0h post-transfection. Almost all mock-transfected cells died within 24\u00a0h of adding puromycin (Fig. 4a). The transfected cells survived longer and the percentage of viable adherent cells was found to be similar at the selected time points (Fig. 4a). Most of the transfected cells were dead 3 days post-transfection regardless of the replicon. Thus, our results demonstrate that high-level expression of Bcl-2 does not protect BHK-21 cells against SFV induced death. Similar results were also obtained when Annexin-V PE conjugate or propidium iodide were used to label apoptotic and necrotic cells (data not shown).\nThe WST-1 assay, used to analyze cell viability, measures mitochondrial activity in cells. Results obtained with this assay were similar to cell survival experiments: expression of Bcl-2 from bicistronic vectors (SFV-d1EGFP-CR-bcl2, SFV-d1EGFP-EMCV-bcl2 or SFV-d1EGFP-PR-bcl2) did not provide any protective effect (Fig. 4b). In fact, viability of cells infected with bicistronic replicons at 48\u00a0h p.i. was reduced, compared to cells infected with SFV-PL-d1EGFP (Fig. 4b). Infection with SFV-d1EGFP-EMCV-bcl2 caused the most important reduction in cell viability, which is in accordance with our previous data. (Fig. 4a). This tendency was also seen when cells infected with SFV-d1EGFP-EMCV-HcRed, SFV-d1EGFP-CR-HcRed and SFV-d1EGFP-PR-HcRed VLPs were analyzed by WST-1 assay (data not shown), indicating that not Bcl-2 expression, but rather the second gene expression unit (especially EMCV-IRES) is responsible for this effect. Taken together, our results suggest that Bcl-2 expression does not have a detectable effect on the viability of SFV-infected BHK-21 cells.\n3.4\nSFV infection does not induce release of cytochrome c from mitochondria\nThe anti-apoptotic effect of Bcl-2 is connected to its ability to prevent the release of cytochrome c from mitochondria (Kluck et al., 1997; Yang et al., 1997). Apoptosis induced by death receptors or by ER stress bypasses this mitochondrial pathway and is, therefore, relatively insensitive to protection by Bcl-2 (Scaffidi et al., 1998). The finding that high-levels of Bcl-2 expression did not have a protective effect against SFV induced death suggests that apoptosis in SFV-infected BHK-21 cells may not involve the mitochondrial pathway. To determine whether the mitochondrial pathway is activated in SFV-infected BHK cells, localization of cytochrome c was visualized by immunofluorescence in BHK-21 cells infected with the SFV-PL-d1EGFP 4 and 24\u00a0h p.i. Localization of cytochrome c in mock-infected BHK-21 cells was mitochondrial as evidenced by dotty localization (Fig. 5A). In cells, treated with the non-selective protein kinase inhibitor staurosporine, cytochrome c staining was diffuse, demonstrating its release from mitochondria (Fig. 5B). In cells infected with SFV-PL-d1EGFP, the cytochrome c staining maintained a granular pattern 4\u00a0h p.i. (Fig. 5C and D), indicating that when shut-off of cellular gene expression starts cytochrome c was not released from mitochondria. Furthermore, the cytochrome c localization pattern was unchanged even at 24\u00a0h p.i. (Fig. 5E and F). We conclude that induction of cell death in SFV replicon-infected BHK-21 cells does not involve the release of mitochondrial cytochrome c. This result is consistent with the absence of a protective effect of Bcl-2.\nSFV replicons lack the coding sequences for structural proteins. To analyze the role of the structural proteins in the release of cytochrome c from mitochondria, BHK-21 cells were infected with SFV4 (moi\u00a0=\u00a01) and localization of cytochrome c was determined at 4, 12 and 24\u00a0h p.i. By 4\u00a0h p.i. infected cells did not release cytochrome c (Fig. 6A and B). At 12 and 24\u00a0h p.i. detection of cytochrome c became more difficult due to extensive cytopathic effects. This includes cell rounding and re-localization of mitochondria into the perinuclear region (Fig. 6C\u2013F); all these effects were more extensive than in SFV-PL-d1EGFP infected cells (Fig. 5). Nevertheless, the localization pattern of cytochrome c remained dotty indicating that its mitochondrial localization was preserved (Fig. 6C\u2013F). Taken together, these results indicate that for both replicon vector and SFV4 infection, BHK-21 cells do not release cytochrome c from the mitochondria.\n3.5\nBcl-2 expression does not protect SFV-infected AT3-neo cells against cell death\nTo analyze whether the absence of cytochrome c release and lack of the protection against SFV induced cell death are specific to BHK-21 cells, two lines of AT3 cells, AT3-neo (Cepko et al., 1984) and AT3-Bcl2 (Levine et al., 1993) cells were also characterized.\nFirstly, AT3-neo cells were infected with SFV4 and cytochrome c distribution was determined. Infected AT3-neo cells showed extensive virus-induced cytopathic effects but as with BHK-21 cells no detectable release of cytochrome c (Fig. 7). Similarly, no detectable release of cytochrome c was observed in AT3-neo or AT3-bcl2 cells infected with SFV-d1EGFP-EMCV-bcl2 (Fig. 8A\u2013D and I\u2013L) or SFV-PL-d1EGFP VLPs (Fig. 8E\u2013H and M\u2013P). Therefore, we conclude that the inability of SFV to induce cytochrome c release from mitochondria was not a BHK-21 cell line-restricted phenomenon.\nSecondly, the viability of the AT3-neo and AT3-bcl2 cells, infected with SFV VLPs was analyzed. It should be noted that we were unable to obtain highly efficient infection of the AT3-neo and especially the AT3-bcl2 cells by use of the SFV VLPs. Typically, up to 20% of AT3-neo and 10% of AT3-bcl2 cells were infected when the amount of VLPs, corresponding to moi\u00a0=\u00a020 in BHK-21 cells, was used for infection; using larger amounts of VLPs did not result in higher percentages of infected cells. Thus, we conclude that AT3 cells are not as susceptible for SFV infection as BHK-21 cells and sorting of EGFP positive (i.e. infected) cells before the viability analysis was required. This revealed that, in the case of AT3-neo cells, expression of Bcl-2 by any SFV replicon vector did not provide any protective effect and almost all infected cells were dead by 48\u00a0h p.i. (Fig. 9a). Thus, the results were highly similar to those obtained in BHK-21 cells. In contrast, we found that almost all AT3-bcl2 cells, infected with SFV VLPs, were viable at 24\u00a0h p.i. and over the next 24\u00a0h numbers of viable cells rapidly increased in all samples. This indicates efficient cell division (Fig. 9b). Since cell sorting ruled out the possibility of non-infected cells being present in analyzed samples, we concluded that AT3-bcl2 cells, infected with SFV replicons were either able to recover from infection or, alternatively, established a persistent infection. Our observation that surviving AT3-bcl2 cells rapidly lost EGFP fluorescence favours the first option. Expression of Bcl-2 by SFV replicons had little or no effect on survival and subsequent division of infected AT3-bcl2 cells. All results taken together we conclude that not bcl-2 expression as such but some other property of AT3-bcl2 cells is likely responsible for this phenomenon.\n4\nDiscussion\nPreviously published data on mechanisms of alphavirus-induced cell death are controversial (Glasgow et al., 1997; Griffin, 2005; Li and Stollar, 2004). On the one hand, induction of apoptotic cell death via the death receptor pathway has been suggested (Li and Stollar, 2004; Nava et al., 1998); on the other hand, it has been shown that over-expression of the anti-apoptotic Bcl-2 can protect against alphavirus-induced apoptosis and pathogenesis (Levine et al., 1993; Lundstrom et al., 1997; Mastrangelo et al., 2000; Scallan et al., 1997). However, in these studies different alphaviruses, strains, cell lines and experimental systems were used. It is possible that mechanisms, by which cell death is induced may differ between viruses, cells etc.\nIn this study, shut-off of cellular translation and cell death in SFV-infected BHK-21 cells were studied using a novel bicistronic replicon vector system. This avoids transient or stable expression of Bcl-2 in host cells. In transiently transfected cells, cell metabolism can be seriously altered by the transfection procedure (Lepik et al., 2003). Stable expression of Bcl-2, known to have oncogenic properties (Reed, 1994; Reed et al., 1991), may lead to adaptation of the cell, which in turn might affect virus replication and the outcome of infection. It is also important to mention that both transient and stable cellular expression are mediated by cellular RNA polymerase II and are, therefore, subjected to the alphavirus-induced transcriptional shutdown, which starts early in infection.\nIn this study, Bcl-2 was expressed using novel vectors containing IRES elements. The use of IRESs has significant advantages over other systems. The first important advantage, as shown by our results (Fig. 2), is that IRES-mediated expression of Bcl-2 can take place directly from incoming genomic RNA. In contrast, expression from the subgenomic promoter requires replication and is activated later on in infection. Early expression of Bcl-2 may be crucial since it is not known when and how cell death is induced. It is possible that expression of Bcl-2 from the subgenomic promoter is too late to protect from cell death. Another important advantage of IRES-mediated expression is the level of expression, which is significantly higher than from the minimal subgenomic promoter (Fig. 2). It is also important to mention that at least for the EMCV-IRES, synthesis of Bcl-2 was resistant to inhibition of cellular translation (Fig. 3D).\nDetection of Bcl-2 protein by immunoblotting in BHK-21 cells infected with VLPs of SFV1 vectors harboring bcl-2 revealed a lower molecular band, which increased in intensity proportional to Bcl-2 expression. This is presumably protein expressed from an internal initiation site (Tsujimoto and Croce, 1986). The truncated protein could also be the product of Bcl-2 cleavage by endogenous caspase-3, resulting in curtailed Bcl-2 with proapoptotic activity (Cheng et al., 1997; Kirsch et al., 1999). The latter localizes to mitochondria and causes the release of cytochrome c, thus, promoting further caspase activation as a part of a positive feedback loop (Kirsch et al., 1999). It has been shown that alphaviruses induce apoptosis in Bcl-2-overexpressing cells by caspase-mediated proteolytic inactivation of Bcl-2 (Grandgirard et al., 1998). Viral capsid protein was suggested to trigger activation of the cell death machinery. However, in this study neither release of cytochrome c nor increase in the proportion of truncated product due to feedback loop action was observed. Infection of cells with VLPs eliminated the possibility that capsid protein alone triggers apoptosis.\nSimultaneous expression of two target proteins was achieved with all the bicistronic SFV expression vectors constructed in this study. The expression level of d1EGFP was slightly reduced for bicistronic vectors, compared to the monocistronic SFV-PL-d1EGFP, independent of the nature of the second gene (bcl-2 or HcRed) (Fig. 2). Interference between the native subgenomic promoter and the duplicated promoter or IRES element may have influenced expression from the first promoter. It is also possible that presence of the IRES elements had some effect on replication. However, infectivity of bicistronic vectors was not altered. Taken together, these results indicate that different IRES elements can be used to construct novel and efficient bicistronic SFV vectors.\nIn this study, we show that early and high-level of Bcl-2 expression, achieved with bicistronic SFV replicon vectors, did not have a detectable effect on host protein synthesis shut-off (Fig. 3) or cell death (Fig. 4). This cannot be attributed to the delayed expression of bcl-2 since its high expression levels were observed at early time points (Fig. 2). These findings are coherent with our data suggesting that infection of BHK-21 cells with SFV replicons or with SFV4 does not cause the release of cytochrome c from mitochondria (Figs. 5 and 6). This effect was not restricted to BHK-21 cells since similar results were also obtained for AT3-neo (Figs. 7 and 8) and AT3-bcl2 (Fig. 8) cells. The release of cytochrome c in SFV-infected BHK-21 or AT3-neo cells did not even occur at late time points. This explains the lack of protective effect by Bcl-2 expression against cell death in infected cells, as observed by us.\nAt the same time, the mechanism of cell death in infected cells remains unknown. We can rule out that binding of virions to the cellular receptor and\/or virus internalisation have crucial roles, since induction of cell death does not depend on whether the cells are infected with virus or transfected by infectious transcripts. It seems most likely that cell death is induced by some non-mitochondrial pathway. This is consistent with previous data, which suggested that apoptosis in alphavirus infected cells uses the death receptor pathway (Nava et al., 1998; Li and Stollar, 2004). However, our findings do not exclude the possibility that cell death in SFV-infected BHK-21 cells may be triggered by the ER pathway.\nA delay of cell death in SFV-infected AT3 cell line expressing bcl-2 has been reported (Scallan et al., 1997). Our results confirm that the AT3-bcl2 cell line is remarkably resistant to infection by SFV VLPs. However, our data is more coherent with the hypothesis that these cells are not just delaying cell death but are also capable to recover from SFV infection. The finding that Bcl-2 expression by SFV replicons did not have any effect on the survival of AT3-neo cells suggests that resistance of the AT3-bcl2 cells to the SFV infection most likely represents an indirect effect of Bcl-2. The Bcl-2 gene is potent oncogene and its constitutive expression significantly changes cell cycle and gene expression. It is indeed evident from data presented in Fig. 9 that AT3-bcl2 cells grow much more rapidly than AT3-neo cells. It could be hypothesized that constitutive expression of Bcl-2 in AT3-bcl2 cells may have resulted in changes protecting cells from virus infection. The most likely candidates for roles of these factors may be the components of the innate immune system and\/or host factor(s) like rat zinc-finger antiviral protein, which provides resistance against alphavirus infection (Bick et al., 2003).","keyphrases":["bcl-2","cell death","semliki forest virus","alphaviruses","at3"],"prmu":["P","P","P","P","P"]}
{"id":"Ann_Biomed_Eng-4-1-2239251","title":"Biomechanical Analysis of Reducing Sacroiliac Joint Shear Load by Optimization of Pelvic Muscle and Ligament Forces\n","text":"Effective stabilization of the sacroiliac joints (SIJ) is essential, since spinal loading is transferred via the SIJ to the coxal bones, and further to the legs. We performed a biomechanical analysis of SIJ stability in terms of reduced SIJ shear force in standing posture using a validated static 3-D simulation model. This model contained 100 muscle elements, 8 ligaments, and 8 joints in trunk, pelvis, and upper legs. Initially, the model was set up to minimize the maximum muscle stress. In this situation, the trunk load was mainly balanced between the coxal bones by vertical SIJ shear force. An imposed reduction of the vertical SIJ shear by 20% resulted in 70% increase of SIJ compression force due to activation of hip flexors and counteracting hip extensors. Another 20% reduction of the vertical SIJ shear force resulted in further increase of SIJ compression force by 400%, due to activation of the transversely oriented M. transversus abdominis and pelvic floor muscles. The M. transversus abdominis crosses the SIJ and clamps the sacrum between the coxal bones. Moreover, the pelvic floor muscles oppose lateral movement of the coxal bones, which stabilizes the position of the sacrum between the coxal bones (the pelvic arc). Our results suggest that training of the M. transversus abdominis and the pelvic floor muscles could help to relieve SI-joint related pelvic pain.\nIntroduction\nThe human body uses an ingenious 3-D framework of bones, joints, muscles, and ligaments for posture and movement. In upright posture, the trunk load passes the sacroiliac joints (SIJ). The orientation of the SIJ surfaces, however, is more or less in line with the direction of loading, which induces high shear forces between sacrum and coxal bones.34 The SIJ have a strong passive, viscoelastic ligamentous system for providing stability. These ligaments are vulnerable for creep during constant trunk load and need to be protected against high SIJ shear forces.19 From a biomechanical point of view, an active muscle corset that increases the compression force between the coxal bones and the sacrum could protect the ligamentous system and support the transfer of trunk load to the legs and vice versa. Interlocking of the SIJ may be promoted by transversely oriented muscles, e.g., M. transversus abdominis, M. piriformis, M. gluteus maximus, M. obliquus externus abdominis, and M. obliquus internus abdominis, which has been described as self-bracing.33\u201335 However, due to the complex lines of action of (counteracting) muscles and ligaments in the pelvic region, it is difficult to demonstrate the contribution of transversely oriented muscles to SIJ stability, in vitro as well as in vivo.\nIn the past, a number of biomechanical models of the lumbosacral region (spine and pelvis) have been developed to study the aetiology of low back pain (LBP) in relation to (over)loading of the lumbar spine1, 9, 11, 36 and the pelvis.10,27 Most of these models dealt with mechanical stability in terms of muscle3,4,18 and compression forces between (lumbar) vertebrae.2,8,12,20,22, 28 A different approach is to relate LBP to overloading of SIJ and nearby ligaments, for example the iliolumbar ligaments.24,30 The load transfer through the SIJ was studied using a static, 3-D biomechanical simulation model based on the musculoskeletal anatomy of the trunk, pelvis, and upper legs.15 This simulation model calculates forces in muscles, ligaments, and joints that are needed to counterbalance trunk weight and other external forces. It was shown that this simulation model underestimated antagonistic muscle activity, but a good agreement was found for agonist muscle activity. The number of passive structures in the model was small, for example no joint capsules were incorporated. Therefore, the model was only valid for postures in which none of the joints were near an end position. The aim of the present study was to determine which muscles have to become active in the 3-D pelvic simulation model when there is an imposed reduction of the vertical SIJ shear force.\nMaterials and Methods\nThe 3-D Simulation Model\nThe present study was performed using the validated, 3-D simulation model as described by Hoek van Dijke et\u00a0al.15 The model is based on the musculoskeletal anatomy of the trunk, pelvis, and upper legs, including muscle and ligament attachment sites, cross-sectional areas of muscles and the direction of muscle, ligament, and joint reaction forces. The geometry of this model was based on structures extracted from MRI slices and from previously published data on lumbar spine8 and upper leg4,17 geometry. Figure\u00a01a illustrates, in frontal and median view, the bones on which muscle and ligament forces act in the simulation model. These are the lowest thoracic vertebra, five lumbar vertebrae, the sacrum, the left and right coxal bones and the left and right femurs. The vertebrae are treated as a single structure. The arrangement of the bones depends on the static posture for which muscle forces are calculated, for example standing with or without trunk flexion. A description of the model equilibrium, its optimization scheme and validation of some of the parameters is presented in the Appendix. In the present study, we focus on the compression and shear forces in the SIJ. These forces are represented as perpendicular vectors. The normal vector of the SIJ surface has an oblique direction (xyz\u00a0=\u00a00.365, \u00b10.924, 0.114). Compression force is defined along this normal vector and can only vary in magnitude. One of the two components of the SIJ shear force was defined in the YZ-plane (xyz\u00a0=\u00a00, \u00b10.123, 0.992). This force is denoted as the vertical SIJ shear force. Directions of the SIJ compression force and the SIJ vertical shear force in the YZ-plane are shown in Fig.\u00a01a, left panel. Figure\u00a01b shows the vectors representing the most important muscle and ligament forces in the pelvic region superimposed on the bones. Bone shapes are for illustration purpose only; they are not part of the simulation model. In total, the model contains 100 vectors for muscle forces, 8 vectors for ligament forces, and 22 vectors for joint forces; see Table\u00a01 for a list of all the structures.\nFigure\u00a01Panel (a) shows the bones on which the muscles, ligaments and joint reaction forces act in the frontal plane (left) and the median plane (right): the lowest thoracic vertebra, five lumbar vertebrae, the sacrum, the left and right coxal bones, and the left and right femurs. The coordinate system is defined with the origin halfway between the rotation centers of the hip joints. Axes: x posterior, y left, z vertical. Panel (b) shows, superimposed on the bones, the vectors representing the most important force components in the frontal plane (left) and the median plane (right), see also Table\u00a01. The labels in this panel refer to a selection of the muscle structures listed in Table\u00a01Table\u00a01List of muscles, ligaments, and joints and the number of vectors describing the forces used in the simulation model on transferring trunk load from lumbar spine via the pelvis to the upper legs (unilateral), see also Fig.\u00a01Name of structureNumber of elementsRemark1M. adductor brevis2Upper and lower muscle2M. adductor longus13M. adductor magnus3Upper, middle and lower muscle4M. biceps femoris15M. coccygeus1Pelvic floor muscle6M. iliococcygeus1Pelvic floor muscle7M. pubococcygeus1Pelvic floor muscle8M. gemellus inferior19M. gemellus superior110M. gluteus maximus2Femur\u2014sacrum and ilium muscle11M. gluteus maximus facia2Ilium\u2014femur and trunk muscle12M. gluteus medius3Upper, middle, and lower muscle13M. gluteus minimus3Upper, middle, and lower muscle14M. gracilis115M. iliacus116M. longissimus117M. iliocostalis118M. multifidus119M. obliquus externus abdominis2Ventral and dorsal muscle20M. obliquus internus abdominis2Ventral and dorsal muscle21M. obturatorius externus122M. obturatorius internus123M. pectineus124M. piriformis125M. psoas2Upper and lower muscle26M. quadratus femoris127M. quadratus lumborum5Sacrum\u2014rib12, L1, L2, L3 and L4 muscle28M. rectus abdominis129M. rectus femoris130M. sartorius131M. semimembranosus132M. semitendinosus133M. tensor fasciae latae134M. transversus abdominis1AIliolumbar ligament1Transversal planeBPosterior sacroiliac ligament1Transversal planeCSacrospinal ligament1DSacrotuberous ligament1IL5-S1 joint3Shear (two directions) and compressionIISI joint3Shear (two directions) and compressionIIIHip joint3Shear (two directions) and compressionIVKnee joint3Shear (two directions) and compressionVPubic symphysis1Compression\nSimulations and Data Analyses\nA first simulation, with the model in standing posture and a trunk weight of 500\u00a0N, showed that the vertical shear SIJ force was 563\u00a0N on each side of the sacrum. To find the muscles that promote sacroiliac joint stability, the maximum value for the vertical SIJ shear force was decreased in steps of 30\u00a0N (\u223c5% of the initial vertical SIJ shear force). Theoretically, lowering of the imposed vertical SIJ shear force to 0\u00a0N could induce a non-physiological equilibrium between muscle, ligament, and joint forces. In addition, when the model was set in 30\u00b0 flexion, the force in the iliolumbar, the sacrotuberal, and the posterior sacroiliac ligaments was 250\u00a0N. This value was set as a maximum physiological ligament force in the simulation model in the upright position to prevent overloading of the pelvic ligaments that were implemented in the model. The following criteria were defined to warrant a physiological solution for muscle and ligament forces.Muscle tension must not exceed 240\u00a0kPa16;Lowering of the maximum vertical SIJ shear force must result in reduction of the total SIJ shear force (combination of vertical and horizontal shear);Ligament force must not exceed 250\u00a0N.\nA muscle was included for further analysis when it produced at least 15% of the maximum muscle stress during the simulation. For all muscles, the maximum muscle stress depended on the calculated minimum muscle stress (see optimization criterion 1 in the Appendix). Two muscle groups were analyzed separately: (1) the muscles that increased at least 80% in force after the first simulation step and (2) the muscles that increased at least 10 times in force after completion of the simulation series.\nResults\nTable\u00a02 summarizes the muscle (de)activation pattern when the maximum vertical SIJ shear force was stepwise decreased. Initially, vertical SIJ shear force was 563\u00a0N (on each side of the sacrum) at a trunk load of 500\u00a0N. The angle between the normal direction of the SIJ surface and the direction of the total SIJ reaction force was 81\u00b0, indicating that mainly vertical shear force acted through the SIJ, see Fig.\u00a02a. Force equilibrium was mainly achieved by activation of M. abdominal oblique (internus and externus), M. iliacus, M. psoas, M. rectus abdominis, M. rectus femoris, M. tensor fasciae latae, and loading of the sacrotuberous ligament.\nTable\u00a02Summary of the structures that stabilize the sacroiliac joints in terms of lowered shearReduction of sacroiliac shearInitialPreset value (N)SIJ (vertical shear)563533503473443413383353323StructuresForce (N)M. adductor longus9181818189M. coccygeus1111241020M. iliococcygeus1111241020M. pubococcygeus126M. gluteus medius (lower)711131415274430M. gluteus medius (middle)58111010102941M. gluteus medius (upper)4290M. gluteus minimus (lower)333347M. gluteus minimus (middle)51011111151012M. gluteus minimus (upper)8171818175111625M. iliacus4747505458688885102M. obliquus externus abdominis2118171412M. obliquus internus abdominis15202023273429M. obturatorius externus1544458M. pectineus8181818186M. piriformis1826282522M. psoas (lower)64504627M. rectus abdominis272729313437517683M. rectus femoris343436394249646250M. sartorius16171819162214M. tensor fasciae latae313133353844588579M. transversus abdominis3556721325382Iliolumbar ligament53250250Posterior sacroiliac ligament26496273147250250250Sacrospinal ligament3815015914714513210674Sacrotuberous ligament20615121SIJ (compression)92121130142154229473607633SIJ (horizontal shear)\u2212132\u2212141\u2212160\u2212154\u2212142\u2212154\u2212208\u2212226\u2212233Total SIJ shear579551528497465441436419398Angle of SIJ reaction force (\u00b0)817876747263433532Maximum muscle tension (kPa)37373942455369125247Included are those muscles that produced at least 15% of the maximum muscle stress after each simulationThe muscles printed italic increased at least 80% in force after the first simulation step. The muscles printed bold increased at least 10 times in force after completion of the simulation seriesFigure\u00a02Directions of the force in the frontal plane exerted by the right ilium through the SIJ on the sacrum as a reaction to trunk load, Ftrunk. Panel (a): initial loading condition without limitation of the vertical shear component (563\u00a0N, see under \u201cinitial\u201d in Table\u00a02). This condition led to loading of the sacrotuberal ligaments, Fsacrotuberal lig. (solid thick line). Panel (b): loading condition with the vertical shear component preset at a 120\u00a0N lower level than the initial value (see under 443\u00a0N in Table\u00a02). This condition led to loading of the sacrospinal ligaments, Fsacrospinal lig. (solid thick line). Panel (c): loading condition with the vertical shear component preset at a 240\u00a0N lower level than the initial value (see under 323\u00a0N in Table\u00a02). In this situation, SIJ compression force increased by \u223c400%, mainly by M. transversus abdominis, Ftransversus abdominis, and the pelvic floor, Fpelvic floor, muscle forces. The location of these muscles is schematically drawn by the thick solid lines, including the M. pubococcygeus, the M. iliococcygeus and the M. coccygeus (as drawn from the mid to the lateral position). It also led to loading of the iliolumbar ligaments to the maximum allowed force Filiolumbar lig. of 250\u00a0N, (solid thick line). 3-D images copyright of Primal Pictures Ltd. http:\/\/www.primalpictures.com\nWhen the maximum vertical SIJ shear force was decreased from 563 to 443\u00a0N in steps of 30\u00a0N, the SIJ compression force increased by about 70%. Force equilibrium was obtained, amongst others, by activation of some of the muscles with a hip flexion component (M. adductor longus, M. iliacus, M. pectineus, and M. sartorius, M. rectus femoris) and some of the counteracting hip extensors (MM. gluteus medius and minimus and M. piriformis). Most of these muscles became (more) active after we lowered the maximum vertical SIJ shear force by 30\u00a0N. This led to unloading of the sacrotuberous ligaments and loading of the sacrospinal ligaments. The angle between the normal direction of the SIJ surface and the direction of the total SIJ reaction force was reduced to 72\u00b0, indicating that a combination of reduced vertical SIJ shear and increased SIJ compression could balance the trunk load on the sacrum, see Fig.\u00a02b.\nFurther stepwise reduction of the vertical SIJ shear force resulted in a sharp rise of the maximum muscle stress. The simulation series ended with exceeding the maximum physiological muscle stress when the vertical SIJ shear force was decreased to about 60% of its initial value. Surprisingly, activation of some of the hip flexors and extensors had decreased or even disappeared. This was not the case for the MM. gluteus medius and minimus. In this simulation, force equilibrium was obtained by activation of the transversely oriented M. transversus abdominis (ventral to the SIJ) and the pelvic floor muscles, i.e., the M. coccygeus, the M. iliococcygeus, and the M. pubococcygeus (caudal to the SIJ). This resulted in further reduction of the angle between the normal direction of the SIJ surface and the direction of the total SIJ reaction force to 35\u00b0, see Fig.\u00a02c. This indicates that the SIJ compression force, which increased by about 400% and the reduced vertical SIJ shear force, now clamped the sacrum between the coxal bones, see Fig.\u00a02c. The MM. gluteus medius and minimus contributed to some extent to this increased compression due to a distinct force component in the transverse direction. To maintain force equilibrium, increased SIJ compression led to loading of the iliolumbar and posterior sacroiliac ligaments to the preset maximum value of 250\u00a0N.\nDiscussion\nIn the present study, the simulation model predicted muscle and ligament forces in the pelvic region when there was an imposed reduction of the vertical SIJ shear force. Initially, the forces acting through the SIJ were mainly vertical shear forces, see Fig.\u00a02a. These forces were not only caused by trunk load, but also by muscles that acted in the longitudinal direction of the spine, for example the M. psoas and M. rectus abdominis. As a result of the forward bending moment, the sacrotuberous ligament was loaded. This large ligament protects the SIJ against excessive flexion of the sacrum relative to the coxal bones. The controlled reduction of the vertical SIJ shear force with 30\u00a0N forced some muscles that act as hip flexors and hip extensors to become active. Due to their transverse orientation, especially the MM. gluteus medius and minimus and M. piriformis contributed to the increased compression force between the coxal bones and the sacrum. However, these muscles did not contribute enough to self-bracing of the SIJ, because the total force through the SIJ still mainly acted in vertical direction. When the vertical SIJ shear was further reduced to about 60% of its initial value, the simulation model predicted that self-bracing mainly resulted from the transverse muscles ventrally (M. transversus abdominis) and caudally (pelvic floor) to the SIJ. In this situation, some of the hip flexors and extensors reduced in activity, for example the M. piriformis. Although the M. piriformis has a transverse orientation and crosses the SIJ, its contribution was minimized by the simulation program because this muscle also induces vertical SIJ shear force. The pelvic floor muscles, the M. coccygeus and M. pubo-, and iliococcygeus, contribute to the stabilization with respect to the sacrum. It has been suggested that this stabilization by force closure has an analogy with a classical stone arc.33 When sideways displacement of both ends of the arc is opposed, mechanical equilibrium of the stones is achieved by compression forces and not by shear forces. In the pelvis, the pelvic floor muscles may help the coxal bones to support the sacrum by compression forces, while shear forces between sacrum and coxal bones are minimized, see Fig.\u00a03. Note that the SI compression force is defined as the force acting perpendicular to the SIJ surface. Therefore, decreasing or increasing this force will not alter the shear forces. The articular surfaces of the SIJ are irregular which results in bony interdigitation in the SI joint space. SIJ shear force calculated in the simulation model thus reflects the combination of real joint friction and friction due to this intermingling of bones. The real joint friction forces may be extremely small considering the extremely low coefficients of friction between the articular surfaces. The majority of the shear force is effectuated as normal contact pressures due to the bony interdigitation in the SI joint space. It was not possible to calculate the percentage of shear in terms of joint friction force. This requires a more detailed description of the SIJ surfaces.\nFigure\u00a03Analogy of pelvic bones supporting the trunk with a classical stone arc. The M. transversus abdominis and the pelvic floor muscles caudal to the SIJ mainly oppose lateral movement of the coxal bones. Spinal loading is transferred mainly by compression forces through the SIJ to the coxal bones and further down to the legs. 3-D images copyright of Primal Pictures Ltd. http:\/\/www.primalpictures.com\nThe simulation model predicts that simultaneous contraction of the M. transversus abdominis and pelvic floor muscles, i.e., the M. coccygeus, the M. iliococcygeus, and the M. pubococcygeus, contribute to lowering of the vertical SIJ shear forces, increasing of the SIJ compression and hence increasing of the SIJ stability. We emphasize that this simulation model was set up to estimate the forces acting in the pelvic region under static conditions and that the outcome of the simulations must be interpreted with caution.5 Nevertheless, in a previous study co-contraction was shown of pelvic muscles and M. transversus abdominis.26 This result and the prediction of our simulation model suggest that a protective mechanism against high SIJ shear forces may exist in humans. This mechanism has been investigated in vivo and in vitro. The contribution of the M. transversus abdominis to SIJ stability was shown in an in vivo study in patients with LBP.25 An in vitro study in embalmed human pelvises showed that simulated pelvic floor tension increased the stiffness of the pelvic ring in female pelvises.23 It is worthwhile to further investigate the contribution of both muscle groups simultaneously, not only during stiffness measurements of the SIJ but also during lumbo-pelvic stability tests based on increased intra-abdominal pressure (IAP). It was shown that the pelvic floor muscles, in combination with abdominal muscles and the diaphragm, may control and\/or sustain IAP to increase lumbar spine stability as well.7,14\nIn the present study, the ligament forces were not allowed to exceed 250\u00a0N. The distribution between muscle and ligament forces depended on the maximum muscle stress as formulated in the first optimization scheme as presented in the Appendix. Increasing the maximum ligament forces might result in a lower maximum muscle stress, which could lead to a different muscle activation pattern to stabilize the SIJ. A small sensitivity test, however, showed that when the ligament forces were allowed to exceed the 250\u00a0N up to 500\u00a0N and in a next step up to 750\u00a0N, the model calculated a similar muscle activation pattern. The outcome of the present study also depended on the choice of optimization criteria and the magnitude of the cross-sectional areas of the muscles. The influence of different criteria was previously investigated for muscle forces in the leg.21 Indeed, various choices led to different calculated forces, but the obtained solutions were qualitatively similar, as was the case in our model. When we developed the model, other optimization criteria were also tested, for example minimization of the sum of muscle forces. However, minimization of the sum of squared muscle stresses yielded the most plausible solutions. The model cannot account for anatomical variations or detailed variation in muscle attachment sites. Obviously, direct comparison between the model predictions and the outcome of in vivo force measurements in the SIJ are not available, so there is no data to confirm the outcome of the present study. Nevertheless, EMG recordings of (superficial) abdominal and back muscles in various postures showed higher M. abdominal oblique internus activity when standing upright than resting on one leg and tilting the pelvic backwards.33 This muscle is considered as one of the self-bracing muscles of the SIJ. It was hypothesized that when standing on one leg, the shear load on the contralateral SIJ is diminished. Posterior tilt of the pelvis with less lumbar lordosis may than lead to less M. psoas major muscle load on the spine meaning less shear load on the SIJ. These findings indirectly support our findings that transversely oriented muscles reduce SIJ shear forces. We emphasize that the present model served as a tool to investigate the general relations between muscle and ligament forces in the pelvic region. The present simulations results may lead to the development of a new SIJ stabilizing training-program to reduce pain induced by high SIJ shear forces. The effectiveness of such a program, however, can only be tested with an intervention study.\nThe simulation model predicted unloading of the sacrotuberous and loading of the iliolumbar and posterior sacroiliac ligaments when the vertical SIJ shear was forced to reduce. This loading of the dorsal ligaments resulted from the absence of transversely oriented muscles at the dorsal side of the SIJ to counterbalance activation of the M. transverse abdominis at the ventral side of the SIJ. Loading of the iliolumbar ligament has been related to LBP.24 It was shown that in sitting position, the stepwise backward movement of an erect trunk (from upright position into a slouch) resulted in forward flexion of the spine combined with backward tilt of the sacrum relative to the pelvis.32 It was shown, that this movement into a sudden or sustained slouch might cause loading of the well-innervated iliolumbar ligaments near failure load.31 The co-contraction that exists between the deep abdominal M. transversus abdominis and the deep back extensor M. multifidus presumably retains lumbo-pelvic stability.13 In the future, we intend to extend the model with co-contraction between the M. transversus abdominis, the M. multifidus, and the pelvic floor muscles to study prevention of (over)loading of pelvic ligaments at different static postures.\nConclusions\nEffective stabilization of the SIJ is essential in transferring spinal load via the SIJ to the coxal bones and the legs. A biomechanical analysis of the upright standing posture showed that activation of transversely oriented abdominal M. transversus abdominis and pelvic floor, i.e., M. coccygeus and M. pubo- and iliococcygeus muscles would be an effective strategy to reduce vertical SIJ shear force and thus to increase SIJ stability. The force equilibrium in this situation induced loading of the iliolumbar and posterior sacroiliac ligaments. The M. transversus abdominis crosses the SIJ and clamps the sacrum between the coxal bones. Moreover, the pelvic floor muscles oppose lateral movement of the coxal bones, which stabilizes the position of the sacrum (the pelvic arc).","keyphrases":["sacroiliac joints","pelvis","pelvic floor muscles","static forces","human posture"],"prmu":["P","P","P","R","R"]}
{"id":"Mod_Rheumatol-4-1-2275302","title":"Gene therapy for arthritis\n","text":"Arthritis is among the leading causes of disability in the developed world. There remains no cure for this disease and the current treatments are only modestly effective at slowing the disease's progression and providing symptomatic relief. The clinical effectiveness of current treatment regimens has been limited by short half-lives of the drugs and the requirement for repeated systemic administration. Utilizing gene transfer approaches for the treatment of arthritis may overcome some of the obstacles associated with current treatment strategies. The present review examines recent developments in gene therapy for arthritis. Delivery strategies, gene transfer vectors, candidate genes, and safety are also discussed.\nIntroduction\nRheumatoid arthritis (RA) is the most common inflammatory disorder, affecting approximately 0.5\u20131% of the North American adult population. It causes significant pathology and functional impairment in affected individuals. RA is a multifactorial disease whose main risk factors include genetic susceptibility, sex and age, smoking, infectious agents, hormones, diet, and socioeconomic and ethnic factors [1]. Those afflicted by RA report a decrease in quality of life measures such as persistent pain, functional disability, fatigue, depression, and an inability to perform daily tasks [2]. RA is also a significant burden on the health care system, averaging between $2,800\u2013$28,500 per patient per year for direct and indirect costs in developed countries [3]. Although the pathogenesis of RA is not completely understood, specific HLA-DR genes, autoantibody and immune complex production, T cell antigen-specific responses, networks of cytokine production, and a hyperplastic synovium have all been shown to play a role.\nRA primarily affects diarthrodial joints of the hands and feet. These joints are normally lined by a thin cell layer (1\u20133 cells) of both type I (macrophage-like) and type II (fibroblast-like) synoviocytes. In RA, the joint is inflamed and the synovium becomes hyperplastic, creating a pannus of synovial tissue comprised of CD4+ T cells, B cells, mast cells, dendritic cells, macrophages, and synoviocytes that invade and destroy nearby cartilage and bone. Neutrophils accumulate in the synovial fluid and also contribute to the destructive processes [4, 5]. Macrophages and fibroblast-like synoviocytes (FLS) secrete inflammatory cytokines, such as TNF\u03b1, IL-1\u03b2, and IL-6, all of which contribute to cartilage and bone destruction. They have been implicated as major contributors to many aspects of RA, including inflammatory cell infiltration, fibrosis, and T cell proliferative responses [6, 7]. These cytokines are thought to act primarily through MAP kinase and nuclear factor \u03baB (NF\u03baB) signaling pathways to activate transcription factors that turn on genes for chemokines and cell adhesion molecules, along with extracellular matrix degrading enzymes like matrix metalloproteinases (MMPs). Chemokines and chemokine receptors have also been linked to RA [6, 8, 9].\nInitial treatment strategies for RA include drugs such as non-steroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids. While providing pain relief and decreased joint swelling, these drugs are unable to stop the progression of RA [10]. This led to the use of small molecules like methotrexate that were demonstrated to slow disease progression. For many decades these disease-modifying agents of rheumatic disease (DMARDs) were the best treatment option for RA. In recent years, a further understanding of the disease process has led to the development of biologic DMARDs primarily aimed at neutralizing the effects of pro-inflammatory cytokines. The most successful agents in this class are anti-TNF-\u03b1 molecules and IL-1\u03b2 blocking agents. Based on improvement criteria set forth by the American College of Rheumatology (ACR), these drugs are more effective than treatment with methotrexate alone. Even so, less than half of the patients show improvement of at least 50% in their ACR scores. In addition, the half-life of these drugs is relatively short and they require frequent systemic administration in order to be effective [11]. Gene transfer strategies have the potential to overcome some of these limitations, potentially leading to increased efficacy and decreased frequency of administration.\nGene delivery strategies\nAlthough the majority of inflammation in RA is localized to the joints, there are systemic components to the disease. Therefore, when developing treatment strategies, one must consider whether the therapy should be delivered locally or systemically. Local administration is attractive because it has less potential for side effects and the treatment is delivered directly to the joint, the main site of inflammation. However, systemic features of the disease would seem to be left untreated. Interestingly, several researchers have observed a \u201ccontralateral effect\u201d in animal models of local gene delivery where the delivered transgene is protective not only to the injected joint, but also to distal, untreated joints [12\u201314]. This effect appears to be independent of trafficking of modified immune cells to distal joints, because ex vivo modified fibroblasts alone are still able to confer a contralateral effect [15]. It is also independent of systemic circulating levels of transgene and non-specific immunosuppression and instead was found to depend on a complex antigen-specific mechanism [16]. Systemic delivery, typically by intravenous administration, would be expected to have a broader therapeutic effect, but is also associated with an increase in side effects and toxicity.\nAnother consideration for gene transfer is whether to employ in vivo or ex vivo delivery strategies. In vivo strategies have the advantage of being relatively easy and less expensive. In addition, many more studies have been performed in animal models looking at in vivo gene delivery. Ex vivo strategies, although expensive and time-consuming, have the advantage of being able to treat and select very specific cells, avoiding the possibility that the gene transfer vector could genetically modify a stem cell population and result in oncogene activation. A phase I clinical trial has been performed using a retroviral vector to deliver IL-1 receptor antagonist (IL-1ra) to cultured autologous synovial fibroblasts [17]. The cells were then injected into the RA patients' joints. After a scheduled arthroplasty 1 week later, significant expression of the transgene was seen in the injected joints. No adverse events were reported. This trial was initiated following previous experiments where antagonists of both IL-1\u03b2 and TNF-\u03b1 were delivered to autologous cultured rabbit fibroblasts ex vivo and injected into arthritic rabbit knee joints, with significant therapeutic benefit [15, 18].\nMost ex vivo studies use fibroblast-like synoviocytes (FLS) as the target cell. These cells have been targeted specifically because they are thought to be directly responsible for cartilage destruction and drive and perpetuate the inflammatory response and autoimmunity [19]. The disadvantages of using this cell type are that FLS have a low proliferation rate, lack highly specific surface markers, and are a non-homogenous population. Future studies may examine other strategies, including gene delivery to T cells, dendritic cells, muscle cells, or mesenchymal stem cells [20].\nGene transfer vectors\nGene transfer vectors can be broadly categorized into two groups: viral and non-viral vectors. In general, viral vectors tend to provide for longer-term gene expression but often come with additional safety concerns, ranging from fears of generating replication competent virus during vector production, random insertion of the transgene into the genome following treatment, or development of a harmful immune response.\nPlasmid DNA\nThe most common non-viral vector used in arthritis studies is plasmid DNA. Plasmid DNA can be delivered by liposomes, gene gun, or direct injection of the plasmid. The use of plasmid DNA tends to be less toxic and less immunogenic than the use of viral vectors and is also easy and relatively inexpensive to produce. However, plasmid DNA often leads to low transfection efficiency and short-term expression of the transgene, lasting only 1\u20132\u00a0weeks [21\u201323]. These limitations make it unlikely that local delivery of plasmid DNA in the joint will be successful.\nThe most success with the use of plasmid DNA in gene transfer for arthritis has been garnered through delivery of transgenes to skeletal muscle. Electrotransfer of soluble TNF-\u03b1 receptor I variants to the tibial-cranial muscle at the onset of collagen-induced arthritis (CIA) led to a decrease in the clinical and histological signs of disease for up to 5\u00a0weeks [24]. Similarly, plasmids encoding cDNA for other anti-inflammatory molecules such as IL-1ra and a soluble TNFR-Fc fusion protein have been demonstrated to improve both macroscopic and microscopic scores of CIA when delivered intramuscularly [25, 26]. Plasmid encoding TGF-\u03b2 delivered to skeletal muscle delayed progression of streptococcal cell wall induced arthritis when administered at the peak of the acute phase and virtually eliminated subsequent inflammation and arthritis when given at the beginning of the chronic phase of the disease [27]. Intramuscular injections of plasmids encoding immuno-modulatory molecules such as IL-4, IL-10, viral IL-10, and soluble complement receptor type I have given similar results [28\u201331]. Intramuscular delivery of a plasmid encoding TIMP-4, and inhibitor of matrix metalloproteinases, completely abolished the development of arthritis in a rat adjuvant-induced arthritis model [32]. Intravenous delivery of a plasmid encoding the heparin-binding domain of fibronectin inhibited leukocyte recruitment and decreased inflammation in CIA [33]. Intra-dermal injection of plasmid encoding IL-10 and intra-peritoneal injection of plasmid IL-10\/liposome complexes have also been demonstrated to delay onset and progression of CIA [34, 35]. The liposome delivered DNA was able to maintain expression for only 10\u00a0days after injection, significantly less than the intramuscular studies mentioned above. Recently, the use of chitosan, a polycationic polysaccharide derived from crustacean shells, has been shown to act as an efficient gene carrier to rabbit knee joints both in vitro and in vivo [36].\nOther non-viral vectors\nOther non-viral gene delivery systems that have potential in the treatment of RA are the artificial chromosome expression (ACE) system and the sleeping beauty (SB) transposon system. The ACE system is attractive because it is non-integrating and can provide stable and long-term gene expression of one or multiple genes. A feasibility study was recently performed in a Mycobacterium tuberculosis rat arthritis model, which demonstrated that rat skin fibroblasts could be modified ex vivo to express a reporter gene from an artificial chromosome. These cells, when subsequently injected into rat joints, demonstrated engraftment into the synovial tissue microarchitecture and detectable transgene expression. The ACE system did not induce local inflammation at the injection site that is often associated with viral vector administration [37]. The sleeping beauty transposon system melds the advantages of both viral and non-viral vectors, allowing for both integration into the genome and long term expression. No studies have yet been performed in arthritis models using the sleeping beauty transposon system, but success has been found in both cancer and hemophilia models, suggesting that it might be have potential to successfully treat arthritis as well [38].\nViral vectors are by far the most widely used vectors for delivering transgenes in arthritic animal models [39]. There are several different viral vectors that have been examined for use in gene transfer for arthritis, including adenovirus, retrovirus, adeno-associated virus (AAV), and lentivirus, each with their respective advantages and disadvantages.\nAdenovirus\nAdenovirus is a non-encapsidated double-stranded DNA virus that can infect non-dividing cells and can be produced at high titers. Many gene-therapy studies have been performed with this vector but it has several limitations that may prevent it from being successful in the clinic. The high prevalence of neutralizing antibodies may prevent successful administration or re-administration. Injected adenovirus also causes a significant inflammatory immune response, which is a safety concern. In addition, adenovirus vectors typically only allow for 1\u20133\u00a0weeks of transgene expression, which would limit its long-term efficacy. Some improvements to adenoviral vectors have recently been made in an effort to improve delivery of transgenes to the synovium. FLS lack the coxsackie-adenovirus receptor (CAR) and are not efficiently transduced by adenovirus. By modifying the fiber knobs on the virus, adenoviral transgene delivery to synoviocytes and synovium was improved dramatically [40, 41]. Other recent improvements include the development of an adenoviral vector with an inflammation inducible promoter [42]. This would allow expression of the transgene during active disease, but expression would turn off once inflammation was brought under control.\nRetrovirus\nRetroviruses, mostly derived from the Moloney murine leukemia virus, have a relatively simple genome and structure. They are enveloped viruses and contain two identical copies of their RNA genome. The key feature of the retroviral life cycle is the ability of the RNA genome to be reverse transcribed into double-stranded DNA, which can then randomly integrate into the genome. They have been mostly used in ex vivo studies and are desirable vectors for several reasons. They can provide for long-term stable expression and their integration into the genome makes it possible to permanently correct a genetic defect [43]. For arthritis in particular, the inflamed synovium appears to be more susceptible to uptake of the virus [18]. The drawbacks to retroviral vectors are that they only infect non-dividing cells and are produced at low titers. The fact that these vectors integrate into the genome randomly is also a concern. In fact, in a recent clinical trial in France using a retrovirus to correct an X-linked SCID disorder, 3 out of 10 children developed leukemia after the vector inserted in or near a known oncogene. As a result, similar trials in the U.S. for this disorder have been halted until more information can be gathered [44, 45]. Future improvements to these vectors, including the development of self-inactivating vectors, which contain no retroviral promoter or enhancer elements, and use of vectors from non-oncogenic retroviruses will hopefully make them safer for clinical use [43].\nLentivirus\nLentivirus vectors are derived from retroviral vectors but have the advantage of infecting non-dividing cells. The most commonly studied lentiviral vectors are derived from either human immunodeficiency virus (HIV) or feline immunodeficiency virus (FIV), although equine anemia infectious virus and visna virus have also been examined [38]. The primary concern with HIV vectors is safety. In contrast, FIV is non-pathogenic in humans and does not cause serologic conversion [46]. Using an FIV vector, TNF-\u03b1 was transduced into primary human FLS with high efficiency. When injected into knees of SCID mice, these cells induced cell proliferation and caused bone and joint destruction [47]. A replication-defective HIV vector encoding endostatin was injected into joints of TNF-\u03b1 transgenic mice and was shown to decrease synovial blood vessel density and decrease the overall arthritis index [48]. Similarly, intra-articular expression of angiostatin inhibited the progression of CIA in mice [49]. A study examining a VSV-G pseudo-typed HIV vector, which has increased host range and stability, demonstrated that a transgene could be efficiently delivered to the synovium of rat knee joints, with transgene expression lasting up to 6\u00a0weeks in immunocompromised animals [50].\nAAV\nOne of the most promising gene transfer vectors is AAV, which is a small, non-enveloped single-stranded DNA virus with broad tissue tropism. It belongs to the Parvoviridae family and has a 4.68kb genome. AAV normally requires adenovirus or herpesvirus to produce active infection. Several serotypes have been identified in primates, with AAV2 being the prototype for most gene transfer studies. Heparan sulfate proteoglycan has been identified as the primary attachment receptor for AAV, with fibroblast growth factor receptor 1 and integrin \u03b1v\u03b25 acting as co-receptors [51]. Although little is known about the details of AAV infection, some of the basic mechanisms have been described. The virus enters the cell by receptor-mediated endocytosis. Acidification of late endosomes leads to AAV release into the cytosol, with subsequent translocation of the virus to a perinuclear region [52\u201354]. The virus then enters the nucleus by an unknown mechanism that is independent of the nuclear pore complex [55]. Following uncoating, the single-stranded genome is converted to a double strand and the viral DNA integrates into chromosome 19 (AAVS1 locus) in a site-specific manner [56, 57].\nAAV is an attractive vector for gene transfer studies for several reasons. It has been shown to deliver transgenes to a wide variety of tissues, has low immunogenicity, and mediates long-term gene expression [51]. Recombinant, replication incompetent AAV vectors have been designed that lack the Rep genes, which are required for integration, so long-term expression with these vectors is thought to be mediated by episomal viral DNA [58]. In addition, AAV vectors have been designed that are able to package double-stranded viral genomes, bypassing a rate-limiting step of viral transduction (second-strand synthesis) and allowing rapid and highly efficient transduction both in vitro and in vivo [59].\nSeveral studies have demonstrated the efficacy of AAV vectors in arthritis models. Primary and recurrent arthritis were suppressed following a single injection of AAV encoding IL-1ra into knee joints of rats with LPS-induced arthritis. Surprisingly, disease-regulated expression of the transgene was observed [60]. Our laboratory has recently observed a similar phenomenon in in\u00a0vitro cultured FLS infected with AAV in which inflammatory cytokines can increase transgene expression in these cells in a regulatable, PI3K-dependent manner. Protesosome inhibition has also been shown to enhance AAV transduction of human synoviocytes both in vitro and in vivo [61]. AAV encoding soluble TNF-\u03b1 receptor type I decreased synovial cell hyperplasia and cartilage and bone destruction in human TNF-\u03b1 transgenic mice injected intra-articularly with the virus [62]. Intra-articular or peri-articular delivery of AAV encoding IL-4 in CIA mice was also shown to decrease paw swelling, protect from cartilage destruction, and delay the onset of CIA [63, 64]. A vIL-10 transgene delivered by AAV under control of a tetracycline inducible promoter decreased the incidence and severity of CIA on a macroscopic, radiologic, and histologic level [65]. More recently, angiostatin, an anti-angiogenic molecule, was demonstrated to efficiently decrease development of CIA in the treated joint when delivered by AAV [66].\nAAV vectors are in clinical trials for the treatment of cystic fibrosis and hemophilia B and preliminary results are promising [67]. Targeted Genetics Corporation (Seattle, WA) is currently conducting a phase I clinical trial (13G01; identifier NCT00126724) to assess the safety of using an AAV2 vector to deliver a soluble TNF receptor-Fc fusion gene in RA.\nGene transfer strategies\nIL-1\u03b2 inhibition\nSeveral gene transfer strategies have been aimed at neutralizing the effects of IL-1\u03b2. IL-1\u03b2 communicates with many different cell types in the joint. Its action on these cells leads to emigration of blood cells to the synovium, increased cartilage destruction, and increased production of other chemokines and pro-inflammatory mediators by macrophages, B cells, and T cells [7]. Neutralization of this key cytokine has proven beneficial in the treatment of RA. Several animal models support using gene transfer to block the effects of this cytokine. Primary and recurrent arthritis were suppressed following a single injection of AAV encoding IL-1ra into knee joints of rats with LPS-induced arthritis [60]. Adenoviral vectors encoding IL-1ra proved more effective than soluble type I TNF receptor-IgG fusion protein at reducing cartilage matrix degradation and decreasing leukocyte infiltration into the joint space of rabbits with antigen-induced arthritis [13]. Rabbit synovial fibroblasts modified ex vivo using a retrovirus encoding IL-1ra demonstrated a chondroprotective and mild anti-inflammatory effect when injected back into the joint after onset of antigen-induced arthritis [18]. This led to a phase I clinical trial using a retroviral vector to deliver IL-1ra to cultured human autologous synovial fibroblasts. The cells were then injected into RA patient joints. After a scheduled arthroplasty 1 week later, significant expression of the transgene was seen in the injected joints, and no adverse events were reported [17].\nIntramuscular injection of plasmid DNA encoding IL-1ra has also been shown to decrease paw swelling and arthritis incidence in a CIA mouse model. Reduced synovitis and cartilage erosion were also seen [26, 68]. In a rat model of bacterial cell wall-induced arthritis, rat synoviocytes were modified ex vivo using a retroviral vector to express IL-1ra. When injected into ankle joints prior to reactivation of arthritis, a decreased severity of arthritis and attenuated destruction of cartilage and bone was observed [69]. In a SCID mouse model, human RA FLS transduced with retrovirus encoding IL-1ra co-implanted with normal human cartilage was able prevent progressive cartilage degradation compared to controls [70]. 3T3 mouse fibroblasts transfected with plasmid encoding IL-1ra were able to prevent the onset of CIA and cartilage destruction when injected into knee joints of CIA mice [12]. Similar effects on arthritis were seen in a rabbit model of antigen-induced arthritis in which a retroviral vector carrying the IL-1ra gene was used to transduce rabbit fibroblasts ex vivo, with subsequent injection of the cells into the knee joint [15]. More recently, the soluble form of interleukin-1 receptor accessory protein (sIL-1RAcP) was delivered to CIA mice either using an adenoviral vector or by injection of plasmid transfected 3T3 cells. In both instances, a profound prophylactic effect on the development of CIA was observed [71].\nTNF-\u03b1 inhibition\nTNF-\u03b1 is another pro-inflammatory cytokine that plays a key role in the pathogenesis of RA. Many of its effects overlap those of IL-1\u03b2 listed above. Currently, therapies aimed at neutralizing this cytokine represent the most successful treatment strategies for RA. Current regimens involve receiving injections (etanercept) or infusions (infliximab) every 2 or 8 weeks, respectively. Gene transfer strategies have the potential to provide longer-term control and may also be delivered locally rather than systemically, potentially minimizing treatment side effects. Several studies in animal models have been performed. After one injection at onset of CIA, intramuscular electrotransfer of plasmids encoding soluble TNF receptor I variants led to a decrease in clinical and histological signs of CIA [24]. Expression lasted up to 5 weeks and was as least as efficient as repeated injections of the recombinant protein etanercept in controlling the disease. A similar study using a retroviral vector to deliver the transgene peri-articularly saw similar results and also observed a decrease in systemic levels of IgG2a antibodies to collagen type II [72].\nElectrotransfer of a plasmid encoding a soluble p75 TNF receptor:Fc fusion protein was also beneficial in a CIA model and was associated with a decrease in the levels of IL-1\u03b2 and IL-12 in the paw [25]. Other studies using electrotransfer or intramuscular injection of a plasmid with a doxycycline-regulated promoter to control expression of a dimeric soluble TNF receptor II molecule saw a therapeutic effect on CIA only when doxycycline was administered [73, 74]. In TNF-\u03b1 transgenic mice, intra-articular delivery of soluble TNF receptor I by adeno-associated virus led to a decrease in synovial cell hyperplasia and cartilage and bone destruction [62]. Similarly, AAV5 encoding sTNFRI-Ig was also able to decrease paw swelling in a rat AIA model when expression was under control of an inflammation responsive promoter, but interestingly, not if expression was under control of the CMV promoter [75].\nSplenocytes from arthritic DBA-1 mice can passively transfer collagen type II-induced arthritis when injected into SCID recipients. If these splenocytes were first modified ex vivo using retroviral vectors to express soluble p75 tumor necrosis factor receptor, the SCID recipients did not develop arthritis, bone erosion, or joint inflammation [76, 77]. Delivery of a rat TNF receptor:Fc fusion protein in a streptococcal cell wall-induced arthritis model by either plasmid or local or systemic administration of AAV vectors encoding the molecule led to decreased inflammation, pannus formation, bone and joint destruction, and mRNA expression of joint pro-inflammatory cytokines [21]. Adenoviral delivery of a soluble TNF receptor type I-IgG fusion protein directly to rabbit knees with antigen-induced arthritis reduced-cartilage matrix degradation and decreased leukocyte infiltration into the joint space, especially when administered in conjunction with a soluble IL-1 type I receptor-IgG fusion protein [13]. The above animal model data has led to the initiation of a phase I clinical trial using AAV vectors to deliver soluble TNF Receptor:Fc fusion protein (Targeted Genetics Corporation).\nIL-18 inhibition\nIL-18 is a pro-inflammatory cytokine that is overexpressed in the synovium of RA patients and correlates with inflammation. Elevated levels of this cytokine are also observed in serum and synovial fluid. Overexpression of an IL-18-binding protein using an adenoviral vector was able to ameliorate arthritis in a CIA model, indicating neutralization of IL-18 may be an effective target in the future treatment of RA [78].\nImmune deviation\nPreviously, an imbalance between Th1 and Th2 cytokines was thought to play a role in the pathogenesis of several inflammatory diseases, including RA. Th1 cells secrete cytokines like IFN-\u03b3 that promote a pro-inflammatory environment, while Th2 cytokines secrete cytokines like IL-4, IL-10, and IL-13 that down-regulate Th1 activity. It was previously felt that overproduction of Th1 cytokines contributed significantly to the pathogenesis of RA. More recently, a new subset of T cells, termed Th17 cells, have been identified [79]. These cells produce, among other cytokines, IL-17, a pro-inflammatory cytokine previously implicated in the pathogenesis of CIA [80]. Dysregulation of Th17 cells and IL-17 overproduction has been implicated in the pathogenesis of inflammatory diseases and the development of severe autoimmunity. In fact, the pathogenesis of RA may be more directly related to an imbalance between Th17 cells and Foxp3-positive regulatory T cells than an imbalance between Th1 and Th2 cells [81]. Regardless of the exact mechanism, several strategies aimed at immune deviation have been successful in animal models of arthritis and are outlined below.\nIL-13 inhibits activated monocytes\/macrophages from secreting a variety of pro-inflammatory molecules. A possible role for this cytokine in the pathogenesis of RA was observed when adenoviral delivery of IL-13 to RA synovial tissues explants led to a decrease in IL-1\u03b2, TNF-\u03b1, IL-8, MCP-1, NAP-78, PGE2, and MIP-1\u03b1 when compared to controls [82]. Subsequent studies demonstrated that adenoviral delivery of IL-13 directly to ankle joints in a rat antigen-induced arthritis model significantly decreased paw size, bony destruction, vascularization, inflammatory cell infiltration, and inflammatory cytokine production [83]. IL-13 overexpression during immune-complex-mediated arthritis significantly decreased chondrocyte death and MMP mediated cartilage destruction, despite the presence of enhanced inflammation [84].\nUsing IL-4 to skew the cytokine profile towards Th2, or perhaps by inhibiting Th17 cell production, has also proven successful. Adenoviral delivery of IL-4 to RA synovial tissue explants demonstrated decrease IL-1\u03b2, TNF-\u03b1, IL-8, MCP-1, and PGE2 in the cultured medium [85]. Intra-articular delivery of adenoviral vectors encoding IL-4 to CIA mice led to an enhanced onset of inflammation but less chondrocyte death and cartilage and bone erosion. Proteoglycan synthesis was enhanced and there was decreased MMP activity [86]. IL-17, IL-12, cathepsin K and osteoprotegrin ligand mRNA levels were also reduced [86, 87]. Kim et al. observed similar effects on CIA upon local and systemic administration of adenoviral vectors encoding IL-4 [88]. AAV-mediated delivery of IL-4 has also prove beneficial in CIA models [63, 64]. Electrotransfer of an IL-4 encoding plasmid prior to CIA onset decreased synovitis and cartilage destruction, with an associated decrease in IL-1\u03b2 in the paw and an increased TIMP2:MMP2 ratio [29]. Similar results were also seen using either gene gun delivery or intra-dermal administration of plasmid encoding IL-4 [89]. Both retroviral and adenoviral delivery of IL-4 in a rat antigen-induced arthritis model had beneficial effects [90\u201392]. Cell-based therapies that deliver IL-4 in arthritis models have also been successful. Injection of fibroblast transfected with plasmid encoding IL-4 decreased histologic evidence of joint inflammation and destruction in a CIA model [93, 94]. Likewise, collagen type II pulsed antigen presenting cells engineered to secret IL-4 down-regulated CIA [95].\nIL-10 is an anti-inflammatory cytokine that has demonstrated benefit in several animal models of RA. Either intramuscular administration via electrotransfer or intra-dermal injection of plasmid encoding IL-10 had beneficial effects on CIA [31, 35]. Similarly, systemic administration of a plasmid IL-10\/liposome mixture decreased signs of CIA after a single intra-peritoneal injection [34]. Viral IL-10 (vIL-10) is homologous to human and mouse IL-10 but while retaining its immunosuppressive function, it lacks many of the immunostimulatory properties of IL-10, and therefore may be a superior treatment option. An adenoviral vector encoding vIL-10 was able to decrease CIA when delivered locally or systemically [14, 96, 97]. Electrotransfer of viral IL-10 decreased histologic evidence of arthritis in an arthrogen collagen-induced arthritis model and was associated with decreased TNF-\u03b1, IL-1\u03b2, and IL-6 transcripts in the joint [30]. A tet-inducible vIL-10 transgene delivered by AAV was able to decrease macroscopic, radiology, and histologic signs of CIA only when doxycycline was administered [65].\nTGF-\u03b2 is a pleiotropic cytokine with many different effects on many different cell types and has been suggested of playing a role in RA. While some of its effects, like immunosuppression, would appear to be beneficial for RA, it has also been associated with pro-inflammatory activity. Not surprisingly, data from animal models using gene transfer support both possibilities, making its true role in RA difficult to decipher. Splenocytes from CIA mice were isolated and infected ex vivo with a retroviral vector encoding TGF-\u03b2. These cells were then injected in the intra-peritoneal cavity 5 days after arthritis onset. Without TGF-\u03b2 expression, an exacerbation of arthritis is normally observed. However, TGF-\u03b2 expressing splenocytes were able to inhibit this exacerbation and also resulted in a decrease in MMP2 activity and a transient reduction in anti-collagen type II antibodies [98]. In a rat streptococcal cell wall-induced arthritis model, intramuscular delivery of plasmid encoding TGF-\u03b2 showed significant decreases in inflammatory cell infiltration, pannus formation, bone and joint destruction, and inflammatory cytokine production [27]. In contrast to the above reports, another study found that injection of adenovirus encoding TGF-\u03b2 into the knees of rabbits with antigen-induced arthritis resulted in significant pathology in the knee joint and surrounding tissue, suggesting that TGF-\u03b2 therapy may not be suitable for treating arthritis in some models [99].\nCTLA-4Ig fusion protein binds to the co-stimulatory molecules B7-1 and B7-2 present on antigen-presenting cells and blocks CD28\/B7 interactions, resulting in decreased T cell activation. It has been shown to ameliorate several experimental autoimmune diseases, including CIA. A single intravenous injection of an adenovirus encoding CTLA-4Ig fusion protein suppressed established CIA as least as efficiently as repeated injections of monoclonal antibody to CTLA-4. Pathogenic cellular and humoral responses were also diminished in adenoviral vector treated group as compared to antibody treated and control groups [100]. CIA could also be inhibited both histologically and clinically by intra-articular administration of a low dose of adenovirus encoding CTLA-4Ig fusion protein [101].\nPromoting apoptosis\nOne of the key features of RA is the increased cellularity of the synovial lining leading to pannus formation, which has been shown to contribute to cartilage invasiveness and bone destruction. Promoting synovial apoptosis has been suggested as a treatment strategy for RA. In a rabbit model of arthritis, intra-articular adenoviral delivery of TNF-related apoptosis-inducing ligand (TRAIL) was able to increase apoptosis in the synovial cell lining, decrease inflammatory cell infiltration, and promote new matrix deposition [102]. Mice injected with collagen type II-pulsed antigen presenting cells engineered to express TRAIL under control of a doxycycline inducible promoter decreased the incidence of CIA and infiltration of T cells in the joint in the presence of doxycycline. In situ TUNEL staining demonstrated TRAIL-induced apoptosis of activated T cells in the spleen [103]. Modulation of TRAIL receptor expression on RA synoviocytes has also been suggested as a gene therapy strategy for the treatment of RA [104].\nInjection of adenovirus-expressing Fas ligand (FasL) into joints of CIA mice induced apoptosis and ameliorated CIA. IFN-\u03b3 production by collagen-specific T cells was also reduced [105]. Ex vivo modified T cells engineered to express FasL were injected into human RA synovial tissue that had been implanted in SCID mice. Analysis of the tissue following treatment demonstrated that synoviocytes and mononuclear cells present in the tissue had been eliminated by apoptosis through a Fas\/FasL interaction [106]. Similar results were observed when adenovirus encoding FasL was injected directly into the implanted tissue [107]. Dendritic cells modified by adenoviral vectors to express FasL were able to suppress CIA when systemically injected and also demonstrated decreased IFN-\u03b3 production from spleen-derived lymphocytes and decreased T-cell proliferation in response to collagen stimulation [108]. Fas-associated death domain protein (FADD) also plays a key role in Fas-mediated apoptosis of synovial cells. It was found that adenoviral vectors expressing FADD could induce apoptosis in synoviocytes both in vitro and in vivo, suggesting that this strategy may be effective in the treatment of RA [109].\nAnti-angiogenesis\nIncreased cellularity of the synovial lining is also associated with neo-vascularization in the local environment of the joint. This angiogenesis is necessary for the development and maintenance of the pannus and also provides nutrients required for the survival and proliferation of infiltrating inflammatory cells [110, 111]. A peptide targeted to the integrins present in the inflamed synovium and associated with angiogenesis was fused to an anti-apoptotic peptide. Systemic administration of this fusion peptide in a CIA model resulted in decreased clinical arthritis and increased apoptosis of synovial blood vessels [112]. 3T3 fibroblasts modified with retroviral vectors to express angiostatin were able to decrease pannus formation and cartilage erosion when injected into knee joints of mice with CIA. Arthritis-associated angiogenesis was also inhibited [113]. AAV and HIV vector-mediated delivery of angiostatin to CIA knee joints were similarly beneficial [49, 66]. The potent anti-angiogenic factor endostatin was able to decrease arthritis and reduce blood vessel density in a human TNF-transgenic mouse model of arthritis when delivered to knee joints using a lentiviral vector [48]. Tie2 has also been demonstrated to play a role in angiogenesis in arthritis. Adenoviral delivery of a soluble receptor for Tie2 resulted in a decreased incidence and severity of CIA, inhibition of angiogenesis, and was associated with decreased bone destruction that appeared to result from a decrease in RANKL [114]. VEGF is another angiogenic factor that promotes synovitis and bone destruction in arthritis. When soluble VEGF receptor I was administered via adenoviral vectors to CIA mice, disease activity was suppressed significantly [115]. Thrombospondins (TSP1 and 2) have also been shown to inhibit angiogenesis and also decrease pro-inflammatory cytokine production in animal models of arthritis [116, 117]. In addition, adenoviral gene transfer of a urokinase plasminogen inhibitor was also able to inhibit angiogenesis in a CIA model [118].\nTargeting matrix degradation enzymes\nMatrix metalloproteinases (MMPs) degrade extracellular matrix components and have been demonstrated to contribute to cartilage degradation in RA. Ribozymes and an antisense construct targeting the destruction of MMP-1 delivered to RA synovial fibroblasts via retroviral vectors decreased MMP-1 production and reduced the invasiveness of RA synovial fibroblasts in a SCID mouse model of RA, suggesting that this may be an effective approach to inhibiting cartilage destruction in RA [119, 120]. Another strategy that has proven successful is to increase the ratio of tissue inhibitors of matrix metalloproteinases (TIMPs) to MMPs. In a rat antigen-induced arthritis model, intramuscular injection of naked DNA encoding TIMP-4 completely abolished the development of the disease [32]. Likewise, adenoviral delivery of TIMP-1 and TIMP-3 to RA synovial fibroblasts significantly decreased their invasiveness both in vitro and in an in vivo SCID mouse model of RA. These molecules were found to act by both decreasing MMP production and reducing cell proliferation [121].\nTargeting NF\u03baB\nThe transcription factor NF\u03baB plays a significant role in the activation of many cytokines that contribute to the pathogenesis of RA. Inhibition of this factor could lead to therapeutic benefit in RA. Injection of decoy oligodeoxynucleotides with high affinity for NFkB into ankle joints of CIA rats significantly decreased joint swelling and joint destruction. The levels of the pro-inflammatory cytokines TNF-\u03b1 and IL-1\u03b2 were also decreased in the treated joints [122]. Another group found that inhibiting NF\u03baB in RA synovial fibroblasts using an adenovirus to deliver a dominant negative inhibitor of NF\u03baB led to an increase in apoptosis upon stimulation with TNF-\u03b1. These cells are normally resistant to apoptosis when stimulated with TNF-\u03b1, suggesting that this strategy may be beneficial in the treatment of RA [123]. In adjuvant arthritis in rats, a dominant negative IkB kinase \u03b2 (IKK\u03b2) was used to inhibit the NF\u03baB pathway. Delivering the molecule intra-articularly using an AAV5 vector resulted in significantly reduced paw swelling and decreased levels of IL-6 and TNF-\u03b1. Bone and cartilage destruction, as well as MMP-3 and TIMP-1 levels were unaffected. The same vector was also able to efficiently transduce ex vivo-cultured biopsies from joints of human RA patients. TNF-\u03b1-induced IL-6 production was significantly decreased in the ex vivo cultures receiving the vector encoding IKK\u03b2 [124].\nOther strategies\nOther molecules that have been demonstrated to play a role in arthritis using gene transfer in various in vitro or animal models are Csk, cathepsin L, fibronectin, galectin-1, p16INK4A, p21Cip1, SOCS3, soluble CR1, superoxide dismutase and catalase, Ras, and prothymosin \u03b1 [28, 33, 125\u2013136]. Vectors and genes used to successfully treat animal models of arthritis are summarized in Table\u00a01.\nTable\u00a01Summary of vectors and genes used in animal models of arthritis\u00a0AdenovirusAAVRetrovirusLentivirusPlasmid DNAGenes demonstrated to successfully treat arthritis in various animal modelsIL-1ra, sTNFRI-Ig fusion protein, sIL-1RacP IL-18-binding protein, IL-13, IL-4, vIL-10, CTLA4-Ig, TRAIL, Csk, IFN-\u03b2, p16INK4A, P21CIP1, prothymosin-\u03b1, VEGF receptor I, Tie2 soluble receptor, FADD, FasL, SOCS3, urokinase plasminogen inhibitor, TIMP-1, TIMP-3, Thromobospondin-1, dominant negative NF\u03baB inhibitorIL-1ra, sTNFR-Ig, sTNFRI, sTNFR:Fc fusion protein, IL-4, IL-10, angiostatin, IKK\u03b2IL-1ra, sTNFRI variants, IL-4, TGF-\u03b2, angiostatin, soluble complement receptor I, superoxide dismutase, catalaseAngiostatin, endostatinIL-1ra, sTNFRI receptor variants, sTNFR:Fc fusion protein, sTNFRII, TGF-\u03b2, IL-4, IL-10, vIL-10, soluble complement receptor I, TIMP-4, fibronectin peptide, sIL-1RAcP\nSafety concerns\nAs with most investigative therapies, one of the most important concerns with regards to gene transfer technologies is the issue of safety. While the FDA has outlined specific strict standards for clinical trial protocols for gene transfer studies, there have been several instances where the therapy has resulted in serious adverse events in patients enrolled in the studies. The most widely known is probably the September 1999 death of Jesse Gelsinger, who had a fatal systemic inflammatory response to adenoviral vector gene transfer [137]. Retroviral vectors have also resulted in insertional mutagenesis leading to a secondary malignancy [44]. Lentiviral vectors may have this potential as well as sharing a similar mechanism, although this has never been demonstrated in clinical studies. More recently, in July of 2007, Jolee Mohr, a patient enrolled in Targeted Genetics\u2019 clinical trial for rheumatoid arthritis using AAV vectors, died several weeks following her second injection of the experimental treatment. The trial is currently suspended, and although the investigation into her death is still ongoing, initial results suggest she died of a systemic fungal infection that may be unrelated to the injected virus. This case and the final results of the investigation are likely to have a large impact on the future of gene therapy trials in the United States, as AAV has been heralded as one of the most promising vectors in the field, precisely because it has, until now, failed to reveal significant safety concerns in previous studies.\nFuture of gene therapy for arthritis\nMuch progress has been made in the past several years in the use of gene therapy for the treatment of arthritis. However, there are many obstacles that must be overcome in order for it to become a viable treatment option. Future studies will need to address improving targeted delivery of vectors, regulating transgene expression, obtaining long-term transgene expression, and improving the safety and efficacy of the vectors already in use before gene therapy becomes a viable clinical therapy for arthritis.\nEven in light of the recent setback in clinical trials utilizing AAV vectors, the authors believe that the future of gene transfer for arthritis will rely heavily upon this vector. Once the safety issues have been clarified, these trials can hopefully move forward. Presently, AAV would seem to be the viral vector that has the best profile in terms of safety, efficacy, and level and length of transgene expression. Alternatively, strategies using siRNA technology or preventing the dysregulation of Th17 cells would appear to be emerging as treatment strategies that may one day outperform the current standards of care for rheumatoid arthritis.","keyphrases":["gene therapy","gene transfer","rheumatoid arthritis","inflammation","cytokines"],"prmu":["P","P","P","P","P"]}
{"id":"Pediatr_Nephrol-3-1-2064942","title":"Mechanisms of progression of chronic kidney disease\n","text":"Chronic kidney disease (CKD) occurs in all age groups, including children. Regardless of the underlying cause, CKD is characterized by progressive scarring that ultimately affects all structures of the kidney. The relentless progression of CKD is postulated to result from a self-perpetuating vicious cycle of fibrosis activated after initial injury. We will review possible mechanisms of progressive renal damage, including systemic and glomerular hypertension, various cytokines and growth factors, with special emphasis on the renin\u2013angiotensin\u2013aldosterone system (RAAS), podocyte loss, dyslipidemia and proteinuria. We will also discuss possible specific mechanisms of tubulointerstitial fibrosis that are not dependent on glomerulosclerosis, and possible underlying predispositions for CKD, such as genetic factors and low nephron number.\nIntroduction\nChronic kidney disease (CKD) occurs in all age groups, with an incidence in children between 1.5 per million and 3.0 per million. Renal developmental abnormalities (congenital abnormalities of the kidney and urinary tract, CAKUT) are the most common causes of CKD in children. Other diseases commonly underlying CKD in children include focal segmental glomerulosclerosis (FSGS), hemolytic uremic syndrome (HUS), immune complex diseases, and hereditary nephropathies, such as Alport\u2019s disease [1]. The incidence of diabetes, especially type\u00a02, is increasing in children. Although CKD secondary to diabetes usually does not develop until adulthood, early structural lesions of diabetic nephropathy start in childhood [2].\nCKD shares a common appearance of glomerulosclerosis, vascular sclerosis and tubulointerstitial fibrosis, suggesting a common final pathway of progressive injury [3]. Adaptive changes in nephrons after initial injury are postulated ultimately to be maladaptive, eventually causing scarring and further nephron loss, thus perpetuating a vicious cycle that results in the end-stage kidney. We will review possible mechanisms of progressive renal damage, which include, but are not limited to, hemodynamic factors, the renin\u2013angiotensin\u2013aldosterone system (RAAS), various cytokines and growth factors, podocyte loss, dyslipidemia, proteinuria, specific mechanisms of tubulointerstitial fibrosis, and possible underlying predispositions for CKD, such as genetic factors and low nephron number.\nSystemic and glomerular hypertension\nSystemic hypertension often accompanies renal disease and may both result from, and contribute to, CKD. Progression of CKD is accelerated by hypertension, and control of blood pressure is key in the treatment of CKD. In addition, the glomerulus has a unique structure, with both an afferent and an efferent arteriole, which permits modulation of glomerular perfusion and pressure without corresponding systemic blood pressure change.\nThe remnant kidney model has been extensively studied to investigate CKD [4]. In this model, one kidney and infarction\/removal of two-thirds of the remaining kidney (i.e. five-sixths nephrectomy) results in progressive hyperperfusion, hyperfiltration, hypertrophy and FSGS [4\u20136]. Additional models with initial podocyte injury, namely the puromycin aminonucleoside and adriamycin models of renal disease, show initial proteinuria and podocyte damage similar to human minimal-change disease, followed by progressive FSGS [7].\nDirect micropuncture studies have demonstrated that single nephron function was increased after renal ablation, and led to the hypothesis that hyperfiltration caused sclerosis, setting in motion a vicious cycle of hyperfiltration and glomerulosclerosis [3, 8]. Maneuvers that decreased hyperfiltration, such as low-protein diet, angiotensin\u00a0I converting enzyme inhibitors (ACEIs), lipid-lowering agents, or heparin, were, indeed, effective in ameliorating glomerular sclerosis. However, in some studies, glomerular sclerosis was decreased without altering glomerular hyperfiltration [9], and glomerular sclerosis occurred in some settings even in the absence of intervening hyperperfusion [10].\nThus, focus was shifted to glomerular hypertension as a key mediator of progressive sclerosis. Maneuvers that increase glomerular capillary pressure, such as therapy with erythropoietin, glucocorticoids, or high-protein diet, accelerated glomerulosclerosis, while decreasing glomerular pressure ameliorated sclerosis. These beneficial effects were particularly apparent in the comparison of agents such as ACEIs that preferentially decrease glomerular pressure even more than systemic BP to non-specific antihypertensive agents [11].\nRenin\u2013angiotensin\u2013aldosterone system\nThe RAAS has been the focus of investigation of progression in CKD because of the efficacy of inhibition of its components in CKD. ACEIs decrease glomerular capillary pressure by preferential dilation of the efferent arteriole [1], likely mediated by both inhibition of angiotensin\u00a0II (AngII) and especially by the effect of ACEIs in augmenting bradykinin, which is degraded by angiotensin I converting enzyme (ACE) [12]. Indeed, angiotensin type 1 receptor blockers (ARBs), which do not have this activity to increase bradykinin, do not preferentially dilate the efferent arteriole or decrease glomerular pressures to the extent of that seen with ACEIs in most experimental studies. However, both ACEIs and ARBs have shown superior efficacy in slowing progressive CKD in experimental models and in human CKD [13\u201316].\nARBs leave the angiotensin type 2 (AT2) receptor active, and may in theory even lead to augmented AT2 effects by allowing unbound AngII to bind to this receptor. The AT2 receptor counteracts some of the classic AT1 receptor actions and thus is mildly vasodilating and mediates growth inhibition and apoptosis [17\u201320]. Apoptosis often is associated with decreased injury, as injured cells are quickly removed without activation of profibrotic cytokines and chemokines. Absence of AT2 receptor actions, either by pharmacological inhibition or by genetic absence, indeed resulted in diminished apoptosis after injury, associated with increased fibrosis [21, 22].\nCombined ACEI and AT1 receptor antagonist treatment could have a theoretic advantage, allowing further blockade of AngII actions while maintaining preferential local availability of the AT2 receptor [23]. In an experimental model, combined ACEI and ARB therapy did not result in added benefit on glomerulosclerosis when compared with single-drug therapy with similar blood pressure control [24, 25]. However, addition of AT2 receptor inhibition to ARB treatment prevented the beneficial effects of ARBs  [26]. A beneficial effect of the AT2 receptor in renal injury was also demonstrated in transgenic mice overexpressing the AT2 receptor. These mice developed less severe injury than did the wild type after subtotal nephrectomy [27]. Results from small clinical studies of human CKD suggest that the combination of ARBs and ACEIs has greater effect in the decrease of proteinuria, not attributable to effects on systemic blood pressure [28, 29]. In a large study of hypertensive patients with diabetic nephropathy and microalbuminuria, combined therapy resulted in greater reduction of  blood pressure and albuminuria than did therapy with either drug alone [30]. In a Japanese study, in addition to decreased proteinuria, the slope of decline of glomerular filtration rate (GFR) improved with combination ACEI and ARB versus monotherapy [31]. However, complete dose-range comparisons of combined therapy with monotherapy were not made in these clinical trials. A recent review of clinical trials with combination therapy with ACEI and ARB in CKD patients support that such combination therapy had increased effects to decrease proteinuria without significantly increasing adverse side effects [16].\nAntifibrotic effects of combination therapy versus monotherapy could include augmented bradykinin and AT2 activity and also decreased urinary transforming growth factor (TGF)-\u03b2 [32]. In addition, there may be greater suppression of the renin\u2013angiotensin system (RAS) with combined therapy, decreasing both ligand generation by inhibition of ACE and binding of any remaining AngII to the AT1 receptor. However, even suprapharmacological doses of ACE inhibition did not achieve complete suppression of the local RAS in experimental models [33]. Similarly, patients receiving ACEIs long term still have measurable ACE in their plasma. These data support the notion that non-ACE-dependent AngII generation by chymotrypsin-sensitive generating enzyme occurs in humans. New directions under investigation include the development of renin antagonists that could obviate these obstacles to optimal inhibition of the RAAS. Renin itself may have direct effects, independent of activation of the RAAS, with renin receptor activity detected on mesangial cells [34].\nMany profibrotic actions of the RAAS are mediated directly by AngII. AngII promotes migration of endothelial and vascular smooth muscle cells, and hypertrophy and hyperplasia of smooth muscle cells and mesangial cells [35, 36]. All components of the RAS are present in macrophages, which may thus serve as yet another source of AngII and also respond to ACEI and ARB. AngII also induces other growth factors, including basic fibroblast growth factor (basic FGF), platelet-derived growth factor (PDGF) and TGF-\u03b2, and plasminogen activator inhibitor-1 (PAI-1), all of which may impact on fibrosis (see below), [37\u201339].\nImportantly, new data indicate that aldosterone has both genomic and non-genomic actions to promote fibrosis, independent of its actions to increase blood pressure by mediating salt retention [40, 41]. Aldosterone enhances angiotensin induction of PAI-1 (see below), and also has direct actions on fibrosis [40]. Conversely, aldosterone receptor antagonism with spironolactone decreased injury [40]. PAI-1 deficiency prevented aldosterone-induced glomerular injury, but interestingly did not alter cardiac or aortic injury in this mouse model, suggesting site-specific and perhaps species-specific mechanisms of aldosterone-PAI-1 mediated fibrosis [42]. In clinical trials, aldosterone antagonism has further decreased proteinuria when added to ACEI and ARB therapy [43, 44]. However, the potential risk of hyperkalemia may limit the ability to add aldosterone antagonism to angiotensin inhibition. Whether these approaches also apply to children with CKD has not been investigated.\nClearly, the RAAS has many non-hemodynamic actions and thus, doses beyond usual antihypertensive doses are potentially of additional benefit. Regression has even been achieved in experimental models with high-dose ACEI\/ARB. A shift in the balance of synthesis\/degradation of extracellular matrix (ECM) must occur to accomplish regression of sclerosis; endothelial cells must regenerate, mesangial cells must regrow, and finally, podocytes must be restored. New glomeruli cannot be generated after term birth in humans. However, remaining segments of non-sclerotic loops can give rise to more open capillary area by lengthening or branching of the remaining capillaries [45\u201348]. Recent experimental data show that regression can, indeed, be induced by high-dose ACEI or ARB or spironolactone, linked to decreased PAI-1, restored plasmin activity and capillary remodeling [25, 49\u201351]. Of note, regression was not associated with increased expression or activity of matrix metalloproteases-2 or -9 or decreased mRNA for TGF-\u03b2 or local decreases in TGF-\u03b2 expression as assessed by in situ hybridization. However, lack of changes in mRNA does not rule out that local changes in TGF-\u03b2 actions could occur, and clearly, in many systems, TGF-\u03b2 has been shown to impact on ECM accumulation. Regression is also possible in human CKD, demonstrated in principle by regression of early diabetic sclerosis and tubulointerstitial fibrosis in patients over a 10-year period when the underlying diabetes was cured by pancreas transplantation [52]. Regression of existing lesions also occurred in IgA nephropathy in response to high-dose corticosteroids and tonsillectomy [53].\nSpecific cytokines\/growth factors and progression of CKD\nNumerous cytokines\/growth factors appear to modulate progression of glomerular and tubulointerstitial scarring. These factors and their roles may differ at the various stages of injury. Altered gene expressions and\/or pharmacologic manipulations in pathophysiological settings have implicated e.g. PDGF, TGF-\u03b2, AngII, basic FGF, endothelin, various chemokines, peroxisome proliferator-activated receptor-\u03b3 (PPAR-\u03b3) and PAI-1, among others, in progressive renal scarring [10, 54\u201356]. Current state-of-the-art approaches with proteomic and array analysis of renal tissue in human CKD and in animal models can identify novel targets and markers, and even mediators of progression [57, 58]. Of these many potential molecules of interest, we will discuss only a few that have been investigated in depth.\nIncreased PAI-1 is associated with increased cardiovascular disease and fibrotic kidney disease [59]. Conversely, PAI-1 could be decreased by inhibition of AngII and\/or aldosterone, and linked to prevention of sclerosis or even regression of existing kidney fibrosis [25, 38, 51, 60]. AngII and aldosterone can also induce PAI-1 expression and subsequent fibrosis independent of TGF-\u03b2 activation [61]. Some of the effects of PAI-1 in promoting fibrosis are independent of its effects on proteolysis. PAI-1 also modulates cell migration, perhaps by its effects on vitronectin interaction [59]. Thus, PAI-1 may in some inflammatory or interstitial disease settings increase fibrosis primarily by enhancing cell migration and epithelial-mesenchymal transition (EMT). In contrast, in the glomerulus, the effects of PAI-1 in the increase of sclerosis may predominantly be due to its ability to modulate ECM turnover [59]. These data support that mechanisms of fibrosis in the interstitium and glomerulus are not identical, and involve complex interactions of parenchymal and infiltrating cells and cytokines, with variable net effects on ECM accumulation.\nTGF-\u03b2 promotes ECM synthesis and is a key promoter of fibrosis. The biological actions of TGF-\u03b2 are complex and depend not only on cell state, but also on the presence of decorin and latency-associated peptide (LAP), both of which can bind and modify its activity [37]. TGF-\u03b2 also induces both PAI-1 and AngII [62]. Animals transgenic for TGF-\u03b2 developed progressive renal disease [63]. Conversely, inhibition of either TGF-\u03b2 or PDGF-B decreased mesangial matrix expansion in the anti-Thy1 model [64, 65]. Animals genetically deficient for TGF-\u03b2 develop lymphoproliferative disease, thought to reflect a loss of TGF-\u03b2 immune regulatory effect [66]. Interestingly, pharmacologic inhibition of TGF-\u03b2 was more effective at lower dose, and with higher dose of anti-TGF-\u03b2 associated with more fibrosis and greater macrophage influx, perhaps also reflecting effects on TGF-\u03b2 immune modulation [67]. TGF-\u03b2 may promote a more fibroblastic phenotype of the podocyte, with loss of differentiation markers and de novo expression of alpha-smooth muscle actin [68]. Although TGF-\u03b2 promotes growth arrest and differentiation of podocytes at low doses, at higher doses, TGF-\u03b2 causes podocyte apoptosis, mediated by Smad\u00a07 signaling [69, 70]. Loss of podocytes (see below) is a key factor contributing to progressive kidney fibrosis.\nPPAR-\u03b3 modifies numerous cytokines and growth factors, including PAI-1 and TGF-\u03b2. PPAR-\u03b3 is a transcription factor and a member of the steroid superfamily [71]. On activation, PPAR-\u03b3 binds the retinoic acid X receptor, translocates to the nucleus and binds to peroxisome proliferator activator response elements (PPREs) in selected target genes, modifying their expression. PPAR-\u03b3 agonists, such as the thiazolidinediones, are most commonly used to treat type 2 diabetes, due to their beneficial effects to increase insulin sensitivity and improve lipid metabolism, and they have been shown to decrease diabetic injury correspondingly in diabetic animal models [72]. Interestingly, PPAR-\u03b3 agonists also have antifibrotic effects in non-diabetic or non-hyperlipidemic experimental models of CKD. PPAR-\u03b3 agonist ameliorated the development of sclerosis in these non-diabetic models, linked to decreased PAI-1 and TGF-\u03b2 and decreased infiltrating macrophages and protection of podocytes against injury [56, 73]. Further study is necessary to determine the specific role each of the above factors plays at varying stages of renal fibrosis.\nPodocyte loss\nPodocytes are the primary target in many glomerular diseases, including FSGS and the experimental models of adriamycin and puromycin aminonucleoside-induced nephropathies [74]. The podocytes are pivotal for maintenance of normal permselectivity, and are a source of matrix in both physiological and pathophysiological settings. The podocyte does not normally proliferate. Loss of podocytes after injury is postulated to be a key factor resulting in progressive sclerosis [74]. This principle was proven in experimental models in mice and rats, where podocyte-specific injury was produced by genetic manipulation of the podocytes to express toxin receptors only on this cell [75, 76]. Injection of toxin then resulted in podocyte loss, the degree of which depended on toxin dose. Animals subsequently developed progressive sclerosis. Of interest, even though only podocytes were initially injured, subsequent injury rapidly also developed in endothelial and mesangial cells, with resulting sclerosis. Even when chimeric mice were genetically engineered so that only a portion of their podocytes was susceptible to the toxin, all podocytes developed injury after toxin exposure [77]. These data show that injury can also spread from the initially injured podocyte to initially intact podocytes within a glomerulus, setting up a vicious cycle of progressive injury at the glomerular level [77].\nThe limited proliferation in the mature podocyte is accompanied by high expression of a cyclin-dependent kinase inhibitor, p27kip1, a rate-limiting step for the growth response of the podocyte [78]. Either too much or too little proliferation of the podocyte in response to genetic manipulation of p27kip1 is postulated be detrimental [79]. Inadequate growth of the podocyte is postulated to give rise to areas of dehiscence and insudation of plasma proteins, which progress to adhesions and sclerosis [80]. Another cyclin-dependent kinase inhibitor, p21, appears to be necessary for development of injury after five-sixths nephrectomy in mice, pointing to the crucial importance of cell growth responses in determining response to injury [81].\nPodocytes normally produce an endogenous heparin-like substance, which inhibits mesangial cell growth; thus, injury may decrease this growth inhibitory effect and allow increased mesangial growth. Podocytes are also the main renal source of angiopoietin-1 and vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen that plays a key role in both physiologic and pathologic angiogenesis and vascular permeability [82]. Overexpression or partial loss of podocyte VEGF results in a collapsing lesion or pre-eclampsia-like endotheliosis lesion, respectively [82].\nPodocyte genes and CKD\nNew studies of the molecular biology of the podocyte and identification of genes mutated in rare familial forms of FSGS and nephrotic syndrome, such as nephrin, WT-1, transient receptor potential cation channel-6 (TRPC-6), phospholipase C epsilon, \u03b1-actinin-4 and podocin, have given important new insights into mechanisms of progressive glomerulosclerosis. The gene mutated for congenital nephrotic syndrome, nephrin (NPHS1) is localized to the slit diaphragm of the podocyte and is tightly associated with CD2-associated protein (CD2AP) [83]. Nephrin functions as a zona occludens-type junction protein, and together with CD2AP, provides a crucial role in receptor patterning and cytoskeletal polarity and also provides signaling function of the slit diaphragm [84]. Mice with CD2AP knockout develop congenital nephrotic syndrome, similar to congenital nephrotic syndrome of Finnish type [85]. Autosomal dominant FSGS with adult onset is caused by mutation in \u03b1-actinin 4 (ACTN4) [86]. This is hypothesized to cause altered actin\u2013cytoskeleton interaction, causing FSGS through a gain-of-function mechanism, in contrast to the loss-of-function mechanism implicated for disease caused by the nephrin mutation [85]. Patients with \u03b1-actinin 4 mutation progress to end-stage by age 30\u00a0years, with rare recurrence in a transplant. TRPC-6 encodes for a cation channel, which is present in several sites including podocytes. TRPC-6 is mutated in some kindreds with familial FSGS with adult onset in an autosomal dominant pattern [87]. Podocin, another podocyte-specific gene (NPHS2), is mutated in autosomal recessive FSGS with childhood onset with rapid progression to end-stage kidney disease [88]. Podocin interacts with the CD2AP-nephrin complex, indicating that podocin could serve in the structural organization of the slit diaphragm. In some series of steroid-resistant pediatric patients with non-familial forms of FSGS, a surprisingly high proportion, up to 25%, had podocin mutations [89, 90]. However, not all patients with nephrotic syndrome caused by mutation are steroid resistant. Diffuse mesangial sclerosis in a large kindred was recently linked to a truncating mutation of phospholipase C epsilon (PLCE1), and two of those patients responded to steroid therapy [91]. However, in two patients with missense mutation of this same gene, FSGS lesions developed, demonstrating that a spectrum of structural abnormalities may arise from varying mutations in the same gene. PLCE1 is expressed in the glomerulus, where it is postulated to play a key role in development, perhaps by interacting with other proteins that are crucial for the development and function of the slit diaphragm.\nWT-1 mutation, which may occur sporadically with only FSGS or be associated with Denys\u2013Drash syndrome, was found in only 5% of steroid-resistant patients [92]. Interestingly, mutations of podocin or WT-1 were not found in relapsing or steroid-dependent pediatric patients [93]. Acquired disruption or polymorphisms of some of these complexly interacting molecules have been demonstrated in experimental models and in human proteinuric diseases. Thus, in puromycin aminonucleoside nephropathy, a model of FSGS, nephrin localization and organization were altered [94]. Similar decreases in nephrin were observed in hypertensive diabetic rat models with significant proteinuria [95]. TRPC-6, a calcium channel, was induced in various non-genetic human proteinuric diseases [96]. Conversely, treatments that ameliorated these experimental models preserved e.g. glomerular nephrin expression, providing further support for a key causal role for slit diaphragm and key podocyte molecules in proteinuria [97]. Whether polymorphisms, compound heterozygosity for mutations or merely altered distribution and\/or expression of any of these proteins contribute to proteinuria or progressive disease in various causes of CKD in humans has not been determined.\nDyslipidemia\nPatients with CKD frequently have dyslipidemia and greatly increased cardiovascular disease risk, even beyond that predicted by lipid abnormalities [98]. Abnormal lipids are important in modulating glomerular sclerosis in rats; however, analogous studies in humans are still evolving [99\u2013102]. Glomerular injury was increased in experimental CKD when excess cholesterol was added to the diets. Glomerular disease has been reported in the rare familial disease, lecithin cholesterol acyltransferase deficiency, and with excess apolipoprotein\u00a0E. However, renal disease is not typical in the more common forms of primary hyperlipidemias. Patients with minimal-change disease or membranous glomerulonephritis, characterized by hyperlipidemia as part of their nephrotic syndrome, usually do not develop glomerular scarring. However, recently, post hoc and meta-analyses of clinical trial data support that abnormal lipids are associated with increased loss of GFR and that treatment with statins may not only benefit cardiovascular disease risk, but also be of benefit for progressive CKD. A post hoc analysis suggests that statins may even slow progression in patients with stage 3\u00a0CKD [102]. These beneficial effects of statins appear to extend beyond their lipid-lowering effects [98, 101].\nProteinuria\nProteinuria is a marker of renal injury, reflecting loss of normal permselecitvity. Further, proteinuria itself has been proposed to contribute to progressive renal injury inflammation [74, 103]. Increased proteinuria is associated with worse prognosis [104]. Whether proteinuria is merely a marker of injury or a contributor to progressive injury has been debated.\nAlbumin can in vitro in tubular cells increase AngII and in turn upregulate TGF-\u03b2 receptor expression [105]. However, in most settings, pure albumin per se is not directly injurious. Other filtered components of the urine in proteinuric states, such as oxidized proteins, appear to be more potent in inducing direct injury of tubular epithelial cells and activating proinflammatory and fibrotic chemokines and cytokines. Complement and various lipoproteins are also present in the urine in proteinuric disease states and can activate reactive oxygen species [101, 106]. Proteinuria may thus alter tubule cell function directly, potentially contributing to a more profibrotic phenotype, and also augment interstitial inflammation, in particular by macrophages. Proteinuria may activate many profibrotic pathways through its ability to increase NF-kB, and also by other pathways. These include for instance complement synthesis from tubules [107].\nInterventions that are particularly effective in decreasing proteinuria, such as the administration of ACEIs or ARBs, also decrease overall end organ injury. Whether these beneficial effects are dependent on the reduction of proteinuria has not been proven, in that these interventions have multiple parallel effects that may all contribute to the decrease of fibrosis [107].\nMechanisms of tubulointerstitial fibrosis\nTubulointerstitial fibrosis classically was thought merely to reflect glomerular injury and resulting whole nephron ischemia in most CKD. Interesting new data point to independent mechanisms of interstitial fibrosis and the importance of the tubulointerstitial lesion in progression. Decreased peritubular capillary density, possibly modulated by decreased VEGF or other angiogenic factors, has been proposed as a mechanism in various progressive renal diseases [108]. Future studies may demonstrate whether these interstitial microvascular lesions are causal or consequential in the development of interstitial injury.\nIncreased numbers of macrophages are closely correlated with both glomerulosclerosis and tubulointerstitial fibrosis and are usually decreased by interventions that decrease fibrosis. These cells are potential sources of numerous cytokines and eicosanoids that affect the glomerulus [109]. Support for this hypothesis is seen with the protective effects of maneuvers that decrease macrophage influx. In a rat model of unilateral ureteral obstruction (UUO), administration of ACEI ameliorated interstitial monocyte\/macrophage infiltration and decreased fibrosis [110]. Studies in \u03b26\u00a0integrin-deficient mice revealed that infiltrating macrophages do not inevitably transduce fibrotic effects; in these mice local activation of TGF-\u03b2 is impaired, and they are protected from fibrosis despite abundant macrophage infiltration [61]. Macrophages may even play a beneficial role in scarring. The specific role of the macrophage AT1a receptor in renal fibrosis was examined in studies of bone marrow transplantation in wild type mice with UUO mice reconstituted with either wild type macrophages or macrophages devoid of the AT1a receptor. There was more severe interstitial fibrosis in mice with the AT1a deficient macrophages, even though fewer infiltrating macrophages were observed, suggesting that the macrophage AT1a receptor functions to protect the kidney from fibrogenesis [111].\nIn human diabetic nephropathy there is an early increase in total interstitial cell volume (which may represent increased cell size and\/or number), preceding the accumulation of interstitial collagen [112]. This is in contrast to the diabetic glomerular lesion, where the expanded mesangial area is largely due to increased matrix accumulation rather than hypercellularity. These interstitial cells could possibly represent interstitial myofibroblasts, postulated to play a key role in interstitial fibrosis. These activated interstitial cells are a major source of collagen synthesis, and increased expression of \u03b1-smooth muscle actin (SMA), a marker of myofibroblasts, predicts progressive renal dysfunction both in human and experimental renal disease.\nThe source of interstitial myofibroblasts is a topic of controversy. Bone marrow-derived or potential renal stem cells may give rise not only to interstitial cells but also to regenerating parenchymal cells [113]. Epithelial\u2013mesenchymal transformation (EMT) is another possible mechanism for generation of interstitial myofibroblasts [114]. This seamless plasticity of cells changing from epithelial to mesenchymal phenotypes exists during early development. EMT may also occur in the adult after injury, contributing approximately half of the interstitial fibroblasts in experimental models [114]. Injured tubular epithelial cells can change phenotype both in vivo and in vitro, with de novo expression of a fibroblast-specific protein (FSP1), and possibly migrate into the interstitium as myofibroblasts. The surrounding matrix and basement membrane underlying the tubular epithelium is disrupted by local proteolysis, modulated by an array of cytokines and growth factors, including insulin-like growth factors\u00a0I and II, integrin-linked kinases, EGF, FGF-2 and TGF-\u03b2 [114]. Several key factors inhibit EMT, including hepatocyte growth factor and bone morphogenetic factor-7, and thus inhibit fibrosis in experimental CKD [114].\nAnatomic and genetic risks for CKD: nephron number and gene polymorphisms\nRisk for development of CKD and its rate of progression varies in differing populations. CKD associated with hypertension and arterio-nephrosclerosis is particularly common in African Americans, and FSGS is more frequently the underlying cause of steroid-resistant FSGS in African Americans and Hispanics than in Caucasians [115, 116]. These varying disease trends in differing ethnic populations could represent both genetic and environmental factors. Low birth weight is epidemiologically linked to increased risk for cardiovascular disease, hypertension and CKD in adulthood. The link is postulated to be due to the decreased nephron number that accompanies low term birth weight, defined as less than 2,500\u00a0g [117, 118]. These fewer nephrons are postulated to be under greater hemodynamic stress, thus contributing to progressive sclerosis. Of interest, low birth weight is much more common in African Americans than in Caucasians and is not accounted for by socioeconomic status [119]. Further, glomerular size in normal African Americans is larger than in Caucasians and could possibly reflect smaller nephron number [120]. In Australian Aborigines, marked increase in incidence of CKD is associated with larger but fewer glomeruli and low birth weight [121, 122]. Mechanisms other than hemodynamic stress that could underlie these differences in normal glomerular populations and also relate to increased incidence of end-stage renal disease include functional polymorphisms of genes that are involved both in renal\/glomerular development and contribute to amplified scarring mechanisms, such as the renin\u2013angiotensin system [10].\nAfrican Americans also have increased severity of renal disease associated with several systemic conditions. The course of lupus nephritis in a prospective trial was more severe in African Americans than in Caucasians, with more extensive crescent formation and interstitial fibrosis and greater likelihood of end-stage renal disease [123]. Even the manifestations of HIV infection in the kidney differ markedly between African Americans and Caucasians: HIV-associated renal disease in African Americans is typically an aggressive collapsing type of FSGS, contrasting lower grade immune-complex-mediated glomerulonephritides in Caucasians with HIV infection and renal disease [124]. Genetic background also modulates susceptibility in experimental models, both to podocyte injury (e.g. only the balb\/c mouse strain is susceptible to adriamycin) and to hypertension injury (e.g. in the five-sixths nephrectomy model, C57Bl mice are resistant, 129Sv\/J mice are susceptible) and even to diabetic injury [125\u2013127].\nThere is also accumulating evidence that specific genes in humans modulate the course and rate of organ damage. Polymorphisms in several genes within the RAAS system, including ACE, angiotensinogen and the angiotensin type\u00a01 receptor, have been linked with cardiovascular and renal disorders, including diabetic nephropathy, IgA nephropathy and uropathies [128\u2013133]. The ACE DD genotype, associated with increased RAS activity, was increased in patients with IgA nephropathy who ultimately experienced progressive decline in renal function during follow-up compared with those whose function remained stable over the same time [134].\nPolymorphisms of TGF-\u03b2 are also implicated in hypertension and progressive fibrosis. The Arg 25 polymorphism may be increased in African Americans, who may also have greater elevation of circulating TGF-\u03b2 when they reach end-stage renal disease than do Caucasians [135].\nThese observations suggest that complex genetic traits can modulate the response of glomerular cells to pathogenic stimuli in experimental models. Whether ethnic differences in development of renal disease in humans reflect contributions of genetic and\/or environmental influences remains to be definitively determined.\nQUESTIONS (Answers appear following the reference list)\nA 6-year-old African American boy presented with generalized edema, 24\u00a0h urine protein excretion of 1.5\u00a0g and normal complements, and serum creatinine of 0.7\u00a0mg\/dl. His blood pressure was 110\/70\u00a0mmHg. His nephrotic syndrome did not respond to an 8-week course of steroids, and a renal biopsy is planned. The most likely diagnosis in this patient is:\nFSGS due to mutation of podocinMinimal-change diseaseFSGS, usual typeDiffuse mesangial sclerosisCollapsing glomerulopathyFor the same patient detailed in question 1, what additional treatment should be initiated at this time to decrease risk of CKD:\nDiureticsSpironolactoneACEIsBeta blockersACEIs and ARBsIn the same patient detailed in the above questions, what parameters would be most important to follow and evaluate for adjustment of therapy:\nEdemaWhite blood cell (WBC) countBlood pressureProteinuriaA 14-year-old Caucasian girl was diagnosed with IgA nephropathy, which on biopsy showed fibrocellular crescents, with focal proliferative and secondary sclerosing lesions of glomeruli. Her urine protein excretion was 1.0\u00a0g in 24\u00a0h. Urinalysis showed frequent red blood cell casts, serum creatinine was 1.2\u00a0mg\/dl and her blood pressure was 120\/93\u00a0mmHg. Which of the following mechanisms are likely to contribute to progression of her CKD:\nPodocyte lossProteinuriaGlomerular hypertensionInfiltrating macrophagesAll of the aboveA 10-year-old Caucasian boy with a history of multiple episodes of steroid-dependent nephrotic syndrome since the age of 4\u00a0years now has proteinuria of 3.8\u00a0g in 24\u00a0h, with unremarkable urinalysis without red blood cell casts; his serum creatinine is 0.6\u00a0mg\/dl, and his blood pressure is 98\/64\u00a0mmHg. He has an increased cholesterol level of 480\u00a0mg\/dl and triglyceride levels are 110\u00a0mg\/dl. What mechanisms of renal injury are likely to be activated in this child:\npodocyte lossproteinuriadyslipidemiaglomerular hypertension(b) and (c)","keyphrases":["angiotensin","podocytes","interstitial fibrosis","glomerulosclerosis","low birth weight","angiotensin i converting enzyme inhibitors (acei)","angiotensin receptors","angiotensin receptor blockers","transforming growth factor (tgf)-beta"],"prmu":["P","P","P","P","P","R","R","R","M"]}
{"id":"Crit_Care-8-2-420020","title":"Clinical review: The implications of experimental and clinical studies of recruitment maneuvers in acute lung injury\n","text":"Mechanical ventilation can cause and perpetuate lung injury if alveolar overdistension, cyclic collapse, and reopening of alveolar units occur. The use of low tidal volume and limited airway pressure has improved survival in patients with acute lung injury or acute respiratory distress syndrome. The use of recruitment maneuvers has been proposed as an adjunct to mechanical ventilation to re-expand collapsed lung tissue. Many investigators have studied the benefits of recruitment maneuvers in healthy anesthetized patients and in patients ventilated with low positive end-expiratory pressure. However, it is unclear whether recruitment maneuvers are useful when patients with acute lung injury or acute respiratory distress syndrome are ventilated with high positive end-expiratory pressure, and in the presence of lung fibrosis or a stiff chest wall. Moreover, it is unclear whether the use of high airway pressures during recruitment maneuvers can cause bacterial translocation. This article reviews the intrinsic mechanisms of mechanical stress, the controversy regarding clinical use of recruitment maneuvers, and the interactions between lung infection and application of high intrathoracic pressures.\nIntroduction\nMechanical ventilation (MV) is a supportive and life saving therapy in patients with acute lung injury (ALI) and\/or acute respiratory distress syndrome (ARDS). Despite advances in critical care, mortality in these patients remains over 40% [1]. During the past decade the possibility that MV can produce morphologic and physiologic alterations in the lung has been recognized [2]. On histopathologic examination, findings in ventilator-induced lung injury (VILI) do not differ from those in ARDS [2]. To minimize this damage, lung protective strategies to avoid overdistension and cyclic collapse and reopening of alveoli have successfully been used in patients with ARDS receiving MV [3,4]. Recruitment maneuvers (RMs) consisting of sustained inflation to open the collapsed alveolar units have been proposed as an adjunct to MV in anesthesia and ARDS [5,6]. In most ARDS patients, however, lung recruitment and overdistension occur simultaneously at higher intrathoracic pressure [7]. Whether RMs can initiate cellular mechanisms of injury in healthy parts of the lung is unknown.\nIn the present review of the literature we describe the intrinsic mechanisms that explain how MV inflicts alveolar damage and the controversy regarding the use of RMs as an adjunct to MV. Finally, we discuss the interactions between lung infection and periodic application of high intrathoracic pressure, both in experimental models of ALI and in patients with ARDS.\nMethod\nTo identify the most relevant English language publications, the Medline database was searched using the following keywords: mechanotransduction, acute lung injury, acute respiratory distress syndrome, mechanical ventilation, ventilator-induced lung injury, overdistension, recruitment maneuvers, and bacterial translocation.\nMany different methods of RM delivery have been proposed in the literature (Table 1). Several investigators have demonstrated that RMs can increase oxygenation and lung volume in collapsed prone lungs. However, the benefits of RMs in terms of oxygenation and lung recruitment in ARDS patients and in experimental models with alveolar flooding or consolidation are unclear.\nIntrinsic mechanism of ventilator-induced lung injury\nThe mechanical stresses produced by MV at high pressure or volumes, and the forces generated by repeated opening and collapse lead to upregulation of an inflammatory response, with release of cytokines and chemokines and activation of neutrophils and macrophages that produce lung damage [2]. Injurious MV can lead to end-organ dysfunction, and the inflammatory cascade also plays a pivotal role in the systemic inflammatory response syndrome and in multiple organ system failure [8-10]. Like all adherent cells, alveolar epithelial cells interact with extracellular matrix through transmembrane adhesion receptors such as integrins. These receptors transmit forces from the surrounding matrix to the cytoskeleton via the focal adhesion complex [11]. When the basement membrane is strained, adherent epithelial cells must change shape and the ratio of their surface (plasma membrane) to their volume must increase. If the plasma membrane is disrupted the intracellular lipid stores are utilized to repair the cell surface. Most breaks are repaired within seconds, usually via a calcium-dependent response [12]. This dynamic remodeling process is the most important determinant of cell wounding [13].\nMechanotransduction is the conversion of mechanical stimuli, such as cell deformation, into biochemical and biomolecular alterations. How mechanical forces can be sensed by cells and converted into intracellular signals is still unclear, but in various experiments it was observed that mechanical stimuli activate the nuclear factor-\u03baB \u2013 a critical transcription factor that is required for maximal expression of many cytokines involved in the pathogenesis of VILI [14]. It is unknown whether a single stimulus such as RMs applied during MV can trigger the above-mentioned pathways of lung injury, and the long-term benefits and safety of RMs will depend on the extent of this effect.\nExperimental evidence on recruitment maneuvers\nIn saline lavaged rabbit lungs, Bond and coworkers [15] found an improvement in respiratory system compliance and oxygenation during high frequency oscillatory ventilation after RMs. In a similar model, Rimensberger and coworkers [16] showed that a single RM resulted in better oxygenation without augmenting histologic injury at positive end-expiratory pressure (PEEP) below the lower inflection point of the respiratory system pressure\u2013volume curve, as compared with the group with PEEP set above the lower inflection point without RM. Furthermore, those investigators showed that a single sustained inflation to 30 cmH2O boosted the ventilatory cycle onto the deflation limb of the pressure\u2013volume curve (Fig. 1). In other words, a RM applied in a recruitable lung increases the amount of recruited tissue at end expiration, favoring tidal ventilation.\nSome data suggest that RMs have different effects depending on the type of lung insult and on the use of various combinations of tidal volume and PEEP. Whether RMs are necessary to prevent alveolar collapse when optimal PEEP is used remains controversial. Van der Kloot and colleagues [17] studied the effects of RMs on gas exchange and lung volumes in three experimental models of ALI: saline lavage, oleic acid, and pneumonia. After application of RMs, oxygenation improved only in the surfactant depletion group when low PEEP was used. At high PEEP in any model, RMs had no effect. Similar effects were observed in the study conducted by Bond and coworkers [15]. Takeuchi and colleagues [18] highlighted the difficulties in maintaining tidal ventilation at high lung volumes. Those investigators showed that, after RMs, PEEP set at 2 cmH2O above the lower inflection point was more effective in maintaining gas exchange and minimizing inflammation and lung injury than was PEEP set at the maximum curvature of the deflation pressure\u2013volume curve. When recruitment is achieved with posture, Cakar and coworkers [19] showed better oxygenation after RMs in the prone than in the supine position, and importantly the benefit was sustained at lower PEEP. In other studies other adjuncts to MV were necessary to keep the lung open after RMs [20]. Lu and coworkers [21] demonstrated that RMs completely reversed the atelectasis, bronchoconstriction, and decrease in arterial oxygen saturation observed after endotracheal suctioning in an anesthetized sheep model.\nIn summary, the beneficial effects of RMs have been demonstrated in animal models of alveolar collapse induced by surfactant depletion. However, the pathobiology of ARDS is more complex and includes an altered vascular barrier function and alveolar flooding or consolidation. Indeed, in animal models other than that involving surfactant depletion, the effect of RMs on lung function is less evident.\nRole of recruitment maneuvers in anesthetized patients\nFormation of atelectasis and airway closure are mechanisms of impaired gas exchange in anesthetized patients with healthy lungs [22]. RMs have successfully been used to reverse collapsed dependent areas in these patients. Rothen and coworkers [22] found that a pressure of 40 cmH2O maintained for 7\u20138 s entirely re-expanded the collapsed lung tissue in anesthetized humans, although the net effect on gas exchange might be rather small if low ventilation\/perfusion areas still coexist at the time of decrease in intrapulmonary shunt. Long-term effects of RMs in anesthetized patients depend on gas composition. Re-expanded lung tissue remained inflated for at least 40 min at low oxygen concentration [5], whereas lung collapse reappears within minutes with pure oxygen [23]. Finally, RMs followed by moderate PEEP may produce physiologic benefits in patients undergoing upper abdominal, thoracic, or laparoscopic surgery [24,25], and in patients prone to develop a moderate degree of lung injury after surgical procedures [26,27].\nRecruitment maneuvers in patients with acute respiratory distress syndrome\nSince the publication of the reports from Amato and coworkers [3] and the Consensus Conference on ARDS [28], the application of periodic RMs in patients with ARDS has gained acceptance among clinicians, although controversy remains. Among the earliest reports providing evidence that RMs improve lung function was that from Pelosi and coworkers [29], who demonstrated that sighs at 45 cmH2O plateau pressure in patients ventilated with PEEP at 14 \u00b1 2.2 cmH2O significantly improved oxygenation, intrapulmonary shunt, and lung mechanics. Foti and colleagues [6] observed that RMs were effective in improving oxygenation and alveolar recruitment only during MV at low PEEP, suggesting that high PEEP better stabilized alveoli and prevented loss of lung volume. In the same line, Lapinsky and coworkers [30] reported beneficial effects on oxygenation, but this effect was sustained only if PEEP was increased after RMs. Lim and colleagues [31] found an improvement in oxygenation that persisted for 1 hour after an 'extended sigh'; this effect was partially lost soon after ventilatory support returned to the baseline PEEP level.\nOther studies have shown a modest and variable effect of RMs on oxygenation when ARDS patients are ventilated with high PEEP. Richard and coworkers [32] demonstrated decreased oxygenation when tidal volume was switched from 10 to 6 ml\/kg with PEEP set above the lower inflection point of the pressure\u2013volume curve. However, increasing PEEP and RMs prevented alveolar derecruitment, and RMs performed in patients already ventilated with high PEEP had minimal effects on requirements for oxygenation support. Similarly, Villagr\u00e1 and colleagues [33], studying the effect of RMs superimposed on a lung protective strategy (tidal volume <8 ml\/kg and PEEP 3\u20134 cmH2O higher than the lower inflection point on the pressure\u2013volume curve), found no effect on oxygenation regardless of the stage of ARDS, and in some patients venous admixture increased during RMs (Fig. 2). This deleterious effect suggested that the RMs increased lung volume by overdistending the more compliant already-opened and aerated alveolar units, favoring blood flow redistribution from overdistended to collapsed lung regions. Furthermore, a negative correlation was found between recruited lung volume induced by PEEP before RMs and RM-induced changes in oxygenation, suggesting that RMs are less effective when the lungs have been near optimally recruited by PEEP and tidal volume. Recently Hubmayr [34] suggested that alveolar flooding is probably the main mechanism of end-expiratory loss of lung aeration in human ARDS. This may explain, at least in part, why RMs are less beneficial in patients with ARDS. Nevertheless, when sudden lung derecruitment occurs in conditions of adequate PEEP ventilation, such as the loss in lung volume produced during secretion aspiration, Maggiore and coworkers [35] observed that suctioning-induced lung derecruitment can be prevented by performing RMs.\nThe cause of ARDS may also influence the response to RMs. In a majority of trauma patients developing ARDS, Johannigman and coworkers [36] found an improvement in oxygenation after RMs in patients receiving MV with low tidal volume and high PEEP. However, when Bein and colleagues [37] analyzed the impact of RMs on intracranial pressure and cerebral metabolism in patients with acute cerebral injury and respiratory failure, they observed an increase in intracranial pressure at the end of RMs and a subsequent reduction in mean arterial pressure resulting in a decrease in cerebral perfusion pressure. Both normalized 10 min after RMs. Grasso and coworkers [38] found that RMs significantly improved arterial oxygenation and lung volume in patients with early ARDS without impaired chest wall mechanics (i.e. with large recruitment potential). Nevertheless, in the group with low chest wall compliance, RM-induced lung overdistension reduced blood pressure and cardiac output, making RMs ineffective and potentially harmful (Fig. 3).\nRMs can also be applied during assisted breathing in non-sedated patients. Patroniti and coworkers [39] applied one sigh per minute to baseline pressure support ventilation in patients with early ARDS. They observed a significant improvement in arterial oxygenation associated with an increase in end-expiratory lung volume and respiratory system compliance during the sigh, suggesting that sighs promote alveolar recruitment. These changes returned to baseline after the sighs were discontinued.\nOther studies emphasize the importance of body posture (supine or prone) on regional distribution of intrapulmonary ventilation and perfusion, and the beneficial effects of prone position in limiting VILI in experimental animals [40]. Lim and coworkers [31] found that the benefit was significantly greater when patients were in the supine position as compared with those in the prone position, suggesting that patients in the prone position have less collapsed lung. These findings were recently confirmed by Pelosi and colleagues [41], who demonstrated that adding cyclical sighs during ventilation in the prone position provided optimal lung recruitment in the early stage of human ARDS.\nFinally, two randomized physiologic pilot studies of RMs superimposed on low tidal volume ventilation and moderate to high PEEP conducted in approximately 100 patients with ALI showed no clear benefits in terms of oxygenation [42,43]. Moreover, RMs were potentially harmful because some patients developed hemodynamic instability, ventilator dysynchrony, and pneumothorax after RM.\nIn summary, RMs can be useful in improving oxygenation in patients receiving MV with low PEEP and low tidal volume. However, in patients with ARDS receiving MV with high PEEP levels, the beneficial effects of RMs disappear. RMs may restore lung volume and oxygenation in endotracheal suctioning-induced lung derecruitment in mechanically ventilated patients diagnosed with ALI\/ARDS. RMs should be avoided in patients with suspected or documented intracranial hypertension, in patients with a stiff chest wall, and in patients in the late stage of ARDS.\nLung infection and mechanical ventilation\nRecent studies suggest that the detrimental effect of MV may be aggravated when lungs are infected or primed with endotoxin. In ex vivo rat lungs, Ricard and coworkers [44] showed that ventilation that severely injures lungs does not lead to release of significant amounts of inflammatory cytokines by the lung in the absence of lipopolysaccharide challenge. Likewise, in experimental studies other investigators have shown that MV predisposes to development of pneumonia [45] and that coexisting MV and infection have a strong impact on the lung because they appear to act synergistically in causing alveolar damage [46]. Finally, when bacteria were injected in animals with previous severe ALI, MV produced a clinical picture closely resembling that of hyperdynamic sepsis in humans [47]. These experimental studies taken together suggest that in the presence of lung infection MV (cyclic positive intrathoracic pressure) predisposes to greater bacterial burden and bacterial translocation from the lung into the systemic circulation than would occur without MV. These effects are particularly important when using ventilatory strategies that apply large transpulmonary pressures (high tidal volume and\/or high alveolar pressures without PEEP) [48] and are partially attenuated when protective ventilatory strategies are used [49].\nRMs can be applied as sighs or as periodic sustained inflations that can damage or transiently alter the integrity of the alveolar\u2013capillary barrier [50,51]. Whether such strategies to improve lung function can result in failure of the alveolar\u2013capillary barrier and promote transient bacterial translocation in humans remains unknown. The amount of recruitable lung parenchyma in patients with ALI\/ARDS receiving MV is a matter of debate, and controversy exists on the use of RMs in such patients for two main reasons. First, consolidation (non-recruitable lung parenchyma) and sticky atelectasis (potentially recruitable) coexist in different amounts in ALI\/ARDS, and cannot be distinguished and quantified at the bedside to inform a decision regarding a recruitment strategy. Second, the amount of lung tissue to be recruited in some ARDS patients is sparse [52]. Therefore, RMs can exert little effect on consolidated lung areas but can cause overdistension in some lung regions where bacteria are compartmentalized at the site of infection or colonization. Because spillover of lung cytokines into the systemic circulation is observed in lung inflammation and is potentiated with MV [53], a similar phenomenon is likely to occur when the concentration of bacteria in the lungs is high enough. In a recent study [54] it was found that high pressure ventilation promoted early translocation of bacteria; however, intermittent RMs applied as a sustained inflation superimposed on low-pressure ventilation without PEEP did not cause translocation of intratracheally inoculated Pseudomonas aeruginosa in rats with previously healthy lungs. However, we do not yet know whether the lung injury model used is valid for human ARDS or the degree of reproducibility of short-term experimental studies in patients receiving MV for days or weeks [55].\nConclusion\nOn the basis of our review of the literature on experimental and clinical studies, considerable uncertainty remains regarding the use of RMs in humans with ARDS. RMs may have a role to play in patients with early ARDS and normal chest wall mechanics because there is great potential for alveolar recruitment, and after disconnections from the ventilator, when sudden loss of lung volume promotes alveolar instability and derecruitment. Recommendations to use RMs as adjuncts during lung protection ventilatory strategies seem unnecessary because sustained improvements in lung function have not been found when the strategies are combined. The presence of lung infection must be considered a major limitation for aggressive RMs because translocation of bacteria and the occurrence of systemic sepsis have been demonstrated in animal models. Finally, large randomized studies do not support the use of RMs in patients with ARDS.\nIn conclusion, the use of RMs cannot be recommended in the light of current knowledge, and if RMs are used they should be restricted to an individualized clinical decision or to a last resort to improve oxygenation and lung mechanics in a severely hypoxemic ARDS patient.\nCompeting interests\nNone declared.\nAbbreviations\nALI = acute lung injury; ARDS = acute respiratory distress syndrome; MV = mechanical ventilation; PEEP = positive end-expiratory pressure; RM = recruitment maneuver; VILI = ventilator-induced lung injury.","keyphrases":["acute lung injury","mechanical ventilation","mechanical stress","lung infection","lung collapse"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_J_Pediatr-3-1-1914296","title":"What is new in surgical treatment of vesicoureteric reflux?\n","text":"In addition to conventional open surgery and endoscopic techniques, laparoscopic correction of vesicoureteric reflux, sometimes even robot-assisted, is becoming an alternative surgical treatment modality for this condition in a number of centres around the world. At least for a subgroup of patients laparoscopists are trying to develop new techniques in an effort to combine the best of both worlds: the minimal invasiveness of the STING and the same lasting effectiveness as in open surgery. The efficacy and potential advantages or disadvantages of these techniques are still under investigation. The different laparoscopic techniques and available data are presented.\nIntroduction\nWhen confronted with the title \u201cWhat is new in surgical treatment of vesicoureteric reflux?\u201d many readers will automatically think of endoscopic techniques with subureteric injection of bulking agents, also known as STING (Subureteral Teflon INjection). Over the years several substances have been advocated as bulking agents, but the original Teflon is no longer in use. (The most commonly used substance nowadays is DefluxR, a dextranomer\/hyaluronic acid copolymer.) However, a technique that has been around for a quarter of a century can hardly be considered for a text on surgical novelties. Instead, this review concentrates on the use of laparoscopic techniques in this setting.\nGeneral considerations about vesicoureteric reflux\nVesicoureteric reflux (VUR) remains one of the most frequent conditions in paediatric urology, although the exact prevalence is largely unknown. VUR can be primary, secondary (e.g. to elevated bladder pressures in neurogenic bladders or dysfunctional voiding) and sometimes intermittent in nature, only disclosing itself when infection has possibly induced a degree of insufficiency of the ureterovesical junction. It is generally assumed that VUR predisposes to urinary tract infections and that surgical treatment of reflux and prophylactic antibiotics are equivalent in terms of preventing infections and renal scarring. The relative merit of these interventions in the natural course of these conditions remains to some extent controversial [32].\nThe importance of voiding dysfunction with detrusor overactivity, underactivity or dysfunctional elimination disorder in the aetiology of VUR should not be underestimated [4, 30] and this has its implications in the treatment offered to these children. Hence bladder training and minimally invasive techniques have acquired a prominent role over the years. Children with VUR and concomitant voiding dysfunction are likely to suffer more breakthrough infections and have lower spontaneous resolution rates and therefore represent a large proportion of the patients undergoing surgical intervention [31].\nAntimicrobials form the mainstay in the treatment of VUR, in combination with other conservative measures, because VUR will spontaneously disappear in a majority of children and rarely gives rise to serious long-term complications [2]. Increasingly however the exact role of prophylaxis is being questioned as well-designed prospective trials are rare [10]. Nevertheless, a small subgroup of patients does pose problems of break-through infections despite all conservative measures and in fact some of them seem prone to renal scarring leading to hypertension and exceptionally even end stage renal failure [16].\nTraditional surgical techniques in the treatment of VUR\nSince the 1950s several surgical techniques have been developed for the correction of VUR. All techniques share the same basic principle of creating an anti-reflux mechanism by increasing the portion of the distal ureter lying in a submucosal tunnel between the detrusor muscle and the bladder mucosa. They offer comparable and very high success rates with few complications [12]. From a purely technical standpoint, these open techniques can basically be divided into two groups. There are those that involve mainly or entirely intravesical ureteral dissection (and hence a need for postoperative bladder drainage) and those that use a purely extravesical approach to the ureter without disconnecting it from the bladder. To the former group belong the techniques of Politano and Leadbetter (1958), Glenn and Anderson (1967), the psoas-hitch technique and the (most widely used) Cohen technique (1975) [6, 11, 13, 25]. In these techniques the ureter is disconnected from the bladder and reimplanted in a new and longer submucosal tunnel from the luminal side of the bladder. In the Cohen technique, a cross-trigonal tunnel is created bringing the ureter to the contralateral side, the other techniques result in a more natural course of the ureter, but are somewhat more prone to complications such as bowel injury or kinking of the ureter. The psoas-hitch technique is generally reserved for more complex situations as in mega-ureters or re-do surgery and is helpful in creating a longer tunnel. The conceptually different extravesical approach was popularized by Lich and Gregoir, reducing postoperative bladder irritation to insignificance, but predisposing to temporary bladder retention when performed bilaterally [14, 21].\nThe more recent and certainly minimally invasive STING technique where bulking agents are injected submucosally has gained wide acceptance. Undoubtedly this is technically a very easy, relatively cheap and patient-friendly treatment modality, tempting many doctors into an increasingly pre-emptive approach to VUR, using it as first-line treatment in cases of (antenatally detected) high-grade reflux even in infants [27]. Success rates, even in low-grade reflux, are clearly lower than in open surgery and a second injection of bulking agent is often necessary [8]. Moreover, prospective randomised trials and long-term results are still not available. The tendency to use this endoscopic technique as an alternative to medical treatment is underscored by the fact that since the Food and Drug Administration (FDA) approval of DefluxR the total number of procedures for reflux has increased, while open surgery rates have remained stable [20].\nAll these facts and tendencies mentioned above in turn suggest that, at least for the foreseeable future, there will remain a group of patients in whom STING is deemed-or proves to be-insufficient. Open surgery on the other hand has its drawbacks as well due to its invasiveness. In an ideal world physicians would be able to define very precisely and at the earliest possible point in time which group of patients with VUR is at increased risk for the complication of pyelonephritic scarring and which group is not. This would in turn allow a very tailored approach to each individual child with pre-emptive surgical measures in the group at risk. Failing this knowledge, the next best thing to aim for is to combine the superior results of time-honoured open procedures like a Cohen reimplantation or Lich-Gregoir operation with the much sought after minimal invasiveness of laparoscopy, possibly with the added ultra-precise tissue handling and dexterity of robotic surgery. These considerations are the driving force of the developments described in this text.\nConventional laparoscopic techniques\nBoth intra- and extravesical laparoscopic treatments have been described in a great variety of techniques. Most series however remain small and follow-up is very limited. Ehrlich et al. and Janetschek et al. were the first to report in 1994 and 1995 on two and six children undergoing laparoscopic Lich-Gregoir anti-reflux surgery for vesicoureteral reflux [7, 17]. The reflux was successfully corrected without morbidity, requiring only a short hospitalisation. Peri-operative ureteral stents were deemed unnecessary. One mild unilateral stenosis did develop later, requiring temporary stenting. Ehrlich et al. described decreased peri- and post-operative pain and improved cosmesis by comparison with open surgery. He suggested that this preliminary report deserved further study. Janetschek et al. on the other hand concluded that the Lich-Gregoir anti-reflux procedure was a complicated one because of the difficult suturing and knot-tying, offering no clear advantage over the conventional procedure. Other teams were very reluctant to join in the efforts to develop this approach for several years to come. The choice for a Lich-Gregoir technique for the first attempts at correction of VUR can be explained by the fact that, at that time, experience with laparoscopy in cavities other than the abdomen was very limited. Five years later Lakshmanan and Fung reported technical modifications to further minimize invasiveness, basically by downsizing ports and instruments and limiting tissue dissection [19]. A more recent paper by Riquelme et al. again reported excellent outcomes in 15 children, even in cases of bilateral reflux and duplex ureters [28]. There was no postoperative voiding dysfunction. Laparoscopic ureteral reimplantation with extracorporeal tailoring and stenting of megaureters combined with a Lich-Gregoir type of extravesical reimplantation was recently reported by Ansari et al. in three children [1].\nAlthough the Cohen procedure was the more widely used in the treatment of VUR, a laparoscopic version thereof was investigated later than the extravesical laparoscopic techniques. The obvious reason is the anticipated difficulties with port placement and the limitations of the intravesical working space. Different approaches were used by Gill et al. and Yeung et al. [12, 33]. Gill et al. combined the use of two suprapubic ports with a transurethral resectoscope for unilateral cases whereas Yeung et al. used three suprapubic ports, more closely copying the open Cohen procedure.\nA recent report by Kutikov et al. on either transvesical laparoscopic cross-trigonal ureteral reimplantation in patients with reflux or a Glenn-Anderson reimplantation in patients with a primary obstructing mega-ureter mentions operative success in 25 of 27 patients with VUR and 4 out of 5 patients with mega-ureters, results that are comparable to the ones obtained in open surgery [18]. Complications were postoperative urinary leak in four patients and ureteral stricture at the anastomosis in two. The authors noted that most complications occurred in the younger patients with small bladder capacities.\nFor completeness two papers on reimplantations in (young) adults can be mentioned. Chung et al. described successful laparoscopic nonrefluxing ureteral reimplantation with a psoas hitch using a submucosal tunnelling technique after submucosal injection of saline under cystoscopy in two adult female patients without postoperative complications [5]. Also in 2006, Puntambekar et al. described laparoscopic extravesical ureteroneocystostomy with psoas hitch in five gynaecologic patients, clearly minimizing the procedural morbidity [26]. Again no intraoperative or postoperative complications occurred.\nGradually more relevant series with larger numbers of patients and longer follow-up are being presented. At the 2007 European Society for Paediatric Urology (ESPU) annual meeting two groups will present their experience in about 80 patients each, with success rates above 90% (http:\/\/www.espu.org).\nRobot-assisted techniques\nOver the last 2\u00a0years a few authors reported robot-assisted laparoscopic techniques using the Da VinciR (Intuitive Surgical, Mountain View, CA) system for the treatment of VUR, adding yet another approach to this rapidly expanding field [3, 23, 24]. They made good use of the experience gained with conventional laparoscopy, adding the advantages of robotics: enhanced dexterity of the instruments, absence of tremor and 3-D vision. The generally used term of \u201crobotic surgery\u201d is to some extent actually misleading because it suggests completely autonomous function of the equipment. In reality it works as a master-slave system, merely transferring the movements of the surgeon\u2019s hands to the tip of the instruments (Fig.\u00a01). The evolution parallels the one seen in conventional laparoscopy, experience having started with the extravesical approach and later moving to intravesical procedures. The sequence of surgical steps of both techniques will briefly be discussed. As stated, they closely mirror the steps in conventional laparoscopy.\nFig.\u00a01Outside view once the draped robotic arms are connected to the laparoscopic ports: the child seems completely \u201cembraced\u201d by the machine\nExtravesical technique\nTo start, a cystoscopic evaluation of the relevant anatomy is carried out. The camera port is then placed in the umbilicus and the two working ports in each lower abdominal quadrant. A small transverse peritoneal incision is made on the laterodorsal side of the bladder where the ureter is retrieved. The ureter is then buried in a trough between the mucosa and detrusor to create the anti-reflux mechanism (Figs.\u00a02 and 3). The bladder catheter is removed already at the end of the procedure unless a significant perforation needing suturing of the mucosa has been made.\nFig.\u00a02Extravesical approach: very gently the detrusor muscle is incised and peeled away until the delicate bladder mucosa starts to bulgeFig.\u00a03Extravesical approach: the completely freed ureter is hinged into the trough to create an anti-reflux valve mechanism\nIn our experience there were no bladder symptoms post surgery in any of the patients. All cases of reflux resolved, but there was one case of \u201cde novo\u201d contralateral low-grade reflux [3]. Later we successfully performed this operation in an adult male patient after a failed subureteral injection (unpublished data). Interestingly, Elmore et al. recently reported on the use of the open Lich-Gregoir technique as salvage in these patients as well [9] and already in 2004 one similar laparoscopic patient was reported by Shu et al. [29]. Both in open and laparoscopic surgery this was a novel approach, meant to avoid the sometimes difficult intravesical dissection due to foreign material after STING. Peters and Borer reported persisting low-grade reflux in 2 of their 24 patients [23].\nIntravesical technique\nOlsen was the first to experiment with a Cohen cross-trigonal ureter reimplantation by laparoscopic access to the bladder in a pig model using the Da VinciR system [22]. In all pigs the reflux disappeared after the procedure. The advantage of the robotic equipment seemed to be the better access to submucosal tunnelling of the ureter and the intravesical suturing of the anastomosis. Peters and Woo in 2005 and Callewaert in 2006 reported their experience with robot-assisted Cohen procedures in six and three paediatric patients respectively [3, 24].\nInitial port placement and closure of the incisions at the end of the procedure were the crucial steps, the rest of the procedure being straightforward. Once inside the bladder the mucosa is circumferentially incised around the ostium using the cautery hook. After both ureters are freed, a submucosal tunnel connecting the most proximal part of the two mucosal incisions is created, using forceps and scissors (Fig.\u00a04). Creation of the submucosal tunnel and reimplantation of the ureters is remarkably easy because of the three dimensional visualisation and great dexterity inside the very small volume of a child\u2019s bladder. The anatomical detail is such that dissection of the plane between the detrusor and mucosa is achieved with more detail than in open surgery. The bladder catheter is left indwelling for 24 to 48\u00a0h.\nFig.\u00a04Intravesical approach: creation of the submucosal tunnel connecting the periureteral incisions. (The jaws of the forceps measure 5\u00a0mm in length)\nWe had one conversion to open surgery out of three cases in our early experience because of port-related problems in a small child [3]. Kutikov et al. using conventional laparoscopy similarly found that the smaller children were more prone to complications and that these procedures were technically more demanding [18]. Peters and Woo on the other hand reported no conversions in a series of six children aged between 5 and 15 [24]. They did however have a case of port-site urinary leakage requiring prolonged bladder drainage.\nPeters and Callewaert each reported one case of persisting low-grade reflux in their initial experience. Unlike the situation in open surgery, bladder spasms remained completely absent and anticholinergics were unnecessary. This fact is highly suggestive of the minimal invasiveness and limited trauma incurred by the bladder wall.\nWhen comparing the robotically assisted intra- and extravesical operations it is our impression that the Lich-Gregoir technique offers some advantages over the intravesical operation: no need for catheters, no haematuria and easier reproducibility. The drawback is that the abdominal cavity needs to be entered. The abdominal cavity even in smaller children is large enough to allow comfortable movement of the instruments, whereas intravesical operations in this patient group can be technically impossible due in part to the relative bulkiness of the robotic instruments.\nConclusion\nTreatment modalities of reflux are evolving rapidly. Conventional or robot-assisted laparoscopic techniques must be considered a possible future alternative to the more traditional ways of treating this condition. There is no proven superiority at this time and experience is limited to a few centres only and relatively small numbers of patients. It is well established that with open surgery very high success rates can be achieved and that morbidity is relatively low and hospitalisation nowadays can be kept short. The first impressions are that morbidity using laparoscopic techniques is lower still and that there is some cosmetic gain, but it is obvious that the most important issue will be whether the long-term success rates are at least comparable.\nMost surgeons agree that robotics certainly add to the precision and ease of the individual surgical steps when compared to conventional laparoscopy, but the financial costs are very high. The intravesical approach using robotics is feasible, but technical difficulties must be taken into account in smaller children. (The same holds true for the conventional laparoscopy.) The extravesical robotic approach clearly seems the more promising, possibly even after failed submucosal injection therapy. Nevertheless we feel that the intravesical approach deserves further pursuing because it may allow surgical correction of other malformations at the level of the bladder neck and ureterovesical junction in a minimally invasive and very precise way.\nIt would be premature to promote laparoscopy as the golden mean between STING and open surgery for a subgroup of reflux patients at this point, as this would imply diverting a large number of patients to a few centres where either the technical laparoscopic expertise or a robotic system is available. However, we remain convinced that in the (near) future laparoscopy will find its place in the care for these patients.","keyphrases":["vesicoureteric reflux","laparoscopy"],"prmu":["P","P"]}
{"id":"Ann_Surg_Oncol-3-1-2077912","title":"Population-Based Study of Islet Cell Carcinoma\n","text":"Background We examine the epidemiology, natural history, and prognostic factors that affect the duration of survival for islet cell carcinoma by using population-based registries.\nIslet cell carcinomas are low- to intermediate-grade neuroendocrine carcinomas of the pancreas. Also known as pancreatic endocrine tumors or pancreatic carcinoid, they account for the minority of pancreatic neoplasms and are generally more indolent than pancreatic adenocarcinoma. Islet cell carcinomas, which arise from islets of Langerhans, can produce insulin, glucagon, gastrin, and vasoactive intestinal peptide, causing the characteristic syndromes of insulinoma, glucagonoma, gastrinoma, and VIPoma. Pancreatic polypeptide is also frequently produced, yet it is not associated with a distinct clinically evident syndrome.\nAlthough the molecular biology of sporadic islet cell carcinoma is less well understood than other more common solid tumors, they can arise in connection with several hereditary cancer syndromes. The best known of these, multiple endocrine neoplasia type 1 (MEN1), is an autosomal-dominant inherited disorder characterized by tumors of the parathyroids, pituitary, and pancreas.1 Less commonly, neuroendocrine (carcinoid) tumors of the duodenum (gastrinomas), lung, thymus, and stomach have also been described.2\nTuberous sclerosis and neurofibromatosis are two other hereditary cancer syndromes associated with the development of neuroendocrine tumors. TSC1\/2 complex inhibits mTOR and is normally expressed in neuroendocrine cells.3 Patients with a defect in the TSC2 gene have tuberous sclerosis and are known to develop islet cell carcinoma.4 Neurofibromatosis is associated with the development of carcinoid tumors of the ampulla of Vater, duodenum, and mediastinum.5,6 The gene responsible for neurofibromatosis 1 (NF1) regulates the activity of TSC2. The loss of NF1 in neurofibromatosis leads to constitutive mTOR activation.7 Finally, islet cell carcinomas also occur in approximately 12% of patients with von Hippel-Lindau disease (vHL).8 The vHL gene is located on chromosome 3p26\u2013p25; inactivation of the vHL gene is thought to stimulate angiogenesis by promoting increased HIF-1\u03b1 activity.\nLittle is known about the epidemiology and natural history of islet cell carcinoma. Although several case series have been reported, there have been few population-based studies. This is in part due the uncommonness of this disease as well as the complexity of its classification. Although pancreatic carcinoid based on ICD-O-3 histology classification (8240\u20138245) has been partially described in studies of carcinoid from the Surveillance, Epidemiology, and End Results (SEER) Program,9 these have been incomplete analyses because most islet cell carcinomas were coded differently in the ICD-O-3 system (8150\u20138155). Survival of patients with islet cell tumors was also described in a recent report on malignant digestive endocrine tumors that was based on data from England and Wales.10 In this population-based study, we have undertaken a comprehensive analysis of patients with islet cell carcinoma identified through the SEER Program database in the United States.\nMETHODS\nThe SEER Program was created as a result of the National Cancer Act of 1971. The goal of the SEER Program is to collect data useful in the prevention, diagnosis, and treatment of cancer. In this study, we used the SEER data based on the November 2005 submission. For incidence and prevalence analyses, registry data was linked to total U.S. population data from 1969 to 2003.11\nSince 1973, the SEER Program has expanded several times to improve representative sampling of minority groups as well as to increase the total sampling of cases to allow for greater precision. The original SEER 9 registries included Atlanta, Connecticut, Detroit, Hawaii, Iowa, New Mexico, San Francisco\u2013Oakland, Seattle\u2013Puget Sound, and Utah. In 1992, four additional registries were added to form the SEER 13 registries, which included the SEER 9 registries, plus Los Angeles, San Jose\u2013Monterey, rural Georgia, and the Alaska Native Tumor Registry. More recently, in 2000, data from greater California, Kentucky, Louisiana, and New Jersey were added to the SEER 13 Program to form the SEER 17 registries. SEER 9, 13, and 17 registries cover approximately 9.5%, 13.8%, and 26.2% of the total U.S. population, respectively. The data set we use here contains information about a total of 4,539,680 tumors from 4,123,001 patients diagnosed from 1973 to 2003.\nIslet cell carcinomas were identified by search for ICO-O-3 histology codes 8150\u20138155, 8240\u20138245, and pancreatic primary site (duodenal gastrinomas were excluded). The included histology codes correspond to the following clinical\/histologic diagnoses: islet cell carcinoma, insulinoma, glucagonoma, gastrinoma, mixed islet cell\/exocrine carcinoma, VIPoma, carcinoid, enterochromaffin cell carcinoid, and adenocarcinoid. The SEER registries include neuroendocrine neoplasms that are considered invasive and malignant (behavior code of 2 or 3 in the International Classification of Diseases for Oncology, 2nd edition [ICD-O-2]). Cases designated as poorly differentiated or anaplastic were excluded. A total of 1310 cases of islet cell carcinoma were included in this study. Cases identified at the time of autopsy or by death certificate only were excluded (36 cases) from survival analyses.\nAlthough a tumor, node, metastasis system classification system has recently been proposed,12 during the period of time that we studied, there was no accepted staging system for islet cell carcinoma. Here, we use the SEER staging system. Tumors in the SEER registries were classified as localized, regional, or distant. In this system, a localized neoplasm was defined as an invasive malignant neoplasm confined entirely to the organ of origin. A regional neoplasm was defined as a neoplasm that has (1) extended beyond the limits of the organ of origin directly into surrounding organs or tissue; (2) involves regional lymph nodes; or (3) fulfills both of the above. A distant neoplasm was defined as a neoplasm that has spread to parts of the body remote from the primary tumor.\nThe comparisons between patients, tumor characteristics and disease extension were based on the \u03c72 test. One-way analysis of variance was used for the comparison of continuous variables between groups. Survival duration was measured by the Kaplan-Meier method and compared by the log rank test. The statistical independence between prognostic variables was evaluated by multivariate analysis by the Cox proportional hazard model.\nSEER*Stat 6.2.4 (Surveillance Research Program, National Cancer Institute) was used for incidence and limited-duration prevalence analyses.13 All other statistical calculations were performed by SPSS 12.0 (Apache Software Foundation 2000). Survival durations calculated by SPSS were also verified by parallel analyses by SEER*Stat. Comparative differences were considered statistically significant when the P value was <.05.\nRESULTS\nFrequency and Incidence\nBetween 1973 and 2003, a total of 101,192 pancreatic neoplasms in 101,173 patients were identified in the SEER 17 registries. Among these, 101,046 neoplasms were classified as malignant and occurred in 101,029 patients. When we restricted the search to codes of neuroendocrine histology, a total of 1385 neoplasms in 1385 patients were identified. We removed 75 patients who were classified as having tumors of poorly differentiated or anaplastic grade. Thus, a total of 1310 patients had pancreatic islet cell carcinomas in the SEER registries. This consisted of 1.3% of all patients with pancreatic cancers.\nBy using linked population files, we calculated the incidence of islet cell carcinoma as a rate per 100,000 per year, age-adjusted to year 2000 U.S. standard population. Because the SEER 9, 13, and 17 registries were linked to different population data sets, we computed the age-adjusted incidence rates in three time periods. The age-adjusted incidence in the SEER 9 registries between 1973 and 1991 was .16 per 100,000. For the SEER 13 registries, from 1992 to 1999, an age-adjusted incidence of .14 per 100,000 was observed. Finally, for the period covered by the SEER 17 registries, from 2000 to 2003, the age-adjusted incidence rate was .12 per 100,000. This suggests that on the basis of the current U.S. population estimate of 302 million, approximately 362 cases of malignant islet cell carcinoma will be diagnosed each year. The number of small benign islet cell tumors may be higher. Detailed incidence data by time period, sex, and race are included in Table\u00a01.\nTABLE\u00a01.Age-adjusted incidence rate of islet cell carcinoma per 100,000 populationaTime period and raceAllMaleFemaleSEER 9 (1973\u20131991)\u00a0\u00a0All races.16 (.15\u2013.18).19 (.17\u2013.22).14 (.13\u2013.16)\u00a0\u00a0White.16 (.14\u2013.17).19 (.16\u2013.21).14 (.12\u2013.16)\u00a0\u00a0African American.25 (.19\u2013.32).29 (.19\u2013.43).22 (.16\u2013.32)\u00a0\u00a0Other.11 (.07\u2013.18).16 (.09\u2013.28).08 (.04\u2013.16)SEER 13 (1992\u20131999)\u00a0\u00a0All races.14 (.13\u2013.16).17 (.15\u2013.20).12 (.10\u2013.14)\u00a0\u00a0White.15 (.13\u2013.17).18 (.15\u2013.21).13 (.11\u2013.15)\u00a0\u00a0African American.11 (.07\u2013.16).12 (.05\u2013.25).10 (.06\u2013.18)\u00a0\u00a0Other.13 (.09\u2013.18).18 (.11\u2013.28).09 (.05\u2013.15)SEER 17 (2000\u20132003)\u00a0\u00a0All races.12 (.10\u2013.13).12 (.11\u2013.15).11 (.09\u2013.13)\u00a0\u00a0White.12 (.11\u2013.13).14 (.12\u2013.16).11 (.09\u2013.13)\u00a0\u00a0African American.14 (.09\u2013.19).10 (.05\u2013.20).16 (.10\u2013.24)\u00a0\u00a0Other.07 (.04\u2013.12).03 (.01\u2013.11).11 (.06\u2013.18)a Rates are per 100,000 population (95% confidence interval) age-adjusted to year 2000 U.S. standard population. Cases were selected by ICD-O-3 histology codes 8150\u20138155, 8240\u20138245, and confirmed pancreatic primary site. Cases designated as poorly differentiated or anaplastic by grade were excluded. Cases of unknown race are excluded from the \u201cother\u201d category (included in \u201call races\u201d).\nLimited Duration Prevalence\nAmong the population sampled by the SEER 9 registries, 28-year limited duration prevalence for islet cell carcinoma on January 1, 2003, was estimated by the counting method to be 227 (95% CI, 199\u2013259). These data were then projected to the general U.S. population. Data were matched by sex, race, and age to the U.S. standard population. The estimated 28-year limited duration prevalence of islet cell carcinomas on January 1, 2003, in the United States was 2705 cases. In comparison, the 28-year limited duration prevalence for all pancreatic neoplasms regardless of histology was 27,201.14 Thus, although islet cell carcinoma represented 1.3% of pancreatic cancer by incidence, it represented 9.9% of cases by the 28-year limited duration prevalence analyses.\nPatient Characteristics\nOf the 1310 patients with islet cell carcinoma identified in the SEER database, there were 619 women and 691 men. The majority (1095 cases) were white. African American and other racial groups accounted for 134 and 78 cases, respectively. Details of patient characteristics are included in Table\u00a02. The other racial groups included American Indian\/Alaskan Natives, and Asian\/Pacific Islanders. In three cases, the race was unknown. We plotted the number of cases by age group at diagnosis in Fig.\u00a01; the peak age distribution was 65 to 69 years. However, the median age at diagnosis was 59 years.\nTABLE\u00a02.Characteristics of 1310 patientsaCharacteristicValueSex, n (%)\u00a0\u00a0Male691 (53)\u00a0\u00a0Female619 (47)Race, n (%)\u00a0\u00a0African American134 (10)\u00a0\u00a0White1095 (84)\u00a0\u00a0Other78 (6)\u00a0\u00a0Unknown3 (.2)\u00a0\u00a0Median (SD) age at diagnosis (y) 59 (15)ICD-O-3 groupings, n (%)\u00a0\u00a0Islet cell1117 (85)\u00a0\u00a0Insulinoma49 (4)\u00a0\u00a0Glucagonoma29 (2)\u00a0\u00a0Gastrinoma73 (6)\u00a0\u00a0VIPoma16 (1)\u00a0\u00a0Mixed histology26 (2)SEER stage, n (%)\u00a0\u00a0Localized179 (14)\u00a0\u00a0Regional295 (23)\u00a0\u00a0Distant711 (54)\u00a0\u00a0Unknown125 (10)Location of primary tumor, n (%)\u00a0\u00a0Head379 (29)\u00a0\u00a0Body103 (8)\u00a0\u00a0Tail278 (21)\u00a0\u00a0Overlapping108 (8)\u00a0\u00a0Unknown442 (34)Year of diagnosis, n (%)\u00a0\u00a01973\u20131988482 (37)\u00a0\u00a01989\u20132003828 (63)a Cases selected from SEER 17 database by ICD-O-3 histology codes 8150\u20138155, 8240\u20138245, and pancreatic primary site. Cases designated as poorly differentiated or anaplastic by grade were excluded.FIG.\u00a01.Age at diagnosis of 1310 cases of islet cell carcinoma. The median and mean (SD) ages at diagnosis are 59 and 58 (15) years.\nWe next examined the effect of race and sex on age at diagnosis. The median age in years at diagnosis for white, African American, and other racial groups was 60 (mean 58, SD 14.9), 55 (mean 54.5, SD 16.5) and 56 (mean 57, SD 16.5), respectively (P = .02, Fig.\u00a02). However, there was no difference in median age at diagnosis based on sex.\nFIG.\u00a02.Age at diagnosis by race. White patients were older at the time of diagnosis (P = .02). The median age at diagnosis for white, African American, and other racial groups was 60 (mean 58, SD 14.9), 55 (mean 54.5, SD 16.5), and 55.5 (mean 57, SD 16.5), respectively.\nTumor Location and Hormone Production\nThe location of the primary tumors within the pancreas was described in 868 cases. In 442 cases, the detailed location data is not known. This is partially because the current coding system allows tumor location to be coded as islet of Langerhans, which gives no information about the location of the tumor within the pancreas. In 379 cases, the primary tumor was located in the head of the pancreas; body, tail, and overlapping groups accounted for 103, 278, and 108 cases, respectively.\nBy ICD-O-3 histology codes, 1117 cases (Table\u00a02) were coded as islet cell or carcinoid (ICD-O-3 = 8150, 8240, 8241). Because these designations can include either serotonin-producing or nonfunctional tumors, it is not possible to determine the secretory status in these cases. Among the known functional tumors, gastrinoma was the most common with 73 cases (22 cases of duodenal gastrinoma not included); insulinoma, glucagonoma, and VIPoma accounted for 49, 26, and 16 cases, respectively. Finally, in 26 cases, the tumors were considered to have mixed endocrine\/exocrine histology (ICD-O-3 = 8154, 8243\u20138245).\nNext, we compared the location of the primary tumors within the pancreas by histological classification. We found significant differences in the pattern of primary tumor localization (P = .029); nonfunctional or serotonin-producing tumors (coded as islet cell and carcinoid tumors) were more likely to be located in the head (44%) than in the body (12%) or tail (31%) or overlapping (14%). Among the functional neoplasms, most insulinomas (57%), glucagonomas (53%), and VIPomas (64%) were located in the tail of the pancreas. Gastrinomas were much more likely to be located in the head of the pancreas (63%).\nTumor Stage\nOf the 1310 cases, 125 (10%) were not staged (Table\u00a02). For the remaining 1185 cases, 179 (14%) were localized; 295 (23%) were classified as regional; and 711 (54%) were classified as distant. When we compared stage by ICD-O-3 histology, we found that most carcinomas were metastatic at the time of diagnosis, including islet cell carcinomas (61%), insulinomas (61%), glucagonomas (56%), and gastrinomas (60%). A smaller percentage of VIPomas (47%) were metastatic (P < .001). This may be the result of the presence of massive diarrhea in VIPoma patients, which may bring them to medical attention earlier. The higher rate of metastases among insulinoma patients observed in this study is likely because most small insulinoma are considered benign and not reported to SEER. Next, we compared stage by tumor location and found that tumors located at the head of the pancreas trended toward a lower rate of distant disease (48% head vs. 57% body, 58% tail, and 60% overlapping) and a higher rate of regional disease (34% head vs. 27% body, 23% tail, 27% overlapping). However, the difference was not statistically significant (P = .063).\nSurvival\nFor survival analyses, we excluded 36 cases that were identified at autopsy or on the basis of death certificates only. The median overall survival for all 1274 remaining cases was 38 months (95% CI, 34\u201343). When compared by the log rank test, SEER stage predicted patient outcome (P < .001). The median survival for patients with localized, regional, and distant islet cell carcinoma was 124 months, 70 months, and 23 months, respectively (Fig.\u00a03). One-year, 3-year, 5-year, and 10-year survival rates are listed in Table\u00a03. The median survival for those cases that were not staged was 50 (95% CI, 34\u201366) months. Relative risk was calculated by Cox proportional modeling. Compared to the group with localized disease, patients with regional and distant disease had 1.56- and 3.50-fold of increased risk of death during the period (1973\u20132005) included in this study.\nFIG.\u00a03.Stage and survival of 1157 patients from the time of diagnosis. Median duration of survival of patients with localized (n = 167), regional (n = 289), and distant disease (n = 558) was 124, 70, and 23 months, respectively (P < .001).TABLE\u00a03.Survival by SEER stageSEER stageMedian survival (mo) (95% CI)Survival rateHRP1 y5 y10 yLocal124 (80\u2013168)88%71%52%1.00 (referent)<.001Regional70 (54\u201386)82%55%38%1.56 (1.17\u20132.07)Distant23 (20\u201326)65%23%9%3.50 (2.71\u20134.54)SEER, Surveillance, Epidemiology, and End Results Program; 95% CI, 95% confidence interval; HR, hazard ratio.\nWe then examined potential prognostic factors for survival duration that were based on data available from the SEER database. For these analyses, we stratified patients by stage. Patients with missing stage data were excluded from the analyses. When compared against the group designated as islet cell or carcinoid by ICD-O-3 histology codes, patients with gastrinoma (P = .001) and VIPoma (P = .044) had longer survival durations after adjusting for the effect of stage (Table\u00a04), while the group with mixed histology experienced worse survival. The difference was not statistically significant, which is likely because of the small number of cases in this category.\nTABLE\u00a04.Cox proportional hazard analyses of potential prognostic factors adjusted for stageaParameterHR (95% CI)P valueICD-O-3 group\u00a0\u00a0Islet cell1.00 (referent)\u00a0\u00a0Insulinoma.935 (.64\u20131.37).728\u00a0\u00a0Glucagonoma1.12 (.68\u20131.84).652\u00a0\u00a0Gastrinoma.57 (.41\u2013.80).001\u00a0\u00a0VIPoma.44 (.20\u2013.98).044\u00a0\u00a0Mixed histology1.18 (.65\u20132.13).596Location of primary tumor\u00a0\u00a0Head1.00 (referent)\u00a0\u00a0Body.74 (.55\u2013.98).037\u00a0\u00a0Tail.82 (.67\u20131.01).056\u00a0\u00a0Overlapping1.32 (1.01\u20131.72).041\u00a0\u00a0Age as a continuous variable1.03 (1.03\u20131.04)<.001Age grouped by median\u00a0\u00a00\u2013591.00 (referent)\u00a0\u00a060+2.16 (1.87\u20132.49)<.001Sex\u00a0\u00a0Female1.00 (referent)\u00a0\u00a0Male1.07 (.93\u20131.23).367Race\u00a0\u00a0African American1.00 (referent)\u00a0\u00a0White1.18 (.93\u20131.50).176\u00a0\u00a0Other1.31 (.89\u20131.92).173Year of diagnosis\u00a0\u00a01973\u201319881.00 (referent)\u00a0\u00a01989\u20132003.79 (.68\u2013.91).001a All analyses have been adjusted for Surveillance, Epidemiology, and End Results Program stage.\nThe location of the primary tumor may have a marked effect on the time of presentation (head tumors may obstruct the bile duct and cause visible jaundice) and resectability, as well as on surgical morbidity and mortality. Therefore, we next examined the prognostic role of the location of the primary tumor within the pancreas (Table\u00a04). In our analyses, tumors located at the head of the pancreas were less likely to be associated with distant metastasis. The rates of distant metastasis for primary tumors located in the head, body, tail, and overlapping locations were 48%, 57%, 58%, and 60%, respectively. These differences were not statistically significant (P = .063). However, once adjusted for stage, primary tumor location in the pancreatic head was associated with a worse prognosis than the pancreatic body (P = .037). Similarly, tumors in the pancreatic head tended to be associated with worse survival compared with those in the pancreatic tail (P = .056); however, the difference was not statistically significant. Patients with primary tumors that were classified as overlapping had the worst prognosis (P = .041). In some cases, an overlapping lesion may indicate a larger primary tumor that covered a larger portion of the pancreas.\nAge is found to be a predictor of outcome in a variety of malignancies. We examined the effect of age at diagnosis on overall survival stratified by stage (Table\u00a04, Fig.\u00a04). In the Kaplan-Meier analysis, we separated the patients into two groups on the basis of median age. We observed decreasing survival with increasing age (P < .001). Similarly, we also examined the effect of age as a continuous variable in Cox proportional hazard modeling and found increasing age to be a predictor of poor outcome (P < .001).\nFIG.\u00a04.Age and survival. For the <59 age group, the median survival durations for localized, regional, and distant disease were 282 (95% CI, 204\u2013360) months, 114 (95% CI, 83\u2013145) months, and 35 (28\u201342) months. For the 60+ age group, the median survival durations for localized, regional, and distant disease were 46 (95% CI, 20\u201372) months, 38 (95% CI, 24\u201352) months, and 13 (95% CI, 10\u201316) months (P < .001).\nNext, we examined the survival duration of patients with islet cell carcinoma by year of diagnosis. There has been an improvement in survival over time. Whether analyzed as a continuous variable by Cox proportional hazard modeling or divided into discrete time durations, the observed difference was statistically significant (P = .001). Sex and race did not significantly affect survival. The details of these analyses are included in Table\u00a04.\nFinally, we performed multivariate survival analyses by Cox proportional hazard modeling. SEER stage, primary tumor localization, ICD-O-3 histology groups, and age at diagnosis were entered into the model. In multivariate analysis, the ICD-O-3 histology group was not a statistically significant predictor of outcome. All other variables retained statistical significance; the most important predictor of outcome was stage. Compared to patients with localized disease, patients with regional (HR = 1.44) and distant (HR = 3.40) disease had decreased survival duration. Location of the primary tumor within the pancreas (P = .032) and age at diagnosis (P < .001) also remained significant predictors of outcome (Table\u00a05).\nTABLE\u00a05.Multivariate survival analysesParameterHR (95% CI)P valueStage<.001\u00a0\u00a0Local1.00 (referent)\u00a0\u00a0Regional1.44(1.05\u20131.99).026\u00a0\u00a0Distant3.40 (2.53\u20134.67)<.001\u00a0\u00a0Age as a continuous variable1.03 (1.02\u20131.04)<.001Location of primary tumor.032\u00a0\u00a0Head1.00 (referent)\u00a0\u00a0Body.81 (.60\u20131.08).146\u00a0\u00a0Tail.87 (.71\u20131.07).175\u00a0\u00a0Overlapping1.26 (.97\u20131.65).089ICD-O-3 group.186\u00a0\u00a0Islet cell1.00 (referent)\u00a0\u00a0Insulinoma.80 (.46\u20131.39).427\u00a0\u00a0Glucagonoma1.08 (.57\u20132.02).822\u00a0\u00a0Gastrinoma.70 (.47\u20131.04).077\u00a0\u00a0VIPoma.39 (.12\u20131.22).105\u00a0\u00a0Mixed histology1.38 (.73\u20132.59).322HR, hazard ratio; 95% CI, 95% confidence interval.\nDISCUSSION\nIn order for us to make advances in the diagnosis and management of patients with islet cell carcinoma, we must improve our understanding of the epidemiology, natural history, and prognostic factors for this relatively rare disease. Because of the rarity of neuroendocrine carcinoma and the lack of a staging system, much of the information previously published has been based on case series and anecdotal experiences. In this study, we take advantage of the vast amount of data collected by the SEER Program to examine the largest series of islet cell carcinomas reported to date. To our knowledge, this study represents the only population-based study of islet cell carcinoma in published literature.\nPrevalence is defined as the number people alive on a certain date in a population that ever had a diagnosis of the disease. In this study, the counting method15 was used to estimate prevalence from incidence and follow-up data obtained from the SEER 9 registries. Complete prevalence can be established by this method by using registries of very long duration. The SEER 9 registries have the longest follow-up duration and contain data suitable for prevalence analyses for the past 28 years. Given the longer survival duration often experienced by patients with neuroendocrine carcinoma, we report only limited duration prevalence data which may somewhat underestimate the complete prevalence. By incidence, islet cell carcinomas account for only 1.3% of all pancreatic cancers. However, because of the better outcome generally experienced by patients with islet cell carcinoma, they represent almost 10% of pancreatic cancers in prevalence analyses.\nWe acknowledge that analyses from the SEER registries underestimate the total number of patients with islet cell tumors. All cases from the SEER database were denoted to be malignant. Thus, it is likely that small, benign-appearing tumors were not included in the SEER registries (for example, insulinomas and small nonfunctioning tumors). Although histologic evidence of invasion of basement membrane defines malignant behavior for most epithelial malignancies, the definition of malignant behavior for pancreatic neuroendocrine neoplasms is more complex. In the absence of malignant behavior such as direct invasion of adjacent organs, metastases to regional lymph nodes or distant sites, it may be difficult to classify an islet cell tumor as benign or malignant. Pancreatic endocrine tumors are classified as benign, uncertain malignant potential, and malignant on the basis of size, the presence or absence of lymphovascular invasion, and the number of mitoses and Ki-67\u2013positive cells by immunohistochemistry. However, there is a considerable overlap among these histopathologic features in benign and malignant neuroendocrine tumors and there is even heterogeneity within different areas of the same tumor. Many small islet cell tumors may have been considered benign or of unclear malignant potential and were excluded from the SEER registries. However, size is likely a function of when a tumor is diagnosed; left untreated, it is likely that most islet cell tumors will eventually grow locally into adjacent structures or soft tissues, and\/or spread to distant organs. Therefore, outside of small insulinomas, all islet cell neoplasms should be considered potentially malignant. Thus, although the SEER registry data provide important information about malignant islet cell tumors, the extent to which it underestimates the frequency of smaller islet cell neoplasms is unknown. In our experience, even small islet cell tumors without clear evidence of invasion or metastasis at the time of initial surgical resection may recur and spread years later.\nWe found a wide distribution of age at diagnosis, with a median of 59 years. Separated by race, white patients were older at the time of diagnosis. When we compared stage by histologic type, we found that VIPomas were less likely to be metastatic at the time of diagnosis. Several findings may have contributed to this observation. One possible explanation is that VIPomas are generally associated with profound watery diarrhea, which may cause patients to seek medical attention earlier than if they had nonfunctional tumors. Second, as previously mentioned, small insulinomas and gastrinomas may have been considered benign (in the absence of invasion of adjacent organs, lymph node metastases, or distant metastases) and therefore not captured for analysis thereby enriching the population of study patients with metastatic functioning tumor.\nIn an earlier publication that was based on data from the SEER Program, Modlin et al.9 described the five-year overall survival of patients with carcinoid tumors to be 59.5% to 67.2%. The survival of patients with islet cell carcinoma seems less favorable. In the present study, we observed a median overall survival of 38 months. This is identical to the median survival observed in a large retrospective series of 163 cases from the University of Texas M. D. Anderson Cancer Center.16 The survival duration of patients with distant metastases was also similar (23 months in current study vs. 25 months in the M. D. Anderson series). We did, however, observe improvements in the outcome of patients with islet cell carcinoma over time. These improvements were observed among all SEER stage groups (data not shown), and are likely due in part to improvements in supportive care.\nBy multivariate survival analysis, we found that stage of disease, primary tumor location, and age at diagnosis were important predictors of outcome. The difference in survival by primary tumor location can be attributed to several possible factors. In this study, patients with tumors classified as having overlapping location had the worst outcome. This is likely because of the larger tumor size in this group. Tumor located at the head of pancreas may be diagnosed earlier because of hyperbilirubinemia resulting from biliary obstruction. This is suggested by our finding of a trend (nonsignificant) for a lower rate of metastastic disease at the time of diagnosis on the basis of tumor location in the pancreatic head. However, adjusted for stage, patients with a tumor located in the pancreatic head were more likely to have a worse outcome than patients with tumors located in the body or the tail of the pancreas. One possible explanation is that tumors arising in the pancreatic head are of greater malignant potential. For example, most insulinomas (57%), glucagonomas (53%), and VIPomas (64%) were located in the tail of the pancreas. Such functioning tumors were also associated with improved survival compared with nonfunctioning tumors. In addition, the fact that tumors in the pancreatic head are more likely to cause biliary obstruction (with a risk for cholangitis), invade the duodenum (resulting in hemorrhage or obstruction), or involve the peripancreatic mesenteric vasculature (resulting in pain, mesenteric foreshortening, and malabsorption) may contribute to local tumor morbidity which may influence survival duration. There is also likely to be a general tendency on the part of physicians to avoid surgical resection of the pancreatic head (pancreaticoduodenectomy or Whipple procedure) when faced with a large tumor or low volume metastatic disease. This is not the case with tumors in the body or tail of the pancreas that can be surgically excised with a distal pancreatectomy. However, it is the pancreatic head tumor that carries the greatest risk for tumor associated morbidity such as hemorrhage and biliary or gastric outlet obstruction.16 To what degree patients with pancreatic head tumors suffer morbidity and mortality from local disease progression rather than distant metastases cannot be determined from this data set.\nFinally, increasing age at diagnosis was a predictor of poor outcome in our study. The differences in survival were seen across all SEER stage groupings. Certainly, pancreatic resection carries considerable operative risks and the medical comorbidities that are associated with advancing age may have precluded some patients from resection while increasing the risk to those taken to surgery. For patients with advanced disease, systemic chemotherapy often includes drugs that are considered toxic.17,18 For example, doxorubicin is recognized to be cardiotoxic, and streptozocin may cause worsening of diabetes. Thus, heart disease and diabetes which are more common among older patients may have limited the use of these drugs.\nAt present, surgery is the only curative treatment for islet cell carcinoma. Surgery should be recommended for most patients in whom cross-sectional imaging suggests that complete resection is possible.19 Although islet cell carcinoma has a better prognosis than adenocarcinoma of the pancreas, the disease remains incurable once multifocal unresectable metastatic disease exists. Although survival beyond 10 years has been described in the literature for some patients with metastatic disease, the survival duration for most with advanced disease is far shorter. Although streptozocin-based chemotherapy can induce objective response in 32% to 39% of patients,17,20 second-line treatment options are limited. Newer approaches such as peptide receptor radiotherapy, systemic agents targeting vascular endothelial growth factor, and mTOR are under development.\nOptimal management of patients with islet cell carcinoma requires an understanding of the disease process and a multimodal approach. A better understanding of the molecular biology of this disease may lead to improved clinical models for predicting outcome and developing novel treatment strategies for this relatively rare but complex disease. Until then, an understanding of the natural history of the disease as provided herein is necessary to allow physicians and patients to accurately assess the risks and potential benefits of treatment alternatives that are based on the extent of disease and the age and performance status of the patient.","keyphrases":["islet cell","epidemiology","survival","pancreatic endocrine tumor","neuroendocrine tumor"],"prmu":["P","P","P","P","P"]}
{"id":"Exp_Eye_Res-2-1-2394572","title":"Pharmacological disruption of the outer limiting membrane leads to increased retinal integration of transplanted photoreceptor precursors\n","text":"Retinal degeneration is the leading cause of untreatable blindness in the developed world. Cell transplantation strategies provide a novel therapeutic approach to repair the retina and restore sight. Previously, we have shown that photoreceptor precursor cells can integrate and form functional photoreceptors after transplantation into the subretinal space of the adult mouse. In a clinical setting, however, it is likely that far greater numbers of integrated photoreceptors would be required to restore visual function. We therefore sought to assess whether the outer limiting membrane (OLM), a natural barrier between the subretinal space and the outer nuclear layer (ONL), could be reversibly disrupted and if disruption of this barrier could lead to enhanced numbers of transplanted photoreceptors integrating into the ONL. Transient chemical disruption of the OLM was induced in adult mice using the glial toxin, dl-alpha-aminoadipic acid (AAA). Dissociated early post-natal neural retinal cells were transplanted via subretinal injection at various time-points after AAA administration. At 3 weeks post-injection, the number of integrated, differentiated photoreceptor cells was assessed and compared with those found in the PBS-treated contralateral eye. We demonstrate for the first time that the OLM can be reversibly disrupted in adult mice, using a specific dose of AAA administered by intravitreal injection. In this model, OLM disruption is maximal at 72 h, and recovers by 2 weeks. When combined with cell transplantation, disruption of the OLM leads to a significant increase in the number of photoreceptors integrated within the ONL compared with PBS-treated controls. This effect was only seen in animals in which AAA had been administered 72 h prior to transplantation, i.e. when precursor cells were delivered into the subretinal space at a time coincident with maximal OLM disruption. These findings suggest that the OLM presents a physical barrier to photoreceptor integration following transplantation into the subretinal space in the adult mouse. Reversible disruption of the OLM may provide a strategy for increasing cell integration in future therapeutic applications.\n1\nIntroduction\nRetinal degeneration is the leading cause of untreatable blindness in the developed world. Current clinical treatments are limited, at best only slowing disease progression and very rarely restoring visual function. Cell transplantation offers a novel therapeutic approach, enabling the replacement of photoreceptor cells lost in the degenerative process. Photoreceptor transplantation may be more feasible than other types of neuronal transplantation, because photoreceptors are stimulated by light and their function is not, therefore, dependent on the reformation of complex afferent connections. Nevertheless, an efferent connection to host second order sensory neurons in the retina is essential for visual function and this is arguably best achieved if the transplanted photoreceptor is fully integrated into the host outer nuclear layer (ONL).\nTransplanted whole retinal sheets, derived from either embryonic or neonatal sources, can survive and differentiate, but frequently fail to integrate and make functional connections within the host neural retina (Ghosh and Ehinger, 2000; Royo and Quay, 1959; Seiler et\u00a0al., 1990; Zhang et\u00a0al., 2003b). Conversely, dissociated neural stem cells, such as those derived from the adult hippocampus, can migrate extensively within the retina when transplanted into an adult or developing recipient, but rarely differentiate into mature retinal phenotypes (Takahashi et\u00a0al., 1998; Young et\u00a0al., 2000). Progenitor cells isolated from dissociated embryonic retinae can differentiate into retinal neurons after transplantation and express photoreceptor-specific markers (Ahmad et\u00a0al., 2000; Chacko et\u00a0al., 2000; Coles et\u00a0al., 2004; Klassen et\u00a0al., 2004; Qiu et\u00a0al., 2005; Yang et\u00a0al., 2002), but migration and integration of these cells into the laminar structure of the host neural retina has remained limited. Better integration has been achieved by transplanting cells into an immature, developing retina, such as that of the neonatal Brazilian opossum, which provides a foetal-like host environment. The ability of transplanted cells to integrate within the host opossum retina declines with host maturation (Sakaguchi et\u00a0al., 2003, 2004). This also coincides with the maturation of glial elements, such as M\u00fcller cells, which form anatomical barriers within the host retina, including the outer limiting membrane (OLM) (MacLaren, 1996).\nWe have recently shown that a significant degree of integration of fully differentiated and functional photoreceptors can be achieved after transplantation into the adult retina, but only if donor cells are post-mitotic photoreceptor precursors. When transplanted into the subretinal space, these cells can migrate into the recipient ONL, form synaptic connections with downstream targets and are light-sensitive (MacLaren et\u00a0al., 2006). However, while the number of integrated photoreceptor cells is sufficient to restore the pupillary light reflex, higher levels of integration are needed to improve visual acuity. Given that immature neurons and progenitor cells are intrinsically capable of migrating and differentiating (Komuro and Rakic, 1998a,b; Nadarajah and Parnavelas, 2002; Parnavelas et\u00a0al., 2002; Pearson et\u00a0al., 2005), it is likely that natural physical barriers, such as the OLM, impede migration. Transient disruption of these barriers at the time of transplantation might be one way of increasing the number integrating into the host retina.\nThe OLM comprises a series of zonula adherens junctional complexes, located between the plasma membranes of photoreceptor inner segments and the apical processes of M\u00fcller glia (Fig.\u00a01a\u2013c). These junctions seal off the light-sensitive photoreceptor inner and outer segments from the rest of the retina, limiting the diffusion of phototransduction cascade components. The OLM is first discernible by post-natal day 5 (P5) in the mouse (Uga and Smelser, 1973; Woodford and Blanks, 1989). Alpha-aminoadipic acid (AAA) is a glutamate analogue (Fig.\u00a01d), which disrupts the OLM by inducing specific toxicity in M\u00fcller glial cells within the mammalian retina (Karlsen et\u00a0al., 1982; Pedersen and Karlsen, 1979). Single intravitreal injections of AAA have been shown to disrupt the OLM irreversibly and to the extent whereby photoreceptors drop out of the ONL to reside amongst outer segments in the subretinal space (Ishikawa and Mine, 1983). In this study, we show that by using an appropriate dose and route of administration, AAA can produce a transient disruption in OLM integrity. Furthermore, OLM disruption can facilitate movement of cells in the opposite direction, significantly enhancing the number of donor photoreceptors integrated into the recipient ONL after transplantation into the subretinal space. These findings suggest that the OLM represents at least one important barrier to cell integration.\n2\nMaterials and methods\n2.1\nAnimals\nC57Bl\/6 and Nrl.gfp+\/+ (Akimoto et\u00a0al., 2006) mice were maintained in the animal facility at University College London. All experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Mice defined as \u201cadult\u201d were at least 6\u00a0weeks, but not more than 3\u00a0months old. Nrl.gfp+\/+ mice were used as donors to provide dissociated retinal progenitor cells for transplantation. Recipients were C57Bl\/6 animals, unless otherwise stated.\n2.2\n\u03b1-Aminoadipic acid formulation and administration\ndl-\u03b1-Aminoadipic acid (AAA; Sigma) was prepared in phosphate-buffered saline (PBS), adjusted to pH 7.5 and sterile-filtered prior to administration. Mice were anaesthetized with a single intra-peritoneal injection of 0.15\u00a0ml of a mixture of Dormitor (1\u00a0mg\/ml medetomidine hydrochloride; Pfizer Pharmaceuticals, Kent UK), ketamine (100\u00a0mg\/ml; Fort Dodge Animal Health, Southampton, UK) and sterile water for injections in the ratio of 5:3:42 for adult mice. AAA was administered by intravitreal, subretinal or subcutaneous injection.\nFor histological assessment, mice were sacrificed at various time points (3\u20134 mice per time-point) and the eyes were fixed in buffered formalin for 48\u00a0h at 4\u00a0\u00b0C. Retinal sections were prepared by overnight dehydration and paraffin embedding (Histocentre). Sections (5\u00a0\u03bcm thick) were cut and affixed to glass slides and stained using standard haematoxylin and eosin protocols.\n2.3\nDissociation of retinal cells and transplantation\nTo investigate the role of AAA-induced disruption of the OLM on cell integration, C57Bl\/6 mice received subretinal transplants of dissociated retinal precursor cells 72\u00a0h or 1\u00a0week (9 mice per time-point) after intravitreal injection (through the inferior pars plana) of AAA (test eye) or PBS (contralateral eye). Dissociated cells were prepared from P 2\u20135 Nrl.gfp+\/+ mice, as described previously (MacLaren et\u00a0al., 2006). Cells were dissociated using a papain-based kit (Worthington Biochemical, Lorne Laboratories, UK) and diluted to a final concentration of \u223c4\u00a0\u00d7\u00a0105 cells\/\u03bcl. Surgery was performed under direct ophthalmoscopy through an operating microscope, as previously described (MacLaren et\u00a0al., 2006). Cell suspensions were injected (1\u00a0\u03bcl) slowly to produce a standard and reproducible retinal detachment in the superior hemisphere. Mice were sacrificed 21\u00a0days after transplantation and eyes were fixed in 4% paraformaldehyde (PFA) in PBS, for cell counts. Retinal sections were prepared by cryo-protecting fixed eyes in 20% sucrose, before cryo-embedding in OCT (TissueTek) and sectioning at 18\u00a0\u03bcm.\n2.4\nHistology and immunohistochemistry\nMice were sacrificed at various time points after AAA administration (3 mice per time-point) and eyes were fixed in 1% PFA in PBS, for immunohistochemistry. Retinal cryosections were permeablized in chilled acetone for 5\u00a0min. Sections were pre-blocked in Tris-buffered saline (TBS) containing normal goat serum (5%), bovine serum albumin (1%) and 0.05% Triton X-100 for 2\u00a0h before being incubated with primary antibody overnight at 4\u00a0\u00b0C. After rinsing with TBS, sections were incubated with secondary antibody for 2\u00a0h at room temperature (RT), rinsed and counter-stained with Hoechst 33342. Negative controls omitted the primary antibody. The following antibodies were used: rabbit anti-ZO-1 (kind gift of K. Matter) and rabbit anti-GFAP (Dako), with an anti-rabbit Alexa-546 tagged secondary antibody (Molecular Probes, Invitrogen). An apoptosis TdT DNA fragmentation kit (ApopTag Red Apoptosis Detection Kit, Chemicon, CA, USA), which stains apoptotic cells red, was used to perform an in situ TUNEL assay on the sections.\n2.5\nElectron microscopy\nMice were sacrificed at various time points after AAA administration. The eyes were fixed, the cornea and lens removed and the eye cups orientated and processed, as previously described (Tschernutter et\u00a0al., 2005). Ultrathin sections were collected on copper grids, (100 mesh, Agar Scientific) contrast stained with 1% uranyl acetate and lead citrate and analysed using a JEOL 1010 transmission electron microscope (80\u00a0kV), fitted with a digital camera for image capture.\n2.6\nConfocal microscopy\nRetinal sections were viewed on a confocal microscope (Zeiss LSM510), as previously described (MacLaren et\u00a0al., 2006). Unless otherwise stated, images show: (i) merged Nomarski and confocal fluorescence projection images of GFP (green) and the nuclear counter stain Hoechst 33342 (blue), and (ii) the same region showing GFP signal only.\n2.7\nIntegrated cell counts\nCells were considered to be integrated if the whole cell body was correctly located within the outer nuclear layer and at least one of the following was visible: spherule synapse, inner\/outer processes and\/or inner\/outer segments, as previously defined (MacLaren et\u00a0al., 2006). The number of integrated cells per eye was determined by counting all the integrated GFP-positive cells in alternate serial sections through each eye. This was doubled to give the mean number of integrated cells per eye. While it is unlikely that a photoreceptor cell body would be present across three sections, given that AAA causes cell swelling in M\u00fcller glia (Ishikawa and Mine, 1983), it is possible that it may also affect other cell types including photoreceptors. The total number of nuclei in the ONL in a volume of 2500\u00a0\u03bcm3 at the site of transplantation was determined in both control and AAA-treated eyes (72\u00a0h and 1\u00a0week prior to transplantation). There was no significant difference in the number of photoreceptor cell bodies between any of the treatment groups (P\u00a0=\u00a00.35, N\u00a0=\u00a06; ANOVA). Thus, AAA treatment is highly unlikely to lead to double-counting of integrated cells.\n2.8\nApoptotic cell counts\nThe number of apoptotic cells was determined by counting all TUNEL-positive profiles in each layer of the retina in alternate serial sections. Only sections that encompassed the site of intravitreal injection were used and are thus not representative of apoptosis in the whole eye.\n2.9\nStatistics\nAll means are stated \u00b1SEM (standard error of the mean), unless otherwise stated. The statistical test used was a two-tailed paired t-test with a significance threshold of P\u00a0<\u00a00.05. N, number of eyes; n, number of sections examined or cells counted, where appropriate.\n3\nResults\n3.1\nDosage and route of AAA administration\nWe sought to determine whether it is possible to achieve a transient, reversible disruption of the OLM in the adult mouse by administration of the glial toxin, AAA. First, we assessed different routes of administration including: intravitreal (20\u00a0\u03bcg\/\u03bcl, N\u00a0=\u00a04), subretinal (10\u00a0\u03bcg\/\u03bcl, N\u00a0=\u00a04) and subcutaneous (0.7\u20132.7\u00a0mg\/g body weight, N\u00a0=\u00a06) injection. The dose of AAA used for each route of administration was ascertained from previous published studies (Ishikawa and Mine, 1983; Pedersen and Karlsen, 1979; Rich et\u00a0al., 1995) Retinae failed to recover normal histological morphology following subretinal injection, while subcutaneous injections resulted in variable morphological changes (data not shown). However, intravitreal administration caused modest and reversible morphological changes (see below). Therefore, AAA was administered to the retina via intravitreal injection, through the inferior pars plana towards the superior hemisphere of the eye, for the remainder of the experiments described.\nIn order to establish the optimum dose of AAA required to induce a transient disruption of the OLM, several doses (20, 100 and 320\u00a0\u03bcg\/\u03bcl, N\u00a0=\u00a03, N\u00a0=\u00a04 and N\u00a0=\u00a04, respectively for each time point) were administered intravitreally and the retinae examined histologically at 6, 24\u00a0h or 3\u00a0weeks post-injection (Fig.\u00a02) and compared with contralateral controls. At 6\u00a0h post-injection of all three doses of AAA, we observed vacuoles at the margin of the OLM that protruded into the inner\/outer segment layer. These were possibly due to a swelling of the M\u00fcller cell apical processes and both the size and number of vacuoles present was dosage dependent (Fig.\u00a02a,d,g). Upon morphological examination at 24\u00a0h post-AAA injection, eyes receiving the low (20\u00a0\u03bcg\/\u03bcl) dose appeared normal (Fig.\u00a02b). Conversely, recovery was not evident at this time following application of either 100 or 320\u00a0\u03bcg doses; vacuoles remained in both, and eyes receiving the highest dose displayed marked morphological abnormalities (Fig.\u00a02e,h). Following administration of these higher doses, the OLM also appeared disrupted, as indicated by the presence of cell bodies in the segment layers (Fig.\u00a02e,h). By 3\u00a0weeks post-administration, retinae receiving 20\u00a0\u03bcg\/\u03bcl AAA appeared normal by morphological assessment, whilst those receiving 100\u00a0\u03bcg\/\u03bcl AAA, exhibited a small number of remaining vacuoles (Fig.\u00a02c,f). In eyes that received the highest dose (320\u00a0\u03bcg\/\u03bcl) retinal morphology remained severely disrupted at 3\u00a0weeks, including a loss of retinal lamination, sustained disruption of the outer limiting membrane and retinal thinning (Fig.\u00a02i). An apparent disruption of the inner limiting membrane was also noted in these eyes, but was not observed with lower doses of AAA.\nThus, the optimum dose of AAA required for transient OLM disruption by intravitreal injection was determined to be 100\u00a0\u03bcg\/\u03bcl. This dose invariably resulted in OLM disruption, as determined by histological assessment, but with consistent recovery of OLM integrity and relatively normal retinal morphology by 3\u00a0weeks. The early morphological effects of AAA in the retina described here concur with the few published studies on subcutaneous and intravitreal injections in mice and rats (Pedersen and Karlsen, 1979; Rich et\u00a0al., 1995). In addition, our findings also demonstrate that these effects can be induced in a reversible manner.\n3.2\nWindow of OLM disruption\nHaving determined the optimal dose of AAA for a reversible and consistent OLM disruption (100\u00a0\u03bcg\/\u03bcl, administered intravitreally), we also wished to identify the time point at which this disruption was maximal. We therefore examined retinal morphology, OLM integrity and apoptosis in treated retinae at 24, 48, 72\u00a0h, 1 and 2\u00a0weeks post AAA administration (N\u00a0=\u00a03 for each time point).\nThe effect of AAA pre-treatment on host photoreceptor morphology was examined in the adult Nrl.gfp+\/+ mouse, which expresses GFP in rod photoreceptors (Akimoto et\u00a0al., 2006; Mears et\u00a0al., 2001; Swain et\u00a0al., 2001). Disruption of the ONL was clearly identifiable by the movement of GFP-labelled cells into the inner and outer segment layers of the retina (Fig.\u00a03a). At 24\u00a0h post AAA injection, photoreceptor morphology was largely normal, with correctly orientated inner and outer segments and only occasional photoreceptor cell bodies in the subretinal space. By 48\u00a0h, the laminar organization of the retina was disrupted and more photoreceptors were displaced from the ONL. Some retinal folds were evident at 72\u00a0h post-injection and the organization of the inner and outer segments was significantly disrupted. Recovery of the retina was first seen at 1\u00a0week post AAA injection, as lamination returned and photoreceptor inner and outer segments regained nearly normal orientation. Photoreceptor organization appeared largely normal by 2\u00a0weeks post injection.\nThe displacement of photoreceptor cell bodies into the subretinal space post AAA administration suggested that the OLM was disrupted. To confirm this, we used antibodies directed against ZO-1, an adherens junction protein located at the OLM (see Fig.\u00a01b,c). Staining showed that the OLM was largely intact at 24 and 48\u00a0h post-AAA administration, with the exception of a few localized areas of disruption (Fig.\u00a03b). The peak of OLM disruption was present at approximately 72\u00a0h post-administration, as demonstrated by a substantial lack of ZO-1 staining at sites where photoreceptors had dropped out of the ONL. By 1\u00a0week, the OLM had largely reformed, with disturbed but continuous adherens junctions seen. Normal staining was seen by 2\u00a0weeks post AAA administration.\nWhile AAA is a glial-specific toxin (Karlsen et\u00a0al., 1982; Pedersen and Karlsen, 1979), we wished to establish whether or not other cell types, particularly photoreceptors, might be affected by the changes induced by AAA (Fig.\u00a03c). Retinal sections encompassing the area of AAA administration were stained with an ApopTag Red Apoptosis detection kit and the number of apoptotic nuclei per section quantified. Apoptosis staining revealed that at 24 and 48\u00a0h post AAA administration, the vast majority of apoptotic nuclei were present in the inner nuclear layer (25\u00a0\u00b1\u00a02 cells per retinal section at 24\u00a0h and 31\u00a0\u00b1\u00a05 cells per retinal section at 48\u00a0h, n\u00a0=\u00a015 sections), most likely M\u00fcller cells. Apoptosis was absent in the ONL at these time-points. By 72\u00a0h, however, the peak of morphological and OLM disruption, some apoptotic cells were present in the ONL (61\u00a0\u00b1\u00a05 cells per retinal section, n\u00a0=\u00a015). Less than 12\u00a0\u00b1\u00a02 apoptotic cells per retinal section remained in the ONL at 1\u00a0week, reducing to 2\u00a0\u00b1\u00a01 cells by 2\u00a0weeks post-administration (n\u00a0=\u00a012). Occasionally, apoptotic profiles were seen in the ganglion cell layer. However, these were most likely the result of the intravitreal injection procedure itself, as similar levels were seen in control PBS injected eyes. Note that little apoptosis was observed in any layers of the retina in the rest of the eye, away from the site of AAA injection, at all times points examined. Neuronal toxicity after d,l- AAA treatment is thought to be a secondary effect due to the loss\/inhibition of the supporting glial cells in the retina or brain (Tsai et\u00a0al., 1996). This concurs with our results, since apoptosis in the ONL was observed only after apoptosis of cells in the INL.\nThe impact of AAA administration on photoreceptor morphology and OLM integrity were further examined using electron microscopy. Retinae were examined 72\u00a0h and 1\u00a0week post AAA administration and compared with PBS injected controls (Fig.\u00a04). In PBS injected control retinae, the OLM was intact and photoreceptor inner and outer segment morphology was normal at 72\u00a0h post-administration (Fig.\u00a04a). Conversely, the integrity of the OLM was lost in many regions of the AAA treated retinae and photoreceptor nuclei were mislocalized in the outer segment layer. The photoreceptor inner and outer segments were significantly disturbed, vacuoles were present and there was a loss of outer segments (Fig.\u00a04b). By 1\u00a0week post AAA treatment, retinae showed significant recovery of OLM integrity, together with fewer vacuoles in the inner segment region and recovery of inner and outer segment organization (Fig.\u00a04c).\nTogether, these findings demonstrate that intravitreal administration of 100\u00a0\u03bcg AAA in the mouse causes a transient, reversible disruption of the OLM approximately 72\u00a0h post injection.\n3.3\nCell integration with OLM disruption\nWe next sought to determine whether or not disruption of the OLM permits greater levels of integration of transplanted photoreceptor precursor cells. To test this, animals received a subretinal injection of early post-natal Nrl.gfp+\/+ donor cells 72\u00a0h after intravitreal administration of AAA, when OLM disruption is maximal. To control for the intravitreal injection, the contralateral eye received a subretinal injection of donor cells 72\u00a0h after intravitreal administration of PBS. Three weeks post-transplantation, retinae were sectioned and the total number of Nrl.gfp cells integrated within the ONL was quantified. Nrl.gfp expression is restricted to rod photoreceptors (Akimoto et\u00a0al., 2006; Mears et\u00a0al., 2001; Swain et\u00a0al., 2001) and provides genetic evidence that any transplanted integrated cells within the ONL are photoreceptors. We have previously demonstrated that these integrated donor cells are light sensitive and form functional synaptic connections with downstream targets in the recipient retina. Transplanted non-GFP-positive cells remained in the subretinal space, forming a cell mass. Very few integrated cells, that were not photoreceptors, have previously been observed in transplants using donor tissue from C57Bl\/6 GFP+\/\u2212 mice (MacLaren et\u00a0al., 2006).\nIn all animals examined, the eyes that received pretreatment with AAA showed a significantly higher number of integrated donor cells within the ONL compared with their contralateral PBS-treated counterparts (AAA-treated 1088\u00a0\u00b1\u00a0172.28 cells, vs. PBS control 523\u00a0\u00b1\u00a0106.49 cells; N\u00a0=\u00a09, P\u00a0=\u00a00.009, paired t-test; Fig.\u00a05a). The number of integrated photoreceptors was also increased compared to previous cell count data from wildtype mice (control 691\u00a0\u00b1\u00a0209.50 cells; N\u00a0=\u00a05). To exclude variability in cell counts resulting from inter-animal variation, the ratio of the number of cells in the AAA treated eye compared with the contralateral control eye was also calculated for each individual mouse. This revealed an average three-fold increase in the number of transplanted photoreceptors for each animal as a result of AAA treatment (3.0\u00a0\u00b1\u00a00.75; N\u00a0=\u00a09; Fig.\u00a05b,c).\nTo determine whether this statistically significant increase in the number of integrated cells coincided with OLM disruption, we also performed cell transplants at 1\u00a0week post-AAA administration when the OLM disruption had largely recovered. Transplantation at this stage showed a mean fold-difference of less than 1.3-fold between the AAA treated eye compared with the control PBS-treated eye (Fig.\u00a05b) and the number of integrated cells was not significantly higher post-AAA administration (AAA-treated 363\u00a0\u00b1\u00a052.22 cells, vs. PBS control 527.78\u00a0\u00b1\u00a094.89; N\u00a0=\u00a09, P\u00a0=\u00a00.34, paired t-test; Fig.\u00a05a).\nWe investigated whether AAA treatment enhanced reactive gliosis as this may affect cell integration. Previous studies have shown that AAA affects astrocytes in both the brain and eye (Ishikawa and Mine, 1983; Khurgel et\u00a0al., 1996) and Rich et\u00a0al. (1995) demonstrated up-regulation of GFAP in M\u00fcller cell processes, after chronic systemic administration of AAA during development (P3\u20139). GFAP is a marker of reactive gliosis and astrocytes, and we examined GFAP immunohistochemistry after AAA treatment and compared it with PBS injected controls. Here the doses of AAA used were much lower than those in the studies cited above and were administered as single intravitreal injections. We observed no difference in GFAP staining between the AAA and PBS treated eyes at either time point, indicating that AAA had little effect on M\u00fcller cell activation or astrocyte toxicity (Supplementary Fig.\u00a01).\nPre-treatment with either AAA or PBS had no identifiable effect on the morphology of integrated photoreceptors. In both the PBS control and the AAA treated retinae, integrated photoreceptors appeared fully differentiated and morphologically indistinguishable from those we have described previously (MacLaren et\u00a0al., 2006) (Fig.\u00a05d).\n4\nDiscussion\nHere we demonstrate that the OLM in the adult mouse retina can be transiently disrupted by the intravitreal administration of AAA. OLM disruption is maximal approximately 72\u00a0h post administration. When combined with precursor cell transplantation, this time point correlates with a significantly enhanced level of transplanted photoreceptor cell integration into the recipient ONL, compared with sham-injected controls. These findings suggest that the OLM represents a natural barrier to the successful integration of photoreceptor precursor cells transplanted into the subretinal space. Consideration of the OLM may therefore be important in any future clinical photoreceptor transplantation strategies directed towards retinal repair.\n4.1\nEffect of AAA on M\u00fcller cells and the OLM\nWe show for the first time that the glutamate analogue, AAA, can be used to induce a transient disruption of both M\u00fcller glial morphology and OLM integrity in the adult mouse retina. AAA appears to disrupt the OLM by exerting a largely M\u00fcller glial-specific transient toxicity (Karlsen et\u00a0al., 1982; Olney, 1982; Pedersen and Karlsen, 1979). The M\u00fcller glia recover well, with only low levels of cell death observed in the first week after AAA administration. Death of M\u00fcller glia following exposure to AAA has previously been observed in the carp retina, following an injection of much larger doses than those used in the present study (Sugawara et\u00a0al., 1990). In addition to M\u00fcller cells, the other glial cell type of the retina, astrocytes, also exhibit toxicity in response to AAA in the brain and eye (Ishikawa and Mine, 1983; Khurgel et\u00a0al., 1996). The mechanism of action of AAA on M\u00fcller glia is uncertain, although the morphological damage includes swelling and nuclear changes (Pedersen and Karlsen, 1979). Suggested modes of action include: inhibition of glutamate uptake, resulting in possible neuroexcitotoxicity (Tsai et\u00a0al., 1996); inhibition of the cystine\/glutamate transporter expressed by M\u00fcller glia, leading to reduced levels of intracellular glutathione and oxidative stress (Kato et\u00a0al., 1993); and uptake of AAA itself by M\u00fcller glia, and subsequent cytotoxicity via metabolic stress (Chang et\u00a0al., 1997; McBean, 1994). The downstream effects of AAA occur in a time specific manner, resulting first in the early gliotoxicity, followed by an apparent secondary neurotoxicity, seen with increased dose (Tsai et\u00a0al., 1996).\n4.2\nOLM disruption and photoreceptor integration\nCells transplanted into the subretinal space of adult mice are capable of correctly integrating within the recipient ONL and forming functional, synaptically-connected photoreceptors, if these cells are at the appropriate post-mitotic precursor stage of development (MacLaren et\u00a0al., 2006). However, the numbers integrating are below that likely to be required for a clinical therapy. Given that immature neurons and neural stem cells are intrinsically capable of migration (Hagg, 2005; Komuro and Rakic, 1998a; Nadarajah and Parnavelas, 2002; Parnavelas et\u00a0al., 2002; Pearson et\u00a0al., 2005), it is likely that barriers exist within the adult retina that impede greater numbers of donor cells from migrating and integrating, as demonstrated by the failure of graft integration in recipients where the host photoreceptor layer is largely intact (Zhang et\u00a0al., 2003a). Extensive migration into the neural retina is largely restricted either to severely degenerated retina (Zhang et\u00a0al., 2004) or, in wildtype animals, to areas where there is significant disruption to the ONL (Ghosh et\u00a0al., 1999; Gouras et\u00a0al., 1994; Zhang et\u00a0al., 1999). Zhang and colleagues concluded that breaks in the OLM and\/or loss of the photoreceptor component of the OLM were necessary for the formation of bridging fibres between graft and host tissues (Zhang et\u00a0al., 2003a). Similarly, localized mechanical disruption of the retina may facilitate migration of dissociated cells from the subretinal space, as observed with neural stem cells (Nishida et\u00a0al., 2000).\nThe OLM consists of unique heterotypic (involving M\u00fcller cells and photoreceptors) or homotypic (between M\u00fcller cells) adherens junctions (Paffenholz et\u00a0al., 1999; Williams et\u00a0al., 1990). We have shown that AAA causes marked morphological changes within M\u00fcller glia, leading to the disruption of these adherens junctions. Furthermore, a number of photoreceptor nuclei became displaced within the segment layer, outside the OLM, which normally acts to retain them (Rich et\u00a0al., 1995). This suggests that if cells can exit the ONL following disruption of the OLM, the converse is also likely\u2014cells in the subretinal space can migrate more readily into the ONL when the OLM is disrupted. Accordingly, pre-treatment with AAA leads to significantly greater numbers of donor cells integrating following transplantation, compared with controls. Importantly, this effect was only seen if cells were transplanted 72\u00a0h after AAA administration, i.e. only when OLM disruption is at its peak.\nOLM disruption causes a significant increase in photoreceptor integration following transplantation. However, the increase (3-fold) is less than might be expected if the OLM were the only factor limiting donor cell migration, suggesting that manipulation of additional factors is likely to be required to optimize integration. A number of factors need consideration in relation to our findings. First, the donor cell population is heterogeneous; only a small proportion of cells in the early post-natal retina will be at the appropriate stage and specification for transplantation (i.e. photoreceptor precursors), so cell number may be a limiting factor. This could be augmented in the future by pre-selecting photoreceptor precursors, provided efficient cell sorting methods can be established. Second, M\u00fcller glia may actually facilitate donor cell migration into the ONL of the recipient retina or play a role in supporting rod differentiation, and the transient toxic effects of AAA may impede or reduce these supportive functions. Thus, while AAA may aid integration by disrupting the integrity of the OLM, it may conversely limit that enhancement by disturbing this supportive glial scaffold. M\u00fcller glia are also known preferentially to support rod process outgrowth (Kljavin and Reh, 1991). However, because all integrated photoreceptor cells in the AAA-treated retinas were morphologically identical to controls and those previously described (MacLaren et\u00a0al., 2006), this suggests that M\u00fcller cells are at least partially dispensable, which is consistent with the observation that AAA-treatment does not affect normal photoreceptor development in early post-natal mice (Rich et\u00a0al., 1995).\nFinally, M\u00fcller glia up-regulate the expression of GFAP and other intermediate filament proteins under stress (Bignami and Dahl, 1979; Bjorklund et\u00a0al., 1985). It is possible that the physiological changes induced by AAA could trigger aspects of the glial scarring pathway, which may subsequently impede the migration of transplanted cells. Recent work investigating retinal cell transplantation by Kinouchi et\u00a0al. (2003) has demonstrated that cell migration, to the ganglion cell layer is enhanced in mice lacking GFAP and vimentin, two intermediate filament proteins found in reactive M\u00fcller glia and astrocytes. M\u00fcller cell morphology was stated to appear normal in these mice and spanned the entire retina. They did not, however, observe increased cell integration into other retinal layers, including the outer nuclear layer (Kinouchi et\u00a0al., 2003).\n4.3\nTherapeutic implications\nThe results described here demonstrate a proof of concept; namely that disruption of the OLM increases the integration of transplanted photoreceptor precursor cells. However, the compound used, AAA or the l-AAA enantiomer have been shown to have other significant effects in the retina, albeit at higher doses than those used in this study. These include M\u00fcller cell necrosis, light insensitivity and suppression of the electroretinographic b-wave (Kato et\u00a0al., 1990; Pedersen and Karlsen, 1979; Sugawara et\u00a0al., 1990). Therefore, AAA is highly unlikely to be of therapeutic value. It will be of considerable interest to identify alternative reagents that can induce a specific, reversible disruption of OLM integrity without impacting on the function of the retina. It is important to note that cystoid macular oedema (CME) is a condition seen in the end stages of many diseases of the outer retina, such as retinitis pigmentosa and diabetic maculopathy. Microscopic examination of pathological specimens has shown that CME represents an intra-cytoplasmic swelling (oedema) of M\u00fcller cells in the foveal region (Yanoff et\u00a0al., 1984) which is similar to the effects of AAA described in this study. Based on the results of our experiments, it is therefore not inconceivable that the OLM disruption resulting from CME may make the diseased human fovea a particularly favourable site for future retinal cell transplantation strategies.","keyphrases":["outer limiting membrane","photoreceptor","mouse","cell integration","stem cells","m\u00fcller cell","retinal transplantation"],"prmu":["P","P","P","P","P","P","R"]}
{"id":"Eur_Spine_J-2-2-1602185","title":"In-vivo demonstration of the effectiveness of thoracoscopic anterior release using the fulcrum-bending radiograph: a report of five cases\n","text":"Thoracoscopic anterior release of stiff scoliotic curves is favored because of its minimally invasive nature. Animal and human cadaveric studies have shown that it can effectively improve spinal flexibility in non-scoliotic spines; however it has not been demonstrated to be effective in actual patients with scoliosis. The fulcrum-bending radiograph has been shown to accurately reflect the post-operative correction. To demonstrate that the flexibility was increased after the anterior release; five patients with idiopathic thoracic scoliosis who underwent staged anterior thoracoscopic release and posterior spinal fusion were assessed using the fulcrum-bending radiograph. The average number of discs excised was four. Spinal flexibility as revealed by the fulcrum-bending technique, was compared before and after the anterior release. The patients were followed for an average of 4 years (range 2.2\u20134.9 years). Fulcrum-bending flexibility was increased from 39% before the thoracoscopic anterior spinal release to 54% after the release (P<0.05). The average Cobb angle before the anterior release was 71\u00b0 on the standing radiograph and 43\u00b0 with the fulcrum-bending radiograph. This reduced to 33\u00b0 on the fulcrum-bending radiograph after the release, and highly corresponded to the 30\u00b0 measured at the post-operative standing radiograph and at the latest follow-up. Previous animal and cadaveric studies demonstrating the effectiveness of thoracoscopic anterior release did not have scoliosis. We are able to demonstrate in patients with adolescent idiopathic scoliosis, that thoracoscopic anterior spinal release effectively improves the spinal flexibility.\nIntroduction\nIn the management of patients with stiff thoracic scoliosis and kyphosis, anterior spinal release is helpful in increasing the spinal flexibility and therefore, the result of deformity correction. Excision of intervertebral discs through open thoracotomy is the preferred method in the past. However, cutting of the chest-wall muscles is associated with complications such as reduced ventilation, post-operative atelectasis, extensive and painful scars, blood loss, and prolonged hospital stay [7].\nVideo-assisted thoracoscopic surgery (VATS) for anterior release of the spine is becoming increasingly more popular due to its minimally invasive nature [1, 19, 25]. However, opponents do not believe that VATS is effective in improving the spinal flexibility as it is difficult to perform a radical discectomy, and to a lesser extent, rib-head excision by thoracoscopy [5, 23]. While VATS supporters have used animals and cadavers to demonstrate the effectiveness of thoracoscopic spinal release [6, 20], these studies were performed in spines that did not have scoliosis.\nThe fulcrum-bending radiograph, obtained with the patient lying sideways hinging over a fulcrum provides a simple and reproducible technique for the in-vivo assessment of spinal flexibility. It can accurately predict before surgery, the amount of correction that can be achieved by modern segmental spinal instrumentations [4, 11, 16, 17]. Using this method, one can assess the effectiveness of an anterior release by either directly comparing the spinal flexibility (as reviewed by the fulcrum-bending radiograph) before and after an anterior release, or indirectly by comparing the predicted correction by the pre-release fulcrum-bending radiograph and the actual correction achieved by the anterior release and posterior fusion. Either way, differences in the measured Cobb angle would be attributable to the effect of the thoracoscopic release.\nIn our institution, a number of thoracoscopic anterior release and posterior fusion surgeries for thoracic idiopathic scoliosis were staged. This gave the opportunity to perform the fulcrum-bending radiograph before and after the anterior release, thereby allowing direct assessments of flexibility changes as a result of the thoracoscopic procedure.\nMaterials and methods\nBetween 1997 and 1999, five patients with idiopathic scoliosis requiring anterior release for stiff thoracic curves were prospectively investigated. The authors define stiff curves as those which have a Cobb angle of more than 40\u00b0 with the fulcrum-bending radiograph; this was arbitrarily used as we felt that the residual curves of over 40\u00b0 gave unacceptable cosmetic results, and therefore a flexibility-modifying procedure would be indicated. The mean age at the time of operation was 23.0\u00a0years (range 13.9\u201335.3). According to the King\u2019s classification [10], there were one type I, two type II, and two type III curves. For the patient with type I curve, both thoracic and lumbar curves were corrected and fused, and for the type II and III curves, only the thoracic curves were instrumented. According to Lenke\u2019s classification [12], there were three type I, one type III, and one type V curve.\nAll the patients underwent an anterior thoracoscopic release, followed by a second-stage posterior instrumented correction, and fusion 1\u00a0week later. The instrumentations used for the posterior spinal fusion were Texas Scottish Rite Hospital (TSRH, n=2; Sofamor-Danek, Memphis, TN, USA), ISOLA (n=2; Acromed Corp., Cleveland, OH, USA), and CD-Horizon (CD-H: n=1; Sofamor-Danek, Memphis, TN, USA) systems. The mean number of thoracic discs resected was four, ranging from 3 to 5 (Table\u00a01). Posterior fixation was done by a pre-dominant hook construct using one upper and two intermediate hooks, and either a hook or pedicle-screw at the lowest fusion level.\nTable\u00a01General patient dataCase 1Case 2Case 3Case 4Case 5King\u2019s typeIIIIIIIIIIIAge (years) at operation21.513.925.435.219.1Levels releasedT7-T10T7-T11T6-T10T7-T12T6-T10Number of discs released34454Instrumentation used TSRHTSRHISOLACDISOLA\nThe thoracoscopic anterior release was performed with the patient under general anesthesia. The technique involved a discectomy by making a large annular window over the convex side of the scoliosis; the whole nucleus and cartilaginous end-plates were removed. Excision of the posterior annulus to the posterior longitudinal ligament is always attempted but is usually successful only at the apical discs. Rib-head excision was not performed.\nThe effectiveness of the thoracoscopic anterior release in increasing spinal flexibility was investigated by two methods. First by a direct comparison of the Cobb angle measured from a pre-release fulcrum-bending radiograph with that of the post-release fulcrum-bending radiograph. Second, by an indirect method, comparing the pre-release fulcrum-bending radiograph with the final post-operative correction. As the pre-operative fulcrum-bending radiograph has been reported to be able to accurately predict the post-operative coronal deformity correction for posterior surgery [4, 11, 17], the difference of the Cobb angles between the pre-operative fulcrum-bending radiograph (which predicted the correction for the posterior surgery alone) and the post-operative standing X-ray (which shows the actual correction achieved by combined anterior release and posterior instrumentation) will indirectly demonstrate the in vivo effect of the anterior thoracoscopic spinal release. Statistical analyses were carried out using the Student\u2019s t-test, with a significance level of (P<0.05).\nThe \u201cfulcrum flexibility\u201d, a measure of the spinal flexibility as revealed by the fulcrum-bending radiograph [11, 17] was calculated based on the following formula:  where FB stands for fulcrum bending.\nThis relationship was used to directly assess the changes in flexibility as a result of the anterior release.\nResults\nThe patients were followed for an average of 4\u00a0years (range 2.2\u20134.9\u00a0years). The mean fulcrum flexibility before the anterior release was 39%; it increased by 15\u201354% after the anterior thoracoscopic release (P<0.05).\nThe mean pre-operative Cobb angle on the postero anterior (PA) standing radiograph was 71\u00b0, the mean pre-operative fulcrum-bending angle was 43\u00b0, the mean post-release fulcrum-bending angle was 33\u00b0, and the actual mean Cobb angle after combined anterior thoracoscopic release and posterior surgery was 30\u00b0. The latter correlated with the post-release fulcrum-bending result (P=0.09), and was significantly different (P<0.05) from the pre-release fulcrum-bending result to suggest that the thoracoscopic anterior release can effectively improve the surgical correction of the coronal curve. On an average, four discs were excised per patient, and as the mean improvement in correction is 13\u00b0, it suggested that resection of one disc resulted in a mean improvement in correction by approximately 3\u00b0 (Table\u00a02, Fig.\u00a01).\nTable\u00a02Measured Cobb angles for each case (For case 5, it was not possible to perform a post-release fulcrum-bending XR because of wound pain)Case 1Case 2Case 3Case 4Case 5MeanPre-operative AP standing 657675786171Pre-release fulcrum bending434145454043Post-release fulcrum bending35284030N\/A33AP standing latest follow-up322830282629Fig.\u00a01\u00a0aPre-operative anteroposterior standing radiograph of case 2, showing a 76\u00b0-curve from T6 to T12. b Pre-release fulcrum-bending radiograph showing a correction to only 41\u00b0. c Fulcrum-bending radiograph taken 1\u00a0week after an anterior thoracoscopic release of four levels, showing an improvement in the flexibility to 28\u00b0. d Post-operative standing radiograph taken 1\u00a0week after the posterior correction, showing the curve correction to 29\u00b0\nThe immediate post-operative Cobb angle of 30\u00b0 (data not shown) was well predicted by the post-release fulcrum-bending radiograph of 33\u00b0. There was no significant change in this correction at the latest follow-up (Table\u00a02).\nDiscussion\nAnterior spinal release by open thoracotomy has been extensively used in the past to help improve spinal flexibility in stiff thoracic scoliosis. Its role has been increasingly taken over by VATS as the latter is associated with a lower morbidity [8, 21]. However, some surgeons feel that an anterior thoracoscopic release is not as effective as an open release. This is because they believe that a successful anterior spinal release requires a rib-head resection and a radical discectomy, which cannot be easily performed through VATS [5, 23]. Although studies using animals and cadavers have demonstrated that a thoracoscopic release can improve spinal flexibility [6, 20], it was not performed on actual patients with scoliosis. Thus, there is no definitive proof in human patients with scoliosis that the thoracoscopic spinal release is effective. Although some clinical studies have demonstrated that combined VATS and posterior spine fusion resulted in good scoliosis correction [2, 21, 22], they did not assess the actual flexibility of the scoliosis and therefore were not able to directly demonstrate that the thoracoscopic release added to the spinal flexibility.\nThe use of the fulcrum-bending radiograph, which reflects the spinal flexibility and accurately predicts the post-operative coronal deformity correction, provides an opportunity to assess the effectiveness of thoracoscopic release in vivo [24, 27, 28] The five cases included in this study had stiff curves, with a pre-operative fulcrum-bending angle of more than 40\u00b0, a mean pre-operative fulcrum flexibility of only 39%, and a mean Cobb angle of 71\u00b0. As these cases had staged surgery, we were able to obtain a post-release fulcrum-bending radiograph for comparison with the pre-release and post-operative Cobb angles. After anterior thoracoscopic release, flexibility was increased from 39 to 54%, thus representing a direct proof that the procedure can improve the spinal flexibility, in our cases by an average of 15%.\nOne patient (Case 5) was unable to lie on the fulcrum to perform a post-release fulcrum-bending radiograph due to wound pain. Nevertheless, case 5 has been included in the analysis, as a comparison of the pre-release fulcrum-bending radiograph with the post-operative radiograph will still provide an indirect evidence on the success of a thoracoscopic release (Table\u00a02). For those who were able to lie on the fulcrum, we found the technique reliable. In this limited series of four cases, the post-release fulcrum-bending radiograph correlated well with the post-operative result.\nIt should be noted that the previous work on the fulcrum-bending radiograph are based on the use of hooks and hybrid systems only [4, 15]. The predictability of this method with reference to the use of pedicle-screw fixation is not known. It is widely believed that pedicle-screw systems give a superior degree of correction when compared to hook systems, and may obviate the need to perform anterior releases. However, there is no published data, which directly compares the two systems, taking into account the spinal flexibility.\nThe authors are aware of two different ways to measure Cobb angles before and after surgery. One method is to determine the Cobb angles in the pre-operative standing radiograph, and then to use the same levels throughout, although in the post-operative radiograph, the same measured levels may no longer be the most \u201ctilted\u201d vertebra. The alternative method is always a measure from the most \u201ctilted\u201d levels even though the levels may change between pre- and post-operative radiographs. While the authors prefer the former method, use of the latter method would not alter the results. Using the presented case as an example (Fig.\u00a01), the pre-operative Cobb angle from T6 to T12 was 76\u00b0, the pre-release Cobb angle from T7 to T10 was 50\u00b0, the post-release Cobb angle from T8 to T10 was 40\u00b0, and the final Cobb angle from T7 to T10 was 40\u00b0.\nThe technique of anterior spinal release is different amongst different surgeons, while some do only a discectomy; others routinely remove the rib-heads, and even disrupt the posterior longitudinal ligament. The use of VATS limits the number of structures that can be easily released due to limitations in visualization and access to all disc levels. This study is not comparing the open thoracotomy and release with the thoracoscopic anterior release; this is because no data is available for the former. Moreover, an evaluation of open release versus thoracoscopic release using the present method is not possible, as patients undergoing the open release would still have a painful wound, which would prevent them from lying sideways over a fulcrum.\nOne potential pitfall of this study is that three different types of implants were used, while the original study on the fulcrum-bending radiograph was based on TSRH [4]. It maybe possible that different implants vary in their ability to correct scoliosis and therefore invalidate the indirect assessment. However, the authors feel that this is unlikely because it has been demonstrated by the same group that there was no significant difference in the ability of different instrumentation systems to correct thoracic scoliosis [18]. Moreover, the direct comparison of fulcrum flexibility before and after an anterior release would still stand.\nThis study was performed in the early part of our experience with thoracoscopic anterior release, hence relatively few discs were released and the surgeries were staged. However, it did serve the purpose of demonstrating that thoracoscopic anterior release does result in an improvement in spinal flexibility. With increasing experience, the authors tend to release five to six discs per patient and the posterior surgery is performed on the same day.\nWith the advent of new techniques and instrumentation, the indications for a thoracoscopic release may change in time. In particular, pedicle-screw systems appear to provide a better correction of large magnitude curves [9, 13, 26]. However, to date, there are no randomized studies comparing hooks versus screw systems, nor a correlation of the correction to spinal flexibility assessment, such as the fulcrum-bending correction index [17, 18]. Additionally, the use of anterior instrumentation systems may mean that such surgeries are carried out as a single-stage anteriorly, avoiding the need for posterior surgery [3, 14].\nIn summary, this is the first study to provide a direct in vivo evidence demonstrating the effectiveness of thoracoscopic anterior release in improving the spinal flexibility in patients with scoliosis.","keyphrases":["anterior release","spinal flexibility","adolescent idiopathic scoliosis","video-assisted thoracoscopic surgery","thoracoscopy"],"prmu":["P","P","P","P","P"]}
{"id":"Qual_Life_Res-3-1-2039790","title":"Different perceptions of the burden of upper GI endoscopy: an empirical study in three patient groups\n","text":"Background Few studies have evaluated patients\u2019 perceived burden of cancer surveillance tests. Cancer screening and surveillance, however, require a large number of patients to undergo potentially burdensome tests with only some experiencing health gains from it. We investigated the determinants of patients\u2019 reported burden of upper gastrointestinal (GI) endoscopy by comparing data from three patient groups.\nIntroduction\nOpportunities for screening and surveillance of premalignant conditions have increased and will increase in the future. However, such interventions can be burdensome, and, as in any screening situation, the number of subjects exposed to this burden is often much higher than the number of subjects experiencing the beneficial health effects of the screening [1].\nUpper gastrointestinal (GI) endoscopy is commonly used to diagnose and treat patients with a range of conditions and symptoms. Complications related to upper GI endoscopy are rare, and it is considered to be a safe procedure [2, 3]. Patients with Barrett esophagus (BE), a premalignant condition mostly without physical symptoms but associated with an increased risk of developing esophageal adenocarcinoma of 0.5% per year, are recommended to undergo regular biennial endoscopic surveillance for early detection of esophageal cancer [4]. All patients participating in surveillance experience the pain and discomfort of biennial upper GI endoscopy, whereas progression to adenocarcinoma occurs only in a minority of BE patients [5\u20138] and undisputable evidence that surveillance prolongs survival is still lacking [9\u201312]. Hence, the patients\u2019 perceived burden of upper GI endoscopy testing needs to be taken in to account in evaluating the health benefits of surveillance of subjects with BE.\nIn some situations, there is a trade-off between the effectiveness of screening (or surveillance) and the test uptake. For example, colorectal cancer screening using sigmoidoscopy is more effective than faecal occult blood testing [13]. At present, this trade-off is not relevant for surveillance of BE because a less burdensome test than upper GI endoscopy is not available, but the recognition that upper GI endoscopy is burdensome may prompt a reconsideration of the frequency of surveillance. Ongoing studies aim to identify groups of BE patients at lower risk of developing esophageal cancer than others, so that offering less frequent surveillance may be warranted [14].\nAt the patient level, empirical data on perceived burden of upper GI endoscopy can be used in the process of informing subjects with BE who consider participation to a surveillance programme. In a general sense, empirical data of the patients\u2019 perceived burden of testing may contribute to subjects\u2019 informed decision-making on participation (or non-participation) to screening or surveillance and hence, to quality of health care [15].\nStudying the determinants of patients\u2019 perceived burden of upper GI endoscopy, e.g. by comparing data from different patient groups, may allow for the identification of patient groups who are likely to experience more pain or discomfort than others. This information can be used in practice guidelines, e.g. on provision of sedation to prevent pain and discomfort, or other types of patient support. Studying determinants of patients\u2019 perceived burden is of additional interest from the perspective of evaluation research. If patients\u2019 perception of the burden of endoscopy differs by the context of e.g., surveillance or diagnostic work-up, the generalisability of data from one context to another is limited.\nOur previous work [16] has shown that BE patients under regular surveillance perceive upper GI endoscopy as burdensome. They experienced anxiety and discomfort, but hardly reported pain or symptoms. We analysed potential determinants of the perceived burden of upper GI endoscopy by comparing BE patients with two additional patient groups, i.e., patients with non-specific upper GI symptoms (NS) and patients with a recent diagnosis of cancer of the upper GI tract (CA).\nMethods\nEthics approval\nThe Medical Ethical Review Board of Erasmus MC\u2014University Medical Center Rotterdam, The Netherlands, approved of the study (MEC 03.1064; October 9, 2003).\nPatients\nPatients undergoing upper GI endoscopy for surveillance of BE were participants of an ongoing trial (CYBAR), whose endoscopic burden was previously reported [16]. Inclusion criteria were: BE segment of 2\u00a0cm or more confirmed by a histological diagnosis of intestinal metaplasia, absence of high-grade dysplasia and carcinoma, willingness to adhere to endoscopic surveillance, ability to read the Dutch language and informed consent.\nPatients with non-specific upper GI symptoms (NS) were referred for endoscopy by their respective GPs because of non-specific upper GI symptoms. They needed to be able to read the Dutch language, provide informed consent, not to have \u201calarm symptoms\u201d such as hematemesis, melena, or dysphagia, and not be diagnosed with BE previously.\nPatients with a recent diagnosis of upper GI cancer (CA) were referred for upper GI endoscopy plus ultrasonography (EUS) to determine therapeutic options. Ability to read the Dutch language and to give informed consent was also required in these patients. Patients were recruited from one academic and two regional hospitals for BE, two regional hospitals for NS and in one academic hospital for CA.\nEndoscopic procedure\nBE and NS patients underwent endoscopy with adult endoscopes (Olympus GIF-Q160, Zoeterwoude, The Netherlands). In the group of cancer patients, a combined endoscopy and EUS was performed with a Olympus GF-UM160. More than 95% of patients received oral anaesthetics (Xylocain 10% spray, Astra Zeneca, Zoetermeer, The Netherlands) preceding the introduction of the endoscope. Additional sedation with 2,5-5\u00a0mg midazolam (Roche, Woerden, The Netherlands) intravenously was offered as a standard procedure to all cancer patients, but was only administered with explicit patient consent. In BE and NS patients this was not standard, but it was administered on a patient\u2019s request. Practice variations between and within countries in the use of sedation for upper GI endoscopy are common [17].\nHypotheses\nPerceived burden of endoscopy was operationalised as pain and discomfort during the procedure, symptoms afterwards and psychological distress over time. We hypothesized that subjects who had previous endoscopies may get used to it to some extent and hence report less burden. Demographic characteristics (age, sex, educational level, employment status, etc) were considered as potential confounders. We expected that BE patients may get used to regular endoscopy to some extent, and that they adhere to surveillance expecting that the test result will be reassuring. Therefore, we expected BE patients to report less discomfort and burden than the patients with non-specific GI symptoms, who had less endoscopy experience. We also expected the BE group to report less burden from the endoscopy than the cancer patients, due to the endoscopy itself (combined with EUS in the cancer patients) and the fact that cancer patients were aware of their generally bad prognosis. Table\u00a01 shows the potential determinants of perceived burden of endoscopy between patient groups.\nTable\u00a01Potential determinants of perceived burden of endoscopy for three patient groupsDeterminantBENSCA Indication for endoscopyRegular endoscopic surveillance for early detection of cancerTo find out if symptoms can be explained by e.g. a hiatal hernia or duodenal ulcer; a life-threatening diagnosis is not expected.To determine whether intentionally curative therapy is possible or notEndoscopy experienceYes, under endoscopic surveillance Limited, ranging from 0 to some Yes (by definition: at least one previous endoscopy needed to diagnose the cancer)Generic health statusGenerally healthy Mild impairment (aspecific gastrointestinal symptoms)Seriously impaired (cancer diagnosis)AgeIn-betweenYoungestOlderSexMore males than females Equal sex distributionPredominantly maleEndoscopy procedureNormal endoscopy procedure, sedation only on patient requestNormal endoscopy procedure, sedation only on patient requestEndoscopy combined with ultrasonography; sedation routinely offered\nQuestionnaires and measurements\nPatients were asked to complete questionnaires at different time points, i.e., one week before the endoscopy (baseline), at the day of endoscopy (just before undergoing it), one week and one month after endoscopy [16]. In order to minimize the questionnaire load for CA patients they received only two questionnaires: the first on the day of endoscopy and the other one week afterwards. Some baseline items had to be included in the \u2018endoscopy day\u2019 questionnaire in the CA group. The content of the questionnaires is described below.\nPain and discomfort \nSeparate items in the questionnaire one week after endoscopy were used to assess pain and discomfort, respectively, as experienced during the procedure, for four steps of the procedure: the introduction of the endoscope, the endoscopy itself, the removal of the endoscope, and the period directly after endoscopy. Subjects were offered three response options (\u2018no\u2019, \u2018quite\u2019 and \u2018very\u2019 painful or discomforting, respectively). Additionally, patients rated the overall burden of undergoing the endoscopy (very, somewhat, not burdensome) [16].\nSymptoms \nWe compared the prevalence of 10 symptoms experienced in the week after endoscopy with the prevalence at baseline. For CA patients, the baseline questions were asked at the day of endoscopy. Presence of throat ache, heartburn, regurgitation, flatulence or feeling bloated, vomiting, hematemesis, dysphagia for solid foods or for of liquids, diarrhea, and constipation, was assessed using four response options (not at all, one day, 2\u20133\u00a0days, 4 or more days) [16].\nPsychological distress (BE and NS patients)\nWe assessed general distress using the Hospital Anxiety and Depression scale (HAD) at all time points [18, 19]. Anxiety and depression scores of this scale range from 0\u201321, with scores of 11 or over indicating clinical, and scores between 8 and 10 indicating borderline anxiety or depression [18, 19]. We analysed the pattern of scores across measurements, assuming scores to return to normal after endoscopy. Scores from a Dutch general population sample (n\u00a0=\u00a01901; mean age\u00a0=\u00a061\u00a0year; 51% female) were available for comparison [19].\nAt baseline and at one week we also measured specific distress with the Impact of Event Scale (IES) [20, 21]. At baseline we assessed intrusive and avoiding thoughts regarding the endoscopy itself, and at one week regarding the communication of the final test result. The total scale ranges between 0 and 75, with scores of 26 or over indicating a high risk of developing a stress disorder [22].\nPsychological distress (CA patients)\nFor CA patients we omitted the HAD and the IES measures regarding the endoscopy itself, because we expected that distress in these patients was already at the top of the scale, making any additional distress caused by the procedure itself indiscernible. The IES to assess specific distress regarding the endoscopy result was included in the questionnaire at the day of endoscopy, because these patients received the endoscopy results earlier than the next questionnaire.\nDemographics and other data\nDemographic data were collected at baseline (at the endoscopy day for CA patients). The EQ\u20135D self-classifier results in a patient\u2019s classification of own health on five domains: mobility, self-care, usual activities, pain, and anxiety and depression (3 response options: no, some, severe\/ complete limitations) and a summary score [23\u201325]. We asked BE and NS patients whether this was their first, second or a later endoscopy. Whether sedation was used during endoscopy was recorded separately.\nAnalyses\nDifferences in demographic and treatment characteristics between patient groups were analysed by Chi-square tests for categorical variables or one-way analysis of variance (ANOVA) for continuous variables.\nThe items for pain and discomfort were combined into summary scores to enable adjustment for confounders and analysis of determinants, by adding up the item responses (0, 1, 2, respectively) of the 4 items (range of pain and discomfort summary scores: 0 (no pain or discomfort) to 8) [16]. The response to the single item rating of overall burden was also treated as a summary score, with a range from 0 (no burden) to two [16]. Because these summary scores had a limited number of possible values and because the data were not distributed normally, we chose to analyse them with proportional odds models [26]. These models produce odds-ratios (ORs) for cumulative probabilities of the outcome variables. Proportional odds models are a variant of simple logistic regressions, but now ORs for dichotomies at all possible cut-off levels are estimated. E.g., for a variable with three possible outcomes 1, 2, and 3, ORs are estimated for (1\u00a0+\u00a02) vs. 3 and for 1 vs. (2\u00a0+\u00a03). The OR presented represents an overall OR, that is assumed to be similar across cut-off levels. Because some of the outcome variables and determinants had 10\u201315% missing data, and there were no reasons for selective missing data, we used multiple imputation (function AregImpute in Splus 6.0) [27] so that all available information in our dataset was used. In multivariate analysis of the determinants of patients\u2019 perceived burden with the proportional odds model, we first adjusted for confounders (age, sex and employment status). Subsequently we evaluated the potential effects of the following determinants on discomfort, pain and overall burden, respectively:patient group (BE, NS or CA). This variable combines the differences in the endoscopy procedure (with or without EUS, sedation) and the indication to undergo the endoscopy. For BE and NS patients, this analysis was refined by additional separate analysis of the effect of the number of previous endoscopies (continuous, truncated at \u226520).baseline generic health status (EQ 5D summary score).whether sedation was administrated or not.baseline HAD anxiety score (not available for the CA patients).The prevalence of symptoms before and after endoscopy was compared using a method analogous to the Wilcoxon test. Responses were ranked and ANOVA was applied to the differences in these ranks [16].\nThe continuous HAD and IES scores were compared over time in SAS version 8.2 with repeated-measures ANOVA, using \u2018Proc Mixed\u2019 with REML and a compound symmetry covariance structure. Models comprised main effects of time (the measurements), confounders, determinants and interactions between determinants and time.\nProportional odds models were estimated with Splus 6.0. All other analyses were conducted in SPSS version 11.0.1.\nResults\nPatients and response\nIn total, 684 patients were eligible for inclusion: 192 BE, 365 NS and 127 CA patients. The overall response rate was 70% with 476 patients completed at least one questionnaire. The response differed by patient group; it was 180\/192 (94%) in BE patients, 214\/365 (59%) in NS patients and 82\/127 (65%) in CA patients. Most BE patient had no dysplasia (78%), 22% had low-grade dysplasia [16]. NS patients were diagnosed with hiatal hernia (45%), non-specific gastritis (25%), reflux esophagitis (20%) and some other diagnoses (e.g. ulcer, polyps; 10%). CA patients underwent endoscopy and EUS for staging of esophageal carcinoma (72%), gastric cancer (26%) or lymphoma (2%).\nDifferences between groups in mean age, sex and employment status were statistically significant (P\u00a0<\u00a00.001) (Table\u00a02). We therefore considered these variables as confounders and controlled for them in further analyses.\nTable\u00a02Patient characteristicsaBENSCADifferbNGroup18021482N.A.476Mean age (sd)62 (12)54 (16)64 (10)<0.001474Sex: male119 (66%)101 (47%)66 (80%)<0.001476EmploymentPaid employment59 (34%)85 (44%)25 (36%)<0.001438Retired87 (50%)65 (34%)38 (54%)Unpaid\/unemployed29 (17%)43 (22%)7 (10%)Civil status Married\/ together134 (77%)137 (69%)57 (80%)0.034444Never married\/ tog.13 (7%)26 (13%)3 (4%)Divorced10 (6%)23 (12%)4 (6%)Widowed18 (10%) 12 (6%)7 (10%)EducationPrimary35 (20%)37 (19%)16 (23%)0.498435Secondary95 (56%)122 (63%)43 (61%)Tertiary40 (24%)35 (18%)12 (17%)HospitalAcademic center (1)37 (21%)082 (100%)N.A.476Regional hospital (3)143 (79%)214 (100%)0Sedation: yes43 (27%)18 (9%)56 (77%)<0.001419Endoscopy numberFirst1 (1%) 99 (59%)Unknown<0.001338Second26 (15%)38 (23%)Third or later144 (84%)30 (18%)EQ\u20135D summary score0.85 (0.18)0.73 (0.22)0.77 (0.21)<0.001433N.A., Not assessedaData for the BE group were published previously [16]b\u03c7- test (categorical variables) or F-test (continuous) for differences between patient-groups\nAbout 84% of the BE patients had had two or more previous endoscopies [16], compared with 18% of the NS patients (P\u00a0<\u00a00.001). Seventy-seven per cent of the CA patients received sedation during endoscopy, compared with 27% of the BE and 9% of the NS patients (P\u00a0<\u00a00.001). The differences in the mean EQ\u20135D summary score were in the expected direction (P\u00a0<\u00a00.001) (Table\u00a02).\nPain and discomfort \nTables\u00a03\u20135 show that the patient groups differed significantly in reported discomfort, pain and overall burden of endoscopy. The p-values shown for the summary scores relate to univariate analysis of differences between the patient groups before adjustment for confounders.\nTable\u00a03Discomfort during upper GI endoscopy as reported by patientsaDiscomfortNotQuiteVerynDifferbIntroducing the endoscope141 (34%)177 (42%)99 (24%)417P\u00a0<\u00a00.001\u00a0\u00a0\u00a0\u00a0NS42 (24%)76 (43%)58 (33%)176\u00a0\u00a0\u00a0\u00a0BE64 (37%)81 (47%)27 (16%)172\u00a0\u00a0\u00a0\u00a0CA35 (51%)20 (29%)14 (20%)69Undergoing endoscopy166 (40%)162 (39%)89 (21%)417P\u00a0=\u00a00.024\u00a0\u00a0\u00a0\u00a0NS62 (35%)63 (36%)51 (29%)176\u00a0\u00a0\u00a0\u00a0BE75 (44%)72 (42%)25 (15%)172\u00a0\u00a0\u00a0\u00a0CA29 (42%)27 (39%)13 (19%)69Removing the endoscope290 (70%)90 (22%)35 ( 8%)415P\u00a0<\u00a00.001\u00a0\u00a0\u00a0\u00a0NS97 (55%)52 (30%)26 (15%)175\u00a0\u00a0\u00a0\u00a0BE144 (84%)24 (14%)3 ( 2%)171\u00a0\u00a0\u00a0\u00a0CA49 (71%)14 (20%)6 ( 9%)69Period immediately after317 (78%)71 (17%)20 ( 5%)408P\u00a0=\u00a00.348\u00a0\u00a0\u00a0\u00a0NS132 (77%)29 (17%)11 ( 6%)172\u00a0\u00a0\u00a0\u00a0BE136 (81%)29 (17%)4 ( 2%)169\u00a0\u00a0\u00a0\u00a0CA49 (73%)13 (19%)5 ( 8%)67Discomfort summary score (range: 0\u20138)Mean (sd)\u00a0\u00a0\u00a0\u00a0All2.35 (2.10)406P\u00a0<\u00a00.001\u00a0\u00a0\u00a0\u00a0NS2.92 (2.36)171\u00a0\u00a0\u00a0\u00a0BE1.88 (1.69)168\u00a0\u00a0\u00a0\u00a0CA2.07 (1.99)67aData for the BE group were published previously [16]bSignificance of differences between three groups as determined by Chi-square test (categorical variables) or proportional odds models for ordinal response data (summary score). No correction for confoundersTable\u00a04Pain during upper GI endoscopy as reported by patientsaPainNotQuiteVerynDifferbIntroducing the endoscope332 (80%)68 (16%)17 (4%)417P\u00a0=\u00a00.050\u00a0\u00a0\u00a0\u00a0NS135 (77%)29 (17%)12 (7%)176\u00a0\u00a0\u00a0\u00a0BE145 (85%)24 (14%)2 (1%)171\u00a0\u00a0\u00a0\u00a0CA52 (74%)15 (21%)3 (4%)70Undergoing endoscopy320 (77%)77 (19%)17 (4%)414P\u00a0<\u00a00.001\u00a0\u00a0\u00a0\u00a0NS141 (81%)23 (13%)10 (6%)174\u00a0\u00a0\u00a0\u00a0BE137 (81%)28 (17%)5 (3%)170\u00a0\u00a0\u00a0\u00a0CA42 (60%)26 (37%)2 (3%)70Removing the endoscope365 (88%)39 (9%)11 (3%)415P\u00a0=\u00a00.098\u00a0\u00a0\u00a0\u00a0NS152 (87%)16 (9%)6 (3%)174\u00a0\u00a0\u00a0\u00a0BE157 (92%)11 (6%)3 (2%)171\u00a0\u00a0\u00a0\u00a0CA56 (80%)12 (17%)2 (3%)70P\u00a0=\u00a00.454Period immediately after350 (84%)52 (13%)15 (4%)417\u00a0\u00a0\u00a0\u00a0NS147 (84%)23 (13%)6 (3%)176\u00a0\u00a0\u00a0\u00a0BE145 (85%)22 (13%)4 (2%)171\u00a0\u00a0\u00a0\u00a0CA58 (83%)7 (10%)5 (7%)70Pain summary score (range: 0\u20138)Mean (sd)\u00a0\u00a0\u00a0\u00a0All0.86 (1.60)413P\u00a0=\u00a00.02\u00a0\u00a0\u00a0\u00a0NS0.91 (1.81)173\u00a0\u00a0\u00a0\u00a0BE0.66 (1.35)170\u00a0\u00a0\u00a0\u00a0CA1.20 (1.60)70aData for the BE group were published previously [16]bSignificance of differences between three groups as determined by Chi-square test (categorical variables) or proportional odds models for ordinal response data (summary score). No correction for confoundersTable\u00a05Overall burden of upper GI endoscopy as reported by patientsaOverall burdenNotQuiteVerynDifferbEndoscopy in general137 (34%)204 (51%)58 (15%)399P\u00a0=\u00a00.007\u00a0\u00a0\u00a0\u00a0NS48 (30%)82 (50%)32 (20%)162\u00a0\u00a0\u00a0\u00a0BE68 (41%)87 (52%)12 ( 7%)167\u00a0\u00a0\u00a0\u00a0CA21 (30%)35 (50%)14 (20%)70Overall burden summary score (range: 0\u20132)Mean (sd)\u00a0\u00a0\u00a0\u00a0All0.80 (0.67)399P\u00a0<\u00a00.001\u00a0\u00a0\u00a0\u00a0NS0.90 (0.70)162\u00a0\u00a0\u00a0\u00a0BE0.67 (0.61)167\u00a0\u00a0\u00a0\u00a0CA0.90 (0.71)70aData for the BE group were published previously [16]bSignificance of differences between three groups as determined by Chi-square test (categorical variable) or proportional odds models for ordinal response data (summary score). No correction for confounders\nTable\u00a06 shows these adjusted differences in summary scores for pair wise comparisons between the groups, and how these are affected by the determinants. NS patients reported significantly more discomfort than BE patients, as demonstrated by the significant OR of 1.69. After adjusting for differences in the number of previous endoscopies, the difference in reported discomfort between NS and BE patients was no longer significant. Similarly, the difference in reported discomfort between NS and BE patients could also be explained by differences regarding the administration of sedation. The differences between the NS and BE groups in the baseline EQ\u20135D summary score and in baseline anxiety scores did not explain the differences in reported discomfort: the ORs remained significant. Reported pain during upper GI endoscopy did not differ between NS and BE groups. The difference in reported overall burden was significant (OR\u00a0=\u00a01.64, P\u00a0=\u00a00.03). This difference became also insignificant after adjustment for the number of previous endoscopies and for sedation.\nTable\u00a06Differences in discomfort summary score, pain summary score and overall burden score, pair wise between patient groups (after correction for age, sex and employment status as confounders); and effects of other determinants than patient group on these differencesDeterminantDiscomfort scorePain scoreOverall burdenORa95% CIbP-valueORa95% CIbP-valueORa95% CIbP-valueNS compared to BE (BE\u00a0=\u00a0reference group)Patient group: NS versus BE1.691.15\u20132.47< 0.011.090.70\u20131.710.701.641.05\u20132.550.03\u00a0\u00a0\u00a0\u00a0+ Number of previous endoscopies1.490.98\u20132.270.061.190.73\u20131.940.501.560.96\u20132.530.07\u00a0\u00a0\u00a0\u00a0+ Baseline EQ\u20135D1.511.02\u20132.230.040.830.53\u20131.320.441.490.96\u20132.330.08\u00a0\u00a0\u00a0\u00a0+ Baseline anxiety1.631.12\u20132.390.011.040.66\u20131.640.861.591.02\u20132.490.04\u00a0\u00a0\u00a0\u00a0+ Sedation1.420.96\u20132.090.081.080.68\u20131.720.731.400.88\u20132.220.15CA compared to BE (BE\u00a0=\u00a0reference group)cPatient group: CA versus BE1.220.75\u20131.990.422.691.51\u20134.77<0.012.371.38\u20134.07<0.01\u00a0\u00a0\u00a0\u00a0+ Baseline EQ\u20135D1.070.66\u20131.740.792.321.29\u20134.18<0.012.101.21\u20133.66<0.01\u00a0\u00a0\u00a0\u00a0+ Sedation2.061.16\u20133.640.012.711.40\u20135.27<0.013.101.68\u20135.74<0.01aOdds ratios were calculated by proportional odds models for ordinal response data. They show the differences between the patient groups, corrected for confounders and the determinant mentionedb95% confidence intervalcNumbers of previous endoscopies and baseline anxiety were not available for CA patients\nCA patients reported significantly more pain (OR\u00a0=\u00a02.69, P\u00a0<\u00a00.01) and overall burden than BE patients (OR\u00a0=\u00a02.37, P\u00a0<\u00a00.01; Table\u00a06). The differences in reported pain could not be explained by differences in baseline EQ\u20135D summary scores or whether sedation had been administrated or not (all ORs remained significant, Table\u00a06). CA and BE patients did not differ in reported discomfort (OR\u00a0=\u00a01.22, P\u00a0=\u00a00.42), but after taking differences in the provision of sedation into account, the difference in reported discomfort became significant (OR\u00a0=\u00a02.06, P\u00a0=\u00a00.01).\nSymptoms\nAfter endoscopy, throat ache was the only symptom that was reported more often than before the procedure (51 vs. 23%; P\u00a0<\u00a00.001). Other symptoms did not increase in frequency. Compared to BE patients, the increase in throat ache was smaller for NS patients and larger for CA patients (P\u00a0<\u00a00.001); 31% of NS patients reported throat ache before and 46% afterwards, compared to12% and 47% of BE patients, and 12% and 70% of CA patients, respectively.\nPsychological distress\nFigure\u00a01 shows unadjusted mean anxiety and depression scores (HAD\u2014not available for CA patients) by patient group over time.\nFig.\u00a01Differences between BE and NS groups in Hospital Anxiety and Depression (HAD) scale scores for general distress before and after upper GI endoscopy (mean scores, no adjustment for confounders)\nAfter adjusting for confounders (repeated measures ANOVA), anxiety levels were similar between the BE and NS groups across measurements, but the pattern differed significantly between them (interaction effect of \u2018group\u2019 with \u2018time\u2019, P\u00a0=\u00a00.01): BE patients reported lower anxiety levels at the start and slightly higher at the end. Determinants (number of previous endoscopies, baseline EQ\u20135D summary score, sedation) did not influence this pattern of anxiety over time (no significant interaction effects with \u2018time\u2019). Anxiety scores of both NS and BE patients were significantly higher at all time points than reported by a general population sample (score\u00a0=\u00a03.9; P\u00a0<\u00a00.001 for each group at each measurement) [19].\nAt all measurements, depression scores were lower in BE than in NS patients (P\u00a0<\u00a00.001). This difference was significantly larger before than after endoscopy (interaction effect of \u2018group\u2019 with \u2018time\u2019, P\u00a0=\u00a00.01). The number of previous endoscopies affected the pattern of depression over time (interaction effect of \u2018number of previous endoscopies\u2019 with \u2018time\u2019 P\u00a0=\u00a00.046) making the pattern of the two groups more similar. Depression scores differed from those reported by the general population sample: BE patients reported significantly lower levels at all measurements, while baseline NS scores were significantly higher (norm score\u00a0=\u00a03.7, P\u00a0<\u00a00.001 for each comparison).\nSpecific distress (IES) scores regarding the endoscopy itself and its outcome were lower in BE patients than in NS patients (mean scores at baseline measurement and 1\u00a0week measurements are BE 5.5 (sd 9.5), NS 12.9 (sd 14.7), and BE 3.5 (sd 7.7), NS 9.4 (sd 14.3) respectively, P\u00a0<\u00a00.001). The determinants did not affect this difference. In both BE and NS patients, specific distress regarding the endoscopy (IES, baseline measurement) was higher than regarding the test result (IES, one week measurement) (P\u00a0<\u00a00.001). High IES-distress scores regarding the endoscopy were seen in 51 patients (14%). CA patients (mean IES score 22.3 (sd 17.8)) had significantly higher distress levels (IES) regarding the test-result than the other patient groups (P\u00a0<\u00a00.001).\nDiscussion\nThis study is the first to investigate determinants of patients\u2019 perceived burden of upper GI endoscopy. Patients undergoing endoscopy for different reasons reported a different burden from the procedure. BE patients who underwent endoscopy as part of regular surveillance, reported the lowest discomfort, pain and overall burden, confirming our hypotheses in this respect. Patients with non-specific GI complaints reported more discomfort from the procedure, while those diagnosed with cancer experienced more pain and both groups reported more overall burden than patients under surveillance for BE. These differences remained significant after adjustment for confounders (age, sex, employment status). Differences in baseline anxiety scores or in baseline general health (EQ\u20135D) did not explain the differences in reported discomfort or pain. Differences in the number of previous endoscopies, and in whether sedation was provided during endoscopy or not, explained part of the differences in reported discomfort between NS and BE patients. Whether sedation was provided or not did not explain the differences in reported pain and overall burden between BE and CA patients.\nThe study also confirms that that upper GI endoscopy is burdensome for all groups of patients: two-thirds of the total group of patients reported discomfort and overall burden from the procedure, and patients were distressed beforehand. These results may however underestimate the actual burden because this empirical study was limited to patients who actually underwent upper GI endoscopy, hence excluding patients who refrained from undergoing endoscopy because of past or anticipated adverse experiences. Another potential limitation of our study results from the differences in response rates between the groups.\nDifferences between patient groups were also found for symptoms resulting from the endoscopy. Of all symptoms explored, only throat ache increased after upper GI endoscopy. CA patients reported a higher increase in throat ache than BE patients and NS patients. As upper GI endoscopy hardly caused any symptoms, we considered an investigation into determinants of these differences to be less interesting and therefore omitted those analyses.\nFurthermore, BE and NS patients differed in the levels of generic (HAD) and specific (IES) distress they reported. Specific distress (IES) was significantly higher in NS patients than in BE patients, both regarding the endoscopy itself and its result. General distress (HAD) also differed between groups: BE patients reported less depression across all measurements and the pattern of anxiety and depression across measurements was different. However, general distress is not necessarily related to the endoscopy. The persistent higher depression scores across different time points suggest that NS patients have more depressive symptoms in general but that this was not related to the endoscopy. The different pattern of anxiety levels before and after endoscopy, however, suggests that the patient groups also differed in endoscopy-related distress. The pattern corroborated the findings of the specific (IES) distress scores: NS patients were more distressed than BE patients before the endoscopy. The investigated determinants did not explain the differences between groups in specific distress or general distress pattern, except for the number of previous endoscopies explaining part of the difference in the depression scores.\nBE patients thus reported less distress and also less pain or discomfort than other patient groups. This was not caused by differences in patient characteristics (age, sex, employment status, baseline anxiety, baseline general health). There are several potential reasons why the reported burden differs. Firstly, BE patients are under regular surveillance and may get used to or adapt to the procedure decreasing its burden. As the number of previous endoscopies explained the lower distress, discomfort and overall burden reported in the BE group, we conclude that getting used, or adapting to endoscopy plays a role.\nSecondly, patients who perceive a greater benefit of the test may weigh its burden differently and consequently report less burden. BE patients potentially have more to gain from early discovery of adenocarcinoma than NS patients, who are usually referred for endoscopy to detect potential explanations for their symptoms, and also more than CA patients for whom endoscopy and ultrasonography are only part of the procedure to determine their treatment options and prognosis. As we did not measure perceived expected benefit of the endoscopy we are not able to determine whether this mechanism is part of the explanation.\nThirdly, the endoscopic procedure was slightly different for the different patient groups. CA patients received sedation more often. Adjusting for this difference into the analysis did not explain the differences in pain and overall burden, whereas the difference in reported discomfort became significant after adjustment for sedation. These results suggest that differences in the proportions of patients receiving sedation during endoscopy did not explain the differences between the groups, and that sedation was provided to those patients who really needed it. The procedure for CA patients also differed; they underwent upper GI endoscopy combined with ultrasonography, and for the combined procedure an endoscope with a slightly larger diameter is used. Our data did not allow us to test separately whether this affected perceived pain and overall burden. Finally, most CA patients had esophageal carcinoma, and this disease may make passing the endoscope through the esophagus more difficult and therefore more painful.\nWe measured general psychological distress (HAD) at different time points; assuming that a pattern of higher distress levels before compared to after endoscopy indicates that the procedure causes distress. As discussed in a previous paper [16], this may be debated for the reason that lower distress levels afterwards may also result from a reassurance effect of patients receiving a negative test result (no serious disease present). Nevertheless, the fact that the specific distress (IES) score relating to the endoscopy was higher than the IES score relating to the test outcome led us to conclude that the prospect of undergoing upper GI endoscopy does indeed increase distress levels.\nEven if upper GI endoscopy causes HAD anxiety and depression scores to be increased before the endoscopy, the relevance of these increased distress levels can be questioned. Anxiety may be a relevant problem with 20% of patients having scores indicating clinical anxiety levels at baseline, while the depression scores are less worrisome (6%). Endoscopy-specific distress (IES) was high in 14% of patients and higher than the distress related to the outcome. Anxiety scores in our study were increased compared to general population scores at all time points. Especially the fact that NS patients remained at increased levels one month after endoscopy makes the comparability of our scores with the population scores questionable [19]. General population scores are not available for procedure specific-distress (IES), as this can only be measured in patients. Considering the cut-off values for clinical scores, the prospect of endoscopy causes moderate distress.\nThe observation that patients under regular endoscopic surveillance may adapt to this invasive procedure should not result in an underestimation of the burden of regular endoscopic surveillance. The search for less invasive surveillance tests should continue, and frequency of surveillance should preferably be established by evidence-based individualized estimates of risk of progression.","keyphrases":["barrett esophagus","endoscopic surveillance","discomfort","anxiety","distress","upper gastrointestinal endoscopy","perceived patient burden"],"prmu":["P","P","P","P","P","R","R"]}
{"id":"Clin_Rheumatol-3-1-2039777","title":"A comparison of the measurement properties of the Juvenile Arthritis Functional Assessment Scale with the childhood health assessment questionnaire in daily practice\n","text":"We compared the measurement properties of a performance test (Juvenile Arthritis Functional Assessment Scale; JAFAS) with a questionnaire-based instrument (Childhood Health Assessment Questionnaire; CHAQ) to measure functional ability in patients with juvenile idiopathic arthritis on the level of individual items. In 28 consecutive children visiting an outpatient paediatrics clinic, the JAFAS (range 0\u201320) and CHAQ (range 0\u20133) were applied, and measures of disease activity and joint range of motion (ROM) were determined. Twenty-eight children with a median age of 10 years and median disease duration of 3.2 years were included. The median JAFAS score was 0, and the median CHAQ score was 0.125. Cronbach\u2019s alpha was 0.92 for the JAFAS and 0.96 for the CHAQ. The Spearman correlation coefficient between the JAFAS and the CHAQ was 0.55 (P < 0.01). With six out of ten items, the JAFAS classified the child as less disabled than with corresponding CHAQ activities. Overall, associations with measures of disease activity and ROM were higher for the CHAQ than for the JAFAS. A performance test (JAFAS) does not appear to have an added benefit over the questionnaire-based assessment (CHAQ) of physical function in a cross-sectional study.\nIntroduction\nIn patients with juvenile idiopathic arthritis (JIA), functional disability can both be evaluated by means of questionnaires and observed performance tests. In a previous study [1], the internal consistency, construct validity and responsiveness of a questionnaire-based instrument, the Childhood Health Assessment Questionnaire [2] (CHAQ), proved to be somewhat better than those of an observed performance test, the Juvenile Arthritis Functional Assessment Scale [3] (JAFAS). As performance tests are time consuming and require specific equipment and trained assessors, it is relevant to know whether they have an added benefit over a questionnaire-based instrument.\nIn the abovementioned, both measures displayed a floor effect [1]. As minor average disability in patients with JIA is nowadays a reality, identifying those tasks that can discriminate among lower levels of disability becomes all the more important. In addition, as performance tests are time consuming and require specific equipment and trained assessors, it is relevant to know whether they have an added benefit over a questionnaire-based instrument.\nThe aim of the present study was therefore to compare the measurement properties of the JAFAS and the CHAQ in an unselected population of children with JIA on the level of individual items.\nMaterials and methods\nStudy design and patient recruitment\nBetween January 2001 and April 2002, 34 consecutive children with JIA were recruited according to the following criteria: age between 7 and 12\u00a0years, diagnosis JIA [4] and no other medical conditions interfering with functional ability. The patients were visiting the outpatient paediatric rheumatology clinic of the Leiden University Medical Centre. The clinic, which has two part-time working paediatric rheumatologists, is a tertiary referral centre for children with rheumatic diseases from the Leiden district and surrounding area (1 million inhabitants). The Medical Ethics Committee approved the study, and all patients and their parents gave written informed consent.\nAssessment methods\nThe JAFAS (range 0\u201320) was developed as an objective measure of functional ability in children with rheumatic diseases between 7 and 18\u00a0years. With the JAFAS, the observed time needed to perform ten activities is compared with a standard \u2018criterion\u2019 time. The JAFAS was administered by one well-trained paediatric physical therapist (Bekkering).\nThe CHAQ, including 30 activities in eight different domains, with a total score ranging from 0 (no limitation) to 3 (maximal limitation), was completed by interviewing the children.\nDisease activity was measured by the erythrocyte sedimentation range (ESR) and joint counts on swollen (JC-swollen) or tender joints (JC-tender), concerning 28 joints included in the Fuchs score [5] plus the ankles (range 0\u201330). The feeling of well-being and the presence of pain were determined by 15-cm Visual Analogue Scales (VAS), with anchors of \u2018no pain\/no discomfort\u2019 on the left and \u2018very severe pain\/severe discomfort\u2019 on the right (pain-VAS and VAS-well-being; final scores converted to scores ranged from 0 to 3). A similar VAS was used for the physician\u2019s evaluation of disease activity (VAS-paediatrician; score range 0\u20133). Limitation in range of motion (ROM) was determined by a joint count on in motion-restricted joints (JC-limitation; range 0\u201330) and the paediatric Escola de Paulista de Medicina ROM scale [6] (pEPM-ROM; score range 0\u20136).\nStatistical analysis\nThe ten JAFAS items were linked with nine corresponding CHAQ items (CHAQ-9). All ten items had a counterpart in the CHAQ; however, the JAFAS items \u2018get in\u2019 and \u2018get out of bed\u2019 matched only one item in the CHAQ (get in and out of the bed). Associations between the JAFAS, the CHAQ-total and CHAQ-9 scores were determined by means of Spearman correlation coefficients (rs). To test the concordance between individual JAFAS and CHAQ-9 items, the score on every item was dichotomised into not limited (0) or limited (\u22651) and mutually compared with Cohen\u2019s Kappa (\u03ba value greater than 0.80 is considered as good) [7]. Internal reliability of the JAFAS, CHAQ-total and CHAQ-9 was determined by calculating Cronbach\u2019s \u03b1 (\u03b1 value of 0.85 is considered good) [8] and item\u2013total correlations. In addition, Spearman correlation coefficients of the JAFAS, CHAQ-total and CHAQ-9 scores with measures of disease activity and ROM were computed.\nResults\nCharacteristics of the patients\nFrom the 33 eligible children who visited the outpatient paediatric rheumatology clinic in the study period, two children refused to participate, and three did not fulfil the inclusion criteria (two children had serious mental retardation and one child had the attention deficit hyperactivity disorder). Thus, 28 children, 12 boys and 16 girls, were included. Their median age was 10.0\u00a0years (range 7.3\u201312.8), and the median disease duration was 3.3\u00a0years (range 0.1\u201310.2). A majority of the children had a polyarticular (nine patients) or oligoarticular pattern (11 patients) of joint involvement. Systemic onset JIA (three patients), arthritis and psoriasis (four patients) and enthesitis type JIA (one patient) were less frequently seen. Twenty (71%) children used anti-rheumatic medication, of whom 17 (68%) used disease-modifying anti-rheumatic drugs and three (11%) used oral corticosteroids.\nThe median scores of the ESR (7.7\u00a0mm\/h, range 2\u201354), the JC-swollen (1.0, range 0\u201328), the JC-tender (0.8, range 0\u20138), the VAS-well-being (0.2, range 0\u20132.5), VAS-pain (0.1, range 0\u20131.5) and VAS-paediatrician (0.2, range 0\u20132.7) point at a relatively low level of disease activity. With respect to joint ROM, the median JC-limitation score was 1.0 (range 0\u201317), and the median EPM-ROM score was 0. 5 (range 0\u201319.5).\nThe median scores of the JAFAS (0, range 0\u201313) and CHAQ (0.125, range 0\u20132.6) indicate, on average, the presence of no and very little functional disability, respectively. The frequency distributions (Fig.\u00a01) of the JAFAS and the CHAQ show that according to the JAFAS, 18 out of 28 patients (65%) had no limitations, whereas according to the CHAQ, 13 out of 28 patients (47%) had no functional disability.\nFig.\u00a01Frequencies of JAFAS and CHAQ scores. No disability JAFAS\u2009=\u20090, CHAQ\u2009=\u20090. Mild disability JAFAS\u2009=\u20091\u20133, CHAQ\u2009=\u20090\u20130.5. Moderate disability JAFAS\u2009=\u20094\u20139, CHAQ\u2009=\u20090.6\u20131.5. Severe disability JAFAS\u2009=\u200910\u201320, CHAQ\u2009=\u20091.6\u20133.0\nReliability and validity\nWith respect to internal reliability, Cronbach\u2019s \u03b1 was 0.91 for the JAFAS, 0.96 for the CHAQ-total score and 0.92 for the CHAQ-9. The item\u2013total correlation was moderate (\u22650.60; p\u2009<\u20090.01) for two out of ten JAFAS and six out of nine matching CHAQ tasks. Besides these six items (dressing, pull on socks, cutting meat, bend down, walk outdoors and climb stairs) selected in this study, the items reach for object, writing, turn door key or water tab, and running were frequently scored as difficult and showed good item\u2013total correlations.\nConcerning the agreement between the two instruments, the JAFAS total score correlated moderately well with both the CHAQ-total score (r\u2009=\u20090.55, p\u2009<\u20090.01) and the computed score of the corresponding CHAQ-9 items (r\u2009=\u20090.56, p\u2009<\u20090.01). On the individual item level, there was excellent agreement [7] (\u03ba\u2009>\u20090.80) with respect to tasks 2 (pull shirt or sweater over head) and 8 (from standing position sit on floor, then stand up). The results of the internal reliability statistics and the associations between the total scores and the corresponding item scores of JAFAS and CHAQ are presented in Table\u00a01.\nTable\u00a01JAFAS and CHAQ scores and their associations (Spearman correlation coefficients) in 28 patients with JIAItemJAFASNumber of patients limited in activityInternal reliability, item\u2013total correlationCHAQNumber of patients limited in activityInternal reliability, item\u2013total correlationConcordance among JAFAS and CHAQ itemsNumber of concordant pairsCHAQ \/JAFASbCohen\u2019s \u03ba1Button shirt\/blouse90.85**Dress, including tying shoelaces and doing buttons70.76**203\/50.30 ns2Pull shirt or sweater over head10.38*Pull on sweater over head10.34 ns280\/01.00**3Pull on both socks20.49**Pull on socks70.64**216\/10.13 ns4Cut food with knife and fork40.70**Cutting meat70.79**215\/20.23 ns5Get into bed00.00Getting in and out of bed30.47*244\/0a6Get out of bed00.00244\/0a7Pick something up off floor from standing position10.38*Bend down to pick up clothing or a piece of paper50.62**244\/00.29*8From standing position sit on floor, then stand up20.52**Stand up from a low chair or floor20.51**280\/01.00**9Walk 50 feet without assistance10.38*Walk outdoors on flat ground50.67**244\/00.29*10Walk up flight of 5 steps10.38*Climb up five steps70.69**226\/00.20 nsTotal scoresCronbach\u2019s \u03b1Cronbach\u2019s \u03b1Spearman\u2019s r10 JAFAS items140.919 CHAQ items180.920.56**total CHAQ score180.960.55***p\u2009<\u20090.05, **p\u2009<\u20090.01aNo statistics are computed because of a constant factorbNumber of pairs with CHAQ\u2009\u2265\u20091 and JAFAS\u2009=\u20090\/Number of pairs with JAFAS\u2009\u2265\u20091 and CHAQ\u2009=\u20090\nConstruct validity\nThe relationship between the JAFAS, CHAQ, CHAQ-9 and measures of disease activity, pain, swelling and limited range of joint motion are shown in Table\u00a02. Both the JAFAS and the CHAQ scores correlated moderately to well with the VAS-paediatrician, JC-swollen, JC-limited joints, pEPM-ROM and CHAQ-pain. Neither the JAFAS nor the CHAQ scores were significantly associated with the JC-tender. The CHAQ showed significant associations with the ESR and CHAQ well-being, whereas the JAFAS did not.\nTable\u00a02Spearman correlation coefficients between JAFAS, CHAQ and other measures in 28 patients\u00a0JAFASCHAQCHAQ (9 items)VAS-paediatrician0.41*0.56**0.34 nsESR0.37 ns0.62*0.75**Joint count on swollen joints0.47*0.65**0.48*Joint count on tender joints0.07 ns0.41*0.09 nsJoint count on limited joints0.44*0.64**0.59**Paediatric EPM-ROM0.50**0.73**0.88**CHAQ\u2013pain0.38*0.69**0.55**CHAQ-well-being0.20 ns0.44*0.48**p\u2009<\u20090.05, **p\u2009<\u20090.01\nDiscussion\nIn parallel with an earlier publication [1], this study demonstrated no advantages of a performance test (JAFAS) as opposed to a questionnaire (CHAQ) to measure functional disability in children with JIA. We found modest correlations between the two instruments and the floor effect with the CHAQ being smaller than with the JAFAS. Moreover, the CHAQ showed a better internal reliability and stronger associations with measures of disease activity and joint ROM than the JAFAS. Tennant et al. [1] reported similar results regarding the internal reliability and validity, in addition to a smaller responsiveness of the JAFAS.\nPossible explanations for the discrepancy between the JAFAS and CHAQ are that the JAFAS is concerned with performance on one specific time point and the speed of performance, whereas the CHAQ refers to the last week and the experience of difficulties, including the need for aids, appliances or other persons. Discordance between observed and reported functional disability has been reported earlier in both children with JIA [9] as well in adults with rheumatoid arthritis [10].\nThe relatively small number of patients and the lack of distribution of the scores over the full ranges of the various outcome measures could limit the external validity of this study. However, the observed low level of functional disability is consistent with results of previous studies in paediatric rheumatology [11].\nThe children\u2019s version of the CHAQ was originally designed and validated with the questionnaire self-administrated by the patients. In this study, the questions were read out and filled in by the investigator to ensure appropriate completion. Although it is likely that with this method, the same results are obtained as with self-administration, a possible influence on the final scoring cannot be totally ruled out.\nGiven the large improvements in medical treatment of JIA and considering the persisting need for valid and responsive measures in clinical trials, a further elaboration of the tasks included in measures of functional ability, reflecting relevant activities in daily life, is needed. Lam et al. [12] showed that by utilizing new response scales as well as adding more challenging questions than those posed by the original Health Assessment Questionnaire (HAQ), the floor effect could be reduced and the sensitivity enhanced. In addition, the excellent internal reliability of the CHAQ-9 score as found in the present study and the observation that some JAFAS and selected CHAQ items were not or only marginally contributing to the final scores suggest that with both instruments, there are opportunities for a reduction in the number of items, in parallel with the recently developed short version of the HAQ in adult rheumatoid arthritis patients [13]. Which items should be included has to be further examined, as the nine CHAQ items this study focusses on are only selected because they corresponded with the JAFAS. With any future research, the conduction of large, prospective follow-up studies is to be advised.","keyphrases":["questionnaire","juvenile idiopathic arthritis","activities of daily living","disability evaluation"],"prmu":["P","P","M","R"]}
{"id":"Matern_Child_Health_J-2-2-1592249","title":"Preconception Healthcare: What Women Know and Believe\n","text":"Objectives: The objectives of this study were to determine if women realize the importance of optimizing their health prior to a pregnancy, whether the pregnancy is planned or not; and to evaluate their knowledge level and beliefs about preconception healthcare. Additionally, we sought to understand how and when women wanted to receive information on preconception health. Methods: A survey study was performed using consecutive patients presenting to primary care practices for an annual well-woman exam. Patients were recruited based on appointment type and willingness to complete the survey at the time of their appointment, but prior to being seen by the physician. Results: A total of 499 women completed the survey. Nearly all women (98.6%) realized the importance of optimizing their health prior to a pregnancy, and realized the best time to receive information about preconception health is before conception. The vast majority of patients surveyed (95.3%) preferred to receive information about preconception health from their primary care physician. Only 39% of women could recall their physician ever discussing this topic. The population studied revealed some significant knowledge deficiencies about factors that may threaten the health of mother or fetus. Conclusions: A majority of women do understand the importance of optimizing their health prior to conception, and look to their Primary care physician as their preferred source for such information. Study participants demonstrated deficiencies in their knowledge of risk factors that impact maternal and fetal health suggesting that physicians are not addressing preconception healthcare during routine care.\nIntroduction\nPreconception care is defined as the promotion of the health and well being of a woman and her partner before pregnancy [1]. The goal of the preconception visit is to identify medical and social conditions that may put the mother or fetus at risk. The concepts of preconception care have been articulated for over a decade\u2014but unfortunately have not become part of the routine practice. Although many studies [2\u20139] document the effectiveness of interventions targeted to increase awareness of preconception folic acid supplementation, however, little evidence links comprehensive preconception health promotion to improved pregnancy outcomes. Only one study using data gathered more than 10 years ago demonstrated a greater likelihood of pregnancy intendedness in a low-income cohort of woman exposed to information on preconception health during routine family planning visits at a community health department [10]. Studies in the United Kingdom [11, 12] of knowledge and attitudes toward preconception care among primary health care teams show widespread consensus among the healthcare workers of the importance of the topic. One of these studies included the attitudes of women of childbearing age and noted that the view of the importance of preconception care was less strongly held by the female population studied [11]. Other studies have focused on more specific topics, such as rubella immunity, folic acid supplementation and glycemic control in diabetics [13\u201315]. Approximately 2% to 3% of all pregnancies result in a neonate with a serious genetic disease or a birth defect that can cause disabilities, mental retardation, and in some cases early death [16]. We are unaware of additional studies assessing the general knowledge and beliefs that women possess about optimizing their health prior to conception, or their preferences for obtaining such information.\nMethods\nA survey study of consecutive patients presenting to primary care practices for an annual well-woman exam was performed in accord with prevailing ethical principles. The selected primary care practices, occupying the same building at the Mayo Clinic Arizona, represented a women\u2019s health general internal medicine practice (5 physicians) and a family medicine practice (9 faculty physicians and 18 family medicine residents). Women were recruited for the study by nursing staff working within the practices, with permission and informed consent completed prior to distributing the survey tool. The Mayo Clinic IRB approved the study. Patients were recruited based on appointment type (e.g., well woman exam, annual Pap exam, and annual preventive medicine exam) and consenting to complete the survey questionnaire at the time of their appointment, but prior to being seen by the physician. The enrollment period was between August 2004 and July 2005. Women were considered eligible for the study if they were between the ages of 18 and 45 years, understood English, and gave their permission. The survey instrument was a four-page questionnaire and required approximately 10 minutes to complete. The survey included questions about demographics, pregnancy intendedness, knowledge and attitudes about preconception care, and personal preferences about sources of health information about preconception care.\nData from each survey was entered into a database at the Research Survey Center, Mayo Clinic, Rochester, Minnesota, and the aggregate data made available for analysis to the research team.\nResults\nA total of 570 women were invited to participate in the study; 58 declined and 13 did not meet eligibility criteria, leaving 499 women who completed surveys for data analysis. The demographic profile of the study population is shown in Table 1. In this study population, the majority (70.6%) of women were not currently attempting to conceive as noted in Table 2. Approximately 5% were actively trying to conceive, with 13.5% and 8.4% considering a pregnancy in either the next 1\u20132 or 3\u20135 years respectively. Interestingly, of the women who had previously been pregnant, pregnancies had actually been planned in only 47.2% of instances. Nearly all women in the study (98.6%) realized the importance of optimizing their health prior to a pregnancy, again as noted in Table 2. However, only 39% could ever recall their physician discussing preconception health. The majority of the women in this study population who were interested in preconception health education preferred the information prior to a pregnancy (74.8%) or at the time of their annual medical exam (11.9%), as displayed in Fig. 1.Table 1Study population demographic (n=499)Age\u2003Range: 18 to 45 years\u2003\u200318 to 25 years24%\u2003\u200326 to 35 years30%\u2003\u200336 to 45 years46%\u2003Mean: 33 yearsEthnicity\u2003White84.8%\u2003Asian3.6%\u2003African-American1.3%\u2003Native American1.4%\u2003Other9.0%Education (highest level)\u200311th grade or less0.4%\u2003Graduated High School7.5%\u2003Some college or technical school33.0%\u2003Graduated college38.5%\u2003Some graduate work6.7%\u2003Graduate degree14.0%Household income\u2003Less than $25,00010.5%\u2003$26,000 to $50,00019.7%\u2003$51,000 to $75,00018.6%\u2003$76,000 to $99,00013.0%\u2003$100,000 to $125,00013.2%\u2003$126,000 to $150,0006.8%\u2003$151,000 to $200,0006.4%\u2003Greater than $200,00011.8%Table 2Conception history and planningPlans about getting pregnant\u2003No plans at present time70.6%\u2003Currently trying4.6%\u2003Considering in next 1 to 2 years13.5%\u2003Considering in next 3 to 5 years8.4%\u2003Have tried, unable to get pregnant2.8%Ever been pregnant?\u2003Yes50.7%\u2003No49.3%If ever pregnant, where previous pregnancies planned?\u2003Yes47.2%\u2003No52.8%Affect of optimizing health of mother and pregnancy?\u2003Has a good effect on the pregnancy98.6%\u2003Has no effect on the pregnancy0.8%\u2003Has a bad effect on the pregnancy0.6%Doctor ever spoken to you about preconception health?\u2003Yes39.0%\u2003No61.0%Are you interested in receiving preconception health education?\u2003Very interested34.8%\u2003Somewhat interested21.6%\u2003Unsure10.1%\u2003Not at all interested33.5%If interested or unsure about education, when would you prefer?`\u2003At the time I become pregnant7.6%\u2003Before I try to get pregnant74.8%\u2003During pregnancy0.7%\u2003Every time I get an annual medical exam11.9%\u2003Unsure5.0%Fig. 1Are you interested in preconception health education?\nThe women who were interested in preconception health education, or unsure of such interest, were asked their preferences for sources for such information (Table 3 and Fig. 1). The vast majority preferred their physician, either a primary care physician (51.3%) or Obstetrician\/gynecologist (44.0%). Only a fraction would primarily seek their information from sources other than their physician.\nThe survey included questions about patient awareness of key preconception risk factors which may influence the outcome of a future pregnancy. The study population demonstrated high awareness of certain risk factors, such as tobacco, alcohol, drug use, and domestic abuse (Table 4). There were, however, opportunities for improvement in basic understanding of the risk to maternal\/fetal health as it relates to fish consumption, exposure to cat litter, and the impact of family and\/or genetic history.\nDiscussion\nPreconception care is the primary prevention of maternal and perinatal morbidity and mortality. It is an important issue in women\u2019s health that is easily overlooked by physicians and patients. The results of this study demonstrate that the vast majority of study participants understood the importance of preconception healthcare and realized that it should be obtained prior to conception. Interestingly, many of the women who expressed an interest in receiving information about preconception healthcare perceived the annual exam as the appropriate venue for exploring this topic. The study participants showed a strong preference for obtaining information about preconception healthcare from their personal physician and the majority eschewed the use of technology such as the Internet as a source of such information. The study revealed that a large percentage of women expected their primary care physician or OB\/GYN physician to address preconception healthcare with them.Table 3Patient preferences for sources of preconception information (Percentage ranking the choice as first preference)Primary care physician51.3%Obstetrician\/gynecologist44.0%Family, friends0.3%Magazine, newspaper0.3%Internet\/world wide web3.1%Other0.7%\nThese data demonstrate that the study population exhibited gaps in knowledge about specific preconception health topics, but confirmed previous findings that women do have an increased awareness of the importance of folic acid supplementation [2\u20139]. Additionally, the study population showed a high awareness of the risks associated with tobacco, alcohol and drug use, but were much less aware of the risks to fetal health associated with fish consumption and exposure to cat litter. Such findings point out the need to continue efforts at increasing public awareness of other modifiable risks to fetal health. Unfortunately, fewer than 40% of the women surveyed recalled discussing preconception healthcare with their physician at the time of their annual exam.\nClearly, there is a gap in patient expectation and delivery of healthcare services in this population of educated middle class women. This study only surveyed women who had chosen to obtain their primary care in Internal Medicine and Family Medicine practices of a private health care facility, suggesting that there may be obstacles to delivering preconception care even in an academic outpatient setting. Potential explanations for this gap in the delivery of preconception healthcare may include: age of patients studied, time constraints in the outpatient setting or insufficient training and\/or content knowledge in preconception healthcare of the physicians. This study demonstrates that an opportunity to better understand the gap in care delivery and to evaluate possible solutions in private middle class settings. More exposure on the part of residents to a curriculum on preconception healthcare may provide an opportunity for physicians in these specialty fields to improve their skills and the delivery of this much-needed care to women.Table 4Preconception health knowledge and opinions (Percentage awarea of risk factor potentially affecting a pregnancy)Consumption of certain fish54.2%Exposure to cat liter64.9%Folic acid use79.6%Impact of family and\/or genetic history84.1%Infectious diseases (need to screen for)89.3%Immunizations (up to date)91.2%Alcohol use95.8%Abuse (verbal, sexual and\/or physical)97.2%Medication use (prescription and nonprescription)97.4%Tobacco use98.2%Illicit drug use98.8%aAgreed or strongly agreed on a five point Likert scale\nOne of the limitations of our study was the homogeneity of our patient population. The majority of our study participants were middle class, Caucasian and had at least some college education. Our findings may not be broadly applicable to women of other socioeconomic backgrounds, ethnicity and educational levels. The lack of diversity of our population reflects the demographics of this community and further study in other populations should be pursued.\nAs all women of reproductive age and potential presenting for continuing care in the primary care setting are candidates for preconception care, the essential and critical role of primary care physicians and providers in the provision of preconception care is apparent. The Centers for Disease Control (CDC) has published their \u201cRecommendations for Improving Preconception Health and Health Care\u201d [17]. These recommendations were developed through a collaborative effort during which the CDC successfully aligned the missions of a number of its external partners and internal programs to ultimately draft these national recommendations for preconception care. These national recommendations can now serve as a roadmap for both graduate medical education and continuing medical education curricula to improve the knowledge and skill of the physician workforce in the delivery of comprehensive preconception health care.","keyphrases":["preconception health","primary care","preconception care","women\u2019s health"],"prmu":["P","P","P","P"]}
{"id":"J_Gastrointest_Surg-3-1-1852384","title":"Factors Affecting Morbidity and Mortality of Roux-en-Y Gastric Bypass for Clinically Severe Obesity: An Analysis of 1,000 Consecutive Open Cases by a Single Surgeon\n","text":"Introduction Determinants of perioperative risk for RYGB are not well defined.\nIntroduction\nObesity is currently the number one public health problem in the United States, affecting one-third of all Americans (http:\/www.surgeongeneral.gov\/topics\/obesity). Approximately 5 to 8% have clinically severe or morbid obesity and are candidates for bariatric surgery.\nThis obesity epidemic has been accompanied by a geometric rise in the number of bariatric surgical procedures. The American Society for Bariatric Surgery (ASBS) estimates that the number of bariatric surgery procedures has increased from approximately 20,000 in 1996 to over 140,000 in 2004 (http:\/\/www.asbs.org). During this period, membership in the ASBS has also increased fivefold, suggesting that many more surgeons are performing these procedures. The most common bariatric procedure performed in the United States is Roux-en-Y gastric bypass (RYGB).1\nThe explosive growth of bariatric surgery has garnered much attention in the media, with much speculation about the risks, both short- and long-term, of RYGB. Unfortunately, to date, little information from large series is available concerning the operative mortality of RYGB and those factors that predict mortality. In 2002, Livingston et al.2 reported an operative mortality of 1.3% in 1,067 patients after open RYGB and found that only age over 55\u00a0years correlated with perioperative mortality. More recently, Fernandez et al.3 analyzed their patient cohort of 1,431 patients having open RYGB and found a 1.9% mortality, which was associated with age, weight, longer limb gastric bypass, and the occurrence of a leak or pulmonary embolism.\nThis report is a multivariate analysis of preoperative mortality and morbidity in 1,000 consecutive open RYGBs performed over a 5-year period by a single surgeon (LF) in at a single institution.\nMaterials and Methods\nBariatric Surgery Program The bariatric surgery program at St. Luke\u2019s\u2013Roosevelt Hospital Center in New York City was initiated in April 1999 and the first operation performed in June 1999. The basis of this report consists of 1,000 consecutive open RYGBs (primary cases and revisions) performed by a single surgeon, LF, between June 1999 and June 2004.\nClinical Protocol and Surgical Technique All patients were evaluated preoperatively and met generally accepted criteria outlined by the NIH at its Consensus Development Conference on Gastrointestinal Surgery for Severe Obesity.4 In addition, all patients were routinely evaluated by a registered dietitian experienced in the treatment of obesity. Mental health and other specialty consultations were only obtained if they were felt to be clinically indicated or were required by an insurance carrier. All patients were evaluated by an attending anesthesiologist before surgery.Routine preoperative studies included: electrocardiogram, chest x-ray, gallbladder ultrasonography, serum electrolytes, glucose, HbA1c, calcium, albumin, lipid profile, liver function tests, complete blood count, platelets, prothrombin time, partial thromboplastin time, INR, and urinalysis. Over time, additional preoperative studies, including serum insulin, iron, ferritin, vitamin B12, 25-OH vitamin D, and thiamine (vitamin B1) were added.RYGB was performed in a standard fashion with the following common elements: (1) open technique; (2) 20\u201330\u00a0ml pouch, nondivided stomach, TA 90B (US Surgical, Norwalk, CT, USA) applied twice; (3) hand-sewn two-layer retrocolic, antegastric gastrojejunostomy, 12\u00a0mm in length, tested with methylene blue under pressure intraoperatively; (4) side-to-side, functional end-to-end jejunojejunostomy with dispSAble GIA (US Surgical) or LC (Ethicon Endosurgery, Sommerville, NJ, USA) 75\u00a0mm staplers, and TA55 (US Surgical) or TX 60 (Ethicon Endosurgery) staplers and routinely oversewn with 3\u20130 silk Lembert sutures. The biliopancreatic limb-length measured 75\u00a0cm along the antimesenteric border for patients with BMI less than 50 and 150\u00a0cm for patients with BMI greater than or equal to 50. The Roux or alimentary limb-length was 150\u00a0cm in all patients. For the initial 14 cases, the stomach was divided using the GIA-100 stapler. This technique was abandoned and switched to the nondivided stomach after a stapler malfunction led to a leak.Closed suction drains were placed in all revisions, patients with BMI\u2009>\u200955, or if clinically indicated (e.g., identification of an intraoperative leak, technically difficult anastomosis). Drains were removed as clinically indicated. Fascia was closed with a running looped #1 PDS (Ethicon, Sommerville) and infiltrated with 0.25% bupivacaine. The subcutaneous space was drained with a #10 Jackson\u2013Pratt drain and the skin was closed with a running subcuticular stitch.The gallbladder was removed if gallstones were documented by preoperative ultrasound. Incisional or umbilical hernias were primarily repaired if encountered, oftentimes through a separate periumbilical incision.Invasive monitoring was not used routinely and Foley catheters were only placed if patients required invasive hemodynamic monitoring or in the case of revisions or when patients had multiple prior abdominal operations. Nasogastric tubes were left in placed as clinically indicated (intraoperative leak identified) or in patients with BMI\u2009>\u200955. All patients received perioperative antibiotics for 24\u00a0h, cefazolin (2\u00a0g intravenously every 8\u00a0h for three total doses) or clindamycin (900\u00a0mg intravenously every 6\u00a0h for four doses). Prophylaxis against deep vein thrombosis consisted of 5,000 units of unfractionated heparin administered subcutaneously every 8\u00a0h and pneumatic compression stockings until ambulatory. All patients were given a PCA pump (patient-controlled analgesia) for pain. Patients were routinely cared for on a regular surgical floor, equipped for severely obese patients. Patients with significant SA or documented CAD were usually observed in the recovery room overnight, and only rarely admitted to the intensive care unit. On the morning of postoperative day\u00a01, patients were routinely studied with gastrograffin upper-GI studies. If no leak was identified, they were given liquids for lunch and advanced to soft food (yogurt, apple sauce, and cottage cheese) for dinner and switched to oral pain medications. Drains were usually removed before discharge. Patients were discharged on POD #2 or #3 as indicated with the following medications: a codeine-derivative for pain, prenatal vitamins, iron polysaccharide, calcium citrate, ursodeoxycholic acid, if the gallbladder was present.After hospital discharge, patients were scheduled to be seen at 2 and 8\u00a0weeks, 6, 12, 18, 24\u00a0months, and yearly thereafter. All routine follow-up appointments included nutritional counseling and those after 2\u00a0weeks included laboratory studies [serum electrolytes, glucose, HbA1c, calcium, albumin, lipid profile, liver function tests, complete blood count, platelets, serum insulin, iron, ferritin, vitamin B12, 25-OH vitamin D, and thiamine (vitamin B1)]. Follow-up was 74% at 1\u00a0year, 68% at 2\u00a0years, 59% at 3\u00a0years, 53% at 4\u00a0years, and 48% at 5\u00a0years.\nClinical Data and Data Analysis Clinical and laboratory data were PC-based database prospectively maintained since the program\u2019s inception in 1999. Data collected included: age, sex, height weight, BMI, race\/ethnicity, payer status, obesity-related comorbidities, operative procedure, duration of stay, mortality, major complications, and death. Complications were classified as systemic (prolonged intubation, deep venous thrombosis, pulmonary embolism, and myocardial infarction\/fatal arrhythmia) or technical (incisional hernia, intestinal obstruction, leak\/perforation, dehiscence, GI bleeding, anastomotic stricture, and anastomotic ulcer). Deaths were analyzed with respect to BMI, demographics, comorbidities, and complications.Superficial wound infections were not included; the incidence of urinary tract infections was not tracked. Nutritional complications were not evaluated because all patients were routinely maintained on vitamins, iron, and calcium supplements postoperatively; it would be impossible to determine the true incidence of any of these nutritional deficiencies.Univariate analyses and logistic regression with SPSS 11.0 were used to determine significance.\nResults\nThe population consisted of 854 women and 146 men. Their demographic characteristics are summarized in Table\u00a01. The prevalence of obesity-related comorbid conditions is shown in Table\u00a02. \nTable\u00a01Demographic Characteristics by Sex and Race\u00a0WomenMenTotalp-ValueAge (years)38\u2009\u00b1\u20091.0 (15\u201373)40\u2009\u00b1\u200911.9 (15\u201365)38\u2009\u00b1\u200911.17 (15\u201373)0.064Weight (kg)134\u2009\u00b1\u200927 (84\u2013263)170\u2009\u00b1\u200943 (81\u2013345)139\u2009\u00b1\u200933 (82\u2013345)<0.01BMI (kg\/m2)51\u2009\u00b1\u200910 (35\u20131,000)55\u2009\u00b1\u200913 (24\u2013116)51\u2009\u00b1\u200910 (24\u2013116)<0.01Caucasian(28%) 238\/853(53%) 78\/14732%<0.01African\u2013American(30%) 253\/853(20%) 29\/14728%0.01Hispanic(42%)358\/853(27%)40\/14740%<0.01Other(0.5%) 4\/853(0%) 0\/1470.4%0.41Table\u00a02Prevalence of Obesity-Related Comorbid Conditions by Sex\u00a0Women (N\u2009=\u2009853) (%)Men (N\u2009=\u2009147) (%)Total (%)p-ValueType II diabetes mellitus177 (21)54 (37)23<0.01Hypertension310 (36)79 (54)39<0.01CAD\/CHF29 (3)26 (18)6<0.01Dyslipidemia376 (44)80 (54)470.02Sleep apnea172 (20)63 (43)24<0.01Asthma135 (16)16 (11)150.12Dyspnea on exertion811 (95)127 (86)48<0.01GERD513 (60)77 (52)590.08Osteoarthritis791 (93)126 (87)92<0.01Urinary stress incontinence430\/(50)5 (3)44<0.01Irregular menses276 (32)NA32NACAD\/CHF coronary artery disease\/congestive heart failure, GERD gastroesophageal reflux disease\nThe most common comorbidites encountered were dyspnea on exertion (94%), joint pain\/arthritis (92%), and gastroesophageal reflux disease (59%). The comorbidites typically associated with systemic disease included hypertension (HTN, 39%), obstructive sleep apnea (SA, 24%), dyslipidemia (46%), and asthma (15%). Approximately 23% of the patient population suffered from type II diabetes mellitus (DM). At time of initial evaluation, 13.0% of this diabetic patient subset had a prior history of insulin-dependent DM, 57.6% had noninsulin dependent DM, and 23% had a previous diagnosis of DM intermittently controlled on diet or were newly diagnosed with DM during their preoperative evaluation. Six percent of the patient population had angiographically documented histories of coronary artery disease (CAD) but were deemed suitable risk by their respective specialists.\nProcedures and Duration of Hospital Stay There were 966 primary RYGB and 34 revisions of failed bariatric procedures (21\u2014VBG, 5\u2014RYGB, 8\u2014other; all performed at outside institutions) to RYGB. The median length of stay for primary procedures was 2.4\u00a0days compared to 3.7\u00a0days for revisions. Average (LOS) for all patients having primary RYGB was 3.8\u00a0days with 87% of the group leaving in 3\u00a0days or less.\nComplications Overall, 91% of the procedures were without systemic or technical complications.The incidence of complications in relation to BMI is summarized in Table\u00a03. Overall, systemic complications occurred rarely, but did not usually correlate with BMI. The most common technical complications were incisional hernia (3.5%), intestinal obstruction (1.9%), and leak\/perforation (1.6%). \nTable\u00a03Incidence of Complications after RYGBComplicationBMI\u2009<\u200950 (%) (N\u2009=\u2009481)BMI\u2009>\u200950 (%) (N\u2009=\u2009515)Total (%)p-ValueSystemic complicationsProlonged intubation3 (0.6)8 (1.5)1.10.16 (NS)Deep venous thrombosis0 (0)2 (0.4)0.2NSPulmonary embolism0 (0)3 (0.6)0.3NSMI\/fatal arrhythmia1 (0.2)1 (0.2)0.2NSTechnical complicationsIncisional hernia10 (2.1)25 (4.8)3.50.019Intestinal obstruction10 (2.1)9 (1.7)1.9NSLeak7 (1.5)9 (1.9)1.6NSDehiscence2 (0.4)2 (0.4)0.4NSGI bleeding requiring transfusion3 (0.6)6 (1.2)0.9NSAnastomotic ulcer2 (0.4)0 (0)0.2NSAnastomotic Stricture6 (1.2)2 (0.4)0.8NSDeath4 (0.8)11 (2.1)1.5.03Thirty-one patients (3.1%) required reoperation within 30\u00a0days of the original procedure. The indications for reoperation within 30\u00a0days were leak\/perforation (11), intestinal obstruction (9), bleeding (4), rule-out leak (2), dehiscence (4), and subphrenic abscess (1).Indications for late operations (>30\u00a0days postoperatively) included incisional hernia repair in 35 patients, intestinal obstruction in 10 patients, and repair of leaks\/perforations in five patients who were experiencing ongoing postoperative complications. No patients required reoperation for refractory anastomatic stricture or ulcer. One patient developed a gastrogastric fistula after repair of an early leak, which was treated expectantly since it was clinically insignificant. No late gastrogastric fistulae were identified.\nDeaths Thirty-day mortality was 1.2%. Overall mortality attributable to surgery was 1.5%. Patients with late deaths due to unrelated events, such as motor vehicle accidents (N\u2009=\u20092) or drug overdoses (N\u2009=\u20091), were excluded and not classified as mortalities in the analysis. Mortality correlated with BMI, with four (0.8%) patients having a BMI\u2009<\u200950 dying compared to 11 (2.1%) patients with BMI\u2009\u2265\u200950 (p\u2009=\u20090.03) (Table\u00a03).Causes of death after RYGB, along with their timing and relationship to BMI are shown in Table\u00a04. Two patients died of fatal arrhythmias on POD #3 and #4. Both were males, ages 48 and 54, with BMIs\u2009>\u200950, DM, CAD, HTN, and SA. Autopsies were performed in both instances and no other precipitating factors were identified. One patient, a 43-year-old woman with a BMI 59, died of a pulmonary embolism (identified at autopsy) at home after 2\u00a0weeks. \nTable\u00a04Causes of Early and Late Deaths Related to BMICause of deathEarly (<30\u00a0days) (N\u2009=\u200911)Late (>30\u00a0days) (N\u2009=\u20094)BMI\u2009<\u200950BMI\u2009\u2265\u200950BMI\u2009<\u200950BMI\u2009\u2265\u200950MI\/arrhythmia0200Pulmonary\/PE0100MSOF 20 leak1212MSOF 20 bowel obstruction0100MSOF cause unknown1100Bleeding complications1200PE Pulmonary embolism, MSOF multisystem organ failureSix patients died from MSOF after postoperative leaks, three from the gastrojejunostomy and three from perforations of the distal small bowel within the common channel. Of these, four occurred and were diagnosed within 48\u00a0h of the initial RYGB and all were emergently explored. All four developed MSOF and expired between 14 and 211\u00a0days postoperatively. Of the remaining two deaths, one patient, 46-year-old woman with a BMI 79 and a history of chronic ventilator dependence due to a paralyzed left hemidiaphragm on chronic steroid therapy developed a late leak and multiple intestinal fistulae and eventually succumbed to MSOF. The other patient, a 43-year-old woman, BMI 43, had undergone a laparoscopic RYGB at another institution that was complicated by a strangulated internal hernia, massive intestinal gangrene, and short-bowel syndrome. She underwent a reversal of her RYGB with reconstruction of her GI tract, but developed multiple small bowel fistulae 2\u00a0weeks postoperatively and died of MSOF several months later.Three patients died from a severe systemic inflammatory response syndrome (SIRS) with MSOF accompanied by extremely high fevers, without any identifiable source. One was a 70-year-old woman with BMI 68 underwent reexploration for an early postoperative small bowel obstruction. The others were a 54-year-old woman with a BMI 47, DM, HTN, and SA and a 37-year-old male with BMI 94 and severe SA and HTN. Each developed SIRS and MSOF with temperatures of 105\u2013107\u00b0F and hyperdynamic circulations. No intraabdominal or other sources were identified despite numerous cultures and radiologic studies.Three patients died of bleeding complications. A 43-year-old woman with a BMI 48 suffered progressive hypotension and tachycardia in the recovery room postoperatively. These symptoms were initially addressed with rehydration allowing the patient\u2019s hematocrit to fall to a level where hypovolemic shock and resulting coagulopathy obscured efforts to surgically control or identify a single source. A 31-year-old woman with a BMI 50 was returned to the OR 4\u00a0h postoperatively for control of bleeding from the small bowel mesentery. After this second procedure, she developed SIRS, temperatures of 106\u00b0F, and MSOF, with no identifiable source of sepsis. A 54-year-old male with a BMI 56 and a history of HTN, CAD, and chronic atrial fibrillation suffered a postoperative myocardial infarction and developed a coagulopathy complicated by a massive intraspenic hematoma after restarting coumadin, which rapidly progressed to anuria and MSOF. Attempts to reverse his anticoagulation and control the bleeding angiographically were unsuccessful.The necessity for reoperation within 30\u00a0days of the original procedure was particularly ominous. Overall, the incidence of death after a second operative procedure within 30\u00a0days was 9\/31 (29%). Two of the 16 patients with a BMI\u2009<\u200950 who required reoperation within 30\u00a0days died (12.5%) compared to 7 out of 15 patients with BMI\u2009\u2265\u200950 (47% p\u2009=\u20090.03). This is similar to the mortality for the entire series where 0.8% for patients with a BMI\u2009<\u200950 died compared to 2.1% for patients with BMI\u2009\u2265\u200950 (p\u2009=\u20090.03). However, among those 15 patients classified as operative deaths for the entire series, 9 (60%) died after their second operative procedure.Logistic regression demonstrated that CAD [LR 7.5 p\u2009<\u20090.01 (95% CI 2.2 to 25.3)], and SA [LR 3.3 p\u2009=\u20090.03 (95% CI 1.1 to 10.1)], followed by age [LR 1.06 p\u2009=\u20090.042 (95% CI 1.00 to 1.12)], were risk factors for death in all patients (Table\u00a05). Although a small sample set, 12.7% of patients with CAD died (7\/55) and 29% of patients with BMI\u2009>\u200950 and CAD and SA died. \nTable\u00a05Logistic Regression Evaluation of Patient Comorbidity as Predictors of Mortality\u00a0p-ValueRelative Risk95% Confidence IntervalAge0.0421.0591.002\u20131.120BMI0.1301.0250.993\u20131.058Female0.6120.7290.215\u20132.474DM0.8520.8890.259\u20133.054HTN0.2572.0850.585\u20137.438CAD0.0017.4462.195\u201325.258Dyslipidemia0.0990.3590.106\u20131.211Asthma0.0703.0650.913\u201310.293SA0.0333.3421.104\u201310.115SOB0.6930.6440.073\u20135.725Although the average BMI of males was slightly greater than that of females (55.2 vs 51.2\u00a0kg\/m2, p\u2009<\u20090.01), the two populations also differed in characteristics other than BMI. The male population had a significantly significant greater prevalence of DM, HTN, CAD, dyslipidemia, and SA. Females had a greater prevalence of pulmonary comorbidities, including asthma and dyspnea on exertion (Table\u00a02).When logistic regression was performed specific to the subsets of sex, males with angiographically demonstrated CAD were 30 times more likely to die [LR 30.1 p\u2009=\u20090.028 (95% CI 1.4 to 631.4)]. Logistic regression did not identify CAD as a significant predictor when the analysis was limited to women. Predictors of death for women include age [LR 1.07 p\u2009=\u20090.033 (95 % CI 1.0 to 1.14) and SA [LR 4.1 p\u2009=\u20090.040 (95% CI 1.07 to 16.2)].Logistic regression was repeated specific to race. When limited to Caucasian patients only, Caucasians with CAD were 58 times more likely to die [LR 58.8 p\u2009<\u20090.01 (95% CI 4.8 to 716.9)] than those Caucasians without CAD. Increasing BMI was also significant among Caucasians. [LR 1.08 p\u2009=\u20090.02 (95% CI 1.01 to 1.16)]. Evaluation of African\u2013American patients demonstrated that only SA was significant [LR 19.1 p\u2009=\u20090.03 (95% CI 1.29 to 282.8)]. Regression of the Hispanic population did not identify a specific factor. One-way ANOVA did not demonstrate any significant differences in prevalence of death or CAD between the three racial groups for the entire population or specific to sex.Hispanic patients had significantly less SA than either the Caucasian or African\u2013American patients (18.1 vs 25.6 and 27.4%, respectively (p\u2009=\u20090.01)). When examined specifically in relation to sex, there were no differences among prevalence of SA in the males. African\u2013American women, however, had the greatest prevalence of SA (26.4%) compared to Caucasian (18.9%) and Hispanic (15.7%) women (p\u2009<\u20090.01).\nDiscussion\nThe tremendous growth of bariatric surgery over the past several years has spawned much interest in its complications and mortality, first in the media, but most recently in the public health arena as well. Health and malpractice insurance carriers as well as governmental agencies and professional societies are evaluating the risks of bariatric surgery and the surgeons that perform it. In several states, insurance companies have stopped covering bariatric surgery at the same time that the Centers for Medicare and Medicaid Services have approved coverage for them. Several malpractice carriers have stopped issuing policies for surgeons performing bariatric procedures while others are categorizing bariatric surgery as a high-risk subspecialty area, similar to obstetrics and neurosurgery, and increasing premiums accordingly. In each of these instances, the overriding fear or consideration appears to be that the risks associated with bariatric surgery are excessively high.\nMuch of the information being utilized in this regard has come form series utilizing pooled data from multiple smaller series or government databases.1,5\u20139 Buchwald et al.5 performed a metaanalysis of 16,944 patients which included 7,074 patients that underwent gastric bypass (open and laparoscopic) in 44 studies with a 30-day mortality rate of 0.5%. Several authors have reported that complication rates of bariatric surgery were inversely correlated with case loads, reporting mortality rates of in the range of 0.1\u20130.5% for surgeons more than 100 or 150 cases per year.6\u20138 Flum et al.9 recently reported 30- and 90-day mortality rates of 2.0 and 2.8%, respectively in Medicare beneficiaries having bariatric surgery, with men having higher rates than women and those over 65\u00a0years of age having higher rates than those younger than 65. Many of these series lack data concerning BMI and comorbidities, making risk assessment difficult or impossible. In one large series from a single-center, Christou et al.10 reported a 30-day mortality of 0.4% in 1,035 patients undergoing RYGB, of whom 820 had open RYGB. No details about preexisting comorbidities or perioperative complications were given.\nThe results of these pooled series differ from those in several large series of open gastric bypasses2,3,11. Livingston et al.2 reported an operative mortality of 1.3% in 1,067 patients after open RYGB. In his series, mean BMI was 53.6\u00a0kg\/m2, mean age 42.3\u00a0years, and the incidence of comorbidities was diabetes (23%), hypertension (48%), and sleep apnea (39%). Only male sex was predictive of severe life-threatening complications; mortality in patients over 55\u00a0years was significantly greater than in patients under 55 (3.5 vs 1.1%, p\u2009<\u20090.05). More recently, Fernandez et al.3 analyzed their patient cohort of 1,431 patients having open RYGB and found a 1.9% mortality, which was associated with age, weight, longer limb gastric bypass, and the occurrence of a leak or pulmonary embolism. In that series, the mean BMI was 53.3, mean age 40.7\u00a0years, and incidence of serious comorbidities was diabetes (19.5%), hypertension (51%), and sleep apnea (33%). In both of these series, the BMI was higher, the patients were older and the incidence of serious comorbidities, such as diabetes, hypertension, dyslipidemia, and sleep apnea, higher. Pories et al.11 reported a 1.5% perioperative mortality rate, with 0.8% dying of sepsis, 0.5% dying of pulmonary embolism, and 0.2% of an unknown cause. Additional information regarding the incidence of leaks, bowel obstructions and other complications was not reported as these were not the focus of the original paper.\nSimilarly, in the present series, the 30-day mortality rate was 1.2% in 1,000 patients with a mean BMI of 52\u00a0kg\/m2. The prevalence of preoperative comorbidities was comparable to the larger series (diabetes\u201423%, hypertension\u201439%, dyslipidemia\u201446%, coronary artery disease\/congestive heart failure\u20145.5%, sleep apnea\u201423.5%, and asthma\u201415%) and generally higher than in the pooled series. The incidence of leaks and postoperative small bowel obstruction in our series was comparable to the other series. The incidence of pulmonary embolism was less than those reported, perhaps related to the use of both pneumatic compression stockings and subcutaneous heparin and early ambulation with a shorter length of stay. Although the incidence of incisional hernia in our series was low (3.5%), this may well be affected by our suboptimal follow-up.\nMultisystem organ failure accounted for 11 deaths in our series (73%). In six patients, this resulted from leaks or perforations; in one it followed a bowel obstruction, in two it followed postoperative hemorrhage and in two patients, the cause was never determined. In each of these instances, the complication was identified early and appropriate treatment and supportive care instituted. In four of the patients, the clinical course of SIRS and MSOF was characterized by extremely high temperatures (>105\u00b0F), with no apparent source ever identified. To our knowledge, this \u201csyndrome\u201d has not been reported, but may be due to the fact that the enormous adipose tissue stores in these patients may act as a \u201cmetabolic sink\u201d, releasing cytokines and other mediators and perpetuating this extreme systemic inflammatory response. Two of the deaths were due to fatal arrhythmias, both in patients with known CAD, who were extensively evaluated preoperatively. The death due to pulmonary embolism occurred after discharge, even though the patient received prophylaxis with both heparin and pneumatic compression stockings in the hospital. The remaining death due to exsanguination was clearly preventable.\nAlthough males were significantly heavier and had higher BMIs than women, sex was not an independent predictor of morbidity. However, the presence of angiographically documented coronary artery disease was particularly ominous in men. Males with angiographically demonstrated CAD were 30 times more likely to die. In women, predictors of death included age and SA, but not CAD. With respect to race, Caucasian males with BMI\u2009>\u200950 and CAD were most likely to die, whereas SA was a predictor in African\u2013American men. There were no predictors in Hispanics. Despite the increased mortality in Caucasian males with CAD, these patients were not candidates for cardiac revascularization and extreme weight loss was the only intervention thought to make a beneficial health impact.\nBased upon our data and those of others2,3,11, it appears that the risk of open RYGB is in the range of 1\u20132%. Risk appears to be adversely affected by increasing BMI and those factors with which it is often associated namely male sex and coronary artery disease. How this compares with the perioperative mortality after laparoscopic RYGB is still unclear because many series of patients having laparoscopic RYGB do not include patients with the highest BMIs, above 60 or 70\u00a0kg\/m2, or patients having revisional surgery.\nA perioperative mortality rate of 1.2% after RYGB compares favorably with that after other common surgical procedures. For example, perioperative mortality after elective surgery for abdominal aortic aneurysms has been reported at 3.1\u20134.7% overall, 1.0\u20132.7% in patients under 65\u00a0years of age, and 3.5\u20135.2% in patients over 65\u00a0years of age.12 Using Medicare data adjusted for high volume surgeons, Birkmeyer et al.13 reported perioperative mortality rates of 4.5% for colectomy, 8.6% for gastrectomy, 8.4% for esophagectomy, 3.8% for pancreatic resection, 4.0% for pulmonary lobectomy, and 10.7% for pneumonectomy. While it is true that all of these patients were over 65\u00a0years of age, the fact still remains that these perioperative mortality rates are all substantially greater than that after RYGB (even in Medicare recipients, as recently reported9) and neither the public, the press, the insurance industry, or various state Departments of Health are appalled or alarmed, or calling for a moratorium on those procedures. This is not meant to suggest that every effort should not be made to lessen the risks of bariatric surgery and to improve operative mortality, but rather to inject some proportionality into the discussion. The importance of such careful analysis of bariatric surgical data, including its limitations, and the need to continue to offer bariatric surgery to those patients for whom it constitutes the best available treatment has recently been emphasized.14 In his 2004 PERSPECTIVE, Surgery for Severe Obesity, Steinbrook15 quotes Robert Brolin, MD, cautioning physicians and the public \u201c...to reconcile the fact that the operation has a real mortality and it will continue to have a real mortality under the best of circumstances. Some of these patients are just profound operative risks for any kind of surgical intervention...The sickest ones are the ones who benefit the most, but they are also the highest risk\u201d.\nConclusion\nRYGB can be performed with acceptable perioperative morbidity in patients over a wide range of BMIs. Patients with BMI\u2009\u2265\u200950 have a higher morality for both initial operations and after reexploration. Age, coronary artery disease, and obstructive sleep apnea correlate with perioperative mortality. These three comorbidities were more prevalent in these patients and may contribute to this finding.","keyphrases":["morbidity","mortality","roux-en-y gastric bypass","obesity","morbid obesity"],"prmu":["P","P","P","P","P"]}
{"id":"Clin_Rheumatol-4-1-2367392","title":"Tracheobronchomalacia due to amyloidosis in a patient with rheumatoid arthritis\n","text":"In this case report, we describe a patient with longstanding rheumatoid arthritis who developed tracheobronchomalacia with fatal outcome. Despite negative antemortem biopsies of abdominal fat and tongue, amyloid was found postmortem in the trachea and appeared to be associated with tracheobronchomalacia.\nIntroduction\nAmyloidosis in longstanding chronically active rheumatoid arthritis (RA) is a well-known complication of this disease [1]. We present a RA patient with amyloidosis, albeit with a unique presenting symptom and unexpected organ involvement.\nCase report\nThe patient was a 69-year-old female with seropositive erosive RA, who had not been treated with disease-modifying antirheumatic drugs since 1984. In March 2005, she presented to another hospital because of progressive dyspnea of several hours, due to a bronchopneumonia. After 2\u00a0days, she developed a stridor. Otolaryngological examination results showed diffuse swelling of the tongue, pharynx and neck, and an extensive tracheobronchomalacia. Treatment consisted of intubation, antibiotics, diuretic therapy, and corticosteroids. She was admitted to our Intensive Care Unit because of progressive swelling and respiratory insufficiency due to tracheobronchomalacia. Physical examination results revealed extensive swelling and a remarkable protrusion of the tongue. Typical rheumatoid joint deformities and rheumatoid nodules were present but no active arthritis. Laboratory investigations revealed a normocytic anemia and a renal insufficiency. The measured creatinine clearance was 12\u00a0ml\/min, with a proteinuria of 0.35\u00a0g\/24\u00a0h. Computed tomography scan of thorax and neck provided no explanation for the tracheobronchomalacia. Clinical suspicion of amyloidosis was not confirmed by biopsies of abdominal fat tissue and of the tongue. Ultimately, the patient succumbed to multiple organ failure. Autopsy revealed AA amyloid in the trachea (Fig.\u00a01), spleen, liver, and perivascularly in the kidneys.\nFig.\u00a01Amyloidosis of the trachea in Congo red\nDiscussion\nAmyloidosis of the trachea is a rare but known disorder, even rarer in patients with systemic diseases (such as Sjogren syndrome) but never associated with tracheobronchomalacia [2\u20134]. We report the first case of an RA patient with tracheobronchomalacia due to AA amyloidosis of the trachea. The diagnosis was only confirmed at postmortem, although several biopsies had been done in life to confirm the clinical suspicion. We refrained from rectal and kidney biopsies because of the relatively high risk of complications associated with these procedures; the absence of diarrhea and the low level of proteinuria suggested a relatively low pretest probability. Therefore, the finding of renal amyloid was surprising. Recently, Uda et al. [5] described a prospective cohort study of patients with RA without clinical signs of kidney disease and amyloidosis of the kidney. At follow-up, the group of patients with amyloid deposition confined to the perivascular region continued to have little proteinuria and a good renal prognosis, compared to those with amyloid deposition in the glomeruli who developed rapidly deteriorating renal function. This patient, however, does show that the disease course in RA patients with perivascular amyloid deposition in the kidney is not benign and may be associated with fatal complications elsewhere.\nIn conclusion, tracheobronchomalacia can be due to amyloid deposition in patients with RA. Amyloidosis should be considered in RA patients with a small amount of proteinuria, and a renal biopsy should be performed, especially as accumulating evidence shows that patients with amyloidosis have a better prognosis when the ongoing inflammation is effectively suppressed [1].","keyphrases":["tracheobronchomalacia","amyloidosis","rheumatoid arthritis","kidney"],"prmu":["P","P","P","P"]}
{"id":"Pediatr_Radiol-3-1-1805044","title":"2005 PRETEXT: a revised staging system for primary malignant liver tumours of childhood developed by the SIOPEL group\n","text":"Over the last 15 years, various oncology groups throughout the world have used the PRETEXT system for staging malignant primary liver tumours of childhood. This paper, written by members of the radiology and surgery committees of the International Childhood Liver Tumor Strategy Group (SIOPEL), presents various clarifications and revisions to the original PRETEXT system.\nIntroduction\nThe PRETEXT system was designed by the International Childhood Liver Tumor Strategy Group (SIOPEL) for staging and risk stratification of liver tumours [1, 2]. PRETEXT is used to describe tumour extent before any therapy, thus allowing more effective comparison between studies conducted by different groups. The system has good interobserver reproducibility [3] and good prognostic value in children with hepatoblastoma [2\u20135], and is the basis of risk stratification in current SIOPEL hepatoblastoma studies. Most other study groups now use the PRETEXT system to describe imaging findings at diagnosis, even if this is not their main staging system.\nCertain limitations of the system have become obvious over the last 15\u00a0years. In addition, there have been significant advances in imaging during this period [6]. This paper is the report of a working party that met in June 2005 to update the PRETEXT system.\nPRETEXT staging is based on Couinaud\u2019s system of segmentation of the liver (Fig.\u00a01) [7]. The liver segments are grouped into four sections as follows: segments 2 and 3 (left lateral section), segments 4a and 4b (left medial section), segments 5 and 8 (right anterior section) and segments 6 and 7 (right posterior section). The term section is used (where other authors use segment or sector) to avoid terminological confusion.\nFig.\u00a01Schematic representations of the segmental anatomy of the liver. a Frontal view of the liver. The numerals label Couinaud\u2019s segments 2 to 8. b The hepatic veins (black) and the intrahepatic branches of the portal veins (grey) are shown. Segment 1 (equivalent to the caudate lobe) is seen to lie between the portal vein and the inferior vena cava. c Exploded frontal view of the segmental anatomy of the liver. The umbilical portion of the left portal vein (LPV) separates the left medial section from the left lateral section (LLS). Segment 1 is obscured in this view. Note that the term \u201csection\u201d has been used in preference to \u201csegment\u201d or \u201csector\u201d (see text). d Transverse section of the liver shows the planes of the major venous structures used to determine the PRETEXT number. The hepatic (blue) and portal (purple) veins define the sections of the liver (2\u20138). This schematic diagram shows how the right hepatic (RHV) and middle hepatic (MHV) veins indicate  the borders of the right anterior section (RAS)  with the right posterior (RPS) and left medial (LMS) sections. Note that the left portal vein (LPV) actually lies caudal to the confluence of the hepatic veins and is not seen in the same transverse image. The left hepatic vein (LHV) runs between segments 2 and 3 and is not used in PRETEXT staging\nIn the original system, the caudate lobe (segment 1) was ignored. The PRETEXT number was derived by subtracting the highest number of contiguous liver sections that were not involved by tumour from four [1]. This number is, very roughly, an estimate of the difficulty of the expected surgical procedure (Table\u00a01). Pedunculated tumours are considered to be confined to the liver and to occupy only the section(s) from which they originate. \nTable\u00a01Definitions of PRETEXT number (see text for PRETEXT number of tumours involving the caudate lobe)PRETEXT numberDefinitionIOne section is involved and three adjoining sections are freeIIOne or two sections are involved, but two adjoining sections are freeIIITwo or three sections are involved, and no two adjoining sections are freeIVAll four sections are involved\nIn addition to describing the intrahepatic extent of the primary tumour(s), the PRETEXT system includes certain other criteria. These assess involvement of the inferior vena cava (IVC) or hepatic veins (designated V), involvement of the portal veins (P), extrahepatic abdominal disease (E) and distant metastases (M).\nThe purpose of the 2005 revision was to improve the original definitions of the PRETEXT stages, to clarify the criteria for \u201cextrahepatic\u201d disease, and to add new criteria (Table\u00a02). The term \u201cextrahepatic\u201d disease is confusing, and these categories will in future be called \u201cadditional criteria\u201d. There is still much to be learned about prognostic factors in the primary malignant liver tumours of childhood. An important goal of these changes, therefore, is to improve our ability to identify prognostic imaging findings, and thereby refine risk stratification. \nTable\u00a022005 PRETEXT staging: additional criteriaCaudate lobe involvementCC1Tumour involving the caudate lobeAll C1 patients are at least PRETEXT IIC0All other patientsExtrahepatic abdominal diseaseEE0 No evidence of tumour spread in the abdomen (except M or N)Add suffix \u201ca\u201d if ascites is present, e.g., E0aE1Direct extension of tumour into adjacent organs or diaphragmE2Peritoneal nodulesTumour focalityFF0Patient with solitary tumourF1Patient with two or more discrete tumoursTumour rupture or intraperitoneal haemorrhageHH1Imaging and clinical findings of intraperitoneal haemorrhageH0All other patientsDistant metastasesMM0No metastasesAdd suffix or suffixes to indicate location (see text)M1Any metastasis (except E and N)Lymph node metastasesNN0No nodal metastasesN1Abdominal lymph node metastases onlyN2Extra-abdominal lymph node metastases (with or without abdominal lymph node metastases)Portal vein involvementPP0No involvement of the portal vein or its left or right branchesSee text for definition of involvement. Add suffix \u201ca\u201d if intravascular tumour is present, e.g., P1aP1Involvement of either the left or the right branch of the portal veinP2Involvement of the main portal veinInvolvement of the IVC and\/or hepatic veinsVV0No involvement of the hepatic veins or inferior vena cava (IVC)See text for definition of involvement. Add suffix \u201ca\u201d if intravascular tumour is present, e.g., V3aV1Involvement of one hepatic vein but not the IVCV2Involvement of two hepatic veins but not the IVCV3Involvement of all three hepatic veins and\/or the IVC\nAlthough the PRETEXT system is principally used for hepatoblastoma, the 2005 revision is intended to be applicable to all primary malignant liver tumours of childhood, including hepatocellular carcinoma and epithelioid haemangioendothelioma. The original SIOPEL risk stratification system for hepatoblastoma has already been modified in the protocols for current SIOPEL studies (Table\u00a03). Firstly, tumour rupture or intraperitoneal haemorrhage at the time of diagnosis (H1, see below) is now a defining criterion of high risk. Secondly, children with alpha-fetoprotein levels of <100\u00a0\u03bcg\/l are also considered to be high risk. The 2005 revision involves no further change in the SIOPEL risk stratification system for hepatoblastoma. \nTable\u00a03Risk stratification in hepatoblastoma for current SIOPEL studiesHigh riskStandard riskPatients with any of the following:Serum alpha-fetoprotein <100\u00a0\u03bcg\/lAll other patientsPRETEXT IVAdditional PRETEXT criteria:\u00a0E1, E1a, E2, E2a\u00a0H1\u00a0M1 (any site)\u00a0N1, N2\u00a0P2, P2a\u00a0V3, V3a\nPRETEXT grouping\nThe traditional approach to radiological segmentation of the liver, based on the paths of the hepatic veins, is an oversimplification. This is partly due to the variability of hepatic venous anatomy [8\u201310]. The main problem, however, is the imperfect correlation with segments defined by the branching pattern of the portal veins [8, 11\u201313]. Although the plane of the right hepatic vein reliably separates the right posterior and anterior sections [9], the left hepatic vein runs to the left of the boundary between the left lateral and medial sections, which is best defined by the plane of the fissure of the ligamentum teres and the umbilical portion of the left portal vein (Fig.\u00a01) [14].\nPRETEXT I\nThis group includes only a small proportion of primary malignant liver tumours of childhood. From the definition of the PRETEXT number, it can be seen that only tumours localized to either the left lateral section or the right posterior section qualify as PRETEXT I (Fig.\u00a02).\nFig.\u00a02PRETEXT I. a The left lateral section (segments 2 and 3) is involved. b The right posterior section (segments 6 and 7) is involved\nPRETEXT II\nMost PRETEXT II tumours are limited to either the right lobe or the left lobe of the liver. Tumours of the left medial or right anterior sections are also PRETEXT II. Multifocal tumours involving only the left lateral and right posterior sections are classified as PRETEXT II; this pattern is very rare. Tumours limited to the caudate lobe were not classifiable under the original PRETEXT system [1]. In the 2005 PRETEXT system these tumours are classified as PRETEXT II (but see also C, below). This is the only change in the PRETEXT numbering system in this revision. There is no change in numbering for tumours involving the caudate lobe and any other part of the liver, which are classified as PRETEXT II (if two or three contiguous sections are free), III (if there are no two contiguous sections free) or IV (if all four sections are involved) (Fig.\u00a03).\nFig.\u00a03PRETEXT II. a Tumour involving only the right lobe of the liver. b A transverse T1-weighted MR image of a child with hepatoblastoma shows that the middle hepatic vein (arrow) is displaced but not involved by the tumour. This is the most common type of PRETEXT II tumour. c Tumour involving only the left lobe of the liver. d Tumour involving only the left medial section. e Tumour involving only the right anterior section. f Multifocal tumours involving only the left lateral and right posterior sections. g The tumour is confined to the caudate lobe (PRETEXT II C1, see text; RPV right portal vein)\nPRETEXT III\nThe unifocal tumours in this category spare only the left lateral or right posterior section. These tumours are relatively common. In children with hepatoblastoma, great care must be taken to distinguish between invasion and compression of the apparently uninvolved section of the liver, because risk stratification (and\/or the need for liver transplantation) may depend on this point. Anterior central liver tumours involve segment 4 and either or both of segments 5 and 8. Although recent advances in surgical technique permit resection of these tumours without trisectionectomy [15], classification as PRETEXT III reflects the difficulty of these operations (Fig.\u00a04).\nFig.\u00a04PRETEXT III. a Extensive tumour sparing only the left lateral section. b Extensive tumour sparing only the right posterior section. c Anterior central liver tumour involving the left medial and right anterior sections. d Contrast-enhanced CT image shows a central liver tumour lying between the left portal vein (white arrow) and the right hepatic vein (black arrow). e Multifocal PRETEXT III tumour, sparing the right anterior section. f Multifocal tumours sparing only the left lateral and right anterior sections. g Multifocal tumours sparing only the left medial and right posterior sections\nMultifocal PRETEXT III tumours may also spare the right anterior or left medial sections, or two non-contiguous sections. These patterns are rare.\nPRETEXT IV\nPRETEXT IV tumours involve all sections of the liver. These tumours are often multifocal. Alternatively, a very large solitary tumour can involve all four sections (Fig.\u00a05).\nFig.\u00a05PRETEXT IV. a Multifocal PRETEXT IV tumours involve all four sections. b Contrast-enhanced CT image of a patient with PRETEXT IV F1 (see text) hepatoblastoma. c Unifocal PRETEXT IV tumours often have a diffuse growth pattern. d Contrast-enhanced CT image of a patient with diffuse PRETEXT IV hepatoblastoma\nC: caudate lobe tumours\nThe caudate lobe and caudate process (segment 1 or segments 1 and 9, depending on the system of nomenclature) can be resected with either the left or right lobe of the liver [7]. For this reason, segment 1 was not considered in the PRETEXT classification in the original system [2]. Modern surgical techniques have made resection of segment 1 safer, but these operations remain difficult. Involvement of the caudate lobe is, therefore, a potential predictor of poor outcome. If any tumour is present in segment 1 on imaging at diagnosis (Fig.\u00a03g), the patient will be coded as C1, irrespective of the PRETEXT group (see above). All other patients should be coded as C0.\nE: extrahepatic abdominal disease\nThe assessment of extrahepatic abdominal disease was one of the most confusing aspects of the original PRETEXT system, and clearly needed revision. Originally, there was a requirement for all extrahepatic abdominal spread of tumour (E+) to be proved by biopsy. Modern imaging techniques are capable, in principle, of identifying extrahepatic abdominal tumour extension in many forms. The frequency and significance of these imaging findings is different for different tumour types, and not all patterns are easily biopsied.\nIn hepatoblastoma, for example, direct extension of tumour into other abdominal organs is unusual. Tumour extension through the diaphragm (Fig.\u00a06) is uncommon, but can be shown quite convincingly by MRI or CT, and biopsy proof may be impractical. In the 2005 revision, patients with direct extension of tumour through the diaphragm or into other organs can be coded as E1 without biopsy proof.\nFig.\u00a06Extrahepatic abdominal tumour extension. This composite of contrast-enhanced CT images in a patient with hepatoblastoma shows growth of the primary tumour through the diaphragm into the thorax (E1). The 2005 PRETEXT system no longer requires biopsy proof for this form of tumour spread\nPedunculated tumours are considered to be confined to the sections from which they arise, and are not extrahepatic disease.\nPeritoneal tumour seeding was originally not included in this category [2]. It probably indicates more advanced abdominal disease than direct extension of the primary tumour. Imaging techniques, especially ultrasonography, can often show even small peritoneal nodules clearly, and the differential diagnosis is very limited. In the 2005 revision, peritoneal nodules will be assumed to be metastases, and will be coded as E2. All other patients should be coded as E0.\nAscites is an unusual finding at presentation in hepatoblastoma, but is more common in hepatocellular carcinoma, where it may be an independent predictor of poor prognosis. For this reason, patients with ascites will be coded as E0a, E1a or E2a as appropriate.\nAbdominal lymph node metastases, which were previously recorded as E+, are now coded as N (see below).\nF: tumour focality\nIn SIOPEL 1, multifocal tumours were identified at the time of diagnosis in 18% of the patients with hepatoblastoma where this information was available [4]. Univariate analysis showed that the 5-year event-free survival was significantly worse for patients with multifocal tumour (40%) than for those with unifocal tumour (72%) [4]. The independent significance of this finding is unclear, as there is clearly an association between multifocality and advanced PRETEXT number. The German Society of Pediatric Oncology and Hematology reported slightly different results [16]. In its HB89 study, 21% of patients had multiple well-defined tumours, and these children had a similar disease-free survival (DFS; 87%) to those with a single tumour (86%). However, in 20% of children the tumour exhibited a diffuse growth pattern (Fig.\u00a05), and these had a significantly worse DFS (21%) [16]. Unfortunately, a diffuse growth pattern is difficult to define, and despite the promise that this finding shows as a potential risk factor, it was decided not to incorporate it in the 2005 PRETEXT revision.\nPatients with one hepatic tumour should be coded as F0. All those with more than one tumour nodule (Figs.\u00a03, 4 and 5), regardless of nodule size or PRETEXT stage, should be coded as F1.\nH: tumour rupture or intraperitoneal haemorrhage\nIt is not uncommon for hepatoblastoma and hepatocellular carcinoma to present with tumour rupture [17, 18]. Originally, these patients were not automatically included as high risk in SIOPEL studies, because of the requirement that extrahepatic disease (E) be proved by biopsy. Although the data to prove this are not currently available, it seems intuitively likely that tumour rupture (usually manifesting as intraperitoneal haemorrhage) is a risk factor, and these patients should be coded as H1. Laparotomy or aspiration of peritoneal blood is not necessary for diagnostic purposes if characteristic imaging and clinical findings (such as hypotension and low haematocrit or haemoglobin level) are present. The presence of peritoneal fluid on imaging alone does not imply tumour rupture (but see E above).\nSince the opening of the SIOPEL 4 study in September 2004, tumour rupture has become a defining feature of high-risk hepatoblastoma in SIOPEL studies. Patients with no evidence of tumour rupture or haemorrhage, and those with only subcapsular or biopsy-related intraperitoneal bleeding, are coded as H0.\nM: distant metastases\nPatients with distant metastases at diagnosis are coded as M1. In hepatoblastoma, these metastases are predominantly found in the lungs. Although the best imaging modality for the identification of lung metastases is currently CT, the defining characteristics of lung metastases in this context have not been specifically studied. It is believed, however, that factors favouring a diagnosis of metastasis include multiple lesions, a rounded, well-defined contour and a subpleural location. In most parts of the world, a single rounded lung lesion with a diameter of >5\u00a0mm in a child with a primary liver tumour is very likely to be a metastasis. Patients with these findings on chest CT scans should be classified as M1. Biopsy is not required for staging purposes, because it is uncommon for other lesions to mimic metastases in this clinical context. The protocols of the SIOPEL studies recommend central radiological review if there is any doubt about the presence of lung metastases.\nOther metastases are infrequently found at diagnosis in hepatoblastoma, but are more common in hepatocellular carcinoma. The imaging findings of brain metastases are usually characteristic, and biopsy is not required.\nBone scintigraphy is recommended for staging in children with hepatocellular carcinoma, but not hepatoblastoma. Abnormal calcium metabolism is common in children with hepatoblastoma, and may cause abnormal uptake on bone scintigraphy, especially in the ribs [19], whereas bone metastases are rare [20]. Biopsy proof is therefore mandatory for suspected bone metastases in hepatoblastoma, unless the findings of cross-sectional imaging are characteristic and the patient is already in the high-risk category for some other reason, such as the presence of lung metastases.\nBone marrow biopsy is not recommended in children with hepatoblastoma, because bone marrow spread is rare [20]. It is not known whether metastases at different sites have different prognostic implications. For statistical purposes, it is therefore recommended that one or more suffixes be added to M1 to indicate the major sites of metastasis: pulmonary (p), skeletal (s), central nervous system (c), bone marrow (m), and other sites (x). A child with lung, brain, and adrenal metastases would therefore be coded as M1cpx. Patients with no evidence of haematogenous metastatic spread of tumour should be coded as M0.\nN: lymph node metastases\nBecause porta hepatis (and other abdominal) lymph node metastases are quite unusual in hepatoblastoma, SIOPEL trials have always required this form of tumour spread to be proved by biopsy. In fact, benign enlargement of lymph nodes is probably not uncommon, and the accuracy of positron emission tomography is not known in this context. Because biopsy of equivocal lymph nodes inevitably carries some risk, the SIOPEL committee actively discourages this. Biopsy may, however, be required if there is significant nodal enlargement (for example short axis >15\u00a0mm) in a child with no other criteria for high-risk hepatoblastoma.\nLymph node metastases are quite common in hepatocellular carcinoma and fibrolamellar carcinoma, and biopsy proof is not required if the imaging abnormality is unequivocal. An arbitrary threshold short axis diameter of 15\u00a0mm is suggested for this purpose.\nChildren with no lymph node metastases by these criteria are coded as N0, those with nodal metastases limited to the abdomen (i.e. caudal to the diaphragm and cranial to the inguinal ligament) as N1, and those with extra-abdominal nodal metastases as N2.\nP: portal vein involvement\nInvolvement of the main portal vein and\/or both major branches has been considered a risk factor in hepatoblastoma, because this has obvious implications for the resectability of the tumour. It is also possible that portal vein invasion detected by imaging is an independent risk factor for tumour recurrence [21]. The original PRETEXT criteria, however, did not specifically define the word \u201cinvolvement\u201d.\nIt is well recognized that a tumour that abuts or displaces a major portal venous branch at imaging performed at diagnosis (Fig.\u00a07) may shrink away from the vein following preoperative chemotherapy. Imaging evidence of complete obstruction or circumferential encasement (Fig.\u00a07) is therefore required to qualify as portal vein involvement. Failure to identify the portal vein or one of its major branches in either its normal position or its expected displaced location, on good quality images, is strong evidence of obstruction. The other form of involvement, portal vein invasion, is not uncommon, and is often best detected by ultrasound (Fig.\u00a07). Various US signs may be present [22, 23], and analogous findings can be seen on CT and MR imaging.\nFig.\u00a07Involvement of the portal and hepatic venous systems. a When the tumour (grey) approaches or abuts the vein (black), there is no venous involvement, even if the vein is partly encased. b Complete obstruction or encasement of the vein is one form of involvement. Obstruction of the inferior vena cava by extrinsic compression, however, does not count as involvement (see text). c Intravascular tumour growth in the portal and\/or hepatic venous systems is not uncommon in children with hepatoblastoma or hepatocellular carcinoma. d Transverse ultrasound image of the right lobe of the liver in a patient with hepatoblastoma. The tumour (white circles) has grown into the right branch of the portal vein (P1a), disrupting the normal \u201cwhite line\u201d of the vein wall (arrows)\nPatients with no imaging evidence of involvement of the main portal vein, its bifurcation, or either of its main branches will be coded as P0. Those who fulfil the original PRETEXT definition of P+ (involvement of the main portal vein, its bifurcation, or both of its main branches), as well as those with \u201ccavernous transformation\u201d of the portal vein will be coded as P2. P2, however, represents very advanced disease. For this reason, the category P1 has been created for patients with evidence of involvement of one major branch of the portal vein. In addition, the detection of portal vein invasion should be marked by the suffix \u201ca\u201d (e.g., P2a).\nV: involvement of the IVC and\/or hepatic veins\nThe same definitions of involvement (venous obstruction, encasement and\/or invasion) used for the portal veins apply to the hepatic veins (Fig.\u00a07). A hepatic vein can be assumed to be involved if it cannot be identified at all, and its expected course runs through a large tumour mass. It is important to look carefully for the hepatic veins, preferably with ultrasonography as well as CT and\/or MRI, as they may be displaced from their expected position by the tumour. Complete obstruction of the IVC can occur with mass effect alone, without any tumour extension to the vein itself. Inability to visualize the IVC, and the presence of an enlarged azygos vein, are not, therefore, sufficient criteria for involvement. Patients with no imaging evidence of involvement of the hepatic veins or IVC will be coded as V0.\nAs for the portal vein, the original classification of involvement (V+) indicated a very advanced level of disease. Intermediate categories have therefore been created. V1 and V2 indicate involvement of one or two main hepatic veins respectively. V3 indicates involvement of either the IVC or all three of the hepatic veins. In addition, the detection of hepatic vein or IVC invasion should be marked by the suffix \u201ca\u201d (e.g., V2a). The presence of tumour in the right atrium automatically makes a patient V3a.\nSIOPEL risk stratification for patients with hepatoblastoma\nThe SIOPEL risk stratification for children with hepatoblastoma is essentially unchanged by this revision. Patients with any one or more of certain criteria (Table\u00a03) are high risk. All other SIOPEL patients are standard risk.\nPresurgical re-evaluation\nAlthough the timing of surgery will depend on the treatment protocol and the patient\u2019s response to therapy, preoperative reimaging is almost always necessary. All of the PRETEXT categories should be reassessed after preoperative chemotherapy, as near as possible to the time of surgery, and recorded as POSTEXT (post-treatment extent of disease). Comparison of surgical findings with POSTEXT will allow prospective assessment of the accuracy of imaging techniques.","keyphrases":["staging","liver","tumour","children","hepatoblastoma"],"prmu":["P","P","P","P","P"]}
{"id":"Trans_R_Soc_Trop_Med_Hyg-1-5-1950430","title":"Associations between mild-to-moderate anaemia in pregnancy and helminth, malaria and HIV infection in Entebbe, Uganda\n","text":"Summary\n1\nIntroduction\nAnaemia in pregnancy contributes to maternal deaths and may also contribute to adverse birth outcomes including intrauterine growth retardation and prematurity, and hence to perinatal morbidity and mortality (WHO, 1991).\nHookworm is an important cause of anaemia in developing countries, but lack of consensus regarding the risks and benefits of treating helminths in pregnancy has, until recently, led to a tendency to exclude pregnant and even breastfeeding women from deworming programmes. In 1994, a call was made for research leading to improved estimates of hookworm infection in women of child-bearing age and for evaluation of interventions that might be beneficial (WHO, 1994) but, to our knowledge, only one placebo-controlled trial of the treatment of hookworm in pregnancy has been reported (Torlesse and Hodges, 2000, 2001).\nMore recently, an Informal Consultation held by the WHO gave consideration to the possible effects of schistosomiasis in pregnancy and it was suggested that anaemia might be among them (Allen et al., 2002; WHO, 2002). Although praziquantel had been widely avoided in pregnant and lactating women, it was noted that there was no evidence from studies in animals, from case reports or from mass treatment campaigns in humans of any major adverse effects. Treatment of schistosomiasis during pregnancy was therefore advocated by the Consultation Committee.\nWe have undertaken a trial designed to examine the effects of maternal helminths and of deworming during pregnancy on the response to immunisation and susceptibility to infection and disease in infancy [ISRCTN32849447] (Elliott et al., 2007). Anaemia is among the secondary outcomes of the trial. The trial is ongoing. In view of current interest in the role of helminths in anaemia during pregnancy, we have examined associations between anaemia and helminths and other major infections (malaria and HIV) among pregnant women at enrolment into the trial.\n2\nMaterials and methods\n2.1\nStudy population and procedures\nThe study area comprises Entebbe Municipality and the adjacent subcounty of Katabi. This area supports semi-urban, rural and fishing communities residing on the Entebbe peninsula in Lake Victoria, Uganda. Women were recruited at the antenatal clinic at Entebbe Hospital between April 2003 and November 2005.\nWomen were assessed for screening at their first antenatal visit, thus initial screening could take place in any trimester of pregnancy. They were eligible for screening if they were well, resident in the study area, planning to deliver their baby at the hospital, willing to participate and willing to know their HIV status. On the screening day, after giving written informed consent, eligible women were interviewed regarding sociodemographic characteristics and risk factors for helminth infection, malaria and HIV and were examined by a midwife. A blood sample was obtained for investigations including haemoglobin (Hb) estimation, examination for microfilariae (mf) and malaria parasites, syphilis and HIV serology.\nScreened women were asked to return for enrolment within 1 month with a stool sample. They were excluded from enrolment if they had a Hb level <8\u00a0g\/dl, clinically apparent severe liver disease, diarrhoea with blood in the stool, an abnormal pregnancy, a history of adverse reaction to anthelminthic drugs or had already participated in the study during an earlier pregnancy. Women were enrolled in the trial when they returned with a stool sample if they were in the second or third trimester and full eligibility was confirmed.\nAll women received routine antenatal care including haematinics and intermittent presumptive treatment for malaria using sulfadoxine\/pyrimethamine. Women were treated for syphilis and provided with nevirapine for prevention of mother-to-child HIV transmission, if indicated. Women who were excluded from the study on grounds of severe anaemia were treated with albendazole and haematinics and referred for transfusion if required.\n2.2\nHaemoglobin estimation and definition of anaemia\nHb was estimated at the antenatal clinic using a colorimetric haemoglobinometer (DHT haemoglobin meter; Developing Health Technology, Barton Mills, UK) with same-day results. The same sample was then sent to the Medical Research Council\/Uganda Virus Research Institute (MRC\/UVRI) laboratories for analysis by a Coulter analyser (Beckman Coulter AC-T 5 diff CP; Beckman Coulter, Nyon, Switzerland). Quality control for the Coulter analyser was provided through the United Kingdom National External Quality Assessment Schemes, with consistently good results. Initial evaluation suggested that the haemoglobinometer results reliably matched the Coulter analyser results (intraclass correlation coefficient (ICC)\u00a0=\u00a00.81, 95% CI 0.74\u20130.88) and the immediately available haemoglobinometer results were used to determine enrolment. Evaluation of results to March 2005 showed less reliability (ICC\u00a0=\u00a00.54, 95% CI 0.51\u20130.58) and it was noted that 37 women had been enrolled with haemoglobinometer results >8\u00a0g\/dl but Coulter analyser values below this cut-off; thereafter, Coulter analyser results were used for enrolment. Coulter analyser results have been used in this analysis.\nIn accordance with WHO criteria, anaemia was defined as Hb\u00a0<\u00a011.2\u00a0g\/dl, i.e. 0.2\u00a0g\/dl above the standard cut-off of 11\u00a0g\/dl to allow for the altitude in Entebbe (1132\u00a0m a.s.l.) (http:\/\/www.sph.emory.edu\/\u223ccdckms\/hbadj2.html) (WHO, 1999a).\n2.3\nParasitology\nExamination of stool samples was performed as described previously (Bukusuba et al., 2004) using the Kato\u2013Katz method (Katz et al., 1972) and charcoal culture for Strongyloides (Friend, 1996). Two Kato\u2013Katz slides were prepared from each sample, each examined within 30\u00a0min for hookworm or the following day for other parasites. Blood was examined for Mansonella by a modified Knott's method (Melrose et al., 2000). Intensity of infection was assessed by egg counts in stool and mf counts in blood. Intensities were categorised as follows: hookworm: light <1000 eggs per gram of stool (epg), moderate 1000\u20133999\u00a0epg, high \u22654000\u00a0epg (WHO, 1994); Schistosoma mansoni: light <100\u00a0epg, moderate 100\u2013399\u00a0epg, high \u2265400\u00a0epg (WHO, 1999b); Trichuris trichiura: light <1000\u00a0epg, moderate 1000\u20139999\u00a0epg, high \u226510\u00a0000\u00a0epg (WHO, 1999b). No standard categories are available for Mansonella intensity, therefore arbitrary categories were defined to obtain approximately equal numbers of participants in each category: light <30\u00a0mf\/ml, moderate 30\u201399\u00a0mf\/ml, high \u2265100\u00a0mf\/ml.\n2.4\nHIV serology\nHIV serology was performed using a rapid test algorithm with same-day results. Testing kits, provided by the Ministry of Health, varied with availability. Most commonly, Determine (Abbott Laboratories, Abbott Japan Co. Ltd., Tokyo, Japan) was used for screening, with positive results confirmed by Unigold (Trinity Biotech plc, Bray, Ireland). Samples with differing results were referred for analysis by non-rapid ELISA tests or were examined using a \u2018tie-breaker\u2019 rapid test, usually Statpack (Chembio Diagnostic Systems, Medford, NY, USA). A proportion of samples, including all those with differing results by rapid test, were re-examined at the MRC\/UVRI laboratories for quality control, with high agreement of the results.\n2.5\nDemographic information and potential risk factors for anaemia\nSocioeconomic data were summarised by developing two indices based on the variables that appeared to describe socioeconomic status most usefully. These were \u2018woman's socioeconomic index\u2019, comprised of education, personal income and occupation, and \u2018household socioeconomic index\u2019, comprised of building materials, number of rooms and items collectively owned. The relationship between potential confounding factors and anaemia and helminths, malaria and HIV were considered, and a diagram describing hypothesised relationships was developed (Figure 1). Age, socioeconomic status and tribe were considered to be possible confounders, with gravidity of potential importance, particularly for malaria, to which primigravidae are known to be particularly susceptible.\n2.6\nData management and statistical analysis\nData were entered using Microsoft Access (Microsoft Corp., Redmond, WA, USA) and analysed using STATA version 8 (Stata Corp., College Station, TX, USA). The initial analysis was based on a binary variable for outcome (anaemic\/not anaemic) and categorical variables for exposure, with binary variables for exposures to helminths, malaria and HIV infection (infected\/not infected). Effects of helminth infection were then investigated in more detail by examining categories of infection intensity (none, light, moderate or heavy). For these analyses, cross-tabulations were made between selected risk factors and the presence of anaemia. Logistic regression was used to estimate unadjusted and adjusted odds ratios (OR). Likelihood ratio tests were used to determine P-values. Continuous variables for anaemia (Hb level) and infection intensity (egg or parasite count) were then used to examine effects of intensity in more detail among infected participants using linear regression. Adjustment was made for the same potential confounders in logistic and linear regression models.\n3\nResults\nA total of 15\u00a0035 women registered at the antenatal clinic during the recruitment period, of whom 11\u00a0783 were assessed for inclusion in the study and 3163 were considered eligible and screened. The commonest reasons for ineligibility were residence outside the study area (6243), unwillingness to have an HIV test (1186), unwillingness to join the study (874) and enrolment during an earlier pregnancy (115). Of the 3163 screened, 2515 were enrolled; 8 of these were subsequently excluded because they had been enrolled during a previous pregnancy. Of the 648 women screened but not enrolled, the majority (596) failed to return for enrolment and only 15 brought a stool sample. Since intestinal helminth infection detected by stool analysis was a focus of interest, this analysis was confined to the 2507 women who were enrolled in the trial and for whom all or almost all relevant data were available.\n3.1\nCharacteristics of the study women\nMaternal age ranged from 14 years to 47 years (mean 23.6 years). The majority were Baganda (49.1%), the predominant tribe of the district. Most (83.8%) were married, with 13.4% single, 0.6% widows and 2.3% divorced or separated. Education varied from none (3.9%), to primary (50.5%), secondary (37.3%) and tertiary (8.4%) and most women were poor (85.1% with a personal income of less than \u00a310 per month). Primigravidae comprised 27.7% of the women studied.\n3.2\nPrevalence of infections and anaemia\nComplete data were obtained for Kato\u2013Katz assays from 2498 women, for Strongyloides assays from 2485 women, for Mansonella from 2499 women and for malaria from 2459 women. The prevalence of hookworm was 44.5%, Mansonella perstans 21.3%, S. mansoni 18.3%, Strongyloides stercoralis 12.3%, T. trichiura 9.1%, Ascaris lumbricoides 2.3%, Trichostrongylus sp. 1.0%, Hymenolepis nana 0.2% and Loa loa <0.1%. Ova of Fasciola hepatica and Dicrocoelium dendriticum were found in samples from one and five women, respectively, but infection was not confirmed in follow-up samples and eggs probably originated from liver or offal in the women's diet; detection of ova of these two species was not considered to indicate infection. The prevalence of asymptomatic Plasmodium falciparum malaria parasitaemia was 10.9% and HIV infection 11.9%.\nThe prevalence of anaemia (Hb\u00a0<\u00a011.2\u00a0g\/dl) was 39.7%. Six women were enrolled in error with Hb\u00a0<\u00a08\u00a0g\/dl by both methods (Coulter analyser results 5.6\u20137.9\u00a0g\/dl). These, as well as the 37 women enrolled prior to March 2005 with haemoglobinometer results above but Coulter analyser results below 8\u00a0g\/dl, have been retained in this analysis.\n3.3\nAssociations between characteristics of pregnant women and anaemia in pregnancy\nRelationships between characteristics of the participating women and anaemia are shown in Table 1. The prevalence of anaemia declined with age. Anaemia was associated with tribe, with Basoga most likely to be anaemic. Anaemia showed no association with women's socioeconomic index, but low household socioeconomic status was associated with anaemia. Primigravidae were more likely to be anaemic than multigravidae.\n3.4\nRelationship between infections and anaemia in pregnancy\nRelationships between infections and anaemia are shown in Table 2. None of the helminths showed an association with anaemia when considered using categorical variables for the presence or absence of infection. Hookworm showed a weak positive association, but this was reduced in the adjusted model.\nMalaria parasitaemia and HIV infection were strongly associated with anaemia; for malaria the effect was unchanged and for HIV it increased after adjusting for potential confounding factors. Malaria was also associated with HIV infection (OR 1.81, 95% CI 1.30\u20132.53; P\u00a0=\u00a00.001); the association between HIV and anaemia was reduced slightly, but not explained, when malaria was added to the model (adjusted OR (AOR), adjusted for age, tribe, socioeconomic index, gravidity and malaria: 2.27, 95% CI 1.74\u20132.97; P\u00a0<\u00a00.001). The likelihood of being anaemic was particularly high among women with both HIV infection and malaria compared with women with neither infection (AOR 6.50, 95% CI 3.27\u201312.95; P\u00a0<\u00a00.001). Comparing those with HIV only to those with neither gave an AOR of 2.30 (95% CI 1.73\u20133.05; P\u00a0<\u00a00.001) and comparing those with malaria only to those with neither gave an AOR of 3.09 (95% CI 2.28\u20134.20; P\u00a0<\u00a00.001).\nHookworm and Mansonella showed positive associations with malaria parasitaemia, and hookworm showed a negative association with HIV infection, but adjusting for these infections had minimal effect on the observed associations between helminths and anaemia (data not shown).\nAttributable fractions for anaemia were 3.1% for hookworm, 12.3% for malaria and 10.2% for HIV infection.\n3.5\nRelationship between anaemia and infection intensity\nAssociations between infections and anaemia were further explored by examining infection intensity (Table 3). The prevalence of anaemia increased with each category of hookworm infection intensity. However, this trend was greatly weakened, showing no evidence of association after adjusting for age, tribe, socioeconomic index, gravidity, malaria and HIV. On the other hand, there was a small negative association between log10 hookworm egg count and Hb in hookworm-infected women (adjusted regression coefficient \u22120.23, 95% CI \u22120.38 to \u22120.09; P\u00a0=\u00a00.002), i.e. women with the highest hookworm intensity (approximately 10\u00a0000\u00a0epg) had, on average, a Hb level 0.69\u00a0g\/dl lower than those with the lowest detectable egg counts (12\u00a0epg). Anaemia prevalence was higher in women with heavy S. mansoni infection than in those with lower intensity infection or no infection, but the number of such women was small and the effect was not statistically significant. Infections with Trichuris were light in all but six of the infected women, therefore associations between anaemia and moderate-to-heavy Trichuris infections could not be examined. There was no evidence of an association between Hb and egg count for women infected with S. mansoni or Trichuris.\nThere was a negative association between log10 malaria parasite count (parasites per 200 white blood cells) and Hb, but this was reduced in the adjusted model (crude regression coefficient \u22120.27, 95% CI \u22120.53 to \u22120.01, P\u00a0=\u00a00.039; adjusted regression coefficient \u22120.17, 95% CI \u22120.45 to 0.11, P\u00a0=\u00a00.206). Hb was not significantly associated with log10 CD4+ T-cell count among HIV-positive women (adjusted regression coefficient 0.51, 95% CI \u22120.17 to 1.19; P\u00a0=\u00a00.120).\n3.6\nAnaemia in women excluded from enrolment\nHb\u00a0<\u00a08\u00a0g\/dl was an exclusion criterion. Among the 648 women who were screened but never enrolled, Hb\u00a0<\u00a08\u00a0g\/dl was the reason given in 17 cases, but Coulter analyser Hb was <8\u00a0g\/dl for a further 37 women excluded for other reasons. The prevalence of anaemia was 281\/648 (43.4%) among those excluded compared with 996\/2507 (39.7%) among those enrolled (P\u00a0=\u00a00.093), giving an overall prevalence of 40.5% among all women screened. Since stool results were only available for 15 of the excluded women, associations with intestinal helminths could not be analysed. Of the 54 women excluded and with Coulter Hb\u00a0<\u00a08\u00a0g\/dl, two had stool results: both had hookworm infections (one light and one moderate intensity) and neither had schistosomiasis. A sensitivity analysis was performed assuming that all women excluded and with Coulter Hb\u00a0<\u00a08\u00a0g\/dl had hookworm. As expected, a slightly stronger association was obtained, but this was again reduced after adjusting for confounding factors (crude OR 1.29, 95% CI 1.10\u20131.51, P\u00a0=\u00a00.001; AOR adjusted for age, tribe, socioeconomic indices and gravidity 1.19, 95% CI 1.00\u20131.41, P\u00a0=\u00a00.047).\nAssociations with malaria and HIV were similar in the excluded group to those in the enrolled group, with crude ORs of 3.73 (95% CI 2.23\u20136.23; P\u00a0<\u00a00.001) and 1.57 (95% CI 1.06\u20132.34; P\u00a0=\u00a00.026), respectively.\n4\nDiscussion\nThis study suggests that, among pregnant women in Entebbe, Uganda, malaria and HIV are more important infectious causes of anaemia than helminths. No association was observed between mild-to-moderate anaemia and any species of helminth, and a weak association between anaemia and increasing intensity of hookworm infection was reduced after adjusting for confounding factors. Anaemia was slightly more common among women heavily infected with S. mansoni, but the number of such women was small.\nIn keeping with the exclusion of women with Hb\u00a0<\u00a08\u00a0g\/dl, excluded women were more likely to be anaemic than enrolled women, but this had minimal impact on the overall estimate of prevalence of anaemia (40.5%). Given the recognised ability of hookworm to cause anaemia (Bondevik et al., 2000; Hotez et al., 2004; Shulman et al., 1996), we conducted a sensitivity analysis to estimate the effect of hookworm if all women with Hb\u00a0<\u00a08\u00a0g\/dl and no stool result had hookworm. The result was in keeping with a possible effect of hookworm, but after adjusting for confounding factors the effect was small. Associations between anaemia and malaria and between anaemia and HIV infection were similar in enrolled and excluded women.\nOur investigations for intestinal helminths used only one stool sample from each woman, meaning that a proportion of women with low-intensity infections will have been misclassified as uninfected and that estimates of intensity will have been imprecise (Hall, 1981; Utzinger et al., 2001). Recent comparable studies have the same limitation (Ajanga et al., 2006; Bondevik et al., 2000; Dreyfuss et al., 2000; Larocque et al., 2005). In our study this may, again, be important for hookworm, where there was a small increase in anaemia with infection intensity, but may not explain the lack of association for schistosomiasis where there was no suggestion of an effect of light-to-moderate infections. Our method of investigation for Mansonella and intensity does not suffer from this limitation: repeat examinations among 1971 women showed 96% agreement for the binary variable (infected\/uninfected), with an ICC for mf\/ml of 0.87 (95% CI 0.85\u20130.88) (unpublished data).\nNot all pregnant women in Entebbe attend the district hospital antenatal clinic, but a community survey undertaken in the study area showed an increase in the proportion choosing this clinic during the recruitment period, to approximately 80%, and most of the personal and socioeconomic characteristics of women choosing, or not choosing, to attend the district hospital clinic were similar (unpublished data). Thus, our results are likely to be reasonably representative of pregnant women in this area.\nRecent studies from different settings give similar results in relation to the effects of hookworm and S. mansoni on anaemia in pregnancy. In Peru (where hookworm and Trichuris are predominant), in Tanzania (S. mansoni and hookworm), in the Democratic Republic of Congo (Ascaris and hookworm) and in Java (Trichuris and hookworm), no association was observed between anaemia and infection with any single species (Ajanga et al., 2006; Kalenga et al., 2003; Larocque et al., 2005; Nurdia et al., 2001). In Peru there was an association between anaemia and higher hookworm intensities, and in Java there was a negative association between serum ferritin and hookworm, suggesting an effect of hookworm on iron status but not anaemia; in Tanzania, in an area of higher S. mansoni prevalence and intensity, a strong association between anaemia and heavy S. mansoni infection was observed. Infection intensity thus appears to be important, with light-to-moderate hookworm or S. mansoni infections having relatively weak effects on Hb levels. However, a second important factor is the underlying nutritional status of the women. In two studies in Nepal, among populations perhaps poorer and less well nourished than ours (as indicated by items owned, anthropometry and vitamin A status (Dreyfuss et al., 2000) and with a traditionally vegetarian diet (Bondevik et al., 2000)), anaemia showed a significant association with hookworm infection (Bondevik et al., 2000; Dreyfuss et al., 2000). The effects of hookworm infection are partially mediated by iron deficiency (Bondevik et al., 2000; Olsen et al., 1998) and in the trial conducted in Sierra Leone iron-folate supplements had a greater benefit for anaemia in pregnancy than treatment with albendazole (Torlesse and Hodges, 2001).\nWe found no suggestion of an association between anaemia and any other helminth species that was common in our environment (Mansonella, Trichuris or Strongyloides). This again is in agreement with other studies (Dreyfuss et al., 2000; Larocque et al., 2005; Nurdia et al., 2001). Larocque et al. (2005) noted a stronger effect of moderate-to-heavy hookworm infection when combined with moderate-to-heavy Trichuris infection. Although there was broad overlap between the confidence intervals for these effects in their analysis, such an effect is plausible, given evidence in children that heavy Trichuris infections (>10\u00a0000\u00a0epg) can be associated with anaemia (Ramdath et al., 1995). In our study, as in that reported by Nurdia et al. (2001), no such heavy Trichuris infections were observed.\nThe strong effects of malaria and HIV contrast with the weak effect of hookworm and the lack of effects of other helminths in this study.\nOf note, women were screened for this study when they were apparently healthy, thus both malaria and HIV infection were largely asymptomatic. The effects of these infections are not mediated by iron deficiency and may override any benefit of good nutrition. The importance of malaria as a cause of anaemia in pregnancy is well established (Shulman and Dorman, 2003). HIV infection is also a recognised cause of anaemia (Belperio and Rhew, 2004), and anaemia may be one mechanism by which it causes adverse birth outcomes (Dairo et al., 2005; McIntyre, 2003).\nThere are compelling reasons for preventing and treating malaria and HIV during pregnancy (Shulman and Dorman, 2003; ter Kuile et al., 2004). Our results highlight that anaemia is among them. On the other hand, our results as well as recent literature suggest that associations between helminth infections and anaemia in pregnancy are weaker, with regional variations that may be based on nutrition and intensity of helminth infection. These findings are relevant when estimating the relative disease burden of helminths and other infections and the relative value of possible interventions in pregnancy. Globally, the majority of helminth infections are of low intensity so, in some settings, the benefit of routine deworming during pregnancy in relation to anaemia may be modest. The effects of deworming during pregnancy on other parameters, including birth outcome, birth weight and long-term effects on health in infancy and childhood, also need to be considered (Christian et al., 2004). The forthcoming results of our ongoing trial of deworming in pregnancy are expected to contribute further to this debate.\nAuthors\u2019 contributions\nAME designed the study; JN and CA carried out interviews and recruited participants; MO and HM carried out clinical assessments; NO and DK carried out laboratory assessments; LM, PW and LAM analysed and interpreted the data; LM, PW, LAM and AME drafted the manuscript. All authors reviewed and approved the final manuscript. LM and AME are guarantors of the paper.\nFunding\nWellcome Trust Career Post fellowship held by Dr Elliott, grant number 064693; PMTCT programme, Ministry of Health, Uganda.\nConflicts of interest\nNone declared.\nEthical approval\nThe Science and Ethics Committee, Uganda Virus Research Institute, the Uganda National Council for Science & Technology, and the London School of Hygiene & Tropical Medicine.","keyphrases":["anaemia","pregnancy","helminth","malaria","hiv","uganda"],"prmu":["P","P","P","P","P","P"]}
{"id":"Osteoporos_Int-3-1-1766476","title":"A multidisciplinary, multifactorial intervention program reduces postoperative falls and injuries after femoral neck fracture\n","text":"Introduction This study evaluates whether a postoperative multidisciplinary, intervention program, including systematic assessment and treatment of fall risk factors, active prevention, detection, and treatment of postoperative complications, could reduce inpatient falls and fall-related injuries after a femoral neck fracture.\nIntroduction\nNearly all hip fractures occur as a result of a fall [1] and many fall again soon after sustaining the fracture [2]. Osteoporosis with low bone mineral density (BMD) puts older people who fall at high risk of sustaining fractures [3, 4]. A first hip fracture is associated with a 2.5-fold increased risk of a subsequent fracture [5]. A population-based study among people aged 85\u00a0years or older showed that 21% of those with a hip fracture had suffered at least two hip fractures [6].\nPrevious research has identified several fall risk factors such as comorbidity, functional disability, previous falls, and use of drugs [7\u201311] but also aging [12, 13] and among the oldest old, male sex [14]. Delirium, which is very common after hip fracture surgery, especially among those with a cognitive decline [15, 16], has been found to be one of the most important risk factors for falls among older people [10].\nMulti factorial intervention strategies among community-living older people can prevent falls [17\u201320] and are the recommendations in fall prevention interventions nowadays [21]. The recommendations in fall prevention programs is that they should include gait training, advice on use of assistive devices, medication reviews, exercise programs including balance training, treatment of hypotension, environmental modification, and treatment of cardiovascular disorders. In long-term care the program recommends also to include staff education [21]. Most fall prevention studies are performed in the community, but multidisciplinary and multifactorial interventions have also been shown to be beneficial in residential care facilities [22].\nFew fall prevention studies have been carried out in hospitals; there have been a few studies with single interventions among older patients in rehabilitation units, without any significant effects [23\u201325]. Recently two studies, one using multiple interventions [26] and one using a multidisciplinary fall prevention approach [27], have demonstrated a reduction in falls. None of these fall prevention studies have focused on hip fracture patients or tried to reduce postoperative complications as a fall prevention measure.\nConsidering the lack of fall prevention studies in hospitals, especially after recent hip fracture surgery, this is an area of interest for study. The aim of this study was thus to evaluate if a postoperative multidisciplinary, multifactorial intervention program could reduce inpatient falls and fall-related injuries in patients with femoral neck fractures.\nMethods\nRecruitment and randomization\nThis study included patients with femoral neck fracture aged \u226570\u00a0years, consecutively admitted to the orthopedic department at the Ume\u00e5 University Hospital, Sweden, between May 2000 and December 2002, and the study was designed according to the CONSORT guidelines [28].\nIn Sweden different surgery methods are used depending on the displacement of the femoral neck fracture. In the present study patients with undisplaced fracture were operated on using internal fixation (IF) and patients with displaced fracture were operated on using hemiarthroplasty (HAP). If patients had severe rheumatoid arthritis, severe hip osteoarthritis, or pathological fracture they were excluded, by the surgeon on duty, because of the need for a different surgery method, such as total hip arthroplasty (THA). Patients with severe renal failure were excluded, by the anesthesiologist, because of their morbidity. Patients being bedridden before the fracture occurred were also excluded.\nIn the emergency room the patients were asked both in writing and orally if they were willing to participate in the study. The next of kin was always asked prior to the inclusion in patients with cognitive impairment. The patients or their next of kin could at any time decline participation. A total of 258 patients met the inclusion criteria; 11 patients declined to participate and 48 patients were not invited to participate because they had sustained the fracture in the hospital or the inclusion routines failed (Fig.\u00a01). These 59 patients were more likely to be men (p\u2009=\u20090.033) and living in their own house\/apartment (p\u2009=\u20090.009), but there was no difference in age (p=0.354) compared to the participating patients. The remaining 199 patients (Table\u00a01) consented to participate. All patients received the same preoperative treatment. \nPatients were randomized, to postoperative care in a geriatric ward with a special intervention program or to conventional care in an orthopedic ward, in opaque sealed envelopes. The lots in the envelopes were sequentially numbered. All participants received this envelope while in the emergency room but the envelope was not opened until immediately before surgery to ensure that all patients received similar preoperative treatment. Persons not involved in the study performed these procedures. \nThe randomization was stratified according to the operation methods used in the study. Depending on the degree of dislocation, the patients were treated with IF using two hook-pins (Swemac Ortopedica, Link\u00f6ping, Sweden) (n=38 intervention vs n\u2009=\u200931 control) or with bipolar hemiarthroplasty (Link, Hamburg, Germany) (n\u2009=\u200957 vs 54). Basocervical fractures (n\u2009=\u20097 vs 10) were operated on using a dynamic hip screw (DHS, Stratec Medical, Oberdorf, Switzerland) and one had a resection of the femoral head due to a deterioration in medical status and one died before surgery (both were in the control group).\nFig.\u00a01Flow chart for the randomized trialTable\u00a01Basic characteristics and assessments during hospitalization among participants in the intervention and control groups. SD standard deviation, ADL activity of daily living\u00a0Intervention (n\u2009=\u2009102)Control (n\u2009=\u200997)p valueSociodemographic\u00a0Age, mean\u00b1SD82.3\u2009\u00b1\u20096.682.0\u2009\u00b1\u20095.90.724\u00a0Females74740.546\u00a0Independent living before the fracture66600.677Health and medical problems\u00a0Stroke (n\u2009=\u2009102\/93)29200.265\u00a0Dementia28360.145\u00a0Previous hip fracture (n\u2009=\u2009102\/96)a16140.829\u00a0Depression (n\u2009=\u2009102\/95)33450.031\u00a0Diabetes (n\u2009=\u2009102\/95)23170.417\u00a0Cardiovascular disease (n\u2009=\u2009101\/93)57530.938Medications on admission\u00a0Number of drugs, mean\u00b1SD5.8\u2009\u00b1\u20093.85.9\u2009\u00b1\u20093.60.867\u00a0Antidepressants 29450.009Sensory impairments\u00a0Impaired hearing (n\u2009=\u200994\/82)42340.667\u00a0Impaired vision (n\u2009=\u200991\/74)37270.584Functional performance before fracture\u00a0Use of roller walker (n\u2009=\u2009101\/93)56520.948\u00a0Use of wheelchair (n\u2009=\u2009101\/93)23160.334\u00a0Previous falls, last month (n\u2009=\u200999\/90)b24250.580\u00a0Walking independently, at least indoors (n\u2009=\u2009101\/94)85850.191\u00a0Staircase of ADL, median (Q1,Q3) (n\u2009=\u200992\/88)5 (1\u20137.75)5 (0.25\u20137)0.859Assessments during hospitalization\u00a0Mini Mental State Examination, mean\u00b1SD (n\u2009=\u200993\/90)17.4\u2009\u00b1\u20098.215.7\u2009\u00b1\u20099.10.191\u00a0Organic Brain Syndrome Scale, mean\u00b1SD (n\u2009=\u200994\/90)10.1\u2009\u00b1\u200910.812.5\u2009\u00b1\u200911.40.148\u00a0Geriatric Depression Scale, mean\u00b1SD (n\u2009=\u200981\/68)5.2\u2009\u00b1\u20093.64.5\u2009\u00b1\u20093.50.271aExcept for the present hip fracturebExcept for the fall that caused the hip fracture\nIntervention\nThe intervention ward was a geriatric unit specializing in geriatric orthopedic patients. The staff worked in teams to apply comprehensive geriatric assessments, management, and rehabilitation [29, 30]. Active prevention, detection, and treatment of postoperative complications such as falls, delirium, pain, and decubitus ulcers was systematically implemented daily during the hospitalization (Table\u00a02). The staffing at the intervention ward were 1.07 nurses\/aides per bed. \nTable\u00a02Main content of the postoperative program and differences between the two groups\u00a0Intervention groupControl groupWard layoutSingle and double roomsSingle, double, and four-bed rooms24-bed ward, extra beds when needed27-bed ward, extra beds when neededThe geriatric control ward was similar to the intervention wardStaffing1.07 nurses\/aides per bed1.01 nurses per bedTwo full-time physiotherapistsTwo full-time physiotherapistsTwo full-time occupational therapists0.5 occupational therapist0.2 dieticianNo dieticianThe geriatric control ward had staffing similar to the intervention wardStaff educationA 4-day course in caring, rehabilitation, teamwork, and medical knowledge including sessions about how to prevent, detect, and treat various postoperative complications such as postoperative delirium and fallsNo specific education before or during the projectTeamworkTeam included registered nurses (RN), licensed practical nurses (LPN), physiotherapists (PT), occupational therapists (OT), dietician, and geriatriciansNo corresponding teamwork at the orthopedic unitClose cooperation between orthopedic surgeons and geriatricians in the medical care of the patientsThe geriatric ward, where some of the control group patients were cared for, used teamwork similar to that in the intervention wardIndividual care planningAll team members assessed each patient as soon as possible, usually within 24\u00a0h, to be able to start the individual care planningIndividual care planning was used in the orthopedic unit but not routinely as in the intervention wardTeam planning of the patients\u2019 individual rehabilitation process and goals twice a weekAt the geriatric rehabilitation unit there was weekly individual care planningPrevention and treatment of complicationsInvestigation as far as possible regarding how and why they sustained the hip fracture, through analyzing external and internal fall risk factorsNo routine analysis of why the patients had fractured their hipsAn action to prevent new falls and fractures was implemented including global ratings of the patients\u2019 fall risk every week during team meetingsNo attempt was made to systematically prevent further fallsCalcium and vitamin D and other pharmacological treatments for osteoporosis were used when indicatedNo routine prescription of calcium and vitamin DActive prevention, detection, and treatment of postoperative complications such as delirium, pain, and decubitus ulcers was systematicAssessments for postoperative complications were made with check-ups for, i.e., saturation, hemoglobin, nutrition, bladder and bowel function, home situation etc., but these check-ups were not carried out systematically as in the intervention groupOxygen-enriched air during the 1st postoperative day and longer if necessary until the measured oxygen saturation was stableUrinary tract infections and other infections were screened for and treatedIf a urinary catheter was used it should be discontinued within 24\u00a0h postoperativelyRegular screening for urinary retention, and prevention and treatment of constipationBlood transfusion was prescribed if B-hemoglobin \u2009<100\u00a0g\/l and \u2009<110 for those at risk of delirium or those already deliriousIf the patient slept badly, the reason was investigated and the aim was then to treat the causeNutritionFood and liquid registration was systematically performed and protein-enriched meals were served to all patients during the first 4 postoperative days and longer if necessaryA dietician was not available at the orthopedic unitNutritional and protein drinks were served every dayNo routine nutrition registration or protein-enriched meals were available for the patientsRehabilitationMobilization within the first 24\u00a0h after surgeryMobilization usually within the first 24\u00a0hThe training included both specific exercise and other rehabilitation procedures delivered by a PT and OT, as well as basic daily ADL performance training, by caring staff. The patients should always do as much as they could by themselves before they were helpedThe PT on the ward mobilized the patients together with the caring staff. The PT aimed to meet the lucid patients every day. Functional retraining in ADL situations was not always given. The OT at the orthopedic unit only met the patients for consultationThe rehabilitation was based on functional retraining with special focus on fall risk factorsThe geriatric control ward had both specific exercise and other rehabilitation procedures delivered by a PT and OT, similar to the intervention ward but did not systematically focus on fall risk factorsHome visit by an OT and\/or a PTNo home visits were made by staff from the orthopedic unit\nThe control ward was a specialist orthopedic unit following the conventional postoperative routines. A geriatric unit, specializing in general geriatric patients, was used for those who needed longer rehabilitation (n\u2009=\u200940). The staffing at the orthopedic unit was 1.01 nurses\/aides per bed and 1.07 for the geriatric control ward. The main content of both the intervention program and the conventional care is described in Table\u00a02.\nThe staffs on the intervention and control wards were not aware of the nature of the present study.\nData collection\nTwo registered nurses were employed and performed the assessments during hospitalization.\nMedical, social, and functional data were collected from the patients, relatives, staff, and medical records on admission. Complications during hospitalization, including falls, length of stay, morbidity, and mortality, were systematically registered in the medical and nursing records. Nurses are obliged by law to document any falls in the records [31]. A fall was defined as an incident when the patient unintentionally came to rest on the floor and included syncopal falls. Numbers of falls and time lapse to first fall after admission were calculated. The Abbreviated Injury Scale (AIS) [32] was used to classify the injuries resulting from a fall. The maximum injury (MAIS) connected with each incident was recorded.\nA few days after surgery, patients were assessed and interviewed regarding their cognitive status using the Mini Mental State Examination (MMSE) [33]. The modified Organic Brain Syndrome Scale (OBS Scale) [34] was used to assess cognitive, perceptual, emotional, and personality characteristics as well as fluctuations in clinical states. Mental state changes were also documented from medical records. Depression during hospitalization was diagnosed due to current treatment with antidepressants and depression screened using the Geriatric Depression Scale (GDS-15) [35] in combination with depressive symptoms observed and registered by the OBS Scale. The patients\u2019 vision and hearing were assessed by their ability to read 3-mm block letters with or without glasses, and their ability to hear a normal speaking voice from a distance of 1\u00a0m. Activities of daily living (ADL) prior to the fracture were measured retrospectively using the Staircase of ADL [36].\nA geriatrician, unaware of study group allocation, analyzed all assessments and documentation, after the study was finished, for completion of the final diagnoses according to the same criteria for all patients.\nThe Ethics Committee of the Faculty of Medicine at Ume\u00e5 University approved the study (\u00a7 00-137).\nStatistical analysis\nThe sample size was calculated to detect a 50% reduction of number of fallers between the intervention and control groups at a significance level of 0.050, based on our previous multifactorial fall intervention study in institutional care [22]. Student\u2019s t-test, Pearson\u2019s \u03c72 test, and the Mann-Whitney U test were performed to analyze group differences regarding basic characteristics and postoperative complications.\nWe analyzed outcomes on an intention to treat basis. The incidence of falls between intervention and control groups was compared in three ways. First, an unadjusted comparison using Pearson\u2019s \u03c72 and Fisher\u2019s exact test regarding number of patients who fell and injuries. Second, the fall incidence rate was compared between intervention and control groups by calculating the fall incidence rate ratio (IRR) using a negative binomial regression, with adjustment for observation time and for overdispersion. Negative binomial regression (Nbreg) is a generalization of the Poisson regression model and is recommended for evaluating the efficacy of fall prevention programs [37]. Third, a Cox regression was used to compare the time lapse to first fall between groups (hazard rate ratio, HRR). The difference in fall risk between groups was further illustrated by a Kaplan-Meier graph.\nBasic characteristics that differed between the intervention and the control groups, corresponding to a p value \u2009<0.150 (depression, antidepressants, and dementia, Table\u00a01), were considered as covariates in the Poisson (Nbreg) and the Cox regression models. However, the inclusion of these variables had only marginal effects on the log-likelihood values of the models as well as on the IRR and HRR values and standard errors for the group allocation variable (intervention or control). In addition, none of the variables showed significant effects on the dependent variable and are therefore not included in the Poisson (Nbreg) and Cox regression analyses.\nPearson\u2019s \u03c72 test and Fisher\u2019s exact test were also used to analyze the associations between falls and days with delirium between the groups.\nAll calculations were carried out using SPSS v 11.0 and STATA 9 statistical software for Macintosh. A p value \u2009<0.050 was considered statistically significant.\nResults\nDuring hospitalization 12 patients in the intervention group sustained 18 falls (range: 1\u20133) and in the control group 26 patients sustained 60 falls (30 falls in the orthopedic unit and 30 in the geriatric control unit) (range: 1\u201311). Among patients with dementia 1 patient sustained a single fall in the intervention group and 11 patients sustained 34 falls in the control group (Table\u00a03). \nTable\u00a03Falls during hospitalization. CI confidence interval, IRR incidence rate ratio\u00a0Intervention (n\u2009=\u2009102)Control (n\u2009=\u200997)p valueNumber of falls1860Postoperative in-hospital days2,8603,685Crude fall incidence rate (number of falls\/1,000\u00a0days)6.2916.28IRR (95% CI)0.38 (0.20\u20130.76)a1.00 (Ref.)0.006Number of fallers12260.007Number of fallers with injuries due to falls3150.002Number of fallers with fractures due to falls040.055Number of falls among people with dementia134IRR (95% CI) among people with dementia0.07 (0.01\u20130.57)a1.00 (Ref.)0.013Number of fallers among people with dementia (n\u2009=\u200928\/36)1110.006aNegative binomial regression analyses adjusted for overdispersion and controlled for dementia, depression, and use of antidepressants\nThe crude postoperative fall incidence rate was 6.29\/1,000\u00a0days in the intervention group vs 16.28\/1,000\u00a0days in the control group. Using a negative binomial regression, the fall incidence was significantly lower in the intervention group, IRR 0.38 (95% CI: 0.20\u20130.76, p\u2009=\u20090.006), and among patients with dementia, IRR 0.07 (95% CI: 0.01\u20130.57, p\u2009=\u20090.013) (Table\u00a03). In Fig.\u00a02, a Kaplan-Meier survival analysis of time lapse to first fall illustrates the difference between the two groups with a significantly reduced fall rate in the intervention group (log rank p value 0.008).\nFig.\u00a02Kaplan-Meir survival graph\nThe difference in fall risk, expressed as time lapse to first fall, was compared between intervention and control groups in a Cox regression (HRR). Including all patients in the calculation, the fall risk was significantly lower in the intervention group, HRR 0.41 (95% CI: 0.20\u20130.82, p\u2009=\u20090.012).\nThere were in total 3 minor or moderate injuries (MAIS 1-2) in the intervention group compared to 15 in the control group according to the AIS. The serious injuries (MAIS 3) were new fractures of which four, two hip fractures, one rib fracture with pneumothorax, and one with multiple skull fractures, occurred in the control group and none in the intervention group (Fisher\u2019s exact test: p\u2009=\u20090.055).\nThree of the patients who fell in the intervention group (25%) and 12 in the control group (46%) fell during a day when they were delirious (p\u2009=\u20090.294). Analyzing the number of falls revealed that 4 of 18 (22%) falls in the intervention group and 27 of 60 (45%) in the control group occurred on a day when the patient was delirious, p\u2009=\u20090.083.\nApart from the falls there were fewer other postoperative complications in the intervention group, such as fewer patients with postoperative delirium (p=0.003) and fewer delirious days (p\u2009\u2264\u20090.001), urinary tract infections (p\u2009=\u20090.005), sleeping disturbances (p\u2009=\u20090.009), nutritional problems (p\u2009=\u20090.038), and decubitus ulcers (p\u2009=\u20090.010). The postoperative in-hospital stay was shorter in the intervention group, 28.0\u2009\u00b1\u200917.9\u00a0days vs 38.0\u2009\u00b1\u200940.6\u00a0days, p\u2009=\u20090.028. Among those ten with the longest postoperative in-hospital stays in the control group there were eight patients with any fall and two had had new fractures.\nDiscussion\nThe present study shows that the number of falls and time lapse to first fall can be reduced during in-hospital rehabilitation after a femoral neck fracture. A multidisciplinary, multifactorial geriatric care program with systematic assessment and treatment of fall risk factors as well as active prevention, detection, and treatment of other postoperative complications resulted in fewer patients who fell, a lower total number of falls, and fewer injuries.\nTo our knowledge this is the first fall intervention study in this group of patients, despite the fact that this is a group of patients with a high fall risk. In general there are few fall prevention studies in hospital settings. Two [26, 27] with positive outcomes in other patient groups and on subacute wards have recently been published. The first one [26] reduced falls at three subacute rehabilitation wards, but the differences were most obvious after 45\u00a0days of observation. Thus the results were not comparable with those from the present study, which included both the acute and rehabilitation hospital stay. The other study [27] resulted in fewer fallers, falls, and injuries on a geriatric ward but the differences disappeared when the results were adjusted for observation time. Those studies used a multidisciplinary approach in their fall intervention similar to that used in the present study, but in the present study we have, in addition, focused on inpatient complications associated with falls such as delirium and urinary tract infections. One of those studies [27] tried to manage the delirious patients using bedrails, alarms, and changing the furniture arrangements for the patients, but no mention was made of any prevention and treatment of the underlying causes of delirium. The use of physical restraints was not included in the intervention program in the present study. The studies above used fall risk assessment tools to recognize those with a high fall risk. In the present study, we used a rehabilitation and care program including assessment of risk factors for falls and global ratings for each patient during team meetings. A critique of fall risk assessment tools is that few have been tested for validity and reliability testing in a new independent sample. When using fall prediction tools in different clinical settings the specificity decreases [38].\nA limitation in the present study is that some falls could have been missed, but we presume that there were very few. For one thing the nurses are obliged to document falls in the records. Also hip fracture surgery patients can hardly get up by themselves after a fall so soon after the surgery and are, therefore, bound to be noticed; but if there were any missing falls there would probably be no difference between the groups. Another limitation is that the fall registration could not be blinded regarding group allocation, but the staffs on each ward were not aware of the comparison with another ward regarding falls and injuries. The study sample is also quite small, but the sample size is calculated according to the results from a previous study [22]. The method of concealment could have been improved, but one strength was that none from the research team performed this procedure and the envelopes were not opened until the intervention was to begin. Other strengths were the intention to treat analyses, the few patients who refused to participate, and that there were no crossover effects due to staff changing wards during the study period.\nOne may speculate that the successful reduction in number of falls in the present study could be a result of the active prevention, detection, and treatment of postoperative complications after surgery. During the period of hospitalization there were differences between the groups regarding some complications associated with falls among older people in residential care facilities and in hospitals, such as delirium and urinary tract infections. The reduction of postoperative delirium can probably explain much of the difference between the groups regarding the numbers of falls and the number of patients who fell. There are studies that have found that delirium is an important risk factor for falls [10]. Demented patients especially are at high risk of developing delirium when they are treated for femoral neck fractures [15, 16] and these patients seemed to have benefited most in this study from the intervention program regarding prevention of postoperative falls. Our findings support an earlier non-randomized study that fewer injurious falls occur when the incidence and duration of delirium was reduced [39].\nThe investigation into why the patients had fractured their hip and why they fell may also have influenced the result, as well as the investigation and rehabilitation concerning external fall risk factors such as the use of walking aids, safe transfers, balance, and mobility. It seems that teamwork and individual care planning alone do not have the same effect on falls, as half the falls in the control group occurred in the geriatric control ward, a ward specializing in geriatric patients where teamwork, as well as individual care planning, is applied.\nIn the community and residential care facilities, interdisciplinary and multifactorial fall prevention studies have shown positive effects on the reduction in the number of falls and injuries [19, 22]. Among those with cognitive decline or dementia there is no evidence that such strategies prevent falls [40, 41], but the present study allowed the conclusion that at least during the in-hospital stay, this group of patients could benefit from such strategies. The reduced number of falls and injuries also probably contributed to the shorter hospitalization seen in the intervention group. The program seems easy applicable both in the acute postoperative care as well in the post-acute rehabilitation settings and except for the staff education there were no increased costs.\nConclusion\nA team applying comprehensive geriatric assessment and rehabilitation, including prevention, detection, and treatment of fall risk factors, can successfully prevent inpatient falls and injuries, even in patients with dementia.","keyphrases":["intervention","hip fracture","in-hospital","accidental falls","elderly"],"prmu":["P","P","P","M","U"]}
{"id":"Anal_Bioanal_Chem-3-1-1592466","title":"Capillary-assembled microchip as an on-line deproteinization device for capillary electrophoresis\n","text":"A capillary-assembled microchip (CAs-CHIP), prepared by simply embedding square capillaries in a lattice polydimethylsiloxane (PDMS) channel plate with the same channel dimensions as the outer dimensions of the square capillaries, has been used as a diffusion-based pretreatment attachment in capillary electrophoresis (CE). Because the CAs-CHIPs employ square-section channels, diffusion-based separation of small molecules from sample solutions containing proteins is possible by using the multilayer flow formed in the square section channel. When a solution containing high-molecular-weight and low-molecular-weight species makes contact with a buffer solution, the low-molecular-weight species, which have larger diffusion coefficients than the high-molecular-weight species, can be collected in a buffer-solution phase. The collected solution containing the low-molecular-weight species is introduced into the separation capillary to be analyzed by CE. This type of system can be used for CE analysis in which pretreatment is required to remove proteins. In this work a fluorescently labeled protein and rhodamine-based molecules were chosen as model species and a feasibility study was performed.\nIntroduction\nOn-line combination of sample pretreatment and electrophoretic separations, for example capillary electrophoresis (CE) and microchip-based capillary electrophoresis (MCE), has been focused on by many researchers, because of the possibilities of integrating complicated pretreatment procedures. During the past decade a variety of approaches have been used to combine pretreatment with CE separations. The \u201csingle capillary approach\u201d, involving position-selective immobilization of functional molecules or polymers inside a capillary, has been used to demonstrate preconcentration or enzymatic reaction before CE separation [1, 2], and in-capillary preconcentration during the electrophoretic process [3\u20138] has provided a simple system enabling direct application of the capillary in commercial equipment. The \u201cmicrochip-based approach\u201d, recently reviewed by many authors [9\u201311], has enabled integration of a variety of complicated pretreatment processes with the separation channel on a single microchip. These approaches have different advantageous features for each purpose. From the standpoint of total system design, however, a \u201ccombination approach\u201d involving the use of a flow-system or a functionalized capillary together with a separation capillary provides a more flexible system designed for particular analyte. Ye et al. reported connection of a trypsin-immobilized capillary with a separation capillary, by means of a home-built chip prepared by lamination of Lexan and Parafilm, and demonstrated on-line digestion and CE separation [12]. Kuban et al. and Fang et al. reported \u201cflow-injection capillary electrophoresis (FI-CE)\u201d comprising a flow-injection system and a separation capillary [13, 14]. This system enabled various pretreatment processes to be combined with CE separation and has since been expanded to the use of MCE to achieve rapid separations. These systems have been reviewed by Chen et al. [15]. Vizioli et al. prepared a monolithic polymer capillary, for selection of histidine-containing peptides, connected it to a CE separation capillary [16]. Fan et al. recently reported combination of an FI-CE system with in-capillary preconcentration using a dynamic pH junction [17].\nIn all the examples mentioned above, combination of a flow-reaction system or chemically functionalized conduits with a separation capillary played an important role, and would provide an attractive integrated device enabling flexible design for combining pretreatment and CE separation. In contrast, we have recently reported a new concept for fabricating a chemically functionalized microchip, called a \u201ccapillary-assembled microchip (CAs-CHIP)\u201d [18]. The microchip is fabricated simply by embedding chemically functionalized square capillaries in a lattice PDMS channel chip which has the same channel dimensions as the outer dimensions of the square capillaries. By using this technique, we have constructed a multi-ion sensing system based on combination of several different ion-sensing capillaries and a valving and sensing system based on thermo-responsive polymer-immobilized capillaries and an enzyme-immobilized capillary [19, 20]. Because the CAs-CHIP enabled easy connection of different functional capillaries, we focused on this device as an on-line sample-pretreatment attachment for CE separation capillaries in a manner similar to the previously reported FI-CE system. In this instance the CAs-CHIP, prepared from a PDMS chip, served both as the device for pretreatment of the sample solution, by use of a variety of chemical processes, and the injection device. Interfacing the flow system and the separation capillary using a PDMS chip has already been reported by Bergstrom et al. [21, 22]. Because the CAs-CHIP employs a square-shaped channel structure, however, the characteristic multilayer flow formed in the square section channel can also be used as a useful pretreatment process which can be integrated. The CAs-CHIP is, therefore, expected to enable the integration of universal pretreatment processes by using different kinds of chemically-functionalized capillary and multilayer flow-based chemical processes; this is usually difficult to achieve using other reported techniques.\nIn this paper, we report preliminary results concerning the preparation of a CAs-CHIP as a deproteinization attachment for CE separation. Deproteinization was achieved by use of the multilayer flow obtained in the PDMS microchannel, and the small molecules separated from the mixed protein sample were injected into the separation capillary connected directly to the CAs-CHIP, to be analyzed by CE. In this work a fluorescently labeled protein and rhodamine-based molecules were chosen as model species, and feasibility study was performed.\nExperimental\nSquare capillaries and reagents\nSquare capillaries of 300\u00a0\u03bcm outer width (flat-to-flat) and 50\u00a0\u03bcm inner width were purchased from Polymicro (Phoenix, AZ, USA). Before use of these capillaries the polyimide coating was removed by heating. Sylgard 184 silicone elastomer was purchased from Dow Corning (Midland, MI, USA). Reagents of the highest grade commercially available were used to prepare the aqueous solutions. Rhodamine B (RB), sulforhodamine (SR), and bovine serum albumin fluorescein conjugate (F-BSA) were purchased from Sigma (St Louis, MO, USA). All reagents were used without further purification. Distilled and deionized water had resistivity greater than 1.7 \u00d7 107 \u03a9 cm\u22121 at 25\u00b0C.\nFabrication of CAs-CHIP-CE system by embedding square capillaries on a PDMS plate and bonding of a PDMS cover\nFigure\u00a01 shows the design of a CAs-CHIP-CE system. The PDMS plate for pretreatment was connected to the inlet of the separation capillary (left side of Fig.\u00a01) and another PDMS plate was used to fix the detection position (right side of Fig.\u00a01). Square capillaries were cut into appropriate lengths and embedded in the lattice microchannel network fabricated on the PDMS plate. The general procedure for fabrication of a lattice microchannel on a PDMS plate has been reported elsewhere [18]. Briefly, a glass mold with a lattice structure was prepared by cutting a 300-\u03bcm depth with 1\u00a0mm pitch, using a dicing saw with an edge 300\u00a0\u03bcm wide. The conventional PDMS molding process using the glass mold was then performed to prepare a PDMS mold. The second molding process using this PDMS mold gave the lattice microchannel network on the second PDMS plate. Plugged capillaries were prepared by introduction of PDMS prepolymer into the square capillaries (inner width 50\u00a0\u03bcm) and cured at 70\u00b0C for more than 5\u00a0h. These plugged capillaries were also cut and used to prepare the designed channel network. After embedding all the capillaries, a PDMS cover was bonded on top. For this, a spin-coated PDMS prepolymer on an acrylic plate (ca. 2\u00a0mm thick) was used as the cover plate, to fill the voids formed between the cover plate and the capillary-embedded PDMS plate [18]. PDMS prepolymer was spin-coated on the acrylic plate at 7000\u00a0rpm, then attached to the capillary-embedded PDMS plate before curing. Bonding was carried out by curing at 60\u00b0C for 12\u00a0h. Figure\u00a02 shows the final CAs-CHIP-CE system with a 12-cm separation capillary. Depending on the resolution required, 12-cm (effective length 10\u00a0cm) and 48-cm (effective length 33\u00a0cm) separation capillaries were used. To apply the high voltage, two plastic vials, as solution reservoirs, were connected at the outlets of the PDMS channel and the separation capillary.\nFig.\u00a01General concept for fabricating a diffusion-based pretreatment\u2013CE separation system using a capillary-assembled microchip (CAs-CHIP). The plugged capillaries indicated as gray parts are actually square capillaries with 50\u00a0\u03bcm square-shaped conduits blocked with PDMS. In this figure, for simplicity, these conduits are not shownFig.\u00a02Photograph of a fabricated CAs-CHIP-CE device\nOperating procedures\nChannels (or capillaries) on the CAs-CHIP-CE system were washed sequentially with methanol, 0.1\u00a0mol\u00a0L\u22121 NaOH, 0.1\u00a0mol\u00a0L\u22121 HCl, and water, then rinsed and preconditioned with 50\u00a0mmol\u00a0L\u22121 phosphate-buffer solution (PBS) at pH 8.1. Sample solution (RB, SR: 0.5\u00a0mmol\u00a0L\u22121, F-BSA: 4\u00a0mg\u00a0mL\u22121) and two buffer solutions for diffusion-based separation and sample injection were introduced by use of syringe pumps with appropriate flow rates (see figure captions). Sample injection was achieved by on\u2013off switching of the syringe pump delivering buffer 2, shown in Fig.\u00a01. Immediately after injection, high voltage was applied manually by use of a standard high-voltage power supply (Matsusada Precision, Shiga, Japan).\nCapturing fluorescence images and laser-induced fluorescence measurement\nFluorescence images of the microchannel were obtained by using an optical\/fluorescence inverted microscope (Eclipse TS100-F, Nikon, Tokyo, Japan). Photographs were captured using a 3CCD color camera (HV-D28S, Hitachi Kokusai Electric, Tokyo, Japan) installed at the front port of the microscope. Fluorescent images were collected using a mercury lamp as a light source and a filter block (G-2A and FITC, Nikon, Tokyo, Japan).\nCE with laser-induced fluorescence (LIF) detection was performed on a home-built system based on an inverted fluorescence microscope (IX70, Olympus, Tokyo, Japan). Light at 488\u00a0nm from an argon ion laser (Newport Spectra Physics Laser Division, Mountain View, CA, USA) was introduced into the microscope. The laser beam was filtered through a 460\u2013490\u00a0nm band-pass filter, reflected by a 510\u00a0nm dichroic mirror, then focused on the detection point by means of a 20\u00d7 objective lens. Fluorescence was collected by use of the same objective lens, filtered through a 515\u00a0nm high-pass filter, and finally detected by use of a CCD camera (Model PMA-11, Hamamatsu Photonics, Shizuoka, Japan). To obtain electropherograms fluorescence at 600\u00a0nm was used throughout.\nResults and discussion\nOptimization of diffusion-based deproteinization\nDiffusion-based separation of chemical species by use of multilayer flow was first reported by Brody et al. [23]. They separated small molecules from a particle-containing sample, or proteins from a biological cell-containing sample [23, 24]. Here we used this technique to separate small molecules from a mixed protein sample. When the sample solution containing small molecules and proteins made contact with the buffer-solution flow, the small molecules, which have larger diffusion coefficients than the proteins, diffused into the buffer solution. In this study, RB and SR were used as models for small molecules and F-BSA as that for a large molecule.\nFigure\u00a03 shows the fluorescence images at the confluence point of two flows and the injection point. Fluorescence images for F-BSA and rhodamine molecules were captured using different optical filters. In this case, buffer 2 was not flowing. As can be seen, when the total flow rate of sample and buffer was very high, neither F-BSA nor rhodamine molecules could reach the separation capillary (Fig.\u00a03a). In contrast, selection of an appropriate total flow rate resulted in selective diffusion of small rhodamine molecules and negligibly small protein diffusion (Fig.\u00a03b). Figure\u00a03b also shows the fluorescence intensity profiles obtained by analysis of the fluorescence images for the rhodamine derivatives. According to this, the fluorescence intensity of the rhodamine-based molecules at the injection point was approximately 40% of the initial fluorescence intensity at the confluence point (see fluorescence intensity at position y at the injection point shown in the fluorescence intensity profile). Because complete diffusion gives 50% signal intensity for rhodamine-based molecules, these results suggest that diffusion of the rhodamine-based molecules was almost complete. In our experiments, surface adsorption of the solutes by the channel surface occurred, because of the hydrophobicity of the PDMS surface. Because these samples were continuously flowing for deproteinization, however, surface adsorption did not seriously affect the deproteinization procedure when sample injection was performed after steady flow was achieved.\nFig.\u00a03Fluorescence images obtained at the confluence and injection points at different flow rates. (a) Total flow rate 10\u00a0\u03bcL\u00a0min\u22121 (sample 5\u00a0\u03bcL\u00a0min\u22121, buffer 1 5\u00a0\u03bcL\u00a0min\u22121). (b) Total flow rate 1\u00a0\u03bcL\u00a0min\u22121 (sample 0.5\u00a0\u03bcL\u00a0min\u22121, buffer 1 0.5\u00a0\u03bcL\u00a0min\u22121). In (b), fluorescence intensity profiles obtained by analysis of the fluorescence images obtained for rhodamine derivatives (SR, RB) are also shown. F-BSA and rhodamine-based molecules were detected by use of different optical filters\nIn this study the molecular weights of RB and SR are 443 and 581, respectively, and that for F-BSA is approximately 73,000 [25]. According to the literature, the diffusion coefficient for a small molecule with a molecular weight of 342 (sucrose) is 5.2\u00d710\u22126 (cm2\u00a0s\u22121) and that for a large molecule with a molecular weight of 69,000 (HSA) is 6.1\u00d710\u22127 (cm2\u00a0s\u22121) [26, 27]. Because these molecular weights are very close to those of the molecules used in this study, an approximately tenfold difference between the diffusion coefficients of the small and large molecules enabled successful separation of small molecules from mixed protein sample solution. Although the difference between diffusion coefficients is a primary principle of diffusion-based separation, it should be noted that the optimum conditions may change, depending on the concentration ratio of the protein and small molecules. Therefore, careful optimization experiments may be required when the concentrations of the small molecules are very low.\nSample injection and electrophoretic separation\nSample injection was performed manually by on\u2013off switching of the syringe pump delivering buffer 2. In the diffusion-based separation and the CE separation mode, three-layer flow composed of sample solution, collection buffer (buffer 1), and buffer 2 occurs in the 300\u00a0\u03bcm square-PDMS channel along the buffer 2 flow channel, as illustrated in Fig.\u00a01. Because buffer 2 prevents introduction of sample solution into the separation capillary, on-off switching of the syringe pump (buffer 2) enables sample injection similar to the \u201cgated injection\u201d reported by Jacobson et al. [28]. Usually, stopping the syringe pump does not immediately stop flow of buffer 2. In this work, therefore, it took several seconds to start injection. This causes diffusion of molecules at the front and rear ends of the sample plug. When sequential sample injections with injection times of 15\u00a0s were performed, however, fairly good reproducibility of 6.5% RSD (peak height; n=5) was obtained. Although this method of injection needs to be improved, good reproducibility led us to use this simple method for further investigation.\nFigure\u00a04 shows detection signals obtained at 10\u00a0cm downstream of the injection point with and without voltage application. When the sample plug is introduced without voltage application, ca. 160\u00a0s, corresponding to a linear flow rate of 0.63\u00a0mm\u00a0s\u22121, was required for detection. The approximate linear flow rate of the pressure-driven flow inside the separation capillary can be estimated from the total flow rate of sample and buffer flow (in total 2\u00a0\u03bcL\u00a0min\u22121), and the ratio of the cross sectional areas of the PDMS channel (300\u00a0\u03bcm square) and the separation capillary (50\u00a0\u03bcm square). Under our experimental conditions the calculated linear flow rate was ca. 0.36\u00a0mm\u00a0s\u22121. Although the order of the calculated value was the same as the experimental value, the difference may be because of the slight difference between the back pressures of the two waste reservoirs. When the high voltage of 5\u00a0kV (417\u00a0V\u00a0cm\u22121) was applied just after injection, the time required for detection was reduced to ca. 70\u00a0s and slight separation was observed. Because the net charge of SR was \u22121 and that of RB was 0, migration time of SR was slightly longer than that of RB. These results indicated that induction of electroosmotic flow and separation by electrophoresis, by voltage application, were confirmed.\nFig.\u00a04Detection of rhodamine-based molecules (RB and SR) downstream of the separation capillary with and without application of high voltage. Flow rates: sample, 1\u00a0\u03bcL\u00a0min\u22121; buffer 2, 1\u00a0\u03bcL\u00a0min\u22121. Injection time: 20\u00a0s\nDeproteinization and electrophoretic separation\nOn the basis of these experiments, deproteinization and electrophoretic separation were achieved. Figure\u00a05 shows the electropherograms obtained at the downstream of the separation capillary with and without diffusion-based deproteinization. When the sample solution containing RB, SR, and F-BSA was introduced into the separation capillary without deproteinization, a broad signal arising from F-BSA was observed after the appearance of the RB and SR peaks (Fig.\u00a05a). This broad signal may be because of the protein adsorption frequently observed in CE, or the multiple labeling of the F-BSA used in this work [29]. Commercially available F-BSA contains 7\u201312 labeling molecules (average) per BSA molecule. Therefore, the electrophoretic mobilities of BSA species with different numbers of labels may cause the broad signals shown in Fig.\u00a05a. In contrast, the electropherogram obtained after deproteinization by use of the multilayer flow had a completely flat baseline after the appearance of two peaks of the rhodamine molecules (Fig.\u00a05b). Thus our system enabled successful diffusion-based deproteinization and CE separation. Although interference from the protein was successfully removed, resolution of RB and SR is still not good in Fig.\u00a05b (resolution: R=1.4). When the longer, 48\u00a0cm, separation capillary (effective length: 33\u00a0cm) was connected to the CAs-CHIP instead of 12\u00a0cm capillary, however, baseline separation of these species after deproteinization was successfully achieved (resolution: R=5.7). In addition, relative standard deviations of peak height and migration time for five sequential measurements were 11.4 and 11.5% for RB and SR, and 1.5 and 1.4% for RB and SR, respectively, indicating that reliable analysis with deproteinization pretreatment was successfully achieved by use of a longer separation capillary.\nFig.\u00a05Electropherograms obtained downstream of the separation capillary with and without diffusion-based deproteinization. Flow rates: sample, 0.5\u00a0\u03bcL\u00a0min\u22121, buffer 1 0.5\u00a0\u03bcL\u00a0min\u22121, buffer 2 1\u00a0\u03bcL\u00a0min\u22121. Injection time 15\u00a0s. Applied voltage 5\u00a0kV (ca. 417\u00a0V\u00a0cm\u22121)\nConclusions\nWe have demonstrated deproteinization and CE separation on a single device using CAs-CHIP technology. Diffusion-based deproteinization in a CAs-CHIP was successfully achieved by choosing an appropriate flow rate, and subsequent CE separation of small molecules was achieved by use of a separation capillary connected directly to the CAs-CHIP.\nBecause diffusion-based separation is, in general, based on dilution of the sample solution, preconcentration before CE separation will be required in the next step. Because the CAs-CHIP is prepared by simply embedding square capillaries, however, further integration of the preconcentration process using chemically-functionalized capillaries, or the on-line preconcentration techniques reported to date, can be also used. These applications are currently under investigation.","keyphrases":["capillary-assembled microchip","deproteinization","capillary electrophoresis","square capillary","polydimethylsiloxane"],"prmu":["P","P","P","P","P"]}
{"id":"Bioinformation-1-1-1891626","title":"AVATAR: A database for genome-wide alternative splicing event detection using large scale ESTs and mRNAs\n","text":"In the past years, identification of alternative splicing (AS) variants has been gaining momentum. We developed AVATAR, a database for documenting AS using 5,469,433 human EST sequences and 26,159 human mRNA sequences. AVATAR contains 12000 alternative splicing sites identified by mapping ESTs and mRNAs with the whole human genome sequence. AVATAR also contains AS information for 6 eukaryotes. We mapped EST alignment information into a graph model where exons and introns are represented with vertices and edges, respectively. AVATAR can be queried using, (1) gene names, (2) number of identified AS events in a gene, (3) minimal number of ESTs supporting a splicing site, etc. as search parameters. The system provides visualized AS information for queried genes.\nBackground\nAlternative splicing (AS) is an important mechanism for functional diversity in eukaryotic cells. AS allow processing \nof one pre-mRNA into different transcripts in a cell type. This results in protein diversity with each protein having \ndistinct function. [1\u20132\n\u20133] To address this problem we used EST (short, single pass cDNA \nsequences generated from randomly selected library clones produced in a high throughput manner from different tissues, \nindividuals and conditions) and mRNA sequences to detect AS variants. The detected variants (using 5,469,433 EST and \n26,159 mRNA sequences) were stored in a database called AVATAR.\nAlthough, AS databases are available in the public domain, not many contain AS information for multiple eukaryotes \n(a comparison summarized in AVATAR web site). Therefore, it is important to document AS information for multiple \neukaryotes. Hence, we developed AVATAR containing AS information for six eukaryotes. Here, we describe AVATAR development, \nits content and utility.\nMethodology\nDataset used\nThe dbEST database (Jan 16, 2004) at NCBI contains nearly 5.4 million human EST sequences and this dataset is used \nin the current analysis. [4] The human genome sequences (CONTIG build 3.4)\nin Genbank format is obtained from NCBI. [5] Gene information and \nmRNA sequence were downloaded from the NCBI RefSeq project.\nIdentification of AS\nThe identification of AS in AVATAR is performed in three steps (described below) as illustrated in Figure 1.\nStep 1:\nAlignment of EST and mRNA with the genome sequence\nEST sequences were aligned to the whole genome sequence using Mugup. [6\n] Mugup is a sequence  alignment program developed in Windows platform. This procedure identified splice sites in \nthe ESTs (Figure 1 panel A and B). The matched regions and gaps correspond to exons and introns, respectively. EST and mRNA \nalignments with scores greater than 94% were used for further analysis.\nStep 2:\nClustering EST and mRNA\nEST and mRNA were clustered according to their location in the genome (Figure 1 panel C). EST and mRNA with overlapping \nregions were then assembled together.\nStep 3:\nDetection of AS sites\nThe mapping of EST alignment with genome sequence to intron positions helps to identify skipped exons and \nincluded exons.\nSearching AVATAR\nAVATAR can be queried using keywords. The keywords include accession number, gene name, gene isoform, gene location, \ncytogenetic locations, chromosome number and number of AS events. The database search produces AS visuals for queried gene.\nUtility to the Biological Community\nAVATAR is a collection of AS information for 6 eukaryotic organisms. The database can be queried simultaneously for 6 \norganisms. It can also be searched using gene names and desired number of AS events. EST sequences are error prone resulting \nin the detection of aberrant transcripts. Frequency of EST alignment at a specific site provides improved detection in AVATAR.\nCaveats\nAS information on paralogous genes in eukaryotic genomes are not included in AVATAR due to the difficulty in identifying \ntheir corresponding chromosomal locations using EST sequences.\nFuture developments\nNew EST sequences are generated in laboratories every day. Hence, it is a time consuming to keep AS databases updated due \nto the growth of genome and mRNA sequences. Hence, we are in the process of developing a computer agent which can update AVATAR \nautomatically. We also plan to include tumor specific AS data.","keyphrases":["database","alternative splicing","est","mrna","human","eukaryotes","protein diversity","sequence alignment"],"prmu":["P","P","P","P","P","P","P","R"]}
{"id":"Eur_Spine_J-4-1-2226191","title":"High failure rate of the interspinous distraction device (X-Stop) for the treatment of lumbar spinal stenosis caused by degenerative spondylolisthesis\n","text":"The X-Stop interspinous distraction device has shown to be an attractive alternative to conventional surgical procedures in the treatment of symptomatic degenerative lumbar spinal stenosis. However, the effectiveness of the X-Stop in symptomatic degenerative lumbar spinal stenosis caused by degenerative spondylolisthesis is not known. A cohort of 12 consecutive patients with symptomatic lumbar spinal stenosis caused by degenerative spondylolisthesis were treated with the X-Stop interspinous distraction device. All patients had low back pain, neurogenic claudication and radiculopathy. Pre-operative radiographs revealed an average slip of 19.6%. MRI of the lumbosacral spine showed a severe stenosis. In ten patients, the X-Stop was placed at the L4\u20135 level, whereas two patients were treated at both, L3\u20134 and L4\u20135 level. The mean follow-up was 30.3 months. In eight patients a complete relief of symptoms was observed post-operatively, whereas the remaining 4 patients experienced no relief of symptoms. Recurrence of pain, neurogenic claudication, and worsening of neurological symptoms was observed in three patients within 24 months. Post-operative radiographs and MRI did not show any changes in the percentage of slip or spinal dimensions. Finally, secondary surgical treatment by decompression with posterolateral fusion was performed in seven patients (58%) within 24 months. In conclusion, the X-Stop interspinous distraction device showed an extremely high failure rate, defined as surgical re-intervention, after short term follow-up in patients with spinal stenosis caused by degenerative spondylolisthesis. We do not recommend the X-Stop for the treatment of spinal stenosis complicating degenerative spondylolisthesis.\nIntroduction\nLumbar spinal stenosis complicating degenerative spondylolisthesis is a common cause for low back pain, neurogenic claudication, and radiculopathy in the elderly population. The majority of the patients will respond well to non-operative treatment modalities. However, in patients that fail to respond to conservative treatment, surgical decompression with or without a posterolateral fusion and instrumentation, may be considered [3, 12]. Unfortunately, these procedures have variable long-term outcomes and are frequently followed by complications, especially in the elderly patients with high co-morbility [2, 10]. Therefore, alternative therapies are being developed, of which the interspinous distraction device is rapidly gaining popularity [4, 9]. Of such, the X-Stop (X-Stop, St. Francis Medical Technologies, Inc\u00ae, Alameda, CA) has been introduced as a minimal invasive surgical procedure to treat symptomatic degenerative lumbar spinal stenosis [4, 9]. Initial results of the treatment of degenerative lumbar spinal stenosis with the X-Stop are promising [8, 13, 14]. Recently, encouraging results have been reported for the treatment of patients with symptomatic lumbar spinal stenosis caused by degenerative spondylolisthesis [1]. However, we observed an alarmingly high failure rate, defined as surgical re-intervention, in a cohort of patients treated with the X-Stop for symptomatic lumbar spinal stenosis caused by degenerative spondylolisthesis. This prompted us to perform a retrospective chart review, and analysis of the radiographs.\nPatients and methods\nWe retrospectively reviewed 12 consecutive patients with symptomatic lumbar spinal stenosis caused by degenerative spondylolisthesis treated with the X-Stop interspinous distraction device. The patients were treated between January 2003 and May 2005. There were 9 female and 3 male patients with a mean age at surgery of 67.5\u00a0years (50\u201383). All patients complained of progressive low back pain throughout the day with neurogenic claudication, radiculopathy and a diminished walking distance. In all patients, neurological examination was judged normal or nonspecific. Anteroposterior, lateral and flexion\/extension plain radiographs, and magnetic resonance imaging (MRI) were performed in all cases. The percentage of degenerative slip was measured on the lateral radiograph and measured according to the method described by Anderson et al. [1]. The anteroposterior dural sac diameter in the axial and sagittal plane T2 sequence was measured on the MRI. A standardized walking and cycling test [6] was performed at the department of physical therapy. A limited walking distance less than 1\u00a0km (0.62 miles) independent of the time needed, was considered positive. After walking the patient had to sit and the pre-walking pain must be reduced in less than 5\u00a0min. Cycling should be unlimited without complaints. Initial treatment consisted on patient education, medications to control pain, and exercise and physical treatments to regain or maintain activities of daily living. Surgical treatment with the X-Stop was considered in patients not improving with conservative care for more than 6\u00a0months.\nAll operations were performed under general anesthesia. The patients were placed in prone position on a Wilson spinal surgery frame (Orthopaedic Systems, Inc., Union City, CA) with the lumbar spine in maximum flexion. Prophylactic antibiotics, cefazolin (cefalosporin, Kefzol\u00ae) 1,000\u00a0mg IV, were administered at the induction of anesthesia, and as a second and third dose 8 and 16\u00a0h post-operatively, respectively. After radiographic identification of the surgical level, a mid-sagittal incision of approximately 4\u00a0cm is made over the spinous processes. The musculature was elevated to the level of laminae and facets. The supraspinal ligament is kept intact. To pierce the interspinous ligament, a curved dilator is inserted in the anterior margin of the interspinous space. Subsequently, a sizing distractor is inserted to determine the appropriate implant size. The X-Stop is inserted into the interspinous space as close to the posterior aspect of the lamina as possible. An adjustable wing is attached to the implant and secured along the midline. Patients were mobilized immediately once they had recovered from the anesthetic effects. They were discharged from hospital within 48\u00a0h.\nClinical follow-up took place at 6 and 12\u00a0weeks and at 12 and 24\u00a0months. All patients underwent a clinical and radiographic examination of the lumbar spine in standing position at each follow-up visit. The mean follow-up was 30.3\u00a0months (13\u201341). In patients with persistent or recurrence low back pain with neurogenic claudication and radiculopathy, a second MRI was made. The endpoint was secondary surgical intervention of the lumbar spine. Statistical analysis, comparing the pre- en post-operative MRI dimensions, was performed using Students\u2019 t-test.\nResults\nThe pre-operative percentage of degenerative spondylolisthesis was less than 30% in all patients, with an average slip of 19.6%\u00a0\u00b1\u00a06.20 (9.6\u201329.7). In 9 out of the 12 patients there was a slip of less than 25% (grade 1) degenerative spondylolisthesis. Bending radiographs revealed mobility at the level of the spondylolisthesis in all patients. MRI showed nerve root compression and impingement of the thecal sac. The mean anteroposterior axial cross-sectional diameter was 7.33\u00a0mm (5.71\u201311.19) and the mean anteroposterior sagittal cross-sectional diameter was 7.32\u00a0mm (5.40\u20138.49).\nThe operations were performed at L4\u20135 in ten of the patients and at both L3\u20134 and L4\u20135 in two patients. A 14-mm diameter X-Stop was implanted in nine levels. In the remaining levels, a 12-mm implant was used three times, and 16 and 10-mm implants both once. No peri-operative complications were observed. Post-operative plain radiographs showed a correct position of the implants in all patients. No fractures of the spinous processes were observed. The post-operative percentage of spondylolisthesis, measured on plain radiographs post-operatively and at final follow-up, remained unchanged in all patients.\nDirect post-operatively, 8 out of 12 patient reported a significant improvement of pain, neurogenic claudication, and radiculopathy. However, four patients did not experience any relief of symptoms following surgery and no improvement at follow-up. At 12\u00a0weeks follow up, two patients, that initially had experienced a relief of symptoms suffered from a recurrence of pain, neurogenic claudication, and radiculopathy. In addition, a third patient experienced a recurrence of symptoms at 24\u00a0months follow-up.\nAll patients with persistent or recurrent symptoms had a post-operative MRI. No statistically significant (P\u00a0>\u00a00.05) difference of spinal stenosis was seen at the effected levels in comparison to the pre-operative values. The mean post-operative anteroposterior axial cross sectional diameter was 6.80\u00a0mm (5.24\u20137.65) and the mean sagittal cross sectional diameter was 6.91\u00a0mm (5.12\u20137.70) (Fig.\u00a01, 2, 3). The pre-operative axial and sagittal cross sectional diameter in these seven patients (7 levels) was not significantly different (P\u00a0>\u00a00.05) from that of the five patients (7 levels) without persistent or recurrent symptoms. Finally, the seven patients with persistent or recurrent symptoms underwent surgical re-intervention. The mean degenerative spondylolisthesis of these seven patients was 17.8%\u00a0\u00b1\u00a06.9. Six of these patients had a pre-operative degenerative spondylolisthesis of less than 25%. One patient had a 27.6% degenerative spondylolisthesis. The X-Stop was removed and a decompression and posterolateral fusion with instrumentation was performed.\nFig.\u00a01a Pre-operative lateral plain radiograph. b Post-operative lateral plain radiograph. X-Stop positioned at the level L4\u20135Fig.\u00a02Pre-operative T2-weighted a transversal and b sagittal MR Image showing lumbar spinal stenosis due to discopathy, facet arthritis, ligamentum flavum hypertrophy and anterolisthesisFig.\u00a03Post-operative T2-weighted a transversal and b sagittal MR Image. No change in canal cross-sectional area and mid-sagittal diameter visible after insertion of the X-Stop at level L4\u20135\nDiscussion\nThe X-Stop interspinous distraction device has shown to be an attractive alternative to conventional surgical procedures in the treatment of symptomatic degenerative lumbar spinal stenosis [4, 9]. It may be questioned, however, if the X-stop will be effective in patients with lumbar spinal stenosis caused by degenerative spondylolisthesis also. To our best knowledge, there is only one study that investigated the clinical effects of the X-Stop in patients with lumbar spinal stenosis caused by degenerative spondylolisthesis [1]. In this study, 42 patients were treated with the X-Stop and compared to 33 patients with non-operative treatment. The indication for treatment was a percentage of slip of less than 25%. An overall clinical success rate of 63.4% was reported in the X-Stop treated patients compared to 12.9% in the non-operative treated patients after 2\u00a0year follow-up. Secondary surgery was required in 5 (11.9%) of the patients in the X-Stop group compared to 4 (12.1%) in the control group.\nUnfortunately, we experienced an extremely high failure rate, defined as surgical re-intervention, in a cohort of patients with lumbar spinal stenosis caused by degenerative spondylolisthesis treated with the X-Stop interspinous distraction device. In our cohort, the average percentage of slip was less than 25%, though in 3 patients the percentage of slip was between 25 and 30%. Surgical re-intervention was required in 7 (58%) patients within 24\u00a0months. Of these, only 1 patient had a slip of more than 25% (27.6%). There was no relation between the severity of the slip and the failures in our cohort. Our indication for re-intervention included recurrence or persistent and unremitting low back pain and persistent or progressive neurogenic claudication with radiculopathy. Both clinical and radiological findings were considered together for diagnosing failure of treatment. Unfortunately, we did not enclose pre- and post-operative outcome measurements. However, since the surgical goal of the X-Stop include pain reduction, improvement of neurological symptoms, and improvement of quality of live, re-intervention was considered as the endpoint for failure.\nIn diagnosing spinal stenosis, thecal sac impingement and nerve root compression are seen on MRI. We observed no improvement of the axial and sagittal diameter of the central canal on the MRI after insertion of the X-Stop. In addition, no relation was found between the severity of the pre-operative spinal stenosis measured on MRI and an eventual secondary surgical intervention. Recently, in a study using positional MRI pre- and post-operatively following insertion of the X-Stop, improvement of the cross sectional area of the dural sack has been observed in 12 patients with symptomatic spinal stenosis [11]. This study, however, did not include patients with spinal stenosis caused by degenerative spondylolisthesis. Unfortunately, we do not have the opportunity to use the positional MRI. It may be hypothesized that the spinal stenosis will be more severe in a standing positional MRI, as a result of the instability in degenerative spondylolisthesis. A limitation of the present study is the lack of objective standards of measurement spinal stenosis on MRI. Nevertheless, all patients in our study showed pre-operative a severe thecal sack impingement at the level of degenerative spondylolisthesis. In addition, the spondylolisthesis, as measured on the lateral standing radiographs, did not show progression or improvement after surgery.\nFrom a biomechanical point of view, it may be questioned if the X-Stop interspinous distraction device provides any stabilizing effect on the affected motion segment and will increase the spinal canal in degenerative spondylolisthesis. It has been shown that the facet joints in patients with degenerative spondylolisthesis demonstrate an increased sagittal orientation [5, 7]. When the facet joints are orientated in a more sagittal plain, the resistance to shear forces is decreased. Obviously, the more sagittal orientation of the L4\u2013L5 segment combined with an interspinous distraction device may result in a progressive forward slip of the superior vertebra, and a progressive narrowing of the spinal canal and lateral recesses. Thus, the presence of a degenerative spondylolisthesis in patients with lumbar spinal stenosis may be considered as a contra indication for the X-Stop.\nIn conclusion, the X-Stop interspinous distraction device showed an extremely high failure rate, defined as surgical re-intervention, after short term follow-up in patients with spinal stenosis caused by degenerative spondylolisthesis. We do not recommend the X-Stop for the treatment of lumbar spinal stenosis with degenerative spondylolisthesis, and we consider a degenerative spondylolisthesis a contra-indication for the X-Stop interspinous distraction device.\nCompeting interests\nNo benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.","keyphrases":["x-stop","lumbar spinal stenosis","degenerative spondylolisthesis"],"prmu":["P","P","P"]}
{"id":"Doc_Ophthalmol-3-1-1784540","title":"Multifocal ERG findings in carriers of X-linked retinoschisis\n","text":"Purpose To determine whether retinal dysfunction in obligate carriers of X-linked retinoschisis (XLRS) could be observed in local electroretinographic responses obtained with the multifocal electroretinogram (mfERG). Methods Nine obligate carriers of XLRS (mean age, 46.2 years) were examined for the study. Examination of each carrier included an ocular examination and mfERG testing. For the mfERG, we used a 103-scaled hexagonal stimulus array that subtended a retinal area of approximately 40\u00b0 in diameter. The amplitudes and implicit times in each location for the mfERG were compared with the corresponding values determined for a group of 34 normally-sighted, age-similar control subjects. Results Mapping of 103 local electroretinographic response amplitudes and implicit times within a central 40\u00b0 area with the mfERG showed regions of reduced mfERG amplitudes and delayed implicit times in two of nine carriers. Conclusions The mfERG demonstrated areas of retinal dysfunction in two carriers of XLRS. When present, retinal dysfunction was evident in the presence of a normal-appearing fundus. Multifocal ERG testing can be useful for identifying some carriers of XLRS.\nIntroduction\nJuvenile X-linked retinoschisis (XLRS) is a hereditary, vitreoretinal degeneration that was initially described in 1898 by Haas [1] and subsequently recognized to have an X-linked recessive inheritance in 1938 by Mann and Mac Rae [2]. It is characterized by decreased visual acuity within the first to second decade of life and cystic-appearing lesions within the fovea [3\u20135]. Approximately 50% of patients will also show peripheral retinoschisis [3\u20135]. A selective or predominant b-wave amplitude reduction on full-field electroretinogram (ERG) testing is a distinctive feature of the disease [6].\nIn other X-linked diseases, such as X-linked retinitis pigmentosa, choroideremia and X-linked ocular albinism, because of the principle of Lyonisation, which predicts a random inactivation of one X chromosome within each cell, female carriers can express some features of the diseases [7\u20139]. As a rule, female carriers of XLRS do not exhibit any clinically apparent fundus abnormalities [3, 4]. Isolated observations of foveal cystic-appearing lesions in female carriers from families with XLRS have been reported. There were six such female carriers noted out of a total of 13 who were examined in two different families with consanguineous marriages reported by two previous studies [10, 11]. Foveal changes, described as wrinkling of the internal limiting membrane, of one eye was reported in one female XLRS carrier from a non-consanguineous marriage by Wu et\u00a0al. [12]. One study did find that 11 out of 11 obligate carriers showed an abnormal rod-cone interaction by psychophysical testing [13]. Nevertheless, carrier detection of XLRS currently can most reliably be successful by identifying genetic mutations in a gene that codes for a protein referred to as retinoschisin (RS1) [14, 15]. In the current study, we evaluated the possible role of multifocal ERG (mfERG) testing as a means of detecting functional abnormalities in obligate carriers of XLRS.\nMethods\nNine obligate carriers (eight parents, one offspring) of XLRS patients were enrolled in the study. They were selected on the basis of their availability and willingness to participate in the study. Each had at least one male family member who was diagnosed as having characteristic XLRS findings by one of the authors (GAF). Five of the nine XLRS patients were confirmed as having the causative gene RS1. None of the carriers had any other medical or ocular conditions that might have affected their retinal function. After each carrier was counseled regarding the study, they signed a written informed consent approved by the Institutional Review Board at the University of Illinois at Chicago. The examinations were conducted in accordance with Health Insurance Portability and Accountability Act regulations.\nAll carriers were examined at the Department of Ophthalmology, University of Illinois at Chicago by two of the authors (GAF, LSK). Best-corrected visual acuity (BCVA) was measured in each carrier with a Snellen projection chart. A dilated fundus examination and slit-lamp biomicroscopy of the lens and anterior segment of both eyes were performed on all carriers as were mfERG (Electro-Diagnostic Imaging, San Mateo, CA) measurements. Multiple retinal areas were stimulated to record local cone responses using a stimulus array of 103-scaled hexagons subtending a retinal area of approximately 40\u00b0 in diameter. Each hexagon was scaled with eccentricity to obtain approximately equal amplitudes of local ERG responses [16]. The luminance of each hexagon was modulated according to a binary m-sequence. Individual stimulus elements were modulated between black (0.45\u00a0cd\/m2) and white (280\u00a0cd\/m2) for a time averaged luminance of 140.23\u00a0cd\/m2 (approximately 3.8 log td). The surround luminance was set at 140\u00a0cd\/m2. The stimulus was displayed on a black-and-white monitor driven at a frame rate of 75\u00a0Hz (Nortec, Plymouth, MN). Each subject\u2019s vision was optimally corrected with a refractor\/camera system for the fixed viewing distance of 40\u00a0cm. To ensure equal magnification of the stimulus array, the distance between a subject\u2019s eye and the refractor\/camera was adjusted for each subject to obtain a sharp image on a control monitor. Each carrier\u2019s pupil was dilated with 2.5% phenylephrine and 1% tropicamide to obtain a pupil diameter of at least 7\u00a0mm. Multifocal ERGs were recorded only for the right eye for each of the XLRS carriers. A Burian-Allen bipolar contact lens electrode (Hansen Ophthalmic Laboratories, Iowa City, IA) was used and grounded to the ipsilateral ear. Before insertion of the contact lens electrode, a carrier\u2019s cornea was anesthetized with 0.5% proparacaine, and the left eye was occluded. The total recording time was approximately 8\u00a0min divided into 32 segments. All carriers were required to maintain fixation during each of 14\u00a0sec segments. Segments with large eye movements, losses of fixation, or blinks were discarded and re-recorded. The raw data were filtered at a bandpass of 10 to 300\u00a0Hz, amplified at a gain of 100,000 (Astro-Med\/Gass model CP11 amplifier), and digitized at 1200\u00a0Hz. Each local response was isolated by a cross correlation between the m-sequence and response cycle according to the VERIS algorithm.\nThe amplitude (a-scale) and implicit time (t-scale) of all local (first-order) mfERG responses were derived using the algorithm of Hood and Li [17]. For the current analysis, raw waveforms were exported from the VERIS 3.0 for each of the 34 (15\u00a0male, 19\u00a0female) age-similar (30\u201379\u00a0years) normally sighted control subjects. Artifact reject and averaging with neighbors were turned off. Template wave forms were constructed for each hexagon from the average of all the control subjects\u2019 data. These averaged templates were fitted using a least-squares fitting procedure to each control subject\u2019s data by stretching horizontally (timing) and vertically (amplitude). For each hexagon, the a-scale values were then averaged across all normal subjects and standard deviations calculated. This was also done separately for the t-scale values. Each carrier\u2019s data were then fit by the control templates and the resulting values for the a-scale and t-scale were plotted for each hexagon. For the a-scale, a number less than one indicates an amplitude lower than for the average control subjects and for the t-scale, a number greater than one indicates a timing delayed compared to the average control subjects. The statistical probability of each value was calculated using the control subjects mean and \u00b13 standard deviations (SD) for the a-scale and t-scale respectively and at each hexagon. The grey hexagons indicate >3\u00a0SD above the normal mean. For the 99.7% confidence level (grey hexagons) five control subjects had one abnormal amplitude and two subjects had two abnormal grey hexagons on the a-scale. For implicit time, six control subjects had one abnormal grey hexagon and two had two abnormal grey hexagons. Therefore, for this analysis, any carrier exhibiting a total of three or more grey hexagons were considered abnormal. A black hexagon indicated that a measurable signal could not be detected from the noise level. None of the 34 control subjects had any hexagons that were depicted as black. We used a criterion of two or more black hexagons for a carrier as highly likely to represent a local abnormality.\nResults\nThe nine XLRS carriers ranged in age from 23\u201370\u00a0years, with a mean age of 46.2\u00a0years. All showed a normal exam of the anterior segment of the eye, including the lens. Visual acuity was 20\/25 or better in each eye. None showed any fundus abnormalities. These findings are summarized in Table\u00a01. Seven of the nine obligate carriers were observed to have varying degrees, from mild to moderate, of situs inversus of their temporal retinal vessels. In ocular situs inversus, the temporal vessels leave the optic disc directed towards the nasal retina before making a sharp temporal turn. The fundus photograph for Carrier #1 with situs inversus is shown in Fig.\u00a01. In a study of fundus findings for XLRS patients, situs inversus was observed in 32% of patients with XLRS [18]. To our knowledge, situs inversus of the retinal vessels has not been emphasized as a possible finding in carriers of XLRS.\nTable\u00a01The age, visual acuity for the right eye (OD) and left eye (OS) and fundus findings for each of the X-linked retinoschisis carriersCarrierAge (years)VA ODVA OSFundus findings13520\/1520\/15Mild situs inversus OU24720\/2020\/20Normal OU32320\/2020\/20 \u22121Normal OU45320\/2020\/20Mild situs inversus OU54820\/2520\/20Situs inversus OU65820\/2020\/20Situs inversus OU73920\/2020\/20Mild situs inversus OU84320\/20 \u2212220\/20Situs inversus inferior vein OS, anomalous branching OS\u00a0>\u00a0OD97020\/20 \u2212220\/20 \u22122Anomalous branching OU, situs inversus superior artery OD, inferior artery OSFig.\u00a01Fundus photograph of XLRS carrier #1 demonstrating situs inversus of the retinal veins\nTwo of nine carriers demonstrated a mosaic pattern of statistically significant amplitude reductions (Figs.\u00a02a, 3a) and implicit time delays (Figs.\u00a02b, 3b). The actual mfERG tracings and hexagon analysis for these two carriers are shown in Figs.\u00a02 and 3.\nFig.\u00a02Multifocal electroretinogram (mfERG) results of X-linked retinoschisis (XLRS) carrier #8 exhibiting a mosaic pattern of retinal dysfunction. MfERG amplitudes (a) and implicit times (b) for the right eye. In this figure, as in figure 3, the white hexagons show regions falling within 2 standard deviations (SD) of the normal mean. The grey hexagons represent locations that are more than 3 SD from the normal mean while the black hexagons indicate a non-measurable response. MfERG waveform traces are illustrated below (c)Fig.\u00a03Multifocal electroretinogram (mfERG) results of XLRS carrier #3 exhibiting a mosaic pattern of retinal dysfunction. MfERG amplitudes (a) and implicit times (b) for the right eye. MfERG waveform traces are illustrated below (c)\nDiscussion\nOur findings support the conclusion that the presence of a functional abnormality on mfERG testing, in the absence of other causes for retinal dysfunction in a female at risk, should suggest the presence of a carrier state for XLRS, which could then be further confirmed by genetic testing. However, a normal mfERG result would not, by itself, rule out the carrier state. Piao and co-workers [19] showed that mfERG cone-mediated responses were more impaired centrally than in the more peripheral retina in their study of seven male patients with XLRS. They also observed a higher frequency of delayed implicit times than reduced amplitudes in their patients. We did not observe either of these features in our two carriers.\nThe presence of situs inversus of the retinal vessels in a female from a family with XLRS is also suggestive of the carrier state. Our findings on mfERG testing demonstrate the effect of Lyonisation on retinal function in carriers of XLRS. A similar finding on mfERG testing was observed in carriers of X-linked retinitis pigmentosa [20]. Multifocal ERG testing may be a useful technique for the detection and monitoring of localized retinal dysfunction in some carriers of XLRS.","keyphrases":["carriers","x-linked retinoschisis","situs inversus","multifocal electroretinography"],"prmu":["P","P","P","M"]}
{"id":"Bioprocess_Biosyst_Eng-2-2-1705532","title":"A next generation, pilot-scale continuous sterilization system for fermentation media\n","text":"A new continuous sterilization system was designed, constructed, started up, and qualified for media sterilization for secondary metabolite cultivations, bioconversions, and enzyme production. An existing Honeywell Total Distributed Control 3000-based control system was extended using redundant High performance Process Manager controllers for 98 I\/O (input\/output) points. This new equipment was retrofitted into an industrial research fermentation pilot plant, designed and constructed in the early 1980s. Design strategies of this new continuous sterilizer system and the expanded control system are described and compared with the literature (including dairy and bio-waste inactivation applications) and the weaknesses of the prior installation for expected effectiveness. In addition, the reasoning behind selection of some of these improved features has been incorporated. Examples of enhancements adopted include sanitary heat exchanger (HEX) design, incorporation of a \u201cflash\u201d cooling HEX, on-line calculation of Fo and Ro, and use of field I\/O modules located near the vessel to permit low-cost addition of new instrumentation. Sterilizer performance also was characterized over the expected range of operating conditions. Differences between design and observed temperature, pressure, and other profiles were quantified and investigated.\nIntroduction\nContinuous sterilization also is known as high-temperature, short-time (HTST) sterilization. A continuous sterilizer heats non-sterile (raw) medium to the desired sterilization hold temperature (typically 135\u2013150\u00b0C), maintains it at constant temperature in an adiabatic holding loop (consisting of a long length of insulated stacked piping connected with U-bends for compactness), then cools it to 35\u201360\u00b0C before transferring flow to a fermenter that has been previously sterilized empty or with a minimal amount of water. The residence time that medium is held at sterilization temperature, tR (min) [calculated from the adiabatic retention loop volume, Vs (L), divided by the system volumetric flowrate, Q (lpm)], is varied by adjusting flowrate and\/or length of the holding loop.\nEnergy is recovered by pre-heating incoming cold medium from 15\u00b0C (worst case) to 120\u00b0C with outgoing sterilized medium that is cooled from its sterilization temperature of 150 to 45\u00b0C prior to entering the process cooler where it is cooled further to 35\u00b0C. Medium is recycled back to a circulation tank (also called a surge or recycle tank) or diverted to the sewer during start up or process upsets (such as a decrease in sterilization temperature or an increase in system flowrate). This circulation tank can be pressurized or non-pressurized with the non-pressurized design approach requiring a second \u201cflash\u201d cooler before returning flow to the recycle tank to avoid flashing. Heating is accomplished indirectly using steam or hot water via a heat exchanger (HEX) or directly by mixing steam with incoming medium (steam injection). Cooling HEXs can use cooling tower\/chilled water, but also may use vacuum to reduce temperature and draw off any accumulated water from direct steam injection. Continuous sterilization systems typically are pre-sterilized with steam by direct injection and\/or with hot water. After attaining steady state with water flow, non-sterile medium feed is introduced. Various media components are sterilized in aliquots and sent to the receiving fermentation vessel with water flushed between them.\nA next generation, pilot-scale continuous sterilization system was designed, installed, started up, and validated. Demolition as well as retrofit was accomplished within an actively operating industrial pilot plant. Despite prior experience with a stick-built, internally designed system, a skid-mounted vendor design was selected consisting of five skids (recovery and heating exchangers, hot water loop and exchanger, retention loop, process and \u201cflash\u201d cooling HEXs, and switching valve station). Sterilized medium, obtained from the system at 40\u2013100\u00a0lpm, typically was aliquoted into 800\u201319,000\u00a0L scale fermenters with lower flowrates being most appropriate for lower fermenter volumes. The design accommodated a range of different media types, including low solids levels below 5\u00a0wt.% and concentrated nutrient solutions.\nThe design evaluated features from related industrial applications of continuous sterilization, including sanitary design advances in spiral HEX fabrication that were considered helpful. Valued design characteristics were flexibility, reliability, and straightforwardness in operation and maintenance. Although some general papers describing continuous sterilizer design are available [10, 35], there have been few, if any, publications linking design and operation, despite the considerable and varied industrial applications of high temperature, short time (HTST) sterilization. This paper describes the design and testing of a next generation HTST continuous media sterilization system, along with the technical rationale behind its features and flexibility.\nBackground\nAdvantages\nAdvantages of continuous sterilization have been outlined in several reports [3, 7, 10, 30, 60, 84]. By far the most noted benefit is energy conservation since continuous sterilization consumes up to 60\u201380% less steam and cooling water for large-scale fermenter media volumes. This economy lies at the high end of this range when continuous sterilization utilizes heat recovery via indirect heat exchange to pre-heat incoming cold medium with hot medium leaving the sterilization hold loop. It thus requires less energy (as well as generates a more uniform demand without peak draws [7]); than the alternative batch sterilization process involving sterilizing the fermenter vessel and its non-sterile contents together. Batch sterilization becomes less efficient with scale since heating and cooling portions of the cycle are longer than the constant hold temperature portion [54, 83], and heat transfer coefficients decrease with scale up [34]. Prior to receiving continuously sterilized medium, the fermenter is sterilized empty or with a small volume of water covering the pH and dissolved oxygen probes. Heat up\/cool down times are substantially shorter, decreasing overall turn-around time [96].\nContinuous sterilization results in gentler treatment of medium compared to batch sterilization, which tends to overheat medium to ensure that vapor space vessel internals achieve sterilization temperatures [1]. Sterilization at higher temperatures for a shorter time generates less degradation of heat-sensitive medium components since spore destruction rates increase faster than nutrient destruction rates as temperature rises [15]. The activation energy for nutrient degradation ranges from 50 to 150\u00a0kJ\/mol, which typically is smaller than activation energies for the thermal death of microorganisms, which range from 250 to 350\u00a0kJ\/mol [67]. Consequently Fo increases more than Ro as temperature rises [1, 52]. Similarly, amino carbonyl or Maillard browning reactions, which form objectionable color and tastes to consumers for pasteurization and adversely affect medium quality (destroy growth factors) for fermentation, are minimized [12, 54]. In the case of polymerized poly (l-lactide) rod implants, lower molecular weight decreases also have been found for autoclave cycles at higher temperatures and shorter times [88].\nContinuous sterilization results in more uniform heat treatment of medium than batch processes since the system operates at steady state [15]. Proteins and carbohydrates can be separately sterilized in multiple sections using several mix tanks with a sterile water flush between them [96]. Since there is no need to agitate unaerated (ungassed) large liquid volumes during batch sterilization, fermenter agitator design can be based on drawing full load during gassed conditions [96]. Some feel that continuous sterilization offers a lower contamination rate relative to batch sterilization since fermenter internals are more easily sterilized in an empty rather than full fermenter [96]; others believe that batch sterilization has a lower risk since there is no need to transfer aseptic media [97]. HTST systems have a high degree of flexibility since a large range of time\/temperature combinations can be selected within equipment design limits. Scale up is linear with media flowrates of 10\u2013100,000\u00a0L\/h reported for HTST systems [36] and up to 30,000\u201350,000\u00a0L\/h for pasteurizers [38]. Time\/temperature exposure profiles accurately reflect sterilization conditions for the media of interest and can be readily modeled. Finally, the ability to design heat exchange equipment to minimize fouling reduces cleanability and maintenance concerns.\nDisadvantages of continuous sterilization primarily are that process control performance is critical since it is necessary to immediately divert flow of any inadequately sterilized medium, halt any further medium sterilization, and resterilize the system. In contrast, for a batch sterilization system upset, often additional hold time can be readily added to the sterilization. Continuous sterilizer systems also use dedicated equipment that usually is not well suited for other purposes.\nApplications\nSeveral relevant background papers on applications of continuous heat treatment of liquids have been published in the food, biowaste, and fermentation fields.\nApplications of continuous sterilization to dairy and other food pasteurization are prevalent in the literature. A continuous pasteurization process with a hold temperature of 72\u00b0C and hold time of 15\u00a0s replaces a batch process with a lower hold temperature of 63\u00b0C for 30\u00a0min [65]. Ultra high temperature (UHT) treatment (120\u2013136\u00b0C), using either direct steam injection or indirect heating, is used to obtain longer preservation (specifically greater log reduction) than pasteurization at 72\u00b0C [32]. Temperatures of 100\u2013145\u00b0C produce extended shelf life milk with a product shelf life of 15\u201330\u00a0days at 7\u00b0C [16]. Direct steam injection for heating feed to its hold temperature for UHT treatment causes less destruction to other milk components owing to rapid heating using injected steam and rapid cooling using a vacuum [43].\nFor dairy applications, often the lethality achieved during heat up and cool down periods is similar in magnitude to that achieved during isothermal hold time [67], and thus needs to be considered in evaluating exposure. The integrated pasteurization effect (PE) is calculated to convert the time, t (min), at different temperatures, T(t) (K), in various sections of the pasteurizer (specifically the heat up, holding, and cooling sections), to the equivalent time at a reference temperature, To, of 72\u00a0C (345\u00a0K) and a reference time, to, of 15\u00a0s (0.25\u00a0min) (Eq. 1):  where Ea is the activation energy, cal\/mol, and R is the universal gas constant of 1.987\u00a0cal\/mol-K. A PE of one corresponds to complete pasteurization at 72\u00b0C for 15\u00a0s [65].\nThe effectiveness of heat treatment in the food industry is established indirectly since it is undesirable to introduce indicator organisms into production equipment. An indicator enzyme such as alkaline phosphatase is used to test proper milk pasteurization after first establishing its relation to pathogen load [28, 65] and pasteurization effect [69]. The behavior of indicator organisms also is examined and rigorously modeled since obtaining accurate kill kinetics at operational conditions can be problematic [72, 80, 87].\nContinuous sterilization also is used for biowaste destruction and decontamination of spent broth, the major byproduct from biotechnology plants [41, 84]. Typical sterilization temperatures vary from as low as 80\u00b0C up to 140\u00b0C, for usually short hold times of 1\u20135\u00a0min. Less aggressive conditions are warranted since organisms being sterilized are active cultures and not dormant spores. Batch systems involve heat up, sterilization, and cool down of waste, all in the same jacketed vessel, and often with direct steam sparging for heating and an external HEX used for cooling to shorten the time cycle [57]. Owing to its higher throughput, continuous sterilization has been applied to biowaste treatment [60], and the prediction that it eventually would be the preferred method of biowaste inactivation [84] has been realized for larger facilities.\nOperational concerns for biowaste treatment are opposite those for media sterilization. Although both processes require achievement of the desired log reduction of live organisms in the feed, biowaste treatment is concerned with live organism leakage into either previously sterilized effluent broth and\/or uncontained cooling water. In contrast, media sterilization\/pasteurization is concerned with live organisms leaking into sterilized media from non-sterile cooling water [98] and\/or non-sterile incoming feed.\nFor fermenter media sterilization applications, continuous sterilization complements continuous fermentation, which can be more productive for certain fermentation processes since it substantially reduces fermenter turn-around time between successive runs [102]. Systems are able to be maintained on-line and ready so that they can continuously sterilize and deliver mid-cycle medium additions directly into active fermentations. Typical sterilization temperatures range from 135 to 150\u00b0C with hold times of 4\u201312\u00a0min. Similarly to the PE value for pasteurization, the Fo value (min) is used to characterize sterilization effectiveness for fermentation medium [18]. It is the time for the actual sterilization hold temperature that is equivalent to exposure to a saturated steam environment of 121\u00b0C, according to Eq. 2:  where T(t) is the sterilization hold temperature, K, t is the incremental sterilization hold time, min, integrated over the start time, ti, to finish time, tf, and Z is the temperature difference (K or \u00b0C) for a one log change in DT (min), the time for a one log reduction in spore concentration. A similar expression can be developed for the analogous impact on nutrient degradation, Ro (min) [17].\nHigh temperature, short time continuous heat treatment also has been evaluated for the viral inactivation of mammalian cell culture medium (hold temperature of 102\u00b0C and hold time of 10\u00a0s) to minimize nutrient degradation [66] and of blood plasma (hold temperature of 77\u00b0C and hold time of 0.006\u00a0s) to maintain protein structure and activity [23].\nKill\/degradation kinetics\nRelative to Escherichia coli, the heat resistance of bacterial spores is 3 million:1, mold spores is 2\u201310:1, and viruses and bacteriophages is 1\u20135:1 [34]. As a first pass, the kinetics of kill and degradation are based on the Arrhenius equation for the thermal death constant, k(t), min\u22121 (Eq. 3) as a function of the incremental sterilization holding time, t:  where T(t), Ea, and R are defined as in Eq. 1 and A is the frequency factor of the reaction, min\u22121. Adherence to strict first order kinetics is not always the case [4, 33], and this model does not incorporate partial germination and\/or heat activation of dormant spores prior to media sterilization reducing their heat resistance [90]. Nevertheless, this simple model is employed for the validation of media sterilization conditions in the fermentation industry.\nUsing the Arrhenius model, the non-temperature dependent activation energy can be calculated from the regressed slope of log of reaction rate constant versus the reciprocal of absolute temperature [67]. Typical values for Bacillus (Geobacilllus) stearothermophilus, an indicator organism commonly used to evaluate heat treatment effectiveness, are 9.5\u00d71037\u00a0min\u22121 for A and 70,000\u00a0cal\/mol for Ea [11].\nThe D value, DT (min), or decimal reduction time, is the time to decrease the population to one-tenth its original number at a specified temperature [15]. The Z value (K or \u00b0C) is number of degrees of temperature rise that causes a tenfold increase in D value [15]. It is obtained by plotting the log of the D value versus the corresponding temperature and calculating the Z value obtained from the reciprocal of the slope of the least squares regression line (Bigelow model) [67]. The Q\u0394T value is the death rate increase for a specified change in temperature, \u0394T [15], and it is calculated from the dependence of the D value (specifically k) on temperature. Both D and Z values are affected by the physicochemical and biochemical properties of the solution to be sterilized (e.g., composition, pH) [21, 51, 52, 85, 101].\nThe Z value for B. stearothermophilus in water is 10\u00b0C, vs. 56\u00b0C for vitamin B1 and 50\u00b0C for vitamin B2 (riboflavin) [15], both notably higher. Correspondingly, the Q10 value for B. stearothermophilus is 11.5, vs. 2.1 for vitamin B1, 2.3 for vitamin B2 (riboflavin), and 3.0 for the Maillard reaction [15], all notably lower. Two models (Arrhenius or Bigelow) can be used to extrapolate death rates for higher temperatures than those measured experimentally since it is difficult to measure kill and degradation kinetics at temperatures above 130\u00b0C with existing equipment [67].\nUsing the overall sterilization hold time or residence time, tR, the log reduction may be obtained according to Eq. 4:  where N(t) is the number of spores surviving heat treatment at incremental sterilization time, t, No is the initial number of spores, and DT is defined below Eq. 2. Assuming D121=3\u00a0min and Z=10\u00b0C (typical values for B. stearothermophilus spores in water [52]), then log reductions for continuous sterilization at 150\u00b0C range from 1,800 to 7,500-fold for the system residence times of 5.4\u201322.5\u00a0min, substantially higher than what is obtainable via batch sterilization. Actual sterilization conditions are selected based on specific medium properties and fermentation process requirements.\nRetention loop flow behavior and its impact\nFlow through a pipe is characterized by the Reynolds number, NRe, given by DV\u03c1\/\u03b7, which is the ratio of inertial to viscous forces. Since the system flow tube diameter, D (cm), does not change, NRe varies with sterilization fluid velocity, V (controlled by system flowrate), fluid viscosity, \u03b7 (cp), and density, \u03c1 (g\/cm3), which is fixed for the selected medium. As flow becomes more turbulent (higher NRe), flow behavior approaches ideal plug flow.\nThere is considerable disparity in the literature regarding the Reynolds numbers associated with laminar and turbulent flow through a tube for various continuous flow sterilization applications. For flow through a tube, laminar flow was below 1,100 and turbulent flow was above 2,100 [95]. Laminar flow was below 2,100, and turbulent flow above 4,000 according to another study [81]. A minimum velocity, which gives turbulent flow is recommended with a Reynolds number of about 3,000 [96] or at least 2,500 [30], but preferably above 20,000 [30]. For a retention loop in a dairy application, Reynolds numbers of 1,130\u20132,300 were considered laminar [67], and Reynolds numbers of 4,800\u20137,080 were considered transitional [74]. Substantially higher Reynolds numbers of 7,200\u20139,400 were considered transitional for the heating and cooling sections of tubular HEXs for a dairy application [67]. Differences in tube roundness and entrance effects may have an influence [68]. System design for NRe well above 10,000, particularly in the holding tube, minimizes potential for inadvertent operation in the laminar flow regime. One potential design approach is to incorporate flow disturbances to induce turbulence at lower NRe. Confirmation of turbulent flow, based on the deviation between ideal and non-ideal plug flow behavior for specific operating conditions, can be determined experimentally.\nContinuous thermal treatment is most uniform when the flow through the retention loop is turbulent since residence times of individual streamlines become less variable. The parabolic velocity profile associated with laminar flow leads to variable residence times [67]. Specifically, for laminar flow the mean velocity of a viscous fluid through a pipe is one-half of the maximum velocity along the axis, and for turbulent flow, the mean velocity is 82% of the maximum value [3]. Thus, there are concerns about laminar flow for viscous solutions in pasteurization (e.g., ice cream mix, egg nog, and liquid egg products) [81].\nNon-ideal flow behavior is problematic since each fluid element spends different lengths of time in the sterilizer hold phase and thus receives different levels of sterilization. Consequently, it is necessary to characterize the residence time distribution to accurately predict lethality [95], specifically the spectrum of times spent in the sterilizer hold tube for different fluid elements. The extent of the distribution dictates the degree of axial or Taylor dispersion, i.e., concentration gradients along the length of the retention loop. [Radial gradients are assumed negligible]. The holding efficiency, tmin\/tm, is evaluated by comparing the minimum holding time, tmin (min), to the mean holding time, tm (min), and the extent of product overheating, tmax\/tm, can be calculated using the ratio of the maximum, tmax (min), to mean holding times [55].\nStimulus-response measurement techniques and data analysis to determine the extent of non-ideal flow have been described comprehensively [61, 62, 95]. After a pulse or step change is introduced, tracer concentration is measured as a function of time by sampling effluent at the system outlet. The total area under the concentration versus time curve then is calculated and used to normalize concentration measurements so they can be readily compared for different tracer tests.\nThe exit age distribution, E(t), delineates the fraction of fluid elements exiting the system having a particular hold time, t. It characterizes instantaneous pulse changes (delta function) in tracer concentration. This curve is normalized by dividing measured C(t) values by the area under the resulting concentration versus time curve to obtain E(t), for which the area under the E(t) curve is always one [61].\nFor E(t) curves, the mean time, tm, is the sum of individual products of time, normalized tracer concentrations, and time interval, \u0394t (min), which is assumed constant [46]. The distribution variance, \u03c32, is given by Eq. 5:  where \nThe dimensionless variance, \u03c32\/tm2, can be used to estimate the dispersion coefficient based on experimental data.\nAnother concentration versus time curve, F(t), is the probability that a fluid element left the system within time, t, or the volume fraction of the outlet stream that has remained in the system for a time less than t [95]. It characterizes behavior resulting from step inputs of tracer with an initial entering concentration, Co. The ratio of C(t)\/Co versus time produces a normalized F(t) curve for which the axes range from 0 to 1 [61].\nThese distributions are related mathematically according to Eqs. 6 and 7:  Integration of E(t) curve to obtain the corresponding F(t) curve via Eq. 7 is accomplished graphically for various \u0394t [95].\nBoth the E(t) and F(t) curves also can be made dimensionless in time by normalizing by the mean hold time, tm. Normalization with respect to time is helpful to compare conditions at different residence times, and normalization with respect to concentration assists in comparing data from different tracer experiments.\nThe sterilization efficiency for a given residence time distribution is given by Eq. 8:  This equation permits quantitative assessment of the sterilization impact from non-ideal flow patterns.\nThe Bodenstein number (or Peclet number or Peclet-Bodenstein number [3, 54]), NBs, is given by VL\/Dz, where Dz is the axial dispersion coefficient, m2\/s, V is the flow velocity, m\/s, and L is the retention loop length, m. This dimensionless group is the ratio of convective transport to axial dispersion [63, 94], and it is used to quantify the extent of axial dispersion. For NBs>>1, there is plug flow with minimal axial mixing and sterilization efficiency is the highest possible [62, 63]. For NBs<<1, axial dispersion is at its worst, with retention loop contents completely mixed along the tube length, and sterilization likely is incomplete. Actual operating conditions fall between these two extremes [63]. The flow system should be designed so that dispersion is minimized with high NBs and high NRe [54], preferably NRe>2\u00d7104. NBs of 3\u2013600 have been reported as typical for continuous sterilizers [30]. The current system\u2019s NBs of about 1.8\u00d7104 is much higher than this range, most likely due to its longer retention loop.\nExperimental distribution data may be used to calculate \u03c32\/tm2, NBs, and then Dz [61, 62] using Eqs. 9 and 10. For Dz\/VL<<1 and a normal (Gaussian) distribution for the E(t) curve: \nAlternatively, the dispersion coefficient and residence time distribution may be inferred from correlations. For turbulent flow, Eq. 10 applies:  where the Fanning friction factor, f, for the retention loop pipe is obtained from correlations based on the ratio of surface roughness, \u03b5, to pipe diameter, D [78]. An experimental correlation for water, where Dz\/VD=0.25 for NRe=105 and Dz\/VD=0.33 for NRe=104 [61], was used to assess the reasonableness of measured Dz\/VL values and to compare NBs values obtained using Eqs. 10 and 11a. Friction factors for the retention loop pressure drop, applicable for both laminar and turbulent flow, were calculated using Eq. 11a [26, 75] and shown in Table\u00a09:  where \nAlternatively these friction factors can be estimated using the Colebrook equation, Eq. 11b [29, 78] and solving iteratively, but this approach was not used:  Rule-of-thumb conditions generating a narrow residence distribution and low dispersion coefficient for flow through a pipe are L\/D>200 and NRe>12,000 [55].\nInfluence of solid content of media\nFor sterilizer feed medium that contains solids, ranging from small amounts to in excess of 10\u00a0vol.% [31], solids must be adequately wetted and dispersed without clumps. Particles flow at different velocities through the retention loop, and temperature distribution within a particle is challenging to monitor. Although it is somewhat straightforward to determine residence time distributions, partial differential equations using finite differences are required to model convective\u2013conductive heat transfer between the fluid and particles [20]. Thermal properties of the surrounding fluid are less critical for heat transfer to particles since the heat transfer coefficient, h (cal\/s-cm2\u2212\u00b0C), between the fluid and particle is limiting [59]. Its effectiveness is shown by the Nusselt number (ratio of total heat transfer to conductive heat transfer), NNu, given by hDp\/K, where Dp (cm) is the particle diameter, and K (cal-cm\/s-cm2\u2212\u00b0C) is the thermal conductivity of fluid at the processing temperature [22, 49]. If the predicted particle temperature profile is hotter than the actual one, it is possible to obtain incomplete inactivation [20].\nThe sterilization challenge of large diameter solids is to avoid selecting hold times\/temperatures that sterilize solids but damage liquid medium components [89]. The time required for particles to attain sterilizing temperature is on the order of microseconds for particles several microns in size (i.e., media bioburden) and seconds for solids several millimeters in size (i.e., raw material particles), highlighting the need to clarify raw materials [3, 30]. Time-temperature integrators have been developed to quantify the heating impact on spores within the entire particle. These indicators are spores immobilized in alginate cubes or polymethylmethacrylate designed to have a mechanical resistance to flow through the system similar to that of actual particles [49, 73]. Other tracers have been found to mimic the flow behavior of microbial cells except when the flow is laminar [2].\nAs the solid content increased from 0 to 30\u00a0w\/w.%, the mean residence time of the liquid phase increased by 40% and flow less resembled plug flow which indicated that the presence of solids can significantly influence liquid phase flow patterns [76]. Consequently, a safe design approach for solids-containing medium sterilization uses the maximum rather than average fluid velocity [8, 9]. Residence time distributions for solid particles also often have more than one peak representing different groups of particles.\nExperimental methods to quantify axial dispersion\nTesting of dispersion has been done using a variety of tracers, most commonly salts and dyes. Experimental mean residence times calculated from salt tracer measurements in skim milk were close to the average holding time [74]. A salt tracer was found to be adequate for low viscosity and Newtonian fluids only; it overestimated thermal exposure in more viscous fluids [81]. Salt tracers can be saturated sodium chloride solutions, but chloride exposure is not desirable for stainless steel [50]; thus, other salts with high aqueous solubilities (sodium sulfate, sodium citrate, and magnesium sulfate) and\/or sodium hydroxide can be substituted. Dye tracers include fast green FCF (Sigma; St. Louis, MO, USA) and basic Fuchsin (no vendor given) [46].\nOther tracers have been based on chemical reactions, specifically sucrose inversion (hydrolysis to glucose and fructose) when heated in an HCl solution at pH 0\u20132 or sulfuric acid at a pH of 2.5 to avoid exposure of stainless steel to chlorides. Changes in optical rotation and freezing point were used to quantify reaction extent [1]. Another tracer used has been the pulse injection of 20\u00a0w\/w% citric acid and subsequent pH measurement [76]. Finally, temperature spikes also have been effective tracers in scraped surface HEXs [44].\nOverview of operation\nA comparison of major changes between the prior and next generation pilot-scale, continuous sterilization systems, as well as expected benefits\/risks or potential drawbacks, are summarized in Table\u00a01. A schematic of major equipment components and their arrangement is shown by Fig.\u00a01. System specifications and design criteria are listed in Table\u00a02. After system design was completed for water, its effectiveness was evaluated for concentrated nutrients, typically sterilized separately from the base medium to avoid Maillard reactions or sterilized just prior to delivery to active fed-batch fermentations to avoid storage in a holding tank. The nutrients and concentrations selected were 55\u00a0wt.% cerelose (glucose monohydrate; CPC International, Argo, IL, USA) and 50\u00a0vol.% glycerol (Superol glycerine; Proctor and Gamble Chemicals, Cincinnati, OH, USA). Physical properties for these test media at various temperatures were estimated for water from [37, 42, DIPPER database tables (dippr.byu.edu)]. Physical properties for 50\u00a0vol.% glycerol and 50\u00a0wt.% cerelose were modeled using Aspen Plus (AspenTech, Cambridge, MA, USA) process simulation software with physical properties database information.\nTable\u00a01Comparison of major changes in large continuous sterilization systemItemPrior designNew designExpected benefit\/Risk or potential drawbackFinal heating method to attain sterilization temperatureDirect steam injectionIndirect heating loopLess dilution, improved stability with respect to source steam fluctuations, no adulteration from plant steam additives\/higher costFlowmeterMagneticCoriolisAbility to sense deionized water\/position of flag criticalNumber of different retention loop lengthsAble to increase\/decrease by two tubes for 16\u201330 tubes (tR=4.0\u201312.5\u00a0min), removable connections at both ends of all tubesFive configurations from 18 to 30 tubes (tR=5.4\u201322.5\u00a0min), removable jumpers at same end of selected tubesFewer fittings\/limited visual inspectionFlowrate turn down60\u2013100\u00a0lpm40\u2013100\u00a0lpmAvoids separate smaller unit\/multiple ranges for tuning controlPressure safety deviceSafety valve onlyRupture disc and safety valve with tell-tale pressure gaugeSanitary disc in process contact, evident when disc blown\/added cost and maintenanceSecond cooler of same size as process cooler (\u201cflash\u201d cooler)AbsentPresentAbility to conduct water sterilization, installed marker for process cooler\/extra expenseBooster pump with pressure control on recuperator outletAbsentPresent (used with centrifugal feed pump only)Sterile media at higher pressure than non-sterile media\/increased complexity of tuning and operationRetention loop insulationInsulated box without packing, 4\u20136\u00b0C temperature dropInsulated box with packing, <2\u00b0C temperature dropMore adiabatic and isothermal\/modest additional expenseHEX plate thickness1\/4\u20333\/16\u2033Lower cost and higher surface area per unit volume\/higher risk of breachHEX aspect ratioHigher velocities\/increased effect of channeling due to gap and drain notchesRecuperator2.73.46Coolers4.762.91Heater0.93 (horizontal cross flow for condensing service)1.31HEX process side channel thickness0.25\u2033 (coolers 0.375\u2033)0.25\u2033 (coolers 0.25\u2033)Higher surface area per unit volume\/higher pressure dropHEX utility side channel thickness (coolers)0.75\u20330.5\u2033Higher surface area per unit volume\/higher pressure dropAspect ratio is the HEX diameter divided by its widthFig.\u00a01Schematic of sterilizer system. a major components, b switching stationTable\u00a02System specifications and design criteriaParametersDesign range (min\u2013max)Sterilization hold temperature (T)135\u2013150\u00b0CRetention loop hold up volume (Vs)540\u2013900\u00a0LFlowrate (to achieve design recuperator HEX heat recovery) (Q)40\u2013100\u00a0lpm at 15\u201360\u00b0C (water)40\u2013100\u00a0lpm at 60\u00b0C (55\u00a0wt.% cerelose)40\u201365\u00a0lpm at 25\u00b0C (55\u00a0wt.% cerelose)40\u201391.5\u00a0lpm at 25\u201360\u00b0C (50 vol.% glycerol)40\u201388\u00a0lpm at 15\u00b0C (50 vol.% glycerol)Flow rates <40\u00a0lpm may not achieve sufficient back-pressure for the selected sterilization temperature to avoid flashingFeed temperature (Tin,cold,ext)15\u201360\u00b0C (water, 50 vol.% glycerol)25\u201360\u00b0C (55\u00a0wt.% cerelose)Feed temperature of 15\u00b0C for 55\u00a0wt.% cerelose insufficient to maintain a solutionResidence time (tR)5.4\u201322.5\u00a0minBack-pressure (P)3.5\u20135\u00a0kgf\/cm2 (typically 4.1\u00a0kgf\/cm2)Sufficient pressures used to avoid flashing\u00a0>2.15\u00a0kgf\/cm2 for 135\u00b0C\u00a0>3.93\u00a0kgf\/cm2 for 150\u00b0CRetention loop temperature drop (900\u00a0L volume and inlet temperature of 150\u00b0C), \u0394T2.0\u00b0C for 40\u00a0lpm, 1.5\u00b0C for 60\u00a0lpm, 1.0\u00b0C for 80\u00a0lpm, and 1.0\u00b0C for 100\u00a0lpmHeat recovery (HR)>70\u201380% depending inlet feed temperature, media type and flowrate\u00a078.9% (100\u00a0lpm water, 60\u00b0C)\u00a075.5% (100\u00a0lpm 55\u00a0wt.% cerelose, 60\u00b0C)\u00a078.9% (91.5\u00a0lpm 50\u00a0vol.% glycerol, 60\u00b0C)\nThe sterilizer had several distinct phases that are depicted by Fig.\u00a02 and described briefly below: System start up included (1) leak tests of the cold system under pressure, (2) flowrate and totalizer accuracy checks versus a decrease in feed tank volume, and (3) leak re-check after raising the system to sterilization temperature. Proper operation and reliability of instrumentation was assured by evaluating all pressure and temperature transmitter and gauge readings for consistency. Prior to system sterilization and before introducing steam or superheated water, draining of process water, supplemented by evacuating with 90\u00a0psig air, was necessary for preventing stress corrosion cracking of HEXs [93].\nFig.\u00a02Overview of sterilizer phases\nThe next phase was steam sterilization using four high point steam injection points, one of which was located at the outlet of retention loop, and setting the hot water heating loop to 135\u00b0C, slightly above the corresponding steam sterilization temperature of 134\u00b0C for the 30\u00a0psig (2.1\u00a0kgf\/cm2) steam supplied. After 2\u00a0h of steam sterilization, the system was transitioned from steam to water carefully (over a period of 1\u00a0h) to maintain sterility, or with less care assuming that water sterilization was planned next. Steam sterilization could be conducted for the system up through the process cooler as well as up through the \u201cflash\u201d cooler (Fig.\u00a01a). Use of the \u201cflash\u201d cooler was the preferred configuration since it provided an extra buffer during steam collapse.\nDuring this next phase of steam-to-water transition, the system flowrate was started with 15\u00a0lpm water flowing to the sewer after the \u201cflash\u201d cooler using the Moyno pump. All steam injection points, such as the medium distribution system, were closed, and the hot water generated by the heater provided sufficient back-pressure. The system was set up for water sterilization (i.e., recuperator non-sterile side bypassed and the \u201cflash\u201d cooler used to cool sterilizing water so that the process cooler could be sterilized), and the hot water loop was set at 150\u00b0C in cascade (set point for water sterilization). As water entered the system, the temperature of the retention loop rose from 133 to 150\u00b0C, while the temperature of the process cooler fell from 148 to 122\u00b0C. (The temperature drop of the process cooler was not a sterility risk since the entering 15\u00a0lpm water flowrate, sterilized at 133\u00b0C, resulted in a sufficient Fo of 965\u00a0min to assure sterility of the retention loop effluent). Loss of incoming water due to boiling while the system was at a lower backpressure was believed minimized by the nearly closed \u201cflash\u201d cooler backpressure valve (expected fill volume 1,215\u00a0L, actual volume 1,218\u00a0L).\nDuring water sterilization, incoming cold water was circulated for two passes (one pass if system was already hot from steam sterilization) at 60\u00a0lpm using the centrifugal inlet feed pump, after it rose to the sterilization inlet hold temperature of 150\u00b0C. It required previously steamed-through system block\/drain valves since users were not comfortable that conduction adequately sterilized through them when closed. The non-sterile side of the recuperator HEX was bypassed to ensure that the sterile side reached sterilization temperature. Cooling water was applied to the \u201cflash\u201d cooler to ensure that the process cooler attained sterilization temperature. For the target sterilization temperature of just below 150\u00b0C and a 60\u00a0lpm water flowrate, the temperature reached about 148.5\u00b0C at the sterile side of the recuperator and 146.5\u00b0C for the sterile side of the process cooler.\nWater sterilization was redone during medium sterilization if medium diversion was necessary owing to system sterility upset. After taking immediate action to divert media away from the production vessel, water re-sterilization was conducted by (1) diverting flow through the \u201cflash\u201d cooler and enabling its pressure control loop, (2) fully opening the process cooler back-pressure valve, (3) conducting water re-sterilization, (4) enabling the process cooler pressure control loop, (5) fully opening the \u201cflash\u201d cooler back-pressure valve, and then (6) resuming medium sterilization. When switching from the \u201cflash\u201d to the process cooler, it was necessary to maintain sterile conditions.\nAfter the system was sterilized and running on water, typically in recirculation mode or emptying into the system sewer, the switching valve station was used to divert flow to distribution. Water now flowed to a waste vessel or the process sewer located near the eventual medium receiving vessel. After conditions stabilized, sterilizer inlet feed was switched from water to medium. Again, after conditions stabilized, flow was switched to the receiving vessel. When the receiving vessel was filled sufficiently, sterilizer effluent was switched back to the waste tank, sterilizer inlet feed was switched back to water, and then effluent switched back to either the recirculation vessel or system sewer. After media sterilization, a thorough water rinse was conducted at sterilization temperature and the system was cooled to 60\u00b0C for cleaning. Alkaline and\/or acid cleaning solutions were used depending on the nature of the soil. After cleaning, the system was cooled and drained completely.\nEquipment design\nThe system\u2019s five skids were designed and fabricated at the vendor\u2019s shop and delivered with only field installation of interconnecting piping required. To minimize design miscommunications, three-dimensional piping models were used for skid piping plans, which were able to be reviewed remotely by the customer. Ball valves were used instead of diaphragm valves for hot temperature service. Hazardous energy control was carefully considered with locking devices installed and valve placement selected for facile equipment isolation and operability. Equipment was citric acid-passivated after installation.\nEach relief device on the process side consisted of a flanged rupture disc (RD) with a pressure indicator and telltale as well as a pressure safety valve (PSV) that reseated after the source of excessive pressure was removed. These devices were placed directly after the positive displacement Moyno (Robbins and Myers; West Chester, PA, USA) system inlet feed pump, recuperator outlet, and booster pump. Discharges were piped to return to the feed tank for safety as well as for medium recovery. Piping was designed such that no PSV devices were required on the process side to minimize risk of system integrity disruption. Sample points were located on the inlet feed (pre-sterilization, prior to recuperator) and sterilized medium outlet (post-sterilization, after process cooler) lines.\nHeat exchangers\nThe chief goals of HEX design are to optimize cost, heat transfer, size, and pressure drop [48]. The type of HEX selected was a spiral, which was preferred over alterative shell and tube, plate and frame, or concentric double pipe designs. A spiral HEX consists of two long, flat, preferably seamless sheets of metal plate, separated by spacers or studs, wrapped around a center core or mandrel which forms two concentric spirals. Alternate ends are welded (both by machine and manually) to create separated flow channels. Hot fluid enters the center (flows inside to outside) and cold fluid enters on the exterior (flows outside to inside) to achieve countercurrent flow. Details of spiral HEX design are described elsewhere [71].\nAdvantages of spirals are chiefly that they require less space per unit of heat transfer surface area [104]. Their continuously curved channel, unrestricted flow path, and presence of spacers increases turbulence due to secondary flow effects which maintain solids in suspension [13, 103]. Fouling is lower than shell and tube designs since cross-sectional velocities increase as channel size decreases, creating a scrubbing effect [13, 19]. (In contrast, as individual tubes of shell and tube exchangers plug, flow is diverted into unplugged tubes.) Spirals are particularly well suited for slurries and many viscous fluids [103]. Specifically, slurries can be processed at velocities as low as 2\u00a0ft\/s (0.61\u00a0m\/s) [71]. Periodic, thorough cleaning can be conducted by simply removing the cover to expose the spiral cavities and cleaning with a high pressure water source.\nEvaluation of thermal effectiveness can be done by calculating the number of thermal transfer units, NTU [25, 100], using Eq. 12:  where AHEX is the heat transfer area, m2, QM is the mass flow rate (kg\/s), U (W\/s\u2212m2-K) is the overall mean heat transfer coefficient between the fluid streams, and Cp (J\/kg-K) is the specific heat capacity of the fluid at constant pressure. This quantity also can be obtained from individual stream temperatures where Tin,hot,ctr is the temperature of the incoming hot stream entering in the center, Tin,cold,ext is the temperature of the incoming cold stream entering on the periphery, Tout,hot,ext is the temperature of the outgoing hot stream exiting on the periphery, and Tout,cold,ctr is the temperature of the outgoing cold stream exiting in the center. In this case, the temperature rise of the cold stream is divided by the log mean temperature difference (LMTD) for the HEX. An NTU of 1.0 correspond to a shell and tube HEX; NTU>1 represents overlap of hot and cold side temperature ranges indicative of spiral HEXs.\nThermal effectiveness also can be evaluated using Eq. 13 to calculate the thermal effectiveness factor, TE [25, 100]:  Overall Eq. 13 represents the change in recuperator cold side stream temperature divided by the temperature difference of streams flowing through its center connections (i.e., incoming, sterilized, hot medium exiting retention loop and outgoing, pre-heated, non-sterile medium).\nFinally, thermal effectiveness can be evaluated by calculating the heat recovery, heat recovery (HR), using Eq. 14:  which represents the heat gained by the incoming cold medium after passing through the recuperator divided by total heat gained by the cold medium after passing through both the recuperator and heater HEXs. Heat recoveries improve with clean HEXs (higher heat transfer coefficient), lower flowrates (permitting more time for heat transfer), and higher inlet feed temperatures (lower medium viscosity which improves heat transfer coefficient). When the temperature difference on both sides of the recuperator is the same, then NTU is replaced by TE, and Eq. 12 cannot be used since \u0394Tln=0 [14].\nFor consistency in comparing design and observed performance over the entire sterilizer system, the value of Tin,hot,ctr (retention loop outlet temperature) used for calculating design values in Eqs. 12, 13, and 14 was assumed to be identical to the retention loop inlet temperature (T), i.e., the retention loop was assumed to be isothermal (adiabatic). The sensitivity of these three parameters to small temperature measurement errors of \u00b11\u00b0C for the expected temperature change of each HEX stream was estimated \u00b15.7% for NTU, +3.5\/\u201312.5% for TE, and \u00b13.0% for HR.\nSpecific limiting performance case scenarios depend on media type and inlet feed temperature. Higher inlet feed temperature (60\u00b0C) is worst case for the cooling HEX since the recuperator removes less heat from sterile medium. Lower inlet feed temperature (15\u00b0C) is worst case for the recuperator since it represents the greatest challenge to HR.\nHeat exchanger design and material selection is important to extending the unit\u2019s lifetime. A pressure rating of 150\u00a0psig was selected to match piping specifications, specifically flange connections. Thicker gauge material permitted wider spacing of studs, directly affecting cost [58], minimized corrosion without significantly reducing heat transfer [13], but decreased HEX surface area per unit volume (Table\u00a01). All HEXs underwent hydrostatic as well as helium leak tests. Studs for spacing were only partially welded around the base so a small crack existed. These crevices were considered unavoidable, and they were accepted since they were shallow enough to permit adequate contact with sterilizing and cleaning fluids.\nHeat exchanger diameter, width, and channel spacing were designed to minimize fouling and deposits by ensuring channel velocities were sufficiently high during operation. Channel widths of \u00bc\u2033 were used, except for the utility sides of cooler HEXs, where a channel width of \u00bd\u2033 was selected to minimize plugging due to cooling water deposits and to reduce pressure drop. Bulk velocities for each process fluid are shown in Table\u00a010.\nA sanitary design was utilized with a continuous sheet for coil formation, a tapered channel transition for the medium inlet and outlet, external bracing of shell connections, back-welding of the center pocket stiffener as much as possible, elimination of additional center stiffeners, and polishing\/cleaning of all internal welds. Process connections were 150\u00a0psig bolted, milled, lap-joint flanges possessing a right angle rather than a bevelled edge to line up directly with the gasket and avoid crevices. A solid 316\u00a0L stainless steel door eliminated a process side weld around the center nozzle required to attach a stainless steel skin to a carbon steel door.\nHeat exchangers initially were designed to include a gap between the spiral face and cover to minimize gouging of the door due to distortion or telescoping. Spirals can \u201cgrow\u201d during thermal cycling and cut into full-face gaskets, if present [71] or door covers if annular gaskets were utilized without a sufficient gap. Telescoping also was minimized by not applying pressure to one HEX side without either the opposite door bolted closed or suitable bracing installed on the open side. An annular gasket initially was selected which when placed in its groove provided a 1\/16\u2033 gap between the exchanger spiral surface and the door. At first, this gap was considered small enough so that any short-circuiting negligibly impacted performance, particularly the recuperator HR. Subsequently, the gap size was realized to be critical, especially for high aspect ratio (\u201cpancake\u201d type) HEXs like the recuperator. The smoothness and straightness of the spiral and door faces (tolerance of \u00b11\/32\u2033) also assured a consistent gap, minimizing bypassing, and maximizing heat transfer; HEXs were modified to be within this tolerance. Observed pressure drops during operation (150\u00b0C inlet retention loop temperature, 100\u00a0lpm water) approached design values for cases where this gap was minimized through installation of compressible gaskets. Both an annular gasket (Gylon 3510; Garlock Sealing Technologies, Palmyra, NY, USA) and a full-face gasket (Gylon 3545) were used to implement a full-face gasket installation with gasket material compressed so as to cut into the spiral face.\nLow point drains were installed to permit complete system drainage with steam barriers applied to sterile process side drain valves to reduce sterility risk. These drains were fed by small \u201cU\u201d shaped notches in each wind of the spiral from the center down to the door drain. These notches were expected to negligibly impact performance relative to the gap. The extent that gaps or notches remained when a pliable, full-face gasket was installed was estimated by qualitative assessment of HEX gravity draining rates, which corresponded to measured pressure drops closer to design values. For the HEX gaskets selected, the remaining water after gravity drain was 20\u201350% of the entire HEX hold up volume, suggesting only very small gaps. This water was removed by subsequent blow-down using a 90\u00a0psig air supply.\nA second cooling HEX was required for water sterilization conducted without a pressurized recirculation vessel. This HEX was cooled with chilled\/cooling water and not by flashing, and it is referred to as a \u201cflash\u201d cooler throughout this paper. Since this pilot scale system was used intermittently, a sterilized system was not continuously maintained by recirculating sterile water between media runs, which often is done in production facilities. This \u201cflash\u201d cooling HEX also was sized to directly replace the process cooling HEX. Since process coolers experienced the highest extent of thermal cycling, they were more likely than the other HEXs to fail based on previous experience. In addition, it was desirable to avoid using chlorine treatments to reduce bioburden in chilled and tower cooling water since this adversely affected stainless steel integrity [98]. Other causes of stress cracking and pitting corrosion during normal operations and cleanouts also existed [55], and their impact needed to be minimized.\nChilled\/cooling water flowrates to cooling HEXs were sized for reasonable exit temperatures to minimize load on the chiller\/cooling tower and reduce deposits that formed at higher outlet cooling water temperatures above 50\u00b0C [106]. The peak design condition was for water sterilization of the system and not production of sterile media. The observed rise in chilled water temperature was within design values when full cooling was applied but not when the control valve restricted flow to attain the desired outlet temperature set point (Table\u00a06). Less water was used for cooling when under control than was assumed in the design since (1) process and \u201cflash\u201d coolers were oversized and (2) media outlet temperature and chilled water flowrate cannot both be specified.\nHot water heating loop\nA hot water or tempered heating loop involves indirect heating without direct steam contact, and it utilizes a HEX, expansion tank, and circulation pump to heat water to above 150\u00b0C. It is more expensive than direct steam injection since additional equipment is required, but hot water loops have some key advantages.\nDirect steam injection can be accomplished with a specialized steam water mixing valve, for example a Pick heater [79]. Although it is more energy efficient since heat up is almost instantaneous, its ability to provide accurate temperature control has been debated. It has a faster response time, can be used with solids-containing media, and is easier to clean and maintain [97], but it is sensitive to source steam pressure and media composition changes. Limited theoretical design information is established for these mixers, although detailed photographic examination of injected steam characteristics in water as a function of flow Reynolds number is available [77]. The key drawback to direct steam injection is process stream dilution, which can be up to 20\u00a0vol.% [83]. Excess water must be removed by subsequent flashing elsewhere in the system or the initial feed concentration must be adjusted. Also, since medium is exposed directly to steam, it may accumulate any additives or iron present in the steam [15]. Finally, there can be additional noise from direct steam injection into flowing liquid in some applications.\nThere is higher energy in steam (2,260\u00a0kJ\/kg energy released from condensing steam) versus the heat capacity, Cp, of water of 4.2\u00a0kJ\/kg\u00a0K, making condensing steam heat content 540-fold higher than hot water heat content on a per degree basis [5]. In addition, injected steam heat transfer coefficients are 60-fold higher than indirect condensing steam heat transfer coefficients [77] and are not reduced by fouling as in a HEX [45]. Consequently, there are advantages of direct steam injection due its higher steam utilization efficiency [97] for high temperature sterilization of milk [82] and beer mash heating [5].\nBased on its advantages for media sterilization, indirect heating via a hot water loop was implemented. A shell and tube 316\u00a0L stainless steel HEX was selected for this application since a spiral HEX was not found to be cost-effective for the size required. The hot water loop utility piping, originally carbon steel, exhibited substantial amounts of iron oxide corrosion due to its operation at higher temperatures. This build-up throughout the hot water loop was subsequently removed by a citric acid wash and piping was replaced by stainless steel. The hot water loop was designed for an operating temperature of up to 160\u00b0C and pressure of 75\u00a0psig using compressed air (>80\u00a0psig) applied to the expansion tank. Installation of a computer limit of 160\u00b0C for the loop temperature was necessary to avoid inadvertent system over-pressurization since the steam control valve opened fully during initial loop heat up.\nInlet retention loop temperature was controlled for these loops rather than outlet temperature, commonly used in pasteurization applications [82], owing to longer loop residence times for medium sterilization applications. Sterilization inlet temperature was controlled in either automatic or cascade mode. In automatic mode, a single loop was used to modulate hot water temperature to control retention loop inlet temperature at the outlet of the heater. In this single loop feedback control, wider periodic fluctuations have been found, but response time is quicker [27]. In cascade mode inlet temperature was used for primary control, and hot water temperature input was used as the secondary control loop. Using cascade control, the slave, inner or secondary loop manipulated the steam control valve to control water outlet temperature from the hot water HEX. The master, outer or primary loop manipulated the secondary (slave) loop set point to control medium outlet temperature on the final heating HEX prior to medium entry into the retention loop. This control has been found to be smoother and more accurate [91], but it has about a twofold longer response time [27]. Steam valve signals were more stable under cascade control with more constant steam flows instead of oscillating between high and low steam supply flow rates as in single loop control. An alternative feed forward control algorithm also has been used in other systems to anticipate process upsets due to load changes and to ensure tight control of product outlet temperature from the retention loop [56], but this strategy was not implemented in the current system.\nThe hot water loop temperature controllers initially were tuned using the Ziegler\u2013Nichols closed loop method [107] for both primary and secondary loops. Tuning constants for the secondary loop were first determined from the ultimate gain (specifically controller gain that causes continuous cycling) and ultimate period (specifically cycle period). Next, the primary loop was tuned with the secondary loop placed in cascade using these constants. The speed of the slave (secondary) loop was slowed down considerably and reset minimized [70] to gain more precise control (\u00b10.1\u00b0C) of inlet temperature in cascade mode. Together with low heat loss over the insulated retention loop, this tuning strategy permitted operation at more uniform sterilizing temperatures. Thus, sterilization temperature effects on subsequent production media performance were quantified more precisely, and operation with a safety factor of several degrees was avoided.\nRetention loop\nThe retention loop (holding tube or box) was composed of thirty, 2\u2033 diameter tubes (schedule 10 pipe with an ID of 2.16\u2033 and wall thickness of 0.11\u2033), each with only one weld over the 40\u00a0ft straight length, plus 29 connecting U-bends. These U-bends were fabricated from 2\u2033 pipe bent by machine that resulted in a minimum thickness at the bend slightly less than normal schedule 10 pipe. The total length was 1,253\u00a0ft (382\u00a0m). The L\/D was 9,318, suggesting that a narrow residence time distribution (and low axial dispersion) was achievable with sufficient turbulent flow. Also, additional mixing at each U-bend owing to its curvature might further reduce axial concentration gradients, although this effect has not been mentioned specifically in the literature.\nThe retention loop was designed to be drainable, and tubes were arranged in two banks in an \u201caccordion-type\u201d fashion on a 0.11 incline. Pipe supports were designed to permit expansion [96]. A high point vent valve was installed in the loop for draining and for bleeding of air during steaming\/filling. (This vent valve did not appear to be required since a negligible amount of air exited the system when it was opened). Jumpers were configurable for variable retention loop volumes of 18 (540\u00a0L), 20 (600\u00a0L), 24 (720\u00a0L), 28 (840\u00a0L), or 30 (900\u00a0L) tubes. All of the jumpers were located on the same end of the retention loop with a removable insulation cover. Removable U-bends were attached using pipe-to-I-line ferrule fitting adaptors with minimal welds and maintaining the inner diameter so that flow was not constricted.\nThe retention loop was required to operate as close to isothermally (adiabatically) as possible. Improved insulation was installed by packing fiberglass blankets inside 2\u2033 thick fiberglass board surrounding the faces between the frame and tubes. This method was preferred over insulating individual tubes since the large insulation thickness required around each tube adversely enlarged overall loop dimensions. The temperature profile along the length of the retention loop was assumed to be linear [35]. For this improved insulation, observed retention loop temperature drops as a function of flowrate compared favorably with design values.\nFlow and pressure control\nFlow and pressure control was composed of five loops that, although not related by software linkages, were closely related operationally. Two flow control valves were installed with one located after the centrifugal inlet feed pump and one after the centrifugal booster pump (Fig.\u00a01a). The system was designed to utilize either a positive displacement (Moyno) or centrifugal inlet feed pump. When the positive displacement pump was used, both flow control valves were held fully open and flow was controlled using the Moyno pump\u2019s variable speed drive. When the centrifugal feed pump was used with the booster pump, booster pump suction pressure was controlled just prior to the booster pump suction and flow was controlled at the booster pump discharge only. (The flow control valve after the centrifugal feed pump was not used since this starved the booster pump suction).\nThree pressure control valves also were installed. One pressure control valve, located on the recuperator inlet piping on the booster pump suction side, was set to maintain a positive pressure differential to avoid leakage on non-sterile feed should a HEX breach develop [sterile side at higher average pressure (0.8\u20131.5\u00a0kgf\/cm2) than the non-sterile side]. The second and third valves were located after the process and \u201cflash\u201d coolers respectively (Fig.\u00a01a) to maintain system pressure above the boiling point, which eliminated noise and potential damage from hammering [96]. To avoid leakage of non-sterile cooling fluids, the pressure on the utility side of the process cooler can be operated slightly below that of the sterile process side [96] by raising the system back-pressure or by reducing the chilled water supply pressure (i.e., by opening the supply to the nearby \u201cflash\u201d cooler).\nPiping and HEX pressure drops were designed to be low so that sufficient back-pressures were attainable at the system outlet to provide adequate protection against flashing. There were limits to the range of suitable temperature and back-pressure combinations that avoided operating close to the fluid flashing point (Table\u00a02). In addition, sufficient system flowrate (>40\u00a0lpm) was necessary to maintain back-pressure and flow consistency to avoid flashing.\nThe tuning strategy for the system began with tuning each flow and pressure loop individually and then operating them together, slowing down the response of the pressure loops as necessary to eliminate interactions. Loops were tuned using the Ziegler\u2013Nichols method [107]; however the loop response with these settings excessively oscillated even before approaching set point. For flow and liquid pressure loops, large proportional bands (i.e., small gain) and fast reset action (i.e., small reset\/integral time) are recommended [6]. Proportional and integral constants only are recommended for most liquid flow control with only integral constants (i.e., floating control) recommended for noisy loops [64]. Consequently, gain and reset time were reduced so that integral control was the dominant action, which reduced oscillations. In addition, booster pump suction pressure control was deliberately detuned to have a slow response so as not to interact with flow and system back-pressure controllers (which themselves did not interact with each other). Thus, both booster pump flow and suction pressure controllers could be used together with no instability. Table\u00a03 shows the tuning constants selected.\nTable\u00a03Optimized tuning constants for sterilization of test mediaParameterKpT1 (min\/repeat)T2 (min)Flow control valve after centrifugal feed pump (40\u2013100\u00a0lpm)0.080.050Pressure control valve on suction of booster pump\u00a040\u201360\u00a0lpm0.050.300\u00a080\u2013100\u00a0lpm0.050.200Flow control valve after centrifugal booster pump (40\u2013100\u00a0lpm)0.0450.050Hot water temperature control of retention loop inlet\u2014sterilization temperature of 135\u2013150\u00b0C cleaning temperature of 60\u201380\u00b0C (40\u2013100\u00a0lpm)Primary (outer)0.360.50.225Secondary (inner)20.020.00.20Pressure control after process cooler\u2014Moyno or centrifugal inlet feed pump\u00a040\u201360\u00a0lpm0.050.100\u00a080\u2013100\u00a0lpm0.050.080Pressure control after \u201cflash\u201d cooler\u2014Moyno or centrifugal inlet feed pump\u00a040\u201360\u00a0lpm0.050.100\u00a080\u2013100\u00a0lpm0.050.080Temperature control of process cooler cooling to 35\u00b0C1.51.00.25Temperature control of \u201cflash\u201d cooler-during sterilization cooling to 35\u00b0C and with 35\u00b0C inlet feed (40\u2013100\u00a0lpm)1.51.00.25Temperature control of \u201cflash\u201d cooler-during cleaning solution cooling to 60\u00b0C and with 60\u00b0C inlet feed\u00a060\u00a0lpm0.481.350.5\u00a080\u2013100\u00a0lpm1.51.00.25(1) Zero T2 values were used for faster (relative to temperature loop) pressure and flow control loops. (2) Slightly different tuning constants required for (a) Moyno and centrifugal feed pumps and (b) 40\u201360 and 80\u2013100\u00a0lpm flowrates to remain within desired \u00b10.1\u00a0kgf\/cm2 back-pressure variation. Tuning constants for 40\u201360\u00a0lpm worked up to 80\u00a0lpm. (3) At 60\u00a0lpm slower tuning required for \u201cflash\u201d cooler when cooling to 60\u00b0C for cleaning (inlet feed of 60\u00b0C) than cooling to 35\u00b0C (inlet feed of 35\u00b0C) during sterilization. (4) Higher T2 value for primary hot water loop relative to its T1 value minimized variations of slower secondary temperature control loop\nSwitching valve station\nThe switching valve station was comprised of several diverter valves to direct the flow of steam, water, or medium to distribution, recycle, condensate trap, or system sewer as desired (Fig.\u00a01b). The system switched according to the following valving arrangements (Fig.\u00a01a, b): (1) system recycle to circulation tank (after passing through \u201cflash\u201d cooler; used for clean-in-place (CIP) and water sterilization), (2) transfer (feed) of sterilized medium to fermenters\/waste tank, (3) system flow to sewer after process cooler, (4) system flow to sewer after \u201cflash\u201d cooler, (5) steam sterilization through process cooler to its condensate trap, and (6) steam sterilization through \u201cflash\u201d cooler to its condensate trap. Simultaneously with pathway switch, a steam barrier was applied to the pathway not being used to maintain sterility.\nIsometric design of the switching station was challenging since several automatic valves with actuators were located in close proximity to reduce dead legs. Actuator size was minimized by sizing appropriately with little excess buffer for the facility instrument air pressure. Limit switches were avoided to save additional space as well as to streamline installation and maintenance costs.\nInstrumentation\nSterilizer instrumentation is described in Table\u00a04. In general, instrumentation mounting was important both for sanitary operation and for accurate instrument measurements. Remotely mounted transmitters were used where needed to extend temperature range suitability of instrument sensors (e.g., flowmeters) and where helpful for space and access reasons. Wherever possible, locally indicating transmitters were selected which permitted operation by a single person since the human\/machine interface (HMI) was upstairs in the facility control room. Transmitters were mounted either in a panel (drawback of additional wiring but able to be factory tested) or on the skid (drawback of crowding skid access but avoids cost of a separate panel).\nTable\u00a04InstrumentationParameterModelFeaturesTemperatureRosemount 3144PD1A1NAM5C2QPX30\u2013200\u00b0C (hot water loop) 0\u2013160\u00b0C (all others)FlowMicromotion R100S128NBBAEZZZZ0\u2013120\u00a0lpmPressureRosemount 3051CG4A22A1AS1B4M5QP0\u201310\u00a0kgf\/cm2 (feed and booster pumps) 0\u20136\u00a0kgf\/cm2 (post-cooler HEXs)ConductivityRosemount 225-07-56-99LC\/54EC-02-090\u2013100\u00a0MS\/cm triclamp connectionRosemount 403VP-12-21-36\/54EC-02-090\u2013100\u00a0\u03bcS\/cm triclamp connectionTemperature controlFisher-Rosemount 1052-V200-3610JSoftware limit of 160\u00b0C for hot water loopFlow controlFisher-Rosemount 1052-V200-3610JFlow control valves usable with either transmitterPressure controlFisher-Rosemount 1052-V200-3610JSoftware adjustment to prevent full closing of system back-pressure valveSteam controlFisher-Rosemount 667-EZ-3582125\u00a0psig unregulated plant steam supplyI\/P transducerMarsh-Bellofram 966-710-1013\u201315\u00a0psig compactSolenoidAsco series 541 multifunction ISO 1 mono stableSpring-return piston actuators\nAccurate measurement of temperature was critical to ensuring that adequate medium sterilization was achieved and permitting reliable Fo and Ro calculations. Typical accuracies reported in HTST pasteurization equipment are \u00b10.5\u00b0C at 72\u00b0C between indicating and recording temperature devices and \u00b10.25\u00b0C at 72\u00b0C between test and indicating devices [92]. This compared favorably with the loop accuracy of \u00b10.21\u00b0C for this system, estimated based on stated vendor accuracy for matched sensors [86].\nPressure loop accuracy was \u00b10.02\u00a0kgf\/cm2. The system back-pressure valve was capable of controlling pressure for flowrates ranging from 40 to 100\u00a0lpm using a computer-controlled maximum output of 80% closure to prevent unintentional shut off when operating with the positive displacement Moyno pump. In contrast, a closure of at least 95% was required when operating with the centrifugal pump since the pump output pressure was lower. A pressure and temperature gauge or transmitter was installed on the inlet and outlet of both sides of all HEXs for assuring adequate heat transfer performance and determining when HEXs required cleaning. These instruments also were important to evaluate individual unit performance for systems of interrelated HEXs during trouble-shooting [39].\nAccurate measurement of volumetric flow was critical to ensuring that medium was properly sterilized for the appropriate residence time. A back-up flowmeter was installed for confirmation. Coriolis meters (Micromotion; Rosemount, Chanhassen, MN, USA), with a meter accuracy of \u00b10.5% of flowrate (loop accuracy of \u00b10.6% of flowrate), were selected rather than magnetic meters. Since flow measurements were based on fluid density, Coriolis meter readings were similar for both deionized and process (city) water (<\u00b10.5\u00a0lpm at 60\u2013100\u00a0lpm) and within expected variations. Coriolis meters also were insensitive to media composition changes, specifically the switch from media to water, assuming these changes negligibly affected fluid density and were not affected by the hydraulics of medium to water switches. However, volumetric flow rate readings were up to 5.5% higher after the recuperator than before it owing to density decreases with temperature for specific medium types. [Mass flowrate (kg\/min) readings were similar]. Finally, since air bubble entrainment altered density readings and thus Coriolis flowmeter readings, a variable speed agitator (5:1 turndown) was installed on the larger non-sterile medium feed tanks. For soluble media, shutting off the agitation also minimized air entrainment.\nProper Coriolis flowmeter installation was critical to performance. The preferred orientation was in a vertical upward flow section of pipe so that the flag filled and drained completely. Alternatives were not attractive: in the horizontal position pointing downwards the flag does not drain, in the horizontal position pointing upwards the flag incompletely fills due to air entrapment, and in the vertical position in a downward flow section of pipe the flag incompletely fills owing to gravity drainage. It also was necessary to secure the surrounding pipe to minimize interfering vibrations.\nConductivity sensors were used on-line to measure changes in the composition upon switching inlet feed stream contents and between inlet and outlet streams. One conductivity meter with a range of 0\u2013100\u00a0\u03bcs\/cm detected DIW with typical conductivities of 2.1\u00a0\u03bcs\/cm during cleaning. Two other conductivity meters, each with a range of 0\u2013100\u00a0Ms\/cm, were located on feed (after the inlet feed pump) and outlet (after the process cooling HEX) of the sterilizer system (Fig.\u00a01a). Similarly to the flowmeters, they were mounted in the vertical flow section to ensure no adverse effects on readings. Conductivity monitoring at the cooling water exit may enable instant detection of a cooling HEX breach [93], but this leak might have to be fairly substantial since cooling water and media conductivities are similar in magnitude.\nControl system\nStrategy\nThe control system strategy utilized minimal sequencing with manual operation preferred both to reduce installation expense and maximize flexibility. The system was composed of about 100 I\/O (input\/output) with about 55% analog input\/output (AI\/AO) and 45% digital input\/output (DI\/DO). The controls were interfaced to an existing Honeywell Total Distributed Control 2000\/3000 hybrid system using newly-installed, dual (redundant) Honeywell High performance Process Manager controllers. Field-mounted (remote) I\/O was installed inside a Nema 4\u00d7 enclosure.\nCalculated values by the control system\nSeveral calculated values were displayed on the HMI to permit alarming and trending. These parameters included the temperature difference (inlet minus outlet) across the insulated retention loop, flowrate difference across the recuperator (upstream minus downstream), conductivity difference (inlet minus outlet), and pressure difference across the recuperator (outlet of hot side minus inlet of cold side). In addition, the totalized volumetric flowrate was calculated based on flowmeter readings rather than using the flow transmitter totalizer signal since implementation of the former was more straightforward.\nValues of Fo and Ro were obtained based on on-line calculation of system residence time. The flowrate measured after the recuperator HEX was used in the calculation. Activation energies, Ea, of 16,800\u201326,000\u00a0cal\/mol used for Ro generally were lower than those of the various spore types of 67,700\u201382,100\u00a0cal\/mol used for Fo [34]. An Ea of 67,700\u00a0cal\/mol was used for Fo [105] and an Ea of 20,748\u00a0cal\/mol was used for Ro [17].\nAn adjustable filtering function [53] could be applied to final calculated Fo and Ro process variable values (PV) to smooth fluctuations caused by pulsations in flow and temperature readings. This function (Eq. 16) had only one user adjustable parameter for the filter value (FV):  With FV set to 1.0 (i.e., no filtering), variations of Fo and Ro were less than 1%. In addition, a user adjustable input permitted entry of proper retention loop volumes, Vs, to ensure accurate residence time, tR, calculations.\nThree methods were used to evaluate this on-line calculation for a simulated continuous sterilization run (Table\u00a05): (a) calculation for 1\u00a0min residence time intervals along the loop length and summation of values over the entire length of pipe, (b) use of the average of retention loop inlet and outlet temperatures in the calculation, and (c) averaging of two separate calculations using inlet and outlet temperatures. Although method a was most accurate, method b was selected since the error was sufficiently small, and implementation was more straightforward. In general, errors were smaller for Ro than Fo. This approach was in contrast to the dairy industry where a worst case lethality has been calculated using the outlet temperature of the insulated retention loop [91].\nTable\u00a05Comparison of calculation methods for Fo and Ro for a simulated continuous sterilization run with a 2\u00b0C temperature drop across the insulated retention loop and tR=10\u00a0min (error calculated relative to method a)MethodFo (min)Error (%)Ro (min)Error (%)aIntegrate at 1\u00a0min residence time intervals439.93Basis31.84BasisbUse average of inlet and outlet loop temperatures437.000.6731.820.06cAverage separate calculations using inlet and outlet loop temperatures445.621.3031.880.13\nTuning constants and control variation\nFor all control loops, proportional\/integral\/derivative (PID) control in the fast mode (PID calculation updated every 0.25\u00a0s) was utilized based on three parameters (definitions are specific to the Honeywell control system): the proportional gain, KP, unitless (reciprocal of the proportional band); the integral constant, T1, min per repeat; and the derivative constant, T2, min. Control tuning constants were developed for water (Table\u00a03) then tested and found to be acceptable for different media (55\u00a0wt.% cerelose, 50\u00a0vol.% glycerol). Slightly different values were optimal for flowrates of 40\u201360\u00a0lpm than for 80\u2013100\u00a0lpm with lower flowrate range constants performing somewhat better than higher flowrate range constants between 60 and 80\u00a0lpm. Variations in these control loops, characterized under various operating conditions, were found to be acceptable. Hot water loop control performance did not change significantly with media type since system disturbances expected for media were likely to be dampened relative to water.\nSystem performance\nWater and media testing\nThe three types of media tested were water [both deionized (DIW) and process (city)], 55\u00a0wt.% cerelose and 50\u00a0vol.% glycerol. Heating of non-sterile feed tanks by external jacket platecoils and recycling of cooled effluent back to the system inlet permitted system testing with feeds of differing temperatures over the range of 15\u201360\u00b0C. \u201cOnce-through\u201d testing was used only for cerelose to reduce Maillard reactions, which were feared to soil sterilizer internals. Water and glycerol were recycled by setting the \u201cflash\u201d cooler temperature to the desired inlet temperature, permitting testing of process cooler performance at or above inlet feed temperatures. The manner in which readings were taken affected assessment of their variability; readings were observed for a few seconds, then a mental average was taken and evaluated to determine whether the bounce was within reasonable limits. Computer system historical trends, which recorded data every 1\u00a0min also were used to assess variability.\nPressure drops were calculated for each HEX for various media considering the temperature effect on the inlet feed stream density. The vendor\u2019s proprietary software was used which did not account for gaps between spiral face and HEX door and assumed a tighter-than-actual stud spacing. Thus, the design HEX pressure drop was likely overestimated. Re-calculation (data not shown) using published equations for the pressure drop across the spiral HEX [71], which also did not account for the gap impact, resulted in estimates somewhat closer to measured values. The retention loop pressure drop was calculated using a retention loop equivalent length of 1,525\u00a0ft (including elbows and pipe-to-tube adapters) and f values according to Table 9.\nCalculated pressure drops were compared to measured values (data not shown). For the 100\u00a0lpm flowrate, most measured values were about 30\u201340% lower than calculated values with the exception of the heater\u2019s cold side which was 2.5- to 3.5-fold higher. For the 40\u00a0lpm flowrate, observed values were substantially higher (6.5- to 10-fold) than calculated values for the heater\u2019s cold side. These results may indicate difficulty in predicting pressure drops for the lower aspect ratio heater HEX (Table\u00a01), particularly at its higher operating temperatures relative to the other HEXs. Measured pressure drops for various test media were reasonably similar.\nSystem temperatures were calculated for each media type and compared with observed values (Tables\u00a06, 7, 8). Design HR, NTU, and TE were calculated assuming no temperature drop across the retention loop (i.e., retention loop inlet temperature equal to hot side recuperator inlet temperature). Negligible heat loss for the retention loop, HEXs, and piping also was assumed; design values would be higher if these losses were included.\nTable\u00a06System performance using water at 100\u00a0lpm (900\u00a0L retention volume unless noted otherwise)ParameterFeed inlet of 15\u00b0CFeed inlet of 25\u00b0CFeed inlet of 60\u00b0CDesignObserved Moyno\/centrifugalDesignDesignObserved Moyno\/centrifugalObserved Moyno 540\u00a0LRecuperator cold side inlet feed (\u00b0C)15.015\/1525.060.059\/5960Recuperator cold side outlet\/heater hot side inlet (\u00b0C)120.0 (126.0)120\/120122.5 (128.0)131.0 (135.0)131\/132131Heater hot side outlet\/retention loop inlet (\u00b0C)150.0150.1\/150.0150.0150.0150.0\/150.0150.01Retention loop outlet\/recuperator hot side inlet (\u00b0C)150.0149.2\/149.1150.0150.0149.2\/149.1149.02Recuperator hot side outlet\/process cooler hot side inlet (\u00b0C)45.7 (39.6)46\/4353.2 (47.6)79.5 (75.4)80\/7877Process cooler hot side outlet (\u00b0C)35.035.0\/35.035.035.035.0\/35.134.8Process cooler cold side inlet (\u00b0C)6.08.6\/8.26.06.08.3\/N\/AN\/AProcess cooler cold side outlet (\u00b0C)12.0 (8.5)41\/5012.0 (11.6)12.0 (11.4)49\/N\/AN\/ARecuperator heat recovery (HR, %) (Eq. 14)77.8 (82.2)77.7\/77.878.0 (82.4)78.9 (83.3)79.1\/80.278.9Recuperator NTUs (Eq. 12)3.46 (4.57)3.49\/3.683.50 (4.62)3.69 (4.93)3.68\/4.054.06Recuperator thermal efficiency (TE) (Eq. 13)3.50 (4.63)3.60\/3.613.55 (4.68)3.74 (5.00)3.96\/4.273.94(1) Water not tested at 25\u00b0C but design is included for comparison. (2) Process cooler inlet temperature reading taken from building chilled water supply temperature. (3) Design numbers calculated based on intermediate temperatures needed to reach 150\u00b0C at the retention loop inlet assuming that most of the load was undertaken by the heating HEX based on a maximum heating loop temperature of 160\u00b0C. Thus, less than the maximum area of the recuperator was utilized in some cases. (4) Design numbers in bold calculated based on 100% utilization of the recuperator surface area and permitting the hot water loop to operate at values less than 160\u00b0CTable\u00a07System performance (900\u00a0L retention loop volume) using 55\u00a0wt.% cerelose at 65 (25\u00b0C inlet temperature) and 100\u00a0lpm (60\u00b0C inlet temperature)ParameterFeed inlet of 25\u00b0CFeed inlet of 60\u00b0CDesignObserved Moyno\/centrifugalDesignObserved Moyno65\u00a0lpm65\u00a0lpm40\u00a0lpm100\u00a0lpm100\u00a0lpm40\u00a0lpmRecuperator cold side inlet feed (\u00b0C)25.02525\/2660.05760Recuperator cold side outlet\/heater hot side inlet (\u00b0C)105.0 (110.5)120126\/125128.0 (127.0)127130Heater hot side outlet\/retention loop inlet (\u00b0C)150.0150.0150.0\/150.0150.0150.0150.0Retention loop outlet\/recuperator hot side inlet (\u00b0C)150.0148.8148.1\/147.9150.0149.1148.1Recuperator hot side outlet\/process cooler hot side inlet (\u00b0C)77.2 (64.5)5447\/5185.2 (86.3)8277Process cooler hot side outlet (\u00b0C)35.035.735.1\/34.335.035.035.8Process cooler cold side inlet (\u00b0C)6.08.27.5\/NA6.07.7 (est)7.7Process cooler cold side outlet (\u00b0C)12.0 (13.3)4042\/NA12.0 (21.1)61.4 (est)45Recuperator heat recovery (HR, %) (Eq. 14)64.0 (68.4)76.080.8\/79.875.5 (74.4)75.377.8Recuperator NTUs (Eq. 12)1.65 (2.17)3.294.58\/4.132.89 (2.72)2.983.99Recuperator thermal efficiency (TE) (Eq. 13)1.78 (2.17)3.304.57\/4.323.09 (2.91)3.173.86(1) Est estimation by using data for 40\u00a0lpm case as a basis. (2) Cerelose after sterilization was dark brown at 150\u00b0C; lighter brown at 135\u00b0C. (3) At 25\u00b0C, 55\u00a0wt.% cerelose forms a cloudy dispersion in feed tank with entrained air (requiring several minutes to dissipate after agitation is stopped) compared to 55\u00a0wt.% cerelose at 60\u00b0C where solution in feed tank is clear. (4) Design numbers in bold (see note 4 of Table\u00a06) for 65\u00a0lpm interpolated from 60 and 80\u00a0lpm casesTable\u00a08System performance (900\u00a0L retention loop volume) using 50\u00a0vol.% glycerol at 88\u00a0lpm using flowmeter after inlet feed pump (15\u00b0C inlet temperature) and 91.5\u00a0lpm (60\u00b0C inlet temperature)ParameterFeed inlet of 15\u00b0CFeed inlet of 60\u00b0CDesignObserved MoynoDesignObserved Moyno\/centrifugal88\u00a0lpm88\u00a0lpm40\u00a0lpm91.5\u00a0lpm91.5\u00a0lpm40\u00a0lpmRecuperator cold side inlet feed (\u00b0C)15.015156060\/6060\/60Recuperator cold side outlet\/heater hot side inlet (\u00b0C)125.8 (116.0)114117131.0 (128.3)128\/128130\/130Heater hot side outlet\/retention loop inlet (\u00b0C)150.0150.0150.0150.0150.0\/150.0150.0\/150.0Retention loop outlet\/recuperator hot side inlet (\u00b0C)150.0149.1147.9150.0149.1\/148.9147.9\/148.1Recuperator hot side outlet\/process cooler hot side inlet (\u00b0C)45.0 (49.0)524882 (81.7)82\/8076\/76Process cooler hot side outlet (\u00b0C)35.034.934.635.035.0 (est)\/60.262.44\/60.2Process cooler cold side inlet (\u00b0C)6.08.17.666.07.8\/7.897.6\/7.83Process cooler cold side outlet (\u00b0C)12.0 (17.6)38.543.012.0 (13.9)45.2 (est)\/7378\/72Recuperator heat recovery (HR, %) (Eq. 14)82.1 (74.8)73.375.678.9 (75.9)75.6\/75.677.8\/77.8Recuperator NTUs (Eq. 12)4.10 (2.97)2.753.193.47 (3.16)3.16\/3.324.14\/4.11Recuperator thermal effectiveness (TE) (Eq. 13)4.58 (2.97)2.823.303.74 (3.16)3.23\/3.253.92\/3.87(1) Glycerol at 60\u00b0C inlet feed temperature cooled to 60\u00b0C outlet temperature. (2) Est estimation by back calculating heat transfer coefficient for cooling using tower water performance data for similar test media\/conditions. Chilled water flowrate varies, which alters heat coefficient, which changes chilled water outlet temperature. Chilled water flowrate and outlet temperature solved iteratively by balancing heat transferred (U A \u0394Tln) with heat absorbed (QMCp \u0394T). (3) Design numbers in bold (see note 4 of Table\u00a06) for 88 and 91.5\u00a0lpm interpolated from 80 and 100\u00a0lpm cases\nObserved temperature profiles, HRs, NTUs, and TEs generally met or were somewhat lower than design depending upon which design basis was utilized. The primary factor causing under-performance was believed to be lack of allowance for the gap that was likely present even with pliable, full-face gaskets installed owing to unavoidable variations in flatness of the HEX spiral and door faces. The observed hot side recuperator inlet temperature from the non-adiabatic retention loop was lower than the isothermal design assumption and thus raised measured values compared with design HR, NTU, and TE. Viscosity decreases with higher temperature resulted in improved performance.\nSystem draining hold up volume\nThe system\u2019s hold up volume was established by running process water into a completely drained and air-blown system. It was determined when water reached a certain section by opening the adjacent downstream drain valve. Measured hold up volumes agreed with calculated ones within reasonable limits but may have been affected by the ability to fill the system completely at the lower flowrates used to obtain these measurements. Overall, the impact on design residence times of these differences was negligible, however.\nInlet feed stream and outlet distribution stream switching\nPrior to testing all instrument air connections to the switching skid were checked for leaks and proper venting. When switching to the distribution manifold (sterilizer feed to fermenters) from either the sewer or recycle flow paths, transient flow and pressure spikes and their effect on inlet and outlet retention loop temperatures were observed and found to be negligible. When sterilizer distribution was switched from the receiving waste tank to the desired fermenter vessels, pressure spikes also had a negligible effect on temperature. Minimal disturbances were observed for actual water-to-media switch over when the system feed was changed from water to 55\u00a0wt.% cerelose and back again. In all of these instances, an acceptable temperature spike was considered to be less than the variation observed during normal flow operation. Since these spikes were negligible, it was not necessary to flush the system appreciably after a switch to regain steady performance. In addition, sewer to recycle, waste to sewer, and fermenter to waste transitions were not potential sterility risks since these typically occurred after sterilized medium transfer was completed.\nHeat losses\nFor this system, the target retention loop temperature drops (Table\u00a02) were met or exceeded for the test media at residence times of 9\u201322.5\u00a0min. In another study, for a retention loop of 50\u00a0mm (2\u2033) diameter, the temperature drop was negligible for short residence times of 4\u00a0s (hold temperature of 85\u00b0C and room temperature of 20\u00b0C) regardless of whether the retention loop was insulated (0.005\u00b0C) or not insulated (0.04\u00b0C) [55]. For longer residence periods of 40\u00a0s, the temperature drop was 0.04\u00b0C for insulated and 0.35\u00b0C for non-insulated cases. These temperature drops are expected to increase with higher residence times and higher holding temperatures. Extrapolating from these data assuming operation at 85\u00b0C, a change of 0.0583\u00b0C\/min was expected, translating to an expected drop of 0.525\u00b0C for a residence time of 9\u00a0min. Since the actual operating temperature was substantially higher at 150\u00b0C, the observed drop of 1\u00b0C appears consistent with these earlier studies.\nInfrared pictures (Inframetrics model PM250 Thermocam; Flir, Boston, MA, USA) of the retention loop and HEXs were taken to evaluate retention loop insulation effectiveness (particularly the removable jumper end) and HEXs heat losses due to lack of insulation. (Radiant heat loss due to thermal radiation was considered negligible). These pictures showed that the retention loop was adequately insulated. Typical HEX surface temperatures were consistent with the temperature profile of Table\u00a06. Since cold fluids enter the HEX on the periphery and hot fluids enter in the center, there was minimal heat loss to the surroundings [100]. In addition, during water sterilization temperature drops were measured for the recuperator\u2019s hot media side (with the inlet cold side bypassed) and the process side of the process cooler (without chilled\/cooling water flow). These drops were <0.3\u00b0C for the recuperator (aspect ratio of 3.5) and about 2.0\u00b0C for the process cooler (aspect ratio of 2.9). Lower heat losses were expected for thicker, lower aspect ratio HEXs and HEXs with more turns [99]. In addition, full-face gaskets minimized heat transfer rates to the HEX cover faces, which reduced heat loss.\nSystem sensitivity\nVariations in inlet retention loop temperature (\u00b10.05\u00b0C), system flowrate (Moyno feed pump- \u00b10.015\u00a0lpm, centrifugal feed pump- \u00b11.0\u00a0lpm), and back-pressure (\u00b10.05\u00a0kgf\/cm2) generally were negligible across the operating range and for the various test media. As expected, flowrate variations were somewhat greater for the centrifugal than for the positive displacement Moyno feed pump. The relative sensitivities of retention loop inlet and outlet temperatures to changes in system back-pressure were found to be negligible. This behavior was improved from the prior direct steam injection design since steam\/water mixing was more volatile and pressure-sensitive.\nFo and Ro\nFor a hold temperature of 150\u00b0C, Fo magnitudes were acceptable ranging from 3,000\u00a0min at 100\u00a0lpm to 6,500\u20137,000\u00a0min at 40\u00a0lpm. At 135\u00b0C, Fo was substantially lower at 150\u00a0min, but still representing a 50-log reduction for spores with a D value of 3.0\u00a0min. For a hold temperature of 150\u00b0C, Ro magnitudes ranged from 50 to 55\u00a0min at 100\u00a0lpm to 125\u2013130\u00a0min at 40\u00a0lpm; at 135\u00b0C, Ro was substantially lower at 20\u00a0min. Acceptability of these Ro values depends on the degree and impact of media degradation for the specific process.\nReproducibility was high at <1.5% for the same inlet feed pump. Fo and Ro differences were <10% between the two inlet feed pumps using the same medium type, residence time, and sterilization hold temperature; these differences were presumed due to small volumetric flowrate changes, thus slightly altering residence times. Different flowmeters controlled flowrate depending on the inlet pump utilized, but the flowmeter after the recuperator was used for the calculation regardless.\nDimensionless groups and axial dispersion\nFor the retention loop, NRe and NBs were calculated for each type of media at various system flowrates and at a sterilization temperature of 150\u00b0C (Table\u00a09), starting with velocity estimation. Process bulk velocities for the retention loop, as well as HEXs, ranged from 0.62\u20131.33\u00a0m\/s at 100\u00a0lpm and 0.24\u20130.47\u00a0m\/s at 40\u00a0lpm (Table\u00a010). The velocity at 40\u00a0lpm was 40% of the value for 100\u00a0lpm. Over the 50\u00a0wt.% cerelose feed temperature range of 25\u201360\u00b0C and the water\/50\u00a0vol.% glycerol feed temperature range of 15\u201360\u00b0C, there was only a very slight change in calculated velocity (data not shown).\nTable\u00a09Key calculated parameters for the retention loop for an inlet feed temperature of 15\u00b0C, sterilization hold temperature of 150\u00b0C, and process cooler set point of 35\u00b0C (L\/D of 9,318 for 900\u00a0L retention loop volume)ParameterWater (40\u2013100\u00a0lpm)55\u00a0wt.% cerelose (40\u201365\u2013100\u00a0lpm)50\u00a0vol.% glycerol (40\u201388\u2013100\u00a0lpm)NRe84,000\u2013210,00053,000\u201386,000\u2013132,00060,600\u2013133,300\u2013151,500V (m\/s)0.305\u20130.7620.335\u20130.518\u20130.8230.335\u20130.701\u20130.792f (Eq. 11a)0.0222\u20130.02040.0232\u20130.0221\u20130.02130.0231\u20130.0212\u20130.0209Dz\/VD=3.57f0.5 [61], Eq. 100.532\u20130.5100.544\u20130.531\u20130.5210.543\u20130.520\u20130.516Dz (m2\/s)0.00890\u20130.02130.0100\u20130.0151\u20130.02350.00999\u20130.0200\u20130.0224NBs (VL\/Dz)17550\u20131830017150\u201317550\u20131790017150\u201317950\u201318100WaterDz\/VD correlation [61]0.33 (NRe=104)\u20130.2 (NRe=105)Dz (m2\/s)0.00550 (NRe=104)\u20130.00837 (NRe=105)NBs (VL\/Dz)28350 (NRe=104)\u201346600 (NRe=105)(1) Surface roughness assumed to be equivalent to commercial steel (\u03b5=4.57\u00d710\u22125\u00a0m, D=0.0549\u00a0m, \u03b5\/D=0.00083) [78]. (2). Inlet temperature variation from 15 to 60\u00b0C only slightly affects NRe (<1%) based on density change of volumetric inlet flow rate. (3) NBs for 540\u00a0L retention loop volume is about 60% of that for 900\u00a0L retention loop volumeTable\u00a010Calculated bulk velocities of HEXs for various test media (based on 40\u2013100\u00a0lpm process flowrate, sterilization temperature of 150\u00b0C, process cooler set point of 35\u00b0C)HEXProcess side velocity (m\/s)Utility side velocity (m\/s)Water (15\u00b0C inlet feed)55.0\u00a0wt.% cerelose (25\u00b0C inlet feed)50\u00a0vol.% glycerol (15\u00b0C inlet feed)Interconnecting piping0.47\u20131.260.47\u20131.330.47\u20131.32N\/ARecuperator (both sides)0.35\u20130.940.34\u20130.940.34\u20130.98N\/AHeater0.24\u20130.620.25\u20130.660.25\u20130.672.0Retention loop0.31\u20130.770.32\u20130.810.32\u20130.80N\/AProcess and \u201cflash\u201d coolers0.35\u20130.870.35\u20130.890.35\u20130.883.3\nCalculated system velocities were compared to expected settling velocity for a solids-containing media such as 5% cottonseed flour (Pharmamedia, Traders Protein; Memphis, TN, USA). According to the manufacturer, 91% of the particles are <74\u00a0\u03bc. (i.e., pass through a 200 mesh screen) and particle density, \u03c1p, is 1,485\u00a0kg\/m3. The particle settling velocity at 150\u00b0C based on a specific gravity of 1,013\u00a0kg\/m3 for a 50\u00a0g\/L solution at 25\u00b0C (measured using a Fisherbrand, Category No. 11-555G hydrometer) was estimated at 0.016\u00a0m\/s for 200\u00a0\u03bc particles, 0.0054\u00a0m\/s for 100\u00a0\u03bc particles, and 0.0016 for 50\u00a0\u03bc particles. These values were 15- to 100-fold lower (depending on the particle size assumed) than the lowest system velocities. Settling velocities were expected to be even lower as the temperature decreased owing to the higher specific gravity of water.\nNRe ranged from 53,000\u2013150,000 regardless of media type (Table\u00a09). Equation\u00a011a was used to obtain f values ranging from 0.0204 to 0.0232 regardless of media type or flowrate, resulting in Dz values (Eq. 10) ranging from 0.0089 to 0.0235 over the flowrate range from 40 to 100\u00a0lpm. NBs values ranged from 17,150 to 18,300 and were relatively insensitive to either medium type or flowrate (Table\u00a09). Using the experimental correlation and applying it to water [61], NBs values were somewhat higher than the calculated values (Table\u00a09), but reasonable considering the data scatter of the correlation itself. Thus, NBs>>1 for expected operational ranges.\nDips for disturbances were shallower for the retention loop outlet than for the inlet and were observed\u00a0about tR minutes later. For example, at 100\u00a0lpm, an inlet temperature dip to 143\u00b0C (from 150\u00b0C) resulted in an outlet temperature dip to only 145.5\u00b0C. Based on the 1\u00b0C steady-state temperature loss observed, an outlet temperature dip to 142\u00b0C was expected, but not realized most likely due to axial dispersion.\nDirect qualitative examination of axial dispersion was accomplished using step change and spike tests for flowrates of 40 and 100\u00a0lpm. For the step change test, water flowed through the sterilizer then the step change was performed quickly by switching from one feed tank at ambient temperature to a second tank at a higher temperature (60\u00b0C). The system was not heated and was operated without backpressure. For the spike (delta or pulse) change, a temperature spike in the hot water loop was created by quickly opening the steam valve fully and then returning it to its original output setting, taking care to maintain the peak temperature below 150\u00b0C. For both types of tests the non-sterile side of the recuperator was by-passed to avoid heat transfer, as well as reducing the piping length\/volume (relative to that of the retention loop) between the switching point and the retention loop entrance (about 50\u00a0ft excluding spiral loops vs. 1,253\u00a0ft, 65\u00a0L vs. 900\u00a0L). It was difficult operationally to perform step and spike changes with sufficient rapidity owing to this hold up. The dispersion results are shown in Fig.\u00a03a and b. Clearly the degree of dispersion increased at the lower flowrate of 40\u00a0lpm (versus 100\u00a0lpm) for both types of tests. However, the absolute value of the dispersion coefficient suggested by these data appears artificially higher than that predicted from the calculations.\nFig.\u00a03Axial dispersion. a pulse, b step change\nSteam-in-place testing\nThe use of biological indicators (BIs) and thermocouples (TCs) was avoided in steam-in-place (SIP) testing since (1) fittings had to withstand higher pressures up to 10\u00a0kgf\/cm2 and (2) the system was physically large precluding use of TCs with attached wires [although Valprobe (GE Kaye Instruments Inc., North Billericia, MA, USA) wireless TCs were one alternative considered]. TCs commonly have been used in pasteurization applications with a lower hold temperature of 72\u00b0C [82] and correspondingly lower pressure. BIs (in the form of feed inoculated with indicator organisms) were not considered desirable for contact with production equipment for pasteurization [47], although studies are underway to determine if non-indicator organisms can be used [82]. Simulations have been conducted (1) using laboratory or pilot scale test apparatuses with inoculated spore solutions, (2) by tracking an indicator enzyme [69] and\/or (3) utilizing rigorous temperature distribution monitoring on laboratory and production equipment, obtaining spore inactivation kinetic data in parallel, then developing models to estimate lethality [47, 82, 92]. Installation of several temperature and pressure indicators for in process monitoring, along with a functional sterility test, was the preferred approach to demonstrate proper operation.\nOperational testing was performed to test both the steam sterilization-to-water transition and water sterilization modes. Regardless of the sterilization method used, prior steam sterilization of the empty system and subsequent switching back pressure control from the \u201cflash\u201d to process cooler was required. Each method had key points requiring extra care- the initial water introduction for the steam sterilization-to-water transition and placing the empty non-sterile side of the recuperator on-line for the water sterilization. Thus, the time and attention required to execute either method was reasonably equivalent.\nUsing the steam sterilization-to-water transition and a 9\u00a0min residence time, three sterility tests employing soluble medium were successfully completed at 150\u00b0C and one at 135\u00b0C to test the Fo range of 150\u20133,000\u00a0min. One sterility test was successfully completed using the water sterilization method. Changes between non-sterilized and sterilized sterility medium were minimal for total dissolved solids (<\u00b13\u20138%) and conductivity (<3% decrease), confirming minimal medium dilution since all heating was done indirectly. Glucose concentration (measured enzymatically by a YSI analyzer, Yellow Springs, OH, USA) decreased by 15\u201325%, likely reflecting glucose complexing with nitrogen during sterilization and not medium dilution. Further tests for different media (50\u00a0vol.% glycerol, 50\u00a0wt.% cerelose, 5\u00a0wt.% Pharmamedia) are to be done as process development requirements dictate.\nClean-in-place (CIP) testing\nCleaning of HEXs to remove fouling and solid accumulations was required both to maintain heat transfer performance and avoid system sterilization problems. After medium was run through the sterilizer, the system was flushed with water while at sterilization temperature for at least one system volume, then flushing was continued as the system cooled. An alkaline cleaning agent (typically 1.5\u00a0vol.% low heat #3; Oakite Products, Bardonia, NY, USA) was added to dissolve medium components as well as any denatured proteins. Besides alkaline cleaning agents, acid cleaning agents (typically 1.2\u00a0wt.% sulfamic acid) were used to dissolve mineral deposits. Also, a high pressure water stream (1,800\u20132,500\u00a0psi pressure) can be used to clean the spiral HEX channels [19, 71], although this required opening HEXs doors which was costly on a routine basis. The system was heated to 60\u201380\u00b0C, rather than sterilization temperatures of 135\u2013150\u00b0C, which past experience indicated to be sufficient for cleaning. An additional set of tuning constants was implemented for these lower cleaning temperatures (Table\u00a03), but overall control was not required to be as tight as during sterilization.\nContact of the cleaning solution with all internal wetted sterilizer parts at a minimum required velocity of 1.5\u00a0m\/s (2.0\u00a0m\/s recommended) [24] was desirable. Although these high velocities were not achievable in the process side (Table\u00a010), system cleanability was acceptable. There were significant concerns about cleaning HEXs with full-face gaskets installed due to the potential for accumulated residue where the spirals contacted the door and underneath the gasket at the center nozzles since the gasket hole was braced only by the spiral face. System cleanliness was evaluated by (1) examining inlet and outlet conductivity differences and comparing values to those for DIW, (2) analyzing rinse water samples for total solids, filtered solids, color, and ultraviolet absorbance, and (3) visual inspection and swabbing internal surfaces for total organic carbon (TOC), including the HEXs. There were no appreciable differences between source and rinse water in these measurements. Residue accumulation was negligible on the process sides of all spiral HEXs, and swab-testing results were less than 25\u00a0ppm TOC for each location examined.\nCleaning approaches utilized in the dairy industry for continuous pasteurization applications are consistent with the above approach. After processing in certain milk pasteurization applications, systems are rinsed with cold water, flushed with 1.5\u20132\u00a0w\/v.% caustic detergent at 85\u00b0C for 30\u00a0min, cooled, drained, then flushed with water a second time [40]. For other dairy applications, the nature of the soil was primarily protein, butter-fat, and minerals [38]. In this latter case, an acid detergent first dissolved minerals and loosened burned on accumulation, which increased soil solubility in subsequent caustic detergent solution. Instead of draining and rinsing the system between detergent switches, caustic has been added directly to the acid solution to minimize energy costs associated with a second heating of the cleaning solution [38]. However, this short cut can result in soil particles already dissolved in acid or caustic solution potentially re-depositing on system surfaces as the pH changes.\nConclusion\nImprovements implemented for a next generation, pilot-scale continuous sterilization system span the design, fabrication, and testing project phases and have been described. Advantages and disadvantages of various system features were evaluated based on literature analysis from fermentation as well as other related applications. Successful realization of these requirements depended on the adoption of an effective project strategy. The selected system vendor had experience primarily with the food industry since there were few new media sterilizers for manufacturing being constructed and even fewer for pilot plant process development use. Thus, it was critical to devote sufficient time to comprehensively determining system requirements. Development of a detailed sequence of operation as the piping and instrument diagram (P&ID) itself was developed ensured alignment of performance expectations. In addition, selection of a system (as well as HEX) vendor who was located nearby facilitated interim progress examinations prior to delivery.\nThe \u201cworst case\u201d design scenarios were determined carefully, ensuring that they did not create unnecessary additional costs. Agreement on the design assumptions and performance requirements was critical, particularly for calculated quantities. Specifically, the entire system operation needed to be evaluated when developing the HEX performance requirements. Interim temperatures and pressures were estimated based on the system\u2019s flow connections and not simply considering each HEX separately. Since the temperature rise in each HEX stream depended on actual flowrate, design calculations were done using expected flowrates and not solely the maximum flowrates that the HEX can support. Finally, a check of calculations for the various design cases ensured they were internally consistent.\nPerformance testing was devised to quantify actual operation versus design expectations. Intermediate pressure and temperature measurements within the system were compared to design calculations to identify performance issues. Communication of acceptable variability to the control and instrument system designer upfront ensured proper test criteria were met and steady state variations were acceptable. Tests were performed and documented for all operational phases. These system tests were considered critical to effectively characterizing the system\u2019s capabilities prior to placing the equipment in service.","keyphrases":["continuous sterilizer","pilot scale","high temperature short time","spiral heat exchanger","start-up"],"prmu":["P","P","R","R","U"]}
{"id":"Virchows_Arch-4-1-2329733","title":"Validation of tissue microarray technology in squamous cell carcinoma of the esophagus\n","text":"Tissue microarray (TMA) technology has been developed to facilitate high-throughput immunohistochemical and in situ hybridization analysis of tissues by inserting small tissue biopsy cores into a single paraffin block. Several studies have revealed novel prognostic biomarkers in esophageal squamous cell carcinoma (ESCC) by means of TMA technology, although this technique has not yet been validated for these tumors. Because representativeness of the donor tissue cores may be a disadvantage compared to full sections, the aim of this study was to assess if TMA technology provides representative immunohistochemical results in ESCC. A TMA was constructed containing triplicate cores of 108 formalin-fixed, paraffin-embedded squamous cell carcinomas of the esophagus. The agreement in the differentiation grade and immunohistochemical staining scores of CK5\/6, CK14, E-cadherin, Ki-67, and p53 between TMA cores and a subset of 64 randomly selected donor paraffin blocks was determined using kappa statistics. The concurrence between TMA cores and donor blocks was moderate for Ki-67 (\u03ba = 0.42) and E-cadherin (\u03ba = 0.47), substantial for differentiation grade (\u03ba = 0.65) and CK14 (\u03ba = 0.71), and almost perfect for p53 (\u03ba = 0.86) and CK5\/6 (\u03ba = 0.93). TMA technology appears to be a valid method for immunohistochemical analysis of molecular markers in ESCC provided that the staining pattern in the tumor is homogeneous.\nIntroduction\nEsophageal carcinoma is the eighth most common type of cancer in the world [13]. Although the recent rise in incidence of esophageal cancer has predominantly been caused by an increase in adenocarcinomas, the majority of esophageal cancer cases globally are squamous cell carcinomas [13]. For both histological types, radical en bloc esophagectomy with an extensive lymph node dissection offers the best chance for cure, leading to an overall 5-year survival rate of around 30% [1, 20].\nWell-known histopathological factors for prognostication of esophageal cancer include the TNM stage, the number of positive lymph nodes, and the presence of extracapsular lymph node involvement [16, 24, 26, 32]. Recently, there has been a growing interest in the prognostic value of molecular markers in (esophageal) cancer [21]. The expression of such markers is often studied by immunohistochemistry on formalin-fixed, paraffin-embedded tumor slides. Tissue microarray (TMA) technology has been developed to enable high-throughput immunohistochemical analyses [14]. By inserting small (diameter e.g. 0.6\u00a0mm) donor tissue core biopsies into a single recipient paraffin block, this technique allows for rapid analysis of large numbers of tissues under standardized laboratory and evaluation conditions without significantly damaging the patient\u2019s tissue. In addition, TMA technology leads to a significant reduction of the amount of consumables used and time needed for interpretation, increasing cost-effectiveness.\nA potential disadvantage compared to full tissue sections is that the donor cores may not be representative for the whole tumor, particularly in case of heterogeneous tumors and heterogeneously expressed molecular markers. Hence, some validation studies have been performed in various cancers using different kinds of antibodies [2, 4, 6, 7, 9, 12, 23, 35]. Although several studies have revealed novel prognostic biomarkers in esophageal squamous cell cancer (ESCC) by means of TMA technology [38, 39, 41], this technique has not yet been validated for these tumors.\nThe aim of the present study was, therefore, to validate TMA technology in ESCC by assessing the concurrence of immunohistochemical staining scores of established molecular markers with various expression patterns between triplicate 0.6\u00a0mm core biopsies of the TMA and their whole tissue section counterparts.\nMaterials and methods\nTMA construction\nFormalin-fixed, paraffin-embedded tissues from thoracic ESCCs of consecutive patients having undergone esophagolymphadenectomy at the authors\u2019 institute between 1989 and 2006 were retrieved from the archives of the Department of Pathology. Patients who received neoadjuvant therapy were excluded from this study. The study was carried out in accordance with the ethical guidelines of our institution concerning informed consent about the use of patient\u2019s materials after surgical procedures.\nBy an experienced pathologist (FtK), three representative tumor regions were marked on one selected hematoxylin and eosin (H&E)-stained section of each tumor, avoiding areas of necrosis. From these three tumor regions, a tissue cylinder with a diameter of 0.6\u00a0mm was punched out of the corresponding paraffin block (\u2018donor block\u2019) and placed into the TMA paraffin block using a manual tissue arrayer (MTA-I, Beecher Instruments, Sun Prairie, USA), which was guided by the MTABooster\u00ae (Alphelys, Plaisir, France). The distribution and position of the cores was determined in advance with the TMA-designer Software (Alphelys-TMA Designer\u00ae, Version 1.6.8, Plaisir, France). Cores of normal esophageal mucosa, lymph node, kidney, liver, spleen, and prostate were incorporated in the tissue array block as internal controls.\nImmunohistochemistry\nFor each marker, a 4-\u03bcm slide of the TMA and one of every selected donor paraffin block were immunohistochemically stained. Table\u00a01 shows the details of all antibodies, dilutions, incubation times and antigen retrieval methods applied in this study.\nTable\u00a01Specification of antibodies used and details of tissue processingPrimary antibodyStaining patternSourceaClone and codeAntigen retrievalDilutionIncubation time (min\/room temperature)DetectionbPositive controlProcedureCK5\/6CytoplasmicChemiconD5\/16 B4EDTA pH\u00a09.01:3,00060Strept ABCBreastAutostainerCK14CytoplasmicNeomarkersLL002EDTA pH\u00a09.01:40060PowervisionBreastAutostainerE-cadherinMembranousZymed4A2C7Citrate autoclave pH\u00a06.01:20060PowervisionBreastAutostainerMIB-1 (Ki-67)NuclearDakoM7240Citrate pH\u00a06.01:10060Strept ABCTonsilAutostainerp53NuclearBiogenexBP53-12Citrate pH\u00a06.01:20060Strept ABCSerous adenocarcinoma of the endometriumAutostaineraBiogenex, San Ramon, CA, USA; Chemicon, Chemicon International, Temecula, CA, USA; Dako, DakoCytomation, Glostrup, Denmark; Neomarkers, Fremont, USA; Zymed, Zymed Laboratories, San Francisco, CA, USA.bStrept ABC is biotinylated horse\u2013antimouse Vector BA-2000, diluted 1:500 in PBS, followed by streptavidin\u2013biotin complex, diluted 1:1,000. Powervision ready to use (Poly-HRP-antiMs\/Rb\/RtIgG biotin-free; ImmunoVision Technologies, Norwell, CA, USA).\nFor all stainings, sections were deparaffinized in xylene for 10\u00a0min followed by dehydration through graded alcohols. Endogenous peroxidase activity was blocked for 15\u00a0min in a buffer solution of pH\u00a05.8 (containing 8.32\u00a0g citric acid, 21.52\u00a0g disodium hydrogen phosphate, 2\u00a0g sodium azide in 1\u00a0l of water) with hydrogen peroxide (0.3%). After antigen retrieval for 20\u00a0min, a cooling down period of 30\u00a0min was followed by incubation with the primary antibody. Depending on the antibody used, slides were incubated with the secondary antibody followed by the streptavidin\u2013biotin complex or slides were directly incubated with Powervision (details of both products shown in the legend of Table\u00a01). Then, the peroxidase reactivity was developed by 3,3\u2032-diaminobenzidine for 10\u00a0min and slides were counterstained with Mayer\u2019s hematoxylin. In between steps, slides were washed with phosphate-buffered saline (pH\u00a07.4).\nImmunohistochemical scoring\nBy two observers (FtK and JB) conjointly, the degree of differentiation and the percentage of immunohistochemically stained tumor cells were determined in all TMA cores and in the full sections of the selected donor blocks. Histologic grade was scored as well-differentiated (G1), moderately differentiated (G2), or poorly differentiated (G3) [37]. Staining of p53 and Ki-67 were marked as negative (<10% of tumor nuclei stained), weakly positive (10\u201350%), or strongly positive (\u226550%) [10, 40]. Cytokeratin (CK)5\/6 and CK14 staining were scored as negative (<10% of tumor cell cytoplasms stained), weakly positive (10\u201380%), or strongly positive (\u226580%). E-cadherin expression was regarded negative when <50% of tumor cell membranes stained and positive when \u226550% stained [29, 30].\nCores were considered lost if <10% of cells contained tumor (\u2018sampling error\u2019) or when <10% of tissue was present (\u2018absent core\u2019). Cases were excluded if two out of three cores were lost. When the scores between the cores of a particular case differed, the most frequent score determined the overall score. In case of three different scores in one case, the middle score was chosen. When only two cores were available with both a different score, the case was excluded from further analysis [11].\nStatistical analysis\nStatistical analyses were performed using the SPSS software for Windows (Version 12.0, SPSS, Chicago, IL, USA). Sixty-four donor blocks (60% of the tumors incorporated in the TMA) were randomly chosen by means of a random selection function of SPSS.\nTo determine the chance-corrected agreement between the immunohistochemical staining scores of TMA cores and large sections, the Cohen\u2019s weighted kappa statistic was calculated. Chance-corrected agreement was considered poor if \u03ba\u2009<\u20090.00, slight if 0\u2009<\u2009\u03ba\u2009<\u20090.20, fair if 0.21\u2009<\u2009\u03ba\u2009<\u20090.40, moderate if 0.41\u2009<\u2009\u03ba\u2009<\u20090.60, substantial if 0.61\u2009<\u2009\u03ba\u2009<\u20090.80, and almost perfect if 0.81\u2009<\u2009\u03ba\u2009<\u20091.00 [17]. The overall agreement was defined as the percentage of correct agreement between the TMA and the donor blocks from the total number of cases [15].\nResults\nOf the 324 (3\u2009\u00d7\u2009108) tumor tissue cores that were transferred into the TMA paraffin block, a median of 295 (91%) was available for immunohistochemical scoring on the 6 TMA slides used in this study (Table\u00a02). Of the 64 randomly selected cases, a median of 176 (92%) of 192 cores (3\u2009\u00d7\u200964) was evaluable on the TMA slides.\nTable\u00a02Overview of the amount of cores that were evaluable, absent or contained too little tumor in all 108 cases and in the 64 randomly selected cases on the TMA slides\u00a0H&ECK5\/6CK14E-cadherinKi-67p53MedianTotal TMA cases (n\u2009=\u2009108)\u00a0No. of evaluable cores293309294306293295295\u00a0Percentage90959194909191\u00a0No. of absent cores207229222221\u00a0Percentage6273776\u00a0No. of cores without tumor11889979\u00a0Percentage4333323Randomly selected TMA cases (n\u2009=\u200964)\u00a0No. of evaluable cores176187176185176176176\u00a0Percentage92979296929292\u00a0No. of absent cores133134131313\u00a0Percentage7272777\u00a0No. of cores without tumor3233333\u00a0Percentage2122222H&E: hematoxylin and eosin\nOn the H&E-stained TMA slide, 49 (76%) of the 64 randomly chosen cases were represented by 3 cores; 14 (22%) by 2 cores. One (1.6%) case was excluded from further analysis because only a single core was available. The agreement in the scores for the grade of differentiation between the TMA cores and the full sections is shown in Table\u00a03. The weighted kappa score was 0.65.\nTable\u00a03Agreement in the degree of differentiation between TMA cores and full sections\u00a0Full sectionG1G2G3Total\u03baTMAG12305\u00a0G2221225\u00a0G3072633\u00a0Total43128630.65G1: well-differentiated, G2: moderately differentiated, G3: poorly differentiated\nFifty-nine (92%) of the 64 randomly selected cases stained for CK5\/6 were represented by 3 cores (Fig.\u00a01); the 5 remaining cases by 2 cores. The immunohistochemical scores of the TMA and the donor blocks are shown in Table\u00a04. Overall agreement in CK5\/6 scores between the TMA and the donor blocks was 98% with a kappa of 0.93.\nFig.\u00a01Example of strong CK5\/6 staining in TMA cores and the corresponding full section. a Three TMA cores representing one tumor; magnification \u00d720. b Enlargement of the middle TMA core depicted in a; magnification \u00d7100. c Part of the slide of the donor block of the same tumor; magnification \u00d7100Table\u00a04Agreement in immunohistochemical scores between TMA cores and full slides stained for CK5\/6 and CK14\u00a0Full sections<10%10\u201380%\u226580%Total\u03baTMAsCK5\/6\u00a0<10%1102\u00a010\u201380%0404\u00a0\u226580%005858\u00a0Total1558640.93CK14\u00a0<10%5218\u00a010\u201380%011213\u00a0\u226580%043438\u00a0Total51737590.71\nFor CK14, two cases were excluded because only one tumor core was left and three cases were also excluded because the two available cores had discrepant immunohistochemical scores. Fifty (85%) of 59 cases had complete agreement (Table\u00a04). Four cases were scored one class higher on TMA when compared with the full sections. Conversely, five other cases were classified lower on TMA with one case two classes lower. Kappa score was 0.71.\nRegarding E-cadherin staining, three assessable cores were present in 89% of the cases; two cores in 11%. Overall agreement in E-cadherin staining scores was accomplished in 72% of cases (Table\u00a05). In one case, a higher score was found on the TMA compared to the full section. In 17 cases, the expression of E-cadherin was scored lower on TMA than on the full sections. The observed kappa was 0.47.\nTable\u00a05Agreement in immunohistochemical scores between TMA cores and full slides stained for E-cadherin\u00a0\u00a0Full sectionsE-cadherin<50%\u226550%Total\u03baTMA\u00a0<50%221739\u00a0\u226550%12425\u00a0Total2341640.47\nThree-core analysis of Ki-67 staining could be performed in 78% of selected cases and two-core analysis in 19%. Two cases were represented by a single tumor core and were, therefore, excluded from further analysis. Ki-67 staining was scored as \u201cmoderate\u201d on both TMA and full sections in 42 (69%) of 61 selected cases. In 79% of cases, the Ki-67 scores of the TMA were similar to that of the full sections. Thirteen cases were discordant (Table\u00a06); kappa was 0.42.\nTable\u00a06Agreement in immunohistochemical scores between TMA cores and full slides stained for Ki-67 and p53\u00a0Full sections<10%10\u201350%\u226550%Total\u03baTMAsKi-67\u00a0<10%2103\u00a010\u201350%342348\u00a0\u226550%06410\u00a0Total5497610.42p53\u00a0<10%193022\u00a010\u201350%0123\u00a0\u226550%033538Total19737630.86\nWith regard to p53 staining, two cores were present in 12 (19%) cases and 3 cores were available in 51 (80%) cases. One case was excluded as it was represented by only one TMA core. Complete agreement was achieved in 87% of the selected tumors (Table\u00a06). In the eight nonconcordant cases, the difference was one class, resulting in a kappa of 0.86.\nDiscussion\nAfter its introduction in 1998, TMA technology has been applied in the immunohistochemical analysis of various malignancies, including squamous cell carcinomas and adenocarcinomas of the esophagus [3, 5, 18, 19, 25, 41]. Although it seems a very attractive method for high-throughput analysis of hundreds of tissues simultaneously, it may have limitations as the evaluation of the marker expression is reduced from full-section analysis to a few tissue cores of only 0.6\u00a0mm in diameter, especially for proteins that are heterogeneously expressed or that are cell cycle-dependent [36]. It is, therefore, essential to assess in each type of cancer individually and for every molecular marker whether TMA technology is feasible and valid [8, 33]. To our knowledge, this has not been done in esophageal cancer.\nIn our TMA containing triplicate cores of 108 ESCCs, a median of 9% of cores was uninformative (6% lost during tissue processing and 3% containing too little tumor), which is comparable to the results reported in other studies [7, 8, 27]. Improper selection of representative tumor areas on the donor block\u2019s H&E slide by the pathologist or incorrect punching of these representative areas out of the donor block can cause tissue cores that contain too little tumor. Possible causes of absent cores are the size and fragility of the tumor tissue used and the aggressiveness of tissue processing applied [31, 35, 42].\nMoreover, the number of available cores on the TMA slide depends on the level at which the TMA paraffin block has been sectioned. The slides stained for H&E, CK14, Ki-67, and p53 were one of the first slides that were cut from our TMA block, whereas sections stained for CK5\/6 and E-cadherin were taken slightly deeper. On these latter sections, a lower number of absent cores was observed (Table\u00a02), showing that not all cores were placed at the exact same level in the TMA block during TMA construction, mainly due to dissimilar thicknesses of the donor paraffin blocks that were used to construct the TMA [34].\nThe agreement in immunohistochemical results of the markers between our TMA and the full sections varied from moderate to almost perfect (\u03ba\u2009=\u20090.42 to 0.93), which is consistent with the results reported in other TMA validation studies [4, 7, 8, 31, 35]. The observed variation in agreement could be due to tumor heterogeneity, topographical variation in the expression pattern of the molecular marker, or to the scoring criteria used [31].\nRegarding tumor heterogeneity, the optimal amount of tissue cores incorporated in the TMA has been a matter of debate. Several validation studies have shown that three cores are highly representative for the full section [6, 11, 12, 28]. The addition of a fourth core did not add to the percentage of agreement in a colorectal cancer TMA [12]. Moreover, the more cores punched per case, the fewer cases can be placed into the TMA reducing throughput. Adding a fourth core may nevertheless be worthwhile in tissues prone to uninformative cores due to small lesions such as dysplasias or carcinomas in situ [42]. In our TMA, the amount of uninformative cores was low (5\u201310%), probably because ESCCs have a large diameter, thereby increasing the chance of obtaining a core containing tumor tissue. Taken together, we consider it justified to utilize three biopsy cores in ESCCs. Nonetheless, using such a low amount of cores requires careful selection of the tumor regions by an experienced pathologist to deal with the heterogeneity of the tumor in the TMA [33].\nThe agreement between TMA and full sections was substantial to almost perfect for CK5\/6 and CK14. Because 91% of cases have shown a very strong expression of CK5\/6 and only 1 of 64 cases showed negative staining, this molecular marker does not subdivide ESCCs and consequently will not be a prognostic marker for this malignancy. CK14 was more evenly distributed over the three scoring groups, but because one case was scored two classes lower on TMA when compared to the full section, kappa was lower when compared to CK5\/6.\nThe relatively moderate concordance in case of Ki-67 may be explained by the fact that almost 80% of cases were situated in one category (staining of 10\u201350% of tumor cells) with 13 discordant cases deviating from this category. E-cadherin also had a moderate concordance, mainly because the relatively faint staining intensity of this molecular maker made its assessment in our TMA very difficult (Fig.\u00a02).\nFig.\u00a02Representative example of E-cadherin staining in TMA cores and the corresponding full section. a Three TMA cores representing one tumor; magnification \u00d720. b Enlargement of the right TMA core depicted in a; magnification \u00d7100. c Part of the slide of the donor block of the same tumor; magnification \u00d7100\nTMA technology was also found to be valid for determining the histologic grade of differentiation in ESCC. Complete agreement between TMA and full sections occurred in 78% (49 out of 63; \u03ba\u2009=\u20090.65) of selected cases, which is high when compared to the 40% agreement achieved in a TMA of bladder cancer [22]. Due to its homogeneous staining pattern, p53 showed excellent concordance (\u03ba\u2009=\u20090.86) in our microarray (Fig.\u00a03).\nFig.\u00a03Representative example of p53 staining in TMA cores and the corresponding full section. a Three TMA cores representing one tumor; magnification \u00d720. b Enlargement of the left TMA core depicted in a; magnification \u00d7100. c Part of the slide of the donor block of the same tumor; magnification \u00d7100\nThe concurrence between the TMA and the full sections is affected by the cut-off values of the immunohistochemical scoring system of the stainings as well [7, 31]. The application of a two-class scoring system in an endometrial cancer TMA improved \u03ba to 1.0 compared to 0.81 with a three-class system [7]. In our study, the two-class scoring system did not substantially affect the kappa (data not shown). Because the E-cadherin expression had a very low intensity in our ESCCs, we have chosen to apply a two-class system. In addition, the cut-off values indicating a strong immunohistochemical expression were set higher in the cytokeratins (80%) than in the other molecular markers (cut-off value 50%) because otherwise practically all tumors would be designated having a strong expression of cytokeratins.\nNow that our esophageal cancer TMA has been validated, it will be used to correlate the expression of various molecular pathways with clinicopathologic data, aiming at detecting markers of prognostic significance and molecular targets for new therapies. Because the agreement between TMA slides and full sections depended on the molecular marker stained for, it should be considered to assess the expression pattern of a marker on a full section first, before staining a TMA slide. When a focal or heterogeneous expression pattern is noticed, it might be more valuable to assess marker expression by means of full sections instead of TMA. On the other hand, when a marker shows a homogeneously diffuse expression pattern, staining a TMA slide does allow for high-throughput screening of tumors. When a prognostic molecular marker has been identified by means of TMA technology, it is recommended to verify the results by full-section analysis.\nIn conclusion, this study has demonstrated TMA technology to be a valid method for immunohistochemical analysis in ESCC with agreement levels for well-known molecular markers with different staining potential between TMA and full sections ranging from moderate to almost perfect.","keyphrases":["squamous cell carcinoma","validation studies","biological markers","esophageal neoplasms","protein microarray analysis"],"prmu":["P","P","M","M","R"]}
{"id":"Pediatr_Nephrol-3-1-1766478","title":"Renal replacement therapy for acute renal failure in children: European Guidelines\n","text":"Acute renal failure (ARF) is uncommon in childhood and there is little consensus on the appropriate treatment modality when renal replacement therapy is required. Members of the European Pediatric Peritoneal Dialysis Working Group have produced the following guidelines in collaboration with nursing staff. Good practice requires early discussion of patients with ARF with pediatric nephrology staff and transfer for investigation and management in those with rapidly deteriorating renal function. Patients with ARF as part of multi-organ failure will be cared for in pediatric intensive care units where there should be access to pediatric nephrology support and advice. The choice of dialysis therapy will therefore depend upon the clinical circumstances, location of the patient, and expertise available. Peritoneal dialysis has generally been the preferred therapy for isolated failure of the kidney and is universally available. Intermittent hemodialysis is frequently used in renal units where nursing expertise is available and hemofiltration is increasingly employed in the intensive care situation. Practical guidelines for and the complications of each therapy are discussed.\nIntroduction\nAcute renal failure (ARF) is uncommon in childhood, but its incidence may be increasing and modalities of treatment changing with an increasing number of children being treated in the intensive care unit (ICU) with multi-organ failure. Traditionally children with ARF with renal involvement were only treated with peritoneal dialysis, but extracorporeal techniques are being increasingly used in ICUs.\nMembers of the European Pediatric Dialysis Working Group reviewed all modalities of renal replacement therapy for ARF in children and developed the following guidelines in collaboration with nursing staff during three meetings and extensive e-mail discussion. There are no randomized trials of renal replacement treatment in children with ARF. The guidelines are based upon published reports and consensus opinion to emphasize good practice.\nARF is recognized when renal excretory function declines rapidly. Rising values of plasma urea and creatinine are usually accompanied by oliguria (<1\u00a0ml\/kg per hour), but occasionally patients may be polyuric. The cause of ARF may be pre-renal, intrinsic, or post-renal (obstructive) problems, and causes differ between neonates and older children [1, 2, 3].\nThe incidence of ARF in children is hard to define, as often renal insufficiency in the newborn and on ICUs is conservatively managed by ICU staff. Outside the neonatal period, ARF is an uncommon condition accounting for 8 referrals per million population per year to one regional pediatric nephrology unit in the United Kingdom [4].\nARF may occur as isolated failure of the kidneys alone, with other organ systems functioning normally, or in association with multiple organ failure. The mortality of the latter group is considerably higher, especially with the growth in pediatric intensive care. For example, the mortality in neonates and infants is 51% after cardiac surgery for congenital heart defects [4], but only 3%\u20136% for children with intrinsic renal disease such as hemolytic uremic syndrome (HUS) in developed countries [5, 6].\nThe case mix in different units treating ARF, and hence mortality and morbidity rates, will therefore vary according to local clinical activity and resources [7, 8]. Many pediatric renal units will be close to pediatric ICUs (PICUs) in hospitals that may offer cardiac surgery, liver transplantation, and specialist treatment for metabolic disorders, oncology patients, etc. [6]. Other renal units may be in hospitals that do not have a PICU on site and conversely there may be hospitals offering pediatric intensive care with no specialist pediatric nephrology service.\nRecommendations\nAll children with ARF require discussion with a pediatric nephrologist. Early transfer for investigation and management is essential in those with rapidly deteriorating renal function or in those with hemodynamic or biochemical disturbances (good practice) [9].\nAll children with ARF as part of multi-organ failure require transfer to a designated regional pediatric ICU where there should be access to pediatric nephrology advice and support (good practice).\nRationale\nSince there are few comprehensive regional pediatric nephrology centers the distances that families may have to travel can be considerable. Children with acute renal impairment may be managed in local hospitals, but it is essential that early referral is made, especially if children have evidence of rapidly deteriorating renal function and require an urgent histological diagnosis to determine if immunosuppressive therapy or other treatment is required. Indications for referral include oligoanuria, especially if associated with fluid overload, hypertension, hyperkalemia, hyponatremia, acidosis, or the need for blood transfusion. Dialysis is often accompanied by early nutritional support and pediatric nephrology units should be equipped to provide the necessary medical and nursing expertise, combined with dietetic and psychosocial support. The latter support is also important if the child is managed conservatively.\nNeonates and premature infants with ARF require transfer to a tertiary neonatal unit with pediatric nephrology team expertise. Patients with ARF and multi-organ failure require prompt transfer to a designated regional PICU.\nThe choice of dialysis therapy for ARF depends upon the clinical circumstances, patient location, and expertise available. Peritoneal dialysis (PD) has generally been considered the preferred therapy if there is isolated failure of the kidneys, such as HUS. It is regarded as a simpler technique that is universally available. However, hemofiltration (HF) and hemodiafiltration (HDF) are increasing in popularity in PICUs where the facilities to perform hemodialysis (HD) may not be available. HD may be the preferred mode of treatment in more-stable patients with adequate vascular access treated on renal units where specialist nurses are available.\nAlthough extracorporeal techniques such as continuous venovenous hemofiltration (CVVH) or continuous venovenous hemodiafiltration (CVVHDF) are used quite frequently in adult ICUs, there is still limited expertise in many PICUs. Such techniques are very dependent on technology and are more costly than PD [10]. They are also dependent upon the availability of appropriate nursing expertise [11]. Such expertise can be developed and maintained in units remote from the pediatric nephrology center by an outreach service using a renal critical care nurse educator [12].\nRecommendation\nThere is no evidence for the optimum level of renal function for starting renal replacement therapy nor for the optimum dialysis modality. Advantages and disadvantages are listed in Table\u00a01. Consideration should be given to establishing national and international databases to collect these data along with patient outcomes [6, 13].Table\u00a01 Advantages and disadvantages of various modalities of renal replacement therapy for acute renal failure (CVVH continuous venovenous hemofiltration, CVVHDF continuous venovenous hemodiafiltration)TypeComplexityUse in hypotensionEfficiencyVolume controlAnticoagulationPeritoneal dialysisLowYesModerateModerateNoIntermittent hemodialysisModerateNoHighModerateYesCVVHModerateYesModerateGoodYesCVVHDFHighYesHighGoodYes\nChoice of therapy\nAcute PD\nThe main advantage of PD is that it is continuous therapy that requires neither anticoagulation nor vascular access, and the technique can be used in hemodynamically unstable patients [14]. Acute PD can be performed in units with no HD expertise and is effective in children of all ages, including neonates [15, 16, 17, 18]. PD has been used in treating acute pancreatitis, tumor lysis syndrome, intoxications, metabolic diseases, and other pathological conditions in children [19, 20, 21, 22]. The choice of PD as therapy has always to be individualized, balancing advantages against disadvantages.\nLimitations in the use of PD\nInborn areas of metabolism in the newborn period lead to acute accumulation of neurotoxic metabolites that can be better removed using techniques such as CVVHDF [23, 24]. The latter technique requires good vascular access, which can still be a major problem in small children [25]. Newborns with respiratory diseases, even if on ventilatory treatment, can be treated with PD provided that the fill and exchange volumes are adapted to the clinical situation. However, caution is necessary in neonates with necrotizing enterocolitis and older children with suspected bowel perforation [26].\nPreparation for PD\nDialysis is only possible if the access provides free flow in and out of the abdomen. The choice is between catheters inserted at the bedside under sedation or the placement of a chronic PD catheter by a pediatric surgeon in the operating theater, or exceptionally at the bedside in the ICU.\nThe rigid Trocath catheter with a stylet has largely disappeared and surgically placed Tenckhoff catheters are reported to have fewer complications [27, 28, 29]. However, small catheters for percutaneous placement using a Seldinger technique are invaluable in providing acute PD rapidly, especially in the neonatal PICU [13, 30].\nBlockage by the omentum is always a risk with PD catheters. If the catheter is to be placed surgically then consideration should be given to partial omentectomy [31].\nIn patients who are having a PD catheter inserted under general anesthetic a cephalosporin antibiotic (20\u00a0mg\/kg) should be given as a single intravenous dose up to 1\u00a0h prior to implantation of the catheter [32]. Any subsequent accidental contamination should result in the use of prophylactic antibiotics, e.g., cefuroxime 125\u00a0mg\/l in the dialysate for 48\u00a0h.\nFor catheters that are inserted percutaneously, prophylactic antibiotics, e.g., cefuroxime 125\u00a0mg\/l, should be added to the dialysis fluid unless the patient is on systemic treatment.\nHeparin, 500\u00a0units\/l, should be prescribed to prevent catheter blockage with fibrin. This is generally maintained for the first 48\u00a0h, and longer if the PD fluid remains slightly bloodstained [33, 34].\nPD prescription\nThis needs to be individualized according to patient size and condition. Automated PD machines are the preferred method for delivering the individualized dialysis prescription and accurately measuring ultrafiltration [30]. Such machines are now available that can deliver dialysis volumes accurately down to 60\u00a0ml with 10-ml increments. Although such machines now have improved accuracy of ultrafiltration measurements, the dead space of the tubing can reduce dialysis efficiency. A manual PD set can be used, using burettes that can accurately measure inflow and outflow, with the PD fluid warmed appropriately [35]. With manual sets, attempts should be made to maintain a closed drainage system, which can help reduce the frequency of peritoneal contamination [36]. Such manual PD sets are commercially available for neonatal patients.\nChoice of dialysis solution\nThe choice of dialysis solution will depend upon the weight, blood pressure, and hydration status of the child, bearing in mind the need to create nutritional space as part of the management strategy [37].\nThe general principle is to commence with the lowest concentration of glucose solution possible (1.36%), with stepwise increments. Care is needed if 3.86% glucose solution is required as (1) rapid ultrafiltration can occur (especially in infants) and (2) hyperglycemia may develop (especially in septic and multi-organ failure patients) leading to hyperosmolarity and loss of effective ultrafiltration.\nIcodextrine solutions need a longer dwell time to obtain significant ultrafiltration and so are rarely indicated in ARF. Lactate-containing dialysis solutions are likely to be replaced by bicarbonate solutions, which are being evaluated in chronic PD. The routine use of bicarbonate solutions should be considered in neonates or in patients with reduced lactate metabolism or with lactic acidosis [38, 39].\nPractical points\nPatients should be connected and automated PD or manual cycles started immediately after catheter implantation. Heparin (500\u00a0units\/l) should be added to the dialysis fluid to prevent fibrin deposition and to improve peritoneal solute permeability [33, 34], but it can be absorbed and care is needed in patients with coagulation disorders.\nDialysis fill volumes of 10\u201320\u00a0ml\/kg (300\u2013600\u00a0ml\/m2) should be used initially, depending on the body size and cycle in and out, until the dialysate becomes clear.\nA PD program with 1-h dwells should be used during the first 24\u00a0h. Shorter cycles can be considered initially if hyperkalemia needs urgent treatment.\nThe program should be adjusted with increasing dwell times and cycle fill volume (if no leakage problems) until the desired fill volume (800\u20131,200\u00a0ml\/m2) is achieved, with adequate ultrafiltration and biochemical control [40].\nHigh intraperitoneal pressure (IPP) can be a problem in the first 2\u20133\u00a0days after surgical catheter insertion. The measurement of IPP may limit the risk of leakage when the fill volumes are being increased and allow optimized pain management, but is not yet in routine use [41].\nInflow\/outflow pain on PD usually diminishes with time. Tidal dialysis is an alternative [42] and bicarbonate dialysis should be considered [43].\nThe amount of ultrafiltration that is prescribed will partly depend upon the volume of oral, nasogastric, or total parenteral nutrition that is required, combined with fluid for drugs. Ultrafiltration may not be enough without the use of 2.27% or 3.86% glucose solutions.\nThe clinical, biochemical, and nutritional status of the patient should be assessed regularly in conjunction with an experienced renal dietitian [44]. Optimal nutrition is necessary to avoid a catabolic state and associated production of blood urea nitrogen and uremic products.\nRationale\nPatients with ARF need constant assessment while on PD, and adequacy should be judged in terms of clinical status, ultrafiltration achieved, and biochemical parameters, particularly urea, creatinine, and bicarbonate levels [40]. Although a link between the dialysis dose and the outcome of adult patients in ARF has been established [45], there are no guidelines as to what constitutes adequate PD in a child with ARF. The aim is to deliver maximum clearance to compensate for the catabolic stress.\nComplications of acute PD\nLeakages can be a difficult problem and are mostly due to a leakage around the catheter. The incidence can be reduced by proper surgical technique when using a Tenkhoff catheter [46] or resuturing around a percutaneous catheter. Fibrin glue injected into the catheter tunnel is a technique under evaluation [47].\nPoor drainage due to mechanical blockage or catheter migration is all too common. Flushing the catheter and preventing fibrin accumulation by increasing the heparin dosage and\/or urokinase is suggested initially [48]. A plain abdominal X-ray is rarely justified, as repeated poor drainage will require catheter relocation. If available, a laparoscopic technique may be used to correct poor drainage or replace the malfunctioning catheter [49].\nHernias can be a problem in neonates and infants, particularly males. They do not usually require interruption of PD and can be repaired electively by laparoscopic or direct measures when the child\u2019s clinical condition has improved or stabilized.\nPeritonitis remains a constant threat, especially if there has been a lot of manipulation of the catheter. The standard features of cloudy PD fluid require urgent attention [50].\nContinuous extracorporeal techniques\nContinuous arteriovenous hemofiltration (CAVH) has largely been replaced by pumped CVVH and CVVHDF, particularly in ICUs [51]. Such continuous renal replacement therapies (CRRT) have expanded the possible role of blood purification in the management of critically ill patients. However, there is a lack of randomized trials in patients with sepsis, and a recent analysis failed to show a benefit for hemofiltration [52]. Studies in adult ICU patients have shown a lower mortality in patients treated with CRRT compared with intermittent HD. However, a recent meta-analysis of studies before 1996 concluded that the evidence was insufficient to draw strong conclusions regarding the mode of renal replacement therapy for ARF in the critically ill [53]. A recent randomized trial in adult ICU patients showed a significant survival advantage when the intensity of ultrafiltration was increased [54].\nPractical guidelines for prescription\nSince the concentration of solutes in the filtrate is the same as in the plasma, biochemistry is controlled by removing large volumes of filtrate and replacing it with electrolyte-containing fluid (HF replacement fluid). As most solutes are distributed within the extracellular and intracellular fluid compartments (total body water), the exchange volume of filtration necessary to control biochemistry relates to total body water. Clinical experience has shown that a turnover of approximately 50% of body weight in 24\u00a0h is usually adequate for CVVH.\nThe extracorporeal circuit requires good central venous access, usually via a dual-lumen catheter, to allow the high blood flows necessary to prevent clotting in the hemofilter. Suggested catheter sizes in French gauge (FG) are:Patient size (kg)Vascular access2.5\u2013106.5-FG dual-lumen (10\u00a0cm)10\u2013208-FG dual-lumen (15\u00a0cm)>2010.8-FG or larger dual-lumen (20\u00a0cm)\nFor neonates a 5-FG dual-lumen catheter may be adequate, and access can be obtained via the umbilical vein [55]. A single-lumen catheter using a \u201csingle needle\u201d for CVVHD in very low birth weight infants has also been described [56], but this method may be compromised by high recirculation rates with most available systems. However, the smaller the access the greater the problems [57]. It is possible to consider placing two small single-lumen catheters in different central veins.\nA low blood flow rate, high hematocrit, and high plasma protein concentration will limit the rate at which filtration can occur and solutes (particularly of higher molecular weight) are removed. For a given blood flow rate, pre-dilution results in higher clearance of solutes than does post-dilution [58], but at the expense of greater use of replacement fluid (approximately 20%\u201350% more). Pre-dilution has the potential for extending filter life.\nAs with HD, the blood volume in the extracorporeal circuit should be less than 10% of the patient\u2019s circulatory volume. Blood flows of 6\u20139\u00a0ml\/kg per min or 8% of circulating blood volume prevents excessive hemoconcentration in the filter. Automated machines with appropriate accuracy for children are recommended for delivering the CRRT prescription safely [59], and have replaced pump-assisted hemofiltration using volumetric pumps [60].\nTo achieve a 50% exchange of total body water in 24\u00a0h, an appropriate filter should be selected with a surface area of no more than the surface area of the patient. Suggested maximum filtration rates are:Patient size (kg)Maximum filtration rate (ml\/h)<8.52508.5\u201320500>202,000Under post-dilution conditions, the filtration rate should never exceed one-third of the blood flow.\nSeveral filter materials are now available. Synthetic membranes have replaced cellulose acetate, as they are more biocompatible, causing less complement reaction and anticoagulation needs. The synthetic polysuphone membranes are also thought to aid convective clearance of solutes through solute drag [61].\nA variety of replacement fluids are available such as lactate, bicarbonate, and buffer-free solutions. Bicarbonate or buffer-free solutions should be used in young infants and those intolerant of lactate. If a commercially available bicarbonate solution were freely available, then this would be the solution of choice. Careful monitoring of electrolytes, glucose, and phosphate is essential, as the constituents vary between the solutions.\nAnticoagulation\nThe goals of anticoagulation are to prevent clotting of the circuit and maintain adequate clearances with minimal risk to the patient. Heparin is the standard anticoagulant in Europe, but the choice of dosage will depend upon the patient\u2019s coagulation status, adequacy of blood flow, and blood viscosity. In most patients, heparin should be administered as an initial bolus (maximum 50\u00a0units\/kg) at the time of connection to the extracorporeal circuit, followed by a continuous infusion of 0\u201330\u00a0units\/kg per hour. The activated clotting time (ACT) or whole blood activated partial thromboplastin time (aPPT) are usually used to monitor treatment. The optimal ACT during hemofiltration is 120\u2013180\u00a0s. The aPPT should be between 1.2 and 1.5 times the respective baseline value. Some patients can be treated without heparin in the circuit [6].\nIn those patients who are severely thrombocytopenic or where there is suspected heparin-induced thrombocytopenia, alternative treatment with prostaglandin infusions or recombinant hirudin [62], a direct thrombin inhibitor, can be considered [63].\nRegional anticoagulation with citrate has been favored by some centers [64, 65]. Sodium citrate chelates ionized calcium necessary for the coagulation cascade and systemic anticoagulation is avoided by infusing calcium through a separate central line. The disadvantages include the possibility of various acidbase and electrolyte disturbances, including hypernatremia, hypocalcemia, and metabolic alkalosis.\nAdjustment of the prescription\nAny formula for the prescription of HF is at best an approximation or starting point, as the needs will be determined by many unmeasured variables, such as the rate of solute production, nutritional intake, and the actual volumes of the extracellular fluid and intracellular fluid compartments.\nIf only fluid removal is required, then relatively low rates of filtration are needed, often referred to as slow continuous ultrafiltration (SCUF). There will be negligible solute removal under these circumstances.\nCorrection of \u201curemia\u201d and electrolyte disturbance requires the turnover of large volumes per kilogram of fluid, typically of the order of 50% of body weight per day for post-dilution and 75% for pre-dilution (approximately 20\u201330\u00a0ml\/kg per hour).\nIn catabolic patients, the clearances achieved with standard CVVH may not be sufficient. Solute removal may be increased by attempting \u201chigh-volume exchange,\u201d but this may be limited by the practical problems of pediatric patients with limitations of vascular access and hemoconcentration in the filter. In these cases, small solute clearances can be maximized by establishing diffusive mass transport via a dialysis circuit. This can be performed with CVVHDF or without an additional major ultrafiltration component (CVVHD). CVVHDF latter technique requires an additional pump to achieve separate control of the dialysate in- and outflow and of the replacement fluid flow. CVVH substitution fluid bags can be used as dialysis fluid. Dialysis fluid flow should be 2\u20133 times the blood flow if maximal efficacy is desired. This setting requires frequent manual bag exchanges and continuous supervision of the system. For practical purposes, the HD component can be added for several hours per day to a CVVH regimen.\nCVVHD has recently been recommended as the method of choice for the treatment of inborn errors of metabolism, since it supplies maximal clearance of ammonium and other neurotoxic metabolites. When CVVHD is unavailable, large volume turnover of body water with CVVH will provide the next best therapy. Rates of up to 100\u00a0ml\/kg per hour have been reported [66]. If possible, the blood pump speed also needs to be increased.\nWhen high turnover and blood flow rates are in use, patients should be carefully monitored for hypothermia, hypokalemia, and circulatory failure. Hypothermia may need to be treated with an external warming blanket and hypokalemia will require replacement. Blood flow should not be increased if the patient develops cardiovascular instability.\nCVVH and extracorporeal membrane oxygenation\nIn the authors\u2032 experience, the best results are achieved when pre-diluted fully automated CVVH is used, attached to the venous (outflow from patient) side of the extracorporeal membrane oxygenation (ECMO) circuit. This appears to reduce problems of shunting blood around the oxygenator and overcomes the problems of the increased hematocrit that may be associated with ECMO. It also reduces the complications of excessive fluid and solute clearances, with a free flow when systemic hemofilters are used in line with the ECMO circuit. When using CVVH in the suggested configuration, the \u201cpigtails\u201d provide access with very little resistance, causing the arterial and venous pressure alarms to activate and shut down the circuit. Therefore, three-way taps are used to create more resistance to flow into and out of the CVVH circuit. When treating neonatal patients, the ECMO circuit increases the extracorporeal blood volume very significantly. Therefore, the blood pump speed should be calculated taking into account the patient\u2019s blood volume and the priming volume of the ECMO circuit.\nComplications of continuous extracorporeal techniques\nComplications of continuous extracorporeal techniques are described in reference [67].\nHypotension\nHemofiltration is most commonly used in sick septic children, many of whom will be on pressor therapy. Indeed, the need for pressor agents gives a poorer prognosis [6]. Care should have been taken to minimize the amount of blood in the extracorporeal circuit and blood priming of the HF circuit may be necessary at the outset. Fluid removal is obviously adjusted according to the patient\u2019s clinical state during the treatment.\nClotting of the filter and lines\nThis is one of the commonest complications and again is related to the patient\u2019s changing clinical status and problems with anticoagulation. This complication occurred in 24% of 89 patients treated with CVVH in a 2-year local audit (B. Harvey, unpublished observations).\nOther potential complications of bleeding, anticoagulation toxicity, and infections appear to be minimal. Air embolism is a rare but preventable complication of extracorporeal circuits, and is greatly reduced with the proper use of automated machinery.\nIntermittent HD\nThe advantages and limitations of intermittent HD are described in reference [68].\nAdvantages\nThe main advantage of HD is the relatively rapid removal of uremic toxins and ultrafiltration of fluid. This makes the technique well suited for acute situations.\nLimitations\nHD is not a continuous therapy and it requires good vascular access as with HF. A purified water supply is also required, as well as anticoagulation, which should always be minimized. The technique might not be applicable for hemodynamically unstable patients. Often the major limiting factor is the availability of expert nursing staff [69], especially in the ICU [70].\nPractical guidelines for prescription\nHD is only possible with good vascular access provided either by a double-lumen HD catheter or a single-lumen catheter of sufficient diameter to achieve flows for single-needle dialysis. Catheter lengths vary from 5\u00a0cm for neonates to 20\u00a0cm for large adolescents.\nBloodline choice depends on the priming (extracorporeal) volume, which traditionally has not exceeded 10% of the blood volume (approximately 80\u00a0ml\/kg).\nDialyzer choice depends on the priming volume and maximum flow rate, with a surface area that should not exceed the child\u2019s surface area and with a urea clearance between 3 and 5\u00a0ml\/kg per min. There is no evidence for dialyzer choice in pediatric practice, but meta-analysis in adult patients with ARF suggested synthetic membranes conferred a significant survival advantage over cellulose-based membranes, but with no similar benefit for recovery of renal function [71].\nBloodline priming is usually performed with isotonic saline. Small babies, anemic patients, and those in an unstable cardiocirculatory condition, require priming with albumin or blood.\nHD catheter care\nAfter the session the catheter should be flushed with isotonic saline and filled with undiluted heparin (1,000\u00a0IU\/ml), with volumes according to manufacturer\u2019s recommendations (usually marked on the catheter itself).\nHD prescription\nThe first session should not exceed 2\u20133\u00a0h, but the standard time is usually 4\u00a0h. Longer sessions are advisable to avoid too-rapid ultrafiltration and disequilibrium syndrome.\nAll children should be dialyzed using volume-controlled machines and with bicarbonate dialysate.\nThe blood pump rate is usually 6\u20138\u00a0ml\/kg per min, but depends upon the catheter and patient size [69].\nThe ultrafiltration target should not exceed 0.2\u00a0ml\/kg per min for acute patients who should be carefully monitored for hypovolemia and hypotension. Sodium profiling is rarely used in pediatric HD practice. Anticoagulation is usually with heparin (50\u2013100\u00a0IU\/kg per session including initial bolus). Reinfusion is usually performed with isotonic saline.\nComplications occurring during acute HD\nFor hypotension, the ultrafiltration should be switched off and isotonic saline infused into the venous line until the blood pressure normalizes; additional 20% albumin 5\u00a0ml\/kg might be helpful.\nHypertension is treated according to standard hypertension protocols available elsewhere [72].\nDisequilibrium syndrome is now a rare event with adequate control of ultrafiltration and stepwise reduction of uremic toxins.\nHypoglycemia should not occur with the use of glucose-containing dialysis fluid.\nIn cases of anemia transfusions are avoided unless patient symptomatic. Erythropoietin may be given intravenously at the end of dialysis (50\u2013200\u00a0IU\/kg) to maintain hemoglobin levels.\nMedications\nThe clearance of drugs on HD or during CRRT needs to be considered. Reference should be made to standard texts [73, 74].","keyphrases":["acute renal failure","guidelines","peritoneal dialysis","hemodialysis","hemofiltration"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_Radiol-3-1-1797072","title":"MR imaging of therapy-induced changes of bone marrow\n","text":"MR imaging of bone marrow infiltration by hematologic malignancies provides non-invasive assays of bone marrow cellularity and vascularity to supplement the information provided by bone marrow biopsies. This article will review the MR imaging findings of bone marrow infiltration by hematologic malignancies with special focus on treatment effects. MR imaging findings of the bone marrow after radiation therapy and chemotherapy will be described. In addition, changes in bone marrow microcirculation and metabolism after anti-angiogenesis treatment will be reviewed. Finally, new specific imaging techniques for the depiction of regulatory events that control blood vessel growth and cell proliferation will be discussed. Future developments are directed to yield comprehensive information about bone marrow structure, function and microenvironment.\nIntroduction\nBone marrow is the fourth largest organ of the human body. Its main function is the hematopoiesis, i.e., it provides the body with erythrocytes, leukocytes and platelets in order to maintain the oxygenation, immune function and auto-restoration of the body.\nMR imaging provides a non-invasive visualization of the bone marrow and may be used to define its cellular or fat content as well as its vascularity and metabolism. Treatment effects due to irradiation, chemotherapy or other new treatment regimens may change one or several of these components, thereby causing local or generalized changes in bone marrow signal intensity on MR images. This article will provide an overview over such treatment-related bone marrow signal changes on MR imaging. In order to recognize these treatment effects, one has to be familiar with the MR imaging features of the normal and non-treated pathologic marrow. Thus, this article will first provide a brief overview over MR imaging features of the normal and non-treated pathologic marrow as a basis for subsequent descriptions of the bone marrow after conservative or new treatments.\nIndications to study treatment effects\nIn patients with hematopoietic malignancies the diagnosis of neoplastic bone marrow infiltration is crucial to determine prognosis and to identify suitable treatment protocols [1]. This diagnosis is usually obtained by iliac crest biopsy, which is mandatory for staging and histopathologic classification of the disease [2\u20134]. In non-Hodgkin\u2019s lymphoma (NHL) a neoplastic bone marrow infiltration indicates the highest stage (stage four) according to the Ann Arbor Classification. In cases of myeloma, the extent of bone marrow infiltration may be associated with any stage of the Salmon and Durie classification [2]. In both instances, iliac crest biopsy may be false negative when the bone marrow infiltration is focal rather than diffuse [3, 4].\nThe role of MR imaging for the depiction of bone marrow infiltration by hematologic malignancies is controversial and depends on the type, stage and clinical course of the malignancy. For patients with multiple myeloma, MR has been established in some centers as an integrative technique for staging and treatment monitoring, since it proved to be highly sensitive for the detection of bone marrow infiltrates and provided important additional information to conventional bone surveys. In several studies, MR showed a higher sensitivity than conventional bone surveys and bone scintigraphy for the detection of focal bone marrow lesions [5, 6]. Thirty-three percent of patients with myeloma were \u201cunderstaged\u201d based on the skeletal survey when compared to MR [7]. And some MR findings have been shown to have direct prognostic relevance: In patients with stage 1 disease according to Salmon and Durie, a pathologic bone marrow MR and normal bone survey was associated with a worse prognosis than a normal MR and normal bone survey [8, 9]. And in patients with stage 3 disease according to Salmon and Durie, a diffuse bone marrow infiltration, as shown on MR images, was associated with a worse prognosis than a multifocal bone marrow infiltration [10, 11].\nFor patients with Hodgkin\u2019s lymphomas and high-grade NHL, FDG-PET has been established as the imaging modality of choice for staging and treatment monitoring and MR imaging should only be considered in selected cases with high risk of bone marrow involvement and equivocal findings on PET or extracompartmental tumor growth. Neither MR nor FDG-PET is meant to replace marrow biopsy, because the histopathologic subtype of lymphoma has to be defined, and because minimally diffuse, microscopic marrow involvement can be false negative with either imaging technique [12]. On the other hand, routinely performed iliac crest biopsies cover only a small portion of the entire bone marrow and also provide potential false-negative findings [3, 4, 12]. Thus, in selected patients with a high risk of bone marrow involvement, MR imaging may contribute clinically significant information. In a study by Hoane et al. in 98 patients with malignant lymphoma, up to one-third of the patients evaluated with routine iliac crest biopsies had occult marrow tumor detectable with MRI [12]. In addition, patients with negative marrow biopsies but pathologic MR were found to have a worse prognosis than patients with negative marrow biopsies and normal MR [13]. Thus, since the diagnosis of bone marrow involvement affects treatment decisions and prognosis, these authors concluded that optimal bone marrow evaluation in such patients should include both biopsy and MR [12]. Another valuable contribution of MR is its capacity to detect soft-tissue paravertebral masses as well as epidural masses and neural foramen invasion, which may accompany vertebral disease. In case of clinical symptoms such as unexplained back pain or new neurological symptoms, MR is the modality of choice to detect paravertebral and epidural masses, which may accompany bone marrow infiltration of the spine.\nFor patients with leukemia, there is currently no role for routine bone marrow evaluation with MR imaging, neither before nor after therapy. Particular questions in these patients that may be addressed by MR are the search for biopsy sites in patients with suspected new or relapsed disease, but negative bone marrow biopsy as well as the diagnosis of treatment complications, such as treatment induced bone marrow infarcts or avascular necroses, when conventional radiographs are negative or equivocal [14].\nIn summary, dedicated MR imaging of the bone marrow in patients with hematologic malignancies is currently restricted to applications in patients with multiple myeloma, selected patients with other malignant lymphomas with a high risk of bone marrow infiltration and\/or extracompartmental tumor growth as well as patients with potential treatment complications. Additional patients with hematologic malignancies may undergo MR imaging for evaluations of other pathologies, outside the bone marrow. And since the bone marrow is depicted on nearly any MR examination throughout the body, the knowledge of the normal and pathologic marrow of these patients before and after therapy is crucial for the radiologist in order to provide a comprehensive diagnosis. Such \u201csecondary\u201d evaluations of the bone marrow may outperform above mentioned primary indications in this patient population.\nTechnique\nStandard techniques to depict the bone marrow at 1\u00a0T and 1.5\u00a0T clinical scanners comprise plain T1-weighted spinecho (SE) or fast-SE (FSE) sequences as well as short TI inversion recovery (STIR) sequences. The T1-weighted, non-fat-saturated spinecho (SE) or FSE sequences are best suited to define the cellular content of the bone marrow and should be included in any protocol for MR imaging of the bone marrow. STIR sequences are useful as a screening sequence to search for abnormalities in the bone marrow, most of which appear with a very high signal intensity on these sequences. There is some debate about the optimal inversion time (TI) for depiction of the bone marrow abnormalities with STIR sequences [15\u201318]: Some authors prefer a TI that causes a suppression of all normal structures and provides a maximal contrast between normal (no signal) and pathologic (high signal) tissues. Other authors prefer a TI, which is slightly higher in order to provide some additional background signal of normal tissues (low signal), and, thus, improved anatomical information. The advantages of STIR sequences are that they provide a very high tissue contrast and that they are insensitive to magnetic field inhomogeneities. The disadvantages are that STIR sequences have a limited signal-to-noise ratio and that the fat suppression technique is non-specific: The signal from tissue or fluid with a T1 similar to that of fat will also be suppressed, for example, mucoid tissue, hemorrhage, proteinaceous fluid, and gadolinium [18].\nSelective fat-suppressed T2-weighted images are an alternative to STIR-images at high field MR scanners. Selective fat saturation is lipid specific, usually provides a higher signal-to-noise ratio than STIR sequences and does not suppress gadolinium-based contrast agent (i.e., can be added after contrast medium administration). However, selective fat saturation is susceptible to magnetic field inhomogeneities. To achieve reliable fat saturation, the frequency of the frequency-selective saturation pulse must equal the resonance frequency of lipid. Inhomogeneities of the static magnetic field will shift the resonance frequencies of both water and lipid, this discrepancy would result in poor fat suppression or - even worse - saturation of the water signal instead of the lipid signal. Static field inhomogeneities inherent in magnet design are relatively small in modern magnets and can be reduced by decreasing the field of view, centering over the region of interest, and autoshimming. However, substantial inhomogeneities can be caused by local magnetic susceptibility differences such as those found at air-bone interfaces or around foreign bodies like metal or air collections [18]. Of note, T2-weighted FSE sequences without fat saturation are probably the worst sequences for evaluation of the bone marrow since both lesions and normal fatty marrow appear hyperintense on these sequences.\nOther MR imaging techniques have been developed to improve the detection and quantification of diffuse bone marrow involvement. These techniques include chemical-shift imaging, bulk T1 relaxation time measurement, and hydrogen 1 spectroscopy [19]. All of these methods were used to measure the fat content or the water\/fat fraction more accurately. However, these measurements have so far not demonstrated clinical significance, and hence these techniques are currently not used for routine imaging.\nDiffusion-weighted MR imaging techniques have been reported to be useful for the differentiation of neoplastic marrow infiltration and pathologic vertebral fractures [20]. Recently, further advanced diffusion-weighted whole body scans have been described for treatment monitoring of patients with leukemia [21]. The technique relies on selective excitation of the water resonance and generation of image contrast that is dependent upon differential nuclear relaxation times and self-diffusion coefficients.\nContrast-agent enhanced scans In most instances, the administration of Gd-based contrast agents is not necessary for evaluation of bone marrow disorders. Administration of Gd-DTPA can be helpful to differentiate cysts and tumors, to differentiate necrotic and viable tumor tissue before a biopsy, in suspected osteomyelitis or in equivocal cases of bone infarcts. If Gd-DTPA-enhanced scans are performed, fat saturated T1-weighted sequences should be used in order to suppress the fatty components of the bone marrow with intrinsic high signal intensity and, thus, to provide an optimal depiction of the Gd-enhancement.\nThe diagnosis of lesion vascularization based on comparisons between plain non-fat-saturated and fat-saturated, Gd-enhanced T1-weighted sequences is no problem, if the investigated focal lesion shows a marked enhancement. However, it may be difficult to determine, if a certain lesion shows minimal or no contrast enhancement based on comparisons of plain non-fat-saturated and fat-saturated Gd-enhanced sequences. In these cases, we recommend to obtain additional Gd-enhanced non-fat-saturated T1-weighted MR sequences with identical pulse sequence parameters as the plain sequences. A subtraction of pre and post sequences can then identify a presence or absence of contrast enhancement of certain focal lesions, listed above.\nAdditional dynamic, contrast enhanced scans after administration of Gd-DTPA have been applied by some investigators in order to generate estimates of the blood volume of the normal or pathologic bone marrow before and after treatment [22, 23]. Such dynamic contrast enhanced MR studies with gadolinium chelates should be performed with rapid acquisition techniques, producing interscan intervals as short as possible, to best measure the rapidly evolving distribution patterns of these small molecular probes (<600 daltons). Although investigated since 1993 [23], dynamic Gd-DTPA-enhanced MR studies have not shown clinical significance so far. In addition to bone marrow perfusion, the amount of contrast agent at any time within the bone marrow depends on several additional inter-related variables including microvessel permeability, endothelial surface area, hydrostatic pressure, osmotic pressure, diffusion, convective forces, interstitial pressure and clearance. Clearly, contrast media kinetics in the bone marrow is not a simple matter and shows a high interindividual and intraindividual variability with respect to the mentioned influencing factors [24\u201326]. This may be one reason why it did not find wide clinical application to date.\nNew, macromolecular contrast media (MMCM) may provide more specific information on bone marrow blood volume and sinus permeability, which may be more useful to study treatment effects. Small molecular Gd-chelates can only estimate the blood volume (immediate post-contrast scans) and extracellular space (later post-contrast scans) of the normal or abnormal bone marrow, because these small molecules permeate readily and non-selectively across normal and abnormal capillaries in the bone marrow. MMCM can also estimate the blood volume based on immediate post-contrast scans. In addition, MMCM are so large that their diffusion across microvessels is affected by the permeability of these vessels. This may be helpful for treatment monitoring of anti-angiogenesis drugs, which specifically and readily decrease microvascular permeability, but which have no immediate effect on the blood volume. Thus, MMCM may be able to predict a response to anti-angiogenic treatment before a subsequent arrest in tumor vessel growth (decrease in blood volume) and before clinical signs of response (laboratory parameters) are apparent [27]. At this time, none of the MMCMs are yet FDA approved, but several of these agents are currently in advanced stages of development for applications in patients (phase II and III clinical trials), such as MS-325\/Vasovist (Epix and Schering, approved in Europe), Gadomer-17 (Schering), SHU555C (Schering), Sinerem\/Combidex (Guerbet\/Advanced Magnetics), B-22956\/1 (Bracco) and Code 7228 (Advanced Magnetics). In order to acquire useful kinetic information from MMCM-enhanced studies, a series of images spanning at least 20\u201330 minutes after contrast medium administration, is required [27]. On the other hand, since the transendothelial diffusion of MMCM is a rather slow process, it is usually sufficient to acquire MMCM-enhanced dynamic data with intervals of one to two minutes or, for specific questions, even to obtain just one delayed contrast enhanced scan. Applications of potential clinical interest will be mentioned in the specific sections below.\nOne new class of contrast agents, particularly noteworthy with respect to bone marrow imaging, is the group of ultra small superparamagnetic iron oxide particles (USPIO). These particulate iron oxide contrast agents are phagocytosed by macrophages in the normal bone marrow, where they induce a T2-shortening effect. The principle is similar to MR imaging of the liver with superparamagnetic iron oxide particles (SPIO). However, SPIO, used for liver imaging have a diameter of >50\u00a0nm, whereas USPIO used for bone marrow imaging have a diameter of <50\u00a0nm. USPIO particles are not taken up in neoplastic marrow infiltrates, which do not contain macrophages [28\u201330]. Thus, USPIO may be used to differentiate hypercellular normal and neoplastic marrow. After infusion of USPIO at a dose of 2.6\u00a0mg\/kg body weight, the normal marrow shows a USPIO induced signal loss as opposed to focal neoplastic infiltrates, which do not show any signal loss, and, thus, stand out as bright lesions [17, 29, 31]. STIR- or T2-weighted fat saturated sequences are best suited for such USPIO-enhanced MR scans. Metz et al. found a significantly increased number of detected focal bone marrow lesions (<1\u00a0cm) in patients with lymphoproliferative disorders on these sequences after administration of USPIO compared to non-enhanced scans [17]. The USPIO Ferumoxtran-10 (Sinerem\/Combidex, Guerbet \/ Advanced Magnetics) is expected to become approved for clinical applications in 2006 in Europe and has already shown its ability to differentiate normal and pathologic hypercellular marrow and to detect multifocal lesions within the bone marrow [17, 31]. Other USPIOs, which are in different stages of preclinical and clinical trials are SHU555C\/Resovist S (Schering), Code 7228 (Advanced Magnetics), VSOP (Ferropharm) and Clariscan (Amersham\/GE).\nFuture imaging developments are likely to generate combined techniques that will maximize the information to be extracted from the image. Tumor location, morphology and function will be integrated. For example, both dynamic contrast-enhanced MR imaging and MR spectroscopy can be acquired in a single diagnostic session to define bone marrow vascular and metabolic characteristics [32].\nNew developments for whole body MR imaging, such as parallel imaging techniques, dedicated coils (Angio-SURF), and the total imaging matrix (Siemens systems, Avanto) may provide a \u201cscreening\u201d of the whole red bone marrow for tumor infiltration within a reasonable time.\nAdditional development is being directed towards combined PET and MR imaging, either by retrospective spatial registration of data from PET and MR images, obtained on separate PET and MR machines or by sequential data acquisitions on combined PET-MR scanners [33, 34]. PET-MR imaging has been described as superior, a least for some applications, compared to PET-CT because of the improved intrinsic soft tissue contrast and potential direct bone marrow depiction provided by MR.\nNormal bone marrow\nThe normal bone marrow undergoes age-related changes of its cellular content with increasing age of the patient. In adults, the normal bone marrow is characterized by a partial or complete fatty conversion and low cellularity, which leads to a relatively high signal intensity on plain T1-weighted images and low signal intensity on STIR- or fat saturated T2-weighted MR images [35\u201338]. In children, the normal bone marrow is highly cellular, which leads to a low signal intensity on plain T1-weighted images and high signal intensity on STIR- or fat saturated T2-weighted MR images. With increasing age, a conversion from this highly cellular marrow in children to fatty marrow in adults occurs with a gradual increase of the bone marrow signal on T1-weighted MR images and a gradual decline of the bone marrow signal on STIR- or fat saturated T2-weighted MR images over time. This conversion also follows a particular distribution pattern within the skeleton: it starts in the peripheral skeleton and progresses centrally [38]. Within long bones, it first involves the epiphyses, then the diaphyses and, finally, the metaphyses [38]. In the vertebrae, it starts in the center, around the venous plexus, and progresses peripherally [38]. In adults, the signal intensity of the normal bone marrow is typically hyperintense to surrounding muscle and intervertebral disks on T1-weighted MR images and hypointense to surrounding muscle on STIR- or fat saturated T2-weighted MR images. The knowledge of this pattern of conversion is useful to differentiate normal cellular marrow from focal or diffuse neoplastic involvement and to recognize treatment effects and tumor recurrence.\nThe enhancement of the normal bone marrow in healthy persons after administration of standard small molecular Gd-chelates can vary greatly (range 3-59%, mean 21%, SD 11%) and decreases with increasing age [26]. As a rule of thumb, a signal Gd-enhancement of less than 40% on T1-weighted MR images was reported to be normal in adults of more than 40 years. However, this threshold is dependent on the applied field strength and pulse sequence parameters. The relative enhancement of both normal and abnormal marrow may be higher with new, more sensitive pulse sequences [25]. Thus, each investigator should establish the specific threshold for normal bone marrow enhancement for the technique used at his particular MR scanner and institution.\nPathologic bone marrow in hematologic malignancies\nNeoplastic infiltration of the bone marrow in MR images results in a replacement of the fatty converted marrow by neoplastic cells, thereby increasing the cellular content of the bone marrow, which results in a prolongation of T1- and T2-relaxation times and subsequent decreased T1-signal and increased T2-signal of the bone marrow. The detection of neoplastic bone marrow infiltrations with MR imaging depends on the quantity and distribution of cellular infiltration.\nThe distribution of neoplastic bone marrow involvement in patients with hematologic malignancies may be focal, multifocal or diffuse. In patients with NHL, a focal or multifocal involvement is more common than the diffuse infiltration pattern. In patients with myeloma, an additional, typical \u201csalt and pepper\u201d or variegated distribution may be observed, most frequently in stage I disease according to Salmon and Durie. In patients with leukemia, the bone marrow is usually involved in a diffuse fashion. A multifocal involvement may be seen in a small proportion of patients, particularly those with AML.\nThe detection of focal, multifocal and \u201csalt and pepper\u201d or variegated infiltrations of the bone marrow in patients with NHL is straight forward: The MR signal intensity of focal bone marrow lesions is typically iso- or hypointense to surrounding muscle and intervertebral disks on T1-weighted MR images and hyperintense to surrounding muscle on STIR- or fat saturated T2-weighted MR images. Since these focal lesions are also associated with an increased angiogenesis, they show an increased signal enhancement compared to the surrounding bone marrow on fat saturated T1-weighted MR images.\nThe detection of a diffuse bone marrow infiltration with MR imaging is limited. Using standard MR scanners and pulse sequences, an infiltration of the bone marrow with more than 30% of neoplastic cells can be readily detected by a diffusely decreased T1-signal and a diffusely increased T2-signal [39]. An infiltration with less than 20% neoplastic cells cannot be distinguished from normal marrow with standard MR pulse sequences. Several authors reported a normal bone marrow MR signal in patients with leukemia, in patients with early stages of bone marrow invasion by lymphoproliferative diseases and even in up to one-quarter of patients with stage III multiple myeloma [40].\nThe administration of Gd-DTPA is not necessary for staging of focal, multifocal and \u201csalt and pepper\u201d lesions, which can be readily detected on plain MR scans. Dynamic Gd-DTPA-enhanced MR scans documented repetitively an increased blood volume (i.e., increased enhancement) of neoplastic infiltrations compared to the normal bone marrow [22, 23, 41]. As one would expect, the Gd-enhancement was significantly higher in marked bone marrow infiltrations than in mild or no infiltration (P<0.05) and the enhancement was higher in lesions with high vessel-density than in lesions with low vessel-density at histology (P=0.01). In addition, a higher enhancement was found in the presence of increased serum immunoglobulins [42]. However, the Gd-DTPA enhancement of focal neoplastic lesions has not shown additional clinically significant information for staging purposes so far. In some cases, Gd-administration may help to diagnose a diffuse bone marrow infiltration. According to studies from Staebler et al., a bone marrow signal intensity increase exceeding 40% indicates a diffuse infiltration in adult patients [43].\nIrradiation\nMR signal changes of the bone marrow during and after irradiation are time and dose dependent: In the acute phase (day 1\u20133 of irradiation), the bone marrow develops an edema, which appears hypointense on T1-weighted MR images and hyperintense on fat-saturated T2-weighted and STIR-images. Contrast enhanced T1-weighted images show a transiently increased enhancement of the bone marrow during this phase. Subsequently (day 4\u201310), focal T1-hyperintense and T2\/STIR-hypointense areas of hemorrhage may occur. The bone marrow ultimately undergoes a conversion to fatty marrow, which closely represents the irradiation field and which appears very bright on T1-weighted MR images (close to subcutaneous fat) and dark on fat suppressed images (Fig.\u00a01). Dependent on the applied dose, this fatty transformation of the bone marrow may be detected with MR imaging as early as 10\u201314 days after therapeutic irradiation [44]. In other cases, the edema may persist for weeks and a fatty conversion may only become apparent months after the irradiation. This fatty conversion is reversible after an irradiation of less than 30\u201340\u00a0Gy and irreversible after an irradiation with more than 40\u00a0Gy. The time frame of reconversion after low dose irradiation has not been specified so far and will be most likely highly variable with respect to potential additional chemotherapy or GCSF-treatment, location and extend of the affected anatomical area and age of the patient. Contrast enhanced scans show a markedly decreased enhancement of the fatty converted bone marrow.\nFig.\u00a01T1-weighted non-enhanced (a) and fat saturated contrast enhanced (b) MR images after radiation therapy of a focal PNET tumor infiltration in L 2. Note the fatty conversion of the bone marrow in the irradiation field, L1 to L3, is only apparent on the non-fat saturated non-enhanced MR image. The tumor in L2 shows a mild Gd-enhancement after radiotherapy on contrast-enhanced scans\nIn addition to these bone marrow signal changes in the irradiation field, several authors also reported an additional small, but measurable fatty conversion of the adjacent bone marrow, outside of the irradiation field. The bone marrow outside the irradiation field showed similar MR signal intensity changes compared to the bone marrow within the irradiation field, but to a much lower degree. Interpretations of these findings differ, the most appealing explanation is a fatty conversion due to scatter irradiation [45, 46].\nIn children, irradiation may also cause impairment or stop in skeletal maturation. Local irradiation may impair the growth at the growth plate of the irradiated bone after doses as little as 1.3\u00a0Gy [47]. In parallel to the above described changes of the bone marrow in general, the metaphyses and growth plates of the affected bones may also show an edema on MR images initially, and, later, a fatty conversion. The severity of impairment in bone growth is dependent on the age of the patient at the time of the irradiation and the administered dose. High doses may lead to marked epimetaphyseal deformities. The affected metaphyses of long bones may show horizontal or longitudinal areas of signal loss of on T1- and T2-weighted MR images, which resemble metaphyseal bands and striations seen on conventional radiographs [47]. Irradiation of the spine may cause a scoliosis, which is typically concave to the irradiation field, if only one side of the spine was irradiated. In children, irradiation of the whole spine may also result in a scoliosis. There is usually an associated impairment of the growth of paraspinal muscles as well as an impaired vertical growth of the irradiated vertebrae. Depending on the applied dose, the bone marrow of the vertebrae may undergo a usually transient or (rarely) persistent fatty conversion and the bone marrow in the peripheral skeleton may show a compensatory hypercellularity.\nIrradiation of the brain in children with acute leukemia may result in growth arrest due to a deficiency in growth hormone [47]. In these patients, the bone marrow usually appears normal (in case of remission of the leukemia) on MR images.\nIn children, irradiation of the proximal femur may cause a slipped capital epiphysis [47]. This may be diagnosed early on MR images by an asymmetry and typical widening of the center or posteromedial region of the affected physis, which is best seen on plain T1-weighted MR images. A potential subsequent closure of the physis may be diagnosed by sequential MRI, it typically develops from the posterior portion anteriorly.\nIn adults, a short-term complication of local irradiation may be the development of an \u201cirradiation osteitis\u201d, which presents as a T1-hypointense, T2\/STIR-hyperintense, inhomogeneous edema on MR images in the irradiation field and which shows a narrow zone of transition to the adjacent, non-irradiated bone marrow. There is no associated extraosseous soft tissue mass. The irradiation osteitis may be associated with insufficiency fractures, which may sometimes be better apparent on MR and sometimes be better apparent on conventional radiographs. Thus, imaging of a suspected irradiation osteitis should always also include conventional radiographs of the affected bone. Irradiation osteitis is rare in children [47].\nOther short-term complications of irradiation are insufficiency fractures and avascular necroses (AVN) in the irradiated bone. Insufficiency fractures are caused by normal stress on weight-bearing bones with an irradiation-induced decreased elastic resistance. Depending on the acuteness of the fracture and the time interval after irradiation, MR may show a more predominant edema or a more predominant fatty conversion of the bone marrow. The fracture line may be seen as a bright line on T2-weighted and contrast enhanced T1-weighted MR images, if it is surrounded by adjacent marrow edema (Fig.\u00a02). AVN is caused by an irradiation induced arteritis with fibrosis and endothelial proliferation, blocked arterial inflow or venous outflow, rise in intramedullary pressure, compromised perfusion, and, finally, anoxia and death of bone marrow cells. The MR imaging findings of AVN are described in detail below.\nFig.\u00a02Coronal fat saturated T2-weighted (3200\/50\u00a0ms) (a), T1-weighted (600\/20\u00a0ms) (b) and fat saturated T1-weighted (600\/20\u00a0ms) contrast-enhanced (c) MR images of the knee of a 16 year-old patient who underwent radiation therapy of the lower femur and poplitea region. Note fatty bone marrow conversion and fracture lines at the distal femur and proximal tibia (arrows) consistent with irradiation induced insufficiency fractures\nAs long-term complications, benign or malignant tumors may occur after irradiation of the bone and bone marrow. Osteochondromas are the most common tumors that may develop after total body irradiation (TBI). The irradiation induced development of osteochondromas is inversely related to the age of the patient at the time of the TBI. In children, osteochondromas developed in about 6\u201318% after TBI. The latency for the development of osteochondromas after TBI is highly variable, but generally shorter than the latency for the development of malignant tumors [48]. On MR imaging, a continuity of the bone marrow space with the lesion and a cartilage cap with a thickness of not more than 3\u00a0mm are criteria to prove a benign osteochondroma and to exclude a malignancy. Of note, sarcomatous degeneration of an irradiation induced osteochondroma is extremely rare, although incidental cases have been reported [49]. Other benign bone tumors that may develop after TBI are fibrous dysplasia and aneurysmal bone cysts [48].\nRadiation-induced sarcoma is a rare late complication of irradiation, which develops after a latency period of about 10 years in the previous irradiation field. Irradiation induced sarcomas are not directly related to the local radiation dose. Osteosarcomas, fibrosarcomas, malignant fibrous histiocytomas, and other sarcomas may occur [50, 51].\nCortisone treatment\nIschemic (avascular) necrosis is a well recognized complication of high dose cortisone treatment, seen in 1\u201310% of patients in the initial treatment phase for leukemias or lymphomas (Fig.\u00a03). In addition, AVN occurs in 10% of long-term survivors of bone marrow transplantation (Fig.\u00a04) who received high doses of steroids for the prevention or treatment of graft-versus-host disease. AVN has been also described after chemotherapy or irradiation [47]. The apparently increasing prevalence of this complication may be due to increasing recognition based on increasing use of MR imaging.\nFig.\u00a03Coronal fat saturated Spinecho (3200\/46\u00a0ms) MR images of the knee joint of a 13 year old boy with ALL after treatment with high dose cortisone. There are multiple bone infarctions in the distal femur and proximal tibia (arrows). MR studies were obtained 14months (a) and 28 months(b) after onset of treatment. Note that the infarcts decrease in sizeFig.\u00a04Conventional radiograph (a), sagittal T1-weighted (600\/20\u00a0ms) (b) and sagittal STIR sequence (4000\/70\u00a0ms, TI: 150\u00a0ms) (c) of the ankle in a patient with a history of chronic myeloid leukemia and bone marrow transplantation, showing multiple infarcts in the distal tibia, the talus and the calcaneus (arrows), which are not visualized on the radiograph\nAVN is caused by vascular insufficiency, compromised bone marrow perfusion, and, finally, anoxia and death of bone marrow cells. Bones with end-arterial vascular supply and poor collaterals are particularly prone to develop an AVN, such as the femoral head, distal femur and proximal tibia, proximal humeri, tali, scaphoid, and lunate bones. Usually, the cartilage is not affected because it is nourished by synovial fluid.\nMRI is the most sensitive non-invasive method for diagnosis of bone marrow infarction. Early forms of AVN are characterized by a diffuse marrow edema. These T1-hypointense and T2-hyperintense areas of edema may be extensive and are non-specific in their MR appearance. Potential causes, which may all be related to steroid administration, are transient osteoporosis, osteomyelitis, occult intraosseous fracture, and stress fracture. However, in patients under high dose steroid treatment, these areas of edema may represent an early stage of AVN and should be considered a marker for potential progression to advanced osteonecrosis. Therefore, careful examinations for osteonecrosis are necessary when bone marrow edema is seen is these patients [52].\nThe classic MR appearance of advanced bone marrow infarction is characterized by a segmental area of low signal intensity in the subchondral bone on plain T1-weighted pulse sequences, outlining a central area of marrow, which may have variable signal intensities [53]. This crescentic, ring-like well defined band of low signal intensity on T1-weighted images is thought to represent the reactive interface between the necrotic and reparative zones and typically extends to the subchondral plate. On T2-weighted images, this peripheral band classically appears hypointense with an adjacent hyperintense line - the \u201cdouble line sign\u201d. The hyperintense inner zone represents hyperemic granulation tissue, the hypointense outer zone represents adjacent sclerotic bone. Though characteristic of AVN, this sign is uncommon with the use of fast spin-echo sequences with or without fat suppression and is not necessary for diagnosis of the disease. There is no need to perform conventional T2-weighted sequences to find this sign. On STIR images, the band-like signal alterations of the bone marrow (corresponding to the \u201cinner zone\u201d) usually appear hyperintense.\nMitchell et al. described four stages of AVN based on MR imaging findings [54]. The knowledge of these stages may be helpful in the recognition of AVN. Class A lesions had signal intensity characteristics analogous to those of fat, i.e., high signal intensity on T1-weighted images and intermediate signal intensity on T2-weighted images. Class B lesions demonstrated signal intensity characteristics similar to those of blood, i.e., high signal intensity on both T1- and T2-weighted images. Class C lesions had signal intensity properties similar to those of fluid, i.e., low signal intensity on T1-weighted images and high signal intensity on T2-weighted images. Class D lesions had signal intensity properties similar to those of fibrous tissue, i.e., low signal intensity on both T1- and T2-weighted images (2). Class A signal intensity tended to reflect early disease, and class D signal intensity tended to reflect late disease. However, these stages may not occur in a chronological order and did not show prognostic significance [53, 54].\nThere is currently no established role for Gd-administration in non-traumatic AVN.\nOf note, in children, the proximal femur epiphyses may show a residual, small subcortical rim of hematopoietic marrow, which typically appears brighter than adjacent muscles on plain T1-weighted MR images. This should not be confused with the \u201cdouble line sign\u201d in early AVN, which is characterized by a subcortical rim, which is iso- or hypointense compared to surrounding muscle.\nThe percentage of the affected weight bearing surface occupied by the AVN is the most reliable factor for predicting outcome [52]. AVN that are entirely circumscribed, and that do not extend cranially to the cortical subchondral margin, have a good outcome, independent of the overall size of the AVN lesion. AVN that do extend to the subchondral margin and are associated with epiphyseal collapse are at risk to result in permanent disability.\nAVN may present in an atypical fashion. For example AVN of the spine typically involves a single vertebrae. The MRI findings of a wedge-shaped lesion with fluid intensity (hyperintense signal, like that of cerebrospinal fluid on T2-weighted images) are characteristic for AVN. However, AVN may involve two contiguous vertebrae and the intervening disc and can then be confused with infective or neoplastic processes [55].\nChemotherapy\nChemotherapy in patients with leukemia results in typical signal changes of the bone marrow on MR images, which reflect the underlying changes in the cellular composition and vascularity of the bone marrow [56\u201358]. During the first week of treatment, the bone marrow sinus becomes dilated and hyperpermeable, leading to an edema. The edematous bone marrow shows a low signal intensity on plain T1-weighted MR images and a high signal intensity on plain T2-weighted and STIR-images. This bone marrow edema was reported to be more pronounced in patients with AML than in patients with ALL, probably reflecting the higher bone marrow toxicity of the chemotherapeutic drugs applied for the treatment of AML (e.g., cytosine arabinoside, which has a known high bone marrow toxicity) as opposed to ALL. Subsequently, a marked decrease in bone marrow cellularity and a fatty conversion of the bone marrow develops, which is characterized by an increase in T1- and T2-relaxation times, an increased signal intensity on plain T1-weighted MR images (Fig.\u00a05) and decreased signal intensity on fat saturated T2-weighted and STIR-images. After successful therapy, a normalization of the MR signal occurs with regeneration of normal hematopoietic cells in the bone marrow. This regeneration often occurs in a multifocal pattern, i.e., within the fatty converted marrow, multiple diffuse small foci of decreased signal on T1-weighted images and increased signal on T2- or STIR-images develop.\nFig.\u00a05Coronal T1-weighted spinecho (600\/20\u00a0ms) images of the pelvis before (a) and after (b) chemotherapy for a fibrosarcoma of the pelvis (arrow). Note a decrease of hematopoietic bone marrow and an increased fatty conversion in the pelvis and bilateral proximal femurs, while the tumor size shrinks\nHodgkin\u2019s and non-Hodgkin\u2019s lymphomas, low- and high-grade lymphomas as well as distinct subgroups of NHL differ markedly in response to treatment. In Hodgkin\u2019s lymphoma, a bone marrow infiltration at diagnosis is rare and expected to resolve after treatment. Patients with residual active bone marrow lesions need additional, dedicated treatment. MR is non-specific in differentiating residual viable from non-viable disease. FDG-PET or (in equivocal cases) biopsy are preferable diagnostic methods to answer these questions. There are about 30 subtypes of NHL. About 30% of patients with NHL develop a diffuse large B-cell lymphoma, an aggressive B-cell lymphoma. About 20% of patients with NHL develop a follicular lymphoma, an indolent B-cell lymphoma [59]. About 6% of patients with NHL develop a mantle cell lymphoma, an aggressive B-cell lymphoma that is often widespread at diagnosis. Low-grade lymphomas are considered incurable and are often followed by \u201cwatchful waiting\u201d. The aggressive high-grade lymphomas are treated with aggressive therapy regimens, chemotherapy, irradiation, often a combination of both and\/or autologous bone marrow transplantation. Bone marrow lesions in these patients are expected to change under therapy on MR images. In general, MR images of patients with Hodgkin\u2019s lymphoma and NHL, including myeloma, show a conversion from hypercellular, hypervascularized to normocellular and less vascularized marrow after chemotherapy. However, the \u201cideal\u201d evolution from hypercellular to normocellular marrow may not occur in all patients with malignant lymphomas. Rahmouni et al. reported that the marrow often returned to normal after treatment when the pattern was diffuse or variegated before treatment [60]. But residual signal alterations of the bone marrow have been also reported after the end of therapy, particularly in patients with a focal pattern before treatment (Fig.\u00a06) [60]. A conversion of a diffuse to a focal MR imaging pattern of marrow involvement, reduction, but not disappearance in lesion size and number, and persistant peripheral lesion enhancement on contrast-enhanced MR images (Fig.\u00a06) have been described in association with a response to standard chemotherapy [58, 60]. In addition, focal bone marrow lesions in patients with lymphoma may show a persistent abnormal signal and\/or a cystic or fatty degeneration: A fatty or cystic conversion of focal lesions has been reported as an indication for a response to treatment (Fig.\u00a07) [61]. Low signal intensity on post-therapeutic T2-weighted images is usually associated with fibrosis and rules out relapse. A persistent intermediate hyperintense signal of focal bone marrow lesions on T2-weighted MR images (non-cystic, non-fatty) has been described in association with treatment induced necrosis and inflammation and was found in both responding and non-responding patients [60].\nFig.\u00a06Sagittal T1-weighted Spinecho sequences (500\u2013 700\/15\u201325\u00a0ms) with (a, c) and without (b, d) contrast before (a, b) and after c, d) chemotherapy for lymphoma. Note decrease in size of the soft tissue component after chemotherapy, yet still significant contrast enhancement in the bone marrowFig.\u00a07Sagittal MR images of a patient with malignant lymphoma and multifocal bone marrow infiltration. Sagittal STIR (a) and contrast enhanced fat saturated T1-weighted fast SE sequences (700\/15\u201325\u00a0ms) were obtained before chemotherapy (a, b). Fat saturated T2-weighted fast SE (4000\/60\u00a0ms) \u00a9, contrast enhanced fat saturated T1-weighted fast SE sequences and non-contrast enhanced T1-weighted SE sequences (600\/20\u00a0ms) were obtained after chemotherapy (c\u2013e). After therapy, the areas of previous tumor infiltration show a decreased signal on T2-weighted images, a decreased contrast enhancement and fatty degeneration (arrows)\nIn patients with myeloma, new or progressive vertebral compression fractures may occur as a complication of treatment response in vertebrae that had extensive marrow disease before treatment [58]. With response to treatment, the tumor mass that had replaced the trabeculae, may resolve and the unsupported vertebrae can collapse. An increasing back pain in patients with myeloma after treatment may be caused by such vertebral compression fractures or tumor progression. Relapse and poor response to treatment are well evaluated with MR imaging. In patients with clinical relapse, new focal lesions, an increase in the size of previously identified focal lesions or a change from focal to diffuse infiltration may be seen. Additional signs of tumor progression are increasing paraosseous soft tissue masses or increased Gd-enhancement of focal lesions after chemotherapy. A progress of focal to diffuse bone marrow neoplasias may be sometimes more difficult to assess, since a diffuse bone marrow infiltration may be indistinguishable from reconverted hematopoietic marrow after e.g., GCSF-treatment. As specified above, a new Gd-enhancement of >40% or lack of USPIO uptake in a hypercellular marrow may indicate a tumor progress or recurrence in these cases.\nA rare form of progression in myeloma under therapy is leptomeningeal spread within the central nervous system, reported in 18 out of a series of 1856 patients [62]. MR findings of leptomeningeal enhancement in the brain or spine helped to establish the diagnosis, which was subsequently confirmed by cytologic analysis of cerebrospinal fluid. Myelofibrosis and amyloidosis can also develop as a consequence of treatment in patients with myeloma. Myelofibrosis can be suggested on MR studies as a conversion of the entire bone marrow to diffuse hypointensity on both T1-weighted and STIR images. Amyloidosis can be seen as focal areas of hypointensity on both T1-weighted and STIR images [63].\nPerfusion studies using MR enhanced with standard small molecular Gd-based contrast agents provide functional information concerning the response of bone marrow neoplasias to chemotherapy. Conventional cytotoxic drugs have direct or indirect effects on angiogenesis and cause a decrease in bone marrow contrast medium uptake within weeks or months [22, 23, 41]. However, though these techniques are clinically available for more than a decade, perfusion studies are used infrequently or not at all in clinical practice since the obtained functional changes in bone marrow vascularity do not or only slightly precede the obvious and readily apparent clinical parameters for treatment response.\nTreatment effects of various chemotherapeutic regimen on the bone marrow in patients with leukemia and lymphomas could be also detected with 31P MR spectroscopy. A treatment induced change in tumor pH with an alkaline shift was related temporally to increases in the phosphodiester\/beta-adenosine triphosphate ratio, and occurred before alterations in tumor size were documented [64]. And interestingly, changes in the 31P MR spectral profile could not only be detected by direct investigations of the bone marrow itself, but also by MR spectroscopy of the serum of the patients. The 31P MR spectral profile of the serum changed in responding patients to resemble that of normal serum with typical, higher peak intensities as compared to non-treated and non-responding patients [65].\nBone marrow reconversion after standard therapy\nAfter successful cytotoxic therapy and\/or irradiation, the normal bone marrow may undergo a reconversion from fatty to highly cellular hematopoietic marrow. This reconversion occurs in a reverse fashion compared to the conversion from hematopoietic marrow to fatty marrow, described above, i.e., the reconversion progresses from the central skeleton to the periphery. Within long bones, it involves first the metaphyses and then the diaphyses. The presence of cellular marrow within the epiphyses in an adult patient with hematologic malignancies is always suspicious for neoplastic infiltration, especially when the rest of the bone marrow did not undergo a complete reconversion. A reconversion of marrow within the epiphyses is only rarely seen, usually in conjunction with an extensive reconversion of the hematoietic marrow of the whole bone.\nIn patients with lymphoma, the reconversion process may be enhanced by administration of granulocyte colony stimulating factor (GCSF), which activates the hematopoietic marrow and decreases the period of aplasia after chemotherapy [66]. The differentiation between this reconverted, highly cellular normal hematopoietic marrow and recurrent tumor after chemotherapy is not possible with conventional MR techniques, since relaxation rates and MR signal characteristics of highly cellular hematopoietic and highly cellular neoplastic bone marrow are similar [67]. Various investigators have addressed this problem, but were not able to differentiate reconverted hypercellular hematopoietic marrow and tumor infiltration using a variety of pulse sequences, static post-contrast MR images, and MR spectroscopy (Fig.\u00a08) [66, 68].\nFig.\u00a08A patient with myeloma at different stages of therapy. a) After chemotherapy and irradiation, the bone marrow of the pelvis and proximal femurs shows a major fatty conversion with small areas of hypointense, cellular marrow of uncertain significance on plain T1-weighted MR images. b) After high-dose chemotherapy and GCSF treatment, several areas of hypercellular, hypointense marrow are seen in the marrow of the pelvis and proximal femurs. The lower row of images shows that these areas appear bright on FS-T2-w images (b1), hypointense on plain T1-w images (b2) and show a marked enhancement on Gd-enhanced T1-w scans (b3). A follow up study, 3 months after the study in (c), shows again a fatty conversion of the bone marrow. In retrospect, the lesions in B were most likely due to reconverted hematopoietic marrow\nDynamic T1-weighted MR images after intravenous bolus injection of standard small molecular Gd-chelates may be helpful in the differentiation between normal hypercellular hematopoietic and neoplastic marrow. When compared with red marrow, enhancement of abnormal bone marrow occurred earlier, was steeper and did not last as long in neoplastic bone marrow infiltrations [69\u201371]. However, in our experience, the Gd-enhancement of the GCSF-treated, markedly hypercellular marrow and neoplastic marrow in patients with hematologic malignancies shows considerable overlap and is of limited clinical value for a definitive differentiation of these entities.\nUSPIO contrast agents may be more useful for such a differentiation of reconverted marrow and tumor recurrence. The pathophysiologic basis for this is the distribution of RES cells in the bone marrow and their ability to phagocytose exogenous iron oxides. After chemotherapy, especially after GCSF treatment, the bone marrow reconversion causes an increased quantity of all hematopoietic cell lines in the bone marrow, including RES cells [72]. In bone marrow neoplasia, on the other hand, the hematopoietic marrow is replaced by tumor cells and the number of RES cells is substantially reduced [73]. Thus, USPIO-enhanced MRI can differentiate these entities by depicting iron oxide-targeted RES cells, which are present in the reconverted hematopoietic marrow, but not present or substantially reduced in focal or multifocal tumor deposits [17, 29, 31]. Before USPIO administration, both, the hypercellular hematopoietic and neoplastic marrow appear of low signal intensity on plain T1-weighted MR images and of high signal intensity on STIR and fat saturated T2-weighted MR images (Fig.\u00a08). After USPIO administration, however, the normal marrow, which takes up the USPIO, shows an iron oxide induced signal loss, whereas focal neoplastic marrow infiltrates, which do not take up the USPIO, stand out as bright lesions on STIR and fat saturated T2-weighted MR images (Fig.\u00a09) [17, 29, 31]. The technique can also be applied in case of a diffuse, marked hypercellularity of the bone marrow (Fig.\u00a010). Diffuse or focal cell components of the normal, non-neoplastic bone marrow, which appear iso- or hypointense to intervertebral disks or skeletal muscle on plain T1-weighted MR images do show a substantial signal loss on USPIO-enhanced STIR images. If this signal loss is not observed, a malignant infiltration is present (Fig.\u00a010). On the other hand, a USPIO-administration is not meaningful in patients with a fatty marrow on T1-weighted MR images, because there are obviously no cells present, which could take up these particulate contrast agents. Thus, USPIO may be applied in specific patients, where a differentiation of hypercellular normal marrow and neoplastic infiltration is warranted.\nFig.\u00a09A 42-year-old patient after recurrent chemotherapy and GCSF treatment with reconverted, hyperplastic hematopoietic marrow and multifocal bone marrow infiltration by lymphoma. Both hematopoietic marrow and focal tumor infiltration show a low signal intensity on plain T1-weighted images (a), as well as an increased signal intensity on plain STIR images (b); however, STIR images after iron oxide administration (c) show a marked signal decrease of the hematopoietic marrow, whereas focal tumors (arrows) do not show any iron oxide uptake; thus, the tumor-to-bone marrow contrast increases substantially (figure from [31])Fig.\u00a010A 57-year-old patient with myeloma after chemotherapy and GCSF treatment. T1-weighted images before (a) show a pathologic fracture of Th 9 (arrow) and a diffuse hypointense signal intensity of the bone marrow in all vertebrae, compatible with a high bone marrow cellularity. Unenhanced STIR images (b) show a diffuse hyperintense bone marrow, also compatible with high bone marrow cellularity. After iron oxide infusion, the hypercellular bone marrow shows only minimal changes in signal intensity on STIR images (c), indicative of a diffuse tumor infiltration. Iliac crest biopsy revealed 80% tumor cells in the bone marrow (figure from [31])\nNew therapy regimens\nAngiogenesis-inhibiting drugs, such as thalidomide, may be useful for treating hematologic malignancies that depend on neovascularization, and these agents were recently added to some treatment regimens for patients with advanced myeloma [74]. The anti-angiogenic therapy is intended to stop cancer progression by suppressing the tumor recruitment of new blood supply. As such, the anti-angiogenic drugs are generally tumorostatic, not cytotoxic. Thus, a successful therapeutic inhibition of angiogenesis can be expected to slow or stop tumor growth, but not to cause tumor regression or disappearance. Accordingly, MR imaging may show a persistent high bone marrow cellularity under anti-angiogenic treatment. Signs of a response to this treatment are rather a delayed, less steep and smaller bone marrow enhancement after intravenous administration of small molecular Gd-chelates compared to pretreatment studies.\nNew macromolecular contrast agents (MMCM) may provide an earlier diagnosis of response or failure of anti-angiogenic treatment than standard small molecular contrast agents. MMCM are more sensitive in the detection of changes in vascular permeability than small molecular contrast agents. And such changes in the permeability of bone marrow sinus occurs earlier than changes in perfusion and blood volume, typically assessed with small molecular contrast agents. In animal models, MMCM-enhanced MRI was able to define anti-VEGF effects of certain angiogenesis inhibitors as early as one day after initiation of therapy [75, 76]. Future studies have to show, if these results can be also obtained in patients and if so, they would be obviously of high clinical significance for treatment monitoring and management.\nIn clinical practice, anti-angiogenic therapy regimens will almost certainly combine anti-angiogenic drugs with cytotoxic drugs. The effect of such combined therapies on the tumor accumulation and therapeutic effect of the individual drugs are complex and currently investigated in several experimental and clinical trials. Again, imaging techniques are the only available tool to provide a non-invasive and serial assessment of such combined therapy regimens. Some angiogenesis inhibitors, such as anti-VEGF antibody, decrease microvessel permeability and thereby reduce tumoral delivery of large molecular cytotoxic drugs, but not small molecular cytotoxic drugs [76]. MRI assays of angiogenesis can monitor such anti-angiogenesis therapy induced changes in tumor microvascular structure and optimize the choice and timing of cytotoxic drug administration. Other inhibitors of angiogenesis, such as anti-angiogenic steroids (tetrahydrocortisol, cortisone acetate), cyclodextrin derivates 460 (cyclodextrin tetradecasulfate), and tetracycline derivates (minocycline), may increase tumor microvascular permeability and thus potentiate certain cytotoxic therapies [77, 78]. In addition, inhibitors of angiogenesis have been shown to effectively potentiate tumor irradiation effects [79]. This possible synergy between these anti-angiogenesis drugs and cytotoxic drugs or irradiation, as a function of apparent tumor microvascular hyperpermeability, can also be interrogated with MMCM-enhanced MR imaging.\nStem cell transplantation\nThe most commonly used therapy for patients with lymphoma is autologous stem cell transplantation. For this, the patient receives a conditioning therapy, then his or her own stem cells are collected by leukapheresis, the patient subsequently receives a high-dose chemotherapy or irradiation and his previously collected stem cells are reinfused. With some types of lymphoma, an autologous transplant may not be possible in case of persistent malignant bone marrow infiltration. Even after purging (treatment of the stem cells in the lab to kill or remove lymphoma cells), reinfusion of some lymphoma cells with the stem cell transplant is possible. It would, therefore, be of high clinical significance to be able to differentiate patients with still viable lymphoma cells in their bone marrow at the time of leukapheresis from patients in true complete remission.\nIn refractory diseases or in aplastic anemia, allogenic marrow transplantation or chord cell transplantation are also used. This much more aggressive procedure is initiated by a conditioning high dose chemotherapy and\/or total body irradiation and subsequent reinfusion of allogenic donor cells, i.e., stem cells from a matched sibling or unrelated donor. Allogenic transplantation has limited applications, because of the need for a matched donor. Another drawback is that side effects of this treatment are too severe for most people over 55 years old. After the conditioning therapy for allogenic marrow transplantation, the patients reach complete aplasia, are usually isolated on a bone marrow transplantation unit and should only undergo MR imaging for vital indications.\nOnly limited studies exist about the evaluation of bone marrow with MR imaging in the setting of bone marrow transplantation in patients with hematologic disorders. Based on these data, the MR imaging characteristics of the bone marrow after autologous or allogenic marrow transplantation are apparently quite similar [80].\nAfter the conditioning therapy and before bone marrow transplantation, the bone marrow would be assumed to be depleted of tumor cells. Ideally, a conversion from focal or diffuse hypercellular marrow to normocellular or fatty marrow would be expected to occur after the induction high-dose chemotherapy. However, MR studies in patients before bone marrow transplantation showed that some patients have persistent focal lesions. Metz et al. described residual focal bone marrow lesions in patients with lymphomas right before leukapheresis [17]. Negendak and Soulen found that patients with residual lesions on MR before bone marrow transplantation had a significantly shorter median time until relapse compared to patients who did not show any residual bone marrow disease on MR images [81]. By contrast, Lecouvet et al. reported that patients with an apparently normal marrow and patients with a persistent pathologic bone marrow right before autologous or allogenic transplantation did not show differences in survival [80]. One reason for this difference in observation may be the limitation of the MR imaging technique to detect and differentiate viable and non-viable tumor cells. Some patients with a \u201cclean\u201d bone marrow may have residual microscopic active tumor cells while macroscopic residual lesions on MR imaging in other patients may or may not be viable. In our opinion, it would be highly significant to investigate MR imaging criteria (e.g., diffusion weighted MR, dynamic MR, new contrast agents, spectroscopy) for the differentiation of such persistent focal bone marrow lesions that may or may not develop to recurrent disease after stem cell transplantation.\nAfter bone marrow transplantation, the evolution of distinct MR signal patterns of the bone marrow has been described [82, 83]. During the first post-transplantation days, the bone marrow shows a decline in signal intensity on T1-weighted images and an increased signal intensity on T2-weighted images, probably due to a treatment-induced edema. Within 3 months from bone marrow transplantation, a characteristic band pattern appears on T1-weighted MR images of the spine. This band pattern consists of a peripheral T1-hypointense zone and a central T1-hyperintense zone. At histologic examination, the peripheral zone corresponded to repopulating hematopoietic marrow and the central zone to marrow fat. This \u201cband pattern\u201d may be seen for several months [82, 83]. Subsequently, the band pattern gradually evolves into a homogeneous appearance of the marrow after successful bone marrow transplantation. On late post-transplant MR studies, years after bone marrow transplantation, the bone marrow shows the fatty conversion of adult marrow. The signal intensity of the post-transplant bone marrow on T1-weighted MR images is usually increased compared to age-matched controls [84].\nIn some patients, residual marrow abnormalities may be observed on MR images after bone marrow transplantation and the administration of high-dose myeloablative chemotherapy, in the same way that they may occur after conventional chemotherapy and conditioning chemotherapy regimens, described above (Fig.\u00a011). For example, sharply defined focal low signal intensity areas of bone marrow on T1-weighted images have been reported in patients who are in complete remission after transplantation [85]. Patients with these abnormalities did not have a poorer outcome than those with normal post-transplantation MR imaging findings [80]. These data show that there is clearly a need for the MR imaging technique to add some functional information to the anatomical data, e.g., by adding spectroscopy, perfusion studies or markers for tumor cell proliferation.\nFig.\u00a011Patient with malignant lymphoma after TBI and bone marrow transplantation: Plain T1-w MR image (center) shows residual hypointense bone marrow lesions in the pelvis and proximal femur. Some of these lesions may be residual marrow abnormalities of uncertain significance. Other lesions, such as the serpiginous lesions in both proximal femurs represent therapy-induced bone infarcts. These lesions show a corresponding serpiginous hyperintense area on STIR images (left) and a minor, serpiginous enhancement on Gd-enhanced T1-w scans (right)\nOf note, iron overload commonly accompanies bone marrow transplantation and may result in a diffusely decreased signal intensity of the liver (reported in 77% of pediatric cases after bone marrow transplantation), spleen (46%) and bone marrow (38.5%) on T2-weighted and STIR MR images (Fig.\u00a012). The degree of hepatic iron overload correlated significantly and splenic iron overload correlated weakly with the number of blood transfusions [86].\nFig.\u00a012A patient with myeloma after bone marrow transplantation: The bone marrow of the lumbar spine shows a diffuse hypointense signal intensity on both plain T1-weighted (left) and fat-saturated T2-weighted (right) MR images\nTBI can cause irradiation induced signal changes and complications, described above (Fig.\u00a011). Long-term complications of TBI, applied as part of the conditioning regimen for a bone marrow transplantation, are the development of osteochondromas or sarcomas.\nIn summary, MR imaging can be a useful tool to aid in the treatment monitoring of patients with hematologic malignancies by monitoring treatment response, detecting treatment complications, differentiating normal and neoplastic hypercellular marrow and by diagnosing residual or recurrent tumor deposits. In order to achieve clinical significance and cost-effectiveness, the MR imaging technique should be clearly tailored to specific patients and the specific questions described in detail above. New MR imaging techniques may serve to depict those molecular pathways and regulatory events that control blood vessel growth and proliferation. Non-invasive monitoring of anti-angiogenic therapies has found success by defining tumor microvascular and metabolic changes, while treatment-related changes in bone marrow morphology tend to occur rather late and are non-specific. Already established in many institutions, future developments will almost certainly include \u201cfusion\u201d or \u201chybrid\u201d imaging methods, such as PET-CT and PET-MR in the treatment monitoring of patients with malignant lymphomas.","keyphrases":["mr imaging","bone marrow","treatment effects","contrast agents"],"prmu":["P","P","P","P"]}
{"id":"Intensive_Care_Med-4-1-2271079","title":"Factors associated with posttraumatic stress symptoms in a prospective cohort of patients after abdominal sepsis: a nomogram\n","text":"Objective To determine to what extent patients who have survived abdominal sepsis suffer from symptoms of posttraumatic stress disorder (PTSD) and depression, and to identify potential risk factors for PTSD symptoms.\nIntroduction\nPosttraumatic stress disorder (PTSD) is the development of psychological and physical symptoms following exposure to one or more traumatic events\u00a0[1, 2]. PTSD symptoms include intrusive recollections (re-experiencing the trauma in flashbacks, memories or nightmares); avoidant and numbing symptoms (including diminished emotions and avoidance of situations that are reminders of the traumatic event); and hyperarousal (including increased irritability, exaggerated startle reactions or difficulty sleeping or concentrating)\u00a0[3]. PTSD symptoms have a\u00a0major impact on life, illustrated by the fact that the patients have a\u00a0reduced quality of life\u00a0[4] and frequently suffer from depression\u00a0[5].\nEvents that typically trigger the development of PTSD include exposure to violent events such as rape, domestic violence, child abuse, war, accidents, natural disasters and political torture, all of which include a\u00a0threat to life\u00a0[6\u20138]. Increasingly PTSD has also been found in patients who have survived a\u00a0major, life-threatening disease and patients who have spent a\u00a0significant amount of time in an intensive care unit (ICU)\u00a0[9\u201313]. Severe peritonitis (or abdominal sepsis) is such a\u00a0disease where typically an episode of acute and severe illness\u00a0[14, 15] is followed by a\u00a0lengthy ICU stay and a\u00a0long recovery period that often includes multiple surgical and non-surgical interventions\u00a0[16\u201322]. This combination of factors could make this patient group particularly vulnerable for developing PTSD symptoms. To date, little is known about the presence and severity of PTSD and possible risk factors in patients recovering from severe peritonitis\u00a0[15].\nTherefore, our aims were to determine the presence and level of symptoms of PTSD in patients surviving abdominal sepsis. In addition, we searched for demographic and disease-related factors associated with higher levels of PTSD symptoms. Identification of such factors may be important to determine possible targets of intervention and to select patients for psychological assessment interviews.\nMethods\nStudy design\nOur study was embedded in an ongoing randomized clinical trial (the RELAP Trial) evaluating two surgical treatment strategies for patients with secondary peritonitis after the initial emergency laparotomy. Patients were enrolled between December 2001 and February 2005 in two academic medical centers and seven regional teaching hospitals in The Netherlands. All patients were followed up for 12 months after initial (index) laparotomy.\nThe study was approved by the medical ethics committee of the Academic Medical Center, Amsterdam. All patients gave informed consent to participate in this study.\nStudy population\nPatients were eligible for the RELAP trial if they had a\u00a0clinical diagnosis of secondary peritonitis requiring emergency laparotomy and an Acute Physiology and Chronic Health Evaluation II (APACHE-II) score above 10. Further details of the study population can be found elsewhere\u00a0[23].\nData collection\nAll self-administered PTSD questionnaires were distributed by mail to patients who survived at least 12\u00a0months following initial emergency laparotomy, with a\u00a0reminder by phone within 2 weeks in the case of no response. After 1\u00a0month without response a\u00a0new questionnaire including a\u00a0reminder letter was sent.\nInstruments assessing the level of PTSD symptoms\nWe used two instruments with good psychometric characteristics\u00a0[24, 25] for measuring PTSD symptoms in research settings: the Post-Traumatic Stress Scale 10\u00a0[26] and the Impact of Event Scale\u2013Revised\u00a0[27, 28].\nThe Post-Traumatic Stress Syndrome Scale 10 (PTSS-10) was originally designed to diagnose PTSD according to the Diagnostic and Statistical Manual of Mental Disorders III (DSM-III) criteria in victims of natural disasters\u00a0[14]. The PTSS-10 is now a\u00a0widely used self-report questionnaire assessing symptoms related to PTSD, particularly in critically ill and ICU patients\u00a0[4, 11, 12]. The PTSS-10 consists of 10 items, each of which ranges from 1 point (none) to 7 points (always). The total score ranges from 10 to 70, with higher scores indicating more symptoms; scores of 35 or above are considered indicative of PTSD\u00a0[11, 29].\nThe Impact of Events Scale\u2013Revised (IES-R) is one of the most commonly used self-report questionnaires for determining PTSD symptomatology following a\u00a0trauma\u00a0[27]. The IES-R consists of 22 items, each ranging from 0 (no problems) to 4 (frequent problems), with the total score ranging from 0 to 66. Scores above 24 points are generally considered indicative of PTSD, with higher scores indicating more symptoms\u00a0[28]. The IES-R has been developed based on DSM-IV criteria and therefore has three distinct subscales, the avoidance subscale (eight questions), the intrusion subscale (eight questions) and the hyperarousal subscale (six questions)\u00a0[28, 30], and is one of the most frequently used self-report questionnaires in both the clinic and in PTSD research\u00a0[27].\nPotential risk factors\nPotential risk factors were selected from previous studies\u00a0[31] examining factors for increased mortality and morbidity\u00a0[17\u201322, 32, 33] in secondary peritonitis supplemented with specific factors mentioned in the PTSD literature\u00a0[6, 9\u201311, 14, 34, 35]. We divided these factors into three distinct categories.\nGeneral patient characteristics included age, gender and the presence of major comorbidity (cardiovascular disease; COPD; renal failure; diabetes; malignancy).\nDisease characteristics and postoperative course included severity of disease measured at the time of initial laparotomy using the APACHE-II score. As several components of the APACHE-II score are already considered in a\u00a0univariate analysis (namely age and comorbidity); we chose to replace the APACHE-II score with the APS score as a\u00a0potential predictor of PTSD\u00a0[36]. The APS comprises only the acute components of the APACHE-II score, without age and comorbidity. Postoperative characteristics included administration of hydrocortisone during ICU stay\u00a0[13, 37], development of acute respiratory distress syndrome (ARDS),\u00a0[4, 12, 20], number of relaparotomies, duration of ICU and hospital stay, the development of a\u00a0disease-related major morbidity during 6 months' follow-up\u00a0[23] and an enterostomy present after 6 months' follow-up.\nTraumatic memories of ICU\/hospital stay were assessed using the four-item adverse experiences questionnaire, which captures four types of traumatic memories of the stay in the ICU or hospital ward: nightmares, fear and panic, pain, and difficulty in breathing\u00a0[13]. Patients scored the frequency of traumatic memories of their stay in the ICU or hospital ward using a\u00a0four-point scale of 0 (never), 1 (sometimes), 2 (regularly) or 3 (often), administered concurrently with the PTSS-10 and IES-R questionnaires after at least 12 months' follow-up. These were subsequently summed and classified into three graded categories of traumatic memories: 0 (no traumatic memories), 1\u20134 (some traumatic memories), more than 4 (many traumatic memories).\nWe also collected data on whether patients had experienced other traumas or whether a\u00a0close family member or friend had experienced a\u00a0trauma within the previous 3 years. We used questions 29 and 30 from the Life Stressor Checklist\u2013Revised\u00a0[38], administered at the same time as the PTSS-10, IES-R and Beck Depression Inventory\u00a0II (BDI-II) questionnaires. Responses were given dichotomously as yes or no, and patients were subsequently asked to specify the event type\u00a0[38]. These questions were asked to determine to what extent the PTSD symptomatology found in this patient group was due to their peritonitis or to other traumatic events.\nData analysis\nWe used two instruments aimed at measuring the presence and severity of PTSD symptoms in our population, each with their own cut-off value. Combining data from two instruments measuring the same construct (PTSD symptoms) may lead to a\u00a0more robust classification of patients. To preserve the natural ordering of patients who scored below the cut-off value on both questionnaires (\u2018low-scoring patients\u2019), patients scoring above the cut-off on only one of these questionnaires (\u2018moderate scoring patients\u2019) and patients scoring above the cut-off on both questionnaires (\u2018high-scoring patients\u2019), we applied ordinal regression modeling. The proportion of patients in each of these three categories is presented with 95% confidence intervals (95% CI) using the method of Wilson\u00a0[39].\nPotential predictors for PTSD symptoms were analyzed using an ordinal logistic regression model. This ordinal regression model is an extension of the binary logistic model and is appropriate when a\u00a0continuous trait is grouped into several categories by using cut-offs\u00a0[40].\nAll potential predictors for PTSD symptoms were first examined in univariate ordinal regression models. Factors with a\u00a0p-value of less than 0.1 were entered in a\u00a0multivariate ordinal logistic regression model. If variables within a\u00a0group of predictors were strongly correlated, only the factor with the strongest univariate relationship and\/or most relevant clinical interpretation was added to the model. Because the literature on PTSD and ICU studies shows them to be clinically relevant, age and gender were always included in the multivariate model regardless of the strength of their associations\u00a0[34].\nIn addition, a\u00a0factor comprised of other non-related traumas that the patient had experienced within the previous 3 years was included in the final model to assess its potential confounding role.\nThe fit and validity of the model was evaluated by checking the discriminatory properties (overlap in risk scores of patients with different outcomes), the proportional odds assumption (test for parallel lines) and calibration (closeness in expected and observed numbers of patients evaluated by an extension of the Hosmer\u2013Lemeshow goodness-of-fit statistic).\nCalibration was checked by comparing expected and observed number of patients in each of the three outcome categories across deciles of expected risk and tested for significance by using an extension of the Hosmer\u2013Lemeshow goodness-of-fit statistic\u00a0[41].\nNomogram: A\u00a0nomogram was developed to visualize the prognostic strength of the different factors from the multivariate model in a\u00a0single diagram. A\u00a0nomogram allows readers to calculate an expected distribution of PTSD symptomatology (\u2018low-scoring\u2019, \u2018moderate-scoring\u2019 and \u2018high-scoring\u2019 patients) based on a\u00a0specific profile of a\u00a0patient. The number of points for each predictor was based on the original coefficient from the multivariate ordinal model. The total number of points derived by specifying values for all predictors was used to calculate the expected probabilities that a\u00a0patient would be a\u00a0\u2018low-scoring patient\u2019, a\u00a0\u2018moderate-scoring patient\u2019 or a\u00a0\u2018high-scoring patient\u2019.\nAnalyses were performed using SAS software version 9.1 (SAS Institute Inc., Cary, NC, USA).\nResults\nOf the total of 132 patients eligible for this study, 108 (80%) patients responded to the questionnaire (Fig.\u202f1). On average the responses were provided approximately 12.5 months following initial emergency laparotomy. There were no significant differences in any of the patient or disease characteristics between respondents and non-respondents.\nFig.\u00a01Flowchart summarizing inclusion and response\nThe median age of patients was 66.8 years and 54% were male. Patients were severely ill, with a\u00a0median APACHE-II score of 14 and a\u00a0median APS score of 6, and 5% had a\u00a0major comorbidity (Table\u202f1). Ninety-six patients (89%) were admitted to ICU: their median ICU-stay was 7 days, and these patients were mechanically ventilated for a\u00a0median of 5 days. Patients were hospitalized for a\u00a0median period of 28 days (IQR 19\u201355\u00a0days). Fifty-one percent of patients also underwent another trauma in the 3 years prior to filling in the PTSD questionnaires.\nTable\u00a01Association between severity of PTSD symptoms (three categories) and patient, disease operative and postoperative characteristics: results from univariate ordinal regression modelsOverallPTSD symptoms\u202fa(n\u202f=\u202f107)Univariate ordinal regression\u202fbNone to mild (n\u202f=\u202f66)Moderate (n\u202f=\u202f30)High (n\u202f=\u202f11)p-valueGeneral patient characteristicsMedian age (IQR)66.8 (57\u201373)70.2 (60\u201374)58.7 (47\u201372) 57.8 (49\u201365)0.004Male gender (%)54%53%53% 64%0.847Major comorbidity present (%)\u202fc53%55%50% 55%0.670Peritonitis and postoperative characteristicsInitial Median APS score (IQR) 6 (4\u20138) 6 (4\u20138) 7 (5\u20139) 8 (3\u20138.5)0.271 Hydrocortisone in first 14 days in ICU (median days) 2 (0\u20137) 1.5 (0\u20138) 1 (0\u20138) 5 (1\u20137)0.749 ARDS 6% 3%10% 9%0.192 One or more relaparotomies67%70%63% 64%0.515 Admitted to ICU89%85%93%100%0.110 Median length of ICU stay (IQR) 7 (4\u201315) 7 (4\u201312) 7 (4\u201319) 9 (6\u201316)0.042 Median ventilation time (IQR) 5 (1\u20138) 4 (1\u20137) 5 (1\u201310) 7 (4\u201313)0.073 Median length of hospital stay (IQR)28 (19\u201355)26 (18\u201347)31 (23\u201360) 56 (19\u201372)0.102Follow-up Disease-related major morbidity at 6-month follow-up15% 9%27% 18%0.068 Enterostomy at 6-month follow-up51%47%55% 70%0.183IQR, Interquartile range; a Three\ngraded outcomes: none to mild, moderate and high; two patients' data are based on only one completed questionnaire;\nb All models were checked for parallel lines to see if an ordinal test for significance\nwas appropriate; c Major comorbidity included cardiovascular disease, COPD, renal\nfailure, diabetes and malignancy\nPrevalence of PTSD symptoms\nThe proportion of \u2018moderate-scoring\u2019 PTSD patients was 28% (95% CI 20\u201337%), whilst 10% (95% CI 6\u201317%) of patients were \u2018high-scoring\u2019 patients (Table\u202f1). Detailed information on depression and PTSD symptoms is presented in the electronic supplementary material (ESM).\nPredictive factors\nResults from the univariate analysis are presented in Table\u202f1 and Table\u202f2, and descriptive details can be found in the ESM.\nTable\u00a02Association between severity of PTSD symptoms (three categories) and other traumatic experiences following peritonitisPTSD symptoms (n\u202f=\u202f105) Univariate ordinal regressionNone to mild (n\u202f=\u202f64)\u202faModerate (n\u202f=\u202f30)\u202faHigh (n\u202f=\u202f11)p-valueTraumatic memories of ICU or hospital stayNightmares39%61% 82%      0.002Fear and panic24%61%100%<\u202f0.001Pain67%70% 82%      0.002Difficulty breathing33%76%100%<\u202f0.001Traumatic memoriesNone (0)41%50% 9%<\u202f0.001Moderate (1\u20134) 7%47% 47%Severe (>\u202f4) 0%18% 82%a Two patients not included in final analysis due to missing data on\ntraumatic memories during ICU or hospital stay\nThe final multivariate model included age, gender, length of ICU stay, disease-related morbidity during the 6-month follow-up, traumatic memories of the ICU or hospital stay and other traumatic factors within the previous 3 years (Table\u202f3).\nTable\u00a03Association between severity of PTSD symptoms and patient, disease operative and postoperative characteristics and other traumatic experiences following peritonitis in a\u00a0multivariate analysisFinal model (n\u202f=\u202f105)\u202faOR95% CIp-valueLowerUpperTen years increase in age 0.740.53 1.04      0.084Female 0.90.94 2.3      0.822Length of ICU stay (log2 transformed) 1.41.1 1.7<\u202f0.003Major disease-related morbidity during 6-month follow-up (including index hospital admittance) 2.10.61 7.11      0.238Traumatic memories of ICU or hospital stayModerate (1\u20134) 4.90.95 24.9      0.058Severe (>\u202f4)55.59.4328.0<\u202f0.001Other trauma within previous 3 years 2.40.94 6.3      0.085a This multivariate ordinal analysis included a test for parallel lines\n(p\u202f=\u202f0.694)\nIn our final model, increasing age was associated with a\u00a0lower likelihood of PTSD symptomatology (OR\u202f=\u202f0.74 per 10 years increase in age, p\u202f=\u202f0.084). Gender was not predictive of PTSD symptoms (OR\u202f=\u202f0.90, p\u202f=\u202f0.82). Disease-related morbidity at the 6-month follow-up (OR\u202f=\u202f2.1, p\u202f=\u202f0.24) was no longer independently predictive of PTSD symptoms. Memories of the ICU\/hospital stay (patients that reported some memories: OR\u202f=\u202f4.9, p\u202f<\u202f0.057; patients that reported many memories: OR\u202f=\u202f55.5, p\u202f<\u202f0.001) were the most prominent independent risk factor for increased PTSD symptomatology. Length of ICU stay was also significantly predictive in the development of PTSD symptomatology in the multivariate model (OR\u202f=\u202f1.4, p\u202f=\u202f0.004). The relative strengths of these relationships are visualized in the nomogram (Fig.\u202f2). In this nomogram, one can calculate for the individual patient given his\/her risk profile the probability that he\/she will score either no to mild, moderate or high PTSD symptoms according to the PTSS-10 and IES-R.\nFig.\u00a02Nomogram for prediction of severity of PTSD symptoms in patients with secondary peritonitis. Graded outcome categories are: none to mild (negative on both instruments), moderate (positive on one instrument), and severe (positive on both instruments)\nThe proportional odds assumption was not violated as indicated by a\u00a0p-value of 0.694 for the test of parallel lines. Calibration of the model (closeness between predicted and observed probabilities) was good, with a\u00a0p-value of 0.987 for the goodness-of-fit test for ordinal models. A\u00a0graphical impression of the model's discriminative ability is shown in Fig.\u202f3. This figure shows that the mean risk score is significantly different between all three PTSD symptom severity categories (p\u202f<\u202f0.001), although there is some substantial overlap in the risk scores between patients from different categories of PTSD symptom severity.\nFig.\u00a03Distribution of total points from nomogram (risk score) for the prediction of the severity of PTSD symptoms with use of the risk factors taken from the multivariate ordinal model. PTSD categories are graded according to severity: none to mild (negative on both instruments), moderate (positive on one instrument), severe (positive on both instruments)\nDiscussion\nThe proportion of patients with \u2018high-scoring\u2019 PTSD symptomatology 12 months after peritonitis was 10%, and the number of \u2018medium-scoring\u2019 patients (28%) was in line with earlier studies measuring PTSD symptoms in critically ill patients who had been admitted to ICU\u00a0[9\u201313, 15, 42]. The prevalence of PTSD recorded in the general population varies between 0.9% and 2.9% (the ESEMeD study)\u00a0[7, 43, 44].\nFrom our study, the following observations can be made. Firstly, the development of PTSD symptoms is not directly related to the severity of the disease at presentation. The APS score at baseline was not predictive for the development of PTSD symptoms. The APS measures the severity of disease score solely on the weight of the acute clinical features and does not incorporate age and comorbidity\u00a0[36].\nThe development of PTSD symptoms was, however, predominantly related to a\u00a0more complicated course of secondary peritonitis. Longer ICU and hospital stays and major disease-related morbidity during the 12-month follow-up were associated with more PTSD symptoms. In concordance with earlier studies\u00a0[15, 34], the strongest predictor of having PTSD symptoms following abdominal sepsis was having traumatic memories and experiences during their initial hospital or ICU stay. These results suggest that the presence of traumatic memories is one of the most relevant aspects for the development of PTSD-related symptoms. Earlier studies also found that subjective interpretation of the intensive care experience emerged as a\u00a0consistent predictor of adverse emotional outcome in both the short and the long term\u00a0[13, 34].\nAge plays a\u00a0critical role in the development of PTSD symptoms. Younger patients are much more likely to develop and report such symptoms. This finding confirms the results of an earlier, retrospective study of a\u00a0different cohort of patients 4\u201310 years after hospital admission for severe peritonitis\u00a0[15]. These findings suggest that older patients are more able to adapt to the limitations that are associated with experiencing such a\u00a0major disease, most likely because they have already experienced a\u00a0co-morbid illness and health-related problems. In contrast to some other studies, gender did not play a\u00a0role in the development of PTSD symptoms\u00a0[34].\nPatients with abdominal sepsis suffering from ARDS did not report more PTSD symptomatology than those without ARDS. In earlier ICU studies, ARDS patients reported considerable PTSD symptoms\u00a0[12, 45]. In our cohort of abdominal sepsis patients we found different predictive factors for PTSD than those found in the ARDS patients\u00a0[10, 12, 15]. Secondary peritonitis in itself may have been severe enough, with ICU admission and extended mechanical ventilation days, to cause PTSD symptoms; therefore, the added risk by ARDS may be moot. Lack of power may also be a\u00a0factor, because the proportion of patients developing ARDS in this study was modest.\nWe did not find an association between hydrocortisone administration during ICU stay and PTSD symptoms within this peritonitis cohort, as has been demonstrated for other critically ill patient groups\u00a0[13, 37, 46, 47]. Hydrocortisone did not protect against developing PTSD symptoms, whereas other studies have found that administration of hydrocortisone during ICU can lead to a\u00a0reduction in PTSD symptoms after discharge. In this study corticosteroid use during only the first 14 days of ICU was included in our analyses. The effect of prolonged use of hydrocortisone or late-stage use during conditional adrenal insufficiency cannot be excluded.\nUnfortunately, due to the acute and life-threatening nature of secondary peritonitis, it was not possible to collect baseline information on PTSD or data on earlier psychological disorders. However, as recommended in a\u00a0recent review by Griffiths and colleagues\u00a0[48], to account for possible earlier traumas we considered information pertaining to comorbid diseases. Furthermore, we collected data on other, non-disease-related traumatic events that had occurred within the previous 3 years. These non-disease-related events were indeed associated with having more PTSD symptoms, and altered the initial ORs of the other factors to the extent that we considered it a\u00a0moderate confounder.\nTiming plays an important role in collecting data on PTSD symptoms in critically ill and ICU patients\u00a0[48]. We set the period for the recording of PTSD symptoms at 12 months for a\u00a0very specific reason; in this severely ill patient group, we did not want to record patient recovery. Past studies have shown that critically ill patients develop PTSD symptoms only after their physical recovery period has passed, hence with a\u00a0delayed onset\u00a0[9].\nAlthough these self-report questionnaires are frequently used, the diagnostic value of such instruments in relation to a\u00a0DSM-IV diagnosis obtained by a\u00a0structured interview is still being researched and discussed\u00a0[49]. Some studies have reviewed the diagnostic value of the questionnaires, but in general these studies were methodologically limited\u00a0[11, 29, 50]. In this study we did not include a\u00a0structured clinical interview for establishing a\u00a0definite DSM-IV criteria diagnosis of PTSD, although this is highly recommended in clinical psychology. However, we feel that the use of questionnaires is more feasible in the ICU\u00a0[48], and patients who report many symptoms on these self-report questionnaires\u00a0[51] can subsequently be referred to an appropriate mental healthcare provider\u00a0[50].\nIn this study we have tried to learn from two questionnaires, one commonly used and validated in particular for critically ill patients, together with one of the most frequently used screening instruments for PTSD, the IES-R. The prevalence of PTSD symptomatology in the present study was based on whether or not a\u00a0patient scored above the cut-offs of the IES-R and the PTSS-10. We employed the two questionnaires as complementary for the detection of PTSD symptoms and not to compare results deduced from both questionnaires separately\u00a0[49]. Combining the results of both questionnaires in our analysis was anticipated to lead to a\u00a0more robust assessment of the factors associated with more PTSD symptoms. Although these two instruments aim to measure the presence of PTSD symptoms, their concordance in classification of patients was not perfect, with 30 patients (28%) being positive on one questionnaire but not on the other. This demonstrates the difficulty in measuring PTSD symptoms by questionnaire, but also means that both questionnaires are informative in their own right. Combining the two instruments may therefore lead to a\u00a0more robust classification of patients based on their level of PTSD symptoms and may be a\u00a0more useful tool in screening patients following ICU stay, while potentially reducing biases due to instrument variation\u00a0[52].\nWe assessed traumatic experiences during ICU\/hospital stay based on the patients' recollections (after 1 year). The patients' perceived traumatic experience may well have contributed to the development of PTSD symptoms, but it is also possible that having PTSD symptoms influenced their perceptions. Future studies should aim to prospectively quantify traumatic experiences during or shortly after ICU stay to draw more causal conclusions, even though this might be difficult in patients with such a\u00a0lengthy recovery period\u00a0[9, 48, 53, 54].\nIn the clinical setting, there is a\u00a0continuing debate on whether to intervene in the more acute peritraumatic psychological processes or in a\u00a0later phase, when symptoms or prodromes of PTSD are observed. By improving our understanding of which factors play an important role in the development of PTSD, we can better prevent PTSD symptoms in high-risk patients and decide when best to intervene. The aim of our predictive model is for it to be used by treating physicians, following the acute episode and phase of secondary peritonitis in which survival and physical recovery are the main concerns, to recognize high-risk PTSD patients. This relatively simple model can aid the surgeon, for instance, during the first outpatient visit in determining which patients are at higher risk for the development of PTSD symptoms. However, before this nomogram can be used to actually predict PTSD symptomatology in clinical practice, it must be externally validated in another cohort of patients with secondary peritonitis.\nIn conclusion, 10% of peritonitis patients report \u2018high\u2019 PTSD symptomatology and another 28% \u2018moderate\u2019 PTSD symptoms. Factors that were related to more PTSD symptoms included younger age, traumatic memories of the period of hospitalization and length of ICU stay. Knowledge of these predictive factors is required to increase awareness, and to develop tailored early treatment options for these high-risk patients our nomogram may assist in identifying patients with PTSD symptoms.\nElectronic supplementary material\nElectronic Supplementary Material (DOC 21K)\nElectronic Supplementary Material (DOC 52K)","keyphrases":["sepsis","posttraumatic stress disorder","ptsd","depression","intensive care","peritonitis","ptss-10","ies-r","bdi-ii"],"prmu":["P","P","P","P","P","P","P","P","P"]}
{"id":"Eur_J_Pediatr-3-1-1802727","title":"The probiotic Escherichia coli strain Nissle 1917 (EcN) stops acute diarrhoea in infants and toddlers\n","text":"In most cases, acute diarrhoea will become self-limiting during the first few days after onset. For young children, however, health risks may develop when the disease lasts longer than 3 days. The purpose of the present trial was to determine whether the stool frequency of infants and toddlers suffering from acute diarrhoea could be normalised more quickly by administering the probiotic Escherichia coli Nissle 1917 (EcN) solution than by administering a placebo. The safety of EcN were also assessed. A total of 113 children (aged 2\u201347 months) with acute diarrhoea (> three watery or loose stools in 24 h) were randomised to either a group receiving the probiotic EcN suspension (n = 55) or a group receiving the placebo suspension (n = 58) in a confirmative, double-blind clinical trial. Depending on the age of patients, 1\u20133 ml per day of verum suspension (108 viable EcN cells per millilitre) or placebo were administered orally. The causes of the diarrhoea were viral rather than bacterial, but they were mainly unspecific infections. The median onset of treatment response (reduction of daily stool frequency to \u2264 three watery or loose stools over at least 2 consecutive days) occurred more rapidly in the children receiving the EcN solution (2.5 days) than in those receiving the placebo (4.8 days), a significant difference (2.3 days; p = 0.0007). The number of patients showing a response was clearly higher (p < 0.0001) in the EcN group (52\/55; 94.5%) than in the placebo group (39\/58; 67.2%). EcN was found to be safe and well-tolerated, and it showed a significant superiority compared to the placebo in the treatment of acute diarrhoea in infants and toddlers.\nIntroduction\nProbiotics are non-pathogenic microorganisms \u2013 mostly of human origin \u2013 which confer health benefits to the host when administered in adequate amounts. They are considered to be a safe and effective part of the first-line therapy for acute diarrhoea in children and adults [12]. In addition, probiotics are able to prevent or improve not only gastrointestinal diseases such as inflammatory bowel disease, irritable bowel syndrome, infectious gastroenteritis or diverticular disease of the colon, but also to act in the prevention of allergic diseases.\nVarious probiotics are commercially available in, for example, Europe, the USA and Japan where they are marketed as functional foods or probiotic drugs. To date, lactobacilli, bifidobacteria and Saccharomyces boulardii are the most commonly marketed probiotic active substances. Certain strains of Escherichia coli are also available in some European countries, the best known example of which is E. coli strain Nissle 1917 (EcN).\nEcN is marketed as a probiotic drug in two galenic presentations for oral use: enteric-coated capsules and a suspension in which 1\u00a0ml contains 108 viable EcN cells. While capsules are mostly used in adults (e.g. Kruis et al. [8]), the use of the suspension form is the most reasonable form in neonates, infants and toddlers.\nThe purpose of the present trial was to examine the efficacy and safety of an EcN suspension administered to infants and toddlers suffering from acute diarrhoea of different causes in terms of normalising the stool frequency.\nMaterials and methods\nInfants and toddlers treated for acute diarrhoea in the paediatric outpatient wards of 11 centres between February and April 2005 were eligible for enrollment in this study. This was a multicentre, prospective, confirmative, randomised, double-blind, placebo-controlled, parallel group clinical trial of phase III. It was carried out in accordance with the requirements of Good Clinical Practice and the Revised Declaration of Helsinki. The study was approved by the Independent Ethics Committee (IEC) of the Federal Agency of Drugs Quality Control, Moscow, Russia, and by the IEC of the State Enterprise Centre of Immunobiological Medicines at the Ministry of Health of Ukraine.\nAcute diarrhoea was defined as more than three watery-to-loose stools per day from an acute episode of non-bloody diarrhoea which did not persist longer than 3 successive days. For reasons of comparability, one of the exclusion criteria was a higher grade of dehydration (loss of body weight >5%; hydration status was surveyed, rehydration was not implemented in low-grade dehydration); the most important inclusion and exclusion criteria are listed in Table\u00a01. The participant was assessed until ascertainment of response, 10\u00a0days at maximum. An overview of the study design is presented in Fig.\u00a01. A stool sample was taken at both the beginning and end of the study and checked for the presence of the following pathogens: Salmonella, Campylobacter, Yersinia, E. coli (ETEC, EPEC, EIEC, EHEC), Shigella, Entamoeba histolytica, Cryptosporidium parvum and Rota-, Adeno- and Noroviruses. \nTable\u00a01Inclusion and exclusion criteriaInclusion criteriaExclusion criteriaAge <4\u00a0years at the time of enrolmentDehydration (>5% loss of body weight)More than three watery or loose non-bloody stools in a 24-h period that had not persisted for more than three consecutive daysParticipation in another clinical trialSigned informed consent by the parentsIntake of EcN within the past 3\u00a0months prior to enrolmentIntake of food supplements or drugs which contain living microorganisms or their metabolic products or components within 7\u00a0days prior to enrolment or during the trialOther antidiarrhoeal drugsAntibioticsBreast-feeding, Premature birthSevere or chronic disease of the bowel or severe concomitant diseasesFig.\u00a01Study design. *Final visit Duration of treatment, which was until ascertainment of response, 10\u00a0days at maximum\nThe parents were asked to maintain a daily record (diary) containing information on the number of stools, stool consistency, admixtures of blood or mucus, frequency of vomiting, abdominal pain and cramps and fluid intake as well as concomitant medication and general state of health. An assessment of general health was also documented during each control visit by the investigator and parents.\nThe randomisation schedule was generated by means of SAS, ver. 9.1 (SAS Institute, Cary, N.C.) based on seed values dependent on a random number generator. The method of randomly permuted blocks was used (block size: 4).\nStudy medication\nThe drug being studied (verum) is a commercially available suspension for oral use that contains non-pathogenic E. coli strain Nissle 1917 (Mutaflor suspension; Ardeypharm, Herdecke, Germany, with 108 viable microorganisms per millilitre). As placebo, we administered an identical preparation consisting of a suspension devoid of the active substance. In accordance with good clinical practice (GCP), identical containers were used in order to guarantee a concealed random allocation both to the parents and the study personnel involved. Depending on the age, daily doses of the study medication (EcN or placebo) were: \nInfants <1\u00a0year1\u00a0ml once dailyToddlers \u22651 to \u22643\u00a0years1\u00a0ml twice dailyToddlers >3 to <4\u00a0years1\u00a0ml three times daily\nThe parents received a diary in which the intake of the trial medication was documented. The investigator checked the entries for completeness and plausibility. The compliance was also evaluated by comparing the amount of trial medication handed out with that returns.\nOutcome\nThe primary effect criterion was the time to response. Treatment response was defined as a reduction in stool frequency to \u2264 three watery or loose stools in 24\u00a0h over a period of at least 2 consecutive days. Secondary effect criteria included the response rate, stool consistency, abdominal pain and cramps, body temperature, frequency of vomiting, occurrence of adverse events and tolerance to the study medication.\nStatistical analysis\nThe study was conducted according to a three-stage group sequential test design (O\u2019Brien\/Fleming type) with possible sample size adaptation after the two planned interim analyses [9]. A time-to-response analysis was performed (Kaplan-Meier method; log-rank test to test the superiority of EcN compared to placebo; overall type-I error rate \u03b1\u2009=\u20090.025; one-sided). The response rates were also computed and compared between treatment groups by means of Fisher\u2019s exact test (one-sided; exploratory). The intention-to-treat (ITT) data set included all randomised patients who took at least one dose of study medication (primary analysis), whereas patients with major protocol violations were excluded from the per-protocol (PP) analysis. The analysis sets were defined in a blind review of the data. The sample size was estimated prospectively using ADDPLAN ver. 3.0. An independent data monitoring committee (IDMC) was responsible for reviewing the results of the interim analyses and giving recommendations. Two interim analyses were performed, resulting in continuation of the study with the pre-planned sample sizes.\nResults\nBaseline data\nA total of 113 infants and toddlers between 2 and 47\u00a0months of age with acute diarrhoea were admitted to the trial. All patients were Caucasian. The patients were randomly allocated to either the EcN group (55 patients) or the placebo group (58 patients) (Fig.\u00a02). No relevant differences between the groups were observed in terms of gender, age, height, weight and BMI of the patients (Table\u00a02). The vast majority of patients had an average body development and a good nutritional status, but reduced appetite was reported. There were also no differences in systolic and diastolic blood pressure, heart rate and body temperature between the two treatment groups at baseline.\nFig.\u00a02Diagram of participant flowTable\u00a02Baseline data for the two treatment groupsEcN (n\u2009=\u200955)Placebo (n\u2009=\u200958)Male gender 32 (58.2%)32 (55.2%)Age (median)21\u00a0months23\u00a0monthsHeight (median) 83\u00a0cm83\u00a0cmWeight (median)12.7\u00a0kg12.6\u00a0kgBMI (median) 17.4\u00a0kg\/m217.2\u00a0kg\/m2Mean duration of diarrhoea1.4\u00a0days1.6\u00a0daysMean stool frequency5.0 per day5.1 per dayPossible causes for the current acute diarrhoea episodePrevious antibiotic treatment2 (3.6%)4 (6.9%)Virus infections 16 (29.1%)19 (32.8%)Bacterial infections9 (16.4%)4 (6.8%)Unspecified infections25 (45.5%)29 (50.0%)Other causes3 (5.5%)2 (3.4%)\nThere was no difference in the duration of the current acute diarrhoea episode between patients in the EcN group and placebo group (EcN: 1.4\u2009\u00b1\u20090.6\u00a0days, mean: 1.0\u00a0days; placebo: 1.6\u2009\u00b1\u20090.6\u00a0days, mean: 2.0\u00a0days). The number of infections during the past 12\u00a0months was \u2264 five in 55\/55 patients (100%) of the EcN group and 56\/58 patients (96.6%) of the placebo group. Infections as a cause of the acute diarrhoea were more often viral than bacterial. However, unspecific infections were the most common (Table\u00a02). The number and proportion of patients with pathogenic microorganisms were slightly higher in the EcN group (27\/55 patients) than in the placebo group (21\/58 patients) at baseline. This difference was not statistically significant.\nData analyses\nAll efficacy analyses were originally designed to be performed on both the ITT and PP data sets. However, as the ITT and PP data sets were identical in this study (n\u2009=\u2009113 patients), only the ITT data are evaluated here. All safety analyses were performed on the ITT data set.\nPrimary objective\nThe median time to response was 2.5\u00a0days in the EcN group and 4.8\u00a0days in the placebo group, i.e. treatment with EcN shortened the duration of diarrhoea by 2.3\u00a0days. Statistical testing revealed that the EcN treatment was significantly superior to that of the placebo in terms of time to response (p\u2009=\u20090.0007; overall p value of the group sequential test design) (Fig.\u00a03). Analysis by centre showed no difference in the number of responders between treatment groups.\nFig.\u00a03Time-to-response curves: Kaplan-Meier analysis (ITT analysis)\nIn total, diarrhoea was stopped in 52\/55 patients (94.5%) in the EcN group and 39\/58 patients (67.2%) in the placebo group within 10\u00a0days. Fourteen patients dropped out of the trials (EcN, n\u2009=\u20091; placebo, n\u2009=\u200913) because of unsuccessful therapy. The diarrhoea did not cease within 10\u00a0days of treatment in two patients of the EcN and six patients of the placebo group.\nSecondary objectives\nAn exploratory comparison showed a significant difference in the number of responders between the treatment groups (p\u2009<\u20090.0001; ratio of rates: 1.406; 95%CI: 1.162\u20131.701). A cumulative presentation of the number of responders on each study day showed a difference between EcN- and placebo-treated patients starting on day 3 [EcN 34\/55 (61.8%) vs. placebo 24\/58 (41.4%); ratio of rates: 1.494; 95%CI: 1.032\u20132.163] (Fig.\u00a04). The difference increased until day 5 [EcN 45\/55 (81.8%) vs. placebo 30\/58 (51.7%); ratio of rates: 1.582; 95%CI: 1.198\u20132.089] and then decreased slightly from day 6 to the end of study [EcN 52\/55 (94.5%) vs. placebo 39\/58 (67.2%); ratio of rates: 1.406; 95%CI: 1.162\u20131.701).\nFig.\u00a04Response rates among the patients receiving the EcN solution (n\u2009=\u200955) and placebo (n\u2009=\u200958) during the course of the study\nPrior to the treatment regimen, almost no infant had a normal stool consistency. During the course of the study the patients of the EcN group showed a more pronounced improvement than their counterparts in the placebo group. The same trend was observed for the disappearance of abdominal pain (28\/30 EcN patients vs. 24\/33 placebo patients) and abdominal cramps (17\/18 EcN patients vs. 21\/26 placebo patients) (Table\u00a03). \nTable\u00a03Improvement of symptoms after treatment\u00a0EcN (%)Placebo (%)Normal stool consistency78.440.5No abdominal pain93.372.7No abdominal cramps94.480.8\nIn addition, the general state of health, as assessed by the investigator during clinical examinations or by the parents by means of the diary, of the patients in the EcN group improved more clearly than that of the patients in the placebo group (data not shown). Body temperature showed an almost identical decrease over time in both treatment groups (EcN: \u22120.5\u2009\u00b1\u20090.4\u00b0C; placebo: \u22120.4\u2009\u00b1\u20090.5\u00b0C). The number of vomiting episodes was very small at baseline in all patients and decreased to 0% in both groups. In principle, body weight and the status of dehydration did not show any changes from baseline to the end of study in either treatment group. Only one patient in the placebo group experienced mild dehydration.\nFrom baseline to study termination, pathogenic microorganisms disappeared in a similar number of patients in both treatment groups (14\/27 patients in the EcN group and 12\/21 patients in the placebo group). In the patients who were free from infectious agents at baseline, pathogenic microorganisms were detected at the end of study in 3\/28 and 6\/37 patients in the EcN and placebo group, respectively.\nTolerance to treatment\nThe study medication was well tolerated. Only 2\/55 patients (3.6%) in the EcN group and 2\/58 patients (3.4%) in the placebo group experienced one adverse event (AE) each. These were rhinitis and abdominal pain in the EcN-treated patients and two cases of acute otitis media in the patients receiving the placebo. According to the European Medicines Agency (EMEA) classification, none of these AEs are rated as \u201cserious\u201d or \u201csevere\u201d (regulatory guidance CPMP\/ICH\/377\/95). The two placebo-treated patients with otitis media were removed from the study due to the AE being intolerable. For the two AEs in patients receiving the EcN treatment, complete recovery was documented. According to the parents, tolerance to treatment was slightly better in the EcN group than in the placebo group, whereas no notable difference was observed by the investigators (Table\u00a04). \nTable\u00a04Tolerance to treatment in the two treatment groups\u00a0Assessed by parentsAssessed by investigators\u00a0EcNPlaceboEcNPlaceboVery good11\/55 (20.0)a4\/58 (6.9)5\/55 (9.1)4\/58 (6.9)Good44\/55 (80.0)53\/58 (91.4)50\/55 (90.9)53\/58 (91.4)Poor0\/55 (0.0)1\/58 (1.7)0\/55 (0.0)1\/58 (1.7)aPercentage is given in parenthesis\nDiscussion\nThe aim of this multicentre, prospective, confirmative, randomised, double-blind, placebo-controlled clinical trial of phase III was to investigate the therapeutic efficacy and safety of orally administered EcN in treating acute diarrhoea in infants and toddlers. The results showed that EcN was superior to the placebo in terms of both time to response and response rate. The difference in median duration of diarrhoea \u2013 2.3\u00a0days \u2013 was statistically significant and also clinically important.\nAcute diarrhoea in children is very often self-limiting within a few days. However, toddlers and young infants are in danger of developing dehydration and a deteriorating general health. Therefore, a fast-tracking antidiarrhoeal treatment would be beneficial. Several investigations have been carried out with probiotics for the treatment of acute gastroenteritis, and different meta-analyses and systematic reviews have been published in this field. All of these have demonstrated the efficacy of probiotics in treating or preventing diarrhoea. On average, the treatment of diarrhoea with lactobacilli, bifidobacteria and\/or S. boulardii shortened the duration of diarrhoea by only 0.5\u20131.5\u00a0days [4, 12, 14, 18, 19].\nSzajewska and Mrukowicz reviewed ten randomised, double-blind, placebo-controlled studies and concluded that the administration of probiotics led to a substantial reduction in the duration of acute diarrhoeal symptoms: an average of 20\u00a0h [18]. Moreover, a meta-analysis of nine clinical trials conducted by D\u2019Souza et al. demonstrated that probiotics effectively prevented antibiotic-associated diarrhoea [4]. The work of van Niel et al. included nine randomised controlled studies with lactobacilli in acute infectious diarrhoea in children. In these studies, the duration of diarrhoea was significantly reduced by an average of 0.7\u00a0days along with the stool frequency [19]. Most recently, McFarland et al. examined the efficacy of probiotics in paediatric diarrhoea by analysing 39 randomised, controlled and blinded clinical trials comprising a total of 41 probiotic treatment arms [12]. Of these, 32 (78%) reported efficacy. The latest meta-analysis of 39 trials by Sazawal et al. showed that probiotics prevented acute diarrhea, with a risk reduction among children off 57% (range: 35\u201371%) [14]. Diarrhoea is one of the best-studied indications for probiotics, and treatment with EcN has been found to stop acute diarrhoea more rapidly than other probiotics. The efficacy of EcN was confirmed by a second multicentre, prospective, randomised, double-blind, placebo-controlled phase III study conducted by our group [7]. In that study, children with prolonged diarrhoea treated with EcN showed a more rapid onset of response to treatment than those treated with placebo (median: 2.4 vs. 5.7\u00a0days; p\u2009<\u20090.0001). There was also a remarkable difference in the response rates, as determined on days 14 (EcN: 93.3%; placebo: 65.8%) and 21 (EcN: 98.7%; placebo: 71.1%), thus showing a statistically significant superiority of EcN on both days (p\u2009=\u20090.0017 and p\u2009<\u20090.001, respectively).\nIn the present trial, high initial response rates in both groups represent the spontaneous healing known for acute gastroenteritis. The superiority of the EcN treatment became increasingly noticeable from 3. The healing process was markedly faster in the EcN-treated patients than in the patients receiving placebo, a result which underlines the high efficacy of this probiotic.\nThe relatively high number of children with unspecific diarrhoea corresponds quite well to the frequent failure to detect the responsible pathogen in routine analyses. This is the reason why the results of this study are not helpful in answering the question whether EcN is more efficient in bacterial or viral diarrhea. This question should be addressed by future studies.\nIn the present study, EcN was safe and well-tolerated. There was no difference between the EcN and placebo treatments in terms of AEs, body weight, stool examinations and the assessment of tolerance. This result is in accordance with experience from clinical trials in premature and fullterm newborns where EcN was not only very safe but improved the microbial intestinal milieu of the treated infants and reduced the risk of acquiring pathogens early in life [3, 10, 11]. It has also been shown that prolonged colonisation with EcN protected infants at an age of 6\u201312\u00a0months from flatulence, diarrhoea or constipation when given immediately on the first 5\u00a0days after birth [16].\nOur understanding of the effects of probiotics and their numerous modes of action has grown substantially in recent years. With regard to gastroenteritis, probiotics may improve symptoms by several mechanisms, including: \ncompetition with pathogens (for adherence to intestinal epithelium, for growth and survival in the gut) and inhibition of pathogen overgrowth;secretion of bacteriostatic\/bactericidal peptides (e.g. colicins, microcins);enforcement of intestinal barrier function and reduction of microbial translocation;modulation of immune responses (local and\/or systemic, e.g. stimulation of secretion of IgA by lymphocytes and defensins by enterocytes).\nAll of these mechanisms of action have been shown for E. coli strain Nissle 1917. The antagonistic activity of EcN against pathogens has been demonstrated in vitro in animal models and in humans [1, 10, 13, 17]. In a pig model of intestinal infection, EcN was able to prevent acute secretory diarrhoea [15]. Among many other strain-specific characteristics [2, 5, 6], EcN exerts an intense immunomodulatory effect in children [3, 11]. Here, EcN was found to stimulate the production of antibodies of mucosa-associated B lymphocytes and the systemic production of antibodies (IgM, IgA) in premature and fullterm children.\nConclusion\nIn summary, EcN showed a significant superiority to placebo in the treatment of acute diarrhoea in infants and toddlers. EcN treatment also improved the general state of health and its administration was safe and well tolerated.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material \nStatistical analysis (DOC 31 kb)","keyphrases":["probiotic","ecn","acute diarrhoea","infants","toddlers","escherichia coli nissle 1917"],"prmu":["P","P","P","P","P","P"]}
{"id":"Eur_Spine_J-2-2-1602181","title":"Os odontoideum with bipartite atlas and segmental instability: a case report\n","text":"We report on the case of a 15-year-old adolescent who presented with a transient paraplegia and hyposensibility of the upper extremities after sustaining a minor hyperflexion trauma to the cervical spine. Neuroimaging studies revealed atlantoaxial dislocation and ventral compression of the rostral spinal cord with increased cord signal at C1\/C2 levels caused by an os odontoideum, as well as anterior and posterior arch defects of the atlas. The patient underwent closed reduction and posterior atlantoaxial fusion. We describe the association of an acquired instability secondary to an os odontoideum with an anteroposterior spondyloschisis of the atlas and its functional result after 12 months. The rare coincidence of both lesions indicates a multiple malformation of the upper cervical spine and supports the theory of an embryologic genesis of os odontoideum.\nIntroduction\nThe craniovertebral junction is a common site for malformations [10]. Clefts of the anterior and posterior arch of the atlas are rare, but well-documented congenital anomalies [1, 2]. Several reports attributed the aetiology of os odontoideum to an either embryologic, traumatic or vascular basis [3\u20135, 8]. We describe the unusual case of a combined midline cleft of the anterior and posterior arch of the atlas associated with os odontoideum leading to atlantoaxial instability with acute myelopathia after a minor trauma. We presume an embryologic genesis of our findings.\nCase report\nA 15-year-old male patient injured his cervical spine in a hyperflexion trauma when performing a somersault on a trampoline. He presented with transient numbness and weakness of both arms. Initial X-rays and computed tomography demonstrated a displaced os odontoideum which reduced on extension and a rostral compression of the cervical spinal cord due to a subluxation of C1 over C2 with narrowing of the spinal canal to 50%. Additionally midline clefts of the anterior and posterior arch of the atlas became evident (Figs.\u00a01, 2). MRI scans revealed increased cord signals at C1\/C2 levels on T2-weighted images and a persistent subdental synchondrosis was visualized on T2-weighted turbo spin echo sequences (Fig.\u00a03). The patient underwent closed reduction and posterior atlantoaxial fusion by sublaminar tension band wiring with autologous bone grafting and transarticular lag screw fixation. Postoperatively all symptoms improved significantly. Radiographs taken 1\u00a0year after the trauma showed a stable fusion of C1\/C2 (Fig.\u00a04). The patient presented with a range of motion at 30\u00b0 of extension, 40\u00b0 of flexion and 50\u20130\u201340\u00b0 of rotation. He was free of symptoms and had returned to his pre-injury status regarding work and leisure activities.\nFig.\u00a01X-rays of the cervical spine in neutral position (a), flexion (b) and extension (c). Ventral subluxation of C1 over C2 in flexion (b), which reduces on extension (c)Fig.\u00a02CT scans reveal midline clefts of the anterior and posterior arch of the atlas (a). Displaced os odontoideum and subluxation of C1 over C2 with narrowing of the spinal canal (b)Fig.\u00a03T2-weighted MRI images with increased cord signals at C1\/C2 levels and a persistent subdental synchondrosisFig.\u00a04Follow-up X-rays 12\u00a0months after trauma. Stable atlantoaxial fusion after sublaminar wiring and transarticular screw fixation in ap (a) and lateral (b) view\nDiscussion\nThe odontoid process and the atlas originate from the first cervical sclerotome. Body of the axis, lateral masses and posterior arch arise entirely from the second cervical sclerotome. The atlas is formed from three primary ossification centres, which develop during the seventh week of gestation. Two centres at the lateral masses extend posteromedially to form the posterior arch usually in the fourth year. Ossification of the anterior arch involves one or two ossification centres, which extend posterolaterally to fuse with lateral masses around the seventh year. The odontoid process separates from the atlas between the sixth and seventh week of intrauterine life and moves caudally to join the body of the axis (Fig.\u00a05).\nFig.\u00a05Scheme of the embryologic development of atlas and axis\nOs odontoideum is an oval or round shaped ossicle of variable size with a smooth cortical border. Several reports attributed its aetiology to either an embryological, traumatic or vascular basis. Failure of fusion of ossification centres in the odontoid process has been considered to be the main aetiology [8]. Considerable evidence on cases of acquired os odontoideum indicates, that occult fractures with subsequent avascular necrosis might result in similar pathology [5]. O\u2019Rahilly et al. [9] studied the cervical spine of 8-weeks-old human embryos and observed that axis consists morphologically of three parts. Two rostral parts form the odontoid process and a caudal part gives rise to the axis body, separated from the middle part by subdental synchondrosis. A segmentation anomaly of the two rostral parts, which never occurred in normal individuals, may result in bipartite dens [3]. Currarino [3] reported 11 cases with complete or partial segmentation defect in mid odontoid, suggesting an embryological anomaly characterized by a complete segmentation of two rostral parts of axis which may explain the congenital os odontoideum.\nMalformations of the atlas are very rare and include both clefts and aplasias [1, 2]. Galindo and Francis [6] reported the incidence of anteroposterior spondyloschisis of the atlas in normal individuals as 0.3%.\nAtasoy et al. [1] reported the first case of bipartite atlas with os odontoideum causing spinal stenosis. Garg et al. [7] presented a report of bipartite atlas with anterior arch aplasia associated to an os odontoideum. They found a small projection on the anterior surface of the dens and concluded that the ossification centre of anterior arch of atlas may fail to separate from future dens resulting in anterior arch aplasia with a small tubercle attached to the anterior surface of the dens. To our knowledge no other case of bipartite atlas with os odontoideum had been reported previously in the English literature.\nIn our case, there was a malformation of the anterior and posterior arch of the atlas and a persistent subdental synchondrosis, both likely to be a result of a congenital failure of fusion of ossification centres. The associated os odontoideum was clinically silent until a traumatic instability occurred which resulted in acute myelopathy.\nOur findings support the theory of a congenital aetiology of os odontoideum. Both the combined anterior and posterior clefts of atlas and os odontoideum are either asymptomatic or, if cervical instability arises, may develop neurological symptoms.\nConclusion\nWe described a rare association of an anterior and posterior midline cleft of the atlas with an os odontoideum in an adolescent. An embryologic genesis is likely. Minor trauma is commonly the cause for the onset of symptoms, which may occur immediately after the injury, be transitory and experienced repeatedly or have a delayed progressive course. If the lesion is reducible, atlantoaxial fusion is recommended.","keyphrases":["os odontoideum","atlantoaxial instability","c1\/c2 fusion","midline cleft of atlas"],"prmu":["P","P","R","R"]}
{"id":"Purinergic_Signal-2-2-2254478","title":"The E-NTPDase family of ectonucleotidases: Structure function relationships and pathophysiological significance\n","text":"Ectonucleotidases are ectoenzymes that hydrolyze extracellular nucleotides to the respective nucleosides. Within the past decade, ectonucleotidases belonging to several enzyme families have been discovered, cloned and characterized. In this article, we specifically address the cell surface-located members of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase\/CD39) family (NTPDase1,2,3, and 8). The molecular identification of individual NTPDase subtypes, genetic engineering, mutational analyses, and the generation of subtype-specific antibodies have resulted in considerable insights into enzyme structure and function. These advances also allow definition of physiological and patho-physiological implications of NTPDases in a considerable variety of tissues. Biological actions of NTPDases are a consequence (at least in part) of the regulated phosphohydrolytic activity on extracellular nucleotides and consequent effects on P2-receptor signaling. It further appears that the spatial and temporal expression of NTPDases by various cell types within the vasculature, the nervous tissues and other tissues impacts on several patho-physiological processes. Examples include acute effects on cellular metabolism, adhesion, activation and migration with other protracted impacts upon developmental responses, inclusive of cellular proliferation, differentiation and apoptosis, as seen with atherosclerosis, degenerative neurological diseases and immune rejection of transplanted organs and cells. Future clinical applications are expected to involve the development of new therapeutic strategies for transplantation and various inflammatory cardiovascular, gastrointestinal and neurological diseases.\nIntroduction\nExtracellular nucleotides modulate a multiplicity of tissue functions including development, blood flow, secretion, inflammation and immune reactions. Indeed, signaling via extracellular nucleotides has been recognized for over a decade as one of the most ubiquitous intercellular signaling mechanisms [1, 2]. Essentially every cell in a mammalian organism leaks or releases these mediators, and carries receptors for nucleotides of which seven ionotropic (P2X) and at least eight metabotropic (P2Y) receptor subtypes have been identified and characterized to date. Whereas P2X receptors respond to ATP, P2Y receptors can be activated by ATP, ADP, UTP, UDP, ITP, and nucleotide sugars, albeit agonist specificity varies between subtypes and the multiple animal species [3]. Depending on the P2 receptor subtype and signaling pathways involved, these receptors trigger and mediate short-term (acute) processes that affect cellular metabolism, adhesion, activation or migration. In addition, purinergic signaling also has profound impacts upon other more protracted responses, including cell proliferation, differentiation and apoptosis, such as seen in atherosclerosis, degenerative neurological diseases and in several inflammatory conditions [2, 4, 5].\nThe effects of extracellular nucleotides appear to overlap, at least in part, with those of vascular growth factors, cytokines (inflammatory), adhesion molecules and nitric oxide (NO). Nucleotide-mediated activation may be also synergistic with polypeptide growth factors (PDGF, bFGF) and insulin, the signaling being mediated via phospholipase C and D, diacylglycerol, protein kinase C, ERKs, phosphatidylinositol 3-kinases (PI3K), MAP kinases (MAPK) and Rho [6\u20138]. The situation concerning extracellular nucleotide-signaling can be suitably contrasted with the unique specificity of peptide hormones or vasoactive factors for often single, defined receptors [9, 10]. Within purinergic\/pyrimidinergic signaling events specificity is dictated by three essential modulatory components: 1) The derivation or source of the extracellular nucleotides [1, 11, 12]; 2) the expression of specific receptors for these molecular transmitters (and for the nucleotide and nucleoside derivatives) [13\u201316] (See also Molecular Recognition Section of National Institutes of Health, http:\/\/mgddk1.niddk.nih.gov\/ also http:\/\/www.ensembl.org\/index.html and http:\/\/www.geocities.com\/bioinformaticsweb\/speciesspecificdatabases.htm), and, 3) select ectonucleotidases that dictate the cellular responses by the stepwise degradation of extracellular nucleotides to nucleosides [17\u201320].\nEnsembles of ectonucleotidases, associated receptors and signaling molecules\nWithin the past decade, ectonucleotidases belonging to several enzyme families have been discovered, cloned and functionally characterized by pharmacological means. Specifically, we refer here to members of the ecto-nucleoside triphosphate diphosphohydrolase (ENTPDase) family (EC 3.6.1.5) as ectoenzymes that hydrolyze extracellular nucleoside tri-and diphosphates and have a defined pharmacological profile. Most notably, in many tissues and cells, NTPDases comprise dominant parts of a complex cell surfacelocated nucleotide hydrolyzing and interconverting machinery. This ensemble includes the ecto-nucleotide pyrophosphatase phosphodiesterases (E-NPPs), NADglycohydrolases, CD38\/NADase, alkaline phosphatases, dinucleoside polyphosphate hydrolases, adenylate kinase, nucleoside diphosphate kinase, and potentially ecto-F1-Fo ATP synthases [21\u201325] that may interact in various tissues and cellular systems.\nThe ectonucleotidase chain or cascade, as initiated by NTPDases can be terminated by ecto-5\u2032-nucleotidase (CD73; EC 3.1.3.5) with hydrolysis of nucleoside monophosphates [26]. Together, ecto-5\u2032-nucleotidase and adenosine deaminase (ADA; EC 3.5.4.4), another ectoenzyme that is involved in purine salvage pathways and converts adenosine to inosine, closely regulate local and pericellular extracellular and plasma concentrations of adenosine [10, 27].\nSeveral of these ectonucleotidase families and additional functions of NTPDases [28\u201330] are addressed elsewhere in this issue in detail. This review focuses on the surface-located mammalian members of the E-NTPDase protein family. It starts with a brief introduction of molecular structure and functional properties, followed by an analysis of the physiological and pathophysiological roles at various sites with an emphasis on vasculature and neural tissues.\nMolecular identities unraveled\nThe literature on the molecular and functional characterization of the E-NTPDase family has been intensively reviewed [18\u201322, 31\u201336] and will not be repeated here in detail. Our intent is to summarize principal properties of the enzymes that will be of use for the reader new to this field.\nEight different ENTPD genes (Table 1 and Fig. 1) encode members of the NTPDase protein family. Four of the NTPDases are typical cell surface-located enzymes with an extracellularly facing catalytic site (NTPDase1, 2, 3, 8). NTPDases 5 and 6 exhibit intracellular localization and undergo secretion after heterologous expression. NTPDases 4 and 7 are entirely intracellularly located, facing the lumen of cytoplasmic organelles (Fig. 1). The molecular identification of individual NTPDase subtypes, genetic engineering, mutational analyses, and the generation of subtypespecific antibodies have not only led to considerable insight into enzyme structure and function. These advances have also defined physiological and pathophysiological functions of NTPDases in a considerable variety of tissues.\nTable 1Nomenclature of mammalian members of the E-NTPDase family and chromosomal localizationProtein nameAdditional namesGene name human, mouseChromosome location human, mouseAccession number human, mouseNTPDaselCD39, ATPDase, ecto-apyrase [43, 44]ENTPD1, Entpdl10q24, 19C3U87967, NMJ309848NTPDase2CD39L1, ecto-ATPase [49, 109, 252]ENTPD2, Entpdl9q34, 2A3AF144748, AY376711NTPDase3CD39L3, HB6 [50, 177]ENTPD3, Entpd33p21.3, 9F4AF034840, AY376710NTPDase4UDPase, LALP70 [253, 254]ENTPD4, Entpd48p21, 14D1AF016032, NMJ326174NTPDase5CD39L4, ER-UDPase, PCPH [137, 255, 256]ENTPD5, Entpd514q24, 12E (12D1)aAF039918, AJ238636NTPDase6CD39L2 [257\u2013259]ENTPD6, Entpd620p11.2, 2G3AY327581, NM_172117NTPDase7LALP1 [260]ENTPD7, Entpd710q24, 19D1 (19C3)aAF269255, AF288221NTPDase8liver canalicular ecto-ATPase, hATPDase [52, 174]ENTPD8, Entpd89q34, 2A3AY430414, AY364442Information is provided for the human genome from GenBank (http:\/\/www.ncbi.nlm.nih.gov) and mouse genome informatics (MGI) for the mouse genome (http:\/\/www.informatics.jax.org\/). Since the mouse genome represents a composite assembly that continues to undergo updates and changes from build to build, the computed map locations may be corrected in the future.a For mouse Entpd5 and Entpd7, the BLAST analysis displayed in Map Viewer indicates a different map location (in brackets) when compared with the mapping data reported on MGI records using cytoband information based on experimental evidence.Fig. 1Hypothetical phylogenetic tree derived for 22 selected members of the E-NTPDase family (NTPDase1 to NTPDase8) from rat (r), human (h) and mouse (m), following alignment of amino acid sequences. The length of the lines indicates the differences between amino acid sequences. The graph depicts a clear separation between surface-located (top) and intracellular (bottom) NTPDases. In addition, the major substrate preferences of individual subtypes and the predicted membrane topography for each group of enzymes is given (one or two transmembrane domains, indicated by barrels). Modified from [59].\nThe presence of ATP and\/or ADP hydrolyzing activity at the surface of many cell types had been recognized for several decades [17, 37\u201340]. However, the molecular identity of the first member of the ENTPDase family (NTPDase1) was not unraveled and determined until the mid-1990s. The prototypic member of the enzyme family had first been cloned and sequenced as a lymphocyte cell activation (CD39) antigen of undetermined function [41]. Final success came from three independent approaches. Handa and Guidotti [42] purified and cloned a soluble ATP diphosphohydrolase (apyrase) from potato tubers and noted that this protein was related not only to similar enzymes of some protozoans, plants and yeast but also to human CD39. They also recognized conserved sequence domains and the relation to members of the actin-hsp70-hexokinase superfamily. This was then followed by the functional expression of human CD39 and the demonstration that this protein was in fact an ecto-apyrase [43]. In parallel, ectonucleotidases (termed ATP diphosphohydrolases) from porcine pancreas and bovine aorta were purified. The partial amino acid sequences for both ATP diphosphohydrolases revealed identity with the cloned cDNA sequence of CD39 [44]. The cDNA was isolated from human endothelial cells and functional, thromboregulatory studies confirmed that the dominant vascular ectonucleotidase (ATP diphosphohydrolase) activity was identical to the previously described and cloned human CD39 [44]. Several internal peptide sequences obtained from the purified human placental ATP diphosphohydrolase [45] revealed that in retrospect this protein was also identical to CD39.\nIt was originally thought that there existed a single ectonucleotidase of the NTPDase type with potential post-translational modifications [46]. However, a close molecular relative was soon cloned that revealed functional properties of an ecto-ATPase (now NTPDase2) rather than of an ecto-ATP diphosphohydrolase [47, 48]. Further human genomic analysis of expressed sequence tags (ESTs) allowed the identifi-cation of additional members of the gene family [49\u201351]. These genes were originally named CD39L(ike)1 to CD39L4. Then followed the identification, cloning and functional expression of all members of the ENTPDase family, the last to date being NTPDase8 [52].\nPotential splice variants have been isolated for the surface-located NTPDase1 and NTPDase2 [for references see 34, 53]. It should be further noted that heterologous expression of potential splice variants does not necessarily result in the formation of a functional protein [54].\nThe initially proposed nomenclature [50] has been somewhat confusing as it did not meet with generally accepted norms for human cell differentiation molecules [55]. While CD39 (now NTPDase1) indeed belongs to the cluster of differentiation antigens, CD39L1 (NTPDase2), CD39L3 (NTPDase3), CD39L4 (NTPDase5) and CD39L2 (NTPDase6) do not. Scientists at the Second International Workshop on Ecto-ATPases proposed that all E-NTPDase family members be termed as NTPDase proteins and classi-fied in order of discovery and characterization [34, 56]. The CD39 nomenclature should fall away for all but the prototypic member NTPDase1 that already has a long history of use in the Immunology and Oncology fields. Further revisions are however inevitable.\nCatalytic properties\nThe individual NTPDase subtypes differ in cellular location and functional properties. The four cell surface-located forms (NTPDase1,2,3,8) can be differentiated according to substrate preference, divalent cation usage and product formation. All surfacelocated NTPDases require Ca2+ or Mg2+ ions in the millimolar range for maximal activity and are inactive in their absence [34, 57]. They all hydrolyze nucleoside triphosphates including the physiologically active ATP and UTP. Notably, the hydrolysis rates for nucleoside diphosphates vary considerably between subtypes (Figs. 1 and 2). Whereas NTPDase1 hydrolyzes ATP and ADP about equally well, NTPDase3 and NTPDase8 reveal a preference for ATP over ADP as substrate. NTPDase2 stands out for its high preference for nucleoside triphosphates and therefore has previously also been classified as an ecto-ATPase [34, 57]. In contrast to NTPDase1 and NTPDase2, murine NTPDase3 and NTPDase8 are preferentially activated by Ca2+ over Mg2+ [52, 58, 59]. Presumably, differences in sequence but also in secondary, tertiary and quaternary structure account for differences between subtypes in the catalytic properties [60, 61].\nFig. 2Cell surface-located catabolism of extracellular nucleotides and potential activation of receptors for nucleotides (P2 receptors) and adenosine (P1 receptors). The figure depicts the principal catalytic properties of members of the E-NTPDase family and of ecto-5\u2032-nucleotidase. NTPDases sequentially convert ATP to ADP + Pi and ADP to AMP + Pi. NTPDase1 is distinct among these enzymes as it dephosphorylates ATP directly to AMP without the release of significant amounts of ADP. Hydrolysis of the nucleoside monophosphate to the nucleoside is catalyzed by ecto-5\u2032-nucleotidase. NTPDases, NPPs and alkaline phosphatase sometimes co-exist and it seems likely that they can act in concert to metabolize extracellular nucleotides. ATP can activate both P2X receptors and subtypes P2Y receptors whereas UTP activates subtypes of P2Y receptors only. After degradation, ADP or UDP may activate additional subtypes of P2Y receptors. The adenosine formed can potentially act on four different types of P1 receptors and is either deaminated to inosine or directly recycled via nucleoside transporters. Bottom: Profiles of nucleotide hydrolysis and substrate formation by plasma membrane-located NTPDases. The figure compares catalytic properties of human and murine NTPDase1,2,3 and 8, following expression in COS-7 cells. The principal catalytic properties of the respective human and murine enzymes are similar. ATP (\u2022), ADP (\u25aa), AMP (\u2246). Modified from [57].\nMembrane-bound NTPDase1 hydrolyzes ATP almost directly to AMP with the transient production of minor amounts of free ADP (Fig. 2). This functional property largely circumvents activation of P2Y-receptors for nucleoside diphosphates. Interestingly, significant amounts of UDP are accumulated when UTP is hydrolyzed by NTPDase1 [57]. In contrast, ADP is released upon ATP hydrolysis by NTPDase2, then accumulates and is slowly dephosphorylated to AMP. On the one hand, this results in the removal of agonists for nucleoside triphosphate-sensitive P2Y-receptors (Fig. 2). On the other hand, it generates agonists for nucleoside diphosphate-sensitive receptors such as platelet P2Y1 and P2Y12 receptors [62]. The actions of NTPDase3 and NTPDase8 result in intermediate patterns of product formation, leading to a transient accumulation of diphosphonucleosides with the simultaneous presence of triphosphonucleosides.\nPrincipal structural features\nThe hallmarks of all NTPDases are the five highly conserved sequence domains known as \u2018apyrase conserved regions\u2019, abbreviated and termed ACR1 to ACR5 [42, 63, 64] that are involved in the catalytic cycle. This notion is supported by a considerable variety of deletion and mutation experiments [for reviews see 30, 34, 64\u201368].\nNTPDases share two common sequence motifs with members of the actin\/HSP70\/sugar kinase superfamily, the actin-HSP 70-hexokinase b-and g-phosphate binding motif [(I\/L\/V)X(I\/L\/V\/C)DXG(T\/S\/G)(T\/S\/G)XX(R\/K\/C)] [42, 47, 69, 70], with the DXG sequence strictly conserved. These motifs are identified in ACR1 and ACR4. Furthermore, there are striking similarities in secondary structure with members of the actin\/HSP70\/sugar kinase superfamily [30, 59, 71]. These proteins are soluble, have ATP phosphotransferase or hydrolase activity, depend on divalent metal ion and tend to form oligomeric structures. In spite of negligible global sequence identity they share the principal structure of two major domains (I and II, possibly resulting from gene duplication) of similar folds on either side of a large cleft. They reveal similar conserved secondary structure topology (\u03b21\u03b22\u03b23\u03b11\u03b24\u03b12\u03b25\u03b13) repeated in each domain and fold into a pocket for substrate binding at the bottom [59]. Presumably, NTPDases share not only secondary structure but also major elements of tertiary structure with members of the actin\/HSP70\/sugar kinase superfamily (Fig. 3). Homology modeling of the NTPDase3 sequence reveals high degrees of structural fold similarity with a bacterial exopolyphosphatase (PDB 1T6C) that further refine structural predictions for members of the E-NTPDase family [30, 72].\nFig. 3Hypothetical membrane topology of a surface-located NTPDase with two transmembrane domains. A comparison of the conserved secondary structure reveals duplicate conservation of two major domains related to subdomains Ia and IIa of actin, and other members of the actin\/HSP70\/sugar kinase superfamily [59]. In contrast to the other members of the superfamily, surface-located NTPDases are anchored to the plasma membrane by terminal hydrophobic domains. The figure takes into account the close distance of the N-and C-terminus of actin at domain I and the binding of ATP (red) in the cleft between domains I and II [80]. These two domains are expected to undergo conformational changes involving movement relative to each other.\nNTPDases readily form homo-oligomeric assemblies. NTPDase1 to NTPDase3 were found as dimers to tetramers [29, 64, 73\u201378]. In contrast to the P2X receptors that share a similar membrane topography, hetero-oligomeric complexes between NTPDases have not been reported, to date. Oligomeric forms reveal increased catalytic activity [73, 75, 76] and the state of oligomerization can affect catalytic properties [77, 78].\nNTPDase1,2,3, and 8 are firmly anchored to the membrane via two transmembrane domains that in the instance of NTPDase1 are important for maintaining catalytic activity and substrate specificity [29, 64, 79]. The two transmembrane domains interact both within and between monomers. They may also undergo coordinated motions during the process of nucleotide binding and hydrolysis [29, 61]. This could in turn induce conformational changes [80] involving movement of the two major domains (I and II) relative to each other (Fig. 3). Alterations in quarternary structure and subunit interactions may thus affect the impact or interaction of ACRs involved in substrate binding and hydrolysis. Whether posttranslational modifications such as protein phosphorylation contribute to this dynamic behavior remains to be investigated.\nFunctional modifications\nBiologically active NTPDase1 is subject to differential forms of surface modification under conditions of oxidative stress that inhibit enzymatic activity, as influenced by unsaturated fatty acids [81, 82]. It also undergoes limited proteolysis that increases enzyme activity and differential glycosylation reactions that appear to be required for membrane expression [64]. Since the surface-located ATP-hydrolyzing members of the NTPDase family pass through the endoplasmic reticulum and Golgi apparatus, the associated catalytic activity might abrogate ATP-dependent luminal functional processes. NTPDase1 becomes catalytically active on reaching the cell surface and glycosylation reactions appear crucial in this respect [83].\nThe N-terminal intracytoplasmic domain of NTPDase1 is palmitoylated. Truncated forms of NTPDase1 lacking the N-terminal intracytoplasmic region and the associated Cys13 residue, are not subject to palmitoylation. This post-translational modification appears to be constitutive and to contribute to the integral membrane association of this ectonucleotidase in lipid rafts [84\u201386]. This raises the possibility that NTPDase1 may be recycled to and from cell membranes via sequential actions of putative palmitoyltransferases and palmitoyl-protein thioesterases [87], in order to fine tune and modulate purinergic signaling responses. In contrast to NTPDase1 and NTPDase3, NTPDase2 does not have the required intra-cytoplasmic Cys to undergo this post-translational modification. The potential multimerization of NTPDase1 [35] may be facilitated by acylation with intermolecular interactions within the cholesterol and sphingolipid-rich microdomains of the plasma membrane [88]. Experiments using endothelial cells from caveolin-1 deficient mice suggest that caveolae are not essential for the enzymatic activity or for the targeting to the plasma membrane of NTPDase1. However, cholesterol depletion results in a strong inhibition of the enzyme [86]. The targeting of palmitoylated NTPDase1 to lipid rafts could influence defined G-protein coupled receptors within this plasmalemmal microenvironment and thus regulate cellular signal transduction pathways. Furthermore, the caveolar co-localization of ecto-5\u2032-nucleotidase, P2 receptors, and NTPDase1 could serve to modulate signaling via both ATP and adenosine at the cell surface and possibly also within endosomal compartments [20].\nTranscriptional regulation of expression\nMembers of the E-NTPDase family are constitutively expressed in many tissues. To date, there is only scattered evidence on promoters and the factors controlling NTPDase expression [22]. The transcription of NTPDase1\/CD39 is constitutive in venous, arterial and certain non-fenestrated microvascular endothelium and certain immune cells e.g., B cells, dendritic cells and defined T-cell subsets [20, 89]. The modulated expression of NTPDase1 has been closely associated with inflammatory cytokines, oxidative stress and hypoxia in vitro and in vivo [19, 90].\nExpression of NTPDase1 is increased in differentiating melanomas followed by a gradual decrease with tumor progression [91] and enhanced NTPDase1 activity of stimulated endothelial and mesangial cells is downregulated by glucocorticosteroids [92]. Activity of \u2018ecto-ATP diphosphohydrolase\u2019 in human endothelial cells in-vitro is increased by aspirin [93] and glomerular \u2018ecto-ATP diphosphohydrolase\u2019 immunoreactivity might well be modulated by estradiol [94].\nTranscription of NTPDase2 in mouse hepatoma cells is inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) [95]. These cells contain both constitutive and TCDD-inducible NTPDase2 activity. The NTPDase2 core promotor reveals constitutive activity that is independent of TCDD [96]. TCDD does not increase expression of NTPDase1, NTPDase3 or other ectonucleotidases and apparently fails to induce NTPDase2 in a variety of other cell lines derived from varying species [97]. In rat Sertoli cells, NTPDase2 is upregulated by follicle-stimulating hormone and cAMP [98] and it is selectively downregulated in biliary cirrhosis [99]. Human epidermoid carcinoma cells increase the cascade for extracellular nucleotide hydrolysis when periodically treated with extracellular ATP, suggesting that the substrate itself may affect the expression of its own hydrolysis chain [100].\nInhibitors\nA considerable number of compounds alter and inhibit extracellular nucleotide hydrolysis by NTPDases. These include non-hydrolysable nucleotide analogues and inhibitors of P2 receptors. Ideally, NTPDase inhibitors should not be P2 receptor agonists or antagonists and not be subject to dephosphorylation by the ectoenzyme [22, 101, 102]. The only commercially available compound reported to effectively inhibit hydrolysis of ATP in a variety of tissues without significantly acting on purinoceptors is the structural analogue of ATP, ARL 67156 (FPL 67156) (6-N, N-diethyl-D-\u03b2,\u03b3-dibromomethylene ATP) [103\u2013105]. Other potential inhibitors include 8-thiobutyladenosine 5\u2032-triphosphate (8-BuS-ATP) [106] and 1-naphthol-3, 6-disulfonic acid (BG0136) [101]. Periodate-oxidized ATP inhibits ecto-ATPase activity in 1312N1 human astrocytoma cells [107] and Gadolinium ions have been found to effectively inhibit the ecto-nucleoside triphosphate diphosphohydrolase from Torpedo electric organ as well as potato apyrase [108].\nIt is noteworthy that the potency of inhibitors can vary considerably between individual members of the E-NTPDase family [109\u2013111]. This necessitates a functional evaluation of each inhibitor for the enzyme investigated in a given tissue or cell type. The failure to develop specific inhibitors remains a major impediment to ongoing discoveries.\nPrincipal functional contexts\nCell surface-located NTPDases are considered to be of major importance for controlling the availability of extracellular nucleotide agonists at P2 receptors. They also contribute to recycling of nucleosides derived from extracellular nucleoside phosphates and metabolic salvage pathways. The number of studies that define a functional impact of individual NTPDases in purinergic signaling in situ is limited and has been dependent to date on global genetic modifications of mice and swine to delete or upregulate the NTPDase or P2 gene of interest [20]. Subtype-specific inhibitors, siRNA approaches, and animals in which the encoding gene can be inactivated or selectively induced in specific tissues will be of major importance.\nThere is increasing experimental evidence that ectonucleotidases compete with P2 receptors for a limited pool of endogenously released nucleotide [112, 113] and \u2014by hydrolyzing released nucleotide \u2014terminate or modulate the function of P2 receptors [114\u2013116]. Portal fibroblasts regulate P2Y receptormediated bile duct epithelial proliferation via expression of NTPDase2 [117] (see liver section, below). NTPDases functionally interact with P2Y-receptors [112] and may also co-localize with these G-proteincoupled receptors (GPCR) in lipid rafts and possibly caveolae [118\u2013121]. The modulatory effects of NTPDases are complex as the enzymes differentially regulate agonist availability in a process that is dependent upon P2 receptor subtype by either degrading ATP\/UTP or by generating ADP\/UDP (Fig. 2).\nRecent experiments suggest that plasma membranebound NTPDases may have functions distinct from their catalytic properties alone. In a yeast two-hybrid system using techniques developed by Zhong for yeast apyrases [122], the N-terminus of human NTPDase1 (used as bait protein) has been shown to interact with truncated Ran Binding Protein M (RanBPM, otherwise known as RanBP9, NM_005493) in the human library screened [122a]. RanBPM contains conserved SPRY (repeats in splA and RyR) domains that appear to be crucial for the interaction with NTPDase1 and is preferentially distributed in human heart tissues [123]. RanBPM is known to interact with Sos and regulate ERK\/Ras signaling. NTPDase1 interacts with RanBPM to directly modulate Ras activation and cellular proliferation in liver regeneration following partial hepatectomy [124].\nThe N-termini of NTPDases also have consensus sequences for protein phosphorylation by protein kinase C [47] that could have additional functional impacts. Furthermore, the C-terminal sequence of NTPDase1 contains a putative PDZ domain (-K-DM-V). This may have utility in determining interactions with select P2Y receptors e.g., the purinergic P2Y1 and P2Y2 receptors that terminate in -D-T-S-L and -D-I-R-L, respectively [125]. PDZ domains are most often found in combination with other protein interaction domains (for instance, SH3, PTB, WW), participating in complexes that facilitate signaling or determine the localization of receptors [126\u2013128].\nFinally, the general membrane topography of NTPDase1 and oligomeric assembly resemble the morphology of channel forming proteins such as P2X nucleotide receptors and members of the epithelial Na+ channel\/degenerin gene superfamily [129]. This raises the question whether, in addition to their catalytic activity, NTPDases could function as channels. Release of ATP from Xenopus oocytes induced by hyperpolarizing pulses requires functional ecto-ATPase activity [130]. To what extent this functional property is shared by the structurally related NTPDase2, NTPDase3 and NTPDase8 has not been investigated.\nVasculature\nThe normal vascular endothelium provides a barrier that separates blood cells and plasma factors from highly reactive elements of the deeper layer of vessel wall. The vessel wall maintains blood fluidity and promotes flow by inhibiting coagulation, platelet activation and promoting fibrinolysis [131]. These properties are governed by important thromboregulatory mechanisms; key biological activities of the vasculature have been already identified and shown to be ecto-nucleotide catalysts that generate the respective nucleosides by phosphohydrolysis [19, 82]. The dominant ectonucleotidases of the vasculature have now more fully been characterized as NTPDases. This important biological property expressed by the endothelium and associated cells is responsible for the regulation of extracellular and plasma levels of nucleotides [20, 44, 82, 132, 133].\nOver the past decade, extracellular nucleotides have been recognized as important mediators of a variety of processes including vascular inflammation and thrombosis with varying impacts in different systems [19]. Adenosine and ATP mediated effects or mechanisms can be implicated in the local control of vessel tone as well as in individual vascular cell migration, proliferation and differentiation. As an example, ATP may be released from sympathetic nerves (see later sections) and results in constriction of vascular smooth muscle through effects mediated by P2X receptors. In contrast, ATP released from endothelial cells during changes in flow (shear stress) or following exposure to hypoxic conditions activates P2Y receptors in a paracrine manner to release NO, resulting in vessel relaxation. Any nucleotide released will be ultimately hydrolyzed to adenosine and will result in vasodilatation via the effects of smooth muscle P1 receptors. P2X receptors also appear on vascular cells and are associated with changes in cell adhesion and permeability [2]. These cellular processes and nucleotidetriggered events are modulated during angiogenesis (Fig. 4) and influence the development of atherosclerosis and restenosis following angioplasty [2, 113, 134\u2013136].\nFig. 4Angiogenesis with expression of NTPDase1 in the vasculature of syngeneic islet transplants. Mouse islets were prepared from wild type and Entpd1 null mice, as described by T. Maki et al. and transplanted under the renal capsule [261]. Islets were harvested at four weeks (n = 4 per group) and stained for NTPDase1 immunoactivity and other markers of EC. Substantially diminished levels of CD31 staining vascular elements were also present in null to Entpd1 null grafts, indicating a defect in new vessel growth (not depicted here). A) Wild type to wild type showing grafted islet vasculature staining for NTPDase1 with adjacent normal renal vascular pattern. B) Wild type to null mouse showing intrinsic vasculature of islet has persisted within the graft and even entered the NTPDase1 null renal parenchyma. C) Null to wild type grafts showing infiltrating macrophages and NTPDase1 positive endothelium migrating from recipient (confirmed by other stains; not shown).\nNTPDase1 is the major ectonucleotidase in the vasculature [112]. Other NTPDases associated with the vasculature are the cell-associated NTPDase2 and the soluble monocyte expressed NTPDase5 [32, 50, 137]. The phosphohydrolytic reaction of NTPDase1 limits the platelet activation response that is dependent upon the paracrine release of ADP and activation of specific purinergic receptors [81, 132, 138]. In contrast, NTPDase2, a preferential nucleoside triphosphatase, activates platelets by converting the competitive antagonist (ATP) of platelet ADP-receptors to the specific agonist of the P2Y1, and P2Y12 receptors. In keeping with these biochemical properties, NTPDase1 is dominantly expressed by endothelial cells and the associated vascular smooth muscle where it serves as a thromboregulatory factor. In contrast, NTPDase2 is associated with the adventitial surfaces of the muscularized vessels, microvascular pericytes of some tissues and organs as the heart and the stromal cells and would potentially serve as a hemostatic factor [62].\nExtracellular nucleotide stimulation of P2 receptors represents components of platelet, endothelial cell and leukocyte activation that culminate in vascular thrombosis and inflammation in vivo [19]. In these inflammatory settings, with oxidant endothelial injury, NTPDase1 biochemical function is substantially, albeit temporarily, decreased because of post-translational changes; reconstitution of vascular NTPDase activity occurs following transcriptional upregulation of CD39 in endothelium [82, 139]. This functional change may relate, at least in part, to alterations in acylation and associated membrane lipid association with consequent disruption of multimer structure. Interestingly, palmitate supplementation may protect against loss of NTPDase activity following cellular activation in vitro [81]. These observations may provide several avenues of research to augment NTPDase activity within the vasculature at sites of injury [134].\nMechanisms of endothelial cell activation by nucleotides\nATP and UTP increases intracellular calcium levels, results in cytoskeletal rearrangements and stimulates phosphorylation of several proteins in human endothelial cells (EC) that are also associated with integrin signaling [140\u2013142]. These include the focal adhesion kinase (FAK) and paxillin, proline-rich tyrosine kinase 2 (Pyk2) (also named related adhesion focal tyrosine kinase, RAFTK) and p38 MAP kinase. Further, UTP preferentially increases EC migration in a PI3-kinase and ERK-dependent manner. Moreover, extracellular nucleotide-mediated EC activation involves cytoskeletal rearrangements and increases in cell motility, comparable to that seen with ligation of integrins by extracellular matrix proteins [143]. These phenotypic changes (seen in both nucleotide-and matrix-mediated activation) are associated with tyrosine phosphorylation of FAK, paxillin and p130 Crk-associated substrate (p130cas) and down-stream activation of p38 MAP kinases. FAK has been implicated to play an important role in integrin-mediated signal transduction pathways [144], suggesting that P2-receptors are implicated in \u2018inside-out\u2019 integrin signaling in EC, as well as platelets [20, 112].\nTherapeutic considerations\nTo test how extracellular nucleotide-mediated signaling influences pathophysiological events, several techniques have been developed and validated to manipulate NTPDase1 expression in the vasculature and to study conditions of inflammatory stress. The first mutant mouse derived and studied concerned the global deletion of the gene encoding the dominant ectonucleotidase NTPDase1 (Entpd1,cd39). The mutant mice exhibit major perturbations of P2 receptormediated signaling in the vasculature and immune systems [19, 89, 145]. These phenomena manifest as hemostatic defects, thromboregulatory disturbances, heightened acute inflammatory responses with a failure to generate cellular immune responses that are all associated with vascular endothelium, monocyte, dendritic cell and platelet integrin dysfunction [20, 112, 134].\nThe therapeutic potential of NTPDase1 to regulate P2 receptor function in the vasculature and mitigate against thrombotic\/inflammatory stress has been further established by the generation of NTPDase1 transgenic mice and swine [20, 146], the use of adenoviral vectors to upregulate NTPDase1 in cardiac grafts [147] and the use of soluble derivatives of NTPDase1 and apyrases [133, 148]. The beneficial effects of administered NTPDases have been determined in several animal models of vascular inflammation [148, 149]. Exogenous infusions of soluble NTPDases are able to rescue Entpd1-deficient mice from systemic toxicity induced by ischemia reperfusion injury and after stroke induction [145, 150].\nAngiogenesis requires the dynamic interaction of endothelial cell proliferation and differentiation with orchestrated interactions between extracellular matrix and surrounding cells (such as vascular smooth muscle and\/or pericytes) [151\u2013153]. NTPDase1 appears crucial in the co-ordination of angiogenic responses in inflammation, organ remodeling and transplantation [20, 134]. For example, in syngeneic pancreatic islet transplantation, the maintenance and revascularization of grafted islets appears dependent upon expression of NTPDase1 by the developing vasculature within the islet (Fig. 4).\nIn summary, multiple experimental studies largely reveal beneficial effects of over-expression of NTPDases within the vasculature, or by their pharmacological administration [20, 133]. Clinical studies of these soluble thromboregulatory factors are in planning [20, 154, 155].\nImmune system\nThere are multiple P2X and P2Y receptor subtypes expressed by monocytes and dendritic cells, whereas lymphocytes express only P2Y receptors [2]. NTPDase1\/CD39 was first described as a B lymphocyte activation marker and also shown to be expressed on activated T cells [156, 157] and dendritic cells [89]. The CD39 enzymatic function on dendritic cells is involved in the recruitment, activation and polarization of naive T cells. ATP is released by CD4+ and CD8+ T cells upon stimulation with Con A or anti-CD3 mAb while CD39 functions as an additional recognition structure on haptenated target immunocytes for HLA-A1-restricted, hapten-specific cytotoxic T cells [156, 157].\nIn cd39 null mice, there are major defects in dendritic cell function antigen presentation and T-cell responses to haptens (type IV hypersensitivity reactions) [19, 89]. Immunocyte-associated CD39 may play an immunoregulatory role by hydrolyzing ATP (and perhaps ADP) released by T cells during antigen presentation and thereby generating adenosine [19, 89, 158]. Ectoenzymes, including ectonucleotidases, are known to play an important role in leukocyte trafficking (for an excellent review on this topic, see [159]).\nRecent work has indicated that regulatory CD4 + ve CD25 + ve T cells (Treg cells) play important roles in the maintenance of immunological reactivity and tolerance [160]. The selective expression of CD39 by Treg and the question whether this ectonucleotidase and\/or extracellular nucleotides influence(s) the function of these interesting cells is a focus of current work.\nDigestive and renal systems\nReleased nucleotides are polarized and do not re-enter cells. They have to be transformed into the corresponding nucleosides that enter cells via specific transporters to rebuild nucleoside pools. If this did not occur, they would be lost from the metabolic pool. The same may pertain to dietary ingestion of nucleotides where NTPDases are potential participants in the digestion of exogenous nucleotides and intestinal function. In addition, extracellular nucleotide and adenosine receptors are highly expressed in the digestive and renal systems, so these molecules are likely to have homeostatic functions [2].\nAn important nucleotide-mediated mechanism that seems common to various epithelia, as well as to hepatocytes, involves the autocrine regulation of cell volume by ATP via P2 receptors [161, 162]. As P2 receptors are expressed by epithelia in a polarized manner and can be linked to several digestive and homeostatic functions [163, 164], the presence of NTPDases in the immediate environment may serve as regulatory switches.\nLiver\nIn the liver, extracellular nucleotides are potentially involved in several functional contexts [161]. There is evidence that extracellular nucleotides regulate glycogenolysis through activation of glycogen phosphorylase and inactivation of glycogen synthase by inhibition of the glucagon effect on cAMP and by the activation of phospholipase D [165, 166]. In addition, nucleotides may be involved in the regulation of canalicular contraction and bile flow [167\u2013169]. Concentrations of canalicular adenine nucleotides in bile samples and effluents from hepatic cell lines are estimated to be around 0.1 to 5 \u00b5M [161, 168]. Hepatocytes and bile duct cells have been shown to interact and communicate via local ATP release in vitro [170]. Extracellular ATP acts as a hepatic mitogen and activates JNK signaling and hepatocyte proliferation both in vitro and in vivo [171].\nSeveral ectonucleotidases are expressed in liver. Of the nucleotide pyrophosphatase\/phosphodiesterases, NPP1 (PC-1) is expressed on the basolateral membrane of hepatocytes while the closely related NPP3 (B10) has a predominant canalicular in distribution [172, 173]. NTPDase1 is highly expressed on larger vessels and more weakly on sinusoids as well as in Kupffer cells [174]. In the quiescent liver, NTPDase2 is expressed by cells of the subendothelium of veins and adventitial cells of arteries, but not in sinusoids. In addition, NTPDase2 is expressed by portal fibroblasts near basolateral membranes of bile duct epithelia [175]. Activated but not quiescent hepatic stellate cells express NTPDase2 at the protein level [176]. Only low expression of NTPDase3 could be demonstrated at the mRNA level in the liver [50, 177].\nNTPDase2 expression in portal fibroblasts, the primary fibroblastic cell type of the portal area, suggests a role in the regulation of bile ductular signaling and secretion [161, 175]. Jhandier et al. tested the hypothesis that portal fibroblast NTPDase2 regulates epithelial cell proliferation. Using co-cultures of cholangiocytes (Mz-ChA-1 human cholangiocarcinoma cells) and primary portal fibroblasts from rat liver, increased NTPDase2 expression decreased cell proliferation, and knockdown of NTPDase2 by siRNA increased proliferation. P2 receptor blockade also attenuated Mz-ChA-1 proliferation [117]. These experiments defined a novel cross-talk signaling pathway between bile duct epithelial cells and underlying portal fibroblasts, regulated by NTPDase2. Because they are the chief fibrogenic cells of the liver, hepatic stellate cells and portal fibroblasts are important targets of liver disease therapy. Loss of NTPDase2 expression in human biliary cirrhosis, as well as in models of bile duct ligation in rat, has been observed. NTPDase2 expression also shifts from the portal area to bridging fibrous bands in cirrhosis with hepatitis C [99].\nFunctional ATPases were previously shown to be associated with bile canalicular plasma membranes by histochemical techniques [178]; the corresponding enzyme was subsequently incorrectly identified as cCAM105 [179\u2013181]. More recent studies revealed that the canalicular ecto-ATPase corresponds to NTPDase8 [52], also referred to as hepatic ATP diphosphohydrolase (ATPDase) [174, 182]. NTPDase8 is the mammalian orthologue of the chicken ecto-ATPDase cloned from oviduct and liver [183, 184]. In tandem with ecto-5\u2032-nucleotidase, NTPDase8 has the potential to regulate the concentration of nucleotides in the hepatic canalicule. The ultimate generation of extracellular adenosine from dephosphorylated ATP not only activates adenosine receptors but also produces the key molecule for purine salvage and consequent replenishment of ATP stores within many cell types [17, 185]. Adenosine transporters are of major importance to organs and cells incapable of de novo nucleotide synthesis such as brain, muscle, intestinal mucosa and bone marrow [167, 186]. As the liver appears to be a major source of purines for these tissues, curtailment of nucleotide loss into the bile may be important to maintain appropriate nucleotide\/nucleoside concentrations within hepatocytes [185]. Thus, dephosphorylation of nucleotides by ectonucleotidases may be critical for appropriate systemic purine homeostasis [167]. The presence of NTPDase8, ecto-5\u2032-nucleotidase and nucleoside transporters in the canalicular domain of hepatocytes would be consistent with an important role of NTPDase8 in purine salvage.\nThe exocrine pancreas\nThe exocrine pancreas secretes digestive enzymes and a HCO3-rich fluid. Acini release ATP and the excurrent ducts express several types of P2 receptors [187, 188]. Thus ATP may function as a paracrine mediator between pancreatic acini and ducts. Ectonucleotidase activity in pancreatic tissues was first detected in the rat in the 1960s [189, 190], followed by analyses in the pig [191, 192]. Cytochemical and biochemical observations have corroborated the association of ATPase activity with zymogen granules [193]. In other studies of small intercalated\/interlobular ducts, NTPDase1 immuno-fluorescence can be localized on the luminal membranes, while in larger ducts it is localized on the basolateral membranes [194]. Upon stimulation with cholecystokinin octapeptide-8 (CCK-8), acinar NTPDase1 relocalized in clusters towards the lumen and is secreted into the pancreatic juice, as an active form associated with particulate fractions [188, 195]. As revealed by electron microscopy, NTPDase2 is located on epithelial cells, myoepithelial cells and the basolateral membrane of acini. Interestingly, NTPDase2 could be also detected at the basolateral surface of endothelial cells [194].\nSalivary glands\nThere are only few studies on the localization of NTPDases in salivary glands. NTPDases might play a role in the transport of electrolytes by modulating the extracellular ATP concentration in the salivary gland ducts. NTPDase1 reveals to be mainly vascular in expression. NTPDase2 was immunodetected on myoepithelial cells and in nerves [194, 196]. The immunolocalization of NTPDases 3 and 8 in salivary glands has not yet been determined.\nKidney\nThe kidney reveals a complex cellular profile of expression for P1 and P2 receptors as well as of ectonucleotidases. Both ATP and adenosine have been invoked in the regulation of tubuloglomerular feedback [197, 198]. This feedback system links the salt concentrations in the tubular fluid at the macula densa to the vascular tone of the afferent arteriole of the same nephron. As depicted by their localization, NTPDases may participate in the regulation of several biological functions of the kidney, including vascular perfusion. In mouse, rat and porcine kidneys, NTPDase1 can be detected in vascular structures, including blood vessels of glomerular and peritubular capillaries [174, 199, 200]. NTPDase2 is detected on the Bowman\u2019s capsules of mouse and rat [199] and NTPDase8 on the luminal side of porcine renal tubules [174]. More recently, an immunohistochemical analysis of various ectonucleotidases of the rat nephron revealed expression of both NTPDase2 and NTPDase3 in the thick ascending limb, the distal tubule and the inner medullary collecting ducts. In addition, NTPDase3 is located in the cortical and outer medullary collecting ducts [201].\nThe nervous system\nAll cell types of the nervous system express nucleotide receptors [2]. It is increasingly apparent that NTPDases are distributed in the nervous system as ubiquitously as are P2 receptors and that these ectoenzymes are directly involved in the control of P2 receptor function in nervous tissues [22, 31, 36]. Signaling via nucleotides is widespread both in the peripheral and central nervous system. Major nucleotide receptormediated functions in the central nervous system include the modulation of synaptic signal transmission [202], the propagation of Ca2+ waves between glial cells [203], or the control and activation of astrocytes and microglia [204, 205]. In addition, ATP can contribute to synaptic signal transmission [36]. In the sympathetic nervous system, ATP acts as a fast neurotransmitter together with catecholamines [206], it is an important mediator of central and peripheral chemosensory transduction, including pain [207] and it is involved in the control of myelination formation of peripheral axons [208].\nCentral nervous system\nATP can be rapidly hydrolyzed to adenosine at brain synapses that in turn activates pre-or postsynaptic receptors, thereby modulating neuronal transmission. Adenine nucleotides undergo conversion to adenosine within a few hundred milliseconds in the extracellular (synaptic) space of rat brain slices [209, 210]. Complex synaptic interactions in the central nervous system may thus be modulated both by the activation of P2 and (after hydrolysis of the nucleotide) P1 receptors that may be located at identical or different cellular targets [202, 211].\nBased on immunoblotting and in situ hybridization, NTPDase1, 2 and 3 are expressed in the mammalian brain [47, 57, 59, 116, 177, 212]. NTPDase1 and 2 have been purified from porcine brain [213, 214]. But the exact cellular allocation of individual subtypes is still a challenge. There is ample evidence from early enzyme histochemical investigations that surface-located catalytic activity for the hydrolysis of nucleoside tri-and diphosphates can be allocated to all cell types of the nervous system [for reviews see 22, 31, 36, 215]. This catalytic activity can be localized to synapses, including the synaptic cleft, at the surface of neurosecretory nerve terminals in the pituitary or at peripheral nerve terminals. These data imply a wide distribution of cell surface-located ATP hydrolyzing activity in the CNS.\nNeurons\nEcto-ATPase activity has been observed in synaptosomal fractions isolated from various sources, implying endogenous ectonucleotidase activity of nerve cells. Biochemical studies on isolated synaptosomes permit the determination of the ratios of ATP to ADP hydrolysis rates as well as the analysis of product formation. Total synaptosome fractions isolated from rat brain cortex and immunopurified cholinergic striatal synaptosomes revealed ratios of 3.4: 1 and 2.1: 1, respectively [216]. ADP was found to transiently accumulate after addition of ATP, and was subsequently metabolized to AMP and adenosine. Similar results were obtained with hippocampal synaptosomes [217]. This strongly argues against a major contribution by NTPDase1 and NTPDase2 and would rather be compatible with a neuronal expression of NTPDase3 (comp. Fig. 2). A recent immunocytochemical study allocates NTPDase3 to neurons including axon-like structures of various brain regions [218].\nAstrocytes, oligodendrocytes, and microglia\nThe ratio of ATP to ADP hydrolysis is clearly different in cultured astrocytes. Astrocytes cultured from cortex or hippocampus display a ratio of 8: 1 [219]. Furthermore, cultured rat cortical astrocytes accumulate ADP from ATP that is only very slowly further degraded to AMP [220]. This would be largely compatible with NTPDase2 as the major ectonucleotidase of cultured astrocytes. Immunocytological investigations of adult rat and mouse brain sections assign NTPDase2 solely to the astrocyte-like stem cells in the subventricular zone of the lateral ventricles and the dentate gyrus of the hippocampus and to astrocytes in few distinct additional brain regions [221, 222]. Thus, cultured astrocytes may reveal functional properties that differ from the in situ situation as they tend to rapidly alter their protein expression profile [223].\nEnzyme histochemistry assigns ecto-ATPase activity to both central and peripheral myelin [31], but fully supplementary immunocytochemical data are lacking. Enzyme histochemical staining for surface-located nucleoside diphosphate activity has long been used to identify microglia in tissue sections of the adult and developing brain [224]. The major microglial ectonucleotidase has been identified as NTPDase1 [225].\nStem cells in the adult mammalian brain\nIn the adult rodent brain, neurogenesis persists in two restricted regions, the subventricular zone (SVZ) of the lateral ventricles and the dentate gyrus of the hippocampus. These regions contain stem cells that give rise to neurons throughout the life span of the animal. Interestingly, these cells share astrocytic properties [226]. They generate highly proliferating intermediate cell types and finally mature neurons. NTPDase2 is highly and selectively expressed by the stem cells (type B cells) of the SVZ [221] (Fig. 5) as well as by the progenitor cells (residual radial glia) of the dentate gyrus [222]. In the presence of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2), SVZ-derived stem cells can be cultured as free floating cellular aggregates (neurospheres). Cultured stem cells express NTPDase2 and functional P2 receptors. Agonists of P2Y1 and P2Y2 receptors augment cell proliferation, whereas inhibition of the receptors attenuates cell proliferation in spite of the presence of mitogenic growth factors [227]. These data suggest that NTPDase2 and nucleotides, together with other signaling pathways, contribute to the control of neurogenesis in the adult mammalian brain.\nFig. 5Detail of arrangement of neuronal stem cells and neuroblasts at the lateral lining of the mouse subventricular zone (SVZ) (triple labeling). A) DAPI staining of all nuclei. Arrow heads mark endymal lining. B) Stem cells (type B cells) immunopositive for NTPDase2 form tube-like sheeths around clusters of migrating immature neurons (type A cells) that immunostain for the microtubule-associated protein doublecortin (DCX) (C). The spaces covered by type A cells remain dark in (B) and are indicated with stars. D) Merge of B) and C). E) Merge of A), B) and C). Bar = 10 \u00b5m. (by courtesy of David Langer, Frankfurt am Main).\nApparently, individual enzyme isoforms govern cell surface-located nucleotide hydrolysis in the various cell types of the central nervous system. This does not exclude however, the possibility that individual cell types express more than one isoform with one of the enzymes predominating. For example, PC12 cells express mRNA for NTPDase1-3. But the ATP\/ADP hydrolysis ratio, the pattern of product formation and the immunocytochemical surface staining suggest that NTPDase3 is the major functional isoform [59, 228]. Similarly cultured normal and immortalized pituitary and hypothalamic cells express NTPDase1-3 [116]. The future planned use of transgenic mice expressing fluorescent protein under the promoter of the respective NTPDase isoform will greatly facilitate the identification of the expression pattern of individual enzyme isoforms in the developing and adult nervous system.\nPeripheral nervous system\nNoradrenaline and ATP are co-released from sympathetic nerve terminals of the guinea pig heart whereby ATP enhances noradrenaline release by a mechanism controlled by ectonucleotidases, possibly NTPDase1 [229]. Interestingly, stimulated sympathetic nerves of the guinea pig vas deferens release not only ATP and noradrenaline but also enzyme activity that degrades ATP to adenosine. The latter exhibits similarities to NTPDases and ecto-5\u2032-nucleotidase but their molecular identity has not been defined [230].\nNTPDase2 associates with immature and non-myelinating Schwann cells of peripheral nerves whereas NTPDase1 immunoreactivity is absent [231]. NTPDase2 is also expressed by the satellite glial cells in dorsal root ganglia and sympathetic ganglia and by the enteric glia surrounding cell bodies of ganglionic neurons of the myenteric and submucous plexus [231].\nSensory systems\nThe most comprehensive investigation of expression of NTPDases within sensory systems concerns the inner ear. Ectonucleotidase activity is associated with the tissues lining the perilymphatic compartment of the cochlea [232, 233]. Immunohistochemical analysis of the murine cochlea has assigned NTPDase1 to the cochlear vasculature and primary auditory neurons in the spiral ganglion, whereas NTPDase2 is associated with synaptic regions of the sensory inner and outer hair cells, supporting cells of the organ of Corti and additional tissue elements [234, 235]. Interestingly, noise exposure induces upregulation of NTPDase1 and NTPDase2 in the rat cochlea [236].\nTaste buds transduce chemical signals in the mouth into neural messages. Taste cells and nerve fibers express P2X2 and P2X3 receptors [237] and various P2Y receptors [238, 239]. Genetic elimination of P2X2 and P2X3 receptors revealed that ATP is a key neurotransmitter in this system [240]. NTPDase2 is expressed at the mRNA level in mouse taste papillae [241]. Immunohistochemistry and enzyme histochemical staining allocate NTPDase2 to type I \u2018glial-like\u2019 cells in the tongue, palate and larynx. Furthermore, NTPDase2 immunostaining is associated with nearby nerves, suggestive of Schwann cells, implying that NTPDase2 may be a regulator in defined taste transmission.\nPathological implications\nCerebral ischemia\nThe interruption of blood flow accompanied by an interrupted supply of oxygen and glucose initiates a sequence of events resulting in structural and functional damage of the nervous tissue, comparable to that seen at other sites of vascular injury [20]. Transient global cerebral ischemia of the rat results in a long-term increase in extracellular nucleotide hydrolysis pathways [242, 243]. Preconditioning delays the postischemic increase in ATP diphosphohydrolase activity [243]. During the days following transient forebrain ischemia, mRNA for NTPDase1 (but not of NTPDase2) and ecto-5\u2032-nucleotidase becomes upregulated in the hippocampus [242], corresponding to the upregulation of the entire ectonucleotidase chain for the hydrolysis of ATP to adenosine. The data suggest that the increased expression of ectonucleotidases in the regions of damaged nerve cells is associated with activated glia, mainly microglia [224].\nThe upregulation of the ectonucleotidase chain is suggestive of an ischemia-induced increased and sustained cellular release of nucleotides. This could have several functional implications. Since microglial cells express the cytolytic P2X7 receptor [244, 245] the activity of these cells may be particularly endangered by increased levels of extracellular ATP. Enhanced activity of NTPDase1 may prevent activated microglia from overstimulation by ATP released from the injured tissue. Alternatively, microglial expression of NTPDase1 might contribute to preventing receptor desensitization on prolonged exposure to elevated ATP levels. The parallel increase in activity of ecto-5\u2032-nucleotidase would facilitate the formation of the final hydrolysis product adenosine that exerts neuro-modulatory and immunomodulatory actions and contributes to the protection of neurons.\nAlterations following plastic changes in the nervous system\nAdditional experiments, analyzing synaptosome fractions, suggest that changes in neural plasticity can be paralleled by changes in ecto-ATPase activity. Enzyme activity is reduced following avoidance learning [246] and status epilepticus [247, 248]. It is altered in two rat models of temporal lobe epilepsy [249], and on pentylenetetrazol kindling [250]. Changes in synaptosomal ectonucleotidase activity have been implicated by a broad variety of additional treatments including acute caffeine treatment [251]. Taken together, these experiments suggest that expression of ectonucleotidases can be altered following a variety of physiological or pathological stimuli, possibly together with that of purine receptors. Further work needs to define the enzyme subtypes involved and the mechanisms underlying the regulation of ectonucleotidase expression.\nConclusions\nThis review summarizes components of extracellular nucleotide-mediated signaling pathways that are impacted upon largely by the E-NTPDase family of ectonucleotidases. Modulated, distinct NTPDase expression appears to regulate nucleotide-mediated signaling in essentially every tissue, including the vasculature and of immune and nervous systems. For example, extracellular nucleotide-mediated vascular endothelial and accessory cell stimulation might have important consequences for platelet activation, thrombogenesis, angiogenesis, vascular remodeling and the metabolic milieu of the vasculature, in response to inflammatory stress and\/or immune reactions.\nNucleotides are also of significant relevance for the communication of nerve cells and glial cells or in the reciprocal signaling between these cells. These purinergic mechanisms might also dictate pathological processes of the nervous system or following vascular injury, thromboregulatory disturbances, and defective angiogenesis with associated perturbations in tissue remodeling and regeneration.\nThere is a wide field for future investigations of the role of nucleotides and ectonucleotidases in other tissues. Increasing interest in this field may open up new avenues for investigation and the development of new treatment modalities for a large variety of diseases, including neurological pathological states, vascular thrombotic disorders including stoke, atherosclerosis and the vascular inflammation seen in transplant-graft failure.","keyphrases":["ntpdase","cd39","vasculature","nervous tissue","apyrase","ecto-atpase","immunology","platelet","liver","ischemia","brain","kidney"],"prmu":["P","P","P","P","P","P","P","P","P","P","P","P"]}
{"id":"Eur_J_Pediatr-3-1-2151776","title":"What is new in pertussis?\n","text":"Despite high vaccination coverage, over the last fifteen years there has been a worldwide resurgence of B. pertussis infection. While classical pertussis in the prevaccine era was primarily a childhood disease, today with widespread vaccination, there has been a shift in the incidence of disease to adolescents and adults. Centers of Disease Control and Prevention (CDC) data from 2004 reveal a nearly 19-fold increase in the number of cases in individuals 10\u201319 years and a 16-fold increase in persons over 20 years. Indeed adolescent and adults play a significant role in the transmission of pertussis to neonates and infants who are vulnerable to substantial morbidity and mortality from pertussis infection. Several explanations have been proposed to explain the increasing incidence of disease, with waning immunity after natural infection or immunization being widely cited as a significant factor. Improving molecular biology diagnostic techniques, namely PCR assays, also accounts for the increasing laboratory diagnosis of pertussis. Expanding vaccination strategies including universal immunization of adolescents, targeted immunization of adults, and in particular, healthcare workers, childcare providers and parents of newborns, will likely improve pertussis control. With pertussis continuing to pose a serious threat to infants, and greatly affecting adolescents and adults, there remains a need to: (a) increase the awareness of physicians as to the growing pertussis problem, (b) standardize diagnostic techniques, and (c) implement various new vaccine strategies to enhance its control.\nIntroduction\nPertussis, an acute infectious illness of the respiratory tract remains endemic in developed nations despite high vaccination coverage [7, 8, 16]. While the early use of whole-cell vaccine was highly effective in reducing the incidence of reported pertussis in the United States in the 1970s, there has been a resurgence of reported pertussis over the last 15 years [8, 16, 31]. Worldwide, there are an estimated 50 million cases occurring annually (90% of which are in developing countries), and there are as many as 400,0000 pertussis-related deaths [16, 31]. It is general consensus, moreover, that the reported incidence of pertussis is considerably lower than its actual incidence [8, 31].\nThough in the prevaccine era pertussis was regarded as a childhood disease affecting primarily young children, pertussis epidemiology in the postvaccine era is different [8, 17]. Infants are the most vulnerable group with the highest rates of complications and mortality, yet adolescents and adults now comprise a significant percentage of cases and a conduit of infection for the infants [8, 13, 17].\nPCR, culture, and serology are the mainstay of the laboratory diagnosis of pertussis, with various factors affecting the sensitivity and specificity of each modality [8, 24, 36]. However, in recent years, PCR has become an increasingly more popular tool and has significantly contributed to the increasing laboratory diagnosis of pertussis [8, 27, 36].\nAdvances have also been made with regard to prevention and disease control, with experts from 17 countries recently establishing the Global Pertussis Initiative (GPI) with the aim of analyzing the status of pertussis and enhancing existing immunization strategies [8, 15].\nEpidemiology of pertussis\nBefore the introduction of the whole-cell pertussis vaccine in the1940s, there were approximately 200,000 cases reported annually in the US [37]. Immunizations reduced disease rates and in 1976 pertussis incidence reached a nadir of 1,010 reported cases [13, 37]. However, since that time, there has been a substantial increase in the number of cases reported [8, 13, 31]. Indeed, over the past 15 years, there has been a marked increase in the incidence of pertussis with reported disease in the US reaching a rate of 8.9 per 100,000 in 2004 with nearly 19,000 provisional reported cases [7, 17]. It is also well established that, despite high vaccination coverage for primary immunization in infants and children, pertussis continues to be a global concern with increased incidence in many countries including Argentina, Australia, Canada, Italy, Japan, the Netherlands, Switzerland and the US [31]. It is also widely noted that in recent years there is a general shift in the age distribution of pertussis, with adults and adolescents an underrecognized but significant source of infection for neonates and infants [8, 13, 15\u201317]. Data from the EUVAC-NET project, a network for the epidemiologic surveillance and control of communicable diseases in the European community, demonstrate that between 1998 and 2002, the rate of disease incidence remained stable at a high rate among children less than 1 year old. Nevertheless, these data indicate that the incidence rate among adults doubled in 5 years [4]. Similarly, Centers for Disease Control and Prevention (CDC) surveillance data from 1990\u20132003 demonstrate that the reported incidence of pertussis among adolescents has substantially increased with a nearly ten-fold rise [17]. Moreover, when comparing pertussis disease rates in 1990\u20131993, recent CDC data from 2004 reveal a nearly 19-fold increase in the number of cases in persons aged 10\u201319\u00a0years and a 16-fold increase in persons over 20\u00a0years [13].\nSeveral factors have been proposed as underlying the increasing incidence of pertussis disease including waning immunity with subsequent atypical disease manifestations, increasing awareness by public health personnel with subsequent enhanced surveillance and improved laboratory diagnostics [8, 17, 31].\nWaning of both vaccine-induced immunity and infection-acquired immunity is widely cited as an important reason for recent epidemiologic trends [7, 8, 36, 37]. While the assessment of the duration of immunity afforded after either natural infection or vaccination is complex, individuals are clearly susceptible to initial infection\/reinfection after vaccination or previous pertussis illness, respectively. Studies vary in their estimation of protection against disease with protective immunity after natural infection waning 7\u201320\u00a0years after illness, and duration of immunity after vaccination waning at approximately 4\u201312\u00a0years in children [36]. Yet, regardless of the precise interval, when individuals do contract pertussis after the waning of their immunity, their disease manifestations are frequently atypical [8, 17, 27]. As such, their illness is often underdiagnosed. Such underdiagnosis poses a potentially serious public-health concern in that those untreated persons with protracted cough continue to unknowingly transmit the disease to others.\nFinally, it has been proposed that the increased incidence rates may also be a function of enhanced surveillance as well as improved and more sensitive diagnostic lab techniques (e.g., PCR), in that such techniques allow for the diagnosis of cases that would probably have been missed in the past [8, 17, 27, 35]. Nevertheless, it is important to note, that the current estimates are likely to be, if anything, an underrepresentation of the true incidence of disease [8, 31]. First, the clinical diagnosis of pertussis is complicated by underconsulting, particularly among adolescents and adults [8]. Second, with prolonged cough often being their only clinical feature, by the time these adolescents and adults finally do seek medical attention, it is often too late to culture or detect the organism by PCR, thus potentially resulting in a missed diagnosis [8, 17, 31, 35]. Moreover, the wide heterogeneity in disease expression, modification of disease by immunization, mixed infection, inconsistent definition, and insensitive nonstandardized, poorly performed, or lack of available laboratory tests, further complicate physician diagnosis [8]. While the classic or \u201ctypical\u201d pertussis may be easily recognized, it is seen less often since general immunization began. Instead, atypical pertussis, usually characterized by the absence of whoop and often a somewhat shorter duration of cough, is more common than classical pertussis among adolescents and adults [8, 17]. And finally, it should be noted that, immunized young children that are PCR positive for B pertussis can be asymptomatic [29, 31].\nRegardless of whether an individual displays classical pertussis signs and symptoms or a more protracted, atypical cough, pertussis may not be suspected because of the misconception among many physicians that pertussis is a childhood disease [8, 17]. Co-occurrence of other infections like Influenza A or B, adenovirus, and RSV may also complicate the clinical diagnosis [8]. And, finally, even when diagnosed, pertussis is often underreported [8]. Indeed, Sutter and Cochi report that in the US, only an estimated 11.6% of pertussis cases were actually reported [17, 30]. Thus multiple institutional, clinical, and laboratory factors diminish the true assessment of pertussis incidence, and the current data clearly are an underestimation of the true burden of disease.\nLaboratory diagnosis of pertussis\nBecause accurate diagnosis of pertussis cannot be made by clinical signs and symptoms alone, there is a need for improved laboratory diagnosis of pertussis [17]. While several laboratory techniques exist for the identification of B. pertussis, namely, culture, serology and PCR, overall, several practical factors may adversely affect the sensitivity of its laboratory diagnosis. Delayed specimen collection, poor specimen collection techniques, specimen transport problems, and lab media contamination are but a few of the practical constraints often influencing the outcomes of the laboratory diagnosis of pertussis. Moreover, previous exposure to the organism, patient\u2019s age, stage of disease, previous antibiotic administration, and immunization are other factors that may have a substantial impact on the sensitivity of the tests. Finally, limited access to diagnostic or laboratory methods, in both developed and developing countries, undoubtedly affects B. pertussis laboratory confirmation [8, 24].\nCulture B. pertussis is a fastidious gram-negative cocobacillus, and its isolation from nasopharyngeal secretions remains the gold standard for diagnosis. Culture requires collection of a posterior nasopharyngeal specimen with a dacron or calcium alginate swab. To increase the yield of positive cultures, specimens should be immediately plated onto selective Regan Lowe agar or Bordet Gengou medium, selective media that are seldom readily available in physician's offices because of their cost and short shelf-life [17, 24]. The main reasons for failure of bacterial growth in culture, from correctly collected and transported specimens, stem from bacterial and fungal contamination and the lack of fresh media [24]. Generally, 7\u201310\u00a0days are required to grow, isolate, and identify the organism, an obvious limitation of the culture method.The timing of obtaining specimens for culture is also of paramount importance and greatly affects its yield. The proportion of patients testing positive for pertussis by culture is highest when the initial specimens are obtained early in the course of illness, i.e., during the early catarrhal phase of the illness when the organism is present in the nasopharynx in sufficient quantity. However, adults and adolescents often present late in the course of their illness, thereby greatly reducing the likelihood of culturing the organism [17, 24]. Studies also demonstrate that proportions of positive cultures decline in patients who have been previously immunized and undoubtedly in those in whom antibiotics have been started. Thus, given the limited \u201cwindow of opportunity\u201d for positive culture, it is important to stress that a negative culture does not exclude pertussis [16]. Finally, it is important to emphasize, that despite its low yield, culture should be attempted, as the bacterial isolates are needed for genotypic and phenotypic analysis.\nPCR The use of PCR for the diagnosis of pertussis is rapidly evolving as it provides a sensitive, rapid means for laboratory diagnosis in circumstances in which the probability of a positive culture is low [8, 17, 32, 35]. Notably, the CDC and World Health Organization (WHO) now include a positive PCR in their lab definition of pertussis [17]. While the sensitivity of PCR also decreases somewhat with the duration of cough and among previously immunized individuals, it is nevertheless a significantly more robust tool for diagnosis for those in the later stages of the disease or for those who have already received antibiotics [17, 35]. Specifically, in their 2005 consensus paper, the European Research Programme for Improved Pertussis Strain Characterization and Surveillance (EUpertstrain) state that the real-time PCR is more sensitive than culture for the detection of B. pertussis, especially after the first 3\u20134\u00a0weeks of coughing and after antibiotic therapy has been initiated [16, 27]. In a prospective study in which nasopharyngeal samples were obtained simultaneously for both PCR and culture, the identification of B. pertussis infections was nearly four-fold higher with PCR [8, 28]. Finally, PCR is an invaluable tool for the diagnosis of pertussis among young infants since the yield of culture is low and serology is problematic in this age group [1, 17].As with culture, important factors for the successful application of PCR in the diagnosis of infection by Bordetella species include proper sample collection and preparation. For example, a Dacron swab with a fine flexible wire shaft, and not calcium alginate, is the recommended swab. After obtaining the nasopharyngeal sample, the swab should be shaken vigorously in saline solution, the swab discarded and the vial sealed for further processing [24]. Appropriate primer selection, amplification conditions, and controls are also essential for effective PCR testing. Primers have been derived from four chromosomal regions and common primers employed in PCR detection systems include IS481, IS1001 PTp1, and PTp2 [8, 24]. Inherent with the high sensitivity, false positive results are a well-recognized problem associated with the PCR diagnosis of pertussis and other respiratory illnesses. While at the present time, PCR is not routinely available and its methods need more standardization, optimization, and quality control, in the future, an internationally accepted standardized kit might be available, which would facilitate the expanded use of PCR for pertussis diagnosis [16, 35].\nSerology Natural infection with B. pertussis is followed by an increase in serum levels of IgA, IgM, and IgG antibodies to specific pertussis antigen whereas the primary immunization of children induces mainly IgM and IgG antibodies. During the past 15 years, ELISAs have constituted the mainstay of serologic diagnosis using specific B. pertussis protein as antigens, and the serologic diagnosis of pertussis is suspected with increases in IgA or IgG antibody titers to pertussis toxin (PT), filamentous hemmaglutinin (FHA), pertactin, fimbriae or sonicated whole organisms in two serum samples collected 2\u20134\u00a0weeks apart [24]. Notably, these antibody responses to FHA are not specific to B. pertussis, but also occur following other Bordetella species; moreover, these antibodies may be cross-reacting epitopes to other bacteria including H. influenzae and M. pneumoniae. Thus, the greatest sensitivity and specificity for the serological diagnosis of B. pertussis infection is by ELISA measurement of IgG and IgA antibodies to PT demonstrating at least a two-fold rise in titer between acute- and convalescent-phase sera [24].Still, the main problem in the serologic diagnosis of B. pertussis by ELISA is the frequent delay in obtaining the acute-phase specimen. In individuals with re-infections, there is a rapid increase in titer such that if a \u201cdelayed\u201d acute-phase sample is obtained, the titer is likely to have already peaked, thereby hampering the detection of the significant titer increase between the acute- and convalescent-phase serum samples [24]. Notably, for those individuals not recently immunized, a single-serum sample ELISA may circumvent the problem, as ill patients will have significantly higher ELISA titers than the geometric mean titers (GMT) of healthy controls [23\u201325]. With this in mind, although a rise in PT IgA is more suggestive of a recent antibody response, it is less consistent than a PT IgG rise; hence, in adolescents and adults, a single high value of IgG or IgA antibodies to PT suggests pertussis infection [17, 24, 35]. Indeed, de Melker et al. demonstrated that an IgG concentration to PT of at least 100 units\/mL in a single serum sample was diagnostic of either a recent or active pertussis infection [10].The serological diagnosis of pertussis among infants also has notable limitations. Some culture-positive patients, particularly infants younger than 3\u00a0months, do not develop measurable antibodies, a finding that calls into question the utility of even obtaining a serum specimen for serology in young infants [8].In summary, despite the shortcomings of serology, a single-sample serology test can be a useful tool, particularly among older patients presenting late in the course of their illness when culture and PCR testing are negative.\nUse of antibiotics in the treatment and prevention of pertussis Antimicrobial agents administered early in the course of disease, i.e., during the catarrhal stage, may ameliorate the disease; although, after the cough is established antibiotics do not have a discernable effect on the course of the illness, but rather are recommended to limit the spread of organisms to other individuals [9].Erythromycin, clarithromycin or azithromycin are now considered first-line agents for treatment (and prophylaxis) of pertussis in individuals 6\u00a0months of age or older (Table\u00a01). The antibiotic choice for infants younger than 6\u00a0months of age, however, requires special attention. The FDA has not yet approved azithromycin or clarithromycin for use in infants younger than 6\u00a0months; however, the 2006 AAP endorsed Red Book lists azithromycin as the preferred macrolide for this age group because of the risk of idiopathic hypertrophic pyloric stenosis associated with erythromycin [9]. Notably however, there was a recent report of infantile hypertrophic pyloric stenosis among two young infants treated with azithromycin for pertussis [26].\nTable\u00a01Recommended antimicrobial therapy and postexposure prophylaxis for pertussis in infants, children, adolescents, and adults [9]AgeRecommended drugsAlternativeAzithromycinErythromycinClarithromycinTMP-SMX<1\u00a0mo10\u00a0mg\/kg per day as a single dose for 5\u00a0daysa40\u201350\u00a0mg\/kg per day in 4 divided doses for 14\u00a0daysNot recommendedContraindicated at <2\u00a0mo of age1\u20135\u00a0moSee aboveSee above15\u00a0mg\/kg per day in 2 divided doses for 7\u00a0days\u22652\u00a0mo of age: TMP, 8\u00a0mg\/kg per day; SMX, 40\u00a0mg\/kg per day in 2 doses for 14\u00a0days\u22656\u00a0mo and children10\u00a0mg\/kg as a single dose on day 1 (maximum 500\u00a0mg); then 5\u00a0mg\/kg per day as a single dose on days 2\u20135 (maximum 250\u00a0mg\/day)See above (maximum 2\u00a0g\/day)See above (maximum 1\u00a0g\/day)See aboveAdolescents and adults500\u00a0mg as a single dose on day 1, then 250\u00a0mg as a single dose on days 2\u201352\u00a0g\/day in 4 divided doses for 14\u00a0days1\u00a0g\/day in 2\u00a0divided doses for 7\u00a0daysTMP, 300\u00a0mg\/day; SMX, 1600\u00a0mg\/day in 2 divided doses for 14\u00a0daysUsed with permission of the American Academy of Pediatrics. Red Book: 2006 Report of the Committee on Infectious Diseases Book, American Academy of Pediatrics, 2006TMP trimethoprim, SMX sulfamethoxazoleaPreferred macrolide for this age because of risk of idiopathic hypertrophic pyloric stenosis associated with erythromycin\nPostexposure prophylaxis The American Academy of Pediatrics\u2019 2006 Red Book recommends that chemoprophylaxis be administered to all household contacts and other close contacts, regardless of age and immunization status. The rationale behind this recommendation is that administration of chemoprophylaxis to asymptomatic contacts within 21\u00a0days of onset of cough in the index patient can limit secondary transmission [9]. Other countries like the United Kingdom limit the use of prophylaxis for the protection of only those with the greatest risk from pertussis, namely, young infants [11, 33]. Notably, an evidence-based review of literature on the use of erythromycin in preventing secondary transmission of pertussis to close contacts concluded that in countries where effective pertussis vaccines are in use, chemoprophyalxis should be limited to those most susceptible to the complications of pertussis (i.e., unimmunized or partially immunized infants) and to those individuals who come in close contact with the latter [11, 12]. Regardless of the policy, the agents, dose, and duration of prophylaxis are the same as for treatment of pertussis.\nPrevention of pertussis: vaccination strategies\nPertussis vaccines licensed for use in infants, children, and adults vary across countries. These vaccines differ both in terms of their active ingredients and in terms of the other diseases for which coverage is provided (e.g., polio, diptheria). For example, Repevax (Sanofi Pasteur) contains diptheria, tetanus, pertussis (acellular, component) as well as inactivated polio, whereas ADACEL contains only tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis.\nVaccination strategies similarly vary from country to country. Over the last several years, many potential immunization strategies have been proposed to improve pertussis control (Table\u00a02). Universal immunization of adolescents and adults, selective perinatal immunization of women who recently gave birth, and close contacts of newborns, are but a few of the strategies that were discussed by the Global Pertussis Initiative (GPI), which first convened in 2001. Specified immunization goals also included improvement of current infant and toddler vaccination programs [15]. The second GPI convened in 2005 and reiterated several intervention strategies to address the ongoing severe pertussis disease among neonates and infants [16].\nTable\u00a02Immunization strategies assessed by GPI participantsa (see [15], Table 1, pg. S70)StrategyPrimary objectivesSecondary objectives1. Universal adult immunizationReduce morbidity in adultsReduce transmission to young infantsDevelop herd immunityReduce morbidity in older children2. Selective immunization of new mothers, family, and close contacts of newbornsReduce transmission to infantsReduce morbidity in adults, particularly young adults3. Selective immunization of health care workersReduce transmission to patientsReduce morbidity in health care workers4. Selective immunization of child care workersReduce transmission to infantsReduce morbidity in child care workers5. Universal adolescent immunizationReduce morbidity in adolescents and young adultsReduce transmission to infantsDevelop herd immunity6. Preschool booster at 4\u00a0years of ageReduce morbidity in 4- to 6-year oldsReduce transmission to infantsDevelop herd immunity7. Reinforce and\/or improve the current infant and toddler immunization strategyReduce morbidity and mortality in infants, toddlers, and childrenReduce overall circulation of pertussisUsed with permission from Lippincott Williams & WilkinsaEntries represent the consensus of opinion of the GPI participants\nImmunization of adolescents As previously noted, the incidence of pertussis among adolescents is increasing and these individuals then serve as a reservoir of infection to unvaccinated or incompletely vaccinated infants [13]. Two Tdap vaccines (Boostrix and ADACEL) are licensed for use in the US. Recently, the CDCs Advisory Committee on Immunization Practices (ACIP) has recommended routine Tdap for adolescents from 11\u201318\u00a0years [3, 13, 16]. Several other countries including Canada, Austria, Australia, France, and Germany have also introduced the universal immunization of adolescents [16]. In Germany, for example, the current immunization schedule recommends DTaP at 2, 3, 4, and 11\u201314\u00a0months, and dTaP at 5\u20136\u00a0years and at 9\u201317\u00a0years [14]. For a complete overview of the pertussis vaccination in other European countries please access the EUVAC.NET website [14]. Future studies will be needed to evaluate the duration of protection afforded and the potential need for an adult booster.\nImmunization of adults The Adult Pertussis Trial (APERT), a study sponsored by the United States\u2019 National Institute of Health (NIH), has recently demonstrated the efficacy of acellular pertussis vaccines in preventing pertussis disease in adults (and adolescents). [16, 20, 21, 34]. To date, only ADACEL is licensed for use in adults, and the recommended adult immunization schedule in the US (October 2006\u2013September 2007) now recommends that Tdap replace a single dose of Td for adults <65\u00a0years who have not previously received a dose of Tdap (either in the primary series, booster or for wound management) [6]. Given the increased public awareness of adolescent and adult pertussis, in conjunction with perhaps an increased awareness about vaccines in general (e.g., HPV and influenza), the general public may be more receptive to a universal adult vaccination against pertussis [16]. The expected benefits of such programs would be to build up herd immunity and reduce disease. Alternatively, the selective vaccination of only those adults at highest risk of transmitting B. pertussis to vulnerable infants is likely to decrease both the incidence and the impact of pertussis on young infants. Regardless of the approach used, successful adult vaccination programs must include education and public awareness.\nCocoon strategy The vaccination of household members, including parents and siblings of newborn infants, has been recently coined the cocoon strategy [16]. Recent studies have demonstrated that parents are frequently the source of pertussis infection to their infants [2, 13, 16, 19, 22]. While implementation of this strategy is expected to lead to only modest reductions in typical adult cases, there is a strong indirect effect on infants and young children. In countries where universal immunization of adults is not yet feasible, many experts consider such targeted immunization as \u201cworthy of implementation\u201d [16]. Presently, the cocoon strategy is recommended in several European countries, including Australia, France, Germany, and Austria [16].\nMaternal vaccination Although there is efficient placental transfer of pertussis antibodies, low maternal levels and rapid decay in newborns render the infants vulnerable to life-threatening pertussis [16, 18]. Maternal immunization during pregnancy might afford some degree of protection to mother and infant during a vulnerable period, and the use of Tdap during pregnancy is currently under consideration.\nNeonatal vaccination Given the resurgence of reported pertussis in infant populations noted in multiple countries, and the high morbidity and mortality in this age group, newborn pertussis immunization is a potentially attractive strategy [5, 16, 19]. It is still unclear, however, if such a strategy will induce sufficient and timely immunity for this targeted immunization. Future trials are needed to address these concerns.\nConclusion\nDespite the increasing awareness of B. pertussis, it continues to affect millions of people worldwide. While classical pertussis was once regarded as a \u201cchild\u2019s disease\u201d, today, pertussis poses a serious threat to infants, and greatly affects adolescents adults, now functioning as reservoirs of infection. While advances in molecular biology have undoubtedly increased the capacity to diagnose pertussis, work is still needed to standardize laboratory techniques. The increased awareness of the pertussis problem among experts and the lay public will hopefully pave the way for the implementation of various vaccine strategies to enhance its control.","keyphrases":["immunization strategies","review","bordetella pertussis","polymerase chain reaction (pcr)"],"prmu":["P","P","R","M"]}
{"id":"Intensive_Care_Med-4-1-2249616","title":"Surviving Sepsis Campaign: International guidelines for management of severe sepsis and septic shock: 2008\n","text":"Objective To provide an update to the original Surviving Sepsis Campaign clinical management guidelines, \u201cSurviving Sepsis Campaign guidelines for management of severe sepsis and septic shock,\u201d published in 2004.\nIntroduction\nSevere sepsis (acute organ dysfunction secondary to infection) and septic shock (severe sepsis plus hypotension not reversed with fluid resuscitation) are major healthcare problems, affecting millions of individuals around the world each year, killing one in four (and often more), and increasing in incidence\u00a0[1\u20135]. Similar to polytrauma, acute myocardial infarction, or stroke, the speed and appropriateness of therapy administered in the initial hours after severe sepsis develops are likely to influence outcome. In 2004, an international group of experts in the diagnosis and management of infection and sepsis, representing 11 organizations, published the first internationally accepted guidelines that the bedside clinician could use to improve outcomes in severe sepsis and septic shock\u00a0[6,\u00a07]. These guidelines represented Phase II of the Surviving Sepsis Campaign (SSC), an international effort to increase awareness and improve outcomes in severe sepsis. Joined by additional organizations, the group met again in 2006 and 2007 to update the guidelines document using a\u00a0new evidence-based methodology system for assessing quality of evidence and strength of recommendations\u00a0[8\u201311].\nThese recommendations are intended to provide guidance for the clinician caring for a\u00a0patient with severe sepsis or septic shock. Recommendations from these guidelines cannot replace the clinician's decision-making capability when he or she is provided with a\u00a0patient's unique set of clinical variables. Most of these recommendations are appropriate for the severe sepsis patient in both the intensive care unit (ICU) and non-ICU settings. In fact the committee believes that, currently, the greatest outcome improvement can be made through education and process change for those caring for severe sepsis patients in the non-ICU setting and across the spectrum of acute care. It should also be noted that resource limitations in some institutions and countries may prevent physicians from accomplishing particular recommendations.\nMethods\nSepsis is defined as infection plus systemic manifestations of infection (Table 1)\u00a0[12]. Severe sepsis is defined as sepsis plus sepsis-induced organ dysfunction or tissue hypoperfusion. The threshold for this dysfunction has varied somewhat from one severe sepsis research study to another. An example of typical thresholds identification of severe sepsis is shown in Table 2\u00a0[13]. Sepsis induced hypotension is defined as a\u00a0systolic blood pressure(SBP) of < 90 mm Hg or mean arterial pressure < 70 mm Hg or a\u00a0SBP decrease > 40 mm Hg or < 2 SD below normal for age in the absence of other causes of hypotension. Septic shock is defined as sepsis induced hypotension persisting despite adequate fluid resuscitation. Sepsis induced tissue hypoperfusion is defined as either septic shock, an elevated lactate or oliguria.\nTable\u00a01Determination of the Quality of Evidence\u2022\u00a0Underlying methodologyARCTBDowngraded RCT or upgraded observational studiesCWell-done observational studiesDCase series or expert opinion\u2022\u00a0Factors that may decrease the strength of evidence1.Poor quality of planning and implementation of available RCTs suggesting high likelihood of\nbias2.Inconsistency of results (including problems with subgroup analyses)3.Indirectness of evidence (differing population, intervention, control, outcomes, comparison)4.Imprecision of results5.High likelihood of reporting bias\u2022\u00a0Main factors that may increase the strength of evidence1.Large magnitude of effect (direct evidence, relative risk (RR) > 2 with no plausible\nconfounders)2.Very large magnitude of effect with RR > 5 and no threats to validity (by two levels)3.Dose response gradientRCT, randomized controlled trial; RR,\nrelative riskTable\u00a02Factors Determining Strong vs. Weak RecommendationWhat should be consideredRecommended ProcessQuality of evidenceThe lower the quality of evidence the less likely a strong recommendationRelative importance of the outcomesIf values and preferences vary widely, a strong recommendation becomes less likelyBaseline risks of outcomesThe higher the risk, the greater the magnitude of benefitMagnitude of relative risk including benefits, harms, and burdenLarger relative risk reductions or larger increases in relative risk of harm make a strong\nrecommendation more or less likely respectivelyAbsolute magnitude of the effectThe larger the absolute benefits and harms, the greater or lesser likelihood respectively\nof a strong recommendationPrecision of the estimates of the effectsThe greater the precision the more likely is a strong recommendationCostsThe higher the cost of treatment, the less likely a strong recommendation\nThe current clinical practice guidelines build on the first and second editions from 2001 (see below) and 2004\u00a0[6, 7, 14]. The 2001 publication incorporated a\u00a0MEDLINE search for clinical trials in the preceding 10 years, supplemented by a\u00a0manual search of other relevant journals\u00a0[14]. The 2004 publication incorporated the evidence available through the end of 2003. The current publication is based on an updated search into 2007 (see methods and rules below).\nThe 2001 guidelines were coordinated by the International Sepsis Forum (ISF); the 2004 guidelines were funded by unrestricted educational grants from industry and administered through the Society of Critical Care Medicine (SCCM), the European Society of Intensive Care Medicine (ESICM), and ISF. Two of the SSC administering organizations receive unrestricted industry funding to support SSC activities (ESICM and SCCM), but none of this funding was used to support the 2006\u20132007 committee meetings.\nIt is important to distinguish between the process of guidelines revision and the Surviving Sepsis Campaign. The Surviving Sepsis Campaign (SSC) is partially funded by unrestricted educational industry grants, including those from Edwards LifeSciences, Eli Lilly and Company, and Philips Medical Systems. SSC also received funding from the Coalition for Critical Care Excellence of the Society of Critical Care Medicine. The great majority of industry funding has come from Eli Lilly and Company.\nCurrent industry funding for the Surviving Sepsis Campaign is directed to the performance improvement initiative. No industry funding was used in the guidelines revision process.\nFor both the 2004 and the 2006\/2007 efforts there were no members of the committee from industry, no industry input into guidelines development, and no industry presence at any of the meetings. Industry awareness or comment on the recommendations was not allowed. No member of the guideline committee received any honoraria for any role in the 2004 or 2006\/2007 guidelines process. The committee considered the issue of recusement of individual committee members during deliberation and decision making in areas where committee members had either financial or academic competing interests; however, consensus as to threshold for exclusion could not be reached. Alternatively, the committee agreed to ensure full disclosure and transparency of all committee members' potential conflicts at time of publication (see disclosures at the end of this document).\nThe guidelines process included a\u00a0modified Delphi method, a\u00a0consensus conference, several subsequent meetings of subgroups and key individuals, teleconferences and electronically based discussions among subgroups and members of the entire committee and two follow-up nominal group meetings in 2007.\nSubgroups were formed, each charged with updating recommendations in specific areas, including corticosteroids, blood products, activated protein C, renal replacement therapy, antibiotics, source control, and glucose control, etc. Each subgroup was responsible for updating the evidence (into 2007, with major additional elements of information incorporated into the evolving manuscript throughout 2006 and 2007). A\u00a0separate search was performed for each clearly defined question. The committee chair worked with subgroup heads to identify pertinent search terms that always included, at a\u00a0minimum, sepsis, severe sepsis, septic shock and sepsis syndrome crossed against the general topic area of the subgroup as well as pertinent key words of the specific question posed. All questions of the previous guidelines publications were searched, as were pertinent new questions generated by general topic related search or recent trials. Quality of evidence was judged by pre-defined Grades of Recommendation, Assessment, Development and Evaluation (GRADE) criteria (see below). Significant education of committee members on the GRADE approach was performed via email prior to the first committee meeting and at the first meeting. Rules were distributed concerning assessing the body of evidence and GRADE experts were available for questions throughout the process. Subgroups agreed electronically on draft proposals that were presented to committee meetings for general discussion. In January 2006, the entire group met during the 35th SCCM Critical Care Congress in San Francisco, California, USA. The results of that discussion were incorporated into the next version of recommendations and again discussed using electronic mail. Recommendations were finalized during nominal group meetings (composed of a\u00a0subset of the committee members) at the 2007 SCCM (Orlando) and 2007 International Symposium on Intensive Care and Emergency Medicine (Brussels) meetings with recirculation of deliberations and decisions to the entire group for comment or approval. At the discretion of the chair and following adequate discussion, competing proposals for wording of recommendations or assigning strength of evidence were resolved by formal voting. On occasions, voting was performed to give the committee a\u00a0sense of distribution of opinions to facilitate additional discussion. The manuscript was edited for style and form by the writing committee with final approval by section leads for their respective group assignment and then by the entire committee.\nThe development of guidelines and grading of recommendations for the 2004 guideline development process were based on a\u00a0system proposed by Sackett in 1989, during one of the first American College of Chest Physicians (ACCP) conferences on the use of antithrombotic therapies\u00a0[15]. The revised guidelines recommendations are based on the Grades of Recommendation, Assessment, Development and Evaluation (GRADE) system \u2013 a\u00a0structured system for rating quality of evidence and grading strength of recommendation in clinical practice\u00a0[8\u201311]. The SSC Steering Committee and individual authors collaborated with GRADE representatives to apply the GRADE system to the SSC guidelines revision process. The members of GRADE group were directly involved, either in person or via e-mail, in all discussions and deliberations amongst the guidelines committee members as to grading decisions. Subsequently, the SSC authors used written material prepared by the GRADE group and conferred with GRADE group members who were available at the first committee meeting and subsequent nominal group meetings. GRADE representatives were also used as a\u00a0resource throughout subgroup deliberation.\nThe GRADE system is based on a\u00a0sequential assessment of the quality of evidence, followed by assessment of the balance between benefits versus risks, burden, and cost and, based on the above, development and grading of a\u00a0management recommendations\u00a0[9\u201311]. Keeping the rating of quality of evidence and strength of recommendation explicitly separate constitutes a\u00a0crucial and defining feature of the GRADE approach. This system classifies quality of evidence as high (Grade A), moderate (Grade B), low (Grade C), or very low (Grade D). Randomized trials begin as high quality evidence, but may be downgraded due to limitations in implementation, inconsistency or imprecision of the results, indirectness of the evidence, and possible reporting bias (see Table 1). Examples of indirectness of the evidence include: population studied, interventions used, outcomes measured, and how these relate to the question of interest. Observational (non-randomized) studies begin as low-quality evidence, but the quality level may be upgraded on the basis of large magnitude of effect. An example of this is the quality of evidence for early administration of antibiotics.\nThe GRADE system classifies recommendations as strong (Grade 1) or weak (Grade 2). The grade of strong or weak is considered of greater clinical importance than a\u00a0difference in letter level of quality of evidence. The committee assessed whether the desirable effects of adherence will outweigh the undesirable effects, and the strength of a\u00a0recommendation reflects the group's degree of confidence in that assessment. A\u00a0strong recommendation in favor of an intervention reflects that the desirable effects of adherence to a\u00a0recommendation (beneficial health outcomes, less burden on staff and patients, and cost savings) will clearly outweigh the undesirable effects (harms, more burden and greater costs). A\u00a0weak recommendation in favor of an intervention indicates that the desirable effects of adherence to a\u00a0recommendation probably will outweigh the undesirable effects, but the panel is not confident about these tradeoffs \u2013 either because some of the evidence is low-quality (and thus there remains uncertainty regarding the benefits and risks) or the benefits and downsides are closely balanced. While the degree of confidence is a\u00a0continuum and there is a\u00a0lack of a\u00a0precise threshold between a\u00a0strong and a\u00a0weak recommendation, the presence of important concerns about one or more of the above factors makes a\u00a0weak recommendation more likely. A\u00a0\u201cstrong\u201d recommendation is worded as \u201cwe recommend\u201d and a\u00a0weak recommendation as \u201cwe suggest.\u201d\nThe implications of calling a\u00a0recommendation \u201cstrong\u201d are that most well-informed patients would accept that intervention, and that most clinicians should use it in most situations. There may be circumstances in which a\u00a0\u201cstrong\u201d recommendation cannot or should not be followed for an individual patient because of that patient's preferences or clinical characteristics which make the recommendation less applicable. It should be noted that being a\u00a0\u201cstrong\u201d recommendation does not automatically imply standard of care. For example, the strong recommendation for administering antibiotics within one hour of the diagnosis of severe sepsis, although desirable, is not currently standard of care as verified by current practice (personal communication, Mitchell Levy from first 8,000 patients entered internationally into the SSC performance improvement data base). The implication of a\u00a0\u201cweak\u201d recommendation is that although a\u00a0majority of well-informed patients would accept it (but a\u00a0substantial proportion would not), clinicians should consider its use according to particular circumstance.\nDifferences of opinion among committee members about interpretation of evidence, wording of proposals, or strength of recommendations were resolved using a\u00a0specifically developed set of rules. We will describe this process in detail in a\u00a0separate publication. In summary, the main approach for converting diverse opinions into a\u00a0recommendation was: 1. to give a\u00a0recommendation a\u00a0direction (for or against the given action). a\u00a0majority of votes were to be in favor of that direction, with no more than 20% preferring the opposite direction (there was a\u00a0neutral vote allowed as well); 2. to call a\u00a0given recommendation \u201cstrong\u201d rather than \u201cweak\u201d at least 70% \u201cstrong\u201d votes were required; 3. if fewer than 70% of votes indicated \u201cstrong\u201d preference, the recommendation was assigned a\u00a0\u201cweak\u201d category of strength. We used a\u00a0combination of modified Delphi Process and Nominal (Expert) Group techniques to ensure both depth and breadth of review. The entire review group (together with their parent organizations as required) participated in the larger, iterative, modified Delphi process. The smaller working group meetings which took place in person functioned as the Nominal Groups. If a\u00a0clear consensus could not be obtained by polling within the Nominal Group meetings, the larger group was specifically asked to use the polling process. This was only required for corticosteroids and glycemic control. The larger group had the opportunity to review all outputs. In this way the entire review combined intense focused discussion (Nominal Group) with broader review and monitoring using the Delphi process.\nNote: Refer to Tables3, 4, and5for condensed adult recommentations.Table\u00a03Initial Resuscitation and Infection IssuesInitial resuscitation (first 6 hours)Strength of recommendation and quality of evidence have been assessed using the GRADE criteria, presented in brackets after each guideline. For added clarity: \u2022 Indicates a strong recommendation or \u201cwe recommend\u201d; \u25cb indicates a weak recommendation or \u201cwe suggest\u201d\u2022\u00a0Begin resuscitation immediately in patients with hypotension or elevated serum lactate\n> 4mmol\/l; do not delay pending ICU admission.(1C)\u2022\u00a0Resuscitation goals:(1C)\u2013 Central venous pressure (CVP) 8\u201312 mm Hg*\u2013 Mean arterial pressure \u2265 65 mm Hg\u2013 Urine output \u2265 0.5 mL.kg-1.hr-1\u2013 Central venous (superior vena cava) oxygen saturation \u2265 70%, or mixed\nvenous \u2265 65%\u25cb If venous O2 saturation target not achieved: (2C)\u2013 consider further fluid\u2013 transfuse packed red blood cells if required to hematocrit of \u2265 30%\nand\/or\u2013 dobutamine infusion max 20 \u03bcg.kg-1.min-1* A higher target CVP of 12\u201315 mm Hg is recommended in the presence of mechanical\nventilation or pre-existing decreased ventricular compliance.Diagnosis\u2022\u00a0Obtain appropriate cultures before starting antibiotics provided this does not significantly\ndelay antimicrobial administration.(1C)\u2013 Obtain two or more blood cultures (BCs)\u2013 One or more BCs should be percutaneous\u2013 One BC from each vascular access device in place > 48 h\u2013 Culture other sites as clinically indicated\u2022\u00a0Perform imaging studies promptly in order to confirm and sample any source of infection;\nif safe to do so.(1C)Antibiotic therapy\u2022\u00a0Begin intravenous antibiotics as early as possible, and always within the first hour\nof recognizing severe sepsis (1D) and septic shock (1B).\u2022\u00a0Broad-spectrum: one or more agents active against likely bacterial\/fungal pathogens\nand with good penetration into presumed source.(1B)\u2022\u00a0Reassess antimicrobial regimen daily to optimise efficacy, prevent resistance, avoid\ntoxicity & minimise costs.(1C)\u25cb Consider combination therapy in Pseudomonas infections.(2D)\u25cb Consider combination empiric therapy in neutropenic patients.(2D)\u25cb Combination therapy no more than 3\u20135 days and deescalation following susceptibilities.(2D)\u2022\u00a0Duration of therapy typically limited to 7\u201310 days; longer if response slow,\nundrainable foci of infection, or immunologic deficiencies.(1D)\u2022\u00a0Stop antimicrobial therapy if cause is found to be non-infectious.(1D)Source identification and control\u2022\u00a0A specific anatomic site of infection should be established as rapidly as possible(1C)\nand within first 6 hrs of presentation(1D).\u2022\u00a0Formally evaluate patient for a focus of infection amenable to source control measures\n(eg: abscess drainage, tissue debridement).(1C)\u2022\u00a0Implement source control measures as soon as possible following successful initial\nresuscitation.(1C)Exception: infected pancreatic necrosis, where surgical intervention best delayed. (2B)\u2022\u00a0Choose source control measure with maximum efficacy and minimal physiologic upset.(1D)\u2022\u00a0Remove intravascular access devices if potentially infected.(1C)Table\u00a04Hemodynamic Support and Adjunctive TherapyFluid therapyStrength of recommendation and quality of evidence have been assessed using the GRADE criteria, presented in brackets after each guideline. For added clarity: \u2022\u00a0Indicates a strong recommendation or \u201cwe recommend\u201d; \u25cb indicates a weak recommendation or \u201cwe suggest\u201d\u2022\u00a0Fluid-resuscitate using crystalloids or colloids.(1B)\u2022\u00a0Target a CVP of \u2265 8 mm Hg (\u2265 12 mm Hg if mechanically\nventilated).(1C)\u2022\u00a0Use a fluid challenge technique while associated with a haemodynamic improvement.(1D)\u2022\u00a0Give fluid challenges of 1000 ml of crystalloids or 300\u2013500 ml of\ncolloids over 30 min. More rapid and larger volumes may be required in sepsis-induced tissue hypoperfusion.(1D)\u2022\u00a0Rate of fluid administration should be reduced if cardiac filling pressures increase\nwithout concurrent hemodynamic improvement.(1D)Vasopressors\u2022\u00a0Maintain MAP \u2265 65 mm Hg.(1C)\u2022\u00a0Norepinephrine or dopamine centrally administered are the initial vasopressors of choice.(1C)\u25cb Epinephrine, phenylephrine or vasopressin should not be administered as the initial\nvasopressor in septic shock.(2C)\u2013 Vasopressin 0.03 units\/min maybe subsequently added to norepinephrine with anticipation\nof an effect equivalent to norepinephrine alone.\u25cb Use epinephrine as the first alternative agent in septic shock when blood pressure\nis poorly responsive to norepinephrine or dopamine.(2B)\u2022\u00a0Do not use low-dose dopamine for renal protection.(1A)\u2022\u00a0In patients requiring vasopressors, insert an arterial catheter as soon as practical.(1D)Inotropic therapy\u2022\u00a0Use dobutamine in patients with myocardial dysfunction as supported by elevated cardiac\nfilling pressures and low cardiac output.(1C)\u2022\u00a0Do not increase cardiac index to predetermined supranormal levels.(1B)Steroids\u25cb Consider intravenous hydrocortisone for adult septic shock when hypotension remains\npoorly responsive to adequate fluid resuscitation and vasopressors.(2C)\u25cb ACTH stimulation test is not recommended to identify the subset of adults with septic\nshock who should receive hydrocortisone.(2B)\u25cb Hydrocortisone is preferred to dexamethasone.(2B)\u25cb Fludrocortisone (50 \u03bcg orally once a day) may be included if an alternative\nto hydrocortisone is being used which lacks significant mineralocorticoid activity. Fludrocortisone is\noptional if hydrocortisone is used.(2C)\u25cb Steroid therapy may be weaned once vasopressors are no longer required.(2D)\u2022\u00a0Hydrocortisone dose should be < 300 mg\/day.(1A)\u2022\u00a0Do not use corticosteroids to treat sepsis in the absence of shock unless the patient's\nendocrine or corticosteroid history warrants it.(1D)Recombinant human activated protein C (rhAPC)\u25cb Consider rhAPC in adult patients with sepsis-induced organ dysfunction with clinical\nassessment of high risk of death (typically APACHE II \u2265 25 or multiple organ failure) if there\nare no contraindications.(2B,2Cforpost-operativepatients)\u2022\u00a0Adult patients with severe sepsis and low risk of death (eg: APACHE II<20 or one\norgan failure) should not receive rhAPC.(1A)Table\u00a05Other Supportive Therapy of Severe SepsisBlood product administrationStrength of recommendation and quality of evidence have been assessed using the GRADE criteria, presented in brackets after each guideline. For added clarity: \u2022\u00a0Indicates a strong recommendation or \u201cwe recommend\u201d; \u25cb indicates a weak recommendation or \u201cwe suggest\u201d\u2022\u00a0Give red blood cells when hemoglobin decreases to < 7.0 g\/dl (< 70 g\/L)\nto target a hemoglobin of 7.0\u20139.0 g\/dl in adults.(1B)\u2013 A higher hemoglobin level may be required in special circumstances (e. g.: myocardial\nischaemia, severe hypoxemia, acute haemorrhage, cyanotic heart disease or lactic acidosis)\u2022\u00a0Do not use erythropoietin to treat sepsis-related anemia. Erythropoietin may be used\nfor other accepted reasons.(1B)\u2022\u00a0Do not use fresh frozen plasma to correct laboratory clotting abnormalities unless\nthere is bleeding or planned invasive procedures.(2D)\u25cb Do not use antithrombin therapy.(1B)\u2022\u00a0Administer platelets when: (2D)\u2013 counts are < 5000\/mm3 (5 \u00d7 109\/L)\nregardless of bleeding.\u2013 counts are 5000 to 30,000\/mm3 (5\u201330 \u00d7 109\/L)\nand there is significant bleeding risk.\u2013 Higher platelet counts (\u2265 50,000\/mm3 (50 \u00d7 109\/L))\nare required for surgery or invasive procedures.Mechanical ventilation of sepsis-induced acute lung injury (ALI)\/ARDS\u2022\u00a0Target a tidal volume of 6 ml\/kg (predicted) body weight in patients with ALI\/ARDS.(1B)\u2022\u00a0Target an initial upper limit plateau pressure < 30 cm H2O.\nConsider chest wall compliance when assessing plateau pressure.(1C)\u2022\u00a0Allow PaCO2 to increase above normal, if needed to minimize\nplateau pressures and tidal volumes.(1C)\u2022\u00a0Positive end expiratory pressure (PEEP) should be set to avoid extensive lung collapse\nat end expiration.(1C)\u25cb Consider using the prone position for ARDS patients requiring potentially injurious\nlevels of FiO2 or plateau pressure, provided they are not put at risk from positional changes.(2C)\u2022\u00a0Maintain mechanically ventilated patients in a semi-recumbent position (head of the\nbed raised to 45 \u00b0) unless contraindicated(1B),\nbetween 30\u00a0\u201345\u00a0(2C).\u25cb Non invasive ventilation may be considered in the minority of ALI\/ARDS patients with\nmild-moderate hypoxemic respiratory failure. The patients need to be hemodynamically stable, comfortable,\neasily arousable, able to protect\/clear their airway and expected to recover rapidly.(2B)\u2022\u00a0Use a weaning protocol and a spontaneous breathing trial (SBT) regularly to evaluate\nthe potential for discontinuing mechanical ventilation.(1A)\u2013 SBT options include a low level of pressure support with continuous positive airway\npressure 5 cm H2O or a T-piece.\u2013 Before the SBT, patients should: \u2013 be arousable \u2013 be haemodynamically stable without vasopressors \u2013 have no new potentially serious conditions \u2013 have low ventilatory and end-expiratory pressure requirement \u2013 require FiO2 levels that can be safely delivered with a face mask or nasal cannula\u2022\u00a0Do not use a pulmonary artery catheter for the routine monitoring of patients with\nALI\/ARDS.(1A)\u2022\u00a0Use a conservative fluid strategy for patients with established ALI who do not have\nevidence of tissue hypoperfusion.(1C)Sedation, analgesia, and neuromuscular blockade in sepsis\u2022\u00a0Use sedation protocols with a sedation goal for critically ill mechanically ventilated\npatients.(1B)\u2022\u00a0Use either intermittent bolus sedation or continuous infusion sedation to predetermined\nend points (sedation scales), with daily interruption\/lightening to produce awakening. Re-titrate if\nnecessary.(1B)\u2022\u00a0Avoid neuromuscular blockers (NMBs) where possible. Monitor depth of block with train\nof four when using continuous infusions.(1B)Glucose control\u2022\u00a0Use IV insulin to control hyperglycemia in patients with severe sepsis following stabilization\nin the ICU.(1B)\u2022\u00a0Aim to keep blood glucose < 150 mg\/dl (8.3 mmol\/L) using a validated\nprotocol for insulin dose adjustment.(2C)\u2022\u00a0Provide a glucose calorie source and monitor blood glucose values every 1\u20132 hrs\n(4 hrs when stable) in patients receiving intravenous insulin.(1C)\u2022\u00a0Interpret with caution low glucose levels obtained with point of care testing, as these\ntechniques may overestimate arterial blood or plasma glucose values.(1B)Renal replacement\u25cb Intermittent hemodialysis and continuous veno-venous haemofiltration (CVVH) are considered\nequivalent.(2B)\u25cb CVVH offers easier management in hemodynamically unstable patients.(2D)Bicarbonate therapy\u2022\u00a0Do not use bicarbonate therapy for the purpose of improving hemodynamics or reducing\nvasopressor requirements when treating hypoperfusion-induced lactic acidemia with pH \u2265 7.15.(1B)Deep vein thrombosis (DVT) prophylaxis\u2022\u00a0Use either low-dose unfractionated heparin (UFH) or low-molecular weight heparin (LMWH),\nunless contraindicated.(1A)\u2022\u00a0Use a mechanical prophylactic device, such as compression stockings or an intermittent\ncompression device, when heparin is contraindicated.(1A)\u25cb Use a combination of pharmacologic and mechanical therapy for patients who are at\nvery high risk for DVT.(2C)\u25cb In patients at very high risk LMWH should be used rather than UFH.(2C)Stress ulcer prophylaxis\u2022\u00a0Provide stress ulcer prophylaxis using H2 blocker(1A) or proton\npump inhibitor(1B). Benefits of prevention of upper GI bleed must be weighed against\nthe potential for development of ventilator-associated pneumonia.Consideration for limitation of support\u2022\u00a0Discuss advance care planning with patients and families. Describe likely outcomes\nand set realistic expectations.(1D)\nI. Management of Severe Sepsis\nA. Initial Resuscitation\nWe recommend the protocolized resuscitation of a\u00a0patient with sepsis-induced shock, defined as tissue hypoperfusion (hypotension persisting after initial fluid challenge or blood lactate concentration equal to or greater than 4 mmol\/L). This protocol should be initiated as soon as hypoperfusion is recognized and should not be delayed pending ICU admission. During the first 6 hrs of resuscitation, the goals of initial resuscitation of sepsis-induced hypoperfusion should include all of the following as one part of a\u00a0treatment protocol:\nCentral venous pressure (CVP): 8\u201312 mm HgMean arterial pressure (MAP) \u2265 65 mm HgUrine output \u2265 0.5 mL.kg\u22121.hr \u22121Central venous (superior vena cava) or mixed venous oxygen saturation \u2265 70% or \u2265 65%, respectively (Grade 1C)Rationale. Early goal-directed resuscitation has been shown to improve survival for emergency department patients presenting with septic shock in a\u00a0randomized, controlled, single-center study\u00a0[16]. Resuscitation directed toward the previously mentioned goals for the initial 6-hr period of the resuscitation was able to reduce 28-day mortality rate. The consensus panel judged use of central venous and mixed venous oxygen saturation targets to be equivalent. Either intermittent or continuous measurements of oxygen saturation were judged to be acceptable. Although blood lactate concentration may lack precision as a\u00a0measure of tissue metabolic status, elevated levels in sepsis support aggressive resuscitation. In mechanically ventilated patients or patients with known pre-existing decreased ventricular compliance, a\u00a0higher target CVP of 12\u201315 mm Hg is recommended to account for the impediment to filling\u00a0[17]. Similar consideration may be warranted in circumstances of increased abdominal pressure or diastolic dysfunction\u00a0[18]. Elevated central venous pressures may also be seen with pre-existing clinically significant pulmonary artery hypertension. Although the cause of tachycardia in septic patients may be multifactorial, a\u00a0decrease in elevated pulse rate with fluid resuscitation is often a\u00a0useful marker of improving intravascular filling. Recently published observational studies have demonstrated an association between good clinical outcome in septic shock and MAP \u2265 65 mm Hg as well as central venous oxygen saturation (ScvO2, measured in superior vena cava, either intermittently or continuously) of \u2265 70%\u00a0[19]. Many recent studies support the value of early protocolized resuscitation in severe sepsis and sepsis-induced tissue hypoperfusion\u00a0[20\u201325]. Studies of patients with shock indicate that SvO2 runs 5\u20137% lower than central venous oxygen saturation (ScvO2)\u00a0[26] and that an early goal directed resuscitation protocol can be established in a\u00a0non-research general practice venue\u00a0[27].\nThere are recognized limitations to ventricular filling pressure estimates as surrogates for fluid resuscitation\u00a0[28,\u00a029]. However, measurement of CVP is currently the most readily obtainable target for fluid resuscitation. There may be advantages to targeting fluid resuscitation to flow and perhaps to volumetric indices (and even to microcirculation changes)\u00a0[30\u201333]. Technologies currently exist that allow measurement of flow at the bedside\u00a0[34, 35]. Future goals should be making these technologies more accessible during the critical early resuscitation period and research to validate utility. These technologies are already available for early ICU resuscitation.\nWe suggest that during the first 6 hrs of resuscitation of severe sepsis or septic shock, if SCVO2 or SvO2 of 70% or 65% respectively is not achieved with fluid resuscitation to the CVP target, then transfusion of packed red blood cells to achieve a\u00a0hematocrit of \u2265 30% and\/or administration of a\u00a0dobutamine infusion (up to a\u00a0maximum of 20 \u03bcg.kg\u22121.min\u22121) be utilized to achieve this goal (Grade 2C).Rationale. The protocol used in the study cited previously targeted an increase in SCVO2 to \u2265 70%\u00a0[16]. This was achieved by sequential institution of initial fluid resuscitation, then packed red blood cells, and then dobutamine. This protocol was associated with an improvement in survival. Based on bedside clinical assessment and personal preference, a\u00a0clinician may deem either blood transfusion (if Hct is less than 30%) or dobutamine the best initial choice to increase oxygen delivery and thereby elevate SCVO2. When fluid resuscitation is believed to be already adequate. The design of the afore mentioned trial did not allow assessment of the relative contribution of these two components (i. e. increasing O2 content or increasing cardiac output) of the protocol on achievement of improved outcome.\nB. Diagnosis\nWe recommend obtaining appropriate cultures before antimicrobial therapy is initiated if such cultures do not cause significant delay in antibiotic administration. To optimize identification of causative organisms, we recommend at least two blood cultures be obtained prior to antibiotics with at least one drawn percutaneously and one drawn through each vascular access device, unless the device was recently (< 48 h) inserted. Cultures of other sites (preferably quantitative where appropriate) such as urine, cerebrospinal fluid, wounds, respiratory secretions, or other body fluids that may be the source of infection should also be obtained before antibiotic therapy if not associated with significant delay in antibiotic administration (Grade 1C).Rationale. Although sampling should not delay timely administration of antibiotics in patients with severe sepsis (example: lumbar puncture in suspected meningitis), obtaining appropriate cultures prior to their administration is essential to confirm infection and the responsible pathogen(s), and to allow de-escalation of antibiotic therapy after receipt of the susceptibility profile. Samples can be kept in the refrigerator or frozen if processing cannot be performed immediately. Immediate transport to a\u00a0microbiological lab is necessary. Because rapid sterilization of blood cultures can occur within a\u00a0few hours after the first antibiotic dose, obtaining those cultures before starting therapy is essential if the causative organism is to be identified. Two or more blood cultures are recommended\u00a0[36]. In patients with indwelling catheters (for > 48 h) at least one blood culture should be drawn through each lumen of each vascular access device. Obtaining blood cultures peripherally and through a\u00a0vascular access device is an important strategy. If the same organism is recovered from both cultures, the likelihood that the organism is causing the severe sepsis is enhanced. In addition, if the culture drawn through the vascular access device is positive much earlier than the peripheral blood culture (i. e., > 2 hrs earlier), the data support the concept that the vascular access device is the source of the infection\u00a0[37]. Quantitative cultures of catheter and peripheral blood are also useful for determining whether the catheter is the source of infection. Volume of blood drawn with the culture tube should be at least 10 mL\u00a0[38]. Quantitative (or semi-quantitative) cultures of respiratory tract secretions are recommended for the diagnosis of ventilator-associated pneumonia\u00a0[39]. Gram stain can be useful, in particular for respiratory tract specimens, to help decide the micro-organisms to be targeted. The potential role of biomarkers for diagnosis of infection in patients presenting with severe sepsis remains at present undefined. The procalcitonin level, although often useful, is problematic in patients with an acute inflammatory pattern from other causes (e. g. post-operative, shock)\u00a0[40] In the near future, rapid diagnostic methods (polymerase chain reaction, micro-arrays) might prove extremely helpful for a\u00a0quicker identification of pathogens and major antimicrobial resistance determinants\u00a0[41].\nWe recommend that imaging studies be performed promptly in attempts to confirm a\u00a0potential source of infection. Sampling of potential sources of infection should occur as they are identified; however, some patients may be too unstable to warrant certain invasive procedures or transport outside of the ICU. Bedside studies, such as ultrasound, are useful in these circumstances (Grade 1C).Rationale. Diagnostic studies may identify a\u00a0source of infection that requires removal of a\u00a0foreign body or drainage to maximize the likelihood of a\u00a0satisfactory response to therapy. However, even in the most organized and well-staffed healthcare facilities, transport of patients can be dangerous, as can placing patients in outside-unit imaging devices that are difficult to access and monitor. Balancing risk and benefit is therefore mandatory in those settings.\nC. Antibiotic Therapy\nWe recommend that intravenous antibiotic therapy be started as early as possible and within the first hour of recognition of septic shock (1B) and severe sepsis without septic shock (1D). Appropriate cultures should be obtained before initiating antibiotic therapy, but should not prevent prompt administration of antimicrobial therapy (Grade 1D).Rationale. Establishing vascular access and initiating aggressive fluid resuscitation is the first priority when managing patients with severe sepsis or septic shock. However, prompt infusion of antimicrobial agents should also be a\u00a0priority and may require additional vascular access ports\u00a0[42, 43]. In the presence of septic shock each hour delay in achieving administration of effective antibiotics is associated with a\u00a0measurable increase in mortality\u00a0[42]. If antimicrobial agents cannot be mixed and delivered promptly from the pharmacy, establishing a\u00a0supply of premixed antibiotics for such urgent situations is an appropriate strategy for ensuring prompt administration. In choosing the antimicrobial regimen, clinicians should be aware that some antimicrobial agents have the advantage of bolus administration, while others require a\u00a0lengthy infusion. Thus, if vascular access is limited and many different agents must be infused, bolus drugs may offer an advantage.\nWe recommend that initial empirical anti-infective therapy include one or more drugs that have activity against all likely pathogens (bacterial and\/or fungal) and that penetrate in adequate concentrations into the presumed source of sepsis (Grade 1B).Rationale. The choice of empirical antibiotics depends on complex issues related to the patient's history including drug intolerances, underlying disease, the clinical syndrome, and susceptibility patterns of pathogens in the community, in the hospital, and that previously have been documented to colonize or infect the patient. There is an especially wide range of potential pathogens for neutropenic patients.\nRecently used antibiotics should generally be avoided. Clinicians should be cognizant of the virulence and growing prevalence of oxacillin (methicillin) resistant Staphylococcus aureus (ORSA or MRSA) in some communities and healthcare associated settings (especially in the United States) when they choose empiric therapy. If the prevalence is significant, and in consideration of the virulence of this organism, empiric therapy adequate for this pathogen would be warranted. Clinicians should also consider whether Candidemia is a\u00a0likely pathogen when choosing initial therapy. When deemed warranted, the selection of empiric antifungal therapy (e. g., fluconazole, amphotericin B, or echinocandin) will be tailored to the local pattern of the most prevalent Candida species, and any prior administration of azoles drugs\u00a0[44]. Risk factors for candidemia should also be considered when choosing initial therapy.\nBecause patients with severe sepsis or septic shock have little margin for error in the choice of therapy, the initial selection of antimicrobial therapy should be broad enough to cover all likely pathogens. There is ample evidence that failure to initiate appropriate therapy (i. e. therapy with activity against the pathogen that is subsequently identified as the causative agent) correlates with increased morbidity and mortality\u00a0[45\u201348].\nPatients with severe sepsis or septic shock warrant broad-spectrum therapy until the causative organism and its antibiotic susceptibilities are defined. Restriction of antibiotics as a\u00a0strategy to reduce the development of antimicrobial resistance or to reduce cost is not an appropriate initial strategy in this patient population.\nAll patients should receive a\u00a0full loading dose of each antimicrobial. However, patients with sepsis or septic shock often have abnormal renal or hepatic function and may have abnormal volumes of distribution due to aggressive fluid resuscitation. Drug serum concentration monitoring can be useful in an ICU setting for those drugs that can be measured promptly. An experienced physician or clinical pharmacist should be consulted to ensure that serum concentrations are attained that maximize efficacy and minimize toxicity\u00a0[49\u201352].\nWe recommend that the antimicrobial regimen be reassessed daily to optimize activity, to prevent the development of resistance, to reduce toxicity, and to reduce costs (Grade 1C).Rationale. Although restriction of antibiotics as a\u00a0strategy to reduce the development of antimicrobial resistance or to reduce cost is not an appropriate initial strategy in this patient population, once the causative pathogen has been identified, it may become apparent that none of the empiric drugs offers optimal therapy; i. e., there may be another drug proven to produce superior clinical outcome which should therefore replace empiric agents.\nNarrowing the spectrum of antibiotic coverage and reducing the duration of antibiotic therapy will reduce the likelihood that the patient will develop superinfection with pathogenic or resistant organisms such as Candida species, Clostridium difficile, or vancomycin-resistant Enterococcus faecium. However, the desire to minimize superinfections and other complications should not take precedence over the need to give the patient an adequate course of therapy to cure the infection that caused the severe sepsis or septic shock.\nWe suggest combination therapy for patients with known or suspected Pseudomonas infections as a\u00a0cause of severe sepsis (Grade 2D).We suggest combination empiric therapy for neutropenic patients with severe sepsis (Grade 2D).When used empirically in patients with severe sepsis, we suggest that combination therapy should not be administered for more than 3 to 5 days. De-escalation to the most appropriate single therapy should be performed as soon as the susceptibility profile is known. (Grade 2D).Rationale. Although no study or meta-analysis has convincingly demonstrated that combination therapy produces a\u00a0superior clinical outcome for individual pathogens in a\u00a0particular patient group, combination therapies do produce in vitro synergy against pathogens in some models (although such synergy is difficult to define and predict). In some clinical scenarios, such as the two above, combination therapies are biologically plausible and are likely clinically useful even if evidence has not demonstrated improved clinical outcome\u00a0[53\u201356]. Combination therapy for suspected known Pseudomonas pending sensitivities increases the likelihood that at least one drug is effective against that strain and positively affects outcome\u00a0[57].\nWe recommend that the duration of therapy typically be 7\u201310 days; longer courses may be appropriate in patients who have a\u00a0slow clinical response, undrainable foci of infection, or who have immunologic deficiencies including neutropenia (Grade 1D).If the presenting clinical syndrome is determined to be due to a\u00a0noninfectious cause, we recommend antimicrobial therapy be stopped promptly to minimize the likelihood that the patient will become infected with an antibiotic resistant pathogen or will develop a\u00a0drug related adverse effect (Grade 1D).Rationale. Clinicians should be cognizant that blood cultures will be negative in more than 50% of cases of severe sepsis or septic shock, yet many of these cases are very likely caused by bacteria or fungi. Thus, the decisions to continue, narrow, or stop antimicrobial therapy must be made on the basis of clinician judgment and clinical information.\nD. Source Control\nWe recommend that a\u00a0specific anatomic diagnosis of infection requiring consideration for emergent source control- for example necrotizing fasciitis, diffuse peritonitis, cholangitis, intestinal infarction \u2013 be sought and diagnosed or excluded as rapidly as possible (Grade 1C) and within the first 6 hours following presentation (Grade 1D).We further recommend that all patients presenting with severe sepsis be evaluated for the presence of a\u00a0focus of infection amenable to source control measures, specifically the drainage of an abscess or local focus of infection, the debridement of infected necrotic tissue, the removal of a\u00a0potentially infected device, or the definitive control of a\u00a0source of ongoing microbial contamination (Grade 1C) (see Appendix\u00a0A for examples of potential sites needing source control).We suggest that when infected peripancreatic necrosis is identified as a\u00a0potential source of infection, definitive intervention is best delayed until adequate demarcation of viable and non-viable tissues has occurred (Grade 2B).We recommend that when source control is required, the effective intervention associated with the least physiologic insult be employed e. g., percutaneous rather than surgical drainage of an abscess (Grade 1D).We recommend that when intravascular access devices are a\u00a0possible source of severe sepsis or septic shock, they be promptly removed after establishing other vascular access (Grade 1C).Rationale. The principles of source control in the management of sepsis include a\u00a0rapid diagnosis of the specific site of infection, and identification of a\u00a0focus of infection amenable to source control measures (specifically the drainage of an abscess, the debridement of infected necrotic tissue, the removal of a\u00a0potentially infected device, and the definitive control of a\u00a0source of ongoing microbial contamination)\u00a0[58]. Foci of infection readily amenable to source control measures include an intra-abdominal abscess or gastrointestinal perforation, cholangitis or pyelonephritis, intestinal ischemia or necrotizing soft tissue infection, and other deep space infection such as an empyema or septic arthritis. Such infectious foci should be controlled as soon as possible following successful initial resuscitation\u00a0[59], accomplishing the source control objective with the least physiologic upset possible (e. g., percutaneous rather than surgical drainage of an abscess\u00a0[60], endoscopic rather than surgical drainage of biliary tree), and removing intravascular access devices that are potentially the source of severe sepsis or septic shock promptly after establishing other vascular access\u00a0[61, 62]. A\u00a0randomized, controlled trial comparing early vs. delayed surgical intervention for peripancreatic necrosis showed better outcomes with a\u00a0delayed approach\u00a0[63]. However, areas of uncertainty, such as definitive documentation of infection and appropriate length of delay exist. The selection of optimal source control methods must weigh benefits and risks of the specific intervention as well as risks of transfer\u00a0[64]. Source control interventions may cause further complications such as bleeding, fistulas, or inadvertent organ injury. Surgical intervention should be considered when lesser interventional approaches are inadequate, or when diagnostic uncertainty persists despite radiological evaluation. Specific clinical situations require consideration of available choices, patient's preferences, and clinician's expertise.\nE. Fluid Therapy\nWe recommend fluid resuscitation with either natural\/artificial colloids or crystalloids. There is no evidence-based support for one type of fluid over another (Grade 1B).Rationale. The SAFE study indicated albumin administration was safe and equally effective as crystalloid\u00a0[65]. There was an insignificant decrease in mortality rates with the use of colloid in a\u00a0subset analysis of septic patients (p = 0.09). Previous meta-analyses of small studies of ICU patients had demonstrated no difference between crystalloid and colloid fluid resuscitation\u00a0[66\u201368]. Although administration of hydroxyethyl starch may increase the risk of acute renal failure in patients with sepsis variable findings preclude definitive recommendations\u00a0[69, 70]. As the volume of distribution is much larger for crystalloids than for colloids, resuscitation with crystalloids requires more fluid to achieve the same end points and results in more edema. Crystalloids are less expensive.\nWe recommend fluid resuscitation initially target a\u00a0CVP of at least 8 mm Hg (12 mm Hg in mechanically ventilated patients). Further fluid therapy is often required (Grade 1C).We recommend that a\u00a0fluid challenge technique be applied, wherein fluid administration is continued as long as the hemodynamic improvement (e. g., arterial pressure, heart rate, urine output) continues (Grade\u00a01D).We recommend fluid challenge in patients with suspected hypovolemia be started with at least 1000 mL of crystalloids or 300\u2013500 mL of colloids over 30 min. More rapid administration and greater amounts of fluid may be needed in patients with sepsis induced tissue hypoperfusion (see initial resuscitation recommendations) (Grade 1D).We recommend the rate of fluid administration be reduced substantially when cardiac filling pressures (CVP or pulmonary artery balloon-occluded pressure) increase without concurrent hemodynamic improvement (Grade 1D).Rationale. Fluid challenge must be clearly separated from simple fluid administration; it is a\u00a0technique in which large amounts of fluids are administered over a\u00a0limited period of time under close monitoring to evaluate the patient's response and avoid the development of pulmonary edema. The degree of intravascular volume deficit in patients with severe sepsis varies. With venodilation and ongoing capillary leak, most patients require continuing aggressive fluid resuscitation during the first 24 hours of management. Input is typically much greater than output, and input\/output ratio is of no utility to judge fluid resuscitation needs during this time period.\nF. Vasopressors\nWe recommend mean arterial pressure (MAP) be maintained \u2265 65 mm Hg (Grade 1C).Rationale. Vasopressor therapy is required to sustain life and maintain perfusion in the face of life-threatening hypotension, even when hypovolemia has not yet been resolved. Below a\u00a0certain mean arterial pressure, autoregulation in various vascular beds can be lost, and perfusion can become linearly dependent on pressure. Thus, some patients may require vasopressor therapy to achieve a\u00a0minimal perfusion pressure and maintain adequate flow\u00a0[71, 72]. The titration of norepinephrine to as low as MAP 65 mm Hg has been shown to preserve tissue perfusion\u00a0[72]. In addition, pre-existing comorbidities should be considered as to most appropriate MAP target. For example, a\u00a0MAP of 65 mm Hg might be too low in a\u00a0patient with severe uncontrolled hypertension, and in a\u00a0young previously normotensive, a\u00a0lower MAP might be adequate. Supplementing end points such as blood pressure with assessment of regional and global perfusion, such as blood lactate concentrations and urine output, is important. Adequate fluid resuscitation is a\u00a0fundamental aspect of the hemodynamic management of patients with septic shock, and should ideally be achieved before vasopressors and inotropes are used, but using vasopressors early as an emergency measure in patients with severe shock is frequently necessary. When that occurs great effort should be directed to weaning vasopressors with continuing fluid resuscitation.\nWe recommend either norepinephrine or dopamine as the first choice vasopressor agent to correct hypotension in septic shock (administered through a\u00a0central catheter as soon as one is available) (Grade 1C).We suggest that epinephrine, phenylephrine, or vasopressin should not be administered as the initial vasopressor in septic shock (Grade 2C). Vasopressin .03 units\/min may be subsequently added to norepinephrine with anticipation of an effect equivalent to norepinephrine alone.We suggest that epinephrine be the first chosen alternative agent in septic shock that is poorly responsive to norepinephrine or dopamine (Grade 2B).Rationale. There is no high-quality primary evidence to recommend one catecholamine over another. Much literature exists that contrasts the physiologic effects of choice of vasopressor and combined inotrope\/vasopressors in septic shock\u00a0[73\u201385]. Human and animal studies suggest some advantages of norepinephrine and dopamine over epinephrine (the latter with the potential for tachycardia as well as disadvantageous effects on splanchnic circulation and hyperlactemia) and phenylephrine (decrease in stroke volume). There is, however, no clinical evidence that epinephrine results in worse outcomes, and it should be the first chosen alternative to dopamine or norepinephrine. Phenylephrine is the adrenergic agent least likely to produce tachycardia, but as a\u00a0pure vasopressor would be expected to decrease stroke volume. Dopamine increases mean arterial pressure and cardiac output, primarily due to an increase in stroke volume and heart rate. Norepinephrine increases mean arterial pressure due to its vasoconstrictive effects, with little change in heart rate and less increase in stroke volume compared with dopamine. Either may be used as a\u00a0first-line agent to correct hypotension in sepsis. Norepinephrine is more potent than dopamine and may be more effective at reversing hypotension in patients with septic shock. Dopamine may be particularly useful in patients with compromised systolic function but causes more tachycardia and may be more arrhythmogenic\u00a0[86]. It may also influence the endocrine response via the hypothalamic-pituitary axis and have immunosuppressive effects.\nVasopressin levels in septic shock have been reported to be lower than anticipated for a\u00a0shock state\u00a0[87]. Low doses of vasopressin may be effective in raising blood pressure in patients refractory to other vasopressors, and may have other potential physiologic benefits\u00a0[88\u201393]. Terlipressin has similar effects but is long lasting\u00a0[94]. Studies show that vasopressin concentrations are elevated in early septic shock, but with continued shock, concentration decreases to normal range in the majority of patients between 24 and 48 hrs\u00a0[95]. This has been called \u201crelative vasopressin deficiency\u201d because in the presence of hypotension, vasopressin would be expected to be elevated. The significance of this finding is unknown. The recent VASST trial, a\u00a0randomized, controlled trial comparing norepinephrine alone to norepinephrine plus vasopressin at .03 units per minute showed no difference in outcome in the intent to treat population. An a priori defined subgroup analysis showed that the survival of patients receiving less than 15 \u03bcg\/min norepinephrine at the time of randomization was better with vasopressin. It should be noted however that the pre-trial rationale for this stratification was based on exploring potential benefit in the 15 \u03bcg or greater norepinephrine requirement population. Higher doses of vasopressin have been associated with cardiac, digital, and splanchnic ischemia and should be reserved for situations where alternative vasopressors have failed\u00a0[96]. Cardiac output measurement to allow maintenance of a\u00a0normal or elevated flow is desirable when these pure vasopressors are instituted.\nWe recommend that low dose dopamine not be used for renal protection (Grade 1A).Rationale. A\u00a0large randomized trial and meta-analysis comparing low-dose dopamine to placebo found no difference in either primary outcomes (peak serum creatinine, need for renal replacement, urine output, time to recovery of normal renal function), or secondary outcomes (survival to either ICU or hospital discharge, ICU stay, hospital stay, arrhythmias)\u00a0[97, 98]. Thus the available data do not support administration of low doses of dopamine solely to maintain renal function.\nWe recommend that all patients requiring vasopressors have an arterial line placed as soon as practical if resources are available (Grade 1D).Rationale. In shock states, estimation of blood pressure using a\u00a0cuff is commonly inaccurate; use of an arterial cannula provides a\u00a0more appropriate and reproducible measurement of arterial pressure. These catheters also allow continuous analysis so that decisions regarding therapy can be based on immediate and reproducible blood pressure information.\nG. Inotropic Therapy\nWe recommend a\u00a0dobutamine infusion be administered in the presence of myocardial dysfunction as suggested by elevated cardiac filling pressures and low cardiac output (Grade 1C).We recommend against the use of a\u00a0strategy to increase cardiac index to predetermined supranormal levels (Grade 1B).Rationale. Dobutamine is the first-choice inotrope for patients with measured or suspected low cardiac output in the presence of adequate left ventricular filling pressure (or clinical assessment of adequate fluid resuscitation) and adequate mean arterial pressure. Septic patients who remain hypotensive after fluid resuscitation may have low, normal, or increased cardiac outputs. Therefore, treatment with a\u00a0combined inotrope\/vasopressor such as norepinephrine or dopamine is recommended if cardiac output is not measured. When the capability exists for monitoring cardiac output in addition to blood pressure, a\u00a0vasopressor such as norepinephrine may be used separately to target specific levels of mean arterial pressure and cardiac output. Two large prospective clinical trials that included critically ill ICU patients who had severe sepsis failed to demonstrate benefit from increasing oxygen delivery to supranormal targets by use of dobutamine\u00a0[99,\u00a0100]. These studies did not target specifically patients with severe sepsis and did not target the first 6 hours of resuscitation. The first 6 hours of resuscitation of sepsis induced hypoperfusion need to be treated separately from the later stages of severe sepsis (see initial resuscitation recommendations).\nH. Corticosteroids\nWe suggest intravenous hydrocortisone be given only to adult septic shock patients after blood pressure is identified to be poorly responsive to fluid resuscitation and vasopressor therapy (Grade 2C).Rationale. One french multi-center, randomized, controlled trial (RCT) of patients in vasopressor-unresponsive septic shock (hypotension despite fluid resuscitation and vasopressors) showed a\u00a0significant shock reversal and reduction of mortality rate in patients with relative adrenal insufficiency (defined as post-adrenocorticotropic hormone (ACTH) cortisol increase 9 \u03bcg\/dL or less)\u00a0[101]. Two additional smaller RCTs also showed significant effects on shock reversal with steroid therapy\u00a0[102, 103]. However, a\u00a0recent large, European multicenter trial (CORTICUS), which has been presented in abstract form but not yet published, failed to show a\u00a0mortality benefit with steroid therapy of septic shock\u00a0[104]. CORTICUS did show a\u00a0faster resolution of septic shock in patients who received steroids. The use of the ACTH test (responders and nonresponders) did not predict the faster resolution of shock. Importantly, unlike the French trial, which only enrolled shock patients with blood pressure unresponsive to vasopressor therapy, the CORTICUS study included patients with septic shock, regardless of how the blood pressure responded to vasopressors. Although corticosteroids do appear to promote shock reversal, the lack of a\u00a0clear improvement in mortality-coupled with known side effects of steroids such as increased risk of infection and myopathy-generally tempered enthusiasm for their broad use. Thus, there was broad agreement that the recommendation should be downgraded from the previous guidelines (Appendix B). There was considerable discussion and consideration by the committee on the option of encouraging use in those patients whose blood pressure was unresponsive to fluids and vasopressors, while strongly discouraging use in subjects whose shock responded well to fluids and pressors. However, this more complex set of recommendations was rejected in favor of the above single recommendation (see Appendix B).\nWe suggest the ACTH stimulation test not be used to identify the subset of adults with septic shock who should receive hydrocortisone (Grade 2B).Rationale. Although one study suggested those who did not respond to ACTH with a\u00a0brisk surge in cortisol (failure to achieve or > 9 \u03bcg\/dL increase in cortisol 30\u201360 mins post-ACTH administration) were more likely to benefit from steroids than those who did respond, the overall trial population appeared to benefit regardless of ACTH result, and the observation of a\u00a0potential interaction between steroid use and ACTH test was not statistically significant\u00a0[101]. Furthermore, there was no evidence of this distinction between responders and nonresponders in a\u00a0recent multicenter trial\u00a0[104]. Commonly used cortisol immunoassays measure total cortisol (protein-bound and free) while free cortisol is the pertinent measurement. The relationship between free and total cortisol varies with serum protein concentration. When compared to a\u00a0reference method (mass spectrometry), cortisol immunoassays may over- or underestimate the actual cortisol level, affecting the assignment of patients to responders or nonresponders\u00a0[105]. Although the clinical significance is not clear, it is now recognized that etomidate, when used for induction for intubation, will suppress the HPA axis\u00a0[106].\nWe suggest that patients with septic shock should not receive dexamethasone if hydrocortisone is available (Grade 2B).Rationale. Although often proposed for use until an ACTH stimulation test can be administered, we no longer suggest an ACTH test in this clinical situation (see #3 above). Furthermore, dexamethasone can lead to immediate and prolonged suppression of the HPA axis after administration\u00a0[107].\nWe suggest the daily addition of oral fludrocortisone (50 \u03bcg) if hydrocortisone is not available and the steroid that is substituted has no significant mineralocorticoid activity. Fludrocortisone is considered optional if hydrocortisone is used (Grade 2C).Rationale. One study added 50 \u03bcg of fludrocortisone orally\u00a0[101]. Since hydrocortisone has intrinsic mineralcorticoid activity, there is controversy as to whether fludrocortisone should be added.\nWe suggest clinicians wean the patient from steroid therapy when vasopressors are no longer required (Grade 2D).Rationale. There has been no comparative study between a\u00a0fixed duration and clinically guided regimen, or between tapering and abrupt cessation of steroids. Three RCTs used a\u00a0fixed duration protocol for treatment\u00a0[101, 103, 104], and in two RCTs, therapy was decreased after shock resolution\u00a0[102, 108]. In four RCTs steroids were tapered over several days\u00a0[102\u2013104, 108], and in two RCTs\u00a0[101,\u00a0109] steroids were withdrawn abruptly. One cross-over study showed hemodynamic and immunologic rebound effects after abrupt cessation of corticosteroids\u00a0[110]. It remains uncertain whether outcome is affected by tapering of steroids or not.\nWe recommend doses of corticosteroids comparable to > 300 mg hydrocortisone daily not be used in severe sepsis or septic shock for the purpose of treating septic shock (Grade 1A).Rationale. Two randomized prospective clinical trials and a\u00a0meta-analyses concluded that for therapy of severe sepsis or septic shock, high-dose corticosteroid therapy is ineffective or harmful\u00a0[111\u2013113]. Reasons to maintain higher doses of corticosteroid for medical conditions other than septic shock may exist.\nWe recommend corticosteroids not be administered for the treatment of sepsis in the absence of shock. There is, however, no contraindication to continuing maintenance steroid therapy or to using stress does steroids if the patient's endocrine or corticosteroid administration history warrants (Grade 1D).Rationale. No studies exist that specifically target severe sepsis in the absence of shock that offer support for use of stress doses of steroids in this patient population. Steroids may be indicated in the presence of a\u00a0prior history of steroid therapy or adrenal dysfunction. A\u00a0recent preliminary study of stress dose level steroids in community- acquired pneumonia is encouraging but needs confirmation\u00a0[114].\nI. Recombinant Human Activated Protein C (rhAPC)\nWe suggest that adult patients with sepsis induced organ dysfunction associated with a\u00a0clinical assessment of high risk of death, most of whom will have APACHE\u00a0II \u2265 25 or multiple organ failure, receive rhAPC if there are no contraindications (Grade 2B except for patients within 30 days of surgery where it is Grade 2C). Relative contraindications should also be considered in decision making.We recommend that adult patients with severe sepsis and low risk of death, most of whom will have APACHE II < 20 or one organ failure, do not receive rhAPC (Grade 1A).Rationale. The evidence concerning use of rhAPC in adults is primarily based on two randomized controlled trials (RCTs): PROWESS (1,690 adult patients, stopped early for efficacy)\u00a0[115] and ADDRESS (stopped early for futility)\u00a0[116]. Additional safety information comes from an open-label observational study ENHANCE\u00a0[117]. The ENHANCE trial also suggested early administration of rhAPC was associated with better outcomes.\nPROWESS involved 1,690 patients and documented 6.1% in absolute total mortality reduction with a\u00a0relative risk reduction (RRR) of 19.4%, 95% CI 6.6\u201330.5%, number needed to treat (NNT):16\u00a0[115]. Controversy associated with the results focused on a\u00a0number of subgroup analyses. Subgroup analyses have the potential to mislead due to the absence of an intent to treat, sampling bias, and selection error\u00a0[118]. The analyses suggested increasing absolute and relative risk reduction with greater risk of death using both higher APACHE II scores and greater number of organ failures\u00a0[119]. This led to drug approval for patients with high risk of death (such as APACHE\u00a0II \u2265 25) and more than one organ failure in Europe.\nThe ADDRESS trial involved 2,613 patients judged to have a\u00a0low risk of death at the time of enrollment. 28 day mortality from all causes was 17% on placebo vs. 18.5% on APC, relative risk (RR) 1.08, 95% CI 0.92\u20131.28\u00a0[116]. Again, debate focused on subgroup analyses; analyses restricted to small subgroups of patients with APACHE II score over 25, or more than one organ failures which failed to show benefit; however these patient groups also had a\u00a0lower mortality than in PROWESS.\nRelative risk reduction of death was numerically lower in the subgroup of patients with recent surgery (n = 502) in the PROWESS trial (30.7% placebo vs. 27.8% APC)\u00a0[119] when compared to the overall study population (30.8% placebo vs. 24.7% APC)\u00a0[115]. In the ADDRESS trial, patients with recent surgery and single organ dysfunction who received APC had significantly higher 28 day mortality rates (20.7% vs. 14.1%, p = 0.03, n = 635)\u00a0[116].\nSerious adverse events did not differ in the studies\u00a0[115\u2013117] with the exception of serious bleeding, which occurred more often in the patients treated with APC: 2% vs. 3.5% (PROWESS; p = 0.06)\u00a0[115]; 2.2% vs. 3.9% (ADDRESS; p < 0.01)\u00a0[116]; 6.5% (ENHANCE, open label)\u00a0[117]. The pediatric trial and implications are discussed in the pediatric consideration section of this manuscript (see Appendix C for absolute contraindications to use of rhAPC and prescribing information for relative contraindications).\nIntracranial hemorrhage (ICH) occurred in the PROWESS trial in 0.1% (placebo) and 0.2% (APC) (n. s.)\u00a0[106], in the ADDRESS trial 0.4% (placebo) vs. 0.5% (APC) (n. s.)\u00a0[116]; in ENHANCE 1.5%\u00a0[108]. Registry studies of rhAPC report higher bleeding rates than randomized controlled trials, suggesting that the risk of bleeding in actual practice may be greater than reported in PROWESS and ADDRESS\u00a0[120, 121].\nThe two RCTs in adult patients were methodologically strong, precise, and provide direct evidence regarding death rates. The conclusions are limited, however, by inconsistency that is not adequately resolved by subgroup analyses (thus the designation of moderate quality evidence). Results, however, consistently fail to show benefit for the subgroup of patients at lower risk of death, and consistently show increases in serious bleeding. The RCT in pediatric severe sepsis failed to show benefit and has no important limitations. Thus, for low risk and pediatric patients, we rate the evidence as high quality.\nFor adult use there is probable mortality reduction in patients with clinical assessment of high risk of death, most of whom will have APACHE II \u2265 25 or multiple organ failure. There is likely no benefit in patients with low risk of death, most of whom will have APACHE II < 20 or single organ dysfunction. The effects in patients with more than one organ failure but APACHE II < 25 are unclear and in that circumstance one may use clinical assessment of the risk of death and number of organ failures to support decision. There is a\u00a0certain increased risk of bleeding with administration of rhAPC which may be higher in surgical patients and in the context of invasive procedures. Decision on utilization depends upon assessing likelihood of mortality reduction versus increases in bleeding and cost (see appendix D for nominal committee vote on recommendation for rhAPC). A\u00a0European Regulatory mandated randomized controlled trial of rhAPC vs. placebo in patients with septic shock is now ongoing\u00a0[122].\nJ. Blood Product Administration\nOnce tissue hypoperfusion has resolved and in the absence of extenuating circumstances, such as myocardial ischemia, severe hypoxemia, acute hemorrhage, cyanotic heart disease, or lactic acidosis (see recommendations for initial resuscitation), we recommend that red blood cell transfusion occur when hemoglobin decreases to < 7.0 g\/dL (< 70 g\/L) to target a\u00a0hemoglobin of 7.0\u20139.0 g\/dL (70\u201390 g\/L) in adults (Grade 1B).Rationale. Although the optimum hemoglobin for patients with severe sepsis has not been specifically investigated, the Transfusion Requirements in Critical Care trial suggested that a\u00a0hemoglobin of 7\u20139 g\/dL (70\u201390 g\/L) when compared to 10\u201312 g\/dL (100\u2013200 g\/L) was not associated with increased mortality rate in adults\u00a0[123]. Red blood cell transfusion in septic patients increases oxygen delivery but does not usually increase oxygen consumption\u00a0[124\u2013126]. This transfusion threshold of 7 g\/dL (70 g\/L) contrasts with the early goal-directed resuscitation protocol that uses a\u00a0target hematocrit of 30% in patients with low SCVO2 (measured in superior vena cava) during the first 6 hrs of resuscitation of septic shock.\nWe recommend that erythropoietin not be used as a\u00a0specific treatment of anemia associated with severe sepsis, but may be used when septic patients have other accepted reasons for administration of erythropoietin such as renal failure-induced compromise of red blood cell production (Grade 1B).Rationale. No specific information regarding erythropoietin use in septic patients is available, but clinical trials in critically ill patients show some decrease in red cell transfusion requirement with no effect on clinical outcome\u00a0[127, 128]. The effect of erythropoietin in severe sepsis and septic shock would not be expected to be more beneficial than in other critical conditions. Patients with severe sepsis and septic shock may have coexisting conditions that do warrant use of erythropoietin.\nWe suggest that fresh frozen plasma not be used to correct laboratory clotting abnormalities in the absence of bleeding or planned invasive procedures (Grade 2D).Rationale. Although clinical studies have not assessed the impact of transfusion of fresh frozen plasma on outcomes in critically ill patients, professional organizations have recommended fresh frozen plasma for coagulopathy when there is a\u00a0documented deficiency of coagulation factors (increased prothrombin time, international normalized ratio, or partial thromboplastin time) and the presence of active bleeding or before surgical or invasive procedures\u00a0[129\u2013131]. In addition, transfusion of fresh frozen plasma in nonbleeding patients with mild abnormalities of prothrombin time usually fails to correct the prothrombin time\u00a0[132]. There are no studies to suggest that correction of more severe coagulation abnormalities benefits patients who are not bleeding.\nWe recommendagainst antithrombin administration for the treatment of severe sepsis and septic shock (Grade 1B).Rationale. A\u00a0phase III clinical trial of high-dose antithrombin did not demonstrate any beneficial effect on 28-day all-cause mortality in adults with severe sepsis and septic shock. High-dose antithrombin was associated with an increased risk of bleeding when administered with heparin\u00a0[133]. Although a\u00a0post hoc subgroup analysis of patients with severe sepsis and high risk of death showed better survival in patients receiving antithrombin, antithrombin cannot be recommended at this time until further clinical trials are performed\u00a0[134].\nIn patients with severe sepsis, we suggest that platelets should be administered when counts are < 5000\/mm3 (5 \u00d7 109\/L) regardless of apparent bleeding. Platelet transfusion may be considered when counts are 5,000\u201330,000\/mm3 (5\u201330 \u00d7 109\/L) and there is a\u00a0significant risk of bleeding. Higher platelet counts (\u2265 50,000\/mm3 (50 \u00d7 109\/L)) are typically required for surgery or invasive procedures (Grade 2D).Rationale. Guidelines for transfusion of platelets are derived from consensus opinion and experience in patients undergoing chemotherapy. Recommendations take into account the etiology of thrombocytopenia, platelet dysfunction, risk of bleeding, and presence of concomitant disorders\u00a0[129, 131].\nII. Supportive Therapy of Severe Sepsis\nA. Mechanical Ventilation of Sepsis-Induced Acute Lung Injury (ALI)\/Acute Respiratory Distress Syndrome (ARDS).\nWe recommend that clinicians target a\u00a0tidal volume of 6 ml\/kg (predicted) body weight in patients with ALI\/ARDS (Grade 1B).We recommend that plateau pressures be measured in patients with ALI\/ARDS and that the initial upper limit goal for plateau pressures in a\u00a0passively inflated patient be \u2264 30 cm H2O. Chest wall compliance should be considered in the assessment of plateau pressure (Grade 1C).Rationale. Over the past 10 yrs, several multi-center randomized trials have been performed to evaluate the effects of limiting inspiratory pressure through moderation of tidal volume\u00a0[135\u2013139]. These studies showed differing results that may have been caused by differences between airway pressures in the treatment and control groups\u00a0[135, 140]. The largest trial of a\u00a0volume- and pressure-limited strategy showed a\u00a09% decrease of all-cause mortality in patients with ALI or ARDS ventilated with tidal volumes of 6 mL\/kg of predicted body weight (PBW), as opposed to 12 mL\/kg, and aiming for a\u00a0plateau pressure \u2264 30 cm H2O\u00a0[135]. The use of lung protective strategies for patients with ALI is supported by clinical trials and has been widely accepted, but the precise choice of tidal volume for an individual patient with ALI may require adjustment for such factors as the plateau pressure achieved, the level of PEEP chosen, the compliance of the thoracoabdominal compartment and the vigor of the patient's breathing effort. Some clinicians believe it may be safe to ventilate with tidal volumes higher than 6 ml\/kg PBW as long as the plateau pressure can be maintained \u2264 30 cm H2O\u00a0[141, 142]. The validity of this ceiling value will depend on breathing effort, as those who are actively inspiring generate higher trans-alveolar pressures for a\u00a0given plateau pressure than those who are passively inflated. Conversely, patients with very stiff chest walls may require plateau pressures higher than 30 cm H2O to meet vital clinical objectives. One retrospective study suggested that tidal volumes should be lowered even with plateau pressures that are \u2264 30 cm H2O\u00a0[143]. An additional observational study suggested that knowledge of the plateau pressures was associated with lower plateau pressures; however in this trial, plateau pressure was not independently associated with mortality rates across a\u00a0wide range of plateau pressures that bracketed 30 cm H2O\u00a0[144]. The largest clinical trial employing a\u00a0lung protective strategy coupled limited pressure with limited tidal volumes to demonstrate a\u00a0mortality benefit\u00a0[135].\nHigh tidal volumes that are coupled with high plateau pressures should be avoided in ALI\/ARDS. Clinicians should use as a\u00a0starting point the objective of reducing tidal volumes over 1\u20132 hrs from its initial value toward the goal of a\u00a0\u201clow\u201d tidal volume (\u2248 6 mL per kilogram of predicted body weight) achieved in conjunction with an end-inspiratory plateau pressure \u2264 30 cm H2O. If plateau pressure remains > 30 after reduction of tidal volume to 6 ml\/kg\/PBW, tidal volume should be reduced further to as low as 4 ml\/kg\/PBW (see Appendix E for ARDSnet ventilator management and formula to calculate predicted body weight).\nNo single mode of ventilation (pressure control, volume control, airway pressure release ventilation, high frequency ventilation, etc.) has been consistently shown advantageous when compared with any other that respects the same principles of lung protection.\nWe recommend that hypercapnia (allowing PaCO2 to increase above its pre-morbid baseline, so-called permissive hypercapnia) be allowed in patients with ALI\/ARDS if needed to minimize plateau pressures and tidal volumes (Grade 1C).Rationale. An acutely elevated PaCO2 may have physiologic consequences that include vasodilation as well as an increased heart rate, blood pressure, and cardiac output. Allowing modest hypercapnia in conjunction with limiting tidal volume and minute ventilation has been demonstrated to be safe in small, nonrandomized series\u00a0[145, 146]. Patients treated in larger trials that have the goal of limiting tidal volumes and airway pressures have demonstrated improved outcomes, but permissive hypercapnia was not a\u00a0primary treatment goal in these studies\u00a0[135]. The use of hypercapnia is limited in patients with preexisting metabolic acidosis and is contraindicated in patients with increased intracranial pressure. Sodium bicarbonate or tromethamine (THAM\u00ae) infusion may be considered in selected patients to facilitate use of permissive hypercarbia\u00a0[147, 148].\nWe recommend that positive end-expiratory pressure (PEEP) be set so as to avoid extensive lung collapse at end-expiration (Grade 1C).Rationale. Raising PEEP in ALI\/ARDS keeps lung units open to participate in gas exchange. This will increase PaO2 when PEEP is applied through either an endotracheal tube or a\u00a0face mask\u00a0[149\u2013151]. In animal experiments, avoidance of end-expiratory alveolar collapse helps minimize ventilator induced lung injury (VILI) when relatively high plateau pressures are in use. One large multi-center trial of the protocol-driven use of higher PEEP in conjunction with low tidal volumes did not show benefit or harm when compared to lower PEEP levels\u00a0[152]. Neither the control nor experimental group in that study, however, was clearly exposed to hazardous plateau pressures. A\u00a0recent multi-center Spanish trial compared a\u00a0high PEEP, low-moderate tidal volume approach to one that used conventional tidal volumes and the least PEEP achieving adequate oxygenation. A\u00a0marked survival advantage favored the former approach in high acuity patients with ARDS\u00a0[153]. Two options are recommended for PEEP titration. One option is to titrate PEEP (and tidal volume) according to bedside measurements of thoracopulmonary compliance with the objective of obtaining the best compliance, reflecting a\u00a0favorable balance of lung recruitment and overdistention\u00a0[154]. The second option is to titrate PEEP based on severity of oxygenation deficit and guided by the FIO2 required to maintain adequate oxygenation\u00a0[135] (see Appendix D.). Whichever the indicator-compliance or oxygenation-recruiting maneuvers are reasonable to employ in the process of PEEP selection. Blood pressure and oxygenation should be monitored and recruitment discontinued if deterioration in these parameters is observed. A\u00a0PEEP > 5 cm H2O is usually required to avoid lung collapse\u00a0[155].\nWe suggest prone positioning in ARDS patients requiring potentially injurious levels of FIO2 or plateau pressure who are not at high risk for adverse consequences of positional changes in those facilities who have experience with such practices (Grade 2C).Rationale. Several smaller studies and one larger study have shown that a\u00a0majority of patients with ALI\/ARDS respond to the prone position with improved oxygenation\u00a0[156\u2013159]. One large multi-center trial of prone positioning for approximately 7 hrs\/day did not show improvement in mortality rates in patients with ALI\/ARDS; however, a\u00a0post hoc analysis suggested improvement in those patients with the most severe hypoxemia by PaO2\/FIO2 ratio, in those exposed to high tidal volumes, and those who improved CO2 exchange as a\u00a0result of proning\u00a0[159]. A\u00a0second large trial of prone positioning, conducted for an average of approximately 8 hours per day for 4 days in adults with hypoxemic respiratory failure of low-moderate acuity, confirmed improvement in oxygenation but also failed to show a\u00a0survival advantage\u00a0[160]. However, a\u00a0randomized study that extended the length of time for proning each day to a\u00a0mean of 17 hours for a\u00a0mean of 10 days supported benefit of proning, with randomization to supine position an independent risk factor for mortality by multivariate analysis\u00a0[161]. Prone positioning may be associated with potentially life-threatening complications, including accidental dislodgment of the endotracheal tube and central venous catheters, but these complications can usually be avoided with proper precautions.\nA) Unless contraindicated, we recommend mechanically ventilated patients be maintained with the head of the bed elevated to limit aspiration risk and to prevent the development of ventilator-associated pneumonia (Grade 1B). B) We suggest that the head of bed is elevated approximately 30\u201345 degrees (Grade 2C).Rationale. The semirecumbent position has been demonstrated to decrease the incidence of ventilator-associated pneumonia (VAP)\u00a0[164]. Enteral feeding increased the risk of developing VAP; 50% of the patients who were fed enterally in the supine position developing VAP\u00a0[162]. However, the bed position was only monitored once a\u00a0day, and patients who did not achieve the desired bed elevation were not included in the analysis\u00a0[162]. A\u00a0recent study did not show a\u00a0difference in in incidence of VAP between patients maintained in supine and semirecumbent positions\u00a0[163]. In this study, patients in the semirecumbent position did not consistently achieve the desired head of the bed elevation, and the head of bed elevation in the supine group approached that of the semirecumbent group by day 7\u00a0[163]. When necessary, patients may be laid flat for procedures, hemodynamic measurements, and during episodes of hypotension. Patients should not be fed enterally with the head of the bed at 0\u00b0.\nWe suggest that noninvasive mask ventilation (NIV) only be considered in that minority of ALI\/ARDS patients with mild-moderate hypoxemic respiratory failure (responsive to relatively low levels of pressure support and PEEP) with stable hemodynamics who can be made comfortable and easily arousable, who are able to protect the airway, spontaneously clear the airway of secretions, and are anticipated to recover rapidly from the precipitating insult. A\u00a0low threshold for airway intubation should be maintained (Grade 2B).Rationale. Obviating the need for airway intubation confers multiple advantages: better communication, lower incidence of infection, reduced requirements for sedation. Two RCTs demonstrate improved outcome with the use of NIV when it can be employed successfully\u00a0[164, 165]. Unfortunately, only a\u00a0small percentage of patients with life threatening hypoxemia can be managed in this way.\nWe recommend that a\u00a0weaning protocol be in place, and mechanically ventilated patients with severe sepsis undergo spontaneous breathing trials on a\u00a0regular basis to evaluate the ability to discontinue mechanical ventilation when they satisfy the following criteria: a) arousable; b) hemodynamically stable (without vasopressor agents); c) no new potentially serious conditions; d) low ventilatory and end-expiratory pressure requirements; and e) FIO2 requirements that could be safely delivered with a\u00a0face mask or nasal cannula. If the spontaneous breathing trial is successful, consideration should be given for extubation (see Appendix E). Spontaneous breathing trial options include a\u00a0low level of pressure support, continuous positive airway pressure (\u2248 5 cm H2O) or a\u00a0T-piece (Grade 1A).Rationale. Recent studies demonstrate that daily spontaneous breathing trials in appropriately selected patients reduce the duration of mechanical ventilation\u00a0[166\u2013169]. Successful completion of spontaneous breathing trials leads to a\u00a0high likelihood of successful discontinuation of mechanical ventilation.\nWe recommendagainst the routine use of the pulmonary artery catheter for patients with ALI\/ARDS (Grade 1A).Rationale. While insertion of a\u00a0pulmonary artery catheter may provide useful information on a\u00a0patient's volume status and cardiac function, potential benefits of such information may be confounded by differences in interpretation of results\u00a0[170\u2013172], lack of correlation of pulmonary artery occlusion pressures with clinical response\u00a0[173], and absence of a\u00a0proven strategy to use catheter results to improve patient outcomes\u00a0[174]. Two multi-center randomized trials: one in patients with shock or acute lung injury\u00a0[175], and one in patients with acute lung injury\u00a0[176] failed to show benefit with the routine use of pulmonary artery catheters in patients with acute lung injury. In addition, other studies in different types of critically ill patients have failed to show definitive benefit with routine use of the pulmonary artery catheter\u00a0[177\u2013179]. Well-selected patients remain appropriate candidates for pulmonary artery catheter insertion when the answers to important management decisions depend on information only obtainable from direct measurements made within the pulmonary artery.\nTo decrease days of mechanical ventilation and ICU length of stay we recommend a\u00a0conservative fluid strategy for patients with established acute lung injury who do not have evidence of tissue hypoperfusion (Grade 1C).Rationale. Mechanisms for the development of pulmonary edema in patients with acute lung injury include increased capillary permeability, increased hydrostatic pressure and decreased oncotic pressure\u00a0[180, 181]. Small prospective studies in patients with critical illness and acute lung injury have suggested that less weight gain is associated with improved oxygenation\u00a0[182] and fewer days of mechanical ventilation\u00a0[183, 184]. Use of a\u00a0fluid conservative strategy directed at minimizing fluid infusion and weight gain in patients with acute lung injury based on either a\u00a0central venous catheter or a\u00a0pulmonary artery catheter along with clinical parameters to guide treatment strategies led to fewer days of mechanical ventilation and reduced length of ICU stay without altering the incidence of renal failure or mortality rates\u00a0[185]. Of note, this strategy was only used in patients with established acute lung injury, some of whom had shock present. Active attempts to reduce fluid volume were conducted only during periods free of shock.\nB. Sedation, Analgesia, and Neuromuscular Blockade in Sepsis\nWe recommend sedation protocols with a\u00a0sedation goal when sedation of critically ill mechanically ventilated patients with sepsis is required (Grade 1B).Rationale. A\u00a0growing body of evidence indicates that the use of protocols for sedation of critically ill ventilated patients can reduce the duration of mechanical ventilation and ICU and hospital length of stay\u00a0[186\u2013188]. A\u00a0randomized, controlled clinical trial found that protocol use resulted in reduced duration of mechanical ventilation, reduced lengths of stay, and reduced tracheostomy rates\u00a0[186].\nA\u00a0report describing the implementation of protocols, including sedation and analgesia, using a\u00a0short-cycle improvement methodology in the management of critically ill patients demonstrated a\u00a0decrease in the cost per patient day and a\u00a0decrease of ICU length of stay\u00a0[187]. Furthermore, a\u00a0prospective before-and-after study on the implementation of a\u00a0sedation protocol demonstrated enhanced quality of sedation with reduced drug costs. Although this protocol also may have contributed to a\u00a0longer duration of mechanical ventilation, ICU discharge was not delayed\u00a0[188]. Despite the lack of evidence regarding the use of subjective methods of evaluation of sedation in septic patients, the use of a\u00a0sedation goal has been shown to decrease the duration of mechanical ventilation in critically ill patients\u00a0[186]. Several subjective sedation scales have been described in the medical literature. Currently, however, there is not a\u00a0clearly superior sedation evaluation methodology against which these sedation scales can be evaluated\u00a0[189]. The benefits of sedation protocols appear to outweigh the risks.\nWe recommend intermittent bolus sedation or continuous infusion sedation to predetermined end points (e. g., sedation scales) with daily interruption\/lightening of continuous infusion sedation with awakening and retitration if necessary for sedation administration to septic mechanically ventilated patients (Grade 1B).Rationale. Although not specifically studied in patients with sepsis, the administration of intermittent sedation, daily interruption, and retitration or systemic titration to a\u00a0predefined end point have been demonstrated to decrease the duration of mechanical ventilation\u00a0[186, 189, 190]. Patients receiving neuromuscular blocking agents (NMBAs) must be individually assessed regarding discontinuation of sedative drugs because neuromuscular blocking drugs must also be discontinued in that situation. The use of intermittent vs. continuous methods for the delivery of sedation in critically ill patients has been examined. An observational study of mechanically-ventilated patients showed that patients receiving continuous sedation had significantly longer durations of mechanical ventilation and ICU and hospital length of stay\u00a0[191].\nSimilarly, a\u00a0prospective, controlled study in 128 mechanically-ventilated adults receiving continuous intravenous sedation demonstrated that a\u00a0daily interruption in the \u201ccontinuous\u201d sedative infusion until the patient was awake decreased the duration of mechanical ventilation and ICU length of stay\u00a0[192]. Although the patients did receive continuous sedative infusions in this study, the daily interruption and awakening allowed for titration of sedation, in effect, making the dosing intermittent. Systematic (protocolized) titration to a\u00a0predefined end point has also been shown to alter outcome\u00a0[186]. Additionally, a\u00a0randomized prospective blinded observational study demonstrated that although myocardial ischemia is common in critically ill ventilated patients, daily sedative interruption is not associated with an increased occurrence of myocardial ischemia\u00a0[193]. Thus, the benefits of daily interruption of sedation appear to outweigh the risks. These benefits include potentially shorter duration of mechanical ventilation and ICU stay, better assessment of neurologic function, and reduced costs.\nWe recommend that NMBAs be avoided if possible in the septic patient due to the risk of prolonged neuromuscular blockade following discontinuation. If NMBAs must be maintained, either intermittent bolus as required or continuous infusion with monitoring the depth of blockade with train-of-four monitoring should be used (Grade 1B).Rationale. Although NMBAs are often administered to critically ill patients, their role in the ICU setting is not well defined. No evidence exists that maintaining neuromuscular blockade in this patient population reduces mortality or major morbidity. In addition, no studies have been published that specifically address the use of NMBAs in septic patients.\nThe most common indication for NMBA use in the ICU is to facilitate mechanical ventilation\u00a0[194]. When appropriately utilized, NMBAs may improve chest wall compliance, prevent respiratory dyssynchrony, and reduce peak airway pressures\u00a0[195]. Muscle paralysis may also reduce oxygen consumption by decreasing the work of breathing and respiratory muscle blood flow\u00a0[196]. However, a\u00a0randomized, placebo-controlled clinical trial in patients with severe sepsis demonstrated that oxygen delivery, oxygen consumption, and gastric intramucosal pH were not improved during profound neuromuscular blockade\u00a0[197].\nAn association between NMBA use and myopathies and neuropathies has been suggested by case studies and prospective observational studies in the critical care population\u00a0[195, 198\u2013201]. The mechanisms by which NMBA's produced or contribute to myopathies and neuropathies in critically ill patients are presently unknown. There appears to be an added association with the concurrent use o NMBA's and steroids. Although no specific studies exist specific to the septic patient population, it seems clinically prudent based on existent knowledge that NMBA's not be administered unless there is a\u00a0clear indication for neuromuscular blockade that can not be safely achieved with appropriate sedation and analgesia''\u00a0[195].\nOnly one prospective, randomized clinical trial has evaluated peripheral nerve stimulation vs. standard clinical assessment in ICU patients. Rudis et al.\u00a0[202] randomized 77 critically ill patients requiring neuromuscular blockade in the ICU to receive dosing of vecuronium based on train-of-four stimulation or clinical assessment (control). The peripheral nerve stimulation group received less drug and recovered neuromuscular function and spontaneous ventilation faster than the control group. Nonrandomized observational studies have suggested that peripheral nerve monitoring reduces or has no effect on clinical recovery from NMBAs in the ICU setting\u00a0[203, 204].\nBenefits to neuromuscular monitoring, including faster recovery of neuromuscular function and, shorter intubation times, appear to exist. A\u00a0potential for cost savings (reduced total dose of NMBAs and shorter intubation times) also may exist, although this has not been studied formally.\nC. Glucose Control\nWe recommend that, following initial stabilization, patients with severe sepsis and hyperglycemia who are admitted to the ICU receive IV insulin therapy to reduce blood glucose levels (Grade 1B).We suggest use of a\u00a0validated protocol for insulin dose adjustments and targeting glucose levels to the < 150 mg\/dl range (Grade 2C).We recommend that all patients receiving intravenous insulin receive a\u00a0glucose calorie source and that blood glucose values be monitored every 1\u20132 hours until glucose values and insulin infusion rates are stable and then every 4 hours thereafter (Grade 1C).We recommend that low glucose levels obtained with point-of-care testing of capillary blood be interpreted with caution, as such measurements may overestimate arterial blood or plasma glucose values (Grade 1B).Rationale. The consensus on glucose control in severe sepsis was achieved at the first committee meeting and subsequently approved by the entire committee (see Appendix G for committee vote). One large randomized single center trial in a\u00a0predominantly cardiac surgical ICU demonstrated a\u00a0reduction in ICU mortality with intensive IV insulin (Leuven Protocol) targeting blood glucose to 80\u2013110 mg\/dl (for all patients relative 43%, and absolute 3.4% mortality reduction, and for those with > 5 day ICU length of stays (LOS) a\u00a048% relative and 9.6% absolute mortality reduction)\u00a0[205]. A\u00a0reduction in organ dysfunction and ICU LOS (from a\u00a0median of 15 to12 days) was also observed in the subset with ICU LOS > 5 days. A\u00a0second randomized trial of intensive insulin therapy using the Leuven Protocol enrolled medical ICU patients with an anticipated ICU LOS of > 3 days in three MICUs\u00a0[206]. Overall, mortality was not reduced but ICU and hospital LOS were reduced associated with earlier weaning from mechanical ventilation and less acute kidney injury. In patients with a\u00a0medical ICU LOS > 3 days, hospital mortality was reduced with intensive insulin therapy (43% versus 52.5%; p = 0.009). However, investigators were unsuccessful in predicting ICU LOS and 433 patients (36%) had an ICU LOS of < 3 days. Furthermore, use of the Leuven Protocol in the medical ICU resulted in a\u00a0nearly three-fold higher rate of hypoglycemia than in the original experience (18% versus 6.2% of patients)\u00a0[205, 206].\nOne large before-and-after observational trial showed a\u00a029% relative and 6.1% absolute reduction in mortality and a\u00a010.8% reduction in median ICU LOS\u00a0[207]. In a\u00a0subgroup of 53 patients with septic shock there was an absolute mortality reduction of 27% and a\u00a0relative reduction of 45% (p = 0.02). Two additional observational studies report an association of mean glucose levels with reductions in mortality, polyneuropathy, acute renal failure, nosocomial bacteremia, and number of transfusions, and suggest a\u00a0glucose threshold for improved mortality lies somewhere between 145 and 180 mg\/dl\u00a0[208, 209]. However, a\u00a0large observational study (n = 7,049) suggested that both a\u00a0lower mean glucose and less variation of blood glucose may be important\u00a0[210]. A\u00a0meta-analysis of 35 trials on insulin therapy in critically ill patients, including 12 randomized trials, demonstrated a\u00a015% reduction in short term mortality (RR 0.85, 95% confidence interval 0.75\u20130.97) but did not include any studies of insulin therapy in medical ICUs\u00a0[211].\nTwo additional multicenter RCTs of intensive insulin therapy, one focusing on patients with severe sepsis (VISEP) and the second on medical and surgical ICU patients, failed to demonstrate improvement in mortality, but are not yet published\u00a0[212, 213]. Both stopped earlier than planned because of high rates of hypoglycemia and adverse events in the intensive insulin groups. A\u00a0large RCT that is planned to compare targeting 80\u2013110 mg\/dl (4.5\u20136.0 mmol\/L) versus 140\u2013180 mg\/dl (8\u201310 mmol\/L) and recruit more than 6,000 patients (Normoglycemia in Intensive Care Evaluation and Survival Using Glucose Algorithm Regulation, or NICE-SUGAR) is ongoing\u00a0[214].\nSeveral factors may affect the accuracy and reproducibility of point-of-care testing of blood capillary blood glucose, including the type and model of the device used, user expertise, and patient factors including hematocrit (false elevation with anemia), PaO2, and drugs\u00a0[215]. One report showed overestimation of arterial plasma glucose values by capillary point-of-care testing sufficient to result in different protocol-specified insulin dose titration. The disagreement between protocol-recommended insulin doses was largest when glucose values were low\u00a0[216]. A\u00a0recent review of 12 published insulin infusion protocols for critically ill patients showed wide variability in insulin dose recommendations and variable glucose control during simulation\u00a0[217]. This lack of consensus about optimal dosing of IV insulin may reflect variability in patient factors (severity of illness, surgical vs. medical settings, etc) or practice patterns (e. g., approaches to feeding, IV dextrose) in the environments in which these protocols were developed and tested. Alternatively, some protocols may be more effective than other protocols. This conclusion is supported by the wide variability in hypoglycemia rates reported with protocols\u00a0[205\u2013207, 212, 213]. Thus, the use of a\u00a0validated and safe intensive insulin protocol is important not only for clinical care but also for the conduct of clinical trials to avoid hypoglycemia, adverse events, and premature termination of these trials before the efficacy signal, if any, can be determined.\nThe finding of reduced morbidity and mortality within the longer ICU length of stay subsets along with acceptable cost weighed heavily on our recommendation to attempt glucose control after initial stabilization of the patient with hyperglycemia and severe sepsis. However, the mortality benefit and safety of intensive insulin therapy (goal to normalize blood glucose) has been questioned by 2 recent trials and we recommend maintaining glucose levels < 150 mg\/dl until recent and ongoing trials are published or completed. Further study of protocols that have been validated to be safe and effective for controlling blood glucose concentrations and blood glucose variation in the severe sepsis population are needed.\nD. Renal Replacement\nWe suggest that continuous renal replacement therapies and intermittent hemodialysis are equivalent in patients with severe sepsis and acute renal failure (Grade 2B).We suggest the use of continuous therapies to facilitate management of fluid balance in hemodynamically unstable septic patients (Grade 2D).Rationale. Although numerous nonrandomized studies have reported a\u00a0nonsignificant trend toward improved survival using continuous methods\u00a0[218\u2013225], 2 meta-analyses\u00a0[226, 227] report the absence of significant difference in hospital mortality between patients who receive continuous and intermittent renal replacement therapies. This absence of apparent benefit of one modality over the other persists even when the analysis is restricted to only randomized studies\u00a0[227]. To date, 5 prospective randomized studies have been published\u00a0[228\u2013232]. Four of them found no significant difference in mortality\u00a0[229\u2013232]. One study found significantly higher mortality in the continuous treatment group\u00a0[228], but imbalanced randomization had led to a\u00a0higher baseline severity of illness in this group. When a\u00a0multivariable model was used to adjust for severity of illness, no difference in mortality was apparent between the groups\u00a0[228]. It is important to note that most studies comparing modes of renal replacement in the critically ill have included a\u00a0small number of patients and some major weaknesses (randomization failure, modifications of therapeutic protocol during the study period, combination of different types of continuous renal replacement therapies, small number of heterogenous groups of patients enrolled). The most recent and largest randomized study\u00a0[232] enrolled 360 patients and found no significant difference in survival between the 2 groups. Moreover, there is no current evidence to support the use of continuous therapies in sepsis independent of renal replacement needs.\nConcerning the hemodynamic tolerance of each method, no current evidence exists to support a\u00a0better tolerance with continuous treatments. Only 2 prospective studies\u00a0[230, 233] have reported a\u00a0better hemodynamic tolerance with continuous treatment, with no improvement in regional perfusion\u00a0[233] and no survival benefit\u00a0[230]. Four other prospective studies did not find any significant difference in mean arterial pressure or drop in systolic pressure between the 2 methods\u00a0[229, 231, 232, 234]. Concerning fluid balance management, 2 studies report a\u00a0significant improvement in goal achievement with continuous methods\u00a0[228, 230]. In summary, current evidence is insufficient to draw strong conclusions regarding the mode of replacement therapy for acute renal failure in septic patients.\nFour randomized, controlled trials have addressed whether the dose of continuous renal replacement affects outcomes in patients with acute renal failure\u00a0[235\u2013238]. Three found improved mortality in patients receiving higher doses of renal replacement\u00a0[235, 237, 238], while one\u00a0[236] did not. None of these trials was conducted specifically in patients with sepsis. Although the weight of current evidence suggests that higher doses of renal replacement may be associated with improved outcomes, these results may not be easily generalizable. The results of 2 very large multicenter randomized trials comparing the dose of renal replacement (ATN in the United States and RENAL in Australia and New Zealand) will be available in 2008 and will greatly inform practice.\nE. Bicarbonate Therapy\nWe recommendagainst the use of sodium bicarbonate therapy for the purpose of improving hemodynamics or reducing vasopressor requirements in patients with hypoperfusion-induced lactic acidemia with pH \u2265 7.15 (Grade 1B).Rationale. No evidence supports the use of bicarbonate therapy in the treatment of hypoperfusion-induced lactic acidemia associated with sepsis. Two randomized, blinded, crossover studies that compared equimolar saline and bicarbonate in patients with lactic acidosis failed to reveal any difference in hemodynamic variables or vasopressor requirements.\u00a0[239, 240] The number of patients with pH < 7.15 in these studies was small. Bicarbonate administration has been associated with sodium and fluid overload, an increase in lactate and pCO2, and a\u00a0decrease in serum ionized calcium; but the relevance of these parameters to outcome is uncertain. The effect of bicarbonate administration on hemodynamics and vasopressor requirements at lower pH as well as the effect on clinical outcomes at any pH is unknown. No studies have examined the effect of bicarbonate administration on outcomes.\nF. Deep Vein Thrombosis Prophylaxis\nWe recommend that severe sepsis patients receive deep vein thrombosis (DVT) prophylaxis with either (a) low-dose unfractionated heparin (UFH) administered b.i.d. or t.i.d. or (b) daily low-molecular weight heparin (LMWH) unless there are contraindications (i. e., thrombocytopenia, severe coagulopathy, active bleeding, recent intracerebral hemorrhage) (Grade\u00a01A).We recommend that septic patients who have a\u00a0contraindication for heparin use receive mechanical prophylactic device such as graduated compression stockings (GCS) or intermittent compression devices (ICD) unless contraindicated (Grade 1A).We suggest that in very high-risk patients such as those who have severe sepsis and history of DVT, trauma, or orthopedic surgery, a\u00a0combination of pharmacologic and mechanical therapy be used unless contraindicated or not practical (Grade 2C).We suggest that in patients at very high risk, LMWH be used rather than UFH as LMWH is proven superior in other high-risk patients (Grade 2C).Rationale. ICU patients are at risk for DVT\u00a0[241]. Significant evidence exists for benefit of DVT prophylaxis in ICU patients in general. No reasons suggest that severe sepsis patients would be different from the general patient population.\nNine randomized placebo controlled clinical trials of DVT prophylaxis in general populations of acutely ill patients exist\u00a0[242\u2013250]. All 9 trials showed reduction in DVT or PE. The prevalence of infection\/sepsis was 17% in all studies in which this was ascertainable, with a\u00a052% prevalence of infection\/sepsis patients in the study that included ICU patients only. Benefit of DVT prophylaxis is also supported by meta-analyses\u00a0[251, 252]. With that in mind, DVT prophylaxis would appear to have a\u00a0high grade for quality of evidence (A). As the risk of administration to the patient is small, the gravity of the potential result of not administering is great, and the cost is low, the grading of the strength of the recommendation is strong. The evidence supports equivalency of LMWH and UFH in general medical populations. A\u00a0recent meta-analysis comparing b.i.d. and t.i.d. UFH demonstrated that t.i.d. UFH produced better efficacy and b.i.d. less bleeding\u00a0[253]. Practitioners should use underlying risk for VTE and bleeding to individualize choice of b.i.d. versus t.i.d.\nThe cost of LMWH is greater and the frequency of injection is less. UFH is preferred over LMWH in patients with moderate to severe renal dysfunction.\nMechanical methods (ICD and GCS) are recommended when anticoagulation is contraindicated or as an adjunct to anticoagulation in the very high-risk patients\u00a0[254\u2013256]. In very high-risk patients, LMWH is preferred over UFH\u00a0[257\u2013259]. Patients receiving heparin should be monitored for development of heparin-induced thrombocytopenia (HIT).\nG. Stress Ulcer Prophylaxis (SUP)\nWe recommend that stress ulcer prophylaxis using H2 blocker (Grade 1A) or proton pump inhibitor PPI (Grade 1B) be given to patients with severe sepsis to prevent upper GI bleed. Benefit of prevention of upper GI bleed must be weighed against potential effect of an increased stomach pH on development of ventilator-associated pneumonia.\nRationale. Although no study has been performed specifically in patients with severe sepsis, trials confirming the benefit of stress ulcer prophylaxis reducing upper GI bleeds in general ICU populations would suggest that 20\u201325% of patients enrolled in these types of trials have sepsis\u00a0[260\u2013263]. This benefit should be applicable to patients with severe sepsis and septic shock. In addition, the conditions shown to benefit from stress ulcer prophylaxis (coagulopathy, mechanical ventilation, hypotension) are frequently present in patients with severe sepsis and septic shock\u00a0[264, 265].\nAlthough there are individual trials that have not shown benefit from SUP, numerous trials and a\u00a0meta-analysis show reduction in clinically significant upper GI bleeding, which we consider significant even in the absence of proven mortality benefit\u00a0[266\u2013269]. The benefit of prevention of upper GI bleed must be weighed against the potential effect of increased stomach pH on greater incidence of ventilator-associated pneumonia\u00a0[270]. Those severe sepsis patients with the greatest risk of upper GI bleeding are likely to benefit most from stress ulcer prophylaxis. The rationale for the preference for suppression of acid production over sulcrafate was based on the study of 1200 patients by Cook et al comparing H2 blockers and sucralfate and a\u00a0meta-analysis\u00a0[271, 272]. 2 studies support equivalency between H2 blockers and PPIs. One was in very ill ICU patients. The second study is larger and demonstrates non-inferiority of omeprazole suspension for clinically significant stress ulcer bleeding\u00a0[273, 274]. No data relating to utility of enteral feeding in stress ulcer prophylaxis exist. Patients should be periodically evaluated for continued need for prophylaxis.\nH. Selective Digestive Tract Decontamination (SDD)\nThe guidelines group was evenly split on the issue of SDD, with equal numbers weakly in favor and against recommending the use of SDD (see appendix H). The committee therefore chose not to make a\u00a0recommendation for the use of SDD specifically in severe sepsis at this time. The final consensus on use of SDD in severe sepsis was achieved at the last nominal committee meeting and subsequently approved by the entire committee (see Appendix H for committee vote).\nRationale. The cumulative conclusion from the literature demonstrates that prophylactic use of SDD (enteral non-absorbable antimicrobials and short-course intravenous antibiotics) reduces infections, mainly pneumonia, and mortality in the general population of critically ill and trauma patients\u00a0[275\u2013286] without promoting emergence of resistant Gram negative bacteria. Post hoc subgroup analyses\u00a0[287, 288] of two prospective blinded studies\u00a0[289, 290] suggest that SDD reduces nosocomial (secondary) infections in ICU patients admitted with primary infections\u00a0[268] and may reduce mortality\u00a0[288]. No studies of SDD specifically focused on patients with severe sepsis or septic shock. The use of SDD in severe sepsis patients would be targeted toward preventing secondary infection. As the main effect of SDD is in preventing ventilator-associated pneumonia (VAP), studies comparing SDD with non-antimicrobial interventions such as ventilator bundles for reducing VAP are needed. Further investigation is required to determine the comparative efficacy of these two interventions, separately or in combination. Although studies incorporating enteral vancomycin in the regimen appear to be safe\u00a0[291, 292, 293] concerns persist about the potential for emergence of resistant Gram positive infections.\nI. Consideration for Limitation of Support\nWe recommend that advance care planning, including the communication of likely outcomes and realistic goals of treatment, be discussed with patients and families (Grade 1D).\nRationale. Decisions for less aggressive support or withdrawal of support may be in the patient's best interest.\u00a0[294\u2013296] Too frequently, inadequate physician\/family communication characterizes end-of-life care in the ICU. The level of life support given to ICU patients may not be consistent with their wishes. Early and frequent caregiver discussions with patients who face death in the ICU and with their loved ones may facilitate appropriate application and withdrawal of life-sustaining therapies. A\u00a0recent RCT demonstrated reduction of anxiety and depression in family members when end-of-life meetings were carefully planned, conducted, included advance care planning, and provided relevant information about diagnosis, prognosis, and treatment\u00a0[297].\nIII. Pediatric Considerations in Severe Sepsis\nWhile sepsis in children is a\u00a0major cause of mortality, the overall mortality from severe sepsis in children is much lower that that in adults, estimated at about 10%\u00a0[298]. The definitions for severe sepsis and septic shock in children are similar but not identical to the definitions in adults\u00a0[299]. In addition to age-appropriate differences in vital signs, the definition of systemic inflammatory response syndrome requires the presence of either temperature or leukocyte abnormalities. The presence of severe sepsis requires sepsis plus cardiovascular dysfunction or ARDS or 2 or more other organ dysfunctions\u00a0[299].\nA. Antibiotics\nWe recommend antibiotics be administered within one hour of the identification of severe sepsis, after appropriate cultures have been obtained (Grade\u00a01D).Early antibiotic therapy is as critical for children with severe sepsis as it is for adults.\nB. Mechanical Ventilation\nNo graded recommendations.\nDue to low functional residual capacity, young infants and neonates with severe sepsis may require early intubation\u00a0[300]. Drugs used for intubation have important side effects in these patients, for example, concerns have been raised about the safety of using etomidate in children with meningococcal sepsis because of adrenal suppression effect\u00a0[301]. The principles of lung-protective strategies are applied to children as they are to adults.\nC. Fluid Resuscitation\nWe suggest initial resuscitation begin with infusion of crystalloids with boluses of 20 mL\/kg over 5\u201310\u00a0minutes, titrated to clinical monitors of cardiac output, including heart rate, urine output, capillary refill, and level of consciousness (Grade 2C).Intravenous access for fluid resuscitation and inotrope\/vasopressor infusion is more difficult to attain in children than in adults. The American Heart Association along with the American Academy of Pediatrics has developed pediatric advanced life support guidelines for emergency establishment of intravascular support encouraging early intraosseous access\u00a0[302]. On the basis of a\u00a0number of studies, it is accepted that aggressive fluid resuscitation with crystalloids or colloids is of fundamental importance to survival of septic shock in children\u00a0[303\u2013308]. Three randomized, controlled trials compare the use of colloid to crystalloid resuscitation in children with dengue shock\u00a0[303, 307, 308]. No difference in mortality between colloid or crystalloid resuscitation was shown.\nChildren normally have a\u00a0lower blood pressure than adults, and fall in blood pressure can be prevented by vasoconstriction and increasing heart rate. Therefore, blood pressure by itself is not a\u00a0reliable end point for assessing the adequacy of resuscitation. However, once hypotension occurs, cardiovascular collapse may soon follow. Hepatomegaly occurs in children who are fluid overloaded and can be a\u00a0helpful sign of adequacy of fluid resuscitation. Large fluid deficits typically exist and initial volume resuscitation usually requires 40\u201360 mL\/kg but can be much higher\u00a0[304\u2013308]. However, the rate of fluid administration should be reduced substantially when there are (clinical) signs of adequate cardiac filling without hemodynamic improvement.\nD. Vasopressors\/Inotropes (should be used in volume loaded patients with fluid refractory shock)\nWe suggest dopamine as the first choice of support for the pediatric patient with hypotension refractory to fluid resuscitation (Grade 2C).In the initial resuscitation phase, vasopressor therapy may be required to sustain perfusion pressure, even when hypovolemia has not yet been resolved. Children with severe sepsis can present with low cardiac output and high systemic vascular resistance, high cardiac output and low systemic vascular resistance, or low cardiac output and low systemic vascular resistance shock. At various stages of sepsis or the treatment thereof, a\u00a0child may move from one hemodynamic state to another. Vasopressor or inotrope therapy should be used according to the clinical state of the child.\nDopamine-refractory shock may reverse with epinephrine or norepinephrine infusion\u00a0[309].\nWe suggest that patients with low cardiac output and elevated systemic vascular resistance states (cool extremities, prolonged capillary refill, decreased urine output but normal blood pressure following fluid resuscitation) be given dobutamine (Grade 2C).The choice of vasoactive agent is determined by the clinical examination. For the child with a\u00a0persistent low cardiac output state with high systemic vascular resistance despite fluid resuscitation and inotropic support, vasodilator therapy may reverse shock\u00a0[310]. When pediatric patients remain in a\u00a0normotensive low cardiac output and high vascular resistance state despite epinephrine and vasodilator therapy, the use of a\u00a0phosphodiesterase inhibitor may be considered\u00a0[311\u2013313]. In the case of extremely low systemic vascular resistance despite the use of norepinephrine, vasopressin use has been described in a\u00a0number of case-reports. Thus far there is no clear evidence for the use of vasopressin in pediatric sepsis\u00a0[314, 315].\nE. Therapeutic End Points\nWe suggest that the therapeutic end points of resuscitation of septic shock be normalization of the heart rate, capillary refill of < 2 secs, normal pulses with no differential between peripheral and central pulses, warm extremities, urine output > 1mL.kg \u22121.hr\u22121, and normal mental status\u00a0[290] (Grade 2C).Capillary refill may be less reliable in a\u00a0cold environment. Other end points that have been widely used in adults and may logically apply to children include decreased lactate and improved base deficit, ScvO2 \u2265 70% or SvO2 \u2265 65%, CVP of 8\u201312 mm Hg or other methods to analyze cardiac filling. Optimizing preload optimizes cardiac index. When using measurements to assist in identifying acceptable cardiac output in children with systemic arterial hypoxemia such as cyanotic congenital heart disease or severe pulmonary disease, arterial-venous oxygen content difference is a\u00a0better marker than mixed venous hemoglobin saturation with oxygen. As noted previously, blood pressure by itself is not a\u00a0reliable end point for resuscitation. If a\u00a0thermodilution catheter is used, therapeutic end points are cardiac index > 3.3 and < 6.0 L.min\u22121.m\u22122 with normal coronary perfusion pressure (mean arterial pressure \u2013 central venous pressure) for age.\u00a0[290] Using clinical endpoints such as reversal of hypotension and restoration of capillary refill for initial resuscitation at the community hospital level before transfer to a\u00a0tertiary center was associated with significantly improved survival rates in children with septic shock\u00a0[305]. Development of a\u00a0transport system including publicizing to local hospitals and transport with mobile intensive care services significantly decreased the case fatality rate from meningococcal disease in the United Kingdom\u00a0[316].\nF. Approach to Pediatric Septic Shock\nFigure 1 shows a\u00a0flow diagram summarizing an approach to pediatric septic shock\u00a0[317].\nFig.\u00a01Approach to Pediatric Shock\nG. Steroids\nWe suggest that hydrocortisone therapy be reserved for use in children with catecholamine resistance and suspected or proven adrenal insufficiency (Grade 2C).Patients at risk for adrenal insufficiency include children with severe septic shock and purpura\u00a0[318, 319], children who have previously received steroid therapies for chronic illness, and children with pituitary or adrenal abnormalities. Children who have clear risk factors for adrenal insufficiency should be treated with stress dose steroids (hydrocortisone 50 mg\/m2\/24hr).\nAdrenal insufficiency in pediatric severe sepsis is associated with a\u00a0poor prognosis\u00a0[320]. No strict definitions exist, but absolute adrenal insufficiency in the case of catecholamine-resistant septic shock is assumed at a\u00a0random total cortisol concentration < 18 \u03bcg\/dL (496 nmol\/L). A\u00a0post 30- or 60-min ACTH stimulation test increase in cortisol of \u2264 9 \u03bcg\/dL (248 mmol\/L) has been used to define relative adrenal insufficiency. The treatment of relative adrenal insufficiency in children with septic shock is controversial. A\u00a0retrospective study from a\u00a0large administrative database recently reported that the use of any corticosteroids in children with severe sepsis was associated with increased mortality (OR 1.9 95% CI 1.7\u20132.2)\u00a0[321]. While steroids may have been given preferentially to more severely ill children, the use of steroids was an independent predictor of mortality in multivariable analysis\u00a0[321]. Given the lack of data in children and potential risk, steroids should not be used in those children who do not meet minimal criteria for adrenal insufficiency. A\u00a0randomized, controlled trial in children with septic shock is very much needed.\nH. Protein C and Activated Protein C\nWe recommendagainst the use rhAPC in children (Grade 1B).Protein C concentrations in children reach adult values at the age of 3 yrs. This might indicate that the importance of protein C supplementation either as protein C concentrate or as rhAPC is even greater in young children than in adults\u00a0[322]. There has been one dose finding, randomized, placebo-controlled study performed using protein C concentrate. This study was not powered to show an effect on mortality rate, but did show a\u00a0positive effect on sepsis-induced coagulation disturbances\u00a0[323]. An RCT of rhAPC in pediatric severe sepsis patients was stopped by recommendation of the Data Monitoring Committee for futility after enrollment of 399 patients. 28-day all cause mortality: 18% placebo group vs. 17% APC group. Major amputations occurred in 3% of the placebo group vs. 2% in the APC group\u00a0[324]. Due to the increased risk of bleeding (7% vs. 6% in the pediatric trial) and lack of proof of efficacy, rhAPC is not recommended for use in children.\nI. DVT Prophylaxis\nWe suggest the use of DVT prophylaxis in post-pubertal children with severe sepsis (Grade 2C).\nMost DVTs in young children are associated with central venous catheters. Femoral venous catheters are commonly used in children, and central venous catheter-associated DVTs occur in approximately 25% of children with a\u00a0femoral central venous catheter. Heparin-bonded catheters may decrease the risk of catheter-associated DVT and should be considered for use in children with severe sepsis.\u00a0[325, 326] No data on the efficacy of unfractionated or low-molecular weight heparin prophylaxis to prevent catheter-related DVT in children in the ICU exist.\nJ. Stress Ulcer Prophylaxis\nNo graded recommendations.\nStudies have shown that the rate of clinically important gastrointestinal bleeding in children occurs at rates similar to adults\u00a0[327, 328]. As in adults, coagulopathy and mechanical ventilation are risk factors for clinically important gastrointestinal bleeding. Stress ulcer prophylaxis strategy is commonly used in mechanically-ventilated children, usually with H2 blockers. Its effect is not known.\nK. Renal Replacement Therapy\nNo graded recommendations.\nContinuous veno-venous hemofiltration (CVVH) may be clinically useful in children with anuria\/severe oliguria and fluid overload, but no large RCTs have been performed comparing CVVH with intermittent dialysis. A\u00a0retrospective study of 113 critically ill children reported that children with less fluid overload before CVVH had better survival, especially in those children with dysfunction of 3 or more organs\u00a0[329]. CVVH or other renal replacement therapy should be instituted in children with anuria\/severe oliguria before significant fluid overload occurs.\nL. Glycemic Control\nNo graded recommendations.\nIn general, infants are at risk for developing hypoglycemia when they depend on intravenous fluids. This means that a\u00a0glucose intake of 4\u20136 mg.kg\u22121.min\u22121 or maintenance fluid intake with glucose 10%\/NaCl containing solution is advised. Associations have been reported between hyperglycemia and an increased risk of death and longer length of stay\u00a0[330]. A\u00a0recent retrospective PICU study reported associations of hyperglycemia, hypoglycemia, and glucose variability with length of stay and mortality rates.\u00a0[331] No studies in pediatric patients (without diabetes mellitus) analyzing the effect of strict glycemic control using insulin exist. In adults, the recommendation is to maintain a\u00a0serum glucose below 150 mg\/dL. Insulin therapy to avoid long periods of hyperglycemia seems sensible in children as well, but the optimal goal glucose is not known. However, continuous insulin therapy should only be done with frequent glucose monitoring in view of the risks for hypoglycemia.\nM. Sedation\/Analgesia\nWe recommend sedation protocols with a\u00a0sedation goal when sedation of critically ill mechanically ventilated patients with sepsis is required (Grade 1D).Appropriate sedation and analgesia are the standard of care for children who are mechanically ventilated. Although there are no data supporting any particular drugs or regimens, it should be noted that propofol should not be used for long term sedation in children because of the reported association with fatal metabolic acidosis\u00a0[332, 333].\nN. Blood Products\nNo graded recommendations.\nThe optimal hemoglobin for a\u00a0critically ill child with severe sepsis is not known. A\u00a0recent multicenter trial reported similar outcomes in stable critically ill children managed with a\u00a0transfusion threshold of 7 gm\/dl compared to those managed with a\u00a0transfusion threshold of 9.5 g\/dL\u00a0[334]. Whether a\u00a0lower transfusion trigger is safe or appropriate in the initial resuscitation of septic shock has not been determined.\nO. Intravenous Immunoglobulin\nWe suggest that immunoglobulin may be considered in children with severe sepsis (Grade 2C).Administration of polyclonal intravenous immunoglobulin has been reported to reduce mortality rate and is a\u00a0promising adjuvant in the treatment of sepsis and septic shock in neonates. A\u00a0recent randomized controlled study of polyclonal immunoglobulin in pediatric sepsis syndrome patients (n = 100), showed a\u00a0significant reduction in mortality, LOS, and less progress to complications, especially DIC\u00a0[335].\nP. Extracorporeal membrane oxygenation (ECMO)\nWe suggest that use of ECMO be limited to refractory pediatric septic shock and\/or respiratory failure that cannot be supported by conventional therapies (Grade 2C).ECMO has been used in septic shock in children, but its impact is not clear. Survival from refractory shock or respiratory failure associated with sepsis is 80% in neonates and 50% in children. In one study analyzing 12 patients with meningococcal sepsis in ECMO, eight of the 12 patients survived, with six leading functionally normal lives at a\u00a0median of 1 yr (range, 4 months to 4 yrs) of follow-up. Children with sepsis on ECMO do not perform worse than children without sepsis at long-term follow-up\u00a0[336, 337].\nAlthough the pediatric considerations section of this manuscript offers important information to the practicing pediatric clinician for the management of critically ill children with sepsis, the reader is referred to the references at the end of the document for more in-depth descriptions of appropriate management of pediatric septic patients.\nSummary and Future Directions\nThe reader is reminded that although this document is static, the optimum treatment of severe sepsis and septic shock is a\u00a0dynamic and evolving process. New interventions will be proven and established interventions, as stated in the current recommendations, may need modification. This publication represents an ongoing process. The Surviving Sepsis Campaign and the consensus committee members are committed to updating the guidelines on a\u00a0regular basis as new interventions are tested and published in the literature.\nAlthough evidence-based recommendations have been frequently published in the medical literature, documentation of impact on patient outcome is limited\u00a0[338]. There is, however, growing evidence that protocol implementation associated with education and performance feedback does change clinician behavior and may improve outcomes in and reduce costs in severe sepsis\u00a0[20,\u00a024,\u00a025]. Phase III of the Surviving Sepsis Campaign targets the implementation of a\u00a0core set of the previous recommendations in hospital environments where change in behavior and clinical impact are being measured. The sepsis bundles were developed in collaboration with the Institute of Healthcare Improvement\u00a0[339]. Concurrent or retrospective chart review will identify and track changes in practice and clinical outcome. Software and software support is available at no cost in 7 languages, allowing bedside data entry and allows creation of regular reports for performance feedback. The Campaign also offers significant program support and educational materials at no cost to the user (www.survivingsepsis.org ).\nEngendering evidence-based change in clinical practice through multi-faceted strategies while auditing practice and providing feedback to healthcare practitioners is the key to improving outcomes in severe sepsis. Nowhere is this more evident than in the worldwide enthusiasm for Phase III of the Campaign, a\u00a0performance improvement program using SSC guideline-based sepsis bundles. Using the guidelines as the basis, the bundles have established a\u00a0global best practice for the management of critically ill patients with severe sepsis. As of November 2007, over 12,000 patients have been entered into the SSC central database, representing the efforts of 239 hospitals in 17 countries. Change in practice and potential effect on survival are being measured.","keyphrases":["surviving sepsis campaign","sepsis","guidelines","severe sepsis","septic shock","infection","sepsis syndrome","grade","sepsis bundles","evidence-based medicine"],"prmu":["P","P","P","P","P","P","P","P","P","R"]}
{"id":"Ann_Gen_Hosp_Psychiatry-2-_-162166","title":"Contribution of psychoacoustics and neuroaudiology in revealing correlation of mental disorders with central auditory processing disorders\n","text":"Background Psychoacoustics is a fascinating developing field concerned with the evaluation of the hearing sensation as an outcome of a sound or speech stimulus. Neuroaudiology with electrophysiologic testing, records the electrical activity of the auditory pathways, extending from the 8th cranial nerve up to the cortical auditory centers as a result of external auditory stimuli. Central Auditory Processing Disorders may co-exist with mental disorders and complicate diagnosis and outcome.\nBackground\nEvaluation of the central auditory nervous system (CANS) is essential in order to obtain information on its anatomical and functional integrity. Both, children and adults may suffer from central auditory processing disorders (CAPD). This fact has been underestimated but as research in this field progresses, it shows that specific mental disorders may be the outcome of a CAPD or that CAPD can co-exist with a neurological or mental disorder [1].\nAssessment of the CANS begun at the mid-1950s with the confirmation by Bocca and his colleagues [2] that CANS disorders do exist and that there are sensitive tests to reveal them. However, at that time acceptance of the new diagnostic methods by the audiologists, who were the first to be interested in this field was limited. This can be attributed to the slow acceptance of each new method before it is fully validated. Better understanding of the anatomy and physiology of the CANS was gained by advances concerning the presence and physiology of neurotransmitters and the accumulation of data on the psychoacoustic and electrophysiologic tests [3]. As a result audiologists started applying the new diagnostic tests more often and appreciated their contribution. Other medical specialties became aware and interested in the disorders of the CANS. These were mainly psychiatry and neurology. The assessment of the CANS is also of great value concerning neuropsychology and special education [4-6].\nAnatomy and physiology of the CANS\nClinical evaluation of central auditory function requires understanding of the anatomy and physiology of the CANS and appreciation of its complexity. The CANS extends from the anterior and posterior cochlear nuclei which are situated on the surface of the inferior cerebellar peduncle to the auditory cortex. In between important structures through which nerve fibers pass are: the trapezoid body, the lateral lemniscus, the inferior colliculus, the medial geniculate body and the acoustic radiation of the internal capsule. The auditory cortex includes the gyrus of Heschl on the upper surface of the superior temporal gyrus, the planum temporale and the Silvian fissure.\nIt is essential to point out that nerve impulses from each ear proceed along auditory pathways on both sides of the brainstem. Both ipsilateral and contralateral pathways are important in ensuring interchange of auditory information. The contralateral pathway exhibits dominance as opposed to the ipsilateral one [7]. Thirty thousand afferent auditory nerve fibers with different range of frequency response are responsible for conveying auditory information to the cortex [8]. Many components of the stimulus are analyzed separately. There is an increasing complexity of the whole process in the auditory cortex. One should keep in mind that, understanding of the exact way of processing the auditory information at the level of the auditory cortex, is still incomplete. It is in this understanding that Phychoacoustics helps as it is the science concerned with the evaluation of the sensation of hearing as an outcome of the sound or speech stimulus.\nComponents of central auditory processing\nCentral auditory processing occurs prior to language comprehension [9]. It consists firstly of auditory discrimination, which is responsible for the ability to group sounds according to how similarly or differently they are heard. Auditory memory is the component responsible for storing and recalling auditory information. Auditory perception concerns the reception and understanding of sounds and words. It plays a significant part in reading skills, managing verbal information, communication and social relationships. Auditory-vocal association consists of the interaction between what is heard and verbal response. Auditory synthesis is responsible for combining sounds or syllables to formulate comprehensible patterns (words) and de-combining words into separate sounds. Auditory-vocal automaticity is the ability to predict how future linguistic events will be heard by utilizing past experience. Auditory figure-ground plays a role in diminishing sounds which are not important while focusing on others [10]. It is due to this component that someone can listen to another person talking in a railway station, where a lot of environmental noise exists.\nMaterial and methods\nThe medline research revealed 564 papers when using the keywords 'auditory deficits' and 'mental disorders'. 79 papers were referring specifically to CAPD in connection with mental disorders, as this is a new term for auditory deficits and one mostly used by audiologists. Auditory deficit is a more general term used mostly by psychiatrists. Both terms refer to the same disorder. It is essential to point out that 25 of the 79 papers are published between 2000 \u2013 2003.\nSchizophrenia is found related to CAPD in 175 papers, 49 of them are published between 2000 \u2013 2003 showing the research focus of the last three years. Learning disabilities were found related to CAPD in 126 papers. Parkinson's disease was related to CAPD in 29 papers. Dyslexia is related to CAPD in 88 papers, 37 of them are between 2000\u20132003. Alzheimer's disease and auditory deficits are connected in 39 papers. The remaining articles are on depression, alcoholism, anorexia and childhood mental retardation, all being related to some extend to CAPD.\nAssessment of the CANS is carried through a great variety of tests that fall into two main categories: psychoacoustic and electrophysiologic testing. Psychoacoustic tests are considered more subjective. Electrophysiologic ones are more objective with the exception of P300 component.\nResults\npsychoacoustic tests\nLearning disabilities, attention deficit disorders and dyslexia are assessed through a great variety of psychoacoustic tests. Age limitations have to be considered [11] and specially designed tests are used for different age groups. When evaluating children who are less than 12 years old an important step is the Pediatric Speech Intelligibility (PSI) Test. This consists of single words and sentences presented with a competing message at varying levels of difficulty [12]. In this test it is essential that performance is adjusted for language age according to previously determined normative data [13]. Evaluation of this test may provide the cause of learning disabilities including dyslexia [14,15].\nChildren older than 12 years old are assessed through a more complex test battery that contains several tests. These tests are based on the stimulation of the auditory system with tones, numbers, syllables, words and sentences. Evaluation is made according to the different components of the auditory processing. One widely used test is that of the dichotic digits which consists of different pairs of numbers presented simultaneously to each ear [16]. The person under examination has to repeat all four numbers regardless of order. This test is easy to use in order to detect the auditory deficit of dyslexia particularly since it does not contain language and phonological parameters [17].\nThe Staggered Spondaic Word Test (SSW) consists of two-syllable spondaic words that are presented simultaneously to each ear [18]. This involves the diagnosis of auditory deficits in attention disorders, autism, learning disabilities and chronic alcoholism [19,20].\nA series of experiments were planned by Nielzen and Olsson on the basis of psychoacoustic handling of auditory stimulation. The results of these psychacoustic experiments show significant differences between a group of schizophrenic patients and a group of reference subjects thus indicating central auditory processing disorders even in a phase of illness remission or during treatment with neuroleptics [21].\nelectrophysiologic tests\nIn all mental disorders assessed with the suspicion of CAPD an objective measure of the peripheral auditory system is mandatory. The Auditory Brainstem Responses (ABR), measure the electrophysiologic activity from the 8th cranial nerve to the medial geniculate body of the brainstem [22]. A very important element of ABR evaluation is the morphology and synchronization of the waveform. One should always begin his evaluation while observing waveform changes on real time [23].\nThe Auditory Middle Latency Responses (AMLRs) provide an electrophysiologic measure of the primary auditory cortex function [24]. The AMLRs can diagnose central auditory processing disorders in children with learning disabilities [25], patients with Alzheimer's disease [26], adult autistic subjects [27,28] patients with Schizophrenia [29] The Auditory P300 Response, which consists of the measure of the hippocampal and auditory cortex function again from an electrophysiological point of view [30]. The P300 response has been considered an endogenous event-related potential. Endogenous responses depend both on the context within which the auditory stimuli are presented and the psychologic condition and attention of the subject. P300 has been used in diagnosing CAPD in patients with dementia of the Alzheimer type [31], in monitoring long-term effects of donepezil in patients with Alzheimer's disease [32], in anorexic patients [33], in children with mental retardation during a selective attention task to auditory stimuli [34] and in first episode and chronic schizophrenia [35]. Mismatch Negativity Response (MMN) is an event-related evoked potential that measures the electrophysiologic activity of the auditory cortex function [36]. The MMN is always elicited 100\u2013250 msecs from stimulus change onset. Its application is in detecting CAPD in alcoholism [37], in Schizophrenia [38-43], in attention deficit and in developmental dyslexia [44].\npsychoacoustic and electrophysiologic testing according to type of lesion\nIn the selection of tests for the evaluation of brainstem lesions the examiner should keep in mind that all psychoacoustic tests have been reported to aid in the diagnosis. According to the studies of Kartz [45] the Staggered Spondaic Words Test may help differentiating brainstem from cortical lesions and upper from lower brainstem lesions. Musiek et al [46] concluded that Auditory Brainstem Responses in combination with either Masking Level Differences or Dichotic Digits Test may be as sensitive in evaluating a group of patients suffering from multiple sclerosis as a seven test battery. Jerger et al [47] reported that for patients suffering from multiple sclerosis the best test battery was a combination of stapedial reflex measures and speech audiometry.\nThe usual finding in central auditory tests regarding cortical lesions is a deficit or impairment in the ear contralateral to the side of lesion. Psychoacoustic tests such as Dichotic Digits and SSW in patients with well documented cortical and hemispheric lesions demonstrate primarily contralateral ear deficits and impairments [48]. Two exceptions that the examiner should always keep in mind are when frequency and duration tests are applied and when compromise of auditory fibers of the corpus callosum has occurred [49].\nRegarding interhemispheric dysfunction, test results may be difficult to evaluate. Representation of auditory information at the cortical level is mostly contralateral as is clearly depicted in dichotic listening situations. When speech responses are required by the subject auditory information from the right ear are projected through to the left hemisphere without the participation of the opposite hemisphere for the production of a speech response. On the contrary auditory stimuli from the left ear must cross the midline through the corpus callosum for the production of a speech response. Patients with split brain disorders subjected to dichotic testing have interestingly demonstrated decreased scores regarding the left ear and enhanced scores in the right ear [50,51].\nConsiderable evidence has been reported that indicates a relation between various learning disabilities, including dyslexia, attention deficit hyperactivity disorder and poor performance scores on central auditory tests Learning disabilities in children might be the expression of various underlying central auditory disorders such as maturational, developmental or neurological as depicted by abnormal CAPD test results [52].\nConclusions\nCANS assessment represents a fascinating field. Cooperation of professionals in psychiatry, neurology, neuropsychology and pediatric psychology, with the otolaryngologist-audiologist is a prerequisite. Central auditory processing disorders may co-exist with various mental disorders such as: learning disabilities, attention deficit hyperactivity disorder, dyslexia, autism, chronic alcoholism, Alzheimer's disease, adult autistic disorder, Schizophrenia, anorexia and mental retardation. Assessing these disorders is difficult due to the complex anatomy and physiology of the CANS. This explains the great variety of existing methods of testing with two main categories: those of psychoacoustic methodology and those based on electrophysiologic measures. Physiology of CANS is still not completely understood and further research is needed on development of new tests and validation of their clinical applicability.\nConflict of interest\nnone declared","keyphrases":["psychoacoustics","mental disorders","central auditory processing disorders"],"prmu":["P","P","P"]}
{"id":"Rheumatol_Int-3-1-2134974","title":"The potential utility of B cell-directed biologic therapy in autoimmune diseases\n","text":"Increasing awareness of the importance of aberrant B cell regulation in autoimmunity has driven the clinical development of novel B cell-directed biologic therapies with the potential to treat a range of autoimmune disorders. The first of these drugs\u2014rituximab, a chimeric monoclonal antibody against the B cell-specific surface marker CD20\u2014was recently approved for treating rheumatoid arthritis in patients with an inadequate response to other biologic therapies. The aim of this review is to discuss the potential use of rituximab in the management of other autoimmune disorders. Results from early phase clinical trials indicate that rituximab may provide clinical benefit in systemic lupus erythematosus, Sj\u00f6gren\u2019s syndrome, vasculitis, and thrombocytopenic purpura. Numerous case reports and several small pilot studies have also been published reporting the use of rituximab in conditions such as myositis, antiphospholipid syndrome, Still\u2019s disease, and multiple sclerosis. In general, the results from these preliminary studies encourage further testing of rituximab therapy in formalized clinical trials. Based on results published to date, it is concluded that rituximab, together with other B cell-directed therapies currently under clinical development, is likely to provide an important new treatment option for a number of these difficult-to-treat autoimmune disorders.\nBackground\nAutoimmunity is widely believed to be fundamental to the development and progression of many rheumatic diseases\u2014rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) being the best-known examples. The function of B cells in autoimmunity is still not fully understood, although evidence is mounting that they play an essential role in the process. In addition to their well-known function in synthesizing antibodies, B cells act in antigen presentation and as critical regulators of the development and function of T cells [21]. B cells are also the source of rheumatoid factor, levels of which are strongly correlated with disease severity in RA [103]. These and other lines of evidence provided the rationale for testing whether B cell depletion would be an effective strategy for treating rheumatic diseases. The availability of rituximab (RITUXAN\u00ae; Genentech\/Biogen-IDEC, South San Francisco, CA, USA), a genetically engineered monoclonal antibody directed against the B cell-specific antigen CD20 [60], enabled this hypothesis to be tested. The first results, demonstrating sustained clinical responses coupled with B cell depletion in 5 RA patients treated with rituximab [30], ignited intense interest in the wider potential of B cell depletion therapy in autoimmune diseases.\nA full-scale clinical trial program led to the approval of rituximab in 2006 for the treatment of RA in patients with an inadequate response to anti-tumour necrosis factor (TNF) therapy. A number of other B cell-directed agents are currently in clinical development. Among the most advanced is epratuzumab, a humanized monoclonal antibody directed against CD22, another B cell-specific marker [90]. Epratuzumab has been tested in patients with Sj\u00f6gren\u2019s syndrome (SS) [89] and results were published recently of an open-label clinical trial involving patients with SLE [29]. Another strategy under investigation is the neutralization of B cell survival factors. BAFF (also known as B lymphocyte stimulator, BLyS) is essential for the survival of B cells and is involved in many other aspects of B cell biology, including germinal center maintenance, isotype switching, and regulation of B cell-specific markers [48]. Belimumab is an anti-BAFF monoclonal antibody that has reached Phase II trials in SLE and RA [27], while atacicept (previously known as TACI-Ig), a recombinant fusion protein that neutralizes both BAFF and APRIL (a related B cell survival factor) [41], has undergone Phase I evaluation in SLE. A more in-depth review was published recently of current B cell-targeted approaches that are being developed to treat autoimmune disorders [31].\nThe aim of this review is to discuss the potential utility of B cell-directed therapy in the management of autoimmune disorders. As the first of these agents to be approved for clinical use, rituximab will be the focus of this article. In addition, since several excellent reviews have been published recently covering the use of rituximab in RA [25, 31, 58], this review will discuss results from the clinical testing of rituximab in autoimmune disorders other than RA. Information from case reports, clinical trials, and other studies was gathered from a search of the Medline database up to and including June 2007.\nClinical use of rituximab\nRituximab has been tested in a wide range of autoimmune conditions, with clinical trials being most advanced in SLE and SS. A summary of the published clinical data in these and other autoimmune disorders is presented in Table\u00a01.\nTable\u00a01Summary of published data from clinical studies of rituximab in autoimmune disorders other than RADisorder\/study typeNo. of ptsTreatment regimenSummary of clinical resultsSystemic lupus erythematosusPhase I\/II dose escalation [59]18Single RTX infusion of 100\u00a0mg\/m2 (low dose [n\u00a0=\u00a06]) or 375\u00a0mg\/m2 (intermediate dose [n\u00a0=\u00a06]), or 4\u00a0weekly RTX infusions of 375\u00a0mg\/m2 (high dose [n\u00a0=\u00a06]) Improved SLAM score at 12\u00a0months in 11\/17\u00a0(65%) evaluable pts Open-label pilot [84]10aRTX (375\u00a0mg\/m2) once weekly for 4\u00a0weeks\u00a0+\u00a0prednisolone (0.5\u00a0mg\/kg\/day for 10\u00a0weeks, tapered by 4\u00a0mg every 2\u00a0weeks thereafter)Partial remission (improvement in renal parameters) in 8\/10\u00a0pts within a median (range) of 2 (1\u20134) months; of these, 5\u00a0pts had complete remission at 3\u00a0months (median); this was sustained for \u226512\u00a0months in 4\u00a0ptsOpen-label pilot [55]24RTX (1,000\u00a0mg)\u00a0+\u00a0CyP (750\u00a0mg): two infusions, 2\u00a0weeks apart Improvements in global and all 8 individual BILAG scores at 6\u00a0months in 23\/24\u00a0pts (96%) Open-label pilot [85]11RTX (375\u00a0mg\/m2) once weekly for 4\u00a0weeks\u00a0+\u00a0CyP (500\u00a0mg) co-administered at first infusion; immunosuppressive therapy at baseline had been unchanged for \u22653\u00a0months prior to study and was continued until Month 6, following which dose reduction was allowed 6 complete and 5 partial responses (follow-up through 2\u00a0yrs) (overall, significant reduction in median BILAG scores) Sj\u00f6gren\u2019s syndromeSingle-centre, open-label Phase II [73]15RTX (375\u00a0mg\/m2) once weekly for 4\u00a0weeksImprovements in subjective and objective parameters of disease activity (salivary and lacrimal gland function) in all 14\u00a0pts who completed the study. Of the 7\u00a0pts with MALT-type lymphoma, 3 had complete remission, while disease was stable in 3\u00a0pts and progressive in 1\u00a0pt. Retrospective [83]16RTX (375\u00a0mg\/m2) once weekly for 4\u00a0weeks (6\u00a0weeks in 1\u00a0pt with lymphoma); 1\u00a0pt with systemic manifestations received RTX 2\u00a0\u00d7\u00a01,000\u00a0mg. All pts received methylprednisone (100\u00a0mg) and either oral certirizine (20\u00a0mg) or dexchloropheniramine (5\u00a0mg) before the RTX infusionEfficacy observed in 9\/11\u00a0pts with systemic manifestations (improvement in systemic symptoms) and in 4\/5\u00a0pts with lymphomas (disease remission) Open-label pilot [26]16RTX (375\u00a0mg\/m2) once weekly for 2\u00a0weeksSignificant improvement in mean VAS scores for fatigue and dryness, tender point count, and quality of life (at Week 12) and for all 4 VAS scores, tender joint count, tender point count, and quality of life (at Week 36) VasculitisCase series [34]9bRTX (500\u00a0mg [375\u00a0mg\/m2 in 1 patient]) once weekly for 2\u00a0weeks (n\u00a0=\u00a03) or 4\u00a0weeks (n\u00a0=\u00a06)Remission (BVAS\u00a0=\u00a00) in 8\u00a0pts and partial remission (BVAS\u00a0=\u00a01) in 1\u00a0pt at 6\u00a0monthsCase series [50]11cRTX (375\u00a0mg\/m2) once weekly for 4\u00a0weeks\u00a0+\u00a0prednisone (\u22641\u00a0mg\/kg\/day, tapering once disease activity improved)Remission (BVAS\/WG\u00a0=\u00a00) in all 11\u00a0pts (10\u00a0pts within 6\u00a0months); tapering of prednisone dose (median\u00a0=\u00a00; range 0\u20131.5\u00a0mg\/kg\/day) in all ptsOpen-label pilot [51]10dRTX (375\u00a0mg\/m2) once weekly for 4\u00a0weeks\u00a0+\u00a0prednisone (\u22641\u00a0mg\/kg\/day, tapering once disease activity improved)Remission (BVAS\/WG\u00a0=\u00a00) in all pts within 3\u00a0months; tapering of prednisone dose to 0 in all pts by 6\u00a0monthsCase series [88]10eRTX (375\u00a0mg\/m2) once weekly for 4\u00a0weeks\u00a0+\u00a0prednisone (\u22642\u00a0mg\/kg\/day, tapering once disease activity improved)Complete response (BVAS\/WG\u00a0=\u00a00) in 9\u00a0pts and partial response (BVAS\/WG\u00a0=\u00a01) in 1\u00a0pt at 6\u00a0months. Follow-up (median 34\u00a0months; range 26\u201345\u00a0months): 3\u00a0pts relapsed but had new sustained response following re-treatmentOpen-label [85]11cRTX (375\u00a0mg\/m2) once weekly for 4\u00a0weeks\u00a0+\u00a0CyP (500\u00a0mg) co-administered at first infusion; immunosuppressive therapy at baseline had been unchanged for \u22653\u00a0months prior to study and was continued until Month 6, following which dose reduction was allowedRemission in 9\/11\u00a0pts (BVAS\u00a0=\u00a00) and partial remission in 1\u00a0pt (BVAS\u00a0=\u00a02); 6\/10\u00a0pts subsequently relapsed but had new sustained response following re-treatment with RTX (2\u00a0\u00d7\u00a01000\u00a0mg, 2\u00a0weeks apart)Case series [8]8dRTX (375\u00a0mg\/m2) once every 4\u00a0weeks for 4 cycles\u00a0+\u00a0standard treatment (CyP 2\u00a0mg\/kg once daily or 15\u201320\u00a0mg\/kg every 18\u201321\u00a0days or methotrexate 0.3\u00a0mg\/kg once weekly) Remission (BVAS\u00a0=\u00a00) in 2\u00a0pts, partial remission in 1\u00a0pt, unchanged disease activity in 3\u00a0pts, and progression in 2\u00a0pts 1\u00a0month after final cycleMyositisOpen-label pilot [56]7fRTX (375\u00a0mg\/m2) once weekly for 4\u00a0weeks\u00a0+\u00a0standard treatment (included azathioprine, corticosteroids, CyP, and intravenous immunoglobulin)Clinical improvement (increased muscle strength relative to baseline [assessed using dynomometry]) in all 6 evaluable pts Idiopathic thrombocytopenic purpuraOpen-label pilot [87]25RTX (375\u00a0mg\/m2) once weekly for 4\u00a0weeksClinical response (rise in platelet counts) at end of therapy without need for further treatment in 13\/25 (52%) pts. Responses were sustained for \u22656\u00a0months in 7\u00a0ptsPooled data from two pilot trials [22]57RTX (375\u00a0mg\/m2) once weekly for 4\u00a0weeks; 17\u00a0pts received prednisone (60\u00a0mg with Infusion 1 and 20\u00a0mg with Infusion 2)Clinical response (rise in platelet counts) at end of therapy without need for further treatment in 31\/57 (54%) pts; 29\/31 responses occurred within 8\u00a0weeks of initiating RTX therapy. 15\/16\u00a0pts with complete clinical response (rise in platelet counts to normal levels) maintained response for \u226512\u00a0months Retrospective national multicenter [13]35RTX (375\u00a0mg\/m2) once weekly for 4\u00a0weeks\u00a0+\u00a0prednisone; 6\u00a0pts received a fixed dose of 500\u00a0mg supplemented by 100\u00a0mg methylprednisone or 50\u2013100\u00a0mg prednisone\u00a0+\u00a0antihistamine prior to RTX infusionClinical response (rise in platelet counts) within 3\u20138\u00a0weeks for 17\/39 (44%) treatments (4\u00a0pts received 2 cycles); pts with complete or partial responses had been in remission for a median of 47\u00a0weeksRetrospective national multicenterg [71]89RTX (375\u00a0mg\/m2) once weekly for 4\u00a0weeks (n\u00a0=\u00a077) or for 1\u20136\u00a0weeks (n\u00a0=\u00a012); 31\u00a0pts received RTX with other therapies (corticosteroids [n\u00a0=\u00a020], IVIG [n\u00a0=\u00a02], corticosteroids\u00a0+\u00a0IVIG [n\u00a0=\u00a03], others [n\u00a0=\u00a06]) Clinical response (rise in platelet counts) in 49\/89\u00a0(55%) pts; 31\u00a0pts maintained response for a median (range) of 9 (2\u201342) months, 12\u00a0pts for >12\u00a0months Thrombotic thrombocytopenic purpuraOpen-label prospective multicenter [35]11RTX (375\u00a0mg\/m2) once weekly for 4\u00a0weeks\u00a0+\u00a0premedication with IV steroids (30\u00a0mg), IV dexchlorpheniramine (10\u00a0mg), and IV paracetamol (1\u00a0g). Patients with acute TTP (n\u00a0=\u00a05) continued plasma infusions for \u22653\u00a0weeks followed by tapering at the onset of remissionClinical remission (regression of visceral ischemic signs and normalization of blood parameters) in all patients with acute TTP; continued remission in patients with disease remission at enrolment (6\u201311\u00a0months\u2019 follow-up). Biologic remission (\u226510% recovery of ADAMTS-13 activity and disappearance of anti-ADAMTS-13 antibodies) in all pts Open-label prospective multicenter [82]25RTX (375\u00a0mg\/m2) once weekly for 4\u00a0weeks\u00a0+\u00a0premedication with IV hydrocortisone (100\u00a0mg), IV dexchlorpheniramine (10\u00a0mg), and oral paracetamol (1\u00a0g) immediately following PEX; PEX was continued until clinical remission was achievedAll patients achieved clinical remission (sustained normal platelet count, absence of clinical manifestations of TTP, and cessation of PEX) in a median of 11\u00a0days after initiating rituximab. ADAMTS-13 activity returned to normal levels in 21\/25\u00a0pts; anti-ADAMTS-13 antibodies disappeared in 23\/25\u00a0pts Retrospective comparative 2-center [45]15RTX (375\u00a0mg\/m2) once weekly for 1\u20138\u00a0weeks\u00a0+\u00a0standard therapy (PEX\u00a0+\u00a0corticosteroids\u00a0+\u00a0various agents added as second-line therapy, if needed) (n\u00a0=\u00a08) or standard therapy alone (n\u00a0=\u00a07) Clinical remission (absence of clinical manifestations of TTP and normalization of blood parameters): 100% (RTX group) vs. 66% (standard therapy group) (p\u00a0=\u00a00.0025)Mixed cryoglobulinemiaOpen-label prospective [80]20hRTX (375\u00a0mg\/m2) once weekly for 4\u00a0weeksComplete response (improvement of clinical signs and decline in cryocrit) in 16\/20 (80%) pts; response was maintained for \u226512\u00a0months in 12\/16\u00a0respondersCase series [101]15iRTX (375\u00a0mg\/m2) once weekly for 4\u00a0weeks\u00a0+\u00a0prednisone (<0.5\u00a0mg\/kg\/day), if already administered at recruitmentImproved clinical symptoms (including cutaneous manifestations, lymphoma features, neuropathic symptoms) in all 15\u00a0ptsCold agglutinin diseaseOpen-label Phase II [11]27RTX (375\u00a0mg\/m2) once weekly for 4\u00a0weeks. Re-treatment (if required): RTX (same regimen) plus interferon-\u03b1 (5\u00a0million units three-times weekly for 20\u00a0weeks) Clinical response (improvement in anaemia, clinical symptoms, and histopathology) in 14\/27\u00a0(52%) pts after first treatment and in 6\/10\u00a0pts after re-treatment; median (range) time to response was 1.5 (0.5\u20134) monthsPhase II multicenter [81]20jRTX (375\u00a0mg\/m2) once weekly for 4\u00a0weeksOne pt showed a complete response (normalization of hemoglobin levels, absence of signs of hemolysis, and loss of clinical symptoms), 8\u00a0pts had a partial response (increase in hemoglobin levels \u22651.0\u00a0g\/dl for \u22651\u00a0month, no need for erythrocyte transfusions, improvement in clinical symptoms); of the 9 responders, 8\u00a0relapsed and 1 remained in remission at 48\u00a0weeksaProliferative lupus nephritis, bANCA-positive microscopic polyangitis (n\u00a0=\u00a02) and ANCA-positive Wegener\u2019s granulomatosis (n\u00a0=\u00a07), cANCA-associated vasculitisdANCA-positive refractory Wegener\u2019s granulomatosis, eANCA-positive microscopic polyangitis (n\u00a0=\u00a02) and ANCA-positive Wegener\u2019s granulomatosis (n\u00a0=\u00a08), f\u00a0Dermatomyositis, g\u00a0Clinical results were obtained from physicians via a questionnaire (original patient data were not analyzed), h\u00a0HCV-positive type II or type III mixed cryoglobulinemia, i\u00a0Type II mixed cryoglobulinemia (HCV-positive [n\u00a0=\u00a012]; associated with SS [n\u00a0=\u00a01]; \u201cessential\u201d disease [n\u00a0=\u00a02]), j\u00a0Idiopathic CAD (n\u00a0=\u00a013) and CAD associated with malignant B-cell lymphoproliferative disease (n\u00a0=\u00a07)ADAMTS-13 a disintegrin-like and metalloproteinase with thrombospondin-like type I motif 13, ANCA anti-neutrophil cytoplasmic antibody, BILAG British Isles Lupus Assessment Group, BVAS Birmingham vasculitis activity score, BVAS\/WG BVAS modified for Wegener\u2019s granulomatosis, CAD cold agglutinin disease, CyP cyclophosphamide, HCV hepatitis C virus, IVIG intravenous immunoglobulin, MALT mucosa-associated lymphoid tissue, PEX plasma exchange, pts patients, RTX rituximab, SLAM systemic lupus activity measure, VAS visual analog scale\nSystemic lupus erythematosus\nTraditional treatments for SLE include nonsteroidal anti-inflammatory drugs, antimalarials, corticosteroids, methotrexate, mycophenylate, and cytotoxic drugs such as cyclophosphamide (often in combination). However, these therapies are associated with many potential side effects and are usually only partially effective in the long term [46]. The wide body of evidence indicating that B cells play a central role in the etiopathology of SLE has focused attention on the potential benefits of rituximab and other B cell-targeted therapies in the disease [33, 57, 78].\nIndividual case reports and case series, together with encouraging results from early phase clinical trials, indicate that rituximab is likely to provide significant clinical benefit for at least a subset of SLE patients. For example, in a dose-escalation study involving 17 patients, significant improvements in the systemic lupus activity measure (SLAM) score were observed in those patients (11\/17) who achieved concomitant profound B cell depletion; efficacy persisted for 12\u00a0months and no significant adverse events were reported [59]. Analysis of some of the patients in this trial revealed that clinical response to rituximab correlated closely with the Fc\u03b3IIIa genotype of individual patients [6], as observed previously in studies involving the rituximab responses of patients with follicular lymphoma [96]. In another open-label study, 23\/24 patients achieved depletion of B cells following treatment with rituximab (two 1,000\u00a0mg infusions of rituximab separated by 2\u00a0weeks); depletion lasted for 3\u20138\u00a0months\u2014except in 1 individual, who remained depleted after 4\u00a0years [55]. Clinical improvements observed in this study occurred in each of the 8 organs\/systems assessed using the British Isles Lupus Assessment Group (BILAG) system. A recent update from the same group\u2014covering a total of 41 patients with a mean (range) follow-up period of 37 (6\u201379) months\u2014reported that one-third of patients remained well following B cell depletion, without the need for immunosuppressive agents [64]. Thirteen patients had been re-treated with rituximab. Three serious adverse events (1 pneumococcal sepsis, 1 severe serum sickness-like reaction, and 1 seizure related to hyponatremia) and 2 deaths (1 involving varicella pneumonitis and the other involving pancarditis) had occurred in this cohort over the 7-year observation period.\nIn another trial involving patients with active or refractory SLE, with a follow-up period of 2\u00a0years, all 11 patients in the study responded to a single course of rituximab, with 6 achieving a full response and 5 a partial response; although relapse was common (64%), re-treatment was rapidly effective [85].\nIn a recently reported case series of six patients with aggressive refractory SLE, rituximab therapy (doses of rituximab and use of combination drugs varied between patients) resulted in partial clinical improvements in five cases [40]. Rituximab has also shown effectiveness in pilot studies involving patients with the common severe complication lupus nephritis [42, 84, 95] and in patients with refractory SLE involving the central nervous system [92].\nAlthough most studies to date indicate that B cell depletion therapy is likely to be useful in SLE, the variability of responses to rituximab therapy observed in SLE trials published to date remains to be explained. Ongoing Phase II\/III randomized controlled trials should provide some insight into this question. In addition, although the overall tolerability of rituximab in SLE appears to be good, the Food and Drug Administration recently issued an alert concerning two spontaneous fatal cases of progressive multifocal leukoencephalopathy (PML) due to JC polyomavirus reactivation in two patients with SLE who had received rituximab therapy [38]. It is unclear whether these cases were related to rituximab treatment, since only two cases have been reported and PML has also been reported in >20 SLE patients not treated with rituximab.\nSj\u00f6gren\u2019s syndrome\nSj\u00f6gren\u2019s syndrome is a chronic autoimmune disorder of the exocrine glands affecting approximately 1% of the adult US population. The syndrome often occurs in the presence of another autoimmune disorder such as RA or SLE [37]. The etiopathology of SS is not fully understood; however, disturbances in B cell biology are considered to play an important role [43].\nA number of case reports and pilot studies have been published that describe the successful treatment of SS with rituximab [2, 73, 76, 93]. In a recent trial involving 16 female patients with systemic complications of primary SS, rituximab therapy led to B cell depletion and decreased levels of various B cell markers; with a median follow-up period of 14.5\u00a0months, clinical efficacy was observed in 4\/5 patients with lymphomas and in 9\/11 patients with other systemic manifestations [83].\nAnother recent study investigated the effects of rituximab (two infusions of 375\u00a0mg\/m2 separated by 1\u00a0week) in 16 patients with primary SS [26]. Rituximab therapy, which was administered using a slow initial rate of infusion without steroid premedication, was well tolerated and overall improvements were observed in subjective parameters of disease activity and in quality of life after both 12 and 36\u00a0weeks\u2019 follow-up.\nResults were presented recently from the first double-blind, randomized, controlled study of rituximab in SS [24]. In this 20-patient pilot study, subjects received either rituximab (two 1,000\u00a0mg infusions separated by 2\u00a0weeks) or placebo. Although patient responses were highly variable and there was a marked placebo effect, a higher proportion of patients in the rituximab group achieved improvement in fatigue (the primary efficacy endpoint) than in the placebo group (48 vs. 20%); this difference was not statistically significant. Significantly greater improvements with rituximab over placebo in the social functioning aspect of the quality of life assessment were also noted.\nVasculitis\nVasculitis refers to a collection of rare inflammatory diseases that involve the blood vessel walls and surrounding interstitium. A subset of these diseases, including Wegener\u2019s granulomatosis (WG), microscopic polyangitis, and Churg\u2013Strauss syndrome, is characterized by the presence of anti-neutrophil cytoplasmic antibodies (ANCAs) [97]. The mainstay of current therapies in vasculitis involves glucocorticoids, cyclophosphamide, and\u2014more recently\u2014methotrexate and azathioprine [54]. However, these approaches are not always effective and are often limited by significant toxicity. B cells have been implicated in the pathogenesis of ANCA-associated vasculitis [20], indicating that rituximab may be an effective treatment option.\nIn addition to a number of individual case reports, results have been published recently from several small open-label trials of rituximab in vasculitis. In a report of a series of nine individual cases of ANCA-positive vasculitis resistant to conventional therapy in which rituximab therapy was attempted, eight patients achieved complete responses, while the other patient showed a partial response [34]. Keogh and colleagues have conducted small, prospective, open-label trials in both ANCA-associated vasculitis and WG. The vasculitis trial involved 11 patients whose disease was either refractory to cyclophosphamide or in whom cyclophosphamide was contraindicated [50]. Following infusions with rituximab, circulating B cells became undetectable in all patients and ANCA titers decreased significantly. Clinical remission was achieved in all patients and was maintained while B cells were undetectable. In ten patients with refractory WG treated with prednisone (1\u00a0mg\/kg\/day) plus rituximab (four consecutive weekly infusions of 375\u00a0mg\/m2) [51], therapy was well tolerated and\u2014after 3\u00a0months\u2014all patients had achieved clinical remission (reduction in disease activity score to 0); in addition, all patients were able to stop glucocorticoids by 6\u00a0months. Following the recurrence of raised ANCA titers, five patients in the trial were successfully re-treated with rituximab. Results were also recently published of long-term follow-up of ten patients with ANCA-associated vasculitis who had been treated with rituximab [88]: patients had received four consecutive weekly doses of rituximab (375\u00a0mg\/m2) and all experienced rapid clinical improvement at 6\u00a0months. Although three patients subsequently relapsed, re-treatment was effective. In addition, ANCA titers decreased significantly in all patients. Of 11 patients with refractory ANCA-associated vasculitis who were treated with rituximab in another recently published pilot study, 10 showed either complete or partial responses to a course of rituximab together with a single dose of cyclophosphamide [85].\nIn contrast to the above findings, one recent study found that rituximab was less effective in a cohort of eight patients with refractory WG [8]. In this trial, rituximab was given every fourth week. Interestingly, all patients in this study had particular granulomatous manifestations, consisting of retro-orbital granulomata (n\u00a0=\u00a05), nodules of the lungs (n\u00a0=\u00a01), and subglottic stenosis (n\u00a0=\u00a02). Although three patients experienced some clinical improvement, ANCA titers were not affected by rituximab therapy (except in a single patient). A smaller Norwegian study had also previously found only temporary responses to rituximab in three patients with WG, two of whom had granulomatous masses [68].\nIn a recent review of published studies in this area, it was concluded that rituximab may be an effective treatment in patients with refractory ANCA-associated vasculitis (with the probable exception of WG patients with retro-orbital granulomas, who tended to be less responsive to rituximab therapy) [98]. Since then, however, case reports have appeared describing the successful use of rituximab in patients with granulomatous involvement [79, 91]. In addition, results from a recent case series of eight WG patients indicated that, while vasculitis symptoms tended to disappear relatively quickly, granulomatous manifestations usually regressed more slowly (sometimes over several months) [14].\nWith regard to other forms of ANCA-associated vasculitis, two individual case reports have been published recently detailing the successful treatment of Churg\u2013Strauss syndrome with rituximab [49, 52].\nThrombocytopenic purpura and other hematologic disorders\nA number of autoimmune disorders of hemostasis, most notably idiopathic thrombocytopenic purpura (ITP) and thrombotic thrombocytopenic purpura (TTP), have been examined for their potential responsiveness to rituximab in several small trials.\nIn a study involving a cohort of 25 patients with chronic ITP that had proved resistant to conventional therapies [87], patients received weekly rituximab at a dose of 375\u00a0mg\/m2 for 4\u00a0weeks. The overall response rate (comprising those with complete, partial, and minor responses) was 52%; responses were sustained for at least 6\u00a0months in 7 patients. Complete and partial responses were associated with rapid normalization of platelet concentrations. A similar initial response rate (54%) was reported from a larger follow-up trial involving 57 patients; sustained responses were observed in 32% of the study participants [22]. Other reports include a multicenter trial in 35 adults with refractory ITP conducted in Denmark, which resulted in a 44% overall success rate based on predefined rises in platelet concentrations [13]. An indirect retrospective survey of findings from 89 ITP patients treated at multiple centers in Spain indicated that rituximab therapy led to sustained responses in 35% of patients with a median follow-up of 9\u00a0months (range 2\u201342\u00a0months) [71].\nA review was published recently of the clinical outcomes of patients with chronic ITP who were re-treated with rituximab following an initial response to therapy [72]. All 9\u00a0second responses recorded in this report were classified as complete. An interesting additional finding was the higher female:male ratio of the nine re-treated patients compared with the population of patients originally treated across the published studies identified, suggesting that female ITP patients are more likely than male patients to respond to rituximab therapy. In a recently published letter, early administration of rituximab was reported to be associated with a higher response rate in chronic ITP [102].\nThe efficacy and safety of rituximab in adults with ITP were the subject of a recently published systematic review [9]. Based on 19 reports (313 patients) deemed eligible for the analysis up to April 2006, rituximab therapy was associated with mean complete response (platelet count >150\u00a0\u00d7\u00a0109 cells\/l) and overall response (platelet count >50\u00a0\u00d7\u00a0109 cells\/l) rates of 44 and 63%, respectively. Significant toxicities including death occurred in 3% of included cases, although the deaths were not necessarily attributable to rituximab therapy. The authors noted the lack of randomized controlled studies of rituximab therapy in ITP.\nA number of studies have also been conducted in patients with refractory or relapsing TTP. In addition to several case reports and small case series [3, 17, 69, 70, 74, 99], results from the first prospective trial have been published [35]. This study recruited 11 patients (6 enrolled during an acute refractory phase and 5 during a remission phase); following rituximab therapy (375\u00a0mg\/m2 once weekly for 4\u00a0weeks), clinical remission was observed in all 6 acute cases, while all 5 patients enrolled during remission remained in clinical remission during the 6\u201311\u00a0month follow-up period. Biologic remission (disappearance of anti-ADAMTS-13 [a disintegrin and metalloproteinase with thrombospondin motif 13] antibodies, which occur in the great majority of patients with acquired TTP [75]) was achieved in all patients 7\u201324\u00a0weeks after the final rituximab infusion.\nAnother more recent study involved 25 patients with acute refractory\/relapsing idiopathic TTP, who were given rituximab in conjunction with plasma exchange (PEX) because of progressive clinical disease [82]. It was reported that all 25 patients in this trial achieved complete clinical and laboratory remission (sustained normal platelet count, absence of clinical manifestations of TTP, and cessation of PEX) in a median of 11\u00a0days following the initiation of rituximab therapy. Restoration of ADAMTS-13 activity and disappearance of anti-ADAMTS antibodies occurred in the vast majority of cases. At the time of publication, it was stated that none of the patients had clinically relapsed, with a median (range) follow-up of 10 (1\u201333) months.\nIn another recent retrospective study, the clinical outcome of patients who had received rituximab (375\u00a0mg\/m2 once weekly for a maximum of 8\u00a0weeks) together with standard therapy (PEX\u00a0+\u00a0corticosteroids) was compared with that of patients who had received standard therapy alone [45]. The remission rate in the rituximab group was significantly greater than that observed in the standard therapy group (100 vs. 66%; P\u00a0=\u00a00.0025). Interestingly, all three of the TTP studies described above reported good tolerability to rituximab therapy.\nThere have also been sporadic case reports describing the successful use of rituximab in a number of other rare hematologic disorders, including Evans\u2019 syndrome [61, 65], mixed type II cryoglobulinemia [15, 80, 101], and cold agglutinin disease [11, 81]. In addition, we have recently reported on the successful use of rituximab in RA patients with life-threatening hemorrhage due to the presence of Factor VIII inhibitor [67]. By contrast, a case report describing the failure of rituximab therapy in a hemophiliac patient with Factor VIII inhibitor has also been published [18]. A recent analysis of published case reports of patients with acquired antibodies to Factor VIII indicated that rituximab therapy was associated with a similar rate of clinical remission (approximately 80%) compared with the standard treatment modality (cyclophosphamide\u00a0+\u00a0prednisolone) [86].\nMyositis\nMyositis comprises a group of inflammatory myopathies, of which polymyositis, dermatomyositis, and inclusion body myositis are the best defined clinically. The etiopathologies of this group of diseases are currently poorly understood, although autoimmunity is thought to play an important role [19].\nThe aim of a recent open-label pilot study involving seven patients with dermatomyositis was to test the hypothesis that B cells play a critical role in this disease [56]. The results of this trial, in which patients received four infusions of rituximab (375\u00a0mg\/m2) at weekly intervals, showed that rituximab therapy was well tolerated and led to significant clinical improvements in the six evaluable patients who completed 1\u00a0year of follow-up. A number of other small pilot studies and case reports have also appeared recently detailing the generally successful use of rituximab in patients with dermatomyositis or polymyositis [7, 12, 16, 28, 53, 62, 66].\nAntiphospholipid syndrome\nAntiphospholipid syndrome (APS), a rare disorder mostly affecting young adults, is defined by the presence of autoantibodies against phospholipids; the main clinical manifestations are venous or arterial thrombosis and obstetric complications, although the link between antiphospholipid antibodies and these clinical features has not been firmly established [39]. The traditional approach to treatment mainly involves the use of anticoagulation therapies. However, data indicating a link between raised circulating CD5+\u00a0B cells and high levels of antiphospholipid antibodies in APS patients [100] suggest that APS may be amenable to B\u00a0cell-directed therapies.\nTo date, only a small number of case reports have been published which detail attempts to manage APS with rituximab. Three of these studies [5, 77, 94] reported successful clinical outcomes following rituximab therapy, while the other [4] reported only a limited effect of rituximab on thrombocytopenia and anticardiolipin antibodies in a patient with primary APS. Although the data are currently limited, the striking clinical successes seen in some patients suggest that pilot studies with rituximab in APS should be conducted in the near future.\nStill\u2019s disease\nAdult-onset Still\u2019s disease (AOSD) is a systemic inflammatory disorder of unknown etiology. Traditional therapies include NSAIDs, corticosteroids, and\u2014more recently\u2014disease-modifying anti-rheumatic drugs [47]. A number of trials have also been conducted with biologic agents (including TNF inhibitors), with some promising results [32].\nOne case report was published recently that described the successful use of rituximab in a patient with AOSD [1]. This report, together with the author\u2019s unpublished observations of two patients with AOSD refractory to cytotoxic agents who benefited from repeated rituximab infusion therapy, suggests that rituximab may be a future treatment option for this disease.\nNeurologic disorders\nAs reviewed recently by Finsterer [36], rituximab has been tested in a number of immune-mediated peripheral neuropathies with promising results. Its potential clinical utility in neurological diseases of the central nervous system such as multiple sclerosis (MS) remains to be explored. Encouragingly, pilot studies have shown that rituximab therapy results in partial depletion of B cells from the cerebrospinal fluid of patients with progressive MS [23, 63]. The results of the first Phase I and II trials of rituximab in progressive MS were presented recently [10, 44]. In the placebo-controlled Phase II study [44], involving 104 patients with relapsing remitting MS, a single course of rituximab (two infusions of 1,000\u00a0mg given 2\u00a0weeks apart) resulted in significantly fewer inflammatory brain lesions and relapses over the 6-month observation period compared with placebo. Rituximab treatment was reported to have been well tolerated.\nConclusions\nRecent advances in our understanding of autoimmunity have opened up new avenues for exploring novel targeted therapies in a wide range of diseases. The role of B cells in many autoimmune disorders is now widely accepted, in many cases through the demonstration that B cell depletion using rituximab can often be very effective clinically. The potential utility of rituximab and other B cell-directed therapies is currently being studied in several of these diseases, including SLE, SS, and vasculitis. Although to date most of the findings have been encouraging, a significant proportion of the information derives from case reports and small case series. Together with the lack of randomized controlled trials in most of the diseases discussed in this review, it is likely that there has been a degree of positive reporting bias. Therefore, until large-scale clinical trial data are available, it would be prudent to proceed with caution regarding the use of rituximab outside its approved indications. Although rituximab tolerability was generally reported as favorable in most of the studies covered in this review, the true incidence of associated adverse events (e.g., serious infections, serum sickness-like reactions, and PML) will only become clear when larger numbers of patients have been treated in each disease entity. Important questions also remain regarding the optimal rituximab dosing modalities for each disease (for example, the dose and frequency of treatment, when re-treatment should be considered, and whether to use combination therapies). Nevertheless, based on the information published to date, it seems likely that B cell depletion therapy, using rituximab and\u2014in the future\u2014agents currently under development, will offer an effective new approach for the management of many of these burdensome and difficult-to-treat conditions.","keyphrases":["biologic therapies","rituximab","cd20","lupus","sj\u00f6gren\u2019s syndrome","vasculitis","thrombocytopenic purpura","b-lymphocytes"],"prmu":["P","P","P","P","P","P","P","U"]}
{"id":"Pediatr_Nephrol-3-1-2064944","title":"Chronic kidney disease in children: the global perspective\n","text":"In contrast to the increasing availability of information pertaining to the care of children with chronic kidney disease (CKD) from large-scale observational and interventional studies, epidemiological information on the incidence and prevalence of pediatric CKD is currently limited, imprecise, and flawed by methodological differences between the various data sources. There are distinct geographic differences in the reported causes of CKD in children, in part due to environmental, racial, genetic, and cultural (consanguinity) differences. However, a substantial percentage of children develop CKD early in life, with congenital renal disorders such as obstructive uropathy and aplasia\/hypoplasia\/dysplasia being responsible for almost one half of all cases. The most favored end-stage renal disease (ESRD) treatment modality in children is renal transplantation, but a lack of health care resources and high patient mortality in the developing world limits the global provision of renal replacement therapy (RRT) and influences patient prevalence. Additional efforts to define the epidemiology of pediatric CKD worldwide are necessary if a better understanding of the full extent of the problem, areas for study, and the potential impact of intervention is desired.\nIntroduction\nMost epidemiological information on chronic kidney disease (CKD) originates from data available on end-stage renal disease (ESRD), the terminal stage of CKD when treatment with renal replacement therapy (dialysis or transplant) becomes necessary to sustain life. Little information is available on the prevalence of earlier stages of CKD, as patients are often asymptomatic. The epidemiological studies that have been performed provide evidence that ESRD represents the \u201ctip of the iceberg\u201d of CKD and suggest that patients with earlier stages of disease are likely to exceed those reaching ESRD by as much as 50 times [1]. Worldwide, the number of patients with CKD is rising markedly, especially in adults, and CKD is now being recognized as a major public health problem that is threatening to reach epidemic proportions over the next decade [2]. In North America, up to 11% of the population (19 million) may have CKD [1], and surveys in Australia, Europe, and Japan describe the prevalence of CKD to be 6\u201316% of their respective populations [3, 4]. In North America alone, more than 100,000 individuals entered ESRD programs in 2003 (adjusted incidence rate: 341 new cases per million population), with a prevalence count of more than 450,000 as of December 2003 (prevalence rate: 1,509 per million population) [5]. Not surprisingly, the cost of treating patients with ESRD is substantial and poses a great financial challenge. The economic cost of North American ESRD programs reached $25.2 billion in 2002, an 11.5% increase over the previous year, and is expected to reach $29 billion by 2010 [2]. Two factors, aging and the global epidemic of type-II diabetes mellitus, are primarily responsible for the increasing incidence of CKD in adults.\nIn contrast, pediatric ESRD patients (<20\u00a0years of age) constitute a very small proportion of the total ESRD population. However, they pose unique challenges to providers and to the health care system, which must address not only the primary renal disorder but the many extrarenal manifestations that affect growth and development as well. In North America, children younger than 20\u00a0years of age account for less than 2% of the total ESRD patient population, and the prevalence of patients aged 0\u201319\u00a0years has grown a modest 32% since 1990. This is in contrast to the 126% growth experienced by the entire ESRD population over the same time period [5]. Nonetheless, CKD in children is a devastating illness, and the mortality rate for children with ESRD receiving dialysis therapy is between 30 and 150 times that of the general pediatric population [6, 7]. In fact, the expected remaining lifetime for a child 0\u201314\u00a0years of age and on dialysis is only 20\u00a0years [6]. Therefore, the diagnostic and therapeutic approach to CKD must emphasize primary prevention, early detection, and aggressive management. Knowledge of the epidemiology of CKD and its associated clinical manifestations is a crucial component of this effort by helping to target key patient populations at risk, by quantifying the extent of the problem, and by facilitating an assessment of the impact of intervention.\nClassification of CKD\nThere is limited information on the epidemiology of CKD in the pediatric population. This is especially true for less advanced stages of renal impairment that are potentially more susceptible to therapeutic interventions aimed at changing the course of the disease and avoiding ESRD. As CKD is often asymptomatic in its early stages, it is both underdiagnosed and, as expected, underreported. This is in part the result of the historical absence of a common definition of CKD and a well-defined classification of its severity. The current CKD classification system described by the National Kidney Foundation\u2019s Kidney Disease Outcomes Quality Initiative (NKF-K\/DOQI) has helped remedy the situation. According to the K\/DOQI scheme, CKD is characterized by stage 1 (mild disease) through stage 5 (ESRD) (Table\u00a01) [8]. By establishing a common nomenclature, staging has been helpful for patients, general health care providers, and nephrologists when discussing CKD and anticipating comorbidities and treatment plans. The classification system has, however, been subject to debate, as it is argued that stages 1 and 2 would be better defined by the associated abnormalities (e.g. proteinuria, hematuria, structural anomalies) rather being classified as CKD, whereas more advanced stages (3 and 4) should be characterized by the severity of the impaired renal solute clearance [9]. Furthermore, and with particular reference to children, the normal level of glomerular filtration rate (GFR) varies with age, gender, and body size and increases with maturation from infancy, approaching adult mean values at approximately 2\u00a0years of age (Table\u00a02). In turn, GFR ranges that define the five CKD stages apply only to children 2\u00a0years of age and older. Finally, although the threshold of GFR reduction where chronic renal failure (CRF) and chronic renal insufficiency (CRI) begins is a matter of opinion, many registries have operationally defined this as a GFR below 75\u00a0mL\/min per 1.73\u00a0m2 [10]. Hence, populations with CRI or CRF are now categorized as those that comprise CKD stages 2\u20134. \nTable\u00a01National Kidney Foundation\u2019s Kidney Disease Outcomes Quality Initiative (NKF-K\/DOQI) stages of chronic kidney disease [8]StageDescriptionGFR (mL\/min\/1.73\u00a0m2)1Kidney damage with normal or increased GFR>902Kidney damage with mild decrease in GFR60\u2013893Moderate decrease in GFR30\u2013594Severe decrease in GFR15\u2013295Kidney failure<15 or dialysisGFR glomerular filtration rateTable\u00a02Normal glomerular filtration rate (GFR) in children and adolescents [8]AgeMean GFR\u00b1SD (mL\/min\/1.73\u00a0m2)1\u00a0week (males and females)41\u2009\u00b1\u2009152\u20138\u00a0weeks (males and females)66\u2009\u00b1\u200925>8\u00a0weeks (males and females)96\u2009\u00b1\u2009222\u201312\u00a0years (males and females)133\u2009\u00b1\u20092713\u201321\u00a0years (males)140\u2009\u00b1\u20093013\u201321\u00a0years (females)126\u2009\u00b1\u200922\nSources of pediatric data\nMost of the existing data on the epidemiology of CKD during childhood concentrates on the late and more severe stages of renal impairment [11, 12] and are not population based in nature [13]. In addition, some methodologically well-designed childhood CKD registries are limited by being restricted to small reference populations [14\u201316]. Finally, direct comparisons of the incidence and prevalence rate of childhood CKD in different geographical areas around the world is difficult due to methodological differences in study age group, characterization of the degree of renal insufficiency, and disease classification.\nIn the United States, data is primarily available from two sources: the registry of the North American Pediatric Renal Trials and Collaborative Studies (NAPRTCS) organization [10] and the United States Renal Data System (USRDS). NAPRTCS was established as a transplant registry in 1987 with a goal of gathering data from the majority of pediatric renal transplant centers in the United States, Canada, Mexico, and Costa Rica. Its registry was expanded in 1992 to include data from patients receiving maintenance dialysis, and in 1994, data was first collected from patients with CRI characterized by a Schwartz estimated creatinine clearance of \u226475\u00a0mL\/min per 1.73\u00a0m2 [17]. Participation in this registry is voluntary and mandates the involvement of a pediatric nephrologist in the provision of care to those patients entered into the registry. As of December 2005, information had been collected on more than 6,400 patients who entered the registry with a diagnosis of CRI [10].\nIn contrast to the NAPRTCS, which only receives data voluntarily submitted by pediatric nephrology centers, the USRDS is a national data system that collects, analyzes, and distributes information about all patients with ESRD in the United States. Thus, USRDS data includes information on both adults and children with stage 5 CKD, which is published as an Annual Data Report (ADR) [5, 6]. This source of information is particularly important from an epidemiological perspective, as approximately one third of children and adolescents with ESRD requiring dialysis or transplantation in the United States are cared for in facilities that primarily serve adults, and thus, they are not included in the NAPRTCS database [18].\nThe recently published data from the ItalKid Project is by far the most comprehensive data on the epidemiology of CKD in children. The ItalKid Project is a prospective, population-based registry that was started in 1990 and includes all incident and prevalent cases of CRF (CCr\u2009<\u200975\u00a0mL\/min per 1.73\u00a0m2) in children (<20\u00a0years) from throughout Italy (total population base: 16.8 million children) [19].\nThe European Dialysis and Transplant Association (EDTA) was established in 1964 to record demographic data and treatment details of patients receiving renal replacement therapy (RRT), including dialysis and renal transplantation. Historically, the EDTA registry gathered data on RRT in children from individual renal units by means of center and patient questionnaires, a process that was subject to underreporting. At the turn of the century, the EDTA office moved to Amsterdam and began collecting data on RRT entirely through national and regional registries and recently reported data on RRT in children from 12 registries located in Europe (vide infra) [20].\nOther regional societies, such as the Japanese Society for Pediatric Nephrology (JSPN), have also provided useful epidemiological information. In Japan, children are screened annually by urinalysis in a nationwide program, an approach that has provided invaluable epidemiological information and the opportunity for establishing clinical trials focusing on early detection and intervention. Epidemiological data is also available from Australia and New Zealand [21].\nIn contrast, epidemiological information from Asia, where 57% of the world\u2019s population resides and a geographic region characterized by a very high proportion of children, is very scant and is primarily based on patients referred to tertiary medical centers [22, 23]. The situation in central and southern Africa or in the Arab countries of North Africa and the Middle East is even more unfortunate, as there are no regional pediatric nephrology societies in place to collect and publish any valid epidemiological data.\nIncidence and prevalence of CKD in childhood\nLarge population-based studies, such as the Third National Health and Nutrition Examination Survey (NHANES III), have made it possible to estimate the incidence and prevalence of CKD in the adult population [1]. According to this report, the prevalence of patients with early stages of CKD (stages 1\u20134; 10.8%) is approximately 50 times greater than the prevalence of ESRD (stage 5; 0.2%). There is no comparable information available in the United States on the prevalence of the earlier stages of CKD in children and its relationship to ESRD. This is, in large part, due to differences in disease etiology for children and adults. Furthermore, the relationship between the prevalence of earlier stages of CKD and the subsequent development of more severe CKD\/ESRD is determined in part by factors unrelated to disease etiology, as was recently shown in a comparison between adult patients in Norway and the United States [4]. Data that do exist on the epidemiology of CKD in children come from a variety of sources.\nPopulation-based data from Italy (ItalKid Project) has reported a mean incidence of preterminal CKD (CCr\u2009<\u200975\u00a0mL\/min per 1.73\u00a0m2) of 12.1 cases per year per million of the age-related population (MARP), with a point prevalence of 74.7 per MARP in children younger than 20\u00a0years of age [19]. The national survey performed in Sweden from 1986 until 1994 included children (ages 6\u00a0months to 16\u00a0years) with more severe preterminal CKD (CCr\u2009<\u200930\u00a0mL\/min per 1.73\u00a0m2) and reported a median annual incidence and prevalence of 7.7 and 21 per MARP, respectively [16]. Similarly, the incidence rate of severe pre-terminal CKD in Lorraine (France) has been estimated as 7.5 per MARP in children younger than 16\u00a0years; the prevalence rate ranged from 29.4 to 54 per MARP [15]. In Latin America, the Chilean survey from 1996 reported incidence and prevalence rates of 5.7 and 42.5 per MARP, respectively, in children younger than 18\u00a0years of age with CCr\u2009<\u200930\u00a0mL\/min\/1.73\u00a0m2, including patients with ESRD [12]. As alluded to above, there are 81.2 million children in the United States younger than 20\u00a0years of age [5], but no data on the incidence or prevalence of preterminal CKD is available.\nDue to a lack of national registries, any semblance of incidence and prevalence data from developing countries primarily originates as reports from major tertiary care referral centers [22\u201327]. The nature of the data depends on local referral practices and accessibility to hospital care. The Jordan University Hospital has estimated the annual incidence and prevalence of severe CKD (CCr\u2009<\u200930\u00a0mL\/min per 1.73\u00a0m2) to be 10.7 and 51 per MARP, respectively, based on their hospital admission rate [26]. A 15-year review of admissions from a university teaching hospital in Nigeria estimated the median annual incidence of severe CKD (CCr\u2009<\u200930\u00a0mL\/min per 1.73\u00a0m2) to be 3.0 per MARP, with a prevalence of 15 patients per million children [27]. In a recent report, data from a major tertiary hospital in India revealed that approximately 12% of patients (n\u2009=\u2009305) seen by the pediatric nephrology service over a 7-year period had moderate to severe CKD (CCr\u2009<\u200950\u00a0mL\/min per 1.73\u00a0m2), and one quarter of these patients had already developed ESRD, highlighting the late diagnosis and referral pattern [23]. Similar data was reported from another tertiary hospital in India where 50% of 48 patients presenting with CRF over a 1-year period had ESRD [22]. Finally, data from a major Iranian hospital collected over 7\u00a0years (1991\u20131998) reported that 11% of pediatric nephrology admissions (n\u2009=\u2009298) were due to severe CKD (CCr\u2009<\u200930\u00a0mL\/min per 1.73\u00a0m2), and one half of the patients advanced to ESRD [25].\nThe incidence rate of ESRD, adjusted for race and gender, is much higher among adults than among children. Data from the USRDS revealed that in pediatric patients younger than 20\u00a0years of age, the annual incidence of ESRD increased marginally from 13 per MARP in the 1988 cohort to 15 per MARP in the 2003 cohort [5]. This is in contrast to the adult incidence rate of 119 per MARP for patients 20\u201344\u00a0years of age and 518 per MARP for those 45\u201364\u00a0years old in the 2003 cohort [5]. As in adults, a higher incidence rate with older patients was also found across the 5-year age groups within the pediatric cohort. The incidence rate was nearly twice as high among children 15\u201319\u00a0years of age (28 per MARP) compared with children 10\u201314\u00a0years of age (14 per MARP), and nearly three times higher than the rate for children 0\u20134\u00a0years of age (9 per MARP). The point prevalence for pediatric patients (adjusted for age, race, and gender) was 82 per million population during 2002\u20132003 [5].\nThe EDTA registry recently reported its cumulative data on 3,184 patients (<20\u00a0years of age) with ESRD who initiated RRT between 1980 and 2000 in 12 European countries [20]. With a total of 18.8 million children between 0\u201319\u00a0years in the countries surveyed, data revealed that the incidence of ESRD rose modestly from 7.1 per MARP in the 1980\u20131984 cohort to 9.9 per MARP over the next 15\u00a0years. In contrast, the prevalence of patients receiving RRT increased from 22.9 per MARP in 1980 to 62.1 per MARP in 2000, providing evidence of improved long-term survival. As in the United States, the incidence of ESRD was highest in the 15\u201319\u00a0year age group, with the exception of the 0- to 4-year age group in Finland who experienced a high incidence of ESRD (15.5 per MARP) secondary to the large number of infants in that country with congenital nephrotic syndrome. The incidence of ESRD in children (<20\u00a0years age) from Australia and New Zealand has remained fairly constant at around 8\u201310 per million population over the past 25\u00a0years, whereas the prevalence of treated ESRD has steadily increased since 1980, from approximately 25 to 50 patients per million population [21].\nThe 1998 Japanese National Registry data reported comparatively lower ESRD incidence and prevalence rates of 4 and 22 per MARP, respectively, for children 0\u201319\u00a0years of age [28] for reasons that are as yet unexplained. However, as in other countries, the prevalence rate of treated ESRD patients among patients aged 15\u201319\u00a0years of age was not only high (34 per million), but seven times higher than that of patients 0\u20134\u00a0years of age (5 per million).\nIn the 2005 ADR from the USRDS, data regarding the incidence and prevalence of ESRD in children was simultaneously published from 37 countries to corroborate the information above and to facilitate international comparisons [5]. The highest incidence rates for children were reported from the United States, New Zealand, and Austria, at 14.8, 13.6, and 12.4 per million population, respectively. As mentioned earlier (vide supra), Japan\u2019s rate for pediatric patients was, in contrast, one of the lowest, even though Japan ranks fourth highest in the world for the incidence of ESRD in adults. The prevalence rate for pediatric ESRD patients was reported to be highest in Italy, at 258 patients per million population; however, this may be partially related to the addition of data from patients ages 20\u201324 to the prevalent group. The second highest prevalence rate for children was reported from Finland, with a rate only 40% of that in Italy but greater than the rates from the United States and Hungary, where they were reported to be 82 and 81 patients per million population, respectively (Fig.\u00a01) [5].\nFig.\u00a01Incidence (left) and prevalence of end-stage renal disease (ESRD) around the world in the 0\u201319 age group in 2003 [5]\nA number of factors influence incidence and prevalence rate variability of childhood ESRD. Factors such as racial and ethnic distribution, type of prevalent renal disease, and quality of medical care available for preterminal CKD patients have a significant impact on patient outcome. As the vast majority of treated ESRD patients come from more-developed countries, which can afford the cost of renal replacement therapy, the huge disparity in the prevalence of ESRD between the more- and less-developed countries probably stems, in large part, from the inadequacy of health-care resource allocation to programs providing renal replacement therapy in underdeveloped countries [29, 30].\nFinally, characterization of the patient population with CKD (both preterminal CRF and treated ESRD) reveals that the incidence and prevalence rates are universally greater for boys than for girls [10, 16, 19, 22, 23, 25\u201327]. Two thirds of patients in the NAPRTCS CRI registry and in the database of the ItalKid Project are males. This gender distribution reflects the higher incidence of congenital disorders, including obstructive uropathy, renal dysplasia, and prune belly syndrome, in boys versus girls. In fact, in the ItalKid Project, males continue to predominate (male:female ratio 1.72) even after excluding patients with posterior urethral valves [19].\nAs for race, the incidence rate for ESRD in black children in North America is two to three times higher than for white children, irrespective of gender [31]. Likewise, the incidence rate of ESRD for the indigenous people of Australia (Aborigines) and New Zealand (Maoris) is disproportionately higher than that experienced by the remainder of the population [32].\nEtiology of CKD\nUnlike adults in whom diabetes and hypertension are responsible for the majority of CKD, congenital causes are responsible for the greatest percentage of all cases of CKD seen in children. However, whereas this is the most common reported etiology from developed countries where CKD is diagnosed in its earlier stages, infectious or acquired causes predominate in developing countries, where patients are referred in the later stages of CKD. These generalizations apart, certain disorders giving rise to CKD are, indeed, more common in some countries than in others.\nIn the CRI registry arm of NAPRTCS, almost one half of the cases are accounted for by patients with the diagnoses of obstructive uropathy (22%), aplasia\/hypoplasia\/dysplasia (18%), and reflux nephropathy (8%) (Table\u00a03). Whereas structural causes predominate in the younger patients, the incidence of glomerulonephritis (GN) increases in those older than 12\u00a0years. Among the individual glomerular causes, only focal segmental glomerulosclerosis (FSGS) accounts for a significant percentage of patients (8.7%), whereas all other glomerulonephritides combined contribute less than 10% of the causes of childhood CKD. For reasons that are as yet not clear, FSGS is three times more common in blacks than in whites (18% vs. 6%) and is particularly common among black adolescents with CKD [10]. \nTable\u00a03Diagnosis distribution of North American Pediatric Renal Trials and Collaborative Studies (NAPRTCS) chronic renal insufficiency (CRI) patients [10]Distributions by diagnosisNumberPercent MalePercent whitePercent blackPercent otherTotal6,40564611920Primary diagnosis\u00a0Obstructive uropathy1,38586612117\u00a0Aplastic\/hypoplastic\/dysplastic kidney1,12562621721\u00a0Other91358631621\u00a0FSGS55757403921\u00a0Reflux nephropathy5365374620\u00a0Polycystic disease25755741115\u00a0Prune belly18597622315\u00a0Renal infarct15553661321\u00a0Unknown16852472032\u00a0HUS1345881711\u00a0SLE nephritis9625274132\u00a0Cystinosis97489235\u00a0Familial nephritis9986611227\u00a0Pyelo\/interstitial nephritis8739642016\u00a0Medullary cystic disease82508497\u00a0Chronic GN7650432928\u00a0MPGN-type I6761481933\u00a0Berger\u2019s (IgA) nephritis6463641620\u00a0Congenital nephrotic syndrome6846461243\u00a0Idiopathic crescentic GN4648522424\u00a0Henoch-Sch\u00f6nlein nephritis406578320\u00a0MPGN-type II297279317\u00a0Membranous nephropathy3348303930\u00a0Other systemic immunologic disease2532403228\u00a0Wilms tumor2854572121\u00a0Wegener\u2019s granulomatosis17769406\u00a0Sickle cell nephropathy13620928\u00a0Diabetic GN1150364518\u00a0Oxalosis66783017\u00a0Drash syndrome610067033FSGS focal segmental glomerulosclerosis, HUS hemolytic uremic syndrome, SLE systemic lupus erythematosus, GN glomerulonephritis, MPGN membranoproliferative GN, IgA immunoglobulin A\nData from the ItalKid Project revealed that hypoplasia with or without urological malformations accounts for as many as 57.6% of all cases of CKD in Italy, whereas glomerular diseases account for as few as 6.8% of cases of CKD in children [19]. Interestingly, when the analysis was restricted to the patient population that had reached ESRD, the relative percentage of glomerular diseases increased from 6.8% to 15.2%, whereas that of hypoplasia decreased from 57.6% to 39.5%, underscoring the discrepancy between the rates of progression of these two entities. Observations from this study have also prompted questions regarding the commonly accepted cause\u2013effect relationship between vesicoureteral reflux (VUR) and kidney disease (reflux nephropathy) and support the hypothesis that both hypoplasia and VUR may be related to similar developmental factors causing congenital disorders of the kidney and urinary tract [33].\nIn the ESRD population reported by the EDTA registry, hypoplasia\/dysplasia and hereditary diseases were the most common causes for ESRD in the 0- to 4-year age group, whereas GN and pyelonephritis became progressively more common with increasing age in the majority of reporting countries [20]. The exception is Finland, where congenital nephrosis (Finnish type) remains the most common cause of ESRD in children younger than 15\u00a0years of age [34]. Somewhat different is the data reported by the Japanese National Registry, which reflects a very high proportion (34%) of cases secondary to GN [FSGS 60% and immunoglobulin A (IgA) nephropathy 17%] in their pediatric ESRD population [28]. Similarly, the Australia and New Zealand Dialysis and Transplant (ANZDATA) registry reported GN to be the most common cause of ESRD in children and adolescents from Australia and New Zealand (42%) [21].\nComprehensive information on the etiology of ESRD from many less-developed countries is unavailable owing to poor data collection and the absence of renal registries. In addition and in contrast to the experience within developed countries, many of these countries continue to suffer from the burden of infectious diseases such as hepatitis C, malaria, schistosomiasis, and tuberculosis, with resultant infection-related GN. One such example is Nigeria, from which a publication on pediatric CKD reported various glomerulopathies as the cause of renal failure in one half of their patients, a third of whom also had nephrotic syndrome [27]. Human-immunodeficiency-virus (HIV)-associated nephropathy in children is another entity that is underreported, and it is a disorder that is likely to increase along with the increasing incidence of HIV in Africa and Asia. Familial Mediterranean fever leading to amyloidosis has been found to be responsible for up to 10% of cases of CKD in Turkish children (n\u2009=\u2009459) [24].\nHereditary disorders are more prevalent in countries where consanguinity is common. One third of Jordanian children with CKD have been diagnosed with hereditary renal disorders such as polycystic kidney disease, primary hyperoxaluria, and congenital nephrotic syndrome [26]. Similarly, one fifth of Iranian children with CKD have been reported to have hereditary disorders such as cystinosis, cystic kidney disease, Alport syndrome, and primary hyperoxaluria [25].\nProgression of CKD\nAlthough the stages of CKD are now reasonably well defined, the natural history of the early stages is variable and often unpredictable. However, most available data demonstrates a slower progression toward ESRD in patients with congenital renal disorders compared with patients with glomerular disease. For this reason, and as alluded to previously, the relative proportion of glomerular diseases increases in groups of patients with more advanced stages of CKD. The progression of established CKD is also influenced by a variety of risk factors, some of which (e.g., obesity, hypertension, and proteinuria) may be modifiable [35\u201337], whereas others, including genetics, race, age, and gender, are not.\nObesity is associated with hypertension, albuminuria, and dyslipidemia, all of which can potentially influence the progression of CKD. The incidence of certain glomerulonephritides, such as FSGS, is higher in obese than in lean individuals [38, 39]. Hypertension together with proteinuria has been shown to be an important risk factor for progression of primary renal disease in children and adults [40, 41], and the renoprotective efficacy of renin angiotensin system (RAS) antagonists, which is in part independent of blood pressure, has been clearly demonstrated in animal models and adults with acquired nephropathies [42\u201346]. Whereas both angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers have been shown to reduce proteinuria in children with CKD, the renoprotective efficacy of these medications in children and their potential impact on the epidemiology of CKD still needs to better delineated, as is currently being addressed by the Effect of Strict Blood Pressure Control and ACE Inhibition on the Progression of Chronic Renal Failure in Pediatric Patients (ESCAPE) trial [47, 48].\nThe clustering of CKD in families is strongly suggestive of a genetic or familial predisposition in some cases [49]. Studies have suggested the presence of links between CKD and various alterations or polymorphisms of candidate genes encoding putative mediators, including the renin\u2013angiotensin system. Additionally, racial factors may play a role in susceptibility to CKD, as there is a strong concordance of renal disease in the families of African Americans with hypertensive ESRD [49]. Not only may there be an increased susceptibility to disease, but there is evidence that the rate of progression of CKD is faster among African American males [50]. Low birth weight in some ethnic communities might be associated with a reduction in the number of nephrons and a subsequent predisposition to hypertension and renal disease in later life [51].\nIrrespective of the underlying kidney disease or presence of additional risk factors, it is clear that the risk of progression to ESRD in childhood is inversely proportional to the baseline creatinine clearance [10, 19]. Additionally, regardless of the initial level of renal insufficiency, puberty seems to be a critical stage for patients with renal impairment, as a steep decline in renal function often occurs during puberty and the early postpuberty period [19]. Whereas the specific reasons are yet to be determined, it is speculated that this pattern of progression may be attributable to an adolescent-specific pathophysiological mechanism, possibly related to sex hormones and\/or the imbalance between residual nephron mass and the rapidly growing body size. Data collected by NAPRTCS has also revealed that patients whose baseline serum albumin was below 4\u00a0g\/dl, inorganic phosphorus above 5.5\u00a0mg\/dl, calcium below 9.5\u00a0mg\/dl, blood urea nitrogen (BUN) above 20\u00a0mg\/dl, or hematocrit below 33% had a significantly higher risk of reaching ESRD (p\u2009<\u20090.001) [10]. Data pertaining to a variety of risk factors potentially associated with the progression of CKD, including those noted above, is being collected by the Chronic Kidney Disease in Children Study (CKiD), a prospective, multicenter initiative funded by the National Institutes of Health designed to follow the course of 540 children with CKD for 2\u20134\u00a0years [52].\nOutcome for children with CKD\nThe outcome of children with severe CKD is highly dependent upon the economy and availability of health care resources. Approximately 90% of treated ESRD patients come from developed countries that can afford the cost of RRT [29]. Despite comparable incidence rates, high mortality in countries that lack resources for RRT results in a low prevalence of CKD patients in those countries. In one of the tertiary care hospitals in India, for example, up to 40% of the ESRD patients opted out of further therapy because of a lack of financial resources [22], and of the 91 patients with ESRD in another hospital, only 15 underwent renal transplantation, 63 received hemodialysis, and the remainder opted out of dialysis or transplantation care secondary to financial constraints [23]. Similar results were recently published from South Africa where only 62% of children (<20\u00a0years of age) with ESRD were accepted by an \u201cAssessment Committee\u201d for RRT as part of a rationing program [30].\nIn countries where RRT is readily available, the most favored renal replacement modality is transplantation in all pediatric age groups. Sixteen percent of children newly diagnosed with ESRD in North America receive a preemptive transplant, and three fourth of children receive a transplant within 3\u00a0years of RRT initiation [5]. Similar figures are reported by the ANZDATA registry [21]. Among Western countries, Spain\/Catalonia has the highest pediatric transplant rate, reaching 15 patients per million population, followed by a rate of 12 patients per million population in the United States and Finland (Fig.\u00a02) [5]. In the United States, white pediatric patients are more likely to receive a renal transplant than are patients from other racial groups.\nFig.\u00a02Percent distribution of prevalent dialysis modality (left) and transplant rates in the 0\u201319 age group in 2003 [5]\nThe distribution of dialysis modalities varies among countries (Fig.\u00a02) [5]. Peritoneal dialysis (PD) in children is highest in Finland, New Zealand, and Scotland, accounting for 73%, 71%, and 67% of prevalent dialysis patients, respectively. Whereas PD is still the favored mode of dialysis in young children, there has been an increase in hemodialysis (HD) utilization since the early 1990s, and HD is now the most common form of dialysis overall for prevalent patients <19\u00a0years of age (Fig.\u00a02) [5]. In the United States, PD is the most frequently used dialysis modality (60% of dialysis patients) according to the NAPRTCS registry [10], whereas HD is more common according to data collected by the USRDS [5]. Once again, this discrepancy reflects in part the fact that many adolescent patients are cared for in adult dialysis units where there is often a preference for HD [18]. Whereas automated PD (APD) is the most frequently used PD modality in children [53], continuous ambulatory PD (CAPD) is commonly used in countries that lack finances and technical support, as reflected in the recent report of the Turkish Pediatric Peritoneal Dialysis (TUPEPD) registry. [54].\nMortality rates remain significantly lower in pediatric patients with ESRD compared with their adult counterparts. Nevertheless, an assessment of the causes of death reflect the excess risk of cardiac and vascular disease and the high prevalence of left ventricular hypertrophy and dyslipidemia among children treated with RRT [55\u201357]. Pediatric patients with glomerulonephritis or those with cystic\/hereditary\/congenital disease have the greatest probability of surviving 5\u00a0years, in contrast to patients who have developed ESRD as a result of secondary GN or vasculitis [5]. Infants on dialysis have a higher mortality rate than do older children, which is likely, at least in part, to be a result of coexisting morbidities [58]. Although substantial improvement has occurred in the long-term survival of children and adolescents with ESRD over the past 40\u00a0years, the overall (dialysis and transplantation) 10-year survival remains at only 80%, and the age-specific mortality rate is still 30\u2013150 times higher than among children without ESRD [6, 7]. It is noteworthy that dialysis is associated with an appreciably higher risk of death compared with renal transplantation; therefore, patients who experience a longer wait for transplantation are more likely to have a worse overall outcome. Not only is the benefit of transplantation evident when one compares transplant recipients to patients deemed \u201cmedically unsuitable\u201d for transplantation, it has also been substantiated in a recent longitudinal study of 5,961 patients \u226418\u00a0years of age, all of whom were placed on the kidney transplant waiting list in the United States [59]. In that study, transplanted children had a lower estimated mortality rate (13.1 deaths\/1,000 patient years) compared with patients on the waiting list (17.6 deaths\/1,000 patient years). Similarly, the 2005 ADR reported that approximately 92% of children initiating therapy with a transplant survive 5\u00a0years compared with 81% of those receiving HD or PD [5]. Finally, the expected remaining lifetime for children 0\u201314\u00a0years of age and on dialysis is only 18.3\u00a0years, whereas the prevalent transplant population of the same age has an expected remaining lifetime of 50\u00a0years [5].\nConclusion\nChildren with CKD comprise a very small but important portion of the total CKD population. Whereas disorders associated with its development are well delineated, the availability of valid and widespread information regarding the epidemiology of CKD in children requires additional efforts, such as the ItalKid Project, in which early identification and longitudinal follow-up are key practices. This information will, in turn, serve as the basis upon which to judge the impact that observational trials such as CKiD and interventional trials such as ESCAPE have on the evolution of CKD during childhood [48].","keyphrases":["chronic kidney disease","children","epidemiology","end-stage renal disease","renal replacement therapy"],"prmu":["P","P","P","P","P"]}
{"id":"Surg_Endosc-4-1-2358937","title":"Perioperative outcome of laparoscopic left lateral liver resection is improved by using a bioabsorbable staple line reinforcement material in a porcine model\n","text":"Hypothesis Laparoscopic liver surgery is significantly limited by the technical difficulty encountered during transection of substantial liver parenchyma, with intraoperative bleeding and bile leaks. This study tested whether the use of a bioabsorble staple line reinforcement material would improve outcome during stapled laparoscopic left lateral liver resection in a porcine model.\nThe success of laparoscopic cholecystectomy has driven the application of minimally invasive techniques to other disease processes, and led to the use of laparoscopy in solid organ surgery [1\u20139]. Since the report of the first laparoscopic liver resection for a 6-cm focal nodular hyperplasia, laparoscopic surgery for the treatment of liver diseases has become more popular [28\u201330]. In 1995, excision of a segment 4 hepatic tumor was reported. The first successful laparoscopic left lateral hepatectomy (segments 2 and 3) in a patient with a benign adenoma was performed in 1996 [8].\nTechnological advances and the development of new equipment have facilitated the growing trend of laparoscopic liver resections [2]. However, laparoscopic liver surgery remains technically challenging. Compared to open surgery, the laparoscopic surgeon is limited by the lack of an effective means to divide substantial liver parenchyma. Transection of the liver can be performed laparoscopically in several different ways, however bile leakage rates of 1\u201316% and bleeding complication rates of 6\u201355% have been reported [9, 30\u201332].\nWe hypothesized that the addition of a bioabsorbable membrane to reinforce standard laparoscopic stapling devices would reduce the bleeding and biliary complications of stapled laparoscopic liver resections. We tested this hypothesis in a prospective survival study of 20 pigs undergoing laparoscopic left lateral liver resections.\nMethods\nStudy design\nA total of 20 female pigs each weighing approximately 40 kg were used for the study. All animals were maintained in accordance with the recommendations of the institutional animal care and use committee at the Mount Sinai School of Medicine. Animals were randomly assigned at the time of surgery to either group A (n\u00a0=\u00a010) in which the stapling devices used for laparoscopic left lateral segmentectomy were reinforced with a bioabsorbable membrane or group B (n\u00a0=\u00a010), in which standard stapling devices were used. Animals were followed prospectively for a 6-week period after which they were sacrificed.\nSurgery\nAll animals were premedicated with ketamine (22 mg\/kg) and atropine (0.04 mg\/kg) and anesthetized with thiopentothal (15 mg\/kg) prior to intubation. Animals were then mechanically ventilated with an initial tidal volume of 10 ml\/kg and a respiratory rate of 15 breaths per minute. The tidal volume was adjusted to maintain arterial PaCO2 of 35\u201340 mmHg during the experiment. Anesthesia was maintained with inhaled isofluorane (1.5%).\nThe pig was placed in a supine position. A standard open technique was used to gain access to the abdomen and a total of five trocars (Karl Storz Endoscopy America, Culver City, CA, USA) were used for the procedure (Fig.\u00a01).\nFig.\u00a01Schematic illustration of trocars placement in the porcine model\nPneumoperitoneum was established to a pressure of 15 mmHg. After inspection of the abdominal cavity with a 10-mm 30\u00b0 laparoscope, the remaining trocars were placed under direct vision. Two 10-mm trocars were used in the left and right upper quadrants. One 12-mm trocar was positioned in the left anterior axillary line for liver retraction. The liver capsule and parenchyma were inspected superiorly and inferiorly and the line of transection was visualized between the liver hilum, along the surface of the entire left liver lobes towards the diaphragm. Subsequently, the left lateral segmentectomy was demarcated (segments 2 and 3 of the liver) within 1 cm along the falciform ligament. If necessary, the diaphragmatic and minor omental attachments to the liver were divided to mobilize the left lobe. Once mobilized, attention was turned to transection of the liver parenchyma. This was accomplished with sequential firings of a 3.5-mm endo GIA stapler (US Surgical, Norwalk, CT). In group A, the stapler cartridges were buttressed with a bioabsorble reinforcement material (Seamguard\u00ae, W.L. Gore, Flagstaff, Arizona) (Fig.\u00a02). In group B the standard stapler cartridges were used. As the liver parenchyma was divided, no attempt was made to isolate the major feeding vessels (major branches of the left hepatic artery and vein) to segments 2 and 3; rather, they were divided en masse with the liver parenchyma. Upon completion of the transection, hemostasis was achieved with the ultrasonic scalpel (Ethicon Endo-Surgery, Cincinatti, Ohio, USA), which works by means of a vibrating blade or scissors and can effectively seal small vessels and bile ducts, and\/or electrocautery. The resected specimen was placed into an endobag and retrieved through the umbilical trocar site that was extended approximately 3.5\u20135.0 cm to accommodate the specimen. The abdomen was irrigated and inspected prior to closure of the trocar sites. No intra-abdominal drains were used.\nFig.\u00a02An absorbable polymer membrane (Bioabsorbable Seamguard, W.L. Gore, Flagstaff, Arizona, USA) is constructed as a buttress mat integrated in the stapler systems\nData collection\nOperative data recorded at the time of surgery included operative time, specimen size, and blood loss as estimated by amount of fluid collected in a suction container minus irrigation fluid used. Animals were prospectively followed with regard to clinical outcome for 6 weeks. Clinical status (temperature, blood pressure\u00a0=\u00a0BP, heart rate\u00a0=\u00a0HR) was examined routinely on a daily basis unless clinical condition mandated additional assessment. Routine blood tests (bilirubin, ALT, AST, alkaline phosphatase, GGT, and complete blood count) were obtained 2 days and 6 weeks postoperatively.\nAnimals were sacrificed at 6 weeks and examined for intra-abdominal abnormalities such as abscess or bile leaks. Standard methylene blue was injected into the biliary tree to examine for active biliary leaks. A standardized pressure-controlled injector using a green sized syringe injected 5\u201310 ml methylene blue into the ligated common bile duct. Subsequently, the cut edge of the liver was examined both macroscopically and microscopically for evidence of biliary leak. The cut edge of the liver was then sent for histopathological analysis.\nStatistical analysis\nTo obtain a 50% reduction rate in postoperative outcome after laparoscopic liver resection an 80% power analysis showed a series of 20 consecutive animals to be operated. Moreover, a 95% confidence interval was obtained to achieve statistical significance. Data were compared between groups with Student\u2019s t-test and chi-square tests.\nResults\nAll animals tolerated the procedure well and were healthy during the entire follow up. Clinical status (temperature, blood pressure, heart rate) examined routinely on a daily basis were not different. All animals resumed normal diet within 1 day and had their first bowel movement within 2\u20133 days. Routine blood tests (bilirubin, ALT, AST, alkaline phosphatase, GGT, white blood cell, hemoglobin, hematocrit) obtained 2 days and 6 weeks postoperatively showed no abnormalities in either group.\nMean operative time was not different between groups (group A 64\u00a0\u00b1\u00a011 min versus group B 68\u00a0\u00b1\u00a09 min, p\u00a0=\u00a0ns). Intraoperative blood loss was significantly higher in group B (25\u00a0\u00b1\u00a05 mL versus 185\u00a0\u00b1\u00a09 mL, p\u00a0<\u00a00.05) (Fig.\u00a03). An average of six 60-mm stapler cartridge firings per animal were used in each group to transect the left lateral liver lobe (p\u00a0=\u00a0ns). Resected specimen size was similar in both groups (average size 10\u00a0\u00d7\u00a08\u00a0\u00d7\u00a04 cm; average weight 0.41 kg). There was no morbidity or mortality in either group. However, two animals in group B were found to have bile collections in the previous operating field at the time of necropsy. When the livers were subjected to methylene blue injection via the common bile duct (CBD), two additional animals in group B were found to have evidence of biliary leak (a total of four of ten animals in group B versus zero of ten in group A, p\u00a0<\u00a00.05).\nFig.\u00a03Intraoperative blood loss in groups A and B\nHistopathological examination of the cut surface of the liver in group A revealed no evidence of the staple line reinforcement material, indicating that it had been totally reabsorbed. There was no bile duct damage seen and only mild mononuclear inflammation was seen in the portal areas. Subcapsular sinusoid dilation and congestion was seen with sharp demarcation between areas of dilation and absence of dilation in a pattern suggestive of fixation artifact although ischemic or anoxic damage cannot be ruled out. In group B marked fibrotic changes and damaged vascular and biliary endothelium were seen (Fig.\u00a04 and 5).\nFig.\u00a04Macroscopic illustration of bile duct damage at the liver\u2019s transection site of group B in which conventional stapling technique was performedFig.\u00a05Microscopic examination (20\u00d7) of resection line of group A (above) and group B (below)\nDiscussion\nWhile new technology is rapidly expanding the realm of laparoscopic surgery to include major hepatic resections, the majority of laparoscopic hepatic operations currently performed are for diagnostic purposes [3\u20135]. One of the major limitations to laparoscopic liver resections is the difficulty encountered in division of substantial liver parenchyma. Commonly employed devices for standard open liver resections including the Cavitron ultrasonic surgical aspirator (CUSA) (Valleylab, Boulder, CO), and the Argon Beam coagulator (Valleylab, Boulder, CO) are available for use in laparoscopy [34\u201336].\nTransecting the liver can be performed in laparoscopically several ways (varying from gasless procedures to hand-assisted or strict laparoscopic liver resections), each technique with its own risk of bleeding and bile leaks. In the past, the percentage of these complications has not changed considerably, remaining between 10% and 50% [9, 31]. Commonly used laparoscopic devices such as the ultrasonic scalpel or the Ligasure (Valleylab, Boulder, CO) are limited in their ability to divide liver parenchyma because of the small size of the active heating area.\nThe addition of various buttressing materials to stapling devices has been tried as a means to improve results and reduce complications rates in both pulmonary and gastrointestinal surgery [17\u201323]. We hypothesized that the addition of a buttressing material to standard laparoscopic stapling devices wouldprovide a novel technique for liver transection. One buttressing material (Seamguard\u00ae, W.L. Gore, Flagstaff, Arizona) is a porous fibrous structure composed solely of synthetic bioabsorbable poly (glycolide:trimethylene carbonate) copolymer. Degraded via a combination of hydrolytic and enzymatic pathways, the copolymer has been found to be both biocompatible and nonantigenic, with a history of use as bioabsorbable sutures, membranes, and other implantable devices. When used for staple line reinforcement, it can be expected to retain mechanical strength for 4\u20135 weeks and be completely absorbed by the end of 6 months [11]. The fact that this material is completely bioabsorbable should reduce concerns over possible long-term complications such as migration, erosion, calcification, and infection. In addition, this synthetic copolymer does not carry the risk of animal source contamination. The histopathological examination of the resection line of both groups showed fibrotic changes in group B in contrast to group A. This may be caused by excess mechanical stress which was avoided in group A due to addition of a staple line reinforcement.\nOur results show that transaction using conventional stapling devices is inadequate but that the addition of a bioabsorbable staple line reinforcement material to standard laparoscopic stapling devices can reduce intraoperative blood loss during transection of liver parenchyma. In addition, there was a reduction in postoperative bile leaks, although in this study these leaks were not clinically significant. Bioabsorbable reinforcement improves on complications and is a viable addition to laparoscopic liver resection.","keyphrases":["liver resections","staple line reinforcement","laparoscopic surgery","complications","bile duct leak","hemorrhage."],"prmu":["P","P","P","P","R","M"]}
{"id":"Eur_J_Epidemiol-3-1-2071962","title":"Sodium and potassium intake and risk of cardiovascular events and all-cause mortality: the Rotterdam Study\n","text":"Background Dietary electrolytes influence blood pressure, but their effect on clinical outcomes remains to be established. We examined sodium and potassium intake in relation to cardiovascular disease (CVD) and mortality in an unselected older population. Methods A case\u2013cohort analysis was performed in the Rotterdam Study among subjects aged 55 years and over, who were followed for 5 years. Baseline urinary samples were analyzed for sodium and potassium in 795 subjects who died, 206 with an incident myocardial infarction and 181 subjects with an incident stroke, and in 1,448 randomly selected subjects. For potassium, dietary data were additionally obtained by food-frequency questionnaire for 78% of the cohort. Results There was no consistent association of urinary sodium, potassium, or sodium\/potassium ratio with CVD and all-cause mortality over the range of intakes observed in this population. Dietary potassium estimated by food frequency questionnaire, however, was associated with a lower risk of all-cause mortality in subjects initially free of CVD and hypertension (RR = 0.71 per standard deviation increase; 95% confidence interval: 0.51\u20131.00). We observed a significant positive association between urinary sodium\/potassium ratio and all-cause mortality, but only in overweight subjects who were initially free of CVD and hypertension (RR = 1.19 (1.02\u20131.39) per unit). Conclusion The effect of sodium and potassium intake on CVD morbidity and mortality in Western societies remains to be established.\nIntroduction\nObservational and experimental data support an independent, positive relationship between sodium intake and blood pressure, most clearly in hypertensive populations [1\u20133]. Potassium intake, on the other hand, has been inversely related to blood pressure [3, 4]. Since hypertension is a strong predictor of cardiovascular disease (CVD), especially stroke, inadequate intake of sodium and potassium is likely to be associated with increased cardiovascular morbidity and mortality [1]. Only recently, population-based studies on dietary salt intake in relation to CVD and non-cardiovascular events have received priority [5]. Alderman et\u00a0al. were among the first to report an increased risk of myocardial infarction with low urinary sodium in treated hypertensive men [6]. In a subsequent analysis of NHANES I data, an inverse association of sodium intake with all-cause and cardiovascular mortality was found [7]. Estimation of salt intake by 24-h dietary recall and other methodological aspects of this analysis, however, have been criticized [8\u201310]. Salt intake was not significantly related to coronary or all-cause mortality in the large cohorts of the Scottish Heart Health Study [11] and the MRFIT trial [12]. A recent systematic review of 11 randomized trials showed no effect of long-term sodium reduction on overall mortality, but this meta-analysis included only 17 fatal events and should be interpreted with caution [13]. He et\u00a0al. showed that high sodium intake was a strong risk factor for congestive heart failure in overweight participants of the NHANES I follow-up study [14], and also predictive for CVD and all-cause mortality in this group [15]. Similarly, in a Finnish cohort, 24-h urinary sodium excretion predicted mortality and risk of coronary heart disease only in the presence of overweight [16]. With regard to incidence of stroke, the Finnish study showed no association with urinary sodium [16]. Stroke mortality was neither predicted by dietary sodium intake in MRFIT [12]. In the WHO Cardiovascular Diseases and Alimentary Comparison (CARDIAC) Study in 24 countries, however, sodium intake appeared to be a risk factor for stroke in men [17]. As a consequence of these inconsistent findings, there is currently no consensus as to the cardiovascular risks of salt intake.\nTobian et\u00a0al. demonstrated a lower risk of hemorrhagic stroke and mortality in hypertensive rats that had been given potassium supplements, an effect that was not mediated by blood pressure reduction [18]. Khaw and Barrett-Connor confirmed this independent protective effect of dietary potassium against stroke in humans [19]. Also in the CARDIAC study [17], the Cardiovascular Health Study [20] and the Nurses Health Study [21] the intake of potassium was inversely related to risk of stroke. Data on dietary potassium in relation to coronary and all-cause mortality in humans are scanty. We examined the relationship of sodium and potassium intake with cardiovascular events and all-cause mortality in the older cohort of the population-based Rotterdam Study.\nMethods\nThe Rotterdam Study\nThis case\u2013cohort analysis formed part of the Rotterdam Study, a population-based prospective study among 7,983 men and women aged 55\u00a0years and older in the Netherlands [22]. The Medical Ethics Committee of the Erasmus Medical Centre Rotterdam approved the study, and written informed consent was obtained from all participants. From August 1990 until June 1993, a trained research assistant collected data on health, medication use, lifestyle, and risk indicators for chronic diseases during a home interview. Subjects were subsequently invited at the study centre for clinical examination and assessment of diet.\nAssessment of diet\nSubjects were interviewed at the study centre by a trained dietician, who used a validated, semi-quantitative food frequency questionnaire [23]. The intake of total energy, alcohol, macronutrients, and a large number of micronutrients was computed using Dutch food composition tables [24]. No information on salt use was obtained and therefore data on dietary sodium were considered unreliable for this analysis.\nClinical examination\nHeight and body weight were measured with the subject wearing indoor clothing without shoes. The body mass index was computed as weight divided by height squared. A trained research assistant measured sitting systolic and diastolic blood pressure twice with a random-zero sphygmomanometer after a 5-min rest, and values were averaged. Hypertension was defined as a systolic blood pressure \u2265160\u00a0mmHg or diastolic blood pressure \u226595\u00a0mmHg or use of antihypertensive medication. Diabetes mellitus was considered present when the subject reported antidiabetic treatment, or when random or post-load plasma glucose levels were 11.1\u00a0mmol\/l or higher. CVD was considered present in case of a verified history of myocardial infarction, stroke, coronary bypass grafting, or percutaneous transluminal coronary angioplasty. Serum total and HDL cholesterol level (mmol\/l) were determined by standard laboratory methods [25].\nAssessment of sodium and potassium excretion\nParticipants collected an overnight urine sample before visiting the research centre and recorded collection times on the jar. They were not aware that samples would be used for estimation of electrolyte intake. At the research centre, volumes were recorded, urines were swirled and 100\u00a0ml samples were taken. Samples were stored in plastic tubes at \u221220\u00b0C for future laboratory determinations. Urinary sodium, potassium and creatinine determinations were performed by Vitros\u00ae 250 (formerly Ektachem 250) Chemistry System (Johnson & Johnson, Ortho-Clinical Diagnostics Inc., Rochester, New York). Determination of electrolytes and creatinine were based on potentiometry and enzymatic conversion, respectively. Urinary sodium and potassium concentrations (mmol\/l) were standardized to 24-h values using recorded collection times and urinary volumes (ml). In addition, urinary sodium\/potassium ratio was computed.\nFollow-up procedures\nThe present analysis is based on follow-up data collected from baseline (1990\u20131993) until 1 January 1998. Informed consent for collection of follow-up data was obtained from 7,802 participants (98%). Information on vital status was obtained at regular intervals from municipal population registries. General practitioners (GPs) used a computerized information system to record fatal and non-fatal events in the research area (covering 85% of the cohort). In the Netherlands, the GP forms the link to all specialized medical care and clinical events are unlikely to be missed by this follow-up procedure. Research physicians verified all information on incident events using GP records and hospital discharge letters. Events were coded independently by two physicians according to the International Classification of Diseases, 10th revision (ICD-10) [26]. Coded events were reviewed by a medical expert in the field, whose judgment was considered definite in case of discrepancies.\nMyocardial infarction comprised ICD-10 code I21 and stroke comprised ICD-10 codes I60-I67. Both fatal and non-fatal incident events were recorded. For the present study, only first events were considered. Events followed by death within 28\u00a0days were classified as fatal. CVD mortality comprised fatal myocardial infarction, fatal stroke, sudden cardiac death and other forms of fatal CVD (ICD-10 codes I20-I25, I46, I49, I50, I60-I67, I70-I74, and R96).\nStudy population\nOf 7,129 subjects who visited the research centre, 6,605 adequately performed a timed overnight urine collection for which collection times were recorded and volumes exceeded 150\u00a0ml. Of those, 5,531 had blood pressure readings and these subjects were eligible for the present analysis. We followed a case\u2013cohort approach for efficiency reasons. Assessment of urinary sodium, potassium and creatinine excretion was performed in all subjects who died (n\u00a0=\u00a0795, including 217 cardiovascular deaths), and in those who experienced a myocardial infarction (n\u00a0=\u00a0206) or stroke (n\u00a0=\u00a0181) during follow-up. A random sample of 1,500 control subjects was taken from the eligible cohort for assessment of electrolyte excretions. Urine samples could not be retrieved for 52 of these subjects, and data on urinary sodium, potassium and creatinine were thus obtained in 1,448 subjects. Dietary data were available for 1,205 subjects (83%) of the random sample, 518 subjects (65%) who died during follow-up, 157 subjects (72%) who died from CVD, 170 subjects (83%) with an incident myocardial infarction and 147 subjects (81%) with an incident stroke. Reasons for missing dietary data were participation in the pilot phase of the Rotterdam Study, low cognitive function, and logistic reasons, as described in more detail elsewhere [23]. Of the random sub-cohort (n\u00a0=\u00a01,448), 783 subjects (54%) were free of CVD and hypertension at baseline.\nData analysis\nPearson correlations were computed to examine inter-relationships between urinary and dietary measures of electrolyte intake and associations with total energy intake.\nThe association of urinary and dietary electrolytes with incident myocardial infarction, incident stroke, cardiovascular mortality and all-cause mortality was evaluated in a case\u2013cohort design with standard Cox proportional-hazards models with modification of the standard errors based on robust variance estimates [27, 28]. We used the method according to Barlow in which the random cohort is weighted by the inverse of the sampling fraction from the source population. Members of the random cohort are included from baseline until failure or censoring, whereas cases outside the cohort are included at the time of their event. For the Cox models we used Proc MI and Proc MIanalyze, in conjunction with Proc Phreg (SAS 8.2).\nRelative risks (RR) with 95% confidence intervals (95%-CI) were computed per 1 standard deviation increase in urinary sodium (mmol\/24\u00a0h), urinary potassium (mmol\/24\u00a0h) and dietary potassium intake (mg\/day), and per 1\u00a0unit increase in urinary sodium\/potassium ratio. Two-sided P-values below 0.05 were considered statistically significant. Adjustment was made for age, sex and, in urinary analyses, for 24-h urinary creatinine excretion (model 1). In a second analysis (model 2), additional adjustment was made for body mass index (kg\/m2), smoking status (current, past, or never), diabetes mellitus (yes\/no), use of diuretics (yes\/no), and highest completed education (three categories). In a third analysis (model 3), dietary confounders were additionally adjusted for, i.e. daily intake of total energy (kJ), alcohol (g), calcium (g), and saturated fat (g). In the analysis for urinary sodium we additionally included urinary potassium in this model, and vice versa.\nAnalyses were repeated after exclusion of subjects with a history of CVD or hypertension to avoid biased risk estimates due to intentional dietary changes. Within this sub-cohort, a predefined stratified analysis of urinary sodium and urinary sodium\/potassium ratio with cardiovascular and all-cause mortality was performed in subjects with a high body mass index (i.e., \u226525\u00a0kg\/m2), using model 3.\nAlso in the sub-cohort free of CVD and hypertension, the distribution of 24-h urinary sodium excretion was divided into quartiles to be able to examine the relationship with all-cause mortality at extreme intakes. Quartiles of urinary sodium (cut-off levels: 66, 105 and 151\u00a0mmol\/24\u00a0h) were entered categorically into the fully adjusted model (model 3), using the lower quartile as the reference.\nResults\nThe study had a median follow-up of 5.5\u00a0years. Baseline characteristics of the study population are shown in Table\u00a01. Randomly selected controls (n\u00a0=\u00a01,448) were expectedly healthier at baseline than cases, as indicated by a lower prevalence of hypertension, diabetes, and CVD.\nTable\u00a01Baseline characteristics of the study populationRandom sampleCasesIncident MIIncident strokeCVD mortalityAll-cause mortalityNo. of subjects1,448206181217795In random sample (%)31312829Age (year)69.2 (8.7)71.0 (8.0)74.0 (8.5)76.8 (8.4)76.9 (8.9)Men (%)4162455149Body mass index (kg\/m2)26.4 (3.8)26.3 (3.4)26.0 (3.3)26.2 (3.8)25.7 (3.8)Smoking status (%)a\u00a0\u00a0\u00a0\u00a0Current2329282326\u00a0\u00a0\u00a0\u00a0Former4148424740\u00a0\u00a0\u00a0\u00a0Never3623292935Alcohol use (%)8174807173Educational level (%)a,b\u00a0\u00a0\u00a0\u00a0Low5861606566\u00a0\u00a0\u00a0\u00a0Intermediate3231343028\u00a0\u00a0\u00a0\u00a0High108656Serum cholesterol (mmol\/l)\u00a0\u00a0\u00a0\u00a0Total6.6 (1.2)6.3 (1.3)6.5 (1.2)6.6 (1.4)6.3 (1.3)\u00a0\u00a0\u00a0\u00a0HDL1.4 (0.4)1.3 (0.4)1.3 (0.4)1.2 (0.4)1.3 (0.4)Blood pressure (mmHg)\u00a0\u00a0\u00a0\u00a0Systolic140 (22)145 (23)149 (24)146 (25)145 (25)\u00a0\u00a0\u00a0\u00a0Diastolic74 (11)74 (12)75 (13)73 (13)73 (14)Hypertension (%)c3744535547Diabetes mellitus (%)d1021222621History of CVD (%)e1735173928Values are means with standard deviations, or percentages; CVD, cardiovascular disease; MI, myocardial infarctionaValues not always add up to 100% due to roundingbHighest achieved level of education; low, primary education, or less; intermediate, secondary general or vocational education; high, higher vocational education, universitycSystolic blood pressure \u2265160\u00a0mmHg or diastolic blood pressure \u226595\u00a0mmHg or use of antihypertensive medicationdPlasma glucose \u226511.1\u00a0mmol\/l or treated with oral antidiabetes medication or insulineVerified history of cardiovascular disease, i.e. myocardial infarction, stroke, coronary bypass-grafting, or percutaneous transluminal coronary angioplasty\nBaseline urinary excretions and dietary intakes are presented in Table\u00a02. In the random sample, 24-h urinary sodium excretion estimated from overnight urine collection was 117\u00a0mmol (i.e., 2.7\u00a0g\/day, which corresponds to a NaCl intake of 6.8\u00a0g\/day). Urinary potassium excretion was 45\u00a0mmol\/24\u00a0h (1.8\u00a0g\/day), which was half the amount estimated by food frequency questionnaire (3.6\u00a0g\/day). The correlation between urinary and dietary potassium was 0.21 (P\u00a0<\u00a00.001).\nTable\u00a02Baseline urinary excretions and dietary intakes of Dutch men and women aged 55\u00a0years and over: The Rotterdam StudyRandom subcohortCasesIncident MIIncident strokeCVD mortalityAll-cause mortalityUrinary excretionaVolume (l\/24\u00a0h)1.4 (0.6)1.4 (0.6)1.4 (0.6)1.3 (0.6)1.3 (0.6)Sodium (mmol\/24\u00a0h)117 (69)124 (68)115 (72)99 (61)107 (66)Potassium (mmol\/24\u00a0h)45 (22)47 (22)45 (23)44 (24)44 (22)Sodium\/potassium2.8 (1.5)2.7 (1.3)2.7 (1.3)2.5 (1.4)2.6 (1.6)Creatinine (mmol\/24\u00a0h)9.2 (4.9)9.8 (4.7)8.4 (4.4)8.1 (4.7)8.1 (4.4)Sodium\/creatinine13.8 (6.6)13.6 (6.1)14.6 (7.1)14.0 (8.0)14.8 (7.9)Potassium\/creatinine5.4 (2.2)5.3 (2.1)5.8 (2.1)6.1 (2.6)6.1 (2.5)Dietary intakebTotal energy (mJ\/day)8.3 (2.1)8.6 (2.2)8.4 (2.2)8.3 (2.0)8.5 (2.2)Saturated fat (g\/day)32 (12)34 (13)34 (13)33 (13)34 (12)Calcium (g\/day)1.1 (0.4)1.1 (0.4)1.1 (0.4)1.1 (0.5)1.1 (0.4)Sodium (g\/day)c2.2 (0.7)2.3 (0.6)2.2 (0.6)2.2 (0.7)2.2 (0.7)Potassium (g\/day)3.6 (0.8)3.7 (0.8)3.6 (0.8)3.6 (0.9)3.6 (0.9)Values are means with standard deviations; CVD, cardiovascular disease; MI, myocardial infarctionaBased on one timed overnight urine samplebDietary data were available for 1,205 subjects of the random sample (83%), 170 MI cases (83%), 147 stroke cases (81%), 157 CVD deaths (72%), and 518 deaths from any cause (65%)cOnly from foods, discretionary sources not included\nRR for cardiovascular events and all-cause mortality per 1-SD increase in 24-h urinary sodium are presented in Table\u00a03. Urinary sodium was not significantly associated with incident myocardial infarction, incident stroke, or overall mortality. For CVD mortality, however, a borderline significant inverse association was observed (RR\u00a0=\u00a00.77 (0.60\u20131.01) per 1-SD, model 3) but the relationship was attenuated after excluding subjects with a history of CVD or hypertension (RR\u00a0=\u00a00.83 (0.47\u20131.44) per 1-SD, model 3). In subjects initially free of CVD, the risk of all-cause mortality was also examined across quartiles of 24-h urinary sodium (median values: 45, 87, 125 and 190\u00a0mmol, respectively). RR in consecutive quartiles, using the lower quartile as the reference, were 0.80 (0.43\u20131.49), 0.66 (0.34\u20131.27) and 0.98 (0.54\u20131.78), respectively (model 3). In a subgroup analysis of CVD free subjects with a body mass index \u226525\u00a0kg\/m2, the association of urinary sodium with CVD mortality or all-cause mortality was neither statistically significant (RR\u00a0=\u00a00.91 (0.44\u20131.89) and RR\u00a0=\u00a01.19 (0.86\u20131.66) per 1-SD, respectively; model 3).\nTable\u00a03Relative risk of urinary sodium with cardiovascular events and all-cause mortality in Dutch men and women aged 55\u00a0years and overAll subjectsaSubjects initially free of CVD and hypertensionaIncident MIRR, model 1b1.13 (0.95\u20131.34)1.04 (0.75\u20131.43)RR, model 2c1.16 (0.98\u20131.39)1.07 (0.77\u20131.50)RR, model 3d1.19 (0.97\u20131.46)1.14 (0.77\u20131.69)Incident strokeRR, model 11.09 (0.89\u20131.33)1.16 (0.84\u20131.61)RR, model 21.09 (0.87\u20131.35)1.15 (0.81\u20131.62)RR, model 31.08 (0.80\u20131.46)1.02 (0.66\u20131.58)CVD mortalityeRR, model 10.74 (0.60\u20130.91)0.84 (0.59\u20131.22)RR, model 20.83 (0.68\u20131.02)0.95 (0.66\u20131.39)RR, model 30.77 (0.60\u20131.01)0.83 (0.47\u20131.44)All-cause mortalityRR, model 10.90 (0.81\u20131.02)1.00 (0.83\u20131.20)RR, model 20.96 (0.84\u20131.09)1.10 (0.91\u20131.34)RR, model 30.95 (0.81\u20131.12)1.12 (0.86\u20131.46)RR, Relative risk with 95% confidence interval per standard deviation increase in urinary sodium (mmol\/24\u00a0h), obtained by Cox proportional hazard analysisaNumber of cases and subjects in random sample given in Table\u00a01bAjusted for age, sex and (for urinary sodium) 24-h urinary creatinine excretioncAs model 1, with additional adjustment for body mass index, smoking status, diabetes, use of diuretics, highest completed educationdAs model 2, with additional adjustment for daily intake of total energy, alcohol, calcium, saturated fat and 24-h urinary potassium excretioneCardiovascular mortality comprises fatal myocardial infarction, fatal stroke, sudden cardiac death and other forms of fatal CVD\nFindings for potassium are presented in Table\u00a04. Urinary potassium tended to be positively associated with incident CVD events or mortality, especially in subjects who were initially free of CVD and hypertension. After full adjustment for confounders (model 3), however, none of these associations were statistically significant. Urinary potassium did neither predict all-cause mortality. For dietary potassium, similar results were obtained except for risk of all-cause mortality that was significantly reduced both in the entire cohort (RR\u00a0=\u00a00.78 (0.65\u20130.94 per 1-SD) and in subjects initially free of CVD and hypertension (RR\u00a0=\u00a00.71 (0.51\u20131.00), model 3).\nTable\u00a04Relationship of urinary and dietary potassium with cardiovascular events and all-cause mortality in Dutch men and women aged 55\u00a0years and overAll subjectsaSubjects initially free of CVD and hypertensionaUrinary excretion (mmol\/24\u00a0h)Dietary intake (mg\/day)Urinary excretion (mmol\/24\u00a0h)Dietary intake (mg\/day)Incident MIRR, model 1b1.10 (0.89\u20131.35)0.98 (0.85\u20131.13)1.15 (0.84\u20131.59)1.14 (0.85\u20131.54)RR, model 2c1.16 (0.94\u20131.43)0.94 (0.81\u20131.09)1.25 (0.94\u20131.74)1.07 (0.78\u20131.46)RR, model 3d1.11 (0.87\u20131.43)0.90 (0.65\u20131.24)1.22 (0.79\u20131.87)1.32 (0.65\u20132.67)Incident strokeRR, model 11.09 (0.87\u20131.36)0.99 (0.84\u20131.17)1.12 (0.79\u20131.60)1.07 (0.79\u20131.43)RR, model 21.12 (0.89\u20131.42)0.99 (0.84\u20131.16)1.15 (0.77\u20131.71)1.20 (0.86\u20131.68)RR, model 31.17 (0.86\u20131.58)1.02 (0.71\u20131.46)1.11 (0.61\u20132.04)1.06 (0.50\u20132.29)CVD mortalityeRR, model 11.13 (0.90\u20131.41)0.97 (0.82\u20131.14)1.63 (1.14\u20132.33)1.23 (0.83\u20131.84)RR, model 21.14 (0.92\u20131.42)0.95 (0.81\u20131.12)1.66 (1.08\u20132.56)1.19 (0.78\u20131.83)RR, model 31.23 (0.94\u20131.60)0.97 (0.72\u20131.31)1.45 (0.84\u20132.54)1.43 (0.67\u20133.03)All-cause mortalityRR, model 11.04 (0.91\u20131.18)0.91 (0.82\u20131.01)1.06 (0.88\u20131.28)0.95 (0.78\u20131.17)RR, model 21.06 (0.86\u20131.31)0.89 (0.80\u20130.99)1.06 (0.86\u20131.31)0.90 (0.73\u20131.12)RR, model 31.08 (0.91\u20131.28)0.78 (0.65\u20130.94)0.95 (0.71\u20131.26)0.71 (0.51\u20131.00)RR, Relative risk with 95% confidence interval per standard deviation increase in urinary or dietary potassium, obtained by Cox proportional hazard analysisaNumber of cases and subjects in random sample given in Table\u00a01bAjusted for age, sex and (for urinary potassium) 24-h urinary creatinine excretioncAs model 1, with additional adjustment for body mass index, smoking status, diabetes, use of diuretics and highest completed educationdAs model 2, with additional adjustment for daily intake of total energy, alcohol, calcium, saturated fat and 24-h urinary sodium excretioneCardiovascular mortality comprises fatal myocardial infarction, fatal stroke, sudden cardiac death and other forms of fatal CVD\nData for urinary sodium\/potassium ratio (Table\u00a05) showed no relationship with CVD events and mortality. When restricting this analysis to CVD free subjects with a body mass index \u226525\u00a0kg\/m2, urinary sodium\/potassium ratio was significantly associated with all-cause mortality (RR\u00a0=\u00a01.19 (1.02\u20131.39) per unit, model 3), but not with CVD mortality (RR\u00a0=\u00a00.86 (0.60\u20131.25)).\nTable\u00a05Relationship of urinary sodium\/potassium ratio with cardiovascular events and all-cause mortality in Dutch men and women aged 55\u00a0years and overAll subjectsaSubjects initially free of CVD and hypertensionaIncident MIRR, model 1b1.03 (0.93\u20131.14)0.92 (0.76\u20131.13)RR, model 2c1.02 (0.92\u20131.13)0.90 (0.73\u20131.10)RR, model 3d1.04 (0.93\u20131.17)0.91 (0.72\u20131.16)Incident strokeRR, model 11.01 (0.89\u20131.13)1.01 (0.83\u20131.23)RR, model 20.99 (0.86\u20131.13)0.99 (0.77\u20131.20)RR, model 30.99 (0.83\u20131.18)0.90 (0.66\u20131.22)CVD mortalityeRR, model 10.88 (0.77\u20131.01)0.85 (0.65\u20131.11)RR, model 20.93 (0.81\u20131.06)0.86 (0.66\u20131.13)RR, model 30.92 (0.80\u20131.07)0.91 (0.65\u20131.27)All-cause mortalityRR, model 10.99 (0.91\u20131.06)1.04 (0.91\u20131.18)RR, model 20.99 (0.92\u20131.08)1.06 (0.93\u20131.22)RR, model 31.01 (0.91\u20131.12)1.13 (0.93\u20131.36)RR, Relative risk with 95% confidence interval per 1\u00a0unit increase in urinary sodium\/potassium ratio, obtained by Cox proportional hazard analysisaNumber of cases and subjects in random sample given in Table\u00a01bAdjusted for age, sex and 24-h urinary creatinine excretioncAs model 1, with additional adjustment for body mass index, smoking status, diabetes, use of diuretics and highest completed educationdAs model 2, with additional adjustment for daily intake of total energy, alcohol, calcium, and saturated fateCardiovascular mortality comprises fatal myocardial infarction, fatal stroke, sudden cardiac death and other forms of fatal CVD\nDiscussion\nIn an unselected population of older Dutch subjects we found no consistent association of urinary sodium and potassium with CVD events or mortality. Dietary potassium estimated by food frequency questionnaire, however, was associated with a lower risk of all-cause mortality. Urinary sodium\/potassium ratio was positively associated with mortality risk, but only in overweight subjects without CVD and hypertension at baseline.\nElectrolyte intake was assessed from one overnight urine collection, which provides a crude estimate of short-term intake [29, 30]. Luft et\u00a0al. examined the utility of nocturnal sodium excretion under controlled intake conditions, in which daily sodium intake was randomly varied [31]. In that study, on a randomly selected day, both 24-h and nocturnal sodium excretion estimated the daily intake reasonably well. Nevertheless, it is likely that misclassification has attenuated the relationships with CVD events and mortality in our study. Incomplete urine collection was partly adjusted for by adding urinary creatinine excretion to the multivariate models. In addition, we examined the urinary sodium\/potassium ratio, which is less influenced by incomplete urine collection. To exclude bias due to dietary changes, we repeated all analyses in a subgroup without CVD or hypertension at baseline.\nSalt intake was not consistently related to CVD or mortality in our study. An explanation for the absence of a positive relationship, apart from regression dilution bias, may be the relatively narrow range of salt intake in the Netherlands and the lack of contrast in exposure within a single population. An increased risk of mortality was observed for high salt intake in overweight Finnish subjects with 24-h urinary excretions close to 200\u00a0mmol (RR\u00a0=\u00a01.56 per 100\u00a0mmol) [16]. However, this could not be confirmed in our analysis of quartiles of sodium intake in relation to overall mortality (RR\u00a0=\u00a00.98 in CVD free subjects with median sodium excretion of 190\u00a0mmol\/24\u00a0h). The absence of a relationship between salt intake and mortality in our study corroborates the findings from the large Scottish Heart Health Study among almost 12,000 middle-aged subjects with 24-h urine samples [11]. Follow-up data of the MRFIT trial neither showed a relationship between dietary sodium intake estimated by 24-h recall and cardiovascular events or mortality [12]. However, other prospective epidemiological studies do suggest that sodium intake is related to morbidity and mortality [6, 7, 15, 16], although this may be confined to specific subgroups with overweight, hypertension or high salt intake. In overweight subjects, we did find a positive relationship between urinary sodium\/potassium ratio and overall mortality (19% increase in risk per unit change in sodium\/potassium ratio).\nA protective effect of potassium intake against stroke, as previously reported [19\u201321], could not be confirmed by our data. We neither observed an association between potassium intake and coronary events. Long-term rather than short-term intake may be relevant and therefore we estimated habitual potassium intake during the preceding year by food frequency questionnaire. Mortality risk was reduced by 29% per 1-SD increase in dietary potassium, although only in subjects initially free of CVD and hypertension. Except for misclassification, although less likely than for sodium, we have no explanation for the absent relationship between potassium intake and CVD events. In the Scottish Heart Health Study, 24-h urinary potassium excretion was inversely related to all-cause mortality and coronary events [11]. Data on potassium intake in relation to mortality, however, are sparse and more prospective population-based studies are needed to draw conclusions. Preferably, the effect of dietary potassium on CVD should be examined in a randomized trial.\nProlonged differences in blood pressure of 5\u00a0mmHg may result in a one-third reduction in stroke and one-fifth reduction in coronary events [32]. Meta-analysis of randomized controlled trials showed that sodium reduction around 2\u00a0g per day could lower blood pressure by 2\u20133\u00a0mmHg, with the effect being twice as large in hypertensives [33]. The World Health Organization recommends that people should consume less than 5\u00a0g of salt (i.e. 2\u00a0g of sodium) per day in order to prevent CVD [34]. From this and other epidemiological studies we conclude that effect of dietary salt on clinical cardiovascular endpoints and overall mortality within the range of intake commonly observed in Western countries has not yet been established. More research is needed to settle the discussion regarding this major public health issue.","keyphrases":["sodium","potassium","mortality","cardiovascular disease","myocardial infarction","stroke","population-based","salt"],"prmu":["P","P","P","P","P","P","P","P"]}
{"id":"Int_J_Hematol-4-1-2276241","title":"Diagnosis of acute myeloid leukemia according to the WHO classification in the Japan Adult Leukemia Study Group AML-97 protocol\n","text":"We reviewed and categorized 638 of 809 patients who were registered in the Japan Adult Leukemia Study Group acute myeloid leukemia (AML)-97 protocol using morphological means. Patients with the M3 subtype were excluded from the study group. According to the WHO classification, 171 patients (26.8%) had AML with recurrent genetic abnormalities, 133 (20.8%) had AML with multilineage dysplasia (MLD), 331 (51.9%) had AML not otherwise categorized, and 3 (0.5%) had acute leukemia of ambiguous lineage. The platelet count was higher and the rate of myeloperoxidase (MPO)-positive blasts was lower in AML with MLD than in the other WHO categories. The outcome was significantly better in patients with high (\u226550%) than with low (<50%) ratios of MPO-positive blasts (P < 0.01). The 5-year survival rates for patients with favorable, intermediate, and adverse karyotypes were 63.4, 39.1, and 0.0%, respectively, and 35.5% for those with 11q23 abnormalities (P < 0.0001). Overall survival (OS) did not significantly differ between nine patients with t(9;11) and 23 with other 11q23 abnormalities (P = 0.22). Our results confirmed that the cytogenetic profile, MLD phenotype, and MPO-positivity of blasts are associated with survival in patients with AML, and showed that each category had the characteristics of the WHO classification such as incidence, clinical features, and OS.\nIntroduction\nThe French-American-British (FAB) classification of acute myeloid leukemia (AML), based on morphological and cytochemical findings, was established in 1976 and has since become the standard classification [1, 2]. However, specific chromosomal and genetic abnormalities that have been extracted from analyses of prognostic factors for AML are recognized as important in selecting treatment strategies and are reflected in the AML classification as factors that are required to establish the disease entity [3]. The 1999 World Health Organization (WHO) classification includes morphological, immunological, cytogenetic, genetic, and clinical features [4\u20136]. The WHO and FAB classifications differ in several aspects. The blast threshold required for a diagnosis of AML was reduced from 30 to 20%, and new AML categories have been added for cytogenetic abnormalities, the presence of multilineage dysplasia (MLD), as well as a history of chemotherapy and subtypes for acute basophilic leukemia, acute panmyelosis with myelofibrosis, and myeloid sarcoma. The WHO classification comprises more subtypes and is more comprehensive than the FAB classification.\nCytogenetic features are important prognostic factors in AML [3, 7\u201312]. However, 11q23 abnormalities have not yet been established as a cytogenetic risk classification. Over 30 partner genes with 11q23 abnormalities have been described, and some reports indicate that patients with t(9;11) have a relatively more favorable prognosis than those with other partner chromosomes\/partner genes [13\u201316].\nIn the present study, we reviewed stained smears of blood and bone marrow from patients who were registered in the Japan Adult Leukemia Study Group (JALSG) AML-97 trial, and classified them into FAB subtypes and WHO categories. We also evaluated their survival on the basis of the WHO classification, the myeloperoxidase (MPO)-positivity of blasts, and cytogenetic findings including 11q23 abnormalities.\nPatients and methods\nPatients\nBetween December 1997 and July 2001, 809 patients aged from 15 to 66 years with untreated AML (excluding M3) were registered from 103 institutions in the AML-97 trial of the JALSG. The patients were diagnosed with AML according to the FAB criteria at each institution. Patients with a history of MDS, hematological abnormalities before the diagnosis of AML, or a history of chemotherapy were not eligible for the AML-97 trial.\nTreatment strategies\nDetails of the JALSG AML-97 treatment protocol are described elsewhere [17]. In brief, all patients underwent induction therapy consisting of idarubicin (3 days) and Ara-C (7 days). Patients who achieved complete remission were randomized into one of two arms of consolidation chemotherapy alone or in combination with maintenance chemotherapy. Patients who were placed into intermediate\/poor risk groups according to the JALSG scoring system [17] and who had an HLA-identical sibling (\u226450 years old) were simultaneously assigned to receive allogeneic hematopoietic stem cell transplantation during their first remission.\nMorphologic and cytochemical analyses\nPeripheral blood and bone marrow smears from registered patients were sent to Nagasaki University for staining with May-Giemsa, MPO, and esterase, and the diagnosis was then reevaluated by the Central Review Committee for Morphological Diagnosis. Patients were subsequently categorized according to the FAB and WHO classifications. Dyserythropoietic features were defined as >50% dysplastic features in at least 25 erythroblasts and dysgranulopoietic features including \u22653 neutrophils with hyposegmented nuclei (pseudo-Pelger\u2013Heut anomaly), and hypogranular or agranular neutrophils (>50% of \u226510 neutrophils). Dysmegakaryopoietic features were defined as \u22653 megakaryocytes that were micronuclear, multiseparate nuclear, or large mononuclear [18].\nWe assessed the ratios (%) of MPO-positive blasts on MPO-stained bone marrow smears using the diaminobenzidine method [19].\nCytogenetic analysis\nCytogenetic analysis was performed at either laboratories in participating hospitals or authorized commercial laboratories. The karyotypes of leukemic cells were collected through the JALSG AML-97 case report forms and reviewed by the Central Review Committee for Karyotyping. The patients were classified into favorable, intermediate, or adverse risk groups based on karyotypes according to results of the Medical Research Council (MRC) AML 10 trial [3]. The favorable risk group included patients with t(8;21) and inv(16), whether alone or in combination with other abnormalities. The intermediate risk group included those with a normal karyotype and other abnormalities that were not classified as either favorable or adverse. The adverse risk group included patients with a complex karyotype with four or more numerical or structural aberrations, \u22125, deletion (5q), and \u22127, whether alone or in combination with intermediate risk or other adverse risk abnormalities.\nStatistical analysis\nThe overall survival (OS) for all patients was defined as the interval from the date of diagnosis to that of death. We applied the Kaplan\u2013Meier method to estimate OS and 5-year survival. We compared survival rates between groups using the log-rank test (Stat View J 5.0). Differences were examined by the Chi-square test using Excel software. All P-values are two-sided, and values <0.05 were considered significant.\nResults\nPatient characteristics\nOf the 809 registered patients, 638 were consistent with the WHO classification. Data were incomplete for 10 of the 638 patients. Table 1 lists the characteristics of the patients. The median age of all 638 patients (390 males and 248 females) was 45 years (range 15\u201366 years). The median values of WBC, hemoglobin (Hb), platelets, and the ratio of blasts in the bone marrow were 13.7 \u00d7 109\/l, 8.3 g\/dl, 52.0 \u00d7 109\/l, and 56.0%, respectively.\nTable 1Patient characteristicsAge (year)45 (15\u201366)Male\/female390\/248WBC count (\u00d7109\/l)13.7 (0.4\u2013709)Hemoglobin (g\/dl)8.3 (3.8\u201317.2)Platelet count (\u00d7109\/l)52 (0\u2013890)Bone marrow blasts (%)56 (6\u201399)Values are presented as the median (range)\nWBC white blood cell\nFAB classification\nTable 2 shows the FAB classification of the 638 patients. Most were classified as M2 (n = 261; 40.9%), followed by M4 (n = 148; 23.2%), and M1 (n = 109; 17.1%) with M0, M4Eo, M5a, M5b, M6, M7, and acute leukemia of ambiguous lineage comprising the remainder in that order.\nTable 2Number of patients according to the FAB classificationSubtypeDescriptionNo. of patients%M0Minimally differentiated acute myeloid leukemia (AML)304.7M1AML without maturation10917.1M2AML with maturation26140.9M4Acute myelomonocytic leukemia (AMMoL)14823.2M4EoAMMoL with eosinophils233.6M5aAcute monoblastic leukemia193.0M5bAcute monocytic leukemia243.8M6Acute erythroleukemia162.5M7Acute megakaryoblastic leukemia50.8Acute leukemia of ambiguous lineage30.5Total638100\nWHO classification and clinical characteristics\nTable 3 shows the patients categorized according to the WHO classification. The first category of AML with recurrent genetic abnormalities accounted for 171 patients (26.8%), 133 (20.8%) were in the second category of AML with MLD, 331 (51.9%) were in the fourth category of AML not otherwise categorized, and 3 (0.5%) were categorized as having acute leukemia of ambiguous lineage. Most patients in the second category were identical to those with a de novo MLD phenotype. We found that 144 patients diagnosed with the MLD phenotype comprised 133 (92.4%) in the second category, 10 (7.0%) with 11q23 abnormalities, and 1 (0.7%) with acute leukemia of ambiguous lineage. Figure 1 shows the OS of each category. The 5-year survival rates of the first, second, and fourth categories were 58.2, 22.5, and 40.9% (P < 0.0001), respectively.\nTable 3Number of patients according to the WHO classificationCategory and subtypeNo. of patients%I. AML with recurrent genetic abnormalities17126.8\nt(8;21)(q22;q22);(AML1\/ETO)11317.7 inv(16)(p13;q22) or t(16;16)(p13;q22);(CBF\u03b2\/MYH11)264.1\nt(15;17)(q22;q12)(PML\/RAR\u03b1)\u2013\u2013 11q23(MLL)abnormalities325.0II. AML with multilineage dysplasia13320.8 Following MDS\u2013\u2013 Without antecedent MDS13320.8III. AML and MDS, therapy-related\u2013\u2013 Alkylating agent-related\u2013\u2013 Topoisomerase type II inhibitor-related\u2013\u2013 Other types\u2013\u2013IV. AML not otherwise categorized33151.9 AML, minimally differentiated253.9 AML without maturation9915.5 AML with maturation10816.9 Acute myelomonocytic leukemia (AMMoL)639.9 AMMoL with eosinophilia50.8 Acute monoblastic leukemia81.3 Acute monocytic leukemia162.5 Acute erythroid leukemia60.9 Acute megakaryoblastic leukemia10.2Acute leukemia of ambiguous lineage30.5Total638100\nFig.1Overall survival of patients categorized according to the WHO classification\nTable 4 compares the clinical features among the WHO categories. The mean values of platelets, WBC, Hb, and the ratio (%) of blasts in bone marrow and of MPO-positive blasts significantly differed, whereas age did not significantly differ. Patients in the second category had a higher platelet count (111.0 \u00d7 109\/l), whereas those with 11q23 abnormalities had a lower count (38.3 \u00d7 109\/l) compared with those of other subtypes.\nTable 4Comparison of clinical findings of patients diagnosed according to the WHO classificationCategoryPlatelets (\u00d7109\/l \u00b1 SE)WBC (\u00d7109\/l \u00b1 SE)Hb (g\/dl \u00b1 SE)Age (year \u00b1 SE)Blasts in bone marrow (%\u00b1SE)MPO positivity of blasts (%\u00b1SE)I\nt(8;21)76.7 \u00b1 56.43 (113)a\n1.4 \u00b1 0.6 (113)7.8 \u00b1 0.2 (113)41.6 \u00b1 1.3 (113)49.9 \u00b1 2.0 (113)93.3 \u00b1 3.3 (108)inv(16)57.8 \u00b1 52.03 (26)6.6 \u00b1 1.2 (26)9.2 \u00b1 0.5 (26)44.5 \u00b1 2.6 (26)50.5 \u00b1 4.1 (26)66.9 \u00b1 6.7 (26)11q2338.3 \u00b1 30.8 (32)4.3 \u00b1 1.1 (32)8.9 \u00b1 0.4 (32)41.6 \u00b1 2.4 (32)56.3 \u00b1 3.7 (32)43.6 \u00b1 6.1 (32)II111.0 \u00b1 121.5 (133)3.0 \u00b1 0.5 (133)8.3 \u00b1 0.2 (133)44.2 \u00b1 1.2 (133)48.0 \u00b1 1.8 (133)34.0 \u00b1 3.1 (126)IV72.8 \u00b1 91.7 (330)5.1 \u00b1 0.3 (331)8.8 \u00b1 0.1 (330)43.8 \u00b1 0.7 (331)65.7 \u00b1 1.2 (328)53.7 \u00b1 1.9 (312)\nP < 0.0001\nP < 0.0001\nP = 0.0004\nP = 0.4077\nP < 0.0001\nP < 0.0001\nSE standard error, WBC white blood cell, MPO myeloperoxidase, Hb hemoglobin\naNumber of patients\nThe WBC count of patients with t(8;21) was 1.4 \u00d7 109\/l and lower than in other subtypes. The MPO-positive rate of blasts among patients with t(8;21) was higher (93.3%) and that of patients in the second category was lower (34.0%), than in other subtypes. All patients were grouped as high- or low-MPO according to \u226550% or <50% of MPO-positive blasts, respectively. A total of 339 patients (53.1%) were classified as high-MPO, 268 (42.0%) as low-MPO, and the MPO status of blasts could not be assessed in 31 (4.9%). Figure 2 shows the OS of patients with high- or low-MPO. The 5-year survival rate for patients with high or low-MPO was 50.7 and 29.6%, respectively (P < 0.0001).\nFig. 2Overall survival of patients with high or low MPO-positive blasts\nCytogenetics\nAll 638 patients were classified into favorable (n = 139; 21.8%), intermediate (n = 413; 64.7%), and adverse (n = 54; 8.5%) cytogenetic risk groups (Table 5). Figure 3 shows the OS according to this stratification. The 5-year survival rates were 63.4, 39.3, and 0.0% in the favorable, intermediate (except for those with 11q23 abnormalities), and adverse risk groups, respectively, and 35.5% in the group with 11q23 abnormalities (P < 0.0001).\nTable 5Distribution of patients classified by cytogenetic riskCytogenetic risk groupNo. of patients%Favorable13921.8\nt(8;21)11317.7 inv(16)264.1Intermediate41364.7 Normal karyotype26741.8 11q23325.0 Ph(+)71.1\nt(7;11)(p15;p15)40.6\nt(6;9)40.6 Other13120.5Adverse548.5 Complex416.4 \u2212720.3 abn350.8 del5q20.3 \u2212510.2 Other30.5Total638100.0\nFig. 3Overall survival of patients stratified according to cytogenetic risk groups. Significant differences were observed between patients with a favorable, intermediate (except 11q23), and adverse karyotype (P < 0.0001)\nThe numbers of patients with or without MLD and high- or low-MPO in each cytogenetic risk group are listed in Table 6. None of those with the MLD phenotype were classified into the favorable risk group, while 129 (89.6%) and 15 (10.4%) of 144 patients with MLD were classified into intermediate or adverse risk groups, respectively. Only 15 patients (4.4%) in the high-MPO group were classified as having an adverse risk, while 11 (4.1%) in the low-MPO group were included in the favorable risk group.\nTable 6Relationship between cytogenetic risk groups and MLD phenotype or MPO-positive rates of blastsFavorable n = 139Intermediate n = 445Adverse n = 54TotalMLD +0129 (89.5%)15 (10.4%)144 \u2212138 (28.2%)292 (59.6%)38 (7.8%)490 Unknown1214MPO High123 (36.3%)201 (59.3%)15 (4.4%)339 Low11 (4.1%)221 (82.5%)36 (13.4%)268 Unknown523331High- and low-MPO indicates a percentage of myeloperoxidase positive blasts \u226550 or <50%, respectively\nMLD multilineage dysplasia\nThe 32 patients with 11q23 abnormalities comprised 11 (34.4%) with t(11;19), 9 (28.1%) with t(9;11), 5 (15.6%) with del(11)(q23), 4 (12.5%) with t(6;11), and 3 (9.4%) with t(11;17). Figure 4 shows the OS of the intermediate risk group. The 5-year survival rate was 44.0% in patients with a normal karyotype, 35.5% in those with 11q23 abnormalities, and 30.6% in other patients including those with t(7;11), t(6;9), and Ph(+) abnormalities, respectively (P = 0.033).\nFig. 4Overall survival of patients with subtypes of intermediate cytogenetic risk. Significant differences were observed between patients with a normal karyotype and those with 11q23 abnormalities (P = 0.033)\nTable 7 shows the relationship between t(9;11) (n = 9) and other 11q23 abnormalities (n = 23). More patients with low-MPO, without MLD, or with the FAB M5 subtype were found in the group with t(9;11) than with other 11q23 abnormalities. The survival rates between the two groups did not significantly differ (P = 0.22, data not shown).\nTable 7Comparison of t(9;11) and other 11q23 abnormalitiesNo. of patientsAuerMPO*MLD*FABMedian age (year)Median survival (day)+\u2212HighLow+\u2212M1M2M4M4EoM5a**M5b\nt(9;11)9091809003060391031.00Other 11q232351813101013131312348520.00Total3252714181022131618344.5531.5High- and low-MPO indicates a percentage of myeloperoxidase-positive blasts \u226550 or <50%, respectively\nMLD multilineage dysplasia* P < 0.05, ** P < 0.01\nDiscussion\nWe attempted to classify selected patients who were reviewed morphologically and had available chromosomal data according to the WHO system. However, our series had some limitations in terms of analysis and patient selection. Although we obtained chromosomal data, genetic data were not available. Patients who were diagnosed with AML M3 or who had t(15;17), a history of MDS, or preceding hematological abnormalities, or who had previously undergone chemotherapy, were not eligible for the present study. However, multicenter trials might have some advantages in diagnosing AML according to the WHO classification, because morphological diagnoses and karyotypes are reviewed by the corresponding institutional committees.\nThe incidence of each category of the WHO classification was similar to those in several reports when patients with t(15;17) and therapy-related AML were excluded [20\u201322]. We and several others have shown that approximately 30% of patients have recurrent genetic abnormalities. Multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) assays have recently been applied to analyze cytogenetic abnormalities [21, 23, 24]. This method might cause the frequency of the first WHO category to increase. Thus, the multiplex RT-PCR assay might have to be incorporated into the WHO system. The JALSG has started a cohort study in which all AML patients in participating hospitals are registered and analyzed according to the WHO classification. That study should clarify the real ratios of the AML subtypes in the WHO classification.\nFew reports have included clinical data with the WHO classification. We found that the platelet count was higher among patients in the second category than in other categories. This supports our previous finding that the platelet count is higher in patients with AML accompanied by the MLD phenotype [25]. Among patients with MLD, none were in the favorable risk group, whereas the intermediate or adverse risk ratios among these patients were 89.6 and 10.4%, respectively. These differences might influence the finding that OS was better among patients without than with MLD (P = 0.0002, data not shown). Previous studies have also associated the MLD phenotype with a poorer outcome, although MLD is not significantly prognostic on multivariate analysis [18, 26], and a German group showed that dysplastic features correlate with adverse karyotypes [26]. Furthermore, patients in the second category had a lower MPO-positive rate of blasts, whereas those with t(8;21) had a higher rate. Patients with high- and low-MPO were more frequently observed in the favorable and adverse risk groups, respectively. Multivariate analysis has shown that MPO is a significant factor affecting OS [19]. We did not assess prognostic factors by multivariate analysis here because the main theme of this study was to categorize patients according to the WHO classification, and we have already examined these in a previous series [18, 19].\nSeveral studies have demonstrated the impact of specific cytogenetic abnormalities on survival in AML [3, 7\u201312, 20\u201322]. The cytogenetic risk groups stratified the AML patients in the present study according to the MRC system, as in these reports [3]. Therefore, we confirmed the clinical usefulness of cytogenetics as the first category of the WHO classification. We found that 32 patients had 11q23 abnormalities. The MRC system revealed that de novo and secondary AML patients with 11q23 abnormalities had an intermediate outcome with an OS rate of 45% at 5 years (n = 60; median age, 17 years) in a younger cohort [3] and an OS rate of 0% at 5 years (n = 11; median age 64 years) in an elderly cohort [7]. In contrast, SWOG\/ECOG trials including adult de novo AML patients (age, 16\u201355 years) assigned those with 11q abnormalities to the unfavorable cytogenetic subgroup [8]. Our data showed that patients with 11q23 abnormalities have an intermediate rather than adverse outcome. The prognostic effect of 11q23 abnormalities might depend on the partner gene. Several studies have shown that 11q23 abnormalities with t(6;11) and t(10;11) are associated with a poor prognosis, whereas t(9;11) is associated with a superior OS and such patients might respond well to intensive treatment, especially when the chemotherapy regimen includes high-dose cytarabine [15, 27\u201330]. The CALGB study has shown that the median OS of 13.2 months among 23 patients with t(9;11) was significantly longer than the 7.7 months among 24 patients with other 11q23 rearrangements (P = 0.009) [30]. In a recent CALGB series of 54 patients with 11q23 abnormalities, 27 patients with t(9;11) had an intermediate outcome and a median OS of 13.2 months, whereas those with t(6;11) or t(11;19) had a poor outcome of 7.2 or 8.4 months [15]. Conversely, Schoch et al. showed that 14 patients with t(9;11) had a median OS of 10.0 months compared with the 12.8 months of 26 patients with other MLL rearrangements, and that the two cytogenetic groups did not significantly differ [13]. Our data showed that nine patients with t(9;11) were more frequently involved in M5. The MPO and MLD features significantly differed between patients with t(9;11) and those with other 11q23 abnormalities. However, the CALGB study found no significant differences in myelodysplastic features between the two cytogenetic groups [30]. In terms of OS, our results showed no significant differences between patients with t(9;11) and those with other 11q23 abnormalities (P = 0.22). Some problems are associated with the analyses of 11q23 abnormalities. We had few patients with these abnormalities, particularly individual translocations, and genetic analysis was not performed. Thus, the prognostic risk of 11q23 abnormalities cannot be concluded from the present study. Nonetheless, these abnormalities were never associated with a favorable risk. To classify 11q23 abnormalities into each prognostic risk group, further investigations and genetic analyses of a large number of patients with 11q23 abnormalities are required.\nThe fourth WHO category, which is not otherwise categorized, accounted for 52% of patients in the present study. Most of them were classified into the intermediate risk group, and no prognostic subdivisions were valuable. Using cytogenetic features as a prognostic factor in groups with a normal karyotype has limitations, and such patients accounted for 64.6% of the intermediate risk group (data not shown). Additional factors are required to stratify these patients. We and several others suggested that differences could be based on molecular genetic analysis [22, 31\u201335]. For example, FLT3 mutations are important biomarkers of a normal karyotype and might be valuable for stratifying the intermediate risk group. Further follow-up studies might also shed light on the roles of FLT3 ITD mutations in the development of AML and aid their use as novel molecular targeting agents against AML [22, 32]. Bienz et al. identified CEBPA mutations, FLT3-ITD, and differing levels of BAALC expression as having independent prognostic significance in patients with a normal karyotype [33]. If these genetic markers can be confirmed as being of clinical significance, genetic analyses will probably be incorporated into the WHO classification.\nIn summary, our results confirmed those of previous studies showing the prognostic significance of cytogenetics, MLD, and MPO-positivity of blasts in AML. Furthermore, we categorized patients with de novo AML according to the WHO classification and showed the clinical characteristics and OS of each category.","keyphrases":["who classification","aml","myeloperoxidase","11q23 abnormalities","multilineage dyplasia"],"prmu":["P","P","P","P","M"]}
{"id":"Purinergic_Signal-3-1-2096770","title":"P2 receptors in atherosclerosis and postangioplasty restenosis\n","text":"Atherosclerosis is an immunoinflammatory process that involves complex interactions between the vessel wall and blood components and is thought to be initiated by endothelial dysfunction [Ross (Nature 362:801\u201309, 1993); Fuster et al. (N Engl J Med 326:242\u201350, 1992); Davies and Woolf (Br Heart J 69:S3\u2013S11, 1993)]. Extracellular nucleotides that are released from a variety of arterial and blood cells [Di Virgilio and Solini (Br J Pharmacol 135:831\u201342, 2002)] can bind to P2 receptors and modulate proliferation and migration of smooth muscle cells (SMC), which are known to be involved in intimal hyperplasia that accompanies atherosclerosis and postangioplasty restenosis [Lafont et al. (Circ Res 76:996\u2013002, 1995)]. In addition, P2 receptors mediate many other functions including platelet aggregation, leukocyte adherence, and arterial vasomotricity. A direct pathological role of P2 receptors is reinforced by recent evidence showing that upregulation and activation of P2Y2 receptors in rabbit arteries mediates intimal hyperplasia [Seye et al. (Circulation 106:2720\u2013726, 2002)]. In addition, upregulation of functional P2Y receptors also has been demonstrated in the basilar artery of the rat double-hemorrhage model [Carpenter et al. (Stroke 32:516\u201322, 2001)] and in coronary artery of diabetic dyslipidemic pigs [Hill et al. (J Vasc Res 38:432\u201343, 2001)]. It has been proposed that upregulation of P2Y receptors may be a potential diagnostic indicator for the early stages of atherosclerosis [Elmaleh et al. (Proc Natl Acad Sci U S A 95:691\u201395, 1998)]. Therefore, particular effort must be made to understand the consequences of nucleotide release from cells in the cardiovascular system and the subsequent effects of P2 nucleotide receptor activation in blood vessels, which may reveal novel therapeutic strategies for atherosclerosis and restenosis after angioplasty.\nIntroduction\nAtherosclerosis is a pathological phenomenon primarily affecting the large conduit arteries, for example the aorta, coronary, carotid iliac, and femoral arteries. The development of atherosclerotic lesions in arteries involves the intimal recruitment of smooth muscle cells (SMC) within the blood vessel wall, and also the infiltration of blood-derived cells [1]. This process necessitates the proliferation and migration of SMC from the underlying media and the endothelial adhesion of leukocytes and their infiltration into the subendothelium. A similar intimal accumulation of SMC also takes place during the postangioplasty restenotic process. Although the factors involved in intimal cell recruitment are not clearly identified, it is becoming evident that endothelial dysfunction is a key factor in the development of vascular disease. Experimental evidence suggests that an intact endothelium plays a central role in maintaining a low proliferative state of SMC under normal conditions [10]. In arterial injury, endothelial cells, SMC, and various blood cells can release chemotactic factors and mitogens, including ATP and other nucleotides (see [4] for review). Activation of P2 nucleotide receptors has been shown to induce not only the proliferation and migration of vascular SMC but also apoptosis, a process involved in the evolution of the atherosclerotic plaque [11]. In addition, P2 receptors mediate both vasorelaxation and vasoconstriction of arteries that may be involved in the vascular remodeling accompanying atherosclerosis and postangioplasty restenosis [5]. A better understanding of the causative agents and mechanisms of proliferation and migration of vascular SMC as well as recruitment of blood-derived cells by the endothelium could lead to prevention, attenuation, or even reversal of intimal thickening, which may dramatically reduce morbidity and mortality from vascular diseases such as atherosclerosis and restenosis after angioplasty. In this respect, a better understanding of the physiological role of P2 receptors in both normal and pathological blood vessels could potentially lead to a breakthrough in the fight against vascular disease.\nP2 receptors in the cardiovascular system\nExtracellular nucleotides bind to cell surface receptors known as P2 receptors, which are present in many tissues. To date, these receptors have been classified into two main families: the P2X receptors that are ligand-gated ion channels comprised of homo- or hetero-oligomers [12] and P2Y receptors that are seven membrane spanning receptors coupled via G proteins (Gq\/11 or Gi\/o) to phospholipase C (PLC) and\/or adenylate cyclase [12\u201314]. In turn, PLC activation generates inositol 1,4,5-triphosphate (IP3), a mediator of Ca2+ release from intracellular stores, and diacylglycerol, an activator of protein kinase C (PKC), whereas adenylate cyclase generates cyclic adenosine monophosphate (cAMP), an activator of protein kinase A (PKA). The cloning of seven P2X (P2X1, P2X2, P2X3, P2X4, P2X5, P2X6, P2X7) and eight P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, P2Y14) receptor subtypes has made it possible to use molecular and pharmacological approaches to study the distribution and functional properties of specific P2 receptor subtypes at the tissue and cellular level.\nP2 receptors in vascular cells\nThe normal arterial wall consists of three layers: the intima, media, and adventitia. The single layer of endothelial cells facing the vessel lumen is a very important component of the vascular wall in terms of releasing both vasodilators such as nitric oxide (NO) and prostacyclin (PGI2) and vasoconstrictors like thromboxane A2 and endothelin. The principal P2Y receptor subtypes that have been functionally characterized in endothelial cells are P2Y1 and P2Y2, but mRNAs for P2Y4 and P2Y6 receptors have also been detected [4]. Endothelium-dependent vasorelaxation has been attributed to the release of NO and PGI2 after binding of nucleotides to P2Y1 and P2Y2 receptors in endothelial cells [15], whereas vasoconstrictor effects in SMC result from the action of nucleotides on P2Y2 and P2X receptors [16, 17]. In most blood vessels, P2Y1 receptors for ADP are present on the endothelium and regulate vasodilatation by Ca2+-dependent (PLC-mediated) activation of nitric oxide synthase (NOS) and generation of endothelial-dependent relaxing factor (EDRF) (see [4] for review). Endothelial prostacyclin production is also stimulated by P2Y1 and P2Y2 receptors, but this seems to play a minimal role in vasodilatation, at least under physiological conditions (see [18] for review). Recent studies have indicated that in the aorta of P2Y2-null mice the endothelium-dependent relaxation by ATP and ATP\u03b3S was inhibited, demonstrating the role of the P2Y2 receptors, but that a relaxation by UTP and UDP was maintained, suggesting the additional involvement of P2Y6 receptors [19]. The majority of the cells in intact blood vessels are SMC, which occupy most of the media, and are involved in vasoconstriction and vasorelaxation of the vessel. P2Y2Rs in SMC mediate the induction of immediate-early and delayed-early cell cycle-dependent genes, consistent with a role for P2Y2Rs in vascular proliferation of SMC [20, 21]. A recent study demonstrated that P2Y2 is the predominant functional receptor that responds to ATP and UTP in rat aortic SMC [22]. In human cerebral arteries, P2Y6 seems to be the predominant subtype and induces vasoconstriction when activated by UDP\/UTP [23, 24]. This is consistent with findings from rat pulmonary and mesenteric arteries [25, 26]. In addition, a recent study on P2X1 knockout mice further supports the prominent contractile effect of the P2Y6 subtype in mesenteric arterial trees [27]. Taken together, it seems that the principal receptor mediating UTP\/UDP-induced contractile responses in blood vessels might be the P2Y6 subtype. Other studies have reported the presence of both P2Y4 and P2Y6 receptors in rat aortic SMC [21, 28, 29]. P2Y1 receptors are expressed in SMC of a number of blood vessel types and like their EC counterparts mediate vasodilatation most likely through the activation of K+ channels (see [18] for review). The presence of several P2X receptor subtypes also has been reported in human saphenous vein SMC, including P2X1, P2X2, P2X4, and P2X7 receptors [30]. The outermost layer of the blood vessel consists of connective tissue and fibroblasts, which have not been appreciated in the regulation of vascular tone. A recent study showed that fibroblasts can migrate into the neointima, suggesting their possible involvement in the development of vascular diseases such as atherosclerosis and restenosis after angioplasty [31]. Since human and rat fibroblasts are known to express P2Y1, P2Y2, P2Y4, P2Y6, P2X3, P2X4, and P2X7 receptors [32], further investigation is needed to establish the role of fibroblast P2 receptors in either physiological or pathophysiological conditions.\nP2 receptors in blood cells\nG protein-coupled P2Y receptors whose activation leads to intracellular calcium mobilization have been observed in neutrophils [4, 33, 34], and turkey erythrocytes express both P2Y1 and P2X7 receptors [35, 36]. ATP and UTP act as secretagogues by binding to P2Y2Rs to enhance exocytosis of primary granules in neutrophils [37]. In macrophages, ATP activates a P2X7 receptor that differs from other ligand-gated ion channel P2X receptors by its ability to generate plasma membrane pores when activated [38]. Human T lymphocytes also have been shown to express P2X7 receptors [39]. Human monocytes and macrophages coexpress P2X1, P2X4, and P2X7 receptors, whereas granulocytes only express P2X7 receptors [40]. P2Y1, P2Y2, and P2Y6 receptors also are expressed in monocytes, B lymphocytes, and polymorphonuclear granulocytes [41]. Human platelets express P2Y1, P2Y12, and P2X1 receptors [42\u201344]. Thus, the diversity of P2 receptor expression in blood cells, as well as endothelial cells, SMC, and fibroblasts, suggests that this receptor superfamily plays a significant role in the regulation of cardiovascular functions.\nP2 receptors regulate nucleotide-induced vascular smooth muscle cell proliferation\nProliferation of SMC is a hallmark of vascular diseases such as atherosclerosis and restenosis following angioplasty. ATP and other nucleotides are released by aggregating platelets and damaged vascular cells, such as endothelial cells and SMC in pathological or stress conditions [45]. Extracellular nucleotides can act via P2X and P2Y receptors to induce acute responses such as the regulation of vascular tone [46]. However, it is generally believed that the ionotropic P2X receptors do not mediate the chronic responses of nucleotides such as cell proliferation. The mitogenic effect of extracellular nucleotides on vascular SMC (VSMC) has been known for years [47]. However, a potent antiproliferative effect of UTP on VSMC also has been reported in human arterial and venous SMC [48]. In either case, the P2 receptor subtype(s) responsible for these effects on the proliferation of VSMC has not been determined. Earlier studies have shown that ATP or UTP increased DNA and protein synthesis in subcultured rat aortic VSMC [49, 50]. In the same cell culture model, however, Malam-Souley et al. [51] were unable to detect increases in DNA synthesis after ATP\/UTP stimulation, although ATP or UTP upregulated the expression of mRNA to several cell cycle progression-related genes. Because P2X receptor agonists were essentially inactive, it was concluded that a P2U-like receptor (now termed P2Y2) was responsible for the mitogenic effects of ATP\/UTP. However, the role of a P2Y4 receptor cannot be excluded, because the nucleotide agonist profile between rat P2Y2 and P2Y4 receptors is essentially indistinguishable [52]. Indeed, Harper et al. [53] suggested that the P2Y4 receptor mediated ATP\/UTP-induced proliferation of rat aortic VSMC. Recent studies indicated that ATP, UTP, or ITP, three agonists of the cloned porcine P2Y2 receptor, increased DNA and protein synthesis and cell number in coronary artery SMC [54]. Indeed, treatment of pig coronary artery SMC with UTP, ATP, or ITP caused a concentration-dependent increase in DNA and protein synthesis and cell number, whereas UDP only caused a small increase in protein synthesis. Intriguingly, ATP was much more potent and efficacious than UTP, ITP, and UDP in increasing DNA synthesis and expression of PCNA, a protein marker of cell proliferation, suggesting that another receptor may contribute to the proliferative response [54]. In vivo experiments have shown that intimal thickening of collared rabbit carotid arteries was greatly enhanced by in situ UTP application and was closely associated with osteopontin expression in medial SMC [6]. Osteopontin is chemotactic for SMC and is associated with arterial smooth muscle cell proliferation [55]. Moreover, both UTP and ATP increased osteopontin expression in cultured SMC, whereas ADP, UDP, and 2-MeSATP were ineffective, which suggests a role for the P2Y2R in which ATP and UTP are equipotent. Direct evidence for involvement of the P2Y2R was provided by inhibition of UTP-induced osteopontin expression in cultured SMC by P2Y2 antisense oligonucleotides [6]. P2Y6 receptors also have been shown to mediate proliferation of SMC in rat aorta [56].\nRole of P2 receptors in the migration of vascular SMC\nRecent studies indicate that the extracellular nucleotides ATP, ADP, UTP, and UDP serve as directional cues for the migration of rat aortic SMC [57]. At identical concentrations, the most powerful migratory response induced by these nucleotides was elicited by UTP. Nucleotide-induced migration of SMC is the consequence of both chemotaxis and chemokinesis and may result either from the activation of one particular P2 nucleotide receptor subtype or from activation of several P2 receptor subtypes. The ability of UTP at submicromolar levels to stimulate migration of SMC supports the hypothesis that this response could have physiological consequences and is essentially mediated by P2Y2 receptor activation without excluding participation of other P2Y receptor subtypes. The difference in the capacity of UTP and ATP to elicit migration of SMC could be due to the inhibition of nucleotide-induced cell migration by adenosine generated from ATP catabolism by cell surface ectonucleotidases. Indeed, ATP and UTP were equally effective in causing migration of SMC when ATP was prevented from degradation by addition of the ectonucleotidase inhibitor \u03b1,\u03b2-methylene-ATP [57]. Several P2Y receptor subtypes could be involved in nucleotide-induced migration of SMC. It has been shown in rat aortic SMC that the P2Y2 receptor is the predominant P2Y receptor subtype [28, 58], whereas lower levels of P2Y4 and P2Y1 receptor mRNA were detected [58]. The very low level of P2Y1 receptor mRNA expression was consistent with the absence of ADP-induced migration of cultured rat aortic SMC, demonstrating that P2Y1 is not involved in this process [58]. In addition, the same study showed that a commercially available solution of hexokinase-treated UDP (UTP-free) induced cell migration equally well as untreated UDP, thereby demonstrating that UDP is chemotactic for aortic SMC by activation of the P2Y6 receptor. Conversely, migration of rat aortic SMC induced by UTP occurred even when UTP degradation by nucleoside diphosphate kinase was inhibited, demonstrating the involvement of P2Y2 and\/or P2Y4 receptor(s) [58].\nThe increased migration of SMC in response to extracellular nucleotides could be related to increases in extracellular matrix (ECM) protein expression. Indeed, previous studies have shown that UTP induces osteopontin expression in rat and rabbit aortic SMC [51, 6]. Increased expression of osteopontin, an RGD-containing ECM protein, is associated with the activation of rat arterial SMC in vitro and in vivo [57]. The increase in osteopontin expression plays a key role in UTP-induced migration of rat aortic SMC, since a monoclonal antibody against osteopontin fully abolished UTP-induced migration [57], whereas an antibody against vitronectin, another ECM protein also involved in migration of human SMC [59], had no effect on the migration of rat aortic SMC [57]. UTP induces increases in osteopontin mRNA expression by increasing both osteopontin mRNA stabilization and osteopontin promoter activity [60]. Recent studies have shown that activation of an AP-1 binding site located 76 bp upstream of the transcription start in the rat osteopontin promoter is involved in UTP-induced osteopontin expression. Using a luciferase promoter deletion assay, Renault et al. [61] identified a new region of the rat osteopontin promoter (-1837 to -1757) that is responsive to UTP. This region contains an NF\u03baB site located at -1800 and an Ebox located at -1768. Supershift electrophoretic mobility shift and chromatin immunoprecipitation assays identified NF\u03baB and USF-1\/USF-2 as DNA binding proteins induced by UTP. Using dominant negative mutants of I\u03baB kinase and USF transcription factors, it was confirmed that NF\u03baB and USF-1\/USF-2 are involved in the UTP-induced expression of osteopontin.\nThis ability of nucleotides to act as chemoattractants for rat arterial SMC in a concentration range potentially found in pathological vessels [62] and the findings of previous studies demonstrating the mitogenic activity of extracellular nucleotides for these cells suggest that nucleotides released from mechanically stretched vascular or damaged cells during the angioplasty process may participate in arterial wall remodeling.\nRole of P2 receptors in nucleotide-induced vascular inflammation\nIn addition to their mitogenic effects, extracellular nucleotides may also cause cell recruitment by inducing lymphocyte and macrophage adhesion to human pulmonary artery endothelial cells as demonstrated in vitro [63]. Nucleotides can also modulate rat aortic smooth muscle cell adhesion and migration by increasing the expression of osteopontin [51, 57], a protein involved in both processes. Moreover, extracellular nucleotides may play a role in intra-arterial attraction of monocytes by inducing an increased expression of monocyte-chemoattractant protein-1 by arterial SMC [21]. Stimulation of P2 receptors is coupled to the release of the proinflammatory cytokines interleukin (IL)-1\u03b2, IL-1\u03b1, IL-8, and tumor necrosis factor (TNF)-\u03b1 (see [4] for review) that are of obvious relevance to inflammation in atherosclerosis. Activation of P2X7 receptors on monocytes\/macrophages enhances release of proinflammatory cytokines that modulate NO production and expression of inducible nitric oxide synthase (iNOS) (see [64] for review), mediators of immune cell activation that is an early step in atherosclerotic lesion development.\nMonocyte recruitment into the vessel wall is a complex process that includes cell rolling, firm attachment, and directed migration. It is now becoming evident that adhesion molecules such as VCAM-1 play an important role in leukocyte adherence to vascular endothelial cells [65, 66]. VCAM-1 expression is induced or upregulated by proinflammatory cytokines such as TNF-\u03b1 and IL-1\u03b2 in cellular components of the arterial wall including endothelial cells, smooth muscle cells, and fibroblasts [67-69]. ATP and UTP have been shown to induce cell-cell adhesion in a human monocyte\/macrophage lineage and neutrophil adherence to human endothelial cell monolayers [63, 70].\nRecent studies have shown that local UTP delivery via an osmotic pump to collared rabbit carotid arteries induced intimal accumulation of macrophages, similar to oxidized low-density lipoprotein (LDL), a response that was mediated by activation of P2Y2 receptors [6]. Leukocyte migration depends on the activities of adhesion proteins (e.g., selectins and integrins) on leukocytes and vascular endothelial cells. We have demonstrated that activation of P2Y2 receptors in endothelial cells causes the expression of VCAM-1 that mediates the adherence of monocytes to vascular endothelium [71], leading to their penetration into the vessel wall to promote arterial inflammation associated with atherosclerosis.\nRecent studies have revealed that a Src homology-3 (SH3) binding domain in the C-terminal tail of the P2Y2 receptor promotes the nucleotide-induced association of Src with the P2Y2 receptor, leading to the transactivation of growth factor receptors, such as the epithelial growth factor (EGF) and vascular endothelial growth factor (VEGF) receptors, and nucleotide-induced upregulation of VCAM-1 [72, 73]. Since leukocyte infiltration and migration are key processes involved in atherosclerosis, these findings suggest that P2Y2 receptors represent a novel target for reducing arterial inflammation associated with cardiovascular disease.\nP2 receptors in vascular apoptosis\nApoptosis (programmed cell death) has been reported to occur in various vascular diseases such as atherosclerosis, restenosis, and hypertension [74, 75]. The major cell types undergoing apoptosis in human atherosclerotic lesions are arterial SMC [11, 76\u201378] and macrophages [79]. In restenosis following balloon angioplasty, there is a peak in the proliferation and apoptosis of rat vascular SMC 14 days postangioplasty [76]. Furthermore, apoptosis of arterial SMC has been described in animal models of intimal thickenings [80] and probably takes part in the normal process involved in the control of hyperplasia. In contrast, apoptosis of SMC in advanced human atherosclerotic plaques may destabilize the fibrous lesion to promote plaque rupture and its clinical consequences.\nAs a mediator of cell-to-cell communication, ATP can trigger a variety of biological responses after being rapidly released in large amounts from various sources, including activated platelets, endothelial cells, nerve terminals, antigen-stimulated T cells, and other cell types following hypoxia, stress, and tissue damage. For example, in human umbilical cord vein endothelial cells (HUVEC), a substantial release of ATP (and UTP) is induced by shear stress [81], which may lead to alterations in the balance between proliferation and apoptosis regulated by P1 adenosine and P2 (particularly P2X7) nucleotide receptors [82]. P2X7 and P1 receptors have been previously linked to apoptosis in other cell types, including immune cells, astrocytes, and thymocytes [83\u201385]. The P2X7R also has been shown to mediate ATP-induced cell death in human embryonic kidney cells [86], human cervical epithelial cells [87], and primary rat cortical neurons [88]. In human arterial SMC, adenosine-induced apoptosis is essentially mediated via the A2b-adenosine receptor subtype and involves a cAMP-dependent pathway [89].\nAs an important constituent of atherosclerotic plaques, fibroblasts share several features with smooth muscle cells. In human fibroblasts, P2X7 was identified as the main nucleotide receptor involved in the high glucose concentration-dependent responses modulated by ATP, including morphological changes, enhanced apoptosis, caspase-3 activation, and IL-6 release [90]. In the immune system, ATP also plays important roles through nucleotide receptors in leukocyte functions. P2X7 receptor-mediated apoptosis has been demonstrated in various types of leukocytes, including a lymphocytic cell line, murine thymocytes, murine peritoneal macrophages, human macrophages, mesangial cells, dendritic cells, and microglial cells [83, 91\u201396]. Extracellular ATP acting via the P2X7 receptor activates the transcription factor NF\u03baB by selectively targeting NF\u03baB p65 (Rel A) in the N9 mouse microglial cell line [97]. It also has been reported that the P2X7 receptor modulates macrophage production of TNF-\u03b1, IL-1\u03b2, and nitric oxide (NO) following lipopolysaccharide (LPS) exposure [98], consistent with a role for the P2X7 receptor in inflammation. In HUVEC, TNF-\u03b1 markedly increases apoptotic cell death via the activation of caspase-3 [74]. Recent reports indicate that ATP\/ADP activate NF\u03baB and induce apoptosis probably through P2X7 receptors in porcine aortic endothelial cells [99]. These studies have provided compelling evidence suggesting a role for P2 and P1 receptor-mediated apoptosis in vascular diseases; however, further studies are needed to determine the precise pathways involved and to accumulate direct evidence that these pathways contribute significantly to the development of atherosclerosis, hypertension, and restenosis.\nModulation of P2 receptors in vascular injury\nExperimental arterial intimal hyperplasia can be induced by balloon angioplasty or by the perivascular placement of a silicone collar around an artery. An influx of leukocytes precedes the migration and proliferation of vascular SMC into the intima in both these models [100]. In normal adult rat aorta, P2Y2 mRNA was found in the endothelial cell lining, while a sustained expression of P2Y2 mRNA was detected in few medial SMC [20]. In contrast, P2Y2 mRNA was detected in all medial SMC of rat fetal aortas and in most of the aortic SMC of intimal lesions after balloon angioplasty, with an overexpression in cells lining the lumen both 1 and 3 weeks after injury.\nIn the collar model, neointimal formation appears to be triphasic [100]. The first phase is characterized by vascular infiltration of leukocytes beginning 2 h after collar placement around a rabbit carotid artery. The second phase begins within 12 h of collar placement and is characterized by medial replication of SMC. The third phase is characterized by the appearance, beginning at day 3 after collar placement, of subendothelial SMC. In situ hybridization with sham-operated rabbit carotid arteries indicated that P2Y2 mRNA expression was localized to CD31-positive aortic endothelial cells and not medial SMC [6]. High levels of P2Y2 mRNA were detected in medial SMC 3 days after collar placement, before appearance of neointima. At day 14, all intimal and medial SMC were P2Y2 positive. Fura-2 digital imaging of single SMC, used to measure changes in myoplasmic calcium concentration in response to P2Y receptor agonists, confirmed an increase in P2Y2 receptor activity. However, the same study showed that P2Y4 mRNA was equivalently expressed in sham-operated and collared arteries or cultured rabbit carotid SMC, whereas P2Y6 mRNA was not detected in carotid arteries or cultured SMC. In a more recent study, it was shown that P2Y2 receptor upregulation occurs in stented porcine coronary artery, a clinically relevant model of arterial injury [54]. P2Y2 receptor mRNA levels were significantly increased in coronary SMC dispersed from stented segments of coronary arteries 3 weeks after stent angioplasty compared with SMC from unstented segments. There was no significant difference observed in levels of P2Y6 mRNA in the stented and unstented artery segments, whereas P2Y4 receptor mRNA was undetectable.\nUpregulation of functional P2Y receptors also occurs in the basilar artery of the rat double-hemorrhage model [7], in the coronary artery of diabetic dyslipidemic pigs [8], and in human atherosclerotic lesions (Seye and Desgranges, unpublished data). It has been proposed that upregulation of P2Y receptors could be a potential diagnostic indicator for the early stages of atherosclerosis [9]. Interestingly, a more recent study showed that high shear stress, associated with vascular diseases, can selectively upregulate P2Y2 and P2Y6 receptors in perfused arterial SMC [101]. P2X1 and P2X4 receptors have been shown to be upregulated in rabbit intimal thickenings [102]. Taken together, these findings strongly suggest that at least some P2 receptor subtypes (most notably P2Y2) are implicated in the development of vascular disease.\nPathophysiological significance of P2 receptor modulation in vascular injury\nMigrating and proliferating SMC in the arterial media, together with infiltrating macrophages and T lymphocytes, are the main cell types that comprise atherosclerotic and restenotic lesions [1]. In the early stages of intimal hyperplasia, SMC are modified from a differentiated, contractile phenotype to an immature, synthetic phenotype, and this enables them to migrate into the intima, to proliferate, and to secrete extracellular matrix components. In many respects, this shift in phenotype is a reversal of the normal differentiation pattern of vascular SMC during fetal and early postnatal life [103, 104]. However, there is still a lack of knowledge concerning the phenotypic regulation of SMC during vasculogenesis and vascular disease. A high level of P2 receptor expression could be related to the altered phenotype of SMC in intimal thickenings. Partially dedifferentiated SMC are found in rat arterial intimal lesions after balloon angioplasty [105, 106], and their phenotype has been compared with that of newborn rat aortic SMC [107]. Interestingly, it has been reported that medial SMC of rat embryonic aorta also exhibit high P2Y2 expression, similar to intimal thickenings [20]. Other studies have shown that P2Y1 and P2Y2 receptor transcripts are strongly upregulated with phenotypic changes in rat SMC, whereas P2X1 mRNA is completely downregulated, and P2Y4 and P2Y6 mRNA levels are unchanged [58, 28]. Taken together, these and other results suggest that P2Y2 receptor expression is upregulated in the entire cell population of intimal thickenings and is closely associated with a poorly differentiated phenotype of SMC. The dramatic increase in P2Y2 mRNA expression observed in balloon angioplasty-induced intimal lesions would suggest increased activity of extracellular nucleotides with consequent enhancement of cell proliferation and vasoreactivity. Indeed, extracellular nucleotides, particularly ATP and UTP, have been shown to induce cell cycle progression and proliferation of cultured arterial SMC [21, 49, 50, 51] and vasoconstriction in the absence of endothelial cells [17, 108, 109]. Since both neointimal hyperplasia and vasoconstrictive remodeling have been found to be involved in postangioplasty restenosis [1, 5, 107, 110], these findings suggest that extracellular nucleotides may play a significant role in this process, at least as long as functional endothelial cells, which regulate intimal thickening [111, 112] and nucleotide-induced vasorelaxation [113, 114], are not regenerated.\nIncreased P2Y2 receptor expression in the neointima may by itself be sufficient to enhance the local effects of extracellular nucleotides on the proliferation of SMC. Although the expression of other P2 receptors has been described in arterial SMC [21, 29, 115], the P2Y2 receptor seems to be more specifically involved in the response of SMC to ATP and UTP [116], particularly in the potentiation of proliferation [21, 50]. The effects of extracellular nucleotides are not only dependent on the nature and number of P2 receptors present on target cells, but also on the local concentrations of nucleotide agonists. Although in vivo concentrations of extracellular nucleotides are difficult to measure, various in vitro experiments suggest that extracellular nucleotides are released from blood and vascular cells exposed to various physicochemical conditions, e.g., stress, hypoxia, and other factors [117, 118, 119] associated with the angioplasty process.\nP2Y2 receptors in SMC are involved in nucleotide-induced constriction of normal arteries [17, 108, 109]. Long lasting alterations in vasomotricity after endothelial cell denudation, resulting in increased sensitivity to vasoconstrictive substances, have previously been demonstrated [113, 114]. It appears that like other receptors for vasoconstrictive factors such as angiotensin II [120], endothelin [121], and platelet-derived growth factor (PDGF) [122], which are overexpressed in neointima, P2Y2 receptors may play an important role in controlling the vasoactive properties of pathological arteries, particularly in chronic constriction at the lesion site that is postulated to be one of the processes leading to postangioplasty restenosis [5, 110].\nConclusion\nP2 receptor subtypes, including P2Y2, P2X1, and P2X4, appear to play a role in responses to endothelial injury that are thought to be key events in the initiation of atherosclerosis and restenosis after angioplasty. P2Y2R upregulation and activation in endothelial cells and SMC promote leukocyte transmigration and intimal thickening in arteries of animal models of vascular injury, suggesting a possible regulatory role for this receptor in the mechanisms leading to neointimal hyperplasia after angioplasty. Although there are no selective P2Y2 receptor antagonists yet available, recent progress in siRNA technology has made it possible to design small RNA interference molecules that can selectively inhibit P2 receptor subtype expression. Such molecules can be efficiently delivered into the vessel wall using adenoviral vectors. In addition, P2 receptor transgenic mice (i.e., mice in which the relevant receptor subtype has been deleted or overexpressed) will be valuable tools to substantiate the role that nucleotides and P2 receptors play in the etiology of cardiovascular disease. Further delineation of the signaling pathways involved in these P2 receptor-mediated processes may help limit or prevent vascular diseases such as atherosclerosis and restenosis after angioplasty.","keyphrases":["atherosclerosis","restenosis","inflammation","proliferation","migration","smooth muscle cell","nucleotide receptors"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"J_Gastrointest_Surg-3-1-1852385","title":"Controversies in the Surgical Management of Sigmoid Diverticulitis\n","text":"The timing and appropriateness of surgical treatment of sigmoid diverticular disease remain a topic of controversy. We have reviewed the current literature on this topic, focusing on issues related to the indications and types of surgery. Current evidence would suggest that elective surgery for diverticulitis can be avoided in patients with uncomplicated disease, regardless of the number of recurrent episodes. Furthermore, the need for elective surgey should not be influenced by the age of the patient. Operation should be undertaken in patients with severe attacks, as determined by their clinical and radiological evaluation.\nMagnitude of the Problem\nDiverticular disease, either diverticulosis or diverticulitis, was regarded as a surgical curiosity in the 19th surgery, but over the past 100\u00a0years, its prevalence in Western countries has increased dramatically. In the US, an individual\u2019s risk for developing diverticular disease approaches 50% by age 60.1 Diverticulitis, defined as inflammation and infection related to diverticula, occurs in 20 to 30% of patients with diverticulosis and is one of the most common indications for gastrointestinal tract-related hospitalizations. One in four of these patients presenting with diverticulitis will require an emergency operation because of perforation, peritonitis, or systemic complications. At present, diverticulitis is the associated diagnosis for one third of all colostomies and\/or colon resections.2 As such, diverticulitis is one of the five most costly gastrointestinal disorders affecting the US population.3\nEtiology\nColonic diverticula tend to develop in the areas of weakness in the colonic wall, most frequently at the sites of penetration of the wall by blood vessels.4 These outpouchings of mucosa and peritoneum are of the pulsion type and are thought to be caused by an increase in the intraluminal pressure within the colonic wall in affected individuals (Fig.\u00a01).\nFigure\u00a01Endoscopic images of diverticuli. Colonoscopy can be rather difficult when several diverticula are encountered because of increased colonic tortuosity and lack of distensibility.\nIt is thought that a low intake of dietary fiber and resultant decrease in stool bulk predisposes those in Western societies to an elevation in colonic pressure. Some authors attribute the high rate of diverticular disease to the development of the roller mills during the last half of the 19th century, causing the grains to be crushed so effectively as to nearly eliminate all of the cellulose from the Western diet.5 Despite significant supporting evidence for fiber and its role in the development of diverticulosis, no study to date has demonstrated that a high fiber diet can reverse this process or reduce the incidence of complications in cases of established diverticulosis.6\nIn addition to dietary intake, other factors have been implicated in the development of diverticular disease. Most studies report that diverticular disease is more common in the elderly, especially elderly women, and in patients who smoke cigarettes or drink alcohol.7 Sigmoid colon specimens from patients with diverticulosis have been found to have increased in vitro sensitivity to acetylcholine, as well as reduced smooth muscle choline acetyltransferase activity and upregulation of smooth muscle muscarinic M3 receptors.8 The significance of these biochemical characteristics still needs to be elucidated, but the differences suggest that there are underlying physiological abnormalities that may predispose to the development and progression of diverticular disease.\nClinical Presentation and Evaluation\nThe clinical presentations of diverticular disease range from asymptomatic diverticulosis, diverticulosis with periodic spasmodic abdominal pain and bloating, diverticulosis with hemorrhage, and finally, diverticulitis. Although diverticula can occur in any portion of the colon, this review will only focus on sigmoid diverticulitis, by far, the most common site for this disease process.\nMost patients with diverticulitis present with symptoms of left lower quadrant abdominal pain, fever, and leukocytosis (Table\u00a01). Additional symptoms of acute sigmoid diverticulitis may include nausea, vomiting, change in bowel habits, urinary frequency, and\/or dysuria.1 In cases of clear-cut diverticulitis based upon the clinical picture, one can manage the patient without any imaging studies. In many cases, especially in those with severe symptoms and potential complicated diverticulitis, computed tomography (CT) scanning should probably be performed. The value of CT scanning is the ability to confirm the diagnosis and confidently stratify the severity of the disease process, differentiating mild, localized inflammation from advanced inflammation with abscess formation and\/or distant extension. \nTable\u00a01Clinical Symptoms of DiverticulitisSymptomsFrequency (%)Left lower quadrant pain93\u2013100Leucocytosis69\u201383Fever57\u2013100Nausea10\u201330Vomiting15\u201325Constipation10\u201330Diarrhea5\u201315Dysuria5\u201320Urinary frequency6\u201325\nBefore the advent of CT, the contrast enema was the primary tool in the evaluation of colonic diverticular disease. However, CT scans have largely replaced barium enema as the preferred imaging modality to evaluate patients with suspected diverticulitis. The use of CT scanning has been justified by several studies from the radiological literature, demonstrating a high sensitivity (97%) and specificity (100%) for diverticulitis (Fig.\u00a02). Contrast enema, on the other hand, has a sensitivity of only 82% and a specificity of 81% for diverticulitis.9Figure\u00a02a Computed tomography scan images of a patient who presented with uncomplicated diverticulitis that was subsequently treated successfully with antibiotics. Note the thickening of the sigmoid colon, yet the lack of any extraluminal fluid or air. b Computed tomography scan images of a patient who presented with complicated diverticulitis and an extraluminal fluid collection that did not resolve with attempted CT-guided drainage an required an eventual sigmoid colectomy.\nClassification\nThere are two commonly utilized classifications of diverticulitis. The European Association for Endoscopic Surgeons developed a classification scheme based upon the severity of its clinical presentation.10 In this system, diverticulitis is divided into symptomatic uncomplicated disease, recurrent symptomatic disease, and complicated disease (Table\u00a02). \nTable\u00a02Clinical Classification of Diverticulitis (Adapted from Kohler et al. 10)GradeClinical DescriptionSymptomsISymptomatic uncomplicated diseaseFever, crampy abdominal pain, CT evidence of diverticulitisIIRecurrent symptomatic diseaseRecurrence of aboveIIIComplicated diseaseHemorrhageAbscessPhlegmonPerforationPurulent and fecal peritonitisStrictureFistulaObstruction\nAnother classification system was developed by Hinchey et al.11 and is used to describe the stages of complicated diverticulitis (Table\u00a03). This scheme allows for good communication among surgeons when it comes to describing the various degrees of diverticular perforation, ranging from a localized perforation with a small abscess to generalized fecal peritonitis. Clearly, the proper surgical approach will vary depending upon the Hinchey stage. \nTable\u00a03Hinchey Classification of Complicated Diverticulitis (Adapted from Hinchey et al.11)StageDescriptionIPericolic or mesenteric abscessIIWalled off pelvic abscessIIIGeneralized purulent peritonitisIVGeneralized fecal peritonitis\nWe will refer to both of these classifications when discussing the appropriate management of this disease. Yet another classification, developed by Ambrosetti et al.12, is based upon the CT findings. The criteria of Ambrosetti et al. are being increasingly utilized to stratify patients into optimal pathways for management (Table\u00a04). Thus, patients with mild disease are likely to be successfully managed with intravenous antibiotics, whereas percutaneous drainage and surgery is generally indicated for cases of complicated diverticulitis.\nTable\u00a04Ambrosetti\u2019s CT Staging of Diverticulitis (Adapted from Ambrosetti et al.12)Mild DiverticulitisSevere DiverticulitisLocalized sigmoid wall thickening (<5\u00a0mm)AbscessInflammation of pericolic fatExtraluminal airExtraluminal contrast\nManagement of Complicated Diverticulitis\nSurgical intervention is rarely indicated in cases of acute diverticulitis because most of these cases will resolve with appropriate antibiotic management. Operations are reserved for cases of complicated diverticulitis, i.e., patients with perforation and peritonitis, abscess formation, fistula, or obstruction. Although this may seem clear-cut, decisions regarding if and when to operate patients with diverticulitis remain a topic of significant debate.\nOperation is clearly indicated when the patient presents with perforation and diffuse peritonitis, whether it is purulent or feculent (Hinchey stages III and IV). However, the ideal surgical procedure in such cases of perforation remains a matter of debate. The possible operations advocated range from a simple washout of the abdomen with drainage, as described in a few case reports from Scotland and France, to primary resection with a Hartmann pouch, primary resection with anastomosis, diverting ileostomy, and finally, a primary resection with anastomosis and no temporary stoma. Of these,1,13,14 American surgeons are most likely to perform the Hartmann procedure, which has been advocated as the standard of care for perforated diverticulitis.1 The Hartmann\u2019s resection has proven to be a safe and effective approach, and is based upon the idea that an anastomosis in the setting of acute infection\/inflammation is dangerous and associated with a high rate of suture line breakdown.\nA simple washout without resection would not be considered an appropriate approach because ongoing infection\/inflammation of the involved bowel is likely to occur. There is a paucity of data to support a minimalist, simple washout approach; there are only 18 case reports in the literature describing the technique and its results.13,14 On the other hand, the practice of routine stomas in operations for acute diverticulitis may not be justified. Belmonte et al.15 looked at 277 consecutive patients treated for acute diverticular disease at the University of Minnesota, both urgently and electively. Of these, 88% had a primary anastomosis, most of them without diversion. They found that primary anastomosis was quite safe, with an overall 4% leak rate. Interestingly, none of these leaks were in their subset of patients with Hinchey stage IV diverticulitis, a group that comprised 9% of their total study population.\nA systematic literature review of 50 studies comparing a Hartmann\u2019s procedure to a primary resection with anastomosis for perforated diverticulitis found 569 reported cases of primary anastomoses. The reported mortality and morbidity in the patients with an anastomosis was the same as in the patients who underwent the Hartmann\u2019s procedure.16 These data suggest that in a select group of patients undergoing surgery in the acute stage of diverticulitis, an anastomosis is probably safe, even in the milieu of feculent peritonitis.\nThese data are intriguing, but must be viewed with caution, especially in the case of a very sick or toxic patient with multiorgan system failure and\/or shock. In the absence of randomized controlled studies, we still recommend the Hartmann\u2019s procedure in patients with significant purulent or feculent peritonitis, and those patients with any instability related to the systemic effects of sepsis. However, in a patient who is clinically stable, a primary anastomosis at the first operation can be performed even in the setting of perforation (Fig.\u00a03).\nFigure\u00a03Gross specimen of the sigmoid colon that was resected from a patient who presented with freely perforated diverticulitis (Hinchey III). Proximal margin extends to the area where the diverticuli end, and the distal margin is at the rectum.\nMention should be made of the meticulous surgical technique that must be used in this situation. The splenic flexure of the colon may need to be mobilized to ensure a tension-free anastomosis. One should imagine the rectum collapsing back into the pelvis with the patient standing upright when deciding on whether the bowel ends are truly free of tension. The margins of resection must be clearly viable with regard to vascularity. Finally, it may be best to avoid the crossed staple lines inherent to the double-stapled technique. Either a double pursestring technique with a stapled end-to-end anastomosis or a standard handsewn anastomosis are preferred when operating in an inflamed milieu.\nPreventive Surgery\nThe question of when to recommend elective, preventive surgery for patients with diverticulitis remains very controversial. Current American Society of Colon and Rectal Surgeons (ASCRS) guidelines suggest preemptive surgery for any patient who has had two attacks of acute diverticulitis, with the intention of preventing another attack that could present with perforation and would necessitate a stoma.1 This recommendation for surgery after the second episode of diverticulitis is based on the data published in 1969 by Parks17 showing that the mortality rate for each subsequent attack of diverticulitis increases from 4.7% during the first admission to 7.8% during each subsequent admission. Parks is also widely quoted for stating that each subsequent episode of diverticulitis is less likely to respond to medical therapy, with a 70% response after the first episode vs 6% response after the third episode.1 However, there are little data to support this concept of poor response to medical treatment in subsequent attacks of diverticulitis. Furthermore, the advent of CT scanning and better antibiotics has improved nonoperative management of these patients. In a modern series of 673 patients with diverticular disease, only 3% of patients required emergency operations during a follow up of 10\u00a0years.18 Another 10-year study of 366 patients showed that recurrence was not associated with an increased rate of either complications or less successful medical management.19\nLooking at the issue from another angle, Somasekar et al.20 reviewed 108 patients admitted with complicated diverticulitis. Almost all of them (104) required emergency surgery. Interestingly, only 26% of these patients were previously diagnosed with diverticular disease and only three patients had been admitted in the past with a prior episode of acute diverticulitis. In other words, only 2.7% of patients in this group would have benefited from an elective resection. Complications would still have occurred in 92.6% of patients in whom these attacks happened de novo.\nThus, it appears that elective resection might have little impact on the incidence of patients requiring emergency procedures because most of these occur with the first attack of diverticulitis. Subsequent attacks of diverticulitis in the same patient seem to be akin to their previous ones, suggesting that specific patients are predisposed to a set pattern of diverticulitis, and once settled into this pattern they stay within it. The threat of the colostomy bag to a patient who has been successfully managed medically during two previous attacks may be unwarranted and misleading.\nIn addition, it is important to recognize that elective surgery for diverticulitis is not without complications. Bookey et al.21 demonstrated that elective diverticular disease resection is associated with higher rates of morbidity and mortality than elective colorectal carcinoma resection, with the mortality rate increasing from 0 to 15% with advancing age. Furthermore, colectomy is not a guaranteed cure for diverticulitis, with recurrence rates varying from 3 to 13%. These rates have improved, however, with the recognition that the chances of recurrence are fourfold higher if a colosigmoid anastomosis is performed, emphasizing the importance of resecting the entire sigmoid colon in an operation for diverticulitis.22\nWith these conflicting data in mind, we maintain that the patients with uncomplicated diverticulitis can be managed nonoperatively regardless of the number of recurrent episodes. Patients who develop complications, such as fistulas, obstruction, or nonresolving smoldering disease, are best managed with surgical resection. Elective surgery may also be offered to patients who have had two or more episodes of severe diverticulitis, as determined by their clinical presentation and CT grade. In addition, elective surgery may be justified in patients with limited access to medical care or in those who are concerned about the negative impact of repeated illnesses with regard to work productivity and\/or psychosocial issues.\nIn elective or semielective circumstances, both open and laparoscopic sigmoid resection with a primary anastomosis have been considered as acceptable methods of treatment.23 Laparoscopy has been shown to be associated with an approximate 10% rate of conversion to open surgery. Interestingly, no direct relationship has been found between the number of attacks of diverticulitis or the timing after an acute attack with regard to complications or conversion rates with laparoscopic colectomy.24\nDiverticulitis in Young Men\nMany authors believe that diverticulitis is a more virulent disease in younger patients. As such, it has been argued that all patients younger than 50 should undergo elective colon resection after an initial attack of diverticulitis.1,25 This argument arose from studies in the pre-CT era, which were replete with data, indicating a high risk of surgical intervention in young patients eventually diagnosed with diverticulitis. Subsequent authors have argued that these earlier studies were flawed because of a significant rate of unnecessary laparotomies in the younger patients because of erroneous preoperative diagnoses of appendicitis.26 Vignati et al.27 were among the first to challenge the concept that diverticulitis in the young is a more virulent disease. These authors surveyed 40 patients under the age of 50 that where treated with intravenous antibiotics and bowel rest and found that at a 5- to 9-year follow-up, none of these patients required colostomies. One third of them did undergo surgery, but most of these procedures where either elective or, if urgent, still conducive to a successful primary anastomosis.\nGuzzo et al.26 performed a retrospective review of 762 patients with diverticulitis treated at their institution from 1990\u20132000 and found that 76% of the patients under age 50 improved with antibiotics and did not require surgery during their first attack. These rates did not differ from the rates of surgery in the elderly patients. Of the patients treated nonoperatively, only four patients had a recurrence requiring surgery at a later time and only one needed a colostomy. Thirty-eight additional patients underwent preemptive elective surgery based upon their surgeon\u2019s recommendation. One hundred fifty-five patients, 60% of the entire group, did not require surgery at all.26\nA prospective study from Switzerland followed 118 patients who had their first attack of diverticulitis and found that recurrence rates in the younger patients were similar to those seen in the older patients, once stratified by their CT severity.28\nBased upon these studies, we believe that young patients should generally be treated using the same criteria as older patients, and that the there is no justification for the routine recommendation for surgery after a single attack of diverticulitis in young patients. Elective preemptive surgery should be reserved for those who had at least two episodes of severe diverticulitis, and this decision should be supported by CT scan documentation of prior complicated disease.\nFistulas\nAs we succeed with the nonoperative treatment of acute diverticulitis, the incidence of fistulas appears to be increasing, reported to occur in approximately 12% of patients.29 Colovesical fistulas account for two thirds of the cases, followed by colovaginal, colocutaneous, and enterocolic cases.30 These patients can present a significant challenge to the surgeon. Some fistulas will close spontaneously as the inflammatory process resolves. Therefore, a selective approach should be used, in which operation is offered to those patients with persistent symptoms after 5\u20136\u00a0months after an acute attack. The most commonly reported symptoms in this group of patients include abdominal pain (43%), pneumaturia (43%), cystitis (40%), fecaluria (38%), diarrhea (15%), and hematuria (5%).31\nIn the operating room, the surgeon should expect a significant desmoplastic reaction and a contained abscess cavity in the area of fistulization. It may be prudent to place ureteral stents before the procedure, although most fistulas to the bladder will be at the dome and away from the trigone region, allowing relatively safe access for identification, dissection, and closure. Most of these cases should be amenable to resection with primary anastomosis, avoiding the need for a temporary stoma.32\nIn expert hands, a colectomy can be accomplished by either an open or laparoscopic approach.33 Some authors suggest that these procedures are best performed by surgeons whose main interest focuses on colon and rectal surgery. A study from McGill University comparing outcomes of surgery for diverticulitis-induced fistulas found that colorectal surgeons performed less diverting Hartmann\u2019s and colostomies (5 vs 27%), and had a lower rate of complications, including wound infections and anastomotic leaks.31 It is not clear, however, whether the data from this small study of 121 patients are applicable to all surgeons in all centers.\nDiverticulitis in the Immunocompromised Patient\nDiverticulitis in immunocompromised patients can be virulent because there is an increased likelihood of free perforation and fecal peritonitis. In addition, the clinical presentation of these patients often underestimates the severity of their disease.34 Marked differences have also been noted in the response of these patients to medical treatment. In the nonimmunocompromised group one should expect that 75% of patients will respond to antibiotics. In contrast, a very large percentage of immunocompromised patients will fail standard, nonoperative treatment.35 As such, most of these patients require urgent surgical intervention, and this is associated with a significantly higher mortality rate: 39 vs 2% in noncompromised patients.35 Given these data, most authors and the ASCRS recommend elective sigmoid resection after the first episode of diverticulitis in immunocompromised patients.1,34\u201336\nConclusion\nThe management of patients with sigmoid diverticulitis is still evolving. We should continue to constantly reassess the surgical dogma regarding the appropriate treatment of this common disease entity. Clearly, a randomized controlled study comparing the Hartmann\u2019s procedure to primary anastomosis in the setting of perforated diverticulitis would be worthwhile. It is becoming increasingly clear that mandatory operations may not be warranted in young patients or those with two episodes of diverticulitis. As in other areas of clinical surgery, we must tailor our treatment to the specific situation for each individual patient.","keyphrases":["surgical management","sigmoid diverticulitis","elective surgery","diagnosis"],"prmu":["P","P","P","P"]}
{"id":"Health_Care_Anal-4-1-2244696","title":"Social Patterning of Screening Uptake and the Impact of Facilitating Informed Choices: Psychological and Ethical Analyses\n","text":"Screening for unsuspected disease has both possible benefits and harms for those who participate. Historically the benefits of participation have been emphasized to maximize uptake reflecting a public health approach to policy; currently policy is moving towards an informed choice approach involving giving information about both benefits and harms of participation. However, no research has been conducted to evaluate the impact on health of an informed choice policy. Using psychological models, the first aim of this study was to describe an explanatory framework for variation in screening uptake and to apply this framework to assess the impact of informed choices in screening. The second aim was to evaluate ethically that impact. Data from a general population survey (n = 300) of beliefs and attitudes towards participation in diabetes screening indicated that greater orientation to the present is associated with greater social deprivation and lower expectation of participation in screening. The results inform an explanatory framework of social patterning of screening in which greater orientation to the present focuses attention on the disadvantages of screening, which tend to be immediate, thereby reducing participation. This framework suggests that an informed choice policy, by increasing the salience of possible harms of screening, might reduce uptake of screening more in those who are more deprived and orientated to the present. This possibility gives rise to an apparent dilemma where an ethical decision must be made between greater choice and avoiding health inequality. Philosophical perspectives on choice and inequality are used to point to some of the complexities in assessing whether there really is such a dilemma and if so how it should be resolved. The paper concludes with a discussion of the ethics of paternalism.\nIntroduction\nApproaches to Screening\nA major component of current public health strategy is the provision of screening programmes to allow prevention and early treatment of serious disease. A characteristic of screening is that it involves the possibility of immediate harm in return for the possibility of future benefit. For example breast screening with mammography has a number of substantial possible immediate harms [26, 38, 50]. In 2001 five percent of mammography screens in the US gave a false positive result leading to further testing [53]. Even when further tests result in a diagnosis of breast cancer, about 20 percent of the cases identified will be of ductal carcinoma in situ which has an uncertain course without treatment [50]. Many such cancers are not life threatening but, once detected, are usually treated as such [53]. Such treatments are unpleasant and damaging to health and women diagnosed with ductal carcinoma in situ have similar anxiety levels to those with early invasive breast cancer [39]. In contrast, only a small number of people, relative to the total screened, have their lives saved, even over a considerable period of time. Estimates suggest that 2451 women aged between 50 and 59 need to be screened as recommended over a five-year period to save one life [41].\nBecause only a few cases of disease will be detected within a healthy population, large numbers need to be screened in order to have an impact on overall population health. Thus high uptake of screening has been encouraged by emphasizing the benefits of participation, reflecting a public health approach to screening [27]. Recently, in the UK, there has been a policy change towards promoting informed choices particularly in screening:There is a responsibility to ensure that those who accept an invitation [to screening] do so on the basis of an informed choice, and appreciate that in accepting an invitation or participating in a programme to reduce their risk of a disease there is a risk of an adverse outcome [32].\nOne of the factors influencing the move to informed choices has been a concern about patient autonomy [16]. The issue of autonomy has become more salient with the rise of \u2018patient-centred medicine\u2019 in reaction to concerns with traditional medical practice and its emphasis on the role of the health professional in medical decision-making. [37]\nInformed Choice in Screening\nInformed choices have been defined as those based on relevant knowledge, consistent with the decision-maker\u2019s values and behaviourally implemented [28]. To make an informed choice participants need information about their personal risks of developing the condition, what having the screening test will be like, accuracy of the test, and what will happen if the screening test is positive [15]. While the place of this information in facilitating informed choices is obvious, the role of values may be less so. People will, however, attach different values to the possible outcomes of screening. For example those who are more and less socially deprived may place a different value on early diagnosis. The consequences of being diagnosed with a serious condition can have very different implications for those who have material resources and those who don\u2019t, even within a universal healthcare system. While those who have a high level of material resources can use those resources to ameliorate the impact of a diagnosis of serious illness, for those who are more socially deprived such a diagnosis may bring the prospect of increasing poverty and uncertainty and they might thus prefer to delay knowledge of an illness for as long as possible [24].\nThere has been no research to describe or evaluate the impact of an informed choice policy in screening. One possible impact is that it will reduce uptake of the screening programme with a greater reduction in some groups, for example those who are more socially deprived and already have lower participation in screening. Such a decline in the uptake of screening could be evaluated negatively as contributing to a decline in the overall health of the population or positively as reflecting an increase in the autonomy of the individual. This paper seeks to describe and evaluate the possible impact of an informed choice policy in screening.\nExplaining Screening Uptake\nUptake of screening is lowest in those who are most socially deprived. This is a consistent finding across different screening programmes and healthcare systems [20, 29, 48]. Lower uptake among the most socially deprived is, at least in part, a consequence of a lack of material resources, such as transport costs to the screening centre. But when the lack of such resources is controlled for in statistical analyses, differences in uptake remain [23]. Psychological characteristics, among other factors, contribute to explaining those differences [48, 52].\nOne such psychological characteristic might be time orientation. Psychologists suggest that people use information about the timeframe in which an event occurs to process information about the event and to make decisions. However, people\u2019s responses to the specific timeframe in which an event might occur, vary. Individuals have preferences for certain timeframes which influence their information processing and evaluation of actions and the possible outcomes of those actions. These preferences are called time orientation.\nWhile a number of different time orientations have been identified [55], evidence for their existence is strongest for two of these, future and present orientation. Those who have high future orientation think more about the future and have an awareness of the effects of current actions on future outcomes compared to those with lower future orientation [42]. Future orientation is associated with the practice of health-related behaviours, such as physical activity and healthy eating, that might involve immediate cost for possible future gain [22, 25, 34, 42]. Those who have high present orientation think more about immediate outcomes of their behaviour than those who are less present orientated [42]. High present orientation is also associated with a limited sense of control and fatalism about life events [42] and unhealthy behaviours, such as substance abuse, which result in immediate rather than future rewards [18, 42]. Future and present orientation are largely independent, of one another with each being associated with different patterns of thought and behaviour [18, 56].\nBecause the possible harms of screening are immediate while the possible benefits occur in the future, time orientation may contribute to explaining screening uptake. Increasing information about the possible harms and limited benefits of screening may therefore have a differential impact on those with different time orientations. Given that time orientation and social deprivation are associated [33, 47], this effect may vary by social deprivation.\nAims\nThe first aim of this study is to describe the possible impact of an informed choice policy on screening uptake by exploring the relationships between social deprivation, present orientation and expectations of participation in screening. The second aim is to evaluate that impact from different ethical perspectives.\nMethod\nDesign\nA questionnaire-based descriptive survey.\nSample\nA total of 300 participants was recruited. The sample was structured to reflect the English population in terms of age and sex with one third of the sample being drawn from each of the North, South and Midlands of England.\nProcedure\nHome-based interviews were conducted by a research agency. Questionnaires were completed by interviewers on behalf of participants.\nMaterials\nIn the first part of the interview information about diabetes screening, based on that used in previous research [34], was presented to participants (Appendix 1), after which the following measures were completed:Expectations of participation in screening. The mean response to three items measuring intention to participate in screening was assessed on five-point response scales [34, 35] giving a measure with good reliability (Cronbach\u2019s alpha: .85).Time orientation. A brief, nine item version of the Stanford Time Perspective Inventory (Crockett et\u00a0al. in submission). The Stanford Time Perspective Inventory (STPI) has been extensively validated and used in the study of health behaviour [9, 21, 42, 56]. This brief STPI consists of nine items, measured on five-point rating scales, comprising two subscales with adequate reliability: five items measuring future orientation (Cronbach\u2019s alpha: .67) and four items measuring present orientation (Cronbach\u2019s alpha: .62).Social deprivation: A brief three item measure asking participants to indicate:\nPossession of educational qualificationsHome ownership (including having a mortgage).\nThose who neither owned their homes nor had any educational qualifications were considered to have the greatest social deprivation (scored as 2); those who either owned their homes or had educational qualifications were considered to have intermediate levels of social deprivation (scored as 1); and, those who both owned their homes and had educational qualifications were considered to have the least social deprivation (scored as 0). This measure was derived from our previous research which indicated that individual level measures of social deprivation showed greater associations with psychological characteristics than did neighbourhood measures such as the Indices of Multiple Deprivation (Crockett et\u00a0al. in preparation).\nAnalysis\nStatistical analyses were conducted using SPSS version 12. Associations between social deprivation, future and present time orientation and expectations of participation in diabetes screening were examined using Spearman\u2019s rank correlations.\nResults\nThe associations between social deprivation, time orientation and expectations of participation in diabetes screening are shown in Table\u00a01.\nTable\u00a01Associations between expectations of participation in screening, social deprivation and present orientation (Spearman\u2019s rho correlation)Present orientationFuture orientationExpectations of diabetes screeningSocial deprivationa\u2212.245***.067.218***Present orientation\u2212.301***\u2212.265***Future orientation.054***\u00a0P\u00a0<\u00a00.001amost deprived scored as 0, intermediate group scored as 1, least deprived group scored as 2\nFuture orientation was not significantly associated with social deprivation (r\u00a0=\u00a0\u2212.067, n\u00a0=\u00a0300, P\u00a0=\u00a0.247) or with expectations of participation in screening (r\u00a0=\u00a0.054, n\u00a0=\u00a0300, P\u00a0=\u00a0.349). However present orientation was significantly associated with both social deprivation (r\u00a0=\u00a0.245, n\u00a0=\u00a0300, P\u00a0<\u00a00.001) and expectations of participation in screening (r\u00a0=\u00a0\u2212.265, n\u00a0=\u00a0300, P\u00a0<\u00a00.001).\nBecause only present orientation showed associations with social deprivation and expectations of participation in screening, further findings are presented for present orientation only. To illustrate the association between social deprivation, uptake of screening and present orientation, two figures were plotted. Figure\u00a01(a) shows the mean expectation of participation in diabetes screening at each level of deprivation. As social deprivation increases, expectations of participation decrease. A one-point difference between those most and those least deprived indicates a substantial effect of social deprivation on expectations of participation in screening. Figure\u00a01(b) shows the mean expectation of participation between those with high and low levels of present orientation, indicating that those with low present orientation express higher expectations of participating in screening. Figure\u00a01(b) does not indicate such large effects as seen in Fig.\u00a01(a) suggesting that other variables must contribute to the impact of social deprivation and expectations of participation in screening.\nFig.\u00a01Expectations of participation in screening and (a) social deprivation, (b) present orientation, High and low present orientation calculated by means of a mean split\nAdditional analyses using another part of the data set presented here indicate present orientation explains part of the association between social deprivation and uptake (Crockett et\u00a0al. in submission).\nSummary\nThe results of this study suggest that present orientation is associated with both social deprivation and with uptake of screening, such that present orientation partially accounts for the relationship between greater social deprivation and lower expectations of participation in screening. The results indicate that psychological factors can contribute to an explanatory framework of screening uptake. This framework suggests that there is an association between social deprivation and present orientation and that decisions about uptake of screening are influenced by the time orientation of those who are invited. This framework further suggests that making the more immediate possible harms of screening more salient, as would happen in an informed choice policy, could reduce uptake of screening in those who are more socially deprived. This framework can be used to identify and evaluate the possible impact of an informed choice policy in screening.\nEvaluation\nThe Possible Impact of Informed Choices on Inequality\nThe study results suggest that the implementation of an informed choice policy, which makes salient the possible immediate harms of participation, might lead to decreases in uptake of screening among those who are more socially deprived. This decrease is unlikely to be matched by a similar decrease among those who are less deprived, not only because they are less present orientated but also because evidence suggests that those who are more educated, and typically less deprived, are more aware of the limited benefits of screening programmes [12].\nA differential decline in uptake of screening is an issue of particular concern. Those who are more socially deprived already have poorer health [7, 17] and are more likely to develop diabetes and its complications [3, 8]. A reduction in uptake of screening among those who are most deprived might widen the existing gap in physical health between those who are more and less socially deprived, running counter to UK government policy of reducing such inequalities [10, 11]. If an informed choice policy does reduce the rate of screening of the most deprived, then there appears to be an ethical dilemma. On the one hand, there can be greater choice but at the ethical cost of increased inequality; on the other hand, greater inequality can be avoided, but at the ethical cost of less informed choice. In the rest of this paper, we aim to describe this dilemma more fully and offer some thoughts about how, in the face of it, an informed choice might be evaluated.\nWe should say at the start that our aims are limited and that we do not try, nor think it possible, to evaluate an informed choice policy fully in the space available. Our discussion is aimed at those who feel an initial ethical pull both toward reducing inequality and toward informed choice. Among those who feel this pull are the UK government, whose policies are explicitly designed to try to achieve both. Thus we do not aim to persuade those libertarians or elitists who oppose taxation-funded screening programs altogether. Nor do we intend the ethical claims we make to be applicable in all times and places.\nIn aiming our discussion at those who are attracted both to equality and choice, we do not suggest that the nature of these values is at all obvious. Indeed, one of our major points will be that any evaluation of an informed choice depends on further specification of these values. It might well turn out, as these values are specified, that the dilemma between choice and equality is merely apparent and that a decision between them in the context of screening does not have to be made.\nAfter the ethical characterization of the choice and equality dilemma, we critique a public health and paternalistic approach to screening that is sceptical about the value of choice.\nIs There a Dilemma?\nAs mentioned, it appears that there is a dilemma for those who value both choice and equality if an informed choice policy reduces the rate of screening of the most deprived without significantly reducing the rates for everybody else. It appears that the health of the most deprived would decline relative to others and thus inequality of health would increase. Whether there is actually a dilemma depends partly on what happens to screening rates in practice, but it also partly depends on the characterization of the ethical values, as we shall now show.\nIn the first place, it is not obvious, even if there are differential impacts on screening rates, that choice would produce greater inequality of health. While maximizing informed choices might result in decreased physical health, by promoting personal autonomy they might increase psychological well-being. Personal autonomy has been related to two specific psychological constructs [51]: perceived control, which has been linked to positive outcomes including coping, personal adjustment and success or failure in a variety of areas of life [46]; and self-efficacy, which is the sense of having mastery over the action needed to achieve a particular end [4]. Self-efficacy is associated with a number of health-related behaviours including uptake of screening [13, 14, 19, 25]. Those who are more socially deprived are not only disadvantaged in terms of physical health but commonly also have lower levels of positive psychological characteristics, such as self-efficacy, which contribute to an individual\u2019s overall well-being and are related to the ability to make autonomous choices in a variety of health and life domains [5].\nThe point here can be made in two different ways. One is to say that health, as a philosophical concept, is about more than narrowly defined physical health and that the psychological benefits of greater choice for the most deprived are gains in health to them. This leaves it moot whether a reduction in their rates of screening is worse for them in terms of their health, and thus moot whether choice increases inequalities in health. A second way, which avoids the complicated conceptual debates about what health is, puts the point as one about the determinants of health. Even on a relatively narrow construal of health as physical health the psychological gains of choice, such as increased sense of control [51], might improve the physical health of the most deprived [1], again leaving it moot whether choice increases inequalities in health.\nEven if there is a conflict between choice and equality of health, it does not follow that there is a conflict between choice and the value of equality. Again, whether there is a conflict depends on further specification of the value. There is a large debate in political philosophy called the `equality of what?\u2019 debate [44]. Fundamentally, however health is characterized and however equality is characterized, health would only be one item in the metric of inequality. Suppose that an informed choice policy causes the health of the most deprived to be lower than it would otherwise have been. The policy also causes their choice to go up. Whether the result is to make them worse off than they would otherwise have been is a complicated question that depends on the relative value of health and choice. Possibly the result should be counted as a gain in equality, since it increases the choice of the most deprived, although possibly not. The point here is that, from the viewpoint of equality, concentrating on inequalities in health is concentrating on only part of the picture.\nNot only is the evaluation of choice complicated by debates about health and the metric of equality, it is also complicated by debates over how to understand the value of equality. It is possible to give only an incomplete and sketchy account of this here. Consider three views: utilitarianism, prioritarianism, and egalitarianism. Utilitarianism recommends policies that maximize aggregate welfare [45]. It would recommend reducing inequalities of health if this would indeed maximize welfare. Many utilitarians believe that greater equality would maximize welfare [6]. As for the policy of informed choice, utilitarians would recommend it if it led to greater welfare than the alternative and oppose it if it reduced welfare. In the next section, on paternalism, we make some points about the effects of choice on welfare.\nPrioritarianism recommends policies that improve the position of the worst off. Prioritarians disagree among themselves about the extent of the priority that should be given to the worst off, for instance whether small gains to the worst off outweigh large gains to everyone else [30, 31]. However prioritarians decide to evaluate gains to the worst off, there is no necessary connection between their view and favouring equality. Improving the position of the worst off might reduce inequalities in health, but it might also increase them [36, 40]. Egalitarians, by contrast, believe that inequality is in itself bad. For them, there is some value in reducing inequality even if it is bad for some and good for no one [49]. This is not to say that egalitarians would, all-things-considered, want greater equality if it produced no gain in anyone\u2019s welfare. Their attitude to inequalities would depend on the other values they hold [36].\nIn the face of this sketch of various positions, it is clear that evaluating any inequality produced by an informed choice policy requires more detail about those effects. That inequality increases does not on its own justify opposing the policy. Suppose inequality increases because the most deprived stay the same, on whatever is the right metric for measuring people\u2019s positions, but the position of others improves. Utilitarians and prioritarians should endorse the policy. Egalitarians might oppose it but also might not, depending on the other values they hold. Suppose the position of the most deprived goes down and others\u2019 positions improve. Egalitarians and prioritarians would probably oppose it, although that might depend on how much the most deprived lose and how much others gain. Utilitarians will weigh up the gains and losses to see what maximizes welfare.\nIn this section we have pointed out some of the complexities of evaluating the policy of informed choice even if we assume some initial ethical commitment to both choice and equality. In particular, we have shown that the initial statement of the dilemma between choice and equality is too crude, and that there might be no dilemma in the end. In describing the complexities we do not want to suggest that a rationally defensible overall evaluation is impossible, only that it is perhaps more difficult than one might think. In the next section, we continue the theme of complexity by pointing to the for a brisk paternalistic public health approach that says that if choice reduces health it must be bad.\nInformed Choice, Public Health, and Paternalism\nIf an informed choice policy really does reduce the screening rates of the most deprived\u2014or, indeed, of the general population\u2014then the argument might be made that the policy is ethically wrong because it would increase morbidity and mortality. The argument might be put in terms of public health, that the disease burden of society would increase, or more directly in paternalistic terms, that individuals would be net worse off for having and making the choice not to be screened, and that either way, an informed choice should not be implemented. While we cannot show decisively that this argument is mistaken, this section points to some of the serious difficulties that face it. We should state at the outset that we understand paternalism in the sense commonly used in political philosophy, where an action is paternalistic only if it is both motivated by concern for the target\u2019s interests and in some way bypasses the target\u2019s own decision-making, for instance by coercing the target or failing to disclose relevant information [54].\nFirstly, at the population level any loss of life resulting from an informed choice policy has to be set alongside the negative impacts of a public health approach to screening including death as a consequence of that approach. Emphasizing the benefits of screening results in limited understanding of the harms of screening, including the possibility of false negatives and in turn this leads to an overestimation of the reliability of screening results by both the public and health professionals [38]. This may lead to symptoms of disease being ignored following a negative screening result, delaying diagnosis and treatment [2, 38].\nEssentially the same points can be made if the argument is put instead in paternalistic terms, that individuals choose against their best interests when they choose against screening. From the point of view of any given individual facing the choice of whether to undergo screening, not being screened carries a certain risk of morbidity and mortality and so does being screened. Given other costs and benefits of screening, it might be quite rationally self-interested for some individuals to decide to undergo screening while others choose against it. It is likely that in many cases those who are more deprived would indeed value increased life expectancy and it would therefore be appropriate to give those from lower socioeconomic groups information about their higher risk of dying from the disease to inform their personal evaluation of the benefits and harms of participation. However, it is also possible that those who are more socially deprived may value increased life expectancy differently to those who are less socially deprived resulting in a preference to delay diagnosis for as long as possible, even if, in the long term this results in a shortened life expectancy [24]. Thus it is much harder than might be thought to show that those who choose against screening must be acting against their better interests.\nMoreover, as we pointed out in the previous subsection, the value of autonomous choice can be intertwined with people\u2019s interests in that having choices can have psychological, and health-promoting, benefits given perceived control and self-efficacy have been linked to good health [1, 5]. This is a further reason to doubt that restricting individuals\u2019 informed choice would promote their overall welfare.\nThe argument from the benefits of screening against informed choice, in either public health or paternalistic forms, arguably also undervalues autonomy. For many political philosophers, and people more generally, being able to make choices for oneself is good in itself. The value of informed choice is not simply in the extent to which is allows us to choose what is really in our interests. In other dimensions of life, such a choice of career or partner, many people want to make their own choices even if they are less good at selecting than some disinterested observer [43]. Perhaps these reasons to value informed choice carry over to the choice of screening even if people do sometimes get it wrong.\nA persuasive paternalistic argument must show that, given the possibility of an informed choice, the people would tend to choose against their interests and that this would justify denying them the choice. We have not shown that no such argument could be made, but we have tried to show the severity of the difficulties that face it.\nConclusions\nThis paper has shown how psychological research can contribute to assessing the possible impact of an informed choice policy on screening uptake. It suggests that an informed choice policy could lead to a decrease in uptake of screening amongst those who are most socially deprived, resulting in decreases in the physical health of this group. From a public health perspective any decrease in physical health is a matter of concern, particularly if that decrease is greater in those who are more socially deprived. From an informed choice perspective such a decrease in uptake could be interpreted as indicating that people are making autonomous choices based not only on good knowledge, but also in line with their own values. Those who are more socially deprived are more present orientated. They therefore value actions that have positive outcomes immediately and thus, once they understand that the benefits of screening are not immediate, may be less likely to participate in the screening programme.\nThese results have been evaluated from different philosophical perspectives on health inequality and on choice. This evaluation has not attempted to provide a definitive assessment of whether the introduction of an informed choice policy in screening can be justified in the light of the likely impact on physical health inequalities outcomes across the population. Rather, the evaluation has sought to describe the way in which philosophical approaches to choice and to health inequality can be used to inform further discussions about choosing an informed choice approach to screening over a public health approach.","keyphrases":["screening","informed choice","public health approach","health inequality","paternalism","autonomy","utilitarianism","prioritarianism","egalitarianism"],"prmu":["P","P","P","P","P","P","P","P","P"]}
{"id":"Oecologia-3-1-2039789","title":"Costs and benefits of induced resistance in a clonal plant network\n","text":"Plant defense theory suggests that inducible resistance has evolved to reduce the costs of constitutive defense expression. To assess the functional and potentially adaptive value of induced resistance it is necessary to quantify the costs and benefits associated with this plastic response. The ecological and evolutionary viability of induced defenses ultimately depends on the long-term balance between advantageous and disadvantageous consequences of defense induction. Stoloniferous plants can use their inter-ramet connections to share resources and signals and to systemically activate defense expression after local herbivory. This network-specific early-warning system may confer clonal plants with potentially high benefits. However, systemic defense induction can also be costly if local herbivory is not followed by a subsequent attack on connected ramets. We found significant costs and benefits of systemic induced resistance by comparing growth and performance of induced and control plants of the stoloniferous herb Trifolium repens in the presence and absence of herbivores.\nIntroduction\nPlants can allocate a limited pool of resources to the three main functions of growth, reproduction and defense, suggesting that increased investments in one function may compromise the others. Empirical studies have shown that constitutive defense can be costly (e.g., tannins, Sagers and Coley 1995; glandular trichomes, Elle et al. 1999). Plant defense theory postulates that inducible defense mechanisms have evolved to reduce these costs by optimizing the temporal match between resource investment into defense and herbivory threats (Herms and Mattson 1992; Agrawal et al. 1999). In the last decades numerous studies have attempted to find costs of induced defense (reviewed in Bergelson and Purlington 1996 and Strauss et al. 2002) but evidence for costs of inducible plant defense remains scarce and inconclusive. More recently, empirical evidence has emerged supporting the allocation cost theory with the help of improved experimental designs, genetic engineering, and biochemical methodology (Baldwin 1998; Van Dam and Baldwin 1998, 2001; Heil and Baldwin 2002).\nInducible resistance is a form of phenotypic plasticity as it allows plants to express an adequate phenotype in response to temporally and spatially variable herbivore damage. Herbivore-induced changes in the phenotypes of plants often relate to trait alterations which reduce the palatability and digestibility of consumed tissue by producing toxic metabolites and\/or by up-regulating the production of a variety of defensive compounds. The ecological viability of induced resistance as an efficient defense strategy depends on the balance of costs and benefits associated with plastic defense induction. Assessing the benefits of induced defense in conjunction with possible costs is a prerequisite for estimating the advantages and disadvantages of plastic defense induction as a potentially adaptive form of phenotypic plasticity (Dudley and Schmitt 1996; Schmitt et al. 1999) and hence for understanding potential selection pressures leading to the evolution of induced plant defenses (Agrawal 2000).\nCosts of defense have been traditionally measured in terms of decreased plant fitness. Allocation costs refer to a direct fitness decrease as a consequence of resource-mediated trade-offs between defense investment and other plant functions. Recent empirical and conceptual work has provided convincing arguments for the notion that defense induction can also affect fitness in an indirect manner, via a multitude of potentially complex ecological interactions (Van Dam and Baldwin 1998, 2001; Heil and Baldwin 2002; Strauss et al. 2002). These costs are commonly referred to as ecological costs.\nAllocation theory suggests that costs of plastic defense induction should be more apparent in low-resource environments than under optimal growth conditions (Herms and Mattson 1992; Bergelson 1994; Bergelson and Purlington 1996; but see van Dam and Baldwin 2001) as the diversion of resources to defense can not easily be compensated for by enhanced resource acquisition. In addition, experiments to detect costs of defense conducted under quasi-optimal conditions are unlikely to reflect realistic situations, and therefore, tend to underestimate plasticity costs. To overcome this problem, several studies have used competitive and\/or low-resource environments to quantify costs of induced defense (Siemens et al. 2003 and studies quoted therein). Additionally, previous studies have shown that controlling the genetic background of plants can substantially enhance the chances to detect costs, by removing confounding effects due to genetic variation in the induced response (Bergelson and Purlington 1996; Strauss et al. 2002).\nStoloniferous plants consist of multiple, genetically identical individuals (ramets) that are interconnected by aboveground horizontal stems (stolons). Resource transport within clonal plant networks has been extensively described in the literature (Pitelka and Ashmun 1985; Marshall 1990; Alpert 1996; Alpert and Stuefer 1997). Nevertheless, the importance of stolon connections for the transport of defense agents is a novel aspect (Stuefer et al. 2004) that has only recently been demonstrated (G\u00f3mez and Stuefer 2006). Ramets of the stoloniferous herb Trifolium repens are able to systemically induce other ramets after local herbivore damage. On the one hand, this form of physiological integration may confer clonal plant networks with considerable benefits by allowing for a fast, specific and efficient early-warning system among interconnected ramets. On the other hand, the potentially large spatial scale of clonal plant networks may also lead to substantial costs if network members become induced without being threatened by herbivores (G\u00f3mez and Stuefer 2006). These costs are due to a potential mismatch in the spatio-temporal scale of plastic defense expression and the dynamics and patterns of herbivore attacks.\nTo assess the potentially adaptive nature of plastic responses, \u201cit is necessary to demonstrate that the phenotype induced in each relevant environment confers higher fitness in that environment relative to alternative phenotypes\u201d (Schmitt et al. 1999). This is analogous to stating that the induced phenotype should incur costs in herbivore-free environments, while defense induction should lead to benefits in herbivore-exposed environments. To quantify costs and benefits we measured traits related to plant fitness and performance of induced and uninduced T. repens plants in the absence and presence of herbivores. Growing induced and uninduced plants in the absence of herbivores allows for a quantification of possible costs of induced resistance, simulating localized damage (e.g., by small herbivores with a low mobility) and the activation of defense in ramets beyond the feeding range of the herbivore. Benefits of induction, however, can only be assessed in the presence of herbivores after an initial attack, thereby simulating a scenario with mobile herbivores showing active foraging behavior beyond the first place of attack.\nIn this study we tested the following specific hypotheses:\nIn the absence of herbivores, systemically induced ramets of clonal plants perform worse than uninduced ramets of the same genotype. This is due to costs of defense induction when defense is not needed.\nIn the presence of herbivores, induced ramets of clonal plants perform better than uninduced plants, due to an enhanced protection through induced defense.\nTo test these hypotheses we grew induced and uninduced (control) plants of the stoloniferous herb T. repens together to expose them to mutual competitive interactions, resembling sub-optimal growing conditions in a sward. To quantify costs and benefits of induced resistance we grew plants in herbivore-free and herbivore-exposed environments, respectively.\nMaterials and methods\nStudy organisms\nFive genotypes of the stoloniferous herb T. repens L. were vegetatively propagated in a greenhouse at a mean temperature of 21\u00b0C\/19\u00b0C (day\/night), and at a 16\u00a0h\/8\u00a0h (light\/dark) photoperiod. The genotypes originated from natural riverine grassland populations situated along the river Waal, The Netherlands. They had been collected 4 years prior to the start of this experiment and were grown under common garden conditions, eliminating possible maternal and environmental carry-over effects.\nThe beet armyworm (Spodoptera exigua H\u00fcbner) used in this study is a generalist caterpillar with a broad host range. The caterpillar colony was maintained at a constant temperature of 24\u00b0C and 16\u00a0h\/8\u00a0h (light\/dark) photoperiod. The larvae were reared on an artificial diet described in Biere et al. (2004).\nPre-growth of plant material\nWe started the experiment with 64 cuttings of each of the five genotypes. The cuttings were planted in pairs in plastic trays (16\u00a0cm\u00a0\u00d7\u00a012\u00a0cm\u00a0\u00d7\u00a05\u00a0cm) using sterilized clay grains as a substrate (Seramis; Masterfoods, Germany). Each tray was fertilized weekly with 50\u00a0ml full-strength Hoagland solution before the start of the experiment. At the beginning of the experiment, all cuttings consisted of a main stolon with at least eight fully developed ramets. If present, side branches were removed immediately before starting the experiment.\nExperimental design\nThe experimental set-up (Fig.\u00a01) to measure costs and benefits of systemic induced resistance (SIR) consisted of four peripheral trays placed around a central tray, which we will refer to as the \u201ccompetition tray\u201d. All trays were of similar dimensions (16\u00a0cm\u00a0\u00d7\u00a012\u00a0cm\u00a0\u00d7\u00a05\u00a0cm). Each of the peripheral trays contained two cuttings with at least eight ramets each. The cuttings in two of those trays received a treatment to induce defense during the entire duration of the experiment (for details see below), while the cuttings in the other two trays remained uninduced (control). Trays receiving the same treatment were placed diagonally opposite each other. The competition tray was placed inside a metal frame (20\u00a0cm\u00a0\u00d7\u00a015\u00a0cm\u00a0\u00d7\u00a020\u00a0cm) covered by mosquito netting (mesh gauge 0.2\u00a0cm2) with four small openings on both longitudinal sides. The two youngest ramets of each cutting were inserted through the mesh openings and allowed to grow (proliferate and root) in the competition tray for 19\u00a0days. We used five T. repens genotypes, each of which was replicated 4 times to measure costs and 4 times to measure benefits of defense induction. All induced and control plants grown together in the same experimental set-up (as described above) belonged to the same genotype. The experimental systems were randomly distributed on greenhouse benches.\nFig.\u00a01Schematic representation of the experimental set-up to measure costs and benefits of systemic induced resistance (SIR) in a clonal plant network. Control (white) and defense-induced (gray) plants grew from four peripheral trays into a common, central competition tray. The circles represent petri dishes used for a continued controlled herbivore attack (defense induction treatment). To measure costs of SIR, plants grew together in the absence of herbivores in the competition tray (upper drawing). To measure benefits, ten caterpillars (wavy black lines) were added to the competition tray (lower drawing). See Materials and methods for more details\nSystemic induction of resistance\nSystemic induction of resistance was achieved through a controlled herbivore attack. One S.exigua larva was confined with two leaves in one petri dish mounted on the plants (G\u00f3mez and Stuefer 2006). The corresponding ramets of uninduced control plants were similarly enclosed in modified petri dishes but without adding any larvae. The controlled herbivore attack was maintained throughout the course of the experiment, starting on the ramet on the eighth position (counting from the tip of the stolon) from each cutting. When the two ramets inside the petri dish had lost at least 50% of leaf tissue, the petri dish was moved forward on the stolon and the adjacent, younger ramet was inserted into the petri dish. Whenever the induction treatment was moved forward on the induced cuttings, a comparable leaf area was removed with scissors from one ramet of each cutting in the control trays. This was done to compensate for the leaf area loss due to caterpillar feeding in the induced plants. Cutting the leaves with scissors does not induce resistance in T. repens (S. G\u00f3mez, unpublished data). The induction treatment started 1 day after the cuttings were placed into the competition tray. If the caterpillar inside the petri dish died, it was replaced by a new one to maintain defense induction.\nIn order to enhance plant interactions, induced and control plants were grown together in the competition tray. Since all plants growing together belonged to the same genotype, induction effects cannot be confounded with genetic differences in plant traits, including competitive ability, between induced and control plants. All measurements described below were performed on ramets growing in the competition trays.\nCosts of SIR\nCosts of defense induction were measured as a reduction in plant performance. Costs can be measured after initial herbivore damage (and consequent defense induction) in the absence of subsequent herbivore attacks. To quantify costs of defense induction we measured the following traits which are known to be closely related to plant performance and fitness: total biomass production, relative biomass allocation to leaves, petioles, stolons, and roots, number and length of the main and side stolons and number of ramets on the main and side stolons. We also measured the petiole length, petiole dry mass, leaf area, leaf dry mass of the fourth and fifth youngest ramets of each cutting.\nBenefits of SIR\nTo quantify benefits of SIR we exposed the plants in the competition tray to a second, controlled herbivory attack (referred to as \u201cherbivory treatment\u201d). We released five fourth instar caterpillars on day 16 in the competition tray and then added two and three more on day 17 and 18, respectively, to achieve substantial levels of herbivore damage. The plants were harvested 19\u00a0days after the start of the experiment. We quantified benefits of induced resistance by scoring herbivory damage in the induced and in the control plants. At the time of harvesting each ramet on the main stolon was classified according to the leaf area consumed. We visually estimated the damage and assigned each ramet a damage category ranging from 0 to 3. The values corresponded to the following amounts of damage: 0\u00a0=\u00a0no damage, 1\u00a0=\u00a01\u201333%, 2\u00a0=\u00a033\u201366% and 3\u00a0=\u00a066\u2013100% of leaf area consumed. We also recorded the position of the damaged ramet on the stolon to investigate possible intra-clonal variation in the damage pattern according to ramet age. In addition to the degree of damage, we measured the dry mass of leaves, petioles, stolons and roots in induced and control plants.\nHerbivore preference test\nOne day before releasing the caterpillars (herbivory treatment) we performed two dual choice tests per competition tray to check whether plants assigned to the defense-induction treatment were systemically induced. For each competition tray we cut off two control and two induced ramets of a similar developmental stage (third-youngest fully expanded leaf). Each control ramet was paired with an induced one and placed together on a moist filter paper in a petri dish to perform a dual choice test. A fourth instar S. exigua caterpillar was placed in the middle and allowed to feed until more than 30% of one of the leaves was consumed or for 48\u00a0h. By means of visual estimates the leaf with the largest area consumed was recorded for each choice test. In 78% of the cases more of the control leaf was consumed (sign test M\u00a0=\u00a023, P\u00a0<\u00a00.0001; n\u00a0=\u00a077) than the induced one, confirming that plants in the competition trays that had received local herbivore damage (defense induction treatment) were induced before the herbivory treatment started.\nStatistical analysis\nCentral competition trays were considered the units of replication in all statistical analyses. To avoid pseudo-replication and a consequent inflation of df (Hurlbert 1984), all traits measured on plants (cuttings) in the competition trays were pooled per treatment (by averaging the four control cuttings and the four defense-induced cuttings, respectively) prior to data analysis. Consequently, our experiment had 20 replicates for measuring costs and 20 replicates for assessing benefits. Competing plants cannot be considered independent from each other as, by definition, they change each other\u2019s environment, growth and development. To take this dependence into account we used a repeated measures design to analyze differences between competing plants that belonged to different treatment groups. Repeated measures analysis explicitly considers intrinsic relationships between treatment groups (Potvin et al. 1990).\nCosts of SIR\nRepeated measures ANOVA was performed to test for costs of defense induction in number and length of the main and side stolons and number of ramets on the main and side stolons, relative biomass allocation to roots, stolons, petioles and leaves and petiole length, petiole dry mass, leaf area, leaf dry mass of the fourth and fifth youngest ramets. Defense induction (induced vs. control) was considered a within-subjects effect, and genotype was treated as a between-subjects effect. Absolute dry masses of roots, stolons, petioles and leaves were analyzed using two-way repeated measures ANOVA (within-subjects effect\u2014defense induction; between-subjects effects\u2014plant genotype and herbivory).\nBenefits of SIR\nThe amount of damage in the herbivory treatment was assessed with doubly repeated measures ANOVA using ramet age and defense induction as repeated factors and genotype as main effect. The analysis included a profile analysis (SAS procedure GLM; profile statement) to test for differences in the degree of damage between adjacent ramets on the stolons. To correct for differences in the developmental stage of different cuttings we used only the six youngest ramets of each cutting in the damage analysis. All analyses were conducted with SAS 9.1 (SAS Institute, Cary, N.C.).\nResults\nCosts of SIR\nTotal dry mass did not differ between control and induced plants (Table\u00a01). However, defense induction caused a significant reduction in petiole dry mass. Additionally, defense induction resulted in a shift in biomass allocation to the different plant parts. Relative biomass allocation to leaves increased significantly after defense induction (Table\u00a02; P\u00a0=\u00a00.01). The percentage of biomass allocated to roots, stolons and petioles did not significantly differ between control and induced plants. (Table\u00a03)\nTable\u00a01Repeated measures ANOVA for effects of genotype, herbivory and defense induction on roots, stolons, petioles, leaves and total dry massSourcedfRootStolonPetiolesLeavesTotalMSFMSFMSFMSFMSFBetween-subject effectsGenotype (Gen)49817.20***3,7104.12***3,74110.97***11,00210.85***60,3778.99***Herbivory (Herb)128.10.2178.60.091,6464.83*4,2344.18*11,9261.78Gen\u00a0\u00d7\u00a0Herb449.30.361240.141050.312920.291,1710.17Error301368993421,0146,714Within-subject effectsInduction (Ind)179.80.815741.974165.11*98.60.371,8790.99Ind\u00a0\u00d7\u00a0Gen453.90.5483.10.2850.70.623671.371,0540.55Ind\u00a0\u00d7\u00a0Herb11061.0757.10.2011.10.141600.601,1430.60Ind\u00a0\u00d7\u00a0Gen\u00a0\u00d7\u00a0Herb41391.405421.861591.952650.993,5251.85Error3099.029281.32681,901*0.01\u00a0<\u00a0P\u00a0<\u00a00.05, ***P\u00a0<\u00a00.0001 Table\u00a02Costs of systemic induced resistance (SIR). Repeated measures ANOVA for effects of genotype and defense induction on relative dry mass allocation to roots, stolons, petioles and leaves on plants without an herbivory treatment in the competition traySourcedfRootsStolonsPetiolesLeavesMSFMSFMSFMSFBetween-subjects effectsGenotype (Gen)429.86.61** 49614.57***22.73.54*35.77.3**Error154.51276.44.9Within-subject effectsInduction (Ind)112.02.060.040.015.31.9235.87.32*Ind\u00a0\u00d7\u00a0Gen42.20.388.21.161.80.6410.92.24Error155.87.12.84.9*0.01\u00a0<\u00a0P\u00a0<\u00a00.05, **0.001\u00a0<\u00a0P\u00a0<\u00a00.01, ***P\u00a0<\u00a00.0001 Table\u00a03Average (\u00b1SE) absolute and relative dry mass allocated to roots, stolons, petioles and leaves of uninduced and induced plants in the absence of a subsequent herbivory treatment (Costs) and in the presence of a subsequent herbivory treatment (Benefits) in the competition trayRoot (mg)Stolons (mg)Petioles (mg)Leaves (mg)Total (mg)CostsUninduced15.6\u00a0\u00b1\u00a03.0 (4.5\u00a0\u00b1\u00a00.7%)103.2\u00a0\u00b1\u00a06.7 (34.9\u00a0\u00b1\u00a01.0%)72.6\u00a0\u00b1\u00a04.8 (24.9\u00a0\u00b1\u00a00.6%)107.5\u00a0\u00b1\u00a08.3 (35.7\u00a0\u00b1\u00a00.7%)298.9\u00a0\u00b1\u00a021.1Induced11.3\u00a0\u00b1\u00a02.1 (3.4\u00a0\u00b1\u00a00.6%)96.1\u00a0\u00b1\u00a06.0 (34.8\u00a0\u00b1\u00a01.0%)67.3\u00a0\u00b1\u00a04.5 (24.2\u00a0\u00b1\u00a00.5%)106.8\u00a0\u00b1\u00a08.3 (37.6\u00a0\u00b1\u00a00.6%)281.6\u00a0\u00b1\u00a019.1BenefitsUninduced14.5\u00a0\u00b1\u00a02.2 (5.1\u00a0\u00b1\u00a00.8%)99.5\u00a0\u00b1\u00a04.8 (37.6\u00a0\u00b1\u00a00.8%)62.8\u00a0\u00b1\u00a03.7 (23.8\u00a0\u00b1\u00a00.8%)90.1\u00a0\u00b1\u00a05.6 (33.5\u00a0\u00b1\u00a00.8%)266.9\u00a0\u00b1\u00a013.2Induced14.8\u00a0\u00b1\u00a03.7 (4.8\u00a0\u00b1\u00a00.9%)95.8\u00a0\u00b1\u00a06.1 (36.8\u00a0\u00b1\u00a01.1%)59.0\u00a0\u00b1\u00a04.5 (22.6\u00a0\u00b1\u00a00.6%)95.1\u00a0\u00b1\u00a07.6 (35.8\u00a0\u00b1\u00a00.6%)264.7\u00a0\u00b1\u00a019.7\nThe number of ramets produced on the main stolon was 7% lower in induced as compared to control plants (Table\u00a04; induction effect P\u00a0=\u00a00.003). The number and length of side stolons and the number of ramets formed on them did not change after defense induction.\nTable\u00a04 Costs of SIR. Repeated measures ANOVA for effects of genotype and defense induction on plant fitness and performance-related traits in the absence of herbivoresSourcedfRamet no. main stolonLength main stolonRamet no. side stolonsLength side stolonsSide stolons numberFourth ramet petiole lengthFourth ramet areaMSFMSFMSFMSFMSFMSFMSFBetween-subjects effectsGenotype (Gen)42.383.41*23.55.4***71.96.72**30.81.6316.012.97***18.014.93***8.5715.36***Error150.694.3110.718.91.231.200.55Within-subject effectsInduction (Ind)11.8012.13**7.572.451.250.250.780.230.150.314.385.22*0.100.23Ind\u00a0\u00d7\u00a0Gen40.171.193.921.271.830.374.101.220.120.240.350.420.090.21Error150.143.084.953.370.510.840.46*0.01\u00a0<\u00a0P\u00a0<\u00a00.05, **0.001\u00a0<\u00a0P\u00a0<\u00a00.01, ***P\u00a0<\u00a00.0001 \nThe fourth and fifth youngest ramets on the main stolon produced petioles 5% shorter in the induced plants (Table\u00a04; fourth ramet P\u00a0=\u00a00.03, fifth ramet P\u00a0=\u00a00.07). Leaf area, leaf dry mass and petiole dry mass measured on those ramets were not significantly affected by the induction treatment.\nBenefits of SIR\nDefense induction had a very strong effect on the amount of damage inflicted by S. exigua larvae on the plants (Table\u00a05; induction effect P\u00a0<\u00a00.0001; Fig.\u00a02). The number of ramets that were partially or fully consumed during the herbivore attack was consistently higher in control than in induced plants. Most of the damaged ramets lost only a small part of their leaf area (1\u20135%). This was consistent for both control and induced plants (Fig.\u00a02). In induced plants up to 44% of the ramets on the main stolon were not damaged, whereas in control plants only 22% of ramets on the main stolon were undamaged.\nTable\u00a05Benefits of SIR. Doubly repeated measures ANOVA for effects of genotype, defense induction and ramet age on leaf area loss due to herbivorySourcedfMSFBetween-subjects effectsGenotype (Gen)41,1022.39\u2020Error15461Within-subjects effectsInduction (Ind)16,84763.92***Ind\u00a0\u00d7\u00a0Gen42442.28Error (induction)15107Age518,988133.0***Age\u00a0\u00d7\u00a0Gen206304.41***Error (age)75142Ind\u00a0\u00d7\u00a0Age51251.22Ind\u00a0\u00d7\u00a0Age\u00a0\u00d7\u00a0Gen201721.67\u2020Error (Ind\u00a0\u00d7\u00a0Age)75103\u2020\u00a00.1\u00a0>\u00a0P\u00a0>\u00a00.05, ***P\u00a0<\u00a00.0001Fig.\u00a02Average damage (\u00b11 SE) inflicted on ramets of the main stolon (the 1st ramet being the youngest and the 6th being the oldest) of control and induced plants in the competition tray after carrying out a controlled herbivore attack (herbivory treatment). Damage categories: no damage (0), 1\u201333% (1), 33\u201366% (2), 66\u2013100% (3). The asterisks above the bars indicate the statistical significance of the result of a profile analysis (SAS procedure GLM; profile statement) to test for differences in the degree of damage between ramets of successive age classes. The amount of damage was significantly higher for control than for induced plants in all age classes. ***P\u00a0<\u00a00.001, ns not significant\nThe herbivory treatment significantly reduced the biomass of leaves and petioles (Table\u00a01; P\u00a0=\u00a00.049 and P\u00a0=\u00a00.036, respectively; Table\u00a03) in both induced and uninduced plants. In the presence of herbivores, induced and uninduced plants had a comparable total biomass. However, induced plants showed a larger percentage of biomass in their leaves (Table\u00a03; repeated measures ANOVA; F\u00a0=\u00a017.44 P\u00a0=\u00a00.0008), suggesting that the induced plants benefitted from increased relative biomass in those organs under attack.\nRamet age, regardless of the induction state, had a very strong effect on herbivore preference (Table\u00a05; age effect P\u00a0<\u00a00.0001). Younger ramets, especially the first and second youngest ones, were heavily preferred over older ones (profile analysis; Fig.\u00a02). The first ramet exhibited particularly severe damage in both induced and control plants (average leaf area consumed\u00a0>\u00a065%; Fig.\u00a02).\nDefense induction had a significant effect on leaf area loss due to herbivory in all ramet age classes (Fig.\u00a02). The degree to which systemic defense induction reduced herbivory damage was similar for ramets of all age classes (Table\u00a05; no age\u00a0\u00d7\u00a0induction effect). There was a marginally significant genotype effect on the feeding of the caterpillars (Table\u00a05; genotype effect P\u00a0=\u00a00.09).\nDiscussion\nOur study provides empirical evidence of significant costs and benefits of SIR in a clonal plant network. In agreement with our hypotheses, induced and control plants showed clear differences in performance and fitness-related traits when grown in the absence and presence of herbivores. In environments without herbivores, induced plants produced fewer ramets, shorter petioles and exhibited a shift in biomass allocation patterns. In environments with herbivores, control plants suffered consistently higher degrees of leaf damage than induced plants. Even though defense induction resulted in changes in plant growth, and significantly affected the amount of damage caused by the herbivores, total plant biomass did not respond as expected under the adaptive plasticity hypothesis (Dudley and Schmitt 1996; Schmitt et al. 1999), as we could not find a significant induction\u00a0\u00d7\u00a0herbivory interaction effect. However, we propose that the differences observed in our study (e.g., reduced ramet production rates and shorter petioles in the cost experiment, decreased amount of leaf damage in the benefits experiment) are likely to translate into substantial differences in plant productivity, and hence biomass, in the longer term.\nCosts of SIR\nBiomass production and allocation\nTotal plant biomass production did not change as a consequence of defense induction, implying that defense induction did not incur direct and immediate productivity costs. After induction, however, biomass allocation shifted significantly towards the leaves. We suggest that this allocation shift may enable plants to better cope with current and future herbivory by reducing resource allocation to those organs that are not currently impacted by herbivore damage. While potentially beneficial in the short term, this response might result in longer term indirect costs due to reduced performance under certain environmental conditions, such as drought, root herbivory and severe root competition. A similar shift in the biomass allocation pattern was observed in Lepidium virginicum plants after defense induction. Induced plants grown at a high density showed a reduction in root biomass and an increase in aboveground biomass (Agrawal 2005). In agreement with our findings, total biomass production was not significantly altered by defense induction in that study. A reduction in belowground biomass was also reported for induced wild parsnip plants. In this case, however, the aboveground biomass did not change significantly after defense induction (Zangerl et al. 1997). Further studies are necessary to assess the generality, functional significance (including costs and benefits) and mechanistic basis of changes in root\u2013shoot allocation in response to induced resistance to herbivory.\nReduction in developmental growth rate\nDefense induction negatively affected plant fitness by reducing the number of ramets produced. This delayed developmental growth rate was expressed as a reduction in the number of ramets on the main stolon produced during the experiment (7.4 ramets on the control and 7.0 on the induced plants). In the shorter run (i.e., time span of this experiment) this effect is unlikely to translate into biomass differences. In the longer run, however, subtle changes in the developmental growth rate are known to result in very major divergences in performance, structure and clonal fitness of stoloniferous plants (Birch and Hutchings 1992a; Birch and Hutchings 1992b; Huber and Stuefer 1997; Stuefer and Huber 1998).\nReduction in petiole length\nDefense induction had significant negative effects on petiole lengths. This effect can have severe performance and fitness consequences for a stoloniferous plant like T. repens, which often grows in dense herbaceous canopies, and which relies on petiole elongation for shade avoidance (Huber 1997). Petiole length largely determines the ability of stoloniferous plants to place their leaves higher up in the canopy (Huber and Wiggerman 1997; Weijsched\u00e9 et al. 2006). Even a small reduction in petiole length could have serious performance costs since differences in the relative position of leaves in herbaceous canopies are likely to be amplified by asymmetric competition for light (Weiner 1990; Pierik et al. 2003). Defense induction may also cause physiological trade-offs which impede the simultaneous expression of plasticity to herbivores and to shading by competitors (Cipollini 2004). A decrease in petiole length as a result of defense induction can hence, compromise the competitive ability of plants and result in an enhanced risk of induced plants being over-shaded by neighbors. A recent study by Kurashige and Agrawal (2005) supports this notion by showing that Chenopodium album plants, which had previously been damaged by herbivores, were able to elongate stems to a similar proportional degree as undamaged plants when grown in competition for light. However, the damaged plants were smaller due to the expression of induced resistance, thereby incurring potential opportunity costs due to asymmetric competition.\nBenefits of SIR\nReduced damage\nOur results provide direct evidence for short-term benefits of having an early-warning system in clonal plant networks. In the presence of herbivores, induced plants suffered considerably less damage than control plants. As many as 50% fewer ramets were attacked in induced plants as compared to controls. Localized damage (defense-induction treatment) resulted in a greater degree of protection against herbivores for ramets further along that main stolon and its side branches. The reduced damage did not translate into a significant effect of defense induction on biomass production, due to the fact that the youngest, usually not fully developed leaves were heavily preferred by the herbivores. The biomass loss due to young leaf consumption is very likely to strongly underestimate the negative effects of herbivory and defense induction on future plant growth and performance. Coleman and Leonard (1995) demonstrated how leaf area consumption, and its consequences for plant performance, can be severely underestimated if the developmental stage of leaves is not taken into account. They showed that a certain amount of damage inflicted on young expanding Nicotiana tabaccum leaves is more detrimental than the same amount received by mature, fully developed leaves. As leaf tissue expanded, the area of the holes increased almost fourfold and the final area of the leaf decreased by approximately 40%. In addition, they observed a 35% decrease in the number and mass of fruits on the plants that received the damage to expanding young tissues. Therefore, an initially small amount of damage inflicted on young developing leaves may have dramatic consequences for plant performance and fitness over time. Similarly, the differences found in our experiment can be expected to result in considerable performance differences between induced and uninduced plants as increased damage and loss of young leaves in uninduced plants will compromise plant productivity by reducing the number of future source ramets.\nOur results show that ramet age largely determines herbivore damage. The first and second ramets were heavily attacked as compared to the rest. This damage, although still large, was significantly reduced in induced plants. The reduction in leaf area loss in induced young ramets likely increases their chance of survival and establishment. Young ramets in clonal plants constitute the most valuable tissue since they represent the future reproductive potential of the plant (Huber and During 2000) and their protection is critical since they are responsible for a high proportion of the future biomass production (Beinhart 1963). We present evidence supporting the hypothesis that an early-warning system after herbivory in a clonal plant network grants vulnerable young offspring ramets with parental support (Stuefer et al. 2004) that non-clonal plants are unable to confer their offspring at the moment of the attack (but see Agrawal et al. 1999).\nOur study provides evidence for significant costs and benefits of systemic defense induction in T. repens. The experimental approach used in this study, however, does not allow for balancing costs and benefits in terms of plant fitness and overall plant performance, because both positive and negative effects of induction reported here, although likely to have significant longer-term effects on productivity and ultimately on fitness, did not have an effect on biomass at the short time scale during which the experiment took place. While our results indicate clear advantages and disadvantages of network induction in the subsequent presence and absence of herbivores, respectively, an accurate and reliable quantification of the cost\u2013benefit ratio should make use of long-term experiments.\nIn conclusion, the present study shows that in the short term, the activation of early-warning responses in clonal plant networks has both costs and benefits. In the absence of herbivores, the performance of the induced phenotype was compromised as compared to the uninduced phenotype in terms of potential competitive ability. In the presence of herbivores, the induced phenotype was favored by suffering considerably less herbivore damage suggesting potential advantages for the phenotype correctly matching its environment. Whether this represents an adaptive value of the induced responses remains to be demonstrated in longer-term studies where the initial small changes observed in our study can be measured directly in terms of fitness. The long-term balance of costs and benefits of induced resistance in clonal plant networks is likely to be strongly context dependent and a function of the match between spatio-temporal aspects of systemic defense expression and the feeding behavior of herbivores.","keyphrases":["plant defense","trifolium repens","physiological integration","adaptive plasticity hypothesis","plant communication"],"prmu":["P","P","P","P","M"]}
{"id":"Mol_Hum_Reprod-1-1-2408934","title":"In utero exposure to low doses of environmental pollutants disrupts fetal ovarian development in sheep\n","text":"Epidemiological studies of the impact of environmental chemicals on reproductive health demonstrate consequences of exposure but establishing causative links requires animal models using \u2018real life\u2019 in utero exposures. We aimed to determine whether prolonged, low-dose, exposure of pregnant sheep to a mixture of environmental chemicals affects fetal ovarian development. Exposure of treated ewes (n = 7) to pollutants was maximized by surface application of processed sewage sludge to pasture. Control ewes (n = 10) were reared on pasture treated with inorganic fertilizer. Ovaries and blood were collected from fetuses (n = 15 control and n = 8 treated) on Day 110 of gestation for investigation of fetal endocrinology, ovarian follicle\/oocyte numbers and ovarian proteome. Treated fetuses were 14% lighter than controls but fetal ovary weights were unchanged. Prolactin (48% lower) was the only measured hormone significantly affected by treatment. Treatment reduced numbers of growth differentiation factor (GDF9) and induced myeloid leukaemia cell differentiation protein (MCL1) positive oocytes by 25\u201326% and increased pro-apoptotic BAX by 65% and 42% of protein spots in the treated ovarian proteome were differently expressed compared with controls. Nineteen spots were identified and included proteins involved in gene expression\/transcription, protein synthesis, phosphorylation and receptor activity. Fetal exposure to environmental chemicals, via the mother, significantly perturbs fetal ovarian development. If such effects are replicated in humans, premature menopause could be an outcome.\nIntroduction\nExposure to environmental compounds (ECs) may alter female reproductive tissues and thus affect the ability of human couples to conceive and maintain a healthy pregnancy (Hruska et al., 2000). ECs include endocrine disrupting compounds [EDCs, e.g. dioxins, polychlorinated biphenyls (PCB) and organochlorine pesticides] that have been\/are used extensively in manufacturing and agriculture. ECs are found ubiquitously within the environment, together with heavy metal pollutants. Human exposure occurs through a variety of routes including the consumption of meat and dairy products, ingestion of water, absorption through the skin and inhalation. The concentrations of ECs within human tissues and the mechanisms through which they elicit effects on reproductive tissues are not well understood. Studies conducted in a variety of animal models demonstrate that the female fetus is particularly sensitive to small endocrine changes (Lovekamp-Swan and Davis, 2003; Henley and Korach, 2006) that have detrimental effects on reproductive function (Miller et al., 2004; Uzumcu and Zachow, 2007). There is uncertainty as to whether women\u2019s fertility and reproductive health have deteriorated or whether the link between exposure to ECs and increased incidence of breast cancer, precocious puberty, premature menopause and decreased fertility is valid (Sharara et al., 1998; Hruska et al., 2000). Nevertheless, the importance of developmental disturbance in disease aetiology is increasingly evident (Heindel, 2006). Determining the incidence of reproductive problems in women (15\u201320% of couples have difficulty conceiving) is confounded by diagnostic, social (e.g. increasing age of women first trying to conceive) and obesity issues (Evers, 2002; Sharpe and Franks, 2002) which complicates linking changes in reproductive function with EC exposure (Akkina et al., 2004; Cesario and Hughes, 2007). Similarly, although the incidence of breast cancer is clearly rising and has a strong links with environmental factors and in utero exposures (Kortenkamp, 2006), there is no general increase in ovarian cancer (Bray et al., 2005). Two conclusions arise from the literature: first, that the development of the female fetus or embryo can be perturbed by exposure to ECs and, second, that the evidence for population trends in humans is mixed and often inconclusive.\nHuman tissues, including the maternal-fetal unit and maternal tissues during gestation, contain levels of ECs that are associated with many in utero effects (e.g. Ikezuki et al., 2002; Younglai et al., 2002; Tsutsumi, 2005; Barr et al., 2007; Chao et al., 2007; Huang et al., 2007; Thundiyil et al., 2007). However, the lack of robust human exposure-phenotype linkage data and multi-factorial effects on reproductive trends hampers our ability to test the hypothesis that in utero exposure to environmental chemicals damages female reproductive development (Foster, 2003). Many ECs have the potential to perturb development (Tabb and Blumberg, 2006; Watson et al., 2007) and epidemiological studies (Toft et al., 2004) suggest that there is a need to study the effects of EC exposure in relevant animal models at real-world rates of exposure, focusing on likely \u2018sensitive\u2019 components and mechanisms of relevant reproductive circuits. Most studies into effects of ECs have focused on rodent models, using single compounds, administered for short periods, often at pharmacological doses. Although essential to understanding of the mechanisms though which such compounds act, these studies are of limited value in the assessment of risks to human developmental health because the patterns of experimental exposure are not representative of normal human exposure. Thus an animal model using a prolonged, \u2018real-life\u2019 (defined as exposure through natural routes, not via specific application, such as injection), in-utero, pattern of EC exposure will be better able to indicate likely effects on humans.\nAn ovine model in which pregnant ewes are exposed to the complex mix of ECs and heavy metals contained in human sewage sludge, following its application to pasture as a fertilizer, is characterized by defects in fetal development (Paul et al., 2005). To determine which chemical classes might be involved in these effects, we measured levels of seven heavy metals, the di(2-ethylhexyl) phthalate (DEHP), nonyl phenol and seven polychlorinated biphenyl (PCB) congeners in tissues of exposed fetuses\/offspring and found concentrations of Cu, Pb, Zn and DEHP to be significantly altered (Rhind et al., 2002, 2005a,b, 2007a,b). Sewage sludge is a relevant source of a chemical cocktail to which humans are exposed as it, in itself, broadly reflects human exposure (Rhind et al., 2002, 2005b; Abad et al., 2005; Oleszczuk, 2006; Martinez et al., 2007) to a complex mixtures of ECs (Groten et al., 2004; Jonker et al., 2004; Robinson and MacDonell, 2004; Koppe et al., 2006). In addition, sheep, like humans, are long-lived and have a relatively long gestation period (145 days), during which time ovine fetuses show a similar timing and sequence of ovarian development to that observed in the human (Pryse-Davies and Dewhurst, 1971; Juengel et al., 2002; Sawyer et al., 2002; De Felici et al., 2005). Unlike the rodent, the ruminant fetal ovary synthesizes estrogens which are important for germ cell development (Pannetier et al., 2006). Therefore, notwithstanding the metabolic and dietary differences between sheep and humans, the long-term, real-life, low-dose, exposure of pregnant sheep to a complex mix of relevant ECs is a good model for the human given that we know that many ECs are gaining access to the materno-fetal unit in pregnant women.\nIn this study, we aimed to determine whether prolonged, low-dose, exposure of the developing fetus, in utero, to maternal dietary EC load adversely affects the developing fetal ovary.\nMaterials and Methods\nAnimals, blood and tissue collection\nThe study was conducted at the Macaulay Institute research station (Hartwood, Lanarkshire, Scotland, UK) using Texel ewes. Animals were maintained on pasture at conventional stocking rates, adjusted according to pasture height, as described previously (Paul et al., 2005), so that animals from the respective treatments were maintained in comparable nutritional states. Neither group received supplementary feeding during their breeding lives. The animals were inspected by a qualified shepherd on a daily basis and routine animal care and vaccination procedures were conducted, as prescribed by best practice protocols.\nDigested sewage sludge was applied twice annually, to each of three replicate 9 ha plots, at a rate of 2.25 metric tons of dry matter per hectare. For the first five applications, the sludge was applied in liquid form as described previously (Rhind et al., 2002). Thereafter, owing to changes in sludge production practices by the UK water authorities, thermally dried sludge pellets were used and applied at similar rates (about 2.25 tones dry matter\/ha\/year). The composition of the sludge, on a dry matter basis, did not differ between the two methods of application. The application of sludge to the surface of the pastures was not designed to conform to the UK recommendations for good practice (SEDE and Arthur Anderson, 2001). According to recognized codes of practice, sludge can only be applied to grazed grassland when it is deep injected into the soil or, if it is applied to the surface, there can be no grazing of that land within the season of application. However, this study was designed to maximize the rate of contamination of the pasture and topsoil and thus to maximize the risk of exposure of grazing animals to the chemical constituents of sewage sludge through their food\u2019. Animals were not allowed to graze the pasture until a minimum of 3 weeks after sludge application, as prescribed by relevant legislation (Parliament, 1989). Control ewes were maintained on similar pasture to which 225 kg of nitrogen\/ha\/year was applied in the form of conventional inorganic fertilizers. The relatively harsh environmental conditions in central Scotland where the farm is located mean that there was no growth of clover or other estrogenic plant species in any of the pastures.\nThe treated and control groups from which the study animals were drawn each comprised 3 replicate subgroups of 5 breeding ewes in each of four age categories (total flock size = 120 ewes). For the present study, subgroups of ewes that were 6 years of age and had been maintained on the respective treatments throughout their breeding lives were drawn from all replicates of each treatment. Ewes from both groups underwent estrous cycle synchronization, using progestagen sponges (Chronolone, 30 mg; Intervet, Cambridge, UK), before being mated to rams of the same genotype and from the same source, to remove the effect of genotypic differences. Estimation of gestational age was based on the knowledge that conception should occur within 48 h of sponge removal when estrus cycle synchronization is used. Animals were euthanized at \u223c110 days of gestation (GD110, equivalent to 27 weeks in the human) according to Schedule 1 protocols as defined by the UK Animals (Scientific Procedures) Act, 1986. Twenty-three female and 24 male fetuses were collected from both sets of ewes (control, n = 10, treatment, n = 7) and all of the resulting female fetal lambs (n = 15 in control, n = 8 in treated groups) were used for the ovary studies detailed below. Maternal and fetal body weight and fetal ovary weight were recorded at slaughter; fetal blood samples were also collected and the serum, isolated by centrifugation, stored at \u221220\u00b0C until required for assay. One ovary from each animal was fixed for 5.5 h in Bouin\u2019s fixative and then transferred to 70% ethanol until analysis. The other ovary from each animal was snap-frozen in liquid N2 and stored at \u221280\u00b0C until analysis. GD110 was selected as a representative developmental stage since the primordial follicle pool has been established and expression of many developmentally important genes, such as growth differentiation factor 9 (GDF9) is maximal (Mandon-Pepin et al., 2003).\nImmunohistochemistry\nSagittal 5 \u00b5m sections of each fetal ovary were cut, floated onto slides and dried at 50\u00b0C overnight. Slides were de-waxed in xylene, hydrated gradually through graded alcohols and washed in water. For all ovaries, slides were prepared containing two randomly selected, non-consecutive sections. At least one slide from each ovary was H&E stained for morphological assessment. Further slides were immunostained for induced myeloid leukaemia cell differentiation protein (MCL1) (pro-proliferative), GDF9 (development of primordial follicles) and phosphorylated histone H3 (pH3), to assess active proliferation and establish a mitotic index (Brenner et al., 2003). Following routine de-waxing, antigen retrieval procedures were used for the MCL1 and GDF9 epitopes [microwaving in 0.01 M citrate buffer (pH 6.0) on full power for 3 \u00d7 5 min]. Immunostaining for MCL1 and GDF9 was carried out using standard peroxidase-based immunostaining protocols (MCL1: chemMate, Dako and GDF9: vector). Slides for MCL1 were placed in a Dako autostainer and incubated with anti-MCL1 antibody (Serotec, 1\/50), for 30 min. Antibody binding was visualized using the ChemMate peroxidase\/DAB detection system (DakoCytomation Ltd., Ely, Cambridgeshire, UK). Slides for GDF9 were incubated overnight in a humid chamber with anti-GDF9 antibody (R & D Systems, 1\/15) and antibody binding visualized using the Vector peroxidase\/DAB detection system for 30 min (Vector Laboratories, Peterborough, UK). A rabbit anti-P-Histone H3 (#06\u2013570; 1:500; Upstate Cell Signalling Solutions, Hampshire UK) was used after antigen retrieval and visualized using a goat biotinylated anti-rabbit antibody, followed by AB complex with horse-radish peroxidase (HRP) and DAB detection (all DAKO, Cambridge, UK). Ovarian sections were analysed for oocyte numbers and follicle size classes using an established follicle classification system (Lundy et al., 1999) by two independent observers (N.J.D. and H.M.) using six fields of view per section. Total numbers of pH3-positive granulosa, stromal, endothelial and surface epithelial cells were counted at \u00d740 and identified at \u00d7400 by M.A. Because of the relatively low incidence of pH3-positive cells, cell numbers were expressed per mm2 of \u00d740 fields of view. In all analyses, sections from 15 control and 8 sewage sludge exposed fetuses were used. Only oocytes with the nucleus clearly visible were included in quantification of oocyte and follicle densities, which was performed in six separate slides containing a total of 12 sections for each fetus (H&E, MCL1 and GDF9).\nProtein extraction\nThe frozen fetal ovaries were processed for one-dimensional and two-dimensional gel electrophoresis, as described in detail previously (Fowler et al., 2007a,b). Briefly, the frozen ovaries (15 control, 8 treated) were blotted on filter paper and combined with lysis buffer (1 mg wet tissue weight: 5 \u00b5l lysis buffer) containing 0.01 M Tris\u2013HCl, pH 7.4, 1 mM EDTA, 8 M Urea, 0.05 M DTT, 10% (v\/v) glycerol 5% (v\/v) NP40, 6% (w\/v) pH 3\u201310 Resolyte (Merk Eurolab Ltd, Poole, Dorset, UK) and protease inhibitor cocktail (Roche Diagnostics, Lewes, UK). The tissues were minced, sonicated in iced water for four 10 min bursts, with 2 min between each sonication, and centrifuged at 50 000g for 20 min at 4\u00b0C. Once the protein content of the final supernatant containing the soluble cellular proteins had been determined (RC-DC assay, Bio-Rad Laboratories Ltd, Hemel Hempstead, UK), the ovary extracts were stored at \u221280\u00b0C until required for further analysis.\nOne-dimensional gel electrophoresis and western blot\nIndividual ovary lysates (15 control and 8 treated) were electrophoresed (30 \u00b5g protein\/lane) on 26-lane one-dimensional gel electrophoresis 4\u201312% Bis\u2013Tris gels (Invitrogen Ltd, Paisley, UK) under reducing conditions and transferred to immobilon-FL membrane [Millipore (UK) Ltd, Watford, UK] as described previously (Lea et al., 2005). SeeBlue plus two molecular weight markers (Invitrogen) were electrophoresed in three lanes of every gel. The membranes were blocked (overnight, 4\u00b0C) with (Odyssey Blocking Buffer, 927\u20134000: LI-COR Biosciences UK Ltd, Cambridge, UK) and were incubated with primary antibodies (in blocking buffer) at 4\u00b0C overnight: (i) BAX (1:200: santa-Cruz Biotechnology Inc, CA, USA, sc-493), (ii) BCL2 (1:200: santa-Cruz, sc-783), (iii) SCF (1:200: santa-Cruz, sc-113126), (iv) CYP17A1 (1:2500: Fowler et al., 2007a), (v) SOD2 (1;2500: abnova Corp, Taipei, Taiwan, H00006648), all combined with an anti-\u03b2-actin load control of differing species (mouse 1:5000 AB6276; rabbit 1:10 000 AB8227, both AbCam Ltd, Cambridge, UK). The protein bands were visualized using an Odyssey infrared fluorescence imager (LI-COR) and the resulting electronic images were analysed using Phoretix-1D Advanced software (Nonlinear Dynamics Ltd, Newcastle upon Tyne, UK) in order to determine the band volumes and molecular weights. This software calculates band volumes, based on constant lane width and automatic band selection, from the raw data of pixel area and intensity that are independent of operator-altered contrast or brightness. The band volumes of \u03b2-actin were compared between groups to check the validity of this load control for ovaries from control and treated groups.\nProteomics\nIn the present study, we have selected proteomic methods as an exploratory tool to uncover novel effects on the ovary; we have previously demonstrated the utility of this approach for heterogeneous tissues (Fowler et al., 2007a,b) and others have used it to characterize responses to endocrine disruption (Alm et al., 2006).\nTwo-dimensional gel electrophoresis\nSoluble proteins were analysed by two-dimensional gel electrophoresis using a small format gel system described previously (Cash and Kroll, 2003; Uwins et al., 2006), using pooled (control versus sewage sludge) lysates comprising equal quantities of protein from each ovary (15 ovaries in the control pool, 8 ovaries in the treated pool). Briefly, in order to ensure the clearest electrophoresis of the protein by two-dimensional gel electrophoresis, sample clean-up was performed using ReadyPrep 2-D Cleanup Kits (Bio-Rad Laboratories Ltd, Hemel Hempstead, Herts, UK) according to manufacturer\u2019s instructions. Hundred microgram of total protein from the pooled lysates were separated in the first dimension on 7 cm pH 4\u20137 immobilized pH gradient gels (GE Healthcare, Uppsala, Sweden). For the second dimension, the proteins were resolved on 10\u201315% gradient polyacrylamide slab gels and proteins were detected using Colloidal Coomassie Blue G250. Four replicate gels were electrophoresed from each lysate pool. Stained gels were scanned using a Molecular Dynamics Personal Densitometer (GE Healthcare) at 50 \u00b5m resolution to generate 12-bit images, which were transferred to Phoretix 2D Analytical software, V 6.01 (Nonlinear Dynamics, Newcastle, UK). The semi-automated routines available in this software were used to detect and quantify protein spots as well as to match the profiles across a gel series. Individual spot volumes are expressed as normalized volumes relative to the total detected spot volume separately for each gel, minimizing potential analytical artefacts from variations in protein loading and migration.\nMass spectroscopic protein identification\nProteins in the gel pieces were digested with trypsin (sequencing grade, modified; Promega UK, Southampton, UK) using an Investigator ProGest robotic workstation (Genomic Solutions Ltd., Huntingdon, UK). Briefly, proteins were reduced with DTT (60\u00b0C, 20 min), S-alkylated with Iodoacetamide (25\u00b0C, 10 min) then digested with trypsin (37\u00b0C, 8 h). The resulting tryptic peptide extract was dried by rotary evaporation (SC110 Speedvac; Savant Instruments, Holbrook, NY, USA) and dissolved in 0.1% formic acid for LC-MS\/MS analysis. Peptide solutions were analysed using an HCTultra PTM Discovery System (Bruker Daltonics Ltd., Coventry, UK) coupled to an UltiMate 3000 LC System [Dionex (UK) Ltd., Camberley, Surrey, UK]. Peptides were separated on a Monolithic Capillary Column (200 \u00b5m i.d. \u00d7 5 cm; Dionex part no. 161409). Eluent A was 3% acetonitrile in water containing 0.05% formic acid, Eluent B was 80% acetonitrile in water containing 0.04% formic acid with a gradient of 3\u201345% B in 12 min at a flow rate of 2.5 \u00b5l\/min. Peptide fragment mass spectra were acquired in data-dependent AutoMS(2) mode with a scan range of 300\u20131500 m\/z, three averages, and up to three precursor ions selected from the MS scan 100\u20132200 m\/z. Precursors were actively excluded within a 1.0 min window, and all singly charged ions were excluded. Peptide peaks were detected and deconvoluted automatically using Data Analysis software (Bruker). Mass lists in the form of Mascot Generic Files were created automatically and used as the input for Mascot MS\/MS Ions searches of the NCBInr database using the Matrix Science web server (www.matrixscience.com). The default search parameters used were: enzyme = Trypsin; Max. Missed cleavages = 1; Fixed modifications = Carbamidomethyl (C); Variable modifications = Oxidation (M); Peptide tolerance \u00b1 1.5 Da; MS\/MS tolerance \u00b1 0.5 Da; Peptide charge = 2+ and 3+; Instrument = ESI-TRAP. Only proteins showing good agreement with mass and pI on the two-dimensional gels, statistically significant MOWSE scores and good sequence coverage were considered to be positive identifications.\nHormone assays\nFetal serum levels of follicle-stimulating hormone (FSH), estradiol and prolactin (PRL) were measured by radioimmunoassays that have been described and validated previously for sheep (McNeilly et al., 1986; Mann and Lamming, 1995; Lincoln et al., 2003; Crawford et al., 2004). The assay standards used, assay sensitivities and intra-assay coefficients of variation were: FSH; NIDDK-FSH-RP2 and NIH-LS18, 0.1 ng\/ml, <10%, estradiol; MAIA estradiol kit (Serono Diagnostics, Fleet, Hants, UK), 0.2 pg\/ml, <12%, PRL; NIDDK-PRL\u2014RP3, 0.5 ng\/ml, 10%). All fetal blood samples were measured in single assays.\nStatistical analysis\nAnalyses were performed using JMP (5.1, Thomson Learning, London, UK). Normality of data distribution was tested with the Shapiro\u2013Wilk test and non-normally distributed data were log-transformed prior to analysis. Morphological and endocrine data and the one-dimensional gel electrophoresis WB band volumes (normalized relative to \u03b2-actin expression separate for each lane) and normalized spot volumes (% of total spot volume for each gel separately) were compared as control versus sewage sludge-exposed using one-way ANOVA. For the proteomics, virtual \u2018average\u2019 gels were prepared so that only spots present in three out of four gels for each group were included and compared to determine differences in spot expression. Spots demonstrating statistically significant differences (ANOVA) in normalized volume were investigated further. Unless stated, otherwise data are presented as mean \u00b1 SEM.\nResults\nMaternal and fetal body weights and fetal ovary weights and endocrinology\nThere was no difference in the body weights, at the time of slaughter, of the ewes pastured on control and sewage sludge-treated fields (83 \u00b1 2 versus 84 \u00b1 1 kg, respectively). In contrast, the female fetuses from the ewes maintained on the sewage-treated pastures were significantly (P = 0.013) lighter (14%) (Table\u00a0I) than the controls [and similar to the male fetuses (Paul et al., 2005)]. Despite difference in body weight, there was no significant (P > 0.05) treatment effect on the ovary weights. When ovary weights were normalized to body weight (mg\/kg), the difference between treated and control animals remained non-significant (treated 22.7 \u00b1 3.0 versus control 18.8 \u00b1 1.1 mg\/kg, P > 0.05). Of the three hormones measured, only PRL was significantly affected by exposure to sewage sludge (reduced 49% compared with controls, P = 0.011) although estradiol concentrations tended to be lower (22%) than the controls.\nTable\u00a0I.\nEffects of sewage sludge on morphological and endocrine characteristics of Day 110 fetal ewes.\nCharacteristic\nControl n = 15\nSludge-exposed n = 8\nFold-change following sludge-exposure\nANOVA\nBody weight (g)\n1593 \u00b1 67\n1365 \u00b1 67\n\u22121.17\nP = 0.013\nOvary weight (mg)\n29.4 \u00b1 2.1\n31.0 \u00b1 4.2\n+1.05\nNS\nFSH (ng\/ml)\n2.1 \u00b1 0.3\n1.4 \u00b1 0.2\n\u22121.50\nNS\nEstradiol (pmol\/l)\n128 \u00b1 54\n100 \u00b1 27\n\u22121.28\nNS\nPRL (ng\/ml)\n3.7 \u00b1 0.7\n1.9 \u00b1 0.3\n\u22121.95\nP = 0.011\nLack of statistical significance (NS) is indicated where P > 0.05.\nEffects of treatment on oocyte and follicle numbers and mitotic index\nThe densities of oocytes and follicles were similar, whether quantified by assessing H&E stained slides or MCL1 and GDF9 positive oocytes (Fig.\u00a01a and b). Total oocyte densities were significantly reduced in the treated ovaries assessed by H&E staining (19% reduced: 18.8 \u00b1 1.3 in treated versus 23.1 \u00b1 1.6 in control, P = 0.042), MCL1 immunostaining (26% reduced: 14.6 \u00b1 2.1 in treated versus 19.6 \u00b1 1.5 in control, P = 0.039, Fig.\u00a01) and GDF9 immunostaining (28% reduced: 13.9 \u00b1 1.9 in treated versus 19.4 \u00b1 1.4 in control, P = 0.034). The ratio of different follicle classes and isolated oocytes was also significantly skewed (P = 0.003 by two-way ANOVA combining treatment with follicle size class) by treatment (Fig.\u00a01e) with a slightly, but significantly, greater proportion of the follicles being further developed following EC exposure: (combining classes 1a, 2 and 3: treated 16.1 \u00b1 1.8 versus control 11.2 \u00b1 0.9, P = 0.040). There were no statistically significant effects of sewage sludge exposure (P > 0.05) on the mitotic index of granulosa cells (0.6 \u00b1 0.3 in treated versus 1.0 \u00b1 0.2 in control), stromal cells (14.1 \u00b1 1.3 in treated versus 13.3 \u00b1 1.0 in control) or surface epithelial cells (0.3 \u00b1 0.2 in treated versus 0.5 \u00b1 0.1 in control), expressed as pH3-positive cells\/mm2. In contrast, sewage sludge-exposed ovaries had significantly (P = 0.014) more pH3-positive endothelial cells\/mm2 (1.2 \u00b1 0.3 treated versus 0.3 \u00b1 0.2 control).\nFigure\u00a01:\nEffect of sewage sludge exposure on fetal ovarian morphology.\nImmunolocalization of (a) MCL1, (b) GDF9 and (c) phosphorylated histone H3 expression in representative Day 110 fetal ovary sections. In (a) and (b), the arrow highlights immunopositive oocytes, whereas in (c), the arrow shows a histone pH3 positive granulosa cell. GDF9 and MCL1 immunopositive oocytes were present in all classes of follicles observed. Quantification (n = 15 control versus 8 treated ovaries) of the MCL1 immunohistochemistry is shown in (d) demonstrating the significant decrease in oocyte density in sewage sludge-exposed fetuses and (e) demonstrating the small but significant increase in the proportion of more advanced follicles in sewage sludge-exposed fetuses. Since the findings were similar between H&E staining and MCL1 and GDF9 positive oocytes, only the MCL1 data are shown to illustrate these results.\nEffects of treatment of markers of apoptosis, developmental signalling, steroidogenesis and response to oxidative stress\nThere were no significant differences in \u03b2-actin band volumes between treated and control groups, demonstrating the validity of this load control. In utero exposure to sewage sludge pasture altered the balance between pro- and anti-apoptotic processes in the fetal ovaries. Whereas BAX was significantly higher in treated ovaries relative to the controls (65%, P = 0.002), BCL2 tended to be lower but this difference did not reach statistical significance (54%, P > 0.05, Fig.\u00a02a and b). Expression of the key ovarian developmental signalling protein, SCF, was not different between controls and sludge-exposed ovaries (Fig.\u00a02c). CYP17 expression tended to be lower (34%) in the treated compared with the control ovaries, but again this difference did not reach statistical significance (Fig.\u00a02d). SOD2 was detected as both the mature 15 kDa form (weakly) and the 25 kDa precursor form (Fig.\u00a02e and f). The precursor form tended to be lower (22%, P > 0.05), but the mature molecule was significantly lower (by 42%) in the treated compared with the control ovaries (P = 0.031).\nFigure\u00a02:\nQuantification of levels of Day 110 fetal ovarian proteins (n = 15 control versus 8 treated ovaries) involved in (a) pro-apoptosis, BAX, (b) anti-apoptosis, BCL2, (c) developmental cell signalling, stem cell factor (SCF), (d) steroidogenesis, CYP17 and (e and f) oxidative stress response, manganese superoxide dismutase (SOD2).\nP-values denote significant differences between control and sewage sludge-exposed fetuses and the load control was \u03b2-actin at 42 kDa.\nEffects of treatment on the fetal ovarian proteome\nThere was little difference in the overall number of robust protein spots between the control (349 \u00b1 9) and sewage sludge (354 \u00b1 5) groups (P > 0.05); however, 147 protein spots showed significant (P < 0.05) differences in normalized volume and presence or absence in the sewage sludge compared with controls, based on a minimum change of \u22651.2-fold, P < 0.05, for inclusion. The differentially expressed protein spots covered a wide range of normalized spot volumes. In the sewage sludge ovarian proteome, a total of 52 spots were up-regulated (1.2\u20134.6-fold increased, P < 0.05), 46 were down-regulated (1.2\u20135.2-fold decreased, P < 0.05), 25 were absent (only present in the control group) and 24 were unique (only present in the sewage sludge group). Nineteen protein spots showing consistent detection, reproducible quantification between replicate gels and statistically significant differences, or very clear uniqueness to one experimental group, were identified by tandem mass spectroscopy (Table\u00a0II and Fig.\u00a03). Six of these proteins were blood protein which showed highly variable alterations in expression (\u22125.2-fold to +3.5-fold, absent to unique) between sewage sludge and control groups, preventing any simplistic links to potential differences in vascularization. The remaining 13 proteins fell into four broad functional categories: (i) cytoskeleton and its regulation, (ii) gene expression, transcription and processing, (iii) protein synthesis and (iv) protein phosphorylation and receptor activity. Pathway Architect software (Stratagene Europe, Amsterdam, The Netherlands) was used to inform on the functional links between the proteins that were seen to exhibit altered expression between the two groups. Six of these proteins were directly linked with translation (Fig.\u00a04a), and SOD2 and BAX are directly associated with DNA fragmentation during apoptosis (Fig.\u00a04b). Graphical representation of an interactions network demonstrates that CALM1 dominates the relationships between many of the differentially expressed proteins (Fig.\u00a05).\nFigure\u00a03:\n2-DE gel electrophoresis based proteomic analysis of Day 110 fetal ovaries (n = 15 control versus 8 treated ovaries) pooled into treated and control groups.\n(a) Representative 2-DE 7 cm gel using a 4\u20137 pH gradient. The numbered arrows show the location of the 19 protein spots identified in Table\u00a0II. Zoom boxes demonstrating representative protein spots showing (b) down-regulation and (c) up-regulation in fetuses exposed to sewage sludge. The histograms are based on averaged (n = 4 gels\/group) spot volume (normalized relative to total spot volumes separately for each gel). Significant differences are based on ANOVA following log-transformation of spot volumes.\nFigure\u00a04:\nBiological processes network of some of the differentially expressed proteins from the treated ovaries, demonstrating key interactions between CALM1 and HNRPH in regulating (a) translation and antagonism between SOD2 and BAX in (b) DNA fragmentation during apoptosis.\nOutput from Pathway Architect.\nFigure\u00a05:\nBiological interactions network of some of the differentially expressed proteins from the treated ovaries.\nCALM1 sits central within the network, interacting with most of the differentially regulated proteins via one or two steps. Ovals with a green background denote small molecules. Output from Pathway Architect software (Stratagene Europe, Amsterdam, the Netherlands).\nTable\u00a0II.\nPositively identified proteins showing differential expression between ovaries at GD110 from 15 control fetuses with 8 fetuses exposed to sewage sludge in utero.\nSpot #\nProtein name GENE SYMBOL\nProtein function and Tissue\/cellular location\nMW (kDa)\npI\nMOWSE score\nAccession number (NCBI)\nFold change (P, ANOVA)\nCytoskeleton and its regulation\n1\nGelsolin [actin depolymerising factor (ADF), brevin] GSN\nPromotes nucleation and severs formed filaments. Expression lost in ovarian carcinoma. Cytoplasm\n80.9\n5.58\n429\n2833344\n\u22122.8 (P = 0.020)\n2\nVinculin VCL\nInvolved in cell adhesion and focal complex assembly with role in actin microfilament attachment to plasma membranes. Cytoskeleton, cell\u2013cell junctions.\n117.2\n5.83\n1050\n4507877\n\u22123.4 (P = 0.043)\n9\nTubulin \u03b1-chain (alpha-tubulin 1) TUBA1\nMajor constituent of microtubules, binds two GTP molecules. Cytoskeleton\n50.6\n4.97\n383\n3502919\n+3.1 (P < 0.001)\nBlood proteins\n5\nSerum albumin precursor ALB\nSecreted protein, typically into plasma, binding molecule, regulates colloidal osmotic pressure\n71.1\n5.82\n251\n162648\n\u22125.2 (P = 0.003)\n10\n97\n+3.0 (P < 0.001)\n12\n189\nAbsent\n8\nAlbumin precursor ALB\n71.1\n5.80\n483\n1387\n+3.5 (P = 0.010)\n16\n646\nUnique\n14\nSerotransferrin precursor (transferrin, siderophilin) TF\nIron-binding transport protein and stimulation of cell proliferation\n79.9\n6.75\n243\n29135265\n\u22123.1 (P = 0.003)\nGene expression, transcription and processing\n3\nDNA-binding pur alpha PURA\nInteracts with RNA and DNA and recruits regulatory proteins to specific nucleic acid sequences, stimulating transcription. Nucleus\n34.8\n5.88\n61\n9652255\n\u22125.0 (P < 0.001)\n7\nHeterogeneous nuclear ribonucleoprotein K (HNRPK) HNRPK\nBinds RNA, interacts with pur alpha to mediate repression of CD43 promoter. Ribonucleoprotein complex\n51.3\n5.14\n598\n74354615\n+3.1 (P = 0.023)\n11\nHeterogeneous nuclear ribonucleoprotein H (HNRNPH) HNRPH1\nPart of complex providing substrates for processing of pre-mRNA. Nucleus\n49.5\n5.89\n475\n10946928\nAbsent\nProtein synthesis\n4\nEndoplasmic reticulum protein ERp29 (ERp31 or ERp28) ERP29\nMolecular chaperone, participates in the folding of secretory proteins in endoplasmic reticulum. Endoplasmic reticulum\n28.9\n5.63\n270\n109658363\n\u22123.2 (P = 0.002)\n6\nCalmodulin (CaM) CALM1\nMediates the control of enzymes and proteins (e.g. protein kinases and phosphatases) by Ca, role in calcium signalling. Cytoplasm, cell membrane\n16.8\n4.09\n224\n115509\n\u22123.1 (P = 0.033)\n17\nTu translation elongation factor (EF-Tu) TUFM\nRole in protein synthesis, promoting GTP-dependent binding of tRNA to ribosomes. Mitochondria\n49.7\n6.72\n235\n111304949\nUnique\n18\nTumor rejection antigen 1 (GP96) (HSP90 family) TRA1 (GRP94, HSP90B1)\nMolecular chaperone during processing, folding and transport of secreted proteins in the endoplasmic reticulum. Endoplasmic reticulum\n92.7\n4.76\n727\n27807263\nUnique\nProtein phosphorylation and receptor activity\n13\nGuanine nucleotide-binding protein G, beta 1 (transductin beta 1 chain) GNB1\nPart of G-protein heterotrimer, transduces transmembrane signalling systems. Cell membrane, cytoplasm\n38.2\n5.60\n384\n6680045\nAbsent\n15\nProtein phosphatase 1D PPM1D (WIP1)\nRequired for relief of p53-dependent checkpoint mediated cell cycle, negatively regulates cell proliferation. Nucleus\n38.0\n5.84\n351\n227436\n+4.6 (P = 0.010)\n19\nS100 calcium-binding protein A11 (similar to) S100A11\nProposed function is calcium-ion binding and signal transduction, negative regulation of cell proliferation. Cytoplasm\n11.5\n6.72\n202\n29135265\nUnique\nDiscussion\nThe aim of this study was to establish whether long-term exposure of pregnant ewes to a cocktail of ECs disrupts female fetal ovarian development. Our results clearly show that long-term dietary exposure to such a cocktail, delivered via application of sewage sludge fertilizer to pasture, significantly disturbs both the fetal ovary and fetal endocrinology. These findings of alterations in the ovary were associated with a mild but significant growth restriction of the female fetuses.\nIn utero exposure of the female fetus to ECs results in a very wide range of morphological changes, varying according to species and nature of the chemicals (reviewed Miller et al., 2004). Although 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) does not produce the reduction in rodent oocyte and follicles numbers that we find (Flaws et al., 1997), PCBs and polycyclic aromatic hydrocarbons (PAHs) do (Flaws et al., 1997; Matikainen et al., 2002). The actions of other chemicals in the sewage sludge with effects on reproduction, such as cadmium (Henson and Chedrese, 2004), must also be taken into account. However, it is unlikely that our results were due to elevated phytoestrogens since studies of phytoestrogens (usually in rodents), such as genistein (e.g. Jefferson et al., 2006), show decreased germ cell apoptosis and reduced breakdown of oocyte nests, quite different from our findings of reduced oocyte density in fetuses exposed to sewage sludge chemicals in utero. This is extremely interesting given that ovarian BAX expression was significantly higher in sludge-exposed fetuses. In the mouse, BAX is a key regulator of follicle numbers and the BCL family is heavily implicated in the mechanisms underlying detrimental effects of selected environmental chemicals on the ovary (Borgeest et al., 2004; Miller et al., 2005; Greenfeld et al., 2007a,b). It is most probable that similar mechanisms act to determine the follicle pool in all mammals. That the anti-apoptotic BCL2 tended to be reduced, although not significantly so, in the sludge-exposed fetuses, suggests a redistribution of the pro- and anti-apoptotic balance more towards apoptosis. These findings agree with our observation that there were no differences in granulosa cell mitotic index and lead to the conclusion that the reduction in follicle numbers that we observed was due to an early loss of germ cells. Should these effects be reproduced in women exposed in utero to ECs as fetuses, it would be likely to result in reduced fertility and earlier onset of menopause. SCF was not affected by exposure to an EC cocktail in sewage sludge demonstrating the subtlety of the effects of a cocktail of chemicals. Clearly, while oocyte density is lower, the pre-granulosa or granulosa cells were producing the same amount of SCF, which is important in early follicle development (Parrott and Skinner, 2000; Jin et al., 2005). Sewage sludge exposure did not significantly affect the mitotic index (density of pH3-positive cells) of any ovarian cell type studied, other than endothelial cells. This suggests an effect on ovarian angiogenesis and supports our conclusion that chemicals in the sewage sludge are acting on the fetus since EDCs are know to have both stimulatory and inhibitory effects on angiogenesis (Tavolari et al., 2006).\nAlthough neither was statistically significant, our findings of reduced levels of circulating estradiol and lowered ovarian expression of CYP17 in sludge-exposed fetuses were consistent with each other and suggest a mild impairment of steroidogenesis in these animals, possibly related to reduced germ cell proliferation (Pannetier et al., 2006) in sewage sludge-exposed fetuses. We quantified SOD2, because reactive oxygen species (ROS) induced by some ECs can damage developing organs (Ahmed et al., 2005). Further, methoxychlor and heavy metals decrease the expression of protective enzymes such as SOD1 and SOD2 in ovarian cells (Gupta et al., 2006; Nampoothiri et al., 2007), which would have developmental and steroidogenic consequences. Our animals exposed to a real-life complex cocktail of ECs revealed a similar trend since the levels of SOD2 were lower than that in the controls and thus their ovarian cells may be more susceptible to oxidative damage than controls. The reduction in SOD2 would also imply reduced inhibition of DNA fragmentation during apoptosis at a time when increased BAX will elevate apoptosis rates (Fig.\u00a04b; Southgate et al., 2006). The fact that phytoestrogens, such as daidzein, reduce ROS-induced toxicity (Tang et al., 2006), contrasts sharply with the effects of exposure to sewage sludge chemicals emphasizing that the observed effects are not due to phytoestrogens.\nExposure to sewage sludge chemicals altered the expression patterns of 42% of the protein spots in the exposed compared with control ovaries. Although this by no means reflects the entire ovarian proteome, it indicates that chemicals in sewage sludge can cause widespread changes in the ovary. Some of these changes will reflect altered cell numbers, but some will reflect changes in pathways involved in ovarian function and development. In addition, proteins may be present in many spots and spots may contain several proteins and so changes in spot volumes reflect complex and sometimes very subtle alterations in the protein profile. Proteomics allows us to investigate global effects of exposure to environmental chemicals without preconceptions about pathways and mechanisms, complementing the targeted study of ovarian morphology and proteins of known importance in reproductive development. We targeted the most consistently, statistically significantly, altered proteins for identification. This addresses gel\u2013gel variation and reduces potential methodological error. In addition, we limited ourselves to proteins showing a \u22652-fold change in expression. Liquid-chromatography-mass spectrometry enabled positive identification of all 19 selected protein spots that fit the selection criteria. Overall, the proteins showing disturbed expression would be consistent with altered transcription and translation, apoptosis\/proliferation and protein production\/actions within the developing ovary. The disturbed proteins could also result in dysregulated receptor and calcium-dependent functions.\nThe most obvious consequence of the altered proteins is the probable disturbance in translation since 6 of the 19 differentially expressed proteins, that we identified, have interactions and effects on translational processes (Fig.\u00a04a). This would have consequences for the expression of gene products during development. The reduction of CALM1 protein in sludge-exposed fetal ovaries was unexpected because up-regulation of CALM1 is characteristic of genomic estrogenic activation (Wang et al., 2004). This, therefore, is evidence that exposure to sewage sludge was not necessarily activating conventional genomic estrogen pathways. Indeed, it is now well known that environmental chemicals operate through a wide range of mechanisms and pathways in addition to genomic estrogen action (Henley and Korach, 2006; Tabb and Blumberg, 2006). The 5-fold reduction of DNA-binding pur-alpha (PURA) in ovaries from fetuses exposed to sewage sludge chemicals is interesting on two counts. First, PURA, which recruits regulatory proteins to RNA and DNA sequences, is involved in oncogenic inhibitory pathways (Johnson et al., 2003), and, second, CALM1 stimulates PURA (Kuo et al., 1999), so a reduction in both proteins suggests a causative link. The location of CALM1 at the node of a biological interactions network that links most of the proteins we observed to be affected by exposure to sewage sludge chemicals (Fig.\u00a05) demonstrates how changes in protein expression could propagate down numerous pathways involved from transcription along to post-translational modification and receptor activity. Relevantly, calmodulin-dependent kinases are implicated in maintaining ovarian steroidogenesis (Seals et al., 2004) and calmodulin binding protein is expressed in fetal germ cells during development (Luers et al., 2002), whereas defects in the caldesmon\/calmodulin system impairs cell cycling and migration (Li et al., 2004). Gelsolin (GSN), 2.8-fold reduced in treated ovaries, was of interest since its expression is reduced in ovarian cancer (Noske et al., 2005). GSN, which modulates cellular motile activities, is widely expressed in the developing embryo (Arai and Kwiatkowski, 1999) and adult ovary (Teubner et al., 1994) where it is important in follicle growth. The role of vinculin (VCL) in a variety of processes, such as granulosa cell differentiation (Kranen et al., 1993), suggests that its down-regulation in our study may have consequences for ovarian development.\nThe HNRNP family of proteins is important in the formation of mRNA and both H and K forms are highly expressed in the primary oocyte (Kamma et al., 1995). HNRNPK, up-regulated in sludge-exposed ovaries, is important for cell spreading (Yoo et al., 2006) and transcriptional responses to DNA damage (Moumen et al., 2005). HNRNPK and PURA act together in transcriptional regulation of leukocytes (Da Silva et al., 2002) and, should both proteins act together on ovarian cells, disruption of transcription would be expected. Pertinently, the HNRNPH homologue, glorund, represses ovarian expression of Drosophila nanos (Kalifa et al., 2006), which is highly conserved and important for germ cell migration in mouse embryos (Tsuda et al., 2003). This contrasts with our study where HNRNPH was greatly reduced, which would suggest increased germ cell numbers. Disturbance of transcription would have multiple effects in many cell types, although redundancy of function could greatly reduce the severity of phenotypic effects.\nThe endoplasmic reticulum is important in protein secretion and many cellular activities, such as response to oxidative stress, so it is interesting that two endoplasmic reticulum chaperone proteins ERP29 and TRA1 (also called GRP94) were reduced and increased, respectively, by exposure to sewage sludge chemicals. Although not well understood, endoplasmic reticulum chaperone proteins are important in developmental and disease processes, including programmed cell death (Rao et al., 2006; Ni and Lee, 2007). TRA1 is abundant in the adult oolemma (Calvert et al., 2003) and regulates the secretion of a key player in ovarian function: insulin-like growth factor (Knight and Glister, 2006; Wanderling et al., 2007). TRA1 is increased in endoplasmic reticulum stress (Hagg et al., 2004) underlining the relevance of this finding. Although ERP29 is redox-inactive (Mkrtchian and Sandalova, 2006), it may be a target for reactive metabolites of toxic chemicals like bromobenzene (Koen and Hanzlik, 2002). The fact that pituitary levels of ERP29 are increased by exposure to estrogen (Blake et al., 2005), but decreased in our sewage sludge-exposed ovaries, may indicate either non-estrogenic effects or differential responses in the ovary. The increase in Tu translation elongation factor (TUFM), which has chaperone activities during protein synthesis (Suzuki et al., 2007), is interesting since mutations in elongation factors have developmental pathologies (Smeitink et al., 2006). Increased PPM1D and decreased GNB1 in the sludge-exposed ovaries imply effects on phosphorylation and receptor activity. PPM1D over-expression amplifies tumorigenesis by suppressing p53 activation (Bulavin et al., 2002) and enhances progesterone receptor activation (Proia et al., 2006). Importantly, progesterone receptor activation reduces primordial follicle assembly (Kezele and Skinner, 2003). This may be part of the mechanism by which sewage sludge-exposed ovaries exhibit reduced density of follicles at Day 110 of gestation.\nThe sludge-exposed female fetuses were 14% lighter than controls which raise the issue of what caused this difference and what effect this apparent very mild growth restriction might have on fetal ovarian development. There were no differences in maternal body weight in our study, which would contradict any conclusion that sludge-exposed fetuses were nutritionally challenged. The literature (reviewed Rhind et al., 2003) suggests that such a mild body weight difference is highly unlikely, in itself, to cause the ovarian developmental perturbation that we report here. For instance, a 50% maintenance diet during gestation resulted in a 32% decrease in lamb body weight and a 23% reduction in fetal ovary weight, but no loss of germ cells (Murdoch et al., 2003). More detailed studies of maternal undernutrition during pregnancy in sheep (e.g. Rae et al., 2001) found that cutting energy provision during pregnancy to 50% induced a trend towards smaller ovaries and significantly fewer more advanced follicles (types 2, 3 and 4) without changing germ cell numbers. This contrasts with our study in which the proportion of more advanced follicle increased, possibly contributing to our finding that ovary weight was not different between controls and sludge-exposed fetuses. Following growth-restricted fetuses into adulthood finds them recovering body mass with apparently normal hypothalamo-pituitary-ovary-axes, although ovulation rates were 20% reduced (Rae et al., 2002; Borwick et al., 2003). In the human, girls born small for gestational stage (i.e. growth-restricted) may have small ovaries and reduced ovulation rates at puberty (Ibanez et al., 2000, 2002), an effect that is at least partly related to insulin resistance (Ibanez and de Zegher, 2006), but probably not due to a reduction in the fetal follicle pool (de Bruin et al., 2001). The ovaries from our sludge-exposed fetuses are very different: no reduction in ovarian weight, but reduced germ cell and follicle densities and a small but significant increase in the proportion of more advanced follicles. Thus, we can conclude that growth restriction was not a major factor and, therefore, the effects of exposure to sewage sludge are much more likely to be due to chemicals in the sludge. That both the sewage sludge exposure in our study and the growth restriction of the sheep fetus (Phillips et al., 2001) have some similar reproductive consequences suggests some commonality in regulatory pathways between the two forms of fetal insult.\nIt is not clear which components of sewage sludge are reaching the maternal and fetal tissues and causing the disturbed fetal development we observed. It is currently impossible to accurately measure many chemicals in the fetal gonad because of the limited tissue quantity available. Therefore, we cannot be confident of actual fetal ovarian exposure to different chemicals in our study. Some tissue EC levels do differ significantly between control and sludge-exposed fetuses (see Introduction), but it is most likely that subtle and complex alterations in the balance between a very wide range of chemicals, probably affecting a variety of body systems and organs, is the key factor in causing the disruption of fetal ovarian development. This is supported by our finding that although maternal smoking during gestation does not lead to any major changes in fetal liver PAH concentrations, the fetuses were correctly allocated to smoking and non-smoking groups by discriminant analysis of liver PAH concentrations (Fowler et al., 2008).\nThe sheep in our study were pastured on fields treated with sewage sludge in a way that may have raised exposure above the levels to which sheep are normally exposed to under current UK\/EC legislation and agricultural guidelines; the treatments were designed to maximize the risk of exposure. It is important to note that our studies are designed to elucidate the effects of prolonged exposure to a real-life cocktail of chemicals and are not designed to determine whether the use of processed human sewage sludge as a fertilizer poses human health risks (under the current best operating practices). With this proviso, we can strongly conclude from our study that long-term exposure to low doses of a complex \u2018real-life\u2019 cocktail of environmental chemicals affects the developing fetal ovary, probably starting very early in fetal life, in such a way as to suggest that similar human fetal exposures could reduce fertility of the resulting women and increase their chances of premature menopause. Future studies should address genetic and endocrine mechanisms involved in these effects and also establish the critical windows of exposure, as well as determining the fertility of resulting adults.\nFunding\nThe Wellcome Trust (080388) to P.A.F.; NHS Research & Development (1664) to P.A.F.; the Scottish Executive Environment & Rural Affairs Department (302132) to S.M.R.","keyphrases":["environmental chemicals","sewage sludge","oocyte","fetal development","granulosa cell"],"prmu":["P","P","P","P","P"]}
{"id":"Diabetologia-3-1-1914278","title":"Perturbation of hyaluronan metabolism predisposes patients with type 1 diabetes mellitus to atherosclerosis\n","text":"Aims\/hypothesis Cardiovascular disease contributes to mortality in type 1 diabetes mellitus, but the specific pathophysiological mechanisms remain to be established. We recently showed that the endothelial glycocalyx, a protective layer of proteoglycans covering the endothelium, is severely perturbed in type 1 diabetes, with concomitantly increased plasma levels of hyaluronan and hyaluronidase. In the present study, we evaluated the relationship between hyaluronan and hyaluronidase with carotid intima-media thickness (cIMT), an established surrogate marker for cardiovascular disease.\nIntroduction\nMacro- and microvascular complications are a major cause of morbidity and mortality in patients with diabetes mellitus. While the macrovascular complications in patients with type 2 diabetes mellitus can partly be attributed to the increased prevalence of classic cardiovascular risk factors such as dyslipidaemia, these risk factors cannot explain the increased prevalence of atherosclerosis in type 1 diabetes mellitus [1\u20133]. Recent data demonstrated that hyperglycaemia itself may play a causative role. Thus, improved metabolic control is associated with a decreased macrovascular event rate [1\u20133]. However, the pathophysiology of glucose-associated atherogenesis remains to be elucidated [4, 5]. In recent years, the glycocalyx has emerged as a potential orchestrator of vascular homeostasis, which closely determines anti-adhesive and barrier properties of the vessel wall [6]. In line, we found that the endothelial glycocalyx is adversely affected by both acute and chronic hyperglycaemia in volunteers and type 1 diabetes patients, respectively [7, 8].\nHyaluronan is a principal constituent of the glycocalyx and removal of the glycocalyx with hyaluronidase has been associated with increased vascular vulnerability towards atherogenic insults [9\u201311]. In animal models of type 1 diabetes, hyaluronidase activity has been shown to be increased and this correlated with increased carotid intima-media thickness (cIMT) [12\u201314]. In line with this, increased accumulation of hyaluronan within the arterial wall in type 1 diabetes patients correlated with vascular changes [15]. We recently found an acute increase in plasma hyaluronan coinciding with glycocalyx perturbation during a normo-insulinaemic\u2013hyperglycaemic clamp in healthy volunteers [7]. Moreover, we observed an inverse correlation between plasma hyaluronan as well as plasma hyaluronidase and glycocalyx volume in patients with type 1 diabetes [8]. In this concept, hyperglycaemia-induced perturbation of hyaluronan metabolism, characterised by increased hyaluronidase activity with subsequent increased plasma hyaluronan levels, may indicate increased vascular vulnerability.\nIn the present study, we set out to evaluate the potential relationship between structural changes of the carotid artery and hyaluronan metabolism in patients with uncomplicated type 1 diabetes.\nSubjects and methods\nWe enrolled non-smoking Europid patients with type 1 diabetes, all without clinical signs of micro- or macrovascular disease. The patients were recruited from the Internal Medicine outpatient clinics of the Academic Medical Center and Onze Lieve Vrouwe Gasthuis in Amsterdam, the Netherlands. The presence of macrovascular disease, defined as ECG abnormalities or a history of cardiac, cerebral or peripheral vascular events, was an exclusion criterion for the study. Moreover, subjects with retinopathy, neuropathy, (micro) albuminuria, or hypertension were excluded from participation. All patients were on multiple daily injections of insulin with no other concomitant medication use. Matched non-smoking controls (selected for this study specifically) were unrelated volunteers of similar age and sex. Investigations of both study groups were randomly performed during the study period. Approval for the study was obtained from the Internal Review Board of the Academic Medical Center Amsterdam and all subjects gave written informed consent. The study was carried out in accordance with the principles of the Declaration of Helsinki.\nAll measurements were performed after an overnight fast and in a quiet and air-conditioned room. Blood pressure was measured in triplicate and the last two measurements were averaged to obtain heart rate and systolic and diastolic blood pressure. The latter were averaged to calculate mean blood pressure. At baseline, blood samples were collected for determination of lipids, high sensitivity C-reactive protein (hsCRP), HbA1c, hyaluronan and hyaluronidase. The markers of hepatic function ASAT and ALAT (aspartate aminotransferase and alanine aminotransferase, respectively) were determined, since chronic liver disease is known to be associated with increased plasma hyaluronan levels [16]. After centrifugation (within 1\u00a0h after collection), aliquots were snap-frozen in liquid nitrogen and stored at \u221280\u00b0C.\nClinical chemistry Total cholesterol, HDL-cholesterol and triacylglycerol were measured by enzymatic methods (Roche Diagnostics, Basel, Switzerland). LDL-cholesterol was calculated using the Friedewald formula. ALAT and ASAT were measured by a pyridoxal-phosphate activation assay (Roche Diagnostics). HbA1c was measured using an HPLC (Reagens Bio-Rad Laboratories BV, the Netherlands) on a Variant II (Bio-Rad Laboratories). Total plasma hyaluronan and hsCRP levels were determined in duplicates by commercial ELISA (Echelon Biosciences, Salt Lake City, UT, USA and Roche, Bern, Switzerland, respectively). Plasma hyaluronidase levels were determined with a previously described assay [8, 17].\nUltrasound B-mode protocol for cIMT measurement B-mode ultrasound imaging was used to visualise three carotid arterial wall segments comprising common carotid, bulb and internal of the left and right carotid arteries according to a previously published protocol [18, 19]. Subjects were scanned in the reclined position following a predetermined, standardised protocol. An Acuson 128 XP\/10\u00a0v (Siemens, Erlangen, Germany) equipped with an L7 linear array transducer and extended frequency software was used. B-mode images were stored as 4:1 compressed jpeg files on a digital still recorder (SONY DKR-700 P). All scans were performed by the same sonographer. To investigate intra-sonographer reproducibility, ten study subjects were scanned in duplicate. These investigations enabled us to provide robust arterial wall thickness measurements (SD of the means of the paired cIMT measurements 0.05\u00a0mm; CV\u2009=\u20099.0%). One image analyst performed the analyses off-line with semi-automated quantitative and qualitative video image analysis software. Both the sonographer and the image analyst were blinded to the clinical status of the subjects. These images provided the cIMT data. Mean cIMT was defined as the mean cIMT of the right and left common carotid, the carotid bulb and the internal carotid far wall segments. For a given segment, cIMT was defined as the average of the right and left cIMT measurements. The per-patient averaged means of the cIMT values of segments was used for the primary analysis.\nStatistical analysis Mean values of continuous variables between type 1 diabetes patients and controls were compared using Student\u2019s t test for independent samples. In the case of a skewed distribution the t test was performed on log-transformed values, while medians and interquartile ranges are presented. Chi-square tests were applied for comparison of distribution of dichotomous data. Correlation between hyaluronan and hyaluronidase was calculated by Spearman\u2019s rank coefficient (two-tailed). In this study our main interest was to find predictors for type 1 diabetes-associated atherosclerosis and vascular dysfunction. The relationship between the dependent variable cIMT on the one hand and other parameters (e.g. plasma hyaluronan) on the other was first explored univariably using linear regression analysis. For clinical variables and variables which revealed statistically significant correlations in the univariate analysis, estimates of cIMT adjusted for confounding were calculated with SPSS version 11.5 (Chicago, IL, USA). In addition, several multivariate models were built to explore the effects of age, sex and the statistically significant variables on cIMT. Throughout, a two tailed p value <0.05 was considered statistically significant.\nResults\nClinical characteristics of the 99 type 1 diabetes subjects and 99 matched controls are listed in Table\u00a01. There was no significant difference between type 1 diabetes subjects and controls with regard to age, sex, BMI, systolic and diastolic blood pressure, liver function tests and cholesterol profile. However, we did observe increased values for HbA1c, heart rate, plasma hsCRP, hyaluronan and hyaluronidase in type 1 diabetes patients. A significant correlation was found between plasma hyaluronan and hyaluronidase activity in type 1 diabetes (r\u2009=\u20090.3, p\u2009<\u20090.05, see Fig.\u00a01a). After exclusion of the highest hyaluronidase activity levels (levels >750\u00a0U\/ml), the correlation between hyaluronan and hyaluronidase levels was still present. Liver function tests (ASAT and ALAT) were not significantly associated with plasma hyaluronan levels in type 1 diabetes.\nTable\u00a01Demographic and baseline parameters of the study cohort\u00a0Type 1 diabetes patientsControlsNumber of participants9999Sex (male\/female)44\/5544\/55Age (years)32.8\u2009\u00b1\u200914.834.9\u2009\u00b1\u200916.4Duration of diabetes (years)16.4\u2009\u00b1\u200911.9\u2013Daily insulin dose (IU) 52.9\u2009\u00b1\u200920.3\u2013Smoking (yes\/no)0\/990\/99BMI (kg\/m2)23.4\u2009\u00b1\u20093.523.3\u2009\u00b1\u20093.6Systolic blood pressure (mmHg)123\u2009\u00b1\u200917125\u2009\u00b1\u200920Diastolic blood pressure (mmHg)72\u2009\u00b1\u2009973\u2009\u00b1\u200912Heart rate (beats\/min)71\u2009\u00b1\u200911**60\u2009\u00b1\u200914 Total cholesterol (mmol\/l)4.9\u2009\u00b1\u20090.94.9\u2009\u00b1\u20091.0LDL-cholesterol (mmol\/l)2.8\u2009\u00b1\u20090.72.9\u2009\u00b1\u20090.8HDL-cholesterol (mmol\/l)1.6\u2009\u00b1\u20090.51.5\u2009\u00b1\u20090.4Triacylglycerol (mmol\/l)0.9 (0.5\u20131.1)1.0 (0.5\u20131.2)ASAT (U\/l)24 (21\u201327)25 (21\u201329)ALAT (U\/l)22 (15\u201328)20 (14\u201324)HbA1c (%)8.3\u2009\u00b1\u20091.6**5.1\u2009\u00b1\u20090.3Hyaluronan (ng\/ml)78\u2009\u00b1\u200943*60\u2009\u00b1\u200918Hyaluronidase (U\/ml)362\u2009\u00b1\u200923**242\u2009\u00b1\u200913hsCRP (mg\/l)2.6 (0.4\u20132.9)*1.1 (0.2\u20132.0)cIMT (mm)0.61\u2009\u00b1\u20090.15**0.53\u2009\u00b1\u20090.12Data are means\u00b1SD, except for triacylglycerol, ASAT, ALAT and hsCRP, which are expressed as median (inter-quartile range).*p\u2009<\u20090.05, **p\u2009<\u20090.01 type 1 diabetes patients vs controlsFig.\u00a01a Relationship between plasma hyaluronan levels and plasma hyaluronidase activity in type 1 diabetes patients. b Relationship between plasma hyaluronan levels and cIMT in type 1 diabetes patients\nMean cIMT was increased in the type 1 diabetes group compared with controls (0.61\u2009\u00b1\u20090.15 vs 0.53\u2009\u00b1\u20090.12\u00a0mm, p\u2009<\u20090.001). In type 1 diabetes subjects plasma hyaluronan levels (Fig.\u00a01b), age, male sex, duration of diabetes and mean blood pressure were positively correlated with mean cIMT in univariate analysis. No dose-dependent relationship between insulin dose, HbA1c levels and mean cIMT was found in these patients. However, upon multivariate linear regression analysis only age and sex remained significantly associated with cIMT (Table\u00a02).\nTable\u00a02Univariate and multivariate associations of cIMT with various risk factors in patients with type 1 diabetesParameterUnivariate \u03b2 coefficientp valueMultivariate \u03b2 coefficientp valueFemale sex \u22120.86*0.005\u22120.048*0.029Age0.007*0.0010.07*0.0001Duration of diabetes 0.008*0.001\u22120.1100.340Daily insulin dose\u22120.0010.109BMI0.0040.433Mean blood pressure0.004*0.004\u22120.0270.726Heart rate0.00010.994Total cholesterol0.0160.366LDL-cholesterol0.0210.321HDL-cholesterol0.0260.416Triacylglycerol\u22120.0540.076ASAT0.0370.615ALAT\u22120.0190.589HbA1c\u22120.0080.441Hyaluronan0.126*0.0010.1160.130Hyaluronidase0.0360.731hsCRP\u22120.0130.306*p\u2009<\u20090.05 type 1 diabetes patients vs controls\nDiscussion\nIn line with expectation, type 1 diabetes patients were characterised by structural changes of the arterial wall. In addition, we observed significant elevations of plasma hyaluronan and hyaluronidase activity levels in type 1 diabetes patients, whereas hyaluronan was correlated to cIMT. These present data imply that disturbances of hyaluronan metabolism may be associated with vascular damage in type 1 diabetes patients.\nIntima-media thickness in type 1 diabetes We observed a significant increase in cIMT in type 1 diabetes patients without micro- or macro-vascular complications compared with controls and confirmed that male sex is associated with an increased cIMT [3]. Despite the cross-sectional design of our study, this finding is compatible with data from the longitudinal DCCT study [3]. LDL-cholesterol was not associated with cIMT progression in the DCCT cohort, underscoring a potential role for hyperglycaemia in diabetic atherogenesis [1, 20, 21]. In contrast to the DCCT findings, we did not find a correlation between cIMT and glycaemic status. This apparent discrepancy may have several explanations. First, a single determination of HbA1c in our study may not be a reliable reflection of glycaemic excursions over the last months to years. In the DCCT, HbA1c was evaluated repetitively in a prospective cohort of type 1 diabetes patients, all receiving an intensive treatment regimen [1, 3]. Under these circumstances, HbA1c was predictive of cIMT progression over the next 4\u00a0years. Second, the elderly patients in our cohort may have been exposed to longer periods of poorly controlled diabetes followed by more intensive treatment regimens only in the last few years. As a consequence, their present HbA1c levels may have underestimated long-term glycaemic exposure.\nHyaluronan metabolism and type 1 diabetes Hyaluronan is the principal constituent of endothelial glycocalyx as well as the extra-cellular matrix [7\u201310, 14, 15, 22]. Since the glycocalyx is a principal determinant of vascular permeability for macromolecules (e.g. lipoproteins), glycocalyx loss may contribute to the increased transvascular leakage of lipoproteins in patients with type 1 diabetes [20]. Indeed exposure of the carotid artery to atherogenic challenges in mice resulted in increased plasma hyaluronan shedding, concomitant loss of endothelial glycocalyx and increased cIMT [23, 24]. In line, we observed that patients with uncomplicated type 1 diabetes have a profound reduction of endothelial glycocalyx, which was associated with increased plasma hyaluronan and hyaluronidase levels [8]. In fact, in the present study we find a positive correlation between plasma hyaluronan and cIMT thickness. Upon multivariate analysis this correlation was lost, most likely due to the fact that age and sex are very closely associated with cIMT, thus attenuating the predictive value of hyaluronan [19]. We did not find a relationship between chronic inflammation as measured by hsCRP and plasma hyaluronan in type 1 diabetes. Therefore further studies are needed to evaluate the effect of other inflammatory markers (e.g. leucocyte count) on hyaluronan metabolism in these patients.Collectively, the present finding implies that hyperglycaemia may elicit alterations in hyaluronan metabolism, which are likely to reflect glycocalyx perturbation. The latter facilitates a wide array of pro-atherogenic effects, including vascular dysfunction and increased permeability of the vessel wall for, for example, lipoproteins. In line, aggregation of calcium with hyaluronan (and other subendothelial glycosaminoglycans) could enhance ectopic calcification of the cIMT, which is increased in type 1 diabetes patients [25, 26]. It would be interesting to investigate whether therapeutic intervention aimed at this disturbed hyaluronan metabolism in type 1 diabetes has the potential to reverse the pro-atherogenic state in these patients [27, 28].\nStudy limitations Several methodological aspects of our study merit caution. We did not determine fasting plasma glucose levels in our type 1 diabetes subjects, therefore we were only able to study markers of chronic hyperglycaemia (HbA1c) on vascular dysfunction. Moreover, the observational nature of our study combined with the use of surrogate markers of future vascular disease can be considered a weakness. However, solid evidence exists that changes in arterial wall function are predictive of cardiovascular outcome [29, 30]. In addition, reproducibility of the measurements was excellent, since a single ultrasound machine was used, one experienced sonographer performed all ultrasonography and images were analysed by a single reader. To reduce variability further, image analysis software automatically investigated each measurement.Finally endothelial glycocalyx also comprises other glycosaminoglycans such as syndecan, chondroitin sulphate and heparan sulphate, which all have their own function [6, 31]. Since hyaluronan and heparan sulphate are important in shear stress-mediated nitric oxide production, which is inextricably entangled with atherosclerosis, further research is needed to explore the role of these other endothelial glycocalyx compounds on diabetes mellitus associated vascular dysfunction.","keyphrases":["hyaluronan","type 1 diabetes mellitus","hyaluronidase","intima-media thickness"],"prmu":["P","P","P","P"]}
{"id":"Anal_Bioanal_Chem-3-1-1839866","title":"Biochemical applications of surface-enhanced infrared absorption spectroscopy\n","text":"An overview is presented on the application of surface-enhanced infrared absorption (SEIRA) spectroscopy to biochemical problems. Use of SEIRA results in high surface sensitivity by enhancing the signal of the adsorbed molecule by approximately two orders of magnitude and has the potential to enable new studies, from fundamental aspects to applied sciences. This report surveys studies of DNA and nucleic acid adsorption to gold surfaces, development of immunoassays, electron transfer between metal electrodes and proteins, and protein\u2013protein interactions. Because signal enhancement in SEIRA uses surface properties of the nano-structured metal, the biomaterial must be tethered to the metal without hampering its functionality. Because many biochemical reactions proceed vectorially, their functionality depends on proper orientation of the biomaterial. Thus, surface-modification techniques are addressed that enable control of the proper orientation of proteins on the metal surface.\nIntroduction\nThe seminal discovery of surface-enhanced Raman scattering (SERS) in the early 70s opened the field of surface-enhanced spectroscopy [1, 2]. The phenomenon has subsequently also been observed at longer wavelengths and, ultimately, led to the realization of surface-enhanced infrared absorption spectroscopy (SEIRAS) [3]. Several reports appeared in the 90s on both practical and theoretical aspects of the phenomenon [4\u20136]. SERS and SEIRAS have lately received attention in the field of biochemistry and biophysics, because of growing interest in bio-nanotechnology [7]. Typical approaches of bio-nanotechnology are constructions of hybrid devices in which bio-molecules, e.g. DNA or proteins, are combined with a solid sensing and\/or actuating substrate, for example as an electrode. With this architecture the whole bandwidth of biological functions can be addressed by exchange of signals with the sensor\/actuator. The concept of the hybrid bio-device is key to the development of biosensors for DNA or proteins, or for immunoassays on a chip, etc. This concept is, moreover, valuable not only for technological progress but also for fundamental studies on proteins and other biologically active materials. Triggering the properties of the adsorbate on the substrate enables functional studies of biomolecules. A critical issue in the design of such a device is assessment of the interface between biomaterial and substrate in which the essential signal relay between the two different materials occurs. This signal relay comprises only small amounts of monolayer molecules at the interfaces which are difficult to detect by conventional Raman and IR techniques. Spectroscopic distinction from the strong background of the bulk is also difficult. This obstacle is overcome by exploiting the \u201coptical near-field effect\u201d of surface-enhanced spectroscopy in which the signal enhancement is restricted to the interface. Characteristic of vibrational techniques, SERS and SEIRA provide a wealth of molecular information on the level of a single chemical bond.\nSERS and SEIRA are complementary techniques, and each has its own advantages and disadvantages. SERS takes advantage of its enormous enhancement factor (of the order of 106\u20131012). The strongest enhancement occurs as a result of the resonance condition if the biomolecule carries a chromophoric co-factor. The fluorescence which often accompanies this may render detection of the Raman spectrum difficult, however. Although the latter is not a problem with SEIRA, the surface-enhancement is only modest (\u223c101\u2013103). SEIRAS probes almost all bands of the adsorbed species as long as the vibrational mode includes a dipole component perpendicular to the surface (surface-selection rule) [5]. Although the enhancement factor of SEIRAS is smaller than that of SERS, the cross-section for IR absorption is several orders of magnitude higher than the corresponding Raman cross-section. Thus, the modest enhancement of SEIRAS may be sufficient for many applications.\nTo study the functionality of proteins by IR spectroscopy, the difference technique has provided an unprecedented amount of molecular information [8\u201310]. The IR spectrum of a protein is recorded in one state\u2014often the resting state\u2014and subtracted from the IR spectrum of another state\u2014an active state or reaction intermediate. The difference spectrum then contains only the vibrational bands associated with the transition from one state to the other. All of the other vibrational bands are cancelled which drastically simplifies interpretation of the vibrational changes. As a consequence, the amplitudes of the difference bands are much smaller than the absorption bands of the entire protein (difference bands may be smaller by a factor of 10\u22124, depending on the size of the protein). Resolving the small difference bands requires acute spectroscopic sensitivity, in particular when surface-enhanced infrared difference spectroscopy (SEIDAS) is performed on a protein monolayer.\nIn this report, we review applications of SEIRA in which biochemical processes were studied. Because SEIRAS was introduced to the field of biomolecules only recently, we consider it worthwhile to start with practical aspects of the method.\nExperimental considerations\nPreparation of thin metal-film substrates\nPreparation of the thin metal film is the critical part of a successful SEIRA experiment. Enhancement by SEIRA is very dependent on the size, shape, and particle density of the selected metal-island film. These properties are easily affected by the experimental conditions during film fabrication, e.g. rate of film deposition, type of the substrate, the substrate temperature, etc. [11].\nSEIRA-active metal islands are usually prepared by high-vacuum evaporation of the metal on to a supporting substrate. Metals such as Au, Ag, Cu, and Pt are vapor-deposited by Ar sputtering, electron-beam heating, or resistive thermal heating of a tungsten basket. The thickness and the rate of deposition are monitored by use of a quartz crystal microbalance (QCM). Controlling of the deposition rate is essential for optimum enhancement. Slow deposition (0.1\u00a0nm s\u22121 or less for deposition of Au or Ag on Si or CaF2) generally results in greater enhancement [11]. This condition also depends on the type of metal, the type of substrate, and the metal film thickness, however, and must therefore be optimized for the system being used. As the morphology of the metal film affects the extent of surface enhancement, templates such as a periodic particle-array film prepared by nanosphere lithography have been used [12, 13]. This approach not only increases the enhancement factor but the reproducibility of signal enhancement will enable quantitative SEIRAS.\nAlthough vacuum evaporation is routinely used, the equipment is costly and not readily available. An alternative means of forming a metal thin film is by chemical (electroless) deposition. Stable SEIRA-active thin films of Au, Pt, Cu, and Ag have been reported on Si or Ge substrates [14\u201317]. The procedure for preparing a thin Au film on a silicon surface is described below: \nThe surface of the Si is covered with 40% w\/v NH4F for a few minutes (typically 1\u20133\u00a0min) to remove the oxide layer and to terminate the surface with hydrogen.After rinsing with water, a freshly prepared 1:1:1 mixture of: \n0.03\u00a0mol L\u22121 NaAuCl40.3\u00a0mol L\u22121 Na2SO3+0.1\u00a0mol L\u22121 Na2S2O3+0.1\u00a0mol L\u22121 NH4Cl, and2% w\/v HFis put on the Si surface for 60\u201390\u00a0s.Although a shiny Au film is formed, the Au surface may still be contaminated with thio compounds from the plating chemicals. These are removed by electrochemical cycling of the potential between 0.1 and 1.4\u00a0V in 0.1\u00a0mol L\u22121 H2SO4 until the cyclic voltammogram of polycrystalline gold appears (broad oxidation peak above 1.1\u00a0V and a sharp reduction peak at 0.9\u00a0V relative to the SCE). One can, instead, apply a dc voltage of +1.5\u00a0V between the Au film and the counter electrode (e.g. a Pt wire) for ca. 1\u00a0min.\nAtomic force microscope (AFM) images of the chemically deposited Au film reveals an island structure similar to that of the vacuum-evaporated Au films, albeit with somewhat larger average diameter of the metal islands [16, 17]. Another advantage of the chemical method is the stronger adhesion of the deposited metal layer to the substrate. This property helps significantly when long-term stability of the metal film is required, as is typical for preparation of biomimetic devices (vide infra).\nAn interesting option is the use of colloidal gold nano particles [18, 19]. Colloidal gold is prepared by reducing tetrachloroauric(III) acid with sodium citrate. It is also commercially available in different particle sizes. Typically, 10\u201350\u00a0nm colloidal gold is chosen. A major difference from the metal thin-film method is that the sample is attached to the colloidal gold suspension before measurement. The colloidal gold is then collected on an optical substrate by filtration or by centrifugation. The sample\/colloid gold is measured either in the transmission configuration (with an IR card) or dried on an ATR prism.\nRemoval of the metal film from the supporting substrate\nThe metal film prepared by vacuum-evaporation is usually poorly adhesive. It is easily wiped off with ethanol or acetone. A metal film prepared by the chemical deposition method adheres much more strongly, and hence may not be removed by wiping or even by polishing with aluminium powder. Such metal films can be dissolved by immersion in a boiling solution of a 1:1:1 mixture of HCl (32%), H2O2 (30%), and H2O.\nGeometry and optical configuration\nThe most widespread optical arrangement employs the so-called metal underlayer configuration (sample\/metal film\/supporting substrate; Fig.\u00a01a). IR-transparent materials are usually used as supporting substrates. Highly refractive materials, for example Si, Ge, and ZnSe, are suitable for internal reflection optical geometry (Fig.\u00a01a, (ii)) and CaF2 and BaF2 are more suitable for transmission geometry (Fig.\u00a01a (i)). For the former, relatively thick metal films (in the range of ten to several hundred nanometers) can be used whereas for the latter geometry the thickness of metal film should be kept to less than 10\u00a0nm, because the island structure starts to merge at higher thickness. As a result, the metal film is no longer transmissive [11, 20].\nFig.\u00a01Schematic diagram of the optical configuration of SEIRAS. (a) Metal underlayer configuration. (b) Metal overlayer configuration. The arrows denote the optical pathway of the IR beam in (i) transmission, (ii) attenuated total reflection, and (iii) external reflection geometry. (c) Spectro-electrochemical cell for SEIRA spectroscopy\nFor internal reflection geometry (Kretschmann attenuated total-reflection geometry), half-cylindrical, half-spherical, or trapezoidal prisms are commonly used. The former two types are advantageous for optical reasons, because the position of the focus on the metal film is not affected by changes in the angle of incidence of the IR beam. Si is commonly used as reflection element, because of its high chemical stability. One disadvantage is that the available spectral range is then limited to >1000\u00a0cm\u22121. Although use of Ge enables use of a wider spectral window (>700\u00a0cm\u22121), it is not suitable for electrochemical experiments at potentials above 0.0\u00a0V (relative to the SCE) in acidic solution (pH<4.0), in which it starts to dissolve. Although Ge matches well with Ag or Cu, combination with Au may cause a decrease in the enhancement factor, because of the formation of a Ge\u2013Au alloy at the boundary which strongly affects the morphology of the island structure of the film.\nNon-IR-transparent material, for example glass, polymers, carbon, and metal, can be used as a substrate only with external reflection which uses the metal overlayer configuration (metal particle\/sample\/supporting substrate, Fig.\u00a01b). In this configuration the thin metal layer (ca. 4\u20137\u00a0nm) is directly evaporated on the sample\/support surface. It should be noted that poorly reflective material is optically favorable, because of reduced distortion of band shape in the corresponding SEIRA spectrum [21, 22]. This configuration is advantageous for detection in trace analysis of a sample adsorbed on glass or a polymer [22]. It is pointed out, however, that external reflection IR spectroscopy is not very useful for functional studies of biomaterial, because of the required presence of bulk water that strongly absorbs the IR probe beam.\nChemical modification of the metal film surface\nIt is crucial to chemically modify the bare metal if direct contact with the metal surface hampers the structure and function of the biomaterial. Strong interaction forces may even induce denaturation of proteins, because their secondary structure is very dependent on weak hydrogen-bonds between amino acids. The use of a chemically modified electrode (CME) is an approach used to tackle these obstacles [23]. The surface of the metal electrode is modified by heterobifunctional crosslinkers that comprise: \na thiol group that is spontaneously adsorbed by the metal electrode;a spacer group with different lengths of alkyl chain; anda functional headgroup pointing toward the bulk solution.\nThe CME surface can be conveniently prepared by the so-called \u201cSAM (self-assembled monolayer) method\u201d in which the SEIRA-active metal substrate is immersed in a solution containing the crosslinker molecule (normally for between 10\u00a0min and 2\u00a0h, depending on size of the molecule). The SAM of the crosslinker is formed spontaneously by quasi-covalent bonding between the sulfur and the metal surface. The substrate is then thoroughly rinsed with the solvent so that a SAM of the crosslinker remains on the metal substrate.\nAdsorption of the protein by the chemically modified electrode (CME) is a suitable model for protein\u2013protein interaction. By varying the properties of the functional headgroup it is possible to mimic the specific conditions of the substrate during the protein\u2013protein or protein\u2013ligand interaction process. For example, use of a carboxyl headgroup mimics the side-chain terminus of Asp or Glu. The negatively charged carboxyl headgroup can specifically interact with positively charged amino acids, for example Lys, His, and Arg residues, on the protein surface. Another approach is to exploit the specific interaction of Ni2+-chelated nitrilotriacetic acid (NTA) with a sequence of histidine residues genetically introduced into the target protein (His-tag) [24, 25]. Details of this approach are given below.\nApplications\nStudies of nucleic acids and DNA\nSEIRAS studies on nucleic acid bases have provided information about the adsorption and orientation of thymine on silver island film [26], and of cytosine [27] and uracil [28] on gold electrodes. These experiments have been expanded to studies of the hydrogen-bonding interaction between complementary bases (adenine (A)\u2013thiamine (T) and guanine (G)\u2013cytosine (C)) as model systems of DNA and RNA. Sato et al. used SEIRAS to study base-pairing between thymidine and 6-amino-8-purinethiol, a thio-derivatized adenine, immobilized on a gold electrode surface [29]. They observed a characteristic thymidine band at 1571\u00a0cm\u22121 adsorbed on the adenine-modified surface by hydrogen-bonding. This base-pairing occurs at potentials higher than 0.1\u00a0V (relative to the SCE), at which the N1 of adenine is deprotonated and orientated toward the solution. This result implies that hydrogen-bonding between the base pairs can be controlled by the applied electrode potential.\nAs an interesting extension of the method, SEIRAS has been used as a diagnostic criterion in cancer research [30, 31]. Although the reported enhancement factor of 3\u20135 only modest [31], the enhanced bands are impossible to observe with conventional IR spectroscopy but are crucial for determining the properties of nucleic acids of tumor tissues and their interaction with anticancer drugs.\nImmunoassays based on SEIRAS\nThere is substantial interest in the development of biosensors in which the biological component is associated with a transducer to provide a means of detecting changes in the biological component. Most current biosensors consist of an enzyme or an antibody which interacts with the substrate or the respective antigen. The enzymes or antibodies are usually immobilized on a platform and the interaction between the enzyme and the substrate (or the antibody and the antigen) is monitored. Biosensors based on surface plasmon resonance (SPR) have received considerable attention in recent years. In this method, the biomaterial is immobilized on the surface of a metal-film-covered optical prism (ATR\u2013Kretschmann configuration), i.e. the same optical principle is applied as with SEIRAS. The advantage of SEIRAS compared with SPR is that the chemical information derived from the vibrational spectrum identifies the nature of the adsorbed species yet the sensitivity of SEIRAS is comparable with that of SPR. The sensitivity of SPR is poor when the refractive index of the adsorbed species is close to that of the solvent, as it is for small organic molecules dissolved in an organic solvent. SEIRAS detects the individual vibrational modes of functional groups of the adsorbate, and the S\/N ratio does not depend on the type of solvent if the vibrational bands of the solvent do not overlap with those of the adsorbate. SEIRAS can, moreover, be used to detect even minute structural changes that occur in the adsorbed layer during the adsorption process.\nBrown et al. have applied SEIRAS to biosensor analysis for determination of antibody\u2013antigen interactions [32]. In this experiment, antibodies for Salmonella (anti-SAL) were immobilized on a gold surface deposited on a silicon wafer. After taking the spectrum of anti-SAL, this \u201cdipstick\u201d sensor was immersed in a solution containing the antigen (SAL). The SEIRA spectra of anti-SAL without SAL has characteristic antibody peaks at 1085 and 990\u00a0cm\u22121. Addition of SAL results in an additional band at 1045\u00a0cm\u22121 which was assigned to the P\u2013O stretching vibration of the phospholipid in the cell wall, which indicates the presence of SAL in the solution.\nInitially, the analyte adsorbed by the \u201cdipstick\u201d sensor was probed by external reflection SEIRA spectroscopy. Later, the authors developed a new method for SEIRAS in which they used a colloidal gold surface as adsorption platform [19]. The colloidal gold\u2013antibody\u2013antigen complexes are left to assemble in solution, taking advantage of the tight binding of the antibodies to colloidal gold which stabilizes the colloid solution. When the sample has been assembled the complexes are collected, either by filtration on a porous polyethylene membrane [19] or by centrifugation on an ATR substrate [18], and are then readily mounted in the FTIR spectrometer. Both \u201cdipstick\u201d and \u201ccolloidal gold\u201d furnish similar spectral quality, although the enhancement of the latter approach is not mentioned. The colloidal gold system is more convenient and less expensive means of preparation than coating by sputtering. Filtration of the colloidal particles on disposable IR cards is also a rapid, easy, and cost-effective means of producing a SEIRA substrate which operates in transmission mode and does not require complicated optical arrangements.\nSEIRAS to probe protein functionality\nThe metal used for surface enhancement, can also be used as an electrode. The enzymatic reactions of many biological systems, especially those of membrane proteins, are driven by an electrochemical gradient across the cell membrane. Such a system can be artificially reproduced on an electrode surface to mimic the physiological properties of a biological membrane.\nElectrochemically induced oxidation and reduction of a monolayer of cytochrome c (Cc), a protein that mediates single-electron transfer between the integral membrane protein complexes of the respiratory chain, is the model system for electron transfer within and between proteins [33]. Electrons are directly injected to and\/or withdrawn from Cc after proper contact has been established with the electrode by means of a suitable surface modifier. Proteins can be attached to such a chemically modified electrode (CME) by electrostatic attraction or covalent interaction. Figure\u00a02 depicts potential-induced difference spectra of Cc that has been electrostatically adsorbed by different types of CME. The positive and negative peaks are indicative of conformational changes associated with the transition from the fully oxidized to the fully reduced state. Details of band assignment have been published [14]. The difference spectra reveal subtle changes of the secondary structure induced by rearrangement of the hydrogen-bonded network among the internal amino acid side-chains surrounding the heme chromophore. Note that the peak positions of the observed Cc bands do not depend on the type of CME layer whereas the relative intensities are strongly dependent on the CME. The former suggests that the internal conformational changes of Cc are not affected by interaction with the modifiers. The latter observation is attributed to the different surface structure, i.e. the orientation or position of the Cc relative to the CME underlayer [14].\nFig.\u00a02Potential-induced redox difference spectra of a cytochrome c monolayer adsorbed on a variety of chemically modified electrodes: (a) mercaptopropionic acid, (b) mercaptoethanol, (c) cysteine, (d) dithiodipyridine. Minor spectral contributions from the surface modifier have been subtracted to selectively reveal the vibrational spectra of the adsorbed cytochrome c [14]\nStudies of molecular and protein recognition\nThe acute sensitivity of SEIRAS is particularly useful when the technique is applied to studies of membrane proteins. Handling of the membrane proteins usually requires great care, because of their sensitivity to degeneration as soon as they are separated from the native lipid bilayer. The large size of the proteins also makes it difficult to control their orientation.\nA successful strategy for immobilization of membrane proteins is to attach the purified protein, by using the selective affinity of a genetically introduced histidine tag (His-tag), to a nickel-chelating nitrilotriacetic acid (Ni-NTA) monolayer (SAM) self-assembled on a chemically modified gold surface. The lipid environment of the surface-anchored membrane protein is subsequently restored by in-situ dialysis of the detergent around the amphiphilic membrane protein. This approach results in orientated immobilization and reconstitution of the native matrix, which enhances the stability of the membrane protein and restores full functionality.\nThe success of the method has been demonstrated by use of cytochrome c oxidase (CcO) [24, 25]. Recombinant CcO (solublized by detergent) from Rhodobacter sphaeroides was immobilized on the stepwise-formed Ni-NTA SAM bound to the gold surface. It is advantageous to follow each surface-modification step by in-situ SEIRAS, which ensures qualitative and quantitative control of each reaction step. The protein is subsequently embedded in the lipid layer by removing the detergent by addition of Bio-Beads. SEIRA spectra reveal the increase of several lipid bands during the reconstitution without any dissociation of protein from the surface. Atomic-force microscopy (AFM) provides direct evidence of the formation of the lipid bilayer. Spots of size \u223c7\u00a0nm appear in the AFM image; this is in accordance with the diameter of a single CcO molecule [34]. It is remarked that the same approach can be used to bind CcO to a rough Ag surface. Integrity of the native structures of the heme cofactors (heme a and heme a3) was shown by surface-enhanced resonance Raman spectroscopy (SERRS) [35, 36].\nFull functionality of the lipid-reconstituted CcO electrode is demonstrated by the fact that a catalytic current resulting from reduction of oxygen, the native property of CcO, is observed when an electron is donated by the adsorbed Cc. In such an experiment orientation of the CcO is controlled by the position of the His-tag on either side of the membrane surface CcO. With molecular genetic techniques, the His-tag is introduced either in the C-terminal tail of subunit I or in the C-terminus of subunit II. By binding CcO through the respective His-tag, the former orients CcO such that the binding site of Cc is exposed to the bulk solution. The latter orientation obstructs the binding site for Cc, because of the barrier imposed by the lipid bilayer. Indeed, SEIRAS, in combination with electrochemistry, reveals that Cc binds and initiates the catalytic reaction of CcO only when the Cc binding site faces the bulk aqueous phase and that Cc does not interact with the oppositely oriented CcO [24, 25].\nInterestingly, the electrochemically induced redox difference surface-enhanced infrared difference absorption (SEIDA) spectrum of the Cc\u2013CcO complex is remarkably similar to that of Cc adsorbed on a carboxy-terminated SAM (mercaptoundecanoic acid, MUA) (Fig.\u00a03c and d). It is noted that SEIDA spectra of Cc are sensitive to the properties of the terminal headgroup of the SAM that it interacts with [14]. The similarity of the spectra of Cc\u2013CcO and Cc\u2013MUA suggests a preponderance of carboxylate residues at the physiological docking site of Cc, i.e., those of the side-chains of aspartate or glutamate units. Ferguson-Miller et al. have, indeed, identified residues Glu148, Glu157, Asp195, and Asp214 (all in subunit II of CcO from R. sphaeroides) as the major interaction partners of Cc [37].\nFig.\u00a03Potential-induced IR difference spectra of: (a) cytochrome c bound to a monolayer of cytochrome c oxidase tethered to a gold surface via His-tag\/Ni-NTA interaction; (b) tethered monolayer of cytochrome c oxidase alone; (c) difference between (a) and (b) which recovers the vibrational contribution from cytochrome c only; (d) cytochrome c adsorbed to a monolayer of mercaptoundecanoic acid\nThe same strategy has recently been applied to immobilization of photosystem 2 (PS2) on an Au surface with the purpose of developing a semi-artificial device for production of hydrogen by photosynthetic oxidation of water [38]. In this report, SEIRA was used to determine the adsorption kinetics of PS2 on the Ni-NTA-modified Au surface. On illumination of the surface-bound PS2 a photocurrent was generated. The action spectrum corresponds to the absorption spectrum of PS2, indicating that the observed photocurrent is caused by photoreaction of PS2.\nThis methodology is a general approach for immobilization of proteins, because the introduction of affinity tags is routine with modern genetic techniques. Other tags beyond the His-tag may also be an option. Thus, orientational control of protein adsorption on a solid surface can be conveniently achieved. An oriented sample is mandatory when the vectorial function of membrane proteins is addressed. Many membrane proteins are asymmetric in their functionality, because they translocate ions or solutes preferentially in one direction or because their stimulant, e.g. ligand, binding partner protein, membrane potential, etc., affects the protein from one side only.\nSummary and outlook\nDespite the accomplishments reported in this article, studies of biomaterials by SEIRA is still in its infancy. Potential-induced difference spectroscopy using SEIRA is a promising possibility for study of the functionality of proteins. The stimulus in such studies is not limited to the electric trigger, as reviewed here, but can also be light illumination, temperature jump, or chemical induction. Many protein functions can be addressed by these means and the mechanism of action of these molecular machines may be resolved down to the level of a single bond.\nThe sensitivity may not be sufficient for functional difference spectroscopy on any protein with SEIDAS, however. As the enhancement factor for SEIRA is modest compared with that for SERS, optimization of the enhancement by proper design of the metal surface is crucial. Although enhancement factors are usually in the range 10\u2013100, factors as high as 1000 have sometimes been reported [16]. To obtain the latter enhancement reproducibly, however, development of homogeneous island metal film preparation under strict topological control will be mandatory.","keyphrases":["electron transfer","protein","self-assembled monolayer","membrane","ftir"],"prmu":["P","P","P","P","P"]}
{"id":"Diabetologia-3-1-1871609","title":"The role of physical activity in the management of impaired glucose tolerance: a systematic review\n","text":"Although physical activity is widely reported to reduce the risk of type 2 diabetes in individuals with prediabetes, few studies have examined this issue independently of other lifestyle modifications. The aim of this review is to conduct a systematic review of controlled trials to determine the independent effect of exercise on glucose levels and risk of type 2 diabetes in people with prediabetes (IGT and\/or IFG). A detailed search of MEDLINE (1966\u20132006) and EMBASE (1980\u20132006) found 279 potentially relevant studies, eight of which met the inclusion criteria for this review. All eight studies were controlled trials in individuals with impaired glucose tolerance. Seven studies used a multi-component lifestyle intervention that included exercise, diet and weight loss goals and one used a structured exercise training intervention. Four studies used the incidence of diabetes over the course of the study as an outcome variable and four relied on 2-h plasma glucose as an outcome measure. In the four studies that measured the incidence of diabetes as an outcome, the risk of diabetes was reduced by approximately 50% (range 42\u201363%); as these studies reported only small changes in physical activity levels, the reduced risk of diabetes is likely to be attributable to factors other than physical activity. In the remaining four studies, only one reported significant improvements in 2-h plasma glucose even though all but one reported small to moderate increases in maximal oxygen uptake. These results indicate that the contribution of physical activity independent of dietary or weight loss changes to the prevention of type 2 diabetes in people with prediabetes is equivocal.\nIntroduction\nGiven the growing prevalence of diabetes and the high economic cost of treating the condition and its comorbities, it is important to find effective ways of targeting those who are most at risk of developing the disease [1]. Prediabetes is the collective term for people with IGT and\/or IFG [2]. Prediabetes is associated with an increased risk of development of type 2 diabetes [3] and cardiovascular disease [4\u20136]. There is good evidence from cross-sectional and longitudinal studies for a link between levels of physical activity and the risk of type 2 diabetes [7\u20139]. However, evidence from intervention studies in high-risk populations is limited, making it difficult to quantify the effectiveness of physical activity in reducing the risk of type 2 diabetes in individuals with prediabetes. Lifestyle intervention studies that have encouraged weight loss through a combination of dietary change and increased physical activity have reduced the risk of type 2 diabetes in individuals with IGT [10\u201314]. However, because physical activity was not usually analysed independently of other variables, such as weight loss, it is difficult to determine the effectiveness of physical activity at protecting against the risk of diabetes in individuals with prediabetes. Therefore, the aim of this systematic review is to establish the effectiveness of physical activity independent of other variables at reducing the risk of diabetes or improving glucose parameters in people with prediabetes.\nMaterials and methods\nSearch strategy MEDLINE (1966 to February week 4, 2006) and EMBASE (1980 to week 8, 2006) were searched for articles examining the effect of an exercise or lifestyle intervention on individuals with prediabetes. The search was carried out using medical search headings (MeSH) and by searching titles and abstracts for relevant words. For example, studies including individuals with prediabetes were found by using the MeSH \u2018prediabetic state,\u2019 \u2018insulin resistance,\u2019 \u2018glucose intolerance\u2019 and \u2018diabetes mellitus\u2019 (subheading \u2018prevention and control\u2019), and by searching titles and abstracts for \u2018prediabetes,\u2019 \u2018impaired glucose tolerance,\u2019 \u2018IGT,\u2019 \u2018impaired fasting glucose\u2019 and \u2018IFG.\u2019 Studies that included an exercise intervention were found by using the MeSH \u2018lifestyle,\u2019 \u2018sports,\u2019 \u2018exercise therapy\u2019 and \u2018physical fitness,\u2019 and by searching titles and abstracts for \u2018exercise,\u2019 \u2018physical activity,\u2019 \u2018physical fitness,\u2019 \u2018resistance training,\u2019 \u2018strength training,\u2019 \u2018circuit training,\u2019 \u2018endurance training\u2019 and \u2018aerobic training.\u2019 In addition, the reference lists of relevant published original articles and reviews were hand-searched.One reviewer (T. Yates) performed the electronic and hand-searches and reviewed the results. Studies that clearly did not meet the inclusion criteria were rejected during the initial review. Where uncertainty existed, the full text of the article was obtained and reviewed. Two reviewers (T. Yates and K. Khunti) independently assessed all potentially relevant studies and performed data extraction. Disagreement was resolved by discussion and, where necessary, third party adjudication.\nSubjects Participants were adults (age \u226518\u00a0years) diagnosed with prediabetes. Prediabetes was defined as IGT and\/or IFG using one of the sets of criteria previously recommended by the WHO [15, 16] or the American Diabetes Association (ADA) [17, 18]. Studies that defined IGT or IFG using other criteria were included if the mean value of the participants\u2019 plasma glucose fell within the range of IGT or IFG as defined by the WHO or ADA criteria (2-h plasma glucose \u22657.8\u00a0mmol\/l and <11.1\u00a0mmol\/l, and fasting glucose <7.8\u00a0mmol\/l for IGT; 2-h plasma glucose  <7.8\u00a0mmol\/l, and fasting glucose \u22655.6\u00a0mmol\/l and <7.0\u00a0mmol\/l for IFG).\nInterventions Interventions that included an exercise programme were included. \u2018Exercise programme\u2019 was taken to mean any intervention that actively promoted and supported physical activity or a structured exercise training regimen. Studies that only provided individuals with brief written or verbal physical activity advice were excluded. Studies investigating the effect of a single or acute episode of exercise were also excluded.\nOutcome measures Only studies with an outcome measure of physical activity and a relevant clinical measure were included. A relevant clinical measure was defined as progression to diabetes or a suitable measure of plasma glucose (2-h plasma glucose for IGT, or fasting glucose for IFG).\nType of study Randomised and non-randomised controlled trials were included.\nAnalysis As the heterogeneity of the type of exercise interventions and outcome measures did not lend itself to quantitative methods of analysis, a systematic narrative review was undertaken. Baseline and follow-up exercise, body mass and glucose parameters were reported using mean\u00b1SEM, or median (interquartile range). Results reporting the SD or the 95% CI were converted to SEM using the formulas  (where t is the t distribution value for a 95% CI), respectively. Where the SEM, SD or 95% CI for the change from baseline to follow-up was not reported, only the mean value is reported because of the potential error involved in calculating SEM for this figure. When available, the relative risk of diabetes in the intervention group compared with the control group was also reported.\nResults\nThe search produced 307 hits, from which 279 potential studies were identified, of these, eight trials met the criteria for inclusion (see Fig.\u00a01). Study details for the eight included studies are shown in Table\u00a01; the main outcomes are presented in Table\u00a02.\nFig.\u00a01Flow diagram of the literature search. Duplicates: where several studies reported on the same trial and cohort, only one published study for each trial was included for the purposes of this review; the included studies were those that reported on the cohort as a whole, had relevant follow-up measures and included the most recently published data. Where a relevant study was identified in more than one publication, the study was included only onceTable\u00a01Characteristics of included studiesAuthors, yearStudy location\/nameStudy designIntervention durationNumber of subjects (men\/women)Inclusion criteriaType of interventionType of dietary interventionType of exercise interventionMethod of physical activity measurementLindstr\u00f6m et al. 2003 [21]Finland\/Finnish Diabetes Prevention StudyRCT3\u00a0years522 (172\/350)IGT (WHO criteria, 1985), age 40\u201364\u00a0years, BMI\u226525\u00a0kg\/m2Exercise and dietWeight reduction through a healthy dietParticipants individually encouraged to increase their overall level of physical activity. Circuit-type exercise sessions were also offered.Self-report\u2014Kuopio 12\u00a0month leisure time physical activity questionnaireKnowler et al. 2002 [12]USA\/Diabetes Prevention Research GroupRCTAverage follow-up 2.8 (range 1.8\u20134.6) years2,161 (680\/1481)aIGT (ADA criteria, 1997), age \u226525\u00a0years, BMI \u226524\u00a0kg\/m2 (\u226522\u00a0kg\/m2 if Asian), fasting plasma glucose \u22655.3\u00a0mmol\/lExercise and dietWeight reduction through a healthy, low-energy, low-fat dietParticipants individually encouraged to accumulate at least 150\u00a0min\/week of moderate intensity exercise.Self-report\u2014Modified Activity Questionnaire and activity logPan et al. 1997 [10]China\/The Da Qing IGT and Diabetes Study RCT6\u00a0years530 (283\/247)IGT (WHO criteria, 1985), age \u226525\u00a0years1. Exercise 2. Exercise and dietWeight maintenance for those with a BMI <25\u00a0kg\/m2 through a healthy energy-balanced diet; weight reduction for those with a BMI \u226525\u00a0kg\/m2 through reduced energy intakeParticipants were encouraged to increase their physical activity to 1 unit per dayb. Those who were aged <50\u00a0years and were able to were encouraged to accumulate 2 units per day.Self-report\u2014type not reportedEriksson and Lindg\u00e4rde, 1991 [24]Sweden\/The 6-year Malm\u00f6 feasibility studyNon-randomised controlled trial5\u00a0years260 (260\/0)IGT (2-h post-challenge glucose values 7\u201311\u00a0mmol\/l, and fasting plasma glucose <7.8\u00a0mmol\/l)Exercise and dietHealthy dietary advice givenParticipants were encouraged to increase their physical activity levels. Participants given the option of training in organised groups for a 6-month period in the first year.VO2max\u2014heart rate response to submaximal workload using bicycle ergometerOldroyd et al. 2006 [19]EnglandRCT2\u00a0years69 (39\/30)IGT (WHO criteria, 1985)Exercise and dietWeight reduction through a healthy, low-energy, low-fat dietParticipants were encouraged to undertake 20\u201330\u00a0min of aerobic activity 2\u20133\u00a0days\/week. In addition, all participants were given a discount at local gyms.Cardiovascular fitness\u2014shuttle testSelf-report\u2014type not reportedMensink et al. 2003 [22]Netherlands\/Study on lifestyle-intervention and impaired glucose tolerance MaastrichtRCT2\u00a0years114 (64\/50)IGT (2-h post-challenge glucose values 7.8\u201312.5\u00a0mmol\/l, and fasting plasma glucose <7.8\u00a0mmol\/l), age >40\u00a0years, BMI \u226525.0\u00a0kg\/m2Exercise and dietWeight reduction through a healthy, low-energy, low-fat dietParticipants were encouraged through goal setting to undertake 30\u00a0min of moderate intensity exercise per day and were given use of free exercise classes.VO2max\u2014incremental exhaustive test using bicycle ergometerLindahl et al. 1999 [23]SwedenRCT1\u00a0year186 (69\/117)IGT (WHO criteria, 1985), age 30\u201360\u00a0years, BMI \u226527\u00a0kg\/m2Exercise and dietWeight reduction through a healthy, low-energy, low-fat diet.Participants were encouraged to increase their physical activity. Supervised exercise sessions were available in the first monthVO2max\u2014heart rate response to submaximal and maximal workload using bicycle ergometerCarr et al. 2005 [20]USARCT2\u00a0years62 (29\/33)IGT (WHO criteria, 1998)Structured exercise and dietParticipants were encouraged to follow the energy-balanced American Heart Foundation Step 2 dietWalking\/jogging at >70% of heart rate reserve for 1\u00a0h on 3\u00a0days\/weekVO2max\u2014modified branching treadmill protocolVO2max, maximal oxygen consumptionaData for lifestyle and control groups onlyb1 unit\u2009=\u200930\u00a0min of mild exercise, or 20\u00a0min of moderate exercise, or 10\u00a0min of strenuous exercise, or 5\u00a0min of very strenuous exerciseTable\u00a02Baseline values and change in main outcomesAuthors, yearBaseline VO2max (l\/min)Change in VO2max (l\/min)aBaseline self-reported physical activity levelsChange in self-reported leisure time physical activityaBaseline body mass (kg)Change in body mass (kg)aBaseline 2-h plasma glucose (mmol\/l)Change in 2-h plasma glucose from baseline (mmol\/l)a Baseline fasting glucose (mmol\/l)Change in fasting plasma glucose (mmol\/l)aRelative risk of diabetes in the intervention group vs the C: group (95% CIs)Lindstr\u00f6m et al. 2003 [21]N\/AN\/AL: 156 (62 to 288) min\/weekL: 61 (\u221233 to 168) min\/weekb`L: 86.7\u2009\u00b1\u20090.8Results at 3\u00a0yearsL: 8.9\u2009\u00b1\u20090.1Change at 1\u00a0yearL: 6.1\u2009\u00b1\u20090.05Change at 1\u00a0year0.4 (0.3\u20130.7)C: 169 (65 to 352) min\/weekC: 6 (\u221291 to 104) min\/weekC: 85.5\u2009\u00b1\u20090.9L: \u22123.5\u2009\u00b1\u20090.3bC: 8.9\u2009\u00b1\u20090.1L: \u22120.9\u2009\u00b1\u20090.1bC: 6.2\u2009\u00b1\u20090.04L: \u22120.2\u2009\u00b1\u20090.04C: \u22120.9\u2009\u00b1\u20090.4C: \u22120.3\u2009\u00b1\u20090.1C: 0.0\u2009\u00b1\u20090.04Change at 3\u00a0yearsChange at 3\u00a0yearsL: \u22120.5\u2009\u00b1\u20090.2L: 0.0\u2009\u00b1\u20090.05C: \u22120.1\u2009\u00b1\u20090.2C: 0.1\u2009\u00b1\u20090.05Knowler et al. 2002 [12]N\/AN\/AL: 15.5\u2009\u00b1\u20090.7\u00a0MET-h\/weekL: 6\u00a0MET-h\/weekb,cL: 94.1\u2009\u00b1\u20090.6L: \u22125.6bL: 9.1\u2009\u00b1\u20090.03NRL: 5.9\u2009\u00b1\u20090.01NR0.4 (0.3\u20130.5)C: 17.0\u2009\u00b1\u20090.9\u00a0MET-h\/weekC: 1\u00a0MET-h\/weekcC: 94.3\u2009\u00b1\u20090.6C: \u22120.1C: 9.1\u2009\u00b1\u20090.03C: 5.9\u2009\u00b1\u20090.02Pan et al. 1997 [10]N\/AN\/AE: 3.4\u2009\u00b1\u20090.2 unitsdE: 0.6 unitsdNRD&E: \u22122.5E: 8.8\u2009\u00b1\u20090.1E: 1.7E: 5.6\u2009\u00b1\u20090.1E: 1.3E: 0.5 (0.2\u20130.9)D&E: 3.1\u2009\u00b1\u20090.2 unitsdD&E: 0.8 unitsdE: \u22120.4D&E: 9.1\u2009\u00b1\u20090.1D&E 1.7D&E 5.7\u2009\u00b1\u20090.1D&E: 1.5D&E: 0.6 (0.3\u20130.9)C: 2.4\u2009\u00b1\u20090.2 unitsdC: 0.1 unitsdC: \u22121.0C: 9.0\u2009\u00b1\u20090.1C: 4.0C: 5.5\u2009\u00b1\u20090.1C: 2.1Eriksson and Lindg\u00e4rde 1991 [24]L: 2.46\u2009\u00b1\u20090.04L: 0.20bN\/AN\/AL: 82cL: \u22123.3bL: 8.2\u2009\u00b1\u200901L: \u22121.1bNRNR0.4 (0.2\u20130.7)C: 2.29\u2009\u00b1\u20090.1C: \u22120.05C: 83cC: 0.2C: 8.3\u2009\u00b1\u20090.1C: 0.1cOldroyd et al. 2006 [19]N\/AN\/ANRNRL: 85.3\u2009\u00b1\u20092.9L: \u22121.8\u2009\u00b1\u20091.1bL: 9.2\u2009\u00b1\u20090.1Change at 1\u00a0yearL: 6.1\u2009\u00b1\u20090.1Change at 1\u00a0yearN\/AC: 85.5\u2009\u00b1\u20092.5C: 1.5\u2009\u00b1\u20090.5C: 9.2\u2009\u00b1\u20090.2L: \u22120.6\u2009\u00b1\u20090.3C: 6.2\u2009\u00b1\u20090.2L: 0.0\u2009\u00b1\u20090.1C: 0.2\u2009\u00b1\u20090.3C: 0.1\u2009\u00b1\u20090.2Change at 2\u00a0yearsChange at 2\u00a0yearsL: 0.2\u2009\u00b1\u20090.3L: 0.3\u2009\u00b1\u20090.1C: \u22120.5\u2009\u00b1\u20090.4C: 0.1\u2009\u00b1\u20090.2Mensink et al. 2003 [22]L: 2.15\u2009\u00b1\u20090.1L: 0.09\u2009\u00b1\u20091.90bN\/AN\/AL: 86\u2009\u00b1\u20091.9L: \u22122.4\u2009\u00b1\u20090.7bL: 8.9\u2009\u00b1\u20090.3Change at 1\u00a0year L: 5.9\u2009\u00b1\u20090.1Change at 1\u00a0yearN\/AC: 2.13\u2009\u00b1\u20090.1C: \u22120.03\u2009\u00b1\u20092.77C: 83.7\u2009\u00b1\u20091.5C: 0.1\u2009\u00b1\u20090.5C: 8.6\u2009\u00b1\u20090.2L: \u22120.9\u2009\u00b1\u20090.3bC: 5.8\u2009\u00b1\u20090.1L: \u22120.1\u2009\u00b1\u20090.1C: 0.3\u2009\u00b1\u20090.3C: 0.1\u2009\u00b1\u20090.1Change at 2\u00a0yearsChange at 2\u00a0yearsL: \u22120.6\u2009\u00b1\u20090.3bL: 0.2\u2009\u00b1\u20090.1C: 0.8\u2009\u00b1\u20090.4C: 0.5\u2009\u00b1\u20090.1Lindahl et al. 1999 [23]L: 2.12\u2009\u00b1\u20090.1eL: 0.21\u2009\u00b1\u20090.1b,eN\/AN\/AL: 86.4\u2009\u00b1\u20091.1L: \u22125.4\u2009\u00b1\u20090.5bL: 7.5L: \u22120.7\u2009\u00b1\u20090.2L: 5.4L: \u22120.5\u2009\u00b1\u20090.1N\/AC: 1.89\u2009\u00b1\u20090.1eC: \u22120.02\u2009\u00b1\u20090.1eC: 83.6\u2009\u00b1\u20091.1C: \u22120.5\u2009\u00b1\u20090.3C: 8.0C: \u22120.3\u2009\u00b1\u20090.3C: 6.1C: \u22120.3\u2009\u00b1\u20090.1Carr et al. 2005 [20]E: 1.93\u2009\u00b1\u20090.1E: 0.16\u2009\u00b1\u20090.04bN\/AN\/AE: 66.5\u2009\u00b1\u20092.9E: \u22121.8\u2009\u00b1\u20090.5bE: 9.2\u2009\u00b1\u20090.2Change at 6\u00a0monthsE: 5.3\u2009\u00b1\u20090.1Change at 6\u00a0monthsN\/AC: 2.02\u2009\u00b1\u20090.1C: \u22120.04\u2009\u00b1\u20090.03C: 69.7\u2009\u00b1\u20092.6C: 0.6\u2009\u00b1\u20090.5C: 9.1\u2009\u00b1\u20090.2E: \u22120.7C: 5.4\u2009\u00b1\u20090.1E: 0.0C: \u22120.1C: 0.1Change at 2\u00a0yearsChange at 2\u00a0yearsE: \u22120.6E: 0.0C: 0.0C: 0.1Data are presented as means\u00b1SD or medians (interquartile range)aChange from baseline values reflect results at final follow-up, unless stated otherwisebp\u2009<\u20090.05 vs C:cValue estimated from graphd1 unit\u2009=\u200920\u00a0min of moderate E: or 10\u00a0min of strenuous E:eFitness measurement taken in a randomly selected subgroup, n\u2009=\u200945C control, D diet, E exercise, L lifestyle, MET-h\/week metabolic equivalent per week, NR not reported, N\/A not applicable\nStudy design Seven of the eight trials involved randomisation of subjects to a treatment group or control group [10, 12, 19\u201323]. The non-randomised trial identified control participants by using individuals who, for various (unstated) reasons, were not enrolled in the intervention programme [24].\nSample size Sample size ranged from 62 to 2,161. Two studies reported a power calculation based on the expected difference in the incidence of diabetes between groups [12, 21], and one reported a power calculation based on the expected difference between groups in the proportion of individuals with IGT at the end of the study [19]. In the latter study, Oldroyd et al. calculated that a total of 100 participants were required to detect a 0.6\u00a0mmol\/l difference in fasting glucose and a 20% difference in the number of individuals with IGT, allowing for a 90% power at a significance of 0.05. Three studies had sample sizes of fewer than 100 participants at follow-up [19, 20, 22].\nInclusion criteria All studies examined in this review included individuals with IGT and excluded those with isolated IFG [10, 12, 19\u201324].\nSex Except for one trial that involved only men (n\u2009=\u2009188) [24], all trials included both men and women. In the included studies a total of 40% of participants were men.\nIntervention conditions Seven of the eight included studies used a multi-component lifestyle intervention [10, 12, 19, 21\u201324], and one used a structured gym-based exercise training intervention [20].Six of the lifestyle intervention studies were based on encouraging individuals to increase their physical activity to approximately 150\u00a0min of exercise of moderate to vigorous intensity per week whilst also encouraging weight loss through a healthy energy-restricted diet [10, 12, 19, 21, 22, 24]. Participants in all six studies received regular encouragement and counselling from a trained dietician at least once every 3\u00a0months throughout the duration of the intervention. Two of the six studies also provided participants with the option of attending supervised exercise classes for some or all of the study duration [21, 24] and one provided discounted access to local gyms [19]. One study determined the effect of diet and exercise separately and in combination [10].One lifestyle intervention included an initial 1-month stay at a wellness centre where individuals were provided with healthy dietary options and encouraged to take part in 2.5\u00a0h\/day of light to moderate intensity exercise using the leisure facilities provided [23]. After the stay at the wellness centre, participants were encouraged to make plans about how they could incorporate healthier habits into everyday life and then received no further contact until follow-up.The structured exercise intervention study used a training protocol of 180\u00a0min per week of aerobic exercise at 70% of heart rate reserve [20]. Exercise training was supervised for the first 6\u00a0months and both groups were encouraged to eat a healthy energy-balanced diet, with those in the exercise training group also being encouraged to eat a diet with a high percentage of energy from carbohydrate [20].\nOutcomes Four studies included the incidence of diabetes as the main outcome [10, 12, 21, 24], and four used 2-h plasma glucose levels as a direct measure of glucose control [19, 20, 22, 23]. All the studies using the incidence of diabetes as their main outcome were based on a multi-component lifestyle intervention (see intervention conditions).\nIncidence of diabetes and physical activity All four of the intervention studies that measured the incidence of diabetes as their primary outcome found a significant reduction in the incidence of type 2 diabetes in the intervention group. Diabetes incidence was reduced by 42\u201363% in this group compared with the control group (see Table\u00a02). The study that investigated the effect of diet and physical activity both separately and in combination found a greater reduction in the incidence of diabetes (46% reduced risk) in the physical activity-only group than in either the combined physical activity and diet group (42% reduced risk) or the diet-only group (13% reduced risk), although the difference between groups was not statistically significant [10]. Three of these four studies relied on self-reported measures of physical activity [10, 12, 21], and of these, only the Diabetes Prevention Program (DPP) [12] and the Finnish Diabetes Prevention Study (FDPS) [21] reported using a validated physical activity questionnaire. All three of the studies relying on self-reported physical activity levels reported non-significant to small changes in physical activity levels in the intervention group. For example, the DPP reported a mean increase in energy expenditure due to leisure time physical activity of around six metabolic equivalent hours per week [12], which is approximately equivalent to walking at a moderate pace for 15\u00a0min\/day [25]. The FDPS reported no significant change in total physical activity levels compared with the control group and an increase of 9\u00a0min\/day in moderate to vigorous physical activity [21], and the Da Qing IGT and Diabetes Study reported no significant change in physical activity levels compared with the control group [10]. The Malm\u00f6 Feasibility Study, which used an objective outcome measure (cardiovascular fitness), reported an 8% increase in maximal oxygen uptake [24].\n2-h post-challenge plasma glucose and physical activity Three of the studies that used the incidence of diabetes as their primary outcome measure also measured 2-h plasma glucose before and after the intervention [10, 21, 24]. The FDPS reported a 0.9\u00a0mmol\/l decrease in 2-h plasma glucose after 1\u00a0year, but no significant change after 3\u00a0years [21]; the Da Qing IGT and Diabetes Study found that 2-h plasma glucose increased in all groups, but the increase in the control group was over twice that in either of the intervention groups [10]; and the Malm\u00f6 Feasibility Study reported a 1.1\u00a0mmol\/l reduction in 2-h plasma glucose in the intervention group [24]. Of the three lifestyle intervention studies that used 2-h plasma glucose levels rather than the incidence of diabetes as the primary indictor of improved glucose tolerance [19, 22, 23], only one reported a significant difference between the groups in terms of 2-h plasma glucose at follow-up [22]. Two of the studies used a measure of cardiovascular fitness as an indicator of physical activity levels [22, 23], and one [19] used distance walked in a shuttle test [26] as a measure of physical activity. Two studies found a small to moderate increase in cardiovascular fitness (<10% increase compared with baseline value) [22, 23], and the study using the shuttle test reported no change in the distance walked during the test [19]. Similarly, the moderate increases in cardiovascular fitness observed in the structured exercise training study were not associated with significant improvements in 2-h plasma glucose compared with the control group [20].\nFasting glucose None of the included studies reported a significant change in fasting glucose in the intervention group compared with the control group at follow-up. One study did not report fasting glucose values [24].\nDiscussion\nEight controlled trials in individuals with IGT were included in this review. Four studies measured the incidence of diabetes as a primary outcome measure, and found that the risk of diabetes was reduced by approximately 50% (range 42\u201363%) in individuals who were encouraged to reduce their body mass through changes in diet and physical activity [10, 12, 21, 24]. Although the promotion of physical activity was an important component of these studies, the effect of exercise independent of other factors on the risk of diabetes in individuals with IGT is still unclear. All but one [10] of the studies included in this review reported significant weight loss among participants. Given that weight loss is known to improve many of the factors associated with IGT, including insulin sensitivity and glycaemic control [27], and considering only modest increases in physical activity were found in these studies, the success of these interventions is likely to be largely explained by weight loss. The apparent success of the exercise-only intervention in the Da Qing IGT and Diabetes Study [10] is likely to be attributable, at least in part, to the significantly higher levels of physical activity at baseline in the exercise intervention group compared with the control group. The separation of physical activity and weight loss may seem an overcorrection given that increased physical activity may encourage weight loss through increased energy expenditure; however, several meta-analyses of controlled trials investigating the effect of physical activity on glycaemic control in individuals with diabetes found that exercise training was not associated with weight loss [28, 29]. Furthermore it is increasingly recognised that at least 60\u00a0min\/day of moderate intensity exercise should be undertaken for the effective management of body mass [30], an amount that none of the interventions included in this review achieved.\nThree of the four studies that investigated the effect of a lifestyle intervention in individuals with IGT on the incidence of type 2 diabetes [10\u201312] relied on self-reported measures of physical activity. Given the limitations of subjective measures of physical activity, particularly when measuring non-structured forms of moderate physical activity such as walking activity [31], these lifestyle intervention studies provide uncertain information about the effect of physical activity in individuals with IGT.\nResults from the lifestyle intervention studies that relied on changes in 2-h plasma glucose rather than the incidence of diabetes as the primary measure of glucose control were inconclusive [19, 20, 23]. Two of the three studies were unsuccessful at improving glucose tolerance [19, 23]. Similarly, the one study that used an aerobic exercise training protocol found no improvements in glucose tolerance as measured by 2-h plasma glucose [20]. However, it did find a significant improvement in insulin sensitivity at both 6 and 24\u00a0months. This suggests that, although the intervention goal of 3\u00a0h\/week of moderate intensity exercise was enough to improve insulin sensitivity, it was not long enough and\/or of sufficient intensity to elicit the necessary magnitude of change in insulin sensitivity for this to be translated into a significant reduction in 2-h plasma glucose.\nOverall, non-significant results were seen in all but two of the studies that measured 2-h plasma glucose before and after the intervention. However, despite the link between 2-h plasma glucose and diabetes risk, it does not follow that the risk of diabetes was unchanged in these studies, as demonstrated by the FDPS, which reported a non-significant change in 2-h plasma glucose over the course of the intervention but a >50% reduction in the risk of diabetes [21]. One reason for this discrepancy is likely to be the poor repeatability of 2-h plasma glucose values [32], and given the relatively small sample sizes in most of these studies, it is possible that improvements in glucose tolerance were not detected using 2-h plasma glucose. The glucose AUC has been identified as a more reliable measure of glucose tolerance than 2-h plasma glucose [33], and is therefore a more sensitive measure of glucose tolerance. One study included in this review measured both 2-h plasma glucose and glucose AUC at baseline and follow-up [20]. It reported that, although 2-h plasma glucose did not change significantly at any of the follow-up time points, there was a significant reduction in the glucose AUC at 6\u00a0months, and a trend towards significance at 24\u00a0months.\nGiven the failure of the lifestyle interventions to substantially increase physical activity levels, and the inconclusive result of the structured exercise training study, the role of physical activity independent of other lifestyle changes in the treatment of prediabetes remains equivocal. However, statistical analysis of the independent effects of physical activity, which has been carried out on some of the lifestyle intervention studies included in this review, show interesting results. For example, the conclusion of the Malm\u00f6 Feasibility Study that cardiovascular fitness and weight loss were equally correlated to improved glucose tolerance is supported by data from the Study on Lifestyle Intervention and Impaired Glucose Tolerance Maastricht (SLIM) [34], and a recent analysis of data from the FDPS found a 49% difference in the risk of diabetes, after adjustment for changes to body mass and diet, when comparing those in the highest and lowest tertiles of moderate to vigorous leisure time physical activity change [35]. Thus, although the overall evidence for the independent effect of physical activity in the management of prediabetes is equivocal, encouraging evidence is starting to emerge in support of the importance of exercise.\nGiven the limitations of the studies included in this review it is not possible to make any recommendations as to the intensity and duration of exercise needed to improve glucose tolerance and\/or reduce the risk of diabetes in individuals with IGT, independently of other lifestyle changes. The equivocal nature of the evidence is reflected in the advice given by the ADA, which recommends that individuals with IGT should include 150\u00a0min\/week of moderate to vigorous intensity exercise as part of a weight management programme [36]. However, the aforementioned analysis of the change in physical activity in the FDPS found that a difference of 246\u00a0min\/week in median values between those in the lowest and the highest tertiles of moderate to vigorous physical activity change was associated with a significant reduction in the risk of diabetes, after adjusting for changes in diet and body mass. However, the difference of 120\u00a0min\/week in median values between the lowest and middle tertiles was not associated with a reduced risk of diabetes [35]. Although this result was obtained by analysing the pooled cohort, and therefore provides little information about the effectiveness of the intervention itself, it does suggest that 150\u00a0min\/week of moderate to vigorous intensity exercise is unlikely to be enough to significantly reduce the risk of type 2 diabetes in individuals with IGT, independently of other lifestyle changes. However, given that this analysis relied on self-reported physical activity levels, further rigorous studies are needed to confirm this.\nAll studies included in this review selected individuals using IGT as an inclusion criteria. Therefore, any conclusions from this review can only be applied to individuals with IGT and it is impossible to determine whether or not exercise may be effective in treating individuals with isolated IFG. However as individuals with isolated IFG account for a minority of individuals with prediabetes [37], conclusions about the effect of exercise on IGT drawn from this review will apply to the majority of individuals with prediabetes.\nIn summary, the majority of studies identified for this review used interventions that encouraged dietary and physical activity to initiate and maintain weight loss in individuals with IGT. Analysis of these studies found that the independent effect of physical activity in reducing the risk of type 2 diabetes in individuals with prediabetes is equivocal. Furthermore, given the limited evidence, no definite conclusion can be drawn either as to the amount of physical activity needed to reduce the risk of diabetes in individuals with prediabetes or the effectiveness of a single-component physical activity intervention compared with more conventional multi-component interventions.\nThus, more evidence from rigorously designed randomised controlled trials with objective measures of physical activity is needed. As the majority of studies promoting lifestyle changes included in this review failed to substantially increase physical activity levels, strategies for effecting increased physical activity in this population also need to be researched thoroughly. Further investigation is also needed into whether exercise is equally effective in treating the different phenotypes of IGT and IFG.","keyphrases":["physical activity","impaired glucose tolerance","type 2 diabetes","prediabetes","exercise","igt","ifg","prevention","impaired fasting glucose"],"prmu":["P","P","P","P","P","P","P","P","P"]}
{"id":"Arch_Orthop_Trauma_Surg-4-1-2324129","title":"Closed reduction and percutaneus Kirschner wire fixation for the treatment of dislocated calcaneal fractures: surgical technique, complications, clinical and radiological results after 2\u201310 years\n","text":"Introduction To reduce complications, a minimally invasive technique for the treatment of dislocated intraarticular fractures of the calcaneus was used. Therefore previously described closed reduction and internal fixation techniques were combined and modified.\nIntroduction\nA fracture of the calcaneus allowed to heal in improper anatomical position leads to static and dynamic malfunctions of the whole foot with consequent limited load bearing capacity and walking ability [4, 29]. The associated pain leads to a significant impairment in quality of life. The goal of therapy for calcaneal fractures is the elimination of pain and restoration of walking ability for patients with normal foot shape and the ability to wear normal footwear.\nAt present, there are multiple operative procedures for the treatment of calcaneal fractures. The procedures can be differentiated by approach, implant type and whether the treatment is one- or two-stages. Recently, open procedures using internal fixation have been favored for surgical therapy of the calcaneus [2, 5, 9, 15, 17, 23, 24, 26]. A possible complication of an open procedure is the disturbance of wound healing with skin and soft tissue necrosis [2] and the possibility of cutaneous flaps [5, 8, 9, 15, 17]. In addition to posttraumatic arthritis in the lower ankle and adjoining joints, there are reports of osteitis of the calcaneus [2, 5, 17]. Advanced osteitis of the calcaneus can require a calcanectomy [15] or amputation of the lower leg [9, 17].\nIn an effort to reduce the complications that can occur with an open procedure, we have combined and modified previously described closed reduction and internal fixation techniques [14, 27, 28] to create a minimally invasive technique.\nThe aim of this study was to evaluate the clinical and radiographic results of our minimally invasive surgical treatment of intraarticular fractures of the calcaneus at 2\u201310\u00a0years postoperatively. We then compared our results to the results of both open and minimally invasive surgical techniques found in the literature.\nMaterial and methods\nPatients\nA total of 88 patients with 92 closed, dislocated and intraarticular fractures of the calcaneus were consecutively treated with a minimally-invasive technique developed at our institution by modifying and combining the procedures of Westhues [27], Poigenf\u00fcrst and Buch [14], and Wondr\u00e1k [28]. All the surgeries were performed without the use of bone graft. The average age at time of calcaneal fracture was 46.1\u00a0years (range 18\u201382\u00a0years) and most patients were male (71.6%). The cause of fracture was a fall from varying heights in 75 (85.2%) of cases and a motor vehicle accident in 13 (14.8%) of cases. From this group of patients, 63 (71.6%) patients with 67 fractures were available for retrospective examination with an average follow-up time of 5.7\u00a0years (range 2\u201310\u00a0years). Twenty-five patients were unavailable for follow-up examination; 12 patients who satisfied with the result of the surgery did not wish to participate in the study due to age or unwillingness to travel to the hospital, 7 patients were at an unknown address and 6 patients deceased.\nExamination\nAll the medical records, radiographs, pre- and post-operative computed tomography scans were available for the entire study group. Traumatic soft tissue damage was determined using the classification method of Tscherne and Oestern [25]. In addition to radiographic evaluation using the calcaneus lateral, calcaneus axial and Broden views at 20\u00b0 and 40\u00b0 [3], all the patients obtained a bilateral calcaneal preoperative CT scan for fracture classification and surgical planning. Fractures were evaluated by the classification scale of Sanders et al. [17, 18]. The duration of surgery, as well as the time the X-ray image intensifier used, were noted as operative data. For postoperative evaluation of the reduction, radiographs of the calcaneus in lateral, axial, Broden 20\u00b0 and 40\u00b0 [3] views were obtained. At the last follow-up evaluation, a CT scan of both calcanei was obtained to examine the geometry of the calcaneus and to evaluate arthritic changes in the lower ankle joint.\nFor a reconstruction of the calcaneal posterior facet to be considered satisfactory a joint deviation of \u22642\u00a0mm had to be seen radiographically on the postoperative radiographs. For the evaluation of the clinical results, the Zwipp score was used [30].\nStatistical analysis\nFor statistical analysis the Fisher\u2019s exact test and the Chi-square test were used.\nPerioperative treatment and surgical technique\nAfter a calcaneal fracture was diagnosed, the lower leg was evaluated. If the soft tissue was in good condition, primary surgery performed. In cases of severe swelling with potential soft tissue damage NSAIDs, local cryotherapy and active movement exercises determined the course of therapy.\nOnce the soft tissue was in good condition, surgery was performed. The patient was placed prone on the traction table under general anaesthesia or spinal anaesthesia without arrest of blood supply. After application of the calcaneus wire for traction in the dorso-cranial plane, the varus or valgus malaligment of the back foot was corrected with the wire positioned orthogonally to the longitudinal axis of the calcaneus. To achieve correct placement, the surgeon pulled at the traction bow with the knee bent along the longitudinal axis of the calcaneus. The subsequent traction in the plantar plane was performed with the leg stretched. Once placed in the proper orientation, the traction bow was connected to the retaining jig of the traction table (traction of 20\u00a0kg). The surgeon then held the heel of the patient with both hands and applied compression both medially and laterally with his thenar muscle to reduce the main medial and lateral fragments.\nWith the use of the X-ray image intensifier, the restoration of B\u00f6hler\u2019s angle and the reduction of the posterior facet were verified. A Steinmann pin was put with the pointed end into a universal chuck and the other end into the dorso-lateral calcaneus beneath the posterior facet with a stab incision (Fig.\u00a01a). For reduction of the posterior facet, the traction wire was used as a hypomochlion (Fig.\u00a01b). The anatomical restoration of the posterior facet was verified with use of the Brod\u00e9n radiographic views (20\u00b0 and 40\u00b0). The reduction of the calcaneus was verified by examining the lateral radiographic view (Fig.\u00a02a). The fragments were fixed with percutaneus Kirschner wires, which were arranged conically into the talus and\/or cuboid (Fig.\u00a02b). All of the main fragments to be reduced were held in place by the Kirschner wires and the arch of foot was held until the bone healed [28]. The wires were bent above the skin level, the wire traction removed and the stab incision closed with sutures.\nFig.\u00a01The Steinmann pin is placed into the dorso-lateral calcaneus above the traction wire (left side). For the reduction of the posterior facet, the traction wire is used as a hypomochlion (right side). The hematome is drained though the stab incisionFig.\u00a02Intraoperative lateral radiograph of a dislocated calcaneal fracture before reduction (left side). After reduction the fragments are fixed with percutaneus Kirschner wires (right side)\nPostoperatively, a dorsal lower leg splint was applied for immobilization and the lower portion of the operative side was elevated. On the first postoperative day, the patient was mobilized on crutches with no weight bearing on the operative side. Depending on pain levels, the patient began with active dorsal extension and plantar flexion in the upper ankle joint. The radiographic controls, (upper ankle joint anterior and lateral, calcaneus axial Brod\u00e9n views [25]) were performed immediately postoperative, and again 2 and 8\u00a0weeks postoperative. After the fracture was healed, the Kirschner wires were removed. Partial weight bearing began after the eighth postoperative week at 20\u00a0kg and was increased up to full weight bearing by the twelfth postoperative week (Fig.\u00a03).\nFig.\u00a03Clinical example. a Preoperative lateral radiograph of an intraarticular, displaced calcaneal fracture (left side). The preoperative CT scan shows a Sanders Type II AC fracture on the coronal reconstruction (right side). b Lateral radiograph taken 2.5\u00a0years postoperatively. The patient had a Zwipp score of 166 points (left side). Anatomical reconstruction of the posterior facet was achieved (right side). This coronal CT image shows the anatomical reconstruction of the posterior facet with no joint deviation\nResults\nFifty-five of the 88 (62.5%) patients suffered exclusively from calcanal fractures. Thirty-three of the 88 (37.5%) patients had multiple injuries. Twenty of the 33 (61%) patients with multiple injuries had local co-injuries such as fractures of the upper ankle joint, tarsus and front-foot. In 83 of the 92 calcaneal fractures, the soft tissue injury was graded as 1\u00b0 or 2\u00b0; in 9 fractures, the soft tissue injury was graded as 3\u00b0. In eight cases, including three patients with multiple injuries, surgery was performed immediately on the day of the trauma.\nLength of surgery averaged 61\u00a0min (range 20\u2013175\u00a0min) and screening time averaged 115\u00a0s (range 20\u2013454\u00a0s). To obtain proper retention of the fracture, between 4 and 7 Kirschner wires were used. The Kirschner wires were removed with or without local anaesthesia at an average of 10\u00a0weeks (range 7\u201315\u00a0weeks) as an outpatient procedure. Full weight bearing was achieved at an average of 15\u00a0weeks postoperative.\nSanders classification\nSanders type II, III and IV fractures were diagnosed (Table\u00a01). All the patients had a joint deviation of at least 2\u00a0mm. Patients with type I non-dislocated fractures were treated conservatively and were therefore not included in this study. Reconstruction of the posterior facet was verified radiographically in 13 of 15 (86.7%) type II fractures, in 47 of 52 (90.4%) type III fractures and in 16 of 25 (64%) type IV fractures immediately postoperatively (Table\u00a01).\nTable\u00a01Patients with immediate postoperative reconstruction of the posterior facet by Sanders fracture classificationSanders fracture classification (N\u00a0=\u00a092)Radiological reconstruction of posterior facet achievedType II15 (16.3%)13 (86.7%)Type III52 (56.5%)47 (90.4%)Type IV25 (27.2%)16 (64.0%)\nThe Zwipp score of 67 patients at the last follow-up evaluation\nThe Zwipp score of the 67 patients available for follow-up averaged 130\/200 points (range 48\u2013186 points) which is considered a good result [30]. Regardless of fracture type, 41 (61.2%) of the 67 patients had a very good or good result, 24 (35.8%) patients had a satisfactory result and 2 (3%) patients had a bad result. The worst clinical results occurred with type IV fractures (Table\u00a02). Thirty (44.8%) patients considered their treatment outcome very good or good, 28 (41.8%) as satisfactory and sufficient and 9 (13.4%) as insufficient.\nTable\u00a02Clinical and radiological results at last follow-up evaluation by Sanders fracture classificationSanders fracture classificationVery good or good resultArthritis in lower ankle jointNormal B\u00f6hler angle achievedOverall6741 (61.2%)33 (49.3%)47 (70.1%)Type II 6 (8.9%)4 (66.7)1 (16.7%)5 (83.3%)Type III 39 (58.2%)29 (74.4%)12 (30.8%)31 (79.5)Type IV 22 (32.8%)8 (36.4%)20 (90.9%)11 (50.0%)\nThirty-seven (55.3%) of the 67 patients had no pain while full weight bearing or could walk at least 4\u00a0h without pain at last follow-up evaluation. Nine (13.4%) patients had constant pain. Thirty-three (49.3%) patients had a range of motion in the upper ankle joint identical to that of the non-affected side. Thirty-four (50.7%) patients had a restricted range of motion of up to 15\u00b0 and more than half (58.2%) of the patients had achieved more than 75% of their total range of motion in the lower ankle joint. Forty-three (64.2%) of 67 patients were able to wear normal shoes while 5 (7.4%) used shoes with an unroll aid. Nineteen (28.3%) patients had obtained orthopaedic shoes on their own at last follow-up.\nTwo (3%) of 67 patients had arthritis of the upper ankle joint at last follow-up. Independent of fracture type 33 (50.2%) patients had arthritic changes in the lower ankle joint and 14 (20.89%) cases had arthritic changes in the calcaneocuboidal joint (Table\u00a02). B\u00f6hler\u2019s angle was restored in 70.1% (47 of 67) of the cases (Table\u00a02). A total of 85.1% (57 of 67) cases had a reduction in calcaneal height to within 10% when compared to the non-operative side. Additionally, a reduction in the length of the calcaneus occurred within 10 in 94% (63 of 67) of cases when compared to the non-operative side. Thirty-eight (56.8%) of the 67 patients had a widening of the calcaneus to within 10% compared to the opposite side.\nComplications\nOf the 92 surgically treated calcaneus fractures, 76 (82.6%) healed without complications. In nine (9.8%) cases, superficial skin infections, perforations of the Kirschner wires and bone dystrophy occurred and healed without any further complications. Significant complications occurred in six (6.5%) cases: three cases had osteitis of the calcaneus, one case had dislocation of the fracture requiring revision surgery and two cases had peroneal tendon impingement. Three cases of osteitis healed through conservative therapy with oral antibiotics and the two patients with impingement of the peroneal tendon refused any operative intervention.\nUsing our method, disturbance of wound healing with skin and soft tissue necrosis requiring operative intervention was not observed. Additionally, no lower leg amputations and no total or partial calcanectomies had to be performed.\nStatistical analysis\nUsing the Chi-square test a statistical significant correlation was found between the fracture type and the incidence of subtalar arthritis (p\u00a0<\u00a00.0268). Using the same test there was no statistical significant correlation found between the fracture type and quality of reduction (p\u00a0>\u00a00.6522), between fracture type and clinical result (p\u00a0>\u00a00.3204) and between fracture type and B\u00f6hler\u2019s angle (p\u00a0>\u00a00.5488). However, a statistical significant correlation was found between the clinical result and subtalar arthritis (p\u00a0<\u00a00.0013) and between the incidence of subtalar arthritis and quality of reduction when using the Fisher`s exact test (p\u00a0<\u00a00.0003).\nDiscussion\nTo correct deformations of the calcaneus, spare the soft tissue and lower the complication rate, indirect and less invasive reduction and fixation techniques to treat calcaneal fractures have been developed [7, 10, 12\u201314, 16, 19, 21, 22]. Besides techniques with percutaneous reduction and internal K-wire fixation [7, 14, 16, 21] percutaneous reduction techniques with external fixation are described in the literature [12, 13, 19, 22]. In our department we have combined and modified previously described closed reduction and internal fixation techniques [14, 27, 28] to create a minimally invasive technique. This method used the lower joint surface of the talus as a guide for remodeling the posterior facet and reconstruction of the calcaneus.\nA multiple of different evaluation scores makes a comparison of the clinical results with published results difficult [11, 17, 20, 30]. Using the Zwipp score, 61.3% of the cases (41 of 67) treated by our modified method had good or very good results regardless of fracture type. In 38.7% (26 of 67) of cases, a satisfactory or bad result was achieved. After open reduction and internal fixation with a plate, Rammelt et al. [15] and Boack et al. [2] reported good and very good results in 65 and 66% of cases, respectively with the use of the \u00b1200 Zwipp score. Boack et al. [2] presented satisfactory or bad clinical results in 34% of cases also using the Zwipp score.\nSanders et al. [18] showed that under application of a lateral approach followed by internal fixation with a plate, the worst clinical results occurred with type IV fractures. In this study the worst clinical results were achieved in the treatment of type IV fractures although within our cases no statistical significant correlation between fracture type and clinical result could be demonstrated (Table\u00a02).\nPatient satisfaction is an essential criterion for the successful treatment of calcaneus fractures. With a comparable follow-up period of 5.4\u00a0years, Thermann et al. [23] reported that 48.3% of patients viewed their treatment outcome as good or very good after open reduction of their calcaneus fractures, 37.3% of patients judged their treatment outcome as satisfactory and sufficient and 14.4% judged their outcome as insufficient. Our treatment method had comparable results with 44.8% of patients considering their treatment outcome very good or good, 41.8% as satisfactory and sufficient and 13.4% as insufficient.\nAt last follow-up evaluation, 64.2% of patients in this study were able to wear normal shoes while 28.4% required orthopaedic shoes. Comparably, in the study by Therman et al. [23], 68.7% of the patients wore normal shoes and 16.8% required orthopaedic shoes.\nOverall, satisfactory mobility of the joints adjoining the calcaneus was achieved with our surgical technique. In approximately half of our patients, complete range of motion in the upper ankle joint was achieved and in the lower ankle joint, approximately 60% of patients had no or low movement restriction. Similar mobility in the upper and lower ankle joints has been reported in the literature after open reduction and osteosynthesis with a plate [5, 23]. However, Buch [6] reported a worse range of motion in the upper and lower ankle joint after performing a percutaneus wire osteosynthesis in 100 calcaneal fractures with a varus or valgus deviation of the back foot occurring in half of the cases. In this study, malpositions of the back foot of more than 5\u00b0 were diagnosed in 6 (9%) cases of the 67 cases. Clinically relevant changes in the axis of the front foot were not observed.\nThe radiological evaluation of the joints adjoining the calcaneus showed arthritic changes in the upper ankle joint in two of our cases. Half of our cases showed arthritic changes in the lower ankle joint and in approximately a fifth of our evaluated cases, arthritic changes were found in the calcaneocuboidal joint. In our study, 66.7% of type-II fractures and 98% of type-III\/IV fractures showed arthritic changes in the lower ankle joint. These results are similar to those presented by Thermann et al. [23], who showed arthritic changes in the lower ankle joint in 65.2% of type-II fractures and in 81.7% of type III\/IV fractures.\nRegardless of fracture type, anatomical restoration of the joint surface or a joint deviation of up to 2\u00a0mm of the posterior facet was achieved postoperatively in 82.1% of cases. A total of 86.7% of type-II fractures and 72.8% of type-III\/IV fractures achieved anatomical restoration of the posterior facet. These results are comparable to those presented in the literature with open reduction and internal fixation with plates or screws [2, 17, 23].\nIn this study, the average length of the surgery was 61\u00a0min. This is considerably less than the average length of surgeries utilizing open reduction and internal fixation of 139 and 168\u00a0min [2, 20].\nIn 9.8% of cases, complications such as superficial skin infections, perforation of the Kirschner wires and bone dystrophy healed with conservative therapy or after removal of the Kirschner wires. This rather insignificant rate of complications is similar to those described in the literature after operative treatment of calcaneal fractures utilizing a minimally invasive technique [7, 16, 21].\nThe rate of significant complications in this study was 6.5%. In two cases, an impingement of the peroneal tendon occurred. One case had a dislocation of the fragments requiring revision surgery. Three cases had infections of the calcaneus and were related to a traumatic soft tissue injury that healed with conservative therapy. With open reduction, skin and soft tissue necrosis [2] with possible cutaneous flaps have been reported in scattered cases [5, 8, 9, 15, 17]. Folk et al. [9] demonstrated that in 25% of cases, wound complications required an additional surgery after open reduction of calcaneus fractures 84% of the time. Abidi et al. [1] reported wound healing disturbances in 33% of cases following open reduction and internal fixation of calcaneal fractures. There have also been reports in the literature of partial or total calcanectomies [15], including amputations, as a result of calcaneal osteitis after open reduction [9, 17]. Sanders et al. [17] considered a lower leg amputation if a patient suffered continual osteitis. In the current study no disturbances of wound healing, skin or soft tissue necrosis that required microsurgical intervention were observed.\nIn this study no statistical significant correlation could be found between fracture type and the ability to restore B\u00f6hler`s angle. However, regardless of fracture type Stulik et al. [21] showed similar results using a comparable minimally invasive technique while Thermann et al. [23] had worse results following open reduction with internal fixation.\nIn our study, there was a slight reduction in the height and length of the operated calcaneus when compared to the non-operated side. With our closed reduction and internal fixation technique, more than half of the cases had a widening of the calcaneus of more than 10% when compared to the opposite side, which is an unsatisfactory result. At the same time only two patients had an impingement of the peroneal tendon that the patients considered tolerable.\nIn summary, we presented a minimally invasive technique for the treatment of intraarticular, dislocated calcaneus fractures and were able to produce results comparable to open techniques with a lower rate of serious complications. In the majority of cases, an almost identical B\u00f6hler angle and geometry of the calcaneus was achieved when compared to the opposite side at the time of last follow-up. Simple removal of the Kirschner wires and shorter surgery time decrease patient stress and must be recognized as an advantage of this minimally invasive technique. Thus, we feel that our minimally invasive technique is a viable alternative for the treatment of intraarticular, dislocated calcaneal fractures.","keyphrases":["dislocated calcaneal fracture","minimal-invasive"],"prmu":["P","U"]}
{"id":"Purinergic_Signal-3-4-2072916","title":"Guanosine reduces apoptosis and inflammation associated with restoration of function in rats with acute spinal cord injury\n","text":"Spinal cord injury results in progressive waves of secondary injuries, cascades of noxious pathological mechanisms that substantially exacerbate the primary injury and the resultant permanent functional deficits. Secondary injuries are associated with inflammation, excessive cytokine release, and cell apoptosis. The purine nucleoside guanosine has significant trophic effects and is neuroprotective, antiapoptotic in vitro, and stimulates nerve regeneration. Therefore, we determined whether systemic administration of guanosine could protect rats from some of the secondary effects of spinal cord injury, thereby reducing neurological deficits. Systemic administration of guanosine (8 mg\/kg per day, i.p.) for 14 consecutive days, starting 4 h after moderate spinal cord injury in rats, significantly improved not only motor and sensory functions, but also recovery of bladder function. These improvements were associated with reduction in the inflammatory response to injury, reduction of apoptotic cell death, increased sparing of axons, and preservation of myelin. Our data indicate that the therapeutic action of guanosine probably results from reducing inflammation resulting in the protection of axons, oligodendrocytes, and neurons and from inhibiting apoptotic cell death. These data raise the intriguing possibility that guanosine may also be able to reduce secondary pathological events and thus improve functional outcome after traumatic spinal cord injury in humans.\nIntroduction\nSpinal cord injury (SCI) occurs in an instant, but its devastating effects last a lifetime at huge personal and economic cost [1]. The spinal cord conveys both afferent sensory and efferent motor information, so disruption of spinal cord function results not only in motor paralysis but also sensory and autonomic impairment distal to the injury [2, 3]. Sensory dysfunction contributes to the generation of pressure sores that, like bladder impairment, are a major source of morbidity and even mortality in those with spinal cord injury [4].\nRestoration of function in longstanding spinal cord injuries is poor, although some limited functional restoration has been reported [5]. Therefore, there is much interest in reducing the extent of the initial damage from traumatic spinal cord injury in the acute phase. Trauma often results in primary damage resulting from mechanical disruption of the nerve axons in the spinal cord that is not amenable to neuroprotective therapy. However, secondary pathological changes involving cascades of biochemical, molecular, and cellular changes can produce even more extensive damage, and these changes are potentially susceptible to therapeutic intervention with neuroprotective agents [3, 6]. Thus, pathological changes occur from the moment of injury and continue for years afterwards and have been divided into three phases: an acute phase, a phase of secondary tissue loss, and a chronic phase [3, 6].\nIn the acute phase, which starts at the moment of injury and extends over the first few days, numerous pathological processes begin. Mechanical injury induces an immediate change in neuronal tracts at the moment of impact, blood flow is reduced, creating substantial ischemic necrosis [7, 8] and a cascade of pathophysiological processes rapidly follows mechanical trauma to the spinal cord, resulting in secondary neuronal damage that can significantly exacerbate the original injury [9]. Traumatic injury to the spinal cord also leads to a strong inflammatory response, with the recruitment of peripherally derived inflammatory cells, including macrophages [10]. Damage to the spinal cord also results in extensive cell proliferation in and around the epicenter, many of which are microglia and macrophages. This acute inflammatory response at the site of the initial lesion is at least partly responsible for secondary spinal cord pathology [11\u201313]. The inflammatory cells (particularly macrophages\/microglia) can mediate tissue damage by producing a variety of cytotoxic factors including interleukins [14], tumor necrosis factor-alpha (TNF-alpha) [15], and reactive nitrogen species [13, 16\u201318]. Neuronal and oligodendroglial cell loss is apparent in the lesion epicenter, and rostral and caudal to it within 4\u00a0h of injury [19]. From days to years after the initial trauma, apoptotic cell death continues, and scarring and demyelination accompany Wallerian degeneration. All these processes contribute to motor and sensory functional deficits [20, 21].\nMany pharmacological agents have been reported to reduce secondary injury and to be neuroprotective in a variety of animal models; these include anti-inflammatory [22\u201324] and antiapoptotic agents [24, 25] and agents that elevate cyclic adenosine monophosphate (cAMP) [26, 27]. None has yet proved effective in ameliorating the effects of acute spinal cord injury in clinical trials in humans.\nThere is increasing evidence that the non-adenine-based purine guanosine acts as an intercellular signaling molecule. It is released from cells and has several diverse effects on cells in vivo and in vitro, particularly trophic effects modulating cellular growth, differentiation, and survival [28, 29]. Guanosine has a number of effects on various cell types that make it a good candidate to test as a neuroprotective agent in acute spinal cord injury since it might potentially interact with several steps of the biochemical and cellular cascade. It is neuroprotective [28\u201332] and stimulates nerve regeneration [5, 33]. It also protects several cell types against apoptosis induced by a variety of agents such as staurosporine [34] and beta-amyloid [35] and has been reported to increase intracellular cAMP [36, 37]. Therefore, in the present study, we assessed whether guanosine might ameliorate tissue damage and enhance functional outcome after acute spinal cord injury.\nMaterials and methods\nAnimals\nAdult female Wistar rats (280\u2013300\u00a0g body weight, Charles River) were maintained in a temperature-controlled vivarium on a 12:12-h light-dark cycle with food and tap water freely available. Rats were handled daily for 2\u00a0weeks before surgery.\nSpinal cord injury induction and experimental design\nSpinal cords were surgically exposed and compressed with modified coverslip forceps to produce a moderate traumatic spinal cord injury [5, 38]. Before surgery, rats were given buprenorphine (0.03\u00a0mg\/kg body weight, subcutaneously) for pain relief. They were then anesthetized with isoflurane (3\u20135%): O2 (1\u00a0l\/min) and a laminectomy was performed at T11\/T12 to expose the spinal cord that was then crushed with modified coverslip forceps [5, 38\u201340]. The forceps were closed slowly (over 2\u00a0s) compressing a 5-mm length of the spinal cord to a thickness of 1.4\u00a0mm for 15\u00a0s. The wound was closed by suturing the muscles and fat pad, and by clipping the skin with stainless steel clips. Postoperatively, the rats were kept quiet and warm.\nTo evaluate the effect of guanosine on acute phase SCI pathophysiology, we studied two groups of rats (n\u2009=\u200924) with SCI. After surgery and prior to treatment, behavioral tests were done for each rat. Two of the total 24 animals were excluded from the study because of incorrect injury (BBB score at the surgery day before treatment below 4 or above 7; in our experience, about 91% of rats with this degree of crush were within the range of 5 to 6 on the BBB scale at first day after injury [38]). The 22 rats were then randomly divided into two groups. Starting 4\u00a0h after surgery, rats received either daily intraperitoneal (i.p.) injections of 8\u00a0mg\/kg guanosine or the same volume of saline containing 0.001\u00a0N NaOH [5] for 2\u00a0weeks. On day 7 after injury one animal in the saline group was euthanized because of a severe bladder infection.\nMotor and sensory functional recovery assessment\nAll rats were handled daily for 2\u00a0weeks preoperatively to acclimatize them to the handling and behavioral testing. After spinal cord compression, the locomotor behavior segmental reflexes and spinothalamic senses of the rats were assessed immediately prior to treatment (day 0) and on days 1, 3, 5, 7, 14, 21, and 28 after the injury. Five tests were used: an open field walking task [5, 41, 42], hind limb placing response [38, 40], foot orienting response [38, 40, 43], an inclined plane test [38, 44, 45], and a hot plate test [46]. Behavioral analyses were performed by individuals who were blinded with respect to treatment groups.\nAn open field walking testing (OFWT) was used to assess the locomotor functional recovery of the hind limb. It was conducted in a child\u2019s circular plastic swimming pool (1.3\u00a0m in diameter [5, 38]). Cagemates (two animals) were placed in the center of the open field. They were observed for 5-min periods and scored for general locomotor ability using the standard BBB scale. The rats were rated on a scale of 0 to 21, 0 being no function and 21 being normal. If the animal stopped moving for a minute, it was placed again in the center of the open field; otherwise it was left undisturbed for the duration of the 5-min test period.\nReflexes in the hind limbs were assessed with hind limb placing response (HLPR) and foot orienting response (FOR). They were each scored on a scale of 0 to 2, 0 indicating no function and 2 indicating full function [38, 40, 43]. Half-scores were assigned if the behavioral response appeared intermediate. Hind limbs were scored separately for each measure. To assess HLPR, we grasped the hind foot between the thumb and forefinger, pulled backwards, and then released the foot. The placement of the foot on the table surface was then scored [38, 40]. The FOR followed Gruner\u2019s [40] protocol modified from the previous descriptions of this reflex [43]. When a rat is raised and lowered by the tail, it shows a characteristic behavior of the hind legs. A normal rat spreads the toes of its hind leg wide apart and generally holds them apart for several seconds. After spinal cord injury, this response is sometimes lost completely, or reduced in magnitude.\nThe inclined plane test (IPT) measured the ability of the rats to maintain their position for 5\u00a0s on an inclined plane, covered by a rubber mat containing horizontal ridges (1\u00a0mm deep, spaced 3\u00a0mm apart, self-made) [44]. The rats were observed as the angle of the surface was increased from 5 to 90\u00b0 at 5\u00b0 intervals. The angle at which the rat could no longer stay in position was the outcome measure.\nHind limb sensory function was tested by ability to perceive a standard application of a controlled stimulus to the trunk below the level of the lesion using a standard testing apparatus hot plate (IITC Life Science Inc., Woodland Hills, CA, USA) [46]. The time taken for the rat to withdraw its hind paws from contact with the hot plate was noted. A cutoff time was about 20\u00a0s after which the animals were removed to prevent thermal injury [46].\nRecovery of lower urinary tract function\nNormal lower urinary tract (LUT) function involves both spinal and supraspinal circuitry that controls urine storage and release [47]. Incomplete SCI results in an initial loss and later partial or complete recovery of LUT function depending on the severity of injury [48, 49].\nAfter SCI the rats were not capable initially of spontaneous micturition, and their bladders were manually expressed twice daily. The volume of expressed urine was measured each time, and the data were used to estimate the initiation of LUT function after SCI.\nHistological analysis\nTissue processing and immunohistochemical staining Twenty-eight days after SCI, animals were killed for histological and immunohistochemical analysis. Rats were deeply anesthetized with sodium pentobarbital (50\u201360\u00a0mg\/kg b\/w, i.p.) and perfused transcardially; first with 100\u00a0ml 0.05\u00a0M phosphate-buffed saline (PBS) containing 0.1% heparin, followed by 300\u2013500\u00a0ml of 4% paraformaldehyde (PFA). The T9 to L1 segments of the spinal cords were removed and incubated in the same fixative solution overnight at 4\u00b0C and then cryoprotected in PBS solution containing 30% sucrose. A segment of each cord, extending from 5\u00a0mm rostral to 5\u00a0mm caudal to the lesion site was embedded in medium (Tissue-Tek\u00ae O.C.T. compound, Sakura Finetek USA, Inc., Torrrance, CA, USA). Serial sections were cut at 20-\u03bcm intervals on a cryostat and mounted onto slides (ColorFrost\/Plus; Fisher, Pittsburgh, PA, USA) for histochemical staining. Some cords were transverse-sectioned for immunohistochemical analysis using the antibodies described in the section below. Longitudinal sections were cut for Luxol fast blue staining, a lipophilic dye used commonly to stain myelin [38].Details of these immunohistochemical procedures have been described previously [5, 38]. Briefly, the cryostat sections were thawed, air-dried, and then incubated in hydrogen peroxide to reduce endogenous peroxidase activity, before being rinsed in PBS. The sections were then incubated in 1% sodium borohydride for 15\u00a0min. After thorough washing with PBS, the sections were treated with PBS\/5% normal goat serum with 0.3% Triton X-100 at room temperature for 30\u00a0min. Overnight incubation with primary antibody was performed in humidified boxes at 4\u00b0C. Macrophages and reactive microglia were detected with a mouse monoclonal antibody against ED-1 (MCA 341R, 1:500; Serotec, Hornby, ON, Canada) and reactive astrocytes (astrogliosis) were determined with rabbit anti-glial fibrillary acidic protein (GFAP) polyclonal antibodies (Zymed\u00ae Lab-SA System Kit, dilution was 1:600, Invitrogen Canada Inc., Burlington, ON, Canada). The following day, sections were rinsed with PBS and incubated with either rhodamine-conjugated or fluorescein isothiocyanate (FITC)-conjugated secondary antibodies. Sections were then rinsed, coverslipped, and examined under a confocal microscope.To determine the spared axons and myelin, double fluorescent immunolabeling was performed, combining mouse monoclonal antibodies against neurofilament (RT-97, 1:10; Developmental Hybridoma Bank) and rabbit anti-myelin basic protein (MBP) polyclonal antibodies (1:50; Chemicon Int., Temecula, CA, USA). For double immunolabeling, sections were developed using a mixture of FITC-conjugated goat anti-rabbit IgG and rhodamine-conjugated goat anti-mouse IgG in 1% normal goat serum and 0.25% Triton X-100; 1:200 (Invitrogen Canada Inc., Burlington, ON, Canada) for 2\u00a0h. Sections were then rinsed, coverslipped, and examined under a confocal microscope. An investigator who was blinded to the treatment groups conducted histological analysis.\nTerminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling assays and quantification For detection of apoptotic cells, a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) stain was performed using the \u201cIn situ Cell Death Detection Kit-Fluorescein\u201d (Roche Molecular Biochemicals, Chemicon Int., Temecula, CA, USA), according to the manufacturer\u2019s instructions. After fixation, tissue sections were incubated in TdT-mediated dUTP-biotin nick end labeling (TUNEL) reaction mixture containing TdT buffer with TdT and biotinylated dUTP in TdT buffer, incubated in a humid atmosphere at 37\u00b0C for 90\u00a0min, and then washed with PBS. The sections were incubated at room temperature (37\u00b0C) for 30\u00a0min with fluorescence-conjugated antibody. The results were analyzed using confocal fluorescence microscopy. TUNEL-positive cells in the lesion site in the spinal cord were quantified by counting positively stained cells. Sections (5\u20137 per animal) taken from the penumbra of the lesion and spaced about 100\u00a0\u03bcm apart were analyzed for each animal (n\u2009=\u20093\u20135 animals per group). Apoptotic cell death was determined by counting the total number of TUNEL-positive nuclei through entire cross sections. Low power sections were digitized and manually outlined using an image analysis system. Any cavities present in the sections were excluded from analysis. Data are expressed as cells per section [5].\nQuantification For cell counting, 5\u20137 sections from each animal (n\u2009=\u20095 for each group) at the lesion site (every third section, 100\u00a0\u03bcm apart) were analyzed. OX-42-positive microglia and TUNEL-positive nuclei were counted through the entire cross section. Data are expressed as the number of immunostained cells per section (mean \u00b1 SEM).\nStatistical analysis\nAll data are presented as mean \u00b1 SEM. The statistical significance of behavioral scores was analyzed by Kruskal-Wallis nonparametric analysis of variance followed by Mann-Whitney U tests. Histological data were evaluated by Student\u2019s t-tests.\nResults\nGuanosine improves neurological function\nOver the course of 4\u00a0weeks after spinal cord injury, control rats (which received saline treatment) recovered occasional weight-supported plantar steps, but no fore-hind limb coordination, and had a mean BBB locomotor score of 9.3\u2009\u00b1\u20090.6 (Fig.\u00a01). In contrast, the guanosine-treated rats recovered to a BBB score of 14\u2009\u00b1\u20090.5 (Fig.\u00a01); they exhibited consistent weight supporting with consistent fore-hind limb coordination. Hind limb locomotor function in guanosine-treated animals was significantly better than in rats that received saline treatment (P\u2009<\u20090.01).\nFig.\u00a01Open field walking test (OFWT) scores from day 0 (the same day as surgery, prior to treatment) to 28\u00a0days after spinal cord injury for groups of saline- and guanosine-treated animals (means \u00b1 SEM). Animals with normal spinal cord function score 21, whereas a score of 0 represents total paralysis. Hind limb locomotor function in guanosine-treated animals (n\u2009=\u200911) was significantly better than in rats that received saline treatment (n\u2009=\u200910; P\u2009<\u20090.01) from day 1 to 28 after injury\nUninjured rats have normal HLPR scores of 2 [38, 40]; they always place an extended hind limb briskly beneath the body in a proprioceptive placing response. Injured rats place their hind limb either partially, or unreliably, or not all, depending on the time since the injury and the treatment. In the present study, 4\u00a0weeks after injury, control rats with saline injection attained a 1.0\u2009\u00b1\u20090.1\u00a0HLPR score (Fig.\u00a02) that was characterized by little or no attempt to place the foot, or leaving the foot extended with its dorsal surface down. In contrast, rats treated with guanosine reached a score of 1.7\u2009\u00b1\u20090.1 (Fig.\u00a02), with the toes of their hind legs spread wide apart, a finding which is significantly better than rats that received saline treatment (P\u2009<\u20090.01). Uninjured rats have a normal FOR score of 2. Saline-treated animals had a FOR score of 1.0\u2009\u00b1\u20090.1 (Fig.\u00a03). These rats extended their hind legs laterally with toe spread but turned their feet outward. When these rats were lowered, they did not orient their feet toward the surface. In contrast, the guanosine-treated rats recovered to a score of 1.7\u2009\u00b1\u20090.1 (Fig.\u00a03), which is significantly different (P\u2009<\u20090.01) from the saline-treated rats.\nFig.\u00a02Hind limb placing response (HLPR) scores from day 0 (the same day as surgery, prior to treatment) to 28\u00a0days after spinal cord injury for groups of saline- and guanosine-treated animals (means \u00b1 SEM). Animals with normal spinal cord function score 2, whereas a score of 0 represents total paralysis. Compared with saline-treated control animals (n\u2009=\u200910), guanosine-treated animals (n\u2009=\u200911) had a significantly better improvement of their hind limb placing responses (P\u2009<\u20090.01) from day 1 to 28 after injuryFig.\u00a03Foot orienting response (FOR) scores from day 0 (the same day as surgery, prior to treatment) to 28\u00a0days after spinal cord injury for groups of saline- and guanosine-treated animals (means \u00b1 SEM). Animals with normal spinal cord function score 2, whereas a score of 0 represents total paralysis. Compared with saline-treated control animals (n\u2009=\u200910), guanosine-treated animals (n\u2009=\u200911) had a significantly better improvement of their foot orienting responses (P\u2009<\u20090.01) from day 1 to 28 after injury\nUninjured rats maintain their position on the inclined plane even at an angle of 90\u00b0. In the present study, saline-treated control rats recovered their ability to maintain their position at 69\u00b0\u2009\u00b1\u20092 by 4\u00a0weeks after injury, whereas guanosine-treated rats were able to maintain their position to a mean incline of 79\u00b0\u2009\u00b1\u20091 (Fig.\u00a04) by 4\u00a0weeks (P\u2009<\u20090.05).\nFig.\u00a04Inclined plane test (IPT) scores from day 0 (the same day as surgery, prior to treatment) to 28\u00a0days after spinal cord injury for groups of saline- and guanosine-treated animals (means \u00b1 SEM). Compared with saline-treated control animals (n\u2009=\u200910), guanosine-treated animals (n\u2009=\u200911) had a significantly better score in their inclined plane test (P\u2009<\u20090.05) from day 1 to 28 after injury\nBoth saline- and guanosine-treated rats were insensitive to the thermal stimulus using the hot plate test during the first 2\u00a0days after injury. There was a gradual recovery of sensory function thereafter with recovery being accelerated in guanosine-treated animals compared to controls (P\u2009<\u20090.05) (Fig.\u00a05).\nFig.\u00a05Post-lesional sensitivity of hind limbs touched by a hot plate from day 0 (the same day as surgery, prior to treatment) to 28\u00a0days after spinal cord injury for groups of saline- and guanosine-treated animals. Values are the means \u00b1 SEM of the average time of withdrawal of left and right hind paws during contact with a hot plate. Compared with saline-treated control animals (n\u2009=\u200910), guanosine-treated animals (n\u2009=\u200911) had a significantly better improvement of their sensory function (*P\u2009<\u20090.05) from day 2 to 14 after injury\nGuanosine accelerated LUT functional recovery following SCI\nIn saline-treated rats the volume of manually expressed urine increased over the first 7\u00a0days after SCI and then decreased as spontaneous micturition was reestablished. On the basis of a previous study [48], the increase in volume of expressed urine during the first week after injury was interpreted as resulting from increased bladder size in the absence of spontaneous micturition. The subsequent decrease in manually expressed urine indicates the initiation of spontaneous micturition (Fig.\u00a06). In guanosine-treated rats there was a significantly lower residual urine volume compared to the saline-treated animals (Fig.\u00a06; P\u2009<\u20090.001). More importantly, the rats that received guanosine recovered their LUT function completely by 7\u00a0days after injury (Fig.\u00a06).\nFig.\u00a06Time course of recovery of lower urinary tract function (spontaneous voiding). Urinary bladders were expressed every 12\u00a0h, and the collected urine volume was measured. Means \u00b1 SEM of the volume for each group. Compared with saline-treated control animals (n\u2009=\u200910), guanosine-treated animals (n\u2009=\u200911) had significantly less urine collected with time after SCI (*P\u2009<\u20090.05; **P\u2009<\u20090.001). The rats that received guanosine had empty bladders by day 7 after injury\nGuanosine attenuates macrophage and microglia activation, but not astrogliosis\nAfter SCI, early inflammatory reactions consisting of neutrophil and macrophage invasion as well as activation of microglia and astrocytes were reported in earlier studies [50, 51]. In the present study, the activated macrophages and microglia were labeled by the ED-1 immunoreaction. Guanosine treatment decreased the number of the ED-1 immunopositive cells compared with saline treatment (Fig.\u00a07a, b, e; P\u2009<\u20090.01). However, no differences were found between the saline and guanosine groups in the number of activated astroglia (astrogliosis) indicated by the GFAP immunostaining (Fig.\u00a07c, d).\nFig.\u00a07Fluorescent immunostaining using antibodies against a marker (ED-1) for macrophages and activated microglia in cross sections of cords from saline-treated (a) and guanosine-treated (b) animals at the lesion site 4\u00a0weeks after injury. There were fewer ED-1-immunolabeled cells in cords of guanosine-treated rats (b, e) compared to the cords of vehicle-treated rats (a, e; P\u2009<\u20090.01). GFAP-immunofluorescent staining of cross sections of spinal cords from vehicle-treated (c) and guanosine-treated (d) animals at the lesion site 4\u00a0weeks after injury showed no difference in immunostaining of GFAP between the cords of guanosine-treated rats (d) and vehicle-treated rats (c). Scale bar\u2009=\u200950\u00a0\u03bcm for all\nGuanosine attenuates apoptotic cell death in lesion site of spinal cord\nIn the present study, apoptotic cell death induced by traumatic spinal cord injury was determined with TUNEL staining. TUNEL-positive nuclei were not uniformly distributed throughout the cross section of the spinal cord but were more numerous in the vicinity of the injury center. Quantification of TUNEL-positive nuclei showed that there were significantly fewer TUNEL-positive nuclei in the spinal cords of guanosine-treated rats than saline-treated control animals (Fig.\u00a08; P\u2009<\u20090.001).\nFig.\u00a08TUNEL-positive apoptotic cells in spinal cord lesions were quantified by counting the total number of TUNEL-positive nuclei through entire cross sections. Compared with the cords from saline-treated animals (a, c), guanosine-treated cords had significantly fewer TUNEL-positive cells (b, c; P\u2009<\u20090.001). Scale bar\u2009=\u200950\u00a0\u03bcm for both a and b\nGuanosine increases axon and myelin sparing\nAxons were labeled with antibodies against RT-97 (a neuronal cytoskeletal protein; Fig.\u00a09a, b) and myelin was labeled with antibodies against MBP (a specific marker for myelin; Fig.\u00a09c, d). The immunohistochemistry showed that there were more axons (red, Fig.\u00a09b) and myelin (green, Fig.\u00a09d) as well as myelinated axons (yellow, Fig.\u00a09f) in the spinal cords from guanosine-treated animals than in the cords from saline-treated control rats (Fig.\u00a09a, c, e), indicating that systemic administration of guanosine is associated with preservation of tissue including neuronal and glial elements.\nFig.\u00a09Immunostaining with antibodies against RT-97 for labeling axons (a, b) and against myelin basic protein (MBP) for central myelin (c, d) at the lesion site demonstrated the spared tissue. Cross sections from saline-treated (a) and guanosine-treated cords (b) demonstrate a neurofilament (NF) immunoreactivity surrounding the lesion site. c, d Cross sections from saline-treated (c) and guanosine-treated cords reveal myelin at the lesion site. e, f Merger of the two images demonstrates NF (red) and MBP (green) double fluorescent immunolabeling. There were more spared axons and more myelin in the cords around the lesion site from guanosine-treated animals compared to the saline-treated group. Scale bar\u2009=\u200950\u00a0\u03bcm for all\nDiscussion\nThese data are the first to demonstrate the ability of guanosine to act as a neuroprotective agent in vivo. These findings have potential clinical relevance. Previous studies have shown that after central nervous system injuries, the concentrations of guanosine are elevated around the injury and in CSF, sometimes for prolonged periods [32, 52]. Given the various trophic and antiapoptotic effects of guanosine in vitro [28, 34, 35], it seemed likely that increasing the concentration of guanosine in the central nervous system after injury in vivo might also be neuroprotective. We found that within 7\u00a0min of systemic administration, guanosine enters and progressively accumulates in the central nervous system (unpublished data), where it is converted to guanine. It remains to be determined whether guanosine, guanine, or both are the active neuroprotective agent. However, guanosine is more readily deliverable than guanine as a potential neuroprotectant.\nThe model of incomplete spinal cord injury that we have used is clinically relevant since about 50% of patients with spinal trauma have incomplete injury [53]. After incomplete spinal cord injury, both reflex and voluntary motor functions below the level of the injury are initially lost; partial recovery occurs over time [41, 54, 55]. The recovery of functions mediated by supraspinally controlled reflexes is slow and incomplete since these require the function of long tracts, many of which are irreversibly damaged by the injury [56, 57]. Recovery of locomotion and limb placement depends on ascending and descending spinal cord tracts, including cortico-, rubro-, reticulo-, vestibulo-, and raphespinal tracts [58]. The functional loss after spinal cord injury in rats involves interruption of descending serotonergic [59], reticulospinal, and other descending spinal tracts that facilitate segmental reflexes [40, 60]. The ascending spinothalamic tracts mediate the perception of pain and temperature below the level of the lesion. Since all of these motor and sensory functions were better in guanosine-treated animals than in control animals, it appears that guanosine preserved the function of multiple long tracts. This was associated with increased axonal survival and myelin preservation.\nNormal micturition requires coordinated activation of the bladder smooth muscle (detrusor) and the striated muscle of the external urethral sphincter, controlled by spinal and supraspinal circuitry [47]. Thus after incomplete spinal cord injury, initially bladder function is lost, but later partially recovers, the extent of recovery depending on the degree of preservation of white matter (and hence the long tracts) at the injury site [48, 49]. Our data revealed that in guanosine-treated rats, residual urine volume was not only much less than in controls from the first day after the spinal cord injury, but that by 7\u00a0days after spinal cord injury micturition had returned to normal. Because guanosine began to exert a neuroprotective effect on both motor and bladder function soon after it was first administered, its neuroprotective effect on each may involve similar mechanisms.\nThe inherent complexity of the biological system coupled with the many potential trophic actions of guanosine make it difficult to determine the mechanisms by which guanosine produces its effects. Nevertheless, neuroprotective effects were observed by 24\u00a0h after spinal cord injury. As guanosine was administered 4\u00a0h post-injury, it must have affected processes that were important between 4 and 24\u00a0h post-injury. Within this time frame after traumatic injury of the spinal cord, both inflammatory responses and apoptosis are prominent. Activation and proliferation of microglia\/macrophages play an important role in the secondary damage following spinal cord injury. Thus, cells at the center of the injury cascade are potential targets for neuroprotective treatments of acute SCI [21, 61]. As guanosine attenuated the activation and proliferation of microglia\/macrophages following SCI, this may at least in part provide an explanation for its neuroprotective effect.\nGuanosine has antiapoptotic effects in vitro [34, 35] and this is another potential mechanism through which guanosine might exert beneficial effects after spinal cord injury. Apoptosis after SCI has been described by many investigators [15, 62\u201367]. In these reports, early apoptosis of neural cells, including neurons, is followed by a delayed wave of predominantly oligodendroglial-programmed cell death in degenerating white matter tracts [63\u201367]. Studies of apoptosis in white matter after injury raise the possibility that glial apoptosis occurs, at least in part, as a consequence of axonal degeneration [68, 69]. However, the presence of activated microglia in contact with apoptotic oligodendrocytes after SCI indicates that this interaction may also activate cell death programs in the oligodendrocyte [70]. Secondary axonal degeneration may then follow [71, 72]. In the present study, guanosine significantly suppressed apoptosis in the injured spinal cords when it was systemically administered daily for 2\u00a0weeks beginning 4\u00a0h after injury. It seems reasonable to postulate that decreasing apoptosis may be an important mechanism through which guanosine improves neurological outcome after SCI.\nAlthough the intracellular pathways through which guanosine suppresses apoptotic cell death following spinal cord injury are not known, a number of intracellular pathways which protect cells against apoptosis have been identified. These include the phosphatidylinositol 3-kinase (PI-3-K)\/Akt\/protein kinase B (PKB) pathway [73, 74] and the mitogen-activated protein (MAP) kinase pathway [75]. Our previous studies in vitro have shown that the antiapoptotic effect of guanosine is mediated by the activation of both PI-3-K\/Akt\/PKB and MAPK pathways [34, 35].\nGuanosine can also increase intracellular cAMP in various cell types [36, 37], and increases in cAMP after spinal cord injury have been shown to increase recovery [26, 27]. However, it appears unlikely that increases in intracellular cAMP are responsible for the effects of guanosine since the principal effect of cAMP in enhancing recovery from spinal cord injury appears to be due to its ability to promote axonal regeneration. In contrast, the neuroprotective effects we observed early after administration of guanosine are unlikely to be explained on the basis of outgrowth of damaged axons. A much longer time frame could be required for this to occur.\nA further possible mechanism by which guanosine might exert its neuroprotective effects is by stimulating cells to release and increase synthesis of various trophic factors, such as basic fibroblast growth factor (bFGF), nerve growth factor (NGF), and neurotrophin-3 (NT-3) [28]. Such trophic factors can contribute to tissue preservation after trauma [67]. The ability of guanosine to stimulate production and release of trophic factors may not only have early effects, but also may contribute to the reduced number of apoptotic cells observed 3\u00a0weeks after the injury.\nThis study is important not only because it is the first to demonstrate the neuroprotective effect of systemically administered guanosine in acute spinal cord injury, but also because it indicates some of the potential mechanisms whereby guanosine may exert its neuroprotective effects in vivo. This work provides a basis for further exploration of the purinergic mechanisms underlying the neuroprotective effects of exogenous guanosine. Furthermore, and of potential clinical importance, is that guanosine was effective when it was administered 4\u00a0h after the injury\u2014a realistic time frame in which to initiate treatment after spinal cord injury in humans.","keyphrases":["guanosine","apoptosis","inflammation","spinal cord injury","cell death","myelin","immunohistochemistry","glia","locomotor and sensory function"],"prmu":["P","P","P","P","P","P","P","U","R"]}
{"id":"Behav_Genet-4-1-2226020","title":"Genetic and Environmental Influences on the Relation Between Attention Problems and Attention Deficit Hyperactivity Disorder\n","text":"Objective The assessment of symptoms of ADHD in children is usually based on a clinical interview or a behavior checklist. The aim of the present study is to investigate the extent to which these instruments measure an underlying construct and to estimate the genetic and environmental influences on individual differences in ADHD. Methods Maternal ratings were collected on 10,916 twins from 5,458 families. Child Behavior Checklist (CBCL) ratings were available for 10,018, 6,565, and 5,780 twins at the ages 7, 10, and 12, respectively. The Conners Rating Scale (4,887 twins) and the DSM interview (1,006 twins) were completed at age 12. The magnitude of genetic and environmental influences on the variance of the three measures of ADHD and the covariance among the three measures of ADHD was obtained. Results Phenotypic correlations range between .45 and .77. Variances and covariances of the measurements were explained mainly by genetic influences. The model that provided the best account of the data included an independent pathway for additive and dominant genetic effects. The genetic correlations among the measures collected at age 12 varied between .63 and 1.00. Conclusions The genetic overlap between questionnaire ratings and the DSM-IV diagnosis of ADHD is high. Clinical and research implications of these findings are presented.\nIntroduction\nAs is the case for all psychiatric disorders, the diagnosis of attention deficit hyperactivity disorder (ADHD) is not based on a specific pathological agent, such as a microbe, a toxin, or a genetic mutation, but rather on the collection of signs and symptoms and evidence of impairment that occur together more frequently than expected by chance (Todd et\u00a0al. 2005). The presence of these symptoms is usually established by direct observation, or by the completion of a clinical interview or questionnaire by the parent or teacher of a child. Instruments vary with respect to the included symptoms, the exact manner of data collection (checklist or interview), and the response format (e.g., yes\/no versus Likert scale). In the present paper, we investigated if (co)variance of the scores on different instruments can be explained by a common underlying construct and to what extent this common factor is influenced by genetic and environmental factors. The focus is on three widely used instruments: the Child Behavior Checklist (CBCL; Achenbach 1991), the Conners Parent Rating Scale-Revised:Short version (CPRS-R:S; Conners 2001), and the Diagnostic and Statistical Manual of Mental Disorders-4th edition (DSM-IV; American Psychiatric Association 1994).\nThe CBCL-Attention Problem scale (CBCL-AP) was developed by means of factor analyses, and includes eleven items. The psychometric properties and methods to establish the reliability of the syndrome are discussed in detail elsewhere (Achenbach 1991). Despite its name, the scale assesses problems related both to attention and hyperactivity. The CBCL has sex- and age-specific norms, which are useful in assessing a child\u2019s risk for ADHD. The CPRS-R:S ADHD-index comprises the 12 best items for distinguishing children with ADHD from children without ADHD as assessed by the DSM (Conners 2001). As with the CBCL, sex- and age-specific norm scores are available, allowing the clinician to determine whether a given child is at risk for ADHD. DSM-IV ADHD is assessed on the basis of 18 symptoms; nine relate to inattention, and nine relate to hyperactivity\/impulsivity. In the DSM framework, ADHD is viewed as a categorical trait; i.e., children either do or do not meet criteria for ADHD. The norms for clinical diagnosis do not vary as a function of sex or age of the child. Table\u00a01 contains the symptoms included in the CBCL-AP scale, the CPRS-R:S ADHD-index and DSM-IV ADHD.\nTable\u00a01An overview of the Child Behaviour Checklist (CBCL), Conners Parent Rating Scale-Revised:Short version (CPRS-R:S), and the Diagnostic and Statistical Manual of Mental Disorders-4th edition symptomsScaleSymptomCBCL Attention ProblemsActs too young for his\/her ageCan\u2019t concentrate, can\u2019t pay attention for longCan\u2019t sit still, restless, or hyperactiveConfused or seems to be in a fogDaydreams or gets lost in his\/her thoughtsImpulsive or acts without thinkingNervous, high-strung, or tenseNervous movements or twitchingPoor school workPoorly coordinated or clumsyStares blanklyCPRS-R:S ADHD-indexInattentive, easily distractedShort attention spanFidgets with hands or feet or squirms in seatMessy or disorganized at home or schoolOnly attends if it is something he\/she is very interested inDistractibility or attention span a problemAvoids, expresses reluctance about, or has difficulties engaging in tasks that require sustained mental effort (such as schoolwork or homework)Gets distracted when given instructions to do somethingHas trouble concentrating in classLeaves seat in classroom or in other situations in which remaining seated is expectedDoes not follow through on instructions and fails to finish schoolwork, chores, or duties in the workplace (not due to oppositional behavior or failure to understand directions)Easily frustrated in effortsDSM-IV ADHDInattentionOften fails to give close attention to details or makes careless mistakes in schoolwork, work, or other activitiesOften has difficulty sustaining attention in tasks or play activitiesOften does not seem to listen when spoken to directlyOften does not follow through on instructions and fails to finish schoolwork, chores, or duties in the workplace (not due to oppositional behavior or failure to understand instructions)Often has difficulty organizing tasks and activitiesOften avoids, dislikes, or is reluctant to engage in tasks that require sustained mental effort (such as schoolwork or homework)Often loses things necessary for tasks or activities (e.g., toys, school assignments, pencils, books, or tools)Is often easily distracted by extraneous stimuliIs often forgetful in daily activitiesHyperactivityOften fidgets with hands or feet or squirms in seatOften leaves seat in classroom or in other situations in which remaining seated is expectedOften runs about or climbs excessively in situations in which it is inappropriateOften has difficulty playing or engaging in leisure activities quietlyIs often \u201con the go\u201d or often acts as if \u201cdriven by a motor\u201dOften talks excessivelyOften blurts out answers before questions have been completedOften has difficult awaiting turnOften interrupts or intrudes on others (e.g., butts into conversations or games)\nAlthough the CBCL, DSM, and CPRS-R:S focus on different symptoms, and are based on distinct assumptions, the scores of these instruments are strongly related. CBCL-AP scores predict the presence of ADHD (Gould et\u00a0al. 1993; Chen et\u00a0al. 1994; Eiraldi et\u00a0al. 2000; Lengua et\u00a0al. 2001; Sprafkin et\u00a0al. 2002; Hudziak et\u00a0al. 2004). In a non-referred sample enriched for ADHD, about 50% of the children with a high CBCL-AP score were diagnosed with ADHD compared to 3% of the children with a low CBCL-AP score (Derks et\u00a0al. 2006). Although these results imply a good convergence between the CBCL and a DSM-IV interview, the relation is clearly less than perfect. The CPRS-R:S ADHD-I was developed for assessing children at risk for ADHD based on a DSM-IV diagnosis (Conners 2001). Conners (2001) showed that the CPRS-R:S ADHD-I is a good screening instrument for DSM-IV ADHD with a sensitivity of 100%, a specificity of 92.5%, and an overall correct classification rate of 96.3%. As far as we know, the relation between CBCL-AP and the CPRS-R:S ADHD-I has not been studied, but given that they are both related to DSM-IV ADHD, these are likely to be correlated.\nGenetic studies of psychiatric disorders are complicated by the lack of clear diagnostic tests (Hudziak 2001). Heritability estimates in epidemiological genetic studies, and the results of gene-finding studies may depend on the exact instrument that is used to assess ADHD. Although a number of papers have established the convergence between CBCL-AP and DSM-IV ADHD, the causal factors underpinning this relationship remain unclear. Is it the result of genetic overlap, environmental overlap, or both? This is an important question, which may determine the progress in gene finding studies. If variance in alternative measures of ADHD is explained by different genes, we would expect disagreement in the results of studies using different instruments. If the same genes explain variance in these measures, the data from studies using different instruments may be combined in order to increase statistical power (Boomsma 1996; Boomsma and Dolan 1998). Assuming that the convergence between different instruments will be less than perfect, part of the variance will be attributable by instrument-specific factors. It is important to investigate the nature of such factors. If the divergence among instruments is merely a matter of measurement error, we would expect no genetic influences on the instrument-specific factors. Genetic influences on the instrument-specific factors, on the other hand, would suggest that the instruments tap partly unique aspects of children\u2019s behavior.\nGenetic and environmental influences on individual differences in behavior can be studied in genetically informative designs, such as the classical twin design. Such studies have shown that genetic influences explain between 55 and 89% of the variance in clinical diagnoses of ADHD (Eaves et\u00a0al. 1997; Sherman et\u00a0al. 1997). Shared environmental influences were nearly always absent. Likewise, about 70\u201380% of the variance in CBCL-AP scores is explained by genetic influences. The remaining variance is explained by non-shared environmental influences (Rietveld et\u00a0al. 2003; Hudziak et\u00a0al. 2000; Gjone et\u00a0al. 1996). Kuntsi and Stevenson (2001) used the Conners Rating Scale to assess symptoms of ADHD and reported a heritability of 72%. A review of genetic studies on AP, HI and ADHD suggested the absence of qualitative and quantitative sex differences in the genetic etiology of parent ratings of ADHD (Derks et\u00a0al. in press).\nInterestingly, in parent ratings, but not in teacher ratings, the DZ twin concordances and correlations are lower than would be expected under a purely additive genetic model. For example, in maternal structured interview reports, the concordance rate is .67 in MZ twins, but .00 in DZ twins (Sherman et\u00a0al. 1997). Similarly, in CBCL ratings, the DZ twin correlations are less than half the MZ correlations (Rietveld et\u00a0al. 2003). In the literature, two explanations are offered for these low DZ correlations. Firstly, the DZ correlation can be less than half the MZ correlation due to the presence of non-additive genetic effects (i.e., genetic dominance) (Lynch and Walsh 1998). Secondly, the low DZ correlation may be explained by social interaction effects, which may be the result of interaction among siblings (i.e., the behavior of a twin influences the behavior of the other twin) or rater bias (i.e., the behavior of a twin is compared to the behavior of the other twin) (Eaves 1976; Carey 1986; Boomsma 2005). In previous studies, support was found both for the presence of genetic dominance (Rietveld et\u00a0al. 2003; Martin et\u00a0al. 2002) and sibling interaction (Simonoff et\u00a0al. 1998; Kuntsi and Stevenson 2001; Vierikko et\u00a0al. 2004; Eaves et\u00a0al. 1997).\nA high heritability of attention problems and ADHD has been reported, irrespective of the instrument that is used. However, based on the findings of univariate studies, we cannot conclude that CBCL, Conners Rating Scale, and DSM ratings measure the same construct, or that they are influenced by the same set of genes. To address this question, multivariate analyses are needed. Although a number of studies have focused on the genetic and environmental influences on either AP or ADHD, only the study of Nadder and Silberg (2001) included multivariate analyses. Nadder and Silberg (2001) analyzed data obtained in a sample of 735 male and 819 female same-sex twin pairs, aged 8\u201316\u00a0years. They modelled the genetic influences on nine measures of ADHD symptomatology, including maternal and paternal DSM-III-R interview data (three dimensions: hyperactivity, inattention and impulsivity), maternal questionnaire data (the Rutter Parental Scale, and the CBCL), and a questionnaire completed by the twin\u2019s teacher. The aim of this study was to determine whether overactivity, inattention, and impulsivity reflect the same underlying genetic liability, while taking method (i.e., instrument-specific) variance into account. In males, 23.7\u201370.1% of the genetic variance was explained by a common factor that loaded on all nine indicators. A second and third factor loaded on the three dimensions of the maternal and paternal interview data, respectively. The remaining variance (0.0\u201365.7%) was explained by factors that were specific to each measure. In females, there was also one factor common to all indicators (explaining 16.2\u201360.2% of the variance), and a second and third factor, which loaded on the three dimensions of the interview data. In contrast to the males, a fourth factor loaded on the three behavioral questionnaires. This factor explained 12.3\u201346.2% of the genetic variance. In total, measurement specific factors explained 0.0\u201373.0% of the genetic variance.\nThe purpose of the present paper is to investigate the construct validity of CBCL-AP, CPRS-R:S ADHD-I, and DSM-IV ADHD. Three questions are addressed. First, what are the phenotypic correlations between the three instruments? Second, do the instruments reflect a common underlying factor? Third, what are the genetic and environmental influences on the common and the instrument-specific factors?\nMethods\nSubjects\nThis study is part of an ongoing longitudinal twin study in the Netherlands. The subjects were all registered at birth with the Netherlands Twin Register (Boomsma et\u00a0al. 2002, 2006; Bartels et\u00a0al. 2007). Mothers of the registered twin pairs receive the CBCL and the CPRS at the ages 7, 10, and 12\u00a0years. A subsample of the twins was selected based on their longitudinal CBCL scores. The mothers of these pairs completed a diagnostic interview. The twins, with an age range of 10\u201313\u00a0years (mean age\u00a0=\u00a011.71; SD\u00a0=\u00a0.77) at the time of the interview, were born between 1989 and 1994. The mean time-span between the completion of the interview and the questionnaires was 4.42 (SD\u00a0=\u00a0.75), 1.82 (SD\u00a0=\u00a0.73), and \u2212.84 (SD\u00a0=\u00a0.63) years for the questionnaires completed at age 7, age 10, and age 12, respectively.\nQuestionnaires were sent to all families that agreed to participate with the research of the Netherlands Twin Registry when the children were born (N\u00a0=\u00a07,828 families; birth cohorts 1989\u20131994) at the ages 7, 10, and 12\u00a0years. At least one measurement is available for 10,916 twins from 5,458 families, so the response rate is 70%. CBCL ratings were available in 10,018 twins at age 7, 6,565 twins at age 10, and 5,780 twins at age 12. CPRS-R:S ratings were available for 4,887 twins at age 12, and DSM-IV interviews were available for 1,006 twins. Complete data were available in 740 twins. The fact that the number of CPRS-R:S ratings is lower than the number of CBCL ratings, can be explained by the fact that the CPRS-R:S was not included for children born before 1991. The number of available questionnaires decreases over time as a result of the longitudinal character of the study (i.e., a number of children in the study had yet to reach the age of 12).\nZygosity diagnosis was based on DNA in 674 same-sex twin pairs. In the remaining same-sex pairs, zygosity was assessed using a 10\u2013item questionnaire. Zygosity determination using this questionnaire is almost 95% accurate (Rietveld et\u00a0al. 2000). Of the 5,458 twin pairs, there were 898 monozygotic male (MZM) pairs, 888 dizygotic male (DZM) pairs, 1,005 monozygotic female (MZF) pairs, 844 dizygotic female (DZF) pairs, and 1,823 dizygotic opposite sex (DOS) pairs.\nSelection for the diagnostic interview\nFor the diagnostic interview, subjects were selected on the basis of their standardized maternal CBCL ratings (T-scores; mean\u00a0=\u00a050, SD\u00a0=\u00a010) at the ages 7, 10, and 12\u00a0years (Derks et\u00a0al. 2006). Subjects were excluded if maternal ratings were available at only one time-point, or if they suffered from a severe handicap, which disrupted daily functioning. Twin pairs were selected if at least one of the twins scored high on AP (affected pairs), or if both twins scored low on AP (control pairs). A high score was defined as a T-score above 60 at all available time-points (age 7, 10, and 12\u00a0years) and a T-score above 65 at least once. A low score was defined as a T-score below 55 at all available time-points. The control pairs were matched with the affected pairs on the basis of sex, cohort, maternal age, and social economic status (SES). T-scores were computed in boys and girls separately. In other words, girls were selected if they scored low or high compared to other girls, and boys were selected if they scored low or high compared to other boys. This procedure resulted in the selection of similar numbers of boys (N\u00a0=\u00a0499) and girls (N\u00a0=\u00a0507).\nMeasures\nThe Child Behavior Checklist (CBCL) (Achenbach 1991) is a standardized questionnaire designed for parents to report the frequency and intensity of their children\u2019s behavioral and emotional problems as exhibited in the past 6 months. It consists of 120 items that measure problem behavior. The items are rated on a 3-point scale ranging from \u201cnot true\u00a0=\u00a00\u201d, \u201csomewhat or sometimes true\u00a0=\u00a01\u201d, to \u201cvery true or often true\u00a0=\u00a02\u201d. The Attention Problem scale contains 11 items. The 2-week test\u2013retest correlation and the internal consistency of this scale are .83 and .67, respectively (Verhulst et\u00a0al. 1996). In the statistical analyses, we included the CBCL ratings at the ages 7, 10, and 12\u00a0years in order to correct for the selection, as explained below.\nThe Conners\u2019 Parent Rating Scale-Revised is a widely used instrument to assess behavior problems in the past month (CPRS-R; Conners 2001; Conners et\u00a0al. 1998). The short version contains 28 items. The items are rated on a 4-point scale ranging from \u201cnot true at all\u00a0=\u00a00\u201d to \u201cvery much true\u00a0=\u00a03\u201d. The CPRS-R:S ADHD-I, which was used in the present study, comprises the best 12 items for distinguishing children with ADHD from children without ADHD as assessed by the DSM-IV (American Psychiatric Association 1994; Conners 2001). The internal consistency of this scale at age 12\u201314\u00a0years is .94 in boys and .91 in girls. The 6\u20138\u00a0weeks test\u2013retest correlation is .72. The Diagnostic Interview Schedule for Children (DISC) (Shaffer et\u00a0al. 1993) is a structured diagnostic interview. It can be used to assess the presence of DSM-IV diagnoses, including ADHD. The Dutch translation is by Ferdinand and van der Ende (1998). The mothers of twins were interviewed by ten experienced research assistants to determine which symptoms of ADHD were displayed by the twins during the last year. Maternal ratings of DISC symptoms in their children were assessed by the same interviewer for each twin in a given pair. We analyzed the total number of symptoms.\nStatistical analyses\nTransformation to categorical data\nThe distributions of the CBCL, CPRS-R:S, and DSM symptom data are characterized by excessive skewness and kurtosis. Derks et al. (2004) showed that bias in parameter estimates due to non-normality of the data may be avoided by using categorical data analysis. In this approach, a liability threshold model is applied to the ordinal scores (Lynch and Walsh 1998). It is assumed that a person is \u201cunaffected\u201d, if his or her liability is below a certain threshold, and that he or she is \u201caffected\u201d, if his or her liability is above this threshold. In the present paper, the scores were recoded in such a way that three thresholds divide the latent liability distribution into four categories, of about equal size. The liability threshold model was identified by constraining the variance of the observed variables at 1.\nThe CBCL AP score was calculated by summing the responses on the 11 items which resulted in a sum score with a possible maximum of 22. The four categories consisted of a score of 0, 1\u20132, 3\u20135, and 6 or higher, respectively. The CPRS-R:S ADHD-I score was calculated by summing the responses on the 12 items, which resulted in a sum score with a possible maximum of 36. The four categories consisted of a score of 0\u20131, 2\u20135, 6\u201311, and 12, or higher, respectively. The DISC sumscore with a range of 0 to 18 was transformed into an ordinal variable with four categories. The four categories were: (i) not affected (0 symptoms); (ii) mildly affected (1\u20132 symptoms); (iii) moderately affected (3\u20135 symptoms); and (iv) highly affected (more than 6 symptoms). The use of this four category variable provides greater resolution, and so better statistical power than the use of a dichotomous variable (ADHD absent versus ADHD present).\nCorrecting for the selection\nDiagnostic interview data were collected only in a subsample of the twins. The probability of selection for the interview depends on a measured variable, namely the twin\u2019s CBCL scores at age 7, 10, and 12. The data of the complete sample may be partitioned as the observed (selected) and missing (unselected) parts. The data are missing at random (MAR) if the probability of missingness depends only on the observed part of the data, and not on the missing part (Little and Rubin 2002). Given that the data are MAR, unbiased parameter estimates can be obtained by full information (i.e., raw data) maximum likelihood estimation of the parameters in a statistical model that includes the variables that were used for selection. It is essential to include all variables that were used for selection, because the probability of missingness should not depend on the missing part of the data, in which case the data would be missing not at random (MNAR) and parameter estimates would be biased. We therefore included the CBCL ratings obtained at the ages 7, 10, and 12\u00a0years in the statistical analyses. All twin pairs in which at least one measure is available are included in the analyses.\nPrevalences\nTo investigate if the prevalences of AP and ADHD depend on the twin\u2019s sex or zygosity, we performed \u03c72-tests with the five ordinal measures as dependent variables and sex and zygosity as independent variables.\nGenetic modeling\nGenetic and environmental influences on variance in ADHD scores were estimated using structural equation modeling. All model fitting was performed on raw data with Mx (Neale et\u00a0al. 2003), a statistical software package well suited for conducting genetic analyses.\nThe influence of the relative contributions of genetic and environmental factors to individual differences in ADHD can be inferred from the differences in correlations across MZ and DZ twin pairs, as MZ and DZ twins differ in their genetic relatedness (Plomin et\u00a0al. 2001). Using the twin method, phenotypic variance may be attributed to additive genetic effects (A), dominant genetic effects (D) or shared environmental effects (C), and non-shared environmental (E) effects. The genetic effects (A and D) correlate 1 in MZ twins, as they are genetically identical. In DZ twins, A correlates .5, and D correlates .25. C correlates 1 in both MZ and DZ twins. E or non-shared environmental effects are, by definition, uncorrelated. Uncorrelated measurement error, if present, is absorbed in the E term. Note that estimating C and D at the same time is not possible in a design using only data from MZ and DZ twins reared together. If the correlations of DZ twins are less than half the correlations of MZ twins, which is the case for maternal ratings of attention problems and ADHD, D is included in the genetic model. The proportion of the variance accounted for by heritability or environmental influences is calculated by calculating the ratio of variance due to A, D, or E to the total phenotypic variance. For instance, let a, d, and e denote the regression coefficients in the regression of the phenotype on the standardized latent variables A, D, and E, respectively. The variance due to A is then a2, and the (narrow-sense) heritability is calculated as a2\/(a2\u00a0+\u00a0d2\u00a0+\u00a0e2).\nSocial interactions may be an additional source of variance. Social interaction effects lead to differences in variances in MZ and DZ twins in continuous data (Carey 1986). Using ordinal data, the presence of an interaction component can be tested by equating the prevalences of AP\/ADHD between MZ and DZ twins. The absence of significant prevalence differences suggests that the presence of sibling interaction or rater bias is considered implausible.\nThree multivariate models were tested: a triangular (Cholesky) decomposition, an independent pathway model, and a common pathway model (Neale and Cardon 1992). The triangular decomposition is the least restrictive model, as no specific hypotheses regarding the covariance matrices of A, D, and E are tested. These matrices are merely assumed to be positive (semi) definite. This is a saturated model that can be used to obtain (otherwise unconstrained) genetic and environmental correlations among traits. In the independent pathway model, common and specific genetic and environmental factors are included. In our data analyses of the five variables, we specified a common factor and five instrument-specific factors for each of the factors A, D, and E, which we denote Ac, Dc, and Ec. An independent pathway model provides a good fit to the data if the covariance between the five variables is due to the common factors Ac, Dc, and Ec. Finally, in the common pathway model, a model that is nested under the independent pathway model, it is assumed that genes and environment explain variance in a latent phenotype. This latent factor, of which the variance is constrained at 1, explains variance in the five variables. In addition, the variance of the five variables is allowed to be influenced by instrument-specific influences of A, D, and E. In other words, the common pathway model would provide a good fit to the data if the covariance between the five variables can be explained by a latent construct.\nBecause the number of twins for whom interview data are available is relatively small, and sex differences in heritability are usually not found, the data from male and female twins were combined in the analyses. To allow for prevalence differences between boys and girls, sex was included as a covariate on the thresholds. The type-I error rate of all statistical tests was set at .05.\nResults\nDescriptives\nThe prevalences for the five measures were compared between MZ and DZ twins and between boys and girls. The first model that was fitted to the data was a fully saturated model. In this model, 90 correlations were estimated, 45 in MZ twins and 45 in DZ twins. In addition, the model included 30 thresholds in each of the following groups: MZ boys, DZ boys, MZ girls, and DZ girls, which results in a total of 120 estimated thresholds. Next, a model was fitted that included a number of constraints on the thresholds. This model included 30 thresholds, 1 sex effect on the thresholds, and 5 zygosity effects on the thresholds (one for each of the five measurements). As this model fitted the data well, it was used as the reference model to test for prevalence differences as a function of zygosity for each of the five measurements. The results of these analyses are summarized in Table\u00a03. Zygosity did not affect the prevalences of the CBCL, CPRS-R:S, and DSM scores. In view of the absence of prevalence differences in MZ and DZ twins, social interaction effects were not included in the genetic model. The model that was used as the reference model to test for sex differences included as free parameters 30 thresholds, 1 zygosity effect on the thresholds, and five sex effects on the thresholds, one for each measurement. The results showed that boys have significantly more problems than girls on all five measurements; therefore, sex was included as a covariate on the thresholds. Because of the use of categorical scores in the present paper, we did not report means and standard deviations of the CBCL, CPRS-R:S and DSM scores. These descriptives can be requested from the corresponding author by interested readers.\nTwin correlations\nThe polychoric correlations between the five measurements are shown in Table\u00a02 for MZ and DZ twins. The MZ (DZ) twin correlations are reported above (below) the diagonal. As expected, the phenotypic correlations (i.e., the correlation between traits within the same individual) are similar in first- and second-born twins and in MZ and DZ twins. The correlations range from .45 to .77, with slightly lower correlations between different assessment methods (e.g., CBCL questionnaire versus clinical interview) than similar assessment methods (e.g., CBCL questionnaire versus CPRS-R:S questionnaire). Equating the correlations of first- and second-born twins at age 12, the phenotypic correlation between CBCL-AP and CPRS-R:S was .75, while the correlations between CBCL-AP and DSM, and CPRS-R:S and DSM were .62. The fact that the cross-twin and the cross-trait cross-twin correlations are higher in MZ than DZ twins indicates that genetic influences contribute to the variance of the three measures and to the covariance between them.\nTable\u00a02Polychoric correlations in monozygotic (above diagonal) and dizygotic (below diagonal) twinsFirst-bornSecond-bornCBCL 7 CBCL 10 CBCL 12 CPRS DSM CBCL 7 CBCL 10 CBCL 12 CPRSDSMFirst-bornCBCL age 71.66.62.51.59.76.54.49.45.45CBCL age 10.701.69.61.59.56.77.58.53.48CBCL age 12.63.741.71.57.48.54.75.58.53CPRS-R:S .56.68.771.60.46.55.62.84.51DSM.51.55.59.681.34.41.46.46.64Second-bornCBCL age 7.31.22.18.15.041.66.63.52.46CBCL age 10.22.35.22.21.01.661.71.64.59CBCL age 12.21.28.34.24.13.60.721.75.58CPRS-R:S .22.27.28.38.08.49.64.741.60DSM.11.16.11.07.13.45.63.67.581Note: CBCL\u00a0=\u00a0Child Behavior Checklist; CPRS\u00a0=\u00a0Conners Parent Rating Scale-Revised:Short version ADHD-index; DSM\u00a0=\u00a0Diagnostic Statistical Manual of Mental Disorders\nGenetic analyses\nA Cholesky decomposition that included additive genetic influences (A), dominant genetic influences (D), and non-shared environmental influences (E) was fitted to the data. The full ADE cholesky decomposition fitted the data well (\u03c72(50)\u00a0=\u00a059.03, P\u00a0=\u00a0.180); see Table\u00a03 for an overview of the model fitting results. Next, an independent pathway model was fitted to the data. Imposition of the independent pathway model for A, D, and E, resulted in a significant deterioration in fit compared to the fit of a cholesky decomposition (\u03c72(15)\u00a0=\u00a042.42, P\u00a0<\u00a0.001). Additional analyses showed that the influence of A and D were consistent with the independent pathway model, whereas the influence of E was not. A model that incorporated an independent pathway model for A and D, and a cholesky decomposition for E fitted well compared to the full cholesky decomposition (\u03c72(10)\u00a0=\u00a016.45, P\u00a0=\u00a0.087). The fit of the common factor model was poor (\u03c72(23)\u00a0=\u00a0259.12, P\u00a0<\u00a0.001). Next, we tested if the instrument-specific influences of A and D could be constrained at zero. The instrument-specific additive genetic factors could not be dropped from the model (\u03c72(5)\u00a0=\u00a091.80, P\u00a0<\u00a0.001). In contrast, the dominant genetic variance could be explained by one common factor (\u03c72(5)\u00a0=\u00a01.06, P\u00a0=\u00a0.96). In other words, the covariance structure of D did not include specific variances. This means that this covariance matrix has rank one, and that the correlations (obtained by standardizing the covariance matrix of D) were all one. Figure\u00a01 provides a graphical representation of the genetic part of the best fitting model and includes the unstandardized factor loadings of the additive genetic and dominant genetic factors.\nTable\u00a03Multivariate model fitting of maternal ratings on CBCL, CPRS-R:S and DSM-IV ratings on attention problems and ADHD in 7-year-old children\u22122\u00a0log\u00a0LLN parWith modeld.f.\u03c72P1. Fully saturated63020.52210\u2013\u2013\u2013\u20132. Thresholds MZ\/DZ free, thresholds boys\/girls equated 63123.54126184103.02.082a. Thresholds CBCL age 7 equated in MZ\/DZ63124.66125211.11.292b. Thresholds CBCL age 10 equated in MZ\/DZ63123.9812521.43.512c. Thresholds CBCL age 12 equated in MZ\/DZ63123.6812521.14.712d. Thresholds Conners age 12 equated in MZ\/DZ63123.6012521.06.812e. Thresholds DSM age 12 equated in MZ\/DZ63126.34125312.80.093. Thresholds boys\/girls free, thresholds MZ\/DZ equated 63108.1812618487.66.373a. Thresholds CBCL age 7 equated63423.1912531315.01<.0013b. Thresholds CBCL age 10 equated63395.1812531287.00<.0013c. Thresholds CBCL age 12 equated63321.3212531213.14<.0013d. Thresholds Conners age 12 equated63388.2412531280.06<.0013e. Thresholds DSM age 12 equated63137.031253128.85<.0014. Cholesky decomposition ADE63147.4110215059.03.184a. Independent pathway model D; Cholesky decomposition AE63149.4097451.99.854b. Independent pathway model A; Cholesky decomposition DE63151.8897454.47.484c. Independent pathway model E; Cholesky decomposition AD63170.06974522.65<.0014d. Independent pathway AD; Cholesky decomposition E63163.869241016.45.094e. Independent pathway model ADE63189.838741542.42<.0014f. Independent pathway AD; Cholesky decomposition E, instrument-specific A factors dropped63255.66874d591.80<.0014g. Independent pathway AD; Cholesky decomposition E, instrument-specific D factors dropped63164.92874d51.06.965. Common pathway model63406.5379423259.12<.001Fig.\u00a01A graphical representation of the unstandardized additive genetic (A) and dominant genetic (D) effects on five measurements of Attention Problems and ADHD. In this figure, a graphical representation of the best-fitting model and the estimated factor loadings is provided for one individual twin. Additive genetic effects correlate 1 in MZ twins and .5 in DZ twins. Dominant genetic effects correlate 1 in MZ twins and .25 in DZ twins. To identify the model, the variances of the five categorical measurements are constrained at 1. CBCL7\u00a0=\u00a0Child Behavior Checklist at age 7; CBCL10\u00a0=\u00a0Child Behavior Checklist at age 10; CBCL12\u00a0=\u00a0Child Behavior Checklist at age 12; CPRS-R:S\u00a0=\u00a0Conners Parental Rating Scale-Revised:Short version at age 12; DSM\u00a0=\u00a0DISC-IV ADHD at a mean age of 12\u00a0years\nAlthough the influence of the nonshared environment was not included in Fig.\u00a01, the fact that the total variances of the five measurements are constrained at 1 in order to identify the model allows a calculation of the additive and dominant genetic variance based on the unstandardized factor loadings. For example, 41% (i.e., .442\u00a0+\u00a0.462) of the variance in the CBCL rating at age 7 is attributable to additive genetic effects, 36% (.602) is attributable to dominant genetic effects. The remaining variance is explained by nonshared environmental effects. The additive genetic variance on the five measurements can be decomposed into variance due to the common factor and variance due to instrument-specific factors. For the CBCL rating at age 7, 19% (.442) of the total variance is attributable to common additive genetic effects, and 22% (.462) is attributable to instrument-specific genetic effects. The influence of common additive genetic effects on the total variance accounts for 36%, 55%, 56%, and 32% for the CBCL at age 10, the CBCL at age 12, the CPRS-R:S, and the DSM, respectively. Likewise, the influences of instrument-specific effects account for 17, 13, 23, and 24% of the variance, respectively.\nTable\u00a04 shows an overview of the standardized influences of A, D, and E on the variance and covariance of the five measurements. The three diagonals of the five by five tables of A, D, and E contained the standardized variance components. The results indicate a high heritability, irrespective of measurement instrument or age. On the off-diagonals in Table\u00a04, one can find the standardized influences of A, D, and E on the covariance between the measurements. For example, the covariance between CBCL7 and DSM is for 51% explained by A, 25% by D, and 24% by E. To obtain the unstandardized amount of variances explained, the standardized influences should be multiplied with the phenotypic covariance between the measures, which is .51 for CBCL7 and DSM. The most interesting comparison is between the data that were collected at approximately the same time. The covariance between the CBCL at age 12 and the DSM is explained largely by genetic effects (68% A, 9% D, and 23% E). Similar results were found for the covariance between CPRS-R:S and the DSM (67% A and 7% D) and for the covariance between the CBCL age 12 and CPRS-R:S (74% A and 8% D).\nTable\u00a04Standardized genetic and environmental influences on the variances and covariances of five ratings of ADHD and attention problemsADECBCL 7CBCL 10CBCL 12CPRS-R:SDSMCBCL 7CBCL 10CBCL 12CPRS-R:SDSMCBCL 7CBCL 10CBCL 12CPRS-R:SDSMCBCL age 7.41.36.23CBCL age 10.39.53 .45.25.16.22CBCL age 12.53.62.68 .26.19.07.21.19.25CPRS-R:S.62.69.74.79 .26.17.08.05.13.14.18.16DSM.51.57.68.67.56.25.17.09.07.04.24.26.23.25.40Note: CBCL\u00a0=\u00a0Child Behavior Checklist; CPRS-R:S\u00a0=\u00a0Conners Parent Rating Scale-Revised:Short version ADHD-index; DSM\u00a0=\u00a0Diagnostic Statistical Manual of Mental Disorders; A\u00a0=\u00a0additive genetic effects; D\u00a0=\u00a0dominant genetic effects; E\u00a0=\u00a0non-shared environmental effects\nTable\u00a05 includes the genetic and environmental correlation matrices in the best-fitting model, which represent the overlap between the genetic and environmental influences on the five measurement instruments. The additive genetic correlations range between .52 and .76. All dominant genetic correlations are 1, which is a result of the absence of specifics in the one-factor model used to model the dominant genetic covariance structure. The non-shared environmental correlations range from .34 to .68.\nTable\u00a05Genetic and environmental correlations of five ratings of ADHD and attention problemsADECBCL 7CBCL 10CBCL 12CPRS-R:SDSMCBCL 7CBCL 10CBCL 12CPRS-R:SDSMCBCL 7CBCL 10CBCL 12CPRS-R:SDSMCBCL age 71.01.01.0CBCL age 10.571.01.01.0.501.0CBCL age 12.62.741.01.01.01.0.56.601.0CPRS-R:S.58.69.761.01.01.01.01.0.34.49.681.0DSM.52.62.68.631.01.01.01.01.01.0.39.52.45.621.0Note: CBCL\u00a0=\u00a0Child Behavior Checklist; CPRS-R:S\u00a0=\u00a0Conners Parent Rating Scale-Revised:Short version ADHD-index; DSM\u00a0=\u00a0Diagnostic Statistical Manual of Mental Disorders; A\u00a0=\u00a0additive genetic effects; D\u00a0=\u00a0dominant genetic effects; E\u00a0=\u00a0non-shared environmental effects\nDiscussion\nThe aim of this study was to determine the extent to which three different instruments, which are commonly used to assess ADHD, attention problems, and hyperactivity, measure a common construct. The instruments considered are two scales based on items from questionnaires (CBCL-AP, and CPRS-R:S ADHD-I), and a DSM-IV ADHD interview. First, we considered the phenotypic correlations. Second, we tested if the variance in the different instruments reflects one common underlying factor. Third, we estimated the genetic and environmental influences on individual differences in ADHD. This is the first study that includes multivariate genetic analyses of behavior rating scales and DSM-IV interview data collected in a large sample of twins of approximately the same age. The CBCL scores collected at age 7 and 10\u00a0years were included only to correct for the selection. In the discussion, we focus mainly on the CBCL, CPRS-R:S and DSM interview data, which were collected at a mean age of 12\u00a0years.\nThe phenotypic correlation between CBCL-AP and the CPRS-R:S ADHD-I was high (r\u00a0=\u00a0.75). The correlations between the CBCL and the DSM and between the CPRS-R:S and the DSM were slightly lower (r\u00a0=\u00a0.62). These lower correlations can both be the result of the different time-points at which the behavior checklists and the DSM interview data were collected (the mean time-span between measurement occasions was 10\u00a0months), the differences in the time frame for the assessment of the items (e.g., 1\u00a0month for the CPRS-R:S, 6\u00a0months for the CBCL, and 1\u00a0year for the DSM), and of instrument or method variance (e.g., interview versus behavior checklists). The genetic analyses show that the covariance between CBCL and CPRS is for 82% explained by genetic factors while the covariance between CBCL and DSM was for 75% explained by genetic factors. Therefore, the higher phenotypic correlation between CBCL and CPRS is not caused by a relatively higher genetic covariance.\nAs noted, the AP scale of the CBCL questions relate to both inattention and hyperactivity\/impulsivity. The fact that the correlation between the CPRS-R:S ADHD-I and DSM-IV ADHD is identical to the correlation between CBCL-AP and DSM-IV ADHD implies that the CPRS-R:S and the CBCL measure ADHD equally well. The description of the eleven item CBCL scale as an inattention scale seems to be too limited, because both the item content and the current results suggest that the CBCL also signals problems related to hyperactivity\/impulsivity.\nAlthough the phenotypic correlations provide an interesting insight regarding the similarities and dissimilarities of the quantitative and qualitative approaches towards child psychopathology, an important question concerns the etiological influences on the variances and covariances. In agreement with previous studies (Eaves et\u00a0al. 1997; Sherman et\u00a0al. 1997; Rietveld et\u00a0al. 2003; Hudziak et\u00a0al. 2000), individual differences in AP and ADHD are mainly explained by genetic factors. An independent pathway model provided a better fit than a common factor model. A common factor model implies a similar structure for the additive genetic, dominant genetic and nonshared environmental influences so the poor fit is probably due to the fact that there are instrument-specific additive genetic factors while these are absent for the dominant genetic factors. As referees of earlier drafts of this paper noted, alternative models might be fit to our data; a model including three common factors (one loading on all ratings, a second loading on CBCL ratings, and a third loading on age 12 ratings, might offer a good solution, but because the structure of A, D, and E differ (with no rating-specific influences for D), we did not fit this model to our data.\nAn independent pathway model allows for the inclusion of common and instrument-specific genetic and environmental factors. The model that provided the best fit to the data included common additive and dominant genetic effects, instrument-specific additive genetic effects, and nonshared environmental effects. The relative influence of common and instrument-specific genetic effects varies by rating. Two third of the additive genetic variance of the CBCL ratings at age 10, age 12, and the CPRS-R:S rating at age 12, was explained by common effects. More specifically, Instrument-specific effects played a more important role in the CBCL ratings at age 7, and in the DSM ratings. For these ratings, the ratio of common and instrument-specific effects was about 50:50. Apparently, the overlapping genes explain less of the variance in these ratings compared to the other ratings, probably as a result of developmental changes in behavior and of method variance (i.e., questionnaire versus interview). The dominant genetic effects overlapped completely between ratings, as the instrument-specific effects could be dropped from the model. Our results show some agreement with the findings of Nadder and Silberg (2001), who fit an independent pathway model to ADHD symptomatology based on maternal and paternal questionnaire and interview data, and to teacher reports. Although their best-fitting model included contrast effects instead of genetic dominance, our finding of both common and specific genetic influences on the questionnaire and interview data on ADHD is supported by their results.\nThe poor fit of the common factor model suggests that the construct validity of the instruments is not perfect. However, it is interesting to consider the implications of the overlap between the sets of genes that explained variance in the three instruments. High genetic correlations imply that the detection of the specific genes that play a role for ADHD, does not depend much on the instrument that is used. At age 12, the additive genetic correlations of the CBCL, CPRS-R:S, and DSM varied between .63 and .76, while the dominant genetic correlations could be constrained at 1. The non-shared environmental correlations are also quite high, and vary between .45 and .68. The dominant genetic correlations of 1 suggest that there is a subset of genes whose effect is not instrument or age dependent. In contrast, the correlations of the additive genetic effects are high but less than perfect. This suggests that the influence of most genes with an additive effect are not sensitive to the particular instrument that is used, although there are some genes that explain variance only in a particular measurement (e.g., CBCL), but not in another (e.g., DSM).\nWhat are the implications of the present findings for gene finding studies? Thus far, five groups have conducted genome-wide linkage scans in an attempt to find genomic regions which are involved in ADHD, and a number of regions that may be of interest have been identified. Linkage peaks with a LOD score above 2 (P\u00a0<\u00a0\u223c.002) were reported at chromosomes 16p13 and 17p11 (Ogdie et\u00a0al. 2003), chromosomes 7p and 15q (Bakker et\u00a0al. 2003), chromosomes 4q, 8q, and 11q (Arcos-Burgos et\u00a0al. 2004), chromosomes 5p and chromosome 12q (Hebebrand et\u00a0al. 2006), and chromosomes 14q32 and 20q11 (Gayan et\u00a0al. 2005). All these studies based diagnosis on DSM-IV (Ogdie et\u00a0al. 2003; Bakker et\u00a0al. 2003; Arcos-Burgos et\u00a0al. 2004; Hebebrand et\u00a0al. 2006) or DSM-III (Gayan et\u00a0al. 2005) criteria. The discrepancy in the results of these five studies could be due to low statistical power. The present study showed that the genetic overlap between behavior checklist scores and the DSM-IV diagnosis of ADHD is high. This implies that the detection of genes, which play a role for ADHD, can be based on questionnaire scores, rather than diagnostic interviews. This will reduce the costs of collecting phenotypic data. Resources may then be reallocated to the collection of genotypic data. An increased number of subjects can be genotyped and the statistical power to detect a QTL will be increased.\nLimitations\nThe results of this study should be interpreted bearing in mind the following limitations. First, further study is required to investigate if the results of the current study, which was based on a Dutch population sample, generalize to population samples outside the Netherlands. Second, clinical diagnoses were based on structured diagnostic interviews with the mother. The results may be different when the assessment of ADHD is based on expert clinical diagnoses. Third, no distinction was made between problems related to inattention and problems related to hyperactivity. Since the CBCL does not distinguish between inattention and hyperactivity (and probably the number of items is too small to reliably measure these two factors) we did not distinguish between the subscales. Fourth, we did not allow for sex differences in the genetic and environmental influences based on the results of univariate studies. Because of the increased statistical power in the multivariate model, it is possible that sex differences do exist. However, due to the categorical nature of the data, and the fact that some of the cells in the contingency tables contain few individuals in a two-group analysis, statistical problems will arise in a four-group analysis. Fifth, as a result of the categorical nature of the data, computational limitations prohibited inclusion of confidence intervals.\nClinical implications\nTwo general approaches towards the measurement of ADHD can be distinguished. In the DSM-IV framework, ADHD is viewed as a categorical trait. Using behavior checklists, children can show variation in a continuum from not affected at all to severely affected. The current study shows that variance in DSM-IV symptoms, the CBCL-AP scale, and the CPRS-R:S ADHD-I is explained mostly by genetic effects. The correlations between the genetic influences on variance in these three measurements of ADHD are high. This implies that different measurements tap the same genetic liability.","keyphrases":["genetics","attention problems","adhd","measurement","twins","multivariate analysis"],"prmu":["P","P","P","P","P","R"]}
{"id":"Breast_Cancer_Res_Treat-3-1-2096637","title":"MRI compared to conventional diagnostic work-up in the detection and evaluation of invasive lobular carcinoma of the breast: a review of existing literature\n","text":"Purpose The clinical diagnosis and management of invasive lobular carcinoma (ILC) of the breast presents difficulties. Magnetic resonance imaging (MRI) has been proposed as the imaging modality of choice for the evaluation of ILC. Small studies addressing different aspects of MRI in ILC have been presented but no large series to date. To address the usefulness of MRI in the work-up of ILC, we performed a review of the currently published literature.\nIntroduction\nInvasive lobular carcinoma (ILC) is the second most common histologic type of breast carcinoma after invasive ductal carcinoma (IDC). In most series ILC constitutes between 5 and 15% of all breast cancers, whereas IDC constitutes between 70 and 90% of all breast cancers [1\u20135]. Probably due to the use of complete hormone replacement therapy the lobular breast cancer component has continuously increased over the past decade from 9.5% in 1987 to 15.6% in 1999 [3].\nPatients are, according to most series, a little older than patients presenting with IDC, especially the fraction of patients presenting with ILC younger than 40 is smaller [1, 5, 6]. Furthermore, the mean tumor size of ILC is slightly larger than in patients with IDC and patient presentation with a tumor larger than 5\u00a0cm occurs more often in cases of ILC [1, 5, 7].\nHistopathologically, ILC are clearly defined: ILCs are constituted from small, relatively uniform cells, very similar to normal endothelial cells. Characteristically, these cells are only loosely cohesive and infiltrate the stroma in single cell file strands along ductuli. This growth pattern, present in 30\u201377% of cases [8], is also known as \u201cIndian filing.\u201d It is probably caused by a typical loss of the adhesion molecule E-cadherin. Often there is very little desmoplastic stromal reaction [8, 9]. The biological characteristics of ILC are usually less alarming than those of IDC: more tumors contain estrogen receptors and progesterone receptors, while expression of Her2\/Neu and p53 are more often normal and axillary lymph nodes are not more often positive, even though ILC are overall larger in size than IDC [1, 7].\nProbably due to the diffuse infiltrative growth pattern, ILC is frequently missed on mammography [5]. Detection is also compromised because ILC often has a density less than or equal to normal fibroglandular breast tissue on mammography [5, 10].\nFor correct treatment of ILC, adequate staging is important. Both mammography and ultrasound tend to underestimate lesion size and are therefore not optimal for staging purposes [5, 11]. This may in part be the reason that higher failure rates of breast-conserving therapy (BCT) in ILC than in IDC are reported [2, 11, 12]. Various authors therefore propose magnetic resonance imaging (MRI) as the modality of choice for the evaluation of ILC. Several small studies addressing the different aspects of the use of MRI in ILC have been presented, but no large series to date. Therefore many questions regarding the use of MRI in ILC remain unanswered.The sensitivity of MRI for breast lesions is approximately 95\u201398%, however, whether this holds true for ILC as well is not clear [13].The morphologic aspects of ILC are not yet well defined, nor is the dynamic behavior of contrast agents in these tumors clearly documented.Moreover, whether the MRI findings are similar to pathologic findings and can thus be used for accurate staging still needs to be established.Finally, the impact of MRI on surgical treatment of ILC should be evaluated.To answer these questions we performed a thorough review of the existing literature regarding the use of MRI in case of ILC and performed meta-analysis whenever possible. We subsequently reviewed the literature on other imaging modalities for this indication in order to evaluate the use of MRI from a clinical perspective.\nMaterials and methods\nSearch strategy\nWe performed a literature search for articles that specifically dealt with the use of MRI in patients with histologic proof of ILC published before 1 April 2006. The Cochrane Library, MEDLINE and the in-progress citations as provided by PubMed were searched using the query: \u201clobular AND (MR OR MRI OR MRT OR magnetic).\u201d These databases were further searched using the \u201cRelated Articles\u201d function in PubMed. The same query was used to browse the web using scholar.google.com.\nFurthermore, the references of all retrieved articles were manually searched for relevant cross-references. Articles in all languages were accepted. All retrieved articles were then compared and from overlapping series of patients only the most recent publication was accepted.\nMany different search terms were used for literature review of other imaging modalities. However, only PubMed was used as search engine.\nEndpoints\nThe study was thus undertaken to answer the following four questions.What is the sensitivity of MRI for ILC?What are the visual characteristics of ILC on MRI?Are the findings on MRI equal to the findings at pathology?What is the impact of MRI on surgical management of ILC?Whenever studies allowed direct comparison between MRI and other imaging modalities, these modalities were also analyzed. Sensitivity was defined as the number of lesions visible on MRI divided by the total number of ILC detected at pathology. We regarded morphology, dynamic curve analysis of contrast behavior, and quantitative dynamic analysis of contrast behavior as three different aspects of tumor appearance and these were thus analyzed separately. A principal distinction between mass-like and non-mass-like lesions was made in the analysis of morphology. Based on the BI-RADS lexicon [14], we defined architectural distortion, regional, segmental, ductal, multifocal, or diffuse enhancement, and multiple enhancing foci as descriptors of non-mass lesions. Nodular or focal enhancement, well-defined, round, irregular or spiculated masses, and dominant masses with small enhancing foci were defined as descriptors of mass-like lesions. Correlation between the findings on MRI and pathology was evaluated for relative tumor size (unifocal versus multifocal disease and single quadrant versus multicentric disease) and absolute tumor size. The impact on surgical management was derived from all changes implemented, based solely on MRI findings. The numbers of correct and incorrect changes were tabulated.\nEligibility criteria\nAll studies that presented a series of at least ten patients with histologic proof of pure ILC, with or without concurrent DCIS and\/or LCIS, were considered eligible. A quality analysis of the study had to be possible, otherwise no abstracts were accepted. Patients with mixed carcinomas of ILC and IDC were excluded. Studies that presented data on both ILC and mixed carcinomas had to allow extraction of the relevant data for ILC only. Every study considered eligible according to these eligibility criteria was then evaluated for all the study endpoints. Specific eligibility criteria for the various considered endpoints are described below.Detection: Studies had to be based on a pathology database and all subsequent patients with ILC who underwent a MRI had to be included. The total number of ILC confirmed at pathology had to be clearly stated as well as the number of lesions found with MRI.Morphology: Studies describing the appearance of ILC visible on MRI were eligible. Separation between mass and non-mass-like lesions had to be possible.Dynamic curve analysis of contrast behavior: Studies that described the enhancement versus time curve were eligible. However, as time to peak and shape of the final phase of the enhancement curve were our main endpoints, these had to be described.Quantitative analysis of contrast behavior: Studies performing quantitative analysis of the contrast-enhancement parameters were eligible.Relative correlation with pathology: Studies presenting data on the unifocal versus multifocal correlation or single quadrant involvement versus multicentric involvement were eligible.Absolute correlation with pathology: Studies comparing sizes measured on MRI with those measured at pathology and presenting a correlation coefficient or sufficient raw data to calculate such a value were eligible.Detection of additional lesions: Any study describing additional lesions apart from the index lesion detected by MRI only with subsequent acquisition of histologic proof of malignancy was considered eligible. Lesions in the ipsilateral breast and the contralateral breast were evaluated separately.Impact on surgical treatment: Studies mentioning all changes in surgical strategy based on MRI findings were eligible.\nStatistics\nThe quality of all included studies was assessed using the QUADAS tool [15]. The latter is a list of 14 items created for quality assessment of studies to diagnostic accuracy. Although not all the included studies specifically evaluate diagnostic accuracy, this tool was judged to be the most appropriate available. Data of all the studies were collected according to the inclusion and exclusion criteria. When at least five studies presented the same type of data or at least 100 patients were included in a smaller series of studies with similar data, we considered meta-analysis and heterogeneity analysis was performed. Dichotomous data with a binomial distribution (e.g., sensitivity) were transformed to the log odds scale because this scale has a normal distribution and is a good approximation to the exact binomial distribution. A disadvantage of this transformation, however, is that the confidence intervals are a little wider and values in the middle of the distribution (e.g., sensitivity closer to 50%) are more heavily weighted in meta-analysis than values close to the upper or lower level. Pearson\u2019s correlation coefficient was transformed to Fisher\u2019s Z for the same reason [16].\nWe calculated Cochran\u2019s Q coefficient and the I2-statistic to assess heterogeneity. Cochran\u2019s Q is a form of the \u03c72-test and provides information about the applicability of pooling the data. The I2-statistic provides a quantitative measure of the amount of heterogeneity and has an upper limit of 100%. Values of the I2-statistic of 25, 50, and 75% can be interpreted as low, moderate, and high heterogeneity, respectively [17]. Meta-analysis of the data using a random effects model was performed when the Q-coefficient showed no significant heterogeneity (p\u00a0>\u00a00.05).\nIn cases where meta-analysis was feasible, the estimate and the 95% CL are expressed. When meta-analysis was not feasible due to severe heterogeneity, only the range of values found in the different studies is mentioned. All calculations were performed using R version 2.3.1 (The R Project for Statistical Computing, www.r-project.org) and the meta package (G. Schwarzer, cran.r-project.org).\nResults\nStudies\nWe identified 21 separate studies that dealt with MRI and ILC [18\u201338]. We further identified four studies that did not deal specifically with ILC and MRI. However, they did present their data in such a fashion that relevant information for ILC only could be extracted for at least ten patients [39\u201342]. Four studies were case-reports and were dropped from the cohort [20, 21, 29, 37]. The study by Bazzocchi et al. [18] was excluded because only eight patients underwent MRI. Leung et al. [27] and Newstead et al. [42] only published their findings in abstract form and were consequently excluded. Table\u00a01 gives an overview of the included studies and their characteristics, including the QUADAS score.\nTable\u00a01Characteristics of the included studiesAuthorsPub. YearaStudy typebNcAge meandAge min.eAge max.fFieldgScan seq.hUni\/bilatiCompressionjMean sizekQUADAS scorelRodenko et al. [32]19961206038842110X11Sittek et al. [34]1998123XXX1220X11Weinstein et al. [36]200111753326922111,712Kim et al. [41]200111254m24m88m22102,1m12Trecate et al. [35]2001128X32812220X9Francis et al. [24]2001222XXX22203,712Qayyum et al. [30]20021135546842110X11Munot et al. [28]20021206139782320X11Yeh et al. [38]200311959427922204,111Kneeshaw et al. [26]20031215743722210X11Quan et al. [31]200316253XX2231X10Bedrosian et al. [39]200312453mXX2001X10Schelfout et al. [33]20041265741743220X11Diekmann et al. [22]2004117XXX0000X10Boetes et al. [19]200413455357822204,910Berg et al. [40]2004229XXX3220X13Kepple et al. [25]20051296251672130X9Fabre Demard et al. [23]2005134XXX2220X11aYear of publication of the original articleb1 indicates retrospective cohort study, 2 indicates prospective cohort studycNumber of patients includeddMean age of all included patients, X denotes not mentionedeAge of respective youngest patient included in the studyfAge of respective eldest patient included in the studygStrength of magnetic field\u20140 denotes unknown, 1 denotes 1\u00a0T, 2 denotes 1.5\u00a0T, 3 denotes both 1\u00a0T and 1.5\u00a0ThType of scan sequence used\u20140 denotes unknown, 1 denotes RODEO, 2 denotes FLASH 3D, 3 denotes otheriUnilateral or bilateral imaging of the breast\u20140 denotes unknown, 1 denotes unilateral, 2 denotes bilateral, 3 denotes both unilateral and bilateral depending on the patientjCompression applied to the breast\u20140 denotes no, 1 denotes yeskMean size of the lesions in centimeters, X denotes not mentionedlNumber of items valid on QUADAS scorings listmValid for whole study population only, not for subpopulation of patients with ILC\nThe applied scan protocols in the included studies are diverse. In general, most studies presented herein used a 1.5-T MRI scanner, although some authors had at least some of their included patients scanned using 1.0\u00a0T machines [33, 34, 40]. Most protocols were based on T1 weighted images made with either a normal FLASH 3D sequence or a FLASH 3D sequence with fat-suppression [19, 20, 23, 24, 26, 31, 33\u201336, 38, 40, 41] or a RODEO sequence with water selective excitation [25, 30, 32]. A number of authors also used T2 weighted sequences [22, 23, 31, 38, 40, 41]. Other differences in scan protocols involve the voxel sizes and temporal resolution. Some authors emphasize high spatial resolution [32, 39] while others prefer high temporal resolution [26] and yet again others performed both types of sequences in succession [30, 38]. Furthermore, single breast coils [26, 30, 32, 36, 41, 43] and double breast coils (all others) were used and sometimes compression was applied to the imaged breast [31, 36, 39]. In most reported studies the scanning protocols evolved over time and are thus not identical for all imaged patients.\nLesion detection\nEight studies provided sufficient data to calculate sensitivity of MRI for ILC [19, 23, 24, 26, 28, 33, 34, 40]. Sensitivity ranged from 83 to 100%. Cochran\u2019s Q was 6.48 (p\u00a0=\u00a00.49), I2 was 0%, indicating homogeneous studies and hence data pooling could be performed. Mean sensitivity was 93.3% (95% CI 88\u201396%). Only the studies by Francis et al. [24] and Berg et al. [40] provided prospective data and are therefore able to show sensitivity in clinical practice. They showed a sensitivity of 95 and 97%, respectively, and were statistically not different from the retrospective studies (two-sided T-test, p\u00a0=\u00a00.78). Seven of these studies also provided data on mammography [Q 31.79 (p\u00a0<\u00a00.001), I2\u00a0=\u00a081%], six on ultrasound [Q 10.92 (p\u00a0=\u00a00.05), I2\u00a0=\u00a054%], and five on clinical examination [Q 29.63 (p\u00a0<\u00a00.001), I2\u00a0=\u00a087%]. Sensitivity of ultrasound could also be computed through meta-analysis and was 83% (95% CI 71\u201391%), although moderate heterogeneity was present. The provided data for mammography and clinical examination were too heterogeneous for meta-analysis and ranged from 34 to 91 and 28 to 94%, respectively. Figure\u00a01 shows the results of each independent study and the overall results.\nFig.\u00a01Forestplot of the sensitivity of the respective modalities for ILC (MMG mammography, US ultrasound, CE clinical examination), the horizontal lines represent 95% confidence intervals. Modalities presented on the right of the authors name have not been tested in the appropriate study. The diamonds at the bottom represent the pooled estimates and their 95% confidence intervals for MRI and US, respectively. Because mammography and clinical examination were too heterogeneous for meta-analysis no pooled estimate is presented for these modalities\nMorphology\nSeven studies described lesion morphology on static MRI images [23, 30, 32, 33, 36, 38, 41]. However, Kim et al. [41] studied morphologic appearances of masses only and therefore did not include non-mass-like lesions. Information provided by their study is therefore only used to evaluate the appearance of masses and not for the principal distinction between mass and non-mass lesions. The terminology used in the literature to describe the lesions is highly variable. Only Yeh et al. [38] consistently used the terminology of the BI-RADS lexicon [14]. The six eligible studies that presented data on morphologic appearance described a total of 133 tumors. However, results are highly variable. The incidence of a mass-like lesion ranged from 31 to 95% [Q 16.44 (p\u00a0<\u00a00.01), I2\u00a0=\u00a070%]. Table\u00a02 shows the appearance of ILC on MRI for all individual studies.\nTable\u00a02Morphologic appearance of ILC on MRIAuthorsNumber of tumorsNon-mass-likeMass-likeRodenko et al. [32]201 (5)19 (95)Weinstein et al. [36]188 (44)10 (56)Qayyum et al. [30]139 (69)4 (31)Yeh et al. [38]2011 (55)9 (45)Schelfout et al. [33]276 (22)21 (78)Fabre Demard et al. [23]3511 (31)24 (69)Numbers between parenthesis represent percentages\nFabre Demard et al. [23] did not specify the lesions beyond the description \u201cmass-like.\u201d Other authors used many different terms to further describe lesions. In the study presented by Rodenko et al. [32], five pre-defined shapes were used, but they described all 19 mass-like lesions as spicular enhancing masses. In the other studies most lesions are described as spiculated masses as well. Schelfout et al. [33] recognized a dominant mass with multiple enhancing foci in eight cases and Yeh et al. [38] described even a round focal mass. In the 12 mass-like cases described by Kim et al. [41], 10 had an irregular shape and 8 were spiculated. Therefore, among the 76 masses, a total of 65 tumors were described as an irregular or spiculated mass. This appears to be the most common type of mass-like presentation in ILC.\nKinetics\nOnly two studies reported on the dynamic curve appearance of ILC [34, 35]. The most apparent similarity between findings was that maximum enhancement is often delayed and wash-out is present in only a minority of lesions. Sittek et al. [34] reported that maximum enhancement was not reached before 2\u00a0min after contrast administration. Trecate et al. [35] noted that a classic pattern of rapid signal increase was only present in 4 of 12 pure ILC, whereas a delayed pattern was observed in the other 8 cases.\nTwo other studies reported on quantitative contrast behavior analysis in ILC [30, 38]. Qayyum et al. [30] reported on a parameter called K21, analogue to the Ktrans parameter as described by Tofts et al. [44]. Yeh et al. [38] evaluated the extraction flow product (EFP), which is a similar analogue but respects the possibility that contrast leakage from the vessels is limited by flow instead of being limited by the permeability surface area product. Both studies did not, however, include sufficient patients to produce meaningful results, other than a high variability in the values of these parameters and the presence in some tumors of enhancement very much like enhancement in normal breast tissue. It was noted that K21 values appeared to be an order of magnitude less in ILC than in IDC lesions.\nCorrelation\nSeveral authors evaluated correlation of the MRI findings with pathology [19, 24\u201326, 28, 32, 33, 40]. Three studies compared unifocality and multifocality between MRI and pathology [26, 32, 33] (Table\u00a03). Overall 5 of 67 cases (7%) were regarded as multifocal, whereas they appeared unifocal at pathology and, vice versa, 2 cases (3%) in one study appeared unifocal at MRI, but were multifocal according to pathology.\nTable\u00a03Relative correlation of unifocality versus multifocality for MRI versus pathologyAuthorsNumber of patientsUF MRIUF PATHMF MRIMF PATHOverestimatedaUnderestimatedbRodenko et al. [32]2091111921Kneeshaw et al. [26]21910121110Schelfout et al. [33]261417121021Total6752UF unifocal, MF multifocal, PATH pathologyaDisease was classified as multifocal on MRI, but was unifocal on pathologybDisease was classified as unifocal on MRI, but was multifocal on pathology\nOverestimation of multifocality based on mammography in 63 patients from these studies occurred in 2 patients (3%), whereas underestimation occurred 25 times (40%) and the lesion was not visible on mammography in another 4 patients (6%).\nTwo of these studies further analyzed single quadrant versus multicentric involvement of the affected breast [32, 33] (Table\u00a04). In the study by Rodenko et al. [32], two cases of single quadrant disease were erroneously classified as multicentric on MRI.\nTable\u00a04Relative correlation of single quadrant versus multicentric involvement for MRI versus pathologyAuthorsNumber of patientsSQ MRISQ PATHMC MRIMC PATHOverestimatedaUnderestimatedbRodenko et al. [32]2091111920Schelfout et al. [33]2621215500Total4620SQ single quadrant, MC multicentric, PATH pathologyaMulticentric involvement was seen on MRI, but involvement of only one quadrant was shown on pathologybInvolvement of only one quadrant was seen on MRI, but on pathology multicentric involvement was shown\nMammography in 42 of these patients resulted in overestimation of disease extent in 1 patient and underestimation in 15. Again, no lesion was visible in four patients.\nBerg et al. [40] further showed a series of 12 patients that underwent MRI. Correct size estimation was performed in seven patients. In one patient an additional focus was missed and in four patients overestimation occurred due to foci of LCIS.\nAbsolute correlation of MRI and pathologic size measurement was performed by six authors [19, 24\u201326, 28, 32]. Rodenko et al. [32] found a Kappa coefficient of 0.77, which represents substantial agreement. The other authors presented Pearson\u2019s correlation coefficients ranging from 0.81 to 0.97 [Q 10.90 (p\u00a0=\u00a00.03), I2\u00a0=\u00a063%]. Correlation coefficients for other modalities were substantially more variable. Presented correlation coefficients in Table\u00a05 are optimized by excluding cases where no abnormalities were seen from the calculations.\nTable\u00a05Correlation of tumor size measured by various modalities compared to pathologyAuthorsMRIMMGUSCENPCC\u039aNPCCKNPCCKNPCCKRodenko et al. [32]200.77315\u22120.081Munot et al. [28]200.97100.66140.67Kneeshaw et al. [26]210.86210.93a210.93a210.47Francis et al. [24]220.87160.79200.56190.89Boetes et al. [19]360.81360.34360.24Kepple et al. [25]330.8890.71MMG mammography, US ultrasound, CE clinical examination, N number of lesions visible on the appropriate modality, PCC Pearson\u2019s correlation coefficient, K Kappa valueaKneeshaw et al. did not provide a correlation coefficient for either MMG or US, but only one for the combined modalities\nBoetes et al. [19] applied a correctness measure of 1.0\u00a0cm to their data and found that MRI underestimated disease extent in 5 of 36 tumors and overestimated extent in 4 cases by more than 1.0\u00a0cm. The data provided by Francis et al. [24] allow a similar calculation. Underestimation occurred in 6 of 22 cases and overestimation occurred in 1.\nAdditional lesions\nFive studies focused on the detection of concurrent additional lesions in the affected breast apart from the index lesion only visible by MRI [22, 23, 31, 33, 36]. In 44 of 146 patients, additional malignant lesions were found [Q 7.20 (p\u00a0=\u00a00.13), I2\u00a0=\u00a044%]. Additional malignant findings only visible on MRI were present in 32% of cases (95% CI 22\u201344%). The results of the individual studies are presented in Table\u00a06.\nTable\u00a06Additional malignant findings in the ipsilateral breast by MRIAuthorsNumber of patientsNumber of additional findingsWeinstein et al. [36]187Quan et al. [31]5111Schelfout et al. [33]269Diekmann et al. [22]179Fabre Demard et al. [23]348Total14644Meta-analysis (%)10032\nEight studies, presented in Table\u00a07, reported on findings in the contralateral breast [19, 22\u201325, 28, 31, 40]. In 12 of 206 patients, unexpected contralateral cancer was discovered exclusively by MRI [Q 2.28 (p\u00a0=\u00a00.94), I2\u00a0=\u00a00%]. Cases where contralateral cancer was also visible on mammography and\/or ultrasound are excluded. Contralateral carcinoma only visible by MRI was present in 7% of patients (95% CI 4\u201312%).\nTable\u00a07Additional findings in the contralateral breast by MRIAuthorsNumber of patientsNumber of contralateral findingsFrancis et al. [24]220Munot et al. [28]202Quan et al. [31]535Diekmann et al. [22]171Boetes et al. [19]342Berg et al. [40]150Kepple et al. [25]140Fabre Demard et al. [23]342Total20612Meta-analysis (%)1007\nEffect on surgical treatment\nSix studies explicitly stated the effect of MRI on the surgical treatment of their patients [23, 26, 28, 31, 32, 39]. In 160 patients with ILC, a total of 44 changes in surgical management occurred [Q 7.90 (p\u00a0=\u00a00.16), I2\u00a0=\u00a037%]. Overall, MRI changed the surgical management in 28.3% of cases (95% CI 20\u201339%). In 24 cases BCT was changed to mastectomy. In nine cases a wider local excision was performed. In the remaining 11 cases the type of change was not further described. Forty-one of 44 changes in surgical management were retrospectively judged necessary based on pathologic findings [Q 1.24 (p\u00a0=\u00a00.94), I2\u00a0=\u00a00%]. Therefore, 88% of all changes were correct (95% CI 75\u201395%). In three cases the change in management was retrospectively judged unnecessary based on pathology. The data of the individual studies are presented in Table\u00a08.\nTable\u00a08Changes in surgical management based solely on MRI findingsAuthorsNumber of patientsNumber of changesCorrect changesIncorrect changesCorrect wider excisionIncorrect wider excisionCorrect mastectomyIncorrect mastectomyRodenko et al. [32]2087171Munot et al. [28]20333Kneeshaw et al. [26]215514Quan et al. [31]51111156Bedrosian et al. [39]241192NANANANAFabre Demard et al. [23]246633Total160444139231Number of changes (%)10028.3Correct changes (%)10088Number of changes and correct changes show the result of meta-analysesNA not available\nRodenko et al. [32] and Kneeshaw et al. [26] both reported one further unnecessary mastectomy based on MRI outcomes. However, these mastectomies would also have been performed based on the mammography findings and are therefore not only due to the MRI. Berg et al. [40] also reported that findings on MRI in 12 patients with ILC would have resulted in two unnecessary mastectomies. However, mastectomies were also indicated according to the ultrasound report. Nonetheless they based their treatment on the mammograms only and therefore these mastectomies were not performed.\nDiscussion\nStudies and quality analysis\nWe included 18 studies, but the highest number of studies that could be used to answer a specific endpoint was 8 (sensitivity and contralateral findings). Strong evidence is therefore lacking and this review is thus a clear call for more substantial research in this area. The overall study quality of all studies is, according to the QUADAS score, reasonably high (lowest score\u00a0=\u00a09\/14). However, this tool does not include the study size in the analysis, which was generally low. The tool places a strong emphasis on the relation of the test to the reference standard (typical for observational studies). In all studies, the reference standard was pathology and therefore always acceptable as gold standard. However, the test results (in this case the MRI reports), were never shielded from the pathologist who performed the pathologic evaluation. In studies that were performed to evaluate the visual characteristics of ILC on MRI a thorough description of the pathological examination was, deservedly so, not included [23, 30, 32, 33, 36, 38, 41]. These studies thus scored a little lower. There are some other drawbacks that must be considered and that are not included in the QUADAS score. Firstly, all but 2 of the included 18 studies were retrospective in nature, and secondly, the applied MRI protocols were largely heterogeneous (see Table\u00a01). However, the presented data were extracted from studies that made use of the various standards in MRI of the breast of the last decade and therefore give a reasonable overview of the overall capability of MRI in ILC imaging in this period.\nSensitivity\nThe sensitivity of physical examination and conventional imaging for ILC of the breast is not optimal. The sensitivity of physical examination for ILC ranges between 65 and 98% [10, 45\u201347], with usually over 50% of patients presenting with palpable abnormalities.\nThe sensitivity of mammography for ILC (BI-RADS 3 or higher) ranges between 81 and 92% in literature [10, 45\u201351]. In a recent study that evaluated intra- and interobserver variability, sensitivity even ranged from 88 to 98% [52], which could be regarded as sufficient. However, ILC often do not appear as a malignant lesion on mammography; approximately 30% is classified as equivocal and sensitivity is then approximately 57\u201359% [51].\nThe overall sensitivity of mammography in the current analysis appears lower than findings in the literature on mammography in ILC. However, equivocal findings may have been classified as undetected lesions in some studies resulting in the overall lower results. Nevertheless, the sensitivities of only 34% found by Berg et al. [40], and 50% found by Munot et al. [28] are on the lower end of the spectrum. Munot et al. [28] did not state which views constituted their mammograms, while Berg et al. [40] made craniocaudal, mediolateral and spot-compression views on a standard mammography machine, which we regard as common practice. A possible explanation for the poor results in the study by Berg et al. [40] may be that they defined an ILC as a focus of tumor, thereby allowing more tumors to be present in one breast, whereas other authors defined this as multifocal or multicentric tumors and thus as detected when at least one lesion was visible on mammography.\nIn literature, the reported sensitivity of ultrasound for ILC ranges between 68 and 98% [47, 53\u201358]. As this range is comparable to the range found in the present evaluation, we are of the opinion that an overall sensitivity of 83% is accurate. However, application of newer high-frequency ultrasound transducers may improve sensitivity. Initial series using 7.5\u00a0MHz transducers show sensitivities of 68% [47] and 78% [56], whereas series that used 10\u201313\u00a0MHz transducers report sensitivities up to 98% [57, 59].\nContrast-enhanced MRI is nowadays widely accepted as the most sensitive modality for detection of malignancy of the breast. Early reports on overall sensitivity of MRI for breast lesions range from 93 to 100% [13, 60\u201363]. Thus, the sensitivity of MRI found for ILC in the studies presented herein and the overall sensitivity of 93.3% calculated from these studies are not different from those known for malignancy in the breast in general. The relatively low heterogeneity of all studies describing lesion detection as well as detection of additional lesions in the ipsi- and contralateral breast show that the applied MRI technique only has a minor impact on the ability of MRI to detect lesions.\nThe overall sensitivity could even be increased to 96% (95% CI 92\u201398%) if an early study is excluded from the analysis [34]. This study reported a sensitivity for ILC of only 83%, a discrepancy that may well be explained by the fact that the slice thickness in this study was 4.2\u00a0mm, thicker than in any of the other presented studies, which could have had a negative impact on sensitivity. Moreover, 15 of 23 patients in their series were scanned with a FLASH 3D sequence with TR 8.4\/TE 3.0, resulting in image acquisition with a phase-shift of water and fat, which might have further decreased their sensitivity, although this was not apparent from their data.\nIt must be taken into account that the acquired sensitivity in all studies was achieved in cases where prior knowledge of the existence of ILC was present. Mostly because of the retrospective nature of the presented studies, but also because the two prospective studies both included their patients on the basis of histological proof of invasive (lobular) carcinoma by core biopsy. It is therefore not possible to formulate conclusions on the sensitivity of MRI for ILC prior to biopsy. In a large multicenter trial by Bluemke et al. [64] overall sensitivity for invasive cancer prior to biopsy was 91%, thus it might be expected that sensitivity for ILC prior to biopsy is also slightly lower. However, in most cases the indication for MRI is assessment of disease extent because of inconclusive findings at mammography or ultrasound. In conclusion, the sensitivity of MRI for ILC is higher than that achieved by any other modality, in direct comparison and validated by literature, and is equal to the overall sensitivity of MRI for malignant lesions of the breast. Only modern ultrasound examinations seem to have the ability to approach the performance of MRI in the detection of ILC [57].\nMorphology\nThe morphologic appearance of ILC on MRI ranged from 69% non-mass-like lesions to 95% mass-like lesions, thereby raising questions concerning the amount of heterogeneity in the description of morphology of lesions by radiologists. In fact, the general agreement on the description of lesion type according to the BI-RADS lexicon is only moderate [14, 65]. In the current analysis, this is even further complicated because most authors did not specifically use the BI-RADS lexicon. Additionally, differences in scan techniques may have further affected the appearance of the lesion. However, in keeping with the above, the classification of lesion type is also highly variable on mammography, where the incidence of mass lesions ranges from 32 to 78% [10, 45, 46, 48, 50, 51, 55].\nThe vast majority of the mass-like lesions described on MRI are irregular or spicular lesions. The eight patients with a dominant mass surrounded by multiple enhancing foci, as described by Schelfout et al. [33], may present noncontiguous foci of disease without visible spiculae due to the absence of desmoplastic reaction, which is a well-known histopathological presentation [8]. In all series only one round mass was described [38], suggesting this to be a very rare presentation for ILC. This is consistent with findings in mammography by Le Gal et al. [10], who described a round mass in only 2% of all patients where a mass was present (4\/174) while the remainder was either classified as a spicular mass (54%) or poorly defined mass (44%).\nMammographic findings would therefore appear to correlate well with MRI findings. However, only one study allows direct comparison [33]: of all lesions visible in this study on both mammography and MRI, 78% (18\/23) were classified as mass-like by MRI, while only 48% (11\/23) were classified as mass-like by mammography. Six masses on MRI were visible as architectural distortion on mammography and two as asymmetric density. In one case a lesion described as spicular mass on mammography was visible on MRI as multiple enhancing foci with interconnecting enhancing strands.\nNon-mass-like ILC in mammography are typically described as architectural distortion or asymmetric density. In some cases microcalcifications are present, although these are often related to concurrent surrounding DCIS, sclerosing adenosis or fibrotic changes and might thus not be related to the presence of ILC [45, 51, 55]. The descriptors currently used for non-mass-like lesions on MRI are diverse and include various types of abnormal enhancement, such as regional, ductal, segmental, and diffuse enhancement. According to Qayyum et al. [30] the morphologic description of ILC on MRI has a good correlation to histopathologic findings. The non-mass-like presentation might specifically occur in cases where ILC grow in the classic pattern with cells arranged in a linear fashion along the ductuli.\nIt may thus be concluded that the appearance of most ILC on MRI and mammography is similar: most ILC are mass-lesions that have clear malignant properties. However, the more diffuse growing tumors are characterized by areas of unexpected enhancement and are more difficult to recognize. In a number of cases where no clear mass is visible on mammography, a mass-like lesion may be found on MRI [33].\nKinetics\nThe relatively late contrast enhancement of ILC apparent in all studies presented here and mirrored by the relatively low values of K21 and EFP in the studies by Qayyum et al. [30] and Yeh et al. [38] must be taken into account when evaluating ILC. Standard subtraction images, generated from the pre-contrast and the first or second post-contrast acquisitions may be inconclusive as maximum enhancement is not achieved at this point in time and the lesion is thus not yet clearly visible. In fact, false-negative MRI in cases of ILC is usually contributed to inadequate enhancement of the tumor [26, 35, 66]. The diffuse and often slow tumor growth, not requiring extensive neovascularization, may partly cause this difficult visualization [1, 67, 68]. This is also clear from the relatively lower amount of vascular endothelial growth factor found in tumors with a lobular histology, which might also indicate a different signaling pathway in the formation of neovascular vessels in ILC, resulting in more mature and thus less leaky capillaries [69], with consequently diminished or absent contrast enhancement.\nCorrelation\nIn the herein presented studies overestimation of lesion extent by mammography is rare, yet underestimation is more rule than exception. This is also confirmed by studies that specifically deal with mammography in cases of ILC. Yeatman et al. [5] showed that mammography underestimated ILC by a mean of 12\u00a0mm. Uchiyama et al. [51] reported 56% of all visible ILC on mammography to be underestimated and Veltman et al. [52] showed 35\u201337% of all ILC to be mammographically understaged.\nUltrasound also tends to underestimate tumor size in the studies presented here. This finding is underlined by Tresserra et al. [70] and more recently by Watermann et al. [71], who documented a structural underestimation of 5.4\u00a0\u00b1\u00a012.2\u00a0mm in cases of ILC versus 1.4\u00a0\u00b1\u00a012.0\u00a0mm for cases of IDC. This might be partly due to the observation that US tends to underestimate larger tumors more than smaller tumors and low grade tumors more than high grade [70], consistent with the finding that ILC usually presents with slightly larger and less aggressive tumors [1, 5, 67, 72]. The current analysis shows that there is good correlation of tumor size measured on MRI compared to pathology. The various studies presented only moderately heterogeneous results.\nIn most cases MRI outperforms mammography and ultrasound in the assessment of disease extent. Most tumors are correctly classified as uni- or multifocal and multicentric disease is only seldom overestimated [19, 32].\nAdditional lesions and effect on surgical treatment\nEspecially important in this analysis is the detection of additional lesions apart from the index lesion in patients with ILC. The co-existence of other invasive malignant lesions apart from the index lesion in the ipsilateral breast in 32% of patients only visualized by MRI is high. Moreover, the detection of contralateral cancer in another 7% of patients by MRI only, seems to make MRI indispensable. These findings are confirmed by the rate of change in treatment of the ipsilateral breast based on MRI. The fact that change in treatment was considered correct, as verified by pathologic findings in the specimen, in 88% of cases shows that ILC is often more extensive than appreciated on conventional imaging.\nHowever, various authors have shown that there is no significant difference in disease free survival (DFS) or overall survival (OS) after BCT or mastectomy in patients with breast cancer. Although some authors report more local recurrence in patients with ILC after BCT [2, 73], most authors showed that there is no difference in DFS or OS after BCT in ILC versus IDC [74, 75]. On the other hand, Yeatman et al. [5] reported a higher rate of conversion from lumpectomy to mastectomy in ILC compared to IDC (17.5% versus 6.9%). More recently, Molland et al. [68] reported similar findings (37.2% versus 22.4%). Hussien et al. [2] even reported failure of BCT in patients with ILC in 63% (34\/54) of patients, resulting in conversion to mastectomy in 76% of failures (26\/34). However, a very recent study by Morrow et al. [76] showed that BCT did not fail more often in patients with ILC when corrected for age and tumor size, although they still observed a trend of more excisions in patients with ILC [OR 1.58 (0.89\u20132.79), p\u00a0=\u00a00.12].\nTo date, there is no evidence suggesting increase in survival for patients with ILC due to the performance of MRI. What is then the added value of MRI? The rate of recurrence 10 years after BCT followed by radiotherapy is between 7 and 18% and is not significantly different from the rate of recurrence in case of IDC [77, 78]. However, in view of the MRI findings (additional malignant lesions in 32% of patients), we can only conclude that in a large number of patients with ILC, surgery is not curative but merely debulking. As recurrence rates are fortunately much lower, we must assume that curative treatment is to be expected from adjuvant therapy. Unfortunately, because there is no possibility to determine which additional findings will respond to adjuvant therapy, the detection of additional lesions on MRI currently still requires a change of treatment when malignancy has been proven by core biopsy. This may further reduce the rate of recurrence in patients with ILC and may even improve survival. However, this requires confirmation in future studies.\nConclusion\nMagnetic resonance imaging has a high sensitivity for ILC, not achieved by other imaging modalities. Therefore MRI is helpful in cases where conventional imaging is inconclusive. Morphology is often mass-like and a typical ILC presents as an irregular or spiculated mass. However, asymmetric enhancement that can be ductal, segmental, regional, or diffuse in nature may be the only sign of tumor. MRI measures disease extent with a high reliability. Although underestimation and overestimation of lesion size by MRI still occurs, it is more accurate than size determination by other modalities, indicating often more extensive tumor burden than expected.\nThe underestimation by other imaging modalities results in more failure of BCT, more re-excisions and more conversion to mastectomy in series where MRI is not used. MRI has an effect on surgical management in that when used to assess disease extent, surgical management was changed in 28.3% of which 88% were judged necessary based on pathology. Larger series of patients are required to confirm the findings of this review; especially evaluation of tumor morphology and dynamic profile seems feasible.","keyphrases":["invasive lobular carcinoma of the breast","magnetic resonance imaging","sensitivity","morphology","additional findings","impact on treatment"],"prmu":["P","P","P","P","P","R"]}
{"id":"Arch_Dermatol_Res-3-1-1950585","title":"Preservation of skin DNA for oligonucleotide array CGH studies: a feasibility study\n","text":"Array-based comparative genomic hybridization (a-CGH) is a promising tool for clinical genomic studies. However, pre-analytical sample preparation methods have not been fully evaluated for this purpose. Parallel sections of normal male human skin biopsy samples were collected and immediately immersed in saline, formalin and a molecular fixative for 8, 12 and 24 h. Genomic DNA was isolated from the samples and subjected to amplification and labeling. Labeled samples were then co-hybridized with normal reference female DNA to Agilent oligonucleotide-based a-CGH 44k slides. Pre-analytic parameters such as DNA yield, quality of genomic DNA and labeling efficacy were evaluated. Also microarray analytical variables, including the feature signal intensity, data distribution dynamic range, signal to noise ratio and background intensity levels were assessed for data quality. DNA yield and quality of genomic DNA\u2014as evaluated by spectrophotometry and gel electrophoresis\u2014were similar for fresh and molecular fixative-exposed samples. In addition, labeling efficacy of dye incorporation was not drastically different. There was no difference between fresh and molecular fixative material comparing scan parameters and stem plot analysis of a-CGH result. Formalin-fixed samples, on the other hand, showed various errors such as oversaturation, non-uniformity in replicates, and decreased signal to noise ratio. Overall, the a-CGH result of formalin samples was not interpretable. DNA extracted from formalin-fixed tissue samples is not suitable for oligonucleotide-based a-CGH studies. On the other hand, the molecular fixative preserves tissue DNA similar to its fresh state with no discernable analytical differences.\nIntroduction\nApplications of new technologies have resulted in major advancements in laboratory medicine [1]. Bringing these advances into clinical practice, however, requires careful evaluation and validation. Array-based comparative genomic hybridization (a-CGH) is an extremely powerful tool that can generate high resolution mapping of chromosomal abnormalities. Advances in microarray technology and bioinformatics have now made a-CGH easily available and affordable [14, 15]. With a-CGH\u2019s potential for clinical application, it is important that guidelines for proper sample preparation and control of quality are developed. Since conventional methods of clinical tissue preparation commonly employ formalin fixation, we studied the suitability of formalin for array CGH studies and compared the results to that of clinical samples that were preserved in a newly developed, molecular-friendly fixative.\nThe study was approved by the University of Miami Institutional Review Board. Three separate normal skin biopsies from one volunteer healthy male were immediately sliced in three parts each 0.1\u00a0\u00d7\u00a00.2\u00a0\u00d7\u00a00.2\u00a0cm. One part was immersed in normal saline solution, one part in a methanol-based molecular tissue fixative, UMFIX (Universal Molecular Fixative, marketed as Tissue-Tek\u00ae Xpress\u2122 Molecular Fixative, Sakura Finetek, Torrance, CA), and the third slice was fixed in 10% neutral buffered formalin. Volume of fixative\/preservative was 150\u00a0ml and incubation was performed at room temperature. After 8 (set 1), 12 (set 2) and 24\u00a0h (set 3), the genomic DNA was extracted from the samples using Puregene DNA Purification System tissue kit (Gentra, Minneapolis, MN). One microliter of extracted DNA solution was diluted in Tris\u2013EDTA (TE) buffer and evaluated on ND-1000 spectrophotometer (NanoDrop Technologies, Rockland, DE). Gel electrophoresis (0.8% agarose gel) was performed to evaluate the quality of genomic DNA.\nDNA yield and quality of genomic DNA as evaluated by spectrophotometry, were similar between the samples (Table\u00a01), although UMFIX samples appeared to have better quality. A260\/A280 ratio determines presence of contaminating proteins and a ratio of more than 1.8 is generally considered to be indicative of high quality sample. All UMFIX samples consistently had a ratio of more than 1.8. We further evaluated the quality of DNA by agarose gel electrophoresis that showed presence of high molecular weight (HMW) genomic DNA band in all samples (Fig.\u00a01a). While formalin-fixed sample showed higher degradation and lower intensity of genomic DNA, UMFIX-exposed samples did not appear degraded.\nTable\u00a01Spectrophotometric result of total DNA yield, DNA purity, and labeling efficiency, for fresh, formalin-fixed and UMFIX-exposed samplesSampleTotal DNA (\u03bcg)A260\/A280A260\/A230Sample-cy5 labeling efficiency (pmol\/\u03bcg)Control-cy3 labeling efficiency (pmol\/\u03bcg)Fresh 13.891.771.4370.8111Fresh 22.941.861.7942.889.6Fresh 32.931.9252.897Mean\u00a0\u00b1\u00a0SD3.25\u00a0\u00b1\u00a00.551.84\u00a0\u00b1\u00a00.071.74\u00a0\u00b1\u00a00.2955.47\u00a0\u00b1\u00a014.1999.20\u00a0\u00b1\u00a010.87UMFIX 16.4161.85278.6110UMFIX 22.71381.861.8449.688UMFIX 34.0121.861.848.281.8Mean\u00a0\u00b1\u00a0SD4.38\u00a0\u00b1\u00a01.881.86\u00a0\u00b1\u00a00.011.88\u00a0\u00b1\u00a00.1158.80\u00a0\u00b1\u00a017.1693.27\u00a0\u00b1\u00a014.82Formalin 15.3661.580.8459.8142.4Formalin 24.04241.671.1141.273.4Formalin 34.4271.932.0836.277.4Mean\u00a0\u00b1\u00a0SD4.61\u00a0\u00b1\u00a00.681.73\u00a0\u00b1\u00a00.181.34\u00a0\u00b1\u00a00.6545.73\u00a0\u00b1\u00a012.4497.73\u00a0\u00b1\u00a038.731 8\u00a0h, 2 12\u00a0h, 3 24\u00a0hFig.\u00a01Result of gel electrophoresis for genomic DNA a HMW genomic DNA is visible in all samples. There is more DNA degradation in formalin-fixed samples. b Formalin-fixed samples do not show uniform strong smear after linear amplification. c PCR for GAPDH shows a 450\u00a0bp amplicon in all samples with a lesser intensity in formalin-fixed samples, L ladder (numbers indicate bp), S fresh control in saline, U UMFIX, F formalin, P positive control, N negative control\nGenomic DNA from the skin samples and the control female DNA (Promega, Madison, WI) were then subjected to amplification according to Agilent\u2019s (Palo Alto, CA) protocol for oligonucleotide array-based CGH for genomic DNA (version 2.0 August 2005). Amplification, of both male genomic DNA and female control DNA, 100\u00a0ng each, was performed using Qiagen REPLI-g Amplification Kit. Amplified DNA was digested during a 2\u00a0h incubation at 37\u00b0C, with Alu I and Rsa I (10\u00a0U\/\u03bcl; 5\u03bcl\/reaction) restriction enzymes (Promega, Madison, WI). Purification of digested DNA was performed with QIAprep Spin Miniprep Kit (Qiagen, Valencia, CA). Digested DNA was subjected to electrophoresis to evaluate quality of amplified DNA by visual inspection of its uniformity and range.\nThe amplification efficiency of DNA from formalin-fixed samples was less than fresh and UMFIX samples, as evidenced by the size and fluorescent intensity of the bands (Fig.\u00a01b).\nWe also used PCR for GAPDH primers to gauge the quality of extracted DNA. PCR was performed using glyceraldehyde-3-phosphate dehydrogenase primers (GAPDH, Clonetech, Palo Alto, CA) using 0.5\u00a0\u03bcg of RNase-treated isolated DNA and Qiagen TaqPCR Mastermix (Qiagen, Valencia, CA). The conditions for DNA PCR were as follows: 95\u00b0C, 15\u00a0min; 35 cycles at 94\u00b0C, 45\u00a0s; 60\u00b0C, 45\u00a0s; 72\u00b0C, 2\u00a0min. As seen in Fig.\u00a01c, a 450\u00a0bp band was detected in all samples, although the intensity was considerably lower in formalin-fixed material.\nAmplified Genomic DNA was then labeled using BioPrime Array CGH Genomic Labeling kit (Invitrogen, Carlsbad, CA). Quality analysis and quantitation was performed with ND-1000 (NanoDrop Technologies, Wilmington, DE) spectrophotometer to calculate the labeling efficiency. Labeled and purified samples were combined with hybridization master mix and applied to Agilent Human Genome CGH Microarray 44B slides for 40\u00a0h at 65\u00b0C. To minimize the impact of environmental oxidants on signal intensities, slides were scanned immediately using Agilent microarray scanner 4800B. Array images were analyzed using Agilent feature extraction software (v8.1) and CGH explorer (v2.51) [9]. Microarray analytical variables, including the feature signal intensity, data distribution dynamic range, signal to noise ratio and background intensity levels, and the number of saturated and undetected features was used to assess microarray quality.\nOne of the steps in microarray quality control is labeling efficacy or dye incorporation. Dye incorporation was not drastically different between fresh and UMFIX-exposed samples but formalin-fixed samples showed less dye incorporation (Table\u00a01). There were no differences between fresh and UMFIX-exposed material with regard to scan parameters (Table\u00a02). Formalin-fixed samples, showed various errors such as over-saturation, non-uniformity in replicates and increased signal to noise ratio. The numbers of non-uniform features were at least ten fold higher in formalin-fixed samples. The signal to noise ratio of replicated probes can be used to evaluate reproducibility of signals. Formalin-fixed samples showed a higher median %CV value, indicating lower reproducibility of signal across the microarray and lower signal to noise ratio. The number of features that were saturated in the scanned image was also significantly higher in formalin-fixed samples.\nTable\u00a02Representative data of microarray scan quality measurementSampleNon-uniform featureReproducibility: non-control replicated probes median %CVSaturated featureRedGreenRedGreenRedGreenFresh 11133797801Fresh 2511394400Fresh 3381355600Mean\u00a0\u00b1\u00a0SD67\u00a0\u00b1\u00a040218\u00a0\u00b1\u00a01405\u00a0\u00b1\u00a016\u00a0\u00b1\u00a0200UMFIX 1352560131300UMFIX 2851797800UMFIX 3361366700Mean\u00a0\u00b1\u00a0SD158\u00a0\u00b1\u00a0170292\u00a0\u00b1\u00a02338\u00a0\u00b1\u00a049\u00a0\u00b1\u00a0300Formalin 11,4023027762851Formalin 21,98715737123610Formalin 33,1351709483260Mean\u00a0\u00b1\u00a0SD2,175\u00a0\u00b1\u00a0882210\u00a0\u00b1\u00a08069\u00a0\u00b1\u00a0309\u00a0\u00b1\u00a03324\u00a0\u00b1\u00a0380Red cy5 labeled samples, green cy3 labeled controls, 1 8\u00a0h, 2 12\u00a0h, 3 24\u00a0h\nThese findings may be attributed to erratic and random fragmentation of DNA in formalin-fixed samples. The fragmentation itself results in increase background noise and aberrant signal intensity due to random hybridization.\nWe further analyzed array scan data by a-CGH explorer software using stem-plot analysis for moving averages. Fresh and UMFIX-exposed sample showed a readable plot with the expected difference in XY chromosome regions; as expected from the female control and male samples. Also, they showed reproducibly amplified and deleted region in our test sample DNA. A-CGH result from formalin-fixed samples was not interpretable (Fig.\u00a02).\nFig.\u00a02Array CGH stem plot of 8-h preserved skin samples\u2014chromosomes are displayed on x-axis and relative ratios on y-axis. Green (control XX female, Cy3 labeled) Red (test XY male, Cy5 labeled). X and Y chromosomes are marked\nArray CGH studies have great potential for clinical application. Since the technique does not utilize live cells, it is considerably more advantageous when compared to conventional karyotyping techniques. Array CGH has its own limitations, such as failure to detect translocations; nevertheless it offers unprecedented spatial resolution [6, 11]. Recently, it has been shown that low-level gene copy number change is associated with changes in expression level of its transcripts [12]. Therefore, results of transcriptomics studies can be used to study DNA markers, which are more stable and easier to study than RNA.\nArray GGH was originally based on BAC (Bacterial Artificial Chromosomes), but more recently it utilizes oligonucleotides, with its ability to provide whole genome-covering resolution. Synthetic oligonucleotides obviously have the advantage because their exact sequence and length are known for each element on the array. Oligonucleotide array CGH (oa-CGH) has the benefit of overcoming the difficulties inherent to BAC arrays, such as the amount of available DNA, clone management, probe identity due to PCR contaminations, and mapping inaccuracies. Using oa-CGH, it is also possible to eliminate another source of error, which is the batch-to-batch variability of Cot-1 DNA, used to block repetitive DNA sequences, since the oligonucleotide probes are designed to be repeat-free [20].\nSamples used in nearly all a-CGH studies have been fresh or fresh-frozen tissue. Such material, although useful in research settings, is impractical and cumbersome to use in clinical practice. Furthermore, because diagnostic biopsy samples are relatively small, the amount of residual tissue for additional ancillary testing may be inadequate. Hence there is a great need to develop test strategies that require minimal amounts of tissue and are robust enough to withstand pre-analytical sample preparation. Simplified schemes for sample preservation that allow reliable histomorphology along with preservation of high quality macromolecules are desirable.\nWe have previously described a novel tissue fixation and processing technique that beside providing adequate histomorphology also preserves high quality HMW RNA, akin to fresh samples [19]. This fixative also protects HMW tissue DNA for use in PCR studies [18]. Here, we further evaluated the suitability of skin tissue DNA for array CGH studies using same methods applied routinely for fresh samples. This study demonstrates that using the novel fixative it is possible to preserve and extract high molecular weight genomic DNA, supported by high labeling efficiency, comparable to that from fresh tissue. Furthermore, no artifacts were seen using this DNA in microarray scanning or analysis for array CGH.\nArray CGH studies have been performed on formalin-fixed tissues with variable and irreproducible results. Most of the prior studies were based on low-resolution BAC arrays without detailed description of DNA quality or array metrics [10, 21]. Besides, there was often inadequate documentation of fixation time, the volumetric ratio of fixative to tissue, or buffering status. Only few studies have adequately addressed analytical aspects of fixation and processing on array CGH. Ghazani et al. [4] studied the effect of formalin fixation on MCF-7 cell line and on one breast cancer tissue sample. They used two different BAC clone arrays; 1.7k for the cell line and 19k for the breast cancer sample [4]. There was no mention of fixative volume or quality of genomic DNA. They showed that long term (1\u00a0week) fixation results in extensive loss of HMW DNA. In cell lines, the concordance between fresh and formalin-fixed samples was around 85%. When genomic DNA was amplified, the concordance decreased to 75%.\nJohnson et al. [5] also studied the effect of formalin fixation on a-CGH using BAC arrays. Our results support their conclusion that analysis of DNA samples on agarose gels does not offer any advantage in prediction of suitability of formalin-fixed tissue samples for a-CGH. They also show that the quality of a-CGH depends on the integrity of DNA samples with the requirement that extracted DNA supports the PCR amplification of an amplicon of 300\u00a0bp or longer. We showed that it is possible to detect amplicons of up to 450\u00a0bp in formalin-fixed sample; (albeit with lower intensity when compared to fresh or UMFIX-exposed samples). The studies by Johnson and others show the possibility of having acceptable results with DNA from formalin-fixed samples using BAC arrays but there are no data that supports its suitability for oa-CGH arrays. This may be due to larger sequence of probes presented in BAC array. Other investigators have shown that using alternative approaches to DNA extraction, quantification, amplification or labeling may produce improved results. However, all other authors do agree that the procedures for improving DNA quality are neither substitute for high quality DNA nor they could obtain consistent results from formalin-fixed samples [7, 10]. This is mostly due to complex chemical effect of formalin on tissue, which is still poorly understood. Formalin has a tissue penetration rate of 2.4\u00a0mm in 24\u00a0h and adequate fixation requires at least a ten to one volumetric ratio of fixative to tissue [3]. Therefore, many clinical specimens are only partially fixed by formalin before processing. Furthermore during processing, they are exposed to formalin and alcohol, introducing formalin and alcohol-related tissue artifacts [13]. Formaldehyde is a dipolar molecule and can react with amino, or imino group of the anionic forms of the amino acids. This reaction is time and temperature dependent [2, 9, 16, 17, 18]. More recent findings show that the deleterious effect of formalin fixation might also result from cumulative action of other reagents and processing conditions. Conversely, no DNA or nucleoside reactions have been reported with ethanol and none would be expected under physiological conditions [8]. By changing the three dimensional structure of the proteins, alcohols prevent protein functions. Therefore, rapid fixation of samples prevents alteration and degradation of biomolecules and preserves them in their native form [11].\nIn summary, we show that by using a new molecular fixative it is possible to preserve skin tissue DNA that is suitable for array CGH studies; identical to fresh tissue. This can be achieved using same methods and protocols used for fresh samples.","keyphrases":["genomics","fixatives","array comparative genomic hybridization","tissue preservation"],"prmu":["P","P","R","R"]}
{"id":"J_Gastrointest_Surg-3-1-1852375","title":"Intestinal Perforations in Beh\u00e7et\u2019s Disease\n","text":"Beh\u00e7et\u2019s disease accompanied by intestinal involvement is called intestinal Beh\u00e7et\u2019s disease. The intestinal ulcers of Beh\u00e7et\u2019s disease are usually multiple and scattered and tend to perforate easily, so that many patients require emergency operation. The aim of this study is to determine the extent of surgical resection necessary to prevent reperforation and to point out the findings of concurrent oral and genital ulcers and multiple intestinal perforations in all patients of our series. During a 25-year study period, information of 125 Beh\u00e7et\u2019s disease cases was gathered. Among the 82 patients who were diagnosed with intestinal Beh\u00e7et\u2019s disease, 22 cases had intestinal perforations needing emergency laparotomy. We investigated and analyzed these cases according to the patients\u2019 demographic characteristics, clinical presentations, laboratory data, and surgical outcome. There were 14 men and 8 women ranging from 22 to 65 years of age. Nine cases were diagnosed preoperatively, and the diagnoses were confirmed in all 22 cases during the surgical intervention. Surgical resection was performed in every patient, with right hemicolectomy and ileocecal resection in 11 cases, partial ileum resection in 8 cases with two reperforations, and ileocecal resection in 3 cases with one reperforation.\nIntroduction\nBeh\u00e7et\u2019s syndrome is a systemic process affecting multiple organ systems1,2. Surgeons need to be aware of the lethal complication of Beh\u00e7et\u2019s disease with intestinal ulcers, which tend to perforate at multiple sites3,4. A review of the literature reveals that involvement of the gastrointestinal tract is not infrequent. Most cases reported in the literature are in the eastern Mediterranean countries and Japan5\u20137. We report here a series of 22 cases of intestinal Beh\u00e7et\u2019s disease with multiple perforations, treated by emergency surgical resections.\nMaterials and Methods\nDuring the 25\u00a0years from July 1979 to June 2004, 125 patients with Beh\u00e7et\u2019s disease were encountered at the Cardinal Tien Hospital and Tri-Service General Hospital, Taipei, Taiwan. Eighty-two patients were diagnosed as having intestinal Beh\u00e7et\u2019s disease, which was based on the Mason\u2013Barnes criteria (Table\u00a01)1,2. Among these patients, 22 had intestinal perforations (see Table\u00a02 for the details of these 22 cases). \nTable\u00a01The Mason\u2013Barnes CriteriaMajor SymptomsMinor SymptomsBuccal ulcerationsGastrointestinal lesionsGenital ulcerationsThrombophlebitisOcular lesionsCardiovascular lesionsSkin lesionsArthritisNeurologic lesionsFamily historyThree major or two major and two minor criteria are required to establish the diagnosis of Beh\u00e7et\u2019s diseaseTable\u00a02Intestinal Perforation in Beh\u00e7et\u2019s Disease Encountered at CTH and TSGH (from 1979 to 2004, n\u2009=\u200922)Case No.Age (years)SexOral UlcerGenital UlcerGI S & SOcular SignsSkin LesionPathergic ReactionArthritis or Arthalgia138M+++\u2212\u2212++245M++++++326F+\u2212++\u2212\u2212447M+++++\u2212528F+\u2212++\u2212++636F++++\u2212\u22127a22M+++\u2212\u2212+842M++++\u2212922M+++\u2212+++1028F++++\u2212\u22121165M+++\u2212+\u221212a23M+++\u2212+\u2212\u22121332F+\u2212+++\u22121424M++++\u2212++1534M+++\u2212\u22121641F+++\u2212+17b38M++++++\u22121833M+++\u2212+\u2212\u22121925M+++\u2212+++2048F++++\u22122129M+++\u2212+\u2212\u22122250F++++\u2212+Plus signs mean that the feature is present; minus signs mean that the feature is not present.CTH = Cardinal Tien Hospital, TSGH = Tri-Service General Hospital, S & S = symptoms and signsaReperforations at ileum after partial resection of ileumbReperforation at ileum after ileocecal resection\nIn 13 of these 22 cases, the diagnosis was confirmed at surgical resection for multiple perforations. Nine of the 22 cases had Beh\u00e7et\u2019s disease with intestinal involvement, which was confirmed preoperatively, six were confirmed by endoscopic examination; two by radiological examination; and one patient had gastrointestinal symptoms of intermittent abdominal pain, diarrhea, and nausea.\nResults\nPatient Characteristics\nThere were 14 men and 8 women in the 22 cases investigated. The ages of the patients with perforated intestinal Beh\u00e7et\u2019s disease ranged from 22 to 65\u00a0years, with a mean age of 35.3\u00a0years. The age at onset of symptoms of Beh\u00e7et\u2019s disease varied from 18 to 64\u00a0years on diagnosis, with a mean age of 33.1\u00a0years.\nIn Table\u00a02, oral ulcers with gastrointestinal symptoms and signs were found concurrently in all 22 cases, genital ulcers in 19 cases, ocular lesions in 12 cases, and skin lesions in 11 cases. The painful oral ulcers (Fig.\u00a01) occurred on oral mucosa, lips and in the larynx. They varied from 2 to 8\u00a0mm in size and invariably healed without scarring. The genital ulcers (Fig.\u00a02) resembled the oral ulcers in appearance and course, except that vaginal ulcers were painless. Four patients had anterior uveitis and eight had a mild relapsing conjunctivitis as their sole ocular lesion. The nodular cutaneous lesions resembled those of erythema nodosum and were chronic and multiple. Most lesions occurred on the chest wall, back (Fig.\u00a03), and legs. Biopsy of dermal subcutaneous lesions had been done in 10 cases. In each of them, a nonspecific vasculitis of subcutaneous capillaries and venules was present (Fig. 4). Pathergic reaction was found positive in 7 of 10 patients.\nFigure\u00a01Buccal ulcer.Figure\u00a02Penile ulcer.Figure\u00a03Nodular cutaneous lesion on the back.Figure\u00a04Vasculitis characterized by lymphocytic and plasmacytic infiltration of perivascular tissue (hematoxylin and eosin; 10\u2009\u00d7\u200940).\nThere were no specific immunologic abnormalities in any of the 16 patients tested (Table\u00a03). The levels of immunoglobulin were variable. IgG was increased in 3 of 16 patients, IgA in 5 patients, and IgM in 3 patients. There was a significant decrease of IgG in two patients and of IgA in one patient. The total hemolytic complement was normal in all 16 serum samples. Alpha-2 globulin was increased in 9 of 16 patients, and gamma globulin was increased in seven patients. \nTable\u00a03Laboratory DataCase No.Immunoglobulins (mg\/dl)Serum Complement (mg\/dl)Globulin (%)IgGIgAIgMC\u20323C\u2032411,9763752501453813.823.821,726245174924012.010.842,1504002401104514.224.651,500590300382510.518.07a7401856090387.814.381,18019514059326.612.292,2704642621274614.016.2111,8503802501905012.523.512a1,30032023588399.615.0142,3504902951804813.325.01668098561503513.021.817b1,65047528076349.412.5181,8002901501054513.823.2192,4185812091664014.428.0211,8803302501803510.520.0221,9853862281683813.824.2Normal range950\u20132,110170\u201341054\u201326247\u201319127\u2013524.8\u201312.18.8\u201322.8aReperforations at ileum after partial resection of ileumbReperforation at ileum after ileocecal resection\nMultiple concurrent penetrating ulcers (Fig.\u00a05) were found in all 22 cases, with multiple perforation sites identified from terminal ileum to the ascending colon (Table\u00a04). The size and number of perforated ulcers were variable, ranging from 0.2 to 6\u00a0cm in size, and 4 to 16 in number. The perforations were found at the ileocecal region and ascending colon in 10 cases, at the terminal ileum in 8 cases, and at the cecum and ascending colon in 4 cases.\nFigure\u00a05Surgical specimen of ileocecal region showing multiple penetrating ulcers.Table\u00a04Operative Findings and Operation Performed in 22 Perforated Intestinal Beh\u00e7et\u2019s Disease PatientsCase No.Location of Perforated UlcersNo. of PerforationsOral\/genital UlcerOperation Performed1Terminal ileum4+\/+Partial resection of the ileum2Terminal ileum6+\/+Partial resection of the ileum3Ileocecal region and ascending colon10+\/\u2212Right hemicolectomy and ileocecal resection4Ileocecal region and ascending colon16+\/+Right hemicolectomy and ileocecal resection5Cecum and ascending colon5+\/\u2212Ileocecal resection6Terminal ileum5+\/+Partial resection of the ileum7aTerminal ileum4+\/+Partial resection of the ileum8Cecum and ascending colon9+\/+Right hemicolectomy and ilececal resection9Terminal ileum8+\/+Partial resection of the ileum10Ileocecal region and ascending colon11+\/+Right hemicolectomy and ileocecal resection11Ileocecal region and ascending colon10+\/+Right hemicolectomy and ileocecal resection12aTerminal ileum5+\/+Partial resection of the ileum13Terminal ileum7+\/\u2212Partial resection of the ileum14Ileocecal region and ascending colon11+\/+Right hemicolectomy and ileocecal resection15Ileocecal region and ascending colon5+\/+Right hemicolectomy and ileocecal resection16Ileocecal region and ascending colon13+\/+Right hemicolectomy and ileocecal resection17bCecum and ascending colon4+\/+Ileocecal resection18Ileocecal region and ascending colon7+\/+Right hemicolectomy and ileocecal resection19Ileocecal region and ascending colon9+\/+Right hemicolectomy and ileocecal resection20Cecum and ascending colon6+\/+Ileocecal resection21Terminal ileum4+\/+Partial resection of the ileum22Ileocecal region and ascending colon12+\/+Right hemicolectomy and ileocecal resectionaReperforations at ileum after partial resection of ileumbReperforation at ileum after ileocecal resection\nOperative Treatment and Outcome\nAll 22 perforated intestinal Beh\u00e7et\u2019s disease cases were confirmed at operation, with nine of them correctly diagnosed preoperatively. Surgical resection of the perforated intestinal ulcers was done in all cases, with right hemicolectomy and ileocecal resection in 11 cases, partial ileum resection in 8 cases, and ileocecal resection in 3 cases. No reperforation occurred in the group of patients who underwent right hemicolectomy and ileocecal resection. However, two reperforations ocurred in patients who underwent partial ileum resection alone and one in the ileocecal resection group.\nThe pathologic study of the resected specimens showed nonspecific inflammatory reactions with the infiltration of lymphocytes and plasma cells as the predominant finding (Fig.\u00a06). Histological sections from the ulcer walls showed changes consistent with a nonspecific ulcerative inflammatory process and infiltration containing both plasma cells and chronic inflammatory cells.\nFigure\u00a06Chronic inflammatory response and perivascular infiltration (hematoxylin and eosin; 10\u2009\u00d7\u200910).\nAfter operation on these 22 patients with Beh\u00e7et\u2019s disease and intestinal perforation, four patients died during the postoperative course due to septic shock, which was present prior to the surgical intervention; three died from complications of hypertension and diabetes mellitus; and three were lost to follow-up. Thus, only 12 patients are still under observation, without evidence of gastrointestinal complications up to this date. The remaining 60 cases of intestinal Beh\u00e7et\u2019s disease, without perforations, are still under surveillance.\nDiscussion\nIn 1937, Beh\u00e7et described a chronic relapsing triple-symptom complex of oral ulceration, genital ulceration, and ocular inflammation5. Over the years, it has become apparent that the process is a systemic recurrent inflammatory disease affecting a number of organs consecutively6. In 1940, Bechgaard first described intestinal involvement in Beh\u00e7et\u2019s disease. Tsukada et al. proposed the term \u201cintestinal Beh\u00e7et\u2019s disease\u201d in 19642,3. Baba et al.4. agreed to this proposal and cited 49 cases of the disease treated from 1975. Since then, the number of operations reported has increased rapidly3, but perforated intestinal Beh\u00e7et\u2019s disease is still rarely reported.\nIn a large review series, Oshima and colleagues reported that 40% of patients with Beh\u00e7et\u2019s disease had gastrointestinal complaints, such as nausea, vomiting, and abdominal pain2\u20134,8,9. The age at onset of these symptoms ranges from 16 to 67\u00a0years, and the male-to-female ratio ranges from 1.5:1 to 2:12,5. Our cases were in accordance with this reported age range and sex ratio. The third decade is the most commonly reported age of onset for Beh\u00e7et\u2019s disease6,8,10,30 and the fourth decade for intestinal Beh\u00e7et\u2019s disease3. In our study, intestinal Beh\u00e7et\u2019s disease occurred at a mean age of 33.1\u00a0years. However, Beh\u00e7et\u2019s disease and intestinal involvement were diagnosed simultaneously in some of these patients, most of whom had already experienced systemic manifestations.\nThe exact cause of this disease still remains an enigma. Current hypotheses include allergic vasculitis of small vessels, autoimmune disease, and immunologic deficiency2,4,11,12. The deposition of immune complexes in the walls of small blood vessels was found by the laboratory results of three of our cases, and this process has been proposed as one of the underlying pathologic mechanisms in intestinal Beh\u00e7et\u2019s disease12.\nSince no clinicopathologic findings are pathognomonic in this disease, the diagnosis is made on the basis of combinations of various clinical symptoms and signs13. Mason and Barnes constructed an elaborate set of major and minor criteria for diagnosis1. They suggested the triad of buccal ulceration, genital ulceration, and eye lesion and skin lesion as major symptoms. The minor symptoms included gastrointestinal lesions, arthritis, thrombophlebitis, cardiovascular lesions, neurologic lesions, and family history. Three major criteria or two major criteria and two minor criteria are necessary for diagnosis. These various symptoms are not usually present at the same time. If we hold the original triple-symptom complex as a prerequisite for the diagnosis, cases may be missed. In 1990, the International Study Group for Beh\u00e7et\u2019s Disease14 introduced a diagnostic criteria requiring the presence of oral ulcerations plus any two of the following: genital ulcerations, typical eye lesions, typical skin lesions, or positive results to a pathergy test. However, some reports have shown that almost 20% of patients with Beh\u00e7et\u2019s disease presented without oral lesions initially15,16. Furthermore, 2\u20135% of patients did not show any oral lesions at all16,17. In our series, all patients had manifestations of concurrent oral ulceration. All perforated cases present oral or genital ulcerations at the same time. Because we warned that patients of intestinal Beh\u00e7et\u2019s disease may have abdominal pain and oral or genital ulcerations concurrently, intestinal perforations should always be kept in mind.\nA phenomenon of pathergy was first described by Blobner in 1937 and was further elaborated by Katzenellenbogen in 1968. It consists of an itradermal test applied to Beh\u00e7et\u2019s disease patients with a sharp needle prick causing skin hypersensitivity, which is characterized by the formation of a sterile pustule 24 to 48\u00a0h after the trauma. Biopsy at the intradermal puncture site is taken 48\u00a0h after for histopathologic evaluation. In a study conducted by Tuzum et al., this reaction was found to be positive in 84% of 58 patients with the disease, as compared to 3% of 90 healthy controls1. A positive pathergic reaction should make us aware of the possibility of the disease in the presence of any of the accepted symptoms of this process. However, the recent results and interpretations of pathergy tests have varied widely according to the technical aspects of the tests18,19 and ethnic differences of the patients.\nThe histological lesions in Beh\u00e7et\u2019s disease are rather uncharacteristic. Nonspecific perivascular infiltrations of plasma cells and lymphocytes are usually found in the cutaneous and mucosal lesions5,20. The intestinal ulcers in Beh\u00e7et\u2019s disease are characterized not only by the absence of the granulomatous formation of Crohn\u2019s disease, but also by deeper penetration of the ulcers to areas nearer to serosa membrane than the ulcers of ulcerative colitis3,4,21. The ulcers tend to be undermined, and the submucosal connective tissues are usually destroyed. The bases of the ulcers are avascular with edema-like swelling and crater-shaped formation around the ulcer margin.2,22\u201324 These ulcers are usually found in the terminal ileum and the cecum, but they may be present at any site throughout the digestive system and tend to perforate at multiple sites25\u201329. The gross pathologic characteristics of our intestinal Beh\u00e7et\u2019s disease included perforations at multiple sites concurrently in variable sizes and configurations, extending from the ileocecal region to ascending colon, in accordance with the reported literature3,4,8,30,31.\nThe medical treatment of the intestinal Beh\u00e7et\u2019s disease remains unsettled. The beneficial effect of steroid therapy has not been convincing in most series2,7,30. It may control the disease initially, but recurrences are common. Topical application of corticosteroids decreases the ocular inflammation, and is also useful in relieving the pain of oral ulcers. Haim and Sherf reported a favorable response to fresh blood and plasma in cases of Beh\u00e7et\u2019s disease, but the nature of the useful component in hematotherapy is unknown5. In our two patients with perforations, steroid therapy was given for 2\u00a0weeks after surgery with favorable outcomes.\nResection of the ileocecal region or the right half of the colon is the usual operation in the treatment of gastrointestinal complications3,4. In our series, perforations at multiple sites were found in all cases; right hemicolectomy and ileocecal resection were performed in 11 cases without reperforation; ileocecal resection in 3 cases with one reperforation; and partial resection of the ileum in 8 cases with two reperforations.\nConclusion\nBecause concurrent oral and genital ulcers were found in all patients in our series, the presentation of this seemingly innocuous clinical manifestation along with gastroinstestinal symptoms should raise the level of suspicion that intestinal involvement and complications of perforations may have already happened. The other constant finding among our 22 patients is that all the intestinal perforations were located between the terminal ileum and the ascending colon. Therefore, to prevent reperforations, wide excision of the terminal ileum with right hemicolectomy is recommended for perforated intestinal Beh\u00e7et\u2019s disease. We found out that the specimens of the resected bowel of the 19 nonreperforated patients all had more than 60\u00a0cm of terminal ileum, but those of the three reperforated cases had less than 60\u00a0cm. Furthermore, the perforation sites were all at 10 to 12\u00a0cm proximal to the anastomosis. This is the main reason we recommend the resection of up to 80\u00a0cm of ileum from the ileocecal valve at the time of right hemicolectomy4,31.","keyphrases":["intestinal perforations","beh\u00e7et\u2019s disease","intestinal ulcers"],"prmu":["P","P","P"]}
{"id":"Domest_Anim_Endocrinol-2-1-2428105","title":"Endometrial expression of the insulin-like growth factor system during uterine involution in the postpartum dairy cow\u2606\n","text":"Rapid uterine involution in the postpartum period of dairy cows is important to achieve a short interval to conception. Expression patterns for members of the insulin-like growth factor (IGF) family were determined by in situ hybridisation at day 14 \u00b1 0.4 postpartum (n = 12 cows) to investigate a potential role for IGFs in modulating uterine involution. Expression in each uterine tissue region was measured as optical density units and data were analysed according to region and horn. IGF-I mRNA was localized to the sub-epithelial stroma (SES) of inter-caruncular and caruncular endometrium. Both IGF-II and IGF-1R expression was detected in the deep endometrial stroma (DES), the caruncular stroma and myometrium. IGFBP-2, IGFBP-4 and IGFBP-6 mRNAs were all localised to the SES of inter-caruncular and caruncular uterine tissue, and in the DES and caruncular stroma, with IGFBP-4 mRNA additionally expressed in myometrium. IGFBP-3 mRNA was only detectable in luminal epithelium. IGFBP-5 mRNA was found in myometrium, inter-caruncular and caruncular SES and caruncular stroma. These data support a role for IGF-I and IGF-II in the extensive tissue remodelling and repair which the postpartum uterus undergoes to return to its non-pregnant state. The differential expression of binding proteins between tissues (IGFBP-3 in epithelium, IGFBP-2, -4, -5 and -6 in stroma and IGFBP-4 and -5 in myometrium) suggest tight control of IGF activity within each compartment. Differential expression of many members of the IGF family between the significantly larger previously gravid horn and the previously non-gravid horn may relate to differences in their rate of tissue remodelling.\n1\nIntroduction\nIn dairy cows, the peri-partum period is critical to future milk production and fertility. Uterine involution involves extensive restructuring of the extracellular matrix alongside mitogenesis and apoptosis [1\u20133]. Initial degeneration of placental cotyledons and maternal caruncles accumulate as tissue debris in the uterine lumen forming a lochial discharge [4]. Contractions of the myometrium aid expulsion of lochia, and also restore uterine size, shape and tone to that of a non-pregnant animal [5,6]. Whilst most of these changes have occurred within 2\u20133 weeks postpartum, involution is not considered complete until about 40\u201350 days postpartum [1]. The previously non-gravid uterine horn returns to a non-pregnant state 10\u201315 days earlier than the previously gravid uterine horn [7]. Histological repair of the endometrium lags physical involution by 10\u201320 days [8], completing when caruncles regenerate epithelium [4]. Microbial contamination of the postpartum uterus is almost universal during the first week postpartum [9]. When pathogenic bacteria are not cleared the uterus becomes infected and inflamed and uterine involution is delayed [1,10]. Clinical endometritis is characterised by the continued presence of a purulent discharge beyond 21 days after calving [1].\nMany processes involved in uterine repair are common to those of wound healing in other tissues (for a review see [11]). Potential mediators of tissue turnover and remodelling in the uterus include cytokines, matrix-degrading enzymes and growth factors [11,12]. The insulin-like growth factors (IGF-I and IGF-II) function in such tissue repair processes. In healing-impaired wounds, the mRNA for IGF-I, IGF-1R, and IGFBP-3 is significantly reduced [13]. The administration of IGF-I to these wounds corrects defective tissue repair [14] and in combination with other growth factors it increases connective tissue regeneration and epithelialisation [15]. Components of the IGF system have been described in the uteri of a variety of species (e.g. humans [16], rodents [17], pigs [18], cattle [19], and sheep [20]). The proliferative and differentiating effects of IGFs on uterine cells are thought to support the growth and regression of uterine tissue throughout the estrous cycle and also the regenerative processes in women following menstruation [16,21]. IGFBP-2 has also been shown to stimulate endometrial cell mitogenesis directly [22].\nAn increased rate of uterine involution is associated with earlier resumption of ovarian activity [23], which is in turn important for increasing pregnancy rate to first service [24]. Conversely, endometrial damage associated with sub-clinical endometritis leads to prolonged intervals to conception, with many cows failing to conceive at all [25]. The mechanisms that regulate uterine involution are not completely understood and, to the best of our knowledge, no previous studies have investigated the uterine IGF system during involution in lactating dairy cows. We postulated that changes in IGF bioavailability may be implicated in the rate of postpartum uterine recovery and thus influence the calving to conception interval and reproductive efficiency. The objective of the study was to determine patterns of mRNA expression for the IGF system within the previously gravid (PG) and previously non-gravid (PNG) uterine horns during the early postpartum period. Samples were obtained at approximately 2 weeks after calving as we hypothesised that this represents a time by which a delay in the normal recovery process may predispose cows to the subsequent development of endometritis.\n2\nMaterials and methods\n2.1\nAnimals and tissue samples\nAll procedures were carried out under license in accordance with the European Community Directive, 86-609-EC. Uteri were collected from 12 multiparous Holstein-Friesian dairy cows (mean parity 4.7) following slaughter at day 14\u00a0\u00b1\u00a00.4 postpartum. The diameters of both horns were measured approximately 5\u00a0cm anterior to the bifurcation of the uterus. Samples of inter-caruncular and caruncular tissue were dissected from the previously gravid and non-gravid uterine horns approximately 1\u00a0cm anterior to the bifurcation of the uterus. A 5\u00a0cm square region was harvested, wrapped in aluminium foil, and frozen in liquid nitrogen-tempered isopentane. Samples were stored at \u221280\u00a0\u00b0C until sectioning.\n2.2\nIn situ hybridisation\nThe in situ hybridisation procedure was performed as described previously [26]. All chemicals were purchased from Sigma\u2013Aldrich Company Ltd. (Poole, Dorset, UK) or VWR International Ltd. (Poole, Dorset, UK) unless otherwise specified. Briefly, sections of 10\u00a0\u03bcm were cut from each uterine tissue sample and thaw-mounted onto SuperFrost\u00ae Plus or POLYSINE\u2122 microscope slides, fixed in 4% (w\/v) paraformaldehyde in 0.01\u00a0M PBS, washed in PBS, and sequentially dehydrated in 70% and 95% ethanol. The oligonucleotide probes for the IGF system were end-labelled with [35S]dATP (Amersham Biosciences UK Ltd., Buckinghamshire, England) using terminal deoxynucleotidyl transferase (Promega UK Ltd., Southampton, England). Tissue sections were subsequently treated with 100\u00a0000\u00a0cpm\u00a0(100\u00a0\u03bcl)\u22121 hybridisation buffer and hybridised overnight at either 42, 45, or 52\u00a0\u00b0C (Table 1). Following incubation, slides were washed in a solution of 1\u00a0\u00d7\u00a0SSC, 2\u00a0g\u00a0l\u22121 sodium thiosulphate at room temperature for 30\u00a0min followed by fresh 1\u00a0\u00d7\u00a0SSC, 2\u00a0g\u00a0l\u22121 sodium thiosulphate at 60\u00a0\u00b0C for 60\u00a0min. Slides were then rinsed in solutions of 1\u00a0\u00d7\u00a0SSC, 0.1\u00a0\u00d7\u00a0SSC, 75% ethanol and 95% ethanol and air-dried before exposure to \u03b2-max hyperfilm (Kodak BioMax MR Film) for either 4 or 5 days. All uterine sections treated with a particular probe were hybridized in the same batch. Sense probes, which were identical in sequence to the respective mRNA targets, were always included as negative controls and any signal from these was regarded as non-specific. Each batch also contained an appropriate positive control tissue, based on previous studies. These were cross-sections of uterus from an estrous ewe for IGF-I and the type 1 IGF receptor [20], IGFBP-1 [27] and IGFBP-6 [28]; ovine placentome for IGF-II and IGFBPs-2, -3 and -4 [29] and ovine intercotyledonary tissue for IGFBP-5 [30].\n2.3\nPhotographic emulsions\nTo aid cellular localisation of hybridised probes, slides previously subject to autoradiography were coated with photographic emulsion LM1 (Amersham Biosciences UK Ltd., Buckinghamshire, England) according to the manufacturer's instructions and stored for 28, 30 or 42 days at 4\u00a0\u00b0C in the dark (Table 1). The slides were developed in 20% phenisol (ILFORD Imaging UK Ltd., Cheshire, England) fixed in 1.9\u00a0M sodium thiosulphate and counterstained with haematoxylin and eosin. All other slides were also stained with haematoxylin and eosin to aid identification of tissue region.\n2.4\nOptical density measurements\nReadings were obtained from at least two sections per tissue for each of the antisense (AS) and sense (S) probes. Autoradiographs were scanned into a computer and optical density (OD) measurements were recorded from digital images. The relative expression of mRNA for components of the uterine IGF system was quantified from the autoradiographs using the public domain NIH ImageJ program (available through the NIH website\u2014http:\/\/www.nih.gov), which calculated the average optical density (OD) over the selected area of film based on a linear grey scale of 0.01\u20132.71. The following tissue layers were each assessed separately: luminal epithelium, sub-epithelial stroma (a layer of dense connective tissue underlying the luminal epithelium), caruncular stroma (the dense connective tissue forming the caruncles), deep endometrial stroma (loose connective tissue between the sub-epithelial stroma and the myometrium) and myometrium. The latter two tissue types were only present in samples collected from the inter-caruncular region. Each tissue type was measured separately on each section. The background OD, from a blank area of film, was also measured and subtracted from both AS and S OD measurements. Finally the S values were subtracted from AS values to give an average OD value for specific hybridisation [31]. The detection limit was taken as an OD value of 0.01.\n2.5\nStatistical analysis\nStatistical analyses were performed using Statistical Package for the Social Sciences (SPSS for Windows, V13.0). Data for uterine diameter measurements at the time of tissue collection were analysed using Student's t-test. OD measurements were obtained from four samples per cow, taken from each of the caruncular and inter-caruncular regions of the previously gravid and non-gravid horns. The effects of uterine horn and tissue region on the level of mRNA expression for each probe were analysed by general linear model analysis. Cow was entered as a random effect. For this purpose, data from uteri in which a particular probe showed no detectable specific hybridisation (OD of <0.01) were given an OD of 0.01, which equated to the lower limit of detection. Results were considered statistically significant when P\u00a0<\u00a00.05.\n3\nResults\nAt the time of tissue collection, the diameter of the previously gravid uterine horn was larger than that of the previously non-gravid uterine horn (56\u00a0\u00b1\u00a06.9 and 31\u00a0\u00b1\u00a03.1\u00a0mm, respectively, mean\u00a0\u00b1\u00a0S.E.M., P\u00a0=\u00a00.005). The spatial distribution of mRNA encoding components of the uterine IGF system is shown in Figs. 1 and 2. The concentrations of mRNA in OD units are summarised in Table 2 according to uterine horn and tissue region and their two-way interactions are illustrated in Figs. 3 and 4. The method used provided a semi quantitative measure of the intensity of mRNA expression in specific cell types.\n3.1\nExpression of the IGFs and IGF type 1 receptor\nIGF-I mRNA was localized to the sub-epithelial stroma (SES) of inter-caruncular and caruncular endometrium in both uterine horns (Figs. 1(A) and 2A). Both IGF-II and IGF-1R expression was detected in the deep endometrial stroma (DES), the caruncular stroma (not shown) and myometrium (Figs. 1(C), (E) and 2C, E).\nOverall expression of IGF-I mRNA was higher in the inter-caruncular than caruncular SES (P\u00a0=\u00a00.001, Table 2). There was a significant horn\u00a0\u00d7\u00a0region interaction (P\u00a0=\u00a00.032), with lower levels of IGF-I transcript in the inter-caruncular SES of the PG compared with the PNG horn (Fig. 3(A)). IGF-II expression was higher in the DES than in the caruncular stroma and myometrium (P\u00a0\u2264\u00a00.001, Table 2). When data from tissue regions were pooled the concentration of IGF-II mRNA did not vary between the PNG and PG horns, but there was a significant horn\u00a0\u00d7\u00a0region interaction (P\u00a0\u2264\u00a00.001). Expression of IGF-II in the DES and caruncular stroma was lower in the PG than PNG horn, whereas within myometrium expression was higher in the PG than PNG horn (Fig. 3(B)). For the IGF-1R, expression was highest in myometrium and similar between DES and caruncular stroma (Table 2 and Fig. 3(C)). Overall, the level of IGF-1R transcript was higher (P\u00a0=\u00a00.030) in the PNG than PG horn (Table 2). The horn\u00a0\u00d7\u00a0region interaction was not significant for uterine IGF-1R mRNA expression.\n3.2\nExpression of IGFBPs\nIGFBP-1 mRNA could not be detected in any uteri examined, despite expression being observed in the ovine estrous uterus which was used as positive control tissue (data not shown). IGFBP-2, IGFBP-4 and IGFBP-6 mRNAs were all localised to the SES of inter-caruncular and caruncular uterine tissue, and in the DES and caruncular stroma (Figs. 1(G), (K), (O) and 2G, K, O). IGFBP-4 mRNA was additionally expressed in myometrium. In contrast, IGFBP-3 mRNA expression was only detected in the luminal epithelium (LE) of both inter-caruncular and caruncular samples (Figs. 1(I) and 2I). IGFBP-5 mRNA was found in myometrium, inter-caruncular and caruncular SES and caruncular stroma (Figs. 1(M) and 2M).\nIGFBP-2 mRNA expression in inter-caruncular and caruncular SES was higher than in DES and caruncular stroma (P\u00a0\u2264\u00a00.001, Table 2). There was no main effect of horn, but there was a horn\u00a0\u00d7\u00a0region interaction (P\u00a0=\u00a00.034). Within caruncular stroma only, the concentration of IGFBP-2 mRNA was higher in the PG than the PNG uterine horn (Fig. 4(A)).\nFor IGFBP-3 mRNA the main effects of uterine horn and tissue region were not significant but there was an interaction (P\u00a0\u2264\u00a00.001). Expression in the inter-caruncular LE was higher in the PNG than PG horn, whereas in the caruncular LE expression was higher in the PG uterine horn (Fig. 4(B)).\nExpression levels of IGFBP-4 mRNA varied between tissue regions, with higher expression in the caruncular than inter-caruncular SES, lowest expression in myometrium, and intermediate signal intensity in the DES and caruncular stroma (Table 2). There was no difference in transcript levels between the PNG and PG uterine horns when regional data were combined (Table 2). Levels of IGFBP-4 mRNA expression were, however, affected by an interaction between uterine horn and tissue region (P\u00a0=\u00a00.024): within DES expression was lower in the PG than PNG uterine horn (Fig. 4(C)).\nExpression of IGFBP-5 mRNA was highest in myometrium, intermediate in the inter-caruncular and caruncular SES and lowest in caruncular stroma (P\u00a0\u2264\u00a00.001, Table 2). When regional expression data were pooled, the PNG uterine horn expressed higher concentrations of IGFBP-5 mRNA than the PG horn (P\u00a0\u2264\u00a00.001, Table 2). There was also a significant effect of the interaction between uterine horn and tissue region (P\u00a0\u2264\u00a00.001). Expression in both the inter-caruncular SES and the caruncular stroma was lower in the PG than PNG uterine horn whereas for the caruncular SES the reverse was true (Fig. 4(D)).\nIGFBP-6 mRNA was expressed at higher concentrations in the inter-caruncular and caruncular SES than in DES and caruncular stroma (P\u00a0\u2264\u00a00.001, Table 2). Transcript levels were higher (P\u00a0\u2264\u00a00.001) in the PNG than PG uterine horn when regional expression data were pooled (Table 2). The interaction between uterine horn and tissue region was significant (P\u00a0=\u00a00.045). Expression in each of the inter-caruncular SES, caruncular SES, DES and caruncular stroma was lower in the PG than PNG horn (Fig. 4(E)).\n4\nDiscussion\nThe rate of uterine involution is an important factor influencing the subsequent fertility of dairy cows [24]. In this study we have investigated for the first time a possible role for the IGF family of proteins in this event in lactating dairy cows. The timing of tissue collection at approximately day 14 postpartum occurred when the PG uterine horn in our group of multiparous cows was larger than the PNG horn. At this stage caruncular tissue is expected to have undergone degeneration and sloughing, but not to have completed re-epithelialisation [2]. In contrast, the inter-caruncular area does not lose its epithelial layer [32] and recovers from pregnancy more quickly [2]. The ongoing process of uterine involution at the time of tissue collection was thus expected to involve tissue regeneration alongside size recovery. An adequate recovery process may be crucial in preventing the uterus, which is heavily contaminated with bacteria following calving [1], from developing endometritis. Samples were analysed using in situ hybridisation. Whilst this approach is considered only semi-quantitative, we have found the technique described here to be highly repeatable. Furthermore, it enables measurement of mRNA concentrations in individual cellular types. This is not feasible in a complex organ such as the uterus using alternative techniques such as RT-PCR, as it is not readily possible to separate different populations of epithelial and stromal cells for RNA extraction.\nIGF-I mRNA was localised to the SES, confirming earlier observations in the cow [33] and sheep [20,34]. Normal wound healing involves a sequence of inflammation, proliferation, and maturation or remodelling [12] and local IGF-I production increases as wound healing progresses [35]. Since IGF-I increases during the late proliferative phase of the human menstrual cycle [36], and is known to stimulate cell proliferation and collagen synthesis during tissue regeneration [14,35], we propose that IGF-I produced by SES may act in an autocrine and\/or paracrine manner to stimulate the proliferation of uterine stroma and epithelium [37,38] during uterine involution.\nIn early pregnancy the bovine endometrium synthesises IGF-II primarily within caruncular stroma [33]. The present study localised IGF-II mRNA at similar concentrations in both the caruncular stroma and myometrium. The strongest expression of IGF-II mRNA was, however, in the DES. Similar results were found in human endometrium [36]. IGF-1R was similarly localised to the DES, caruncular stroma and myometrium, confirming earlier observations in the bovine uterus [33]. Since the effects of IGF-II are probably mediated by the IGF-1R (for a review see [39]), the co-localisation of IGF-II and IGF-1R transcripts supports a local action for IGF-II in uterine repair and regeneration within both endometrial and caruncular stroma [16,21]. Stromal IGF-II may also act in a paracrine manner to stimulate epithelial cell proliferation [22]. The interaction between IGFs and their receptors in muscle growth and regeneration has been comprehensively reviewed by Florini et al. [40]. In myometrium, IGF-II may stimulate muscle growth and regeneration [40], and potentially increases muscle strength [41]. These actions would support myometrial contractions that return the uterus to its non-pregnant size, shape and tone [5]. Since the PG horn has to contract from a larger size at parturition, the higher concentration of IGF-II in the PG myometrium supports the proposal that IGF-II assists postpartum uterine size recovery. In rat myometrium the IGF-1R is up-regulated in the early postpartum period [42].\nThis study failed to detect IGFBP-1 mRNA in any postpartum uteri. During the estrous cycle IGFBP-1 expression is low at estrous and relatively higher during the luteal phase [33] concurrent with progesterone production. Since the cows in this study had yet to establish ovulatory cycles, then the uterus would not have been recently exposed to progesterone stimulation [43]. In ruminants IGFBP-1 appears to be involved in pregnancy recognition [33,44] rather than, as this study shows, postpartum uterine events.\nIn agreement with previous studies [33,44], the expression of uterine IGFBP-2 mRNA was localised to the SES and at relatively lower levels in the DES and caruncular stroma. In the cyclic cow IGFBP-2 mRNA levels increased during the luteal phase, concomitant with the highest levels of plasma progesterone [19]. Other studies have shown that human endometrial cells constitutively synthesise and secrete IGFBP-2 in vitro\n[45] and in response to estradiol [46]. In the bovine mammary gland [47], IGF-I may stimulate IGFBP-2 expression and protein secretion, whereas in fetal visceral glomerular epithelial cells isolated from human kidneys IGFBP-2 production was stimulated by IGF-II [48]. The exact mechanisms regulating postpartum uterine IGFBP-2 mRNA expression thus requires further investigation. IGFBP-2 is presumably modulating uterine involution indirectly by regulating the bioavailability of IGF-I and IGF-II and the interaction of these ligands with their receptors [39]. The precise action of IGFBP-2 also remains uncertain. Both stimulatory [22] and inhibitory [48] actions of IGFBP-2 on IGF-stimulated epithelial cell proliferation have been suggested. Alternatively or additionally, IGFBP-2 could modulate uterine cell growth directly [22].\nIn contrast to all the other binding proteins investigated, the expression of IGFBP-3 mRNA was confined to the luminal epithelium, again agreeing with earlier work in the cyclic animal [33]. Epithelial IGFBP-3 may regulate local IGF bioavailability [39] or transport IGFs across this cell layer [49,50] for secretion into the uterine lumen. Removing excess IGF from the endometrium would prevent the IGF-1R from being down-regulated [51]. Alternatively, since IGFBP-3 associates with cell surfaces, then it may store IGFs [52] in the uterus and further promote IGF-stimulated tissue repair [37,39] as proposed for other physiological systems [53].\nIGFBP-4 mRNA was detected in multiple uterine tissue compartments. The localisation in SES and caruncular stroma has also been found in the pregnant ewe [54] and synthesis in the DES and myometrium agrees with studies in the human and pregnant bovine uterus [44,55]. IGFBP-4 is generally considered inhibitory to IGF actions [56]. IGFBP-4 does not appear to bind to the cell surface or extracellular matrix, but can cross the endothelium [39], indicating that IGFBP-4 may clear endometrial IGFs.\nIGFBP-5 mRNA was localised to caruncular stroma and myometrium, in agreement with previous studies in the cow [33] and sheep [34], and transcript was also detected in the SES. With a lack of detectable IGFBP-2, IGFBP-3, and IGFBP-6 mRNA alongside relatively low levels of IGFBP-4 mRNA in the myometrium, the abundance of IGFBP-5 in this tissue suggests this binding protein as the primary regulator of local IGF bioavailability [57]. In the rat myometrium, IGFBP-5 mRNA is significantly up-regulated after parturition, which is suggested to support tissue remodelling during involution [42]. It is also possible that within the myometrium, IGFBP-5 is directly stimulating muscle cell survival during myogenesis [57,58]. In stromal fibroblasts, IGFBP-5 can adhere to the extracellular matrix, which decreases its affinity for IGF and can potentiate IGF-stimulated DNA synthesis [39,59]. Furthermore, IGFBP-5 may stimulate local tissue growth independently of IGF [39,59].\nIGFBP-6 was localised to SES and at lower levels in DES and caruncular stroma, similar to the non-pregnant ovine uterus [28]. This expression pattern parallels that of IGFBP-2 mRNA and may suggest these two binding proteins are co-regulated [60]. IGFBP-6 has a markedly higher affinity for IGF-II [61] and so the major function of IGFBP-6 is probably to regulate IGF-II actions [62,63]. Furthermore, IGFBP-6 is generally considered to inhibit the effects of IGF-II, including cell proliferation and differentiation [59]. The importance of controlling uterine IGF bioactivity has been demonstrated in the human uterus where low levels of IGFBP-6 and higher levels of IGF-II are associated with uterine leiomyomas (fibroids), compared with normal endometrium [64].\nMany members of the IGF family showed differential expression between the two uterine horns. Expression of IGF-I, the IGF-1R and IGFBP-6 was at an overall lower level in the previously gravid horn whereas IGFBP-4 mRNA expression was lower in the DES only. IGF-II, IGFBP-3 and IGFBP-5 expression showed horn by region interactions, with mRNA concentrations reduced for some regions but increased for others. These differences may reflect the temporal misalignment of the horns in their rate of tissue remodelling, with the PG horn lagging behind the PNG horn by up to 15 days [7].\nIn conclusion, the IGF system is significant to uterine function and the synthesis of these growth factors in the postpartum uterus indicates a role in uterine involution. This study has shown that in the postpartum bovine uterus IGF-I synthesis was localised to sub-epithelial stroma, whilst maximum concentrations of IGF-II and IGF-1R mRNA were in the endometrial stroma and myometrium, respectively. The uterine tissue compartments expressed different profiles of IGF-binding proteins, indicating that IGF bioavailability and bioactivity is differentially regulated throughout the regenerating endometrium. IGFs are presumably supporting the tissue repair that follows parturition, similar to that of normal wound healing [12]. We propose that myometrial IGF-II synthesis stimulates tissue recovery in an autocrine manner, which may assist the uterus returning to its non-pregnant shape and size. Although IGFs may be key physiological mediators of endometrial repair, other growth factors and cytokines are undoubtedly also important in this process [11,65].\nConflicts of interest\nThe authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.","keyphrases":["involution","igf","igfbp","uterus","bovine"],"prmu":["P","P","P","P","P"]}
{"id":"Evid_Based_Complement_Alternat_Med-3-4-1697739","title":"The Use of Herbal Medicine in Alzheimer's Disease\u2014A Systematic Review\n","text":"The treatments of choice in Alzheimer's disease (AD) are cholinesterase inhibitors and NMDA-receptor antagonists, although doubts remain about the therapeutic effectiveness of these drugs. Herbal medicine products have been used in the treatment of Behavioral and Psychological Symptoms of Dementia (BPSD) but with various responses. The objective of this article was to review evidences from controlled studies in order to determine whether herbs can be useful in the treatment of cognitive disorders in the elderly. Randomized controlled studies assessing AD in individuals older than 65 years were identified through searches of MEDLINE, LILACS, Cochrane Library, dissertation Abstract (USA), ADEAR (Alzheimer's Disease Clinical Trials Database), National Research Register, Current Controlled trials, Centerwatch Trials Database and PsychINFO Journal Articles. The search combined the terms Alzheimer disease, dementia, cognition disorders, Herbal, Phytotherapy. The crossover results were evaluated by the Jadad's measurement scale. The systematic review identified two herbs and herbal formulations with therapeutic effects for the treatment of AD: Melissa officinalis, Salvia officinalis and Yi-Gan San and BDW (Ba Wei Di Huang Wan). Ginkgo biloba was identified in a meta-analysis study. All five herbs are useful for cognitive impairment of AD. M. officinalis and Yi-Gan San are also useful in agitation, for they have sedative effects. These herbs and formulations have demonstrated good therapeutic effectiveness but these results need to be compared with those of traditional drugs. Further large multicenter studies should be conducted in order to test the cost-effectiveness of these herbs for AD and the impact in the control of cognitive deterioration.\nIntroduction\nAlzheimer's disease (AD) is characterized as a progressive neurodegenerative disorder and considered as prominent cause of dementia in the elderly. The main characteristics of this disease are difficulties in household handling routine and cognitive and emotional disturbance in the elderly. The treatment of AD is a clinical challenge. With the development of cholinesterase inhibitors and a N-methyl-d-aspartate antagonist (memantine), good perspectives have emerged in controlling the symptoms of AD. Therapeutic decisions have to be guided by clinical studies and should consider the physiopathogenesis and epidemiology of the disease.\nThe main objective of these clinical trials is to reduce the Behavioral and Psychological Symptoms of Dementia (BPSD) and to improve cognition and the functional activity status, thus reducing the impairment of instrumental activities of the daily living (IADLs) and to lower the institutionalization rates (nursing home placement). Unfortunately, only a limited number of trials have dealt with this topic and with follow-up periods shorter than two years. In spite of the absence of sufficient therapeutic effectiveness in mild and moderate AD, these drugs are still considered as the first line of treatment for AD (1). Studies of cost-effectiveness suggest that memantine (2,3) and donepezil (4) are useful in the reduction of institutionalized care and\/or cognitive impairment in patients with AD. Recently, two clinical trials showed no improvement of the cognitive deficit (5,6) or reduction in the institutionalization rate (6).\nSearching for alternatives, many herbal products have been tested and employed in the treatment of AD, but with different clinical responses (7). The assessment of these drugs through randomized controlled trials should be useful to identify effective products in the treatment of AD.\nMethods\nSearching at MEDLINE (during April 2006, PubMed), LILACS (Latin American and Caribbean Health Science Literature: 40th edition, May 2001, the last research was performed in April 2006); Cochrane Library (issue 1, 2006); Dissertation Abstract (USA, during April 2006); ADEAR (Alzheimer's Disease Clinical Trials Database, until April 2006); National Research Register (1\/2006); Current Controlled trials (the last research was performed in October 2005); PsychINFO Journal Articles (during the year of 2006); relevant web sites; and scanning of reference list of relevant articles. There were no language or publication restrictions.\nSearch for keywords in MeSH (medical subject heading (MeSH) with the words \u2018Alzheimer disease, dementia, cognition disorders\u2019 was performed first. In the second part, the keywords were \u2018Herbal\u2019 and \u2018Phytotherapy\u2019. The crossover results of the two searches were evaluated by the Jadad's measurement scale (8).\nInclusion criteria: Three investigators independently reviewed all of the articles found. The articles were selected using the criteria listed below:\nThe studies should be randomized; double-blind and controlled (with a control group and a treatment group).\nStudies should establish methodological procedures in the crossover or be conducted at the same time.\nIn the case of being crossover, a washout period of at least 7 days was required.\nPatients included in the researches had their diagnosis rated into three degrees as follows: mild, moderate and severe forms of AD, according to the criteria from the National Institute of Neurological and Communicative Disorders and Stroke\u2014AD and Related Disorders Association (NINCDS-ADRDA) (9). The models used were as follows: Mini-mental beginning values between 10 and 26 (initial and mild group) or <10 (initial group).\nClinical trials should last for at least 1 month (4 weeks).\nDetailed description of the herbal product used.\nNeuropsychiatry symptoms progression should be measured with numerical score using the Assessment Scale (ADAS-noncog, range of score, 0\u201370), NPI (Neuropsychiatric Inventory, range of score 0\u2013120), Clinical Global impression of change or Behavioral rating scale for Geriatric patients.\nThe final score should be quantified using a combination of ADL and IADL methodological procedures.\nExclusion criteria: The herbal product has already been target of a quantified systematic review study. In this case, only the results of the studies will be considered.\nJadad's measurement scale: Methodological quality was assessed using a scale developed and validated by Jadad et al. (8). This scale assesses the completeness of reporting using three items with a five points maximum score. If the allocation into groups is explicitly randomized, item 1 is scored. A bonus point is given if an adequate method to generate the random sequence is described. If there is an explicit statement that the study is double-blind Item 2 is scored. A bonus point is given if the method is described and adequate. Item 3 is scored if there is either an explicit statement that all patients included were also analyzed or if the number and reasons for dropouts in all groups are given separately. For being classified as adequately reported a trial should score at least three of five points, a cut-off point is recommended by the author of the scale (10).\nAll extraction and quality assessments were performed by at least two independent reviewers using standard forms developed for each review. Disagreements were documented and discussed with final decisions made by the principal reviewer.\nResults\nTwo herbs and two herbal formulations were identified to have effectiveness in the treatment of cognitive disturbance of AD in the systematic review: Salvia officinalis (11), Melissa officinalis (12), and Yi-Gan San (13) and Ba Wei Di Huang Wan (BDW) (14). The main characteristics of the study are described in Table 1.\nGingko biloba was previously identified in one meta-analysis (15), and only the conclusions of the study will be considered. Another study will be conducted with huperzine A, a product derived from a Chinese herb Huperzia serrata, to evaluate the safety and efficacy in the treatment of AD in a multicenter randomized controlled trial of its effect on cognitive function (16).\nThe studies of Salvia (11), Melissa (12), Yi-Gan San (13) and BDW (14) have reached Jadad's measurement scale of \u2265 3. The researches had a follow up of 1 month (Yi-Gan San) (13), 2 months (BDW) (14) and 4 months (Salvia and Melissa) (11,12). All samples studied were composed of patients with initial mild symptoms judged as AD. Two studies compared herbal medicines and control samples, using intention to treat [Salvia (11) and Melissa (12)].\nNone of the studies evaluated the institutionalization rate or compared the active principle with the current therapies with Acetyl Cholinesterase Inhibitor or memantine.\nDiscussion\nThe results of this systematic review identified four studies with methodological quality assessing S. officinalis (11), M. officinalis (12), Yi-Gan San (13) and BDW (14). The last two are composed of formulations with different phytoactive agents. These herbs and formulations presented efficiency in reducing the mild and moderate symptoms of AD.\nGingko biloba presented statistically significant mild effectiveness in the treatment of cognitive deficit in AD. The meta-analysis study of Cochrane (13) concluded that additional controlled studies would be necessary in order to recognize cognitive improvement with the use of gingko. There is still need of a prospective study with an appropriate duration and representative sample to identify if G. biloba reduces the development of AD (17). Another plant with a large application perspective is H. serrata, after multicenter trial confirmation underway (16).\nMelissa and Yi-Gan San showed reduction in the cognitive deficits and a good sedative effect in patients with AD (12,13). Previous clinical studies showed that the extract of M. officinalis reduces laboratory-induced stress (18) and might have benefits in mood improvement (19\u201321). The use of these herbs and formulations should be well tolerated, (22) and adverse effects have not yet been reported (23). Further studies should be conducted to compare the current therapies for AD and the use of these herbal remedies in controlling the symptoms of AD.\nThe action mechanisms of these herbs and formulations are not well known. It has been suggested that the chemical composition of the essential oil of the Melissa and Salvia leaf extracts are monoterpene aldehydes, polyphenol flavonoids (including rosmarinic acid) (24) and monoterpene glycosides (25). All of these components have many observable effects in vitro, which include powerful anti-oxidative activity (26,27) and an affinity to nicotinic and muscarinic receptor in the human cerebral cortex (28). This last mechanism is of special interest, as modulation of cholinergic systems should play a role in improving the cognitive function, especially in AD.\nYi-Gan San and BDW are mixes of many herbal ingredients. Yi-Gan San is a mixture of 7 different dried plants, many of them (Unticariae sinensis and Angelicae root) with possible actions in the serotoninergic and gaba system. BDW consists of 8 herbs: Rehmannia glutinosa Libosh. var purpurea Makino, Cornus officinalis Sieb et Zucc (Cornaceae), Dioscorea batatas Decne root (Dioscoreaceae), Alisma orientale Juzep rhizome (Alimataccae), Poria cocos Wolf, Paeonia suffruticosa Andr. (Paeoniaceae), Cinnamomum cassia Blume (Lauraceae) and Aconitum carmichaeli Debx. (Ranunculaceae). Studies have suggested that BDW enhances the choline acetyltransferase activity and increases the acetylcholine content of the frontal cortex in a murine model (29,30).\nMajor methodological limitations of the four studies are small size of samples and short-term duration. There is no description of the chemical composition and\/or possible active principles of the different products employed in studies Involving Yi-Gan San (13) and BDW (14) formulation (Table 1). In the study with BDW (13), Sepia sp. and face powder were used as placebo; however, the way that the shellfish formulation was performed was not described. In the control group of the study using the Yi-Gan-San (14) formulation, 25 mg per day of tiapride hydrochloride were introduced. This drug is a substituted derivative with selective dopamine D2-receptor antagonist properties. This intervention (contamination) occurred in 44% of the individuals and was responsible for the symptoms, among them dizziness.\nGenerally, crude herbal drugs are natural products and their chemical composition depends on several factors such as geographic source of the plant material, climate in which it was grown, and time of harvest. Commercially available herbal medicinal products also vary in their content and concentration of chemical constituents from batch to batch and when products containing the same herbal ingredient are compared between manufacturers. Even when herbal products are standardized for content of known active or marker compounds to achieve more consistent pharmaceutical quality, variations in the concentrations of other constituents can be observed. The use of a protocol such as the Consolidated Standards of Reporting Trials (CONSORT), composed of 22 items, will probably minimize the limitations of RTC with phytotherapic agents (31).\nThe use of herbal medicines in the treatment of AD should be compared with the pharmacological treatment currently in use. Such studies should include the identification of the active principle in order to improve the validation of the clinical trial. Further large-scale, multicenter studies are necessary to determine the effectiveness of these substances in the cognitive deterioration of AD. Until then, this review provides some evidence of the benefit of Melissa, Salvia, Yi-Gan San and BDW in the treatment of AD.","keyphrases":["herbs","alzheimer's disease","systematic review","dementia","elderly","cognitive impairment","randomized clinical trial"],"prmu":["P","P","P","P","P","P","R"]}
{"id":"Bioinformation-1-10-1896055","title":"Integrative analysis of the mouse embryonic transcriptome\n","text":"Monitoring global gene expression provides insight into how genes and regulatory signals work together to guide embryo development. The fields of developmental biology and teratology are now confronted with the need for automated access to a reference library of gene-expression signatures that benchmark programmed (genetic) and adaptive (environmental) regulation of the embryonic transcriptome. Such a library must be constructed from highly-distributed microarray data. Birth Defects Systems Manager (BDSM), an open access knowledge management system, provides custom software to mine public microarray data focused on developmental health and disease. The present study describes tools for seamless data integration in the BDSM library (MetaSample, MetaChip, CIAeasy) using the QueryBDSM module. A field test of the prototype was run using published microarray data series derived from a variety of laboratories, experiments, microarray platforms, organ systems, and developmental stages. The datasets focused on several developing systems in the mouse embryo, including preimplantation stages, heart and nerve development, testis and ovary development, and craniofacial development. Using BDSM data integration tools, a gene-expression signature for 346 genes was resolved that accurately classified samples by organ system and developmental sequence. The module builds a potential for the BDSM approach to decipher a large number developmental processes through comparative bioinformatics analysis of embryological systems at-risk for specific defects, using multiple scenarios to define the range of probabilities leading from molecular phenotype to clinical phenotype. We conclude that an integrative analysis of global gene-expression of the developing embryo can form the foundation for constructing a reference library of signaling pathways and networks for normal and abnormal regulation of the embryonic transcriptome. These tools are available free of charge from the web-site http:\/\/systemsanalysis.louisville.edu requiring only a short registration process.\nBackground\nAnimal development is fashioned by conserved signaling pathways that orchestrate morphogenesis, pattern formation, and cell differentiation - complex processes operating jointly in\ndifferent parts of an embryo and in stages associated with sequential gene activation. Monitoring local and temporal changes in gene expression can provide insight into how genes\nand regulatory signals work together to guide development. [1] This knowledge is important for understanding the pathogenesis\nof birth defects and to the central problems of defining precursor target cell susceptibility and the causal mechanisms of abnormal development triggered by diverse environmental and genetic\nperturbations to maternal-fetal unit. [2]\nProfiling gene expression on a global scale has become an important source of information for biological knowledge discovery. Despite well-known challenges confronting\ntechnology development, the analysis of global gene expression data can reveal themes in the biologically robust response patterns in gene activity. [3] Gene Expression Omnibus (GEO)\nis one of the main national repositories for high-information content transcript data from microarray analysis and serial analysis of gene expression (SAGE). [4] GEO has grown from\n18,235 records in June 2004 to 115,415 records in December 2006, reflecting an average growth of over 100 new entries per day. A subset of this information describes the embryo proper\nand can be mined for major biological themes in developmental health and disease. Using keyword searches and trend analysis to mine PubMed and Medline literature databases, the public\navailability of embryo-based microarrays currently numbers 500-600 (564), mostly studies on mouse embryos and differentiating human cell lines.\nThe increasing volume of gene expression data on local and temporal states confronts the developmental biologist with the need for reference libraries and information management\nsystems to handle optimal-scale gene-expression signatures and facilitate biological knowledge discovery. For example, Lamb et al., [5] created a prototype reference collection of geneexpression\nsignatures from cultured human cells exposed to bioactive molecules, which serves as a platform for pattern-matching software to establish a \u2018connectivity map\u2019 between\ndrugs, genes, and diseases. Another example is the integrative analysis of multi-study tumor profiles. [6,7]\nThe emerging database model for tumor classification based on molecular abundance profiles has implied a 67-gene core \u2018common transcriptional program\u2019 in multiple cancers. [8] In developmentteratogenesis,\na preliminary meta-analysis across microarray studies in the mouse embryo returned a gene-expression signature of 512 developmentally regulated genes of which 16%\n(~82-genes) changed during exposure to teratogenic agents. [2] Given the promises and pitfalls of computational methods for\nsolving gene expression problems, automated access to a reference collection of gene-expression signatures to benchmark the programmed (genetic) and adaptive (environmental)\nregulation of the embryonic transcriptome is scientifically needed.\n\u2018Birth Defects Systems Manager\u2019 (BDSM) is a knowledge management system that provides custom software to mine public microarray data for interesting patterns across\ndevelopmental stages, organ systems, and disease phenotypes. [2,9]\nThis open resource enables: consolidation of communal data and metadata relevant to developmental health and disease; interactions with current builds of national databases and data\nrepositories; efficient algorithms for cross-species annotation of symbolic gene annotations using the NCBI sequence homologybased annotations for corresponding homologues and\northologues; specific queries across experiments to facilitate secondary analysis; and data formats interoperable with analysis software for phenetic clustering, chromosomal mapping, gene\nontology classification, pathway evaluation, and network identification.\nSince a comprehensive reference collection of gene-expression signatures for developing structures must be constructed from highly-distributed data, the present study was designed to\nempower BDSM with tools for seamless data integration: MetaSample, MetaChip, and CIAeasy. These tools are accessible\nat http:\/\/systemsanalysis.louisville.edu requiring only a short\nregistration process to BDSM. A field test of the prototype run with published microarray data illustrates proof of concept for integrative analysis of the mouse embryonic transcriptome.\nMethodology\nDataset collections\nSearch of PubMed using keywords \u2018embryo\u2019 and \u2018microarray\u2019 returned 495 records of which 193 actually used the technology to study developing animal systems. GEO data sets (GDS) narrowed the list to 47 nonredundant\nmicroarray datasets, and including the keyword \u2018teratogen\u2019 added a few more datasets, for a total of 564 public microarrays on the embryo. Raw and\/or processed microarray\nsample-data files and associated metadata were parsed onto the server using LoadBDSM. [9] BDSM currently holds 25\ndevelopmental series containing 537 samples that are derived from the public domain and 3 series containing 43 samples which are private. These data represent 15 developing organ\nsystems, 6 chemical exposures, and 5 drug interventions across 42 development stages.\nTracking provenance\nFor the present study, we restricted the analysis to 160 arrays in the BDSM library based on wellannotated experiments published on normal mouse\nembryogenesis using an Affymetrix technology platform. These conditions are identified by GEO Series Accession number (GSE) and\/or literature citation as follows: preimplantation\nmouse embryo (GSE1749)  [10]; heart (GSE1479) GD10 - GD18 [11]; nerve (GSE972) GD9.5 - birth  [12]; \novary (GSE1359) and testis (GSE1358) between GD11.5 - birth  [13]; orofacial region\n(GSE1624)  [14] and secondary palate  [15] between GD13-15.\nThe platforms for these series included: MG-U74Av2 (12488 probes), MG-U74Bv2 (12478 probes), MG430Av2 (45104 probes), MOE430Av2 (22690 probes), and MOE430Bv2 (22576\nprobes). Internal annotation of Affymetrix probe identifiers was performed to standardize gene labels across samples and improve cross-platform interoperability, as discussed previously. \n[2]\nData integration\nIndividual microarray sample-data files from the aforementioned developmental series were integrated using QueryBDSM, a module that for merging pre-processed,\nnormalized samples from the BDSM library. Three metaanalysis tools written in PHP were designed to compare and analyze expression data across multiple chips and platforms:\nMetaSample, MetaChip, and CIAeasy. The workflow schema is diagrammed in Figure 1. Individual sample-data files are\nselected from the BDSM library and added to a queue for integration. The formatted input files are tab-delimited expression ratios of probes (rows) x samples (columns).\nQueryBDSM determines the number of distinct microarray platforms in the sample queue and merges the data as follows: if all samples come from the same microarray platform (number\n=1), then QueryBDSM automatically runs MetaSample to create a merged-table having \u2018columns\u2019 of normalized expression data (samples) in \u2018rows\u2019 derived from the platform, with unique probe\nidentifiers (ProbeID) expanded to include GenBank accession (GeneID), and symbolic gene name (Gene Symbol). If multiple platforms are represented by samples in the queue (number >1),\nthen QueryBDSM automatically runs MetaChip. MetaChip merges data when probe identifiers are different but represent the same annotation, such as across microarray platforms or\nphylogenetic species. The probe identifiers from each platform are converted to UniGene ID and then merged accordingly, with associated expression data, for those genes common across the\ndatasets. The probes are annotated based on reverse-engineering the sequence homology-based annotations from GenBank. [2]\nFor this purpose the system uses data flat files downloaded monthly from the HomoloGene and UniGene databases of NCBI. [16]\nIn contrast to MetaSample and MetaChip, automated tools for merging samples, CIAeasy must be specified explicitly to\ncompare datasets for the same samples on different platforms. CIAeasy was created from the ADE-4 for R statistical\ncomputing software package [17] and is adapted from \u2018coinertia analysis\u2019 (CIA) for microarrays. [18] With CIAeasy\nusers can perform CIA from the BDSM web site without detailed knowledge of R language programming. Since samples are aligned on a common space, CIA extracts\ninformation about the joint trends in expression patterns of genes independent of probe or sequence annotation. [18]\nCIAeasy automatically computes successive orthogonal axes with correspondence analysis and returns the percentage of total variance explained by each eigenvector to find the\nstrongest trends in the co-structured datasets.\nData analysis\nOne of the problems confronting meta-analysis is the normalcy and spread of expression data. In order to derive expression ratios from the Affymetrix data, we computed a\nreference denominator, averaged for each gene at the earliest stage in a developmental series. These ratios were transformed to logarithm base 2 (log2) to produce a continuous spectrum of\nvalues without biasing between up- and down-regulated genes and making the spread more normal. Each microarray set was centered to median of 0.00 and standardized by scaling to an\naverage standard deviation of 0.5. [2] The merged data were imported to GeneSpring v 7.2 using the UniGene cluster ID as\nthe unique gene identifier. The data were clustered using Pearson correlation for the gene tree and two-sided Spearman Confidence for the developmental conditions. Functional\nannotation used the NIH\/NIAID Database for Annotation, Visualization and Integrated Discovery (DAVID). [19] The\nhighest-ranking biological themes were stratified by Gene Ontology (GO) terms.\nDiscussion\nImplementation of QueryBDSM\nFor proof of concept we examined samples from GEO data source GSE1391, a series describing global gene-expression profiles of the mouse embryo\nduring preimplantation stages.  [10] Samples included in this series represent development of the oocyte through fertilization\n(1-cell embryo), activation of the zygotic genome (2-cell embryo) and first differentiation (8-cell embryo) leading to divergent embryonal (inner cell mass) and trophectodermal\n(placental) lineages of the blastocyst. Biological replicates (3-4) arrayed at each stage used three different Affymetrix platforms: MOE430Av2 (22690 probes), MOE430Bv2 (22576 probes), and\nMG-U74Av2 (12488 probes). Gene-expression profiles were normalized to the \u2018oocyte\u2019 in each platform as the earliest stage in the series. Derived data are log2-scale expression values\ncomputed from the ratio of signals to the oocyte reference.\nUsing QueryBDSM, we merged datasets from the different samples to create three distinct datasets for the MOE430Av2, MOE430Bv2, and MG-U74Av2 platforms. Statistical\n(ANOVA) analysis, run at high stringency with Benjamini-Hochberg correction applied, returned 4417 probes (alpha = 0.0001), 1614 probes (alpha = 0.0001), and 2400 probes (alpha\n= 0.001) that were differentially regulated. Aside from 34 probes that overlapped between the first two datasets, different genes were detected across these diverse platforms. Hierarchical\nclustering revealed two basic trajectories of gene-expression in all three platforms (Figure 2). One expression cluster contained genes that increased at the 2-cell stage to the blastocyst stage,\nand the other cluster contained genes that decreased over these stages (not shown). These probes were mapped to the 307 reference pathways in the KEGG: Kyoto Encyclopedia of Genes\nand Genomes (http:\/\/www.genome.ad.jp\/kegg\/) library to\nidentify metabolic themes. The top significant KEGG pathways showed concordance between platforms MOE430Av2 and MGU74Av2 (Table 1), whereas MOE430Bv2 detected different\npathways. Some pathways had marginal P-values with individual analysis that became significant in datasets joined by MetaChip (e.g., Adherens Junction, Tight Junction pathways).\nThis illustrates the strength of the meta-analysis approach.\nWe next used QueryBDSM to merge samples from platforms MOE430Av2 and MG-U74Av2 to illustrate the MetaChip and CIAeasy tools. These chips contained 13022 and 9562 nonredundant\nUniGene cluster identifiers, respectively, of which 7278 were common between the two platforms when passed through MetaChip. Statistical (ANOVA) analysis with the same\nparameters as before returned 3324 genes that were differentially regulated in this data subset. The top significant KEGG pathways for the combined 430A-U74A MetaChip are\nconcordant with the individual analysis (Table 1). CIAeasy was used to compare the joint trend between 4417, 1614, and 2400 probes from MetaSample, resulting in a high-level of similarity\nbetween these three platforms (Figure 2). Of the 6812 nonredundant probes between MOE430Av2 and MG-U74Av2 (4417 + 2400), only five probes were common. Annotating the probes which were not in common gave 4551 unique DAVID\nidentifiers. Again, meta-analysis picked up most of the significant KEGG pathways identified in either of the singular analyses performed earlier and a few additional metabolic pathways (Table 1).\nComparative bioinformatics analysis across developing systems\nAn obvious limitation in combining data from several different platforms is that as more platforms are included, fewer representative genes are found to be common amongst all\nplatforms. This problem increases when considering less comprehensive arrays, older arrays with outdated probe annotations, or arrays across animal species. Staging the entry of\ndata from the most versatile arrays first can lessen the problem of losing information when data are combined across platforms; however, in some data mining efforts the discriminating power\ngained by increasing samples and conditions might outweigh the loss of information. For example, multi-platform datasets have been found to discriminate tumor classification by expression\nprofile with as few as 25 genes. [6] For this reason it may be possible to benchmark developmental stages using a limited\nnumber of genes across many diverse platforms. To illustrate this point we used BDSM-derived data to compare expression profiles across six unrelated studies and developing systems. We\nconstructed a virtual meta-chip for probes common to all five technology platforms represented in these studies, yielding 346 genes. Unsupervised clustering and Pearson correlation of the\ngene-expression profiles correctly ordered the samples first by organ system and then by developmental sequence within each system (Figure 3). Within this hierarchy commonalities and\ndifferences across organ systems were evident for the patterns of expression in subsets of genes. Unfortunately, the number of genes returned from 346 by statistical (ANOVA) analysis of\neach individual system, or by K-means clustering of the entire matrix, was too small for insightful functional annotation. QueryBDSM is a simple and efficient solution that can be used\nto construct a self-evolving reference collection of geneexpression profiles from highly-distributed data on mouse development. Although robust mapping of biological themes and pathways that are expressed at particular developmental\nstages is straightforward when the same technology platform is considered  [2] fuzzy-clustering methods will be needed when multiple platforms are considered.\nThe MetaSample and MetaChip components of QueryBDSM are also available as standalone tools under the MetaBDSM module. In this way, the user can upload data outside the BDSM library.\nThe user supplies details of the technology platform, organism, and information about the file format. Once all the required fields are entered and submitted, the files are checked for unique\nheaders. Columns can be dropped by selecting the appropriate checkboxes. Clicking the Continue button combines the datasets with expression data and unique identifiers only for genes\ncommon between the datasets. These tools have been tested on Internet Explorer 6.0 or greater and Mozilla-based browsers, such as Netscape 6.0 and Firefox.\nOther tools are available to assemble data when the platform is same, such as Microarray data assembler. [20] This Excel-based\nprogram inherits Excel's limitation from file sizes and number of samples (256 columns and 65,000 data points) whereas the MetaSample and MetaChip tools do not have this limitation.\nThese tools create temporary tables in the Oracle database and join them using the functionality of Oracle before putting it into a text file, reducing restrictions on the number of samples and\nsize of files. Although users can theoretically combine 100 files at a single time, it is not recommended to load more then 25 files at a time.\nConclusion\nThe representation of experimental samples as developmentally contiguous groups is expected to yield a novel mosaic view of gene-expression signatures and genetic dependencies. Although\nsufficient data exists for data-mining efforts to begin, the ultimate goal of an unabridged reference collection must be viewed as a long-term effort. Regarding the embryo, a search of\nOVID MedLine using keywords \u2018embryo\u2019 OR \u2018teratogen\u2019 (136,146 records) AND \u2018microarray\u2019 OR \u2018SAGE\u2019 (16,906 records) returned 343 total records. At the current rate of 564\nmicroarrays per 343 publication records (factor = 1.64), the trajectory of embryo-based microarray publications projects GEO to hold in excess of 1,476 microarrays relevant for\nembryogenesis or teratogenesis by the year 2010.\nAs studies unravel gene-expression signatures, the key principles in teratology \u2013 namely, chemical effects on biological mechanisms, dose-response relationships, factors underlying\ngenetic susceptibility, stage-dependent responses, and maternal influences, can be framed in a systems biology context to address an \u2018experience database\u2019 for ranking pathways and\nnetworks by strength of association with anatomical landmarks and developmental abnormalities.  [2] The BDSM resource\nwould parallel efforts toward molecular diagnostics in cancer biology (http:\/\/www.oncomine.org\/), which includes data sets\nprofiling human tumor samples. [21] Since interpreting geneexpression signatures in birth defects will be predicated on\nposterior (prior) knowledge about developmental health and disease, an important payoff from this bioinformatics effort is to recognize and characterize how these biological states emerge\nfrom adaptation or adverse regulation of the embryonic transcriptome.","keyphrases":["integrative analysis","mouse","embryo","transcriptome","expression","birth defects"],"prmu":["P","P","P","P","P","P"]}
{"id":"Pediatr_Nephrol-3-1-1989763","title":"Nutrition in children with CRF and on dialysis\n","text":"The objectives of this study are: (1) to understand the importance of nutrition in normal growth; (2) to review the methods of assessing nutritional status; (3) to review the dietary requirements of normal children throughout childhood, including protein, energy, vitamins and minerals; (4) to review recommendations for the nutritional requirements of children with chronic renal failure (CRF) and on dialysis; (5) to review reports of spontaneous nutritional intake in children with CRF and on dialysis; (6) to review the epidemiology of nutritional disturbances in renal disease, including height, weight and body composition; (7) to review the pathological mechanisms underlying poor appetite, abnormal metabolic rate and endocrine disturbances in renal disease; (8) to review the evidence for the benefit of dietetic input, dietary supplementation, nasogastric and gastrostomy feeds and intradialytic nutrition; (9) to review the effect of dialysis adequacy on nutrition; (10) to review the effect of nutrition on outcome.\nThe importance of nutrition in normal growth\nNormal growth can be divided into four important phases: prenatal, infantile, childhood and pubertal. Nutrition is important at all phases of growth, but particularly so during the infantile phase because the rate of growth is higher than at any other time of life (other than prenatally) and is less dependent on growth hormone (GH) than during other phases. Rate of growth gradually decreases from >25\u00a0cm\/year at birth to an average of 18\u00a0cm\/year at age 1 year and 10\u00a0cm\/year by the age of 2. Half of adult height is achieved by the age of 2 years, so that irrecoverable loss of growth potential can occur during this phase. At birth, 170\u00a0kcal\/day are stored in new tissue, falling to 50\u201360 at 6 months, 30\u201340 by 1 year and 20\u201330 by the age of 2 years. During the childhood phase, growth becomes more dependent on the GH\/insulin-like growth factor-1 (IGF-1) axis; growth rate decelerates continuously until the pubertal phase. The pubertal phase results from the coordination of GH and sex steroid production. Together they have an anabolic effect on muscle mass, bone mineralization and body proportions. It is another phase of rapid growth so that nutrition can again modify the genetic growth potential [1].\nMethods of assessing nutritional status\nNormal nutrition can be defined as maintenance of normal growth and body composition. Although it is agreed that nutritional assessment is important in chronic renal failure (CRF), there is no single or easy definition or measure of inadequate nutritional status: measurement of nutritional parameters are complicated in CRF because of salt and water imbalances and the potential inappropriateness of using age matched controls in a population that is short and may be delayed in puberty; it has been suggested that it is more appropriate, therefore, to express measures relative to height age (age at which the child\u2019s height would be the 50th centile) and\/or pubertal stage. The reader is referred to extensive and excellent reviews on this subject [2, 3].\nAnthropometric measures\nThe most commonly used assessment of nutrition is height and weight, along with head circumference in younger children, plotted on percentile charts. Anthropometric and nutritional measures are usefully expressed as a score of the number of standard deviations (SDs) from the mean for a normal population of the same age [e.g. height or weight SD score (HtSDS, WtSDS), also called z scores]. This allows comparison with the normal population and helps follow progress in the individual patient. However, although a normal rate of growth can be considered to represent adequate nutrition, weight loss and alterations in body composition occur before height velocity is affected [2], and poor growth can occur due to reasons other than nutrition (see below).\nAnother way of expressing the relative weight and height is the body mass index (BMI, Ht\/Wt2), which is important because extremes are associated with increased morbidity and mortality [4]. Because BMI varies considerably throughout childhood, reaching a trough at 4\u20136 years of age, it has been suggested that it should be calculated according to height age [5]. It must be borne in mind that BMI does not distinguish between fat mass and fat-free mass (FFM) and an appropriate BMI for age (whether height age or chronological age) does not necessarily indicate ideal body composition; weight gain may be due to the laying down of excess fat rather than a balance of fat and lean tissue.\nSkinfold thickness is a measure of subcutaneous fat and mid-arm circumference (MAC) is a reflection of muscle mass and may therefore be more useful in determining body composition than the calculation of BMI alone. Decreased values have been found in children with CRF [6\u20139]. However, both are rather unreliable tools because consistent measurements are difficult, values may not be representative of visceral fat and fat or muscle mass, oedema will influence values, regional fat and muscle distribution may be different in CRF and values vary according to age in normal children.\nDietary assessment\nThe paediatric renal dietician is crucial to the successful management of nutrition in children with renal disease. Monthly review has been recommended for under twos on dialysis, and three- to four-monthly in those over that age [10]; and six-monthly or one- to three monthly in children with moderate and severe CRF, respectively [3]. The purpose is to prevent the development of malnutrition. Children entirely dependent on enteral feeds may need to be seen more often, particularly in infancy when feed adjustments may be necessary as often as weekly. Assessment of intake in children taking an oral diet can depend on prospective dietary diaries, usually over 3 days, or retrospective recall. It has been estimated that 5.9 contacts per patient (in clinic or by phone) per month in children <5 years of age and 3.1 in children >5 years of age are necessary to successfully support families of children on peritoneal dialysis (PD). This intensive input resulted in improvement of HtSDS and WtSDS from \u22121.2 and \u22121.32 to \u22121.14 and \u22120.73, respectively, over a 3-year period [11]. Protein intake can also be calculated using well established formulae (protein catabolic rate, nPCR [2]).\nSerum albumin\nSerum albumin has been identified as a surrogate marker for nutritional status and morbidity\/mortality in patients with end-stage renal failure (ESRF). Patients <18 years of age initiating dialysis with hypoalbuminemia are at a higher risk for death: in 1,723 children each fall of serum albumin by 1\u00a0g\/dl at the start of dialysis was associated with a 54% higher risk of death. This was independent of other potential confounding variables [12].\nAlthough serum albumin may be a reflection of nutrition, low levels may be due to haemodilution, nephrotic syndrome or chronic infection\/inflammation [13]. To remove the effect of fluid overload, therefore, it has been suggested that if practical, levels should be checked post dialysis [14]. Chronic inflammation in itself will lead to malnutrition [15]. Low serum albumin is more common in children on PD: low levels were present in 35.9% of assays in 39 children on PD over a 2-year period compared with none in 32 children on haemodialysis (HD), even though protein intake (estimated by nPCR) was similar, averaging 1.1\u00a0g\/kg per day. Thus, children maintained on PD are at greater risk of protein malnutrition compared with peers treated with HD [16]. This may be due in part to losses in PD fluid: average losses of free amino acids (AA) vary with transporter status from 0.02 to 0.03\u00a0g\/kg\/day [17]. There is an inverse correlation between body weight and surface area and peritoneal protein loss, such that infants have nearly twofold greater peritoneal protein losses per metre-square body surface area than those weighing more than 50\u00a0kg. Such protein losses in infants impair normal growth and may contribute to permanent loss of growth potential [18].\nDual energy X-ray absorptiometry (DEXA) and other methods\nA whole body DEXA scan can estimate fat mass, lean mass and bone mineral density (BMD), but is affected by body water content. Other methods include bioelectrical impedance analysis (BIA) [19], total body potassium, densitometry and in vivo neutron activation analysis, but these are predominantly used as research tools [2].\nNutritional requirements for normal children\nRecommended daily amounts (RDA) and recommended intakes (RI) for energy, protein and nutrients vary between countries. Regardless of the national dietary recommendations that are used it is important to consider that these are estimates of requirements for normal healthy populations of people and are not recommendations for absolute intakes for individuals. They serve as a guide for the energy and nutrients that an individual may require for normal growth, maintenance, development and activity. Requirements for any particular nutrient will differ between individuals.\nThe United Kingdom dietary reference values (DRV) [20] are for infants fed artificial formulas and for older infants, children and adults consuming food. DRVs are not set for breast-fed babies, as it is considered that human milk provides the necessary amounts of nutrients. In some cases the DRVs for infants aged up to 3 months who are formula-fed are in excess of those which would be expected to derive from breast milk; this is because of the different bioavailability of some nutrients from breast and artificial milk.\nThe DRV for energy intake is assumed to be normally distributed and is expressed as the estimated average requirement (EAR). For protein and other nutrients, requirements are expressed as reference nutrient intakes (RNI), set at 2 SDs above the average. Therefore, intakes of protein and nutrients above this amount will almost certainly be adequate for all individuals in a population. For some nutrients where there is insufficient data to establish DRVs with great confidence safe intakes are set\u2014a level or range of intake at which there is no risk of deficiency.\nDaily DRVs for energy, protein and some nutrients are given in Table\u00a01. If a diet for a normal child is adequate in calcium, iron and vitamin C and the child is receiving adequate energy from a mixed diet, then most other nutrients are likely to be taken in adequate amounts. \nTable\u00a01UK dietary reference values for normal populations of children [20] (mo months, yr years)AgeEARRNIRNIRNIRNIEnergy (kcal)Protein (g)Calcium (mmol)Iron (mg)Vitamin C (mg)0\u20133 mo115\u2013100\/kg2.1\/kg13.11.7254\u20136 mo95\/kg1.6\/kg13.14.3257\u20139 mo95\/kg1.5\/kg13.17.82510\u201312 mo95\/kg1.5\/kg13.17.8251\u20133 yr95\/kg1.1\/kg8.86.9304\u20136 yr90\/kg1.1\/kg11.36.1307\u201310 yr1,970\/day28.3\/day13.88.730Boy 11\u201314 yr2,220\/day42.1\/day25.011.335Girl 11\u201314 yr1,845\/day41.2\/day20.014.835Boy 15\u201318 yr2,755\/day55.2\/day25.011.340Girl 15\u201318 yr2,110\/day45.0\/day20.014.840\nNutritional requirements for children with CRF, on dialysis and post transplant\nEnergy\nIt is unlikely that energy requirements for children with CRF are different from those of normal children, and energy intakes below the EAR will contribute to growth failure. Restoration of normal energy requirements to 100% EAR allows for catch-up growth in children under 2 years and shows some benefit in older children (see section on Dietary supplements). If recurrent vomiting resulting from abnormal gastric motility and delayed gastric emptying is not treated, energy intake may need to be increased by up to 30% daily to replace lost feed and food in order to preserve growth; once vomiting is controlled growth is maintained on normal energy requirements [21]. To ensure an adequate energy intake it is advisable to use the EAR for height age if the child is below the 2nd centile for height.\nThere is no evidence that intakes for children on dialysis should exceed those for normal children [10], though dietary energy intake may need to be reduced for children on PD to compensate for the energy derived from dialysate glucose, estimated at 8\u201312\u00a0kcal\/kg\/day if there is excessive weight gain [7, 22].\nPost-transplant energy requirements should match those of normal children, though care needs to be taken with increased appetite in response to steroid administration. Up to 13% of transplanted chidren become obese [23, 24]. A reduction in energy intake is indicated where there is excessive weight gain and will help correct any dyslipidaemia.\nProtein\nProtein intakes in CRF must provide at least 100% RNI if protein is not to become the limiting factor for growth. Inadequate protein will impact on body composition with a preponderance of fat rather than lean tissue. Adequate energy must be given to promote deposition of protein [25]. To ensure an adequate protein intake, it is advisable to use the RNI for height age if the child is below the 2nd centile for height.\nIn children undergoing PD, protein intake must provide at least 100% RNI plus an allowance for both replacement of transperitoneal losses and replacement of daily nitrogen losses in order to achieve positive nitrogen balance [7, 18, 22]. There are few studies describing the optimal amount of protein for children on PD and the existing data does not include all age groups. Taking this into account, and the wide variability in transperitoneal losses of protein, recommended intakes for protein for populations of children on PD may be considered generous. Routine assessment of growth, albumin and urea levels will determine the required intake for the individual child. For chronic haemodialysis, the Kidney Disease Outcomes Quality Initiative (K\/DOQI) recommends the RDA for age plus an increment of 0.4\u00a0g\/kg\/day to achieve positive nitrogen balance [10]. This recommendation is based on work done in adults on HD who failed to maintain nitrogen balance on 1.1\u00a0g protein\/kg\/day [26]. Published recommended intakes for the US and the UK shown in Tables\u00a02 and 3 are for populations and may need adjustment for the individual. \nTable\u00a02US recommended dietary protein for children on maintenance dialysis [10]\u00a0Age (yr)RDA (g\/kg\/day)Protein intake for HD (g\/kg\/day)Protein intake for PD (g\/kg\/day)Infants0\u20130.52.22.62.9\u20133.00.6\u20131.01.62.02.3\u20132.4Children1\u201361.21.61.9\u20132.07\u2013101.01.41.7\u20131.811\u2013141.01.41.7\u20131.8Males15\u2013180.91.31.4\u20131.5Females15\u2013180.81.21.4\u20131.5Table\u00a03UK guidelines on dietary protein for children on maintenance dialysis [27]\u00a0AgeRNI (g\/kg\/day)Protein intake for HD (g\/kg\/day)Protein intake for PD (g\/kg\/day)Infants0\u20133 mo2.12.52.8\u20132.94\u201312 mo1.5\u20131.61.92.2\u20132.3Children1\u20133 yr1.11.51.8\u20131.94 yr\u2013puberty1.01.4\u20131.51.7\u20131.9Pubertal1.01.3\u20131.41.6\u20131.8Post\u2013pubertal0.91.2\u20131.31.4\u20131.5\nPost-transplant protein requirements should match those of normal children.\nVitamins and minerals\nLittle is known about the requirements of children with CRF. It would be reasonable to give the RNI for vitamins, minerals and micronutrients as for normal children, with the exception of calcium, phosphate, magnesium, sodium and potassium, which may be deranged and must be determined for the individual child. UK RNIs are shown in Table\u00a04. \nTable\u00a04Micronutrient guidelines for children with CRF [28]\u00a0InfantsChildrenThiamin (mg)0.2\u20130.30.5\u20131.0Riboflavin (mg)0.40.6\u20131.3Niacin (mg)3.88\u201318Vitamin B6 (mg)0.2\u20130.70.7\u20132.0Vitamin B12 (\u03bcg)0.3\u20130.50.5\u20131.5Folic acid (\u03bcg)*50\u201350070\u20131000Vitamin C (mg)2525\u201340Vitamin A (\u03bcg)*350350\u2013700Vitamin D (\u03bcg)*7\u20138.5\u2013Zinc (mg)4.0\u20135.05.0\u20139.5Copper (\u03bcg)0.2\u20130.30.3\u20131.0\n*Vitamin A - care should be taken not to give excessive amounts of vitamin A, as the resultant high serum levels can lead to hypercalcaemia, anaemia and hyperlipidaemia [29]. A common practice guideline is not to exceed 200% of the RNI from the diet and\/or supplements.\n*Vitamin D - there is no need to achieve the RNI for vitamin D, as it cannot be converted to the activated form (as which it is usually supplemented). Recent recommendations for adults are to assess 25 and 1, 25-vitamin D levels and replace as needed, but there are no equivalent recommendations for children.\n*Folic acid - hyperhomocysteinaemia is an independent risk factor for cardiovascular disease [30, 31]. Additional folic acid may be given to effectively lower plasma homocysteine levels. A common practice guideline is to supplement when the glomerular filtration rate (GFR) is <40\u00a0ml\/min\/1.73\u00a0m2, but the doses used are arbitrary.\nInfants: 250\u00a0\u03bcg\/kg to maximum of 2.5\u00a0mg daily\nChildren 1\u20135\u00a0years: 2.5\u00a0mg daily\nChildren >5\u00a0years: 5\u00a0mg daily\nIn adults on PD, blood concentrations of some water soluble vitamins (C, B6 and folic acid) are reported to be low. This is due to a combination of inadequate intake, increased transperitoneal losses and increased needs. In children supplements of these vitamins have been given with the result that blood concentrations have met or exceeded normal values [32, 33]. Accordingly, based on these authors\u2019 recommendations and the RNIs above, the following intakes are suggested [27]:\nVitamin C: 15\u00a0mg (infants)-60\u00a0mg (children) daily\nVitamin B6: 0.2\u00a0mg (infants)-1.5\u00a0mg (children) daily\nFolate: 60\u00a0\u03bcg (infants)-400\u00a0\u03bcg (children) daily\nThese amounts may well be met from food, feeds and nutritional supplements so it is important to assess the dietary contribution before routinely giving medicinal supplements. Whilst adequate vitamin C needs to be given to offset dialysate losses excessive intakes should be avoided, as the resulting elevated oxalate levels may lead to cardiovascular complications [34]. If folic acid is given to lower plasma homocysteine this will more than compensate for dialysate losses of folate. The K\/DOQI recommends that supplementation should be considered if dietary intake alone does not meet or exceed the dietary reference intake, if blood vitamin levels are below normal values, or if there is clinical evidence of deficiency [10].\nThere are no reported specific micronutrient requirements for children on HD and post transplant and 100% of the RNIs can be considered the goal for these children.\nCalcium and phosphate\nControl of phosphate, calcium and parathyroid hormone (PTH) levels is necessary to prevent renal bone disease. Dietary phosphate may need to be restricted when the GFR falls below the normal range, and almost always when below 50\u00a0ml\/min\/1.73\u00a0m2. The following common practice guidelines will help maintain serum phosphate within acceptable reference ranges, although phosphate binders may also be necessary.\nInfants <10\u00a0kg: <400\u00a0mg daily\nChildren 10\u201320\u00a0kg: <600\u00a0mg daily\nChildren 20\u201340\u00a0kg: <800\u00a0mg daily\nChildren >40\u00a0kg: <1,000\u00a0mg daily\nIron, copper and zinc\nAnaemia can be prevented by the prescription of iron supplements and erythropoietin (rhuEPO). Advice to increase dietary haem iron and reduce the inhibition of non-haem iron absorption by: phytates in wholegrains and legumes; polyphenols in tea, coffee and cocoa; calcium in dairy products; and simultaneous administration of phosphate binders and antacids should be given. It may be necesary to give intravenous iron if stores are low. Low dietary intakes of copper and zinc are reported [35] in children on PD. The K\/DOQI recommends the intake of these to be monitored every 4\u20136 months and supplements given if necessary [10].\nReports of spontaneous nutritional intake in children with CRF\nSeveral studies have demonstrated decreased spontaneous intake in children with CRF. Four-day weighed dietary records from 50 children with a GFR <65\u00a0ml\/min\/1.73\u00a0m2 and 93 healthy children showed an energy intake 76\u201388% of the RI in CRF patients and 90%\u201393% in controls. Protein intake was 2.1\u20133.1\u00a0g\/kg per day in controls and 1.6\u20132.7\u00a0g\/kg per day in CRF patients, so that overall, the energy intake was 10% and the protein intake 33% lower in CRF patients than in healthy children [36]. Other studies have shown low energy intake (87% RNI) but high protein\/energy ratio, protein (223%), carbohydrate (73%), fat (110%), polyunsaturated (55%), monounsaturated (129%) and saturated fatty acid (111%), with relative distribution of calories of 15% from proteins, 48% from carbohydrates and 37% from lipids in 15 children with moderate CRF [14]; and RIs in 82 children with CRF were <86% for energy and >161% for protein [12].\nAlthough energy intake is low, it may be proportionate to body weight: 4-day food records from 120 children with CRF found an energy intake of 80% of RI for age, decreasing with increasing age. However, this was in the normal range when factored by body weight. The protein intake was 153% of the RDA [37].\nIntake deteriorates with severity of CRF: energy intakes correlated negatively with GFR in 95 children with CRF, and fell to 85% of EAR when the GFR was <25\u00a0ml\/min\/1.73\u00a0m2 [38]. Nitrogen balance studies have been performed in 19 children on PD. Protein intake was 1.64\u00a0g\/kg\/day (126% of the RDA), and the calorie intake reached 75% of RDA. Nitrogen losses were 0.205\u00a0g\/kg\/day, and nitrogen balance was positive in three quarters of studies, correlating with nitrogen and calorie intake [39].\nIntake also decreases with time: 3-day semi-quantitative dietary diaries in 51 children with a GFR <75\u00a0ml\/min\/1.73\u00a0m2 assessed over 2 years showed protein intake to decrease by 0.4\u00a0g\/kg per day, and calcium by \u221220% RNI [40]. Energy and protein intake (energy more than protein), all anthropometric measurements and plasma proteins and AAs were low in 24 children on continuous ambulatory PD (CAPD), particularly in those <10 years of age [13].\nLow intakes of calcium [35\u201338], zinc [35, 37, 40, 41] and vitamins [37, 40, 42] are also reported.\nThe epidemiology of nutritional disturbances in renal disease, including height, weight and body composition\nHeight and weight\nIt is during the infantile phase of growth that the most significant loss of height potential can occur, but also it is the phase during which there is the greatest potential for catch-up with nutritional intervention. Many infants with CRF are already growth-retarded by the time they are first seen in a paediatric nephrology service: a loss of HtSDS from birth of \u22121.68 SD (up to \u22125 SD\/year) has been reported [43]. Approximately one-third of the reduction in height occurs during foetal life and one-third during the first 3 months, accompanied by a similar decline in head circumference [44\u201346]. Mean HtSDS below the lower limit of normal has been reported in most studies [43\u201346]. However, catch-up can occur, even on dialysis [47\u201349]. Poor nutritional status is associated with starting PD at a younger age [50]. Interestingly, infants who grew well continued with catch-up in early childhood [43, 47].\nDuring the childhood phase, growth in CRF usually parallels the centiles but without catch-up [43, 46]. The North American Pediatric Renal Transplant Cooperative Study (NAPRTCS) CRF database (<20 years of age, GFR <75\u00a0ml\/min\/1.73\u00a0m2) has shown a mean HtSDS that has changed little since 1996 when it was \u22121.5 in 1,725 patients, to \u22121.4 (one third >\u22121.88) in 3,863 patients in 1998 and \u22121.4 in 4,666 patients in 2001 [51\u201353]. European study group data on 321 children aged 1\u201310 years with congenital CRF reported a mean HtSDS of \u22122.37. Increasing severity of CRF adversely affects growth: when the patients were divided into those with a GFR greater or less than 25\u00a0ml\/min\/1.73\u00a0m2, the HtSDS was \u22121.65 and \u22122.79, respectively [44]. Reports of growth on dialysis vary from improvement [54], to no change [55] to declining HtSDS [56], with a worsening of nutritional status in children dialysed for more than a year [50].\nThe pubertal phase of rapid growth is another period when loss of height potential can occur. Early studies demonstrated that puberty was delayed, with an irreversible decline in height SDS, particularly in patients on dialysis [57, 58]. However, others have reported normal pubertal progression and growth [43, 46, 59].\nBody composition\nChildren with CRF and short stature are significantly protein-depleted for age, although not for height: 17 patients, mean age 12.9 years had total body nitrogen (TBN) and TBN\/height of 54% and 63%, respectively, when predicted from age, but 100% when predicted from height. Energy and protein intakes were 65% and 172% of RDA, respectively. This suggests that chronic energy deficiency may contribute to impaired protein deposition which, in turn, may be important in the pathogenesis of growth failure in CRF [25].\nIt has been suggested that the ratio of the length of trunk to limb is low in CRF, suggesting a disproportionately greater effect of disease and\/or treatment on spinal growth [60] although not all have found this [61].\nBIA in children starting PD showed an improvement of hydration and nutrition after 6 months, although levels remained below normal, suggesting that introduction of dialysis should not be left until malnutrition has developed [62, 63]. DEXA studies of 20 PD patients receiving the RDA for energy and a daily protein intake of 144.3% and 129.9% RI at months one and six showed an increase in BMD, bone mineral content (BMC) and FFM. However, the daily protein intake showed a negative correlation with these parameters and also plasma bicarbonate, suggesting that a high protein intake may negatively affect bone mineralization and FFM by its effect on acid-base status [64].\nDisturbances in plasma and intracellular AAs have been found in CRF. Muscle isoleucine and valine levels and the valine\/glycine ratio were low in children with CRF who were short but had no other signs of malnutrition (normal skinfold thickness, MAC and serum proteins) [65]. Levels have been studied in ten children on CAPD. Although plasma levels of essential AAs (EAAs) were low, only muscle intracellular leucine and valine were low, whereas both plasma and intracellular levels of some non-essential AAs were high. No correlations were found between plasma and muscle AAs and indicators of nutritional status, except muscle branched-chain AA levels with BMI [66].\nThe pathological mechanisms underlying poor appetite, abnormal metabolic rate and endocrine disturbances in renal disease\nPoor appetite is common in children with CRF and may in part be due to abnormal taste sensation [67]. Appetite is also affected by cytokines, and their roles in CRF, along with their effect on metabolic rate, have been extensively and excellently reviewed [68, 69]. Malnutrition may be an inappropriate term in CRF because it infers that dietary replacement would be curative (which is not always the case in CRF), and the term cachexia, which implies replacement of muscle with fat and declining plasma proteins, may be more appropriate [68, 69]. Cachexia may result not only from anorexia but also from acidosis and inflammation (which are common in CRF), which cause elevated levels of cytokines such as leptin, TNF-\u03b1, IL-1 and IL-6. These act through the hypothalamus to affect appetite and metabolic rate and maintain the constancy of fat stores.\nLeptin is produced by adipocytes and is probably the most important cytokine involved in this process. When body fat falls, levels of leptin decline and the brain responds by increasing appetite and metabolic efficiency; in contrast, when leptin levels rise, food intake is decreased and metabolic rate increases. However, leptin is excreted by the kidney and not cleared by dialysis, so levels can be paradoxically high in malnourished patients, contributing further to decreased food intake and increased metabolic rate. Serum leptin levels were >95th percentile in 45% of 134 children with varying severity of CRF, and correlated positively with their percentage body fat and GFR and negatively with spontaneous energy intake [70]. Leptin levels also correlate with CRP, a marker of inflammation, and with insulin resistance [67, 69]. Leptin signalling in the brain occurs through the hypothalamic melanocortin receptors, and may offer a new area for therapeutic intervention in the cachexia of CRF. The part played by the short-term regulators of satiety, such as ghrelin (which stimulates appetite), is not yet fully understood [68, 69].\nAbnormalities of the GH\/IGF-1 axis occur in CRF. Whether they are primarily due to CRF itself or secondary to malnutrition is controversial [71, 72]. GH levels are normal to high and IGF-1 levels are low in both CRF and malnutrition [73]. IGF-1 decreases according to nutritional status in children on HD [71]. Leptin levels correlate positively with IGF-1 and negatively with GH in children with starvation [73]. However, in CRF this relationship is disrupted: in 17 children on HD with energy and protein intakes of 40\u201370\u00a0kcal\/kg per day and 1\u20131.54\u00a0g\/kg per day, respectively, who had reduced anthropometric measurements, although IGF-I levels were low, leptin levels were high [74].\nEvidence for the benefit of dietetic input, dietary supplementation, nasogastric and gastrostomy feeds and intradialytic feeding\nThere are very few controlled trials, so recommendations are based on the evidence available. Some, but not all, reports have been able to establish a relationship between nutritional intake and growth. Growth velocity correlated positively with energy intake in 17 children with CRF [75], and with energy but not protein intake in 15 children on CAPD [76]. Growth velocity was inversely correlated with dietary protein intake and positively correlated with caloric intake both before the initiation of rhGH therapy and after the first year of treatment in 31 children on dialysis [77]. The advantage of input from a dietitian has been specifically demonstrated in two studies [11, 40], but it is likely that in all studies dietetic input was necessary.\nDietary supplements\nBecause of the importance of nutrition in the first two years of life it might be expected that enteral nutrition would be most effective at this age. Most studies have therefore concentrated on this age group, and there is evidence to show that nutritional supplementation is of benefit. Eight studies have demonstrated an improvement in growth [21, 47, 78\u201383], three showed an initial decline followed by stabilisation [84\u201386], three showed no effect on growth [11, 87, 88] and one showed a decline in HtSDS [89]. All but one study [86] used feeds administered by nasogastric or gastrostomy tubes and aim for at least the EAR for energy and RNI for protein, with a protein supplement for dialysis.\nAn early study of nasogastric feeding in 14 children weighing <10\u00a0kg demonstrated a benefit on HtSDS and WtSDS in 11 [78]. Twenty-six children aged <2 years with a GFR <26\u00a0ml\/min\/1.73\u00a0m2 were treated with a whey-based infant formula (supplemented with fat and\/or carbohydrate) which provided 100% of the RNI for protein for Ht age and 100% of the EAR for energy for chronological age. HtSDS increased from \u22122.9 to \u22122.1 over 2 years [21]. A similar feed resulted in improvement in HtSDS from \u22122.34 at 6 months of age to \u22121.93 at 2 years in 24 infants with a GFR <20\u00a0ml\/min\/1.73\u00a0m2, and from \u22122.17 to \u22121.24 in 13 infants on dialysis over a similar time-frame, although a protein supplement for dialysis was included [47]. A further study from the same centre showed an increase in HtSDS from \u22121.8 to \u22120.8 at 2 years in 20 infants on PD [79]. Three studies, each reporting the results of nasogastric feeding in three young children with CRF, have shown benefit in eight out of the nine children [80\u201382]. It is important not to restrict the salt and water content of the diet in the salt-wasting polyuric infant: a feed providing just over the RDA for calories and protein diluted to 0.3\u20130.5\u00a0kcal\/ml with additional 2\u20134\u00a0mEq of sodium\/100\u00a0ml in 24 infants resulted in improvement of HtSDS by 1.37 SD at 1 year and 1.82 SD at 2 years [83].\nThree studies have shown an initial decline followed by normalisation of growth. Twelve infants with CRF dropped to a HtSDS of \u22122 by 12 months of age and then stabilised at that level [84]. Decline in HtSDS was arrested in eight children <2.5 years of age after starting gastrostomy feeds [85] and stabilised after 3 months in the only study using supplementation without nasogastric or gastrostomy feeds [86]. In three studies, enteral feeds made no impact on growth: there was no change in infants with CRF [87], HD [88] or PD [11]. In one study, a decline in HtSDS occurred in 82 children <2 years of age at the start of dialysis [89].\nWhether supplemental feeds benefit children over 2 years of age has been challenged [90]. Six studies have included older children [21, 80, 81, 85, 89, 91]. Of these there was an improvement in growth in three [80, 81, 91]. Dietary advice and supplements of glucose polymer and vitamins (as Ketovite) were given to 65 children aged 2\u201316 years with a GFR <75\u00a0ml\/min\/1.73\u00a0m2 if their EAR and RNI, respectively, fell below 80% as assessed by annual 3-day dietary diaries. Mean HtSDS was maintained in those with a GFR of 25\u201375 and significantly increased in children with a GFR <25\u00a0ml\/min\/1.73\u00a0m2. There was an increase in HtSDS and\/or BMI SDS in all the patients on supplements, and change in energy intake correlated with change in HtSDS in those with a GFR <25\u00a0ml\/min\/1.73\u00a0m2 [91]. Three children with a GFR of 20\u201325\u00a0ml\/min\/1.73\u00a0m2 fed overnight by nasogastric tube for 11\u201316 months with increasing amounts until weight gain occurred improved their HtSDS [81], as did three children with CRF over the course of a year [80]. Two studies have shown no change in HtSDS. Nine children aged 2\u20135 years, treated with a whole protein enteral feed supplemented with fat and\/or carbohydrate showed no change in HtSDS (\u22122.3 to \u22122.0) [21], as did seven children in given gastrostomy feeds [85]. One study has demonstrated an ongoing decline in HtSDS in children aged 2\u20135 years at the start of dialysis, 14 of whom were given supplements and 20 not [89].\nComplications of gastrostomy are uncommon, but include gastro-colic fistulae, paraoesophageal herniae and, in children on PD, post-surgical peritonitis and an increased risk of exit site infection and dialysis catheter removal from infection, a risk that might be reduced if open rather than percutaneous surgery is used [92, 93]. After removal the track usually closes spontaneously [94]. There have been concerns that enteral tube feeding precludes the development of normal feeding behaviour [95]. However, other studies have shown that, despite long term nasogastric or gastrostomy feeding, oral feeding is resumed in the majority of children after successful transplantation [21, 96]. Positive reinforcement at feeding times using behavioural therapy techniques allowed five infants who had PD and nasogastric tube feeding initiated in the first month of life, and who showed persistent food refusal, to convert to oral feeding [97]. Our impression is that spontaneous oral intake increases with long-term tube feeding; indeed, over a period of 31 months energy intake from the feed did not increase, implying that oral intake had improved over this time to support the demonstrated growth [21]. Reports of the use of Nissen fundoplication are principally from one group [21, 47, 54, 79, 88, 92], making assessment of its effect difficult, although results of growth from this centre are good.\nEssential aminoacid (EAA) supplements\nSerum EAAs, carnitine and total protein levels have been demonstrated to be low in CRF, particularly in patients on PD [98]. It has been suggested, therefore, that a low protein diet supplemented with EAAs might benefit growth by ensuring adequate AA intake without protein toxicity. However, results have been inconclusive. No improvement in growth was seen in seven children with severe CRF who were given half the protein RDA for height age as EAAs for 6\u20138 months [99]. Ten children with CRF managed for 3 years using a strict low protein diet supplemented by a mixture of the keto and amino forms of the EAAs and histidine showed a significant increase in height and weight velocity [100]. HtSDS improved from \u22121.93 to \u22121.37 over 30 months in 20 patients with a GFR <50\u00a0ml\/min\/1.73\u00a0m2 on a diet of 0.6\u00a0g\/kg of protein supplemented with ketoacids [101]. Ten children on HD given AA supplementation (0.25\u00a0g\/kg body weight i.v.) with and without carnitine (25\u00a0mg\/kg body weight i.v.) had no overall improvement in AA levels [102].\nAmino acid-containing peritoneal dialysis solutions\nExcessive glucose absorption and dialysate AA and protein losses contribute to malnutrition in children on PD. It has been suggested, therefore, that using an AA dialysate might both decrease glucose load and replace AA losses. AAs are absorbed in proportion to the concentration difference between dialysate and plasma; after a 1% AA exchange in seven children on CAPD, the rise in plasma levels of AAs correlated with the ratio of the amount of AA in the bag to the basal plasma concentration. The amount of AA absorbed was 66% after 1\u00a0h, and 86% after 4\u00a0h and 6\u00a0h [103, 104].\nHowever, there is no evidence for any long-term nutritional benefit: eight children on CAPD who had a first morning exchange of 1% AA dialysate instead of dextrose for 12\u201318 months had no improvement in any plasma or anthropometric parameter of nutrition; plasma urea increased. Plasma EAAs, which had been low, improved but the intracellular pool of free AAs, measured in polymorphonuclear leucocytes did not improve [105]. Two randomised prospective cross-over studies of 3 months AA or dextrose dialysate for three months have been performed, both in seven growth-retarded children either on CAPD [106] or continuous cycling PD (CCPD) [107]. In the children on CAPD there was no nutritional benefit from the AA dialysis [106]. The children on CCPD received dextrose dialysate overnight, plus a single daytime dwell of either AA dialysate or dextrose dialysate. Appetite, calorie and protein intake improved and total body nitrogen increased in half the children during AA dialysis. However, total plasma protein and albumin did not change and fasting AAs after 3 months of AA dialysis were comparable to baseline; plasma urea concentrations were higher [107]. High plasma urea may be due to inadequate protein synthesis in the absence of glucose. Ten children underwent overnight CCPD using a 3:1 ratio of glucose to AA solutions simultaneously during the night. Glucose absorption was 33.7% and AA absorption was 55.2% of the infused amount, and although plasma AA levels were high for the entire ambulatory PD (APD) treatment the plasma urea levels did not increase suggesting that the AAs were being used for protein synthesis with this regimen [108].\nDisadvantages compared with glucose include cost, and reports of fluid removal are variable. However, equal amounts of urea and creatinine are removed, normoglycaemia is maintained and there are no reported adverse clinical or biochemical effects, other than a slight increase in plasma urea [106, 107, 109].\nIntradialytic parenteral nutrition (IDPN)\nThere are only four studies of IDPN during HD in children so it is difficult to draw conclusions about its effectiveness. Losses of AAs occur into the dialysate during HD and depend on their plasma concentrations and molecular weights. AAs were added to the dialysate of three children in increasing concentrations. Plasma nonessential AAs were not affected, but EAAs improved [110]. Four malnourished children on HD were given IDPN as AAs (8.5% solution), glucose (10% to 15% dextrose), and 20% fat emulsion at every dialysis session (three times a week) for 7\u201312 weeks. Oral intake improved and, although weight did not improve during treatment, it did so subsequently. Albumin did not change [111]. The weights of three children improved after 6 weeks of IDPN; again, albumin did not improve [112]. Nine patients who on HD had a >10% weight loss and were <90th percentile of ideal body weight received thrice weekly IDPN. In six, BMI increased in the first 5 months and PCR increased, whereas serum albumin did not change; those who did not gain weight were considered to have psychosocial causes for their malnutrition [113].\nNutritional causes of poor growth not related to energy and protein\nSodium\nRequirements for sodium vary according to the type of renal disease. Congenital structural abnormalities often result in an obligatory urinary loss of salt and water. Such children may become chronically salt- and water-depleted and need sodium supplementation and free access to water as salt wasting impairs growth [83]. On the other hand, children with glomerular disease need to restrict their sodium intake. Young children on PD may need sodium supplementation as considerable sodium losses can occur in dialysate.\nAcidosis\nAcidosis is associated with a catabolic state, suggesting that acidosis-related protein wasting could contribute to growth retardation [114, 115]. Correction of acidosis improves serum albumin, catabolic rate and growth [83, 116, 117].\nAnaemia\nAnaemia is a well recognised cause of poor appetite. However, studies that include blinded, placebo-controlled trials have found that despite subjective increases in appetite, there were no consistent improvements in dietary intake or anthropometric measures observed during rhuEPO treatment [118\u2013122].\nVitamin D\nThe dose of calcitriol prescribed to control hyperparathyroidism must be balanced against its potential to depress the activity of chondrocytes causing adynamic bone disease. Large doses impair growth, even if intermittent [123, 124], but the frequency of administration does not affect growth if small doses are used [125, 126].\nGrowth hormone\nThere may be cases when, despite at least 6 months of adequate nutrition, growth continues to be poor. GH may be offered in these circumstances.\nThe effect of dialysis adequacy on nutrition\nSeveral studies have looked to see whether increasing dialysis dose benefits appetite, protein intake, nutrition and growth. Twenty-one children on CCPD showed an improvement in HtSDS when aiming for a Kt\/V of >2 and a creatinine clearance of >60\u00a0l\/week\/1.73\u00a0m2 compared with 1.7 and 40\u00a0l\/week\/1.73\u00a0m2 [127].\nHowever, in some, but not all studies, there would appear to be a ceiling above which no further benefit occurs. The nPCR and serum albumin were assessed according to Kt\/V in 15 patients on HD. Serum albumin levels were normal. The nPCR was lowest in patients with a Kt\/V <1.3, but increasing over 1.6 did not improve nPCR further, suggesting that although adequate dialysis needs to be achieved in order to ensure good protein intake, high dialysis doses are of no further benefit [128]. However, in 12 children taking 90.6% and 155.9% of their requirements for energy and protein, respectively, and receiving HD with a Kt\/V of 2.00 and a urea reduction ratio of 84.7%, there was an improvement in HtSDS of +0.31 SD\/year and pubertal growth was normal, suggesting that for HD, increasing dialysis dose does improve growth [129].\nIn PD, but not HD, there was an inverse relationship between albumin level and Kt\/V, suggesting that increasing PD dose may reach a point of no further benefit due to increasing albumin losses in PD fluid [130]. PD causes an influx of glucose which can contribute to obesity. High transporter status was associated negatively with HtSDS and positively with BMI SDS in 51 children on PD. Large dialysate volumes also affected BMISDS [131].\nImproving dialysis dose can be achieved by the addition of an icodextrin daytime dwell to overnight PD. A cross-over study in eight children of overnight PD with or without addition of a daytime dwell with 1,100\u00a0ml\/m2 icodextrin for a week showed an improvement in weekly dialysis creatinine clearance from 35 to 65\u00a0l\/week\/1.73\u00a0m2 and Kt\/V from 1.99 to 2.54. However, protein and calorie intake did not improve and peritoneal albumin loss and serum albumin did not change, but there was increased loss of AAs, although plasma AA levels did not change [132].\nResidual renal function (RRF) has an important positive effect on clearance and growth. Mean HtSDS improved from \u22121.78 to \u22121.64 over a year of PD in 12 patients with RRF, but declined from \u22121.37 to \u22121.90 in 12 patients without. Weekly Kt\/V was not different, and only the native kidney Kt\/V and creatinine clearance correlated with growth, suggesting that clearance obtained by PD cannot be equated with that obtained by native kidneys [133]. Eleven of 20 patients on PD with a minimum total Kt\/V of 2.1, daily protein intake of 3.25\u00a0g\/kg\/day and HtSDS of \u22122.3 improved their HtSDS by 0.55, while in nine it declined by \u22120.50. Variables affecting growth were nitrogen balance and residual Kt\/V [134].\nThe role of nutrition in the outcome of children with CRF\nMalnutrition is associated with increased mortality. The association with plasma albumin levels has already been discussed [12]. Of 2,306 children, those with a HtSDS less than \u22122.5 at the start of dialysis had a twofold higher risk of death [135]. In 1,949 children with ESRF, each decrease in height by 1 SD was associated with a 14% increase in risk for death, and there was a U-shaped association between BMI and death [4]. Part of this may be due to an increased risk of infection in malnourished patients [136].\nIn conclusion, commencement of careful nutritional support early in the course of disease may improve not only growth but also mortality in children of all ages with CRF.\nMultiple choice questions\n(Answers appear following the reference list)\nFor each question answer true or false: \nDuring the phases of growth \nRate of growth is highest during prenatal life50% of final height is achieved by the age of 2 yearsThe infantile phase of growth is principally dependent on growth hormoneGrowth rate stays the same throughout the childhood phase of growthA pubertal growth spurt can occur without the development of secondary sexual characteristicsAssessment of nutritional status in CRF and on dialysis \nIt may be more appropriate to express measures of growth according to height age rather than chronological ageThe height standard deviation score (HtSDS) is the number of standard deviations from the mean for a normal population of the same age and sexThe BMI is the Ht\/Wt2 and indicates the proportion of fat mass to fat free massLow serum albumin is always an indication of malnutritionPeritonealprotein losses in dialysate are twofold greater in relation to body surface area in infants than in those >50\u00a0kg in weightNutritional requirements \nThe estimated protein requirement for a normal healthy 30-week-old girl weighing 6.0\u00a0kg (0.4th centile) and 59.0\u00a0cm in length (<0.4th centile) is >2.1\u00a0g\/kg\/dayHer estimated energy requirement is 150\u00a0cal\/kg\/dayThe prescribed dietary protein intake for the same child on PD would be 2.8\u20133.0\u00a0g\/kg\/dayChildren with CRF or on dialysis need a calorie intake that exceeds the EAR for height ageSupplements of vitamin A are necessary in children on dialysisAppetite and metabolic rate \nAbnormal taste sensation can occur in CRF and on dialysisLeptin is produced by adipocytes and levels are high in CRF and on dialysisHigh leptin levels cause an increase in food intake and metabolic rateGH levels are normal or high and IGF-1 levels are low in CRFGH levels are normal or high and IGF-1 levels are low in malnutritionDietary supplementation \nSalt restriction is important in all children with CRFNutritional supplementation has not been shown to benefit children over two years of ageIncreasing dialysis dose in PD may increase peritoneal dialysate protein losses and contribute to obesitySodium supplementation may be necessary in children on PDGastrostomy placement is preferable before PD commences","keyphrases":["nutrition","dialysis","growth","chronic renal failure"],"prmu":["P","P","P","P"]}
{"id":"Pediatr_Radiol-3-1-1891641","title":"Doppler waveforms of the ureteric jet: an overview and implications for the presence of a functional sphincter at the vesicoureteric junction\n","text":"This paper is a comprehensive review of the Doppler waveform appearance of ureteric jets. Six jet waveform patterns have been identified: monophasic, biphasic, triphasic, polyphasic, square and continuous. Details of the physical properties of jet patterns and their changes under various physiological conditions are illustrated. The immature monophasic ureteric jet pattern is common in infancy and early childhood up to around 4 years of age. This pattern is also noted to have a high incidence in older children with urinary tract infection\/vesicoureteric reflux, nocturnal enuresis and in other special physiological conditions such as in children undergoing general anaesthesia, in women during pregnancy, and in patients who have had ureteric transplantation. A hypothesis of dual myogenic and neurogenic components is proposed to explain the mode of action of the vesicoureteric junction (VUJ). The implication of this hypothesis is that it alters the scientific basis of the understanding of the VUJ. Furthermore, the application of colour Doppler US to ureteric jets may provide a non-invasive technique to study the physiology or pathophysiology of the VUJ in humans. This might shed light on new novel approaches to the monitoring and treatment of diseases related to VUJ function.\nIntroduction\nWhen the bolus of urine being transmitted through the ureter reaches the terminal portion, it is ejected forcefully into the bladder through the vesicoureteric junction (VUJ). This creates a jet of urine that can be seen within the urinary bladder during cystoscopy and grey-scale ultrasonography (US). Ureteric jets are also occasionally visible during intravenous urography (IVU) and voiding cystourethrography (VCU).\nIn 2-D, grey-scale real-time US imaging the jet can be visualized as a stream or burst of low-intensity echoes emerging from the ureteric orifice. Each ureteric jet usually lasts for few seconds and is fast enough to produce a frequency shift; therefore the ureteric jet can be demonstrated by colour Doppler US. Colour Doppler US is in fact the easiest method for demonstrating the jet. It is also amenable to further characterization using a pulse-wave Doppler waveform. The US appearance of the jet has been documented in a number of studies and the jet can be consistently demonstrated in both humans [1, 2] and animals such as the dog [3].\nThis review is based on a number of previous studies that involve US scanning of a total of 2,128 subjects, which include a normal population of 1,341 subjects. The characteristics of ureteric jets in a normal population of 1,341 subjects are described and the effect of age, gender and bladder-filling (based on a subgroup of 102 normal adult females) are discussed. A summary of jet patterns seen in children with urinary tract infection\/vesicoureteric reflux (VUR) (n\u2009=\u200998) [4] and nocturnal enuresis (n\u2009=\u2009511) [5] is also presented. Special groups, including pregnant women (n\u2009=\u2009107) [6], anaesthetized children (n\u2009=\u200916) [7] and subjects with ureteric transplantation (n\u2009=\u200955) [8] are also discussed to look at changes of ureteric jet patterns under specific physiological conditions. Overall the observations suggest a possible correlation of different jet patterns with functional sphincteric action of the VUJ. A hypothesis of dual myogenic and neurogenic components is proposed to explain the mode of action of the VUJ.\nDiscussion\nBasic ureteric jet patterns on Doppler US\nJequier et al. [9] were the first to describe the ureteric jet. In their study of children, they demonstrated that ureteric jets had both crescendo and decrescendo forms. The jet waveforms ranged from a single to as many as four \u201chumps\u201d. Cox et al. [2] noted that the number of peaks (the \u201chumps\u201d of Jequier et al.) in the ureteric jet varied from one to four while Wu et al. [10] found only two or three peaks. Both the latter studies involved only adults. To the best of our knowledge, ours is the only group that has studied in detail ureteric jet patterns in a cohort of subjects with a wide age range, including a large paediatric population.\nSix basic patterns have been identified according to the number of peaks within a single ureteric jet: monophasic, biphasic, triphasic, polyphasic, square and continuous [11] (Fig.\u00a01). Among these, the square and continuous waveforms represent modified waveforms under the state of forced diuresis and they are deliberately avoided in most of the study analysis. The biphasic, triphasic and polyphasic waveforms are grouped under the category of mature complex jets while the monophasic jet is classified as the immature jet. We have shown that the majority of the population have a complex mature waveform of the ureteric jet while the immature monophasic waveform has a significantly higher incidence in young children. This is discussed in more detail below.\nFig.\u00a01Six patterns of the ureteric jet: a monophasic, b biphasic, c triphasic, d polyphasic, e square, and f continuous\nThe initial slope, duration and maximum velocity (peak velocity) of the strongest jet are measured on every Doppler waveform for quantification purposes. Significant differences have been found in the above physical parameters among the four jet patterns monophasic, biphasic, triphasic and polyphasic. The monophasic jet has the shortest duration, lowest velocity and smallest initial slope [11].\nOccasional modification features of the ureteric jet\nSome interesting features have been observed in the jet patterns of some normal subjects. These features include (a) the presence of breaks, (b) a multispike pattern, and (c) change of angle of the jet between the beginning and the end. The number of cases with the above features is too small for statistical analysis; however, we sought to describe them in detail for completeness of the whole spectrum of ureteric jet patterns. These features also provide indirect supportive evidence for the hypothesis of functional sphincteric action at the VUJ and are discussed further in the final section.\nBreaks\nThe presence of break is defined as the total absence of Doppler signal between peaks within a single ureteric jet waveform (Fig.\u00a02). Breaks were identified in 5.7% (149\/2,629 ureters) of the study population. Comparing different groups of subjects, it has been found that most breaks occur in adult females when their bladder is at maximum capacity, i.e. when the subjects experience an urgent desire to micturate (33.3% of adult females, P<0.05, chi squared test).\nFig.\u00a02Break within a ureteric jet\nMultispike pattern\nA multispike pattern is defined as the pulsations noted within a single jet waveform as the result of pulsation transmitted from adjacent arteries (Fig.\u00a03). The incidence of a multispike pattern was found to be 1.9% (50\/2,629) in the normal population and is more commonly observed when the bladder is extremely full (forced diuresis).\nFig.\u00a03A multispike pattern of a ureteric jet\nChange in angle of the jet\nA change in the angle within a single jet waveform is illustrated given in Fig.\u00a04. The incidence of change in angle of the ureteric jet in the whole normal population was 4.3% (113\/2,629).\nFig.\u00a04Change in angle of the ureteric jet (a) at the beginning and (b) at the end of the ureteric waveform\nUreteric jet pattern and physical properties of jets in normal subjects\nGeneral properties\nDirection of flow Dubbins et al. [1] and Elejalde and de Elejalde [12] have found on both US and MRI that the ureteric jet is usually directed anteriorly or anteromedially (with or without crossing of the jets), while others have found ureteric jets directed in a more vertical direction or perpendicular to the bladder base [2, 9, 13\u201316]. Our US findings are in agreement with the above on the ureteric jet direction of flow.\nMean jet velocity In our studies, the mean velocity of ureteric jets in children was found to be 34.03\u00a0cm\/s for the monophasic pattern and 61.82\u00a0cm\/s for the complex pattern (Tables\u00a01 and 2). These values are higher than those reported previously. The mean jet velocity previously reported in children aged from 26\u00a0days to 17\u00a0years varies between 18 and 31.6\u00a0cm\/s [9, 17, 18]. The discrepancy between the findings of our study and those of previous studies can be explained by different proportions of children of different ages. The mean velocity in adults in our cohort was found to be 57.65\u00a0cm\/s for the monophasic pattern and 78.89\u00a0cm\/s for the complex pattern. These values are similar to those reported previously [2, 18, 19]. \nTable\u00a01Mean values of jet parameters in children and adults with the monophasic pattern\u00a0Right sideLeft sideChildrenAdultsP valueChildrenAdultsP valueNumber of ureteric jets83188018Initial slope (cm s\u22122)211.82195.540.60256.55281.100.87Velocity (cm s\u22121)34.0357.65<0.0138.6663.93<0.01Duration (s)1.171.91<0.011.171.90<0.01Table\u00a02Mean values of jet parameters in children and adults with the complex pattern\u00a0Right sideLeft sideChildrenAdultsP valueChildrenAdultsP valueNumber of ureteric jets293910296892Initial slope (cm s\u22122)293.32271.210.09264.48309.130.13Velocity (cm s\u22121)61.8279.89<0.0161.9773.83<0.01Duration (s)5.266.92<0.015.157.03<0.01\nMean jet duration In our studies, the mean jet duration in children was 1.17\u00a0s for the monophasic pattern and 5.26\u00a0s for the complex pattern. In adults, the mean jet duration was 1.91\u00a0s for the monophasic pattern and 6.9\u00a0s for the complex pattern (Tables\u00a01 and 2). These values are similar to those reported previously. In adults, previously reported jet durations range from 3.5 to 15\u00a0s [2, 16, 18, 20], while in children previously reported mean jet durations in two different series were 2.77\u00b11.53\u00a0s [9] and 1.8\u00b10.2\u00a0s [18].\nLaterality difference in ureteric jets In general, there are no significant differences in waveform pattern, initial slope, velocity and duration of ureteric jets between the right and left sides in both children and adults. This is in agreement with the study of Matsuda and Saitoh [18]. There were two exceptions in our cohort, which might not have clinical significance. Boys were found to have a higher incidence of the monophasic waveform in jets on the left side compared with girls (P<0.01, chi-squared test; Table\u00a03), while adult males have a lower velocity on the left than the right (P<0.01, paired sample T-test) (Table\u00a04). \nTable\u00a03Waveform patterns in children and adults in relation to sex (total of 2,629 ureteric jets in 1,341 subjects)\u00a0ChildrenAdultsFemaleMaleFemaleMaleRightLeftRightLeftRightLeftRightLeftNumber of ureteric jets166164211215567560373373PatternMonophasic28 (16.9%)19 (11.6%)55 (26.1%)61 (28.4%)14 (2.5%)12 (2.1%)4 (1.1%)6 (1.6%)Biphasic54 (32.5%)53 (32.3%)59 (28.0%)63 (30.2%)219 (38.6%)224 (40%)110 (29.5%)89 (23.9%)Triphasic54 (32.5%)53 (32.3%)57 (27.0%)54 (25.1%)207 (36.5%)183 (32.7%)134 (35.9%)122 (32.7%)Polyphasic29 (17.5%)37 (22.6%)40 (19.0%)34 (15.8%)120 (21.2%)126 (22.5%)120 (32.2%)148 (39.7%)Square1 (0.6%)1 (0.6%)0 (0%)0 (0%)5 (0.9%)13 (2.3%)2 (0.5%)1 (0.3%)Continuous0 (0%)1 (0.6%)0 (0%)1 (0.5%)2 (0.4%)2 (0.4%)3 (0.8%)7 (1.9%)Table\u00a04Mean values of jet parameters in children and adults in relation to sex\u00a0ChildrenAdultsFemaleMaleFemaleMaleRightLeftRightLeftRightLeftRightLeftNumber of ureteric jets165162211214560545368365Initial slope (cm s\u22122)290.73261.06264.22264.31268.12274.72271.93234.07Velocity (cm s\u22121)56.3157.0255.7257.6069.9468.8494.0180.82Duration (s)4.644.564.214.206.376.437.527.69\nEffect of age on ureteric jets\nThe distribution of the four basic patterns (monophasic, biphasic, triphasic, polyphasic) among children and adults has been previously described by our group. We have found a strikingly larger proportion of monophasic waveforms in children than in adults in a population of 1,010 subjects [11].\nIn this review, the above finding is substantiated after expanding the study population to 1,341 normal subjects. Children are found to have a higher incidence of the immature pattern than adults. The incidences of the monophasic waveform in children and adults are 22% and 1.9% on the right side, and 21.1% and 1.9 % on the left side, respectively (P<0.01, chi-squared test; Table\u00a05). This immature pattern occurs constantly in the first 6\u00a0months of life [4]. In our previously reported cohort, the immature monophasic pattern in children changed to a mature complex pattern at a mean age of 4.54\u00a0years, which probably reflects the mean age of VUJ maturity in general. There is no significant gender difference for the mean age of VUJ maturity in children: boys show VUJ maturity at a mean age of 4.88\u00a0years and girls at a mean age of 4.34\u00a0years (P>0.05, simple Z test) [21].\nAdults have higher velocity (P<0.01, independent sample t test) and longer duration of the ureteric jet (P<0.01, independent sample t test) than children in both the monophasic and complex patterns. This finding has also been reported by Matsuda and Saitoh [18], suggesting that the stroke volume of urine in children is less than that in adults. The initial slope of the ureteric jet, however, shows no significant difference between children and adults (all P>0.05, independent sample t test; Tables\u00a01 and 2).\nEffect of gender on ureteric jets\nIn the adult population, males have a higher incidence of the polyphasic waveform than females (P<0.01 for both right and left sides, chi-squared test; Table\u00a03). Male subjects also have a higher velocity (P<0.01 for both sides, independent sample t test) and longer duration of the ureteric jet than females (P<0.01 for both sides, independent sample t test; Table\u00a04). In children, however, no significant differences in velocity, duration, initial slope or number of peaks within a single jet was observed between boys and girls (all P>0.05, chi-squared and independent sample t test; Tables\u00a04 and 5). \nTable\u00a05Incidence of monophasic jet in children and adults (total of 2,629 ureteric jets in 1,341 subjects). Note that the total number of ureteric jets is less than double the number of subjects as jets in some subjects could not be satisfactorily demonstrated on both sides by Doppler US\u00a0Right sideLeft sideChildrenAdultsP valueChildrenAdultsP valueNumber of ureteric jets377940379933Monophasic pattern83 (22%)18 (1.9%)0.0180 (21.1%)18 (1.9%)0.01\nEffect of bladder filling status on ureteric jets\nIn a subgroup of 102 adult females we demonstrated how jet patterns are affected by different bladder filling status. Jet characteristics are compared between two different time intervals: (1) when bladder volume was small and rate of diuresis was low, and (2) when bladder volume was large and rate of diuresis more active. We found that 42.2% of the subjects showed no change in the number of peaks within a single jet waveform, 28.9% showed a decrease and 26.5% showed an increase, and 2.4% had square and continuous jet patterns when the bladder was very full [11]. Among the group with changes in the number of peaks, 3.4% showed a change from a monophasic to a complex pattern and 3.9% showed a change from a complex to a monophasic pattern. In all subjects, the initial slope, velocity and duration of the jet were not affected by different stages of bladder filling (all P>0.05, paired sample t test; Table\u00a06). Even though bladder filling was shown to have little effect on whether the subjects had an immature or complex pattern, we standardized the protocol of bladder filling in all our subsequent Doppler US studies of ureteric jets. Scanning was started in most subjects 20\u00a0min after water intake because the jet frequency is reasonably high at that time while the bladder is not yet too distended to make the subject feel uncomfortable. \nTable\u00a06Mean values of jet parameters in 102 subjects under different stages of bladder filling. Only the jet on the right side is shown for comparison as there was no significant difference between the two sidesJet parameterBladder statusP valueNot fullMaximally fullInitial slope (cm s\u22122)245.27209.440.39Velocity (cm s\u22121)62.8158.300.37Duration (s)6.246.230.81\nCharacteristics of ureteric jet under specific physiological conditions\nPregnancy (physiological)\nA total of 107 pregnant women and 375 non-pregnant women were investigated. The occurrence of the monophasic waveform was significantly higher in the pregnant women than in the non-pregnant women (18.7%, 41.1% and 1.6% at 20\u00a0weeks\u2019 gestation, 32\u00a0weeks\u2019 gestation, and 3\u00a0months postpartum, respectively, vs. 1.9% in the non-pregnant women) [6].\nGeneral anaesthesia (pharmacological)\nOur previous study has documented loss of the complex jet pattern after general anaesthesia. A total of 16 children undergoing surgery under general anaesthesia were recruited. Before anaesthesia, 14 of them showed a complex pattern and two showed a monophasic pattern. After anaesthesia, all showed a monophasic waveform [7].\nUreteric transplantation following renal transplantation\nOur previous study has shown that transplanted ureters do not have the normal regulatory function at the VUJ, but inherent peristalsis is retained [8]. From a comparison of 55 transplant patients and 817 healthy subjects, we found that the Doppler waveforms of transplanted ureters are distinctly different from those of healthy adult ureters. Basically, only two patterns were identified from transplanted ureters: more commonly a short monophasic waveform (66.1% vs. 2.6% in the health ureters), and less commonly a longer multiphase pattern that does not resemble the patterns of the healthy ureter.\nUreteric jet characteristics in paediatric conditions\nLiterature review\nPrevious studies have attempted to relate the ureteric jet seen during IVU in UTI, VUR and bladder neck obstruction. The studies of Kalmon et al. [22] and Nevin et al. [23] have suggested that identification of ureteric jets in IVU studies do not exclude VUR, but Kuhns et al. [24] found an association between the jet sign and absence of VUR. They postulated that the jet sign is produced by peristalsis through the ureteric sphincter. There might be an abnormal increase in the intravesical volume and pressure in the presence of reflux, thus preventing ureteric peristalsis so that no jet can be seen. Ekl\u00f6f and Johanson [25], however, disagreed with this hypothesis, finding a lower incidence of visible jet in subjects without radiological proof of VUR (5.7% compared with 32% in the study of Kuhns et al.). Although infrequent, significant ipsilateral VUR is observed even when a ureteric jet is detected (10.7% [25] vs. 5.3% [24]). Ekl\u00f6f and Johanson [25] concluded, therefore, that the low rate of bilateral jets detected on IVU restricts the potential clinical value of jet detection on IVU as an indicator of the absence of gross VUR and that voiding cystourethrography is necessary in the radiological work-up of children with UTI.\nMarshall et al. [17] agreed with the view of Kuhns et al. [24] that visualization of a ureteric jet on IVU should not exclude VUR. In addition, they found a strong correlation between relatively lateral positioning of the orifice and the presence of VUR. The mean velocity of the ureteric jet is not related to VUR. A midline-to-orifice distance of >7\u00a0mm has been proposed as the cut-off for predicting VUR. The more laterally positioned the ureteric orifice, the more likely it is that reflux will occur [17].\nSubsequent studies largely agree with the concept that the ureteric jet is just a normal physiological phenomenon. Gothlin [26] found that the ureteric jet can be identified in subjects with and without UTI. Neither sex nor age has an effect on jet visualization. He therefore concluded that the jet is just a physiological phenomenon and the roentgenographic finding is normal.\nThere are a number of previous studies evaluating characteristics of the ureteric jet based on Doppler US. Jequier et al. [9] found that the Doppler waveform parameters of jet direction, duration, frequency, velocity and shape do not help in predicting VUR. A lateral ureteric orifice is not seen in normal patients, but is identified in subjects with VUR and other urinary tract disorders.\nGudinchet et al. [27] found no difference between refluxing and non-refluxing ureters with regard to ureteric jet length, angle, and midline-to-orifice distance. They concluded that these parameters cannot be used to predict recurrence of reflux in children after endoscopic subureteric collagen injection (SCIN) for the treatment of VUR.\nIn two recent studies carried out by our group, we found a high correlation between the immature monophasic jet pattern in children with specific urinary disease entities. The details are discussed in the following sections.\nChildren with VUR and UTI\nUreteric jets of 241 healthy children and 98 children with UTI were studied. The incidence of monophasic jet (immature pattern) was 29% in healthy children overall, but varied greatly according to age. The immature pattern was universal in the first 6\u00a0months of life, but was markedly reduced to below 15% in late childhood. This immature pattern was more commonly seen in children with UTI (73.5%) and VUR (90.5%) than in healthy controls of the same age [4].\nChildren with nocturnal enuresis\nA comparison was made between 511 children presenting with primary nocturnal enuresis and 266 age-matched normal controls. The incidence of immature monophasic jet was significantly greater in enuretic children (19.2% in both sides) than in normal children (6.4% on the right side and 8.3% on the left side). Furthermore, the immature waveform was more commonly seen in the enuretic group with a markedly thickened bladder wall and multiple urodynamic abnormalities [5].\nTaking all the observations together, we postulate a hypothesis concerning the mode of action of the VUJ as illustrated in the last section.\nHypothesis of an active sphincter at the VUJ\nThere has been controversy regarding the anatomy and function of the VUJ for a long time. There are three schools of thought for the antireflux mechanism at the VUJ. In the first theory the VUJ is thought to be governed by a passive valve mechanism dependent on the length and obliquity of the intravesical ureter [28, 29]. In the second theory the VUJ is considered to possess mixed active and passive valvular action. In addition to the anatomical factors, the distal ureter also shows antireflux ureteric peristaltic activity so that contraction of the ureter can prevent retrograde leakage of the intraluminal contents [30\u201332]. In the third theory the VUJ is considered to be able to act as a sphincter. Noordzij and Dabhoiwala [33] have proposed a sphincteric function for the VUJ which might be activated by the intrinsic muscular meshwork of the trigonal region of the bladder complementing a purely passive antireflux mechanism.\nTaking into consideration all the features observed in previous Doppler US studies of the ureteric jet, we postulate that the human VUJ can act as a functional sphincter. Only a monophasic ureteric peristaltic wave is demonstrated by M-mode study of the ureter [11] while the waveform of the ureteric jet emanating from the VUJ becomes more complex in pattern. Because the waveform of the ureteric jet is modified, an active sphincter mechanism is probably present at the VUJ [11]. Because the VUJ shows sphincteric activity, different patterns of ureteric jet can be identified under different physiological and pathological conditions with occasional modifications.\nWe hypothesize that dual components are present regarding the mode of action in the active functional sphincter. The first component is the \u201cmyogenic\u201d (primary or \u201cimmature\u201d) and the second component is \u201cneural\u201d (secondary or \u201cmature\u201d). The distal ureteric muscle and possibly part of the detrusor muscle may contribute to the functional sphincteric action at the VUJ. We postulate that the monophasic jet pattern is the result of contraction caused by the myogenic component of the VUJ, while the complex pattern is the result of modulation of the myogenic component of the jet by the neural component in response to the distal intraureteric pressure. The mode of the functional sphincteric action of the VUJ and the subsequent ureteric jet waveform vary depending upon whether or not the neural component is active.\nIn normal adults and in children reaching a certain age of maturity, the neural component modulates the myogenic component and complex patterns can thus be seen. When the neural component is absent, for example in a small immature child, under general anaesthesia or in certain pathological conditions, only the myogenic component is functioning, and thus the jet pattern reverses to the monophasic pattern.\nIn renal transplantation patients with ureteric reimplantation, the normal VUJ mechanism in the transplanted ureter is completely lost and the ureteric jet patterns observed are completely different from the patterns obtained from the native VUJ in normal subjects [8]. These distinct patterns could be explained by loss of both the normal myogenic and the neural components of the sphincter action.\nAlthough the neural component governing the ureteric jet pattern is either present (resulting in a complex pattern) or absent (resulting in a monophasic pattern), the characteristics (initial slope, velocity, and duration) of the monophasic and complex pattern within the same age group remain distinct. Although there is a trend for a longer duration and higher peak velocity of the ureteric jet with increasing age, this could be explained by a larger bolus of urine in each jet in adults than in smaller children.\nThe neural component governing the sphincteric action of the VUJ appears to be affected under various physiological and pathological conditions. We have observed a marked increase in the incidence of a monophasic jet pattern in pregnant women. However, for those pregnant women who retain the complex jet pattern, the characteristics of the complex waveform remain similar to those seen in non-pregnant women [6]. The above observation suggests that the neural mechanism of the VUJ is lost in some pregnant women, but the underlying reason remains obscure.\nIn subjects undergoing general anaesthesia, the ureteric jet waveforms revert to the monophasic pattern with no difference in initial slope, velocity, and duration of the waveform when compared with the normal population [7]. This suggests that the ureteric jet in anaesthetized subjects is under myogenic influence solely, while the neurogenic influence is temporarily suspended. Histochemical study of the VUJ [34] has shown the muscular components of the VUJ to be innervated by both adrenergic and cholinergic nerves. However, because several drugs were simultaneously administered during general anaesthesia in our small cohort, the exact pharmacological action of these drugs on the neurogenic pathway could not be clearly determined.\nThe presence of VUR is highly correlated with the immature monophasic waveform [4]. This observation suggests that the complex jet pattern is associated with a more efficient antireflux mechanism than the simple monophasic waveform that is more primitive or immature. In those children with UTI, but without VUR, a high correlation with the immature monophasic pattern is still observed. It is not clear whether the monophasic pattern is a risk factor for or the consequence of UTI. However, the association of the monophasic jet waveform with VUR and UTI might partially explain why pregnant women with a higher incidence of the monophasic jet pattern are also more prone to VUR and UTI [4]. A higher incidence of the immature jet pattern is also found in children with nocturnal enuresis [5]. This suggests that there is a lower level of maturity of the VUJ in a proportion of enuretic children. This group of children also has more deranged parameters on urodynamic studies.\nThe hypothesis of dual components in the sphincteric action of the VUJ might also help explain some of the jet phenomena that we have described previously. The multispike pattern in the ureteric jet resulting from pulsations transmitted from adjacent arteries could probably be explained by premature relaxation of the VUJ that precedes the ureteric jet proper so that the transmitted arterial pulse becomes dominant. Premature relaxation of the VUJ is also likely to be governed by the neural mechanism seen in forced diuresis.\nThe modification involving breaks in the jet is predominantly observed when the bladder is maximally full. Under these circumstances the intravesical pressure would be very high, which might impose a countering effect on the pressure wave of the ureteric jet emitted from the VUJ. Breaks might therefore appear within the jet waveform when the jet velocity drops significantly to zero flow on entering the bladder.\nIn summary, based on all the above observations, we postulate that the human VUJ can act as a functional sphincter with two possible components: (1) a myogenic component which has a simple \u201copen and close\u201d action that gives rise to the monophasic jet pattern, and (2) a neural component that modulates the monophasic waveform into a more complex pattern. Further anatomical study to determine the exact nature of the sphincteric muscle governing VUJ function is warranted. The major implication of this overview of ureteric jet patterns is a change of concept for the human VUJ. Rather than being a passive valve, the VUJ functions as an active sphincter. This might lead to a novel approach to the management of VUR, UTI and enuresis in children which could replace traditional treatment.\nConclusion\nThis review has provided a comprehensive understanding of the physiological pattern of ureteric jets and contributes to our knowledge of the pathophysiology of urinary dysfunction in disease entities such as UTI, VUR and primary enuresis. The application of this technique in future studies might lead to novel approaches to the monitoring and prognosis of these conditions and more evidence-based treatment of related diseases.","keyphrases":["doppler","ureteric jet","vesicoureteric junction","children","ultrasound"],"prmu":["P","P","P","P","U"]}
{"id":"Biochem_Pharmacol-1-5-1920586","title":"Inhibition of the HERG potassium channel by the tricyclic antidepressant doxepin\n","text":"HERG (human ether-\u00e0-go-go-related gene) encodes channels responsible for the cardiac rapid delayed rectifier potassium current, IKr. This study investigated the effects on HERG channels of doxepin, a tricyclic antidepressant linked to QT interval prolongation and cardiac arrhythmia. Whole-cell patch-clamp recordings were made at 37 \u00b0C of recombinant HERG channel current (IHERG), and of native IKr \u2018tails\u2019 from rabbit ventricular myocytes. Doxepin inhibited IHERG with an IC50 value of 6.5 \u00b1 1.4 \u03bcM and native IKr with an IC50 of 4.4 \u00b1 0.6 \u03bcM. The inhibitory effect on IHERG developed rapidly upon membrane depolarization, but with no significant dependence on voltage and with little alteration to the voltage-dependent kinetics of IHERG. Neither the S631A nor N588K inactivation-attenuating mutations (of residues located in the channel pore and external S5-Pore linker, respectively) significantly reduced the potency of inhibition. The S6 point mutation Y652A increased the IC50 for IHERG blockade by \u223c4.2-fold; the F656A mutant also attenuated doxepin's action at some concentrations. HERG channel blockade is likely to underpin reported cases of QT interval prolongation with doxepin. Notably, this study also establishes doxepin as an effective inhibitor of mutant (N588K) HERG channels responsible for variant 1 of the short QT syndrome.\n1\nIntroduction\nDiverse cardiac and non-cardiac drugs are associated with prolongation of the rate-corrected QT (QTc) interval of the electrocardiogram and with a risk of the potentially fatal arrhythmia Torsade de Pointes (TdP) [1\u20134]. The majority of such agents exert a common action of inhibiting the cardiac rapidly activating delayed rectifier K+ current (IKr). IKr is a major determinant of ventricular action potential repolarization and, thereby, of the QTc interval [2,4,5]. The pore-forming subunit of IKr channels is encoded by HERG (human ether-\u00e0-go-go-related gene [6,7]). HERG channels appear to have a larger pore cavity than other (Kv) six transmembrane domain K+ channels and possess particular aromatic amino-acid residues in the S6 region of the channel [5,8\u201310]. These features combine to confer a high susceptibility to pharmacological blockade upon the HERG channel. Indeed, the association between drug-induced QTc interval prolongation and pharmacological blockade of HERG channels is sufficiently strong that drug-screening against recombinant HERG channels is now an important component of cardiac safety-pharmacology during drug development [11\u201313].\nDoxepin is a tricyclic antidepressant (TCA) structurally related to amitriptyline and imipramine that combines antidepressant and sedative actions [14]. Initially doxepin was suggested to have better cardiac safety than other TCA drugs [15]. However, a subsequent review of the clinical and animal data [16] and a study in depressed patients [17] found no evidence that doxepin had fewer cardiovascular effects than other TCAs. Moreover, similar to other TCAs, doxepin can be cardiotoxic in overdose [14]. Adverse cardiac effects associated with doxepin include premature ventricular complexes (PVCs) and wide QRS complexes on the electrocardiogram. Furthermore, there are a number of documented cases of QT interval prolongation with doxepin [18\u201320]. For example, in overdose doxepin has been associated with a greatly prolonged QTc interval (reaching 580\u00a0ms) and TdP [19], whilst QT interval prolongation and syncope have been reported for doxepin in combination with methadone and \u03b2 blocker use [20]. At present there is no information on the basis for QTc prolongation with doxepin. However, other TCAs including imipramine and amitryptiline have been demonstrated to inhibit recombinant HERG channels [21\u201323] and we hypothesised that doxepin is also likely to act as an inhibitor of HERG channel current (IHERG). The present study was conducted to test this hypothesis and to characterise the nature of any observed IHERG blockade.\n2\nMethods\n2.1\nMaintenance of mammalian cell lines stably expressing wild-type and mutant HERG channels\nExperiments on wild-type HERG were performed on a cell line (Human Embryonic Kidney; HEK 293) stably expressing HERG (donated by Dr Craig January, University of Wisconsin [24]), except for those in Fig. 8; these utilised a cell line stably expressing lower levels of HERG developed in this laboratory, for use with high external [K+] (for comparison with the F656A mutant, see Section 2.2 below). Cell lines stably expressing HERG and its mutants, F656A and Y652A were maintained as described previously [25]. Cell lines stably expressing the S631A [26] and N588K [27] mutants were made from appropriately mutated HERG sequences using previously described methods [25]. Cells were passed using a non-enzymatic dissociating agent (Splitase, AutogenBioclear) and plated out onto small sterilised glass coverslips in 30\u00a0mm petri dishes containing a modification of Dulbecco's modified Eagle's medium with Glutamax-1 (DMEM; Gibco, Gibco\/Invitrogen, Paisley, UK), supplemented with 10% fetal bovine serum (Gibco), 400\u00a0\u03bcg\u00a0ml\u22121 gentamycin (Gibco) and 400\u00a0\u03bcg\u00a0ml\u22121 geneticin (G418; Gibco). Treatment of the mutant cell lines was identical to treatment of the wild-type cell line except that cultures were maintained with 800\u00a0\u03bcg\u00a0ml\u22121 of hygromycin. The cells were incubated at 37\u00a0\u00b0C for a minimum of two days prior to any electrophysiological study.\n2.2\nExperimental solutions\nWhole-cell patch-clamp measurements of wild-type (WT) and mutant IHERG were made at 37\u00a0\u00b1\u00a01\u00a0\u00b0C. Once in the experimental chamber cells were superfused with a standard extracellular Tyrode's solution containing (in mM): 140 NaCl, 4 KCl, 2.5 CaCl2, 1 MgCl2, 10 glucose, 5 HEPES, (titrated to pH 7.45 with NaOH). Similar to other studies from our laboratory (e.g. [28,29]), for experiments employing the S6 mutant F656A (which shows comparatively low levels of channel expression [8,25]) and its WT control, the external solution contained 94\u00a0mM KCl (the NaCl concentration was correspondingly reduced). Experimental solutions were applied using a home-built, warmed, solution delivery system that exchanged the solution surrounding a cell in <1\u00a0s. Doxepin powder (Sequoia Research Products and Sigma-Aldrich) was dissolved in Tyrode's solution to produce initial stock solutions of either 10 or 50\u00a0mM, which were serially diluted to produce working solutions ranging from 0.1\u00a0\u03bcM to 1\u00a0mM.\nThe pipette dialysis solution for IHERG measurement contained (in mM): 130 KCl, 1 MgCl2, 5 EGTA, 5 MgATP, 10 HEPES (titrated to pH 7.2 with KOH) [28,29]. Patch-pipettes were heat-polished to 2.5\u20134\u00a0M\u03a9. No correction was made for the \u2018pipette-to-bath\u2019 liquid junction potential, which was measured to be \u22123.2\u00a0mV.\n2.3\nExperiments on rabbit isolated ventricular myocytes\nOne series of experiments was performed to investigate blockade by doxepin of native IKr from adult ventricular myocytes (Results, Fig. 2). For these, male New Zealand white rabbits (2\u20133\u00a0kg) were killed humanely in accordance with UK Home Office legislation. Ventricular myocytes were then isolated by a combination of mechanical and enzymatic dispersion, using previously described methods [30,31]. Pipette and external solutions for IKr measurement were identical to those described above for IHERG measurement.\n2.4\nElectrophysiological recording and analysis\nWhole-cell patch-clamp recordings were made using Axopatch 200 or 200B amplifiers (Axon Instruments) and a CV201 head-stage. Between 75 and 80% of the pipette series resistance was compensated. Voltage-clamp commands were generated using \u2018WinWCP\u2019 (John Dempster, Strathclyde University), Clampex 8 (Axon Instruments), or \u2018Pulse\u2019 software (HEKA Electronik). Data were recorded either via a Digidata 1200B interface (Axon Instruments) or an Instrutech VR-10B interface and stored on the hard-disk of a personal computer. The voltage-protocols employed for specific experiments are described either in the relevant \u2018results\u2019 text, or are shown diagrammatically on the relevant figures; unless otherwise stated in the text, the holding membrane potential between experimental sweeps was \u221280\u00a0mV. Data are presented as mean\u00a0\u00b1\u00a0S.E.M. Statistical comparisons were made using, as appropriate, paired and unpaired t-tests or one-way analysis of variance (Anova) (Prism 3 or Instat, Graphpad Inc.). P values of less than 0.05 were taken as significant; ns\u00a0=\u00a0no statistically significant difference.\nThe following equations were used for numerical analysis and graphical fits to data:\nThe extent of IHERG inhibition by differing concentrations of doxepin was determined using the equation:where \u2018Fractional block\u2019 refers to the degree of inhibition of IHERG by a given concentration of doxepin; IHERG\u2013DOXEPIN and IHERG\u2013CONTROL represent current amplitudes in the presence and absence of doxepin.\nConcentration\u2013response data were fitted by a standard Hill equation of the form:where IC50 is [DOXEPIN] producing half-maximal inhibition of the IHERG tail and h is the Hill coefficient for the fit.\nHalf-maximal voltage values for IHERG activation were obtained by fitting IHERG tail-voltage (I\u2013V) relations with a Boltzmann distribution equation of the form:where I is the IHERG tail amplitude following test potential Vm, Imax the maximal IHERG tail observed during the protocol, V0.5 the potential at which IHERG was maximally activated, and k is the slope factor describing IHERG activation. Data from each individual experiment were fitted by this equation to derive V0.5 and k values in \u2018control\u2019 and with doxepin. The resultant mean V0.5 and k values obtained from pooling values from each experiment were then used to calculate the mean activation relations plotted in Fig. 3.\nParameters describing voltage-dependent inactivation of IHERG were derived from fits to voltage-dependent availability plots with the equation:where \u2018inactivation parameter\u2019 at any test potential, Vm, occurs within the range 1\u20130, V0.5 is the voltage at which IHERG was half-maximally inactivated and k describes the slope factor for the relationship.\n3\nResults\n3.1\nDoxepin produces concentration-dependent inhibition of WT IHERG\nIHERG was elicited by the protocol shown in the inset of Fig. 1A, which is a standard protocol used to study IHERG pharmacology in this laboratory (e.g. [28,29,32]). Membrane potential was stepped from \u221280 to +20\u00a0mV for 2\u00a0s followed by a 4-s step to \u221240\u00a0mV to elicit IHERG tails. A brief (50\u00a0ms) pulse to \u221240\u00a0mV preceded the step to +20\u00a0mV in order to monitor the instantaneous current without activation of IHERG (peak outward IHERG tails on repolarization to \u201340\u00a0mV were compared with the instantaneous current during the 50\u00a0ms pulse to \u221240\u00a0mV, in order to measure IHERG tail amplitude). This voltage protocol was applied repeatedly (at 20\u00a0s intervals) prior to and during the application of doxepin. Fig. 1A shows a representative recording of IHERG in control, 5\u00a0min after the addition of 10\u00a0\u03bcM doxepin and 5\u00a0min following wash-out of the drug. Doxepin produced a substantial inhibition of IHERG; this was largely reversible upon washout (to 73\u00a0\u00b1\u00a09% of control in six cells to which 10\u00a0\u03bcM doxepin was applied). Fig. 1B shows records for the same cell before and at increasing times during doxepin exposure, indicating that blockade was maximal within 4\u20135\u00a0min of drug application. Fig. 1C shows a plot of mean\u00a0\u00b1\u00a0S.E.M. fractional block of IHERG tails by four different concentrations of doxepin, fitted by Eq. (2). The IC50 for doxepin inhibition of IHERG with this protocol was 6.5\u00a0\u00b1\u00a01.4\u00a0\u03bcM and the Hill coefficient for the fit was 1.0\u00a0\u00b1\u00a00.2. IHERG blockade by doxepin was not accompanied by statistically significant changes to the time-course of deactivation of the time-dependent component of IHERG tails on repolarization to \u221240\u00a0mV: bi-exponential fitting of the time-dependent tail current decline yielded fast time-constants of deactivation (\u03c4fast) of 203\u00a0\u00b1\u00a015 and 273\u00a0\u00b1\u00a021\u00a0ms in control solution and 10\u00a0\u03bcM doxepin, respectively (n\u00a0=\u00a06; p\u00a0>\u00a00.05, paired t-test), and slow time-constants of deactivation (\u03c4slow) of 1164\u00a0\u00b1\u00a0141 and 1499\u00a0\u00b1\u00a0131\u00a0ms (p\u00a0>\u00a00.1). Doxepin did not influence significantly the relative proportions of deactivating current described by the fast and slow time-constants of deactivation (the proportion of deactivating current fitted by the \u03c4fast was \u223c0.6 in both control and doxepin).\nIt has been suggested that pharmacological inhibition of IHERG by some drugs may vary between different stimulus protocols [33]. Therefore, we also investigated IHERG blockade by doxepin using an action potential (AP) voltage waveform [32,34]. The voltage-command used (shown in the lower panel of Fig. 2A) was a previously acquired, digitised AP from a rabbit ventricular myocyte. This was applied repeatedly (at 4\u00a0s intervals) from a holding potential of \u221280\u00a0mV [32]. The upper panel of Fig. 2A shows traces in the absence and presence of doxepin. Peak outward IHERG during the repolarizing phase of the AP was inhibited 60\u00a0\u00b1\u00a012% (n\u00a0=\u00a05) by 10\u00a0\u03bcM doxepin, which does not differ significantly from the extent of IHERG tail current blockade by this concentration obtained using the protocol shown in Fig. 1 (62\u00a0\u00b1\u00a04%; n\u00a0=\u00a06; p\u00a0>\u00a00.8; unpaired t-test).\nIn order to determine effects of doxepin on native IKr, three drug concentrations (1, 10 and 100\u00a0\u03bcM) were applied to ventricular myocytes under whole-cell patch clamp. The command protocol for these experiments (similar to [32]) is shown in Fig. 2B and IKr tails were monitored on repolarization from +20 to \u221240\u00a0mV. Under our conditions, the outward tail currents observed on repolarization to \u221240\u00a0mV were almost completely abolished by 1\u00a0\u03bcM dofetilide (91\u00a0\u00b1\u00a03% blockade; n\u00a0=\u00a07), verifying that these were carried by IKr with little or no contamination from overlapping currents. Fig. 2C shows representative IKr tails on repolarization from +20 to \u221240\u00a0mV in the absence and presence of 100\u00a0\u03bcM doxepin; at this concentration the current was largely abolished. Fig. 2D contains mean\u00a0\u00b1\u00a0S.E.M fractional block data for the tested doxepin concentrations, fitted by Eq. (2). The derived IC50 for doxepin inhibition of native IKr tails was 4.4\u00a0\u00b1\u00a00.6\u00a0\u03bcM, in good agreement with the observed inhibitory potency of doxepin on IHERG.\n3.2\nVoltage dependence of IHERG blockade by doxepin\nVoltage dependence of IHERG blockade by doxepin was determined by the application, in control and doxepin-containing solutions, of a series of 2\u00a0s duration depolarizing commands to a range of test potentials up to +40\u00a0mV [32]. Successive command pulses were applied at 20\u00a0s intervals. Representative control currents at selected command voltages are shown in Fig. 3Ai; currents from the same cell following equilibration in 10\u00a0\u03bcM doxepin are shown in Fig. 3Aii (upper traces; lower traces in each panel show corresponding voltage commands). At all test potentials, IHERG was inhibited by doxepin. For each of five similar experiments, fractional inhibition of IHERG tails following each voltage command was calculated using Eq. (1); the mean\u00a0\u00b1\u00a0S.E.M. fractional block of IHERG tails is plotted against command voltage in Fig. 3B. Also shown in Fig. 3B are voltage-dependent activation relations for IHERG in control solution and in the presence of doxepin (see Section 2). The derived mean V0.5 and k values were: control V0.5\u00a0=\u00a0\u221221.0\u00a0\u00b1\u00a03.1; doxepin V0.5\u00a0=\u00a0\u221224.0\u00a0\u00b1\u00a02.2, p\u00a0>\u00a00.1; control k\u00a0=\u00a05.9\u00a0\u00b1\u00a00.2; doxepin k\u00a0=\u00a07.5\u00a0\u00b1\u00a01.7, p\u00a0>\u00a00.3, with the IHERG activation relations in control and doxepin closely overlying one another (Fig. 3B). Fractional block of IHERG showed no statistically significant dependence on voltage over the range from \u221240 to\u00a0+\u00a040\u00a0mV (p\u00a0>\u00a00.2; Anova).\nThe voltage dependence of IHERG availability\/inactivation was assessed using a 3-step protocol similar to those used in previous IHERG investigations from our laboratory ([32,35], and shown schematically as an inset to Fig. 3C). Mean data from five experiments were corrected for deactivation [35] and the resulting values were plotted to give availability plots in the absence and presence of doxepin (Fig. 3C); the data-sets were fitted with Eq. (4) (see Section 2). In control and doxepin the inactivation V0.5 values were, respectively, \u221237.9\u00a0\u00b1\u00a02.4 and \u221243.5\u00a0\u00b1\u00a04.1\u00a0mV (ns, paired t-test), with corresponding k values of \u221216.2\u00a0\u00b1\u00a02.4 and \u221217.3\u00a0\u00b1\u00a08.8\u00a0mV (ns, paired t-test). Thus, doxepin did not alter the voltage-dependence of IHERG inactivation. Although there was a trend towards an acceleration in the time-course of IHERG inactivation, this did not attain statistical significance (inactivation time\u2013constant of 3.3\u00a0\u00b1\u00a01.2\u00a0ms in control versus 1.4\u00a0\u00b1\u00a00.6\u00a0ms in doxepin, p\u00a0>\u00a00.1; obtained from exponential fits to the inactivating phase of the current elicited during the third step of the protocol, following a brief hyperpolarizing step to \u221280\u00a0mV).\n3.3\nTime dependence of IHERG inhibition by doxepin\nGating-dependence of IHERG inhibition by doxepin was investigated further by the use of two protocols to examine the time-dependence of development of IHERG blockade. The first protocol used a sustained (10\u00a0s) depolarizing step to 0\u00a0mV from a holding potential of \u221280\u00a0mV (shown in Fig. 4A). This protocol was applied first in the absence of doxepin to elicit control IHERG. It was then discontinued whilst cells were equilibrated in 10\u00a0\u03bcM doxepin (for 7\u00a0min), after which it was re-applied. The first current trace recorded on resumption of stimulation was used to determine development of fractional blockade throughout the applied depolarization. Fig. 4A shows representative traces in control and doxepin; these traces diverged rapidly following depolarization, suggesting that IHERG blockade developed rapidly with time. Fig. 4B shows plots of mean\u00a0\u00b1\u00a0S.E.M. fractional block of IHERG at various time intervals throughout the applied depolarization (main panel; n\u00a0=\u00a08) and on an expanded time-scale to show development of blockade over the first 0.5\u00a0s (inset). These plots show clearly that IHERG blockade developed rapidly on depolarization, with little change in blockade after 200\u2013300\u00a0ms following the onset of the voltage-command. The time-course of development of blockade was well-described by a mono-exponential fit to the data, with a rate constant (K) for the fit of 16.33\u00a0s\u22121 (equivalent to a time-constant (1\/K) of 61\u00a0ms).\nAlthough the protocol used in Fig. 4 is well suited for examining the development of IHERG inhibition over a period of seconds following membrane depolarization, it is less well suited for accurate assessment of blockade of IHERG immediately following membrane depolarization than are protocols based on tail current measurements [29,32]. Therefore, a second protocol was also used [29]. Membrane potential was held at \u2013100\u00a0mV and, from this, 10 and 200\u00a0ms duration depolarizations to +40\u00a0mV were applied, each followed by a period at \u221240\u00a0mV to monitor IHERG tails [29]. Fig. 5A shows representative currents from the same cell activated by 10\u00a0ms (Fig. 5Ai) and 200\u00a0ms (Fig. 5Aii) commands in the absence and presence of doxepin. Some IHERG tail inhibition was evident following the 10\u00a0ms duration command, with further blockade evident after the 200\u00a0ms command. Fig. 5B shows mean fractional block of IHERG tails for five similar experiments. Whilst blockade for 200\u00a0ms commands was significantly greater than that for 10\u00a0ms commands (p\u00a0<\u00a00.02), the occurrence of some blockade with only a very brief duration depolarizing command is concordant with either a very rapidly developing gating-dependent blockade on depolarization or with a contribution of closed-channel block to the overall effect of doxepin [25,29,36].\n3.4\nEffect of inactivation-attenuating mutants on IHERG inhibition by doxepin\nIn order to investigate further the gating-dependence of IHERG blockade by doxepin, experiments were performed using two inactivation-attenuating HERG mutants: S631A and N588K. Residue S631 is located towards the outer mouth of the HERG channel pore, and the S631A (serine\u00a0\u2192\u00a0alanine) mutation has been reported to shift IHERG inactivation by \u223c+100\u00a0mV [26]. Residue N588 is located in the external S5-Pore linker of the channel, and the N588K (asparagine\u00a0\u2192\u00a0lysine) mutation, which is responsible for one form of the recently identified genetic short QT syndrome [27], has been reported to shift IHERG inactivation by \u223c+60 to +100\u00a0mV [35,37]. The effect of each mutation on the potency of IHERG blockade by doxepin was determined using the same protocol as was used to establish concentration-dependence of WT IHERG blockade (Fig. 1). Fig. 6Ai shows representative currents in control and 10\u00a0\u03bcM doxepin for WT\u2013HERG, whilst Fig. 6Aii and Aiii show similar records for S631A\u2013HERG and N588K\u2013HERG, respectively. In contrast to WT\u2013HERG, for both S631A\u2013HERG and N588K\u2013HERG the IHERG tail magnitude was substantially smaller than the maximal current during the voltage-command (Fig. 6Aii and Aiii, respectively) reflecting the greatly attenuated IHERG inactivation of these HERG mutants [26,35,37]. Both mutant channels retained the ability to be inhibited by doxepin. Exponential fits to currents activated on depolarization to +20\u00a0mV for both mutants showed similar time-courses of current activation in control solution and following equilibration with 10\u00a0\u03bcM doxepin (S631A control: 60.6\u00a0\u00b1\u00a05.5\u00a0ms; doxepin: 64.5\u00a0\u00b1\u00a08.0\u00a0ms, p\u00a0>\u00a00.5; N588K control: 49.8\u00a0\u00b1\u00a06.2; doxepin: 41.7\u00a0\u00b1\u00a09.9\u00a0ms, p\u00a0>\u00a00.5; n\u00a0=\u00a05 for both). Three doxepin concentrations (1, 10 and 100\u00a0\u03bcM) were tested to obtain concentration-response data. Fig. 6B shows concentration-response relations (determined using Eq. (2)) for IHERG tail inhibition for both S631A\u2013HERG and N588K\u2013HERG, with corresponding data for WT\u2013HERG plotted for comparison. Although the S631A and N588K mutations produced small and modest increases, respectively, in the IC50 derived from the fits to the concentration-response relations (from 6.6\u00a0\u03bcM for WT to 8.6\u00a0\u03bcM for S631A\u2013HERG and 12.6\u00a0\u03bcM for N588K\u2013HERG), these differences did not attain statistical significance (p\u00a0>\u00a00.05; Anova). These observations indicate the IHERG blockade by doxepin was not highly sensitive to attenuation of HERG channel inactivation.\n3.5\nSensitivity of IHERG inhibition by doxepin to the S6 mutations Y652A and F656A\nTwo aromatic amino-acid residues, Y652 and F656, have been shown to be important components of the drug-binding site for a variety of HERG channel blockers [5,9,38]. Accordingly, we investigated whether or not doxepin inhibition of IHERG was sensitive to mutation of either residue, adopting a similar approach and protocols to those used in other recent IHERG pharmacology studies [29,32,39\u201341]. Fig. 7 shows the effects on doxepin inhibition of IHERG of the mutation Y652A (tyrosine\u00a0\u2192\u00a0alanine). The experimental voltage protocol used (Fig. 7Aiii) was identical to that used for WT IHERG in Fig. 1 and to investigate the S631A and N588K mutants in Fig. 6. Representative traces showing the effect of 10\u00a0\u03bcM doxepin on WT IHERG and Y652A IHERG are shown in Fig. 7Ai and Aii. WT IHERG was inhibited substantially by this doxepin concentration (Fig. 7Ai). In contrast, inhibition of Y652A IHERG was noticeably reduced (Fig. 7Aii; 28\u00a0\u00b1\u00a03% peak tail current inhibition compared to 62\u00a0\u00b1\u00a04% for WT IHERG; p\u00a0<\u00a00.001). Four other doxepin concentrations (30, 100, 300\u03bcM and 1\u00a0mM) were also tested in order to construct a concentration response relation for inhibition of Y652A IHERG tails (Fig. 7B). A fit to the data with Eq. (2) (dashed line) yielded an IC50 for inhibition of Y652A IHERG of 27.8\u00a0\u00b1\u00a08.8\u00a0\u03bcM (with a Hill coefficient of 0.9\u00a0\u00b1\u00a00.3), which represented a modest, though significant (p\u00a0<\u00a00.05), \u223c4.2 fold increase in IC50 over that for WT IHERG.\nIHERG carried by F656A\u2013HERG channels was studied using the protocol shown in the inset of Fig. 8Aiii and inward tails were measured at \u2212120\u00a0mV [25,28,29]. Initial experiments employed a doxepin concentration of 100\u00a0\u03bcM (shown in Fig. 1 to produce extensive inhibition of WT IHERG with a standard [K+]e). Representative traces showing effects of this concentration on WT and F656A IHERG are shown in Fig. 8Ai and Aii. The F656A mutation produced a modest attenuation of IHERG inhibition by this concentration of doxepin. However, as observed previously for some other drugs (e.g. [28]), the extent of inhibition of WT IHERG by 100\u00a0\u03bcM doxepin under conditions of high [K+]e (see Section 2) was significantly smaller (56\u00a0\u00b1\u00a05%) than that observed with the same concentration in the experiments with a standard [K+]e in Fig. 1 (93\u00a0\u00b1\u00a04%; p\u00a0<\u00a00.001). Therefore, the effects of two further concentrations of doxepin (500\u00a0\u03bcM and 1\u00a0mM) were also examined. Fig. 8B shows bar-chart plots of the mean levels of inhibition of WT and F656A IHERG by the three doxepin concentrations. These concentrations resulted in progressive increases in the level of blockade of WT IHERG. In contrast, although Anova comparison of the data with the three drug concentrations confirmed that doxepin inhibition of F656A IHERG was concentration-dependent (p\u00a0<\u00a00.001), the observed concentration-dependence was unusual: there was no statistically significant difference between the observed level of blockade of F656A IHERG between 100 and 500\u00a0\u03bcM, whilst at 1\u00a0mM the observed level of inhibition of F656A IHERG was markedly increased (p\u00a0<\u00a00.001 compared to each of 100 and 500\u00a0\u03bcM, Bonferroni post-test) and approached that of WT IHERG. Taken together, the data with the three doxepin concentrations suggest that the F656A mutation exerted some influence on the ability of doxepin to inhibit IHERG, though the F656A data did not appear to follow a conventional monotonic concentration dependence.\n4\nDiscussion\nDespite a strong association between TCA use and QT interval lengthening [42], and although doxepin itself has been linked with both QT interval prolongation and TdP [18\u201320], the effects of this drug on HERG K+ channels have not hitherto been reported. Moreover, whilst IHERG blockade has been investigated previously for the TCAs imipramine and amitriptyline [21\u201323], to our best knowledge the present study is the first in which molecular determinants of IHERG inhibition have been investigated for any member of the TCA family.\n4.1\nCharacteristics of IHERG blockade by doxepin\nPreviously, imipramine has been reported to inhibit IHERG recorded in experiments using a mammalian expression system with an IC50 of 3.4\u00a0\u03bcM [23] whilst an IC50 of 10\u00a0\u03bcM was reported for amitriptyline [21]. A different study of amitriptyline, using the Xenopus oocyte expression system, reported IC50's of \u223c3.3\u20134.8\u00a0\u03bcM (depending on [K+]e) [22]. Thus, the potency of doxepin as an IHERG inhibitor found here (IC50 of 6.5\u00a0\u03bcM) is broadly comparable to that seen previously for these other two TCAs. However, in terms of the characteristics of observed IHERG blockade, doxepin appears to be closer to imipramine than to amitriptyline: amitriptyline block of IHERG has been reported to show significant voltage-dependence [22] whereas imipramine showed only weak voltage-dependence [23]. Moreover, no significant effects of doxepin on the voltage-dependence of activation or inactivation were seen in this study. Unfortunately, comparative data for the TCAs imipramine and amitriptyline are lacking [21\u201323] and, therefore, a direct comparison between doxepin and these agents cannot be made in this regard. However, the atypical tetracyclic antidepressant maprotiline has recently been reported to show no alteration to the voltage-dependence of activation and inactivation [43], though another study contradicted this with regard to activation [44]. The lack of a significant leftward shift in the voltage-dependence of inactivation with doxepin in our study suggests that this agent does not act to stabilise IHERG inactivation, and the time-course of WT IHERG inactivation was not significantly accelerated by the drug. Moreover, the results with inactivation-attenuating mutations to two residues from distinct parts of the channel (S631A: outer mouth of the channel pore; N588K: in the S5-Pore linker) provide evidence that IHERG inactivation does not play an obligatory role in doxepin's binding to the channel. This is further supported by unaltered levels of WT IHERG blockade with progressive depolarization over voltages at which IHERG was maximally activated, but over which inactivation increased (Fig. 3C and D). Doxepin is quite distinct in this regard from a number of other drugs, including the archetypal high affinity HERG blocking methanesulphonanilide drugs E-4031 and dofetilide, for which IHERG inactivation exerts a strong influence on blocking potency \u2013 either as a direct consequence of inactivation-state dependent block, or due to conformational changes during inactivation facilitating optimal orientation of S6 helical residues to which drugs bind [10,45\u201348].\nBlockade of IHERG by imipramine has been reported to develop rapidly during a sustained depolarization, with a component of inhibition visible even for comparatively brief depolarizing voltage commands [23]. These features of imipramine's action correspond well with those seen for doxepin in the present study. They are also similar to those reported for the serotonin-selective reuptake inhibitors (SSRIs) fluvoxamine and citalopram [25,49], but differ significantly from the methanesulphonanilides, for which little blockade is observed immediately upon depolarization, with blockade then increasing progressively during the maintained depolarization [24,49,50]. The lack of a strong dependence of HERG channel blockade by doxepin on IHERG inactivation suggests that gating-dependent blockade by this drug is likely to arise predominantly to activated\/open channels. Accordingly, the observed time-dependence of IHERG inhibition by doxepin in this study is consistent with either a mixed state-dependence of blockade (with components of both closed and open channel blockade) or with the presence of a very rapidly developing component of activation-dependent inhibition immediately on depolarization.\nFor the majority of drugs that have been studied, one or both of the Y652 and F656 aromatic amino acid residues in the S6 helices of the HERG channel comprise key elements of the drug binding site [5,9,38,51]. For example, the Y652A and F656A mutations increased the IC50 for HERG blockade by the methanesulphonanilide MK-499 by 94-fold and 650-fold, respectively, and that for terfenadine by \u223c100-fold [8]. In comparison, the \u223c4-fold increase in IC50 for doxepin produced by the Y652A mutation in this study, is rather modest, suggesting that this residue is less influential for binding of doxepin than for either of these high affinity blockers. The unusual concentration dependence seen with the F656A mutation indicated that at a high doxepin concentration of 1\u00a0mM IHERG blockade was little affected by mutation of this residue, whilst at concentrations of 100 and 500\u00a0\u03bcM significant attenuation of blockade occurred with no significant increase in block at 500\u00a0\u03bcM compared to 100\u00a0\u03bcM. It was not possible to obtain an adequate fit of these data with Eq. (2). A cautious interpretation of the lack of a conventional concentration-dependence of F656A\u2013HERG inhibition by doxepin is that blockade may depend partly, but incompletely, on this residue. Neither the Y652A nor F656A mutations attenuated blockade by doxepin concentrations producing very high levels (>90%) of blockade of WT IHERG, suggesting that neither residue is absolutely obligatory for doxepin binding to HERG channels to occur. Whilst unusual, this is not unprecedented; IHERG block by both the SSRI fluvoxamine and the antiarrhythmic agent dronedarone has been reported to be only partially attenuated by the Y652A and F656A mutations [25,28]. Comparative data for other TCAs are lacking, though recently these residues have been implicated in IHERG inhibition by the tetracyclic drug maprotiline [43,44]; one of these two studies obtained IC50 values for the Y652A and F656T mutants, with respective (modest) 3-fold and 7-fold increases in IC50\n[43]. Whilst obligatory molecular determinants of doxepin-binding to HERG remain to be found, the fact that IHERG inhibition by doxepin developed progressively over several minutes following rapid external solution exchange is consistent with the drug crossing the cell membrane to reach its site of action. The reduced IHERG inhibition by doxepin of WT IHERG in the presence of raised [K+]e may also be of significance. Since IHERG inhibition by doxepin appears not to be critically dependent on channel inactivation, decreased blockade with high [K+]e is unlikely to result from any effect of [K+]e on IHERG inactivation. Rather, reduced inhibition in high [K+]e may be accounted for by an interference with drug-binding due to an electrostatic repulsion or \u201cknock-off\u201d process [28,52], consistent with drug binding to the channel at a site close to the ion conduction pathway. It remains to be determined whether this site would need to reside within the channel pore. However, in a limited series of experiments using the D540K mutant [53], we did not find evidence that doxepin can readily unbind on hyperpolarization-induced channel opening (data not shown); though not all drugs that bind within the pore exhibit marked \u2018untrapping\u2019 [54]. Nevertheless, given the presence of a significant component of IHERG blockade with brief depolarization and incomplete attenuation of inhibition by the Y652A and F656A mutations, we cannot exclude the possibilities that a proportion of the observed blockade with doxepin involves binding outside of the channel pore, or binding to closed HERG channels.\n4.2\nClinical significance of IHERG blockade by doxepin\nTCAs are associated with QTc interval lengthening in clinical use [42], and IHERG inhibition by doxepin observed in the present study is consistent both with documented cases of QT interval prolongation and TdP with doxepin [18\u201320] and with QT interval increases seen in anaesthetised guinea-pigs receiving doxepin infusion [15]. Our study was conducted at a physiologically relevant temperature and the extent of IHERG blockade by doxepin was similar between ventricular action potential and conventional voltage step protocols. A lack of relief of IHERG blockade on membrane hyperpolarization for the D540K mutant (mentioned in Section 4.1, above) is suggestive of relatively poor drug unbinding at negative voltages in the maintained presence of drug, whilst the data shown in Fig. 5 indicate that at 10\u00a0\u03bcM (the same concentration as used for the AP clamp experiment in Fig. 2) doxepin was able to bind and inhibit IHERG rapidly on membrane depolarization to a positive voltage. These factors may combine to account for the similar levels of IHERG inhibition by doxepin observed with step and AP clamp protocols. Although we did not co-express HERG with MiRP1, a putative \u03b2 subunit suggested to be necessary to recapitulate native IKr\n[55], it has recently been suggested that MiRP1 is unlikely to interact with HERG outside of the cardiac conduction system [51] and, additionally, the pharmacological sensitivity of HERG channels expressed in mammalian cells without MiRP1 co-expression has been found to be similar to that of native IKr\n[56]. This notion is reinforced by the close concordance of inhibitory potency of doxepin on IHERG and native IKr in our experiments (IC50 values of 6.5 and 4.4\u00a0\u03bcM, respectively). A question therefore arises as to the relationship between the potency of IHERG\/IKr blockade seen in this study and plasma concentrations of doxepin in patients. As a class, the TCAs are lipophilic and are known to become concentrated in some tissues, including the myocardium [57]. In the case of doxepin, one experimental study has reported doxepin concentration in cardiac muscle to be 41-fold higher than plasma levels [58]. This makes it difficult to extrapolate with accuracy from known plasma concentrations to likely levels of IHERG\/IKr blockade by doxepin in vivo. The therapeutic plasma level of doxepin is thought to be between 50 and 250\u00a0ng\/ml (0.16\u20130.8\u00a0\u03bcM), although a wide variety of recommendations from university psychiatric departments and laboratories (up to 1000\u00a0ng\/ml; 3.2\u00a0\u03bcM) have been reported [59]. Whilst IHERG (or IKr) blockade at the lower end of this range might be anticipated to be modest, inhibition at higher concentrations would be significant and, taking into account also potential cardiac accumulation, the observed potency of IHERG inhibition by doxepin in this study is likely to be clinically relevant, particularly in overdose. Such an effect may be exacerbated in individuals exhibiting pre-existing QT interval prolongation (congenital or acquired), electrolyte abnormalities or with impaired drug metabolism. Thus, as for other IHERG blocking medications, caution is warranted in its use in patients with pre-existing QT interval prolongation or with risk factors likely to exacerbate the effects of IHERG blocking medications.\nThe findings of this study have further clinical relevance in a second, perhaps less expected, respect. The attenuated-inactivation N588K\u2013HERG mutant used in this study has been shown recently to underlie the SQT1 familial form of the recently identified genetic \u2018Short QT syndrome\u2019, which carries a risk of cardiac arrhythmia and sudden death [27,60]. Pharmacological approaches to correcting the QT-interval of SQT1 patients are currently very limited. These patients are comparatively insensitive to Class III IKr\/HERG blocking drugs [27,61] and the N588K\u2013HERG blocking potencies of the IKr\/HERG blockers E-4031and D-sotalol are reduced \u223c12\u201320-fold compared to their effects on WT\u2013HERG [62,63], presumably due to a role (direct or otherwise) of channel inactivation in facilitating drug binding to the HERG channel. To date, only the Class Ia antiarrhythmic drug quinidine has been found both to inhibit N588K\u2013HERG effectively and to correct the QT interval in such patients [27,62,63]; however, very recently, another Class Ia antiarrhythmic, disopyramide, has been shown to be effective against N588K\u2013HERG in vitro [63]. The present study identifies doxepin as both an IHERG-blocker for which channel inactivation does not play a major role in drug binding and as an additional drug that is an effective inhibitor of N588K\u2013HERG. Whilst the sedative effects of doxepin may make it unsuitable as a corrective treatment for SQT1 patients, our findings prompt the question as to whether chemical structures related to doxepin might feasibly offer viable IHERG blocking agents in SQT1.","keyphrases":["herg","potassium channel","antidepressant","doxepin","rapid delayed rectifier","qt interval","arrhythmia","short qt syndrome","torsade de pointes","i kr","long qt syndrome","qt-prolongation"],"prmu":["P","P","P","P","P","P","P","P","P","M","M","U"]}
{"id":"Diabetologia-4-1-2292427","title":"What is the mechanism of microalbuminuria in diabetes: a role for the glomerular endothelium?\n","text":"Microalbuminuria is an important risk factor for cardiovascular disease and progressive renal impairment. This holds true in the general population and particularly in those with diabetes, in whom it is common and marks out those likely to develop macrovascular disease and progressive renal impairment. Understanding the pathophysiological mechanisms through which microalbuminuria occurs holds the key to designing therapies to arrest its development and prevent these later manifestations.\nIntroduction\nThe associations between microalbuminuria, cardiovascular disease and progressive renal impairment are well described, but how these are linked mechanistically is something of a conundrum [1]. Here we focus on the pathogenesis of microalbuminuria in patients with diabetes, in whom it occurs commonly and has particular significance. In type 1 diabetes the prevalence gradually increases from onset of disease (6% after 1\u20133\u00a0years), reaching over 50% after 20\u00a0years [2]. In type 2 diabetes the prevalence is 20\u201325% in both newly diagnosed and established diabetes [3]. However, it is also instructive to review the general epidemiology of microalbuminuria, including those conditions with which it is associated and those for which it is a risk factor. Such an analysis reveals generalised endothelial dysfunction as a common denominator in microalbuminuria in both the general and diabetic populations. In 1989, this observation led to the hypothesis that a common process underlies both microalbuminuria and generalised endothelial dysfunction in diabetes. This process was suggested to be the dysregulation of enzymes involved in metabolism of extracellular matrix, the \u2018Steno hypothesis\u2019 [4].\nNearly 20\u00a0years on from the Steno hypothesis, the determinants of selective glomerular permeability to proteins at the cellular and molecular level are much better understood. In particular, the importance of podocyte-specific proteins in the regulation of selective permeability has been recognised. Similarly, much is now known about the biochemical derangements important in the pathogenesis of diabetic complications. We draw together these elements to consider the pathophysiological mechanisms through which diabetes exerts its effects on glomerular permeability in the initiating stages of diabetic nephropathy, i.e. at or before the appearance of microalbuminuria. These early changes establish the milieu in which the more advanced changes of overt diabetic nephropathy develop. Defining the mechanistic links from biochemical derangements to the appearance of increased urinary albumin highlights key elements in the pathophysiological pathway of the development of both diabetic nephropathy and micro- and macrovascular disease elsewhere.\nWe hold with the established view that increased transglomerular passage of albumin is the major source of microalbuminuria [5]. While other hypotheses have been advanced, for example, failure of tubular reuptake of albumin, none are sufficiently robust to seriously challenge this position.\nIn both general and diabetic populations, conditions associated with endothelial damage predispose to microalbuminuria\nIn the general (non-diabetic) population, hypertension is the major risk factor for microalbuminuria, and the prevalence of microalbuminuria in essential hypertension is around 25%. Individuals with essential hypertension who develop microalbuminuria have a higher incidence of biochemical disturbances, implying that hypertension per se may not be the cause of microalbuminuria, but, rather, these additional derangements [6]. Microalbuminuria is strongly associated with vascular disease in hypertensive patients, suggesting that it is a marker of vascular and\/or endothelial damage in this condition [7].\nThe insulin resistance syndrome describes a clustering of disorders the underlying pathology of which is thought to be related to insulin resistance and\/or endothelial dysfunction [8]. Microalbuminuria is associated with several of the disturbances found in the insulin resistance syndrome, including endothelial dysfunction and obesity, in addition to type 2 diabetes. Proinflammatory cytokines produced by visceral adipocytes (adipokines) have recently emerged as important mediators of the increased cardiovascular risk associated with the insulin resistance syndrome. These adipokines represent a possible link from insulin resistance and obesity to microalbuminuria in the non-diabetic population.\nMicroalbuminuria can be detected in patients undergoing major surgery, particularly when complicated by sepsis [9], and is associated with other inflammatory states, including rheumatoid arthritis and inflammatory bowel disease [10]. Microalbuminuria can also be detected in a significant proportion of the normal non-diabetic, normotensive population (6.6% in one study [11]), where it also associates with cardiovascular disease. Male sex [11] and hormone replacement therapy in women [12] seem to increase susceptibility to microalbuminuria, and although the basis for this is not clear, the fact that men have a higher incidence of vascular disease in general implies a common aetiology.\nHypertension is approximately twice as frequent in individuals with diabetes as in those without, and hypertensive individuals are predisposed to the development of diabetes [13]. Hypertension is certainly a major determinant of microangiopathy in diabetes, but the relationship between hypertension and microalbuminuria in diabetes is complex. Hypertension and microalbuminuria often coexist in diabetic patients, and reducing blood pressure reduces microalbuminuria in type 1 diabetes [14]. However, it is unclear whether hypertension contributes to the development of microalbuminuria in diabetes. At least in type 1 diabetes, hypertension and microalbuminuria appear to develop together: in longitudinal studies there is no evidence that hypertension develops before microalbuminuria [15]. This is more difficult to demonstrate in type 2 diabetes, perhaps because of the heterogeneity of the disease.\nOne third of patients with type 1 diabetes develop advanced nephropathy, and the renal status of probands of diabetic patients makes a difference of nearly 50% in risk [16]. A family history of hypertension also predisposes to microalbuminuria in diabetes [17]. These observations suggest a genetic predisposition in individuals with type 1 diabetes who develop diabetic nephropathy, but as yet no gene has been identified. Perhaps most significant is that this and other evidence suggests that the susceptibility of type 1 diabetic patients to diabetic nephropathy is increased in response to increased genetic risk of insulin resistance [18]. Consequently, it is the combination of this genetic risk with hyperglycaemia and its sequelae that leads to microalbuminuria and, eventually, diabetic nephropathy.\nThus, in the diabetic as well as in the general population the risk factors for the development of microalbuminuria can be grouped into those associated with vascular disease, including endothelial dysfunction, inflammation and insulin resistance. This implies that microalbuminuria may also, at least in these situations, result from endothelial dysfunction. This does not preclude significant damage to other components of the vessel wall, including basement membrane, pericytes, matrix components (collagen, elastin) and vascular smooth muscle cells. However, as the principal regulators of vascular permeability, endothelial cells are likely to be involved in systemic increases in permeability.\nMicroalbuminuria is a risk factor for macro- and microangiopathy, including advanced diabetic nephropathy\nMicroalbuminuria is a significant risk factor for cardiovascular mortality in type 1 [19] and 2 diabetes [20], as well as in the non-diabetic population [21]. In type 1 diabetes, microalbuminuria is closely associated with microangiopathy elsewhere (e.g. retinopathy). Although this association is also present in type 2 diabetes, in a proportion of these patients microalbuminuria does not appear to reflect generalised microvascular damage [22].\nMicroalbuminuria predicts the development of overt diabetic nephropathy in type 1 and 2 diabetes; however, the relationship in type 2 diabetes is less clear because of the greater heterogeneity of this condition and the presence of other risk factors for microalbuminuria in these, usually elderly, patients [23]. Microalbuminuria invariably precedes overt diabetic nephropathy, and although microalbuminuria may regress spontaneously in a proportion of cases, it remains the best documented predictor for high risk of development of diabetic nephropathy in both type 1 and type 2 diabetes [24].\nThe relationship between microalbuminuria and vascular disease suggests a common causality, as established risk factors explain at most a small part of these associations [25]. Unifying mechanisms, such as generalised endothelial dysfunction or inflammation, are therefore implicated [8]. Indeed, AER is correlated with endothelial dysfunction in type 1 and type 2 diabetes, and in the non-diabetic population [26]. Markers of chronic low-grade inflammation, including C-reactive protein (CRP), are also correlated with microalbuminuria in type 1 and type 2 diabetes [27], and increasing evidence from the non-diabetic population indicates the importance of inflammation in the pathogenesis of cardiovascular disease [28].\nGeneralised and glomerular endothelial dysfunction\nMarkers of endothelial dysfunction, including elevated serum von Willebrand factor (vWF) and increased transcapillary albumin escape rate, are present before the onset of microalbuminuria in type 1 diabetes and worsen in association with it [27, 29]. Type 2 diabetes is often complicated by the presence of other risk factors for vascular disease, and discerning the contribution of hyperglycaemia and its sequelae to endothelial dysfunction in this condition is more difficult. While in some type 2 diabetic patients microalbuminuria may occur in the absence of evidence of endothelial dysfunction, in others, vWF levels predict its development [30]. The close association between endothelial dysfunction and microalbuminuria in type 1 diabetes may underlie the predictability of development of diabetic nephropathy and the greater susceptibility to micro- and macrovascular disease in other organs.\nAs endothelial dysfunction is an important antecedent of microalbuminuria in both types of diabetes (albeit with a less predictable relationship in type 2), it provides an attractive explanation for the association between microalbuminuria and vascular disease in diabetes (Fig.\u00a01). But to what extent can endothelial dysfunction be said to cause microalbuminuria? As the glomerular endothelium is exposed to the same diabetic milieu as other endothelia, it is highly likely that it is also dysfunctional. This begs the question of how glomerular endothelial dysfunction could lead to microalbuminuria. To address this we now turn to consider the structure and function of the glomerular filtration barrier (GFB).\nFig.\u00a01The relationship between hyperglycaemia, insulin resistance, endothelial dysfunction, macrovascular disease and microalbuminuria in type 1 and type 2 diabetes. Proposed major pathways are represented by red arrows; those of less certain significance by black arrows. The diagram illustrates, for the example, a possible mechanism for the increased risk of microalbuminuria in patients with type 1 diabetes and a susceptibility to insulin resistance. Particularly in type 2 diabetes, other pathways, not directly involving endothelial dysfunction, are likely in the pathogenesis of macrovascular disease and may also contribute to microalbuminuria (broken arrows)\nThe glomerular filtration barrier is a complex biological sieve\nUnlike other capillaries, glomerular capillaries have a high permeability to water (hydraulic conductivity) yet, like other capillaries, are relatively impermeable to macromolecules. These fundamental permeability properties depend on the unique, three-layer structure of the GFB: the endothelium with its glycocalyx, the glomerular basement membrane (GBM) and podocytes (glomerular epithelial cells; Fig.\u00a02) [31].\nFig.\u00a02Representation of a cross-section through the GFB showing the three-layer structure consisting of glomerular endothelium and glycocalyx, glomerular basement membrane (GBM) and podocyte foot processes. Albumin, represented by orange ellipses, does not pass through the normal GFB in significant amounts\nGlomerular endothelial cells Glomerular endothelial cells are highly specialised cells with regions of attenuated cytoplasm punctuated by numerous fenestrae, circular transcellular pores 60\u201380\u00a0nm in diameter [31, 32]. Initially these fenestrations were thought of as empty and to therefore provide little barrier to the passage of proteins. Standard fixation protocols for electron microscopy do not preserve the glycocalyx, but newer fixation techniques have allowed the demonstration of a glomerular endothelial glycocalyx of 200\u2013400\u00a0nm in thickness [33, 34]. This glycocalyx covers both fenestral and inter-fenestral domains of the glomerular endothelial cell luminal surface.Studies of the systemic endothelial glycocalyx are instructive here. The glycocalyx is a dynamic, hydrated layer largely composed of glycoproteins and proteoglycans with adsorbed plasma proteins. Heparan sulphate proteoglycans (HSPGs) are largely responsible for the negative charge characteristics of the glycocalyx. Removal of the glycocalyx increases vascular protein permeability, providing evidence that it hinders the passage of macromolecules [35, 36]. Compared with those without, capillaries with fenestrations are much more permeable to water and small solutes, but not to proteins. These characteristics can only be explained by the glycocalyx [37].Therefore, the presence of a significant glomerular endothelial glycocalyx implies that the glomerular endothelium significantly contributes to the barrier to macromolecules [31, 32, 38]. Experimental data support this view: in mice treated with glycocalyx-degrading enzymes the distance between luminal lipid droplets and the glomerular endothelium was decreased, and this was accompanied by an increase in AER [39]. In rats, under normal perfusion conditions albumin is confined to the glomerular capillary lumen and endothelial fenestrae [40]. Endothelial glycocalyx has the correct anatomical distribution (on the surface of endothelial cells, including in fenestral openings) to explain this distribution. Reactive oxygen species (ROS), which are known to disrupt the glycocalyx [41], cause torrential proteinuria without any identifiable structural changes in the GFB using standard electron microscopy techniques [42]. Furthermore, the ability of human glomerular endothelial cell glycocalyx to form a permeability barrier to macromolecules can be directly demonstrated in vitro [43].\nThe GBM The GBM is a basal lamina specialised for the structural requirements of the GBM and its filtration function. It is a hydrated meshwork of collagens and laminins to which negatively charged HSPGs are attached. Traditional concepts have therefore characterised the GBM as a charge-selective barrier. However, more recent analyses indicate that it only makes a small direct contribution to the barrier to protein passage [31].\nPodocytes Podocytes, or, more specifically, their interdigitating foot processes, form the outer layer of the GFB (Fig.\u00a02). The gaps between adjacent foot processes, the \u2018filtration slits\u2019 (25\u201360\u00a0nm), are spanned by the slit diaphragm. This is a molecular structure thought to form the most restrictive barrier to the passage of water and macromolecules. The effect of mutations in podocyte-specific proteins (e.g. nephrin mutations result in congenital nephrotic syndrome) indicate the importance of podocytes in resisting the passage of protein [44]. However, exactly how podocytes and their foot processes contribute to selective permeability is not yet clear. Electron microscopy studies have suggested that the slit diaphragm has a porous structure with a pore half-width of 2\u00a0nm. However, this figure is too small to account for the experimentally observed passage of solutes of various radii, emphasising that our understanding of slit diaphragm function is incomplete [45]. Podocyte biology has been reviewed in detail elsewhere [44].\nPassage of albumin across the normal GFB\nFiltration Water filters across the GFB via gaps through or between cells rather than through the cell cytoplasm. Therefore, the high hydraulic conductivity of the GFB depends on the presence of fenestrae and filtration slits. Fifty per cent of the hydraulic resistance of the GFB is afforded by the two cell layers (endothelium and podocyte foot processes), and 50% by the GBM [46].\nSolute flux Solute flux (including albumin) from the glomerular capillary occurs by a combination of convection (i.e. being swept along by the filtration of water) and diffusion [31]. The rate of convection of a solute depends on the filtration rate and on its reflection coefficient. Reflection coefficients are related to solute size, larger solutes having higher reflection coefficients. A value of 1 indicates that a molecule is totally excluded. The rate of diffusion of a solute depends on the concentration gradient and its diffusivity (solute permeability). The diffusivity of a solute decreases with increasing size. A measure of overall flux of a solute is given by the sieving coefficient: the filtrate to plasma concentration ratio. The sieving coefficient for albumin across the normal GFB is <0.001 [47]. The ratio of the contribution of convective to diffusive flux (the Peclet number) increases with increasing molecular size. That is, for small molecules, diffusion dominates; for larger molecules, convection dominates. The Peclet number for albumin across the normal GFB is near to unity, so both diffusion and convection contribute [48]. Hence, an increase in GFR will result in an increase in the convective flux of albumin. However, despite this, the best estimates indicate that even a 50% increase in filtration rate would only increase urinary albumin in the sub-microalbuminuric range, as the majority of the excess albumin is reabsorbed by the tubules [47]. Therefore, for albumin flux to increase sufficiently to produce microalbuminuria (assuming normal tubular reuptake), the GFB must be physically altered in such a way as to increase the sieving coefficient of albumin across it.Models have been developed to correlate the macromolecular sieving properties of the GFB with the structure and properties of its components [31]. These indicate that the GFB functions as a whole, with each layer having an important contribution to selective permeability. Whereas hydraulic resistances are essentially additive, sieving coefficients are multiplicative. This relationship means that a change in one element significantly affects the overall protein permeability to the same degree, i.e. a 10% change in permeability of any one layer will produce a 10% change in overall GFB permeability. Importantly, this means that, regardless of which layer is most restrictive, a change in the permeability of any layer of the GFB could potentially account for microalbuminuria.\nStructural alterations in the GFB associated with microalbuminuria in diabetes\nGlomerular structural changes typical of diabetic nephropathy are commonly established by the time microalbuminuria becomes apparent [49, 50]. However, the changes seen are heterogeneous, and all may be found in normoalbuminuric diabetic patients [51]. Early changes described include an increase in glomerular size, GBM thickening, mesangial expansion and broadening of podocyte foot processes [49\u201351]. The increase in glomerular size is due both to mesangial expansion and to enlargement in glomerular capillaries. The latter occurs at least in part through angiogenesis, an endothelium-dependent process [52, 53]. Glomerular structural changes are less marked in type 2 diabetes, with only a third conforming to the classical pattern observed in type 1 diabetes [22].\nSpecific assessment of structural changes in glomerular endothelial cells and associated glycocalyx in diabetes has not been performed. Evidence is emerging, however, that total systemic glycocalyx volume is reduced by acute hyperglycaemia in humans [54]. Furthermore, type 1 diabetic patients have decreased systemic glycocalyx volume, and this correlates with the presence of microalbuminuria [55]. GBM thickening alone, without change in composition, does not significantly affect its protein permeability characteristics.\nBroadening of podocyte foot processes (effacement) is an indicator of podocyte injury but generally correlates poorly with degree of proteinuria [56]. Indeed, proteinuria may occur in the complete absence of structural changes to podocytes [57]. This is the case with diabetic microalbuminuria, which can occur in the absence of such changes, at least in type 2 diabetes [58]. Some have suggested that podocyte loss occurs in early diabetes and that this would contribute to the filtration barrier defect [51]. Others have argued that significant early podocyte loss does not occur [59]. A likely reconciliation is that the lower proportion of podocytes seen in early disease is due to a relative increase in mesangial and endothelial cells, while podocyte loss occurs at a later stage.\nFunctional alterations in GFB selective permeability associated with microalbuminuria in diabetes\nIncreased flux of albumin across the GFB in diabetic microalbuminuria can be confirmed in experimental models using isolated glomeruli [60] and by inhibition of tubular reabsorption of albumin [61]. Analysis of the permeability of the GFB to molecules of varying size and charge can be used to estimate whether the increased flux of albumin is due to loss of size or charge selectivity of the GFB. In animal models of diabetes the defect is primarily in charge selectivity [61]. In clinically healthy non-diabetic individuals with microalbuminuria, loss of both size and charge selectivity of the GFB can be demonstrated [62]. However, in type 1 diabetes, defects in charge selectivity occur earlier than loss of size selectivity [63]. Similarly, in Pima Indians with type 2 diabetes, microalbuminuria is associated with loss of charge selectivity. Loss of size selectivity is seen only in those developing macroalbuminuria [58].\nWe have already noted that physical alteration of the GFB is necessary to increase its permeability to albumin. Therefore, this confirmation of changes in GFB selective permeability in diabetic microalbuminuria in the absence of clearly identified structural correlates suggests that the key changes are yet to be elucidated. These considerations and the predominance of defects in charge selectivity point to alterations in the negatively charged glomerular endothelial glycocalyx as the missing link. We now move to consider what aspects of the diabetic milieu are responsible for these GFB changes and how they might cause endothelial, including glycocalyx, dysfunction.\nMetabolic pathways and effectors from hyperglycaemia to microalbuminuria\nThere is now overwhelming evidence that hyperglycaemia is the major initiating factor in the pathogenesis of diabetic complications, including microalbuminuria. However, most adverse effects of glucose are mediated indirectly through diverse metabolic pathways. Four major hypotheses have highlighted the roles of AGEs, increased activity of the polyol pathway, activation of protein kinase C and increased flux through the hexosamine pathway (Fig.\u00a03). Activation of these pathways in turn causes dysregulation of a number of effector molecules which cause cellular damage and dysfunction. The roles of these pathways and effectors have been studied in detail in overt diabetic nephropathy, but the importance of these elements individually in its initiation and the appearance of microalbuminuria are less clear.\nFig.\u00a03Pathways to microalbuminuria in diabetes. Hyperglycaemia, through increased mitochondrial superoxide production, dysregulates key intracellular metabolic pathways. These in turn lead to the production of effectors that directly cause glomerular endothelial cell (GEnC) dysfunction (particularly of the glycocalyx) and disturb podocyte\u2013endothelial cell communication. This results in microalbuminuria. Progression of these lesions and development of other glomerular changes, including podocyte damage, lead to overt diabetic nephropathy\nFor example, TGF\u03b2 is an effector molecule with a clear role in the progression of established diabetic nephropathy, but there is no evidence that it is important in the mechanism of proteinuria per se. It is a key pro-fibrotic mediator, and in type 1 and type 2 diabetes, TGF\u03b2 is elevated in serum, urine and glomeruli from an early stage of disease. Its levels correlate with the degree of mesangial expansion, interstitial fibrosis and renal insufficiency, but not with microalbuminuria. Similarly, in animal models, blockade of TGF\u03b2 signalling inhibits the development of these pathological features, apart from the microalbuminuria [64, 65].\nHere we focus on selected intermediaries and effectors that have an identifiable role in microalbuminuria. In large part, dissection of these pathways and elucidation of their role in glomerular disease has necessarily relied on tissue culture or animal models. Such are described where human studies are lacking or where they provide additional significant insights.\nROS Brownlee has proposed oxidative stress as a unifying mechanism whereby the above-mentioned four pathways are inter-linked in the pathogenesis of diabetic complications (Fig.\u00a03) [66]. Hyperglycaemia increases oxidative stress through overproduction of superoxide and other ROS by the mitochondrial electron transport chain. ROS have direct cellular effects (e.g. they increase activity of nuclear factor \u03baB [NF\u03baB], a key inflammatory regulator), increase oxidative stress and interact with the above four pathways. Normalisation of mitochondrial superoxide production blocks all four of these pathways implicated in hyperglycaemic damage and corrects a variety of hyperglycaemia-induced phenotypes in target cells of diabetic complications. Endogenous superoxide dismutase normally neutralises excess superoxide but is overwhelmed in the diabetic state.As well as forming a nexus of metabolic pathways dysregulated by hyperglycaemia, ROS can also be considered as effectors through direct cellular actions. ROS decrease glomerular HSPG production [67], directly disrupt the endothelial glycocalyx [68], interfere with nitric oxide bioavailability and activate NF\u03baB.Glomerular ROS production is increased in experimental diabetes [69] and transgenic overexpression of superoxide dismutase attenuates renal injury, including increases in AER [70]. A superoxide dismutase mimetic also ameliorates increases in glomerular permeability both in vivo and ex vivo in non-diabetic models [71]. Little work has yet been done on the use of antioxidants in human diabetic nephropathy, but in vitro work confirms the importance of ROS in cells of the GFB. Podocytes produce ROS in response to high glucose [72], and genetic overexpression of superoxide dismutase prevents inhibition of endothelial nitric oxide synthase (eNOS) activity by hyperglycaemia in endothelial cells [73].\nVascular endothelial growth factor Vascular endothelial growth factor (VEGF) is a key regulator of vascular permeability and angiogenesis and is implicated in the pathogenesis of diabetic retinal neovascularisation. In the glomerulus it is produced in large amounts by podocytes and is thought to be important in maintaining glomerular endothelial cell fenestrations [74]. In rat models of type 1 diabetes, VEGF is upregulated in podocytes throughout the course of disease [75]. VEGF inhibition attenuates glomerular hypertrophy and albumin excretion and prevents upregulation of eNOS production in glomerular endothelial cells [76]. Similar effects are observed in some, but not all, models of type 2 diabetes [77].In human type 1 diabetes, serum VEGF concentrations vary according to glycaemic control, and higher levels are associated with microvascular complications, including microalbuminuria [78]. In type 2 diabetes, VEGF is upregulated early in the course of disease and urinary VEGF levels are correlated with microalbuminuria [79]. Further studies have confirmed initial upregulation of VEGF signalling in type 2 diabetes followed by a downregulation as podocyte loss and sclerosis develops [80].VEGF has the potential to induce the new vessel growth seen in early diabetic nephropathy [53] and to alter the permeability characteristics of the endothelium. Indeed, there is increasing evidence of the importance of precise control of glomerular VEGF for normal GFB function: both transgenic podocyte-specific under- or overexpression result in glomerular abnormalities, including glomerular endothelial changes and proteinuria [81]. Retardation of albuminuria in experimental diabetes by angiogenesis inhibitors further implies the importance of angiogenesis and\/or endothelial-related processes in the development of diabetic microalbuminuria [52, 82].\nThe growth hormone\/IGF system Dysregulation of the growth hormone\/IGF system can be detected early in experimental diabetes and is associated with both glomerular hypertrophy and microalbuminuria [83]. All components of the growth hormone\/IGF system can be detected at the mRNA level in the normal kidney. However, as detailed histochemical studies have not been performed, the exact location of system components with the glomerulus and the likely role of particular cell types cannot be defined.The rapid renal growth in experimental diabetes is preceded by a rise in the renal concentration of IGF-1 [84]. Somatostatin analogues ameliorate the increase in IGF-1 and renal hypertrophy and reduce AER [85]. In human type 1 diabetes, serum growth hormone levels and urinary IGF-1 levels are elevated and correlate with microalbuminuria, while an IGF-1 gene polymorphism modifies the risk of development of microalbuminuria [86]. The somatostatin analogue octreotide reduces macroalbuminuria and endothelial dysfunction in type 2 diabetes [87].Taken together, this evidence points to a role for the growth hormone\/IGF system early in human diabetic nephropathy. However, there are few clues to its contribution to the pathogenesis of microalbuminuria at the structural level. IGF-1 activates intracellular intermediates, including NF\u03baB, and an IGF-1 receptor inhibitor suppresses VEGF production, suggesting that VEGF is a downstream mediator of the effects of IGF-1 [88].\nProinflammatory cytokines and adipokines TNF\u03b1 is a proinflammatory cytokine with diverse actions, including increased production of endothelial cell adhesion molecules and IL-6, which in turn regulates CRP. TNF\u03b1 also directly increases endothelial permeability and disrupts the glycocalyx [41, 89]. In experimental diabetes, TNF\u03b1 levels rise in urine and the renal interstitium prior to the onset of albuminuria and correlate with it [90], as they do in human type 2 diabetes [91]. IL-6 levels also correlate with albuminuria in type 1 and 2 diabetes [91, 92]. CRP, often thought of as a downstream marker of inflammation, is elevated in both type 1 and 2 diabetes [27, 93] and correlates strongly with cardiovascular disease.Adipokines, which include leptin and adiponectin (and, arguably, TNF\u03b1 and IL-6), also have the potential to contribute to the development of microalbuminuria in both non-diabetic and diabetic populations. Enlargement of fat cells in obesity is associated with a generalised proinflammatory state, including increased levels of TNF\u03b1 and IL-6, and hypersecretion of adipokines, with the exception of adiponectin, which is downregulated [94]. Leptin induces vascular permeability and synergistically stimulates angiogenesis with VEGF [95]. Its serum levels correlate strongly with nephropathy in type 2 diabetes [96]. Adiponectin, unlike other adipokines, appears to have protective effects in the vasculature in general by reducing endothelial cell activation and inflammation.What do these changes in regulation and expression of these mediators tell us about the mechanism of microalbuminuria? It is clear that ROS and oxidative stress have a central role, given their importance in various metabolic pathways and direct cellular effects, including in the disruption of endothelial glycocalyx, which may be relevant in pathogenesis of microalbuminuria. There is strong evidence for a role of VEGF early in the course of diabetic nephropathy, probably through upregulation of production by podocytes and actions on glomerular endothelial cells disrupting their contribution to the GFB. The importance of dysregulation of angiogenic factors is emphasised by the protective effects of angiogenesis inhibitors. The growth hormone\/IGF system is also dysregulated early in diabetes, and it appears that IGF-1 is an important contributor to microalbuminuria through VEGF. Involvement of inflammatory mediators again points to a role for glomerular endothelial cells through endothelial activation, and hence, an increase in permeability through disruption of the glycocalyx.\nConclusions\nIn summary, the various avenues of study of diabetic microalbuminuria reviewed converge on the glomerular endothelium. Our analysis therefore leads to the conclusion that this is the site of the initial damage that leads to the development of microalbuminuria in diabetes. The most important aspect of this damage is disruption of the endothelial glycocalyx through actions of mediators dysregulated by the diabetic milieu. Key players include ROS, VEGF and proinflammatory cytokines (Fig.\u00a04). Disturbance of endothelial cell\u2013podocyte communication contributes to and amplifies the endothelial lesion. Progression of microalbuminuria to overt nephropathy is accompanied by predictable structural changes in the glomerulus, including podocyte damage and loss. This is the result of the ongoing diabetic milieu and disturbed cell\u2013cell communication, but is also secondary to increased penetration of the GFB by serum proteins [97].\nFig.\u00a04Proposed mechanism of glomerular filtration barrier damage leading to diabetic microalbuminuria. High glucose causes dysregulation of mediators including TNF\u03b1 and enhanced production of ROS, which directly damage the glomerular endothelial glycocalyx leading to microalbuminuria. Increased levels of pro-angiogenic molecules, including VEGF and inflammatory mediators, induce an activated and more permeable glomerular endothelial cell phenotype\nThus recent evidence remains broadly supportive of the Steno hypothesis [4] but it points specifically to disturbance of the endothelial glycocalyx as the common process underlying both microalbuminuria and generalised endothelial dysfunction. While it would be overly simplistic, particularly in type 2 diabetes, to suggest that this is the only factor involved (Fig.\u00a04), it follows nevertheless that microalbuminuria is an indicator of generalised endothelial dysfunction. Most interestingly, these conclusions imply that glycocalyx dysfunction is involved in the pathogenesis of other vascular disease, both microvascular and macrovascular. Therefore, therapies aimed at protecting or repairing endothelial cells and their glycocalyx would be expected to retard these diseases. Reduction or reversal of microalbuminuria implies resolution of generalised endothelial dysfunction and has potential to be a useful indicator of successful reduction of overall cardiovascular risk [98].","keyphrases":["microalbuminuria","diabetes","podocyte","glomerular filtration barrier","glycocalyx","glomerular endothelial cell"],"prmu":["P","P","P","P","P","P"]}
{"id":"Environ_Manage-4-1-2242854","title":"Headwater Influences on Downstream Water Quality\n","text":"We investigated the influence of riparian and whole watershed land use as a function of stream size on surface water chemistry and assessed regional variation in these relationships. Sixty-eight watersheds in four level III U.S. EPA ecoregions in eastern Kansas were selected as study sites. Riparian land cover and watershed land use were quantified for the entire watershed, and by Strahler order. Multiple regression analyses using riparian land cover classifications as independent variables explained among-site variation in water chemistry parameters, particularly total nitrogen (41%), nitrate (61%), and total phosphorus (63%) concentrations. Whole watershed land use explained slightly less variance, but riparian and whole watershed land use were so tightly correlated that it was difficult to separate their effects. Water chemistry parameters sampled in downstream reaches were most closely correlated with riparian land cover adjacent to the smallest (first-order) streams of watersheds or land use in the entire watershed, with riparian zones immediately upstream of sampling sites offering less explanatory power as stream size increased. Interestingly, headwater effects were evident even at times when these small streams were unlikely to be flowing. Relationships were similar among ecoregions, indicating that land use characteristics were most responsible for water quality variation among watersheds. These findings suggest that nonpoint pollution control strategies should consider the influence of small upland streams and protection of downstream riparian zones alone is not sufficient to protect water quality.\nIntroduction\nNonpoint source pollution is a serious problem that degrades surface waters and aquatic ecosystems. Loading of nutrients, sediment, and other pollutants from the landscape may compromise the integrity of freshwaters (Hunsaker and Levine 1995). In particular, excessive inputs of nitrogen and phosphorus result in eutrophication and fundamental changes in trophic state of lakes and streams (Carpenter and others 1998; Dodds and others 2002; Dodds 2006) and the impairment of surface waters for uses such as drinking, recreation, and support of aquatic life (Dodds and Welch 2000). These problems are pervasive; almost 40% of classified stream miles in the United States may be impaired, with diffuse pollutants responsible for a large percentage of impairments (U.S. Environmental Protection Agency [EPA] 2000). In response to these problems, research has focused on identifying and testing practices that reduce excessive pollutant loading and help restore the health of aquatic ecosystems.\nThe development of remote sensing and geographic information systems (GIS) technologies has facilitated quantitative assessment of landscape influences on aquatic ecosystems and watershed-scale approaches to the study of water quality (Johnson and Gage 1997). Watershed land cover is strongly correlated with water chemistry parameters, especially nutrient concentrations (e.g., Hunsaker and Levine 1995; Johnson and others 1997; Jones and others 2001; Osborne and Wiley 1988; Sliva and Williams 2001). Riparian land use may be particularly influential and, in some cases, a better predictor of in-stream water quality than land cover in the entire catchment (Johnson and others 1997; Osborne and Wiley 1988). Intact riparian zones provide water quality benefits and help preserve the biological integrity of watersheds (Gregory and others 1991).\nIn areas such as the Midwestern United States large-scale land use conversion has resulted in some of the worst water pollution in the United States (U.S. EPA 2000) and imperilment of many native aquatic species (Fausch and Bestgen 1997). Establishing or protecting riparian zones or large watershed areas that mitigate impacts of human land use on water quality may be costly or politically difficult, particularly in areas where much of the land is privately owned. In such instances, it is essential that scientists and managers identify areas within watersheds where protection would produce the most substantial water quality benefits, and prioritize these areas for protection. Geographic information systems are ideally suited to provide such identification because landscape analyses encompass the full range of spatial scales across which stream processes are regulated (Allan and others 1997) and allow for multiscale examinations of riparian (e.g., Johnson and Gage 1997) or headwater impacts on water quality.\nWe examined relationships between riparian and whole watershed land cover and water chemistry metrics in streams in Kansas at spatial scales ranging from several kilometers to the entire watershed, with the objective of testing areas where land use may strongly affect water quality in downstream reaches of the watershed (herein referred to as \u201cdownstream water quality\u201d). We hypothesized that land use adjacent to small headwater streams would have a disproportionately large impact on water quality, because these streams provide the predominant hydrologic contributions to the watershed (Lowrance and others 1997), and substantial in-stream nutrient processing and retention in upland streams and rivers can regulate downstream water quality (Alexander and others 2000; Peterson and others 2001).\nNatural geological and topographic features also influence surface water quality at landscape scales, in addition to anthropogenic factors such as land use conversion (Johnson and others 1997; Sliva and Williams 2001). To assess regional differences related to these features, we compared riparian-water chemistry relationships among four U.S. EPA level III ecoregions. Ecoregions denote general similarities in ecosystem types, serve as a spatial framework for research, assessment, and management of ecosystems (Omernik 1995), and can correspond well with principal factors that may influence surface water quality (e.g., Brown and Brown 1994; Rohm and others 2002). We assessed the degree to which relationships between surface water quality and land cover were affected by landscape heterogeneity (as indicated by ecoregions) by evaluating regional variation in riparian-water chemistry relationships. To our knowledge, no previous studies have examined the importance of headwater riparian zones, compared to other riparian areas within watersheds, at these scales of analysis across multiple watersheds.\nMethods\nSixty-eight small watersheds (mean watershed area, 280 km2; range, 19\u20131400 km2) were identified in four level III U.S. EPA ecoregions (U.S. EPA 1998a) across eastern Kansas (Fig.\u00a01). These ecoregions also represent 4 of the 14 regions developed for the National Nutrient Strategy (U.S. EPA 1998b), which were classified by both anthropogenic and natural characteristics (i.e., geology, geomorphology, land use, soils, vegetation) associated with nutrient concentrations in streams. Sites were selected across the four ecoregions so results would not be as tied to within-ecoregion characteristics. Sites were chosen from those regularly sampled by the Kansas Department of Health and Environment within the ecoregions such that the watersheds did not cross ecoregion boundaries and none of the sites were nested.\nFig.\u00a01Location of study watersheds in Kansas, grouped by level III U.S. EPA ecoregion, and example of land cover classification scheme, in which riparian and catchment land cover was quantified for the subcatchment of each stream segment in the watersheds\nTwenty-four watersheds were located in the Flint Hills (FH) ecoregion, characterized by rolling hills, coarse soils, and relatively intact tracts of tallgrass prairie predominantly used as cattle pasture. Because of topography and geology, little of this region has been converted to cropland agriculture. Eighteen watersheds were located in the Central Irregular Plains (CIP), characterized by irregular topography, loam soils, and a variety of land use types, including cropland agriculture, tallgrass prairie, and oak-hickory forests. Fourteen watersheds were located in the Western Corn Belt Plains (WCBP), a region that was historically covered with tall and mixed-grass prairie but has now been almost entirely converted to cropland agriculture. Finally, 12 watersheds were located in the eastern part of the Central Great Plains (CGP) ecoregion, characterized by reduced topography, mixed-grass prairie, and large tracts of cropland agriculture. Criteria for inclusion in the study were as follows: (1) watersheds were sampled for water chemistry parameters a minimum of 12 times, and (2) watersheds were entirely contained within one U.S. EPA level III ecoregion. Watersheds were located across a precipitation gradient, with average rainfall ranging from 610 to 1016 mm\/year. No watersheds were chosen that had very large livestock feeding operations or municipal point sources. The few smaller feeding operations (\u223c1000 animals) included were in all cases at least 0.1 km upstream of the stream chemistry site, and the total area of these operations was included in the analysis (see section Statistical Analyses, below).\nRelationships between riparian land cover and water chemistry parameters were assessed at four spatial scales (Fig.\u00a02). Riparian land cover throughout entire watersheds was quantified to examine cumulative impacts on water quality. Because small streams exert a large influence on downstream water quality (Alexander and others 2000; Peterson and others 2001), we examined correlations between riparian land cover adjacent to only the smallest (first-order) streams and water chemistry parameters sampled in downstream reaches of these watersheds. In addition, we examined localized riparian impacts on water quality by quantifying riparian land cover both 2 and 4 km upstream of the sampling site. The results of the above analyses were compared to correlations between water chemistry parameters and catchment-scale land cover at both the watershed and the first-order streams scales. In this way, we assessed the relative impact of riparian land cover on water chemistry parameters, compared to catchment land cover. Temporal variation was explored by partitioning water chemistry data seasonally, which allowed for examination of riparian-water chemistry relationships during both high and base flow conditions.\nFig.\u00a02Riparian land cover assessed at four spatial scales: (A) land cover in the whole watershed, (B) land cover adjacent to the first-order streams of watersheds, and (C) land cover 2 and 4 km upstream of the water chemistry sampling point\nWe examined a subset of 39 study watersheds where water chemistry measurements were taken on a fourth-order reach of stream to directly compare the influence of riparian land cover on streams of similar sizes within watersheds. Riparian land cover was quantified by stream order (Strahler 1957) and correlated with downstream water chemistry values separately, so comparisons could be made between stream sizes. In addition, we analyzed riparian land cover-water chemistry relationships among ecoregions to determine if differences existed, or if these relationships held constant across ecosystem types. These analyses also help to show that watershed size and natural factors captured by ecoregions (geology, precipitation, elevation, gradient, etc.) did not confound the interpretations of land use effects.\nWater Chemistry Data\nWater chemistry data were collected and analyzed by the Kansas Department of Health and Environment (KDHE) as part of their stream chemistry monitoring network (KDHE 2000). Total nitrogen (TN), nitrate (NO3\u2212), ammonium (NH4+), total phosphorus (TP), total suspended solids (TSSs), atrazine (AT), fecal coliform bacteria (FC), and dissolved oxygen (DO) data were used to assess the impact of riparian land cover on water chemistry. Samples are collected every 2 months between 0900 and 1700 hr at each site on a rotational schedule. Extreme weather (river icing, very high floods) precludes sampling occasionally. Water chemistry samples were collected from the thalweg of each stream, frozen, and stored in acid-washed bottles in the dark, prior to analysis. All TN, NH4+, and TP samples were analyzed within 28 days of collection, NO3\u2212 samples were analyzed within 48 hr of collection, TSS and AT samples were analyzed within 7 days of collection, FC samples were analyzed within 24 hr of collection, and DO measurements were taken in the field using a membrane electrode probe. Total nitrogen and phosphorus were analyzed by a colorimetric automated phenate method, following digestion by metal-catalyzed acid and persulfate techniques, respectively (U.S. EPA 1983). Nitrate was analyzed by ion chromatography; NH4+, by semiautomated colorimetry; TSS, by a residue, nonfilterable and TSSs method; and AT, by gas chromatography (U.S. EPA 1983). Fecal coliform bacteria samples were analyzed by a membrane filter procedure (APHA 1992). Field duplicate samples and internal spikes were used to assess the reliability and recovery efficiencies of the assays.\nWater chemistry data for NO3\u2212, NH4+, TP, TSS, AT, FC, and DO were collected from 1990 to 2001 for all study watersheds. Total nitrogen data were collected from January 2000 to May 2003 for 57 of the 68 study watersheds. Collection of TN data began in 2000 to assist establishment of nutrient criteria for Kansas\u2019 surface waters. For all analyses, mean concentrations of TN, NO3\u2212, NH4+, TP, TSS, AT, and FC were taken for each watershed across sampling dates. Minimum and maximum DO concentrations were quantified by averaging minimum and maximum concentrations by year for all years in which at least five samples were taken, then taking a mean of these concentrations across years.\nTo examine temporal variability in riparian-water chemistry relationships, we first classified seasons using mean monthly discharge measurements (1990\u20132001) from 30 USGS gauging stations across the study region. Seasons were classified as the month or months in which 0%\u201325%, 26%\u201350%, 51%\u201375%, and 76%\u2013100% of the annual water volume across the region was discharged. Mean water chemistry concentrations of NO3\u2212, NH4+, TP, TSS, AT, and FC were taken for each of the four seasons. Insufficient data prevented analysis of total nitrogen and DO for temporal differences.\nDigital and Land Cover Data\nDigital stream networks were derived for each watershed using 30-m digital elevation models, ARCGIS (Arcview version 8.2, 2002), and ArcHydro (Maidment, 2002) software. This method accounts for permanent streams and all but the smallest intermittent streams. Catchment area above each KDHE monitoring site was delineated using catchment-processing tools in ArcHydro software. Using the same processing tools, a subcatchment was delineated for each stream segment of the watersheds. A stream segment was defined as a section of stream from its upstream confluence to its downstream confluence with other tributaries. By overlaying catchment and subcatchment layers with digitized riparian and catchment land cover data, we quantified land cover for each watershed and watershed subcatchment (Fig.\u00a01).\nRiparian land cover was classified from the Kansas Riparian Areas Inventory dataset (NRCS 2001). The riparian ecotone in this dataset was defined as the 33 m adjacent to the stream and was digitized at a 1:24,000 scale from USGS Digital Orthophotograph Quarter Quadrangles that reflected land cover conditions in 1991. Land cover was identified from the beginning of the period of water chemistry sampling. Large socioeconomic changes did not occur in Kansas over this time period (e.g., only \u223c10% population increase). This dataset contained 11 land cover classes (animal production area (holding pens or feeding areas), barren land, cropland, crop\/tree mix, forest, grassland, grass\/tree mix, shrub\/scrub land, urban land, urban\/tree mix, water), and riparian areas were classified by the land cover type occurring in \u226551% of the 33-m ecotone. Of the 11 land cover classes, 3 (shrub\/scrub land, barren land, and animal production area) did not account for more than 1% of the riparian land cover in any watershed and were not included in the analyses. The remaining eight classifications were aggregated into five categories (cropland, forest, grassland, urban land, and water) following the level I classification scheme developed by Anderson et al. (1976). Water was not included as a land cover type in analyses. While this scheme can create problems with colinearity, the primary goal of this paper was to determine the best-fit model at different spatial scales within the watershed. Colinearity influences the ability to ascribe causation by individual categories of land use (e.g., cropland, urban, forest, or grassland), but this was not the primary goal of our analysis.\nCatchment land cover was classified from the Kansas Land Cover dataset (KARS 1993). This dataset was digitized at a 1:100,000 scale from Landsat Thematic Mapper imagery and, also, contained 11 land cover classes that reflected conditions in 1991. Land cover classes were reclassified in the same way as the riparian dataset. Comparison of the riparian dataset to a 33-m \u201cbuffer\u201d clipped from the catchment dataset showed highly significant correlations (average Kendal \u03c4 correlation = 0.93, p\u00a0<\u00a00.01) between the two datasets for all land cover types. Information on permitted point sources and confined livestock feeding operations within watersheds was obtained from KDHE and incorporated into GIS to ensure that point sources were not in close proximity to sampling sites.\nStatistical Analyses\nForward stepwise linear regression models were used to predict water chemistry parameters with land cover data (animal production area [holding pens or feeding areas], barren land, cropland, crop\/tree mix, forest, grassland, grass\/tree mix, shrub\/scrub land, urban land, urban\/tree mix, water) at four spatial scales (watershed, first-order streams, 2 km upstream, 4 km upstream). Separate regressions were done at each scale. F-values of 1 and 0 were used as thresholds to include and exclude land cover classifications from regression models. We investigated the predictive ability of riparian land cover independent of catchment effects by examining partial correlations (r) among riparian land cover classifications that were significant predictors in regression models and water chemistry parameters, controlling for predictor catchment land cover classifications. Analysis of variance (ANOVA) was used to test for differences among ecoregions. Since ecoregions were correlated with land use, slopes of relationships were compared among ecoregions at all four spatial scales using general linear model (GLM) analysis of variance (ANCOVA) to assess whether riparian-water chemistry relationships held constant across ecoregions. Results of comparisons of intercepts on these data were presented in a prior publication (Dodds and Oakes 2004). Least-squares means were used to compare slopes of regression lines. Slopes represent the fundamental response to anthropogenic effects (most relevant to this paper) and intercepts indicate the baseline nutrient or pollutant level. Response data appeared normally distributed and were not transformed prior to analyses. All relationships among the data were plotted and no clear outliers or leveraged relationships were observed.\nResults\nRiparian-Water Chemistry Relationships\nStrahler ordering showed the smallest (first-order) digitized streams on average comprised >60% of the stream miles within study watersheds, with larger streams accounting for sequentially fewer percentages of stream miles. Across all studied watersheds, riparian land cover was a significant predictor of among-site variation in water chemistry concentrations at the watershed and first-order streams scales, particularly for nutrients (Table\u00a01). Less variance was explained at local scales represented as riparian cover 2 or 4 km upstream from the sampling site (Fig.\u00a03). Total nitrogen, TP, and NO3\u2212 were the parameters with the greatest R2 values related to riparian land cover, and all three had slightly greater R2 values using land cover adjacent to first-order streams of watersheds than using riparian land cover across the whole watershed.\nTable\u00a01Multiple regression models showing correlations between water chemistry parameters and riparian land cover in both the whole watersheds the first-order streams of watershedsWater chemistry parameterCropForestGrasslandUrban InterceptR2Watershed\u00a0\u00a0TN\u22120.4400.2601.932 0.355\u00a0\u00a0NO3-N0.6230.490\u22120.5000.525\u00a0\u00a0NH4-N\u22120.466\u22120.6620.2030.327\u00a0\u00a0TP0.2640.7120.0950.507\u00a0\u00a0AT0.4280.5580.171\u00a0\u00a0FC0.3781621.5700.199\u00a0\u00a0DO (max)0.50812.0850.247First order\u00a0\u00a0TN0.3880.5760.5510.406\u00a0\u00a0NO3-N0.6500.538\u22120.0330.606\u00a0\u00a0NH4-N\u22120.445\u22120.6830.1950.304\u00a0\u00a0TP0.3200.7800.0870.634\u00a0\u00a0AT0.4130.6050.158\u00a0\u00a0FC0.458798.8320.198\u00a0\u00a0DO (max)0.52212.1130.261Note. Significant regression coefficients are presented, illustrating the magnitude and direction of importance of land cover classes in models. TN analyses based on 57 watersheds; all other analyses based on 68 watersheds. Nutrient parameters and dissolved oxygen expressed as milligrams per liter, atrazine (AT) expressed as micrograms per liter, microbiological parameters expressed as colony forming units\/100 ml, and land cover classifications expressed as percentages. All values reported were significant at p\u00a0<\u00a00.05Fig.\u00a03Variance in water chemistry variables (R2 values) accounted for by (A) land cover in the riparian ecotone (33 m) at multiple scales and (B) catchment land cover at two scales, using multiple linear regression analyses. TN analyses based on 57 watersheds; all other analyses based on 68 watersheds. Bars for R2 values were not plotted when there was not a significant relationship (p\u00a0>\u00a00.05)\nRiparian land cover 2 and 4 km upstream explained no significant variance in TP concentrations, and riparian land cover 2 km upstream of the sampling point explained no significant variance in AT concentrations. Total suspended solids and minimum DO concentrations did not have significant relationships with riparian cover in any analyses and are not discussed further in this section.\nCatchment land cover showed similar relationships to water chemistry parameters as riparian land cover (Fig.\u00a03). In all comparisons between catchment and riparian land cover, the magnitude of differences was small. Partial correlations indicated that riparian land cover classifications were still significantly correlated with some water chemistry parameters after controlling for variance explained by catchment land cover classifications that were significant predictors in regression models (Table\u00a02). Removal of the effect of land use cover by using partial correlations can actually remove riparian effects from the overall correlation so these data should not be interpreted to suggest that riparian cover only explains a small portion of the variance in water quality.\nTable\u00a02Partial correlations among nutrient concentrations and riparian land cover classificationsWater chemistry parameterCatchment land cover Riparian land coverrp-valueWatershed\u00a0\u00a0TNGrass, forestGrass, urbanGrass = \u22120.060.687Urban = 0.200.134\u00a0\u00a0NO3\u2212Crop, urbanCrop, urbanCrop = 0.220.071Urban = 0.480.000\u00a0\u00a0NH4+Grass, forestGrass, forestGrass = \u22120.030.803Wood = \u22120.110.370\u00a0\u00a0TPCrop, urban, forestCrop, urbanCrop = 0.040.779Urban = 0.580.000First order\u00a0\u00a0TNCrop, grassCrop, urbanCrop = 0.250.068Urban = 0.330.013\u00a0\u00a0NO3\u2212Crop, urbanCrop, urbanCrop = 0.260.033Urban = 0.500.000\u00a0\u00a0NH4+Grass, forestGrass, forestGrass = \u22120.000.994Forest = \u22120.080.543\u00a0\u00a0TPCrop, urbanCrop, urbanCrop = 0.040.724Urban = 0.680.000Note. Correlations controlled for catchment land cover classifications that were significant predictors in regression models and were used to partition additional variance explained by riparian land cover from variance explained by catchment land cover. Partial correlations (r) for which riparian crop land (crop), forest, grassland (grass), and urban land (urban) explained >30% of the variation in water chemistry parameters among sites (see Table\u00a01 and Fig.\u00a03) are presented\nTemporal Variation\nExamination of regional discharge patterns revealed that 25% of annual water volume was discharged from January to April, 50% by June, 75% by August, and the remainder in the August\u2013December time period. Thus, the periods of January\u2013April, May, June\u2013July, and August\u2013December were designated as seasons in temporal analyses. Seasons in which a quarter of annual water volume was discharged in 1 or 2 months (i.e., May, June\u2013July) represented periods of high flow and high connectivity across the landscape, while seasons encompassing more than 2 months (January\u2013April, August\u2013December) represented predominantly base flow conditions (with most of the upper reaches of the first-order streams dry).\nMost water chemistry parameters exhibited temporal changes in the degree that they were statistically related to riparian land cover. Total P and NH4+ were significantly correlated with riparian land cover in all seasons except May (Fig.\u00a04); in particular, riparian land cover at both the watershed and the first-order streams scales explained most variance in TP concentrations in January\u2013April compared to other seasons. Conversely, AT and FC concentrations were best explained during the high flow period of May, and did not have significant relationships with riparian land cover during some base flow seasons. Nitrate exhibited comparatively less temporal variation; riparian land cover at the watershed scale explained a minimum of 30%, and at the first-order streams scale a minimum of 45%, of among-site variance in NO3\u2212 concentrations across seasons.\nFig.\u00a04Temporal variation (R2 values) in relationships between water chemistry parameters and (A) total riparian land cover in watersheds and (B) riparian land cover adjacent to the first-order streams of watersheds. Seasons were designated from quartiles of annual discharge occurring across the study region. Total nitrogen and DO were not analyzed for temporal differences (see Methods). Bars for R2 values were not plotted when there was not a significant relationship (p\u00a0>\u00a00.05)\nA particularly interesting aspect of these data is that even when first-order streams are not very likely to flow (August\u2013December), the riparian land cover around them yielded somewhat greater R2 values than did the whole watershed riparian cover for TP and NO3\u2212.\nImpact of Stream Size\nDifferent stream sizes were used in the analyses to this point. To control for this a subset of sites was chosen from which data were taken only for fourth-order streams. Total N and NO3\u2212 were most closely correlated with first-order riparian land cover (Fig.\u00a05). In general, the most variance was explained by riparian land cover adjacent to first-order streams and less variance was explained by riparian cover near larger-order streams closer to sampling sites. Atrazine and maximum DO concentrations were not significantly correlated with riparian land cover near streams of any size in this subset of watersheds.\nFig.\u00a05Variance in water chemistry variables (R2 values) explained by riparian land cover adjacent to different sized streams within watersheds. Analyses performed with a subset of 39 fourth-order watersheds; TN analyses preformed with 38 fourth-order watersheds. Comparisons between riparian land cover and AT and maximum DO concentrations were not significant and are not presented\nEcoregion Effects\nANOVA indicated some variation in TN, NO3\u2212, NH4+, TP, FC, and maximum DO concentrations among ecoregions. Comparison of least-squares means showed TN and NO3\u2212 concentrations were significantly different (p\u00a0<\u00a00.05) among all ecoregions except the CGP and CIP (Fig.\u00a06). The Western Corn Belt Plains was the only ecoregion that exhibited significantly different NH4+, FC, and maximum DO concentrations, which were all higher than mean concentrations in other ecoregions.\nFig.\u00a06Mean values for selected water chemistry parameters and riparian cropland, grouped by ecoregion (WCBP, Western Corn Belt Plains; CGP, Central Great Plains; FH, Flint Hills; CIP, Central Irregular Plains). TN data for WCBP were available for only 3 of 12 study watersheds. Significant differences are labeled with different letters; error bars represent 1 SE\nThe Flint Hills was the only ecoregion that exhibited significantly different TP concentrations, which were lower than those of other ecoregions. Atrazine concentrations did not differ significantly among ecoregions.\nThe percentage of riparian land in agricultural production also varied by ecoregion and closely mirrored nutrient concentrations. Least-squares means comparing slopes of regression lines between water chemistry parameters and significant predictor land cover classifications among the four ecoregions showed that slopes were generally similar across all water chemistry parameters (e.g., Fig.\u00a07), and differences that did exist most often occurred when comparing the Flint Hills to other ecoregions (Table\u00a03).\nFig.\u00a07Example of typically observed relationships between riparian land cover and water chemistry parameters among the four ecoregions analyzed (WCBP, Western Corn Belt Plains; CGP, Central Great Plains; FH, Flint Hills; CIP, Central Irregular Plains). The percentage of riparian cropland in the watersheds is plotted versus in-stream NO3\u2212 concentrations. Slopes of regression lines fitted through each of the four ecoregions were not significantly differentTable\u00a03Comparisons of least-squares means using general linear model analyses to assess differences in slopes of riparian-water chemistry relationships at four spatial scales, across level III U.S. EPA ecoregionsSpatial scaleResponse variableEcoregions with different slopesp-valueWatershedTPFH & CGP0.010First orderTNFH & CGP0.019First orderTPFH & CGP0.007First orderTPCIP & CGP0.007First orderTPCIP & WCBP0.0412 km upstreamTNFH & CGP0.0222 km upstreamNO3\u2212FH & CIP0.0142 km upstreamNO3\u2212FH & WCBP0.0024 km upstreamNO3\u2212FH & WCBP0.0014 km upstreamFCFH & WCBP0.023Note. Significantly different slope comparisons between Central Great Plains (CGP), Central Irregular Plains (CIP), Flint Hills (FH), and Western Corn Belt Plains (WCBP) ecoregions are listed. All other comparisons were not significantly different at p\u00a0<\u00a00.05\nDiscussion\nLand Cover-Water Chemistry Relationships\nRiparian and whole watershed land cover was significantly correlated with water quality metrics, particularly nutrient concentrations. Land cover explained greater variance at landscape scales (watershed and first-order streams) than riparian cover at local scales (2 and 4 km upstream of sampling), which is consistent with the idea that nutrient loading and retention occurs at larger spatial scales (Allan and others 1997). Given that NO3\u2212 uptake lengths are often less than 2 km in this region (O\u2019Brien and others 2007), it is possible that local riparian cover would influence NO3\u2212 concentrations, but the effect was small. Differences in correlations between nitrogen species (NO3\u2212 and NH4+) may have occurred because NO3\u2212 inputs from the watershed are often greater than NH4+ inputs (Peterson and others 2001) and NH4+ is a preferred nitrogen source for aquatic organisms that can use inorganic N and cycles more quickly than NO3\u2212 (Dodds and others 2000). Seasonal differences in relationships between riparian land cover and both NH4+ and TP may be attributable to their strong relationship to particulate dynamics (Johnson and others 1997). Phosphate and NH4+ both adsorb readily to sediments, and are primarily transported into streams via surface runoff (Novotny and Olem 1994).\nWe wanted to remove the potential problem that the proportion of length of first order streams would vary by stream order. But if we only used our fourth-order sites, then we had about half the total number of sites and our statistical power decreased. Thus we analyzed the subset of fourth-order stream sites (Fig.\u00a05) to be certain that our results were not an artifact of sampling sites occurring at different order streams. Since our results were similar with this subset, all other analyses used the full dataset.\nVariation in nutrient concentrations during high flow may have resulted from \u201cpulses\u201d of sediment-bound nutrients entering from the landscape which were not effectively captured by our method of analyzing mean seasonal concentrations. This could explain the lack of correlation between riparian land cover and NH4+ and TP in May compared to other seasons. Conversely, the primary mode of NO3\u2212 transport to surface water is generally via subsurface flow (Hill 1996), and this consistent connectivity to the landscape may explain the comparatively low temporal variability seen in riparian-NO3\u2212 relationships.\nDiscrepancies in numbers of sampling dates and sites made it difficult to directly compare riparian-TN relationships with those of other parameters. However, we felt it was important to include TN in these analyses because of its importance in establishing nutrient criteria (Dodds and Welch 2000) and because other available parameters, such as dissolved inorganic nitrogen, can be unsuitable substitutes (Dodds 2003). A disproportionate number of watersheds for which TN data were not available were primarily agricultural and contained some of the highest observed concentrations of both NO3\u2212 and TP; the absence of these sites in TN analyses may explain why TN was not as strongly correlated with riparian land cover compared to NO3\u2212 or TP.\nNonnutrient water chemistry parameters had weaker correlations with riparian land cover. Although AT, FC, and maximum DO concentrations were significantly correlated with riparian land cover, relationships were weak across all spatial and temporal scales and preclude conjecture into the mechanisms underlying correlations. Lack of correlation between TSS and riparian land cover contrasts with results of previous studies (Johnson and others 1997; Sliva and Williams 2001) and, as with relationships observed in sediment-bound nutrients, may be a function of averaging TSS concentrations into one measurement.\nAlthough permitted livestock operations and other point sources were not substantial in each watershed, point sources falling below Kansas\u2019 permitting regulations (e.g., confined livestock operations under 300 animals) were likely present in some watersheds and may have accounted for unexplained variance in the observed relationships. Our results were consistent with previous studies (Johnson and others 1997; Jones and others 2001; Osborne and Wiley 1988; Sliva and Williams 2001), suggesting that agricultural and\/or urban lands were the most important predictors of water quality variability.\nMaintaining buffers or other passive land uses in headwater streams may effectively reduce diffuse pollution downstream. The importance of these streams and their riparian zones is due in part to their sheer numbers; small streams often comprise the majority of stream miles within a drainage network (Horton 1945; Leopold and others 1964), and in this study the smallest (first-order) streams on average comprised more than 60% of the stream miles in the study watersheds. Riparian land cover near the first-order streams of watersheds explained greater variance in TN, NO3\u2212, and TP concentrations than did riparian land cover immediately upstream from sampling sites. First-order riparian land cover was statistically related to most water quality measures, even when all potential correlation related to watershed land cover was controlled for. Our results suggest that headwater riparian areas could have an important impact on downstream water quality.\nOur study was correlative in nature and does not unequivocally confirm causation. Such an approach is required at the spatial scales of our study. Previous work suggests several possible causes for our observed associations. First, lower-order streams have the greatest potential for interactions between water and the adjacent landscape (Lowrance and others 1997). Second, the large benthic surface area-to-volume ratio of small streams favors rapid in-stream uptake, processing, and retention of nitrogen (Alexander and others 2000; Dodds and others 2000; Peterson and others 2001), which in larger streams increases in proportion to depth (Alexander and others 2000) or discharge (Wollheim and others 2001). Because high nitrogen inputs may overwhelm this ability (O\u2019Brien and others 2007; Wollheim and others 2001), riparian zones adjacent to small streams may be particularly important in regulating nutrient inputs and allowing natural in-stream processes to significantly impact nutrient concentrations.\nSeveral studies have addressed the relative importance of riparian versus whole catchment land use in regulating water quality. Reports in the literature have been mixed; some researchers (Hunsaker and Levine 1995; Sliva and Williams 2001) found that catchment land cover was better correlated with water quality, while others (Osborne and Wiley 1988; Johnson and others 1997) reported that land cover in the riparian ecotone was more influential. Likewise, although partial correlations indicated riparian land cover classifications were significantly related to TN, NO3\u2212, and TP even after accounting for catchment effects, this did not hold true for all water chemistry parameters. Overall, it is difficult to separate the effects of land cover in the riparian ecotone and land cover in the catchment because they are highly correlated, and in many altered landscapes, riparian land cover may simply reflect the dominant catchment land cover types.\nSignificant partial correlations between riparian land cover and TN, NO3\u2212, and TP concentrations correspond with previous work (e.g., Karr and Schlosser 1978; Lowrance and others 1997) identifying riparian zones as key regulators of nutrient inputs to surface waters. These results, in addition to strong relationships among water quality metrics and riparian land use that have been previously reported at both field (e.g., Karr and Schlosser 1978; Peterjohn and Correll 1984) and landscape (e.g., Johnson and others 1997; Osborne and Wiley 1988) scales, suggest that intact riparian zones could influence landscape impacts on surface water quality.\nEcoregion Effects\nThe finding that slopes of the relationships were not significantly different in most ecoregion comparisons may be attributable to several factors. Exceptionally variable relationships could preclude the statistical power to determine differences. It is possible that the study regions were not sufficiently distinct to allow detection of differences in riparian interactions, although this is unlikely given their previous classification as both separate ecoregions (U.S. EPA 1998a) and nutrient regions (U.S. EPA 1998b). Because U.S. EPA ecoregion designations encompass human impacts such as land use in addition to natural geological, climatic, and soil characteristics (Omernik 1995), observed intraregion differences in riparian land cover classification and nutrient concentrations were expected. However, since land use is often a dominant factor regulating surface water quality (Hunsaker and Levine 1995; Johnson and others 1997; Osborne and Wiley 1988), riparian-water chemistry relationships would be expected to remain relatively constant across ecoregions if designations were partially dependent on land use, as was the case in this study.\nConclusions\nThe data suggest that riparian cover near sampling sites is generally less well correlated with water quality parameters than riparian cover or land use in first-order streams. Because watershed cover and riparian land use were correlated, it is difficult to determine how important first-order riparian cover is related to water quality. Our results suggest a statistically significant effect of riparian cover of first-order streams on water quality because partial correlations among riparian land cover classifications were significant predictors in regression models when controlling for predictor catchment land cover classifications. We take the conservative approach in our interpretation, but it is possible that riparian cover has much stronger effects than whole-watershed land cover and that most of the correlation is driven by riparian effects. The effect of first-order land cover may not be too surprising; first-order streams make up the majority of stream length in watersheds. Our approach shows that a correlation with land uses in small headwater streams does hold, and holds even in seasons when many of the first-order stream channels are not flowing.","keyphrases":["water quality","riparian zones","nonpoint source pollution","geographic information systems","headwater streams","watershed management"],"prmu":["P","P","P","P","P","R"]}
{"id":"Arch_Dermatol_Res-3-1-1839867","title":"The relevance of the IgG subclass of autoantibodies for blister induction in autoimmune bullous skin diseases\n","text":"Autoimmune bullous skin diseases are characterized by autoantibodies and T cells specific to structural proteins maintaining cell\u2013cell and cell\u2013matrix adhesion in the skin. Existing clinical and experimental evidence generally supports a pathogenic role of autoantibodies for blister formation. These autoantibodies belong to several IgG subclasses, which associate with different functional properties and may thus determine the pathogenic potential of IgG antibodies. In pemphigus diseases, binding of IgG to keratinocytes is sufficient to cause intraepidermal blisters without engaging innate immune effectors and IgG4 autoantibodies seem to mainly mediate acantholysis. In contrast, in most subepidermal autoimmune blistering diseases, complement activation and recruitment and activation of leukocytes by autoantibodies are required for blister induction. In these conditions, tissue damage is thought to be mainly mediated by IgG1, but not IgG4 autoantibodies. This review summarizes the current knowledge on the pathogenic relevance of the IgG subclass of autoantibodies for blister formation. Characterization of the pathogenically relevant subclass(es) of autoantibodies not only provides mechanistic insights, but should greatly facilitate the development of improved therapeutic modalities of autoimmune blistering diseases.\nIntroduction\nAutoimmune blistering diseases are associated with an autoimmune response directed to structural proteins mediating cell\u2013cell and cell\u2013matrix adhesion in the skin [62, 66]. Both autoantibodies and autoreactive T cells have been found in patients with these organ-specific autoimmune diseases. However, blister induction is mainly mediated by autoantibodies. Autoimmune blistering diseases are classified based on the ultrastructural site of deposition of immunoreactants and on the molecular target of autoantibodies. Diseases of the pemphigus group are associated with autoantibodies to epidermal components mediating cell\u2013cell adhesion and are characterized by acantholytic blisters within the epidermis [39, 71]. Tissue-bound and circulating autoantibodies to the dermal\u2013epidermal junction are characteristic immunopathological features of subepidermal autoimmune bullous diseases [62, 85]. Target antigens of autoantibodies have been identified for the majority of autoimmune blistering diseases. In most of these diseases, the pathogenicity of autoantibodies is supported by clinical observations and extensive experimental evidence [62].\nAntibodies are effector molecules of the adaptive immune system secreted by plasmablasts and long-lived plasma cells. Antibody responses are physiologically mounted following an infection or vaccination and protect against various pathogens. Occasionally, in the setting of an autoimmune disease, antibodies to autologous structures may develop and cause different forms of tissue damage. The immunopathology induced by autoantibodies, similar to the immunity mediated by antibodies to pathogens, relies on several mechanisms of action of antibodies, including direct mechanisms, which are mediated by the antibody\u2019s variable regions (e.g., by steric hindrance and signal transduction), and indirect mechanisms, which are triggered by the constant regions of antibodies. For the latter, (auto)antibodies typically interact through their Fc portions with other factors of the innate immune system, including the complement system and inflammatory cells [62].\nAntibodies of the IgG isotype predominate in the systemic immune response, as reflected in serum immunoglobulin concentration, and activate a wide range of effector functions. Four subclasses of IgG are defined, originally from the antigenic uniqueness of their heavy chains, which are products of distinct genes [20, 27, 77]. The subclasses are designated as IgG1, IgG2, IgG3 and IgG4 in order of their serum concentration \u223c60, 25, 10 and 5%, respectively. Although the heavy chains show >95% sequence homology, each IgG subclass expresses a unique profile of effector activities [35, 56, 59, 76, 80, 82]. Protein antigens characteristically provoke IgG1 and IgG3 responses and these isotypes are able to activate all types of Fc receptors and the C1 component of complement. The IgG4 subclass may be characteristic of chronic antigen stimulation, as in autoimmune disease; it has restricted Fc receptor activating abilities and does not activate C1q. The IgG2 subclass often predominates in responses to carbohydrate antigens; it has restricted Fc receptor and C1 activating abilities [35, 56, 80, 82].\nThe pathogenic potential unfolded by autoantibodies is determined not only by their specificity and affinity, but also by their isotype. Autoantibodies against cutaneous proteins in autoimmune blistering diseases belong to different IgG subclasses. This paper summarizes the current knowledge on the relevance of IgG subclasses for tissue injury in autoimmune bullous diseases.\nPemphigus diseases\nPemphigus designates a group of life-threatening-autoimmune blistering diseases characterized by intraepithelial blister formation caused by loss of cell\u2013cell adhesion [39, 54, 71]. IgG autoantibodies in patients with pemphigus seem to mediate their pathogenic functions independently of their Fc portions [62]. Patients\u2019 IgG autoantibodies are pathogenic in C5-deficient mice and F(ab\u2019)2, Fab, and scFv fragments of autoantibodies induce acantholysis by passive transfer in wild type mice showing that complement activation or other Fc-mediated effects are not required for pathogenicity [4, 21, 45, 55].\nNumerous studies clearly demonstrated that tissue bound and circulating autoantibodies in pemphigus patients mainly belong to the IgG1 and IgG4 subclasses [2, 5, 8, 9, 16\u201318, 23, 28, 36, 38, 40, 49, 61, 79]. The IgG subclass distribution of autoantibodies in a representative pemphigus patient is shown in Fig.\u00a01. While it is generally agreed upon the subclass distribution of IgG autoantibodies, the relevance of autoantibodies of different IgG isotypes for acantholytic blistering in pemphigus is still a matter of debate. IgG4 autoantibodies, known to have poor complement- and leucocyte-activating properties, predominate in pemphigus vulgaris and foliaceus. While several studies suggest a pathogenic role of IgG4 in pemphigus, the capacity of IgG1 autoantibodies to induce acantholysis has not yet been ruled out. In patients with active pemphigus vulgaris, IgG4 autoantibodies against desmogleins were found to predominate [5, 17, 23, 40]. The transplacental transfer of these autoantibodies in mothers with pemphigus induces acantholytic skin disease in neonates [52]. In endemic pemphigus foliaceus the early antibody response in normal subjects living in the endemic area and in patients before the onset of clinical disease is mainly IgG1. Acquisition of an IgG4 response seems to be a key step in the development of clinical disease [78]. Clinical and experimental data suggests that IgG1 differ from IgG4 autoantibodies in terms of both their epitope specificity and pathogenic potential [9, 42]. A monoclonal IgG4 antibody against desmoglein 3 generated from a patient with active pemphigus vulgaris induces acantholysis in cultured skin and when injected in neonatal mice [86]. The fact that IgG4 antibodies purified from patients with fogo selvagem, an endemic form of pemphigus foliaceus, are pathogenic in mice further supports the notion that IgG4 is pathogenic and complement activation is not required for blister formation [57], but does not exclude a pathogenic potential of IgG autoantibodies belonging to other subclasses. Indeed, in several pemphigus foliaceus patients, only IgG1 autoantibodies were found that caused intraepidermal blisters by passive transfer into neonatal mice [29]. This observation clearly shows that autoantibodies of different subclasses may display blister-inducing activity. In addition, in paraneoplastic pemphigus, autoantibodies against desmoglein 3 mainly belong to the IgG1 and IgG2 subclasses suggesting that the IgG subclass per se is not a direct determinant of the antibodies\u2019 pathogenic potential in pemphigus [24].\nFig.\u00a01IgG subclass distribution of circulating pemphigus autoantibodies. A 1:10 dilution of serum from a patient with pemphigus foliaceus was incubated with 6\u00a0\u03bcm-thick cryostat sections of normal human skin for 30\u00a0min at room temperature. Bound antibodies, visualized using an FITC-labeled antibody specific to human IgG, were of a IgG1 and d IgG4 subclasses. In contrast, no binding of b IgG2 and c IgG3 autoantibodies was evidenced\nExperimental evidence using a murine model of pemphigus vulgaris shows that, similar to human pemphigus, the autoimmune response in mice is biased toward non-complement fixing autoantibodies. In this model, in immunodeficient mice infused with splenocytes from desmoglein-deficient mice immunized against this antigen, IgG autoantibodies are produced by homeostatically expanded antigen-specific B cells under T cell control and mice develop a phenotype reminiscent of pemphigus vulgaris. These IgG autoantibodies predominantly belong to the IgG1 subclass, which is a non-complement fixing antibody in the mouse [3, 50]. These results demonstrate that non-complement-fixing autoantibodies can induce acantholysis and suggest a similar mechanism in patients, but they do not exclude a pathogenic potential of patients\u2019 IgG1 autoantibodies in pemphigus.\nIn conclusion, in pemphigus diseases the autoantibodies mainly belong to the IgG4 and IgG1 subclasses. Extensive experimental evidence demonstrates the blister-inducing potential of IgG4 autoantibodies. The pathogenic activity of autoantibodies of other subclasses seems likely, but needs further investigation.\nSubepidermal autoimmune blistering diseases\nBullous pemphigoid and pemphigoid gestationis\nBullous pemphigoid is an autoimmune blistering disease characterized by subepidermal blisters and associated with linear deposits of C3 and IgG at the epidermal basement membrane zone. Autoantibodies in bullous pemphigoid are directed against two hemidesmosomal antigens, BP230 and BP180\/type XVII collagen [87]. Pemphigoid gestationis, also referred to as herpes gestationis, is a subepidermal blistering disease associated with pregnancy and characterized by linear deposition of C3 and, to a lesser extent of IgG at the dermal\u2013epidermal junction, as detected by immunofluorescence microscopy [62, 66]. The autoimmune response in bullous pemphigoid and pemphigoid gestationis is mainly directed against epitopes clustered within the immunodominant 16th non-collagenous (NC16) A region of type XVII collagen [48, 65, 88].\nExperimental evidence generally supports the pathogenic role of autoantibodies against type XVII collagen for blister formation. Data from passive transfer animal models strongly suggest that antibodies to type XVII collagen are directly involved in the pathogenesis of bullous pemphigoid [44, 84]. In addition, in an ex vivo model utilizing cryosections of human skin, it has been demonstrated that binding of autoantibodies to the immunodominant NC16A domain of type XVII collagen, is the first critical step in subepidermal blister formation [31, 64].\nAnalysis of the subclass distribution of IgG autoantibodies in the skin of patients with bullous pemphigoid by immunofluorescence microscopy, revealed IgG4 as being the predominant subclass of autoantibodies in bullous pemphigoid, followed by IgG1 autoantibodies, while IgG2 and IgG3 autoantibodies were found only occasionally [1, 10\u201312, 22, 61, 83]. In addition, serum autoantibodies binding to the dermal\u2013epidermal junction by immunofluorescence microscopy also mainly belong to the IgG4 and IgG1 subclasses [10, 11, 61, 83]. Subsequent molecular analysis of IgG autoantibodies by immunoblotting and ELISA generally confirmed the predominance of IgG1 and IgG4 autoantibodies reactive with type XVII collagen and BP230 (Fig.\u00a02) [6, 19, 32, 41, 69].\nFig.\u00a02IgG1 and IgG4 autoantibodies mainly target type XVII collagen in bullous pemphigoid. Immunoblot analysis of serum from a bullous pemphigoid patient with recombinant type XVII collagen revealed that reactivity against its immunodominant domain consists mainly of IgG1 (lane 1) and IgG4 (lane 4), and less IgG2 (lane 2) and IgG3 (lane 3) autoantibodies\nIn contrast to bullous pemphigoid, in pemphigoid gestationis tissue-bound and circulating autoantibodies seem to mainly belong to the IgG1 and IgG3 subclasses [14, 37]. However, a recent study challenged these reports revealing IgG4 as the predominant IgG subclass of tissue-bound autoantibodies in pemphigoid gestationis patients [53], a pattern similar to the one found in bullous pemphigoid. Further studies should solve this contradiction.\nData from several studies in patients suggested a pathogenic role of IgG1 autoantibodies for blister formation (briefly reviewed in [43]). In a recent study, ELISA analysis showed that autoantibodies against the N-terminus of the extracellular domain of type XVII collagen predominantly belong to the IgG1 subclass. More importantly, a NC16A-specific IgG1 response was predominant in the acute phase of bullous pemphigoid, while IgG4 was predominantly detected in bullous pemphigoid patients in remission [32]. Using immunoaffinity purified IgG subclasses, it has been shown that IgG1, but not IgG4 autoantibodies from bullous pemphigoid patients activate the complement system in vitro (Fig.\u00a03) [46, 72]. This observation is in line with the currently accepted view that IgG4 is unable to activate the classical pathway of complement. However, until recently it was unclear which IgG subclass is actually pathogenic in bullous pemphigoid. Using the ex vivo cryosection model, we demonstrated that, in addition to IgG1, IgG4 autoantibodies are also able to activate leukocytes and to induce leukocyte-dependent tissue damage (Fig.\u00a04) [46]. Our results are in line with recent studies demonstrating that both polyclonal human IgG1 and IgG4 from patients with Wegener\u2019s granulomatosis and chronic urticaria can activate leukocytes [33, 70]. Although the pathogenic potential of IgG4 autoantibodies was significantly lower compared to IgG1, IgG4 autoantibodies, which generally predominate, may activate the inflammatory cells already recruited into the upper dermis by complement-fixing IgG1 autoantibodies and thus amplify the recruitment of additional leukocytes and the extent of blister formation. Therefore, when associated with IgG1 and\/or IgG3 autoantibodies, IgG4 may significantly contribute to the pathology induced by autoantibodies in antibody-induced granulocyte-mediated autoimmune blistering diseases [46].\nFig.\u00a03IgG4 autoantibodies, in contrast to IgG1, do not fix complement to the dermal\u2013epidermal junction in bullous pemphigoid. Cryosections of normal human skin were incubated with serum and immunoaffinity purified IgG1 and IgG4 antibody preparations from a bullous pemphigoid patient and, subsequently, treated with normal human serum as a source of complement. Both a serum and b purified IgG1 autoantibodies fixed complement C3 at the dermal\u2013epidermal junction in a linear fashion. c In contrast, incubation of cryosections with IgG4 specific for the dermal\u2013epidermal junction does not result in C3 deposition (all magnifications, \u00d7200)Fig.\u00a04IgG4 autoantibodies from bullous pemphigoid patients induce dermal\u2013epidermal separation in sections of human skin. Dermal\u2013epidermal separation in sections of normal human skin is induced by a IgG1 and b IgG4 autoantibodies from a bullous pemphigoid patient. c IgG antibodies from a healthy control (NHS) do not induce subepidermal splits (all magnifications, \u00d7200)\nSeveral reports suggested that binding of bullous pemphigoid antibodies to keratinocytes triggers a signal transduction [58, 73\u201375]. Bullous pemphigoid autoantibodies trigger a signal-transducing event that leads to expression and secretion of interleukin-6 and interleukin-8 from human cultured keratinocytes [58]. A series of studies from another group demonstrated that IgG1 autoantibodies from bullous pemphigoid patients and rabbit IgG against type XVII collagen induces Ca2+ release from intracellular storage sites [73\u201375]. Interestingly, complement activation by these IgG1 autoantibodies did not result in lysis of keratinocytes [73]. While the relevance of these findings is not yet fully understood, IgG2 and IgG4 patients\u2019 autoantibodies were found to inhibit the transient increase of intracellular Ca2+ induced by bullous pemphigoid IgG1 antibody [74].\nMucous membrane pemphigoid\nMucous membrane pemphigoid is a heterogeneous disease with regard to the clinical phenotype and the target antigens. Different target antigens have been identified in mucous membrane pemphigoid, including BP180, laminins 5 (epiligrin) and 6, \u03b24 integrin [62, 66]. In general, autoantibodies in mucous membrane pemphigoid mainly belong to the IgG4 and IgG1 subclasses [7, 34]. Interestingly, in anti-epiligrin cicatricial pemphigoid, autoantibodies against laminin 5 almost exclusively belong to the IgG4 subclass [34]. Consistent with these findings, sera from patients with anti-laminin 5 IgG autoantibodies do not fix C3 to the epidermal basement membranes and do not induce leukocyte-dependent dermal\u2013epidermal separation in vitro [34, 60]. These data suggest that complement activation does not play a major role in this disease and subepidermal blisters in these patients may develop via a direct effect of anti-laminin 5 IgG itself [34, 60].\nDiseases associated with autoimmunity against type VII collagen\nEpidermolysis bullosa acquisita is a chronic subepidermal blistering disease characterized by circulating and tissue-bound antibodies targeting the non-collagenous domain 1 (NC1) of type VII collagen. The pathogenic relevance of antibodies against type VII collagen is supported by compelling evidence: (1) autoantibodies from patients with epidermolysis bullosa acquisita were shown to recruit and activate leukocytes ex vivo resulting in dermal\u2013epidermal separation in cryosections of human skin [60, 63], (2) antibodies against type VII collagen induce subepidermal blisters when passively transferred into mice [67, 81], (3) immunization with recombinant autologous type VII collagen induces an autoimmune response to this protein resulting in a blistering phenotype closely resembling human epidermolysis bullosa acquisita [68].\nTissue-bound and circulating antibodies in epidermolysis bullosa acquisita patients mainly belong to the IgG1 and IgG4 subclasses [7, 15, 26, 47]. A similar distribution of IgG subclasses of autoantibodies is found also in SJL mice immunized against murine type VII collagen [68]. In these mice, while both non-complement-fixing IgG1 and complement-fixing IgG2a and IgG2b autoantibodies are produced after immunization, IgG2a\/b autoantibodies seem to induce blistering [68].\nSystemic lupus erythematosus and inflammatory bowel diseases may be also associated with autoantibodies against type VII collagen [13, 25, 30, 51]. However, in contrast to epidermolysis bullosa acquisita, autoantibodies from patients with bullous systemic lupus erythematosus and inflammatory bowel diseases mainly belong to IgG2 and IgG3, respectively [30, 51]. The pathogenic relevance of autoantibodies against type VII collagen in inflammatory bowel diseases has not yet been addressed [51]. IgG autoantibodies from patients with bullous systemic lupus erythematosus were shown to induce leukocyte-dependent dermal\u2013epidermal separation in cryosections of human skin ex vivo [30]. This findings suggest that the presence of complement-fixing autoantibodies is not a strict requirement for blistering in patients.\nConclusion and perspectives\nThe polyclonal antibody response against structural skin proteins in autoimmune bullous diseases is heterogeneous, but shows a skewing in subclass distribution of autoantibodies. The strong bias toward production of IgG4 autoantibodies in these organ-specific autoimmune diseases suggests chronic antigenic stimulation. In pemphigus, IgG4 autoantibodies that dominate the autoimmune response are clearly pathogenic. However, IgG1 autoantibodies also likely possess blister-inducing potential that requires further investigation. In subepidermal autoimmune blistering diseases, the effector functions of autoantibodies are important for blistering. Thus, in bullous pemphigoid and epidermolysis bullosa acquisita, complement-fixing IgG1 autoantibodies may show a significantly higher pathogenic potential when compared with IgG4 autoantibodies. Characterization of the blister-inducing capacity of different subclasses of autoantibodies in autoimmune bullous diseases will not only provide relevant mechanistic insights, but should also greatly facilitate the development of improved therapeutic modalities of autoimmune blistering diseases. Detailed knowledge on the pathogenic IgG isotype(s) will serve as a basis for the development of IgG subclass-specific immunoapheresis, skewing autoantibody production toward non-pathogenic subclasses by immunotherapy or blocking of complement or leukocytes activation by targeting specific IgG subclasses. A promising approach is represented by interventions aimed at inhibiting the production of autoantibodies in general or skewing the production of autoantibodies toward non-pathogenic subclasses. The molecular targets of these approaches may include different cytokines (e.g., IL-12 and IL-17) and their activity could be modulated using inhibitory antibodies, small peptide inhibitors or peptidomimetics as well as immunization with the autoantigen together with adjuvants known to induce a Th2 immune response.","keyphrases":["igg subclasses","complement","autoimmune bullous diseases"],"prmu":["P","P","P"]}
{"id":"Diabetologia-3-1-1794135","title":"Adiponectin receptor genes: mutation screening in syndromes of insulin resistance and association studies for type 2 diabetes and metabolic traits in UK populations\n","text":"Aims\/hypothesis Adiponectin is an adipokine with insulin-sensitising and anti-atherogenic properties. Several reports suggest that genetic variants in the adiponectin gene are associated with circulating levels of adiponectin, insulin sensitivity and type 2 diabetes risk. Recently two receptors for adiponectin have been cloned. Genetic studies have yielded conflicting results on the role of these genes and type 2 diabetes predisposition. In this study we aimed to evaluate the potential role of genetic variation in these genes in syndromes of severe insulin resistance, type 2 diabetes and in related metabolic traits in UK Europid populations.\nIntroduction\nAdiponectin, encoded by the gene ADIPOQ (also known as 30-kDa adipocyte complement-related protein, Acrp30, APM-1, APM1, ACDC, and gelatin-binding protein-28 or GBP28), is an adipokine with insulin-sensitising [1, 2] and anti-atherogenic actions [3]. Its levels correlate strongly with insulin sensitivity in humans and animal models, and increasing levels of plasma adiponectin produce a sensitising effect to the biological action of insulin [4]. Several genetic reports have detected association between adiponectin gene variants and obesity, insulin resistance, type 2 diabetes, and adiponectin levels [5\u20139].\nRecently, two adiponectin receptors were identified, adiponectin receptor 1 (ADIPOR1), cloned from a human skeletal muscle expression library, and adiponectin receptor 2 (ADIPOR2), identified using computational tools by Yamauchi et al. [10]. In mice, Adipor1 is expressed ubiquitously, with higher levels in skeletal muscle, and has a higher affinity for the globular form of adiponectin. Adipor2, on the other hand, is most abundant in the liver and preferentially binds the full-length form of adiponectin [10]. In contrast, both human ADIPOR1 (375aa) and ADIPOR2 (311aa) were predominantly expressed in skeletal muscle [10, 11].\nIn Mexican Americans, glucose-tolerant individuals with a family history of type 2 diabetes were reported to exhibit significantly lower levels of mRNA for ADIPOR1 and ADIPOR2 in skeletal muscle than subjects without a family history of diabetes. mRNA levels of both receptors were also reported to positively correlate with glucose disposal [11]. It is possible therefore that lower expression or altered function of the receptors would predispose to increased insulin resistance and type 2 diabetes. In fact, common variants in the ADIPOR1 gene were recently tested for association in a case\u2013control study with white and African American individuals, but no association was reported [12]. Two additional studies have evaluated the role of adiponectin receptor variants and risk of type 2 diabetes. In the Old Order Amish population, two intronic variants in ADIPOR1 were reported to associate with risk of type 2 diabetes, while in ADIPOR2 an extended haplotype block was associated with increased risk of disease [13]. In contrast, in a Japanese population no associations between polymorphisms in adiponectin receptor genes and risk of type 2 diabetes were detected [14]. More recently, studies in French and Finnish populations reported no association between ADIPOR1 single nucleotide polymorphisms (SNPs) and type 2 diabetes [15, 16], although evidence for association between rs767870 in ADIPOR2 and type 2 diabetes in a French population has been suggested [15]. In light of these studies, and the potential role of these receptors in insulin action and diabetes, we sought to identify and investigate the effects of genetic variants in these genes in UK populations.\nSubjects and methods\nParticipants\nSevere insulin resistance cohort A cohort of human patients with severe insulin resistance (SIR cohort) was collected at the University of Cambridge, UK. The inclusion criteria for this cohort were: (1) fasting insulin >150\u00a0pmol\/l or exogenous insulin requirement >200\u00a0U\/day; (2) acanthosis nigricans; and (3) BMI <33\u00a0kg\/m2. In the present study, 129 patients from this cohort were screened for mutations in exons and splice junctions of ADIPOR1 and ADIPOR2 genes. Cambridge Local Research Ethics Committee approval was obtained, and informed consent was received from all individuals before participation.\nCambridgeshire Case\u2013Control Study The Cambridgeshire Case\u2013Control Study has been described previously [17]. Briefly, this population-based case\u2013control study consists of 552 type 2 diabetes patients and matched control subjects. DNA was available from 516 cases and control subjects for this study. The cases were a random sample of Europid men and women with type 2 diabetes, aged 47 to 75\u00a0years, from a population-based diabetes register in a geographically defined region in Cambridgeshire, UK. The presence of type 2 diabetes in these participants was defined as onset of diabetes after the age of 30\u00a0years without use of insulin therapy in the first year after diagnosis. The control participants were individually matched to each of the diabetic subjects by age, sex and geographical location, but not by BMI. Potential control subjects with HbA1c levels greater than 6% were excluded, as this subgroup could have contained a higher proportion of individuals with previously undiagnosed diabetes. Ethical approval for the study was granted by the Cambridge Local Research Ethics Committee.\nEPIC-Norfolk participants This is a nested case\u2013control study within the EPIC-Norfolk prospective cohort study; both the case\u2013control and full cohort study [18, 19] have been described in detail previously. Briefly, the case\u2013control study consists of 417 incident type 2 diabetes cases and two sets of 417 control subjects, matched on age, sex, time in study and family physician, with the second set additionally matched for BMI. A case was defined by a physician\u2019s diagnosis of type 2 diabetes, with no insulin prescribed within the first year after diagnosis, and\/or HbA1c >7% at baseline or the follow-up health check. Controls were selected from those in the cohort who had not reported diabetes, cancer, stroke or myocardial infarction at baseline, and who had not developed diabetes by the time of selection. Potential control subjects with measured HbA1c levels >6% were excluded. DNA was available for this analysis from 354 cases and 741 control subjects. Ethical approval for the study was granted by the Norwich Local Research Ethics Committee.\nEly Study This is a population-based cohort study of the aetiology and pathogenesis of type 2 diabetes and related metabolic disorders in the UK [20]. It uses an ethnically homogeneous Europid population, in which phenotypic data were recorded at the outset and after 4.5\u00a0years. The cohort was recruited from a population sampling frame with a high response rate (74%), making it representative of the general population for this area in Eastern England. This analysis included 1,721 men and women, aged 35\u201379\u00a0years and without diagnosed diabetes, who attended the study clinic for a health check between 2000 and 2004. Of these, 1,005 were attending a follow-up health check, while the remaining 716 were newly recruited in 2000 from the original population sampling frame. Participants attending the health check underwent standard anthropometric measurements and a 75-g oral glucose tolerance test. Plasma glucose was measured using the hexokinase method. Plasma insulin was measured by two-site immunometric assays with either 125I or alkaline phosphatase labels. Cross-reactivity with intact proinsulin was less than 0.2% and interassay CVs were less than 7%. Ethical approval for the study was granted by the Cambridge Local Research Ethics Committee.\nPCR and sequencing\nGenomic DNA from patients was randomly preamplified in a GenomiPhi reaction (GE Healthcare UK, Chalfont St. Giles, UK) prior to amplification with gene-specific primers. Primers were designed using Primer3 software [21] to cover all coding exons and splice junctions. PCR primers and expected product sizes are described in Electronic supplementary material (ESM) Table 1. Following PCR, performed using standard conditions, products were purified using exonuclease I and shrimp alkaline phosphatase (USB Corporation, Cleveland, OH, USA), and bi-directional sequencing was performed using a DNA sequencing kit (Big Dye Terminator 3.1; Applied Biosystems, Foster City, CA, USA). Sequencing reactions were run on ABI3700 capillary machines (Applied Biosystems) and sequences were analysed using Mutation Surveyor version.2.20 (SoftGenetics LLC, State College, PA, USA).\nGenotyping\nSNP selection All SNPs with a minor allele frequency greater than 2% in our SIR cohort were selected for genotyping. To increase coverage in areas not re-sequenced, a number of dbSNPs were selected in an attempt to eliminate gaps, between genotyped SNPs, of greater than 2.5\u20133\u00a0kb on average (additional SNPs were selected prior to HapMap phase I data release). SNP choice was based on the following criteria: (1) all putative non-synonymous SNPs in dbSNP were selected irrespective of whether or not there was frequency or validation information for the SNP; (2) SNPs with frequency information were selected if their minor allele frequency was greater than or equal to 5%; (3) for SNPs with no frequency information the choice was based on whether the SNP was a double-hit SNP, had been validated by-cluster or by-submitter; and finally (4) some SNPs with no validation information were included to try and eliminate gaps of greater than 2.5\u20133\u00a0kb between SNPs selected for genotyping.\nGenotyping and quality control Samples were arrayed on 96-well plates with three replicates and one water control per plate. For the case\u2013control populations, cases and control samples were randomly distributed across each 96-well plate, with approximately the same number of cases and controls per plate. Genotyping of samples was performed in 384-well plates at the Wellcome Trust Sanger Institute, Cambridge, using an adaptation of the homogenous MassExtend protocol for the MassArray system (Sequenom, San Diego, CA, USA) [22]. Assay results for case\u2013control are described in ESM Table 2. The following criteria were used to pass assays resulting from genotyping: (1) call rates had to be greater than or equal to 90% (in one case a call rate of 88% was accepted); (2) concordance rates between duplicate samples had to be greater than or equal to 98%; (3) minor allele frequency had to be greater than or equal to 1% in the genotyped populations; (4) agreement with Hardy\u2013Weinberg equilibrium was tested separately in cases and controls using a \u03c72 goodness-of-fit test, and if p\u2009<\u20090.01 in controls, the assay was failed, while if p\u2009<\u20090.01 in cases, the assay was flagged but included in primary analysis. Assays that failed quality control were excluded from further analysis. In total we analysed results from nine SNPs in ADIPOR1 and 15 SNPs from ADIPOR2.\nStatistical analysis\nAll analyses used SAS 8.02 (SAS Institute, Cary, NC, USA) or Stata 7.0 (Stata Corporation, College Station, TX, USA) statistical programs, unless otherwise stated. All used genotypes were in Hardy\u2013Weinberg equilibrium. The pair-wise linkage disequilibrium (LD) coefficient for the controls (r2) was calculated for genotyped SNPs and is represented in Fig.\u00a01. For each SNP, two primary models were used to assess association with diabetes and quantitative traits, the linear trend (additive model) on 1df and the general model on 2df. Since the results from these analyses were not materially different, we present only the results from the linear trend test. Tests for association were performed by logistic regression combined in the two case\u2013control populations adjusting for age, sex and population. Between-study heterogeneity was tested by log-likelihood ratio tests. Quantitative trait analysis was undertaken in the Ely Study population. Association between fasting plasma insulin, fasting and 2-h post-load plasma glucose, 30-min insulin increment (30-min insulin minus fasting insulin over 30-min glucose in an OGTT), BMI and genotype was tested in a multiple regression model, adjusted for age and sex.\nFig.\u00a01Genomic structure and pair-wise marker LD in ADIPOR1 (a) and ADIPOR2 (b). The location of SNPs identified in this study and\/or genotyped is represented along the gene (bold type: SNPs selected for genotyping, and failed or monomorphic SNPs). Exons are represented in boxes (black for coding and open for untranslated). Introns and flanking sequences appear as lines. The pair-wise marker LD measured by r2 statistics is shown below the genomic structures and indicated by the shade of grey blocks (white to black) and the r2 value. a, b, isoforms a and b in ADIPOR2\nResults\nTo evaluate whether genetic variation in ADIPOR1 and ADIPOR2 contributed to severe insulin resistance in humans, we sequenced exons and splice junctions in both genes in 129 individuals from our SIR cohort. These individuals were unrelated and had a variety of syndromes of severe insulin resistance [23]. We identified 13 and 29 polymorphisms in ADIPOR1 and ADIPOR2 respectively, none of which altered the protein sequence of either gene (ESM Table 3). This suggests that no functional mutations were identified in these genes. We next tested whether common variants at these genes impacted on type 2 diabetes predisposition or related quantitative traits in UK Europid populations. We selected all variants with a minor allele frequency greater than 2% in the SIR cohort and supplemented our SNP selection with additional variants from the dbSNP database plus SNPs with significant association results in other published studies. Thirteen polymorphisms from ADIPOR1 and 23 polymorphisms from ADIPOR2 were selected for genotyping in two type 2 diabetes case\u2013control studies (n=2,127) and one metabolic quantitative trait study (n=1,721) (Fig.\u00a01). Those SNPs that passed our genotyping quality control criteria (described in methods) were used to investigate the degree of LD in control individuals across ADIPOR1 and ADIPOR2. In total nine SNPs in ADIPOR1 and 15 SNPs in ADIPOR2 were included in the analysis. LD was measured by r2 statistic and is depicted in Fig.\u00a01. Under both general and linear trend models no evidence was found for statistically significant associations between SNPs and disease risk (Table\u00a01). In ADIPOR1, SNPs rs2275738, rs2275735 and rs10581 were removed from the quantitative trait analysis as they were out of Hardy\u2013Weinberg equilibrium (p<0.01). For the remaining SNPs there was also no evidence for association of the SNPs tested with BMI, fasting and 2-h glucose levels, fasting insulin or 30-min insulin incremental response (ESM Table 4). In ADIPOR2 a few SNPs showed nominally significant association with BMI and 2-h glucose levels. However, these results are likely to be chance findings given the number of statistical tests performed (ESM Table 4). \nTable\u00a01ADIPOR1 and ADIPOR2 SNP association results with type 2 diabetesGene\u00a0SNPAlleleCasesControlsOdds ratiop value trendp value homogeneity1\/211 (%)12 (%)22 (%)11 (%) 12 (%) 22 (%)(95% CI)ADIPOR1rs6666089G\/A378 (47.6)340 (42.8)76 (9.6)536 (46.2)525 (45.2)100 (8.6)1.00 (0.86,1.15)0.96010.750rs1539355A\/G374 (46.6)346 (43.1)82 (10.2)558 (45.9)547 (45.0)111 (9.1)1.02 (0.89,1.18)0.73500.649rs2275738C\/T277 (34.4)379 (47.1)149 (18.5)395 (34.0)551 (47.4)216 (18.6)0.97 (0.85,1.1)0.61090.496IVS3+85C\/G707 (92.1)61 (7.9)0 (0)1046 (92.6)82 (7.3)2 (0.2)1.03 (0.73,1.45)0.87210.654rs2275735C\/T763 (92.0)66 (8.0)0 (0)1152 (93.5)79 (6.4)1 (0.1)1.24 (0.88,1.75)0.22390.228rs1342387C\/T249 (30.4)414 (50.5)156 (19.0)362 (29.5)634 (51.7)231 (18.8)0.96 (0.85,1.1)0.59090.603rs10581G\/A741 (93.8)46 (5.8)3 (0.4)1079 (95.7)46 (4.1)3 (0.3)1.35 (0.92,1.97)0.12810.466rs7539542C\/G373 (47.4)336 (42.7)78 (9.9)554 (46.9)510 (43.2)117 (9.9)1.00 (0.87,1.15)0.95660.193rs2185781C\/T532 (63.6)256 (30.6)49 (5.9)789 (63.8)398 (32.2)50 (4.0)1.08 (0.92,1.26)0.34250.495ADIPOR2rs1029629A\/C353 (44.6)372 (47.0)67 (8.5)537 (44.9)528 (44.1)132 (11.0)0.94 (0.81,1.08)0.34970.265rs11061971A\/T228 (28.1)409 (50.4)174 (21.5)349 (29.5)589 (49.7)246 (20.8)1.05 (0.93,1.20)0.43940.092rs4766415A\/T207 (25.6)411 (50.7)192 (23.7)323 (27.1)586 (49.2)282 (23.7)1.04 (0.92,1.19)0.51590.074rs767870A\/G575 (71.3)214 (26.5)18 (2.2)857 (72.3)310 (26.1)19 (1.6)1.08 (0.9,1.3)0.38430.095rs2286384G\/C214 (25.7)429 (51.5)190 (22.8)329 (26.8)618 (50.3)282 (22.9)1.03 (0.9,1.17)0.68370.037*rs2286383C\/T237 (28.6)418 (50.4)174 (21.0)368 (30.1)602 (49.2)253 (20.7)1.05 (0.92,1.19)0.45980.068I290C\/A618 (75.2)193 (23.5)11 (1.3)940 (77.9)252 (21.0)14 (1.2)1.18 (0.97,1.44)0.09850.118rs9805042C\/T603 (75.8)180 (22.6)12 (1.5)900 (79.0)225 (19.8)14 (1.2)1.17 (0.96,1.43)0.12820.051rs2286382G\/A791 (94.5)45 (5.4)1 (0.1)1159 (94.2)71 (5.8)0 (0)0.98 (0.67,1.43)0.90540.664rs12342C\/T370 (44.6)376 (45.3)84 (10.1)543 (44.3)540 (44.1)142 (11.6)0.96 (0.84,1.1)0.54610.398rs1044471C\/T227 (27.8)420 (51.5)169 (20.7)334 (27.6)592 (49.0)282 (23.3)0.93 (0.82,1.05)0.25000.115rs2286380A\/T621 (77.0)176 (21.8)10 (1.2)947 (79.0)238 (19.8)15 (0.0125)1.12 (0.91,1.37)0.27620.155rs13219T\/C251 (30.2)403 (48.4)178 (21.4)369 (30.1)618 (50.4)238 (19.4)1.04 (0.91,1.18)0.56100.276rs2286379T\/C259 (31.0)399 (47.7)178 (21.3)376 (30.6)614 (50.0)239 (19.4)1.03 (0.91,1.17)0.64270.269rs3815325G\/A490 (60.6)273 (33.7)46 (5.7)712 (58.7)437 (36.0)64 (5.3)0.96 (0.82,1.11)0.57850.26311, homozygous for allele 1, 12, heterozygous, 22, homozygous for allele 2. Genotype counts are shown with frequency (%), odds ratios are shown per allele 2 with significance calculated for the linear trend (p value trend). Homogeneity in results from association between EPIC and Cambridge Case\u2013Control populations was tested (p value homogeneity)*p values\u2009\u2264\u20090.05\nDiscussion\nThe current study, including sequencing of 129 patients with syndromes of severe insulin resistance and genotyping of both population-based type 2 diabetes case\u2013control studies (n=2,127) and a metabolic quantitative trait study (n=1,721), suggests that ADIPOR1 and ADIPOR2 genetic variants are unlikely to be major risk factors for type 2 diabetes and insulin resistance in UK Europid populations.\nAlthough sequencing of ADIPOR1 and ADIPOR2 genes in a cohort of patients with syndromes of severe insulin resistance (n=129) led to the identification of 42 polymorphisms, including 21 novel rare variants, none altered the protein sequence. Given that this group of patients comprises a heterogeneous cohort representative of a variety of syndromes of extreme insulin resistance, the lack of variants affecting the protein sequence suggests that functional mutations in the genes ADIPOR1 and ADIPOR2 are not major causes of extreme insulin resistance in humans.\nFor ADIPOR1, our data in case\u2013control studies are consistent with previous reports showing that SNPs in this gene are not associated with type 2 diabetes risk in Europid [12, 15, 16], African [12] or Japanese populations [14]. This is in contrast to evidence from the Old Order Amish, where an association of rs2275738 (and rs2275737 which is in perfect LD with it) and rs1342387 with type 2 diabetes risk has been reported [13]. Since the Amish represent an isolated population, it is possible that variants in ADIPOR1 play a role in type 2 diabetes predisposition among them, which is not apparent in more heterogeneous populations. However, given that both reported associated SNPs are present in all populations, it is unlikely that either is the true causal variant, although they could be detecting, through LD, the effect of an untested SNP. Alternatively, given the relative small sample sizes used and lack of adjustment for multiple testing, the authors may have reported a false\u2013positive association.\nFor ADIPOR2 the data are less consistent. While no evidence of association between ADIPOR2 SNPs and type 2 diabetes was present in a Japanese population [14], an association has been suggested between SNP rs767870 and type 2 diabetes risk in French populations [15]. Our data, and those from the Old Order Amish [13], do not support this finding. Notably, in the French population, meta-analysis of rs767870, including 1,380 individuals with type 2 diabetes and 1,496 controls, demonstrated allelic association of nominal significance only (p=0.02), while the most significant result was under a recessive model (p=0.0018). Our study has only 23% power to detect such small recessive effects (odds ratio 1.3) with an allele frequency of 0.15, and this could explain our discrepant result. Further large scale studies of this SNP in additional populations will be required to elucidate its role in conferring risk of disease. A haplotype was also reported to increase risk of diabetes in the Old Order Amish [13]. However, when we performed haplotype analyses in both ADIPOR1 and ADIPOR2, we were unable to detect any significant associations with diabetes risk (data not shown), and this discrepancy might also be accounted for by differences in the populations studied.\nWith regard to quantitative metabolic traits, to date two studies have reported nominally significant results with insulin sensitivity and body size [16, 24]. The first study showed nominally significant associations between two tightly linked SNPs (rs6666089 and an intronic \u20131927 SNP) in ADIPOR1 and decreased insulin sensitivity and increased HbA1c levels [24]. We did not test association with HbA1c, but have not replicated any association with insulin sensitivity (including with SNP rs6666089) as assessed by fasting insulin measurements. Recently Kantartzis and colleagues reported that the association between rs6666089 and insulin sensitivity is observed only in more obese, but not in lean individuals [25]. This dependence on the degree of adiposity could explain some of the discrepant results observed for this SNP, if there are substantial differences in mean BMI between populations tested. To explore this hypothesis, we performed SNP\u00d7BMI interaction tests (using BMI both as a continuous trait and splitting the population above and below the median) on all ADIPOR1 SNPs tested. Our data provided no evidence for such an interaction (data not shown). We also specifically tested for association between rs6666089, and measures of insulin and glucose in participants with BMI above and below 25. Again we found no statistically significant difference between the two groups (p>0.3). Therefore, it is unlikely that BMI differences between populations are at the root of our discrepant results. Of note, our data cannot exclude possible small effects of this SNP on insulin sensitivity, in particular if this effect is only observed in subjects with higher BMI. The second study suggested there was evidence of association between two markers (rs10920534 and rs2275738) and BMI, but this evidence came from men only. Furthermore, three other markers (rs10920534, rs12045862 and rs7539542) were reported to associate with fasting and 2-h insulin levels, particularly in men at baseline [16]. We did not test rs10920534, but did test rs6666089 (D\u2032=r2=1 with rs10920534) and found no evidence for SNP\u00d7sex interaction on any of the quantitative traits we analysed. Our data also did not replicate the sex effect of SNP rs7539542 on insulin measurements. Although we did not test SNP rs12045862, previously published data was conflicting. Thus while the C allele was suggested to be associated with higher 2-h insulin levels in men (p=0.027), in women the T allele was associated with higher levels (p=0.029) [16]. Given that neither we, nor others [24], have found evidence for sex\u00d7SNP interaction effects on measures of insulin sensitivity, we suggest that further confirmatory studies are required to test this hypothesis.\nUntil recently, remaining uncertainty regarding the identity of the true physiological receptors for adiponectin [26] had hampered interpretation of the functional relevance of polymorphisms in ADIPOR1 and ADIPOR2 with respect to adiponectin\u2019s insulin sensitising effects. However, a recent yeast two-hybrid screen identified an ADIPOR1 interacting molecule, APPL1, thought to mediate many of the effects of adiponectin [27]. This molecule was shown to interact with both ADIPOR1 and ADIPOR2 in an adiponectin-sensitive manner, and was shown to mediate many of adiponectin\u2019s insulin-sensitising effects. This suggests that ADIPOR1 and ADIPOR2 could be therapeutic targets for drug development and should renew interest in association studies, such as those we present here, testing polymorphisms in ADIPOR1 and ADIPOR2 for effects on type 2 diabetes risk and metabolic traits.\nIn summary, sequencing of ADIPOR1 and ADIPOR2 genes in a cohort of patients with syndromes of severe insulin resistance (n=129) suggests that functional mutations in these genes are not a major cause of extreme insulin resistance in humans. Furthermore, testing of common genetic variants (n=24) did not find evidence for association of these genes with type 2 diabetes risk (n=2,127) or with five additional quantitative metabolic traits (n=1,721). These data suggest that ADIPOR1 and ADIPOR2 variants are unlikely to be major risk factors for type 2 diabetes and insulin resistance in UK Europid populations, although more detailed analyses of gene variants may be required to exclude a potential minor role of these genes in insulin resistance and glucose homeostasis.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material. \nTable\u00a01\nPrimer sequences and PCR product sizes used for sequencing ADIPOR1 and ADIPOR2 (DOC 46\u00a0kb)\nTable\u00a02\nGenotyping assay results for case-control studies (DOC 61\u00a0kb)\nTable\u00a03\nSNPs detected by sequencing ADIPOR1 and ADIPOR2 in severely insulin-resistant patients (DOC 78\u00a0kb)\nTable\u00a04\nADIPOR1 and ADIPOR2 SNP association results with quantitative traits (DOC 215\u00a0kb)","keyphrases":["insulin resistance","association studies","type 2 diabetes","adipor1","adipor2","polymorphisms"],"prmu":["P","P","P","P","P","P"]}
{"id":"Oecologia-3-1-2039837","title":"Geographic and seasonal patterns and limits on the adaptive response to temperature of European Mytilus spp. and Macoma balthica populations\n","text":"Seasonal variations in seawater temperature require extensive metabolic acclimatization in cold-blooded organisms inhabiting the coastal waters of Europe. Given the energetic costs of acclimatization, differences in adaptive capacity to climatic conditions are to be expected among distinct populations of species that are distributed over a wide geographic range. We studied seasonal variations in the metabolic adjustments of two very common bivalve taxa at European scale. To this end we sampled 16 populations of Mytilus spp. and 10 Macoma balthica populations distributed from 39\u00b0 to 69\u00b0N. The results from this large-scale comprehensive comparison demonstrated seasonal cycles in metabolic rates which were maximized during winter and springtime, and often reduced in the summer and autumn. Studying the sensitivity of metabolic rates to thermal variations, we found that a broad range of Q 10 values occurred under relatively cold conditions. As habitat temperatures increased the range of Q 10 narrowed, reaching a bottleneck in southern marginal populations during summer. For Mytilus spp., genetic-group-specific clines and limits on Q 10 values were observed at temperatures corresponding to the maximum climatic conditions these geographic populations presently experience. Such specific limitations indicate differential thermal adaptation among these divergent groups. They may explain currently observed migrations in mussel distributions and invasions. Our results provide a practical framework for the thermal ecophysiology of bivalves, the assessment of environmental changes due to climate change and its impact on (and consequences for) aquaculture.\nIntroduction\nA fundamental step forward in predicting the ecological and economic consequences of climate change would be to identify the mechanistic link between the physiology of species and climatic variations. How and to what extent climatic variations cause stress in eurythermal bivalve species is still not fully understood and is expected to differ among taxa. In general, for organisms that maintain their body temperature by absorbing heat from the environment (ectotherms), temperatures that are too high can act as a stressor in two ways. On the one hand, it may cause the denaturing of sensitive proteins. This damage can be minimized by the actions of heat-shock proteins, which increase the thermostability of proteins and chaperone cellular processes (Feder and Hofmann 1999; Lyons et al. 2003). On the other hand, excessive temperatures may cause oxygen limitation, due to a limited respiratory capacity, resulting in a maximum respiration rate at a specific temperature, beyond which anaerobic metabolic pathways are utilized and respiration rates usually drop drastically (P\u00f6rtner 2001, 2002). The temperature that corresponds to this respiratory maximum is referred to as the breakpoint temperature. Since breakpoint temperatures tend to correlate with the maximum habitat temperatures of several marine ectotherms (Somero 2002), climate-change-induced shifts in the distributions of these species may be due to their respiratory limitations.\nThe metabolic rate of an ectotherm is proportional to its respiration rate. To remain energy-efficient, and for protection against oxygen shortage during the warmer seasons, organisms need to adjust their metabolic energy requirements to their maximum food uptake and oxygen consumption rate. Seasonal variation in the respiratory response to temperature reveals how organisms adjust throughout the annual cycle. While several terrestrial and aquatic mollusks apply the strategy of metabolic down-regulation during the summer (Buchanan et al. 1988; McMahon 1973; McMahon and Wilson 1981; Storey and Storey 1990; Wilson and Davis 1984), others do not (McMahon et al. 1995). The level of metabolic down-regulation is also reflected by the temperature quotient (Q\n10) of the metabolic rate, i.e., the sensitivity of the organisms\u2019 metabolism to changes in body temperature. Experimental work performed by Widdows (1976) has demonstrated that when thermal fluctuations approach and exceed breakpoint temperatures, this sensitivity usually decreases. Such reduced sensitivities have been found for field populations of M. edulis (Newell 1969) and M. balthica (Wilson and Elkaim 1991) sampled from high-shore habitats in summertime. Taken together, the thermal sensitivity of the metabolic rate (Q\n10) is expected to decrease towards the warm end of the species distribution range.\nLately, invasions (Geller 1999; Wonham 2004) and northward introgression (Luttikhuizen et al. 2002) have been reported for Mytilus spp., and a range contraction for M. balthica (Hummel et al. 2000). Aiming to bridge the gap between the observed migrations of these species and the changing climate in Europe, we studied seasonal adjustments to temperature in bivalve metabolism at the European scale, from 39\u00b0 to 69\u00b0N. The analysis of patterns in the extent of metabolic acclimatization across widely distributed populations will reveal how core populations differ from marginal populations, presenting species-specific responses to cold winters and hot summers near the upper and lower edges of temperature-induced distribution ranges. Such latitudinal gradients may provide a powerful tool that can be used to understand the temperature-dependent distributions of species and to predict their adaptive tolerance to climate change. In macrophysiological studies, the possibility of differential adaptation to regional climates among distinct populations should be taken into account. Based on neutral genetic variation, both European mussels and clams can be subdivided into three main genetic groups (Hummel 2006; Daguin et al. 2001; Luttikhuizen et al. 2003; Skibinski 1985). Uncertainty exists about the nomenclature of European Mytilus species. Traditionally, mussels from the Mediterranean Sea and the coast of the Iberian Peninsula are referred to as Mytilus\ngalloprovincialis (Lamarck). Mussels from the English Channel, the North Sea coast, and the Atlantic coast of Norway and Iceland are named Mytilus edulis (L.), and the mussels from the Central Baltic Sea are called Mytilus trossulus (Gould), which is based upon their genetic resemblance to Mytilus trossulus from the Atlantic coast of Canada (Varvio et al. 1988). However, the morphological characteristics of the different holotypes do not diagnostically separate the three genetic groups found in European mussels. In addition, broad hybridization zones (Daguin et al. 2001) and deep introgressions (Luttikhuizen et al. 2003) have been reported; a genetic characteristic of a single species with separated clades. To avoid confusion, we will not use these species names in this study, but rather refer to them as Mytilus spp., with reference to their geographic distributions, i.e., a Baltic Sea group, a North Sea group, and a Mediterranean Sea and Bay of Biscay group. Also, different genetic groups have been distinguished for M. balthica (Hummel 2006; Luttikhuizen et al. 2003) that have never been described as different species. The coupling of phylogenetic and ecophysiological analyses is urgently needed to understand and predict current and future migrations of these bivalve taxa and their clades.\nMethods\nFieldwork\nTo study the marginal and core populations of both taxa, including different genetic groups, we defined 21 research sites of interest along the European coastline, and sampled 11 M. balthica populations and 16 Mytilus spp. populations, respectively. All sampling stations are numbered (1\u201321) in Fig.\u00a01. Whenever a station name is mentioned in this text, its number is given between parentheses. The sampling stations were located in the coastal areas of the different sea basins that represent much of the European coastline (including the Mediterranean Sea, the Bay of Biscay, the North Sea and the Baltic Sea), and a variety of microhabitats. The sampling stations in the Mediterranean Sea are characterized by a high and stable salinity (38\u201340\u00a0PSU). In the Bay of Biscay and North Sea estuaries, the ambient salinity at the sampling stations fluctuated, generally varying between \u223c35 and \u223c25\u00a0PSU. Due to elevated river runoff, oligohaline conditions may have occurred occasionally at these stations. The Baltic Sea stations were distributed along the Baltic salinity gradient. While the ambient salinity is still \u223c15\u00a0PSU in the Mecklenburg Bight (7), it is around 6\u20137 in the Gulf of Gdansk (6) and Ask\u00f6 (5), and has decreased to 3\u00a0PSU in Ume\u00e5 (3). Although high and low peak temperatures occur at the intertidal sampling stations in the Bay of Biscay and the North Sea, water temperatures were intermediate compared to the warmer Mediterranean Sea and the colder Baltic Sea sites (Fig.\u00a02). In summer, the Baltic Sea warms rapidly, reaching temperatures that are comparable to North Sea conditions. In the tidal estuaries of the Bay of Biscay and the North Sea, sampling was carried out at mid-shore level. Lacking significant tidal movements, Baltic Sea populations of Mytilus spp. and M. balthica, and Mediterranean Sea populations of Mytilus spp. were sampled at a water depth of 0.5\u20131.0\u00a0m. During the period July 2003\u2013May 2005 16 populations (i.e., 10 Mytilus spp. and 6 M. balthica) were visited seasonally and the others only once or twice. During each sampling occasion, mussels were sampled from hard substrate, and clams were sieved from the sediment. For Mytilus spp. populations, mean shell lengths ranged from 28\u00a0mm (SD: 2.0) for specimens sampled in the Gulf of Gdansk (6) to 33\u00a0mm (SD: 3.5) for mussels sampled from the Santa Giusta Lagoon (21). Mean shell lengths of mussels collected from the other populations was in the same range, and standard deviations were <3.5. About 95% of all sampled mussels fell within a size range of 25\u201335\u00a0mm. For M. balthica mean shell lengths ranged from 12\u00a0mm (SD: 1.5) for clams sampled from the Mecklenburg Bight (7) to 16\u00a0mm (SD: 1.8) for clams sampled at Point d\u2019Aiguillon (15). Mean shell lengths of all other clam populations were within this range, standard deviations did not exceed 2.8\u00a0mm, and 95% of all sampled clams fell within the size range of 10\u201319\u00a0mm. After sampling, the collected animals were stored in foam boxes (clams were offered sediment from the field to bury) and transported to a nearby laboratory, where they were kept in (constantly aerated) aquaria, under ambient field conditions (\u00b13\u00a0\u00b0C). Measurements were carried out within 24\u00a0h after sampling.\nFig.\u00a01Map of research area with 21 research sites. The coastal area is subdivided into three parts representing the approximate distributions of the three genetic groups found in Mytilus spp. and M. balthica. Light gray indicates the Baltic Sea group, medium gray the North Sea group and dark gray the Mediterranean Sea and Bay of Biscay group (hybridization and introgression zones are not indicated). The white and the gray circles represent mussel and clam populations, respectively (see legend). Site no.\/name: 1, Rykyavik; 2\/3, Ume\u00e5 a\/b; 4, Fallvikshamn; 5, Ask\u00f6; 6, Gulf of Gdansk; 7, Lomma; 8, Mecklenburg Bight; 9, Grevelingenmeer; 10, Westerschelde estuary; 11, Granville; 12, Le Vevier; 13, Brest; 14, Loire estuary; 15, Point d\u2019Aiguillon; 16, Bidasoa estuary; 17, Mundaka estuary; 18, Vias plage; 19, Marseille; 20, Gulfo di Oristano; 21, Santa Giusta lagoon\nFig.\u00a02Annual sea surface temperature (SST) regimes for the sampling stations in the Baltic Sea, the North Sea, the Bay of Biscay, and the Mediterranean Sea. Lines and error\nbars represent monthly averages and standard deviations, based on measurements taken by satellite twice daily, at 9\u00a0a.m. and 2\u00a0p.m., during the period 2003\u20132005. This data were taken from the NASA JPL website (NASA Jet Propulsion Laboratory 2005)\nTemperature profiles\nField temperature profiles were obtained between April 2004 and May 2005 with temperature loggers (HOBO Water Pro\u00ae, Onset Computers, Bourne, MA, USA) at research sites 5, 7, 9, 10, 15, 16 and 18, with a resolution of one measurement per 30\u00a0min. The loggers were positioned in the direct vicinity of the animals. Logger output was compared with the sea surface temperature (SST) profiles obtained by satellite (NASA Jet Propulsion Laboratory 2005). Mean habitat temperature of the shallow water and intertidal habitats showed a constant relation with SST. In summer SST was about two degrees lower than the mean values calculated from the logger data at all sites, while in winter the SST was slightly lower for the Atlantic and Mediterranean sites. For the Baltic Sea sites, winter SSTs were similar to the logger data. Using the relation between SST and logger data, we estimated the acclimatization temperature of each mussel and clam population for each sampling occasion. This acclimatization temperature is defined in this study as the mean water temperature for the period of 30\u00a0days before sampling. We assume that this is a proper indication of the temperature to which the animals should be well adjusted.\nRespiration rates\nWithin 24\u00a0h after sampling, groups of 3\u20136 mussels and 7\u201315 clams were gently removed from their aquaria, and without further acclimation transferred to respiration chambers of volume 264 and 154\u00a0ml, respectively. The chambers were positioned in a thermostated tank to maintain a constant temperature during the incubations (\u00b10.3\u00a0\u00b0C). The chambers were filled with filtered habitat water, previously aerated to 100% oxygen saturation. Chamber lids contained Clark-type electrodes to record the change in oxygen tension in the water. In this way, 2\u20136 replicate measurements were taken per population and temperature after each sampling occasion. Control measurements were carried out using the same experimental setup, without animals. The total number of M. balthica specimens used from the populations that were sampled seasonally ranged from 475 from the Westerschelde estuary (10) to 685 from the Mecklenburg Bight (8). For Mytilus spp. the number of experimental animals ranged from 104 from Point d\u2019Aiguillon (15) to 322 from Ask\u00f6 (5). To avoid light-induced stress in M. balthica, the chambers were made out of tanned Plexiglas. Respiration rates were measured at 3, 10, 17, 24 and 31\u00a0\u00b0C. Measurements continued until the oxygen tension in the chambers had decreased by 20\u201330%. After each measurement, experimental animals were frozen at \u221220\u00a0\u00b0C and subsequently lyophilized for 72\u00a0h to a stable weight. From the dried specimens, valves were removed and the soft tissue dry-weights determined to the nearest mg, after which the mass-specific respiration rates and the temperature quotients were calculated. In addition, we estimated the respiration rates and Q\n10 values at the acclimatization temperature, per population, per season. Both, respiration rates and Q\n10 values were based on the rates that corresponded to the seven-degree temperature interval (for instance 10\u201317\u00a0\u00b0C) nearest to the acclimatization temperature. The respiration rates were assessed by linear interpolation, and the Q\n10 values were calculated with the following equation:\n. Here k\n2 and k\n1 are the respiration rates measured at the higher and the lower temperatures, t\n2 and t\n1, respectively.\nStatistical analysis\nThe following working hypothesis was formulated: H0: \u201cSeason\u201d or \u201cgenetic group\u201d have no effect on the metabolic temperature dependence of M. balthica or Mytilus spp. Because temperature is not the only the factor limiting ectotherm metabolism, commonly used statistical methods such as correlations and regressions are not very well suited for testing this relationship (Blackburn et al. 1992; Cade et al. 1999; Thompson 1996). Estimating the function along the upper edge of this distribution would describe the evolutionary relation between temperature and metabolism in our species. Data points scattered below this \u201cslope of upper bounds\u201d (Blackburn et al. 1992) are responses induced by other limiting factors. To estimate this slope of maximum respiration rate at a given temperature, we carried out a main axis regression analysis (after Thompson et al. 1996). The first step in this is a general regression through all data points. Subsequently, the data are divided into points that fall below and above the line of least squares. All data points that were found above are then used to fit a second regression that again divides the data into two subsets; etc. This was repeated three times. The regression lines were forced through the x, y point (\u22122, 0), assuming that at the approximate freezing point of seawater bivalve aerobic metabolism is near to zero. The final regression line for M. balthica populations was based on 12 data points, with an r\n2 of 0.95. For Mytilus spp. populations, the final regression was based on seven data points with an r\n2 of 0.99. The distance between measured respiration rates and this upper slope describes the extent of metabolic down-regulation in the populations. These rate-deviations (distances) were estimated for all populations and used for statistical comparison. ANOVAs based on rate deviations were carried out with \u201cseason\u201d or \u201cgenetic group\u201d as independent variables. For the \u201cgenetic group\u201d analysis, only data points from the temperature range at which all three groups were sampled were included, i.e., the temperature range for the \u201cgenetic group\u201d comparison for M. balthica was 9\u201318\u00a0\u00b0C, and for Mytilus ssp. 9\u201315\u00a0\u00b0C. Bonferroni\u2019s multiple comparison test was used to test for specific differences among seasons or genetic groups.\nResults\nRespiration rates at experimental temperatures\nFor each population sampled seasonally, mean respiration rates at experimental temperatures are given in Fig.\u00a03 for Mytilus spp. and in Fig.\u00a04 for M. balthica. Standard deviations among replicate measurements were usually less than 20% of mean values at experimental temperatures near ambient conditions. At high experimental temperature (31\u00a0\u00b0C), variation among replicate measurements could be higher, with standard deviations occasionally exceeding 50% of the mean values. This was caused by the fact that some groups of specimens exhibited high respiration rates, while other groups consumed almost no oxygen.\nFig.\u00a03Seasonal variation in the respiratory response to an experimental temperature range for ten European Mytilus spp. populations. Station names and degrees north are given in the graphs. Numbers in square boxes refer to the station names presented in Fig.\u00a01. See legend for explanation of the symbols\nFig.\u00a04Seasonal variation in the respiratory response to an experimental temperature range for six European M. balthica populations. Station names and degrees north are given in the graphs. Numbers in square boxes refer to the station names presented in Fig.\u00a01. See legend for explanation of the symbols\nThe respiration rate of mussels usually declined between 24 and 31\u00a0\u00b0C (Fig.\u00a03). Exceptions were some cold-acclimatized populations that showed maximum rates between 17 and 24\u00a0\u00b0C [e.g., the Ask\u00f6 (5), Gulf of Gdansk (6), and Grevelingenmeer (9) populations sampled in January]. Peaks in respiration rates between 1.5 and 2.5 (mg O2\/g (dry weight)\/h) at 24\u00a0\u00b0C were frequently observed in springtime (April\u2013May) and occasionally in other seasons. Respiration rates never exceeded 1.5 (mg O2\/g (dry weight)\/h) in mussels from the Mecklenburg Bight (6) and the Gulf of Gdansk (5).\nThe highest respiration rates for the M. balthica specimens were mainly observed at 31\u00a0\u00b0C; the maximum temperature applied in this study (Fig.\u00a04). Especially in the populations from the Westerschelde estuary (10) and Point d\u2019Aiguillon (15), respiration rates never decreased at high experimental temperatures. However, in the Baltic populations, outside the summer period maximum rates were observed at a lower experimental temperature. In Ume\u00e5 (3), respiration rates measured in January and April declined at experimental temperatures exceeding 10\u201317\u00a0\u00b0C.\nRespiration rates at acclimatization temperatures\nThe respiration rates of both taxa increased with acclimatization temperature (Fig.\u00a05). In winter and springtime, deviations from the upper edge slope were small for both Mytilus spp. and M. balthica. At acclimatization temperatures of >12\u00a0\u00b0C, an increasing number of data points were scattered at increasing distance from the upper edge slope, and the seasonal comparisons (ANOVA\u00a0+\u00a0Bonferroni) revealed that rate deviations were mainly restricted to measurements taken in summer and autumn (see column bar plots, superimposed in Fig.\u00a05A,B). These data also revealed genetic group specific differences between the respiration rate and the acclimatization temperature for Mytilus spp. (P\u00a0<\u00a00.05); (Fig.\u00a05C). Maximum acclimatized respiration rates occurred between 8 and 14\u00a0\u00b0C in the Baltic Sea group, 15\u201319\u00a0\u00b0C in the North Sea group and 20\u201324\u00a0\u00b0C in the Mediterranean Sea and Bay of Biscay group. The rate deviations found for the Mediterranean Sea and Bay of Biscay group differed significantly from those found for the Baltic Sea group. Several data points related to the North Sea group were found to fit the reaction norm of the Mediterranean Sea and Bay of Biscay group between 15 and 20\u00a0\u00b0C. Thus, the rate deviations of these two groups could not be distinguished (P\u00a0>\u00a00.05). No genetic group specific differentiation was observed for M. balthica (Fig.\u00a05D).\nFig.\u00a05A\u2013DRespiration rates as a function of the acclimatization temperature. The black lines are the upper edge slopes. These graphs include measurements from all research sites presented in Fig.\u00a01. In A and B, different labels represent different sampling seasons (see legend in A). In B and C respiratory response to temperature is presented per genetic group. The gray diamonds, white triangles and black circles represent the Baltic Sea group, the North Sea group and the Mediterranean Sea and Bay of Biscay groups, respectively. Superimposed are the rate deviations calculated per season and per genetic group, in which BS indicates the Baltic Sea group, NS the North Sea group, MS the Mediterranean Sea group and BC the Bay of Biscay group. *P\u00a0<\u00a00.05 and **P\u00a0<\u00a00.01 refer to the statistical differences for a given season compared to \u201cJanuary\u201d in A and B and for a given group compared to \u201cBS\u201d in C and D\nThermal sensitivity of the metabolic rate\nSeasonal variation in thermal sensitivity of the metabolic rate shows that Q\n10 values of greater than four were exclusively found in winter and spring (Fig.\u00a06). For M. balthica populations, such high Q\n10 values were found at low (January) and high (April) latitudes. In April, Q\n10 values decreased significantly with latitude. Maximum Q\n10 did not exceed 3.0 in July and October. July values were about one for clams from the Westerschelde estuary (10) and Point d\u2019Aiguillon (15). The clam population at Ask\u00f6 (5) also exhibited relatively low values (<2). Q\n10 values of greater than four were only observed in January and April for mussels (Fig.\u00a06). A significant decrease in Q\n10 values with latitude was observed in January (P\u00a0=\u00a00.0083), with the lowest values observed (Q\n10\u00a0=\u00a01.2) for the population from Gulfo di Oristano (20), and the highest values for the population from the Gulf of Gdansk (6). In April, this trend vanished, mainly due to the reduced Q\n10 values of mussels from the northernmost sampling stations. During the following seasons, July and October, mean Q\n10 values decreased further and latitudinal variation disappeared.\nFig.\u00a06Seasonal and latitudinal variations in mean Q\n10 values of M. balthica (circles) and Mytilus spp. (diamonds) populations. Trend lines represent linear regressions. r\n2 and P values are given in each graph\nAs a function of the acclimatization temperature, both low and high Q\n10 values were found at low ambient temperatures (Fig.\u00a07A,B). The maximum Q\n10 values observed at a given temperature decreased with increasing acclimatization temperature for both taxa. To illustrate this, we fitted lines of maximum Q\n10 values as a function of temperature. These lines crossed the Q\n10\u00a0=\u00a01 line at \u223c23\u00a0\u00b0C for M. balthica and at \u223c24\u00a0\u00b0C for Mytilus spp. (Fig.\u00a07A,B, line a). In addition to the maximum Q\n10 values for all Mytilus spp. populations, group-specific lines could be drawn (lines a\u2013c in Fig.\u00a07A). This resulted in different intercepts for the Q10\u00a0=\u00a01 line: at \u223c17\u00a0\u00b0C for the Baltic Sea group, at \u223c19\u00a0\u00b0C for the North Sea group and at \u223c24\u00a0\u00b0C for the Bay of Biscay and Mediterranean Sea group. No differentiation of the maximum thermal sensitivity of the metabolic rate was observed for M. balthica populations (Fig.\u00a07B).\nFig.\u00a07A\u2013B Q10 values as a function of the acclimatization temperature, presented per genetic group. The diamonds, triangles and circles represent the Baltic Sea group, the North Sea group and the Mediterranean Sea and\/or Bay of Biscay group, respectively. These graphs include measurements taken from all research sites. Lines of maximum Q10 values are fitted for M. balthica (B; a) and for Mytilus spp. (A) per genetic group: a the Mediterranean Sea and Bay of Biscay group, b the North Sea group, and c the Baltic Sea group\nDiscussion\nRates at high experimental temperatures\nWhen exposed to experimental temperatures of 24 or 31\u00a0\u00b0C, respiration rates in both Mytilus spp. and M. balthica may exceed 2.0 (mg O2\/g (dry weight)\/h). Such high rates were mostly observed in winter and spring, probably due to the low activation energy required for enzyme-catalyzed reactions at this time of the year (Hochachka and Somero 2002). The population from the Mecklenburg Bight (7) exhibited exceptionally high respiration rates for Baltic Sea mussels in the summer. In general, these high respiration rates measured at high experimental temperatures are not expected to occur in the field. When the summer starts, and peaks in habitat temperatures (24\u00a0\u00b0C+) occur in the field, the amplitude of the respiratory response at these higher temperatures declined. We propose that this modulation of metabolic thermal sensitivity is a protection mechanism that prevents excessive metabolic rates at high ambient temperatures. At the northern Baltic Sea station (3), the high thermal sensitivity of the metabolic rate did not result in high respiration rates in wintertime (which includes April in this area). Rates declined above 10\u00a0\u00b0C, indicating that the respiratory capacity was low and that acclimatization to increasing temperatures in springtime requires physiological changes in these clams.\nComparing both taxa, their respiratory responses to the experimental temperature range were rather different. The most striking difference is the regular occurrence of the breakpoint temperature between 24 and 31\u00a0\u00b0C in Mytilus spp., which was mostly absent for M. balthica (Fig.\u00a08A,B; in these graphs we summarize all respiration rates assessed for all populations during all seasons between 2003 and 2005). This breakpoint temperature may reflect the thermal tolerance limits of marine ectotherms. It has been suggested that species with lower breakpoint temperatures are less tolerant to high temperatures (Hochachka and Somero 2002; P\u00f6rtner 2002). Interestingly, despite its apparent breakpoint temperature between 24 and 31\u00a0\u00b0C, Mytilus spp. seem to tolerate higher environmental temperatures than M. balthica, given the geographical distribution of mussels, which reaches as far south as northern Africa (Comesana et al. 1998). Nonetheless, the lethal temperatures of both taxa under submerged conditions are comparable. The LT50 values for both species were 30\u201331\u00a0\u00b0C in a 24-h experiment using mussels and clams that were previously acclimatized to 20\u201325\u00a0\u00b0C (Kennedy and Mihursky 1971; Wallis 1975).\nFig.\u00a08A\u2013B Mean respiratory responses to temperature of Mytilus spp. (B; diamonds) and M. balthica (A; circles). Data are presented as the averages of all measurements taken (all populations and all seasons, 2003\u20132005). The numbers next to the labels are the number of replicate measurements taken, and the dashed lines indicate the standard deviations\nRates at acclimatization temperatures\nThe relations between the mean acclimatized respiration rates of the studied taxa and temperature are based on specimens from a great variety of microhabitats. Therefore, we expect these data to give a representative overview of metabolic temperature dependence in acclimatized mussels and clams. A narrow relation between temperature and acclimatized respiration rates in winter and springtime suggests that variations in the metabolic rate during this time of the year were directly dependent on temperature, and not limited by other physiological or environmental variables. A temperature-limited metabolic rate was also reflected by the high thermal sensitivity of the metabolic rate of some of the populations during those seasons. Only a few observations confirmed that the constant increase in respiration rates with acclimatization temperature can continue in summer and autumn, when most data points were found to be scattered below the upper edge slope. We suggest that the upper edge slopes represent the \u201cmetabolic scopes\u201d of these taxa. However, since measurements are based on groups of specimens, respiration rates of single individuals can be higher.\nThe relatively high acclimatized respiration rates (1.0\u20131.5 mgO2\/g\/h) most probably result from metabolic up-regulation, related to elevated rates of digestion and protein synthesis. The present study did not find any acclimatized routine respiration rates that exceeded 1.5 (mg O2\/g\/h) in mussels or clams. In general, respiration rates exceeding 1.5 mg O2\/g(dry weight)\/h can be considered to be high and will rarely occur under ambient conditions. Also, in other studies no acclimatized routine respiration rates higher than 1.5 mg O2\/g(dry weight)\/h have been reported for M. balthica (McMahon and Wilson 1981; Wilson and Elkaim 1991; Hummel et al. 2000) or Mytilus spp. (Arifin and Bendel-Young 2001; Bayne and Widdows 1978; Tedengren et al. 1999; Thompson 1984). Towards tolerance limits, the arising breakpoint temperature will force down the \u201cmetabolic scope,\u201d as indicated by the mussel populations from the Santa Giusta lagoon (21) and Gulfo di Oristano (20) in July, and for clams from Point d\u2019Aiguillon (15) in July and October. Physiological rates that are acclimatized to (near) breakpoint temperatures are rarely described for ectotherms.\nThe relatively low respiration rates at acclimatization temperatures >12\u00a0\u00b0C are interpreted as metabolic down-regulation resulting from both intrinsic and extrinsic factors (Brokordt et al. 2000; Burkey 1971; Velasco and Navarro 2003). The extremely low respiration rate of mussels from the Bidasoa estuary (16) in January was an exception, presumably caused by a temporary drop in ambient salinity (from 34 to 9\u00a0PSU). Such hypo-osmotic conditions are known to induce a metabolic depression in Mytilus spp. (Newell 1969; Storey and Storey 1990).\nIn general, Baltic Sea populations exhibited relatively low respiration rates, intermediate respiration rates were found in the North Sea populations, and relatively high metabolic rates in the Bay of Biscay and Mediterranean Sea populations. These observations fall together with the low growth rates in bivalves from the Baltic Sea, intermediate growth rates in North Sea populations, and the highest growth rates found in the Bay of Biscay and in some Mediterranean Sea populations (Bachelet 1980; Fuentes et al. 1998, 2000; Gangnery et al. 2004; Hummel et al. 1998; Peteiro et al. 2006; Westerborn et al. 2002). This indicates that mean routine respiration rates, measured at ambient temperatures, do indeed reflect the metabolic rate and ultimately the physiological performance of bivalves.\nSeparate curves describing the respiratory response to acclimatization temperatures were found for the three genetic groups of Mytilus populations. This resulted in group-specific respiration rates at a given temperature, which was significantly lower for the Baltic Sea group that exhibited reduced metabolic rates at intermediate temperatures (12\u201315\u00a0\u00b0C), where populations from the other genetic groups mostly exhibited optimal respiration rates (see Fig.\u00a05c). In line with our earlier conclusion that the observed suboptimal respiration rates represent metabolic adjustment to limiting extrinsic factors (including energy\u2013substrate availability), we expect that these group-specific response curves mainly reflect the food conditions in the respective sea-basins during the warmer months of the year. This assumption is supported by the seasonal variation in chlorophyll concentrations The chlorophyll concentrations in the Baltic Sea decline (Heiskanen and Lepp\u00e4nen 1995) at thermal conditions that correspond to the maximum annual chlorophyll concentrations in the North Sea and the Bay of Biscay (Colebrook 1979) and their adjacent estuaries (Rybarczyk et al. 1993; van Bergeijk et al. 2006). It also explains the exceptionally high respiration rates of some populations from the North Sea group at high habitat temperatures, e.g., specific dynamic action, facilitated by high ambient food availability. Still, differential genetic adaptation to temperature may add to the observed physiological differences, and cannot be excluded as an explanation.\nThe relation between the respiration rate and the acclimatization temperature shows great overlap upon comparing Mytilus spp. with M. balthica. Further comparison with other studies demonstrates that seasonal variation in the acclimatized respiration rates obtained from two Mytilus populations from England (Bayne and Widdows 1978) fits the relation described in this study. The respiratory performances of other bivalve species, such as Cerastoderma edule (Newell and Bayne 1980), Ostrea edulis (Beiras et al. 1994), or Dreissena polymorpha (Sprung 1995), were highly comparable as well. This great similarity in respiratory performance among European bivalve species suggests that they share a comparable evolutionary relation with temperature.\nSensitivity of the metabolic rate to temperature changes\nWe hypothesized that the sensitivity to temperature of the metabolic rate would decrease towards more southern localities, especially in the warmer seasons. Such latitudinal clines were only observed in January for Mytilus spp., and in April for M. balthica populations. In October no specific pattern was observed for Mytilus populations, since the Q\n10 values were low in all populations. This may be related to limiting food conditions in autumn. Absence of significant latitudinal clines in July was caused by cline-interruption, which corresponded to the geographic transition of one genetic group to the next. These genetic group specific clines became especially apparent in mussels when the Q\n10 was presented as a function of the acclimatization temperature (Fig.\u00a06).\nThe obtained Q\n10 values decreased with increasing acclimatization temperature for three reasons. First, metabolic down-regulation for energetic balancing results in a reduced Q\n10. Second, Q\n10 will decrease near breakpoint temperatures. Third, during thermal fluctuations that involve high peaks in habitat temperature, ectotherms will minimize the sensitivity of their metabolic rate, avoiding excessive rates when exposed to elevated temperatures (Peck et al. 2002; Widdows 1976; Wilson and Elkaim 1991). Thus, the increasing abundance of relatively nutrient-poor and thermally dynamic habitats causes a gradual shrinking of the ecological niche of these bivalve taxa towards the warm end of their distribution range. The group-specific clines discussed in the preceding paragraph indicated differential adaptation to temperature among Mytilus spp. populations. Since these bivalve species have great dispersal capacities, strongly coupled to hydrodynamic circulation (Gilg and Hilbish 2002), genetic divergence within these species requires geographic isolation. During isolation in different climatic regions, selective genetic variation may have evolved at the same spatial scale as the observed neutral genetic variation. This may explain why the biogeography of these genetic groups is associated with European temperature gradients. Physiological studies revealed that mussels from different genetic groups exhibit different growth rates when hatched under similar conditions (Beaumont et al. 2004; Hillbish et al. 1994). Differential thermal adaptation has recently been demonstrated for different mussel species from the West coast of North America as well (Fields et al. 2006). The differential adaptation to temperature indicated by our results, combined with the strong spatial variability of the coastal climate in the Bay of Biscay (Puillat et al. 2004), may explain the broad and mosaic-like transition from one mussel-group present in this area to the next (Bierne et al. 2003).\nQ10 values lower than one only occur beyond the breakpoint temperature, where the metabolic rate decreases with increasing temperature. Since thermal conditions beyond breakpoint temperatures are not beneficial for bivalve performance and are most probably lethal when extended, it is most interesting to observe that the southernmost Mytilus spp. populations in our research area exhibited Q10 values of less than one at acclimatization temperatures in July 2004. In the Santa Giusta Lagoon (21), the monthly mean water temperature reached 27\u00a0\u00b0C, exceeding the breakpoint temperature of mussels. Very few bivalves survived the summer of 2004 in the Santa Giusta lagoon (21), indicating that thermal tolerance limits were indeed crossed under field conditions. Survival of temperature-induced stress depends on its duration and on the physiological status and condition of the organisms. Widdows and Bayne (1971) found that mussels can cope with relatively high temperatures so long as they can regularly recover in water at a suitably low temperature. This is an important strategy that allows survival of temporary heat exposure, e.g., when exposed to the air during low tide in summer. The range and limits of this relation for mussels or clams have not been studied in depth yet.\nIn conclusion, we demonstrated that the respiratory responses to temperature of two European bivalve taxa are greatly dependent on seasonal variations in temperature. These responses, obtained throughout all seasons and at a large geographic scale, fit together in a framework when presented as a function of the acclimatization temperature. This framework is useful in both the fundamental and the applied sciences, facilitating the interpretation of respiration rates measured under ambient conditions, and the further development of ecophysiological theory. We observed that the maximum thermal sensitivity of the metabolic rate decreases with increasing acclimatization temperature, crossing a threshold (Q\n10\u00a0=\u00a01) in Mytilus spp. at the maximum acclimatization temperatures observed in the field. Whether the temperature quotient will become less than one in M. balthica populations when the acclimatization temperature exceeds 23\u00a0\u00b0C cannot be answered with any certainty, since no breakpoint temperature is observed in the experimental southern populations under or near ambient conditions.\n(Onto)genetic adaptation to regionally different climates implies that climate change will affect not only marginal populations via their metabolic rates, but all genetic groups are expected to shift northward with increasing temperatures. Although the dispersal capacity of mussel larvae and the active transport of juvenile and adult mussels for aquaculture purposes support range shifts with the speed of climate change, other ecological and physiological variables and their impact on the environment need to be investigated to predict the fate of mussels and other bivalve populations under changing climatic conditions. For M. balthica populations, no differential adaptation to temperature was observed among divergent groups, which leads us to expect that the direct impact of climate-induced temperature changes will be restricted to the southernmost populations.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\n(DOC 57 kb)","keyphrases":["metabolic rate","climate change","respiration rate","distribution range","thermal tolerance"],"prmu":["P","P","P","P","P"]}
{"id":"Int_J_Cardiovasc_Imaging-3-1-2048827","title":"Assessment of normal tricuspid valve anatomy in adults by real-time three-dimensional echocardiography\n","text":"Background The tricuspid valve (TV) is a complex structure. Unlike the aortic and mitral valve it is not possible to visualize all TV leaflets simultaneously in one cross-sectional view by standard two-dimensional echocardiography (2DE) either transthoracic or transesophageal due to the position of TV in the far field.\nIntroduction\nThe tricuspid valve (TV) is a multi-component complex structure [1]. In classic anatomic studies the anterior, septal and posterior TV cusps were described [2, 3]. Unlike the aortic and mitral valve it is not possible to visualize all TV cusps simultaneously in one cross-sectional view by standard transthoracic two-dimensional echocardiography (2DE) [4]. During transesophageal 2DE small changes in transducer angle, probe position and rotation may bring to light some additional TV details [5, 6]. However, because of the position of the TV in the far field in relation to probe, transesophageal 2DE can still only provide limited information and can also not visualize all TV cusps simultaneously. In three-dimensional (3D) transesophageal image reconstruction and intracardiac echocardiography studies this goal could be achieved but at the cost of some procedural risks and an increase in procedural duration [7, 8]. Real-time three-dimensional echocardiography (RT3DE) can visualize the atrio-ventricular valves from both the ventricular and atrial side in detail without these limitations [9]. This study aimed to apply RT3DE for quantitative and qualitative assessment of normal TV anatomy.\nSubjects and methods\nIn one hundred patients (mean age 30\u00a0\u00b1\u00a09\u00a0years, 65% males) the TV was examined by transthoracic RT3DE after an informed consent. All patients had sinus rhythm and normal right-sided heart (normal right ventricular dimensions and function, normal right atrial dimension, trivial or absent tricuspid regurgitation and normal tricuspid valve function). Patients with good 2DE image quality only were included. RT3DE was done with a commercially available ultrasound system (Philips Sonos 7500, Best, The Netherlands) attached to a X4 matrix array transducer capable of providing real-time B-mode images. The 3D data set was collected within approximately 5\u201310\u00a0s of breath holding in full volume mode from an apical window and transferred for off-line analysis with TomTec software (Unterschleissheim, Munich, Germany). Data analysis of 3D images was based on a two-dimensional approach relying on images obtained initially from the apical 4-chamber view. The images were adjusted to put the TV in the center of interest. To exclude non-relevant tissue, the TV was sliced between the two narrowest lines by which all parts of the TV leaflets were still in between. The TomTec software allows in this way visualization of the short-axis TV view in a 3D display (see Fig.\u00a01). RT3DE gain and brightness were adjusted to improve delineation of anatomic structures. The following points were checked for visualization: 1) tricuspid annulus diameter and area, 2) TV leaflets (number, mobility, thickness and relation to each other), 3) TV area, and 4) TV commissures (antero-septal, antero-posterior, and postero-septal) including the position of their closure lines. All these structures were classified according to a subjective 4-point scale for image quality (1\u00a0=\u00a0not visualized, 2\u00a0=\u00a0inadequate, 3\u00a0=\u00a0sufficient and 4\u00a0=\u00a0good).\nFig.\u00a01TomTec quadri screen display of the tricuspid valve. The upper two images represent two-dimensional views created from the 3D data set (4-chamber, left and orthogonal view, right). The lower left image represents a two-dimensional short axis view and the lower right image represents the 3D image\nFor quantitative assessment of TV the following RT3DE data were obtained: 1) TV annulus diameter defined as the widest diameter that could be measured from an end-diastolic still frame, 2) maximal TV annulus area obtained from an end-diastolic still frame and measured by manual planimetry, 3) TV area defined as the narrowest part of the TV at the time of maximal opening and measured by manual planimetry, and 4) TV commissural width obtained from a late-diastolic still frame using zoom function to avoid underestimation. The images were optimized for each commissure along its plane to measure the maximal width of the angle formed by the two adjacent TV leaflets.\nTo identify the TV leaflets visualized by the standard 2DE images the TomTec quad screen display was used. As seen in Fig.\u00a01, this screen contains four images; the upper two images are 2DE images perpendicular to each other, the lower two images are a short-axis 2DE image and a RT3DE image. From the properly chosen two-dimensional image a mid-diastolic frame was selected to visualize the TV leaflets just separated from each other. Each leaflet was defined by a marker, after which this marker position was compared with the RT3DE image to detect which leaflet was shown in the 2DE images. Analysis of images was done by two experienced echocardiographers (AMA, JSM) independently. Each one dealt with the full volume image as acquired from echo machine and the selection of cut plane, angulation and gain setting adjustment were dependable on his experience.\nStatistical analysis\nAll data obtained by RT3DE were presented as mean\u00a0\u00b1\u00a0SD. Interobserver and intraobserver agreements for the visualization score were estimated using kappa values for each morphologic feature and classified as poor (kappa\u00a0<\u00a00.4), moderate (kappa 0.4 to 0.7), or good (kappa\u00a0>\u00a00.7). Interobserver and intraobserver variabilities for RT3DE measurements was assessed according to the Bland and Altman method in a randomly selected group of 50 patients [10] Table\u00a01.\nTable\u00a01Scores for real-time three-dimensional echocardiography visualization of TV structuresScoreTV annulusTV leafletsTV areaTV commissuresGood (4)60%80%55%50%Sufficient (3)30%10%30%20%Inadequate (2)10%10%15%20%Not visualized (1)0%0%0%10%Mean score3.5\u00a0\u00b1\u00a00.73.7\u00a0\u00b1\u00a00.63.4\u00a0\u00b1\u00a00.73.1\u00a0\u00b1\u00a01.0Median score3.03.03.02.5Abbreviations: TV\u00a0=\u00a0tricuspid valve\nResults\nAcquisition and analysis of the RT3DE data was performed in approximately 10\u00a0min per patient. The TV could be visualized in 90% of patients enface from both ventricular and atrial aspects in relation to adjacent cardiac structures. In these 90 patients detailed analysis of the TV was performed including tricuspid annulus shape and size, TV leaflets shape, size, and mobility, and commissural width.\nTricuspid annulus\nTricuspid annulus visualization was good in 54 patients (60%), sufficient in 27 patients (30%), and inadequate in 9 patients (10%). As seen in Fig.\u00a02, tricuspid annulus shape appeared as oval rather than circular. Tricuspid annulus diameter and area could be measured in 63 patients (70%), normal values were 4.0\u00a0\u00b1\u00a00.7\u00a0cm and 10.0\u00a0\u00b1\u00a02.9\u00a0cm2, respectively.\nFig.\u00a02Oval-shaped Tricuspid annulus (the line represent the tricuspid annulus diameter, the dots demark the area)\nTricuspid valve leaflets\nVisualization of the three TV leaflets (in motion) was good in 72 patients (80%), sufficient in 9 patients (10%), and inadequate in another 9 patients (10%). The anterior leaflet was the largest and most mobile of the three leaflets and had a nearly semicircular shape. The septal leaflet was the least mobile and had a semi-oval shape. Its position was parallel to the interventricular septum. The posterior leaflet was the smallest one with variable shape. It was clearly separated from the septal leaflet in all patients but in 10% of patients it was hard to discriminate the posterior leaflet from the anterior leaflet even during maximal TV opening.\nFrom the RT3DE data set all standard two-dimensional TV cross-sections (apical 4-chamber, parasternal short-axis and parasternal long-axis right ventricular inflow) were simulated. As seen in Fig.\u00a03, in the apical 4-chamber view in all patients the septal leaflet was seen adjacent to the septum and the anterior leaflet was seen adjacent to the right ventricular free wall. In the parasternal short-axis view, the posterior leaflet was seen adjacent to the right ventricular free wall in 92% of patients and in the remaining 8% no leaflet could be obtained although modification of the cut plane downward could identify this leaflet. In this view the leaflet adjacent to the aorta was the anterior in 52% and the septal leaflet in 48%. In the parasternal right ventricular inflow view the leaflets seen were identical to the apical 4-chamber view with in all patients the septal leaflet seen adjacent to the septum and the anterior leaflet seen adjacent to the right ventricular free wall.\nFig.\u00a03Identification of the tricuspid valve leaflets seen on two-dimensional imaging. Below the 2D images, percentage of leaflet identification in each standard view depending the RT3DE images\nTricuspid valve area\nVisualization of the triangular shaped TV area was good in 50 patients (55%), sufficient in 27 patients (30%), and inadequate in 13 patients (15%). As seen in Fig.\u00a04, the anterior and septal leaflets formed the TV area\u2019s angle and the small posterior leaflet formed its base. TV area could be measured in 77 patients (86%) and mean TV area in these patients was 4.8\u00a0\u00b1\u00a01.6\u00a0cm2.\nFig.\u00a04Triangular shape TV area and commissural views\nTricuspid valve commissures\nAs seen in Fig.\u00a04, the three TV leaflets were separated from each other by three commissures. The commissures and the direction of closure lines were good visualized in 45 patients (50%), sufficient in 18 patients (20%), inadequate in 18 patients (20%), and not visualized in 9 patients (10%). TV commissural width could be obtained in 63 patients (70%), mean commissural width in these patients was 5.4\u00a0\u00b1\u00a01.5\u00a0mm for the antero-septal commissure, 5.2\u00a0\u00b1\u00a01.5\u00a0mm for the postero-septal commissure, and 5.1\u00a0\u00b1\u00a01.1\u00a0mm for the antero-posterior commissure, respectively. Visualization and measurement of commissures was relatively easy for the antero-septal commissure and most difficult for the antero-posterior commissure. All measurements are listed in Table\u00a02 as absolute value and indexed to body surface area.\nTable\u00a02Normal (absolute and index) values of tricuspid valve annulus (diameter and area), Tricuspid valve area and the width of the 3 commissuresParameterAbsolute valueIndex valueTricuspid annulus diameter 4.0\u00a0\u00b1\u00a00.7 (cm)2.2\u00a0\u00b1\u00a00.4 (cm\/m2)Tricuspid annulus area 10.0\u00a0\u00b1\u00a02.9 (cm2)5.5\u00a0\u00b1\u00a01.6 (cm2\/m2)Tricuspid valve area 4.8\u00a0\u00b1\u00a01.6 (cm2)2.7\u00a0\u00b1\u00a00.9 (cm2\/m2)Antero-septal commissure 5.4\u00a0\u00b1\u00a01.5 (mm)2.9\u00a0\u00b1\u00a00.8 (mm\/m2)Postero-septal commissure5.2\u00a0\u00b1\u00a01.5 (mm)2.9\u00a0\u00b1\u00a00.7 (mm\/m2)Antero-posterior commissure 5.1\u00a0\u00b1\u00a01.1 (mm)2.8\u00a0\u00b1\u00a00.6 (mm\/m2)\nInterobserver variability\nThe visualization score between two observers was good for the TV annulus (kappa value 0.91) and TV leaflets (kappa value 0.71) and moderate for the TV commissures (kappa value 0.59). As seen in Fig.\u00a05, good interobserver correlations were found for measurement of TV annulus (r\u00a0=\u00a00.98, P\u00a0<\u00a00.0001) and TV area (r\u00a0=\u00a00.95, P\u00a0<\u00a00.0001) and fair correlation was found for TV commissural width (r\u00a0=\u00a00.51, P\u00a0<\u00a00.001). In the same Figure, the interobserver agreement for TV annulus diameter (mean difference \u22120.28\u00a0\u00b1\u00a01.20\u00a0mm, agreement: 2.12, \u22122.68), for TV area (mean difference: 0.17\u00a0\u00b1\u00a00.52\u00a0cm2, agreement: 1.21, \u22120.87), and for mean TV commissural width (mean difference: 0.01\u00a0\u00b1\u00a00.62\u00a0mm, agreement: 1.25, \u22121.24) is displayed.\nFig.\u00a05Interobserver correlations (top) and Bland\u2013Altman analysis (bottom) of TV annulus, leaflets, and commissures\nIntraobserver variability\nThe visualization score by the first observer at 2 separate settings was good for the TV annulus (kappa value 0.92) and TV leaflets (kappa value 0.73) and moderate for the TV commissures (kappa value 0.58). Intraobserver agreement was (mean difference \u22120.26\u00a0\u00b1\u00a01.15\u00a0mm, agreement: 2.14, \u22122.56) for TV annulus diameter, (mean difference: 0.15\u00a0\u00b1\u00a00.50\u00a0cm2, agreement: 1.15, \u22120.85) for TV area, and (mean difference: 0.02\u00a0\u00b1\u00a00.60\u00a0mm, agreement: 1.22, \u22121.18) for the mean TV commissural width.\nDiscussion\nTwo-dimensional echocardiography is a valuable imaging modality for the functional assessment of TV [11\u201313]. However, with 2DE it is not possible to visualize all TV cusps simultaneously in one cross-sectional view nor can detailed anatomical information of the TV annulus, leaflets, and commissures be provided. Previous studies and case reports described visualization of TV by RT3DE [9, 14] in abnormal states, while this study applied RT3DE for the morphological assessment of the normal TV anatomy. RT3DE allowed analysis of TV annulus, leaflets and commissures in the majority of patients. Beside this morphologic description, quantitative assessment could be obtained. However, it should be noticed that only patients with good 2DE image quality underwent RT3DE. In our experience these patients represent over 50% of the total number of patients referred to our echocardiographic laboratory. Nevertheless, RT3DE allowed TV analysis to a level quite comparable to that recently reported by others for the mitral valve leaflets [15].\nOne of the salient findings in our study was the identification of the TV leaflets as seen in the routine 2DE views. It is still a matter of controversy in echocardiographic textbooks. In one well known echocardiographic textbook [16], the leaflet seen in the apical 4-chamber view adjacent to the right ventricular free wall was described as being the anterior or posterior leaflet depending on the exact rotation and angulation of the image plane. However, in our study this leaflet was consistently found to be the anterior leaflet (see Fig.\u00a06 for explanation), as described in another textbook [17]. Also, in both these echocardiographic textbooks [16, 17] in the parasternal short-axis view the leaflet adjacent to the right ventricular free wall was described as being the anterior. However, as shown in Fig.\u00a03 and explained in Fig.\u00a06, in all patients in whom a leaflet could be identified in our study it was the posterior one.\nFig.\u00a06Surgical view of the heart valves demonstrating the range of the two-dimensional echocardiographic 4-chamber and short-axis planes\nIn our study tricuspid annulus diameter (and area) could be reliably obtained with RT3DE. Tricuspid annulus measurement is of critical importance in the TV surgical decision-making process if a patient is operated for mitral valve disease and has concomitant TV regurgitation [18, 19]. In addition, TV area could be reliably obtained and this may have important implications for the diagnosis of tricuspid stenosis [20, 21]. Visualization of commissures and measurement of its width were obtained with difficulty, in particular for the antero-posterior commissure. Commissural width also showed weak interobserver correlation. This may be due to differences in the commissural levels and tissue dropout. For proper assessment of the three commissures, more cut planes with different angles are needed. However, assessment of commissural width may also be a valuable tool for the diagnosis, follow up, and selection of therapeutic strategy of tricuspid stenosis. All our RT3DE measurements were consistent with the measurements described in anatomical studies [2, 3]. Our data may potentially take RT3DE a step further into clinical routine (providing accurate TV measurements) and may enhance the understanding of TV valve morphology during the cardiac cycle (Fig.\u00a07). Also, the detailed assessment by RT3DE may affect the therapeutic decision of various TV abnormalities and thus expand the abilities of non-invasive cardiology [22].\nFig.\u00a07Visualization of the 3 TV leaflets during valve closure (A), at early diastole (B), and at late diastole (C)\nLimitation of study\nThe main limitation of this study is that RT3DE data were not compared with a \u201cgold standard\u201d such as magnetic resonance imaging, autopsy or surgical findings. Also, RT3DE images more critically depend on 2DE image quality and could be obtained only in patients with sinus rhythm during hold breath that limits its application to all. The study included patients with narrow age range (21\u201339\u00a0years), and thus the normal findings in this study is confined to this age group and could not be applied to both extremities (<21 and >39\u00a0years).\nConclusion\nThree-dimensional imaging of the TV is feasible in a large number of patients. RT3DE may add to functional 2DE data in description of TV anatomy and providing highly reproducible and actual reality (anatomic and functional) measurements.","keyphrases":["normal tricuspid valve","real-time three-dimensional echocardiography","tricuspid valve anatomical structure"],"prmu":["P","P","R"]}
{"id":"Diabetologia-4-1-2170456","title":"Duration of breast-feeding and the incidence of type 2 diabetes mellitus in the Shanghai Women\u2019s Health Study\n","text":"Aims\/hypothesis The aim of this study was to examine the association between lifetime breast-feeding and the incidence of type 2 diabetes mellitus in a large population-based cohort study of middle-aged women.\nIntroduction\nThe prevalence of type 2 diabetes mellitus has been increasing rapidly worldwide [1], making knowledge of risk factors and protective factors associated with type 2 diabetes mellitus essential for the development of prevention strategies.\nResults from animal and human studies suggest an improvement in glucose and insulin sensitivity during lactation [2\u20135]. Data from two large cohorts in the USA also indicate that longer duration of breast-feeding may reduce the risk of type 2 diabetes mellitus by improving glucose homeostasis [6].\nWe examined the association between lifetime breast-feeding and the incidence of type 2 diabetes mellitus in a large population-based cohort study of middle-aged women, the Shanghai Women\u2019s Health Study (SWHS).\nMethods\nStudy population The SWHS is a population-based prospective cohort study of middle-aged women (40\u201370\u00a0years old) conducted in seven urban communities of Shanghai, China. Details of the SWHS survey have been reported elsewhere [7]. From a total of 81,170 women who were invited to participate, 75,221 were recruited (92.7% participation rate). Reasons for non-participation were refusal (3.0%), absence during the enrolment period (2.6%) and other reasons (health, hearing, speaking problems; 1.6%). After exclusion of women younger than 40\u00a0years or older than 70\u00a0years at the time of interview (n\u2009=\u2009278), 74,942 women remained for the study. Participants completed a detailed survey as part of an in-person interview, which included assessment of dietary intake, physical activity, measurement of anthropometrics and other lifestyle factors. The original questionnaires, in Chinese, are available from the corresponding author upon request. Protocols for the SWHS were approved by the Institutional Review Boards of all institutes involved in the study and written informed consent was obtained prior to interview. Biannual in-person follow-up of all living cohort members was conducted by in-home visit from 2000 to 2002 and from 2002 to 2004, with a response rate of 99.8 and 98.7%, respectively; only 934 participants were lost to follow-up.\nOutcome ascertainment Incident type 2 diabetes mellitus was identified through follow-up surveys. A total of 1,561 parous women reported a type 2 diabetes mellitus diagnosis since the baseline survey. For the current study we considered a case of type 2 diabetes mellitus to be confirmed if the participants reported having been diagnosed with type 2 diabetes mellitus and met at least one of the following criteria: fasting glucose level \u22657\u00a0mmol\/l on at least two separate occasions or an oral glucose tolerance test with a value \u226511.1\u00a0mmol\/l and\/or use of hypoglycaemic medication (i.e. insulin or oral hypoglycaemic drugs). All tests were performed as part of patients\u2019 primary care. Of the self-reported cases, a total of 869 participants met the study outcome criteria and are referred to herein as confirmed cases of type 2 diabetes mellitus. We performed analyses restricted to confirmed cases as well as analyses including all cases of type 2 diabetes mellitus.\nBreast-feeding duration assessment Information on each pregnancy was obtained during the in-person interview. This included date and outcome of each pregnancy and whether and for how long the participant breastfed each child. Based on this information, we calculated the duration of breast-feeding (months and years) and duration of breast-feeding per live birth (years).\nMeasurement of potential confounders Anthropometric measurements were taken at baseline recruitment according to a standard protocol by trained interviewers who were retired medical professionals [8]. Self-reported body weight history was obtained for ages 20 and 40\u00a0years and at baseline recruitment. We calculated BMI (weight in kg divided by the square of height in m), WHR (waist circumference divided by hip circumference) and standardised weight change (difference between measured weight at baseline and weight at 20\u00a0years divided by the interval between study recruitment and 20\u00a0years; kg\/year).A structured questionnaire was used at the baseline survey to collect information on socio-demographic factors such as age, level of education (none, elementary school, middle\/high school, college), family income in yuan per year (<10,000, 10,000\u201319,999, 20,000\u201329,999, >30,000), occupation (professional, clerical, manual labour\/other, housewife\/retired), smoking (smoked at least one cigarette per day for more than 6\u00a0months continuously) and alcohol consumption (had ever drunk beer, wine or spirits at least three times per week). History of diseases such as diabetes, cancer, cardiovascular disease and high blood pressure was also collected.Information on physical activity was obtained using a validated questionnaire [9]. The questionnaire evaluated exercise and sport participation, daily activity and daily commuting round-trip journey to work. We calculated the metabolic equivalents (METs) for each activity, using a compendium of physical activity values [10]. We derived a quantitative estimate of overall non-occupational activity (MET-h per day).Dietary intake was assessed through an in-person interview using a validated food frequency questionnaire at the baseline recruitment survey and at the first follow-up survey [11]. The Chinese food composition tables [12] were used to estimate energy intake (kJ\/day).\nStatistical analysis Person-years for each participant were calculated as the interval between the baseline recruitment to one of the following: diagnosis of type 2 diabetes mellitus; censored at death; or completion of the second follow-up survey.The Cox proportional hazards model was used to assess the effect of breast-feeding on the incidence of type 2 diabetes mellitus. Tests for trend were performed by entering the categorical variables as continuous parameters in the models. In all models, we adjusted for the following potential confounding variables: age, BMI, WHR, total energy, physical activity, number of live births (entered as continuous variables), income level, education level, occupation, smoking status, alcohol consumption status and presence of hypertension at baseline (as categorical variables).We also derived a propensity score by regressing breast-feeding (yes\/no) on risk factors for type 2 diabetes mellitus (age, daily energy intake, BMI, WHR, smoking, alcohol consumption, physical activity, hypertension, income, education level, occupation status, oral contraceptive use, vegetable intake, legume intake, meat intake and staples intake). We then examined the association between breast-feeding (yes\/no) and type 2 diabetes mellitus by including the propensity score in the Cox proportional hazard model. All analyses were performed using SAS (version 9.1, SAS Institute, Cary, NC, USA), and all tests of statistical significance were based on two-sided probability. A p value of less than 0.05 was considered statistically significant.\nResults\nThe average age of the entire SWHS cohort (n\u2009=\u200974,942) was 52.1 (SD\u2009=\u20099.1). The average age at first pregnancy was 25.5\u00a0years (median: 25.7\u00a0years) and the median number of live births was 1.0. A total of 3.3% women had no children, 54.4% had only one child, 21.2% had two children, 10.5% had three children, 6.2% had four children and 4.3% had five or more children. The incidence of diabetes mellitus was 6.0 cases per 1,000 person-years at risk.\nThere were 62,095 parous women in this study with no prior history of type 2 diabetes mellitus, cancer or cardiovascular disease at study recruitment; of these 50,700 (81.65%) reported having breastfed their children. The average number of live births was 1.7, the average number of months of breast-feeding was 14.6 and the average number of years of breast-feeding per child was 0.6. The number of live births was associated with months of breast-feeding (Spearman correlation coefficient, 0.70; p\u2009<\u20090.001). Age-standardised characteristics of the study population by duration of breast-feeding are shown in Table\u00a01. Women with a longer duration of breast-feeding were older, had less education, lower income and were less likely to be employed at the time of the survey or to hold a professional job. Duration of breast-feeding was also associated with smoking and higher physical activity in this population. We did not find an association between differences in participation in exercise or prevalence of hypertension at baseline and duration of breast-feeding (p\u2009>\u20090.05).\nTable\u00a01Age-standardised characteristics of parous women in the Shanghai Women\u2019s Health Study by duration of breast-feeding\u00a0Duration of breast-feeding (months)0>0 to 6>6 to 11>11 to 35\u226536p value for trendaWomen (n)11,39510,46314,95818,1447,135Cases of diabetes (n)225163253585335Person-years (n)53,03848,77069,74183,87032,535<10\u00a0years since last pregnancy (%)26.921.928.918.63.7Age, median (Q25\u2013Q75)45 (42\u201349)45 (42\u201349)45 (42\u201349)54 (49\u201361)64 (62\u201367)<0.001Energy intake, mean kJ\/day (SE)6,875 (14)6,927 (15)6,920 (13)6,985 (11)6,738 (21)<0.001Live births, median (Q25\u2013Q75)1 (1\u20131)1 (1\u20131)1 (1\u20131)2 (1\u20132)4 (3\u20135)<0.001BMI, mean kg\/m2 (SE)23.4 (0.03)23.2 (0.03)23.6 (0.03)24.2 (0.02)24.7 (0.04)<0.001WHR, mean (SE)0.80 (0.0005)0.8 (0.0005)0.80 (0.0005)0.81 (0.004)0.82 (0.007)<0.001Weight gain, mean kg\/year (SE)0.34 (0.003)0.31 (0.003)0.33 (0.003)0.36 (0.003)0.37 (0.005)<0.001Weight at age 20\u00a0years, median kg (Q25\u2013Q75)48 (45\u201353)49 (45\u201353)50 (45\u201354)50 (45\u201354)50 (45\u201355)<0.001Smoker (%)1.7 (1.5\u20131.9)1.4 (1.2\u20131.9)1.5 (1.3\u20131.7)2.4 (2.3\u20132.5)3.6 (3.2\u20134.0)<0.001Ever drinker (%)1.9 (1.7\u20132.1)2.1 (1.9\u20132.3)2.1 (1.9\u20132.3)2.4 (2.3\u20132.5)1.9 (1.7\u20132.1)<0.001Exercise (%)30.5 (29.5\u201331.5)32.9 (31.9\u201333.9)34.3 (33.3\u201335.3)33.7 (32.7\u201334.7)31.4 (30.4\u201332.4)0.18High physical activityb (%)22.8 (22.0\u201323.6)22.1 (21.3\u201322.9)24.4 (21.6\u201325.2)27.3 (26.9\u201327.7)41.2 (40.2\u201342.2)<0.001Education (%)None11.0 (12.4\u201311.6)8.5 (7.9\u20139.1)9.6 (9.0\u201310.2)17.8 (17.4\u201318.2)41.2 (40.2\u201342.2)<0.001Elementary38.2 (37.2\u201339.2)34.4 (33.4\u201335.4)39.2 (38.2\u201340.2)44.3 (43.8\u201344.8)46.9 (47.9\u201348.9)Middle\/high school32.5 (31.5\u201333.5)34.3 (33.3\u201335.3)32.4 (31.4\u201333.4)27.1 (26.7\u201327.5)8.6 (8.0\u20139.2)College18.3 (17.5\u201319.1)22.7 (21.9\u201323.5)18.8 (18.0\u201319.6)10.8 (10.5\u201311.1)3.3 (2.9\u20133.7)Household income (%)<10,000 yuan14.0 (13.4\u201314.6)11.6 (11.0\u201312.2)12.6 (12.0\u201313.2)15.6 (15.3\u201315.9)22.1 (21.3\u201322.9)<0.00110,000\u201319,999 yuan38.8 (37.8\u201339.8)35.8 (34.8\u201336.8)37.1 (36.1\u201338.1)38.0 (37.5\u201338.5)38.7 (37.7\u201339.7)20,000\u201329,999 yuan29.4 (28.5\u201330.3)31.3 (30.3\u201332.3)30.4 (29.4\u201331.4)28.6 (28.2\u201329.0)27.8 (27.0\u201328.6)>30,000 yuan17.8 (17.0\u201318.6)21.4 (20.6\u201322.4)19.8 (19.0\u201320.6)17.8 (17.4\u201318.2)11.4 (10.8\u201312.0)Occupation (%)Professional21.6 (20.8\u201322.4)25.9 (25.1\u201326.7)22.2 (21.4\u201323.0)16.4 (15.6\u201317.2)6.8 (6.2\u20137.4)<0.001Clerical12.8 (12.2\u201313.4)12.4 (11.6\u201312.8)13.2 (12.6\u201313.8)13.7 (13.4\u201314.0)19.5 (18.7\u201320.3)Manual labour\/others21.8 (21.0\u201322.6)21.0 (20.2\u201321.8)23.1 (22.3\u201323.9)23.6 (23.2\u201324.0)20.0 (19.2\u201320.8)Housewife\/retired43.8 (42.8\u201344.8)40.7 (39.7\u201341.7)41.6 (40.6\u201342.6)46.3 (45.8\u201346.8)53.6 (52.6\u201354.6)Hypertension (%)18.4 (17.6\u201319.2)18.3 (17.5\u201319.1)18.8 (18.0\u201319.6)19.8 (19.4\u201320.2)16.6 (15.8\u201317.4)0.12Data are presented as n, percent, median (Q25\u2013Q75, interquartile range) and mean (SE), and are directly standardised in years to the age distribution of the populationMeans of energy intake, BMI, WHR and standard weight are adjusted by ageWeight at 20\u00a0years and number of live births were not normally distributed and could not be adjusted for ageap values for trend were calculated by: proportional odds model for prevalence of population characteristics; ANOVA test for daily energy intake, BMI, WHR and standard weight gain; and Kruskal\u2013Wallis test for age, number of live births and weight at 20\u00a0yearsbHigh physical activity: participants in the upper quartile of total METs\nWomen who had breastfed tended to have a lower risk of type 2 diabetes mellitus [relative risk (RR)\u2009=\u20090.88; 95% CI, 0.76\u20131.02; p\u2009=\u20090.08] than women who had never breastfed, in analyses adjusted for age, daily energy intake, BMI, WHR, number of live births, occupation, income levels, education, smoking, alcohol consumption, physical activity and presence of hypertension (Table\u00a02). When we adjusted the analysis by a propensity score of predictors of type 2 diabetes mellitus, the RR was 0.81 (95% CI, 0.70\u20130.94; p\u2009<\u20090.01).\nTable\u00a02Associations between type 2 diabetes mellitus and duration of breast-feeding for all participants, Shanghai Women\u2019s Health Study\u00a0%Cases per person-yearsRR95% CIp value for trendBreast-feedingNo18.35225\/53,0381.00Yes81.651,336\/234,9160.880.76\u20131.02Duration of breast-feeding\u00a0Years\u00a0\u00a0018.35225\/53,0381.000.01\u00a0\u00a0>0\u20130.9947.99416\/118,5110.880.75\u20131.04\u00a0\u00a0>0.99\u20131.9917.31343\/57,4180.890.75\u20131.06\u00a0\u00a0>1.99\u20132.997.57242\/26,4510.880.71\u20131.07\u00a0\u00a0>2.99\u20133.994.15148\/14,9290.750.59\u20130.96\u00a0\u00a0\u226544.63187\/17,6060.680.52\u20130.90\u00a0Years per child\u00a0\u00a0018.35225\/53,0381.000.11\u00a0\u00a0>0\u20130.4915.10200\/43,6150.910.75\u20131.10\u00a0\u00a0>0.49\u20130.9944.20659\/127,4530.870.74\u20131.02\u00a0\u00a0\u2265122.35477\/63,8480.870.78\u20131.03Values are adjusted for age, daily energy intake, BMI, WHR, number of live births, smoking, alcohol consumption, physical activity, education, income, occupation and hypertension\nThe duration of breast-feeding and duration of breast-feeding per child were associated with a lower risk of type 2 diabetes mellitus (Table\u00a02). The fully adjusted RR for 0, >0 to 0.99, >0.99 to 1.99, >1.99 to 2.99, >2.99 to 3.99 and \u22654\u00a0years of breast-feeding with the risk of type 2 diabetes mellitus were 1.00, 0.88, 0.89, 0.88, 0.75 and 0.68. The fully adjusted RR between years of breast-feeding per child and type 2 diabetes mellitus risk were 1.00, 0.91, 0.87 and 0.87 (p\u2009=\u20090.11 for trend) for 0, >0 to 0.49, >0.49 to 0.99 and \u22651\u00a0years of breast-feeding per number of births. Associations between months of breast-feeding and incidence of type 2 diabetes mellitus are presented in Table\u00a03. BMI and WHR are the two major confounding factors for the association between the duration of breast-feeding and risk of type 2 diabetes mellitus. Both BMI and WHR were weakly correlated with breast-feeding (r\u2009=\u20090.22 and 0.22, respectively). The correlation between BMI and WHR was 0.46. The association between the duration of breast-feeding and diabetes mellitus was accentuated and the trend was only marginally significant when we adjusted the model for BMI. Additional adjustment for WHR resulted in the trend becoming statistically significant (p\u2009=\u20090.04). The association between breast-feeding and type 2 diabetes mellitus did not change much after adjustment for number of live births. When we restricted the analysis of breast-feeding and incidence of type 2 diabetes to confirmed cases of type 2 diabetes mellitus, we found similar patterns of association (Table\u00a04).\nTable\u00a03Incidence of type 2 diabetes by duration of breast-feeding, Shanghai Women\u2019s Health Study\u00a00>0 to 6\u00a0months>6 to 11\u00a0months>11 to 35\u00a0months\u226536\u00a0months\u00a0RR95% CIRR95% CIRR95% CIRR95% CIRR95% CIp value for trendModel 11.000.810.66\u20130.990.880.74\u20131.071.080.92\u20131.271.030.84\u20131.250.20Model 21.000.840.69\u20131.030.890.74\u20131.070.990.85\u20131.180.880.71\u20131.080.75Model 31.000.840.69\u20131.030.880.73\u20131.050.900.76\u20131.050.760.62\u20130.930.06Model 41.000.870.71\u20131.060.910.76\u20131.090.980.83\u20131.150.840.68\u20131.030.39Model 51.000.890.71\u20131.060.890.74\u20131.070.890.76\u20131.050.750.61\u20130.920.04Model 61.000.870.71\u20131.060.890.75\u20131.070.890.75\u20131.050.730.58\u20130.910.05Model 1 age-adjusted only; Model 2: age plus other confounders, but not BMI, WHR or number of live births; Model 3: model 2 plus BMI; Model 4: model 2 plus WHR; Model 5: model 2 plus BMI and WHR; Model 6: model 5 plus number of live birthsTable\u00a04Associations between type 2 diabetes mellitus and duration of breast-feeding, Shanghai Women\u2019s Health Study (confirmed cases of diabetes only)\u00a0%Cases per person-yearsRR95% CIp valueBreast-feedingNo18.40128\/52,8481.000.17Yes81.60741\/233,8360.870.72\u20131.06Duration of breast-feeding\u00a0Months\u00a0\u00a0018.40128\/52,8481.000.17\u00a0\u00a0>0\u2013616.9186\/48,6170.800.61\u20131.05\u00a0\u00a0>6\u20131124.21159\/69,5580.980.77\u20131.24\u00a0\u00a0>11\u20133529.11314\/83,3980.840.68\u20131.05\u00a0\u00a0>3611.37182\/32,2630.740.55\u20131.01\u00a0Years\u00a0\u00a0018.40128\/52,8481.000.01\u00a0\u00a0>0\u20130.9948.20297\/11,8170.910.73\u20131.13\u00a0\u00a0>0.99\u20131.9917.25200\/57,1560.900.72\u20131.13\u00a0\u00a0>1.99\u20132.997.4899\/26,2420.720.54\u20130.95\u00a0\u00a0>2.99\u20133.994.1065\/14,8090.720.52\u20131.01\u00a0\u00a0>44.5778\/17,4540.670.46\u20130.96\u00a0Years per child\u00a0\u00a0018.40128\/52,8481.000.23\u00a0\u00a0>0\u20130.4915.13116\/43,4430.940.73\u20131.21\u00a0\u00a0>0.49\u20130.9944.21359\/126,9160.840.69\u20131.04\u00a0\u00a0>122.25266\/63,4470.880.70\u20131.11Values are adjusted for age, daily energy intake, BMI, WHR, number of live births, smoking, alcohol consumption, physical activity, education, income, occupation and hypertension\nIn analyses restricted to women who reported having been pregnant within the last 10\u00a0years (Table\u00a05), the RR for type 2 diabetes mellitus by the duration of breast-feeding per number of live births was 1.00, 1.01, 0.74 and 0.65; (p\u2009=\u20090.03 for trend) for 0, >0 to 0.49, >0.49 to 0.99 and \u22651\u00a0years of breast-feeding per number of live births. Among women who had been pregnant within the last 10\u00a0years, the RRs of type 2 diabetes mellitus for 0, >0 to 0.99, >0.99 to 1.99, >1.99 to 2.99, >2.99 to 3.99 and \u22654\u00a0years of breast-feeding were 1.00, 0.80, 0.78, 0.58, 0.47 and 0.45 (p\u2009=\u20090.04 for trend). The RRs of type 2 diabetes mellitus for 0, >0 to 0.99, >0.99 to 1.99, >1.99 to 2.99, >2.99 to 3.99 and \u22654\u00a0years of breast-feeding among women who had been pregnant within the last 5\u00a0years were 1.00, 0.87, 0.68, 0.44, 0.69 and \u22650.49 (p\u2009=\u20090.02 for trend; data not shown in tables). In addition, we found that the RRs for diabetes mellitus associated with \u226512\u00a0months of breast-feeding were 0.66 (95% CI, 0.45\u20130.95), 0.63 (95% CI, 0.40\u20130.98), 0.49 (95% CI, 0.28\u20130.85) and 0.46 (95% CI, 0.26\u20130.86), respectively, when analyses were conducted with time since last pregnancy defined as \u226413, \u226410, \u22645 and \u22642\u00a0years (data not shown). In analyses restricted to women who had not been pregnant in the last 10\u00a0years, we still found inverse associations between breast-feeding and risk of type 2 diabetes mellitus, although the associations between months of breast-feeding and years of breast-feeding per child were not significant. The association between years of breast-feeding and type 2 diabetes mellitus was of marginal significance. The RRs of type 2 diabetes mellitus for 0, >0 to 0.99, >0.99 to 1.99, >1.99 to 2.99, >2.99 to 3.99 and \u22654\u00a0years of breast-feeding were 1.00, 0.90, 0.94, 0.94, 0.79 and 0.72 (p\u2009=\u20090.06 for trend). Similar trends were found when the analysis was restricted to confirmed cases of type 2 diabetes mellitus. Finally, we found that the beneficial effect associated with the duration of breast-feeding was more evident among women over 60\u00a0years of age (data not shown).\nTable\u00a05Duration of breast-feeding and risk of type 2 diabetes mellitus, Shanghai Women\u2019s Health Study\u00a0Time since last pregnancy\u226410\u00a0years since last pregnancy>10\u00a0years since last pregnancyRR95% CIp value for trendRR95% CIp value for trendAll participants\u00a0Months\u00a0\u00a001.000.051.000.20\u00a0\u00a0>0\u201360.900.57\u20131.420.880.70\u20131.10\u00a0\u00a0>6\u2013110.830.55\u20131.250.910.74\u20131.12\u00a0\u00a0>11\u2013350.630.40\u20130.980.940.79\u20131.13\u00a0\u00a0\u2265360.660.30\u20131.480.760.60\u20130.97\u00a0Years\u00a0\u00a001.000.041.000.06\u00a0\u00a0>0\u20130.990.870.60\u20131.240.900.75\u20131.08\u00a0\u00a0>0.99\u20131.990.680.43\u20131.090.940.77\u20131.13\u00a0\u00a0>1.99\u20132.990.440.21\u20130.920.940.76\u20131.17\u00a0\u00a0>2.99\u20133.990.690.29\u20131.620.790.61\u20131.02\u00a0\u00a0\u226540.490.18\u20131.350.720.54\u20130.96\u00a0Years per child\u00a0\u00a001.000.031.000.45\u00a0\u00a0>0 \u20130.491.000.64\u20131.590.910.74\u20131.13\u00a0\u00a0>0.49\u20130.990.740.51\u20131.070.900.76\u20131.07\u00a0\u00a0\u226510.650.41\u20131.050.920.76\u20131.10Confirmed diabetes only\u00a0Months\u00a0\u00a001.000.261.000.34\u00a0\u00a0>0\u201360.800.41\u20131.530.810.60\u20131.09\u00a0\u00a0>6\u2013110.960.55\u20131.650.980.75\u20131.27\u00a0\u00a0>11\u2013350.690.39\u20131.220.870.69\u20131.11\u00a0\u00a0>360.600.23\u20131.570.780.56\u20131.08\u00a0Years\u00a0\u00a001.000.101.000.06\u00a0\u00a0>0\u20130.990.900.55\u20131.480.910.72\u20131.16\u00a0\u00a0>0.99\u20131.990.800.44\u20131.440.930.72\u20131.19\u00a0\u00a0>1.99\u20132.990.440.18\u20131.090.760.56\u20131.03\u00a0\u00a0>2.99\u20133.990.530.17\u20131.590.770.54\u20131.09\u00a0\u00a0>40.530.16\u20131.730.710.48\u20131.04\u00a0Years per child\u00a0\u00a001.000.251.000.44\u00a0\u00a0>0\u20130.490.970.52\u20131.820.940.71\u20131.25\u00a0\u00a0>0.49\u20130.990.810.49\u20131.330.860.68\u20131.08\u00a0\u00a0>10.730.40\u20131.340.920.71\u20131.17Values are adjusted for age, daily energy intake, BMI, WHR, number of live births, smoking, alcohol consumption, physical activity, education, income, occupation and hypertension\nDiscussion\nIn this large, prospective population-based study of middle-aged Chinese women, breast-feeding was associated with a reduced risk of type 2 diabetes mellitus [13] independently of known risk factors for type 2 diabetes mellitus.\nOur study adds to the limited data on the association between breast-feeding and risk of type 2 diabetes mellitus in mothers. Longer duration of breast-feeding was associated with reduced incidence of type 2 diabetes mellitus in two large US cohorts of young and middle-aged women, the Nurses\u2019 Health Studies I and II [6]. Similar to our results, the RRs in these studies for parous women with 0, >0 to 3\u00a0months, >3 to 6\u00a0months, >6 to 11\u00a0months, >11 to 23\u00a0months and \u226523\u00a0months of breast-feeding were 1.00, 0.98, 1.03, 0.96, 0.92 and 0.88 (p\u2009=\u20090.02 for trend) for middle-aged women and 1.00, 1.04, 0.91, 0.87, 0.88 and 0.67 (p\u2009<\u20090.01 for trend) for younger women. The RR for women who had breastfed compared with women who had never breastfed was 0.97 (95% CI, 0.91\u20131.02) for middle-aged women and 0.90 (95% CI, 0.77\u20131.04) for younger women. In agreement with our findings, these studies also found that the protection conferred by breast-feeding appeared to wane with time since last birth and that longer duration of breast-feeding per pregnancy was associated with a greater benefit. However, among women with gestational diabetes, breast-feeding was not associated with lower risk of type 2 diabetes mellitus later in life [6]. To our knowledge, no other studies have examined the long-term association between breast-feeding and subsequent development of type 2 diabetes mellitus.\nIn the Nurses\u2019 Health Studies I and II, analyses were adjusted for participants\u2019 birthweight and BMI at 18\u00a0years, as the investigators found that duration of breast-feeding was inversely related to BMI at 18\u00a0years [6]. In our study, we only had information on birthweight for a subset of participants. The correlation coefficients between duration of breast-feeding and both birthweight of participants and BMI at 20\u00a0years were 0.02 and 0.12, respectively. We chose to adjust for current BMI and WHR in our analysis, rather than BMI at 20\u00a0years or weight at 20\u00a0years, as BMI and WHR were strongly associated with type 2 diabetes mellitus. In addition, measurements used to calculate BMI and WHR in our study were taken by trained professionals at the time of the interview, while weight at 20\u00a0years was self-reported. In analyses adjusted for BMI at 20\u00a0years, the fully adjusted RRs for 0, >0 to 0.99, >0.99 to 1.99, >1.99 to 2.99, >2.99 to 3.99 and \u22654\u00a0years of breast-feeding were 1.00, 0.91, 1.02, 1.09, 0.77 and 0.73 (p\u2009=\u20090.30 for trend). We conducted additional analyses adjusting for standardised weight gain (kg per year) since age 20\u00a0years and weight at age 20\u00a0years. The fully adjusted RRs for 0, >0 to 0.99, >0.99 to 1.99, >1.99 to 2.99, >2.99 to 3.99 and \u22654\u00a0years of breast-feeding were 1.00, 0.90, 0.96, 0.99, 0.76 and 0.68 (p\u2009=\u20090.08 for trend).\nIt has been suggested that breast-feeding may protect against type 2 diabetes mellitus by facilitating weight loss, although the association between breast-feeding and weight loss remains inconclusive [14\u201320]. In our population, the age-adjusted means of standard weight gain since age 20\u00a0years by months of breast-feeding were 0.34, 0.31, 0.33, 0.36 and 0.37\u00a0kg per year for 0, >0 to 6\u00a0months, >6 to 11\u00a0months, >11 to 35\u00a0months and \u226536\u00a0months of breast-feeding, respectively (p\u2009<\u20090.01). Thus, weight loss is unlikely to be the reason for the inverse association between breast-feeding and type 2 diabetes mellitus observed in our study.\nAnother possible mechanism is that breast-feeding may improve insulin sensitivity and glucose intolerance. In a study of both breast-feeding and non-breast-feeding non-diabetic women, insulin levels and insulin\/glucose ratios were lower, while carbohydrate use and total energy expenditure were higher in the breast-feeding group [21]. Data from studies of women with gestational diabetes suggest that breast-feeding affects insulin and glucose homeostasis. In a study of 809 Latina women, breast-feeding was associated with improved glucose tolerance, fasting glucose and total area under the glucose tolerance curve [2], while in another study of 26 white women (14 breast-feeding, 12 non-breast-feeding), the breast-feeding group had a higher disposition index, indicating more efficient pancreatic beta cell function [3]. Data from animal studies also suggests that in the post-partum period, breast-feeding is associated with a decrease in insulin resistance. Blood glucose levels were reduced 20% and insulin levels were reduced 35% in lactating rats compared with non-lactating rats [4], while in another study, a 12-fold increase in insulin uptake was observed in the mammary glands of lactating rats, as well as a marked decrease in the plasma half-life of insulin [5]. Breast-feeding may also influence pituitary hormones [22] and may induce long-term changes in the hypothalamic-pituitary axis [23].\nOur study has several strengths. Our population is representative of urban Shanghai. The high follow-up rates minimised the possibility of selection bias. In addition, the prevalence of breast-feeding in this population was very high (81.62%), and there was a high number of live births (of the total number of women in our study at baseline, 74,942 or 96.4% were parous). In addition, the extensive information on potential confounders and the large study size allowed us to examine the effect of duration of breast-feeding on the development of type 2 diabetes mellitus in detail.\nThe major limitation of the study is reliance on self-reported diabetes, which is an important factor to consider when interpreting these results. A recent report suggested that the prevalence of diabetes was under-diagnosed in Shanghai [13]. We are not aware of any programme for systematic screening for diabetes in our study area. At baseline recruitment, we conducted a urinary glucose test for all cohort members who donated a urine sample (88.2% of participants). We found that 1% of participants who reported never having been diagnosed by a physician as having diabetes had a positive urinary glucose test. These participants were excluded from the current analysis. However, it is possible some other type 2 diabetes cases remained undiagnosed in our study. Similarly, self-reported diabetes may also include some false positive cases. Misclassification of diabetes could weaken the association between duration of breast-feeding and the risk of type 2 diabetes mellitus. To address the possibility of surveillance bias, we conducted analyses restricted to women with confirmed diabetes and found similar results.\nIn our study, duration of breast-feeding was correlated with parity. In a recent study from the UK, having more children was associated with a higher risk of diabetes in women [24]. However, the association was attenuated after adjustment for BMI and socioeconomic factors. Similarly to the UK study, we found parity to be associated with lower income and education levels, higher BMI, WHR and standard weight gain, as well as smoking and alcohol consumption in this population. In age-adjusted analyses the incidence of diabetes was positively associated with number of children. The RRs of diabetes were 1.00, 1.38, 1.32, 1.31 and 1.62 for one, two, three, four and five or more children (p\u2009<\u20090.01). When we adjusted the analysis for BMI and WHR, parity was no longer associated with incidence of type 2 diabetes. We found that the association between breast-feeding and type 2 diabetes did not change much after adjustment for number of live births. Before and after inclusion of the number of live births in the analysis, the RRs of type 2 diabetes mellitus for 0, >0 to 6\u00a0months, >6 to 11\u00a0months, >11 to 35\u00a0months and \u226536\u00a0months of breast-feeding were 1.00, 0.87, 0.89, 0.89 and 0.75 (p\u2009=\u20090.04 for trend) and 1.00, 0.87, 0.89, 0.89 and 0.73 (p\u2009=\u20090.05 for trend), respectively.\nBreast-feeding could also reflect differences in other risk factors in our population that might not have been completely controlled for or measured in our study. We applied a propensity score adjustment, including a wide array of lifestyle and dietary intake variables, and found little change in the pattern of associations between breast-feeding and type 2 diabetes mellitus (data not shown). Nevertheless, residual confounding from unmeasured or less than perfectly measured confounders, such as socioeconomic status at the time of breast-feeding, is still a concern. Thus, our results should be interpreted with caution, particularly given that the association was relatively weak and that the reduced risk was limited to participants with a longer duration of breast-feeding (\u226536\u00a0months). In addition, over-adjustment due to the inclusion of possible mediating variables such as BMI and WHR in the model may also be a concern. In our study population, breast-feeding was weakly associated with BMI and WHR (r\u2009=\u20090.22 and r\u2009=\u20090.22, respectively). Further studies conducted in other populations and focused on the underlying biological mechanisms are warranted.\nIn summary, we found that breast-feeding duration was inversely associated with the risk of type 2 diabetes mellitus in this population. Together with results from two other large US cohorts and some clinical evidence of improved glucose homeostasis in breast-feeding women, these data suggest that breast-feeding may reduce the risk of type 2 diabetes mellitus in middle-aged women.","keyphrases":["type 2 diabetes mellitus","middle-aged women","parous women","lifelong breast-feeding"],"prmu":["P","P","P","M"]}
{"id":"Virchows_Arch-3-1-2039817","title":"Primary retroperitoneal mucinous cystadenoma with sarcoma-like mural nodule\n","text":"Primary retroperitoneal cystadenomas are extremely rare. This is the first report in literature to describe a primary retroperitoneal cystadenoma with a sarcoma-like mural nodule. A 45-year-old woman complained of a left-sided abdominal mass. A computed tomography scan revealed a cystic mass with a mural nodule, which seemed to originate from the tail of the pancreas. At laparotomy the cyst was not adhered to the pancreas but localized retroperitoneally. Histologic examination showed a mucinous cystadenoma with only foci of borderline malignancy with a mural \u201csarcoma-like\u201d nodule. In view of the surgical and histopathological findings, the mucinous cystadenoma was regarded as primary retroperitoneal. This case demonstrates that in the era of radiological preoperative refinement, pathological diagnosis remains of utmost importance, especially for rare cases.\nIntroduction\nMucinous cystadenomas of the ovary are clinically and histopathologically well-established and common tumors. Primary retroperitoneal mucinous cystadenomas are extremely rare. Such tumors are histologically similar to ovarian mucinous cystadenomas. Their histogenesis is still unclear. We report a case of primary retroperitoneal mucinous cystadenoma with foci of borderline malignancy containing a mural \u201csarcoma-like\u201d nodule.\nCase report\nClinical history\nA 45-year-old, para 2, woman presented at the emergency room with a 3-week history of left-sided abdominal pain. She had felt a mass in the left lower quadrant 2\u00a0days before. Her clinical history included endometriosis and a car accident. The mass was progressive but not painful. Apart from the palpable mass of 15\u00a0cm in the left lower abdomen, physical examination was unremarkable. Ultrasonography demonstrated a 15-cm cystic mass with a 3.8-cm nodule in its wall. The uterus was normal in size, and internal ultrasonography showed small ovaries. Carcinoembryonic antigen, cancer antigen (CA) 125, and CA 19-9 levels were within normal limits. The next day, a contrast-enhanced computed tomography scan of the abdomen revealed a 15-cm left-sided cystic mass, which seemed to originate from the tail of the pancreas (Fig.\u00a01a). The cystic mass showed a 4-cm nodule in its wall (Fig.\u00a01b) and was suspected for a cystic papillary adenocarcinoma. At laparotomy, the cyst was not adhered to the pancreas and could be easily separated from its location near the tail without opening the pancreatic capsule. Vascularization appeared to arise from the mesentery of the left colon. It was localized in the retroperitoneal space extending caudally from the spleen to the lower abdomen with medial displacement of the left colon. Total resection of the cyst was performed, and the specimen was sent for histopathological examination. Further inspection showed two normal ovaries. Her postoperative recovery was uneventful. One year after surgery, the patient was without signs of recurrence or metastasis.\nFig.\u00a01Contrast enhanced CT scan shows images of a pancreatic cyst (arrowheads) originating from the tail of the pancreas (a) and a mural nodule within the wall of the cystic mass (b)\nMaterials and methods\nThe specimen was fixed in 4% buffered formalin. Representative samples were routinely processed and embedded in paraffin blocks. Four-micrometer-thick sections were stained with hematoxylin and eosin and with parallel routine immunohistochemical procedures. The antigens tested by immunohistochemistry were: pan-keratin, keratin Cam 5.2, cytokeratin 7, cytokeratin 10, cytokeratin 18, cytokeratin 20, epithelial membrane antigen, vimentin, desmin, actin, myosin, CD34, CD68, CD99, CD117, S-100 protein, and bcl-2.\nPathological findings\nThe specimen consisted of a unilocular cyst measuring 20\u2009\u00d7\u200911\u00a0cm with a smooth surface. The content was a watery mucinous material. The wall was thin with a smooth gray-white inner surface and contained a circumscribed bean-shaped solid mural nodule of 3.5\u2009\u00d7\u20093.5\u2009\u00d7\u20092.5\u00a0cm, which showed a brown-yellow and focally hemorrhagic cut surface. Microscopically, most of the cyst was lined by single-layered tall columnar cells, abundant clear cytoplasm, and small basally located nuclei (Fig.\u00a02a).\nFig.\u00a02Cyst wall with typical tall columnar mucous-secreting epithelium and fibrous wall (a). Low cellular proliferation with some stratification of the cells and slight nuclear atypia (b). Heterogenous proliferation of pleomorphic cells, spindle cells, osteoclast-like giant cells, and some mononuclear cells with some pigment (c). Detail of sarcoma-like nodule (d)\nOccasionally (over less than 1% of the surface), the epithelium showed slightly atypical proliferation with glandular budding, tufting of the epithelium, decreased cytoplasmic mucin, some stratification of slightly irregular nuclei, and an occasional mitoses (Fig.\u00a02b). There was no infiltrative growth but foci of borderline malignancy. The nodule was well circumscribed without vascular invasion and consisted of a heterogenous population of spindle-shaped cells, pleomorphic cells with bizarre nuclei, mixed mononuclear inflammatory cells, benign osteoclast-like giant cells, and foci of hemorrhage. There were mitotic figures, including some atypical forms (Fig.\u00a02c and d). The sarcoma-like cells proved to be keratin negative (Table\u00a01).\nTable\u00a01Immunohistochemical results of the sarcoma-like cellsAntigenResultPan-keratin\u2212Keratin Cam 5.2\u2212Cytokeratin 7\u2212Cytokeratin 10\u2212Cytokeratin 18\u2212Cytokeratin 20\u2212Epithelial membrane antigen\u2212Vimentin+Desmin\u2212Actin+\/\u2212 (some)Myosin\u2212CD34\u2212CD68+CD99\u2212CD117\u2212S-100 protein\u2212bcl-2+\/\u2212 (some)\nDiscussion\nMucinous cystadenomas can be located in the ovaries, pancreas, and in the retroperitoneum. The mucinous cystadenoma presented was localized retroperitoneally near the pancreas but was clearly not adhered to it. Because normal-appearing ovaries were found, the cystadenoma was thought to be primary retroperitoneal.\nAccording to the literature, symptoms are nonspecific, and most patients complained of an abdominal distension or mass with or without pain [9]. Mucinous cystadenomas were relatively large, varying from 10 to greater than 20\u00a0cm in diameter, which is large enough to cause symptoms like abdominal fullness [9]. Preoperative diagnosis is very difficult, not because the tumors are often overlooked in the differential diagnosis but also because no sensitive methods or reliable markers are available [2]. As retroperitoneal mucinous cystadenomas are histologically similar to mucinous cystadenomas of the ovary, the ultrasonographic image pattern is in general of no help in distinguishing between ovarian and retroperitoneal origin. In our case, the diagnosis of the retroperitoneal mucinous cystadenoma could not be established preoperatively by ultrasonography or computed tomography. Although ultrasonography, computed tomography, or magnetic resonance imaging can detect retroperitoneal cysts, the diagnosis of mucinous cystadenoma is seldomly made preoperatively. The usual preoperative differential diagnosis consists of ovarian cyst, cystic mesothelioma, cystic lymphangioma, nonpancreatic pseudocyst, and renal cyst [4, 9, 18]. Although aspiration is a good method for delineating the nature of the cyst, cytologic analysis of the aspirated fluid frequently fails to reveal the type of epithelial cells lining the cyst. Therefore, exploratory laparotomy with complete excision of the cyst is usually indicated both for diagnosis and treatment [2].\nRetroperitoneal mucinous cystadenomas are histologically similar to mucinous cystadenomas of the ovary. The histogenesis of these tumors is still unclear. Four main hypotheses have been proposed [2, 14]. According to the first three hypotheses, the tumor arises either from ectopic ovarian tissue, although ovarian tissue was only rarely found [14], from a teratoma in which the mucinous epithelium has overgrown all other components or from urogenital remnants. The most widely accepted theory suggests coelomic metaplasia as the causal agent, whereby tumors arise from invagination of the peritoneal mesothelial layer that undergoes mucinous metaplasia with cyst formation [3, 9]. Such origin rather than from ectopic ovarian tissue is supported by the occurrence of such a tumor in a male patients [5, 8, 10, 17].\nPrimary mucinous tumors of the retroperitoneum are very uncommon. These tumors can be classified into three clinicopathologic types: mucinous cystadenoma, mucinous cystic tumor of borderline malignancy, and mucinous cystadenocarcinoma. Our case was diagnosed as a primary retroperitoneal mucinous cystadenoma with only foci of borderline malignancy and a mural \u201csarcoma-like\u201d nodule.\nMural nodules have been described in ovarian and pancreatic mucinous cystic tumors [7, 16]. Mural nodules may be malignant representing anaplastic carcinoma, containing a predominant population of cytokeratin-positive cells with high-grade malignant nuclei, or a genuine soft tissue-type sarcoma [7, 15, 16]. Benign pseudosarcomatous mural nodules are composed of a heterogeneous cell population of epulis-type giant cells, atypical spindle cells with bizarre nuclei and mitotic figures, mixed inflammatory cells, and signs of hemorrhage and necrosis. In these cases, immunohistochemical staining shows a weakly or focally cytokeratin positivity in the pseudosarcomatous cells.\nWe performed a literature review using Embase and Medline starting in 1966 and identified approximately 45 cases of retroperitoneal mucinous cystadenoma and 25 cases of mucinous cystadenocarcinoma. Only eight cases of mucinous cystadenoma with borderline malignancy have been reported (Table\u00a02). Thus, our case is the ninth case of a retroperitoneal mucinous cystadenoma of borderline malignancy. By our knowledge, however, combination with a mural \u201csarcoma-like\u201d nodule has not been described earlier in the literature. The patient should be followed. However, in this case, long-term follow-up seems not warranted regarding the only focal aspect (<1% of the surface) of borderline malignancy of the cyst and the benign reactive nature of the mural nodule.\nTable\u00a02Cases of primary retroperitoneal mucinous cystadenomas of borderline malignancyStudy (year)Age (years)SexSymptomSize, imageTumor markerPreoperative diagnosisHistoryPathologyExtracystic extensionTherapyOutcomeNagata et al. [11] (1987)41FAbdominal swelling, pain12\u2009\u00d7\u200910\u2009\u00d7\u20099 cmUDUDUDMCAborNoTRUDBanerjee and Gough [1] (1988)47FAbdominal mass10\u00a0cm (US) next to spl.NDLt adrenal tumorApp HystMCAborNoTR, resection spl.\u2009+\u2009lt adrenalNEDMotoyama et al. [10] (1994)63MAbdominal pain6\u00a0cm (US), under rt kidneyHigh CEA in cystic fluidRt renal cystNRMCAborNoNDNEDPearl et al. [13] (1996)33FAbdominal swelling, painLarge, unilocular lt (CT)NDNDNRMCAborNoLRNED, 10 monthsPapadogiannakis et al. [12] (1997)33FAbdominal mass13\u2009\u00d7\u20099\u00a0cm (US\u2009+\u2009CT)NDMesenteric cystNRMCAborNoTRNED, 12 monthsChen et al. [2] (1998)48FAbdominal fullness15\u2009\u00d7\u200913\u2009\u00d7\u20099\u00a0cm (CT)NDMesenteric cystNRMCAborNoLRNED,8 monthsGutsu et al. [6] (2003)41FFlank pain, abdominal distension21\u2009\u00d7\u200916\u00a0cm rt (CT)NDRetroperitoneal cystNRMCAborNoTRNED, 18 monthsMatsubara et al. [9] (2005)36FAbdominal distension12\u2009\u00d7\u20098\u00a0cm rt (CT)CA 125:51 CA 19-9: 55Ovarian cystNRMCAborNoTR, App, MyoNED, 6 monthsPresent case (2007)45FAbdominal pain15\u00a0cm (US\u2009+\u2009CT)CEA, CA125, CA19-9: normalCystic papillary adenocarcinoma or mucinous cystic neoplasmEndomMCAborNoTRNED, 12 monthsF Female; M male, UD unknown data, NR not remarkable, MCAbor mucinous cystadenoma borderline malignancy, TR tumor resection, LR laparoscopic resection, NED no evidence of disease, US ultrasonography, spl. spleen, App appendectomy, Hyst hysterectomy, Myo myomectomy, Endom endometriosis, rt right, lt left","keyphrases":["mucinous cystadenoma","mural nodule","retroperitoneum"],"prmu":["P","P","P"]}
{"id":"Environ_Health_Perspect-115-3-1849945","title":"Cumulative Lead Dose and Cognitive Function in Adults: A Review of Studies That Measured Both Blood Lead and Bone Lead\n","text":"Objective We review empirical evidence for the relations of recent and cumulative lead dose with cognitive function in adults.\nIn the development of the adult lead management guidelines (see Kosnett et al. 2007), a number of health outcomes adversely affected by lead exposure were discussed. Cognitive function was an important consideration of because of the growing number of studies in this area and increasing concern that cognitive function in adulthood may be affected by relatively low lead doses. In this article, we systematically review recent evidence concerning recent and cumulative lead dose and adult cognitive function.\nMeasurement of lead dose\nIn reviewing studies of the health effects of lead, it is critical to understand the available lead biomarkers in terms of how they represent external exposure (in terms of timing, duration, magnitude, and accumulation); how they are influenced by metabolic factors (organ distribution, compartmental dynamics, the influence of physiologic factors); and how the combination of these considerations affects inferences regarding the health effects of lead (Hu et al. 2007). We conclude from these important methodologic issues that the most informative recent epidemiologic studies of lead\u2019s impact on health are those that were able to derive estimates of both recent and cumulative lead exposure for each study participant. To achieve this end with the greatest precision and accuracy, such studies have incorporated measurements of lead in both blood (whole blood, using standard chemical assays such as graphite furnace atomic absorption spectroscopy) and bone [using noninvasive in vivo K-shell X-ray fluorescence (KXRF) instruments].\nBlood lead levels measured in epidemiologic studies with valid instruments and standardized calibration and quality control procedures have been reported in the literature for > 35 years. Bone lead levels measured by in vivo KXRF were begun in some research laboratories in the 1980s, but it was not until the mid-1990s that reports began to emerge of KXRF-measured bone lead levels in relation to potential health indicators from epidemiologic studies with sufficient sample sizes (for example, \u2265 100 subjects) to have substantial statistical power. Thus, in this review we summarize all studies to date that measure cognitive function and both blood and bone lead levels (or acceptable surrogate for cumulative lead dose).\nPublished reviews of relevance to this review\nWe begin our review with a discussion of three other reviews on the topic of lead dose and cognitive function (Balbus-Kornfeld et al. 1995; Goodman et al. 2002; Meyer-Baron and Seeber 2000). Balbus-Kornfeld et al. (1995) reviewed the evidence on cumulative lead exposure and cognitive function from studies published from 1976 to 1991. Among 21 unique studies that were identified at the time of the authors\u2019 review, none used a biomarker of cumulative dose. Of the four longitudinal studies, all were small (mean sample size in the analysis of 47 lead-exposed subjects), with relatively low follow-up rates and relatively short durations of follow-up. The authors thus concluded that the available literature provided inadequate evidence to conclude whether cumulative exposure or absorption of lead adversely affected cognitive function in adults.\nGoodman et al. (2002) and Meyer-Baron and Seeber (2000) are reviewed here because they had generally opposite conclusions, which led to considerable controversy and discussion (Goodman et al. 2001; Schwartz et al. 2002; Seeber and Meyer-Baron 2003; Seeber et al. 2002). The Goodman et al. (2002) article was funded by the German Battery Association, apparently in anticipation of consideration in Germany of lowering the blood lead standard in lead workers (Seeber and Meyer-Baron 2003). Goodman et al. (2002) reviewed 22 studies published between 1974 and 1999 with the expressed aim of evaluating associations between moderate blood lead levels and neurobehavioral test scores after occupational exposure to lead. Studies were included if the central tendency for blood lead levels was < 70 \u03bcg\/dL, the numbers of exposed and unexposed were reported, and test score arithmetic means and measures of variability were reported for exposed and unexposed workers (Goodman et al. 2002). The authors concluded that none of the individual studies were conclusive or adequate in providing information on the effects of lead on cognitive function and called for prospective studies that would evaluate cognitive function before and after exposure. There was no discussion about whether examining relations of blood lead levels with cognitive function was the most relevant question if the hypothesis was that cumulative lead dose was most important to cognitive function. There was little explicit discussion of whether lead may have acute effects as a function of recent dose, and chronic effects as a function of cumulative dose, or how this could be assessed by review of epidemiologic studies.\nMeyer-Baron and Seeber (2000) performed a meta-analysis of 12 studies using selection criteria similar to Goodman et al. (2002) but also with the requirement for reporting means and standard deviations of dependent variables (Meyer-Baron and Seeber 2000). They concluded that there were obvious neurobehavioral deficits at current blood lead levels < 40 \u03bcg\/dL. Again, the focus was on associations with blood lead levels, and there was little formal discussion about which lead biomarker was most relevant to hypotheses about how cumulative lead dose may influence cognitive function. Thus, this is the first review to evaluate epidemiologic studies that distinguish between the acute effects of recent dose from the chronic effects of cumulative dose.\nMethods\nMethodologic considerations for relations of lead dose and cognitive function\nMany methodologic issues of relevance to the epidemiologic investigation of lead and cognitive function have been addressed elsewhere in this minimonograph (Hu et al. 2007). When evaluating the associations of cumulative lead dose with cognitive function, it is important to acknowledge that nonoccupational sources of lead exposure were present for all members of the general population, including lead workers throughout the early part of this century until public health interventions progressively removed lead from gasoline and many consumer products during the 1970s and 1980s (Agency for Toxic Substances and Disease Registry 1999; Annest et al. 1983; Pirkle et al. 1998). Lead remains a low-level and ubiquitous neurotoxicant in the environment and is found in measurable levels in all individuals (Hoppin et al. 1995). Thus, current tibia lead levels represent a mix of occupational and environmental exposures. This review does not try to determine whether the main source of lead was occupational or environmental but rather focuses on whether lead in blood or bone is associated with adverse cognitive outcomes in adults.\nIdentification of studies\nWe conducted a systematic literature review of the association between blood and bone lead biomarkers and cognitive functioning in adults. Our aim was to select studies that compared markers of both recent and cumulative lead dose in their relations with cognitive function. Both occupationally and environmentally exposed adult populations were included. We searched the PubMed (National Library of Medicine 2006) and PsycINFO databases (American Psychological Association 2006) for epidemiologic studies using keywords such as blood, bone, lead, cumulative, cognitive, and neurobehavior. There were no date or language restrictions. From the identified publications and relevant review articles, we examined reference lists to locate additional studies that measured both recent and cumulative lead dose. This includes blood lead levels, bone lead levels, or a surrogate measure of cumulative lead dose such as integrated blood lead (IBL), area under the curve of blood lead levels over time, or the product of blood lead level and employment time. Studies were not considered for the review if they a) contained no original research, b) were conducted on nonhuman subjects, c) were case reports, d) contained no standardized neurocognitive assessment outcomes, or e) lacked measures of both recent and cumulative lead dose.\nData abstraction\nWe abstracted data from articles meeting the selection criteria. Study quality was assessed with the following criteria: a) exposure was assessed at an individual level; b) exposure was assessed with a biomarker; c) cognitive outcomes were objective, standardized tests; d) statistical adjustment for potential confounders including age, sex (in studies with both men and women), and education; e) data collection was similar in exposed and nonexposed participants; f ) time period of study was the same in exposed and nonexposed participants; and g) there was a detailed description of the approach to data analysis. We decided not to try to derive a pooled estimate across studies of the associations of lead dose biomarkers with cognitive function because of differences in methods for subject selection, blood and bone lead measurements, neurobehavioral outcomes, approach to regression modeling, and presentation of results across studies. Pooled estimates from metaanalysis also can be highly influenced by decisions regarding how and whether to pool certain results. We thus decided to present details for each study and discuss them in turn.\nResults\nOverview of evidence\nWe identified three main types of studies that reported cross-sectional or longitudinal associations of blood and bone lead levels with cognitive function. These were of a) environmentally exposed individuals in the general population, b) workers with current occupational exposure, and c) former lead workers without current occupational exposure to lead. We have summarized these studies in Table 1, provided details in Table 2, and discuss them in order below.\nStudies of adults without occupational lead exposure\nWe identified six articles from three studies [i.e., residents near a lead smelter, the Normative Aging Study (NAS), and the Baltimore Memory Study] that evaluated subjects with mainly environmental exposure to lead (Tables 1 and 2). One study of young adults 19\u201329 years of age compared 257 individuals with high childhood blood lead levels from exposure 20 years previously from a lead smelter to 276 age- and sex-matched controls. This study found impairment on many cognitive tests among the highly exposed group, but minimal association on most tests with tibia lead levels measured during young adulthood (Stokes et al. 1998).\nFour articles from the NAS reported associations of blood and bone lead levels in a cohort of older men. One of these articles (Payton et al. 1998) was a first report that examined scores on a large battery of cognitive tests of a small sample (n = 141) of NAS participants. This was subsequently followed up with a report on a much larger number of NAS participants (n = 1,089 with blood lead levels and n = 760 with bone lead levels, 412\u2013515 of whom took different tests twice approximately 3.5 years apart) (Weisskopf et al. 2007). Cross-sectional analyses in the original report found that increased blood lead levels across a relatively low range of levels [mean \u00b1 SD = 5.5 \u00b1 3.5 \u03bcg\/dL) were a stronger predictor, compared with tibia or patella lead levels, of poorer performance on tests of speed, verbal memory, vocabulary, and spatial copying skills. However, this was not confirmed in the larger, cross-sectional analysis, except possibly for scores on a vocabulary test (Weisskopf et al. 2007). Conversely, in longitudinal analyses, the larger study found more decline over time on almost all cognitive tests associated with both higher patella and higher tibia bone lead levels, with the associations reaching statistical significance for pattern comparison and spatial copying skills. An earlier, similar longitudinal analysis by Weisskopf et al. (2004) in this same population reported that patella lead levels were significantly associated with a decline in Mini-Mental State Examination (MMSE; Folstein et al. 1975) score over time. A slightly smaller association was observed with tibia lead levels, whereas no association was observed with blood lead levels. In cross-sectional analyses of the same population, higher blood lead levels were a stronger predictor of poorer performance on the MMSE, as were higher patella and tibia bone lead levels (Payton et al. 1998; Wright et al. 2003).\nIn a study of almost 1,000 persons 50\u201370 years of age randomly selected from the general population in the Baltimore Memory Study (BMS), a cross-sectional analysis showed that relatively low current blood lead levels were not associated with cognitive domain scores. However, moderate tibia lead levels (mean ~ 19 \u03bcg\/g) were significantly associated with worse performance in all seven cognitive domains (Shih et al. 2006). Thus, in the environmental studies of older adults, the most consistent findings across studies are associations between bone lead levels and cognitive function. The associations in the BMS were cross-sectional, whereas the predominant associations in the NAS were with change in cognitive function over time, although a significant cross-sectional association with MMSE score was also observed in this sample. Taken together, these data suggest that at environmental exposure levels, the effects of cumulative exposure are more pronounced than recent effects of current exposure. The absence of associations in the Stokes et al. (1998) study could be because of the younger age of studied subjects, the very low current blood and tibia lead levels, or the inadequacy of tibia lead in the third decade of life to estimate early life dose (Hoppin et al. 2000).\nStudies of occupationally exposed workers\nFifteen articles were identified of workers with current or past occupational exposure to lead. Eight of these studies used a surrogate measure of cumulative lead dose (i.e., IBL) rather than a direct measure of lead in bone. Among these studies, which compared blood and IBL lead dose, when the lead exposure was primarily current (e.g., relatively high blood lead levels), most studies found an association between increasing blood lead values and worse cognitive function (Barth et al. 2002; Bleecker et al. 1997; Lucchini et al. 2000). However, studies in which the exposure was primarily in the past demonstrated that surrogate measures of cumulative dose were a stronger predictor of worse cognitive function compared with blood lead levels (Bleecker et al. 2005; Chia et al. 1997; Lindgren et al. 1996). Studies that used bone lead levels as a direct indicator of retained cumulative lead dose are summarized below.\nOne study of currently exposed lead workers in South Korea (n = 803) found strong and consistent associations of blood lead levels with worse cognitive function after adjustment for covariates, but tibia lead levels were not as consistently associated (Schwartz et al. 2001). The same null findings for bone lead levels were observed in two smaller studies, one with male smelter workers (n = 57) in whom finger bone (mixed trabecular and cortical tissue) lead levels were measured (Osterberg et al. 1997). The second article describes the study of a sample of 54 storage battery workers in whom tibia and calcaneus lead levels were measured (Hanninen et al. 1998). This is the only study published to date to report an association between IBL and cognitive outcomes in which there was a lack of an association with bone lead levels. Both these studies used early XRF techniques (e.g., KXRF with cobalt-57) with higher limits of detection that have not been commonly used since, and this use makes the findings more difficult to interpret. Bleecker et al. (1997), in a study similar to the one by Schwartz et al. (2001), reported stronger and more consistent associations of blood lead measures and neurobehavioral test performance compared to tibia lead levels.\nIn the South Korean lead workers with current occupational exposure, a longitudinal analysis was performed to separate recent lead dose (measured as blood lead levels) from cumulative lead dose (measured as tibia lead levels), and acute effects from chronic effects in 575 subjects with complete data across the three study visits (Schwartz et al. 2005). The authors reported significant cross-sectional associations of blood lead levels with lower executive ability and manual dexterity test scores, with some evidence also for a longitudinal association of changes in blood lead levels with neurobehavioral decline. Tibia lead levels were more consistently associated with longitudinal declines in manual dexterity, executive abilities, neuropsychiatric symptoms, and peripheral sensory functioning than change in blood lead levels. The authors concluded that lead was associated with worse cognitive function in two ways: an acute effect of recent dose and a chronic effect of cumulative dose. The authors also discussed that contrasting associations with blood and tibia lead levels could be due to the following: a) tibia and blood lead levels are biologically related and blood lead is in equilibrium with bone lead stores; b) the error in measurement of tibia lead levels is larger than that for blood lead; c) controlling for cross-sectional associations could obscure longitudinal ones; and d) lead in blood reflects recent external exposure, and is in equilibrium with bone lead stores, possibly taking away explained variance from bone lead associations via this correlation in cross-sectional analyses.\nResults of a cross-sectional analysis of former organolead workers showed that higher peak tibia lead levels (range, \u20132.2 to 105.9 \u03bcg\/g) were related to poorer functioning on a number of cognitive tests, including those assessing manual dexterity, executive ability, verbal intelligence, and verbal memory (Stewart et al. 1999). In a longitudinal analysis in this same population, among 535 lead workers exposed a mean of 16 years before, increases in peak tibia lead levels [mean \u00b1 SD = 22.6 \u00b1 16.5 \u03bcg\/g] but not in blood lead levels predicted declines over time in these same domains in addition to visual memory (Schwartz et al. 2000). This finding indicates that even many years after high lead exposure, and in the absence of high current lead exposure, cumulative lead dose may exert progressive effects on cognitive functioning (Links et al. 2001).\nLead exposure and psychiatric symptoms\nSeveral lines of evidence suggest that increased blood lead levels are associated with psychiatric symptoms in adults, such as depression, anxiety, irritability, and anger. For example, a cross-sectional analysis of 107 occupationally exposed individuals showed increased rates of depression, confusion, anger, fatigue, and tension as measured by the Profile of Mood States (POMS; McNair et al. 1971) among those with blood levels > 40 \u03bcg\/dL (Baker et al. 1983). Maizlish et al. (1995) found that current and cumulative measures of blood lead levels in currently exposed lead workers were associated with tension, anxiety, hostility, and depression measured by the POMS questionnaire. Lindgren et al. (1996) examined the POMS\u2019 factor structure in retired lead smelter workers and showed that the resulting \u201cgeneral distress\u201d factor was significantly related to IBL but not to current blood lead level.\nIn occupationally exposed South Korean lead workers, tibia lead levels were significantly associated with more depressive symptoms measured by the Center for Epidemiologic Studies Depression scale (CES-D; Radloff 1977) after adjusting for age, sex, education, job duration, and blood lead level (Schwartz et al. 2001). However, only one recent study has examined a direct measure of cumulative dose with bone measurements in a community sample (Rhodes et al. 2003). These authors used the Brief Symptom Inventory (BSI; Derogatis and Melisaratos 1983) to show that patella bone lead levels were associated with an increased risk of anxiety and depression sub-scale scores. The logistic regression estimate for the phobic anxiety subscale was statistically significant (p < 0.05), as well as for the combined measure of all three BSI subscales (anxiety, depression, and phobic anxiety).\nPsychiatric symptoms, specifically symptoms of depression, potentially share the same neural substrates with components of cognition, and thus may be important to late-life cognitive functioning. Compared with nondepressed elderly individuals, depressed elderly perform more poorly on tests involving attention, memory encoding, and retrieval. However, intelligence tests are more resistant to these effects of depression (Arnett et al. 1999; Naismith et al. 2003; Weingartner et al. 1981). Depressive symptoms (as measured by the CES-D) are positively associated with both the risk of Alzheimer disease and a steeper rate of cognitive decline (Wilson et al. 2002). Because late-life symptoms of depression are closely associated with dementia, investigators have put forth a number of hypotheses that suggest depression a) may be a risk factor for cognitive decline, b) has risk factors in common with dementia, c) is an early reaction to declining cognition, and d) influences the threshold at which dementia emerges [for review see Jorm (2000)]. The exact temporal and mechanistic relation remains unclear. Regardless of the exact relation between depressive symptoms and cognitive function, however, the assessment of the impact of lead exposure on these outcomes is not compromised. Whatever the associations with these outcomes, they would still be attributed to lead\u2014that is, even if depressive symptoms lead to worse cognitive performance, and lead leads to symptoms of depression, the cognitive impairment as a result of that depression could still be considered part of the total effect of lead.\nLead\u2013gene interactions\nIn the former organolead worker studies discussed above, possessing at least one apolipoprotein E (APOE) \u025b 4 allele magnified the negative cross-sectional association of tibia lead levels with performance on the cognitive domains of executive ability, manual dexterity, and psychomotor skills (Stewart et al. 2002). No direct effects of the APOE \u025b 4 allele were observed on cognitive function in this study, presumably because of the sample\u2019s younger age (range, 41\u201373 years). Other studies have found that APOE \u025b 4 modifies dementia outcome in individuals with previous traumatic head injury, suggesting that APOE \u025b 4 plays a role in recovery from brain insults (Mayeux et al. 1995), which may be extended to include insult from lead exposure.\nDiscussion\nSummary of evidence for a causal relationship\nThe literature on associations of recent and cumulative dose biomarkers with cognitive function has grown impressively since the 1995 review (Balbus-Kornfeld et al. 1995). We believe sufficient evidence exists to conclude that there is an association between lead dose and decrements in cognitive function in adults. Overall, while the association between blood lead levels and cognitive function is more pronounced in occupational groups with high current lead exposures, associations between bone lead levels and cognitive function are more evident in studies of older subjects with lower current blood lead levels, particularly in longitudinal studies of cognitive decline.\nConsistency of associations\nFollowing is a summary of the findings from each of the three types of populations. First, cross-sectional studies of currently exposed lead workers showed that associations of blood lead levels and cognitive function were clearer than the associations for tibia, patella, or calcaneus lead levels, perhaps because the acute effects of recent dose in an occupational setting masked the chronic effects of cumulative lead dose. Second, previously exposed occupational populations demonstrated a stronger association between cumulative lead dose measured in tibia bone with cognitive deficits compared with blood lead levels. The two studies that deviated from these otherwise consistent findings may not have had sufficient power to detect any associations (n < 60). Last, studies of environmentally exposed adults who had notably higher exposures in the past suggest that bone lead level is more consistently associated with performance on cognitive tests than is blood lead level. The domains associated with lead dose do not differ in general by lead biomarker (blood, tibia, patella). The cognitive domains consistently associated with each biomarker in both environmental and occupational studies on adults include verbal and visual memory, visuospatial ability, motor and psychomotor speed, manual dexterity, attention, executive functioning, and peripheral motor strength. Comparisons of lead and psychiatric symptom associations in previously and currently exposed samples lend credence, although perhaps at higher thresholds than for cognitive outcomes, that neurobehavioral functioning is consistently associated with blood lead when exposure is currently high (e.g., occupational) and bone lead when exposure is primarily from past chronic exposure.\nThese associations exist in multiple settings, including both occupational and non-occupational, in men and women, and in populations with diversity by socioeconomic status and race\/ethnicity. This reduces the likelihood of associations by statistical chance or due to unmeasured confounding. However, this consistency cannot completely rule out the possibility of uncontrolled confounding or effect modification (Martin et al. 2006; Shih et al. 2006). In addition, in studies of general populations with diversity by socioeconomic status and race\/ethnicity, the ability to disentangle social, cultural, and biological factors from the \u201cindependent\u201d influence of lead dose may be a futile exercise (Weiss and Bellinger 2006).\nStrength of association\nThe strength of associations between lead and cognitive function is strong and can be compared to the influence of age on cognitive function. The comparative magnitude of these effects has been reported in several studies. In currently exposed lead workers, cross-sectional associations showed that a 5-\u03bcg\/dL increase in blood lead was equivalent to an increase of 1.05 years in age (Schwartz et al. 2001). The magnitude of cross-sectional associations with tibia lead levels in the BMS was moderate to large. A proportion comparison of the direct effect of age and the direct effect of tibia lead levels on cognitive outcomes demonstrated that the magnitude of the association with tibia lead levels was moderate to large, equivalent to 22\u201360% of the magnitude of the age effect in its relations with cognitive domain scores. Specifically, an interquartile range increase in tibia lead levels was equivalent to 2\u20136 more years of age at baseline across all seven domains (Shih et al. 2006).\nLongitudinal analyses in the NAS observed that an interquartile range higher patella lead level was approximately equivalent to that of aging 5 years in relation to the baseline MMSE score (Weisskopf et al. 2004) and an interquartile range higher bone (patella or tibia, depending on the specific cognitive outcome) lead level was approximately equivalent to that of aging 1 year in relation to the baseline test scores on a battery of cognitive tests (Weisskopf et al. 2007).\nSpecificity\nLead has adverse effects on many other health outcomes in addition to cognitive function. This is not surprising given lead\u2019s numerous biologic effects, including calcium agnonism and antagonism (Ferguson et al. 2000), binding to sulfhydryl and carboxyl groups on proteins, and activation of nuclear transcription factors (Ramesh et al. 2001), for example. It is thus not surprising that lead\u2019s toxicity is not specific to the brain and we do not believe this lack of target organ specificity diminishes the inference for a causal relationship between lead and cognitive dysfunction.\nTemporal relationship\nAssociations between lead biomarkers and cognitive outcomes have been demonstrated in both cross-sectional and longitudinal studies. In several of the longitudinal studies, change in cognitive function was explicitly modeled in relation to preceding lead dose or in relation to change in lead dose. In either case, the temporality condition is met. In addition, as bone lead is a measure that ascertains prior dose, even in cross-sectional analyses, analysis of bone lead with cognitive test scores evaluates lead dose that preceded current cognitive performance; thus, while cognitive assessment is cross-sectional, dose assessment is retrospective and cumulative. This again would minimize concerns about incorrect temporal relations.\nBiological gradient (dose\u2013effect relations)\nNearly all reviewed studies found a dose\u2013effect relation for blood lead, bone lead, or both. Existing studies do not allow determination of a threshold dose for either blood lead or bone lead or the shape of the dose\u2013effect relationship at low dose levels. Associations have been observed in populations with mean blood lead levels as low as 4.5 \u03bcg\/dL (Wright et al. 2003) and mean tibia lead levels as low as 18.7 \u03bcg\/g (Shih et al. 2006).\nBiologic plausibility and experimental data\nLead adversely affects the brain in a variety of ways. Lead is thought to increase oxidative stress, induce neural apoptosis, influence neurotransmitter storage and release, and damage mitochondria. The ability of lead to substitute for calcium allows it to affect calcium-mediated processes and pass through the blood\u2013brain barrier. It may also interfere with zinc-dependent transcription factors, altering the regulation of genetic transcription (Zawia et al. 2000). Animal studies indicate that the accumulation of lead in the brain is generally uniform (Widzowski and Cory-Slechta 1994), although the hippocampus and limbic system, prefrontal cerebral cortex, and cerebellum are clearly principal sites of the effects of lead (Finkelstein et al. 1998). Low lead levels in rats produce structural changes in the hippocampus (Cory-Slechta 1995), a brain region critical for learning and memory (Eichenbaum 2001), which is consistent with the finding of learning and memory deficits in lead-exposed individuals.\nBlood lead level is a measure of current biologically active lead burden and is therefore a better marker of the acute effects of recent lead dose. These are likely to be effects on neurotransmission and calcium enzyme-dependent processes such as synaptic plasticity. This could lead to circulating blood lead impairing, for example, information storage and retrieval mechanisms or processing speed, which have been suggested to impair performance on cognitive tests (Salthouse 1996a, 1996b). Lead levels in bone are a measure of cumulative dose over decades as well as a source of lead in the body that is available for mobilization into blood, especially during periods of increased bone turnover (e.g., pregnancy, puberty). Although lead stored in bone is not directly harmful to the brain, the cumulative effects of chronic lead exposure are likely to be related to oxidative stress and neuronal death and could impair cognitive function, for example, by reducing the capacity of specific regions to process information, or by impairing diffuse ascending projection systems such as the midbrain cholinergic and dopaminergic cells.\nLead may also influence cognitive function indirectly through its effects on blood pressure, hypertension, or homocysteine levels. Increased homocysteine levels, a well-known risk factor for cardiovascular disease, have also been associated with risk for poorer cognitive functioning (Dufouil et al. 2003; Schafer et al. 2005a) and risk for dementia (Hogervorst et al. 2002; McCaddon et al. 2003; Selley 2003). Homocysteine is neurotoxic to the central nervous system by influencing neurotransmitter synthesis, and causing excitotoxicity and cell death (McCaddon and Kelly 1992; Parnetti et al. 1997). Blood lead levels were associated with homocysteine levels as well, although the direction of causality has yet to be determined (Guallar et al. 2006; Schafer et al. 2005b). Both blood and bone lead levels have been linked with blood pressure and hypertension in community-based samples of older adults (Martin et al. 2006; Nash et al. 2003) and occupationally exposed populations (Glenn et al. 2003, 2006). Hypertension has also been identified as a potential risk factor for dementia (Birkenhager and Staessen 2006; Hayden et al. 2006; Skoog and Gustafson 2006). Thus, lead may indirectly play a role in cognitive declines by way of poor vascular health.\nWe believe the effect modification by APOE genotype offers strong biologic plausibility to the inference that lead causes cognitive dysfunction (Stewart et al. 2002). The APOE \u025b 4 allele is a risk factor for lateonset Alzheimier disease (Corder et al. 1993; Meyer et al. 1998; Saunders et al. 1993), hippocampal atrophy (Moffat et al. 2000), and senile plaques (Zubenko et al. 1994). It appears that the APOE \u025b 4 allele lowers the age of onset of the disease and accelerates age-related cognitive decline (Meyer et al. 1998). Mechanistically, APOE \u025b 4 is involved in the recovery response of injured nerve tissue (Poirier and Sevigny 1998), with the APOE \u025b 4 allele having reduced ability to promote growth and reduced antioxidant properties (Miyata and Smith 1996; Teter et al. 1999; Yankner 1996). The interaction of APOE genotype with tibia lead level may be related to an impaired ability to counteract injury from lead exposure among APOE \u025b 4 carriers.\nAnother recent study also offers biologic plausibility. In the former organolead workers, tibia lead level was associated with the prevalence and severity of white matter lesions on brain MRI, using the Cardiovascular Health Study white matter grading system (Stewart et al. 2006). Tibia lead level was also associated with smaller volumes on several regions of interest ranging from large (e.g., total brain volume, lobar gray and white matter volumes) to small (e.g., cingulate gyrus, insula, corpus callosum). As volume can decline because of changes in cell number, synaptic number or density, or other changes in cellular architecture, these findings reinforce evidence that lead may cause a persistent change in the brain that is associated with progressive declines in cognitive function.\nPublic health implications\nThe removal of lead from gasoline, paint, and most other commercial products has succeeded in dramatically reducing environmental sources of lead exposure, and this has been reflected by the parallel declines in mean blood lead levels in Americans over the same time frame. However, lead has accumulated in the bones of older individuals, and especially those of lead workers exposed at the continued higher levels encountered in lead-using workplaces. Thus, past use of lead will continue to cause adverse health effects even when current exposures to lead are much lower than in the past. Lead in bone is not directly harmful to the central nervous system, and most of the structural and neurochemical damage is likely to have occurred decades ago. Nevertheless, lead in bone might serve as a source from which lead can be mobilized into blood, and potentially cross the blood\u2013brain barrier. The chronic effects of lead may account for a proportion of cognitive aging; future research will be able to determine whether the chronic effects of cumulative lead dose alter the trajectory of normal cognitive aging. Research efforts should be directed to development of preventive interventions for both lead-associated cognitive decline with aging from past exposures, as well as the mobilization of current bone lead stores into the circulatory system leading to new health effects.\nCognitive aging occurs in conjunction with the normal biological aging process. It remains to be determined whether lead affects cognitive aging in adults by permanently reducing brain circuitry capacity thereby lowering baseline cognitive functioning, or by inducing steeper declines in cognitive functioning, leading to abnormal cognitive aging. It may be that lead influences cognitive health through its relationship with depressive symptoms, hypertension, or homocysteine levels, all of which influence cognitive impairment and risk of dementia. Future investigations should explicitly account for these complex causal pathways, and also determine whether chronic effects of cumulative lead dose increases the risk for such clinically relevant syndromes as mild cognitive impairment (Petersen et al. 1999).","keyphrases":["lead","cognitive function","adults","blood","bone","neurobehavior"],"prmu":["P","P","P","P","P","P"]}
{"id":"Eur_J_Pediatr-3-1-1820762","title":"ADAMTS13 phenotype in plasma from normal individuals and patients with thrombotic thrombocytopenic purpura\n","text":"The activity of ADAMTS13, the von Willebrand factor cleaving protease, is deficient in patients with thrombotic thrombocytopenic purpura (TTP). In the present study, the phenotype of ADAMTS13 in TTP and in normal plasma was demonstrated by immunoblotting. Normal plasma (n = 20) revealed a single band at 190 kD under reducing conditions using a polyclonal antibody, and a single band at 150 kD under non-reducing conditions using a monoclonal antibody. ADAMTS13 was not detected in the plasma from patients with congenital TTP (n = 5) by either antibody, whereas patients with acquired TTP (n = 2) presented the normal phenotype. Following immunoadsorption of immunoglobulins, the ADAMTS13 band was removed from the plasma of the patients with acquired TTP, but not from that of normal individuals. This indicates that ADAMTS13 is complexed with immunoglobulin in these patients. The lack of ADAMTS13 expression in the plasma from patients with hereditary TTP may indicate defective synthesis, impaired cellular secretion, or enhanced degradation in the circulation. This study differentiated between normal and TTP plasma, as well as between congenital and acquired TTP. This method may, therefore, be used as a complement in the diagnosis of TTP.\nIntroduction\nVon Willebrand factor (VWF) is a glycoprotein that plays a key role in the primary hemostatic process by inducing platelet adhesion and aggregation at sites of vascular injury under conditions of high shear stress. The main source of circulating VWF is the endothelium, from which it is secreted in the form of ultra-large multimers (ULVWF) [53]. ULVWF multimers are biologically very active [2, 31] and, upon release, undergo processing into smaller multimers in normal individuals. This occurs on the surface of endothelial cells [10]. VWF defects may potentially lead to both bleeding and thrombotic disorders: defective VWF secretion, intravascular clearance, multimer assembly, or increased proteolytic degradation may lead to different types of von Willebrand disease. On the other hand, dysfunctional VWF proteolysis may lead to the thrombotic disorder thrombotic thrombocytopenic purpura (TTP) [40].\nTTP is a thrombotic microangiopathy (TMA) characterized by microangiopathic hemolytic anemia, thrombocytopenia, fever, neurological and renal manifestations. Chronic recurrent TTP has been associated with the presence of ULVWF in the plasma [30]. ULVWF multimers are capable of inducing increased platelet retention in children with TTP [21]. These observations, along with the finding of VWF and platelet-rich (but fibrin-poor) thrombi in the microcirculation of the heart, brain, kidneys, liver, spleen, and adrenals in TTP patients [3], led to the conclusion that ULVWF multimers are responsible for the disseminated platelet thrombi occurring in TTP and that their degradation to smaller VWF multimers is impaired due to the deficiency of a VWF-cleaving protease [15].\nRecently, the VWF-cleaving protease was purified [12, 13, 16, 50] and the encoding gene sequenced, linking the protease to the ADAMTS (a disintegrin-like and metalloprotease with thrombospondin-type-1 motif) family of metalloproteases [27]. The protease, named ADAMTS13, cleaves VWF at the 1605Tyr-1606Met peptide bond in the A2 domain, yielding the 140-kD and 176-kD VWF fragments present in normal plasma [13, 50]. Cleavage is made possible by a conformational change in VWF due to shear stress in the circulation, which exposes the cleavage site, making it susceptible to proteolysis [55]. ADAMTS13 activity is severely deficient (<5% of normal plasma activity) in TTP patients [6], either due to a mutation in the ADAMTS13 gene in the congenital form of TTP or due to auto-antibodies in the acquired form [14, 27, 52]. Autosomal recessive hereditary TTP (also termed the Upshaw-Schulman syndrome) typically presents during the neonatal period or early childhood (<10\u00a0years of age), but may also manifest during adolescence and adulthood. Recurrent TTP episodes may occur as often as every third week. TTP recurrences are associated with cerebral vascular accidents in approximately 30% of cases, and these episodes may lead to neurological complications. Renal manifestations may be mild or may result in acute renal failure due to hemoglobinuria and TMA. About 20% of patients progress to end-stage renal failure [28].\nHemolytic uremic syndrome (HUS) is a similar microangiopathic disorder characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure [5]. Two forms of HUS have been described: D+ or typical (diarrhea-associated) HUS and D- or atypical (non-diarrhea-associated) HUS. D+ HUS occurs after infection with Shiga-like toxin producing bacteria, typically, enterohemorrhagic Escherichia coli. The patients are usually children presenting with abrupt onset of diarrhea, followed by the development of HUS 2\u201310\u00a0days later. A prothrombotic state precedes the acute renal failure [8], but the pathogenetic mechanism is, as yet, unclear. It is assumed that bacterial virulence factors gain access to the circulation, circulate on blood cells, activate platelets, and reach the kidney, where the endothelium is injured [36, 47]. D- HUS is associated with mutations in certain complement regulatory factors, such as factor H, factor I, and membrane co-factor protein (CD46). The mutations lead to activation of the complement system on host endothelial cells [29, 58]. The resulting vascular damage may lead to the formation of thrombotic lesions in the kidneys.\nAlthough HUS patients are, typically, young children with a history of diarrhea and acute renal failure, the clinical manifestations of HUS and TTP often overlap, making differentiation between the two syndromes based solely on clinical presentation difficult. ADAMTS13 antigen levels can differentiate between HUS and TTP, as they are severely deficient in patients with congenital TTP and normal to moderately reduced in HUS [51]. Assays for ADAMTS13 activity can, therefore, differentiate between TTP (congenital and acquired) and HUS [26, 48].\nSeveral ADAMTS13 assays are available today based on antigen detection and activity [15, 17, 25, 38], showing the presence of the protease (by enzyme-linked immunosorbent assay, ELISA) and its bioactivity in normal plasma and the lack of protease and activity in the plasma from patients with congenital TTP. These assays have also shown that patients with acquired TTP have auto-antibodies that neutralize the activity of ADAMTS13. The present study utilized a different method, immunoblotting, and two anti-ADAMTS13 antibodies against specific domains, to investigate the presence of ADAMTS13 antigen in normal plasma, TTP plasma (congenital and acquired), and in heterozygous carriers of ADAMTS13 mutations, demonstrating the presence of ADAMTS13 and its size in normal plasma, the lack thereof in congenital TTP, and auto-antibody-bound protease in acquired TTP.\nMaterials and methods\nSubjects\nCitrated plasma was available from patients with congenital (n\u2009=\u20095) and acquired (n\u2009=\u20092) TTP. The patient data are presented in Table\u00a01. The ADAMTS13 activity level was assayed as previously described [15, 17]. \nTable\u00a01Clinical and laboratory data regarding thrombotic thrombocytopenic purpura (TTP) patientsPatient no.SexAge at debut Age at sampling (years)Current age (years)Symptoms during episodesNo. of episodesADAMTS13 activity levelADAMTS13 mutationADAMTS13 inhibitor Reference1aM2\u00a0d1619Jaundice, hemolytic anemia, thrombocytopenia, macroscopic hematuria, pathological urinalysis, fever, neurological symptoms, elevated serum creatinine>5<5%4143insAbcNone [4, 20, 43]2aM5.3\u00a0y1718Jaundice, hemolytic anemia, thrombocytopenia, fever, neurological symptoms, pathological urinalysis>5<5%4143insAbcNone[4, 20, 43]3F20\u00a0m1523Hemolytic anemia, thrombocytopenia, hematuria, epileptic attacks, slightly elevated serum creatinine>5<5%P353Ld, P457LeNone[4]4M3\u00a0y79Hemolytic anemia, thrombocytopenia, purpura, pathological urinalysis4<5%P671Lf, 4143insANone[43]5M2\u00a0d3939Jaundice, hemolytic anemia, petechiae, thrombocytopenia, transitory neurological deficits and aphasia, elevated creatinine>5<5%4143insAbNone\u20136F54\u00a0y7075Recurrent hemolytic anemia and thrombocytopenia, reduced consciousness, pathological urinalysis>5<5%NA0.5\u00a0U\/ml\u20137F25\u00a0y4244Thrombocytopenia, hemolytic anemia, elevated creatinine during viral infection and pregnancy2<5%NA0.2\u00a0U\/ml\u2013aPatients 1 and 2 are siblingsbPatients 1, 2, and 5 are homozygous for the ADAMTS13 mutationc4143insA leads to a mutation in the second CUB domaindP353L is a mutation in the disintegrin-like domaineP457L is a mutation in the cysteine-rich domainfP671L is a mutation in the spacer domainNA: not assayed\nThe study also included the parents of patients 1\u20134. The parents of patients 1 and 2 are both heterozygous for the 4143insA mutation, and have protease activity levels of 20% (mother) and 50% (father), as assayed by the VWF multimeric structure analysis [15]. The parents of patient 3 are heterozygous for the P353L (mother) and P457L (father) mutations, and both have 50% ADAMTS13 activity. The parents of patient 4 are heterozygous for P671L (mother) and 4143insA (father), and have 50% ADAMTS13 activity. All parents are clinically unaffected.\nPlasma samples from 20 healthy adult volunteers were used as controls. The study was conducted with the approval of the ethics committee of Lund University and the plasma samples were collected with the informed consent of the patients, their parents, and the controls.\nPlasma samples\nAt the time of sampling, the patients were treated regularly with fresh frozen plasma or Octaplas (Octapharma, Stockholm, Sweden; patients 1\u20135). Patient 6 was treated with plasma infusions every sixth week. Patient 7 did not receive any plasma treatment at sampling. All blood samples were obtained at least three weeks (patients 1\u20134 and 6) or one week (patient 5) after the last treatment. Venous blood from patients and controls was collected, and the plasma obtained as previously described [20].\nAnti-ADAMTS13 antibodies\nA polyclonal anti-peptide antibody was raised in New Zealand white rabbits against a unique sequence in the second CUB domain (AA1413-1427) and affinity-purified against the peptide. Antibody specificity was tested by ELISA (plates coated with the peptide) and by immunoblotting with purified plasma ADAMTS13 [50] under reducing (SDS-PAGE) and non-reducing (dot blot) conditions.\nThe monoclonal antibody A10 [56], directed against the disintegrin-like domain, was used to confirm the results of the polyclonal antibody. The polyclonal antibody reacted with ADAMTS13 under reducing (reduced by the addition of 2-mercaptoethanol to disrupt disulfide bonds in the protease) and non-reducing conditions, whereas the monoclonal antibody reacted with ADAMTS13 only under non-reducing conditions.\nImmunoblot analysis for the detection of ADAMTS13 in plasma\nThe plasma samples (1:20) were subject to SDS-PAGE under reducing (for blotting with the polyclonal antibody) and non-reducing conditions (for blotting with the monoclonal antibody) [19, 58]. Purified plasma ADAMTS13 (1:100) was used as the control for the polyclonal antibody. Immunoblotting was performed with rabbit anti-ADAMTS13 IgG 1.6\u00a0\u03bcg\/ml followed by goat anti-rabbit IgG HRP (DakoCytomation, Carpinteria, CA) 1:2000, or with mouse anti-ADAMTS13 IgG 0.6\u00a0\u03bcg\/ml followed by goat anti-mouse IgG HRP (DakoCytomation, Carpinteria, CA) 1:2000. The signal was detected by chemiluminescence. The specificity of the signal obtained with the polyclonal antibody was tested by preincubation with a 50-fold molar surplus of blocking peptide followed by immunoblotting with the blocked antibody. The specificity of the secondary antibodies was tested by omission of the primary antibodies.\nImmunoblotting with the polyclonal antibody revealed, in addition to ADAMTS13, two unspecific bands at 130\u00a0kD and 170\u00a0kD, which were identified as C3 and alpha-2-macroglobulin. These proteins were removed by incubating the plasma samples with protein A-sepharose coupled rabbit anti-C3 IgG and rabbit anti-alpha-2-macroglobulin IgG. The results obtained using the polyclonal antibody show samples from which these proteins have been removed.\nIn order to investigate the presence of ADAMTS13-autoantibody complexes in the plasma from patients with acquired TTP, samples were passed onto protein G-sepharose (Amersham Biosciences, Buckinghamshire, UK) prior to immunoblotting. Normal plasma (n\u2009=\u20092) was used for comparison.\nResults\nDetection of ADAMTS13 in plasma samples from normal individuals\nNormal plasma under reducing conditions revealed a single immunoreactive band at 190\u00a0kD when blotted against the polyclonal antibody (Fig.\u00a01a). Purified plasma ADAMTS13 showed a similar band under the same conditions (Fig.\u00a01a). The monoclonal antibody detected an immunoreactive band at 150\u00a0kD in normal plasma under non-reducing conditions (Fig.\u00a01b). Preincubation of the polyclonal antibody with the blocking peptide abolished the ADAMTS13 band in normal plasma (data not shown). Immunoblots in which the primary antibodies had been omitted showed no bands (data not shown).\nFig.\u00a01a\u2013gDetection of ADAMTS13 in normal and thrombotic thrombocytopenic purpura (TTP) plasma. a The polyclonal antibody detected a single band at 190\u00a0kD in normal plasma (NP) under reducing conditions. Purified plasma ADAMTS13 in the right lane showed a similar band. b Normal plasma under non-reducing conditions revealed an immunoreactive band at 150\u00a0kD when blotted against the monoclonal antibody. c Immunoblotting with the polyclonal antibody revealed that patients 1\u20135 with congenital TTP all lacked the ADAMTS13 band, whereas patients 6 and 7 with acquired TTP presented a normal expression pattern. Normal plasma (NP) was run on the same gel for comparison. d These results were confirmed by the monoclonal anti-ADAMTS13 antibody. e Immunoblot using the polyclonal antibody. Immunoadsorption of immunoglobulins from the plasma samples of patients 6 and 7 with acquired TTP lead to the simultaneous removal of the ADAMTS13 band, indicating that ADAMTS13 is complexed with the anti-ADAMTS13 auto-antibodies. In contrast, the ADAMTS13 band remained visible in normal plasma (NP) treated similarly. f A schematic presentation of the mechanism by which the removal of immunoglobulins leads to the removal of the ADAMTS13 antigen from the plasma of patients with acquired TTP. The plasma sample contains ADAMTS13 (filled circles), auto-antibodies to ADAMTS13 (\u2919), and various other plasma proteins (open circles). Immunoblotting of the plasma sample prior to the removal of immunoglobulins detects the presence of ADAMTS13 antigen. Passage of the plasma sample through a protein G-sepharose column leads to the binding and removal of all immunoglobulins from the sample. Since ADAMTS13 is bound to the anti-ADAMTS13 auto-antibodies, it is removed along with them. Immunoblotting of the flow-through shows no ADAMTS13 band. g Immunoblot with the monoclonal antibody showing a normal ADAMTS13 band in the plasma of the parents, which are all heterozygous for one mutated allele and are clinically unaffected. Lane 1: the mother of patients 1 and 2, 4143insA; lane 2: the mother of patient 3, P353L; lane 3: the father of patient 3, P457L; lane 4: the mother of patient 4, P671L; lane 5: the father of patient 4, 4143insA. Normal plasma (NP) was run on the same gel for comparison\nDetection of ADAMTS13 in plasma samples from TTP patients\nThe plasma samples from all patients with congenital TTP, regardless of the mutation, lacked the ADAMTS13 band (Fig.\u00a01c). Patients 6 and 7 with acquired TTP revealed the same ADAMTS13 protein expression pattern as the controls (Fig.\u00a01c). These results were obtained using both the polyclonal (Fig.\u00a01c) and the monoclonal antibody (Fig.\u00a01d). When immunoadsorption of plasma immunoglobulins was carried out prior to immunoblotting, the ADAMTS13 band remained visible in normal plasma, but was completely removed from the samples of the patients with acquired TTP (Fig.\u00a01e), probably due to its association with the immunoglobulin inhibitor.\nDetection of ADAMTS13 in the parents of the TTP patients\nThe parents of the TTP patients are all carriers of one ADAMTS13 mutation and one normal allele. All parents presented a normal ADAMTS13 phenotype using both the polyclonal (data not shown) and the monoclonal antibody (Fig.\u00a01g).\nDiscussion\nIn the present study, we detected ADAMTS13 in plasma using a polyclonal and a monoclonal antibody. This assay was capable of distinguishing TTP patients from normal individuals, as well as differentiating between congenital and acquired TTP. Plasma from the patients with congenital TTP lacked the ADAMTS13 antigen. In contrast, the plasma of patients with acquired TTP expressed a normal ADAMTS13 phenotype.\nPrevious studies describing the ADAMTS13 phenotype in normal plasma by immunoblotting with other specific anti-ADAMTS13 antibodies have shown immunoreactive bands of the same molecular weight using similar conditions [33, 46]. The ADAMTS13 antigen in patients with congenital and acquired TTP has recently been shown by ELISA, demonstrating low to undetectable ADAMTS13 levels in patients with congenital TTP [11, 38] and decreased, but mostly detectable, levels in patients with acquired TTP [11, 38, 45]. In the present study, the ADAMTS13 phenotype in TTP patients is described by immunoblotting, confirming the lack of ADAMTS13 antigen in the plasma of patients with congenital TTP and the presence of circulating complexes in acquired TTP. Furthermore, we showed that heterozygous carriers of the ADAMTS13-related mutations who, thus, have reduced ADAMTS13 bioactivity have a normal phenotype.\nThe plasma of the patients with congenital TTP did not present the ADAMTS13 band. This may be due to altered synthesis, secretion or antigenicity, or due to increased breakdown of the protease in plasma. The fact that two antibodies directed to two different domains in ADAMTS13 were unable to detect the protease band makes altered antigenicity less likely to be the cause for the lack of the ADAMTS13 band in these patients. Previous studies have shown impaired secretion of the 4143insA (patients 1, 2, 4, and 5) [35, 42, 44] and P353L (patient 3) [42] mutants from cells, thus, indicating that the protease may accumulate intracellularly, at least in some patients with congenital TTP. This may be due to a missing cell sorting signal, as in the case of 4143insA [44] or due to conformational changes in the protein, impairing its secretion. Similar findings regarding two other ADAMTS13 mutations, V88M and G239V, have been recently reported [34]. A total lack of ADAMTS13 activity in the plasma is thought to be incompatible with life [27], thus, the patients may have very low amounts of ADAMTS13 activity in their plasma, which we were unable to detect with this method.\nThe patients with acquired TTP presented with a normal ADAMTS13 band, which is consistent with the fact that ADAMTS13 protease genotype and expression are normal, but their activity is lower, due auto-antibodies [11, 14, 41, 45, 52]. The finding that the immunoadsorption of immunoglobulins from the plasma of the patients with acquired TTP also led to the removal of the ADAMTS13 antigen from their samples indicates that the protease is complexed with the auto-antibodies in the circulation of these patients, and that this occurs even during clinical remission.\nThe majority of the ADAMTS13 assays available today detect ADAMTS13 activity levels in plasma (Table\u00a02). This is performed either by detecting the VWF products resulting from ADAMTS13 cleavage (assays 1\u20135) or by measuring the residual VWF activity (assays 6\u20137). The VWF substrate utilized in these assays may be high molecular weight VWF (plasma-derived or recombinant; assays 1\u20134) or VWF domains or short synthetic peptides (assay 5). Two multicenter studies evaluating methods 1, 3\u20135, and 6\u20137 showed that all assays were able to detect severely ADAMTS13-deficient plasma samples and indicated that methods 1, 6, and 7 were the most consistent and reliable methods [48, 49]. A recent smaller study evaluating the FRETS-VWF73 method (assay 5) showed that this is a reliable assay which provides results in good accordance with other methods [26]. \nTable\u00a02ADAMTS13 assaysASSAYPRINCIPLEREFERENCEDetection of ADAMTS13 activityDetection of VWF cleavage products 1. VWF multimer structure analysisDetection of the breakdown of high-molecular-weight VWF[15, 23] 2. Immunoblotting of VWF Detection of cleavage products of native VWF or recombinant VWF domains[37, 50] 3. IRMADetection of VWF cleavage products[32] 4. Flow assayDetection of the breakdown of ULVWF-platelet strings attached to endothelial cells[1] 5. Various methods using VWF domains or short synthetic VWF  peptides as the substrate, such as the FRETS-VWF73 assayDetection of cleavage products of the VWF domains or VWF peptides[9, 18, 22, 25, 59\u201361]Detection of VWF residual activity 6. Collagen bindingDetection of VWF binding to collagen; binding correlates to VWF multimer size[17] 7. Ristocetin cofactor activityDetection of platelet aggregates; VWF ability to induce platelet aggregates in the presence of ristocetin correlates to multimer size[7]Detection of ADAMTS13 antigen and auto-antibodies 8. ELISADetection of ADAMTS13 antigen or anti-ADAMTS13 auto-antibodies[11, 38, 39, 41, 54] 9. ImmunoblottingDetection of anti-ADAMTS13 auto-antibodies[57] 10. Present assay (immunoblotting)Detection of ADAMTS13 antigen and size and (indirectly) of auto-antibodies\u2013Mutation analysis 11. PCRDetection of mutations in the ADAMTS13 gene[27]IRMA: immunoradiometric assay; FRET: fluorescence resonance energy transfer; FRETS-VWF73: a 73-amino-acid-long synthetic peptide which provides a minimal substrate for ADAMTS13 [24] that has been made fluorogenic; PCR: polymerase chain reaction\nFew recent studies have shown that ADAMTS13 antigen was detectable in plasma by ELISA (assay 8 in Table\u00a02). ELISA assays are a valuable complement to the ADAMTS13 activity assays, and offer a fast analysis of the ADAMTS13 antigen levels in plasma samples using antibodies directed to ADAMTS13. These assays are able to distinguish ADAMTS13-deficient TTP plasma from normal plasma samples. Other methods are capable of detecting anti-ADAMTS13 auto-antibodies (Table\u00a02, assays 8\u20139). Mutational analysis can be carried out by polymerase chain reaction (PCR) in order to detect mutations in the ADAMTS13 gene (assay 11 in Table\u00a02).\nIn the present study, we developed a qualitative, semi-quantitative assay capable of detecting ADAMTS13 antigen and anti-ADAMTS13 auto-antibodies in plasma and, thus, distinguishing between TTP and normal plasma, as well as distinguishing between congenital and acquired TTP. All patients with congenital TTP, regardless of their ADAMTS13 mutations, presented with undetectable levels of ADAMTS13 antigen, whereas patients with acquired TTP presented a normal phenotype. Heterozygous carriers of ADAMTS13 mutations also revealed a normal ADAMTS13 antigen band. Although not a quantitative method like the above-mentioned ELISA assays, the present assay offers the advantage of not only demonstrating the presence or absence of ADAMTS13 antigen, but also the molecular size of ADAMTS13 present in plasma samples. Furthermore, this assay was able to show the presence of anti-ADAMTS13 antibodies indirectly by performing immunoadsorption of plasma immunoglobulins prior to immunoblotting. In conclusion, this assay is able to distinguish between normal and TTP plasma, and also between congenital and acquired TTP. It is easy to perform and can be used at any hospital laboratory routinely using SDS-PAGE electrophoresis and immunoblotting, and offers a complement to existing ADAMTS13 methods in the diagnosis of TTP.","keyphrases":["adamts13","plasma","thrombotic thrombocytopenic purpura","von willebrand factor","von willebrand factor cleaving protease","immunoblotting"],"prmu":["P","P","P","P","P","P"]}
{"id":"Pediatr_Nephrol-3-1-1805051","title":"Iron therapy for renal anemia: how much needed, how much harmful?\n","text":"Iron deficiency is the most common cause of hyporesponsiveness to erythropoiesis-stimulating agents (ESAs) in end-stage renal disease (ESRD) patients. Iron deficiency can easily be corrected by intravenous iron administration, which is more effective than oral iron supplementation, at least in adult patients with chronic kidney disease (CKD). Iron status can be monitored by different parameters such as ferritin, transferrin saturation, percentage of hypochromic red blood cells, and\/or the reticulocyte hemoglobin content, but an increased erythropoietic response to iron supplementation is the most widely accepted reference standard of iron-deficient erythropoiesis. Parenteral iron therapy is not without acute and chronic adverse events. While provocative animal and in vitro studies suggest induction of inflammation, oxidative stress, and kidney damage by available parenteral iron preparations, several recent clinical studies showed the opposite effects as long as intravenous iron was adequately dosed. Thus, within the recommended international guidelines, parenteral iron administration is safe. Intravenous iron therapy should be withheld during acute infection but not during inflammation. The integration of ESA and intravenous iron therapy into anemia management allowed attainment of target hemoglobin values in the majority of pediatric and adult CKD and ESRD patients.\nIntroduction\nIron-restricted erythropoiesis is a common clinical condition in patients with chronic kidney disease (CKD). The causes underlying this pathology and the subsequent contribution of absolute or functional iron deficiency to renal anemia include: \nInadequate intake of dietary ironBlood loss during the extracorporeal procedure in hemodialysis patientsBlood loss from the gastrointestinal tract (bleeding)(Too) frequent diagnostic blood testsInadequate intestinal iron absorption and inhibition of iron release from macrophages (anemia of chronic disease)Increased iron requirements during therapy with erythropoiesis-stimulating agents (ESAs).\nIn iron deficiency without anemia, reduction in iron storage is not sufficiently large enough to decrease the hemoglobin level. In CKD patients with absolute iron-deficient anemia, however, iron deficit is so severe that it aggravates renal anemia. Iron supplementation is mandatory in the majority of patients with end-stage renal disease (ESRD), particularly in those receiving ESA therapy.\nEvaluation of iron status\nIn patients with normal kidney function, absolute iron deficiency is characterized by low serum ferritin concentration (<30\u00a0\u03bcg\/l). The ferritin cut-off level for absolute iron deficiency in CKD patients is 100\u00a0\u03bcg\/l [1] by the experience that chronic inflammation increases serum ferritin levels approximately three-fold. The Kidney Disease Outcomes Quality Initiative (K\/DOQI) guidelines recommend serum ferritin levels >200\u00a0\u03bcg\/l for the adult hemodialysis patient population [2]. The European Best Practice Guidelines define the optimal range for serum ferritin as 200\u2013500\u00a0\u03bcg\/l in adult patients with ESRD [1]. A normal ferritin level (\u2265100\u00a0\u03bcg\/l) cannot exclude iron deficiency in uremic children [3, 4], but a serum ferritin <60\u00a0\u03bcg\/l is a specific predictor of its presence [5]. An upper ferritin level of 500\u00a0\u03bcg\/l is recommended for adults and children with CKD [2]. Serum ferritin is an indicator of storage iron. Iron deficiency is accompanied by reductions in serum iron concentration and transferrin saturation (TSAT) and by elevations in red cell distribution width, free erythrocyte protoporphyrin concentration, total serum iron binding capacity (TIBC), and circulating transferrin receptor [6]. Serum soluble transferrin receptor, however, reflects ongoing erythropoiesis but not iron availability in ESA-treated chronic dialysis patients [7]. Typically, TSAT (the ratio of serum iron to TIBC) is 15% or less (normal 16\u201340%) with iron deficiency, but TSAT also decreases in the presence of acute and chronic inflammation (functional iron deficiency). TSAT is raised with bone marrow dysfunction due to alcohol, cancer chemotherapy, or a megaloblastic process. TSAT is also affected by diurnal variations, being higher in the morning and lower in the evening [8]. Even a TSAT\u2009>\u200920% or a serum ferritin level\u2009>\u2009200\u00a0\u03bcg\/l does not exclude iron deficiency in ESRD patients. In a study by Chuang et al. [9], 17% of iron-deficient hemodialysis patients had serum ferritin levels greater 300\u00a0\u03bcg\/l. Clinically, functional iron deficiency is confirmed by the erythropoietic response to a course of parenteral iron and is excluded by the failure of erythroid response to intravenous iron administration [10].\nErythrocyte and reticulocyte indices, such as the percentage of hypochromic red blood cells and the reticulocyte hemoglobin content (CHr) provide direct insight into bone marrow iron supply and utilization. Determination of the percentage of hypochromic red blood cells, i.e., those with a cellular hemoglobin concentration <28\u00a0g\/dl, provides important information on functional iron deficiency in ESA-treated dialysis patients [11]. Tessitore et al. [12] found that hypochromic red blood cells >6% are the best marker to identify adult ESRD patients who will have the best response to intravenous iron. CHr has been proposed as a surrogate marker of iron status and as an early predictor of response to iron therapy in adult dialysis patients [13, 14]. Combined use of CHr and high-fluorescence reticulocyte count predicts with a very high sensitivity and specificity the response to intravenous iron in adult dialysis patients [8]. There are, however, only few studies in the pediatric renal literature on the use of CHr [15, 16]. In children with ESRD, an increase from baseline CHr levels was observed in response to oral and intravenous iron, but cut-off values for the use of CHr in the pediatric CKD population are not clear. This measure has proven to be of value with adult ESRD patients.\nDetection of both absolute and functional iron deficiency is important because iron deficiency is the most common cause of hyporesponsiveness to ESAs. In clinical practice, an increased erythropoietic response to iron supplementation is the most widely accepted reference standard of iron-deficient erythropoiesis. For pharmacological therapy of iron deficiency, both oral and parenteral iron preparations are available. Intravenous iron is more effective than oral iron supplementation, at least in CKD patients. Iron is not only a prerequisite for effective erythropoiesis but also an essential element in all living cells. Elemental iron serves as a component of oxygen-carrying molecules and as a cofactor for enzymatic processes. Its redox potential, however, limits the quantity of iron that can be safely harbored within an individual.\nOral iron therapy\nOral iron is best absorbed if given without food. Side-effects of oral iron therapy include constipation, diarrhea, nausea, and abdominal pain. In the treatment of iron deficiency with ferrous sulphate, the usual adult dose is one 300\u00a0mg tablet (containing 60\u00a0mg elemental iron) three to four times daily. The pediatric dose is 2\u20136\u00a0mg\/kg per day of elemental iron in 2\u20133 divided doses [17, 18]. Intestinal iron absorption is enhanced in patients with iron deficiency and declines with the correction of iron deficiency and reaccumulation of iron stores. If side-effects limit compliance, the medication can be administered with food, or the dose can be reduced. One 500-mg ferrous sulphate dose nightly at bedtime may be an effective therapy in adults [19].\nUremia is a chronic inflammatory state [20, 21]. Even in the absence of overt infection or inflammation, many ESRD patients show increased levels of acute-phase proteins, such as C-reactive protein (CRP), ferritin, fibrinogen, and\/or interleukin-6 (IL-6), associated with low serum albumin levels [22]. The interaction of proinflammatory cytokines with hepcidin in mediating functional iron deficiency may explain why CKD patients have high ferritin levels, poor intestinal iron absorption, and disturbed iron release from the reticuloendothelial system [23].\nIn the duodenum and proximal jejunum, the nonheme dietary Fe3+ is reduced to Fe2+ by the cytochrome b-like ferrireductase Dcytb. Fe2+ is gathered from the lumen of the intestine and crosses the apical enterocyte brush border membrane through the divalent metal transporter-1 (DMT1). The expression of both Dcytb and DMT1 is strongly affected by the iron concentration within the enterocyte. Circulating levels of hepcidin negatively regulate intestinal iron absorption by the enterocyte DMT1. Hypoxia, anemia, iron deficiency, and\/or stimulated erythropoiesis strongly down-regulate hepatic hepcidin release, allowing intestinal iron absorption, while iron overload or inflammation\/infection stimulates hepcidin production, resulting in inhibition of intestinal iron absorption. Hepcidin controls the whole-body iron content. It also inhibits the release of iron by the iron exporter ferroportin (iron-regulated transporter-1) located along the entire basolateral membrane of enterocytes and also in the intracellular vesicular compartment of tissue macrophages. Hepcidin is primarily produced in the liver in response to acute-phase reactions. Any further expression depends on the degree of hepatic iron storage (for review, see [24]). Thus, the inflammatory state associated with uremia and less uremia per se is predominantly responsible for poor intestinal iron absorption in ESRD patients. In iron-depleted peritoneal dialysis patients with normal CRP values, high-dose oral iron is well absorbed [25].\nThe European Pediatric Peritoneal Dialysis Working Group recommended that anemia treatment should aim for a target hemoglobin concentration of at least 11\u00a0g\/dl accomplished by administration of ESA and iron. Oral iron should be preferred in pediatric peritoneal dialysis patients [18]. The majority of pediatric hemodialysis patients are also supplemented with oral iron. The 2001 North American Pediatric Renal Transplant Cooperative Study (NAPRTCS) annual report showed that 84% of pediatric peritoneal dialysis and 72% of pediatric hemodialysis patients at 12\u00a0months of dialysis were receiving oral iron therapy [26].\nIntravenous iron therapy\nSince oral iron therapy is often not sufficient in ESRD patients, parenteral administration of iron is necessary to optimally care for these patients. Intravenous iron can be given safely to CKD patients [27\u201334] as long as the therapy is performed according to international recommendations and guidelines [1, 2]. This therapy is unequivocally superior to oral iron supplementation [35]. All forms of intravenous iron may be associated with acute adverse events [1, 2]. Potential risk factors associated with intravenous iron therapy include acute allergic reactions such as rash, dyspnoea, wheezing, or even anaphylaxis, as well as long-term complications caused by the generation of powerful oxidant species, initiation and propagation of lipid peroxidation, endothelial dysfunction, propagation of vascular smooth muscle cell proliferation, and\/or inhibition of cellular host defense. Allergy is believed to relate to dextran moiety. Iron dextran therapy is associated with a higher risk for serious type I reactions compared with newer intravenous iron products. Iron sucrose carries the lowest risk for hypersensitivity reactions [36]. In our clinical experience with more than 100,000 intravenous injections of iron sucrose and ferric gluconate within the last 15\u00a0years, we detected no significant differences in efficacy or adverse events between both intravenous iron preparations. Serious reactions to iron dextran are unpredictable and possibly life threatening. Labile- or free-iron reactions are more frequent with nondextran forms [37]. Recommended doses of iron sucrose or ferric gluconate appear safe, at least in adult CKD patients [32, 34, 38]. Parenteral therapy with iron sucrose or ferric gluconate is also safe and effective in the management of anemia in adult hemodialysis patients sensitive to iron dextran [29, 39]. Iron sucrose safety data are sparse in the pediatric CKD literature [2]. It should be considered that the iron load administered intravenously to CKD patients based on international recommendations is more than ten times less than the iron load by repeated blood transfusions at times when no ESA therapy was available for ESRD patients.\nIron deficiency in CKD patients develops primarily during the correction of renal anemia by ESA treatment. Approximately 150\u00a0mg of iron is necessary for an increase of 1\u00a0g\/dl in hemoglobin level. In adult hemodialysis patients, annual blood losses up to 4\u00a0l of blood, equivalent to 2\u00a0g iron, should be considered [40]. Thus, intravenous iron prevents iron-restricted erythropoiesis during ESA therapy. Parenteral treatment strategies depend on the availability of iron products in respective countries. Hemodialysis patients should receive at least one dose of intravenous iron every 2\u00a0weeks [1]. Careful monitoring of iron status is mandatory in order to avoid iron overload. In patients with anemia of chronic disease (and inflammation), a major part of iron administered intravenously is transported into the reticuloendothelial system, where it is not readily available for erythropoiesis [41].\nIntravenous iron therapy is underused in pediatric ESRD patients. Chavers et al. [42] compared anemia prevalence in US Medicare pediatric and adult dialysis patients treated with ESAs from 1996 to 2000. Prevalence of anemia (defined as hemoglobin values less than 11\u00a0g\/dl) was found in pediatric and adult hemodialysis patients during 54.1% versus 39.8% patient years as well as in pediatric and adult peritoneal dialysis patients during 69.5% versus 55.1% patient years, respectively. The percentage of patient years with intravenous iron was low, especially for pediatric peritoneal dialysis patients: 33.9% (age group 0\u20134\u00a0years) and 71% (age group 5\u201319\u00a0years) versus 0.3% and 19.4% among pediatric hemodialysis patients in these age categories, respectively. Among pediatric hemodialysis and peritoneal dialysis patients, intravenous iron was not administered among 34% and 85% of patient years [42]. Data obtained from the US Centers for Medicare and Medicare Services on hemodialysis patients in an age range between 12 and <18\u00a0years indicate that 37% of these patients are anemic, defined as hemoglobin <11\u00a0g\/dl. Dialyzing <6\u00a0months, a low serum albumin, and a mean TSAT <20% were identified as predictors of anemia in these children. Despite the prescription of iron supplements in almost all pediatric patients, there was evidence for low TSAT and\/or low ferritin in many children. In this study, approximately 60% of all children received intravenous iron therapy [43].\nAn international multicenter study investigated the safety and efficacy of two dosing regimens (1.5\u00a0mg\/kg or 3\u00a0mg\/kg) of ferric gluconate during eight consecutive hemodialysis sessions in iron-deficient pediatric hemodialysis patients receiving concomitant ESA therapy. Efficacy and safety profiles were comparable, with no unexpected adverse events with either dose [16]. Initial recommended ferric gluconate therapy is 1.5\u00a0mg\/kg for eight doses for iron-deficient pediatric hemodialysis patients and 1\u00a0mg\/kg per week for iron-replete pediatric hemodialysis patients, with subsequent dose adjustments made according to TSAT and\/or ferritin levels [16, 44]. In children, iron sucrose in a high dose (5\u00a0mg\/kg) should be given over 90\u00a0min and in a low dose (up to 2\u00a0mg\/kg) over 3\u00a0min [45]. An other recommendation for pediatric patients is to inject 6\u00a0mg iron\/kg per month during iron deficiency, with subsequent dose adjustments according to serum ferritin [46]. Nonrandomized intravenous iron (1\u20134\u00a0mg\/kg per week) trials in children on hemodialysis and nondialyzed or transplanted children showed an increase in hemoglobin or hematocrit and a decrease in ESA requirements between 5% and 62% per week or per dose of ESA [3, 4, 47, 48]. De Palo et al. [49] reported an excessive increase in hemoglobin with severe hypertension in children on maintenance hemodialysis after the first month of darbepoetin alpha therapy combined with intravenous ferric gluconate in a very high dose of 10\u201320\u00a0mg\/kg per week. The patients had already been on erythropoietin therapy for at least 6\u00a0months with adequate iron status (serum ferritin 220\u2009\u00b1\u2009105\u00a0\u03bcg\/l; TSAT 24.2\u2009\u00b1\u200911.5%). The complications observed in this study are not surprising, as such a high-dosed intravenous iron therapy is not justified, either in adult or in pediatric dialysis patients with \u201cadequate iron status\u201d. The current practice of intravenous iron therapy in pediatric hemodialysis patients is often performed on extrapolation from adult data and not based on data obtained from prospective multicenter trials performed in children [50].\nEven if exposure to intravenous iron may lead to oxidative stress, renal injury, infection, and\/or cardiovascular disease, the magnitude of these complications is not really clear. The overall risk\u2013benefit ratio favors the use of intravenous iron in CKD patients in order to optimize erythropoiesis and prevent iron deficiency [51]. Intravenous iron therapy is still underutilized in the adult hemodialysis population [52], but its use increased from 1997 to 2002. Ferric gluconate and iron sucrose have become the predominant form of intravenous iron therapy [53].\nIron and inflammation\/infection\nIntravenous iron therapy may adversely impact CKD patients via a potentiation of systemic inflammation. In animals, even a single ultra-high-dosed intravenous injection of available iron preparations (2\u00a0mg iron for mice with a body weight of 25\u201335\u00a0g, corresponding to 5,000\u00a0mg iron for a 75-kg adult patient) does not independently raise plasma levels of tumour necrosis factor-\u03b1 (TNF-\u03b1). Systemic inflammation experimentally induced by intraperitoneal endotoxin injection (2 or 10\u00a0mg\/kg in mice) resulted in a dramatic increase in plasma TNF-\u03b1 levels. Interestingly, 2\u00a0h following concomitant injection of endotoxin and ferric gluconate (2\u00a0mg) or iron dextran (2\u00a0mg), a decrease of plasma TNF-\u03b1 levels was observed. In contrast, combined endotoxin and iron sucrose injection resulted in a further increase in plasma TNF-\u03b1 compared with endotoxin alone [54]. However, a 75-kg CKD patient will neither receive 5,000\u201325,000\u00a0mg endotoxin intraperitoneally nor concomitant 5,000\u00a0mg iron sucrose injection intravenously in order to demonstrate that under these artificial conditions, TNF-\u03b1 mRNA and TNF-\u03b1 release are stimulated. It is therefore of particular importance that relevant clinical studies demonstrated that intravenous iron sucrose therapy within recommended doses may even display anti-inflammatory effects [55, 56].\nIntravenous iron sucrose therapy affects positively circulating cytokine levels in hemodialysis patients: IL-4 levels increase, while TNF-\u03b1 levels decrease [55]. There is a direct correlation between IL-4 and TSAT but an inverse correlation between TNF-\u03b1 and TSAT. Hemoglobin levels increase with an increase of IL-4 and a decrease of TNF-\u03b1, while ESA dose decreases with an increase of IL-4 and a decrease of TNF-\u03b1 [55]. In other words, adequately dosed intravenous iron therapy in hemodialysis patients results in down-regulation of proinflammatory immune effector pathways and stimulation of the expression of the anti-inflammatory cytokine IL-4. By these mechanisms, in addition to its well-known stimulatory effects on erythropoiesis, iron therapy contributes to an increase in hemoglobin levels and to a decrease in the need of ESAs. The anti-inflammatory properties of intravenous iron therapy have also been demonstrated in patients with rheumatoid arthritis [56].\nIn contrast, iron-mediated weakening of the Th-1 immune effector function (estimated by lowered TNF-\u03b1 production) with a subsequent strengthening of Th-2-mediated immune effector function (estimated by increased IL-4 production) is an unfavorable condition for ESRD patients in the case of an acute infection or malignant disease [55]. Moreover, intravenous administration of iron increases the availability of this essential nutrient for microorganisms [57] associated with an increased incidence of infectious complications in ESRD patients. Teehan et al. [58] followed 132 hemodialysis patients for up to 1\u00a0year after the initiation of intravenous iron therapy for the outcome of bacteremia. Iron-replete patients (those with a TSAT value\u2009\u2265\u200920% and a ferritin level\u2009\u2265\u2009100\u00a0ng\/ml) had a significantly higher risk of bacteremia (hazard ratio 2.3 in the univariate analysis and 2.5 in the multivariate analysis) compared with adult hemodialysis patients who were not iron replete [58]. Inhibition of intracellular killing of bacteria by polymorphonuclear leukocytes (PMNL) due to iron sucrose therapy in high-ferritin hemodialysis patients has been reported [59]. Peritoneal dialysis patients receiving high-dose intravenous iron sucrose also displayed short-term inhibition of bacterial killing by PMNL [60]. Finally, iron sucrose as well as ferric gluconate inhibit in vitro migration of PMNL through endothelial cells [61]. All these data suggest a risk for infectious complications, at least in patients overtreated with iron.\nHowever, clinical studies on intravenous iron therapy in ESRD patients reported controversial results [62\u201364]. According to the published cohort study, among 32,566 hemodialysis patients, there was no association between iron administration and mortality. This study by Feldman and coworkers [64] supports intravenous administration of iron \u22651,000\u00a0mg over 6\u00a0months if needed to maintain target hemoglobin levels. This is, however, an adult and not a pediatric recommendation. Intravenous iron therapy should be withheld in the presence of acute infection until the infection has successfully been treated and resolved [65]. Intravenous iron is ineffective and may increase the virulence of bacterial and viral pathogens. On the other hand, ESRD patients with chronically infectious complications may develop absolute iron deficiency if iron supplementation is withheld over months. In such a situation, iron should be administered intravenously as soon as ferritin levels drop below 100\u00a0\u03bcg\/l (personal opinion). ESA-stimulated erythropoiesis of chronically infected adult ESRD patients may benefit from low-dose intravenous iron supplementation (10\u201320\u00a0mg iron sucrose or ferric gluconate per hemodialysis session), even if serum ferritin is normal or slightly elevated. The level of serum ferritin at which ESRD patients are considered to be in an iron overload state is still not defined. Inflammatory states should not be considered indiciations to withhold the benefits of intravenous iron therapy in general [65]. However, a clinical problem is the diagnosis of chronic anemia associated with inflammation and true iron deficiency, as serum ferritin concentration increases rather than decreases.\nIron and kidney function\nIntravenous administration of 100\u00a0mg iron sucrose in CKD patients caused transient proteinuria and tubular damage [37], but ferric gluconate did not (125\u00a0mg infused over 1\u00a0h or 250\u00a0mg infused over 2\u00a0h) [66]. Induction of passive Heymann nephritis in rats resulted in a marked increase in nonheme iron content of kidney cortex and tubules, while a iron-deficient diet caused a significant reduction of nonheme iron level in glomeruli and also a significant reduction of proteinuria in these animals [67]. Pediatric thalassemia patients have a high prevalence of renal tubular abnormalities, probably caused by the anemia and increased oxidative stress induced by excess iron deposits. Significantly higher levels of urinary N-acetyl-beta-d-glucosaminidase, malondialdehyde, and beta-2-microglobulin were found in these children compared with normal children [68]. Under artificial experimental conditions (intravenous injection of 2\u00a0mg iron sucrose or ferric gluconate into mice with a body weight of 25\u201335\u00a0g), induction of monocyte chemoattractant protein-1 (MCP-1) in renal and extrarenal tissues has been observed. Since MCP-1 has profibrotic properties, implications for CKD progression in case of intravenous iron therapy has been suggested [69]. However, in a recent article, Mircescu et al. [70] reported that intravenous iron sucrose therapy (200\u00a0mg elemental iron per month for 12\u00a0months) resulted in an increase in hemoglobin from 9.7\u2009\u00b1\u20091.1 to 11.3\u2009\u00b1\u20092.5\u00a0g\/dl in nondiabetic patients with CKD and a mean glomerular filtration rate (GFR) of 36.2\u2009\u00b1\u20095.2\u00a0ml\/min per 1.73\u00a0m2, estimated by the formula of Cockcroft and Gault. The majority of these CKD patients had preexisting iron deficiency (mean ferritin 98.0\u00a0\u03bcg\/l, range 24.8\u2013139.0\u00a0\u03bcg\/l). An important finding of this study was that GFR (final values at the end of the study 37.2\u2009\u00b1\u20090.9\u00a0ml\/min per 1.73\u00a0m2) remained completely stable over a period of 12\u00a0months despite 2,400\u00a0mg of intravenous iron sucrose administration. The CKD patients had relatively high blood pressure (140\u2009\u00b1\u200932\/82\u2009\u00b1\u200920\u00a0mmHg at baseline), which did not change throughout the investigation [70]. Agarwal [71] found that a single dose of 100\u00a0mg iron sucrose results in a transient increase of MCP-1 in plasma and urine of CKD patients. Those who believe that 100\u00a0mg iron sucrose administered to CKD patients may negatively affect kidney function should simply administer a lower intravenous iron dose, e.g., 50\u00a0mg iron sucrose intravenously.\nIron and oxidative stress\nZager et al. [72] compared in vitro parenteral iron toxicity induced by three commercially available iron preparations (iron dextran, ferric gluconate, iron sucrose) using renal tubular cells and renal cortical homogenates. Each test agent induced massive and similar degrees of lipid peroxidation. Under the in vitro conditions used, iron sucrose caused markedly higher cell death than ferric gluconate, and ferric gluconate caused higher cell death than iron dextran. This relative toxicity profile was also observed in cultured aortic endothelial cells. Again, it should be stressed that the study of Mircescu et al. [70] demonstrated that intravenous iron sucrose therapy administered within international recommendations (none of the 58 CKD patients exceeded serum ferritin of 500\u00a0\u03bcg\/l) does not cause a decline in kidney function in CKD patients over a period of 1\u00a0year.\nIntravenous iron therapy may enhance symptoms of oxidative stress [73\u201375]. Dr\u00fceke et al. [75] demonstrated that advanced oxidation protein products (AOPPs) correlated with iron exposure and carotis artery intima thickness in dialysis patients. In hemodialysis patients, oxidative stress as a result of intravenous iron therapy caused serum albumin oxidation [76]. Ferric gluconate modifies fibrinogen and \u03b22-microglobulin as a marker of oxidative stress in adult hemodialysis patients [77, 78]. Intravenous administration of 100\u00a0mg iron sucrose in CKD patients increased malondialdehyde as a marker of lipid peroxidation [37]. Hemodialysis patients with ferritin levels above 650\u00a0\u03bcg\/l showed an enhanced oxidative burst in PMNL [59]. However, not all studies found evidence for enhanced oxidative stress caused by parenteral iron therapy in ESRD patients. Hemodialysis therapy per se was found to cause a significant increase in peroxide concentration. Interestingly, this rise in plasma total peroxides was not additionally influenced by concomitant intravenous injection of 100\u00a0mg iron sucrose [79]. These data confirm increased oxidative stress associated with hemodialysis [80]. Whether intravenous iron therapy results in an additional oxidative stress reaction needs to be further evaluated.\nIncreased blood levels of non-transferrin-bound iron (NTBI) and\/or its redox-active part have been reported in adult ESRD patients receiving intravenous iron therapy [79, 81, 82]. Intravenous infusion of 300\u00a0mg iron sucrose in ESRD patients also caused peripheral vasodilation, which was confirmed by increased forearm blood flow. NTBI and redox-active iron were considered to be, at least in part, responsible for endothelial dysfunction observed in ESRD patients. However, an increase in NTBI and redox-active iron caused by intravenous iron sucrose infusion did not influence vascular reactivity to intra-arterial acetylcholine, glycerol-trinitrate, or L-N-mono-methyl-arginine (L-NMMA) [82].\nVitamin C and iron\nESRD patients undergoing regular hemodialysis or hemodiafiltration may develop vitamin C deficiency [83]. Vitamin C deficiency may cause oxidative stress and vascular complications as well as impairment of intestinal iron absorption and iron mobilization from iron stress. Moretti et al. [84] measured iron absorption in young women from test meals fortified with isotopically labeled ferric pyrophosphate and ferrous sulfate. The addition of ascorbic acid at a molar ratio of 4:1 to iron increased iron absorption from ferric pyrophosphate to 5.8% and that from ferrous sulfate to 14.8%. In the fasting state, ferrous ascorbate is better absorbed than ferric hydroxide-polymaltose complex [85]. High-dose oral vitamin C may increase intestinal aluminium absorption [86]. Oxidative stress can cause hyporesponsiveness to ESA therapy in ESRD patients. Vitamin C may improve erythropoiesis through its antioxidative properties [87]. Intravenous ascorbic acid therapy facilitates iron release from inert deposits, resulting in a decrease of soluble transferrin receptor and an increase of TSAT [88]. In contrast, oral vitamin C supplementation (250\u00a0mg three times per week for 2\u00a0months) did not influence oxidative\/antioxidative stress and inflammation markers in adult hemodialysis patients [89].\nIn adult hemodialysis patients on maintenance intravenous iron sucrose therapy, intravenous administration of 500\u00a0mg ascorbic acid three times a week for 6\u00a0months resulted in an increase of TSAT and hemoglobin in approximately 65% of the patients [90]. In contrast, neither oral nor intravenous ascorbic acid changed TSAT or hemoglobin levels in a study performed by Chan et al. [91]. Ascorbic acid increases the intracellular labile iron pool and iron mobilization to transferrin in human hepatoma HepG2 cells only in the presence of iron sucrose but not in the presence of iron dextran or ferric gluconate [92]. Several studies reported an increase in hemoglobin and\/or a decrease in adult ESA dose during adjuvant ascorbic acid therapy three times per week in ESRD patients [93\u201399]. Measurements of plasma oxalate concentration are needed in ESRD patients supplemented with ascorbic acid [100]. Studies on vitamin C and iron in children with ESRD are not available so far.\nIron therapy after kidney transplantation\nAnemia is observed in 21\u201339.7% of adult renal transplant patients [101\u2013104]. The prevalence may be even higher in pediatric transplant recipients: 84.3% of children were anemic in the first month after kidney transplantation, and prevalence of anemia was not below 64.2% between 6\u00a0months and 6\u00a0years after transplantation. Iron deficiency was identified in 27\u201356% of children between 1 and 60\u00a0months posttransplantation [105]. Fourteen pediatric and young adult renal transplant recipients received single iron gluconate infusions ranging from 1.9 to 6.4\u00a0mg\/kg. The mean hemoglobin level increased significantly from 10.1\u2009\u00b1\u20091.6 to 11.4\u2009\u00b1\u20092.1\u00a0g\/dl following ferric gluconate therapy. Adverse events were observed in three children [106]. A recent study by Morii and coworkers showed that oral coadministration of ferrous sulphate markedly decreased the absorption of mycophenolate mofetil in healthy Japanese subjects [107]. However, a randomized crossover trial failed to confirm this observation in European transplant patients receiving long-term mycophenolate mofetil therapy [108]. In line with this observation, an in vitro study showed that iron ions did not interact with mycophenolate mofetil [109].\nConclusions\nThe integration of ESA and intravenous or oral iron therapy into standard anemia management resulted in target hemoglobin levels (as established by international guidelines) in the vast majority of ESRD patients [110]. Correction of renal anemia reduced morbidity and mortality as well as hospitalization in ESRD patients. It also improved quality of life, cognitive function, and physical activity. Using a balanced approach to iron supplementation within international recommendations allowed the attainment of benefits of intravenous iron therapy at storage iron levels far below those generally seen with transfusions in the pre-ESA era [110].","keyphrases":["erythropoiesis-stimulating agents","iron supplementation","iron status","inflammation","infection"],"prmu":["P","P","P","P","P"]}
{"id":"Diabetologia-3-1-1914292","title":"Advanced glycation end products cause increased CCN family and extracellular matrix gene expression in the diabetic rodent retina\n","text":"Aims\/hypothesis Referred to as CCN, the family of growth factors consisting of cystein-rich protein 61 (CYR61, also known as CCN1), connective tissue growth factor (CTGF, also known as CCN2), nephroblastoma overexpressed gene (NOV, also known as CCN3) and WNT1-inducible signalling pathway proteins 1, 2 and 3 (WISP1, \u22122 and \u22123; also known as CCN4, \u22125 and \u22126) affects cellular growth, differentiation, adhesion and locomotion in wound repair, fibrotic disorders, inflammation and angiogenesis. AGEs formed in the diabetic milieu affect the same processes, leading to diabetic complications including diabetic retinopathy. We hypothesised that pathological effects of AGEs in the diabetic retina are a consequence of AGE-induced alterations in CCN family expression.\nIntroduction\nDiabetic retinopathy is a major complication of diabetes and a leading cause of blindness [1, 2]. Despite recent progress in understanding the pathogenesis of diabetic retinopathy, further research is warranted, as the disease remains neither preventable nor curable.\nDiabetic retinopathy is preceded by an asymptomatic preclinical phase, in which a microangiopathy develops which is characterised by diffusely increased vascular permeability and capillary basement membrane thickening, resulting from excess accumulation of extracellular matrix components [3\u20135]. In later stages of preclinical diabetic retinopathy, endothelial cell and pericyte loss leads to vascular cell death and the development of acellular capillaries. Experimental prevention of basement membrane thickening has been shown to ameliorate these retinal vascular changes [6, 7]. Thus in galactose-fed rats, a model of diabetes, downregulation of fibronectin synthesis partly prevented retinal basement membrane thickening and also reduced pericyte and endothelial cell loss [6]. Combined downregulation of mRNA levels of the extracellular matrix components fibronectin, collagen type IV alpha 3 (Col4a3) and laminin beta 1 (Lamb1) not only prevented increases in their protein levels, but also reduced vascular leakage in the retina of rats with streptozotocin-induced diabetes [7]. These findings suggest that basement membrane thickening is not just an epiphenomenon of the diabetic state, but may be instrumental in the progression of sight-threatening diabetic retinopathy. Modulation of basement membrane thickening may therefore have a preventive effect on the development of diabetic retinopathy.\nHowever, the mechanisms leading to diabetes-induced basement membrane thickening remain largely unknown. One of the postulated mechanisms is the formation of AGEs in the diabetic milieu. Inhibition of AGE formation by aminoguanidine has been shown to protect against retinal capillary basement membrane thickening [8]. AGE have also been shown to induce extracellular matrix synthesis in the diabetic rat kidney. A similar induction of extracellular matrix synthesis was also shown to be mediated by connective tissue growth factor (CTGF, also known as CCN2) [9, 10], a member of the family of proteins referred to as CCN (for cystein-rich protein 61 [CYR61, also known as CCN1], CTGF and nephroblastoma overexpressed gene [NOV, also known as CCN3]). CTGF leads to accumulation of extracellular matrix by induction of collagen, fibronectin and laminin synthesis, as well as decreased proteolysis of extracellular matrix components as a result of increased production of tissue inhibitors of metalloproteases (TIMP) [9, 11\u201318]. Recently, we observed that Ctgf+\/\u2212 mice (lacking one functional allele for Ctgf) are protected from diabetes-induced basement membrane thickening of retinal and kidney glomerular capillaries ([19]; P. Roestenberg, F .A. Van Nieuwenhoven, R. Verheul et al., unpublished results).\nWe hypothesised in the present study that AGE-induced basement membrane thickening observed in the retina is at least partly mediated by CTGF. To establish the role of CTGF in the AGE-induced production of vascular basement membrane components and their mediators, we investigated the effects of aminoguanidine on the levels of CTGF, the other CCN family members (CYR61, NOV and WNT1-inducible signalling pathway protein 1, 2 and 3 [WISP1, \u22122, and \u22123, also known as CCN4, \u22125 and \u22126]) [20\u201322] and vascular basement membrane-related molecules in the retina of rats with streptozotocin-induced diabetes, as well as in the retina of mice infused with AGE.\nMaterials and methods\nAnimals All animal studies were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All experiments involving rats were reviewed and approved by the ethics committee for animal care and use of the Free University Medical Centre, Amsterdam, the Netherlands. All experiments involving mice were carried out in accordance with British Home Office regulations.\nStreptozotocin-induced diabetic rat model Adult Wistar rats (Charles River, Maastricht, the Netherlands), weighing approximately 250\u00a0g, were randomly divided into three experimental groups: a control group (n\u2009=\u200914), a diabetic group (n\u2009=\u200916) and a diabetic group treated with aminoguanidine hydrogen carbonate (n\u2009=\u200914; Fluka, Buchs, Switzerland). Diabetes was induced by a single i.p. injection of 60\u00a0mg\/kg streptozotocin (Sigma, St Louis, MO, USA). Immediately prior to use, streptozotocin was dissolved in cold 0.1\u00a0mol\/l citrate buffer, pH\u00a04.5. Control rats received a single i.p. injection of 0.1\u00a0mol\/l citrate buffer only. Aminoguanidine was administered from day 1 at a dose of 1\u00a0g\/l in drinking water. Serum glucose levels and body weight were monitored at the start and end of the experiment. Diabetes was verified by a serum glucose level >13.9\u00a0mmol\/l. At 6\u00a0weeks, half of the rats were randomly selected from the three experimental groups and killed with a lethal dose of pentobarbital sodium (i.p.). At 12\u00a0weeks the remaining rats were killed. Eyes from each rat were rapidly enucleated, one being snap-frozen in liquid nitrogen and stored at \u221280\u00b0C, while the contralateral eye was fixed in 4% paraformaldehyde. Additionally, blood samples were collected and plasma levels of N\u025b-(carboxymethyl)lysine (CML) were measured by stable-isotope dilution tandem mass spectrometry [23].\nIn vivo administration of exogenous AGE Female C57BL\/6 mice (10\u201312\u00a0weeks old) were randomly assigned to two groups of equal size and injected i.p. with either native mouse serum albumin (MSA) or glycoaldehyde-modified MSA (10\u00a0mg\/kg) daily for seven consecutive days. At 3\u20134\u00a0h after the final injections, mice were killed, eyes enucleated and retinas dissected freshly, before being snap-frozen in liquid nitrogen.\nPreparation of AGE-modified albumin The glycoaldehyde-modified MSA preparation was made according to Nagai et al. [24]. Following dialysis against PBS, endotoxin was removed using an endotoxin-removing column (Pierce, Rockford, IL, USA). Glycoaldehyde-MSA and native MSA were passed three times through separate columns to ensure that all contaminating endotoxin was removed. Analysis of the CML content of glycoaldehyde-MSA and native MSA was performed using gas chromatography\u2013mass spectrometry. The lysine content of the samples was analysed by cation exchange chromatography and the values for CML were corrected for lysine loss and expressed as mmol CML\/mol lysine as previously reported [25].\nRNA isolation and mRNA quantification Snap-frozen rat and mouse eyes were allowed to thaw in ice-cold RNAlater (Ambion, Austin, TX, USA). The anterior chambers of the eyes were removed and the retinas were carefully dissected. Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The amount of total retinal RNA isolated was approximately 12\u00a0\u03bcg per retina (spectrophotometric measurements at 260\u00a0nm), with no significant differences between the experimental groups. The integrity of the RNA samples was verified using an automated electrophoresis system (Experion; Bio-Rad, Hercules, CA, USA). All samples had sharp ribosomal RNA bands with no sign of degradation.A 2-\u03bcg aliquot of total RNA was treated with DNAse I (amplification grade; Invitrogen) and reverse-transcribed into first strand cDNA with Superscript II and oligo(dT)12\u201318 (Invitrogen).Details of the primers are given in the Electronic supplementary material (ESM) (ESM Table\u00a01). Specificity of the primers was confirmed by a nucleotide\u2013nucleotide BLAST (http:\/\/www.ncbi.nlm.nih.gov\/blast\/index.shtml) search. The presence of a single PCR product was verified by both the presence of a single melting temperature peak and detection of a single band of the expected size on a 3% agarose gel.Real-time quantitative PCR (qPCR) was performed in a sequence detection system (ABI Prism 5700; Applied Biosystems, Foster City, CA, USA). For each primer set a mastermix was prepared consisting of 1\u00d7 SYBR Green PCR buffer (Eurogentec, Seraing, Belgium), 3\u00a0mmol\/l MgCl2, 200\u00a0\u03bcmol\/l each of dATP, dGTP and dCTP, 400\u00a0\u03bcmol\/l dUTP, 0.5\u00a0U AmpliTaq Gold (Eurogentec) and 2\u00a0pmol primers. All cDNA samples were diluted 1:10 and amplified using the following PCR protocol: 10\u00a0min at 95\u00b0C, followed by 40 cycles of 15\u00a0s at 95\u00b0C and 60\u00a0s at 60\u00b0C and a melting program (60\u201395\u00b0C). Relative gene expression was calculated using the equation: R=E\u2212Ct, where E is the mean efficiency of all samples for the gene being evaluated and Ct is the cycle threshold for the gene as determined during real-time PCR. All qPCR experiments were performed at least twice.Real-time qPCR data from the mouse experiments were normalised using 18S rRNA, which was determined to be stably expressed in all experimental groups. For the rat experiments, no suitable housekeeping genes that were not regulated by the diabetic background could be found. Therefore, the rat data were normalised using the relative starting amounts of cDNA, which was determined using a novel technique recently developed in our laboratory (J. M. Hughes, I. Klaassen, W. Kamphuis, C. J. F. Van Noorden and R. O. Schlingemann, unpublished results). In brief, reverse transcription reactions were carried out in duplicate with one set of reactions containing the normal dNTP mix and the parallel set of reactions containing a dNTP mix with \u03b1-32P-labelled dCTP. From each sample 4\u00a0\u03bcl of the \u03b1-32P-labelled dCTP-incorporated cDNA were pipetted on to separate nitrocellulose filters, which were allowed to air-dry. After washing with 0.1\u00a0mol\/l phosphate buffer, radioactivity of the filters was measured using a scintillation counter (Beckman Coulter, Fullerton, CA, USA).\nWestern blotting Protein was isolated from paraformaldehyde-fixed retinal tissue as described by Shi et al. [26]. In brief, retinas were dissected from the 4% paraformaldehyde-fixed rat eyes and pooled in 1.5\u00a0ml Eppendorf vials in antigen-retrieval buffer (20\u00a0mmol\/l Tris, 2% SDS, pH 7). The pooled samples were then dissociated using a pestle and incubated at 100\u00b0C for 20\u00a0min followed by 2\u00a0h at 60\u00b0C. Supernatant fractions were collected after centrifugation at 4\u00b0C for 15\u00a0min at 10,000g. Protein concentrations were determined with a bicinchoninic acid protein assay kit (Perbio, Etten-Leur, the Netherlands) and adjusted to 2.5\u00a0\u03bcg\/\u03bcl. For SDS-PAGE and western blots, proteins were separated using 13% mini gels under reducing conditions. Following gel electrophoresis, proteins were transferred to a nitrocellulose filter (Whatman Schleicher & Schuell, Brentford, Middlesex, UK) using a semi-dry transfer cell (Bio-Rad). At the end of the transfer, the filter was blocked in blocking buffer (1% non-fat skimmed milk powder, 1% BSA, 1\u00a0mmol\/l NaN3 in Tris-buffered saline and 0.05% Tween) overnight at 4\u00b0C while being gently rocked. The filter was incubated in blocking solution for 2\u00a0h at room temperature, with the relevant antibodies as defined below. Following three washes in TBS\/0.05% Tween-20, the blots were incubated in blocking solution with horseradish peroxidase-conjugated goat\u2013anti-rabbit or goat\u2013anti-mouse antibodies for 1\u00a0h at room temp. After extensive washing, blots were developed using a chemiluminescent kit (SuperSignal West Pico; Perbio). Filters were exposed to X-ray film (Kodak-Biomax, Herts, UK).Primary antibodies (ESM Table\u00a02) were diluted with 0.3% skimmed milk powder in TBS\/Tween and horseradish peroxidase-conjugated goat\u2013anti-rabbit or goat\u2013anti-mouse (Perbio) was diluted 1:20,000. Intensity of bands was quantified by densitometry using AlphaEase software (AlphaInnotech, San Leandro, CA, USA).\nImmunohistochemistry Cryostat sections (10-\u03bcm thick) were stained using an indirect immunoperoxidase procedure as previously described [27]. Primary antibodies are listed in ESM Table\u00a02. Primary antibody was omitted for negative controls. Indirect immunoperoxidase staining was performed using histostaining reagents (Powervision; ImmunoVision, Daly City, CA, USA) for all sections except those incubated with the TIMP1 antibody. The TIMP1 sections were indirectly stained using horseradish peroxidase-labelled rabbit\u2013anti-goat antibody (P0160; Dako, Glostrup, Denmark).\nStatistics CML data were log10 transformed to obtain a normal distribution. Significant differences (p\u2009<\u20090.05) in glucose and CML plasma levels, and in gene expression levels among groups were calculated with single ANOVA. The Bonferroni post hoc test was used to perform pairwise comparisons of groups.\nResults\nGlucose and CML levels in control and diabetic rats Induction of diabetes and the degree of hyperglycaemia in streptozotocin-treated rats was established by serum glucose levels (Fig.\u00a01a). Streptozotocin treatment resulted in a three- to fourfold increase in serum glucose concentration after 6 and 12\u00a0weeks irrespective of aminoguanidine treatment.\nFig.\u00a01Plasma glucose (a) and CML (b) levels in control and diabetic rats at 6 and 12\u00a0weeks of streptozotocin-induced diabetes. White bars, control rats; black bars, diabetic rats; cross-hatched bars, diabetic rats treated with aminoguanidine. *p\u2009<\u20090.05 and *** p\u2009<\u20090.001 for difference between experimental and control group. The aminoguanidine group was only significantly different from the groups with diabetes at 12\u00a0weeks (\u2020p\u2009<\u20090.05). The error bars show the standard deviation for each groupPlasma levels of CML were measured to determine the efficacy of aminoguanidine treatment. CML plasma levels were elevated by twofold at 6 and 12\u00a0weeks after streptozotocin-treatment (Fig.\u00a01b). Aminoguanidine treatment had no effect on CML levels at 6\u00a0weeks, but at 12\u00a0weeks the CML levels were decreased by approximately 25%.\nCCN family gene expression in control and diabetic rats After 6\u00a0weeks of diabetes, Cyr61 mRNA levels in the diabetic retina were increased by threefold against control retina. Treatment with aminoguanidine reduced Cyr61 expression to levels that were not significantly different from control levels (Fig.\u00a02). CYR61 protein was mainly localised in the ganglion cell layer (Fig.\u00a03). No differences in staining patterns were found between experimental groups.\nFig.\u00a02Gene expression of CCN family members. Fold change, compared with control values, in retinal mRNA levels of CCN family members in streptozotocin-induced diabetic rats at 6 (white bars) and 12\u00a0weeks (cross-hatched bars) after streptozotocin-induction and in aminoguanidine-treated, streptozotocin-induced diabetic rats at 6 (black bars) and 12\u00a0weeks (grey bars) of diabetes. *p\u2009<\u20090.05 for difference between experimental group and control group; \u2020p\u2009<\u20090.05 for difference between aminoguanidine-treated diabetic group and diabetes-only groupFig.\u00a03Immunohistochemical staining patterns of a CYR61, b CTGF and c TIMP1 in control rat retinas. Intense staining of CYR61 and CTGF was present in large cell bodies of the ganglion cell layer (GCL) and weak staining in the inner plexiform layer (IPL). Intense uniform immunostaining of TIMP1 was found in the GCL and weak staining in the IPL. INL inner nuclear layer, OPL outer plexiform layer, ONL outer nuclear layer, RCL rod and cones layer, RPE retinal pigment epithelium. Magnification: \u00d7150Ctgf mRNA levels were elevated by twofold at 12\u00a0weeks of streptozotocin-induced diabetes. Aminoguanidine treatment almost completely prevented this increase (Fig.\u00a02). Western blotting showed a 1.8-fold increase in CTGF protein levels in the retina of diabetic rats at 12\u00a0weeks, whereas aminoguanidine treatment also prevented this effect (Fig.\u00a04). CTGF immunostaining was mainly found in the ganglion cell layer and was more diffuse throughout the outer plexiform layer, inner nuclear layer and inner plexifrom layer (Fig.\u00a03). Differences in staining between experimental groups were not observed.\nFig.\u00a04CTGF protein levels in retina of control and diabetic rats. a Western blots of CTGF and GAPDH as loading control. Samples were pooled for each group. A prominent band of CTGF protein is present in the 12-week diabetic group (12D; n\u2009=\u20098), whereas protein bands were similar in all other groups (control rats at 6 [6C; n\u2009=\u20096] and 12 [12C; n\u2009=\u20098] weeks, diabetic rats at 6 [6D; n\u2009=\u20098] weeks and diabetic rats treated with aminoguanidine at 6 [6AG; n\u2009=\u20097] and 12 [12AG; n\u2009=\u20097] weeks). b The blots were quantified by densitometry and expressed as a ratio of CTGF:GAPDHWisp1 and Wisp3 mRNA levels were low in retinas of all groups of rats. The levels never differed more than 1.4-fold between experimental and control groups. Due to small standard deviations, significant differences were found (Fig.\u00a02), but it is doubtful whether these small differences are biologically meaningful. Nov and Wisp2 mRNA expression levels were too low to be detected in all groups of rats.\nExpression of transforming growth factor beta 1 and 2 in control and diabetic rats To determine whether: (1) transforming growth factor beta (TGFB), an upstream regulator of CTGF production, is induced by diabetes; and (2) this induction is prevented by aminoguanidine in rat retina, we examined the mRNA levels of its two most common isoforms, Tgfb1 and Tgfb2. Tgfb1 mRNA expression was decreased by approximately 30% in retina of rats with diabetes for 6\u00a0weeks (Fig.\u00a05). Whether this difference is biologically relevant remains to be determined. In all other experimental groups, Tgfb1 and Tgfb2 mRNA levels were similar to control levels (Fig.\u00a05). Immunohistochemical analysis of TGFB1 revealed a vascular pattern of staining in all experimental groups (Fig.\u00a06).\nFig.\u00a05Fold change of Tgfb1 and Tgfb2 expression in diabetic rats at 6 (white bars) and 12\u00a0weeks (cross-hatched bars) after streptozotocin-induction and in aminoguanidine-treated diabetic rats at 6 (black bars) and 12\u00a0weeks (grey bars) after streptozotocin-induction. Tgfb1 was significantly decreased in the diabetic rats after 6\u00a0weeks (*p\u2009<\u20090.05 vs control). This difference was not observed in the aminoguanidine-treated group at 6\u00a0weeks. Tgfb2 expression was not significantly altered at either time pointFig.\u00a06Immunohistochemical staining patterns of TGFB1 (a, d), laminin (b, e) and fibronectin (c, f) in retina of control rats (a\u2013c) and rats 12\u00a0weeks after streptozotocin-induced diabetes (STZ) (d\u2013 f). Immunostaining of TGFB1, laminin and fibronectin was confined to the retinal microvasculature and did not notably differ between control and streptozotocin sections. Magnification: \u00d7 150\nExpression of extracellular matrix molecules in control and diabetic rats CTGF and CYR61 are known modifiers of the extracellular matrix. Therefore, we investigated expression patterns of various extracellular matrix components. Col4a3 mRNA levels in the rat retina were elevated by threefold after 6\u00a0weeks of diabetes. Aminoguanidine treatment inhibited this induction of Col4a3 mRNA levels by 30% (Fig.\u00a07). Lamb1 mRNA levels showed a 1.5-fold increase in 12-week diabetic rats, which was virtually unaffected by aminoguanidine treatment. Fibronectin mRNA levels were not affected by streptozotocin-induced diabetes. Timp2 mRNA levels were not affected either, but Timp1 mRNA levels were elevated by 2.5-fold in retina of 12-week diabetic rats, this increase being completely prevented by aminoguanidine treatment (Fig.\u00a07). Laminin and fibronectin were localised immunohistochemically in microvessels in rat retina (Fig.\u00a06). This staining pattern was similar in all groups. TIMP1 immunostaining was restricted to the ganglion cell layer in all groups of rats (Fig.\u00a03).\nFig.\u00a07Gene expression of extracellular matrix components. Fold change of extracellular matrix gene expression as indicated in diabetic rats at 6 (white bars) and 12\u00a0weeks (cross-hatched bars) after streptozotocin-induction and in aminoguanidine-treated diabetic rats at 6 (black bars) and 12\u00a0weeks (grey bars) after streptozotocin-induction. *p\u2009<\u20090.05 for difference between experimental and control group; \u2020p\u2009<\u20090.05 for difference between aminoguanidine-treated diabetic group and diabetes-only group\nCCN family mRNA expression in control and AGE-treated mice AGE-treated MSA infusion of mice induced an increased retinal expression of Cyr61 and Ctgf mRNA by 3.7-fold and twofold, respectively, compared with control mice (Fig.\u00a08). Wisp1 and Wisp3 mRNA expression was not affected by AGE-treated MSA infusion (data not shown).\nFig.\u00a08Relative mRNA levels of Cyr61 and Ctgf in retinas of control (white bars) and AGE-treated (black bars) mice, depicted as fold change in comparison with control mice. *p\u2009<\u20090.05 for effect of AGE treatment on Cyr61 and Ctgf mRNA levels\nSummary of results In the retina of rats with streptozotocin-induced diabetes, mRNA levels of the CCN family members Cyr61 and Ctgf were increased threefold at 6\u00a0weeks and twofold at 12\u00a0weeks, respectively, whereas expression of all other CCN family members was not notably affected. CTGF protein levels in retina were also elevated twofold at 12\u00a0weeks of diabetes. In the aminoguanidine-treated diabetic rats these increases were partly counteracted by treatment with this AGE inhibitor. In line with these findings, treatment of mice with exogenous AGE induced elevated retina mRNA levels of Cyr61 and Ctgf, but not of the other CCN family members. In parallel, mRNA levels of some extracellular matrix components were also increased in the retina of diabetic rats, an effect also prevented by aminoguanidine treatment.\nDiscussion\nWe present here a comprehensive expression analysis of the CCN family of fibrosis-inducing cytokines in the retina of rats with streptozotocin-induced diabetes. Messenger RNA and protein levels of CYR61 and CTGF, both known to be capable of modulating the extracellular matrix, were increased in diabetic rats, whilst the AGE inhibitor aminoguanidine attenuated these effects of diabetes. We also found that exogenously administered AGEs are capable of inducing Cyr61 and Ctgf expression in the adult mouse retina in vivo. Taken together, these data present evidence that AGEs are both necessary and sufficient to cause increased levels of CYR61 and CTGF in the diabetic retina.\nExpression of CTGF at the mRNA or protein level has previously been demonstrated in vivo in normal and diabetic rat [28] and human retina [29], as well as in cultured retinal microvascular cells [30] and astrocytes [31]. Our data on CTGF are in agreement with a previously reported twofold increase in Ctgf expression in the diabetic rat retina [28]. In our study, immunostaining with a polyclonal anti-CTGF antibody showed staining in the ganglion cell layer [28] and diffusely throughout the larger part of the inner rat retina. In contrast, in a previous study of the human diabetic retina employing a monoclonal anti-CTGF antibody, CTGF was detected in microglia and pericytes in the microvasculature of the inner retina [29]. These varying patterns of CTGF protein distribution may be species-related or due to differences in specificity of the antibodies used.\nGene expression analysis was also performed on the other five known members of the CCN family. We found Cyr61 expression in the normal adult rat retina and upregulation in the diabetic retina. In addition to CTGF, this suggests a possible role for CYR61 in the development of diabetes-related retinal sequelae. The lack of detectable Nov and Wisp2 expression argues against a role for these two proteins in normal or diabetic retina. This is in agreement with a previous study, which demonstrated that Wisp2 mRNA was not present in tissues of the adult rat [32]. Expression of Wisp1, which is much less studied but known to suppress cancer cell growth in vivo [33], was slightly decreased in diabetic retina, whereas Wisp3, a cell growth suppressor and inhibitor of angiogenesis [34], was increased. The significance of these findings remains unclear, as the functions of the CCN family members are complex. Still, such opposing actions may be reflective of a regulatory balance or may indicate that some CCN family members are redundant, although this is currently not considered to be the case in other tissues [35].\nThe role of CTGF in diabetic renal pathology has been clearly established. CTGF is responsible for mesangial expansion [36] and increased extracellular matrix deposition [37, 38] as observed in early stages of diabetic nephropathy. Therefore, CTGF and\/or CYR61 may have a similar role in diabetic retina and be responsible for the thickening of microvascular basement membranes observed in early stages of diabetic retinopathy.\nWe also therefore examined expression patterns of the genes encoding several extracellular matrix-related molecules. Expression of the basement membrane component Col4a3 was found to be increased concomitantly with Cyr61 expression after 6\u00a0weeks of diabetes, an increase significantly reduced in aminoguanidine-treated diabetic rats. At 12\u00a0weeks of diabetes, Timp1 and Lamb1 expression demonstrated a concomitant increase with Ctgf, which was significantly attenuated in the aminoguanidine-treated group (not statistically significant for Lamb1). These findings suggest a causal role of AGEs in the diabetes-induced production of CTGF, CYR61, COL4A3 and TIMP1 in the retina. Whether increased Lamb1 expression is also mediated by AGEs remains to be determined. However, this possibility is supported by a previous study showing that protein and mRNA levels of the ribosomal protein SA (previously known as laminin receptor 1 [67\u00a0kD, ribosomal protein SA]) are upregulated by AGEs in cultured retinal microvascular endothelial cells [39].\nAGE CML plasma levels, used in our study as a marker of AGE formation in the rat diabetes model, were not altered at 6\u00a0weeks in diabetic rats, making an effect of AGEs at this time point questionable. However, it should be noted that CML is merely one of many types of AGE known to be generated under hyperglycaemic conditions [40] and that aminoguanidine has been shown to decrease serum AGE levels in diabetic rats as early as 6\u00a0weeks after diabetes induction [41].\nIn previous studies in the diabetic rat kidney, Ctgf and fibronectin gene expression both increased after 32\u00a0weeks of streptozotocin-induced diabetes [10]. These changes were prevented by aminoguanidine treatment. As AGE accumulation in the diabetic kidney was prevented by aminoguanidine, those authors surmised that the anti-fibrotic effects of aminoguanidine could be at least partially mediated by a decrease in CTGF expression [10]. Our study may have been too short to observe an increase of fibronectin in the diabetic retina, but otherwise our results are in line with these findings in kidney. Additional studies will be necessary to further elucidate the ability of CYR61 and CTGF to directly modulate these extracellular matrix molecules in retinal vascular cells.\nTGFB, considered to be the most important fibrotic factor, has been shown to upregulate CTGF in many cell types in vitro [42\u201344] and in vivo [45, 46]. Although our findings indicate that TGFB production is not increased in the diabetic retina, a role for TGFB in the observed upregulation of CTGF cannot be ruled out, as the regulation of TGFB bioavailability is complicated and not solely dependent on the level of TGFB production [47].\nWe have recently demonstrated that vascular endothelial growth factor (VEGF) increases expression of Ctgf and Cyr61 in the rat retina in vivo as well as in retinal vascular endothelial cells in vitro (E. J. Kuiper, J. M. Hughes, I. M. C. Vogels et al., unpublished results). As AGEs are known inducers of VEGF in retinal cells [48, 49], it is possible that the increases in CTGF and CYR61 observed in our animal models are the result of AGE-induced VEGF.\nA major finding of our study was the ability of aminoguanidine to attenuate the increase in Ctgf expression observed in the diabetic rat retina. Tikellis et al. [28] have reported that perindopril, an ACE inhibitor, prevents increased Ctgf expression in diabetic rat retina [28]. This suggests that both interventions may affect a common molecular mechanism leading to the upregulation of CTGF. As ACE inhibition has been shown to prevent AGE accumulation in diabetic tissues [50], it is feasible that inhibition of AGE formation could be a common molecular mechanism allowing both ACE inhibitors and aminoguanidine to inhibit the increase of retinal CTGF.\nIn summary, this study provides the first evidence that, in addition to CTGF, the CCN family molecules CYR61, WISP1 and WISP3 play possible roles in the development of early stages of experimental diabetic retinopathy. At the very least, these results warrant further study into the functional aspects of these molecules in the eye, and of how these aspects pertain to the development of diabetic retinopathy. Additionally, we demonstrate for the first time that AGEs directly upregulate both CTGF and CYR61 levels in the retina in vivo and that aminoguanidine inhibits these diabetes-induced increases. This provides the first evidence that CTGF and CYR61 are downstream effectors of AGEs in the diabetic retina and implicates them as possible targets for future intervention strategies.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.","keyphrases":["advanced glycation end products","extracellular matrix","cystein-rich protein 61","connective tissue growth factor","diabetic retinopathy","basement membrane","experimental","aminoguanidine","diabetes mellitus","gene expression regulation"],"prmu":["P","P","P","P","P","P","P","P","M","R"]}
{"id":"Histochem_Cell_Biol-4-1-2386530","title":"Structure and function of mammalian cilia\n","text":"In the past half century, beginning with electron microscopic studies of 9 + 2 motile and 9 + 0 primary cilia, novel insights have been obtained regarding the structure and function of mammalian cilia. All cilia can now be viewed as sensory cellular antennae that coordinate a large number of cellular signaling pathways, sometimes coupling the signaling to ciliary motility or alternatively to cell division and differentiation. This view has had unanticipated consequences for our understanding of developmental processes and human disease.\nIntroduction\nAlthough in 1835, Purkinje and Valentin (1835) published the first treatise to include studies of mammalian cilia, the history of what may be considered modern research on mammalian cilia does not extend back more than a few years longer than the history of this journal. Motile mammalian cilia attracted the attention of microscopists before the advent of electron microscopy, because they moved and were obviously similar in this motion to protozoan cilia. Nevertheless, several light microscope structures whose later fine structure appearance (and molecular biology) had little in common with cilia were nevertheless labeled cilia, including the stereocilia of the hair cells, which are in fact modified microvilli. In 1954, the electron microscope observations of Fawcett and Porter (1954) definitively characterized the 9\u00a0+\u00a02 pattern of the cytoskeleton, the axoneme, of motile mammalian and other cilia. They showed that this structure was enclosed by an extension of the cell membrane, the ciliary membrane. The axoneme of true cilia was always an extension of a basal body and related to the nine-fold construction of the centriole. By 1956, Porter (1957) and De Robertis (1956) realized that cilia were found as sensory structures in, for example mammalian photoreceptors, where the central pair of microtubules in the axoneme was absent, and the cilia were nonmotile, hence 9\u00a0+\u00a00. Finally, in the early 1960s, a number of electron microscopy studies showed that solitary 9\u00a0+\u00a00 cilia, now termed primary cilia, are present on many differentiated cells of tissues of the mammalian and vertebrate body, including kidney cells, fibroblasts, neurons and even Schwann cells (Barnes 1961; Sorokin 1962; Grillo and Palay 1963).\nAlthough specific antibodies against detyrosinated and acetylated tubulin became available to detect primary cilia in immunofluorescence microscopy (Piperno et al. 1987; Cambray-Deakin and Burgoyne 1987), primary cilia were generally neglected as interesting cell organelles. The biggest problem was that primary cilia had no demonstrable function, even though considerable circumstantial evidence suggested that they were some form of chemical or mechanical sensor. On this basis, Poole et al. (1985) proposed that primary cilia of connective tissue cells functioned as a cellular cybernetic probe, or in current terminology, as a cellular global positioning system. Roth et al. (1988) then demonstrated that primary cilia on cultured kidney epithelial cells could be bent by flow, and Wheatley (1995) suggested that if primary cilia in renal epithelia did not function properly in sensing flow, there would be pathophysiological consequences. Nevertheless, many cell biologists dismissed the primary cilium as vestigial.\nWhile early studies of mammalian cilia focused on showing that the mechanism and biochemistry of motility was virtually identical to that discovered in other cilia, more recent studies have examined the way that cilia are built. These studies have led to novel insights for both motile and nonmotile, including primary, cilia. All cilia can now be viewed as sensory cellular antennae that coordinate a plethora of cellular signaling pathways, and this view has had unanticipated consequences for our understanding of developmental processes and disease. A recent review of mammalian cilia structure and function covering many of the topics presented here, in further detail, is found in Satir and Christensen (2007).\nThe mechanism of ciliary motility and primary ciliary dyskinesia\nThe basic mechanism of ciliary motility is now reasonably well understood, although many details of how waveform is generated and propagated remain obscure. While much of the information about this mechanism was derived from protistan and invertebrate organisms (Satir 1985), the basic principles clearly apply to mammalian cilia of the respiratory and reproductive tract, and to brain ependymal cilia. An electron micrograph of cilia of the mouse oviduct is shown in Fig.\u00a01.\nFig.\u00a01Classic transmission electron micrograph of mouse oviduct cilia. Cross-sections show the 9\u00a0+\u00a02 axoneme of motile cilia (asterisk). The axoneme grows from a basal body, with a basal foot (arrowhead) pointing in the direction of the effective stroke. The transition zone between basal body and axoneme contains the ciliary necklace (arrow). (From Dirksen and Satir 1972, unpublished, with permission)\nCiliary motility is caused by the relative sliding of the nine outer axonemal doublets, operating as two opposing sets, one (doublets 1\u20134) to produce the effective stroke (or principal bend in flagella), one (doublets 6\u20139) to produce the recovery stroke (or reverse bend), powered by ATP and a set of molecular motors, the axonemal dyneins. The axonemal dyneins are arranged in two sets of arms: the outer dynein arms (ODAs) consisting of two heavy-chain dynein isoforms in mammalian cilia, packaged with intermediate and light chains, with four identical ODAs, each aligned along 96-nm long doublet microtubule (Nicastro et al. 2006), and the more complex inner dynein arms (IDAs). The IDAs, more centrally located, consist of at least seven heterodimeric and monomeric heavy-chain dynein isoforms per 96\u00a0nm. Generally, the ODAs and IDAs function together, but the ODAs principally regulate beat frequency, in part by cAMP-dependent phosphorylation of an ODA regulatory light chain, while the IDAs control beat form. Both frequency and form change by changing the rate of sliding and the switching of activity between the doublet sets. Sliding is converted into propagated bending, most efficiently by control of IDA activity via phosphorylation and mechanical interaction involving the radial spoke, central pair complex.\nIn effect, the 9\u00a0+\u00a02 axoneme is a nanomachine composed of perhaps over 600 proteins in molecular complexes, many of which also function independently as nanomachines. The enzymes that control axonemal response, for example protein kinase A, are structurally part of the complexes. Strong support for this mechanism of ciliary motility comes from studies of paralyzed and other mutants of protistan cilia, where a structural defect and a corresponding genetic mutation can directly be correlated with a change in ciliary beat. Similarly, strong support for the applicability of the mechanism to human cilia comes from studies, mainly of ciliary structural defects and genetic mutations associated with impaired mucociliary function in the respiratory system, which produces sinusitis and bronchiectasis. The genetic diseases are now called primary ciliary dyskinesia (PCD). Clinical features of PCD, which include chronic rhinitis\/sinusitis, otitis media, male infertility and an increased incidence of hydrocephalus, point to physiological processes where ciliary motility is essential (Afzelius 2004). As might be anticipated, prominent causes of PCD are mutations that affect the assembly or function of the dynein arms\u2014most commonly a mutation in DNAH5, a gene encoding a dynein heavy chain of the ODAs (Olbrich et al. 2002).\nAny mutation affecting the ciliary beat mechanism will potentially produce PCD, depending on the severity of beat impairment. An instructive example is hydin, a component of the central pair projection complex that interacts with the radial spokes in the switching mechanism between doublet sets. Hydin-deficient flagella of Chlamydomonas become intermittently paralyzed, stopping for long times at the end of the effective or recovery strokes, where the direction of beat is reversed (Lechtreck and Witman 2007). Mice defective in Hydin develop hydrocephalus and die shortly after birth. Lechtreck et al. (2008) analyzed ciliary structure and motility in these mice. The central pair projection assigned to the hydin complex was missing. Mutant cilia were unable to bend properly and frequently stalled, implying that because central pair structure was defective, interactions between the central pair and the radial spokes that affected dynein arm switching were abnormal, and consequently cilia-generated flow was impaired. Motility of tracheal cilia was also impaired. One would predict that human mutations in HYDIN are also likely to result in hydrocephalus or a form of PCD.\nMany people diagnosed with PCD have situs inversus totalis that is reversal of left\u2013right asymmetry of body organs such as the heart. This is now known to be produced, because fluid flow produced by motile cilia at the embryonic node is defective (Hirokawa et al. 2006). The usual flow pattern produced by the cilia is unidirectional right-to-left, and this activates a left-side-only gene cascade, probably by impinging on primary cilia at the left side of the node (McGrath et al. 2003). The nodal cilia are mainly missing the central pair of axonemal microtubules, hence, although motile, are often 9\u00a0+\u00a00, but with some unique ODA dynein isoforms (Supp et al. 1999). It is not known whether the response of the primary cilia at the node is to mechanical displacement (flow sensing) or if it involves morphogens produced in membrane parcels in the node (Tanaka et al. 2005). Morphogen-containing multimembrane vesicles are a feature of nodal response that could also apply respiratory and other ciliated epithelia, where flow occurs.\nWhile mouse mutants or knockouts that are missing cilia develop situs inversus, hydin mutants do not show this phenotype, presumably because their motility is sufficient to generate appropriate nodal flow. In a similar way, by examining the PCD phenotype of mutations in various ciliary proteins, identified by proteomics, it should be possible to dissect details of the function of the protein in the mechanism of beat generation (Avidor-Reiss et al. 2004; Badano et al. 2006; Blacque et al. 2005).\nPrinciples of ciliogenesis\nA major breakthrough in the understanding of the importance of cilia in the body has come from the study of Chlamydomonas ciliogenesis. In that cell, the cilia are traditionally called \u2018flagella\u2019. Ciliogenesis involves transport of materials into, along and out of the cilium, directly visualized as \u2018intraflagellar transport\u2019 or IFT (reviewed by Rosenbaum and Witman 2002). IFT requires molecular motors, several kinesins for anterograde transport and a specialized cytoplasmic dynein for retrograde transport. Coupling the transported cargo to the molecular motors that move along the outer doublets of the axoneme are two protein complexes (Cole 2003) comprising about 19 IFT proteins. Remarkably, this machinery is conserved almost universally wherever cilia are built, and orthologs of the motors and IFT proteins are found in sensory cilia and their derivatives, such as the mammalian photoreceptor (Baker et al. 2003) and in primary cilia, as well as in protistan motile cilia. However, motile cilia of mammalian tissues often assemble in mass (Gaillard et al. 1989; Dirksen 1991). Centriologenesis of hundreds of basal bodies occurs from a fibrogranular mass in the cytoplasm and cilia sprout from the cell surface. It is unclear whether classical IFT is the major mechanism of ciliogenesis in respiratory epithelium, or what modifications of the process occur.\nWheatley (1969), Fonte et al. (1971) and Archer and Wheatley (1971) studied ciliogenesis of primary cilia of established cell lines in vitro. Primary cilia grow when the cells become confluent and reach stationary phase. Cells resorb their cilia shortly before entering mitosis. Postmitotic cells reassemble primary cilia in G1 and maintain the cilium as the cells enter growth arrest (G0) and undergo differentiation. In contrast to motile cilia, the primary cilium emanates uniquely from the distal end of the existing mother centriole of the centrosome, which migrates to the cell surface during growth arrest. Ciliogenesis by IFT is initiated while the centrosome is positioned at the Golgi apparatus near the nucleus, while extensive IFT and elongation of the cilium take place after docking of the nascent cilium at the cell surface, where microtubule pairs are quickly assembled to form the mature axoneme. Two recent and extensive reviews of the complex interactions between IFT particles, molecular motors, centrosomal proteins and other microtubule-associated proteins in ciliogenesis are presented in Pedersen et al. (2008) and Blacque et al. (2008).\nCiliopathies\nThe first human illness that could be linked to primary cilia in epithelial cells was polycystic kidney disease (PKD). One of the orthologs of IFT88 is the mammalian protein polaris mutated in a transgenic mouse Tg737orpk, which was a model for the study of PKD. Chlamydomonas IFT88 mutants are defective in ciliogenesis, and Pazour et al. (2000) demonstrated that, similarly, cilia of the mouse kidney were abnormally short or missing, which suggested that PKD might be a ciliary disease. This conclusion was strengthened by two succeeding discoveries. Mechanical deflection of kidney tubule epithelial cells induces an increase in intracellular calcium (Praetorius and Spring 2001), and the PKD proteins, polycystins 1 and 2 in Ca2+-signaling, localize to the ciliary membrane (Pazour et al. 2002; Yoder et al. 2002). Many other proteins whose functions are disrupted in cystic diseases, e.g. nephrocystins, have now been localized to the cilium or at the ciliary basal body, supporting the conclusion that the primary cilium in kidney tubules partly functions as a mechanosensor that, upon bending, activates a series of signaling pathways in the cilium to control development and homeostasis of the tissue (for reviews, see Yoder et al. 2007; Hildebrandt and Zhou 2007), as originally envisaged by Roth et al. (1988) and Wheatley (1995). Thus, even if primary cilia are present, unless these proteins are targeted appropriately to the cilia or the ciliary basal body, pathology develops.\nIt is now clear that the ciliary membrane is a privileged and specialized compartment for receptor signaling and that the polycystins are only one type of many membrane proteins that must be targeted to the primary cilium to function properly. Other carefully documented examples include the receptor tyrosine kinase PDGFR\u03b1 (Schneider et al. 2005) and the patched receptors for hedgehog signaling (Rohatgi et al. 2007) that control cell growth and differentiation processes. Further, essential downstream components in PDGFR\u03b1 and hedgehog signaling as well as signaling in neurotransmission, Wnt pathways, extracellular matrix interaction and osmolyte transportation uniquely localize to the cilium and\/or to the ciliary centrosome (Gerdes et al. 2007; Corbit et al. 2008; for reviews, see Singla and Reiter 2006; Michaud and Yoder 2006; Christensen et al. 2007; Christensen and Ott 2007). In many of these signaling systems, the cellular response is initiated by receptor activation in the primary cilium. Deficiencies in placement of receptors and their immediate downstream signaling components in the cilium or at the ciliary base results in ciliopathy, including cystic kidney, pancreatic and liver diseases, retinitis pigmentosa, cancer, defective neurogenesis, Bardet-Biedl syndrome, polydactyly, anosmia and other developmental defects. In the Tg737 mouse, these defects lead to early perinatal death. As shown in Fig.\u00a02, primary cilia with hedgehog receptor pathway signaling are found on human embryonic stem cells (Kiprilov et al. 2008), which suggests that ciliary signaling is involved in differentiation from the beginning of embryogenesis. In embryogenesis, signaling through the primary cilium is necessary for normal development, probably because such signaling regulates the balance between cell division, polarity, migration, differentiation and apoptosis for many tissues.\nFig.\u00a02Primary cilia of human embryonic stem cells. Immunofluoresence microscopy using acetylated \u03b1 tubulin antibody (tb) reveals the presence of primary cilia (arrows) on human embryonic stem cells. In the absence of stimulation, the hedgehog receptor \u2018patched\u2019 (Ptc) colocalizes with the acetylated \u03b1 tubulin all along the ciliary membrane. Red and green channels are displaced in the images to define colocalization more clearly. Nuclei are stained with DAPI (blue). Upon stimulation, as part of the signaling cascade, Ptc leaves the cilium and the smoothened receptor (Smo) enters to activate the hedgehog signaling cascade. Asterisk marks the ciliary base. (From Kiprilov et al. 2008, with permission, courtesy of The Journal of Cell Biology)\nPrimary cilia in adult tissues\nPrimary cilia persist in many differentiated cells, including kidney tubule epithelial cells, fibroblasts and neurons, after organogenesis is complete and cell division rates fall. Presumably, as semipermanent structures, the cilia function as mechano- or chemosensors and as a cellular global positioning system to detect changes in the surrounding environment, to initiate cellular replacement after damage, for example. To test this hypothesis, techniques are being used to knock out primary cilia or ciliary proteins in specific tissues of adult organisms.\nDavenport et al. (2007) have completed one of the first of these studies, using an inducible system in adult mice to disrupt IFT in several different ways, causing loss of primary cilia. Respiratory motile cilia are probably more stable and relatively unaffected. Surprisingly, when primary cilia are lost from all adult tissues, the devastating abnormalities and lethality seen after embryonic loss of cilia are not observed. PKD eventually develops a year after induction. The same delay has been reported by Piontek et al. (2007) after adult-specific knockout of polycystin-1. This delay correlates well with a greatly reduced rate of cell division and a different pattern of gene expression in the mature kidney and could explain the increase in human PKD with age. Such changes in cell proliferation rate may relegate the primary cilium of adult tissues to a less immediate role, involving long-term homeostasis of the tissue. Because signaling through primary cilia is coupled to cell cycle events, for example in PDGFR\u03b1\u03b1 signaling in fibroblasts, long-term disruption of ciliary signaling could be a factor in oncogenesis.\nOne adult tissue responds immediately to ciliary knockout however\u2014that is nervous tissue, most specifically neurons in the hypothalamus. Knockout of all adult primary cilia in the mouse, or specifically only of primary cilia on POMC neurons, leads to hyperphagia\u2014compulsive and excessive eating\u2014leading to obesity. Obesity then causes numerous secondary defects resembling type II diabetes. These defects do not occur if the knockout mice are kept on a restricted diet. Eating behavior is regulated by the hormone leptin. These fndings suggest that the leptin receptor might be located in the membrane of the primary cilia of the POMC neurons.\nThe hypothalamic hormone somatostatin is a negative regulator of leptin. While leptin leads to reduced eating, somatostatin increases eating behavior. The somatostatin receptor sst 3 is localized to primary cilia in hypothalamic neurons (Stepanyan et al. 2007). The results of Davenport et al. (2007) suggest that, much like the patched and smoothened receptors work in hedgehog signaling, the leptin receptor and a somatostatin receptor could work in a Yin-Yang relationship within the POMC primary cilium (Satir 2007).\nWhatever the precise cell biological explanation of the relationship between ciliary knockout and hyperphagia, the effect of knockout is behavioral. We might expect that mutations in other proteins of neuronal primary cilia could lead to other behavioral responses. There are reports that the rages that fueled the famous Hatfield-McCoy feud of Appalachia probably were driven by a family suffering from Von Hippel-Landau (VHL) syndrome. VHL protein controls ciliogenesis and is localized to cilia (Schermer et al. 2006). Perhaps a more ciliocentric view of neuronal activity affecting recurrent or addictive actions is warranted.\nConclusions\nBeginning with important electron microscopic studies and culminating in immunolocalization combined with molecular genetic technology, much has been learned about motile, sensory and primary cilia in mammals. Defects in building the primary cilium or mutations in ciliary membrane or axonemal proteins lead to ciliopathies, important human diseases. The cilium has moved to a prominent place in studies of embryogenesis and tissue differentiation and maintenance. There are hints that the fundamental cell biology of cilia will also be important in oncogenesis, aging diseases and human behavioral disorders. The strides of the past half century in understanding this organelle have been impressive, and the promise of discovery in the next half century is compelling.","keyphrases":["motility","primary cilia","signaling","motile cilia","ciliopathies","sensory organelles"],"prmu":["P","P","P","P","P","R"]}
{"id":"Diabetologia-4-1-2270365","title":"Joint effects of HLA, INS, PTPN22 and CTLA4 genes on the risk of type 1 diabetes\n","text":"Background\/hypothesis HLA, INS, PTPN22 and CTLA4 are considered to be confirmed type 1 diabetes susceptibility genes. HLA, PTPN22 and CTLA4 are known to be involved in immune regulation. Few studies have systematically investigated the joint effect of multiple genetic variants. We evaluated joint effects of the four established genes on the risk of childhood-onset type 1 diabetes.\nIntroduction\nThe risk of complex diseases such as type 1 diabetes is generally thought to be influenced by multiple genetic and non-genetic factors, and it has been hypothesised that interactions between genes, or epistasis, are very common for such diseases [1]. The presence of interactions could be one of the reasons why searching for susceptibility loci for many diseases has been less successful than expected [2]. When moving from monogenic diseases to complex diseases, it seems reasonable to assess more than one locus at a time, although models become increasingly complex as the number of loci increases [3].\nWhereas few or no common genetic variants have been firmly established for most common diseases [4], there are now at least four genetic loci that are established as causally involved in the aetiology of type 1 diabetes. They give us a unique possibility to evaluate gene\u2013gene interactions among established susceptibility genes. Specific allelic combinations of DRB1, DQA1 and DQB1 in the human leucocyte antigen (HLA) complex, variants in the insulin gene (INS), the cytotoxic T lymphocyte antigen-4 gene (CTLA4) and the protein tyrosine phosphatase, non-receptor type 22 gene (PTPN22) have been repeatedly associated with type 1 diabetes susceptibility [5\u20138] using different approaches. All established loci are thought to be involved somehow in immune regulation, but details of the mechanisms relating the polymorphisms to risk of type 1 diabetes are in most cases poorly understood.\nEvaluating the joint effects of genes contributes important information for risk prediction, and is also thought to provide information about biological interactions, although the latter is controversial and more complex than commonly thought [2, 3, 9].\nPrevious studies have assessed interaction between HLA and INS and reported divergent results [10\u201318]. The reported results of the joint effect of HLA and INS are confusing not only because they have shown diverging results but also because the definitions and terminology of interactions are not consistent [19]. The interpretation of statistical interaction depends on the choice of scale used to measure the effects [2]. Although additivity of risks is often taken as independence [20], multiplicativity of risk is sometimes also taken as independence [21]. The joint action of HLA and INS has variously been described as being multiplicative [14, 15], additive [13], providing evidence of interaction [15], and non-interacting [13, 14, 17].\nFew studies have investigated the more recently established susceptibility loci PTPN22 and CTLA4 in the context of joint effects on the risk of type 1 diabetes. The studies that have been done have mainly concluded that there is a non-interaction [22, 23] effect, but here also the results have diverged [24\u201327].\nThe aim of our study was to assess the joint effects of the four established susceptibility loci HLA, INS, CTLA4 and PTPN22 in type 1 diabetes, using a consistent approach with both population-based case-control and family trio designs and with large sample sizes.\nMethods\nParticipants We analysed two independent type 1 diabetes data sets. One nuclear family set consisted of 421 trios with mother, father and one child diagnosed in Norway with type 1 diabetes before age 15\u00a0years (225 [53.4%] of the affected children were boys). The families were collected between 1993 and 1997. In families with more than one affected sibling, only the proband was included in the analyses. The case-control data set consisted of 1,331 type 1 diabetes patients (51.9% boys and 48.1% girls) and 1,625 (51.4% boys and 48.6% girls) control participants aged <15\u00a0years. In analyses involving age of disease onset, we divided the data sets according to age of disease onset of the affected child into three groups (0\u20134.9, 5\u20139.9 and \u226510\u00a0years). The controls were randomly selected from the official population registry among children born between 1985 and 1999 and recruited in 2001, as previously described [28]. The patients in the case-control material were from the Norwegian Childhood Diabetes Registry consecutively recruited between 1997 and 2000 [29] and between 2002 and 2005. The type 1 diabetes patients and their family members were recruited by the Norwegian Childhood Diabetes Study Group, including all paediatric departments in Norway. All type 1 diabetes patients were diagnosed according to EURODIAB criteria [30]. The study was approved by the local ethics committee, and informed consent was obtained from all participants or their parents.\nGenomic DNA extraction and genotyping In the majority of the type 1 diabetes case-control samples we used DNA extracted from buccal cells [31]. In all remaining samples, DNA was extracted from peripheral whole blood using a salting-out protocol. Genotyping of HLA-DRB1, \u2212DQA1 and \u2212DQB1 was performed using PCR-SSOP (sequence-specific oligonucleotide probing) mainly following published methods [32], or PCR-SSP (sequence-specific primer) [33, 34], or using time-resolved fluorescence technology in the Delfia assay (Perkin-Elmer Life Sciences, Turku, Finland). HLA genotypes were grouped into four risk categories based on DQB1, DQA1 and DRB1 genotypes, including DRB1*04 subtyping. The majority of DRB1*04-DQA1*0301-DQB1*0302 haplotypes in Norway are DRB1*0401 or \u22120404 (constituting >94% of the haplotypes) [35]. In the present study the other rare subtypes are referred to as DRB1*04XX. Because of the almost complete linkage disequilibrium between INS-VNTR allele classes and the \u221223\u00a0HphI polymorphism, we genotyped \u221223\u00a0HphI (rs689) as a marker for the INS-VNTR. The \u221223\u00a0HphI A allele corresponds to the VNTR class I and the \u221223\u00a0HphI T allele correspond to the VNTR class III. In PTPN22 we genotyped the single nucleotide polymorphism (SNP) Arg620Trp (rs2476601). The SNP JO27_1 (rs11571297) was genotyped in CTLA4. SNP genotyping was performed by TaqMan allelic discrimination assays on an ABI 7900HT DNA Analyzer (Applied Biosystems, Foster City, CA USA). Primer and probe sequences are shown in Electronic Supplementary Material (ESM) Table\u00a01. The PCR conditions are available on request.\nData analysis The HLA haplotypes were grouped as high risk, intermediate risk, neutral risk and low risk according to the following criteria: high-risk category, DRB1*0401\/04XX-DQA1*03-DQB1*0302\/DRB1*03-DQA1*05-DQB1*0201 (DR4-DQ8\/DR3-DQ2); low-risk category, all genotypes with at least one DQB1*0602 allele; intermediate-risk category, DRB1*0404-DQ8\/DR3-DQ2, DR3-DQ2\/DR3-DQ2, DR4-DQ8\/DR4-DQ8 (with the exception of DRB1 0404-DQ8 homozygotes, which were grouped as neutral), DRB1*0401 or 040XX-DQ8\/X (X\u2260DQB1*0602 or DR3-DQ2). The remaining haplotypes were grouped in the neutral-risk category. For assessment of two-locus joint effects, we pooled genotypes of INS, PTPN22 and CTLA4 as follows: INS class I\/I genotypes were compared with I\/III together with III\/III genotypes. The PTPN22 TT and CT genotypes were compared with CC. CTLA4 (JO27_1) TT genotypes were compared with TC and CC genotypes. Not all individuals were genotyped for non-HLA polymorphisms because of lack of DNA. We did not exclude individuals with some missing genotypes to prevent the loss of important information when looking at joint effects between polymorphisms. The available numbers of individuals for each analysis are seen in the tables. Data were presented using stratified 2\u2009\u00d7\u20092 tables and analysed using logistic regression models including interaction (product) terms, in SPSS for Windows (version 14.0; SPSS, Chicago, IL, USA). In addition to formal analyses treating HLA categories as categorical in the logistic regression, we also tested for interactions when treating HLA category as a continuous variable coded 1, 2, 3, 4, thus maximising the power under alternative models where the effect of a non-HLA locus (as measured by the odds ratio [OR]) was assumed to decrease (or increase) (logit) linearly over the four HLA risk categories (test for interaction with one degree of freedom). Case-only analyses were used to estimate interaction parameters and to test for deviation from multiplicative effects using logistic regression [36]. In addition to the increased power obtained by utilising all cases (from case-control and trio materials) simultaneously, case-only analyses have increased power by making the implicit assumption that there is no association between the two loci in the population; i.e. the OR for their association is 1.0 in the population. Thus, under reasonable assumptions the case-only analysis makes the most efficient use of data to assess deviation from multiplicative models. In the case-control analysis, likelihood ratio tests comparing nested logistic regression models were used as global tests for interaction. The transmission disequilibrium test [37] was performed using the UNPHASED application implemented in the UNPHASED software version 2.4 [38]. For the trio data, 95% confidence intervals for the relative risk were estimated using conditional logistic regression in UNPHASED. Receiver operating curve (ROC) and confidence bounds for the area under the curve were estimated assuming a non-parametric distribution and analyses were done using SPSS version 14.0. Genotypes were added sequentially in order of likelihood ratio (or equivalently by the absolute risk conferred by a given genotype combination estimated using Bayes\u2019 formula). Two four-locus genotype combinations were absent among cases in our material, and a very low value for the estimated absolute risk was imputed for these to allow inclusion in the ROC curve estimation with all four loci simultaneously. A p value <0.05 was considered to be statistically significant.\nResults\nThe single-locus main effects are shown in ESM Table\u00a02 (case-control data) and ESM Table\u00a03 (trios). Compared with the neutral HLA risk category, the high-risk category showed a strong association with type 1 diabetes, with OR 20.6; for the intermediate-risk category the OR was 5.7 and for the low-risk category it was 0.09. INS, PTPN22 and CTLA4 also showed an association with type 1 diabetes, as expected. The transmission of the risk allele in the nuclear families confirmed the associations in INS, PTPN22 and CTLA4 (JO27_1), although with borderline significance for JO27_1 (ESM Table\u00a03).\nJoint effect of HLA and PTPN22 ORs for the effect of PTPN22 varied across the HLA risk categories and were significant in some of the subgroups. The ORs were smaller for the risk-conferring HLA genotypes, indicating negative deviation from a multiplicative model. A global test of interaction (with 3\u00a0df) between HLA and PTPN22 in the logistic regression model confirmed a significant interaction (p\u2009=\u20090.024). In the trio data, the relative risk conferred by the PTPN22 T allele was similar in the strata defined by HLA group, with no evidence for deviation from a multiplicative model (ESM Table\u00a04). A case-only analysis among all cases from the case-control and family materials (ESM Table\u00a05) supported a significant negative deviation from multiplicative effects, with weaker ORs conferred by PTPN22 in the HLA risk categories (3\u00a0df test for interaction; p\u2009=\u20090.028). When treating HLA-encoded risk as a continuous variable in the analysis (1\u00a0df), the interaction was even more statistically significant (p\u2009=\u20090.003). There was no association between HLA and PTPN22 among the controls (3\u00a0df test; p\u2009=\u20090.19). We tried to fit the case-control data to an additive odds model using generalised linear models in STATA (version 9), as described by Skrondal [39]. However, convergence was not obtained, suggesting that the data did not fit well to an additive model.\nJoint effect of HLA and INS The 3\u00a0df test for interaction between INS and HLA was not statistically significant (p\u2009=\u20090.67). There was also no statistically significant deviation from a multiplicative model in the trio data (test for interaction, p\u2009=\u20090.5) (ESM Table\u00a04) or in the case-only analysis (3\u00a0df test; p\u2009=\u20090.49) (ESM Table\u00a05); even when treating HLA-encoded risk as a continuous variable in the analysis there was no significance (1\u00a0df test, p\u2009=\u20090.12). There was also no association between INS and HLA among controls, as expected (3\u00a0df test; p\u2009=\u20090.41).\nJoint effect of HLA and CTLA4 The ORs for CTLA4 in the different HLA categories (Table\u00a01) indicated no deviation from a two-locus multiplicative model (3\u00a0df test; p\u2009=\u20090.53). This was also the case in the trio data set (ESM Table\u00a04) and was supported by the case-only analysis (ESM Table\u00a05; 3\u00a0df test; p\u2009=\u20090.57). Again, there was no association between the two loci among controls (3\u00a0df test; p\u2009=\u20090.21).\nTable\u00a01Interaction between HLA-INS (-23HphI), HLA-PTPN22 (Arg620Trp) and HLA-CTLA4 (JO27_1) in the case\u2013control data set using logistic regressionHLA categoryaNon-HLA genotypesCasesControlsOR95% CITest for interaction (p value)n (%)n (%)INSb0.67High riskI-I218 (25.2)20 (2.8)1.560.76\u20133.21III+98 (28.6)14 (2.5)1Intermediate riskI-I412 (47.6)122 (17.1)2.101.52\u20132.89III+160 (46.8)99 (17.4)1Neutral riskI-I221 (25.5)346 (48.5)2.341.74\u20133.15III+81 (23.7)299 (52.6)1Low riskI-I14 (1.6)225 (31.6)3.260.92\u201311.54III+3 (0.9)156 (27.5)1Total12071281PTPN220.024High riskTT\u2009+\u2009TC89 (23.2)8 (3.1)1.260.55\u20132.88CC237 (27.8)27 (2.6)1Intermediate riskTT\u2009+\u2009TC177 (46.1)55 (21.2)1.310.91\u20131.86CC406 (47.7)164 (15.6)1Neutral riskTT\u2009+\u2009TC111 (28.9)121 (46.7)2.441.80\u20133.31CC199 (23.4)536 (51.0)1Low riskTT\u2009+\u2009TC7 (1.8)75 (29.0)3.271.18\u20139.06CC9 (1.1)323 (30.8)1Total12351309CTLA40.53High riskTT122 (24.9)14 (3.6)0.900.45\u20131.84TC-CC210 (27.9)22 (2.5)1Intermediate riskTT241 (49.2)65 (16.5)1.591.14\u20132.23TC-CC346 (46.0)148 (16.7)1Neutral riskTT121 (24.7)212 (53.8)1.341.01\u20131.77TC-CC187 (24.9)439 (49.7)1Low riskTT6 (1.2)103 (26.1)1.800.63\u20135.18TC-CC9 (1.2)275 (31.1)1Total12421278n, Number of cases\/controlsaHLA risk categories: high risk, DQA1*03-DQB1*0302\/DQA1*05-DQB1*0201 (DQ8\/DQ2), where DRB1\u22600404; low risk, at least one DQB1*0602 allele independent of genotype on the other allele; intermediate risk, DRB1*0404-DQ8\/DR3-DQ2, DR3-DQ2\/DR3-DQ2, DR4-DQ8\/DR4-DQ8 (excluding homozygous DRB1 0404), DRB1*0401 or 040x-DQ8\/X (X\u2260DQB1*0602 or DR3). The remaining haplotypes were grouped in the neutral risk category (see Methods)bRepresents genotypes III\/III and I\/III\nJoint effects of non-HLA loci There was also no indication of deviation from multiplicative two-locus joint effects of PTPN22-INS, INS-CTLA4 or PTPN22-CTLA4 in the case-control data (Table\u00a02) or in the case-only analysis (ESM Table\u00a05) (all p\u2009>\u20090.39). For the trios, the test for interaction between PTPN22 and CTLA4 showed p\u2009=\u20090.046 (ESM Table\u00a06). Taken together with the analysis of the case-control and the case-only data, this weighs against any deviation from a multiplicative two-locus joint effect also of CTLA4 and PTPN22.\nTable\u00a02Interaction between INS-PTPN22, INS-CTLA4 and PTPN22-CTLA4 in the case-control data set using logistic regressionNon-HLA genotypesCasesControlsOR95% CITest for interactiona (p value)n (%)n (%)INSPTPN\u00a0\u00a0\u00a0\u00a00.67I-ITT+TC274 (72.9)156 (58.4)1.741.38\u20132.18I-ICC594 (71.6)587 (54.8)1\u00a0III+TT+TC102 (27.1)111 (41.6)1.891.38\u20132.58III+CC236 (28.4)485 (45.2)1\u00a0Total (n)\u00a01,2061,339\u00a0\u00a0INSCTLA4\u00a0\u00a0\u00a0\u00a00.42I-ITT354 (73.1)222 (53.9)1.511.23\u20131.86I-ITC-CC520 (71.0)492 (54.8)1\u00a0III+TT130 (26.9)190 (46.1)1.310.99\u20131.73III+TC-CC212 (29.0)405 (45.2)1\u00a0Total (n)\u00a01,2161,309\u00a0\u00a0PTPNCTLA4\u00a0\u00a0\u00a0\u00a00.78TT+TCTT143 (29.7)75 (17.7)1.461.04\u20132.05TT+TCTC-CC239 (31.7)183 (20.1)1\u00a0CCTT339 (70.3)348 (82.3)1.41.15\u20131.67CCTC-CC514 (68.3)729 (79.9)1\u00a0Total (n)\u00a01,2351,335\u00a0\u00a0The low-risk genotypes were used as reference (CC, TC-CC, TC-CC)aLikelihood ratio tests of whether the OR conferred by one locus is significantly different over strata defined by genotypes in the other locusn, Number of cases\/controls\nJoint effects of more than two susceptibility loci We also tested models with all three-way and four-way interactions involving the four susceptibility loci using logistic regression (categorising all loci in two groups: increased risk genotypes or not), but none of the multi-way interactions were statistically significant (all p\u2009>\u20090.29). The simultaneous distribution of risk genotypes at all four loci among cases and controls is shown in ESM Table\u00a07. The results show that the more risk loci an individual carries, the higher the relative risk, but the presence or absence of HLA risk loci influences the relative risk much more than the other loci, as expected. For instance, carrying risk genotypes at all three non-HLA loci but not at HLA is associated with a much lower risk than HLA risk genotypes together with low-risk genotypes at all three other loci. The relative risk (OR) conferred by simultaneously carrying high- or moderate-risk HLA and risk genotypes at all the three other loci compared with non-risk-associated genotypes at all four loci was 61. The expected relative risk under a strict multiplicative model involving all four loci was 123 (multiplying all four single-locus effects by each other). The relatively small number of individuals simultaneously carrying all risk genotypes indicates that the observed negative deviation from a four-way multiplicative model was not statistically significant, in accordance with the formal test cited above.\nROC curve Another way to assess the predictive utility of combinations of genetic risk markers is the ROC curve [40]. This utilises the genotypes of all included individuals and assesses the combination of sensitivity and specificity of different combinations of genotypes. ROC curves for HLA alone, pairwise combinations of HLA and non-HLA loci, and multiple genotypes (Fig.\u00a01) showed an area under the curve of 0.82 for HLA alone, which was only marginally increased by adding non-HLA loci.\nFig.\u00a01ROC curve for HLA genotypes in four categories and for combinations of genotypes defined by HLA and non-HLA susceptibility loci. The area under the curve (95% confidence interval) was 0.820 (0.803\u20130.836) for HLA (dark blue line), 0.828 (0.811\u20130.844) for HLA+CTLA4 (purple line), 0.835 (0.819\u20130.851) for HLA+PTPN22 (grey line), 0.840 (0.824\u20130.855) for HLA+INS (green line), 0.848 (0.833\u20130.863) for HLA+INS+PTPN22 (yellow line) and 0.852 (0.837\u20130.867) for HLA+INS+PTPN22+CTLA4 (red line). Turquoise dashed line, reference line\nAge of disease onset and sex We found no significant deviation from a multiplicative model concerning age\u2013locus and sex\u2013locus interaction for any of the genes. This was confirmed in the trio families and case-only analysis (ESM Tables\u00a08 and 9).\nDiscussion\nThe present study is a comprehensive evaluation of joint effects of the four most well established type 1 diabetes susceptibility genes in both a large case-control series and family material. The relative risk conferred by PTPN22 was stronger in the lower-risk HLA categories than in the high-risk HLA category, while all other two-locus combinations (HLA-INS, HLA-CTLA4, INS-CTLA4, INS-PTPN22 and PTPN22-CTLA4) were consistent with multiplicative models. Although model-free methods have been developed for gene\u2013gene interaction studies, such as multifactor dimensionality reduction (see [1] and references therein), these methods are designed for the detection of novel susceptibility loci, which was not the goal of our investigation.\nTwo of the three previous studies of the joint effect of PTPN22 and HLA were in accordance with our results [25, 27] while the other study found no deviation from a multiplicative model [22]. It should be noted that the interaction between PTPN22 and HLA found in the case-control material and case-only analysis was not replicated in our trio data. One of the reasons for this could be lower statistical power in the trio data. Using the Quanto program ([41]; http:\/\/hydra.usc.edu\/gxe), we found that we had more than 80% power to detect significant two-way gene\u2013gene interaction if the true interaction parameter was 0.5. For the trio design we would need as many trios as we had cases in the case-control study to obtain a similar power. Our number of trios was only about a third of the number of cases in the case-control study, with consequently lower power. The case-only design is known to be the most efficient to detect interaction under certain assumptions. For instance, we had >99% power to detect interaction if the true interaction parameter was 0.5.\nThe few studies concerning two-locus interaction effects between HLA and CTLA4 and among non-HLA genes [22] have generally indicated multiplicative effects, which is in accordance with our results. Some previous studies have found that the relative risk conferred by INS was similar in subgroups defined by HLA susceptibility genes [11, 12], while three studies have indicated that the effect was stronger in the low risk-HLA categories [16\u201318], a finding that was only partially supported by our data. On the other hand, one relatively small study has found that the effect of INS was confined to the high-risk HLA-DR4 group [10].\nThe reason for diverging results of the joint effect of established type 1 diabetes susceptibility genes in the literature could be that the studies have been performed with varying sample sizes and with different study designs. Linkage studies [14, 15], case-control association studies [10, 12, 13, 16] and family trio designs [16, 18, 22, 25] have all been used in these studies. Smaller studies might be inadequate to reveal significant interactions. Different criteria for categorising the HLA risk groups could potentially influence the result of the joint effect of HLA and other type 1 diabetes susceptibility genes. However, our conclusions were not affected by alternative classification of HLA risk groups, such as into DR4-DQ8 vs DR3-DQ2 carriers (data not shown). Studies in different populations and ethnic groups have indicated some heterogeneity in HLA-associated risk of type 1 diabetes and it is also possible that gene\u2013gene interactions may vary across populations. However, despite the observed variations in population risk of type 1 diabetes and in HLA haplotype frequencies across populations, the relative predisposing effects of HLA haplotypes seem to be consistent across populations [42]. In our study all the patients were diagnosed before 15 years of age. The fact that the relative risks associated with both risk genotypes and low-risk genotypes seem to diminish with age above 15\u00a0years [43] raises the question whether gene\u2013gene interactions may also differ in different age-groups.\nAlthough no preventive intervention is available for type 1 diabetes today, prediction of disease is an important part of strategies for prevention, both for recruitment of participants for research studies and for identification of target populations for future preventive interventions. Understanding the interacting effect of the established type 1 diabetes susceptibility genes will increase this possibility. In a multiplicative model the relative risk (RR) for a person holding a high-risk genotype at both loci compared with a person with low risk at both loci will be RRlocus1\u2009\u00d7\u2009RRlocus2. In terms of absolute risk differences, a doubling or tripling of risk due to INS or PTPN22 would be greater for a person with a high-risk HLA genotype than it would for someone with a low-risk HLA genotype. The absolute risk for persons with a given genotype can be estimated by multiplying the average cumulative incidence in the population (0.42% cumulative risk up to age 15\u00a0years in Norway [44]) by the ratio of genotype frequency in patients and genotype frequency in controls [18], or using Bayes\u2019 formula. For instance, for a person with a low-risk HLA genotype, a high- or low-risk INS genotype will define whether the estimated absolute risk is approximately 0.01% or 0.028% (absolute risk difference, 0.027%), whereas for those with the high-risk HLA genotype INS will define an estimated risk of approximately 3.0% or 4.7% (absolute risk difference 1.7%).\nAs discussed in the general setting by Janssens et al. [40], increasing the number of susceptibility loci considered simultaneously generally increases the predictive value for disease. The downside is that the proportion of the population simultaneously carrying multiple risk alleles becomes minute even with a moderate number of susceptibility polymorphisms, and that even with relatively large data sets, as in our study, the absolute risk estimate becomes imprecise. The high-risk HLA genotype is carried by fewer than 3% of population controls, but confers a very high risk of disease. Several practical and scientific aspects of prediction should be considered when evaluating the utility of different prediction regimes. The ROC curve analysis confirms that, despite the higher absolute risk for those few with combinations of several risk markers, adding non-HLA genetic markers only marginally increases the utility of the prediction over that of HLA alone. While up to six susceptibility loci in addition to those studied here have recently been established in type 1 diabetes [45], the magnitude of the effect for each additional locus is very much smaller than that of HLA and even smaller than that of INS and PTPN22, suggesting that they are likely to add only marginally to the prediction of disease in individuals. Furthermore, an informal assessment of the number needed to be genetically screened in order to obtain a cohort of high-risk individuals, which will give rise to a given number of cases of type 1 diabetes, and the costs connected to the genotyping also suggest limited cost-effectiveness in adding non-HLA genetic markers to the prediction regime (data not shown).\nIn conclusion, in this comprehensive study of interactions among established type 1 diabetes susceptibility genes, we found that the joint effect of HLA and PTPN22 was significantly less than multiplicative in the case-control material, while a multiplicative model could not be rejected for HLA-INS, HLA-CTLA, PTPN-INS, INS-CTLA4 and PTPN-CTLA4. Despite near-multiplicative effects for most loci, and the fact that groups with very high relative risk of type 1 diabetes can be identified by testing for multiple susceptibility genes, only a small proportion of the population (and cases with type 1 diabetes) simultaneously carry HLA and multiple non-HLA susceptibility genotypes.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nESM Table\u00a01\n(PDF 14.3 kb)\nESM Table\u00a02\n(PDF 110 kb)\nESM Table\u00a03\n(PDF 69.2 kb)\nESM Table\u00a04\n(PDF 51.2 kb)\nESM Table\u00a05\n(PDF 31.9 kb)\nESM Table\u00a06\n(PDF 48.8 kb)\nESM Table\u00a07\n(PDF 25.9 kb)\nESM Table\u00a08\n(PDF 68.0 kb)\nESM Table\u00a09\n(PDF 25.1 kb)","keyphrases":["ins","hla","ptpn22","ctla4","genes","type 1 diabetes","interaction"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"J_Comp_Physiol_A_Neuroethol_Sens_Neural_Behav_Physiol-4-1-2323032","title":"Mechanics of the exceptional anuran ear\n","text":"The anuran ear is frequently used for studying fundamental properties of vertebrate auditory systems. This is due to its unique anatomical features, most prominently the lack of a basilar membrane and the presence of two dedicated acoustic end organs, the basilar papilla and the amphibian papilla. Our current anatomical and functional knowledge implies that three distinct regions can be identified within these two organs. The basilar papilla functions as a single auditory filter. The low-frequency portion of the amphibian papilla is an electrically tuned, tonotopically organized auditory end organ. The high-frequency portion of the amphibian papilla is mechanically tuned and tonotopically organized, and it emits spontaneous otoacoustic emissions. This high-frequency portion of the amphibian papilla shows a remarkable, functional resemblance to the mammalian cochlea.\nIntroduction\nThe anatomy and physiology of the amphibian ear show both remarkable resemblances and striking differences when compared to the mammalian auditory system. The differences between the human and the amphibian auditory system are too significant to warrant direct generalizations of results from the animal model to the human situation. However, studying hearing across species helps to understand the relation between the structure and function of the auditory organs (Fay and Popper 1999). Thus, we hope and expect that the knowledge gained about the amphibian auditory system fits into our understanding of auditory systems in general.\nOver the course of history, a number of diverse amphibian species developed. Currently only three orders remain: anurans, urodeles, and caecilians. Their evolutionary relationship, as well as the evolutionary path of the individual orders, is still under debate. However, they are generally grouped into a single subclass, Lissamphibia of the class Amphibia (Wever 1985).\nThe ancestral lineage of amphibians separated from the mammalian lineage, approximately 350 million years ago, in the paleozoic era. Many of the important developments in the auditory systems emerged after the ancestral paths separated (Manley and Clack 2003). This implies that shared features, like the tympanic middle ear, developed independently in different vertebrate lineages.\nThe anurans -frogs and toads- form the most diverse order of amphibians. The living species are classified into two suborders, Archaeobatrachia and Neobatrachia (Wever 1985). Both within and between these suborders, there is a large variation in the anatomy and physiology of auditory systems. The most thoroughly studied species belong to the family Ranidae, as is reflected in the work referenced in this paper.\nThe hearing organs of anurans are often falsely assumed to be more primitive than those of mammals, crocodiles, and birds. The relatively simple structure and functioning of the amphibian ear offer an excellent possibility to study hearing mechanisms (e.g., Ronken 1990; Meenderink 2005). On the other hand, the sensitivity of the frog inner ear, which appears to be able to detect (sub)angstr\u00f8m oscillations (Lewis et\u00a0al. 1985), shows that the frog ear functions as a sophisticated sensor.\nWhile the ears of most vertebrate species contain one dedicated acoustic end organ, the frog ear has two, the amphibian papilla and the basilar papilla.\n1 Like in other vertebrates, these organs contain hair cells for the transduction of mechanical waves into electrical (neural) signals. In mammals, birds and lizards, the hair cells are set on a basilar membrane. The frog inner ear lacks such a flexible substrate for its sensory cells. The hair bundles of the frog\u2019s auditory organs are covered by a tectorial membrane, as they are in all terrestrial vertebrates except for some lizards species (Manley 2006).\nIn mammals, the mechanical tuning of the basilar membrane is the primary basis for frequency selectivity. In the absence of the basilar membrane, the frog\u2019s auditory organs must rely solely on the tectorial membrane and on the hair cells themselves for frequency selectivity.\nRecently, Simmons et\u00a0al. (2007) and Lewis and Narins (1999) published reviews of the frog\u2019s ear anatomy and physiology. In the current paper, we focus on the mechanics of the inner ear, specifically on the mechanics of the tectorial membrane. Only one publication exists on direct mechanical\/acoustical measurements of structures in the frog inner ear (Purgue and Narins 2000a). Therefore, many of our inferences will result from indirect manifestation of inner ear mechanics, as observed in anatomical, electro-physiological and otoacoustic-emission studies. Nevertheless, these studies provide a consistent view of the mechanics of the anuran inner ear.\nAnatomy\nMiddle ear\nThe ears of most terrestrial vertebrates can be divided into three principal parts: the outer ear, the middle ear and the inner ear. In mammals, the outer ear consists of a pinna and an ear canal, which terminates at the tympanic membrane. In most frog species the outer ear is absent,\n2 and the tympanic membrane is found in a bony ring, the tympanic annulus, in the side of the skull.\nThe tympanic membrane defines the distal boundary of the middle ear cavity. This air-filled cavity is spanned by the ossicular chain, which serves to transfer vibrations of the tympanic membrane to the oval window of the inner ear. In the frog, the ossicular chain consists of two structures, the extra-columella and the columella (J\u00f8rgensen and Kanneworff 1998; Mason and Narins 2002a). The cartilaginous extra-columella is loosely connected to the center of the tympanic membrane. Medially, it flexibly connects to the partially ossified columella. The columella widens to form a footplate at its medial end, where it attaches to the oval window of the inner ear. Acoustic stimuli primarily enter the inner ear through the oval window.\nThe middle-ear\u2019s primary function is to compensate for the impedance mismatch between the air and the fluid-filled inner ear. There are two contributions to this compensation (Jaslow et\u00a0al. 1988; Werner 2003). The first contribution results from the small area of the oval window relative to the area of the tympanic membrane. This causes a concentration of the external force exerted on the tympanic membrane. The second contribution involves a lever action of the columella footplate. The footplate attaches to the otic capsule along its ventral edge. This bond is suggested to be the location of the hinging point of the middle ear lever in the frog (J\u00f8rgensen and Kanneworff 1998; Mason and Narins 2002b). The lever action serves as a force amplification mechanism and contributes to the impedance matching between the outside air and the fluids in the inner ear. Both effects result in pressure amplification between the tympanic membrane and the columella footplate, thus overcoming the impedance mismatch between air and the inner-ear fluids.\nAn additional bony disk, the operculum, is flexibly attached to the oval window in amphibians. The presence of an operculum in anurans is unique among vertebrates. The operculum\u2019s position in the oval window can be modulated through the m. opercularis, which also connects it to the shoulder girdle.\nThe function of the operculum is not entirely clear. Possibly, it serves to transfer substrate vibrations to the inner ear (Lewis and Narins 1999; Mason and Narins 2002b). The putative path for these vibrations includes the front limbs, the shoulder girdle and the m. opercularis (Hetherington 1988; Wever 1985).\nAlternatively, the operculum-columella system is proposed to protect the inner ear\u2019s sensory organs from excessive stimuli. This protection hypothesis takes two different forms. Wever (1985) suggests that the operculum and the columella footplate can be locked together through muscle action. In this manner, the flexibility of the connection to the oval window decreases and the input impedance increases, which in turn decreases the input signal amplitude of the pressure wave in the inner ear. It has also been suggested that the action of the m. opercularis could uncouple the operculum and the footplate (Mason and Narins 2002b). This would allow the operculum to move out of phase with the footplate. The out-of-phase motion could absorb part of the inner ear fluid displacement caused by the motion of the footplate. Effectively this creates an energetic by-pass and decreases the amplitude in the inner ear.\nA tympanic middle ear, as described above, is considered to be the typical situation (Jaslow et\u00a0al. 1988), which can be found in the family Ranidae. However, a wide range of variations in middle ear structures is found across species. In some species, a bony disk occupies the tympanic annulus rather than a membrane, for example, Xenopus leavis (Wever 1985), and there are a number of \"earless\" frogs. The tympanic membrane and the tympanic annulus are absent in these species. A functioning inner ear and a partial middle ear usually exist, although the middle ear cavity may be filled with connective tissue (e.g., Telmatobius exsul, Jaslow et\u00a0al. 1988), or not exist at all (e.g., species in the Bombina family, Hetherington and Lindquist 1999; Wever 1985). Remarkably, some of these \"earless\" frogs have a mating call and exhibit neurophysiological responses (Bombina bombina, Walkowiak 1988; Atelopus, Lindquist et\u00a0al. 1998) at typically auditory frequencies, which implies they have another path for the transfer of airborne sound to the inner ear (Jaslow et\u00a0al. 1988), for example, through the lungs (Narins et\u00a0al. 1988; Lindquist et\u00a0al. 1998; Hetherington and Lindquist 1999).\nInner ear\nThe inner ear in the frog has two membranous windows: the oval window and the round window. As mentioned above, acoustic energy primarily enters the inner ear through the oval window. The round window is the main release point of this energy (Purgue and Narins 2000a). A similar lay-out can be found in other terrestrial vertebrates. However, the round window of the frog does not open into the middle ear as it does in mammals. Rather, it can be found in the top of the mouth cavity, under a lining of muscle tissue.\nWithin the inner ear, there are two intertwined membranous compartments: the perilymphatic and the endolymphatic labyrinths (see Fig.\u00a01). The perilymphatic labyrinth connects to both the oval window and the round window. Starting at the oval window and going medially, it passes through a narrow foramen, and widens into the otic cavity, forming the periotic cistern. Continuing medially it narrows again into the periotic canal. This canal connects the periotic cistern with the perilymphatic space at the round window (Purgue and Narins 2000b). Fig.\u00a01Schematic drawing of a transverse section through the frog ear (adapted from Wever 1985). The division into the middle, and inner ear is indicated above the image; a selection of features is indicated in the image. The colored arrows indicate the paths of vibrational energy: green arrows represent the columellar path, red arrows the putative opercular path, and blue arrows indicate the path through the inner ear after combination of the columellar and opercular paths. The grey areas represents endolympatic fluid, dark yellow perilymphatic fluid. The green areas indicate the tectorial membranes in the papillae. (Color figure is available in the online version)\nBetween the lateral perilymphatic cistern and the round window, part of the endolymphatic space can be found. The endolymphatic space also includes the semi-circular canals located dorsally from the otic system. It contains the sensory organs of hearing and balance. In the frog inner ear, there are eight sensory epithelia (Lewis and Narins 1999; Lewis et\u00a0al. 1985), located as follows:\nthree cristae in the semi-circular canals, which are sensitive to rotational acceleration of the head, and one each in:\nthe utricule, which detects linear acceleration,the lagena, which detects both linear acceleration and non-acoustic vibrations (Caston et\u00a0al. 1977),the sacculus, which is sensitive substrate vibrations up to approximately 100\u00a0Hz, and also detects high level low-frequency airborne sound, (Narins 1990; Yu et\u00a0al. 1991)the amphibian papilla, which detects low-frequency acoustic stimuli (Feng et\u00a0al. 1975), andthe basilar papilla, which is sensitive to high-frequency airborne stimuli (Feng et\u00a0al. 1975). Hair cells are the sensory cells in all of these organs. Like all hair cells, these cells have a stereovillar bundle on their apical surface. Deflection of the bundle as a result of an acoustical vibration or a mechanical acceleration, initiates an ionic transduction current into the cell. This initial current causes a cascade of ionic currents, eventually resulting in the release of neurotransmitter at the basal surface of the cell. The released neurotransmitter triggers neural activity in the nerve fiber dendrites that innervate to the basal portion of the hair cell (Pickles 1988; Yost 2000; Keen and Hudspeth 2006).\nAs in most vertebrates, a tectorial membrane covers the sensory cells of the auditory end organ. This membrane is a polyelectrolyte gel, which lies on the stereovilli (Freeman et\u00a0al. 2003). The function of the tectorial membrane is not well understood, and may vary between classes. However, since the stereovilli in most vertebrate ears connect to this membrane, it obviously plays an important role in the conduction of acoustic vibrations to the hair cells.\n3\nBasilar papilla\nThe basilar papilla is found in a recess that opens into the saccular space at one end, and is limited by a thin contact membrane at the other. The contact membrane separates the endolymphatic fluid in the papillar recess from the perilymphatic fluid at the round window (Lewis and Narins 1999; Wever 1985). The recess perimeter is roughly oval in shape; in the bullfrog, Rana catesbeiana, its major axis is approximately 200\u00a0\u03bcm long, while the minor axis measures approximately 150\u00a0\u03bcm (Van Bergeijk 1957). In the leopard frog, Rana pipiens pipiens, it is of similar size (personal observation, RLMS & JMS).\nThe oval perimeter of the lumen is formed from limbic tissue; a substance unique to the inner ear, and similar to cartilage (Wever 1985). The sensory epithelium is approximately 100\u00a0\u03bcm long. It occupies a curved area that is symmetrical in the major axis of the elliptical lumen. It contains approximately 60 hair cells (measured in Rana catesbeiana), from which the stereovilli protrude into the lumen and connect to the tectorial membrane (Frishkopf and Flock 1974). Typically the orientation of the hair cells, as defined by the direction to which the v-shape of the stereovilli bundle points (Lewis et\u00a0al. 1985), is away from the sacculus in Ranidae.\nThe tectorial membrane spans the lumen of the papillar recess. It occludes about half the lumen, and consequently takes an approximately semi-circular shape when viewed from the saccular side (Frishkopf and Flock 1974; Wever 1985). The membrane has pores at the surface closest to the epithelium, into which the tips of the hair bundles project (Lewis and Narins 1999).4\nAmphibian papilla\nThe amphibian papilla can be found in a recess, that extends medially from the saccular space and, in frogs with derived ears, bends caudally to end at a contact membrane. Like the basilar papilla\u2019s contact membrane, the membrane separates the endolymphatic fluid in the papilla recess from the perilymphatic fluid at the round window.\nThe sensory epithelium is set on the dorsal surface of this recess (Lewis and Narins 1999). The epithelium itself has a complex shape; it consists of a triangular patch at the rostral end, and an s-shaped caudal extension towards the contact membrane (see Fig.\u00a02). The exact shape and length of the caudal extension varies across species, with the most elaborate extensions occurring in species of the family Ranidae (Lewis 1984), while some species lack the s-shaped extension altogether (Lewis 1981). Fig.\u00a02Schematic drawing of the amphibian papilla of the bullfrog, Rana catesbeiana, (adapted from Lewis et al. 1982), rotated to match orientation of Fig.\u00a01). TM Tectorial membrane, AP amphibian papilla. a General overview of the AP; the dashed outline indicates the location of the sensory epithelium, b hair cell orientation in the sensory epithelium; dashed line indicates the position of the tectorial curtain. The numbers along the perimeter indicate the characteristic frequency of the auditory nerve fibers connecting to that site (in Hz)\nIn the epithelium, the hair cell orientation follows a complicated pattern (see Fig.\u00a02b). In the rostral patch the cells are orientated towards the sacculus. On the rostral half of the s-shaped extension, they are oriented along the s-shape. However, on the caudal half, the orientation rotates 90\u00b0 to become perpendicular to the s-shape (Lewis 1981).\nAn elaborate tectorial membrane is found on the hair bundles. A bulky structure covers the rostral patch, while the membrane gets thinner along the caudal extension (Lewis et\u00a0al. 1982). A tectorial curtain spans the papilla recess approximately halfway between the sacculus and the contact membrane (Shofner and Feng 1983; Wever 1985). The curtain, also called the sensing membrane (Yano et\u00a0al. 1990), spans the entire cross-section of the lumen. A small slit in the tectorial curtain may function as a shunt for static fluid pressure differences (Lewis et\u00a0al. 1982).\nResponse of the auditory end organs\nAs mentioned in the section \u201cAnatomy\u201d, the oval window serves as the primary entry point of acoustic energy into the inner ear; the round window presumably serves as the primary release point. After the energy passes through the oval window, it enters the periotic cistern. Between this relatively large perilymphatic space and the round window there are two possible routes: through the endolymphatic space, or through the periotic canal, bypassing the endolymphatic space and the sensory organs altogether (Purgue and Narins 2000a). The bypass presumably serves to protect the sensory organs against low-frequency over stimulation (Purgue and Narins 2000b).\nThe vibrational energy that ultimately stimulates the auditory end organs predominantly may enter the endolymphatic space through a patch of thin membrane in its cranial wall near the sacculus. This entry-point was identified by Purgue and Narins (2000b), by mechanically probing the perimeter of the endolyphatic space. After entering the endolymphatic space, the energy may pass either through the basilar papilla\u2019s or through the amphibian papilla\u2019s lumen to the round window.\nMeasurements of the motion of the respective contact membranes show that there is a frequency-dependent separation of the vibrational energy between paths through the amphibian and the basilar papilla (Purgue and Narins 2000a; see Fig.\u00a03c). The accompagnying dynamic model of the energy flow through the bullfrog\u2019s inner ear (Purgue and Narins 2000b) indicates that this separation may occur based on the acoustic impedances of the paths. Fig.\u00a03Overview of measurements of the frog inner ear; comparison between Rana (left) and Hyla (right). The dashed lines indicate the separation between the amphibian papilla and the basilar papilla. a,  b Distributions of characteristic frequencies of auditory nerve fibers in Rana pipiens pipiens, and Hyla cinerea. c Example of the response of the contact membrane in R. catesbeiana; black line represents the amphibian papilla, open markers the basilar papilla. d, e Distributions of spontaneous otoacoustic emissions in ranid species (combined data from R. pipiens pipiens and R. esculenta), and hylid species (combined data from H. cinerea, H. chrysoscelis, and H. versicolor). f Example of stimulus frequency otoacoustic emissions in R. pipiens pipiens at indicated stimulus levels. g, h Examples of DP-grams measured in Rana pipiens pipiens, and Hyla cinerea. a, b, d, e, g and h are taken from Van Dijk and Meenderink (2006). There they were reproduced from Ronken (1990), Capranica and Moffat (1983), Van Dijk et\u00a0al. (1989, 1996), Meenderink and Van Dijk (2004), and Van Dijk and Manley (2001), respectively. c is taken from Purgue and Narins (2000a), and f is an adapted presentation of data from Meenderink and Narins (2006) (graph created with data provided by Dr. Meenderink)\nThe perilymphatic path through the periotic canal may serve as a shunt for acoustical energy to the round window. As its impedance exponentially increases with frequency, low-frequency vibrations will most effectively utilize this path. The endolymphatic path, on the other hand, presumably has a relatively constant impedance throughout the frog\u2019s auditory range. The respective lumina of the amphibian and basilar papilla have a frequency-dependent impedance of their own. According to the model mentioned above, these impedances are dominated by the characteristics of the contact membranes (Purgue and Narins) (2000b). The respective peak displacements of the contact membranes correspond to the detected frequencies in the associated organs (Purgue and Narins 2000a).\nBasilar papilla\nThe basilar papilla\u2019s tectorial membrane is presumably driven by a vibrating pressure gradient between the the sacculus and the basilar papilla\u2019s contact membrane. No reports have been published on direct measurements of the mechanical response of the tectorial membrane, or on the basilar papilla\u2019s hair bundle mechanics. However, the hair cell orientation in the basilar papilla implies that the tectorial membrane\u2019s primary mode of motion is to and from the sacculus.\nAuditory nerve fiber recordings from the frog basilar papilla show a frequency selective response (see Fig.\u00a04 for examples of tuning curves). The range of characteristic frequencies in nerve fibers from the basilar papilla is species dependent. In the leopard frog, they are approximately between 1,200 and 2,000\u00a0Hz (Ronken 1990); in the bullfrog they are slightly lower, between 1,000 and 1,500\u00a0Hz (Shofner and Feng 1981; Ronken 1991). In the Hyla-family, the characteristic frequencies appear to be significantly higher; in Hyla cinerea, the green treefrog, they range from 2.8 to 3.9\u00a0kHz (Ehret and Capranica 1980; Capranica and Moffat 1983), and in Hyla regilla roughly from 2 to 3\u00a0kHz (Stiebler and Narins 1990; Ronken 1991). Where studied in other species, the characteristic frequencies of the basilar papilla\u2019s nerve fibers fall roughly within the bounds defined by the bullfrog at the low end and the green treefrog at the high end (Scaphiopus couchi: approximately 1\u22121.5\u00a0kHz, Capranica and Moffat 1975; Ronken 1991; Eleutherodactylus coqui: approximately 2\u22124\u00a0kHz, Narins and Capranica 1980, 1976; Ronken 1991; Physalaemus pustulosus group: around 2.2\u00a0kHz, Wilczynski et\u00a0al. 2001). Fig.\u00a04Tuning curves measured in the auditory nerve in R. catesbeiana (unpublished measurements by JMS & PvD, 1992; various specimens). The numbers in the graph indicate  values\nIn each individual frog, the tuning curves of the auditory nerve fibers appear to have a nearly identical shape and characteristic frequency (Ronken 1990; Van Dijk and Meenderink 2006). This suggests that the entire basilar papilla is tuned to the same frequency. Because of this collective tuning, characterized by one characteristic frequency and a single tuning-curve shape throughout the organ, the basilar papilla may be referred to as a \"single auditory filter\". In comparison, the mammalian cochlea, and the anuran amphibian papilla (see below), consist of a combination of a large number of auditory filters (Pickles 1988).\nThe quality factor, Q10dB (e.g., Narins and Capranica 1976; Shofner and Feng 1981), is lower than that of other vertebrate hearing organs in the same frequency range, (Evans 1975; see Fig.\u00a05), and ranges from approximately 1 to 2 in both the leopard frog and the bullfrog (Ronken 1991; see Fig.\u00a05). For other anuran species, the ranges are somewhat different, with the lowest minimum values (approximately 0.5) reported for Hyla regilla, and the highest maximum values (approximately 2.8) in Scaphiopus couchi. Thus, the basilar papilla\u2019s frequency selectivity is relatively poor. Fig.\u00a05Comparison of the filter quality factor  versus the characteristic frequency (CF, in kHz) of nerve fibers from the cat cochlea (adapted from Evans 1975) and the leopard frog (adapted from Ronken 1991). In the leopard frog graph, the triangular symbols correspond to nerve fibers from the amphibian papilla; the circles to fibers from the basilar papilla. The black line indicates the upper limit of the amphibian papilla\u2019s frequency domain. The grey area in the upper (cat) graph corresponds to the area of the lower (frog) graph. The loops indicate the approximate perimeter of the fiber populations in the lower graph for the amphibian papilla and the basilar papilla.\nAs illustrated in Fig.\u00a03, there is no correspondence between the range of characteristic frequencies in the basilar papilla and the range of spontaneous otoacoustic emission frequencies (Van Dijk and Manley 2001; Van Dijk and Meenderink 2006; Van Dijk et\u00a0al. 2003; Meenderink and Van Dijk 2004, 2005, 2006; Meenderink and Narins 2007). Since it is generally assumed that otoacoustic emissions of a particular frequency are generated at the detection site for this frequency, this suggests that the basilar papilla does not generate spontaneous emissions. However, it does emit distortion product otoacoustic emissions (Van Dijk and Manley 2001), and stimulus frequency otoacoustic emissions (Palmer and Wilson 1982; Meenderink and Narins 2006). The peak amplitudes of the distortion product otoacoustic emissions match the characteristic frequencies of the auditory nerve fibers innervating the basilar papilla (Meenderink et\u00a0al. 2005).\nThe amplitude and phase characteristics of the distortion product otoacoustic emissions can be qualitatively modeled by assuming the basilar papilla to be a single passive non-linear auditory filter (Meenderink et\u00a0al. 2005). Thus, nerve fiber recordings, otoacoustic emission measurements and a model based on these measurements show that the basilar papilla functions as a single frequency band auditory receptor. This frequency band is relatively broad, and the center frequency may depend on species and individual animals.\nThe hypothesis that considers the basilar papilla as a single resonator was originally put forward by (Van Bergeijk 1957). He investigated the mechanical response of the tectorial membrane in a scale model consisting of a thin rubber tectorium spanning a lumen in a stiff wall. A number of different vibration modes existed in this model. Although Van Bergeijk\u2019s model is vastly oversimplified, the basic idea that the mechanical tuning of the tectorial membrane may be the basis of the basilar papilla\u2019s frequency selectivity is still viable.\nAmphibian papilla\nAs in the basilar papilla, the tectorial membrane in the amphibian papilla is presumably driven by a vibrating pressure difference between the sacculus and the round window. Due to the more elaborate tectorial membrane and the more complex pattern of hair cell orientations, the motion of the membrane may be expected to be more complex than that of the basilar papilla\u2019s tectorial membrane. The tectorial curtain is in the sound path through the papilla, and presumably plays a role in conveying vibrations to the tectorial membrane and the hair bundles.\nElectrophysiological recordings from and subsequent dye-filling of single fibers of the auditory nerve show that the amphibian papilla has a tonotopic organization (Lewis et\u00a0al. 1982). The fibers innervating the triangular patch have low characteristic frequencies, down to approximately 100\u00a0Hz. The frequencies increase gradually along the caudal extension. In the bullfrog, the upper frequency is about 1000\u00a0Hz; an overview of the tonotopic organization is given in Fig.\u00a02b.\n5\nThe frequency selectivity of the amphibian papilla\u2019s nerve fibers is similar to that of mammalian auditory nerve fibers with the same characteristic frequency. This is in contrast to the significantly poorer frequency selectivity in the basilar papilla\u2019s nerve fibers (Ronken 1990; Evans 1975; see also Fig.\u00a05).\nIn the low-frequency, rostral part of the papilla, the hair cells are electrically tuned (Pitchford and Ashmore 1987; Smotherman and Narins 1999). This tuning stems from the electrical properties of the cell membrane\u2019s ion channels. The hair cell tuning characteristics parallels the tonotopy of the single nerve recordings. Therefore, frequency selectivity in the rostral part of the amphibian papilla appears to be primarily determined by the electrical characteristics of the hair cells.\nHowever, there is a fundamental discrepancy between the tuning characteristics of the hair cells and the auditory nerve fibers. While hair cells exhibit a second-order resonance (Pitchford and Ashmore 1987) auditory neurons display a higher order filter characteristic (Lewis 1984). Nevertheless, due to the parallels in the tonotopic organization, the assumption that the frequency selectivity is determined by the electrical tuning seems viable for the rostral part of the amphibian papilla. The higher-order responses in the neural signal may result from coupling between hair cells, which may be mechanical, for instance through the tectorial membrane.\nNeurons innervating the rostral portion of the amphibian papilla display non-linear two-tone suppression similar to that in other vertebrates (Capranica and Moffat 1980; Benedix et\u00a0al. 1994). Another manifestation of non-linear behavior can be found in the response to noise: second-order Wiener kernels of low-frequency neurons show off-diagonal components, which are an indication of non-linearity (Van Dijk et\u00a0al. 1994, 1997). The spectro-temporal receptive fields constructed from these Wiener kernels exhibit suppressive side bands besides the main characteristic frequency band of the fiber (Lewis and Van Dijk 2004).\nHair cells caudal to the tectorial curtain do not display electrical resonance (Smotherman and Narins 2000). Therefore, the tuning of this high-frequency, caudal region of the papilla must result from the mechanical properties of the tectorial membrane and the hair cells.\nBased on the hair cell orientation, displayed in Fig.\u00a02b, the tectorial membrane motion in the amphibian papilla is expected to be far more complex than in the basilar papilla. Assuming that the hair bundles are orientated in such a way that they are maximally deflected by the connected tectorial membrane, the rostral patch of the membrane should be moving to and from the sacculus, if the appropriate stimuli are presented. The rostral part of the s-shaped extension is moving along its major axis, whereas the extension caudal to the tectorial curtain should be moving in a transverse direction.\nThe amphibian papilla appears to be the only source of spontaneous otoacoustic emissions in the frog inner ear (Van Dijk et\u00a0al. 1989, 1996; Long et\u00a0al. 1996; Van Dijk and Manley 2001; Fig.\u00a03d-e). The frequency distribution of these emissions corresponds to the range of best frequencies of the neurons projecting to the portion of the amphibian papilla caudal to the tectorial curtain. It is generally assumed that an otoacoustic emission of a specific frequency is generated at the location in the inner ear where that frequency is detected. Under this assumption, the presence of spontaneous otoacoustic emissions indicates that the caudal portion of the amphibian papilla exhibits spontaneous activity. Presumably, this activity is related to active amplification of input signals in this area.\nThe caudal region of the amphibian papilla is also involved in the generation of distortion product otoacoustic emissions (Van Dijk and Manley 2001; Meenderink and Van Dijk 2004), and stimulus frequency otoacoustic emissions (Meenderink and Narins 2006). The distortion product otoacoustic emissions from the amphibian papilla are more vulnerable to metabolic injuries than those from the basilar papilla (Van Dijk et\u00a0al. 2003). Also, both the spontaneous (Van Dijk et\u00a0al. 1996) and distortion product (Meenderink and Van Dijk 2006) otoacoustic emissions display a clear dependence on body temperature. These results combine to indicate that the s-shaped extension of the amphibian papilla caudal to the tectorial curtain functions as an active hearing organ.\nDiscussion\nOur aim in this review is to outline what is known about the mechanical response properties of the amphibian and basilar papilla. Only one published report exists of the direct mechanical measurements of structures associated with these papillae (Purgue and Narins 2000a). The measurements show that the response of the contact membrane is frequency dependent for each papilla. The movement of the contact membrane may be assumed to reflect the fluid motion within the respective papilla. The contact membrane of the amphibian papilla shows a maximum response when the ear is stimulated with relatively low acoustic frequencies, while the basilar papilla contact membrane exhibits a maximum response to higher frequencies.\nThe amphibian and the basilar papilla are the only hearing organs found in terrestrial vertebrates in which the hair cells are not on a flexible basilar membrane. Instead the hair cells are embedded in a relatively stiff cartilaginous support structure. Any frequency selective response, therefore, most likely originates from the mechanical or electrical properties of the hair cells, or the mechanical properties of the tectorial membrane, or a combination of these factors. Since there are no direct mechanical measurements of either the hair cells in the papillae or the tectorial membranes, we cannot come to any definite conclusions regarding their properties. However, the available morphological and functional data allow for some hypotheses.\nThe most conspicuous functional characteristic of the amphibian papilla is its tonotopic organization (Lewis et\u00a0al. 1982). Rostral to the tectorial curtain, the hair-cell orientation is essentially parallel to the tonotopic axis. In this low-frequency region of the amphibian papilla, the tectorial membrane apparently moves in a rostro-caudal direction. In contrast, the hair-bundle orientation suggests that the tectorial-membrane motion is perpendicular to the tonotopic axis in the high-frequency, caudal region of the papilla. The tectorial membrane\u2019s caudal end, therefore, appears to vibrate in a markedly different direction than its rostral end.\nIn the low-frequency region of the amphibian papilla, the hair cells display electrical tuning. The tuning properties of the hair cells parallel the tonotopic organization are measured from the afferent nerve fibers (Pitchford and Ashmore 1987). This strongly suggests that the tuning characteristics of the nerve fibers are primarily determined by the electrical hair-cell resonances. The auditory nerve-fiber recordings reflect the presence of high-order filtering (Lewis 1984), whereas hair cells essentially function as second-order resonances. It is, therefore, likely that coupling between the hair cells shapes the frequency responses in the nerve fibers. Such coupling may be mechanical, for example, by the tectorial membrane, or electrical, or a combination of mechanical and electrical.\nHair cells in the high-frequency, caudal region do not display any electrical resonance (Smotherman and Narins 1999). This implies that the frequency selectivity must be based on mechanical tuning, probably by the tectorial membrane. The caudal region of the amphibian papilla shares some notable characteristics with the mammalian cochlea (see also Lewis 1981): the papilla is elongated, and it exhibits a tonotopic gradient along the long axis;the orientation of the hair cells is perpendicular to the tonotopic axis, indicating that the hair cells are stimulated most efficiently by a deflection perpendicular to the tonotopic axis;frequency selectivity, very probably, relies on mechanical tuning;frequency selectivity is similar, with Q10dB-values ranging from 0.8 to 2.2; andboth spontaneous and distortion product otoacoustic emissions are generated. These emissions are physiologically vulnerable. The presence of spontaneous otoacoustic emissions shows that at least part of the amphibian papilla\u2019s caudal extension functions as an active hearing organ. In this respect it is similar to the mammalian cochlea and other vertebrate hearing organs (Lewis and Narins 1999). One active mechanism in the mammalian cochlea is the prestin-mediated active somatic length changes in the outer hair cells (Brownell et\u00a0al. 1985; Yost 2000; Zheng et\u00a0al. 2000; Liberman et\u00a0al. 2002; Dallos 2003). However, this mechanism is probably exclusively present in mammalian outer hair cells. Active hair bundle movements have been reported as an alternative active mechanism in anuran saccular hair cells (Martin and Hudspeth 1999; Martin et\u00a0al. 2003; Bozovic and Hudspeth 2003); this mechanism may be present in the auditory organs as well. Although the fundamental active mechanism may differ between species, the functional result seems to be very similar across vertebrates: high auditory sensitivity and good frequency selectivity (Manley 2000).\nThe basilar papilla seems to function in a much simpler manner. Both neural recordings and otoacoustic emission measurements suggest that it functions as a single auditory filter. Since the hair cells in the basilar papilla are unlikely to be electrically tuned, its frequency selectivity most likely results from mechanical tuning, probably via the tectorial membrane.\nThe basilar papilla is remarkable in that no spontaneous otoacoustic emissions have been recorded in its frequency range. The absence of such emissions can either be caused by the fact that they are not generated within the papilla, or by the fact that the transmission of such emissions to the tympanic membrane is inhibited. However, distortion product otoacoustic emissions can be recorded in this range (e.g., Van Dijk and Manley 2001). This implies that the outward transmission is not inhibited, and therefore that spontaneous emissions are most likely not generated within the basilar papilla.\nFurthermore, the amplitude of the basilar papilla\u2019s distortion product otoacoustic emissions depends less on temperature than that of the amphibian papilla\u2019s (Meenderink and Van Dijk 2006). Also, emissions from the basilar papilla are less sensitive to the disruption of oxygen supply (Van Dijk et\u00a0al. 2003). Apparently, emissions from the basilar papilla are relatively independent of the metabolic rate, and therefore, it has been suggested that the basilar papilla is not an active hearing organ (Vassilakis et\u00a0al. 2004; Van Dijk and Meenderink 2006).\nIn conclusion, the frog inner ear takes an exceptional place among the hearing organs of terrestrial vertebrates. It includes two auditory end organs, which both lack the basilar membrane present in every other terrestrial vertebrate species. Instead the hair cells are embedded in a relatively stiff structure. They are stimulated by the motion of the tectorial membrane. Although the basilar and amphibian papilla are similar in this respect, they appear to function by different mechanisms. In fact, even within the amphibian papilla two distinctly different functional regions can be identified. The low-frequency portion, rostral to the tectorial curtain, contains hair cells that exhibit electrical tuning. The hair cells are most sensitive to deflection along the tonotopic axis, thus this is presumably the tectorial membrane\u2019s direction of vibration. By contrast, the region caudal to the tectorial curtain shows more similarities to, for example, the mammalian cochlea: the hair cell orientation is perpendicular to the tonotopic axis, and the presence of spontaneous otoacoustic emissions suggests that it functions as an active hearing organ. Finally, the basilar papilla is yet different: it appears to function as a single passive auditory filter. Thus the frog inner ear includes two auditory end organs with three functional regions.","keyphrases":["anuran","auditory system","amphibian","frog","inner ear mechanics"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_J_Appl_Physiol-4-1-2267484","title":"Modulation in voluntary neural drive in relation to muscle soreness\n","text":"The aim of this study was to investigate whether (1) spinal modulation would change after non-exhausting eccentric exercise of the plantar flexor muscles that produced muscle soreness and (2) central modulation of the motor command would be linked to the development of muscle soreness. Ten healthy subjects volunteered to perform a single bout of backward downhill walking exercise (duration 30 min, velocity 1 ms\u22121, negative grade \u221225%, load 12% of body weight). Neuromuscular test sessions [H-reflex, M-wave, maximal voluntary torque (MVT)] were performed before, immediately after, as well as 1\u20133 days after the exercise bout. Immediately after exercise there was a \u221215% decrease in MVT of the plantar flexors partly attributable to an alteration in contractile properties (\u221223% in electrically evoked mechanical twitch). However, MVT failed to recover before the third day whereas the contractile properties had significantly recovered within the first day. This delayed recovery of MVT was likely related to a decrement in voluntary muscle drive. The decrease in voluntary activation occurred in the absence of any variation in spinal modulation estimated from the H-reflex. Our findings suggest the development of a supraspinal modulation perhaps linked to the presence of muscle soreness.\nIntroduction\nIn an exercise inducing muscle damage, inadequate neural drive can be an attempt of the neuromuscular system to protect the muscle-tendon unit from additional damage (Strojnik and Komi 2000; Nicol et al. 2006). This inadequate neural drive could be the result from a combination of three factors: the conscious and unconscious will of the subject to reduce the exercise intensity; an inability of the motor cortex to generate sufficient output to maximally activate the muscle; and\/or a decreased transmission of the supraspinal input to the muscle by the spinal motor axons.\nA decrement in H-reflex amplitude has been observed immediately after an exhausting voluntary contraction of a single muscle group (i.e., Duchateau et al. 2002; Garland and McComas 1990; Kuchinad et al. 2004). It has been proposed that this decline in the transmission of the action potentials from the Ia afferent to the \u03b1-motoneuron may be a consequence of a presynaptic inhibition mediated by group III and IV afferents (Bigland-Ritchie et al. 1986; Duchateau et al. 2002; Garland and McComas 1990; Garland 1991; Woods et al. 1987) induced by muscle damage (Avela et al. 2006). Furthermore, increased group III and IV muscle afferent inputs could induce H-reflex depression when muscle soreness progresses as muscle pain is believed to reflect activity in group III and IV muscle afferents (O\u2019Connor and Cook 1999). But only a few studies have observed a decrease in H-reflex amplitude after exercise of multiple muscle groups as occurs for example in running (Avela et al. 1999; Bulbulian and Bowles 1992; Racinais et al. 2007b) raising the question of whether spinal modulation occurs after whole body exercise. Recently it has been shown that walking backward induces muscle soreness in the muscles of the lower limb (Nottle and Nosaka 2005). Accordingly, this exercise model allows the study of the effects of muscle soreness on alteration in neural drive. Thus the goal of this study was to determine whether the impaired exercise performance of muscles with delayed onset muscle soreness (DOMS) is due to an alteration in neural drive related to spinal modulation.\nMethods\nSubjects\nTen healthy subjects (eight males and two females; age 27\u00a0\u00b1\u00a01 years; weight 68\u00a0\u00b1\u00a02\u00a0kg; height 174\u00a0\u00b1\u00a02\u00a0cm; data in mean\u00a0\u00b1\u00a0SEM) gave informed, written consent to participate in this study. The procedures complied with the Helsinki declaration for human experimentation and were approved by the local Ethics Committee. None of the subjects suffered from muscle soreness or ankle injuries. Subjects were asked to avoid caffeine intake within the 8-h preceding the test and to avoid all vigorous activity during the 24-h preceding the test. Subjects were also asked to refrain from analgesic intake all along the protocol, which could have disturbed DOMS perception.\nExperimental procedures\nSubjects visited our laboratory on four consecutive days (Fig.\u00a01). The first day consisted of a neuromuscular test session (described subsequently) followed by a backward walking exercise (description below) followed by further neuromuscular testing. On the second, third and fourth days, subjects returned to the laboratory at the same hour of day that they had finished the walking exercise and performed the neuromuscular testing.\nFig.\u00a01Experimental design. Thin arrow indicates stimulation at Hmax intensity, simple thick arrow indicates stimulation at Mmax intensity, double thick arrow indicates doublet at Mmax intensity\nNeuromuscular tests\nThe neuromuscular tests are described in Fig.\u00a01. All the neuromuscular tests began with the determination of the stimulation intensity required to elicit a maximal H-reflex (Hmax). Afterwards, three Hmax (interspaced by 20\u00a0s) and three maximal M-wave amplitudes (Mmax) interspaced by 8\u00a0s were elicited from the relaxed muscle. The amplitude of the three twitches evoked at Hmax and Mmax intensities were averaged for subsequent analysis in both the soleus and gastrocnemius medialis. Thereafter, subjects were instructed to perform three maximal voluntary torque (MVT) contractions of the plantar flexor muscles, each for 5\u00a0s. Subjects were verbally encouraged to perform maximally. A superimposed stimulus (Hmax intensity) was evoked during each MVT plateau to obtain the H-reflex during contraction (Hsup). Then, another superimposed stimulus (Mmax intensity) was evoked in order to obtain a superimposed M-wave (Msup) during voluntary contraction. Finally, a doublet (two electrically evoked twitches, 10\u00a0ms apart, Mmax intensity) was evoked during each plateau (superimposed twitch) and another doublet was evoked 4\u00a0s after each MVT (potentiated twitch). The ratio of the amplitude of the superimposed twitch torque over the amplitude of a twitch evoked at rest 4\u00a0s after the MVT was used to assess the level of voluntary activation (VA) (Allen et al. 1995). According to the twitch interpolation method (Allen et al. 1995), the percentage of VA was calculated as follow: VA (%)\u00a0=\u00a0(1\u00a0\u2212\u00a0Superimposed Twitch\/Potentiated Twitch)\u00a0\u00d7\u00a0100.\nMuscle soreness assessment\nA subjective evaluation of the extent of DOMS in the plantar flexor muscles was performed before each neuromuscular test by completing two subjective scales for evaluation of DOMS. The first was a visual scale of 9\u00a0cm without any graduation (horizontal line ranging from no pain at the left to extreme pain at the right). The second was a Lickert scale with seven items (from 0: no pain to 6: severe pain limiting movement, Vickers 2001).\nBackward downhill walking exercise\nSubjects exercised by walking on a motorized treadmill (S2500, HEF Techmachine, France) for 30-min at a constant velocity of 1\u00a0ms\u22121 with a negative grade of \u221225%. To increase the eccentric loading on the plantar flexor muscles, the walk was performed backward (Nottle and Nosaka 2005) whilst wearing a vest loaded with an additional weight equivalent to 12% of body weight.\nMeasurement and calculations\nTorque measurement\nThe MVT of the plantar flexor muscles was recorded by a dynamometric pedal (Captels, St Mathieu de Treviers, France). Subject position was standardized with hip, knee and ankle angulations of 90\u00b0, and foot securely strapped on the pedal.\nEvoked potentials\nThe tibial nerve was stimulated with a cathode electrode with a diameter of 9\u00a0mm placed in the popliteal cavity (Contr\u00f4le Graphique Medical, Brie-Comte-Robert, France). Subjects were in a standardized position with motionless head (Zehr 2002) and a standardized environment (i.e., same time-of-day, silent room, constant lighting). Furthermore, a constant pressure was applied to the electrode with the use of a strap. This was controlled by an air pressure-recorder (Kikuhime, TT MediTrade, Soro, Denmark) located under the strap. The anode (10\u00a0cm\u00a0\u00d7\u00a05\u00a0cm, Medicompex, Ecublens, Switzerland) was positioned distal to the patella. Electrical stimulations (400\u00a0V, rectangular pulse of 0.2\u00a0ms) were delivered by a high-voltage stimulator (Digitimer DS7AH, Digitimer, Hertfordshire, England). The amperage was adjusted for each subject during the familiarization session. During this first session, the amperage was increased progressively (10\u00a0mA increment) until a plateau in twitch mechanical response [peak twitch (Pt)] and Mmax were observed. With increasing stimulation intensity, the H-reflex response initially increased progressively before decreasing and then disappearing. Thereafter, the intensity needed to obtain Hmax was adjusted by 1\u00a0mA. The stimulation intensity needed to obtain Hmax was determined before each test session but with a simplified procedure based on the knowledge of the intensity used during the first test session. This adjustment seemed necessary for the H-reflex in view of the important variation occurring in Hmax for a small variation in stimulation conditions (e.g., intensity, localization of the cathode).\nRecordings\nEvoked Pt torque was recorded in relaxed muscle by the same ergometer than MVT. Both MVT and Pt were measured with the knee at 90\u00b0 to reflect the changes occurring in the soleus. Reflex waves for both the soleus, which provides the highest responses due to the activation of the slow twitch fibre by H-reflex (Buchthal and Schmalbruch 1970), and the gastrocnemius medialis, which is particularly susceptible to be affected by the walking exercise were recorded with 9\u00a0mm diameter bipolar Ag\/AgCl electrodes (Contr\u00f4le Graphique Medical, Brie-Comte-Robert, France) with an inter-electrode distance of 25\u00a0mm. The reference electrode was placed on the wrist. Low impedance between the two electrodes (<5\u00a0k\u03a9) was obtained by abrading and washing the skin with emery paper and cleaning with alcohol. Signals were amplified and filtered (band pass 30\u2013500\u00a0Hz, gain\u00a0=\u00a01,000), and recorded at high frequency (2,000\u00a0Hz). The compound muscle action potentials were recorded using MP30 hardware (Biopac Systems Inc., Santa Barbara, CA, USA) and dedicated software (BSL Pro Version 3.6.7, Biopac Systems Inc., Santa Barbara, CA, USA). The same equipment was also used to drive the stimulator.\nCalculation\nThe Pt may be considered as an index of the contractile properties and Mmax amplitude represents an index of sarcolemmal excitability. Because no evidence exists to show that data recorded at rest reflects the responses of the neuromuscular system during contraction, Hsup amplitude was recorded during MVT to complement Hmax amplitude at rest. To ensure that any changes in the evoked Hmax and Hsup amplitudes were not due to changes at the muscle fiber membrane or neuromuscular junction (Cupido et al. 1996), we normalized these recordings to the M-wave amplitude recorded under the same testing conditions, that is the Hmax\/Mmax and Hsup\/Msup ratios. These ratios may be considered as a global index of the spinal modulation produced by presynaptic inhibition, motoneuron excitability, collision in antidromically activated axons and Renshaw cell inhibition, acting individually or in concert.\nStatistical analysis\nEach variable was tested for normality using the Skewness and Kurtosis tests with acceptable Z values not exceeding \u00b11.5. With the assumption of normality confirmed, parametric tests could be performed. The effect of the walking exercise was analyzed for each variable by a one-way analysis of variance for repeated measures (five test sessions). The contrast method was applied as post hoc to further investigate the effect of both the exercise and the recovery. Because VA level failed to display a normal distribution, a Friedman test was used instead of the ANOVA. Statistical analyses were performed with Systat software (Systat, Evanston, IL, USA). Data are reported as mean\u00a0\u00b1\u00a0SEM and the level of statistical significance was set at P\u00a0<\u00a00.05.\nResults\nMaximal voluntary torque, voluntary activation and contractile properties\nThe MVT significantly changed across the 4 days following the walking test (F4,36\u00a0=\u00a03.8, P\u00a0<\u00a00.02, Fig.\u00a02a). Post hoc analysis showed a significant decrease in MVT after the walking exercise and which persisted during the next two days (F1,9\u00a0=\u00a012.33, P\u00a0<\u00a00.01). A significant recovery in MVT was observed on the third day (48-h versus 72-h after: F1,9\u00a0=\u00a09.64, P\u00a0<\u00a00.02).\nFig.\u00a02Evolution of voluntary torque (a), voluntary activation (b), electrically evoked peak twitch (c) and subjective delayed onset muscle soreness (d, black rectangle: analogic visual scale, white rectangle: Lickert scale) across the experimental sessions. Data in mean\u00a0\u00b1\u00a0SEM, Asteriskindicates value or group of values significantly different from the other values of the graph (P\u00a0<\u00a00.05)\nIn line with the evolution observed in MVT, post hoc analysis showed a significant decrease in VA level after the walking exercise (pre versus post-exercise: P\u00a0<\u00a00.02, Fig.\u00a02b) which failed to recover by 48-h (post-exercise versus 24-h and 48-h after exercise, NS). However there was a significant recovery by 72-h (post-exercise versus after 72-h, P\u00a0<\u00a00.005).\nThe electrically evoked Pt also displayed a significant variation following walking exercise (F4,36\u00a0=\u00a014.07, P\u00a0<\u00a00.001, Fig.\u00a02c). Post hoc analysis revealed a significant decrease in Pt after the exercise (F1,9\u00a0=\u00a058.34, P\u00a0<\u00a00.001) followed by a significant recovery thereafter (F1,9\u00a0=\u00a024.76, P\u00a0<\u00a00.001).\nSubjective DOMS\nThe subjects feeling of DOMS increased significantly in the days following the walking exercise (F4,27\u00a0>\u00a028, P\u00a0<\u00a00.001 for both scales used, Fig.\u00a02d). Post hoc analysis displayed significantly higher subjective DOMS for the 3 days following exercise compared to the termination of exercise (F1,9\u00a0>\u00a035, P\u00a0<\u00a00.001, for both scales). Muscle soreness reached a maximum 48-h after exercise and began to recover by 72-h (48-h versus 72-h after exercise: F1,9\u00a0>\u00a012, P\u00a0<\u00a00.01 for both scales).\nEvoked potentials\nAn example of evoked potentials recorded in a representative subject is displayed in Fig.\u00a03 and the mean values are displayed in Table\u00a01. The walking exercise failed to induce significant changes in the evoked potentials both at rest (Mmax, F4,36\u00a0=\u00a01.60 and 0.60 for soleus and gastrocnemius medialis respectively, NS) and during the voluntary contraction (Msup, F4,36\u00a0=\u00a00.46 and 2.20 for soleus and gastrocnemius medialis respectively, NS). Furthermore, the reflex waves calculated both at rest (Hmax\/Mmax ratio) and during contraction (Hsup\/Msup ratio) also did not change significantly (all F4,36\u00a0<\u00a00.98, NS, Table\u00a01).\nFig.\u00a03Example of evoked potentials recorded in a representative subject. Each drawing represents the average of three recordings obtained on a relaxed muscleTable\u00a01Evolution of maximal compound action potential electrically evoked at rest (Mmax) and during MVT (Msup), normalized H-reflex at rest (Hmax\/Mmax) and during MVT (Hsup\/Msup)ExerciseRecoveryStatistical analysisBeforeAfter24-h48-h72-hSoleusMmax (mV)7.356.807.837.747.18NS0.530.490.80.580.75Msup (mV)8.878.139.058.918.75NS1.140.761.130.971.29Hmax\/Mmax ratio0.360.370.380.370.36NS0.100.090.100.100.09Hsup\/Msup ratio0.390.450.390.340.34NS0.100.100.080.080.06Gastrocnemius medialisMmax (mV)6.616.106.426.836.50NS1.070.760.641.00.92Msup (mV)9.407.048.247.958.14NS1.291.060.810.892.26Hmax\/Mmax ratio0.180.190.220.190.19NS0.050.050.050.050.05Data in mean\u00a0\u00b1\u00a0SEM\nDiscussion\nThe downhill walking exercise induced a significant decrease in the MVT of the plantar flexor muscles. Immediately after the walking exercise, the torque decrement of \u221215% (Fig.\u00a02a) appeared to be caused partly by an alteration in muscle contractile properties (i.e., \u221223% for Pt, Fig.\u00a02c). This alteration is typically referred to as \u201cperipheral fatigue\u201d [for a review, see Millet and Lepers 2004]. Furthermore, this peripheral fatigue was also associated with a decrease in VA (i.e., \u22125.3%, Fig.\u00a02b) suggesting the concomitant existence of a \u201ccentral modulation\u201d [for a review, see Gandevia 2001]. The first finding of this study is that the maximum voluntary torque failed to recover before the third day (i.e., \u221212% and \u221210% after 24-h and 48-h of recovery, respectively, Fig.\u00a02a) whereas the measure of the (peripheral) contractile properties had recovered significantly within the first 24\u00a0h after exercise (P\u00a0<\u00a00.01, Fig.\u00a02c). This delayed recovery in MVT appeared to be mainly associated with a decrease in voluntary muscle activation (Fig.\u00a02b). The time course of change in VA presents similarities with the time course of torque changes.\nA significant decrease in the VA reaching the muscle has previously been observed after prolonged (Millet et al. 2002, 2003) and short-duration (Racinais et al. 2007a) fatiguing exercise, but our data showed that a backward downhill walking exercise did produce a decrease in muscle activation persisting for a few days after the exercise. It has been suggested that this central component could explain as well the reduction in force production by the respiratory muscles after heavy exercise (Verin et al. 2004) and represents a protective mechanism in order that peripheral muscle fatigue does not exceed a critical threshold (Amann et al. 2006). This central protection of the muscle from further peripheral fatigue and damage will be performed at the expense of a truly maximal performance in which all the motor units are activated (Gandevia et al. 1996).\nIt has recently been suggested that a \u201ccentral governor\u201d in the brain regulates the extent of skeletal muscle recruitment during exercise (Noakes et al. 2005). According to this theory, the sensation of fatigue is the conscious interpretation of these homoeostatic control mechanisms during prolonged exercise (Noakes et al. 2005). Our results could partly support this theory but with the proviso that the modulation of central activation in this experiment appears to be related not only to fatigue immediately after exercise but also to the development of DOMS the days following the exercise. However, regulation of muscle recruitment by the brain (i.e., supraspinal regulation) is not the only system that could explain the observed decrease in skeletal muscle activation since reflex pathways need also to be considered (i.e., spinal modulation). Previous results showed impairment in VA after eccentric exercise when VA was estimated by nerve stimulation but not by cortical stimulation (Prasartwuth et al. 2005). That suggests that the VA deficit lies within these two site of stimulations, i.e., in the motor cortex or at spinal level (Prasartwuth et al. 2005), rather than a governor upstream to the motor cortex.\nIn the present study, we used H-reflex amplitude as a tool to evaluate the spinal modulation (Aagaard et al. 2002; Schieppati 1987) to provide a differentiation within these possible regulatory mechanisms. The DOMS produced by our experimental protocol would have induced an increased discharge of group III and IV muscle afferents (Avela et al. 1999) and thus a pre-synaptic inhibition of the transmission from the Ia afferent stimulation to the \u03b1-motoneurons (Avela et al. 2006; Bigland-Ritchie et al. 1986; Duchateau et al. 2002; Garland and McComas 1990; Garland 1991; Woods et al. 1987). The extent to which this inhibition occurs will depend on whether the input producing the inhibition is ongoing or has ceased. But, in theory, increased group III and IV muscle afferent input induced by the DOMS should induce H-reflex depression as the soreness progresses. Indeed, muscle pain is believed to reflect activity in group III and IV muscle afferents (O\u2019Connor and Cook 1999). However, our results failed to show significant variations in the evoked reflex-wave amplitudes throughout the experiment (i.e., Hmax\/Mmax and Hsup\/Msup ratios) suggesting that the motoneuron pool excitability was well preserved.\nA number of previous studies have demonstrated a significant H-reflex decrease in the exercised muscle group after an exhausting voluntary contraction of that muscle group (i.e., Duchateau et al. 2002; Garland and McComas 1990; Kuchinad et al. 2004). But only a few studies have observed this decrease after more generalized muscular exercise such as running, (Avela et al. 1999; Bulbulian and Bowles 1992; Racinais et al. 2007b). Our results support these findings by showing that after a 30-min downhill walking exercise, electrically evoked reflex-wave activity was not decreased. Thus we conclude that non-exhausting walking exercise, sufficient to induce significant DOMS seems not to induce alteration of the spinal modulation.\nSince the persistence of a decrease in VA during several days in this study could not be explained by spinal modulation, it seems likely that a supraspinal component must have played a part. Accordingly the changes in VA that occurred at the same time that the subjective symptoms of DOMS suggest a supraspinal regulation of muscle recruitment. Indeed it has been known for some time that exercise performance is regulated at least in part by supraspinal factors. For example, Bigland-Ritchie (1981) showed that central fatigue during repeated isometric contraction is minimized by exhorting the subject to produce a \u201csuper\u201d effort at the end of each voluntary contraction. Our results add to that interpretation by suggesting the possibility that supraspinal modulation can also occurs after locomotory activity such as walking, even without exhaustion.\nAs we have already argued, the observed increase in DOMS could represent the conscious interpretation of an increased discharge of group III and IV muscle afferents (O\u2019Connor and Cook 1999). Even though we failed to observe a significant alteration of the spinal modulation at the time of increased muscle soreness, this would not prove that the discharge of these afferents had not increased since it is still unclear whether group III and IV muscle afferents induce a post-exercise decrease in motoneurons excitability in healthy humans. Indeed, previous studies have shown that maintained firing of ischaemically sensitive group III and IV muscle afferents does not influence the altered muscle responses to cortical or corticospinal stimulation observed after fatiguing exercise (Andersen et al. 2003; Gandevia et al. 1996; Taylor et al. 2000). All these findings might suggest that, after exercise, increased output from group III and IV muscle afferents may not directly inhibit the motoneurons but may act upstream of the motor cortex to impair voluntary descending motor drive (Taylor et al. 2006).\nAccordingly, a novel contribution of this study is that VA significantly recovered on the third day, when the DOMS displayed also a significant decrement (F1,9\u00a0>\u00a012, P\u00a0<\u00a00.01, for the both scales). Although this temporal relationship does not prove causality, this finding could suggest a relationship between the persistence of the decrease in VA and the subjective symptoms of DOMS in these subjects. This observation is consistent with the data of Le Pera et al. (2001) showing that muscle pain could induce a long-lasting depression in motor activation. Their data suggest that this inhibition in motor system excitability could be linked to a decreased excitability of the motor cortex as well as spinal modulation (Le Pera et al. 2001). However, Prasartwuth et al. (2005) observed a different time course in muscle soreness after eccentric exercise and changes in VA leading these authors to suggest that muscle pain did not directly cause the change in voluntary drive.\nThese different data emphasised the complexity of the relation within muscle soreness and voluntary muscle drive. It has been recently observed that motoneuron excitability in elbow flexors, but not extensors, was able to recover when ischemia is maintained after fatiguing contractions (Martin et al. 2006), a finding that suggests differential influences of group III and IV muscle afferents on different motoneuron pools (Martin et al. 2006). In this study concerning the plantar flexors, we observed some statistical similarities within DOMS and VA (i.e., lowest value of VA at 48-h when DOMS was the highest and significant recovery for the both at 72-h). However, from a functional point of view, VA returned at control level at 72-h whereas DOMS at 72-h was not less than DOMS at 24-h, and VA decreased after exercise when DOMS was weak. That suggests that subjective DOMS of the plantar flexors can not be considered as an objective indicator of VA capability.\nConclusion\nIt was recently reported that a 90\u00a0min bout of flat running exercise produced a modification in spinal modulation (Racinais et al. 2007b). The present study showed that a 30-min downhill walking exercise failed to induce the same modification. However there was a significant decrease in VA during maximal voluntary contractions performed during the days after eccentric exercise that produced DOMS. This suggested the occurrence of a supraspinal modulation of muscle activation during this period when muscle contractile properties had fully recovered following the eccentric exercise. Furthermore, the persistence of the decrement for several days suggests that this modulation was not caused by an acute exercise-related physiological or biochemical alteration in the motor cortex, so-called central fatigue but more likely represents a modulation which may be partly linked to the muscle soreness.","keyphrases":["eccentric","exercise","neuromuscular","doms","central fatigue"],"prmu":["P","P","P","P","P"]}
{"id":"Ann_Surg_Oncol-3-1-2039838","title":"A Comparison Between Radioimmunotherapy and Hyperthermic Intraperitoneal Chemotherapy for the Treatment of Peritoneal Carcinomatosis of Colonic Origin in Rats\n","text":"Background Cytoreductive surgery (CS) followed by heated intraperitoneal chemotherapy (HIPEC) is considered the standard of care for the treatment of patients with peritoneal carcinomatosis (PC) of colorectal cancer (CRC). These surgical procedures result in a median survival of 2 years at the cost of considerable morbidity and mortality. In preclinical studies, radioimmunotherapy (RIT) improved survival after CS in a model of induced PC of colonic origin. In the present studies we aimed to compare the efficacy and toxicity of CS followed by adjuvant RIT in experimental PC to the standard of care, HIPEC.\nPeritoneal carcinomatosis (PC) of colorectal cancer (CRC) frequently is an end stage of colorectal cancer, occurring in 5\u201350% of the patients, either synchronous or metachronous.1 If untreated, patients suffering from PC have a median survival of only 6 months.2 Survival is significantly improved by radical surgical debulking procedures (cytoreduction) in combination with intraperitoneal chemotherapy, either in combination with normothermia or hyperthermia (HIPEC).3\u20137 The median survival after cytoreductive surgery and HIPEC is 13\u201334 months,4,8 and the 5-year survival rate is 19\u201327% at the cost of considerable morbidity and mortality rates of up to 23% and 4%, respectively.6,9\nIn the latest reported clinical trial on adjuvant RIT in the setting of colon cancer, Liersch et al. reported results of a Phase II trial with 131I-labeled anti-CEA antibody labetuzumab administered to patients after complete resection of colorectal liver metastases. This study, where RIT was applied in an adjuvant setting to complete resection, resulted in a promising 5-year survival rate of 51.5%.10 Radioimmunotherapy using radiolabeled monoclonal antibodies directed against tumor-associated antigens may therefore be an attractive anticancer therapy in patients with small volume disease.\nWe therefore have studied the application of RIT as adjuvant therapy following cytoreductive surgery (CS) in the setting of PC. In previous studies regarding PC of CRC in a rat model we showed that RIT could be an effective adjuvant treatment after CS. The efficacy of adjuvant RIT in combination with CS was investigated and compared with no treatment, CS only, and RIT only. The results of this study showed a significantly improved survival of animals treated with CS followed by RIT (median 88 days) compared with those treated with CS only (median 51 days) and RIT only (median 61.1 days).11 Based on the encouraging results, showing the observed increase in survival that was achieved with low-dose RIT and concomitant low toxicity, we now aimed to compare the efficacy of this treatment to that of today\u2019s standard of care, HIPEC,12,13 in a preclinical setting.\nMATERIALS AND METHODS\nAnimal Model of Peritoneal Carcinomatosis\nWAG\/Rij rats (10\u201312 weeks old, body weight 240\u2013290 g, Harlan Horst, The Netherlands) were housed under nonsterile standard conditions (temperature, 20\u201324\u00b0C; relative humidity, 50\u201360%; 12-h light\/dark cycle) in filter-topped cages (two rats per cage), with free access to food (Ssniff, Bio Services Uden, The Netherlands) and water. Rats were accustomed to laboratory conditions for at least 1 week before experimental use. Peritoneal carcinomatosis was induced by intraperitoneal inoculation of 2.0 \u00d7 106 CC-531 colon cancer cells, as described previously.14 All experiments were approved by the local Animal Welfare Committee of the Radboud University Nijmegen and were carried out in accordance with the Dutch Animal Welfare Act of 1997.\nOperative Procedure\nPrior to the laparotomy, all rats were given 10 mL of saline to prevent hypovolemia. Surgical procedures were performed under general anaesthesia using isoflurane 3%, O2 and N2O 1:1. Thirty minutes prior to and once daily until the third day postoperatively, rats were given buprenorphine (5\u00a0\u03bcg, 0.1 mL\/rat\/day) for analgesia. During the operation, rats were placed on a warmed mattress to limit body heat loss. All rats underwent a midline laparotomy. After opening the abdomen the extent of intraperitoneal tumor growth was scored semiquantitatively, 0 (no macroscopic tumor), 1 (little; located at 1\u20132 sites with a diameter of 1\u20132 mm), 2 (moderate; located at 1\u20132 sites and a diameter 2\u20135 mm), or 3 (abundant; located at multiple sites and\/or diameter >5 mm) in all four quadrants of the abdomen. The sum of the tumor scores of all sites represented the peritoneal cancer index (PCI).11\nSubsequently, CS, including a routine omentectomy, was performed in all rats. Irresectable tumor deposits were cauterized using an electrocoagulation device. After completion of the surgical cytoreduction, the abdominal wall was closed in two layers using continuous Vicryl 3\/0 sutures for the muscular component and iron wound clips for the skin in animal treated with CS only or CS + RIT.\nMonoclonal Antibody, Radiolabeling, and RIT\nThe murine MG1 monocolonal antibody (MAb), an anti-CC531 IgG2a monoclonal antibody that recognizes a 80 kDa cell surface antigen and localizes preferentially in tumors when injected in rats bearing CC-531 tumors,15 was purchased from Antibodies for Research Applications BV (Gouda, The Netherlands). Labeling of the antibody with 177Lu was carried out as previously described.11 In brief, the MAb was conjugated with 2-(4-isothiocyanatobenzyl)-diethylenetriaminepenta-acetic acid (ITC-DTPA) (Macrocyclics, Dallas, TX) and subsequently labeled with 177Lutetium (IDB Holland, Baarle Nassau The Netherlands) and purified by gel filtration on a PD10 column (Amersham, Pharmacia Biotech, Maarsen, The Netherlands). The purified 177Lu-MG1 preparation was diluted in PBS with 0.5% BSA for injection, the specific activity of the administered 177Lu-MG1 preparation was 0.4 MBq\/\u03bcg. The labeling procedure using 177Lu was performed under strict metal-free conditions.\nRIT (185 \u03bcg MG1\/ rat, radiolabeled with 74 MBq 177Lu in 3.0 mL) was intraperitoneally injected immediately after surgery, as this was determined to be the most optimal time for adjuvant administration.16\nMitomycin-C\nMitomycin-C (MMC) was obtained from Nycomed Christiaens BV (Breda, The Netherlands) as a powder in glass vial (40 mg\/vial). Immediately before use, MMC was dissolved in 0.9% sodium chloride to the appropriate concentrations.\nHIPEC Procedure\nFollowing CS, while the abdomen was still exposed, two multiperforated catheters (Argyle, Sherwood Medical, Ireland) were inserted laterally through the abdominal wall and subsequently fixed in the abdominal cavity. The inflow drain was placed in the right paracolic gutter, the outflow drain in the left subdiaphragmatic space. The intraperitoneal temperature was monitored with an intra-abdominal thermometer (PTFE Insulated thermocouple, VWR International, Amsterdam, The Netherlands), at the site with generally the highest tumor load (omentum). In addition, a thermometer was placed inside the rectum. After placement of the catheters, the abdominal wall was closed using a continuous suture (Ethilon 3.0, Johnson & Johnson, Ethicon) (Fig.\u00a01). During the HIPEC procedure, rats were removed from the warmed mattress to prevent general hyperthermia. The perfusion system was filled with 250 mL saline, containing 4 mg MMC (Mitomycin-C Kyowa, Christiaens). The perfusate was heated in a tube coil using a thermostatically regulated water bath set to a temperature of 48\u00b0C and infused into the peritoneal cavity by a roller pump (Ismatec IPS-8, Ismatec SA, Glattbrugg, Switzerland) for the duration of 60 minutes at 10 mL\/min. Abdominal inflow temperature was set at 44\u00b0C. In order to achieve a uniform heat distribution, gentle massage of the abdomen was applied throughout the duration of the HIPEC procedure. After completion of the perfusion, the abdominal cavity was flushed with warmed (37\u00b0C) saline for a period of 10 minutes. The abdomen was opened again to remove the catheters. Subsequently, the abdomen was closed as described previously.\nFIG.\u00a01.HIPEC Perfusion System; MMC Mitomycin C Kyowa 16 mg\/L perfusate. Adapted from Ref. 19. Reproduced with permission.\nIntraperitoneal Distribution of MMC and Dose Determination\nPrior to the therapy experiment with HIPEC, we investigated the intraperitoneal distribution of the perfusion fluid using a methylene blue stained perfusate. The perfusate was administered in the same fashion as in the therapeutic experiment. After completion, the abdominal cavity was inspected for the presence of blue dye in all quadrants on both parietal as well as visceral peritoneum of the intra-abdominal organs and the diaphragm. Subsequently, a study to determine the dose of MMC that resulted in acceptable toxicity was performed in nine animals (three animals per group). Animals underwent a laparotomy including an omentectomy and complete bowel inspection followed by heated perfusion of the abdominal cavity with MMC at 4 mg\/L or 16 mg\/L. Control rats underwent laparotomy and an omentectomy only. Body weight and physical condition were monitored during 6 days following the procedure to assess treatment-related toxicity.\nTreatment Efficacy\nSeven days after intraperitoneal tumor induction with 2.0 \u00d7 106 CC-531 tumor cells, 45 rats, 15 per treatment group, were randomly assigned to undergo either CS only, CS + RIT or CS + HIPEC. The operative procedures and application of the adjuvant therapies were performed as described previously. Toxicity of the treatment was determined clinically and by weighing the rats. Body weight was expressed as relative body weight compared with the body weight on the day of surgery. Survival was scored, and at autopsy the extent of tumor growth was determined.\nFollow-Up\nThe primary endpoint was 16-week survival. As part of monitoring the physical condition during the immediate postoperative period, the general condition was monitored and the body weight was measured daily during the first 2 weeks. When the humane endpoint (HEP) was reached (signs of massive hemorrhagic ascites, physical inactivity or signs of intra-abdominal tumor growth with invalidating consequences), rats were killed by O2\/CO2-administration and immediately dissected. The HEP was determined by an experienced biotechnician who was blinded to the therapeutic regimen. At the time of the HEP rats were generally lethargic, showing signs of advanced PC as the presence of ascites. At dissection, the intraperitoneal tumor growth was scored as described previously. At 16 weeks postoperatively, the study was terminated and the remaining rats were euthanized and dissected. In case of absence of macroscopic tumor, all relevant organs, including the greater momentum, the mesentery, and the diaphragm were removed for histopathological staining to determine tumor presence microscopically. Sections were stained using hematoxylin & eosin (H&E) and\/or immunohistochemical staining using the murine MG1 antibody in combination with a horse-anti-mouse IgG antibody, HRP conjugated (Vector Laboratories Inc., Burlingame, CA, USA).\nStatistical Analysis\nStatistical analysis was performed using SPSS (Chicago, IL) software and Graphpad Prism (Graphpad Software Inc., San Diego CA) for analysis. Comparison of dichotomous values was done using chi-square or Fisher\u2019s Exact test. Nonparametric testing was performed using two-way ANOVA testing. Survival curves were analyzed using Kaplan\u2013Meier curves and compared by means of the log-rank test. Posttesting using Bonferroni was applied to correct for multiple groups. All tests were two-sided; the level of statistical significance was set at a P value of <.05.\nRESULTS\nIntraperitoneal Distribution of HIPEC and Dose Determination\nThe intraperitoneal distribution of the perfusate administered according to the previously described procedure, showed a distribution pattern amongst all quadrants, including the diaphragm bilaterally and at the mesenterial root (Fig.\u00a02).\nFIG.\u00a02.Intraperitoneal distribution of methylene blue stained perfusate.\nThe applied dose of 16 mg MMC\/L resulted in a maximum mean weight loss of 13.7 \u00b1 2.9% at 4 days postoperatively. In addition, the first 3 days following the heated perfusion, animals were lethargic and suffered from diarrhea from day 2 until day 4 postoperatively. In contrast, the maximum weight loss in the 4 mg\/L was 8.3 \u00b1 2.9% at day 3 and 7.5 \u00b1 2.3% at day 3 in the control group (Fig.\u00a03). None of the animals died during the immediate postoperative period. Based on these observations, HIPEC, when administered at a dose of 16 mg\/L for the duration of 60 minutes at the given temperature, was considered to be the maximal tolerable dose to be used for the HIPEC procedure.\nFIG.\u00a03.The relative body weight of Wag\/Rij rats after exploratory laparotomy (control) and heated intraperitoneal chemotherapy (HIPEC) given immediately postoperatively in different doses. Data represent means \u00b1 standard error of the mean (SEM).\nOperative Procedure\nPreoperative body weight did not differ between groups, P = .52 (Table\u00a01) At laparotomy, tumor nodules were present in the omentum, liver hilum, the mesentery, and gonadal fatpads (1\u20133 mm diameter). Median PCI score at time of surgery was 5 (range 4\u20138) and was similar in all experimental groups. After surgical cytoreduction, residual disease remained in situ in 7 rats after cauterization and was equally distributed among the groups (P = .84). The surgical procedures without adjuvant therapy took 20\u201330 minutes per animal.\nTABLE\u00a01.Treatment group characteristicsPCIMedian (range)CSCS + HIPECCS + RITPreoperative body weight (g)266 (251\u2013287)264 (245\u2013285)262 (244\u2013276)Tumor score per site\u00a0\u00a0Greater omentum2 (2\u20133)2 (1\u20132)2 (1\u20132)\u00a0\u00a0Liver hilum1 (0\u20131)1 (0\u20131)1 (0\u20131)\u00a0\u00a0Perisplenic0 (0\u20131)0 (0)0 (0)\u00a0\u00a0Mesentery1 (0\u20132)1 (0\u20132)1 (0\u20132)\u00a0\u00a0Gonadal fatpads0 (0\u20132)0 (0\u20132)1 (0\u20132)\u00a0\u00a0Diaphragm0 (0\u20131)0 (1)0 (0\u20131)\u00a0\u00a0Parietal peritoneum1 (0\u20131)1 (0\u20131)1 (0\u20131)\u00a0\u00a0Total5 (4\u20138)5 (4\u20136)5 (4\u20138)Resection macroscopically complete\u00a0\u00a0Yes121313\u00a0\u00a0No322Treatment group characteristics (peritoneal cancer index; PCI) found during laparotomy before the administration of the adjuvant therapy.CS, cytoreductive surgery; HIPEC, heated intraperitoneal chemotherapy; RIT, radioimmunotherapy. PCI is expressed as median and range.\nThere was no intraoperative mortality. However, one rat in the CS + HIPEC and one rat in the CS + RIT group were euthanized after 2 and 9 days, respectively. The animal in the CS + HIPEC group showed massive weight loss as a result of bowel necrosis and subsequent perforation, the cause of death of the animal in the CS + RIT group remained unclear. The median intra-abdominal temperature during the HIPEC procedure, measured at the site where the greater omentum was removed, was 41.0\u00b0C (range 40.4\u201341.6\u00b0C). In contrast, the median rectal temperature was 34.6\u00b0C (range 34.1\u201334.8\u00b0C) (Fig.\u00a04).\nFIG.\u00a04.The recorded intra-abdominal and rectal temperature during the HIPEC procedure. Data represent means \u00b1 standard error of the mean (SEM).\nCS and CS + RIT were well tolerated, whereas animals in the CS + HIPEC groups showed signs of physical discomfort; animals of the latter group were generally lethargic and showed pilo erection two days following the procedure. In addition, these animals all suffered from diarrhea up to 4 days after the HIPEC procedure. The relative body weight after of the various treatment groups is depicted in Fig.\u00a05. Maximum body weight loss after CS or CS + RIT was similar (7.3 \u00b1 2.6% vs 9.3 \u00b1 1.8% 4 days postoperatively, P > .05). Rats that received adjuvant HIPEC had a maximum body weight loss of 12.3 \u00b1 1.7%, which was significantly higher than that after CS alone (P < .001) or CS + RIT (P < .001). Rats generally gained weight from the fifth postoperative day onward. In the HIPEC group, however, postoperative mean body weight remained significantly lower than that of the animals in the CS group, until 5 weeks postoperatively.\nFIG.\u00a05.The relative body weight of Wag\/Rij rats with small peritoneal CC-531 tumors in the first 14 days after cytoreductive surgery (CS) only, CS + radioimmunotherapy given immediately postoperatively (RIT) or heated intraperitoneal chemotherapy (HIPEC) given immediately postoperatively. Data represent means \u00b1 standard error of the mean (SEM).\nTreatment Efficacy\nDuring the experiment, 29 animals were euthanized because of massive amounts of ascites that resulted from intraperitoneal tumor growth. The mean amount of ascites at the humane endpoint was 31 \u00b1 22.6 mL, 26.5 \u00b1 23.8 mL, and 26.4 \u00b1 22.6 mL in the CS, RIT, and HIPEC groups, respectively (P = .82). At the time of death, mean PCI in the CS, CS + HIPEC, and CS + RIT groups was 18 (range 9\u201322), 12 (range 5\u201315), and 18 (range 16\u201319), respectively, with significant differences between the CS + HIPEC and both other treatment groups (P < .001 for both comparisons).\nThe survival curves of the various treatment groups are depicted in Fig.\u00a06. Median survival of the rats that were treated with CS only was 57 days (range 36\u2013112). Adjuvant HIPEC resulted in a median survival of 76 days (range 33\u2013112), P = .17, when compared with CS only. Median survival of the rats that were treated with CS followed by the adjuvant administration of RIT was improved to a median survival of 97 days (range 49\u2013112), P < .001 compared with CS only. When compared with CS followed by adjuvant HIPEC, the adjuvant administration of RIT to surgery did not result in an improved survival (P = .33).\nFIG.\u00a06.Kaplan\u2013Meier survival curves for Wag\/Rij rats with small peritoneal CC-531 tumors after cytoreductive surgery (CS), CS + RIT (RIT) or CS + HIPEC (HIPEC).\nAt the endpoint of the study, 16 weeks after CS, 14 animals (two animals in the CS group, five animals in the CS + HIPEC group, and seven animals in the CS + RIT group) were still alive, without any physical signs of intraperitoneal tumor growth.\nOf these 14 animals that were still alive 16 weeks after surgery, one animal in the CS + HIPEC group and three animals in the CS + RIT group showed macroscopic evidence of tumor at dissection. In the remaining 10 animals (two in the CS alone group, four in the HIPEC group, and four in the RIT group) not even microscopic tumor presence was found.\nDISCUSSION\nIn the present study, adjuvant radioimmunotherapy after cytoreductive surgery for peritoneal carcinomatosis of colorectal origin in rats significantly improved survival, whereas HIPEC did not. In addition, the application of HIPEC was associated with considerably more toxicity as compared to RIT.\nThe treatment of peritoneal carcinomatosis was studied with CC-531 syngeneic tumors that grew intraperitoneally in Wag\/Rij rats. This model is highly reproducible, and the growth and distribution pattern throughout the abdominal cavity are similar to the human entity of PC.14 Cytoreduction performed at 7 days after tumor reduction resulted in minimal residual disease (<1 mm). In this setting, both HIPEC and RIT result in maximum therapeutic efficacy.12,13,17\nThe MG1 MAb preferentially localizes in the CC-531 tumors,11 with only minor localization in thymus, lymph node, salivary gland tissue, and skin.15177Lu was selected as the radionuclide for RIT of minimal residual disease because of its high tumor uptake and adequate physical properties including a medium-energy \u03b2-emission with a maximum penetration range in tissue of 2.5 mm. In our previous studies we have used the combination of 177Lu-MG1 radionuclide-antibody. These studies demonstrated the combination to be highly effective for the improvement of survival in the model of PC as described previously.11,16 Moreover, in previous experiments we have shown that radioimmunotherapy with a radiolabeled irrelevant antibody is less effective by far compared with therapy with a radiolabeled specific antibody.18 HIPEC has been studied in only a few preclinical studies.19\u201322 These studies showed that its use was associated with a decreased tumor load compared with control groups.19 However, in these studies, HIPEC was associated with a considerable toxicity, indicated by lethargy, marked body weight loss, and bacterial translocation.23 These results on toxicity are in corroboration with the results of our study and mimic the clinical effects of HIPEC. Clinical studies with postoperative intraperitoneal chemotherapy are associated with a high mortality and morbidity.12\nOf 16 published reports on the use of HIPEC in the clinical setting, 13 reports described the administration of MMC.12 In vitro, MMC has also shown to inhibit growth of CC531 cells in a concentration a thousand-fold lower than the concentration used in the present experiment.24 The applied dose of 16 mg\/L MMC in our study is within the range of doses applied in clinical practice (5\u201320 mg\/L25) and is higher than described in other preclinical studies (2.25 mg\/L19 and 4 mg\/L22). In addition, in the present study HIPEC was applied as an adjuvant treatment to cytoreduction, whereas Pelz et al.19 applied HIPEC as monotherapy with subsequent killing of the animals after only 10 days.\nThe intraperitoneal distribution of MMC during the perfusion was studied before the start of the actual experiment and showed equal distribution among all quadrants. The effect of the perfusion technique on distribution differences and their associated differences in survival have never been studied clinically. Glehen and colleagues performed a large clinical study in 506 patients that were treated with both the open and closed abdomen perfusion technique. The authors reported no differences in survival between both perfusion techniques.6 This observation was confirmed by the study of Sugarbaker and colleagues.26\nRIT with 74 MBq of the 177Lu-MG1 radionuclide-antibody in 3 mL has previously been shown to be an effective treatment for experimentally induced PC of colonic origin when administered intraperitoneally.11,16 The biodistribution of intraperitoneally injected 111In-labeled MG1 was studied by Koppe et al. and showed a preferential uptake of the radiolabeled antibody in the tumor.11\nThe extraperitoneal temperature of 48\u00b0C necessary to obtain inflow temperatures of 44\u00b0C would not have had a negative influence on the cytotoxicity of MMC, since Ahrar et al.27 showed that only temperatures exceeding 60\u00b0C decreased its cytotoxic effect. On the other hand, one has to bear in mind the fact that the additive effect of hyperthermia in HIPEC in the clinical setting has not yet been proven in a randomized trial. Elias et al. and Glehen et al. both reported the use of early postoperative chemotherapy (EPIC), without hyperthermia, ranging from day 1 to day 5 after surgery, and HIPEC. Elias et al. found no significant difference in survival. Similarly, in the multicenter study in 506 patients of whom 53.3 and 24.3 % underwent HIPEC and EPIC alone, no significant difference was found in survival between the two treatment groups.6 The duration of the heated perfusion, 60 min. in this experiment, is in concordance with the recent consensus statement regarding Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy in the Management of Peritoneal Surface Malignancies of Colonic Origin, stating that the perfusion should last 60\u2013120 minutes.13 These data on the dose of MMC, the used perfusion time and temperature, together with our results that showed antitumor effect in the model of induced PC from CC531 cells (significantly lower PCI at HEP in favor of CS + HIPEC), we can conclude that the HIPEC model used in our study was able to induce regression of PC of colonic origin.\nIn the latest reported clinical trial on adjuvant RIT in the setting of colon cancer, Liersch et al. reported results of a Phase II trial with 131I-labeled anti-CEA antibody labetuzumab administered to patients after complete resection of colorectal liver metastases. This study, where RIT was applied in an adjuvant setting to complete resection, resulted in a promising 5-year survival rate of 51.5%.10 The results of this study is in accordance with the conclusion of a recent review on the use of RIT to treat colon cancer.17 In this review, the authors state that the time may have come for clinical trials in which RIT is added to standard regimens to establish the place of this treatment modality as an adjuvant treatment after CS.\nTo our knowledge, the present study is the first study comparing the use of RIT and HIPEC in an adjuvant setting to CS for the treatment of PC in colorectal cancer. Our preclinical studies indicate that the application of RIT immediately following CS can improve survival in rats with PC of CRC. Moreover, from the present study we conclude that the use of adjuvant RIT is an effective treatment with low toxicity. When compared with today\u2019s standard of care, HIPEC, RIT was at least as effective. RIT consisted of an activity dose of 74 MBq of 177Lu-labeled MG1per rat, resulting in only minor toxicity, whereas the theoretical MTD of 177Lu-labeled antibodies in 250 g rats could be approximately 150 MBq.11\nThere are, however, some challenges to the clinical applications of adjuvant RIT. For example, after cytoreductive surgery, patients are transferred to the intensive care unit. Optimal patient care has to be balanced with radiation safety issues for the medical staff. Previously, we reported on the optimal time interval between RIT and CS.16 In that study, we showed that RIT should be administered as soon as possible after CS, with a window of opportunity for RIT administration up to 4 days after surgery. It can therefore be envisioned that for radiation safety reasons the therapy should be given not before discharge of the patient from ICU and removal of the abdominal drains.\nOur study therefore justifies the consideration of intraperitoneal radioimmunotherapy after cytoreductive surgery in case of peritoneal carcinomatosis of colorectal cancer. In clinical studies, this approach should be compared with HIPEC.\nCONCLUSION\nThis study showed that RIT adjuvant to CS significantly improved survival compared to CS alone in a rat model of PC of CRC, whereas the contemporary gold standard, HIPEC, did not cause a significant improvement in survival. This improvement of survival was associated with a decreased level of treatment-related toxicity compared with HIPEC. Adjuvant radioimmunotherapy might therefore be an alternative adjuvant treatment after cytoreductive surgery of PC of colorectal origin in a clinical trial setting.","keyphrases":["radioimmunotherapy","peritoneal carcinomatosis","cytoreductive surgery","heated intraperitoneal chemotherapy","adjuvant","colon cancer"],"prmu":["P","P","P","P","P","P"]}
{"id":"Int_J_Colorectal_Dis-4-1-2386750","title":"Surgical and pathological outcomes of laparoscopic surgery for transverse colon cancer\n","text":"Purpose Several multi-institutional prospective randomized trials have demonstrated short-term benefits using laparoscopy. Now the laparoscopic approach is accepted as an alternative to open surgery for colon cancer. However, in prior trials, the transverse colon was excluded. Therefore, it has not been determined whether laparoscopy can be used in the setting of transverse colon cancer. This study evaluated the peri-operative clinical outcomes and oncological quality by pathologic outcomes of laparoscopic surgery for transverse colon cancer.\nIntroduction\nSince its first report [1], laparoscopic colon surgery has been controversial with regard to its use for colorectal cancer. Several prospective randomized trials including the COLOR and CLASSIC studies have demonstrated that laparoscopic-assisted surgery for colorectal cancer resulted in a shorter hospital stay, reduced analgesic use, and earlier recovery to bowel movement [2\u20136]. Moreover, the COST study established the long-term oncological safety of laparoscopic-assisted surgery for colon cancer, and currently the laparoscopic approach is accepted as an alternative to open surgery for colon cancer [7]. However, transverse colon cancer was excluded from prior randomized controlled trials. The reasons for exclusion of transverse colon cancer include the following: difficulty in deciding the appropriate operative procedure and extent of lymph node dissection, as well as technical difficulties with the laparoscopic identification, ligation, and lymph node dissection around the middle colic vessels. Therefore, there continues to be debate on whether to use laparoscopic surgery for transverse colon cancer.\nMaterials and methods\nBetween August 2004 and November 2007, the medical records of all patients who underwent laparoscopic surgery for colorectal cancer were reviewed. Laparoscopic colorectal cancer resection was started August 2004 in our clinic. Pathologic confirmation, colonoscopy, barium enema, computed tomography (CT), ultrasonography, and chest PA were performed for diagnosis in all patients preoperatively. All patients with colorectal adenocarcinoma admitted to our clinic were considered for laparoscopic surgery. Exclusion criteria for laparoscopic surgery were as follows: (1) patients with colorectal cancer obstruction and failure of stent insertion, (2) patients with colorectal cancer perforation requiring emergency surgery, (3) patients with T4 colorectal cancer lesion that could not be resected laparoscopically, and (4) patients with compromised cardio-pulmonary function in whom pneumoperitoneum under general anesthesia was contraindicated. In this study, transverse colon cancer was defined as lesions between the hepatic flexure and splenic flexure in the colon, requiring ligation of the middle colic vessels at their origin. CT or barium enema was performed in all patients with colon cancer preoperatively for localization of the tumor. If radiological localization was unclear, preoperative colonoscopic Indian ink tattooing or endoscopic clipping was performed.\nThe procedure used for the transverse colon cancer was chosen based on the location of the tumor. A tumor located at the hepatic flexure or within 10\u00a0cm distal to hepatic flexure was treated by an extended right hemicolectomy, and a tumor located at the splenic flexure or within 10\u00a0cm proximal to the splenic flexure was treated by an extended left hemicolectomy. A tumor located between the above two lesions was treated by a transverse colectomy. An extended right hemicolectomy was defined as lymphadenectomy simultaneously with ligation of ileocolic, right colic, and middle colic vessels at their origins; an extended left hemicolectomy was defined as lymphadenectomy simultaneously with ligation of left colic and middle colic vessels at their origins. A transverse colectomy was defined as lymphadenectomy simultaneously with ligation of middle colic vessels at their origins. Extra-corporeal anastomosis was performed in all cases of laparoscopic surgery for transverse colon cancer. All patients started a diet after passing flatus. To evaluate the postoperative surgical outcomes and oncological quality of laparoscopic surgery for transverse colon cancer, we compare the age, gender, body mass index (BMI), operating time, blood loss, time to passing flatus, time to start of diet, hospital stay, surgical morbidity, surgical mortality, conversion to open surgery, tumor size, distal resection margin, proximal resection margin, radial margin, and number of harvested lymph nodes between the transverse colon cancer group (TCC) and other site colon cancer group (OSCC). In this study, a single surgeon (YS Lee) performed all operations. Comparisons between the two groups were made by applying the independent samples t test and \u03c72 test. Differences were considered to be significant for P value\u2009<\u20090.05.\nResults\nPostoperative clinical outcomes\nThree hundred and twelve patients underwent colorectal cancer resection in our clinic during these periods. Among them, 44 patients underwent conventional open surgery according to exclusion criteria (five cases of stent failure or colon obstruction, five cases of emergency operation, 12 cases of far advanced tumor, 12 cases of recurrent cancer operation, seven cases of patient\u2019s refusal, and three cases for old age with high risk for pneumoperitoneum). There was no transverse colon cancer in the conventional open surgery group. A total of 268 patients underwent laparoscopic resection for colorectal cancer. Of the 268 patients, 140 patients who underwent laparoscopic resection for rectal cancer were excluded in this study. Finally, 128 patients who underwent laparoscopic resection for colon cancer were enrolled in this study. Of the 128 patients, 34 patients had transverse colon cancer, counting for 10.8% of total colorectal cancers, and 94 patients had other sites of colon cancer. In the TCC group, extended right hemicolectomy was performed in 18 cases, transverse colectomy was performed in eight cases, and extended left hemicolectomy was performed in eight cases. In the OSCC group, right hemicolectomy was performed in 38 cases, left hemicolectomy was performed in five cases, and anterior resection was performed in 51 cases. There were no statistical differences in age, gender distribution, BMI, operating time, intraoperative blood loss, time to flatus, time to start of diet, and hospital stay between patients with TCC and OSCC (Table\u00a01). Four patients in the OSCC group had a major complication, one had colon injury, and two had anastomosis leak. A simple laparoscopic closure was performed in the case of colon injury, and two patients with anastomosis leak underwent re-operation. Minor complications that occurred in four cases in the OSCC group (one case of ileus, two cases of port site minor infection, and one case of urinary retention) and two cases in the TCC group (one case of ileus and one case of atelectasis) were treated successfully conservatively. One case among the TCC group and three cases in the OSCC group were converted to open surgery; all of these converted cases were due to tumor-related factors, T4 lesion, or huge tumor. There was no surgical mortality in this study.\nTable\u00a01Clinical characteristics of patients\u00a0TCC (N\u2009=\u200934)OSCC (N\u2009=\u200994)P valueAge (years)64.1\u2009\u00b1\u200911.362.5\u2009\u00b1\u200912.1NSSex (M:F)15:1946:48NSBody mass index (kg\/m2)23.5\u2009\u00b1\u20093.024.0\u2009\u00b1\u20093.1NSOperating time (min)211.1\u2009\u00b1\u200952.2 (140\u2013360)a220.4\u2009\u00b1\u200994.3 (60\u2013620)aNSBlood loss (ml)100.0\u2009\u00b1\u200983.8 (0\u2013400)a114.2\u2009\u00b1\u2009145.2 (0\u2013700)aNSTime to pass flatus (days)2.8\u2009\u00b1\u20090.82.6\u2009\u00b1\u20091.0NSDiet start (days)4.2\u2009\u00b1\u20091.84.2\u2009\u00b1\u20092.8NSHospital stay (days)11.4\u2009\u00b1\u20094.111.2\u2009\u00b1\u20096.0NSTCC Transverse colon cancer, OSCC other site colon cancer, NS not significantaValues are ranges.\nOncological quality by pathologic outcomes\nThere were no statistical differences in the tumor size, proximal resection margin, distal resection margin, radial margin, and number of harvested lymph nodes between patients with TCC and OSCC (Table\u00a02).\nTable\u00a02Pathological outcomes of patients\u00a0TCC (N\u2009=\u200934)OSCC (N\u2009=\u200994)P valueTumor size (cm)5.2\u2009\u00b1\u20092.5 (0.7\u201311)a4.6\u2009\u00b1\u20092.4 (0.1\u201314)aNSPRM (cm)19.5\u2009\u00b1\u200910.2 (10.0\u201325.0)a15.3\u2009\u00b1\u200911.0 (4.2\u201325.0)aNSDRM (cm)13.9\u2009\u00b1\u20096.9 (6.0\u201325.0)a11.6\u2009\u00b1\u20095.3 (3.0\u201332.0)aNSNo. of lymph nodes24.4\u2009\u00b1\u200911.7 (3\u201359)a21.1\u2009\u00b1\u20098.4 (3\u201359)aNSRadial margin (cm)0.9\u2009\u00b1\u20090.8 (0\u20133.0)a1.0\u2009\u00b1\u20090.9 (0\u20133.5)aNSTCC Transverse colon cancer, OSCC other site colon cancer, DRM distal resection margin, PRM proximal resection margin, NS not significantaValues are ranges.\nDiscussion\nSince laparoscopic colon resection was first reported in 1991 [1], laparoscopic surgery has been widely employed for various benign colorectal disease such as benign mass, diverticular disease, inflammatory bowel disease, rectal prolapse, and now increasingly for colorectal malignant disease. Evidences of the safety and efficacy of the laparoscopic surgery for colorectal cancer had been reported from several prospective randomized controlled studies and meta-analysis of several trials, which favored the laparoscopic surgery for colorectal cancer over conventional open surgery due to its many short-term benefits such as shorter hospital stay, reduced use of analgesics, and earlier recovery of bowel movements [2\u20136, 8\u201313]. Eventually, long-term oncological safety of laparoscopic colon cancer resection was established, and the laparoscopic approach was accepted as an alternative to open surgery for colon cancer by the COST study [7].\nTransverse colon cancer occurs in about 10% of cases of colorectal cancer, and it often presents a challenge for the choice of the best surgical procedure based on the location of the tumor and extent of lymph node dissection. There could also be technical difficulties with laparoscopic identification, ligation, and lymph node dissection around the middle colic vessels depending on the surgeon\u2019s experience. Because of these reasons, transverse colon cancer was excluded from almost every prior prospective randomized trial. Therefore, there is continued debate over the validity of laparoscopic surgery for transverse colon cancer. The major controversy about laparoscopic surgery for transverse colon cancer lies on whether or not it is feasible to perform sufficient extent of lymph node dissection around the middle colic artery laparoscopically. As experiences of laparoscopic surgery are accumulating and surgical techniques and instruments are developing, we consider that the extent of laparoscopic lymph node dissection for transverse colon cancer is not less than the extent of conventional lymph node dissection for transverse colon cancer.\nSome of the difficulties with laparoscopic surgery for transverse colon cancer that need to be resolved include the following: First, precise localization of carcinoma of the transverse colon is important; this is because the extent of resection and lymph node dissection depends on the location of the tumor in the transverse colon. Diminished tactile guidance can make localization of the tumor in the transverse colon more difficult in small tumor. There are a number of methods used to localize the tumor. Preoperative barium enema is useful for localization of large and advanced tumor, but radiological localization is inconclusive and difficult in early cancer. In such cases, colonoscopic tattooing with Indian ink or placing endoscopic clips is very effective. Using endoscopic clip at the time of preoperative colonoscopy and using fluoroscopy for localization, the clip migration could be the main problem, and using fluoroscopy in the operating theater is troublesome and time consuming. However, in the case of hepatic flexure or splenic flexure colon cancer, by checking X-ray just after placing the endoscopic clip, we could precisely localize both flexure tumors easily. Intraoperative colonoscopy can be used for localization of the tumor, but it is also time consuming, and moreover, it can cause colonic insufflation that makes laparoscopic surgery difficult [14]. Properly placed tattoos are long lasting and can be placed at the time of diagnostic colonoscopy [15]. In the event of tattoo failure, one can use intraoperaitve colonoscopy for localization easily. In this study, we performed preoperative colonoscopic tattoo in three cases to localize the mid transverse colon cancer, colonoscopic clipping in three cases of hepatic flexure colon cancer, and barium enema in the other cases. There was no case with non-localization. We think that colonoscopic tattooing is effective in small mid transverse colon cancer, and endoscopic clipping is effective in small hepatic or splenic flexure colon cancer. Second is the laparoscopic identification, ligation, and lymph node dissection around the middle colic vessels. Fujita et al. reported laparoscopic techniques that can be used for identification of middle colic vessel. They suggested that the ventral aspect of the caudal portion of superior mesenteric vein be exposed, and the exposed vessel brought cephalad toward the caudal portion of the pancreas, with identification of the middle colic vessels [16]. Baca et al. introduced the \u2018window technique\u2019 for lymphadenectomy with simultaneous resection of the vascular stem [17]. Ichihara et al. introduced the technique of rotation of mesocolon to identify middle colic artery [18]. The authors have identified the middle colic vessels using surgical techniques introduced by Fujita in most of the cases, and the in case of slender patients we could directly identify pulsating middle colic artery by stretching the transverse mesocolon by traction of both ends of the transverse colon by the first assistant. Identifying middle colic vessels, excessive traction of the transverse mesocolon by the first assistant may cause tearing of the vein and bleeding. Moreover, the length of the gastrocolic trunk of Henle is relatively short, and attempt to control bleeding from the gastrocolic trunk of Henle can injure the superior mesenteric vein [19]. It is especially important for a laparoscopic colorectal surgeon to recognize the variable drainage of the right colic and middle colic vein to the gastrocolic trunk of Henle and to have a precise knowledge of superior right colic vein anatomy. Third is the low incidence of transverse colon cancer. Consecutive cases are needed for overcoming the learning curve. Therefore, it might take surgeons longer time to become experienced in the techniques used for laparoscopic transverse colon cancer resection.\nIn this study, there were no significant differences between patients with TCC and OSCC in terms of operating time, blood loss, resection margin, and number of lymph nodes. Schlachta et al. reported that the operating time was longer in the TCC group compared to the OSCC group, and the number of harvested lymph nodes in the TCC group was greater than in the OSCC group because of the extended resection and larger specimens, as well as the need to attend to the middle colic vessels in patients with TCC [20]. However, in this study, there was no statistical difference in operating time and number of harvested lymph nodes, as well as the surgical outcomes between the two groups. We think that operating time could be similar in the TCC and OSCC groups in experienced hands, and actually, the extent of lymph node dissection in transverse colon cancer is not too wide than that of other site colon cancer, so there were no statistical differences in pathological outcomes between the two groups in this study.\nThere was one case of colon injury proximal to anastomosis caused by electrocautery during the operation in the OSCC group; this was treated by a laparoscopic simple closure on postoperative day 3. Three cases of anastomosis leak, one case of right hemicolectomy, and two cases of anterior resection occurred in the OSCC group. A patient with anastomosis leak was diagnosed by clinical symptoms or sign, fecal or purulent discharge from drain, fever, leukocytosis, and peritoneal irritation sign. Radiologic study using a water-soluble dye was not performed in this study. Of the three cases, one patient with a leak of anterior resection was treated conservatively successfully, and two patients with leak cases underwent laparoscopic re-operation. All other minor complications were successfully treated conservatively.\nConversion to open surgery occurred in one case in the TCC group and in three cases in the OSCC group; these differences were not significant. Factors related to the tumor were the cause of conversion to open surgery. One case in the TCC group was converted to open surgery due to T4 lesion in the transverse colon cancer that invaded the anterior wall of the stomach. In the OSCC group, one case with a larger tumor that was hard to handle by laparoscopy, and two cases of sigmoid colon cancer that invaded the uterus were converted to open surgery. However, T4 colon cancer is not a contraindication for laparoscopy if en bloc resection could be performed with the laparoscopy. In those cases converted to open surgery, laparoscopic en bloc resection was impossible.\nThis study has some weak points. First, the number of patients with transverse colon cancer is too small, making up 10% of the total colorectal cancer resection and 12% of the total laparoscopic colorectal resection in this study. The second is that although the data in this study were collected prospectively, the data were not derived from a larger case series and this study was not randomized controlled. The third is that the mean follow-up period was too short to evaluate the oncological outcomes (15.9\u00a0months; range 1\u201340\u00a0months). We think that large-scale prospective controlled trials and long-term analysis are mandatory to overcome these limitations and confirm the oncological safety of laparoscopic transverse colon cancer surgery. We are going to report a long-term analysis after a long-term follow-up involving more cases prospectively.\nConclusions\nThe results of this study show no significant differences with regard to surgical outcomes and oncological quality by pathologic outcomes between patients in the OSCC and TCC groups. Further investigations with large-scale prospective studies and long-term analysis of laparoscopic surgery for transverse colon cancer are mandatory to establish the oncological safety of laparoscopic surgery for transverse colon cancer.","keyphrases":["transverse colon","colon cancer","laparoscopy"],"prmu":["P","P","P"]}
{"id":"Biochem_Biophys_Res_Commun-1-5-1899526","title":"An outwardly rectifying anionic background current in atrial myocytes from the human heart\n","text":"This report describes a hitherto unreported anionic background current from human atrial cardiomyocytes. Under whole-cell patch-clamp with anion-selective conditions, an outwardly rectifying anion current (IANION) was observed, which was larger with iodide than nitrate, and with nitrate than chloride as charge carrier. In contrast with a previously identified background anionic current from small mammal cardiomyocytes, IANION was not augmented by the pyrethroid tefluthrin (10 \u03bcM); neither was it inhibited by hyperosmolar external solution nor by DIDS (200 \u03bcM); thus IANION was not due to basal activity of volume-sensitive anion channels. IANION was partially inhibited by the Cl\u2212 channel blockers NPPB (50 \u03bcM) and Gly H-101 (30 \u03bcM). Incorporation of IANION into a human atrial action potential (AP) simulation led to depression of the AP plateau, accompanied by alterations to plateau inward calcium current, and to AP shortening at 50% but not 90% of complete repolarization, demonstrating that IANION can influence the human atrial AP profile.\nThe electrophysiological behaviour of cardiac myocytes from mammalian hearts is determined by the combined activity of a range of different cation and anion channel types. The reversal potential for chloride (Cl\u2212) ions in the heart (ECl) lies between \u223c\u221260 and \u221240\u00a0mV [1]. Negative to ECl outward Cl\u2212 movement generates depolarizing ionic current, whilst positive to ECl inward Cl\u2212 movement generates repolarizing ionic current. Therefore, the activation of Cl\u2212 channels can influence both the resting membrane potential and the duration of cardiac action potentials (APs) ([1\u20133] for reviews). Several different anion channel types have been identified that may contribute to cardiac physiology and pathophysiology [1\u20133]. Of the cardiac anion channel currents thus far identified, the three major types are: (i) a cystic fibrosis transmembrane conductance regulator (CFTR) current-activated through cAMP-dependent phosphorylation (ICl,cAMP; e.g. [4\u20136]); (ii) a stretch- or swelling-activated Cl\u2212 current (ICl,Swell; e.g. [7\u20139]) and (iii) a Ca2+-activated Cl\u2212 current (ICl,Ca; e.g. [10\u201312]).\nRecently, an outwardly rectifying anionic background current (IAB) has been identified in cardiac myocytes from two commonly studied model species (rat and guinea-pig) using whole-cell patch-clamp measurements [13,14]. IAB is distinct from previously identified Cl\u2212 currents as it has a distinct permeability sequence  and is insensitive to the stilbene diphosphonate Cl\u2212 channel inhibitor DIDS, to cell swelling and to intracellular Ca2+ and cAMP [13,14]. IAB can also be differentiated from other major cardiac anion currents as it can be activated by the pyrethroid agent tefluthrin [14]. Anion substitution experiments have provided evidence that IAB can influence AP duration (APD) [13]. There is some disagreement as to whether or not a basally active anionic current exists in human atrium [15,16] and there is no information as to whether humans exhibit an IAB with the characteristics of that seen in small mammal hearts. Therefore, the present study was undertaken to determine whether or not IAB exists in adult human cardiac myocytes. The resulting findings indicate the presence in human atrial myocytes of an outwardly rectifying anionic background current (IANION). Notably, the IANION observed in this study has the potential to contribute to human atrial electrophysiology, but is distinct from both the IAB recorded previously from myocytes from small mammal hearts [13,14] and from outwardly rectifying stilbene diphosphonate-sensitive anionic currents recorded previously from human atrium [17].\nMethods\nAtrial myocyte isolation\nThe study was approved by the local Central and South Bristol Research Ethics Committee and was conducted in accordance with the principles of the Declaration of Helsinki. Human right atrial appendages were obtained, with consent, from 32 patients (27 males, 5 females, average age 69.7\u00a0\u00b1\u00a01.7\u00a0years) undergoing coronary artery bypass surgery. Single human atrial myocytes were isolated from right atrial appendages by mechanical and enzymatic dispersion. Tissue samples were quickly immersed in cardioplegic solution (see Table 1; solution G, 100% O2, ice cold). The samples were chopped into small chunks and washed with an EGTA-containing solution (see Table 1; solution H) gassed with 100% O2 for 15\u00a0min at 37\u00a0\u00b0C. The chunks were then incubated in the same solution from which EGTA was excluded and protease type XXIV (3\u00a0U\/ml, Sigma) and collagenase type V (250\u00a0U\/ml, Sigma) were added. The medium was continuously gassed with 100% O2 at 37\u00a0\u00b0C. After 15\u00a0min, the incubation medium was substituted for the same solution containing collagenase only. Myocytes were progressively released from the chunks into the supernatant and their yield monitored under a microscope. The suspension was washed in enzyme-free solution and the myocytes were stored at room temperature until use (within \u223c8\u00a0h of cell isolation).\nElectrophysiology\nSolutions used. Experimental solutions for the investigation of anionic current were similar to those used previously to study IAB\n[14]; the composition of all solutions used is given in Table 1. Osmolarity values given for each of the solutions listed in Table 1 were measured using a micro-osmometer employing a freezing-point method (Advanced Instruments, Norwood, MA, USA). Myocytes used in whole-cell voltage-clamp experiments were superfused (20\u201325\u00a0\u00b0C) with a standard Hepes-buffered Tyrode\u2019s solution (see Table 1; solution A) until the whole-cell recording configuration had been obtained. For isolation of background anion current, sodium-free Tyrode\u2019s solutions were used (solutions B\u2013E) in which Na was replaced by N-methyl-d-glucamine (NMDG), with one of several possible dominant anions: solution B, chloride; solution C, aspartate; solution D, iodide; solution E, nitrate. All drugs used were added to solution E from stock solutions made in dimethyl sulfoxide (DMSO) with an exception of N-(2-naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene)glycine hydrazide (Gly H-101), which was solved in distilled water. The hyperosmotic external solution (solution F) was prepared by adding 70\u00a0mM sucrose to solution E. A Cs-based pipette solution (solution I) was used for all experiments. Solution I was sodium-free to prevent contamination of chloride currents by the sodium\u2013calcium exchanger current.\nDrugs. Diisothiocyanostilbene-2,2\u2032-disulfonic acid (DIDS, final concentration 200\u00a0\u03bcM), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, final concentration 50\u00a0\u03bcM) and tefluthrin (TEF, final concentration 10\u00a0\u03bcM) were purchased from Sigma Chemical Co. (Poole, UK). N-(2-naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene)glycine hydrazide (Gly H-101, final concentrations of 10 and 30\u00a0\u03bcM) was purchased from Merck (Frankfurt, Germany). All the drug-containing solutions were protected from light throughout.\nElectrophysiological recording\nIn electrophysiological experiments, junction potential changes were minimized by immersing the reference Ag\/AgCl electrode in a 3\u00a0M KCl solution with a continuous agar bridge (4% agar in 3\u00a0M KCl). Borosilicate glass pipettes (Harvard Apparatus, UK) were pulled using a vertical two-step Narishige PP-830 microelectrode puller (Narishige, Japan) and had a tip resistance of 5\u20137\u00a0M\u03a9 when filled with the pipette solution. During anion substitution experiments, background anion current was elicited from voltage-clamped myocytes (superfused with solutions B, C, D and E in the whole-cell configuration) by depolarizing ramps from \u221290 to +70\u00a0mV from a holding potential of \u221250\u00a0mV (ramp rate of 0.32\u00a0V\u00a0s\u22121; sweep duration 1.03\u00a0s). A holding potential of \u221250\u00a0mV was used to inactivate the Na+-current and T-type Ca2+-current.\nRecordings were made using an Axopatch 200A amplifier, and data were recorded on computer using pClamp v. 9.0 software (Axon Instruments, Forster City, CA). Data were analyzed using the Clampfit program of pClamp v. 9.0.\nMean values of averaged original signals over five command pulses were used for statistical analysis. Hyperpolarizing voltage steps of \u221220\u00a0mV and 5\u00a0ms duration were applied at 20\u00a0Hz to record the capacitance transients required for direct integration and the calculation of cell capacitance. The statistical significance between control and the drug periods or  and other anions were determined by the Paired Student\u2019s t-test using either Microsoft Excel or GraphPad Prism v. 4.0. The statistical significance between the normal and hyperosmotic  solutions was calculated with two-way ANOVA test using GraphPad Prism v. 4.0. Statistical significance was considered to refer to the 95% level of confidence (p\u00a0<\u00a00.05).\nHuman atrial action potential simulations\nThe Courtemanche et al. human atrial action potential (AP) model [18] was modified to incorporate a formulation for IANION based on the experimental data obtained with  and Cl\u2212 in Figs. 1 and 2. Readers are referred to [18] for the general equations required to set up the model. The following equation was used to simulate anionic background current (IANION)where EANION represents the current reversal potential and gANION is the conductance of IANION. By fitting Eq. (1) to experimental data shown in Fig. 1B and scaled to the mean data shown in Fig. 2A, we obtained gANION\u00a0=\u00a00.37\u00a0pS\/pF, EANION\u00a0=\u00a0\u221245.64\u00a0mV, c\u00a0=\u00a00.87, d\u00a0=\u00a08.4\u00a0\u00d7\u00a010\u22124\u00a0mV\u22121 for the -sensitive IANION, and gANION\u00a0=\u00a00.19\u00a0pS\/pF, c\u00a0=\u00a00.94, d\u00a0=\u00a02.5\u00a0\u00d7\u00a010\u22124\u00a0mV\u22121 for the Cl\u2212-sensitive IANION.\nResults and discussion\nThe voltage protocol used for these experiments was similar to that used previously to study IAB present in cardiomyocytes from small mammal hearts [14] and is shown as an inset to Fig. 1A. From a holding potential of \u221250\u00a0mV, ascending voltage ramps were applied between \u221290 and +70\u00a0mV. This protocol was applied to cells superfused first with aspartate (Asp)-containing solution (solution C) and then with different superfusates containing more permeant anions. Fig. 1A shows an example of the net current traces obtained from a cell superfused serially with solutions containing Asp\u2212, Cl\u2212,  and I\u2212. Both inward and, particularly, outward current components were greater with Cl\u2212,  and I\u2212 than with Asp\u2212 in the external superfusate. Fig. 1B shows current traces for the same cell, obtained by subtracting the current in Asp\u2212 from that with each of the more permeant anions. With each of Cl\u2212,  and I\u2212, the Asp\u2212-sensitive difference current showed marked outward rectification. Fig. 1C compares the mean outward current amplitude at +60\u00a0mV (normalized to membrane capacitance) for the three anions. Compared to , the observed current was significantly greater with I\u2212 and smaller with Cl\u2212 as charge carrier. These observations support the presence in human atrial cells of a basally active, anionic current (IANION); however, the relative current amplitudes with the three permeant anions differ from those observed previously with for the IAB observed in myocytes from guinea-pig and rat hearts, where IAB was largest with  (permeability sequence ; [13,14]).\nFig. 2A shows the mean IANION\u2013voltage relation for 19 atrial cells, with  as the major external anion (with IANION measured as the \u00a0\u2212\u00a0Asp\u2212 difference current). The mean current\u2013voltage relation for the resulting current showed clear outward rectification, with an observed reversal potential (Erev) for IANION in these experiments of \u221245.7\u00a0\u00b1\u00a02.2\u00a0mV (obtained by pooling Erev values from individual experiments). Previous studies provide evidence that human atrial cells exhibit ICl, Swell (e.g. [15,17,19\u201321]). Therefore, in order to determine whether or not IANION could be attributed to basal activity of channels mediating ICl,Swell, Asp\u2212 to  substitutions were also made using hyperosmolar external solution [14]. The mean data from eight such experiments are shown in Fig. 2B. There was no statistically significant difference between the plotted densities of IANION from the I\u2013V relation obtained in hyperosmolar solution and that shown in Fig. 2A, suggesting that IANION is distinct from ICl,Swell.\nIn order to characterize further IANION from human atrial myocytes, the sensitivity of the current to a range of pharmacological interventions was tested. Fig. 2C summarises the effects of the various interventions (expressed as % changes in -sensitive current at +60\u00a0mV). The stilbene disulphonate DIDS failed to inhibit IANION at a concentration (200\u00a0\u03bcM) that would be anticipated to inhibit ICl,Swell\n[19,21]. On the other hand, tefluthrin (10\u00a0\u03bcM), which we have previously reported to activate the IAB seen in myocytes from guinea-pig hearts [14], failed to alter significantly the amplitude of IANION from human atrial myocytes. Together with the relative IANION amplitudes in I\u2212, Cl\u2212 and , the lack of effect of tefluthrin indicates that IANION is distinct from the previously reported rat\/guinea-pig IAB\n[13,14]. Moreover, the lack of significant inhibition of the current by DIDS or hyperosmolar solution makes the IANION observed in the present study distinct from ICl,Swell\n[1] and from an osmolarity- and stilbene-sensitive outwardly rectifying chloride current recently reported by Demion and colleagues [17]. NPPB (50\u00a0\u03bcM) produced a partial, statistically significant (p\u00a0<\u00a00.05) inhibition of IANION. The glycine hydrazide Cl\u2212 channel inhibitor Gly H-101 failed to produce a significant blockade of IANION at 10\u00a0\u03bcM (\u223c7-fold greater than the reported IC50 for CFTR channel inhibition at +60\u00a0mV [22]); but produced partial attenuation of the current at 30\u00a0\u03bcM (\u223c20-fold the reported IC50 for CFTR [22]). Evidence for the presence of CFTR (ICa,cAMP) in human atrial cells is mixed [1,15,19\u201321,23], with a number of studies failing to observe the current in response to \u03b2-adrenergic stimulation, forskolin or cAMP (e.g. [15,19\u201321]). Previous work has failed to find evidence for ICl,Ca in human atrial myocytes [24] and, moreover, the presence of EGTA in the pipette dialysate (Table 1, solution I) and external [Ca2+] replacement in our experiments would have inhibited any [Ca2+]i-activated conductances on membrane depolarization. Therefore, the IANION seen here appears to differ not only from guinea-pig and rat IAB\n[13,14] but also from the three major reported cardiac anion conductances in: (i) being basally active and (ii) its overall pharmacological profile and sensitivity to anion substitution.\nIn order to gain insight into the physiological role of IANION, the current was incorporated into human atrial AP simulations as outlined in the \u2018Methods\u2019. Fig. 3A shows the simulated APs (at an AP frequency of 1\u00a0Hz) from the Courtemanche et al. model [18] both without (Control) and with inclusion of IANION, whilst Fig. 3B shows the corresponding current profiles during the time-course of the AP. With either  and Cl\u2212 as charge carrier, incorporation of IANION into the model produced shortening of AP duration at 50% repolarization (APD50; the measured APD50 values were 184, 165 and 159\u00a0ms for Control, Cl-sensitive IANION and NO3-sensitive IANION, respectively). In contrast, APD90 was comparatively unaffected (the measured APD90 was \u223c305\u00a0ms under each condition) and resting potential also changed relatively little (with resting potential values of \u221281, \u221279 and \u221278\u00a0mV, respectively, for Control, IANION with Cl\u2212 and IANION with ). The more marked effect of IANION inclusion at less negative potentials during AP repolarization is concordant with the outwardly rectifying nature of the current. An additional observation made from the AP simulations is that incorporation of IANION also influenced the profile of L-type calcium current (ICa,L) during the AP plateau: the initial rapid component of ICa,L was unaffected by IANION incorporation, but the sustained component during the AP plateau showed a modest reduction. Thus, both an increase in repolarizing current carried by IANION and the consequent decrease in the sustained component of ICa,L combined to lead to AP plateau depression and abbreviation of APD50. The results shown in Fig. 3 demonstrate clearly that IANION is able to influence human atrial AP repolarization. Further work is now warranted to determine both the extent to which the incorporation\/omission of IANION influences the susceptibility of human atrial cells and tissue to arrhythmia and to pursue the underlying identity and regulation of this novel background conductance.","keyphrases":["anion","anion","background current","atrial myocyte","heart","patch-clamp","action potential","cardiac","atrium","anionic","computer modelling"],"prmu":["P","P","P","P","P","P","P","P","P","P","R"]}
{"id":"Psychopharmacologia-4-1-2270918","title":"Acute neuropsychological effects of MDMA and ethanol (co-)administration in healthy volunteers\n","text":"Rationale In Western societies, a considerable percentage of young people expose themselves to 3,4-methylenedioxymethamphetamine (MDMA or \u201cecstasy\u201d). Commonly, ecstasy is used in combination with other substances, in particular alcohol (ethanol). MDMA induces both arousing as well as hallucinogenic effects, whereas ethanol is a general central nervous system depressant.\nIntroduction\nIn Western societies, a considerable proportion of young people expose themselves to 3,4-methylenedioxymethamphetamine (MDMA or \u2018ecstasy\u2019; Gross 2002; Parrott 2001; Tancer and Johanson 2007). Ecstasy has gained widespread use in the \u2018club\u2019 scene, typically all-night parties with loud music and intense lights (Winstock et al. 2001). The average dose of ecstasy used recreationally is reported to be around 80\u201390\u00a0mg of MDMA with considerable individual variation (Tanner-Smith 2006). Ecstasy users are generally multidrug users who have experience with various recreational drugs and use these in combination with ecstasy (Gouzoulis-Mayfrank and Daumann 2006b). Probably due to its availability, alcohol remains one of the most co-used substances (Barrett et al. 2005). As the use of alcohol is known to induce impairment of cognitive function and decrease the awareness of this impairment, this can lead to dangerous behaviour like driving under influence (Lamers and Ramaekers 2001; Riley et al. 2001).\nMDMA acts primarily by releasing serotonin (5-hydroxytryptamine (HT)) from pre-synaptic 5-HT terminals. It reverses the direction of the reuptake transporter and increases 5-HT levels at the post-synaptic receptors (Liechti and Vollenweider 2000; Mlinar and Corradetti 2003; Pifl et al. 1995). MDMA is also a potent releaser of dopamine and (nor)adrenaline (Colado et al. 2004; Liechti and Vollenweider 2001).\nMDMA is rapidly absorbed following oral administration. Within 30\u00a0min, MDMA is detectable in the blood. Plasma levels peak at 1\u20132\u00a0h after drug administration, and maximum behavioural and subjective effects occur around 1\u20132\u00a0h and have declined by 4\u00a0h in spite of persisting plasma levels (de la Torre et al. 2004; Green et al. 2003). Increasing the dose does not result in a proportional rise in plasma concentrations, which is indicative of non-linear pharmacokinetics (de la Torre et al. 2000).\nThe behavioural effects of MDMA resemble but are not restricted to effects of psychostimulants (e.g. amphetamines or \u2018speed\u2019) as well as hallucinogenics (e.g. lysergic acid or \u2018lysergic acid diethylamide\u2019), although MDMA\u2019s most characteristic effects are described as an increase in empathy and friendliness (Vollenweider et al. 2002). This led to MDMA being categorized as an \u2018entactogen,\u2019 as coined by Nichols and Oberlender (1990).\nMost research into the cognitive effects of MDMA in humans has focused on the long-term effects, where only memory was consistently found to be impaired (Verbaten 2003; Verkes et al. 2001). Our review of the acute effects of MDMA in humans showed that cognitive effects were assessed only in a limited number of studies, using diverse tests and generally addressing only certain aspects of neuropsychological function. As such, no consensus on MDMA\u2019s cognitive effects could be reached (Dumont and Verkes 2006). Since then, reports on the effects of MDMA generally confirmed previous findings (Kuypers et al. 2006; Kuypers et al. 2007; Ramaekers et al. 2006; Tancer and Johanson 2007). Interestingly, two studies reported effects of MDMA on memory, which had not been assessed previously. These reports showed acute impairment of immediate and delayed recall of words as well as spatial memory by MDMA (Kuypers and Ramaekers 2005, 2007).\nDrinks containing ethanol, commonly referred to as alcohol, are widely available and regularly used in Western society. Ethanol is chiefly a central nervous system (CNS) depressant. It inhibits both excitatory and inhibitory post-synaptic potentials by potentiating the action of gamma-aminobutyric acid at its receptor (Suzdak et al. 1988). Reports of the cognitive effects of combined use of MDMA and ethanol in humans have been sparse in the literature. Studies that were performed assessed psychomotor function, attentional performance and subjective effects (Hernandez-Lopez et al. 2002; Kuypers et al. 2006; Ramaekers et al. 2006). In general, MDMA and ethanol had no or opposite effects on effect measures, and as such co-administration did not exacerbate single-drug effects.\nIn the current study, we employed a series of tests sensitive to changes in all common neuropsychological domains induced by several pharmacological compounds, including amphetamines (Wezenberg et al. 2004).\nIt is generally acknowledged that the combined use of alcohol with other CNS-depressant drugs may enhance the effects of ethanol or of the other drugs. MDMA, however, has stimulant effects while ethanol is a sedative agent, suggesting that the effects of co-administration are diminished rather than augmented compared to the effects following single administration. This hypothesis was investigated during acute co-administration of MDMA and ethanol in healthy volunteers.\nMaterials and methods\nStudy design\nThis study utilised a four-way, double-blind, randomised, crossover, placebo-controlled design. Sixteen volunteers were randomly assigned to one of four treatment sequences. Each volunteer received a capsule containing either 100-mg MDMA or placebo and an ethanol\u2013placebo infusion (target blood alcohol concentration (BAC) of 0.6\u2030) with a washout of 7\u00a0days between each treatment.\nStudy outline\nSubjects arrived in the morning and were admitted to the study after a negative urine drug screen (opiates, cocaine, benzodiazepines, amphetamines, methamphetamines and delta-9-tetrahydrocannabinol), as well as a negative alcohol breath test and recording of signs and symptoms of possible health problems. A light breakfast was offered. Drug administration was scheduled at 1030\u00a0hours and the alcohol infusion was started at 1100\u00a0hours for a duration of 3\u00a0h. At 1130\u00a0hours, subjects performed the psychological test battery as described below. Specific test times are reported in Table\u00a01. Subjects received lunch at 1400\u00a0hours and were sent home at 1700\u00a0hours after a medical check. Adverse events where recorded throughout the study day. Vital signs were monitored using a Datascope\u00ae Accutorr Plus\u2122 cardiovascular monitor and Braun\u00ae type 6021 ThermoScan during the study day. The data presented in this report are a subset of a larger data set, which will be reported elsewhere.\nTable\u00a01TimelineNeuropsychological testsDescriptionTime (h:m)Drug administration0:0018-word list immediate recallImmediate recall of 18-word list1:00SDSTTranslate symbols to digits with key present in 90\u00a0s1:05SDRTTranslate symbols to digits from memory1:08Pursuit taskKeep dot within moving circle1:10Tangles taskTangled line leads to which target?1:13Switch taskFollow, possibly conflicting, instructions (choice between left or right)1:1718-word list delayed recallDelayed recall of 18-word list1:2218-word list delayed recognitionRecognise words of 18-word list memorised earlier among 18 distracters1:23Point taskKeep pen steady in air, measures tremor1:25Visual analogue scales16 100-mm scales for subjective experiences1:30Times are relative to drug administration.\nSubjects\nSixteen healthy volunteers (nine male, seven female), regular users of ecstasy and alcohol, aged 18\u201329\u00a0years and within 80\u2013130% of their ideal bodyweight, were recruited through advertisement on the internet and at local drug testing services. They were all in good physical and mental health as determined by assessment of medical history, a medical, electrocardiogramme and clinical, haematological and chemical blood examination. Previous drug use was assessed using a structured interview. Fifteen volunteers were right handed and one was left handed. The study was approved by the local Medical Ethics Committee. All subjects gave their written informed consent before participating in the study and were compensated for their participation. Subject demographics and drug history are reported in Table\u00a02.\nTable\u00a02Volunteer demographics\u2013drug history\u00a0MeanSDMinMaxAge (years)22.12.918.029.0Education (years)16.51.61218Height (cm)174.712.3147.0189.1Weight (kg)67.512.445.788.4Opiates0.10.301LSD2.56.6025Amphetamines37.381.10250Ecstasy94.6138.414431Cannabis1,174.31,665.5205,840Cocaine33.7105.70400Alcohol2,367.91,981.6505,200Solvents3.613.3050Barbiturates0000Benzodiazepines18.657.30216Psilocybin6.910.4030Drug quantities mentioned are lifetime drug exposures, not further specified.\nOne subject had a mild adverse reaction (local vascular reaction) to the alcohol infusion and one subject did not refrain from drug use; both (one male, one female) were excluded from further participation and results obtained were not included in the data analysis.\nDrugs and dosages\nMDMA (or matched placebo) was given as a capsule in a single dose of 100\u00a0mg via oral administration (dose range; 1.1\u20132.2\u00a0mg\/kg). MDMA was obtained from Lipomed AG, Arlesheim, Switzerland and encapsuled according to Good Manufacturing Practise by the Department of Clinical Pharmacy, UMC St Radboud, Nijmegen, the Netherlands. MDMA 100\u00a0mg orally is a relevant dose in the range of normal single recreational dosages. Previous experiments in humans used doses up to 150\u00a0mg without serious adverse events. Ethanol (or matched placebo) was administered continuously by intravenous infusion of 10% ethanol in glucose solution resulting in an ethanol blood concentration of 0.6\u2030 with a duration of 3\u00a0h as described below.\nAlcohol clamping\nTo standardise alcohol delivery and maintain a constant alcohol blood concentration over time, an intravenous ethanol clamp was used. Ethanol was administered by infusion of a 10% ethanol in glucose solution for a duration of 3\u00a0h. The infusion rate was calculated using frequent breath alcohol concentrations measurements, according to a previously designed algorithm (Amatsaleh et al. 2006a). Breath alcohol concentration was assessed using a HONAC AlcoSensor IV\u00ae Intoximetre.\nAn intravenous administration route was chosen, ensuring standardisation of the rate and bioequivalence of ethanol administration. This is an important pre-requisite for predictable pharmacokinetics of ethanol. The process was semi-automated using a computer spreadsheet programme, which uses measured breath alcohol concentrations to calculate the infusion rate needed to maintain the ethanol level at 0.6\u00a0mg\/mL. This is a relevant dose equivalent to peak levels of approximately two to three units of alcoholic beverages. In many European countries driving is prohibited at BAC above 0.5\u2030. This limit has been confirmed by a report that shows that at an average BAC of 0.6\u2030 psychomotor performance is significantly impaired (Amatsaleh et al. 2006b). A BAC of 0.6\u2030 is equivalent to approximately two to three alcoholic beverages commonly used in social settings in Western society, which is considered to be a safe and relatively moderate dose, despite its significant CNS effects.\nMDMA blood analysis\nFor the assessment of serum levels of MDMA, blood samples were collected 90\u00a0min after drug administration from each subject on each study day. Venous blood samples (10\u00a0ml) were collected into heparinised tubes, centrifuged immediately at 4\u00b0C for 15\u00a0min. Plasma was split into aliquots of 2\u00a0mL (to prevent over-freezing\u2013thawing), frozen rapidly using liquid nitrogen and stored at \u221280\u00b0C. Samples were analysed for MDMA and MDA concentration by the Toxicology unit of the Leyenburg hospital, The Hague, the Netherlands.\nNeuropsychological tests, apparatus and procedure\nThe performance on all neuropsychological tests was recorded by means of a digitising tablet (WACOM UD-1218-RE), a laptop computer, a pressure-sensitive pen (which could also be used as a cursor) and test forms. The x and y coordinates of the pen tip on and up to 5\u00a0mm above the digitiser were sampled with a frequency of 200\u00a0Hz and a spatial accuracy of 0.2\u00a0mm. The time schedule of the tests is summarised in Table\u00a01.\nTo familiarise the subjects with the tests and procedures, they were invited to the hospital to perform a practise session within 1\u00a0week before the actual study days. All tests had five equivalent versions for four test days and one practise day, test versions were counterbalanced over test days.\nExecutive function\nSwitch task This test is a reaction time task measuring simple as well as complex reaction time, assessing executive performance (Baker and Letz 1986). After a random period of 0.75 to 1.75\u00a0s, two rectangular fields appeared on both sides of a circle in the centre of the screen. Only one of the two fields provided the subjects with information, either a colour, an arrow or both. The other non-informative field always had a neutral grey colour. Five conditions were subsequently presented to subjects. If only green fields appeared, subjects had to move as fast as possible into the green field. If green and red fields appeared, subjects had to move into the green field and away from the red field as soon as they appeared.\nIf green fields with a left or right arrow were presented, subjects were to move into the direction of the arrow. Green and red fields with a left or right arrow indicated that subjects were to follow the direction of the arrows in the green field, but the opposite direction of the arrows in the red field. Finally, the first condition was repeated. All conditions contained 20 trials except condition four in which there were 40 trials (total\u2009=\u2009120 trials). The outcome measures were the mean reaction times per condition. The last condition is a repetition of the first to check for possible changes in attention.\nMemory\nEighteen-word list A verbal memory test based on the classic Auditory Verbal Learning Test (Vakil and Blachstein 1993) was used. A variant was made consisting of a list of 18 words. The classic test uses 15 words. A longer wordlist was chosen, however, to prevent ceiling effects. The list was presented verbally three times. Under normal circumstances, subjects are supposed to remember an increasing number of words after each trial. Directly after each presentation, and after an interval of 20\u00a0min, subjects were asked to recall as many words as possible. After the delayed recall trial, a list of 36 words was presented from which they were asked to recognise the 18 words previously presented. The incorrect words were distracters and resembled the correct words in a semantic or phonologic manner. Responses were either correct positive (when a word that was recognised was indeed part of the list presented during immediate recall) or false positive (when a word was recognised but was not part of the list presented during immediate recall, e.g. the word was a distracter). The outcome measure was the number of correctly recalled or recognised words for the average of the three immediate recall trials, the delayed recall trial and the delayed recognition trial.\nSymbol digit recall test The symbol digit recall test (SDRT) followed directly after the Symbol Digit Substitution test (SDST), which is discussed in the last paragraph of this section. After subjects had finished the SDST, they were shown the symbols of the SDST without the translation key, one at a time, and asked to produce the corresponding numbers. This test is based on an extended procedure of the SDST to measure incidental learning (Kaplan et al. 1991). The outcome measure was the number of correctly translated symbols.\nPsychomotor function\nPursuit task To measure implicit procedural learning, a computerised version of the rotor pursuit task was used. This test is based on the classic rotary pursuit task (Ammons 1951). It is a continuous motor task. Subjects had to follow the movement of a large target stimulus on the computer screen with a cursor by moving the pen over the XY tablet. The speed of the target gradually increased when the cursor was contained within the target but decreased considerably when it was not. The target followed a spatially predictable circular path over the screen. The outcome measure for this test was the total number of rotations within 2\u00a0min.\nPoint task The point task, a measure for tremor, required subjects to try to keep the cursor inside a very small circle for 1\u00a0min, while avoiding contact between the pen and the test form. The outcome measure for this test was the deviation from the target.\nVisuospatial and visuomotor function\nTangle task The tangle task required the subject to visually track a particular line winding through two to four other lines. On subsequent trials, the tangles increased in complexity; they got longer and made more 90\u00b0 turns. The paper form had a start area and five target areas, numbered 1 to 5, which reflect the maximum target areas on the screen, starting with only three target areas.\nThis test is modelled after the visualisation test from the \u2018kit for factor referenced cognitive tests.\u2019 It was selected by the US NAVY to study environmental and other time-course effects and has good task stability and reliability (Bittner et al. 1986). The outcome measures are the reaction time per trial and the number of correct trials in 2\u00a0min.\nAttention\nSymbol Digit Substitution test This test is a version of the subtest from the Wechsler Adult Intelligence Scale (Wechsler 1981). Subjects had to substitute the nine symbols for the digits 1\u20139 on the basis of a given translation key. The outcome measure was the total number of digits completed in 90\u00a0s.\nAccording to Hege et al. (1997) this task measures many cognitive components, e.g. visuospatial scanning, intermediate memory, perceptual motor speed and speed of cognitive processing. Therefore, subsequent analyses were performed in order to attempt and disentangle these cognitive processes. Based on pen pressure, movement trajectories were defined as either pen-up periods or pen-down periods. This allowed for subsequent analysis of matching times and movement (writing) times in the Symbol Digit Substitution test. For the motor component, the mean writing times were computed. For the more cognitive component, the mean matching times were computed. These analyses have been previously performed (Sabbe et al. 1999; Wezenberg et al. 2005).\nSubjective\nSubjective effects were recorded using the Bond and Lader (Visual Analogue) Mood Rating Scale (BLMRS). This inventory was completed at the end of each neuropsychological test battery on each study day.\nThe BLMRS scale consisted of 16 lines, each 10\u00a0cm in length, with opposite terms at each end of the line (alert\u2013drowsy, calm\u2013excited, strong\u2013feeble, muzzy\u2013clear-headed, well coordinated\u2013clumsy, lethargic\u2013energetic, contented\u2013discontented, troubled\u2013tranquil, mentally slow\u2013quick witted, tense\u2013relaxed, attentive\u2013dreamy, incompetent\u2013proficient, happy\u2013sad, antagonistic\u2013amicable, interested\u2013bored, withdrawn\u2013gregarious). Subjects were asked to indicate which item was more appropriate by marking the line. The outcome measure was the distance to the marker on each scale. These scale scores were then aggregated to scores for \u2018calmness,\u2019 \u2018alertness\u2019 and \u2018contentedness\u2019 as described by Bond et al. (1974).\nStatistical analyses\nStatistical evaluation (using SPSS 11.5 for Windows) was performed with general linear model repeated-measures analysis of variance. Main and interaction effects were tested using a two-factor (\u2018ethanol\u2019 and \u2018MDMA\u2019), two-level (absent versus present) multivariate model.\nThe analysis of the data was based on Maxwell and Delaney (2004) and Kirk (1995). First the presence of interaction (non-additivity) was tested with alfa\u2009=\u20090.05. When the interaction was not statistically significant we proceeded by testing the main effects, each at alfa\u2009=\u20090.05. In the case of a significant interaction, we proceeded by testing simple main effects of each drug, i.e. MDMA vs. placebo and ethanol vs. placebo.\nResults\nSubject demographics are summarised in Table\u00a02. Out of 16 subjects, 14 completed the study procedure. One subject had a mild adverse reaction (local vascular reaction which subsided with infusion stop) to the alcohol infusion and one subject did not refrain from drug use; both were discontinued from study participance and data already obtained were not included in statistical analysis. Only significant results are mentioned in this section, unless stated otherwise.\nMDMA blood concentration 90\u00a0min after administration did not differ for MDMA single vs. MDMA and ethanol co-administration and was on average 196\u00a0\u03bcg\/L (SD\u2009=\u200983\u00a0\u03bcg\/L). Blood alcohol concentration was maintained at an average of 0.54\u2030 (SD\u2009=\u20090.07\u2030).\nExecutive function\nExecutive function (switch task) did not show any significant main or interaction effects.\nMemory function\nMemory function was assessed by the 18-word list (outcome measures were \u2018immediate recall,\u2019 \u2018delayed recall\u2019 and \u2018recognition,\u2019 see Fig.\u00a01) as well as the SDRT. Immediate recall was impaired only by ethanol (F(1, 12)\u2009=\u20098.71, p\u2009=\u20090.011).\nFig.\u00a01Memory effects (18-word list), Immediate: immediate recall, average score of three trials of correctly recalled verbally presented words, Delayed: correctly recalled verbally presented words 20\u00a0min after presentation, Recognition: correctly recognised verbally presented words containing 18 distracters, 20\u00a0min after presentation (mean and SEM). Immediate recall was impaired only by ethanol (F(1, 12)\u2009=\u20098.71, p\u2009=\u20090.011). Delayed recall was impaired by MDMA (F(1, 12)\u2009=\u200910.447, p\u2009=\u20090.007) as well as by ethanol (F(1, 12)\u2009=\u200916.031, p\u2009=\u20090.002); recognition was not affected by any drug condition\nDelayed recall as assessed by the 18-word list was impaired by MDMA (F(1, 12)\u2009=\u200910.447, p\u2009=\u20090.007) as well as by ethanol (F(1, 12)\u2009=\u200916.031, p\u2009=\u20090.002). The SDRT, also a test for delayed recall, showed a similar pattern of impairment by MDMA (F(1, 12)\u2009=\u20095.300, p\u2009=\u20090.038) as well as by ethanol (F(1, 12)\u2009=\u20097.654, p\u2009=\u20090.016).\nPsychomotor function\nPsychomotor function was assessed with tests for tremor (point task), accuracy (pursuit task) and speed (SDST motor time, see Fig.\u00a02); other SDST results are reported in the section \u201cAttention.\u201d Ethanol impaired psychomotor speed as reflected in the increase in SDST motor time (F(1, 12)\u2009=\u20099.295, p\u2009=\u20090.009).\nFig.\u00a02Psychomotor effects: SDST writing time (mean, SEM). Ethanol increased writing times (F(1, 12)\u2009=\u20099.295, p\u2009=\u20090.009)\nVisuospatial and visuomotor function\nVisuospatial and visuomotor function were measured with the tangle task, subdivided into \u2018total number correctly solved\u2019 and \u2018reaction time,\u2019 and did not show any significant effects, although a trend of impairment by MDMA (F(1, 12)\u2009=\u20093.966, p\u2009=\u20090.068) was observed.\nAttention\nAttention was assessed with the SDST task; the outcome measures were \u2018motor time\u2019 (see \u201cPsychomotor function\u201d), \u2018matching time\u2019 (Fig.\u00a03) and \u2018total number correctly substituted.\u2019 The time required to match symbols to the corresponding numbers showed a significant MDMA and ethanol interaction (F(1, 12)\u2009=\u20096.214, p\u2009=\u20090.027). Tests for simple main effects revealed that both single-drug conditions reduced attention compared to placebo (ethanol F(1, 13)\u2009=\u20096.248, p\u2009=\u20090.027; MDMA F(1, 13)\u2009=\u20096.822, p\u2009=\u20090.022; see Fig.\u00a03).\nFig.\u00a03Attention effects: SDST matching time, i.e. time needed for translation (mean and SEM). A significant MDMA by ethanol interaction was found (F(1, 12)\u2009=\u20096.214, p\u2009=\u20090.027)\nSubjective effects\nSubjective effects are depicted in Fig.\u00a04. Feelings of \u2018contentedness\u2019 where increased significantly by MDMA only (F(1, 12)\u2009=\u20094.710, p\u2009=\u20090.049).\nFig.\u00a04Subjective effects (aggregated Bond and Lader scores, mean and SEM). Feelings of \u2018Contentedness\u2019 where increased significantly by MDMA only (F(1, 12)\u2009=\u20094.710, p\u2009=\u20090.049) A significant interaction effect (F(1, 12)\u2009=\u20097.358, p\u2009=\u20090.018) was found for feelings of \u2018Alertness.\u2019 Feelings of \u2018Calmness\u2019 were reduced only by MDMA (F(1, 12)\u2009=\u200920.259, p\u2009=\u20090.001)\nA significant interaction effect (F(1, 12)\u2009=\u20097.358, p\u2009=\u20090.018) was found for feelings of \u2018alertness.\u2019 Tests for simple main effects revealed that ethanol but not MDMA significantly decreased feelings of alertness compared to placebo (F(1, 13)\u2009=\u200950.613, p\u2009<\u20090.001). Feelings of \u2018calmness\u2019 were reduced only by MDMA (F(1, 12)\u2009=\u200920.259, p\u2009=\u20090.001).\nDiscussion\nThis study demonstrates that the effects of 100-mg MDMA, commonly known as ecstasy, on cognitive function are no greater than the effects of a relatively low dose of ethanol. This is remarkable as these results suggest that the effects of 100-mg MDMA are comparable to the peak effects of two to three alcoholic beverages. Co-administration of these compounds did not result in any significant cognitive impairments beyond those observed after administration of only ethanol. The use of moderate amounts of alcohol is common in Western societies and, although impairing cognitive function, socially accepted, while ecstasy use remains very controversial. Of course, our findings only relate to the acute neuropsychological implications of ecstasy use and not to the physiological and long-term effects, which rightfully remain topics of discussion (Gouzoulis-Mayfrank and Daumann 2006a; Nutt 2006; Parrott 2007).\nDrug effects observed in this placebo-controlled crossover study were moderate. Co-administration was well tolerated as indicated by the subjective scores, which were comparable to those found after single administration of MDMA. An interaction of MDMA and ethanol was found for subjective alertness scores. Ethanol, as expected, reduced subjective alertness, while MDMA co-administration reversed the reduction of subjective alertness by ethanol. In the present study, MDMA by itself did not significantly affect subjective alertness, although this effect has been consistently reported in other studies and is a well-known effect of amphetamines. However, MDMA did significantly reduce subjective calmness, i.e. subjects felt more excited after MDMA use. Probably, the Bond and Lader Mood Rating Scale is not well suited for the assessment of subjective effects of psychoactive drug effects and future studies should employ more appropriate subjective drug effect measures such as the Profile Of Mood States (de Wit et al. 2002).\nWhen considering the results for each neuropsychological domain, executive function was not affected by any drug condition. A previous study showed impairment of executive function by ethanol but not MDMA, although ethanol impaired performance in only one out of three tests of executive function (Lamers et al. 2003). The BAC in this study was 0.3\u2030 at the time of testing compared to 0.56\u2030 in our current study, suggesting a lack of sensitivity of the test employed in the current study.\nThe above-mentioned previous study also reported visuospatial and visuomotor impairment by MDMA but not by ethanol. Although not significant, our current results show a similar pattern where MDMA showed a trend of impairment of visuospatial and visuomotor function, whereas ethanol did not.\nPsychomotor function was impaired only after ethanol administration (SDST motor time, see Fig.\u00a02). The majority of studies addressed in our review of acute effects of MDMA in humans (Dumont and Verkes 2006) did not report any change in psychomotor function after MDMA either. However, increased psychomotor function after MDMA has also been found (Lamers et al. 2003; Ramaekers et al. 2006). These studies administered 75\u00a0mg instead of 100\u00a0mg. Possibly, the effects of MDMA are biphasic, with a low dose of MDMA exhibiting more amphetamine-like effects, e.g. arousal, increasing performance, whereas higher doses may elicit more hallucinogenic effects and impair performance (Liechti et al. 2001; Solowij et al. 1992).\nAs mentioned above, MDMA co-administration reversed the ethanol-induced feelings of sedation, although MDMA was unable to reverse the psychomotor impairment induced by ethanol. This dissociation between subjective and objective sedation confirms previous findings by Hernandez-Lopez et al. (2002).\nSeveral studies assessed MDMA\u2019s effect on attention using the Digit Symbol Substitution Task (DSST), although no significant effects were found (Cami et al. 2000; Farre et al. 2004; Kuypers and Ramaekers 2005). One study reported decreased DSST performance after ethanol as well as after ethanol and MDMA co-administration but no effect of MDMA (Hernandez-Lopez et al. 2002). Our findings confirm these findings to a large extent. We found no main effects of MDMA or ethanol on attention, although an interaction of ethanol and MDMA for \u2018matching time\u2019 (time required to match the number to the corresponding symbol) was found. Co-administration of MDMA and ethanol increased \u2018matching time\u2019 comparable to the increase observed after both MDMA and ethanol single administration, compliant with our hypothesis of competitive mechanisms of action of both drugs (see Fig.\u00a03).\nStudies investigating the long-term effects of MDMA consistently found memory to be affected (Verbaten 2003). In the present report, almost all memory measures showed quantitatively comparable impairment for each drug condition (see Fig.\u00a01), although the effect of MDMA on immediate recall did not reach statistical significance. Only delayed recognition was not impaired in any drug condition. These findings suggest a deficit in the retrieval of verbal information encoded in memory, rather than impairment in the storage of information. Our findings are similar to the results of a previous study on MDMA-induced effects on memory (Kuypers and Ramaekers 2005). In this previous study, no memory impairment was observed after methylphenidate administration, a pronounced dopamine and norepinephrine releaser, suggesting the involvement of serotonin in memory impairment. Several other studies also have shown serotonin-mediated modulation of memory function through interaction with the cholinergic neurotransmitter system, although the details of this complicated interaction remain elusive (Cassel and Jeltsch 1995; Garcia-Alloza et al. 2006; Meneses 2007). Generally, subjects stated that they were well aware of their impaired memory after MDMA.\nBAC was on average 0.56\u2030. At this level, driving is prohibited by law in many European countries because of its interference with normal functioning. Although the effects were moderate, ethanol impaired cognitive performance in various tests. Similar moderate effects were observed with MDMA 100\u00a0mg, considered to be slightly above the average recreational dose (Tanner-Smith 2006). This might be considered surprising for a drug with reported robust subjective stimulating and hallucinogenic properties. However, because the effects caused by a single dose of 100-mg MDMA were comparable to the effects of a BAC of 0.56\u2030, this dose should by inference be considered unacceptable in motorised traffic.\nArguably, the moderate drug effects as found in this study could be explained by \u2018missing\u2019 the time of the maximal drug effects. Although the average MDMA blood concentration reported here (196\u00a0\u03bcg\/L) is comparable to Cmax of 100-mg MDMA (199.8\u00a0\u03bcg\/L) as reported by de la Torre et al. (2000), MDMA concentration was assessed at the end of the testing procedure. However, Hernandez-Lopez et al. (2002) found significant effects at 60\u00a0min as well as 90\u00a0min after drug administration, arguing against the suggestion of \u2018missing\u2019 peak drug effects.\nThe circumstances in which these substances are normally used cannot be fully recreated in the laboratory and this may have suppressed the effects of both substances. It is not unlikely that these substances show enhanced effects when tested under typical circumstances and surroundings. Recently, Parrott et al. (2006) concluded that the increase in physical activity and body temperature typically experienced when using MDMA enhance MDMA effects. Ball et al. (2006) demonstrated that a familiar surrounding increased MDMA-induced locomotor response as well as single-neuron activity in rats, compared to unfamiliar surroundings. Therefore, the psychosocial context in which MDMA is used, along with the different expectations and behaviour, probably influences its effects (Sumnall et al. 2006). It is unlikely, however, that this affects the quality of the interactions of MDMA and ethanol.\nIn conclusion, co-administration of MDMA and ethanol did not impair cognitive function significantly more than MDMA or ethanol administration alone. The most prominent effect of (co-)administration of MDMA and ethanol was an impairment of memory. Ethanol also impaired psychomotor function. Although the impairment of performance by each drug condition was relatively moderate, this significant impairment of cognitive function should be considered intolerable in motorised traffic and other cognitively demanding situations as confirmed by previous research and as defined by law. However, the effects of these drugs in the concentrations used in the present study on established neuropsychological tests appear to be smaller than one would assume based on their reputation.","keyphrases":["acute","neuropsychologic","effects","mdma","ethanol","healthy volunteers","ecstasy","alcohol","interaction"],"prmu":["P","P","P","P","P","P","P","P","P"]}
{"id":"Eur_J_Clin_Pharmacol-4-1-2426926","title":"Efficacy and safety of disodium ascorbyl phytostanol phosphates in men with moderate dyslipidemia\n","text":"Objective This study investigated the efficacy, safety, tolerability, and pharmacokinetics of a novel cholesterol absorption inhibitor, FM-VP4, comprising disodium ascorbyl sitostanol phosphate (DASP) and disodium ascorbyl campestanol phosphate (DACP).\nIntroduction\nWorldwide, cardiovascular disease (CVD) is the most common cause of death, with atherosclerotic vascular disease as the underlying cause. It has been well established that elevated plasma concentrations of low-density lipoprotein cholesterol (LDL-C) lead to an increased risk of atherosclerosis and coronary heart disease [1]. Dietary modification can improve the lipid profile and the potential risk of CVD, but for a significant portion of the population, treatment with lipid-lowering agents is necessary to reduce blood cholesterol effectively. Statins are the primary class of drugs used for managing LDL-C and are the most potent agents in lowering LDL-C. In fact, the lowering of LDL-C by statin therapy has been shown to decrease the number of fatal and nonfatal myocardial events in both primary and secondary prevention studies [2\u20136]. However, statins have not provided the final answer in terms of CVD prevention, and their use is associated with side effects, albeit rare, at higher doses. Therefore, cholesterol-lowering agents that act via other mechanisms are of great interest.\nPlant sterols, or phytosterols, are naturally occurring compounds in vegetable oil, seeds, and nuts that are structurally related to cholesterol. Daily intake of 2,000\u00a0mg of plant sterols or their saturated counterparts, plant stanols, or phytostanols, has been shown to decrease LDL-C by 9\u201314% without affecting high-density lipoprotein cholesterol (HDL-C) concentrations [7, 8]. Plant sterols and stanols are thought to decrease plasma cholesterol concentrations by inhibiting cholesterol absorption in the intestine [9]. When cholesterol absorption is decreased, the hepatic cholesterol pool is reduced, resulting in enhanced cholesterol synthesis by the liver. At the same time, LDL receptors are upregulated, with ensuing lower LDL-C concentrations in plasma [10].\nA new water soluble plant stanol derivative, disodium ascorbyl phytostanol phosphate (FM-VP4, Fig.\u00a01) has been developed as a cholesterol-absorption inhibitor. FM-VP4 consists of a mixture of sitostanol and campestanol to which ascorbate is covalently bound via a phosphodiester linkage. It has been shown to inhibit the in vitro uptake of micellar [3H]-cholesterol by approximately 50% in human and rat enterocyte cell lines [11, 12]. In vivo, FM-VP4 led to a dose-related inhibition of [3H]-cholesterol absorption in rats, as shown by a maximally 80% reduced area under the concentration time curve (AUC) of orally administered micellar [3H]-cholesterol [13]. Furthermore, the LDL-lowering activity of FM-VP4 was observed in a broad range of LDL-sensitive species, including gerbils [14, 15], hamsters [16], and apolipoprotein E (ApoE)-deficient transgenic mice [17]. In hamsters, FM-VP4 was a more potent LDL-lowering agent than unesterified plant stanols [16], and in ApoE deficient mice, the LDL-lowering effect of FM-VP4 was correlated with retardation of atherosclerotic plaque development [17]. Other effects of FM-VP4 included the decrease of plasma triglyceride levels and the reduction of abdominal fat or body-weight gain in gerbils [14, 15] and mice [18]. In all of these cases, FM-VP4 showed no side effects to the animals tested.\nFig.\u00a01Chemical structure of FM-VP4, composed of disodium ascorbyl campestanol phosphate (R=CH3) and disodium ascorbyl sitostanol phosphate (R=C2H5)\nPreclinical data suggested that FM-VP4 is a potent cholesterol-lowering agent with no significant toxic effects. Here, we report the first human study designed to assess the efficacy, safety, tolerability, and pharmacokinetics of single and multiple doses of this water-soluble plant stanol analogue.\nSubjects and methods\nSubjects\nSubjects were recruited via advertisements in local newspapers. The study protocol was carefully explained before subjects were asked to give their written informed consent. The study protocol was approved by the Institutional Review Board of the Academic Medical Centre. Subjects were eligible if they were male, 18\u201375\u00a0years old, healthy as reviewed by medical history and physical examination, had LDL-C concentrations \u22653.0\u00a0mmol\/L during one of the screening visits or at baseline and triglyceride (TG) concentration \u22644.5\u00a0mmol\/L at the first visit, had a body mass index (BMI) <35\u00a0kg\/m2, and did not use any steroids, \u03b2-blockers, corticosteroids, thiazide diuretics, or antiepileptics. Subjects with a history of hypertension, arterial diseases, diabetes mellitus type I or II, hypothyroidism, obstructive biliary disorders, pancreatitis, collagen disorders, or autoimmune disease were excluded, as were subjects with history of malignancy during the previous 3 years, significant hepatic, renal, cardiac, or cerebral disease, or plasma levels of hepatic transaminases higher than two times the upper limit of normal (ULN). Subjects of phase 1 were not allowed to use any lipid-lowering drugs at inclusion, and the use of plant sterol- or stanol-containing products had to be discontinued at the inclusion visit. Subjects of phase 2 had to discontinue the use of plant sterol or stanol products or fish oils at the inclusion visit and statin treatment 6\u00a0weeks (40\u00a0days) before the start of study treatment. Thirty men participated in the phase 1 trial and 100 men in phase 2.\nDrugs\nTwo types of tablets were used: 100\u00a0mg oval FM-VP4 tablets or a matching placebo. Both investigational products were supplied through Forbes Medi-Tech Inc. (Vancouver, Canada). FM-VP4 is a semisynthetic esterified plant stanol derivative produced as a disodium salt. The two major components of FM-VP4 are disodium ascorbyl sitostanol phosphate (DASP) and disodium ascorbyl campestanol phosphate (DACP) (Fig.\u00a01), which are present in the proportion of approximately 2:1, respectively.\nDesign\nThis single-center, double-blind, placebo-controlled, dose-escalation trial comprised two parts: In phase 1, 30 men received a single dose of FM-VP4. Five subjects were assigned to each dose group (100, 200, 400, 800, 1,600, or 2,000\u00a0mg), and within each group, one subject was randomly assigned to placebo. Once a complete cohort of five subjects was treated and the safety parameters had been reviewed, the following dose was initiated. In the subsequent phase 2 trial, 100 men were treated for 28\u00a0days (4\u00a0weeks). Twenty-five subjects were assigned to each dose group (100, 200, 400, or 800\u00a0mg\/day), and within each group, five subjects were randomly assigned to placebo. The first five subjects in each cohort were hospitalized for 5 days. Escalation to the next dosing level was only allowed once these five subjects completed treatment and all results and safety data were evaluated.\nPhase 1 A week before treatment, subjects visited the hospital for screening. Informed consent was obtained, and subjects underwent a physical examination. A fasting blood sample was taken to measure lipids, biochemistry, and hematology. Within 3 days before treatment, subjects visited the hospital for another blood sample, and urinalysis and electrocardiogram (EKG) were performed. If subjects met all inclusion criteria, they were hospitalized for 24 h on the day of treatment. In the morning, subjects\u2019 weight, supine blood pressure (BP), and heart rate were measured, and a predose fasting blood sample was taken for baseline safety parameters and pharmacokinetics. Subsequently, subjects were administered one to 20 tablets containing 100\u00a0mg FM-VP4 each or placebo. Tablets were swallowed with 250\u00a0mL of water. Breakfast followed 30 min later. Weight, supine BP, and heart rate were recorded at 3, 6, 9, and 12\u00a0h after dosing. A blood sample for pharmacokinetics was taken 6 and 12\u00a0h after dosing. Any spontaneous complaints were recorded as adverse events and closely monitored. Subjects were detained overnight under observation, and 24\u00a0h after dosing, another blood sample was taken for safety parameters and pharmacokinetics. Subjects\u2019 weight, supine BP, and heart rate were measured, and urinalysis and an EKG were performed. Once the EKG, biochemistry, and hematology of the 24-h postdosing sample had been reviewed, subjects were discharged from the hospital. They returned to hospital 48, 96, and 144 or 168\u00a0h (6 or 7\u00a0days) after treatment for weight, supine BP, and heart rate measurements and for a blood sample to measure safety parameters and pharmacokinetics. At the last visit, a final physical examination was performed.\nPhase 2 Four to 8\u00a0weeks before treatment, subjects visited the hospital for screening. Informed consent was obtained, and subjects underwent a physical examination. A fasting blood sample was taken to measure lipids, biochemistry, and hematology. Subjects were instructed to follow a diet adapted from the National Cholesterol Education Program (NCEP) Step 1 diet during the entire study, including the 4- to 8-week run-in period. Consumption of plant sterol- or stanol-containing products and the use of fish oils had to be discontinued at the screening visit, and statin treatment had to be discontinued 6\u00a0weeks before treatment with FM-VP4. If they met all inclusion criteria, subjects visited the hospital halfway through the run-in period and within 3 days before study treatment for baseline blood lipids, biochemistry, and hematology measurements. At the latter visit, urinalysis was performed and an EKG recorded.The first five subjects per dose cohort, of which four were on active treatment and one on placebo, were hospitalized for 5\u00a0days. Each morning, subjects\u2019 weight, supine BP, and heart rate were measured, and a predose fasting blood sample was taken for safety parameters and pharmacokinetics. Subsequently, subjects were administered one to four tablets containing 100\u00a0mg FM-VP4 each, depending on the dose cohort, or placebo. Tablets were swallowed entirely with up to 100\u00a0mL of water. Breakfast followed 30\u00a0min later. All doses were divided and given twice per day; another one to four tablets were administered 30\u00a0min before dinner. The morning and evening doses were packaged in separate bottles. Subjects in the 100-mg group received one FM-VP4 tablet and one placebo tablet. Supine BP and heart rate were also recorded daily after lunch and dinner. Any spontaneous complaints were recorded as adverse events and closely monitored. Subjects were observed overnight and discharged 5\u00a0days after the first dosing. All subjects visited the hospital for efficacy and safety measurements once per week during and 14\u00a0days after treatment. At the last visit, a final physical examination and an EKG were performed. Compliance was calculated based on the number of tablets supplied to the patient minus the number returned.\nPlasma analyses\nComplete blood count, fibrinogen, and the biochemical profile [alanine aminotransferase (ALT) and aspartate aminotransferase (AST), bilirubin, creatine kinase (CK), creatinine, glucose, and C-reactive protein (CRP)] were assessed using the local hospital laboratory. Changes in laboratory parameters were considered abnormal in case of ALT or AST levels greater than three times ULN, a CK level greater than five times ULN, a creatinine increase of \u226540\u00a0\u03bcmol\/L compared with baseline, a creatinine level >177\u00a0\u03bcmol\/L, white blood cell count <3\u2009\u00d7\u2009109\/L, and a decrease in hemoglobin of at least 1.5\u00a0g\/dL compared with baseline. Thyroid-stimulating hormone (TSH) was measured at the first screening visit. Urinalysis for blood, glucose, protein, pH, and specific gravity was performed by dipstick within 3\u00a0days before and 1\u00a0day after dosing in phase 1 and within 3\u00a0days before, after 4\u00a0weeks of treatment, and at the last visit of phase 2. Urinalysis by wet microscopic slide was performed if the dipstick analysis was abnormal. Lipid analyses were performed by an external laboratory (MRL International, Zaventem, Belgium) by using standardized procedures, and LDL-C was calculated using the Friedewald equation [19]. The results were kept blinded to the investigators.\nPlasma was stored at \u221280\u00b0C for further analyses. ApoB, ApoAI, and lipoprotein a [Lp(a)] as well as vitamin E and vitamin A were analyzed in one run after the study had been completed. ApoAI, ApoB, and Lp(a) levels were determined by nephelometry with a Beckman Array (Mijdrecht, the Netherlands) according to the manufacturer\u2019s instructions, and vitamin E and vitamin A were measured by high-performance liquid chromatography (HPLC) with fluorescence detection using a Chromsep Glass, 100*3\u00a0mm, inertsil 5, ODS-3 column (Varian-Chrompack, Middelburg, the Netherlands).\nAll DASP and DACP plasma concentrations were assayed by a validated liquid chromatography\/mass spectrometry\/mass spectrometry method (LC\/MS\/MS) based on a previously described method [20]. The lower limit of quantification (LLOQ) was 30.8\u00a0ng\/mL. Data analysis was carried out using Microsoft Excel 97, and pharmacokinetic data was analyzed using Pharsight WinNonlin software, version 3.1 build 168 (Pharsight WinNonlin, Mountain View, CA, USA) based on noncompartmental kinetics analysis. For phase 1, concentrations of DASP and DACP in plasma were measured at baseline and 6, 12, 24, 48, 96, and 144 or 168\u00a0h after treatment. DASP and DACP concentrations were plotted against time, and the half-lives of the compounds were estimated by the method of residuals. The area under the DACP and DASP concentration time curve (AUC 0-t) was estimated by the trapezoidal rule [21]. In phase 2, trough concentrations of DASP and DACP were measured at baseline, after 8 and 28\u00a0days of treatment, and 14\u00a0days after treatment (day 42). In subjects who were hospitalized for the first 5\u00a0days of treatment, trough DASP and DACP concentrations were also measured each morning of hospitalization (day 1 through day 5).\nPlant sterols and stanols \u03b2-sitosterol, campesterol, sitostanol, and campestanol were assessed by selective ion monitoring gas chromatography mass spectrometry (SIM-GC-MS). High-purity solvents were purchased from Merck, Germany. Bis-(Trimethylsilyl)trifluoroacetamide (BSTFA) was obtained from Sigma (Steinneim, Germany) and pyridine from Pierce (Rockford, IL, USA). Beta-sitosterol (24\u03b2-ethylcholesterol), \u03b2-sitostanol (24\u03b1-ethyl-5\u03b1-cholestan-3\u03b2-ol), and campesterol (24\u03b1-methyl-5-cholesten-3\u03b2-ol) were purchased from Sigma, and stigmasterol from Lacoclau AB, Sweden. For sterol extraction, 500\u00a0\u03bcl of plasma was mixed with 100\u00a0\u03bcl 0.01\u00a0mg\/ml stigmasterol and saponified for 60\u00a0min at 60\u00b0C in 1\u00a0ml of 4% (w\/v) KOH in 90% ethanol. After saponification, the samples were mixed with 1\u00a0ml of water and extracted two times with 2\u00a0ml of hexane. The pooled hexane extracts were dried under nitrogen and derivatized with 50\u00a0\u03bcl BSTFA\/pyridine (v\/v 5:1) at 60\u00b0C for 60\u00a0min. For SIM-GC-MS, 2\u00a0\u03bcl of derivative mixture were delivered by automatic injection to an HP-5890 gas chromatograph split-injection port (1:20) leading to a 0.2\u00a0mm\u2009\u00d7\u200925\u00a0m Chrompack CP-sil 19 CB (WCOT Fused Silica) capillary column. The injection port contained a glass wool liner. The carrier gas was helium at a linear rate of 1\u00a0ml\/min. The oven temperature started at 120\u00b0C and was raised to 260\u00b0C at 20\u00b0C\/min, then to 280\u00b0C at 2\u00b0C\/min, and finally to 300\u00b0C at 40\u00b0C\/min and held for 5\u00a0min. An HP-5989B mass spectrometer was used as detector. Measurements were done in the electron impact mode at 70\u00a0eV with an ion source temperature of 250\u00b0C. The quadropole temperature was 150\u00b0C. Mass spectrometric data were collected in the elected ion mode at m\/z=396 and 357 for \u03b2-sitosterol, m\/z=488 and 373 for \u03b2-sitostanol, m\/z=382 and 343 for campesterol, m\/z=369 and 384 for campestanol, and m\/z=255 and 394 for stigmasterol. Calibration curves were constructed by mixing 100\u00a0\u03bcl of 0.01\u00a0mg\/ml stigmasterol with a series of 0- to 500-\u03bcl samples of a standard solution containing 10\u00a0\u03bcmol \u03b2-sitosterol, 2\u00a0\u03bcmol\/l sitostanol, and 20 \u03bcmol\/l campesterol.\nStatistical analyses\nThe primary efficacy variable was the percent change in LDL after 4\u00a0weeks of treatment compared with baseline levels in phase 2. Differences in percentage LDL-C changes between the five treatment groups were calculated using analyses of variance (ANOVA) in SAS. P\u2009<\u20090.05 was considered statistically significant. If there was a significant difference between dose groups by ANOVA, each active dose was compared with placebo by a one-sided t test, with p\u2009<\u20090.025 being statistically significant. This procedure was also performed for percentage changes in total cholesterol (TC), HDL-C, and TG. For ApoB, ApoAI, Lp(a), vitamin A, vitamin E, and plant sterols, only ANOVA was performed to test differences in percentage changes between the five treatment groups. Safety data are presented descriptively.\nResults\nIn phase 1, 30 male volunteers completed the study in accordance with the protocol. There were no withdrawals. The majority of subjects (93%) were of Caucasian descent. Mean subject age was 50.4\u00a0(range 24\u201363) years. In phase 2, 101 male subjects were enrolled, but one subject withdrew consent prior to receiving study medication due to personal reasons and therefore contributed no safety or efficacy data. The remaining 100 subjects completed the study. The majority of subjects (89%) were Caucasian, with the remaining being Asian or another race. Mean subject age was 53.7\u00a0(range 23\u201375) years. As calculated from returned tablets, the mean percentage of received tablets was 97%. One subject in the 800-mg group had a compliance <80%. All other patients received >80% of the planned number of tablets during the treatment period. Five subjects received statin treatment within 40\u00a0days before the first dose of study treatment (29\u201335\u00a0days). Mean weight ranged between 83.9\u2009\u00b1\u200913.1 and 84.6\u2009\u00b1\u200913.0\u00a0kg during the entire study.\nAdverse events\nIn phase 1, 23 treatment-emergent adverse events were reported by 16 subjects. Of these events, five (21.7%) occurred in the 100-mg group, three (13.0%) in both the 200- and 400-mg groups, four (17.4%) in the 800-mg group, two (8.7%) in the 1,600-mg group, and three (13.0%) in both the 2,000\u00a0mg and placebo groups. All reported events were mild. The most common events were dizziness, headache, and fatigue. Other adverse events were loose stools, vasovagal attack, influenza, upper respiratory tract infection, elevated bilirubin, elevated BP, arthralgia, difficult micturition, polyuria, and pharyngolaryngeal pain. Three events were considered to be possibly related to the study drug. One was reported by a subject receiving the placebo treatment, whose bilirubin concentration increased from 12\u00a0\u03bcmol\/L on the morning of treatment (baseline) to 25\u00a0\u03bcmol\/L 24\u00a0h after treatment but decreased to normal 7 days after treatment. The other two possibly related events were reported in the 800- and 1,600-mg groups, and both consisted of one episode of loose stools on the day of treatment.\nIn phase 2, 67 subjects reported one or more treatment-emergent adverse events: 12 in the 100-mg group, 14 in the 200-mg group, 11 in the 400-mg group, 15 in the 800-mg group, and 15 in the placebo group. Most events were mild, and four subjects reported a moderate event. The most frequent event was headache, which was reported by a total of 19 subjects and by two (400-mg group) to five subjects (100-mg and 200-mg groups) in each of the five groups. The four adverse events that were moderate included two subjects with headache in the 800-mg group, one subject with an elevated CK level in the 400-mg group, and one subject with epilepsy in the placebo group. No subjects discontinued study treatment due to a treatment-emergent adverse event. One subject in the 800-mg group did not take the study medication for 3.5\u00a0days due to nausea and diarrhea, which was not considered to have been related to the study drug. Once the subject recommenced treatment, no further treatment-emergent adverse events were reported.\nA total of 24 subjects reported events that were considered to be drug-related: eight in the 100-mg group, one in the 200-mg group, two in the 400-mg group, six in the 800-mg group, and seven in the placebo group. The most commonly reported event was flatulence, which was reported by three subjects in the 100-mg groups, one in the 400-mg group, two in the 800-mg group and one in the placebo group. There were no differences in the incidence of treatment-emergent adverse events between active and placebo groups.\nBlood pressure, heart rate, and EKG analysis\nThere was no effect of FM-VP4 on BP or heart rate during phase 1. In phase 2, the mean systolic BP was slightly decreased after 4 weeks of treatment (a maximum average of 4% in the 100-mg group; data not shown), but there was no relationship between this decrease and the dose of FM-VP4. Diastolic BP did not change. All pre- and postdose EKGs were normal in both phases of the trial.\nLaboratory analyses\nIn phase 1, one subject in the placebo group showed a bilirubin concentration increase from 12\u00a0\u03bcmol\/L on the morning of treatment (baseline) to 25\u00a0\u03bcmol\/L 24\u00a0h after treatment, as described in \u201cAdverse events.\u201d Seven days after treatment, bilirubin concentration was reduced to 13\u00a0\u03bcmol\/L. There were no other clinically significant changes in hematology, biochemistry, plant sterols, or urinalysis measurements, and there were no differences between treatment groups. One subject who received 100\u00a0mg of FM-VP4 showed a decrease in white blood cell (WBC) count from 6.2\u2009\u00d7\u2009109\/L on the morning of treatment (baseline) to 2.9\u2009\u00d7\u2009109\/L 24\u00a0h after dosing. Seven days after dosing, the WBC count recovered to 7.3\u2009\u00d7\u2009109\/L. This event was not considered to be clinically significant.\nNine subjects had abnormal laboratory values during phase 2. Four laboratory abnormalities were considered clinically significant and were reported as adverse events. Two subjects showed a decrease of WBC count. One subject in the 800-mg group had a level of 2.2\u2009\u00d7\u2009109\/L at screening, but those levels were increased to 5.8\u2009\u00d7\u2009109\/L at the following visit and remained normal during the rest of the trial. This event was not considered clinically significant. A subject in the 100-mg group had a WBC count of 3\u2009\u00d7\u2009109\/L at screening, and levels fluctuated between 2.8\u2009\u00d7\u2009109\/L and 4.6\u2009\u00d7\u2009109\/L during the study. Two subjects showed an increase in CK levels that was greater than five times the ULN. In one of these patients, who was in the 400-mg group, the event was recorded as an adverse event of moderate severity. In the other subject, who was in the placebo group, the event was not recorded as an adverse event, as the patient had performed vigorous exercise the previous day. Both subjects continued with medication, and both had CK levels within normal limits at subsequent measurements. Five subjects showed a decrease in hemoglobin of a least 1.5\u00a0g\/d (0.93\u00a0mmol\/L): one each in the 100-mg, 200-mg, and 400-mg groups, and two in the placebo group. However, those decreases occurred only once, and levels were restored to normal at the subsequent visit. Therefore, those changes were not considered clinically significant.\nThere were no differences between treatment groups in the changes of vitamin A and vitamin E concentrations at week 4 compared with baseline (p=0.32 and p=0.38, respectively; data not shown).\nOverall, there were few abnormal laboratory values, and no trends were observed over time. There appeared to be no relationship between laboratory parameters and the dose of FM-VP4 administered.\nEfficacy\nIn phase 1, changes in lipids and lipoproteins were not statistically compared between the seven treatment groups, as only one dose of FM-VP4 was administered.\nIn the phase 2 run-in period, mean LDL-C levels were 3.94\u2009\u00b1\u20090.71\u00a0mmol\/L at 4\u20138\u00a0weeks before baseline, 4.19\u2009\u00b1\u20090.78\u00a0mmol\/L halfway through the run-in period, and 4.16\u2009\u00b1\u20090.76\u00a0mmol\/L at baseline. The absolute lipoprotein levels at baseline and after 4 weeks of treatment per dose group and the mean percentage change in LDL-C levels are presented in Table\u00a01. The percent changes of LDL-C by visit are also depicted in Fig.\u00a02. ANOVA analyses showed a borderline statistically significant difference in the percentage change in LDL-C and HDL-C, and pairwise comparisons between each active dose and placebo revealed that 400\u00a0mg of FM-VP4 reduced LDL-C significantly (p=0.02). In the placebo group, LDL-C increased by 2.7%, and in the 400-mg\/day group LDL-C reduced by 6.5%. There were no statistically significant differences in percentage change of LDL-C changes between the other active doses and placebo, and there were no statistically significant differences in percentage change of HDL-C between any of the active doses and placebo (Table\u00a01). When statistical analysis was carried out on a per-protocol basis and subjects who were noncompliant (n=1) or who received statins within 40\u00a0days before study treatment (n=5) were excluded, a dose-response in percentage change from baseline in LDL-C was observed: changes in the placebo group and in the 100-, 200-, 400-, and 800-mg\/day groups were 3.7%, 0.9%, \u22124.1%, \u22126.9%, and \u22126.2%, respectively. When using these per-protocol data, decreases in the 400- and 800-mg\/day groups were significantly different from placebo (p=0.007 and p=0.01, respectively). The percentage change in ApoB at week 4 compared with baseline differed significantly between treatment groups (p 0.007). Absolute changes in ApoB were 0.017, 0.063, \u22120.058, \u22120.047, and \u22120.038\u00a0g\/L in the placebo, 100-, 200-, 400-, and 800-mg\/day group, respectively. There were no differences between treatment groups in ApoAI and Lp(a) changes at week 4 compared with baseline (data not shown).\nTable\u00a01Plasma lipid and lipoprotein concentrations before and after 4 weeks of treatment with placebo or with 100\u00a0mg, 200\u00a0mg, 400\u00a0mg, or 800\u00a0mg\/day of FM-VP4 (disodium ascorbyl campestanol phosphate and disodium ascorbyl sitostanol phosphate in phase 2 (n=20 per dose)\u00a0Dose level (mg)P valueaPlacebo100\u00a0mg200\u00a0mg400\u00a0mg800\u00a0mgmmol\/LTotal cholesterolBaseline6.36\u2009\u00b1\u20090.656.40\u2009\u00b1\u20090.876.40\u2009\u00b1\u20090.886.04\u2009\u00b1\u20091.056.02\u2009\u00b1\u20090.90Day 286.50\u2009\u00b1\u20090.726.49\u2009\u00b1\u20091.126.26\u2009\u00b1\u20090.785.81\u2009\u00b1\u20091.055.90\u2009\u00b1\u20090.90% Change2.6%1.2%\u22121.7%\u22123.8%\u22121.7%0.09LDLBaseline4.29\u2009\u00b1\u20090.634.17\u2009\u00b1\u20090.584.40\u2009\u00b1\u20090.774.02\u2009\u00b1\u20090.933.94\u2009\u00b1\u20090.83Day 284.38\u2009\u00b1\u20090.794.28\u2009\u00b1\u20090.784.18\u2009\u00b1\u20090.633.75\u2009\u00b1\u20090.883.73\u2009\u00b1\u20090.81% Change2.7%2.9%\u22124.2%\u22126.5%b\u22124.6%0.05HDLBaseline1.37\u2009\u00b1\u20090.321.34\u2009\u00b1\u20090.381.20\u2009\u00b1\u20090.251.24\u2009\u00b1\u20090.301.21\u2009\u00b1\u20090.27Day 281.37\u2009\u00b1\u20090.371.29\u2009\u00b1\u20090.341.28\u2009\u00b1\u20090.311.22\u2009\u00b1\u20090.271.26\u2009\u00b1\u20090.37% Change\u22120.1%\u22123.6%6.7%\u22121.2%4.1%0.04TGBaseline1.56\u2009\u00b1\u20090.771.86\u2009\u00b1\u20091.301.77\u2009\u00b1\u20090.721.73\u2009\u00b1\u20090.721.92\u2009\u00b1\u20090.68Day 281.65\u2009\u00b1\u20090.861.95\u2009\u00b1\u20091.161.76\u2009\u00b1\u20091.081.86\u2009\u00b1\u20090.951.97\u2009\u00b1\u20091.00% Change8.2%16.3%0.7%7.5%9.7%0.8All values are mean\u2009\u00b1 standard deviationLDL low-density lipoprotein, HDL high-density lipoprotein, TG triglyceridesaDifferences between all treatment groups were analyzed by using analysis of variance. If the difference between treatments was statistically significant, then each active treatment group was individually compared with placebobP\u2009<\u20090.025 (one-sided) as compared with the change in the placebo groupFig.\u00a02Mean low-density lipoprotein cholesterol levels during 28\u00a0days of treatment with placebo or 100, 200, 400, and 800\u00a0mg\/day FM-VP4 (disodium ascorbyl campestanol phosphate and disodium ascorbyl sitostanol phosphate) and after 14\u00a0days of follow-up\nPharmacokinetics\nIn the 100- and 200-mg cohorts of phase 1, the majority of plasma samples had DASP and DACP concentrations below detection level. Therefore, the AUC 0\u2192t and the plasma elimination half-live (t1\/2) values were not evaluable, and for that reason, those data are not presented. For the groups of 400\u00a0mg or higher, the peak DACP level was reached 6\u201324\u00a0h after FM-VP4 administration (tmax), and DASP tmax was reached 12\u201324\u00a0h postdose with the exception of one subject in the 1,600\u00a0mg group who had a tmax of 49\u00a0h (Table\u00a02). Mean t1\/2 of all eligible subjects was 57\u00a0h (2\u20133\u00a0days), which ranged from 16 to 134\u00a0h for DACP and from 29 to 90\u00a0h for DASP.\nTable\u00a02Pharmacokinetic parameters of disodium ascorbyl campestanol phosphate (DACP) and disodium ascorbyl sitostanol phosphate (DASP) in 24 subjects after a single dose of 400, 800, 1,600, or 2,000\u00a0mg FM-VP4 (DACP and DASP) in phase 1Dose level (mg)Numbert1\/2 (h)Cmax (ng\/mL)tmax (h)AUC 0\u2192\u221e (h.ng\/mL)AUC 0\u2192t (h.ng\/mL)DACP\u00a0\u00a0400479.6\u2009\u00b1\u200942.3100.6\u2009\u00b1\u200925.510.7\u2009\u00b1\u20093.08,734\u2009\u00b1\u20093,8645,942\u2009\u00b1\u20092,646\u00a0\u00a0800446.2\u2009\u00b1\u200921.8144.5\u2009\u00b1\u200943.612.1\u2009\u00b1\u20090.17,672\u2009\u00b1\u20092,4795,787\u2009\u00b1\u20092,045\u00a0\u00a01,600435.9\u2009\u00b1\u200911.1175.6\u2009\u00b1\u2009106.57.9\u2009\u00b1\u20093.211,736\u2009\u00b1\u200911,4559,314\u2009\u00b1\u200910,910\u00a0\u00a02,000464.0\u2009\u00b1\u200928.3190.9\u2009\u00b1\u200947.218.3\u2009\u00b1\u20096.417,179\u2009\u00b1\u20095,68511,414\u2009\u00b1\u20094,318DASP\u00a0\u00a0400477.3\u2009\u00b1\u200911.6247.9\u2009\u00b1\u200955.112.1\u2009\u00b1\u20090.128,142\u2009\u00b1\u20097,15321,115\u2009\u00b1\u20095,255\u00a0\u00a0800452.2\u2009\u00b1\u200919.0339.7\u2009\u00b1\u200986.912.1\u2009\u00b1\u20090.128,872\u2009\u00b1\u20098,18322,907\u2009\u00b1\u20099,382\u00a0\u00a01,600453.0\u2009\u00b1\u200914.9344.3\u2009\u00b1\u2009225.824.9\u2009\u00b1\u200921.639,029\u2009\u00b1\u200938,42830,243\u2009\u00b1\u200930,624\u00a0\u00a02,000443.6\u2009\u00b1\u200921.8495.9\u2009\u00b1\u200989.018.5\u2009\u00b1\u20096.635,642\u2009\u00b1\u200916,58429,204\u2009\u00b1\u200914,549Mean\u2009\u00b1\u2009standard deviationt1\/2 half-life in plasma, Cmax peak concentration, tmax time to reach peak concentration, AUC0\u2192t area under the concentration time curve where t is the last time of blood sampling at 168\u00a0h (7\u00a0days) after treatment\nAUC0\u2192t strongly correlated with the dose (400\u20132,000\u00a0mg) of FM-VP4 for DACP (R2\u2009=\u20090.93) and DASP (R2\u2009=\u20090.90). However, AUC increased in a lower than dose-proportional manner. Between 400\u00a0mg and 2,000\u00a0mg, DACP and DASP AUC increased approximately twice and 1.3 times, respectively, when the dose was increased fivefold (Table\u00a02).\nIn phase 2, trough DACP and DASP concentrations were at or near steady-state levels by day 8 in the subset of subjects per active dose group who were hospitalized and sampled during the first week of dosing (Fig.\u00a03). The mean concentrations for all subjects in each cohort on day 8 were about the same as the mean concentrations on day 28, at the end of the treatment period. At the end of the follow-up period, 14\u00a0days after the final dose, DASP was present in the plasma of most or all of the subject plasma samples in the 200-, 400-, and 800-mg groups. DACP was mainly present in plasma samples of most subjects in the 800-mg group. The majority of plasma samples for DACP in the 100- and 200-mg groups and for DASP in the 100-mg group were below detection limit 14\u00a0days after follow-up.\nFig.\u00a03Mean trough concentrations of disodium ascorbyl sitostanol phosphate (DASP) and disodium ascorbyl campestanol phosphate (DACP) during 28\u00a0days of dosing and 14\u00a0days postdosing follow-up. The dots of days 2\u20135 represent the 16 hospitalized subjects on FM-VP4 (DACP and DASP), whereas the dots of days 8, 28, and 42 represent all 80 subjects on FM-VP4. DASP and DACP were not present in plasma at day 1 (baseline), and DACP concentrations were mostly below detection limit during the first 5 days of intake in the 100-mg group. The mean levels of DASP in the 100-mg group and DACP in the 200-mg group were below the lower limit of quantification (LLOQ) because some of the subjects had levels below the LLOQ, which were regarded as 0\u00a0ng\/ml\nDASP and DACP concentrations increased in a less than dose-proportional manner compared with baseline on days 8 and 28, as well as on day 42, 14\u00a0days after treatment.\nPlant sterol and stanol concentrations\nIn phase 1, concentrations of campestanol and sitostanol did not change within 24\u00a0h and 7\u00a0days after a single dose of FM-VP4. Also, concentrations of campesterol and \u03b2-sitosterol were not affected after a single dose of FM-VP4 (data not shown).\nIn phase 2, sitostanol and campestanol apparently rose in a dose-dependent fashion over the 28-day time period, with the maximal percent change from baseline being 87% for campestanol and 178% for sitostanol in the 800-mg group (Table\u00a03). Campesterol and sitosterol did not change from baseline to day 28 (Table\u00a03).\nTable\u00a03Concentrations of plant sterols and sterols in 100 subjects after 4 weeks of treatment with 100, 200, 400, or 800\u00a0mg\/day FM-VP4 (disodium ascorbyl campestanol phosphate and disodium ascorbyl sitostanol phosphate) or placebo in phase 2Plant stanol or sterolDose level (mg)P valuePlacebo (n\u2009=\u200920)100 (n\u2009=\u200920)200 (n\u2009=\u200920)400 (n\u2009=\u200920)800 (n\u2009=\u200920)ng\/mLSitostanolBaseline77.9\u2009\u00b1\u200933.379.6\u2009\u00b1\u200930.890.4\u2009\u00b1\u200932.975.0\u2009\u00b1\u200928.363.8\u2009\u00b1\u200919.2Day 28102.5\u2009\u00b1\u200939.2104.2\u2009\u00b1\u200938.3132.5\u2009\u00b1\u200956.3178.3\u2009\u00b1\u200963.8168.3\u2009\u00b1\u200944.6% Change41\u2009\u00b1\u20095837\u2009\u00b1\u20094648\u2009\u00b1\u200937159\u2009\u00b1\u200999178\u2009\u00b1\u200983<0.0001CampestanolBaseline116.0\u2009\u00b1\u200984.691.0\u2009\u00b1\u200936.6104.3\u2009\u00b1\u200981.7100.7\u2009\u00b1\u200957.283.4\u2009\u00b1\u200918.9Day 28140.9\u2009\u00b1\u200977.3107.1\u2009\u00b1\u200936.6134.1\u2009\u00b1\u200978.9156.2\u2009\u00b1\u200972.5150.2\u2009\u00b1\u200937.9% Change39\u2009\u00b1\u20097021\u2009\u00b1\u20092243\u2009\u00b1\u20095070\u2009\u00b1\u20096187\u2009\u00b1\u2009570.002SitosterolBaseline2,998.3\u2009\u00b1\u20091,842.53,579.3\u2009\u00b1\u20092,084.32,715.0\u2009\u00b1\u200910,725.82,704.3\u2009\u00b1\u20091,071.22,276.3\u2009\u00b1\u2009993.2Day 283,036.0\u2009\u00b1\u20091,164.93,782.1\u2009\u00b1\u20092,377.92,744.9\u2009\u00b1\u20091,256.13,138.4\u2009\u00b1\u20091,632.72,316.9\u2009\u00b1\u2009734.4% Change35\u2009\u00b1\u20091475\u2009\u00b1\u2009202\u2009\u00b1\u20092317\u2009\u00b1\u20094010\u2009\u00b1\u2009380.61CampesterolBaseline3,275.2\u2009\u00b1\u20092,017.23,699.1\u2009\u00b1\u20091,914.82,850.4\u2009\u00b1\u20091,289.02,791.1\u2009\u00b1\u20091,403.22,370.8\u2009\u00b1\u20091,036.6Day 283,212.3\u2009\u00b1\u20091,375.93,870.2\u2009\u00b1\u20092,252.63,002.7\u2009\u00b1\u20091,439.23,031.5\u2009\u00b1\u20091,500.12,416.9\u2009\u00b1\u2009917.6% Change16\u2009\u00b1\u2009804\u2009\u00b1\u2009226\u2009\u00b1\u20091912\u2009\u00b1\u2009358\u2009\u00b1\u2009350.91Mean\u2009\u00b1\u2009standard deviation. Differences between all treatment groups were analyzed by using analysis of variance\nDiscussion\nIn this study, we showed that a single dose of 100\u20132,000\u00a0mg as well as 4-week treatment in doses of 100\u2013800\u00a0mg of FM-VP4 administered to moderate dyslipidemic men was well tolerated and safe. Furthermore, 4-week treatment of FM-VP4 reduced LDL-C levels by 6\u20137% compared with baseline or by 9\u201311% compared with placebo.\nThe main treatment\u2014emergent adverse events were dizziness, headache, and fatigue after a single administration and headache after multiple-dose administration of the drug. All symptoms resolved spontaneously, and subjects receiving placebo also reported these symptoms. As there was no difference in the incidence between active and placebo groups, it is unlikely that the treatment-emergent adverse events were due to FM-VP4.\nNo drug-related abnormalities could be identified by laboratory safety tests. In phase 1, one subject receiving 100\u00a0mg of FM-VP4 showed a decrease in WBC count 24\u00a0h after treatment, but this was considered unrelated to the study drug and returned to normal 7\u00a0days after treatment. In phase 2, one subject in the 100-mg group and one in the 800-mg group also showed low WBC counts, but these abnormalities were already present at baseline. One subject in the placebo group but also one in the 400-mg group showed increased CK levels at one visit. However, we think this is no reason for concern. First of all, the patient in the 400-mg group had no complaints. Second, the levels had returned to normal by the next visit without medication discontinuation. Because no adverse events have been reported for plant sterols in general [8], we think this single CK increase was likely to be a chance finding. In addition, five subjects from various dose groups showed a hemoglobin decrease at one of the visits. These decreases were nonpersistent, and levels had returned to normal by subsequent visits. No EKG or hemodynamic abnormalities were detected after administration of FM-VP4. Also, no liver or kidney abnormalities were identified. Thus, in terms of safety tests, there was no evidence that treatment with FM-VP4 caused any acute or delayed toxicity.\nIn phase 1, DACP and DASP were absorbed slowly into plasma, with a tmax of approximately 12\u00a0h for both components. Clearance was also rather slow, with the average elimination t1\/2 of 57\u00a0h. Furthermore, t1\/2 from phase 1 can be used to estimate the time to reach steady-state concentration levels by calculating three times t1\/2, which is 3\u2009\u00d7\u200957=171\u00a0h, or 7.1\u00a0days. This estimated time to reach steady state corresponds with phase 2 data that showed a steady state after approximately 8\u00a0days, as the mean trough concentration at day 8 was similar to the mean trough concentration after 4 weeks of treatment.\nThe low plasma concentrations of DACP and DASP suggest low bioavailability of FM-VP4 in humans. This is supported by findings with other plant sterol structures. The bioavailability from 600\u00a0mg unesterified soy plant stanols was only 0.04% for sitostanol and 0.15% for campestanol [22], and in another study, the absorption of campestanol from a margarine spread containing 540\u00a0mg campestanol as fatty acid esters was 5.5% in healthy subjects [23]. In both studies, absorption of plant sterols was measured by intravenous injection and oral administration of labeled isotopes in the fasting state [22, 23]. As we have not administered labeled FM-VP4 intravenously, we were not able to estimate the bioavailability of FM-VP4 in humans and to investigate whether the esterified ascorbate group affects its absorption. Future studies with FM-VP4 are needed to assess the bioavailability of FM-VP4 in humans.\nRepeated doses of FM-VP4 in phase 2 appeared to increase sitostanol levels in plasma by up to 178% and campestanol by 87%, whereas corresponding sterol components did not change. This rise in campestanol and sitostanol appeared to increase with dose but in a less than proportional manner. Furthermore, the difference in increase from baseline between sitostanol and campestanol was consistent with the difference in plasma concentrations for DASP and DACP of approximately 2:1, which was most clear in the 400- and 800-mg groups. We speculate that conversion of DASP and DACP to free stanols may have occurred in vivo or that FM-VP4 alters the metabolism of plant stanols. Nevertheless, the absolute increase in total stanol levels remains relatively small, as the stanol contribution is <5% of total levels (Table\u00a03). Therefore, the clinical relevance of such increases is probably limited.\nBased on our data, we may speculate on the mechanism by which FM-VP4 exerts its LDL-C-lowering effect. Plant sterols and stanols are thought to compete with cholesterol for incorporation into mixed micelles, the vehicles that transport sterols to the enterocyte. In the case of FM-VP4, the solubility of plant stanols for the mixed micelles has been improved by the addition of the hydrophilic ascorbyl residue to the hydrophobic campestanol and sitostanol tail through a phosphodiester linkage (FM-VP4). In fact, this new structure allowed self-assembly into micelle structures in aqueous media in the absence of bile salts [24]. Although the assembly of micelles by means of FM-VP4 has never been directly compared with other types of plant sterols, the incorporation of plant stanols and sterols as well as cholesterol into mixed micelle normally requires the addition of bile salts in vitro and in vivo. Furthermore, in animals, FM-VP4 was shown to be more potent than unesterified plant stanols in a direct comparison when administered in the diet [16]. The results of the current clinical trial in humans showed that FM-VP4 lowered LDL-C by 6\u20137% compared with baseline, whereas the control group showed a 3\u20134% increase in plasma LDL-C levels. This means that compared with placebo, FM-VP4 showed LDL-C decreases of 9\u201311% when administered at 400\u2013800\u00a0mg\/day over 4\u00a0weeks. This effect was also reached with plant sterol and stanol esters, but at doses that were two to three times higher than the currently used doses of FM-VP4 [8]. Thus, the higher solubility of FM-VP4 into mixed micelles may lead to a more efficacious competition with cholesterol for incorporation into the micelles, and as a consequence, a lower dose of FM-VP4 is required to realize an equal LDL-lowering effect compared with plant sterol and stanol esters. However, the optimal treatment dose and duration, schedule and formulation for FM-VP4 have yet to be determined. On the other hand, it has also been suggested that, in addition to competition with cholesterol for incorporation into the micelles, plant sterol and stanol esters also may activate liver X receptor (LXR) target genes within the enterocyte [25]. It is unknown whether DACP and DASP activate such genes within the enterocyte or whether there may be systemic effects or additional mechanisms to decrease cholesterol absorption. Further research is needed to elucidate the mechanism by which several types of plant sterol and stanol esters lower LDL-C levels.\nIn conclusion, this study demonstrated that single and multiple doses of FM-VP4 for 4\u00a0weeks are safe and well tolerated by moderately hypercholesterolemic subjects. Furthermore, the higher doses of FM-VP4 significantly reduce LDL-C levels by 6\u20137% compared with baseline or by 9\u201311% when compared with placebo. The pharmacokinetics showed that DACP and DASP are absorbed and cleared slowly but that the absolute quantity of drug absorbed is low, as suggested by the low plasma concentrations of DACP and DASP. This study suggests that FM-VP4 merits further investigation as an alternative for treating hyperlipidemia.","keyphrases":["safety","pharmacokinetics","fm-vp4","plant sterols","hypercholesterolemia"],"prmu":["P","P","P","P","U"]}
{"id":"Invert_Neurosci-4-1-2413115","title":"Crystal structures of Lymnaea stagnalis AChBP in complex with neonicotinoid insecticides imidacloprid and clothianidin\n","text":"Neonicotinoid insecticides, which act on nicotinic acetylcholine receptors (nAChRs) in a variety of ways, have extremely low mammalian toxicity, yet the molecular basis of such actions is poorly understood. To elucidate the molecular basis for nAChR\u2013neonicotinoid interactions, a surrogate protein, acetylcholine binding protein from Lymnaea stagnalis (Ls-AChBP) was crystallized in complex with neonicotinoid insecticides imidacloprid (IMI) or clothianidin (CTD). The crystal structures suggested that the guanidine moiety of IMI and CTD stacks with Tyr185, while the nitro group of IMI but not of CTD makes a hydrogen bond with Gln55. IMI showed higher binding affinity for Ls-AChBP than that of CTD, consistent with weaker CH\u2013\u03c0 interactions in the Ls-AChBP\u2013CTD complex than in the Ls-AChBP\u2013IMI complex and the lack of the nitro group-Gln55 hydrogen bond in CTD. Yet, the NH at position 1 of CTD makes a hydrogen bond with the backbone carbonyl of Trp143, offering an explanation for the diverse actions of neonicotinoids on nAChRs.\nIntroduction\nNicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate fast-acting excitatory cholinergic neurotransmission in vertebrates and invertebrates. Each nAChR molecule consists of five subunits; acetylcholine (ACh) binds at extracellular ligand-binding domain (LBD) at the subunit interfaces, and a central, cation-permeable ion channel opens transiently in response to ACh and agonists (Karlin 2002; Sine and Engel 2006). Each subunit possesses an N-terminal extracellular domain with a conserved di-cysteine loop, four transmembrane regions (TM1\u2013TM4 of which TM2 provides most of the channel-lining residues), and a large intracellular loop between TM3 and TM4 (Karlin 2002). Usually, nAChRs are made up of \u03b1 and non-\u03b1 subunits (Fig.\u00a01a), the \u03b1 subunits being defined by adjacent cysteines in loop C of LBD. The ACh binding site is located at the LBD interfaces and formed by loops A, B and C of the \u03b1 subunit and loops D, E and F which are normally located in non-\u03b1 subunit (Corringer et al. 2000; Karlin 2002; Lindstrom 2003). However, \u03b17, \u03b18 and \u03b19 subunits can form functional homo-pentamers (Couturier et al. 1990; Elgoyhen et al. 1994; Gerzanich et al. 1994) (Fig.\u00a01a), whereas \u03b19 and \u03b110 subunits form \u03b1 hetero pentamers (Elgoyhen et al. 2001). When only \u03b1 subunits are present, they donate not only loops A\u2013C, but also loops D\u2013F.\nFig.\u00a01Nicotinic acetylcholine receptors (nAChRs) and the acetylcholine binding protein (AChBP). a Schematic representation of neuronal nicotinic acetylcholine receptors and acetylcholine binding protein. b Chemical structures of natural nicotinic ligands and neonicotinoids [imidacloprid (IMI) and clothianidin (CTD)] used in this study. c Multiple sequence alignment of AChBP with the ligand binding domain of nAChR subunits. Lymnaea stagnalis AChBP (Ls-AChBP) and N-terminus LBD sequences of insect (species names are colored magenta) and vertebrate (species names are colored black) nAChRs are aligned using ClustalW2 program (Larkin et al. 2007) with default parameters and the details are manually adjusted. Secondary structure elements from the Ls-AchBP\u2013IMI complex are indicated above the multiple alignments. Amino acids involved in the interactions with neonicotinoids are highlighted in bold with yellow and cyan backgrounds in the principal (+)- and complementary (-)-sides, respectively. Amino acid residue number from methionine for each protein was indicated at the top of each sequence\nIn mammals, nAChRs play central roles in neuromuscular and inter-neuronal cholinergic neurotransmission. Mutations in muscle nAChR subunits account for many cases of congenital myasthenia syndrome (Engel et al. 2003) and also multiple pterygium (Morgan et al. 2006), whereas mutations in neuronal nAChR subunits (\u03b14,\u03b22) can produce epilepsies (Aridon et al. 2006; Steinlein 2004). It has also been shown that cholinergic neurotransmission is reduced in Alzheimer\u2019s (Marutle et al. 1999) and Parkinson\u2019s diseases as well as in schizophrenia (Woodruff-Pak and Gould 2002). Further, in some cases of Alzheimer\u2019s disease, loss of nAChRs has been reported (Court et al. 2001). Thus there is a growing interest in nAChRs as potential drug targets (Arneric et al. 2007; Dani and Bertrand 2007).\nIn the insect nervous system, where ACh is the primary excitatory neurotransmitter, nAChRs are present at densities comparable to those in the electric organ of the electric fish, Electrophorus electricus (Sattelle 1980), and several classes of insecticides (cartap, bensultap, thiocyclam, spinosad and neonicotinoids) target insect nAChRs (Narahashi 2000; Raymond-Delpech et al. 2005; Millar and Denholm 2007). Of these, neonicotinoids (Fig.\u00a01a) are increasingly used not only for controlling crop pests worldwide, but also for animal health application such as flea and louse controls. Imidacloprid (IMI; Fig.\u00a01b), the first insecticide with billion dollars per annum sales, is a partial agonist of native (Deglise et al. 2002; Ihara et al. 2006; Tan et al. 2007) as well as of recombinant (Ihara et al. 2003) nAChRs expressed in Xenopuslaevis oocytes. On the other hand, clothianidin (CTD; Fig.\u00a01b) and its analog evoke supra-maximal responses (with reference to ACh responses) not only in the Drosophila D\u03b12\/chicken \u03b22 hybrid nAChRs (Ihara et al. 2004) expressed in Xenopus oocytes, but also in native Drosophila central cholinergic neurons (Brown et al. 2006). Single channel recording has shown that a CTD analog induces the opening of the Drosophila nAChR channels at the largest conductance state more frequently than ACh, offering a possible explanation for its super-agonist action (Brown et al. 2006). Contrasting with these actions, some neonicotinoids (Ihara et al. 2006; Salgado and Saar 2004), and bis-neonicotinoids containing two neonicotinoid units joined together by an alkyl chain (Ihara et al. 2007a), antagonize the acetylcholine-induced responses of native insect neurons.\nNeonicotinoids show higher affinity for insect nAChRs, accounting, at least in part, for their selective toxicity to insects over vertebrates (Matsuda et al. 2001, 2005; Tomizawa and Casida 2005). Our studies using site-directed mutagenesis combined with two-electrode voltage-clamp electrophysiology have shown that the X residue in the YXCC motif of loop C (Shimomura et al. 2004), the region upstream of loop B (Shimomura et al. 2005) and basic residues in loop D (Shimomura et al. 2002, 2003, 2006) contribute to the high neonicotinoid sensitivity of insect nAChRs (see Fig.\u00a01c for loop positions).\nThe acetylcholine binding protein (AChBP) from snail, Lymnaea stagnalis (Ls-AChBP) was discovered in glial cells as a water soluble protein modulating synaptic ACh concentration (Smit et al. 2001), and the X-ray crystal structure of the protein was described at the same time (Brejc et al. 2001). Subsequently, X-ray crystal structures of Ls-AChBP homologs from Aplysia californica (Ac-AChBP) (Bourne et al. 2005) and Bulinus truncatus (Bt-AChBP) (Celie et al. 2005b) were reported. Both Ls- and Ac-AChBPs show similarities to the extracellular domain of neuronal \u03b17 nAChRs and form homopentamers in solution (Smit et al. 2001). Notably, all the six regions (loops A\u2013F) that make up the ligand binding site are conserved in AChBPs. Thus AChBPs are considered as surrogates of the LBD in nAChRs and therefore have been employed to elucidate the mechanism for interactions with low molecular weight ligands as well as peptide toxin antagonists (Bourne et al. 2005; Celie et al. 2004, 2005; Dutertre et al. 2007; Hansen et al. 2005; Hansen and Taylor 2007; Ulens et al. 2006). Employing the crystal structures of AChBPs, we have previously modeled LBDs in complex with IMI to show that basic residues in loop D may interact electrostatically with the nitro group of neonicotinoids (Shimomura et al. 2006). Yet, details of neonicotinoid\u2013nAChR interactions are poorly understood. Therefore, we have crystallized the Ls-AChBP in complex with two commercial neonicotinoids, IMI and CTD.\nMaterials and methods\nMaterials\nLymnaea stagnalis derived from the stocks of Vrije Universeit (Amsterdam) was provided from Professor Sakakibara at Tokai University. IMI and CTD were gifts from BayerCropscience Co. in Japan. 1-(6-Bromopyridylmethyl)-2-nitroiminoimidazolidine (bromine-substituted imidacloprid; Br-IMI) and N-(2-bromo-thiazol-5-ylmethyl)-N\u2032-methyl-N\u2032\u2032-nitroguanidine (bromine-substituted clothianidin; Br-CTD) were synthesized according to the published methods (Maienfisch et al. 2000; Nishiwaki et al. 2000).\nProtein preparation\nThe cDNA of Ls-AChBP was amplified by RT-PCR from L. stagnalis and was cloned into the pPICZ\u03b1 B vector (Invitrogen) at the Pst I site, with an L1A mutation introduced to facilitate cloning. The Ls-AChBPs were expressed in yeast, Pichia pastris X-33 using EasySelect Pichia Expression Kit (Invitrogen). Secreted proteins were purified with Q-Sepharose column (GE Healthcare) immediately following concentration and buffer-exchange using Vivaflow200 (Zartorius), and then treated with 50\u00a0U\/mg protein of peptide-N-glycosidase F (Wako Pure Chemical Industries) at 37\u00b0C for 24\u00a0h to remove the glycosyl-chain at Asn67. The protein samples were subsequently purified with Mono Q and Superdex 200 columns (GE Healthcare). Purified proteins were confirmed to be the samples of interest by N-terminal sequencing and mass spectrometry. Purified proteins were buffer-exchanged against 20\u00a0mM Tris\u2013HCl buffer with 0.02% NaN3 (pH 8.0).\nEvaluation of equilibrium binding of neonicotinoids\nThe equilibrium binding of neonicotinoids with Ls-AChBP was determined by quenching of intrinsic tryptophan fluorescence. Ls-AChBP at 600\u00a0nM in binding site concentration in 20\u00a0mM Tris\u2013HCl buffer with 0.02% NaN3 (pH 8.0) was incubated with each ligand at various concentrations on a 96-well plate. Ls-AChBP was excited at 280\u00a0nm, and emission intensity was recorded at 342\u00a0nm using Varioskan microplate reader (Thermo Fisher Scientific) at room temperature. Data were fitted by non-linear regression according to the following equation using Prism Software version 4.03 (GraphPad Software): where \u0394F and \u0394Fmax represent quenching of fluorescence at a ligand concentration X and maximum quenching of fluorescence at saturation, respectively. F0 and [Protein] are fluorescence measured in the absence of ligands and the concentration of Ls-AChBP, respectively.\nCrystallization and X-ray data collection\nPurified Ls-AChBPs (6.0\u00a0mg\/mL) were incubated with 0.5\u00a0mM neonicotinoids at 4\u00b0C for 1\u00a0h prior to crystallization. Ls-AChBP-neonicotinoid complex crystals were obtained by vapor diffusion method at 20\u00b0C with 1:1 ratio of protein to reservoir solution containing 0.2\u00a0M Na citrate pH 5.7, 15\u201322% PEG3350 and about 0.5\u00a0mM of either IMI, CTD, Br-IMI or Br-CTD. The crystals were flash-cooled in liquid nitrogen after soaking into the cryoprotectant solutions containing 0.2\u00a0M Na citrate pH 5.7, 25% PEG3350, 20% glycerol and about 0.5\u00a0mM of each neonicotinoid. X-ray diffraction data sets were collected at 90\u00a0K using either Bruker AXS DIP6040 detector at BL44XU (Yoshimura et al. 2007) or ADSC QUANTUM 210 detector at BL44B2 (Adachi et al. 2001) beamlines in SPring-8, and processed with Mosflm (Leslie 1992) or HKL2000 (Otwinowski and Minor 1997). In order to identify the positions of the sulfur atom at the thiazole ring of CTD as well as the bromine atom of Br-IMI and Br-CTD, anomalous data from the crystals complexed with these neonicotinoids were collected at wavelength of 1.75\u00a0\u00c5 and 0.919\u00a0\u00c5 (Table\u00a01), respectively.\nTable\u00a01X-ray diffraction data collection and refinement statistics for Ls-AChBP\u2013neonicotinoid complexesIMICTDBr-IMIBr-CTDCTD (S ano)aData collection\u00a0BeamlinebBL44XUBL44B2BL44B2BL44B2BL44B2\u00a0Wavelength (\u00c5) 0.9000.9190.9190.9191.75\u00a0Space groupP65P65P65P65P65\u00a0Cell dimensions a, c (\u00c5)75.0, 351.074.6, 351.074.8, 350.974.5, 351.274.6, 351.3\u00a0Resolution (\u00c5)15.8\u20132.5650.0\u20132.7050.0\u20132.7050.0\u20132.9050.0\u20132.90\u00a0Rsym (%)c, d8.0 (41.3)8.5 (38.9)9.7 (34.1)11.8 (34.1)8.7 (39.5)\u00a0I\/\u03c3c8.1 (1.7)20.7 (4.0)47.0 (10.7)45.5 (10.9)60.8 (9.32)\u00a0Completeness (%)c94.8 (96.7)100 (100)100 (100)100 (100)99.5 (96.2)\u00a0Redundancyc2.6 (2.6)5.7 (5.7)23.0 (23.3)22.5 (23.2)21.6 (20.5)Refinement\u00a0Resolution (\u00c5)15.8\u20132.5850.0\u20132.70\u00a0No. of reflections3303130121\u00a0R\/Rfree (%)e20.3\/27.720.2\/27.0\u00a0Bond length (\u00c5)\/angles (deg)f0.008\/1.30.008\/1.3\u00a0Average B factor41.841.6\u00a0PDB accession codeg2ZJU2ZJVSee text for abbreviations of the neonicotinoid namesaData set for detection of anomalous peaks at sulfur atomsbAll data sets were collected at SPring-8cValues in parentheses refer to data in the highest resolution shellsdRsym\u00a0=\u00a0\u03a3|I-\u00a0<\u00a0I>|\/\u03a3I, where I is the observed intensity and <I> is the average intensity from multiple observations of the symmetry-related reflectionseR\u00a0=\u00a0\u03a3||Fo| \u2013 |Fc|| \/ \u03a3|Fo|. Rfree is an R factor of the CNS refinement evaluated for 5% of reflections that were excluded from the refinementfRMS deviations from ideal values were calculatedgThe atomic coordinates and structure factors have been deposited in the Protein Data Bank\nStructure determination and refinement\nThe structure of the IMI complex was solved by molecular replacement with Phaser (McCoy 2007) using the coordinates of an Ls-AChBP in complex with nicotine (PDB entry: 1UW6) (Celie et al. 2004) as a search model. Subsequently, the refined coordinates of the IMI complex were used to solve the structure of the CTD complex. Refinements and manual model building were performed with CNS version 1.2 (Br\u00fcnger et al. 1998) and Coot (Emsley and Cowtan 2004), respectively. The non-crystallographic-symmetry restraints between the subunits were initially applied for the refinements and removed at later steps. The details of data collection and refinement statistics are provided in Table\u00a01. Figures are generated by PyMOL (DeLano Scientific LLC), Bobscript (Esnouf 1999) and Raster3D (Merritt and Bacon 1997).\nResults and discussion\nStructure analysis\nIn this study, we have crystallized the Ls-AChBP in complex with IMI and CTD to elucidate the mechanism underpinning their selective and diverse actions on nAChRs. Both complexes were crystallized in the same space group with one pentamer in an asymmetric unit. Ligand-omit and anomalous electron density maps clearly indicated AChBP in the crystals bound neonicotinoids (see Fig.\u00a03a, b). The positions and orientations of the 2-chloropyridine ring in IMI and 2-chloro-1,3-thiazole ring in CTD were modeled to satisfy the observation of Br- and S-anomalous peak positions. The X-ray crystal structures of the Ls-AChBP complexed with IMI and CTD were refined at 2.58 and 2.70\u00a0\u00c5 resolutions, respectively (Table\u00a01).\nThe neonicotinoid-bound Ls-AChBPs form the same homopentameric structure (Fig.\u00a02a, b) as those determined for the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-bound apo form (Brejc et al. 2001) and the other ligand-bound forms (Celie et al. 2004, 2005; Hansen et al. 2005; Ulens et al. 2006). Typically, each subunit has a secondary structure composed of an N-terminal 12-residue \u03b1-helix followed by \u03b2-strand-rich parts as shown previously for other Ls-AChBPs. Root mean square differences (rmsd) of C\u03b1 atoms between the IMI- or CTD-complex and the nicotine-complex (Celie et al. 2004) were observed as 0.975 and 0.758\u00a0\u00c5, respectively. In two structures determined in the present study, neonicotinoids occupied all five ligand-binding sites that are formed between the two adjacent subunits (Fig.\u00a02a, b). The neonicotinoids occupy broadly the same position as HEPES and (-)-nicotine within the pocket formed by \u201cA-F loop\u201d regions. Although \u201cloops A, B, D and E\u201d are mainly composed of \u03b2-strands and only loops C and F have no canonical secondary structure, we use these designations to facilitate comparison with previous studies on nAChRs (Corringer et al. 2000; Matsuda et al. 2001, 2005). The principal side [(+)-chain] of the ligand-binding site possesses residues from loops A, B and C which are located in \u03b1 subunits, whereas the complementary side [(-)-chain] contains loops D, E and F, which are donated by non-\u03b1 subunits in \u03b1\/non-\u03b1 heteropentamers. Overall, the structures of the IMI and CTD complexes were very similar with C\u03b1 rmsd of 0.597\u00a0\u00c5. However, the loop C region has a specific conformation in each complex. For example, compared with the IMI complex, loop C of the CTD complex takes a \u201cclosed conformation\u201d, showing approximately 4\u00a0\u00c5 shift of the positions of the C\u03b1 atoms (Fig.\u00a02c). The variations in loop C conformation were often observed between different chains within the same Ls-AChBP pentamer in complex with neonicotinoids (e.g. subunits A and C of the CTD complex, data not shown). Conformational change in loop C is evident when comparing structures with the apo form Ls-AChBP complexed with HEPES (PDB entry code: 1UX2) (Fig.\u00a02c). The Thr155-Asp160 loop region of the (-)-chain upstream of loop F also indicates a conformational change in the ligand-binding site. These results suggest that induced-fit movement of loop regions is essential for recognition of ligands including neonicotinoids. The detailed binding interactions of neonicotinoids will be considered in relation to their key components, the aromatic ring and the guanidine\/related moieties.\nFig.\u00a02Overall view of crystal structure of Ls-AChBP complexed with imidacloprid (IMI). The pentameric structure of Ls-AChBP is viewed from the top (a) corresponding to an extracellular surface of nAChRs and from the side (b). IMI molecules bound to Ls-AChBP are shown in space-filling model, whereas carbons, nitrogens and oxygens are colored green, blue and red, respectively. Two subunits, A and B chains, are colored by pink and blue, respectively. c The structure of A\u2013B domain boundary of the Ls-AChBP complexed with IMI (red) is compared with that of the Ls-AChBP complexed with CTD (blue) and the apo form (light brown) complexed with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (PDB entry code: 1UX2) (Celie et al. 2004). Ligands are presented by stick models\nInteractions with aromatic rings of insecticides\nWhen complexed with Ls-AChBP, the pyridine ring of IMI was buried deep in the binding site, interacting with loop E segments from the (-)-chain (Fig.\u00a03c). Recently, Tomizawa et al. (2008) has reported that two azido-neonicotinoid probes containing the photolabile group in the pyridine ring labeled Tyr164 in loop F of the (-)-chain and Tyr192 in loop C of the (+)-chain in the Ls-AChBP. However, these two distinct binding modes were not observed in our models, neither in the IMI complex, nor in the CTD complex of the Ls-AChBP. The nitrogen atom of the pyridine ring forms a hydrogen bond with the amide group of Met114 and the carbonyl group of Leu102 in loop E via a water molecule, resembling the observations for both the Ls-AChBP-nicotine complex (Celie et al. 2004) and Ac-AChBP-epibatidine complex (Hansen et al. 2005).\nFig.\u00a03Electron density maps of bound ligands and their interactions with the loop E region. The annealed Fo\u2013Fc omit maps (blue) for imidacloprid (IMI) (a) and clothianidin (CTD) (b) bound to the Ls-AChBP are contoured at 4\u03c3 and drawn with the final models of the native and non-Br-neonicotinoids. In addition, the anomalous maps of bromine (purple) from the Br-neonicotinoid-complexes and sulfur atoms (yellow) from the native-neonicotinoid-complexes for ligands, which are contoured at 5 and 1.5\u03c3, respectively, are overlaid. Interactions of the loop E region with pyridine moiety of IMI (c) and thiazole moiety of CTD (d) are shown in the binding site of the Ls-AChBP. In each ligand and amino acid, chlorine, nitrogen, oxygen and sulfur atoms are colored purple, blue, red and yellow, respectively. The hydrogen bonds are presented by orange broken lines\nThe chlorine atom was located in the vicinity of a hydrogen bond bridge between the backbone C\u00a0=\u00a0O of Leu112 and the backbone amide NH of Arg104. As a result, it made van der Waals contact the peptide backbones of these two amino acids. The aromatic ring of CTD also contacts with the loop E segment in a manner similar to IMI (Fig.\u00a03d). The nitrogen atom of the thiazole ring of CTD, in a similar fashion to the nitrogen atom of the pyridine ring of IMI, formed hydrogen bond with the backbone carbonyl of Leu102 and the backbone amide group of Met114 via water molecule (Fig.\u00a03d).\nNicotine, which is active not only on insect, but also on vertebrate nAChRs, has also been shown to interact with the backbone of loop E in a similar manner to neonicotinoids (Celie et al. 2004). Therefore interactions with the backbone are insufficient to explain the selective actions of neonicotinoids. On the other hand, Met114 in loop E was located in the vicinity of the nitroimine group (Fig.\u00a03c, d) which plays a key role in selective insect nAChR\u2013neonicotinoid interactions (Ihara et al. 2003). Some insect \u03b1 subunits possess either basic or hydrogen bondable residues favorable for interactions with the nitro group such as those corresponding to Met114 (Fig.\u00a01c). Hence, if two adjacent \u03b1 subunits form a ligand-binding site, such residues may contribute to the neonicotinoid sensitivity of insect nAChRs.\nInteractions with guanidine and related moieties\nThe crystal structures of the Ls-AChBP bound with nicotine or carbamylcholine (Celie et al. 2004) and Ac-AChBP in the presence of epibatidine (Hansen et al. 2005), as well as quantum chemical calculations (Cashin et al. 2005), suggested that the protonated nitrogen atom in nicotinic agonists undergoes cation\u2013\u03c0 interactions with Trp143 in loop B. In line with this notion, it was earlier demonstrated that the electron-deficient guanidine moiety of neonicotinoids is likely to contact with Trp143 (Matsuda et al. 2005; Tomizawa and Casida 2005). However, the guanidine moiety of IMI and CTD was found to stack with the aromatic ring of Tyr185 in the crystal structures of CTD- as well as IMI-Ls-AChBP complexes (Fig.\u00a04a, d). Since Tyr185 is conserved throughout vertebrate and invertebrate nAChR \u03b1 subunits (Fig.\u00a01c), this residue itself is not involved directly in determining the selective interactions with neonicotinoids. Yet the interactions with Tyr185 contribute to the affinity for neonicotinoids. The stacking level for CTD appears to be weaker than that for IMI (Fig.\u00a04a, b). In addition, the stacking interaction resulted in CH\u2013\u03c0 contacts with Trp143 of the methylene (CH2\u2013CH2) bridge in IMI, which was not seen in CTD, accounting, at least in part, for the higher binding potency of IMI (Kd\u00a0=\u00a01.57\u00a0\u00b1\u00a00.21\u00a0\u03bcM, n\u00a0=\u00a03) compared to that of CTD (Kd\u00a0=\u00a07.26\u00a0\u00b1\u00a01.13\u00a0\u03bcM, n\u00a0=\u00a03) on the Ls-AChBP as evaluated by quenching of the protein fluorescence.\nFig.\u00a04Imidacloprid (IMI) and clothianidin (CTD) binding to Lymnaeastagnalis AChBP (Ls-AChBP). a IMI\u2013Ls-AChBP complex. b CTD\u2013Ls-AChBP complex. The binding site is located at the interface between two adjacent subunits, as drawn in green for (+)-chains and in cyan for (-)-chain (see text). The subunits EA and CD interfaces are shown for the binding sites for IMI (a) and CTD (b), respectively. Orange broken lines depict hydrogen bonds. In each ligand and amino acid, chlorine, nitrogen, oxygen and sulfur atoms are colored purple, blue, red and yellow, respectively\nAnother difference between CTD and IMI is that the former exposes a hydrogen bond donor NH at position 1, whereas the latter lacks it. We suggested earlier that this NH may form a hydrogen bond with the backbone carbonyl of Trp143 in loop B (Ihara et al. 2007b). As suggested, the NH of CTD made a hydrogen bond with the backbone carbonyl of Trp143 from the (+)-chain in the crystal structures of the Ls-AChBP (Fig.\u00a04b), although it was observed at only 2 of 5 interfaces. Such hydrogen bond formation may assist CTD in keeping contact with nAChRs when large conformational changes occur in LBD to open the central channel (Miyazawa et al. 2003; Unwin 2005), accounting, at least in part, for higher agonist efficacy of CTD compared to IMI and ACh in certain native and recombinant nAChRs (Brown et al. 2006; Ihara et al. 2004).\nInteractions with loop D and selectivity of neonicotinoids\nWe have previously shown that substitution of a glutamate for the glutamine corresponding to Gln55 of Ls-AChBP (Fig.\u00a01c) markedly reduced the maximum response to IMI and nitenpyram of the chicken \u03b17\u00a0nAChR, whereas mutation of the same glutamine to arginine enhanced it, slightly shifting the concentration\u2013response curve to the left (lower concentrations). In complete contrast, the same mutation reduced the (-)-nicotine sensitivity of the \u03b17, whereas substitution with a glutamate enhanced it (Shimomura et al. 2002). Therefore, it was postulated that this glutamine residue is likely to be located in close proximity to the nitro group of neonicotinoids, and that marked changes in the \u03b17 response to neonicotinoids as well as (-)-nicotine are the results of electrostatic interactions between the added residues and the nitro group. Consistent with this hypothesis, Gln55 faced the nitro groups of IMI and CTD in the crystal structure of the Ls-AChBP within a hydrogen bondable distance in the AChBP\u2013IMI complex (<3.3\u00a0\u00c5 in 2 of 5 interfaces in the crystal structure of the Ls-AChBP) (Fig.\u00a04). For CTD, however, the distance between the Gln55 and the nitro group was longer than 3.5\u00a0\u00c5, excluding or minimizing a contribution of hydrogen bonding to the binding potency of CTD.\nAll vertebrate nAChRs, perhaps apart from those containing either human or rat \u03b24 subunit, are likely to show reduced sensitivity to neonicotinoids due to the lack of the basic residues in loop D. The human \u03b24 subunit (Fig.\u00a01c), as well as rat \u03b24 subunit (sequence not shown), possesses a lysine as residue corresponding to Gln55 of Ls-AChBP. However, the vertebrate \u03b24 subunits possess a glutamate residue as that corresponding to Thr57 in Ls-AChBP which is located in the vicinity of Gln55 (Fig.\u00a05). This glutamate residue is postulated to interfere electrostatically with the neighboring basic residue\u2013neonicotinoid interactions, thereby reducing the neonicotinoid sensitivity of nAChRs containing the \u03b24 subunits.\nFig.\u00a05Interactions between loops C, D and F. IMI-binding site of Ls-AChBP is presented by especially focusing on loops C, D and F. The binding site at the interface of the subunits C and D is drawn in green for (+)-chains and in cyan for (-)-chain (see text). In each ligand and amino acid, chlorine, nitrogen, oxygen and sulfur atoms are colored purple, blue, red and yellow, respectively. Orange broken lines depict hydrogen bonds\nRole of loops C and F in their interactions with neonicotinoids\nWe have found that replacement by glutamate of the proline corresponding to Ser186 in the YXCC motif in loop C of the Drosophila D\u03b12 subunit reduces the IMI sensitivity of the D\u03b12\u03b22 hybrid nAChR, whereas its reverse mutation in the \u03b14\u03b22 nAChR enhanced sensitivity (Shimomura et al. 2004), suggesting a contribution of the loop C residue to the neonicotinoid sensitivity of nAChRs. However, another view has been proposed in which the serine or threonine residue corresponding to Ser186 from (+)-chain of Ls-AChBP in loop C of insect nAChRs (see Fig.\u00a01c) forms a hydrogen bond with the nitro group of neonicotinoids, thereby enhancing the binding potency of neonicotinoids (Tomizawa et al. 2007). In the crystal structures of Ls-AChBP, Ser186 made hydrogen bonds with Glu163 and Tyr164 in loop F of (-)-chain but not with the nitro group of IMI (Fig.\u00a05). This hydrogen bond web, as well as the basic residues in loop D, seems to play an important role in determining the neonicotinoid sensitivity of nAChRs. In most heteromeric vertebrate nAChRs, \u03b1 subunits possess acidic residues as those corresponding to Ser186 in loop C, resulting in electrostatic repulsion of the acidic residues in loop F of \u03b2 subunits corresponding to Glu163 in Ls-AChBP (Fig.\u00a01c). On the other hand, insect nAChR \u03b1 subunits do not possess such an acidic residue in loop C (Fig.\u00a01c). Hence, it is conceivable that amino acids not only from \u03b1 subunits, but also from non-\u03b1 subunits of insect nAChRs, contact more effectively with neonicotinoids than those of vertebrate nAChRs. In this context, Tyr164 may also contribute indirectly to the neonicotinoid sensitivity of insect nAChRs because vertebrate non-\u03b1 subunits possess phenylalanine residue incapable of assisting the formation of the hydrogen-bond-network at this position.\nIn conclusion, we have for the first time elucidated the crystal structures of the Ls-AChBP in complex with two commercial neonicotinoids IMI and CTD. Met114 in loop E and Gln55 in Loop D closest to the nitro group in neonicotinoids, suggesting that corresponding residues in insect nAChRs may play important roles in determining neonicotinoid sensitivity. Ser186 in loop C was found to form hydrogen bonds with Gln163 as well as Tyr164 in loop F, suggesting that these hydrogen bondable residues can also indirectly influence the selective interactions with neonicotinoids. CTD forms hydrogen bond with the backbone carbonyl of Trp143 in loop B and shows weaker hydrophobic contacts with Trp143 compared with other neonicotinoids tested, which may altogether lead to its unique actions on nAChRs. Although there are sequence and structural similarities between AChBPs and nAChRs, there are also important differences. Future structural studies on AChBP mutants engineered to resemble more closely insect or human nAChRs could help to enhance even further our understanding of neonicotinoid actions and selectivity. Nevertheless, we have provided new insights into the molecular basis of differential actions of neonicotinoid molecules. These results may be of value in the design of even safer new crop protection agents.","keyphrases":["crystal structures","neonicotinoids","nicotinic acetylcholine receptors","ion channels","acetylcholine binding protein (lymnaea stagnalis)"],"prmu":["P","P","P","P","R"]}
{"id":"Environ_Manage-2-2-1705480","title":"Multiscale Drivers of Water Chemistry of Boreal Lakes and Streams\n","text":"The variability in surface water chemistry within and between aquatic ecosystems is regulated by many factors operating at several spatial and temporal scales. The importance of geographic, regional-, and local-scale factors as drivers of the natural variability of three water chemistry variables representing buffering capacity and the importance of weathering (acid neutralizing capacity, ANC), nutrient concentration (total phosphorus, TP), and importance of allochthonous inputs (total organic carbon, TOC) were studied in boreal streams and lakes using a method of variance decomposition. Partial redundancy analysis (pRDA) of ANC, TP, and TOC and 38 environmental variables in 361 lakes and 390 streams showed the importance of the interaction between geographic position and regional-scale variables. Geographic position and regional-scale factors combined explained 15.3% (streams) and 10.6% (lakes) of the variation in ANC, TP, and TOC. The unique variance explained by geographic, regional, and local-scale variables alone was <10%. The largest amount of variance was explained by the pure effect of regional-scale variables (9.9% for streams and 7.8% for lakes), followed by local-scale variables (2.9% and 5.8%) and geographic position (1.8% and 3.7%). The combined effect of geographic position, regional-, and local-scale variables accounted for between 30.3% (lakes) and 39.9% (streams) of the variance in surface water chemistry. These findings lend support to the conjecture that lakes and streams are intimately linked to their catchments and have important implications regarding conservation and restoration (management) endeavors.\nIntroduction\nSurface water chemistry is regulated by a complex suite of processes and mechanisms operating at varying spatial and temporal scales. Early work by lake ecologists focused on the importance of geographic position as a strong predictor of lake water chemistry. For instance, in the early 1900s, Thienemann (1925) and Naumann (1932) developed lake trophic classification schemes that basically recognized differences between lowland, nutrient rich (eutrophic) and alpine, nutrient poor (oligotrophic) ecosystems. Although lake ecologists were early to appreciate the importance of adjacent land type on lake-water chemistry, stream ecologists have addressed the terrestrial\u2013aquatic linkage concept more formally, with streams being regarded as \u201copen systems that are intimately linked with their surrounding landscapes\u201d (e.g., Hynes 1975). However, lake ecologists have recently revisited the landscape position hypothesis and formalized paradigms that recognize more explicitly the importance of landscape position and its significance for describing among-lake variance (e.g., Kratz and others 1997; Soranno and others 1999; Riera and others 2000).\nThe surrounding landscape (catchment) with its distinct geology, hydrology, and climate clearly influences the physico-chemical features of a specific water body (e.g., Omernik and others 1981; Osborne and Wiley 1988; Allan 1995; Kratz and others 1997; Soranno and others 1999; Riera and others 2000), and several studies have highlighted the links between surface water chemistry and catchment characteristics, particularly in relation to sensitivity to nutrient enrichment and acidification (Vollenweider 1975; Sverdrup and others 1992; Hornung and others 1995). Indeed, water chemistry, both within and among lakes or streams, is considered to be driven by factors acting on both regional and local scales. Regional factors such as climate, geology and weathering are interrelated with other factors such as soil type and land cover\/use, whereas local factors, like the input and retention of organic matter, are related to the vegetation type and topographical relief. Hence, a priori, a close linkage is expected between regional- and local-scale factors. Geographic proximity alone is, however, often not sufficient to predict the physical and chemical characteristics of individual streams or lakes, as differences in external processes such as stream hydrology or lake morphometry and water retention time as well as internal processes such as nutrient cycling, and strengths of interactions with the surrounding landscape may singly or in concert confound the importance of regional-scale factors.\nAlthough a number of studies have addressed the importance of land use\/type on surface water chemistry, few studies have simultaneously focused on the importance of local and regional factors as determinants of surface water chemistry, and fewer still have addressed the similarities and differences of lake and stream ecosystems. To our knowledge, only one study (Essington and Carpenter 2000) has simultaneously studied the response of stream and lake ecosystems. These authors showed that streams and lakes were surprisingly similar in nutrient cycling, in particular when adjustments were made for water residence time. By concurrently studying stream and lake ecosystems, we hope to better our understanding of the processes and mechanisms that drive surface water chemistry in these different, but certainly not ecologically isolated ecosystems.\nWe hypothesize that both streams and lakes are strongly linked to the surrounding landscape, and that spatial variation in surface water chemistry is regulated by non-mutually exclusive factors acting on various hierarchical scales depending on landscape type and\/or geographic position. Here, we study the effect of regional and local-scale factors on three commonly measured water chemical variables. Acid neutralizing capacity (ANC) was selected to indicate the effect that catchment geology and weathering might have on buffering capacity. Total phosphorus (TP) was selected for its key role in driving ecosystem productivity and because it is biologically active (i.e., is expected to decrease along, e.g., lake chains). Finally, total organic carbon (TOC) was used as a surrogate measure of the importance of allochthonous input from the boreal catchments. The sites used in this study are often natural brown-water systems, with high contents of humic substances.\nWe attempted to (1) identify and quantify possible sources of variation in surface water chemistry of boreal streams and lakes, (2) determine which environmental factors and which spatial scales are most important in determining the surface water chemistry of boreal streams and lakes, and (3) determine similarities\/differences in the factors driving stream\/lake water chemistry.\nMethods\nStudy Site\nThe data set used in this study consists of 390 streams and 361 lakes sampled as part of the Swedish national stream and lake survey in autumn 2000 (Johnson and Goedkoop 2000; Wilander and others 2003) (Fig.\u00a01). A number of factors suggested that this dataset was sufficiently robust for examining among-site similarities\/dissimilarities in surface water chemistry of boreal streams and lakes. Firstly, streams and lakes were selected randomly; thus, the samples should be representative of the population of streams and lakes sampled. In selecting lakes, only lakes with surface areas >4 ha were included, and two size classes were used for stratifying stream sites (catchment area classes of 15 to 50 and 50 to 250 km2). Because we were interested in obtaining a depth-integrated measure of surface water chemistry, lakes were sampled during autumn turnover. Hence, sampling started in the northernmost parts of the country and progressed southwards. A more detailed description of stream and lake selection is given in Wilander and others (2003). In this study, we were interested in understanding the effects of local and regional-scale variables on the expected natural variability of selected water chemistry variables. Thus, sites deemed to be affected by liming, acidification (lakes: critical load exceedence of S and N > 0; Rapp and others (2002)) and agriculture\/silviculture (catchments with more than 25% defined as arable and affected by clear-cutting, respectively) were not included in this dataset.\nFig.\u00a01Location of the 361 lakes and 390 streams used to assess the influence of geographic position, and regional and local scale factors on surface water chemistry\nThe streams and lakes can be classified as relatively small (mean stream width = 5 m; mean lake area = 3.27 km2), nutrient poor, ranging from clear to brownwater ecosystems (mean stream abs 420 nm = 0.188; mean lake abs 420 nm = 0.149). The streams and lakes were distributed fairly evenly across the country. Streams were generally situated at a somewhat lower altitude than lakes (201 m a.s.l. for streams and mean altitude = 331 m a.s.l. for lakes). The catchment area of streams was also smaller (mean catchment area = 64 km2) than that of lakes (257 km2), because streams with catchments >250 km2 were not included in the national stream survey.\nWater Chemistry\nA single, midstream or midlake (approximately 0.5 m depth) water sample was collected in autumn 2000. All water chemistry analyses were done by the SWEDAC (Swedish Board for Accreditation and Conformity Assessment) certified laboratory at the Department of Environmental Assessment, Swedish University of Agricultural Sciences following international (ISO) or European (EN) standards when available. ANC is a measure of the buffering ability of lakes and streams against strong acid inputs. This metric was chosen because it includes humic substances and compensates for their natural variation, i.e., the effect of acid deposition is more pronounced than in other acidification indicators such as pH or sulfate concentration.\nIndependent Variables\nDuring sampling, sites were classified according to (aquatic) substratum particle size and vegetation; six substrate classes (ranging from silt\/clay to block), two classes of detritus (coarse and fine), and 10 classes of riparian land use and vegetation were classified using four categories as: 0%, <5%, 5\u201350%, and >50% coverage (Table\u00a01). For streams, 50-m reaches (sampling site) of relatively homogeneous substratum were chosen, and the riparian vegetation designated at a 5-m-wide zone on both sides of the sampling site was classified as above. For lakes, 10-m long and 5-m wide littoral areas of relatively homogeneous substratum were chosen and riparian vegetation, designated at a 50-m long and 5-m wide shoreline zone, was classified as above.\nTable\u00a01Dependent and independent variables used in RDAVariableUnitLakes (N = 361)Streams (N = 391)a) DependentChemistryAcid neutralizing capacity (ANC)meq l\u221213.36 (0.09\u22120.74)0.51 (0.15\u22120.99)Total phosphorus (TP)\u03bcg l\u2212113.17 (2\u221228)27.42 (2\u221267)Total organic carbon (TOC)mg l\u221219.13 (2.02\u221216.6)10.57 (2.2\u221221.08)b) IndependentExplained variabilityGeographic positionLakesStreamsLatitudeDecimal degreesAltitudem a.s.l.18.5%(2)2.7%(3)Ecoregions*Dummy variable\u00a0\u00a0Arctic\/alpineDummy variable\u00a0\u00a0Northern borealDummy variable\u00a0\u00a0Southern borealDummy variable\u00a0\u00a0BoreonemoralDummy variable\u00a0\u00a0NemoralDummy variableRegional factors\u00a0\u00a0Mean annual discharge (Q)m3 s-11.3%(5)\u00a0\u00a0Wet & Dry NHx deposition\u00a0\u00a0Wet & dry non-seasalt Mg depositionCatchment land use\/cover\u00a0\u00a0Urban areas%\u00a0\u00a0Forested areas%\u00a0\u00a0Alpine treeless land cover%55.4%(1)17.3%(2)\u00a0\u00a0Glacier%\u00a0\u00a0Open freshwater bodies%\u00a0\u00a0Marsh\/mires%\u00a0\u00a0Arable land%4.6%(3)68%(1)\u00a0\u00a0Pasture%\u00a0\u00a0Alpine forested areas%Local factorsPhysical properties of sample site\u00a0\u00a0Stream width mM2.7%(4)\u00a0\u00a0Lake areakm21.5%(5)\u00a0\u00a0Water temperature\u00b0CAquatic substrate**Classified 0\u22123\u00a0\u00a0Boulder (>250 mm)Classified 0-3\u00a0\u00a0Block (200\u2013250 mm)Classified 0-3\u00a0\u00a0Cobble (60\u2013200 mm)Classified 0-3\u00a0\u00a0Pebble (20\u201360 mm)Classified 0-3\u00a0\u00a0Silt\/clay (0.02 mm)Classified 0-3\u00a0\u00a0Coarse detritusClassified 0-3\u00a0\u00a0Floating leaved vegetationClassified 0-34.6%(4)\u00a0\u00a0Fine leaved submerged vegetationClassified 0-3\u00a0\u00a0PeriphytonClassified 0-3\u00a0\u00a0Fine dead woodClassified 0-3Riparian land use\/cover\u00a0\u00a0Deciduous forestClassified 0-3\u00a0\u00a0HeathClassified 0-3\u00a0\u00a0Arable landClassified 0-3\u00a0\u00a0AlpineClassified 0-3\u00a0\u00a0PastureClassified 0-3\u00a0\u00a0MireClassified 0-3\u00a0\u00a0Canopy coverClassified 0-3a) Chemistry variables (n = 3) with mean values and 10th and 90th percentiles in parentheses. b) Environmental variables (n = 38), divided into three subsets, included in the analyses. Also shown are the first five variables (explained variability in %) that could best explain the variability in ANC, TP, and TOC, using RDA and stepwise forward selection with the order of selection shown in parentheses. Note: the middle boreal ecoregion was insignificant in the Monte Carlo permutation test and excluded from the analysis*Six major ecoregions according to the Nordic Council of Ministers (1984)**Classified as percent coverage where no coverage 0 = 0%, very low coverage 1 = <5%, medium coverage 2 = 5\u201350%, high coverage 3 = >50%\nCatchments were classified as percentage land use\/vegetation cover according to the same land use categories used for riparian zones. Hence, catchment land use\/cover ranged from 0% (all classes) to 100%. Thereby, maximum urban areas in catchments were 10.2% (lakes) and 26.3% (streams), forested areas covered 99.8% in both lake and stream catchments, and alpine treeless cover was very high with 99.7% (lakes) and 99.9% (streams). Glacier areas comprised only 2.3% of total lake catchment areas, but covered 26.6% of stream catchments; other open freshwater bodies in the catchment comprised 19.4% of lake and 28.9% of stream catchments. Maximum marsh or mire land cover was 82.9% for lake and 67.4% for stream catchments, whereas pasture comprised 18.1% (lakes) and 14.2% (streams). Maximum alpine forested area cover was higher in lake (98.7%) than in stream catchments (65.6%), and maximum arable land covered 24.4% of lake and 24.6% of stream catchments.\nEcoregion delineation of Sweden was obtained from the Nordic Council of Ministers (1984). The ecoregions range from the nemoral region in the south to the arctic\/alpine complex in the north. The nemoral region is characterized by deciduous forest, mean annual temperatures >6\u00b0C, and a relatively long growth period (180\u2013210 d). In contrast, the arctic\/alpine complex in the north is characterized by relatively low mean annual temperatures (<2\u00b0C) and short growth periods (<140 d). Geographic position descriptors (longitude, latitude, altitude), ecoregion delineation, discharge, deposition variables, land use\/vegetation cover descriptors, physical properties (stream width, lake area) as well as aquatic substrate descriptors resulted in a dataset of 60 environmental (independent) variables.\nStatistical Analyses\nFirst, detrended correspondence analyses were conducted to obtain the gradient length of both the stream and lake chemistry data. Because the gradient lengths were in both cases \u22641.5 SD, the linear method redundancy analysis (RDA) was used to study the effects of environmental variables representing geographic position and regional- and local-scale factors on stream and lake water chemistry. Moreover, preliminary analyses of water chemistry (total phosphorus concentration) and catchment land use (% agriculture) did not reveal any step changes between the northern and southern regions. RDA was performed on a correlation matrix and is a form of direct gradient analysis (like Principal Components Analysis). In a first step in RDA, the entire set of 60 environmental variables was tested to determine the significance of individual variables using a Monte Carlo permutation test (with 999 unrestricted permutations). Variables that were not significantly correlated with the three water chemistry variables or that were found to co-vary with other environmental variables (i.e., variance inflation factors >100) were removed (n = 22) from the data set.\nVariance Partitioning\nThe remaining 38 explanatory variables were grouped into three subsets to yield ecologically interpretable variance components as follows: (1) variables describing the geographic position (G) of the water body, (2) regional scale (R), and (3) local scale (L) variables (Fig.\u00a02, Table\u00a02). The variation partitioning technique used has been previously described by Borcard and others (1992) and hence we will not go into detail here. In brief, the procedure allows for the variance in the explanatory data set to be partitioned into different variable components through the use of covariables (i.e., variables whose influence is partialled out of the analysis). Initially, this technique was used to partition variation in ecological data sets into environmental and spatial components (e.g., \u00d8kland and Eilertsen 1994) and has been extended by incorporating three sets of explanatory variables (e.g., Anderson and Gribble 1998).\nFig.\u00a02Venn diagram (hypothetical model) showing the unique variation, the partial common variation, and the common variation of the three subsets G, R, and L representing the environmental dataTable\u00a02The procedure of variation partitioning of water chemistry (n = 3) in streams (n = 390) and lakes (n = 361) explained by three sets of environmental variables, geographic (G), regional (R), and local (L) in partial redundancy analysis (pRDA)RunEnvironmental variableCovariable\u03bbstreams\u03bblakes1GRLNone0.7510.6512GeoR&L0.0180.0373R&LNone0.7330.6144R&LGeo0.1730.1845GeoNone0.5780.4676RegG&L0.0990.0787G&LNone0.6520.5738G&LReg0.0550.1169RegNone0.6960.53510LocalG&R0.0290.05811G&RNone0.7210.59312G&RLocal0.2700.22113LocalNone0.4800.430a\u03bb = computed eigenvalue in RDA. These numbers are used to calculate the explanatory power of each component (see Table\u00a03)Table\u00a03Calculation of explanatory power of each component in the variance partitioning modelVariation explained by factorsAbbreviation (see Figs.\u00a02 & 3)Calculation (no. of run, Table\u00a02)\u03bbstreams\u03bblakesGeographicG20.0180.037RegionalR60.0990.078LocalL100.0290.058Geographic & regionalGR12\u20136\u201320.1530.106Geographic & localGL8\u20132\u2013100.0080.021Regional & localRL4\u20136\u2013100.0450.048Geographic, regional & localGRL7\u20138\u2013(12\u20136\u20132)\u2013(4\u20136\u201310)0.3990.303Total explainedTotX10.7510.651UnexplainedUXTotV\u2212TotX0.290.349Total varianceTotV1.01.0aAbbreviations refer to the legend in Fig.\u00a02. The figures in the calculation column refer to the runs in Table\u00a02Fig.\u00a03Sources of variation in lake and stream water chemistry, respectively. Column labels indicate the variation (%) in acid neutralizing capacity, total phosphorus, and total organic carbon accounted for by each subset and their combinations\nThe total variance explained and the unique contributions of each subset and their joint effects were obtained by the following: (1) RDA was run with all three subsets as environmental variables and no covariables to obtain a measure of the total variance, (2) partial RDA was run with one of the three subsets as environmental variables and no covariables, and (3) partial RDA was run with one of the three subsets as environmental variables constrained by the remaining two groups as covariables and reverse. The third step was repeated three times and each subset was treated as environmental variables constrained by the remaining subsets as covariables. This procedure resulted in four runs of RDA for each subset combination or a total of 13 runs of RDA were done for the full set of analyses for each ecosystem (Table\u00a02). With three subsets of environmental data, the total variation of water chemistry was then partitioned into seven components including covariance terms (Fig.\u00a02, Table\u00a03). The variation explained by these subsets is subtracted from the total variation (1.0 in case of RDA) to obtain the unexplained variation.\nStepwise RDA\nStepwise RDA with forward selection was performed with all 38 environmental variables as independent variables and the 3 water chemical variables (ANC; TP and TOC) as dependent variables to determine the best predictors (high R2 values). In this procedure, selected variables are run as co-variables and subsequent variables (step 2 and on) need to explain a significant amount of the residual variance (tested by Monte Carlo permutation).\nRedundancy analyses and partial RDA were done using CANOCO for Windows Version 4.5 (Ter Braak and Smilauer 1997\u20131998). Prior to all statistical analyses (RDA), chemical and deposition variables, stream width, lake area, and altitude were log-transformed and proportional catchment land use\/vegetation cover variables were arcsine square-root transformed to achieve normal distribution (SAS).\nResults\nVariance decomposition using redundancy analysis showed that all independent variables combined explained more than 65% of the total variation in stream and lake surface water chemistry (Table\u00a03). The amount of variation explained was somewhat higher for streams (\u03bbstreams = 0.751) compared to lakes (\u03bblakes = 0.651). The largest proportion of variance was explained by the interaction between all three scale factors (Fig.\u00a03).\nBoth stream and lake surface water chemistry was more influenced by regional-scale factors than either by geographic position or local-scale factors. However, the unique variance explained by geographic position, regional- or local-scale variables was low (<10%) (Fig.\u00a03). For streams, the unique variance explained by regional-level variables (9.9%) was substantially higher than that explained by local-scale variables (2.9%) or geographic position (1.8%). Similarly, for lakes the unique variance explained by regional-scale variables (7.8%) was higher than that explained by local-scale variables (5.8%) and that explained by geographic position (3.8%). Geographic position and regional-scale factors (G&R) were better predictors of surface water chemistry than regional and local (R&L) or geographic position and local (G&L) factors. The strongest interaction was found between geographic position and regional-scale variables. For streams, the interaction between geographic position and regional-scale variables (G&R) explained 15.3% of the variance in stream chemistry. For lakes, the G&R interaction explained 10.6% of the variance in lake chemistry. The relation between geographic position and local-scale variables was much weaker, in particular for streams. The G&L interaction explained 0.8% of the variance in stream and 2.1% of the variance in lake chemistry. The amount of variance explained by the interaction between regional- and local-scale variables was 4.5% for streams and 4.8% for lakes.\nOrdination of stream chemistry and environmental variables showed that the primary RDA axis represented a latitudinal gradient (Fig.\u00a04a). Eigenvalues for the first and second RDA axes were 0.685 and 0.056, respectively. Streams situated in alpine forested or alpine treeless catchments were placed on the right side of the ordination, whereas lowland streams situated in pasture and arable landscape in the south (e.g., boreonemoral ecoregion, eco5) with high wet and dry deposition of NHx (WDNHx) were placed to left. ANC and TP were strongly associated with pasture and arable land use and high WDNHx. TOC concentration was positively correlated with forested catchments and habitats with high amounts of coarse detrital matter and negatively correlated with mean annual discharge (Q) and, like lake-TOC, unrelated to arctic\/mountainous characteristics. The second RDA axis was related to glacial land cover and whether the stream was located in the southern boreal ecoregion (eco4).\nFig.\u00a04RDA biplot of environmental factors and ANC, TP, and TOC of (A) streams and (B) lakes. 1 = riparian pasture cover; 2 = floating leaved vegetation; 3 = riparian deciduous forest cover; 4 = riparian alpine cover; 5 = riparian heath cover; 6 = boulder; 7 = block; 8 = pebble; 9 = periphyton; 10 = cobble; 11 = fine leaved submerged vegetation; 12 = water temperature; 13 = wet & dry non\u2013sea salt Mg deposition; 14 = riparian arable cover (streams), alpine forest (lakes); eco1 = arctic\/alpine ecoregion; eco2 = northern boreal ecoregion; eco4 = southern boreal ecoregion; eco5 = boreonemoral ecoregion; = nemoral ecoregion; WDNHx = Wet & Dry NHx deposition; c_detritus = coarse detritus; f_detritus = fine detritus; FWD = fine wooded debris (substrate); Q = annual mean discharge\nAll three lake chemistry variables were negatively correlated with the first RDA axis (Fig.\u00a04b). Eigenvalues for the first and second RDA axes were 0.599 and 0.056, respectively. The first RDA axis represented gradients in latitude and catchment\/ecoregion. Lakes situated in alpine, treeless catchments at high latitude and altitude were situated to the right, whereas sites situated in forested catchments or catchments with pasture or arable land use were placed to the left in the ordination. Both ANC and TP were positively correlated with the amount of catchment classified as pasture and arable. Moreover, many of these lakes were situated in the boreonemoral ecoregion (eco5), with high wet and dry deposition of NHx (WDNHx). In contrast, lake water total organic carbon (TOC) was associated with forested catchments, with high amounts of coarse detrital matter (c_detritus). The second RDA axis represented gradients in the amount of catchment classified as mire (or bog), in particular the importance of local factors such as substrate type, water temperature, and riparian mire and fine wooded debris (FWD).\nStepwise RDA of stream and lake chemistry as dependent variables and the \u201csingle\u201d variables of geographic position and regional and local environmental variables showed that all variables accounted for 65% (lakes) and 75% (streams) of the total variance. The amount of alpine treeless areas in the catchment was the single most important predictor of lake water chemistry (explaining 55.4% of the explained variance). The second variable selected was altitude (18.5%, i.e., the amount of residual variance explained after running the first variable selected, \u201calpine treeless areas in the catchment,\u201d as a covariable), followed by the amount of arable land in the catchment (4.6%), percent coverage of floating leaved vegetation in the littoral (4.6%), and lake surface area (1.5%). For streams, the five best single predictors of water chemistry were the amount of arable land in the catchment (68%), followed by the amount of alpine treeless areas in the catchment, altitude (2.7%), stream width (2.7%), and mean annual discharge Q (1.3%).\nDiscussion\nLakes and streams are often perceived as structurally and functionally different ecosystems, and indeed major dissimilarities do exist regarding differences in water movement. For example, streams are characterized by unidirectional, turbulent flow and high flushing rates, whereas lake chemistry is more affected by the timing and frequency of turnover events (e.g., polymictic to dimictic mixing in boreal lakes). Furthermore, obvious differences in nutrient cycling and recycling are expected due to the relative importance of benthic vs. pelagic productivity (Essington and Carpenter 2000). The surface water chemistry of streams is considered to be tightly linked to catchment characteristics, with geomorphology determining soil type and availability of ions through weathering (e.g., Allan 1995). Lakes, on the other hand, have until recently been perceived as separate entities, more isolated than streams from the surrounding landscape (e.g., Kratz and others 1997; Soranno and others 1999; Riera and others 2000; Quinlan and others 2003). Clearly, terrestrial\u2013aquatic linkages are important predictors of surface water chemistry for both streams and lakes, but the strength of this interaction should vary with geologic and hydrologic settings. Thus, the major difference between the River Continuum Concept (Vannote and others 1980) and the concept of lake landscape position (Kratz and others 1997) probably lies in large differences in water residence times between streams and lakes. Given the differences in water movement, in particular flushing rates, one might expect that streams and lakes differ in the external drivers that affect water chemistry. Surprisingly, our findings do not support this conjecture; the major part of the variation in water chemistry in both streams and lakes was explained by all components (i.e., geographic position as well as regional- and local-scale variables), followed by the combination (or interaction) of geographic position and regional-scale factors. These findings support the premise that variability in surface water chemistry is driven by interactions between geographic position and regional factors. Our finding, however, that regional factors alone accounted for a large part of variation in ANC, TP, and TOC indicates the pivotal role that catchment land use\/cover plays in determining surface water chemistry.\nThe finding that the surface water chemistry of streams and lakes could be partly predicted by regional-scale variables, in particular catchment land use (e.g., arable) agrees with the findings of several earlier studies (e.g., Schonter and Novotny 1993; Allan and others 1997). Johnson and others (1997) showed, for example, that urban land use and rowcrop agriculture were important factors in explaining variability in stream water chemistry. Similarly, Hunsaker and Levine (1995) were able to explain more than 40% of the variance in total nitrogen using landscape metrics. In our study, we were interested in analyzing \u201cnatural\u201d variability, so we removed sites judged to be affected by agriculture (i.e., sites with >25% of their catchments classified as arable were not included). Hence, our finding that the amount of arable land in a catchment explained nearly 70% of the variability in stream water chemistry was not expected using these data, and implies that even a small-scale agricultural land use within a catchment may affect phosphorus concentration. The importance of a riparian zone has been proposed to be less important in explaining among-site differences in heavily managed catchments (Omernik and others 1981). These authors suggested that the total amount of agriculture and forest in a catchment are more important predictors of water chemistry than the vegetation composition of the riparian zone. Our finding that less than 6% of the variation in surface water chemistry was explained by local factors alone (such as the presence of a riparian zone) supports this conclusion. Furthermore, in contrast to regional-scale factors, only a small amount of the variation was \u201chidden\u201d in joint effects or interaction terms between regional and local (<8%) and between geographic and local (<3%) variables. Hence, other factors not considered here, such as where the land use is located in the catchment and in relation to the water body, presumably need to be considered. Indeed, studies of small scale or local factors have been shown to be important in modifying larger scale effects, e.g., several studies have shown the ameliorative influence of a vegetated riparian zone (e.g., Cooper 1990; Osborne and Kovacic 1993).\nRedundancy analysis showed that the variability in both stream and lake water chemistry was explained by the similar regional- and local-scale variables. For example, as discussed above, the proportion of arable land use in the catchments was a strong predictor of stream water chemistry (68%), followed by alpine, treeless land cover (17.3%). For lake water chemistry, the amount of alpine, treeless land cover was a good predictor (55.4%), followed by altitude (18.5%) and catchment arable land use (4.6%). Clearly, several of the variables in different \u201clocal\u201d and \u201cregional\u201d components covary. For instance, the amount of alpine treeless land cover in the region\/catchment and stream width are presumably correlated with altitude. However, as demonstrated here, regional factors were better predictors of stream and lake water chemistry and thus contribute largely to the explanatory power of the covariation components.\nAll three water chemistry variables were strongly correlated with variables representing a latitudinal gradient; for example, sites in the south are more well buffered and nutrient rich compared to sites in the north. This distinct north\u2013south gradient in water chemistry was not unexpected, but can be easily explained by major landscape-level differences between the northern and southern parts of the country. For instance, the legacy of historical processes on present-day distribution patterns of vegetation is clearly visible in Sweden. At approximately 60\u00b0N latitude, a marked difference in vegetation occurs, and this ecotone (limes norrlandicus) basically delineates the transition of broad-leaved (e.g., English oak and elm) and coniferous mixed (e.g., Scots pine and spruce) forests in the south from the boreal pine and spruce forests in the north (Nordic Council of Ministers 1984). In addition, the limes norrlandicus ecotone is also correlated with the highest postglacial coastline or the highest level the sea reached after the last ice age and below which fluvial sediments have been deposited. Hence, these two landscape-scale discontinuities in vegetation and soil type can have profound importance for surface water chemistry. Finally, broad-scale climatic differences also exist between the northern and southern parts of the country, which are manifested in differences in discharge regimes. For example, streams in the south are dominated by autumn and winter rains, whereas streams in the north are dominated by snowmelt-driven peaks in runoff during spring (Anonymous 1979).\nGiven the profound differences in climate, geomorphology, and vegetation between the northern and southern parts of the country, we anticipated discernible differences in the factors driving surface water chemistry. Indeed, the finding that landscape position is important in explaining variability in surface water chemistry has been shown in previous studies (e.g., Johnson 1999), and supports the use of ecoregions to partition natural variability. Ecoregions have been suggested as appropriate ecological units for classification because they are generally perceived as being relatively homogeneous, having similar climate, geology, and other environmental characteristics (Wright and others 1998), and hence are considered as relatively good predictors of spatial patterns of surface water chemistry (e.g., Landers and others 1988; Larsen and others 1988). However, to be an appropriate classification tool, an ecoregion should minimize within and maximize among region variability, and, ideally, knowledge of how both natural and human-induced variability affect ecosystem processes should be known in order to fully assess the adequacy of ecoregions for partitioning natural variability. For example, it is well known that catchment management practices can have profound effects on surface water quality. For instance, whether a catchment is forested (promotes infiltration, high transpiration, and reduces runoff), clear-cut (results in lower infiltration and transpiration and increased runoff), or reforested may singly or in concert affect the water chemistry of aquatic ecosystems (Foster and others 2003).\nThe results of this study showed the importance of interactions between variables acting on multiple spatial scales on among-lake and stream water chemistry. Somewhat surprising was the finding that the major drivers were similar between lakes and streams, despite the obvious differences in ecosystem types. For instance, in streams nutrients are spiralling downstream, whereas in lakes, nutrient retention is relatively high, depending on lake size and morphometry. Obviously, the chemical composition of a surface water body is a product of a series of mechanisms and processes acting along a scale continuum, i.e., from broad (geographic) to small (local) scales. Moreover, the environmental characteristics of a specific habitat are not random, but are considered to be controlled by macro-scale geomorphic patterns (Frissell and others 1986). Building on this premise, a conceptual framework has been developed where the aquatic (stream) organism assemblage at a site can be seen as a product of a series of filters (e.g., from continental to microhabitat), with each species occurring at a site having passed through these filters (e.g., Tonn and others 1990; Poff 1997). Similarly, surface water chemistry of a particular site is also constrained to some extent by a number of environmental filters. Small-scale systems develop within the constraints set by broad-scale systems of which they are part, and likewise local-scale processes and conditions are generated by broad-scale, geographic patterns and conditions.\nThe idiosyncrasies of both ecosystems might be suppressed by the effects of large-scale factors. Geographic position functions as a template determining both regional and local factors. At the catchment level, geology controls soil type, weathering determines ion concentrations (and buffering capacity), and climate determines vegetation type (and land use). However, changes in land use (e.g., afforestation of arable to urban) and\/or vegetation cover (e.g., deforestation or afforestation of arable land) are sources of catchment variation that might generate high amounts of variability or noise, making it difficult to tease apart components of natural variation from the effects of anthropogenic impact on surface water chemistry. This inherent catchment variation is probably responsible for the large amount of variation explained by regional factors, which may hide the effects of individual features of lakes and streams appearing similar in their response to environmental factors and influences. However, another caveat in addressing issues of \u201cscale-effects\u201d is that the spatial resolution at which observations are made can confound interpretation of scale-related processes (e.g., Minshall 1988; Manel and others 2000). For example, environmental variables such as nutrient concentrations and hydrology are more influenced by regional-scale processes, whereas other variables such as in-stream vegetation cover are more influenced by local control mechanisms (e.g., Allan and others 1997). Our findings of the importance of interactions between geographic position and regional- and local-scale variables support this conclusion.","keyphrases":["anc","total phosphorus","toc","geographic position","spatial scale","variation partitioning","partial rda","lentic","lotic"],"prmu":["P","P","P","P","P","P","P","U","U"]}
{"id":"Knee_Surg_Sports_Traumatol_Arthrosc-4-1-2226194","title":"Does ligament balancing technique affect kinematics in rotating platform, PCL retaining knee arthroplasties?\n","text":"The goal of this prospective, randomized, blinded trial was to determine if ligament balancing techniques for rotating platform TKA affect postoperative knee kinematics. Sixteen patients with unilateral rotating platform TKA consented to participate in this institutional review board approved study. Eight patients were randomly selected to receive ligament balancing with an instrumented joint spreader device and eight patients received ligament balancing using fixed thickness spacer blocks. A single plane shape matching technique was used for kinematic analysis of static deep knee flexion and dynamic stair activities. There were no differences in knee kinematics between groups during static deep flexion activities. The spreader group demonstrated kinematics more similar to the normal knee during the ascending phase of the dynamic stair activity. Knee kinematics in static knee flexion were unaffected by ligament balancing technique, while knees balanced with the spreader demonstrated a medial pivot motion pattern during stair ascent. This medial pivot motion pattern may improve long-term results by more closely replicating normal knee kinematics.\nIntroduction\nRotating-platform total knee arthroplasty (TKA) has become increasingly popular because this type of design provides good tibiofemoral conformity and low contact stresses without imposing rotational constraint [28]. These designs have been used for well over 20\u00a0years with excellent survivorship [10]. Recent kinematic studies of rotating platform knee arthroplasties have shown excellent stability in extension, but frequent anterior translation of the femur with respect to the tibia in flexed postures [6]. These anterior femoral translations may reduce maximum weightbearing flexion [4] and implant longevity [9], and therefore merit further study.\nTibiofemoral translations are influenced by ligament balance [4, 16, 21, 30], muscle and external forces, and implant design. Ligament balance is thought to play a particularly important role in the function of rotating-platform knee arthroplasties, and numerous balancing techniques have been reported [8, 12, 13, 18]. However, no well designed clinical studies of ligament balance and knee kinematics have been reported.\nBy performing a prospective, randomized, blinded trial of two ligament balancing techniques for rotating platform TKA, we sought to determine if ligament balancing technique affected postoperative knee kinematics. We hypothesized that ligament balancing with a calibrated spreader\/balancer would provide better controlled knee kinematics, specifically reduced anterior femoral translations with flexion, than ligament balancing with fixed thickness spacer blocks.\nMaterials and methods\nSixteen patients with unilateral osteoarthritis of the knee and with no history of knee injuries or trauma consented to participate in this prospective, randomized, blinded, and institutional review board approved study. All subjects received the same rotating platform, PCL-retaining total knee prosthesis (TC-PLUS SB Solution, Plus Orthopedics AG, Rotkreuz, Switzerland, Fig.\u00a01). The subjects were randomly assigned to two groups preoperatively: eight knees received the prosthesis using a ligament balancing technique employing fixed thickness spacer blocks (control group), while the other eight knees received the same prosthesis employing a calibrated spreader\/balancer device to equalize the joint gaps and ligament balance in flexion and extension (spreader group) (Fig.\u00a02).\nFig.\u00a01All patients received a rotating platform total knee arthroplasty (TC-PLUS SB Solution, Plus Orthopedics, Rotkreuz, Switzerland)Fig.\u00a02One group of knees was treated using fixed thickness spacer blocks for ligament balancing (control group, left) and the other group was treated using a calibrated tensioning device (spreader group, right)\nAll surgeries were performed by the senior surgeon (FK) at South\u2013West London Elective Orthopaedic Centre, Epsom, United Kingdom. All study subjects were operated in the supine position under spinal anesthesia and sedation, and each was administered prophylactic antibiotic prior to inflation of the tourniquet. Standard extramedullary and intramedullary instrumentation were used in all knees for preparation of the tibia and femur, respectively. Standard sequential soft-tissue releasing techniques [22, 29] were utilized in the control and spreader groups, which included resection or release of (1) the anterior fibres of PCL, (2) medial and posteromedial capsule, (3) medial osteophytes, and (4) superficial MCL. In the control group spacer blocks were used in extension and 90 degree flexion to guide soft tissue releases to create balanced and equal flexion\u2013extension gaps. In the spreader group a balancer device (laminar spreader, Plus Orthopedics AG) (Fig.\u00a02) was used in extension and 90\u00a0degree flexion to guide soft tissue releases to create balanced and equal flexion\u2013extension gaps. Soft-tissue balance was assessed at 0 and 90\u00a0degrees of flexion with patella equally subluxed during measurements in both spreader and spacer block groups in order to accommodate appropriate measuring device into the joint space. A standard force of 20\u00a0N was applied to the medial and lateral jaws of the balancing device during this technique [23]. The posterior cruciate ligament (PCL) was retained in all knees with a bone block on the proximal tibia, recessing anterior fibers when necessary to achieve suitable balance.\nStudy subjects were assessed with pre-operative plain anteroposterior and lateral weightbearing radiographs of the knee and immediately postoperative non-weightbearing anteroposterior and lateral weightbearing radiographs. Patients were assessed post-operatively and over an average follow-up time of 11\u00a0\u00b1\u00a03\u00a0months (range: 7\u201315\u00a0months). The Knee Society Score [14] was employed as the scoring instrument. There were no differences between the control and spreader groups for height, weight, age, sex distribution, preoperative deformity or preoperative clinical scores (Table\u00a01).\nTable\u00a01Patient demographics and clinical assessments (mean\u00a0\u00b1\u00a01SD)ControlSpreaderP valueAge at operation (years)71.0\u00a0\u00b1\u00a08.472.2\u00a0\u00b1\u00a06.60.96Height (cm)167\u00a0\u00b1\u00a07.6165\u00a0\u00b1\u00a07.80.65Weight (kg)75.0\u00a0\u00b1\u00a022.670.3\u00a0\u00b1\u00a012.90.72Sex (M\/F)3\/54\/41.0aVarus\/valgus distribution8\/07\/11.0aPre-op knee score42.1\u00a0\u00b1\u00a010.350.0\u00a0\u00b1\u00a011.70.13Pre-op function score50.0\u00a0\u00b1\u00a018.955.0\u00a0\u00b1\u00a020.40.50Post-op knee score90.5\u00a0\u00b1\u00a05.993.5\u00a0\u00b1\u00a01.80.51Post-op function score81.3\u00a0\u00b1\u00a023.488.1\u00a0\u00b1\u00a015.10.72Follow-up (months)10.3\u00a0\u00b1\u00a03.111.3\u00a0\u00b1\u00a02.30.44aFisher\u2019s exact test\nFollow-up consisted of clinical and fluoroscopic assessment performed at Mayday University hospital, Croydon, United Kingdom. Fluoroscopic imaging (Siemens Polystar TOP, Siemens AG, Munich, Germany) consisted of (1) weightbearing maximum flexion lunge activity, (2) kneeling on a padded bench to maximum comfortable flexion and (3) a 4 cycles of a step-up\/down on a 25\u00a0cm step. For the stair activity, the subjects faced the same direction throughout the cycle, therefore, the step-down was a backward motion that reversed the step ascent motion. Patients were instructed on the study activities prior to recording, and were given an opportunity to practice until comfortable. Lateral fluoroscopic views of the knee were recorded in the maximally flexed positions for the lunge and kneeling activities, as were four repeat trials of step-up\/down on the stair. The fluoroscopic images were recorded at 15 frames per second onto an S-VHS VCR. Views of calibration targets also were acquired for distortion correction and optical calibration.\nThe three-dimensional (3D) positions and orientations of the implant components were determined using model based shape matching techniques [3, 5], including previously reported techniques, manual matching, and image space optimization routines (Fig.\u00a03). The fluoroscopic images were digitized and corrected for static optical distortion. The optical geometry of the fluoroscopy system (principal distance, principal point) was determined from images of calibration targets [3, 5]. The implant surface model was projected onto the geometry corrected image, and its 3D pose was iteratively adjusted to match its silhouette with the silhouette of the subject\u2019s TKA components. The results of this shape matching process have standard errors of approximately 0.5\u00b0 to 1.0\u00b0 for rotations and 0.5\u20131.0\u00a0mm for translations in the sagittal plane [3, 5].\nFig.\u00a03Model based shape matching techniques are used to determine the three-dimensional pose of the arthroplasty components from fluoroscopic images. The fluoroscopic image shows the outlines, in red, of the implant surface models superimposed in their registered positions. The images along the right margin show medial, lateral, coronal and transverse views of the implant components\u2019 relative orientations\nThe relative motions of the femoral and tibial components were determined from the 3D pose of each TKA component using the convention of Tupling and Pierrynowski [26]. The locations of condylar contact were estimated as the lowest point on each femoral condyle relative to the transverse plane of the tibial baseplate. Anteroposterior translations of the condyles were computed with respect to the anteroposterior midpoint of the tibial baseplate. Motion of the mobile bearing was not analyzed since the mobile bearing insert was not visible in the X-ray images and could not be tracked without addition of metallic markers.\nResearchers were unblinded to subject group membership only after all kinematic data had been produced. Statistical comparisons of the fluoroscopic images were performed (SPSS ver 13, SPSS Inc., Chicago, US) using two-way repeated measures ANOVA with post hoc pair-wise comparisons (Tukey\/Kramer) at a 0.05 level of significance. All other parameters were evaluated using non-parametric tests.\nResults\nBoth the Knee Score and the Function Score were slightly lower for the control group (Table\u00a01). This tendency existed pre-operatively but was not statistically significant.\nFor the maximum kneeling activity, no significant differences were found in knee angles or translations (Table\u00a02). Maximum implant flexion for the control and spreader groups averaged 102\u00b0\u00a0\u00b1\u00a013\u00b0 and 108\u00b0\u00a0\u00b1\u00a010\u00b0 (P\u00a0=\u00a00.34), respectively. Tibial component valgus for the control and spreader groups averaged 0\u00b0\u00a0\u00b1\u00a02\u00b0 and \u22121\u00b0\u00a0\u00b1\u00a02\u00b0 (P\u00a0=\u00a00.56), respectively. Tibial external rotation for the control and spreader groups averaged \u22125\u00b0\u00a0\u00b1\u00a07\u00b0 and \u22125\u00b0\u00a0\u00b1\u00a06\u00b0 (P\u00a0=\u00a00.87), respectively. Medial tibial contact was located 2.7\u00a0\u00b1\u00a012.2 and 1.8\u00a0\u00b1\u00a08.2\u00a0mm (P\u00a0=\u00a00.87) posterior to the midline of the tibial plateau for the control and spreader groups, respectively. Lateral tibial contact was located at 10.5\u00a0\u00b1\u00a011.4 and 11.1\u00a0\u00b1\u00a011.8\u00a0mm (P\u00a0=\u00a00.93) posterior to the midline of the tibial plateau for the control and spreader groups, respectively.\nTable\u00a02Knee pose during maximum flexion kneeling (mean\u00a0\u00b1\u00a01SD)GroupFlexion (\u00b0)Valgus (\u00b0)Tibial Ext. Rot. (\u00b0)Medial AP (mm)Lateral AP (mm)Control102.0\u00a0\u00b1\u00a012.80.1\u00a0\u00b1\u00a02.1\u22124.7\u00a0\u00b1\u00a07.4\u22122.7\u00a0\u00b1\u00a012.2\u221210.5\u00a0\u00b1\u00a011.4Spreader107.9\u00a0\u00b1\u00a010.1\u22120.5\u00a0\u00b1\u00a01.8\u22125.3\u00a0\u00b1\u00a06.3\u22121.8\u00a0\u00b1\u00a08.2\u221211.1\u00a0\u00b1\u00a011.8P value0.34\u00a00.56\u00a00.87\u00a00.87\u00a00.93\u00a0\nFor the maximum lunge activity, no significant differences were found in knee angles or translations (Table\u00a03). Knee flexion for the control and spreader groups averaged 95\u00b0\u00a0\u00b1\u00a015\u00b0 and 102\u00b0\u00a0\u00b1\u00a011\u00b0 (P\u00a0=\u00a00.36), respectively. Tibial component valgus for the control and spreader groups averaged 0\u00b0\u00a0\u00b1\u00a01\u00b0 and \u22121\u00b0\u00a0\u00b1\u00a02\u00b0 (P\u00a0=\u00a00.62), respectively. Tibial external rotation for the control and spreader groups averaged \u22129\u00b0\u00a0\u00b1\u00a06\u00b0 and \u22126\u00b0\u00a0\u00b1\u00a07\u00b0 (P\u00a0=\u00a00.29), respectively. Medial tibial contact was located 0.3\u00a0\u00b1\u00a08.2\u00a0mm and 6.7\u00a0\u00b1\u00a07.7\u00a0mm (p\u00a0=\u00a00.1.5) posterior to the midline of the tibial plateau for the control and spreader groups, respectively. Lateral tibial contact was located 16.5\u00a0\u00b1\u00a08.7\u00a0mm and 16.8\u00a0\u00b1\u00a09.9\u00a0mm (p\u00a0=\u00a00.96) posterior to the midline of the tibial plateau for the control and spreader groups, respectively.\nTable\u00a03Knee pose during maximum flexion lunge (mean\u00a0\u00b1\u00a01SD)GroupFlexion (\u00b0)Valgus (\u00b0)Tibial Ext. Rot. (\u00b0)Medial AP (mm)Lateral AP (mm)Control95.3\u00a0\u00b1\u00a015.1\u22120.1\u00a0\u00b1\u00a01.4\u22129.6\u00a0\u00b1\u00a05.9\u22120.3\u00a0\u00b1\u00a08.2\u221216.5\u00a0\u00b1\u00a08.7Spreader101.6\u00a0\u00b1\u00a010.6\u22120.6\u00a0\u00b1\u00a02.1\u22125.7\u00a0\u00b1\u00a07.3\u22126.7\u00a0\u00b1\u00a07.7\u221216.8\u00a0\u00b1\u00a09.9P value0.360.620.290.150.96\nFor the stair activity, knees in the spreader group exhibited more posterior medial (P\u00a0=\u00a00.04, RM-ANOVA) and lateral (P\u00a0<\u00a00.005, RM-ANOVA) condylar contact than the control knees. There was no difference in average tibial rotation between the two groups, and no pair-wise comparisons at specific flexion ranges resulted in significant differences (Fig.\u00a04). On average, both groups of knees had approximately 2\u00b0 tibial internal rotation at 0\u00b0 flexion, and rotated to 7\u00b0 tibial internal rotation at 80\u00b0 flexion. Medial contact was observed to remain at approximately 2\u00a0mm posterior to the AP midpoint from 0\u00b0 to 50\u00b0 flexion, then moved anterior to 80\u00b0 flexion. The control group showed greater anterior translation of medial contact from 50\u00b0 to 80\u00b0 flexion than did the spreader group. Lateral contact was more posterior in the spreader group throughout the stair activity. Both groups showed posterior translation of lateral contact of 2\u20133\u00a0mm from 0\u00b0 to 30\u00b0 flexion, with very little net translation from 30\u00b0 to 80\u00b0 flexion.\nFig.\u00a04Knee motions during the stair activity differed between the control and spreader groups. Condylar positions were significantly more posterior in the spreader group. There were no significant differences in tibial rotation, nor were there significant pair-wise differences for rotations or translations\nTibiofemoral kinematics during the step activity also were compared using average centers of rotation (COR) for femoral motion with respect to the tibial base-plate (Fig.\u00a05) [6]. The COR provides a concise measure of femoral AP translation\u2014if the COR is central (close to 0%), the femur rotates about the center of the tibia with little AP translation. A medial COR (between 0% and +50%) indicates the femur translates posterior with external rotation during flexion. A lateral COR (between \u221250 and 0%) indicates the femur translates anterior with external rotation during flexion. For the entire step-up\/down cycle, the centers of rotation were at 0% (central) and 13% (medial) (P\u00a0=\u00a00.058) for the control and spreader groups, respectively. When step-up kinematics were compared, the spreader group showed a COR located more medially (28%) than the control group (0%, P\u00a0<\u00a00.05, Table\u00a04). There was no difference in COR for step-down kinematics (Table\u00a04). Both groups of knees showed tibial internal rotation with knee flexion, 8.5\u00b0 and 7.6\u00b0 for the control and spreader groups, respectively. These differences were not statistically significant.\nFig.\u00a05Average centers of rotation for the entire stair activity were in the center of the tibial plateau (0%) for the control group (left) and to the medial side (13%) for the spreader group (right). This difference was not significant (P\u00a0=\u00a00.058)Table\u00a04Medial\/lateral center of rotation (COR) during the stair activity (mean\u00a0\u00b1\u00a01SD)GroupExtension phase (%)Flexion phase (%)Control\u22123\u00a0\u00b1\u00a019\u22124\u00a0\u00b1\u00a02Spreader28\u00a0\u00b1\u00a041\u22123\u00a0\u00b1\u00a03P value<0.05>0.05\nNone of the subjects demonstrated valgus or varus angles larger than 2 degrees during motion, consequently there was no obvious evidence of condylar lift-off.\nDiscussion\nOne goal of TKA is to reproduce normal knee kinematics. Ligament and soft-tissue balance are thought to play critical roles in obtaining optimal kinematic behavior. Theoretical merits of many balancing techniques and instruments have been discussed [13, 25, 27, 30]. This prospective, randomized and blinded study evaluated two ligament balancing techniques with posterior cruciate retaining rotating platform total knee arthroplasty to determine if balancing technique affected knee kinematics. Randomizing patients for surgical treatment and blinding the investigators to group membership until after all data had been processed reduced the potential for selection, measurement, and interpretation bias to affect the study findings. All subjects demonstrated satisfactory knee function based on clinical scores, there were no clinical complications in any knee, and no evidence of condylar lift-off was found during dynamic activity.\nThe two knee groups exhibited no significant differences in knee kinematics for the weightbearing lunge and passive kneeling activities. This similarity is not unexpected given the posterior cruciate was retained in all knees. It is interesting to note that tibiofemoral AP position in these knees appears to differ from previous reports with mobile bearing knee arthroplasties. Banks et al. [4] reported lunge kinematics for a mixed group of rotating platform and rotating-and-translating arthroplasties during the same lunge activity, and observed 102\u00b0 average flexion, 7.7\u00b0 average tibial internal rotation, and 2.2\u00a0mm posterior femoral position with respect to the tibial AP midpoint. The control and spreader groups exhibited approximately the same knee angles, but 8.4 and 11.8\u00a0mm posterior femoral position, respectively. Greater posterior femoral translation with flexion is suggestive of more physiologic posterior cruciate ligament function and knee mechanics, although there is insufficient information to attribute those translations specifically to surgical technique, implant design, or a combination of factors. The amount of tibial rotation observed in the flexed postures is similar to other previous reports for knee arthroplasties [6, 15] but is much smaller than the amount of tibial rotation observed in healthy knees in similar postures [1, 20].\nLigament balancing technique did affect knee kinematics during the dynamic stair activity. Condylar contact locations were observed to remain more posterior on the tibia and to have a more medial center of rotation during step-up in the spreader group. These findings suggest the spreader balancing technique provided more normal balance or stability to the medial compartment of the knee, resulting in less medial contact translation during the stair activity. Medial contact in the control group was observed to move anterior with flexion on the stair activity, indicating greater functional laxity in that compartment.\nSimple comparisons of knee kinematics across groups are possible using the center of rotation characterization. A medial center of rotation has been described in the healthy normal knee [2, 17]. The spreader group showed a medial center of rotation during stair ascent, indicating medial contact did not move significantly while lateral contact moved anterior with knee extension and femoral internal rotation. The control group showed a center of rotation close to the middle of the tibia for the stair activity, indicating that the femur rotated internally during knee extension with little AP translation (medial contact moved posterior and lateral contact moved anterior with extension). The center of rotation in the spreader group knees differed between the ascending (medial COR) and descending (central COR) phases of the step-up\/down activity (Table\u00a04). This suggests that the spreader balancing technique provided greater anterior medial stability than the technique employing fixed thickness spacer blocks, but posterior medial stability was equivalent between the two balancing techniques. Banks and Hodge [7] reported on a mixed group of 44 rotating platform and rotating-and-translating mobile bearing knee arthroplasties during the same stair activity, and found average tibial rotations of 9\u00b0 and average centers of rotation at \u221219% (lateral). These motions were associated with anterior femoral translation with flexion, which has been observed in numerous knee arthroplasty designs [11, 19, 24]. The knees in the present study showed similar amounts of tibial rotation, but both groups showed centers of rotation that were more medially located. Thus, the knees in this study exhibited less anterior femoral translation with flexion compared to the knees in the previous report, suggesting both balancing techniques provided beneficial tibiofemoral stability compared to the group average of well-functioning mobile-bearing knee arthroplasties.\nThis double-blinded prospective randomized study used fluoroscopic kinematic measurements to determine if two ligament balancing techniques would affect knee motions in several activities. Kinematics in flexion were similar, with both groups showing a more posterior femoral position than previously has been reported for similar implant designs. Knees operated with a spreader\/balancer device showed a more medial center of rotation in ascending a stair activity, and both groups showed average centers of rotation that were more medial than previously had been reported for similar implant designs. Kinematics closer to the normal knee may yield improved knee performance and implant longevity. However, these kinematic differences are clinically insignificant upon short-term follow up, and their long-term significance remains to be studied.","keyphrases":["rotating platform","tka","knee kinematics","spreader balancing device","soft tissue balancing","randomized control trial"],"prmu":["P","P","P","R","R","R"]}
{"id":"Dig_Dis_Sci-3-1-2140097","title":"Twenty-Four Hour Tonometry in Patients Suspected of Chronic Gastrointestinal Ischemia\n","text":"Background and aims Gastrointestinal tonometry is currently the only clinical diagnostic test that enables identification of symptomatic chronic gastrointestinal ischemia. Gastric exercise tonometry has proven its value for detection of ischemia in this patients group, but has its disadvantages. Earlier studies with postprandial tonometry gave unreliable results. In this study we challenged (again) the use of postprandial tonometry in patients suspected of gastrointestinal ischemia.\nIntroduction\nIn patients presenting with postprandial pain, especially when associated with weight loss and positive history for cardiovascular diseases, chronic gastrointestinal ischemia (CGI) should be among the differential diagnosis [1\u20133]. Vascular anatomic abnormalities can be demonstrated with duplex ultrasound or angiography. However, a stenosis does not necessarily imply ischemia, due to the abundant collateral circulation. We have recently demonstrated that gastric exercise tonometry (GET) allows differentiation between patients with and without gastrointestinal ischemia. GET showed an accuracy of 87% in detection of gastrointestinal ischemia. The patients selected for treatment using GET, are likely to benefit from revascularization techniques [4\u20139].\nGastric (exercise) tonometry is not widely accepted as a diagnostic technique for gastrointestinal ischemia due to the lack of familiarity with this approach and its time-consuming nature. Twenty-four hour gastrointestinal tonometry with meals is more familiar (resembling 24-h pH measurement) and easier to perform. Over 90% of CGI patients report postprandial, while only 60% report exercise-related, complaints. Ischemic pain after meals in CGI is caused by insufficient increase of postprandial blood flow to balance the increased metabolic demand of the gastrointestinal tract [10]. This indicates that tonometry directly after meals would be the most physiologic approach of measuring ischemia in these patients. However, earlier studies showed unreliable results using tonometry after meals, related to insufficient suppression of gastric acid secretion and dilution effects [11\u201313]. We therefore started by testing standard meals in vitro and performed a study with healthy subjects using these meals and high-dose proton pump inhibition (PPI) as an optimal gastric acid suppressant [14]. In this study we retrospectively evaluated the additional value of prolonged gastrointestinal tonometry in a group of patients suspected for possible CGI.\nMethods\nPatients with unexplained chronic abdominal symptoms who were referred for suspected CGI were included in this study. More-common causes of chronic abdominal symptoms had been excluded previously by appropriate diagnostic evaluation. All patients had imaging of the splanchnic arteries [intra-arterial digital substraction multiplane abdominal angiography (DSA) and duplex ultrasound scanning] and GET. Along with this standard diagnostic work up, patients had twenty-four hour (24\u00a0h) tonometry testing, directly following GET.\nGastric exercise tonometry (GET)\nGET was performed using a standardized protocol, before, during, and after 10\u00a0min of submaximal exercise, as described previously with both gastric and jejunal catheters [15]. A maximal gradient was calculated between stomach and arterial PCO2. The criteria for a positive GET (all three required), established in healthy volunteers and a patient cohort, were: (1) a gradient of >0.8\u00a0kPa in the stomach after exercise, (2) an increase in gastric PCO2, and (3) an arterial lactate <8\u00a0mmol\/l [8,15].\nTwenty-four hour tonometry testing\nA gastric and jejunal tonometer catheter (8 French, Datex Ohmeda, Helsinki, Finland) and a gastric pH meter (pHersaflexTM, internal reference, Medical Measurement Systems, Enschede, the Netherlands) were inserted nasogastrically using fluoroscopy. Intravenous infusion of omeprazole was started with a bolus of 80\u00a0mg in 30\u00a0min, followed by 8\u00a0mg\/h, using an infusion pump (Perfusor compact\u00ae, B Braun Melsungen AG, Melsungen, Germany). The catheters were connected, respectively, to the Tonocap (Datex Ohmeda, Helsinki, Finland) and the pH recording device (Medical Measurement Systems, Enschede, the Netherlands). The Tonocaps were connected to a computer on which a data-collection program automatically registered the gastric and jejunal PCO2 level every 10\u00a0min. The gastric pH was automatically recorded and stored in a datalogger (Medical Measurement Systems, Enschede, the Netherlands), which also allows for real-time reading of the gastric pH.\nAs soon as the gastric pH was >4.0 for \u226530\u00a0min, the first meal was started (t\u00a0=\u00a00\u00a0min). All patients had meals at standard times: breakfast I (08:00), dinner (12:00), liquid compound meal I (15:00), bread meal (18:00), liquid compound meal II (21:00), and breakfast II (08:00 the next day). The breakfast, bread, and dinner meals were standardized. The liquid compound meal consisted of two packages of 200\u00a0ml each (Nutridrink\u00ae, Nutricia, The Netherlands). The contents and caloric density of each meal used are presented in Table\u00a01. The patients were instructed to eat their meals within 15\u00a0min. The consumption of small amounts of liquids (noncarbonated) was allowed and noted, consumption of alcohol-, acid-, and CO2-containing beverages was strictly prohibited. Due to the limited length of the catheters, the subjects were only capable of performing very minor exercise and were allowed to lie down in supine position from 22:00.\nTable\u00a01Composition characteristics of the various standard mealsMealCompositionkcal\/gBreakfast Fat (16%), proteins (22%), carbohydrates (62%)1.7DinnerFat (16%), proteins (47%), carbohydrates (37%)2.2Bread mealFat (10%), proteins (19%), carbohydrates (71%)1.8Compound solutionFat (35%), proteins (16%), carbohydrates (49%)1.5Percentages of delivered energy (En%); kcal\u00a0=\u00a0kilocalories; g\u00a0=\u00a0gram.\nDiagnosis and treatment\nThe results of all diagnostic procedures were discussed in a multidisciplinary team. In this team a gastroenterologist, a vascular surgeon, and an interventional radiologist discussed the symptoms, medical history, physical examination, and all diagnostic evaluations, with the exception of the results on 24-h tonometry. The latter therefore did not influence the consensus diagnosis. The multidisciplinary team decided for every patient: (1) no splanchnic stenosis, (2) splanchnic stenosis and no ischemia or (3) splanchnic stenosis and ischemia (i.e. chronic gastrointestinal ischemia, CGI). The gold standard for the diagnosis of chronic gastrointestinal ischemia was a positive outcome after successful revascularization at (long-term) follow-up. The outcome of GET, and consensus diagnosis of the multidisciplinary team were compared to the results of the 24-h tonometry testing.\nDefinition of a positive (abnormal) 24\u00a0h tonometry\nThe cut-off values established in the previous healthy subjects study were used to define the criteria for the results on 24-h tonometry [9]. These cut-off values were, for the stomach: 12.1, 11.4, and 11.3\u00a0kPa for the breakfast (or bread meal), dinner, and compound solution meals, respectively; in the jejunum these threshold values were, respectively, 12.0, 13.6, and 10.6\u00a0kPa. The criteria for a positive finding (abnormal result) on 24-h tonometry were: (1) pathologic responses after three or more (standard) meals, or (2) a combination of one or two pathologic responses after (standard) meals combined with a median PCO2 > 8.0\u00a0kPa, measured in between meals.\nStatistics\nData were expressed as mean (standard deviation) or median (range) when appropriate. The data of the ischemic and non-ischemic patients were compared using Student\u2019s t-test or \u03c72 testing. Sensitivity, specificity as well as positive and negative predicted values of 24-h tonometry were calculated with the consensus diagnosis as the gold standard.\nResults\nPatient characteristics\nIn a period of three\u00a0years (2002\u20132005), in 37 patients referred for suspected of CGI, 24-h tonometry along with the standard work-up was performed. Of these, 33 (89%) patients had complete work-up and were included in this study. Mean age was 54 (22\u201382) years, with eight males and 25 females. Significant splanchnic stenosis were found in 23\/33 (69%) patients. A significant single vessel splanchnic stenosis was found in 14\/33 (42%) patients [13 celiac artery (CA) and one superior mesenteric artery (SMA)]. A significant stenosis of two splanchnic arteries was found in 9\/33 (27%) patients (all CA and SMA stenosis).\nAll 33 patients had chronic abdominal pain for a mean of 35\u00a0months (range 3\u2013120), 24\/33 (73%) patients had pain following meals, 11\/33 (33%) patients reported pain during or after exercise, 9\/33 (27%) patients reported both pain following meals and during, or after, exercise, and 23\/33 (70%) patients reported weight loss. The mean weight loss was 11\u00a0kg (range 3\u201328\u00a0kg) in 17\u00a0months (range 2\u2013120\u00a0months); see Table\u00a01.\nGastric exercise tonometry\nIn 18\/31 (58%) patients a gradient of >0.8 was found by GET. In 14\/18 (78%) patients this increased gradient was defined as abnormal using the three criteria as previously defined. In four patients a gradient >0.8\u00a0kPa was not caused by ischemia: three patients had persistent acid production, and one performed excessive exercise (leading to false positive findings) [8].\nConsensus diagnosis of the multidisciplinary team and results after treatment\nAccording to the team the diagnosis of no stenosis (and no ischemia), stenosis but no ischemia, and stenosis with ischemia (CGI) was diagnosed in, respectively, 12 (36%), four (12%) and 17 (52%) patients.\nFifteen patients diagnosed with CGI had treatment: 10 patients surgical and five patients stent-placement therapy. Three patients had no treatment: two patients preferred a conservative approach and one patient proved inoperable due to comorbidity. After a mean follow-up of 55\u00a0months (49\u201385), 12 out of 15 (80%) patients were free of complaints, one patient died immediately after surgical revascularization (multiple organ failure), one patient had partial improvement, and one patient had persistent complaints. The latter patient had a celiac artery release, and was diagnosed as having no CGI after follow-up; this patient had no abnormalities on 24-h tonometry testing; see Table\u00a02.\nTable\u00a02Patient characteristics, results of diagnostic tests and conclusionNrAgeSexStenosisPP painPE painGET resultsConsensus diagnosisTreatmentOutcome complaintsFinal conclusion24-h tono results161FNone+\u22120,5No stenosis, no ischemia\u2013No stenosis, no ischemiaNormal236MCA+\u22120,4No ischemia\u2013No ischemiaNormal355FCA+\u22120,7aCGISurgeryFreeCGIAbnormal476MNone\u2212\u22120,6No stenosis, no ischemia\u2013No stenosis, no ischemiaNormal547MNone\u2212+1,6bNo stenosis, no ischemia\u2013No stenosis, no ischemiaNormal642FCA\u00a0+\u00a0SMA\u2212\u22121,5CGISurgeryFreeCGIAbnormal765FCA\u00a0+\u00a0SMA\u2212\u22122,0CGIConservativeCGIAbnormal877FCA\u00a0+\u00a0SMA+\u22122,2CGISurgeryDied post-op.CGIAbnormal972FSMA++1,8CGIStentFreeCGIAbnormal1041MNone+\u22122,8cNo stenosis, no ischemia\u2013No stenosis, no ischemiaNormal1172FCA+\u22121,4CGIStentPartial relieveCGIAbnormal1267FCA\u2212\u22120,9cNo stenosis, no ischemia\u2013No ischemiaNormal1340FCA\u2212+1,1cCGISurgeryUnchangedNo ischemiaNormal1482MCA\u00a0+\u00a0SMA++1,0CGIConservativeCGINormal1554FCA++0,9CGIConservativeCGINormal1626MCA++1,0CGISurgeryFreeCGIAbnormal1758MNone++2,2cNo stenosis, no ischemia\u2013No stenosis, no ischemiaNormal1822FCA+\u22120,7No ischemia\u2013No ischemiaNormal1942FNone+\u22121,1dNo stenosis, no ischemia\u2013No stenosis, no ischemiaNormal2048FCA+\u22121,7CGISurgeryFreeCGINormal2151FCA+\u22120,7No ischemia\u2013No ischemiaNormal2243FCA\u00a0+\u00a0SMA++0,5eCGISurgeryFreeCGIAbnormal2354FCA\u00a0+\u00a0SMA\u2212\u22121,5CGISurgeryFreeCGINormal2476FCA\u00a0+\u00a0SMA+\u22120,6fCGIStentFreeCGIAbnormal2553MNone+\u22122,0cNo stenosis, no ischemia\u2013No stenosis, no ischemiaNormal2653FCA++1,1CGIStentFreeCGIAbnormal2750FCA\u2212\u22121,3CGISurgeryFreeCGIAbnormal2861FCA\u00a0+\u00a0SMA++1,7CGIStentFreeCGIAbnormal2963FNone+\u22120,8No stenosis, no ischemia\u2013No stenosis, no ischemiaNormal3024FCA++1,3CGISurgeryFreeCGIAbnormal3174FCA\u00a0+\u00a0SMA\u2212\u22120,8No ischemia\u2013No ischemiaNormal3241FNone+\u22121,2dNo stenosis, no ischemia\u2013No stenosis, no ischemiaNormal3363FNone+\u22120,0No stenosis, no ischemia\u2013No stenosis, no ischemiaAbnormalPP\u00a0=\u00a0postprandial, PE\u00a0=\u00a0post-exercise, M\u00a0=\u00a0male, F\u00a0=\u00a0female; CA\u00a0=\u00a0celiac artery, SMA\u00a0=\u00a0superior mesenteric artery; GET\u00a0=\u00a0gastric exercise tonometry, result presented as gradient (in kPa); 24-h tono\u00a0=\u00a0twenty-four hour tonometry; CGI\u00a0=\u00a0chronic gastrointestinal ischemia.a\u00a0false negative GETb\u00a0acid production during GETc\u00a0false positive GETd\u00a0no CO2 raise during GETe\u00a0abnormal jejunal gradient during GETf\u00a0minor exercise during GET\nTwenty-four hour tonometry\nThe 24-h tonometry was well tolerated in all patients, no medical or technical problems occurred. In 28\/33 (85%) patients tonometric measurements were performed in both stomach and jejunum, in 5\/33 (15%) patients only stomach measurements were performed (in all five patients placement of jejunal tonometry catheter failed); see Fig.\u00a01. In 8\/33 (24%) patients a dose reduction of the compound solution was necessary, due to patient\u2019s inability to consume the normal dosage. The overall gastric acid suppression was good, with a gastric pH > 4 during 94.8% (range 71\u2013100%) of the time. Pathological peaks during 24-h tonometry coexisting with periods of pH\u00a0<\u00a04, were defined as non-pathologic peaks.\nFig.\u00a01\u00a0Individual curves of results of 24-h tonometry in a non-ischemic (A) and an ischemic patient (B). Individual curves of a non-ischemic patient (A) and an chronic gastrointestinal ischemia patient (B); on the horizontal axis the time from 0 to 24\u00a0h, on the vertical axis PCO2 from 0 to 20 in kilopascal (kPa); curves: PCO2 values measured every 10\u00a0min in stomach\n(\u25a1), jejunum\n(\u00d7) and four meals spread over the 24\u00a0h period (*)\nThe fasting baseline of stomach and jejunal PCO2 measurements were significantly higher in the ischemic patients compared to the non-ischemic patients group. The jejunal PCO2 peaks after breakfast and dinner were significantly higher in the ischemic patients compared to the non-ischemic patients; see Tables\u00a03 and\u00a04.\nTable\u00a03Results of 24-h tonometry in ischemic and non-ischemic patientsCGI pts.Non-ischemic pts.Peak\u0394-peakMean PCO2Peak\u0394-peakMean PCO2StomachB10.6 (3.9)4.0 (3.4)\u20138.5 (2.7)2.6 (2.0)\u2013D9.9 (1.9)3.7 (1.5)\u20138.5 (2.3)3.3 (2.5)\u2013CS10.4 (3.0)3.3 (2.2)\u20138.1 (2.6)1.8 (1.2)\u2013Fasting\u2013\u20137.7 (1.4)a\u2013\u20136.8 (0.7)\u00a0\u00a0\u00a0\u00a0Day\u2013\u20136.9 (1.1)\u2013\u20136.5 (0.7)\u00a0\u00a0\u00a0\u00a0Night\u2013\u20138.2 (1.8)\u2013\u20136.9 (0.8)JejunumB11.6 (3.2)b3.2 (1.5)\u20138.8 (1.4)2.1 (0.8)\u2013D12.2 (3.4)c3.7 (2.0)\u20139.0 (1.7)2.2 (0.7)\u2013CS10.6 (2.2)2.5 (1.6)\u20139.0 (1.9)1.5 (1.0)\u2013Fasting\u2013\u20138.9 (1.6)d\u2013\u20137.4 (0.7)\u00a0\u00a0\u00a0\u00a0Day\u2013\u20138.8 (1.3)\u2013\u20137.5 (0.9)\u00a0\u00a0\u00a0\u00a0Night\u2013\u20138.9 (1.9)\u2013\u20137.5 (0.8)CGI\u00a0=\u00a0chronic gastrointestinal ischemia; PCO2\u00a0=\u00a0carbon dioxide in kilopascal; B\u00a0=\u00a0breakfast, D\u00a0=\u00a0dinner, CS\u00a0=\u00a0compound solution meal; a\u00a0P\u00a0=\u00a00.02, b\u00a0P\u00a0=\u00a00.005, c\u00a0P\u00a0=\u00a00.04, d\u00a0P\u00a0=\u00a00.03Table\u00a04Results of different tests compared to final diagnosisPatientsFinal diagnosisGET24-h tonometryCombination GET\u2013\u201324-h tonometryCGI 1714 (82%)13 (76%)17 (100%)No ischemia1611 (69%)15 (94%)16 (100%)Data presented as number of patients with positive predictive value (PPV) and negative predictive value (NPV); GET\u00a0=\u00a0gastric exercise tonometry; CGI\u00a0=\u00a0chronic gastrointestinal ischemia; GET\u00a0=\u00a0gastric exercise tonometry\nUsing the previously defined criteria, 13\/17 patients with CGI and 15\/16 patients without ischemia were correctly identified with 24-h tonometry. The calculated test properties show a sensitivity of 76% and a specificity of 94%, a positive predictive value (PPV) of 76% and a negative predictive value (NPV) of 94% for detection of ischemia by 24-h tonometry alone. Combining the results of GET and 24-h tonometry, 17\/17 patients with CGI and 16\/16 patients without ischemia could be correctly identified (sensitivity of 100% and specificity of 100%).\nComparing patients with single- and multi-vessel ischemia, or patients with or without postprandial and\/or exercise-related complaints, no significant differences in diagnostic accuracy were found.\nDiscussion\nThe results of this retrospective study indicate that 24-h gastrojejunal tonometry is feasible and may be clinically useful in diagnosing chronic gastrointestinal ischemia. The measurements were easy to perform, generally well tolerated and no complications occurred.\nThe fasting baseline PCO2 in both stomach and jejunum was significantly higher in the ischemic patients group compared to the non-ischemic patients. This difference might be explained by the continuous compromised arterial blood flow of the mucosa of the stomach (and\/or jejunum) in the ischemic patients group. The significant higher maximum peak after the breakfast and dinner and the borderline significant higher peak after the compound solution meal (P\u00a0=\u00a00.07 and 0.052 for stomach and jejunum, respectively) support the theory that, with a maximal metabolic oxygen demand, mucosal ischemia is apparent and detectable using tonometry.\nUsing the cut-off values predicted by the previously performed healthy subjects study, the positive predictive value of 24-h tonometry seems very promising [14]. Comparing the results of detection of ischemia for 24-h tonometry and GET, no significant differences were found. Theoretically it might be expected that 24-h tonometry is more accurate in patients with postprandial complaints, and GET in patients with exercise-predominant complaints, but this was not found in this small patient series. In five patients the results of GET were incorrect (four false-positive and one false-negative result), whereas the 24-h tonometry (retrospectively) correctly predicted the presence (one patient) or absence (four patients) of gastrointestinal ischemia in these patients.\nOne of the major advantages of 24-h tonometry over GET is the fact that all patients are suitable for 24-h tonometry, in contrast to GET, where patients have to perform submaximal exercise, which is not always possible due to age, concomitant disease, and\/or compromised general condition of the patient. Another advantage of the 24-h tonometry is the familiarity with 24-h pH measurement, which is a widely accepted diagnostic tool in gastroenterology. Moreover 24-h tonometry testing is more easily standardized than GET, especially regarding the level of exercise, which is difficult to manage and may cause false positive findings during or after excessive exercise [15]. In this study, the 24\u00a0h tonometry was not repeated in a standard fashion after treatment. In two patients who had successful, anatomical and clinical, revascularization 24-h tonometry was repeated, and showed normalization in both patients.\nIn theory, the compound solution meal (low volume, high calorie content) should be the ideal test meal for provocation of gastrointestinal ischemia. The use of standard meals with large metabolic demand, like the compound solution meal, called for a dose reduction in several patients. These patients already had severely impaired food intake and could hardly tolerate (larger) meals. This dose reduction might have influenced the outcome of the 24-h tonometry, the borderline significant differences between CGI and non-ischemic patients after compound solution meals might be explained by this effect. The results of tonometry after meals have to be interpreted with care. Unsuccessful suppression of acid production and meal-related production of CO2 may influence the results of tonometry. Using optimal acid suppression medication and standardized meals, these effects can be minimized, but not completely ruled out. In this study gastric pH measurement was used to control acid suppression. Furthermore, duodenogastric reflux of jejunal contents and\/or pancreatic juices are quite common and might theoretically influence intragastric and -jejunal PCO2 levels in a major way, leading to false positive tonometry findings [16].\nIn conclusion, this retrospective study shows that 24-h tonometry is feasible, safe, and has a very promising diagnostic accuracy for the detection of gastrointestinal ischemia. Using high-dose PPI acid suppression and standard meals, and previously established normal values, 24-h tonometry identifies gastrointestinal ischemia with an acceptable accuracy. The definitive role of 24-h tonometry in the diagnosis of chronic gastrointestinal ischemia has to be established in (future) prospective studies.","keyphrases":["splanchnic stenosis","carbon dioxide","chronic splanchnic syndrome","chronic mesenteric ischemia","gastric tonometry","small bowel tonometry"],"prmu":["P","P","M","R","R","M"]}
{"id":"Neurosurg_Rev-4-1-2279160","title":"Pure endoscopic endonasal odontoidectomy: anatomical study\n","text":"Different disorders may produce irreducible atlanto-axial dislocation with compression of the ventral spinal cord. Among the surgical approaches available for a such condition, the transoral resection of the odontoid process is the most often used. The aim of this anatomical study is to demonstrate the possibility of an anterior cervico-medullary decompression through an endoscopic endonasal approach. Three fresh cadaver heads were used. A modified endonasal endoscopic approach was made in all cases. Endoscopic dissections were performed using a rigid endoscope, 4 mm in diameter, 18 cm in length, with 0 degree lenses. Access to the cranio-vertebral junction was possible using a lower trajectory, when compared to that necessary for the sellar region. The choana is entered and the mucosa of the rhinopharynx is dissected and transposed in the oral cavity in order to expose the cranio-vertebral junction and to obtain a mucosal flap useful for the closure. The anterior arch of the atlas and the odontoid process of C2 are removed, thus exposing the dura mater. The endoscopic endonasal approach could be a valid alternative to the transoral approach for anterior odontoidectomy.\nIntroduction\nRemoval of the odontoid process is a procedure often required for the treatment of the basilar impression with compression of the brain stem or cervical spinal cord due to irreducible atlanto-axial translocation. Different disorders may produce atlanto-axial dislocation such as congenital malformation, chronic inflammation, metabolic disorders and trauma.\nThe transoral approach is the most favoured approach to the odontoid process and it is largely used for the surgical treatment of extradural and also intradural disorders of the cranio-vertebral junction [6\u20139, 11\u201313, 15, 16, 22\u201326]. Despite the fact that such an approach provides a direct route to the odontoid process, it presents several disadvantages such as the deepness of the surgical corridor, the sometimes required splitting of the soft palate, the risk of tongue and teeth damage and, in case of dural opening, the increased risk of post-operative CSF leakage and meningitis.\nBased on the experience of endoscopic endonasal pituitary surgery [3, 17], some works have recently reported anatomical studies and clinical applications of a modified endoscopic endonasal approach for the removal of the dens. These studies show the potential applications of the endoscopic endonasal approach for the surgical management of suprasellar, parasellar and retroclival pathologies [1, 2, 4, 5, 10, 14, 18\u201321].\nThis anatomic study describes the extended endoscopic endonasal approach to the cranio-vertebral junction, with particular attention to the reconstruction of the surgical route.\nMaterial and methods\nFor this anatomic study, three fresh cadaver heads were dissected; an extended endoscopic endonasal approach to the cranio-vertebral junction was performed in all cases.\nEndoscopic dissections were performed using a rigid endoscope (Karl Storz GmbH, Tuttlingen, Germany), 4\u00a0mm in diameter, 18\u00a0cm in length, with 0 degree lenses.\nThe endoscope was connected to a light source through a fiberoptic cable and to a camera fitted with 3CCD sensors. The video-camera was connected to a 21\u201d monitor supporting the high resolution of the 3CCD technology.\nResults\nThe procedure started with the introduction of the endoscope into a nasal vestibule through a lower trajectory as compared to the one employed for reaching the sellar region. Along such trajectory, the first structures to be visualized were the nasal septum medially, the inferior turbinate and the middle turbinate laterally. The inferior margin of the middle turbinate led to the choana which represented the main landmark of the approach.\nBy advancing the endoscope through the choana it was possible to identify the ostium of the Eustachian tube laterally, the rhinopharynx posteriorly, the soft palate inferiorly and the inferior wall of the sphenoid sinus superiorly; the latter representing the superior limit of the surgical approach. Angling the endoscope to the contralateral nasal cavity, it was possible to visualize the ostium of the contralateral Eustachian tube. The ostia of the two Eustachian tubes represented the lateral limits of this approach (see Fig.\u00a01a,b).\nFig.\u00a01a, b Entering the choana, the rhinopharinx and the Eustachian tube have been bilaterally visualized (iwsphs inferior wall of sphenoid sinus, ET Eustachian tube, Rphx rhinopharinx)\nIn order to expose the cranio-vertebral junction the mucosa of rhinopharynx was incised along its lateral limits at the edge with the ostia of the Eustachian tube and along the inferior wall of the sphenoid sinus superiorly (see Fig.\u00a02a). The mucosa, the longus capitis and longus colli muscles were gently dissected downward as a single layer, thus creating a muscle-mucosal flap (see Figs.\u00a02b and 3). Proceeding from the inferior wall of the sphenoid sinus to the soft palate of the lower clivus, the atlanto-occipital membrane, the anterior arch of C1 and the body of C2 were visualized.\nFig.\u00a02a The mucosa of the rhinopharynx has been incised in order to create a mucosal flap. b The muscles longus capitis and colli have been dissected together with the mucosa in order to expose the cranio-vertebral junction. (ET Eustachian tube, Rphx rhinopharinx, NS nasal septum, iwsphs inferior wall of sphenoid sinus, C clivus, aom atlanto-occipital membrane, C1 atlas, mmf muscle-mucosal flap)Fig.\u00a03Schematic drawing showing the muscle-mucosal flap and the lower structures (iwsphs inferior wall of sphenoid sinus, ET Eustachian tube, C1 atlas, mmf muscle-mucosal flap, C2 axis, D dens)\nIntroducing the endoscope in the oral cavity it was possible to reach the dissected mucosa of rhinopharynx and to transpose the muscle-mucosal flap into the oral cavity (see Fig.\u00a04a,b).\nFig.\u00a04a The muscle-mucosal flap is transposed in the oropharinx. b Schematic drawing showing the exposure of the cranio-vertebral junction after replacing the flap. (Ophx oropharynx, mmf muscle-mucosal flap, T tongue)\nThis manoeuver permitted an adequate endonasal exposure of the cranio-vertebral junction without removing the mucosa of the rhinopharinx, which provides a useful autologous material for closure of the surgical field.\nReintroducing the endoscope into the nasal cavity, the anterior arch of the atlas was removed and the dens with the apical and alar ligaments were exposed (see Fig. 5a,b). The dens was then thinned with a microdrill, separated from the alar and apical ligaments, and finally removed (see Fig.\u00a06a,b).\nFig.\u00a05a The anterior arch of the atlas has been removed. b The dens has been exposed. (C clivus, aom atlanto-occipital membrane, C1 atlas, dm dura mater, al alar ligament, D dens)Fig.\u00a06a The dens has been drilled. b After removal of the odontoid process the transverse ligament and the underling tectorial membrane are visible. (iwsphs inferior wall of sphenoid sinus, ET Eustachian tube, dm dura mater, D dens)\nAt this point the transverse ligament was identified; it was removed with the tectorial membrane, a double layer membrane positioned behind the transverse ligament, in order to expose the dura mater. At the end of the procedure the muscle-mucosal flap was replaced into the nasal cavity, thus closing the surgical field (see Fig.\u00a07).\nFig.\u00a07The muscle-mucosal flap is replaced in the rhinopharinx (iwsphs inferior wall of sphenoid sinus, ET Eustachian tube, mmf muscle-mucosal flap)\nIn this study, the endoscopic endonasal approach to the cranio-vertebral junction has been performed using both the one-nostril and the two-nostril technique, without removal of inferior and\/or middle turbinate, nasal septum or other nasal structures. Although the procedure can be performed through only one nostril, the binostril technique provides, without any additional surgical trauma, a better manoeuverability of the surgical tools and the possibility to work with \u201cthree hands\u201d. As a matter of fact, this technique permits a free-hand use of the endoscope in one nostril, held by the assistant, and the use of the other nostril or both nostrils for the insertion of the surgical instruments.\nFurthermore, in the case of a narrow nasal cavity, it is valuable to perform a unilateral middle turbinectomy and removal of the posterior third of the nasal septum to enlarge the surgical corridor.\nDiscussion\nDifferent pathological disorders may produce atlanto-axial translocation with ventral compression of the brain stem or spinal cord. The most common are congenital malformations, such as Arnold Chiari malformation type II, chronic inflammation, such as rheumatoid arthritis, genetic transformation, such as Down\u2019s syndrome and trauma, such as type II odontoid fracture. Some of these patients are candidate to the resection of the odontoid process for anterior decompression. The indication for odontoid resection is irreducible atlanto-axial subluxation, associated with severe spinal cord compression causing progressive myelopathy.\nThe most favoured approach to the odontoid process is the transoral approach. This approach provides a direct route to the surgical field, without any neurovascular manipulation and passing through the oropharynx, without injuring major neurovascular structures. The main limitation to this approach is the difficulty of dural closure and the subsequent higher risk of CSF leak and meningitis. For this reason the trans-oral approach is mainly used for extradural lesions [7, 8, 11\u201313, 15, 22\u201324], although some studies have reported its application for the surgical treatment of intradural pathology of the lower clivus and ventral cranio-cervical junction [6, 9, 16, 25, 26]. Other minor disadvantages are, however, related to this approach: the split of the soft palate and even of the hard palate is often performed in the case when rostral extension of the approach is required; tongue swelling may occur for prolonged compression; there is risk of damaging the teeth with retractors; velopharyngeal insufficiency may develop; and there is the necessity of nasal feeding in the postoperative stay.\nRecently, increased diffusion in the use of the endoscope for transsphenoidal pituitary surgery [3, 17] led some studies to explore the possibility of applying the endoscopic endonasal approach in the surgical treatment of skull base lesions other than pituitary tumors. In recent years some works have reported anatomical studies and surgical experience in the endoscopic endonasal approach to different areas of the midline skull base, from the olfactory groove to the cranio-vertebral junction [1, 2, 4, 5, 10, 14, 18\u201321].\nThanks to the properties of the endoscope itself, the endonasal approach provides a wider view of the surgical field and a close-up vision, when compared with the transoral microscopic approach. Furthermore, the minimal invasiveness of the endoscopic endonasal route may reduce some morbidities related to the transoral approach. In fact, it is no longer necessary to use mouth retractors, prolonged compression of the tongue or split of the soft palate, and even considering the necessity of a middle turbinectomy or removal of the posterior portion of the nasal septum to enlarge the surgical corridor, these adjunctive manoeuvres do not usually produce morbidity to the patient. These manoeuvres are often performed in the endonasal extended approaches to the area around the sella in live patients and do not cause any respiratory problems.\nThe possibility of performing an odontoidectomy through the nose is strictly related to the level of the C1\u2013C2 junction. In fact, in the case of a low junction, below the level of the hard palate, it is virtually impossible to remove the odontoid process with an endonasal approach. On the contrary, in the case of a high position of the atlas-axis junction, the dens is more easily reached and removed through the nasal cavities.\nOdontoidectomy may be considered one of the most complicated manoeuvres for the transoral approach, in which the split of the soft and even hard palate is often necessary. Thus, this approach could be evaluated for those cases in which a transoral removal is considered more difficult.\nHowever, this kind of approach still presents some of the main problems of the transoral approach. The first problem concerns the risk of CSF leak and subsequent meningitis. Although the endoscope, thanks to its close-up and multi-angled vision, has a greater chance of detecting an occasional CSF leak, it is quite hard to suture the dura and the nasopharynx mucosa with conventional suturing tools through the nose. For this reason, in our anatomical study, we have created a muscle-mucosal flap, comprehensive of the entire muscular and mucosal tissue covering the ventral cranio-vertebral junction. This flap, as shown, is transposed into the oral cavity during the bone\u2019s removal and replaced in its original site at the end of the procedure. Due to the difficulty of anchoring the flap with suture, it is only distended on the defect and its borders are apposed on the corresponding lines of incision. The mucosa of the inferior wall of the sphenoid sinus could be stripped to favour the adherence of the flap and fibrin glue could be used to seal the edges. The creation of a peduncolated muscle-mucosal flap permits a more physiological reconstruction of the surgical corridor and, furthermore, the vascularization of the flap that directly continues with the oropharynx, facilitates a rapid healing. An endoscopic control should be performed one month after surgery to check the recreating integrity of the rhinopharynx mucosa.\nThe second problem of the transoral approach concerns the stability of the cranio-vertebral junction. The removal of the odontoid process with its ligaments can destabilize the cranio-vertebral junction.\nThe third problem concerns the haemostasis that is often difficult in the extended endonasal approaches. Bleeding control may become difficult with bipolar coagulation because the endonasal approach presents a long and narrow corridor, with a limited working space between the tips of the bipolar forceps. Nevertheless, specific bipolar forceps (TAKE-APART bipolar forceps; Karl Storz GmbH, Tuttlingen, Germany) have been used to work through the nose in a safe and effective way as well.\nConclusions\nThis cadaver study has been performed to demonstrate the possibility of an anterior decompression of the upper cervical cord through an endoscopic endonasal approach. Similar to the transoral approach, the endoscopic endonasal approach provides a direct route to the surgical target, but it seems related to less morbidity. For clinical applications of this approach, the most common surgical problems are the risk of CSF leak and meningitis, the instability of the cervico-medullary junction and the bleeding control. The creation of a muscle-mucosal flap may represent a valid modality for closure of the surgical field. For application in live surgery, dedicate surgical instruments, such as endonasal bipolar forceps, high-speed low-profile drill and surgical guidance systems are needed.\nIn selected cases this approach could be considered a valid alternative to the transoral microscopic approach for the resection of the odontoid process of C2. Obviously, it should be performed only by surgeons very skilled in endoscopic endonasal surgery and in endoscopic cadaver-dissections, while cooperation in a team with ENT surgeons is recommended.","keyphrases":["odontoid process","cranio-vertebral junction","endoscopy"],"prmu":["P","P","U"]}
{"id":"Exp_Neurol-2-1-2288636","title":"Effects of low-frequency stimulation of the subthalamic nucleus on movement in Parkinson's disease\n","text":"Excessive synchronization of basal ganglia neural activity at low frequencies is considered a hallmark of Parkinson's disease (PD). However, few studies have unambiguously linked this activity to movement impairment through direct stimulation of basal ganglia targets at low frequency. Furthermore, these studies have varied in their methodology and findings, so it remains unclear whether stimulation at any or all frequencies \u2264 20 Hz impairs movement and if so, whether effects are identical across this broad frequency band. To address these issues, 18 PD patients chronically implanted with deep brain stimulation (DBS) electrodes in both subthalamic nuclei were stimulated bilaterally at 5, 10 and 20 Hz after overnight withdrawal of their medication and the effects of the DBS on a finger tapping task were compared to performance without DBS (0 Hz). Tapping rate decreased at 5 and 20 Hz compared to 0 Hz (by 11.8 \u00b1 4.9%, p = 0.022 and 7.4 \u00b1 2.6%, p = 0.009, respectively) on those sides with relatively preserved baseline task performance. Moreover, the coefficient of variation of tap intervals increased at 5 and 10 Hz compared to 0 Hz (by 70.4 \u00b1 35.8%, p = 0.038 and 81.5 \u00b1 48.2%, p = 0.043, respectively). These data suggest that the susceptibility of basal ganglia networks to the effects of excessive synchronization may be elevated across a broad low-frequency band in parkinsonian patients, although the nature of the consequent motor impairment may depend on the precise frequencies at which synchronization occurs.\nIntroduction\nThere is extensive evidence that neuronal activity is abnormally synchronized at low frequencies in Parkinson's disease (PD) and in animal models of parkinsonism (reviewed in Gatev et al., 2006; Hammond et al., 2007; Uhlhaas and Singer, 2006). However, this does not, by itself, prove that pathological synchrony is mechanistically important in parkinsonism. More persuasive evidence would be the impairment of voluntary movement by the artificial synchronization of neural activity in the basal ganglia. Such synchronization is possible by stimulating deep brain electrodes implanted for the treatment of PD at low frequencies, rather than at those frequencies above 100\u00a0Hz used for therapeutic benefit. Electrical stimulation of surgical targets like the subthalamic nucleus (STN) simultaneously activates neural elements in the vicinity of the electrode and this synchronous activity is then propagated onwards, as evinced by evoked pallidal (Brown et al., 2004; Hashimoto et al., 2003), cortical (MacKinnon et al., 2005) and muscular activity (Ashby et al., 1999, 2001).\nSo far there have been several reports of the impairment of movement by stimulation of the STN at frequencies \u2264\u00a020\u00a0Hz in patients with PD. Moro et al. (2002) and Chen et al. (2007) studied finger tapping during DBS at 5\u00a0Hz and 20\u00a0Hz, respectively, and found this to be slowed. However, Timmermann et al. (2004), using the motor United Parkinson's Disease rating scale (UPDRS), failed to confirm a worsening during DBS at 5 and 20\u00a0Hz, but did find increased bradykinesia with stimulation at 10\u00a0Hz. Another study evaluated tapping performance over several frequencies within the same patients, but only found weak effects that involved relative rather than absolute impairments in motor performance, superimposed upon an overall tendency for movement to improve with increasing stimulation frequency (Fogelson et al., 2005). Accordingly, it is unclear whether stimulation at any or all frequencies \u2264\u00a020\u00a0Hz impairs movement and if so, whether effects are identical across this broad frequency band. The issue is an important one, as although spontaneous synchrony tends to occur at frequencies centered around 20\u00a0Hz in PD (Hammond et al., 2007), it occurs at rather lower frequencies in the 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP) primate model of PD (Goldberg et al., 2004; Raz et al., 1996, 2000).\nHere we contrast the effects of STN stimulation and thereby extrinsically imposed synchronization at a number of frequencies \u2264\u00a020\u00a0Hz, to establish whether all such frequencies impair movement and, if so, whether they impair movement in the same way. To this end we studied performance in a simple finger tapping task, as this is objective and correlates with motor impairment (Giovannoni et al., 1999; Rabey et al., 2002), and considered changes in task execution according to baseline performance (Chen et al., 2006a).\nMaterials and methods\nPatients and surgery\nTwenty patients participated with informed consent and the permission of the local ethics committees (5 females, mean age 59.5\u00a0\u00b1\u00a01.4\u00a0years; mean disease duration 13.5\u00a0\u00b1\u00a01.0\u00a0years). Their clinical details are summarized in Table 1. Fourteen of these patients had also been recorded at least 6\u00a0months previously in a different paradigm involving stimulation at 20\u00a0Hz, 50\u00a0Hz and 130\u00a0Hz (Chen et al., 2007). Implantation of bilateral STN DBS electrodes was performed in all subjects for treatment of Parkinson's disease at least 6\u00a0months prior to study (mean 34.7\u00a0\u00b1\u00a05.9\u00a0months). The DBS electrode used was model 3389 (Medtronic Neurological Division, Minneapolis, USA) with four platinum\u2013iridium cylindrical surfaces (1.27\u00a0mm diameter and 1.5\u00a0mm length) and a centre-to-centre separation of 2\u00a0mm. Contact 0 was the most caudal and contact 3 was the most rostral. The intended coordinates at the tip of contact 0 were 10\u201312\u00a0mm from the midline, 0\u20132\u00a0mm behind the midcommissural point and 3\u20135\u00a0mm below the anterior commissural\u2013posterior commissural line. Adjustments to the intended coordinates were made in accordance with the direct visualization of STN in individual stereotactic MRI (Hariz et al., 2003) and, in the patients operated in Taiwan (n\u00a0=\u00a07), the results of microelectrode recordings. Correct placement of the DBS electrodes in the region of the STN was further supported by: [1] effective intra-operative macrostimulation; [2] post-operative T2-weighted MRI compatible with the placement of at least one electrode contact in the STN region; [3] significant improvement in UPDRS motor score during chronic DBS off medication (22.7\u00a0\u00b1\u00a03.0) compared to UPDRS off medication with stimulator switched off (52.6\u00a0\u00b1\u00a04.8; p\u00a0<\u00a00.00001, paired t-test). One patient was excluded due to the absence of significant improvement in UPDRS motor score during chronic DBS and another one due to missing clinical data.\nProtocol\nAll patients were assessed after overnight withdrawal of antiparkinsonian medication, although the long action of many of the drugs used to treat PD meant that patients may still have been partially treated when assessed. They were studied when the stimulator was switched off and during bilateral STN stimulation at 5\u00a0Hz, 10\u00a0Hz and 20\u00a0Hz. The stimulation types were assessed in pseudo-randomized order across patients, as was the presentation order of trials within a stimulation type. Stimulation contacts, amplitude and pulse duration were the same as utilized for therapeutic high frequency stimulation in each subject (see Table 1). There was no evidence of capsular spread during stimulation, as determined by clinical examination. Patients were not informed of the stimulation type. We did not stimulate one side at a time to avoid possible functional compensation by the non-stimulated side. We waited 20\u00a0min after changing between conditions before testing. This is sufficient time to elicit about 75% of DBS effects (Temperli et al., 2003).\nTask\nThe task was repetitive depression of a keyboard key as fast as possible by rapid alternating flexion and extension of the index finger at the level of the metacarpophalangeal joint (Chen et al., 2006a, 2007). Tapping was performed in two runs of 30\u00a0s, separated by \u223c\u00a030-s rest and each hand tested separately (giving four runs per condition). Data from one side were rejected as these were collected contralateral to a previous unilateral pallidotomy (case 18 in Table 1). The number of taps made with the index finger in 30\u00a0s was recorded and the run from each pair with the best performance selected for analysis, as this was less likely to be affected by fatigue, or the effects of impaired arousal\/concentration.\nStatistics\nThe results of the tapping task in patients were analyzed according to their baseline performance (e.g. without stimulation). The lower limit of normal baseline performance was obtained by testing ten healthy age matched control subjects (20 sides, 4 males, mean age 57\u00a0years, range 52\u201364\u00a0years) using the same tapping task. The mean tapping rate in this control group was 162\u00a0taps\/30\u00a0s. The lower limit of the normal range (e.g. mean\u00a0\u2212\u00a0[2\u00a0\u00d7\u00a0standard deviation]) in this control group was 127\u00a0taps per 30\u00a0s. The 35 tapping sides studied in the 18 patients were accordingly divided into those with baseline performance within normal limits (n\u00a0=\u00a017; the mean tapping performance across this group, 157\u00a0taps\/30\u00a0s, was still lower than the mean tapping performance in healthy subjects) and those with baseline tapping rates lower than normal limits (n\u00a0=\u00a018; mean tapping performance 58\u00a0taps\/30\u00a0s). The rationale behind this approach was to select those sides (with baseline performance within normal limits) in which any deleterious effects of DBS would not be overshadowed by the beneficial effects of DBS-induced suppression of spontaneous pathological activity or limited by floor effects due to major baseline impairment (Chen et al., 2006a, 2007). Four subjects had sides distributed across the two groups of differing baseline tapping performance. Tapping rates and coefficients of variation were normally distributed (one-sample Kolmogorov\u2013Smirnov tests, p\u00a0>\u00a00.05). Repeated-measures ANOVAs with within-subjects simple contrasts (comparing different frequencies of stimulation to no stimulation) were performed in SPSS (SPSS for Windows version 11, SPSS Inc., Chicago, IL, USA). Mauchly's test was used to determine the sphericity of the data entered in the ANOVAs, and where data were non-spherical Greenhouse\u2013Geisser corrections applied. Means\u00a0\u00b1\u00a0standard error of the means are presented in the text.\nResults\nLow-frequency stimulation had no reliable clinical effect and did not consistently induce tremor, mobile dyskinesia, or dystonic posturing. Four patients experienced dystonic postures during the experiment and one had increased tremor. However, these effects were seen for overlapping stimulation frequencies. We divided the tapping sides into two groups according to whether or not tapping performance off DBS was within normal limits established on 20 sides in 10 healthy age-matched subjects (see Materials and methods). ANOVA of tapping scores with factors FREQUENCY (four levels: 0, 5, 10 and 20\u00a0Hz) and BASELINE TAPPING PERFORMANCE (two levels: within normal limits and less than normal limits) demonstrated a within-subjects interaction between FREQUENCY and BASELINE TAPPING PERFORMANCE (F[3,48]\u00a0=\u00a04.224, p\u00a0=\u00a00.01). Data were further analyzed with separate ANOVAs in each baseline tapping performance group. In those patients with baseline tapping performance within normal limits, ANOVA with the factor FREQUENCY confirmed that the latter was a significant main effect (F[3,48]\u00a0=\u00a03.777, p\u00a0=\u00a00.016). Within-subjects contrasts indicated that tapping during 5 and 20\u00a0Hz stimulation was worse than during no stimulation (F[1,16]\u00a0=\u00a06.385, p\u00a0=\u00a00.022 and F[1,16]\u00a0=8.793, p\u00a0=\u00a00.009, respectively). The average deterioration in tapping rate during 5 and 20\u00a0Hz stimulation compared to no stimulation (0\u00a0Hz) in this group was 11.8\u00a0\u00b1\u00a04.9% and 7.4\u00a0\u00b1\u00a02.6%, respectively (Fig. 1). There was a trend towards a decreased tapping performance at 10\u00a0Hz compared to 0\u00a0Hz (F[1,16]\u00a0=\u00a03.578, p\u00a0=\u00a00.077). There was no effect of FREQUENCY in patients with baseline tapping performance below normal limits (ANOVA, F[1.9,32.8]\u00a0=\u00a02.202, p\u00a0=\u00a00.128).\nWe also analyzed the variability in tapping as measured by the coefficient of variation (CV) of the time intervals between successive taps on those sides with baseline tapping performance within normal limits. ANOVA with the factor FREQUENCY (four levels: 0, 5, 10 and 20\u00a0Hz) revealed a significant effect of FREQUENCY (F[3,48]\u00a0=\u00a03.408, p\u00a0=\u00a00.025). Within-subjects contrasts indicated that the CV increased during 5 and 10\u00a0Hz stimulation compared with no stimulation (F[1,16]\u00a0=\u00a05.144, p\u00a0=\u00a00.038 and F[1,16]\u00a0=\u00a04.852, p\u00a0=\u00a00.043, respectively). The average increase of the CV during 5 and 10\u00a0Hz stimulation compared to 0\u00a0Hz in this group was 70.4\u00a0\u00b1\u00a035.8% and 81.5\u00a0\u00b1\u00a048.2%, respectively (Fig. 2). There was no difference between the CV at 20\u00a0Hz compared to 0\u00a0Hz (F[1,16]\u00a0=\u00a00.871, p\u00a0=\u00a00.365). There was, however, a trend for the CV with 5\u00a0Hz stimulation to exceed that with 20\u00a0Hz stimulation (t-test; p\u00a0=\u00a00.059).\nDiscussion\nWe have shown that STN DBS at a variety of low frequencies can slow distal upper limb movements in PD patients with relatively preserved baseline tapping function at the time of study. The effect was present with DBS at 5\u00a0Hz and 20\u00a0Hz in line with previous studies (Chen et al., 2007; Fogelson et al., 2005; Moro et al., 2002), and there was a trend towards a similar effect with stimulation at 10\u00a0Hz (Timmermann et al., 2004). These effects were apparent when tapping sides were separately analyzed according to whether the level of baseline performance was within or outside of normal limits, in line with previous studies suggesting that deleterious effects of DBS are more evident on those sides with relatively preserved baseline performance (Chen et al., 2006a, 2007). The effect was not apparent during stimulation on those sides with impaired baseline performance, either because of confounding, albeit mild, suppressive effects of low-frequency DBS on spontaneous pathological oscillations or because of floor effects (Chen et al., 2007; Fogelson et al., 2005).\nIn principle, then, the susceptibility of basal ganglia\u2013cortical loops to the effects of excessive synchronization may be elevated across a broad low-frequency band in parkinsonian patients. Accordingly, the relatively different frequency ranges of pathological synchronization in patients and MPTP-treated primates (Hammond et al., 2007) may be more indicative of the resonance properties of basal ganglia networks in the different situations, rather than any fundamental differences in the mechanism of bradykinesia. However, it must be stressed that this is a generalization, and although synchronization at different frequencies may conspire to disturb movement, there may still be subtle differences in the way movement is impaired. This is brought out by the differential effects of low-frequency stimulation on the variation in tapping intervals, evident in differences in the coefficient of variation and hence independent of any differences in tapping rate. Only DBS at 5 and 10\u00a0Hz increased temporal variability, whereas DBS at 20\u00a0Hz selectively decreased tapping rates without changing tapping variability.\nThe implication is that basal ganglia networks are involved in processing related to the temporal patterning and regularity of movement and that these circuits may be particularly susceptible to disruption by pathological synchronization at frequencies \u2264\u00a010\u00a0Hz. In support of basal ganglia involvement in the temporal patterning of movement, PD patients have increased temporal variability in finger tapping (Giovannoni et al., 1999; Shimoyama et al., 1990), and temporal variability in motor performance is a very early feature of Huntington's disease (Hinton et al., 2007). Indeed, Flowers considered increased variability of movement in both time and space to be one of the core components of motor dysfunction in PD, along with a basic slowness of movement, and a difficulty in initiating and maintaining movement (Sheridan et al., 1987). This variability in motor performance may also relate to the phenomenon of freezing. No overt freezing episodes were observed during tapping in our patients, but an increased variability of stride has been shown in PD patients experiencing freezing of gait independent of frank freezing episodes (Hausdorff et al., 2003).\nHowever, a primary disturbance of temporal patterning is not the only potential interpretation for the increased variability seen during stimulation at 5\u00a0Hz and 10\u00a0Hz. Tremor was not seen during low-frequency stimulation (except in one patient), in agreement with Timmermann et al. (2004), nor were there any obvious and consistent dyskinesias. Nevertheless, it is possible that synchronization at frequencies \u2264\u00a010\u00a0Hz induced subtle hyperkinesias that led to increased temporal variability across taps. A previous case report describes dyskinetic movements induced by STN DBS at 5\u00a0Hz (Liu et al., 2002a), and there is increasing evidence that excessive synchronization over 4\u201310\u00a0Hz within basal ganglia circuits may be related to both levodopa-induced dyskinesias in PD (Alonso-Frech et al., 2006) and mobile elements of dystonia (Chen et al., 2006b; Liu et al., 2002b; Silberstein et al., 2003). Relevant in this regard, a recent study demonstrated an increased variability of speech rate in patients treated with l-DOPA and suggested that this effect was related to dyskinesia (De Letter et al., 2006). Variability of swing movement was also observed in the gait of dyskinetic CP children (Abel et al., 2003).\nIn summary, our results provide further evidence that DBS of the STN over a relatively broad band of low frequencies can impair movement, in line with other more circumstantial evidence of an association between low-frequency synchrony in basal ganglia\u2013cortical loops and altered movement (see recent reviews by Gatev et al., 2006; Uhlhaas and Singer, 2006; Hammond et al., 2007). The present results also raise the important possibility that the detailed profile of motor abnormalities evident in extrapyramidal diseases depends to some extent on the precise frequencies at which pathological synchronization occurs. Indeed, some differences in the details of the effects of pathological synchrony at different frequencies might be anticipated, given the evidence for selective tuning of basal ganglia\u2013cortical sub-circuits (Fogelson et al., 2006).","keyphrases":["parkinson's disease","synchronization","basal ganglia","dbs"],"prmu":["P","P","P","P"]}
{"id":"Arthritis_Res_Ther-5-3-165040","title":"Degeneration of the intervertebral disc\n","text":"The intervertebral disc is a cartilaginous structure that resembles articular cartilage in its biochemistry, but morphologically it is clearly different. It shows degenerative and ageing changes earlier than does any other connective tissue in the body. It is believed to be important clinically because there is an association of disc degeneration with back pain. Current treatments are predominantly conservative or, less commonly, surgical; in many cases there is no clear diagnosis and therapy is considered inadequate. New developments, such as genetic and biological approaches, may allow better diagnosis and treatments in the future.\nIntroduction\nBack pain is a major public health problem in Western industrialized societies. It causes suffering and distress to patients and their families, and affects a large number of people; the point prevalence rates in a number of studies ranged from 12% to 35% [1], with around 10% of sufferers becoming chronically disabled. It also places an enormous economic burden on society; its total cost, including direct medical costs, insurance, lost production and disability benefits, is estimated at \u20ac12 billion per annum in the UK and 1.7% of the gross national product in The Netherlands [1,2].\nBack pain is strongly associated with degeneration of the intervertebral disc [3]. Disc degeneration, although in many cases asymptomatic [4], is also associated with sciatica and disc herniation or prolapse. It alters disc height and the mechanics of the rest of the spinal column, possibly adversely affecting the behaviour of other spinal structures such as muscles and ligaments. In the long term it can lead to spinal stenosis, a major cause of pain and disability in the elderly; its incidence is rising exponentially with current demographic changes and an increased aged population.\nDiscs degenerate far earlier than do other musculoskeletal tissues; the first unequivocal findings of degeneration in the lumbar discs are seen in the age group 11\u201316 years [5]. About 20% of people in their teens have discs with mild signs of degeneration; degeneration increases steeply with age, particularly in males, so that around 10% of 50-year-old discs and 60% of 70-year-old discs are severely degenerate [6].\nIn this short review we outline the morphology and biochemistry of normal discs and the changes that arise during degeneration. We review recent advances in our understanding of the aetiology of this disorder and discuss new approaches to treatment.\nDisc morphology\nThe normal disc\nThe intervertebral discs lie between the vertebral bodies, linking them together (Fig. 1). They are the main joints of the spinal column and occupy one-third of its height. Their major role is mechanical, as they constantly transmit loads arising from body weight and muscle activity through the spinal column. They provide flexibility to this, allowing bending, flexion and torsion. They are approximately 7\u201310 mm thick and 4 cm in diameter (anterior\u2013posterior plane) in the lumbar region of the spine [7,8]. The intervertebral discs are complex structures that consist of a thick outer ring of fibrous cartilage termed the annulus fibrosus, which surrounds a more gelatinous core known as the nucleus pulposus; the nucleus pulposus is sandwiched inferiorly and superiorly by cartilage end-plates.\nThe central nucleus pulposus contains collagen fibres, which are organised randomly [9], and elastin fibres (sometimes up to 150 \u03bcm in length), which are arranged radially [10]; these fibres are embedded in a highly hydrated aggrecan-containing gel. Interspersed at a low density (approximately 5000\/mm3 [11]) are chondrocyte-like cells, sometimes sitting in a capsule within the matrix. Outside the nucleus is the annulus fibrosus, with the boundary between the two regions being very distinct in the young individual (<10 years).\nThe annulus is made up of a series of 15\u201325 concentric rings, or lamellae [12], with the collagen fibres lying parallel within each lamella. The fibres are orientated at approximately 60\u00b0 to the vertical axis, alternating to the left and right of it in adjacent lamellae. Elastin fibres lie between the lamellae, possibly helping the disc to return to its original arrangement following bending, whether it be flexion or extension. They may also bind the lamellae together as elastin fibres pass radially from one lamella to the next [10]. The cells of the annulus, particularly in the outer region, tend to be fibroblast-like, elongated, thin and aligned parallel to the collagen fibres. Toward the inner annulus the cells can be more oval. Cells of the disc, both in the annulus and nucleus, can have several long, thin cytoplasmic projections, which may be more than 30 \u03bcm long [13,14] (WEB Johnson, personal communication). Such features are not seen in cells of articular cartilage [13]. Their function in disc is unknown but it has been suggested that they may act as sensors and communicators of mechanical strain within the tissue [13].\nThe third morphologically distinct region is the cartilage end-plate, a thin horizontal layer, usually less than 1 mm thick, of hyaline cartilage. This interfaces the disc and the vertebral body. The collagen fibres within it run horizontal and parallel to the vertebral bodies, with the fibres continuing into the disc [8].\nThe healthy adult disc has few (if any) blood vessels, but it has some nerves, mainly restricted to the outer lamellae, some of which terminate in proprioceptors [15]. The cartilaginous end-plate, like other hyaline cartilages, is normally totally avascular and aneural in the healthy adult. Blood vessels present in the longitudinal ligaments adjacent to the disc and in young cartilage end-plates (less than about 12 months old) are branches of the spinal artery [16]. Nerves in the disc have been demonstrated, often accompanying these vessels, but they can also occur independently, being branches of the sinuvertebral nerve or derived from the ventral rami or grey rami communicantes. Some of the nerves in discs also have glial support cells, or Schwann cells, alongside them [17].\nDegenerated discs\nDuring growth and skeletal maturation the boundary between annulus and nucleus becomes less obvious, and with increasing age the nucleus generally becomes more fibrotic and less gel-like [18]. With increasing age and degeneration the disc changes in morphology, becoming more and more disorganized (Fig. 2). Often the annular lamellae become irregular, bifurcating and interdigitating, and the collagen and elastin networks also appear to become more disorganised (J Yu, personal communication).\nThere is frequently cleft formation with fissures forming within the disc, particularly in the nucleus. Nerves and blood vessels are increasingly found with degeneration [15]. Cell proliferation occurs, leading to cluster formation, particularly in the nucleus [19,20]. Cell death also occurs, with the presence of cells with necrotic and apoptotic appearance [21,22]. These mechanisms are apparently very common; it has been reported that more than 50% of cells in adult discs are necrotic [21]. The morphological changes associated with disc degeneration were comprehensively reviewed recently by Boos et al. [5], who demonstrated an age-associated change in morphology, with discs from individuals as young as 2 years of age having some very mild cleft formation and granular changes to the nucleus. With increasing age comes an increased incidence of degenerative changes, including cell death, cell proliferation, mucous degeneration, granular change and concentric tears. It is difficult to differentiate changes that occur solely due to ageing from those that might be considered 'pathological'.\nBiochemistry\nNormal discs\nThe mechanical functions of the disc are served by the extracellular matrix; its composition and organization govern the disc's mechanical responses. The main mechanical role is provided by the two major macromolecular components. The collagen network, formed mostly of type I and type II collagen fibrils and making up approximately 70% and 20% of the dry weight of the annulus and nucleus, respectively [23], provides tensile strength to the disc and anchors the tissue to the bone. Aggrecan, the major proteoglycan of the disc [24], is responsible for maintaining tissue hydration through the osmotic pressure provided by its constituent chondroitin and keratan sulphate chains [25]. The proteoglycan and water content of the nucleus (around 50% and 80% of the wet weight, respectively) is greater than in the annulus (approximately 20% and 70% of the wet weight, respectively). In addition, there are many other minor components, such as collagen types III, V, VI, IX, X, XI, XII and XIV; small proteoglycans such as lumican, biglycan, decorin and fibromodulin; and other glycoproteins such as fibronectin and amyloid [26,27]. The functional role of many of these additional matrix proteins and glycoproteins is not yet clear. Collagen IX, however, is thought to be involved in forming cross-links between collagen fibrils and is thus important in maintaining network integrity [28].\nThe matrix is a dynamic structure. Its molecules are continually being broken down by proteinases such as the matrix metalloproteinases (MMPs) and aggrecanases, which are also synthesized by disc cells [29-31]. The balance between synthesis, breakdown and accumulation of matrix macromolecules determines the quality and integrity of the matrix, and thus the mechanical behaviour of the disc itself. The integrity of the matrix is also important for maintaining the relatively avascular and aneural nature of the healthy disc.\nThe intervertebral disc is often likened to articular cartilage, and indeed it does resemble it in many ways, particularly in the biochemical components present. However, there are significant differences between the two tissues, one of these being the composition and structure of aggrecan. Disc aggrecan is more highly substituted with keratan sulphate than that found in the deep zone of articular cartilage. In addition, the aggrecan molecules are less aggregated (30%) and more heterogeneous, with smaller, more degraded fragments in the disc than in articular cartilage (80% aggregated) from the same individual [32]. Disc proteoglycans become increasingly difficult to extract from the matrix with increasing age [24]; this may be due to extensive cross-linking, which appears to occur more within the disc matrix than in other connective tissues.\nChanges in disc biochemistry with degeneration\nThe most significant biochemical change to occur in disc degeneration is loss of proteoglycan [33]. The aggrecan molecules become degraded, with smaller fragments being able to leach from the tissue more readily than larger portions. This results in loss of glycosaminoglycans; this loss is responsible for a fall in the osmotic pressure of the disc matrix and so a loss of hydration.\nEven in degenerate discs, however, the disc cells can retain the ability to synthesize large aggrecan molecules, with intact hyaluronan-binding regions, which have the potential to form aggregates [24]. Less is known of how the small proteoglycan population changes with disc degeneration, although there is some evidence that the amount of decorin, and more particularly biglycan, is elevated in degenerate human discs as compared with normal ones [34].\nAlthough the collagen population of the disc also changes with degeneration of the matrix, the changes are not as obvious as those of the proteoglycans. The absolute quantity of collagen changes little but the types and distribution of collagens can alter. For example, there may be a shift in proportions of types of collagens found and in their apparent distribution within the matrix. In addition, the fibrillar collagens, such as type II collagen, become more denatured, apparently because of enzymic activity. As with proteoglycans, the triple helices of the collagens are more denatured and ruptured than are those found in articular cartilage from the same individual; the amount of denatured type II collagen increases with degeneration [35,36]. However, collagen cross-link studies indicate that, as with proteoglycans, new collagen molecules may be synthesized, at least early in disc degeneration, possibly in an attempt at repair [37].\nOther components can change in disc degeneration and disease in either quantity or distribution. For example, fibronectin content increases with increasing degeneration and it becomes more fragmented [38]. These elevated levels of fibronectin could reflect the response of the cell to an altered environment. Whatever the cause, the formation of fibronectin fragments can then feed into the degenerative cascade because they have been shown to downregulate aggrecan synthesis but to upregulate the production of some MMPs in in vitro systems.\nThe biochemistry of disc degeneration indicates that enzymatic activity contributes to this disorder, with increased fragmentation of the collagen, proteoglycan and fibronectin populations. Several families of enzymes are capable of breaking down the various matrix molecules of disc, including cathepsins, MMPs and aggrecanases. Cathepsins have maximal activity in acid conditions (e.g. cathepsin D is inactive above pH 7.2). In contrast, MMPs and aggrecanases have an optimal pH that is approximately neutral. All of these enzymes have been identified in disc, with higher levels of, for example, MMPs in more degenerate discs [39]. Cathepsins D and L and several types of MMPs (MMP-1, -2, -3, -7, -8, -9 and -13) occur in human discs; they may be produced by the cells of the disc themselves as well as by the cells of the invading blood vessels. Aggrecanases have also been shown to occur in human disc but their activity is apparently less obvious, at least in more advanced disc degeneration [29,30,40].\nEffect of degenerative changes on disc function and pathology\nThe loss of proteoglycan in degenerate discs [33] has a major effect on the disc's load-bearing behaviour. With loss of proteoglycan, the osmotic pressure of the disc falls [41] and the disc is less able to maintain hydration under load; degenerate discs have a lower water content than do normal age-matched discs [33], and when loaded they lose height [42] and fluid more rapidly, and the discs tend to bulge. Loss of proteoglycan and matrix disorganization have other important mechanical effects; because of the subsequent loss of hydration, degenerated discs no longer behave hydrostatically under load [43]. Loading may thus lead to inappropriate stress concentrations along the end-plate or in the annulus; the stress concentrations seen in degenerate discs have also been associated with discogenic pain produced during discography [44].\nSuch major changes in disc behaviour have a strong influence on other spinal structures, and may affect their function and predispose them to injury. For instance, as a result of the rapid loss of disc height under load in degenerate discs, apophyseal joints adjacent to such discs (Fig. 1) may be subject to abnormal loads [45] and eventually develop osteoarthritic changes. Loss of disc height can also affect other structures. It reduces the tensional forces on the ligamentum flavum and hence may cause remodelling and thickening. With consequent loss of elasticity [46], the ligament will tend to bulge into the spinal canal, leading to spinal stenosis \u2013 an increasing problem as the population ages.\nLoss of proteoglycans also influences the movement of molecules into and out of the disc. Aggrecan, because of its high concentration and charge in the normal disc, prevents movement of large uncharged molecules such as serum proteins and cytokines into and through the matrix [47]. The fall in concentration of aggrecan in degeneration could thus facilitate loss of small, but osmotically active, aggrecan fragments from the disc, possibly accelerating a degenerative cascade. In addition, loss of aggrecan would allow increased penetration of large molecules such as growth factor complexes and cytokines into the disc, affecting cellular behaviour and possibly the progression of degeneration. The increased vascular and neural ingrowth seen in degenerate discs and associated with chronic back pain [48] is also probably associated with proteoglycan loss because disc aggrecan has been shown to inhibit neural ingrowth [49,50].\nDisc herniation\nThe most common disc disorder presenting to spinal surgeons is herniated or prolapsed intervertebral disc. In these cases the discs bulge or rupture (either partially or totally) posteriorly or posterolaterally, and press on the nerve roots in the spinal canal (Fig. 1). Although herniation is often thought to be the result of a mechanically induced rupture, it can only be induced in vitro in healthy discs by mechanical forces larger than those that are ever normally encountered; in most experimental tests, the vertebral body fails rather than the disc [51]. Some degenerative changes seem necessary before the disc can herniate; indeed, examination of autopsy or surgical specimens suggest that sequestration or herniation results from the migration of isolated, degenerate fragments of nucleus pulposus through pre-existing tears in the annulus fibrosus [52].\nIt is now clear that herniation-induced pressure on the nerve root cannot alone be the cause of pain because more than 70% of 'normal', asymptomatic people have disc prolapses pressurizing the nerve roots but no pain [4,53]. A past and current hypothesis is that, in symptomatic individuals, the nerves are somehow sensitized to the pressure [54], possibly by molecules arising from an inflammatory cascade from arachodonic acid through to prostaglandin E2, thromboxane, phospholipase A2, tumour necrosis factor-\u03b1, the interleukins and MMPs. These molecules can be produced by cells of herniated discs [55], and because of the close physical contact between the nerve root and disc following herniation they may be able to sensitize the nerve root [56,57]. The exact sequence of events and specific molecules that are involved have not been identified, but a pilot study of sciatic patients treated with tumour necrosis factor-\u03b1 antagonists is encouraging and supports this proposed mechanism [58,59]. However, care must be exercised in interrupting the inflammatory cascade, which can also have beneficial effects. Molecules such as MMPs, which are produced extensively in prolapsed discs [30], almost certainly play a major role in the natural history of resorbing the offending herniation.\nAetiology of disc degeneration\nDisc degeneration has proved a difficult entity to study; its definition is vague, with diffuse parameters that are not always easy to quantify. In addition, there is a lack of a good animal model. There are significant anatomical differences between humans and the laboratory animals that are traditionally used as models of other disorders. In particular, the nucleus differs; in rodents as well as many other mammals, the nucleus is populated by notochordal cells throughout adulthood, whereas these cells disappear from the human nucleus after infancy [60]. In addition, although the cartilage end-plate in humans acts as a growth plate for the vertebral body, in most animals the vertebrae have two growth plates within the vertebral body itself, and the cartilage end-plate is a much thinner layer than that found in humans. Thus, although the study of animals that develop degeneration spontaneously [61,62] and of injury models of degeneration [63,64] have provided some insight into the degenerative processes, most information on aetiology of disc degeneration to date has come from human studies.\nNutritional pathways to disc degeneration\nOne of the primary causes of disc degeneration is thought to be failure of the nutrient supply to the disc cells [65]. Like all cell types, the cells of the disc require nutrients such as glucose and oxygen to remain alive and active. In vitro, the activity of disc cells is very sensitive to extracellular oxygen and pH, with matrix synthesis rates falling steeply at acidic pH and at low oxygen concentrations [66,67], and the cells do not survive prolonged exposure to low pH or glucose concentrations [68]. A fall in nutrient supply that leads to a lowering of oxygen tension or of pH (arising from raised lactic acid concentrations) could thus affect the ability of disc cells to synthesize and maintain the disc's extracellular matrix and could ultimately lead to disc degeneration.\nThe disc is large and avascular and the cells depend on blood vessels at their margins to supply nutrients and remove metabolic waste [69]. The pathway from the blood supply to the nucleus cells is precarious because these cells are supplied virtually entirely by capillaries that originate in the vertebral bodies, penetrating the subchondral plate and terminating just above the cartilaginous end-plate [16,70]. Nutrients must then diffuse from the capillaries through the cartilaginous end-plate and the dense extracellular matrix of the nucleus to the cells, which may be as far as 8 mm from the capillary bed.\nThe nutrient supply to the nucleus cells can be disturbed at several points. Factors that affect the blood supply to the vertebral body such as atherosclerosis [71,72], sickle cell anaemia, Caisson disease and Gaucher's disease [73] all appear to lead to a significant increase in disc degeneration. Long-term exercise or lack of it appears to have an effect on movement of nutrients into the disc, and thus on their concentration in the tissue [74,75]. The mechanism is not known but it has been suggested that exercise affects the architecture of the capillary bed at the disc\u2013bone interface. Finally, even if the blood supply remains undisturbed, nutrients may not reach the disc cells if the cartilaginous end-plate calcifies [65,76]; intense calcification of the end-plate is seen in scoliotic discs [77], for instance. Disturbances in nutrient supply have been shown to affect transport of oxygen and lactic acid into and out of the disc experimentally [78] and in patients [79].\nAlthough little information is available to relate nutrient supply to disc properties in patients, a relationship has been found between loss of cell viability and a fall in nutrient transport in scoliotic discs [80,81]. There is also some evidence that nutrient transport is affected in disc degeneration in vivo [82], and the transport of solutes from bone to disc measured in vitro was significantly lower in degenerate than in normal discs [65]. Thus, although there is as yet little direct evidence, it now seems apparent that a fall in nutrient supply will ultimately lead to degeneration of the disc.\nMechanical load and injury\nAbnormal mechanical loads are also thought to provide a pathway to disc degeneration. For many decades it was suggested that a major cause of back problems is injury, often work-related, which causes structural damage. It is believed that such an injury initiates a pathway that leads to disc degeneration and finally to clinical symptoms and back pain [83]. Animal models have supported this finding. Although intense exercise does not appear to affect discs adversely [84] and discs are reported to respond to some long-term loading regimens by increasing proteoglycan content [85], experimental overloading [86] or injury to the disc [63,87] can induce degenerative changes. Further support for the role of abnormal mechanical forces in disc degeneration comes from findings that disc levels adjacent to a fused segment degenerate rapidly (for review [88]).\nThis injury model is also supported by many epidemiological studies that have found associations between environmental factors and development of disc degeneration and herniation, with heavy physical work, lifting, truck-driving, obesity and smoking found to be the major risk factors for back pain and degeneration [89-91]. As a result of these studies, there have been many ergonomic interventions in the workplace [91]. However, the incidence of disc degeneration-related disorders has continued to rise despite these interventions. Over the past decade, as magnetic resonance imaging has refined classifications of disc degeneration [5,92], it has become evident that, although factors such as occupation, psychosocial factors, benefit payments and environment are linked to disabling back pain [93,94], contrary to previous assumptions these factors have little influence on the pattern of disc degeneration itself [95,96]. This illustrates the tenuous relationship between degeneration and clinical symptoms.\nGenetic factors in disc degeneration\nMore recent work suggested that the factors that lead to disc degeneration may have important genetic components. Several studies have reported a strong familial predisposition for disc degeneration and herniation [97-99]. Findings from two different twin studies conducted during the past decade showed heritability exceeding 60% [100,101]. Magnetic resonance images in identical twins, who were discordant for major risk factors such as smoking or heavy work, were very similar with respect to the spinal columns and the patterns of disc degeneration (Fig. 3) [102].\nGenetic predisposition has been confirmed by recent findings of associations between disc degeneration and gene polymorphisms of matrix macromolecules. The approach to date has been via searching for candidate genes, with the main focus being extracellular matrix genes. Although there is a lack of association between disc degeneration and polymorphisms of the major collagens in the disc, collagen types I and II [103], mutations of two collagen type IX genes, namely COL9A2 and COL9A3, have been found to be strongly associated with lumbar disc degeneration and sciatica in a Finnish population [104,105]. The COL9A2 polymorphism is found only in a small percentage of the Finnish population, but all individuals with this allele had disc degenerative disorders, suggesting that it is associated with a dominantly inherited disease. In both these mutations, tryptophan (the most hydrophobic amino acid, which is not normally found in any collagenous domain) substituted for other amino acids, potentially affecting matrix properties [103].\nOther genes associated with disc generation have also been identified. Individuals with a polymorphism in the aggrecan gene were found to be at risk for early disc degeneration in a Japanese study [106]. This mutation leads to aggrecan core proteins of different lengths, with an over-representation of core proteins able to bind only a low number of chondroitin sulfate chains among those with severe disc degeneration. Presumably these individuals have a lower chondroitin sulfate content than normal, and their discs will behave similarly to degenerate discs that have lost proteoglycan by other mechanisms. Studies of transgenic mice have also demonstrated that mutations in structural matrix molecules such as aggrecan [107], collagen II [108] and collagen IX [109] can lead to disc degeneration. Mutations in genes other than those of structural matrix macromolecules have also been associated with disc degeneration. A polymorphism in the promoter region of the MMP-3 gene was associated with rapid degeneration in elderly Japanese subjects [110]. In addition, two polymorphisms of the vitamin D receptor gene were the first mutations shown to be associated with disc degeneration [111-114]. The mechanism of vitamin D receptor gene polymorphism involvement in disc degeneration is unknown, but at present it does not appear to be related to differences in bone density [111,112,114].\nAll of the genetic mutations associated with disc degeneration to date have been found using a candidate gene approach and all, apart from the vitamin D receptor polymorphism, are concerned with molecules that determine the integrity and function of the extracellular matrix. However, mutations in other systems such as signalling or metabolic pathways could lead to changes in cellular activity that may ultimately result in disc degeneration [115]. Different approaches may be necessary to identify such polymorphisms. Genetic mapping, for instance, has identified a susceptibility locus for disc herniation, but the gene involved has not yet been identified [116].\nIn summary, the findings from these genetic and epidemiological studies point to the multifactorial nature of disc degeneration. It is evident now that mutations in several different classes of genes may cause the changes in matrix morphology, disc biochemistry and disc function typifying disc degeneration. Identification of the genes involved may lead to improved diagnostic criteria; for example, it is already apparent that the presence of specific polymorphisms increase the risk for disc bulge, annular tears, or osteophytes [112,117]. However, because of the evidence for gene\u2013environment interactions [97,114,118], genetic studies in isolation are unlikely to delineate the various pathways of disc degeneration.\nNew therapies\nCurrent treatments attempt to reduce pain rather than repair the degenerated disc. The treatments used presently are mainly conservative and palliative, and are aimed at returning patients to work. They range from bedrest (no longer recommended) to analgesia, the use of muscle relaxants or injection of corticosteroids, or local anaesthetic and manipulation therapies. Various interventions (e.g. intradiscal electrotherapy) are also used, but despite anecdotal statements of success trials thus far have found their use to be of little direct benefit [119]. Disc degeneration-related pain is also treated surgically either by discectomy or by immobilization of the affected vertebrae, but surgery is offered only to one in every 2000 back pain episodes in the UK; the incidence of surgical treatment is five times higher in the USA [93]. The success rates of all these procedures are generally similar. Although a recent study indicated that surgery improves the rate of recovery in well selected patients [120], 70\u201380% of patients with obvious surgical indications for back pain or disc herniation eventually recover, whether surgery is carried out or not [121,122].\nBecause disc degeneration is thought to lead to degeneration of adjacent tissues and be a risk factor in the development of spinal stenosis in the long term, new treatments are in development that are aimed at restoring disc height and biomechanical function. Some of the proposed biological therapies are outlined below.\nCell based therapies\nThe aim of these therapies is to achieve cellular repair of the degenerated disc matrix. One approach has been to stimulate the disc cells to produce more matrix. Growth factors can increase rates of matrix synthesis by up to fivefold [123,124]. In contrast, cytokines lead to matrix loss because they inhibit matrix synthesis while stimulating production of agents that are involved in tissue breakdown [125]. These proteins have thus provided targets for genetic engineering. Direct injection of growth factors or cytokine inhibitors has proved unsuccessful because their effectiveness in the disc is short-lived. Hence gene-therapy is now under investigation; it has the potential to maintain high levels of the relevant growth factor or inhibitor in the tissue. In gene therapy, the gene of interest (e.g. one responsible for producing a growth factor such as transforming growth factor-\u03b2 or inhibiting interleukin-1) is introduced into target cells, which then continue to produce the relevant protein (for review [126]). This approach has been shown to be technically feasible in the disc, with gene transfer increasing transforming growth factor-\u03b2 production by disc cells in a rabbit nearly sixfold [127]. However, this therapy is still far from clinical use. Apart from the technical problems of delivery of the genes into human disc cells, the correct choice of therapeutic genes requires an improved understanding of the pathogenesis of degeneration. In addition, the cell density in normal human discs is low, and many of the cells in degenerate discs are dead [21]; stimulation of the remaining cells may be insufficient to repair the matrix.\nCell implantation alone or in conjunction with gene therapy is an approach that may overcome the paucity of cells in a degenerate disc. Here, the cells of the degenerate disc are supplemented by adding new cells either on their own or together with an appropriate scaffold. This technique has been used successfully for articular cartilage [128,129] and has been attempted with some success in animal discs [130]. However, at present, no obvious source of clinically useful cells exists for the human disc, particularly for the nucleus, the region of most interest [131]. Moreover, conditions in degenerate discs, particularly if the nutritional pathway has been compromised [65], may not be favourable for survival of implanted cells. Nevertheless, autologous disc cell transfer has been used clinically in small groups of patients [132], with initial results reported to be promising, although few details of the patients or outcome measures are available.\nAt present, although experimental work demonstrates the potential of these cell-based therapies, several barriers prevent the use of these treatments clinically. Moreover, these treatments are unlikely to be appropriate for all patients; some method of selecting appropriate patients will be required if success with these therapies is to be realized.\nConclusion\nDisorders associated with degeneration of the intervertebral disc impose an economic burden similar to that of coronary heart disease and greater than that of other major health problems such as diabetes, Alzheimer's disease and kidney diseases [1,133]. New imaging technologies, and advances in cell biology and genetics promise improved understanding of the aetiology, more specific diagnoses and targeted treatments for these costly and disabling conditions. However, the intervertebral disc is poorly researched, even in comparison with other musculoskeletal systems (Table 1). Moreover, the research effort in, for instance, the kidney in comparison with that in the disc is completely disparate to the relative costs of the disorders associated with each organ and the number of people affected. Unless more research attention is attracted to interverterbal disc biology, little will come from these new technologies, and back pain will remain as it is at present \u2013 a poorly diagnosed and poorly treated syndrome that reduces the quality of life of a significant proportion of the population.\nCompeting interests\nNone declared.\nAbbreviations\nMMP = matrix metalloproteinase.","keyphrases":["back pain","genetics","epidemiology"],"prmu":["P","P","P"]}
{"id":"Eur_J_Pediatr-3-1-2042511","title":"What\u2019s new in using platelet research? To unravel thrombopathies and other human disorders\n","text":"This review on platelet research focuses on defects of adhesion, cytoskeletal organisation, signal transduction and secretion. Platelet defects can be studied by different laboratory platelet functional assays and morphological studies. Easy bruising or a suspected platelet-based bleeding disorder is of course the most obvious reason to test the platelet function in a patient. However, nowadays platelet research also contributes to our understanding of human pathology in other disciplines such as neurology, nephrology, endocrinology and metabolic diseases. Apart from a discussion on classical thrombopathies, this review will also deal with the less commonly known relation between platelet research and disorders with a broader clinical phenotype. Classical thrombopathies involve disorders of platelet adhesion such as Glanzmann thrombastenia and Bernard-Soulier syndrome, defective G protein signalling diseases with impaired phospholipase C activation, and abnormal platelet granule secretion disorders such as gray platelet disorder and delta-storage pool disease. Other clinical symptoms besides a bleeding tendency have been described in MYH9-related disorders and Duchenne muscular dystrophy due to adhesion defects, and also in disorders of impaired Gs signalling, in Hermansky Pudlack disease and Chediak Higashi disease with abnormal secretion. Finally, platelet research can also be used to unravel novel mechanisms involved in many neurological disorders such as depression and autism with only a subclinical platelet defect.\nIntroduction\nNormal hemostasis prevents spontaneous bleeding and traumatic hemorrhage by a coordinated sequence of cellular and biochemical reactions to the ultimate formation of a stable platelet-fibrin aggregate [20]. Platelets, under normal circumstances, circulate in close contact to the endothelial cell lining of the vessel wall, and respond to vascular damage by adhering to subendothelial structures. Platelet adhesion is the first step in the hemostatic plug formation [11]. The major platelet receptors involved in this process are the von Willebrand factor (vWF) receptor GP(glycoprotein)Ib\/IX\/V, the collagen integrin receptor \u03b12\u03b21, and the fibrinogen integrin receptor \u03b1IIB\u03b23 (Fig.\u00a01a). Subsequent platelet spreading is conducted by cytoskeleton proteins including the structural subunit of the microtubules, the \u03b1\u03b2-tubulin heterodimer, filamin and actin (Fig.\u00a01a). The cytoskeleton is responsible for the shape of the resting platelet and carries out contractile events such as the secretion of granules and retraction of clots by activated cells.\nFig.\u00a01a Schematic model of the main components involved in platelet adhesion and the cytoskeleton proteins. Platelet adhesion and its subsequent activation by calcium release is mainly regulated by the platelet receptor \u03b1IIb\u03b23 after binding to fibrinogen or the RGD domain of vWF, the main vWF receptor GPIb\/IX\/V and the collagen receptor \u03b12\u03b21. Microtubules together with the cytoplasmic, actin-rich cytoskeleton are responsible for the platelet structure. Different actin binding proteins have been identified in platelets such as filamin A, myosin and dystrophin. b Schematic model of G protein signal transduction in platelets regulated by Gq for platelet activation by the ultimate step of calcium release. Gi and Gs further influence the platelet activation by respectively inhibiting and stimulating the intracellular cAMP formation. c Schematic model of platelet secretion. The second amplification step in platelet activation is the release of alpha and dense granules in platelets guarantying irreversible platelet activation\nPlatelet adhesion also initiates multiple intracellular G protein-coupled signalling pathways (Fig.\u00a01b). Stimulation of Gq by different ligands such as adenosine diphosphate (ADP) and thromboxane A2 (TXA2) results in platelet activation by stimulating phospholipase C and a release of calcium from the intracellular stores. This platelet activation process is enhanced when Gi is activated and inhibited when Gs is activated both by modulating the intracellular cAMP level.\nPlatelet adhesion and activation eventually results in secretion from platelet organelles (Fig.\u00a01c) [37]. Resting platelets circulate as discoid anuclear cells and consist of a lipid bilayer and an internal dense tubular system, where calcium is sequestered. The platelet cytoplasm contains mitochondria, glycogen particles, lysosomes, and the platelet-specific storage granules: the \u03b1-granules and dense granules (Fig.\u00a02). The \u03b1-granules contain proteins such as platelet factor 4, \u03b2-thromboglobulin, platelet derived growth factor, fibrinogen, fibronectin, thrombospondin, plasminogen activator inhibitor I and vWF. Dense bodies are rich in serotonin, ATP, ADP and calcium.\nFig.\u00a02Electron microscopy (original magnification \u00d722,500) of platelets showing the dense tubular system (DTS), microtubules (MT), open canalicular system (OCS), alpha granules (G), glycogen (Gly) and the dense bodies (DB)\nThe study of platelet adhesion, G protein signalling and secretion is particularly useful for our understanding of several clinical disorders (Table\u00a01). The most obvious reason is of course the study of platelet defects in patients with an isolated platelet disorder leading to bleeding or thrombosis in order to gain additional information on the different pathways involved in platelet function. The analysis of human disorders in which the defective platelet phenotype is just one part of its clinical spectrum to unravel novel biological and genetic mechanisms involved in the disease. Also, using platelet functional and morphological studies can be used as a tool to find novel pathways involved in more complex disorders usually caused by more than one gene defect. Recently, it became obvious that the molecular pathways involved in more complex human disorders such as diabetes type 2 or neurological disorders such as schizophrenia, migraine, bipolar disorder, and depression can also be better understood by studying platelet signalling and secretion. Due to space limitations, it is impossible to give a full overview of all human disorders studied today by means of platelet research. This review describes some well-known as well as some less common disorders to illustrate how platelet research contributes to the understanding of thrombopathies [13, 35] as well as the broader future of this research outside its classical field of thrombosis and hemostasis (Table\u00a01).\nTable\u00a01Syndromic and non-syndromic platelet defects and the implicated genes according to the defective platelet pathwayType of platelet defectIsolated platelet disordersDisorders including a platelet defectDisorders studied by functional platelet assaysAdhesion and cytoskeletal defectsGlanzmann thrombasteniaMay-Hegglin anomaly, Fechtner syndrome, Epstein syndrome, and Sebastian syndromeNeurological disorders as bipolar disorder, schizophrenia, depression, autismProlonged bleeding timeAll: macrothrombocytopenia, prolonged bleeding timeNo other clinical problemsAll: leucocyte inclusions, Epstein,Fechtner: nephritis, deafness, cataractsITGB3 or ITGA2MYH9Bernard-Soulier SyndromeDuchenne Muscular DystrophyMacrothrombocytopenia, prolonged bleeding timeProlonged bleeding after surgeryNo other clinical problemsMuscle degenerationGPIb\u03b1, GPIb\u03b2 or GPIXDMDG protein signalling defectsADP P2Y12 receptorInducible Gs\u03b1 hyperfunction syndromeSubclinical platelet defectProlonged bleeding timeProlonged bleeding time after traumaNo other clinical problemsBrachydactyly, increased alkaline phosphatase and neurological or growth retardationP2Y12XL\u03b1sThromboxane TXA2 receptorPACAP overexpressionProlonged bleeding timeProlonged bleeding timeNo other clinical problemsMental retardation and hypogonadismTXA2RPACAPSecretion defectsGray platelet disorderHermansky Pudlack diseaseNeurological defect?Prolonged bleeding timeProlonged bleeding timeNo other clinical problemsAlbinism, lysosomal defect?HSP 1\u20138Delta storage Pool diseaseChediak Higashi diseaseProlonged bleeding timeProlonged bleeding timeNo other clinical problemsAlbinism, immunological lethal defect?LYSTThe following implicated genes are indicated in : ITGB3 integrin beta3; ITGA2 integrin alpha2; GPIb\u03b1 glycoprotein Ibalpha; GPIb\u00df glycoprotein Ibbeta; GPIX glycoprotein IX; P2Y12 purinergic receptor 12; TXASR thromboxane A2 receptor; MYH9 nonmuscle myosin heavy chain 9; DMD Dystrophin; XL\u03b1s extra-large stimulatory G protein alpha subunit; PACAP pituitary adenylate cylase-activating peptide; HSP 1\u20138 Hermansky Pudlack genes 1 through 8; LYST lysosomal trafficking regulator\nWhen considering type of platelet tests\nA clinical platelet-based bleeding problem is of course the main reason to investigate platelet function and morphology. The diagnostic approach to easy bruising or a suspected platelet-based bleeding disorder includes a careful history and physical examination of the patient as well as different laboratory investigations such as the Ivy bleeding time, platelet aggregation tests, ATP secretion, platelet adhesion by the platelet function analyzer (PFA100) and platelet morphology by electron microscopy [13, 40]. Reviewing the medical history can already establish whether the disorder is hereditary or acquired. The specific clinical findings useful in the differential diagnosis of coagulation versus platelet-based disorders are summarized in Table\u00a02. Frequently, mucocutaneous bleedings characterize abnormalities of platelet function. In contrast, hemorrhage into synovial joints and deep muscular hemorrhage are signs of severe hereditary coagulation disorders and very rare events in disorders of platelets, vessels or acquired coagulation disorders. Inherited disorders of platelet function are further subdivided based on the functions or responses that are abnormal and therefore can belong to different subgroups including abnormal platelet adhesion, signalling and secretion [36]. Platelet-based bleeding disorders are usually classified according to abnormalities of platelet function, platelet number (thrombocytopenia) or both [36].\nTable\u00a02Clinical presentation of coagulation and platelet-based bleeding disordersClinical symptomsDisorders of coagulationDisorders of plateletsPetechiae, epistaxisRareCharacteristicSuperficial ecchymosesCommon: large and solitaryCharacteristic: small and multipleBleeding from superficial cuts and bruisesMinimalPersistant: often profuseDelayed bleedingCommonRareDeep dissecting hematomasCharacteristicRareHemarthrosisCharacteristicRare\nFunctional and morphological platelet studies in patients with mainly a neurological, metabolic or another clinical problem but no obvious bleeding problem are usually not performed for diagnostic purposes but rather for research aims. In such patients, novel insights are expected to result from the platelet research studies, which are still preliminary today but in the future hopefully will help to better define when to ask for what type of platelet tests in a given patient.\nThrombopathies\nGlanzmann thrombasthenia and the Bernard-Soulier syndrome (BSS) are two rare inherited disorders of platelet adhesion. Glanzmann thrombasthenia (MIM 273800) is an autosomal recessive disorder, characterized by prolonged bleeding time and abnormal clot retraction [30, 31, 35]. The hallmark of this disease is severely reduced or absent platelet aggregation in response to various physiological platelet agonists such as ADP, thrombin and collagen. The defect is caused by mutations in one of the integrin genes, ITGA2 or ITGB3, encoding the \u03b1IIb\u03b23 receptor complex. Lack of expression or qualitative defects in \u03b1IIb\u03b23 results in a disturbed interaction between activated platelets and adhesive glycoproteins (fibrinogen at low shear and vWF at high shear) that bridge adjacent platelets during platelet aggregation (Fig.\u00a01a). The Bernard-Soulier syndrome (MIM 231200) is caused by abnormalities in the GP Ib\/IX\/V receptor complex due to mutations in the genes for GPIb\u03b1, GPIb\u03b2 or GPIX (but there are no reports of BSS affecting the GPV gene) [22]. It is an autosomal recessive disorder with moderate to severe macrothrombocytopenia, decreased platelet survival and often a spontaneous bleeding tendency. The bleeding events can be severe but are usually controlled by platelet transfusion. Most heterozygotes, with a few exceptions, do not have a bleeding diathesis. BBS platelets aggregate normally in response to physiological agonists (ADP and collagen), have a weak response towards low concentrations of thrombin and do not agglutinate when platelet rich plasma is stirred with ristocetin or botrocetin [22].\nDefects in G protein signaling resulting in an isolated platelet defect [36] are expected to be caused by a mutant G protein-coupled receptor (GPCR) since these can be cell-specific while the G proteins and their downstream effectors in this pathway are ubiquitously expressed. A dominantly inherited mutation (Arg60Leu) in the Gq-coupled TXA2 receptor was described in patients with a mild bleeding disorder characterized by defective platelet aggregation responses to TXA2 and its analogues (MIM 188070) [15]. In cultured cells, the Arg60Leu mutant was shown to impair phospholipase C (PLC) activation. Patients can be heterozygous (with some PLC activation left) or homozygous (without PLC activation) for this mutation but all have a life-long history of mucosal bleeding and easy bruising but no episodes of major bleeding such as hematuria, gastrointestinal bleeding or hemarthrosis [14]. The Gi-coupled ADP receptor P2Y12 (Fig.\u00a01b) is responsible for the sustained, full aggregation response to ADP. P2Y12 deficiency (MIM 609821) is an autosomal recessive bleeding disorder characterized by excessive bleeding, prolonged bleeding time and abnormalities that are very similar to those observed in patients with secretion defects (reversible aggregation in response to weak agonists and impaired aggregation towards low concentrations of collagen and thrombin), except for the severely impaired response to ADP [2]. Study of the heterozygous P2Y12 defect revealed platelets that undergo a normal first wave of ADP induced aggregation but abnormal ATP secretion with different agonists [3, 42].\nA defective platelet secretion is described for patients with absent alpha granules (gray platelet syndrome) or abnormal dense granules (delta-storage pool disease, \u03b4-SPD) [37]. Gray platelet syndrome or \u03b1-SPD (MIM 139090) owes its name to the fact that the typically enlarged platelets devoid of \u03b1-granule staining, present with a gray color in a Wright-stained blood smear [32]. Most cases are sporadic though some family studies suggest an autosomal dominant inheritance. Affected members have a life-long history of mucocutaneous bleeding, which may vary from mild to moderate in severity, prolonged bleeding time, mild thrombocytopenia, abnormally large platelets and an isolated reduction of the platelet \u03b1-granule content. The molecular defect(s) in \u03b1-SPD have not yet been defined and further insights into the molecular mechanisms responsible for platelet exocytosis (as the SNARE proteins) will help in the search for causes of human platelets secretory disorders. \u03b4-SPD (MIM 185050) may present as an isolated platelet function defect or can be associated with a variety of other congenital defects (see further). \u03b4-SPD is characterized by a bleeding diathesis of variable degree, mildly to moderately prolonged skin bleeding time (fully related to the amount of ADP or serotonin contained in the granule), abnormal platelet secretion induced by several agonists and a reduced platelet aggregation. The \u03b4-SPD platelets have decreased levels of the dense granule contents: ATP and ADP, serotonin, calcium and pyrophosphate (Fig.\u00a01c). It was estimated that 10\u201318% of patients with a congenital abnormality of the platelet function have \u03b4-SPD [12]. The inheritance pattern is autosomal recessive in some families while autosomal dominant in others but the molecular players responsible for \u03b4-SPD are still unknown.\nHuman disorders comprising a platelet defect\nDefects in platelet adhesion and subsequent platelet activation can also be due to an alteration in the platelet cytoskeletal organization, which consists of the microtubules and F-actin coupled to myosin, filamin and dystrophin [29]. Mutations in these widely expressed proteins result in a broader clinical phenotype. May-Hegglin anomaly (MIM 155100), Fechtner syndrome (MIM 153640), Epstein syndrome (MIM 153650), and Sebastian syndrome (MIM 605249) are characterized by macrothrombocytopenia, with or without different types of leukocyte inclusions, which can only be differentiated by an accurate ultrastructural examination [38]. In addition, patients with Epstein or Fechtner syndrome also suffer from nephritis, deafness, and congenital cataracts. Recently it became obvious that Sebastian platelet syndrome, May-Hegglin anomaly, Fechtner and Epstein syndrome have mutations in the same gene MYH9, encoding the 224-kD nonmuscle myosin heavy chain 9 polypeptide [26]. This gene is expressed in platelets, monocytes, granulocytes, the kidney, the auditory system but also in a lot of other tissues. MYH9 deficiency results in an alteration of the composition and agonist-induced reorganization of the platelet cytoskeleton [1, 6]. The cytoskeletal defect could also explain the abnormal platelet formation from megakaryocytes, resulting in thrombocytopenia and giant platelets in MYH9 deficiency. Why patients with May-Hegglin anomaly, Fechtner syndrome, Epstein syndrome and Sebastian platelet syndrome have different signs and symptoms in other tissues than their common defect in platelets still remains to be elucidated.\nDuchenne muscular dystrophy (DMD) is an X-linked recessive disease (MIM 310200) characterized by progressive degeneration of muscle resulting in early death from respiratory or cardiac failure. DMD is caused by mutations in the dystrophin gene, a 427-kDa membrane-associated cytoskeletal protein. Evidence for a role of dystrophin in platelets started with the observation that DMD patients tend to bleed more during spinal surgery for scoliosis than do patients during the same surgery with other underlying conditions [7]. Other C-terminal isoforms of dystrophin due to differential promoter usage and\/or alternative splicing at the 3\u2032-end of the gene have been identified in platelets (Dp71), the retina (Dp260) and in the peripheral (Dp116) and central nervous systems (Dp140). It is well established that platelets contain a complex membrane cytoskeleton that resembles, at least in part, the cytoskeleton found in muscle, but a role for dystrophin during platelet activation still remains to be clarified [18, 27]. Recent studies showed a role for dystrophin in normal controls during platelet spreading and adhesion by regulating the \u03b12\u03b21 receptor but this was not studied in DMD patients [4, 5]. Another study describes a normal platelet function in DMD patients and blames the selective defect of primary hemostasis in DMD to impaired vessel reactivity [43].\nPatients with an abnormal signal transduction are a heterogeneous group combined of defects in platelet G protein-coupled receptors (GPCR), the G proteins, and their effectors. Due to an extreme complex regulation between those key components (Fig.\u00a01b), the incidence of this class of defects is definitely underestimated and the underlying molecular defects for the signaling problems are still largely unknown. Platelet Gs activity is easily determined using the platelet aggregation-inhibition test which gives a value for the Gs activity based on the inhibition of platelet aggregation by the rapid generation of cAMP after incubation with different Gs agonists such as prostacyclin or prostaglandin (Fig.\u00a01b). A congenital Gs hyperfunction syndrome was described in three patients of two unrelated families due to a paternally inherited functional polymorphism in the extra-large stimulatory G-protein gene (XL\u03b1s) and its overlapping cofactor ALEX [8]. This XL\u03b1s variant is associated with Gs hyperfunction in platelets, leading to an increased trauma-related bleeding tendency but is also accompanied by neurological problems and brachydactyly (MIM 139320). A subsequent study revealed eight additional patients who paternally inherited the same XL\u03b1s polymorphism presenting with platelet Gs hyperfunction, brachydactyly, increased alkaline phosphatase and neurological problems or growth deficiency [9, 17].\nMegakaryocytes and platelets express the Gs-coupled VPAC1 receptor, for which both the pituitary adenylyl cyclase-activating polypeptide (PACAP) and the vasointestinal peptide (VIP) are specific agonists. Studies in two related patients with a partial trisomy 18p revealed three copies of the PACAP gene and elevated PACAP concentrations in plasma. The patients suffer from multiple neurological (epilepsy, hypotonia, convulsions, mental retardation, tremor, psychotic, hyperactive behavior), gastro-intestinal (diarrhea, vomiting) and endocrinological (hypoplasia of the pituitary gland, hypogonadotropic hypogonadism) problems and have a pronounced bleeding tendency (MIM 102980) [10]. The basal cAMP level in the patients' platelets was strongly elevated, providing a basis for the strongly reduced platelet aggregation. The VPAC1 signalling pathway also mediates megakaryocyte maturation and platelet formation (unpublished results). Patients with PACAP overexpression have a mild thrombocytopenia, a normal platelet survival, relatively small platelets and their bone marrow examination reveals almost no mature megakaryocytes.\nThere exist two rare syndromic forms of the \u03b4-SPD: the Hermansky-Pudlak syndrome (HPS) and the Chediak-Hygashi syndrome (CHS). HPS (MIM 203300) consists of several genetically different autosomal recessive disorders, which share the clinical manifestations of oculocutaneous albinism, bleeding, and lysosomal ceroid storage resulting from defects of multiple cytoplasmic organelles: melanosomes, platelet-dense granules, and lysosomes hypopigmentation [25, 36]. HPS can arise from mutations in at least eight different genes known to date (HSP1 to HSP8), all coding for proteins involved in the formation, trafficking or fusion of intracellular vesicles of the lysosomal lineage [44]. CHS (MIM 214500) is also an autosomal recessive disorder, characterized by variable degrees of oculocutaneous albinism, large peroxidase-positive cytoplasmic granules in hematopoietic and non-hematopoietic cells, \u03b4-SPD, recurrent infections, neutropenia, and an accelerated chronic lymphohistiocytic infiltration phase. The only known CHS-causing gene, LYST, codes for a large protein of unknown function but it seems that CHS is a disease of vesicle trafficking [16]. Most CHS patients present in early childhood and die before the age of 7\u00a0years unless treated by bone marrow transplantation [21]. About 10\u201315% of patients exhibit a much milder clinical phenotype and survive to adulthood but develop progressive and often fatal neurological dysfunction.\nHuman disorders examined by functional and morphological platelet assays\nSome mainly polygenetic disorders can also be studied by using platelets although the patients only present with a subtle subclinical platelet phenotype. It is not easy to define disorders such as diabetes type 2 or some neurological diseases according to a defective adhesion, G protein signalling and secretion since the platelet defect is not yet well studied and usually overlaps different pathways. This part of the review will only briefly focus on the use of platelet research in our understanding of neurological disorders. It has been known for years that certain cellular functions are very similar in platelets and in neurosecretory cells [33] but the link between functional platelet studies and neurological defects is novel. A platelet and a neuron contain both mitochondria and dense core vesicles in which transmitters (such as serotonin) are stored. Platelets release serotonin upon activation (Fig.\u00a02c) and the neuronal membrane is facilitated by a calcium dependent excitation-excretion coupling mechanism. In addition, both platelets and neurons contain functional neurotransmitter and neuromodulator receptor sites on their outer membrane such as adrenoceptors, serotonin receptors and serotonin transporters. More recently it was shown that platelets also express GABA and glutamate receptors [24, 34]. Serotonin uptake and release by platelets and serotonin plasma levels have been quantified and found to be altered in patients with bipolar disorder, schizophrenia, depression, aggression, autism, migraine, etc. [19, 23, 28]. Many epidemiological studies try to link these changes in activity of the serotonin transporter or changes in the density and responsiveness of the serotonin 2A receptor and the alpha2 adrenoceptor on the platelet membrane of these patients with genetic polymorphism in the corresponding genes. As for other epidemiological studies solid evidence for any linkage is not obvious but it is irrefutable that platelet studies have been invaluable in enabling an insight to be gained into the role of serotonin in a number of psychiatric and neurological disease [39, 41]. Major advances are expected of platelet research for this field in the near future since it is now obvious that besides the serotonin pathway, which was only the tip of the iceberg, many other pathways are chaired between platelets and neurons as well as the many gene products responsible for the regulation of granule formation, transport, secretion and endocytosis.\nConclusions\nPlatelet research is an expanding field originally studying isolated thrombopathies caused by the imbalance between thrombosis and hemostasis but more recently being able to bring novel insights in our understanding of human pathology in other clinical disciplines such as neurology, endocrinology and metabolic diseases. Platelets are easily accessible cells, and different techniques are possible to study platelet function and morphology under basal and activated conditions. Defects in platelet adhesion, G protein signalling and secretion can result from mutations in platelet-specific genes leading to isolated thrombopathies or from mutations in widely expressed genes leading to broader clinical phenotype including a platelet defect. In addition to using platelet research for diagnostic purposes, these platelet functional and morphological studies can also be used for research aims. From the close collaboration between clinicians of different disciplines, geneticists and the functional platelet research unit, novel insights in the pathogenesis of different human disorders are to be expected in the near future by using this strategy.","keyphrases":["platelets","adhesion","secretion","g protein signalling","cytoskeleton"],"prmu":["P","P","P","P","P"]}
{"id":"Int_Arch_Occup_Environ_Health-4-1-2175021","title":"Coping and sickness absence\n","text":"Objectives The aim of this study is to examine the role of coping styles in sickness absence. In line with findings that contrast the reactive\u2013passive focused strategies, problem-solving strategies are generally associated with positive results in terms of well-being and overall health outcomes; our hypothesis is that such strategies are positively related to a low frequency of sickness absence and with short lengths (total number of days absent) and durations (mean duration per spell).\nIntroduction\nA strong association exists between ill health and sickness absence, particularly for long absence spells (Marmot et al. 1995; Hensing et al. 1997). However, the decision of an employee to go on sick leave or to stay at work is not just the result of his or her (ill) health status alone (Aronsson et al. 2000; Rosvold and Bjertness 2001; Sandanger et al. 2000; Whitaker 2001; Anonymous 1979; Johansson and Lundberg 2004) but depends also on a number of demographic, social, and economic determinants (Johansson and Lundberg 2004; Voss et al. 2001; Eshoj et al. 2001). For instance, age (Sandanger et al. 2000), gender (Evans and Steptoe 2002), marriage (Mastekaasa 2000), level of education (Eshoj et al. 2001), salary (Chevalier et al. 1987), and sickness absence history (Landstad et al. 2001) are known to be associated with sickness absence behaviour. In addition, the way the individual deals with stressful situations (at work) is likely to affect his or her decision to report ill. In this article we focus on the role of this kind of so-called employee coping behaviour.\nThe relationship between coping and illness behaviour has been a major research focus over the past two decades (Somerfield and McCrae 2000). A variety of conceptual coping-frameworks have been proposed and numerous measures have been developed to assess ways of coping (McWilliams et al. 2003). Pioneering work in the field of coping has been carried out by Folkman and Lazarus (1980) who define coping as \u201cthe cognitive and behavioral efforts made to master, tolerate, or reduce external and internal demands and conflicts among them\u201d. In their opinion, coping has to be considered as a behaviour that is primarily determined by environmental demands, that is, coping is an individual response to a stressful environment. In contrast, other scholars (Holahan et al. 1996; Moos and Holahan 2003) consider coping primarily as a trait or as a resource. The former refers to a relatively stable personal characteristic: that is, similar coping strategies are used across a wide variety of situations (Parker and Endler 1992; Carver and Scheier 1994). The latter refers to the use of particular social and personal characteristics: that is, personal resources on which the individual may draw upon when dealing with stressful situations (Pearlin and Schooler 1978). This trait- or dispositional approach of coping implies a stable coping style or a coping resource regularly used.\nAs early as four decades ago, Kahn et al. (1964) distinguished between two general coping strategies: problem-solving strategies and reactive\u2013passive strategies. Their idea of two general coping strategies has been worked out by Lazarus and Folkman (1984) in what nowadays is probably the most popular and widely accepted conceptualization of coping behaviour. Problem solving-coping refers to active strategies that are directly targeted at solving the problem at hand, whereas reactive\u2013passive focused coping refers to those strategies that reduce the negative emotions that are evoked by the stressful situation (Elfering et al. 2005).\nMuch research on coping strategies reveals that both reactive\u2013passive strategies and avoidance strategies result in psychological and physical symptoms (Terry et al. 1996; Pisarski et al. 1998; Penley et al. 2002), whereas active, problem-solving coping generally has a positive impact on well-being and overall health outcomes (Penley et al. 2002). However, in their recent review, Austenfeld and Stanton (2004) criticized this popular and almost generally accepted conclusion. They identified over a hundred articles examining the relationship between reactive\u2013passive coping and adjustment (Stanton et al. 2002b) and found that hardly any of the coping instruments contained the same set of coping strategies, which made it practically impossible to aggregate the findings. Furthermore, the association between reactive\u2013passive strategies and psychological and physical symptoms appeared to be related to the way these strategies had been operationalized (Stanton et al. 2002a). It appeared that corruption of the original coping items as well as the use of item formulations that include the expression of emotional distress or self-deprecation result in spurious correlations.\nStudies on coping and sickness absence are scarce. Kristensen (1991) was among the first to investigate this relationship and he asserted that sickness absence itself should be regarded as coping behaviour reflecting the individual\u2019s perception of health or illness. Sickness absence itself, in his opinion, is a functional coping strategy, used by employees to reduce work-related strain by avoiding the workplace and thus creating for themselves the opportunity for recuperation. Kristensen was one of the first not to primarily focus on determinants of sickness absence, but rather tried to understand sickness absence from a coping perspective. By doing so, he went beyond existing concepts of coping by considering sickness absence \u201ca type of coping behaviour\u201d (Kristensen 1991). As he stated: \u201csickness absence can well be a rational coping behaviour seen in the light of a person\u2019s wish to maintain his\/her health and working capacity: as such it is the opposite of withdrawal behaviour\u201d. Clearly, this approach differs from considering coping as a personality trait or resource.\nIn the present study, coping is conceptualized and measured as a trait or disposition i.e. it is assumed that individuals tend to use rather similar coping strategies across a wide variety of situations. The Utrecht coping list (UCL) (Schreurs et al. 1993) was selected to assess the employees\u2019 coping style. This well-validated self-report questionnaire is the most widely used coping inventory in the Netherlands, both in research and in practice (Schreurs et al. 1993; Schaufeli and Van Dierendonck 1992; Norberg et al. 2005; Buitenhuis et al. 2003). Like the COPE questionnaire of Carver et al. (1989), the UCL asks individuals how they deal with stressful situations; that is, how often they engage in various exertions encountering problems or unpleasant occurrences. The UCL distinguishes between five coping styles that can be grouped together into two higher-order coping styles: active, problem-solving and a reactive\u2013passive style (Schaufeli and Van Dierendonck 1992). Hence, the UCL offers the possibility to investigate employees\u2019 coping styles at a more detailed level, at the same time taking into account the conceptual distinction between problem-solving and reactive\u2013passive coping.\nSickness absence has been measured in terms of frequency, (total) length of sickness absence, (mean) duration of sickness absence spells as well as by the sickness absence free interval. These sickness absence measures are defined in accordance with recommendations of Hensing et al. (1998) who pleaded for a more standardized international description of sickness absence measures. In their literature review, Hensing et al. pointed out the multi-interpretability of sick leave indicators and recommended basic measures to encompass the full spectrum of the sickness absence phenomenon to make studies more accessible for international comparisons. Recently, a study by Landstad et al. (2001) confirmed this line of reasoning by concluding that different forms of absenteeism need to be studied simultaneously, in order to distinguish changes in sickness absence pattern correctly.\nIn summary then, the aim of the study is to examine the role of coping styles in sickness absence. Based on the fact that, contrary to reactive\u2013passive strategies, problem-solving strategies are generally associated with positive results in terms of well-being and overall health outcomes, our hypothesis is that such strategies are positively related to a low frequency of sickness absence and with short lengths and durations. Reactive\u2013passive strategies, on the other hand, are not expected to be related to sickness absence.\nSubjects and methods\nStudy population and participants\nParticipants were employees of a large Dutch telecom company. An occupational health survey was sent to all 7,522 employees, including an assessment of coping strategies (response rate 51%; N\u00a0=\u00a03,852). Sickness absence of the participants was followed up for 1\u00a0year after the survey. Due to missing sickness absence data, the sample was reduced to 3,628 employees [3,302\u00a0men (mean age 44.7\u00a0years, SD\u00a0=\u00a07.5) and 311 women (mean age 39.7\u00a0years, SD\u00a0=\u00a08.7)]. A description of the sample is shown in Table\u00a01. During the first quarter after the start of the study, 64% of the participants have not been absent because of sickness, whereas, 7% of the participants have been absent for more than 14\u00a0days (length).\nTable\u00a01Demographics and absenteeism of participants Variable\u00a0Male91%\u00a0Age, mean (SD) (min\u2013max) years44.2 (7.7) (22\u201363)Marital status\u00a0Married or cohabiting79%\u00a0Single17%\u00a0Divorced or separated4%Educational level\u00a0Lower vocational education27%\u00a0Intermediate vocational education50%\u00a0Higher vocational education and university21%\u00a0Missing\/something else2%Working years present job\u00a01\u00a0year30%\u00a0>1\u20135\u00a0year43%\u00a0>5\u201310\u00a0year14%\u00a0>10\u00a0year14%Sickness absence first quarter\u00a00\u00a0days 64%\u00a01\u20137\u00a0days22%\u00a08\u201314\u00a0days6%\u00a0>14\u00a0days7%Function\u00a0Blue collar (executive)41%\u00a0Office workers (administrative)30%\u00a0Supervisors6%\u00a0Consultants16%\u00a0Managerial staff7%\nCompared to non-participants, participants were predominantly male, older, better paid, and were less absent for sickness (see Table\u00a02).\nTable\u00a02Demographics and absenteeism of participants and non-participantsParticipants (n\u00a0=\u00a03,628)Non-participants (n\u00a0=\u00a03,670)T Test p\u03c72pGender (%women)8.614.10.000Age, mean (SD) in years44.2 (7.7)40.7 (9.3)0.000Salary (%)0.000\u00a0Low40.653.2\u00a0Medium42.933.3\u00a0High16.513.5Absenteeism\u00a0Length mean (SD) days14.9 (39.9)22.9 (59.3)0.000\u00a0Frequency 1.20 (1.31)1.31 (1.46)0.000\nMeasures\nCoping style\nWe assessed the coping strategy of the participants using the shortened 19-item version of the original 30-item Utrecht Coping List (UCL) (Schreurs et al. 1993). This questionnaire was designed to measure the coping strategies people use in stressful situations, either life events or daily hassles. Each item is rated on a four-point Likert scale ranging from one (never) to four (very often).\nThe UCL includes five dimensions; (1) active problem-focusing (five items, e.g. thinking of different possibilities to solve a problem), (2) seeking social support (5 five items, e.g. seeking comfort and sympathy), (3) palliative reaction pattern (four items, e.g. looking for distraction), (4) avoidance behavior (three items, e.g. complying to avoid problematic situations) and (5) expression of emotions (two items, e.g. showing frustrations). The first three coping styles were found to cluster into a second-order active problem-solving factor, whereas both final styles clustered into a reactive\u2013passive factor (Schaufeli and Van Dierendonck 1992). According to the test manual, the internal consistencies as well as the test\u2013retest reliability are satisfactory (Schreurs et al. 1993). In order to assess the factorial validity of the shortened UCL in our employee sample, a confirmative factor analysis was carried out.\nSickness absence \nSickness absence data were taken from the sickness absence records of the employees filed in the database of ArboNed, an occupational health service (OHS) serving the telecom company. All spells of absence for medical reasons were centrally reported and registered by the executive manager of the company. Absence spells longer than 2\u00a0weeks were verified by an occupational physician by inviting the employee on sick leave for an interview. Therefore, the validity of the absence data is assumed to be high.\nMeasures used are (1) (total) length of sickness absence in current and new spells during the study period (1\u00a0year) per sick listed person (i.e. total number of days absent) (2) frequency of sickness absence (new sick-leave spells during the study period (1\u00a0year) and (3) (mean) duration of sickness absence (sick-leave days in new spells during the study period (1\u00a0year) per spell). The duration of sickness absence is classified into more or less than 7\u00a0days. In our sample, short-term sickness (less than 7\u00a0days) accounts for 75% of the absences and mainly represents minor ailments. Finally, we assessed the median time before the onset of a new sick leave period after the occupational health survey.\nStatistical analysis\nConfirmatory factor analysis (CFA), using the AMOS 5 software program (Arbuckle 2003) was used to test the fit of two competing models: M1 that assumes that all 19 items load on one general coping factor, and M2 that assumes that the items load on the five hypothesized correlated factors. Maximum likelihood estimation methods were used and the input for each analysis was the covariance matrix of the items. The goodness-of-fit of both models was evaluated using the \u03c72 goodness-of-fit statistic and the root mean square error of approximation (RMSEA). However, \u03c72 is sensitive to sample size so that the probability of rejecting a hypothesized model increases when sample size increases, even if the difference between the fitted model and the \u201ctrue\u201d underlying model is very small. To overcome this problem, the computation of relative goodness-of-fit indices is strongly recommended (Bentler 1990). Three relative goodness-of-fit indices were computed: the normed fit index (NFI), the non-normed fit index (NNFI) and the comparative fit index (CFI). The latter is particularly recommended for model comparison purposes (Goffin 1993). For all relative fit-indices, as a rule of thumb, values greater than 0.90 are considered as indicating a good fit (Byrne, 2001, pp. 79\u201388), whereas values smaller than .08 for RMSEA indicate acceptable fit (Cudeck and Browne 1993). Next, Cronbach alphas were calculated for the UCL-subscales.\nIn a next step, scale scores for different coping strategies were calculated and transformed into scale scores ranging from 0 to 100. Finally, tertiles of the distribution of the 0\u2013100 scale scores were used to distinguish between low-, medium- and high levels of the coping strategies.\nTo examine the relationship between coping and sickness absence, odds ratios and corresponding 95% confidence intervals were calculated using logistic regression analysis.\nStepwise multiple logistic regression analysis was used to study the (confounding) influence of sociodemographic factors and other determinants on the relationship between coping and sickness absence. The magnitude of the (confounding) effects was assessed by calculating the proportion of the excess risk (OR minus 1.0) explained when fitting these terms in the model.\nFinally, the period between the health surveillance and the onset of a new period of absenteeism was evaluated using survival analysis. Since we wish to estimate the probability of absenteeism at a designated time interval (conditional probability) the Kaplan\u2013Meier methodology (Kaplan and Meier 1958) has been applied. With this statistical technique, means, medians and confidence intervals of the \u2018survival\u2019 (in this study: the onset of absenteeism) are calculated without making assumptions about the survival distribution.\nResults\nUCL factor structure\nAs can be seen in Table\u00a03, confirmatory factor analysis (EFA) corroborated the underlying five-factor structure of the short form of the UCL. More particularly, all fit-indices of M2\u2014the hypothesized model with five correlated factors\u2014sufficed their respective criteria, except NNFI that approached its criterion of 0.90. The mean correlation between the five factors was 0.24, ranging from \u22120.04 to 0.45. Moreover, the fit of M2 was superior to that of M1 that assumed that all items load on one undifferentiated coping factor (\u0394\u03c72\u00a0=\u00a010146.22; df\u00a0=\u00a010; P\u00a0<\u00a00.001). Hence the factorial validity of the UCL-15 was demonstrated.\nTable\u00a03Fit indices of one-factor (M1) and two-factor (M2) models of coping (UCL-19)Model\u03c72dfGFIAGFIRMSEANFINNFICFIM11030.291520.690.610.140.430.360.43M2184.071420..950.930.060.900.880.90Null model17976.601710.540.490.17\u2013\u2013\u2013GFI goodness of fit index, AGFI adjusted goodness of fit index, RMSEA root mean square estimate of approximation, NFI normed fit index, NNFI non-normed fit index, CFI comparative fit index; all \u03c72, P\u00a0<\u00a00.001\nThe Cronbach alphas for the subscales avoidance behaviour, expression of emotions, seeking social support, active problem-focusing and palliative reaction in this study were 0.67, 0.65, 0.76, 0.81 and 0.68, respectively. Although some values are slightly below 0.70, which is recommended for established scales, all values are well above 0.60, which is deemed satisfactory for newly developed scales (Nunnaly and Bernstein 1994).\nSickness absence and demographics \nAs can be seen from Table\u00a04, length (total number of days absent) and duration (mean duration per spell) of sickness absence are associated with gender (i.e. women), being divorced or single, having an intermediate or lower education, a shorter period working in the present (current) job, lower salary, higher age, and a history of sickness absence both for length and frequency. Likewise, a higher frequency of sickness absence was associated with gender (i.e. women), being divorced, an intermediate salary and a history of sickness absence both for length and frequency. In our sample there is no association between absence frequency and level of education, the period working in the current job, or age.\nTable\u00a04Associations of demographics and sickness absenceSickness absenceLength >14\u00a0daysDuration\u00a0>7\u00a0daysFrequency >2xOR95% CIOR95% CIOR95% CIGender\u00a0Woman1.001.001.00\u00a0Man0.490.38\u20130.620.660.51\u20130.850.420.32\u20130.55Married\u00a0Married1.001.001.00\u00a0Single0.830.66\u20131.031.251.03\u20131.521.050.82\u20131.36\u00a0Divorced1.731.24\u20132.412.180.54\u20138.811.541.04\u20132.28Education\u00a0University1.001.001.00\u00a0Higher vocational education0.880.59\u20131.331.130.72\u20131.781.000.63\u20131.59\u00a0Interm. vocational education1.421.00\u20132.001.931.30\u20132.861.160.78\u20131.72\u00a0Lower vocational education2.071.45\u20132.962.841.90\u20134.241.300.86\u20131.96Present (current) job\u00a0>10\u00a0years1.001.001.00\u00a05\u201310\u00a0years0.980.74\u20131.281.020.78\u20131.341.040.74\u20131.46\u00a0<5\u00a0years0.670.54\u20130.830.610.49\u20130.760.910.69\u20131.19Salary\u00a0Low 4\u201361.001.001.00\u00a0Intermediate 7\u201390.500.43\u20130.600.490.41\u20130.591.711.28\u20132.28\u00a0High >90.380.30\u20130.490.330.25\u20130.441.070.79\u20131.44Age\u00a0<35\u00a0years1.001.001.00\u00a035\u201345\u00a0years1.381.07\u20131.781.541.17\u20132.021.200.90\u20131.60\u00a0>45\u00a0years1.481.16\u20131.881.791.38\u20132.320.930.70\u20131.22History sickness absence in days (length) 1\u00a0year before\u00a001.001.001.00\u00a01\u201371.571.24\u20132.011.190.95\u20131.503.072.18\u20134.31\u00a08\u2013143.682.85\u20134.742.652.08\u20133.396.304.44\u20138.95\u00a0> 149.727.75\u201312.24.263.43\u20135.2912.99.40\u201317.8History frequency (1\u00a0year before)\u00a00x1.001.001.00\u00a01\u20132x2.772.26\u20133.411.911.58\u20132.323.962.91\u20135.40\u00a0>2x8.666.81\u201311.023.833.04\u20134.8417.4712.6\u201324.2n ranges between 3,575\u20133,606 due to missing values\nSickness absence and ways of coping\nAs displayed in Table\u00a05, a greater length (total number of days) of sickness absence is predicted by low- or medium-active problem-focusing, avoidance behaviour and a medium- or high palliative reaction. The frequency and the duration of sickness absence are associated in a similar way, however, the latter showing a relation with low seeking social support rather than a palliative reaction. Table\u00a05b summarizes the significant associations between various sickness absence measures and ways of coping (Table\u00a05). It can be seen from this table that the crude ORs of the active and avoidant coping styles show the most consistent patterns of associations across all sickness absence measures.\nTable\u00a05Associations of coping and sickness absenceSickness absenceLength\u00a0>14\u00a0daysDuration\u00a0>7\u00a0daysFrequency >2xOR95% CIOR95% CIOR95% CIProblem-solvingActive problem-focusing\u00a0Low1.001.001.00\u00a0Medium0.840.70\u20130.990.830.69\u20130.990.840.68\u20131.04\u00a0High0.610.49\u20130.750.690.56\u20130.860.780.53\u20130.87Seeking social support\u00a0Low1.001.001.00\u00a0Medium0.980.82\u20131.170.970.80\u20131.161.01\u22120.80\u20131.26\u00a0High0.920.75\u20131.120.810.66\u20130.991.150.90\u20131.45Palliative reaction\u00a0Low1.001.001.00\u00a0Medium1.221.01\u20131.491.150.94\u20131.411.431.13\u20131.81\u00a0High1.331.11\u20131.591.190.99\u20131.431.401.12\u20131.74Reactive\u2013passiveAvoidance behaviour\u00a0Low1.001.001.00\u00a0Medium1.110.92\u20131.321.140.94\u20131.371.220.98\u20131.51\u00a0High1.351.10\u20131.651.321.07\u20131.631.391.09\u20131.67Expression of emotions\u00a0Low 1.001.001.00\u00a0Medium1.050.87\u20131.281.210.99\u20131.480.910.72\u20131.14\u00a0High1.190.95\u20131.501.130.89\u20131.441.290.99\u20131.69Summary table\u00a05Sickness absenceLengthDurationFrequencyProblem-solvingActive problem-focusingXXXSeeking social support\u2013X\u2013Palliative reactionX\u2013XReactive\u2013passiveAvoidance behaviourXXXExpression of emotions\u2013\u2013\u2013\nSickness absence, and demographics and ways of coping\nOf course, the question arises whether or not the association between coping and sickness absence could be explained by previous sickness absence and by demographics. Therefore, Table\u00a06 displays the ORs for the three sickness absence measures with coping strategies after adjustment for previous sickness absence and the demographics mentioned in Table\u00a04.\nTable\u00a06Odds ratios (95% CI) for sickness absence (length\u00a0>\u00a014\u00a0days, frequency >2x, duration >7\u00a0days) associated with different coping styles measured at the start of a 1-year follow-up study (n\u00a0=\u00a03575)AdjustmentsLength >14\u00a0daysDuration >7\u00a0daysFrequency >2xOR95% CIOR95% CIOR95% CIProblem-solvingActive problem- focusingNo adjustments (crude OR)0.63(0.51\u20130.77)0.71(0.57\u20130.80)0.69(0.54\u20130.89)History sickness absence length0.71(0.56\u20130.88)0.78(0.63\u20130.97)0.71(0.55\u20130.93)History sickness absence length\u00a0+\u00a0gender (female)0.72(0.58\u20130.90)0.79(0.63\u20130.98)0.73(0.55\u20130.95)History sickness absence length\u00a0+\u00a0gender\u00a0+\u00a0salary (high)0.77(0.61\u20130.97)0.86(0.69\u20131.07)0.74(0.57\u20130.98)History sickness absence length\u00a0+\u00a0gender\u00a0+\u00a0salary\u00a0+\u00a0education (high)0.79(0.63\u20130.99)0.88(0.71\u20131.10)0.74(0.57\u20130.98)History sickness absence length\u00a0+\u00a0gender\u00a0+\u00a0salary\u00a0+\u00a0education\u00a0+\u00a0marital status (married)0.79(0.62\u20130.99)0.880.70\u20131.10)0.74(0.57\u20130.98)Seeking social supportNo adjustments (crude OR)0.90(0.73\u20131.09)0.80(0.65\u20130.98)1.14(0.90\u20131.45)History sickness absence length0.95(0.78\u20131.16)0.81(0.66\u20131.00)1.06(0.83\u20131.37)History sickness absence length\u00a0+\u00a0gender (female)0.83(0.67\u20131.03)0.78(0.63\u20130.97)1.01(0.78\u20131.30)History sickness absence length\u00a0+\u00a0gender\u00a0+\u00a0salary (high)0.87(0.70\u20131.08)0.83(0.67\u20131.02)1.03(0.80\u20131.33)History sickness absence length\u00a0+\u00a0gender\u00a0+\u00a0salary\u00a0+\u00a0education (high)0.88(0.71\u20131.10)0.84(0.68\u20131.04)1.03(0.80\u20131.34)History sickness absence length\u00a0+\u00a0gender\u00a0+\u00a0salary\u00a0+\u00a0education\u00a0+\u00a0marital status (married)0.88(0.71\u20131.10)0.84(0.68\u20131.04)1.03(0.80\u20131.34)Palliative reactionNo adjustments (crude OR)1.32(1.10\u20131.58)1.19(0.99\u20131.43)1.37(1.10\u20131.72)History sickness absence length1.24(1.02\u20131.50)1.16(0.96\u20131.41)1.18(0.93\u20131.50)History sickness absence length\u00a0+\u00a0gender (female)1.20(0.99\u20131.45)1.14(0.94\u20131.38)1.15(0.91\u20131.46)History sickness absence length\u00a0+\u00a0gender\u00a0+\u00a0salary (high)1.20(0.99\u20131.46)1.14(0.94\u20131.38)1.15(0.91\u20131.45)History sickness absence length\u00a0+\u00a0gender\u00a0+\u00a0salary\u00a0+\u00a0education (high)1.21(1.00\u20131.47)1.15(0.95\u20131.40)1.15(0.91\u20131.46)History sickness absence length\u00a0+\u00a0gender\u00a0+\u00a0salary\u00a0+\u00a0education\u00a0+\u00a0marital status (married)1.22(1.00\u20131.48)1.16(0.96\u20131.41)1.15(0.91\u20131.46)Reactive\u2013passiveAvoidance behaviourNo adjustments (crude OR)1.36(1.11\u20131.66)1.33(1.07\u20131.64)1.37(1.07\u20131.75)History sickness absence length1.24(0.99\u20131.54)1.29(1.04\u20131.60)1.37(1.05\u20131.78)History sickness absence length\u00a0+\u00a0gender (female)1.23(0.99\u20131.53)1.28(1.03\u20131.59)1.36(1.04\u20131.77)History sickness absence length\u00a0+\u00a0gender\u00a0+\u00a0salary (high)1.22(0.98\u20131.52)1.27(1.02\u20131.57)1.35(1.04\u20131.75)History sickness absence length\u00a0+\u00a0gender\u00a0+\u00a0salary\u00a0+\u00a0education (high)1.21(0.97\u20131.51)1.26(1.02\u20131.57)1.35(1.03\u20131.75)History sickness absence length\u00a0+\u00a0gender\u00a0+\u00a0salary\u00a0+\u00a0education\u00a0+\u00a0marital status (married)1.22(0.97\u20131.52)1.27(1.02\u20131.58)1.35(1.03\u20131.75)Expression of emotionsNo adjustments (crude OR)1.17(0.97\u20131.40)1.01(0.83\u20131.22)1.41(1.13\u20131.75)History sickness absence length1.07(0.88\u20131.31)0.98(0.80\u20131.19)1.34(1.06\u20131.68)History sickness absence length\u00a0+\u00a0gender (female)1.06(0.87\u20131.29)0.97(0.79\u20131.18)1.32(1.05\u20131.67)History sickness absence length\u00a0+\u00a0gender\u00a0+\u00a0salary (high)1.07(0.87\u20131.30)0.98(0.80\u20131.19)1.33(1.05\u20131.67)History sickness absence length\u00a0+\u00a0gender\u00a0+\u00a0salary\u00a0+\u00a0education (high)1.08(0.88\u20131.31)0.99(0.81\u20131.20)1.33(1.05\u20131.67)History sickness absence length\u00a0+\u00a0gender\u00a0+\u00a0salary\u00a0+\u00a0education\u00a0+\u00a0marital status (married)1.08(0.88\u20131.31)0.99(0.81\u20131.20)1.33(1.05\u20131.67)All odds ratios are based each time on the same 3,575 employees without missing values on each variable in the model \nLength\nAdjustment for sickness absence history increases the excess risk to be absent for more than 14\u00a0days in one year by 22% for active problem-focusing (thus, sickness absence history reduces the effect of active coping), while reducing it by 25 and 33% for palliative reaction and avoidance coping, respectively. After adjustment for gender and sickness absence history, the excess risk for length in addition to palliative reaction and sickness absence history decreases by 17%.\nThe excess risk for length adjusted for salary in addition to active problem-focusing, sickness absence history and gender increases by 18%. In summary, adjusted for several confounding variables, the length of sickness absence is effectively influenced by active problem-focusing and palliative reaction.\nFrequency\nAdjustment for sickness absence history barely minimizes the risk for frequency by coping considering active problem-focusing and avoidance behaviour. For palliative coping, the reduction for the excess risk amounts to 51%. When adjusted for gender, in addition to sickness absence history, the risk of high frequency in association with palliative reaction reduces by another 16%. In sum, adjusted for several confounding variables the frequency of sickness absence is effectively influenced by active problem-focusing, avoidance behaviour and expression of emotions.\nDuration\nAdjustment for sickness absence history reduces the excess risk of active problem focusing by 24%, of seeking social support and palliative reaction by 16%, and of avoidance behaviour by 12%. Adjustment for gender in association with seeking social support affects the excess risk of duration by 16%. In summary, adjusted for several confounding variables, the duration of sickness absence is effectively influenced by active problem focusing, avoidance behaviour and seeking social support.\nEffects on the onset of a new period of absenteeism\nDuring the first year, the median time before the onset of a new episode of absenteeism is significantly shorter for those low in active problem-focusing, high in avoidance, and high in a palliative response. For the two remaining coping styles, no significant results were found (Table\u00a07). This means that employees who are used to solving problems actively instead of avoiding problems or engaging in alternative behaviours, enter sick leave later, the next time.\nTable\u00a07Kaplan\u2013Meier: the relation between different coping styles and the onset of absenteeism in the year after coping assessmentLog rankMedian (days)SE95% CIStatdfSignProblem-solvingActive problem-focusing\u00a0Low1527137\u2013167\u00a0Medium17013145\u2013195\u00a0High17614149\u20132039.4420.01Seeking social support\u00a0Low16810148\u2013188\u00a0Medium1658148\u2013182\u00a0High15517122\u20131880.4520.80Palliative reaction\u00a0Low18214155\u2013209\u00a0medium15512132\u2013178\u00a0High1467131\u201316113.6520.00Reactive\u2013passiveAvoidance behaviour\u00a0Low18211160\u2013204\u00a0Medium15111130\u2013172\u00a0High14410125\u201316314.620.00Expression of emotion\u00a0low16510146\u2013184\u00a0Medium16711145\u2013189\u00a0High15612133\u20131790.9820.61\nDiscussion \nIn accordance with our hypothesis, and after adjustment for potential confounders, employees with an active problem-solving coping strategy are less likely to drop out because of sickness absence in terms of frequency, length (total number of days absent, longer than 14\u00a0days), and duration (mean duration per spell, more than 7\u00a0days) of sickness absence. This positive effect is observed in the case of \u2018seeking social support\u2019 only for duration of sickness absence, and in the case of \u2018palliative reaction\u2019 only for length and frequency of sickness absence. In contrast, an avoidant coping style, representing a reactive\u2013passive strategy, significantly increases the likelihood of frequent absences, as well as the duration of sickness absence. Expression of emotions, representing another reactive\u2013passive strategy, has no effect on sickness absence. The median time before the onset of a new episode of absenteeism, finally, is significantly extended for active problem-solving and reduced for avoidance and for a palliative response.\nIn summary, we conclude that in accordance with our hypothesis, a problem-solving coping strategy, in contrast to a reactive\u2013passive coping strategy, significantly reduces sickness absence. This result seems to corroborate other research findings that showed that problem-solving coping is associated with well being and overall health outcomes (Kohn 1996). On the other hand, our results are at odds with research findings that document a positive relationship between reactive\u2013passive coping and health (Austenfeld and Statton 2004; Coyne and Racioppo 2000). Austenfeld and Statton (2004) have argued that the negative effect of reactive\u2013passive coping on health may partly be attributed to the operationalizaton of this construct, and therefore recommended a clear description of the reactive\u2013passive coping items used. The idea is that reactive\u2013passive coping can be separated into two factors, namely emotional expression and emotional processing (Lazarus 1993). The former factor is an active attempt to acknowledge, explore meanings or come to an understanding of one\u2019s emotions. Items measuring emotional processing, however, focus on the acknowledgement of emotions, the validness and importance of feelings, the delving into the feelings. Especially, emotional processing has a positive association with health, although how the influencing occurs is still unclear. The items that tap reactive\u2013passive coping in the UCL refer to the expression of emotions and not to their processing. This probably explains the indifferent and negative effect on sickness absence by \u2018emotional expression\u2019 and \u2018avoidance behaviour\u2019, respectively. A second possible explanation can be that reactive\u2013passive strategies have a positive relationship to health but not necessarily with sickness absence.\nOur study partly refutes the assumption of Kristensen (1991) that sickness absence is a coping strategy by itself. Kristensen claimed that employees who use sickness absence as a coping strategy would experience less work-related strain, especially in jobs with poor decision latitude. Accordingly, because they are no longer exposed to their stressful jobs, employees would recuperate during sickness absence, especially in the case of psychosomatic symptoms. In our study, sickness absence history that can be considered a proxy of the coping strategy of sickness absence had only a minor impact on sickness absence given a general coping style. And although the effect is less strong, the measured coping strategies of the UCL still have an effect on sickness absence.\nThe favourable outcome of problem-solving coping in relation to sickness absence can be attributed to being engaged in active transactions between person and environment with the aim of alleviating stress-inducing situations (Lazarus 1993; Huizink et al. 2002; Roesch and Weiner 2001). Efforts to remove the stressor, gathering information, and finding possible solutions for the problems are a few examples. In general, these strategies are associated with self-confidence and perceived control, and are observed in individuals who are persistent and assertive, self-efficacious, and less anxious and depressed (Heppner 1988; Heppner and Baker 1997).\nTwo factors in the evaluation of problem-solving coping should be commented upon. Men are believed to be more likely to confront a problem with active coping, whereas women are believed to exhibit a more reactive\u2013passive response (Pearlin and Schooler 1978; Hamilton and Fagot 1998). For instance, a meta-analysis of Tamres et al. (2002) showed that compared to men, women are more likely to use indirect strategies that involve verbal expression or to seek emotional support. Huizink et al. (2002), however, argue that the presumed effectiveness of problem-solving strategies is based on the assumption that male-gender role behaviour is superior. She suggests that studies, as a result of gender bias, have failed to identify other styles of coping as potentially effective. In our study, however, considering several styles, the adjustment for gender barely affects the influence of coping on sickness absence measures.\nAnother complicating factor in the evaluation of the effectiveness of problem-solving coping may be that reviewers group several distinct coping behaviours under this one single coping category in an effort to simplify the findings (Tamres et al. 2002). For instance, problem-solving coping may be composed of different behaviours. This is underscored by our finding that different problem-solving strategies have different outcomes on sickness absence. Seeking social support, for example, affects only duration (marginally), whereas active problem-focusing affects length, duration and frequency. The difference in outcome of different sickness absence measures in the case of seeking social support may be clarified by Stansfeld et al. (1997) who argues that social support may influence absence-related behaviour and encourage a person to take absence at a time of illness. Contrarily, one may postulate that social support also shortens sickness absence. Both postulations may result in the absence of a substantial effect.\nTo the authors\u2019 knowledge, this is the first study with four sick leave outcome measures in relation to coping that reveals a more comprehensive picture of changes in the sick leave pattern. In line with Isacsson et al. (1992), we can conclude that \u201cadding more measures gives a more comprehensive picture of sickness absenteeism and of differences between groups\u201d. For instance, the present study demonstrates a relation between a palliative coping reaction and length of sickness absence in contrast with the duration of sick leave. Without the differential pattern for sickness absence, the differential effects of several coping strategies would remain invisible. Another, and perhaps even more important, argument to use different measures of sickness absence is the accessibility of this study for international comparisons in future research.\nFinally, the multi-factorial aetiology of sickness absence requires discussion. Alexanderson (1998) pointed out that different disciplines and scientific traditions deal in different ways with absenteeism. In medical science, for instance, the focus of research is on occurrence, etiology and intervention, whereas the focus in medical sociology is on interacting factors within a pre-circumscribed model. She and other authors (Whitaker 2001; Alexanderson 1998), therefore categorized the many factors of sickness absence in three levels: macro\/national level (Alexanderson 1995) (e.g. insurance systems), organizational level (Jeurissen and Nyklicek 2001; Vahtera et al. 1996) (e.g. job demands, resources) and individual level (e.g. gender, education). Recognizing this phenomenon, our analyses were adjusted for several known risk factors at the level of the individual. Since the present study was conducted in one Dutch company, the influence of organizational and socioeconomic factors was equally present in all groups and in this sense controlled for.\nA strong point of our study is the detailed way in which sickness absence is assessed, using objective archival data. Thus far, relatively little attention has been paid to the implications of different quantitative measures of sickness absence. Moreover, a prospective design was used that allowed for predicting future sickness absenteeism.\nA limitation of the study is the non-recurring measurement of coping in our study. Therefore, we cannot rule out the possibility that sickness absence might influence the way employees cope with stressful situations. Although, coping styles, as measured with the UCL, have proven to be relatively stable in time (Norberg et al. 2005), reversed causation cannot be ruled out.\nA second limitation could be the Cronbach\u2019s alpha of some subscales of the UCL (slightly below the 0.70). However, the criterion of 0.70 is an arbitrary value that is not universally accepted as the minimum level of acceptability. As an example of the arbitrariness of this criterion, Nunnally (1967) mentioned that \u03b1s ranging from 0.50\u20130.60 would be acceptable, but in the second edition of that book he suggests that 0.70 is the minimally acceptable value\u2014without further justification (Nunnally 1978). Moreover, the minimally required degree of reliability is a function of the research purpose; for individual-level, diagnostic research \u03b1 should be much higher than for the basic, group-level research reported in our study (Peterson 1994). Hence we used a minimum threshold for coefficient \u03b1 of 0.65 as was recently proposed by De Vellis (2003).\nIn spite of these limitations, the results of the present study support the notion that problem-solving coping and reactive\u2013passive strategies are inextricably connected with frequency, duration, length and onset of sickness absence. Especially \u2018active problem-focusing\u2019 decreases the chance of future sickness absence.","keyphrases":["coping","sickness absence","frequency","length","duration","ucl"],"prmu":["P","P","P","P","P","P"]}
{"id":"J_Autism_Dev_Disord-4-1-2226079","title":"Brief Report: Normal Intestinal Permeability at Elevated Platelet Serotonin Levels in a Subgroup of Children with Pervasive Developmental Disorders in Cura\u00e7ao (The Netherlands Antilles)\n","text":"This study investigated the relationship between platelet (PLT) serotonin (5-HT) and intestinal permeability in children with pervasive developmental disorders (PDD). Differential sugar absorption and PLT 5-HT were determined in 23 children with PDD. PLT 5-HT (2.0\u20137.1 nmol\/109 PLT) was elevated in 4\/23 patients. None exhibited elevated intestinal permeability (lactulose\/mannitol ratio: 0.008\u20130.035 mol\/mol). PLT 5-HT did not correlate with intestinal permeability or GI tract complaints. PLT 5-HT correlated with 24 h urinary 5-hydroxyindoleacetic acid (5-HIAA; p = .034). Also urinary 5-HIAA and urinary 5-HT were interrelated (p = .005). A link between hyperserotonemia and increased intestinal permeability remained unsupported. Increased PLT 5-HT in PDD is likely to derive from increased PLT exposure to 5-HT. Longitudinal studies, showing the (in)consistency of abnormal intestinal permeability and PLT 5-HT, may resolve present discrepancies in the literature.\nIntroduction\nAutism has been linked to gastrointestinal (GI) disturbances (White 2003). It is, however, questionable whether GI anomalies in children with autism are specific (Erickson et\u00a0al., 2005). An increase of chronic diarrhea, constipation, abdominal bloating and food regurgitation has been found in some studies, but could not be confirmed in more recent studies (reviewed by Erickson et\u00a0al. 2005). Increased GI permeability, as established through the differential sugar absorption test (SAT), was demonstrated in 9\/21 (43%) (D\u2019Eufemia et\u00a0al., 1996) and 19\/26 (76%) (Horvath & Perman, 2002) of patients diagnosed with autism. The SAT measures the integrity of the intestine by the ingestion of two indigestible saccharides that after GI uptake become fully excreted in urine. One of these (usually lactulose) passes the intestinal wall through paracellular transport (\u2018leakage\u2019), while the other (usually mannitol) passes by paracellular and transcellular transport. The urinary lactulose\/mannitol ratio is used as a measure of intestinal integrity and permeability (van Elburg et\u00a0al., 1995). In addition, a recent study found a high prevalence of congenital GI anomalies (adjusted odds ratio 5.1, 95% confidence interval 1.8\u201314.1), notably pyloric stenosis, in autism, which may be linked to the high rate of GI dysfunction reported by their parents (Wier, Yoshida, Odouli, Grether, & Croen, 2006).\nThe implication of the GI tract in autistic pathophysiology warrants more detailed investigation of the gut-brain axis (Erickson et\u00a0al., 2005). Especially the role of serotonin (5-hydroxytryptamine; 5-HT), as a messenger within this axis (Gershon, 2005), deserves attention. Many different aspects of the 5-HT system in autism have already been studied (Burgess, Sweeten, McMahon, & Fujinami, 2006; Croonenberghs, Verkerk, Scharpe, Deboutte, & Maes, 2005; Janusonis, 2005; Mulder et\u00a0al., 2004). A recent report (Mulder et\u00a0al., 2004) on platelet (PLT) 5-HT in PDD showed PLT hyperserotonemia in approximately 36% of patients with autism and in 58% of patients with PDD not otherwise specified (NOS). Using mixture-modeling analysis Mulder et\u00a0al. (2004) derived an empirical cut-off value that enabled dichotomization of patients with PDD into normo- and hyperserotonemic. Extensive behavioral assessments did, however, not show significant correlates with PLT 5-HT or hyperserotonemic status.\nA common (developmental) factor, causing both an autistic brain and deregulated 5-HT release from the GI tract, years after birth, may be involved in the etiology of PDD (Janusonis, 2005). The primary site of the hyperserotonemia in autism is likely to be located in the GI tract. Serotonin is a biogenic amine that derives from the essential amino acid tryptophan (Tryp) by hydroxylation and subsequent decarboxylation. The GI tract contains about 80% of bodily 5-HT, which is unevenly distributed among the enterochromaffin cells (90\u201395%) and neurons (5\u201310%) (Houghton, Atkinson, Whitaker, Whorwell, & Rimmer, 2003). The main functions of 5-HT are in smooth muscle contraction, blood pressure regulation, and peripheral and central neurotransmission. Serotonin localized in the basolateral stores of enterochromaffin tissue is released upon neuronal, chemical or mechanical stimulation. Several 5-HT receptors control GI motility, sensation and secretion (Gershon, 2005). Following its release, 5-HT is removed from the interstitial space by 5-HT selective reuptake transporters (Gershon, 2005). However, part of the 5-HT enters the portal blood and systemic circulation where it is either rapidly taken up and accumulated by PLT, or metabolized by the liver, lung and kidneys into its major metabolite 5-hydroxyindoleacetic acid (5-HIAA) (Houghton et\u00a0al., 2003). Platelets store and transport the majority (99%) of circulating 5-HT (Ortiz, Artigas, & Gelpi, 1988).\nElevated PLT 5-HT levels observed in subgroups of patients with PDD, may be related to increased GI motility. This notion is supported by higher PLT 5-HT in patients with diarrhea predominant irritable bowel syndrome (d-IBS), as compared with healthy controls (Houghton et\u00a0al., 2003), although this was not consistently found (Atkinson, Lockhart, Whorwell, Keevil, & Houghton, 2006). Patients with d-IBS have augmented GI motility, which is likely to cause increased exposure of their circulating PLT to 5-HT (Atkinson et\u00a0al., 2006; Gershon, 2005; Houghton et\u00a0al., 2003). Increased PLT 5-HT is also observed in patients with carcinoid tumors (Kema et\u00a0al., 2001). Carcinoid tumors derive from enterochromaffin cells and are characterized by high 5-HT production with diarrhea as a frequent symptom (Modlin, Kidd, Latich, Zikusoka, & Shapiro, 2005). Consequently, measurement of PLT 5-HT is used as a sensitive marker for the early diagnosis and the subsequent follow-up of patients with carcinoid tumors (Kema et\u00a0al., 2001).\nThe aim of the present study was to investigate whether the subgroup of children with PDD having increased PLT 5-HT levels, is the same as the one exhibiting increased intestinal permeability as established by a SAT.\nMethods\nPatients\nParents of patients with PDD (n\u00a0=\u00a031) according to the DSM-IV TR (American Psychiatric Association, 1994) were asked for the participation of their affected children via the local patient society and pediatricians. Oral and written informed consent were obtained. Information regarding comorbidity, medication, nutritional supplements and the prevalence of GI related complaints was obtained from medical records and with the aid of assisted questionnaires. The study was performed in Cura\u00e7ao (The Netherlands Antilles) in the summer of 2004. All collected urine and blood samples were transported in dry ice to the Netherlands for further analyses in the University Medical Center Groningen (UMCG). The study was approved by the Medical Ethical Committee of the St. Elisabeth Hospital in Cura\u00e7ao.\nSugar Absorption Test\nThe SAT was performed according to van Elburg et\u00a0al. (1995). Shortly, the patients ingested a sugar (lactulose, mannitol and sucrose) containing test fluid after an overnight fast. All urine voidings during the following 5\u00a0h were collected and pooled. Urinary sugars were analyzed by gas chromatography as previously described (Jansen, Muskiet, Schierbeek, Berger, & van der Slik, 1986). A urinary lactulose\/mannitol (L\/M) ratio above 0.090 was considered to be indicative for abnormal GI integrity\/increased intestinal permeability (van Elburg et\u00a0al., 1995).\nSerotonin Assays\nFor estimation of 5-HT turnover and exposure of PLT to 5-HT, we examined 24\u00a0h urinary excretion of 5-HIAA and total 5-HT (Kema et\u00a0al., 2001). For this, parents were asked to abstain their child completely from 5-HT containing foods (e.g. banana, pineapple, kiwi, walnuts) during collection and during the preceding 12\u00a0h. The volumes of the urine samples were measured before storing at \u221220\u00b0C. Urinary 5-HIAA and total 5-HT concentrations were determined as previously reported (Kema et\u00a0al., 2001). Urinary 5-HIAA values were evaluated with the use of age-dependent reference values (American Association for Clinical Chemistry, 2005).\nNon-fasting venous blood (for serum antibodies) and EDTA-anticoagulated blood (all other assays) were collected from children with PDD. EDTA-anticoagulated blood was placed on melting ice. Hematological indices were measured immediately after sampling. Within 1\u00a0h after collection a 1:1 mixture of K2EDTA and Na2S2O5 was added to PLT-rich plasma (PRP) to prevent oxidation of indoles. Plasma and serum were stored at \u221280\u00b0C. Simultaneous analysis of indoles [Tryp, 5-hydroxytryptophan (5-HTP), 5-HT and 5-HIAA] in PRP was performed as previously described (Kema et\u00a0al., 2001). PLT 5-HT data were compared with both a local cut-off value of 5.4\u00a0nmol\/109 PLT (Meijer, Kema, Volmer, Willemse, & de Vries, 2000) and an empiric cut-off value of 4.55\u00a0nmol\/109 PLT (Mulder et\u00a0al., 2004). The local cut-off value represents the 97.5th percentile of a reference group of healthy adults, that is employed in our laboratory for the diagnosis of carcinoid tumors. The empirical cut-off value represents the bottom of the valley of the PLT 5-HT bimodal distribution, as exhibited by patients with PDD. This value allows optimal classification into those who are normoserotonemic and hyperserotonemic. Platelet-rich-plasma Tryp data were evaluated with the use of age-dependent reference values (American Association for Clinical Chemistry, 2005).\nExclusion of Celiac Disease\nSerum IgA anti-endomysium titers and HLA genotype were assessed to rule-out celiac disease, which is an established cause of increased GI permeability (van Elburg, Uil, Mulder, & Heymans, 1993).\nStatistics\nAll data were analyzed using the Statistical Product and Service Solutions package, version 11.5 (SPSS Inc. Chicago). Data were tested for normality using the Shapiro\u2013Wilk W test. Group comparisons (normo- and hyperserotonemic) were performed with the Mann\u2013Whitney U test (non-parametric). Spearman (non-parametric) tests were used to evaluate correlations at \u03b1\u00a0=\u00a00.05, to minimize type-II errors.\nResults\nPatients\nWe enrolled the first 24 (77%) of the 31 patients with PDD whose parents agreed to participate. Patient characteristics are reported in Table\u00a01. The parents reported 13\/23 (57%) of their affected children to have one or more GI symptoms.\nTable\u00a01Characteristics of patients with pervasive developmental disorders in Cura\u00e7ao tested for platelet serotonin and intestinal permeabilityParameterGender (male\/female)18 (75%) \/ 6 (25%)Age (years)9.9 (\u00b13.9)DSM-IV TR diagnoses\u00a0\u00a0\u00a0\u00a0299.00 (autistic disorder)8 (33%)\u00a0\u00a0\u00a0\u00a0299.80 (PDD-NOS)16 (67%)Ethnicity\u00a0\u00a0\u00a0\u00a0Caucasian8 (33%)\u00a0\u00a0\u00a0\u00a0African\u2013American13 (54%)\u00a0\u00a0\u00a0\u00a0Other3 (13%)Comorbidity\u00a0\u00a0\u00a0\u00a0Epilepsy5 (22%)\u00a0\u00a0\u00a0\u00a0Allergy2 (9%)\u00a0\u00a0\u00a0\u00a0Asthma1 (4%)\u00a0\u00a0\u00a0\u00a0Intestinal yeast infection1 (4%)Medication for comorbidity9 (38%)Nutritional supplements (vitamins\/\u03c93-oils)9 (38%) \/ 2 (4%)Diet (gluten and casein free)a2 (9%)Physical complaints related to GI tracta13 (57%)\u00a0\u00a0\u00a0\u00a0Nausea0 (0%)\u00a0\u00a0\u00a0\u00a0Vomiting1 (4%)\u00a0\u00a0\u00a0\u00a0Diarrhea4 (17%)\u00a0\u00a0\u00a0\u00a0Constipation4 (17%)\u00a0\u00a0\u00a0\u00a0Bloating and gaseousness8 (35%)an\u00a0=\u00a023. PDD-NOS pervasive developmental disorder\u2014not otherwise specifiedData represent number (percentage) or mean (\u00b1SD) for 24 patients, unless otherwise specified \nBecause of cleanliness problems, no urine samples were obtained from one patient, while from another we only received urine for SAT. Blood sampling from yet another patient was problematic. Consequently, our study comprised urine for SAT from 23\/24 patients, 24\u00a0h urine from 22\/24 patients and blood samples from 23\/24 patients. Table\u00a02 shows the indices related to 5-HT turnover and intestinal permeability of these patients.\nTable\u00a02Indices of serotonin (5-HT) turnover and intestinal permeability in patients with pervasive developmental disorders in Cura\u00e7aoParameterReference values<RV\u2265RVPLT 5-HT (nmol\/109 PLT)a3.4 (2.0\u20137.1)<4.55b6 (26%)<5.4c4 (17%)Tryp (\u03bcmol\/l)a50.0 (\u00b110.9)2\u201318y: 0\u201379d0 (0%)0 (0%)2y: 35\u201373d}6y: 37\u201376d3 (13%)0 (0%)16y: 54\u201393d5-HIAA (\u03bcmol\/24\u00a0h)e8.4 (3.9\u201336.4)3\u20138y: 2.1\u201329.3d}9\u201312y: 5.2\u201332.9d1 (5%)1 (5%)13\u201317: 4.7\u201334.0d>18y: 5.2\u201336.6d5-HT (nmol\/24\u00a0h)e305 (\u00b192)Lactulose (mmol\/5\u00a0h)f0.024 (\u00b10.11)Mannitol (mmol\/5 h)f1.32 (\u00b10.59)L\/M ratio (mol\/mol)f0.019 (\u00b10.007)<0.090g23 (100%)0 (0%)aPlatelet-rich plasma, n\u00a0=\u00a023bEmpiric cut-off derived from Mulder et\u00a0al. (2004)cUpper reference value derived from apparently healthy adults Meijer et\u00a0al. (2000)dFrom reference American Association for Clinical Chemistry (2005)eAbout 24\u00a0h urine, n\u00a0=\u00a022fAbout 5\u00a0h urine from sugar absorption test, n\u00a0=\u00a023gFrom reference van Elburg et\u00a0al. (1995)Data represent number (percentage), median (range) or mean (\u00b1SD).RV; reference value; PLT, platelet; Tryp, tryptophan; 5-HIAA, 5-hydroxyindoleacetic acid; L\/M, lactulose\/mannitol\nSugar Absorption Test\nIntestinal permeability, reflected by the L\/M ratio [median (range) 0.017\u00a0mol\/mol (0.008\u20130.035)], indicated that none of the patients had increased intestinal permeability (i.e. L\/M ratio\u00a0\u2265\u00a00.090).\nSerotonin Assays\nPLT 5-HT [median (range): 3.4 (2.0\u20137.1)\u00a0nmol\/109 PLT] was elevated in 4 (range: 5.7\u20137.1 nmol\/109 PLT) and 6 [range 4.6\u20137.1\u00a0nmol\/109 PLT] patients if compared to the local cut-off value ((Meijer et\u00a0al., 2000); <5.4\u00a0nmol\/109 PLT) or the empirical cut-off value ((Mulder et\u00a0al., 2004); <4.55\u00a0nmol\/109 PLT), respectively. The sole patient exhibiting detectable levels of plasma 5-HIAA (26.0\u00a0\u03bcmol\/l), also exhibited increased PLT 5-HT (6.4\u00a0nmol\/109 PLT). However, this patient did not exhibit increased urinary total 5-HT or 5-HIAA excretions. Urinary excretion of 5-HIAA was within normal range.\nExclusion of Celiac Disease\nNone of the patients were positive for serum IgA anti endomysium and 8\/23 (35%) patients had a genotype positive for either HLA-DQ2 (n\u00a0=\u00a05) or HLA-DQ8 (n\u00a0=\u00a03). Based on the results of serology, none of the patients seemed to have celiac disease, although we did not perform further tests to exclude this.\nStatistics\nThere was no correlation between PLT 5-HT and L\/M ratio (p\u00a0=\u00a0.663; r\u00a0=\u00a0\u2212.098). Patients exhibiting GI tract complaints did not have higher PLT 5-HT, higher L\/M ratios, or higher 5-HIAA and total 5-HT 24\u00a0h urinary excretions (p\u00a0>\u00a0.4). Platelet 5-HT correlated with 24\u00a0h urinary 5-HIAA excretion (p\u00a0=\u00a0.034; r\u00a0=\u00a0.465). Also the 24\u00a0h excretion rates of 5-HIAA and total 5-HT showed a positive correlation (p\u00a0=\u00a0.005; r\u00a0=\u00a0.580).\nDiscussion\nIn this study of children with PDD we did not observe a relation between PLT 5-HT and intestinal permeability, as derived from the urinary L\/M ratio. The number of children with PDD exhibiting increased PLT 5-HT was lower compared with reports of others. For instance, the recent study of Mulder et\u00a0al. (2004) showed 23\/81 (28%) of Dutch children with PDD to exhibit increased PLT 5-HT, using the same analytical method and a cut-off value of 5.4\u00a0nmol\/109 PLT. Also our data on intestinal permeability contrast with previous reports (D\u2019Eufemia et\u00a0al., 1996; Horvath et\u00a0al., 2002) showing that 43\u201376% of children with autism have increased intestinal permeability, as established by a SAT. A weakness of the current study is its small size and the lack of a local age- and gender matched control group. We have, on the other hand, no indications for deviant reference values for PLT 5-HT or L\/M ratios in Cura\u00e7ao, as compared with The Netherlands. Age and gender do not appear to affect PLT 5-HT (Mulder et\u00a0al., 2004), but it must be noted that PLT 5-HT reaches highest levels during childhood and gradually decreases during adulthood (Flachaire et\u00a0al., 1990). Dependent on the cut-off values employed, we nevertheless found 4 and 6 patients with increased PLT 5-HT. Neither of these patients had abnormal intestinal permeability or L\/M ratios residing in the upper range of normality. However, the positive correlation between PLT 5-HT levels and 24\u00a0h urine 5-HIAA, and also the relation between urinary 5-HIAA and urinary 5-HT, suggest that, also in PDD, exposure of PLT to 5-HT determines PLT 5-HT and its consistently found increase in a subgroup. It is possible that increased PLT 5-HT and GI permeability are not consistent features of children with PDD in time. Long term, e.g. monthly, monitoring of a well defined patient and control group may shed more light on this potential source of variance as a cause of the conflicting results found by several investigators. Differences in the activity of the 5-HT transporter based on genetic polymorphisms are unlikely, since these seem to have minor effects, if any, on PLT 5-HT levels (Mulder, 2006).\nIn conclusion, the finding of a subgroup of children with PDD exhibiting hyperserotonemia was replicated. None of the children exhibited increased intestinal permeability, while PLT 5-HT was unrelated to both intestinal permeability and GI symptoms. Additional studies are needed to elucidate the etiology of increased PLT 5-HT in PDD and to establish its relation with intestinal pathology, if any.","keyphrases":["permeability","platelets","serotonin","pervasive","gastrointestinal","child development disorders"],"prmu":["P","P","P","P","P","M"]}
{"id":"Appl_Microbiol_Biotechnol-3-1-2043089","title":"Assessment of technological options and economical feasibility for cyanophycin biopolymer and high-value amino acid production\n","text":"Major transitions can be expected within the next few decades aiming at the reduction of pollution and global warming and at energy saving measures. For these purposes, new sustainable biorefinery concepts will be needed that will replace the traditional mineral oil-based synthesis of specialty and bulk chemicals. An important group of these chemicals are those that comprise N-functionalities. Many plant components contained in biomass rest or waste stream fractions contain these N-functionalities in proteins and free amino acids that can be used as starting materials for the synthesis of biopolymers and chemicals. This paper describes the economic and technological feasibility for cyanophycin production by fermentation of the potato waste stream Protamylasse\u2122 or directly in plants and its subsequent conversion to a number of N-containing bulk chemicals.\nIntroduction\nPlants have the ability to use the incident sunlight for the biosynthesis of a tremendous variety of compounds that may contain a number of functionalized atoms or groups. An important group of these functionalized compounds are proteins and especially the individual amino acids that contain one or more nitrogen atoms. When starting from crude oil or naphtha, the incorporation of functionalities (e.g., \u2212NH2) into derived bulk chemicals (such as 1,2-ethanediamine and 1,4-butanediamine) requires considerable amounts of energy and catalysts. However, some amino acids appear to be very suitable starting materials for highly functionalized bulk chemicals (Scott et al. 2007).\nThe biorefinery concept is a rapidly emerging field of research and commercial activities aiming at the integral use of all components of agricultural crops. In addition to the main product such as starch or oil, also, other side stream fractions including protein, free amino acid, and fiber fractions have high potential for valorization. An example of such waste stream fraction, Protamylasse\u2122 that remains after starch and protein extraction from potato and its possible application as a substrate for microbial fermentation and production process for cyanophycin (Elbahloul et al. 2005a,b, 2006), will be described in detail in the current paper.\nCyanophycin (multiarginyl-poly[l-aspartic acid]; CGP, cyanophycin granule peptide) is a non-ribosomal protein-like polymer which consists of equimolar amounts of aspartic acid and arginine arranged as a poly-aspartic acid backbone to which arginine residues are linked to the \u03b2-carboxyl group of each aspartate by its \u03b1-amino group. In nature, cyanophycin is produced by most, but not all, cyanobacteria as a temporary nitrogen reserve material during the transition of cells from the exponential phase to the stationary phase. The polymerization reaction is catalyzed by only one enzyme, which is referred to as cyanophycin synthetase (CphA). Because of the low polymer content and the slow growth of cyanobacteria resulting in only low cell densities, cyanobacteria are not suitable for large-scale production of cyanophycin. Therefore, the cphA genes from a number of cyanobacteria have been expressed in several bacteria, and, more recently, also in plants. Furthermore, the polymer isolated from recombinant strains contained lysine as an additional amino acid constituent.\nNow that cyanophycin can be produced in sufficient amounts by pilot scale fermentations for studying its material properties, it appears of biotechnological interest because purified cyanophycin can be chemically converted into a polymer with a reduced arginine content, which might be used like poly-aspartic acid as a biodegradable substitute for synthetic polyacrylate in various technical processes. In addition, cyanophycin might also be of interest for other applications when the hitherto unknown physical and material properties of this polymer will be revealed. On the other hand, cyanophycin is a convenient source of the constituent amino acids that may be regarded as nitrogen-functionalized precursor chemicals.\nIn the current paper, conditions will be discussed for the technological and economic feasibility of cyanophycin production by microbial fermentation and by cyanophycin production directly in plants. The conditions for fermentative cyanophycin production will be based upon the use of cheap substrates derived from agricultural waste streams and the possible cyanophycin production simultaneously with other fermentation products like ethanol. This aspect is denoted process integration.\nBiorefinery and its place in the production of chemicals\nThe depletion in fossil feedstocks, increasing oil prices and the ecological problems associated with CO2 emissions, are forcing the development of alternative resources for energy, transport fuels, and chemicals: the replacement of fossil resources with CO2 neutral biomass.\nPotentially, biomass may be used to replace fossil raw materials in several major applications: heat, electricity, transport fuels, chemicals, and other industrial use. Each of these groups represents about 20% of the total fossil consumption in the industrialized countries (Oil Market Report of the International Energy Agency 2004). Large variations in the cost of these products at the wholesale level, based on their energy content, are evident (Table\u00a01). When one considers the contribution to costs by the raw materials (expressed per GJ end product), large differences are also seen. Heat can be produced from coal for around 3\u00a0\u20ac\/GJ due to utilizing inexpensive feedstocks with high conversion efficiency (about 100%), while the raw material costs for electricity is double (6\u00a0\u20ac\/GJ) due to a conversion yield of about 50%. Most notable is the high raw material costs for chemicals. Here, expensive raw materials (oil) are used with low(er) conversion yields (Sanders et al. 2005, 2007).\nTable\u00a01Different applications and contributions of biomassContributionIntegral cost prices (\u20ac\/GJ end product)Raw material cost fossil (\u20ac\/GJ)Percentage of total energy in the Netherlands (3.000 PJ) consumed per application (%)Heat43 (Coal)\u00b120Electricity226 (Coal)\u00b120Transport fuel108 (Oil)\u00b120Average bulk chemicals7530 (Oil)\u00b120Rest of industry\u00b120\nTo obtain a good net income for biomass, an effective biorefinery system is required for the separation of the harvested crop into fractions for use in (several of) these applications. These may be used directly as the desired product or undergo conversion by chemical, enzymatic, and\/or microbial means to obtain other products. Biorefinery systems are well established for a number of crops. For example, soybeans are the raw materials for large biorefineries to produce oil (for biofuels), proteins and valuable nutraceuticals.\nLess well explored is the use of biomass to make industrial chemicals. Effort to produce chemicals with constant quality and performance (such as lactic acid) has been addressed, but has mainly focused on the use of carbohydrates as raw materials and use of biotechnology for conversion. However, for effective biorefinery approaches, other biomass fractions should also be considered for the production of chemicals. It is anticipated that the substitution of petrochemical transportation fuels with biofuels will rise significantly in the coming years. This means that a rise in the production of biodiesel will lead to large volumes of glycerol as a residual stream, and indeed, some companies are already investigating the use of glycerol to produce chemicals. While the awareness of large volumes of glycerol from biofuel production is apparent, one should not overlook other waste streams. Indeed, from biofuel production, an immense concomitant waste stream of protein will also be generated. Sources of proteins and amino acids are not limited to those generated from biofuel production, but also from other industries such as potato starch production. For example, during AVEBE\u2019s processing of potatoes for starch extraction, the main waste stream is Protamylasse\u2122, which mainly contains sugars, organic acids, proteins, and free amino acids and has currently no major market use. Some of the amino acids present in such sources could be very suitable raw materials for preparing (highly) functionalized chemicals traditionally prepared by the petrochemical industry (Sanders et al. 2007).\nGenerally, the conversion of crude oil products utilizes primary products (ethylene, etc.), and their conversion to materials or (functional) chemicals makes use of co-reagents such as ammonia and various process steps to introduce functionalities such as \u2212NH2. Conversely, many products found in biomass, such as proteins and amino acids, often contain these functionalities. Therefore, it is attractive to exploit this to bypass the use, and preparation, of co-reagents as well as eliminate various process steps by utilizing suitable biomass-based precursors for the production of chemicals. Thus, the production of chemicals from biomass takes advantage of the biomass structure in a more efficient way than the production of fuels or electricity alone and can potentially save more fossil energy than producing energy alone (Scott et al. 2007). When used in combination with environmentally sound production and processing techniques across the whole biomass production chain, i.e., from cultivation and harvest, its (pre)treatment and conversion to products, the use of biomass is considered a sustainable alternative to conventional feedstocks, which is reflected by sound economic advantages in both raw material and investment costs.\nGeneral introduction on NRPs and especially cyanophycin\nCyanophycin (also referred to as CGP, cyanophycin granule polypeptide) that belongs to the family of bacterial poly-amino acids together with poly-\u03b3-glutamic acid and poly-\u025b-lysine, was discovered in 1887 by Borzi (1887) during microscopic studies of cyanobacteria and was later found in all groups of cyanobacteria (Oppermann-Sanio and Steinb\u00fcchel 2002). The cyanophycin molecule structure is related to that of poly(aspartic acid)s, but, unlike synthetic poly-aspartic acid, it is a comb-like polymer with \u03b1-amino-\u03b1-carboxy-linked l-aspartic acid residues representing the poly(\u03b1-l-aspartic acid) backbone and l-arginine residues bound to the \u03b2-carboxylic groups of aspartic acids (Simon and Weathers 1976; for recent review, see Obst and Steinb\u00fcchel 2004; Fig.\u00a01). Cyanophycin is synthesized by most, but not all, cyanobacteria as a temporary nitrogen reserve material during the transition of cells from the exponential phase to the stationary phase (Mackerras et al. 1990). At neutral pH and physiological ionic strength, cyanophycin is insoluble and deposited in the cytoplasm as membraneless granules (Lawry and Simon 1982).\nFig.\u00a01Chemical structure of the cyanophycin monomer\nCyanophycin isolated from Cyanobacteria is highly polydisperse and shows a molecular weight range of 25\u2013100\u00a0kDa as estimated by sodium dodecylsulphate polyacrylamide gel electrophoresis corresponding to a polymerization degree of 90\u2013400 (Simon 1971; Simon and Weathers 1976). Cyanophycin is a transiently accumulated storage compound which is synthesized under conditions of low temperature or low light intensity. Its accumulation can be artificially enhanced by the addition of chloramphenicol as an inhibitor of ribosomal protein biosynthesis (Simon 1973). Cyanophycin plays an important role in the conservation of nitrogen, carbon, and energy and, as indicated by its biosynthesis in presence of chloramphenicol, is non-ribosomally synthesized by CphA. Cyanophycin is accumulated in the cytoplasm of cyanobacteria as membraneless granules (Allen and Weathers 1980) in the early stationary growth phase (Mackerras et al. 1990; Liotenberg et al. 1996). When growth is resumed, for example due to a change in cultivation conditions, cyanophycin is reutilized by the cells (Mackerras et al. 1990). Krehenbrink et al. (2002) and Ziegler et al. (2002) showed that cyanophycin occurs even in heterotrophic bacteria like Acinetobacter sp. and Desulfitobacterium hafniense and therefore confirmed the wide distribution of this biopolymer and its function in nature as a general storage compound.\nCyanophycin is of biotechnological interest because the purified polymer can be chemically converted into a polymer with reduced arginine content (Joentgen et al. 1998), which might be used like poly-aspartic acid as a biodegradable substitute for synthetic polyacrylate in various technical processes. In addition, cyanophycin might also be of interest for other applications if the unknown physical and material properties of this polymer are revealed. Because of the low polymer content and the slow growth of cyanobacteria resulting in only low cell densities, cyanobacteria are not suitable for large-scale production of cyanophycin (Schwamborn 1998), and sufficient amounts of cyanophycin were hitherto not available.\nThe polymerization reaction is catalyzed by only one enzyme, which is referred to as CphA (Ziegler et al. 1998). The cphA genes from Anabaena variabilis ATCC 29413, Anabaena sp. strain PCC7120, Synechocystis sp. strain PCC6803, Synechocystis sp. strain PCC6308, Synechococcus elongatus, Synechococcus sp. strain MA19 and others were cloned and expressed in Escherichia coli (Aboulmagd et al. 2000; Berg et al. 2000; Hai et al. 1999; Oppermann-Sanio et al. 1999; Ziegler et al. 1998). More recently, heterologous expression of cphA was also demonstrated at a small scale in recombinant strains of Ralstonia eutropha, Corynebacterium glutamicum, and Pseudomonas putida (Aboulmagd et al. 2001). Whereas in cyanobacteria the molecular mass of the polymer strands ranged from 25 to 100\u00a0kDa (Simon 1976), the polymer from recombinant strains harboring cphA as well as in vitro-synthesized polymer exhibited a much lower range (25 to 30\u00a0kDa) and polydispersity. Furthermore, it was found that the polymer isolated from recombinant strains contained lysine as an additional amino acid constituent (Aboulmagd et al. 2001; Ziegler et al. 1998). Recently, the results of a detailed in silico analysis of the occurrence of enzymes involved in cyanophycin metabolism was published (F\u00fcser and Steinb\u00fcchel 2007).\nRecently, the earlier postulated instability of recombinant E. coli strains employed for cyanophycin production was also confirmed in both DH1 and DH5\u03b1. This instability may be caused by loss of the plasmid during fermentation. However, as cyanophycin production continues in cultures that rapidly appear to loose the ampicillin resistance employed for selection and plasmid maintenance, other explanations are also under consideration such as competition for Arg and Asp by both cyanophycin and the ampicillin resistance protein and the theoretical possibility that the ampicillin resistance protein could be trapped into the cyanophycin granule or at least be made inaccessible to the ampicillin.\nDue to the wide knowledge of its metabolism and available genetic tools, E. coli is one of the most commonly used bacterial hosts for the production of recombinant proteins (Lee 1996). Several expression systems have been developed for technical-scale production of recombinant proteins in E. coli based on the regulated trp, lac, or lambda PL promoter (Hannig and Makrides 1998). The cultivation of recombinant E. coli strains harboring cphA from Synechocystis sp. strain PCC6803 at the 500-l scale for the production of cyanophycin has been described (Frey et al. 2002). As the previously described method for the purification of cyanophycin (Simon and Weathers 1976) is not applicable to a large scale, a simplified method for isolation of the polymer at the technical scale was elaborated.\nBiosynthesis of cyanophycin was extensively studied in the 1970s by Simon and coworkers (Simon 1971, 1976; Simon and Weathers 1976). Later, this led to the identification of cyanophycin synthetase enzymes and the encoding genes (cphA) in various organisms (Ziegler et al. 1998; Aboulmagd et al. 2000; Berg et al. 2000; Hai et al. 2002). Subsequently, the enzymes involved in the degradation of cyanophycin by intracellular cyanophcyinases of cyanobacteria (cphB) and extracellular depolymerases, like hydrolase (cphE) and cyanophycinase (cphI) genes, were identified (Obst et al. 2002, 2004; Obst and Steinb\u00fcchel 2004). Elbahloul et al. (2005a,b) have found that inactivation of the cyanophycinase gene in Acinetobacter resulted in significantly less cyanophycin accumulation than the wild type presumably due to a shortage of cyanophycin primer molecules. On the contrary, cyanophycin is highly resistant against hydrolytic cleavage by proteases such as trypsin, pronase, pepsin, carboxypeptidases B, carboxypeptidase C, and leucin-aminopeptidase, and cyanophycin is also resistant against arginases (Simon and Weathers 1976).\nBecause the cyanophycin synthetase genes (cphA) of many cyanobacteria, and recently, also of other microorganisms were identified, cloned, and heterologously expressed in other bacteria (Aboulmagd et al. 2001) conferring the ability to produce comparably large amounts of cyanophycin (up to 50% of CDW) in a much shorter period of time (1\u20132\u00a0days) as compared to cyanobacteria (about 4\u00a0weeks) and cyanophycin production was demonstrated at the 30- to 500-l scale (Aboulmagd et al. 2001; Frey et al. 2002; Vo\u00df and Steinb\u00fcchel 2006), the interest in cyanophycin as a potential raw material has constantly increased over the last few years. Improvement of fermentation conditions, feeding regimes, and the possibility to produce cyanophycin now by the employment of many genetically engineered bacteria with industrial relevance like R. eutropha, C. glutamicum, or P. putida (Aboulmagd et al. 2001) in complex but also in defined media make it appear likely that further improvement of cyanophycin production in bacteria will be achieved during future studies. These studies may include elementary mode analyses (Diniz et al. 2006) or more conventional approaches involving experimental design using fermenter arrays and principal component analyses.\nIn conclusion, large-scale fermentation processes for cyanophycin production and downstream processing are available for a number of different microorganisms able to grow in different substrates, including the potato waste stream Protamylasse\u2122, and also, low cyanophycin yields were reported in plants.\nProduction and economic aspects of fermentative cyanophycin production\nAn important contribution to sustainability can be made by the use of a considerable plant waste stream for the production of renewable, biodegradable, and biocompatible polymers and\/or valuable chemicals that are now produced on large scale from petroleum. Some of the polymer classes to be developed may be expected to replace some existing mineral petroleum-based polymers as soon as competitive production prices can be obtained and\/or supporting measures will be taken to promote the use of renewable resources. On the other hand, completely novel types of biopolymers may be developed for completely novel applications.\nAVEBE, located in the northern part of The Netherlands, is the largest potato-starch-producing company in the world involved in extraction, processing, and sales of starch and starch-derived products. During processing of potatoes for starch extraction, the main waste stream is Protamylasse\u2122. Annually, AVEBE produces about 120,000\u00a0m3 of Protamylasse\u2122 containing about 70,000\u00a0tons of dry matter, mainly consisting of sugars (14,000\u00a0tons), organic acids (13,300\u00a0tons), proteins and free amino acids (18,000\u00a0tons). The amino acids arginine and aspartic acid amount to about 1,000\u00a0tons each and lysine to about 700\u00a0tons. Currently, there is no proper outlet for this Protamylasse\u2122 other than low value epandage (e.g., by Bos Agra-Service, NL in which the salts present, such as potassium, are used as fertilizer), whereas rough calculation shows that the total intrinsic gross value of the valuable components in the Protamylasse\u2122 is about 45 million euros. A number of research objectives is now in progress to add substantial value to the entire potato starch production chain (and thus its economical feasibility). Protamylasse\u2122 may be considered a model for other agricultural waste streams, such as grass juice and beet residue. It is not certain whether concentrations of all medium components in the Protamylasse\u2122 will be optimal to sustain microbial growth and cyanophycin production, and it is anticipated that additional medium components need to be identified and\/or tailor-made production strains constructed. This will require a detailed analysis of Protamylasse\u2122 components before and after the cyanophycin production phase.\nTo calculate the economic feasibility of cyanophycin production using Protamylasse\u2122, several aspects need to be included as follows:\nNecessary steps from potato starch and protein extraction, yielding Protamylasse\u2122, to the final purified product cyanophycin (which is considered an intermediate product for derived polymer types and N-containing bulk chemicals) include: shipment to a fermentation plant, dilution to 5\u20136% (v\/v) using tap water, removal of potato particles by filtration, disposal (or alternative use) of particle fraction (60% DM), loading of step up fermenters [to allow an microbial inoculum concentration of 10% (v\/v) per step; e.g., 10\u2192100\u21921,000\u219210,000\u2192100,000\u00a0l], sterilization, cooling, addition of ampicillin, inoculation, (batch) fermentation, addition of acid and\/or base for pH control, cell harvesting and concentration, disposal or recycling of spent Protamylasse\u2122, cell disruption (not for E. coli), cyanophycin extraction using pH\u00a02, neutralization, cyanophycin crystallization, precipitation, purification, and storage.Yearly Protamylasse\u2122 supply, 120,000\u00a0m3; 70,000\u00a0tons (60% DM)Protamylasse\u2122 dry matter composition: amino acids (18,000\u00a0tons), sugars (14,000\u00a0tons), organic acids (13,300\u00a0tons), ash (22,200\u00a0tons). For further details on the composition, see Elbahloul et al. (2005a, b).Small-scale fermentation process data: Protamylasse\u2122 concentration, 5\u20136% (v\/v); fermenter volume, 25\u00a0l; strain E. coli DH1 (pMa\/c5-914::cphA); temperature 37\u00b0C; maximal OD and cyanophycin yield reached after 15\u00a0h; optimal pH\u00a07.5\u20138.0; biomass yield 5\u201310\u00a0g\/l (CDW); cyanophycin content, 25% (w\/w); cyanophycin composition, Asp, 50%; Arg, 45%; Lys, 5%; E. coli occasionally (5\u201310%) incorporates lysine in stead of arginine; poly(Asp\u2013Arg) is non-soluble in water at neutral pH; poly (Asp\u2013Arg\u2013Lys) is soluble in water.\nAssuming that most of the solid particles will be removed from the Protamylasse\u2122 by filtration, a yearly amount of 48,000\u00a0m3 of Protamylasse\u2122 liquid juice will become available, which is used at a 5% dilution during fermentation, thus, providing a yearly amount of 960,000\u00a0m3 of diluted fermentation broth. This volume would be enough to run 9,600\u2009\u00d7\u2009100\u00a0m3 fermenter volumes. Further assuming a 1\u00a0week\u2019s run time (including cleaning, sterilization, fermentation, and harvest) a park of, e.g., 185\u2009\u00d7\u2009100\u00a0m3 or preferably 20\u2009\u00d7\u20091,000\u00a0m3 fermenter units (step up units included) could be operated continuously. With the current E. coli biomass yield of 5\u00a0g\/l (CDM) with a cyanophycin content of 25% (w\/w DM), this would yield a yearly amount of 1,200\u00a0tons of purified cyanophycin. At an estimated market price of \u20ac 1,000 per ton cyanophycin, the Protamylasse\u2122 juice fraction (40% v\/v) would yield a yearly income of only 1.2 million euros. However, by only increasing the amount of microbial biomass from the actual 5\u00a0g\/l (CDM, E. coli) to a realistic value of 100\u00a0g\/l (CDM, S. cerevisiae) with the same cyanophycin content of 25% (w\/w DM), this yearly income could be raised to 24 million euros. If within the same fermentation run via so-called process integration, in addition to cyanophycin, ethanol also [yielding from 960,000\u00a0m3 at 5% (v\/v) about 48,000\u00a0m3 ethanol and an additional yearly income of 27.4 million euros at $2.8 per US gallon, \u20ac 0.57 per liter) could be produced using semi-aerobic fermentation with S. cerevisiae, this yearly income could be further raised to about 50 million euros. The Protamylasse\u2122 particle fraction can, in principle, also be used for cyanophycin and ethanol production. Preliminary calculations using the model of Golden Grain Energy, LLC (http:\/\/sec.edgar-online.com\/ 2004\/06\/14\/0001104659-04-016859\/Section7.asp) suggest that the break-even point could roughly be reached within about 3\u20135\u00a0years.\nFurther process improvements may be obtainable and are necessary to make this process economically feasible. These obtainable figures provide estimates as to the investments and costs that will be necessary to start a cyanophycin production fermentation facility. However, before fermentative cyanophycin production on industrial scale can be started, a number of bottlenecks should be overcome. Table\u00a02 lists current bottlenecks and proposed measures for optimal and economically feasible cyanophycin production.\nTable\u00a02Economical and technological bottlenecks and proposed measuresBottleneckProposed measure(s)Investments, including costs for fermentation and downstream processing equipmentThe calculation provided here suggests that these may be acceptableCosts for the production of cyanophycin, cyanophycin-derived products and for downstream processing of biomassConstruction of a sufficiently productive microbial strain to convert or simply utilize constituents of plant waste streams like Protamylasse\u2122 and to incorporate these compounds, presumably amino acids, into the cyanophycin polymer chain during cyanophycin biosynthesisPhenotypic instability of E. coli production strains used until now, DH1 and DH5\u03b1, containing plasmid pMa\/c5-914::cphA6803Construction of stable strains with integrated copies of the cyanophycinsynthesis genesLow biomass yields of the E. coli strains usedSince not all components present in the current source of Protamylasse\u2122 may have the proper concentration for current laboratory strain(s), an optimization may require the addition of substrates other than Protamylasse\u2122, for example other plant waste streams. Sufficient provision of amino acids like arginine should be ensured during the production phaseOptimization of microbial biomass formationBy using yeasts as alternative production organisms biomass yields could be increased to 100\u00a0g\/l CDM for S. cerevisiae [factor 20\u00d7] or 150\u00a0g\/l CDM for Pichia pastoris [factor 30\u00d7] if in Protamylasse\u2122 the same yields can be obtained as in dedicated growth mediaSub-optimal fermentation processesFermentation technology and feeding regimes have to be developed for optimum amino acid utilization or biosynthesis from Protamylasse\u2122 or other plant waste streamsGeneration of valuable side stream particle fraction of Protamylasse\u2122Alternative use of the side stream particle fraction of Protamylasse\u2122, e.g. by using cyanophycin producing filamentous fungiCo-production with, e.g., ethanolWhen using S. cerevisiae as the production organisms and (semi-) anaerobic fermentation both cyanophycin and ethanol could possibly be produced during the same runCosts for cyanophycin extractionDevelopment of alternative cheap cyanophycin extraction methods using, e.g., hydro-cyclone equipment for the non-soluble fractionCost-efficient production of cyanophycin in plantsThe transfer of the bacterial cyanophycin synthetase gene (cphA) into eukaryotic hosts, mostly plants and its effective expression in suitable organs or cell compartments is a major step (see below)Efficacy of downstream processingDownstream processing has to be adapted and optimized for cyanophycin or cyanophycin derivatives containing biomass, which will be either bacterial cells or eukaryotic (mostly plant) cells or tissuesLack of insight in possible modifications of cyanophycin, their impact on cyanophycin properties and market potentialThe diverse possibilities to modify the cyanophycin molecule chemically or enzymatically has to be exhaustingly explored to identify all potential key applications for cyanophycin-derived products and to find the most suitable products with regard to market potential and the possibility of their commercializationLack of knowledge concerning properties of known cyanophycin synthetases and their genetic engineeringThe possibility to modify the active sites of the cyanophycin synthetases in order to change its substrate specificity and to allow the production of cyanophycin derivatives has to be determinedInsufficient insight in all possible applications for cyanophycin as a polymer or as a starting material for chemical synthesesThe exploitation of cyanophycins and cyanophycin-derived molecules as substitutes for well established industrial products or as renewable raw materials has to be determined precisely\nCyanophycin production in plants\nTransgenic plants can be utilized to produce renewable resources for industrial purposes in a CO2-neutral, environmentally acceptable, and competitive way. Poly-3-hydroxybutyrate (PHB) was the first plastic-like compound produced in plants (Poirier et al. 1992), followed by, e.g., poly-3-hydroxyalkanoate (PHA; Poirier 2002) and medium chain-length PHA in potato (Romano et al. 2003, 2005) and showed the feasibility to produce biopolymers in plants. Recently, it has been shown that it is also possible to produce cyanophycin in plants (Neumann et al. 2005). For this, the Thermosynechococcus elongatus BP-1 cyanophycin synthetase gene was expressed constitutively under a 35S promoter in tobacco and potato plants. It was shown that approximately 1.14 and 0.24% dry weight could be accumulated in the cytosol of tobacco and potato leaves, respectively. The size (35\u00a0kDa), amino acid composition (Asp\/Arg\/Lys\u2009=\u20091:1.05:0.1), and structure of the plant-produced polymer was similar to that produced in transgenic E. coli expressing the same gene; however, the amount and molecular weight of the cyanophycin produced in plants was much lower than that observed in bacteria (up to 50% dry weight and 125\u00a0kDa in bacteria). The experiments have provided proof of concept for the potential of producing cyanophycin in plants.\nProduction of the cyanophycin biopolymer in potato is of high interest to the potato starch industry. Production in this plant does not require any additional infrastructure. After processing of the potatoes, cyanophycin can be isolated from the Protamylasse\u2122. However, for commercial application, the efficiency of cyanophycin accumulation in potato has to be significantly improved.\nNeumann et al. (2005) already indicated that directing the cyanophycin synthetase into several other compartments, such as the chloroplasts, could lead to increased accumulation of cyanophycin. However, chloroplasts in the cyanophycin-producing cells differ morphologically from wild-type chloroplasts. There are fewer and smaller grana stacks, and the growth rate is slower. One of the possible explanations for these properties is depletion of amino acid resources as a result of cyanophycin production (Neumann et al. 2005). The pioneering experiments by Neumann et al. (2005) have opened up a new field for the production of cyanopycin in agricultural crops and show that more research is needed before being introduced into agricultural production (Conrad 2005).\nAdditional strategies\nPriming cyanophycin elongation\nIn in vitro studies, it has been shown that cyanophycin synthetase works more efficiently with a (\u03b2-Asp\/Arg)3\/Arg primer. In planta production of such a primer may enhance cyanophycin biosynthesis. Cyanophycinases encoded by the chpB and chpE genes can degrade cyanophycin into arginine\u2013aspartic acid dipeptides (Asp\u2013Arg) which cannot be used as primer for cyanophycin biosynthesis.\nThe cphI gene encodes a plant-type asparaginase able to hydrolyze \u03b2-Asp\/Arg bonds and that, thus, may be responsible for the last step in cyanophycin degradation. Bacterial studies have indicated that cphI expression contributes to a higher cyanophycin level.\nIt might be possible to use a poly-Asp backbone as primer for cyanophycin biosynthesis. This peptide can be produced by ribosomal protein biosynthesis. The gene should be under the control of a low-level promoter to prevent the production of many peptides, and thus, the production of many low molecular weight polymers.\nOptimization of amino acid biosynthesis\nIt has been shown that cyanophycin synthetase uses arginine and aspartic acid as its major amino acid source but that it also can incorporate lysine in the cyanophycin polymer (Berg et al. 2000). It is unclear how this affects the properties of the polymer. Based on the chemical composition of Protamylasse\u2122 and the affinity of the enzyme for arginine and lysine, it can be proposed that lysine accounts for 1.5% of the total cyanophycin. To reduce this amount, three strategies are possible, i.e., improve biosynthesis of arginine, reduce the level of lysine, and\/or transform lysine into arginine.\nIt is possible that the availability of substrates (Asp and Arg) in plants is limiting or off-balance. Therefore, it is important to identify the organs that have the highest concentrations of available substrates and to investigate whether the substrate supply can be enhanced by introduction of genes involved in substrate production.\nComparison of economics of cyanophycin production by fermentation or in plants\nAs for some other commodities, depending on production price, market volume and final product price (Fig.\u00a02) for cyanophycin specialty product applications, fermentation is the preferred production method (roughly below 20.000\u00a0tons annually), whereas for bulk quantities of cyanophycin, the production directly in plants is preferred (above 20,000\u00a0tons). Therefore, for the production of cyanophycin-derived bulk quantities of nitrogen-containing chemicals, plants are considered the best production organisms, whereas for specialty polymers, fermentation may be the preferred production technology.\nFig.\u00a02Cyanophycin production in planta or by fermentation. Gray square: raw material costs, filled square: fermentation costs, open square: recovery and purification costs\nAssuming that a typical fermentative production of a bulk product, such as lysine, citric acid or glutamic acid costs about \u20ac\u00a01,500 per ton and that these costs consist of: \u20ac\u00a0500 for the raw materials, \u20ac\u00a0500 for the fermentation process, and \u20ac\u00a0500 for recovery and purification. The advantage of producing in plants is that both the raw material costs and the fermentation costs can almost be neglected. On the other hand, recovery costs could be much higher. For the sake of the reasoning, it is assumed that recovery costs for cyanophycin production in plants will be the same as in the case of fermented production, i.e., \u20ac\u00a0500 per ton. In case of a fermentation process, a typical production volume for a company would be in the order of magnitude of 100,000\u00a0tons\/year. The turnover would then be 150 million euros per year (i.e., \u20ac\u00a01,500 per ton \u00d7 100,000\u00a0tons\/year). In the case of production in plants, as raw material and fermentation costs are negligible, cost savings would be 100 million euros per year. This would be the maximum advantage, as this calculation does not include any costs for the production of the crop nor any additional costs for biorefining of the crop and treatment of side products. A similar reasoning would bring a maximum advantage for a product with a typical company production volume of 20,000\u00a0tons per year, such as the case with a medium-sized monomer. In this case, cost savings per ton would be higher, but as the volume is much smaller, the maximum advantage could be around 70 million euros. For specialty products with a volume of 300\u00a0tons per year (enzymes), a maximum advantage is estimated to be in the order of 5\u201310 million euros. For pharmaceutical production with a volume of 10\u00a0tons per year, the advantage would be around 0.5\u20131.5 million euros, as the volume would be small and production costs would mainly be ascribed to recovery.\nOther plant side streams\nIn addition to the possible exploitation of Protamylasse\u2122, a large number of other plant side streams may be used for cyanophycin production, including grass juice, which remains after protein extraction or beet or cane molasses remaining after sugar extraction. It is foreseen that with the production of biodiesel and bioethanol, large volumes of side streams will become available, all containing major protein quantities. Such streams will include dried distillers grain and solubles (DDGS) from corn and wheat, press cake from palm oil and rape seed oil. These streams all have in common a very low cost price, which implies that when used as a microbial growth medium, a major contribution to fermentation process costs (about 30%) can be eliminated. However, it is important to note that none of any waste stream (to be) considered has exactly the same chemical or element composition as the production organism and its product(s), including, e.g., CO2. This implies that in each fermentation process to be developed using plant waste streams as substrates, limiting components should be supplemented. For the identification of such limiting components, fermentation test runs should be performed, preferably supported by statistic analyses such as elementary mode analysis (Diniz et al. 2006). Only after performing such exercises can reliable estimates of integral project costs be obtained.\nCyanophycin-derived bulk chemicals\nCyanophycin can be hydrolyzed to its constituent amino acids, aspartic acid, and arginine. These amino acids may be utilized directly in food and pharmaceutical applications. However, based on the chemical structure of these amino acids and the presence of functionalized nitrogen-containing groups, it is possible to anticipate their conversion to a number of industrial chemicals, including:\nArginine may be converted to 1,4-butanediamine. 1,4-diaminobutane, derived from petrochemistry, is currently used as a co-monomer in the production of nylon-4,6. The volume of production is not known, but is estimated to be in the range of 10,000s\u00a0tons per year with a value of >\u20ac\u00a01,600 per ton.Conversion of aspartic acid to acrylonitrile is also envisaged. Using current petrochemical technology, the current worldwide market volume of acrylonitrile is 2.7\u20135\u2009\u00d7\u2009106 tons per year and a price of about \u20ac\u00a0800\u20131,000 per ton.\nOther chemicals which could be obtained from cyanophycin but are currently prepared from fossil resources include, e.g., 1,4-butanediol and urea.\nThe production of cyanophycin by plants will drastically reduce the cost price, potentially below \u20ac\u00a01,000 per ton. This will enable the production of functionalized bulk chemicals such as 1,4-diaminobutane, and possibly, also acrylonitrile. The transition from mineral oil to plant-based precursor production has a considerable impact. The use of ammonia for the incorporation of nitrogen into chemicals is very important, but also very energy-intensive. Therefore, if the incorporation of nitrogen can be realized in systems based on plant (rest) streams in the form of protein or amino acid precursors, then this will yield considerable energy savings.\nCyanophycin-based biopolymers\nPoly-aspartic acid is derived from cyanophycin after the hydrolytic removal of arginine. This polymer has properties that are very similar to poly-acrylic acid. The cost price of this polymer can be set on 1,000\u20ac\/ton in the cost calculations for arginine here above. As the volumes of these products will be similar, only 1,000\u00a0tons\/year will be manufactured. Higher market prices might be obtained for special applications in food and\/or pharmaceutical applications. This might change the cost structure of arginine in a way that market volumes might double or triple.\nCyanophycin as such might have applications as a polymer. Furthermore, derivatives obtained by enzymatic\/genetic and\/or chemical modifications might give valuable properties. Without thorough investigations, we cannot anticipate on the value of these polymers. Utilization in this area can be expected after 10 to 12\u00a0years after the beginning of the proposed research approaches for research and development.\nPossible cyanophycin modifications, applications for bulk chemicals and for polymers\nBy incorporating other amino acids, different types of polymers can be made. So far, several cyanophycins have been produced in recombinant strains of E. coli up to 50% dry weight. However, especially for health care medical and food packaging applications, E. coli may not be the best commercial production organism. Therefore, the development of alternative food-grade production organisms is also one of the objectives of the current activities. One suitable candidate may be the bakers yeast Saccharomyces cerevisiae (and others: see above). In addition, being stable at a pH between 3 and 9, cyanophycin can also be hydrolyzed in concentrated volumes into its pure components, arginine and aspartic acid. This would make the whole process a novel biological extraction procedure for the selected amino acids.\nOutlook\nGiven the anticipated cost development for fossil energy carriers and environmental regulations, the chemical industry is facing increasing financial pressure and is thus looking for possibilities to broach new resources as a basis for polymer production. Important considerations in this search are to lower energy costs and prices of raw material and to develop cheaper and more sustainable production processes.\nUnlike poly-\u03b3-glutamic acid and poly-\u025b-lysine, cyanophycin has not been commercialized yet. Cyanophycin can be broken down into the individual amino acids that can be used as building blocks in various industrial processes. Because of its homogeneous structure and composition, the cyanophycin polymer and its derivatives also appear to be good candidates as starting materials for the production of nitrogen-rich commodity products, which are based on nitrogen-rich chemicals, like, for example, nylons. For example, for poly(aspartic acid) which is the polymer backbone of cyanophycin, various applications have been developed ranging from water-softening or detergent applications to applications in the paper, building material, petroleum or leather industry, in cosmetics, as well as many dispersant applications.\nCyanophycin can be chemically converted into a polymer with a reduced arginine content, which might be used like poly-aspartic acid as a biodegradable substitute for synthetic polyacrylate in various technical processes (Schwamborn 1998). Thus, cyanophycin may find applications in cyanophycin-derived bulk chemicals and in cyanophycin-based biopolymers.\nIt can be expected that economical activities can be developed within the following areas, such as: fermentation industry, biopolymer production, processing, modification and product development (also for medical technology), packaging industry, food and feed supplementation industries and, last but not least, state-of-the-art technology (which, in turn, will attract additional financial sources and economical activities). It should be emphasized, however, that the mentioned applications are still uncertain and that these are so far only potential applications.\nOn the one hand, this development will lead to the substitution of chemicals that are now produced at the cost of fossil raw materials, such as oil. As oil may be depleted in about 50\u00a0years, and as there seems to be a correlation of the use of fossil raw materials with climate changes, it is essential to develop alternatives. The anticipated alternatives can be produced by fermentation and, in principle, by plant production systems, and so, giving a new economic and knowledge intensive value to the fermentation industry and\/or to agriculture. On the other hand, novel types of polymers will be developed that do not simply replace existing applications but that will enter novel product markets.\nElements of the contents of this paper are included in a dedicated patent application (Elbahloul et al. 2006).","keyphrases":["cyanophycin","biorefinery","bulk chemicals","n-functionality","protamylasse","non-ribosomal","plant waste, rest stream"],"prmu":["P","P","P","P","P","P","R"]}
{"id":"Knee_Surg_Sports_Traumatol_Arthrosc-3-1-2042026","title":"Analysis of Oxford medial unicompartmental knee replacement using the minimally invasive technique in patients aged 60 and above: an independent prospective series\n","text":"We present the outcome of an independent prospective series of phase-3 Oxford medial mobile-bearing unicompartmental knee replacement surgery. Eight surgeons performed the 154 procedures in a community-based hospital between 1998 and 2003 for patients aged 60 and above. Seventeen knees were revised; in 14 cases a total knee replacement was performed, in 3 cases a component of the unicompartmental knee prosthesis was revised, resulting in a survival rate of 89% during these 2\u20137 years follow-up interval. This study shows that mobile-bearing unicompartmental knee replacement using a minimally invasive technique is a demanding procedure. The study emphasises the importance of routine in surgical management and strict adherence to indications and operation technique used to reduce outcome failure.\nIntroduction\nModifications over the past 15\u00a0years have improved unicompartmental knee replacement surgery, as indicated in recent reports on the procedure [1\u20134]. The designers [5] (the originators) of the Oxford unicompartmental knee prosthesis (Biomet, Warsaw, IN) reported in 1998 a 97.7% cumulative survival rate of 10\u00a0years. An independent series with a 15-year survival analysis claimed a 94% cumulative survival rate [6]. The outcome was dependent on proper patient selection, surgical techniques and implant design, [4, 7] and the results have been attributed to improvements in these factors. The procedure is now performed through a short incision from the medial pole of the patella to the tibial tuberosity. Using this approach, there is little damage to the extensor mechanism, the patella is not dislocated, and the suprapatellar synovial pouch remains intact. As a result, patients recover more quickly. Patients achieve knee flexion, straight leg-raising, and independent stair-climbing three times faster than after total knee replacement (TKR) and twice faster than after open unicompartmental knee replacement surgery [8]. The minimally invasive procedure has been shown to be reliable and effective [9].\nBecause of the favourable published clinical results, surgeons at the Martini Hospital in Groningen, the Netherlands, began using the Oxford knee prosthesis in 1998. The goal of this independent prospective study for patients 60\u00a0years of age and above was to compare and evaluate the clinical midterm results of the Oxford phase-3 unicompartmental knee replacement using the minimally invasive technique in a community hospital.\nMaterials and methods\nBetween December 1998 and 2003, 154 successive Oxford unicompartmental knee replacements were performed in patients 60\u00a0years of age and above (Table\u00a01). Of these, 132 patients underwent unilateral surgery, 10 patients underwent bilateral surgery on separate occasions, and 1 patient underwent concomitant bilateral surgery in the same OR session. There were 86 women; the average patient\u2019s age was 69.2\u00a0years (range 60\u201393\u00a0years). All patients gave informed consent before their inclusion in this prospective study. Five patients had secondary osteoarthritis because of previous trauma. The remaining patients had primary osteoarthritis.\nTable\u00a01Oxford phase-3 unicompartmental knee replacementCriteriaResultsNumber of patients132Number of knees154Left\/right knee (%)53.8\/46.2Age (mean\/range, in years)69.2 (60\u201393)Gender (M\/W)57 (40%)\/86 (60%)BMI30.7\u00a0\u00b1\u00a04.9Follow-up range2\u20137\u00a0years\nStandardised anteroposterior radiographs were obtained with the patient in a weight-bearing position (standing), and lateral radiographs were obtained with the patient in a non-weight-bearing position (the patient lying horizontally). The radiographs were examined for loosening or radiolucency around the femoral and tibial components, and the anatomical axis of the limb was measured. The imaging criterion for no increased risk for loosening of the bone was a <2\u00a0mm thick radiolucent line [10]. The presence of osteoarthritic changes in the nonreplaced compartment was graded according to the Ahlback classification of osteoarthritis (Table\u00a02) [11]. These procedures were performed by eight senior staff surgeons over the study period. Mean preoperative range of motion was 122.9\u00a0\u00b1\u00a08.9\u00b0 of flexion and \u22120.7\u00a0\u00b1\u00a04.5\u00b0 of extension.\nTable\u00a02The Alhback radiological scoring system for estimating the severity of OAGrade 0 Normal Grade 1 Joint narrowing Grade 2 Joint obliteration Grade 3 Bone destruction <5\u00a0mm Grade 4 Bone destruction >5\u00a0mmGrade 5 Subluxation\nThe results (preoperative, intraoperative, and follow-ups at 3\u00a0months, 6\u00a0months, and 1\u00a0year) were prospectively recorded with a historical record, procedure record, Knee Society score, SF-36 questionnaire (short form consisting of 36 questions), and the Western Ontario McMaster (WOMAC) score. Knee Society score ratings of excellent (90\u2013100 points) and good (80\u201389 points) indicated success. The preoperative scores of the patients are presented in Table\u00a03.\nTable\u00a03Scoring results of the non-revised patientsScoringResultsKnee society scoreKnee score\u00a0Preoperative39.2 (SD 18.2)\u00a0Postoperative89.4 (SD 14.0)Function\u00a0Preoperative55.8 (SD 14.3)\u00a0Postoperative77.1 (SD 24.7)Total score\u00a0Preoperative47.6 (SD 12.3)\u00a0Postoperative83.4 (SD 16.8)WOMAC scorePain\u00a0Preoperative50.3 (SD 18.7)\u00a0Postoperative78.6 (SD 21.5)Stiffness\u00a0Preoperative51.2 (SD 22.6)\u00a0Postoperative71.2 (SD 20.8)Function\u00a0Preoperative50.6 (SD 20.7)\u00a0Postoperative76.2 (SD 20.4)SF-36 questionnaireFunction\u00a0Preoperative35.7 (SD 17.6)\u00a0Postoperative56.1 (SD 24.5)Physical\u00a0Preoperative28.2 (SD 37.2)\u00a0Postoperative57.2 (SD 44.3)Pain\u00a0Preoperative32.7 (SD 19.2)\u00a0Postoperative59.8 (SD 26.5)Health\u00a0Preoperative63.7 (SD 22.2)\u00a0Postoperative61.4 (SD 21.7)Social function\u00a0Preoperative52.6 (SD 17.1)\u00a0Postoperative64.5 (SD 17.6)Emotional\u00a0Preoperative64.5 (SD 44.6)\u00a0Postoperative70.5 (SD 40.7)Mental health\u00a0Preoperative73.7 (SD 17.9)\u00a0Postoperative75.1 (SD 18.8)SD standard deviation\nPreoperative weight-bearing radiographs showed that the knees had an average femorotibial alignment of 2.4\u00b0 of valgus (range 8\u00b0\u20133\u00b0 of varus). Thirty-seven knees had grade-1 Ahlback osteoarthritis [11] in the lateral compartment on the preoperative radiographs, and one had grade-2 Ahlback osteoarthritis. The preoperative skyline view of the patellofemoral joint showed no bone loss with eburnation and longitudinal grooving in all the cases.\nAll medial compartment arthroplasties were performed using the minimally invasive technique and under tourniquet control. The discharge criteria were control of immediate postoperative pain and the ability to flex the operated knee to a minimum of 90\u00b0 with no lack of extension. All complications and revisions were reported, and a revision was defined as any surgical procedure resulting in removal or exchange of any of the prosthetic components.\nResults\nAt the time of follow-up, two patients who had no known revisions were lost for the follow-up. The remaining 130 patients were available for follow-up. At the final follow-up, June 2006, revision TKR was performed in 14 knees and a prosthetic component was exchanged in three knees. An overview of the revisions is given in Table\u00a04.\nTable\u00a04Revisions of Oxford phase-3 knee replacement surgeryIncidenceRevision of a component of UKA3\u00a0Revision of the mobile bearing1\u00a0Revision of the femoral component and the bearing1\u00a0Revision of the tibial component and the bearing1Conversion to a TKR14Reason for revision to a TKA\u00a0Inappropriate indication1\u00a0Misalignment and loosening5\u00a0Infection1\u00a0Progression of osteoarthritis in lateral compartment4\u00a0Persisting anteromedial pain >1\u00a0year3\nOne bearing was replaced because of luxation after a hyperflexion trauma. A new bearing of the same size was inserted, and no recurrence of luxation was seen at follow-up. In another case of luxation of the bearing, the femoral component, and the bearing were changed 9 months after the primary surgery. The fixation of the femoral component in this case was insufficient. The multiple small drill holes were not made, and there was no cement in the large drill hole. With flexion, the loose femoral component moved distally, causing luxation of the bearing. The tibial component and bearing revision was performed seven months after the primary surgery because of misalignment of this tibial component. With flexion, there was impingement of the bearing with the tibial component, causing a clicking sensation and rotation of the bearing.\nIn one case, there was grade-2 Ahlback osteoarthritis [11] in the lateral compartment on the preoperative radiograph. This patient had no relief of preoperative pain, and the knee underwent TKR 18\u00a0months after the primary surgery. In five cases loosening of the components occurred; misalignment of the components is probably caused by impingement of the bearing. One patient had a deep Staphylococcus aureus infection, and a two-stage procedure was performed leading to a TKR. In four cases of revision, progression of osteoarthritis was seen in the lateral compartment with reported pain on the lateral side. These patients had a mean postoperative anatomical axis, femorotibial alignment of 18.6\u00b0. This overcorrection causes overloading of the lateral compartment with progression of arthritis in that compartment.\nThree patients with persisting anteromedial pain underwent revision. In two cases, no cause was found, and in both pain persisted after TKR. In the third case, the synovial biopsy showed synovitis villonodularis pigmentosa, and after the TKR this patient was pain-free. Except for the two patients with persisting anteromedial pain, all patients with a conversion to TKA were pain-free. No special augmentations or revision prosthetic components were necessary in these procedures; there were no bone defects that required the use of particulate autograft or allograft, and primary cruciate-retaining TKA was used in the revisions.\nPostoperative complications occurred after the primary unicompartmental knee replacements. One patient had a traumatic medial tibia plateau fracture 4\u00a0weeks postoperatively, which was treated conservatively. Another patient developed hemarthrosis that required extended hospitalisation; this was resolved with conservative treatment. There was one deep infection, and no deep venous thrombosis was reported.\nAt the time of the most recent follow-up, average flexion was 125.8\u00a0\u00b1\u00a013.8\u00b0, with two patients achieving <90\u00b0 flexion. The average flexion deformity\/extension was 0.3\u00a0\u00b1\u00a02.2\u00b0.\nThe postoperative scores of those patients who did not undergo revision (140 knees) at the latest follow-up are presented in Table\u00a03. The Knee Society score total was 83.4. All three WOMAC scores improved. For the SF-36, the function, physical, and pain scores showed an improvement in the outcome; the other scores remained approximately the same.\nThe final follow-up radiographs showed an average anatomical axis, femorotibial alignment of 8.8\u00b0 of valgus (range 4\u00b0\u201322\u00b0 of valgus). The knees were corrected by an average of 6.4\u00b0 (range 2\u00b0\u201314\u00b0). This relative overcorrection gives increased stress on the lateral compartment. Signs of osteoarthritis progression in the uninvolved tibiofemoral compartment on the radiograph at the last follow-up were noted in 43 knees (grade-1 Ahlback osteoarthritis in 39 knees and grade-2 Ahlback osteoarthritis in four knees). No grade-3 or -4 changes were noted. At final radiographic evaluation, no component showed evidence of loosening. No knees had >2\u00a0mm of tibial cement-bone radiolucency. There were no radiolucent lines seen at the posterior aspect of the femoral components.\nSeventeen knees were revised, resulting in a survival rate of 89% in these 2\u20137\u00a0years of follow-up interval.\nDiscussion\nThe purpose of this prospective study was to evaluate midterm durability of Oxford unicompartmental knee replacement surgery for patients 60\u00a0years of age and older. We acknowledge that the present study has the limitations of a midterm follow-up. However, longer follow-up for this phase-III version with the minimally invasive technique is not possible, because the current version has been available only since 1998 [7]. Besides, most technical failures occur within the first 2\u00a0years [12].\nIn these 2\u20137\u00a0years of follow-up interval, 11% of unicompartmental knee arthroplasties in all patients needed revision\u2014a survival rate of 89%. These results are considerably lower compared to the designer [5] series or the independent series [6].\nThe primary need for revision surgery could be attributed to indication and technical failures. Thirteen of the 17 revisions were probably related to human error, the remaining four are in one case a hyperflexion trauma and luxation of the bearing, one case with deep infection, and two cases with unexplained persisting anteromedial pain. Misalignment of the components was the primary cause of technical failure. With the minimally invasive technique, the visual field is restricted, making mobile-bearing unicompartmental knee replacement surgery a demanding procedure. Introduction of the minimally invasive option makes the terms surgical technique and pitfalls actual again.\nFor the remaining 113 patients (140 knees) who did not undergo revision, the Knee Society score, WOMAC and SF-36 questionnaires showed an improvement in the outcome. All three scores indicated less pain and improvement in function, as confirmed by an average clinical average flexion of 126\u00b0 at the latest follow-up. The Knee Society score total of 83.4 indicates a successful outcome.\nOver the 7-year period of our study, eight senior surgeons performed the operation with an average of <10 procedures a year per surgeon. All surgeons attended the instructional course organized by the designer group. There is no evidence for a learning curve in our study. The outcome should be attributed to the number of operations performed. As a result of the relatively low survival rate of this study, the number of senior surgeons performing the procedure in this hospital is now reduced to two.\nConclusion\nCareful patient selection, surgeon experience, and proper instrumentation and surgical technique are important factors in mobile-bearing unicompartmental knee replacement surgery [13, 14]. For unicompartmental replacement surgery, long-term results are related to the number performed by the unit [14]. The surgeon should be well versed in the routine, indications, and technique of this procedure to minimise failure rates.","keyphrases":["unicompartmental","knee","replacement","mobile bearing"],"prmu":["P","P","P","R"]}
{"id":"Purinergic_Signal-3-4-2072925","title":"New insights into purinergic receptor signaling in neuronal differentiation, neuroprotection, and brain disorders\n","text":"Ionotropic P2X and metabotropic P2Y purinergic receptors are expressed in the central nervous system and participate in the synaptic process particularly associated with acetylcholine, GABA, and glutamate neurotransmission. As a result of activation, the P2 receptors promote the elevation of free intracellular calcium concentration as the main signaling pathway. Purinergic signaling is present in early stages of embryogenesis and is involved in processes of cell proliferation, migration, and differentiation. The use of new techniques such as knockout animals, in vitro models of neuronal differentiation, antisense oligonucleotides to induce downregulation of purinergic receptor gene expression, and the development of selective inhibitors for purinergic receptor subtypes contribute to the comprehension of the role of purinergic signaling during neurogenesis. In this review, we shall discuss the participation of purinergic receptors in developmental processes and in brain physiology, including neuron-glia interactions and pathophysiology.\nIntroduction\nDuring the last two decades, evidence for the participation of ATP as neurotransmitter in neuronal signaling was collected by Drs. Surprenant [1] and Silinsky [2]. Purine-sensitive receptors were first classified as P1 G-coupled receptors which are activated by adenosine and P2 receptors, responding to stimulation of ATP [3]. Based on receptor cloning and studying of receptor-induced signal transduction, P2 receptors were divided into P2X receptors as ATP-gated ion channels and P2Y G protein-coupled receptors [4].\nThe expression of purinergic receptors has been identified during development and differentiation processes [5\u201310]. Nucleotides exert a synergic effect on cell proliferation in association with growth factors, chemokines, or cytokines in early stages of development [11\u201313] by parallel activation of the MAP kinase pathway and\/or by transactivation of growth factor receptors [14, 15].\nThe complete role of ATP action in developmental processes still needs to be elucidated. It is known that ATP activates purinergic receptors resulting in many cases in increases of intracellular free calcium concentration [Ca2+]i. Changes in [Ca2+]i are involved in several events of differentiation and the embryogenesis process [16, 17]. Spitzer et al. [18] showed that naturally occurring patterns of Ca2+ transients encoded neuronal differentiation. Distinct frequency patterns of [Ca2+]i elevations were sufficient to promote neuronal differentiation, including physiological neurotransmitter receptors expression [19]. ATP and UTP are the main purinergic agonists activating P2X or P2Y receptors. These nucleotides can be rapidly degraded in the extracellular space by ectoenzymes to ADP or UDP, subsequently activating distinct P2Y receptors, or be finally degraded to adenosine, which is known to induce physiological responses via activation of P1 G protein-coupled receptors [20] (Fig.\u00a01).\nFig.\u00a01Purine-induced signaling pathway involves the activation of P1 adenosine and P2 purinergic receptors and purine hydrolysis by ectonucleotidases. The scheme demonstrates purinergic receptor activity present in glia-glia, neuron-glia, and neuron-neuron interaction during neurogenesis as well as in the metabolism of the adult brain\nIn this review article, we shall discuss the roles of purinergic signaling in neurogenesis such as cell cycle control during neural progenitor proliferation and differentiation as well as in maintaining physiology of neurons and glial cells and the involvement of purinergic receptors in pathophysiology. In addition, we shall outline state-of-the-art approaches used in investigation of P2 receptor function in physiological processes such as the use of antisense oligonucleotides, generation of knockout animals, and identification of new purinergic receptor subtype-selective drugs.\nStudy of purinergic receptor function during in vitro differentiation\nDuring the development of the mammalian nervous system, neural stem cells and their derivative progenitor cells generate neurons by asymmetric and symmetric divisions [21]. P2 receptors were shown to be one of the first functionally active membrane receptors in chick embryo cells during gastrulation, in which ATP caused rapid accumulation of inositol triphosphate and Ca2+ mobilization in a similar way as acetylcholine (Ach) did via activation of muscarinic acetylcholine receptors, whereas other endocrine-acting substances such as insulin and noradrenaline (NA) induced much weaker effects in terms of intracellular calcium signaling [22, 23]. The induction of transient fluctuation in [Ca2+]i also denominated as calcium wave signaling allows for a coupling of spatial and temporal information. Thus, calcium waves have been proposed to play a role in mapping of neuronal networks [24] and to modulate neurogenesis during embryonic cortical development [25].\nNeurotransmitters are prominent candidates for transcellular signals that could influence the development of embryonic neurons as they surround neural cells throughout brain development [26\u201329]. In addition, functional ligand-gated ionic channel receptors have been identified in neural progenitor cells prior to establishing cortical and subcortical synapses [30, 31]. In this context, the extracellular signaling mechanisms controlling the various transition steps involved in adult neurogenesis are still poorly understood. One approach used to identify the function of P2 receptors during development and differentiation is the use of in vitro models for neuronal and glial differentiation such as embryonic and adult neural progenitor cells (NPC), also known as neural stem cells (NSC), embryonic stem (ES), and embryonal carcinoma (EC) cells.\nES cells are obtained from the inner mass cell of the blastocyst. The differentiation of these cells closely resembles the in vivo process and, therefore, provides stable models for embryonic growth and development [32, 33]. ATP promotes cell proliferation acting through P2X3, P2X4, P2Y1, and P2Y2 receptors in murine ES cells [34]. Tissue-nonspecific alkaline phosphatase (TNAP) was also detected in these cells and used as a marker for their undifferentiated stage [35].\nThe neuronal differentiation of EC cells, originated from irradiated embryo cells [36], also resembles early neuronal development in vivo. P19 mouse EC cells express stem cell-specific marker proteins and their phenotypic changes in specific differentiation stages are similar to those of stem cells [37]. Recently, our laboratory [38] has determined gene and protein expression of P2 receptor subtypes throughout in vitro neuronal differentiation of P19 cells as well as in the undifferentiated cell stage suggesting the participation of purinergic signaling in initiating and directing differentiation. Differential expression and activity of P2Y1, P2Y2, P2Y4, P2X2 subtypes and P2X6 subunits were reported during neuronal maturation of P19 cells [38, 39]. As direct evidence for participation of purinergic receptors in neuronal differentiation, the presence of the antagonists pyridoxalphosphate-6-azophenyl-2\u2032,4\u2032-disulfonic acid (PPADS), reactive blue 2, or suramin during differentiation of P19 neural progenitor cells (NPC) to P19 neurons resulted in reduced activity of cholinergic and glutamate NMDA receptors in differentiated P19 cells, pointing at a participation of P2Y1, P2Y2, and P2X2 receptors.\nOther in vitro neuronal and glial differentiation models used to understand the purinergic signaling are neural stem cells or progenitor cells which are isolated from the subventricular region (SVZ) located in the lateral ventricles (type B cells) or in the subgranular region of the gyrus dentatus of the hippocampus (residual radial glia) or even from the subcortical parenchyma of the cerebral cortex of embryonic and adult brain [40\u201342]. These regions in the adult brain act as neural stem cell reservoirs. These cells are already advanced in their differentiation stage when compared to ES or EC cells. Since NSC and NPC are capable of differentiating in both functional neurons and glial cells, they possess potential therapeutic applications such as ES cells in regeneration therapy following neuronal loss.\nThese NPC differentiate into olfactory, cerebellar, and retinal neurons [40] in the presence of growth factors, neurotransmitters, vasoactive peptides in vivo [43], and growth factors such as epidermal growth factor (EGF), fibroblast growth factor 2 (FGF-2), and leukemia inhibitory factor (LIF) in vitro. When exposed to a high concentration of FGF-2 in suspension, proliferating NPC form tridimensional cell aggregates denominated as neurospheres, which following induction to differentiation express neuronal marker proteins such as \u03b2-III-tubulin, microtubule-associated protein-2 (MAP-2), and synaptophysin [44] and express P2X3 and P2X7 receptors which may contribute to early [Ca2+]i transients as prerequisites for further differentiation [41]. Shukla et al. [45] identified functional P2 receptors in adult mouse hippocampal progenitors in situ and the nucleoside triphosphate-hydrolyzing ectoenzyme (NTPDase) in type B cells of the SVZ [46] and in hippocampal progenitor cells. In adult murine NPC of SVZ, P2Y1 receptor activity mainly contributes to [Ca2+]i transients with some participation of P2Y2 receptors. The presence of the specific P2Y1 receptor antagonist MRS 2179 resulted in diminished cell proliferation in neurospheres due to reductions of [Ca2+]i transients. Similar results were obtained with NPC from SVZ of P2Y1 receptor knockout mice [47]. P2Y1 receptor-deficient mice are viable; however, they have deficits in platelet aggregation [48]. It is suggested that the purine signaling underlies autocrine or paracrine mechanisms and P2Y1 and P2Y2 receptors are important for NPC differentiation [47]. These models are useful tools to study the roles of P2 receptor signaling in early stages of development and differentiation. The importance of ATP release and purinergic signaling has not only been demonstrated in developmental progenitor cell expansion and neurogenesis, but also in persistent progenitor cells of the adult brain [49].\nExpression of purinergic receptors during development of the central nervous system\nPurinergic signaling pathways are also involved in embryonic neurogenesis in much the same way as already discussed for in vitro differentiation models. ATP mediates elevation of [Ca2+]i and proliferation of immortalized human stem cells from embryonic telencephalon and mouse embryonic neurospheres [50, 51]. Ca2+ waves through radial glial cells in slices of the embryonic rat ventricular zone are mediated by P2Y1 receptors. Disrupting Ca2+ waves between embryonic NPC reduced ventricular zone cell proliferation during the peak of embryonic neurogenesis [25].\nATP directly contributes to modulate network-driven giant depolarizing potentials in the rat hippocampus during early stages of postnatal development [52]. In the developing hippocampal system a trophic role of ATP and the involvement of P2 receptor subtypes in shaping interneuronal connections during neuronal differentiation have been suggested [53]. Alterations of the regulation of embryonic growth by purinergic receptors might be involved in the onset of morphological malformations [54]. During rat postnatal development ectonucleotidase activity in the cerebral cortex steadily increases, reaching maximum values at 21 days of age [55]. Several P2Y and P2X receptors were shown to be dynamically expressed in the pre- and postnatal central and peripheral nervous system [56\u201359]. ATP inhibited motor axon outgrowth during early embryonic neurogenesis, most likely through the P2X3 receptor, and it was speculated that P2X7 receptors might be involved in programmed cell death during embryogenesis [58].\nFrom all of the studied P2X receptors, homomeric P2X2 receptors were the first expressed in the rat central nervous system (CNS) on embryonic day 14 (E14) [56]. On E14, heteromeric receptors were formed by P2X2\/3 receptor subunits. P2X3 receptor immunoreactivity was detected in cranial motor neurons as early as on E11, when neurons exited the cell cycle and started axon outgrowth, as well as postnatally on days 7 and 14 (P7 and P14) [56, 60]. Moreover, expression of P2X3-containing heteromeric receptors and other subunits was developmentally regulated in nucleus ambiguous motoneurons [61]. From E14 onwards P2X7 receptors were also expressed in the embryonic brain. For instance, in primary cultures of human fetal astrocytes basal levels of P2X7 receptor mRNA transcription and protein expression were detected [62]. Sperl\u00e1gh et al. [63] have demonstrated that ATP regulates glutamate release via activation of P2X7 receptors. P2X7 receptor-induced excessive glutamate release alters Ca2+ homeostasis, subsequently resulting in activation of the apoptosis-related caspase cascade [64].\nP2X receptor expression was downregulated in Purkinje cells and deep cerebellar nuclei at P21 and P66 rat embryonic stages, with the exception of P2X5 receptors whose immunoreactivity in granular cells was increased [65]. Evidence for participation of P2X receptors in different developmental processes such as neurite outgrowth (involving P2X3 receptors), postnatal neurogenesis (related to P2X4 and P2X5 receptor expression), and cell death (possibly involving P2X7 receptors) was collected. However, P2X1 and P2X6 receptor subunits may not play a role in neuronal development [58].\nNeocortical neurons from 2-week-old rats possess a quite elaborated purine-triggered signaling system which includes both P2Y and P2X receptor activation [66]. Weissman et al. [25] showed that [Ca2+]i waves and subsequent ATP release, with consequent P2Y1 receptor activation, accompanied radial glial cell-derived neurogenesis in cultured slices of the developing rat forebrain, as mentioned above. Moreover, the importance of calcium signaling for differentiation of NPC has been studied [67, 68], and direct evidence for the participation of P2Y1 receptor-activated pathways in the early development has been provided by Scemes et al. [69]. P2Y receptors (particularly the P2Y1 subtype) were widely expressed in the embryonic rat brain as early as on E11 [57]. There was a marked decrease in the concentration of mRNA coding for P2Y1 receptors and upregulation of mRNA transcription coding for P2Y2 receptors in freshly isolated astrocytes of developing rat hippocampus [57].\nFunctional interactions between neurons and glia: a physiological overview\nAn increasing amount of evidence, initiated by the neuron-glia unit idea proposed by Hyden [70], indicates that glial cells, once referred to as a simple support portion in the CNS, are now considered indispensable functional partners of neurons [71], both in physiological and pathological conditions. However, many questions remain unanswered: (1) how glia detects and interacts with neural function; (2) does neuron-glia signaling play a significant role in synaptic transmission and plasticity; and (3) how glial cells can communicate with other glial cells.\nAnother important subject related to the interaction between glia and neurons emerges in neurogenesis. There is now a general agreement that the adult mammalian nervous system possesses many characteristics of astrocytes. The importance of glia in neuronal development was confirmed in a recent study showing that the number of GFAP (glial fibrillary acidic protein)-containing cells was reduced following transgenic targeting of adult mouse subependymal and subgranular zones, resulting in an almost complete loss of neurogenesis [72, 73]. In addition to assisting migration of neurons to their correct position and managing neurite outgrowth to their final communication targets [74, 75], glial cells have become an essential key for understanding neuronal differentiation by promoting initial stem cell proliferation and instructing undifferentiated cells to adopt a neuronal fate [76, 77].\nIn the mature brain, the proximity of astrocytes to neuronal synapses or to the blood-brain barrier makes these cells appropriate to control water diffusion and ion concentration in extracellular spaces [71, 78]. In particular, astrocytes regulate homeostatic environment and neurotransmitter levels by functional syncytium, in which gap junctions and specific membrane carriers play an important role [79\u201381]. In addition, glial cells produce and release a vast number of neurotrophins, including fibroblast growth factor, nerve growth factor, and transforming growth factor, which directly interfere in neuron physiology and coordinate developmental processes [71, 82\u201385].\nATP release and degradation, connecting adenosinergic and purinergic systems\nAs already mentioned, it is well documented that glial cells may directly alter neuronal activity by releasing neurotrophins and consequently modulating neurotransmitter release in the synapse [86, 87]. One of the main mechanisms connecting the neuron-glia system is believed to be mediated by the release of glutamate from glial cells [88, 89]. In this context, growing evidence indicates that purinergic receptor ligands are widely involved in the cell-cell signaling mechanism by acting as neurotransmitters or neuromodulators released by glial cells to control synaptic transmission in the CNS, as part of multiple functions of astrocytes [22, 90, 91] (Fig.\u00a01).\nATP is an ideal molecule for cell signaling due to its intrinsic properties such as its small size, diffusing molecule rate, instability and low concentration in the extracellular environment, and impossibility to cross the plasma membrane [92, 93]. These properties imply the presence of particular pathways for ATP release that could be associated with cellular excitation\/response and cell-cell signaling [94, 95]. First, ATP may be stored in synaptic vesicles alone or with other neurotransmitters and then released, as a classic synaptic mechanism in the peripheral or central nervous system [96, 97]. Second, a nonvesicular mechanism of ATP release could be observed through gap junction hemichannels, ATP-binding cassette proteins, P2X7 receptor pores in glial cells, and via chloride channels [98\u2013101]. Third, ATP could be released due to cytolysis or cell damage. While this is not a physiological mechanism, it takes place following biological trauma and contributes to pathological conditions [102].\nSubsequent to these mechanisms, the metabolism of the released ATP is regulated by a vast number of different families of ectonucleotidases in the synaptic cleft, including the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) and the ectonucleotide pyrophosphatase phosphodiesterase (E-NPP) which catalyze the degradation of ATP to ADP or AMP. The degradation to adenosine is mediated by ecto-5\u2032-nucleotidase (E-5\u2032-NT) and alkaline phosphatase [91, 103] (Fig.\u00a01). Consequently, the reaction products resulting from the ATP hydrolysis may bind to P2 receptors, in the case of ADP, or to P1 receptors in the case of adenosine [104].\nThe adenosinergic receptor ligand adenosine is recognized as an important regulator of cellular homeostasis in the CNS and may be involved in the prevention or induction of apoptosis [105]. The reduction of ectonucleotidase activity in certain pathological conditions provided additional evidence for the accumulation of ATP in the extracellular environment [20]. Therefore, the complexity of the communication of neural and nonneural cells expands the functional significance by the interaction of the purinergic receptors in association with a variety of neurotransmitter systems.\nATP-mediated neuron-glia signaling\nNovel studies in the purinergic field began to converge with glial research as it became more widely accepted that ATP is released through synaptic vesicles and thus accessible to perisynaptic glial cells, allowing them to detect neuronal activity. In particular, glial cells are responsive to ATP, as all types of glia, such as astrocytes, oligodendrocytes, microglia, and Schwann cells, express purinergic receptors [91]. In Schwann cells and oligodendrocytes, ATP-mediated signaling predominantly occurs through P2Y receptors, which in turn trigger intracellular Ca2+ release [106, 107]. However, the function of P2X1\u20136 receptors in astrocytes remains unclear, although P2X-mediated currents could be detected in astrocyte cells in culture, and P2X7 receptors are widespread in these cells with possible contribution to pathological conditions [108, 109].\nGlial cells express many types of neurotransmitter receptors and conventionally are considered to be nonexcitable [110, 111]. However, a surprising observation was reported by Dani et al. [112] that synaptic transmission may propagate to glial cells as calcium waves, inducing membrane depolarization and regulating neurotransmitter release. These properties of glial cells suggest possible rapid communications between neurons and glia during synaptic transmission. This glial communication mechanism allows the released ATP to act onto adjacent astrocytes and neurons, thus supporting the propagation of Ca2+ waves in glial syncytium [113]. For example, in neuronal-glial cocultures prepared from hippocampus, ATP secreted by astrocytes was shown to inhibit glutamatergic synapses through activation of P2Y receptors [114].\nThe glial communication mechanism based on Ca2+ wave propagation could be inhibited by P2 receptor blockers or enzymes that rapidly hydrolyze extracellular ATP [115]. A stimulation applied to a single astrocyte in cocultures of rat forebrain astrocytes and associated neurons caused an elevation of [Ca2+]i and induced Ca2+ wave propagation in dorsal spinal cord through P2Y1 receptor activation [116] and glutamate release [117]. This new finding provided a parallel mechanism of intercellular communication that could allow astrocytes to detect synaptic function, propagate the information through neighboring glial cells, and then influence synaptic function in a distant part of the nervous system.\nPurinergic receptor-calcium signaling in glial cells plays important roles during CNS development. P2X1, P2X4, and P2X7 receptors were expressed in microglia at rat embryonic stage (E16) [59]. Moreover, changes in P2X4 receptor expression in microglial cells during postnatal development of the rat cerebellum have been reported. P2X5 receptor immunoreactivity was also upregulated in microglia and granular cells. Both P1 and P2 receptors contribute to the modulation of oligodendrocyte (OP) development, since they have been shown to exert similar effects on OP proliferation and differentiation [118].\nThe majority of the studies of ATP action have been concerned with the short-term P2 receptor signaling that occurs in neurotransmission and in secretion [119]. Furthermore, there is increasing evidence that purines and pyrimidines can have trophic roles in neuritogenesis [120, 121], regeneration [122], and proliferation [123]. However, some purines by themselves have limited trophic effects in a few types of cells; they appear to be much more effective as neuritogenic agents when they are combined with other trophic factors, such as NGF. For instance, inosine and 5\u2032AMP alone do not elicit neurite extensions in PC12 cells [124].\nHeine et al. [53] demonstrated that P2 receptor activation induced fiber outgrowth in organotypic cocultures in rat hippocampus. Fiber outgrowth was inhibited in the presence of the purinergic antagonist PPADS, suggesting the involvement of P2 receptors. In another study, the synergistic interaction between bFGF and ATP was reported on DNA synthesis in primary cultures of rat cortical astrocytes. ATP and bFGF induced a twofold and tenfold incorporation of [3H]thymidine into astrocytes, respectively, but when ATP and bFGF were added at the same time a 50-fold increase in [3H]thymidine incorporation was observed [12].\nNeuroprotection\nATP can activate P2X7 receptors in astrocytes to release glutamate, GABA, and also ATP which might regulate the excitability of neurons in certain pathological conditions [125]. It has been suggested that astrocytes can sense the severity of damage in the CNS by the amount of ATP released from damaged cells and that extracellular ATP concentration and the corresponding subtype of activated astrocytic P2 receptor modulate the tumor necrosis factor-\u03b1 (TNF-\u03b1)-mediated inflammatory response [126]. After mechanical brain injury, the administration of PPADS facilitated the recovery of pathologically changed electroencephalograms [127]. These results suggest that interference with the ATP-induced excitatory responses could provide neuroprotection and possible therapeutic consequences.\nEvidence for a neuroprotective role was also found for the adenosine A1 receptor in hippocampus. This cerebral region is highly sensitive to hypoxia and ischemia. The study of the action of hypoxia on synaptic transmission in hippocampal slices has suggested that substances being released during hypoxia, such as GABA, ACh, and even glutamate, may also play neuroprotective roles. However, the actions of these neurotransmitters become evident only when activation of P1 receptors is impaired, suggesting a critical role for this receptor during hypoxic events. These substances can operate in a redundant or even overprotective manner, acting as a substitute for some adenosine actions when the nucleoside is not operative [128].\nNeuroimmune interactions\nMicroglia, the immune cells of the CNS, can be activated by purines and pyrimidines to release inflammatory cytokines such as IL-1, IL-6, and TNF-\u03b1. However, hyperstimulation of the immune reaction in the brain may accelerate neuronal damage. The P2X7 receptor is considered to have a potentially pivotal role in the regulation of various inflammatory conditions. ATP selectively suppresses the synthesis of the inflammatory protein microglial response factor through calcium influx via P2X7 receptors in microglia [129], which also leads to enhancement of interferon-\u03b3 (IFN-\u03b3)-induced type II nitric oxide synthase (NOS) activity [130, 131]. P2X7 receptor activity also participated in ATP-induced IL-1 release from macrophages and microglia that had been primed with substances such as bacterial endotoxin [132] and was shown to stimulate the transcription of nuclear factor \u03baB, TNF-\u03b1 [133], the stress-activated protein kinases (SAPK)\/JNK pathway [134], and the production of 2-arachidonoylglycerol, which is also involved in inflammation induction by microglial cells.\nP2Y rather than P2X7 receptors seem to have a major role in the IL-6 production by microglial cells [135]. ATP evoked the release of plasminogen [136] and IL-6 [135]. The stimulation of microglia by either ATP or BzATP revealed neurotoxic properties and the involvement of the P2X7 receptor has been reported in excitotoxic\/necrotic and apoptotic degeneration [109].\nNeurological disorders\nEpilepsy Several anti-epileptic agents reduce the ability of astrocytes to transmit Ca2+ waves, raising the possibility that blockade of ATP-induced [Ca2+]i transients in astrocytes by purinergic receptor antagonists could offer new treatments for epileptic disorders. Antiepileptic effects of adenosine are mostly due to the well-known inhibitory actions of P1 receptors on synaptic transmission in the hippocampus. However, as recently pointed out, adenosine actions are not limited to presynaptic actions on glutamate release [137]. The intraventricular injection of high doses of ATP in rats evoked severe chronic-tonic convulsions, whereas lower doses of ATP or adenosine elicited a kinetic state with muscle weakness [138]. P2X2 and P2X4 receptor expression in the hippocampus of seizure-prone gerbils was significantly reduced compared with that of normal gerbils [139]. GABAA receptors mediated modulation of expression of both P2X2 and P2X4 receptors, which may play an important role in the regulation of seizure activity in the gerbil hippocampus [139]. P2X7 receptors are thought to play a definite, but not yet well defined role in epilepsy. Treatment with the GABAB receptor agonist baclofen and antagonist phaclofen resulted in increased and decreased P2X7 receptor expression in hippocampus, respectively [140]. These purinergic receptor responses were interpreted as compensatory responses to the modulation of GABAB receptor function [140]. It is noteworthy to mention that this positive relationship between P2X and GABAA receptors was also reported for the spinal cord [141] and dorsal root ganglia (DRG) [142]. In these populations of neurons, ATP-mediated P2X receptor function may participate in neuronal transmission accompanied by GABA-mediated actions [139].\nPain The heteromeric channel comprised of P2X2 and P2X3 subunits was expressed almost exclusively in a subset of primary afferents implicated in nociception [143\u2013145]. It has been observed that mechanical allodynia is reduced in mice with deleted P2X3 receptor genes [146, 147] in agreement with data obtained in rats that have been treated with intrathecal antisense oligonucleotides reducing expression of P2X3 receptors [148] or with the selective antagonist for P2X3 and P2X2\/3 receptors A-317491 [148, 149]. P2X3 receptor knockout mice showed additional defects in afferent pathways.The P2X4 receptor is also implicated in pain sensation. Activation of dorsal horn microglia and tactile allodynia developing several days after ligation of a spinal nerve were greatly reduced when gene expression of P2X4 receptor in the dorsal horn had been inhibited by the presence of intrathecal antisense oligonucleotides [150]. Accordingly, intraspinal administration of microglia following induction of expression and activity of P2X4 receptors produced tactile allodynia in naive rats. Intrathecal administration of cultured brain microglia produced allodynia, but only when the cells had been pretreated with ATP [150]. The inhibition of P2X4 receptor activity in microglia might be a new therapeutic strategy for pain induced by nerve injury.\nAlzheimer\u2019s disease Alzheimer\u2019s disease (AD) is caused by extracellular deposition of amyloid \u03b2-peptide, which can damage neurons, leading to their dysfunction and death [151]. ATP and, in particular, aluminum-ATP promoted the formation of thioflavin T-reactive fibrils of \u03b2-amyloid and an unrelated amyloidogenic peptide, which could be blocked by suramin [152].Microglial cells are believed to contribute to the progression of AD and are known to release proinflammatory neurotoxic substances. Extracellular ATP, acting through the P2X7 receptor, can alter \u03b2-amyloid peptide-induced cytokine secretion from human macrophages and microglia and thus may be an important modulator of neuroinflammation in AD [153]. P2X7 receptors mediate superoxide production in primary microglia, and the expression of this receptor subtype was specifically upregulated around \u03b2-amyloid plaques in a transgenic mouse model of AD [154].In contrast to the control human brain, the P2Y1 receptor was colocalized with a number of characteristic AD structures such as neurofibrillary tangles, neuritic plaques, and neuropil threads in the hippocampus and cortex [155]. In general, control brain tissue exhibited a greater and more abundant level of P2Y1 receptor immunostaining than AD tissue did, probably due to severe neuronal cell degeneration in most AD brains. The intense P2Y1 receptor staining observed over pathological AD structures might imply that this receptor is involved either directly or indirectly in signaling events mediating neurodegeneration of pyramidal cells. Alternatively, P2Y1 receptors might have other diverse signaling roles, possibly involved in the production of intracellular tau deposits or might even serve to stabilize these tangle structures in some way [156].\nIschemia\/hypoxia Under pathological conditions of hypoxia or ischemia, extracellular purine nucleotides leak from damaged cells and thereby may reach high concentrations in the extracellular space [157]. A direct participation of extracellular ATP and P2 receptors in ischemic stress has been reported in various cellular systems [157\u2013160]. For example, P2X2 and P2X4 receptor expression in neurons and microglia, respectively, in the hippocampus of gerbils was upregulated following transient global ischemia [161]. Increased P2X7 receptor expression in astrocytes, microglia, and neurons appears to contribute to the mechanisms of cell death caused by in vivo and in vitro ischemia [162, 163]. Following induction of ischemia P2X7 receptor mRNA transcription and protein expression were elevated in cultured cerebellar granule neurons and organotypic hippocampal cultures [163]. Hence, the P2X7 receptor is apparently an important element in the mechanisms of cellular damage induced by hypoxia\/ischemia. In many cell types, the activation of the P2X7 receptor led to rapid cytoskeletal rearrangements, such as membrane blebbing and cell lysis [164]. P2Y1 receptors are intensely expressed in Purkinje cells in deep layers of the cerebral cortex and in ischemia-sensitive areas of the hippocampus [165]. In conclusion, extensive evidence demonstrates a postischemic time- and region-dependent upregulation of P2X2,4,7 and P2Y1 receptor subtypes in neurons and glial cells and suggests a direct role of P2 receptors in the pathophysiology of cerebral ischemia in vitro and in vivo.\nTrauma and axotomy P2 receptors are suggested to be involved in neuronal reactions after axotomy. Colocalization and temporal coactivation of purinergic and nitrergic markers support this idea, indicating possible interactions between these two systems [166]. Following peripheral nerve lesions, P2X3 receptor expression in DRG neurons was changed [167]. The increased expression of P2X3 receptor mRNA in intact neurons indicates a role of this subtype in the post-injury pathomechanism in primary sensory neurons [167]. After spinal cord injury, large regions of the peritraumatic zone were characterized by a sustained process of pathologically high ATP release [168]. Spinal cord neurons express P2X7 receptors, and exposure to ATP led to high-frequency spiking, irreversible increases in [Ca2+]i and cell death. The administration of P2 receptor antagonists (PPADS, oxATP) after acute impact injury significantly improved functional recovery and diminished cell death in the peritraumatic zone [168]. The involvement of P2X1 and P2X2 receptors in neuronal reactions after hemicerebellectomy was also described [169]. Furthermore, neuronal NOS and P2 receptors were colocalized and showed temporal coactivation after cerebellar lesions, indicating a close relationship between these two systems [166]. In addition, in this mixed model of differentiation and axotomy, the colocalization of ataxin-2 (ax-2, involved in resistance to degeneration phenomena, which may be lost after mutation)-immunopositive cells and P2X2 receptors was demonstrated in neurons, and post-lesional induction of P2X1 receptor and ax-2 immunoreactivity was reported as well [170]. In vivo treatment of P2Y2 receptor-expressing sciatic nerves with ATP-\u03b3S increased expression levels of the growth-associated protein 43 (GAP-43) as a marker for axonal growth in wild-type but not in P2Y2 \u2212\/\u2212 mice [171].Possible therapeutic manipulations to modulate astrocytic proliferation and to diminish glial scar formation in the adult brain and during development include the use of drugs known to interfere with nucleotide synthesis. Pekovic et al. [172] showed that treatment with the purine nucleoside analogue ribavirin (Virazole; 1-\u03b2-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) downregulates the process of reactive gliosis after sensory motor cortex lesion of the adult brain and facilitates re-establishing synaptic connections with the denervated cells at the lesion site. This may be a useful approach for improving neurological recovery from brain damage. The antiproliferative effect of ribavirin is due to the inhibition of de novo nucleic acid synthesis after depletion of GTP and dGTP pools with consequent impairment of specific transduction pathways.\nATP-induced effects on cell cycle progression\nThere is evidence showing that extracellular ATP enhances the expression of cell cycle regulating proteins [173, 174]. Progression of the cell cycle is highly controlled. Cyclins are synthesized and degraded in a synchronous way due to changing transcription or proteolysis rates, thereby directing the periods of the cellular cycle. Cyclins interact with cyclin-dependent kinases (cdks) resulting in activation of their kinase activity, phosphorylating their targets and themselves, and regulating the specific progression of the cell cycle through checkpoints [175].\nProliferation rates in mammalians are largely determined during the G1 phase of the cell cycle. The relevant proteins include three D-type cyclins (D1, D2, and D3) that, in different combinations, bind to and allosterically regulate one of two cdk subunits, cdk4 and cdk6, as well as the E-type cyclins (E1 and E2), which govern the activity of a single catalytic subunit, cdk2 [176]. Various combinations of D-type cyclins are expressed in different cell types, whereas cyclin E-cdk2 complexes are ubiquitously expressed [177].\nTwo families of cdk inhibitors regulate the activity of G1-type cyclins-cdks complexes: the Ink4 family (p16, p15, p18, and p19), which blocks the activity of cyclin D-cdk4-6 complexes, and the Cip\/Kip family (p21, p27, and p57), which preferentially inhibits cyclin E-cdk2 complexes and also acts as a scaffold for the catalytically active cyclin D-cdk4-6 complexes. In addition to cyclins and cdks, mitogen-activated protein kinase (MAPK) is also believed to have a role in induction of cell proliferation. Therefore, cyclin D-dependent kinases may play a role in controlling the cell cycle of embryonic and maybe neural progenitor cells. In addition MAPK is also believed to have a role in induction of cell proliferation. Extracellular ATP induces Ca2+-dependent MAPK activation via stimulation of P2 receptors in neonatal rat astrocytes [178]. On the other hand, cell proliferation is associated with activation of diverse proteins. Positive regulators include cyclins and their partners with catalytic activity (cdks), which are essential for progression of the cells through each phase of the cell cycle and various cell cycle checkpoints [179, 180]. The regulation of cyclin D1 expression is also mediated by the Ras\/ERK signaling pathways [181, 182]. Raf\/MEK\/ERK and PI3-K\/Akt signaling pathways can act in synergy to promote the G1-S phase cell cycle progression in both normal and cancer cells [183, 184]. The promoter for cyclin D1 contains an AP-1 site, and the ectopic expression of either c-fos or c-jun induces cyclin D1 mRNA expression [185, 186]. In many cell types, phosphatidylinositol (PI)-3-kinase-dependent signaling pathways also regulate cyclin D1 expression [187]. It was also reported that the control of the cell cycle regulatory proteins was dependent on PI3-kinase and p44\/42 MAPK pathways, indicating that extracellular ATP alone is sufficient to induce cell cycle progression beyond the G1 phase of the cell cycle. These findings also suggest that, once P2 receptors are activated, protein kinase C (PKC) transmit signals to the nucleus through one or more of the MAPK cascades, which may include Raf-1, MEK, and ERK, and stimulate transcription factors such as myc, max, fos, and jun. Moreover, MAPKs are upstream regulators of cdk2 and cdk4 expression. It has been reported that p44\/42 MAPK phosphorylation is essential and sufficient for the increase in cdk2 [188, 189] and decrease in p27Kip1 expression [190, 191]. However, Delmas et al. [192] provided evidence that p44\/p42 MAPK activation triggers p27Kip1 degradation independently from cdk2\/cyclin E in NIH 3T3 cells. As described above, ATP regulation of the MAPK and cdk-cyclin complex has not been elucidated in other types of cells [193].\nIt is documented in the literature that purinergic receptor inhibitors interfere with the S phase of the cell cycle. Neurospheres treated with the purinergic receptor antagonists reactive blue 2 or suramin are mostly in S phase (5.7\u2009\u00b1\u20090.3% or 8.4\u2009\u00b1\u20092.3%) when compared to untreated control neurospheres with 16.4\u2009\u00b1\u20091.8% of the cells being in S phase. Moreover, neurosphere cultures treated with suramin or reactive blue 2 showed an increase in the expression of the tumor suppressor p27 as a strong regulator of cell division [49].\nThe discussed findings led to the suggestion that extracellular ATP plays an important physiological role during mammalian embryonic development by stimulating proliferation of ES cells, and therefore P2 receptor agonists and antagonists might provide novel and powerful tools for modulating embryonic cell functions. In conclusion, P2X and P2Y purinergic receptors can promote proliferation of ES cells as well as of progenitor cell types by a mechanism by that ATP induces increases in [Ca2+]i, leading to activation of PKC, PI3-kinase\/Akt, p38, and p44\/42 MAPK, followed by an alteration in the cdk-cyclin complex with p21 and p27, which are involved in stimulation of cell proliferation.\nPharmacological approaches\nMost purinergic receptors do not have specific inhibitors. Therefore, P2 receptor agonists and antagonists acting on most of the purinergic receptor subtypes are widely used in experimental approaches to study biological functions of these receptors. Such approaches are feasible, since these compounds mostly have higher affinities to some P2 receptor subtypes than to other ones. As an example, we have used suramin, PPADS, and reactive blue 2 to study the participation of P2Y1, P2Y2, and P2X2 receptors in neuronal differentiation of P19 EC cells [38].\nOne possible approach towards a subtype-specific inhibitor would be based on results from P2 receptor structure determination. Using site-directed mutagenesis it has been possible to understand which amino acids are involved in ATP binding and to identify allosteric sites in purinergic receptors. The knowledge obtained on location and structural features of ligand and inhibitor binding sites is used in rational based drug design of selective purinergic subtype antagonists. Alternatively, combinatorial libraries formed by vast amounts of possible ligands can be employed for discovery of subtype-specific inhibitors.\nA-317491 was identified as a specific inhibitor for P2X2\/3 and P2X3 receptors. In the presence of A-317491 both thermal hyperalgesia and mechanical allodynia were attenuated after chronic nerve constriction injury in which P2X3 homomeric and P2X2\/3 heteromeric receptor activities were involved. Although active in chronic pain models, A-317491 was ineffective in reducing nociception in animal models of acute postoperative pain and visceral pain indicating that P2X3 and P2X2\/3 receptor activation may not be a major mediator of acute postoperative or visceral pain [149]. MRS 2179 (2\u2032-deoxy-N6-methyladenosine 3\u2032,5\u2032-bisphosphate) was discovered as a specific inhibitor of P2Y1 receptor activity [194]. This compound has an efficient antithrombotic action in which P2Y1 receptors are involved [195].\nBased on structure design or combinatorial library approaches specific agonists or antagonists may be discovered for other purinergic receptor subtypes. For instance, the SELEX (systematic evolution of ligands by exponential enrichment) technique provides a particularly promising approach for the discovery of such compounds. This technique is based on the reiterative presentation of a partial random RNA or single-stranded DNA library to a protein preparation containing a particular purinergic receptor subtype. RNA or DNA molecules bound to a target site on the receptor are displaced from the receptor and eluted by addition of an excess concentration of an unspecific purinergic receptor antagonist and amplified by reverse transcription polymerase chain reaction (PCR) or PCR to restore the library used for the next in vitro selection cycle. Using this approach, it was possible to identify inhibitors specific for isoforms of a target protein [196]. Our group prepared membrane protein fractions of 1321N1 cells stably transfected with rat P2X2 receptors and coupled them onto an immobilized artificial membrane (IAM) as matrix for affinity chromatography. The equilibrium binding to the receptor and competition between ATP and the purinergic antagonists suramin and 2\u20323\u2032-O-(2,4,6-trinitrophenyl) adenosine 5\u2032-triphosphate (TNP-ATP) were analyzed by a chromatographic assay using [\u03b1-32P]-ATP as a radioligand. Our data indicate that suramin does not compete with ATP for the ligand binding site and TNP-ATP is a competitive antagonist, as already shown by Trujillo et al. [197]. Moreover, this chromatographic assay can be used in in vitro selection procedures for RNA aptamers binding to P2X2 receptors from a combinatorial SELEX RNA library [198]. The development of a subtype-specific P2X receptor antagonist by using the SELEX technique or another combinatorial library-based approach shall serve as proof of principle and encourage further works to obtain such specific antagonists for all P2 receptor subtypes as tools for elucidating their biological functions and for possible therapeutic applications.\nConclusion\nP2 receptor function is involved in most physiological processes and participates in neurotransmission in the CNS. Results obtained with mouse ES and P19 EC and neural progenitor cells suggest an important role of purinergic signaling in early embryogenesis, especially in cell proliferation, migration, and differentiation, with different subtypes of receptors participating in these processes. Our understanding of the biological functions of specific P2 receptor subtypes during CNS development and in the adult brain has increased due to the availability of knockout animals and specific inhibition of gene expression or activity of purinergic receptor subtypes. The importance of P2 receptor signaling in neuroprotection, neuroimmunity, and guiding neuronal differentiation, especially in glial and microglial cells, has been related to purinergic receptor expression. Most importantly, specific agonists and antagonists for individual P2 receptor subtypes are both needed for studying their involvement in biological processes. The discovery of such selective compounds will elucidate yet unknown biological functions of P2 receptor subtypes as well as open new avenues for therapeutic approaches to disease states in which purinergic receptor activity is involved.","keyphrases":["knockout animal","atp","neurotransmitter","neural stem cells","p19 embryonal carcinoma cells"],"prmu":["P","P","P","P","R"]}
{"id":"Ann_Hematol-3-1-1914243","title":"CD34-related coexpression of MDR1 and BCRP indicates a clinically resistant phenotype in patients with acute myeloid leukemia (AML) of older age\n","text":"Clinical resistance to chemotherapy in acute myeloid leukemia (AML) is associated with the expression of the multidrug resistance (MDR) proteins P-glycoprotein, encoded by the MDR1\/ABCB1 gene, multidrug resistant-related protein (MRP\/ABCC1), the lung resistance-related protein (LRP), or major vault protein (MVP), and the breast cancer resistance protein (BCRP\/ABCG2). The clinical value of MDR1, MRP1, LRP\/MVP, and BCRP messenger RNA (mRNA) expression was prospectively studied in 154 newly diagnosed AML patients \u226560 years who were treated in a multicenter, randomized phase 3 trial. Expression of MDR1 and BCRP showed a negative whereas MRP1 and LRP showed a positive correlation with high white blood cell count (respectively, p < 0.05, p < 0.001, p < 0.001 and p < 0.001). Higher BCRP mRNA was associated with secondary AML (p < 0.05). MDR1 and BCRP mRNA were highly significantly associated (p < 0.001), as were MRP1 and LRP mRNA (p < 0.001) expression. Univariate regression analyses revealed that CD34 expression, increasing MDR1 mRNA as well as MDR1\/BCRP coexpression, were associated with a lower complete response (CR) rate and with worse event-free survival and overall survival. When adjusted for other prognostic actors, only CD34-related MDR1\/BCRP coexpression remained significantly associated with a lower CR rate (p = 0.03), thereby identifying a clinically resistant subgroup of elderly AML patients.\nIntroduction\nClinical resistance to chemotherapy in acute myeloid leukemia (AML) is often associated with the expression of (membrane) transport-associated multidrug resistance (MDR) proteins. Expression of P-glycoprotein (P-gp), encoded by the MDR1 gene, is an independent adverse prognostic factor for response and survival in de novo AML [1\u20134]. Moreover, it has been shown that besides P-gp, also the MDR-related protein (MRP1\/ABCC1) and the lung resistance-related protein (LRP), also designated as the major vault protein (MVP), are expressed in AML. However, the prognostic significance of the latter resistance proteins has not been settled [3, 5\u20137]. Some years ago, a new drug resistant protein, i.e., the breast cancer resistance protein (BCRP\/ABCG2), which is the equivalent of the mitoxantrone (MXT) resistant protein and the placental ABC transporter (ABCP), was found to be expressed in AML [8\u201313]. The precise role of either resistance proteins among poor risk AML such as in patients of older age has not been established. This study prospectively investigated the relevance of MDR1, MRP1, LRP, and BCRP messenger RNA (mRNA) expression in combination with known prognostic characteristics like CD34 expression, white blood cell (WBC) count, and secondary AML as possible denominators of response and survival in patients with AML aged 60+ who were treated in the same clinical trial.\nPatients and methods\nPatients\nA group of 154 patients with AML aged 60\u00a0years or older were included in the present study. All patients were enrolled between May 1997 and February 1999 in an international, multigroup, randomized phase 3 trial performed under auspices of the Dutch\u2013Belgian Hemato-Oncology Cooperative Group and the UK Medical Research Council [14]. In that trial, 419 eligible white patients \u226560\u00a0years with previously untreated de novo and secondary AML (M0\u2013M2 and M4\u2013M7 according to the French\u2013American\u2013British [FAB] classification [15]) were randomized to receive two cycles of induction chemotherapy consisting of daunorubicin (DNR) and cytarabine (ara-C) with or without the P-gp inhibitor PSC-833 (Valspodar, Amdray\u00ae; Novartis Pharma, Basle, Switzerland). Patients in both arms in complete remission after these two cycles were to receive one consolidation consisting of ara-C, MXT, and etoposide. Inclusion criteria, clinical characteristics, treatment, and outcome of the phase 3 trial have been previously reported [14].\nBone marrow (BM) aspirates had been collected at diagnosis for the analysis of P-gp function and expression, as described previously [14]. Selection of patients for our study was based on availability of sufficient purified AML blast samples in our tissue bank, which was the case for 154 patients.\nThis study was approved by the ethics committees of the participating institutions and was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all patients before randomization.\nMethods\nBM aspirates were obtained in heparinized tubes. Mononuclear BM cells were collected by Ficoll Hypaque density gradient centrifugation (density 1.077\u00a0g\/m3; Pharmacia, Uppsala, Sweden). To obtain purified samples with more than 85% of blasts, T-cell depletion and adherence depletion was performed as previously described [16]. Cells were cryopreserved in Dulbecco modified Eagle medium (DMEM; Gibco, Paisley, UK) supplemented with 10% dimethyl sulfoxide (Merck, Darmstadt, Germany) and 20% fetal calf serum (FCS; Gibco) and stored in liquid nitrogen. On the day of the experiments, BM cells were thawed. Cells were washed and resuspended in DMEM supplemented with 10% FCS. Before RNA and DNA isolation, cells were washed with phosphate-buffered saline (Gibco).\nMDR1, MRP1, LRP, and BCRP mRNA analysis\nThe drug resistance proteins were analyzed using the methods that we previously reported [11]. In brief, total RNA was isolated using the TRISOLV\u2122 extraction as described by the manufacturer (Biotecx, Houston, TX). RNA was aliquoted and stored at \u221280\u00b0C. RNA samples were analyzed for RNA integrity by gel electrophoresis. cDNA was synthesized by the use of the TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA), diluted, aliquoted, and stored at \u221280\u00b0C. Quantitative RT-PCR was used to measure the mRNA expression levels of MDR1, MRP1, LRP, and BCRP by Taqman-chemistry on an ABI PRISM 7700 sequence detector (Applied Biosystems) using two endogenous reference genes, i.e., glyceraldehyde-3-phosphate dehydrogenase and porphobilinogen deaminase.\nDefinition of endpoints\nThe clinical endpoints have been defined previously [14]. In brief, complete response (CR) was defined as a normocellular BM with <5% blasts, no Auer rods, and no evidence of extramedullary involvement. Because data on peripheral blood recovery within 60\u00a0days were not always available, they were not considered as a criterium for CR. Patients who relapsed or died within 28\u00a0days after CR were considered as not having achieved a CR. Event-free survival (EFS) was calculated from the date of randomization until no CR on induction therapy, relapse after CR, or death in CR, whichever came first. Patients who did not reach CR were considered failure for EFS at 1\u00a0day after randomization. Disease-free survival (DFS) was determined for all patients who achieved CR on induction therapy and was calculated from the date of CR until relapse or death, whichever came first. Overall survival (OS) was measured from randomization until death from any cause. Patients who were still alive at the date of last contact were then censored.\nStatistical analysis\nThe original phase 3 trial had been designed to detect with a power of 80% an increase in 2-year EFS from 9.5% in the control arm (without PSC-833) to 18% in the PSC-833 arm (two-sided significance level \u03b1\u2009=\u20090.05) and included 419 eligible patients.\nmRNA data were obtained from a subset of 154 patients with sufficient BM samples in our tissue bank available for analysis. Baseline parameters of interest were MDR1, MRP1, LRP, and BCRP mRNA expression. Clinical endpoints were CR rate, EFS, DFS, and OS.\nBaseline characteristics of patients with or without mRNA expression data available were compared using the Fisher exact test or the Pearson \u03c72 test in case of discrete variables, whichever appropriate, or the Wilcoxon rank-sum test in case of continuous variables. The association between patient baseline characteristics and mRNA expression levels was analyzed using the Pearson \u03c72 test of the Spearman rank correlation test, whichever was appropriate. The prognostic value of mRNA levels with respect to CR rate was determined using logistic regression [17] whereas the impact of MDR1, MRP1, LRP, and BCRP on EFS, DFS, and OS was analyzed with Cox regression analysis [18]. For this purpose, the natural logarithm of the mRNA expression levels of the four resistance genes were included in the analyses because of the very skewed distribution of the original mRNA levels. In addition, the outcome of patients with coexpression of MDR1 and BCRP was evaluated to confirm the poor prognosis of AML with MDR1\/BCRP coexpression reported by Benderra et al. [19] in patients with a median age of 45\u00a0years or older. These patients were defined as having mRNA levels of these two drug resistance genes equal to or higher than the median. Their outcome was compared to the other patients with at least one of the MDR1 and BCRP mRNA expression levels below the median. Logistic regression and Cox regression analyses were performed unadjusted, as well as adjusted for other prognostic factors, i.e., secondary AML, natural logarithm of WBC count, square root of percentage CD34+ cells, and cytogenetic risk (favorable\/intermediate versus unfavorable versus unknown), as well as for treatment arm in the phase 3 trial, as about half of the patients had been randomized to receive PSC-833 in addition to their chemotherapy. Kaplan\u2013Meier curves [20] were generated to illustrate survival and were compared using the log-rank test [21]. All reported p values are two-sided and, in view of the exploratory nature of these analyses, were calculated without adjustment for multiple testing. p values\u2009\u2264\u20090.05 were considered statistically significant.\nResults\nIn the phase 3 trial, a total of 419 untreated patients with AML aged 60\u00a0years and older were randomized to receive two induction cycles with or without PSC-833. As reported, no difference was found between both treatment arms as regards CR rate (54% in the PSC-833 arm versus 48% in the control arm, p\u2009=\u20090.22), 5-year EFS (7 versus 8%, p\u2009=\u20090.53) nor DFS (13 versus 17%, p\u2009=\u20090.06) and OS (10% in both arms, p\u2009=\u20090.52) [14]. We previously reported the role of functional MDR1 expression with respect to clinical outcome in these patients.\nIn 154\/419 of the patients, sufficient BM cells were available in our tissue bank to investigate the mRNA expression level of the drug resistance genes MDR1, MRP1, LRP, and BCRP. This subgroup consisted of a representative group according to age, gender, CD34 expression, cytogenetics, and FAB classification (Table\u00a01). In this test group, a higher WBC count at diagnosis was observed than in the other 265 patients, and relatively more patients had been randomized to the PSC-833 arm (57 versus 45%, p\u2009=\u20090.02). There was no significant difference in the levels of MDR1, MRP1, LRP, nor BCRP mRNA expression between the two treatment arms (data not shown). The CR rate and survival endpoints were also similar in both patient groups (Table\u00a01). However, patients with mRNA data in the PSC-833 arm had a higher CR rate (61 versus 40%, p\u2009=\u20090.02), whereas this was 54 versus 48% (p\u2009=\u20090.22) in all 419 patients. \nTable\u00a01Comparison between patients with or without data available for expression of the drug resistance genes\u00a0Drug resistance genes evaluated\u00a0Yes N (%)No N (%)Total N (%)pNumber of patients154265419Patient characteristicsMedian age, (range)67 (60\u201385)68 (58\u201385)67 (58\u201385)0.52Sex0.26\u00a0Male86 (56)163 (62)249 (59)\u00a0Female68 (44)102 (38)170 (41)Secondary AML31 (20)73 (28)104 (25)0.09Median WBC count (\u00d7109\/l; range)19.1 (0.1\u2013389)5.6 (0.5\u2013300)8.9 (0.1\u2013389)0.001N146243389Median % CD34+, (range)32.5 (0.1\u201397.9)29.7 (0.1\u201393.7)30.3 (0.1\u201397.9)0.50N152157309Cytogenetic risk classificationa0.12\u00a0Favorable3 (3)2 (1)5 (2)\u00a0Intermediate90 (80)132 (73)222 (76)\u00a0Unfavorable19 (17)47 (26)66 (23)\u00a0No data42 (n.i.)84 (n.i.)126 (n.i.)Treatment arm randomized0.02\u00a0DNR\/ara-C66 (43)145 (55)211 (50)\u00a0DNR\/ara-C +PSC-83388 (57)120 (45)208 (50)Treatment outcomeCR rate, % (95% CI)52 (44\u201360)50 (44\u201356)51 (46\u201356)0.73EFS, % (95% CI)0.72\u00a01\u00a0year23 (17\u201330)23 (18\u201328)23 (19\u201327)\u00a05\u00a0years9 (5\u201314)7 (4\u201311)8 (5\u201311)DFS, % (95% CI)0.81\u00a01\u00a0year38 (27\u201348)39 (31\u201348)39 (32\u201345)\u00a05\u00a0years17 (10\u201326)14 (9\u201321)15 (11\u201321)OS, % (95% CI)0.31\u00a01\u00a0year42 (34\u201350)41 (35\u201346)41 (36\u201346)\u00a05\u00a0years14 (9\u201320)8 (5\u201312)10 (7\u201314)The results indicate that, apart from WBC count, there are no differences between the two subgroups.N number of patients with data (if not available for all patients); n.i. not included when calculating percentagesaClassification of cytogenetic abnormalities only for 293 patients with successful cytogenetics. Favorable risk: t(8;21), inv(16) or t(16;16). Unfavorable risk: the presence of monosomies or deletions of chromosomes 5 or 7, abnormalities of the long arm of chromosome 3(q21;q26), t(6;9), abnormalities involving the long arm of chromosome 11 (11q23), or complex cytogenetic abnormalities (defined as at least three unrelated cytogenetic abnormalities in one clone). Patients who did not meet the criteria for favorable or unfavorable risk were classified as intermediate risk [14].\nThe mRNA expression levels of the resistance genes were not significantly associated with the age of the patients (Table\u00a02). MRP1 and LRP expression showed a strong positive correlation with WBC count. Negative associations of MDR1 and BCRP with WBC count were observed. A significant positive association was found between CD34 and MDR1 and also with BCRP mRNA expression. No significant correlation was found between MRP1 nor LRP, and CD34 expression (Table\u00a02). Interestingly, secondary AML cases had a significantly higher expression of BCRP (p\u2009<\u20090.05) and lower MRP1 and LRP levels (both p\u2009<\u20090.01, Table\u00a02). In the vast majority of our patients, also P-gp efflux and expression data were available. Function and expression data and MDR1 mRNA expression levels were highly correlated (p\u2009<\u20090.001), which was published recently [22]. \nTable\u00a02Association between clinical patient characteristics and the mRNA expression of the four drug resistance genes and MDR1\/BCRP coexpression\u00a0mRNA expression ofMDR1MRP1LRPBCRPMDR1\/BCRP coexpressionCharacteristic\u00a0Age0.15\u22120.01\u22120.090.090.07(153)(153)(153)(137)(147)\u00a0Secondary AML0.06\u22120.22**\u22120.21**0.19*0.12(153)(153)(153)(137)(147)\u00a0WBC count\u22120.17*0.28***0.36***\u22120.36***\u22120.35***(145)(145)(145)(131)(139)\u00a0CD34+0.54***0.14\u22120.080.17*0.27**(151)(151)(151)(135)(145)\u00a0Unfavorable 0.11\u22120.05\u22120.23*0.130.10\u00a0Cytogenetic risk(111)(111)(111)(98)(106)Unfavorable cytogenetic risk was defined by the presence of monosomies or deletions of chromosomes 5 or 7, abnormalities of the long arm of chromosome 3(q21;q6), t(6;9), abnormalities involving the long arm of chromosome 11 (11q23), or complex cytogenetic abnormalities (defined as at least three unrelated cytogenetic abnormalities in one clone)Each cell displays the Spearman rank correlation coefficient between two variables and, between brackets, the number of patients with both variables available.*p\u2009<\u20090.05; **p\u2009<\u20090.01; ***p\u2009<\u20090.001\nIn this cohort of patients of higher age with AML, MDR1 and BCRP were highly associated (p\u2009<\u20090.001), just as were MRP1 and LRP mRNA (p\u2009<\u20090.001; Fig.\u00a01). A negative association was found between BCRP and MRP1 and between BCRP and LRP (both p\u2009<\u20090.001; Fig.\u00a01). The 40 patients with coexpression of BCRP and MDR1 had significantly higher CD34 expression (median 39.5% [range 0.1\u201397.7%] versus 25.9% [range 0.1\u201397.9%]; p\u2009=\u20090.001) and a lower WBC count (median 4.5 [range 0.8\u2013300]\u00d7109\/l versus 28.1 [range 0.1\u2013389]\u00d7109\/l; p\u2009<\u20090.001). No significant correlation of MDR1, BCRP, or coexpression of MDR1 and BCRP was found with unfavorable cytogenetics (p\u2009=\u20090.4; Table\u00a02).\nFig.\u00a01Association between MDR1, MRP1, LRP, and BCRP mRNA expression levels. Each dot represents the expression of two drug resistance genes in one patient. The Spearman rank correlation coefficient has been calculated, along with the corresponding p value. Both the x- and y-axis have a logarithmic scale trim(X)* indicates that the 2.5% smallest and largest values of X have been shrinked; r, Spearman rank correlation coefficient; and p, p value. The results show a significant positive correlation between MDR1 and BCRP mRNA expression as illustrated by the p value and correlation coefficient. In addition, MRP1 and LRP are highly associated. BCRP shows a negative correlation with MRP1 and LRP\nTo assess the clinical relevance of the four resistance genes, their expression was evaluated with regard to CR rate and survival data, respectively. The median follow-up of the 25 patients still alive was 58\u00a0months (range, 1\u201380\u00a0months). Univariate logistic regression analysis showed that higher MDR1 mRNA expression predicted for a lower CR rate (log[MDR1]: odds ratio [OR]=0.75, 95% confidence interval [CI] 0.61\u20130.93, p\u2009=\u20090.009), whereas MRP1, LRP, and BCRP mRNA were not associated with CR (Fig.\u00a01). MDR1 expression was also associated with a worse EFS (log[MDR1]: hazard ratio [HR]=1.14, 95% CI 1.03\u20131.27, p\u2009=\u20090.01) and OS (log[MDR1]: HR\u2009=\u20091.16, 95% CI 1.05\u20131.29, p\u2009=\u20090.004). Similar results were also obtained for MDR1\/BCRP coexpression (Table\u00a03; Fig.\u00a02). When the analyses were performed with adjustment for other prognostic factors, as described in the \u201cStatistical analysis\u201d, only MDR1\/BCRP mRNA coexpression remained significantly associated with a lower CR rate (OR\u2009=\u20090.37, 95% CI 0.15\u20130.91, p\u2009=\u20090.03), whereas a trend was observed for worse EFS (Table\u00a03). On the other hand, higher CD34 expression was significantly associated with a lower CR rate (square root[CD34]: OR\u2009=\u20090.86, 95% CI 0.76\u20130.98, p\u2009=\u20090.02) and with worse EFS (HR\u2009=\u20091.12, 95% CI 1.06\u20131.19, p\u2009<\u20090.001), DFS (HR\u2009=\u20091.19, 95% CI 1.09\u20131.30, p\u2009<\u20090.001), and OS (HR\u2009=\u20091.17, 95% CI\u2009=\u20091.10\u20131.25, p\u2009<\u20090.001). \nTable\u00a03Prognostic value of drug resistance gene expression w.r.t. CR rate, EFS, DFS from CR, and OS\u00a0CR rateEFSDFSOSOR95% CIpHR95% CIpHR95% CIpHR95% CIpMDR1\u00a0Univariate0.750.61\u20130.930.0091.141.03\u20131.270.011.130.97\u20131.300.111.161.05\u20131.290.004\u00a0Adjusted0.770.58\u20131.030.081.050.91\u20131.210.480.950.77\u20131.180.671.000.87\u20131.160.97MRP1\u00a0Univariate1.060.83\u20131.350.631.020.90\u20131.150.791.070.89\u20131.290.471.110.97\u20131.260.12\u00a0Adjusted1.220.90\u20131.660.201.000.87\u20131.150.981.120.92\u20131.370.261.050.91\u20131.210.54LRP\u00a0Univariate1.160.94\u20131.430.160.950.86\u20131.060.360.980.84\u20131.140.790.970.87\u20131.080.60\u00a0Adjusted1.220.93\u20131.610.150.990.87\u20131.120.831.060.89\u20131.270.520.980.86\u20131.120.78BCRP\u00a0Univariate0.840.66\u20131.060.141.040.91\u20131.180.580.950.77\u20131.160.600.960.84\u20131.100.58\u00a0Adjusted0.790.59\u20131.06 0.120.990.86\u20131.140.920.840.66\u20131.060.140.900.77\u20131.050.19MDR1\/BCRP co-expression\u00a0Univariate0.380.18\u20130.800.011.631.11\u20132.370.011.650.90\u20133.010.111.471.00\u20132.160.05\u00a0Adjusted0.370.15\u20130.92 0.031.530.98\u20132.380.061.370.67\u20132.820.391.160.74\u20131.830.51Results of logistic (for CR rate) and Cox regression (for survival) analyses, either univariate (=unadjusted) or adjusted for treatment arm, secondary AML, WBC count (natural logarithm), % CD34+ (square root), and cytogenetic risk (favorable\/intermediate versus unfavorable versus unknown), are shown for each of the four drug resistance genes MDR1, MRP1, LRP, and BCRP (natural logarithm of mRNA expression levels) and for MDR1\/BCRP co-expression.Fig.\u00a02Survival of elderly AML patients with and without coexpression of MDR1 and BCRP mRNA. a Event-free survival, b disease-free survival, c overall survival. pos indicates patients with coexpression of MDR1 and BCRP; and other, patients without coexpression\nDiscussion\nThis is the first comprehensive analysis of the effect of the major classical MDR genes in a cohort of elderly patients with AML homogeneously treated in a prospective clinical trial [14]. A wide range of expression of the various resistance genes was observed, consistent with previous studies and with comparable median values [9\u201311, 23]. Our results show that MRP1, LRP, and BCRP are not associated with CR rate or survival endpoints in patients with AML aged 60\u00a0years or older, indicating that the clinical relevance of the expression of these genes is limited in this patient population. This study confirms previous reports, which showed the unique prognostic role of MDR1 expression\u2014which was however highly correlated with CD34 expression\u2014in drug resistance in elderly AML (Table\u00a03), in contrast to the prognostic value of MRP1 expression in AML, which has shown conflicting results, whereas currently, LRP is no longer thought to be important for clinical drug resistance [4, 5, 7, 24\u201327]. Recently, two studies in, respectively, 40 and 31 adult AML patients showed no effect of BCRP gene expression on CR rate, whereas OS was lower in patients with the highest BCRP expression [10, 23]. Damiani et al. [28] showed that BCRP expression did not influence achievement of complete remission in AML patients with a median age of 53\u00a0years and normal karyotype, however, BCRP expression was associated with higher relapse rate. In 59 children with de novo AML, a higher BCRP expression was observed in patients who did not reach CR, but this was not translated in poorer survival [29]. Benderra et al. [19] indicated that BCRP gene expression was an adverse prognostic factor for CR in a group of 149 relatively younger adult AML patients but only in patients treated with DNR and MXT and not with idarubicin. In our cohort of elderly AML patients who were all treated with DNR, whereas MXT was given as consolidation therapy after reaching CR, a significant correlation of BCRP mRNA expression with lower CR rate could not be shown.\nOur study confirms that BCRP and MDR1 are coexpressed in AML patients with higher age as has been suggested previously from studies in smaller groups of relatively younger AML patients [9\u201311, 28]. Until now, only two studies have evaluated the clinical value of coexpression of MDR1 and BCRP in a sufficient number, although relatively younger adult AML patients [19, 28]. Benderra et al. [19] showed that CR rate was only 45% in the patients with coexpression of BCRP and MDR1 (+\/+) in comparison with 66% in the MDR1\/BCRP\u2212\/+ and +\/\u2212group and 90% in the MDR1\/BCRP\u2212\/\u2212group (p\u2009=\u20090.003). Moreover, a significantly lower DFS and OS were found in the MDR1\/BCRP+\/+group. Damiani et al. [28] found a trend towards a higher relapse rate in the small group of BCRP+\/MDR1+patients, indicating that this represents a robust resistant AML phenotype, consistent with our findings in elderly AML. The recent finding that BCRP and MDR1 expression was mainly found in the most resistant group of AML, using gene expression profiling, underscores the role of these drug resistance genes in AML [30].\nHowever, this study shows, that the prominent prognostic role of CD34 expression in elderly AML should be emphasized, as higher CD34 expression was adversely associated with all clinical endpoints. MDR1 and BCRP but not MRP1 and LRP mRNA expression were found to be associated with high CD34 expression in these elderly AML patients, which may explain why MDR1 was no longer significant for CR rate, EFS, and OS when adjusted for other prognostic variables including CD34. In the past, MDR1 expression has been linked to the CD34-positive hematopoietic stem cell compartment of the leukemia subtype. In two other studies in younger AML patients, no overexpression (on mRNA and protein level) of BCRP in the CD34-positive blast population of clinical AML samples was found [13, 19]. In contrast, earlier studies in mice demonstrated high levels of BCRP and MDR1 expression in normal hematopoietic stem cells [31\u201334]. Previously, BCRP expression in subsets of stem cells has been reported, indicating that high BCRP expression may exist in CD34+\/CD38\u2212 cells or in CD34+\/CD33\u2212cells [12, 35]. The differential expression of BCRP and MDR1 in specific subsets of hematopoietic stem cells is consistent with the side population phenotype as proposed by Goodell et al. [36] who claim that BCRP expression can be separated from those expressing the other ABC proteins. This would suggest that BCRP is expressed in even less differentiated hematopoietic stem cells than MDR1 [19]. In our study in AML, these immature subsets could not be separately investigated, however, the unique BCRP\/MDR1+\/+subgroup of patients reflects an immature leukemic cell type that has a very resistant phenotype in vivo, illustrated by a low CR rate and poor outcome (Table\u00a03; Fig.\u00a02).\nThis is the first study in which a correlation was found between secondary AML and a high expression of BCRP mRNA but not the other resistance proteins. In addition to our previous report that BCRP is frequently upregulated in patients with AML at relapse, we now demonstrate that expression of BCRP is representative of secondary AML, which is especially observed in elderly patients [11, 29]. Recently, Ross [37] suggested that MDR modifiers may be of benefit for patients with multiple dysplastic features. This may suggest that BCRP is upregulated in diseases in which exposure to xenobiotics during life plays an etiologic role.\nWe conclude that coexpression of CD34-related coexpression of MDR1 and BCRP reflects a clinically resistant subgroup of elderly AML. In this age group, only BCRP is correlated with secondary AML. As such, the development of new treatment strategies for elderly AML patients may focus on modulation of drug resistance targeting both BCRP and MDR1.","keyphrases":["mdr1","bcrp","genes","lrp","mrp1","elderly aml"],"prmu":["P","P","P","P","P","P"]}
{"id":"Qual_Life_Res-4-1-2238788","title":"Effectiveness of health-related quality-of-life measurement in clinical practice: a prospective, randomized controlled trial in patients with chronic liver disease and their physicians\n","text":"Background This study assessed the effectiveness of computerized measurement and feedback of health-related quality of life (HRQoL) in daily clinical practice in patients with chronic liver disease.\nIntroduction\nHealth-related quality of life (HRQoL), or psychological, social, and physical functioning [1], has become an important outcome measure in medical care. Standardized assessment of HRQoL preceding each consultation may potentially provide physicians with valuable information. Several studies have shown that physicians vary in their ability to elicit psychosocial information or that they underestimate patients\u2019 HRQoL [2\u20135]. Furthermore, various studies have shown that when communication with the physician encompasses both physical and psychosocial issues, patients have better treatment compliance, are more satisfied with the consultation, and report less symptoms [6\u20138].\nNevertheless, relatively few studies have assessed the value of HRQoL measurement in clinical practice. Some have shown positive results with regard to acceptance by patients and physicians or a significant increase in the identification and\/or discussion of HRQoL issues [9\u201314]. Less consistent and favorable results have been obtained with regard to the effectiveness of standardized HRQoL measurement in actually improving HRQoL or psychosocial outcomes. Even though decreased depression [15], improved overall and emotional functioning [10], improved mental health [16], and a decrease in disease-specific debilitating symptoms of patients undergoing chemotherapy [13] have been associated with HRQoL measurement in clinical practice, several other studies found no significant improvement in HRQoL or psychosocial outcomes [9, 17\u201320]. A possible explanation might be that the majority of existing studies assessing the effectiveness of HRQoL measurement in clinical practice with regard to patients\u2019 psychosocial functioning or HRQoL have included oncological patients or patients from general practice. Oncological patients can be considered a special group due to the life-threatening nature of the disease. Patients from general practice, on the other hand, may be too diverse and often present with generally minor complaints, which may hamper the discovery of beneficial effects. Both groups impede generalization of results to other chronic patient populations.\nTwo important studies [9, 10] used designs in which physicians were part of both the control and the experimental group, either by using a crossover design (physicians were first assigned to one group, then crossed over to the other group halfway through the study) [9] or by assigning patients rather than physicians to the different groups [10]. This may possibly have caused bias. Two systematic reviews have stressed the need for further research evaluating the effectiveness of repeated measurements of HRQoL in clinical practice [18, 20] and the need for further research to help health care professionals identify patients who would benefit most from such interventions [20].\nThe study reported here differs from previous studies by including a patient population with chronic liver disease (CLD) in order to study the effects of HRQoL use in clinical practice in a population that is more representative of other patients with a chronic disease. CLD is one of the most prevalent diseases in the world. The most common causes of CLD, hepatitis B virus (HBV) and hepatitis C virus (HCV), have been estimated to affect 360\u00a0million and 200\u00a0million people worldwide, respectively (http:\/\/www.epidemic.org, 4-12-2006). In addition, alcohol is another main cause of end-stage liver disease worldwide and the second most common reason for liver transplantation in the United States [21]. CLD is a serious disease that is associated with significant physical and psychological symptoms such as impaired cognition, hepatic coma, fluid in the abdomen, abdominal pain, joint pain, fatigue, depression, and anxiety [22\u201328]. Not surprisingly, HRQoL in patients with CLD has been shown to be impaired [29, 30]. CLD is an appropriate example of a typical chronic disease, with patients experiencing substantial comorbidity and possibly mortality, as is the case in other chronic diseases such as kidney disease and chronic obstructive pulmonary disease.\nOur study also differs from previous studies by assessing the benefits of HRQoL measurement for patients with different demographic characteristics (e.g., men and women, young and old), which is essential for determining which patients are most likely to benefit from HRQoL measurement in clinical practice, a point recently reiterated in a systematic review on this topic [20]. In addition, in our study, physicians rather than patients were assigned to the control or the experimental group. This assigning of physicians to only one group prevents bias of physicians being focused on discussing HRQoL when seeing patients in the control group.\nThe aims of the study were twofold: the first was to assess the effectiveness of real-time computerized measurement of HRQoL in various patients with CLD and presentation of the results to physicians before the consultation in terms of improvement in patient HRQoL, patient management, and patient satisfaction with the consultation by means of a randomized trial with repeated measurements. The second aim was to assess hepatologists\u2019 experiences with the availability of real-time HRQoL patient data and to measure the possible effect(s) it had on their consultations.\nPatients and methods\nPatient recruitment\nThis study was performed at the Department of Gastroenterology and Hepatology of the Erasmus Medical Centre, Rotterdam, where HRQoL measurement on a regular basis was implemented for the duration of 1\u00a0year. All patients older than 17\u00a0years of age with CLD visiting the department between September 2004 and January 2005 were invited to participate. Written information about the study was sent to the patients 3\u00a0days before their consultation at the outpatient department. Patients interested in participating informed their physician, who consequently directed them to the researcher for further explanation of the study and to sign informed consent. For this effectiveness study, we included all patients with two or more measurement moments. All physicians working at the Department of Hepatology participated. The protocol was in accordance with the ethical guidelines of the modified 1975 Declaration of Helsinki and approved by the Medical Ethics Committee of the Erasmus MC.\nStudy objectives\nThe primary aim of this study was to assess the effectiveness of computerized measurement of HRQoL in clinical practice. The primary outcome measures were patients\u2019 generic HRQoL (physical and mental component score separately) and disease-specific HRQoL. Secondary outcome measures were patient satisfaction with the consultation and patient management. The secondary aim of this study was to assess hepatologists\u2019 experiences with the availability of real-time HRQoL patient data.\nStudy design and intervention\nPhysicians\nPhysicians were randomly assigned to either the experimental or control group by means of a restricted randomization procedure called blocking. To ensure an equal number of physicians in each group, it was decided to include six in the experimental group and five in the control group. We used a random sequence table to assign physicians to one of the conditions. Due to the nature of the intervention, it was impossible to blind physicians to group assignment.\nPhysicians in the experimental group were able to obtain an instant computerized graphical output of HRQoL patient data, which also included data from previous measurement moments so that changes in patients\u2019 HRQoL could be monitored (Fig.\u00a01). Prior to the study, physicians received instructions from a psychologist with expertise in the field of HRQoL measurement on how to interpret this output. First, physicians were shown the questionnaires in order to familiarize them with the content. Second, they were informed that the red line in the graph was the average score of patients with CLD on the Short Form-36 (SF-36) measuring generic HRQoL and that scores under this line were to be considered low. They were also told that the average score of healthy people on this questionnaire was 50. The physicians were instructed to interpret the disease-specific Liver Disease Symptom Index 2.0 (LDSI 2.0) at item level, with scores ranging from 1 (not at all) to 5 (to a large extent). The physicians were asked to use the HRQoL data in all consultations for 1\u00a0year. No recommendations for specific responses were given. Instead, they were instructed to use their clinical experience to choose an appropriate treatment. After seeing a participating patient, physicians in both groups completed a checklist about the content of the consultation. Physicians in the control group conducted their consultations as usual.\nFig.\u00a01Example of the graphical output of patients\u2019 health-related quality of life as presented to physicians in the intervention group. A score of 50 is the average score of a healthy norm population. The dashed line represents the mean score for patients with chronic liver disease\nPatients\nThrough the random assignment of physicians, patients were indirectly allocated to either group. Patients were initially blinded to the group assignment. All patients participating in the study completed a computerized generic- and disease-specific HRQoL questionnaire and the first part of a pen-and-paper questionnaire on patient satisfaction with the consultation before each consultation at the outpatient Department of Hepatology for 1\u00a0year. They also completed the second part of the satisfaction questionnaire after the consultation. More specific information on the content of the questionnaires is provided in \u201cStudy measures\u201d. To ascertain good questionnaire completion, a researcher was always available to answer questions about the computer and\/or questionnaires at the patient\u2019s request.\nStudy measures\nHRQoL\nDisease-specific HRQoL: This was assessed by means of the LDSI 2.0, which measures severity and hindrance of nine symptoms: itch, joint pain, pain in the right upper abdomen, decreased appetite, jaundice, fatigue, depressed mood, worries about family situation, and fear of complications [24]. Because of time constraints, only items measuring symptom severity were included in this study (n\u00a0=\u00a09). The physicians were instructed to interpret the questionnaire at item level, with scores ranging from 1 (not at all) to 5 (to a large extent). For data analysis, a total score, ranging from 9 to 45, was computed by summing the scores of each item. The reliability of the LDSI 2.0 is good (internal consistency \u03b1\u00a0>\u00a00.79), as is the construct validity [30].\nGeneric HRQoL: This was assessed by means of the Short Form-12 version 1 (SF-12). The SF-12 produces a Physical Component Summary (PCS) and Mental Component Summary (MCS), representing physical and emotional functioning, respectively. The mean score of the PCS and MCS in the general population is 50 [standard deviation (SD)\u00a010] with higher scores representing better HRQoL. Mean scores and SD of the PCS and MCS of CLD patients was calculated from a large database (n\u00a0=\u00a01,175) [29, 31] (PCS: mean\u00a043.2, SD\u00a010.7; MCS: mean\u00a044.4, SD\u00a012.8). These means were used as a reference point (red line) in the graphical representation for physicians so they could easily identify patients scoring below average within the CLD group. The SF-12 has been shown to be reliable between test and retest (MCS r\u00a0=\u00a00.76, PCS r\u00a0=\u00a00.89), and median relative validity estimates of 0.67\u20130.97 for the PCS and MCS, respectively, have been found [32].\nPatient satisfaction with the consultation\nPatients\u2019 satisfaction with the consultation was measured with the QUOTE-Liver, a newly developed questionnaire consisting of 20 items that assesses the discrepancy between patients\u2019 needs\/expectations (importance: measured before the consultation) and the actual care that they receive (performance: measured after the consultation). The internal consistency of the overall QUOTE-Liver was excellent (\u03b1\u00a0=\u00a00.90), as was the face validity: all patients (n\u00a0=\u00a0152) in the validation study and three psychologists and a hepatologist agreed that the items of the QUOTE-Liver adequately reflected the most important aspects of care for CLD patients. Construct validity, as measured by the correlation between a visual analog scale (VAS) measuring overall satisfaction and the total score on the QUOTE-Liver was good (r\u00a0=\u00a00.69; P\u00a0<\u00a00.01). Content validity was also good: none of the 152 patients in the validation study suggested new items to be included (Gutteling et\u00a0al. 2006, unpublished). A reduced version consisting of the nine items ranked by patients as most important and the two liver-disease-specific items was used in our study. Using a formula applied for all QUOTE-Liver instruments (10-importance\u00a0\u00d7\u00a0performance), a total satisfaction score can be computed ranging from 0 to 10, with 0 meaning not satisfied at all and 10 meaning completely satisfied [33].\nPatient management\nThe effect of the intervention on patient management was measured by means of a checklist that physicians completed after each consultation with a study participant, including the question: \u201cHave you changed your treatment in any way?\u201d and a subquestion: \u201cIf so, what have you done?\u201d followed by several options: \u201cPrescription of antidepressants,\u201d \u201cReferral to psychosocial care,\u201d \u201cAltering the frequency of consultations,\u201d and \u201cOther.\u201d\nPhysicians\u2019 experiences\nExperiences of physicians with the experimental condition were assessed through the checklists that they completed after each consultation with a study participant, asking the question: \u201cDid you find the HRQoL information useful? Why?\u201d with the answering options: \u201cYes, it provided new information,\u201d \u201cYes, it saved time,\u201d \u201cYes...,\u201d \u201cNo, the patient is doing well,\u201d \u201cNo, I know this patient well enough,\u201d \u201cNo, the patient tells me a lot,\u201d \u201cNo...\u201d. Also, a semistructured interview was conducted 6\u00a0months into the study and at the end of the study. The interview included questions about the effort to request HRQoL information, the usefulness of the information, whether the availability of HRQoL information increased the duration of the consultation, and whether participating patients addressed HRQoL issues more often than patients who did not participate. Physicians were also asked whether there were certain subgroups of patients whose HRQoL information they found particularly useful. Opinions of physicians in the control group toward possible future availability of HRQoL information during the consultation were assessed by means of the same semistructured interview at 6\u00a0months only.\nStatistical analysis\nSample size\nA nonclustered power analysis based on a medium effect size (Cohen\u2019s D\u00a0=\u00a00.50) with a 5% significance level and 80% power indicated that at least 64 patients were needed in each group to detect a statistically significant difference.\nData selection\nFor patients who were included in both groups because they had consultations with physicians from the control group as well as physicians from the experimental group during the year of the study, data from the condition in which they had most often been was included (n\u00a0=\u00a033). For patients who had been in both conditions equally (n\u00a0=\u00a019), all data were excluded. The first measurement moment of all patients (T1) was considered a baseline measure, as no HRQoL data had yet been presented to the physicians.\nData analysis\nDifferences on the variables gender, diagnosis, disease severity, and age between participants and nonparticipants were assessed by means of \u03c72 tests or t tests. The same was done for assessing differences between patients in the control group and the intervention group. Scores of participating patients on measurement moments (T2\u2212Ti) were summarized into one overall score per variable in the study. Univariate analyses of variance were performed in SPSS 11.0. Fixed factors were age, gender, disease severity, presentation of HRQoL data to the clinicians (feedback), and interactions between these variables. Differences in diagnoses between patients in both groups were controlled for by entering one propensity score of the variable diagnosis as a covariate in the analyses. Propensity scores were especially designed for situations in which study participants could not be randomly assigned to groups, and their characteristics were therefore not balanced among the groups. A propensity score was defined as the conditional probability of assignment to a certain treatment group given a set of observed pretreatment characteristics and was usually estimated by means of a logistic regression analysis [34]. Thereby, the background characteristic(s), in this case diagnosis, was reduced to one single score, the propensity score. We calculated the propensity score by entering the different diagnoses (HBV, HCV, cholestatic liver disease, pretransplantation, posttransplantation, autoimmune hepatitis, and other) as dummy variables (M-1) in a logistic regression analysis. The unstandardized logistic regression weights were then multiplied by the relevant dummy variable and summed, together with the constant. This score was used in the univariate analysis to adjust for baseline confounding.\nUnivariate analyses of variance were performed for each outcome variable (disease-specific HRQoL and generic HRQoL MCS and PCS) separately. A forward technique was used in which the main effects of the fixed factors were assessed in the first block, and the interactions between feedback of HRQoL data and each of the other fixed factors (age, gender, severity of the disease) were explored in the second block. Differences between the two groups on patient management variables and satisfaction with the consultation were assessed by means of Mann\u2013Whitney tests.\nHepatologists\u2019 experiences with the availability of real-time patient HRQoL data was assessed by means of semistructured interviews and checklists. These data were of a descriptive nature and are presented as such.\nResults\nCharacteristics of patients and physicians in the study\nOf the 587 patients that agreed to participate in the study, 181 completed the questionnaires more than once. Of these, 19 were included in the experimental and control conditions equally often and were therefore excluded from the analyses. One hundred and sixty-two patients (control group n\u00a0=\u00a080, experimental group n\u00a0=\u00a082) were included (Fig.\u00a02). Differences in age, gender, diagnosis, and disease severity between patients in the study and nonrespondents are presented in Table\u00a01. Demographic characteristics of the 162 patients are presented in Table\u00a02. Patients in the control and experimental groups were comparable, except for the variables diagnosis and disease severity (Table\u00a02). In the analyses, these differences between conditions were controlled for. All physicians working at the Department of Hepatology (n\u00a0=\u00a011, ten men) agreed to participate. Their mean age was 39\u00a0(range 27\u201355) years, and their average working experience was 8.7\u00a0(range 0\u201327)\u00a0years.\nFig.\u00a02Patients in the studyTable\u00a01Differences in age, gender, diagnosis, and disease severity between patients in the study and nonrespondentsPatients in the analyses (n\u00a0=\u00a0162)Patients excluded from the analyses (n\u00a0=\u00a0165)P valuePatients excluded from the study (n\u00a0=\u00a0260)P valueAge, mean (range)47.5 (20\u201375)48.6 (20\u201381)0.5247.6 (18\u201380)0.92Gender, n (%)\u00a0\u00a0\u00a0\u00a0Male96 (59)87 (53)0.24136 (52)0.21\u00a0\u00a0\u00a0\u00a0Female66 (41)78 (47)124 (48)Diagnosis, n (%)\u00a0\u00a0\u00a0\u00a0Hepatitis B22 (13)25 (15)0.0449 (19)0.00\u00a0\u00a0\u00a0\u00a0Hepatitis C23 (14)24 (15)56 (22)\u00a0\u00a0\u00a0\u00a0Cholestatic liver disease11 (7)22 (13)32 (12)\u00a0\u00a0\u00a0\u00a0Pretransplantation11 (7)7 (4)1 (0)\u00a0\u00a0\u00a0\u00a0Posttransplantation62 (38)48 (29)55 (21) \u00a0\u00a0\u00a0\u00a0Autoimmune hepatitis12 (8)11 (7)18 (7) \u00a0\u00a0\u00a0\u00a0Other21 (13)28 (17)49 (19)Disease severity, n (%)\u00a0\u00a0\u00a0\u00a0No cirrhosis101 (62)105 (64)0.43159 (61)0.96\u00a0\u00a0\u00a0\u00a0Compensated cirrhosis42 (26)45 (27)69 (27) \u00a0\u00a0\u00a0\u00a0Decompensated cirrhosis19 (12)15 (9)32 (12)Differences were assessed by means of \u03c72 tests (except for age: t test). Reference group for comparison of both P values is the group of patients in the analysesTable\u00a02Characteristics of patients included in the data analysisControl group (n\u00a0=\u00a080)Experimental group (n\u00a0=\u00a082)P valueGender, n (%)\u00a0\u00a0\u00a0\u00a0Women38 (48)28 (34)0.08\u00a0\u00a0\u00a0\u00a0Men42 (52)54 (66)Age, mean (range)47.5 (21\u201374)47.6 (20\u201374)0.98Diagnosis, n (%)\u00a0\u00a0\u00a0\u00a0Hepatitis B1 (1)20 (25)0.00\u00a0\u00a0\u00a0\u00a0Hepatitis C7 (9)16 (19)\u00a0\u00a0\u00a0\u00a0Cholestatic liver disease4 (5)6 (7)\u00a0\u00a0\u00a0\u00a0Pretransplantation5 (6)3 (4)\u00a0\u00a0\u00a0\u00a0Posttransplantation43 (54)23 (28)\u00a0\u00a0\u00a0\u00a0Autoimmune hepatitis6 (7)6 (7)\u00a0\u00a0\u00a0\u00a0Other14 (18)8(10)Disease severity, n (%)\u00a0\u00a0\u00a0\u00a0No cirrhosis44 (55)56 (68)0.01\u00a0\u00a0\u00a0\u00a0Compensated cirrhosis16 (20)22 (27)\u00a0\u00a0\u00a0\u00a0Decompensated cirrhosis20 (25)4 (5)Differences were assessed by means of \u03c72 tests (except for age: t test)\nDescriptives\nThe number of times that patients in the control and experimental groups completed the questionnaires varied between two and 11 (Table\u00a03). Mean scores of patients at T1 and T2\u2212Ti on the outcome variables generic HRQoL and disease-specific HRQoL are presented in Table\u00a04.\nTable\u00a03Questionnaire completion rate of patients in the control and experimental groupsNumber of times questionnaires were completedTotal (n)234568911Control (n)2229117712180Experimental (n)451895221082Table\u00a04Patients\u2019 adjusted means and 95% confidence intervals at T1 and T2\u2212TiT1P valueT2\u2212TiP valueControl ExperimentalControlExperimentalOverallSF-12 PCS41.5 (39.0\u201343.9)45.6 (42.0\u201349.3)0.0642.0 (39.6\u201344.4)44.8 (41.4\u201348.3)0.19SF-12 MCS43.4 (40.3\u201346.5)46.0 (41.4\u201350.6)0.3543.8 (41.0\u201346.5)44.8 (40.8\u201348.8)0.69LDSI 2.021.2 (19.0\u201323.4)18.9 (15.7\u201322.2)0.2720.4 (18.6\u201322.2)18.8 (16.1\u201321.4) 0.31Male patientsSF-12 PCS10.2 (37.1\u201343.3)47.0 (42.9\u201351.2)0.1041.3 (38.2\u201344.2)45.7 (41.7\u201349.7)0.29SF-12 MCS41.6 (37.7\u201345.4)45.6 (40.4\u201350.8)0.4941.2 (37.8\u201344.6)46.7 (42.1\u201351.2)0.02LDSI 2.022.8 (20.0\u201325.5)18.1 (14.4\u201321.8)0.1021.4 (19.2\u201323.6)18.0 (15.0\u201321.0)0.14Female patientsSF-12 PCS42.7 (39.2\u201346.3)44.2 (39.8\u201348.7)42.8 (39.4\u201346.2)44.0 (39.7\u201348.2)SF-12 MCS45.2 (40.7\u201349.6)46.4 (40.8\u201352.0)46.3 (42.4\u201350.2)42.9 (37.9\u201347.8)LDSI 2.019.6 (16.4\u201322.8)19.8 (15.8\u201323.8)19.4 (16.9\u201322.0)19.5 (16.3\u201322.7)Older patientsSF-12 PCS41.5 (38.4\u201344.6)44.6 (40.7\u201348.6)0.4940.4 (37.4\u201343.3)43.4 (39.9\u201347.5)0.72SF-12 MCS41.5 (37.6\u201345.4)46.3 (41.4\u201351.3)0.2641.2 (37.8\u201344.7)45.9 (41.6\u201350.3)0.03LDSI 2.022.8 (20.0\u201325.5)19.1 (15.6\u201322.7)0.3122.1 (19.9\u201324.3)18.1 (15.3\u201321.0)0.04Younger patientsSF-12 PCS41.4 (37.9\u201344.9)46.7 (42.2\u201348.6)43.6 (40.3\u201347.0)45.9 (41.6\u201350.3)SF-12 MCS45.3 (40.9\u201349.7)45.7 (40.0\u201351.3)46.3 (42.5\u201350.2)43.6 (38.7\u201348.6)LDSI 2.019.6 (16.5\u201322.7)18.7 (14.7\u201322.8)18.8 (16.2\u201321.3)19.4 (16.1\u201322.6)The means at T1 and T2\u2212Ti were obtained from the univariate analyses of variance with fixed factors: age, gender, severity of the disease, study group (control or experimental), and interactions between these variables. Differences in diagnoses between patients in both groups were controlled for. The significance level reflects the group for which the largest difference on the variable was foundSF-12 Short Form-12, PCS Physical Component Summary, MCS Mental Component Summary, LDSI 2.0 Liver Disease Symptom Index 2.0 \nEffects of the experimental condition on patients\u2019 HRQoL and satisfaction with the consultation\nDisease-specific HRQoL\nThere was no main effect for the experimental condition on disease-specific HRQoL. There was a statistically significant interaction effect for the variables age and feedback of HRQoL data on the outcome variable disease-specific HRQoL (Table\u00a05): older patients (>48\u00a0years of age, as determined by the median split) in the experimental group had significantly lower total scores on the LDSI 2.0 (meanAdj\u00a0=\u00a018.1, 95% CI: 15.3\u201321.0) (F\u00a0=\u00a04.18; P\u00a0<\u00a00.05), indicating better disease-specific HRQoL, than other patients, especially older patients in the control group (meanAdj\u00a0=\u00a022.1, 95% CI: 19.9\u201324.3). This difference between older patients in the experimental group and the control group on disease-specific HRQoL is equivalent to a Cohen\u2019s D of 0.51, reflecting a medium difference [35].\nTable\u00a05Interaction effects between age, gender, disease severity, and feedback on the outcome variable disease-specific HRQoL, controlled for diagnosisSourceF valuedfP valueR2Corrected model2.11100.03Intercept599.8310.00Diagnosis (propensity score)1.8010.180.08Gender0.0410.85Disease severity3.3920.04Age0.8410.36Feedback1.0510.31Gender\u00a0*\u00a0Feedback2.1710.140.12Severity\u00a0*\u00a0Feedback0.1520.86Age\u00a0*\u00a0Feedback4.1810.04Dependent variable: mean total score of the Liver Disease Symptom Index   2.0 [disease-specific health-related quality of life (HRQoL)] for the measurement moments T2...Ti\nGeneric HRQoL\nMental Component Summary score\nNo main effect for the experimental condition on mental HRQoL was found. However, a significant interaction effect for the variables age and feedback of HRQoL data was found. Older patients in the experimental group had higher scores on the SF-12 MCS (meanAdj\u00a0=\u00a045.9, 95% CI: 41.6\u201350.3) (F\u00a0=\u00a04.62; P\u00a0<\u00a00.05), reflecting better HRQoL, than other patients, especially older patients in the control group (meanAdj\u00a0=\u00a041.3, 95% CI: 37.8\u201344.7) (Table\u00a06). Furthermore, a significant interaction effect was found for the variables gender and feedback of HRQoL data, with male patients in the experimental group showing higher scores on the SF-12 MCS (meanAdj\u00a0=\u00a046.7, 95% CI: 42.1\u201351.2) (F\u00a0=\u00a06.10; P\u00a0<\u00a00.05) than other patients, especially male patients in the control group (meanAdj\u00a0=\u00a041.2, 95% CI: 37.8\u201344.6) (Table\u00a06).\nTable\u00a06Univariate analysis of variance with the variables age, gender, disease severity, and feedback on the outcome variable mental generic HRQoL, controlled for diagnosisSourceF valuedfP valueR2Corrected model1.65100.10Intercept1337.0510.00Diagnosis (propensity score)1.3410.250.03Gender0.1410.71Disease severity0.4020.67Age0.6510.42Feedback0.1610.69Gender\u00a0*\u00a0Feedback6.1010.020.10Severity\u00a0*\u00a0Feedback0.1320.88Age\u00a0*\u00a0Feedback4.6210.03Dependent variable: mean total score of Short Form-12 Mental Component Summary (SF-12 MCS) [generic mental-health-related quality of life (HRQoL)] for the measurement moments T2...Ti\nPhysical Component Summary score\nNo significant main effect or interaction effects were found for the variables feedback of HRQoL data and age, gender, and disease severity on the SF-12 PCS.\nPatients\u2019 satisfaction with the consultation\nThe scores on patient satisfaction did not differ significantly between the experimental and control groups (z\u00a0=\u00a0\u22121.20, P\u00a0=\u00a00.23). Also, no interaction effects of age, gender, and\/or disease severity were found on this outcome variable.\nEffects of the experimental condition on the consultation and on patient management\nPhysicians in the experimental group requested information of their patients in 92% of consultations, and they discussed it with their patients in 58% of consultations. They indicated finding the HRQoL information useful in 45% of consultations, which is generally in accordance with the percentage of patients in the experimental group scoring below average on the MCS (39%) and PCS (42%). They mostly found the HRQoL useless when a patient was doing well. Physicians in the experimental group indicated significantly more often than physicians in the control group that they spent more time than usual discussing psychosocial issues (30.7% vs. 6.6% of consultations, z\u00a0=\u00a0\u22126.65; P\u00a0<\u00a00.001). Treatment policy was altered significantly more often in the experimental group (11% of consultations vs. 1% of consultations in the control group; z\u00a0=\u00a0\u22123.73, P\u00a0<\u00a00.001). Most commonly, frequency of consultations was increased (n\u00a0=\u00a05). Other alterations concerned prescription of medication [3], increased attention for physical complaints [4], referral to psychosocial care [1] or occupational health physician [1], and increased attention to explanations\/reassurance [2].\nPhysicians\u2019 experiences with the availability of HRQoL information in clinical practice\nExperiences of physicians in the experimental group at 6\u00a0months and at the end of the study did not differ. All physicians in the experimental condition found the HRQoL information useful, except for one older physician who claimed to know his patients very well. They indicated being better able to understand some of their patients through the extra information that was provided by the questionnaires. These physicians did not perceive requesting the information as an extra effort on their part. Furthermore, they did not think that using the information lengthened their consultations. All physicians in the experimental group indicated that they wanted to continue using the HRQoL information in the future. Physicians in the control group were similarly positive toward the possible availability of HRQoL information during their consultations in the future, on the condition that it would not be time consuming. This specifically concerned patients awaiting liver transplantation, patients with hepatitis C, and nonnative speakers (mostly patients with hepatitis B).\nDiscussion\nComputerized, real-time measurement of HRQoL at our busy outpatient Department of Hepatology and presentation of the results to physicians before each consultation did not show a main effect on patients\u2019 overall HRQoL. However, secondary analyses showed that the HRQoL measurements positively affected disease-specific HRQoL and generic mental HRQoL of older patients (>48\u00a0years of age) with CLD and also generic mental HRQoL of male CLD patients. The results of our study are among the first to show a beneficial effect of presenting HRQoL data to physicians in clinical practice. Most other studies have failed to show evidence for the actual improvement in HRQoL or psychosocial outcomes [9, 17\u201320]. Of the studies that did find a beneficial effect, one showed a decrease in disease-specific debilitating symptoms [13], and another showed improved emotional functioning [10], which is in line with findings of our study. It should be noted that due to the cross-sectional data analyses, a causal relationship between intervention and HRQoL could not be demonstrated. Future studies should address this in further detail.\nOur study found no differences between patients in the experimental and control groups with regard to satisfaction with the consultation, which is in line with findings from previous studies [9, 36, 37]. The lack of observed differences between the study groups may have been due to high levels of satisfaction, resulting in a ceiling effect.\nThis study was among the first to show a significant difference in patient management between experimental and control groups, with physicians in the experimental group mostly reporting a significant increase in the frequency of consultations. Our findings were statistically significant and in accordance with the findings of a systematic review [20] and subscribe to the increasingly acknowledged importance of using HRQoL information for the improvement of physician consultations [38]. However, it should be noted that even though the differences in patient management between control experimental groups were statistically significant, the absolute numbers were small. Therefore, the results should be interpreted cautiously, and further studies using more elaborate methods of data collection\u2014for instance, monitoring patients\u2019 medical records or administering more detailed checklists\u2014are recommended.\nPhysicians\u2019 experiences with using HRQoL information during the consultation were generally positive; requesting the information was not considered an extra effort on their part, and they found the information especially useful for certain groups of patients, such as those awaiting liver transplantation, those with hepatitis C, and nonnative speakers. All physicians but one found the information useful for at least some (45%) of their patients. Physicians indicated finding the information least useful when patients were doing well in terms of HRQoL or when they knew the patient well. These generally positive experiences are in accordance with findings from previous studies [9\u201314], which assessed oncologists\u2019 attitudes toward using HRQoL information in clinical practice. The confirmation of these results in hepatologists suggests that HRQoL information may also be well accepted by physicians treating patients with other chronic conditions. Another result of our study was that when HRQoL information was available, more time was spent discussing psychosocial issues and more treatments were altered. Interview and checklist data were contradictory regarding the duration of consultations when HRQoL information was available. In a previous study in which the duration of consultations was timed, no increase in consultation time was found [14]. Future studies should shed more light on whether the availability of HRQoL information increases the length of consultations in hepatology.\nThe strength of our study lies in the analyses performed, where benefits for specific groups of liver patients were explored by entering interactions between gender, age, disease severity, and feedback of HRQoL data, rather than solely investigating main effects between the intervention and control groups. Also, this study included patients with CLD rather than patients with cancer or patients from general practice, making it especially relevant to a more general population of patients with a chronic illness.\nWe are aware of several limitations of this study. First, physicians rather than patients were randomly assigned to either the intervention or control group. Randomization is a complicated issue in these kinds of implementation studies, and both methods are subject to limitations. An important advantage of the randomization of physicians is that the control group was not biased toward mentioning HRQoL topics more often than usual. Future studies using the same design but including more physicians are needed to further explore possible main effects of HRQoL measurement on patients\u2019 overall HRQoL. A second limitation was the high number of nonparticipants. Part of the explanation may lie in the fact that patients were responsible for contacting their physician if they were interested in participating in the study. In addition, the number of non-Dutch-speaking patients visiting the department is relatively large (hepatitis B, for example, is most common among people from North Africa). These patients were also invited to participate but were less likely to respond. The relatively large number of patients who completed the questionnaires only once may be explained by the small window of opportunity to complete the questionnaires before each consultation. In addition, for such implementation endeavors, cooperation of all staff members is essential, and future research should explore this further. A last limitation of this study was that the checklists used to assess consultation content were not very detailed. This was done on purpose, as longer inventories would have compromised physician participation. However, considering the positive outcomes of this study, it is advisable that future studies consider ways to obtain a more detailed view of how the HRQoL information affects consultation content, for example, by recording consultations.\nIn conclusion, although a main effect of the intervention was not found, this study showed a beneficial effect of implementation of HRQoL measurement in clinical practice on the HRQoL of older and male patients with CLD and on patient management. Nevertheless, the study had several shortcomings, and further studies are needed to substantiate these findings. Physicians\u2019 experiences with the availability of HRQoL information were positive, especially for patients awaiting liver transplantation, patients with hepatitis C, and nonnative speakers. They expressed an interest in continued use of HRQoL information. These results advocate the continued use of measuring HRQoL in a clinical practice of hepatology. Including older patients and male patients, who have been shown to benefit most from such a procedure, should be aimed for.","keyphrases":["liver","quality of life","hepatology","implementation"],"prmu":["P","P","P","P"]}
{"id":"Purinergic_Signal-3-4-2072922","title":"Adenosine A1 receptor: Functional receptor-receptor interactions in the brain\n","text":"Over the past decade, many lines of investigation have shown that receptor-mediated signaling exhibits greater diversity than previously appreciated. Signal diversity arises from numerous factors, which include the formation of receptor dimers and interplay between different receptors. Using adenosine A1 receptors as a paradigm of G protein-coupled receptors, this review focuses on how receptor-receptor interactions may contribute to regulation of the synaptic transmission within the central nervous system. The interactions with metabotropic dopamine, adenosine A2A, A3, neuropeptide Y, and purinergic P2Y1 receptors will be described in the first part. The second part deals with interactions between A1Rs and ionotropic receptors, especially GABAA, NMDA, and P2X receptors as well as ATP-sensitive K+ channels. Finally, the review will discuss new approaches towards treating neurological disorders.\nIntroduction\nThe vertebrate central nervous system (CNS) is characterized by a dynamic interplay between signal transduction molecules and their cellular targets. Modulation of synaptic transmission by metabotropic or ionotropic receptors is an important source of control and dynamical adjustment in synaptic activity.\nRecent studies have provided new insights into the role of ligand-gated ion channels in modifying synaptic transmission. Along with a growing list of different types of pre- and postsynaptic ionotropic receptors and the cell types that express them, there have also been advances in characterizing the modulatory mechanisms of the receptors that link to receptor activation. This is important due to the convergence of data from biochemical, molecular, and electrophysiological studies, implicating ionotropic receptors in the effects of psychoactive and addictive drugs.\nG protein-coupled receptors (GPCRs) make up the largest and most diverse family of membrane receptors in the human genome, relaying information on the presence of diverse extracellular stimuli to the cell interior. An estimated 1% of the mammalian genome encodes for GPCRs, and about 450 of the approximately 950 predicted human GPCRs are thought to be receptors for endogenous ligands [1]. The manipulation of transmembrane signaling by GPCRs may constitute the most important therapeutic target in medicine. Nearly 40% of all current therapeutic drugs target GPCRs [2].\nAll known GPCRs share a common architecture of seven membrane-spanning helices connected by intracellular and extracellular loops. Drugs acting on GPCRs have been classified as agonists, partial agonists, or antagonists based on a \u201ctwo-state model of receptor function.\u201d Since experimental evidence pointed out the impossibility of explaining the operation of GPCRs without considering dimers as the minimum structure for many GPCRs the \u201ctwo-state dimer receptor model\u201d was developed based on the communication between the two subunits of the receptor dimmer [1, 3, 4]. This model is an extension of the \u201ctwo-state model of receptor function\u201d but considers dimeric structures able to bind one molecule to the orthosteric center in each monomer.\nGPCR signaling is subject to extensive negative regulation through receptor desensitization, sequestration, and downregulation, termination of G protein activation by GTPase-activating proteins, and enzymatic degradation of second messengers. Additional protein-protein interactions positively modulate GPCR signaling by influencing ligand binding and specificity.\nMultiprotein complexes mediate most cellular functions. In neurons, these complexes are directly involved in the neuronal transmission, which is responsible for learning, memory, and developments. The first publication in this direction came from H\u00f6kfelt\u2019s group in 1983. The publication describes how substance P may modulate the high-affinity serotonin (5-HT) binding site in a spinal cord membrane preparation [5]. Over the past decade, the number and outcomes of interactions between receptors have increased continuously [6]. Recent studies have demonstrated close physical interactions where activation of one receptor affects the function of the other.\nAdenosine is an endogenous purine nucleoside that has evolved to modulate many physiological processes. Extracellular adenosine mostly originates from release of intracellular adenosine and from release and extracellular breakdown of cAMP and ATP by ecto-5\u2032-nucleotidase and phosphodiesterase [7]. Cellular signaling by adenosine occurs through four known adenosine receptor subtypes (A1Rs, A2ARs, A2BRs, and A3Rs), all of which are seven-transmembrane-spanning GPCRs. Of the four known adenosine receptors, A1Rs and A2ARs are primarily responsible for the central effects of adenosine, especially in modulating synaptic transmission [8]. Adenosine can act on A1Rs to depress transmitter release and neuronal sensitivity to the transmitter [9, 10]. As a result, the A1Rs are important in the regulation of synaptic plasticity, playing a role in determining the amplitude of long-term potentiation or long-term depression [11].\nThere are numerous reviews that describe regulation of brain adenosine levels, adenosine receptors, their cellular and subcellular localization, signaling pathways, and function in the brain under physiological and pathophysiological conditions as well as selective receptor agonists and antagonists. Using A1Rs as a paradigm of GPCRs, this review focuses on how receptor-receptor interactions contribute to regulatory processes within the central nervous system.\nConsidering the various types of receptors, one may expect to find three principle paths of receptor interaction: (1) interactions between ionotropic receptors, (2) interactions between a metabotropic receptor and an ionotropic receptor, and (3) interactions between metabotropic receptors. The examples mentioned below stem from the second and third type of interaction. Interactions with metabotropic dopamine receptors as well as A2A, A3, NPY, and P2Y1 receptors will be described in the first part. The second part deals with interactions between A1Rs and ionotropic receptors, especially the GABAA, NMDA, and P2X receptors as well as ATP-sensitive K+ channels. Finally, new approaches for neurological disorders will be discussed.\nFunctional interactions with metabotropic receptors\nTwo forms of GPCR classification exist. There is the historical division into three main families: (1) rhodopsin-like family which includes adenosine receptors, (2) secretin-like family, and (3) metabotropic glutamate receptor-like family. The families share some basic similarities\u2014the seven-transmembrane-spanning domains, intracellularly located C terminus, and extracellularly residing N terminus. Differences between the families arise in the length of the intracellular and extracellular termini and amino acid sequences, disulfide bridge linking, and conserved domains. Five different groups can be classified by applying phylogenetic analyses. The GRAFS system distinguishes between glutamate, rhodopsin, adhesion, fizzled\/taste, and secretin-like GPCRs [12].\nThe agonist binding on the receptor results in coupling to heterotrimeric G proteins and regulates a variety of cell responses. In brief, an exchange of G protein-bound GDP to GTP occurs, and the heterotrimer dissociates into the \u03b1 subunit and the \u03b2\u03c7 dimer. The resulting products activate or inhibit effectors independently from each other. Currently, 16 different genes encode G protein \u03b1 subunits, five genes encode \u03b2 subunits, while 14 genes encode \u03c7 subunits [13]. The \u03b1 subunits can be categorized into four basic groups: the stimulatory  family couples to adenylate cyclase and increases cAMP levels, whereas the inhibitory  family acts in the opposite way. Moreover, the  family activates the phospholipase  (), and lastly, the  family which regulates Rho proteins.  dimers are capable of triggering effects on inward rectifier K+ channels (GIRK1\u20134), voltage-dependent Ca2+ channels (VDCC), and phospholipase A2 (PLA2), , and the Na+\/H+ exchanger (NHE1). Thus, it is not surprising that GPCRs are such interesting candidates in current drug research with their amazing potential in affecting signaling events. A single GPCR possesses the potential to activate more than just one signaling pathway [12]; for example, activation of A1Rs includes coupling to  and increasing of IP3 level [14, 15]. Furthermore, homodimerization and heterodimerization are common paths of interaction and have been described in A1Rs and the A2Rs several times [16\u201318]. In addition, functional interactions on the A1R without receptor assembling have already been revealed [19, 20], or are currently being elucidated. The next paragraphs will deal with a few selected examples of this limitlessly wide topic.\nRelationship between A1Rs and A3Rs\nThe A3R was the latest receptor subtype of the adenosine receptor family to be identified [21], and its functional role is still controversially discussed. Several findings indicate neuroprotective as well as neurotoxic action depending on experimental approach [22\u201329]. A3Rs couple to inhibition of adenylyl cyclase as well as to activation of PLC, and to elevation of inositol triphosphate levels [30, 31]. Furthermore, an increase in intracellular Ca2+ levels due to release from intracellular stores and Ca2+ influx has been described [32, 33]. One interesting example of A3Rs\u2019 functional role is their involvement in acute neurotoxic situations and interplay with A1Rs. Dunwiddie et al. [34] reported on the potential of A3Rs to modify responses via A1Rs in the hippocampus. The activation of hippocampal A3Rs induced a desensitization of A1Rs on combined superfusion of Cl-IB-MECA and adenosine. This phenomenon was thought to reduce the protective effects of endogenous adenosine caused by the lack of sensitivity of A1Rs. Further investigations on pyramidal cells of the rat cingulate cortex did not confirm Dunwiddie et al.\u2019s assumption [35]. In this brain area, A1Rs and A3Rs did not show any interaction. The receptor subtypes were unable to affect each other. The discrepancy was taken to be a genetic phenomenon, such as alternative splicing of the rat A3R transcript causing distinguished pharmacological and functional properties in the brain. Furthermore, Hentschel et al. [36] demonstrated the involvement of A3Rs in inhibition of excitatory neurotransmission during hypoxic conditions, indicating a neuroprotective action of endogenously released adenosine on A3Rs in addition to A1Rs. Lastly, Lopes et al. [37] attempted to define the possible role of A3Rs in the rat hippocampus using experiments similar to those of Dunwiddie et al. in non-stressful and stressful situations, with particular attention to whether A3Rs control A1Rs. These data suggested that no interaction between the two receptor subtypes exist, but confirm that A3Rs do not affect synaptic transmission on superfusion with A3R agonist Cl-IB-MECA or A3R antagonist MRS 1191. The authors pointed out that Cl-IB-MECA binds to A1Rs even at low nanomolar concentrations. Thus, the existence of an interaction between A1Rs and A3Rs has to wait for reliable ligands.\nAntagonistic interaction between A1Rs and A2ARs\nA2ARs are widely distributed in the CNS, but local and subcellular differences in allocation exist. They show high levels in all subregions of the striatum and in the globus pallidus. A2ARs are also expressed in neurons in the neocortex and limbic cortex, but at a density a twentieth of that found in basal ganglia [38]. Colocalization of A1Rs and A2ARs was approved for glutamatergic nerve terminals in the hippocampus [39]. In the striatum, A1R\/A2AR heteromers were found on synapses with spines of medium spiny neurons and integrated in the presynaptic membrane of glutamatergic terminals that represent the cortical-limbic-thalamic input [18]. A1Rs and A2ARs modulate excitatory synaptic transmission, albeit in an opposite manner. A1R activation inhibited glutamatergic synaptic transmission mainly through presynaptic inhibition of glutamate release, while A2ARs have been shown to facilitate glutamatergic synaptic transmission [40\u201342]. At first sight, stimulating A1Rs and inhibiting A2ARs may have a neuroprotective influence on the mature CNS. However, problems arise due to long-term desensitization of A1Rs. A2ARs do not upregulate after antagonist administration, but have a low abundance in hippocampal and cortical areas compared with A1Rs [40, 43, 44]. A1Rs and A2ARs cannot be regarded in isolation from one another since cross talk between the subtypes has been described several times [16, 17, 45\u201347]. A2AR activation by agonists caused A1R desensitization resulting in decreased binding affinity for CPA in the hippocampus in young adult rats. Controlling A1Rs by A2ARs was mediated by protein kinase C in a cAMP-independent manner. A2AR activation was seen to play a role in fine-tuning A1Rs by attenuating the tonic effect of presynaptic A1Rs located on glutamatergic nerve terminals [46, 47]. In the striatal system, A1R\/A2AR heteromers became prominent to show an antagonistic reciprocal interaction [18]. As in the hippocampus, A2AR stimulation decreased the affinity of A1Rs for agonists. The A1R\/A2AR heteromer allows adenosine to perform a detailed modulation of glutamate release [16, 48]. Regarding A1Rs and A2ARs, basal conditions generate a low tone of endogenous adenosine and cause A1R activation, in contrast to situations of increased adenosine where A2AR activation becomes dominant. When adenosine concentrations rise, as during anoxia, likely also time appears to be important in regulating A2A receptor activity, which means A2A receptors are \u201cactive\u201d under prolonged stimulation [49]. Finally, activation of the A1R\/A2AR heteromer contributes to A2AR signaling when adenosine levels are elevated and may provide a mechanism to facilitate plastic changes in the excitatory synapse [18].\nInteractions between adenosine and dopaminergic system\nDopamine is an important transmitter in basal ganglia and is noted for influencing motor activity, playing an important role in Parkinson\u2019s disease. Adenosine-dopamine interactions are complex and cannot be limited on functional considerations of A1Rs. Intramembrane heteromeric receptor-receptor interactions and the involvement of A2ARs in influencing dopaminergic signaling have to be mentioned due to the implications in the treatment of Parkinson\u2019s disease. Gin\u00e9s et al. [50] described the formation of functionally interacting heteromeric complexes between dopamine D1 receptors (D1Rs) and A1Rs in mouse fibroblast Ltk\u2212 cells cotransfected with respective cDNAs. Coaggregation occurred when cells were pretreated with R-PIA as A1R agonist, but was decreased by combined pretreatment with R-PIA and SKF-38393, a D1R agonist. Furthermore, the D1R agonist-induced cAMP accumulation was reduced by combined pretreatment of D1R- and A1R agonist, but remained unaffected when given alone, respectively. The results confirmed an antagonistic interaction between A1Rs and D1Rs that had already been observed by Ferr\u00e9 et al. [51] in behavioral studies using reserpinized mice and rabbits. In vivo and in vitro data on adenosine-dopamine interactions were mostly obtained from investigations in the basal ganglia and limbic regions [52, 53] due to the high abundance of A1Rs, A2ARs, D1Rs, and D2Rs in these areas and their involvement in the pathology of Parkinson\u2019s disease. The antagonistic interaction of combined receptor activation seems to distinguish between adenosine and dopamine receptor subtypes. While A1Rs communicate mainly with the D1R subtype in strionigral-strioentopenduncular neurons, A2AR and D2R interaction occurs in striopallidal neurons. Studies on mice and monkeys pretreated with MPTP suggest that some degree of dopaminergic activity is needed to obtain adenosine antagonistic-induced motor activity. Furthermore, blockade of dopaminergic neurotransmission counteracts the antagonistic effect induced by adenosine [54]. Sufficient endogenous adenosine is present interstitially in the substantia nigra pars reticulata to control dopaminergic effects. The effects of adenosine are absent when dopaminergic influence is suppressed [53]. Thus, it seems that monotherapy with A2AR antagonists may only be useful in the early stages of Parkinson\u2019s disease, but could support a therapeutic treatment with dopamine agonists in advanced stages. A promising approach using these therapeutic strategies can be seen in istradefylline, an A2AR antagonist that has since successfully passed clinical trials [55]. However, A1R blockade may also contribute to an increased dopamine release but this effect seems without clinical relevance.\nRelationship between A1Rs and NPY\nNeuropeptide Y (NPY) is one of the most abundant neuropeptides and exerts various functions on at least six GPCR subtypes (Y1Rs-Y5Rs, y6Rs). Immunohistochemical investigations revealed the appearance of the Y1R and Y5R subtypes in the rat frontal cortex [56\u201358]. Activation of NPY receptors results in an inhibition of excitatory synaptic transmission, while a presynaptic influence on cortical neurons has been postulated [59]. NPY receptors affect pertussis toxin-sensitive G proteins, which inhibit adenylyl cyclase and decrease cAMP levels. Inhibitory and facilitating effects on K+ and Ca2+ mobilization have also been observed [60]. Receptor-receptor interactions between Y1Rs have already been described, such as the antagonistic interaction with galanin receptors in the hypothalamus of the rat and their functional relevance for food intake. In contrast, a facilitatory interaction between the two receptors exists in the amygdala which may be of relevance for fear-related behavior [61].\nIn the CNS, A1Rs and NPY receptors share some similarities in distribution. Both A1Rs and Y1Rs are located on neurons of the prefrontal cortex, and their activation inhibits glutamatergic neurotransmission [62]. This is evidence for potential interaction between A1Rs and Y1Rs that may modulate long-term desensitization of A1Rs during pathophysiological situations. To investigate possible functional interactions, postsynaptic potentials (PSPs) were generated by electrical field stimulation on pyramidal neurons of layer V in the rat cingulate cortex as described by Brand et al. [35] and Hentschel et al. [36]. The Y1R agonist [F7,P34]pNPY inhibited the amplitude of PSPs. The inhibitory effect was reversible and reproducible, indicating that no desensitization appeared (Fig.\u00a01a). An additional decrease in PSP amplitude was observed when NPY was superfused in combination with the A1R agonist CPA (Fig.\u00a01b).\nFig.\u00a01Effect of the selective Y1 agonist [F7,P34]pNPY as well as neuropeptide Y (NPY) alone and in combination with selective A1R agonist N6-cyclopentyladenosine (CPA) on the amplitude of postsynaptic potentials (PSPs) evoked by electrical field stimulation (0.2 Hz, 2\u00a0ms) in layer I of the rat cingulate cortex (Sichardt et al., unpublished results). Intracellular recordings were performed in rat brain slices using glass microelectrodes placed in pyramidal cells of layer V. a [F7,P34]pNPY superfused for 5\u00a0min inhibits reversibly the PSPs by 34.6\u2009\u00b1\u20098%. Y1 antagonist BIBP3226 itself reduces the PSP by 23\u2009\u00b1\u200910%, whereas [F7,P34]pNPY has no inhibitory effect in the presence of the antagonist. b NPY depresses PSPs by 28.6\u2009\u00b1\u20090.7%. The combined superfusion of NPY and CPA resulted in an additional depression of the PSPs by 48.1\u2009\u00b1\u20095%. The depressant effects of the two agonists were reversible during washout. Data are expressed as mean \u00b1 SEM from n\u2009=\u20093 independent experiments. *p\u2009<\u20090.05 significant vs control; #p\u2009<\u20090.05 significant vs NPY alone\nThe additional inhibition induced by CPA was in the same range as that found with CPA alone (48.1\u2009\u00b1\u20095% vs 55\u2009\u00b1\u20093%). NPY inhibited PSPs after blockading CPA-mediated inhibitory effects by DPCPX. No significant changes existed before and after blockading A1Rs (Fig.\u00a02). The results suggest that no interaction between A1Rs and Y1Rs exist. Each neuromodulator contributes to inhibitory regulation of excitatory neurotransmission. Regarding the desensitization of A1Rs but not of Y1Rs, this may be important under pathophysiological conditions with increased adenosine concentration in the synaptic cleft.\nFig.\u00a02Effect of neuropeptide Y (NPY) on the PSPs alone and in combination with the selective A1R agonist N6-cyclopentyladenosine (CPA) after preincubation with the selective A1R antagonist 1,3-dipropyl-8-cyclopentylxantine (DPCPX) (Sichardt et al., unpublished results). The experimental procedure was similar to that shown in Fig.\u00a01. In the presence of NPY, PSPs were decreased by 35.4\u2009\u00b1\u20097%. Combined superfusion of NPY and CPA after preincubation with DPCPX decreased PSPs by 32.7\u2009\u00b1\u20098%. The depression was reversible during washout. Data are expressed as mean \u00b1 SEM from n\u2009=\u20095 independent experiments. *p\u2009<\u20090.05 significant vs control; #p\u2009<\u20090.05 significant vs superfusion of CPA and DPCPX\nInteraction of A1Rs and P2Y1Rs\nP2Y1Rs have been cloned and characterized in several species including human and rat, whereas mRNA was detected in various regions of the brain. The receptor subtype can be activated by ATP, but ADP as a degradation product of ATP is a more potent endogenous agonist. Cellular signaling differs between A1Rs and P2Y1Rs since A1Rs couple to Gi\/0 and P2Y1Rs to Gq\/G11. In fact, P2Y1Rs can be assumed to exert stimulatory effects in cells. P2Y1R signaling occurs in non-neuronal and non-muscular cell types, as well as on neurons in the CNS [63] where a colocalization of A1Rs and P2Y1Rs was demonstrated immunohistochemically in rat brain cortex, hippocampus, and cerebellum [64]. In 1996, Ikeuchi et al. reported the activation of an undefined P2YR by adenosine in patch clamp and calcium imaging experiments on hippocampal neurons [65]. Furthermore, extensive heteromerization experiments have been conducted on cotransfected HEK293 cells using immunoprecipitation, Western blotting, and bioluminescence resonance energy transfer (BRET). Receptor binding experiments in combination with cAMP assays have also been described [64, 66\u201370]. These respective studies confirmed heteromerization associated with changes in the agonist binding and signaling compared to monomer properties. The binding for selective A1R agonists was decreased while the A1R antagonist binding remained unaffected. Interestingly, ADP binding was blocked by DPCPX but not by the P2Y1R antagonist, suggesting an altered binding pocket on the A1R\/P2Y1R complex. The G protein-coupling was sensitive to pertussis toxin and revealed a Gi\/0 status for the heteromer. Although colocalization of A1Rs and P2Y1Rs in several brain areas has been demonstrated, there is still a lack of functional investigations. Nevertheless, the physiological relevance of this interaction has been postulated as follows: costorage and release of ATP with neurotransmitters, such as glutamate or noradrenaline, occurs in the CNS [71\u201373]. ADP, the degradation product of ATP, acts as an A1R agonist due to the activation of the A1R\/P2Y1R complex and contributes to the inhibitory modulation of excitatory synaptic transmission via adenosine acting on A1Rs. This interaction can be assumed as an additional mechanism for influencing and fine-tuning synaptic neurotransmission.\nFunctional interaction with ionotropic receptors\nNeuronal excitability is regulated by voltage and ligand-gated ion channels. Ionotropic receptors also referred to as ligand-gated ion channels (LGICs) are a group of intrinsic transmembrane ion channels that open and close in response to binding of a chemical messenger, as opposed to voltage-gated ion channels or stretch-activated ion channels. Ion channels are regulated by a ligand and are usually very selective to one or more ions such as Na+, K+, Ca2+, or Cl\u2212. These receptors located at synapses convert the chemical signal of presynaptically released neurotransmitter directly and very quickly into a postsynaptic electrical signal. Many LGICs are additionally modulated by allosteric ligands by channel blockers, ions, or membrane potential.\nNicotinic acetylcholine receptor serves as the prototypical LGIC [74] and consists of a pentamer of protein subunits with two binding sites, which, when bound, alter the receptor configuration and cause an internal pore to open. This pore, permeable to Na+ ions, allows them to flow down their electrochemical gradient into the cell. With a sufficient number of channels opening at once, the intracellular Na+ concentration rises to the point at which the positive charge within the cell is sufficient to depolarize the membrane, and an action potential is initiated [75]. Many important ion channels are ligand-gated, and they show a great degree of homology at the genetic level. The LGICs are classified into three superfamilies; the first\u2014the Cys-loop receptor family\u2014is subdivided into the anionic GABAA and glycine receptors on the one hand, and cationic 5-HT3 serotonin and nicotinic acetylcholine receptors on the other. The second group\u2014ionotropic glutamate receptors\u2014consists of NMDA, kainate, and AMPA receptors. The third group covers the ATP-gated channels\u2014the P2X receptors [76].\nAdenosine is known to inhibit glutamatergic neurotransmission by activation of presynaptic A1Rs [35]. This is probably due to reduction of the calcium influx, possibly by modulating both P\/Q- and N-type presynaptic voltage-dependent calcium channels, which in turn controls transmitter release [77]. Furthermore, A1Rs have long been known to mediate neuroprotection by reduction of excitatory effects at the postsynaptic level [10, 78, 79]. In addition to its direct presynaptic and postsynaptic actions on neurons, A1R interaction with NMDA [80\u201382, 84], GABAA [85\u201388], and P2X receptors [80, 89\u201391] contributes to fine-tuning neuromodulation via adenosine.\nInteraction between A1Rs and NMDA receptors\nGlutamate is the major excitatory neurotransmitter in the mammalian central nervous system. In most brain areas, glutamate mediates fast synaptic transmission by activating ionotropic receptors of the AMPA, kainate, and NMDA type. Additionally, NMDA receptors play a critical role in synaptic plasticity, synaptic development, and neurotoxicity. Recent studies suggest that some NMDA-mediated actions are altered or mediated by adenosine. Synaptic currents mediated by glutamate in rat substantia nigra pas reticulata neurons were reduced by adenosine acting via A1Rs. The inhibitory action was not mediated by a postsynaptic site since adenosine did not block currents evoked by local application of glutamate [86].\nNMDA is known to increase the extracellular level of adenosine via bidirectional adenosine transporters or from released adenine nucleotides degraded by a chain of ectonucleotidases [92, 93]. On the other hand, endogenous adenosine present in the extracellular fluid of hippocampal slices tonically inhibits NMDA receptor-mediated dendritic spikes as well as AMPA\/kainate receptor-mediated synchronized EPSPs by activation of A1Rs in CA1 pyramidal cells [81]. In line with these results, it has been shown that the tonic activation of A1Rs by ambient adenosine depressed field potentials in the striatum. The effect of adenosine in the striatum [84] or hippocampus [94] has not been found in A1R knockout mice and clearly demonstrates the involvement of A1Rs. The involvement of A1Rs was also supported by experiments using the selective receptor ligand 2-CA. In isolated rat hippocampal pyramidal cells [95] and in bipolar cells of the retina [96], 2-CA decreased inward currents induced by iontophoretic application of NMDA.\nAnother interesting interaction concerns NMDA preconditioning to protect against glutamate neurotoxicity. The A1R antagonist 8-CPT has been shown to prevent neuroprotection evoked by NMDA preconditioning against glutamate-induced cellular damage in cerebellar granule cells [83]. In this study, the functionality of A1Rs was not affected by NMDA preconditioning, but this treatment promoted A2AR desensitization in concert with A1R activation [83]. These results are in line with other studies indicating that adenosine downregulates excitatory and inhibitory synaptic transmission in several brain areas through activation of A1Rs and A2AR [97, 98]. Furthermore, activation of A1Rs mediates reversal of long-term potentiation (LTP) produced by brief application of NMDA in hippocampal CA1 neurons [99].\nTaken together, there are several ways in which adenosine may interact with NMDA-induced cellular events. Adenosine can affect glutamatergic transmission via both presynaptic and postsynaptic mechanisms by activating A1Rs [78]. NMDA receptors and A1Rs interact to downregulate glutamate release presynaptically in pyramidal cells of the cingulate cortex [35], neurons of the hippocampus [100], and striatal neurons [84]. Another putative mechanism is related to postsynaptic A1Rs; adenosine elevates the threshold to open NMDA receptor-operated channels by antagonizing membrane depolarization [101].\nInteraction between A1Rs and GABAA receptors\nFast synaptic inhibition in the brain and spinal cord is largely mediated by GABAA receptors that are also targeted by drugs such as benzodiazepines, barbiturates, neurosteroids, and some anesthetics. The modulation of their function will have important consequences for neuronal excitation [102]. One accepted means of modifying the efficacy is a functional interaction with adenosine. Adenosine may have an effect on either presynaptic GABA release in interneurons and\/or on postsynaptic GABAA receptors in projection neurons. The site of action may be studied electrophysiologically by inducing fast inhibitory postsynaptic potentials (IPSPs) or application of GABA directly onto the cell. Adenosine and selective A1R agonist CHA reduced the amplitude of the fast IPSP in lateral amygdala slice preparations. The effect of CHA was blocked by DPCPX, indicating the involvement of A1Rs. Additionally, adenosine did not block currents evoked by local application of GABA [85]. Thus, the modulatory effect of adenosine on the GABAergic neurotransmission appears to take place on a presynaptic site by inhibiting GABA release from nerve terminals [85, 86]. The assumption that the activation of A1Rs can presynaptically modulate inhibitory postsynaptic responses agrees with findings in several brain areas, such as the thalamus [87], suprachiasmatic and arcuate nucleus [88], and substantia nigra pars compacta [86].\nThere is some evidence that activation of A1Rs is also involved in GABAA receptor downregulation, implying a facilitation of the neurotransmission on a postsynaptic site. GABA but not adenosine evoked an inward current in rat sacral dorsal commissural neurons (SDCN). The GABA-induced current was significantly reduced be adenosine. CHA and DPCPX, but not selective ligands for A2ARs, mimicked or blocked the inhibitory effect of adenosine, respectively [103]. Adenosine and muscimol induced a concentration-dependent reduction in the amplitude of population potentials in hippocampal slices. Additionally, adenosine potentiated the ability of muscimol to inhibit evoked potentials, which were blocked by the A1R-selective antagonist 8-CPT. The effects of adenosine as well as muscimol were reduced by the chloride channel blocker DIDS, indicating the ability of adenosine to regulate the GABAA chloride channel by activation of A1Rs [104].\nSebastiao\u2019s group studied the mechanisms by which GABA modulates adenosine-mediated effects and found that endogenous GABA exerts an inhibitory effect through GABAA receptors via a predominant adenosine-mediated action in the hippocampus. The authors concluded that there is an A1R-mediated ability to inhibit synaptic transmission [19]. Further, this study showed that the blockade of GABAergic inhibition induced the release of NO, which was able to potentiate the inhibitory action of adenosine. They therefore suggested that the modulation of the A1R-mediated response by activation of GABAA receptors occurs indirectly via NO [19].\nActivation of GABAA receptors is effective in limiting neuronal ischemic damage [105] and endogenous adenosine that arises during hypoxia, and acts neuroprotectively partly by activating A1Rs [36]. Therefore, the contribution and potential interactions of GABA and adenosine as modulators of synaptic transmission during hypoxia has been investigated. Activation of A1Rs inhibits the release of GABA from the ischemic cerebral cortex in vivo [106]. In contrast, the administration of an A1R agonist in the hippocampus failed to affect the release of GABA during ischemia [107]. In the light of these controversial results, the role of the two neuromodulators during hypoxia was investigated in the CA1 area of rat hippocampal slices using selective A1R antagonists [108]. Indeed, activation of A1R and GABAA receptors is partly involved in the inhibition of synaptic transmission during hypoxia. The action of GABA becomes evident when A1Rs are blocked. Regarding the desensitization of A1Rs during hypoxia [109, 110], it may be assumed that GABAA-mediated inhibition of the synaptic transmission is evident when the A1R is desensitized or downregulated [108].\nComodulation by A1Rs and GABAA receptors was also suggested in acute cerebellar ethanol-induced ataxia. Using GABAA and A1R agonists and antagonists, respectively, a functional similarity between GABAA receptors and A1Rs has been shown even though both receptor types are known to couple to different signaling systems [111]. This provides conclusive evidence that A1Rs and GABAA receptors both play a comodulatory role in ethanol-induced cerebellar ataxia without any direct interaction.\nFunctional interaction between A1Rs and P2X receptors\nP2X receptors are ligand-gated ion channel receptors; seven subunits (P2X1-P2X7) have been identified [63]. The P2X receptor subunits show many differences in localization, pharmacology, kinetics, and signaling pathways [112, 113]. The P2X1 to P2X6 receptors have 379\u2013472 amino acids, with a predicted tertiary structure of transmembrane segments, a large extracellular loop and intracellular C  and N termini. The P2X2, P2X4, and P2X4\/P2X6 receptors appear to be the predominant neuronal types [91]. These subunits may occur as homooligomers or as heterooligomeric assemblies of more than one subunit. The P2X7 receptor has a similar structure, but with a much larger intracellular C terminus. This contrasts strikingly with any of the other known ligand-gated ionotropic receptors [114]. P2X7 subunits do not form heterooligomeric assemblies, but are involved in mediating apoptosis and necrosis in glial cells and possibly neurons.\nInteraction between adenosine receptor-mediated and P2 receptor-mediated effects have been shown to occur in neuronal and non-neuronal cells [80]. Both adenosine and ATP induce astroglial cell proliferation and formation of reactive astrocytes [89]. In hippocampus, adenosine and ATP are released on stimulation and are potent neuronal transmission inhibitors [115, 116]. It should be pointed out that the interpretation of effects induced by both is difficult since ATP is degraded enzymatically to adenosine [38]. Adenosine is formed by extracellular catabolism of released ATP via the ectonucleotidase pathway [90, 117]. The role of the ectonucleotidases in forming adenosine is difficult to study since this system is extremely efficient, and it is difficult to block an enzyme system. The experimental paradigm used by Cunha et al. [118] demonstrates that ATP has to be converted outside the cell into adenosine to exert its inhibitory effects on hippocampal synaptic transmission. The inhibitory effect of ATP was not modified by the P2 receptor antagonist suramin, but was attenuated by the ecto-5\u2019-nucleotidase inhibitor and was nearly prevented by the adenosine A1R antagonist DPCPX, whereas dipyridamole, an inhibitor of adenosine uptake, potentiated the inhibitory effect of ATP [118]. These results offer evidence for localized catabolism of adenine nucleotides followed by substrate channeling to A1Rs. This localized catabolism may mask the adenosine-mediated ATP effect [119]. Recently it was demonstrated that the exogenous application of ATP or ATP\u03c7S reduced the hippocampal neurotransmission. The inhibitory effect was blocked by the selective A1R antagonist DPCPX and was potentiated by different ecto-ATPase inhibitors [120]. These results suggest that the synaptic inhibition may consist of an inhibitory purinergic component of ATP itself in addition to degradation to adenosine.\nInteraction with neuronal ATP-sensitive K+ channels\nATP-sensitive K+ channels (KATP) are widely expressed in the cytoplasmic membrane of neurons and couple cell metabolism to excitability [121]. These channels are regulated by the intracellular ATP\/ADP ratio [122] and modulated by many endogenous mediators, including adenosine, via A1Rs. Activation of A1Rs inhibited the activity of inspiratory neurons in the brainstem by opening KATP in neonatal mice [123]. A1R stimulation promotes KATP activity in principal dopamine neurons in the substantia nigra pars compacta [124] and hippocampus [125]. In contrast, one recent study has demonstrated that adenosine induces internalization of KATP, resulting in a decrease in KATP-mediated response in the hippocampus [126]. The discrepancy might be due to the additional activation of A2ARs by adenosine located in hippocampus, but not in the substantia nigra pars compacta [127]. In addition to the inhibitory effect on the presynaptic site, the activation of A1Rs acts as an inhibitory modulator to electrical activity on the postsynaptic site, and this effect has been attributed to enhancement of KATP activity. The modulating effect on the membrane potential may differ depending on the brain regions, as neuronal KATP is heterogeneous in different neurons.\nA1R interactions\u2014new approaches for neurological disorders\nBy activation of its receptors, adenosine regulates many pathophysiological processes, particularly in excitable tissues of the brain (Fig.\u00a03). Its widespread functions in the body include regulation of seizure susceptibility [128, 129], neuroprotection [40], regulation of pain perception [130], sleep induction [131], and involvement in Parkinson\u2019s disease [132]. There is increasing evidence that the functional interaction of A1Rs with other neuronal receptors may contribute to fine tuning in synaptic transmission, and A1R agonists may represent a useful therapeutic approach for the treatment of some neurological disorders by regulating homeostasis in transmitter systems. However, the use of A1R agonists has not proved clinically useful due to mainly cardiovascular side effects as well as low brain permeability. Pioneering experimental approaches have been evaluated using focal drug delivery in epilepsy models. One experimental study has used intraventricular implantation of an adenosine-releasing synthetic polymer [128]. In a later study, Guttinger et al. [133] used encapsulated C2C12 myoblasts that were engineered to release adenosine by disruption of their adenosine kinase gene [133]. The local delivery of adenosine by implanted cells appears to be a promising strategy for the control not only for affecting seizure activity but also other neurodegenerative diseases with dysregulated synaptic neurotransmission.\nConcluding remarks\nAs a consequence of its ubiquitous distribution and because of its linkage to the energy pool, adenosine has evolved as an important messenger in extracellular signaling. Modifications in extracellular adenosine levels with subsequent alterations in the activation of its receptors interferes with the action of other receptor systems. Figure\u00a04 summarizes possible interactions of A1Rs with metabotropic receptors, Fig.\u00a05 shows the interactions between A1Rs and ionotropic receptors as well as with KATP, and Fig.\u00a03 shows the neurological disorders where A1R interactions may play a role.\nFig.\u00a03Schematic representation of possible interactions of A1Rs with metabotropic receptors. Heteromerization between presynaptically located A1Rs and A2ARs, D1Rs, and P2Y1Rs causes changes in influencing glutamate release by adenosine. Cross talk between A1Rs and P2Y1Rs contributes mainly to triggering fast attenuation of transmitter release, whereas ADP acts as a ligand on the heteromer. During elevated adenosine levels, A2AR signaling becomes dominant in the A1R\/A2AR complex, providing enhancement of glutamate release. The A1R\/D1R heteromer requires both adenosine and dopamine to be activated and inhibits transmitter releaseFig.\u00a04Schematic representation of possible interactions of A1Rs with ionotropic receptors contributing to the fine-tuning of neurotransmission. Adenosine acting via presynaptic A1Rs may attenuate the influx of Ca2+ through voltage-dependent calcium channels and thus decrease the release of glutamate and GABA, which inhibits or facilitates the activation of postsynaptically located NMDA receptors, respectively. Adenosine acting through postsynaptic A1Rs may activate KATP, which leads to hyperpolarization of postsynaptic neurons and inhibits directly the activity of NMDA and GABAA receptorsFig.\u00a05Neurological disorders where A1R interactions may play a role\nThere is evidence that various regulatory mechanisms exist as well as multiple mechanisms that act independently of each other on the same cell depending on the brain region and cell type. The overaction and redundancy principle ensures transmitter homeostasis under pathophysiological conditions in a special time window. The function of adenosine receptors in the regulation of the synaptic transmission is complex.\nThe key receptor in regulation of the neuronal transmission may be the A2AR, whereas the interaction of the A1R with metabotropic and ionotropic receptors as well as with KATP serves as fine-tuning to inhibit synaptic transmission, as mentioned by Sebastiao and Ribeiro [80]. A1Rs may play a nonessential role in normal physiology as demonstrated in mice lacking the A1Rs [134]. However, they play an important protective role under pathophysiological conditions especially during hypoxia. The activation initiates a fast inhibition of the glutamatergic neurotransmission and the receptor interactions may contribute to its maintenance or can support the A1R-mediated effects.\nMost of our knowledge on receptor-receptor interactions involving the A1R results from experiments on cell cultures, slice preparation or, to a lesser extent, from in vivo experiments where regulation can be studied in principle or new drug targets can be characterized. These findings may contribute to a better understanding of disturbances in transmitter homeostasis. As our understanding of the complexity of receptor signaling and interaction develops, we may well gain new perspectives in new drug development. The clinical relevance of the testing models has often been questioned, however. Discordance between studies on cells and animal and human studies may be due to bias or failure of models to mimic clinical disease to an adequate degree. There are new techniques such as neuroimaging, nanotechnology, siPCR, and new selective receptor ligands that will help to overcome some of these aspects in the near future.","keyphrases":["adenosine","receptor interactions","g protein-coupled receptors","ionotropic receptors","adenosine receptors","neurotransmission"],"prmu":["P","P","P","P","P","P"]}
{"id":"Biotechnol_Lett-3-1-1914260","title":"Expression of alternansucrase in potato plants\n","text":"Alternan, which consists of alternating \u03b1-(1\u21923)\/\u03b1-(1\u21926)-linked glucosyl residues, was produced in potato tubers by expressing a mature alternansucrase (Asr) gene from Leuconostoc mesenteroides NRRL B-1355 in potato. Detection of alternan was performed by enzyme-linked immunosorbent assay in tuber juices, revealing a concentration between 0.3 and 1.2 mg g-1 fresh wt. The Asr transcript levels correlated well with alternan accumulation in tuber juices. It appeared that the expression of sucrose-regulated starch-synthesizing genes (ADP-glucose pyrophosphorylase subunit S and granule-bound starch synthase I) was down-regulated. Despite this, the physico-chemical properties of the transgenic starches were unaltered. These results are compared to those obtained with other transgenic potato plants producing mutan [\u03b1-(1\u21923)-linked glucosyl residues] and dextran [\u03b1-(1\u21926)-linked glucosyl residues].\nIntroduction\nProduction of novel polymers in plants by genetic modification is a great opportunity to obtain plants with unique properties that cannot be generated by conventional breeding (Kok-Jacon et\u00a0al. 2003). In addition, modifications of native polymers in planta could also generate crops with added nutritional, environmental or commercial value. For instance, production of biodegradable plastics in crops such as flax offers new perspectives for the replacement of oil-derived plastics (Wr\u00f3bel et\u00a0al. 2004). Another example is the production of a freeze-thaw-stable potato starch exhibiting novel physicochemical properties, thereby increasing the number of industrial applications (Jobling et\u00a0al. 2002).\nAlternan is a unique polymer which is produced by three Leuconostoc mesenteroides strains: NRRL B-1355, NRRL B-1498 and NRRL B-1501 (Jeanes et\u00a0al. 1954). Alternan synthesized by L. mesenteroides NRRL B-1355 is mediated by the alternansucrase ASR (EC 2.4.1.140) which is a large glucansucrase of 2,057 amino-acids (Arg\u00fcello-Morales et\u00a0al. 2000). Its C-terminal domain (also referred to as glucan-binding domain or GBD) exhibits short repeats specific for ASR, which could contribute to its distinct features (Jane\u010dek et\u00a0al. 2000). The resulting polymer has a unique structure with alternating \u03b1-(1\u21923)\/\u03b1-(1\u21926)-linked glucose residues, present for 46% and 54%, respectively. Due to this structure, alternan is a highly soluble and low viscous polymer, which is resistant to microbial and mammalian enzymes making it suitable for the production of ingredients for functional foods such as prebiotics (C\u00f4t\u00e9 1992). Also, novel industrial applications were investigated by hydrolyzing native alternan polymers with isolates of Penicillium bacterial strains, creating potential replacers of commercial gum arabic (Leathers et\u00a0al. 2002; 2003). Furthermore, ASR is an attractive enzyme because of its efficiency in bond formation, which is higher than that of the dextransucrase (DSRS) (Richard et\u00a0al. 2003). In addition, mutated ASR enzymes showed a high efficiency in glucosylating acceptor molecules (cellobiose, \u03b1-alkylglucosides) in comparison to native ASR and DSRS enzymes, which might enable novel industrial applications (Arg\u00fcello-Morales et\u00a0al. 2001; Richard et\u00a0al. 2003; Luz Sanz et\u00a0al. 2006).\nIn this work, we describe the production of alternan in potato tubers by expressing ASR. Modification of starch structure was envisaged with ASR, because of its high acceptor reaction efficiency. The effect of ASR on starch biosynthesis was studied at the microscopical, molecular and biochemical level, and compared to the effects of the dextransucrase (DSRS) and mutansucrase (GTFI), producing less soluble polymers, such as dextran and mutan that are mainly composed of \u03b1-(1\u21926) and \u03b1-(1\u21923)-linked glucose residues, respectively (Kok-Jacon et\u00a0al. 2005a, b).\nMaterials and methods\nConstruction of binary plant expression vector containing the Asr gene\nAn expression cassette containing the patatin promoter (Wenzler et\u00a0al. 1989), the chloroplastic ferredoxin signal peptide (FD) from Silene pratensis (Pilon et\u00a0al. 1995) fused to the NOS terminator was cloned into the pBluescript SK (pBS SK) plasmid, resulting in pPF that was used as starting material for cloning the alternansucrase (Asr) gene. A mature Asr gene from L. mesenteroides NRRL B-1355 (Arg\u00fcello-Morales et\u00a0al. 2000; AJ250173) was ligated in frame between the signal peptide FD and the NOS terminator. The mature Asr gene was amplified by PCR, with a forward primer containing a SmaI restriction site (5\u2032-CATCAGGGCCCCGGGGATACAAAT-3\u2032) and a reverse primer containing a NruI restriction site (5\u2032-CTCCTTTCGCGAATCCTTCCCTTA-3\u2032) using the proofreading Pfu turbo DNA polymerase (2.5 units\/\u03bcl; Stratagene, UK) and cloned into the SmaI\/NruI restriction sites of pPF, resulting in pPFAsr. FD and the fused Asr gene were completely sequenced in one direction by Baseclear (The Netherlands) to verify the correctness of the construct. pPFAsr was digested with SacI and SalI and subsequently ligated into a pBIN20 binary vector (Hennegan and Danna 1998), resulting in pPFA (Fig.\u00a01).\nFig.\u00a01Schematic representation of pPFA binary vector used for potato plant transformation\nTransformation and regeneration of potato plants\npPFA was transformed into Agrobacterium tumefaciens strain LBA 4404 using electroporation (Takken et\u00a0al. 2000). Internodal stem segments from the tetraploid potato genotype (cv. Kardal (KD)) were used for Agrobacterium-mediated transformation, which was performed as described by Kok-Jacon et\u00a0al. (2005a).\nStarch isolation\nPotato tubers were peeled and homogenized in a Sanamat Rotor (Spangenberg, The Netherlands). The resulting homogenate was allowed to settle overnight at 4\u00b0C and the potato juice was decanted and stored at \u221220\u00b0C for characterization of soluble alternan. The starch pellet was washed three times with water, air-dried at room temperature for at least three days and stored at room temperature.\nImmunological detection of alternans in tuber juices and gelatinized starches\nPresence of alternans was investigated with enzyme-linked immunosorbent assay (ELISA) as described by Kok-Jacon et\u00a0al. (2005a), using monoclonal anti-\u03b1-(1\u21926) dextran antibodies (45.21.1 (groove-type; IgA\/Kappa) and 16.4.12EBI (cavity-type; IgA\/Kappa)) (Wang et\u00a0al. 2002) with tuber juices and gelatinized starches. The monoclonal anti-\u03b1-(1\u21926) dextran antibodies detect structures containing both internal and terminal epitopes of \u03b1-(1\u21926) dextran which can be applicable for the detection of \u03b1-(1\u21926) linked glucose residues present in alternan (Sharon et\u00a0al. 1982; Dr Denong Wang, personal communication).\nExpression analysis of Asr and genes involved in starch biosynthesis using semi-quantitative and real-time quantitative RT-PCR analysis\nRNA was isolated from 3\u00a0g (fresh weight) of potato tuber material from selected transgenic lines according to Kuipers et\u00a0al. (1994).\nSemi-quantitative and real-time quantitative RT-PCR\u2019s were performed as described by Kok-Jacon et\u00a0al. (2005a). AsrRT primers, 5\u2032-ACCGGTTCCATCAACTAATAAT-3\u2032 and 5\u2032-GACATCTCGGAAGGATCCC-3\u2032 (Tm\u00a0=\u00a055\u00b0C, 35 cycles) were based on the Asr gene sequence (Arg\u00fcello-Morales et\u00a0al. 2000). RNA sample from Karnico potato tubers expressing a sense\/antisense GBSSI cDNA inverted-repeat construct referred to as RVT34-77 (Heilersig 2005) was used as a positive control, because its GBSSI expression level was completely down-regulated.\nDetermination of morphological and physicochemical starch properties\nAnalysis of starch granule morphology was performed by light microscopy and scanning electron microscopy (SEM) as described by Kok-Jacon et\u00a0al. (2005a).\nMedian values of the granule size distribution (d50), gelatinization analysis, amylose content, starch content, chain length distributions (HPSEC, HPAEC) were determined as described by Kok-Jacon et\u00a0al. (2005a).\nResults\nDetection of alternan in transgenic potato juices\nTo enable plastidic protein targeting, the mature Asr gene was fused to the ferredoxin (FD) signal peptide (Gerrits et\u00a0al. 2001). The resulting gene fusion was inserted between the patatin promoter (Fig.\u00a01) allowing high-tuber expression (Wenzler et\u00a0al. 1989) and the Nos terminator sequence. At the FD\u25b2Asr fusion, two mutations were present because a SmaI restriction site was engineered at this position (VTAM\u2193ATYKVTLITK\u25b2ADT became VTAM\u2193ATYKVTLITP\u25b2GDT, in which \u2193 represents the splice site for amyloplast entry and \u25b2 the gene fusion). Furthermore, differences from the published ASR sequence (Arg\u00fcello-Morales et\u00a0al. 2000) were found at three positions (Y208H, D221G and G1092S), but these did not affect conserved residues. After Agrobacterium-mediated plant transformation, thirty independent transgenic potato clones were obtained using the Kardal (KD) genotype. Five plants of each transgenic clone were grown in the greenhouse from which the tubers were pooled for further characterization. KDAxx referred to the transformed potato plant serie in which A represents the Asr gene and xx the clone number. The untransformed genotype is referred to as KD-UT.\nDetection of alternan was performed by analyzing tuber juices of the transformants with ELISA using anti-dextran antibodies (Wang et\u00a0al. 2002). Alternan was detected in 4 out of 29 tubers (about 14%) in a concentration ranging from 0.3 to 1.2\u00a0mg\u00a0g\u22121 fresh\u00a0wt (Fig.\u00a02) in the transformants KDA16, KDA19, KDA27 and KDA13. As expected, no alternan was found in KD-UT plants. According to the tuber juice results, the KDA transformants were divided in three classes: (\u2212), (+) and (++), representing no, intermediate (\u22641\u00a0mg\u00a0g\u22121\u00a0FW) and high (>1\u00a0mg\u00a0g\u22121\u00a0FW) levels of alternan, respectively. All the transformants containing alternan and two from the (\u2212) class were selected for further characterization: KDA13 (++), KDA16 (+), KDA19 (+), KDA27 (+), KDA1 (\u2212) and KDA24 (\u2212). RNA was isolated from potato tubers and subjected to RT-PCR analysis. The expression levels were determined for the Asr and Ubi3\u00a0genes, of which the latter is used as a control because of its constitutive expression (Garbarino and Belknap 1994) (Fig.\u00a03). Heterologous Asr gene expression was detected in the expressers KDA13, KDA16, KDA19, KDA27. No Asr mRNA was detected in the (\u2212) class transformants and in the KD-UT plants. The Asr expression levels correlated well with the ELISA results described above.\nFig.\u00a02Detection of alternans accumulated in potato juices by ELISA using anti-dextrans antibodies. Based on the alternan concentration [in mg\u00a0g\u22121\u00a0fresh wt (FW)], three categories of transformants were made, where (\u2212), (+) and (++) represent no, intermediate and high alternan accumulation, respectively. Transgenic clones indicated with grey bars were selected for further characterizationFig.\u00a03RT-PCR analysis of the selected KDA transformants and KD-UT tuber RNA. The upper panel shows the PCR products using the primers designed on the Asr sequence. The lower panel shows the PCR products using the primers designed on the Ubi3 sequence that served as an internal control. pPFAsr plasmid: positive control\nAlternan accumulation does not interfere with plant, tuber and starch morphologies\nAsr expressing plants (green parts and tubers) did not exhibit any morphological changes in comparison to KD-UT plants (data not shown). In addition, starch morphology of Asr expressing plants was quite similar to that of KD-UT. With SEM, a rough surface was present on some of the (++) class transformant granules (Fig.\u00a04B, F), but was considered as not significant when compared to dextran- (Fig.\u00a04C, G) and mutan- (Fig.\u00a04D, H) accumulating plants. In general, starch granules from the (+) and (\u2212) class transformants were similar to those of the KD-UT (data not shown). Starch granules comparable to those illustrated in Fig.\u00a04(F) were scored by analyzing a population of 100\u00a0granules in triplicate for each selected transformant (data not shown). KDA13, belonging to the (++) class transformant, exhibited (12%\u00a0\u00b1\u00a01.0) of altered starch granules, followed by the (+) class transformant [KDA19 (9.3%\u00a0\u00b1\u00a00.6); KDA27 (8.3%\u00a0\u00b1\u00a00.6)]. For the (\u2212) class transformant and KD-UT, the frequency of altered granules was lower, which was around the 7%.\nFig.\u00a04SEM analysis of starch granules (\u00d7350: upper panel) and (\u00d71,000: lower panel) from KD-UT (A, E) compared to that of selected transformants producing foreign polymers with decreasing water-solubility (KDA13 that produces alternan (B and F; ++: highly soluble (S)), KDD30 that produces dextran (C and G; +: soluble (L)) and KDIC15 that produces mutan (D and H; \u2212: insoluble (I)). Degrees of polymer solubility were defined according to Robyt (1996) in which class S = more soluble referring to glucans precipitated by 40\u201344% (v\/v) ethanol, L\u00a0=\u00a0less soluble referring to glucans precipitated by 34\u201337% ethanol and I\u00a0=\u00a0water-insoluble\nThe physicochemical properties and starch content of KDA transformants remain unchanged\nMedian granule size (d50), gelatinization characteristics (T0 and \u0394H), amylose and starch content measurements were performed on selected transformants (Table\u00a01). From these results, it can be seen that no consistent changes were detected for the different classes of transformants. Furthermore, chain length distribution experiments (HPSEC and HPAEC) were also done, particularly because ASR exhibits a high acceptor reaction efficiency. After complete debranching of starch with isoamylase, no consistent changes were found with HPSEC and HPAEC in comparison to KD-UT starches (data not shown). In addition, debranched starches, which were further treated with \u03b1-amylase, were analyzed with HPAEC in order to detect the presence of novel structural elements on starch molecules such as alternating \u03b1-(1\u21923)\/\u03b1-(1\u21926) linkages. Again, no consistent changes were detected with HPAEC in comparison to KD-UT starches (data not shown).\nTable\u00a01Summary of granule size (d50), gelatinization characteristics (To, \u0394H), amylose and starch content measurements of starches from the selected transformants and KD-UT. Data (\u00b1SD) are the average of two or three independent measurementsTransformantsd50 (\u03bcm)*T0 (\u00b0C)\u2020\u0394H (kJ\/g)\u2021Amylose content(%)Starch content (mg\/g FW)KD-UT26.5 (\u00b10.3)67.9 (\u00b10.1)14.5 (\u00b10.1)22.3 (\u00b10.2)214.8 (\u00b1117.5)KDA1 (\u2212)24.4 (\u00b10.2)68.1 (\u00b10.1)17.0 (\u00b10.1)22.2 (\u00b10.2)103.4 (\u00b166.3)KDA24 (\u2212)25.0 (\u00b10.2)68.0 (\u00b10.1)16.3 (\u00b11.2)21.3 (\u00b10.4)86.7 (\u00b141.9)KDA16 (+)24.9 (\u00b10.3)67.9 (\u00b10.2)16.4 (\u00b11.3)22.2 (\u00b10.1)140.0 (\u00b188.2)KDA19 (+)27.9 (\u00b10.2)67.7 (\u00b10.0)15.2 (\u00b10.1)23.0 (\u00b10.2)137.1 (\u00b138.2)KDA27 (+)22.8 (\u00b10.7)67.7 (\u00b10.2)16.2 (\u00b10.5)22.2 (\u00b10.4)289.3 (\u00b139.7)KDA13 (++)24.0 (\u00b10.1)67.8 (\u00b10.1)16.0 (\u00b10.7)22.2 (\u00b10.5)107.2 (\u00b149.4)* Median value of the granule size distribution\u2020 Temperature of onset of starch gelatinization\u2021 Enthalpy released\nExpression levels of AGPase and GBSSI genes are down-regulated in the (+) and (++) KDA class\nThe expression levels of key genes involved in starch biosynthesis such as sucrose synthase (SuSy), ADP-glucose pyrophosphorylase subunit S (AGPase), starch synthase III (SSIII), starch branching enzyme I (SBEI) and granule-bound starch synthase I (GBSSI) were monitored by real-time quantitative RT-PCR (Fig.\u00a05). All these genes seemed to be down-regulated, particularly the AGPase and GBSSI genes. In most cases, the extent of AGPase and GBSSI down-regulation corresponded well with the amount of alternan that was accumulated in the potato tubers. However, AGPase down-regulation did not correlate with a reduction in starch content for the (++) transformants (107.2\u00a0\u00b1\u00a049.4\u00a0mg\u00a0g\u22121\u00a0FW) when compared to KD-UT (214.8\u00a0\u00b1\u00a0117.5\u00a0mg\u00a0g\u22121\u00a0FW). Concerning GBSSI, the down-regulation was about 20 times less than for the transformant RVT34-77 in which GBSSI is completely inhibited. Typically, no reduction in amylose content was observed for the KDA transformants (Table\u00a01), irrespective of their GBSSI messenger RNA level. Thus, the observed reduction in GBSSI expression for the (+) and (++) KDA classes were significant within the selected transformants, but relatively small with respect to the RVT34-77 transformant.\nFig.\u00a05Real-time quantitative RT-PCR analysis of KDA24 (\u2212), KDA27 (+) and KDA13 (++) transformants and KD-UT tuber RNA using the following specific primers: SuSy, sucrose synthase; AGPase, ADP-glucose pyrophosphorylase subunit S; SSIII, starch synthase III; SBEI, starch branching enzyme I; GBSSI, granule-bound starch synthase I. RNA levels for each gene were expressed relative to the amount of Ubi3 RNA, as described in materials and methods. RNA sample from Karnico potato tubers expressing a sense\/antisense GBSSI cDNA construct exhibiting a complete GBSSI down-regulation (RVT34-77), was used as a positive control\nDiscussion\nThis report is the first study on the production of alternan in potato tubers. Their presence in potato juices was demonstrated by ELISA using anti-dextran antibodies. Expression of ASR did not interfere with plant growth and development, and tuber and starch yield penalties were not observed. These results were similar to those obtained with the dextransucrase (DSRS) expression (Kok-Jacon et\u00a0al. 2005a), but not to those obtained with the mutansucrase (GTFI) expression in which the tuber phenotype was significantly affected (Kok-Jacon et\u00a0al. 2005b).\nThe amount of alternan accumulated in potato tubers (1.2\u00a0mg\u00a0g\u22121\u00a0fresh wt) was lower than that of dextran (1.7\u00a0mg\u00a0g\u22121\u00a0fresh wt) (Kok-Jacon et\u00a0al. 2005a). It might be possible that the large size of the mature ASR (2,057 amino-acids (a.a) when compared to DSRS with only 1,527\u00a0a.a.) might reduce the efficiency with which the enzyme is transported through the amyloplast membrane. However, such explanation needs to be approached with caution because the presence of alternansucrase in the amyloplast was not directly evidenced, as no ASR antibodies were available to us. Interestingly, it has been shown that the size of ASR can be reduced (by removal 82% (632\/767\u00a0a.a.) of the C-terminal GBD) without compromising its activity (Joucla et\u00a0al. 2006). If the size of the protein is indeed a critical factor, than this truncated variant may be a useful tool to enhance alternan synthesis in the amyloplast. Such an approach was already employed successfully for the Streptococcus downei mutansucrase GTFI (Kok-Jacon et\u00a0al. 2005b). We have directed a mature and a GBD-truncated GTFI protein to potato amyloplasts, and found that the truncated form synthesized a larger amount of mutan, with much more pronounced effect on starch granule morphology.\nAlthough ASR is known to be efficient in catalyzing acceptor reactions (Richard et\u00a0al. 2003; C\u00f4t\u00e9 and Sheng 2006), no evidence was found for the covalent attachment of novel, alternan-based structural elements to starch molecules. Also with dextransucrase and mutansucrase we have not been able to introduce different glycosyl linkage patterns in starch (Kok-Jacon et\u00a0al. 2005a, b). To this end, acceptor reactions of glucansucrases with starch or maltodextrins are not studied in much detail. It has been observed that the efficiency of acceptor reaction decreases with increasing length of maltodextrins (reviewed in Kok-Jacon et\u00a0al. 2003). We had anticipated that the nascent starch polymers would be poor acceptors for the glucansucrases. However, during starch biosynthesis potential acceptors (small maltodextrins) are thought to be generated through the action of, for instance, debranching enzymes (or isoamylases). If such a small acceptor is mutanylated, alternanylated, or dextranylated at the non-reducing end, then these novel structures might be incorporated into starch polymers through the action of certain transferases such as, for instance, branching enzyme. Apparently, this does not happen, or at a very low (undetectable) frequency, but the reason for this is unclear.\nStarch morphology in the ASR transformants was not significantly altered in comparison to that of dextran and mutan-accumulating plants (Fig.\u00a04). This might be related to the fact that alternan is more water-soluble than dextran and mutan. An indication of the water-solubility of the three polysaccharides is given in Fig.\u00a04; the more ethanol is required for precipitation, the higher the water-solubility. The water-solubility decreases in the order of alternan, dextran and mutan. We hypothesize that the co-synthesis of water-insoluble mutan and starch leads to co-crystallization of the two polymers, as a result of which the granule is packed in a less orderly fashion. This comparison should be approached with caution. For alternan and dextran, the observed differences in starch morphology may also be related to the fact that more dextran than alternan was accumulated in the potato tubers; for mutan, we have not been able to quantify the amount accumulated in the tubers. Therefore, it can not be excluded that the observed effects are related to the amount of foreign polymer produced. Interestingly, co-synthesis of levan, a water-soluble fructosyl-based polymer, and starch resulted in a dramatically altered starch granule morphology (Gerrits 2000). However, it should be noted that much higher levels of levan, which were estimated to be 66\u00a0mg\u00a0g\u22121\u00a0fresh wt (Gerrits et\u00a0al. 2001; Cairns 2003), were produced in comparison with alternan (1.2\u00a0mg\u00a0g\u22121\u00a0fresh wt) or dextran (1.7\u00a0mg\u00a0g\u22121\u00a0fresh wt), and that the starch granules contained approximately 5% of levan. This result contrasts with that of alternan- and dextran-accumulating plants in which foreign polymers were only found in the stroma. Taking together the results of potato transformants expressing glucan- or levansucrases in amyloplasts, it seems that the site of accumulation of the foreign polymer (granule or stroma), the solubility of the foreign polymer, and the amount of foreign polymer that is actually produced are important factors in determining starch granule morphology.","keyphrases":["alternan","transgenic potato","glucansucrase","polymer solubility"],"prmu":["P","P","P","P"]}
{"id":"Qual_Life_Res-3-1-2039822","title":"Reliability and validity of functional health status and health-related quality of life questionnaires in children with recurrent acute otitis media\n","text":"In this study the reliability and validity of generic and disease-specific questionnaires has been assessed focusing on responsiveness. This is part of a study on the effects of recurrent acute otitis media (rAOM) on functional health status (FHS) and health-related quality of life (HRQoL) in 383 children with rAOM participating in a randomized clinical trial. The following generic questionnaires were studied: 1. RAND general health rating index, 2. Functional Status Questionnaire (FSQ Generic and FSQ Specific), 3. TNO-AZL Infant Quality of Life (TAIQOL), and the following disease-specific questionnaires: 1. Otitis Media-6 (OM-6), 2. Numerical rating scales (NRS) for child and caregiver (NRS Child and NRS Caregiver), and 3. a new Family Functioning Questionnaire (FFQ). Reliability was good to excellent (Cronbach\u2019s \u03b1 range 0.80\u20130.90, intraclass correlation coefficient range 0.76\u20130.93). Moderate to strong correlations were found between the questionnaires as well as between questionnaires and relevant clinical indicators (r = 0.29\u20130.49), demonstrating construct validity. Discriminant validity for children with few versus frequent episodes of acute otitis media per year was good for most questionnaires (P < 0.004) but poor for the otitis media-related subscales of the TAIQOL (P = 0.10\u20130.97) and both NRS (P = 0.22 and 0.48). Except for the TAIQOL subscales, change scores were significant (P < 0.003) for generic and disease-specific questionnaires. Effect sizes were somewhat higher for disease-specific compared to generic questionnaires (0.55\u20130.95 versus 0.32\u20130.60) except for the TAIQOL subscales, which showed very poor sensitivity to change. Anchor-based methods resulted in a somewhat larger range of estimates of MCID than distribution-based methods. Combining distribution-based and anchor-based methods resulted in similar ranges for the minimally clinical important differences for generic and disease-specific questionnaires: 2\u201315 points on a 0\u2013100 scale. Apart from the generic TAIQOL subscales, both generic and disease-specific questionnaires used in this study showed good psychometric qualities and responsiveness for use in clinical studies on children with rAOM.\nIntroduction\nAcute otitis media (AOM) is a common childhood infection with a peak incidence occurring between 6 and 12\u00a0months of age. Five to fifteen percent of all children, depending on their age, suffer from recurrent acute infections of the middle ear (4 or more episodes per year) [1\u20134]. Repetitive episodes of pain, fever and general illness during acute ear infections [5\u20138] as well as worries about potential long-term sequelae such as hearing loss and disturbed language development [9\u201313] may all compromise the quality of life of the child and its family [14\u201316]. Although several questionnaires have been used in assessing the effects of recurrent acute otitis media (rAOM) in children, lack of true health-related quality of life (HRQoL) questionnaires as well as incomplete data on their reliability and validity mean that our current knowledge on the subject is limited for both research and clinical practice [17].\nAssessment of functional health status (FHS) and HRQoL, as defined in Table\u00a01 [18\u201326], has become increasingly important in clinical trials on the effectiveness of treatment in paediatric chronic conditions. The validation of FHS and HRQoL questionnaires, however, has so far mainly focused on reliability and construct validity. Responsiveness has been assessed for only a few paediatric HRQoL questionnaires for conditions other than otitis media [27\u201331]. In order to evaluate treatment effects on FHS and HRQoL meaningfully, questionnaires are needed that are not only reliable and valid but also responsive to changes in FHS and HRQoL. In adult studies, various strategies have been used to assess responsiveness, which is defined as the ability to detect clinically important change over time and therefore involves both the assessment of sensitivity to change and the assignment of meaning to that change [32, 33]. Since none of these strategies is without limitations, we will try to assess the responsiveness of FHS and HRQoL questionnaires by using multiple strategies, categorized into distribution-based and anchor-based methods.\nTable\u00a01Definitions of health-related quality of life and functional health statusHealth-related quality of life:Level of satisfaction a person inputes to those aspects of his or her life that are affected by the effects of illness and its treatment [18\u201320]. Incorporation of a person\u2019s valuation of his life distinguishes HRQoL from other measures of well-being [21, 22].Functional health status:Reflection of the (severity of) signs and symptoms and the adequacy of daily functioning across various life-domains in an individual with a certain health condition [23\u201326].\nDistribution-based methods express the amount of change relative to the amount of random variance of a questionnaire [34, 35], whereas anchor-based methods enhance interpretability of changes in questionnaire scores by linking meaning and clinical relevance to change scores [34, 36].\nBoth generic and disease-specific questionnaires have been used in studies of paediatric FHS or HRQoL. Generic questionnaires span a wide spectrum of quality of life components, bridging various health states and populations. Disease-specific questionnaires on the other hand, assess health-related issues specific to particular conditions and may be able to detect small changes that are often small but clinically important; these provide a more detailed assessment of HRQoL, but cannot be used for comparisons across health conditions [37\u201339]. Both questionnaires are often combined in order to profit from the merits of both types. However, there have been few head-to-head comparisons between generic and disease-specific HRQoL measurement questionnaires in the setting of randomized controlled trials (RCT) [40].\nThe current RCT on the effectiveness of pneumococcal vaccination in children with rAOM will address both the issues of using generic versus disease-specific questionnaires and responsiveness in evaluating treatment effects on HRQoL in RCTs. The results will lead to recommendations regarding the applicability of these questionnaires in clinical studies in children with rAOM.\nMethods\nSetting and procedure\nFHS and HRQoL were assessed in 383 children with rAOM participating in a double-blind randomized, placebo-controlled trial on the effectiveness of pneumococcal conjugate vaccination versus control hepatitis vaccination. The study was conducted at the paediatric outpatient departments of a general hospital (Spaarne Hospital Haarlem) and a tertiary care hospital (University Medical Center Utrecht). Children were recruited for this trial through referral by general practitioners, paediatricians, or otolaryngologists, or were enrolled on the caregiver\u2019s own initiative from April 1998 to February 2001.\nStudy population\nInclusion criteria: children were aged between 12 and 84\u00a0months and suffering from rAOM at study entry; defined in this study as having had at least 2 episodes of physician diagnosed AOM in the year prior to study entry. Exclusion criteria were conditions with a known increased risk for AOM such as: known immunodeficiency (other than IgA or IgG2 subclass deficiency), cystic fibrosis, immotile cilia syndrome, cleft palate, chromosomal abnormalities (like Down syndrome) or severe adverse events upon vaccination in the past.\nAt each scheduled visit, two research physicians (C.N.M.B. and R.H.V.) collected data regarding the number of episodes of AOM (based on parental report at baseline and on physician report during follow-up), upper respiratory tract infections, and pneumonia. Information about the medical treatment, and ear, nose, and throat surgery in the preceding 6\u00a0months was also collected. The primary caregivers completed questionnaires assessing FHS and HRQoL of their child and family during the clinic visits at baseline and at 7, 14, and 26\u00a0months follow-up. Caregivers were requested to have the same person complete the questionnaires each time and to rate their child\u2019s FHS and HRQoL with regard to their recurrent episodes of acute otitis media. Informed consent was obtained from caregivers of all children before study entry. Medical ethics committees of both participating hospitals approved the study protocol.\nQuestionnaires\nFour generic questionnaires (RAND, FSQ Generic, FSQ Specific, TAIQOL) and one disease-specific questionnaire (OM-6) were used to assess FHS and HRQoL of the children in the study. Additionally, two disease-specific one-item numerical rating scales (NRS Child and NRS Caregiver) were used to obtain a global rating of HRQoL of the child and of the caregiver, respectively, related to rAOM. For the assessment of the impact of rAOM on family functioning a newly composed disease-specific questionnaire the Family Functioning Questionnaire (FFQ), was used to assess the impact of rAOM on family functioning. Table\u00a02 summarises the characteristics of the questionnaires [14, 41\u201357].\nTable\u00a02Characteristics of FHS and HRQoL questionnaires used in this studyQuestionnairesType; number of items; scaleConstruct(s) measuredApplication in other studiesGenericRANDFHS; 7; LikertGeneral health: current health; previous health; resistance to illnessLow-birth-weight children; survivors of childhood cancer; asthmatic children [42, 44, 47, 48]FSQ genericFHS; 14; LikertAge appropriate functioning and emotional behaviourLow-birth-weight children; survivors of childhood cancer; asthmatic children [41, 45\u201347, 49\u201351]FSQ specificIdem to FSQ Generic, measuring general impact of illness on functioning and behaviourTAIQOLHRQoL; 35\/46*; LikertSleeping, appetite, lung problems, stomach problems, skin problems, motor functioning, problem behaviour, social functioning, communication, positive mood, anxiety, livelinessLow-birth-weight children, childen with chronic illness, children with chronic OME [51, 54]Disease-specificOM-6FHS; 6; LikertPhysical suffering; hearing loss; speech impairment; emotional distress; activity limitations; caregiver concernsChildren with recurrent AOM; children with chronic OME [14, 55\u201357]NRS Child NRS CaregiverHRQoL; 1; index 0\u2013100HRQoL; 1; index 0\u2013100Global well-being of child related to AOM episodesGlobal well-being of parent related to child\u2019s AOM episodesChildren with recurrent AOM or chronic OMEnone [14]Family Functioning Questionnaire (FFQ)FHS; 7; LikertParents: sleep deprivation; change of daily or social activities; emotional distress. Family: cancelling family plans or trips. Siblings: feeling neglected; demanding extra attention.None*\u00a046 items when age\u00a0>\u00a015\u00a0months\nGeneric questionnaires\nThe RAND general health-rating index (RAND) and the Functional Status Questionnaire (FSQ) had already been translated and validated for Dutch children by Post et\u00a0al. [41, 42] (Table\u00a02). The RAND assesses general health perceptions of caregivers regarding their child [43]. The FSQ consists of two parts: one measuring functional limitations in general, not necessarily related to illness (FSQ Generic) and the other (paradoxically named FSQ Specific) measuring functional limitations that are attributable to any illness [43]. Functional limitations in both versions of the FSQ are mainly expressed as behavioural problems. During the course of the study, a new Dutch questionnaire on generic HRQoL became available: the TNO-AZL Infant Quality of Life (TAIQOL) questionnaire [51, 53]. For this reason, from July 1999 the TAIQOL was added to the previously selected set of questionnaires. Although the full, original version of the TAIQOL has been applied during the study, only those subscales from the TAIQOL are discussed that, based on their content, were assumed to be sensitive to the consequences of AOM. The following subscales tap functional items that are often affected by AOM (OM-related): \u2018Sleeping\u2019, \u2018Appetite\u2019, \u2018Liveliness\u2019, \u2018Problem behaviour\u2019, \u2018Positive mood\u2019, and \u2018Communication\u2019 (items about speech and language capacity) which are 6 of the 12 subscales in the TAIQOL. Although the TAIQOL has been developed for children aged up to 5\u00a0years, we also used the questionnaire in children aged 6\u20137\u00a0years, as no appropriate alternative was available during the study.\nDisease-specific questionnaires\nTo measure disease-specific FHS, the Otitis Media-6 (OM-6) [14, 55] was translated into Dutch according to principles of backward\u2013forward translation [58\u201361]. This six-item questionnaire covers both acute and long-term functional effects of otitis media in children on FHS.\nA new questionnaire has been developed to assess the impact of rAOM in children on their caregivers and siblings: the FFQ. The content of the FFQ was based on previous work by Asmussen et\u00a0al. [15, 62] on the impact of rAOM on family well-being. A panel of paediatric otorhinolaryngologists and paediatricians from our study sites selected the items most relevant according to their clinical experience. The FFQ is composed of six questions covering effects of the child\u2019s rAOM on caregiver and family activities and two questions assessing these effects on emotional behaviour of the other siblings. The Likert-scale was used as a response format and was analogous to that of the RAND and OM-6 in our study, ranging from score 1\u20134.\nFurthermore, two numerical rating scales (NRS) (0\u2013100) were used, the NRS Child and the NRS Caregiver (see Table\u00a02). The NRS Child [14] was translated into Dutch using the same principles of backward\u2013forward translation that have been applied to translation of the OM-6. The newly created NRS Caregiver was modelled upon the NRS Child and added to the previously selected set of questionnaires from July 1999. The NRS caregiver has been created in this study, following the example of the NRS child which was created by Rosenfeld et\u00a0al. [14].\nFinally, the Dutch version of the OM Functional Status Questionnaire specific (OMFSQ [52]) was included as an anchor for responsiveness (instrument description in section on responsiveness).\nQuestionnaire application\nQuestionnaires were completed in a randomly selected, but fixed order during the follow-up assessments to prevent possible influence of order effects [63, 64]: RAND, FSQ Generic and Specific, OM-6, NRS Child, FFQ, TAIQOL, OMFSQ, NRS Caregiver. For all questionnaires higher scores indicate the presence of a better HRQoL or FHS. To allow comparisons between scores on the questionnaires, all scores were linearly transformed into 0\u2013100 Scales. For each questionnaire, the evaluation period was the 6\u00a0weeks before completion.\nStatistical analyses\nFloor and ceiling effects\nFloor and ceiling effects were estimated for the baseline-assessment of each questionnaire by calculating percentages of respondents that had minimum and maximum scores, respectively. Questionnaires should exhibit minimal floor and ceiling effects to be optimally able to detect difference and change.\nReliability\nFirst, internal consistency was assessed by calculating Cronbach\u2019s alpha, which should be above 0.70 for each questionnaire or subscale [65]. Inter-item correlations of questionnaires were assessed to reveal item redundancy or \u2018hidden\u2019 subscales that may erroneously yield a high overall Cronbach\u2019s alpha.\nFor the assessment of test\u2013retest reliability, a subset of caregivers attending the outpatient ward from February 2000 to June 2001 (n\u00a0=\u00a0160) was given a second set of the same questionnaires (retest) to complete at home. The time frame for completion was 2\u00a0weeks after the first set of questionnaires was filled out during the outpatient visit at 14\u00a0months (first test). Children with AOM at the first test were excluded, since differences in their scores could be due to real change and interfere with the assessment of reliability.\nFor the assessment of test\u2013retest reliability, a time-interval of 2\u201314\u00a0days is often considered long enough to prevent recall bias and too short for relevant change to occur in chronic disease [66]. Test\u2013retest reliability was computed as the intraclass correlation coefficients (ICC) between the two sets of questionnaires. An ICC of 0.80 was considered the required minimum for good reliability [65, 67].\nConstruct and discriminant validity\nIn order to demonstrate construct validity, hypotheses were formulated about the strength of correlations between questionnaires. A higher percentage of correct predictions indicates stronger support for construct validity. A correlation of 0.10\u20130.30 was defined as weak, 0.30\u20130.50 as moderate, and >0.50 as strong [68]. The correlation between FSQ Generic and NRS Caregiver was predicted to be weak since they were expected to assess two different constructs. Moderate to strong correlations (r\u00a0>\u00a00.40) were predicted between RAND and NRS Caregiver. Moderate to strong correlations were also expected between OM-6 and FSQ Specific, NRS Child, NRS Caregiver and FFQ, as all assess otitis media-related HRQoL or FHS. The correlation between FSQ Generic and FSQ Specific was expected to be strong (r\u00a0>\u00a00.50). The remaining correlations among the questionnaires were expected to be moderate (Table\u00a05). Additionally, correlations between questionnaire scores and frequency of physician visits for upper respiratory tract infections as well as frequency of AOM episodes in the preceding 6\u00a0months were calculated. Since distributions of questionnaire scores were skewed, correlations were assessed using Spearman\u2019s rho.\nDiscriminant validity was assessed by dichotomizing the study participants in children with 2\u20133 versus 4 or more episodes of otitis media per year. Based on clinical and immunological data, children with 4 or more AOM episodes per year are considered as \u2018otitis prone\u2019 [2, 69\u201371], reflecting a sub-group with an increased rate of upper respiratory tract infections, related medical interventions and compromised child functioning [72, 73]. It was assumed that this group would perform significantly poorer than children with 2\u20133 otitis media episodes per year on all questionnaires, which was assessed by independent sample Mann\u2013Whitney tests.\nResponsiveness\nSince pneumococcal conjugate vaccination showed no clinical effectiveness when compared to the control vaccine [74], the intervention could not be used as an external criterion of change. Data of both vaccine groups were pooled instead for the assessment of responsiveness to spontaneous remission. The clinical experience of a panel of 5 experts in the field of otitis media, formed the basis for defining a reduction of 2 or more episodes of AOM per child per year as the external criterion for change while a reduction of 1 episode or less identified no change. Responsiveness was evaluated for two intervals: from 0 to 7\u00a0months and from 7 to 14\u00a0months follow-up. The observed change in these episodes was multiplied by 12\/7 (1,714) to get the estimated change per year.\nThe first step in the assessment of responsiveness was to explore the ability of questionnaires to detect change at all, i.e., its sensitivity to change. Secondly, meaning and clinical relevance of the change score were determined in accordance with recent recommendations, using both distribution- and anchor-based methods [36, 75\u201377]. Distribution-based methods express the amount of change relative to the amount of random variance of a questionnaire [34, 35]. Some ratios of change to random variance have, often empirically, been found to represent a minimally clinical important difference. Anchor-based methods enhance interpretability of changes in questionnaire scores by linking meaningful and clinically relevant indicators to change scores [34, 36].\nThe assessment of responsiveness will be described in further detail below.\nSensitivity to change\nSensitivity to change was assessed by calculating both the statistical significance of change scores using a paired t-test or Wilcoxon matched pairs test (for skewed distributions), and effect sizes (ES) using Guyatt\u2019s responsiveness statistic [78] for changed subjects. In this statistic, the observed change that occurred in changed subjects is related to the observed random change or random error, in unchanged subjects. A parametric effect size was computed as: mean change score changed group\/SD (change score unchanged group); a nonparametric effect size was computed as: median change score changed group\/interquartile range (change score unchanged group)).\nAccording to the benchmarks of Cohen [79], an effect size of 0.2 represents a small change, 0.5 a moderate change and 0.8 or higher represents a large change. For skewed distributions Wilcoxon matched pairs test was used to calculate the significance of change.\nClinical relevance of change scores\nThe interpretation of change is often assessed by calculating the minimally clinical important difference (MCID), which is the smallest difference in a questionnaire total or domain score that patients perceive as beneficial [80]. The MCID can be computed from both distribution-based and anchor-based methods. Several estimates of the MCID from both methods are reported, to assess the likely range of the MCID for each questionnaire.\nInterpretation of change\u2014distribution-based methods (ES-MCID and SEM-MCID)\nThe main distribution-based methods for assessing the MCID are the Effect Size and the Standard Error of Measurement. A change in questionnaire scores corresponding to the effect size of Guyatt\u2019s Responsiveness Statistic with values of 0.3\u20130.5 has been found to be consistent with other (empirical) estimates of the MCID [36, 81\u201383]. In this study the change in questionnaire scores corresponding with an effect size of 0.3 is used as benchmark of MCID (ES-MCID). A change of one Standard Error of Measurement (1-SEM) has empirically been found to correspond with the MCID of a questionnaire [77, 84\u201386]. The 1-SEM of a questionnaire links reliability of an instrument to the variance of scores in a population as reflected in its formula: 1-SEM\u00a0=\u00a0SD (change scores unchanged subjects)\u00a0*\u00a0\u221a(1-ICC). It is an estimate of what part of the observed change may be due to random measurement error by including distribution of scores (SD) and instrument reliability (ICC). Change larger than the SEM therefore is considered \u2018real\u2019 change. The SEM is here used as an estimate of the MCID (SEM-MCID). The ES-MCID and SEM-MCID support the interpretation of measured change, as they reflect the smallest change that is substantially larger than the random variability in the study population which is based on the standard deviation of the unchanged subjects.\nInterpretation of change\u2014anchor-based methods\nAnchor-based methods require an independent standard, the anchor, that in itself is easily interpretable and that is at least moderately correlated (>0.3) with the questionnaire being assessed. Changes in questionnaire scores were compared with change in two clinically relevant anchors: the AOM frequency (incidence of AOM episodes per child) and the AOM severity assessed with the Dutch version of the OM-Functional Status Questionnaire specific (OM-FSQ) [52]. The OM-FSQ was used as an anchor for responsiveness. It consists of three questions assessing clinical AOM severity: earache, sleeping problems, and other signs and symptoms (irritability, fussiness, fever) that may indicate the presence of an ear infection. In our population, the OM-FSQ demonstrated high internal consistency (Cronbach\u2019s \u03b1\u00a0=\u00a00.88) and good test\u2013retest reliability (ICC\u00a0=\u00a00.94). The OM-FSQ correlated weakly with the NRS Child (Spearman\u2019s rho\u00a0=\u00a00.18), but moderately with the RAND (0.36), FSQ Generic (0.37), and NRS Caregiver (0.34), and strongly with the FSQ Specific (0.52), OM-6 (0.73) and FFQ (0.61).\nIn relation to the AOM frequency, an expert panel in the field of otitis media considered a reduction of 2 episodes per year as a small or minimal clinically important change, whereas a change of 3 to 4 episodes per year was considered moderate to large. In the study of Alsarraf et al. [52], the OM-FSQ total score was about 62 on a scale of 0\u2013100 during an episode of AOM, increasing to 92 at 6\u00a0weeks and to 90 at 12\u00a0weeks after an episode of AOM with higher scores reflecting less severe ear-related symptoms. Therefore, a score change of 10\u201320 on the 0\u2013100 scale of the OM-FSQ in the current population was considered to be a small clinically relevant change in AOM severity, a score change of 30\u201350 as moderate to large. Anchor-based estimates of the MCID were computed as the change in questionnaire scores associated with small changes in AOM frequency and OM-FSQ.\nFor all analyses the Statistical Package for the Social Sciences (SPSS) version 10.1 was used.\nResults\nPopulation\nThe population characteristics summarized in Table\u00a03 show that the majority of children suffered from 4 or more AOM episodes per year, and half of them suffered from chronic airway problems or atopic symptoms. Most children had undergone one or more ENT surgeries. Overall they seemed to suffer from more severe disease than the average child with 2\u20133 middle ear infections, as stated earlier.\nTable\u00a03Characteristics of study population*Mean or % (n\u00a0=\u00a0383)SD or 95% CIAge (months)34(19.7)Male gender62%(57\u201367)In the year prior to inclusionNumber of AOM episodes\/year5.0(2.7)2\u2013337%(32\u201342)4\u2013531%(26\u201336)6 or more32%(27\u201337)Impaired hearing**35%(30\u201340)Language or speech problems**22%(18\u201326)History ofChronic airway problems or atopic symptoms ***51%(46\u201356)Adenoidectomy47%(42\u201352)Tympanostomy tubes51%(46\u201356)Other ear-, nose-, and throat surgeries2%(0.6\u20133)Antibiotic prophylaxis15%(11\u201319)Ever had speech-therapy9%(6\u201312)*\u00a0at inclusion in the study**\u00a0reported by the caregiver***\u00a0asthma, wheezing, hayfever, or eczema\nFloor and ceiling effects\nGenerally, the questionnaires demonstrated no floor-effects. However, Table\u00a04 shows that some questionnaires (FSQ Specific and FFQ) and most TAIQOL subscales showed moderate to large ceiling effects, which indicates that measurement of improvement may be limited while it may actually be present.\nTable\u00a04Floor and ceiling effects*, internal consistency and test\u2013retest reliability of the questionnairesMinimum score (%)Maximum score (%)Internal consistency Cronbach\u2019s \u03b1 n\u00a0=\u00a0383**Test\u2013retest reliability ICC*** n\u00a0=\u00a0106GenericRAND000.810.89FSQ generic020.800.92FSQ specific0210.860.89TAIQOLN.A.N.A.0.72\u20130.900.76\u20130.90\u00a0\u00a0\u00a0\u00a0Sleeping2120.900.83\u00a0\u00a0\u00a0\u00a0Appetite0220.860.82\u00a0\u00a0\u00a0\u00a0Positive mood0800.900.81\u00a0\u00a0\u00a0\u00a0Liveliness0.6810.880.76\u00a0\u00a0\u00a0\u00a0Problem behaviour140.860.85\u00a0\u00a0\u00a0\u00a0Communication 0.4530.880.82Disease-specificOM-60140.850.89NRS child23N.A.0.83FFQ0.5270.900.93NRS caregiver00N.A.0.81*\u00a0percentage of respondents with minimum (floor effect) and maximum (ceiling effect) scores**\u00a0n\u00a0=\u00a0169 for the TAIQOL subscales and NRS Caregiver***\u00a0Intra-class Correlation Coefficient\nReliability\nCronbach alpha coefficients were adequate to high (range 0.72\u20130.90) for the TAIQOL subscales and high (range 0.80\u20130.90) for all other questionnaires. The calculation of inter-item correlations revealed no \u2018hidden\u2019 subscales or item redundancy (i.e., individual correlations are too high, with possible loss of content validity) (Table\u00a04).\nIn order to assess test\u2013retest reliability, 126 (79%) of 160 approached caregivers completed a second set of questionnaires of which 113 (71%) were completed within 2\u00a0weeks. Seven children with AOM at the time of the outpatient visit (test 1) were excluded, resulting in 106 sets for analysis (Table\u00a04). ICCs were moderate to high for all questionnaires (range 0.81\u20130.93) and most TAIQOL subscales (range 0.76\u20130.90), but in the borderline range for the TAIQOL subscale \u2018Liveliness\u2019 (0.76).\nConstruct and discriminant validity\nTable\u00a05 reflects the calculated correlations between the questionnaires, which ranged from moderate to strong for the RAND, FSQ Generic, FSQ Specific, OM-6, and FFQ. These outcomes show that 14 (67%) of the hypothesized correlations were correct. False predictions were mainly made about the NRS Child and NRS Caregiver, as the correlations with other questionnaires were generally expected to be at least moderate, but were found to be weak. Disease-specific questionnaires (OM-6, NRS Child, FFQ and NRS Caregiver), showed moderate correlations (Spearmans\u2019 rho 0.39\u20130.49) with the frequency of AOM episodes in the preceding 6\u00a0months. Moderate correlations (Spearmans\u2019 rho 0.29\u20130.48) were also found between global FHS (RAND) and the disease-specific questionnaires on the one hand and the number of physician visits for all upper respiratory tract infections (URTIs), a more global indicator of illness, on the other hand (Table\u00a06).\nTable\u00a05Construct validity: calculated correlations\u00a0*\u00a0 between the questionnaires**RANDFSQ genericFSQ specificOM-6NRS childFFQNRS caregiverRAND1.000.520.490.340.330.430.49FSQ generic1.000.800.370.250.430.24FSQ specific1.000.490.260.520.24OM-61.000.230.740.28NRS child1.000.220.47FFQ1.000.39NRS caregiver1.00*\u00a0Spearman correlation coefficients were calculated**\u00a0appropriately \u00e0 priori predicted correlations are bold-printedTable\u00a06Construct validity\u2014\u2018correlations* between questionnaire scores and frequency of physician visits for URTI** and of AOM** episodes\u2019Frequency of physician visits for URTIFrequency of AOM episodes***GenericRAND\u22120.48\u22120.31FSQ generic\u22120.20\u22120.07#FSQ specific\u22120.27\u22120.12##Disease-specificOM-6\u22120.32\u22120.41NRS child\u22120.41\u22120.49FFQ\u22120.29\u22120.39NRS caregiver\u22120.41\u22120.40*\u00a0Spearmans\u2019rho correlation coefficients were calculated**\u00a0URTI: upper respiratory tract infection; AOM: acute otitis media***\u00a0All correlations P\u00a0<\u00a00.001, except for #\u00a0(P\u00a0=\u00a00.16) and ##\u00a0(P\u00a0=\u00a00.02)\nThe RAND, FSQ Generic, FSQ Specific, OM-6 and FFQ were able to discriminate between children with moderately recurrent AOM (2\u20133 episodes per year) and \u201cotitis-prone\u201d children with severe, recurrent AOM (4 or more episodes per year) (Table\u00a07). However, neither the two numerical rating scales (NRS Child and NRS Caregiver) nor the otitis media-related subscales of the TAIQOL discriminated between these two groups.\nTable\u00a07Discriminant validity: scores of children with 2\u20133 vs. 4 or more AOM episodes in the preceding year*2\u20133 AOM episodes\u22654 AOM episodesMann\u2013Whitney P-valueGenericRAND21.119.60.004FSQ generic76.572.20.002FSQ specific83.978.40.001TAIQOL\u00a0\u00a0\u00a0\u00a0Sleeping66.260.70.10\u00a0\u00a0\u00a0\u00a0Appetite74.773.20.44\u00a0\u00a0\u00a0\u00a0Liveliness93.291.30.81\u00a0\u00a0\u00a0\u00a0Positive mood92.092.50.97\u00a0\u00a0\u00a0\u00a0Problem behaviour64.860.90.24\u00a0\u00a0\u00a0\u00a0Communication83.884.50.69Disease-specificOM-618.917.0<0.001NRS child5.25.40.48FFQ84.978.5<0.001NRS caregiver6.66.20.22Calculated by Mann\u2013Whitney test*\u00a02\u20133 episodes means moderate and >4 episodes means serious AOM\nResponsiveness\nAccording to our external criterion of change (a reduction of 2 or more episodes of AOM per year), 270 children (70%) of 383 were classified as \u2018changed\u2019 for the first interval (0\u20137\u00a0months) and 126 children (33%) for the second interval (7\u201314\u00a0months). The two intervals differed considerably regarding the reduction of AOM incidence; during the 0\u20137\u00a0months follow-up the mean incidence per child decreased by 1.8 AOM episodes, whereas during 7\u201314\u00a0months follow-up the mean decrease was 0.35 episodes [74].\nSensitivity to change\nSensitivity to change, expressed as significant mean change and effect size, is presented in Table\u00a08. Except for most TAIQOL subscales, generic as well as disease-specific questionnaires yielded significant change scores during both follow-up periods, ranging from 4.9 to 28.3 on a 0\u2013100 scale. Absolute change scores for the first follow-up period generally were larger (range 0.4\u201328.3) than for the second period (range \u22122.8\u201314.2).\nTable\u00a08Sensitivity to change: mean change-scores* and effect sizes** for changed subjectsMean change-scoreEffect size\u2014GRS0\u20137\u00a0months#7\u201314\u00a0months0\u20137\u00a0months7\u201314\u00a0monthsn\u00a0=\u00a0270***P-valuen\u00a0=\u00a0126****P-valuen\u00a0=\u00a0270***n\u00a0=\u00a0126****GenericRAND10.2<0.0017.7<0.0010.600.54FSQ Generic7.0<0.001 4.90.0010.370.29FSQ specific9.1<0.0016.0<0.0010.370.32TAIQOL\u00a0\u00a0\u00a0\u00a0Sleeping9.9<0.0017.10.030.370.36\u00a0\u00a0\u00a0\u00a0Appetite6.80.0010.01.00.280.00\u00a0\u00a0\u00a0\u00a0Problem behaviour0.40.80\u22122.80.330.020.13\u00a0\u00a0\u00a0\u00a0Positive mood1.50.303.90.110.060.25\u00a0\u00a0\u00a0\u00a0Liveliness2.30.191.60.510.220.11\u00a0\u00a0\u00a0\u00a0Communication2.90.121.70.320.160.11Disease-specificOM-616.6<0.00111.5<0.0010.600.73NRS child28.3<0.00114.2<0.0010.910.64FFQ13.6<0.0018.0<0.0010.550.60NRS caregiver19.20.0039.10.0030.950.57*\u00a0calculated with paired t-test**calculated with Guyatt\u2019s responsiveness statistic (GRS)*** n\u00a0=\u00a0114 for TAIQOL subscales and NRS Caregiver; #\u00a0follow-up interval**** n\u00a0=\u00a051 for TAIQOL subscales and NRS Caregiver\nThe effect sizes for the generic FHS questionnaires ranged from small to moderate (0.29\u20130.60). For the generic TAIQOL subscales however, the effect sizes were lower, ranging from almost zero for the subscales \u2018Appetite\u2019 (0,0), \u2018Problem behaviour\u2019 (0.02) and \u2018Positive mood\u2019 (0.06) to small for \u2018Sleeping\u2019(0.37) and \u2018Liveliness\u2019 (0.22). Effect sizes for the disease-specific questionnaires were moderate to large (0.55\u20130.95). For the questionnaires the ES were quite similar for the first (0\u20137\u00a0months) and second intervals (7\u201314\u00a0months), whereas for the second interval absolute change scores were smaller.\nThe TAIQOL was excluded from further analyses on the interpretation of change, due to its poor sensitivity to change.\nInterpretation of change\u2014distribution-based methods\nMinimally clinical important differences (MCIDs) calculated with distribution-based methods are presented in Table\u00a09. During the first interval, ES-MCIDs using an effect size of 0.3 as benchmark were somewhat smaller for generic questionnaires, ranging from 5.0 to 7.4 on a 0\u2013100 scale, than those for disease-specific questionnaires ranging from 6.1 to 9.4. During the second interval, however, ES-MCIDs for generic and disease-specific questionnaires were comparable (range 4.0\u20136.7), indicating that for both types of questionnaires similar change scores are needed in order to be clinically relevant.\nTable\u00a09Responsiveness\u2014distribution-based indices for minimally clinical important difference (MCID) using 0.3 Effect Size (ES) and one standard error of measurement (SEM)ES\u2014MCID*SEM\u2014MCID**0\u20137\u00a0months#7\u201314\u00a0months0\u20137\u00a0months7\u201314\u00a0monthsGenericRAND5.04.35.34.5FSQ generic5.75.15.44.8FSQ specific7.45.67.85.9Disease-specificOM-68.34.78.85.0NRS child9.46.712.58.9FFQ7.44.06.13.3NRS caregiver6.14.88.36.6*\u00a0MCID using 0.3 effect size as benchmark; #\u00a0follow-up interval**\u00a0MCID using one-SEM as benchmark\nExcept for the NRS Child and NRS Caregiver, the SEM-MCIDs were quite comparable with the ES-MCIDs for both generic and disease-specific questionnaires. Assuming that the estimated MCIDs using either an effect size of 0.3 or a one-SEM as benchmark are correct, our results suggest that the range for the distribution-based MCID for generic as well as disease-specific questionnaires corresponds with a change of 3 - 9 points on a 0\u2013100 scale (see Table\u00a09).\nInterpretation of change\u2014anchor-based methods\nChanges in AOM frequency (AOM incidence per child per year) were compared to the magnitude of change scores on the FHS and HRQoL questionnaires. A small change of 2 AOM episodes, which is considered a MCID, in AOM frequency corresponded with 3\u201310 points change on a 0\u2013100 scale for the generic questionnaires (Graph 1a), and with 5\u201315 points change for disease-specific questionnaires, except for the NRS Child during the 0\u20137\u00a0months interval with 29 points change.\nGraph\u00a01\u00a0Responsiveness\u2014change-scores per questionnaire corresponding with an anchor-based responsiveness index: (a) AOM frequency; \n(b) AOM severity (OM-FSQ score)\nLikewise, a small improvement in AOM severity corresponded with change scores ranging from 2\u201310 points on a 0\u2013100 scale for the generic questionnaires and with change scores from 4\u20138 points for the disease-specific questionnaires, except again for the NRS Child with 16 and 17 points change (Graph 1b).\nChange scores corresponding with moderate to large changes in AOM frequency and severity are also presented in Graph 1a, b. Comparing small change with moderate to large change shows that, overall, the larger the change in AOM severity or frequency, the larger the magnitude of the change score on the questionnaires. However, this trend was not true for the FSQ Generic and the disease-specific NRS Child (e.g., a small change in AOM severity equalized a change score of 17 on the NRS Child, whereas a moderate-large change equalized a change score of 13).\nComparison of anchor- and distribution-based methods\nComparing the results of the anchor-based methods with those of the distribution-based methods (Graph 2) showed that generic questionnaires (RAND, FSQ Generic, and FSQ Specific), disease-specific questionnaires (OM-6 and FFQ) and the NRS Caregiver yielded quite similar estimates of the MCID for both methods (3\u20139 points on a 0\u2013100 scale for distribution and 2\u201315 points for anchor-based methods) as well as for both follow-up periods (4\u201315 points for 0\u20137\u00a0months interval, 2\u20138 points for 7\u201314\u00a0months interval). Averaging these distribution-based and anchor-based estimates of MCID yields a point-estimate MCID for generic questionnaires of 6.0 (range 2\u201310) and for disease-specific questionnaires of 7.3 (range 3\u201315) on a 0\u2013100 scale (excluding the NRS Child, as it had much larger estimates for the MCID).\nGraph\u00a02\u00a0Minimally clinical important difference (MCID) per questionnaire according to distribution-based (ES-MCID and SEM-MCID) and anchor-based (AOM frequency and AOM severity) methods\nDiscussion\nIn this study, the reliability and validity of generic as well as disease-specific FHS and HRQoL questionnaires have been assessed in the setting of a RCT concerning children with recurrent AOM. Most generic (RAND, FSQ-Generic and FSQ-Specific) and disease-specific (OM-6 and FFQ) questionnaires showed similar, good to excellent reliability and adequate construct and discriminant validity. Construct validity was poor for the numerical rating scales (NRS Child and NRS Caregiver), and discriminant validity was low to moderate for both NRS and the subscales of the TAIQOL considered to be otitis media-related (Tables\u00a04, 5, 6 and 7).\nGeneric as well as disease-specific questionnaires proved to be sensitive to change in the incidence of AOM (Table\u00a08). The effect sizes were found to be ranging from small to moderate for both generic and disease-specific questionnaires (Table\u00a08). The MCIDs for generic and disease-specific questionnaires were quite similar in terms of responsiveness (Table\u00a09 and Figure 1 & 2). However, most otitis media-related subscales of the TAIQOL, the only true HRQoL questionnaire, proved insensitive to change.\nReliability and validity\nResults on internal consistency and test\u2013retest reliability of the RAND, FSQ Generic, FSQ Specific, TAIQOL and OM-6 found in this study, were comparable with those of previous studies using these questionnaires [14, 41, 42, 51, 52]. The consistency of results across different paediatric populations supports the reliability of these questionnaires. Similar to the poor discriminant validity in this study of the otitis media-related TAIQOL subscales, Fekkes et al. [51] found the TAIQOL subscales \u2018Problem behaviour\u2019, \u2018Positive mood\u2019, and \u2018Liveliness\u2019 discriminated neither between healthy and preterm children nor between healthy and chronically ill children. The ability of the RAND, FSQ Generic and FSQ Specific to discriminate between children who differed in AOM frequency, on the other hand, supported their discriminant validity previously found in children with asthma and healthy children [41, 42]. However, the heterogeneity of methods used limits the comparability of results regarding validity of this study with those from previous studies.\nThe FFQ and NRS Caregiver are newly composed questionnaires to assess the influence of recurrent AOM on the caregiver and family. The FFQ demonstrated excellent reliability and validity, meeting the minimal required reliability coefficients of 0.90 for individual assessment [65, 87]. The strong correlation with the OM-6 supports its complementary usefulness in FHS and HRQoL assessment in children with rAOM. Results of the NRS Caregiver, however, were similarly poor as those observed for the NRS Child, which needs further exploration. Their global, single-item assessment of HRQoL may be too crude to reflect subtle differences in HRQoL [88, 89]. On the other hand, comments of the caregivers indicated that some of them may have misunderstood the NRS test-instructions. This is supported by the fact that improvement of construct validity occurred during follow-up assessments, presumably due to learning effects after reading the instructions a second time.\nResponsiveness\nSo far, little attention has been given to the responsiveness of the questionnaires used in our study. Only Rosenfeld et al. [55] assessed effect sizes for the OM-6 (using a standardized response mean) that were much larger (1.1\u20131.7) than the ones found in this study. This may be explained by the use of different identifiers of change. Rosenfeld et al. [55] used an intervention with expected clinical effectiveness, for which proxies were not blinded, as indicator of change. Since pneumococcal vaccination proved to be clinically ineffective [74], treatment could not be used as an external criterion for change. Instead, a change of 2 or more AOM episodes per year was used as criterion to identify changed subjects. In addition, social desirability and expectancy bias may have influenced the outcome of the study of Rosenfeld et al. [55]\nAlthough clinical criteria such as change in the incidence of AOM episodes have been suggested as adequate alternative criteria to identify change [34], the choice for any external criterion for change remains somewhat arbitrary. It is a surrogate measure that often only reflects one aspect of the QoL construct. The poor responsiveness of the TAIQOL subscales \u2018Behavioural problems\u2019, \u2018Positive mood\u2019 and \u2018Liveliness\u2019, for example, may indicate that our clinical indicator is less suitable as external criterion for change in emotional and behavioural functioning. However, considering the overall poor responsiveness of the twelve TAIQOL subscales (results not shown), it seems more obvious that poor responsiveness in itself mainly applies for these three subscales as well.\nSeveral studies have supported the empirically found link between one SEM and the MCID for HRQoL questionnaires [75, 81, 85, 86]. In this study the MCIDS based on the value of one-SEM largely corresponded with a MCID that was estimated using 0.3 ES as a benchmark, which is in further support of the one-SEM as an indicator of MCID (Table\u00a09). However, it should be realized that the SEM as well as the ES are both only statistical indicators, which relate change to random (error) variance. Interestingly, the anchor-based methods yielded similar estimates for the MCIDs (Graphs 1a, b, 2), which is in agreement with recent observations that one-SEM equals anchor-based MCID in patients with moderately severe illness [90]. By applying and comparing multiple methods as well as two evaluation periods, we have not only been able to demonstrate consistency in responsiveness but also to give ranges for minimally clinical important changes instead of point-estimates. As there is no \u2018golden standard\u2019 for the assessment of responsiveness in FHS and HRQoL measurement, a range of scores gives a more realistic reflection of responsiveness than a point-estimate. Point estimates can be misapplied by users who are either unaware of the limited precision of data used for estimating the MCID or who are unaware of the intrinsic limitations of dichotomising what is actually a continuum.\nGeneric versus disease-specific questionnaires\nAlthough generic questionnaires are generally expected to be less sensitive to differences in FHS or HRQoL than disease-specific questionnaires [19, 37, 91, 92], in this study most disease-specific questionnaires performed only marginally better than the generic questionnaires on the discriminant validity test. Likewise, the responsiveness of generic questionnaires, and their usefulness as measures of outcome in randomized trials has been questioned [21]. Although in some studies generic measures indeed were found to be less responsive to treatment effects than specific measures [93\u201396], other studies did find comparable responsiveness [97\u201399]. In this study, only the smaller effect sizes for the FSQ Generic and FSQ Specific may indicate that sensitivity to responsiveness of generic questionnaires is somewhat poorer than that of disease-specific questionnaires. Possibly, this higher sensitivity at the start of the study reflects the higher incidence of symptoms and functional limitations that are specific to AOM, whereas during the study AOM incidence decreases and consequently AOM symptoms become less prominent compared to other health problems. Overall, the generic questionnaires appeared to be as sensitive to clinical change as disease-specific questionnaires, except for the TAIQOL.\nFor the FSQ Generic and FSQ Specific, but not for the RAND which assesses general health perceptions, sensitivity to differences and change in FHS could be explained by their content, as they include many physical and emotional behaviour items that may be affected by rAOM. The more relevant a questionnaire is to a particular condition, the more sensitive it is likely to be. The sensitivity of the RAND, assessing general health and resistance to illness, may indicate that it meets the perceptions of the caregivers of children with rAOM in thinking that their overall health is worse compared with other children. It also may reflect the significant co-morbidity like chronic airway problems and atopic symptoms in the study population (Table\u00a03).\nThe reasons for the poor performance of the TAIQOL with regard to both discriminant validity and sensitivity to change are not obvious. Possibly the subscale scores represent each an aspect of HRQoL that is too limited to be sensitive to differences or change. Combining the subscales to more comprehensive constructs may then improve sensitivity. In addition, each item of the TAIQOL consists of two questions; a question about FHS is followed by the request to rate the child\u2019s well-being in relation to this health status. Response shift bias may have modified the caregivers\u2019 expectations about how their child feels in line with the child\u2019s changing health, that is caregivers may rate their child\u2019s well-being as better than it actually is as they adapt to the situation. Studies on factors that may influence sensitivity to change or responsiveness besides the type of questionnaire (generic versus disease-specific), such as questionnaire structure and content, disease severity, co-morbidity and other population characteristics, are needed.\nBias and generalisibility\nThere are several issues that need to be considered when interpreting the current results. First, frequency of AOM episodes at enrolment was based on proxy report, whereas during the trial only physician-diagnosed episodes were counted. The number of AOM episodes in the year prior to inclusion is likely to be overestimated by proxies [100], resulting in the underestimation of HRQoL change scores because they may have evaluated the situation as worse than it objectively was in the first place. However, if such a recall-bias regarding AOM frequency was in fact present, it may also have influenced caregivers\u2019 reflection on subjective measures such as FHS and HRQoL, which results in realistic or even overestimated change scores. However, estimating responsiveness for the interval of 7\u201314\u00a0months, in which AOM frequency was not affected by recall bias since al episodes were physician diagnosed, yielded similar results. This indicates that recall bias appears not to have influenced responsiveness substantially.\nSecondly, in assessing test\u2013retest reliability, two different modes of questionnaire administration were used: completion at the clinic versus home completion. The possible intention to give more socially desirable answers at the clinic as well as other effects such as being more distracted when filling in the questionnaires at home, may have caused differences in questionnaire scores between the first (test) and second (retest) assessment. Although this impact may be larger for single item questionnaires such as the NRSs compared to multiple item questionnaires, and might explain their somewhat smaller ICCs, the impact on the ICCs appears to be small.\nThirdly, during the trial, 8 children (4.2%) in the pneumococcal vaccine group and 13 (6.7%) in the control vaccine group were lost to follow-up. One child switched from the control to the pneumococcal vaccine group. It is unlikely that these small numbers of dropouts and crossovers influenced the trial results.\nFurthermore, indices of validity and reliability are not fixed characteristics of FHS and HRQoL questionnaires but are influenced by the study design, intervention, and study population in particular. Our study population had significantly severe ear disease with frequent episodes and was older than the average child with AOM. Assessment of reliability and validity of the questionnaires in populations with less severe disease may present more ceiling effects and lack of discriminant validity. Therefore, the results of this study should only be generalized to paediatric populations with moderately to seriously severe recurrent acute ear-infections at an older age (approximately 14\u201354\u00a0months).\nFinally, of all questionnaires in this study, only the FFQ demonstrated a reliability that meets the minimal required reliability coefficients for individual assessment of HRQoL. Although some authors suggest to use FHS and HRQoL questionnaires for individual assessment in clinical practice as well [31], we do not support this approach. It is suggested that routine use of these questionnaires would facilitate detection and discussion of psychological issues and help guide decisions regarding, for example, referral. However, considering the complexity and many pitfalls of reproducibility and responsiveness assessment, individual use of HRQoL and FHS questionnaires as part of the follow-up of individuals is not reliable nor valid.\nRecommendations for clinical use\nIn conclusion, generic (RAND, FSQ Generic and FSQ Specific) as well as disease-specific (OM-6, FFQ, and, to a lesser extent, NRS Caregiver) questionnaires demonstrated similar and high reliability and adequate construct and discriminant validity as well as responsiveness to justify use in clinical studies of children with rAOM. However, NRS as used in this study may be less adequate for assessment of HRQoL in this population. The TAIQOL, the only true generic HRQoL questionnaire, unfortunately showed a poor discriminant validity and sensitivity to change, needing extensive revision before further use in clinical outcome studies in children with otitis media. Using both a generic questionnaire (RAND or FSQ) and the OM-6 in clinical studies regarding FHS in children with rAOM is recommended, as it would combine the merits of both generalisability and sensitivity in outcome assessment and facilitate head-to-head comparisons of their performance in various paediatric populations with OM.\nMore studies are needed assessing responsiveness of paediatric QoL questionnaires by multiple, distribution as well as anchor-based, methods to increase our appreciation of minimal clinically important changes in various paediatric conditions. Further studies on factors such as questionnaire structure and content, disease severity, co-morbidity and other population characteristics that may influence sensitivity to change or responsiveness besides the type of questionnaire (generic versus disease-specific) may increase our appreciation of the complex dynamics in HRQoL and FHS assessment.","keyphrases":["reliability","validity","functional health status","quality of life","acute otitis media","responsiveness","childhood infection"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Purinergic_Signal-3-4-2072921","title":"Mapping P2X and P2Y receptor proteins in striatum and substantia nigra: An immunohistological study\n","text":"Our work aimed to provide a topographical analysis of all known ionotropic P2X1\u20137 and metabotropic P2Y1,2,4,6,11\u201314 receptors that are present in vivo at the protein level in the basal ganglia nuclei and particularly in rat brain slices from striatum and substantia nigra. By immunohistochemistry-confocal and Western blotting techniques, we show that, with the exception of P2Y11,13 receptors, all other subtypes are specifically expressed in these areas in different amounts, with ratings of low (P2X5,6 and P2Y1,6,14 in striatum), medium (P2X3 in striatum and substantia nigra, P2X6,7 and P2Y1 in substantia nigra) and high. Moreover, we describe that P2 receptors are localized on neurons (colocalizing with neurofilament light, medium and heavy chains) with features that are either dopaminergic (colocalizing with tyrosine hydroxylase) or GABAergic (colocalizing with parvalbumin and calbindin), and they are also present on astrocytes (P2Y2,4, colocalizing with glial fibrillary acidic protein). In addition, we aimed to investigate the expression of P2 receptors after dopamine denervation, obtained by using unilateral injection of 6-hydroxydopamine as an animal model of Parkinson\u2019s disease. This generates a rearrangement of P2 proteins: most P2X and P2Y receptors are decreased on GABAergic and dopaminergic neurons, in the lesioned striatum and substantia nigra, respectively, as a consequence of dopaminergic denervation and\/or neuronal degeneration. Conversely, P2X1,3,4,6 on GABAergic neurons and P2Y4 on astrocytes augment their expression exclusively in the lesioned substantia nigra reticulata, probably as a compensatory reaction to dopamine shortage. These results disclose the presence of P2 receptors in the normal and lesioned nigro-striatal circuit, and suggest their potential participation in the mechanisms of Parkinson\u2019s disease.\nIntroduction\nIt is now well established that the arrangement of ionotropic P2X and metabotropic P2Y receptors [1, 2] on a cell membrane is a very dynamic process, often related to developmental or physiopathological conditions. Moreover, it is common knowledge that multiple P2 proteins are simultaneously recruited on a cell membrane for triggering biological functions. As a consequence, P2 receptors are rightly considered more than the sum of their single entities and must be therefore regarded as a complex network of cooperating receptors. Under this perspective, a numerical model was also introduced, the combinatorial receptor web model, which explains the biological efficacy of combining an assorted array of different P2 proteins on a given cell, in order to integrate, upgrade, guarantee and optimize specific receptor-dependent functions [3]. \nThis trend of course applies to the central nervous system (CNS) as well, where in situ hybridization of P2\u00a0mRNA subtypes and immunohistochemistry of P2 proteins shows, for instance, wide but heterogeneous simultaneous distribution of both P2X [4\u201311] and P2Y [12\u201315] classes of receptors. In particular, P2X2,4,6 and P2Y1 subtypes are abundant and widespread approximately in the entire brain, while P2X1 protein is enriched in the cerebellum, P2X3 in the brain stem, and P2X7 is largely prejunctional. The hippocampus concurrently expresses all P2X and, moreover, P2Y1,2,4,6,12 receptor subtypes. Particularly in the basal ganglia (BG), neostriatal medium-spiny neurons and cholinergic interneurons highly express P2X2 and P2Y1 receptors, but it appears that they become functional only under certain, as yet unknown, conditions [16]. Moreover, P2X2 receptor protein was described in substantia nigra pars compacta (SNC) [17], whereas both protein and mRNA were described in SNC and striatum [18]. Finally, only very low levels of P2X4,6 mRNAs were detected in substantia nigra (SN) and striatum [19]. \nBy functional analysis, ATP release was demonstrated from cultured embryonic neostriatal neurons [20], and ATP-evoked potassium currents in rat striatal neurons were shown to be mediated by P2 receptors [21]. ATP was also proved to increase extracellular dopamine levels in rat striatum through stimulation of P2Y subtypes [22], although it was claimed to inhibit dopamine release in the neostriatum [23]. Extracellular ATP via P2 receptors was finally reported to induce neurotoxicity in vitro [24] and in vivo [25] in the striatum. Besides P2 receptors on neurons, in BG there is also evidence of P2 receptors on, and release of ATP from, glial cells. P2Y12 subtype is present, for instance, on oligodendrocytes in striatum and SN [26], and P2X7 receptor is upregulated on microglia in striatum after middle cerebral artery occlusion [27]. In spite of these results, there is a general paucity of studies addressing the cellular distribution of all P2 receptor proteins in BG.\nOur work thus aimed to provide the complete topographical analysis of known P2X and P2Y subtypes that are present in rat striatum and SN in vivo, and to investigate the dynamic presence of P2 proteins after the induction of experimental parkinsonism by dopamine-denervation achieved by using the unilateral 6-hydroxydopamine (6-OHDA) rat model. By upgrading the current map of P2 receptors expressed in the brain, our study discloses the potential impact of these receptors in the normal and lesioned nigro-striatal circuit.\nMaterials and methods\nHistological procedures\nWistar rats (Harlan, Udine, Italy) were anesthetized by i.p. injections of sodium pentobarbital (60\u00a0mg\/kg), and transcardially perfused with saline (0.9 % NaCl) followed by 4% paraformaldehyde, in phosphate buffer (PB, 0.1\u00a0M pH 7.4). Each brain was immediately removed, post-fixed in the same fixative for 2\u00a0h, and then transferred to 30% sucrose in PB at 4\u00b0C, until it sank. The experimental protocol used in this study was approved by the Italian Ministry of Health and was in agreement with the guidelines of the European Communities Council Directive of November 24, 1986 (86\/609\/EEC) for the care and use of laboratory animals. All efforts were made to minimize the number of animals used and their suffering.\nDouble immunofluorescence\nTransverse sections (40-\u03bcm thick) were cut on a freezing microtome and were processed for double immunofluorescence studies. Non-specific binding sites were blocked with 10% normal donkey serum in 0.3% Triton X-100, in phosphate buffered saline (PBS) for 30\u00a0min at room temperature. The sections were incubated in a mixture of primary antisera for 24\u201348\u00a0h in 0.3% Triton X-100 in PBS. Rabbit anti-P2r (1:300, Alomone, Jerusalem, Israel) was used in combination with either mouse anti-calbindin-D-28K (1:200, Sigma, Mi, Italy), mouse anti-tyrosine hydroxylase (TH, 1:500, Sigma), mouse anti-parvalbumin (1:200, Chemicon International, Temecula, CA, USA), mouse anti-glial fibrillary acidic protein (GFAP) (1:400, Sigma), mouse anti-myelin basic protein (MBP, 1:200, Chemicon International), mouse anti-neurofilament H non-phosphorylated (SMI 32, 1:500, Sternberger Monoclonals, Lutherville, MD, USA), mouse anti-neurofilament H and M non-phosphorylated (SMI 33, 1:500, Sternberger Monoclonals), mouse anti-neurofilament 160 (NF160, 1:500, Sigma) or goat anti-neurofilament-L protein (NF-L, 1:100, Santa Cruz, Mi, Italy). The secondary antibodies used for double labeling were Cy3-conjugated donkey anti-rabbit IgG (1:100, red immunofluorescence, Jackson Immunoresearch, West Baltimore Pike, PA, USA), Cy2-conjugated donkey anti-mouse IgG (1:100, green immunofluorescence, Jackson Immunoresearch) or Cy2-conjugated donkey anti-goat IgG (1:100, green immunofluorescence, Jackson Immunoresearch). \nThe sections were washed in PBS three times for 5\u00a0min each, and then incubated for 3\u00a0h in a solution containing a mixture of the secondary antibodies in 1% normal donkey serum in PBS. After rinsing, the sections were mounted on slide glasses, allowed to air dry and coverslipped with gel\/mount anti-fading medium (Biomeda, Foster City, CA, USA).\nConfocal microscopy\nDouble- or triple-label immunofluorescence was analyzed by means of a confocal laser scanning microscope (CLSM) (LSM 510, Zeiss, Arese, Mi, Italy) equipped with argon laser emitting at 488\u00a0nm, helium\/neon laser emitting at 543\u00a0nm, and helium\/neon laser emitting at 633\u00a0nm. Specificity of the antibodies was positively proved by performing confocal analysis in the absence of the primary antibodies, but in the presence of either anti-rabbit or anti-mouse secondary antibodies. Specificity was further confirmed for the P2r antiserum by performing immunoreactions in the simultaneous presence of the P2r neutralizing immunogenic peptides.\nIsolation of cerebral areas and protein extraction\nWistar rats were anesthetized by i.p. injections of sodium pentobarbital (60\u00a0mg\/kg) and, after decapitation, brains were removed. Each brain was transversally cut on a vibratome (300\u00a0\u03bcm). The specific cerebral areas were isolated with the aid of a dissection microscope and homogenized in RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS containing protease inhibitors). After short sonication, the homogenates were incubated on ice for 1\u00a0h and centrifuged at 14,000\u00a0r.p.m. for 10\u00a0min at 4\u00b0C. Protein quantification was performed in the supernatants by Bradford colorimetric assay (Biorad, Milan, Italy).\nWestern blot analysis\nEqual amounts of cell lysate (20\u201330\u00a0\u03bcg of protein from each cerebral area) were separated by electrophoresis on 10\u201312% SDS-PAGE and transferred to nitrocellulose membranes Hybond-C extra (Amersham Biosciences, Cologno Monzese, Italy). The filters were pre-wetted in 5% non-fat milk in TBS-T (10\u00a0mM Tris pH 8, 150\u00a0mM NaCl, 0.1% Tween 20) and hybridized overnight with P2X1,2,4 antisera (Alomone, 1:500), P2X5 and P2Y4\/14 (1:200), P2Y6 (1:300) or P2Y2 (1:400). The antisera were immunodetected with an anti-rabbit HRP-conjugated antibody (1:5,000) and developed by ECL chemiluminescence (Amersham Biosciences), using Kodak Image Station (KDS IS440CF).\nAnti-P2r specificity\nThe polyclonal P2r antisera used in this study were raised against P2r highly purified peptides (identity confirmed by mass spectrography and amino acid analysis, as indicated in the certificate of analysis provided by the manufacturer), corresponding to specific epitopes not present in any other known protein. The specificity of the P2r signals was moreover assessed by incubating Western blots either in the absence of the primary antiserum, or in the presence of the primary antiserum together with the neutralizing P2r immunogenic peptides (\u03bcg protein ratio 1:1 between peptide and antiserum).\n6-OHDA lesion and Nissl staining\nDeeply anesthetized rats (45\u00a0days old, about 150\u00a0g body weight) were injected with 8\u00a0\u03bcg\/4\u00a0\u03bcl 6-OHDA in saline 0.1% ascorbic acid in the medial forebrain bundle (stereotaxic coordinates ap\u2009=\u2009\u22124.4; l\u2009=\u2009+1.2; vd\u2009=\u2009\u22127.8, see also Paxinos et al. [28]) at a rate of 0.38\u00a0\u03bcl\/min. Fifteen days later, the lesioned rats were tested with 0.05\u00a0mg\/kg s.c. of the D1\/D2 dopamine agonist apomorphine, in order to verify the efficacy of the 6-OHDA lesion, and contralateral turns to the lesion were counted for 40\u00a0min. Only those rats that made at least 200 contralateral turns were used for the study. It has been previously demonstrated that rats meeting this screening criterion have greater than 95% depletion of striatal dopamine [29]. At 1.5\u00a0months after the 6-OHDA lesion, rats were used for immunohistological experiments (n\u2009=\u20093). In order to evaluate cell damage, 40-\u03bcm rat brain sections were mounted onto gelatinized slides. They were dehydrated through alcohols, and then rehydrated and stained in 2% cresyl violet for 45\u00a0min. Following deionized water rinses, the slides were dehydrated in a standard alcohol series, cleared in xylene, and coverslipped.\nResults\nP2X and P2Y receptor proteins in rat striatum\nWe describe in this work the cellular and subcellular in vivo distribution of P2X and P2Y receptors in transverse sections of adult rat striatum, showing by double immunofluorescence confocal analysis that the various P2 subtype proteins are distinguished by different degrees of expression and are not uniformly distributed throughout the entire tissue (Fig.\u00a01).\nFig.\u00a01P2X and P2Y receptor proteins in rat striatum. Transverse sections through the striatum of adult rats were processed for double immunofluorescence studies. Rabbit polyclonal antisera against P2X1,2,4 and P2Y4 receptors (red Cy3 immunofluorescence) were used in combination with antibodies against neuronal or glial markers (green Cy2 immunofluorescence). Panel A P2X1: confocal images illustrate clear colocalization of P2X1 receptor with neurofilament-light protein (NF-L). The merged field of inset a shows absence of colocalization between the neuronal GABAergic marker calbindin (green) (a calcium-binding protein expressed mainly in medium spiny neurons of the striatum) and P2X1 receptor (red). The merged field of inset b shows the merged field of P2X1 (red) and MBP (green) overlapping immunoreactivities at higher magnification. Panel B P2X2: double immunofluorescence demonstrates that P2X2 receptor immunoreactivity (red) colocalizes with calbindin protein (green). The insets e and f show colocalization with the neuronal GABAergic marker parvalbumin (green) (a calcium-binding protein that is expressed in interneurons of the striatum). Panel C P2X4: red immunofluorescence for P2X4 protein merges with the green signals of the three types of neurofilament proteins: NF-L (inset g, merged field), NF160 (inset i, merged field) and SMI 32 and, moreover, with parvalbumin (inset h, merged field) and calbindin (inset i, merged field). Panel D P2Y4: red P2Y4 immunoreactivity is present on calbindin-positive neurons (green), on parvalbumin-positive neurons (green) (insets n\u2013q), and on GFAP-positive astrocytes (inset o, merged field). Western blot analysis also confirms the presence of receptor proteins P2X1,2,4 (insets c in panel A, d in panel B, m in panel C, respectively) and P2Y4 (inset r in panel D) in striatum. Specificity of the P2 receptor signals was assessed by incubations of the primary antisera with the corresponding neutralizing immunogenic peptides (\u03bcg protein ratio 1:1 between peptide and antiserum). Scale bars are 10\u00a0\u03bcm in A; 100\u00a0\u03bcm in inset a; 2\u00a0\u03bcm in inset b; 50\u00a0\u03bcm in B and in insets e and f; 20\u00a0\u03bcm in C; 10\u00a0\u03bcm in insets h, i, l; 5\u00a0\u03bcm in inset g; 50\u00a0\u03bcm in D; 5\u00a0\u03bcm in inset n; and 20\u00a0\u03bcm in inset o. Similar results were obtained in at least four independent experiments\nIn particular, a strong P2X1 receptor immunoreactivity (red) confers a patchy appearance to the striatum, being localized mainly in white matter, while sparing the projecting calbindin-positive GABAergic neurons that are highly enriched in gray matter (Fig.\u00a01A, inset a, green). Moreover, P2X1 protein immunofluorescence is present on NF-L positive, transversally oriented neuronal fibers, although the merged field provides only partial colocalization between the two signals (Fig.\u00a01A). In addition, the high magnification analysis (Fig.\u00a01A, inset b) of P2X1 (red) and MBP (green) immunoreactive signals shows that P2X1 receptor is surrounded by MBP, proving the presence of P2X1 protein on myelinated fibers. Due to the close vicinity of the two signals, overlapping yellow immunofluorescence is also observed. Finally, P2X1 receptor in striatum is recognized by Western blot analysis as a single protein band of 60\u201365\u00a0kDa, additionally abolished in the presence of the P2X1 receptor\u2013neutralizing immunogenic peptide (Fig.\u00a01A, inset c).\nConversely, an abundant P2X2 receptor immunoreactivity (red) is found in gray matter of striatum (Fig.\u00a01B), while sparing the bundles of white matter. Specific receptor immunolabeling is present not only on the highly expressed calbindin-positive projecting GABAergic neurons, but also on the fewer parvalbumin-positive GABAergic interneurons (Fig.\u00a01B, insets e, f). By Western blot analysis, we show that P2X2 receptor is present in striatum under two isoforms of about 45 and 32\u00a0kDa, furthermore, it is abolished in the presence of the P2X2 receptor\u2013neutralizing immunogenic peptide (Fig.\u00a01B, inset d).\nP2X3 receptor immunostaining in striatum is of medium intensity (Table\u00a01), and mainly localizes on GABAergic neurons of gray matter (data not shown).\nTable\u00a01Map of P2 receptor proteins in striatum and substantia nigra\u00a0StriatumSubstantia nigraP2X1++++++P2X2++++++P2X3++++P2X4++++++P2X5++++P2X6+++P2X7++++P2X1+++P2X2++++++P2X4++++++P2X6++++P2X11\u2013\u2013P2X12++++++P2X13\u2013\u2013P2X14\u2013+++Relative abundance of all P2X and P2Y receptor proteins was analyzed by confocal immunofluorescence microscopy, as described in \u201cMaterials and methods\u201d. The intensity of the specific immunostaining was scored as follow: \u2013 = not detected; + = just sufficient to evaluate presence and outline of positive cells; ++ = adequate to assess morphological features of cell bodies and\/or cellular processes; +++ = very bright\nP2X4 receptor signal is instead very copious in white matter, although present on a few fibers of gray matter as well (Fig.\u00a01C, red). It partially colocalizes with all types of heavy-, light- and medium-chain neurofilament proteins (merged fields): SMI 32 (green), NF-L (inset g), and NF160 (inset i). Moreover, we find P2X4 protein also on GABAergic interneurons (inset h) and GABAergic spiny neurons (inset l). By Western blot analysis, we demonstrate that P2X4 receptor is present in striatum as a single band of about 60\u00a0kDa, moreover, it is abolished in the presence of the P2X4 receptor\u2013neutralizing immunogenic peptide (Fig.\u00a01C, inset m).\nP2X5,6,7 and P2Y1 receptor immunoreactivities in striatum are very weak (Table\u00a01) in gray matter, although totally absent from white matter under our experimental conditions (data not shown).\nThe P2Y2 receptor is highly expressed in striatum on axons of white matter and astrocytes of gray matter (Table\u00a01). Moreover, it is detected as a double protein band in the 55\u201365\u00a0kDa range (data not shown).\nA strong P2Y4 receptor immunoreactivity is present only in gray matter of striatum, localized on both types of GABAergic neurons: calbindin-positive (Fig.\u00a01D) and parvalbumin-positive (insets n\u2013q). Nevertheless, the receptors are also widespread throughout the striatum on astrocytes, as shown by colocalization with the GFAP marker (inset o). By Western blot analysis, we prove that P2Y4 receptor is present in striatum as a double band of about 42\u00a0and 85\u00a0kDa (inset r), likely corresponding to the monomeric and dimeric aggregation states of the receptor [30, 31].\nWhile P2Y6 receptor is barely detectable (Table\u00a01) on GABAergic neurons in striatum (data not shown), P2Y11,13,14 receptor proteins were not identified by any means under our experimental conditions (Table\u00a01). Finally, P2Y12 receptor in striatum (Table\u00a01) is abundantly expressed only on oligodendrocytes and myelin sheets, as previously shown [26].\nP2X and P2Y receptor proteins in substantia nigra\nWe conducted a parallel analysis on the cellular and subcellular in vivo distribution of P2X and P2Y receptors in transverse sections of adult rat SN. We showed by double immunofluorescence confocal analysis that the different P2 receptor proteins possess more comparable levels of expression with respect to the striatum, and are also more uniformly, although differently, distributed throughout the entire SNC and SNR (Fig.\u00a02). In particular, strong signals for ionotropic P2X2,5 (red, Fig.\u00a02A,B), P2X1,4 (Table\u00a01), metabotropic P2Y6,14 (red, Fig.\u00a02C,D) and P2Y4 (Table\u00a01), or moderate signals for P2X3,6 and P2Y1 receptors (Table\u00a01) are present on dopaminergic neurons (TH-positive) of SNC. Moreover, P2Y2 and P2Y12 receptors are abundantly expressed in SN (Table\u00a01), but P2Y2 is expressed on axons and astrocytes, and P2Y12 only on oligodendrocytes and myelin sheets [26]. Conversely, in SNR, a weak P2X\/Y receptor immunoreactivity is limited to sparse neuronal bodies, likely identified as GABAergic neurons by colocalization with parvalbumin (data not shown). The presence at the tissue level in SN of ionotropic P2X2,5 (insets a in panel A, and b in panel B of Fig.\u00a02, respectively) and metabotropic P2Y6,14 (insets c in panel C, and d in panel D of Fig.\u00a02, respectively) proteins is confirmed by Western blot analysis performed in all cases in the presence of specific receptor\u2013neutralizing immunogenic peptides. Similarly to the striatum, immunoreactive signals for P2Y11,13 receptors were not identified by any means under our experimental conditions (Table\u00a01).\nFig.\u00a02P2X and P2Y receptor proteins in rat substantia nigra. Double immunofluorescence visualized by confocal analysis was performed in transverse sections through the substantia nigra of adult rats. Strong signals for ionotropic P2X2,5 and metabotropic P2Y6,14 (red Cy3 immunofluorescence) are present on dopaminergic neurons (TH-positive, green Cy2 immunofluorescence) of substantia nigra pars compacta (SNC), whereas in substantia nigra pars reticolata (SNR) P2X\/Y immunoreactivity is limited to sparse neuronal bodies. Western blot analysis confirms the presence in substantia nigra of receptor proteins P2X2,5 (insets a in panel A, and b in panel B, respectively) and P2Y6,14 (insets c in panel C, and d in panel D, respectively). Specificity of the P2 receptor signals was assessed by incubation of the primary antisera with the corresponding neutralizing immunogenic peptides (\u03bcg protein ratio 1:1 between peptide and antiserum).Scale bars in all panels are 50\u00a0\u03bcm. Similar results were obtained in at least four independent experiments\n6-Hydroxydopamine modulates the expression of selected P2 receptors in striatum and substantia nigra\nNo contralateral rotation as a sign of motor deficit was reported in rats before being 6-OHDA-lesioned, but was instead detected after the lesion rotation (data not shown), together with loss of dopaminergic TH-positive neurons only from the ipsilateral hemisphere of SNC (Fig.\u00a03A and insets a, b).\nFig.\u00a036-Hydroxydopamine modulates the expression of selected P2 receptor proteins in striatum and substantia nigra. Staining of rat substantia nigra after 6-hydroxydopamine treatment. Panel A Conventional microscopy images of Nissl staining shows several dopaminergic neurons (arrows) in the contralateral control hemisphere, which are lost (asterisks) in the ipsilateral lesioned hemisphere. Specific ipsilateral dopaminergic lesion of substantia nigra pars compacta (SNC) was also visualized by confocal TH-immunostaining (green) (insets a, b). Panel B Confocal merged yellow images show upregulation of P2X1 receptor protein (red) in parvalbumin-positive GABAergic neurons (green) in the lesioned side of substantia nigra pars reticolata (SNR) of 6-hydroxydopamine-treated rats. Panel C Confocal merged yellow images show a drastic increase in GFAP-positive astrocytes (green) in the lesioned side of 6-hydroxydopamine-treated rats and, correspondingly, an augment of P2Y4 signal (red) (inset c). Scale bars are 100\u00a0\u03bcm in A, B and in insets a, b; 20\u00a0\u03bcm in C; and 10\u00a0\u03bcm in inset c. Similar results were obtained in at least three independent experiments\nConcomitantly, we prove that dopamine denervation in the 6-OHDA-lesioned rat generates a significant and selective rearrangement of P2 receptor proteins. Whereas the expression pattern and immunofluorescence intensities of P2X1,4, P2Y2 (colocalizing with all neurofilaments and present in white matter on fibers projecting from the cortex), and P2Y12 (present on oligodendrocytes of white matter) remain constant in both ipsi- and contralateral hemispheres after 6-OHDA treatment (as well as in control animals), all other P2X and P2Y receptors are decreased on parvalbumin- and calbindin-positive GABAergic neurons of deafferented ipsilateral striatum (but not contralateral and in control animals), as measured by semiquantitative analysis (Table\u00a02) (n\u2009=\u20093).\nTable\u00a02Map of P2 receptor modulation after dopamine denervation\u00a0Ipsilateral Striatum Ipsilateral SNP2X1=\u2191GABAP2X2\u2193GABA\u2193THP2X3\u2193GABA\u2193TH, \u2191GABAP2X4\u2193GABA\u2193TH, \u2191GABAP2X5=\u2193THP2X6=\u2193TH, \u2191GABAP2X7==P2X1=\u2193THP2X2==P2X4\u2193GABA\u2191GFAPP2X6=\u2193THP2X11==P2X12==P2X13==P2X14=\u2193THRelative increase (\u2191) or decrease (\u2193) in P2X and P2Y receptor proteins analyzed by confocal immunofluorascence microscopy in striatum and SN after treatment in rat in vivo with 6-hydroxydopamine (ipsilateral), and in control (not lesioned) brain hemisphere (contralateral).TH= presence in dopaminergic neurons,GABA= presence in GABAergic neurons,GFAP= presence in astrocytes\nSimilarly, all P2X and P2Y receptors are lost in the lesioned (but not contralateral) substantia nigra pars compacta, consequent to the degeneration of the majority of TH-positive dopaminergic neurons (Table\u00a02). Conversely, P2X1 (Fig.\u00a03B) and P2X3,4,6 (Table\u00a02) receptors present on GABAergic neurons, and P2Y4 receptors on astrocytes augment their expression only in ipsilateral substantia nigra pars reticulata adjacent to the lesioned pars compacta. In this same area, a phenomenon of astrogliosis is also induced, as detected by more abundant expression of GFAP-positive astrocytes (Fig.\u00a03C).\nDiscussion\nBecause the roles of ATP in the CNS have received less attention until recently, often due to lack of appropriate research tools, our knowledge of the functional qualification of P2 receptors in the brain is limited, although rapidly improving. As a group of nuclei interconnected with cerebral cortex, thalamus and brainstem, and associated with a variety of functions, such as motor control, cognition, emotions and learning, the BG [32] is an area that deserves thorough analysis. Our work was aimed at mapping in vivo the presence of P2 receptor subtypes in the BG nuclei of striatum and SN by immunofluorescence-confocal and Western blotting techniques. The specificity of the highly sensitive molecular probes used for the detection of all known P2X and P2Y receptor proteins has been previously validated [33, 34]. In addition, we undertook an analysis that excluded possible cross-reactivity for all antisera used. \nOur results not only establish that the majority of P2X (P2X1\u20137) and P2Y (P2Y1,2,4,6,11\u201314) receptors so far cloned from mammalian tissues are found in striatum and SN, but also prove their distinctive localization on neurons and\/or glial cells. In detail we show that, with the exception of only P2Y11 and P2Y13 receptors (whose immunoreactivity was not identified by any means under our experimental conditions), all other subtypes are specifically localized in striatum and SN (both pars compacta and reticulata), although with different levels of expression, rated as low (P2X5,6 and P2Y1,6,14 in striatum), medium (P2X3 in striatum and SN; P2X6,7 and P2Y1 in SN) and high. Moreover, while we show a prevalence of P2 receptors on neurons (P2X1,4 and P2Y2 colocalizing with neurofilament light, medium and heavy chains) with features that are either dopaminergic (P2X2\u20135 and P2Y1,4,6,14 colocalizing with TH, in SN) or GABAergic (P2X2\u20134 and P2Y4 colocalizing with parvalbumin and calbindin, in striatum), we also describe their expression on astrocytes (P2Y2,4 in striatum and SN, colocalizing with GFAP), microglia (P2X7, colocalizing with OX42) [27], and oligodendrocytes (P2Y12, colocalizing with MBP and RIP) [26]. By confirming previous autoradiographic studies [35, 36], our results therefore prove the widespread but diversified P2-receptor protein distribution in striatum and SN, and extend to these nuclei the great level of biological complexity and molecular sophistication pertaining to P2 receptors [3]. \nAlthough the configuration of receptor subunits required for assembly into functional cation channels gated by extracellular ATP in different regions of the CNS comprising the BG is not known yet, colocalization of so many different P2X subtypes in striatum and SN is definitely compatible with heteromultimeric assembly of ionotropic subunits. Since a growing body of biochemical and biophysical evidence now indicates that the propensity to form homo- and especially hetero-multimers is frequent also for G protein-coupled receptors [37] comprising the P2Y subtypes [30, 31], the concurrent expression in striatum and SN of as many metabotropic receptors could explain once more a complex hetero-oligomeric architecture. Nevertheless, the biological phenomenon of redundancy could also justify the simultaneous presence of multiple P2 receptor subtypes in these nuclei, with the final outcome of increasing the structural and pharmacological heterogeneity of these brain regions. Finally, the composite architecture of P2 receptors that we depicted in striatum and SN might likely also signify a multipart mechanism of receptor cooperative behavior (Volont\u00e9 et al., personal communication) that sustains the concomitant level of complexity of this brain area in several tasks, such as planning and modulation of movement pathways, cognitive processes involving executive functions, reward and addiction. These possibilities are, of course, not mutually exclusive.\nStriatal neurons, including the most abundant medium spiny neurons, receive convergent synaptic modulation from nigral dopaminergic neurons and from cortical glutamatergic projections [38]. The present study showing that lesions of nigral dopaminergic neurons do not significantly affect purinergic receptors present on axons of striatum white matter, but do generate a significant overall decrease in P2X and P2Y receptor proteins from striatal spiny neurons and GABAergic interneurons, thus confirms and extends the involvement of P2 receptors and extracellular ATP to the cortex-basal ganglia circuit [21]. Since dopaminergic denervation affects not only the nigrostriatal dopaminergic pathway but, as a consequence, the corticostriatal glutamatergic pathway with an increase in glutamatergic transmission [39\u201341] and extracellular glutamate levels in the striatum [42], the reduced P2 receptor protein expression that we demonstrate in striatum gray matter could thus not only be a direct effect of the nigrostriatal inhibition, but also a cause of de-inhibitory mechanisms occurring in the corticostriatal circuit. In this regard, it is common knowledge that extracellular ATP participates in excitatory neurotransmission in the CNS [43], that release of extracellular ATP occurs in CNS under both normal and pathological conditions [44] and, not least, that glutamate release is induced by extracellular ATP in CNS glutamatergic neurons [45].\nNeurons of the pars compacta responsible for dopamine production in the brain, which we have shown here to completely lose their array of P2 receptors as a consequence of neurodegeneration induced by 6-OHDA treatment, receive inhibiting signals also from neurons of the pars reticulata that produce GABA [46]. Loss of dopamine neurons in the SNC, one of the main pathological features of Parkinson\u2019s disease leading to a marked reduction in dopamine function in the brain, thus also impedes the inhibitory pathway of SNR, with a consequent overactivation of GABAergic neurons. Our findings that specific expression of both ionotropic P2X1,3,4,6 receptors on GABAergic neurons and metabotropic P2Y4 receptors on astrocytes is remarkably increased in SNR after dopamine denervation thus probably reflects a parallel compensatory overreaction of GABAergic neurons to dopamine shortage. One possible explanation is that purinergic mechanisms might thus play a crucial role in the fine-tuned regulation not only of dopaminergic and glutamatergic cross-talk in striatum, as it occurs in nucleus accumbens [47], but also of GABAergic and dopaminergic interplay in SN, as it occurs in the mesolimbic neuronal circuit [48]. This is consistent with the overall versatile functions accomplished by P2 receptors in the CNS under both normal and pathological conditions [43, 44, 49] and, in particular, with the intermediary role in oligodendrocyte-to-neuron [26], Bergmann glia-to-neuron, and neuron-to-neuron communication [50] proposed for P2 receptors in various brain regions.\nIn summary, the importance of our work is twofold. We first provide the complete topographical analysis of all known P2X and P2Y receptor subtypes expressed in vivo at their protein levels in rat striatum and SN, which, when considered alongside functional studies, supports a key role for extracellular ATP as a cotransmitter\/neuromodulator in these brain areas. Then, we prove that dopamine denervation in the 6-OHDA animal model of Parkinson\u2019s disease generates a significant rearrangement of P2 receptor proteins in these nuclei, therefore disclosing the participation of P2 receptors in the lesioned nigro-striatal circuit. While requiring further investigation, our findings indicate a potential but noteworthy pharmacological and therapeutic novel outcome for Parkinson\u2019s disease.","keyphrases":["rat brain","tyrosine hydroxylase","6-hydroxydopamine","parkinson\u2019s disease","purinergic receptors","\u03b3-aminobutyric acid"],"prmu":["P","P","P","P","P","M"]}
{"id":"Diabetologia-4-1-2270360","title":"Best practice guidelines for the molecular genetic diagnosis of maturity-onset diabetes of the young\n","text":"Aims\/hypothesis Mutations in the GCK and HNF1A genes are the most common cause of the monogenic forms of diabetes known as \u2018maturity-onset diabetes of the young\u2019. GCK encodes the glucokinase enzyme, which acts as the pancreatic glucose sensor, and mutations result in stable, mild fasting hyperglycaemia. A progressive insulin secretory defect is seen in patients with mutations in the HNF1A and HNF4A genes encoding the transcription factors hepatocyte nuclear factor-1 alpha and -4 alpha. A molecular genetic diagnosis often changes management, since patients with GCK mutations rarely require pharmacological treatment and HNF1A\/4A mutation carriers are sensitive to sulfonylureas. These monogenic forms of diabetes are often misdiagnosed as type 1 or 2 diabetes. Best practice guidelines for genetic testing were developed to guide testing and reporting of results.\nIntroduction\nMaturity-onset diabetes of the young (MODY) describes the dominantly inherited disorder of non-insulin-dependent diabetes typically diagnosed before 25\u00a0years that was first recognised by Tattersall [1, 2]. MODY is the most common form of monogenic diabetes, accounting for an estimated 1\u20132% of diabetes in Europe [3, 4], but is often misdiagnosed as type 1 or type 2 diabetes.\nThe term MODY is used to describe a group of clinically heterogeneous, often non-insulin-dependent forms of diabetes that are defined at the molecular genetics level by mutations in different genes. All show dominant inheritance and are disorders of beta cell dysfunction, but variable features include the age at onset, severity of the hyperglycaemia (and hence risk of complications) and associated clinical features. The most recent classification of diabetes by the American Diabetes Association and the World Health Organization recognises these discrete subtypes of MODY [5].\nMutations in the GCK and HNF1A genes are the most frequent cause of MODY in all populations studied. They account for approximately 70% of cases (see Table\u00a01). The ratio of GCK to HNF1A mutations varies between countries because of different recruitment strategies for genetic testing; blood glucose screening in young, asymptomatic individuals will identify a higher proportion of GCK mutations.\nTable\u00a01Genes in which mutations cause MODY\u00a0Gene symbol (other symbol)GCKHNF1A (TCF1)HNF4APDX1 (IPF1)NEUROD1HNF1B (TCF2)ProteinGlucokinaseHepatocyte nuclear factor-1 alphaHepatocyte nuclear factor-4 alphaInsulin promoter factor-1Neurogenic differentiation 1Hepatocyte nuclear factor-1 betaChromosome locus7p1312q24.3120q13.1213q12.22q31.317q12Gene Accession no.NM_000162.2NM_000545.4NM_000457.3aNM_000209.2NM_002500.2NM_000458.1OMIM * (Gene)138079142410600281600733601724189907OMIM # (Phenotype)125851600496125850606392606394137920Mutation frequency (%) (not known in ~20% of cases)20\u20135020\u201350~5<1<1~5The Genbank reference sequence NM_000457.3 refers to the full length P1 transcript, which uses exons 1a and 1b, and not 1d. For the full length P2 transcript, which includes exon 1d, there is a human mRNA sequence AY680697 but no refseq. The convention is to use NM_000457.3 for exons 1a\/1b and 2\u201310, with AY680697 for exon 1d only. HNF4A mutation descriptions in the literature use the translation start codon reported by Chartier et al. in 1994 [45]. However, an alternative start codon nine amino acids upstream was proposed in 1996 [46] and this alternative start codon is used in NM_000457.3. Hence the convention is to report HNF4A mutations using the amino acid methionine at codon 10 in NM_000457.3 as the start codon, with the A of this codon as the first nucleotideaNomenclature for the HNF4A gene is complicated because the gene encodes nine isoforms expressed from two promoters. The liver-specific P1 promoter drives the expression of transcripts 1\u20133, which include exons 1a and 2\u201310, and transcripts 4\u20136, which include exons 1a, 1b, 1c and 2\u201310. Transcripts 7\u20139 are expressed from the pancreatic (P2) promoter located approximately 46\u00a0kb upstream of the HNF4A transcription start site and exhibit splicing of the upstream exon 1d to exon 2, without the inclusion of sequences from either exons 1a, 1b or 1c\nHeterozygous loss-of-function GCK mutations result in mild, stable hyperglycaemia from birth. Microvascular complications are rare, reflecting the fact that HbA1c is normally just above the upper limit of the normal range. Treatment with oral hypoglycaemic agents or insulin is not needed because it rarely changes HbA1c [6]. A genetic diagnosis is important for the small number of children misdiagnosed with type 1 diabetes and treated with insulin [7]. The identification of GCK mutations in women with gestational diabetes can be useful for obstetric management, since their babies who do not inherit the mutation are at risk of macrosomia [8], and it can guide follow-up in the mothers.\nTranscription factor mutations in the HNF1A or HNF4A genes cause a similar progressive diabetic phenotype although the penetrance of HNF4A mutations is lower (S. Ellard and A. T. Hattersley, unpublished data). Sensitivity to sulfonylureas means that some patients can transfer from insulin to oral agents [9, 10]. A low renal threshold for glucose is a feature of HNF1A mutations [11] and may provide a useful method of screening at-risk family members during childhood [12].\nMutations identified in the GCK, HNF1A and HNF4A genes include missense, nonsense, splicing, small deletions\/insertions\/duplications, and splice site and promoter region mutations [13, 14]. Partial and whole deletions have recently been reported in HNF1A and GCK [15]. The location of mutations within the HNF1A gene influences the age at diagnosis; the average age at diagnosis for patients with exon 1\u20136 mutations that affect all three HNF1A isoforms is younger than for those with mutations in exons 8\u201310 that affect only isoform HNF1A(A) [16, 17].\nRarer forms of MODY include heterozygous mutations in PDX1 (also known as IPF1; [18, 19]) and NEUROD1 [20, 21], but analysis of these genes is not usually included in routine molecular genetic testing for MODY. Dominantly inherited syndromic forms of diabetes may also be described as MODY subtypes. The renal cysts and diabetes syndrome results from HNF1B mutations, and other features include renal abnormalities, female genital malformations, hyperuricaemia, pancreatic atrophy and abnormal liver function tests [22\u201324]. Mutations in the CEL variable number tandem repeat cause a syndrome of diabetes and pancreatic exocrine dysfunction [25]. These syndromes and maternally inherited diabetes and deafness caused by the mitochondrial m.3243A\u2192G mutation are not included in these guidelines since testing is guided by the non-endocrine pancreatic or extra-pancreatic clinical features.\nA molecular genetic diagnosis of a GCK, HNF1A or HNF4A mutation is important because it confirms a diagnosis of MODY, classifies the subtype, predicts the likely clinical course and may change the patient\u2019s treatment. First-degree relatives will be at 50% risk of inheriting the mutation and asymptomatic individuals may be offered predictive genetic testing (after appropriate genetic counselling) in order to provide reassurance (for those shown not to carry the mutation) or regular blood glucose monitoring with early diagnosis and appropriate treatment (for mutation carriers).\nMethods\nA group of European clinicians and scientists met on 22 May 2007 at a workshop to formulate best practice guidelines for molecular genetic testing in MODY. Discussions focused on clinical criteria for selection of patients for testing, methodologies, interpretation of results and reporting those results to the referring clinicians.\nA draft document was posted on 24 August 2007 and an online editing tool was used by participants to produce consensus guidelines.\nResults\nClinical criteria for testing\nMild fasting hyperglycaemia: testing for GCK mutations The finding of raised fasting blood glucose in the range of 5.5\u20138\u00a0mmol\/l is unusual in children and young adults. This always raises concern that they may be about to develop type 1 diabetes or the patient has type 2 diabetes. However, a considerable proportion of young, non-obese patients with persistent mild fasting hyperglycaemia will have a heterozygous mutation in the GCK gene. In a cohort of 82 children with incidental hyperglycaemia, 43% had GCK mutations [26]. The phenotype associated with GCK mutations is remarkably similar for all mutations. The following features suggest a diagnosis of a GCK mutation:\nThe fasting hyperglycaemia is \u22655.5\u00a0mmol\/l (98% patients), persistent (at least three separate occasions) and stable over a period of months or years [27].HbA1c is typically just above the upper limit of normal and rarely exceeds 7.5%.In an OGTT the increment [(2\u00a0h glucose) \u2212 (fasting glucose)] is small (71% of patients in the large European study reported by Stride et al. [27] had an increment <3\u00a0mmol\/l). An increment of 4.6\u00a0mmol\/l is often used to prioritise testing and corresponds to the 90th centile (S. Ellard and A. T. Hattersley, unpublished data).Parents may have \u2018type 2 diabetes\u2019 with no complications or may not be diabetic. On testing, one parent will usually have a mildly raised fasting blood glucose (range of 5.5\u20138\u00a0mmol\/l) unless the mutation has arisen de novo. Testing of apparently unaffected parents\u2019 fasting glucose is important when considering a diagnosis of a glucokinase mutation.\nGestational diabetes: testing for GCK mutations Mutations of GCK cause mild fasting hyperglycaemia throughout life and this is often diagnosed during pregnancy when routine testing is performed. Since these patients have consistently raised fasting blood glucose levels, their babies who do not inherit the mutation may be macrosomic [28]. The diagnosis of a GCK mutation is important, not only as the child may subsequently be picked up as having a raised fasting blood glucose and this may lead to concern about type 1 diabetes, but also because the guidelines given to the mother are different from the normal \u2018pre-type 2\u2019 diabetic phenotype as they will not deteriorate with time. The following criteria identify when GCK testing is appropriate [29]:\nPersistently raised fasting blood glucose in the range of 5.5\u20138\u00a0mmol\/l before, during and after pregnancy.An increment of <4.6\u00a0mmol\/l on at least one OGTT (either during or after pregnancy).A parent may have mild type 2 diabetes but often this has not been detected and so the absence of family history should not exclude the diagnosis.\nChildren and young adults with diabetes and a strong family history of diabetes: testing for HNF1A mutations The possibility of monogenic diabetes should be considered whenever a parent has diabetes even if they are thought to have type 1 or type 2 diabetes. The most common form of MODY is caused by HNF1A mutations. The clinical characteristics of patients with HNF1A mutations include:\nYoung-onset diabetes (typically before 25\u00a0years old in at least one family member).Non-insulin-dependent outside the normal honeymoon period (3\u00a0years), e.g. not developing ketoacidosis in the absence of insulin, good glycaemic control on less than the usual replacement dose of insulin, or detectable C-peptide measured when on insulin with glucose >8\u00a0mmol\/l.Family history of diabetes (at least two generations). This may be insulin treated and considered to be \u2018type 1\u2019 diabetes or \u2018type 2\u2019 diabetes. At least two individuals within the family would typically be diagnosed in their 20s or 30s. There may also be an affected grandparent, although often these are diagnosed after 45\u00a0years. OGTTs in early stages tend to show a very large glucose increment, usually >5\u00a0mmol\/l [27]. Some individuals may have a normal fasting level but a value within the diabetic range at 2\u00a0h.The absence of pancreatic islet autoantibodies.Glycosuria at blood glucose levels <10\u00a0mmol\/l is often seen, as these patients have a low renal threshold [11].Marked sensitivity to sulfonylureas resulting in hypoglycaemia despite poor glycaemic control before starting sulfonylureas [9, 30].Several features suggesting monogenic diabetes rather than a diagnosis of young-onset type 2 diabetes should be considered: no marked obesity or evidence of insulin resistance in diabetic family members, absence of acanthosis nigricans and whether the family is from an ethnic background with a low prevalence of type 2 diabetes (e.g. of European descent).\nChildren and young adults with diabetes and a strong family history of diabetes: testing for HNF4A mutations Diabetes caused by mutations in the HNF4A gene is considerably less common (Table\u00a01) than HNF1A mutations. The clinical characteristics are similar, except there is not a low renal threshold and the age of diagnosis may be later [31]. HNF4A mutations should be considered when HNF1A analysis does not detect a mutation but the clinical features are strongly suggestive of HNF1A. Patients are often sensitive to sulfonylureas [32]. HNF4A mutations are associated with macrosomia (approximately 56% of mutation carriers) and transient neonatal hypoglycaemia (approximately 15% of mutation carriers) [33, 34]. The possibility of HNF4A mutations should be considered when diabetic family members have marked macrosomia (>4.4\u00a0kg at term) or if diazoxide-responsive neonatal hyperinsulinism has been diagnosed in the context of familial diabetes.\nBabies with diazoxide-responsive neonatal hyperinsulinaemic hypoglycaemia and a strong family history of diabetes: testing for HNF4A mutations Mutations of HNF4A are a cause of neonatal hypoglycaemia that remits during infancy or early childhood, with diabetes developing later in life [34]. Macrosomic babies with diazoxide-responsive hyperinsulinism and a strong family history of diabetes (see characteristic 3 in the section above entitled Children and young adults with diabetes and a strong family history of diabetes: testing for HNF1A mutations) should be considered for HNF4A mutation screening.\nTesting methodology\nThe mutation screening methodology should be described in the report [e.g. sequencing, denaturing high-performance liquid chromatography (dHPLC), conformation-sensitive capillary electrophoresis (CSCE)] together with the sensitivity. PCR primers should be checked for primer binding site single nucleotide polymorphisms (SNPs; a useful tool is available at http:\/\/ngrl.man.ac.uk\/SNPCheck\/index.html). Gene dosage analysis may be useful if a diagnosis of MODY is strongly suspected and no mutation is found on mutation screening.\nInterpretation of results\nThe textbox includes recommended interpretations for the most common reporting scenarios.\nReporting\nEach laboratory has its own reporting format and general guidance on reporting is available from the European Molecular Genetics Quality Network (http:\/\/www.emqn.org), the UK Clinical Molecular Genetics Society (http:\/\/www.cmgs.org) and the Swiss Society of Medical Genetics (http:\/\/www.ssgm.ch). A one page report is the preferred format.\nThe report should state the methodology and specify the gene, exons and\/or mutations tested for. If promoter sequences are examined then the report should specify the nucleotides analysed. An estimation of the assay sensitivity is particularly useful for pre-screening techniques such as dHPLC, CSCE etc. The use of mutation nomenclature approved by the Human Genome Variation Society (http:\/\/www.hgvs.org\/mutnomen) is strongly recommended. The gene accession number (with version) is required in order to describe mutations unambiguously (see Table\u00a01). The A nucleotide of the ATG start codon is numbered +1.\nReports describing novel variants should state that the variant is novel and include the evidence in support of pathogenicity. This might include the absence from a large series of ethnically matched controls or MODY patients (testing of 210 normal chromosomes is necessary to achieve at least 80% power to detect a polymorphism present in 1% of the population [35]). Testing of other affected relatives is recommended in order to check for co-segregation and to calculate the LOD score in suitable sized pedigrees (LOD scores of \u22651 or \u22653 are suggestive or conclusive of linkage, respectively).\nFor missense variants the evidence for pathogenicity might include conservation across species and a significant amino acid substitution. Several programs are available that predict the pathogenicity of a missense variant based upon amino acid conservation (SIFT; http:\/\/www.blocks.fhcrc.org\/sift\/SIFT.html) or the structure and function of the protein (PolyPHEN; http:\/\/www.genetics.bwh.harvard.edu\/pph) but they should be used to supplement other pieces of evidence rather than in isolation.\nBoth missense and silent variants can affect splicing if the mutation is within an exon splicing enhancer or exon splicing silencer. Splice predictor software programs (http:\/\/www.fruitfly.org or http:\/\/rulai.cshl.edu\/cgi-bin\/tools\/ESE3\/esefinder.cgi?process=home) may aid interpretation. Base substitutions affecting the conserved splice donor (GT) site, splice acceptor (AG) site or the conserved A nucleotide within the branch site are highly likely to be pathogenic but splice predictor software may be useful in the interpretation of other intronic variants. Analysis of patient mRNA is often informative but lymphoblastoid cell lines are usually required because of the low levels of expression of the MODY genes in blood. Sequence analysis of RT-PCR products amplified from lymphoblastoid cell mRNA has demonstrated exon skipping, retention and the use of cryptic splice sites for a variety of intronic mutations in the GCK, HNF1A and HNF1B genes [36\u201338].\nNovel promoter variants may be investigated by examination of known transcription binding sites or by in vitro transfection experiments [39\u201342]. They may also alter mRNA expression levels, which may be measured by allele-specific real-time PCR [43].\nPolymorphisms\nSome laboratories include details of polymorphisms detected in the report. The reasons for doing this include: (1) making all data available to the requesting clinician based on the rationale that a polymorphism may later be reclassified as a mutation; and (2) identifying heterozygous SNPs excludes a gene deletion involving the exon(s) in question.\nHowever, this information can cause confusion or even misinterpretation of the result (A. T. Hattersley, unpublished data) and it certainly adds to the length of the report. While in some cases there may be reports in the literature of an association with type 2 diabetes or reduced insulin secretion, these polymorphisms do not cause MODY and we recommend that they should be excluded from the report.\nTreatment\nIndividual treatment recommendations are outside the jurisdiction of a molecular genetics report since this is the referring clinician\u2019s responsibility. It is useful to include an appropriate reference if there is evidence in the literature for a particular treatment (e.g. low-dose sulfonylureas in HNF1A\/4A MODY) associated with the genetic diagnosis.\nOther issues\nGenetic counselling should be provided for all asymptomatic individuals requesting predictive testing. We recommend that unaffected relatives are offered a biochemical test first (fasting blood glucose for GCK mutations or OGTT for HNF1A\/HNF4A mutations). If the biochemical test is consistent with a diagnosis of diabetes or hyperglycaemia then the genetic test will be diagnostic, not predictive.\nFor families requesting predictive testing for children too young to provide informed consent, referral to a specialist clinical genetics unit (or equivalent) is strongly recommended. Reasons for testing children include (1) to remove the uncertainty around the child\u2019s status, and (2) to assist with management, as a negative test would mean that monitoring of blood glucose\/glycosuria would not be necessary [44].\nConclusions\nMolecular genetic testing is useful in patients with MODY because it confirms a diagnosis of monogenic diabetes, predicts likely clinical course, defines risk for relatives and determines treatment.\nAt the present time, molecular genetic testing for MODY is relatively expensive and phenotypic selection prior to testing is normal practice. With the development of new technologies it is likely that these costs will decrease in time and that the analysis of genes associated with monogenic diabetes may become routine for all newly diagnosed patients. In the meantime we hope that these guidelines will be useful in determining which patients should be offered testing, and in the interpretation and reporting of the test results.\nBelow is the link to the electronic supplementary material.\nESM\n(PDF 12.9\u00a0kb)","keyphrases":["best practice","maturity-onset diabetes of the young","gck","hnf1a","hnf4a","mody","monogenic diabetes"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Intensive_Care_Med-4-1-2228379","title":"Short-term beneficial effects of methylene blue on kidney damage in septic shock patients\n","text":"Objective We previously demonstrated that upregulation of renal inducible nitric oxide synthase (iNOS) is associated with proximal tubule injury during systemic inflammation in humans. In this study we investigated the short-term effect of methylene blue (MB), an inhibitor of the NO pathway, on kidney damage and function in septic shock patients.\nIntroduction\nRefractory septic shock remains the major cause of death in noncoronary intensive care units, especially when accompanied by multiple organ failure, with an estimated mortality rate of 50\u201360%\u00a0[1]. The incidence of acute renal failure in refractory septic shock is approx. 40\u201350%\u00a0[2]. Nitric oxide has emerged as an important contributory factor to the pathogenesis of septic shock. We previously demonstrated that induction of renal NO is associated with proximal tubule injury during systemic inflammation in humans\u00a0[3]. NO stimulates soluble guanylate cyclase (sGC) by binding to its heme moiety, which generates cyclic guanosine monophosphate (cGMP)\u00a0[4]. In the kidney NO and cGMP production are associated with lipopolysaccharide-induced renal proximal tubular cell toxicity\u00a0[5]. Selective sGC inhibition during septic shock in rats resulted in an attenuation of renal dysfunction\u00a0[6], indicating that blocking sGC may be a\u00a0potential therapeutic strategy to treat septic shock-associated renal failure.\nMethylene blue (MB) binds to sGC, blocks cGMP production and has the ability to scavenge NO and to inhibit NO synthases\u00a0[7, 8]. Although several controlled and uncontrolled clinical studies showed beneficial effects of MB on the hemodynamic instability during septic shock (reviewed in\u00a0[9]), there are no studies on the putative protective renal effects. We examined the short-term effects of continuous infusion of low-dose MB (1\u202fmg\/kg per hour) in patients with septic shock on urinary excretion of the acute kidney injury markers, cytosolic glutathione S-transferases (GSTs) present in proximal tubule (GSTA1-1) and distal tubule (GSTP1-1)\u00a0[10].\nMaterial and methods\nPatients\nNine patients received a\u00a04\u202fh continuous infusion of 1\u202fmg\/kg per hour MB (1% w\/v) provided by the VieCuri Medical Center pharmacy (for inclusion criteria see Electronic Supplementary Material, ESM). Arterial blood and catheterized urine were collected at several time points during the first 24\u202fh. Clinical parameters, the severity of illness using Acute Physiology and Chronic Health Evaluation II (APACHE II), and Sepsis-Related Organ Failure Assessment (SOFA) were recorded.\nChemical assays\nBiochemical parameters were determined by routine clinical chemistry. Hemoglobin, methemoglobin, and bilirubin were measured to assess possible side effects of MB, such as hemolytic anemia and methemoglobinemia\u00a0[11\u201313]. Total amount of the stable NO metabolites, nitrate and nitrite, measure of NO radicals production, and the amounts of GSTA1-1 and GSTP1-1 in urine were assayed as described\u00a0[3]. The blue color in urine due to MB excretion did not affect the chemical assays.\nStatistical analysis\nValues are given as mean\u202f\u00b1\u202fS.E. or as median (25\u201375% range) depending on their distribution. Differences between experimental groups were tested by analysis of variances for repeated measures. A\u00a0two-tailed p-value less than 0.05 was considered statistically significant.\nResults\nPatients\nSeven patients ultimately died in the intensive care unit, one of refractory shock (within 12\u202fh) and six patients because of multiple organ failure. In the latter group two died within 7\u202fdays and the remaining four within 28\u202fdays after intervention. The mean calculated predicted mortality rate was 61%, and all patients had at least three organ failures, reflecting a\u00a0mean SOFA score of 11.1\u202f\u00b1\u202f0.9. The median stay at the intensive care unit was 16\u202fdays (range 7\u201324); the two survivors stayed 89\u202fdays (52\u2013121) in hospital. Pathogenic organisms isolated by culture and site of infection are illustrated in the ESM. Median C-reactive protein was 178\u202fmg\/l (118\u2013189); all patients had lactic acidemia (median 2.7\u202fmmol\/l, range 2.1\u20133.7) and thrombocytopenia (68\u202f\u00d7\u202f109\/l, 50\u2013104). The median MAP increased slightly by 5\u202fmmHg (2\u201311) from 69 (65\u201370) at baseline to 74 (68\u201382) 3\u202fh after the start of MB (p\u202f<\u202f0.05) with no change in norepinephrine infusion rate. Methemoglobinemia or hemolytic anemia did not develop after MB (data not shown). All MB-treated patients showed blue-coloring of urine and skin.\nMB attenuates NO formation\nThe concentration NO metabolites in plasma was higher in septic shock patients than in to healthy volunteers\u00a0[3] but did not change after MB administration (Fig.\u202f1a). In contrast, MB significantly attenuated the urinary excretion of NO metabolites by a\u00a0median 90% (75\u201395%, p\u202f<\u202f0.05) from baseline (233\u202f\u03bcmol\/mmol creatinine, 112\u2013536) to 6\u202fh (37, 10\u201387) after the start of MB (Fig.\u202f1b). At 4h the MB infusion was stopped, after which the median excretion of urinary NO metabolites increased by 135% (65\u2013795%, p\u202f<\u202f0.05) within 6\u201324\u202fh after MB treatment (Fig.\u202f1b).\nFig.\u00a01NO metabolites in plasma and urine and the urinary excretion of tubular injury markers glutathione S-transferase (GST) A1-1 and P1-1. NO metabolite levels in plasma (a, n\u202f=\u202f9) and urine (b, n\u202f=\u202f8) and levels of GSTA1-1 (c, proximal tubule, n\u202f=\u202f8) and GSTP1-1 (d, distal tubule, n\u202f=\u202f8) were measured in urine at various times after MB administration in septic shock patients. The urinary excretion of NO metabolites and GSTs was corrected for creatinine excretion. Data are expressed as median with 25\u201375% range and analyzed by analysis of variance with repeated measures over the two time periods. *p\u202f<\u202f0.05 vs. baseline, # p\u202f<\u202f0.05 vs. 6\u202fh after MB treatment\nMB attenuates kidney damage\nAll patients showed impaired renal function with oliguria and mild proteinuria (Table\u202f1). One patient suffered from anuria during the first day. Six patients required continuous venovenous hemofiltration renal replacement therapy with a\u00a0flow of 35\u202fml\/kg (for median 8.5\u202fdays, 4\u201313), of which 3 during the MB infusion. The creatinine clearance improved by median 51% (18\u2013173%, p\u202f<\u202f0.05) during the first 24\u202fh after MB but was still strongly impaired (Table\u202f1). The urinary excretion of both GSTA1-1 and GSTP1-1 was elevated in all septic shock patients, indicating both proximal and distal renal tubule damage. During the first 6\u202fh of MB urinary excretion of GSTA1-1 and GSTP1-1 was attenuated by median 45% (10\u201370%) and 70% (40\u201385%) vs. baseline (Fig.\u202f1c, d, p\u202f<\u202f0.05). After ending MB infusion urinary excretion of GSTA1-1 and GSTP1-1 increased again, although not significantly (Fig.\u202f1c, d).\nTable\u00a01Kidney function parameters of septic shock patients (n\u202f=\u202f8) treated with MBMedianRangeTotal urine volume, 0\u201324\u202fh (ml)495169\u2013885Protein excretion, 0\u201324\u202fh (mg\/day)342245\u2013434Creatinine clearance (ml\/min) Baseline 8.2 4.2\u201317.4 24\u202fh 10.6* 9.6\u201314.8Blood urea nitrogen Baseline 17.8 10.8\u201320.0 24\u202fh 17.3 10.4\u201322.5Fractional excretion of sodium (%) >\u202f2%  Baseline (n\u202f=\u202f0)\u2013\u2013  24 h (n\u202f=\u202f3) 3.1 2.6\u20133.6 <\u202f1%  Baseline (n\u202f=\u202f8) 0.6 0.3\u20130.7  24 h (n\u202f=\u202f5) 0.4 0.2\u20130.5p\u202f<\u202f0.05 vs. baseline\nDiscussion\nSeveral clinical studies in septic shock patients have investigated the effects of MB on the heart, vascular wall, and lungs\u00a0[9]. This is the first report demonstrating that MB attenuates kidney damage in human septic shock. To determine the effect of NO pathway inhibition on renal damage we examined the urinary excretion of early tubular injury markers and found that MB inhibited the NO pathway and preserved the integrity of renal tubules. After termination of MB infusion these parameters returned to their elevated pretreatment levels. Both plasma concentrations (more than twofold) and urinary NO metabolites levels in our septic shock patients were much higher than in healthy volunteers, as demonstrated earlier\u00a0[3]. Hydrocortisone may have inhibited iNOS activation, however, the patients received a\u00a0continuous infusion of hydrocortisone that started before MB infusion and continued during the 24\u202fh period. Therefore the observation that urinary NO metabolite excretion was attenuated only in the first 6\u202fh after the start of MB suggests that this effect is not related to steroids. Although urinary NO metabolite excretion was attenuated, we did not find a\u00a0reduction in plasma NO metabolites. This is in contrast with an earlier report, in which patients received a\u00a0bolus injection of MB prior to the continuous infusion\u00a0[14].\nRenal failure in septic shock patients is a\u00a0complex and multifactorial disease process. During septic shock the systemic vasodilation increases renal sympathetic activity and angiotensin concentration which results in intrarenal vasoconstriction with sodium and water retention and decreased glomerular filtration rate\u00a0[2]. We previously demonstrated that induction of renal iNOS, constitutively expressed in the kidney\u00a0[15], is associated with proximal tubule injury during systemic inflammation in humans\u00a0[3]. As a\u00a0result of its active secretory transport function and role in urine concentration, the proximal tubule is a\u00a0susceptible target and often the first site of damage\u00a0[16]. Therefore inhibition of peroxynitrite formation from excessively produced NO and superoxide\u00a0[17] by MB may be beneficial for the kidney during septic shock, possibly explained by the local accumulation of MB in renal proximal tubules\u00a0[18]. Global hemodynamic variables can influence renal function, however, only a\u00a0small but statistically significant increase was found for mean arterial pressure, whereas other global hemodynamic parameters did not change during MB treatment.\nSince the detailed nature of our investigation, obviously these methods are not feasible in a\u00a0large-scale clinical intervention study. The most elegant way to examine the effects of MB would be in a\u00a0randomized, placebo-controlled cross-over study. However, with such critically ill patients this design may be considered unethical. We deliberately chose a\u00a0subgroup of severe septic shock patients with a\u00a0high chance of sepsis-induced renal damage for two reasons: first, this is the group of patients in which MB is used as a\u00a0\u201clast resort therapy\u201d and, second, to demonstrate the putative beneficial effects of MB on renal damage. With an estimated standard deviation of 36% in urinary GST excretion, 80 patients would be needed to demonstrate with 80% power a\u00a010% reduction in renal injury. This number of patients was not feasible for our investigations. Therefore we decided to determine the parameters before, during and after MB infusion during a\u00a024\u202fh period, which allows each patient to serve as own control. Because of the observational nature and limited size of the present study and the heterogeneity of the patient population, our findings warrant conformation on hard endpoints by a\u00a0larger clinical trial. However, in our view, first a\u00a0long-term study is necessary to assess the safety of chronic MB administration in septic patients with refractory shock. Promising effects of MB were found in a\u00a0trial with vasoplegic patients after cardiac surgery treated with MB\u00a0[19], in which a\u00a0reduction in both mortality and incidence of renal failure was observed.\nIn conclusion, short-term infusion of MB in septic patients with refractory shock, is associated with a\u00a0decrease in NO production and an attenuation of the urinary excretion of renal tubular injury markers.\nElectronic supplementary material\nElectronic Supplementary Material (DOC 26K)\nElectronic Supplementary Material (DOC 37K)","keyphrases":["acute kidney injury","glutathione s-transferase","inducible nitric oxide synthase expression","nitric oxide metabolites"],"prmu":["P","P","R","R"]}
{"id":"Purinergic_Signal-3-4-2072928","title":"Involvement of P2X and P2Y receptors in microglial activation in vivo\n","text":"Microglial cells are the primary immune effector cells in the brain. Extracellular ATP, e.g., released after brain injury, may initiate microglial activation via stimulation of purinergic receptors. In the rat nucleus accumbens (NAc), the involvement of P2X and P2Y receptors in the generation of microglial reaction in vivo was investigated. A stab wound in the NAc increased immunoreactivity (IR) for P2X1,2,4,7 and P2Y1,2,4,6,12 receptors on microglial cells when visualized with confocal laser scanning microscopy. A prominent immunolabeling of P2X7 receptors with antibodies directed against the ecto- or endodomain was found on Griffonia simplicifolia isolectin-B4-positive cells. Additionally, the P2X7 receptor was colocalized with active caspase 3 but not with the anti-apoptotic marker pAkt. Four days after local application of the agonists \u03b1,\u03b2meATP, ADP\u03b2S, 2MeSATP, and BzATP, an increase in OX 42- and G. simplicifolia isolectin-IR was observed around the stab wound, quantified both densitometrically and by counting the number of ramified and activated microglial cells, whereas UTP\u03b3S appeared to be ineffective. The P2 receptor antagonists PPADS and BBG decreased the injury-induced increase of these IRs when given alone and in addition inhibited the agonist effects. Further, the intra-accumbally applied P2X7 receptor agonist BzATP induced an increase in the number of caspase-3-positive cells. These results indicate that ATP, acting via different P2X and P2Y receptors, is a signaling molecule in microglial cell activation after injury in vivo. The up-regulation of P2X7-IR after injury suggests that this receptor is involved in apoptotic rather than proliferative effects.\nIntroduction\nMicroglial cells are the principal intrinsic immune competent cells of the brain. Resting microglial cells with a ramified morphology become activated under almost all pathological conditions, e.g., mechanical or chemical injury or inflammation. They change into ameboid-shaped motile phagocytotic and cytotoxic cells, which invade the site of injury and initiate repair or defense processes by secreting proteases, neurotrophic factors, and inflammatory mediators [1, 2]. This suggests that microglial cells are able to sense signal molecules entering the extracellular space by disturbances of brain homeostasis and promoting their transformation into the active state [3, 4]. Further, microglia express major histocompatibility complex antigen class II and may perform phagocytotic activity to clear cellular debris [4\u20137].\nOne of the most prominent compounds released by injury is the purine nucleotide ATP. Extracellularly applied ATP induces the activation of a cation conductance and a subsequent increase in intracellular Ca2+ [8]. ATP was shown to stimulate microglial cells to respond with rapid movements of their processes [9]. Further, ATP also induced the outgrowth of processes and membrane ruffling in cultured systems [10, 11]. Dependent on the concentration of ATP applied to in vitro systems, microglial cells secrete various biologically active substances, such as the cytokines tumor necrosis factor-\u03b1 (TNF-\u03b1) [12], interleukin (IL)-1\u03b2 [13, 14], or IL-6 [15]. Boucsein and colleagues [16] have shown that in the presence of purinergic ligands, the lipopolysaccharide-induced release of TNF-\u03b1, IL-6, IL-12, and macrophage inflammatory protein-1\u03b1 was attenuated. In patch-clamp investigations with ATP-induced Ca2+ wave recordings of astrocytes, a current response in an adjacent microglial cell was observed, suggesting that microglia can sense the activity of astrocytes [17].\nAll these data suggest that a variety of ATP-sensitive ionotropic (P2X) and metabotropic (P2Y) receptors is expressed at resting and\/or activated microglial cells [18\u201322]. The functional expression of ATP receptors on cultured microglia is well known [18, 23, 24]. The basic problem in studying this type of glial cells is that any manipulation ex vivo or in vitro will promote their transformation into the active form. We used an in vivo model of a stab wound injury in the rat nucleus accumbens (NAc) to observe the P2 receptor-mediated response of microglial cells to ATP released by injury. This technique enables local administration of receptor-specific purinergic ligands by microinfusion. Furthermore, our investigations focused especially on the P2X7 receptor subtype on microglial cells in vivo and the functional consequences of injury-induced stimulation. The possible involvement of P2X7 receptors in apoptotic and proliferative processes in microglia and astrocytes, respectively, was also investigated.\nMaterial and methods\nMaterials\nKetamine hydrochloride (Ketanest; Ratiopharm, Ulm, Germany), xylazine hydrochloride (Rompun; Bayer, Leverkusen, Germany), and thiopental natrium (Trapanal, Altana Pharma, Konstanz, Germany) were used for anesthesia. For intra-accumbal microinjection, 2-methylthioATP (2MeSATP), \u03b1,\u03b2-methyleneATP (\u03b1,\u03b2meATP, both RBI; Natick, MA, USA); adenosine 5\u2032-(\u03b2-thio)diphosphate (ADP\u03b2S), 2\u2032,3\u2032-O-(4-benzoyl-benzoyl)ATP (BzATP), brilliant blue G (BBG), and 5-bromo-2\u2032-deoxyuridine (BrdU) (all from Sigma, Deisenhofen, Germany); uridine 5\u2032-O-(\u03b3-thio)triphosphate (UTP\u03b3S, Inspire Pharmaceuticals Inc., Durham, NC, USA); pyridoxal-5\u2032-phosphate-6-azophenyl-2\u2032,4\u2032-disulphonic acid (PPADS; Biotrend, K\u00f6ln, Germany); and artificial cerebrospinal fluid [ACSF (in mM): 126 NaCl; 2.5 KCl; 1.2 NaH2PO4; 1.3\u00a0MgCl2, and 2.4 CaCl2; pH 7.4] were used.\nAntibodies directed against glial fibrillary acidic protein (GFAP; mouse anti-GFAP, Sigma, Deisenhofen, Germany; rabbit anti-cow GFAP, DakoCytomation, Hamburg, Germany); BrdU (mouse anti-BrdU, Clone Bu20a; DakoCytomation), active caspase 3 (rabbit, Promega, Madison, WI, USA); OX 42 (CD 11b, mouse anti-rat, complement receptor type 3; Serotec, Oxford, UK); (a) lectin from Bandeiraea simplicifolia (Griffonia simplicifolia) isolectin B4 (BSI-B4, peroxidase labeled) and (b) lectin from B. simplicifolia (G. simplicifolia) isolectin B4 (GSA-B4, biotin conjugated; Sigma, Deisenhofen, Germany, each); phosphorylated serine\/threonine kinases (pAkt1\/2\/3; rabbit, Ser 473, raised against the short amino acid sequence containing phosphorylated Ser 473 of Akt 1, Akt 2, and Akt 3 of mouse origin, recommended for the detection of Ser 473 phosphorylated Akt 1 and corresponding phosphorylated Akt 2 and Akt 3 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); and the following P2 receptor antisera were used: rabbit anti-P2X1, anti-P2X2, and anti-P2X4,5,6 (Alomone Labs, Jerusalem, Israel) as well as rabbit anti-P2X7 receptor-subtype (intracellular C-terminus binding, Alomone Labs), anti-P2X3 (guinea pig; Neuromics Inc., Northfield, MN, USA). Furthermore, a goat anti-rat ecto-P2X7 receptor antibody (a kind gift of Dr. M. Kim; [25]) was used for immunofluorescence double-labeling studies. Additionally, anti-P2Y1, anti-P2Y2, anti-P2Y4, anti-P2Y12 (rabbit; Alomone Labs, Jerusalem, Israel), anti-P2Y6 (rabbit; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-P2Y1 (rabbit, GlaxoSmithKline, Brentford, Middlesex, UK) receptor antibodies were tested. The secondary carbocyanine (Cy)2- and Cy3-conjugated IgGs as well as Cy2- and Cy3-conjugated streptavidin (Jackson ImmunoResearch, West Grove, PA, USA) were used. For histochemical detection, 3,3\u2032-diaminobenzidine hydrochloride (DAB; Sigma Chemical Co., St. Louis, USA) was applied.\nAnimals\nMale Wistar rats (280\u2013320\u00a0g) were housed under a 12-h light, 12\u00a0h-dark cycle and allowed access to lab food and water ad libitum. All procedures using animals were approved by the committee of Animal Care and Use of the relevant local governmental body in accordance with the law of experimental animal protection.\nSurgery\/microinjection\nAnesthetized rats were fixed in a stereotaxic frame. At the coordinates AP\u2009=\u20091.7\u00a0mm (rostral to bregma), L\u2009=\u20091.5\u00a0mm (lateral to the sagittal suture), and V\u2009=\u20096.5\u00a0mm (below the surface of the hemisphere), the injection cannulae connected to a syringe pump via an FEP tubing was inserted into the NAc [26]. By microinfusion, rats received ACSF, which was used as control as well as vehicle for the following P2 receptor agonists: 2MeSATP (nonselective), \u03b1,\u03b2meATP (P2X1,3), ADP\u03b2S (P2Y1,11,12,13), UTP\u03b3S (P2Y4,6), given as 0.1\u00a0nmol each, and BzATP (preferential P2X7) 0.3\u00a0nmol. As antagonists, PPADS (30\u00a0pmol; nonselective) and BBG (1\u00a0pmol, P2X7) were applied. When the effects of the P2 receptor agonists were pharmacologically proven, the microinfusion of the respective antagonists preceded infusion of the agonist and antagonist mixture. As a proliferation marker, BrdU (0.1\u00a0nmol) was applied together with the antagonist, the agonist, or ACSF alone. Test substances were injected in a volume of 1\u00a0\u03bcl at a rate of 12\u00a0\u03bcl\/h.\nImmunocytochemical studies and double-immunofluorescence studies\nImmunocytochemical and double-immunofluorescence studies were done as previously described [26, 27]. After a postinjection time of 2\u00a0h and 4\u00a0days, the rats were transcardially perfused under thiopental sodium anesthesia with paraformaldehyde (2%) in sodium acetate buffer (pH 6.5; solution A) followed by paraformaldehyde (2%)\/glutaraldehyde (0.1%) in sodium borate buffer (pH 8.5; solution B). The brains were immediately removed and stored overnight in solution B without glutaraldehyde. Serial coronal sections (50-\u03bcm thick) from the NAc were obtained using a vibratome (Leica, Typ VT 1000S, Nussloch, Germany) and collected as free-floating slices (0.1\u00a0M Tris; pH 7.6).\nImmunocytochemical studies\nBSI immunoreactivity\nFree-floating sections were rinsed with 0.05\u00a0M Tris-buffered saline (TBS, pH 7.6) and were treated with 1% hydrogen peroxide for 30\u00a0min to inactivate endogenous peroxide activity. Immunolabeling was performed with lectin from BSI-B4 (1:200) in TBS containing 2% bovine serum albumin overnight at 4\u00b0C, followed by washing in 0.05\u00a0M Tris buffer (pH 8.0). Peroxidase activity was visualized with DAB (0.07%) containing nickel ammonium sulphate (1%) and hydrogen peroxide, which renders a black reaction product.\nActive caspase 3 immunoreactivity\nFree-floating sections were rinsed with 0.1\u00a0M TBS (pH 7.4) and treated with 1% hydrogen peroxide for 25\u00a0min to inactivate endogenous peroxide activity. Immunoreactivity (IR) was studied with rabbit anti-active caspase 3 (1:500) in TBS containing 10% normal horse serum (NHS) and 0.1% Triton X-100 overnight at 4\u00b0C, followed by biotinylated horse anti-rabbit IgG (1:100, Vector Labs. Burlingame, CA, USA) and preformed streptavidin\/biotin-peroxidase complex (1:125, StreptABComplex; DakoCytomation) for 2\u00a0h. DAB (0.05%) served as chromogen.\nGFAP\/BrdU immunoreactivity\nThe procedure was applied to free-floating slices as previously described [26]. Briefly, the GFAP staining procedure was carried out with rabbit anti-cow GFAP antiserum (1:600) and biotinylated protein A (1:400; Calbiochem, La Jolla, CA, USA). To detect the astroglial marker, the streptavidin\/biotin technique based on a StreptABComplex (1:125) and DAB (0.05%) as chromogen were used. Mitotic astrocytes were identified by immunostaining of the incorporated BrdU. After DNA denaturation (2\u00a0N HCl) and neutralization (borate buffer; 0.15\u00a0M; pH 8.5), the slices were incubated with a mouse monoclonal antibody against BrdU (1:75), followed by incubation with horse biotinylated anti-mouse immunoglobulins (1:100; Vector Labs., Burlingame, CA, USA) and with ABC Elite Kit (1:50; Vectastain; Vector). Peroxidase activity was visualized with DAB (0.07%) containing nickel ammonium sulphate (1%) plus cobalt chloride (1%) (DAB-Ni\/Co) and hydrogen peroxide, which renders a black reaction product. For GFAP\/BrdU double-staining experiments to characterize mitogenic changes, slices were first processed for anti-GFAP labeling followed by BrdU immunolabeling.\nDouble-immunofluorescence studies\nAfter washing with TBS (0.05\u00a0M; pH 7.6) and blocking with normal goat serum (1% NGS) in TBS, slices were incubated in an antibody mixture of mouse anti-OX 42 (CD11b; 1:100) and rabbit anti-P2X receptor antibodies (P2X1 1:400; P2X2 1:400; P2X4 1:400; P2X5 1:400; P2X6 1:400; P2X7 1:1,000) or P2Y receptor antibodies [P2Y1 1:400; P2Y2 1:1,000; P2Y4 1:1,000; P2Y12 1:400 (Alomone Labs); P2Y1: 1:1,500 (GlaxoSmithKline); P2Y6 1:200 (Santa Cruz Biotechnology, Inc.)] with 0.1% Triton X-100 in 1% NGS in TBS for 48\u00a0h at 4\u00b0C. The secondary antibodies employed for simultaneous localization of the two primary antisera were Cy2-conjugated goat anti-mouse IgG (1:500) and Cy3-conjugated goat anti-rabbit IgG (1:800), respectively. Cy2-conjugated goat anti-guinea pig IgG (1:400) and Cy3-conjugated goat anti-mouse IgG (1:1,000) were used for visualization of guinea pig anti-P2X3 (1:1,000). Sections were washed three times for 5\u00a0min each in 1% NGS [or 5% fetal calf serum (FCS) for guinea pig anti-P2X3] in TBS and then incubated for 2\u00a0h in a solution containing a mixture of the secondary antibodies with 1% NGS (5% FCS) in TBS.\nGSA-B4 and P2X or P2Y double labeling was performed in two steps: first the incubation of the slices with GSA-B4 (1:300), followed by treatment with Cy2-conjugated streptavidin (1:350); and second, by incubation with rabbit P2X\/Y antibodies followed by Cy3-conjugated goat anti-rabbit IgG (1:800) as described above. The ecto-P2X7 receptor antibody (goat anti-rat ecto-P2X7 receptor, 0.12\u00a0\u03bcg\/ml [25]) was combined with rabbit anti-caspase 3 (1:500), rabbit anti-pAkt (1:600), or rabbit anti-P2X7 (C-terminal, 1:1,000) and subsequent detection with Cy2-conjugated donkey anti-goat IgG (1:1,000) and Cy3-donkey anti-rabbit IgG (1:1,000). Incubation of slices with goat anti-rat ecto-P2X7 (1:600), rabbit anti-active caspase 3 (1:500), or rabbit anti-pAkt (1:600) and GSA-B4 (1:300) was followed by treatment with Cy3-conjugated donkey anti-goat (1:1,000) or Cy3-conjugated goat anti-rabbit IgG (1:400) and Cy2-conjugated streptavidin (1:350). Control experiments were carried out without the primary P2 receptor antibody or by preadsorption of the antibody with the peptides used for their generation. When slices were incubated with PBS instead of the primary antibody or with primary antibody serum, which had been preabsorbed with peptide antigen for 1\u00a0h before use, no immunofluorescence with either of the control procedures was observed. After intensive washing and mounting on glass slides, all stained sections were dehydrated in a series of graded ethanol, processed through n-butylacetate, coverslipped with entellan (Merck, Darmstadt, Germany), and analyzed by light microscopy or confocal laser scanning microscopy.\nConfocal microscopy\nDouble immunofluorescence was investigated by a scanning confocal microscope (LSM 510, Zeiss, Oberkochen, Germany) equipped with an argon laser emitting at 488\u00a0nm (yellow-green Cy2-immunofluorescence) and a helium\/neon laser emitting at 543\u00a0nm (red Cy3-immunofluorescence).\nQuantification and statistical analysis\nChanges in BSI-B4-IR were determined by densitometry (in regions 1 and 2, Fig.\u00a01a) using a digital image analyzer (Diana II) in combination with an advanced image data analyzer (AIDA 2.0). Results are expressed as percentage of the BSI-B4-IR of the ACSF-treated sides in the same regions.\nFig.\u00a01a A rat brain slice including needle tracts and regions of interest within the nucleus accumbens (NAc) in which immunoreactivity (IR) and cells were evaluated (1 core 1; 2 core 2; according to [29]). b BSI-B4-marked microglial cells after stab-wound injury (overview). c Quantification of the number of activated microglial cells after injury and microinjection of P2-receptor-agonists 2MeSATP, ADP\u03b2S, \u03b1,\u03b2meATP (0.1\u00a0nmol each), and BzATP (0.3\u00a0nmol) after a postinjection time of 4\u00a0days. Values are expressed as percentage of ACSF-treated controls and represent mean \u00b1 SEM of six animals per group. d\u2013f BSI-B4-IR of microglial cells in the NAc of rats, illustrating changes in the glial morphology: d A great number of resting (process-bearing) microglial cells (arrows) under control conditions; e, f changes in the number of resting microglial cells (arrow) and activated microglial cells (arrowhead) after different treatment conditions (scale bar\u2009=\u2009200\u00a0\u03bcm). g Quantification of the effects of agonists 2MeSATP, ADP\u03b2S, \u03b1,\u03b2meATP (0.1\u00a0nmol each), and BzATP (0.3\u00a0nmol) alone and in combination with PPADS (0.1\u00a0nmol\/0.03\u00a0nmol each) or BBG (1\u00a0pmol) on BSI-B4-IR of microglial cells in brain slices of the NAc of the rat after a postinjection time of 4\u00a0days. Data are expressed as percentage of the ACSF-treated side and represent the mean \u00b1 SEM of six animals per group (*p\u2009<\u20090.05, vs. ACSF group; +p\u2009<\u20090.05, agonist vs. antagonist\/agonist group)\nAdditionally, the number of BSI-B4-labeled ramified and activated microglial cells as well as GFAP and GFAP\/BrdU double-labeled cells were counted under a light microscope (Axioskop; Zeiss) with a 20x objective within a square (0.5\u2009\u00d7\u20090.5\u00a0mm) in region 1 (Fig.\u00a01a). Each value represents five replications for each condition. The results were expressed as a percentage of ACSF-treated sides of the same regions. The individual groups were compared with one-way analysis of variance (ANOVA) using Bonferroni t test. A probability level of 0.05 or less was considered to be statistically significant.\nResults\nImmunocytochemistry\nAfter mechanical injury (Fig.\u00a01a,b), a characteristic BSI-B4 immunolabeling around the stab wound was observed in comparison with untreated controls, decreasing in relation to the distance from the lesion. In untreated rats, a great number of resting (process-bearing) microglial cells was present (Fig.\u00a01d, arrows), whereas after stab-wound injury, changes in the number of resting microglial cells with retracted processes (Fig.\u00a01e, arrow) and activated microglial cells (Fig.\u00a01f, arrowhead) were observed. Quantification of BSI-B4-immunolabeling in the NAc (region 1; Fig.\u00a01c) of rats 4\u00a0days after stab-wound injury indicated a variable influence of P2 receptor agonists on the number of activated cells. Whereas \u03b1,\u03b2meATP had no effect, 2MeSATP was more potent in tendency than was ADP\u03b2S in increasing the number of activated microglial cells. BzATP, which was injected into the NAc in a higher dose than were the previous ligands [28], also induced a marked enhancement of activated microglial cells (Fig.\u00a01c). UTP\u03b3S was without effect, both on the number of activated microglia and on resting microglia (not shown).\nQuantification of BSI-B4-IR in the NAc (region 1 plus 2) by densitometry showed a significant increase for all agonists used with the exception of UTP\u03b3S (not shown) when compared with ACSF-treated animals. The effects of \u03b1,\u03b2meATP, ADP\u03b2S, and 2MeSATP could be inhibited by pretreatment with PPADS and those of BzATP by pretreatment with BBG (Fig.\u00a01g). PPADS and BBG given alone inhibited and failed to alter BSI-B4-IR, respectively.\nDouble-immunofluorescence studies at P2X and P2Y receptor subtypes\nMultiple immunofluorescence labeling in combination with laser scanning microscopy was used to characterize the P2X\/Y receptor subtype expression after injury in comparison with untreated controls. Under control conditions, no P2X or P2Y (with the exception of P2Y1) receptor-IR was coexpressed on single GSA-B4-positive cells. The mechanical injury associated with the introduction of the injection cannula resulted in the expression of P2X1,2,4,7 and P2Y1,2,4,6,12 receptors in the lesioned area, observed earliest 2\u00a0h until 4\u00a0days after injury. An up-regulation of immunolabeling of P2X1,2,4,7 receptor subtypes was unequivocal; examples are shown in Fig.\u00a02 (a\u2013i). In the affected area, P2Y1,2 and P2Y12 fluorescence labeling was observed on GSA-B4-positive cells with low intensity. P2Y4 labeling appeared preferentially on process-bearing microglial cells (not shown). P2X2-IR (Fig.\u00a02c,d), P2X4 (Fig.\u00a02e,f), and P2Y6-IR (Fig.\u00a02l,m) were found on both resting and especially on activated microglial cells, the latter being identified by the lack of processes. No immunofluorescence was observed for P2X3,5,6 receptor subtypes on either type of microglial cells before and after stab wounding.\nFig.\u00a02Confocal images of examples of double immunofluorescence labeling to characterize the colocalization of P2X1,2,4,7 and P2Y1,6,12 receptor subtypes on a\u2013f OX42- or g\u2013o GSA-B4-labeled microglial cells in the NAc of rats 4\u00a0days after stab-wound injury (thin arrow process-bearing microglial cell; thick arrow activated microglial cell) (scale bars: a, b\u2009=\u200920\u00a0\u03bcm; c, d\u2009=\u2009100\u00a0\u03bcm; e\u2013i\u2009=\u200920\u00a0\u03bcm; j, k\u2009=\u2009100\u00a0\u03bcm; l, m\u2009=\u200920\u00a0\u03bcm; n, o\u2009=\u200950\u00a0\u03bcm)\nInjury-induced prominent expression of the P2X7 receptor was observed on microglial cells by double labeling with GSA-B4 (Fig.\u00a02g\u2013i) and also on astrocytes, as described earlier [29]. The two different P2X7 receptor antibodies, one directed against the extracellular domain of the rat P2X7 receptor [25] and the other against its C terminus (C-terminal P2X7) (Alomone Labs) labeled the same cells (Fig.\u00a03a,b; thin arrow) but also different cells (Fig.\u00a03c,d; thick arrow). Additionally, the ecto-P2X7 antibody labeled GSA-positive cells (data not shown).\nFig.\u00a03Apoptosis or proliferation after P2X7 receptor stimulation after stab-wound injury in the NAc of rats 4\u00a0days after injury. a\u2013d Colocalization of P2X7-IR detected by antibodies directed against the C-terminus (C-term) (b, d) and the ectodomain (a\u2013d) of the rat P2X7. e\u2013j Confocal images of double-labeling studies: Coexpression of active caspase 3 on single GSA-B4-positive cells (e, f) and active caspase 3 and the ecto-P2X7 receptor subtype (g, h). No double labeling of pAkT on GSA-B4-labeled microglial cells 4\u00a0days after injury (i, j) (scale bars: b\u2013g\u2009=\u200920\u00a0\u03bcm). k Quantification of the number of active caspase 3-positive cells in region 1 (see Fig.\u00a01a). Values are expressed as percentage of controls and represent the mean \u00b1 SEM of six animals per group. (*p\u2009<\u20090.05, vs. ACSF group; +p\u2009<\u20090.05,agonist vs. antagonist\/agonist group; #p\u2009<\u20090.05, antagonist vs. antagonist\/agonist group)\nPossible role of the P2X7 receptor after injury in vivo\nTo find out whether the P2X7 receptor is related to apoptotic and\/or proliferative processes after injury, we used the early apoptotic marker active caspase 3 and the proliferation marker BrdU. After BzATP microinjection, a significant increase in the number of active caspase-3-positive cells was found in comparison with ACSF-treated animals (Fig.\u00a03k). This effect was reduced by pretreatment with the P2X7 receptor antagonist BBG. Further, BBG given alone decreased the number of active caspase 3-immunopositive cells in comparison with ACSF-treated rats (Fig.\u00a03k).\nDouble immunofluorescence-labeling showed colocalization of active caspase 3 at single GSA-B4-positive cells in the NAc of rats (Fig.\u00a03e,f). Furthermore, colocalization of the ecto-P2X7 receptor subtype on caspase-3-positive cells was also documented (Fig.\u00a03g,h). Additionally, a possible coexpression of the anti-apoptotic and possibly proliferation-promoting pAkt on GSA-B4-positive cells was studied 4\u00a0days after injury. However, no colocalization of pAkt with GSA-B4-positive cells (Fig.\u00a03i,j) was found by immunofluorescence labeling. Eventually, no colocalization of P2X7 and pAkt was observed to this time point (data not shown).\nA possible influence of P2X7 receptor stimulation on the increase in cell number of activated and ramified microglial cells after BzATP stimulation was also studied. Changes in the number of activated microglia are shown in Fig.\u00a01c. By quantification of ramified (resting) microglial cells in region 1 (Fig.\u00a01a), an increase to 123.8\u2009\u00b1\u20095.3% in comparison with ACSF-treated rats was found, similar to that after treatment with 2MeSATP (124.0\u2009\u00b1\u20097.8%). For comparison, no characteristic changes were found after microinfusion of ADP\u03b2S (108.3\u2009\u00b1\u200911,6%) and \u03b1,\u03b2meATP (101.7\u2009\u00b1\u20096.9%). Besides the effects of BzATP on the number of active and resting microglial cells, no influence of BzATP treatment on the number of GFAP (specific marker for fibrous astrocytes)-positive cells (BzATP 1.7\u2009\u00b1\u20097.3%; BBG 0.4\u2009\u00b1\u20093.0%; BzATP\/BBG 0.96\u2009\u00b1\u20094.8%) was found in comparison with ACSF-treated rats. There was also no effect on the number of GFAP\/BrdU double-labeled cells (proliferating cells) (BzATP 14.8\u2009\u00b1\u20094.5%; BBG 8.8\u2009\u00b1\u200911.2%; BzATP\/BBG 15.9\u2009\u00b1\u20095.9%) in comparison with ACSF-treated rats.\nDiscussion\nThe results presented here demonstrate the involvement of various P2 receptors in microglial response to the placement of cannulas and microinjection, both resulting in tissue damage and thereby in high extracellular ATP concentrations [30]. Microglial cells stimulated by ATP may participate both in neurodegeneration (apoptosis and\/or necrosis) and neuroprotection.\nCultured microglial cells from mouse or rat brain were shown to respond to ATP with the activation of a cationic conductance, accompanied by an increase in cytosolic Ca2+ [8], followed by changes of microglial morphology [31, 32]. The exposition of cultured microglial cells to ADP or ATP further induced membrane ruffling and markedly enhanced chemokinesis, a prerequisite to perform tissue surveillance in the brain [33].\nIn vivo, microglial cells are homogenously distributed within the NAc characterized by rod-shaped somata with thin and highly ramified processes extended in all directions. Injury by stab wound caused microglial cell activation around the lesion track, characterized by swollen somata and thicker processes. The additional administration of P2X and P2Y receptor agonists enhanced the occurrence of activated microglial cells detected by their BSI-IR. The observed changes suggest that the stimulation of P2X1,3 receptors has only minor relevance in the process of microglia activation, whereas in agreement with the subsequent pharmacological investigations, involvement of P2X7 and P2Y1,2,4,6,12 receptors in this process is highly probable.\nWhereas P2X1\u20137 receptor-IR was absent in the nonaffected NAc, the expression of P2X1,2,3,4,7 receptors but not of P2X5,6 receptors was detected after the stab-wound lesion. P2X2,4,6 receptors are widely expressed in central and peripheral neurones as well as other excitable cells [22, 34, 35], and the P2X7 receptor is expressed in macrophage-type cells such as microglia but also mediates purinergic responses in astrocytes and neurons [36]. Changes in microglial expression of P2X receptors (P2X1\u20137) during postnatal development of the rat have been described [37]. Obviously, microglial cells are involved in long-term trophic events such as cell proliferation, differentiation, movement, and death during development [37].\nUpregulation of P2X1-IR at microglial cells, as found in this study, was described at activated astrocytes after stab-wound injury in the adult rat NAc [29]. Further, the P2X2 receptor was found to be situated at activated microglia in our study and previously at astroglial cells [29]. After ischemia in vivo, P2X2 and P2X4-receptor-IR in the hippocampus became up-regulated in different cellular phenotypes [38]. P2X2 was expressed on neuronal cell bodies and fibers in hippocampal pyramidal cells, whereas intensive P2X4-IR was localized at microglial cells. Confocal microscopic analysis with OX 42 [39] at organotypic cultures showed that P2X4 and P2X7 are expressed on microglia, whereas P2X1 and P2Y1,2,12, although present in the slices, do not colocalize at microglia, and the P2X6 receptor is absent. P2X4 receptors seem to be functionally relevant for microglial chemotaxis, which was suppressed by the knockdown of the P2X4 receptor in cultured microglia by ribonucleic acid (RNA) interference through the lentivirus vector system [33].\nAfter spinal-nerve injury, P2X4 receptor expression strikingly increased at the ipsilateral site in hyperactive microglia but not in neurons or astrocytes [40]. Intraspinal administration of P2X4 receptor antisense oligodeoxynucleotide decreased induction of P2X4 receptors and suppressed tactile allodynia after nerve injury.\nThe major P2X receptor expressed at microglial cells is the P2X7 receptor, characterized in several reports in vitro [13, 16, 41]. This receptor subtype appeared to be strongly coexpressed with the microglial marker GSA-B4 after the mechanical lesion used in our study, suggesting an outstanding role of this receptor in injury-induced microglial responses. It is thought to be closely related to the immune functions of microglia, such as production of inflammatory cytokines, superoxide, and nitric oxide [42]. The P2X7 receptor is reported to be little susceptible to desensitization in the presence of ATP, which makes it quite different from other purinergic receptors, including the P2X4 receptor, which can be desensitized within a few minutes [23, 43]. Up-regulation of microglial P2X7 receptors has been observed in several pathological models, such as in vivo ischemia [41, 44, 45], as well as multiple sclerosis and amyotrophic lateral sclerosis of the human spinal cord [46]. The P2X7 receptors were also specifically up-regulated around \u03b2-amyloid plaques in a mouse model of Alzheimer\u2019s disease (Tg2576) [47] and in human proliferative vitreoretinopathy on M\u00fcller glial cells [48].\nWithin the P2Y receptor family, in our study, the P2Y1 receptor was markedly expressed on microglial cells of untreated rats and was up-regulated in concert with P2Y2,4,6,12 receptors after injury. In the rabbit retina, microglial cells express functional P2Y1 receptors [49]. Activation of these receptors stimulates phenotypic alterations characteristically for microglial activation.\nOur data also demonstrate a strong colocalization of the P2Y6 receptor with GSA-IR. In addition to the microglial function to build a barrier between injured and healthy tissue, these cells are able to clear dead cells or dangerous debris from brain tissue. This phagocytotic process was shown to be triggered by activation of P2Y6 receptors by the endogenous agonist UDP [50].\nSeveral authors report on ATP-induced migration of microglial cells to the affected sites of the brain [9, 10, 51\u201353]. Expression of the P2Y12 receptor on microglial cells after injury seems to be strongly related to the motility of this cell type. In primary cultured microglial cells, exposition to ADP or ATP induced membrane ruffling and markedly enhanced chemokinesis, which could be blocked by the P2Y12 receptor antagonist cangrelor (AR-C69931MX), suggesting that Gi\/o-coupled P2Y receptors are involved [10]. The high dynamic of intact microglial processes as well as their chemotactic response to injury or local injection of ATP was shown in mouse cortex using time-lapse, two-photon laser imaging [9]. Investigations in P2Y12 receptor-deficient mice gave further support that P2Y12 receptors are required for the nucleotide-evoked chemotaxis [33, 54]. Eventually, expression of P2Y12 receptors was observed in native and axotomized facial nuclei, and the number of P2Y12-expressing cells increased following facial nerve axotomy, considered to be a crucial component in the regeneration cascades of motor neurons [55].\nThe published data confirm that not all known P2 receptors can be detected by their immunoreactivity. However, the mRNA of the P2X1\u20137 [29] and P2Y1,2,4,6,12 subtypes [27] was detectable in the NAc of untreated rats. The P2 receptors present on microglia may be differently involved in the processes of their activation, as shown by the inhibition of BSI-B4-IR using PPADS and BBG. It can be suggested by pharmacological studies that P2Y1 and P2X7 receptors may play a predominant role in the response of microglia to the consequences of a stab wound in the NAc.\nRetinal detachment and application of ADP\u00dfS onto control retinas induced phenotype alterations of microglial cells (decrease of soma size, retraction of cell processes) and had no influence on microglial cell density [49]. In the stab-wound model, stimulation of P2 receptors with agonists preferring P2Y1 and P2X7 receptor subtypes promoted the morphological changes of resting to activated microglial cells.\nP2X7 receptors are ATP-gated ion channels; their sustained activation leads to cytolysis in an apoptotic fashion in the microglia [56]. P2X7 receptor-mediated apoptosis involves activation of the proteolytic pathway of caspase activation, which leads to nuclear DNA damage [57]. In our study, immunocytochemistry showed expression of P2X7 and active caspase 3 at GSA-positive cells after injury without agonist application. The active caspase 3 has been defined as the main executioner of programmed cell death and as an early apoptotic marker [58]. The microinfusion of BzATP increased the number of caspase-3-positive cells, suggesting an involvement of P2X7 receptors in apoptotic changes after traumatic events. The BzATP-induced increase in ramified and activated microglia (and proliferating astrocytes) was only marginal.\nThe phosphoinositide-3-kinase\/serine-threonine-kinase Akt (PI3-K\/Akt) pathway is associated with cell proliferation, promoting cell survival and inhibiting apoptosis [59]. Because in the present study performed at 4\u00a0days after injury no coexpression of pAkt with GSA and P2X7 was found, the evidence for a preferential apoptotic pathway triggered by P2X7 receptors is unlikely.\nUp-regulation of P2 receptors after mechanical injury might be both the cause and the result of the induction of apoptotic\/necrotic cell death activating microglial cells with harmful or beneficial effects. The present results from in vivo investigations implicate that ATP via both P2X and P2Y receptor-stimulation acts as a signaling molecule in microglial cells in vivo. An up-regulation of P2X7-IR after injury suggests involvement of this receptor subtype in apoptotic rather than in proliferative events.","keyphrases":["injury","p2 receptors","microglia","cns","trauma"],"prmu":["P","P","P","U","U"]}
{"id":"J_Mol_Med-3-1-2121654","title":"Modulation of muscle contraction by a cell-permeable peptide\n","text":"In contrast to immortal cell lines, primary cells are hardly susceptible to intracellular delivery methods such as transfection. In this study, we evaluated the direct delivery of several cell-permeable peptides under noninvasive conditions into living primary adult rat cardiomyocytes. We specifically monitored the functional effects of a cell-permeable peptide containing the 15 amino acid N-terminal peptide from human ventricular light chain-1 (VLC-1) on contraction and intracellular Ca2+ signals after electrical stimulation in primary adult cardiomyocytes. The transducible VLC-1 variant was taken up by cardiomyocytes within 5 min with more than 95% efficiency and localized to sarcomeric structures. Analysis of the functional effects of the cell-permeable VLC-1 revealed an enhancement of the intrinsic contractility of cardiomyocytes without affecting the intracellular Ca2+. Therefore, peptide transduction mediated by cell-penetrating peptides represents not only a unique strategy to enhance heart muscle function with no secondary effect on intracellular Ca2+ but also an invaluable tool for the modulation and manipulation of protein interactions in general and in primary cells.\nIntroduction\nTherapeutical delivery of protein or peptides into living primary cells is hampered by the lack of a suitable and efficient method for the introduction of macromolecules. Most commonly used transfections methods work in immortalized cells but mostly fail in primary cells or require specialized and time-consuming protocols, i.e., production of viral vectors. Moreover, the limited life span of primary cells often does not provide sufficient time for expression of gene products and subsequent analysis of their effects. Physical methods such as microinjection and bead loading are highly invasive procedures and are not tolerated by several cell types. It has been known for quite some time that basic proteins like histones or poly-ornithine as well as stretches of basic amino acids within proteins aid the uptake of proteins into mammalian cells [14, 17, 20, 30]. In the 1990s, transducible peptides like penetratin-1 from the homeodomain of Antennapedia [11] and amino acids 48\u201357 from the transactivator of transcription of human immunodeficiency virus (HIV)-1 [14] were identified and exploited to introduce drugs or biological macromolecules into mammalian cells. Together with other native and synthetic transducing peptides, they are referred to as cell-penetrating peptides (CPPs) [12]. However, it became evident that the invasive methods used to analyze the transduction mediated by CPPs or effects of interconnected cargoes had led to overestimated transduction results and overrated effects of cargoes fused to CPPs [27]. Nevertheless, CPPs mediate the introduction of fused cargoes into living cells, with cargo-dependent mechanistical differences. One uptake mechanism can be described as a slow adsorptive endocytosis and is preferentially used by globular proteins, whereas small compounds like peptides linked to CPPs favor a rapid membrane potential-dependent uptake with overall intracellular availability of the transduced species [33]. CPP-mediated peptide transduction has recently been used to deliver cardioprotective peptides derived from protein kinase C [4, 7\u20139, 24], the antiapoptotic BH4 peptide derived from the apoptosis regulator protein Bcl-xL [26, 28] and a Nox2-derived peptide [34] into isolated cardiomyocytes or whole hearts. We have tested the feasibility of this approach to study muscle function in living adult primary cardiomyocytes. Therefore, we targeted the interaction between actin and the essential myosin light chain (MLC-1) of cardiac type II myosin by a peptide competition approach. Transgenic overexpression of the N terminus of the human ventricular MLC-1 (residues 1\u201315; hereafter termed VLC-1), which binds to actin and targets actin\/MLC-1 interaction, significantly increased the magnitude and kinetics of the contraction of isolated perfused hearts [21]. We fused the same human VLC-1 peptide to the TAT CPP (VLC-1-TAT) and investigated its uptake, intracellular distribution, and functional consequences in primary living adult rat cardiomyocytes.\nMaterials and methods\nIsolation of adult rat cardiomyocytes Primary cultures of cardiomyocytes from male WKY rats aged 3\u00a0months were performed as described previously [1].\nPeptides The peptides consisted of either l-amino acids (capital letters) or of d-amino acids (lower case letters). In case of d-amino acid peptides, functional motifs were synthesized as retro-all d-variants (underlined) to maintain their functionality. Peptides were either labeled with the fluorophore 5,6-carboxy-tetramethylrhodamine (TAMRA) or fluorescein isothiocyanate (FITC). TAT (TAMRA- or FITC-rrrqrrkkrg), PTD4 [23, 33] (TAMRA-araqraaaray), and TAT-p21 (TAMRA-rrrqrrkkrgaaAGRKRRQTSMTDFYHSKRRLIFSa-amide) [33] were labeled directly at their N termini with the indicated fluorophores (Peptide Speciality Laboratories, Heidelberg). In the case of VLC-1 (MAPKKPEPKKSSAKA-C-TAMRA-ME-amide) and VLC-1-TAT (MAPKKPEPKDDAKAPAGRKKRRQRRR-C-TAMRA-ME-amide; Biosyntan, Berlin) the TAMRA fluorophore was coupled via an additional cysteine at the C terminus leaving the N terminus free for the interaction with actin.\nUptake of transducible peptides and controls To analyze the transduction ability of different compounds into living adult ventricular cardiomyocytes, the primary cells were plated into a laminin-coated \u03bc-slide eight-well ibiTreat (ibidi, Martinsried) or four-well labtek (Nunc, Wiesbaden) microscopy observation chamber. The respective peptides and controls were added directly to Hank\u2019s balanced salts solution buffered with 10\u00a0mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid at pH\u00a07.4 (HBSS) or medium and gently shaken to yield a final concentration of 10\u00a0\u03bcM in case of the peptides and 5,6-TAMRA-fluorophore. Trypan blue (Sigma-Aldrich) was added to a final concentration of 0.5% (v\/v) to the HBSS buffer. For the experiments summarized in Fig.\u00a01 confocal images were acquired 1\u00a0h after addition of the respective compounds to living cardiomyocytes. To study the intracellular localization of transducible peptides in Fig.\u00a03, after 15\u00a0min of peptide incubation, the medium was removed, and the cells were washed twice with the HBSS buffer. For subsequent confocal image collection, the cells were kept in medium or buffer. To study the effect of the peptide on muscle contraction and intracellular Ca2+ (Fig.\u00a04), cardiomyocytes were electrically stimulated (see below) to permit the access of the peptides to all actin molecules with potential binding sites. During confocal image collection, the electric stimulation was temporarily switched off.\nFig.\u00a01Peptide uptake and intracellular distribution in living adult cardiomyocytes. To study the uptake capacity of different compounds into primary rat cardiomyocytes, different fluorescent compounds were directly added for 1\u00a0h to the medium or buffer of freshly isolated cardiomyocytes. After the incubation period, confocal images of the cells were taken without exchange of the medium and after removal of the fluorescent solution and exchange against the fresh medium. Internalization into healthy adult cardiomyocytes occurred after application of 10\u00a0\u03bcM of the transducible peptides FITC-TAT shown at low (a) and high magnification (b) and TAMRA-TAT-p21 shown at low (c) and high magnification (d) but not with application of 10\u00a0\u03bcM of a basic control peptide PTD4 (e). Moreover, 0.5% of the vital dye trypan blue (f) or 10\u00a0\u03bcM of the TAMRA-fluorophore (g) itself were not able to enter healthy cardiomyocytes (rod-shaped cells in culture). Scale bar, 10\u00a0\u03bcm. Stars indicate the nuclei and arrowheads the nucleoli (N) in the higher magnification images. PC Phase contrast, NP nucleoplasm\nMicroscopy, image acquisition, and analysis Confocal images were acquired with a Zeiss laser scanning microscope LSM510 Meta mounted on an Axiovert 200M inverted microscope using a 63\u00d7 phase-contrast oil immersion plan-apochromat objective NA1.4 or a 100\u00d7 phase-contrast oil immersion plan-neofluar objective NA1.3. For all settings, the main beam splitter was HFT UV\/488\/543\/633, and the specific parameters for the single fluorophores were: FITC, excited at 488\u00a0nm light, detected with a 500\u2013530-nm bandpass filter; TAMRA or rhodamine excited at 543\u00a0nm, detected with 565\u2013615 bandpass filter; and trypan blue, excited with 633\u00a0nm, detected with 650 longpass filter. Phase-contrast images were recorded with excitation at 488\u00a0nm and detection in the transmission channel. Laser power for observation was typically 1\u20135% (488\u00a0nm, 25\u00a0mW) and 50\u201360% (543\u00a0nm, 1\u00a0mW) unless otherwise indicated. Settings were adjusted in a way that image pixels were not over- or underexposed with the range indicator function in the Zeiss LSM image acquisition and examiner software version 3.2.\nMeasurement of cardiomyocyte shortening and Ca2+ transients Attached cardiomyocytes were washed with HBSS. Cells were loaded with Fura-2-AM for 30\u00a0min at room temperature in the dark. The dye solution was removed, and cells were left on HBSS for another 15\u00a0min. Only cardiomyocytes of optically intact rod-shaped morphology with clear cross-striation were analyzed. Cardiomyocytes were electrically stimulated until a stable steady-state contraction and Fura-2 signal could be monitored.\nCell shortening and Fura-2 signals were simultaneously measured at 30\u00b0C on an Ionoptix Contractility and Fluorescence System (Ionoptix). Cardiomyocytes were electrically stimulated with bipolar pulses of 5\u00a0ms duration at 1\u00a0Hz. Cell shortening, expressed as percentage of resting cell length, was measured using the video-edge technique at a sampling rate of 240 per second. Ca2+ transients were monitored as ratio of fluorescence emission at 510\u00a0nm was obtained by alternate excitation at 340 and 380\u00a0nm (340\/380 ratio). Data files from 15 consecutive beats recorded at intervals were averaged for analysis. Subsequently, the cardiomyocytes were incubated with the peptides for 15\u00a0min without electrical stimulation. The peptide was then removed by replacing the peptide-containing HBSS buffer with normal HBSS buffer without the peptide. The cardiomyocytes were subsequently electrically stimulated, and both the shortening and Fura-2 signals were simultaneously recorded. The effect of the peptide on shortening amplitude and the Fura-2 signal was expressed in percent change compared to the steady-state signals obtained in the preincubation period.\nResults and discussion\nWe recently demonstrated by live-cell confocal microscopy that the TAT CPP was able to shuttle fused peptides into primary blood cells and subsequently modulated their functional properties [10]. In this study, we tested whether TAT CPP could penetrate and if a peptide derived from the N terminus of VLC-1 fused to TAT is able to modulate the contractile function of primary adult cardiomyocytes.\nDifferent basic peptides were incubated with freshly prepared cultures of cardiomyocytes, and peptide internalization was analyzed live by laser-scanning confocal microscopy. The fluorescently labeled minimal transduction domain TAT CPP of the transactivator of transcription from HIV-1 was readily taken up into cardiomyocytes and displayed a strong affinity to the nuclear compartment (Fig.\u00a01a). It could also be visualized in a punctate pattern throughout the entire cardiomyocyte (Fig.\u00a01a). To determine whether the TAT CPP is able to shuttle an attached peptide into cardiomyocytes, we used the TAMRA-labeled TAT-p21WAF\/CIP (TAT-p21) fusion peptide [33], which in addition to the transducing moiety contains 26 amino acids derived from the C terminus of human p21WAF\/CIP protein. The uptake of this cargo peptide (assessed by appearance in nucleoli) occurred on a timescale of 5 to 10\u00a0min after peptide application. Its pattern of localization inside the cardiomyocytes was similar than that seen for TAT alone (Fig.\u00a01c). Basic amino acid residues are a prerequisite for the transduction ability of CPPs [13, 25, 35], and peptides containing a minimum of six basic amino acids have been shown to traverse biological membranes [5, 18]. As a nontransducing control peptide, we chose the TAMRA-labeled peptide PTD4, which contains three arginines in total [23, 33]. More than 1\u00a0h after application of this peptide to the cardiomyocyte culture, none of the healthy intact cardiomyocytes took up the fluorescent peptide (Fig.\u00a01e). Similar results were obtained for the vital dye trypan blue (Fig.\u00a01f) and the TAMRA fluorophore (Fig.\u00a01g). Approximately 5% of cardiomyocytes in the cultures were dead or harmed (visible in the phase-contrast images as not rod shaped and with no striations). These cells were flooded with both the CPPs as well as any other control dyes (Fig.\u00a01c,e\u2013g). The cell-permeable TAT-derived peptides displayed a similar enrichment in the nuclear compartment with strong accumulation inside nucleoli (arrowheads) as can be seen in the higher magnification images (Fig.\u00a01b,d). This intranuclear distribution is basically determined by the TAT moiety, which includes a nuclear localization sequence [22, 29, 33].\nHaving shown that CPP TAT was able to shuttle fused peptides into living cardiomyocytes, we designed a transducible peptide comprising the actin-binding element of the ventricular isoform of human MLC-1 (VLC-1), i.e., the N-terminal residues 1\u201315 (VLC-1 peptide) connected to TAT, termed VLC-1-TAT (Fig.\u00a02). The hearts of transgenic animals harboring minigenes encoding for the VLC-1 peptide revealed enhanced myosin motor activity and positive inotropic effects [21]. A possible molecular mechanism for the increase in myosin motor activity and contractility is illustrated in the scheme in Fig.\u00a02. MLC-1 isoforms of vertebrate striated muscle play a regulatory role in myosin motor function: The N-terminal part of MLC-1 contains a sticky element (SE), which consists of several positively charged amino acids that bind to the C terminus of actin [3, 6, 16, 31]. The 46 N-terminal amino acids of MLC-1 have previously been modeled on the basis of contemporary crystallographic data [2]. Interactions between the sticky MLC-1 element and actin serve as a negative modulator and decrease the activity of the myosin motor domain. Intracellular delivery of a synthetic sticky MLC-1 element by TAT-mediated transduction therefore would antagonize MLC-1\/actin interaction. We predict this would increase myosin motor activity and enhance the shortening amplitude without change of the systolic free Ca2+ activation level of intact cardiomyocytes.\nFig.\u00a02Molecular hypothesis for the enhancement of cardiomyocyte contractility by the cell-permeable VLC-1 peptide. a Primary sequence of VLC-1-TAT peptide color-coded orange for the VLC-1- and yellow for the TAT-moiety. b Schematic sliding filament model displaying actin in violet and the MHC in red. The higher magnification shows the catalytic domain (CD) and the neck domain (ND) of one molecule of the MHC. MLC proteins are colored green with the N-terminal SE inside the essential MLC-1 protein highlighted in red. Upon intramolecular conformational changes in the CD of MHC, the ND swings out, exposing the SE of MLC-1 to an actin molecule in direction to the Z-line. Treatment of cardiomyocytes with the transducible TAT peptide (yellow ball) fused to the SE-peptide (orange) blocks potential binding sites of the native MLC-1 N terminus. After the MHC rearrangement, the MLC-1 protein can no longer interact with actin and downregulate the myosin motor activity. The scheme is based on the pre- and postpower stroke model [19] developed with respect to crystallographic data on the Dicytostelium myosin motor domain [15, 32]\nTo first test whether the VLC-1 peptide itself might be taken up by isolated cardiomyocytes because of its basic charge, VLC-1 and VLC-1-TAT were applied for 15\u00a0min to freshly isolated cardiomyocytes under resting conditions or, in another set of experiments, constant electric stimulation (1\u00a0Hz, 20\u00a0V). After the fluorescent peptides were washed away, the cells were analyzed live by confocal laser-scanning microscopy (Fig.\u00a03a). VLC-1 alone was not internalized into healthy cardiomyocytes and stained only compromised cells (upper panel, Fig.\u00a03b). To ensure that low concentrations of intracellular VLC-1 were not missed, the laser intensity was increased to its maximum, and still no fluorescent signals were measured (lower panel, Fig.\u00a03b). In contrast, VLC-1-TAT reached the intracellular compartments of more than 95% of adult cardiomyocytes (Fig.\u00a03c).\nFig.\u00a03Transduction and intracellular localization of VLC-1-TAT in isolated adult cardiomyocytes. a Scheme for the application of the VLC-1-derived peptides and confocal imaging. Peptides were applied at a concentration of 1\u00a0\u03bcM, incubated for 15\u00a0min to freshly isolated cardiomyocytes. After removal of the peptide solution, the cells were washed twice and kept in buffer or medium for direct live-cell confocal imaging microscopy. Subsequently, the transduced cardiomyocytes were electrically stimulated with 1\u00a0Hz for maximally 1\u00a0h. b The negative control peptide TAMRA-labeled VLC-1 stained only dead cardiomyocytes (low magnification, upper panel) but failed to get internalized into healthy cardiomyocytes even when 100% laser power was used to detect potentially weak signals (lower panel). c Application of TAT-VLC-1 to living cardiomyocytes led to rapid internalization of the peptide (upper panel). The lower panel shows transduced VLC-1-TAT at higher magnification in an overlay of the phase contrast and the TAMRA-fluorescence. The arrow indicates the direction of the intensity profile of gray and red intensities in the line scan for the localization analysis of VLC-1-TAT, which was present at sarcomeric structures with enrichment at the actin-containing I-bands (light bands in phase-contrast image)\nUnlike the localization of the TAT and TAT-p21 peptides (Fig.\u00a01), the VLC-1-TAT was not detected in the cell nucleus. Detailed analysis of the intracellular distribution of transduced VLC-1-TAT and correlation of the fluorescent signals with the phase contrast images (merge image, lower panel, Fig.\u00a03c) demonstrated that the peptide associates with sarcomeric structures. It showed strong enrichment at the I-bands representing the nonoverlapping zones of the thin actin filaments with the thick myosin filaments (linescan, lower panel, Fig.\u00a03c). After transduction, the VLC-1-TAT peptide remained inside the sarcomeric compartments of the cardiomyocytes for at least 1\u00a0h after transferring the loaded cardiomyocytes into the peptide-free buffer solution.\nUsing the same incubation protocol, we studied the effects of the cell-permeable VLC-1 peptide on muscle function by simultaneously monitoring the shortening amplitude and the systolic and diastolic cytoplasmic Ca2+ fluctuations (measured by Fura-2 fluorescence) of paced adult rat cardiomyocytes (Fig.\u00a04a). As shown in Figs.\u00a04b and c, 1\u00a0\u03bcM, but not 0.5\u00a0\u03bcM, of VLC-1-TAT significantly increased the shortening amplitude of adult cardiomyocyte, while the cytosolic Ca2+ signal remained unchanged. The same concentrations of control TAT peptide alone did not change the contractile state or the Fura-2 signals if compared to the basal state during the preincubation period. Higher concentrations of both control and test peptides induced deleterious effects on the cardiomyocyte contraction. It is interesting to note that not only the maximal amplitude of shortening rose but also the maximal rate of shortening and the maximal relaxation velocity increased significantly. Because the Fura-2 signal remained unchanged, the selective effect of the VLC-1-TAT peptide on the contraction parameters suggests a selective and direct effect on the function of the myosin cross-bridges. The Ca2+-sensitizing effect of the VLC-1-TAT peptide could be predicted from the mechanism of action, as the inhibition of the MLC-1\/actin interaction may rise selectively myosin motor activity and therefore muscle contraction at a given activating free Ca2+ concentration, i.e., an inotropic effect without additional recruitment of Ca2+. It should be noted that the VLC-1-TAT peptide left the intracellular Ca2+-handling system unchanged, as seen by the fact that the kinetic parameters of the Fura-2 fluorescence signals remained normal (Fig.\u00a04c).\nFig.\u00a04Functional effects of cell permeable VLC-1-TAT in living adult cardiomyocytes. a Original registration of cell length (top) and cytosolic Ca2+ signals (Fura-2 signal, expressed as ratio R between 340 and 380 excitation; bottom) of electrically paced adult rat cardiomyocytes before (basal) and after incubation with 1\u00a0\u03bcM VLC-1-TAT or 1\u00a0\u03bcM TAT as control. b Statistical evaluation of the effects of different concentrations of transducible peptides (0.5, 1\u00a0\u03bcM) on fractional shortening (FS; given in percent of basal contraction obtained before incubation with the peptide), maximal rate of FS (+dFS\/dtmax), and maximal relaxation rate (-dFS\/dtmax; both given in percent of basal values obtained before peptide incubation) of control peptide (TAT) and VLC-1-TAT. c Statistical evaluation of the effects of different concentrations of transducible peptides (0.5, 1\u00a0\u03bcM) on peak Fura-2 fluorescence signal (expressed as ratio R between 340 and 380 excitation, given in percent of basal R340\/380 obtained before incubation with the peptide), maximal rate of R340\/380 (+dR340\/380\/dtmax), and maximal rate of fluorescence decline (-dR340\/380\/dtmax; both given in percent of basal values obtained before peptide incubation) of control peptide (TAT) and VLC-1-TAT. Values are means\u2009\u00b1\u2009SEM, n\u2009=\u20096\u20139 different cardiomyocytes at each peptide concentration used. Double asterisk, p\u2009<\u20090.01; triple asterisk, p\u2009<\u20090.001 (t test for paired values)\nIn summary, we demonstrate by live-cell microscopy that CPPs and CPPs fused to peptides were taken up by adult cardiomyocytes with high efficiency and localize to their targets inside the cytoplasm and\/or nucleus. The cell-permeable peptide VLC-1-TAT accumulated in the actin-containing I-band of the sarcomeres and was able to enhance the contractility of isolated adult cardiomyocytes without changing the myoplasmic Ca2+ levels. Importantly, the fact that VLC-1-TAT did not affect intracellular Ca2+ concentration and that it only has targets in striated muscle cells makes this peptide uniquely suited as a novel potential therapeutic tool to modulate heart function. VLC-1-TAT is a powerful new drug candidate to improve the contractile state of the failing heart.","keyphrases":["cardiomyocytes","ventricular light chain-1 (vlc-1)","peptide transduction","cell penetrating peptide (cpp)","drug delivery"],"prmu":["P","P","P","R","R"]}
{"id":"Eur_J_Appl_Physiol-4-1-2226001","title":"Identification of elderly fallers by muscle strength measures\n","text":"For efficient prevention of falls among older adults, individuals at a high risk of falling need to be identified. In this study, we searched for muscle strength measures that best identified those individuals who would fall after a gait perturbation and those who recovered their balance. Seventeen healthy older adults performed a range of muscle strength tests. We measured maximum and rate of development of ankle plantar flexion moment, knee extension moment and whole leg push-off force, as well as maximum jump height and hand grip strength. Subsequently, their capacity to regain balance after tripping over an obstacle was determined experimentally. Seven of the participants were classified as fallers based on the tripping outcome. Maximum isometric push-off force in a leg press apparatus was the best measure to identify the fallers, as cross-validation of a discriminant model with this variable resulted in the best classification (86% sensitivity and 90% specificity). Jump height and hand grip strength were strongly correlated to leg press force (r = 0.82 and 0.59, respectively) and can also be used to identify fallers, although with slightly lower specificity. These results indicate that whole leg extension strength is associated with the ability to prevent a fall after a gait perturbation and might be used to identify the elderly at risk of falling.\nIntroduction\nOne-third of those over 65\u00a0years of age fall at least once per year. About 50% of them will suffer recurrent falls (Masud and Morris 2001). Even in the large group of relatively fit and healthy elderls, falls are common (Stel et al. 2003a). Consequences of falls are known to contribute substantially to the prevalence of health problems (Lord and Dayhew 2001), health care costs (Stevens et al. 2006), and to lost quality of life (Cumming et al. 2000). Prevention of falls is therefore imperative and should address the people at risk and the physiological causes of falling (Lord et al. 2003).\nNumerous epidemiological studies have shown that falls are associated with many risk factors (for overviews see e.g., Lord et al. 2001). Reduced muscle strength, especially of the lower limbs, has been indicated as one of the most important risk factors for falls (Lord et al. 2003; de Rekeneire et al. 2003; Moreland et al. 2004; Rao 2005; Rubenstein 2006; Skelton et al. 2002). Experimental studies can provide causal verification and insight into the contribution of muscle strength to fall incidence in standardized situations. For example, tripping over an obstacle, which accounts for up to 60% of falls (Berg et al. 1997; Roudsari et al. 2005), can be induced and measured in older adults in experimental settings (Pavol et al. 1999; Pijnappels et al. 2005c; Schillings et al. 2005). Previous findings provided insight into the (neuro)physical requirements of preventing a fall after tripping over an obstacle (for overviews see van Die\u00ebn et al. 2005; Pijnappels et al. 2007). It was shown that older adults were less able to successfully recover balance than younger adults, due to lower maximum ankle moments and lower rates of moment generation around all leg joints during the push-off phase of balance recovery (Pijnappels et al. 2005c). These results suggest that leg strength may be the limiting factor in preventing a fall.\nPavol et al. (2002) have investigated the relation between muscle strength and the outcome of an induced trip. In a group of older adults, they compared isolated ankle, knee, and hip flexion and extension strength between those who recovered from a trip, and those who fell. Although some strong adults fell, presumably due to their high walking velocity, they also found that muscle strength was lower in several other fallers.\nIf low muscle strength indeed is a physiological cause of falls, this raises the question whether people with a high risk of falling can be identified by means of relatively simple maximum muscle strength measures. The aim of this study was to find the best predictor of falls after a gait perturbation in a standardized situation, from a range of muscle strength measures in older adults.\nThere are numerous maximum strength capacity measures that have been assessed in aged populations, for example to evaluate training interventions (Ferri et al. 2003; Reeves et al. 2005b; Verschueren et al. 2004) or to compare fallers with non-fallers (Gehlsen and Whaley 1990; Pavol et al. 2002). We focused on the most common measures by use of a dynamometer, isometric ankle and knee extension moment and rate of moment generation. In addition to isolated joint moments, total lower limb push-off force can be measured by a leg press dynamometer (Gehlsen and Whaley 1990). Performance on a maximum vertical jump could be a more functional test used to assess maximum lower limb strength (Izquierdo et al. 1999; Runge et al. 2004). Finally, hand grip strength is thought to reflect general body strength and has been used as predictor of falls in epidemiological studies (Pluijm et al. 2006).\nElderly volunteers performed these maximum strength capacity tests and we measured their ability to prevent a fall after being tripped. We hypothesized that maximum muscle strength measures can be used to differentiate fallers from non-fallers. If so, this would not only allow identification of potential fallers, but also indicate specific limiting factors to target with exercise-based interventions.\nMethods\nSeventeen healthy older adults participated [10 women: age 71 (SD 4.5)\u00a0years, mass 75 (SD 9)\u00a0kg, height 1.68 (SD 0.09)\u00a0m]. All participants were fit and had no orthopedic, neuromuscular, cardiac or visual problems. The Ethics Committees the VU Medical Center and of the Manchester Metropolitan University approved the procedure and all subjects gave their written, informed consent before participation.\nCapacity measures\nParticipants performed a series of static and dynamic maximum force generating capacity tests. First, isometric ankle plantar flexion moment and knee extension moment (maximum as well as rate of moment development) were assessed using a dynamometer. In view of constraints on equipment and subject availability, nine of the participants were tested using an isokinetic dynamometer (Cybex Norm, New York, USA) (Reeves et al. 2004) and eight using a custom-built dynamometer (de Ruiter et al. 2004) (Fig.\u00a01a, b). Isometric knee extension moment was measured in both settings with a knee joint angle of 90\u00b0 (0\u00b0\u00a0full extension) and a hip angle of 85\u00b0 (0\u00b0\u00a0supine position). Isometric plantar flexion moment was measured using the Cybex with the knee in full extension and the ankle at 0\u00b0 (anatomical position). In the custom ankle dynamometer, the knee joint was fixed at 90\u00b0 and to obtain a similar gastrocnemius muscle length compared to that during the tests using the Cybex, the ankle angle was set at 20\u00b0 dorsiflexion (see equation in Reeves et al. 2005a, p. 2280). Plantar flexion moment of the right leg (push-off leg in the tripping experiments) and knee extension of the left leg (landing leg for tripping) were measured. Measurements resulted in comparable values between settings and were repeated 3 times with 3\u00a0min rest between contractions. Subjects first performed a maximum isometric contraction by increasing their effort in a linear ramp fashion, so that maximum moment was reached within 2\u00a0s and then maintained for 3\u20134\u00a0s. From these measurements, the maximum moments (anklemax and kneemax) were calculated, relative to body mass. Next, subjects were instructed to increase their moment from 0 to at least 70% of their maximum as quickly as possible, without counter movement. Moment onset (2\u00a0N\u00a0m above baseline) was determined and rate of moment development (anklertd and kneertd) was calculated over the first 100\u00a0ms after moment onset and expressed relative to body mass.\nFig.\u00a01Pictures of the experimental settings for a right plantar flexion moment measurements, b left knee extension moment measurements, c leg press push-off force measurements, d jumping measurements, and e tripping experiments\nMaximum leg press push-off force of each lower limb was measured for all subjects using the same leg press fitness instrument (MR Systems, Haarlem, the Netherlands) (Fig.\u00a01c). Subjects performed a one-legged (horizontal) push-off, starting with the ankle joint angle at 0\u00b0 and the knee joint angle at 30\u00b0. This position was comparable to the configuration of the support limb during tripping (Pijnappels et al. 2005a, c). The fore-foot was positioned on a 10\u00a0\u00d7\u00a010\u00a0cm force transducer (AMTI M3-1000, Watertown, USA) that was mounted in the push-off platform. Subjects first performed a maximum isometric contraction with the sled fixed and subsequently a maximum dynamic contraction with the weight stack set equal to body mass, so the only external resistance was the mass and inertia of the body and sled. Both conditions were repeated 3 times for both legs with a 3\u00a0min rest between contractions. The maximum horizontal leg press push-off force (legpressmax) was calculated from the isometric contractions and the rate of force development (legpressrfd) was calculated from the dynamic contractions over 100\u00a0ms after onset of force generation, averaged over both legs and expressed relative to body mass.\nFor the jumping measurement, subjects performed a maximum two-legged counter movement jump (Fig.\u00a01d). Position data of markers placed bilaterally on the trochanter major were measured by Optotrak cameras (Northern Digital, Waterloo, Canada). From the best of two attempts, maximum jump height (jumph) was calculated as the vertical distance between the highest averaged hip position during jumping and averaged hip height during normal standing.\nHand grip strength was measured using a digital strain-gauge dynamometer (Takei TKK 5401, Takei Scientific Instruments, Tokyo, Japan). Subjects performed two maximum force trials with each hand. The maximum values of the right and left hand (in kg) were added together (handf).\nTripping measurements and falls\nThe ability to prevent a fall after a gait perturbation was determined by tripping measurements (Pijnappels et al. 2005c). Subjects walked at a self-selected velocity over a 12\u00a0m walkway. Fourteen 15\u00a0cm high obstacles were hidden at the left side and seven obstacles were hidden at the right side of the walkway, over a total distance of 1.5\u00a0m (Fig.\u00a01e). In 2\u20135 of 40 trials, one of the obstacles suddenly appeared (based on online kinematic data) to catch the subject\u2019s left swing leg at mid-swing, usually causing an elevating strategy for balance recovery. This strategy involves the limb contacting the obstacle being elevated over the obstacle (Eng et al. 1994; Schillings et al. 2000), whilst the contralateral support limb provides push-off. Subjects were aware that they could be tripped on either leg, but did not know whether and where an obstacle would appear in the ongoing trial. They wore a safety harness that moved along a track above the walkway. Subjects were classified as fallers based on visual detection of full body support by the harness (checked by video), in more than 50% of the tripping trials.\nStatistical analysis\nPearson\u2019s coefficients of correlation between the capacity measures were calculated. In case of high correlations between the dependent variables, as we found, the power of a multivariate analysis of variance (MANOVA) decreases when the expected effect sizes are reasonably large and consistent in the same direction (Cole et al. 1994). We therefore focused on separate univariate ANOVAs to determine which capacity measures differed between fallers and non-fallers. The variables that appeared to be determinants of falls from the ANOVAs were used in discriminant analyses, in order to find the variable(s) best discriminating between fallers and non-fallers and to determine sensitivity and specificity. In addition, a cross-validation was performed to assess the generalizability of the predictive models; each case was classified by the functions derived from all other cases (leave-one-out-classification). SPSS statistical software was used and the level of significance was set at p\u00a0<\u00a00.05.\nResults\nTripping measurements and falls\nSeven of the participants (all women) were fully supported by the safety harness in more than half of the tripping trials. Six of them fell in all trials; the other fell in three out of five trials. Non-fallers were never fully supported by the safety harness. The averaged walking velocity was 1.38 (SD 0.17)\u00a0m\/s and did not differ between fallers and non-fallers (p\u00a0=\u00a00.488).\nCapacity measurement as predictor for falls\nlegpressmax, jumph, and handf were significantly correlated with almost all other capacity measures (Table\u00a01). Leg press push-off force was not significantly different between the left and right leg of the subjects (p\u00a0=\u00a00.496 for legpressmax and p\u00a0=\u00a00.149 for legpressrfd), there was no significant interaction with groups (p\u00a0=\u00a00.133 for legpressmax and p\u00a0=\u00a00.919 for legpressrfd), and the values were highly correlated between legs (overall, r\u00a0=\u00a00.813, p\u00a0<\u00a00.001 for legpressmax and r\u00a0=\u00a00.829, p\u00a0<\u00a00.001 for legpressrfd).\nTable\u00a01Correlation coefficients between capacity measuresanklertdkneemaxkneertdlegpressmaxlegpressrfdjumphhandfanklemax0.63b0.230.320.76b0.370.61b0.40anklertd10.360.70b0.68a0.400.65b0.51akneemax10.72b0.53a\u22120.020.340.71bkneertd10.57a0.360.55a0.78blegpressmax10.330.82b0.59alegpressrfd10.290.23jumph10.69bSignificant correlations are in bold.ap\u00a0<\u00a00.05bp\u00a0<\u00a00.01\nAll but one of the univariate ANOVAs resulted in significant differences, indicating several measures to be different between fallers and non-fallers (Fig.\u00a02). Ankle and knee capacity measures (both maximum and rate of moment development), maximum leg press force, jump height, and grip strength were significantly lower for fallers compared to non-fallers.\nFig.\u00a02Averaged group differences in capacity measures between fallers and non-fallers. Maximum (max) and rate of moment development (RTD) of plantar flexion moment (anklemax and anklertd) and knee extension moment (kneemax and kneertd), maximum and rate of force development (RFD) of leg press push-off force (legpressmax and legpressrfd), jump height (jumph), and hand grip strength (handf). Significant differences between groups are indicated with p-values\nThese seven variables were entered in a stepwise discriminant analysis, which resulted in a significant model (p\u00a0=\u00a00.001) with legpressmax as the only predictor. This model resulted in 94% correctly classified cases and a cross-validation of 88% (Table\u00a02). For comparison, discriminant analyses with the ankle and knee capacity tests (stepwise) and with jump height and grip strength as single predictors also resulted in significant predictive models, with slightly lower predictive values (Table\u00a02). Figure\u00a03 presents the distribution of individual jumph,, and handf values, both in relation to legpressmax.\nTable\u00a02Predictive variables resulting from stepwise discriminant analyses and cross-validation on capacity measuresCapacity measuresPredictive variablepDiscriminant analysis (sensitivity\/specificity%)Cross-validation (sensitivity\/specificity%)legpressmax, anklemax, anklemax, kneemax, kneertd, jumph, handflegpressmax0.00186\/10086\/90aanklemax, anklertd, kneemax, kneertdanklemax, kneemax0.00771\/9071\/90jumphjumph0.00286\/9086\/80handfhandf0.00386\/8086\/80aBest predictive modelFig.\u00a03Distribution of the individual values of maximum leg press push-off force (legpressmax) with jump height (jumph) and hand grip strength (handf) for fallers and non-fallers. Crosses indicate the incorrect classified cases (x for legpressmax, + for jumph and handf)\nDiscussion\nIt is important to identify individuals most at risk of falling, because they should be considered with priority for receiving targeted exercise interventions aimed at reducing the incidence of falls. This study investigated the possibility to identify fallers from maximum strength measures that could be applied in clinical settings. The results showed that participants who fell after a gait perturbation could indeed be identified based on these measures. Especially, a classification model based on maximum leg press push-off force yielded high sensitivity and specificity in cross-validation.\nThe subjects in this study were relatively fit and healthy older adults. Nevertheless, muscle strength varied greatly between subjects, with coefficients of variation between subjects ranging from 23 to 60% for all strength measures. Pavol et al. (2002) found that not only people with low muscle strength, but also the stronger people had a higher risk of falling after a trip. This was explained by a higher walking velocity in the stronger group, which increases the demands for adequate balance recovery. These subjects mainly used a lowering strategy, which implies that they were tripped at late swing (Eng et al. 1994; Pijnappels et al. 2005b). Since the trip was induced manually, it may be that faster walkers were more likely to be tripped in late swing. It is therefore unclear whether velocity or instant of trip initiation was responsible for a number of stronger subjects falling. In our study, however, walking velocity was not different between fallers and non-fallers and in the range of \u201cfast walkers\u201d of the study of Pavol, even though we did not standardize it and explicitly instructed subjects to walk at their normal and comfortable velocity. Furthermore, we were able to trip all subjects at exactly mid-swing (when the toe height is minimal and a trip is most likely in daily life). This implies that the effect of the trip by impact with the obstacle was equal over subjects and could not affect the outcome of the trip. Yet, this also may suggest that instant of trip initiation and in a more heterogeneous group, differences in preferred walking velocity must be taken into account when attempting to identify fallers.\nAlthough we were able to trip our subjects repeatedly, three of the fallers and one non-faller did not complete the whole protocol up to five tripping trials, due to discomfort. Nevertheless, the fallers fell in all trials and the non-faller did not fall in four tripping trials. Hence, the number of tripping trials did not affect the classification of the participants.\nHip extension and knee flexion strength were not measured, but might also contribute to fall prediction, as the rate of development of hip extension and knee flexion moments during push-off after tripping were found to be lower in older fallers compared to young adults (Pijnappels et al. 2005c). Since especially hip extension capacity is quite difficult to determine (especially in a clinical setting) and given limitations in the total number of capacity measurements to be performed in a single session, these variables were not included in the present study.\nDuring the push-off phase of a tripping reaction, the knee is generating an internal flexion moment while extention (Pijnappels et al. 2005a). Preliminary data suggest that a high extension moment is required in the leg making ground contact at landing (van Die\u00ebn et al. 2005) and a limited capacity to generate sufficiently high knee extension moments might thus increase the fall risk. In this study, fallers could not position their recovery foot adequately and were therefore fully supported by the safety harness, mostly even before landing. This underlines that most of the balance recovery takes place during push-off by the support limb. However, as falls can also occur after landing (Pavol et al. 2001), the requirements for landing need further investigation.\nFallers and non-fallers were classified in this study based on their ability to prevent a fall after an experimentally induced gait perturbation. In daily life, there are many various ways in which people fall. Furthermore, this experimental study included a small number of participants, which might have influenced the predictive values. A prospective study with a larger cohort is necessary to generalize the results of this study to the prediction of falls in daily life.\nOverall, sufficient leg extension strength is required for adequate balance recovery after tripping. This might explain why a capacity measure that involves extension of all lower limb joints (i.e., leg press push-off) yielded the best identification of fallers. This one-legged task is highly similar to the actual movement during the push-off phase of tripping (Pijnappels et al. 2005a, c).\nJumping also requires rapid moment generation and was therefore expected to be a good and practical measure of whole lower limb strength. Studies have shown that maximum jumping forces are lower in the elderly (Izquierdo et al. 1999; Runge et al. 2004) and related to balance performance (Izquierdo et al. 1999). Jump height indeed resulted in good sensitivity and specificity. This task was less similar to the actual tripping action than the leg press and does not solely reflect strength, as it also requires complex coordination of many joints. One might argue that the older fallers, who were all women, may have been more afraid to jump maximally than their counterparts. However, jump height was strongly correlated to maximum leg press push-off force, which was measured in a secure and comfortable setting. Jumping performance, therefore, more likely reflects lower limb strength than an effect of gender or fear.\nFall incidents and the ensuing injury process are multi-factorial. Muscle strength is only one of many factors that associate with falls and that has to be taken into account when identifying one's fall risk. Yet, this particular factor has been indicated as one of the main causal factors for falls and is essential in risk profiles. For example, in the physiological profile approach by Lord et al. (2003) muscle force is tested in lower extremity muscle groups. However, these tests are not based on empirically demonstrated causal relations. Our experimental study allowed investigating the relation between strength measures and gait perturbations in a standardized situation. It can be questioned whether the findings in this study on tripping over an obstacle in mid-swing can be transferred to other fall mechanisms that occur in daily life. Yet, leg extension force has also been indicated to play an important role in other fall mechanics, such as slipping (Chambers and Cham 2007; Tang and Woollacott 1999). The results of this study may therefore contribute to improvement of risk profiles for identification of high-risk fallers, particularly by using relatively simple but specific measures of maximum strength.\nFor clinical use, a maximum vertical jump test might be more practical than the leg press test, as it requires little instrumentation. Rittweger et al. (2004) demonstrated that such a test is feasible also for subjects over the age of 80 and that reliability is high.\nHand grip strength is a common clinically used strength capacity measure. It has been shown in epidemiological studies to be related to leg extension strength and to be associated to recurrent falling in daily life (Stel et al. 2003b). Our data indeed showed that grip strength is significantly correlated with lower limb capacities and that it can be used to identify fallers from non-fallers. Although hand grip strength is easy to measure and assumed to be related to whole body strength, one should bear in mind that this measure may be less accurate as a predictor for falls it is not task specific for balance recovery.\nWith respect to fall prevention, the associations found in this study suggest that maximum whole leg extension force is an important target in conducting exercise-based interventions. In a pilot study, we showed that lower limb resistance training has the potential to improve balance recovery, as older trainers improved more in moment generation upon tripping than controls (Pijnappels et al. 2007). It can be questioned whether fall prevention training should focus on strength training alone, as task-specific training of motor skills has been shown to be important (Bieryla et al. 2007; Owings et al. 2001; Troy and Grabiner 2005). A combination of both strength and balance training is most likely to be most beneficial for reduction of fall incidence (Gillespie et al. 2003). Finally, it should be noted that training effects are only valuable if task-specific requirements can be achieved and it can be questioned whether this is the case in older and more frail elderly. Therefore, resistance training may be particularly useful in the group of relatively fit and healthy elderly (Barry and Carson 2004; Hunter et al. 2004; Latham et al. 2004; Macaluso and De Vito 2004), which might prevent these people from becoming recurrent fallers.\nConclusion\nRelatively simple and accessible measures of maximum strength did identify elderly fallers from non-fallers after a standardized gait perturbation. The capacity to generate maximum extension force by the whole leg (e.g., in a leg press apparatus or during jumping) resulted in the best classification of older fallers and non-fallers. Follow up studies on larger cohorts with a wide range of muscle strength and walking velocities are necessary to generalize these results towards a valid prediction of fall risk.","keyphrases":["perturbation","classification","ageing","fall risk","physical capacity"],"prmu":["P","P","P","P","M"]}
{"id":"Eur_Spine_J-4-1-2226193","title":"Clinical effect of continuous corrective force delivery in the non-operative treatment of idiopathic scoliosis: a prospective cohort study of the triac-brace\n","text":"A prospective cohort study of skeletally immature idiopathic scoliotic patients treated with the TriaC brace. To determine if the TriaC brace is effective in preventing curve progression in immature adolescent idiopathic scoliotic patients with a very high risk of curve progression based on reported natural history data. The aim of the newly introduced TriaC brace is to reverse the pathologic transverse force pattern by externally applied and continuously present orthotic forces. In the frontal plane the force system used in the TriaC brace is similar to the force system of the conventional braces. However, in the sagittal plane the force system acts only on the thoracic region. In addition, the brace allows upper trunk flexibility without affecting the corrective forces during body motion. In a preliminary study it is demonstrated that the brace prevents further progression of both the Cobb angle and axial rotation in idiopathic scoliosis. Skeletally immature patients with idiopathic scoliosis with curves between 20 and 40\u00b0 were studied prospectively. Skeletally immature was defined as a Risser sign 0 or 1 for both boys and girls, or pre-menarche or less than 1-year post-menarche for girls. Curves of less than 30\u00b0 had to have documented progression before entry. The mean age of the patients at the start of treatment was 11.3 \u00b1 3.1 years. All measurements were collected by a single observer, and all patients were followed up to skeletal maturity. Treatment was complete for all participants when they had reached Risser sign 4 and did not show any further growth at length measurements. This was at a mean age of 15.6 \u00b1 1.1 years, with a mean follow-up of 1.6 years post bracing. In our study a successful outcome was obtained in 76% of patients treated with the TriaC brace. Comparing our data to literature data on natural history of a similar cohort shows that the TriaC brace significantly alters the predicted natural history. The current study demonstrates that treatment with the TriaC brace reduces the scoliosis, and that the achieved correction is maintained in some degree after skeletal maturity is reached and bracing is discontinued. It also prevents further progression of the Cobb angle in idiopathic scoliosis. The new brace does not differ from the conventional braces as far as maintaining the deformity is concerned.\nIntroduction\nThe purpose of this paper is to present the results of a prospective study of a new orthotic device for the non-operative treatment of idiopathic scoliosis. Data are provided in a format to facilitate comparison with natural history data.\nThe main concern in patients with idiopathic scoliosis relates to curve progression and the resulting cosmetic deformity. The risk of curve progression is correlated primarily to periods of rapid skeletal growth [3, 5, 6, 11, 14, 41, 42]. Factors related to growth potential, such as patient age at the time of diagnosis, status of menarche, and Risser sign, have been shown to be important predictors of the progression of scoliosis [3, 15, 29, 39]. In addition to future skeletal growth, curve magnitude and curve shape are further predictors of progression of idiopathic scoliosis. Large initial curves, thoracic curves, and double major curves are more likely to progress [3, 29, 39].\nCurrently bracing is the accepted nonoperative treatment to prevent curve progression in mild to moderate scoliosis during the growth period. A prospective multicenter study, performed by the Scoliosis Research Society, reported that brace treatment has a significant effect on curve progression of idiopathic scoliosis [36]. However, others doubt the effectiveness of braces [12, 16, 19, 32]. Just as for the natural history of untreated curves, Lonstein and Winter [30] found a relationship between the final outcome of brace treatment and curve factors and factors that predict future skeletal growth. In biomechanical theories describing the patho-physiological mechanism of scoliosis, spinal growth is thought to be the main driving force responsible for curve progression [35, 42, 44, 47, 48].\nThroughout history, external devices have been used to correct deformities and immobilize the spine. Brace designs have changed periodically over the years, but most modifications have solely focused on improved efficacy and failed to acknowledge the importance, especially to teenagers, of physical appearance. This age group resists acting or looking different from their peers, which obviously occurs when a visible brace is worn. Modern materials, lower profiles, and reduced wearing times have been tried in attempts to reduce resistance to and the emotional difficulties encountered with wearing braces.\nThe aim in the design of a new orthosis was to create a system which is at least as effective as the TLSO\u2019s, with improved comfort for the wearer. The newly introduced brace should reverse the transverse force pattern by externally applied and constantly present orthotic forces without limiting the normal body motions of the patient and an increased comfort level for the patient.\nConstruction and working principle of the TriaC orthosis\nThe name TriaC is based on the three C\u2019s of Comfort, Control, and Cosmetics. Currently, the orthosis is produced by Somas International, St Anthonis, the Netherlands. For this orthosis, the choice has been made to apply continuous correction forces on the chest with the aim to prevent curve progression during the growth period. A basic requirement for such a brace is that the brace force must be able to follow the main body motions of the patient. To achieve this goal required the use of a flexible coupling, connecting the thoracic and lumbar parts of the orthosis. The thoracic part and lumbar sections each generate their own corrective force on the body of the patient. Both parts are connected to each other by a flexible coupling that is placed on the opposite side of the thoracic force pad (Fig.\u00a01). This coupling enables the patient to bend forward, backward and sideways while the correction forces are maintained (Fig.\u00a02). The thoracic force pad is located just below the shoulder blade and applies a corrective force in a lateral\u2013anterior direction. The lumbar force pad is placed between the pelvis and the lower ribs and acts on the lumbar muscles in a lateral direction. A third counter force on the hip region ensures that the orthosis is in equilibrium.\nFig.\u00a01The TriaC orthosis, with a thoracic part, a lumbar part and a flexible couplingFig.\u00a02The flexible coupling\nPlacing a flexible coupling between the thoracic and the lumbar part of the orthosis determines the unique force pattern on the patient both with respect to the location of the forces as with respect to the relation between the forces (Fig.\u00a02). Modelling and clinical use have shown that the TriaC is suitable for all curve types, with the exception of curves with an apex at the 12th thoracic and the 1st lumbar vertebra. For these curve type the Lumbar force pad must be placed at the location of the flexible coupling which is not possible in the current construction of the orthosis. The construction as described, makes the orthosis force driven. When the patient moves her or his body in any direction (e.g. flexion, extension, rotation or lateral bending), the force pattern exerted by the orthosis moves along with the patient and thus is maintained constant. For this reason we defined the orthosis as \u201cdynamic\u201d because the orthosis moves dynamically along with the body motions of the patient.\nIn contrast, conventional orthoses are displacement driven. A rigid shell shaped in the desired corrected position of the patient applies a corrective force only when the thorax of the patient presses against the pads of the brace. When the body of the patient moves away from these pads the rigid brace is not able to follow the body motion of the patient and therefore the brace no longer generates forces on the patient. Therefore, we defined these orthosis as \u201cstatic\u201d.\nMaterials and methods\nFrom 1997 to 2005 all consecutive patients who met the inclusion criteria were treated nonoperatively with the TriaC orthosis and studied prospectively. The indication for treatment was a progressive curve with a Cobb angle between 25 and 40\u00b0. All curves, except curves already at >30\u00b0, had to show documented progression of at least 5\u00b0. Patients had to be skeletally immature, defined as a Risser sign 0 or 1 for both boys and girls; in addition, girls are pre-menarche or less than 1-year post-menarche. All the curves had to be flexible as demonstrated by at least a 40% correction on the bending radiograph at the first visit. Patients with the apex of the curve at T12 and L1 were excluded, as were patients with a systemic disease that could possibly influence the outcome of the study (Table\u00a01).\nTable\u00a01The in and exclusion criteria for the treatment with the TriaC orthosisInclusion criteria\u00a0Idiopathic scoliosis with a Cobb-angle between 20 and 40\u00b0\u00a0Skeletally immature\u00a0\u00a0Risser 0\u20131 status\u00a0\u00a0\u00a0Pre-menarche\u00a0\u00a0\u00a0Post-menarche <1\u00a0year\u00a0Primary thoracic apex between the 7th and 11th thoracic vertebra\u00a0Primary lumbar apex between the 2nd and 5th lumbar vertebraFlexible spinal column as evidenced by at least 40% correction on bending filmsExclusion criteria\u00a0Idiopathic scoliosis <20\u00b0 and >40\u00b0\u00a0Other types of scoliosis\u00a0Skeletal age >Risser 1\u00a0Rigid curves\u00a0Thoraco-lumbar curves with an apex at the 12th thoracic and the 1st lumbar vertebra\u00a0Patients with a systemic disease which could influence the study parameters\nAll patients who met the in- and exclusion criteria were treated with the TriaC orthosis. An informed consent form was obtained from all parents of the subjects in regard of the treatment with a new orthosis. According to the medical ethics committee no informed consent was necessary in case of the follow-up schedule because there is no difference with the patients treated with a Boston brace in our clinic.\nIf during the treatment period the orthosis failed to stop progression and the curve increased significantly, the patient was placed into a Boston Brace. This procedure was chosen to get a first indication whether a Boston brace would be able to prevent curve progression in patients where the TriaC failed.\nFailure was defined as either a Cobb angle of >45\u00b0, i.e. the generally accepted indication for surgery, or if the curve progressed 5\u00b0 or more compared with the measurement at study entry [16, 24\u201331]. During the nonoperative treatment patients were checked by an orthotist at the outpatient clinic every 4\u00a0months, where the subjective compliance was documented. At every visit a PA and lateral radiographs were made. The patients were allowed to wean of the braces at skeletally maturity, i.e. when the radiographs showed Risser sign 4 or, for girls, 2\u00a0years post-menarche and patients did not show any further growth at length measurements. At every visit standing and sitting height of the patient was recorded as an additional measurement, and these had to show no further increase. Because the TriaC is designed as a dynamic orthosis, it possibly preserves the muscle status of the patient. As a result the weaning process could be faster than with rigid Boston type orthoses. In this study, weaning was generally complete within 4\u00a0weeks, whereas for rigid braces this process may take up to one year. After terminating the treatment patients were seen once a year with an AP and lateral X-ray.\nRadiographic analysis\nAll radiographs were obtained in a standing position. The posterior\u2013anterior projection was used to minimize the radiation dose to the breast. All radiographs were made, using a standardized protocol. Anatomical vertebral landmarks are identified and manually marked on a 21\u00a0in. high resolution monitor using a pointer [46]. Following the placement of the markers the Cobb angle is calculated, using automatic detection of the apex and end vertebrae [8].\nIn this article, only the Cobb-angle data are used for comparison with literature data on the natural history.\nResults\nOut of a total of 212 patients that were treated at the University Hospital of Groningen during the study period, 63, or 30%, met all the in- and exclusion criteria and form the study group. There were seven patients (11%) with a single curve and 56 (89%) with a double curve. The group included six boys and 57 girls. The mean age of the patients at the start of treatment was 11.3\u00a0\u00b1\u00a03.1\u00a0years. The treatment was complete when the patients had reached Risser 4 with a mean age of 15.6\u00a0\u00b1\u00a01.1\u00a0years. The mean Cobb angle of the primary curves before the brace treatment was started was 30.2\u00b0\u00a0\u00b1\u00a07.5\u00b0. The secondary curves had a mean Cobb angle of 22.3\u00b0\u00a0\u00b1\u00a0\u00a06.4\u00b0.\nThe distribution of the location of the apex vertebra of the primary curves is shown in Fig.\u00a03.\nFig.\u00a03The distribution of the apex level of the primary curves in the study group\nThe patient group was divided into a success group and a failure group whereby failure was defined as a Cobb angle of >45\u00b0 or curve progression of more than 5\u00b0 compared to baseline [2, 7, 9, 18, 21, 33, 34, 36, 40].\nThe X-rays showed a mean initial correction of 22\u00a0\u00b1\u00a026% for the primary curves, and a mean correction of and 28\u00a0\u00b1\u00a035% for the secondary curves. The large variation was primarily caused by the big difference between the initial corrections seen in patients that were considered a success in comparison to patients that were considered a failure. In the success group the initial correction of the primary curve was 34\u00a0\u00b1\u00a017% whereas the initial correction in the failure group was \u221216\u00a0\u00b1\u00a017%. For the entire study group the success rate is 76% (48 successes) for a failure rate of 24% (15 failures). In the single curve group the mean Cobb angle at start of the treatment was 35\u00b0\u00b1\u00a011. The mean initial correction achieved in this group was 23\u00a0\u00b1\u00a09%. There were no failures in this group. Although this difference is remarkable compared to the double curved group the single curve group is too small to draw conclusions.\nIn Table\u00a02 an overview of the success rate is shown, divided per initial Risser sub group (Risser 0 and Risser 1) and divided by the initial Cobb angle at start of the treatment.\nTable\u00a02Number of successes\/total patients in subgroupsCobb angleSuccess rateRisser 0Risser 120\u201329\u00b076% (16 of 21 patients)30\u201339\u00b074% (26 of 35 patients)86% (6 of 7 patients)Total75% (42 of 65 patients)86% (6 of 7 patients)\nThe actual number of failures in our patient population was 15 out of 63 patients, or 24%. All patients in whom the brace failed went on to surgery, including the nine patients who were also treated with a Boston style brace after the TriaC brace had failed. A more detailed view of the correction of the primary Cobb angle is presented in Fig.\u00a04. In this figure the mean correction percentage of the Cobb angle is shown as a function of the percentage of the total treatment brace period. The treatment period is considered to be at 0% at the start of the treatment and at 100% at the time the brace is discontinued.\nFig.\u00a04Percent correction of the Cobb angle over the entire TriaC treatment period in the success group\nIt is shown that it was not possible to completely maintain the initial correction for the whole treatment period. At the end of treatment, the mean correction of the Cobb angle in the success patients had decreased to 19\u00a0\u00b1\u00a013%. This decrease in correction does not appear to follow a linear pattern. When a relation between the time that the patient wears the brace, and the loss of correction in the brace is assumed the correlation is only (very) weak (\u03c1\u00a0=\u00a0\u22120.33). The mean correction at the first visit after discontinuation of the brace the treatment was 20\u00a0\u00b1\u00a015%. All patients in whom the brace treatment was a success were followed with a mean follow-up of 1.6\u00a0years and longest follow-up of 6\u00a0years post-bracing. Figure\u00a05 shows the mean correction at different follow-up periods postbracing.\nFig.\u00a05The mean correction during follow-up after termination of successful brace treatment\nBecause the orthosis does not affect the lateral curvature no significant changes were expected. The analysis of the sagittal curve is displayed in Fig.\u00a06.\nFig.\u00a06Lateral curve during treatment\nDiscussion\nBraces are the oldest recorded method of treatment for spinal injury and deformity. The primary goal in treating patients with scoliosis deformities is to stabilize the curves to prevent further progression of the deformity. Closely related to this is the goal of achieving correction of the spinal deformity, although this is not part of the therapeutic regimen for every patient. Brace treatments do not generally correct the scoliosis, but prevent further progression, i.e. bracing has a \u201cholding effect\u201d [49]. In most published studies, the brace treatment has been considered a failure if the patient needed operative stabilisation or if the curve progressed 5\u00b0 or more after the initiation of treatment [2, 7, 9, 18, 21, 30, 33, 34, 40]. However, these should not be the only criteria to determine whether brace treatment is successful. The treatment should also improve the patient\u2019s outcome when compared with the expected natural history.\nThe aim of this study was to investigate whether the TriaC-brace affects the natural history of idiopathic scoliosis. Bunnell [3], studied the natural history of idiopathic scoliosis in a group of patients with similar characteristics to our study group with respect to curve magnitude and age at the first visit. He showed that for the group as a whole 68% show a curve progression of 5\u00b0 or more and only 34% of the patients showed a curve progression of 10\u00b0 or more. The latter progression rate is in the same range as the failure rate reported in most of the retrospective studies, regarding overall brace effectiveness. However, the progression rates are not equal for every sub group of scoliotic patients. Lonstein and Carlson [29] reported that patients with Risser 0 or 1 maturity and curves larger than 20\u00b0 are three times more likely to experience curve progression than those with curves smaller than 20\u00b0. For those with curves exceeding 20\u00b0, patients with Risser 0 or 1 maturity also are three times more likely to experience curve progression than are patients with Risser 2\u20135 maturity. They classified Risser 0\u20131 patients with curves between 20 and 29\u00b0 as high risk (40\u201370%) and Risser 0\u20131 patients with curves between 29 and 39 as very high risk (70\u201390%). Bunnell [3] also found Risser sign and curve size to be good prognostic factors for curve progression in untreated idiopathic scoliosis. In his series patients with a Risser 0 at the time of diagnosis had a 68% risk of progression 10\u00b0 or more. This risk was decreased to 52% in those who had a Risser sign of 1 or 2 and was further decreased to 18% for those with Risser 3 or 4. Nachemson et al. [36] reported that 66% of skeletally immature female patients with untreated idiopathic curves between 25\u00b0 and 35\u00b0 will experience curve progression greater than 5.\nAll patients in our study started at Risser 0\u20131 maturity and had a curve above 30\u00b0 or, if they had an initial curve below 30\u00b0 had to show documented progression of \u22655\u00b0 before enrolment in this study. Wever et al. [47] showed that there is a strong correlation between the curve progression and spinal growth of the patients. Usually the spinal growth is highest around the beginning of the menarche and therefore we only included female patients when they were before or less than 1\u00a0year after menarche. By using these inclusion criteria we aimed to include only patients at the highest risk for curve progression.\nIn order to evaluate whether the TriaC brace alters the natural history of the high-risk patients, we estimated the natural progression rate of our patient group based on the literature as discussed. Our estimates are based on the calculation of a progression factor for all patients in the cohort as suggested by Lonstein and Carlson [29]. According to their algorithm, all our patients individually would have a change of 70\u2013100% of progression of their curves. Based on these data we chose the low end of this range and assumed that in our patient group 70% of the curves would progress without treatment. The actual number of failures in our patient population was 15 out of 63 patients, or 24%, which is a significantly different from the expected number of 44 (P\u00a0<\u00a00.001).\nThe validity of the sample size was evaluated by calculating the 95% confidence interval for the failure rate, which was between 11 and 36% (7\u201323 patients). Thus, the difference in outcomes remains statistically significant. These data show that the TriaC brace significantly altered the predicted natural history of curve progression. The comparison of the results of this study and other brace studies reported in the literature is difficult because of differences in sample size, skeletal maturity and curve magnitudes at the initiation of bracing. Furthermore, some reports excluded those patients who were not compliant with their bracing regimen. These large variations render the value of a statistical comparison of the results doubtful [51]. By applying the inclusion criteria used in this study to the literature data, a more uniform group has been created, making the statistical comparison more reliable. Nachemson et al. [36] prospectively compared 111 adolescent idiopathic scoliotic female patients with curves between 25\u00b0 and 35\u00b0, who were treated with an underarm plastic brace, to 129 patients who were not treated. All patients were followed for 4\u00a0years. The patient\u2019s thoracic or thoracolumbar curves were during this period evaluated for curve progression of more than 5\u00b0. Of the brace treated patients, 23 patients where lost to follow-up. Using survivorship analysis, a successful outcome was estimated in 74% of patients treated with a brace, compared to 34% of those who had no treatment; this difference was significant (P\u00a0<\u00a00.0001). In a retrospective study Lonstein and Winter [30] evaluated the result of brace treatment in a group of 1,020 patients over 35\u00a0years. From these patients a sub group of 177 had a curve between 20 and 29\u00b0 and a Risser sign of 0\u20131. Using the natural history data of a similar group of non treated patients from their previous report [29] they where able to show in this sub-group a significant difference (P\u00a0=\u00a00.0001, chi-square test) of the failure rate between the group of treated patients and not treated patients. The not treated patients had a predicted failure rate of 68% and the failure rate of the braced patients in the sub group was 40%. We used the same report to predict the failure rate in our cohort. Although our results are promising, it is known that 5% of patients with a curve of less than 30\u00b0 still progress to surgery during mature life [34]. We will follow our patients in whom the TriaC treatment effectively stopped curve progression before the surgical cut-off of 45\u00b0 during mature life.\nBesides affecting the lateral curve, most traditional braces reduce lordosis and kyphosis by tilting the pelvis. The purpose of pelvic tilt is to move the lumbar spine closer to the correction pads within the brace. According to Lindh et al. [28] reducing the lumbar lordosis may automatically lead to a reduction of the scoliosis as a result of a coupling mechanism between sagittal and lateral motions of the vertebra [38]. However, reduction of lumbar lordosis will also reduce thoracic kyphosis. The reduction of thoracic kyphosis is an unwanted effect, as reduced thoracic kyphosis already is an integral component of the scoliotic deformity [50]. Schaal et al. therefore, emphasise the need for a system that diminishes the effect of pelvic tilt on the thoracic kyphosis [43]. In the Triac brace the choice has been made to continuously apply corrective forces with the aim of reversing the deforming forces, without affecting the lordosis of the spine [37]. Growth is a continuous process and therefore, the correction forces should be applied continuously as well, even during the normal body motions of the patient.\nIn order to meet this requirement a flexible coupling, connecting the thoracic and lumbar parts of the brace is incorporated in the device. Most importantly, the forces exerted by the brace must be applied in such a way that they are maintained during all body motions of the patient. The new brace incorporates three separate functional elements: frame, springs and pelottes. The springs generate the orthotic forces, which are distributed by the frame and transmitted to the skin by the pelottes. The flexible coupling connects the thoracic and lumbar frame parts resulting in the application of constant forces that cannot be modified by the patients themselves (Fig.\u00a07). The strength of the externally applied forces was chosen on the basis of literature data [10, 20, 27] Fig.\u00a08.\nFig.\u00a07TriaC-braceFig.\u00a08Pre-brace Cobb-angle: 30\u00b0 and 19\u00b0 apical axial rotation. In the brace Cobb-angle: 17\u00b0 and 9\u00b0 apical axial rotation, sagittal curvature unchanged\nThe TriaC-brace provides a good primary correction of idiopathic scoliosis. There was a mean initial correction within the brace of 22\u00a0\u00b1\u00a026% for the primary curves and 28\u00a0\u00b1\u00a035% for the secondary curves, which is slightly less than reported in the literature [2, 30, 33, 34]. In the beginning of the study, we were not certain how patients would respond to the application of constant forces. Therefore, we started very carefully using relatively low forces. As we found that patients tolerated these forces well we subsequently increased the amount of force which resulted in improved initial corrections. According to the literature, achieved corrections gradually are lost following the termination of the brace treatment [7, 23, 30, 34, 49].\nHowever, being efficacious is not all that is required of a brace; it should also be as comfortable as possible and cosmetically acceptable so that teenagers who require this treatment will actually use it. Non-acceptance of a brace by patients is a real and serious problem. Houghton et al. [24] placed a hidden transducer in their braces and found that actual compliance was considerably less than was reported by the patient; and only 20% of patients wore the brace as prescribed. Modern materials, lower profiles and reduced wearing times have all been tried to improve compliance and reduce the emotional difficulties experienced with brace wear. According to some authors, there is little difference in effect between part-time (12\u201316\u00a0h) and full-time (23\u00a0h) wearing of a brace [13, 21]. Kahanowitz [25] reported such findings but only if the pre-brace Cobb-angle were less than 35\u00b0; if larger than this more than 50% of curves would progress to such an extent that surgery became necessary.\nThe current study demonstrates that the TriaC brace reduces the scoliosis, and the achieved correction is maintained during brace treatment. It prevents further progression of the Cobb angle in idiopathic scoliosis. The new brace does not differ from the conventional braces as far as maintaining the deformity in the coronal plane is concerned. This is expected as the force system of the TriaC brace in the frontal plane is in accordance with the force system in the conventional braces.\nThe new brace offers more comfort to the patient and a better cosmetic appearance. With TriaC brace there are no restrictions regarding daily or sporting activities, and it can be worn with all types of clothing.\nSummary\nCompared with published studies on the natural history of idiopathic scoliosis, use of the Triac brace appeared to significantly improve the course of curves between 20 and 40\u00b0 in skeletally immature individuals. Control or net correction of idiopathic scoliotic curves was achieved in 76% of patients.\nThe new dynamic brace offers more comfort to the patient and a better cosmetic appearance.","keyphrases":["scoliosis","bracing","natural history","biomechanics"],"prmu":["P","P","P","P"]}
{"id":"Eur_J_Nucl_Med_Mol_Imaging-4-1-2100432","title":"Functional sex differences in human primary auditory cortex\n","text":"Background We used PET to study cortical activation during auditory stimulation and found sex differences in the human primary auditory cortex (PAC). Regional cerebral blood flow (rCBF) was measured in 10 male and 10 female volunteers while listening to sounds (music or white noise) and during a baseline (no auditory stimulation).\nIntroduction\nPreviously, investigators tried to relate sex differences in behaviour and cognition to differences in brain anatomy, function, or connectivity. A well-documented example of sex differences in the brain is the difference in overall brain size. We now know that the size of the male brain is on average 8\u201310% larger than the female brain [1\u20133]. It is also thought that, compared to the female brain, there is more hemispheric asymmetry in the anatomy of the male brain [4\u20137], which is supported by the finding that in the male brain, functions are represented more unilaterally [8\u201312].\nSexual dimorphism has also been reported for the anatomy and function of the auditory cortex. The human auditory cortex is located in the superior temporal lobe. The primary auditory cortex (PAC) is situated in the medial two-thirds of the transverse temporal gyrus, also called Heschl\u2019s gyrus (Fig.\u00a01). It is surrounded by secondary and associative auditory regions, which cover the lateral part of the transverse temporal gyrus and extend to the superior temporal plane. The primary auditory region was designated as area 41 by Brodmann [13], the secondary auditory regions are areas 42 and 22 [14]. An example of sexual dimorphism in the anatomy of the auditory system is in the study by Rademacher et al. [15] who reported that both left and right primary auditory cortices are larger in females than in males, although an earlier study found no differences [7]. Anatomical reports also show larger and more symmetrical auditory association cortices in females than in males [7, 16]. However, many studies emphasize that there is intersubject variability regarding the size and location of the primary and secondary auditory cortex [17\u201321].\nFig.\u00a01Spatial distribution of significant increases in brain activation in men and women when comparing the auditory processing of noise, music, and a baseline. Activations are superimposed on an anatomical MRI template of SPM2. Clusters are significant at p\u2009<\u20090.05 FDR corrected for multiple comparisons. L=left hemisphere, y\u2009=\u2009\u221220 and y\u2009=\u2009\u22126 means a coronal plane, respectively, 20\u00a0mm and 6\u00a0mm posterior to the anterior commissure, z\u2009=\u20098 means a horizontal plane 8\u00a0mm dorsal to the anterior commissure, z\u2009\u2009=\u2009\u22121 means a horizontal plane 1\u00a0mm ventral to the anterior commissure. a Contrasting music with noise, women showed activation in the secondary auditory areas only, whereas men showed activation in both PAC and secondary auditory areas. b Comparing music to the baseline, both women and men showed bilateral activation in the PAC and secondary auditory areas. c Comparing noise to the baseline, women showed bilateral activation in the PAC. In men, on the other hand, no significant activation was found. The differences between men and women in a and c are significant (see Table\u00a02)\nFunctional sex differences in auditory processing are widely studied in the light of language. Females depend less on their left hemisphere for language processing than males in some studies [22], whereas in other studies the opposite was found [23, 24] or no sex difference could be detected [25, 26]. Whether a sex difference in language processing can be detected might depend on the nature of the task [10].\nSex-based influence on activation patterns in auditory regions is also found when manipulating the amount of background noise [27], and auditory working memory tasks induce different activation patterns in males and females [28]. These studies suggest that combining men and women in auditory neuroimaging studies may obscure or bias results.\nHowever, most of these studies focus mainly on functional sex differences in higher order (associative) brain areas, thereby ignoring possible sex differences in primary auditory cortices. Previously, a sex difference in activation of the PAC during lipreading was found [29]. However, it has not been investigated whether nonspeech sounds can induce different activity in the PAC in both sexes.\nIn the present study, we investigated potential sex differences in the activity of the primary auditory region, using different auditory stimuli.\nMaterial and methods\nSubjects\nTwenty healthy, right-handed volunteers participated in this study (10 males, 10 females). The mean age for men was 23\u00a0years (range 20\u201325) and for women 22\u00a0years (range 19\u201327). All volunteers gave written informed consent according to the Declaration of Helsinki and the Medical Ethics Committee of the University Medical Center Groningen approved the study. None of the subjects had any history of psychiatric or neurological disorders. Prior to the scanning, subjects were tested for their hearing thresholds using standard audiometric measures. All subjects had normal hearing thresholds (<20\u00a0dBHL, 0.25\u20138\u00a0kHz), and only small intersubject variations in hearing thresholds were observed.\nData acquisition\nRegional cerebral blood flow (rCBF) was measured using radioactive water ([15O]-water, half-life 122\u00a0s) as a tracer. A Siemens Ecat Exact HR+PET scanner, operated in three-dimensional mode with a 15.5\u00a0cm axial field of view, acquired 63 slices simultaneously. Each subject was scanned 12 times to measure the distribution of [15O]-water with a 10 minute interval between two scans to allow for decay. Each scan was performed after an intravenous bolus injection of 500\u00a0MBq of [15O]-water per scan. Except for the first scan, scanning started 30\u00a0s prior to injection, to account for background activation. Scanning continued for 120 seconds. The activity measured during this period was summed and used as a measure of rCBF. A scan specific calculated attenuation correction was performed to minimize interscan displacement-induced variance [30]. All subjects were scanned at fixed times on fixed weekdays, and male and female subjects were addressed to the scan dates randomly.\nExperimental design\nThree conditions were used in this study: baseline (no auditory stimulation), white noise (at an intensity level of 75\u00a0dBSL), and music (music of the movie \u201cThe Piano\u201d at 75\u00a0dBSL). Both music and noise have a wide frequency range, stimulating a large number of haircells in the cochlea and hence a large portion of the cortical auditory areas, but noise has a continuous and uniform frequency spectrum whereas music is a dynamic stimulus. Each condition was presented four times in a random order. For stimulus presentation we used a clinical audiometer (Interacoustics, model AC30), a Tandberg Educational tape recorder, and E.A.RTone 3A insert phones (with E.A.Rlink eartips), which have a flat frequency response between 100 and 4000\u00a0Hz, measured in a Zwislocki-coupler. Stimuli were presented binaurally. Ten seconds before injection of radioactive water, the stimulus was started. Because the tracer reaches the brain approximately 10 seconds after injection, subjects were exposed to the stimulus for 20 seconds before the distribution of [15O]-water in the brain starts. Subjects were instructed to close their eyes, not to move during the scans, and to listen to the auditory stimuli. Before each scan we informed the volunteers that the scan was about to start. Immediately after each scan the volunteers were questioned about the scan (did they hear the stimulus? were they uncomfortable or distracted?). During scanning we monitored the subjects with infrared cameras.\nData analysis\nThe 2002 version of Statistical Parametric Mapping (SPM2: software from the Wellcome Department of Cognitive Neurology, London, UK) was used for spatial transformations (realignment, transformation into standard stereotactic space, and smoothing with an isotropic Gaussian kernel of 8\u00a0mm FWHM), and statistical analysis [31]. An ANOVA estimated the following parameters: two groups (male and female), three conditions (baseline, noise, and music) and the mean perfusion to normalize for global flow differences (multigroup, conditions, and covariates). Each scan was scaled to a mean global activity of 50\u00a0ml\/100\u00a0ml\/min. Hypotheses about regionally specific condition effects were tested to compare the estimates by using linear compounds or contrasts. The resulting set of voxel values for these contrasts constituted the associated SPM of the t-statistics.\nThe significance threshold used for the analysis of the two groups separately (male\u2013female) was p\u2009<\u20090.05 (false discovery rate (FDR) corrected for multiple comparisons [32]) with an extent cluster threshold of more than 8 voxels. We used AMIDE software (http:\/\/amide.sourceforge.net\/) for colour scaling and display of the results on the anatomical MRI template of SPM2. For maximum statistical sensitivity and for testing the significance of the sex related differences, we conducted a region of interest (ROI) analysis in our a priori hypothesized areas, i.e. the left and right PAC, using the SPM anatomy toolbox [33] and the MarsBaR toolbox (MARSeille Bo\u00eete \u00c0 R\u00e9gion d\u2019int\u00e9r\u00eat [34]). Specifically, we created anatomical ROIs based on the three-dimensional probabilistic cytoarchitectonic maps from the SPM anatomy toolbox brains [18, 20, 33]. To compensate for differences in stereotactic space between SPM and the anatomy toolbox, a linear transformation was applied to the anatomical ROIs. MarsBaR was then used to conduct the statistical analyses on these ROIs. The statistical procedure in MarsBaR is the same as in SPM, but instead of analysing on a voxel-by-voxel basis like SPM does, all voxels in a region are averaged, and hence inferences about the whole region can be made. Also in MarsBaR, contrasts were considered significant at p\u2009<\u20090.05.\nResults\nMusic versus noise\nFor this contrast, in females the SPM-analysis resulted in significant bilateral activation clusters with a maximal significant voxel in the secondary auditory areas. In men much larger bilateral clusters were found covering not only the secondary auditory areas but also the PAC (Fig.\u00a01a and Table\u00a01). This indicates that males have a much larger activation in the PAC during music than during noise. The voxel-wise analysis of SPM did not reveal any significant activation differences between the two sexes.\nTable\u00a01Overview of brain areas with statistically significant cerebral blood flow changes\u00a0\u00a0SideRegionTalairach coordinatesNumber of voxelst-valuexyzMusic vs noiseFemalesRightBA 2261\u22126\u221211,4597.77LeftBA 22\u221251\u221210\u221213105.02MalesRightBA 41, 42, 22522\u221233,2347.87LeftBA 41, 42, 22\u221251\u2212802,1907.87Music vs baselineFemalesRightBA 41, 42, 2257\u22124\u221212,7508.85LeftBA 41, 42, 22\u221248\u22121731,8667.97MalesRightBA 41, 42, 2251\u22121022,47510.36LeftBA 41, 42, 22\u221248\u22121212,3068.67Noise vs baselineFemalesRightBA 4146\u22122154466.49LeftBA 41\u221240\u22122354636.24MalesNo suprathreshold clustersBaseline vs noiseFemalesNo suprathreshold clustersMalesRightBA 92050299685.20Baseline vs musicMalesNo suprathreshold clustersFemalesNo suprathreshold clustersThe region, covered by the whole cluster and noted in Brodmann areas, and the number of voxels in the cluster are described [14]. BA 41 corresponds to the PAC, BA 22 and 42 correspond to secondary auditory areas. Only the stereotaxic (Talairach) coordinates and t-value of the maximum of the cluster are given. The significance threshold was p\u2009\u2009<\u20090.05 FDR-corrected for multiple comparisons; extent threshold was 8 voxels; voxel size was 2\u2009\u00d7\u20092\u2009\u00d7\u20092\u00a0mm. Brain regions were identified using the Talairach atlas and the stereotactic atlas of the human brain of Mai et al. [52, 53]\nThe region of interest analysis in the PAC showed that females do have a larger activation in the PAC during music than during noise (p values 0.005 and 0.001 for the left and right PAC, respectively). But the difference between music and noise is much smaller than in males in both the left and right PAC (p values 0.016 and 0.008, respectively, Table\u00a02, Fig.\u00a02). No significant deactivations were found (i.e. noise versus music).\nTable\u00a02Region of interest analysis of the left and right primary auditory cortex (PAC)\u00a0P-valueLeft PACRight PACMusic vs noise females0.005*0.001*\u00a0Males<0.001*<0.001*\u00a0Females minus males0.9840.992\u00a0Males minus females0.016*0.008*Music vs baseline females<0.001*<0.001*\u00a0Males<0.001*<0.001*\u00a0Females minus males0.7210.779\u00a0Males minus females0.2790.221Noise vs baseline females<0.001*<0.001*\u00a0Males0.016*0.005*\u00a0Females minus males0.042*0.034*\u00a0Males minus females0.9580.966One-sided statistical significance for various contrasts was tested for men and women separately as well as for the differences between both. *Significant at p\u2009<\u20090.05Fig.\u00a02Regional cerebral blood flow relative to the baseline of each group, based on all voxels in the left and right PAC (with a global mean flow of 50\u00a0ml\/100\u00a0g\/min). Error bars indicate the 90% confidence interval of the mean across subjects per condition; the confidence interval of the baseline is also given\nTo determine whether this sex difference can be attributed to the processing of either music or noise, we compared these two stimuli with a baseline without experimental auditory stimulation.\nMusic versus baseline\nFor this contrast, the SPM-analysis showed large comparable activation clusters covering primary and secondary areas in both males and females (Fig.\u00a01b and Table\u00a01). The ROI analysis showed that there were no significant differences in the activation of the PAC between both sexes for this contrast (Table\u00a02, Fig.\u00a02). Again, no significant deactivations were found (baseline versus music).\nNoise versus baseline\nComparing noise to the baseline, the SPM-analysis showed two significant bilateral activation clusters with maxima in the PAC for the female group. In contrast, no significant activations were found in the male brain at a corrected level of p\u2009<\u20090.05 (Fig.\u00a01c and Table\u00a01). Only when the data were analysed at an uncorrected threshold of p\u2009<\u20090.01, a small activation appeared in the PAC of males, primarily on the right side (Fig.\u00a03). The ROI-analysis confirmed in men the involvement of the PAC while processing noise, showing significant bilateral increases of blood flow, but these activations were significantly less than in women in the left (0.042) and right PAC (p\u2009=\u20090.034) (Table\u00a02).\nFig.\u00a03Spatial distribution of significant increases in brain activation in men and women for the comparison of noise to the baseline. Clusters are significant at p\u2009<\u20090.01, uncorrected for multiple comparisons. L=left hemisphere, y\u2009=\u2009\u221220 means a coronal plane 20\u00a0mm posterior to the anterior commissure, z\u2009=\u20098 means a horizontal plane 8\u00a0mm dorsal to the anterior commissure. In contrast to Fig. 1c, at an uncorrected level men do show activation in PAC, but it is much smaller than in the female group\nThe voxel-wise analysis of SPM also revealed a significant deactivation in the male group. This deactivation was located in the right dorsolateral part of the prefrontal cortex extending to the posterior part of the middle frontal gyrus, covering primarily BA 9 (Fig.\u00a04 and Table\u00a01). In contrast, no significant deactivation was found in the female group.\nFig.\u00a04Spatial distribution of significant decreases in brain activation in men and women for the comparison of noise to the baseline. Clusters are significant at p\u2009<\u20090.05, FDR corrected for multiple comparisons. x\u2009=\u200920 means a sagittal plane 20\u00a0mm on the right of the anterior commissure, z\u2009=\u200929 means a horizontal plane 29\u00a0mm dorsal to the anterior commissure. Only men showed a significant deactivation in the dorsolateral prefrontal cortex\nDiscussion\nOur data demonstrate a sex difference in regional cerebral blood flow in the left and right primary auditory cortex (PAC) when comparing auditory processing of music and noise. The PAC was more activated by music than by noise in both men and women. But this difference between the two stimuli was significantly higher in men than in women. To determine whether this sex difference can be attributed to the processing of music, noise, or both, we compared the two stimuli with a baseline of no auditory stimulation. Comparing music with the baseline resulted in extensive activation of the primary and secondary auditory cortex in both sexes, but no significant sex difference was found. On the other hand, a sex difference was detected in the processing of noise, because females activated their PAC significantly more than males. The finding that sex differences in auditory processing can already be detected at the level of the primary cortex is very important, because it is often assumed that sex differences act exclusively upon higher-order cortical areas.\nThere appears to be a mismatch between the SPM and ROI analyses because there is a difference in power between the two analysing methods. The ROI analysis pools the data of all voxels in the PAC and gives a reduced standard error, resulting in a higher t-value and hence more power [34]. Although the SPM analysis showed no activation at all for the male group and a very significant activation for the female group, it did not have enough power to detect a significant difference between the two groups. Because of the enhanced sensitivity of an ROI analysis, this analysis did show a significant sex difference. Of course, caution has to be made when pooling data to improve the sensitivity and is therefore only allowed in a priori defined regions, in this case the PAC.\nThe question is whether the sex difference found is caused by the experimental conditions or by other factors like anatomical differences or methodological errors. The reported differences in activation of the PAC stand or fall by a correct identification of this area in the region of interest analysis. Several authors reported substantial intersubject variability in size and location of the PAC [17\u201321]. To overcome this problem of variability in size and location of the PAC, we employed linear resizing and shearing as well as nonlinear warping of the brain to normalize the individual brains into a standardized stereotactic frame. In addition, a Gaussian smoothing filter of 8\u00a0mm was used to remove residual variance in brain structure that remains after the stereotactic normalization. Furthermore, we used probability maps, based on the cytoarchitecture of 10 subjects, to identify the PAC [18, 20, 33]. Other studies suggested that the volume of the PAC is bilaterally larger in females than in males [15]. It is therefore necessary that the region of interest depicts the PAC of both men and women. This is the case, because the probability maps of the SPM anatomy toolbox are based on the cytoarchitecture of five male and five female brains [18, 20, 33]. For these reasons we believe that possible intersubject or intergroup differences in size and location of the PAC do not bias our results.\nIf the reported sex and contrast dependent differences in rCBF patterns are not caused by underlying anatomical differences, metabolic differences, or methodological errors, they must have been induced by the experimental stimulus. During the baseline condition, subjects had to lie quietly in the scanner and no auditory stimulus was applied. Without auditory stimulation and a specific task, the variance in rCBF might increase, resulting in less statistical power when comparing conditions to the baseline. However, as shown in Fig.\u00a02, the confidence interval for the model parameters was very similar for the three conditions. In addition, the confidence interval was also very similar for the two sexes. This means that the different contrasts tested have similar statistical power. Considering the significant sexual dichotomy in the processing of music versus noise and the results when comparing either the music or noise with the baseline condition, we conclude that males and females differ in the processing of noise. Thus our data demonstrate a sexual dichotomy in auditory processing. But which mechanism could explain this sexual dichotomy? A key to answering this question is the role of the prefrontal cortex, which we found to be deactivated in men when listening to noise. In humans, the prefrontal cortex is engaged in diverse cognitive processes including cognitive control, working memory, and attention [35]. For example, Gisselg\u00e5rd et al. [36, 37] investigated the influence of irrelevant speech on working memory tasks and revealed a functional link between auditory and prefrontal regions. Tzourio et al. demonstrated that prefrontal areas are engaged in auditory tasks that involve sustained or selective auditory attention [38]. In the present study, no explicit (attention) task was implemented. Subjects lay passively in the scanner and were instructed to listen to the auditory stimuli. While listening to an insignificant stimulus like noise, males deactivated the prefrontal attention areas as compared to silence. Females, on the other hand, had no deactivation of the attention areas and had a higher activation in the primary auditory cortex. Deactivation of the prefrontal regions was only seen in the right hemisphere, which is consistent with Tzourio et al. who stated that a right hemisphere dominance exists for attention [38]. The present results suggest a relationship between activation of PAC and prefrontal cortex. From literature, anatomical evidence exists concerning auditory\u2013prefrontal connections. Studies on monkeys have shown that the prefrontal cortex is reciprocally connected with auditory association areas (i.e. belt and parabelt in the monkey brain) [39]. The secondary and primary auditory areas are reciprocally connected (c.f. [40]). More recent studies on monkeys identified two auditory\u2013prefrontal processing streams: dorso and ventrolateral auditory streams [41\u201343]. Although, one should be cautious when comparing human and nonhuman primate brains, a similar organization of several parts of the human and monkey prefrontal cortex has been reported [44, 45].\nTo summarize, we know from literature that the auditory and prefrontal regions are anatomically and functionally linked, and our data show a sexual dichotomy in the (de)activation of both regions. Apparently, the male and female brains handle an insignificant stimulus like noise differently, and we speculate that this is done by a different engagement of the auditory\u2013prefrontal attention network. Namely, differences in attention result in a different deactivation of the right prefrontal cortex, which in turn modulates the activation of the PAC and thus explains the sex differences found in the activation of the PAC. This corresponds with previous findings that sex differences exist in the frontal\u2013temporal network, namely, males have higher intrahemispheric functional connectivity of frontal and temporal areas than females [46]. It is also known from ERP studies that sex differences exist in orienting attention to auditory stimuli [47]. It must be noted that even though our data indicate differences in the auditory system, no independent behavioural data regarding the attention levels during scanning are available. Hence, the present experimental design only allows us to speculate about the correlation between the different deactivation of the prefrontal cortex and differences in attention. Further research is needed to fully clarify the role of attention on PAC activation and to determine whether our results can be repeated. Furthermore, other auditory conditions like pure tones, noise bursts, and speech (sense and nonsense) sounds are needed to fully map the influence of sex on central auditory processing and to investigate lateralization differences between the sexes.\nTo our knowledge, this is the first time that a sexual dichotomy in the function of the PAC is demonstrated using nonspeech sounds. Previously, a sex difference in the PAC was demonstrated while lip reading [29, 48]. These studies showed only female activation in the PAC during lip reading because they associate the absent speech sound with the visual lip movements. Males on the other hand, did not display activation in the PAC and focus on the present visual lip image itself. The current study shows that even simple sounds induce different activation patterns, especially in the PAC. Other sexual dichotomies in the central auditory system have been reported. For example, sex differences in auditory feedback loops of the corticofugal network have been found in which men showed more suppression of repeated acoustic stimuli than females. This sex difference in auditory gating is probably the result of differential neuronal inhibition to repeated stimulation [49]. Likewise, the stronger spontaneous otoacoustic emissions (SOAEs) in females are thought to originate from a relatively larger amount of efferent inhibition in males [50]. This efferent innervation would start in the olivary complex and terminate in the outer hair cells of the cochlea [50].\nAlthough the present sexual dichotomy would be consistent with the concept of evolutionary advantages in a hunter\u2013gatherer society (c.f. [51]) where the inhibition of constant irrelevant stimuli in men may facilitate them to focus their attention to a single task, e.g. hunting, this concept remains speculation.\nIn conclusion, a very significant sexual dichotomy was found in the activation of the PAC with different types of acoustic stimuli (noise and music) together with sex differences in deactivation of prefrontal areas. It is known that the auditory and prefrontal regions are anatomically and functionally linked, and the prefrontal cortex is engaged in auditory attention tasks. Hence, we hypothesize that differences in attention might result in a different involvement of the right prefrontal cortex, which in turn modulates the activation of the PAC. This shows that sex influences brain activity already at the level of primary sensory cortex and that in functional imaging studies on primary sensory cortical areas, sex cannot be ignored.","keyphrases":["pet","brain","neuroimaging","hearing","gender"],"prmu":["P","P","P","P","U"]}
{"id":"Purinergic_Signal-4-2-2377316","title":"GTP avoidance in Tetrahymena thermophila requires tyrosine kinase activity, intracellular calcium, NOS, and guanylyl cyclase\n","text":"Guanosine 5'-triphosphate (GTP) is a chemorepellent in Tetrahymena thermophila that has been shown to stimulate cell division as well as ciliary reversal. Previous studies have proposed that GTP avoidance is linked to a receptor-mediated, calcium-based depolarization. However, the intracellular mechanisms involved in GTP avoidance have not been previously documented. In this study, we examine the hypothesis that GTP signals through a tyrosine kinase pathway in T. thermophila. Using behavioral assays, enzyme immunosorbent assays, Western blotting, and immunofluorescence, we present data that implicate a tyrosine kinase, phospholipase C, intracellular calcium, nitric oxide synthase (NOS) and guanylyl cyclase in GTP signaling. The tyrosine kinase inhibitor genistein eliminates GTP avoidance in Tetrahymena in behavioral assays. Similarly, pharmacological inhibitors of phospholipase C, NOS, and guanylyl cyclase all eliminated Tetrahymena avoidance to GTP. Immunofluorescence data shows evidence of tyrosine kinase activity in the cilia, suggesting that this enzyme activity could be directly involved in ciliary reversal.\nIntroduction\nChemorepellents are molecules that inhibit or repel the migration of individual cells. In protozoans, chemorepellents are hypothesized to serve a defense mechanism, helping the organism to avoid predation and environmental hazards. For example, a damaged Tetrahymena thermophila cell may leak cytosolic purines such as adenosine triphosphate (ATP) or guanosine 5'-triphosphate (GTP) into the surrounding environment, causing other cells to avoid the area, thereby avoiding the source of damage to the original cell and increasing the survival rate of the species [1, 2]. It is possible that cytosolic purines may be secreted by these organisms as a means of communication between cells (analogous to paracrine secretion) or in order to trigger second-messenger pathways within the cell doing the secretion (analogous to autocrine secretion). At this time, secretion of purine nucleotide by Tetrahymena has not been documented, although Tetrahymena have been shown to secrete a number of enzymes and peptides.\nIn T. thermophila, extracellular GTP is a known chemorepellent [1, 3, 4]. GTP causes depolarization in the somatic membrane [2], which has been proposed to trigger opening of the voltage-dependent calcium channels in the ciliary membrane [2]. The increase in Ca2+ concentration causes the cilia to reverse their direction [1], resulting in a disruption of normal swimming. This \u201cavoidance reaction\u201d [1] is marked by jerky movements, backward swimming, or spinning. This chemokinetic response involves a series of avoidance reactions, resulting in a \u201cbiased random walk,\u201d which orients the organism away from the source of the repellent [3]. Avoidance to chemorepellents may be quantified by observing the response of single cells exposed to repellent using a dissection microscope.\nT. thermophila avoid micromolar concentrations of GTP [1]. An in vivo binding assay using [32P] GTP showed the KD for GTP binding to intact T. thermophila is 21\u2009\u00b1\u20092.5\u00a0nM. GTP binds to its receptor with high affinity, and binding is saturable as well as reversible [1], all of which are characteristic of membrane receptors. Fluorescent staining with 2\u2032-(or 3\u2032)-O-(2,4,6-trinitrophenyl) (TNP)-GTP has shown GTP binding on the cilia and cell surface of T. thermophila [4]. However, the second messenger pathways involved in GTP signaling have not been previously described.\nRecently, Iwamoto and Nakaoka [4] found that addition of extracellular GTP to the medium induces cell division in T. thermophila, a result consistent with a tyrosine kinase signaling mechanism, as tyrosine kinases are involved in many major mitotic pathways. Tyrosine phosphorylation has previously been shown to modulate the interaction of calmodulin with target proteins, including protein phosphatase 2B (calcineurin) and neuronal nitric oxide synthase (nNOS) in an in vitro assay [5]. Tyrosine kinase activity has also been shown to activate phospholipase C, NOS (via protein kinase C), and guanylyl cyclase in the rat ileum [6]. Tyrosine kinases are frequently associated with activation of phospholipase C-\u03b3, and with release of calcium from internal stores via the IP3 pathway [7].\nIn our current study, we examined the hypothesis that GTP signals through a tyrosine kinase pathway in T. thermophila. Our data confirm this hypothesis. In addition to tyrosine kinase signaling, we found that phospholipase C, intracellular calcium stores, NOS, and guanylyl cyclase are necessary for behavioral avoidance to occur. All of these second messengers are consistent with the existence of a tyrosine kinase signaling pathway in T. thermophila.\nMaterials and methods\nCell cultures\nT. thermophila, strain B2086, a generous gift from T.M. Hennessey (SUNY-Buffalo) were used for all of the experiments. Cells were grown at 25\u00b0C in the axenic medium of Dentler [8] without shaking. Two-day-old cell cultures were used for all assays described below.\nChemicals and solutions\nBehavioral assays were carried out in a buffer of pH 7.0 containing 10\u00a0mM Trizma base, 0.5\u00a0mM MOPS, and 50\u00a0\u03bcM CaCl2. All repellents and inhibitors used were dissolved in this buffer. Compounds that were insoluble in aqueous solutions were first dissolved in a small quantity of dimethylsulfoxide (DMSO) and then diluted 1:1,000 or more into the buffer and vortexed vigorously. The chemorepellent guanosine-5\u2032-O-(3-thio)triphosphate (GTP-\u03b3-S) was obtained from Alexis Biochemicals (San Diego, CA, USA), along with the G-protein inhibitor pertussis toxin. The tyrosine kinase inhibitor genistein was obtained from Acros Organics (New Jersey, USA). Calphostin C [a protein kinase C (PKC) inhibitor], diadzein a control for genistein inhibition), guanosine-5\u2032-O- (2-thiodiphosphate) (GDP-\u03b2-S) (a G-protein inhibitor), Rp-8-Bromoguanosine-3\u2032,5\u2032-cyclic monophosphorothioate (Rp-8-Br-cGMPs) (a competitive inhibitor of PKG), 1-(6-(17\u03b2-3-methoxyestra-1,3,5(10)-trien-17-yl) amino)hexyl)-1H-pyrrole-2,5-dione (U73122) (a phospholipase C inhibitor), NG-monomethyl-L-arginine (L-NMMA), and N-(3-(Aminomethyl)benzyl)acetamidine (1400W) (NOS inhibitors), 4H-8-bromo-1,2,4-oxadiazolo(3,4-d) benz(b)(1,4)oxazin-1-one (NS2028) (a PKG inhibitor), and the Quantizyme nitric oxide (NO) assay kit were purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA, USA). The polyclonal antiphosphotyrosine antibody was purchased from Upstate (Lake Placid, NY, USA); 30X Tris-buffered saline (TBS) and 30X TBS\/nonidet P40 (NP40) were obtained from Modern Biology (West Lafayette, IN, USA). The guanosine 3\u20323;5\u2032monophosphate (cGMP) emzyme immunosorbent assay (EIA) kit was purchased from Cayman Chemical (Ann Arbor, MI, USA). Sodium metavanadate (a phosphatase inhibitor) and theophylline (a phosphodiesterase inhibitor) were obtained from Sigma Chemical (St. Louis, MO, USA). All other chemicals were purchased from Sigma as well.\nBehavioral assays\nIn vivo behavioral assays were carried out as previously described [1, 9, 10]. Briefly, cells were washed in buffer, and 300\u00a0\u03bcl of cell suspension was transferred to the first well of a three-well spot microtiter plate. Cells were then individually transferred by micropipette to the second well, which contained 300\u00a0\u03bcl of the test concentration of inhibitor dissolved in buffer. After at least 10\u201315\u00a0min of incubation time, individual cells were transferred to the third well, which contained 300\u00a0\u03bcl GTP-\u03b3-S and the test concentration of inhibitor. Each cell was briefly observed (1\u20135\u00a0s) for signs of avoidance. Avoidance behavior was characterized by jerky, backward movements; swimming in small, tight circles; sudden reversal of swimming direction; or any deviation from the normal helical swimming pattern of T. thermophila.\nWestern blots\nWestern blots were performed as follows: For each sample, 10\u00a0ml of the same 2-day-old culture was washed 3\u00d7 in behavioral buffer; 100\u00a0\u03bcM GTP-\u03b3-S (the concentration required to elicit 100% avoidance) was added to the tube of cells marked GTP. The control tube received only buffer; no GTP was added. Both tubes were immediately extracted with 0.1% sodium dodecyl sulphate (SDS) in the presence of 1\u00a0mM sodium orthovanadate for 30\u00a0min at 4\u00b0C. Extracts were spun at 16,000\u00a0g at 4\u00b0C for 30\u00a0min to precipitate insoluble components. Supernatant was collected, and 5\u00d7 loading buffer was applied in preparation for SDS polyacrylamide gel electrophoresis (PAGE); 100\u00a0\u03bcl of each sample was applied to a 10% SDS-PAGE. Amount of protein loaded onto the gel was standardized using the BIO-RAD protein assay; in addition, a control, Coomassie-stained gel, was run to show that equal amounts of protein were loaded into each well. Proteins were transferred from the SDS-PAGE to nitrocellulose for 30\u00a0min at 150\u00a0mA. Blots were then blocked overnight in a 3% solution of powdered milk. Blots were incubated with a 1:1,000 dilution of polyclonal phosphotyrosine antibody in the presence of 3% powdered milk for 1.5\u00a0h, washed 3\u00d7 in TBS, then incubated with a 1:100 dilution of alkaline phosphate conjugated anti-rabbit antibody for 1.5\u00a0h in the presence of 3% powdered milk. Blots were then washed 3\u00d7 with TBS and incubated for 5\u00a0min in an alkaline phosphatase buffer containing 100\u00a0mM NaCl, 5\u00a0mM MgCl2, 100\u00a0mM Trizma base, pH 9.5 with hydrogen chloride (HCl). Alkaline phosphatase buffer was then removed, and blots were developed using the Sigma-Fast system until bands were visible.\nEIA assays\nNO assays were carried out using 2-day-old cell cultures. Cells were washed twice in buffer and diluted to a final concentration of approximately 720 cells\/ml. Cells were exposed to 100\u00a0\u03bcM GTP-\u03b3-S for approximately 30\u00a0s, then spun down in a bench-top microcentrifuge for 30\u00a0s. Control cells were spun under identical conditions except that no GTP was added to the solution. Supernatant from both sets of cells was analyzed for NO using a kit from BIOMOL according to the manufacturer\u2019s instructions.\ncGMP assays were carried out using 2-day-old cell cultures. Cells were concentrated to approximately 7.66\u2009\u00d7\u2009106 cells\/ml in order to maximize cGMP production. Cells were exposed to 100\u00a0\u03bcM GTP for approximately 30\u00a0s, then immediately lysed by freezing in liquid nitrogen and thawing. Theophylline (1\u00a0mM) was added to the lysate to inhibit phosphodiesterases. Lysate was spun at 16,000\u00a0g for 30\u00a0min. Control cells were treated in identical fashion except for the addition of GTP. Supernatant from both sets of cells was assayed for cGMP using a kit from Cayman Chemicals according to the manufacturer\u2019s instructions.\nImmunofluorescence\nTen milliliters of 2-day-old cells were starved overnight in behavioral buffer then washed 3\u00d7 in behavioral buffer and concentrated down into 2\u00a0ml. Mucocysts were removed by adding Alcian Blue GX to a final concentration of 0.05%, then adding nine volumes of 0.25% proteose peptone [11]. Mucocysts were allowed to precipitate by standing the tube in a rack for 10\u00a0min. Supernatant (including cells) was removed from the top of the tube. The cells were then washed 3\u00d7 in behavioral buffer to remove any remaining mucocysts or Alcian Blue and concentrated down into 3\u00a0ml total; 1\u00a0ml of cells was set aside as the control cells. Another milliliter of cells was incubated with 100\u00a0\u03bcM GTP-\u03b3-S. A third aliquot was incubated with 100\u00a0\u03bcg\/ml genistein prior to GTP treatment. Immediately, all three aliquots of cells were fixed in 3.7% formaldehyde (from 37% stock diluted into behavioral buffer) with gentle shaking for 20\u00a0min. Cells were washed 3\u00d7 in 1\u00d7 TBS\/NP40 (Modern Biology) then incubated with a 1:100 dilution of polyclonal antibody in 1\u00d7 TBS\/NP40 for 1.5\u00a0h. Cells were again washed 3\u00d7 in 1\u00d7 TBS\/NP40 then incubated in a 1:100 dilution of fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (ICN Biomedicals) for 1.5\u00a0h. Cells were washed 3\u00d7 in 1\u00d7 TBS before being viewed under a Nikon microscope at 400\u00d7. Cells were photographed using a SPOT digital camera using a 10-s exposure for each photograph.\nResults\nGTP-\u03b3-S is a chemorepellent in Tetrahymena,, with a minimum concentration of 100\u00a0\u03bcM required for 100% avoidance (Fig.\u00a01). This concentration was used in the inhibition studies that follow as well as in our biochemical studies. We used GTP-\u03b3-S in our experiments rather than GTP to minimize the effects of the ecto-ATPase upon the assays [3]. However, the activity of apyrases and other ectoenzymes may still have degraded some of the GTP-\u03b3-S before it could reach its intended receptor. The concentration of GTP-\u03b3-S required in order to achieve 100% avoidance in this study was 5\u00d7 higher than that previously published [1]; however, considering that the cell line and growth conditions were different than those published earlier, this finding is not unexpected. The EC50 of GTP-\u03b3-S in this study was approximately 30\u00a0\u03bcM.\nFig.\u00a01Guanosine-5\u2032-O-(3-thio)triphosphate (GTP-\u03b3-S) is an effective chemorepellent in Tetrahymena. In vivo behavioral bioassays (see \u201cMaterials and methods\u201d) were used to show the concentration dependencies for avoidance reactions to GTP-\u03b3-S. The percentage of cells showing avoidance was determined by observation of a single cell after transfer to the test solution. Each trial consisted of ten cells, which were individually scored as to whether or not avoidance occurred. Each point represents the mean \u00b1 standard deviation (SD) of at least three trials. Error bars, representing the standard deviation, are shown for each point. Minimum concentration required to give 100% avoidance was 100\u00a0\u03bcM for GTP-\u03b3-S. This concentration was used in the inhibition studies that follow. The EC50 of this compound was approximately 30\u00a0\u03bcM\nTo determine whether a tyrosine kinase activity was involved in GTP avoidance, we used the tyrosine kinase inhibitor, genistein (Fig.\u00a02). Genistein itself did not cause avoidance reactions, nor was it toxic to the cells. Baseline avoidance of \u223c 20% [9] to 100\u00a0\u03bcM GTP-\u03b3-S was seen at a genistein concentration of 100\u00a0\u03bcg\/ml. The inhibition constant (IC)50 of genistein was approximately 40\u00a0\u03bcg\/ml. Another phytoestrogen, daidzein, served as a control for nonspecific side effects of genistein. As seen in Fig.\u00a02, this compound had no measurable effect on avoidance at the tested concentrations, suggesting that the inhibitory effect of genistein was indeed due to its tyrosine kinase inhibition and not attributable to some side effect of the drug.\nFig.\u00a02The tyrosine kinase inhibitor, genistein, blocks avoidance to 100\u00a0\u03bcM guanosine-5\u2032-O-(3-thio)triphosphate (GTP-\u03b3-S) in Tetrahymena. The phytoestrogens genistein (closed circles) and daidzein (open circles) were used as inhibitors of avoidance. Baseline avoidance was seen at a genistein concentration of 100\u00a0\u03bcg\/ml. The IC50 of genistein was approximately 40\u00a0\u03bcg\/ml. Daidzein served as a control for nonspecific effects of genistein and had no measurable effect on avoidance at the concentrations tested. Data shown represent the mean +\/\u2212 standard deviation (SD) for three or more trials\nAs an additional control for specificity, we tested whether genistein had any effect on the transduction of another chemorepellent, adenosine 5\u2032-O-(3-thio)triphosphate (ATP-\u03b3-S). We used 150\u00a0\u03bcM ATP-\u03b3-S, a concentration that normally causes 100% avoidance in T. thermophila [12]. When cells exposed to ATP-\u03b3-S were first incubated for 10\u201315\u00a0min in 100\u00a0\u03bcg\/ml genistein, no measurable effect on avoidance was seen. Cells continued to avoid 150\u00a0\u03bcM ATP-\u03b3-S at a rate of 96.6\u2009+\u20095.8% (n\u2009\u2265\u20093). Genistein also failed to affect avoidance to the chemorepellent pituitary adenylate cyclase-activating polypeptide (PACAP). When testing 0.1\u00a0\u03bcM PACAP, which normally causes 100% avoidance in T. thermophila [9], we found that prior incubation with 100\u00a0\u03bcg\/ml genistein did not markedly affect avoidance. Cells continued to avoid 0.1\u00a0\u03bcM PACAP at a rate of 96.6\u2009+\u20095.8% (n \u2265\u20093). These data suggest that tyrosine kinase activation is specifically related to GTP exposure rather than being a general response to chemorepellent exposure.\nIn an attempt to localize the phosphotyrosines within the cell, immunofluorescence experiments were performed using a polyclonal antiphosphotyrosine antibody. Three separate trials were done with this antibody, and representative photographs are compiled in Fig.\u00a03a\u2013c. GTP-exposed cells (Fig.\u00a03b) showed higher overall fluorescence intensity than did the controls and genistein-treated GTP-exposed cells (Fig.\u00a03a,c). Ciliary staining was seen in the GTP-exposed cells, along with punctuate staining of the cytosol. The control and genistein-treated GTP-exposed cells showed the same cytosolic staining pattern, but ciliary staining was not detected in these control groups, perhaps because the overall fluorescence level of these cells was so low.\nFig.\u00a03Guanosine 5'-triphosphate (GTP) exposure increases tyrosine kinase activity in Tetrahymena. a\u2013c Immunofluorescence labeling of phosphotyrosines in Tetrahymena is evidence of tyrosine kinase activity. Control (a), GTP-exposed (b), and genistein-treated GTP-exposed cells (c) were fixed and labeled with a polyclonal antiphosphotyrosine antibody. GTP-exposed cells showed higher fluorescence intensity than did control cells and cells treated with the tyrosine kinase inhibitor genistein. In addition, GTP-exposed cells showed ciliary staining in addition to punctuate staining of the cytosol. Total magnification 400\u00d7. Cell length \u223c 50\u00a0\u03bcm. d Western blot of whole-cell extract obtained from control and GTP-exposed cells using a polyclonal antiphosphotyrosine antibody shows increased phosphorylation levels in extract taken from GTP-exposed cells (lane 2) relative to extract from control cells (lane 3). A 66-kDa phosphoprotein stained similarly in both the GTP and control lanes, whereas bands of 42, 35, and 21\u00a0kDa were more heavily stained in the GTP lane relative to the control lane. Molecular weight markers are shown in the first lane (molecular weights in kDa). A Coomassie-stained sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) run as a loading control showed equivalent staining of proteins in all lanes (not shown)\nTo determine which proteins were being phosphorylated by the tyrosine kinase in response to GTP-\u03b3-S, we also performed several Western blots of whole-cell extract obtained from control and GTP-\u03b3-S -treated cells using the polyclonal antiphosphotyrosine antibody as a probe. Protein extracts were standardized so that lanes were loaded equally, and a control, Coomassie-stained gel, was also run to control for lane-loading artifacts (data not shown). A representative Western blot is pictured in Fig.\u00a03d. Both GTP-treated and control cells showed similar staining at 66\u00a0kDa. However, increased phosphotyrosine levels were seen in the GTP-\u03b3-S-treated cells at 42, 35, and 21\u00a0kDa, consistent with tyrosine kinase activity. These bands were also present in the control cell extract; however, the phosphorylation level in these lanes was so low that they were barely detectable with our staining procedure. Additional bands were also seen in both the GTP-exposed and the control lanes; however, they were also too light to be measurable. A more sensitive assay, such as chemiluminescence based Western blot detection, might help to give a more complete profile of the phosphotyrosine-containing proteins shown in this procedure.\nIn many cells, activation of a tyrosine kinase activates phospholipase C-\u03b3. We used the phospholipase C inhibitor, U73122, to determine whether phospholipase C might be involved in GTP avoidance. GTP avoidance was effectively eliminated at a U73122 concentration of 1\u00a0\u03bcM. This concentration was ten times lower than that used to inhibit phospholipase C in molluscan ciliary cells [12]. The IC50 of this compound was approximately 0.001\u00a0\u03bcM. Phospholipase C activates the PKC pathway through the generation of diacylglycerol (DAG) and inositol 1,4,5 trisphosphate (IP3). PKC is then activated by DAG binding in the presence of calcium. To determine whether PKC was involved in GTP avoidance, we used the PKC inhibitor calphostin C, which competes for DAG binding to PKC. In the presence of 10\u00a0\u03bcM calphostin C, avoidance to 100\u00a0\u03bcM GTP-\u03b3-S was reduced only to 96.6\u2009+\/\u2212\u20095.8%, compared with 100% avoidance in control cells. This concentration of calphostin C eliminated avoidance to 150\u00a0\u03bcM ATP-\u03b3-S [13]. Because calphostin C had no measurable effect on GTP avoidance, we concluded that PKC activy is not required for GTP signaling. It is very likely that the two second messengers, IP3 and DAG, are generated by phospholipase C and that PKC is activated, but that PKC targets cellular substrates that are not directly involved in ciliary reversal.\nBecause calcium signaling is often associated with tyrosine kinase pathways and because calcium influx is associated with GTP exposure [2], we decided to eliminate calcium rom cells using the membrane-permeable calcium chelator 1,2-bis-(o-Aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra (acetoxymethyl) ester (BAPTA-AM). As seen in Fig.\u00a05a, exposure to this calcium chelator eliminated avoidance in a concentration-dependent manner, with baseline avoidance seen at a BAPTA-AM concentration of 5\u00a0\u03bcg\/ml. This result was certainly not surprising, as all known chemorepellents that have been studied electrophysiologically in Tetrahymena have elicited depolarizations [2, 14, 15] that have been proposed to be linked to calcium. Because our data indicated that phospholipase C was involved in GTP avoidance, we decided to test the hypothesis that internal calcium stores may also contribute to avoidance, consistent with the liberation of IP3 from the plasma membrane. We used an inhibitor of the endoplasmic reticulum (ER) calcium ATPase, thapsigargin, to deplete ER calcium stores to determine whether these stores were necessary for avoidance. Baseline avoidance to GTP was seen at a thapsigargin concentration of 1,000\u00a0nM (Fig.\u00a05b). The IC50 of thapsigargin was approximately 1\u00a0nM.\nNO is another second messenger sometimes associated with tyrosine kinases [5, 17] and often associated with calcium signaling [5, 18]. In addition, NO has been found to be a critical second messenger in the Tetrahymena response to both ATP [13] and PACAP-38 [18]. To determine whether NO was involved in GTP signaling, we used the NOS inhibitors 1400W (Fig.\u00a06a) and L-NMMA (Fig.\u00a06b). Both inhibitors blocked avoidance to 100\u00a0\u03bcM GTP-\u03b3-S in Tetrahymena, with baseline avoidance being reached at 100\u00a0nM for 1400W (Fig.\u00a06a) and 50\u00a0\u03bcM for L-NMMA (Fig.\u00a06b). The IC50 of 1400W was near 0.1\u00a0nM, whereas L-NMMA was approximately 3\u00a0\u03bcM.\nTo confirm the activity of NOS in GTP avoidance, we used the Quantizyme NO assay to determine whether there was a difference in NO metabolism between control and GTP-stimulated cells. Cells stimulated with 8\u00a0mg\/ml GTP had a 146\u2009+\/\u2212\u20094.7% increase in NO levels over control cells, consistent with the inhibitor studies (n\u2009=\u20093).\nIn many cells, NO activates guanylyl cyclase, which then produces cGMP. Previous studies by Christensen et al. [20] described an NO-linked, possibly soluble guanylyl cyclase activity in T. thermophila. More recent studies, reviewed by Linder and Schultz [20], described a membrane-associated and calcium-dependent guanylyl cyclase in these organisms. To determine whether any guanylyl cyclase isoform was involved in GTP avoidance, we used NS2028, an inhibitor of guanylyl cyclase (Fig.\u00a07a), as well as Rp-8-bromo-cGMPs, a competitive inhibitor of cGMP (Fig.\u00a07b), in behavioral assays. NS2028 and Rp-8-bromo-cGMPs both blocked avoidance to 100\u00a0\u03bcM GTP-\u03b3-S in Tetrahymena. Baseline avoidance was obtained at 10\u00a0nM NS2028 (Fig.\u00a07a) and at 50\u00a0\u03bcM Rp-8-bromo-cGMPs (Fig.\u00a07b). The IC50 of NS2028 was approximately 0.05\u00a0nM and of Rp-8-bromo-cGMPs near 7\u00a0\u03bcM. To confirm these results biochemically, we performed an EIA assay to determine whether GTP exposure resulted in elevated cGMP levels within Tetrahymena. Exposure to 5\u00a0mg\/ml GTP resulted in a 1,119\u2009+\/\u2212\u20094.2% increase above control cGMP levels (n\u2009\u2265\u20093), consistent with the hypothesis that GTP avoidance is linked to a guanylyl cyclase.\nBased on the data we collected, we put together a provisional model of GTP signaling (Fig.\u00a08). In our model, GTP binds to a receptor, which activates tyrosine kinase while interacting with a membrane calcium channel, causing a depolarization. Phospholipase C is activated, generating IP3, which allows calcium efflux from ER stores. The high cytosolic calcium interacts with some (unknown) isoform of NOS, guanylyl cyclase, and other calcium-binding proteins, ultimately resulting in ciliary reversal. Whereas some of the relationships between second messengers are still unknown, the model has been put together to stimulate thought and discussion and to make some order out of a complex pathway.\nDiscussion\nThe data we have compiled here are consistent with the hypothesis that the previously described GTP receptor [1] signals through a tyrosine kinase (Fig.\u00a02, 3). The GTP signaling pathway also involves phospholipase C (Fig.\u00a04), intracellular calcium (Fig.\u00a05), NOS (Fig.\u00a06), and guanylyl cyclase (Fig.\u00a07). The proposed GTP signaling pathway is outlined in Fig.\u00a08.\nFig.\u00a04The phospholipase C inhibitor 1-(6-(17\u03b2-3-methoxyestra-1,3,5(10)-trien-17-yl) amino)hexyl)-1H-pyrrole-2,5-dione (U73122) blocks avoidance to 100\u00a0\u03bcM guanosine-5\u2032-O-(3-thio)triphosphate (GTP-\u03b3-S) in Tetrahymena. Baseline avoidance was reached at a U73122 concentration of 1\u00a0\u03bcM. The inhibition constant IC50 of this compound was near 0.001\u00a0\u03bcM. Data shown is mean\u2009+\/\u2212\u2009standard deviation (SD) of three or more trialsFig.\u00a05Intracellular calcium is required for guanosine 5'-triphosphate (GTP) avoidance in Tetrahymena. a The membrane-permeable calcium chelator 1,2-bis-(o-Aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra (acetoxymethyl) ester (BAPTA-AM) blocks avoidance to 100\u00a0\u03bcM GTP-\u03b3-S in Tetrahymena. Baseline avoidance was reached at a BAPTA-AM concentration of 5\u00a0\u03bcg\/\u03bcl. The inhibition constant IC50 of this compound was near 0.5\u00a0\u03bcg\/\u03bcl. Data shown is mean\u2009+\/\u2212\u2009standard deviation (SD) of three trials. b The endothelial reticulum (ER) calcium adenosine triphosphatase (ATPase) inhibitor thapsigargin also blocks avoidance to 100\u00a0\u03bcM guanosine-5\u2032-O-(3-thio)triphosphate (GTP-\u03b3-S) in Tetrahymena.. Baseline avoidance was reached at a thapsigargin concentration of 1,000\u00a0nM. The IC50 of this compound was near 1 nM. Data shown is mean\u2009+\/\u2212\u2009standard deviation (SD) of three or more trialsFig.\u00a06Nitric oxide synthase (NOS) activity is required for guanosine 5'-triphosphate (GTP) avoidance in Tetrahymena. a The NOS inhibitor 1400W blocks avoidance to 100\u00a0\u03bcM guanosine-5\u2032-O-(3-thio)triphosphate (GTP-\u03b3-S) in Tetrahymena. Baseline avoidance was reached at a 1400W concentration of 100\u00a0nM. The inhibition constant IC50 of this compound was near 0.1\u00a0nM. Data shown is mean\u2009+\/\u2212\u2009standard deviation (SD) of three trials. b The NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) also blocks avoidance to 100\u00a0\u03bcM GTP-\u03b3-S in Tetrahymena. Baseline avoidance was reached at an L-NMMA concentration of 10\u00a0\u03bcM. The inhibition constant IC50 of this compound was near 3\u00a0\u03bcM. Data shown is mean\u2009+\/\u2212\u2009standard deviation (SD) of at least three trialsFig.\u00a07Guanylyl cyclase activity is required for guanosine 5'-triphosphate (GTP) avoidance in Tetrahymena. a 4H-8-bromo-1,2,4-oxadiazolo(3,4-d) benz(b)(1,4)oxazin-1-one (NS2028), an inhibitor of guanylyl cyclase, blocked avoidance to 100\u00a0\u03bcM guanosine-5\u2032-O-(3-thio)triphosphate (GTP-\u03b3-S) in Tetrahymena. Baseline avoidance was obtained at 10\u00a0nM. The inhibition constant IC50 of NS2028 was approximately 0.05 nM. Data shown is mean + standard deviation (SD) of three trials. b Rp-8-Bromoguanosine-3\u2032,5\u2032-cyclic monophosphorothioate (Rp-8-Br-cGMPs), a competitive inhibitor of guanosine 3\u20323;5\u2032monophosphate (cGMP), also blocked avoidance to 100\u00a0\u03bcM GTP-\u03b3-S in Tetrahymena. Baseline avoidance was obtained at 50\u00a0\u03bcM. The IC50 of Rp-8-Br-cGMPs was approximately 7\u00a0\u03bcM. Data shown is mean + standard deviation (SD) of three or more trialsFig.\u00a08Current model of guanosine 5'-triphosphate (GTP) signaling. Based on data obtained in this experiment, GTP appears to signal through a receptor linked to a tyrosine kinase. Phospholipase C, intracellular calcium release, nitric oxide (NO) production, and guanylyl cyclase are all involved in GTP avoidance. Some of the relationships between pathway components remain unknown. This model is based on the data presented in this paper, along with data obtained from the literature [26, 32, 33]\nGTP is the first chemorepellent we have studied that does not signal through a G-protein-linked receptor, unlike ATP [13] and PACAP [18, 21]. In contrast to other repellents, GTP signaling is unaffected by either GDP-\u03b2-S or pertussis toxin (unpublished data). GTP is also the only one of these three repellents whose signaling is eliminated by genistein (Fig.\u00a02; see \u201cResults\u201d). Previously published cross-adaptation data [2] indicated that GTP and ATP signal through different receptors. These data are consistent with the previously published literature and add the observation that both receptors and second-messenger pathways for these two repellents appear to be different.\nMultiple target proteins are phosphorylated in response to GTP exposure (Fig.\u00a03d), yet the proteins remain unidentified except for molecular weight. Phosphotyrosine immunolocalization does not help us identify these proteins; however, the ciliary staining (Fig.\u00a03b) opens up the possibility of direct interaction between tyrosine kinase activity and ciliary reversal. This is worthy of note, as Christensen et al. [22] report a similar increase in ciliary phosphotyrosine labeling after exposure to the chemoattractant insulin. However, in their report, the main protein that was phosphorylated was a 66\u00a0kDa protein, which they discovered was homologous to the insulin receptor. In the case of GTP exposure, we saw no increase in labeling of the 66\u00a0kDa protein (Fig.\u00a03d). Taken together, these data raise the possibility that there are multiple tyrosine kinase pathways that act on cilia to increase forward swim speed in the case of a chemoattractant, or to cause ciliary reversal in the case of a chemorepellent. Becausxe there were multiple phosphotyrosine bands in our Western blot (Fig.\u00a03d) and ciliary as well as cytosolic staining in our immunofluorescence (Fig.\u00a03b), we see that GTP exposure has both ciliary and cytosolic effects. Ciliary reversal is seen immediately upon GTP exposure; however, other effects of GTP exposure, such as mitosis, are seen hours after GTP exposure [4]. Perhaps the cytosolic target proteins are involved in other, longer-term effects of GTP, such as mitosis.\nMany tyrosine kinase pathways involve phospholipase C. In various Tetrahymena species, evidence for phospholipase C involvement has been seen in stomatin-induced differentiation [24] and in commitment to cell survival or death [25]. Studies by Leondaritis and Galanopoulou [26] suggest that Tetrahymena possess a functional phosphatidylinositol signaling system that is similar to higher eukaryotes. Indeed, in our previous studies of PACAP [10] and ATP [13], phospholipase C involvement is also implicated. In our current study, we found that U73122 effectively eliminated GTP avoidance (Fig.\u00a04), indicating that phospholipase C signaling is involved in behavioral avoidance. However, our calphostin C data (see \u201cResults\u201d) indicate that PKC, a common downstream target of phospholipase C, is not required for GTP avoidance. IP3, a second messenger generated by phospholipase C, is likely involved in GTP signaling. Although we could not test IP3 involvement directly using our behavioral assay, the fact that the ER calcium ATPase inhibitor thapsigargin eliminated GTP avoidance (Fig.\u00a05b) is indirect evidence that IP3 may be binding to the ER and causing release of calcium from internal stores.\nCalcium is a ubiquitous signaling molecule and has been implicated in ciliary reversal in Tetrahymena. It was no surprise, then, that BAPTA-AM eliminated GTP avoidance (Fig.\u00a05a), as it has previously been shown to eliminate avoidance to PACAP [18] and ATP [13]. Calcium is likely to play multiple roles in signaling. One possible scenario is the direct interaction of calcium with ciliary motor proteins. For example, inner arm dynein 1 (I1) is required for calcium-dependent ciliary reversals in T. thermophila [27]. In this study, a \u201ccalcium sensor,\u201d which interacts with I1, was postulated. This calcium sensor protein remains to be isolated.\nIntracellular calcium may also play other, less direct, roles in GTP avoidance. Indeed, in the protozoan parasite Leishmania donovani, both NOS and guanylyl cyclase are activated by intracellular calcium [28]. Both NOS (Fig.\u00a06) and guanylyl cyclase (Fig.\u00a07) activity are required for GTP avoidance. Previous studies that implicate NOS and guanylyl cyclase in Tetrahymena [13, 18, 19] do not indicate the type of NOS or guanylyl cyclases involved, though Christensen et al. [19] give evidence for a soluble guanylyl cyclase. In L. donovani, there is evidence for a constitutive, neuronal-type NOS that then activates a soluble guanylyl cyclase [28]. Both of these enzymes are activated by intracellular calcium. Our data would be consistent with a similar mechanism in Tetrahymena, as proposed in our model of GTP signaling (Fig.\u00a08). However, neither a soluble guanylyl cyclase nor an nNOS isoform have been purified from Tetrahymena to date.\nCalcium-binding proteins, such as calmodulin, may also be involved in GTP signaling in Tetrahymena, either by interacting with enzymes that generate second messengers or by interacting with the molecular motors responsible for ciliary reversal. Previous studies [29, 30] indicate that calmodulin activates a ciliary guanylyl cyclase in this organism. Our studies leave open the possibility that this cyclase, activated by calcium\/calmodulin, is involved in GTP signaling. In addition to this enzyme, Hirano-Ohnishi and Watanabe [31] reported a calmodulin-dependent phosphorylation of ciliary beta tubulin in Tetrahymena, which they believe to be involved in ciliary reversal. Elongation factor 1\u03b1, which functions in protein translation, also interacts with ciliary calmodulin [32]. Whereas this would not necessarily function in ciliary reversal, it may modulate some of the longer-term effects of GTP. Finally, Ueno et al. [33] report that ciliary calmodulin interacts with proteins that are homologues of radial spoke proteins in Chlamydomonas, further implicating calmodulin directly in ciliary reversal. We have added some of these proteins to our model of GTP signaling (Fig.\u00a08), though there are doubtless more proteins to be discovered.\nIn this study, we used behavioral assays and pharmacological inhibitors to explore the intracellular mechanisms employed in GTP signaling in T. thermophila. Because pharmacological inhibitors often have unknown side effects, we used multiple inhibitors when available. Some of these inhibitors had to be used at higher concentrations in our study than have been previously published for mammalian cells. Perhaps this is because of the differences in the structure of the Tetrahymena proteins compared with their mammalian counterparts. Recognizing the potential pitfalls of relying solely upon pharmacological data, we supplemented behavioral assays with other kinds of tests, such as Western blots, immunofluorescence, and EIA when available. In all cases, the additional tests confirmed the data we originally obtained using pharmacological inhibitors.\nTaken together, our data indicate that a number of enzymes are involved in GTP signaling. However, further experimentation is needed to discern the interrelationships between enzymes in the pathway and to answer the myriad questions that remain. For example, are guanylyl cyclase and NOS truly activated by calcium in Tetrahymena? Would calcium chelation abolish their activity? Is there a soluble guanylyl cyclase in Tetrahymena? What NOS isoforms do Tetrahymena possess? Is tyrosine phosphorylation required for either NOS or guanylyl cyclase activity? Are any additional proteins, particularly ciliary proteins, involved in GTP signaling? Are all of the components of the pathway located in the cilia? These and many other questions remain to be answered. One question we have begun to answer is whether intracellular calcium is required for tyrosine kinase activity. Recent immunofluorescence shows no difference in phosphotyrosine levels between GTP-treated cells and GTP-treated cells that had been pretreated with BAPTA-AM (unpublished data). Similarly, calcineurin (PP2B) does not appear to be involved in GTP signaling, as ascomycin treatment had no effect on GTP avoidance (unpublished data). Endothall, a PP2A inhibitor, did inhibit avoidance (unpublished observation), consistent with observations by Deckman and Pennock [33] that PP2A is involved in ciliary reversal. Further experimentation will help us determine the interrelationship of pathway components and better establish the mechanism by which chemorepellents signal in Tetrahymena.","keyphrases":["chemorepellent","extracellular nucleotide","purine metabolism"],"prmu":["P","R","R"]}
{"id":"Ann_Surg_Oncol-3-1-1914276","title":"Pathological and Biological Differences Between Screen-Detected and Interval Ductal Carcinoma in situ of the Breast\n","text":"Background The incidence of ductal carcinoma in situ (DCIS) has risen dramatically with the introduction of screening mammography. The aim was to evaluate differences in pathological and biological characteristics between patients with screen-detected and interval DCIS.\nWith the introduction of widespread screening mammography, the incidence rates of ductal carcinoma in situ (DCIS) have risen dramatically in Western Europe and North America.1\u20133 DCIS now accounts for nearly 20% of all screen-detected breast malignancies.4\nAs a consequence, treating physicians are confronted with a cumulative caseload because it is not known how many women with screen-detected DCIS will develop an invasive carcinoma in their lifetimes. The proportion of untreated cases of DCIS that would progress to invasive malignancy has been difficult to evaluate, because DCIS is usually excised when detected. Because DCIS is a nonobligatory precursor to invasive carcinoma, and, therefore, has a relatively benign nature, screen-detected DCIS has been argued to represent an overdiagnosis.5,6 This argument is supported by autopsy studies in which the median prevalence of DCIS was 8.9%, suggesting some cases do not progress to clinically significant lesions in a patient\u2019s lifetime.7 On the contrary, patients with DCIS treated with biopsy alone in the premammography era had a higher rate of subsequent occurrences (14\u201350%) of invasive breast cancer than expected.8,9 Large clinical trials, in which patients had been treated with lumpectomy alone, have also indicated that DCIS can recur as invasive ductal carcinoma.10,11\nScreen-detected DCIS is more often presented as linear branching microcalcifications on mammography than symptomatic DCIS.12 The screen-detected group in the previously mentioned study had a larger proportion of patients with comedocarcinoma. Therefore, it was suggested that linear branching microcalcifications were related with a more aggressive type of DCIS.12 This is confirmed in other reports that have indicated that linear branching microcalcifications on mammography are associated with high grade DCIS.13,14\nWe believe that screen-detected DCIS is more often associated with suspicious microcalcifications representing high-grade DCIS, which has been detected before it has had the chance to progress to invasive cancer. Therefore, it is hypothesized that screen-detected DCIS is biologically more aggressive than interval DCIS. To compare screen-detected DCIS with interval DCIS in such a retrospective study, the clinicopathological and biological characteristics of both groups were evaluated for differences. Screen-detected DCIS was classified as DCIS detected by screening mammography, when the examination from two years earlier failed to reveal an abnormality. Interval DCIS was classified as DCIS detected within the two-year interval between two subsequent screening rounds, when the earlier examination failed to reveal an abnormality. Age, tumor size, and pathological grade were studied for their known relation with local recurrence. Finally, the expression of established prognostic biomarkers in breast cancer was studied by immunohistochemistry for estrogen receptor (ER), progesterone receptor (PR), Her2\/neu, p53, and cyclin D1.\nPATIENTS AND METHODS\nPatients and Tumors\nThe Dutch screening program for breast cancer has been gradually implemented in the North Netherlands since 1991. It offered biennial mammography to women 50\u201369 years old, and since 1999 women 70\u201374 years old have also been included. Women received mammography in the cranio-caudal and medio-latero-oblique direction for each breast. Two radiologists evaluated the mammograms by a double independent reading.\nFrom January 1992 to December 2001, 128 consecutive patients were treated for pure DCIS at our institution. To identify patients for inclusion in the study, all women who had actually attended the screening program at least two subsequent rounds with a two-year interval at the time of diagnosis were considered as attenders. Patients who had skipped one or more screening rounds previous to the diagnosis and patients who had not been attending the program at all were considered nonattenders. Patients\u2019 records were checked to obtain this information, and if there was no information regarding the participation of the screening program at the time of diagnosis the general practitioner was consulted. Out of the 128 consecutive patients, 102 attenders and 26 nonattenders could be identified. For immunohistochemistry, patients were selected on the availability of sufficient paraffin-embedded tissue. Thirteen out of the 26 nonattenders and 74 out of the 102 attenders remained, respectively, for evaluation of Her2\/neu overexpression, estrogen receptor (ER) expression, progesterone receptor (PR) expression, p53 expression, and cyclin D1 expression using tissue microarray analysis as part of a project protocol that had been approved by the medical ethics committee. The patients in the study-group (n = 74) were divided into two groups. Patients with DCIS that had been detected by screening mammography were classified as screen-detected patients, when the examination two years earlier failed to reveal an abnormality (n = 54). Patients with DCIS that had been detected within the two-year interval between two subsequent screening rounds were classified as interval patients, when the earlier examination failed to reveal an abnormality (n = 20).\nMammography and Pathological Assessment\nMammographic and pathological characteristics were derived from mammography and pathological reports, respectively. Data were delivered and evaluated anonymously. If data were missing, mammography and pathological slides were reevaluated. Mammographic appearances were scored as microcalcifications, a mass, a combination of the two, or as occult. Microcalcifications were scored as fine granular, coarse granular, or as linear branching. Pathological size had been estimated, and the grade had been scored according to the European Pathologists Working Group (EPWG15) and according to the Van Nuys classification.16\nTissue Microarray Construction\nSlides from all blocks were evaluated for representative areas with DCIS, and tissue microarrays were prepared as described earlier.17 In brief, the most representative area of DCIS was marked on the original hematoxylin and eosin (H&E) stained section. With this marked section as an orientation, three 0.6-mm punches were taken from the selected area in the donor blocks and mounted in a recipient block containing approximately 110 biopsies, using a manual tissue microarray device (Beecher Instruments, Silver Springs, MD). The presence of DCIS in the arrayed samples was verified on hematoxylin eosin stained sections.\nImmunohistochemistry\nFor immunohistochemistry, 3 \u03bcm sections of the paraffin-embedded tissue arrays were deparaffinized in 2 changes of xylene for 5 minutes each and gradually rehydrated through changes of graded ethanol from 100% to distilled water. Antigen retrieval methods and antibodies are summarized in Table\u00a01. The endogenous peroxidase reaction was blocked by incubating the sections in 3% perhydrol for 30 minutes. Primary antibodies were diluted in phosphate-buffered saline (PBS) containing 1% bovine serum albumin and incubated at room temperature for 1 hour. Samples were then washed in PBS and incubated with secondary and tertiary antibodies. For visualization of the antibody-antigen complex, the diaminobenzidine tetrahydrochloride\/peroxidase reaction was used. After a final wash with distilled water, sections were counterstained with hematoxylin. Sections were dehydrated through rising concentrations of ethanol and mounted. Immunohistochemistry was successful in 81\/87 cases for Her2\/neu staining, 73\/87 cases for ER staining, 71\/87 for PR and cyclin D1 staining, and 70\/87 for p53 staining.\nTABLE\u00a01.Antigen retrieval methods and antibodiesAntibodyCloneSupplierDilutionAntigen retrievalSecondary antibodySupplierTertiary antibodySupplierER6F11VentanaaTris\/HCL 0.1M (pH 9.5) 30\u2019 98\u00b0C microwaveRAMBIODakoSARBIODakoPR1A6VentanaaTris\/HCL 0.1M (pH 9.5) 30\u2019 98\u00b0C microwaveRAMBIODakoSARBIODakoHer-2\/NeuCB11VentanaaTris\/HCL 0.1M (pH 9.5) 30\u2019 98\u00b0C microwaveRAMBIODakoSARBIODakop53BP-53-12-1Biogenix1:800Tris\/HCL 0.1M (pH 9.5) 30\u2019 98\u00b0C microwaveRAMBIODakoSARBIODakoCyclin D1SP4Neomarkers1:50Tris\/HCL 0.1M (pH 9.5) 30\u2019 98\u00b0C microwaveRAMBIODakoSARBIODakoa Prediluted by supplier.ER, estrogen receptor; PR, progesterone receptor;; RAMBIO, rabbit anti-mouse biotin; SARBIO, swine anti-rabbit biotin.\nEvaluation of Immunohistochemical Staining\nAll slides stained for molecular markers were read by two authors (MdR and BvdV). The slides were randomly reviewed by a third author (JW) and in case of disagreement between the other two authors. ER, PR, and p53 were graded based on the percentage of cells showing positive nuclear staining in the ducts with DCIS. ER and PR were considered positive if nuclear staining was present in \u226510% of the cases, and p53 was considered positive in case of a substantial percentage of positively stained nuclei (>30%). Her2\/neu expression was graded as recommended by the HercepTestTM scoring guidelines: 0: no staining at all or membrane staining in <10% of tumor cells; 1+: a faint\/barely perceptible partial membrane staining in >10% of the tumor cells; 2+: weak to moderate complete membrane staining in >10% of tumor cells; 3+: strong complete membrane staining in >10%. Her-2\/neu was considered to be overexpressed if the score was 3+. Cyclin D1 expression was scored using a semiquantitative system as described by Vos et al.18 This system was based on the staining intensity scored as 0 (none), 1 (weak), 2 (moderate), and 3 (strong), and the percentage of positive tumor cell nuclei scored as 0 (0%), 1 (1\u201325%), 2 (25\u201350%), 3 (50\u201375%), and 4 (>75%). The cyclin D1 staining score was calculated as the sum of the intensity and the percentage of positive tumor cells.\nStatistical Analysis\nDifferences in clinicopathological characteristics between screen-detected and interval patients with DCIS in the study group, and between the study group and the excluded group of patients were analyzed by chi-square analysis. Differences in clinicopathological and biological characteristics between the study group and the nonattenders were also analyzed by chi-square analysis. Differences in age were tested by using the Mann-Whitney U test. Univariate analyses, investigating differences in pathological and biological features, was performed by logistic regression, using screen detection as a dependent variable. Multivariate analyses were performed with a logistic-regression model. The elimination of variables in a stepwise manner identified the statistically significant pathological and biological parameters. A p value of \u2264.050 was considered as significant. All calculations were performed with SPSS 12.01 (SPSS inc., Chicago, IL).\nRESULTS\nTable\u00a02 shows the clinicopathological characteristics of the patients in the study group (n = 74). Screen-detected DCIS was less often symptomatic than interval DCIS (p < .001). Five patients (25%) in the interval group had no objective signs on presentation; all patients had felt a lump in the breast that could not be verified on clinical examination. On mammography, microcalcifications were more often seen in screen-detected DCIS (p = .002). Screen-detected DCIS was more often presented as linear branching (44.9%) and coarse granular (55.9%) microcalcifications than interval DCIS (p<0.001). High-grade DCIS (Van Nuys, 53.1%, p = .025) was also more often observed in screen-detected DCIS.\nTABLE\u00a02.Clinicopathological characteristics of the patients in the study group and differences between screen-detected and interval patientsClinicopathological characteristicsScreen-detected n = 54Interval n = 20p-valueaAge (mean), years58.960.7.187Family history of breast cancer.055\u00a0\u00a0Yes8 (14.8)7 (35)\u00a0\u00a0No46 (85.2)13 (65)Signs\u00a0\u00a0Palpable mass5 (9.3)6 (30)<.001\u00a0\u00a0Nipple discharge2 (3.7)9 (45)\u00a0\u00a0Mastodynia1 (1.9)0 (0)\u00a0\u00a0No objective signs46 (85.1)5 (25)Mammography.002\u00a0\u00a0Microcalcifications46 (85.2)10 (50)\u00a0\u00a0Mass2 (3.7)6 (30)\u00a0\u00a0Combination mc\u2019s and mass6 (11.1)4 (20)Microcalcifications <.001\u00a0\u00a0Linear branching22 (44.9)4 (28.6)\u00a0\u00a0Coarse granular27 (55.1)4 (28.6)\u00a0\u00a0Fine granular0 (0)6 (42.9)Mammographic size.183\u00a0\u00a0\u22642 cm21 (42.6)12 (60)\u00a0\u00a0>2 cm33 (57.4)8 (40)\u00a0\u00a0BCS23 (42.6)8 (40).841\u00a0\u00a0Mastectomy31 (57.4)12 (60)Tumor size.787\u00a0\u00a0<16mm19 (35.2)6 (30)\u00a0\u00a016\u201340mm17 (31.5)8 (40)>40 mm18 (33.3)6 (30)Grade (EPWG).229\u00a0\u00a014 (7.5)4 (20)\u00a0\u00a0229 (53.7)11 (55)\u00a0\u00a0321 (38.9)5 (25)Grade (Van Nuys).025\u00a0\u00a014 (7.4)6 (30)\u00a0\u00a0224 (44.4)9 (45)\u00a0\u00a0326 (53.1)5 (25)Mann-Whitney U test.Values between parentheses are percentages.BCS, breast conserving surgery; EPWG, European Pathologist Working Group.a Chi-square analyses.\nDifferences in clinicopathological characteristics between the study-group (n = 74) and the group of patients that had been excluded (n = 28) because of insufficient paraffin embedded tissue are outlined in Table\u00a03. There was no marked difference in age, microcalcifications, tumor size, and pathological grade according to the EPWG classification between both groups. The excluded group of patients represented a relatively large proportion of low-grade DCIS according to the Van Nuys classification (p < .001) in comparison with the TMA group.\nTABLE\u00a03.Comparison of clinicopathological characteristics between the study group and the group of patients that were excluded because of insufficient paraffin-embedded tissueClinicopathological characteristicsStudy group n = 74Excluded n = 28p-valueaAge (mean)59.661.5.381Microcalcifications (n = 86).063\u00a0\u00a0Linear branching26 (41.3)5 (25)\u00a0\u00a0Coarse granular31 (49.2)9 (45)\u00a0\u00a0Fine granular6 (9.5)6 (30)Tumor size.458\u00a0\u00a0<16mm25 (33.8)7 (25)\u00a0\u00a016\u201340mm25 (33.8)9 (32.1)\u00a0\u00a0>40 mm24 (32.6)12 (42.9)Grade (EPWG).184\u00a0\u00a018 (10.8)6 (21.4)\u00a0\u00a0240 (54.1)10 (35.7)\u00a0\u00a0326 (35.1)12 (42.9)Grade (Van Nuys)<.001\u00a0\u00a0110 (13.5)15 (53.6)\u00a0\u00a0233 (44.6)6 (21.4)\u00a0\u00a0331 (41.9)7 (25)Mann-Whitney U test.Values between parentheses are percentages.EPWG, European Pathologist Working Group.a Chi-square analyses.\nThere were no differences in clinicopathological and biological characteristics between the study group and the nonattenders (n = 13), except the fact that nonattenders were younger than the patients in the study group (55.9 years versus 59.6 years; p = .042).\nTable\u00a04 displays the relation among pathological characteristics, biological marker expression, and mode of detection in the study group. Univariate logistic regression analysis indicated that in screen-detected DCIS Her2\/neu is more often overexpressed (odds ratio [OR] = 6.5; 95% confidence interval [CI] 1.3\u201331.0; p = .020). Interval DCIS is more frequently positive for PR staining (OR = 0.3; 95% CI 0.1\u20131.0; p = .042) and is related to low pathological grade according to the Van Nuys classification (OR = 7.3; 95% CI 1.6\u201333.3; p = .010). In multivariate logistic regression, including pathological grade according to the EPWG and Van Nuys classification, Her2\/neu overexpression and PR expression in the model, Her2\/neu overexpression was the only independent indicator for screen-detected DCIS (OR = 12.8; 95% CI 1.6\u2013104.0; p = .018).\nTABLE\u00a04.Univariate analysis of pathological and biological characteristics in screen-detected versus interval DCIS in the study groupPathological and biological featuresScreen-detected n = 54Interval n = 20OR by screen-detected95% CIp-valueTumor size\u00a0\u00a0<16mm19 (35.2)6 (30)0.80.20.744\u00a0\u00a016\u201340mm17 (31.5)8 (40)1.10.20.827\u00a0\u00a0>40 mm18 (33.3)6 (30)1Grade (EPWG)\u00a0\u00a014 (7.5)4 (20)3.8000.50.377\u00a0\u00a0229 (53.7)11 (55)1.7030.70.124\u00a0\u00a0321 (38.9)5 (25)1Grade (Van Nuys)\u00a0\u00a014 (7.4)6 (30)7.31.6\u00a0\u00a0224 (44.4)9 (45)1.60.50.416\u00a0\u00a0326 (53.1)5 (25)11.60.010Her2\/neu (n = 68)\u00a0\u00a0Positive29 (60.4)2 (10)6.51.3\u201331.00.020\u00a0\u00a0Negative19 (39.6)18 (90)1ER(n = 62)\u00a0\u00a0Positive33 (75)16 (88.9)0.4\u00a0\u00a0Negative11 (25)2 (11.1)10.1\u20131.90.236PR (n = 60)\u00a0\u00a0Positive18 (42.9)13 (65)0.3\u00a0\u00a0Negative24 (57.1)5 (35)10.1\u20131.00.042p53 (n = 60)\u00a0\u00a0Positive10 (24.4)4 (21.1)0.8\u00a0\u00a0Negative31 (75.6)15 (78.9)10.2\u20133.10.776Cyclin D1 (n = 60)\u00a0\u00a0Positive29 (69.1)13 (72.2)0.90.3\u20132.90.806\u00a0\u00a0Negative13 (30.9)5 (27.8)1Univariate analysis using logistic regression.Figures in parentheses are percentages.OR, odds ratio; EPWG, European Pathologist Working Group; ER, estrogen receptor; PR, progesterone receptor. 95% CI, 95% confidence interval.\nDISCUSSION\nApproximately 1 in every 1300 screening mammography examinations leads to a diagnosis of DCIS.4 Data from a large trial and service screening programs in the United Kingdom, the Netherlands, Australia, and the United States have demonstrated that a woman attending prevalence screen has a 19 times greater chance of having a progressive DCIS or an invasive tumor diagnosed than of having a nonprogressive DCIS diagnosed.19 It is questioned what to do with the high detection rate of screen-detected DCIS. It was hypothesized that screen-detected DCIS is biologically more aggressive than interval DCIS because suspicious microcalcifications, detected by the screening program, will probably more frequently represent high-grade DCIS. Therefore screen-detected DCIS was characterized pathologically and biologically to determine whether screen-detected DCIS differed from interval DCIS. The results of this study indicate that screen-detected DCIS is pathologically (OR = 7.3; 95% CI 1.6\u201333.3; p = .010) and biologically (OR = 12.8; 95%CI 1.6\u2013104.0; p = .018) more aggressive than interval DCIS. Indeed screen-detected DCIS was related with more suspicious microcalcifications (p < .001). DCIS detected by a prevalence screen was pathologically and biologically comparable to DCIS detected in later rounds (data not shown) suggesting prevalence and incident cases to both be of clinical relevance.\nThe relative incidence of high-grade DCIS in our series of screen-detected patients was 53%, which is comparable to the incidence of high-grade DCIS in a screening population from the Netherlands Cancer Institute (47%).20 Much data point out that poorly differentiated or high-grade DCIS lesions have a greater potential to progress to invasive disease than low-grade DCIS.21 High grade is also an independent risk factor of local recurrence after lumpectomy for DCIS, and approximately 50% of these recurrences are invasive cancers.22,23 There are no studies available comparing screen-detected with interval DCIS in a group of patients that had all been attending the screening program. Reports on screen-detected DCIS regarding histopathological grade vary markedly describing a higher incidence of low grade,24 no difference,25 or a higher incidence of high grade12,26,27 in screen-detected DCIS. In these reports screen-detected DCIS is compared to symptomatic DCIS or to DCIS detected in a period before the screening program was introduced. In the present study, a higher incidence of high-grade lesions, which were classified according Van Nuys (OR = 7.3; 95% CI 1.6\u201333.3; p = .010) classification, was found in screen-detected DCIS, indicating a higher malignant potential in screen-detected DCIS. These results are consistent with the results from the studies of Evans et al.26 and Kessar et al.27\nDCIS lesions from patients in the study group were compared to DCIS lesions from patients who had not attended the screening program. Out of the 26 nonattenders, there were only 13 patients from whom sufficient paraffin-embedded tissue was available. Using chi-square analysis there were no differences in pathological and biological characteristics between the two groups. The difference in age could be explained by the fact that the nonattenders group also contained patients under 50-years of age. From these analyses it seems that DCIS in nonattenders is not pathologically and biologically more aggressive than DCIS in attenders of the screening program. However, because of the very small number of patients in the nonattenders group no hard conclusions can be drawn.\nHer2\/neu overexpression has been found to correlate with various pathologic and biological factors believed to be associated with more aggressive behavior; high grade, presence of necrosis, ER and PR negativity, and overexpression of Ki-67 (indicating an increased proliferation rate) are features that are strongly related with Her-2\/neu overexpression.28\u201330 The report of Walker et al.24 displayed a Her-2\/neu expression of 59% in symptomatic DCIS and of 42% in mammographically detected DCIS. Another study by Idvall et al.25 reported no difference in Her-2\/neu expression between DCIS before and after introduction of mammographic screening. In this present study Her2\/neu overexpression was the only independent feature to be related with screen-detected DCIS in multivariate analysis (OR = 12.8; 95%CI 1.6\u2013104.0; p = .018), which indicates a more aggressive profile of screen-detected DCIS when compared to interval DCIS.\nAlthough the statistical methods used were univariate and multivariate analysis, the numbers in both groups are small, which explains the broad 95% CI. The small numbers are due to the selection of patients in this study for study period, attendance of screening rounds, and availability of sufficient paraffin-embedded tissue. Clearly, further studies with larger populations are needed to elucidate the relative significance of the Her2\/neu overexpression in women with screen-detected DCIS. Although 28 out of 102 patients were excluded because of lack of sufficient paraffin-embedded tissue, there is no reason to assume that this exclusion results in a significant selection bias. Apart from pathological grade according to Van Nuys, there were no differences in clinicopathological characteristics between the study group and the group of excluded patients. The group of excluded patients displayed a relatively large amount of low-grade DCIS according to Van Nuys (53.6%, p < .001), which is mainly derived from the interval group (n = 10, data not shown). If all patients would have been included the relation of pathological grade and probably Her2\/neu expression with screen-detected patients would be even more significant. The inclusion of patients that actually took part in the screening program was necessary to analyze differences between screen-detected and interval DCIS, which, to our knowledge, has not been performed previously.\nExpression of ER, p53, and cyclin D1 was not related to screen-detected or interval DCIS. There was, however, a relation between screen-detected DCIS and PR negativity in univariate analysis (OR = 0.288; 95%CI 0.087\u20130.957; p = .042). Other studies could not demonstrate a relation of screen-detected DCIS with the expression of the aforementioned markers.12,24,25 In a review by Boland et al.31 ER and PR positivity are related to low-grade DCIS, whereas p53 and cyclin D1 expression are associated with high grade. The fact that screen-detected DCIS is associated with PR negativity provides indirect evidence for the presence of a more aggressive tumor biology. Obviously, PR expression is related to Her2\/neu expression, because PR expression was not significant in multivariate analysis.\nUltimately, the question is how we should interpret these findings. The authors think the results from this study represent no evidence to alter patient management and screening recommendations. Instead, they should rather be regarded as support of current clinical practice in DCIS of the breast. They confirm that every DCIS should be treated until we are able to identify DCIS that will progress to invasive cancer if left untreated.\nIn conclusion, since the advent of screening, the increased incidence of DCIS has raised concerns about the possibility of overdiagnosis of DCIS. This study has shown that screen-detected DCIS has a more aggressive tumor profile than interval DCIS. Therefore, screen-detected DCIS should not be regarded as an overdiagnosis per se, and every woman diagnosed with DCIS by mammographic screening should be treated properly according to existing guidelines or standards of care.","keyphrases":["screening","ductal carcinoma in situ","immunohistochemistry","biological markers","breast neoplasm"],"prmu":["P","P","P","P","M"]}
{"id":"Eur_J_Pediatr-4-1-2234442","title":"Mucopolysaccharidosis type II (Hunter syndrome): a clinical review and recommendations for treatment in the era of enzyme replacement therapy\n","text":"Mucopolysaccharidosis type II (MPS II; Hunter syndrome) is a rare X-linked recessive disease caused by deficiency of the lysosomal enzyme iduronate-2-sulphatase, leading to progressive accumulation of glycosaminoglycans in nearly all cell types, tissues and organs. Clinical manifestations include severe airway obstruction, skeletal deformities, cardiomyopathy and, in most patients, neurological decline. Death usually occurs in the second decade of life, although some patients with less severe disease have survived into their fifth or sixth decade. Until recently, there has been no effective therapy for MPS II, and care has been palliative. Enzyme replacement therapy (ERT) with recombinant human iduronate-2-sulphatase (idursulfase), however, has now been introduced. Weekly intravenous infusions of idursulfase have been shown to improve many of the signs and symptoms and overall wellbeing in patients with MPS II. This paper provides an overview of the clinical manifestations, diagnosis and symptomatic management of patients with MPS II and provides recommendations for the use of ERT. The issue of treating very young patients and those with CNS involvement is also discussed. ERT with idursulfase has the potential to benefit many patients with MPS II, especially if started early in the course of the disease.\nIntroduction\nFirst described by Major Charles Hunter in 1917 [15], mucopolysaccharidosis type II (MPS II or Hunter syndrome; OMIM +309900) is an X-linked recessive disease caused by deficiency of the lysosomal enzyme iduronate-2-sulphatase (I2S). This enzyme cleaves O-linked sulphate moieties from the glycosaminoglycans (GAGs) dermatan sulphate and heparan sulphate as the first step in their degradative pathway. The clinical phenotype of MPS II (Fig.\u00a01) is characterised by progressive pathological lysosomal storage of GAGs in nearly all cell types, tissues and organs.\nFig.\u00a01Appearance of a child with mucopolysaccharidosis type II (Hunter syndrome) at (a) 6\u00a0years of age and (b) 12\u00a0years of age, illustrating the \u2018typical\u2019 clinical phenotype at the more severe end of the disease spectrum. The manifestations of the syndrome and the rate of progression, however, may vary widely among individual patients\nOropharyngeal and tracheo-bronchial deposition of GAGs leads to severe airway obstruction due to macroglossia, supraglottic narrowing, and tracheomalacia. This obstructive anatomy and physiology leads to sleep apnoea and airway obstruction. As the disease progresses, the respiratory system is further compromised by pulmonary restriction secondary to the effects of the disease on the thoracic skeleton. Deposition of GAGs in the heart, liver and spleen leads to cardiomyopathy, cardiac valve dysplasia and hepatosplenomegaly. Bone and joint involvement results in severe skeletal deformities and limitations of joint mobility. In patients with CNS involvement, severe learning difficulties and progressive neurological decline occur. The clinical manifestations of MPS II generally lead to death in the first or second decade of life, although in the more attenuated form of MPS II, death may occur in early adulthood, and some patients have survived into their fifth and sixth decades of life.\nUntil recently, there has been no effective therapy for MPS II. Care has been palliative, focusing on management of the multiple clinical symptoms. Haematopoietic stem cell therapy (HSCT) has been attempted in some patients [36], but the long-term results have been unsatisfactory. The recent introduction of enzyme replacement therapy (ERT) with recombinant human I2S (idursulfase), however, has raised the possibility that the burden of GAG storage can be reduced and that the progressive tissue and organ damage associated with MPS II can be slowed or even prevented. The availability of ERT thus requires a greater awareness and understanding of the disease amongst a range of medical specialists and primary care physicians, so that early diagnosis can be made and treatment started before organ damage becomes irreversible.\nThis article gives an overview of the clinical manifestations, diagnosis and symptomatic management of patients with MPS II and provides, for the first time, recommendations for the use of ERT.\nIncidence and inheritance\nData from The Netherlands and Germany indicate that the incidence of MPS II is 1.3 per 100,000 male live births [1, 23]. Two-thirds of patients present with CNS involvement, representing the more severe end of the disease phenotype. There have been very few reports of recurrent or common mutations within the gene encoding I2S. As a consequence, genotype\u2013phenotype correlations have been difficult to establish, with the exception of those patients who present with large deletions, which are most often associated with more severe disease [38].\nAs MPS II is an X-linked recessive condition, it would not be expected to affect females. Despite this, a few girls have been reported with MPS II [35]. Affected females generally have low levels of I2S activity and an attenuated clinical phenotype, although the somatic abnormalities can be severe in some individuals. In affected heterozygous females, skewed inactivation of the X-chromosome prevents expression of the normal allele [35].\nMolecular basis of MPS II\nMPS II is the only known X-linked MPS disorder. The human gene encoding I2S has been mapped to Xq28. It contains nine exons spread over 24\u00a0kb. Individuals with major deletions or rearrangements in the gene usually have severe MPS II. An I2S-like pseudogene, comprising copies of exons 2 and 3 and intron 7, is located about 20\u00a0kb from the active gene [34]. A recurring rearrangement is due to recombination between the intron region of the gene and a homologous region near exon 3 of the pseudogene, with inversion of the intervening DNA [3]. Very large deletions of the I2S locus may extend to adjoining genes, as the neighbouring DNA is gene rich. A contiguous gene syndrome involving fragile X mental retardation (FMR1 and FMR2) genes could possibly explain the unusual phenotypes seen in some severely affected patients [33]. Over 150 different mutations, resulting in a spectrum of disease phenotypes, have been described.\nGeneral disease manifestations and diagnosis\nTreatment of possible disease complications can greatly improve quality of life. Early diagnosis is therefore essential, particularly with the recent introduction of ERT. Table\u00a01 shows the common presenting features of patients with MPS II, together with the range of specialists who are likely to be involved in diagnosis and care.\nTable\u00a01Presenting features of patients with mucopolysaccharidosis type II (Hunter syndrome) and the specialists likely to be involved in diagnosis and treatmentPresenting featureSpecialistRecurrent upper respiratory tract infections, recurrent otitis media, developmental delay, hepatosplenomegaly, joint restriction, coarse faciesPaediatricianPaediatric neurologistOtolaryngologistRecurrent ear infections, abdominal distension, stiff jointsPrimary care physicianUmbilical and inguinal hernias with recurrenceSurgeonHip dysplasia, arthropathyOrthopaedic specialistUpper airway obstruction, sleep apnoea, recurrent ear infections requiring tube placementOtolaryngologistPapilloedema in the absence of raised intracranial pressureOphthalmologistValvular thickening on echocardiographyCardiologistJoint pain and restrictionRheumatologistPapular pearly rash across the scapulaeDermatologistRestrictive joint range of motionPhysiotherapistCarpal tunnel syndromeHand surgeonCervical myelopathyNeurosurgeon\nClinical spectrum of MPS II\nMPS II is a variable, progressive, multisystem disorder. In most patients, symptoms are severe and death occurs at an early age. In other patients the disease has a more chronic and protracted course. The age of presentation of MPS II is also variable, as are the presenting signs and disease complications. Figure\u00a02 shows the age at onset of the main signs and symptoms of the disease in a cohort of 82 patients enrolled in HOS, the Hunter Outcome Survey. This is a multinational long-term outcomes survey to investigate the natural history of MPS II and the safety and efficacy of ERT with idursulfase [8].\nFig.\u00a02Reported age at onset and prevalence of clinical features in 82 patients with mucopolysaccharidosis type II (Hunter syndrome) enrolled in HOS, the Hunter Outcome Survey\nThe traditional classification of patients into \u2018mild\u2019 or \u2018severe\u2019 subtypes, on the basis of length of survival and the presence or absence of CNS disease, is a gross simplification. The disorder should rather be regarded as a continuum between two extremes (severe and attenuated). It is important to note that individuals who are diagnosed with an attenuated form of the disease may still have symptoms and complications that lead to significant morbidity and disability, and may present with mild to moderate learning difficulties. Although the clinical course for the more severely affected patients is relatively predictable, there is considerable variability in the clinical phenotype and progression of the more attenuated form of the disease.\nIndividuals with attenuated MPS II are most often diagnosed between the ages of 4 and 8\u00a0years. For the majority of these affected patients, the clinical course is very similar to the intermediate form of MPS I (MPS IH\/S, Hurler\u2013Scheie disease). Survival to adulthood is common, but death often occurs between the ages of 20 and 30\u00a0years from cardiac or respiratory disease (Fig.\u00a03). A few patients are less severely affected. In these, life expectancy can be near normal, and such affected males may have children [9].\nFig.\u00a03Mortality by age of patients with mucopolysaccharidosis type II (Hunter syndrome) in the UK. Data are from the Society for Mucopolysaccharide Diseases (MPS Society) and are based on patients registered with the MPS Society between 1950 and October 2006\nPatients with the more severe form of MPS II exhibit a chronic and progressive disease involving multiple organs and tissues. They appear normal at birth, although they tend to be heavy, some have inguinal or umbilical hernias and there is an increased incidence of Mongolian blue spots [25]. The age at diagnosis is usually between 18 and 36\u00a0months, compared with 9\u00a0months for severe MPS I [6]. Death from a combination of neurological deterioration and cardiorespiratory failure usually occurs in the mid-teenage years.\nPhysical appearance\nThe typical patient with attenuated MPS II is short with some change of facial features. Significant arthropathy and connective tissue involvement lead to joint contractures. A combination of hepatomegaly and lax abdominal muscles leads to abdominal prominence.\nIn the more severely affected patients, the typical facial features become apparent in the first 3\u00a0years. They are most likely caused by a combination of storage in the soft tissues of the orofacial region and underlying facial bone dysostosis. Thickening of the alae nasi, lips, ear lobules and tongue becomes progressively more obvious. The patients often develop a plethoric, rosy-cheeked appearance. Thickening of the calvaria results in macrocephaly. Facial and body hypertrichosis is often seen and the scalp hair becomes coarse, straight and thatch-like. Patients also develop specific, pearly, papular skin eruptions, which are usually first seen around the scapulae, but then spread to involve the trunk and upper thighs. These skin eruptions, first noted in Hunter\u2019s original presentation, are said to be pathognomic for MPS II [32].\nDevelopment\nInfants with MPS II appear normal at birth, and early developmental milestones may also be normal, even in the presence of significant somatic disease. Developmental outcomes, however, are highly variable. Significant psychosocial problems occur in affected teenagers and young adults [40]. Even in patients with attenuated disease, cranial magnetic resonance imaging (MRI) scans are often grossly abnormal, with extensive white matter changes as well as dilated perivascular spaces, despite apparently normal intellectual skills [27].\nPatients with more severe MPS II also appear normal at birth, and early developmental milestones may be normal. Some patients fail hearing screening tests in the first year, and speech delay is not unusual in more severely affected patients. By 18\u201324\u00a0months, developmental delay is usually apparent. Most patients make very slow progress after this stage, with a developmental plateau beginning between 3 and 5\u00a0years of age. Unlike children with severe MPS I, who are usually placid, more severely affected children with MPS II can be hyperactive and aggressive. By the time of death in their second decade, most patients with CNS involvement are severely mentally handicapped and dependant on care providers for all their needs.\nBiochemical diagnosis\nAssessing urinary GAGs (heparan and dermatan sulphates) is the usual first screening test for MPS II, and can be quantitative (measurement of total urinary uronic acid) or qualitative (GAG electrophoresis). Although neither test can offer a definitive diagnosis, abnormal measurements indicate the likely presence of an MPS disorder. The sensitivity of these tests, however, is not perfect, particularly when measurements are made on samples of dilute urine. Definitive diagnosis is established by enzyme assay in leukocytes, fibroblasts or plasma, using substrates specific for I2S. Another sulphatase should be measured in conjunction, in order to exclude multiple sulphatase deficiency. This is a much rarer disorder than MPS II and is generally associated with a very poor prognosis. It should be noted that the amount of enzyme activity measured in vitro from cellular extracts does not indicate where an individual falls within the MPS II spectrum of disease severity.\nPrenatal diagnosis\nPrenatal testing is available for foetuses at risk of MPS II. This is generally carried out by enzyme assay of I2S in uncultured chorionic villi, allowing early testing and rapid diagnosis of affected foetuses. Very low activities have been measured in some pregnancies with a (heterozygous) female foetus, emphasising the need to combine an enzyme assay with foetal sex determination [7]. Molecular-genetic prenatal testing can be performed if the mutation is known in the family.\nCarrier testing\nPrevious methods of carrier detection based on an I2S assay in either plasma or hair roots are now known to be unreliable, as there is considerable overlap between the normal and heterozygous ranges. The only definitive test for determining carrier status is based on DNA analysis. In a small number of families, however, the mutation remains elusive, despite full sequencing of the I2S locus.\nSymptomatic treatment\nEven with the introduction of ERT, patients with MPS II still require supportive symptomatic treatment from a wide range of specialists. A comprehensive initial assessment of each patient at diagnosis should therefore be undertaken, and should be followed by regular reviews. Supportive management and the anticipation of possible complications can greatly improve the quality of life of affected individuals and their families. Family members should be offered genetic counselling, and contact with other affected families, patients and support groups can be helpful.\nSkeletal abnormalities\nOrthopaedic complications, caused by a combination of direct bone involvement and severe arthropathy, can lead to significant disability. The destructive arthropathy, which especially affects the hip joints, is a feature of the skeletal disease in some patients and may be due to secondary events occurring within chondrocytes and\/or osteoblasts as a result of storage.\nProgressive arthropathy may affect all joints and leads to severe restriction of motion. The hip joints appear to be particularly vulnerable and severe erosive hip dysplasia can be especially disabling. Poor hand function, due to the characteristic claw-hand deformity, carpal tunnel syndrome and interphalangeal joint stiffness, is also common.\nAbnormal joint function is largely a result of both metaphyseal deformities and thickened joint capsules. Secondary erosive joint disease is particularly disabling and is extremely difficult to manage medically and\/or surgically. The deformed acetabulum and pelvis makes prosthetic surgery a challenge, and many patients become wheelchair-bound because of hip pain. The role of physical therapy in MPS II is not well studied, but range-of-motion exercises appear to offer some benefit in preserving joint function and should be started at an early age. If significant restriction of joint movement has already occurred, range-of-motion exercises may slow further progression.\nCardiovascular system\nCardiac abnormalities detected by echocardiography are common in patients with MPS II. Valvular involvement, with thickening and stiffening of the valve leaflets, commonly leads to mitral and aortic regurgitation and\/or stenosis [24]. Cardiomyopathy is much less common but may be associated with an increased risk of cardiac arrhythmia [14]. Valve replacement surgery may be necessary, and annual cardiac evaluation with echocardiography is essential. Bacterial endocarditis prophylaxis should be used where appropriate. The prevalence and age at onset of the main cardiovascular signs and symptoms as reported in HOS are shown in Table\u00a02.\nTable\u00a02Prevalence and reported age at onset (median and 10th\u201390th percentiles) of the main cardiovascular manifestations of mucopolysaccharidosis type II (Hunter syndrome) in a cohort of 82 patients in HOS, the Hunter Outcome SurveyCardiac manifestationnPrevalence (%)Age at onset (years)Valvular disease34536.2 (2.9\u201313.8)Murmur32526.4 (3.7\u201312.3)Cardiomyopathy497.6 (5.2\u201327.3)Any cardiovascular sign\/symptom49726.0 (2.9\u201313.7)\nRespiratory disease and upper airway manifestations\nChronic recurrent rhinitis and persistent copious nasal discharge without obvious infection are common. Enlargement of the tonsils and adenoids, a narrowed trachea, tracheomalacia, thickened vocal cords, redundant tissue in the upper airway, and an enlarged tongue can contribute to upper airway complications [4, 26, 39]. The upper airway involvement leads to noisy breathing, particularly at night, and is probably a major component of obstructive sleep apnoea, which is a common complication in the later stages of the disease [16]. Because of the airway disease, there is a high risk associated with anaesthesia in these patients [17].\nIn patients with attenuated MPS II, rhinorrhoea tends to improve with age, but upper airway obstruction and sleep apnoea become more troublesome with increasing age. A progressive restrictive respiratory defect also becomes apparent with increasing age, mainly due to progressive changes in the thoracic skeleton.\nSleep studies should form part of the regular assessment schedule, and significant episodes of hypoxia should be managed by continuous or bilevel positive airway pressure devices. Severely affected patients often find such treatment difficult to tolerate. In these patients, supplemental oxygen alone may be an acceptable alternative, but should be used with caution in patients with documented hypercapnia.\nTonsillectomy and adenoidectomy are frequently performed to correct Eustachian tube dysfunction and to decrease airway obstruction. Severely affected patients also tend to have frequent ear infections and constant rhinorrhoea. Early placement of ventilating tubes is recommended in severely affected individuals.\nGastrointestinal system\nProtuberance of the abdomen and hernias caused by progressive hepatosplenomegaly are common. Although organ size can become massive, storage of GAGs in the liver and spleen does not lead to either liver or splenic dysfunction. Patients are prone to periodic bouts of watery diarrhoea, which occur without apparent cause and are not associated with malabsorption. Rectal biopsies in affected patients have demonstrated storage within gut neural cells, and an autonomic cause for episodes of diarrhoea has been postulated [10]. With age, loss of muscle strength and physical inactivity lead to constipation.\nAbdominal hernias should be repaired surgically, though recurrence can occur. Diarrhoea can be controlled by diet and the use of antimotility drugs.\nCentral nervous system\nCommunicating high pressure hydrocephalus, which is common in MPS I, is rare in MPS II. Other CNS complications, such as seizures, are more common. They are usually tonic\u2013clonic in nature and respond to standard anticonvulsant treatment.\nProgressive compression of the spinal cord with resulting cervical myelopathy due to thickening of the dura (hypertrophic pachymeningitis cervicalis) and hyperplasia of the transverse ligament is common [21]. In addition, MRI scans of the craniocervical junction will often show deposition of GAG around the tip of the odontoid process. Most patients have a well-formed odontoid process and atlanto-axial subluxation is usually not a feature of MPS II. Cervical myelopathy may initially present as reduced activity, exercise intolerance or difficulty in rising from a sitting position. If it remains untreated, irreversible cord damage can occur.\nHypertrophic pachymeningitis cervicalis and cervical compression secondary to hyperplasia of the transverse ligaments should be quickly and aggressively treated in patients with attenuated disease. Early and careful cervical decompression performed by an experienced team may prevent severe and devastating consequences.\nPeripheral nervous system\nCarpal tunnel syndrome is common in patients with attenuated forms of MPS II and should be promptly investigated and treated. Nerve conduction studies should be conducted to monitor patients, as the typical symptoms of compression (pain, tingling or numbness) do not occur [13]. Surgical decompression of the median nerve at an early stage of involvement results in either partial or complete improvement in most patients.\nVisual problems\nAlthough corneal opacity has been reported [31], it is not generally a feature of MPS II and helps to distinguish the disorder clinically from severe MPS I and MPS VI. In addition glaucoma is rarely present, even in severely affected patients. Retinal degeneration resulting in decreased peripheral vision and poor dark adaptation is common, but often difficult to investigate fully in a severely affected patient. Disc oedema, uveal effusions and epiretinal membranes have all been reported as part of the variable ocular pathology [2, 20, 37].\nElectroretinography confirms retinal degeneration in many patients, but a disturbance of vision apart from nyctalopia (\u2018night blindness\u2019) is rare. Chronic papilloedema without evidence of raised intracranial pressure is common and may be due to deposition of GAGs within the sclera causing pressure on the optic nerve at the interscleral level [2].\nRegular, annual, ophthalmological assessment is required and should include a measure of intraocular pressure. Spectacles should be prescribed as appropriate.\nHearing\nHearing loss is common in patients with severe MPS II and is correlated with the severity of somatic disease. Manifestations leading to hearing loss include frequent middle ear disease secondary to Eustachian tube dysfunction, dysostosis of the ossicles of the middle ear, scarring of the tympanic membrane, and damage to the eighth nerve. Audiological abnormalities usually consist of conductive deafness early in the course of the disease but as the child ages a sensorineural element appears and most patients have combined deafness once the disease is fully established [29]. Moderate to severe hearing loss develops in most adults with attenuated MPS II. In most patients there is a mixture of both conductive and sensorineural deafness [22]. Appropriate regular audiology assessment and provision of the correct auditory aids are required.\nEducation and behaviour\nGiven the intellectual involvement in the more severely affected patients, it is important that infants with MPS II are provided with a stimulating environment to encourage as much learning as possible during the early stages, as some skills may be retained during the later period of general deterioration. For patients with more attenuated forms of the disease, deafness and physical limitations may impact on education. Educational assessments of special needs should be undertaken to ensure that an appropriate educational environment is provided. This may be in a mainstream school with additional help or within a special school system.\nMany patients with severe MPS II exhibit hyperactivity and aggression. Psychological assessment, behaviour management and the use of medication should be considered in these patients. The hyperactivity generally responds poorly to methylphenidate, and over-sedation is a risk with other forms of medication.\nAnaesthetic complications\nAll patients with MPS II present major anaesthetic risks, and death can result if appropriate precautions are not taken [12]. Patients should only undergo general anaesthesia in centres staffed with anaesthesiologists experienced in these disorders. The most important complications relate to the following.\nDysostosis multiplex leading to rigidity of the neck and spine. Although instability of the spine is rare, significant cervical compression may be present in patients with attenuated disease. This can result in sudden neurological deterioration if excessive neck manipulation is performed during attempts to intubate the patient.Induction may be difficult because of an inability to maintain an adequate airway.Intubation may require smaller-than-anticipated endotracheal tubes and a narrow trachea and thickened vocal cords will impede view. It is recommended that a paediatric pulmonologist or ear, nose and throat specialist should be present during intubation. Fibre-optic laryngotracheoscopy is usually essential.Extubation can also be hazardous for these patients, and there is an increased incidence of post-obstructive pulmonary oedema.Recovery from anaesthesia may be slow, and post-operative airway obstruction is always a risk.\nEnzyme replacement therapy\nRecombinant I2S (idursulfase; Elaprase, Shire Human Genetic Therapies, Cambridge, MA, USA) has recently been licensed for treatment of patients with MPS II in the United States, European Union and Switzerland. Idursulfase is produced in a continuous human cell line and is a purified form of the natural lysosomal enzyme I2S. Mannose-6-phosphate (M6P) residues on the oligosaccharide chains of the glycoprotein enzyme allow specific binding of idursulfatase to M6P receptors on the cell surface, leading to cellular internalisation and targeting of the enzyme to lysosomes, and subsequent catabolism of accumulated GAGs.\nPhase I\/II trial\nTwelve patients were enrolled in a randomized, double-blind, placebo-controlled trial of idursulfase for 24\u00a0weeks, followed by an open-label extension study.\nUrinary GAGs were reduced within 2\u00a0weeks of initiating idursulfase treatment and remained low for the 48\u00a0weeks of the study (P\u2009<\u20090.0001). Both liver and spleen volumes were decreased at 24\u00a0weeks (P\u2009<\u20090.01) and 48\u00a0weeks (P\u2009<\u20090.001). The distance that patients could walk in 6\u00a0min (6-min walk test) increased by an average of 48\u00a0m after 48\u00a0weeks (P\u2009=\u20090.013). Six patients developed IgG antibodies that did not appear to influence the clinical response to idursulfase [18].\nPhase II\/III trial\nA multinational, randomized, double-blind, placebo-controlled trial was performed to evaluate the safety and efficacy of idursulfase, 0.5\u00a0mg\/kg administered weekly, compared with placebo. Additionally, the trial evaluated idursulfase, 0.5\u00a0mg\/kg every other week, compared with placebo. Ninety-six patients were randomized to one of three groups with each patient receiving a total of 52 infusions of either idursulfase, idursulfase alternating weekly with placebo, or placebo. The primary efficacy endpoint of the trial was a composite of two clinical measures \u2013 forced vital capacity and the 6-min walk test.\nPatients receiving the weekly dosing regimen of idursulfase showed a statistically significant improvement in the primary efficacy endpoint (P\u2009<\u20090.005) compared with placebo. Patients receiving the alternate-week dosing regimen of idursulfase also showed a statistically significant improvement (P\u2009<\u20090.05) compared with placebo [19].\nTreatment with idursulfase was generally well tolerated. The most common adverse events observed were associated with the clinical manifestations of MPS II. Of the adverse events considered possibly related to idursulfase, infusion-related reactions were the most common and were generally mild. There were two patient deaths during the study, both of which were considered unrelated to treatment with idursulfase. IgG and IgM antibodies were observed in the idursulfase-treated patients at some point during the course of the study. When antibodies occurred, there was a transient increase in urinary GAGs, presumably due to a neutralising effect. This did not correlate with any of the clinical variables measured (Shire HGT, data on file). No IgE antibodies were observed and no patient withdrew from the trial due to an adverse event considered related to idursulfase [19].\nCurrent treatment with idursulfase\nIdursulfase is given by weekly intravenous infusion over 3\u00a0h at a dose of 0.5\u00a0mg\/kg diluted in an appropriate volume of saline (according to weight). Patients may receive premedication with antipyretics and\/or antihistamines at the discretion of the prescribing physician. Patients who have experienced infusion-related reactions should be premedicated for subsequent infusions. Consideration should be given to the possibility of sodium overload in small infants, and, if necessary, the total volume of the infusion can be decreased to 50\u00a0ml. Although the treatment is generally safe and infusion-related reactions are no more frequent than with other protein-based therapies, anaphylactoid reactions have been reported.\nEarly response to ERT\nThe response to ERT appears to depend on the severity of the individual\u2019s condition and the age at which the treatment begins. In our experience, the first sign of efficacy in most young (prepubertal) patients is usually an enhanced feeling of wellbeing and greater energy, manifesting as an increased ability to take part in normal daily activities. By 2\u00a0months, most patients have urinary GAG levels approaching the normal range and there is usually evidence of a reduction in size of the liver and spleen. After a further month of therapy there is often an improvement in soft-tissues joint contractures and the beginning of a growth spurt. By 6\u00a0months of treatment, most patients have improved the distance that they can achieve in the 6-min walk test and most show stabilisation (if not improvement) in pulmonary function tests. Most of the improvement occurs in the first 12\u201318\u00a0months of treatment. After this period of time, gains are very slow and the condition of most patients will generally have stabilised, although physicians still need to be vigilant and look actively for possible disease-related complications.\nERT for particular patient groups\nTreating very young patients\nExperience of treating children under the age of 5\u00a0years with idursulfase is limited, as the clinical trials enrolled patients above this age so that the patients would be able to comply with and be able to cooperate with repeated pulmonary function and endurance testing. The following is therefore based on our personal clinical experience. Treating patients at a very young age leads to practical problems with the infusions. These may be resolved with indwelling vascular access devices. Outcomes are likely to be better in patients treated from a very young age, but there are currently no recommendations on how to monitor the response to treatment, especially in patients either too young to cooperate with the tests used in the clinical trials (pulmonary function tests and 6-min walk test) or in patients who are not yet showing symptoms of the disease. This situation would be improved by identifying a suitable biomarker that would reflect the disease burden and that would respond promptly to therapy.\nWe believe that these challenges should not prevent the introduction of treatment in patients under the age of 5\u00a0years after discussion with parents. Careful clinical follow-up, however, will be necessary in these patients, accompanied by regular (annual) developmental assessments. As of October 2006, there were 12 patients below 5\u00a0years of age in HOS.\nTreating patients with severe CNS involvement\nIdursulfase is not expected to cross the blood\u2013brain barrier and therefore would not be expected to lead to any improvement in CNS dysfunction in patients with severe MPS II. In most patients there is likely to be some initial benefit in terms of enhanced respiratory function due to airway improvement and reduction in size of the liver and spleen. In addition, joint mobility may improve as a result of ERT on peri-articular soft tissue. The burden of weekly intravenous therapy may be significant in some patients with very severe CNS disease. The authors believe that patients with severe CNS involvement may be offered the possibility of treatment for a \u2018trial\u2019 period of 12\u201318\u00a0months, after which time a decision should be made as to whether to continue. Decisions on whether to start and continue treatment should be made only after detailed discussion with the parents.\nHome therapy\nMore than 90% of patients treated with idursulfase have had no infusion-related reactions and, in those who do, the reactions are mostly minor and respond promptly to reducing the rate of infusion and using antipyretics and antihistamines for future treatments. Under these circumstances, it would appear that idursulfase is a therapy that could be administered safely in the patient\u2019s home after a minimum of 12 infusions in hospital.\nDepending on the labelling and clinical practices in particular countries, a logical approach would appear to be to consider home therapy for those patients with minimal or no obstructive airway disease (as judged by sleep study and pulmonary function tests). It should be stressed, however, that ERT during respiratory infections or other intercurrent illnesses are contraindicated. Treatment should be postponed under such circumstances, regardless of whether treatment is given in the hospital or home setting.\nManagement of infusion reactions\nInfusion-related reactions appear to be of two types: those occurring during the infusion and \u2018late\u2019 reactions occurring 12\u00a0h or more after the infusion.\nThe classic reactions occurring during the infusion, with fever, chills and urticaria, respond to temporarily stopping the infusion, administering paracetamol (acetominophen) and antihistamines, and restarting the infusion after at least 30\u00a0min but at a slower rate. Before subsequent infusions, patients should be premedicated with acetominophen and antihistamines 1\u00a0h before the infusion. If the reactions continue despite this premedication, consideration should be given to pretreatment with corticosteroids; for example, prednisolone, 1\u00a0mg\/kg, 12\u00a0h and 1\u00a0h before infusion.\nLate reactions typically consist of a sunburn-type rash and mild wheezing. The rash can be managed as above, but the wheezing requires bronchodilator treatment and, possibly, oxygen supplementation. All patients who have significant infusion-associated reactions should have specific immune testing. Reducing the rate of infusion is mandatory in these patients.\nTo assess the efficacy of ERT in individual patients and to ensure the appropriate management of possible infusion-related reactions, it is recommended that the treatment of patients receiving ERT is overseen by experienced physicians in centres with expertise in treating lysosomal storage diseases.\nHaematopoietic stem cell therapy\nThe potentially beneficial effect of HSCT on lysosomal storage diseases is thought to be due to the replacement of deficient macrophages with marrow-derived donor macrophages (Kupffer cells \u2013 pulmonary, splenic, nodal, tonsillar and peritoneal macrophages \u2013 and microglial cells) which constitute an ongoing source of enzyme capable of gaining access to various storage sites. HSCT has been successful in modifying the course of the disease in patients with severe MPS I and MPS VI. In a single small study of HSCT in MPS II, however, the results of treatment were not positive, with nine of ten patients continuing to exhibit progressive neurodegeneration [36].\nThe future\nWhilst ERT is likely to benefit many patients with MPS II, the problems associated with severe CNS involvement are unlikely to be solved by intravenous ERT. Other methods of treatment, including alternative routes of enzyme administration, will therefore need to be studied. Direct administration of enzyme into the cerebrospinal fluid, either into the ventricle or lumbar space, has not prevented progressive neurological deterioration in another lysosomal storage disorder, type II Gaucher disease [28]. This approach has not been attempted in MPS II, and formal studies are required, perhaps with the addition of immunosuppression to prevent antibody formation. Currently, there are no suitable methods for infusion directly into the brain, and repeated intracerebral injections would not be practical. It may be that to treat CNS disease more effectively, peripheral ERT may have to be combined with other approaches, such as cell-based therapies, perhaps involving gene enhancement or replacement.\nGene therapy is the logical approach for treating disorders of the CNS, and animal work on other MPS disorders is beginning to show promise [5, 30]. Choice of vector, the use of immunosuppression and the route of administration are all issues that need addressing before the first human clinical trials of gene therapy involving MPS disorders are performed.\nUse of nanoparticles containing engineered myoblasts expressing I2S has been reported. Scaling-up studies, however, are required to evaluate the feasibility of such devices [11].\nConclusion\nMPS II (Hunter syndrome) is a severe progressive multisystemic disorder that has the potential to cause disease in most body systems and is usually fatal in the second or third decade of life. Ideally, management should be centralised in major medical centres with access to all medical specialties. Management is multidisciplinary and a holistic approach to the patient is required, especially for those who have severe neurological involvement. ERT is an important new therapy that has the potential to help many patients, providing that it is started early in the course of the disease. CNS disease remains a major challenge and an innovative approach to treatment will be needed if this is to be addressed fully.","keyphrases":["mucopolysaccharidosis type ii","hunter syndrome","enzyme replacement therapy","idursulfase","treatment recommendations"],"prmu":["P","P","P","P","R"]}
{"id":"Breast_Cancer_Res_Treat-3-1-2001219","title":"Letrozole in advanced breast cancer: the PO25 trial\n","text":"Tamoxifen has been a standard first-line endocrine therapy for post-menopausal women with hormone-responsive advanced breast cancer, but more than half of patients fail to respond and time to progression is less than 12 months in responders. The third-generation aromatase inhibitors were developed to provide more effective alternatives to tamoxifen. In the Femara Study PO25, post-menopausal women with advanced breast cancer were randomized to receive letrozole 2.5 mg (n = 453) or tamoxifen 20 mg (n = 454) given orally daily until progressive disease occurred. Patients were permitted to cross over to the other treatment at progression. In the primary efficacy analysis, median time to progression (TTP) was significantly longer with letrozole than with tamoxifen (9.4 months vs. 6.0 months, respectively; P < 0.0001). The objective response rate (ORR) was significantly higher for letrozole than for tamoxifen (32% vs. 21%; P = 0.0002). Prospectively planned analyses of the intent-to-treat population showed that letrozole significantly improved overall survival (OS) compared with tamoxifen over the first 24 months of the trial. An exploratory analysis of patients, who did not cross over, indicated a median OS benefit of 14 months for letrozole compared with tamoxifen. Letrozole is the only third-generation aromatase inhibitor that has demonstrated significant improvements in ORR, TTP, and early OS.\nIntroduction and rationale\nThe treatment goals for advanced or metastatic breast cancer (MBC) are to delay disease progression and to prolong survival [1, 2] and to optimize patient care in terms of ameliorating symptoms, thereby improving or maintaining quality of life [3\u20135]. Although treatment may include surgery and radiation therapy for the treatment of locally advanced tumors or isolated metastases, systemic therapies (endocrine, cytotoxic, biologic, and palliative) are the foundation of disease management [6, 7]. Systemic therapy for patients with advanced breast cancer should be tailored according to specific tumor biology, particularly with respect to hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) status, the growth rate of disease, presence of visceral metastases, history of prior therapy and response, susceptibility to treatment-related toxicity, and individual patient preference [7\u201314]. Systemic therapy can prolong survival and enhance patient quality of life but is not curative [1]. Consequently, minimally toxic endocrine therapies are generally preferred to cytotoxic therapy as initial therapy for patients with hormone-responsive tumors [6, 15].\nSince the 1980s, endocrine therapy with tamoxifen was well established as a standard first-line treatment for post-menopausal women with advanced breast cancer, even though estrogen receptor (ER) expression was not always used routinely to select patients for endocrine therapy [16\u201318]. The first-generation aromatase inhibitor aminoglutethimide or a progestin such as megestrol acetate has provided a reasonable second-line alternative [19\u201322]. The objective response rate (ORR) to tamoxifen was shown to be in the range of 25%\u201345% [16, 17, 19, 21, 23\u201332], indicating that more than half of the patients with advanced breast cancer are intrinsically resistant to tamoxifen. Furthermore, the short median time to treatment failure (TTF), in the range 6\u20138\u00a0months, demonstrates a relatively rapid emergence of resistance in patients initially sensitive to tamoxifen [19, 27]. Loss of ER expression appears to be the dominant mechanism of de novo resistance, and most ER\/progesterone receptor negative (PgR\u2212) tumors do not respond to tamoxifen [18, 33\u201336]. However, the majority of patients who develop acquired tamoxifen resistance still express ER at the time of progression [37, 38] and may respond to alternative endocrine therapies [39].\nThe third-generation aromatase inhibitors letrozole, anastrozole, and exemestane were developed in the search for more effective therapeutic alternatives to tamoxifen. Aromatase inhibitors prevent estrogen synthesis by potently inhibiting the aromatase enzyme, which converts androgens to estrogen [40]. Unlike tamoxifen, the aromatase inhibitors do not have any partial estrogen-agonist activity [41] and are less susceptible to the emergence of resistance associated with long-term estrogen deprivation [42]. The development and mechanism of action of aromatase inhibitors is described in detail in the article by Dr. Bhatnagar in this supplement.\nStudies of aromatase inhibitors in the second-line setting\nThe initial randomized controlled trials of third-generation aromatase inhibitors were conducted in patients with advanced breast cancer in whom tamoxifen had failed (i.e., second-line setting). Letrozole, anastrozole, and exemestane all demonstrated evidence of clinical superiority to megestrol acetate in the second-line setting [43\u201347]. Thus, the individual trials demonstrate a trend or even a significant difference in favor of the third-generation aromatase inhibitors in one or more efficacy end points; in addition, the aromatase inhibitors were shown to be associated with improved tolerability versus comparator endocrine therapy in these randomized trials.\nOne trial demonstrated a significantly higher ORR for letrozole (2.5\u00a0mg dose) compared with megestrol acetate (24% vs. 16%, respectively; P\u00a0=\u00a00.04) and a trend toward longer time to progression (5.6 vs. 5.1\u00a0months, P\u00a0=\u00a00.07) [45]. In this trial, low-dose letrozole (0.5\u00a0mg) was associated with similar efficacy outcomes compared with megestrol acetate. However, in another similarly designed trial with letrozole versus megestrol acetate, overall response rates with the two doses of letrozole (0.5 and 2.5\u00a0mg) and with the comparator were similar (21%, 16%, and 15%, respectively). In this trial, low-dose letrozole was superior to megestrol acetate in terms of time to progression (TTP) (P\u00a0=\u00a00.044) and survival (P\u00a0=\u00a00.053). Differences in the distribution of baseline variables may explain the different outcomes in the two trials in terms of the superiority of letrozole over megestrol acetate according to dose [48]. Letrozole was significantly better tolerated than megestrol acetate, specifically in terms of serious adverse experiences, discontinuation due to poor tolerability, cardiovascular side effects, and weight gain [45].\nThird-generation aromatase inhibitors have demonstrated greater potency and selectivity than the first-generation compound aminoglutethimide [49]. Two doses of the most potent aromatase inhibitor letrozole (2.5\u00a0mg and 0.5\u00a0mg) [49] were compared with aminoglutethimide in a randomized controlled trial in the second-line setting and demonstrated superior efficacy and improved safety [50]. The higher dose of letrozole showed a trend (P\u00a0=\u00a00.06) toward superior ORR (19.5%) compared with aminoglutethimide (12.4%). Letrozole 2.5\u00a0mg was also significantly superior in TTP, TTF, and overall survival (OS). Fewer patients taking letrozole experienced adverse events than those taking aminoglutethimide (33% vs. 46%) [50]. Letrozole has also been compared with anastrozole in a randomized, unblinded trial in the second-line setting in patients with MBC. The trial showed that letrozole was associated with a statistically higher ORR than anastrozole (19.1% vs. 12.3%, respectively; P\u00a0=\u00a00.013), whereas TTP (the major end point), TTF, and clinical benefit and duration of response were similar between the two agents [51]. Both letrozole and anastrozole were well tolerated, and a similar incidence of adverse events was observed in the two groups.\nThese studies generated the hypothesis that letrozole might have superior efficacy to tamoxifen as first-line therapy for advanced breast cancer. A large clinical trial (Femara Study PO25) was therefore conducted to compare the efficacy and tolerability of letrozole with those of tamoxifen as first-line therapy in post-menopausal women with advanced breast cancer [52]. This review will describe the results of the PO25 trial, highlighting the evidence for the superiority of letrozole over tamoxifen as first-line endocrine therapy in this setting.\nTrial design and patients\nThe Femara Study PO25 was the largest phase 3 trial conducted in the advanced breast cancer setting [52, 53]. This randomized, double-blind, double-dummy trial was powered for superiority and needed to enroll approximately 900 patients to demonstrate a 20% reduction in the risk of progression with the more effective treatment. To achieve the recruitment target, the trial was conducted in 201 centers in 29 countries. Local ethics review boards approved the protocol, and all patients gave written informed consent before study enrollment.\nRandomized trial design\nPatients were randomized to receive letrozole 2.5\u00a0mg or tamoxifen 20\u00a0mg given orally daily until progressive disease occurred. Patients were permitted to cross over from 1 treatment arm to the other in a double-blind fashion if their first-line treatment was discontinued because of progressive disease or for any other reason (Fig.\u00a01). Patients in whom endocrine therapy was discontinued were subsequently treated as clinically indicated, using chemotherapy, trastuzumab, and bisphosphonates. The crossover design was an integral part of the study, and it probably affected the assessment of OS.\nFig.\u00a01Study design\nPatient population\nPost-menopausal women with advanced breast cancer, defined as stage IIIB locally advanced disease, locoregionally recurrent disease that was not amenable to surgery or radiotherapy, or metastatic disease, were eligible for inclusion in the trial. All patients presented with measurable or assessable tumors and were candidates for endocrine therapy. Patients had estrogen receptor-positive (ER+) and\/or progesterone receptor-positive (PgR+) tumors or unknown HR status. One prior chemotherapy regimen for the treatment of metastatic disease was permitted, but recurrence during or within 12\u00a0months of adjuvant antiestrogen therapy and any prior endocrine therapy for advanced breast cancer precluded enrollment.\nEnd points\nThe primary end point was TTP, defined as the interval between date of randomization and the earliest date of disease progression. Disease progression was determined on the basis of tumor progression (an increase of 25% or more in measurable lesions, an estimated increase of the same magnitude of nonmeasurable lesions, or the appearance of new lesions), treatment discontinuation with evidence of clinical deterioration due to breast cancer, death due to breast cancer, or death of unknown cause (with documented evidence of clinical deterioration due to breast cancer) while receiving treatment or within 6\u00a0weeks of discontinuation of treatment.\nThe secondary end points were ORR, duration of overall response, rate and duration of clinical benefit, TTF, time to response (TTR), time to chemotherapy (TTC), safety, and OS. ORR was defined as the proportion of patients who achieved a complete response (CR) or a partial response (PR), confirmed by a second evaluation 1\u20133\u00a0months later. The duration of overall response was defined for patients with CR or PR, as the interval between date of randomization and the earliest date of disease progression. The rate of clinical benefit was defined as proportion of patients who achieved CR or PR or who stabilized (NC) for at least 24\u00a0weeks; the duration of clinical benefit was defined for patients who achieved CR or PR or NC as the interval between date of randomization and the earliest date of disease progression. TTF was defined as the interval between date of randomization and the earliest date of disease progression, withdrawal, lost to follow-up, or death. TTR was defined for CR or PR patients as the interval between randomization and the earliest documentation of response, and TTC was defined as the total duration of endocrine therapy. The duration of OS was defined as the interval between randomization and death for any reason.\nExploratory analyses of OS were performed. The first analysis included all patients with censoring at crossover, whereas the second included only patients with no crossover. The latter group predominantly comprised of patients with \u201cnonresponsive\u201d disease (patients who responded to first-line therapy are more likely to be crossed over later at progression), whereas the former included \u201cnonresponsive\u201d as well as \u201cresponsive\u201d patients.\nEfficacy\nThe characteristics of the 907 patients included in the intent-to-treat (ITT) population were well balanced between the letrozole and tamoxifen arms. The median age of the patients was 65\u00a0years (range 31\u201396\u00a0years) in the letrozole arm and 64\u00a0years (range 31\u201393\u00a0years) in the tamoxifen arm. Patients were predominantly white (86%), and 92% had Karnofsky performance status (KPS) scores of 80\u2013100. The majority (93%) of the study population had metastatic disease. Soft tissue lesions were the dominant metastatic site in one quarter of patients and were present in 63% and 61% of patients in the letrozole and tamoxifen arms, respectively. Bone metastases were the dominant metastatic site in approximately 30% of patients and were present in 54% and 50%, respectively. Visceral metastases were the dominant site in 43% of patients in the letrozole arm and 46% of the patients in the tamoxifen arm. Most patients (71% in the letrozole arm and 66% in the tamoxifen arm) had not received any prior chemotherapy, and few had received chemotherapy for advanced disease (9% and 11%, respectively). The majority of patients (109 of 167) treated with adjuvant tamoxifen received at least 2\u00a0years of therapy, and the treatment-free interval between stopping adjuvant therapy and entering the study was more than 2\u00a0years in 126 of 167 patients. Of the 907 patients included in the ITT efficacy population, 467 crossed over to the other treatment arm, 75 continued on first-line therapy without progression, and the remainder terminated first-line treatment without crossover (Fig.\u00a02).\nFig.\u00a02Patient disposition\nLetrozole was superior to tamoxifen for all primary and secondary efficacy end points, including a prospectively planned survival analysis at 1- and 2-year follow-up [53].\nTime to progression\nIn the primary efficacy analysis, the median TTP was significantly longer with letrozole than with tamoxifen (9.4\u00a0months vs. 6.0\u00a0months, respectively; P\u00a0<\u00a00.0001) (Fig.\u00a03) [53]. Of patients in the letrozole arm, 359 (79%) progressed, compared with 387 (85%) in the tamoxifen arm. The hazard ratio of 0.72 represents a 28% reduction in the risk of disease progression with letrozole (P\u00a0=\u00a00.0001).\nFig.\u00a03Time to progression at median follow-up of 32\u00a0months for patients on first-line letrozole versus tamoxifen. Reprinted from ref. [53] with permission from the American Society of Clinical Oncology\nThe significant improvement in TTP with letrozole was confirmed in supportive multivariate analysis of prospectively defined baseline covariates, including receptor status, prior adjuvant tamoxifen therapy, and dominant site of metastatic disease [52, 54]. The analysis showed that the risk of progression was increased by the presence of either visceral or bone metastases as the dominant site of metastatic disease compared with soft tissue as dominant site. In the multivariate analysis, the significant improvement in TTP with letrozole over tamoxifen (hazard ratio 0.70; 95% confidence intervals [CI] 0.60, 0.81; P\u00a0=\u00a00.0001) was similar to the benefit observed in unadjusted analysis and was significant for each individual covariate (P\u00a0=\u00a00.0001) [53]. Median TTP values for letrozole and tamoxifen in the different subgroups are shown in Table\u00a01. In patients with nonvisceral metastases, the risk for progression was 25% lower with letrozole than with tamoxifen, whereas in patients with visceral metastases, excluding the liver, the risk for progression was 34% lower and the median TTP was almost twice as long with letrozole than with tamoxifen [54]. Although TTP was shortest for patients with liver lesions, the risk for progression was still 36% lower with letrozole than with tamoxifen in this subgroup [54].\nTable\u00a01Time to progression in different patient subgroups [57]SubgroupLetrozoleTamoxifenDominant disease site: soft tissuen113115Median TTP12.1\u00a0months6.4\u00a0monthsDominant disease site: bonen145131Median TTP9.5\u00a0months6.3\u00a0monthsDominant disease site: visceran195208Median TTP8.3\u00a0months4.6\u00a0monthsPatients who had n9483\u00a0\u00a0\u00a0\u00a0received priorMedian TTP8.9\u00a0months5.9\u00a0months\u00a0\u00a0\u00a0\u00a0antiestrogenHazard ratio (95% CI)a0.60 (0.43, 0.84)HR-positiven294305Median TTP9.4\u00a0months6.0\u00a0monthsHazard ratio (95% CI)a0.69 (0.58, 0.83)HR-unknownn159149Median TTP9.2\u00a0months6.0\u00a0monthsHazard ratio (95% CI)a0.77 (0.60, 0.99)TTP, time to progression; CI, confidence interval; HR, hormone receptoraHazard ratios\u00a0<\u00a01.0 indicate superiority for letrozole relative to tamoxifen\nPatients with prior adjuvant antiestrogen therapy benefited from letrozole in line with the total group, as did patients irrespective of positive or unknown receptor status of the primary tumor.\nA prospectively planned analysis by patient age (<70\u00a0years and \u226570\u00a0years) also demonstrated that median TTP was significantly longer for letrozole than for tamoxifen in both age groups (8.8\u00a0months vs. 6.0\u00a0months, respectively, in the younger group and 12.2\u00a0months vs. 5.8\u00a0months in the older group) [55].\nResponse to therapy\nLetrozole was associated with a significantly better response to therapy compared with tamoxifen [52, 53]. ORR was significantly higher for letrozole than for tamoxifen (32% vs. 21%; P\u00a0=\u00a00.0002), and the corresponding rate of CRs was also significantly higher for letrozole (9% vs. 3%; P\u00a0=\u00a00.0004). The rate of treatment failure was lower with letrozole (75%) than with tamoxifen (85%), and median TTF was significantly prolonged (9.0\u00a0months vs. 5.7\u00a0months, respectively; P\u00a0<\u00a00.0001).\nA supportive multivariate analysis of ORR, adjusted for the same covariates as used for the TTP analysis, showed that prior adjuvant tamoxifen, as well as visceral or bone metastases as the dominant site of metastases, significantly decreased the probability of achieving a response. The analysis also confirmed that letrozole significantly increased the probability of achieving a CR or PR compared with tamoxifen (odds ratio 1.80, 95% CI 1.32\u20132.47; P\u00a0=\u00a00.0002) and that the superiority of letrozole remained statistically significant for each of covariates (P\u00a0=\u00a00.001) [52]. ORRs achieved with letrozole and tamoxifen in the different subgroups are shown in Table\u00a02.\nTable\u00a02Objective response rate in different patient subgroups [57]SubgroupLetrozoleTamoxifenDominant disease site: soft tissuen113115ORR50%34%Dominant disease site: bonen145131ORR23%15%Dominant disease site: visceran195208ORR28%17%Patients who hadn8483\u00a0\u00a0\u00a0\u00a0received priorORR26%8%\u00a0\u00a0\u00a0\u00a0antiestrogenOdds ratio (95% CI)a3.85 (1.50, 9.60)HR-positiven294305ORR33%22%Odds ratio (95% CI)a1.78 (1.20, 2.60)HR-unknownn159149ORR30%20%Odds ratio (95% CI)a1.79 (1.10, 3.00)ORR, objective response rate; CI, confidence interval; HR, hormone receptoraOdds ratios >1.0 indicate superiority for letrozole relative to tamoxifen\nOverall survival\nThe median OS was 34\u00a0months for the letrozole group and 30\u00a0months for the tamoxifen group (P\u00a0=\u00a00.53). Prospectively planned analyses of the ITT population showed that letrozole significantly improved OS compared with tamoxifen over the first 24\u00a0months of the trial [53]. A Kolmogorov-Smirnov analysis to compare the survival distributions in the 2\u00a0arms [56] demonstrated a significant difference in favor of letrozole between 6 and 20\u00a0months (P\u00a0=\u00a00.003) and showed that the maximum difference in survival occurred at 14\u00a0months; at this time point, there were 85 deaths (19%) in the letrozole arm compared with 132 deaths (29%) in the tamoxifen arm. In addition, repeated log-rank tests performed at 6-month intervals indicated that survival was significantly greater with letrozole between 6 and 24\u00a0months (6\u00a0months: P\u00a0=\u00a00.0167; 12\u00a0months: P\u00a0=\u00a00.0038; 18\u00a0months: P\u00a0=\u00a00.0010; 24\u00a0months: P\u00a0=\u00a00.0246) (Fig.\u00a04). The OS curves for the letrozole and tamoxifen groups crossed at around 36\u00a0months, at which time point most patients had either crossed over to the other study drug or had switched to different second-line treatments [53].\nFig.\u00a04Letrozole versus tamoxifen: patients alive at 6-month intervals. Reprinted from ref. [48] with permission from Elsevier\nAdditional exploratory analyses were therefore performed to determine the influence of crossover on OS. The crossover design was an integral part of the trial and, as with all crossover designs, had a confounding influence on the assessment of OS. Second-line endocrine therapy is generally less effective than first-line treatment in patients responsive to first-line therapy [16]; therefore, evaluation of OS may be impaired if the second-line treatment is actually more effective than the original first-line treatment. Furthermore, patients who are responsive to first-line therapy are more likely to cross over than are patients with nonresponsive disease who do not obtain benefit from first-line therapy.\nApproximately 50% of patients crossed over to the other treatment arm (Fig.\u00a02), and almost all of the crossovers had occurred by 36\u00a0months. The median time to crossover was longer for patients initially randomized to the letrozole arm (17\u00a0months for letrozole to tamoxifen vs. 13\u00a0months for tamoxifen to letrozole). The median OS from initial randomization, censoring time to death at crossover, was 42\u00a0months (95% CI 36\u00a0months to not estimable) for letrozole and 30\u00a0months (95% CI 27 to <36\u00a0months) for tamoxifen [53]. The superior efficacy of letrozole compared with tamoxifen was also indicated by an analysis of mortality rates and OS following crossover to the alternate treatment. The analysis showed that the mortality rate was substantially reduced (47% vs. 63%, respectively), and OS improved in patients who crossed over to second-line letrozole compared with those who crossed over to second-line tamoxifen (31\u00a0months; 95% CI 22\u201340\u00a0months vs. 19\u00a0months; 95% CI 17\u201324\u00a0months, respectively) [53].\nThis OS analysis included all patients censored at the time of crossover (i.e., both \u201cnonresponsive\u201d and \u201cresponsive\u201d patients). A second exploratory efficacy analysis of OS included only patients who did not cross over to the other arm and thus predominantly comprised patients with nonresponsive disease. This second analysis, limited to the patients who did not cross over to the alternate drug at progression, indicated a median OS benefit of 14\u00a0months for letrozole (35\u00a0months; 95% CI 29\u201343\u00a0months) compared with tamoxifen (20\u00a0months; 95% CI 16\u201326\u00a0months) [57].\nTime to chemotherapy\nHormone therapy is the preferred treatment strategy for patients with hormone-responsive advanced breast cancer, except for those individuals with rapidly progressive disease for whom initial chemotherapy is indicated [15]. Extending the TTC is thus an important goal with hormone therapy and can maintain quality of life without having a detrimental effect on outcome. In the PO25 trial, TTC was significantly longer for patients whose initial treatment was letrozole compared with those initially randomized to receive tamoxifen (16.3 vs. 9.3\u00a0months; P\u00a0=\u00a00.005).\nSafety\nBoth letrozole and tamoxifen were well tolerated [52, 53]. The incidence of adverse effects related to study drug during first-line treatment was similar for letrozole (38%) and tamoxifen (37%). Hot flushes (16% and 13%, respectively), nausea (6% and 6%, respectively), and hair thinning (5% and 3%, respectively) were the most common treatment-related adverse events reported. Bone fractures of any etiology occurred in 5.3% of patients in the letrozole group, compared with 4.2% in the tamoxifen arm, resulting in fracture rates per patient-year of treatment of 0.0427 and 0.0451, respectively [52].\nA quality-adjusted time without symptoms or toxicity (Q-TWiST) follow-up study assessed the trade-offs between progression-free survival and toxicity in the ITT population from the PO25 trial [58]. The Q-TWiST approach quantitatively adjusts periods in which treatment toxicities or symptoms of disease progression are present to reflect the potentially reduced value for the patient; this methodology divides the survival time of the patient into various health states, assigns utility states to each, and compares treatments based on OS experience [59]. The Q-TWiST analysis of the clinical trial data from the PO25 trial showed that the longer TTP with letrozole compared with tamoxifen is achieved without increased time with adverse events (2.2 vs. 2\u00a0months, respectively), resulting in a significantly greater quality-adjusted survival for patients on letrozole (2.5-month advantage; P\u00a0<\u00a00.0001) [58].\nTime to worsening of KPS (decrease of \u226520 points) was significantly delayed for first-line letrozole compared with first-line tamoxifen (hazard ratio 0.62; P\u00a0=\u00a00.001) [54]. A subset analysis according to sites of metastases demonstrated that in patients with visceral metastases without liver involvement (mostly lung metastases), significantly fewer letrozole patients (14%) than tamoxifen patients (30%) experienced deteriorations in their KPS scores by \u226520 points [54]. However, KPS was relatively insensitive to change in these first-line patients.\nCost-effectiveness\nIn addition to its clinical superiority over tamoxifen, economic analyses have also shown that letrozole is highly cost-effective as first-line endocrine therapy in post-menopausal women with advanced breast cancer [60\u201362]. A follow-up analysis of patient data from the PO25 trial calculated the cost-effectiveness of first-line letrozole and tamoxifen by determining the ratio: difference in costs of breast cancer care to the difference in life years (LYs) between the two treatments [60]. The mean costs of care were $7323 and $5468 for letrozole and tamoxifen, respectively, representing $1855 in incremental costs with first-line letrozole. Mean LYs to death or to the end of first- or second-line hormonal therapy were 1.54 and 1.29 for patients randomized to first-line letrozole or tamoxifen, respectively. Thus, the incremental cost per LY saved with first-line letrozole vs. tamoxifen was $7420 (1855\/0.25\u00a0=\u00a07420) (2.5\u201397.5 percentiles $6470\u2013$14,865).\nIn another economic analysis conducted in the United Kingdom, data from the PO25 trial were used to estimate the effectiveness of treatment [61]. The analysis showed that the mean cost of providing first- and second-line hormonal therapy was GBP4765 for first-line letrozole and GBP3418 for first-line tamoxifen (a difference of GBP1347). Since patients receiving first-line letrozole gain an additional 0.228 LYs, or 0.158 quality-adjusted life years (QALYs), the cost-effectiveness analysis showed that first-line hormonal therapy with letrozole gains additional LYs at a cost of GBP5917, whereas the cost per additional QALY gained is GBP8514, which is well within the accepted cost range.\nThe PO25 trial data were also used in a Canadian analysis that compared the cost-effectiveness of letrozole, anastrozole, and tamoxifen [62]. The analysis showed an incremental cost per quality-adjusted progression-free year of CAN$12,500 and CAN$19,600 for letrozole and anastrozole, respectively, relative to tamoxifen. The authors concluded that both letrozole and anastrozole are economically acceptable alternatives to tamoxifen.\nConclusions\nThe Femara Study PO25 has provided evidence from a well-powered, randomized, controlled trial to show that letrozole provides a significant advantage in OS compared with tamoxifen as first-line treatment of patients with advanced breast cancer [53]. Letrozole is the only aromatase inhibitor to demonstrate consistent superiority over tamoxifen in this setting [53, 54].\nRandomized first-line therapy trials of anastrozole, as part of the TARGET study [63\u201366], and exemestane in the EORTC study [67, 68] have provided evidence of clinical equivalence or superiority to tamoxifen in post-menopausal women with advanced breast cancer. However, none of these trials demonstrated statistically significant improvements in all three end points (ORR, TTP, and OS) for the aromatase inhibitor compared with tamoxifen. The PO25 study was the largest of these randomized trials in the first-line setting and demonstrated extremely strong clinical benefits, evidenced by significant superiority in TTP and ORR, with letrozole compared with tamoxifen as first-line hormone therapy. The benefits of letrozole were observed in all patient subgroups, defined by prior antiestrogen therapy, dominant site of metastatic disease, HR status (positive or unknown), and age [52\u201355]. Furthermore, letrozole is the only aromatase inhibitor associated with an OS advantage for the first-line setting indication at 1-year and 2-year follow-up [53]. As demonstrated in the exploratory analysis of patients who did not cross over to the alternative treatment arm, letrozole prolonged OS by 14\u00a0months compared with tamoxifen. Thus, for every 100 patients treated with hormone therapy, eight more will be alive at 1\u00a0year if they receive letrozole instead of tamoxifen.\nIn conclusion, third-generation aromatase inhibitors are effective and well tolerated. Letrozole should be considered as the first-line endocrine treatment in post-menopausal women with hormone-sensitive advanced or MBC. Of the available agents, only letrozole has demonstrated significant improvements in ORR, TTP, and early OS.","keyphrases":["letrozole","advanced breast cancer","tamoxifen","aromatase inhibitors","postmenopausal"],"prmu":["P","P","P","P","U"]}
{"id":"Matern_Child_Health_J-2-2-1592157","title":"Prevalence of Risk Factors for Adverse Pregnancy Outcomes During Pregnancy and the Preconception Period\u2014United States, 2002\u20132004\n","text":"Objectives: To assess the prevalence of risk factors for adverse pregnancy outcome during the preconception stage and during pregnancy, and to assess differences between women in preconception and pregnancy. Methods: Data from the 2002 and 2004 Behavioral Risk Factor Surveillance System, United States, were used to estimate the prevalence of selected risk factors among women 18\u201344 in the preconception period (women who wanted a baby in the next 12 months, and were not using contraception, not sterile and not already pregnant) with women who reported that they were pregnant at the time of interview. Results: Major health risks were reported by substantial proportions of women in the preconceptional period and were also reported by many pregnant women, although pregnant women tended to report lower levels of risk than preconception women. For example, 54.5% of preconception women reported one or more of 3 risk factors (frequent drinking, current smoking, and absence of an HIV test), compared with 32.0% of pregnant women (p < .05). The difference in the prevalence of these three risk factors between preconception and pregnancy was significant for women with health insurance (52.5% in preconception vs. 29.4% in pregnancy, p < .05), but not for women without insurance (63.4% vs. 52.7%, p > .05). Conclusions: Women appear to be responding to messages regarding behaviors that directly affect pregnancy such as smoking, alcohol consumption and taking folic acid, but many remain unaware of the benefits of available interventions to prevent HIV transmission and birth defects. Although it appears that some women reduce their risk for adverse pregnancy outcomes after learning of their pregnancy, the data suggest that a substantial proportion of women do not. Furthermore, if such change occurs it is often too late to affect outcomes, such as birth defects resulting from alcohol consumption during the periconception period. Preconception interventions are recommended to achieve a more significant reduction in risk and further improvement in perinatal outcomes.\nIntroduction\nAlthough the US has achieved substantial gains in improving the health of women and children, and infant mortality has reached record low levels, the US presently ranks 27th among established market economies in infant mortality [1]. Four recurring causes account for more than half of all infant deaths: birth defects, disorders related to short gestation and preterm birth, maternal complications of pregnancy (including complications of the placenta, cord and membranes), and sudden infant death syndrome [2]. In recent years some causes of infant mortality have increased, particularly in the percentage of births that were preterm and of low birth weight [3]. In 2002, congenital anomalies, low birth weight, preterm delivery, and maternal complications of pregnancy accounted for 14,263 (50.9%) of the 28,034 infant deaths [2].\nRelatively little is known about the risk factors underlying the continued increase in these adverse outcomes. Adequate prenatal care has long been considered as an opportunity to reduce such risks. However, despite increases in access to and utilization of early prenatal care, interventions and efforts directed at addressing such risk factors fall short of their goal. Indeed, the effects of such efforts may have reached their peak, and new approaches may be necessary. Reviews on selected risk factors indicate that a large proportion of women enter pregnancy with pre-existing risks for adverse pregnancy outcomes. Although some women tend to take action to reduce their risk as soon as they learn that they are pregnant, the extent of pregnancy related change in risk factors varies considerably and often does not occur early in pregnancy when teratogenic effects are more pronounced. Moreover, post-pregnancy relapse is high [4]. For example in the period 1996\u20131998 the reported reduction in the use of alcohol, tobacco and illicit drugs in the first trimester was 46, 28, and 28% respectively [4]. Such information is important to the emerging emphasis on preconception care as a complementary approach to reduce risks to pregnancy. This paper provides nationally-representative estimates on risks during the preconception period and describes the apparent reductions in risk achieved during pregnancy for all known risk factors for which data are available.\nData\nThe estimates presented herein are based on data from the 2004 Behavioral Risk Factor Surveillance System (BRFSS) [5]. However, because not all risk indicators that were considered were included in the 2004 BRFSS, 2002 data were used for a small number of risk indicators for which the 2002 BRFSS provided the most recently available data. The BRFSS is an ongoing annual telephone survey of the non-institutionalized adult civilian population aged 18 years and older conducted in each state. The survey obtains information on a wide range of modifiable risk behaviors. In 2004 the median response rate for the BRFSS state surveys was 52.7 percent. This rate represents an estimate of the percentage of eligible respondents that completed telephone interviews, and is computed based on procedures recommended by the Council of American Survey Research Organizations (http:\/\/www.casro.org\/resprates.cfm.) In comparison with other national surveys, BRFSS data appear to be of good quality [5]. Additional technical information is available online at http:\/\/www.cdc.gov\/brfss\/.\nThe survey includes core modules asked in all states, rotating modules asked every second year in all states, and optional modules that are included only in some states. Being a general survey, the BRFSS lacks information on some factors of interest to the present topic, for example month of pregnancy for pregnant women, or information on prenatal care. Further, the BRFSS is not designed specifically to study all known risk behaviors at preconception or pregnancy. In addition, as an interview survey, the survey relies on self-reported data which contain an unknown level of reporting error.\nIdentification of women in the preconception group in the BRFSS is made possible by questions introduced in the \u201cfamily planning section\u201d of the questionnaire. The availability of these questions makes it possible to compare risk behaviors among women about to become pregnant with those who are already pregnant on a large and nationally-representative sample of women. Women in the preconception period were identified as those who reported that they wanted a baby in the next 12 months, were not using contraception, were not sterile and were not already pregnant. Women who were pregnant were identified based on the response to the question \u201cTo your knowledge are you now pregnant?\u201d Age of gestation was not assessed in this survey. Data were aggregated across states to make national estimates. For 2004 this resulted in a total national U.S. sample of 70,917 women aged 18 to 44 years, of whom 2308 (3.4%) were classified into the preconception period, and 2998 (4.7%) reported that they were pregnant at the time of their interview. The corresponding number of women in the 2002 are 61,284 women 18\u201344, with 2204 (3.7%) in the preconception period, and 2556 (4.6%) pregnant women. The analysis on folic acid and vitamins are based on surveys in 12 states in 2004. The results for these items are generalizable to the populations of these 12 states (shown in Table 2) but not to the entire United States population.\nTable 1Comparison of age and race\/ethnicity, 2004 Women in preconception and pregnant (BRFSS) and Births (Vital Statistics data)BRFSSPreconception womenPregnant womenVital statisticsPercentCIPercentCIbirths PercentAge group\u200318\u2013192.00.97.52.27.1\u200320\u20132416.33.326.53.026.1\u200325\u20132923.43.126.52.727.9\u200330\u20133428.43.126.22.824.4\u200335\u20133918.52.510.21.512.0\u200340\u20134411.42.23.10.92.6100.0100.0Race\/ethnicity\u2003NH White64.93.960.13.456.8\u2003NH Black11.92.511.51.913.6\u2003Hispanic16.03.523.03.422.5\u2003Others7.22.15.51.67.2100.0100.0100.0Note. For BRFSS estimates, 95% confidence interval = Percent +\/\u2212 CI. Vital statistics data: Hamilton BE, Martin JA, Ventura SJ, Sutton PD, Menacker F. Births: Preliminary data for 2004. National vital statistics reports; vol 54 no 8. Hyattsville, Maryland: National Center for Health Statistics. 2005.Table 2Health risk indicators by pregnancy status Women 18\u201344, 2004 and 2002 BRFSSPreconception womenPregnant womenPercentCIPercentCI2004 data\u2013nationwidea\u2003General Health\u2003\u2003Poor\/fair general health status8.32.16.41.7\u2003\u200314+ days in past month mental health not good*12.82.49.61.9\u2003\u2003No health plan*18.83.511.92.3\u2003\u2003No dental visit past year28.33.230.23.1\u2003\u2003Told had diabetes*2.00.90.70.4\u2003HIV\u2003\u2003Don\u2019t know about prevention of MTC HIV transmission38.23.534.13.0\u2003\u2003Never tested for HIV*34.93.424.43.0\u2003\u20031 or more HIV risk category*4.31.46.91.8\u2003Alcohol\/smoking\u2003\u2003Any alcohol in past month*53.93.710.71.8\u2003\u2003Average 1 or more drink per day, past month*6.02.00.80.5\u2003\u2003Binging: Any occasions of 5+ drinks in past month*10.72.11.90.9\u2003\u2003Frequent drinking: binging or 1 or more drinks per day*12.92.52.20.9\u2003\u2003Current smoker*19.42.78.41.5\u2003Obesity\u2003\u2003Overweight, body mass index (BMI) >2546.03.6NA\u2003\u2003Obese, BMI >3022.43.1NA2004 data, 12 states onlyb,c\u2003Folic acid\u2003\u2003Don\u2019t know about folic acid for birth defects prevention46.16.938.45.6\u2003\u2003Don\u2019t take vitamins of any kind*36.97.110.23.6\u2003\u2003Don\u2019t take folic acid or multivitamin*38.47.017.54.5\u2003\u2003Don\u2019t take folic acid or multivitamin daily*44.86.919.94.7Nutrition (2002 BRFSS)d\u2003Fewer than 5 servings\/fruit and vegetables*74.93.164.73.5\u2003Fewer than 1 servings\/fruit and vegetables3.31.12.91.3Note. 95% confidence interval = Percent +\/\u2212 CI.aUnweighted number of observations: Preconception women (2308), Pregnant Women (2998).bUnweighted number of observations: Preconception women (607), Pregnant Women (756).cStates: AZ, CO, FL, KY, MN, MT, NC, ND, NE, TX, VA, WI.dUnweighted number of observations: Preconception women (2204), Pregnant Women (2556).*p < .05, preconception vs. pregnant women.**HIV risk: Any statement is true: in past year illegal drug injection, treated for STD, exchange for money\/drugs, uprotected anal intercourse.\nStatistical methods\nThe percentage of women exposed to 21 risk indicators was identified for women in 2 groups: women in the preconception period, and pregnant women (Table 1). The categories of risk indicators for which data are available include perceived general and mental health, lack of medical insurance, knowledge of HIV prevention and practices, alcohol and tobacco use, obesity, nutrition including folic acid uptake for the prevention of birth defects, and presence of chronic medical conditions.\nBecause these are not longitudinal data it was not possible to observe behavior change in individual women. Rather the prevalence of risk factors was compared between preconception and pregnant women as a proxy measure of pregnancy-related risk reduction [6], although is is possible that other factors could influence the differences between preconception and pregnant women. The statistical significance between preconception and pregnant women of the percentage reporting each risk factor, was assessed using a t-test. All statistical analysis was based on weighting factors designed to produce unbiased estimates, and statistical tests were adjusted for complex sample design using the software for survey data analysis (SUDAAN, Research Triangle Institute, Research Triangle Park, NC).\nResults\nComparisons with vital statistics data on births in 2004 indicate that the 2004 BRFSS respondents in the preconception stage and pregnant when interviewed were distributed very similarly by age and race-ethnicity to women giving birth in 2004 (Table 1). Overall, for many risk factors, pregnant women reported lower prevalence of risk than preconception women (Table 2). Percentages reporting alcohol consumption and smoking were much lower for pregnant women than preconception women. For example, 53.9% of preconception women reported any use of alcohol in the past month, compared with 10.7% of pregnant women, 19.4% of preconception women reported current smoking in contrast to 8.4% of pregnant women, and, in the 12 states that included the question, 44.8% of preconception women reported not taking vitamins with folic acid versus 19.9% of pregnant women.\nFor some risk indicators, no statistically significant difference was observed between women in the preconception period and pregnant women. The lack of awareness about methods to prevent mother-to-child HIV transmission (PMTCT) was not significantly different between preconception women and pregnant women (38.2% vs. 34.1%). However, a small but significantly higher percentage of pregnant women reported HIV risk (6.9%) compared to preconception women (4.3%), which is perhaps related to sexual risk behaviors being causally linked to both HIV and to unplanned pregnancies.\nOf concern, is that although a lower percentage of pregnant women compared with preconception women had never been tested for HIV, about one-fourth of pregnant women remained untested, despite national recommendations that call for all pregnant women to be tested for HIV [7].\nIn the case of chronic diseases such as diabetes, the lower level of disease among pregnant women may indicate self-selection of diabetic women to avoid pregnancy. Of note, nearly one fifth of the women in preconception period were obese (defined as having a body mass index or BMI of greater than 30) and twice that number were overweight (BMI of greater than 25) (Table 2). Most preconception and pregnant women did not report consuming the recommended 5 servings of fruits and vegetables per day [8], although the percentage was lower for pregnant women (64.7%) compared with women in the preconception period (74.9%). Indicators of general well being and recent dental care were not different between women in preconception period and pregnancy.\nA composite measure of 3 basic pregnancy risks was formed, frequent alcohol consumption, current smoking and lack of an HIV test, in order to use the BRFSS data to illustrate the size of the target populations for preconception care. These are risk factors for which we have data that are generalizable to the US population, and for which interventions or information are widely available or promoted. The results indicate that 54.5% of preconception women reported one or more of these 3 risk factors, which represents approximately 1 million preconception women (Table 3); 32.0 percent of pregnant women had one or more of these risks, which is equivalent to 835,000 pregnant women. Table 3 also shows this composite risk factor by 2 measures of access to health care, whether the woman had a health plan or insurance and whether she had a personal physician. Pregnant women had a lower percent at risk in all categories except for those without health insurance. Among women without insurance, the percentage reporting risk was not significantly lower for pregnant women, compared with preconception women. This suggests that women with poorer access to health care, as indicated by no health insurance, are not as likely as others to adopt healthier behaviors when they become pregnant.\nTable 3Percentage and estimated number reporting any of 3 risks for pregnancy outcomea for preconception and pregnant women aged 18\u201344 Women 18\u201344, 2004 BRFSSPreconception women (Estimated number)Currently pregnant women (Estimated number)PercentCI1000sCINPercentCI1000sCINTotal54.5*3.71,02494228632.03.18351012949No health plan\/insurance63.411.72225232852.710.315650303Others52.5*3.780279195729.43.2679882646No personal doctor64.1*9.02785944846.39.223473467Others51.6*3.974573183728.62.9601712482Note. 95% confidence interval = Percent +\/\u2212 CI and Estimated number +\/\u2212 CI. Estimated number is the estimated number of women in the category with one of the risk factors.aAny of 3 risk factors: frequent drinking, current smoker, no HIV test.*difference between preconception and pregnant women significant, p < .05.\nDiscussion\nThe 2004 BRFSS provides an opportunity to measure the extent of risk behaviors on a relatively large sample of women who are planning to become pregnant. These nationally-representative survey data indicate that major health risks were reported by substantial proportions of US women in the preconception period. The data suggest that among those at risk during preconception, the majority continued the risk into pregnancy. Women appear to respond to messages regarding behaviors that can improve pregnancy outcomes such as consuming of folic acid, and the reduction in smoking, alcohol consumption, but many remain unaware of the benefits of available interventions to prevent HIV transmission and birth defects. The fact that more than one-third of preconception and pregnant women were not aware of methods to prevent mother-to-child HIV transmission and did not know about the benefits of taking folic acid during pregnancy suggests that education and public information programs could have some effect in reducing risk behaviors.\nRisk reduction appears to be lower among women who have no health insurance. Other data are not available for comparison of the overall burden of risk for adverse pregnancy outcomes among women in the preconception period or pregnancy. However, data on selected indicators presented here for pregnant women are comparable to the reported range from other studies [4, 6, 8\u201311].\nThe finding that the percentage who report some risk behaviors is significantly lower among pregnant women compared with women in the preconception period may indicate the desire of many pregnant women to adopt healthier behaviors to achieve the best possible outcome for their pregnancy. Examination of three well known risk factors for which interventions are available (frequent alcohol consumption, current smoking, or never having been tested for HIV) illustrates that risks for adverse pregnancy outcomes exist among more than half of women in the preconception period and among one-third of pregnant women. Nationwide, this represents approximately one million women in the preconception period and 835,000 pregnant women.\nThe data from the BRFSS are subject to potential errors associated with survey research such as exclusion from the sampling frame, non-response and reporting errors. BRFSS is a telephone-based survey and might not be representative of the small percentage of the population without telephones. The BRFSS methodology contains steps to ensure accurate estimation, including the use of weighting factors to compensate for non-response rates. In addition, estimates from BRFSS data have been found to be consistent with data from other surveys [5]. The preconception and pregnant women identified were distributed very similarly to women giving birth in 2004 by age group and race\/ethnicity, providing support for the representativeness of the data. In this report, the reported risk behavior of women in the preconception stage and pregnant women have been compared as a proxy for the type of behavior change occurring between preconception and pregnancy, but other factors could affect the actual change in behavior among individual women.\nFor known risk factors, progress in efforts to reduce such risks among pregnant women has been slow. For example, as indicated in data collected on earlier rounds of the BRFSS, alcohol consumption among pregnant women has remained relatively steady over the past decade [10]. Even among those who reduce consumption of alcohol, tobacco, or drugs, post pregnancy relapse of such behaviors have been found to be very high [4]. Of the 17 maternal and infant health objectives included in the Healthy people 2010 objectives, progress has been made toward the target in 8 objectives [12]. Little positive progress has occurred in the areas of maternal death, fetal alcohol syndrome, and low birth weight.\nTo close the gap in reduction of poor maternal and child health outcomes, current maternal and child health initiatives should be complemented with alternate approaches, including more emphasis on preconception health promotion strategies among women of childbearing age. Recent experience in the prevention of alcohol-exposed pregnancy and prevention of neural tube defects through folic acid supplementation has shown that such efforts are useful. In 2003, the Project CHOICES Research Group reported the findings of a feasibility study designed to provide prevention counseling to women at high risk for an alcohol\u2013exposed pregnancy [13]. Brief interventions were found to be effective in reducing hazardous alcohol use in adults in a number of well-controlled studies. Interventions generally consist of advice, feedback, goal setting, and follow-up for further assistance and support [14]. The use of a counseling style referred to as \u201cmotivational interviewing\u201d has also proven to be effective in reducing problem drinking [15]. Results of the initial follow-up assessment, suggest that this approach can be effective among women of childbearing age [13], and a larger efficacy trial is now underway.\nTo summarize, our results were consistent with a high prevalence of risk factors in the preconception period, and a high degree continuation of risk into pregnancy. This underscores the need for new approaches to complement current prevention efforts. Given that women of childbearing age in the United States have on an average 6.4 visits to physicians each year [16], it may be feasible to inform them and engage them for interventions. Such contacts may provide an opportunity to assess and advise on many elements of health mentioned here. Our finding of high risk levels among women who had no insurance or no personal doctor strongly suggests that ensuring access to such services will remain the key determinant of maximum uptake of known intervention services. Medicaid and other funding programs can play an important role to address the access gap\u2014whether during, before, or after pregnancy. Attempts to improve the health of would-be mothers not only improve pregnancy and infant health, but also help reduce the long-term impact of many risk factors on the women themselves.","keyphrases":["pregnancy","preconception","riskfactors"],"prmu":["P","P","U"]}
{"id":"Dev_Genes_Evol-4-1-2265772","title":"Glutamine synthetase gene expression during the regeneration of the annelid Enchytraeus japonensis\n","text":"Enchytraeus japonensis is a highly regenerative oligochaete annelid that can regenerate a complete individual from a small body fragment in 4\u20135 days. In our previous study, we performed complementary deoxyribonucleic acid subtraction cloning to isolate genes that are upregulated during E. japonensis regeneration and identified glutamine synthetase (gs) as one of the most abundantly expressed genes during this process. In the present study, we show that the full-length sequence of E. japonensis glutamine synthetase (EjGS), which is the first reported annelid glutamine synthetase, is highly similar to other known class II glutamine synthetases. EjGS shows a 61\u201371% overall amino acid sequence identity with its counterparts in various other animal species, including Drosophila and mouse. We performed detailed expression analysis by in situ hybridization and reveal that strong gs expression occurs in the blastemal regions of regenerating E. japonensis soon after amputation. gs expression was detectable at the cell layer covering the wound and was found to persist in the epidermal cells during the formation and elongation of the blastema. Furthermore, in the elongated blastema, gs expression was detectable also in the presumptive regions of the brain, ventral nerve cord, and stomodeum. In the fully formed intact head, gs expression was also evident in the prostomium, brain, the anterior end of the ventral nerve cord, the epithelium of buccal and pharyngeal cavities, the pharyngeal pad, and in the esophageal appendages. In intact E. japonensis tails, gs expression was found in the growth zone in actively growing worms but not in full-grown individuals. In the nonblastemal regions of regenerating fragments and in intact worms, gs expression was also detected in the nephridia, chloragocytes, gut epithelium, epidermis, spermatids, and oocytes. These results suggest that EjGS may play roles in regeneration, nerve function, cell proliferation, nitrogenous waste excretion, macromolecule synthesis, and gametogenesis.\nIntroduction\nSome types of annelids exhibit remarkable regenerative abilities that are comparable to planarians. However, the regeneration mechanisms are thought to be quite different between planarians and annelids. Whereas planarians regenerate via totipotent stem cells (neoblasts) that are widely distributed throughout their bodies (Reddien and Alvarado 2004), annelid regeneration is thought to occur mainly through the dedifferentiation and subsequent redifferentiation of cells without any contribution from totipotent stem cells (Thouveny and Tassava 1998). In this respect, annelids regenerate in a manner similar to that of amphibians (Carlson 1998) but on a much larger scale. The elucidation of the annelid regeneration mechanisms is thus expected to provide valuable information that may allow us in the future to explore strategies to enhance the regenerative capabilities in vertebrates.\nIn our recent study, we have proposed the oligochaete annelid Enchytraeus japonensis as a new model system for regeneration studies (Myohara et al. 1999). E. japonensis has striking features that are highly advantageous in this regard: (1) It reproduces asexually by dividing its body into several fragments, which then regenerate a complete individual within 4\u20135\u00a0days, (2) artificially amputated fragments can also regenerate new individuals in the same manner, and (3) both asexual and sexual reproduction can be artificially induced in the laboratory (Myohara et al. 1999).\nAs an initial strategy toward the elucidation of the molecular mechanisms underlying annelid regeneration, we previously performed complementary deoxyribonucleic acid (cDNA) subtraction cloning using regenerating fragments of E. japonensis and intact animals (Myohara et al. 2006). Filter array screening subsequently revealed that about 38% of the forward-subtracted cDNA clones we isolated contained genes that were upregulated during regeneration. These clones hybridized with probes derived from regenerating fragments but not with probes from intact worms. Of these clones, 279 were then sequenced and found to contain 165 different sequences (79 known and 86 unknown). Among these 279 regeneration-upregulated clones, as many as 13 (4.7%) were found to encode glutamine synthetase, a ubiquitous and key enzyme in nitrogen metabolism (Kumada et al. 1993).\nBecause of its abundance in our subtracted cDNA library, we cloned the full-length sequence of E. japonensis glutamine synthetase (EjGS). By reverse transcriptase\u2013polymerase chain reaction (RT-PCR), we subsequently revealed that the expression levels of the glutamine synthetase gene (gs) were consistently high during the regenerative process, with a gradual decline seen only in the very late stages, and our preliminary analysis by whole mount in situ hybridization further revealed strong gs expression in the blastemal tissues of the regenerating fragments (Myohara et al. 2006). As these previous results suggested an important role for gs in annelid regeneration, in the present study, we have examined the detailed expression pattern of gs in regenerating fragments and intact worms and considered the possible roles of this enzyme in the biology of E. japonensis. This is the first detailed report of the sequence and expression of gs in annelids.\nMaterials and methods\nAnimals\nE. japonensis worms were reared at 24\u00b0C in 1.1% (w\/v) plain agar medium in disposable Petri dishes and were fed with rolled oats as previously described (Myohara 2004). Under these conditions, the worms grow continuously to about 10\u00a0mm long, consisting of 50\u201360 segments, and reproduces asexually by fragmentation every 2\u00a0weeks. To induce sexual reproduction, starved worms were cultured in wet leaf mold and refed with rolled oats as previously described (Myohara 2004).\nMolecular analysis\nThe full-length cDNA of EjGS (accession number AB109095) has been isolated in our previous study (Myohara et al. 2006). Multiple alignments of the entire amino acid sequences of the glutamine synthetase proteins from representative species, which were obtained through GenBank\/EMBL\/DDBJ Data Bank and chosen to cover a wide range of phylogenic groups, were performed using ClustalW software (Thompson et al 1994). To avoid errors resulting from mixing of possible pseudogenes, only data originating from full-length messenger ribonucleic acid (mRNA) sequences were used.\nIn situ hybridization\nBoth sense and antisense digoxigenin (DIG)-labeled RNA probes were synthesized using the DIG RNA labeling kit (SP6\/T7, Roche) as previously described (Myohara et al. 2006). Whole-mount in situ hybridizations were also performed as previously described (Myohara et al. 2006) with some modifications to enhance the signals. Briefly, hybridization was carried out overnight with 1\u00a0\u03bcg\/ml of gs DIG-labeled RNA probe at 55\u00b0C. An anti-DIG alkaline phosphatase (AP) conjugate antibody (Roche) was preabsorbed by incubation for 2.5\u00a0h with fixed and rehydrated worms and used at a dilution of 1:2,000. In situ hybridization of paraffin sections (6\u00a0\u03bcm) was carried out according to Hoshino et al. (1999) and Abe et al. (2003) with a few modifications. Briefly, sections were hybridized with DIG-labeled probes at a concentration of 100\u00a0ng\/ml in probe diluent (Genostaff, Tokyo, Japan) at 60\u00b0C for 16\u00a0h. Washes were then carried out using 5\u00d7 Hybri-wash (Genostaff), and an anti-DIG AP conjugate antibody (Roche Applied Science, Tokyo, Japan) was then applied at a 1:1,000 dilution in Tris-buffered saline with 0.1% Tween 20 for 2\u00a0h. After an overnight coloring reaction with nitro blue tetrazolium\/5-bromo-4-chloro-3-indolyl-phosphate (Roche), the sections were counterstained with a Kernechtrot stain solution (Muto Pure Chemicals, Tokyo, Japan), dehydrated, and mounted in Malinol (Muto Pure Chemicals).\nResults and discussion\nComparisons between E. japonensis glutamine synthetase and other known glutamine synthetases\nA full-length 2,717-bp cDNA encoding gs was previously isolated from regenerating E. japonensis in our laboratory (Myohara et al. 2006). The sequence of this gene was found to contain a putative open reading frame of 360 amino acids with a calculated molecular mass of 40,509\u00a0Da. Because this was the first report in annelids of a full-length sequence for gs, we performed a database search in the present study to determine whether similarities existed with the gs genes from other organisms. BLASTP analysis revealed that the deduced glutamine synthetase protein in E. japonensis (EjGS) was highly similar to known class II glutamine synthetases along its entire amino acid sequence (Fig.\u00a01), which is well conserved across species (Kumada et al. 1993). Within the EjGS sequence, we identified a beta-grasp (catalytic) and a noncatalytic domain, five conserved regions that are common to both prokaryotes and eukaryotes, ligands for Mn2+ ions, and an adenosine triphosphate-binding site (Pesole et al. 1991). An amino acid alignment between EjGS and the gs proteins from five invertebrates, sea anemone (Cnidaria), C. elegans (Nematoda), Pacific oyster (Mollusca), fruit fly (Arthropoda), sea urchin (Echinodermata), and four vertebrates (zebrafish, clawed frog, chicken, and mouse) showed that the overall amino acid identities between EjGS and its counterparts in various other animal species were within a range of 61\u201371% (Fig.\u00a01).\nFig.\u00a01Amino acid alignment of E. japonensis glutamine synthetase (EjGS) with representative GSs from various other species. The species and protein ID of the GSs listed here are as follows: Enchytraeus japonensis (BAE93509.1), sea anemone Aiptasia pallida (AAR36878.1), nematode Caenorhabditis elegans (NP_001041010.1), Pacific oyster Crassostea gigas (CAD90162.1), fruit fly Drosophila melanogaster (AAZ41780.1), sea urchin Paracentrotus lividus (AAC41562.1), zebrafish Danio rerio (AAH66735.1), clawed frog Xenopus laevis (AAH46681.1), chicken Gallus gallus (AAA48783.1), and mouse Mus musculus (AAH15086.1). Amino acid residues are indicated in blue when greater than or equal to 70% of these residues are identical in the ten representative species. The beta grasp domain, catalytic domain, and the regions that are conserved in prokaryotes and eukaryotes are underlined by broken single lines, broken double lines, and thick single lines, respectively. Ligands for Mn2+ ions are indicated by arrowheads. The ATP-binding sites are boxed. The protein length and overall amino acid identities with EjGS are indicated at the end of each sequence\nExpression of the gs gene in the blastema of regenerating E. japonensis\nBy whole-mount in situ hybridization analysis, we detected strong gs expression in the blastemal region in regenerating fragments of E. japonensis (Fig.\u00a02a). This expression was detectable from the early stages of the regenerative process in the cell layer covering the wound (Fig.\u00a02b,k) and was found to persist in the epidermal cells during the formation and elongation of the blastema (Fig.\u00a02c,d). There was also a notable concentration of this transcript in the region corresponding to the prostomium anlagen in the anterior blastema (Fig.\u00a02e) and in the growth zone in the posterior blastema (Fig.\u00a02l). In situ hybridization analyses with paraffin-embedded sections additionally revealed that the gs expression pattern was mainly restricted to the epidermal cells in the early blastema (Fig.\u00a02h) but did occur also in the inside tissues of the elongated blastema, where it was detected in the presumptive regions of the brain, ventral nerve cord, and buccal cavity (stomodeum), in addition to the prostomium and epidermal cells (Fig.\u00a02i).\nFig.\u00a02Glutamine synthetase mRNA expression during regeneration in E. japonensis. Transcripts were detected using whole-mount in situ hybridization (a\u2013g and k\u2013o) or in situ hybridization analysis of paraffin-embedded sections (h\u2013j) using an antisense gs riboprobe, with the exception of the lower specimen shown in a. The anterior is to the left, and the ventral side is down in each image. Arrowheads indicate the amputated sites. a Whole-body images of regenerating fragments at about 1\u00a0day after amputation with the anterior blastema and an intact tail. The lower specimen was hybridized with a sense probe. b\u2013f Lateral views of anterior blastemas at about 1 (b), 1.5 (c), 2 (d), 2.5 (e), and 4\u00a0days (f) after amputation. g Lateral view of an intact head. h\u2013i Sagittal sections of anterior blastemas at about 1.5 (h) and 2\u00a0days (i) after amputation. j Sagittal section of an intact head. k\u2013m Lateral views of posterior blastemas at about 0.5 (k), 1 (l), and 1.5\u00a0days (m) after amputation. n\u2013o Ventral views of intact tails in an actively growing (n) and a full-grown individual (o). ba Brain anlage, br brain, bc buccal cavity, ch chloragocyte, ea esophageal appendage, ep epidermal cell, es esophagus, gl gut lumen, gz growth zone, m mouth, pc pharyngeal cavity, pp pharyngeal pad, pr prostomium, py pygidium, st stomodeum, va ventral nerve cord anlage, vn ventral nerve cord. Scale bars, 50\u00a0\u03bcm\nIn the later stages of E. japonensis regeneration, by which time segmentation and organogenesis have almost completed, we observed that the gs expression levels in the blastemal regions had become reduced (Fig.\u00a02f,m). In the fully formed intact head, however, the expression of this gene was detectable in the prostomium, brain, anterior end of the ventral nerve cord, epithelial cells lining the buccal and pharyngeal cavities, pharyngeal pad, and esophageal appendages (Fig.\u00a02g,j; for E. japonensis morphology, see Schmelz et al. 2000). In intact tails, gs expression was also evident in the growth zone in actively growing worms (Fig.\u00a02n) but was absent in full-grown individuals in which the growth zone had become indistinguishable (Fig.\u00a02o). These observations are in agreement with the RT-PCR data included in our previous report, which showed that the expression levels of gs were maintained at high levels during the early stages of regeneration, decreased temporarily in the later stages of this process, and were upregulated again in the fully formed intact worms (Myohara et al. 2006).\nExpression of the gs gene in nonblastemal regions\nIn accordance with the abovementioned RT-PCR results, we found by in situ hybridization analysis in the present study that gs expression was not restricted to the blastemal regions (Figs.\u00a02a and 3a) but that less intense but clear expression could be observed also in the nephridia (Fig.\u00a03a,b), chloragocytes (Figs.\u00a02a, 3a\u2013c), gut epithelium (Fig.\u00a03c), epidermis of the nonblastemal regions of regenerating fragments (Fig.\u00a03a), and also in intact worms (Fig.\u00a03d,e). Nephridia are the excretory organs of oligochaete annelids and the chloragocytes (chloragogen cells) are specialized peritoneal cells of the intestine that participate in the metabolism, storage, and excretion of glycogen and lipids, thus resembling the vertebrate liver in terms of function (Jamieson 1981; Edwards and Bohlen 1996). The gs expression levels in these tissues were observed to be variable among the specimens examined and also among regions within the same specimen (Figs.\u00a02a and 3c). We were unable to find any correlation between this pattern and a specific gut region or specific condition of our specimens, although an uneven distribution of glutamine synthetase has been reported previously in the mammalian and fish digestive tracts (James et al. 1998; Mommsen et al. 2003). Intense gs expression was also found in the spermatids in the seminal vesicles and in oocytes in the ovisacs in sexually mature worms (Fig.\u00a03e).\nFig.\u00a03Glutamine synthetase mRNA expression in nonblastemal tissues. Transcripts were detected by in situ hybridization on paraffin-embedded sections (a\u2013c and e) or by whole-mount in situ hybridization (d) using an antisense gs riboprobe, except for the right-hand specimen in a. The anterior is to the left, and the ventral side is down in each image. a Sagittal sections of regenerating fragments at about 0.5\u00a0days after amputation. The right-hand specimen was hybridized with a sense probe. Arrowheads indicate the amputated sites. b\u2013c Sagittal sections of intact worms. d Whole-mount in situ hybridization analysis of an intact worm showing gs expression in the epidermal cells. e Sagittal section of a sexually mature worm. ch Chloragocyte, ep epidermal cell, ge gut epithelium, gl gut lumen, ne nephridium, oo oocyte, sc spermatocyte, st spermatid, sp sperm. Scale bars, 50\u00a0\u03bcm\nGeneral comparisons between the distribution of glutamine synthetase in different organisms\nGlutamine synthetase is found in a wide range of organisms, including microorganisms, plants, and animals and is known to be expressed in a tissue-specific and developmentally controlled manner. In murine organs, glutamine synthetase activity is highest in the liver, followed in descending order by the epididymis, testes, uterine tube, stomach, kidney, brain, and adipose tissues (van Straaten et al. 2006). High glutamine synthetase expression in the brain, kidney, and liver has also been reported in fish (Murray et al. 2003). In Xenopus, gs transcripts have been detected in the ovary, kidney, eye, muscle, and embryo but not in the brain, heart, liver, stomach, or testis (Hatada et al. 1995). In the case of invertebrates, little information is currently available regarding the expression profile of this enzyme and is restricted to arthropods; gs expression has been reported in mosquito midgut epithelium, ovary, and fat body (Smartt et al. 2001) and in crustacean neural tissues (Linser et al. 1997; Allodi et al. 2006; Sullivan et al. 2007). Our present study, therefore, provides novel information regarding the expression of this enzyme in nonarthropod invertebrates, and our results are in general agreement with those of previous reports; that is, in full-grown E. japonensis, gs is expressed in (1) chloragogen tissues, which correspond to the vertebrate liver in terms of functionality (Jamieson 1981), (2) nephridia, which are the annelid counterparts of the kidney and uterine tube in vertebrates (Jamieson 1981), (3) the brain and ventral nerve cord, (4) the prostomium where large numbers of sensory cells including photoreceptor cells are present (Jamieson 1981; Edwards and Bohlen 1996), (5) the epithelium of the digestive tract, and (6) spermatids and oocytes.\nPossible functions of glutamine synthetase in E. japonensis\nRegeneration The possible involvement of glutamine synthetase in the regeneration of damaged tissue has been reported in only a few cases. These include the observed increase in gs expression after peripheral motor nerve injury in mouse (Toki et al. 1998) and also in Xenopus tail regeneration in which gs expression was found to be upregulated and could be detected in the terminal vesicle of the regenerating spiral cord (Tazaki et al. 2005). Our present results, together with our previously reported findings, strongly suggest the involvement of glutamine synthetase also in annelid regeneration. In the previous study, we have shown that glutamine synthetase is one of the most abundantly expressed upregulated genes during regeneration in E. japonensis (Myohara et al. 2006), and in the present study, our detailed expression analysis by in situ hybridization has revealed strong gs expression in the blastemal regions of regenerating E. japonensis. It has been reported previously in Xenopus that embryos abundantly express glutamine synthetase in the developing nervous system during the gastrula and neurula stages (Hatada et al. 1995). In accordance with this finding, we observed in the present analyses that gs is expressed in the presumptive regions of the brain and ventral nerve cord anlagen in the blastema during E. japonensis regeneration. This further suggests the involvement of glutamine synthetase in annelid neural regeneration.\nNerve function Glutamine synthetase has been shown to play an important role in glutamate transmitter degradation in the nervous system (Derouiche and Frotscher 1991) and is used as a specific glial cell marker in the neural retina (Vardimon et al. 1993). In intact E. japonensis, the expression of this enzyme was evident in the brain and ventral nerve cord, suggesting that it has a nervous system function. Its expression in the prostomium may also be attributable to the distribution of numerous sensory cells, probably including photoreceptor cells, in this region (Jamieson 1981; Edwards and Bohlen 1996).\nCell proliferation Glutamine synthetase is the only enzyme that can synthesize glutamine, which provides nitrogen for the synthesis of amino acids, purines, pyrimidines, and other compounds required in many critical cellular events including proliferation (Meister 1980). In the rat intestinal epithelium, high concentrations of glutamine synthetase protein and mRNA have been found in the highly proliferative crypt region (Roig et al. 1995). Hence, the high gs expression levels that we observed in blastemal cells and in the growth zone in actively growing worms could be explained, at least in part, by a high demand for glutamine during the intense cell proliferation that would be ongoing in these areas.\nDetoxification Glutamine synthetase is also important for the detoxification of excess glutamate and ammonia by converting them to glutamine, which is nontoxic and can be stored in tissues or circulate throughout the body without causing any harm (Meister 1980). A comparison between the cellular concentrations of glutamine synthetase in various murine organs in a previous report has also revealed that the highest concentrations of this enzyme positively correlate with the cells that have detoxifying functions (van Straaten et al. 2006). The gs expression that we observed in the nephridia and chloragocytes in E. japonensis herein could therefore be ascribed to the involvement of these tissues in excretion of nitrogenous waste in oligochaete annelids (Jamieson 1981).\nMacromolecule synthesis Glutamine is known to be required for the synthesis of certain macromolecules that are present in mucus, such as the hexosamines that lubricate and protect the gut mucus (Weiss et al. 1999). The mucus secreted by epidermal cells covering the earthworm body is also essential for locomotion, gas exchange, and protection in these animals (Jamieson 1981), and hexosamines have been found to be a component of their cuticles (Muir and Lee 1970). We speculate therefore that the gs transcripts in the E. japonensis epidermal cells have a role in mucus and\/or cuticle synthesis in intact and regenerating individuals. In addition, the gs expression found in epithelial cells of the buccal and pharyngeal cavities may be attributed to cuticle synthesis in these tissues (Jamieson 1981).\nGametogenesis Glutamine synthetase expression in the ovary has been reported in Xenopus (Hatada et al. 1995) and in the yellow fever mosquito (Smartt et al. 2001). In oligochaete annelids, the testes and ovaries are small organs located in the ventral part of the genital segments (Edwards and Bohlen 1996). The spermatogonia that are formed in the annelid testes pass into the seminal vesicles, which contain male cells at all stages of development. Oogonia in annelids are formed in the ovaries and divide to form oocytes, which are shed from the ovaries into the ovisacs. In E. japonensis, gs transcripts were detectable in spermatids in the seminal vesicles and in oocytes in the ovisacs but not in the testes or ovaries, suggesting that glutamine synthetase may have a role in the later stage of gametogenesis.\nIn summary, in the present study, we have characterized an annelid gs gene and shown that it is expressed in a tissue-specific manner in regenerating and intact E. japonensis. To further elucidate the actual functions of this gene, additional studies including RNAi gene knockdown experiments will be needed in the future. Ongoing investigations in our laboratory of other regeneration-associated upregulated genes, several of which are novel (Myohara et al. 2006), should also shed important new light on the network of biological pathways involved in the regenerative processes in E. japonensis.","keyphrases":["glutamine synthetase","gene expression","regeneration","enchytraeus japonensis","oligochaete annelid"],"prmu":["P","P","P","P","P"]}
{"id":"Clin_Rheumatol-4-1-2367388","title":"Antioxidant intervention in rheumatoid arthritis: results of an open pilot study\n","text":"There is evidence that reactive oxygen species play a causal role in auto-immune diseases, such as rheumatoid arthritis (RA). Despite the supporting evidence for a beneficial effect of antioxidants on clinical characteristics of RA, the right balance for optimal effectiveness of antioxidants is largely unknown. To determine the potential beneficial effects of an antioxidant intervention on clinical parameters for RA, an open pilot study was designed. Eight non-smoking female patients with rheumatoid factor + RA and a Disease Activity Score (DAS 28) higher than 2.5 were enrolled in the study. Patients had to be receiving stable non-steroidal anti-inflammatory drug treatment and\/or \u2018second line\u2019 medication for at least 3 months. The pilot group consumed 20 g of antioxidant-enriched spread daily during a period of 10 weeks. The intervention was stopped after 10 weeks and was followed by a \u2018wash-out\u2019 period of 4 weeks. At t = 0, t = 10 weeks and t = 14 weeks, patients\u2019 condition was assessed by means of DAS. In addition, standard laboratory analyses were performed, and blood-samples for antioxidants were taken. The antioxidant-enriched spread was well tolerated. All laboratory measures of inflammatory activity and oxidative modification were generally unchanged. However, the number of swollen and painful joints were significantly decreased and general health significantly increased, as reflected by a significantly improved (1.6) DAS at t = 10 weeks. The antioxidant effect was considered beneficial as, compared to the scores at t = 0, the DAS significantly reduced at t = 10 weeks. Increase of the DAS (0.7) after the \u201cwash-out period\u201d at t = 14 confirmed a causal relation between changes in clinical condition and antioxidants. This open pilot study aimed to assess the clinical relevance of an antioxidant intervention as a first step in assessing potential beneficial effects of antioxidants on rheumatoid arthritis. These conclusions need to be validated in a larger controlled study population.\nIntroduction\nIn spite of the great advances that have been made in the development of new drugs for the treatment of patients with rheumatoid arthritis (RA), many patients are interested in alternative treatments like dietary therapy. Although the aetiology of rheumatoid arthritis is still unknown, the inflammation resulting from the immunological reaction is quite well described. It is known that neutrophil granulocytes, macrophages and lymphocytes are activated, and that reactive oxygen and nitrogen species (RS) are produced [1, 2]. These RS can react with lipid, protein and nucleic acids and are thought to be of importance for the aetiology and chronicity of the inflammatory rheumatic diseases [3, 4]. One approach to counteract this oxidative stress situation is the use of antioxidants as therapeutic agents. There is some evidence for a positive effect of antioxidants on clinical symptoms of RA [5, 6]. However, this evidence is weak, and information about the most effective antioxidants, antioxidant doses or combinations is lacking The intervention studies that have been conducted have tested vitamin E exclusively [6, 7].\nAgainst this background, an open (uncontrolled) pilot study was designed to assess if effects of antioxidants on the clinical parameters of patients with RA could be confirmed and if these effects would associate with changes in a selected number of molecular markers related to human antioxidant defence system status. Further, a mix of antioxidants was to be tested rather than a single high-dosed compound.\nMaterials and methods\nThe open study was approved by the ethical committee. Eight patients with rheumatoid factor + RA, as defined by the ARA revised criteria were enrolled in the study. To avoid influences of gender, only female patients participated. Inflammatory disease activity was defined as a Disease Activity Score (DAS) higher than 2.5. Inclusion criteria included non-smoking and no serious co-morbidity. Patients had to be receiving stable non-steroidal anti-inflammatory drug (NSAID) treatment and\/or \u2018second line\u2019 medication for at least 3\u00a0months. Those taking antioxidant supplements were excluded from the study. During a period of 10\u00a0weeks, patients consumed 20\u00a0g of antioxidant-enriched margarine daily. The spread contained a mix of a-tocopherol (400\u00a0mg), lycopene (10\u00a0mg), palm oil carotenoids (5\u00a0mg; mainly \u03b1-carotene) and lutein (10\u00a0mg). Further, patients received vitamin C (200\u00a0mg daily) as a supplement. \nThe intervention of 10\u00a0weeks was followed by a \u2018wash-out\u2019 period of 4\u00a0weeks. At t\u2009=\u20090, t\u2009=\u20095\u00a0weeks, t\u2009=\u200910\u00a0weeks and t\u2009=\u200914\u00a0weeks, patients\u2019 condition was assessed by means of morning stiffness, visual analogue scale pain, general health score and DAS 28-score (including the following parameters: pain, swelling of joints, erythrocyte sedimentation rate (ESR), general feeling of well-being). At t\u2009=\u20090, t\u2009=\u200910\u00a0weeks and at t\u2009=\u200914\u00a0weeks, blood was collected, and standard clinical laboratory analyses (ESR, C-reactive protein, hemoglobin, leucocytes, thrombocytes, serum values of creatinine and transaminases) were performed. In addition, blood antioxidant status and markers of antioxidant capacity [ferric reducing ability of plasma (FRAP)] and oxidative stress (8-epi F2-isoprostane) levels were determined. Determination of vitamin A, not present in the spread, was used as a control. Serum was collected immediately after blood sampling and stored in liquid nitrogen. The potential antioxidant effect was defined as beneficial if the DAS score would significantly improve >1.2 at t\u2009=\u200910\u00a0weeks as compared to the scores at t\u2009=\u20090. At t\u2009=\u200914\u00a0weeks, a \u2018wash-out\u201d effect was assessed using the same parameters. Return of the clinical condition of the pilot group to that at t\u2009=\u20090 may indicate a causal relation between changes in clinical condition and antioxidant intake, although a placebo-controlled intervention would be better required for this assessment.\nStatistical analyses were done using a paired t test.\nResults\nThe antioxidant-enriched spread was well tolerated. None of the patients withdrew from the study because of adverse reactions. The number of swollen and painful joints were significantly decreased, and general health significantly increased at t\u2009=\u200910\u00a0weeks. This was shown by a significantly decreased DAS (\u22121.6) at 10\u00a0weeks. After the \u201cwash-out period\u201d, the DAS score was increased again by 0.6 (Figs.\u00a01 and 2).\nFig.\u00a01Clinical condition by Disease Activity ScoreFig.\u00a02Mean Disease Activity Score\nThe standard laboratory measures of inflammatory activity were relatively unchanged. Levels of plasma vitamin E, lutein, lycopene and alpha-carotene were significantly increased at 10\u00a0weeks. No significant changes were seen in vitamin A and vitamin C levels (Fig.\u00a03).\nFig.\u00a03Plasma antioxidant status in eight patients\nMarkers of antioxidant capacity and oxidative stress displayed trends for differences, but none of these reached statistical significance (Fig.\u00a04).\nFig.\u00a04Markers of antioxidant capacity and oxidative stress\nDiscussion\nThis pilot study was conducted to obtain some first indications for potential beneficial effects of an antioxidant intervention on clinically relevant parameters for rheumatoid arthritis. In our present study, a significant reduction in Disease Activity Score (DAS) was observed following antioxidant intervention of 10\u00a0weeks. This was accompanied by significant increases in blood levels of antioxidants administered. The reduction of DAS is remarkable as all patients had an active (high initial mean DAS of 5.8) longstanding disease and had tried all kinds of disease-modifying anti-rheumatic drugs, including combination therapy. The unchanged level of vitamin A, not present in the spread, reinforces the significance of the increased levels of the blood antioxidant status. As NSAIDs can influence the absorption of vitamin C, this could explain the non-significant changes of the serum concentration of this antioxidant.\nA limited number of clinical intervention studies is available which support the therapeutic or prophylactic activity of antioxidants in the pathogenesis of RA. These studies were all conducted with an oral intake of 1,200\u00a0mg\/day of vitamin E. Edmonds et al. [6] reported that the antioxidant a-tocopherol significantly reduced pain parameters in a placebo-controlled double-blind trial following a 3-week supplementation period. Results from a randomised double-blind parallel group comparison study with a-tocopherol and diclophenac showed that the clinical parameters assessed, e.g. morning stiffness, Ritchie joint index, grip strength and pain, were significantly reduced by vitamin E, with similar effectivity and less side effects as compared to regular drug therapy by diclofenac [7]. Results from other intervention studies have generally been in line with these observations [8, 9].\nIn our study, not only the effect of vitamin E on the inflammatory response and clinical symptoms was evaluated but also the interactions with bioavailable natural antioxidants such as carotenoids, vitamin C and others. In view of the limited number of controlled studies, the supporting evidence for beneficial effects of antioxidants on clinical characteristics of rheumatoid arthritis may be considered limited but promising. Another point is that the potential mechanism of action of antioxidants in rheumatoid arthritis needs further attention. The aspect of damage by RS and loss of critical functions can be analysed by use of \u2018molecular markers\u2019. These markers may include antioxidant status, products which arise as a consequence of oxidative damage to lipid, protein and DNA and tissue damage (as assessed by lactic dehydrogenase release) or cytokine levels (cq anti-TNF\u03b1).\nAlthough markers are very informative from a mechanistic point of view, a drawback is that none of them has been validated against clinical conditions of patients with RA. In this study, a trend for reduced levels of measures of antioxidant capacity and of \u2018footprints\u2019 of oxidative stress was observed, e.g. statistically non-significant increase in FRAP and reduction in F2-isoprostane levels. Furthermore, these markers showed a trend for correlation with clinical conditions. The data indicate that statistically significant effects may be observed when the number of patients enrolled is increased.\nThis open pilot study therefore should be viewed as a first step in assessing potential beneficial effects of the antioxidants tested on rheumatoid arthritis. The data indicate there may be potential in using mixes of antioxidants in the treatment of RA alongside drug treatment so that drug dosage may be reduced while hopefully retaining efficacy. Further, multi-drug\/antioxidant therapy could be used to reduce drug dose and thus side effects of treatment.\nIn conclusion, our present pilot study indicated that intervention with antioxidant-enriched margarine in RA results in consistent and significant relief of clinical symptoms. Furthermore, increases in blood antioxidant status and indications for effects on oxidative stress markers were observed. These data are promising and indicate need for a double-blind, placebo-controlled randomised human trial to establish effect and demonstrate causality.","keyphrases":["antioxidants","intervention","rheumatoid arthritis","clinical parameters"],"prmu":["P","P","P","P"]}
{"id":"Mcgill_J_Med-11-1-2322926","title":"Anterior cruciate ligament reconstruction: a look at prosthetics - past, present and possible future\n","text":"Biological tissue autograft reconstruction using the patellar tendon or quadrupled semitendinosus\/gracilis tendons has become the most popular procedure in surgical treatment of a ruptured ACL. This article provides a review of the history of the use of prosthetics with respect to ACL reconstruction grafts including Carbon Fibre, Gore-Tex and Dacron prosthetics as well as the Leeds-Keio Artificial Ligament and the Kennedy Ligament Augmentation Device (LAD). Emphasis is placed on the Ligament Advanced Reinforcement System (LARS) as preliminary investigations of its use have been encouraging. Significant progress has been made recently with respect to the understanding of ACL anatomy, composition, biomechanics, and healing processes, leading to innovative techniques using approaches based in tissue engineering principles and computer \u2013 assisted surgery. While research into improved ACL treatment options continues, the synthesis of recent advancements provides a new optimism towards the regeneration of an ACL mirroring its original stability, function, and longevity.\nINTRODUCTION\nThe anterior cruciate ligament (ACL) is the most frequently injured ligament in the knee and consequently, the majority of research into knee ligament injuries has been directed towards the ACL. While the collateral knee ligaments exhibit strong healing potential and generally respond well to conservative treatment, the anterior cruciate ligament has a poor intrinsic healing ability due to the fact that it is enveloped by synovial fluid and lacks significant vascularization (1). Surgical reconstruction is therefore the most frequent mode of treatment pursued when the ACL is torn. The patients who experience ACL injuries are significantly younger and more active than those who experience many other orthopaedic injuries. The need for reconstruction options that exhibit longevity in the face of great stresses is therefore imperative (2). Historically, options for surgical treatment have included primary repair with or without synthetic augmentation and reconstruction using either biological tissue grafts or prosthetic ligaments. Primary repairs with or without augmentation have tended to fail at restoring stability to the knee and are not a common treatment option today (3). Likewise, prosthetic replacements have traditionally tended to be inadequate due to post-surgical complications arising from wear and degeneration. Hence, biological tissue autograft reconstruction using the patellar tendon or quadrupled semitendinosus\/gracilis tendons has become the most popular procedure in surgical treatment of an ACL rupture. However, the frequency of significant anterior knee pain post-surgically and high occurrence of flexion contracture and crepitation in tendon autografts have kept research interests focused on the further development of prosthetic ligament implants. A significant number of early research endeavours into prosthetic ACL replacements failed due to a poor understanding of the biomechanical and physiological properties of the ACL. Tremendous gains in understanding have been made in these areas, with significant progress being made in understanding the inter-dependence between ACL anatomy, tissue composition, biomechanics, and the healing processes. Unfortunately, to date, no prosthesis has proven itself as a viable alternative to the patellar or hamstring tendon autografts, currently used in over 90% of ACL reconstructions. This article reviews some of the major historical milestones in ACL reconstruction technology and looks forward to the continuing evolution of this technology. The literature used for this review was obtained using the PubMED database with keywords \u201canterior cruciate ligament\u201d searched in tandem separately with \u201cprosthetics\u201d and \u201ctissue engineering.\u201d It should be noted that many of the studies cited in this review obtained funding from orthopaedic and biomaterial companies that sponsored the implant.\nSURGICAL REPAIR: ADVANTAGES AND DISADVANTAGES OF BIOLOGICAL TISSUE GRAFTS\nBone-patellar tendon-bone (BPTB) and semitendinosus\/gracilis tendon autografts are currently the most common grafts used in ACL reconstruction. While the BPTB autografts were the former \u201cgold standard\u201d, recent studies have identified the quadruple semitendinosus graft as a comparable option for ACL reconstruction (4). Advances in hamstring graft fixation have led to similar strengths of fixation between BPTB and semitendinosus\/gracilis grafts (5). Both techniques now offer a high degree of strength and stiffness in the reconstructed ligament. Additionally, use of the patient\u2019s own tissue eliminates the risk of rejection and good to excellent results are reported in 85\u201390% of patients receiving either of these grafts for ACL reconstructions. Nonetheless, with patellar tendon autografts, many patients experience impaired function and significant morbidity at the donor site including secondary anterior knee pain, patellar tendonitis, infrapatellar contracture, and patellar fracture. Likewise, hamstring weakness and saphenous nerve injury can be seen secondary to hamstring harvest in semitendinosus\/gracilis autograft ACL reconstruction (6).\nTo avoid complications resulting from donor site morbidity in autograft reconstructions, allograft reconstruction has also been examined. While eliminating the donor site morbidity, the use of allografts is not currently considered advantageous due to a limited donor tissue supply, delayed biological incorporation, risks of disease transmission and tissue rejection. In an attempt to overcome these concerns, research into the use of synthetic prosthetic ligaments began over 30 years ago and continues today in hopes of eliminating donor site morbidity and reducing the risk of disease transmission and supply shortages. One of the main obstacles in finding an adequate prosthetic replacement for the ACL is the longevity of the graft, with autogenous tissue consistently proving to be a more durable and long-lasting replacement than the many biomaterials that have been applied to ACL replacement (7).\nPROSTHETIC BEGINNINGS AND PAST USE\nThe first attempts at a synthetic ACL reconstruction were conducted by Alwyn-Smith in 1918 using silk sutures; they however failed within 3 months (8). Reconstruction with a prosthetic ligament made of polytetrafluoroethylene (PTFE) with embedded carbon or aluminum oxide fibers (Proplast) was first reported in 1973 (9). Results with this system yielded an average time to breakage of just over 1 year. A report by James et al. suggests that only 52% of reconstructions utilizing this graft yielded satisfactory results (10). Satisfactory results include all grafts that patients were content with and had not ruptured at time of lastest follow-up.\nCurrent classification systems for synthetic replacements of the ACL include grafts, ligament augmentation devices, or total prosthetics. Grafts (polyethylene, PTFE), typically fixed at both ends, were the initial focus of synthetic ACL replacement and were meant to provide stability to the ACL-deficient-knee until secondary reconstruction procedures gained popularity (11). Similarly, ligament augmentation devices (polypropylene, polyester) were intended to provide immediate protection for autogenous tissue grafts until revascularization was complete and the ingrown tissue was capable of withstanding local tensile and compressive forces. Unfortunately, these devices may in fact accomplish the opposite of their intended purpose by stress-shielding autogenous tissue, preventing it from developing adequate mechanical strength. Fixed at both ends like a graft, the total prosthesis was intended as a permanent ACL replacement without any soft-tissue ingrowth that would allow the patient to begin aggressive rehabilitation immediately as no tissue maturation or revascularization was required. While the implantation of a full-strength ACL replacement generally led to excellent short-term results, long-term efficacy results were poor due to wear and ensuing rupture of the prosthesis (11).\nCombining the mechanical focus provided by these prior prosthetic approaches with tissue engineering principles has led to the development of a scaffold design intended to serve as a foundation for soft tissue in-growth. Scaffolds may be permanent or may be 31 Vol. 11 No. 1 Anterior Cruciate Ligament Reconstruction: A Look at Prosthetics intended to gradually degenerate as the host tissue replaces them. Problems associated with the biological incorporation of scaffolds include variability of tissue in-growth, immature degeneration of the implant and insufficient maturation of the host tissue resulting in an inability of the scaffold to withstand inherent mechanical stresses placed on the ACL. Current directions in this area involve the use of tissue engineering focusing on developing a mechanically and biologically functional matrix for the scaffold and the use of in vitro mechanical signals to guide new tissue development (11). Table 1 summarizes the advantages of the various prosthetic ACL graft options that have been attempted and are discussed below.\nCARBON FIBRE PROSTHETICS\nSome of the earliest attempts to discover an appropriate alternative to the biological graft resulted in a variety of carbon fibre prostheses that varied greatly with respect to material and implantation technique. In 1977, Jenkins et al. were the first to use a flexible carbon fibre implant, and suggested that the carbon fibre induced the formation of tendon in animals and humans (12). However, subsequent studies reported the generation and migration of carbon wear particles into the joint space and regional lymph nodes following implantation of the prosthesis (12). To combat this problem, attempts were made to coat the carbon fibre with collagen and absorbable polymers. Of particular interest was a carbon fibre implant proposed by Alexander et al. coated with a co-polymer of polylactic acid (PLA) and polycapralactone (13). The carbon filaments were thought to act as a scaffold for tissue ingrowth by evenly distributing and reducing the interfacial stress between the implant and soft tissue attachment, while the PLA\/polycapralactone would protect the fibre during implantation. Over time, the PLA was meant to resorb and the carbon fibres degrade as new tissue developed, thereby encouraging normal tissue regeneration without permanently replacing it (13). A 24-month study involving 82 patients was conducted by Weiss et al. to further examine the PLA coated prosthesis using a variety of subjective and objective measures (pain, stability, function, and isokinetic strength testing), which revealed significant improvements over the duration of the study (14). Arthroscopic evaluation demonstrated collagenous tissue ingrowth, confirmed by histological studies, to be composed of Type I and III collagen in similar proportions to that found in normal healing ligamentous tissue (14).\nA similar design was used in the Surgicraft ABC prosthetic ACL (Surgicraft Ltd., Redditch UK) composed of carbon and polyester fibres oriented in a partial braid by a zig-zag pattern. However, a study by O\u2019Brien et al. with an average follow-up of 34 months showed only 11 of 31 knees (41%) had good results defined as a Lysholm score greater than 76. The Lysholm knee score is an outcome measure that assesses knee function on a 100 point scale. The authors also noted unacceptable stretching and complete rupture as major complications and concluded that the implant is unsuitable for clinical use (15).\nGORE-TEX PERMANENT PROSTHESIS\nThe Gore-Tex ligament prosthesis is composed of a single long fiber of expanded polytetrafluoroethylene (PTFE) arranged into loops. Extensive mechanical testing has shown that the resulting ultimate tensile strength is about 3 times that of the human ACL and the results from cyclical creep tests and bending fatigue testing seem to identify Gore-Tex as the strongest synthetic ACL replacement in terms of pure material stability (16). Bolton and Bruchman reported that 129 out of 130 patients receiving a high strength PTFE ligament showed improved knee stability at 15 months or less (16). Glousman et al. reported an initial improvement upon physical examination and subjective scores. However, at mean follow-up of 18 months, they reported a progressive loosening of the prosthesis (17). Similarly, Woods et al. presented 2- and 3-year follow-up of Gore-Tex ACL reconstructions and showed a similar pattern of early improvement post-operatively, but deterioration over time. They reported an overall failure rate of 33% at 3-year follow-up (18). Indelicato et al.\u2019s follow-up of Gore-Tex implant ACL reconstructions showed a 90% success rate at 2 years versus only a 76% success rate at 3 years or more (19). Despite similar reports describing complications with the Gore-Tex ACL prosthesis, subjective results in several studies remain acceptable at 60\u201380 % (17, 19). The Gore-Tex ACL prosthesis is currently FDA approved for use in patients who have had a failed autogenous intra-articular graft procedure.\nDACRON\nWith its success as a vascular surgery implant (20), various forms of Dacron grafts have been developed as a scaffold for ACL replacements. The implant is a composite of four tightly woven polyester strips wrapped in a sheath of loosely woven velour, designed to minimize abrasion of the graft and act as a scaffold for fibrous tissue ingrowth. A report by Lukianov et al. reviewed the short-term follow-up (mean 28 months) of 41 patients who underwent ACL reconstruction with the Stryker Dacron ligament prosthesis (3). Seventy-five percent of the patients were found to have a negative Lachman, anterior drawer, and pivot shift at their most recent follow-up. However, Richmond et al. reported failure rates of 37.1% in a study of Dacron reconstruction with mean long-term follow-up of 50 months (21). Likewise, Barrett et al. reported higher failure rates of 47.5% after a four-year follow-up period (22). A clinical study by Lopez-Vazquez et al. examining ACL reconstruction with a Dacron prosthesis showed a similar deterioration of results after the first post-operative year (23). With the initial short-term strength shown by these grafts offset by their poor long-term stability, Dacron grafts should not be considered a viable alternative for ACL reconstruction.\nLEEDS-KEIO ARTIFICIAL LIGAMENT\nWith the desire to design a graft that combined the properties of a permanent prosthesis and a tissue-promoting scaffold, Fujikawa and Seedhom developed the Leeds-Keio artificial ligament: a polyester mesh-like structure anchored to the femur and tibia with bone plugs (24). This mesh was intended as a scaffold for soft tissue ingrowth through the intra-articular and extra-articular sections of the ligament, eventually uniting the bone plugs. The implant was considered sufficiently flexible to be effective with a maximal tensile strength of approximately 2100 Newtons (N), which significantly exceeds that of the average young adults\u2019 natural ACL (about 1730 N) (24). Initial descriptions from the inventors also described minimal articular wear with the ligament (24). The inventors of this graft have reported successful clinical results with arthroscopic observations documenting neoligamentous tissue within the implanted Dacron scaffold. Other investigators, however, have reported the ingrowth of non-aligned fibrous tissue (i.e. non-neoligamentous tissue) within the device after implantation and suggested that the Leeds-Keio ligament did not serve as a true scaffolding graft, but instead behaved as a permanent load-bearing prosthesis, subject to long-term failure in the joint (25).\nMcLoughlin and Smith presented a 3.8 year follow-up study of 25 patients implanted with the Leeds-Keio ligament for chronic ACL instability. They reported a low complication rate and considerable success in the elimination of instability after finding good post-operative results with the anterior drawer test using an arthrometer at 90\u00b0 of flexion (26). Nevertheless, ensuing long-term follow-up studies showed a deterioration of results after the first post-operative year and a large number of long-term graft ruptures despite excellent early results in stability testing and on the Lysholm scale (27). These findings were similar to earlier results reported by Schindhelm, who found that good early results in a sheep model were not maintained (28). Due to the number of long-term graft ruptures and the lack of long-term stability provided, the Leeds-Keio ligament is no longer suitable for reconstruction of the human ACL (27, 28).\nKENNEDY LIGAMENT AUGMENTATION DEVICE (LAD)\nKennedy et al. introduced the concept of the Ligament Augmentation Device (LAD) in 1980 (29). The graft, composed of a band-like braid of polypropylene, was originally developed to reinforce the area of pre-patellar tissue considered to be a weak area of autogenous patellar tendon grafts. Use of this prosthesis employed the MacIntosh\/Marshall transfer of a portion of the rectus femoris tendon, pre-patellar tissue, and central third of the patellar tendon in an over-the-top fashion (30). Originally, the graft was developed to protect the autogenous tissue graft from excessive stresses during the initial remodelling phase (characterized by degeneration and revascularization), allowing for earlier resumption of pre-operative activity levels (29). Research has shown that the percentage of load accepted by the LAD varies according to the type of graft employed and method of reconstruction. Comparisons of the patellar tendon and semitendinosus\/gracilis LAD composite grafts revealed that the LAD will accept approximately 28% and 45% of the applied load, respectively (31). To prevent excessive stress shielding that would otherwise prevent the autogenous tissue from developing a normal functional tensile strength, the LAD was attached to the bone at only one end. Despite a promising beginning, the suture interface between the LAD and the graft was identified as the weak link of the composite.\nWhether collagen fibres become truly incorporated into the LAD remains a controversial issue. Most histological evidence has been derived from animal studies and thus may not be fully indicative of results in humans. Nevertheless, this evidence seems to point to adequate longitudinal collagenization of the graft with inconclusive results on whether or not collagen ingrowth within the LAD has occurred (32). Furthermore, as an intra-articular foreign body, the LAD has been reported to induce an inflammatory response characterized by foreign body giant cells and macrophages in the surrounding tissue. A review by Kumar et al. reported that the majority of complications seen following use of the LAD were characterized as effusions and reactive synovitis, likely a result of LAD-induced inflammatory response (23). The decline in use of the aforementioned MacIntosh\/Marshall transfer, combined with the weak graft-prosthetic suture interface and propensity of the LAD to cause high rates of post-operative synovitis have resulted in a lack of widespread use of the device.\nTHE FUTURE: LIGAMENT ADVANCED REINFORCEMENT SYSTEM (LARS) ARTIFICIAL LIGAMENT\nThe Ligament Advanced Reinforcement System (LARS) (Arc-sur-Tille, France) artificial ligament consists of fibres made of polyethylene terephthalate (PET). An intra-osseous segment is composed of longitudinal fibres bound together by a transverse knitted structure while an intra-articular segment is composed of parallel longitudinal fibres twisted at 90\u00b0. The main innovation of this artificial ligament lies in its ability to mimic the natural ligamentous structure and reduce shearing forces by orientating the free fibers of the intra-articular portion of the graft clockwise or counter-clockwise for use in right and left knees, respectively. Furthermore, the PET fibres of the intra-articular segment are designed to encourage tissue ingrowth due to the porosity of the material, allowing ingrowth from the surrounding osseous tunnels. Ideally, such tissue ingrowth between the ligament fibres would contribute to the viscoelasticity of the graft and protect against friction at the opening of the bony canal and between the fibres themselves (1).\nA study by Lavoie et al. examined ACL reconstruction with the use of the LARS artificial ligament. Thirty-eight of forty-seven patients suffered from chronic ruptures of the ACL, while nine others presented with acute or subacute ruptures at a mean follow-up of 21.9 months. Six patients had previously had an unsuccessful ACL reconstruction. The Knee Osteoarthritis Outcome Score (KOOS) was administered to assess patients\u2019 opinions regarding their knee, while a modified International Knee Documentation Committee (IKDC) scoring system was used to examine knee stability. The IKDC form initially consisted of seven knee-related parameters that were each rated as either normal, nearly normal, abnormal, and severely abnormal. The worst score amongst the seven categories determined the final score. The form was later modified in 2001 to include subjective factors such as symptoms, sports activities, and ability to function. The Tegner activity scale, a subjective ten point activity scale, was obtained to assess patient activity levels. A Telos radiographic stress system was used to examine anterior knee displacement, which involves obtaining radiographs with the knee in ninety degrees of flexion and a posterior force applied to the knee. When results are compared to the normal knee, this test helps in the assessment of post-operative laxity in the reconstructed knee which may indicate that the graft is failing. None of the patients presented symptoms of synovitis, but longer follow-up time is required to properly draw conclusions with respect to patient outcome. Although Tegner scores improved significantly following surgery, no patients returned to pre-injury activity levels. Data obtained by the KOOS demonstrated patient satisfaction ranged from 73.5 to 93.0 %. In comparison to the uninjured knee, post-operative Telos stress radiography and the Lachman test indicated an average posterior-anterior displacement of 7.3 mm for the involved knee (28).\nA more complete examination of the LARS artificial ligament was provided in a subsequent study by the same authors. Nau et al. conducted a two-year follow-up randomized controlled trial that compared the BPTB autograft with the LARS artificial ligament method of ACL reconstruction in 53 patients with chronic instability (1). Like the preceding study, assessments included clinical examination, anterior laxity testing, as well as the KOOS, IKDC, and Tegner scores. In particular, IKDC evaluation revealed little significant difference between the two methods. Follow-up values for instrumented laxity testing were greater in the LARS group. While similar overall results were obtained for both groups, these results may suggest that a full return to activity may be hastened by using the LARS artificial ligament rather than the conventional BPTB technique (1).\nAnother recent study by Talbot et al. examined the use of the LARS artificial ligament for ligament reconstruction in knee dislocations (2). Twenty patients were included with a mean follow-up of 27.4 months. Each patient was evaluated using the Lysholm score, underwent clinical examination to identify ligamentous laxity and range of motion, and completed the ACL quality of life (ACL-QoL) questionnaire. The mean Lysholm score was 71.7, which is lower than scores reported by several other studies (range of 74.7 to 91.3) investigating the outcomes of knee dislocations treated with surgery as reported by Fanelli (35). Following the same trend, Telos radiometry revealed a mean residual laxity of 5 mm in patients post-surgery, which is also greater than these previously reported results. The average range of motion post-surgery was 118\u00b0 with a mean fixed flexion contracture of 2\u00b0 (2).\nAlthough preliminary investigations into the use of the LARS artificial ligament have been encouraging, concerns regarding the risk of rupture remain and must be addressed through long-term follow-up studies.\nTISSUE ENGINEERING ADVANCES\nPermanent synthetic prostheses are capable of duplicating the mechanical and structural properties of the ACL. However, they generally tend to lose strength with time. Tissue-based or tissue-aided implants offer the additional possibility of the restoration of normal joint kinematics while the mechanical behaviour of these implants is expected to improve over time as tissues are remodelled within the knee (36). An ideal ACL scaffold must meet the immediate functional mechanical demands within the reconstructed knee, however, they must also degrade at a rate similar to that of tissue ingrowth. Accordingly, the ACL scaffold should lose its mechanical integrity while allowing the remodelled tissues to gain strength and accept an increasing amount of the mechanical demands placed on the ACL. Current research into this novel tissue-engineering approach has focused on seeding either collagen-based scaffolds or synthetic biodegradable polymers with a variety of different cell types. In hopes of stimulating early healing, reducing biomaterial-related inflammatory response and improving neoligament formation, several researchers have sought to adopt a cell sheeting technique to improve the performance of the synthetic ACL scaffold.\nSeveral groups have conducted experiments on this tissue-engineered approach to ACL ligament reconstruction using both fibroblast-seeded synthetic scaffolds and collagen-based prosthetics (37, 38). Bellincampi et al. measured the ingrowth characteristics of rabbit fibroblasts on skin and ACL scaffolds (39). Subsequent in vivo studies suggested that fibroblast-seeded collagen scaffolds were viable after re-implantation into the donor rabbit. The major limitations of these approaches are the allogenicity of the collagen scaffolds, often leading to further complications. Collagen-based constructs also suffer from batch-to-batch variability, making consistent reproduction of these prostheses difficult. Collagen does not offer the same flexibility for modification that is reported with the technology of biodegradable polymers (6).\nAs an alternative to the scaffolds made of non-degradable polymers, investigators have begun to examine biodegradable materials that would provide immediate stabilization to the repaired ligament but would also act as a scaffold for the ingrowth and\/or replacement by host cells. Cao et al. described the generation of neo-tendons in a nude mice model by implanting polyglycolic acid (PGA) scaffolds seeded with bovine tendon fibroblasts in the subcutaneous space of athymic mice (40). Using a similar system, Koski et al. reported the formation of ligament-like structures when fibroblasts isolated from bovine cruciate ligaments were seeded onto PGA scaffolds and implanted subcutaneously in nude mice (6). In both studies, the tissue developed histological characteristics similar to normal tendon and ligament over time.\nOuyang et al. reported that bone marrow stromal cells (bMSCs) seeded onto poly-lactic and -glycolic acid (PLGA) scaffolds grew as a multi-layer of cells intertwined in a collagen matrix synthesized by the cells 35 Vol. 11 No. 1 Anterior Cruciate Ligament Reconstruction: A Look at Prosthetics themselves. They also noted that the cell sheet formed faster than scaffolds seeded with terminally differentiated cells, such as fibroblasts and smooth muscle cells (41). They reported that degradation of the scaffolds occurred over time, as indicated by a decrease in failure load. Altman et al. seeded 6-cord silk scaffolds with human bone marrow stromal cells and cultured them for 14\u201321 days. Their results similarly suggested a slow degradation of the scaffolds. However, fatigue analysis and subsequent regression analysis revealed an expected matrix lifetime equivalent to 1 year in vivo. While these reports are an estimate, the authors suggest that this is a marked improvement compared to similar studies using collagen scaffolds (36).\nThe future of tissue engineering may also require a significant contribution from cell-specific growth factors influencing the maturation and homeostasis of the healing response of ligament tissue. Studies have suggested that individual growth factors may have an important effect on cell division and enhance ligament healing, while others have pointed to the synergistic effect of a combination of growth factors as important to cell outgrowth in ACL explants (42, 43). In a study observing the effects of several growth factors on the cell migration, proliferation and collagen production in human ACL cells, Murray et al. suggested that specific dose-response relationships may exist for the optimal activity of each growth factor (44). These authors observed that the addition of transforming growth factor (TGF)\u03b2-1 led to an increased cell population, as well as increased collagen and smooth muscle actin production in human ACL cells cultured on top of a collagen-glycosaminoglycan scaffold. Despite the complex nature of the inter-dependent factors at play during the recovery and rehabilitative period following an ACL reconstruction, much of the literature today suggests that tissue engineering techniques will lead to a new generation of ACL replacements, capable of regenerating a mechanically robust and natural ACL.\nCOMPUTER-ASSISTED ACL RECONSTRUCTION\nIn an attempt to improve the accuracy and reproducibility of ACL graft placement, several groups have turned to computer-assisted surgery in an attempt to reduce the incidence of graft failure (45, 46). These systems are capable of modeling the placement and predicting the impingement of an ACL graft based on intra-operative anatomical landmarks and signals received at an opto-electric camera. These studies reported that experience level did not affect the placement of the tunnels. Computer-assisted ACL replacement reduces variance in tunnel placement and allows residents and less experienced surgeons to limit complications and control tunnel positioning (46). Based on the success of navigation systems for total knee and hip replacements, the use of computer-assisted ACL reconstruction may lead to similarly dramatic improvements in technical and functional outcomes.\nCONCLUSION\nSatisfactory prosthetic replacement of the ACL has been a focus of orthopaedic research endeavours for the past thirty years. Desires to provide immediate mobility and strength to the ACL-deficient knee while avoiding the donor site morbidity caused by the commonly used autograft surgical techniques continually drive new research initiatives. Most of the grafts that have been developed to date have failed due to unsatisfactory long-term physiologic and functional performance. Most permanent ACL prostheses are prone to creep, fatigue, and mechanical failure within several years after implantation (40). Tissue ingrowth scaffolds and ligament augmentation devices require further refinement to provide effective mechanical support while avoiding stress-shielding of the host tissue. In view of these factors, prosthetics are not widely used today in ACL reconstruction, and autogenous tissue grafts remain the gold standard used by the majority of surgeons. Perhaps development of resorbable, tissue-inducing and cell-seeded biomaterials will improve the long-term biomechanical performance of the reconstructed anterior cruciate ligament. Advances in tissue engineering combined with developments in molecular biology and gene therapy may couple with the rapid gains in computer-assisted surgery to provide improved options for the ACL-deficient knee, with a greater potential to restore its pre-injury state.","keyphrases":["anterior cruciate ligament","prosthetics","tissue engineering","biomaterials","synthetic grafts"],"prmu":["P","P","P","P","R"]}
{"id":"Eur_J_Appl_Physiol-3-1-2039775","title":"Distribution of motor unit potential velocities in short static and prolonged dynamic contractions at low forces: use of the within-subject\u2019s skewness and standard deviation variables\n","text":"Behaviour of motor unit potential (MUP) velocities in relation to (low) force and duration was investigated in biceps brachii muscle using a surface electrode array. Short static tests of 3.8 s (41 subjects) and prolonged dynamic tests (prolonged tests) of 4 min (30 subjects) were performed as position tasks, applying forces up to 20% of maximal voluntary contraction (MVC). Four variables, derived from the inter-peak latency technique, were used to describe changes in the surface electromyography signal: the mean muscle fibre conduction velocity (CV), the proportion between slow and fast MUPs expressed as the within-subject skewness of MUP velocities, the within-subject standard deviation of MUP velocities [SD-peak velocity (PV)], and the amount of MUPs per second (peak frequency = PF). In short static tests and the initial phase of prolonged tests, larger forces induced an increase of the CV and PF, accompanied with the shift of MUP velocities towards higher values, whereas the SD-PV did not change. During the first 1.5\u20132 min of the prolonged lower force levels tests (unloaded, and loaded 5 and 10% MVC) the CV and SD-PV slightly decreased and the MUP velocities shifted towards lower values; then the three variables stabilized. The PF values did not change in these tests. However, during the prolonged higher force (20% MVC) test, the CV decreased and MUP velocities shifted towards lower values without stabilization, while the SD-PV broadened and the PF decreased progressively. It is argued that these combined results reflect changes in both neural regulatory strategies and muscle membrane state.\nIntroduction\nDiverse laboratory conditions have been used in surface electromyography (sEMG) studies in order to gain insights into the neural regulatory strategies and muscle membrane alterations. The influence of force load on sEMG can be investigated by using force tasks or position tasks. The majority of studies have been performed as force tasks, which means that the subject controls the effort by maintaining a target force while the limb position is fixed. During the position tasks, in contrast, an inertial load is applied while the subject controls a target limb position. Both force and position tasks can be performed in static or dynamic conditions. Examples of the static force tasks are the well-known isometric experiments, with higher and lower force levels. In the recent past, force tasks in dynamic conditions have been performed sporadically, such as the cycling experiments by Pozzo et al. (2004) and Farina et al. (2004). Since Hunter et al. (2002) found that, with the same load torque, position tasks resulted in a shorter endurance time than force tasks, suggesting different regulatory mechanism for the both type of tasks, the position task studies gained field. Most position task experiments have been performed in static conditions, evaluating the underlying physiological phenomena during force versus position tasks (Hunter et al. 2002, 2003; Hunter and Enoka 2003; Rudroff et al. 2005, 2007). Potvin (1997) has described the changes in the sEMG during position tasks in dynamic conditions. Previous findings suggest that, as compared with force tasks, position tasks induce greater synaptic input into the motor neurons (Mottram et al. 2005a) and greater adaptation in the motor unit discharge (MacGillis et al. 2003).\nChanges in muscle activity during (static and dynamic) position tasks have been assessed using two of the three traditional sEMG parameters, the power spectrum and the global sEMG amplitude. However, the third parameter, the mean muscle fibre conduction velocity (CV), has been lacking. Spectral estimates are generally accepted in fatigue experiments as equivalents of CV because they highly correlate with the CV\u2019s changes (Bigland-Ritchie 1981; Eberstein and Beatie 1985; Arendt-Nielsen and Mills 1985). But this correlation holds true only for the constant forces and isometric conditions; thus, a replacement of CV by power spectrum assessments does not always seem feasible (Farina et al. 2002; Broman et al. 1985). The favour of CV above power spectrum is, furthermore, that it renders direct and absolute values of conduction velocity, and is less sensitive to the anatomical local relationships, such as depth of the motor unit (MU) in relation to the muscle and skin surface (Farina et al. 2002). Yet the limitation of these global CV measurements remains the lack of sensitivity to the changes at the level of an individual motor unit potential\/ motor unit.\nTo accommodate with these limitations of CV, researchers have recently been trying to disentangle the propagation velocities of individual motor unit potentials (MUPs) from sEMG. One of the methods is the inter-peak latency method (IPL) proposed by Lange et al. (2002). The principle comprises calculating conduction velocities of the MUPs from the latencies between paired MUPs of two differential sEMG signals obtained parallel to the muscle fibres, and the distance between the recording electrodes. The negative peaks of two paired MUPs are then the elements determining the interpeak latency. As motor unit propagation velocity reflects the intrinsic physiological properties of a MU, such as fast-twitch or slow-twitch type (Buchthal et al. 1973; Andreassen and Arendt-Nielsen 1987), the IPL method renders many diverse MUP velocities. Lange at al. (2002) proposed using the standard deviation of MUP velocities as an additional measure that offers information about muscle fibre properties. Changes in these velocities during prolonged effort may indicate, for example, slowing\/fatigue of the activated motor units and\/or appearance of fast\/newly recruited motor units. Such shifts in the activated MUs\u2019 populations were shown by Houtman et al. (2003) by eliciting the MUP velocities with the IPL method and presenting their distribution in histograms. The IPL method has not been applied much. It yields insights into the diversity of MUP velocities and thereby the underlying changes in the MU activity. The method is simple and does not require expensive apparatus or software. When compared with techniques that assess the propagation patterns of MUPs by multi-channel\/spatial resolution sEMG (Masuda and Sadoyama 1986; Rau et al. 1997), the IPL method is unable to distinguish and follow individual MUPs belonging to the specific MUs. \nIn the present study, the sEMG signal was described with four parameters derived from the IPL method: (1) the mean muscle CV which was the average of the obtained MUP velocities; the two statistical distribution variables, which were: (2) the within-subject MUP velocities\u2019 skewness [Sk-peak velocity (PV)] and (3) the within-subject MUP velocities\u2019 standard deviation (SD-PV), and (4) the peak frequency (PF), a variable expressing the amount of MUP activity (number of peaks\u00a0=\u00a0MUPs) per second.\nThe aim of the present study was to investigate with the four parameters the changes in MUPs\u2019 velocities of the biceps brachii muscle (BB) during prolonged dynamic position tasks, in dependence of (low) force and duration. It is chosen for the dynamic position tasks as study design because their physiology promised the finding of a large variety of MUP velocities (great amount of activity due to the position tasks character, and diversity because of the recruitment\/derecruitment changes within the dynamic cycle). Whole cycles of movement with their concentric and eccentric phases were analyzed together in order to evaluate a total of the sEMG activity with its evolution over time. To highlight the initial changes with the effect of force on it, the changes during the first 14.4\u00a0s of the dynamic tasks were evaluated separately. Additively, short static position tasks were performed in order to show the (early) changes on force, without any influence of movements on the signal.\nMethods\nSubjects\nThe study involved short static and prolonged dynamic experiments. Forty-one healthy and physically active males (24.8\u00a0\u00b1\u00a06.7\u00a0years, from 17 to 48) (mean\u00a0\u00b1\u00a0SD) volunteered for the first experiment and 30 randomly chosen subjects from that group (25.4\u00a0\u00b1\u00a07.6\u00a0years, from 18 to 42) participated in both experiments. Exclusion criteria were drug abuse and the practice of bodybuilding. Three from a total of 44 subjects were excluded because of the impossibility to obtain a required correlation coefficient between the sEMG signals used to estimate the parameters\u2019 values. The experimental protocol was conducted according to the Helsinki Declaration and approved by the local ethics committee. All participants gave written informed consent.\nExperimental set up\nMaximal voluntary contraction (MVC) of elbow flexors was measured at least 5\u00a0days before the experiment with a hand-held dynamometer (Lameris Instruments, Utrecht, The Netherlands). During the MVC measurements subjects were sitting upright. The shoulder was slightly abducted and flexed at 45\u00b0, the elbow was firmly sustained and flexed at 90\u00b0, and the forearm was supinated. The dynamometer was applied to the wrist by the break method (van der Ploeg and Oosterhuis 1991). The peak hold was switched off and the force was kept for at least 3\u00a0s. The mean of three maximal values was taken as a MVC. The MVC was assessed with elbow at 90\u00b0, although the tests were performed at the elbow angle of 135\u00b0 (Philippou et al. 2004).\nDuring the experiment subjects were sitting in a chair. The upper arm was slightly abducted and comfortably supported at 45\u00b0 of the shoulder flexion, the forearm was free. When the elbow was stretched, the line of the upper arm-forearm was at 45\u00b0 in relation to horizontal. When the elbow was flexed to the angle of 135\u00b0, the forearm was horizontal. The forearm was supinated during static and dynamic tests.\nStatic tests\nSubjects were asked to hold the forearm horizontally (elbow angle was then 135\u00b0). A visual bar helped to maintain the correct (horizontal) position of the forearm. In the loaded tests a sack filled with lead and sand was placed in the palm. Three levels of force were applied in blocks that were 3\u00a0min apart: unloaded, loaded 10 and 20% MVC. A block consisted of three tests (three repetitions at the same level of force); every test lasted for 3.8\u00a0s and was within a block separated 30\u00a0s from one another.\nDynamic tests\nAll participants of the dynamic tests underwent previously static tests, separated by 5\u00a0min. Subjects were asked to swing the forearm from the stretched (elbow angle 180\u00b0) to horizontal position (elbow angle 135\u00b0), thus moving over an angle of 45\u00b0. They did it within a rate of 40 beats per minute (one up-and-down movement in one beat), given by a metronome sound. The visual bar indicated the horizontal position to which the lower arm returned after being stretched. Four force levels were applied: unloaded, and loaded 5, 10 and 20% MVC. The tests lasted for 4\u00a0min and were separated by 5\u00a0min.\nEMG recording\nMeasurements were performed on the short head of the BB of a dominant arm. A surface electrode array consisted of three golden-coated electrodes (Harwin, P25-3526), diameter 1.5\u00a0mm, insulated in synthetic material plate, with a 10\u00a0mm distance between the electrodes (Sadoyama et al. 1985). The skin was cleaned with 95% ethanol. The electrode array was placed parallel to the muscle fibres (Sollie et al. 1985). The proximal electrode was positioned exactly on the distal one-quarter point of the upper arm, measured between the coracoid and the elbow crease. This place of the electrode was about halfway between the endplate zone and the tendon, securing a sufficient distance from the endplate (Sadoyama et al. 1985; Masuda and Sadoyama 1987). Bipolar derivation was made from the proximal to distal direction. The optimal electrode position was controlled by both the observation of the signal on the monitor and the estimation of a correlation coefficient (CC) of the two sEMG signals, which was accepted at r\u00a0>\u00a00.7 for unloaded, r\u00a0>\u00a00.85 for 5% MVC and r\u00a0>\u00a00.9 for higher loaded tests. (During the static and dynamic tests, the maximal CC for unloaded arm was usually lower than that for loaded arm, which was consistent with Hogrel et al. 1998, who have estimated for an unloaded arm r\u00a0>\u00a00.7). The ground electrode was placed on the lateral upper arm, slightly proximal from the derivation electrode. The temperature sensor was medial on the upper arm. Two obtained signals were differentially amplified (gain 2,000\u201310,000\u00d7) and band pass filtered at 2\u2013250\u00a0Hz by EMG apparatus (Viking IV, US).\nData processing\nThe signals were simultaneously A\/D converted (sampling 10\u00a0kHz, 12\u00a0bits acquisition). Data were stored on a personal computer. The signal was analyzed with LabVIEW 6.1 that also facilitated a partial on-line analysis. The peak selection and the correlation coefficient assessments for both static and dynamic tests were performed on 0.2\u00a0s epochs. In the static tests, measurements were taken every second during 0.8\u00a0s (comprising 4 epochs of 0.2\u00a0s). A static test was of 3.8\u00a0s duration and was repeated three times for each force level. The statistical analyses were performed on the data of these three repeated tests taken together. In the dynamic tests, the data were assembled every 30\u00a0s during 14.4\u00a0s (comprising 72 epochs of 0.2\u00a0s). The test duration was 4\u00a0min.\nPeak selection\nThe basic principle was that of Lange et al. (2002). The software was custom designed and written in LabView. The orientation of the signals is up-negative. 1st step: finding zero line. 2nd step: finding the peak-to-peak amplitude of the largest MUP in an epoch of 0.2\u00a0s. 3d step: finding a peak-decline structure. The peak-decline is defined as a structure with a decline of \u226520% compared with the largest MUP amplitude of a 0.2 epoch, over \u22644\u00a0ms (\u226440 sample points). 3d step: finding a peak. A peak is the highest (most negative) point previous to the decline, and must be \u226510\u00a0\u03bcV (the threshold of the noise level). 4th step: finding a pair peak. A pair peak is a peak in the second signal with the properties as previous, found in a time window between 1.49 and 4\u00a0ms after the peak of the first signal. This window is chosen assuming the physiological CV values of 2.5\u20136.67\u00a0m\/s. 5th step: excluding double peak. If a peak from the first signal matches two different peaks from the second signal, then the first peak from the second signal is true. If during the fatiguing tests the CV severely diminishes, then the low limit of velocity is put at 1.3\u00a0m\/s, making a window of 1.49\u20137.68\u00a0ms. As an objective pragmatic criterion to this change a lowering of the peak frequency with \u226430% was assumed, as compared with the first value of the 20% MVC test.\nThe parameters\nThe following calculations were performed: (1) basic calculation of PV, following the IPL method; (2) mean CV, expressed as an average value of the PVs; (3) within-subject skewness of the peak velocities (Sk-PV), expressed as a skewness of a PVs\u2019 population of a subject; (4) within-subject standard deviation of the peak velocities (SD-PV), expressed as a standard deviation of a PVs\u2019 population of a subject; and (5) peak frequency (PF), expressed as a number of peaks per second.\nStatistics\nOne-way analysis of variance (ANOVA) with repeated measures on force was used to compare the dependent variables for static tests (three levels) and for the initial values of the dynamic tests (four levels). A two-way ANOVA with repeated measures on force ( four levels) and time (nine levels) was used to compare variables during the dynamic tests. In the case of interactions between force and time, to describe changes over time, when appropriate, four separated ANOVAs were performed with smaller time windows of 0\u201360, 60\u2013120, 120\u2013180 and 180\u2013240\u00a0s. To be assured that the dependent variables met parametric assumptions, plots of residues were produced with SPSS program, model control as suggested by Kutner et al. (2005, p. 1157). No relevant deviations of model were detected. Pearson correlation coefficients were calculated to evaluate associations between variables. A level of P\u00a0<\u00a00.05 was used to identify statistical significance.\nResults\nSubjects\u2019 physical characteristics are presented in the Table\u00a01. The force of the biceps correlated positively with the upper arm circumference (r\u00a0=\u00a00.484, P\u00a0<\u00a00.01). No association was found between either force or upper arm circumference and the sEMG variables. The average skin temperature increased during the dynamic tests by 1.65\u00b0C (P\u00a0<\u00a00.001); it did not change during the static tests.\nTable\u00a01Characteristics of 41 participants to the static tests; 30 of them participated in the dynamic tests as wellNMinimumMaximumMeanSDAge (years)41164824.76.7Height (cm)41166.0197.0183.28.0Weight (kg)4160.095.074.39.2Force (N) right38148.5346.5246.141.9Force (N) left (for left-handed)3262.3267.3265.12.5Skin thickness (mm)411.44.22.4.7Circumference upper arm (cm)4124.031.027.52.0Length of biceps (cm)a4130.5039.5035.42.0Length lower arm\/radius (cm)b3324.030.026.41.6Length lower arm\/palm (cm)c3330.036.033.11.6Initial skin temp. (\u02daC)4130.833.832.3.8Temp. upper arm before dyn. tests (\u02daC)3030.834.232.3.9Temp. upper arm after dyn. tests (\u02daC)3032.235.733.91.1Room temp. (\u02daC)4120.024.022.31.0a\u00a0From the coracoid to the elbow creaseb\u00a0From the lateral epicondyle to the wrist creasec\u00a0From the lateral epicondyle to the middle of the palm\nStatic tests\nMean muscle fibre conduction velocity (CV)\nHistograms in Fig.\u00a01 show an example of a PVs\u2019 population in one subject during the static tests at three levels of force: unloaded, 10 and 20% MVC. Figure\u00a02a presents the averages of the CVs over 41 subjects, calculated from the subject\u2019s PVs. The CV of the unloaded test was the lowest (3.92\u00a0\u00b1\u00a0SD 0.27\u00a0m\u00a0s\u22121) and it increased with augmenting force levels (effect of force P\u00a0<\u00a00.001). A positive correlation existed between the CVs of unloaded test with 10% MVC, and 10% with 20% MVC (all r\u00a0>\u00a00.505, P\u00a0<\u00a00.05).\nFig.\u00a01Distribution of peak velocities of one subject during short static tests at three force levels: unloaded, and loaded 10 and 20% MVC. Note the shift of the velocities as a whole to the higher values with increasing level of forceFig.\u00a02Behaviour of peak velocities (PVs) as effect of force, expressed with four parameters. a Mean conduction velocity (CV); b skewness of within-subject PVs; c SD of within-subject PVs (SD); and d number of peaks per second (peak frequency\u00a0=\u00a0PF). Averages and standard errors are given, obtained from 41 subjects in short static tests at three levels of force: unloaded, and loaded 10 and 20% of maximal voluntary contraction. With increasing forces, the CV and amount of activity (PF) increases, accompanied with augmenting proportion of fast peaks (the skewness value diminishes). However, the spread of peak velocities within an individual (SD) does not change\nSkewness of peak velocities (Sk-PV)\nFigure\u00a02b presents the averages of the within-subject PVs\u2019 skewnesses over 41 subjects (see also the histograms of PVs in Fig.\u00a01). In all the tests a moderate positive skewness was found, which indicates a relative excess of lower velocities at the distribution scale. With increasing force the Sk-PV significantly diminished, indicating that the proportion of higher velocities increased (effect of force P\u00a0<\u00a00.05). The PVs\u2019 population as a whole shifted towards higher values with increasing forces too, which can be seen on the histograms in Fig.\u00a01.\nStandard deviation of peak velocities (SD-PV)\nThe averages of the within-subject\u2019s standard deviations of PVs are shown in Fig.\u00a02c. The SD-PV did not change when the force levels increased (effect of force n.s.).\nPeak frequency (PF)\nFigure\u00a02d shows the averages of PF in the static tests. The PF increased with increasing forces (effect of force P\u00a0<\u00a00.001).\nDynamic tests\nMuscle fibre conduction velocity (CV)\nHistograms in Fig.\u00a03 show the PVs estimated from one subject during dynamic tests at four levels of force: unloaded, loaded with 5, 10 and 20 MVC. Figure\u00a04a shows the averages of CV calculated from the subject\u2019s PVs over 30 subjects. As in the static tests, the initial CV of the dynamic tests increased with level of force (effect of force P\u00a0<\u00a00.001). Further, a positive correlation was found between the CV of the static tests and the initial CV of the respective dynamic tests (all r\u00a0>\u00a00.409, P\u00a0<\u00a00.05). The lowest initial CV was that of the unloaded test with about 4 (3.6\u20134.5) m\u00a0s\u22121 and the highest was at 20% MVC with approximately 4.6 (4.0\u20135.15) m\u00a0s\u22121, increasing from the unloaded to 20% MVC test with 14\u00a0\u00b1\u00a09.6%. During all the dynamic tests the CV significantly declined (effects of time for the unloaded test P\u00a0<\u00a00.02; for other tests P\u00a0<\u00a00.001), whereby the decline was steeper with larger forces (interaction between force and time, P\u00a0<\u00a00.001). At the three lowest force levels (unloaded, and loaded 5 and 10% MVC), the CV had two phases: a decline phase lasting for about 120\u2013150\u00a0s and a steady phase continuing to the end of a test. The steady state values approached the level of the unloaded test. During the 20% MVC test, however, the CV continued to decline, without a stable phase. Twelve of 30 subjects (40%) reported fatigue during the 20% MVC test and terminated the task prematurely between 90 and 210\u00a0s. The CV decreased for the fatigued subjects from 4.6\u00a0\u00b1\u00a00.3 (4.2\u20135.0) to 3.6\u00a0\u00b1\u00a00.3 (3.0\u20134.1) m\u00a0s\u22121 and for the continuing subjects from 4.5\u00a0\u00b1\u00a00.3 (4.0\u20135.15) m\u00a0s\u22121 to 3.8\u00a0\u00b1\u00a00.4 (3.0\u20134.7) m\u00a0s\u22121. Neither the absolute initial CV nor the end CV differed significantly between the groups (P\u00a0=\u00a00.497 and P\u00a0=\u00a00.124, respectively). However, the relative decline of the CV tended to be larger for the fatigued subjects compared with the continuing subjects, for fatigued being about \u221220 (\u22127 to \u221235)% and for continuing \u221215 (+7 to \u221228)%; P\u00a0=\u00a00.074.\nFig.\u00a03Changes in the distribution of peak velocities (PVs) over time for different levels of force. The PVs are obtained from one subject (the same as in Fig.\u00a01 for static tests) during prolonged dynamic contractions at four levels of force: unloaded, and loaded 5, 10 and 20% of maximal voluntary contraction (MVC). Every histogram represents a number of PVs within a period of 14.4\u00a0s. Initially (at time zero), a global shift of peak velocities is visible towards higher values when forces augment. During the unloaded, and loaded 5 and 10% MVC tests, slower peaks are moderately increasing and faster peaks are diminishing. During the 20% MVC test, the peak velocities shift considerably as a whole towards lower regions, and the amount of peaks visibly diminishesFig.\u00a04Effects of force and time on the behaviour of peak velocities (PVs), expressed with four parameters. a Mean conduction velocity (CV); b skewness of within-subject PVs; c SD of within-subject PVs (SD); and d number of peaks per second (peak frequency\u00a0=\u00a0PF). Averages and standard errors are given, obtained from 30 subjects during prolonged dynamic tests at four levels of load: unloaded, and loaded 5, 10 and 20% of maximal voluntary contraction (MVC). Note the difference in the decline pattern of the CV and the PF: the mean muscle conduction velocity declines over time for all levels of force, while the amount of activity remains stable for the three lower force level tests (unloaded, and loaded 5 and 10% MVC). In the higher force level test (20% MVC), the CV starts declining immediately, while the PF declines first gradually and later on steeply. Note the stable SD values from about 90\u00a0s for the three lower force tests, while the SD of the higher force test (20% MVC) clearly increases\nSkewness of peak velocities (Sk-PV)\nHistograms in Fig.\u00a03 show the distribution of PVs of one subject and Fig.\u00a04b presents the averages of the within-subject\u2019s skewnesses of the PVs over 30 subjects. In the initial phase, consistent with the static tests, the PVs were most positively skewed (=skewed in favour of lower velocities) in the unloaded test, and the skewness diminished with increasing forces (effect of force P\u00a0<\u00a00.001). That means that lower PVs dominated in the unloaded test and the proportion of higher PVs increased when forces augmented. Thus, in the 20% MVC test, the initial PVs approached a normal distribution. In addition, with increasing forces the PVs as a whole group seem to shift towards higher values, as can be seen in the histograms Fig.\u00a03a\u2013d at time zero. During the tests, the skewness increased again, except for the unloaded test, indicating growing proportion of lower PVs over time and decreasing amount of higher PVs (histograms Fig.\u00a03a\u2013c). The larger the forces the steeper increase of skewness (for all tests together: effect of time P\u00a0<\u00a00.001; interaction between force and time P\u00a0=\u00a00.005; effect of time in unloaded test P\u00a0=\u00a0n.s.; for the tests 5, 10 and 20% MVC interaction between force and time P\u00a0<\u00a00.001). During the last 2\u00a0min of the 5 and 10% MVC tests the Sk-PV stabilized. In the 20% MVC test, however, the positivity still tended to increase up to the end of the test (over the time windows 120\u2013180 and 180\u2013240\u00a0s: effect of time for the 5, 10 and 20% MVC tests, n.s.; interaction between force and time, P\u00a0=\u00a00.106; for the 5 and 10% MVC tests the effect of time, n.s.; for 20% MVC test over 120\u2013180\u00a0s n.s., over 180\u2013240\u00a0s, P\u00a0=\u00a00.052). At the end of the 20% MVC test, the whole population of PVs appeared to shift towards the lower values too, as can be seen at the last two histograms in the Fig.\u00a03d. \nTaken together, in the initial phase of activity, the proportion of fast peaks increased with increasing force. In the prolonged tests loaded up to 10% MVC, the proportion of fast peaks declined again over the first 2\u00a0min and then stabilized at about the level of the unloaded test. During the 20% MVC test, however, the proportion of fast peaks still tended to decline up to the end of the test, accompanied with a growing amount of slow peaks.\nStandard deviation of peak velocities (SD-PV)\nThe averages of SD-PVs of 30 subjects are presented in Fig.\u00a04c. The initial SD-PV was for all force levels similar (P\u00a0=\u00a00.65), which resembled the static tests. The values in the dynamic tests were significantly higher compared with those of respective static tests (paired sample t test for the unloaded, 10 and 20% MVC tests, respectively P\u00a0=\u00a00.014; P\u00a0=\u00a00.027 and P\u00a0=\u00a00.001). During the tests the SD-PV changed significantly with time, depending on the force level (for all tests effect of time P\u00a0=\u00a00.011, interaction between force and time P\u00a0<\u00a00.001). The course of the SD-PV had two phases which were different for the three lower force levels (unloaded, 5 and 10% MVC), compared with 20% MVC. In the three lower force levels, the SD-PV first declined over about 90\u00a0s and then stabilized (interaction between force and time over 0\u2013240\u00a0s P\u00a0=\u00a0n.s.; effect of time over the time windows 0\u201360\u00a0s P\u00a0<\u00a00.001, 60\u2013120\u00a0s P\u00a0=\u00a00.075; 120\u2013180 and 180\u2013240\u00a0s for both P\u00a0>\u00a00.343). However, during the 20% MVC test, the decline, which lasted for approximately 60\u00a0s, was followed by an extreme increase (effect of time over 0\u2013240\u00a0s P\u00a0<\u00a00.001; effect of time over 0\u201360\u00a0s P\u00a0=\u00a00.019; over 60\u2013120\u00a0s, which was in opposite direction, P\u00a0=\u00a00.019, and 120\u2013180 and 180\u2013240\u00a0s P\u00a0<\u00a00.05). This pattern of results can also be seen in the histograms Fig.\u00a03d.\nPeak frequency (PF)\nFigure\u00a04d shows averages of PF over 30 subjects. The initial PF rose with increasing force levels (effect of force P\u00a0<\u00a00.001). Then, during the tests at three lowest force levels (unloaded, 5 and 10% MVC) the PF remained stable. But during the 20% MVC test the PF significantly diminished, at the beginning gradually and from about 120\u00a0s steeply (interaction between force and time for all the four tests, P\u00a0<\u00a00.001; for the three lowest force levels, P\u00a0=\u00a00.234; effect of time for the three lowest force levels P\u00a0=\u00a00.541; effect of time for 20% MVC P\u00a0<\u00a00.001). There was much variability among subjects in the size of decline in 20% MVC test. For those who were able to complete the test, the PF continued to decline up to the end, with exception of one subject in whom the PF increased instead. At 240th\u00a0s the PF of the continuing subjects was reduced by \u221235 (\u221278 to +3)%.\nDiscussion\nChanges in the distribution of MUP velocities as an effect of (low) force and duration were described with four parameters: (1) the global parameter of mean CV, (2) the within subject skewness of a population of MUP velocities; (3) the within-subject standard deviation of MUP velocities and (4) the amount of MUP activity, expressed as MUP frequency. First we will comment on the four parameters. Next, using these parameters, we will discuss the main findings.\nThe four parameters\nThe CV parameter renders a mean value of the motor unit potentials\u2019 propagation velocities. The CV will increase or decrease, depending on the type of the activated (fast-twitch and slow-twitch) motor units. It will also change with the alterations in muscle membrane potential, which influences the depolarisation\/repolarisation processes. For example, it decreases in muscular fatigue (Stalberg 1966; Milner-Brown and Miller 1986), and increases with a smaller interstimulus interval, such as that due to the rising rate coding (Gydikov and Christova 1984; Radicheva et al. 1986; Nishizono et al. 1989). Because of the lack of studies, it is not possible to compare the CV values with those of any other position tasks experiments. However, the estimates are consistent with those of the studies using static and dynamic force tasks, especially with those of Lange et al. (2002), obtained with the inter-peak latency method.\nSkewness is used as an sEMG parameter in the present study for the first time. This statistical measure of deviation from a normal distribution, in this case expresses the proportion between slower and faster MUPs within an individual. It will increase with the growing proportion of slow\/tonic\/fatigue resistant MUs and will decrease with the augmenting proportion of fast\/phasic\/fatigable MUs. All the estimates were moderately positively skewed, which indicates a relative excess of lower MUP velocities.\nThe within-subject standard deviation of MUP velocities, a variable introduced by Lange et al. (2002) shows the spread of MUP velocities. For a fresh and healthy muscle, it will render information about diversity of the participating MUs. In a fatigued muscle, when membrane propagation is slowing, the SD-PV will broaden as a result of the temporal dispersion of velocities. Further, the SD-PV can be expected to narrow when the same velocities repeat, such as in a higher discharge rate of a certain group of MUs. In the brief static position tasks, the standard deviations were larger than those previously estimated by Lange and colleagues (2002) in the staticforce tasks, with respectively 0.55\u20130.62 and 0.3\u20130.52\u00a0m\u00a0s\u22121. This difference can be due to the different type of tasks, as data are available suggesting that different excitatory\/inhibitory inputs to the motor neurons play a part in the position tasks and the force tasks (Rudroff et al. 2005). \nIn the present study, the SD-PVs of the dynamic tests were larger than those of the static tests. This difference can have different explanations. With every contraction of a dynamic cycle, the muscle fibres\u2019 diameter increases, leading to higher fibre propagation velocities (in a part of a cycle) (Arendt-Nielsen et al.1992). This problem was partially restrained by using a small movement angle of 45\u00b0. However, the most important role in the increase of SD-PVs\u2019 during dynamic contractions might be played by the cyclic changes in the motor units\u2019 discharge characteristics. Several studies deliver the supporting data. For example, during dynamic contractions the amount of activity differs between the concentric and eccentric phases, suggesting different regulatory strategies for the two phases (Potvin 1997). Previous studies have also shown that the rate of MU discharge is related to the movement\u2019s velocity, and the (angle) velocity alters depending on the elbow angle (Gillis 1972; Milner-Brown et al. 1973a; Potvin 1997). Thus, the discharge rates will alter through a cycle. In addition, eccentric movements are shown to further the activation of high-threshold (fast propagating) motor units (Komi and Tesch 1979; Nardone et al. 1989).\nPeak frequency (MUP frequency) expresses the amount of MUPs in a time. To our knowledge, it is used as sEMG parameter in the present study for the first time. The PF is comparable with the zero crossing parameter (Lynn 1979; Masuda et al. 1982; Hagg 1981). It is argued that the diminishing zero crossings\u2019 number during prolonged exercises indicates decrease in MUs\u2019 activity, as a sign of fatigue (Inbar et al 1986; Hagg and Suurk\u00fcla 1991). Lange et al. (2002) mentioned a number of MUPs obtained during 1.5\u00a0s measurements in static force tasks, which renders the frequency of about 4\u20135 MUPs\/s for 10% MVC test, and about 8 MUPs\/s for 20% MVC test. These values are much lower than ours with 37, 47 and 50 MUPs\/s (for respectively unloaded, loaded 10 and 20% MVC tests) in static position tasks. The findings are consistent with the interpretation that during position tasks more motor units are being recruited compared with force tasks, and the discharge rate of MUs is higher (Mottram et al. 2005a).\nThe initial changes on increasing force levels (in the static and dynamic tests)\nThe effects of force on the behaviour of MUPs in the short static tests and the initial phase of the prolonged dynamic tests were similar. With increasing forces the CV grew higher and MUP frequency increased. In the population of MUP velocities, not only the proportion of fast MUPs increased (see the skewness in Fig.\u00a02b), but also the velocities as a whole shifted towards higher values (histograms in Figs.\u00a01, 3a\u2013d at time zero). Despite of the changes in the skewness, the standard deviation of MUP velocities remained unaltered.\nThe increases of the CV with increasing force are in accordance with the previous findings inforce tasks (Naeije and Zorn 1983; Broman et al. 1985; Sadoyama and Masuda 1987; Zwarts and Arendt-Nielsen 1988; Lange et al. 2002). It is generally accepted that these increases are caused by activating high threshold\/fast\/phasic motor units when demands of force are augmented (Henneman et al. 1965; Milner-Brown et al. 1973b; Gantchev et al. 1992; Gazzoni et al. 2001). This explanation was supported by the increasing proportion of fast MUPs found in the present study. However, the global shift of MUP velocities towards higher values may be induced by either replacing slow MUs by fast ones, or by increasing the propagation velocity of the muscle membrane due to the rising rate coding (Radicheva et al. 1986; Nishizono et al. 1989; Van der Hoeven and Lange 1994). Little is known about changes in the within-subject standard deviation of MUP velocities. Only Lange and colleagues (2002) mentioned (inforce tasks), contrary to our results, increases between 10 and 50% MVC (and no increases between 50 and 100%). The experiments of Lange et al. and the present short static experiments were both isometric, and the duration of the contraction did not differ much (our 3.8 vs. their 1.5\u00a0s). In fact, the two studies only differed in the type of task (position tasks applied by us vs. force tasks by Lange et al.). This task difference may play a part in the discrepancy of the standard deviation, as the excitatory and inhibitory inputs for the two tasks are supposed to be different (Rudroff et al. 2005). Thus, for the two tasks different types of motor units (with different velocities) may be activated.\nIn short, increases of mean CV with increasing forces in the initial phase of muscle activity may be a result of both recruitment of fast\/phasic motor units and faster membrane propagation.\nChanges in the prolonged dynamic tests\nTests loaded below 20% MVC\nThe main feature of the prolonged tests loaded 5 and 10% MVC were changes in the CV, skewness and standard deviation over the first 90\u2013120\u00a0s, followed by stabilizing. Thus, the CV first declined (with steeper decline for higher forces) and then stabilized at approximately the level of the unloaded test (Fig.\u00a04a). The MUP velocities, which at the beginning of tests were shifted towards the higher values with increasing forces, re-shifted over the tests back to the lower values (skewness variable in Fig.\u00a04b and histograms in Fig.\u00a03a\u2013c). Subsequently, the MUP velocities stabilized nearly at the level of the unloaded test too. The standard deviation narrowed first and later on stabilized at a new level (Fig.\u00a04c). The MUP frequency held steady over these tests at the primary level determined by the used force (Fig.\u00a04d). We suggest that this pattern of results may reflect an emerging equilibrium between phasic and tonic MU activity. \nNo comparison is possible between the parameters used here and those in any other prolonged position tasks study. The decline of CV during contractions at low forces was in contradiction with the studies in static force tasks, which reported increases during sustained isometric contractions at forces of 10\u201325% MVC (Zwarts and Arendt-Nielsen 1988; Arendt-Nielsen et al. 1989; Krogh-Lund and Jorgensen 1991; Krogh-Lund and Jorgensen 1992; Krogh-Lund 1993). This discrepancy may be caused by either\/or both the different contraction types (static vs. dynamic) or different tasks (force vs. position tasks). Increases of the CV in prolonged isometric contractions at low force levels are supposed to be due to the recruitment of fast (anaerobic) MUs in response to the hindered blood flow (Crenshaw et al. 1997; Zwarts et al 1987). In the dynamic conditions, the blood supply is assumed to be undisturbed, so the aerobic MUs can be activated. The decline of the CV followed by stability, along with the changes in the skewness, suggest that, within the cyclically fluctuating activity, the global amount of initially recruited fast\/fatigable\/anaerobic MUs may successively diminish and the proportion of slow\/fatigue resistant\/aerobic MUs may augment. The maintaining activity of slow MUs is in accordance with the hypothesis that tonic\/fatigue resistant (aerobic) MUs remain active through the whole muscular action (Grimby and Hannerz 1968; Hagg and Suurkula 1991). \nOn the other hand, the position character of the present tasks may have contributed to the discrepancy between the decline of CV in the present experiments and increases in previous studies, as firstly, the discharge characteristics of the same motor unit differ between the force and position tasks, and secondly, motor units show greater discharge adaptation during the position tasks (Mottram et al. 2005a, b; MacGilles et al. 2003). The evolution of the standard deviation parameter, with its narrowing followed by stabilizing, fits in with the idea that the rate coding may temporarily increase and consecutively adapt, resulting in a new balance.\nThe MUP frequency variable, expressing the amount of MU activity produced as a result of recruitment and rate coding did not change during these tests. This suggests that all the changes in recruitment and rate coding do not, in principle, affect the total amount of MU activity. \nAll subjects were able to complete the tests, and the sEMG parameters became stable in the course of time as well. Thus, one can assume that the three tests at lowest force levels were non-fatiguing. Taken together, the results of these apparently non-fatiguing dynamic position tasks suggest that following the initially increased activation of fast MUs, the proportion shifts after about 2\u00a0min in favour of slower MUs. The amount of activity seems to remain stable throughout the duration of the tests.\nThe test at 20% MVC\nThe changes encountered during the prolonged test at 20% MVC differed clearly from those at lower forces (Fig. 4). During the 20% MVC test, the CV dropped below the level of the unloaded test. At the same time the proportion of low MUP velocities increased (skewness increased), and finally the velocities\u2019 population made a global move from higher towards lower values, while their standard deviation broadened clearly. The MUP frequency, in contrast with that of non-fatiguing tests, progressively diminished (histogram in Figs.\u00a03d, 4d). \nThe CV\u2019s decreases were in accordance with those shown by Farina et al. (2004) during fatiguing dynamic force tasks of vastus medialis muscle. The behaviour of the skewness and standard deviation suggests that a large majority of the MUPs became extremely slow at the end. This general slowness best matches the slowing of the muscle membrane propagation, which is generally accepted as sign of muscular fatigue (Milner-Brown et al. 1986; Miller et al. 1987). The diminishing MUP frequency can be due to the synchronisation of discharges (Datta and Stephens 1990; Semler and Nordstorm 1999) and diminishing motor unit activity (Hagg 1981; Hagg and Suurkulla 1991). The non-linear decline pattern of MUP frequency, first slow and later on steep, suggests successively exhausting available MUs\u2019 reserves, resulting in lower firing frequencies and failing recruitment. Accordingly, many subjects reported fatigue and stopped exercising. \nIn short, during these apparently fatiguing dynamic position tasks, a global slowing of MUP velocities appears, suggesting a fatigued muscle membrane. The amount of MU activity seems to diminish progressively and finally the recruitment stops.\nSome aspects of the method must be explained. The load was applied to the palm, whereas the MVC was assessed from the wrist. By applying load to the palm, we intended to mimic natural circumstances, such as holding something in the hand. Assessment of the MVC from the palm was not feasible, however, due to the relative weakness of the wrist\u2019s flexors compared with the elbow flexors, which influenced the estimates. A reasonable alternative was to measure the MCV from the wrist. Distances measured over the forearm and the palm (Table\u00a01) enabled calculation of the real exerted load torque, which was about 20\u201325% larger than the used one. The MVC was assessed at the elbow angle of 90\u00b0 despite performing the tests at the angle of 135\u00b0. The reason was that, when applying the dynamometer at the wrist with high forces, the elbow angle being at 135\u00b0, subjects tended to overstretch the wrist and experienced pain. This was not the case at the 90\u00b0 angle. The MVC values at 135 and 90\u00b0 were similar, which was consistent with the findings of Philippou et al. (2004), so it was chosen for assessments at 90\u00b0.\nIn conclusion, we present a set of parameters derived from the interpeak latency method, which yields information about changes in MUP velocities\u2019 distribution and amount of MUP activity. Skewness, standard deviation and peak frequency parameters appear to corroborate the results of a global muscle conduction velocity. Together they could contribute to quantifying the dynamics of motor unit activity and membrane fatigue. The interconnected results may be useful in ergonomics (for assessment of fatigue) and in sports (for eliciting specific capabilities, such as explosive or endurance capabilities).","keyphrases":["motor unit potential","dynamic contraction","skewness","position task","surface electromyography","conduction velocity distribution"],"prmu":["P","P","P","P","P","R"]}
{"id":"J_Abnorm_Child_Psychol-3-1-1915644","title":"Multiple Determinants of Externalizing Behavior in 5-Year-Olds: A Longitudinal Model\n","text":"In a community sample of 116 children, assessments of parent-child interaction, parent-child attachment, and various parental, child, and contextual characteristics at 15 and 28 months and at age 5 were used to predict externalizing behavior at age 5, as rated by parents and teachers. Hierarchical multiple regression analysis and path analysis yielded a significant longitudinal model for the prediction of age 5 externalizing behavior, with independent contributions from the following predictors: child sex, partner support reported by the caregiver, disorganized infant-parent attachment at 15 months, child anger proneness at 28 months, and one of the two parent-child interaction factors observed at 28 months, namely negative parent-child interactions. The other, i.e., a lack of effective guidance, predicted externalizing problems only in highly anger-prone children. Furthermore, mediated pathways of influence were found for the parent-child interaction at 15 months (via disorganized attachment) and parental ego-resiliency (via negative parent-child interaction at 28 months).\n\u00a0\nExternalizing behavior problems \u2013 including aggressive, destructive, and delinquent behavior \u2013 represent the most common type of mental health problems in children. Longitudinal studies have shown that the stability of externalizing problems is relatively high from preschool age into adolescence (Moffit, Caspi, Dickson, Silva, & Stanton, 1996; Smith, Calkins, Keane, Anastopoulos, & Shelton, 2004). Moreover, early externalizing problems are predictive of other forms of psychopathology and often interfere with the child\u2019s personal, social and academic development (Campbell, 1995, 2002; Moffitt, 1993). Although developmental psychopathologists now generally assume such problems to have their roots in children\u2019s early developmental histories, early identification of children at serious risk of developing such pathology is hampered by a lack of insight into the determinants leading to later maladaptive outcomes (Tremblay, 2006).\nAlthough various theoretical models of the development of externalizing problems in the very first years of life (Campbell, Shaw, Gilliom, 2000; Moffitt, 1993, Greenberg, 1999; Sameroff & Chandler, 1975) differ in the emphasis they place on the role of certain etiological factors, they agree in the assumption that multiple factors from various domains additively and interactively contribute to the emergence and maintenance of externalizing problems. Taken together, four domains of factors have been proposed to contribute to their development: (a) parent-child interaction and parent-child attachment, (b) child characteristics (e.g., temperament and cognitive abilities), (c) parental characteristics (e.g., personality), and (d) contextual characteristics (e.g., socioeconomic status, partner support, and stressful life events).\nTo date, very few studies \u2013 all focusing on high-risk samples \u2013 have longitudinally examined the joint contribution of the various key factors to the onset of externalizing problems in the very first years of life (Aguilar, Sroufe, Egeland, & Carlson, 2000; Erickson, Sroufe, & Egeland, 1985; Lyons-Ruth, Alpern, & Repacholi, 1993; Shaw, Owens, Vondra, Keenan, & Winslow, 1996). Ours is the first study to assess predictors hypothesized to be the most important from all of the aforementioned domains both in infancy (i.e., at age 15 months) and in toddlerhood (i.e., at age 28 months) to predict the emergence of later externalizing behavior problems (i.e., at age 5 years) in a non high-risk community sample. All of the abovementioned earlier studies of the multiple determinants of externalizing behavior used samples characterized by one or more risk factors. The sample studied by Shaw et\u00a0al. (1996) included only low-SES families. Aguilar et\u00a0al. (2000) and Erickson et\u00a0al. (1985) studied low-SES samples as well, but these were also characterized by high degrees of life stress and a large proportion of single-parent families. And the low-SES sample studied by Lyons-Ruth et\u00a0al. (1993) was characterized by such additional risk factors as a relatively high incidence of a history of maternal psychiatric hospitalization, single parenthood, and child maltreatment. It remains to be seen whether similar results will also be found in studies with community samples. That is mainly because earlier research has shown that the predictive power of risk factors may increase in the presence of other risk factors (Farrington, 1995). Consequently, it is possible that factors that predict externalizing problems in samples with one or more risk factors fail to do so in samples with lower levels of risk, such as the present sample. In light of this, studying the early predictors of externalizing problems in a community sample constitutes an important extension of the existing research evidence collected in high-risk samples.\nThe criteria on which we based our selection of the various predictors and our hypotheses regarding their joint contribution to the development of externalizing problems \u2013 led by various interrelated theoretical models \u2013 are described per domain in the following paragraphs.\nParent-child interaction and parent-child attachment\nFrom a transactional perspective (Sameroff & Chandler, 1975), externalizing problems are assumed to emerge and to stabilize or change in children\u2019s continuous interactions with their immediate environment, particularly in the interactions with their primary caregivers. Repeated observations of parental and child behaviors during parent-child interactions are thus indispensable to gain more insight into the early development of externalizing behavior problems. Multiple dimensions of parental behavior in parent-child interaction have been found to be associated with behavior problems in children at various ages. A lack of positive parenting behaviors, such as the expression of sensitivity, warmth, involvement, acceptance, and positive guidance, was reported to be related to externalizing problems in preschoolers (Brophy & Dunn, 2002; C\u00f4t\u00e9, Vaillancourt, LeBlanc, Nagin, & Tremblay, 2006; Gardner, 1987; Pettit, Bates, & Dodge, 1997). In addition, high levels of negative parental control such as harsh discipline, intrusiveness, negativity, and hostility also proved associated with externalizing problems (Belsky, Woodworth, & Crnic, 1996; Rubin, Burgess, Dwyer, & Hastings, 2003; Campbell, Pierce, Moore, & Marakovitz, 1996). Based on the above evidence, we expected both a lack of positive parenting behaviors and negative parent-child interactions to predict externalizing behavior problems at age 5.\nAnother frequently mentioned risk factor and closely related to the quality of parent-child interaction, is the quality of attachment between infant and caregiver. By the end of the infant\u2019s first year, four main patterns of infant-parent attachment \u2013 one secure and three insecure \u2013 can be distinguished that have been found to reflect the history of the caregiver\u2019s responses to the child\u2019s attachment behaviors (Ainsworth, Blehar, Waters, & Wall, 1978; Main & Solomon, 1986, 1990). Ainsworth and colleagues (1978) originally proposed three \u201corganized\u201d patterns of attachment. Presumably as a result of a history of caregiver sensitive responsiveness to their signals and needs, securely attached (B) infants use the caregiver as a secure base from which to explore the environment. Avoidant (A) infants are characterized by a tendency to minimize their attachment behaviors while under stress in the presence of the caregiver, probably resulting from earlier experiences with a caregiver who tended to reject or ignore the child\u2019s expression of attachment behaviors. Infants with a resistant attachment (C), featuring both clinging and angry behaviors towards the caregiver when under stress, are presumed to maximize the expression of attachment behavior as an adaptation to their caregiver\u2019s inconsistent responsiveness. Main and Solomon (1990) later added a fourth disorganized (D) pattern typified by seemingly contradictory attachment behaviors that is thought to reflect the breakdown or absence of a strategy for the infant to use the caregiver as a secure base in times of stress (Main & Solomon, 1990). Disorganized attachment is thought to ensue from extremely unpredictable or frightening behavior on the part of a maltreating and\/or traumatized parent (cf. Lyons-Ruth & Jacobvitz, 1999).\nWith regard to the predictive value of attachment security versus insecurity for the later occurrence of externalizing problems, the empirical evidence is rather inconsistent. Although attachment insecurity was found to be associated with externalizing problems in high-risk samples as well as in community samples in studies that did not include the disorganized attachment classification (Erickson et\u00a0al., 1985; Fagot & Kavanagh, 1990; Munson, McMahon, & Spieker, 2001; Shaw et\u00a0al., 1996), other studies did not report such a relationship (Bates, Maslin, & Frankel, 1985; Goldberg, Corter, Lojkasek & Minde, 1990). However, in contrast to the other insecure attachment categories, disorganized attachment has consistently been found to be related to the later development of externalizing problems (for reviews, see Lyons-Ruth and Jacobvitz, 1999, and Van IJzendoorn, Schuengel, and Bakermans-Kranenburg, 1999; also see Lyons-Ruth et\u00a0al., 1993; Vondra, Shaw, Swearingen, Cohen, & Owens, 2001).\nIn view of the above, we expected infant attachment insecurity and particularly attachment disorganization to be related to elevated levels of externalizing behavior at age 5. Considering that attachment patterns have been found to reflect the history of parent-child interactions, we expected the association between the early parent-child interaction and later externalizing problems to be at least partially mediated by the quality of the infant-parent attachment.\nChild characteristics\nSeveral child characteristics can prompt the development of externalizing behavior, both directly and indirectly by affecting the quality of parent-child interactions. A considerable body of research has shown associations between externalizing problems and child temperamental characteristics with a crucial role for negative emotionality denoting negative mood, irritability, and high-intensity reactions like anger (for a review, see Sanson, Hemphill, & Smart, 2004). In contrast, temperamental inhibition and fear\/shyness may act as a protective factor as these characteristics have been demonstrated to be negatively associated with later externalizing problems (Lacourse, Nagin, Vitaro, C\u00f4t\u00e9, Arsenault, & Tremblay, 2006; Sanson, Oberklaid, Prior, Amos, & Smart, 1996; Schwartz, Snidman, & Kagan, 1996). Nevertheless, the relationship between child temperament and externalizing behavior is not always clear or direct. Etiological models of externalizing behavior increasingly propose that children with temperamental difficulties are more vulnerable to negative rearing influences than children without such difficulties (Belsky, 1997; Moffitt, 1993). Several recent studies have substantiated this latter assumption (Bates, Pettit, Dodge, & Ridge, 1998; Belsky, Hsieh, & Crnic, 1998; Hemphill & Sanson, 2001; Lacourse et\u00a0al., 2006; Leve, Kim, & Pears, 2005). In line with these findings, we anticipated high levels of child negativity and low levels of fearfulness in infancy and toddlerhood, particularly in interaction with parenting, to predict the development of externalizing problems.\nAnother contributory child characteristic is low cognitive or linguistic ability (Burt, Hay, Pawlby, Harold, & Sharp, 2004; Dekker, Koot, Van der Ende, & Verhulst, 2002; Moffitt, 1993). Therefore, we hypothesized that child cognitive ability at 15\u00a0months would be negatively related to the occurrence of externalizing problems at age 5 years.\nBoys and girls have been found equally likely to exhibit externalizing behavior problems up until the age of about four years. By school entry, however, boys tend to exhibit up to 10 times the rate of externalizing problems for girls (C\u00f4t\u00e9, et\u00a0al, 2006; Mesman, Bongers, & Koot, 2001; Rubin et\u00a0al., 2003). Accordingly, at age 5 we expected the boys in our sample to score higher on externalizing behavior than the girls.\nParental characteristics\nLittle research has focused on parental personality as a potential predictor of externalizing problems in children. The available studies only focused on aspects of parental psychopathology like antisocial personality (Shaw, Vondra, Hommerding, Keenan, & Dunn, 1994) or maternal depression (Cummings & Davies, 1994; Shaw, Gilliom, Ingoldsby, & Nagin, 2003). Less attention has been paid to the effects of more or less adaptive parental characteristics, which is why we included parental ego-resiliency as a potential predictor of externalizing problems. Block and Block (1980) defined ego-resiliency as a \u201cresourceful adaptation to changing circumstances,\u201d \u201cflexible invocation of the available repertoire of problem-solving strategies,\u201d and \u201cthe ability to maintain integrated performance while under stress\u201d (p. 48). As such, ego-resilient individuals should be particularly well-equipped to cope with the often stressful task of parenting. And \u2013 as outlined before \u2013 higher quality parenting is hypothesized to predict lower levels of externalizing behavior in children. In the 15-month assessment (Van Bakel & Riksen-Walraven, 2002a), parental ego-resiliency was found to be related to the infants\u2019 socioemotional development, partially via the quality of the parent-infant interaction. Based on the above and in line with the 15-month findings, we expected higher levels of parental ego-resiliency to predict lower levels of externalizing behavior in the children at age 5, and we expected this relation to be mediated by lower quality parent-child interactions.\nContextual characteristics\nBesides parental and child attributes, specific characteristics of the social and economic context in which the parent-child interaction is embedded may also contribute to the development of externalizing problems (Belsky, 1984; Greenberg, Speltz, & DeKlyen, 1993). We included three characteristics of the child-rearing context that earlier studies have shown to be associated with externalizing problems: (1) high incidence of stressful life events (Aguilar et\u00a0al., 2000), (2) a lack of partner support (Jouriles, Murphy, Farris, Smith, Richters, & Waters, 1991; Shaw et\u00a0al., 1996), and (3) low socio-economic status (SES; C\u00f4t\u00e9, et\u00a0al, 2006; Shaw et\u00a0al., 1996). It needs to be stressed, though, that in previous research the effects of sociodemographic factors like SES proved modest compared to the effects of parental or child risk factors (for a review, see Loeber and Dishion, 1983; also see Shaw et\u00a0al., 1996). Nevertheless, based on earlier findings, we expected stressful life events, partner support, and SES to contribute to the development of externalizing behavior problems.\nIn sum, the aim of the present study was to predict the occurrence of externalizing behavior problems in a sample of 5-year-old children on the basis of various parental, child, dyadic, and contextual factors assessed at 15 and 28 months and at age 5. We expected the following specific factors to independently or interactively contribute to the development of externalizing problems: (a) a low quality of parent-child interaction at 15 and 28 months; (b) disorganized parent-infant attachment; (c) child temperament (i.e., high anger proneness and low fearfulness) at both 15 and 28 months and particularly in interaction with low quality parenting; (d) low levels of child cognitive ability at 15\u00a0months; (e) low parental ego-resiliency; (f) high incidence of stressful life events between 15\u00a0months and 5\u00a0years; (g) lack of partner support; and (h) low SES. Furthermore, the contribution of parental ego-resiliency to child externalizing behavior was expected to be mediated by the quality of parent-child interaction, and the contribution of the early parent-child interaction by the quality of infant-parent attachment.\nMethod\nParticipants\nThe original 15-month sample (M\u00a0=\u00a015.1; SD\u00a0=\u00a00.25) consisted of 129 healthy children (67 boys, 62 girls) and their primary caregivers. The children were recruited on the basis of the records from health-care centers in the Dutch city of Nijmegen. During nine consecutive months, all families (n\u00a0=\u00a0639) with a 15-month-old baby from various socioeconomic backgrounds were sent a recruitment letter with a brief description of the research procedures and an invitation to participate in this study that aimed to \u201cgain more insight into children\u2019s development in the first years of life\u201d. If they met the two specified eligibility criteria (i.e., sufficient fluency in Dutch and child without serious health problems) and were interested in participation, they were requested to return a response card. Of the 174 families who responded, 129 parent-child dyads (the maximum attainable given the time and resources available for the project) were randomly selected for the study. Of the infants, 73 were first-borns and 56 had one or more older siblings. The sample included 123 two-parent families and six single-parent families. In three families, the father was the primary caregiver and in the remainder of the families this was the mother. The percentages of single parents and fathers acting as primary caregivers are representative of families in the Netherlands with children in this age group. The primary caregivers were between the ages of 22 and 47 years at the time of recruitment. The level of education for the primary caregivers was indicated on a seven-point scale from low (elementary school) to high (college degree or more) with a mean of 4.95 (SD\u00a0=\u00a01.77); the category equivalent of 5 is high school degree. Of the 129 15-month-olds, 114 children (61 boys, 53 girls) participated in the second wave of measurements at age 28 months (M\u00a0=\u00a028.3; SD\u00a0=\u00a00.30) and 116 (62 boys, 54 girls) in the third assessment at age 5 years (M\u00a0=\u00a063.6 months; SD\u00a0=\u00a01.16). At this third assessment, there were 107 two-parent families, nine single-parent families, and again three families with the father as the primary caregiver.1\nTo determine whether there was selective drop-out or not, the 116 families participating in the 5-year assessment were compared to the 13 families that did not take part. Independent t-tests for all the major 15-month study variables revealed a significant difference for parental ego-resiliency only, t(126)\u00a0=\u00a02.08, p\u00a0<\u00a0.05, with a tendency for primary caregivers with lower levels of ego-resiliency to drop out.\nProcedure\nThe 15-month data were available from an earlier study focusing on the determinants of parenting and infant development (Van Bakel & Riksen-Walraven, 2002a). The 15-month assessment involved data collection during a single home visit and a single visit of the primary caregiver and infant to the research center. During the home visit, the primary caregiver completed a Q-sort and a set of questionnaires assessing child temperament, parental ego-resiliency, partner support, and additional background information. Next, the parent-child interaction was videotaped during the performance of four instructional tasks lasting three to four minutes each. At the subsequent assessment at the research center, the child\u2019s cognitive ability was assessed and the quality of\u2009the infant-parent attachment with an abbreviated version of the Strange Situation (Ainsworth et\u00a0al., 1978). For a more detailed description of the 15-month data collection, see Van Bakel & Riksen-Walraven (2002a, 2002b)\nThe 28-month assessment was conducted during a single home visit. The primary caregiver was interviewed about stressful life events since the first assessment and asked to complete a questionnaire to assess child temperament. The parent-child interaction was videotaped during four instructional tasks comparable to those performed at 15\u00a0months.\nAt the age-5 assessment, the primary caregiver was interviewed at home about stressful life events since the previous assessment and asked to complete a questionnaire to assess child behavior problems. The child\u2019s teacher completed a questionnaire to assess any behavior problems and asked to return this by mail, which was done by all but one teacher.\nInstruments and measures\nQuality of parent-child interaction (at 15 and 28\u00a0months)\nThe videotaped parent-child interaction episodes were rated using five 7-point scales (Erickson et\u00a0al., 1985) to assess the quality of parental interactive behavior: (1) supportive presence or the provision of emotional support; (2) respect for the child\u2019s autonomy or nonintrusiveness; (3) effective structure and limit setting; (4) quality of instructions; and (5) hostility. Subsequently, child interactive behavior was rated on four 7-point scales (Erickson, et\u00a0al., 1985): (I) negativity or anger, dislike, and hostility towards the parent; (II) avoidance of interaction with the parent; (III) compliance to suggestions and directions given by the parent; and (IV) positive affect expressed towards the parent. Each interaction episode at 15\u00a0months was rated independently by two trained observers and each interaction episode at 28 months by four independent observers. Based on 25 cases (19%) for the 15-month assessment and 20 cases (18%) for the 28-month assessment, the interrater reliabilities expressed as adjusted kappas were all above 0.83. Evidence for the validity of the scales has been provided in various studies (see Van Bakel & Riksen-Walraven, 2000a, for a review).\nTo obtain robust dyadic measures for parent-child interaction, the ratings of parental and child interactive behavior were factor analyzed together, for the 15- and 28-month assessment separately, using varimax rotation. Scree plots of eigenvalues indicated the emergence of two clear and comparable factors at both ages. The two factors explained 68% of the variance in parent-child interaction at 15\u00a0months, and 71% of the variance at 28 months. The first factor, labeled effective guidance, was marked by high loadings on the following parent-child interaction variables (factor loadings for 15 and 28\u00a0months within parenthesis): effective structure and limit setting (.89; .95), high quality of instructions (.62; .76), and supportive presence (.79; .48) on the part of the parents; and compliance (.53; .84) and low avoidance (\u2212.52; \u2212.65) on the part of the child. The second factor, labeled negative interaction, was characterized by high loadings on the following variables (factor loadings for 15 and 28\u00a0months within parenthesis): parental hostility (.74; .85); low respect (\u2212.63; \u2212.78); child negativity (.85; .67); and an increasing lack of positive affect in both partners as evident from low parental supportive presence (\u2212.48; \u2212.77) and low child positive affect (\u2212.35; \u2212.54).\nQuality of parent-child attachment (at 15 months)\nAn abbreviated version of the Strange Situation Procedure (Ainsworth et\u00a0al., 1978), i.e., including one as opposed to two separation-reunion situations, was used to assess the quality of infant-parent attachment. A similarly abbreviated version of the Strange Situation has been found to be valid for the assessment of attachment quality both within normal (Lewis, Feiring, McGuffog, & Jaskir, 1984; Waters, Wippman, & Sroufe, 1979) and within clinical samples (Willemsen-Swinkels, Bakermans-Kranenburg, Buitelaar, Van IJzendoorn, & Van Engeland, 2000). Two trained coders (the second author and D.C van den Boom from the University of Amsterdam) rated the videotaped separation-reunion episodes and classified the infants as Secure (B), Avoidant (A), Resistant (C), or Disorganized\/ disoriented (D) consistent with the directions provided by Ainsworth et\u00a0al. (1978) and by Main & Solomon (1990). Intercoder reliability on 20 cases (16%) was adequate, with 95% agreement on the main classifications.\nChild temperament (at 15 and 28 months)\nChild temperament was evaluated using the Toddler Behavior Assessment Questionnaire (TBAQ; Goldsmith, 1994) The TBAQ comprises 111 items, organized in five 7-point scales. For the present study, two scales were used that have earlier been found associated with externalizing problems, i.e., Anger proneness (28 items, Cronbach\u2019s alpha .89 and .88 for 15 and 28\u00a0months, respectively) and Social fear (19 items, Cronbach\u2019s alpha .77 and .84 for 15 and 28\u00a0months, respectively).\nCognitive ability (at 15 months)\nA Dutch version of the Bayley (1969) Mental Scale of Infant Development (Van der Meulen & Smrkovsky, 1983) was used to assess the child\u2019s level of cognitive functioning at 15\u00a0months, expressed in the standardized Mental Developmental Index (MDI, M\u00a0=\u00a0100; SD\u00a0=\u00a015).\nParental ego-resiliency (at 15 months)\nPrimary caregivers rated their own ego-resiliency using a Dutch translation of the California Adult Q-set (CAQ; Block, 1961, 1978) The CAQ consists of 100 descriptive statements that sample a broad domain of personal and interpersonal characteristics and functioning. The primary caregivers were asked to sort each statement into one of nine possible categories ranging from \u201cleast characteristic\u201d to \u201cmost characteristic\u201d in terms of salience for themselves. An ego-resiliency score was then attained by correlating the Q-sort description for each parent with the criterion profile provided by experts for a prototypically ego-resilient person (see Block, 1991). The ego-resiliency scores could range from +1.00 (very ego-resilient) to \u20131.00 (very ego-brittle).\nStressful life events (at 28 months and 5 years)\nA semi-structured interview was used to obtain information regarding stressful life events occurring between the three assessments. The questions\/items were derived from Saranson, Johnson, and Siegel\u2019s (1978) Life Experiences Survey and Coddington\u2019s (1972) Life Events Scale for Children. Both measures have sound psychometric properties and have been widely used in international research (Abela, 2001; Johnston, 1996). Stressful events that were likely to have a negative impact on the child\u2019s development were selected for inclusion (e.g., loss of a loved one, serious physical or mental illness on the part of a parent or the child, divorce). All items required a \u2018yes\u2019 or \u2018no\u2019 response. The total number of stressful life events between 15 and 28\u00a0months and 28 months and age 5 were taken together to constitute the stressful life event scores between 15\u00a0months and age 5.\nPartner support (at 15 months)\nA subscale of a Dutch questionnaire to assess family problems (VGP; Koot, 1997) was used to assess the support the primary caregiver received from the partner. The subscale comprises five statements gauging partner support during child rearing, such as \u201cMy partner supports me in my role as a parent\u201d and \u201cMy partner and I agree about child rearing.\u201d Cronbach\u2019s alpha was 0.82. Single parents were also asked to complete the questionnaire when they were still in contact with the other parent or were living with a new partner. Otherwise, they were assigned a minimum score. Evidence supporting the validity of the subscale has been reported in various studies (Van Bakel & Riksen-Walraven, 2002a; Van Zeijl et\u00a0al., 2006).\nSocioeconomic status (SES at 15 months)\nSES scores were assigned on the basis of the level of education (along a 7-point scale) and level of occupation (along a 6-point scale) for both parents. SES scores based on education and occupation of both parents have frequently been used in other studies (e.g., Shaw et\u00a0al., 1996). The levels of education and occupation for the two parents were first standardized and then summed to derive a single SES score. For single parents, the level of education and occupation for the primary caregiver were summed to compute the SES score (cf. Shaw et\u00a0al., 1996).\nExternalizing problems as rated by parents and teachers (5 years)\nTo assess externalizing problems at age 5, the parents completed the Dutch version of the Child Behavior Checklist for ages 4\u201318 (CBCL\/4\u201318; Achenbach, 1991a; Verhulst, Van der Ende, & Koot, 1996). The Aggressive behavior subscale (20 items) and the Delinquent behavior subscale (13 items) from the CBCL\/4\u201318 were summed (combined \u03b1\u00a0=\u00a0.86) to determine the externalizing score for the child at 5 years. Teachers completed the teacher version of the CBCL (TRF, Achenbach 1991b); the externalizing score was based on the scores for the subscales Aggressive behavior (25 items) and Delinquent behavior (9 items) which were summed (combined \u03b1\u00a0=\u00a0.94) to derive an externalizing score for each child.\nResults\nResults are presented in two sections. In the first section we report the results of preliminary analyses, i.e., the distributions of the various predictor and outcome variables as well as their intercorrelations, and the parental and teacher ratings of the children\u2019s externalizing behaviors are compared and related to each other. The second section reports the main results of the study, namely the prediction of externalizing behavior at age 5 from the various predictors at earlier ages. First, hierarchical regression analysis is conducted to estimate the unique and interactive contributions of the multiple predictors to the variance in Externalizing scores at age 5. Next, the hypothesized mediated pathways are tested. Finally, path analysis using AMOS 5.0 (Arbuckle, 2003) is applied to test the complete longitudinal model comprising all the direct, moderated, and mediated pathways of influence between the predictors at the various ages and age 5 externalizing behavior.\nPreliminary analyses\nTable 1 presents the means and standard deviations for the study variables as well as their intercorrelations. The distribution of children across attachment categories (64% B, 14% A, 8% C, and 14% D) did not differ from the distribution reported by Van IJzendoorn et\u00a0al. (1999) for \u201cnormal\u201d, i.e., middle-class, non-clinical groups in North America. The scores for child cognitive ability were normally distributed, with a mean score close to the population mean of M\u00a0=\u00a0100. The distribution of the scores for ego-resiliency was in line with the findings of earlier studies and the distribution of scores for partner support was mildly to moderately skewed to the left but showed sufficient variation. Table 1 also shows significant stability for the two parent-child interaction measures as well as for child social fear and anger proneness from 15 to 28\u00a0months. For the predictor variables, no differences were found between boys and girls.\nTable 1Intercorrelations, means and standard deviations for the study variables1234567891011121314151617181915-month predictors1. Effective guidance\u20132. Negative interaction.00\u20133. B vs. non-B attachmenta.38**-.46**\u20134. A vs. non-A attachmenta\u2212.27**.22*\u2212.55**\u20135. C vs. non-C attachmenta\u2212.05.11\u2212.40**\u2212.12\u20136. D vs. non-D attachmenta\u2212.22*.33**\u2212.53**\u2212.16\u2212.12\u20137. Child social fear.42**-.03.19*\u2212.16\u2212.06\u2212.05\u20138. Child anger proneness.04.02.02.06\u2212.00\u2212.09.35**\u20139. Child cognitive ability.31**\u2212.09.09\u2212.07.05\u2212.08.18.12\u201310. Child sex.10\u2212.01.10.03\u2212.15\u2212.05.08.07.15\u201311. Parental ego-resiliency.23*\u2212.07.17.07\u2212.28**\u2212.09\u2212.00\u2212.07.19*\u2212.05\u201312. Partner support.16\u2212.03.16.02\u2212.21*\u2212.07\u2212.06\u2212.01.12.05.17\u201313. SES.05\u2212.17\u2212.02.02.10\u2212.07.11\u2212.02.14\u2212.02.17\u2212.07\u201328-month predictors14. Effective guidance.32**\u2212.06.21*\u2212.23*.15\u2212.18.25*.10.41**.04.09\u2212.07.19\u201315. Negative interaction\u2212.09.63**\u2212.34**.11\u2212.05.41**\u2212.11\u2212.02\u2212.07\u2212.08\u2212.28**\u2212.10\u2212.29**.00\u201316. Child social fear.21*.14\u2212.04\u2212.15.10.13.47**.16.03.15\u2212.15\u2212.17.05.17.02\u201317. Child anger proneness\u2212.01\u2212.05\u2212.02.15.01\u2212.13.23*.56**.09.07\u2212.02\u2212.15.08\u2212.05\u2212.15.14\u20135-year predictor18. Life events 15 mo-5 yr.02.19*\u2212.06\u2212.15.10.16\u2212.04.12\u2212.12\u2212.03\u2212.25*\u2212.35**\u2212.10.10.19.16.22*\u20135-year outcome19. Externalizing behavior\u2212.22*.31**\u2212.33**\u2212.02.05.45**\u2212.18\u2212.02\u2212.09\u2212.25**\u2212.25**\u2212.27**\u2212.18\u2212.17.51**\u2212.11.11.23*\u2013M0.00b0.00b0.640.140.080.143.683.38103.900.470.448.320.00c0.00b0.00b3.383.611.480.00cSD1.001.000.480.350.270.340.850.7216.930.500.192.052.811.001.000.800.721.311.73Note. N\u00a0=\u00a0105\u2013111.aAttachment was dummy coded as B (=1) vs. ACD (=0); A (=1) vs. BCD (=0); C (=1) vs. ABD (=0); and D (=1) vs. ABC (=0).bThe parent-child interaction variables are factor scores with a mean score of 0.00 and a standard deviation of 1.00.cSES and Externalizing behavior are composed variables which were first standardized and then summed.*p\u00a0<\u00a0.05. **p\u00a0<\u00a0.01Table 2Hierarchical regression results for predicting age 5 externalizing scores (Total Model)Hierarchical regression resultsBlockBSE B\u03b2\u0394R2(%)1D versus non-D attachmenta1.880.42.36**22.2Negative interaction 28 months0.650.15.35**10.7Sex (male)b0.610.26.18*4.3Anger proneness 28 months0.470.18.20**4.4cPartner support\u22120.140.06\u2212.17*2.62Interaction termsEffective guidance \u00d7 Anger proneness 28 months\u22120.330.14\u2212.19**3.3R2 final model =47.6%F(6,94) final model =14.21**aD vs. non-D coded as D\u00a0=\u00a01 and non-D\u00a0=\u00a00.bSex was coded as 0 (girls) or 1 (boys).cAlthough anger proneness was not significantly correlated with externalizing behavior, it had a significant Beta weight. This effect seems due to a suppressor variable. As recommended by Tabachnick & Fidell (1989), predictors were systematically deleted from the regression equation to identify which variable is the suppressor, indicating that D versus non-D attachment acted as the suppressor variable.*p\u00a0<\u00a0.05. **p\u00a0<\u00a0.01.\nThe mean score on the Externalizing scale was 9.97 (SD\u00a0=\u00a06.35; N\u00a0=\u00a0111) for the CBCL\/4\u201318 with 22% (n\u00a0=\u00a024) of the children scoring in the clinical range (T-score of \u226563; Achenbach, 1991a). For the TRF, the mean score was 7.50 (SD\u00a0=\u00a09.10; N\u00a0=\u00a0111) with 16% (n\u00a0=\u00a018) of the children scoring in the clinical range (T \u2265 63; Achenbach, 1991b). Both the CBCL and TRF Externalizing scores were significantly higher than those reported for 5-year-olds in the Dutch norm population for the CBCL\/4\u201318 (M\u00a0=\u00a08.23, SD\u00a0=\u00a06.37; Verhulst et\u00a0al., 1996) and the TRF (M\u00a0=\u00a04.99, SD\u00a0=\u00a06.58; Verhulst, Van der Ende, & Koot, 1997), t (185)\u00a0=\u00a02.26, p\u00a0<\u00a0.05 for the CBCL\/4\u201318, and t (185)\u00a0=\u00a02.51, p\u00a0<\u00a0.05 for the TRF. A paired-samples t-test showed parents to report significantly more externalizing behaviors than school teachers, t (110)\u00a0=\u00a03.24, p\u00a0<\u00a0.01. The correlation between the CBCL and TRF scores was .51, p\u00a0<\u00a0.001. To obtain a robust measure of externalizing behavior, a composite Total Externalizing score was computed by first standardizing and then summing the children\u2019s Externalizing scores on the CBCL and TRF. For sex differences on this measure, a t-test revealed a significantly higher score for boys on Total Externalizing (Mboys\u00a0=\u00a00.41, SD\u00a0=\u00a01.93; Mgirls\u00a0=\u00a0\u22120.44, SD\u00a0=\u00a01.35, t (109)\u00a0=\u00a02.70, p\u00a0<\u00a0.01).\nPredicting age 5 externalizing scores\nHierarchical regression analysis\nTo examine the unique and interactive contributions of the predictors to the variance in Externalizing scores at age 5, hierarchical multiple regression analysis was conducted with Total Externalizing as the dependent variable and the predictors at the different ages as the independent variables. The predictors were entered in the first block. In the second block, the interaction terms hypothesized in the introduction to predict Externalizing scores were entered, i.e., child temperament (2 measures) \u00d7 parent-child interaction (2 measures) at both 15 and 28\u00a0months, and child cognitive development\u00a0\u00d7 parent-child interaction (2 measures) at 15\u00a0months. The interaction terms were computed according to the procedure recommended by Aiken and West (1991). To limit the number of interaction terms in the regression analysis, only those interaction terms that significantly predicted externalizing behavior at age 5 when examined in isolation were included in the regression analysis (see also Leve et\u00a0al., 2005), i.e., Effective guidance \u00d7 Anger proneness at 28 months and Negative interaction \u00d7 Anger proneness at 28 months. Table 2 presents the results for the final model.\nThe overall model proved significant and explained 47.6% of the variance in the Total Externalizing scores. In the first block, D versus non-D attachment at 15\u00a0months explained a significant 22% of the variance in the Total Externalizing scores at age 5. Negative interactions at 28\u00a0months explained an additional 11% of the variance, child sex accounted for another 4%, just like anger proneness at 28 months, and partner support at 15\u00a0months contributed another 3% to the regression equation. In the second block, Effective guidance \u00d7 Anger proneness at 28 months explained another 3% of the variance in Total Externalizing scores at age 5.\nFig. 1Child externalizing behavior scores in relation to effective guidance for children high and low on anger-proneness\nTo examine the nature of the interaction effect of effective guidance and anger proneness at 28 months on externalizing behavior at age 5, the relationship between effective guidance and externalizing behavior was determined for children with high (1 SD above the mean) and low (1 SD below the mean) levels of anger proneness, following the procedures of Aiken and West (1991). The regression lines for high and low anger prone children are plotted in Fig. 1. The simple slope of effective guidance was significant at high levels of anger proneness (B\u00a0=\u00a0\u2212.55, t(107)\u00a0=\u00a0\u22122.56, p\u00a0<\u00a0.05), but not at low levels of anger proneness (B\u00a0=\u00a0.19, t(107)\u00a0=\u00a01.01, ns.). Thus, a lack of effective guidance was associated with more externalizing behavior for highly anger prone children but not for low anger prone children.\nMediational analyses\nNext, the hypothesized mediated pathways between the predictors and age 5 externalizing behavior were tested using regression analysis, according to the procedure outlined by Baron and Kenny (1986). According to this procedure, three conditions must hold in order to establish mediation: (a) the predictor is significantly associated with the outcome, (b) the predictor is significantly associated with the mediator, and (c) the mediator is significantly associated with the outcome. If these conditions hold in the predicted direction, mediation is proven when the effect of the predictor on the outcome is shown to decrease when the mediator is also entered as a predictor in the regression equation.\nFirst, we tested whether D versus non-D attachment mediated the effect of the parent-child interaction (two measures) at 15\u00a0months on age 5 externalizing behavior. For both hypothesized pathways, the three abovementioned conditions for establishing mediation were met (see Table 1 for correlations). Furthermore, the effect of effective guidance at 15\u00a0months on externalizing behavior (\u03b2\u00a0=\u00a0\u2212.22, p\u00a0<\u00a0.05) dropped to a nonsignificant level (\u03b2\u00a0=\u00a0\u2212.13, ns) when D versus non-D attachment was controlled for (Sobel test: z\u00a0=\u00a0\u22122.11, p\u00a0<\u00a0.05), showing that D versus non-D attachment completely mediated the effect of effective guidance at 15\u00a0months on externalizing behavior. The effect of negative interactions at 15\u00a0months on externalizing behavior (\u03b2\u00a0=\u00a0\u2212.31, p\u00a0<\u00a0.001) decreased but remained significant when D versus non-D attachment was controlled for (\u03b2\u00a0=\u00a0\u2212.18, p\u00a0<\u00a0.05); Sobel\u2019s test showed the decrease to be significant (z\u00a0=\u00a02.77, p\u00a0<\u00a0.01). These findings indicate that negative interactions at 15\u00a0months are associated with age 5 externalizing behavior both directly and via D versus non-D attachment.\nNext, we tested whether the effect of parental ego-resiliency on child externalizing behavior was mediated by the quality of the parent-child interaction (two measures) at 15 and 28\u00a0months. As evident from Table 1, the three conditions for establishing mediation only held for one of the four possible pathways, namely for the pathway mediated by negative interactions at 28 months. For this pathway, multiple regression showed the effect of parental ego-resiliency on externalizing behavior (\u03b2\u00a0=\u00a0\u2212.25, p\u00a0<\u00a0.01) to decrease (Sobel test: z\u00a0=\u00a0\u22122.63, p\u00a0<\u00a0.01) to a nonsignificant level (\u03b2\u00a0=\u00a0\u2212.12, ns) when negative interactions at 28 months was controlled for. These results indicate that the effect of parental ego-resiliency on children\u2019s externalizing behavior was completely mediated by negative parent-child interactions at 28 months.Fig. 2Final path model summarizing the pattern of direct, moderated, and mediated relations between predictors at 15 and 28\u00a0months and age 5 externalizing behavior. Values given are standardized path coefficients. *p\u00a0<\u00a0.05. **p\u00a0<\u00a0.01\nPath analysis\nFinally, path analysis using AMOS 5.0 (Arbuckle, 2003) was applied to test the complete longitudinal model for predicting age 5 externalizing behavior including: (1) all the direct and moderated relations between the predictors and age 5 externalizing behavior found in the regression analysis; (2) the three significant mediated pathways resulting from the mediational analyses; and (3) the longitudinal relations between the 15- and 28-month assessments of the two parent-child interaction factors (i.e., effective guidance and negative parent-child interactions). A good model fit is indicated by a nonsignificant \u03c72 statistic, a Bentler comparative fit index (CFI) of .95 or above, and root mean square error of approximation (RMSEA) of .05 or below. The analysis of the initial model yielded a significant fit (\u03c72\u00a0=\u00a021.86, df\u00a0=\u00a023, p\u00a0=\u00a0.53, CFI\u00a0=\u00a01.00, RMSEA\u00a0=\u00a0.00), indicating that the model fits the data well.2 The final model, including the standardized path coefficients, is depicted in Fig. 2.\nDiscussion\nThe present study is one of the first attempts, and the first with a community sample, to include the theoretically most important parental, child, dyadic, and contextual characteristics, longitudinally assessed in both infancy and toddlerhood, in the prediction of externalizing behavior problems at age 5. Data were gathered using multiple sources (parents and teachers) and multiple methods, including questionnaires, standardized tests and extensive observations of the parent-child interaction. Predictors from all four domains were found to be associated with children\u2019s externalizing behavior at age 5, which is in accordance with the results of earlier studies using high-risk samples (Aguilar et\u00a0al., 2000; Erickson et\u00a0al., 1985; Lyons-Ruth et\u00a0al., 1993; Shaw et\u00a0al., 1996). The longitudinal model that emerged from our analyses provides more insight into the pattern of direct, moderated and mediated influences that work together to shape the development of externalizing behavior problems across the first five years of life.\nAs emphasized in the introduction, the present study was conducted on a non high-risk sample, in contrast to the above prior longitudinal studies that were all conducted on high-risk samples. After recruitment, when the children were 15\u00a0months of age, the sample appeared to be representative of the Dutch population of families with young children and therefore was not considered as a high-risk sample (see Van Bakel & Riksen-Walraven, 2002a). At the 5-year assessment, the selective drop-out of families with relatively low ego-resilient parents seemed to make the risk status of the remaining sample even lower. In this light, our finding that the children had higher CBCL and TRF externalizing scores than the relevant normative Dutch samples is puzzling. It may be that the level of externalizing behavior problems has increased over the last few years; unfortunately, no recent normative data are available. An alternative explanation may be that in middle-class communities, families with concerns about their children\u2019s development are more likely to volunteer for research studies. Because relevant information is not available for the present sample, this remains an issue for further study.\nThe only hypothesized predictors that were not associated with later externalizing behavior were SES and child temperament and cognitive ability at 15\u00a0months. The lack of association between SES and externalizing behavior is in line with earlier studies that also reported parental and child characteristics to be more consistently associated with externalizing behavior than contextual characteristics (Shaw et\u00a0al., 1996). Restriction of range in SES scores in our sample is not a likely explanation for the null findings given that the sample seems to be fairly representative of the Dutch population of families with children in the same age range, as indicated above. Yet, it may be that the variation of SES in the Netherlands is less than in other countries, such as the USA. Further research is recommended to examine how well the model that our analyses generated applies in community samples from other countries or for ethnically diverse samples of parents and children. The present nonsignificant relations between infant temperament and cognitive ability on the one hand and externalizing behavior on the other are in line with the results of two other recent longitudinal studies, both using high-risk samples (Aguilar et\u00a0al., 2000; Brennan, Hall, Bor, Majman, & Williams, 2003), that also failed to find an association between early temperamental and neuropsychological risks on the one hand and persistent aggressive behavior on the other. Given that \u2013 as argued in the introduction \u2013 the predictive power of risk factors may be higher in high-risk samples than in lower-risk samples, it is not surprising that variables that failed to predict externalizing behavior in high-risk samples also failed to predict such problems in a lower-risk sample like ours. With regard to the predictive power of risk factors in relation to sample risk status, it should be noticed that comparing the predictive power of risk factors in studies differing in sample risk status is not the most elegant way to address this question. A more direct way to test whether risk factors interact with sample risk status is by testing for interactive effects in a sample with a greater range of risk (see, for example, Lacourse et\u00a0al., 2006).\nAlthough temperament assessed in infancy did not interact with the parent-child interaction in the prediction of externalizing behavior, temperament assessed in toddlerhood did. More specifically, a parent-child interaction characterized by a lack of effective guidance in toddlerhood predicted externalizing behavior only in highly anger-prone toddlers but not in less anger-prone toddlers. This finding seems to support Belsky\u2019s (1997) differential susceptibility hypothesis that not all children are similarly affected by the same rearing experience. It should be kept in mind, however, that our effective guidance factor not only includes parental behavior, but child behavior as well. With the same reserve, our findings appear to also be in line with the results of studies that showed child negative emotionality to interact with parenting in predicting externalizing problems (Belsky et\u00a0al., 1998; Leve et\u00a0al., 2005). Rather than an association with negative parent-child interactions and hence in contrast to earlier reports, our findings showed temperament to interact with a different dimension of the parent-child interaction, namely a lack of effective guidance. This is not to say that negative parent-child interactions as such carried no weight in the prediction of externalizing problems in the present study. Quite the contrary, the factor proved to be one of the most powerful predictors of externalizing behavior problems in the present study and this was true for all the children in our sample and not just for a subsample of temperamentally difficult children.\nOur results on the interaction between temperament and parenting in the development of externalizing problems extend those of earlier studies by showing that a specific temperament by parenting interaction may work particularly in a specific developmental phase. That the interaction between anger proneness and effective guidance proved to predict externalizing problems particularly for toddlers makes sense when interpreted from a developmental psychopathology perspective: establishing autonomy and learning to comply with social rules and expectations are major developmental tasks for toddlers that challenge the parents\u2019 ability to provide effective guidance by imposing structure and setting limits while at the same time remaining emotionally supportive of the child. As a result of the child\u2019s striving for autonomy, the second year is marked by parent-toddler conflicts. Toddlers that are prone to show angry behaviors in such discord situations are particularly in need of effective guidance in order to prevent their lack of emotional and behavioral self-control to further escalate and evolve into externalizing behavior problems.\nOur finding that D attachment was among the strongest predictors of externalizing behavior at age 5 supports the results of previous research (Lyons-Ruth et\u00a0al., 1993; Munson et\u00a0al., 2001; Shaw et\u00a0al., 1996; Vondra et\u00a0al., 2001). Disorganized attachment has been found to reflect a history of disturbed parent-child interaction on the one hand and to predict later externalizing problems on the other (cf. Lyons-Ruth & Jacobvitz, 1999). The results of our path analysis have shed more light on the nature of the contributions of the parent-child interaction and parent-child attachment to the development of externalizing behavior problems. First of all, our path analysis showed D attachment to mediate the relations between the two aspects of parent-child interaction (i.e., effective guidance and negative interaction) at 15\u00a0months and externalizing behavior at age 5. In addition, the results showed negative parent-child interactions at 28 months to contribute significantly to later externalizing problems beyond D attachment at 15\u00a0months. These findings are in line with earlier findings by Erickson et\u00a0al. (1985) and support Greenberg\u2019s (1999) model that considers parent-child interaction and parent-child attachment as two different domains that contribute independently to a child\u2019s development but that also reciprocally influence one another.\nThe results of this study may have clinical and policy implications. Many studies have shown that children born in disadvantaged environments are at risk for developing externalizing problems later in life. The present study showed that, also for children living in presumably lower-risk families, certain characteristics in infancy and toddlerhood indicate an increased risk of externalizing problems at preschool age. It should be kept in mind, however, that our sample may have been more at risk than originally thought, because a relatively large proportion of children turned out to have externalizing behavior scores above the subclinical cutoff. An important practical implication of our findings is that one of the most powerful predictors identified in the present study, i.e., negative parent-child interactions, is relatively easy to observe already in infancy and proves to be highly stable, which makes it an interesting candidate for inclusion in early community mental health screening procedures. And the specific interaction of the other parent-child interaction factor (i.e., effective guidance) with temperamental anger proneness in toddlerhood in predicting later externalizing problems also suggests that paying more attention to early parent-child interactions may be useful in early detection of children at risk and in preventive intervention programs for parents in community mental health care. But more research is needed, of course, to further explore these possible applications.\nIt is important to also point out limitations of the present study. First, although it is a strong point that data were collected using multiple methods and multiple sources, parental report was the sole source of information regarding four different predictors of externalizing problems, namely infant temperament, parental ego-resiliency, partner support, and stressful life events. Thus, response bias may have partially accounted for the results. What speaks against this, however, is that the four predictors were mostly uncorrelated with each other (see Table 1). Only stressful life events was significantly correlated with two other predictors (i.e., ego-resiliency and partner support), which may explain why negative life events did not independently contribute to the explanation of later externalizing problems beyond the other two predictors. Another limitation is that the present study focused on the role of children\u2019s experiences with their primary caregivers in the development of externalizing problems, which leaves questions regarding the possible effects of the children\u2019s experiences with other caregivers and with their peers, for example in child-care centers (cf. Gevers Deynoot-Schaub & Riksen-Walraven, 2006). A third limitation of the study is that we did not have additional information about earlier externalizing behavior and about characteristics of the children and families prior to our first 15-month assessment at our disposal. Future research that includes observations earlier in infancy may further improve our understanding of the mechanisms involved in the early development of externalizing problems. A fourth limitation lies in the relatively small sample size. The sample was small by epidemiological standards, particularly with regard to evaluating mediator and moderator effects. And as a fifth and final limitation of the present study it should be mentioned that we based our assessments of the quality of infant-parent attachment on an abbreviated version of the Strange Situation that includes one as opposed to two separations from the parent. Despite the evidence supporting the validity of the abbreviated procedure (see Method section), it is possible that we have underestimated the number of D children given that in the regular Strange Situation Procedure children may be classified as disorganized based on their reunion behavior after the second separation. Anyhow, the present study shows that D attachment, even when assessed with the abbreviated Strange Situation, was a powerful predictor of later externalizing behavior problems.\nIn sum, the longitudinal model our analyses generated provides more insight into the complex interplay among parental, child, dyadic and contextual characteristics that together shape the development of externalizing behavior from age 15\u00a0months onwards. Future research should reveal whether the predictive power of the model can be improved, for instance by including earlier measurements and by taking into account the children\u2019s early experiences with the other parent and with their caregivers and peers in child-care facilities.","keyphrases":["externalizing behavior","longitudinal model","parent-child interaction","disorganized attachment","early predictors"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_Radiol-4-1-2270369","title":"Prevalence of anatomical variants and coronary anomalies in 543 consecutive patients studied with 64-slice CT coronary angiography\n","text":"The aim of our study was to assess the prevalence of variants and anomalies of the coronary artery tree in patients who underwent 64-slice computed tomography coronary angiography (CT-CA) for suspected or known coronary artery disease. A total of 543 patients (389 male, mean age 60.5 \u00b1 10.9) were reviewed for coronary artery variants and anomalies including post-processing tools. The majority of segments were identified according to the American Heart Association scheme. The coronary dominance pattern results were: right, 86.6%; left, 9.2%; balanced, 4.2%. The left main coronary artery had a mean length of 112 \u00b1 55 mm. The intermediate branch was present in the 21.9%. A variable number of diagonals (one, 25%; two, 49.7%; more than two, 24%; none, 1.3%) and marginals (one, 35.2%; two, 46.2%; more than two, 18%; none, 0.6%) was visualized. Furthermore, CT-CA may visualize smaller branches such as the conus branch artery (98%), the sinus node artery (91.6%), and the septal branches (93%). Single or associated coronary anomalies occurred in 18.4% of the patients, with the following distribution: 43 anomalies of origin and course, 68 intrinsic anomalies (59 myocardial bridging, nine aneurisms), three fistulas. In conclusion, 64-slice CT-CA provides optimal visualization of the variable and complex anatomy of coronary arteries because of the improved isotropic spatial resolution and flexible post-processing tool.\nIntroduction\nSince the beginning of the 1990s, a variety of non-invasive techniques have been introduced in coronary artery imaging in an attempt to replace invasive conventional coronary angiography (CCA). These techniques have shown promising results, although they were considered inadequate for large-scale clinical implementation. Furthermore, advanced modalities such as magnetic resonance (MR) and electron-beam computed tomography (EBCT) are still not widely available on the territory [1\u20136]. The introduction of multislice computed tomography coronary angiography (MSCT-CA) allowed the detection of significant coronary artery stenosis. Improved performance of 64-slice CT equipment, characterized by isotropic spatial resolution and faster temporal resolution, provided a valid alternative to CCA in selected patient populations [7\u201311].\nMSCT-CA is currently considered the ideal tool to three-dimensionally visualize the complex and tortuous anatomy of coronary arteries [12, 13]. Previous studies with four- and 16-slice CT-CA demonstrated that anomalous coronary arteries may be defined [14\u201316]. However, to the best of our knowledge, 64-slice studies with large patient series have not yet been published. In the present study, a large patient population who underwent 64-slice CT-CA was reviewed to assess the prevalence of coronary artery variants and anomalies.\nMaterials and methods\nPopulation\nA total of 543 consecutive patients (389 male, 154 female, mean age 60.5\u2009\u00b1\u200910.9, range 21\u201387\u00a0years), who underwent 64-slice CT-CA from 27\/07\/2004 to 28\/02\/2006 in our department, were reviewed for coronary artery variants and anomalies. The majority of patients (n\u2009=\u2009476) were scheduled for CCA because of suspected or known coronary artery disease (CAD). The other 67 patients were addressed to 64-slice MSCT-CA after CCA to determine the three-dimensional image of a suspected origin or course anomaly. The indication for MSCT-CA were: atypical angina (n\u2009=\u2009111), typical angina with inconclusive stress test (n\u2009=\u2009162), presence of risk factors and high risk of major coronary events (n\u2009=\u200946), proximal stent patency follow-up (n\u2009=\u2009112) and by-pass patency follow-up (n\u2009=\u200945). The Institutional Review Board approved the study protocol.\nCT scan and reconstruction parameters\nAll examinations were performed with a 64-slice CT scanner (Sensation 64, Siemens, Forcheim, Germany) with the following parameters: slices\/collimation 32\/0.6\u00a0mm, rotation time 330\u00a0ms, effective temporal resolution (with 180\u00b0 algorithm) 165\u00a0ms, 120\u00a0kv, 900\u00a0mAs, table feed\/s 11.63\u00a0mm, effective slice thickness 0.6\u00a0mm, reconstruction increment 0.3\u00a0mm, field of view (FOV) 140\u2013180\u00a0mm, isotropic voxel resolution of 0.4\u2009\u00d7\u20090.4\u2009\u00d7\u20090.4\u00a0mm.\nPatients with heart rate >70\u00a0bpm received 100\u00a0mg of metoprolol per os 1 h prior the examination. A bolus of 100\u00a0ml of high iodinated contrast material (400\u00a0mg\/ml iomeprol, Iomeron 400, Bracco, Milan, Italy) was injected into an antecubital vein of the right arm with a flow rate of 5\u00a0ml\/s, followed by a 40-ml saline chaser. A bolus-tracking technique was used for the synchronization between arterial passage of contrast material and MSCT-CA.\nData were reconstructed by retrospective gating in end-diastolic phase (from -300 to -450\u00a0ms before the peak of the subsequent R wave) or end-sistolic phase to better image the right coronary artery (RCA).\nImage and data analysis\nAll CT examinations were reviewed by three radiologists (L.L.G., R.M. and F.A.) with a level 3 expertise in cardiac CT [17], who loaded the datasets off-line into a dedicated workstation (Leonardo, Siemens, Germany). A total number of 559 examinations were performed and only 13 scans were considered not assessable by the readers in consensus due to poor image quality (severe breathing, triggering or motion artefacts).\nAll data were analysed with post-processing tools such as multiplanar reconstructions (MPR), curved MPR (cMPR), maximum intensity projections (MIP), and volume rendering (VR) to three-dimensionally image the complex anatomy of the coronary artery tree. Disagreement was solved by a consensus reading. Segments were classified according to the American Heart Association (AHA) scheme. Variants considered were: the coronary dominance (right, left, balanced), the variable origin of the conus branch and sinus node artery, the left main (LM) length, the presence of the intermediate branch, the number of diagonal and marginal branches. Anomalies of origin and course, intrinsic coronary anomalies (myocardial bridging, aneurisms >1.5\u00a0mm) and termination anomalies (fistulas) were checked. Prevalence data of single coronary artery variants and anomalies were collected.\nResults\nOur cohort results were heterogeneous because of the multi-ethnic Dutch population (immigrants 11%, Table\u00a01). \nTable\u00a01Our population resulted heterogeneous because of the multiethnic Dutch populationEthnic group% (n)The Netherlands88.95 (483)Middle East Asia3.31 (18)South-East Asia2.94 (16)East Europe1.84 (10)South Europe1.10 (6)South America0.92 (5)Africa0.92 (5)\nMost of coronary segments were identified, although with a variable rate due to different diameters (Table\u00a02). The anatomical variants of the coronary artery tree are extremely frequent (Table\u00a03). According to the literature, the dominance was right in 86.6% (n\u2009=\u2009470), left in 9.2% (n\u2009=\u200950), balanced in 4.2% (n\u2009=\u200923) [18]. The LM trunk (segment 5) presented a variable length (mean 112\u2009\u00b1\u200955\u00a0mm, range 17\u2013601\u00a0mm, median 106\u00a0mm): <1\u00a0cm (n\u2009=\u2009226, 41.6%), 1\u20132\u00a0cm (n\u2009=\u2009257, 47.3%), and >2\u00a0cm (n\u2009=\u200938, 7%) (Fig.\u00a01). The LM trunk was absent in 22 cases (4.1%) due to split origin of the left coronary artery (LCA) (n\u2009=\u200918, 3.3%) or other origin anomalies (n\u2009=\u20094, 0.7%). A variable number of diagonal branches was observed: one diagonal branch in 136 cases (25%), two diagonal branches in 270 cases (49.7%), and more than two in 130 cases (24%) (Fig.\u00a02). No diagonal branches were visualized in just seven cases (1.3%). Marginal branches of the left circumflex (LCX) artery were observed in the 99.4% (n\u2009=\u2009540): one in 191 cases (35.2%), two in 251 cases (46.2%), and more than two in 98 cases (18%) (Fig.\u00a02). \nTable\u00a02Segments visualized according to the American Heart Association classificationSegments% (n)199.8 (542)299.3 (539)397.8 (531)492.4 (502)595.9 (521)6100 (543)7100 (543)897.8 (531)998.7 (536)1073.7 (400)11100 (543)1299.4 (540)1397.2 (528)1464.3 (349)1572.4 (393)16a21.9 (119)aSegment 16 refers to the intermediate branchTable\u00a03Prevalence of coronary artery variants (RCA right coronary artery, LAD left anterior descending artery, LCX left circumflex, LM left main, ND not detected)Variants\u00a0Patients % (n)Conus branchFrom proximal RCA64.1 (348)From ostial RCA22.3 (121)From aorta11.6 (63)ND2 (11)Sinus node arteryFrom RCA65.4 (355)From LCX16.6 (90)From RCA and LCX9.2 (50)From LCX and pulmonary artery0.2 (1)From aorta0.2 (1)ND8.4 (46)LM length<1\u00a0cm41.6 (226)1\u20132\u00a0cm47.3 (257)>2\u00a0cm7 (38)Intermediate branch21.9 (119)Diagonal branches from LADND1.3 (7)125 (136)249.7 (270)>224 (130)Septal branches from LAD93 (505)Marginal branches from LCXND0.6 (3)135.2 (191)246.2 (251)>218 (98)Fig.\u00a01a LM length. b The separate origin of the LAD and LCX might cause technical difficulties during coronary angioplasty due to poor visualization. c\u2013e The LM may present variable lengthFig.\u00a02Variable number and course of diagonal (a\u2013c) and marginal branches (d\u2013f).The intermediate branch courses along the anterior wall of the left ventricle with a variable pattern (g\u2013i)\nWhen the intermediate branch supplies the vascularization of the antero-lateral wall of the left ventricle, a decreased number of diagonal branches (segments 9 and 10) was observed: one, 38.6%; two, 43.7%; more than two, 14.3%; none, 3.4% (Fig.\u00a02).\nFurthermore, CT-CA may visualize smaller branches, such as the conus branch artery (532\/543, 98%), the sinus node artery (497\/543, 91.6%), and the septal branches (505\/543, 93%). The conus branch artery may arise from the right coronary artery (RCA) (64.1%), in proximity with the RCA ostium (22.3%) or from the aorta (11.6%). The sinus node artery may originate from the RCA (355\/543, 65.4%), from LCX (90\/543, 16.6%), from RCA and LCX (50\/543, 9.2%), from LCX and pulmonary artery (0.2%), or from aorta (0.2%) (Fig.\u00a03).\nFig.\u00a03The variable origin of the conus branch artery (arrow): from RCA (a), in proximity with the ostium (b), and from aorta (c). The variable origin of the sinus node artery (arrowhead): from RCA (d), from LCX (e), or both pathways may be present (f)\nCoronary anomalies were observed in the 18.4% of our population (n\u2009=\u2009100). Patients presented single or associated coronary anomalies (Table\u00a04). \nTable\u00a04Prevalence of coronary artery anomalies (LM left main artery, PDA posterior descending artery)Coronary anomaliesPatients % (n)Myocardial bridging10.9 (59)Absent LM3.3 (18)Rotation of the aortic root with normal coronary origin from the sinuses of Valsalva2.6 (14)Coronary aneurysms1.6 (9)Anomalies of origin and course1.5 (8)Fistulas0.5 (3)Early take-off of PDA0.5 (3)\nSingle or multiple myocardial bridging was visualized in 59 patients (10.9%) (Fig.\u00a04). Forty-three anomalies of origin and course were found with the following distribution: absence of left main artery occurred in 18 patients (3.3%), origin anomalies of the RCA and the LCA caused by rotation of the aortic root between 45\u00b0 and 90\u00b0 (with normal coronary origin from the sinuses of Valsalva) in 14 patients (2.6%), anomalies of origin and course (with anomalous origin from the sinuses of Valsalva) in eight patients (1.5%), early take-off of the posterior descending artery in three patients (0.5%). The anomalies of origin and course encountered were: three retroaortic LCX (two arising from RCA, one from the right sinus of Valsalva), two inter-arterial LCAs from RCA, two inter-arterial RCAs from the left sinus of Valsalva, one septal RCA from left sinus of Valsalva (Fig.\u00a05).\nFig.\u00a04Examples of myocardial bridging (arrowhead). Myocardial bridging of mid-LAD displayed by MPR (a) and VR (b) images. Another case of myocardial bridging depicted by conventional angiogram in systole (c), not visualized in diastolic image (d), and clearly displayed by VR image (e)Fig.\u00a05Anomalies of origin and course (arrowhead). VR (a) and MIP (b) images of a LCA arising from the RCA with a septal course as confirmed by conventional angiogram (c). VR (d) and MIP (e) images of a RCA arising from the left sinus of Valsalva with an interarterial course, and corresponding conventional angiogram (f). VR (g) and cMPR (h) images of a stented retroaortic LCX arising from the right sinus of Valsalva and corresponding conventional angiogram (i)\nCoronary aneurisms (>1.5\u00a0mm, if compared with the normal vessel diameter) were identified in nine patients (1.6%) (Fig.\u00a06). Coronary fistulas were observed in three patients (0.5%) (Fig.\u00a07).\nFig.\u00a06Examples of coronary aneurisms. Aneurisms of LCX and LAD displayed by VR (a) and MIP (b) images, and corresponding conventional angiogram (c). Aneurisms of the RCA depicted by VR image (d) and vessel tree isolation (e), confirmed by the conventional angiogram (f)Fig.\u00a07Abnormal termination of coronary arteries. Fistula between the RCA and the coronary sinus depicted by VR (a), coronary tree isolation (b), and MPR images (c). Fistula between the LAD and the right ventricle displayed by VR (d) and coronary tree isolation images (e), and corresponding conventional angiogram (f)\nDiscussion\nThe wrong interpretation of a coronary variant or anomaly might cause technical difficulties during interventional procedures or lead to clinical misdiagnosis or major complications might occur during graft surgery. The need for an accurate anatomical evaluation of the coronary artery tree is relevant during angioplasty, due to revascularization purposes [19]. Coronary anomalies are often asymptomatic and may be accidentally discovered. Given the increase of interventional procedures, the detection of coronary anomalies is becoming of major clinical importance [20]. The coronary anomalies cannot be considered just rare aspects because they may often lead to relevant clinical consequences [21].\nIn an attempt to clarify the variability of the coronary artery tree, Angelini et al. [18] proposed these definitions: normal, any morphological feature observed in >1% of an unselected population; normal variant, an alternative, relatively unusual, morphological feature seen in >1% of the population; and anomaly, a morphological feature (number of ostia, proximal course, termination) rarely encountered (<1%) in the general population. However, the incidence of coronary anomalies is relevant not only for conceptual and educational purposes but, more importantly, for public health issues, given that 5.6% of the total American population could have some kind of coronary anomaly [18]. Moreover, the 19% of sudden deaths in young athletes are related to these anomalies [22]. That is a reason why the diagnosis of coronary anomalies should be a healthcare priority.\nTo date, despite some limitations, CCA has been the \u201cgold standard\u201d for the diagnosis of coronary anomalies. Selective catheterization and subsequent interpretation of vessel anatomy may be difficult in CCA because the operator is not aware of an atypical location of the vessel orifice. Therefore, the diagnosis of a coronary anomaly is often established on the impossibility of finding the coronary arteries in their normal anatomical position. Finally, interpretation of the courses of anomalous coronary arteries may be erroneous because CCA is two-dimensional and cannot provide enough information about the complex three-dimensional vessel anatomy [18, 23, 24]. The study with the largest number of cases, performed in North America at the Cleveland Clinic on 126,595 patients who underwent coronary angiography, reported a 1.3% incidence [25]. In recent years, other techniques in cardiologic diagnostic imaging have been developed, such as trans-thoracic echocardiography (TTE), trans-oesophageal echocardiography (TEE), magnetic resonance angiography (MRA), EBCT and MSCT [1\u20134].\nTTE, which is used mainly in paediatric radiology, does not always provide reliable diagnostic results. When performed on adult patients, it proves difficult to obtain diagnostic images owing to the interposition of the bones of the ribcage (ribs and sternum), pulmonary parenchyma and subcutaneous adipose tissue [5, 26].\nData reported in the literature suggest that TEE is more sensitive than TTE in identifying coronary anomalies and assessing their course, although it remains an invasive technique (i.e. insertion of a probe down the oesophagus and a varying degree of sedation according to patient tolerance required to perform the examination) characterised by a significant level of operator dependence and therefore impossible to perform as a screening test [6]. In addition, both echocardiography techniques are able to assess only the proximal tract of the coronary arteries, and therefore, their diagnostic capabilities are limited to only a part of coronary arteries [6].\nMRA is a highly promising technique since no ionising radiation is used. In the study of the origin of the coronary arteries, MRA can provide more complete information than CCA, particularly in patients with other concomitant congenital cardiac anomalies [1, 27]. The main limitation of MRA is incomplete visualisation of the coronary vessels, particularly their distal tracts. This limits the diagnostic capabilities for the assessment of fistulas, the origin of coronary arteries other than the aortic sinuses (i.e. from the pulmonary artery) and collateral vessels. However, MRA provides optimal functional assessment of complex congenital heart disease including anomalies of great vessels, and cardiac chambers and valves (the procedures may include evaluation of ventricular mass and volumes, quantification of valvular disease, and contrast enhancement) [12].\nRopers et al. [2] firstly studied the ability of contrast-enhanced EBCT to identify anomalous coronary arteries and their course with good accuracy. Recent advances in MSCT-CA equipments have continuously improved the quality of non-invasive coronary artery imaging. Various studies have demonstrated a high accuracy of coronary angiography with MSCT-CA for the diagnosis of CAD. In particular, the high negative predictive value of 64-slice CT allows to reliably exclude significant coronary artery stenoses [7\u201311]. Given the high sensitivity and negative predictive value of the technique, MSCT could represent a non-invasive alternative to CCA in patients prior to cardiac valve surgery. By selecting only those patients with coronary significant lesions to undergo CCA, MSCT-CA could avoid cardiac catheterization in a large number of patients without CAD [28]. The latest advance of CT technology is represented by the dual-source CT coronary angiography (DSCT-CA), with improved temporal resolution of 83\u00a0ms. Scheffel et al. [29] firstly demonstrated that DSCT-CA provides high diagnostic accuracy for assessment of CAD in a high pre-test probability population with extensive coronary calcifications and without heart rate control.\nThe advantages of MSCT lie primarily in its high level of diagnostic and anatomical accuracy. The technique offers excellent spatial resolution with the possibility of performing a flexible post-processing (i.e. MPR, MIP, and VR). Previous studies with four- and 16-slice CT-CA confirmed that the complex and tortuous coronary anatomy can be readily visualized and anomalous coronary arteries may be defined [14\u201316]. To the best of our knowledge, that is the first 64-slice CT-CA with large patient population, which evaluates the prevalence of coronary arteries variants and anomalies.\nThe AHA classification is the currently used scheme to identify the coronary artery segments and refers to 16 segments, in the attempt to standardize the remarks [30]. Another classification was reported by the BARI group, with emphasis to the coronary dominance pattern [31\u201333].\nAccording to literature data, the dominance was right in 86.6% (n\u2009=\u2009470), left in 9.2% (n\u2009=\u200950) and balanced in 4.2% (n\u2009=\u200923) [18]. The majority of coronary segments was identified according to the American Heart Association scheme.\nThe intermediate branch was present in 119 patients (21.9%), slightly less than reported in the literature [34]. However, the need for reporting the intermediate branch is stressed by the correlation between its presence and the decreased number of diagonal branches observed.\nFurthermore, the septal branches of the left anterior descending artery (LAD) were detected in the 93% (505\/543). The opportunity of reporting septal branches of LAD must be taken into account because of the hemodinamic relevance of these vessels.\nThe prevalence of coronary anomalies was estimated at the 18% (100\/543) in our population. This prevalence rate is higher than reported in the literature. However, our department is a major centre for cardiovascular pathologies and many patients were enrolled in non-invasive cardiovascular research projects. Myocardial bridging occurred in the 10.9%, a prevalence more comparable with autopsy rates, than with angiographic series [35]. The split of LCA also had a prevalence higher than reported in the literature [18]. Eighty-five patients with not significant CAD presented 14 coronary anomalies (16.5%). Four hundred and fifty-eight patients with significant CAD (with >50% stenosis, during stent or by-pass follow-up) presented 86 coronary anomalies (18.8%). Therefore, the prevalence was similar in the two groups. However, a bias is given by the fact that 67 patients were addressed to 64-slice CT-CA after CCA to determine the three-dimensional imaging of a suspected anomaly. After excluding these 67 patients, the prevalence of coronary anomalies was higher in the CAD patients (11.4% vs 4.2%). The prevalence of myocardial bridging resulted higher in the CAD group (7.4% vs 2.8%).\nThere are several limitations in our study. The first one is inherent to the heterogeneous population consisting of various ethnic groups (immigrants 11%) with a substantial age range (21\u201387\u00a0years). The age may affect the development of the collateral vascularization and influence the percentage of segments visualized. A second limitation is related to the high prevalence of origin anomalies, due to the fact that 67 patients were addressed to 64-slice CT-CA after CCA to determine the three-dimensional imaging of a suspected anomaly. As opposed to MRA, which also permits the non-invasive evaluation of coronary anomalies (proximal tracts), MSCT-CA requires radiation and a contrast agent. However, the high spatial and temporal resolution make it reasonable to use MSCT-CA as one of the first-choice imaging modalities in the work-up of known and suspected coronary anomalies [12, 13]. The high radiation exposure should be a matter of concern and debate in young patients. In these patients, the first-choice imaging modality could be MRA. In the case of suspected complex congenital heart disease (including anomalies of great vessels, and cardiac chambers and valves), the use of MRA would be highly advisable, since the optimal evaluation of ventricular and valvular function is provided [12, 13].\nCompared with 16-slice CT-CA, 64-slice CT-CA provides improved temporal resolution and isotropic spatial resolution which allow optimal three-dimensional visualization of the variable and complex anatomy of coronary arteries. Sixty-four-slice CT-CA may non-invasively define normal anatomical variants from potentially dangerous anomalies and support the clinical management of referring cardiologists and cardiac surgeons.","keyphrases":["anatomical variants","anomalies","multislice computed tomography","coronary artery circulation"],"prmu":["P","P","P","M"]}
{"id":"Pediatr_Nephrol-4-1-2335291","title":"Treatment of the neurogenic bladder in spina bifida\n","text":"Renal damage and renal failure are among the most severe complications of spina bifida. Over the past decades, a comprehensive treatment strategy has been applied that results in minimal renal scaring. In addition, the majority of patients can be dry for urine by the time they go to primary school. To obtain such results, it is mandatory to treat detrusor overactivity from birth onward, as upper urinary tract changes predominantly start in the first months of life. This means that new patients with spina bifida should be treated from birth by clean intermittent catheterization and pharmacological suppression of detrusor overactivity. Urinary tract infections, when present, need aggressive treatment, and in many patients, permanent prophylaxis is indicated. Later in life, therapy can be tailored to urodynamic findings. Children with paralyzed pelvic floor and hence urinary incontinence are routinely offered surgery around the age of 5 years to become dry. Rectus abdominis sling suspension of the bladder neck is the first-choice procedure, with good to excellent results in both male and female patients. In children with detrusor hyperactivity, detrusorectomy can be performed as an alternative for ileocystoplasty provided there is adequate bladder capacity. Wheelchair-bound patients can manage their bladder more easily with a continent catheterizable stoma on top of the bladder. This stoma provides them extra privacy and diminishes parental burden. Bowel management is done by retrograde or antegrade enema therapy. Concerning sexuality, special attention is needed to address expectations of adolescent patients. Sensibility of the glans penis can be restored by surgery in the majority of patients.\nIntroduction\nThe incidence of spina bifida worldwide still ranges from 0.3\u20134.5 per 1,000 births. Renal scarring and renal failure have been important issues in spina bifida literature over the years, with reported death due to renal failure up to 20% in the first year of life. The incidence of renal damage is nearly 100% in patients with an overactive pelvic floor (detrusor\/sphincter dyssynergia; DSD) when not adequately treated. It is important to realize that renal damage starts early in life, within the first 6\u00a0months. In contrast to a few decades ago, we can now assure parents of children with a neurogenic bladder that renal function can be preserved provided they comply with the treatment regime [1\u201314].\nTo prevent renal damage, urological and nephrological treatment of a neurogenic bladder should start immediately after birth. Modern treatment provides the opportunity to convert a high-pressure bladder with functional urethral obstruction based on neuropathic detrusor\/sphincter dyssynergia into a low-pressure reservoir that is safe for the upper urinary tracts [15, 16]. The objectives in the urological management of patients with spina bifida are (1) preservation of renal function; (2) quality of life, preferably with urinary dryness by school age; and (3) independence at an older age with respect to bladder and bowel management. Finally, sexuality in the spina bifida patients is an underestimated problem.\nInitial approach for the management of spina bifida\nTo preserve renal function, low bladder pressure must be maintained from birth. The status of pelvic floor activity must be assessed shortly after birth to ascertain whether a child is at risk for high detrusor pressures. At first presentation after birth, inspection of the anal sphincter, closed or open, gives an impression of the status of the pelvic floor: overactive or paralyzed. Approximately 50% of children with spina bifida aperta and 25% of children with occult spinal dysraphism have a detrusor\/sphincter dyssynergia that carries a serious risk of early upper urinary tract damage by high bladder pressures and urinary tract infections (UTIs). It is important to realize that after closure of the back, pelvic floor behavior can change from paralyzed to overactive in the first 2\u20133\u00a0months of life. That is a reason to delay the first urodynamic study (UDS) until 2\u00a0months after birth.\nClean intermittent catheterization\nIn principle, all newborn patients are put on clean intermittent catheterization (CIC), oxybutynin, and chemoprophylaxis (trimethoprim 2\u00a0mg\/kg once daily) immediately after closure of the back. By doing so, safe pressures in the lower urinary tract can be obtained in most patients. In most patients, bladder volume and compliance remain satisfactory for years. In patients with DSD, this approach has also reduced the need for bowel augmentation of the bladder from 90% to less than 5%. Of course, it is still a matter of discussion whether it is necessary to perform CIC from birth onward in all patients. In patients with proven pelvic floor paralysis, it is also possible to wait until after surgery to make the patient continent. However, it can be a tedious affair to teach CIC to an 8-year-old. Parents of these children are advised to catheterize at least twice daily to maintain the habit of CIC. CIC was introduced for neurogenic bladders in 1972 [17\u201320]. Parents and other caregivers carry out CIC in the first 8\u20139 years of life: if the children show sufficient dexterity, they take up this task themselves after this age. After birth, CIC is carried out with self-lubricating 8-F catheters. The size of the catheter depends on the patient\u2019s age; the aim is always to use the largest possible catheter to obtain optimal bladder emptying.\nAntimuscarinic therapy\nOxybutynin is best started together with CIC immediately after closure of the back. Other antimuscarinic agents have not yet been registered for pediatric use. This subject is more elaborately described in the section \u201cOveractive detrusor\u201d.\nInfection prophylaxis\nIn principle, all patients are put on low-dose chemoprophylaxis, mostly trimethoprim 2\u00a0mg\/kg per day. In case of breakthrough infections, nitrofurantoin or ciprofloxacin can be used. Symptomatic infections are treated intravenously with amoxicillin\/clavulanic acid and gentamicin until the result of bacterial culture is known. Many centres stop prophylaxis after the age of 1 year, and approximately 50% of patients seem to do well without prophylaxis. Scientific proof is lacking on this subject. To obtain such proof, we are doing a multicenter randomized study with half of the patients on prophylaxis and half without.\nSpecific needs\nOveractive pelvic floor\nFrom birth, patients with an overactive pelvic floor generally become dry with the help of oxybutynin and CIC. Every year, UDS are carried out to check bladder activity, capacity, and compliance. Patients are given ultrasound scans for upper-tract dilatation and renal growth. A wait-and-see policy is adopted when bladder capacity is normal for age and end-filling detrusor pressures are 30\u00a0cms H2O or less. When bladder compliance is insufficient, with end-filling pressures of more than 30\u00a0cms, an autoaugmentation or detrusorectomy can reduce the pressure. Augmentation by ileocystoplasty or colocystoplasty is inevitable in patients with serious overactivity in combination with poor bladder compliance and low capacity. Both operations can be performed at any age and can be combined with other procedures to obtain better urinary continence to facilitate CIC and\/or to reduce UTIs (reflux) [21].\nParalyzed pelvic floor\nPatients with a paralyzed pelvic floor are incontinent for urine. Their upper urinary tracts are safe as long as the paralytic pelvic floor is left untreated. They need bladder-neck surgery to become dry. If bladder capacity and compliance are insufficient, surgery can be combined with autoaugmentation of the bladder (detrusorectomy) or (rarely) with clam ileocystoplasty or colocystoplasty.\nOveractive detrusor muscle\nIf UDS reveals detrusor overactivity, patients are treated with antimuscarinics to increase bladder capacity, even if detrusor pressures are safe. Life-long suppression of detrusor overactivity is required in patients with an overactive neuropathic bladder. In some patients, overactivity can be treated surgically by detrusorectomy or ileocystoplasty. In spina bifida, rhizotomies (interruption of spinal roots) are not yet being used routinely to cure overactivity, but they seem to be a promising alternative. Ideally, we would like to start a protocol with neonatal rhizotomies during the first back closure, but so far, practical and ethical considerations have prevented us from doing this. Antimuscarinic therapy is the gold standard for pharmaceutical therapy of neuropathic detrusor overactivity. Oxybutynin has proven to be inexpensive and effective and can be taken orally, intravesically, and transdermally. Several new antimuscarinic agents have been introduced, which may prove valuable in the future.\nRepeated injection therapy of the bladder with 300 U of botulinum toxin can be an alternative to antimuscarinic therapy. This therapy effectively suppresses detrusor contractions for 6\u20139\u00a0months. Injections need to be repeated at a 6- to 9-month interval. Adverse effects, even after repeated injection therapy, have not been reported. The long-term effects have yet to be established [22, 23].\nSurgical procedures\nTiming of surgery\nThere is no age-related contraindication for any operation. Thus, indication for surgery is made in mutual agreement with the child\u2019s parents or with the patients themselves after the age of 11 years. The combination of high bladder pressures and vesicoureteral reflux can sometimes force intervention as early as the first few months of life. We combined antireflux surgery, bladder autoaugmentation, and transvaginal sling suspension in a 3-month-old girl with febrile breakthrough infections, with good clinical and urodynamic result after follow-up for more than 10 years. Initially, reserves existed on doing sling suspensions before puberty, especially in boys, out of fear of introducing obstruction during puberty based on prostate growth. Over the years, it has been proven that puberty can safely be passed after sling suspension of the bladder neck, both in girls and boys.\nParental burden can be an indication for performing a catheterizable stoma. If a child weighs 20\u00a0kg, five daily transfers for CIC can be too much for a parent with lower back pain. Finally, the patient\u2019s privacy can be an important argumentation for construction of a stoma for CIC. Parents are increasingly reluctant for any caregiver to help their child perform CIC transurethrally, as this involves exposure of the genitalia to strangers several times a day.\nSurgery for incontinence\nPatients with a paralytic pelvic floor need bladder-neck surgery to achieve continence. There are many surgical options. Our standard approach is an abdominoperineal puboprostatic sling procedure in boys and a transvaginal sling procedure in girls. If there is some persistent leaking after a sling procedure, this is cured by injecting a bulking agent in the bladder neck. Optimal results are obtained if the injection needle is passed into the bladder neck by means of a suprapubic puncture with transurethral endoscopic visual control. So far, we have mainly used silicon grains in povidone (Macroplastique\u00ae) as a bulking agent. The polymer Deflux\u00ae offers a good alternative because it is easier to inject. However, in our experience, Deflux does not work if CIC needs to be carried out through the same channel. A recent survey, yet unpublished, of the results of 76 sling suspensions revealed an 80% success rate.\nSling suspension in boys\nThere is a great variety in the reported results regarding sling suspension of the bladder neck in boys with neuropathic sphincter incompetence, especially when compared with girls. The plane between the bladder neck\/prostate and the rectum must be developed to allow the sling to be wrapped around the bladder neck. There are three possible techniques, each with their specific advantages and disadvantages. First, from the abdominal wound, the pelvic diaphragm can be opened left and right so that a pathway can be bluntly dissected for the sling around the bladder neck. A disadvantage of this technique is the risk of passing into the prostatic urethra with the right angle that is used to feel the way around during surgery. Experienced urologic surgeons do not take this argument seriously, but in practice, most surgeons perform this operation with the relatively low frequency of only a few cases a year. The second possible technique is the abdominoperineal approach to develop the plane between the bladder neck and the urethra. The plane between the rectum and the bladder neck is exposed through a perineal incision. The most important advantages of this procedure are the short time, approximately 20 min, needed to develop the plane around the bladder neck and the fact that the bladder does not need to be touched at all [24].\nA third method to find the path around the bladder neck has been proposed by Lottmann et al. The complete bladder is dissected from the peritoneum and the rectum until only the urethra and both ureters with the vascular pedicles are left. The sling can easily be passed around the bladder neck, with direct visual control and exactly at the level of the bladder neck [25]. It is difficult to determine which of these three approaches is the best. All three have enthusiastic followers. At our department, we have had two urethral defects treated by two experienced surgeons in a small series of the abdominal approach going directly around the bladder neck. We have had one urethral defect leading to bladder-neck closure in 76 patients operated on abdominoperineally. Our experience with the Lottmann approach is limited to three successful cases and, because of operation time and for fear of limited bladder vascularization, we abandoned this approach.\nAnother important subject is the amount of tension that needs to be put on the sling. In the past, we tried to regulate tension by measuring urethral and leak-point pressures during surgery, but there was no significant positive outcome. Nowadays, we determine sling tension by measuring the ability to pass a relatively large Foley catheter past the sling; for example, a 12-F catheter in a 7-year-old boy. In some patients with anal atresia after prior rectal pull-through surgery, it may be necessary to adopt the abdominal approach to pass around the bladder neck. Abdominoperineal operations in 14 patients, published in 1999, led to continence in all 14 patients; one patient required a subsequent injection of a bulking agent to help achieve continence [24]. It is important to note that in this series, the erectile function of the penis proved to be preserved after sling suspension.\nFalse routes are a risk after sling suspension. Out of approximately 50 male patients, we had to construct a catheterizable stoma in five: four as a result of a false route, and one because of a huge congenital prostatic cyst.\nSling suspension in girls\nIn general, sling suspension of the bladder neck in girls leads to good results concerning urinary continence. There are also several ways to find the way around the bladder neck in girls. The standard well-known technique is to identify the level of the bladder neck by feeling the balloon of a transurethral Foley catheter, and, subsequently, developing the plane between the bladder neck and the anterior vaginal wall from above. This procedure can be very difficult in girls with severe spine deformities. There is always a certain risk of entering the urethra or the bladder neck when passing around. This is also the case in patients with a normal anatomy. To avoid this risk, in postpuberty female patients, many surgeons opt for incising the vaginal wall at the level of the bladder neck and bringing the sling around under direct visual control. We chose to put the sling through the vagina, first only in prepubertal girls and later in all patients. This method has two major advantages: there is no risk of bladder-neck lesions and only a small risk of sling erosion [26]. In spina bifida patients, the risk of sling erosion is higher than in patients with stress incontinence, because the sling must be tightened more strongly in patients with neuropathic sphincter incompetence.\nAlternatives\nThere are many surgical alternatives for reaching urinary continence. Kropp and Pippi Sale developed operations to create a flap valve from the bladder neck. Young\/Deese\/Leadbetter, Tanagho, and Mitchell procedures lengthen the urethra into the bladder. Several authors prefer American Medical Systems (AMS) artificial sphincters to help reach continence. The majority of patients still need CIC after AMS sphincter prosthesis [27]. We found that Burch-type colposuspension was insufficient for spina bifida girls to reach urinary continence. This is related to the huge intra-abdominal pressure that can result from transfers from the wheelchair.\nSeveral groups do not opt for urinary continence at an early age and try to maintain safe leak-point pressures by regular dilatation of the female urethra, sometimes also of the male urethra, after making this possible with a perineal urethral stoma. In some patients, they construct a cutaneous vesicostomy for the first few years and perform an ileocystoplasty when the stoma is closed.\nAlternatives in dealing with a neurogenic bladder\nWhen cerebral function is bad and the patient has no dexterity, diapers can also be chosen. For this choice, a low-pressure bladder is necessary. Good results are obtained by cutaneous vesicostomy. In girls, low pressures can be achieved by making a vesicovaginal fistula by endoscopic cutting into the vagina between the ureteric orifices. Temporary low pressures can be achieved by overdistension of the female urethra. In boys, endoscopic external sphincterotomy in the 12 o\u2019clock position produces low outlet resistance for 2\u20133 years and needs to be repeated when bladder pressures rise.\nCatheterizable stomas\nThe need for transfers from a wheelchair to do CIC is the most important indication to make a catheterizable stoma. Sometimes in boys, the impossibility of carrying out CIC transurethrally after false routes results in the need for a stoma. Parents increasingly indicate their child\u2019s privacy as a reason for asking for a catheterizable stoma. In patients with a large bladder capacity for their age, a continent stoma can be constructed from a bladder tube. Patients with a normal bladder capacity can be treated by cutaneous appendicovesicostomy or by an ileal tube (Monti procedure) [28]. The direction of peristalsis from the appendix (Fig.\u00a01) needs to go in the direction of the bladder to avoid mucus spotting in the clothes. When ileal or colonic bladder augmentation is performed at the same time, the continent stoma can be constructed from the side or middle part of the gut used for the augmentation. In some patients, a ureter can be used as a catheterizable stoma. The intravesical tunnel of the tube in the bladder should be at least 2\u00a0cm long, as there is a risk that the tunnel will end up being too short, especially in case of an extravesical implantation of an appendix.\nFig.\u00a01The appendix can be used to make a stoma to the bladder for intermittent catheterization\nIn the literature, after construction of a catheterizable stoma, complications are described in as many as 50% of patients. These complications are mostly temporary. Stomal stenosis, mostly at skin level, is a frequently occurring temporary complication that can be avoided with a silicon ace stopper left behind in the skin portion of the stoma for 6\u00a0months between catheterizations. Stomal leakage of urine can be a frustrating complication. Endoscopic treatment with bulking agents can be tried, but lengthening of the intravesical tunnel or reimplantation of the stoma will often be needed. Leakage of the stoma occurs more often in midline stomas (umbilicus) than in stomas placed on the right side of the lower abdominal wall. The reason is probably that the lateral stoma passes through the rectus muscle and is thus occluded during increases in abdominal pressure due to rectus contraction. In midline stomas we now also pass the stoma through the median margin of one rectus muscle. Stoma stenosis at the level of entrance into the bladder can sometimes be treated endoscopically with success. In those patients, formal reimplantation of the stoma into the bladder will frequently be needed as well.\nAutoaugmentation or detrusorectomy of the bladder\nIn general, not too much extra capacity should be expected, but high-end filling pressures based on low bladder compliance can be reduced to safe values by detrusorectomy. In selected patients that undergo surgery to become dry, we also do an autoaugmentation (Fig.\u00a02) to try and get patients off antimuscarinic therapy, with success in approximately 50% of cases. An important factor for success is identification, opening, and marking the adventitial layers of the bladder separately before the detrusorectomy starts, and the meticulous closure of this layer at the end of the procedure. This means that before the start of the detrusorectomy, the detrusor has been freed of all adventitial layers. If a mucosal leak occurs during the operation, this is closed with Tissuecoll\u00ae. To prevent shrinkage and scarring, cycling of the bladder has to start immediately after detrusorectomy. This can be done by maintaining a 20-cm H2O pressure on day 1, 30\u00a0cm on day 2, followed by clamping the catheter on day 3 for 2 h, and finally by opening the catheter every 3 h to empty the bladder on following days. In this way, optimal expansion of the detrusorectomized bladder can be obtained [21]. Several authors have reported success by expanding the bladder over a balloon for several days while ensuring drainage of urine by ureteral catheters. We have no experience with this procedure but assume it could work well if there is leakage. Others have successfully covered the bare part of the mucosa with a demucolized patch of sigmoid colon. Covering with peritoneum or omentum has been reported to fail.\nFig.\u00a02The bladder can be augmented by removing the detrusor muscle. A sling is used to improve bladder outlet resistance\nClam cystoplasty\nClam cystoplasty (Fig.\u00a03) is indicated when the bladder capacity is too small to reach acceptable numbers of catheterization per day and\/or end-filling pressures are too high to be safe for the upper tracts. Clam cystoplasty is performed with either ileum or colon. In general, 25\u00a0cm of ileum are used, opened antimesenterically, and constructed into a U-shaped cap. The last 20\u00a0cm of the ileum are not used, to prevent malabsorption of vitamins. If there is a short ileal mesentery, the sigmoid colon can easily be used. It is important to avoid the formation of an hourglass bladder by opening the clam anteriorly up to the bladder neck and posteriorly up to the trigone. In children, we prefer to bring the augmented bladder extraperitoneally by closing the peritoneum around the vascular pedicle. This extraperitonealization of the bladder may initially make it more difficult to reach sufficient bladder capacity, but it seems to reduce the risk of spontaneous perforation of the bladder, which is a prominent risk in children because of their tendency toward bad CIC compliance during puberty. During a period of several years, we had two spontaneously leaking ileal bladders out of six that had not been placed extraperitoneally, and no spontaneously leaking\/rupture in more than 40 other patients with extraperitoneal ileal bladder.\nFig.\u00a03The bladder can be augmented by using a bowel segment\nBowel management and surgery\nFor the first 2 years of life, a wait-and-see policy is adopted in bowel management, often supported by chronic use of laxatives. At the age of 3 years, bowel management can be performed successfully in the vast majority of patients with retrograde colonic enemas. The rectum is filled with 20\u00a0ml\/kg tap water through a plastic cone that passes approximately 2\u00a0cm into the anus. In the rare case where patients have trouble emptying their rectums with retrograde enemas, we offer an antegrade colonic enema stoma [29, 30]. A patient\u2019s choice can be an indication for an antegrade colonic enema stoma. As a separate procedure, the simplest way to carry out an antegrade colonic enema stoma is to use laparoscopy to bring the tip of the appendix to the abdominal skin (Fig.\u00a04). Alternatives are an open procedure with open abdomen for other surgery or, in the absence of an appendix, to create a transverse tube from the colon (left or right) and a submucosal tunnel to prevent leakage. Many pediatric surgeons and pediatric gastroenterologists have had good experiences with several types of buttons that are put into the colon.\nFig.\u00a04The appendix can be used to make an antegrade colonic enema stoma\nIf these procedures are carried out, treatment of spina bifida patients can be very successful. In our experience between 1988 and 2001, the incidence of renal scarring in 144 patients was 2.1%, and urinary dryness in patients who opted to be dry was nearly 80% [14].\nSurgery to improve sexuality in male patients\nSexuality can be frustrating for male spina bifida patients, because in most cases, they can have intercourse but penis sensibility is absent. To overcome this, in patients with a lesion under the level of L1, we have connected the ilioinguinal nerve with the dorsal nerve of the penis, resulting in glans sensibility and erogenous feeling in most patients. Patients report a positive effect on sexuality. Our experience with the first three patients has been published, and by now, of 15 cases operated, 12 report normal sensibility of the glans penis [31].\nConclusion\nKidney-function preservation and early urinary dryness are important factors for optimal quality of life for spina bifida patients. Another important factor is patient independence regarding his or her bladder and bowel management. For optimal treatment of this difficult group of patients, a multidisciplinary team is needed consisting of members from all the necessary medical specialties, including rehabilitation specialists, physical therapists, specialized nurses, and social workers.\nQuestions\n(Answers appear following reference list)\nConcerning renal scarring and renal failure in spina bifida literature:\nReported death rate due to renal failure is up to 20% in the first year of lifeReported renal failure rate is up to 20% in the first year of lifeReported death rate due to renal failure is up to 20% in pubertyReported renal failure rate is up to 20% in pubertyConcerning clean intermittent catheterization (CIC):\nCIC is best started immediately after closure of the spinal defectCIC is best started immediately after the age of 8\u20139 yearsCIC is best started not earlier than after bladder neck surgeryCIC is best started not earlier than after several urinary infections and reported renal scarringInfection prophylaxis in spina bifida patients:\nAll patients need low-dose chemoprophylaxisFifteen percent of all patients need low-dose chemoprophylaxisScientific proof of the need for infection prophylaxis is still lackingNo patients need low-dose chemoprophylaxisIn case of hyperactive detrusor muscle in a patient with spina bifida:\nCentral side-effects occur less frequently if antimuscarinic therapy is administered orallyAntimuscarinic therapy is the gold standard for pharmaceutical therapyRepeated injection therapy of the bladder with only 3 U of botulinum toxin can be an alternative to antimuscarinic therapyAntimuscarinic therapy has positive effects on perspiration and body-temperature regulationIn modern management of spina bifida, endpoint targets are:\nKidney-function preservationDrynessFecal continenceAll the above\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nESM 1\n(MPG 72.8 MB)","keyphrases":["spina bifida","incontinence","renal function","dryness","antimuscarinic","bladder augmentation","surgical treatment","bladder-neck sling"],"prmu":["P","P","P","P","P","P","R","R"]}
{"id":"J_Med_Internet_Res-1-suppl1-1761819","title":"SYD1\/444: Assessing the Readership of the UK National Database of Telemedicine\n","text":"Introduction The National Database of Telemedicine (NDTM) is a Web site containing information resources on UK telemedicine projects and related work. Its purpose is to provide a source of information to anyone researching the field or proposing a trial or a larger scale implementation of Telemedicine. The NDTM Web site was launched on 27th October 1998 and publicised extensively. Before and after its launch, the Web site has been kept up to date with information about new projects and modifications to the details kept about existing ones. It is clearly important for such a resource to keep track of who is reading it. We may need to tailor information to a particular audience, or bring the Web site to the attention of people who may not yet have found it. This paper describes a mechanism for tracking usage of the Web site that provides more information than conventional approaches.","keyphrases":["telemedicine","internet","hypermedia","readership analysis"],"prmu":["P","U","U","M"]}
{"id":"Intensive_Care_Med-3-1-2040486","title":"The first demonstration of lactic acid in human blood in shock by Johann Joseph Scherer (1814\u20131869) in January 1843\n","text":"Lactic acid was first found and described in sour milk by Karl Wilhelm Scheele (1742\u20131786) in 1780. The German physician\u2013chemist Johann Joseph Scherer (1841\u20131869) demonstrated the occurrence of lactic acid in human blood under pathological conditions in 1843 and 1851. In this article we honour the forgotten observations by Scherer and describe the influence of Scherer's finding on further research on lactic acid at the end of the 19th century. We conclude that Scherer's 1843 case reports should be cited as the first description of lactic acid in human blood after death and also as the first demonstration of lactic acid as a pathological finding in septic and haemorrhagic shock. Carl Folwarczny was, in 1858, the first to demonstrate lactic acid in blood in a living patient.\nIntroduction\nLactic acid was first found and described in sour milk by the Swedish chemist Karl Wilhelm Scheele (1742\u20131786) in 1780\u00a0[1]. The Swedish chemist J\u00f6ns Jakob Berzelius (1779\u20131848) found lactic acid in fluid extracted from meat in 1808\u00a0[2, 3], and the German chemist Justus von Liebig (1803\u20131873), who established the world's first school of chemistry at Giessen, proved that lactic acid was always present in muscular tissue of dead organisms\u00a0[4]. In 1859, Emil Heinrich du Bois-Reymond (1818\u20131896) published several articles on the influence of lactic acid on muscle contraction\u00a0[5\u20139]. Araki and Zillessen found that if they interrupted oxygen supply to muscles in mammals and birds, lactic acid was formed and increased\u00a0[10\u201314]. This was the first demonstration of the relationship between tissue hypoxia and the formation of lactate. The occurrence of increased lactic acid in blood (hyperlactataemia) nowadays reflects severe illness, in which the increased blood lactate levels may result from both anaerobic and aerobic production or from a\u00a0decreased clearance.\nIt was the German physician\u2013chemist Johann Joseph Scherer who first demonstrated the occurrence of lactic acid in human blood under pathological conditions after death in 1843 and 1851\u00a0[15, 17], and Carl Folwarczny in 1858 who first demonstrated lactic acid in blood of a\u00a0living patient. In this article we wish to honour Scherer's forgotten observations and describe the influence of his finding on further research on lactic acid at the end of the 19th century.\nBiography of Johann Joseph Scherer\nBorn on 18 March 1814 in Aschaffenburg, Germany, Scherer studied medicine, chemistry, geology and mineralogy at the university of W\u00fcrzburg between 1833 and 1836. He obtained his PhD in medicine and surgery in 1838 with a\u00a0thesis entitled \u201cVersuche \u00fcber die Wirkung einiger Gifte auf verscheidene Thierclassen\u201d (Experiments on the action of some poisons on several classes of animals). He practised medicine in Wipfeld, but inspired by the chemist Ernst von Bibra (1806\u20131878) he completed his studies in chemistry at the University of Munich between 1838\u20131840\u00a0[18]. In 1840 he was employed at the laboratory of Justus Liebig at Giessen, and became professor at the medical faculty in 1842, professor of organic chemistry in 1847, and later professor of general, anorganic and pharmaceutical chemistry. His work especially concerned quantitative research on blood and urine in pathological conditions. In 1843 he published his book \u2018Chemische und Mikroskopische Untersuchungen zur Pathologie angestellt an den Kliniken des Julius-Hospitales zu W\u00fcrzburg\u2019 (Chemical and microscopic investigations of pathology carried out at the Julius Clinic at W\u00fcrzburg)\u00a0[15] (Fig.\u202f1), in which he described 72 case reports, giving details on clinical course, diagnosis, and results obtained during autopsy and analysis of body fluids. Scherer died on 17 February 1869\u00a0[18].\nFig.\u00a01Title page of Scherer's 1843 book\nThe 1843 cases\nIn one chapter in his 1843 book entitled 'Untersuchungen von krankhaften Stoffen bei der im Winter 1842\u20131843 in W\u00fcrzburg und der Umgegend herrschenden Puerperal-Fieber-Epidemie' (Investigations of pathological substances obtained during the epidemic of puerperal fever which occurred in the winter of 1842\u20131843 in and around W\u00fcrzburg) Scherer described the cases of seven young women who all died peripartum.\nOne of the women, the 23-year-old primipara Eva Rumpel, gave birth to a\u00a0healthy child on 9 January 1843. The same night she developed a\u00a0painfully swollen abdomen and became ill, feverish, and sweaty, with rapid pulse and severe thirst. The initiated treatment was bloodletting and clystering. The next evening she deteriorated, became delirious, with anxious breathing, a\u00a0tense abdomen, cold extremities and rapid pulse, finally losing consciousness. Again, bloodletting followed. At 4:30 a.m., 36\u202fh after the onset of the first symptoms, she died. During autopsy, severe purulent endometritis, vaginal pus, pulmonary oedema, and shock liver and shock spleen were found. The blood that was obtained directly from the heart was chemically analysed, in which lactic acid was found. Most likely this unfortunate woman had died from a\u00a0fulminant septic shock caused by group A\u00a0haemolytic streptococci (Streptococcus pyogenes). Scherer diagnosed this case as perimetritis with secondary peritonitis.\nAnother patient, the 28-year-old, 7 months pregnant (second pregnancy) Margaretha Gl\u00fcck, was, after being icteric, nauseous, vomiting and complaining about epigastric pain for 8 days, admitted to the lying-in birth clinic on 6 February 1843. Four days later she was transferred to the hospital with severe nosebleeds and generalised exanthema or purpura. In the evening she suffered from severe gastric bleeding and epistaxis, showing rapid pulse, cold extremities and dizziness. The next morning, she was transferred back to the birth clinic, where she gave birth to a\u00a0premature child (30 weeks) and suffered from a\u00a0severe post-partum fluxus. She was again transferred to the hospital with the following symptoms: cold clammy skin, tachycardia, severe lochia and persistent exanthema or purpura, but without signs of an acute abdomen. During the night of February 11, she became aphasic and restless, followed by chills and profound sweating. On the morning of February 13, she further deteriorated and bilirubinuria was detected. The next day she was comatose, finally developed rattling breathing and convulsions. Death occurred during the following night. Autopsy revealed a\u00a0small intracerebral haematoma, normal lungs without pulmonary oedema, ascites and an anaemic, foul smelling uterus filled with purulent and decayed tissue and pus. Blood was also obtained directly from the heart during autopsy and lactic acid was found.\nIn this case we could think of a\u00a0haemorrhagic shock and cerebral haemorrhage due to clotting disorders possibly resulting from either acute fatty liver of pregnancy\/HELLP syndrome, idiopathic thrombocytopenic purpura, thrombotic microangiopathy (TTP\/HUS) or DIC. The case was most likely complicated by a\u00a0sepsis (endometritis). Scherer himself diagnosed this case as septic endometritis.\nIn the conclusions of his 1843 book, Scherer attached high importance to the fact that he found lactic acid in cases of puerperal fever, which he had not found before in healthy persons. He held the opinion that lactic acid was formed in blood during bodily deterioration in severe diseases like puerperal fever. Lactic acid was thus described for the first time in human blood and was demonstrated for the first time as a\u00a0symptom of septic and haemorrhagic shock.\nIn the same period a\u00a0junior obstetrician in Vienna, Ignaz Philipp Semmelweis (1818\u20131865), discovered in 1847 that physicians carried infectious particles on their hands from the mortuary to the obstetrical clinic, causing puerperal fever and puerperal sepsis, and he introduced a\u00a0successful method for its prevention. Louis Pasteur (1822\u20131894) found in 1879 that infection with streptococci was the most important cause of puerperal fever\u00a0[16].\nThe 1851 article\nScherer worked closely with the famous pathologist Rudolf Virchow (1821\u20131902) on several projects (Fig.\u202f2). In 1851 Virchow performed an autopsy on a\u00a0patient who had died from leukaemia and offered Scherer blood from this patient for analysis. The results of this analysis were published the same year in the \u2018Verhandlungen der Physikalisch-Medicinischen Gesellschaft in W\u00fcrzburg\u2019 \u00a0[17]. Virchow and Scherer had previously studied the spleens of patients who died from leukaemia, and were curious if they could find the same results in the blood. Scherer reached the conclusion that: the blood of this patient contains: \u201cAmeisens\u00e4ure, Essigs\u00e4ure und Milchs\u00e4ure, die gleichfalls von mir schon fr\u00fcher als in der Milzfl\u00fcssigkeit vorkommend bezeichnet wurden\u201d (Formic acid, acetic acid, and lactic acid, as also found by me previously in fluids from the spleen).\nFig.\u00a02Johann Joseph Scherer (left) and Rudolf Virchow (right) in 1849\nFurther research\nScherer's observations inspired others to conduct further research, primarily in patients with leukaemia\u00a0[19\u201322], but also in patients with other conditions and diseases and in animal experiments with dogs and rabbits\u00a0[23]. While Scherer found lactic acid in blood obtained after death during autopsy, Mosler and K\u00f6rner\u00a0[19] mention an observation made by Carl Folwarczny, published in the Allgemeinen Wiener Medicinischen Zeitung in 1858, where blood was withdrawn from a\u00a0leukaemia patient during life, analysed according to Scherer's method, and found positive for lactic acid. In addition, Carl Folwarczny described in 1863 in his 'Handbuch der Physiologischen Chemie'\u00a0[24] that lactic acid can be found in the blood of patients with leukaemia, septicaemia (pyaemia) and in conditions leading to septicaemia like puerperal fever, the latter probably after Scherer's observations.\nIn an extensive article, the Berliner physician Georg Salomon\u00a0[25], who had serious doubts that the occurrence of lactic acid in blood was mostly related to leukaemia, proved in 1878 that lactic acid was also present in the blood of patients who were suffering and died from other diseases. He studied blood obtained during autopsy from cadavers, but also blood from patients obtained by bloodletting or cupping, and in some cases he compared the blood before and after death. He was able to demonstrate lactic acid in the blood of patients suffering from leukaemia, (pernicious) anaemia, congestive heart failure, chronic obstructive pulmonary disease, pleuritis, pericarditis, pneumonia and several solid malignant tumours.\nGaglio\u00a0[26] is often erroneously mentioned as the first author to find lactic acid in blood\u00a0[27\u201329]. He was able to demonstrate lactic acid in fresh arterial blood withdrawn from dogs and rabbits after bloodletting. Berlinerblau\u00a0[30] confirmed these observations in mammalian and venous human blood. Both Gaglio and Berlinerblau, however, neglected previous research, as indignantly described by Salomon in 1888 [\u201cIch erlaube mir, den Inhalt meiner Arbeiten, die von Gaglio nur ganz fl\u00fcchtig, von Berlinerblau gar nicht ber\u00fchrt sind, in K\u00fcrze zu reproduciren\u201d (I take the liberty of summarizing the contents of my work, which was mentioned only briefly by Gaglio and not at all by Berlinerblau)]\u00a0[31].\nThe Japanese chemist Trasaburo Araki showed that the amount of lactic acid in exhausted muscle results from muscle activation\u00a0[11]. Irisawa\u00a0[32], inspired by the results obtained by Salomon and Gaglio, obtained fresh blood of 11 dying patients with serious conditions. In six cases he found hyperlactataemia, in four cases normal values. He speculated on the aetiology of hyperlactataemia, the most plausible cause being the severe hypoxia during the dying process. In an experiment in which he made a\u00a0dog anaemic for several days, he found a\u00a0rise in lactic acid levels during the time leading up to death.\nIn Cambridge (UK), Walter Morley Fletcher (1873\u20131933) and Frederick Gowland Hopkins (1861\u20131947) worked together on the metabolic changes occurring in muscular contractions and rigor mortis under anaerobic conditions, and found that lactate was the product of carbohydrate metabolism\u00a0[33]. Their classic 1907 paper demonstrated rigorously that muscle contraction is accompanied by the anaerobic formation of lactic acid, which is removed aerobically, at a\u00a0rate depending on the level of exposure to oxygen\u00a0[34].\nPoul Astrup and John Severingshaus mentioned Scherer's 1851 article as first demonstration of lactic acid in blood, but overlooked the 1843 cases and Folwarczny's work\u00a0[35]. In conclusion, Scherer's 1843 case reports\u00a0[15] should be cited as the first description of lactic acid in human blood and also as the first demonstration of lactic acid as a\u00a0pathological finding in septic and haemorrhagic shock. Folwarczny, in 1858, was the first to demonstrate lactic acid in blood in a\u00a0living patient.","keyphrases":["lactic acid","shock","johann joseph scherer","lactate","leukaemia","historical article"],"prmu":["P","P","P","P","P","M"]}
{"id":"Anal_Bioanal_Chem-4-1-2287205","title":"Reversed-phase liquid chromatography coupled on-line to estrogen receptor bioaffinity detection based on fluorescence polarization\n","text":"We describe the development and validation of a high-resolution screening (HRS) platform which couples gradient reversed-phase high-performance liquid chromatography (RP-HPLC) on-line to estrogen receptor \u03b1 (ER\u03b1) affinity detection using fluorescence polarization (FP). FP, which allows detection at high wavelengths, limits the occurrence of interference from the autofluorescence of test compounds in the bioassay. A fluorescein-labeled estradiol derivative (E2-F) was synthesized and a binding assay was optimized in platereader format. After subsequent optimization in flow-injection analysis (FIA) mode, the optimized parameters were translated to the on-line HRS bioassay. Proof of principle was demonstrated by separating a mixture of five compounds known to be estrogenic (17\u03b2-estradiol, 17\u03b1-ethinylestradiol and the phytoestrogens coumestrol, coumarol and zearalenone), followed by post-column bioaffinity screening of the individual affinities for ER\u03b1. Using the HRS-based FP setup, we were able to screen affinities of off-line-generated metabolites of zearalenone for ER\u03b1. It is concluded that the on-line FP-based bioassay can be used to screen for the affinity of compounds without the disturbing occurrence of autofluorescence.\nIntroduction\nIn recent years, the presence of endocrine-disrupting compounds (EDCs) in the environment has become a major issue of concern in view of their effects on both human health and ecosystem integrity. The classes of substances that possess estrogenic activities include diverse manmade and natural chemicals (e.g., pesticides, plasticizers, polychlorinated biphenyls, phytoestrogens, natural hormones) and pharmaceuticals (e.g., ethinylestradiol, tamoxifen) [1, 2]. EDCs can interfere with the endocrine system by binding to the estrogen receptors \u03b1 (ER\u03b1) or \u03b2 (ER\u03b2) and mimicking the action of the naturally produced hormone estrogen or blocking the receptor for this hormone in target cells, thereby preventing its action [3]. Therefore, it is important to screen compounds and compound mixtures for their ability to bind to one of these receptors. Alternatively, the task of screening for compounds containing estrogenic and antiestrogenic properties is also very important from medical and pharmaceutical points of view [4].\nSeveral biodetection methodologies have meanwhile been developed to determine the bioaffinities of ligands towards ER\u03b1. These assays include relatively cumbersome radioligand binding assays [5], fluorescence enhancement based assays [6] and fluorescence polarization (FP) based binding assays [7]. A major drawback of these assays is that, in the presence of cross-reactive compounds, the response provided by the bioassay reflects the sum of the concentrations of all individual compounds and their affinities for the ER\u03b1. Moreover, matrix components, which are sometimes present in far higher concentrations than the analyte(s), interfere with the binding assay signals, thereby leading to false-positive or false-negative results [8]. Therefore, none of these methods can be applied for the identification of individual ER\u03b1 binding ligands in mixtures of compounds, as is the case with natural compound extracts, biological samples containing different EDCs, and drug metabolite mixtures.\nHowever, the introduction of the on-line high-resolution screening (HRS) technology enabled the screening of individual compounds in mixtures by coupling a separation technology, usually gradient HPLC, to post-column biochemical detection assays on-line [9\u201314]. Oosterkamp et al. [10] developed a HRS system to screen mixtures for compounds with an affinity for ER\u03b1. A similar setup has been used to screen natural plant product extracts for ER\u03b1 and ER\u03b2 binding activity [15] and to identify and screen cytochrome P450-derived metabolites of tamoxifen with ER\u03b1 affinity [2, 16]. The reported HRS ER\u03b1 bioaffinity assays are based on the increase of the fluorescent signal of the tracer compound coumestrol upon binding to the ER\u03b1 ligand binding domain (LBD). When the tracer is displaced by a compound with ER\u03b1 affinity that elutes from the HPLC column, it is seen as a negative peak in the baseline of the ER\u03b1 bioassay. Coumestrol, the fluorescent probe, has short excitation (340\u00a0nm) and emission (410\u00a0nm) wavelengths when bound to the ER\u03b1 LBD [6]. Since there are many potentially interfering fluorescent compounds that can be excited at 300\u2013350\u00a0nm and emit at 360\u2013430\u00a0nm in biomatrices, coumestrol is not appropriate for the precise and selective detection of EDCs in biosamples containing phytoestrogens such as flavonoids, coumestans or lignans. One solution to this problem is to use another tracer compound, thus making it possible to monitor the bioassay signal at higher excitation and emission wavelengths. However, no tracer compounds are currently available which also display the necessary fluorescence enhancement upon binding to the ER\u03b1, thus making it impossible to use them in a HRS ER\u03b1 setup. Another option is fluorescence polarization (FP) detection. FP is based upon the principle that interaction of a receptor with a fluorophore-containing ligand increases the effective size of the fluorophore through the formation of a receptor ligand complex. The increase in effective size reduces the rate of fluorophore rotation and increases the polarization over that of the unbound fluorophore [7]. FP instruments measure the rate of fluorophore rotation in solution rather than fluorescence intensity [17]. The polarization value is independent of the fluorophore concentration and intensity and has the advantage that it is minimally affected by autofluorescence, solution opacity or color, and is more tolerant to fluorescence quenching and light scattering [18].\nThe objective of the present study was to develop and evaluate the use of FP detection in combination with the on-line HRS technology to provide information about the ER\u03b1 affinities of individual compounds in complex mixtures. The assay is based on competition for the ER\u03b1 LBD between eluting compounds and a fluorescein-labeled estradiol derivative (E2-F), which was synthesized based on tracer structures described by Parker et al. [7] and Ohno et al. [19] for off-line FP-based ER assays. After synthesis of the tracer E2-F, the ER\u03b1 binding assay was first optimized in FP platereader format and subsequently transferred to flow-injection analysis (FIA) mode with FP detection by using an on-line FP detector cell, which was recently constructed by Kool et al. [20] to measure GPCR-mediated modulation of cAMP production. Finally, after optimization of the assay in FIA mode, the on-line FP detection system was coupled to a gradient HPLC system. In this so-called HRS setup we separated mixtures of known estrogenic compounds\u2014e.g., 17\u03b2-estradiol, 17\u03b1-ethinylestradiol and the phytoestrogens coumestrol, coumarol and zearalenone\u2014and screened the individual compounds for ER\u03b1 affinities. We also showed that this HRS ER\u03b1-FP system was suitable for screening off-line-generated cytochrome P450 (Cyt P450) metabolites of zearalenone for ER\u03b1 affinity. We conclude that we were able to incorporate an on-line FP detector cell into a HRS system to screen for the ER\u03b1 affinities of individual compounds in complex mixtures without incurring interference from the autofluorescence of the compounds.\nMaterials and methods\nMaterials\nTween 20, 17\u03b2-estradiol (E2), estrone (E1), 17\u03b1-ethinyl estradiol (EE2), diethylstilbestrol (DES), estriol, zearalenone, fluorescein-5-isothiocyanate (5-FITC) and coumestrol were obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands). ELISA blocking reagent was purchased from Roche (Mannheim, Germany). Dry THF was freshly distilled from LiAlH4. All other chemicals were of analytical grade and obtained from standard suppliers.\nSynthesis\nFluorescein-labeled estradiol derivative (E2-F) The fluorescein-labeled estradiol derivative (E2-F) was synthesized in analogy to methods described previously [7, 19] from estrone as the starting material (Fig.\u00a01). To a solution of estrone (1, 700\u00a0mg, 2.59\u00a0mmol) in dry DMF (3\u00a0mL), imidazole (250\u00a0mg) was added. Subsequently, tert-butyl dimethylsilyl chloride (tBDSC) dissolved in dry THF (2\u00a0mL) was slowly added and the reaction mixture was stirred at room temperature for 3\u00a0h. The resulting cloudy white mixture was extracted with ethyl acetate (EtOAc). The organic extract was washed with water once, with brine twice and then with water once again. The organic extract was dried over MgSO4, filtered, and concentrated by rotary evaporation at 45\u00a0\u00b0C. Recrystallization was performed using a mixture of 15\u00a0mL MeOH and 5\u00a0mL DCM to afford 2 (667.2\u00a0mg, 67%). Under an atmosphere of dry nitrogen, 3-butyn-1-ol (1.35\u00a0g) was dissolved in dry THF (27\u00a0mL) and cooled to \u221278\u00a0\u00b0C. n-Butyllithium (BuLi) in hexane (16.9\u00a0mL, 0.027\u00a0mol) was added dropwise at \u221278\u00a0\u00b0C. Compound 2 (3.60\u00a0g, 9.36\u00a0mmol) dissolved in dry THF (25\u00a0mL) was carefully added dropwise at \u221278\u00a0\u00b0C and the solution was allowed to warm to room temperature and was thereafter stirred at 40\u00a0\u00b0C for an additional 60\u00a0min. The remaining BuLi was neutralized by carefully adding 5\u00a0mL EtOAc\/MeOH (1:1). Extraction was performed with EtOAc, as described above. The crude product was purified by column chromatography on silica gel (10% EtOAc in hexane) to afford 3 (1.62\u00a0g, 38%). To a solution of 3 (1.6\u00a0g, 3.52\u00a0mmol) dissolved in dry THF (25\u00a0mL) was added triethylamine (TEA, 718\u00a0\u03bcL) followed by MeSO2Cl (395\u00a0\u03bcL, 5.1\u00a0mmol) while stirring at 0\u00a0\u00b0C. Afterwards the solution was allowed to warm to room temperature. After 10\u00a0h the reaction mixture was extracted with DCM. The organic extract was treated as described above and concentrated by rotary evaporation to afford 4 (1.80\u00a0g, 96%). Under an atmosphere of dry nitrogen, NaN3 (8.0\u00a0g) was added to a solution of 4 (1.80\u00a0g, 3.38\u00a0mmol) dissolved in dry DMPU (25\u00a0mL) while stirring rapidly. After stirring the solution for three\u00a0days, the resulting reaction mixture was extracted with EtOAc as described above to afford 5 (1.36\u00a0g, 84%). To a solution of 5 (1.36\u00a0g, 2.83\u00a0mmol) in MeOH (70\u00a0mL) and TEA (16\u00a0mL), 1,3-propane dithiol (12\u00a0mL) was added. The solution was stirred o\/n at room temperature and was concentrated by rotary evaporation. The concentrated solution dissolved in 100\u00a0mL hexane was purified by column chromatography on silica gel. Elution of the purified product 6 (1.08\u00a0g, 84%) was performed using EtOAc\/MeOH (1:1). To remove the protective tert-butyl dimethylsilyl group, TBAF.H2O (300\u00a0mg) was added to a solution of 6 (258\u00a0mg, 0.57\u00a0mmol) dissolved in dry THF (4\u00a0mL) at 0\u00a0\u00b0C. After stirring for 1\u00a0h and allowing the solution to warm to room temperature, the crude product was extracted with DCM. The product was purified with Flash Master chromatography with an EtOAc\/MeOH gradient to afford 7 (176\u00a0mg, 91%). Fluorescein-5-isothiocyanate (5-FITC, 7\u00a0mg, 17.8\u00a0\u03bcmol) was added to a solution of 7 (5\u00a0mg, 14.7\u00a0\u03bcmol) dissolved in dry DMF (500\u00a0\u03bcL) and dry pyridine (25\u00a0\u03bcL). The solution was stirred o\/n in the dark at room temperature. The crude product was extracted with DCM and purified first with Flash Master chromatography (silicagel-based; EtOAC\/MeOH gradient) and then with preparative C18 HPLC to afford 8 (E2-F, 8.02\u00a0mg, 75%). 1H NMR (400.13\u00a0MHz, CD3OD): \u03b4 8.04 (1\u00a0H, d, J\u2009=\u20091.9\u00a0Hz), 7.70 (1\u00a0H, dd, J\u2009=\u20098.2, 1.9\u00a0Hz), 7.10 (1\u00a0H, d, J\u2009=\u20098.2\u00a0Hz), 7.01 (1\u00a0H, d, J\u2009=\u20098.5\u00a0Hz), 6.89 (1\u00a0H, d, J\u2009=\u20098.9\u00a0Hz), 6.88 (1\u00a0H, d, J\u2009=\u20098.9\u00a0Hz), 6.66 (2\u00a0H, d, J\u2009=\u20092.3\u00a0Hz), 6.58 (2\u00a0H, dd, J\u2009=\u20098.9, 1.9\u00a0Hz), 6.47 (1\u00a0H, dd, J\u2009=\u20098.5, 2.6\u00a0Hz), 6.42 (1\u00a0H, d, J\u2009=\u20092.6\u00a0Hz), 3.80 (2\u00a0H, t, J\u2009=\u20096.6\u00a0Hz), 2.70 (1\u00a0H, d, J\u2009=\u20091.9\u00a0Hz), 2.65 (2\u00a0H, t, J\u2009=\u20096.6\u00a0Hz), 2.1\u20132.3 (3\u00a0H, m), 1.9\u20132.0 (2\u00a0H, m), 1.65\u20131.85 (4\u00a0H, m), 1.25\u20131.50 (5\u00a0H, m), 0.85 (3\u00a0H, s). ESI-MS: m\/z 729 (M + H)+.\nFig.\u00a01Synthesis of a fluorescein-labeled estradiol (E2-F) derivative (for abbreviations, see \u201cExperimental\u201d section)\n7-Hydroxy-3-p-hydroxyphenylcoumarine (coumarol) The synthesis of coumarol was analogous to the synthesis of benzene-hydroxylated-3-aryl-coumarins described by Buu-Hoi et al. [21]. Briefly, 2,4-dimethoxybenzaldehyde was reacted with p-methoxyphenyl-acetonitrile to form \u03b1-p-methoxyphenyl-\u03b2-(2,4-dimethoxyphenyl)-acrylonitrile. The formed product formed was demethylated using pyridine hydrochloride to form the final product coumarol. 1H NMR (400.13\u00a0MHz, DMSO-d6): \u03b4 10.57 (1\u00a0H, s), 9.68 (1\u00a0H, s), 8.00 (1\u00a0H, s), 7.56 (1\u00a0H, d, J\u2009=\u20098.6\u00a0Hz), 7.53 (2\u00a0H, d, J\u2009=\u20098.7\u00a0Hz), 6.81 (2\u00a0H, d, J\u2009=\u20098.7\u00a0Hz), 6.80 (1\u00a0H, dd, J\u2009=\u200910.8, 2.7\u00a0Hz), 6.73 (1\u00a0H, d, J\u2009=\u20092.2\u00a0Hz). ESI-MS: m\/z 255 (M + H)+.\nBiomaterials\nMicrosomes Rat liver microsomes were prepared from phenobarbital (PB)-induced rat liver, as described previously by Roosenboom et al. [22].\nEstrogen receptor \u03b1 The ER\u03b1 ligand binding domain (LBD) was expressed in Escherichia coli according to the method of Eiler et al. [23], but without estradiol in the medium. The concentration of ER\u03b1 LBD (250\u00a0nM) was measured by determining the Bmax value (by titration with radiolabeled estradiol) [24]. The Bmax value was measured as the maximum amount of ligand binding extrapolated to a very high concentration of ligand.\nMicrosomal incubations\nMicrosomal PB-induced rat liver incubations were carried out in a total volume of 500\u00a0\u03bcL. The reaction mixture consisted of 2.5\u00a0mM MgCl2 in 100\u00a0mM potassium phosphate buffer (pH\u20097.4), 100\u00a0\u03bcM zearalenone and 2.6\u00a0mg\/mL rat liver microsomes. The reactions were initiated by the addition of a NADPH regenerating system, resulting in final concentrations of 0.1\u00a0mM NADPH, 0.3\u00a0mM glucose-6-phosphate, and 0.4\u00a0U\/mL glucose-6-phosphate dehydrogenase. Incubation was carried out at 37\u00a0\u00b0C for 120\u00a0min and the reaction was terminated by the addition of 50\u00a0\u03bcL 10% HClO4. Following centrifugation at 14,000\u00a0rpm for 10\u00a0min, 450\u00a0\u03bcL of supernatant was applied to a strata_X 33\u00a0\u03bcm Polymeric Sorbent 200\u00a0mg \/ 3\u00a0mL (Phenomenex, Amstelveen, The Netherlands) column for SPE sample preparation. Before sample application the column was conditioned with 2\u00a0mL MeOH and subsequently equilibrated with 2\u00a0mL H2O. The sample was washed with 2\u00a0mL 5% (v\/v) of aqueous methanol and elution was performed using 2\u00a0mL 50% (v\/v) of MeOH in ACN. After evaporation, the residues were dissolved in 1\u00a0mL 40% (v\/v) of aqueous methanol.\nInstrumentation\nA NOVOstar+ microplate fluorometer from BMG LABTECH GmbH (Offenburg, Germany) was used for the FP-based microplate reader assay. The excitation wavelength was set at 485\u00a0nm, the emission wavelength at 520\u00a0nm. Black-bottomed PP-96-well microtiter plates from Greiner Bio-one (Alphen a\/d Rijn, The Netherlands) were used for all microplate reader experiments.\nThe setup we used was adapted from the homogeneous coumestrol-based ER\u03b1 affinity detection system described by Kool et al. [2]. For the on-line FP assay, two Knauer K-500 HPLC pumps (Berlin, Germany) were used to control the 120-mL superloops, made in-house, containing receptor (ER\u03b1) and tracer ligand (E2-F), respectively, and one Knauer K-500 HPLC pump was used to deliver the injected samples. The ER\u03b1 and E2-F solutions were prepared in sodium phosphate buffer (10\u00a0mM; pH 7.4) containing 150\u00a0mM NaCl and 0.4\u00a0mg\/mL ELISA blocking reagent. Both superloops were kept on ice. Flow restrictors were inserted between the pumps and the superloops to ensure proper operation of the pumps at low flow rates. The flow restrictors consisted of natural PEEK tubing and resulted in back pressures of approximately 50 bar. The pressure limits of the pumps were set 20 bar higher than the working pressure, and VICI Jour backpressure regulators (Schenkon, Switzerland) were inserted after the superloops to prevent damage from possible clogging of the system. A Gilson 234 autoinjector (Villiers-le-Bel, France) equipped with a Rheodyne (Bensheim, Germany) six-port injection valve (different injection loops) was used to inject samples. To maintain the reaction coils at a constant temperature, a Shimadzu CTO-10AC column oven (Duisburg, Germany) was integrated into the system. An Agilent 1100 (Waldbronn, Germany) series fluorescence detector (\u03bbex 498\u00a0nm; \u03bbem 522\u00a0nm) was used to monitor fluorescence polarization, as described by Kool et al. [20]. Briefly, the standard 8\u00a0\u03bcL FLD cell was modified with parallel-placed (both mounted in the detector cell) fluorescence polarization filters from Melles Griot (Didam, The Netherlands). One filter (12.5\u00a0mm visible pol. 350\u2013650\u00a0nm 03FPG019) was placed in the pathway of the excitation beam and one (Mount. Sheet Pol. 20.D- 16.9CA 03FPG001) in the pathway of the emission beam. All hardware was integrated into one HRS system by Kiadis B.V. (Groningen, the Netherlands), and was controlled by software developed by Kiadis B.V.\nCoumestrol-based microplate reader assay of ER\u03b1 affinity\nThe microplate reader assay for determining ER\u03b1 binding based on the competition between fluorescent coumestrol and estrogenic compounds previously described by Gurer-Orhan et al. [6] was used to plot a competitive binding curve for E2-F.\nFP-based microplate reader assay of ER\u03b1 affinity\nThe microplate reader assay was used to optimize the FP ER\u03b1 receptor affinity detection (RAD) system in FIA and HPLC modes. This assay is based on the competition between estrogenic compounds and the newly synthesized fluorescein-labeled estradiol derivative (E2-F) for binding to the ER\u03b1 LBD. Displacement of E2-F was measured as the decrease in fluorescence polarization intensity. Different concentrations of E2-F tracer were tested first. Since Tween 20, a detergent also used by Kool et al. in their coumestrol-based HRS ER\u03b1 assay [2], can improve the resolution of the assay in HRS mode, and since organic modifiers are necessary for the optimal performance of the gradient HPLC system, the next step in the optimization process was to vary these parameters. The experiments were carried out at 24\u00a0\u00b0C with well volumes of 250\u00a0\u03bcL binding buffer [potassium phosphate buffer (10\u00a0mM; pH 7.4) containing 0.4\u00a0mg\/ml ELISA blocking reagent]. The ER\u03b1 LBD was introduced into the binding assay buffer at a final concentration of 1.64\u00a0nM. The E2-F stock solution was prepared in DMSO and dilutions were made in binding buffer. Concentrations ranging from 0.42\u00a0nM to 3.31\u00a0\u03bcM were investigated for E2-F optimization. For the experiments to investigate the detergent and organic modifier effects, the final concentration of E2-F in the assay was 83.3\u00a0nM. Aliquots of 25\u00a0\u03bcL of organic modifier or detergent at various concentrations were pipetted into a 96-well plate that already contained either 75\u00a0\u03bcL binding buffer (equal to 0% displacement) or 75\u00a0\u03bcL 33.3\u00a0\u03bcM E2 in binding buffer (equal to 100% displacement). The 96-well plate was gently but thoroughly mixed after pipetting 75\u00a0\u03bcL E2-F into each well. Finally, 75\u00a0\u03bcL of the ER\u03b1 LBD was pipetted into the wells. After an incubation time of 60\u00a0min at room temperature, the fluorescence polarization (FP) was recorded at 485\u00a0nm excitation and 520\u00a0nm emission. All experiments were performed in quadruplicate. The difference in FP (\u0394FP) was calculated by subtracting the FP value found at 100% displacement from the FP value found when no E2 was present under different conditions.\nFP-based ER\u03b1 receptor affinity detection (RAD) in flow-injection analysis (FIA) mode\nA schematic overview of the RAD system in FIA mode is depicted in Fig.\u00a02. In this system the flow from a superloop containing ER\u03b1 LBD is continuously mixed with flow from a superloop containing E2-F. Compound samples (10\u00a0\u03bcL) are introduced into the system by flow injection into a carrier solution (100\u00a0\u03bcL\/min). During optimization, the carrier solution was water. Injected compounds and ER\u03b1, added to the carrier stream via an inverted Y-type mixing union at a flow rate of 100\u00a0\u03bcL\/min, were allowed to bind in a Tefzel reaction coil (25\u00a0\u03bcL). This mixture was combined with the flow of the E2-F solution of 100\u00a0\u03bcL\/min. The final equilibrium between ER\u03b1, compound, and E2-F was established in a knitted 0.5\u00a0mm i.d. PTFE reaction coil (250\u00a0\u03bcL). The temperatures of both reaction coils were maintained constant at 37\u00a0\u00b0C. Fluorescence polarization (FP) detection took place directly after the second reaction coil. When the binding of E2-F is decreased due to competition from an eluting ER\u03b1 ligand, depolarization occurs. This decrease in fluorescence polarization is a measure of the affinity of the ligand towards ER\u03b1.\nFig.\u00a02Schematic overview of the ER\u03b1 fluorescence polarization receptor affinity detection (RAD) system in high-resolution screening (HRS) mode. Samples are injected with an autoinjector (A.I.) and are separated using a gradient reversed-phase high-performance liquid chromatography (HPLC) system controlled by P1 and P2, HPLC gradient pumps. After the column the flow is split by a flow spitter, S (split ratio 1:9). HPLC elution is monitored by UV or fluorescence detection. A makeup gradient, controlled by makeup gradient pumps P3 and P4, is introduced and eluting compounds are mixed in the first reaction coil with ER\u03b1 delivered by superloop-1 (SL-1). In the second reaction coil, the probe ligand E2-F is added to the mix by superloop-2 (SL-2). ER\u03b1 affinity detection is performed using a fluorescence detector (FLD). In FIA mode the samples are delivered directly by a carrier solution into the bioassay\nFP-based ER\u03b1 receptor affinity detection coupled on-line to gradient HPLC\nCoupling of the homogeneous ER\u03b1 FP RAD assay to HPLC was performed in a similar way to that described recently by Kool et al. [14] for a Cyt P450 affinity assay. When operating the ER\u03b1 FP RAD system in HPLC mode (Fig.\u00a02), two pumps were used to control the HPLC gradient and, directly after the HPLC column, the HPLC eluent was split into a 1:9 ratio where nine-tenths of the flow was directed to either an UV (Agilent 1100 series) or fluorescence detector (Agilent 1100 series). The choice of detector depended on the compounds to be analyzed. To compensate for increasing the organic modifier concentrations before the delivery of the HPLC eluent to the ER\u03b1 FP RAD system, two pumps were used to supply a makeup flow consisting of an opposite H2O\/MeOH gradient compared with the HPLC gradient in order to keep the MeOH concentration in the assay constant at 10%. One-tenth of the total makeup flow was combined with one-tenth of the HPLC eluent and this flow was directed to the ER\u03b1 FP RAD at 160\u00a0\u03bcl\/min. The remaining part of the flow in the ER\u03b1 FP RAD was the same as described above, with the exception that the HPLC flow combined with the makeup flow replaced the carrier solution flow.\nFor HPLC analysis, test compounds were dissolved in 50% MeOH in water. For coumestrol, a linear gradient from 75% MeOH to 99% MeOH in 7.5\u00a0min, constant for 47.5\u00a0min, and back to 75% MeOH in 1\u00a0min was applied. The total flow of the HPLC pumps was 162\u00a0\u03bcL\/min and detection took place by measuring UV at 254\u00a0nm. For the mixture containing estriol, coumestrol, coumarol, E2 and zearalenone, constant elution at 55% MeOH for 40\u00a0min, followed by a linear gradient from 55% MeOH to 70% MeOH in 50\u00a0min, constant for 50\u00a0min, and back to 55% MeOH in 1\u00a0min was applied. The total flow rate of the HPLC pumps was 229\u00a0\u03bcL\/min and detection took place using UV at 254\u00a0nm. For zearalenone and its cytochrome P450-generated metabolites, a linear gradient from 40% MeOH to 99% MeOH in 40\u00a0min, constant for 30\u00a0min, and back to 40% MeOH in 1\u00a0min was applied. The total flow rate of the HPLC pumps was 162\u00a0\u03bcL\/min and detection took place using a fluorescence detector (\u03bbex 279\u00a0nm; \u03bbem 440\u00a0nm). All HPLC separations were carried out on a 150\u2009\u00d7\u20094.6\u00a0mm i.d. Luna C18(2) column protected with a 2.0\u2009\u00d7\u20095.0\u00a0mm i.d. C18 guard column (Phenomenex, Amstelveen, The Netherlands). The HPLC column was thermostated at 25\u00a0\u00b0C.\nResults and discussion\nThe aim of this research was to combine a recently developed on-line FP detector cell (Kool et al. [20]) with high-resolution screening (HRS) technology to develop a sensitive screening platform for measuring the ER\u03b1 affinities of individual components in mixtures of compounds without incurring interference from autofluorescence at wavelengths below 450\u00a0nm. We first synthesized a suitable tracer compound and used this tracer to set up a FP-based ER\u03b1 binding assay in FP microplate reader format. The optimized assay conditions for the FP microplate reader format were transferred to an on-line bioassay format in FIA mode. In this mode we subsequently optimized the on-line assay conditions and validated them by measuring IC50 values of known estrogenic compounds in both microplate reader format and in FIA mode. We used the optimized conditions in FIA mode to couple the ER\u03b1 bioassay on-line to a gradient HPLC system. In HPLC mode we measured the ER\u03b1 bioaffinities of different estrogenic compounds and screened mixtures with five known estrogenic compounds. Finally, we measured the individual ER\u03b1 bioaffinities of zearalenone and its metabolites generated off-line by Cyt P450 in HPLC mode.\nSynthesis of E2-F\nSynthesis of the fluorescein-labeled derivative E2-F (Fig.\u00a01) was relatively straightforward. The synthetic route described in this paper makes use of readily available chemicals. The yields of intermediate products are relatively high except for the product of the second reaction step, in which compound 2 is converted into 3. However, the required reactants for this step are not expensive, which makes it possible to perform this step on a large scale. The last and most expensive step involving the coupling of 5-FITC to 7 still gives a yield of 75% after extraction and purification. Overall, we conclude that this synthetic protocol offers a good alternative to the synthetic scheme described in the literature [7, 19] that is used to produce a fluorescein-labeled E2 derivative. We describe a seven-step synthesis with an overall yield of 12% compared with an eight-step synthesis in the literature with a total yield of 7% [19].\nTo test whether the newly synthesized E2-F had affinity for the ER\u03b1, a competitive binding curve for E2-F and ER\u03b1 LBD was constructed (data not shown) using the coumestrol-based microplate reader assay described by Gurer-Orhan et al. [6]. E2-F was able to displace coumestrol from the ER\u03b1 binding site, resulting in an IC50 value of 28.2\u00a0nM. This value is consistent with KD values found for similar tracers, which were also in the nanomolar range [7, 19]. Therefore, we concluded that the synthesized E2-F had affinity for the ER\u03b1 and could be used to further develop a FP-based microplate reader assay.\nFP-based microplate reader assay of ER\u03b1 affinity\nThe first step in the optimization of the FP-based microplate reader ER\u03b1 affinity assay was the investigation of the influence of E2-F tracer concentration upon the FP-based microplate reader assay response of ER\u03b1-ligands. To achieve this we compared the FP signal obtained with 0% ligand displacement (no E2 present in the incubation) with the FP signal with 100% ligand displacement (33.3\u00a0\u03bcM E2 present in the incubation). We also incubated different concentrations of E2-F tracer with a fixed concentration of ER\u03b1. The difference in fluorescence polarization [\u0394FP in millipolarization (mP)] was plotted against decreasing E2-F concentrations (Fig.\u00a03). From this figure it can be seen that \u0394FP increases with decreasing tracer E2-F concentrations until it reaches a maximum at 166\u00a0nM. However, it should also be noted that when the E2-F tracer concentration decreases, the error bars increase, which means that the S\/N ratios become worse. For the ER\u03b1-binding assay, we preferred to use a tracer concentration which is not too high, because high tracer concentrations make it necessary to use high concentrations of test compounds as well in order to displace the tracer from the receptor, which results in a decreased assay sensitivity. However, if we use a tracer concentration which is too low, this will also result in unfavorable S\/N ratios. Based upon our results and the KD values of 10.4 and 3\u00a0nM found by Ohno et al. [19] and Parket et al. [7] respectively for structurally similar fluorescein-labeled E2 derivatives, we decided to use an E2-F tracer concentration of 83.3\u00a0nM for further optimization experiments. At this concentration the ER\u03b1 response was high, which makes it easier to detect the tracer\u2013receptor complex in an on-line assay, with a low S\/N ratio, and with the concentration in the same range as the KD value, which makes it possible to determine IC50 values more accurately.\nFig.\u00a03FP-based microplate reader tracer concentration optimization. Incubations in the presence of ER\u03b1 ligand E2 (33.3\u00a0\u03bcM; equal to 100% tracer displacement) were compared with incubations without E2 (equal to 0% tracer displacement) at decreasing E2-F tracer concentrations\nThe second step in the optimization process was the investigation of the influence of the detergent Tween 20 and the solvents methanol (MeOH), acetonitrile (ACN) and isopropanol (iPrOH) on assay performance using the conditions as optimized in the previous step. Experiments revealed that Tween 20 was not compatible with the ER\u03b1 FP assay (data not shown). At very low concentrations (0.04%), the presence of Tween 20 in the assay caused a large decrease in \u0394FP between E2-F bound to ER\u03b1 (0% displacement; absence of E2) and E2-F displaced by 33.3\u00a0\u03bcM E2 (100% displacement). Although the addition of Tween 20 can give improved resolution in terms of peak width [25], we decided not to use it due to the large decrease in the difference in polarization. In Fig.\u00a04, the effect of increasing concentrations of MeOH, ACN and iPrOH on \u0394FP is shown. It can be seen from this figure that \u0394FP decreases with increasing organic modifier concentrations. \u0394FP decreased to almost zero for ACN at the highest concentration. For MeOH and iPrOH, however, a signal difference between the E2-F bound to ER\u03b1 and displaced by E2 could still be observed at the highest modifier concentration. Oosterkamp et al. [10] have stated that denaturation of the estrogen receptor occurs upon prolonged contact with organic solvents such as MeOH and ACN. It is therefore reasonable to assume that when the organic modifier is present at higher concentrations, the denaturation process for ER\u03b1 LBD increases, which results in smaller fluorescence polarization signal differences.\nFig.\u00a04FP-based microplate reader solvent optimization. Incubations in the presence of ER\u03b1 ligand E2 (33.3\u00a0\u03bcM; equal to 100 % tracer displacement) were compared with incubations without E2 (equal to 0% displacement) at increasing MeOH, ACN and iPrOH concentrations at an E2-F concentration of 83.3\u00a0nM\nFP-based ER\u03b1 receptor affinity detection (RAD) in flow-injection analysis (FIA) mode\nThe optimized microplate reader assay conditions were transferred to the on-line ER\u03b1 RAD system in FIA mode (Fig.\u00a02). In this system, compounds introduced by a carrier solution are allowed to bind to the ER\u03b1 LBD in a first reaction coil, and the E2-F tracer is introduced after this coil. The tracer compound can interact with the unbound receptor in a second reaction coil and detection takes place after the second reaction coil using FP detection. In a FIA setup, carrier solution replaces the gradient HPLC system, thereby allowing the direct injection of compounds and the optimization of on-line assay conditions before final on-line coupling to HPLC. The influence of the ER\u03b1 LBD and E2-F tracer concentrations and the corresponding excitation and emission wavelengths were investigated in FIA mode in order to optimize the performance of the FP ER\u03b1 RAD system. The influences of ACN and MeOH, later used in HPLC mode too, on the FP ER\u03b1 RAD system performance were also tested in the FIA mode. All measurements were performed in duplicate.\nER\u03b1 LBD and E2-F tracer optimization Different receptor concentrations (ER\u03b1 LBD) were tested in combination with different tracer concentrations (E2-F) by varying the flows of SL-1 (containing 10\u00a0nM ER\u03b1 in binding buffer) and SL-2 (containing 1\u00a0\u03bcM E2-F in binding buffer). The total flow rate was kept constant by introducing a compensating superloop containing only binding buffer. Water was used as carrier solution in order to inject 10\u00a0\u03bcL 100\u00a0\u03bcM E2 in 50% MeOH samples (100% ligand displacement) in duplicate followed by 10\u00a0\u03bcL injections of 50% MeOH (0% displacement) at a flow rate of 250\u00a0\u03bcL\/min. The results obtained with these experiments are depicted in Fig.\u00a05. This figure shows that as the E2-F tracer concentration increases the FP signal increases too, until it reaches a maximum. Using the present FIA FP ER\u03b1 RAD setup, we were able to determine the KD and maximum response (Rmax) values for the E2-F tracer compound (Table\u00a01) at different ER\u03b1 concentrations. The KD values found were of the same order as observed by Ohno et al. [19] and Parker et al. [7] for an analogous FP tracer compound. It can be seen that  Rmax increases linearly with higher ER\u03b1 concentrations, as was expected. Based upon these results, we conclude that the FP ER\u03b1 RAD in FIA mode worked well, and we continued by investigating the influence of the organic modifier upon the assay performance.\nFig.\u00a05Determination of KD and maximum response (Rmax) for E2-F in FIA mode. Different concentrations of E2-F were incubated with three different ER\u03b1 concentrationsTable\u00a01KD and maximum response (Rmax) determination for E2-F in FIA mode[ER\u03b1] (nM)KD (nM)Rmax (AUC)a0.743.6\u2009\u00b1\u200912.42.17\u2009\u00d7\u20091061.526.4\u2009\u00b1\u20095.04.04\u2009\u00d7\u20091063.034.5\u2009\u00b1\u20095.39.38\u2009\u00d7\u2009106aRmax was determined by integrating the decrease in FP signal, and arbitrary units were chosen to display this decrease\nOrganic modifier concentration Although we also investigated the influence of iPrOH upon the ER\u03b1 bioaffinity assay performance in the FP-based microplate reader format, we decided to evaluate only MeOH and ACN in FIA mode based upon the fact that we planned to use only these two solvents in HPLC mode. Different concentrations of MeOH and ACN were used as the carrier solution in order to inject 10\u00a0\u03bcL 100\u00a0\u03bcM E2 ligand in 50% MeOH samples (100% ligand displacement) in duplicate followed by 10\u00a0\u03bcL injections of 50% MeOH (0\u00a0\u03bcM E2 ligand; 0% displacement) into the system. The ER\u03b1 LBD concentration in the second reaction coil was 2.7\u00a0nM, the E2-F tracer concentration 110\u00a0nM and the total flow rate 300\u00a0\u03bcL\/min. Increasing the concentration of MeOH decreased the S\/N ratio in FIA mode (Fig.\u00a06). A similar effect was observed with ACN. A possible explanation for this decreasing effect is the partial denaturation of the estrogen receptor due to the presence of high concentrations of organic modifier. Oosterkamp et al. [10] have described this phenomenon during the optimization of the ER\u03b1 coumestrol-based RAD system when a decrease in response was observed upon the contact of the ER\u03b1 with organic solvents such as MeOH and ACN. One advantage of a higher organic modifier concentration is that higher flow rates can be used in HPLC gradients, which may result in shorter analysis times and higher resolutions. Another advantage is that high concentrations of organic modifier prevent lipohilic compounds from adsorbing to the walls of the reaction coils and other tubing, which may increase resolution. Based upon our results during FP FIA setup, we concluded that both MeOH and ACN might be used in the on-line gradient HPLC system.\nFig.\u00a06Effect of MeOH and ACN reaction coil concentrations upon the assay performance in FIA mode. The S\/N ratios of the E2 injections equalling 100% tracer displacement are displayed\nExcitation and emission wavelengths When the excitation and emission wavelengths were varied, the optimal FP signal responses in FIA mode were obtained at an excitation wavelength of 498\u00a0nm and an emission wavelength of 522\u00a0nm (data not shown). These wavelengths were therefore used to further optimize the on-line FP system in FIA mode.\nCompetitive receptor binding curves To validate the ER\u03b1 FP RAD system, competitive binding curves for five known estrogenic test compounds\u2014DES, E2, EE2, tamoxifen and coumestrol\u2014were constructed using the FP-based microplate reader assay, and these values were compared with the IC50 values obtained using the ER\u03b1 FP setup in FIA mode. The ER\u03b1 LBD concentration was 1.6\u00a0nM and the E2-F tracer concentration was 80\u00a0nM for the microplate reader assay. In FIA mode, the ER\u03b1 LBD concentration in the second reaction coil was 2.7\u00a0nM and the E2-F concentration was 110\u00a0nM. All five test compounds displayed sigmoidal concentration\u2013response curves during both the microplate reader setup and in FIA mode, and from these curves we were able to determine the IC50 values (Table\u00a02). The values found in the two different setups were similar for each individual compound and in agreement with values obtained from the literature [7, 26]. We thus demonstrated that the FP-based microplate reader assay and the ER\u03b1 FP setup in FIA mode could be employed to determine the IC50 values of a wide range of typical ER\u03b1-binding ligands accurately. The next step in the process of developing the HRS platform was to couple the FIA ER\u03b1 FP system to a gradient HPLC system on-line.\nTable\u00a02IC50 values determined in FIA mode and in microplate reader assay formatCompoundFIA mode IC50 (M \u00b1 SEM; n\u2009=\u20093)Microplate setup IC50 (M \u00b1 SEM; n\u2009=\u20093)Literature data IC50 (M)DES6.9\u2009\u00b1\u20090.3\u2009\u00d7\u200910\u221296.1\u2009\u00b1\u20090.6\u2009\u00d7\u200910\u221293.5\u2009\u00d7\u200910\u22129 aTamoxifen1.2\u2009\u00b1\u20090.1\u2009\u00d7\u200910\u221271.3\u2009\u00b1\u20090.4\u2009\u00d7\u200910\u221271.9\u2009\u00d7\u200910\u22127 aCoumestrol1.8\u2009\u00b1\u20090.2\u2009\u00d7\u200910\u221272.5\u2009\u00b1\u20090.3\u2009\u00d7\u200910\u221273.6\u2009\u00d7\u200910\u22127 bE26.7\u2009\u00b1\u20090.8\u2009\u00d7\u200910\u221293.3\u2009\u00b1\u20090.3\u2009\u00d7\u200910\u221295.6\u2009\u00d7\u200910\u22129 aEE21.6\u2009\u00b1\u20090.2\u2009\u00d7\u200910\u221293.5\u2009\u00b1\u20090.8\u2009\u00d7\u200910\u221292.3\u2009\u00d7\u200910\u22129 bIC50 values of five estrogenic compounds towards the ER\u03b1, as measured with the RAD system in FIA mode and with the microplate reader assay format. Data from literature a[7], b[26] are also shown. DES, diethylstilbestrol; E2, 17\u03b2-estradiol; EE2, 17\u03b1-ethinyl estradiol\nFP-based ER\u03b1 receptor affinity detection coupled on-line to HPLC\nFollowing assay optimization in the microplate reader and FIA modes, the ER\u03b1 FP RAD assay was coupled on-line to gradient HPLC. In this HRS setup the flow eluting from an HPLC column was coupled directly to the FP RAD system, which enables compound separation prior to the screening of individually eluting compounds for their ER\u03b1 affinities. This allowed us to screen for compounds with ER\u03b1 affinities in mixtures. To test the performance of the on-line HRS FP ER\u03b1 system, different concentrations of the phytoestrogen coumestrol were injected first. A negative peak in the ER\u03b1 bioaffinity trace was observed at a retention time of 40\u00a0min, which is the exact time when coumestrol eluted from the HPLC column (data not shown). As expected, the negative bioaffinity peaks became larger upon injections of higher concentrations of coumestrol. The highest negative peaks were observed when all of the E2-F was displaced by coumestrol. Higher concentrations of coumestrol only caused more tailing of the bioaffinity peaks. These measurements illustrate that the ER\u03b1 bioaffinity of the phytoestrogen coumestrol could be measured sensitively and efficiently with the new E2-F based HRS ER\u03b1 FP system, which is not possible due to the occurrence of autofluorescence using the coumestrol-based ER\u03b1 HRS system developed by Oosterkamp et al. [10].\nRepresentative superimposed chromatograms of different concentrations of a mixture of five known estrogenic compounds\u2014coumestrol, coumarol, estriol, 17\u03b2-estradiol and zearalenone\u2014are depicted in Fig.\u00a07. Although some baseline drift in time is observed over two hours, it is quite possible to detect the ER\u03b1 bioaffinities of all five individual peaks in the mixture. The explanation for the baseline drift is most likely that the post-column makeup HPLC gradient does not totally compensate for the effect of variations in the organic modifier concentration. The FP ER\u03b1 RAD system is sensitive to changes in the organic modifier concentration, as shown during FIA optimization, and this can lead to baseline drifts.\nFig.\u00a07ER\u03b1 FP RAD trace of a mixture of five estrogenic compounds injected into the ER\u03b1 FP RAD HPLC system (injected compounds are as follows: estriol (1) [3.75\u00a0nmol; 49.5\u00a0min], coumestrol (2) [3.75\u00a0nmol; 53.7\u00a0min], coumarol (3) [37.5\u00a0nmol; 80.4\u00a0min], 17\u03b2-estradiol (4) [3.75\u00a0nmol; 102.8\u00a0min] and zearalenone (5) [12.5\u00a0nmol; 112.5\u00a0min]\nTo illustrate another application of the on-line HRS ER\u03b1 FP system, metabolites of the phytoestrogen zearalenone, which were anticipated to show affinity for the ER\u03b1, were analyzed in the present FP-based HRS ER\u03b1 system. For this purpose, zearalenone was metabolized by phenobarbital (PB)-induced rat liver microsomes in an off-line fashion, followed by analysis with the FP-based HRS system. The results are depicted in Fig.\u00a08. The UV trace of the t\u2009=\u20090\u00a0min incubation shows that zearalenone (Z) elutes at 51.4\u00a0min. The corresponding bioaffinity trace shows that zearalenone has affinity for ER\u03b1. The FLD trace of the t\u2009=\u2009120\u00a0min incubation shows that zearalenone is metabolized by rat liver microsomes into at least three major metabolites (M1: 33.5, M2: 39.5 and M3: 43.2\u00a0min). The corresponding bioaffinity trace demonstrates that all three major metabolites (M1\u20133) have affinity for ER\u03b1. LC\/MS measurements were performed to identify the three major metabolites. M1 had a molecular ion at an m\/z value of 333, which likely corresponds to a monohydroxylated zearalenone metabolite. M2 had a molecular ion at an m\/z value of 317, which is the exact same m\/z value as that of zearalenone itself. This can be explained by the relocation of the double carbon\u2013carbon bond next to the aromatic ring. However, more research must be performed to confirm this. M3 had a molecular ion at an m\/z value of 319, which corresponds well to the m\/z value of the zearalenone metabolite zearalenol described in the literature [27]. The metabolism of zearalenone in vitro by rat liver microsomes has been described very recently by Pfeiffer et al. [28], and they confirm the findings of M1 and M3. The present Cyt P450 metabolism experiment illustrates that it is possible to screen metabolic mixtures of phytoestrogenic compounds for ER\u03b1 affinity using the present FP-based HRS ER\u03b1 setup. However, no further structural elucidation of the zearalenone metabolites has been performed, because the goal of the present experiment was only to show that the on-line FP HRS ER\u03b1 assay can be used to screen for parent compounds and metabolites of phytoestrogens with ER\u03b1 affinity.\nFig.\u00a08a\u2013ba HPLC FLD chromatograms of zearalenone (Z) incubated for 0 and 120\u00a0min with PB-induced rat liver microsomes. M1, M2 and M3 are metabolites formed during the 120-min incubation period. b The corresponding ER\u03b1 FP affinity traces. The structure of zearalenone is depicted in the figure\nWe conclude that we succeeded in developing, for the first time, an on-line FP-based HRS platform to screen for ER\u03b1 affinity using the novel on-line FP detector cell recently described in a FIA mode by Kool et al. [20]. The sensitivity of the FP-based HRS platform is comparable to that of the coumestrol-based HRS platform, and no interference from fluorescence was observed at wavelengths below 450\u00a0nm. The novel on-line FP detector cell can in principle be applied to other HPLC-based bioaffinity assays which require FP detection. The present HRS ER\u03b1 FP system offers a novel technology for investigating the metabolic profiles of drugs and other compounds such as phytoestrogens which cannot be measured by the coumestrol-based HRS system due to the occurrence of autofluorescence from the test compounds.","keyphrases":["fluorescence polarization","high-resolution screening (hrs)","estrogen receptor \u03b1","phytoestrogens","receptor affinity detection (rad)","on-line bioaffinity assay"],"prmu":["P","P","P","P","P","R"]}
{"id":"Obes_Surg-4-1-2226018","title":"Psychosocial Functioning, Personality, and Body Image Following Vertical Banded Gastroplasty\n","text":"Background In addition to increased risks of morbidity and mortality, extreme obesity is substantially associated with psychosocial problems. Therefore, the ultimate goal of bariatric surgery should not only be reducing weight and counteracting comorbid conditions but also improving psychosocial functioning. In addition to being an important goal of bariatric surgery, enhanced psychosocial functioning may motivate patients to adhere to adequate health behavior to maintain the surgically established weight loss.\nIntroduction\nObesity is a major public health concern. Its prevalence is increasing globally [1]. In addition to an increased risk of morbidity and mortality caused by medical conditions [2], extreme obesity is associated with debilitating psychological and social consequences especially in patients seeking surgery [3\u20136]. Psychological consequences of extreme obesity are, among others, anxiety, depression, low self-esteem, and negative body image [5\u201310]. Socially, the extremely obese have to deal with prejudice, discrimination, and social isolation [11].\nBecause only surgical treatment results in long-term weight reduction and the improvement or cure of comorbidities, including reduction of psychopathology [3\u20136], the number of procedures performed has more than doubled within a few years [12]. However, studies show great variation in outcome. In contrast to research showing large and long-lasting improvements, other studies suggest that postoperative improvements lag behind functioning of normative groups or show a decline over the years [4, 5].\nEnhanced psychosocial functioning is an important goal of bariatric surgery. It may motivate patients to adhere to adequate health behavior to maintain the surgically established weight loss [13]. In addition to medical\u2013somatic and surgical\u2013technical factors, success of bariatric surgery is especially influenced by the extent of successful adjustment of eating patterns, which, for a substantial part, can be attributed to psychological factors. Therefore, success after bariatric surgery should not only reflect weight loss and improvement or cure of comorbid conditions but also improvements in eating behavior, psychosocial variables, and quality of life [14]. However, in contrast to physical changes, psychosocial changes are not studied as systematically or diligently yet [15]. To facilitate the identification of the psychological variables that are related to success and the development of treatment interventions to enhance adjustment and success, a better understanding of patients\u2019 psychosocial functioning after bariatric surgery is needed.\nMaterials and Methods\nThe present study was designed to increase our understanding of patients\u2019 changes in psychosocial functioning, personality, and body image during the first 2\u00a0years after vertical banded gastroplasty (VBG). The main research questions were: what specific improvements can be achieved, is postoperative functioning noticeable different from functioning in relevant normative groups, and do all patients profit? These questions were examined based on data collection preoperatively and 6, 12, and 24\u00a0months after VBG.\nProcedures\nThe restrictive operation technique used in our hospital is hand-assisted laparoscopic vertical banded gastroplasty, with a complete division between the vertical staple lines. VBG is a relative simple, safe, and cost-effective intervention with fewer side effects than other procedures [16]. Not withstanding this, mixed long-term results, such as weight regain and dietary problems, have been reported [17]. Contrary to the situation in the USA, in The Netherlands, the numbers of VBGs that have been performed in the last 5\u00a0years is rather stable, despite the fact that the number of adjustable gastric banding procedures is showing an enormous increase.\nMaterials\nEarlier studies of psychosocial factors in the surgical treatment of extreme obesity have not provided consistent findings, which may, in part, be caused by the employment of a wide variety of non-standardized assessment procedures of questionable reliability and validity [11]. In the present study, we assessed preoperative as well as postoperative psychosocial functioning, personality, and body image using a semi-structured interview and three standardized questionnaires. As interview-based psychological studies may be questionable, we will report here only on the questionnaire data. Preoperatively as well as on three follow-up assessment points, patients filled in the Symptom Checklist-90 (SCL-90) [18], the Dutch Personality Questionnaire (NVP) [19], and the Body Attitude Test (BAT) [20]. The SCL-90 is a multidimensional psychopathology indicator with eight subscales and one summarized score (psychoneuroticism); its psychometric qualities are satisfactory to good [21]. In addition to neuroticism, the NPV measures six personality characteristics; its psychometric qualities are also satisfactory to good [22]. The BAT measures subjective body experience and the attitude towards one\u2019s own body; it has three subscales and one summarized total score, is commonly used in Europe, and is a reliable and valid questionnaire [20].\nPatients\nIn the context of standard preoperative evaluation, 131 patients who underwent VBG in our hospital between January 2001 and October 2004 were psychologically assessed. In addition to the inclusion criteria determined by IFSO [23], we required 10% preoperative weight loss with a minimum of 12\u00a0kg. From these 131 patients, 104 (79.4%) could be included in our study; others did not respond to our repeated requests for follow-up or sending back questionnaires. The studied patient group consisted of 91 (87.5%) female and 13 (12.5%) male patients. Preoperatively, they had a mean age of 38.4\u2009\u00b1\u20098.3\u00a0years, an average body mass index (BMI) of 45.4\u2009\u00b1\u20095.1, and a mean percentage excess weight of 104.5\u2009\u00b1\u200922.1%.\nStatistical Analyses\nStatistical analyses were performed using the SPSS 14.0-packet (SPSS for Windows, Rel. 14.0. 2005, SPSS Inc., Chicago). To study changes over time in weight, psychosocial functioning, personality, and body image, mixed between-within subjects analyses of variance for repeated measures were performed (ANOVA; time as within-subjects factor, gender as between-subjects factor). To identify waning of changes, Bonferroni post hoc equations were used. To compare patients\u2019 questionnaire scores to normative data, we used one-sample t tests. To identify heterogeneity in postoperative psychosocial functioning, descriptive statistics were used using summarized scales whenever possible for reasons of clarity. In reporting excess weight (EW) and excess weight loss (EWL), we followed the guidelines of Deitel and Greenstein [24].\nResults\nWeight Loss\nOver time, using ANOVA, substantial and significant changes in mean BMI, EW, and EWL were found. From preoperative status to 2\u00a0years after surgery, BMI and EW significantly (P\u2009\u2264\u20090.001) decreased 13.5 points and 58.6%, respectively, which represent a large size (partial \u03b72\u2009=\u20090.83) [25]. After 2\u00a0years, using the criterion that an operation can be conceived of as successful when EWL \u226550% [26], 61% of the patients had a successful operation. Preoperatively, 91.3% of the patients were extremely obese; in contrast, 2\u00a0years after surgery, only 12% was extremely obese.\nPsychosocial Functioning\nOur earlier literature review suggests that psychopathology is related to extreme obesity, manifesting itself mostly as depression or anxiety [11].\nIn assessing psychosocial functioning, we used the SCL-90 to assess short-term psychosocial functioning [18] and the NPV neuroticism scale to measure long-term neuroticism [19]. With respect to all aspects, except for hostility, significant changes over time were found (for most of the comparisons, P\u2009\u2264\u20090.001; for neuroticism, P\u2009\u2264\u20090.01; for sleeping problems, P\u2009\u2264\u20090.05). For the majority of alterations, large effect sizes were found [25]. Post hoc analyses showed that compared to the preoperative status, for most aspects of psychosocial functioning, scores were significantly lower on all follow-up assessment points, reflecting better psychosocial functioning. However, depressive symptoms and neuroticism were only significantly lower 6\u00a0months and 1\u00a0year after surgery; sleeping problems were lower only 6\u00a0months after surgery. For most aspects of psychosocial functioning, there was only a significant decrease in scores from preoperative status to 6\u00a0months postoperatively, however, not between 6\u00a0months and 1\u00a0year or between 1 and 2\u00a0years postoperatively (Table\u00a01).\nTable\u00a01Postoperative changes in psychosocial functioning (mean\u2009\u00b1\u2009SD)SCL-90Preoperative6\u00a0months1\u00a0year2\u00a0yearsPartial \u03b72Anxiety14.3\u2009\u00b1\u20095.011.7\u2009\u00b1\u20092.4***12.1\u2009\u00b1\u20093.912.6\u2009\u00b1\u20095.6.23Phobic complaints9.3\u2009\u00b1\u20093.67.4\u2009\u00b1\u20091.1***7.5\u2009\u00b1\u20091.67.9\u2009\u00b1\u20093.1.20Depression24.8\u2009\u00b1\u20098.620.8\u2009\u00b1\u20096.3***20.9\u2009\u00b1\u20097.123.1\u2009\u00b1\u200911.3.18Somatic complaints21.2\u2009\u00b1\u20097.916.2\u2009\u00b1\u20093.5***17.0\u2009\u00b1\u20095.217.9\u2009\u00b1\u20096.7.29Obsessive15.0\u2009\u00b1\u20095.011.6\u2009\u00b1\u20092.7***12.0\u2009\u00b1\u20093.112.7\u2009\u00b1\u20095.1.28Sensitivity28.7\u2009\u00b1\u200910.423.0\u2009\u00b1\u20096.4***22.8\u2009\u00b1\u20096.124.2\u2009\u00b1\u200910.8.25Hostility7.3\u2009\u00b1\u20091.77.2\u2009\u00b1\u20092.07.1\u2009\u00b1\u20091.57.3\u2009\u00b1\u20092.4.06Sleeping problems5.7\u2009\u00b1\u20092.94.6\u2009\u00b1\u20092.3*4.9\u2009\u00b1\u20092.55.5\u2009\u00b1\u20093.1.09Psychoneuroticism139.5\u2009\u00b1\u200941.0113.0\u2009\u00b1\u200922.7***114.8\u2009\u00b1\u200926.4122.8\u2009\u00b1\u200945.2.30N93939393Neuroticism (NPV)11.98\u2009\u00b1\u20098.98.5\u2009\u00b1\u20097.4**8.0\u2009\u00b1\u20096.69.3\u2009\u00b1\u20098.8.15N94949494Because of listwise exclusion, n\u2009=\u200993 (SCL-90) or 94 (NPV) on all moments; Obsessive: obsessive compulsive behavior; Sensitivity: mistrust and interpersonal sensitivity.***P\u2009<\u20090.001**P\u2009<\u20090.01*P\u2009<\u20090.05\nFurthermore, we compared patients\u2019 mean scores on psychosocial functioning to corresponding scores obtained in relevant normative groups, reflecting the general population [18, 19]. Compared to those normative groups, mean preoperative scores for psychosocial functioning were significantly higher, except for hostility. However, with respect to preoperative neuroticism, no significant difference was found. Postoperatively, there was a mixed picture. In spite of these inconsistencies, the overall picture revealed that on most subscales and most follow-up assessment points, patients\u2019 mean scores were not significantly different from the corresponding scores obtained in the normative groups. Two years after surgery, patients\u2019 mean scores on measures of sleeping problems and somatic complaints were again significantly higher than the normative group\u2019s scores. As to neuroticism, patients\u2019 postoperative mean scores were significantly lower than the normative group\u2019s scores on all follow-up assessment points. Despite these differences, all postoperative patients\u2019 scores fell in the below mean, mean, or above mean categories of the normative groups\u2019 scores.\nPersonality\nBecause personality substantially influences health behavior, personality characteristics may be relevant for adjustment to surgery [27]. According to earlier literature reviews, extremely obese individuals can be described as, among others, immature persons having a poor impulse control and a low self-esteem [28]. With respect to postoperative personality change, mixed results have been reported. For instance, some studies suggest that patients still have more pathology than comparison groups [13], whereas other research suggests that personality remains largely unchanged [3].\nIn assessing personality with the NPV [19], over time, we only found, with a large effect size [25], a significant (P\u2009\u2264\u20090.01) change in self-esteem. Post hoc analyses revealed that compared to the preoperative status, self-esteem was significantly higher 1\u00a0year postoperatively. Six months after surgery, there was a tendency to significance (P\u2009=\u20090.06). Two years postoperatively, however, no significant difference was found. Also, no significant differences were found between follow-up assessment points (Table\u00a02).\nTable\u00a02Postoperative changes in personality characteristics (mean\u2009\u00b1\u2009SD)NPV subscalesPreoperative6\u00a0months1\u00a0year2\u00a0yearsPartial \u03b72Social anxiety8.9\u2009\u00b1\u20097.96.8\u2009\u00b1\u20096.57.0\u2009\u00b1\u20096.57.5\u2009\u00b1\u20097.1.0.07Rigidity24.4\u2009\u00b1\u20097.324.0\u2009\u00b1\u20097.924.2\u2009\u00b1\u20097.423.7\u2009\u00b1\u20097.80.03Hostility14.0\u2009\u00b1\u20098.514.1\u2009\u00b1\u20098.614.0\u2009\u00b1\u20098.915.3\u2009\u00b1\u20099.30.05.Egoism9.7\u2009\u00b1\u20094.710.6\u2009\u00b1\u20095.19.9\u2009\u00b1\u20094.810.4\u2009\u00b1\u20095.00.04Dominance13.9\u2009\u00b1\u20096.415.3\u2009\u00b1\u20097.515.3\u2009\u00b1\u20097.215.0\u2009\u00b1\u20097.30.06Self-esteem27.9\u2009\u00b1\u20096.029.7\u2009\u00b1\u20095.329.9\u2009\u00b1\u20095.5*28.6\u2009\u00b1\u20096.70.14N94949494Because of listwise exclusion, n\u2009=\u200994 on all moments.*P\u2009<\u20090.01\nWe did not find many significant personality changes, but compared to normative data [19], we did find some significant differences. Compared to a relevant normative group, reflecting the general population, patients\u2019 mean preoperative scores on social anxiety, dominance, and self-esteem were not significantly different. However, they were after surgery, albeit not on all follow-up assessment points and not always for both sexes. Postoperatively, patients\u2019 scores on social anxiety were lower, whereas their scores on dominance and self-esteem were higher than the corresponding scores obtained in the normative group. On scales of Hostility and Egoism, preoperatively, patients had significant lower mean scores; postoperatively, this was only the case for some small subgroups. On the Rigidity measure, patients had significantly lower mean scores on all measurement moments preoperatively as well as postoperatively. All patients\u2019 mean scores fell in the below mean, mean, or above mean categories of the normative group\u2019s scores.\nBody Image\nSome studies suggest that especially for female patients, the desire to improve their physical appearance and avoidance of embarrassment are the most common motivators for bariatric surgery [29]. Also, improved body image would be one of the main reasons for post-surgical psychological improvement, better social integration, and enhanced quality of life [30]. Weight loss after bariatric surgery leads to marked improvements in body image and attractiveness and to less shame. However, with time, some patients still feel overweight, are discontent with the increasing skin folds [13], or still may have more concern with their body shape and size than controls [31].\nIn our earlier study, we found that preoperatively, patients reported significantly higher body dissatisfaction and negative body image than general population samples [28]. In the present study, using the BAT [20], over time, we found significant (P\u2009\u2264\u20090.001) changes in negative appreciation of body size, lack of familiarity with one\u2019s own body, general body dissatisfaction, and the summarized BAT score, reflecting improved body image and large effect sizes [25]. Post hoc analyses showed that compared to the preoperative status, scores were significantly (P\u2009\u2264\u20090.001) lower on all three follow-up assessment points. We found significant decreases between preoperative status and 6\u00a0months postoperatively, however, not between 6\u00a0months and 1\u00a0year or between 1 and 2\u00a0years after surgery (Table\u00a03). Also, in contrast to the preoperative situation, on all three follow-up assessment points, body image was significantly correlated to BMI, reflecting a more positive body image when BMI was lower (r varied from 0.37, 6\u00a0months postoperatively, to 0.29, 1\u00a0year postoperatively, and 0.31, 2\u00a0years after surgery).\nTable\u00a03Postoperative changes in body image (mean\u2009\u00b1\u2009SD)\u00a0Preoperative6\u00a0months1\u00a0year2\u00a0yearsPartial \u03b72Negative appreciation of body size26.2\u2009\u00b1\u20095.814.9\u2009\u00b1\u20097.1*14.4\u2009\u00b1\u20098.214.2\u2009\u00b1\u20098.20.59Lack of familiarity with one\u2019s own body16.5\u2009\u00b1\u20097.27.5\u2009\u00b1\u20094.7*7.1\u2009\u00b1\u20094.87.5\u2009\u00b1\u20096.10.52General body dissatisfaction15.2\u2009\u00b1\u20093.68.1\u2009\u00b1\u20094.6*8.0\u2009\u00b1\u20094.68.3\u2009\u00b1\u20095.00.61Total BAT score64.0\u2009\u00b1\u200915.436.9\u2009\u00b1\u200915.6*35.8\u2009\u00b1\u200916.235.9\u2009\u00b1\u200917.40.63N97979797Because of listwise exclusion, n\u2009=\u200997 on all moments.*P\u2009<\u20090.001\nAlthough we found significant improvements in body image over time, on all moments, preoperatively as well as postoperatively, and on all scales, patients\u2019 mean scores were significantly (P\u2009\u2264\u20090.001 or P\u2009\u2264\u20090.01) higher than those of (female students) normative groups [20, 32].\nHeterogeneity in Postoperative Psychosocial Functioning\nMost studies report broad psychosocial improvements after bariatric surgery. However, a significant minority of extremely obese patients do not benefit from surgery [4]. Some patients may even have difficulties in adapting psychosocially to the various consequences of bariatric surgery [13].\nIn the present study, in addition to a large variation in EWL, we found substantial variance with respect to changes in psychosocial functioning. Especially with respect to psychoneuroticism and neuroticism, most patients reported postoperative improvements; however, this was not the case for all patients. For instance, in the case of psychoneuroticism, 77 out of 99 patients (77.8%) reported improvement after 6\u00a0months; in contrast, 21 patients (21.2%) had higher scores, and one patient did not change. After 24\u00a0months, approximately two thirds of the patient group (64.7%) still had better scores on psychoneuroticism, whereas one third (31.4%) showed signs of an increase in psychoneuroticism.\nIn the case of personality characteristics, a mixed picture was found; however, in general, there were only small differences between patients reporting positive or negative changes. Regarding body image, almost all patients reported improvements, and these were quite stable over time.\nDiscussion\nAfter vertical banded gastroplasty, in addition to substantial and significant weight loss, patients also changed for the better in body image and most domains of psychosocial functioning. With respect to personality, only a significant improvement in self-esteem was found. Unfortunately, there was substantial variance in improvements, and some improvements waned over time.\nMost of our findings are in line with other studies, with respect to weight loss [33], as well as regarding improvements in psychosocial functioning and body image [7, 8]. The fact that we could not demonstrate many significant changes in patients\u2019 personality is in contrast to the results of some studies [34], but is consistent with the suggestion that there is no obese personality [35]; furthermore, because of the stability of personality, these findings might be expected. The impact of bariatric surgery seems to be more in the areas of symptoms of psychopathology.\nJust like others, we found a decline of some psychosocial improvements over the years [13]. Various explanations have been suggested for waning psychosocial improvements, for example, stabilization or rising of patients\u2019 weight, disappointment that life did not improve dramatically after substantial loss of weight, or difficulties in adapting psychologically to the consequences of bariatric surgery [13]. Also, our results concerning patients\u2019 postoperative heterogeneity are in accordance with earlier findings [36]. These outcomes underscore the suggestion that vertical banded gastroplasty, just like other methods of bariatric surgery, is not equally effective for each patient.\nAlthough our study has some assets, such as the employment of standardized, reliable questionnaires and the focus on psychosocial functioning in a wide variety of domains of life, some weaker points could also be indicated. For instance, we were not able to retain all patients of our preoperative sample. This problem is also identified by others who suggest that patients\u2019 participation in psychological follow-up is much lower than in follow-up visits for other specialists of the bariatric team [37].\nAlthough we found mixed results, the overall psychosocial improvements provide additional justification for vertical banded gastroplasty as a surgical treatment option for extreme obesity. However, as some improvements wane over time and not all patients profit in the same way, surgery alone may not be sufficient to sustain success. Consequently, additional behavioral treatment may be necessary.","keyphrases":["psychosocial functioning","personality","body image","vertical banded gastroplasty","bariatric surgery","psychopathology","morbid obesity"],"prmu":["P","P","P","P","P","P","R"]}
{"id":"Osteoporos_Int-3-1-1820757","title":"Links between cardiovascular disease and osteoporosis in postmenopausal women: serum lipids or atherosclerosis per se?\n","text":"Introduction and hypothesis Epidemiological observations suggest links between osteoporosis and risk of acute cardiovascular events and vice versa. Whether the two clinical conditions are linked by common pathogenic factors or atherosclerosis per se remains incompletely understood. We investigated whether serum lipids and polymorphism in the ApoE gene modifying serum lipids could be a biological linkage.\nIntroduction\nOsteoporotic fractures and acute cardiovascular events remain the predominant contributors to morbidity and mortality among the elderly. Postmenopausal women with osteoporosis are at increased risk for acute cardiovascular events independent of their age and cardiovascular risk profile, and the increase in risk is proportional to the severity of osteoporosis at the time of the diagnosis [1]. Bone mineral density (BMD) at the hip is inversely correlated with the severity of aorta calcification (AC) and hence low hip BMD can be a surrogate marker of the atherosclerotic burden in elderly women [2]. Large cohort studies demonstrate that the rate of bone loss in metacarpals, proximal femur, and calcaneus is proportional to the progression of AC [3\u20135]. Finally, advanced AC is associated with increased risk of osteoporotic fractures [5, 6]. Collectively, numerous epidemiological observations document an overlap between the pathogenesis of the two diseases [7, 8].\nAn attractive concept is that cardiovascular disease and osteoporosis are linked by a common denominator acting in parallel on both vascular and bone cells [9, 10]. Plausible candidates of such linking factors are serum lipids, which in addition to their established role in atherogenesis, were also shown to act on osteoblasts [11\u201313] and osteoclasts [14] under experimental conditions. Although some smaller cross-sectional studies found associations between lipid profile and BMD in humans [15\u201317], neither large population-based studies (e.g., NHANES III) nor prospective studies support these initial observations [18, 19]. A possible explanation the discrepancies could be that the true driver of the association is obstructive vascular disease, meaning that the association of lipids to bone is not detectable before the severity of intraluminal lesions reaches a critical grade when blood supply to the anatomical region is considerably hampered. Until now there have been no epidemiological studies that investigated associations of lipids and lipoprotein components with bone independent of a direct surrogate of the atherosclerotic burden.\nThe aim of the present study was to test this hypothesis and investigate associations of a wide array of serum lipids and lipoproteins as well as of a genetic determinant of lipid profile with different surrogate measures of osteoporosis (BMD, fractures) and cardiovascular disease (AC, acute events) in 1176 Danish postmenopausal women providing full-set of data for such analyses.\nSubjects and methods\nStudy population\nThe study population consisted of 1176 generally healthy postmenopausal women participating in the PERF study. Details on recruiting have been described elsewhere [20]. This subpopulation was selected based on those whose BMD and body composition were measured by the same DXA scanner. The population characteristics were not statistically significant from the total PERF population indicating that the results reported herein are generally applicable to elderly Caucasian women 60\u201385\u00a0years old. None of the women received any approved antiresorptive treatment or any other drugs with known effect on bone mass for more than 1\u00a0year. Forty-two patients reporting ongoing treatment with lipid-lowering drugs was excluded when analyzing associations of allelic variation in the ApoE gene with lipid profile.\nAll women gave written their informed consent to participation and the study was carried out in compliance with the Helsinki Declaration II and the European Standards for Good Clinical Practice. The study protocol was approved by the local ethical committee.\nDemographic characteristics, risk factors, and clinical events\nBody weight and height were measured to the closest 0.1\u00a0kg and 0.1\u00a0cm, respectively, in women wearing light indoor clothing and no shoes. Body mass index (BMI) was calculated as body weight (in kg) divided by height squared (in m). Information about age, years since menopause, smoking habits, regular alcohol and coffee consumption, weekly exercise, prevalent diabetes and cardiovascular diseases (stroke, coronary events, intermittent claudication, unstable angina) were collected during personal interview using a questionnaire [20].\nBone mineral density and fat mass measurement\nBone mineral density (BMD) of the lumbar spine L1-4 and total hip was measured by dual energy X-ray absorptiometry (DEXA) using a QDR-2000 scanner (Hologic Inc., Waltham, MA, USA). Daily phantom scans were performed each morning for proper quality control. Body composition also was measured by the same whole-body scanner. The two components measured were total body fat mass (TFM) in kilograms and total lean mass in kilograms [21]. Total body fat mass was divided into two depots: central fat mass (CFM, subcutaneous and visceral fat mass of the trunk) and peripheral fat mass (PFM, subcutaneous fat mass of the legs and arms). Body fat distribution was expressed by the CFM\/PFM ratio, analogous to the well-known waist-to-hip ratio [22].\nFracture diagnosis\nLateral X-rays of the thoracic and lumbar spine were performed using standard X-ray equipment. Vertebral deformities from T4 to L4 were assessed by digital measurements of vertebral deformations using the Image Pro Image Analyzer software (version 4.5 for windows, Media Cybernetics Inc., Silver Spring, MD, USA). The ratio of the anterior and posterior heights of each vertebral body was determined and a difference between the anterior and posterior edges exceeding 20% was considered as a radiographic vertebral fracture. Information on prevalent non-vertebral fractures (wrist, hip, humeral fracture, rib, ankle, and foot) was collected during personal interview and later verified by X-rays or hospital discharge summaries.\nGrading of aorta calcification\nAC was assessed on lateral radiographs. Briefly, calcified deposits in the lumbar aorta adjacent to each lumbar vertebra (L1\u2013L4) were assessed separately for the anterior and posterior wall of the aorta using the midpoint of the inter-vertebral space as the boundaries. Each wall of each segment was graded for the presence of calcified deposits with a score from 0 to 3 (0: no deposits, 1: less than one-third of the aortic wall, 2: one-third to two-thirds of the aortic wall. 3: more than two-thirds of the aortic wall covered with calcified deposits). The sum of the scores of individual aortic segments both for the anterior and posterior walls, termed as anterior-posterior severity score and was used to describe the overall severity of AC in the lumbar aorta. Maximum score possibly given was 4\u00d72\u00d73=24. The same investigator, who was blinded for all other results of the individual participants, carried out the evaluations. Intra-rater correlations between repeated measurements were in the range of r=0.92\u201398 (n=50). \nLaboratory parameters\nBlood samples were collected in the morning after fasting overnight (>12\u00a0h). Serum total cholesterol, triglyceride, and high-density lipoprotein (HDL) cholesterol were determined by enzymatic assay using a Vitros-250 (Johnson & Johnson, Taastrup, Denmark). LDL cholesterol was calculated by Friedewald formula (LDL\u2212C\u2009=\u2009TC\u2212HDL\u2212C\u22120.45\u2009\u00d7\u2009TG). Lipoprotein(a), ApoA1, and ApoB were measured by the Cobas Mira (Hoffman-La Roche, Mannheim, Germany) automatic blood analyzer.\nGenotyping\nDNA was isolated from peripheral blood samples. ApoE epsilon 2, epsilon 3 and epsilon 4 alleles were determined by enzymatic amplification of genomic DNA followed by restriction enzyme treatment of the amplified products (Ossendorf and Prellwitz, Qiangen News 2000, issue1, 11\u201313). Briefly, amplified products were treated with AflII and HaeII, respectively, and subjected to electrophoretic separation in agarose gels. The sequences of the amplification primers were 5\u2019-ACTGACCCCGGTGGCGGAGGAGACGCGTGC-3\u2019 and 5\u2019-TGTTCCACCAGGGGCCCCAGGCGCTCGCGG-3\u2019. One of these primers was deliberately designed with a mismatch (underlined) to create a non-polymorphic AflIII site permitting assessment of the efficiency of the digestion process.\nStatistical analysis\nResults are presented as mean\u2009\u00b1\u2009SD, unless otherwise indicated. Confounders of hip BMD and AC were identified by univariate correlation analysis. We classified ApoE genotypes into two categories marked by absence or presence of the \u025b4 allele. General linear models (GLM) tested the differences in selected dependent variables after adjustment for possible confounders. Similar approach was used to identify factors showing differences between women with or without fractures. Logistic regression models were established to assess the age-dependent and age-independent relative risk of wrist, hip, or vertebral fractures in women with advanced aorta calcification or cardiovascular disease. Statistical analyses were carried out using the SPSS data analysis software (version 12, SPSS Inc., Chicago, IL). All statistical tests were two-tailed and differences were considered significant if the p-value was less than 0.05.\nResults\nDemographic characteristics\nDemographic characteristics of the total population are shown in Table\u00a01. Less than one fifth of the population with an average age of 69.3\u00a0years had one or more manifestations of osteoporosis in form of a low BMD T-score equal or below \u22122.5 and\/or a fracture. In terms of cardiovascular status, the mean severity score of AC was 3.0\u2009\u00b1\u20093.8 and the number of women with history of an acute cardiovascular event was 50 (4.3%). \nTable\u00a01Demographic characteristics in the study populationCharacteristics and equivalent valuesNo. of women1176Age (years)69.3\u2009\u00b1\u20096.4Age at menopause (years)48.7\u2009\u00b1\u20095.0BMI (kg\/m2)26.4\u2009\u00b1\u20094.4Current smoking (%)24.6%Regular intake of alcohol (%)55.2%Serum total cholesterol (mmol\/l)6.3\u2009\u00b1\u20091.0Serum triglyciders (mmol\/l)1.4\u2009\u00b1\u20090.7Serum HDL (mmol\/l)1.7\u2009\u00b1\u20090.4Serum LDL (mmol\/l)4.0\u2009\u00b1\u20090.9Serum ApoA1 (mmol\/l)203.9\u2009\u00b1\u200937.1Serum ApoB (mmol\/l)120.3\u2009\u00b1\u200924.5Serum Lp(a) (mmol\/l)31.9\u2009\u00b1\u200934.6ApoE \u025b4 allele carrier367, 31.2%Total hip BMD (g\/cm2)0.80\u2009\u00b1\u20090.1Lumbar spine BMD (g\/cm2)0.92\u2009\u00b1\u20090.2Hip BMD T-score \u2264\u22122.5 (no. of women,%)147, 12.5%Spine BMD T-score \u2264\u22122.5 (no. of women,%)179, 15.2%Vertebral fracture (no. of women, %)204, 17.6%Hip fracture (no. of women,%)16, 1.4%Aorta calcification3.0\u2009\u00b1\u20093.8Cardiovascular disease (no. of women,%)50, 4.3%Data shown are mean\u00b1SD.\nThe ApoE genotype frequencies were 0.9% (n\u2009=\u200911) for ApoE \u025b2-2, 12.7% (n\u2009=\u2009149) for ApoE \u025b2-3, 2.5% (n\u2009=\u200929) for ApoE \u025b2\u20134, 55.2% (n\u2009=\u2009649) for ApoE \u025b3\u20133, 26.6% (n\u2009=\u2009313) for ApoE \u025b3\u20134, and 2.1% (n\u2009=\u200925) for ApoE \u025b4\u20134, respectively. When classifying ApoE genotypes into absence of ApoE \u025b4 allele and presence of ApoE \u025b4 allele, 31.2% (n\u2009=\u2009367) of women were ApoE \u025b4 allele carriers. These genotype proportions did not deviate significantly with those expected under conditions of Hardy-Weinberg equilibrium (X2\u2009=\u20094.55; df\u2009=\u20093; p\u2009=\u20090.21), and they were similar to those previously reported for Danish populations [22, 23].\nCorrelates of hip and spine BMD and AC\nIn univariate analyses, summarized in Table\u00a02, AC showed direct correlation with age, CFM\/PFM ratio, smoking, treated hypertension, and inverse association with physical exercise, regular alcohol consumption, BMI, and TFM%. On the other hand, BMI, TFM% and CFM\/PFM ratio were significant direct, whereas age, years since menopause, and smoking were inverse correlates of both spine and hip BMD. Treated hypertension was significantly associated with spine BMD, whereas the severity score of AC was a significant inverse correlate of hip BMD (p\u2009<\u20090.05). \nTable\u00a02Independent correlates of aorta calcification, hip BMD, and spine BMD (multiple regression models)\u00a0Aorta CalcificationHip BMDSpine BMDAge0.35 (p\u2009<\u20090.001)\u22120.23 (p\u2009<\u20090.001)\u22120.03 (p\u2009=\u20090.45)YSM\u22120.06 (p\u2009=\u20090.16)\u22120.06 (p\u2009=\u20090.10)\u22120.08 (p\u2009=\u20090.04)BMI\u22120.09 (p\u2009=\u20090.07)0.48 (p\u2009<\u20090.001)0.45 (p\u2009<\u20090.001)TFM%\u22120.04 (p\u2009=\u20090.36)\u22120.10 (p\u2009=\u20090.04)\u22120.21 (p\u2009<\u20090.001)CFM\/PFM ratio0.08 (p\u2009=\u20090.02)\u22120.004 (p\u2009=\u20090.85)0.05 (p\u2009=\u20090.21)Smoking0.19 (p\u2009<\u20090.001)\u22120.05 (p\u2009=\u20090.10)\u22120.01 (p\u2009=\u20090.66)Exercise\u22120.03 (p\u2009=\u20090.25)0.04 (p\u2009=\u20090.17)0.04 (p\u2009=\u20090.21)Treated hypertension0.08 (p\u2009=\u20090.006)0.006 (p\u2009=\u20090.81)0.05 (p\u2009=\u20090.09)Alcohol consumption\u22120.08 (p\u2009=\u20090.007)0.06 (p\u2009=\u20090.04)0.07 (p\u2009=\u20090.02)Apo e4\u22120.009 (p\u2009=\u20090.77)0.01 (p\u2009=\u20090.76)\u22120.03 (p\u2009=\u20090.22)Triglyceride0.02 (p\u2009=\u20090.63)0.03 (p\u2009=\u20090.43)0.04 (p\u2009=\u20090.32)HDL-C0.05 (p\u2009=\u20090.48)\u22120.06 (p\u2009=\u20090.29)0.02 (p\u2009=\u20090.74)LDL-CExcl.0.02 (p\u2009=\u20090.73)\u22120.10 (p\u2009=\u20090.10)Total cholesterol0.05 (p\u2009=\u20090.43)Excl.Excl.ApoA1\u22120.10 (p\u2009=\u20090.04)0.05 (p\u2009=\u20090.36)0.05 (p\u2009=\u20090.39)ApoB0.10 (p\u2009=\u20090.14)\u22120.03 (p\u2009=\u20090.60)0.10 (p\u2009=\u20090.85)Aorta Calcification\u22120.09 (p\u2009=\u20090.002)0.04 (p\u2009=\u20090.20)R\u2009=\u20090.44, SEE\u2009=\u20093.32 P\u2009<\u20090.001R\u2009=\u20090.55, SEE\u2009=\u20090.09 P\u2009<\u20090.001R\u2009=\u20090.34, SEE\u2009=\u20090.13 P\u2009<\u20090.001Excl.: excluded from the model due to strong co-linearity.\nThe independent role of the ApoE \u025b 4 allele\nResults of comparing lipids and lipoproteins between women with or without the ApoE \u025b4 allele are summarized in Table\u00a03. After adjustment for confounders, women with an ApoE \u025b4 allele had significantly higher serum total cholesterol, triglycerides, LDL-C, and ApoB levels than women without this allele. Furthermore, presence of the ApoE \u025b4 allele was associated with significantly lower HDL-C. In contrast, the differences in serum ApoA1 and Lp(a) did not reach statistical significance (Table\u00a03). Finally, there were no significant differences in adjusted spine or hip BMD or the severity of AC (p\u2009>\u20090.05). \nTable\u00a03Lipid and bone profile in women with or without an ApoE epsilon 4 allele\u00a0Absence of ApoE epsilon 4 allele (n\u2009=\u2009809)Presence of ApoE epsilon 4 allele (n\u2009=\u2009367)P valueSerum total cholersterol* (mmol\/l)6.24\u2009\u00b1\u20090.036.51\u2009\u00b1\u20090.05<0.001Serum triglycides* (mmol\/l)1.36\u2009\u00b1\u20090.021.48\u2009\u00b1\u20090.030.005Serum HDL* (mmol\/l)1.72\u2009\u00b1\u20090.011.65\u2009\u00b1\u20090.020.006Serum LDL* (mmol\/l)3.91\u2009\u00b1\u20090.034.19\u2009\u00b1\u20090.05<0.001Serum ApoA1* (mmol\/l)205.8\u2009\u00b1\u20091.3201.3\u2009\u00b1\u20092.00.06Serum ApoB* (mmol\/l)117.3\u2009\u00b1\u20090.8127.2\u2009\u00b1\u20091.3<0.001Serum Lp(a)* (mmol\/l)31.9\u2009\u00b1\u20091.329.2\u2009\u00b1\u20092.00.24Total hip BMD (g\/cm2)*0.803\u2009\u00b1\u20090.0040.804\u2009\u00b1\u20090.0050.89Spine BMD (g\/cm2)*0.924\u2009\u00b1\u20090.0050.915\u2009\u00b1\u20090.0080.32Hip fracture1.7%0.5%0.17Vertebral fractures16.4%19.3%0.24Severity score of AC**2.81\u2009\u00b1\u20090.142.97\u2009\u00b1\u20090.210.13Cardiovascular disease4.0%4.9%0.44Values are mean\u00b1SEM.*Adjustment for age, years since menopause, BMI, TFM%, CFM\/PFM ratio, current smoking, physical exercise, and regular alcohol consumption.**Adjustment for the aforementioned confounders + treated hypertension.\nAssociations of lipids with BMD and AC\nIn univariate analysis (Table\u00a02), total cholesterol and triglycerides were common correlates of spine BMD, hip BMD, and AC. HDL-C and ApoA1 were common correlates of AC and hip BMD (p\u2009<\u20090.05), whereas LDL-C, but not ApoB, was a common correlate of AC (p\u2009<\u20090.05).\nTo obtain insights into the direct contribution of lipids to the variation of hip and spine BMD and the severity of AC, we established multiple regression models. The independent contributors to the variation in these surrogate markers are indicated in Table\u00a02. Lipids were not independent contributors to the variation in BMD when assessed in the presence of the severity of AC and other confounders. However, ApoA1 levels did seem to contribute to the variation in AC. \nTable\u00a04Characteristics of women with or without at least one vertebral fracture\u00a0Without vertebral fractureWith vertebral fracturep-valueAge68.9\u2009\u00b1\u20096.471.3\u2009\u00b1\u20096.4<0.001YSM12.7\u2009\u00b1\u20098.415.5\u2009\u00b1\u20098.6<0.001BMI26.4\u2009\u00b1\u20094.426.3\u2009\u00b1\u20094.4NSTFM%41.3\u2009\u00b1\u20097.640.7\u2009\u00b1\u20097.8NSCFM\/PFM ratio0.88\u2009\u00b1\u20090.230.90\u2009\u00b1\u20090.24NSRegular exercise73.8%67.6%0.08ApoE \u025b4 allele +30.5%34.8%NSTotal cholesterol6.32\u2009\u00b1\u20091.006.31\u2009\u00b1\u20091.04NSTriglycerides1.42\u2009\u00b1\u20090.691.31\u2009\u00b1\u20090.540.02HDL-C1.68\u2009\u00b1\u20090.431.71\u2009\u00b1\u20090.39NSLDL-C3.98\u2009\u00b1\u20090.934.01\u2009\u00b1\u20090.96NSApoA1203.8\u2009\u00b1\u200937.8204.6\u2009\u00b1\u200933.4NSApoB120.6\u2009\u00b1\u200924.5119.1\u2009\u00b1\u200924.5NSLp(a)31.7\u2009\u00b1\u200934.932.5\u2009\u00b1\u200933.5NSSpine BMD0.93\u2009\u00b1\u20090.150.87\u2009\u00b1\u20090.14<0.001Hip BMD0.82\u2009\u00b1\u20090.110.75\u2009\u00b1\u20090.11<0.001Hip fracture1.0%2.9%0.04Wrist fracture11.4%14.6%0.04AC score2.8\u2009\u00b1\u20093.73.5\u2009\u00b1\u20093.40.03CVD3.6%7.4%0.02There were no significant differences in the frequency of smoking, regular alcohol, coffee, milk or seasonal vitamin D consumption, type 2 diabetes, treated hyperlipidemia or treated hypertension\nComparison of women with or without fractures\nThere were altogether 293 subjects who had an osteoporotic fracture after the menopause (i.e., prevalent hip, vertebral, or wrist fracture). Of the 204 women with at least one vertebral fracture, 33 also had a history of wrist fracture and 16 reported a history of hip fracture. Prevalence of non-vertebral fractures was significantly higher in women with compared with those without vertebral fracture(s). Characteristics of women with or without vertebral fractures are summarized in Table\u00a04. Women with fractures were on average older and had a tendency for being physically less active. However, no significant differences in BMI, body adiposity, or body fat distribution were apparent. In addition, no significant differences in a wide array of lifestyle factors were apparent.\nWhen comparing the lipid profile of women with or without osteoporotic fractures after adjustment for potential confounders (i.e., age, years since menopause, BMI, TFM%, CFM\/PFM ratio, smoking, ApoE \u025b4 allele, physical exercise, alcohol consumption, and severity score of AC), only triglycerides showed significant differences between the two groups (p\u2009<\u20090.05). Thus, triglycerides in women with and without fractures were 1.31\u2009\u00b1\u20090.04\u00a0mmol\/l and 1.42\u2009\u00b1\u20090.03\u00a0mmol\/l, respectively. When analyzing fracture types separately, the difference between women with or without hip or wrist fractures were not statistically significant.\nThe relative risk of hip or vertebral fracture in women with severe AC or CVD\nTo assess the implications of atherosclerotic vascular disease for osteoporotic fracture risk, we calculated the odds ratio of the different types of fractures in women with severe AC (\u22656) and\/or manifest CVD. As indicated in Table\u00a05, after adjustment for age, the risk of hip fractures but not wrist or vertebral fractures was increased in subjects with advanced vascular disease. \nTable\u00a05Relative risk of different types of osteoporotic fractures in women with severe aorta calcification (score\u2009\u2265\u20096) and\/or manifest cardiovascular disease (n\u2009=\u2009282)\u00a0\u00a0Odds ratio (95% CI)p-valueHip fracture N\u2009=\u200916Crude4.4 (1.6\u201312.9)0.001Age-adjusted3.0 (1.1\u20138.8)0.04Vertebral fracture N\u2009=\u2009204Crude1.2 (0.9\u20131.7)0.27Age-adjusted1.0 (0.7\u20131.4)0.93Wrist fracture N\u2009=\u2009145Crude1.4 (1.0\u20132.1)0.06Age-adjusted1.2 (0.8\u20131.7)0.46\nDiscussion\nIn the present study, we investigated whether serum lipid and lipoproteins represent a biological linkage between osteoporosis and cardiovascular disease. The main findings were as follows: 1) neither allelic polymorphism in the ApoE gene nor serum lipids per se were independent correlates of BMD at any skeletal site, 2) ApoA1 levels were independent correlates of AC, which seem to play a role in the determination of hip BMD, 3) decreased triglycerides were independently associated with vertebral fractures, but not with non-vertebral fractures (hip and wrist), and 4) advanced vascular disease was a significant contributor to risk of hip fractures, but not to vertebral or wrist fractures. These observations suggest that the role of lipids as linking factors between cardiovascular disease and osteoporosis is indirect via promotion of atherogenesis, which in turn can contribute to bone loss, especially at skeletal sites with end-arterial blood supply. Antiatherogenic measures can also be expected to contribute to decreasing the burden of hip fractures among the elderly.\nUnivariate analysis pointed out several common correlates of BMD and atherosclerosis. Of these factors, aging, the duration of the menopause, BMI, and TFM% seemed to have reciprocal impact on BMD and AC. In multiple regression models, whereas BMI continued to correlate directly, TFM% became an inverse correlate of BMD. Accordingly, while weight-bearing may have a favourable impact on bone metabolism, adipose tissue accumulation exert an adverse influence. These findings corroborate the recent findings of Hsu YH et al. [24]. Although traditional views considered obesity a protective factor due to endogenous estrogen production, these more recent findings emphasize that other factors deriving from adipose tissue, e.g., adipokines, inflammatory cytokines, and perhaps even lipids\/lipoprotein metabolism are also to be taken into account. In addition, these findings also draw attention to the need of proper adjustment for overall adiposity and body fat distribution when addressing the direct implication of selected potential mediators.\nTo address whether serum lipids represent a biological linkage between osteoporosis and vascular disease, one approach is to investigate whether polymorphism in a gene with known modulator effects on serum lipid profile is associated with variation in the severity of AC and BMD. Similar to previous observations [25, 26], we found that presence of the \u025b4 variant allele in the ApoE gene was independently associated with higher levels of total cholesterol, LDL-C and ApoB. In addition, we also observed subtle, yet statistically significant differences in serum triglycerides and HDL-C levels. Nevertheless, we were not able to document significant associations of the \u025b4 allele with either BMD or AC score. In male or mixed populations, the \u025b4 allele was reported as a risk factor for atherosclerosis [27, 28], suggesting that the association might be gender-specific for males. Total cholesterol and LDL-C are important predictors of cardiovascular risk in men, but less so in postmenopausal women in whom triglycerides and HDL-C carry greater importance [29, 30]. Another explanation to the apparent discrepancies could be geographic variation in the relative importance of allelic variation in the ApoE gene. In support, a study in Finnish men reported higher importance of the \u025b3 allele for atherogenesis compared with the \u025b4 allele [27]. Similar discrepancies exist concerning the relative importance of this gene polymorphism for the determination of bone mass. Although there are reports suggesting that presence of the \u025b4 allele is a contributor to accelerated bone loss and\/or increased risk of osteoporotic fractures [31, 32], not all studies confirm this finding, particularly those undertaken in women [33\u201335]. Collectively, ApoE polymorphism with primary influence on total cholesterol, LDL-C, and apoB levels does not seem to provide a major biological linkage between the pathogenesis of osteoporosis and CVD.\nExperimental observations proposed LDL-C and HDL-C as common modulators of bone cells and vascular smooth muscle cells [11\u201314]. On the clinical side, smaller cross-sectional studies found associations between lipid profile and BMD [15\u201317]. However, none of these studies adjusted systematically for a direct DEXA measure of body fat distribution and\/or smoking habits, which are important confounders of lipid profile. Our multiple regression analyses could not reveal significant independent associations of any of the considered lipid and lipoprotein components with hip or spine BMD. These findings are in line with the largest population-based observational study, the NHAEMS III survey including more than 13,000 subjects [19]. In further support, a recent longitudinal analysis of results from the Framingham study cohort reported insignificant direct influence of serum lipids for changes of BMD during a 25-year observational period [18]. Collectively, these observations do not seem to nurture the concept that serum lipids and lipoproteins exert clinically detectable direct effects on bone mineralization in elderly women.\nTo further assess the potential implications of serum lipids and lipoproteins for osteoporosis, we also compared the profile of postmenopausal women with or without osteoporotic fractures. Interestingly, serum triglycerides were significantly lower in women with at least one vertebral fracture compared with those with no fractures, while no differences were seen between those with or without an osteoporotic non-vertebral fracture (i.e., hip or wrist fracture). This finding is similar to that reported by Yamaguchi et al. [15]. Since triglycerides were not associated with spine BMD, their direct contribution to fracture risk, if any, is via modulation of bone quality or bone strength. Given the cross-sectional design of this study, we cannot exclude the possibility that a confounder or confounders of triglyceride levels, which was not considered herein is the actual modulator of bone fragility and hence fracture risk.\nImportant finding of the multiple regression analysis was that the severity of AC was an independent correlate of hip BMD with no simultaneous independent contribution of lipids. This observation provides evidence that obstructive vascular disease rather than lipids\/lipoproteins per se that drive bone loss from the hip. In a previous study, we showed that women with intermittent claudication have not only severe AC, but also very low BMD [2]. The direct contribution of atherosclerosis is also supported by the observation showing that asymmetric vascular disease is accompanied by asymmetric demineralization of this skeletal site [36]. Moreover, studies show that the rate of demineralization at the hip is significantly associated with the rate of atherogenesis [5] and even future risk of cardiovascular events [37]. On the other hand, we recently demonstrated in a large population-based cohort of postmenopausal women that accelerated atherogenesis is an independent predictor of hip fracture risk. Histological investigations of explants from elderly patients with femoral neck fractures indicate that both the larger (a. iliaca ext and a. femoris profunda) and the small vessels in the ligamentum teres supplying this skeletal site are frequently obstructed by atheromatous lesions [38, 39]. These observations provide strong support for the important role of atherosclerosis for the pathogenesis of osteoporosis in the proximal femur.\nIn summary, the results of the present observational study provide further evidence for the independent association of peripheral vascular disease with osteoporosis in the proximal femur. Since the association of lipids and lipoproteins to BMD and non-vertebral fractures is not independent of the severity of AC, it seems unlikely that these metabolites exert direct and clinically significant effects on bone turnover in postmenopausal women. Their contribution is via promotion of atherogenesis, in which regard ApoA1 levels seem to take a leading role. The remaining issue to be clarified is which genetic or environmental factors underlie the association of low triglycerides levels to vertebral fractures.","keyphrases":["osteoporosis","lipids","cardiovascular events","aorta calcification","apoe polymorphism"],"prmu":["P","P","P","P","P"]}
{"id":"J_Neurol-3-1-1915648","title":"Does modafinil enhance activity of patients with myotonic dystrophy?\n","text":"We performed a double-blind placebo-controlled crossover study in 13 patients with myotonic dystrophy to address the question whether modafinil, known to improve hypersomnolence in myotonic dystrophy, may improve levels of activity as well. We used the Epworth Sleepiness Scale as a measure of hypersomnolence and a structured interview of the patient and the partner or housemate as a measure of activity. We additionally used a restricted form of the RAND-36 to relate a possible improvement of activity to perceived general health. We confirmed earlier positive findings of modafinil regarding reduced somnolence (p = 0.015), but no significant effects were seen regarding activity levels (p = 0.2 for patients\u2019 self-reports and 0.5 for partners\u2019 reports).\nIntroduction\nMyotonic dystrophy (MD) is a multi-system disorder, the most well known symptoms being muscle weakness and myotonia. Distressing daytime sleepiness and diminished spontaneous activity, often referred to as inertia, reduced initiative, inactivity or apathy, are frequently reported. These latter symptoms often seem to cause more hindrance in daily life, both to patients and their spouses, than muscle weakness itself. Hypersomnia can even be present when there is virtually no weakness [1]. Recent reports [2, 3, 4] have demonstrated that modafinil has a beneficial effect on daytime sleepiness in MD. Although the relationship between excessive sleepiness and the lack of spontaneous activity is not clear, it seemed reasonable to suppose that increasing vigilance might result in an increase in spontaneous activity. The present study intended to answer the following questions: firstly, do patients with MD undertake more activities when using modafinil; secondly, does any such improvement relate to a change in somnolence or to another disease characteristic?\nPatients and methods\nThirteen outpatients (5 males) participated in a randomized double-blind crossover placebo controlled study. Their mean age was 43.5\u00a0years (SD: 13.9\u00a0years). Age of onset of symptoms was before 12\u00a0years of age in 3 patients; all three lived independently at the time of study, although they needed professional social support with respect to their household or daily activities. Three patients were employed in highly responsible jobs. The remaining patients had been considered unfit for normal paid employment for reasons related to their disorder. They were mainly involved in housekeeping. With the exception of two elderly men with considerable weakness of the legs, weakness had little impact on activities of daily living (ADL) functions. Even these two men were ambulant, although they used a wheelchair regularly. Twelve patients had a partner or housemate.\nMedication was given during two periods of 14\u00a0days, separated by a one-week washout period. The study was preceded by a two-week period free of all drugs with an exception being made for contraception. Patients were randomized for either placebo first or modafinil first. The modafinil dose was 200\u00a0mg per day for the first week. The patients were instructed to double the dose during the second week of each period if they perceived an insufficient effect.\nThe main outcome measure was an increase in spontaneous activity, assessed using a novel structured interview of both the patient and the partner or housemate, if present. These interviews took place at home after the first period, and by telephone after the second one. After the first medication period the patient and the partner\/housemate were asked to compare the level of activity with that of the preceding two (-baseline-) weeks. After the second medication period they were asked to compare that period with the baseline weeks.\nThe following issues were addressed, and scores attributed:\nhave you, during the past two weeks, been more active than during the baseline period?no (0 points)to some degree (1 point)definitely (2 points)can you give one (1 point), two (2 points) or three (3 points) substantial and observable examples of activities\/specific actions you undertook that you would otherwise not have done?\nThe range of the score was therefore zero to five points. The partner was asked the same questions with respect to the patient. During the interview patients and partners were asked whether they had guessed which medication, modafinil or placebo, the patient had used in the past period. If so, they were asked what made them believe so. The answers were not used in the assessment of medication effects, but served to estimate possible unblinding. The RAND-36 questionnaire was used to measure possible changes in subjective general health [5]. This was filled in before the start and after the end of each medication period. The questionnaire was restricted to issues considered relevant to the study: General Health, Role Limitations by emotional and physical problem, Social Functioning, Vitality, and Mental Health. The Epworth Sleepiness Scale (ESS, [6] was used to assess possible changes in hypersomnolence.\nAfter completion of the trial the remaining capsules in the medication boxes were counted to assess compliance. The institutional Committee of Medical Ethics had approved the study. Patients gave written informed consent after study information was provided orally and in writing at the patients\u2019 home.\nResults\nAll patients completed the trial. Medication compliance was good: only three patients had omitted one dose each. The only reported side effect was slight headache in one patient using modafinil. Ten patients doubled the dose of both modafinil and placebo after the first week, meaning that results largely concern a daily dose of 400\u00a0mg modafinil. More often than not both patients (67%) and their partners\/housemates (77%) correctly guessed when they had been taking either modafinil or Placebo, usually on the basis of a \u2018decreased sleepiness\u2019 and\/or \u2018increased activity\u2019.\nThe structured interview regarding activity and actions did not show significant differences between modafinil and placebo (p = 0.2 for patients and p = 0.5 for partners\/housemates).\nThe RAND-36 questionnaires revealed a poor perception of general health for the whole group with a mean value of 29 points out of 100 (range 0\u201350) on the General Health rating.\nThe ratings were virtually identical for each patient over the four assessments (p = 1,Wilcoxon test). The perception of Role Limitations varied widely: mean 66 out of 100 (range 0\u2013100). A medication related change was not observed (p = 0.7, Wilcoxon test). This also held for the perception of Social Functioning (p = 0.6), Vitality (p = 0.2) and Mental Health (p = 0.5). The ESS revealed a significant improvement with modafinil, in that the mean score decreased from 10.5 (range: 3\u201318) to 6.8 points (range: 1\u201315); for placebo the corresponding values were 10.5 (range: 3\u201318) and 10.7 (range: 2\u201317) points (p = 0.015, Wilcoxon test). There was no suggestion of a difference in outcome between patients with high and those with low scores. There was no significant relationship between the increase in activity\/actions as perceived by the patient\/ partner and indicated by the structured interview, and changes in perceived hypersomnolence as measured by the ESS (p = 0.38, Spearman\u2019s test).\nDiscussion\nThe present study confirmed the beneficial effect of modafinil on excessive sleepiness in MD, but did not detect a concomitant effect on spontaneous activity as measured by a structured interview of the patients and their partners. This interview, not formally validated, was designed to reflect a clinically relevant and observable increase in daily activity by asking for specific actions. Examples might be that patients went to the theatre after a busy day, when they would otherwise have postponed such a visit, or cleaning up the shed. By asking for specific actions we hoped to distinguish actions from the mere feeling of being active or the intention to become so.\nThe study was small, leaving open the possibility that minor changes have been missed. The study was also focused on short-term effects and thus it is not able to detect changes of behaviour that take more time to become manifest, but we believe that a fortnight is long enough to detect relevant improvements in activity as defined above. A further consideration is the unblinding we have observed, which was most probably due to a correct perception of an effect on somnolence. This might have confounding effects on the interpretation of intended double-blind studies of modafinil on symptoms other than hypersomnolence. In the present study this does not seem to have happened, as the effect on hypersomnolence was neither related to perceived improved activity, nor to perceived aspects of general health. That many patients and partners reported more activity in addition to less sleepiness when asked why they thought that modafinil or placebo had been used, might be the result of the expectations implied in the aim of the study as discussed with the participants. The structured interview did not detect this increased activity, which we feel speaks in favour of its validity.\nIn a previous study of 11 patients modafinil improved excessive daytime sleepiness in MD, measured with the Multiple Sleep Latency Test and the Epworth Sleepiness Scale [2]. Possible effects on spontaneous activity were not considered. MacDonald et al. [3], in a double-blind cross-over placebo-controlled study of 40 patients with a time-scale identical to ours confirmed the reduction of somnolence as measured by Epworth and Stanford Sleepiness Scales. They also found modafinil-induced decreased fatigue-inertia, and increased vigor-activity, as measured by the Profile of Mood States. The latter findings might predict improved observed activity, but the study did not include this issue and our study did not demonstrate such effect. Using the RAND-36 they also observed enhanced measures of energy and perception of health, but no changes in the other items of the test; the former effects were not confirmed in our study. Talbot et al. [4] performed a similar double-blind cross-over study of 19 patients selected for hypersomnolence (ESS 10 or more points), using the ESS, a Modified Maintenance of Wakefulness test (MWT), a steering simulator, the Short Form 36 and an \u201cactivity diary\u201d as measures. They found a reduction of sleepiness, especially in the MWT, less convincingly in the ESS. The other tests did not show significant changes. Data from the activity diaries are not given.\nIt is apparent that the symptoms referred to as inertia, reduced initiative, inactivity or apathy are hard to define and even harder to measure. Recently, van der Werf et al. inferred that the lack of correlation between fatigue scores and sleepiness in MD suggests that different pathophysiological mechanisms underlie these clinical manifestations [7]). We believe that our findings point in the same direction.","keyphrases":["modafinil","myotonic dystrophy","hypersomnolence","levels of activity","rand-36"],"prmu":["P","P","P","P","P"]}
{"id":"Antonie_Van_Leeuwenhoek-3-1-2140093","title":"Domain analysis of lipoprotein LppQ in Mycoplasma mycoides subsp. mycoides SC\n","text":"The lipoprotein LppQ is the most prominent antigen of Mycoplasma mycoides subsp. mycoides small colony type (SC) during infection of cattle. This pathogen causes contagious bovine pleuropneumonia (CBPP), a devastating disease of considerable socio-economic importance in many countries worldwide. The dominant antigenicity and high specificity for M. mycoides subsp. mycoides SC of lipoprotein LppQ have been exploited for serological diagnosis and for epidemiological investigations of CBPP. Scanning electron microscopy and immunogold labelling were used to provide ultrastructural evidence that LppQ is located to the cell membrane at the outer surface of M. mycoides subsp. mycoides SC. The selectivity and specificity of this method were demonstrated through discriminating localization of extracellular (i.e., in the zone of contact with host cells) vs. integral membrane domains of LppQ. Thus, our findings support the suggestion that the accessible N-terminal domain of LppQ is surface exposed and such surface localization may be implicated in the pathogenesis of CBPP.\nIntroduction\nMycoplasma mycoides subsp. mycoides small colony type (SC) is an extra-cellular pathogen living in close association with host cells. It causes contagious bovine pleuropneumonia (CBPP), an acute, subacute or chronic infection of the respiratory system in cattle with a mortality rate of up to 30%, causing severe losses in livestock production, in particular on the African continent (Provost et\u00a0al. 1987; Food and Agriculture Organization of the United Nations 2003).\nLipoproteins are usually strongly antigenic membrane proteins known to play a central role in interactions between bacteria and eukaryotic cells, particularly with respect to adhesion, and to stimulate the release of pro-inflammatory cytokines (M\u00fchlradt and Frisch 1994; Herbelin et\u00a0al. 1994; Brenner et\u00a0al. 1997; Marie et\u00a0al. 1999; Calcutt et\u00a0al. 1999; Belloy et\u00a0al. 2003). Lipoproteins have been put forward as possible virulence factors of pathogenic mycoplasmas (Dyson and Smith 1997; Vilei et\u00a0al. 2000; Pilo et\u00a0al. 2003).\nMembrane lipoprotein LppQ is the predominant antigen of M. mycoides subsp. mycoides SC and shows the strongest signal on immunoblots containing total antigen from this pathogen reacted with serum from cattle that have suffered from CBPP. It induces a specific, early and persistent immune response in infected animals (Abdo et\u00a0al. 2000). LppQ is encoded as a precursor (of 445 amino acids) with a consensus sequence for prokaryotic signal peptidase II and a lipid attachment site (Figure 1). The leader sequence of LppQ shows a typical transmembrane structure with a significant helix formation capacity (Abdo et\u00a0al. 2000). LppQ was shown to be a membrane protein by Triton X-114 phase partitioning and lipidation was demonstrated by palmitic acid radiolabelling (Abdo et\u00a0al. 2000). The C-terminal part of LppQ was found to possess repeated integral membrane structures rich in hydrophobic and aromatic amino acids, which have pore formation potential, and immunoblot analysis showed that the C-terminal domain possesses no particular immunogenicity, as serum derived from cattle naturally infected with M. mycoides subsp. mycoides SC did not react against it (Abdo et\u00a0al. 2000). In contrast, the N-terminal domain of LppQ has three strongly hydrophilic domains and was shown to be the origin of the strong antigenic response against LppQ in naturally infected cattle. From these data, Abdo and collaborators deduced that the N-terminal part of LppQ is exposed at the outer surface of M. mycoides subsp. mycoides SC (Abdo et\u00a0al. 2000).\nFig.\u00a01Structure of lipoprotein LppQ. (Panel A) LppQ protein sequence. Repeats are indicated by dashed arrows. The symbol # corresponds to the signal peptidase II cleavage site, which allows for the obtainment of mature LppQ protein. (Panel B) The 10 C-terminal repeats rich in hydrophobic and aromatic residues, as identified with the MEME software, which build the transmembrane region of LppQ. Black background indicates conserved residues. The consensus sequence is shown. (Panel C) Model for protein topology of LppQ in the membrane lipid-bilayer of M. mycoides subsp. mycoides SC. Repeat numbers and amino acid positions for the beginning of mature LppQ (28), the beginning of C-terminal portion (193) and the protein end (445) are indicated. The N-terminal part is supposed to be anchored in the lipid-bilayer by the lipid anchor of the Cys28 residue. The three hydrophilic domains of the N-terminal part, which were found with significant scores for coiled-coil tertiary structure (Abdo et\u00a0al. 2000), are depicted as globular items. The C-terminal part consists of a transmembrane region built up of the 10 repeated integral membrane structures, whose consensus sequence is shown at the bottom\nDue to the strong antigenicity of the extracellular N-terminal part of LppQ and its specificity for M. mycoides subsp. mycoides SC, a recombinant peptide comprising amino acids 22-218 of the LppQ precursor protein was used to develop a specific and sensitive ELISA assay for the serological detection of CBPP in cattle (Bruderer et\u00a0al. 2002). Furthermore, LppQ seems to be involved in the inflammatory processes of M. mycoides subsp. mycoides SC, as cattle immunized with the recombinant peptide showed an increased susceptibility to infection with M. mycoides subsp. mycoides SC and exhibited more severe symptoms of CBPP than unvaccinated animals (Nicholas et\u00a0al. 2004).\nAs LppQ is apparently involved in the molecular mechanisms of pathogenicity of M. mycoides subsp. mycoides SC (Nicholas et\u00a0al. 2004; Pilo et\u00a0al. 2006), we have investigated the topology of LppQ in M. mycoides subsp. mycoides SC by means of high resolution field emission scanning electron microscopy (SEM), with particular respect to the predicted localization of LppQ on the mycoplasmal cell surface. This technique allows proteins to be localized on mycoplasmas unequivocally and with high accuracy and resolution (Pilo et\u00a0al. 2005). Here, we provide evidence that the N-terminal part of LppQ is located on the surface of the mycoplasmal cell membrane and is accessible from the extracellular side while the C-terminal part of LppQ is not exposed.\nMaterials and methods\nBioinformatic analysis\nPrediction of membrane topology was carried out by using the softwares TMpred (Hofmann and Stoffel 1993; http:\/\/www.ch.embnet.org\/software\/TMPRED_form.html), TopPred (von Heijne 1992; http:\/\/bioweb.pasteur.fr\/seqanal\/interfaces\/toppred.html) and Phobius (K\u00e4ll et\u00a0al. 2004; http:\/\/phobius.cgb.ki.se\/). Protein secondary structure was predicted with the MINNOU software available on the web page http:\/\/minnou.cchmc.org\/ (Cao et\u00a0al. 2006). Determination of the C-terminal repeats of lipoprotein LppQ was assessed by the MEME software version 3.5.4 at http:\/\/meme.sdsc.edu\/meme\/meme.html (Bailey and Elkan 1994).\nM. mycoides subsp. mycoides SC strain and sample preparation\nM. mycoides subsp. mycoides SC strain Afad\u00e9 was used in the present study. This strain was isolated in 1968 at Farcha Laboratory, N\u2019Djamena, Chad, from a cow from Afad\u00e9, Northern Cameroon, which suffered from acute CBPP. The strain Afad\u00e9 is known to be highly virulent (Vilei and Frey 2001) and was used previously for experimental infection studies (Abdo et\u00a0al. 1998). It was grown in standard mycoplasma medium (Axcell Biotechnologies, St. Genis l\u2019Argenti\u00e8re, France) by inoculation of 20\u00a0\u03bcl of a frozen stock culture into 3\u00a0ml of medium and incubation for 3\u00a0days at 37\u00b0C. Growth and handling of live M. mycoides subsp. mycoides SC were performed in a biological safety laboratory fulfilling the BSL-3 containment safety standards.\nCrude lysates were prepared by resuspending harvested mycoplasmas in TES buffer (10\u00a0mM Tris\u2013HCl, 1\u00a0mM EDTA, 0.8% NaCl, pH 8.0) at a concentration of approximately 109\u00a0cells\/ml (Abdo et\u00a0al. 2000).\nProduction of antibodies against recombinant LppQ peptides\nThe polyhistidine-tagged recombinant peptides LppQ-N\u2032 (amino acids 22 to 218) and LppQ-C\u2032 (amino acids 214 to 422) from the N- and C-terminal portions of LppQ, respectively, were produced in Escherichia coli strain BL21(DE3) harbouring the respective plasmids as reported previously (Abdo et\u00a0al. 2000; Bruderer et\u00a0al. 2002). Recombinant peptides were polyhistidine-tagged at both their N- and C-termini. Purification of the peptides was performed by Ni2+ chelation chromatography and the purity of the peptides was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).\nPolyclonal monospecific sera directed against LppQ-N\u2032 and LppQ-C\u2032 were obtained by subcutaneous immunization of rabbits with 160\u2013200\u00a0\u03bcg of purified recombinant polyhistidine-tagged peptides in 500\u00a0\u03bcl of 50\u00a0mM PBS buffer (50\u00a0mM Na2HPO4\/NaH2PO4, 140\u00a0mM NaCl, pH 8.0) mixed with 500\u00a0\u03bcl of Adjuvant 10 (Gerbu Biotechnik GmbH, Gaiberg, Germany). Booster immunizations with 40 and 20\u00a0\u03bcg of protein were performed 2- and 4-weeks later, respectively. The rabbits were bled 10\u00a0days after the last booster immunization and antisera were stored at \u221220\u00b0C.\nImmunoglobulin G (IgG) fractions were purified from the two rabbit antisera with the HiTrap Protein G kit (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer\u2019s instructions. Immunoglobulins from pre-immune sera were also purified. The purity of all IgGs was analyzed by SDS-PAGE. Protein concentrations were determined by using the method of Bradford (1976).\nImmunoblot analysis\nWhole cell antigens of strain Afad\u00e9 were separated by 5\u201315% gradient SDS-PAGE (Ausubel et\u00a0al. 1999) and blotted onto a nitrocellulose membrane with a pore size of 0.2\u00a0\u03bcm (Bio-Rad Laboratories AG, Reinach, Switzerland). The membrane was blocked with 1% skim milk powder in TBS buffer (100\u00a0mM Tris\u2013HCl, 150\u00a0mM NaCl, pH 7.5) for 60\u00a0min at room temperature. Strips were incubated with corresponding IgGs at a concentration of 1\u20132\u00a0\u03bcg\/ml (diluted 1:2000) overnight at 4\u00b0C and washed three times with TBS buffer for 5\u00a0min. Serum obtained from a cow that was infected experimentally with M. mycoides subsp. mycoides SC strain Afad\u00e9 (Abdo et\u00a0al. 1998; diluted 1:100) or previously prepared (Abdo et\u00a0al. 2000) rabbit hyperimmune sera directed against LppQ-N\u2032 and LppQ-C\u2032 (diluted 1:1000) were used as controls. The strips were incubated for 90\u00a0min at room temperature with alkaline phosphatase-labelled goat anti-rabbit IgG (H\u00a0+\u00a0L) (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA) diluted 1:2000 or monoclonal antibody anti-bovine IgG (Sigma, Buchs, Switzerland) diluted 1:5000 in skim milk buffer. The strips were washed three times with TBS buffer. The colour reaction was initiated with 0.3\u00a0mg\/ml nitroblue tetrazolium (NBT) (Roche Diagnostics, Rotkreuz, Switzerland) and 0.15\u00a0mg\/ml 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Roche Diagnostics) in alkaline buffer (7\u00a0mM Na2CO3, 3\u00a0mM NaHCO3, 1\u00a0mM MgCl2, pH 9.6) and stopped with distilled water.\nPreparation of M. mycoides subsp. mycoides SC for immunogold labelling\nCoverslips that had been coated with gold or platinum and treated with poly-l-lysine (Sigma) to promote cell adhesion were immersed into 3\u00a0ml of fresh standard medium in multiwell plates. Thereafter, 100\u00a0\u03bcl of mycoplasma culture were added and the cultures were incubated for 5 to 6\u00a0days at 37\u00b0C until stationary phase. Coverslips with adhering mycoplasmas were washed three times with PBS buffer (2.9\u00a0mM NaH2PO4, 7.1\u00a0mM Na2HPO4, 137\u00a0mM NaCl, pH 7.4) at 37\u00b0C and fixed with 4% paraformaldehyde in PBS for 30\u00a0min at room temperature. After fixation, samples were washed with PBS buffer and blocked with 1% BSA and 0.2\u00a0M glycine in PBS for 15\u00a0min at room temperature. Thereafter, mycoplasma cells were incubated with anti-LppQ-N\u2019 or anti-LppQ-C\u2019 IgGs diluted 1:100 (30\u00a0\u03bcg\/ml) in PBS containing 1% BSA overnight at 4\u00b0C. After another washing step with PBS buffer for 5\u201310\u00a0min, mycoplasmas were labelled with 15\u00a0nm colloidal gold-conjugated goat anti-rabbit antibody (British Biocell International, Cardiff, UK) diluted 1:50 in PBS for 90\u00a0min at room temperature. Coverslips were then washed with 0.1\u00a0M cacodylate buffer (pH 7.4) and processed for SEM.\nScanning electron microscopy\nCoverslips were incubated in 1.33% osmium tetraoxide with 0.11% ruthenium red in 0.13\u00a0M cacodylate buffer (pH 7.4) for 15\u00a0min, washed with 0.1\u00a0M cacodylate buffer, dehydrated through an ascending ethanol series and dried by evaporation of hexamethyldisilazane (Sigma) as described previously (Stoffel et\u00a0al. 1993; Stoffel et\u00a0al. 2002; Stoffel and Friess 2002). Finally, they were mounted on metal stubs with a conductive adhesive (Provac AG, Balzers, Liechtenstein).\nSamples were examined without further metal coating. Secondary electron micrographs and corresponding backscattered images were obtained with a fully digital field emission scanning electron microscope DSM 982 Gemini (Zeiss, Oberkochen, Germany) at an accelerating voltage of 5\u00a0kV, a working distance of 6\u20138\u00a0mm and a magnification from 50,000 to 100,000\u00d7.\nControl experiments included omission of primary antibody as well as the use of a rabbit anti-calcitonin antibody (Anawa, Zurich, Switzerland) and of IgGs from rabbit pre-immune sera as irrelevant substitutes for the anti-LppQ primary antibodies.\nResults\nStructure of the C-terminal domain of lipoprotein LppQ\nConventional predictors of membrane topology available online did not envisage the C-terminal domain of LppQ to be membrane-associated. However, determination of repeats in the LppQ protein sequence revealed that its C-terminal domain, represented by the last 250 amino acids, consists of a region built up of 10 repeated units rich in hydrophobic and aromatic residues (Figure\u00a01). These repeat units of 25 amino acids were all found to adopt a particular secondary structure consisting of helices of approximately 10 amino acids flanked upstream and downstream by amino acids forming random coils (not shown).\nMonospecificity of IgG preparations\nOn immunoblots containing total antigen of M. mycoides subsp. mycoides SC strain Afad\u00e9, purified IgG preparations directed against the N-terminal and against the C-terminal halves of LppQ each identified a single protein band with an apparent molecular mass of 48\u00a0kDa, which corresponds to the calculated size of 49.1\u00a0kDa of LppQ (Figure\u00a02). Reference rabbit hyperimmune sera directed against LppQ-N\u2032 and LppQ-C\u2032 collected almost a decade ago gave identical results. Control experiments with IgGs purified from pre-immune sera did not detect any corresponding antigen (Figure\u00a02).\nFig.\u00a02Immunoblot analysis of total antigen of M. mycoides subsp. mycoides SC with anti-LppQ-N\u2032 and anti-LppQ-C\u2032. Approximately 10\u00a0\u03bcg of total antigen of strain Afad\u00e9 per lane was separated by 5\u201315% gradient SDS-PAGE, transferred onto a nitrocellulose membrane and probed with the serum from a cow infected experimentally with strain Afad\u00e9 (Abdo et\u00a0al. 1998), or with IgGs from rabbit pre-immune sera or from antisera against LppQ-N\u2032 and LppQ-C\u2032, respectively. Reference rabbit hyperimmune sera directed against LppQ-N\u2032 and LppQ-C\u2032 obtained almost a decade ago (Abdo et\u00a0al. 2000) were also used. The arrowhead indicates the position of LppQ on the immunoblots\nMorphology of the mycoplasmas\nElectron microscopy showed cultivated M. mycoides subsp. mycoides SC to be basically spherical in shape. They usually occurred as single cells but pairs or small groups of up to five organisms were also noted. Cell diameter varied between 200 and 500\u00a0nm and the cell surface was slightly grainy and irregular (Fig.\u00a03A, C, E). Between fully grown microorganisms, small granular debris were observed.\nFig.\u00a03Scanning electron micrographs of M. mycoides subsp. mycoides SC with anti-LppQ-N\u2032 and anti-LppQ-C\u2032 antibodies. Secondary electron microscope images (A, C, E) reveal the spherical shape and rough cell surface of the mycoplasmas. Corresponding backscattered electron micrographs (B, D, F) show indirect immunolabelling with 15\u00a0nm colloidal gold antibodies directed against the N-terminal extracellular domain of LppQ (B). In contrast, LppQ is not detected with anti-LppQ-C\u2032 antibody (D). Labelling is abolished when an anti-calcitonin antibody is used as an irrelevant primary antibody (F)\nImmunogold labelling\nLabelling of mycoplasmas on coverslips with anti-LppQ-N\u2019 antibodies yielded a SEM signal of moderate to high density at the cell surface (Fig.\u00a03B). Specific labelling of granular debris was also present. In contrast, labelling with anti-LppQ-C\u2032 antibodies was absent in most of the cases (Fig.\u00a03D) and appeared to be insignificant and coincidental in few cases (Table\u00a01), as also observed in the three control experiments whereby labelling was investigated in the absence of primary antibodies and when pre-immune serum or anti-calcitonin antibodies were used (Fig.\u00a03F). Overall, there were significant differences between the number of particles labelled per cell by using anti-LppQ-N\u2019 antibodies and those labelled in the other four experiments (P\u00a0<\u00a00.0001; Table\u00a01)\nTable\u00a01Statistical analysisStatistical dataTreatmentAnti-LppQ-N\u2019Anti-LppQ-C\u2019Anti-calcitoninPre-immune serumNo primary antibodySingle cells investigated2748192023Labelled particlesTotal33317675Min20000Max314332Average per cella12.33\u00a0\u00b1\u00a07.580.35\u00a0\u00b1\u00a00.840.32\u00a0\u00b1\u00a00.820.35\u00a0\u00b1\u00a00.810.22\u00a0\u00b1\u00a00.52P-valueb<0.0001<0.0001<0.0001<0.0001a\u00b1 Standard deviationbCalculated by the analysis of variance (anova) method: single factor. Differences between the number of particles labelled per cell by using anti-LppQ-N\u2019 antibodies and those labelled in the other four experiments were analysed\nDiscussion\nThe goal of the present study was to discriminate the distribution of extracellular vs. integral membrane domains of lipoprotein LppQ in M. mycoides subsp. mycoides SC at the ultrastructural level. Amino acid sequence analysis for hydrophobicity by the method of Hopp and Woods (1981) revealed the N-terminal portion of LppQ to be particularly hydrophilic (Abdo et\u00a0al. 2000). Thus, the lipidated N-terminal domain was predicted to be exposed at the extracellular surface of the plasma membrane (Abdo et\u00a0al. 2000). On the other hand, the C-terminal portion consists of a region built up of 10 hydrophobic repeated units, and not nine as reported previously (Abdo et\u00a0al. 2000). The repeat units were found to consist each of approximately 10 amino acids forming helices delimited on both sides with random coils, i.e. conformations without a regular secondary structure, and were not predicted to be membrane-associated by bioinformatic analysis. Since transmembrane helices are mostly composed of hydrophobic amino acid and the degree of hydrophobicity of the C-terminal domain of LppQ is clearly significant, the C-terminal portion was expected to be an integral membrane domain (Fig.\u00a01). It has to be noted at this point that lipoproteins generally do not possess additional membrane-spanning domains (MSD) beyond their N-terminal lipid anchors. However, there are relevant examples from Gram-positive bacteria, which contradict this general statement about lipoproteins and substantiate instead the occurrence of such additional MSDs. These examples include lipoproteins CtaC and QoxA of Bacillus subtilis (Bengtsson et\u00a0al. 1999; Antelmann et\u00a0al. 2001), as well as the proven lipoprotein of Mycoplasma pneumoniae F0F1 ATPase (Pyrowolakis et\u00a0al. 1998).\nHigh resolution scanning electron microscopy in combination with immunogold labelling revealed extracellular epitopes with antibodies against LppQ-N\u2032 but no signal above the background of the three negative controls was detected with the antibody against LppQ-C\u2032. These results substantiate the predicted localization of the two domains, as they unambiguously corroborate the accessibility of the N-terminal domain at the extracellular side of the plasma membrane. The failure to immunolabel the C-terminal epitopes is an indication that they may be imbedded in the membrane. However, one cannot rule out the possibility that such epitopes (i) are buried in the centre of a globular protein, (ii) are masked by other surface components or (iii) are not recognized in their native forms by rabbit antibodies produced by inoculation of the recombinant peptide. Beyond providing ultrastructural evidence for the location of the hydrophilic and hydrophobic domains of LppQ, these results also demonstrate that immunogold labelling in scanning electron microscopy allows for subtle topographical discrimination and that selective visualization of peptides being exposed at the outer surface of microorganisms can be achieved with high accuracy and reliability.\nThe domain analysis of LppQ, which demonstrates the outstanding surface localization of the strongly hydrophilic N-terminal part of the lipoprotein, may explain the strong humoural response elicited by this lipoprotein during infection of cattle by M. mycoides subsp. mycoides SC (Abdo et\u00a0al. 2000). It has to be demonstrated yet whether LppQ is also able to induce any cell-mediated immune response, as it is more often the case for cytoplasmic proteins. Absence of a cell-mediated immune response would explain the lack of protection in animals immunized with purified recombinant LppQ. In this respect, it should be noted that immunization of cattle with the purified recombinant N-terminal domain of LppQ elicited inflammatory processes of M. mycoides subsp. mycoides SC and exacerbated the clinical signs of CBPP after experimental infection compared to non-immunized animals (Nicholas et\u00a0al. 2004).\nConclusions\nDirect visualization of the N-terminal part of LppQ at the outer surface of M. mycoides subsp. mycoides SC, as demonstrated herein, provides evidence for the contention that LppQ is located to the extracellular side of the plasma membrane of mycoplasmas and that it is easily accessible by the humoural immune system of infected animals upon contact of host cells with M. mycoides subsp. mycoides SC.","keyphrases":["domain analysis","lipoprotein lppq","mycoplasma mycoides subsp. mycoides sc","contagious bovine pleuropneumonia (cbpp)","immunogold labeling","scanning electron microscopy (sem)"],"prmu":["P","P","P","P","P","P"]}
{"id":"Doc_Ophthalmol-3-1-1896293","title":"ISCEV standard for clinical pattern electroretinography\u20142007 update\n","text":"The pattern electroretinogram (PERG) is a retinal response evoked by viewing a temporally alternating pattern, usually a black and white checkerboard or grating. The PERG is important in clinical and research applications because it provides information both about retinal ganglion cell function and, because the stimulus is customarily viewed with central fixation, the function of the macula. The PERG can therefore facilitate interpretation of an abnormal pattern VEP by revealing the retinal responses to a similar stimulus to that used for the VEP. However, practitioners may have difficulty choosing between the different techniques for recording the PERG that have been described in the literature. The International Society for Clinical Electrophysiology of Vision published a standard for clinical PERG recording in 2000 to assist practitioners in obtaining good quality reliable responses and to facilitate inter-laboratory communication and comparison. This document is the scheduled revision of that standard.\nIntroduction\nThe pattern electroretinogram (PERG) is a retinal biopotential evoked when a temporally modulated patterned stimulus (checkerboard or grating) of constant mean luminance is viewed. The PERG is most often evoked by alternating contrast-reversal of a black and white square checkerboard pattern with central fixation. It may be altered in dysfunction confined to the macula or to the retinal ganglion cells, which do not significantly affect the a- and b-waves of the conventional full-field ERG, and thus the PERG receives clinical and research attention in both neurological and ophthalmological practice. Clinically, the PERG can be used in a patient with an abnormal visual evoked potential to establish whether a retinal (macular) disorder is present, and thus differentiate between macular and optic nerve dysfunction as a cause for the VEP abnormality. It can also directly demonstrate retinal ganglion cell dysfunction.\nHowever, the PERG is a very small signal, typically in the region of 2\u20138\u00a0\u03bcV across a normal population, and PERG recording is technically more demanding than standard flash ERGs. The amplitude values recorded will depend upon stimulus characteristics and the electrodes being used. The recordings published in the literature vary considerably in technique and technical quality; this document is intended to guide new or existing users to the most appropriate techniques for recording a standard clinical PERG.\nThe International Society for Clinical Electrophysiology of Vision (ISCEV) initially published \u201cPERG Guidelines\u201d [1], intended as a guide to practice and to assist in interpretation of PERGs. Those guidelines were subsequently revised to a PERG standard [2] and the present document is the scheduled revision of that standard. The standard PERG represents a minimum protocol for recording a PERG with straightforward technical procedures that should allow reproducible responses to be recorded under defined conditions. As a minimum, it is intended that the standard method and responses be widely used, but in addition to rather than to the exclusion of other paradigms. Common additional PERG techniques are also briefly described in this document, and individual laboratories are encouraged to use additional stimuli and protocols tailored to their own requirements.\nThe standard is based upon equipment and analytic capabilities currently available in most neurophysiological or ophthalmological electrodiagnostic clinics. This document addresses recording conditions and technology specific to the PERG, and presumes that the reader already has basic understanding and skills in clinical electrophysiology. Although much of the document will apply equally to adults and children, the standard is not necessarily appropriate to paediatric applications. The standard will be reviewed by ISCEV in a further four years.\nWaveform nomenclature and measurement\nThe waveform of the PERG evoked by pattern-reversal stimuli depends on the temporal frequency of the stimulus. By convention, positivity is displayed upward.\nTransient PERG\nThe ISCEV standard PERG is a transient response i.e. a response that is effectively complete before the next contrast reversal. Transient recording allows separation of the PERG components. At low temporal frequencies (<6 reversals per second (rev\/s); equivalent to\u00a0<3\u00a0Hz) transient PERGs are obtained (Fig.\u00a01). The PERG waveform in normal subjects usually consists of a small initial negative component with a peak time of approximately 35\u00a0ms, N35, which is followed at 45\u201360\u00a0ms by a much larger positive component (P50). This positive component is followed by a larger negative component at 90\u2013100\u00a0ms (N95).\nFig.\u00a01A normal PERG. The amplitude of P50 in a normal subject is usually between 2.0 and 4.0\u00a0\u03bcV\nFor the transient PERG, amplitude measurements are made between peaks and troughs: the P50 amplitude is measured from the trough of N35 to the peak of P50. In some patients the N35 is poorly defined; in these cases N35 is replaced by the average baseline between time zero and the onset of P50. The N95 amplitude is measured from the peak of P50 to the trough of N95. It should be recognised that measured in this way, N95 amplitude includes the P50 amplitude and P50 that of N35. It can be argued that baseline to peak measurements may be more meaningful. However, the P50 and N95 components may not be independent. At the time of writing there are no peer-reviewed data attempting to resolve these issues, and given the wealth of data already published using peak-to-peak measures, they remain the standard measures for PERG amplitudes.\nThe times to peaks in the waveform (implicit time) should be measured from the onset of the contrast-reversal to the peak of the component concerned; it should be noted that the highest absolute amplitude point on a waveform will not always be appropriate for the definition of the peak if there is contamination from muscle activity or other artifacts. The peak should be designated where it would appear on a smoothed or idealised waveform (see Fig.\u00a01). The peak times or implicit times are often (erroneously) referred to as latencies. Correctly, latency refers to the delay prior to the onset of a response, not to the maximum activity of a peak.\nBasic technology\nStandard equipment for visual stimulus generation, amplification of physiological signals, and the recording and storing of electrophysiological data, is required for PERG testing. Information about the calibration of equipment and measurement of the parameters specified in this standard appears in the ISCEV Calibration Guidelines [3].\nElectrodes\nRecording electrodes\nClinical ERG electrodes that contact the cornea or nearby bulbar conjunctiva should be used as the active electrodes to record standard PERGs. Electrodes that degrade image quality on the retina (this includes all contact lens electrodes) must not be used. Thin conductive fibres and foils can usually be positioned without topical anaesthesia. Electrode integrity should be checked prior to insertion, to meet guidelines for each electrode type. It is recommended not to measure impedance in situ unless explicitly specified by the particular equipment manufacturer. Electrodes should be carefully positioned to minimise instability (a major source of artifact or interference). Those who perform the test should be aware of possible causes of artifact.Fibre electrodes are best positioned in relation to the upper margin of the lower eyelid. Some place the electrode in the lower conjunctival fornix (under the lower eyelid); such a position may reduce trial-to-trial variability but will also result in a lower amplitude PERG. Optimum stability is achieved by tethering the electrode at the nasal canthus.Foil electrodes should be positioned directly under the centre of the pupil so that there is minimal or no movement of the electrode when the patient blinks. This is best achieved by having the foil curve over the lower eyelashes without contacting them, and then tethering the lead to the cheek. The junction of the electrode and lead should form as straight a line as possible and the junction should not touch the skin.Loop electrodes should be hooked into the lower fornix. Loops should be folded so that the contact windows on otherwise insulated wire are positioned on the bulbar conjunctiva, about 5\u00a0mm under the limbus. Loop electrodes should not touch the cornea. To achieve this, the limbs of the loop should diverge widely (15\u201320\u00a0mm) before entering the fornix. The lead is then taped to the cheek.\nThe appropriate techniques for individual electrode types are very important to achieve stable and reproducible PERG recordings. Additional sources should be consulted in relation to the specific electrode used.\nSurface (skin) active electrodes should not routinely be used for recording the standard PERG; a surface electrode positioned on the lower eyelid will record PERGs of lower amplitude than those recorded from an electrode in contact with the eye. Surface electrode recordings may however be useful when a corneal electrode is contraindicated or in paediatric practice. The use of a surface electrode to record the PERG is a variation from the standard and should be noted in the report.\nReference electrodes\nSeparate surface reference electrodes should be placed on the skin near the ipsilateral outer canthus of each eye. Mastoid, earlobe or forehead locations may result in contamination of the PERG from cortical potentials or the fellow eye. If monocular PERG recording is performed, the electrode in the occluded eye may be used as a reference.\nGround electrodes\nA separate surface electrode should be attached and connected to the amplifier \u201cground input\u201d; the forehead would be a typical location, but other locations are acceptable as the location of the ground electrode should not affect the standard PERG.\nSurface electrodes\nThe skin should be prepared with a suitable cleaning agent, and a suitable conductive paste used to ensure good electrical connection. The impedance between the skin electrodes used for reference and ground, measured on the subject, should be less than 5\u00a0k\u03a9. Since the electrode in the eye will have very low impedance, a low impedance of the reference electrode is also important to obtain recordings as free as possible from mains (line frequency) interference.\nElectrode cleaning and sterilisation\nElectrodes should be cleaned and sterilised according to local health and safety regulations. The ISCEV Standard for full-field flash ERGs describes the appropriate care of ERG electrodes [4].\nStimulus parameters\nThis standard specifies the protocol for basic clinical PERG recording. Laboratories may choose to test more conditions or parameters than are described herein.\nField and check size\nA black and white reversing checkerboard should be used for the standard PERG. It is not necessary to use a square stimulus field, but the aspect ratio between the width and the height of the stimulus field should not exceed 4:3. The mean of the width and the height of the stimulus field should be 15\u00b0 (\u00b13\u00b0) with a check size of 0.8\u00b0 (\u00b10.16\u00b0).\nLuminance\nPERGs are difficult to record with low stimulus luminance, and a photopic luminance level for the white areas of greater than 80\u00a0cd\u00a0\u00b7\u00a0m\u22122 is required. The mean luminance of the stimulus screen must be constant during checkerboard reversals (i.e. no transient luminance change).\nContrast\nThe contrast between black and white squares should be maximal (close to 100%) for the standard PERG and not less than 80%. The contrast and luminance used should be reported.\nFrame rate\nRaster-based CRTs are typically used to present the pattern stimuli. The frame rate of the CRT is a significant stimulus parameter for PERGs, and a frequency of 75 Hz or greater should be used.\nBackground illumination\nThe luminance of the background beyond the checkerboard field is not critical when using the standard PERG technique providing dim or ordinary room lighting is used; ambient lighting should be the same for all recordings. Care should be taken to keep bright lights out of the subjects\u2019 direct view. Pupil diameter should be recorded.\nReversal rate\nThe standard transient PERG should be obtained using a reversal rate of 4 rev\/s\u00a0\u00b1\u00a00.8 rev\/s (i.e. 2\u00a0Hz\u00a0\u00b1\u00a00.4\u00a0Hz).\nCalibration\nAll stimulus parameters including luminance and contrast should be calibrated either locally or by the manufacturer and regular recalibration is advised [3].\nRecording equipment\nAmplification systems\nAC-coupled amplifiers with a minimum input impedance of 10\u00a0M\u03a9 should be used. Amplification systems must be electrically isolated from the patient according to the current safety standards for medical recording systems. The frequency response of bandpass amplifiers should include the range from 1\u2013100\u00a0Hz; analogue notch filters (that suppress signals at the alternating current line frequency) should not be used. Some users may encounter severe electromagnetic interference from the stimulus display that makes it difficult to obtain satisfactory recordings with these filter settings. Ideally, such interference should be eliminated by shielding or modifying the equipment; rearranging the electrode leads may be of benefit.\nAveraging and signal analysis\nSignal averaging is necessary because of the small amplitude of the PERG. The analysis period (sweep time) for the standard PERGs should be 150\u00a0ms or greater. Some laboratories assess baseline stability by displaying two successive responses on the same trace.\nArtefact rejection\nComputerised artifact rejection is essential. The limits for rejection should be set at no higher than\u00a0\u00b1\u00a050\u00a0\u03bcV, and preferably lower. The amplifiers must return to baseline rapidly following artifactual signals to avoid inadvertent storage of non-physiological data.\nSampling rate\nA minimum sampling rate of 1,000\u00a0Hz (1\u00a0ms per point) is recommended. See the calibration standard (3) for further information.\nData display systems\nDisplay systems must have adequate resolution to represent accurately the characteristics of this small amplitude signal. Ideally the recording system provides simultaneous display of the input signal and the accumulating average. In the absence of a simultaneous display, the system should allow a rapid alternation between displaying the input signal and displaying the current average so that the quality of the input signal can be adequately monitored. Even with a computerised artifact rejection system, it is important that the input signal be continuously monitored for baseline stability and the absence of amplifier blocking.\nClinical protocol\nPreparation of the patient\nPositioning\nThe patient should be as comfortable as possible with their head in a stable position against a head-rest. A chin rest is inappropriate and should not be used.\nPupils\nThe PERG should be recorded without dilatation of the pupils, to preserve accommodation and thus retinal image quality.\nFixation\nA fixation mark in the centre of the screen at a node of the checkerboard is essential. If there are any doubts about the quality of fixation in an individual patient, an effective method is to give the patient a (laser) pointer and have them point at the middle of the screen throughout. Excessive blinking during recording should be discouraged, pauses may be advantageous.\nRefraction\nBecause of the nature of the stimulus, PERG examination should be performed with optimal visual acuity at the testing distance. Patients should wear the appropriate optical correction for the test distance.\nMonocular and binocular recording\nProper positioning of recording and reference electrodes will permit either monocular or binocular recording of the PERG. Binocular recording is recommended for the standard PERG because it is generally more stable, it reduces examination time and it allows fixation by the better eye in cases of asymmetric visual loss. Monocular stimulation is required to record the PERG and the VEP simultaneously.\nRecording\nA minimum of 100 artefact-free sweeps should be collected and averaged for a standard PERG. More sweeps, perhaps as many as 300, will be needed when the PERG is small or undetectable or in a \u201cnoisy\u201d subject. At least two trials for each stimulus condition should be obtained to confirm reproducibility (i.e. at least one replication). It may be beneficial to superimpose repeated PERG recordings to evaluate quality and reproducibility.\nPERG reporting\nReporting\nIt is recommended that all reports contain measurements of P50 and N95 amplitude (see above), and P50 peak time (the peak of N95 is often rather broad precluding accurate peak time measurement of this component). All reports should also contain the stimulus parameters (luminance, contrast, and field size), and normal ranges for the laboratory concerned. Pupil size should be noted. Whenever practical, reporting of PERG results should include representative waveforms with appropriate amplitude and time calibrations, marks for the N35, P50 and N95 components, and should show replications.\nNormative data\/reference ranges\nAt present there are no standard international reference ranges for PERG measurements. Each laboratory should establish normal values for its own equipment and patient population. It should be noted that there are PERG changes with age.\nAdditional tests\nLarge field PERGs\nFor some applications, such as glaucoma assessment, a larger field, such as 30\u00b0, may be more appropriate. Further, some practitioners find the improved signal-to-noise ratio obtained with a larger field may have advantages; users may wish to consider recording a large field PERG in addition to the standard response.\nSteady-state PERG\nAt higher temporal frequencies, i.e. above 10 rev\/s (5\u00a0Hz), the successive waveforms overlap and a \u201csteady-state\u201d PERG is evoked. There are situations in which the steady-state PERG is useful and some laboratories favour it for glaucoma studies. Since little extra time is required, laboratories may wish to consider recording it in addition to the transient response.\nFor steady-state PERG, a reversal rate of approximately 16 rev\/s (8\u00a0Hz) is recommended. The steady-state PERG waveform is roughly sinusoidal, and interpretation of steady-state PERGs requires measurement of amplitude and phase shift (relative to the stimulus) of the second harmonic by Fourier analysis. The presence of a significant first harmonic indicates technical problems. For correct interpretation the analysis period must be an integer number of stimulus cycles, preferably greater than 6. Steady-state PERG recording without the capability for such analysis is not recommended.\nReporting of steady-state PERGs should include the amplitude and phase shift of the PERG at the reversal rate (i.e. at the second harmonic of the stimulus rate in Hz).","keyphrases":["pattern electroretinogram","electroretinogram","macula","clinical electrophysiology","retina"],"prmu":["P","P","P","P","P"]}
{"id":"Anal_Bioanal_Chem-4-1-2045123","title":"A new highly specific and robust yeast androgen bioassay for the detection of agonists and antagonists\n","text":"Public concern about the presence of natural and anthropogenic compounds which affect human health by modulating normal endocrine functions is continuously growing. Fast and simple high-throughput screening methods for the detection of hormone activities are thus indispensable. During the last two decades, a panel of different in vitro assays has been developed, mainly for compounds with an estrogenic mode of action. Here we describe the development of an androgen transcription activation assay that is easy to use in routine screening. Recombinant yeast cells were constructed that express the human androgen receptor and yeast enhanced green fluorescent protein (yEGFP), the latter in response to androgens. Compared with other reporters, the yEGFP reporter protein is very convenient because it is directly measurable in intact living cells, i.e., cell wall disruption and the addition of a substrate are not needed. When yeast was exposed to 17\u03b2-testosterone, the concentration where half-maximal activation is reached (EC50) was 50 nM. The relative androgenic potencies, defined as the ratio between the EC50 of 17\u03b2-testosterone and the EC50 of the compound, of 5\u03b1-dihydrotestosterone, methyltrienolone, and 17\u03b2-boldenone are 2.3, 1.4, and 0.15 respectively. The results presented in this paper demonstrate that this new yeast androgen bioassay is fast, sensitive, and very specific and also suited to detect compounds that have an antiandrogenic mode of action.\nIntroduction\nThere is concern that chemicals in our food, water, and environment affect human health by disrupting normal endocrine function, possibly leading to reproductive failure in humans and tumors in sensitive tissues [1, 2]. This relates to chemicals with previously unknown hormonal properties, like certain pesticides and plasticizers, but also to compounds used in pharmaceutical preparations, eg., oral contraceptives and tablets for hormone-replacement therapy, the endogenous steroids excreted in urine of man and domestic animals and potentially also compounds used for their growth-promoting properties in animals. Of all endocrine disruptors, environmental estrogens are the most studied [3]. However, recent studies show a crucial involvement of the androgen receptor in abnormal sex development. The presence of pollutants with adverse effects on human androgen receptor (hAR) has been reported from paper-mill effluents and as a result of intensive farming [4, 5]. Xenoandrogenic exposure-related disorders include testicular cancer, hypospadias, cryptorchidism, and poor sperm and very recently prepubertal gynecomastia was linked to both estrogenic and antiandrogenic effects of lavender and tea tree oil [6].\nChemical and immunological methods are commonly used to detect steroid hormones in food, clinical practice, environmental samples, or doping control. Owing to the great variety of chemicals with hormonelike activity, these methods have the drawback that they only quantify the compound of interest and are not able to determine biological activity of unknown compounds and their metabolites, this in contrast to biological assays. Receptor-based transcription activation assays can be used to detect all compounds having affinity for a given receptor [7, 8]. In contrast to receptor binding assays, receptor gene bioassays also include the transactivation step and can distinguish between receptor agonists and receptor antagonists [9]. This feature is very helpful in detecting both known and unknown compounds.\nSeveral assays have been developed for this purpose, using both mammalian and yeast cells. In general, transcription activation assays based on mammalian, or more particular human, cell lines have been shown to be more sensitive than yeast-based assays, and may be able to identify compounds that require human metabolism for activation into their active state. Metabolic conversion can either activate or inactivate some compounds [10], whereas the relatively low metabolic capacity of yeast ensures that the test reflects the activity of the original compound. In addition, yeast-based assays have several other advantages. These include low costs, easy handling, lack of known endogenous receptors that may compete with the receptor activity under investigation (no crosstalk), and the use of media that are devoid of steroids [11\u201313]. Furthermore, yeast cell assays are extremely robust and survive extracts from dirty sample matrices such as sediments, urine, and feed [14\u201316]. Especially in the case of androgens, the lack of known endogenous receptors in yeast is a great advantage compared with mammalian cell lines, as androgen responsive elements (AREs) can also be activated by the progesterone receptor and the glucocorticoid receptor. To avoid potential crosstalk in mammalian cell lines, a lot of efforts was expended to construct an ARE that is specific and no longer inducible by the progesterone and glucocorticoid receptor [17\u201319]. However, up till now such an ARE does not exist and it is doubtful whether it will be found, as the consensus progesterone responsive element\/glucocorticoid responsive element is equal to the consensus ARE. Moreover, the glucocorticoid receptor is normally expressed in all mammalian cell types. So far this has resulted in cell lines that are not specific for androgens and that also respond to gestagens or glucocorticoids [20\u201322].\nThis paper reports the development of a new yeast androgen bioassay by creating a stably transfected yeast strain that expresses yeast enhanced green fluorescent protein (yEGFP) as a measurable reporter protein in response to androgens. The lack of known endogenous receptors in yeast enabled us to use the strong nonspecific consensus ARE sequence, which is actually a common hormone responsive element that is recognized by the androgen, progesterone and glucocorticoid receptors and can therefore not be used in mammalian cell lines expressing more than one of these receptors. Exposures to 17\u03b2-testosterone, 17\u03b2-estradiol, progesterone, dexamethasone, and other compounds were performed in 96-well plates in order to demonstrate the suitability and specificity of this new yeast androgen bioassay. Additionally, flutamide and several brominated flame retardants were tested for their antagonistic mode of action and the results were compared with a yeast androgen bioassay expressing \u03b2-galactosidase as a reporter protein.\nMaterials and methods\nChemicals\nChemicals and methods to prepare the growth media, to perform PCR, to isolate DNA, and to transform bacteria and yeast were as described earlier [23]. Corticosterone, dexamethasone, 17\u03b1-estradiol, 17\u03b2-estradiol, estrone, flutamide, 4-hydroxytamoxifen, medroxyprogesterone 17-acetate, and progesterone were obtained from Sigma (St. Louis, MO, USA). The following compounds were obtained from Steraloids (Newport, RI, USA): 17\u03b2-boldenone, diethylstilbestrol, 5\u03b1-dihydrotestosterone, 17\u03b1-ethynylestradiol, 17\u03b2-testosterone and 17\u03b2-trenbolone. Tetrahydrogestrinone (THG) was a gift from M. Thevis (DSHS, Cologne, Germany). Copper sulfate and dimethyl sulfoxide (DMSO) were obtained from Merck (Darmstadt, Germany) and methyltrienolone was obtained from PerkinElmer (USA). All restriction endonucleases and corresponding buffers were obtained from New England Biolabs (Hitchin, UK) and the yeast \u03b2-galactosidase assay kit was from Pierce Biotechnology (Rockford, IL, USA). 2,4,6-Tribromophenol (TBP), BDE-39, and the hydroxyl derivative 4-OH-BDE-17 were synthesized at the Wallenberg Laboratory (Stockholm University, Sweden).\nYeast strains\nThe yeast Saccharomyces cerevisiae (CEN.PK 102-5B, K20, URA3\u2212, HIS3\u2212, LEU\u2212) host strain was a gift from H. Sillj\u00e9 (University of Utrecht, The Netherlands). The yeast androgen bioassay with \u03b2-galactosidase as a marker was kindly provided by D.P. McDonnell (Duke University, USA).\nPlasmids\nThe p403-GPD and p406-CYC1 yeast expression vectors were obtained from the American Type Culture Collection (ATCC, Rockville, Maryland, USA). The pyEGFP3 plasmid was a gift from A.J. Brown (Stanford University, USA).\nConstruction of the p403-GPD-hAR expression vector\nThe yeast cells provided by McDonnell were grown overnight and chromosomal DNA was isolated. This DNA was used to serve as a template for the PCR to obtain the complementary DNA (cDNA) of hAR. Full-length hAR cDNA was obtained using the Expand High Fidelity PCR system (Boehringer Mannheim) and an Eppendorf Mastercycler gradient. The sequence of the 5\u2032-primer was 5\u2032-GCTCTAGAATGGAAGTGCAGTTAGGGCTGGG-3\u2032, containing a restriction site for XbaI just before the ATG start codon. The sequence of the 3\u2032-primer was 5\u2032-GCGGATCCTCACTGGGTGTGGAAATAGATGGG-3\u2032, containing a restriction site for BamHI just after the TGA stop codon. This PCR generated a full-length double-stranded (ds) cDNA of 2,763 bp of the hAR gene with a 5\u2032-XbaI and a 3\u2032-BamHI restriction site.\nThe 2,763-bp full-length hAR PCR product was isolated from a 1% low-melt agarose gel, cleaved with XbaI and BamHI and ligated into the corresponding site of the p403-GPD yeast vector. Plasmid digestion and PCR controls revealed several good clones.\nConstruction of the p406-ARE2-CYC1-yEGFP reporter vector\nA set of complementary oligonucleotides (a and b), each with two consensus ARE sequences (in bold), were synthesized. A solution with both cDNA oligonucleotides, 2.5\u00a0\u03bcM of each, was heated at 95\u00a0\u00b0C and cooled down to room temperature in 2\u00a0h. This set gave a ds DNA with a 5\u2032-SacI sticky end and a 3\u2032-MscI blunt end.Sa: 5\u2032-AAAGTCAGAACAGCATGTTCTGATCAAATCTAGAAGATCCAAAGTCAGAACAGCATGTTCTGATCAAACTCGAGCAGATCCGCCAGGCGTGTATATATAGCGTGGATGG-3\u2032Sb: 5\u2032-CCATCCACGCTATATATACACGCCTGGCGGATCTGCTCGAGTTTGATCAGAACATGCTGTTCTGACTTTGGATCTTCTAGATTTGATCAGAACATGCTGTTCTGACTTTAGCT-3\u2032.\nThis ds DNA was cloned into the corresponding site of the p406-CYC1 vector. Subsequently, yEGFP [24] obtained from a HindIII\/SalI double digestion of pyEGFP was cloned in the corresponding HindIII\/SalI sites of the p406-ARE2-CYC1 reporter construct. Plasmid digestion and PCR controls revealed several good clones.\nTransformation of yeast cells\nTransformation of yeast K20 host strain (Ura\u2212, His\u2212, and Leu\u2212) was performed by the lithium acetate protocol as described earlier [23]. First, the yeast was transformed with the p406-ARE2-CYC1-yEGFP reporter vector, integrated at the chromosomal location of the uracil gene via homologous recombination. Therefore, prior to transformation, the reporter vector was linearized by cutting with StuI, which has a unique restriction site in the URA3 marker gene. Transformants were grown on minimal medium plates containing l-leucine and l-histidine (MM\/L plates). This yeast reporter strain was then transformed with the p403-GPD-hAR expression vector, which was linearized by cleavage with NsiI, which has a unique restriction site in the HIS3 marker gene (histidine). Transformants were grown on MM\/L plates and PCR controls were used to select clones that contain the p406-ARE2-CYC1-yEGFP reporter and the p403-GPD-hAR expression construct.\nPCR controls\nPCR controls were performed on the reporter-receptor transformants. Yeast chromosomal DNA of transformants was isolated and PCR controls were performed. PCR I was performed with a 5\u2032-primer on the backbone of the reporter plasmid and a 3\u2032-primer on the ARE2 sequence. The sequence of the 5\u2032-primer was 5\u2032-AGCGAGTCAGTGAGCGAGGAAG-3\u2032 and the sequence of the 3\u2032-primer was 5\u2032-TGCTGTTCTGACTTTGGATC-3\u2032. PCR II was performed with a 5\u2032-primer on the CYC1 (cytochrome c oxidase) promoter of the reporter plasmid and a 3\u2032-primer on the CYC1 terminator. The sequence of the 5\u2032-primer was 5\u2032-TCTATAGACACACAAACACAA-3\u2032 and the sequence of the 3\u2032-primer was 5\u2032-GGGAGGGCGTGAATGTAAG-3\u2032. PCR III was performed with the primers that were also used to obtain the full length cDNA of the hAR (see \u201cConstruction of the p403-GPD-hAR expression vector\u201d).\nStreamlined yEGFP assay with the yeast androgen bioassay\nThe day before running the assay, a single colony from a MM\/L agar plate was used to inoculate 10\u00a0mL of the selective MM\/L medium. This culture was grown overnight at 30\u00a0\u00b0C with vigorous orbital shaking. At the late log phase, the yeast androgen receptor biosensor was diluted in the selective MM\/L medium to an optical density (OD) at 604\u00a0nm between 0.08 and 0.12. For exposure, aliquots of 200\u00a0\u03bcL of this diluted yeast culture were pipetted into each well of a 96-well plate and 2\u00a0\u03bcL of a 17\u03b2-testosterone or other stock solution in DMSO was added. DMSO-only controls were included in each experiment and each sample concentration was assayed in triplicate. Exposure was performed for 24\u00a0h at 30\u00a0\u00b0C and orbital shaking at 125\u00a0rpm. Fluorescence was measured at 0 and 24\u00a0h directly in a CytoFluor multiwell plate reader (Series 4000, PerSeptive Biosystems) using excitation at 485\u00a0nm and measuring emission at 530\u00a0nm. The fluorescence signal was corrected with the signals obtained with MM\/L containing DMSO solvent only. Densities of the yeast culture were determined by measuring the OD at 630\u00a0nm, but this was only done to check whether a sample was toxic for the yeast cells. For the calculation of the relative androgenic potency (RAP) of the compounds in the yeast androgen bioassay, the data of a complete dose\u2013response curve were fitted using the equation  (Slide write Plus, version 6.00). This is equivalent to , where response is the measured fluorescence signal, [agonist] is the concentration of the test compound, and EC50 is the concentration of the test compound giving half-maximum response.\nlacZ-based yeast androgen bioassay: \u03b2-galactosidase assay\nAn agar plate containing the selective growth medium, consisting of a yeast nitrogen base with dextrose (2%), lysine (36\u00a0mg\/L), tryptophan (48\u00a0mg\/L), uracil (24\u00a0mg\/L), and adenine (41\u00a0mg\/L), was inoculated with the yeast androgen receptor cytosensor from a frozen -80\u00a0\u00b0C stock (20% glycerol v\/v). The plate was incubated at 30\u00a0\u00b0C for 24\u201348\u00a0h and then stored at 4\u00a0\u00b0C. The day before running the assay, a single colony of the yeast was used to inoculate 10\u00a0mL of the selective growth medium. This culture was grown overnight at 30\u00a0\u00b0C with vigorous orbital shaking at 225\u00a0rpm. At the late log phase, the yeast androgen receptor cytosensor was diluted in growth medium to an OD of 0.06 at 604\u00a0nm, and CuSO4 (0.05\u00a0mM) was added to induce the expression of the hAR. For exposure in 96-well plates, aliquots of 200\u00a0\u03bcL of this diluted yeast culture were pipetted into each well and 2\u00a0\u03bcL of stock solutions in DMSO was added. Exposure was performed for 24\u00a0h at 30\u00a0\u00b0C and 125\u00a0rpm, and the \u03b2-galactosidase activity was measured with a commercial yeast \u03b2-galactosidase assay kit from Pierce (Rockford, IL, USA). This kit uses o-nitrophenyl \u03b2-d-galactopyranoside as a substrate, and the solution turns yellow upon hydrolysis of \u03b2-d-galactopyranoside to o-nitrophenol and galactose. The yellow o-nitrophenol is measured in a Biotek (Winooski, VT, USA) model ELx 808 series ultra microplate reader at 405\u00a0nm. Densities of the yeast culture were determined by measuring the OD at 630\u00a0nm. The measured response at 405 nm was corrected for the OD at 630\u00a0nm.\nResults and discussion\nA recombinant yeast cell was constructed that expresses the hAR and yEGFP as a reporter protein in response to androgens. Both the receptor construct as well as the reporter construct were stably integrated into the yeast genome by the use of yeast-integrating plasmids. For the construction of the reporter vector the p406-CYC1 plasmid, containing the URA3 marker gene, was used. Two consensus AREs were placed in the SacI\/MscI site of the truncated CYC1 promoter in a way that the \u2212254 to \u2212147 XhoI-SphI part of the CYC1 promoter was restored [23]. High expression levels of the androgen receptor were obtained by placing the cDNA of the hAR gene behind the strong constitutive yeast glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter in the p403-GPD plasmid. This plasmid contains the HIS3 marker gene. Transfected strains were checked with PCR. The correct and specific functioning of the yeast androgen bioassay was studied by exposures to 17\u03b2-testosterone and other compounds and the results were compared with results obtained with the lacZ-based yeast androgen bioassay provided by McDonnell. In addition, the antiandrogenic properties of several brominated flame retardants were investigated.\nPCR controls\nA number of different PCR controls were carried out to check the integration of the vectors into the yeast genome. Figure\u00a01 shows the gel electrophoresis results of these PCR controls. PCR I (Fig.\u00a01a) was performed with primers on the backbone of the p406 plasmid and on the ARE2 sequence. As expected, it gave the specific 360-bp band with the p406-ARE2-CYC1-yEGFP reporter vector and the DNA that was isolated from the yeast androgen biosensor. The negative controls, performed with the empty p406-CYC1 plasmid and with the DNA that was isolated from the empty yeast host (the nontransfected yeast cell), showed no PCR bands. PCR II (Fig.\u00a01a) was performed with primers on the CYC1 promoter and the CYC1 terminator. As expected, it gave the specific 873-bp band with the reporter vector and the DNA that was isolated from the biosensor, because both contain the reporter construct with the yEGFP that was ligated between the CYC1 promoter and CYC1 terminator. The negative controls, performed with the empty p406-CYC1 plasmid and with the DNA that was isolated from the empty yeast host, did not show the reporter-specific 873-bp band. However, this PCR generated a 435-bp band with the DNA of the empty yeast host and the biosensor. This 435-bp band corresponds to the CYC gene of the yeast host itself and is therefore also a specific band. PCR III (Fig.\u00a01b) was performed with the primers on the hAR gene. As expected, it gave the specific 2,763-bp band with the p403-GPD-hAR expression vector and the DNA that was isolated from the biosensor. In the negative control, performed with the DNA that was isolated from the yeast host, the receptor-specific 2,763-bp band was not present. These PCR controls demonstrate that all specific PCR bands can be seen, thus demonstrating that our yeast androgen bioassay contains the p403-GPD-hAR expression vector and the p406-ARE2-CYC1-yEGFP reporter vector, both stably integrated in the yeast genome.\nFig.\u00a01PCR controls. The PCR controls were performed as described in \u201cPCR controls.\u201d a Lanes 1, 6 and 11 contain a 100-bp ladder. PCR I was performed with primers on the backbone of the p406 plasmid and on the ARE2 sequence. Lanes 2\u20135 are PCR I on the p406-ARE2-CYC1-yEGFP reporter vector, the DNA that was isolated from the yeast transformant, the empty p406-CYC1 plasmid, and the DNA that was isolated from the empty yeast host (the nontransfected yeast cells), respectively. PCR II was performed with primers on the CYC1 promoter and the CYC1 terminator. Lanes 7\u201311 are PCR II on the reporter vector, the DNA from the yeast transformant, the empty p406-CYC1 plasmid, and the DNA from the empty yeast host respectively. b Lane 1 contains a 1-kb ladder. PCR III was performed with the primers on the human androgen receptor gene. Lanes 2\u20134 are PCR III on the p403-GPD-hAR expression vector, the DNA from the yeast transformant, and the DNA from the empty yeast host, respectively. yEGFP yeast enhanced green fluorescent protein\nDose\u2013response curves obtained with the new yeast androgen bioassay\nThe dose\u2013response curves for several natural and synthetic androgens are shown in Fig.\u00a02. 5\u03b1-Dihydrotestosterone, 17\u03b2-testosterone and 17\u03b2-boldenone caused a dose-related increase in the production of yEGFP, demonstrating that these compounds are potent androgens. The bioassay showed a limit of detection of 3\u00a0nM for 5\u03b1-dihydrotestosterone with a dynamic range from 3 to 500\u00a0nM and very low standard deviations (les than 3%). The figure also shows that 17\u03b2-estradiol and progesterone give a response. The female hormone 17\u03b2-estradiol gives a full dose\u2013response curve, but the maximum of the curve is reached at a 500 times higher concentration than that of 17\u03b2-testosterone and is less steep. Progesterone gives a response, but the maximum response is only about 35% of that of 17\u03b2-testosterone and is reached at a 25 times higher concentration. Both 17\u03b2-estradiol and progesterone are known to possess androgenic properties. Progesterone displays low binding to the androgen receptor [25] and shows androgenic effects in vivo [26]. According to [27], 17\u03b2-estradiol and progesterone showed androgenic activity in ten out of 11 and seven out of nine mammalian cell reporter gene (MCRG) systems, respectively. The corticosteroids corticosterone and dexamethasone showed no response in our assay.\nFig.\u00a02Response of the yeast androgen biosensor to different substances. Exposure to 17\u03b2-testosterone, 5\u03b1-dihydrotestosterone, progesterone, dexamethasone, 17\u03b2-estradiol, and 17\u03b2-boldenone was started by adding to 200\u00a0\u03bcL of a yeast culture a 2-\u03bcL aliquot of a stock solution of the compound in dimethyl sulfoxide (DMSO). Fluorescence was determined after 24\u00a0h as described in \u201cStreamlined yEGFP assay with the yeast androgen bioassay.\u201d Fluorescence signals are the mean of a triplicate with the standard deviation (SD). 17\u03b2-T 17\u03b2-testosterone, DHT 5\u03b1-dihydrotestosterone, Prog progesterone, Dex dexamethasone, 17\u03b2-E2 17\u03b2-estradiol, Bold 17\u03b2-boldenone\nTable\u00a01 shows the calculated EC50, ie., the concentration giving a half-maximum response, and the RAP, defined as the ratio between the EC50 of 17\u03b2-testosterone and the EC50 of the compound, for several compounds. The yeast androgen bioassay showed good sensitivity towards all androgens tested, with the following range of potencies: 5\u03b1-dihydrotestosterone > 17\u03b2-trenbolone > methyltrienolone > tetrahydrogestrinone > 17\u03b2-testosterone > 17\u03b2-boldenone > medroxyprogesterone acetate > 17\u03b2-estradiol > progesterone. Steroids representative for other hormone receptors, like estrone, 17\u03b1-estradiol, 17\u03b1-ethynylestradiol, and diethylstilbestrol for the estrogen receptor and corticosterone and dexamethasone for the glucocorticoid receptor, showed no agonistic response. Only 17\u03b2-estradiol, progesterone and medroxyprogesterone acetate gave a clear agonistic response. However, these compounds are known to exert androgenic effects.\nTable\u00a01EC50 concentrations and relative androgenic potencies (RAP) of compounds in the yeast androgen biosensor expressing yeast enhanced green fluorescent protein in response to androgensCompoundQualitative response for AR agonismaCommentsbEC50 (nM) in the yeast androgen bioassaycRAPd17\u03b2-Testosterone Positive (11\/11)Strong AR agonist761.05\u03b1-DihydrotestosteronePositive (21\/21)Strong AR agonist, weak ER agonist332.317\u03b2-Boldenone5100.1517\u03b2-TrenbolonePositiveBinds strongly to AR521.5MethyltrienolonePositive (8\/8)AR agonist541.4TetrahydrogestrinoneAR agonist651.217\u03b2-EstradiolPositive (10\/11)AR agonist and antagonist, strong ER agonist9,0000.0084EstronePositive (2\/2)AR agonist, strong ER agonistNRNR17\u03b1-EstradiolNegative (1\/1)ER agonistNRNR17\u03b1-EthynylestradiolNegative (1\/1)Strong ER agonistNRNRDiethylstilbestrolNegative (2\/2)Strong ER agonistNRNR4-HydroxytamoxifeneNegative (1\/1)ER antagonistNRNRProgesteronefPositive (7\/9)1,7000.045Medroxyprogesterone acetate Positive (4\/4)Weak AR agonist1,5000.051CorticosteroneNegative (1\/1)Binds weakly to ARNRNRDexamethasonePositive (3\/4)AR agonistNRNRFlutamideNegative (5\/5)AR antagonistNRNR2,4,6-Tribromophenol NRNRBDE-19NRNR4-OH-BDE-17NRNRAR androgen receptor, ER estrogen receptor, NR no responseaQualitative response for AR agonism across all mammalian cell reporter gene studies (data obtained from [27])bComments obtained from [27]cThe EC50 is the concentration giving half-maximum response.dThe RAP is defined as the ratio between the EC50 of 17\u03b2-testosterone and the EC50 of the compound.eThis compound was toxic to yeast above 30\u00a0\u03bcM.fThese compounds reach a maximum response that is lower than 70% of the maximum response obtained with 17\u03b2-testosterone. The maxima obtained with 4-androstenedione and progesterone are about 40 and 35%, respectively.\nCompared with the NIH publication [27], there are a few discrepancies. According to [27], estrone is an androgen receptor agonist that showed androgenic activity in two out of two MCRG systems. However, some mammalian cells are able to convert estrone into 17\u03b2-estradiol and vice versa. This conversion is ascribed to 17\u03b2-hydroxysteroid dehydrogenase 3 and this enzyme is responsible for the high relative estrogenic potency (REP) of estrone in the estrogen bioassay with the T47-D breast cancer cells (ER-CALUX test). In that test, the estrogenic potency of estrone was equal to that of 17\u03b2-estradiol and a REP of 1.0 was reported for estrone [10]. This probably explains why estrone gave a positive result for androgenic activity in two MCRG systems, but gave a negative result in our yeast androgen bioassay. Yeast is obviously not able to convert estrone into 17\u03b2-estradiol. The reported REP of 0.2 in our yeast estrogen biosensor corresponds nicely with the in vivo potency of this compound [28]. Dexamethasone was also described as an androgen receptor agonist in the NIH publication, showing androgenic activity in three out of four mammalian assays, but gave a negative result in our yeast androgen bioassay. However, these MCRG systems use an MMTV-Luc reporter construct or an ARE-Luc reporter construct. Both the MMTV and the ARE sequence are recognized by the glucocorticoid receptor and this means that the response found in these MCRG systems is probably due to crosstalk, as the glucocorticoid receptor is normally expressed in all cell types.\nDose\u2013response curves obtained with the lacZ-based yeast androgen bioassay\nFigure\u00a03 shows the dose\u2013response curves for several natural and synthetic androgens obtained with the lacZ-based yeast androgen bioassay. The assay was simplified in our laboratory by scaling it down to a 96-well format and the use of a \u03b2-galactosidase assay kit. With increasing \u03b2-galactosidase activity, the density of the yeast culture, measured at 630\u00a0nm, dropped to about 50% (data not shown). Therefore, the measured \u00e2-galactosidase activity was corrected for the OD of the yeast culture. Table\u00a02 shows the calculated EC50 and the corresponding RAP values. The data demonstrate that there is a good correlation between the EC50 values for androgens determined in our laboratory and those reported by Gaido et al. [29] in 1997. It seems that the lacZ-based yeast androgen bioassay, in terms of EC50 values, is 5\u201310 times more sensitive than our new bioassay. However, there was almost no difference in the limit of detection. Although the curves in the \u03b2-galactosidase assay go up at around 0.3\u00a0nM and in the yEGFP assay at around 1\u00a0nM, the limit of detection is about 3\u00a0nM for 5\u03b1-dihydrotestosterone in both assay types. This is mainly because the standard deviations are much higher in the \u03b2-galactosidase assay. However, the curves in the \u03b2-galactosidase assay are much steeper and are thus responsible for the lower EC50 values. The steeper curves are probably due to using an enzyme as a marker, \u03b2-galactosidase compared with a yEGFP marker protein, and the expression of the RSP5 cofactor that enhances transcription activation in the lacZ-based yeast androgen bioassay. The dynamic range for 5\u03b1-dihydrotestosterone in the McDonnell assay was from 3 to 100\u00a0nM and is slightly smaller than the range of the new bioassay (3\u2013500\u00a0nM). There were also no great differences in the RAP values determined, although methyltrienolone was more potent in the lacZ-based bioassay. However, the new yEGFP bioassay is less sensitive for 17\u03b2-estradiol and progesterone: RAPs of 0.008 and 0.045, respectively, and for the latter no full dose\u2013response curve, compared with RAPs of 0.1 and 0.068 and full dose\u2013response curves in the lacZ-based bioassay. This means that our new bioassay is more specific for detecting compounds with a pure androgenic mode of action. The main difference between the lacZ-based and our yEGFP biosassay is that protein RSP5, which is a counterpart of the mammalian RPF1, is overexpressed in the lacZ-based bioassay in order to enhance transcriptional efficacy. However, this cannot explain the observed difference with methyltrienolone , as according to Imhof and McDonnell [30], this did not alter the potency or specificity of the assay. In addition and in contrast to the lacZ-based yeast androgen bioassay there were no differences in the density of the yeast culture measured at 630\u00a0nm upon exposure to different compounds that induced yEGFP expression, which indicates a decreased growth of yeast cells exposed to androgens in the lacZ-based yeast androgen bioassay. The only other known yeast androgen bioassay, one that uses luciferase as a reporter protein, displays similar characteristics in terms of specificity and was 5\u201310 times more sensitive in terms of EC50 values [15]. However, this assay needs the correction of the same yeast strain that only and stably expressed luciferase as an external control to correct for and normalize the aspecific responses caused by variation in cell vitality due to matrix and analyte toxicity. Unorrected dose\u2013response curves for 17\u03b2-testosterone displayed more than 10 times higher EC50 values. An earlier assay described by Lee et al. [31] in 2003 uses \u03b2-galactosidase, but only expresses the hinge-ligand binding domain of the androgen receptor.\nFig.\u00a03Response of the McDonnell yeast androgen bioassay to different substances. Exposure to 17\u03b2-testosterone, 5\u03b1-dihydrotestosterone, progesterone, dexamethasone, 17\u03b2-estradiol, and 17\u03b2-boldenone was started by adding to 200\u00a0\u03bcL of a yeast culture a 2-\u03bcL aliquot of a stock solution of the compound in DMSO. The \u03b2-galactosidase activity was determined after 24\u00a0h and corrected for the optical density at 630\u00a0nm as described in \u201clacZ-based yeast androgen bioassay: \u03b2-galactosidase assay.\u201d Signals are the mean of a triplicate with the SDTable\u00a02EC50 concentrations and RAPs of compounds in the yeast androgen bioassay expressing \u03b2-galactosidase in response to androgensCompoundEC50 (nM)a by Gaido et al. [29]EC50 (nM)b in our laboratoryRAP17\u03b2-Testosterone4.711.51.05\u03b1-Dihydrotestosterone3.54.92.317\u03b2-BoldenoneND700.217\u03b2-TrenboloneND130.9MethyltrienoloneND3.73.1TetrahydrogestrinoneND11.51.017\u03b2-Estradiol 86.1950.117\u03b1-EstradiolNDNRNRProgesterone89.31700.068CorticosteroneNDNRNRDexamethasoneNDNRNRND not determinedaValues obtained by Gaido et al. [29], using the McDonnell yeast androgen bioassaybValues determined in our laboratory, using the McDonnell yeast androgen bioassay (see \u201cDose\u2013response curves obtained with the lacZ-based yeast androgen bioassay\u201d)\nAntiandrogenic activity\nThe specificity of the new yeast androgen bioassay was further demonstrated by the ability of antiandrogens to suppress the induction of yEGFP. Figure\u00a04 shows the antiandrogenic activity of the known antagonist flutamide and three brominated flame retardants, BDE-39, TBP, and 4-OH-BDE-17. The antagonistic properties were examined by coexposure with a concentration of 5\u03b1-dihydrotestosterone that induced a submaximal response (50\u00a0nM). None of these four compounds were able to show an agonistic response (Table\u00a01), but Fig.\u00a04 clearly shows that all three were able to inhibit the response induced by 5\u03b1-dihydrotestosterone . The IC50 value was about 1\u00a0\u03bcM for flutamide, and TBP and 4-OH-BDE-17 were about as potent, while BDE-39 was clearly less antiandrogenic. Similar results were obtained with a human cell line. Only TBP was less potent in that test, but cytotoxicity of TBP could not be excluded [32].\nFig.\u00a04Inhibition of a submaximal response obtained by 5\u03b1-dihydrotestosterone with flutamide and three brominated flame retardants (BFRs). Coexposure to a concentration of 5\u03b1-dihydrotestosterone that induced a submaximal response was started by adding to 200\u00a0\u03bcL of a yeast culture, 1\u00a0\u03bcL of a 5\u03b1-dihydrotestosterone and 1\u00a0\u03bcL of the BFR stock solution in DMSO. Fluorescence was determined after 24\u00a0h as described in \u201cStreamlined yEGFP assay with the yeast androgen bioassay.\u201d Fluorescence signals are the mean of a triplicate with the SD. TBP 2,4,6-tribromophenol\nConclusions\nA recombinant yeast cell was constructed that expresses the hAR and yEGFP as a reporter protein in response to androgens. Compared with other yeast androgen bioassays, this new biosassay showed a similar limit of detection and dynamic range. However, the measurement of the fluorescence (yEGFP) can be followed as a function of incubation time and is easier, quicker, and cheaper than the measurement of the \u03b2-galactosidase or the luciferase activity, which needs cell wall disruption and\/or the addition of expensive substrates. Owing to the ease of the yEGFP measurement, standard deviations are generally less than 3%. Moreover, the assay seems to be more robust and more specific for detecting compounds with a pure androgenic mode of action.\nBrominated flame retardants with suspected antiandrogenic properties were able to inhibit the response obtained with 5\u03b1-dihydrotestosterone, the most potent endogenous androgen, demonstrating that this yeast androgen bioassay is suited to detect compounds with both agonistic and antagonistic characteristics.\nAs all compounds tested were able to show either their agonistic or their antagonistic properties, neither the cell wall nor the cell membrane seemed to be an obstacle. As for the yeast estrogen bioassay, we validated this new yeast androgen bioassay according to international criteria, eg., the determination of the decision limit (CC\u03b1) and the detection capability (CC\u03b2). The decision limit is then used to distinguish negative and suspect samples [16] (unpublished results). The assay was proven to be useful to detect the new designer steroid tetrahydrogestrinone in human urine [33] while prohormones with an androgenic mode of action, e.g., dehydroepiandrosterone, are not active in yeast-based bioassays and need metabolic activation before they can be detected [34]. Future work will include the validation of the assay for urine and feed and the screening of prohormones with and without metabolic activation. To mimic the in vivo metabolic activation, liver slices, liver cell lines, liver S9 enzymes, and pure enzymes will be used.","keyphrases":["antagonists","receptor","metabolism","crosstalk","brominated flame retardants","saccharomyces cerevisiae"],"prmu":["P","P","P","P","P","P"]}
{"id":"Anal_Bioanal_Chem-2-2-1764597","title":"Advances in analytical techniques for polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans and dioxin-like PCBs\n","text":"Analytical techniques for the determination of polychorinated dibenzo-p-dioxins (PCDD), polychlorinated dibenzofurans (PCDF) and dioxin-like PCBs (DLPCB) are reviewed. The focus of the review is on recent advances in methodology and analytical procedures. The paper also reviews toxicology, the development of toxic equivalent factors (TEF) and the determination of toxic equivalent quantity (TEQ) values. Sources, occurrence and temporal trends of PCDD\/PCDF are summarized to provide examples of levels and concentration ranges for the methods and techniques reviewed.\nIntroduction\nIn this review, we describe the current state-of-the-art for the determination of the chlorinated dibenzo-p-dioxins (dioxins), chlorinated dibenzofurans (furans), and dioxin-like polychlorinated biphenyls (DLPCBs, or coplanar PCBs). Only a brief summary of the historical development of methods for these compounds is given here; all of the most important earlier work has been summarized in previous reviews [1\u20134].\nThe characteristics of modern methods for dioxin\/furan\/DLPCB determination are derived from several factors related to their chemical, physical and toxicological properties. The exceptionally high toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) meant from the outset that very low detection limits (DLs) were required; consideration of their hydrophobic nature and concern over chronic rather than acute toxicity effects meant that bioaccumulation was of increased concern, which pushed the need for even lower DLs. An understanding of the environmental levels, transport and fate of these compounds is important if we wish to understand the significance of these compounds as well as the low detection limits and broad range of detection required; therefore, a section on levels, transport and fate of these compounds is included in this review. A variety of different analytical methods and techniques have been developed to increase sensitivity and selectivity and to reduce analysis times. To fully understand the analytical methods developed and their application, the references cited in Table\u00a05 should be examined. Table\u00a05 also illustrates the wide range of sample types and concentration ranges of dioxins and DLPCBs detected in the global environment, which explains why so many variations on the basic analytical methodology exist.\nAnother factor in method development and application was the large amount of litigation that occurred because of the detection of these compounds in humans as well as the environment. Methods of analysis were required to generate data that could withstand scrutiny in a court of law, which led to the necessity of using methods based on isotope dilution\u2013HRMS techniques. In addition, the rapid growth in high-quality standards and reference materials contributed to substantial improvements in the accuracy and precision of modern methods.\nThe development of analytical methods was also influenced by the need for regulators to be able to evaluate the combined toxicity of a number of compounds that have similar toxic properties to 2,3,7,8-TCDD, but widely varying potencies. This development is so important to the development of analytical methods that we have included a section below on toxicology and TEFs. The bottom line for dioxin methods was that definitive separation of the 2,3,7,8-substituted dioxins\/furans and DLPCBs from a large number of other congeners with almost identical physical and chemical properties, as well as numerous other potentially interfering compounds, was essential.\nBrief background on dioxin determination\nChlorinated dibenzo-p-dioxins (dioxins) have been of concern for decades because of their toxic properties, as described below. A structurally similar series of compounds, the chlorinated dibenzofurans (furans), have similar chemical properties and toxic effects, and are generally determined as a group with the dioxins. In recent years there has been a growing trend to include a specific subgroup of the polychlorinated biphenyls (PCBs): the so-called dioxin-like PCBs (DLPCBs). This has been added to methods along with the dioxins and furans. Figure\u00a01 shows the structures of 2,3,7,8-TCDD, 2,3,7,8-TCDF and PCB-126. The carbon numbering system is marked next to each carbon to indicate substitution positions. Dioxin and furan congeners substituted in the 2,3,7 or 8 position are toxic, while PCBs substituted in the 3,3\u2032,4,4\u2032,5 or 5\u2032 position and no or only one 2- or 2\u2032-substitution are considered to be dioxin-like and to exhibit dioxin toxicity (see Table\u00a01).\nFig.\u00a01Basic structures of the chlorinated dibenzo-p-dioxins (dioxins), chlorinated dibenzofurans (furans) and polychlorinated biphenyls (PCBs). For the dioxins and furans, congeners with chlorine substitution at the 2,3,7 and 8 positions are considered toxic. Of the 75 possible dioxin and 135 possible furan structures, only 17 have 2,3,7,8-substitutionTable\u00a01World Health Organization toxic equivalent factors (TEFs) for humans\/mammals, fish and birds [37]CongenerHumans\/MammalsFishBirds2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)1111,2,3,7,8-Pentachlorodibenzo-p-dioxin (PeCDD)1111,2,3,4,7,8-Hexachlorodibenzo-p-dioxin (HxCDD)0.10.50.051,2,3,6,7,8-Hexachlorodibenzo-p-dioxin (HxCDD)0.10.10.011,2,3,7,8,9-Hexachlorodibenzo-p-dioxin (HxCDD)0.10.10.11,2,3,4,6,7,8-Heptachlorodibenzo-p-dioxin (HpCDD)0.010.001<0.0011,2,3,4,5,6,7,8-Octachlorodibenzo-p-dioxin (OCDD)0.0001<0.00010.0012,3,7,8-Tetrachlorodibenzofuran (TCDF)0.10.0511,2,3,7,8-Pentachlorodibenzofuran (PeCDF)0.050.050.12,3,4,7,8-Pentachlorodibenzofuran (PeCDF)0.50.511,2,3,4,7,8-Hexachlorodibenzofuran (HxCDF)0.10.10.11,2,3,6,7,8-Hexachlorodibenzofuran (HxCDF)0.10.10.11,2,3,7,8,9-Hexachlorodibenzofuran (HxCDF)0.10.10.12,3,4,6,7,8-Hexachlorodibenzofuran (HxCDF)0.10.10.11,2,3,4,6,7,8-Heptachlorodibenzofuran (HpCDF)0.010.010.011,2,3,4,7,8,9-Heptachlorodibenzofuran (HpCDF)0.010.010.011,2,3,4,5,6,7,8-Octachlorodibenzofuran (OCDF)0.0001<0.00010.0013,4,4\u2032,5-Tetrachlorobiphenyl (PCB 81)0.00010.00050.13,3\u2032,4\u2032,4\u2032-Tetrachlorobiphenyl (PCB 77)0.00010.00010.052\u2032,3,4,4\u2032,5-Pentachlorobiphenyl (PCB 123)0.00010.0000050.000012,3\u2032,4,4\u2032,5-Pentachlorobiphenyl (PCB 118)0.0001<0.0000050.000012,3,4,4\u2032,5-Pentachlorobiphenyl (PCB 114)0.0005<0.0000050.00012,3,3\u2032,4,4\u2032-Pentachlorobiphenyl (PCB 105)0.0001<0.0000050.00013,3\u2032,4,4\u2032,5-Pentachlorobiphenyl (PCB 126)0.10.0050.12,3\u2032,4,4\u2032,5,5\u2032-Hexachlorobiphenyl (PCB 167)0.00001<0.0000050.000012,3,3\u2032,4,4\u2032,5-Hexachlorobiphenyl (PCB 156)0.0005<0.0000050.00012,3,3\u2032,4,4\u2032,5\u2032-Hexachlorobiphenyl (PCB 157)0.0005<0.0000050.00013,3\u2032,4,4\u2032,5,5\u2032-Hexachlorobiphenyl (PCB 169)0.010.000050.0012,3,3\u2032,4,4\u2032,5,5\u2032-Heptachlorobiphenyl (PCB 189)0.0001<0.0000050.00001\nOriginally, attention was focused on the most toxic member of these substances, 2,3,7,8-TCDD. Gas chromatographic columns were soon developed that could separate 2,3,7,8-TCDD from the other 21 TCDD isomers in sample extracts [5], and other columns that could fortuitously separate the groups of congeners from each other by degree of chlorination (i.e., all tetrachlorinated dioxins\/furans were eluted, followed by all of the pentachlorinated dioxins\/furans, followed by the hexachlorinated, then the heptachlorinated, then the octachlorinated [6]. Many early publications reported a so-called congener group determination, where the total concentrations of all of the isomers from each group containing the same number of substituent chlorines were quantified and summed. Under this approach, 11 concentrations would be reported: total tetrachlorinated dioxins (T4CDDs), total pentachlorinated dioxins (P5CDDs), total hexachlorinated dioxins (H6CDDs), total heptachlorinated dioxins (H7CDDs), the octachlorinated dioxin (OCDD), the same dibenzofuran groups (T4CDFs, P5CDFs, H6CDFs, H7CDFs, OCDF), and 2,3,7,8-TCDD. This type of determination is often incorrectly termed a total homolog determination. Although \u201chomolog\u201d is still used today to refer to dioxins or furans that have different numbers of chlorine atom substituents, the chlorinated dioxin or furan series are not homologous, because the different members of the series are not formed by adding the same structural unit (Cl atom), but by substituting Cl for H in higher members of these series. The various chlorinated dioxins are correctly termed \u201ccongeners\u201d (members of a like series), as are the chlorinated dibenzofurans.\nIt was clear over 30\u00a0years ago that gas chromatography\u2013mass spectrometry (GC\u2013MS) was the instrumental method of choice for dioxin and furan determinations. The great tunable selectivity of mass spectrometry achieved by the ability to monitor specific characteristic ions in the mass spectrum of a compound combined with the retention time-matching ability of a gas chromatogram made GC\u2013MS far superior to other detection systems. Even with the use of GC\u2013MS, the exceptionally great toxicity of 2,3,7,8-TCDD meant that equally exceptionally low detection limits were required for this compound in real environmental samples. At such DLs as parts-per-trillion (ppt:10\u221212\u00a0g 2,3,7,8-TCDD per g of sample) or parts-per-quadrillion (ppq: 10\u221215\u00a0g 2,3,7,8-TCDD per g of sample), the principal barrier to successful analysis of samples was the separation of TCDD from the bulk sample matrix and from all other organic chemicals in the matrix. The keys to success in this endeavor were the number of separation steps in the entire analytical scheme, and their effectiveness. These separation steps included the following: \nRepresentative sampling (not included in this review);Separation of TCDD from the sample matrix (extraction); transfer of TCDD to an appropriate organic solvent;Separation of TCDD from all other organic coextractives (clean-up; can involve several distinct steps);Separation of TCDD from other relatively nontoxic tetrachlorinated dioxin isomers (gas chromatography);Separation and recording of characteristic TCDD molecular fragments using a mass spectrometer.\nAlthough an impressive degree of optimization of each of these steps has occurred over the past decade, this basic approach was formulated in the mid-1970s. A paper published in 1973 showed how high-resolution mass spectrometry (HRMS) was used to determine 2,3,7,8-TCDD in various sample types at the 1.0\u00a0ppt DL [7].\nModern dioxin\/furan determination\nIn recent years, methods have built on the early developments referred to above, and have achieved a high degree of sophistication and greatly improved accuracy and precision. The various stages of development of dioxin\/furan determination can be generically described as follows:\n1970s to 1980s The principal concern was to ensure correct identification of TCDD; the majority of analyses reported total concentrations of dioxin and furan (tetrachlorinated to octachlorinated) congener groups, and\/or 2,3,7,8-TCDD; very few reliable analytical standards or certified reference materials were available; few laboratories worldwide could perform this work;\n1980s to 1990s Fused silica open tubular GC columns predominantly used for this application; quality control for GC\u2013MS methods enhanced greatly by the use of the isotope-dilution technique; GC-HRMS was accepted as \u201cgold standard\u201d method; introduction of \u201ctoxic equivalent factors (TEFs)\u201d made it imperative to improve speciation of all dioxins\/furans with chlorine substitution at the 2,3,7 and 8 ring positions; 10\u201320 or so laboratories worldwide were proficient in this work;\n1990s to 2000s 13C-isotope labeled standards of all 2,3,7,8-substituted dioxins\/furans available, as well as wide array of certified or consensus real-matrix reference materials; reliable methods available for precise, accurate isomer-specific determination of all 2,3,7,8-substituted dioxins\/furans at ppt to ppq concentrations in virtually any sample matrix; most results for regulatory work reported as \u201ctoxic equivalent quantity (TEQ)\u201d concentrations; coplanar PCBs began to be included in TEQ calculations; >100 laboratories worldwide are proficient in this work;\nFuture focus Detection limits are low enough for most regulatory purposes, but may need to be pushed lower for bioaccumulation studies and determination of subtle effects from these compounds like changes in sex ratios; future improvements will be difficult due to ubiquitous background levels of these compounds and potentially interfering compounds at such low DLs; biggest challenges could be to lower the high cost and to improve the speed of analysis; bioanalytical methods may help, but are still of limited success after over 20\u00a0years of development; multidimensional orthogonal methods, such as two-dimensional chromatography (GC\u00d7GC, LC\u00d7GC ) or tandem mass spectrometry (MS\/MS), may increase selectivity and also reduce detection limits, allowing for an increased number of analytes in a single analysis.\nToxicology and TEFs\nDioxin-like halogenated aromatic hydrocarbons (HAHs) provoke an extraordinarily broad spectrum of toxic effects in vertebrate laboratory animals. Exposure to a few micrograms per kg body weight of the most potent congener, 2,3,7,8-TCDD, leads to (among numerous effects) loss of body weight, liver necrosis, immune impairment, reproductive toxicity, teratogenesis and cancer in many laboratory species [8\u201311]. A single TCDD dose of 1 \u03bcg\/kg is lethal to guinea pigs. In human populations exposed to HAHs by occupation or by industrial accident, the reported adverse health effects include reproductive disorders [12], developmental toxicity [13, 14], insulin insensitivity [15] and cancer [16, 17]. However, there is considerable controversy and uncertainty about the degree of risk to human health from dioxin-like compounds, particularly regarding cancer risk [18, 19]. There is no question, however, that TCDD can cause the severe skin disorder known as chloracne in some highly-exposed individuals [20]; chloracne is considered to be the \u201challmark\u201d of human dioxin toxicity.\nIf we understand the mechanism by which a chemical produces its toxic effects, we are in a better position to assess the risk that the chemical poses to humans and to other species. Extensive research over the past quarter-century has demonstrated that all major toxic effects of dioxin-like chemicals are mediated by their binding to a soluble intracellular protein, the aryl hydrocarbon receptor (AHR) [21\u201323]. The AHR\u2019s normal function is to regulate transcription of multiple genes that are important in development, physiologic function and adaptive responses to xenobiotic chemicals [24, 25]. Dioxin-like chemicals appear to exert their toxicity by dysregulating expression of key genes that are under control of the AH receptor [23].\nThroughout most of its history, the science of toxicology has needed to cope with the effects of only one chemical at a time. HAHs present an unusual challenge to the toxicologist because \u201creal-world\u201d HAH exposure is not to single compounds; rather, HAH exposure involves complex mixtures of dozens to hundreds of congeners from the dioxins (75 possible), furans (135 possible) and PCBs (209 possible).\nThe fact that dioxin-like compounds work through a common mechanism, the AHR [10, 23], fostered development of the \u201ctoxic equivalent factors\u201d (TEF) approach to assessing the toxicological potency of HAH mixtures [26]. The TEF approach is based on a proposal first made by the Ontario Ministry of the Environment in 1984 [27]. In this approach, 2,3,7,8-TCDD, the most potent congener, is assigned a TEF of 1.0, and all other congeners are assigned TEFs that reflect their toxic potency relative to that of TCDD. For example, 2,3,7,8-tetrachlorodibenzofuran and 3,3\u2032,4,4\u2032,5-pentachlorobiphenyl (PCB126) each are assigned a TEF of 0.1, whereas octachlorodioxin has a TEF of 0.0001, reflecting its very low toxicity compared with that of 2,3,7,8-TCDD. The toxicity of a mixture is stated as TEQ (TCDD equivalents) and is assumed to be equal to the sum of the concentration of individual congeners multiplied by their potencies (TEFi) [28]: \nNote that the TEF approach applies only to agents that are dioxin-like in that their toxic effects are mediated by the AHR. Noncoplanar PCBs have different mechanisms of action and are not accommodated in the TEF approach. For simplicity it is assumed that all dioxin-like congeners produce toxic responses that are qualitatively the same and that the congeners differ only in their potency to produce these adverse effects, but this simplifying assumption should not be viewed as being true in a literal sense. It is true, however, that the toxic potencies of congeners within the dioxin-like category vary over several orders of magnitude.\nBy definition, in order to calculate the total TEQ of a mixture we need to know the concentration of each dioxin-like component in the mixture. Hence the need for sensitive and reliable congener-specific chemical analyses. Regulatory decisions and risk management need to proceed even in the absence of perfect information. The TEQ approach is a useful simplifying strategy to allow risk assessment to move forward for exposures that involve complex mixtures, but it includes several assumptions [28\u201330]. There are two key issues: \nWhat is the proper TEF for each congener?What is the predominant mode of interaction between congeners when present in a mixture?\nThe first question\u2014what is the proper TEF for a particular congener?\u2014has not been as straightforward as was initially envisioned. The main difficulty is that the estimated relative potency (REP) of congeners differs depending on what endpoint is measured, in what tissue and in what animal species. The relative potency estimates that underlie TEFs are derived from highly heterogeneous data sets [27] that require judgment and interpretation rather than providing numbers that can simply be plugged into an equation. The TEFs currently used in risk assessment are not based on a single biological endpoint. Rather, current TEFs were assigned in 1997 by an expert panel of the World Health Organization (WHO) who took into account multiple biochemical and toxicologic endpoints. The highest weight was given to studies done in mammalian systems in vivo (rather than in vitro), to chronic studies (rather than acute), and to studies that measured a toxic outcome (rather than a biochemical response). The relative potency estimates and resulting TEFs derived by the WHO panel are now undergoing refinement [27].\nThe second question\u2014how do components in a complex HAH mixture interact?\u2014is also not easily answered. The central premise in the equation used to calculate TEQ is that the toxic effects of components in the mixture are additive; i.e., that the overall toxicity of the mixture is the sum of the concentration of each congener multiplied by its TEF. There is experimental evidence to support the assumption of additivity [31, 32]. However, principles of receptor pharmacology predict that compounds which compete for the same receptor site may antagonize each other so that the overall response is less than additive. From the viewpoint of risk, of course it would be desirable for the toxicity of mixtures to be less than the sum of toxicities of individual components. Several studies have found antagonism between chemicals that act at the AH receptor site [29, 33, 34]\u2014in other words, the standard TEQ approach in these circumstances overestimates the toxic potency of the mixture. It also is possible that components in a mixture might synergize to produce a response that is greater than that predicted by the additive model. Fortunately there is limited evidence of such an unwanted scenario for the toxicity of dioxin-like chemicals [35].\nIt is important to recognize that TEF values for some congeners can differ by tenfold or more among different animal species. For example, the TEF for 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin is ten-fold higher in fish than in birds (Table\u00a01) [37]. When assessing risks to wildlife from exposure to HAH mixtures, we need to be aware that TEF values for most HAH congeners have been rigorously determined within only a very few nonhuman, nonrodent species. Risk estimates may be erroneous or misleading if TEF values from one animal species are assumed to be the same for other species.\nCongener-specific chemical assays for dioxin-like compounds have dramatically improved in sensitivity and reliability over the past two decades. Nonetheless, such assays remain expensive. Therefore, economical alternatives have been sought which would permit estimates of the potential toxicity of mixtures to be made. Bioassays such as the \u201cchemically activated luciferase gene expression\u201d assay (CALUX) assess the overall biological activity of a mixture and do not require knowledge of the identity or concentration of individual components of that mixture. The CALUX cell bioassay is predicated on the AHR being the mediator of toxicity. It tests sample extracts for their ability to increase expression of a luciferase reporter gene that is under the control of the AH receptor and thereby provides an index to the total equivalents of dioxin-like chemicals (CALUX\u2013TEQ). As with chemical assays, sample extraction and sample clean-up are critical stages in bioassays such as CALUX. For some purposes, bioassays such as CALUX can provide a useful complement or alternative to congener-specific chemical assays. See Windal et al. [36] for an excellent review of the properties and applications of CALUX.\nModern methods of analysis\nThe \u201cgold standard\u201d for the analysis of dioxins and furans uses classical extraction techniques such as Soxhlet [38], liquid\/liquid extraction [38], solid-phase extraction (SPE) [40, 41], or the more recent pressurized fluid extraction [42\u201344]. Once the extract has been transferred to a suitable solvent, a three-stage (silica, alumina and carbon) open-column clean-up is followed by GC\u2013HRMS (employing isotope dilution) as the analytical finish. Minor variations on this method have been used since the late 1970s and it continues to be the method of choice for regulatory and legal purposes. A number of these methods are listed in Table\u00a02. \nTable\u00a02Regulatory methods for the analysis of dioxins, furans and DLPCBsMethodAnalytes\/CommentsReferenceUSEPA 1613Seventeen 2,3,7,8-substituted dioxins and furans and congener group totals in water and wastewater. Uses isotope dilution\u2013GC\u2013HRMS[38]USEPA 1668a209 PCB congeners. 12 WHO dioxin-like PCBs by GC\u2013HRMS, the remaining 197 by GC\u2013MS[39]USEPA 23Seventeen 2,3,7,8-substituted dioxins and furans and congener group totals in incinerator stack gasses. Uses isotope dilution\u2013GC\u2013HRMS[45]USEPA 8290 (SW-846)Seventeen 2,3,7,8-substituted dioxins and furans and congener group totals in materials and waste. Uses isotope dilution\u2013GC\u2013HRMS[46]ISO 18073Equivalent to USEPA 1613, also allows GC\u2013MS as an alternate detection method[47]ISO 17858Twelve WHO dioxin-like PCBs in environmental matrices by GC\u2013HRMS [48]EN 1948Seventeen 2,3,7,8-substituted dioxins and furans and congener group totals in stationary sources by isotope dilution\u2013GC\u2013HRMS[49]MOE 3418Seventeen 2,3,7,8-substituted dioxins and furans including congener group totals and 12 WHO dioxin-like PCBs by GC\u2013HRMS. Uses isotope dilution\u2013GC\u2013HRMS[50]ENVCAN 1\/RM\/19Seventeen 2,3,7,8-substituted dioxins and furans and congener group totals in pulp and paper effluents by isotope dilution\u2013GC\u2013HRMS[51]JIS K0312Seventeen 2,3,7,8-substituted dioxins and furans including congener group totals in wastewater by isotope dilution\u2013GC\u2013HRMS[52]JIS K0311Seventeen 2,3,7,8-substituted dioxins and furans including congener group totals in incinerator stack gasses by isotope dilution\u2013GC\u2013HRMS[53]\nOverview of analytical process The generic steps of the analytical process were listed above in the \u201cIntroduction\u201d, except for the objectives, and data evaluation. These are mentioned here because of their great importance, but will not be discussed in detail. It is important to be clear about the objectives of dioxin\/furan\/DLPCBs determinations, because there are several choices in methodology\u2014associated with substantially different costs\u2014that generate data of differing precision and accuracy [54]. For example, for rapid on-site analysis for the purpose of directing a site remediation project, a substantially different methodology can be used compared to that required for legal action. Data evaluation will also not be discussed, but the authors would simply like to make the point that in spite of significant advances in software and data systems, the low-level determination of dioxins, furans and DLPCBs still requires detailed manual examination by experienced analysts of the raw data generated by GC\u2013HRMS systems. A discussion of sampling is beyond the intended scope of this review, but it may often be the case that the imprecision of sampling is similar to or significantly greater than the combined imprecision of the remaining steps of the analytical process.\nSample extraction methods See [55]. Classical extraction techniques like liquid\/liquid extraction and Soxhlet extraction are described in detail in the methods listed in Table\u00a02 and will not be addressed in this review. A number of alternative extraction techniques are summarized in Table\u00a03. Supercritical fluid extraction (SFE) uses a gas above the critical point (the combination of temperature and pressure where the gas has liquid-like properties enabling enhanced extraction capability) to extract analytes from the matrix. The main advantage with SFE is that the extracting gas can be evaporated and so solvent disposal is not required. SFE behaves significantly differently to classical solvent extraction and recoveries can be reduced or enhanced. Pressurized liquid extraction (PLE), also known as accelerated solvent extraction (ASE) evolved from SFE. Solvents were added to SFE extractions as modifiers in order to mimic classical liquid extractions. SFE without extraction gas and only solvent modifier best matched classical extraction recoveries. This led to the development of PLE. Microwave-assisted extraction (MAE or MASE) uses closed vessels to increase pressure and extraction efficiency. Polar solvents like acetone or water are required to supply heat for extraction. Sun et al. [56] reported the analysis of wet samples by MAE, indicating that significant time was saved by the reduced sample drying requirements. \nTable\u00a03Alternative extraction techniquesMatrixTechnique ReferenceSoil\/sediment\/solidPLE[4244]SFE[58]MASE[59]AqueousSPMD, passive sampling[60, 61]SPE[40, 41]AirSwipe\/biofilms[62]Passive sampling[63, 64]Solid-phase extraction can significantly reduce analysis times and solvent usage for the extraction of water and waste water samples, even with significant particulate loadings [40]. Particles collected on C18 extraction disks can be extracted quantitatively without Soxhlet or PLE extraction. Water samples have also been analyzed using semi-permeable membrane devices (SPMD). SPMD sampling is a form of passive sampling that can be used for water, air or sediments. Namiesnik [57] has recently reviewed passive sampling techniques in environmental samples. Passive sampling is based on the free flow of analytes from the sampling medium to the collecting medium. This technique eliminates the requirement for power, and is a composite sampling procedure that can reduce analysis cost because of reduced sampling events and analyte loss during shipping and storage as the analyte is on a trapping medium. Swipe tests of biofilms on surfaces can also be used to monitor ambient conditions, especially after fires or from fugitive emissions.\nSample extract clean-up methods Sample preparation of dioxin extracts is one of the most challenging in analytical chemistry. Many coextractable and potentially interfering compounds present in the raw extract must be removed in order provide maximum concentration factors (106 to 108) to meet DLs. A number of classical adsorbents can be used, including silica, alumina, Florisil and carbon. Most current procedures are based on the \u201cDow\u201d [65] and \u201cSmith-Stallings\u201d [66] procedures. These procedures are described in detail in the references given in Table\u00a02 above. Disposable C18\/silica SPE cartridges [67] have been used for rapid clean-up of biological materials.The analysis of dioxins is very costly and time-consuming. Over the past 20\u00a0years, a number of modifications have been made to automate sample extraction and sample extract preparation to reduce analysis times and to attempt to reduce costs. Focant [42] has recently reviewed the automated sample preparation of biological samples. Multicolumn automated systems like the FMS (fluid management system) automated Power Prep system were developed to prepare human serum extracts, and have subsequently been used in a number of applications, including biota, water and food. Interchangeable cartridges (silica, alumina, carbon) can be used in different configurations [68, 69]. The addition of a PLE interface preceding the Power Prep enables the sample to be extracted and cleaned in the same automated run. Food samples (three) have been extracted, cleaned and analyzed in ten\u00a0hours using this system [68]. An alternate method reported by Thal [70] used an automated gel permeation chromatographic (GPC)\/carbon system. The GPC removes coextractables like lipids from the extract, which is then processed on activated carbon to separate the planar from the nonplanar compounds. This is based on a system reported by Norstrom et al. [71]. Van Beuzekom used an automated microwave solvent extraction system (MASE) coupled to a liquid chromatograph for the analysis of fine particles from fire emissions [59]. Nording [72] was able to modify a PLE extraction cell to insert the sample matrix above AX21 carbon packing in order to perform extraction and clean-up in the cell as a screening method. Results compared well with GC\/HRMS confirmatory analysis.Sample preparation schemes are developed to remove most interferences from the sample extract. Unfortunately, because many compounds have similar physical and chemical properties, a considerable number of interferences often still remain in the cleaned sample extracts. Some examples of interferences on selected DLPCBs using a 5% phenyl column are listed below [50].\nPCB congenerPotential interferencePCB 81PCB 87PCB 77PCB 110PCB 123PCB 149PCB 126PCBs 178 and 129PCB 156PCB 171PCB 157PCB 201\nGas chromatography columns and methods PCB interferences can be eliminated or removed physically as in MOE Method 3418, or by analyzing extracts on multiple columns (US EPA Method 1668). A number of analyte-specific columns have been developed to reduce both dioxin and PCB interferences and reduce the need for multicolumn analysis. The standard 5% phenyl columns exhibit multiple coelutions for PCBs (see above) and dioxins. The Rtx-Dioxin2 [73, 74] column has very few interfering compounds. BPX-DXN exhibits very low bleed and enhanced separation over 5% phenyl columns [75]. Liquid crystal columns [76, 77] provide outstanding separation for 2,3,7,8-substituted dioxins and furans, but suffer from high bleed and low column temperature maximum limits.Fast GC is accomplished by reducing inner column diameters and phase thickness. The number of theoretical plates per meter is increased significantly. If the phase ratio (inner column diameters\/phase thickness) remains constant, the chromatography does not change and separations in micobore columns can be achieved much faster because of the shorter column lengths [78\u201380]. MacPherson [81] reported the use of simultaneous dual parallel column analysis of dioxins\/furans\/coplanar PCBs (Fraction A) on a 40\u00a0m DB-5 column and ortho-substituted PCBs (Fraction B) on a 20\u00a0m DB-5 column in under 45\u00a0min. Extracts were separated using activated carbon\/silica. This combination can meet GC\u2013HRMS QC specifications (e.g., US EPA 1613).Two-dimensional chromatography [82\u201384] has been investigated by a number of researchers in order to solve coelution issues from single-column analysis. A number of column combinations have been used to solve coelutions for dioxins and PCBs [85, 86]. GC\u00d7GC produces very narrow peaks that can provide significantly enhanced sensitivity, increased column capacity and reduced analysis times. It requires fast detection which pushes the limits of HRMS capability. A number of applications have been reported using TOFMS [85, 86], ECD [87, 88] ITMS [89] LRMS [90] and HRMS [178] as detectors for GC\u00d7GC. The potential for enhanced chromatographic selectivity and reduced analysis times has created a lot of interest in this area.\nMass spectrometry detection methods See [91, 92]. HRMS has been the definitive detection method since the beginning of dioxin analysis (in 1973) [7]. Many detection techniques have been investigated [93], but none can match the selectivity and sensitivity of HRMS [94\u201397]. Tandem mass spectrometry as hybrid\/MS [98], triple quadrupole MS\/MS [99, 100] or ion trap MS\/MS [85, 101] have been used to analyze dioxins and furans. MS\/MS is more selective than HRMS for dioxins in most cases, because the parent molecule loses COCl, weighing 63\u00a0amu. No other halogenated organic has been shown to fragment in this way. The sensitivities of MS\/MS instruments are typically less than HRMS, but with slight adjustments to sample size and final extract volume, GC\u2013HRMS QC specifications can typically be achieved. Eppe [102] achieved 5:1 signal to noise with the injection of 200\u00a0fg of 2,3,7,8-TCDD. Unfortunately, the enhanced selectivity observed with MS\/MS analysis of dioxins is not experienced with PCBs. The loss of Cl2 from the parent molecule is not unique to polychlorinated compounds and typically interfering peaks can be detected in the MS\/MS chromatograms of PCBs.Electron ionization (EI) with reduced electron energy (\u223c35\u00a0eV) is the typical method of ionization used in dioxin\/DLPCB analysis. The reduced electron energy focuses ion current towards the molecular ion, reduces the number of fragment ions, and subsequently enhances sensitivity. Negative ion chemical ionization (NICI or NCI) is also a low-energy ionization technique [103], which produces mass spectra with little fragmentation, resulting in enhanced parent ion signals. NCI analysis of dioxins, furans and PCBs has recently been investigated by Chernestsova using a variety of reagent gases [104]. Detection limits for PCBs are equivalent to or lower than for EI with a trend of increasing signal strength with increasing degree of chlorination. Similar results were observed for dioxins, except for 2,3,7,8-TCDD which is up to two orders of magnitude less sensitive in NCI than EI [105]. The molecular anion of 2,3,7,8-TCDD readily fragments to the chloride anion. Unfortunately, many other coextractables also fragment to the chloride anion, significantly reducing selectivity, and as a result NCI has not been used routinely for the analysis of dioxins and furans.\nAlternate methods of analysis Due to the challenging sample preparation, selectivity and ultra-low detection requirements, the cost of dioxin analysis is much higher than any other analytical method. Researchers have been searching for alternate low-cost methods for years. Immunoassay [106\u2013110] and bioassay [111\u2013114] methods can reduce costs by 50% or more and have been used to analyze food samples and samples collected from hazardous sites where very fast analysis is needed. The relative potencies for bioassays and cross-reactivities for immunoassay methods roughly mimic toxic equivalent factors used in mass spectrometric methods, enabling results to be obtained in TEQ directly. The main disadvantages of these types of methods are the inability to use labeled internal standards to correct for recovery and the determination of congener profiles for source apportionment. Spiked samples, similar to standard addition quantitative analysis, permit recoveries to be estimated, overcoming this problem. Recent publications have shown that bio\/immuno methods compare well with standard GC\/HRMS methods in terms of sensitivity, selectivity and accuracy [115\u2013118].Most analytical laboratories are now being challenged to analyze for multiple analyte groups, including polybrominated diphenyl ethers (PBDEs), polychlorinated naphthalenes, as well as dioxins, furans and DLPCBs. Multicomponent methods have been developed for a variety of matrices [119\u2013122] in order to reduce costs accrued by multiple separate extractions, clean-up and instrumental analyses. Analysts must exercise caution when adding additional analyte groups to existing methods. Modifying methods to increase the number of analytes can result in compromised (less than ideal) conditions for a number of analytes, giving rise to potential interferences and\/or reduced recoveries for these analytes.\nQuality control and data reporting Dioxin methods inherently include a number of quality control standards and samples. Isotope dilution (ID) methods are typically used with 13C-labeled internal standards. ID standards are added prior to extraction, allowing correction for recovery over the multistage clean-up procedures. The isotopically labeled standards can also act as markers for the identification of native analytes in samples. As the ID standards are often added at concentrations 10\u2013100 times those expected for the analytes, the standards can act as \u201ccarriers\u201d to improve recoveries of analytes at ultra-trace concentrations and can offset losses due to adsorption. The isotopically enriched standards aid in method development\/validation, as they can be added at various stages of the analytical process to track down problems associated with analyte recovery or method bias.Until recently very few reference materials were available for method validation and performance evaluation. CRMs for sediments [123] and biota [124, 125] are now available.Uncertainty in analytical data is an important method attribute. The calculation of uncertainty is carried out using data from a number of sources, including instrument precision, linearity, fortified matrices, MDLs, spiked samples and interlaboratory data [126]. Uncertainties for dioxins, furans and dioxin-like PCBs are listed in Table\u00a04 in percent (%) [127]. Uncertainties for dioxins and furans range between 15 and 20% for most analytes. The two most significant errors in the analysis are the error in the standards (\u00b110%) quoted by the manufacturer and the error of calibration (typically \u00b120%) that is accepted by the analyst. Other errors, such as mass and volumetric determinations, should be less than 5%. Uncertainties are elevated for water samples over soil and biota due to the greater amount of labware that contacts the sample and the influence of particles on the homogeneity of the sample. Similar levels of uncertainty are observed for dioxin-like PCBs, except for PCB 105 and 118. These congeners are typically present at background levels of 20 and 50 pg, respectively, in the sample. Analyte losses from the extraction and sample preparation steps are corrected by the isotope dilution analytical technique and should not be significantly greater than the error in the addition of the surrogate internal standard. \nTable\u00a04Uncertainty of dioxin, furan and DLPCB measurements in selected matrices, reported in percent (%) [127]ParameterCAS No.Soil N=59Biota N=34Water N=35Veg N=122,3,7,8-TCDD1746-01-6202619211,2,3,7,8-PeCDD40321-76-4251714211,2,3,4,7,8-HxCDD39227-28-6151318161,2,3,6,7,8-HxCDD57653-85-7171830291,2,3,7,8,9-HxCDD19408-74-3192131241,2,3,4,6,7,8-HpCDD35822-46-9122238261,2,3,4,5,6,7,8-OCDD3268-87-9151831202,3,7,8-TCDF51207-31-9152337231,2,3,7,8-PeCDF57117-41-6152032232,3,4,7,8-PeCDF57117-31-4171631271,2,3,4,7,8-HxCDF70648-26-9131529171,2,3,6,7,8-HxCDF57117-44-9141334211,2,3,7,8,9-HxCDF72918-21-9161335222,3,4,6,7,8-HxCDF60851-34-5211631241,2,3,4,6,7,8-HpCDF67562-39-4161332191,2,3,4,7,8,9-HpCDF55672-89-2111537161,2,3,4,5,6,7,8-OCDF39001-02-021271533N=56N=26N=10PCB 77 3,3\u20194,4\u2019-TCB32598-13-3241414PCB 81 3,4,4\u2019,5-TCB70362-50-423169PCB 105 2,3,3,\u20194,4\u2019-PeCB32598-14-4287127PCB 114 2,3\u2019,4,4\u2019,5-PeCB74472-37-0191717PCB 118 2,3\u2019,4,4\u2019,5-PeCB31508-00-6259049PCB 123 2\u2019,3,4,4\u2019,5-PeCB65510-44-3232214PCB 126 3,3\u2019,4,4\u2019,5-PeCB57465-28-8281516PCB 156 2,3,3\u2019,4,4\u2019,5-HxCB38380-08-419239PCB 157 2,3,3\u2019,4,4\u2019,5\u2019-HxCB69782-90-7322210PCB 167 2,3\u2019,4,4\u2019,5,5\u2019-HxCB52663-72-6211213PCB 169 3,3\u2019,4,4\u2019,5,5\u2019-HxCB32774-16-6251511PCB 189 2,3,3\u2019,4,4\u2019,5,5\u2019-HpCB39635-31-9141712When assessing the overall error and\/or uncertainty for determinations of dioxins and DLPCBs, two important factors are often overlooked. First, the sampling error is not investigated in many studies. This is very difficult to assess properly because it requires many more analyses to be performed, and the cost of this work is often prohibitive. Second, a growing trend is to report results as total TEQs, and sometimes the concentration data for individual congeners\u2014used to perform the TEQ calculation\u2014may not be reported at all. Also, more than one set of toxic factors exist, and some papers do not report which set of factors were used or the format (e.g., ND=0, ND=1\/2) in the TEQ calculation. Baccarelli [128] has recently reviewed the reporting of TEQ values where non-detect values are used in the TEQ calculation. It should be remembered that the toxic factors used are themselves approximations, and may contribute significantly to the degree of uncertainty in the interpretation of results. Another unfortunate trend is that reconstructed chromatograms and\/or congener patterns from analyses are being reported less and less. These patterns can be useful when assessing possible sources of the dioxins\/DLPCBs detected.\nSources, occurrence and temporal trends in PCDD\/Fs\nAlcock and Jones [129] have previously reviewed the occurrence and temporal trends in PCDD\/Fs in industrialized countries including Germany, the United States, Sweden, the Netherlands and the United Kingdom. This review covered trends in sediments, air, vegetation, soils, sewage sludge, livestock tissue and milk, wildlife and human tissues. The authors reported a general global temporal trend in PCDD\/F inputs to the environment; PCDD\/F contamination increased in the 1930s\/1940s, reaching a maximum in the 1960s\/1970s, and subsequently declined into the 1990s as a result of actions to reduce emissions. There is general agreement in the temporal trend data between European and North American studies; however, some European studies have not determined the post-1970 declines in PCDD\/F contamination evident in North America. The US EPA [130] estimated a 75% decrease in PCDD\/F emissions over the period 1987\u20131995. P\u00e4pke [131] reported declines ranging from 50\u201370% for PCDD\/F levels in human adipose tissue, blood and breast milk in Germany over the period 1980\u20131996.\nThere is also now a considerable body of literature reporting the presence of preindustrial baseline trace levels of PCDD\/F contamination in soils and sediments prior to the turn of the nineteenth century [132\u2013138]. Natural sources of PCDDs\/Fs can include forest fires, volcanic activity, and other natural combustion processes [139, 140]. Although natural formation of PCDD\/Fs had been postulated much earlier, some previous studies may have been compromised as a result of extraneous contamination of field samples or in the laboratory [133, 135, 136]. These measurements are significant in determining the relevance of natural sources vs. anthropogenic sources in order to assess requirements for further reductions in global PCDD\/F emissions. However, it is also evident that any PCDD\/F loadings from natural sources are dwarfed by contemporary emissions from anthropogenic sources.\nPCDD\/F contamination in the North American Great Lakes has been recently reviewed by Norstrom [141], including occurrence, geographical distribution and temporal trends in air, water, sediments, fish, seabirds, snapping turtles and humans. Patterns and concentrations of PCDD\/Fs in sediment indicate that atmospheric inputs dominate in Lake Superior, lower Lake Michigan and Lake Erie. Inputs from the Saginaw River to Lake Huron, and from the Fox River to upper Lake Michigan, are responsible for additional PCDD\/PCDF loading to these areas beyond atmospheric deposition. Lake Ontario continues to be heavily impacted by input of PCDD\/Fs, particularly 2,3,7,8-TeCDD, from the Niagara River [142].\nAccording to Alcock and Jones [129], studies of sediment cores from the Great Lakes represent the bulk of sediment temporal trend data for PCDD\/Fs (e.g., [143]); sediment cores are particularly useful when studying the chronology of PCDD\/F contamination (e.g., [144\u2013147]). North American Great Lakes sediment core and biomonitoring data generally show that PCDD\/F contamination peaked in most lakes in the late 1960s to early 1970s, followed by rapid order of magnitude declines in the mid to late 1970s [144, 146\u2013148]. The downward trend leveled off in some lakes in the 1980s, but appears to have continued after the late 1990s, presumably in response to remedial actions and reductions in PCDD\/F emissions to the atmosphere [141]. Pearson et al. [148] reported that PCDD\/F accumulation rates in the early 1990s ranged from <0.03\u00a0ng cm\u22122\u00a0y\u22121 for Lake Superior to 0.39\u00a0ng cm\u22122\u00a0y\u22121 for Lake Ontario; these rates represent 30\u201370% of maximum accumulation in the 1960s to 1970s. In his review, Norstrom noted the lack of comprehensive congener-specific PCDD\/F data for Great Lakes sediment cores [141].\nCohen et al. [149] modeled atmospheric transport and deposition of PCDD\/Fs to the Great Lakes using a United States and Canadian air emissions inventory based on data from 5,700 point sources and 42,600 area sources (Fig.\u00a02). They identified municipal waste incineration, iron sintering, medical waste incineration and burning of hazardous waste in cement kilns as the most significant sources based on 1996 emissions data. Source areas up to 2,000\u00a0km removed from the Great Lakes were significant contributors. Combustion sources continue to be the predominant contributor to global ambient air levels of PCDD\/Fs [150]. Subsequent long-range transport of combustion emissions are thought to be primarily responsible for the global ubiquity of PCDD\/Fs [144, 151]. Fiedler recently reviewed global inventories of PCDD\/Fs using national air inventories from 23 countries to formulate a global air flux estimated range of 7,500\u201313,000\u00a0g TEQs per annum [152]. Brzuzy and Hites estimated total global deposition from the atmosphere to land at 12,500\u00b11,300\u00a0kg y\u22121 [151]. A study published in 2000 by Wagrowski and Hites using tree bark and soil samples estimated global deposition from the atmosphere to land at 2\u201315\u00a0t y\u22121 [153].\nFig.\u00a02Geographical distribution of United States and Canadian dioxin emissions for 1996. Reproduced with permission from Cohen et al. [149]\nThe recent report by Fiedler also showed sources of PCDDs\/Fs in developed countries to be different from developing countries [152]. In developed countries, industrial activities are the primary source category, while open-burning activities are the dominant source category in developing countries. However, burn barrels and other household garbage burning methods are estimated to emerge as the greatest source of PCDD\/F emissions in North America subsequent to implementation of new industrial air emission standards [154]. Open-burning processes include forest fires, pre- and post-harvest burning in agriculture, burning in landfills, and backyard burning. High releases were also estimated from incineration of medical waste. Emissions from municipal solid waste incineration (MSWI) were estimated to account for 70% of the PCDD\/F burden from industrial sources in the United Kingdom in 1995 [129]; other significant sources included emissions from metal industries and coal combustion. Industrial emissions account for roughly 90% of the total inventory in the United Kingdom [155]. Other combustion sources contributing to PCDD\/F emissions in industrialized countries include metal reclamation, wood burning, chemical fires, automobile exhaust, cement kilns and cigarette smoke. Non-combustion sources include chemical production (pesticides, perchloroethylene, pentachlorophenol), metal production, and pulp and paper mills. Recent data on PCDD\/F emissions for individual industrialized countries has been reported for the Netherlands [156], the United Kingdom [157], the United States [130] and Canada [158]. A global PCDD\/F emission inventory was reported by the United Nations Environmental Program [159]. The multimedia occurrence of PCDD\/Fs globally in industrialized countries is fairly well documented; however, data are generally far less common than for other chemicals such as PCBs and organochlorine pesticides. UNEP [160] has produced a report on global trends in persistent toxics, including PCDD\/Fs. Table\u00a05 shows selected global data for levels of PCDD\/Fs in a variety of matrices, based on information cited in the UNEP report. \nTable\u00a05Selected global environmental concentrations of PCDD\/FsMatrixRegionConcentration RangeReferenceRemarksAirEurope<1\u00a0fg TEQ\/m3Typical ruralAirEurope15,000\u00a0fg TEQ\/m3Contaminated areaAirAsia7\u20131,486\u00a0fg TEQ\/m3AirSouth America3\u2013394\u00a0fg TEQ\/m3FreshwaterKorea0.001\u20131.061\u00a0pg TEQ\/LFreshwaterJapan0.012\u201348\u00a0pg TEQ\/L2,116 sitesSeawaterJapan0.005\u20133.9\u00a0pg TEQ\/m312 sitesSeawaterJapanND\u20130.4\u00a0pg TEQ\/m321 sitesSoilSeveso, Italy0.91\u201316\u00a0pg TEQ\/gSoilGermany1\u20135\u00a0pg TEQ\/g[143]RuralSoilGermany30,000\u00a0pg TEQ\/g[143]ContaminatedSoilGermany10\u201330\u00a0pg TEQ\/g[143]UrbanSoilThe Netherlands2.2\u201316\u00a0pg TEQ\/g[143]RuralSoilThe Netherlands98,000\u00a0pg TEQ\/g[143]ContaminatedSoilUnited Kingdom<1\u201320\u00a0pg TEQ\/g[143]RuralSoilUnited Kingdom1,585\u00a0pg TEQ\/g[143]ContaminatedSoilNew Zealand0.17\u20131.99\u00a0pg TEQ\/g[143]RuralSoilNew Zealand260\u20136,670\u00a0pg TEQ\/g[143]UrbanSedimentsLake Ontario91\u00a0pg TEQ\/g[165]Lakewide averageSedimentsPo River1\u201311\u00a0pg TEQ\/g[166]SedimentsLake Baikal0.03\u00a0pg TEQ\/gSedimentsJapan16.1\u201350.7\u00a0pg TEQ\/gSedimentsNew Zealand0.081\u20132.71\u00a0pg TEQ\/gMarine sedimentsNorth Sea5.5\u201317.2\u00a0pg TEQ\/gMarine sedimentsGulf of Finland101,000\u00a0pg TEQ\/gHighly contaminatedMarine sedimentsCatalan coast0.4\u20138\u00a0pg TEQ\/g[167]Marine sedimentsJapan0.012\u201349.3\u00a0pg TEQ\/gVegetationGermany0.53\u20131.64\u00a0pg TEQ\/gPine needlesVegetationAustria0.3\u20131.9\u00a0pg TEQ\/gSpruce needlesBirds and animalsIndia19\u201324\u00a0pg\/g fat[168]EagleBirds and animalsIndia150\u2013200\u00a0pg\/g fat[168]OspreyBirds and animalsIndia9.2\u2013270\u00a0pg\/g fat[168]Spotted OwletBirds and animalsIndia1,300\u20132,700\u00a0pg dioxins\/g fat[168]Spotted Owlet liverBirds and animalsIndia620-1,000\u00a0pg furans\/g fat[168]Spotted Owlet liverFishGreat Lakes<1\u201363\u00a0pg TEQ\/g[169]FishGreat Lakes<1\u201359\u00a0pg TEQ\/g[170]Dioxin TEQFishGreat Lakes<1\u2013182\u00a0pg TEQ\/g[170]PCB TEQFishFinnish coast165\u2013329\u00a0pg TEQ\/g lipid[171]Herring muscleFishAdriatic Sea0.23\u2013329\u00a0pg TEQ\/g lipid[171]Mackerel>Red mullet>AnchovyFishJapan Sea0.10\u20130.95\u00a0pg TEQ\/gCodMarine mammalsBird Island2\u00a0pg TEQ\/g blubber[172]Fur sealsMarine mammalsCanadian Arctic2\u201323\u00a0pg TEQ\/g ww[173]Polar bearsMarine mammalsJapan Sea0.71\u201313\u00a0pg TEQ\/g wwWhaleMarine mammalsJapan Sea17\u2013360\u00a0pg TEQ\/g wwWhale fatMarine mammalsAustralia0.1\u20132.6\u00a0ng\/g wwBottlenose dolphinFoodEurope0.3\u20132.5\u00a0pg TEQ\/g fat[174]Milk and dairy productsFoodNorth America0.3\u20130.9\u00a0pg TEQ\/g fat[174]Milk and dairy productsFoodSouth America0.01\u20132.8\u00a0pg TEQ\/g fat[174]Milk and dairy productsFoodAsia0.3\u20131.8\u00a0pg TEQ\/g fat[174]Milk and dairy productsHuman milkThe Netherlands17.09\u201321.29\u00a0pg TEQ\/g fatHuman milkUkraine8.38\u201310.16\u00a0pg TEQ\/g fatHuman milkNorway7.16\u20137.43\u00a0pg TEQ\/g fatHuman milkCzech Republic7.44\u201310.73\u00a0pg TEQ\/g fatHuman milkBulgaria5.08\u20137.11\u00a0pg TEQ\/g fatBlood\/serum\/plasmaGreat Lakes27.5\u00a0ng TEQ\/L[175]Recreational fishersBlood\/serum\/plasmaIsrael26.6\u201332\u00a0ng TEQ\/g fat[176]Blood\/serum\/plasmaA So, Vietnam16.6\u201345.9\u00a0pg TEQ\/g lipid[177]Blood\/serum\/plasmaNew Zealand12.8\u00a0pg TEQ\/g serum fat[143]1,834 samplesAll citations are UNEP (2003) [160] or individual UNEP Regional Assessments unless otherwise noted. ND denotes \u201cnot detected\u201d; ww denotes \u201cwet weight\u201d\nProfiles of PCDD\/Fs in abiotic matrices including air, soils, and sediments in some areas can reflect the source emission profile. However, a confounding factor in attributing PCDD\/F contamination to specific sources can be a contrast in profiles between source samples and corresponding profiles in environmental sinks, e.g., soils and sediments, due to degradation, weathering, or transformation processes [161]. Eitzer and Hites observed a \u201cconsistent and systematic change\u201d from a combustion source profile to a sediment profile as a result of long-range transport and depositional processes [162]. Tysklind et al. attributed differences in PCDD\/F source profiles and air profiles to transformation processes including photolytic degradation or hydroxyl radical reactions [163]. Koester and Hites reported that wet and dry deposition are important mechanisms for scrubbing PCDD\/Fs from the atmosphere, and that these mechanisms contribute to the predominance of OCDD in sediments [164]. Ultimately, the PCDD\/F profile in environmental samples can be influenced by both source type and environmental processes.\nStudies of congener and homolog profiles for source apportionment have been carried out in abiotic matrices; biota have a tendency to accumulate primarily the 2,3,7,8-substituted compounds due to non-2,3,7,8-substituted congeners being metabolized [129, 161]. There are numerous examples of studies of the variation of PCDD\/F congener and homolog profiles with source (e.g., [161, 144\u2013147]); these studies have also illustrated how different sources of PCDD\/Fs have influenced temporal trends, primarily according to production, use and disposal of chlorinated organics. Principal component analysis (PCA) of profiles of source and sink samples have also proven to be a valuable tool for source apportionment [146, 147, 161, 163].","keyphrases":["polychlorinated dibenzofurans","dioxin-like pcbs","pcdd","pcdf","dlpcb","review","toxicity","environmental levels","analysis","extraction","mass spectrometry","sample preparation","polychlorinated dibenzo-p-dioxins"],"prmu":["P","P","P","P","P","P","P","P","P","P","P","P","R"]}
{"id":"Clin_Auton_Res-3-1-1797061","title":"Diversity of sympathetic vasoconstrictor pathways and their plasticity after spinal cord injury\n","text":"Sympathetic vasoconstrictor pathways pass through paravertebral ganglia carrying ongoing and reflex activity arising within the central nervous system to their vascular targets. The pattern of reflex activity is selective for particular vascular beds and appropriate for the physiological outcome (vasoconstriction or vasodilation). The preganglionic signals are distributed to most postganglionic neurones in ganglia via synapses that are always suprathreshold for action potential initiation (like skeletal neuromuscular junctions).\nIntroduction\nThe sympathetic nervous system supplies blood vessels throughout the body with vasoconstrictor axons that control local blood flow and contribute to total peripheral resistance. Signals arising in the central nervous system send specific controls to different tissues according to the functional response for each organ by means of reflexes and patterned regulation from higher centres [14]. The sympathetic outflow arises in the thoracolumbar cord, where it is particularly vulnerable to spinal cord injuries, being damaged directly if the lesion involves regions between T1 and L3 or the ventral roots at these levels, and being disconnected from the higher control centres by injuries to descending pathways in the cervical cord. The degree of disordered visceral and vascular function that ensues depends on the level and the severity of trauma in each individual.\nThe great majority of all sympathetic preganglionic neurones are vasoconstrictor in function and make synapses in ganglia of the paravertebral chain from which they project through grey rami and spinal nerves to the vasculature of the head, neck, trunk and limbs. Because the sympathetic outflow is restricted to thoracolumbar segments, preganglionic neurones in the upper thoracic segments control vessels in the head and neck and those in the lowest thoracic and upper lumbar segments control vessels in the pelvic organs and hindlimbs. Thus, for example, postganglionic neurones in the superior cervical ganglia receive inputs from T1-4 segments [25] whereas inputs to neurones in the lower lumbar paravertebral chain projecting to the hindlimbs mostly arise in the last two segments of the thoracolumbar sympathetic outflow [1]. Many visceral vasoconstrictor neurones also lie in the paravertebral chain and send their axons to the viscera via the various splanchnic nerves. The properties of these pathways supplying the vasculature and their modification after spinal cord injury (SCI) are the main topics of this review.\nPatterns of natural activity in sympathetic vasoconstrictor pathways\nImpulses travelling in sympathetic vasoconstrictor pathways arise in preganglionic neurones by integration of excitatory and inhibitory signals that arise supraspinally, intersegmentally, and segmentally. In vasoconstrictor pathways, the patterns of ongoing or tonic firing of preganglionic neurones originate in the rostral ventrolateral medulla [3]. However many descending excitatory glutamatergic axons and inhibitory axons containing gamma-amino-butyric acid and\/or glycine that arise both locally and supraspinally impinge on preganglionic neurones [17]. These inputs may be modulated by amines and peptides co-released from these pathways [26]. The preganglionic neurones integrate reflex signals from e.g. arterial baroreceptor and chemoreceptors via the brainstem, cutaneous and visceral nociceptors at spinal levels, signals arising from higher levels of the CNS, etc. Strong reciprocal links between vasomotor and respiratory centres in the brainstem, together with blood pressure swings related to breathing, mean that vasoconstrictor activity tends to be linked to the respiratory cycle as well as the cardiac cycle [8, 10, 19, 32].\nThe final output frequency and pattern of discharge in individual sympathetic axons is modulated by the membrane properties of the preganglionic neurones. Action potentials in these neurones are triggered by the summed depolarization produced by many small excitatory inputs [5, 12, 29] combined with inhibitory synaptic potentials arising from both local spinal interneurones and direct descending inhibitory pathways [6, 7]. The frequency of preganglionic discharge is limited to short bursts at <10\u00a0Hz by a large Ca2+-activated K+ conductance (and ensuing afterhyperpolarization) that follows each action potential, together with a prolonged transient (A-type) K+ current [4, 29]. The average ongoing preganglionic discharge in vasoconstrictor pathways in anaesthetized animals is 0.5\u20131\u00a0Hz [14].\nThe natural activity of individual postganglionic neurones projecting to different target tissues has been described by recording extracellularly spontaneous and evoked signals from sympathetic axons in peripheral nerves projecting to a known target tissue [14, 35]. Ongoing activity of individual units is recognized by the spike configuration and the unit functionally characterized by its responses to natural stimuli. Physiologically relevant responses are recorded from e.g. presumed sudomotor axons in cutaneous nerves immediately preceding secretion from sweat glands. Perhaps surprisingly, when simultaneous recordings are made from skin and muscle vasoconstrictor axons, the responses are generally opposite. For example, baroreflex inhibition is powerful in muscle vasoconstrictor (MVC) axons but weak or absent in cutaneous vasoconstrictor (CVC) axons. Chemoreceptor activation stimulates MVCs but inhibits CVCs, nociceptor stimuli excite CVCs but inhibit MVCs [13, 14]. These differences reflect what is known about blood flow through skin and muscle, with the former involved primarily in temperature regulation and the latter in regulation of peripheral resistance at rest. Similar differences in skin and muscle vasoconstrictor axons are observed in microneurographic recordings from conscious humans in which the excitatory effects of emotion on CVCs and mental stress on MVCs are revealed [33, 34]. The importance of these observations is that they make the concept of a generalized sympathetic vasoconstrictor \u201ctone\u201d inappropriate.\nWhen this could be studied, the patterns and frequencies of preganglionic discharge have convincingly parallelled those of the postganglionic projections in the same pathway. Thus the average postganglionic discharge frequency in vasoconstrictor pathways is 0.5\u20131\u00a0Hz [13, 14].\nTransmission of vasoconstrictor signals through sympathetic ganglia\nThe similarity between pre- and postganglionic discharge raises questions about the purpose of the synapses in sympathetic ganglia. Each preganglionic vasoconstrictor neurone branches to form synapses with multiple postganglionic neurones in several adjacent ganglia in the paravertebral chain. However only a subset of inputs (generally those that arise in one spinal segment) transmits the centrally derived command signal to the postganglionic neurones. This results because, in individual postganglionic neurones, transmission occurs in an all-or-none manner from a single input, as at the skeletal neuromuscular junction [21]. By recording intracellularly, and recruiting preganglionic inputs using graded stimuli, each postganglionic neurone can be shown to receive one \u201cstrong\u201d excitatory input (rarely 2 or 3) that is always suprathreshold for action potential initiation, as well as several subthreshold excitatory inputs. (There are no inhibitory synapses in sympathetic ganglia.) As the safety factor for transmission at most ganglionic synapses is very high, high concentrations of nicotinic antagonists are necessary to block ganglionic transmission. The important role of the strong input has been confirmed in intracellular recordings in vivo [22, 30], which show that subthreshold inputs rarely summate, due to both the low preganglionic firing frequency and the large afterhyperpolarization after the action potential (again due to a Ca2+-activated K+ conductance) triggered by the strong input. In practice, subthreshold inputs appear largely redundant. The total number of preganglionic inputs parallels the number of dendrites of the postganglionic neurone and these increase with animal size [27]. It is anticipated that each human paravertebral neurone receives at least 20 synaptic inputs, but that, in most cases, only one of these is functionally effective.\nThe connectivity described above means that ganglionic synapses in vasoconstrictor pathways simply relay the centrally-derived signals to the periphery without alteration. Thus the ganglia act primarily as distributors of the central pattern of impulses, each preganglionic axon activating many postganglionic neurones. Teleologically, this can be seen to reduce the need to provide large numbers of preganglionic neurones in the spinal cord to control the extensive vasculature throughout the body. However, although the preganglionic axons diverge to contact many ganglion cells (\u223c150 in the rat, [28], the redundancy of many of these contacts means that the functional autonomic equivalent to the motor unit is much smaller (only \u223c15 in rat, perhaps >100 in humans).\nChanges in vasoconstrictor outflow after spinal cord injury\nIn microneurographic recordings from skin and muscle nerves in conscious relaxed intact humans (Fig.\u00a01A), recordings from sympathetic axons in muscle nerves show bursts of activity in rhythm with the arterial pulse wave that wax and wane with respiration [31], reflecting the cyclic inhibition of MVCs by baroreceptor input. Such bursts are rarely recorded in CVCs where ongoing activity is largely unsynchronized. During simultaneous recordings from skin and muscle fascicles, brief electrical stimulation of skin leads to a burst of activity in the CVCs but inhibition of ongoing bursts in MVCs. This reflects the reflex activation of CVCs and inhibition of MVCs by nociceptor stimuli.\nFig.\u00a01Extracellular recording of multiunit sympathetic activity in skin and muscle nerves of conscious humans. (A) from intact individual; (B) from a subject with complete spinal cord transection at C6. At arrows, the skin over the forearm was stimulated electrically. Note the absence of ongoing activity in both nerves and the synchronous activation by the stimulus after spinal cord injury. Modified from Stjernberg et al. (1986)\nSuch paired recordings from leg nerves of people with thoracic spinal cord injury, showed completely different responses (Fig.\u00a01B). The cardiac related bursts were absent and the signals from both nerves showed little background activity. Electrical stimuli to the skin produced identical brief bursts of activity in both skin and muscle nerves. These observations reflect the removal of both descending sympathetic drive with periodic baroreceptor inhibition and the conversion of the nociceptor-evoked reflex in MVCs from inhibitory to excitatory. This reversal of the spinal nociceptor reflex has also been demonstrated in chronic spinal cats [15]. It should be noted that sympathoexcitation following nociceptor stimulation was transient without evidence of enhancement or prolongation of the reflex sympathetic discharge although the vasoconstriction was prolonged.\nChanges in connectivity in paravertebral ganglia after destruction of preganglionic neurones\nThe consequences for the remaining sympathetic pathways of damage to preganglionic neurones or their axons by a spinal injury can be studied in experimental animals by partially denervating the paravertebral ganglia and observing the types of connection made by the remaining terminals. Extensive collateral sprouting of the remaining preganglionic axons occurs [16, 23]. Transection of the descending lumbar paravertebral chain just above the last segmental preganglionic outflow left postganglionic neurones in the next more caudal lumbar ganglion with <1 input, the great majority being subthreshold (Ireland 1999); the residual preganglionic input in the last sympathetic segment formed strong inputs on only 10% of the neurones. After 4\u20135\u00a0weeks, however, 70% of neurones in the same ganglion had a large strong input as well as \u223c2 subthreshold inputs. Thus. the remaining preganglionic neurones had sprouted and preferentially formed strong inputs, even on neurones that were completely denervated by the lesion. New strong inputs must have formed on some neurones with a peripheral function that differed from that of the original preganglionic input [16]. The formation of such aberrant strong inputs after SCI might explain more generalized vasoconstriction, or the activation of sudomotor neurones by reflexes normally directed at peripheral vessels.\nAutonomic dysreflexia\nWhile modified vascoconstrictor reflexes in spinally-injured people can be mimicked in experimental animals, they do not predict the dramatic and life-threatening rises in arterial blood pressure that are known as autonomic dysreflexia. These uncontrolled hypertensive episodes are usually triggered by overdistension of bladder or bowel but many also arise from nociceptor activation from a pressure sore or other unheeded injury below the spinal cord injury [20, 36]. Such episodes are usually relieved by removal of the stimulus but, if the trigger is unknown, treatment with vasodilators requires hospitalisation which is inconvenient and expensive. Other symptoms of dysreflexia are excessive sweating and muscle spasms. Dysreflexia is typically seen only if the lesion is at or above T5-6.\nIt is generally believed that the unrestrained hypertension results from the absence of descending inhibitory control by baroreceptors and clearly the loss of this reflex control is highly relevant. The reason that T5 is critical as the cut-off for dysreflexic episodes is presumably because baroreflex vagal slowing of heart rate and inhibition of intact sympathetic pathways only to the head, neck and arm vasculature are not sufficient to buffer increases in vascular resistance below the injury. The simultaneous activation of both cutaneous and muscle vascular beds below the injury would contribute a large increase in peripheral resistance, and the effect would be more dramatic if the visceral vasculature is also excited reflexly. There is a prevailing view that the splanchnic circulation must be involved as it is controlled largely from below T5 [36].\nAnimal experiments have suggested that primary afferent nociceptor inputs (containing calcitonin gene-related peptide but not Substance P) sprout within the cord around the lesion [36], probably due to the release of neurotrophins by inflammatory cells in the segments around the lesion site. However, as mentioned above, sympathetic discharge following brief stimuli in spinally-injured people is not particularly large or prolonged, suggesting that the expansion of afferent connections is not responsible for much enhancement of discharge. Thus the mechanisms underlying the pronounced vasoconstriction during AD have been unclear.\nChanges in vascular reactivity after spinal cord injury\nIf the sympathetic discharge after SCI is very much reduced and the reflex discharges provoked by e.g. bladder stimulation are not particularly marked, why does dysreflexia develop? The prolonged vasoconstriction might be explained if neurally evoked vasoconstriction was in some way potentiated. This might result from the potentiated release of transmitter per impulse, as has been shown at ganglionic [9] and central [24] synapses when ongoing activity is blocked. To test this idea, isolated vessel segments were mounted in a myograph and contractile responses evoked by short trains of supramaximal transmural electrical stimuli at 0.1\u20135\u00a0Hz, frequencies within the physiological range. Vessels were isolated from rats with spinal transection at T4 or T8, leaving all parts of the sympathetic pathways intact, and compared with those from age-matched and sham-operated controls. After 2 or 8\u00a0weeks, the responses of cutaneous and splanchnic arteries to stimulation of the perivascular nerves were greatly enhanced (Fig.\u00a02).\nFig.\u00a02Contractile responses of segments of (A) cutaneous (tail) artery and (B) splanchnic (mesenteric) artery isolated from (above) control rats and (below) rats 7\u20138\u00a0weeks after spinal transection at (A) T8 and (B) T4. Trains of the same number of transmural electrical stimuli at different frequencies (indicated by bars at bottom) were presented to each vessel to activate the sympathetic postganglionic terminals. Responses in vessels from spinalized rats were greatly enhanced compared with controls. Redrawn from (A) Yeoh et al. (2004a) and (B) Brock et al. (2006)\nTransmission in small diameter blood vessels is known to occur primarily at neurovascular junctions on the outer layers of smooth muscle cells [11, 18]. Even when the relation between the postganglionic varicosities and the effector tissue is not direct, it has been shown in all preparations so far investigated that responses to nerve-evoked and to exogenous transmitters do not involve the same receptors or postreceptor mechanisms [11]. The enhanced effect of endogenous neurotransmitters on blood vessels after spinal transection might involve one or more of a number of mechanisms (increased norepinephrine (NE) release, decreased NE clearance, increased junctional receptor sensitivity, increased postreceptor responsiveness), only some of which can be tested directly. Rather unexpectedly, the cellular mechanisms underlying the enhanced responsiveness to NE released from postganglionic nerve terminals were not the same for each vessel. In the rat tail artery, in which contractile responses to nerve stimulation were enhanced more than 10-fold, most of the changes in neurovascular transmission were postsynaptic, involving an increased reactivity of the smooth muscle [38]. There was some maintained increase in sensitivity of \u03b12- but not \u03b11-adrenoceptors to exogenous NE. In contrast, in the mesenteric artery, where transmission was increased 5-fold, most of the changes were presynaptic and involved a decrease in the activity of the NE transporter that takes the released NE back up into the postganglionic terminals [2]. Differences between the vessels in the ability of \u03b1-adrenoceptor and purinoceptor antagonists to inhibit nerve-evoked responses imply that the mechanisms of neurovascular transmission are selective for different vascular beds.\nTo test whether the enhanced responsiveness resulted from the removal of ongoing activity in the postganglionic axons, activity to the tail artery was prevented in normal animals by cutting the lumbar paravertebral chain to disconnect the preganglionic input to the postganglionic neurones (decentralization, Fig.\u00a03(2)). Neurovascular transmission in the tail artery was enhanced after decentralization in the same way as after spinal transection [37]. Notably, sensitivity of the vascular muscle to \u03b11-adrenoceptors was not modified. Even after denervation of the tail artery (by cutting the postganglionic nerves, Fig.\u00a03(3)) to remove the perivascular terminals (and thus the NE transporter), the responses to phenylephrine were much more enhanced relative to control than those to another \u03b11-agonist, methoxamine, that is not a substrate for the NE transporter. These observations emphasize the significant role of the postganglionic nerve terminals in reducing the effective concentration of catecholamines applied to the outside of vascular preparations.\nFig.\u00a03Diagram showing sites of lesions used to study long term effects on neurovascular transmission in arterial vessels of rats. (1) Transection of thoracic spinal cord without damage to preganglionic neurones. (2) Transection of paravertebral chain to remove preganglionic inputs (decentralization). (3) Transection of postganglionic nerves to denervate artery. Segments of artery were removed from the animals after 2\u20138\u00a0weeks and contractile responses to stimulation of perivascular sympathetic nerves were recorded during exposure to adrenoceptor antagonists and other drugs. that selectively interfere with neurovascular mechanisms\nOverall, the data so far suggest that blood vessels in spinalized animals would constrict more powerfully to even short bursts of sympathetic activity evoked reflexly below the lesion. As well as being consistent with the complaints of many spinally-injured people about cold feet and legs, and the common difficulties in healing pressure sores, this modified vascular reactivity could contribute strongly to the development of autonomic dysreflexia in humans.\nConclusions\nDespite many years of investigation, we still know remarkably little about the way in which neurally-released transmitters, including NE, lead to constriction of vascular smooth muscle. It is now clear that, as for many autonomically-innervated tissues, the mechanisms are not the same as those activated by exogenous transmitters. The work discussed here emphasizes the diversity of mechanisms that underlie the neural control of arterial vessels in different vascular beds and the plasticity of both innervation and effector tissues after lesions to the nervous system. Marked changes can occur even when both pre- and postganglionic components of the sympathetic innervation are undamaged. While the findings in experimental animals need to be confirmed in spinally injured people, they may help us to devise better ways to improve their rehabilitation and long-term maintenance. In addition, we may need to revise our thinking about the role of sympathetic activity in the regulation of the cellular processes involved in neurovascular transmission, even in intact individuals.","keyphrases":["vasoconstriction","sympathetic nervous system","sympathetic ganglia","autonomic dysreflexia","norepineph- rine","autonomic nervous system"],"prmu":["P","P","P","P","U","R"]}
{"id":"Soc_Psychiatry_Psychiatr_Epidemiol-3-1-2039804","title":"Prevalence and predictors of health service use among Iraqi asylum seekers in the Netherlands\n","text":"Background A long asylum procedure is associated with higher prevalence rates of psychiatric disorders, lower quality of life, higher disability and more physical health problems. Additional knowledge about health seeking behavior is necessary to guide governments and health professionals in their policies.\nIntroduction\nMental health service use differs among populations and geographical areas [3, 15, 25]. Refugees and asylum seekers have high prevalence rates of psychiatric disorders [6, 12] and adequate use of health services is important. Literature on this issue is growing, but still limited [8, 9, 16]. Health policies, including accessibility of service for asylum seekers differ between European Union countries [19]. In the Netherlands refugees have direct access to the general practitioner, while asylum seekers can only enter the curative health care system after a screening by a nurse or doctor in the asylum seeker center. This system has been criticized [18], but others claim that these professionals, involved in triage after receiving training in cultural competence, may prevent inadequate referrals.\nHealth service use, according to the Anderson behavioral model [1, 2], is a function of three sets of variables: predisposition, enablement and need. Predisposition includes demographic factors such as age, gender, religion, cultural factors, social network, and support. Enabling factors facilitate service use and include individual social functioning, availability of services, and costs. The third set, i.e. the need variables, consists of health related factors. Both objective (type and severity of a health problem), and subjective health indicators (disability and perceived health) belong to the need variables. McCracken et al. [15] used the above model in a community survey on depression in five European countries, and found that severity of depression, lower perceived health status, social dysfunctioning, and low level of social support were significant predictors of use. Kamperman et al. [11] used the model in a study among migrants in Amsterdam and found that there were migrant-specific mechanisms in health care consumption. Higher levels of acculturation and lower level of cultural traditionalism increased the use of mental health care facilities.\nIn these health service use studies, a variety of potential predictors, such as psychopathology, physical diseases, physical, and mental well-being, have been included. But disability and quality of life were not included. Nor is there a study that compared groups from the same country of origin, that differ in length of stay in a host country.\nIn a national community study among Iraqi asylum seekers (n\u00a0=\u00a0294) in the Netherlands [12\u201314] we measured prevalence rates of psychiatric disorders, quality of life, disability, and physical health problems in relationship with pre- and post-migrations stressors. The study focuses on the risks of a long asylum procedure and showed that asylum seekers who stayed more than 2\u00a0years in the Netherlands had significantly higher overall prevalence rates of psychiatric disorders (66.2%), than those who arrived recently (42.0%). A long asylum procedure was an important independent risk factor for a psychiatric disorder with an odds ratio of 2.16 (CI 1.15\u20134.08). This ratio was higher than the odds ratio for premigration stressors, such as exposure to human rights violations. In addition the \u2018long stay\u2019 group had significantly lower quality of life, higher disability, and higher levels of physical health complaints. In the Anderson model these health indicators can be considered as need factors.\nThe aim of this paper is to examine patterns and predictors of health service use and their relationship with length of stay. The research questions are: (a) what is the prevalence of service use of Iraqi asylum seekers in the Netherlands, (b) is a long asylum procedure associated with a higher prevalence of service use, (c) what is the relationship between psychopathology and service use, and (d) which predisposing and other need variables predict service use. We hypothesize that a long asylum procedure is associated with a higher prevalence of service use and that higher levels of psychopathology are related to higher levels of service use.\nMethods\nA comprehensive description of used methods is provided in a previous article [12]. A summary is given below.\nFrom the entire population of adult Iraqi asylum seekers, two groups were selected based on their length of stay in the Netherlands. Personal data on these groups was obtained from the Agency for the Reception of Asylum Seekers (COA). Group 1 was selected on the criterion that persons had been living in the Netherlands for less than 6\u00a0months (between September 2000 and November 2001). From the randomly selected 362 respondents, data could be used from 143 interviews. Group 2 was selected on the criterion that they were living in the Netherlands for at least 2\u00a0years. On the chosen date of May 31st, 1999, the COA found that 2,352 Iraqi asylum seekers fulfilled this criterion. From the randomly selected 474 respondents, data could be used from 151 interviews.\nThe questionnaire about service use\u2014in the 2\u00a0months prior to the interview\u2014included regular services as well as alternative services. The studied regular services are: out-patient services: preventive healthcare (nurse\/doctor in center), primary healthcare (general practitioner), generic healthcare (medical specialist, non-psychiatric), social care (social worker), psychiatric services (mental health professional); in-patient services: hospital admission physical health, hospital admission mental health; use of drugs (any drugs, hypnotics, anxiolytics and analgetics). Examples of drug names were given to be sure the drugs were put in the right category. As mentioned above, in the Netherlands medical staff is present in all asylum seekers centers. They perform health assessments of recently arrived asylum seekers, facilitate entry into primary health care, and refer to the general or mental health services. Their services can be classified as gateway services [7]. The studied alternative services are: use of religious helpers or rituals, and herb-doctors or herbs.\nRespondents\u2019 predisposition to service use was measured by age, gender, religion, ethnicity, and length of stay (membership Group 1 or Group 2). Enabling factors were not measured: regular health services for asylum seekers are available and accessible in the Netherlands without financial obstacles. Need factors include: psychiatric disorders, physical health, quality of life, and disability, while post-migration living problems (PMLP) were added as a special set of need variables. Psychiatric disorders were measured with the WHO-CIDI, version 2.1 [28] and cluster diagnoses were used in the analyses. Physical health was assessed with 22 items, dealing with: perceived physical health, physical handicaps, chronic physical diseases (e.g. lung disease, epilepsy, diabetes), and chronic physical complaints (e.g. stomach problems, joint problems, headache more than 3\u00a0months). Quality of life (Qol) was assessed with the WHOQOL-BREF [22]. The first two single questions i.e.: \u2018How would you rate your quality of life?\u2019 (\u2018overall Qol\u2019) and \u2018How satisfied are you with your health?\u2019 (\u2018Qol perceived general health\u2019) were used in the current study. Disability (Brief Disability Questionnaire, VonKorff [26] was measured in two dimensions: total disability (total of score of 11 items on physical and social role impairments), and the total number of days with serious impairment in the last month (BDQ-days). Post-Migration Living Problems (PMLP) were assessed with a checklist, adapted from Silove et al. [21]. The 24 items were clustered, based on a factor analysis [13] as family issues, discrimination, asylum procedure, socioeconomic living conditions, and religious aspects. The items \u2018lack of work\u2019 and \u2018work below level\u2019 loaded on different factors and were analyzed as separate items.\nThe used Iraqi-Arabic composite questionnaire is based on a Palestinian-Arabic version [4, 5] and was culturally validated and translated with the help of a focus group. Oral interviews were taken by trained Iraqis.\nStatistics\nDifferences between the two groups with respect to socio-demographics, psychiatric disorders, service use, and drug consumption were calculated with the \u03c72-test. Univariate relationships between predisposing and need variables were assessed with a correlation matrix. Ethnicity, religion, and marital status did not show a significant (P\u00a0<\u00a00.05) correlation with any of the health services. All other variables were entered into multivariate logistic regression analyses as: predisposing variables (study group, sex and age), need variables (one or more psychiatric disorders, overall quality of life, perceived quality of general health, disability (2 items), physical health (2 items), and a special set of need variables: the PMLP (7 variables). The same independent variables were used in each analysis, in line with the Anderson model. The dependent\/outcome variables were: use of preventive service (nurse\/doctor in the center), general practitioner, medical specialist (non-psychiatrist), social worker, mental health professional, and use of any drugs. We used a three-step procedure: in the first step each set of variables (predisposing factors, need factors and PMLP) was analyzed separately; in the second step all variables entered one analysis, that way the risk of one variable was corrected for the risks of all other variables. In step one and two, the entire dataset (n\u00a0=\u00a0294) was used. In the third step the analysis was done for Group 1 and Group 2 separately in order to assess the differences of predictors between asylum seekers that had recently arrived (Group 1) and those who had stayed for more than 2\u00a0years in the asylum procedure (Group 2). Adjusted Odds Ratios (ORs), 95% confidence intervals (CIs), and P values were calculated (only the ratios with confidence intervals higher or lower than 1 are shown in the tables). Differences were considered significant at P\u00a0<\u00a00.05. All analyses were performed with SPSS version 10 [20].\nResults\nSocio-demographics, health and health related variables\nThe two study groups differed on several socio-demographic characteristics (Table\u00a01). Group 1 contained more subjects younger than 24\u00a0years of age, and more females. The average time of stay in the Netherlands of Group 2 was more than 3 years. On the characteristics literacy, social status in Iraq, and psychiatric problems in the family, the two groups did not differ (not shown in the table, c.f. [12]. Group 2 had higher scores on prevalence of psychiatric disorders, disability and physical complaints, and lower quality of life score (Table\u00a01). Except social-religious items, all clustered and non-clustered post-migration living problems were significantly higher in Group 2.\nTable\u00a01Socio-demographic, health characteristics and post-migration living problems in at random samples of Iraqi asylum seekers arrived <6\u00a0months (Group 1) versus >2\u00a0years (Group 2) in the Netherlands, 2000\u20132001VariablesGroup 1 (n\u00a0=\u00a0143)Group 2 (n\u00a0=\u00a0151)Total (n\u00a0=\u00a0294)P-valueStay in months (mean, SD)2.51 (1.16)36.77 (6.30)20.12 (17.76)P\u00a0<\u00a00.0005, t(292)\u00a0=\u00a063.66Sex (%)P\u00a0<\u00a00.0005, \u03c72(1)\u00a0=\u00a027.31\u00a0\u00a0\u00a0\u00a0Male49.778.864.6\u00a0\u00a0\u00a0\u00a0Female50.321.235.4Age (%)P\u00a0=\u00a00.003, \u03c72(4)\u00a0=\u00a016.35\u00a0\u00a0\u00a0\u00a018\u201324\u00a0years21.79.315.3\u00a0\u00a0\u00a0\u00a025\u201334\u00a0years42.049.045.6\u00a0\u00a0\u00a0\u00a035\u201344\u00a0years14.725.820.4\u00a0\u00a0\u00a0\u00a0>45\u00a0years21.715.818.7One or more psychiatric disorder (%)42.066.254.4P\u00a0<\u00a00.0005, \u03c72(1)\u00a0=\u00a017.44Overall quality of life (mean, SD)a2.88 (0.99)2.23 (1.14)2.55 (1.11)P\u00a0<\u00a00.0005, Z(294)\u00a0=\u00a0\u22125.29Perceived Qol general health (mean, SD)b3.06 (1.15)2.74 (1.27)2.89 (1.22)P\u00a0=\u00a00.017, Z(294)\u00a0=\u00a0\u22122.39Physical and role disability (mean, SD)c17.31 (7.43)19.25 (6.77)18.30 (7.15)P\u00a0=\u00a00.020, t(292)\u00a0=\u00a0\u22122.34Days of disability (mean, SD)d5.37 (8.24)7.68 (9.17)6.56 (8.80)P\u00a0=\u00a00.024, t(292)\u00a0=\u00a0\u22122.27Physical diseases (mean, SD)e0.85 (1.18)0.84 (0.98)0.85 (1.08)n.s.Physical complaints (mean, SD)e0.83 (1.38)1.62 (1.58)1.23 (1.54)P\u00a0=\u00a00.0005, t(292)\u00a0=\u00a04.52Post-migration living problems\u00a0\u00a0\u00a0\u00a0Family related issues (mean, SD)f36.54 (26.55)52.81 (24.24)44.90 (26.62)P\u00a0<\u00a00.0005, t(292)\u00a0=\u00a0\u22125.49\u00a0\u00a0\u00a0\u00a0Discrimination (mean, SD)f2.05 (7.92)11.17 (21.01)6.74 (16.65)P\u00a0<\u00a00.0005, t(292)\u00a0=\u00a0\u22124.87\u00a0\u00a0\u00a0\u00a0Asylum proc. related issues (mean, SD)f48.58 (25.35)60.13 (23.23)54.51 (24.97)P\u00a0<\u00a00.0005, t(292)\u00a0=\u00a0\u22124.07\u00a0\u00a0\u00a0\u00a0Socioeconomic living cond. (mean, SD)f22.35 (19.16)32.48 (21.08)27.55 (20.76)P\u00a0<\u00a00.0005, t(292)\u00a0=\u00a0\u22124.30\u00a0\u00a0\u00a0\u00a0Socio-religious aspects (mean, SD)f12.96 (17.46)14.48 (17.19)13.74 (17.31)n.s.n.s.: not significant P\u00a0\u2265\u00a00.05aScale of 1 (very bad)\u20135 (very good)bScale of 1 (very bad)\u2013100 (very good)cRange: 11 (no impairment at all)\u201333 (serious impairment)dRange: 0\u201331 (number of days of serious impairment in last months)eRange: diseases: 0\u201312; complaints: 0\u20136fRange: 0 (not worried)\u2013100 (extremely worried about the issues)\nHealth service use\nTable\u00a02 shows the prevalence of service use in the 2\u00a0months prior to the interview. Overall, the most frequently used service was the preventive healthcare service, followed by the general practitioner. Sixteen (5.4%) respondents visited a mental health professional. Group 1 visited the preventive healthcare services in the center more often than Group 2 (72.0% vs. 39.7%) and Group 2 visited the mental health services more often than Group 1 (9.3% vs. 1.4%). More than 39% of the respondents used drugs, 31.0% used analgetics. Overall drug consumption was higher in Group 2 (45.7% vs. 32.2%). Use of alternative services and treatments was very low, and there was no difference between the two groups: contact religious helper (2.7%), use of religious rituals or treatment (5.1%), contact with herbal doctor (0.7%), use of herbal treatment (0.3%).\nTable\u00a02Health service use and other help seeking behavior in at random samples of Iraqi asylum seekers arrived <6 months (Group 1) and >2 years (Group 2) in the Netherlands, 2000\u20132001Use of services last 2\u00a0monthsGroup 1 n\u00a0=\u00a0143 (%)Group 2 n\u00a0=\u00a0151 (%)Total n\u00a0=\u00a0294 (%)P valueUse of any health servicea76.966.271.4P\u00a0=\u00a00.042, \u03c72(1)\u00a0=\u00a04.119bUse of any out-patient (o-p) service74.152.362.9P\u00a0<\u00a00.005, \u03c72(1)\u00a0=\u00a014.992bUse of any o-p curative service38.536.437.4n.s.Use of preventive o-p service\u00a0\u00a0\u00a0\u00a0Nurse\/doctor in center72.039.755.4P\u00a0<\u00a00005, \u03c72(1)\u00a0=\u00a031.004bUse of o-p curative service\u00a0\u00a0\u00a0\u00a0General practitioner32.925.829.3n.s.\u00a0\u00a0\u00a0\u00a0Medical specialist in hospital12.617.915.3n.s.\u00a0\u00a0\u00a0\u00a0Social worker5.66.66.1n.s.\u00a0\u00a0\u00a0\u00a0Mental health worker1.49.35.4P\u00a0=\u00a00.003, \u03c72(1)\u00a0=\u00a08.846Use of in-patient service\u00a0\u00a0\u00a0\u00a0Hospital admission physical health1.44.02.7n.s\u00a0\u00a0\u00a0\u00a0Hospital admission mental health0.00.70.3n.sUse of any drugs32.245.739.1P\u00a0=\u00a00.018, \u03c72(1)\u00a0=\u00a05.643\u00a0\u00a0\u00a0\u00a0Use of anxiolytics10.522.516.7P\u00a0=\u00a00.006, \u03c72(1)\u00a0=\u00a07.649\u00a0\u00a0\u00a0\u00a0Use of hypnotics11.921.216.7P\u00a0=\u00a00.032, \u03c72(1)\u00a0=\u00a04.578\u00a0\u00a0\u00a0\u00a0Use of analgetics23.837.731.0P\u00a0=\u00a00.010, \u03c72(1)\u00a0=\u00a06.707n.s.: P\u00a0>\u00a00.05aIncludes all regular and alternative services, including drugs, religious rituals\/treatment and herbsbGroup 1 more than group 2, see text\nRelationship between psychopathology and service use\nTable\u00a03 shows the univariate relationships between having \u2018one or more psychiatric disorder\u2019 and service use. Overall, respondents with psychopathology used significantly more services (70.0% vs. 54.5%), both curative and preventive ones. There was no significant difference in use of the services of the general practitioner, this was the case in both groups. Respondents with psychopathology visited a medical specialist (non-psychiatrist) much more often in Group 1, but not in Group 2 (P\u00a0>\u00a00.05).\nTable\u00a03Use of services in respondents with and without one or more psychiatric disorder in Iraqi asylum seekers arrived <6\u00a0months (Group 1), and >2\u00a0years (Group 2) in the Netherlands, 2000\u20132001One or more psychiatric disorderGroup 1 n\u00a0=\u00a0143Group 2 n\u00a0=\u00a0151Total n\u00a0=\u00a0294Yes n\u00a0=\u00a060, %No n\u00a0=\u00a083, %Yes n\u00a0=\u00a0100, %No n\u00a0=\u00a051, %Yes n\u00a0=\u00a0160, %No n\u00a0=\u00a0143, %Use of services last 2 months\u00a0\u00a0\u00a0\u00a0Use of any health servicea88.368.7*76.047.1*80.660.4*\u00a0\u00a0\u00a0\u00a0Use of any out patient (o-p) service86.765.1*60.037.3*70.054.5*\u00a0\u00a0\u00a0\u00a0Use of any o-p curative service53.327.7*43.023.5*46.926.1*Use of preventive o-p service\u00a0\u00a0\u00a0\u00a0Nurse\/doctor in center83.363.9*47.025.5*60.649.3Use of o-p curative service\u00a0\u00a0\u00a0\u00a0General practitioner41.726.529.019.633.823.9\u00a0\u00a0\u00a0\u00a0Medical specialist (non-psychiatrist)26.72.4*21.011.823.16.0*\u00a0\u00a0\u00a0\u00a0Social worker8.33.610.00.0*9.42.2*\u00a0\u00a0\u00a0\u00a0Mental health worker1.71.213.02.0*8.81.5*Use of any drugs45.022.9*58.021.6*53.122.4*\u00a0\u00a0\u00a0\u00a0Anxiolytics18.34.8*31.05.9*26.35.2*\u00a0\u00a0\u00a0\u00a0Hypnotics18.37.2*29.05.9*25.06.7*\u00a0\u00a0\u00a0\u00a0Analgetics31.718.148.017.6*41.917.9**P\u00a0<\u00a00.05aIncludes all regular and alternative services, including drugs, religious rituals\/treatment and herbs\nThe use of drugs in respondents with psychopathology was higher in both groups, compared to those without psychopathology. Almost 60% of the respondents in Group 2 with psychopathology used drugs, versus 21.6% of those without psychopathology. Also the use of analgetics was higher, especially in Group 2 (48% vs. 17.6%).\nPredictors of service use\nIn the \u2018step 2\u2019 analyses (Table\u00a04), with all the independent variables in one analysis, the use of preventive healthcare services was predicted by Group 1 membership, low perceived quality of general health, and physical diseases. Visits to a general practitioner were predicted by age, low perceived quality of general health, and days of disability in the last month. Use of the services of a medical specialist (non-psychiatrist) was predicted by having one or more psychiatric disorders, high overall quality of life, low perceived quality of general health and physical diseases. Use of a social worker was predicted by low total disability and days of disability in the last month. Two variables predicted service use of a mental health worker: Group 2 membership, and low perceived quality of general health. Use of any drugs was predicted by age, and low perceived quality of general health. Use of analgetics (not in table) was predicted by age, lower perceived quality of general health, and physical diseases. The variables sex, physical complaints, and post-migration living problems (PMLP) did not predict any type of service use in the step 2 analyses (Group 1 and 2 together).\nTable\u00a04Multivariate logistic regression of predisposing and need (incl. PMLP: post-migration living problems) variables related to service use of Iraqi asylum seeker in the Netherlands (N\u00a0=\u00a0294), 2000\u20132001ServiceNurse\/Doctor centerGeneral practitionerMedical spec. (non-psychiatry)Social workerMental health workerUse of any drugsORaCI (95%)ORaCI (95%)ORaCI (95%)ORaCI (95%)ORaCI (95%)ORaCI (95%)Predisposing factors\u00a0\u00a0\u00a0\u00a0Group 2 membershipb0.180.08\u20130.375.561.08\u201328.69\u00a0\u00a0\u00a0\u00a0Age (older age)1.031.00\u20131.051.051.02\u20131.08Need factors\u00a0\u00a0\u00a0\u00a0One or more psychiatric disorder1.341.18\u20132.76\u00a0\u00a0\u00a0\u00a0Overall Quality of life1.801.18\u20132.76\u00a0\u00a0\u00a0\u00a0Perceived quality of general health0.440.30\u20130.640.510.36\u20130.730.540.35\u20130.840.350.17\u20130.770.490.33\u20130.71\u00a0\u00a0\u00a0\u00a0Disabilityc0.890.80\u20130.99\u00a0\u00a0\u00a0\u00a0Disability daysd1.041.001\u20131.081.071.01\u20131.15\u00a0\u00a0\u00a0\u00a0Physical diseases1.481.01\u20132.161.061.02\u20131.112.071.41\u20133.05PMLPaOR: Odds ratios are adjustedbGroup 2 membership >2\u00a0year in asylum procedurecDisability: total score of Brief Disability QuestionnairedDisability days in last month\nTables\u00a05 and 6 show the \u2018step 3\u2019 analyses for Group 1 (<6\u00a0months) and Group 2 (>2\u00a0years in the Netherlands) separately. In both groups a lower score on perceived quality of general health predicted almost all types of health services as well as drug use. In Group 1, but not in Group 2, use of the services of a medical specialist (non-psychiatrist) was also predicted by physical diseases, and by having a psychiatric disorder (Table\u00a05). In Group 2, but not in Group 1, two PMLP (asylum procedure and socio-economic living conditions) predicted visits to the nurse\/doctor in the center, the general practitioner, and\/or a medical specialist (Table\u00a06). In Group 2, but not in Group 1, number of days of disability in the last month and physical complaints predicted the use of analgetics (not in table).\nTable\u00a05Multivariate logistic regression of predisposing and need (incl. PMLP: post-migration living problems) variables related to service use of Iraqi asylum seeker, <6\u00a0months in the Netherlands (Group 1: N\u00a0=\u00a0143), 2000\u20132001ServiceNurse\/doctor centerGeneral practitionerMedical spec. (non-psychiatry)Mental health workerUse of any drugsORaCI (95%)ORaCI (95%)ORaCI (95%)ORaCI (95%)Predisposing factors\u00a0\u00a0\u00a0\u00a0Age (older age)1.061.01\u20131.10Need factors\u00a0\u00a0\u00a0\u00a0One or more psychiatric disorder1.931.18\u20133.16\u00a0\u00a0\u00a0\u00a0Overall quality of life2.491.05\u20133.163.161.29\u20137.75\u00a0\u00a0\u00a0\u00a0Perceived quality of general health0.250.11\u20130.560.550.31\u20130.970.410.18\u20130.960.290.12\u20130.74\u00a0\u00a0\u00a0\u00a0Disability daysb1.081.01\u20131.15\u00a0\u00a0\u00a0\u00a0Physical diseases2.651.33\u20135.282.711.33\u20135.54PLMPaOR: Odds ratios are adjustedbDisability days in last monthTable\u00a06Multivariate logistic regression of predisposing and need (incl. PMLP: post-migration living problems) variables related to service use of Iraqi asylum seeker, >2\u00a0years in the Netherlands (Group 2: N\u00a0=\u00a0151), 2000\u20132001ServiceNurse\/doctor centerGeneral practitionerMedical spec. (non-psychiatry)Mental health workerUse of any drugsORaCI (95%)ORaCI (95%)ORaCI (95%)ORaCI (95%)ORaCI (95%)Predisposing factors\u00a0\u00a0\u00a0\u00a0Age (older age)1.041.00\u20131.09Need factors\u00a0\u00a0\u00a0\u00a0Perceived quality of general health0.54034\u20130.870.430.25\u20130.720.540.31\u20130.960.410.18\u20130.880.580.37\u20130.93\u00a0\u00a0\u00a0\u00a0Disabilityb0.920.86\u20130.99\u00a0\u00a0\u00a0\u00a0Disability daysc1.071.01\u20131.13\u00a0\u00a0\u00a0\u00a0Physical diseases0.560.32\u20130.992.161.26\u20133.79PMLP\u00a0\u00a0\u00a0\u00a0Asylum procedure1.381.04\u20131.830.680.51\u20130.92\u00a0\u00a0\u00a0\u00a0Socio-economic l.c.1.191.02\u20131.381.231.00\u20131.50aOR: Odds ratio\u2019s are adjustedbDisability: total score of Brief Disability QuestionnairecDisability days in last month\nDiscussion\nThe main findings of this study are: There is a high overall service use among Iraqi asylum seekers in the Netherlands. The hypothesis that a long asylum procedure is associated with higher levels of service use, is not confirmed by the results, except for mental health service use and drug use. Psychopathology is related to a higher level of service use (second hypothesis), but when corrected for the influence of other predisposing and need factors, other factors, such as: high role and functional disability, and low perceived quality of general health, are more important predictors. Moreover having one or more psychiatric disorder(s) predicts the use of a medical specialist (non-psychiatrist), but does not predict mental health service use. The overall use of mental health service use is very low compared to the high prevalence of psychiatric disorder: over 80% of the asylum seekers with a psychiatric disorder used any health service, but only 8.8% visited a mental health service.\nNext paragraphs will discuss the four research questions.\nPrevalence of service use, relationship with length of stay\nThe preventive healthcare services are the most frequently visited services in both groups. As explained earlier (see \u201cMethods\u201d), in the Netherlands medical staff is present in all asylum seeker centers. Shortly after arrival, all asylum seekers are supposed to get a preventive medical screening by the doctor in the center doing triage for e.g. tuberculosis and AIDS. This probably explains the higher use of this service by Group 1. Gerritsen et al. [9] found that 63.7% of the asylum seekers from Iran, Afghanistan and Somalia reported a visit to the preventive healthcare services in the center. Their average length of stay was 3.4\u00a0years, comparable with Group 2. The rate of the Iraqi group in this study is much lower: 39.7%, suggesting a difference in use of this service between asylum seekers with different origins.\nIn contradiction to the hypothesis, the use of a general practitioner in Group 2 was not higher compared to Group 1. The use (30%) is even lower compared to the general Dutch population, which is 42%, and even more so compared to immigrants: 51% [27]. Also Van Oort et al. [24] found that asylum seekers visit the general practitioner less often than the general Dutch population: number of contacts per year 3.5 vs. 4.5. So, despite the higher prevalence of health problems, asylum seekers make less use of the general practitioner. Furthermore, use of a medical specialist is not higher in Group 2. This is explained by the fact that in the Dutch system a patient can only visit a medical specialist after referral by a general practitioner.\nDrug consumption is significantly higher in Group 2 (45.7%), compared to Group 1 (32.2%), confirming the hypothesis. Gerritsen et al. [9] found an even higher rate (57.8%). The high use of analgetics is striking (see later).\nUse of alternative services is very low. Maybe Iraqi asylum seekers are not interested in these services, but even in case they would be, these services are probably not easily available and accessible. We found no other studies on this issue among Iraqi refugees\/asylum seekers.\nRelationship psychopathology and service use\nThe findings indicate a huge unmet need for mental health care. About 30% of the asylum seekers with a psychiatric disorder did not visit any service, and more than 90% of the asylum seekers with a psychiatric disorder did not visit a mental health service (Table\u00a03). However 60.6% visited a nurse\/doctor in the center and 33.8% the general practitioner. Both services are important in the pathway to mental health care (see later).\nWe hypothesized that higher levels of psychopathology would be related with higher service use. In the univariate analyses this hypothesis stands, except for use of a general practitioner. Also, there are differences between the groups. The hypothesis is strongly confirmed when we consider drugs use. The high use of analgetics in Group 2 might be explained by the high levels of pain disorders (11.3%) and physical health complaints (66.2%) in this group. However other explanations are possible. Van Dijk et al. [23] did a qualitative study among 22 asylum seekers and concluded that it seems that \u201cthe prescription of paracetamol has become a symbol for the lack of interest of and the rejection by the health care system\u201d. Their study reports dissatisfaction with the services of the nurse\/doctor in the center, as well as with the services of the general practitioner.\nPredictors of service use\nAfter correcting the risks for all other included risk factors in multivariate analyses, low perceived quality of general health was the only significant predictor for mental health service use. Psychopathology, disability, nor physical complaints were significant predictors, while these factors were found to be important predictors in other studies (e.g. [8, 17]). Psychopathology, however, was a predictor for higher use of a medical specialist (non-psychiatrist). Our findings lead to the following hypothesis: (1) asylum seekers present themselves with physical rather than with mental problems (we know [14] that the level of physical complaints is high), (2) the staff in the center and the general practitioner do not recognize the mental health problems, and if they do (3) only a few patient are referred for adequate mental health care. This hypothesis is supported by the findings in the study of Van Oort et al. [24]. They found that in only 6% of the cases \u2018mental health problems\u2019 was recorded as reason of visiting the medical staff in the center, and only 2% of the referrals to the general practitioners were because of mental health reasons. The general practitioner diagnosed a mental health problem in 10% of the cases, and of those only 21.4% were referred to a mental health service (while 33.3% was referred to a medical specialist).\nThe mismatch between type of health problem and type of health service use seems to be less pronounced in Group 2: in the analyses per group (Tables\u00a05, 6) psychopathology did not predict the use of a medical specialist in this group.\nA curious finding is that overall quality of life has a positive relationship with the use of medical specialist. There might be a parallel with the phenomena that low social support is a risk factor for psychopathology [10], but high social support predicts health service use in some studies [15].\nSeveral post-migration living problems (PMLP) predict health service use in Group 2. Especially worries about socio-economic living conditions increase the need for healthcare. The findings do not support the idea that asylum seekers look for recognition of their health problems in order to get a resident permit: there is no relationship between PMLP and mental health service use and there is a negative relationship with use of a medical specialist.\nConclusion\nThis study has shown that Iraqi asylum seekers have a low level of mental health service use, despite the high levels of psychiatric disorders and other health indicators, especially within the group that stayed in the asylum procedure for over 2\u00a0years.\nMoreover, there is a mismatch between the type of health problem and the type of health service use: asylum seekers with a psychiatric disorder make more use of non-mental health service. There is room for improvement of the \u2018gate way\u2019 preventive services in detection and referrals of patients with mental health problems. The study results suggest that this service is a barrier rather than a facilitator in the pathway to mental health care. The general practitioner should be more involved and consulted. Mental health institutions are recommended to start and\/or improve consultation and (assertive) care programs.","keyphrases":["service use","iraq","asylum seekers","psychiatric disorders","drug consumption"],"prmu":["P","P","P","P","P"]}
{"id":"Ann_Hematol-4-1-2324130","title":"Immunoglobulin and free light chain abnormalities in Gaucher disease type I: data from an adult cohort of 63 patients and review of the literature\n","text":"Gaucher disease type I, the most common lysosomal storage disorder, is associated with immunoglobulin abnormalities. We studied the prevalence, risk factors, pathogenesis, and effect of enzyme relation therapy (ERT) on gammopathies in an adult Gaucher disease type I cohort (N = 63) and related the results to a review of the currently available literature. Polyclonal gammopathies and monoclonal gammopathy of undetermined significance (MGUS) in our adult GD I cohort were found in 41% and 19% of patients. These results are similar to the data from the literature and correspond to the increased risk of multiple myeloma (MM) that has been described. The prevalence of MGUS in our cohort increased with age but was not associated with disease severity or exposure time. The serum levels of free light chains of immunoglobulins were measured and were not found predictive for the development of MGUS or MM. Levels of pro- as well as anti-inflammatory cytokines, growth factors, and chemokines, especially those involved in inflammation and B-cell function, are disturbed in GD I, with the most impressive and consisting elevations for interleukin-10 and pulmonary and activation-regulated chemokine. A beneficial effect of ERT on the occurrence and progression of gammopathies was suggested from longitudinal data.\nIntroduction\nGaucher disease type I (GD I, OMIM #230800) is the most common lysosomal storage disorder. The disease is characterized by a deficiency of the lysosomal enzyme glucocerebrosidase (glucosylceramidase), which leads to the accumulation of glucocerebroside in macrophages. The lipid laden macrophages are called Gaucher cells and are mainly found in liver, spleen, and bone marrow, resulting in the most important clinical features hepatosplenomegaly, skeletal disease, and cytopenia [1, 2]. Since 1991, GD I can be effectively treated with recombinant glucocerebrosidase (enzyme replacement therapy, ERT, imiglucerase; Genzyme, Cambridge, MA, USA). More recently, substrate reduction therapy (miglustat, Actelion Pharmaceuticals, Basel, Switzerland) has been registered for more attenuated disease.\nAn associated feature of GD I is the high frequency of polyclonal and monoclonal gammopathies [3\u201322]. In the general population, polyclonal hypergammaglobulinemia is a common clinical finding, especially in the setting of chronic inflammation [23], resulting from an overproduction of immunoglobulins by plasma cells or B-lymphocytes. An abnormal increase of monoclonal immunoglobulins or fragments thereof (M protein) can be found in pre-malignant conditions such as MGUS (monoclonal gammopathy of undetermined significance) and malignancies of the B-cell lineage such as multiple myeloma (MM), primary amyloidosis, Waldenstr\u00f6m macroglobulinemia, chronic lymphocytic leukemia, and B-cell lymphoma. MGUS is found in approximately 3.2% of adults over the age of 50, increasing to 7.5% at the age of 85 [24]. Of these individuals, 1% per year shows progression to MM or a related malignancy. Accordingly, in GD I the risk of MM is strongly increased, as has been reported in recent studies [25\u201329]. The risk increases with advancing age [19, 26], but whether disease severity is a risk factor is currently unknown.\nThe mechanism explaining the relationship between GD and immunoglobulin abnormalities is far from understood. Studies have mainly focused on cytokines as the mediators between Gaucher cells, surrounding macrophages and B cells. Plasma levels of several pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-\u03b1, interleukin (IL)-1, and IL-6, as well as the anti-inflammatory IL-10, were found to be increased in GD to variable extents [4, 30\u201333]. IL-6 and IL-10, as well as hepatocyte growth factor (HGF), are also involved in the pathogenesis of MM [34]. Other interesting factors, known to be highly elevated in GD patients, are pulmonary and activation-regulated chemokine (PARC) [35], which is involved in initiating the adaptive immune response. Two studies have reported on the effect of ERT, with some decrease in polyclonal gammopathies [6] and a decline in monoclonal M-protein level in a single case [36].\nA relatively new method to identify and monitor monoclonal gammopathies is by quantifying immunoglobulin free light chains (FLC), circulating \u03ba and \u03bb chains that are not bound to the immunoglobulin heavy chains, using a highly sensitive automated immunoassay [37\u201339]. An abnormal FLC ratio is an important independent risk factor for progression of MGUS to MM or a related disorder [40, 41]. In addition, measuring FLC is a sensitive additional tool to identify monoclonal gammopathies [42]. In GD I, in which the incidence of MGUS and MM is highly increased [26], the FLC assay may provide a useful diagnostic tool for early detection of MGUS and MM.\nIn this study, we present new data on the prevalence of polyclonal and monoclonal gammopathies from a large adult GD I cohort and review the currently available literature. Second, we studied whether age, disease severity, and exposure time are risk factors for the development of monoclonal gammopathies, and if this risk is subsequently decreased by the administration of ERT. In addition, for the first time, we investigated the role of FLC as a predictor of the development of monoclonal gammopathies in GD I. Finally, the pathogenesis of immunoglobulin abnormalities in GD patients was studied by means of pro- and anti-inflammatory cytokine, chemokine, and growth factor levels and is discussed in relation to previous literature.\nMaterials and methods\nPatients and study design\nThe files were reviewed and archived serum samples were collected of all adult GD type I patients that were known from the outpatient clinic for inherited metabolic disorders at the Academic Medical Centre (AMC), Amsterdam, The Netherlands (N\u2009=\u200963). The diagnosis GD was based on measurement of deficient glucocerebrosidase activity in leukocytes and genotyping [43]. Of the 63 patients, 50 received ERT according to an individualized dosing protocol [44] and two patients received substrate reduction therapy. Data on age, sex, splenectomy, genotype, severity score index, (SSI [45]), creatinine, urea, chitotriosidase, and the presence and type of immunoglobulin class of monoclonal proteins were collected. In patients with a monoclonal gammopathy, exposure time was defined as the number of years between the diagnosis GD I and the diagnosis monoclonal gammopathy or start of ERT, whichever came first. In patients without a monoclonal gammopathy, exposure time was calculated as the number of years between the diagnosis GD I and start of therapy, or, in patients not receiving therapy, the first visit to our clinic.\nMGUS was defined as the presence of a monoclonal immunoglobulin component in the serum (as determined by protein electrophoresis in agar\/agarose gels and immunofixation) at a concentration of less than 30\u00a0g\/l (IgG, IgA, or IgM) and normal serum calcium and creatinine [46]. Although histology is required to distinguish osteolytic lesions caused by MM from those caused by GD, we decided to classify long-existing lesions as being GD related. Bone marrow examinations were only performed when a previously unknown M protein was discovered or in case of a sharp rise in the levels of immunoglobulins or FLC. In case of MM, stage was determined according to the Durie\u2013Salmon [47] and International Staging System (ISS) criteria [48].\nWe investigated whether levels of FLC could be predictive for the development of MGUS and whether these levels were related to disease severity. Therefore, we measured FLC levels in serum before start of enzyme replacement therapy in ten patients with mild GD (SSI\u2009\u2264\u20098) and ten patients with severe GD (SSI\u2009\u2265\u20099), all without a monoclonal gammopathy.\nIn addition, we studied whether changes in cytokine levels and in FLC and immunoglobulins at baseline and during ERT were related to the presence or absence of a monoclonal gammopathy. Therefore, the group of patients with a monoclonal gammopathy was matched for age, sex, spleen status, SSI, and the use of ERT to a control group of GD patients without a monoclonal gammopathy. Serial measurements of levels of IL-6, IL-10, and PARC at baseline and after 12, 24, and a median of 114\u00a0months of ERT (range 40\u2013143, whatever was the last determination) were performed. These were related to changes in immunoglobulins and free light chains. Hepatocyte growth factor (HGF) was measured only at baseline. One patient did not receive therapy; the first hospital visit was used as T0.\nAssays\nThe standard enzyme activity assay for chitotriosidase with 4-MU-chitotriose (Sigma, St. Louis, MO, USA) as a substrate was performed at pH 5.2 [49]. Serum \u03ba and \u03bb FLC were measured using FREELITE reagents (The Binding Site Ltd, Birmingham, England) on a BNII nephelometer (Dade-Behring, Deerfield, IL, USA) [37\u201339]. Monoclonal FLC were identified as values for \u03ba or \u03bb that exceeded the reference ranges (\u03ba 6.2\u201330.2\u00a0mg\/l, \u03bb 9.1\u201340\u00a0mg\/l) and produced an abnormal \u03ba\/\u03bb ratio (<0.3 or >1.57). Elevated concentrations of FLC without an abnormal \u03ba\/\u03bb ratio indicate a polyclonal or oligoclonal increase of FLC. Levels of IgG, IgA, and IgM were measured by immunoturbidimetric assay (Roche Tina-quant). IL-6, IL-10, HGF, and PARC were measured by specific enzyme-linked immunosorbent assays (ELISAs), according to the instructions of the manufacturer (IL-6 and IL-10: Sanquin, Amsterdam, The Netherlands; HGF: R&D Systems, Minneapolis, MN, USA; PARC: Biosource International, Camarillo, CA, USA).\nStatistics\nValues in patients are given as medians and ranges. Differences between patient groups were analyzed by the Mann\u2013Whitney U test or by \u03c7-square test. Linear mixed models were used to assess longitudinal changes. Correlations were tested by the rank-correlation test (Spearman coefficient, \u03c1). P\u2009<\u20090.05 was considered to represent a statistical difference.\nSearch strategy\nTo compare the prevalence of poly- and monoclonal gammopathies in our cohort with earlier case series and to study the relative risk of MM, we performed a MEDLINE search combining the MESH terms Gaucher disease with either hypergammaglobulinemia, paraproteinemias, and\/or multiple myeloma. Literature on cytokines, chemokines, and growth factors in relation to the development of immunoglobulin abnormalities in GD were searched combining the MESH terms Gaucher disease with cytokines and\/or chemokines. Case reports were excluded and only studies that included at least ten patients were selected.\nResults\nPatients\nThe 63 patients had a median age of 53\u00a0years (range 25\u201383). Thirty-two of patients (51%) were men, and 23 (37%) were splenectomized. At baseline, median SSI was 8 (range 3\u201319). The majority of patients had a genotype containing N370S (95%), with N370S\/L444P being the most common combination (40%). All patients had creatinine and urea values within the normal range.\nAt the first assessment, 24 (38%) patients had normal immunoglobulin levels, 26 (41%) patients had a polyclonal gammopathy, 12 (19%) patients had MGUS, and one patient had MM (Table\u00a01, #2). During follow-up (range 5\u201316\u00a0years), none of the patients with normal immunoglobulin levels or a polyclonal gammopathy developed a monoclonal gammopathy.\nTable\u00a01Characteristics of Gaucher disease type I patients with a monoclonal gammopathyPatient no.SexSxAge in 2007 or at deathTherapyMG typeIg type1MN55ERTMGUS, progression to amyloidosis and MMFree \u03ba2MY71ERTMMIgG\u03ba3FN46ERTMGUS, progression to amyloidosisIgG\u03bb4MY60ERTMGUSIgG\u03ba5MY67ERTMGUSIgG\u03bb6MY59ERTMGUSIgA\u03bb, IgG\u03ba7FN67ERTMGUSIgA\u03ba8MN56ERTMGUSIgG\u03bb9MY65ERTMGUSIgG\u03ba, IgG\u03bb, IgA\u03ba10MN56ERTMGUSIgM\u03ba, IgM\u03bb11FN63NoMGUSIgG\u03bb, IgM\u03ba12FY75ERTMGUSIgG\u03ba13MN51ERTMGUSIgG\u03bbM Male, F female, Sx splenectomy, ERT enzyme replacement therapy, MG monoclonal gammopathy, MM multiple myeloma, MGUS monoclonal gammopathy of undetermined significance, Ig immunoglobulin\nOf the 12 patients with MGUS, two developed MM and\/or amyloidosis after approximately 2\u00a0years of ERT (#1 and 3). Patient #1 was diagnosed with MM and amyloidosis 25\u00a0months after start of ERT. Pulse therapy with dexamethasone was started. Three months after the diagnosis the patient died from cardiac failure. At 24\u00a0months after start of ERT, patient #3 was diagnosed with amyloidosis. She received melphalan and prednisone, but died 12\u00a0months later from cardiac failure. Patient #2 was diagnosed with MM stage IA before start of ERT. During follow-up, IgG levels gradually increased. After 7\u00a0years of ERT, treatment with reduced dose melphalan and prednisone was started, which resulted in unacceptable cytopenia. Subsequently, thalidomide, 100\u00a0mg daily, was started with beneficial effect and stable disease parameters for the last 3\u00a0years.\nOf the ten remaining patients with non-progressive MGUS, six had a monoclonal, three had a biclonal, and one a triclonal gammopathy. The most common immunoglobulin type was IgG\u03ba (Table\u00a01; results previously presented in part in [26]).\nPatients with a monoclonal gammopathy were significantly older than patients without a monoclonal gammopathy (Table\u00a02). Exposure time to GD, defined as time from diagnosis until first assessment at our clinic, and severity of disease measures, assessed by SSI and chitotriosidase at baseline, were comparable.\nTable\u00a02Baseline characteristics of Gaucher type I patients with a monoclonal gammopathy vs patients without a monoclonal gammopathy\u00a0Without MGWith MGPNo. of patients5013\u00a0Age in 2007 or at death51 (25\u201383)60 (46\u201375)0.003No. of male patients23 (46%)9 (69%)NSYears of exposure11 (0\u201337)18 (1\u201344)NSNo. of splenectomies17 (34%)6 (46%)NSSSI at baseline7 (3\u201319)12 (4\u201316)NSChitotriosidase (nmol\/ml h) at baseline16,703 (5,409\u2013132,199)22,534 (6,417\u201362,122)NSData reflect absolute numbers (and percentage) or median (and range).MG Monoclonal gammopathy, NS not significant, SSI severity score index\nA search for studies on monoclonal and polyclonal gammopathies in GD I resulted in five series [4, 6, 16, 19, 21]. Polyclonal gammopathies were reported in 14\u201364% and monoclonal gammopathies in 1\u201335% of GD I patients (Table\u00a03).\nTable\u00a03Studies on the prevalence of monoclonal and polyclonal gammopathies in type I Gaucher diseaseReferenceNo. of Gaucher patientsAgePolyclonal gammopathiesMonoclonal gammopathiesde Fost et al. [26]6325\u20138326 (41%)12 (19%)Pratt et al. [19]169\u2013706 (38%)4 (25%)Shoenfeld et al. [21]2524\u20137815 (60%)2 (8%)Marti et al. [16]2341.8\u2009\u00b1\u20091810 (43%)8 (35%)Allen et al. [4]2223\u20136514 (64%)3 (14%)Brautbar et al. [6]50716\u20138114\u201325%5 (1%)Data reflect absolute numbers (and percentage), or, in the study of Marti et al., mean \u00b1 standard deviation.\nFive studies have described the prevalence and\/or relative risk of MM in GD patients [25\u201329]. Table\u00a04 summarizes the data from these studies. The frequency of MM in these cohorts was 0.4\u20134.0%, with significantly elevated relative risks of 5.9\u201351.1.\nTable\u00a04Studies on the prevalence and relative risk of multiple myeloma in type I Gaucher diseaseReferenceNo. of patientsEthnic backgroundAgeControl groupNo. of patients with MMRR of MMde Fost et al. [26]131Mixed50\u2009\u00b1\u200914Dutch Cancer Registry2 (1.5%)51.1 (95% CI: 6.2\u2013184)Lee [25]239MixedNot givenNone5 (2%)NDShiran et al. [28]48Jewish54\u2009\u00b1\u200920511 individuals from the same region2 (4%)NDZimran et al. [29]505Jewish38\u2009\u00b1\u200921Israeli Cancer Registry2 (0.4%)NDRosenbloom et al. [27]2,510Mixed33US Cancer Registry10 (0.4%)5.9 (95% CI: 2.8\u201310.8)Data on age reflect mean \u00b1 standard deviation; data on number of patients with multiple myeloma reflect absolute numbers (and percentage).MM Multiple myeloma, RR relative risk, 95% CI 95% confidence interval, ND not done\nImmunoglobulin and FLC levels\nIn 20 GD patients from the Dutch cohort without a monoclonal gammopathy (ten with mild disease, SSI\u2009\u2264\u20098, and ten with severe disease, SSI\u2009\u2265\u20099), FLC were measured before start of therapy (Fig.\u00a01a). One patient was found to have a slightly abnormal ratio, and six patients had an increase in one or both FLC, with a normal ratio. There were no significant differences in \u03ba or \u03bb FLC-levels between patients with severe- and patients with mild GD I. During follow-up (range 10\u201316\u00a0years), none of the patients developed a monoclonal gammopathy.\nFig.\u00a01FLC levels in Gaucher disease type I patients without a monoclonal gammopathy with mild disease (SSI\u2009\u2264\u20098) and severe disease (SSI\u2009\u2265\u20099) (a). FLC levels in Gaucher disease patients with a monoclonal gammopathy and matched Gaucher disease controls (b). MG Monoclonal gammopathy. The normal range for \u03ba was 6.2\u201330.2\u00a0mg\/l and for \u03bb was 9.1\u201340\u00a0mg\/l. The normal ratio for \u03ba\/\u03bb was 0.3\u20131.57\nFLC levels were measured in all GD I patients with a monoclonal gammopathy (Table\u00a01, #1\u201313) and matched GD I controls (#co1\u2013co13). No serum was available of patient #12, resulting in two groups of 12 patients. Baseline immunoglobulin levels were not available in one patient with a monoclonal gammopathy (#11) and five patients from the control group (#co4, #co5, #co7, #co11, and #co13). At baseline, the patients who already had or would develop MM and\/or amyloidosis (#1\u20133) had strongly abnormal FLC \u03ba\/\u03bb ratios (Fig.\u00a01b). Of the nine patients with MGUS, one (#7) had an abnormal FLC \u03ba\/\u03bb ratio and four showed elevated levels of FLC \u03ba or \u03bb, but with a normal FLC ratio. The remaining four patients with MGUS had both FLC levels as well as a FLC \u03ba\/\u03bb ratio within the normal range. Six of the patients from the control group had FLC levels and a FLC \u03ba\/\u03bb ratio within the normal range, and six had elevated levels of one or both chains of whom only one (#co6) patient showed an abnormal FLC \u03ba\/\u03bb ratio. During follow-up (range 6\u201315\u00a0years), none of the patients from the control group developed a monoclonal gammopathy.\nCytokines, chemokines, and growth factors\nAt baseline, IL-6 levels were within the normal range in all but four patients (Fig.\u00a02). There was no significant difference in IL-6 levels between patients with or without a monoclonal gammopathy (median (range) 5.9\u00a0pg\/ml (1.2\u2013118.4) and 2.2\u00a0pg\/ml (1.0\u201357.2), respectively). The majority of patients (17\/24) showed elevated IL-10 levels, especially in the group of patients with a monoclonal gammopathy, although not significantly different from the patients without a monoclonal gammopathy (17.1\u00a0pg\/ml (3.9\u2013419.8) and 6.9\u00a0pg\/ml (1.4\u2013299.3), respectively, P\u2009=\u20090.2). PARC levels were elevated in all GD patients, without a difference between patients with or without a monoclonal gammopathy (1,400.0\u00a0pg\/ml (348.9\u20132817.3) and 1,078\u00a0pg\/ml (411.4\u20133,552.0), respectively). HGF was within the normal range in most (16\/22) patients, without a difference between patients with or without a monoclonal gammopathy (1,751\u00a0pg\/ml (926\u20134,791) and 936\u00a0pg\/ml (424\u20139,382), respectively).\nFig.\u00a02Baseline levels of IL-6, IL-10, PARC, and HGF in Gaucher disease patients with a monoclonal gammopathy and matched Gaucher disease controls. Patients with multiple myeloma and\/or amyloidosis are marked by an arrow. Dotted lines reflect the upper limit of the normal range. IL Interleukin, HGF hepatocyte growth factor, PARC pulmonary and activation-regulated chemokine, MG monoclonal gammopathy\nA literature search resulted in seven studies on cytokines, chemokines, and growth factors in GD I patients (Table\u00a05). In one of these studies, mRNA levels were measured [32], while in the remaining studies, plasma levels were studied. If possible, the number of patients with cytokine levels exceeding the reference range was determined. In studies in which normal ranges were not provided [4, 32], we defined the normal range as mean \u00b1 2SD based on the values of the control group. This was not possible in the study of Barak et al. [30], in which no detailed data were provided.\nTable\u00a05Studies on plasma levels of cytokines, chemokines, and growth factors in Gaucher type I patientsReferenceNo. of GD I patientsAgeCytokinesResults, no. of patients (%) with elevated levelsP value for difference with mean values of the control groupde Fost et al. [26]2225\u201383IL-64\/24 (17%)NDIL-1017\/24 (71%)PARC24\/24 (100%)HGF6\/22 (27%)Michelakakis [33]25NGTNF-\u03b116\/25 (64%)NDAllen et al. [4]2223\u201365IL-1\u03b2Not detectableNSTNF-\u03b14\/11 (36%)NSIL-619\/22 (86%)P\u2009=\u20090.0001IL-1013\/13 (100%)P\u2009<\u20090.0001Lichtenstein et al. [32]18NGIL-1\u03b2 mRNA5\/19 (26%)P\u2009=\u20090.0337TNF-\u03b1 mRNA2\/19 (11%)NSIL-6 mRNA3\/19 (16%)NSIL-8 mRNA3\/19 (16%)NSHollak et al. [31]2916\u201366M-CSF24\/28 (86%)P\u2009<\u20090.001sCD1425\/27 (93%)P\u2009<\u20090.0005IL-825\/27 (93%)P\u2009<\u20090.0005IL-60\/27 (0%)NSTNF-\u03b10\/27 (0%)NSBarak et al. [30]215\u201339IL-1\u03b2Mean levels elevatedP\u2009=\u20090.01IL-1RAMean levels elevatedP\u2009=\u20090.01sIL-2RMean levels elevatedP\u2009<\u20090.001IL-6Mean levels elevatedP\u2009<\u20090.01IL-8Not elevatedNSTNF-\u03b13\/21 (14%)NSBoot et al. [35]5512\u201367PARC55\/55 (100%)P\u2009<\u20090.0001Altarescu et al. [56]1211\u201367TNF-\u03b11\/12 (8%)NSNG Not given, ND not done, NS not significant\nLongitudinal changes\nIn general, at no time point was there a statistically significant difference in plasma levels of any of the factors (i.e., IgG, IgA, IgM, FLC \u03ba and \u03bb, IL-6, IL-10, and PARC) between patients with and patients without a monoclonal gammopathy (Fig.\u00a03). In addition, using mixed model analysis, in neither of the two patient groups, a significant decrease or increase could be established in plasma levels of any of the measured factors. The only exception was PARC, which decreased in all patients, except for one patient who did not use therapy (#11). Changes in PARC levels could not be related to changes in levels of immunoglobulins or free light chains. There were no strong correlations (i.e., rho\u2009>\u20090.6) between any of the factors.\nFig.\u00a03Longitudinal changes in plasma levels of immunoglobulin heavy and free light chains, IL-6, IL-10, and PARC. Dotted lines reflect the normal range. Numbers reflect Gaucher disease patients with a monoclonal gammopathy as described in Table 1 (#1\u201313) and matched Gaucher disease controls (#co1\u2013co13) without a monoclonal gammopathy. IL Interleukin, PARC pulmonary and activation-regulated chemokine\nMore specifically, immunoglobulin levels remained stable or decreased in all patients, except for patient #11, who showed an increase in IgM. This patient was different from other MGUS patients because she did not receive therapy. Free light chain levels were more variable, but clearly FLC levels of patients who were to develop or already suffered from MM and\/or amyloidosis remained high during ERT (#1\u20133). Although in most patients no trends could be distinguished, a consistent decrease in free light chains and immunogobulins was seen for example in patient 7, who had an IgA\u03ba M protein. This patient responded very well to ERT, but other patients with excellent clinical responses did not always show a similar decrease in M-protein levels. No relationship was found between clinical response and decrease in either levels of immunoglobulins or FLCs.\nThe course of cytokines did not show a relationship with changes in either FLC or immunoglobulin levels, although in the untreated patient with increasing levels of IgM (#11), also an increase in IL-6 and PARC was noted. On the contrary, a spontaneous decrease in IL-6 was found in patient #2, without therapy for his MM. In patient #1, IL-10 showed a clear decrease after start of ERT but before the diagnosis amyloidosis and MM was made.\nDiscussion\nIn addition to the classical symptoms of GD I, such as hepatosplenomegaly, skeletal disease, and cytopenia, there is increasing evidence for existence of co-morbidities, including increased risk of cancer and abnormal immunoglobulin profiles. In fact, polyclonal gammopathies and MGUS occurred in 41% and 19%, respectively, of our adult Gaucher cohort, which is in line with findings from the literature (14\u201364% and 1\u201335% of patients [4, 6, 16, 19, 21]). In addition, we established that the relative number of biclonal (3\/10) and triclonal (1\/10) gammopathies within the MGUS patients was especially high considering that in general, biclonal gammopathies occur in 3% of subjects with MGUS [24], while triclonal gammopathies occur only very sporadically [50]. The types of immunoglobulin heavy and light chain immunoglobulins were comparable to the findings for MGUS in a non-GD population, with a predominance of IgG\u03ba [24]. As in the general population, the prevalence of MGUS in our cohort increased with age, but there was no relationship with disease severity measures, as assessed by exposure time, baseline SSI, and chitotriosidase levels. A relationship with age was first suggested by Pratt et al. [19], who investigated 16 patients aged 9 to 70\u00a0years and established an MGUS in four patients, all being over 50\u00a0years of age. Indirectly, he also showed an association with severity of disease, since all patients with splenomegaly had either a monoclonal or polyclonal gammopathy.\nConsidering the relatively high risk of monoclonal gammopathies in GD, the question was raised whether sensitive measuring of FLC would be useful for the early detection of monoclonal gammopathies in GD I patients. In a non-GD population, MGUS patients showed an abnormal FLC ratio in 44% of cases, which was an important risk factor for progression to MM or a related disorder [40, 41]. We found that the three GD patients who had or would soon develop a B-cell malignancy could easily be identified by their strongly abnormal FLC \u03ba\/\u03bb ratios. It was however, not possible to identify GD patients with MGUS on the basis of abnormal FLCs since 44% of GD patients with MGUS had both FLC levels as well as a FLC \u03ba\/\u03bb ratio within the normal range. A slightly abnormal ratio was not predictive for progression to MM or MGUS in the single patient without a monoclonal gammopathy, even after 15\u00a0years of follow-up. None of the patients had a decrease in renal function, implicating that any FLC increase was indeed due to increased production by plasma cells.\nMGUS patients are at risk for developing MM at a rate of 1% of patients per year. Accordingly, the prevalence of MM in GD I cohorts of 0.4\u20131.5% clearly exceeds the general prevalence of 0.02% (deduced from The Surveillance, Epidemiology, and End Results (SEER) Program of the National Cancer Institute (NCI)). The assumed diversity in relative risk of developing MM (5.9 and 51.1) can simply be explained by age differences in the cohorts: the highest number of MM patients was clearly established in the highest age groups.\nIn a recent consensus report on hematological manifestations of Gaucher disease, the higher incidence of multiple myeloma in Gaucher disease is acknowledged, and it is recommended that Gaucher patients should have their immunoglobulin profile determined at diagnosis and monitored every 2\u00a0years (patients <50\u00a0years) or every year (patients >50\u00a0years) [51].\nHypotheses on the pathogenesis of immunoglobulin disorders in GD have mainly focused on the accumulated glucocerebroside as the causative agent for chronic stimulation of macrophages surrounding Gaucher cells. Cytokines of macrophagic origin could in turn stimulate B cells, leading to a polyclonal gammopathy which may eventually transform, directly or via MGUS, into MM. Interestingly, plasma cells can be found in close contact with Gaucher cells, suggestive of local interaction (Fig.\u00a04).\nFig.\u00a04Bone marrow aspirate showing plasma cells surrounding a Gaucher cell in a patient with Gaucher disease type I and multiple myeloma\nIn our study, we measured IL-6, IL-10, and HGF, three factors important for differentiation and\/or proliferation of plasma cells. In addition, IL-6 and IL-10 are also involved in inflammation and in abnormal bone remodeling in MM, aspects that are also present in GD. We found elevated IL-10 levels in most patients, especially those with a monoclonal gammopathy, and predominantly normal levels of IL-6 and HGF. Previous studies have also described increased IL-10 levels [4]. However, findings on IL-6 in GD I have been variable [4, 30\u201332]. Inconsistent results have also been reported for the pro-inflammatory cytokines TNF-\u03b1 and IL-1\u03b2 and the chemokine IL-8 [4, 30\u201333, 52]. Although not included in this study, increases have been described for the growth factor M-CSF (monocyte\/macrophage-colony stimulating factor), the monocyte\/macrophage activation marker sCD14 (soluble CD14) [31], sIL-2R (soluble IL-2 receptor), and IL-1RA (IL-1 receptor antagonist) [30]. The most impressive increase, both in our study as well in as in the study by Boot et al. [35], has been found for PARC, a chemokine that is assumed to be involved in B-cell differentiation by recruiting T cells and CD-38 negative mantle zone B lymphocytes to antigen-presenting cells.\nIn addition to the in vivo studies, it has been described that murine macrophages that were stimulated in vitro by glucocerebroside release the pro-inflammatory lymphocyte-activating factor (LAF = IL-1) in a dose-responsive manner. This effect was not seen on incubation with galactocerebroside, sphingomyelin, and ceramidetrihexoside [53]. Possibly, only minor glycolipid accumulation is sufficient for the development of B-cell derangement. In fact, a glucocerebrosidase deficient mouse (L444P homozygote) showed evidence of B-cell proliferation, as well as elevated serum IgG levels, even though storage cells were not found [54].\nThe heterogeneity in plasma cytokine levels that have been found could be explained by differences in patient populations and cytokine assays. In addition, it is not known whether plasma levels adequately reflect tissue levels. Nevertheless, clearly both pro- as well as anti-inflammatory cytokine levels are disturbed in GD I. It is unlikely that Gaucher cells directly induce inflammation, since Gaucher cells have a phenotype resembling anti-inflammatory alternatively activated macrophages. More likely, pro-inflammatory cytokines are produced by surrounding macrophages, with Gaucher cells compensating for the inflammatory compounds [55].\nNow that it is generally accepted that ERT effectively reduces hepatosplenomegaly, cytopenia and skeletal disease, the focus should be shifted to new challenges, including the prevention and treatment of Gaucher associated co-morbidities. Novel data from our study suggest a beneficial effect of ERT on the occurrence and severity of gammopathies in GD patients. First, none of the patients developed MGUS during ERT. We did see progression of MGUS to amyloidosis and MM in two patients. However, since both patients developed these diseases relatively shortly after start of ERT, one could envision that irreversible changes leading to malignant transformation had already taken place, and could not be corrected by ERT. Second, we found that immunoglobulin levels either decreased or remained stable in both patients with and without a monoclonal gammopathy. The only patient with a strong increase in immunoglobulins (IgM) was a patient not using ERT. A clear influence of ERT on levels of FLC, IL-6, and IL-10 was not found in our study. A previous report also found a decrease in immunoglobulin levels during ERT, although only in patients with a polyclonal gammopathy, and not in patients with a monoclonal gammopathy [6]. Decreases in the levels of IL-10 [4], M-CSF, sCD1431, and PARC [35] have been described. Nevertheless, to draw firm conclusions on a possible positive effect of ERT on gammopathies in GD I, our presumptions should be confirmed in larger studies. This would require an international multicenter effort that should aim to establish whether indeed these long-term complications can be prevented by ERT and whether this would justify the early start of treatment.\nIn summary, there is a high prevalence in GD I of both polyclonal as well as monoclonal gammopathies, including MM. The risk of these diseases increases with age. Mechanisms causing gammopathies remain to be elucidated, but include the disturbance of cytokine levels involved in inflammation and B-cell function. ERT is likely to have a beneficial effect in preventing the occurrence and the progression of gammopathies.","keyphrases":["free light chains","gaucher disease","mgus","multiple myeloma","cytokines"],"prmu":["P","P","P","P","P"]}
{"id":"Cancer_Causes_Control-2-2-1705538","title":"Breast cancer risk factors in relation to breast density (United States)\n","text":"Objectives Evaluate known breast cancer risk factors in relation to breast density.\nIntroduction\nNumerous studies have shown that breast density, as assessed through mammography, is an important breast cancer risk factor [1\u20135]. Relative to the lowest classification of breast density (fatty tissue), women with the highest classification (extreme density) may have a 2- to 6-fold increased risk of breast cancer [5\u20138]. In addition to its role in breast cancer risk, breast density reduces mammographic accuracy [9\u201312], potentially increasing the risk of a later stage breast cancer diagnosis.\nPrevious studies indicate that established breast cancer risk factors, including family history of breast cancer, age at first birth, parity, and postmenopausal hormone use, have similar associations with breast density. In contrast, the influence of age at menarche, which in most studies is inversely related to breast cancer risk [e.g., 13\u201315], remains uncertain. Some studies have found positive associations [6, 16, 17], at least one suggests an inverse association [18], and others found no relation between age at menarche on breast density [19, 20].\nIn an effort to clarify inconsistent results from previous studies, we evaluated established breast cancer risk factors in relation to breast density in a large population of women enrolled in a statewide mammography registry. Our intention was to determine whether characteristics associated with breast cancer risk were also related to breast density, a finding that would be consistent with the notion that density mediates breast cancer risk. We were particularly interested in assessing the influence of menarcheal age.\nMethods\nThe New Hampshire Mammography Network (NHMN) registers all consenting women who undergo mammography at participating mammographic facilities in our state. Details of the registry have been described previously [21, 22]. For the present study, potentially eligible women were NH residents of ages 30\u201389, who had at least one mammogram registered in the NHMN from 1 May 1996 to 20 June 2002.\nThe epidemiological data used in this analysis arose from three sources: a self-administered questionnaire completed by the patient, a patient intake form administered face-to-face by the radiologic technologist, and a standardized clinical assessment form completed by the radiologist. The questionnaire collected height, weight, place of birth, ethnicity, marital status, education, insurance coverage, reason for the current visit, past history of clinical breast examinations and mammography, age at menarche, parity, and age at first birth. The questionnaire also queried women regarding the date of their last menstrual period and history of gynecological surgery. This information was used to classify women as premenopausal (still having periods naturally) or postmenopausal (periods had stopped permanently) either naturally, because of chemotherapy\/radiation, or surgery). The patient intake form obtained date of birth, family history of breast cancer (in the subject\u2019s mother, sister, daughter, or other relative), personal history of breast cancer, history of breast procedures, type of exam conducted at current visit, examination outcomes, recommendation for further work-up or follow-up, and current use of postmenopausal hormone therapy (HT). The clinical assessment form obtained the type of exam conducted at current visit, breast density, examination outcomes and recommendation for further work-up or follow-up. All three forms are completed during the woman\u2019s first NHMN mammography visit. Patient intake and clinical assessment forms are also completed at subsequent mammography visits, and the questionnaire is updated as possible.\nBreast density, the outcome variable, estimates the proportion of fibroglandular tissue in the breast, relative to fat. Breast density was recorded on the standardized clinical assessment form by interpreting radiologists using the American College of Radiology Breast Imaging Reporting and Data System\u00ae (BI-RADS\u00ae) classification (1\u00a0=\u00a0fatty, 2\u00a0=\u00a0scattered density, 3\u00a0=\u00a0heterogeneously dense, and 4\u00a0=\u00a0extremely dense) [23]. In the event of discordance in the density of the right and left breast, the woman was classified according to the higher density classification. Breast density readings were available for 162,933 (95.4%) of the 170,815 women who had at least one mammogram recorded in the registry.\nTo optimize temporal correspondence between the women\u2019s characteristics and the classification of breast density, the statistical analyses were, when possible, based on the woman\u2019s breast density on the date of the first recorded mammogram. When data for variables (other than HT use) were unavailable for the date of the mammogram, we searched forward in the NHMN records to retrieve replacement information corresponding to a subsequent mammography visit. Informative forward searches retrieved information from subsequent mammographic encounters occurring, on average, within 24\u00a0months of the index mammogram, and reduced missing values by 3\u20139%.\nCurrent body mass index (BMI; kg\/m2) was missing for 18,195 women, and the analyses were confined to 144,018 women for whom this measure was available. Included in the analytic sample were 131,480 (91%) women with a screening mammogram, 10,885 (8%) with a diagnostic mammogram, and 1,653 (1%) for whom the reason for the mammogram was not recorded. The majority of women, 136,283 (95%) had no personal history of breast cancer, 6,033 (4%) had a prior history of breast cancer based on NHMN records or the patient intake form, and 1,702 (1%) had unknown breast cancer status.\nWe used unconditional logistic regression analyses to generate odds ratios (OR) and 95% confidence intervals (CI) [24] for the association between factors and breast density, dichotomized as heterogeneously\/extremely dense (dense) or fatty\/scattered density (not dense). Statistical significance required a probability value of <0.05 (two-sided test). OR were computed using the cutpoints shown in the tables. Tests of trend and the corresponding OR were based on the categorical (age at menarche) or the continuous form of the variable (age, BMI, age at first birth, parity), in accordance with the method of data collection.\nBecause breast density was inversely associated with age (p for trend <0.0001) and current BMI (p for trend <0.0001), terms for these variables, using the continuous form, were included in all models. We found no evidence of confounding by the other variables shown in Table\u00a01 (fully adjusted OR were within 10% of those adjusted for age and BMI). Model building began with terms representing the main effects, and included interaction terms involving age, BMI, and menopausal status as suggested by visual inspection of the stratified analyses. The presence of statistical interactions was formally tested using likelihood ratio tests. The interaction term representing BMI was defined as BMI \u226530 (high BMI), versus <30 (low BMI). The final multivariable model, based on all women, contained terms for age and BMI in their continuous form, BMI (high, low), family history of breast cancer, age at menarche, age at first birth, parity, menopausal status, current use of HT, and terms for the interactions involving BMI (high, low) and age at menarche, age at first birth, and parity, a term for the interaction involving age and current HT use, and a term for the interaction between age at first birth and menopausal status. We repeated the analyses in parous women, in women with a screening mammogram, and in women who did not have a personal history of breast cancer.\nTable\u00a01Distribution of women\u2019s characteristics by breast density, dichotomized as dense versus not denseCharacteristics of study sample (n\u00a0=\u00a0144,018)Not denseDensen%n%Age (years)\u00a0\u00a0\u00a0\u00a030\u2013396,728810,01116\u00a0\u00a0\u00a0\u00a040\u20134924,9703129,95448\u00a0\u00a0\u00a0\u00a050\u20135921,5372713,59722\u00a0\u00a0\u00a0\u00a060\u20136915,160195,4339\u00a0\u00a0\u00a0\u00a070\u20137910,349133,0395\u00a0\u00a0\u00a0\u00a080\u2013892,47637741\u00a0\u00a0\u00a0\u00a0All women81,2205662,79844Education\u00a0\u00a0\u00a0\u00a0<High school6,88782,7034\u00a0\u00a0\u00a0\u00a0High school graduate27,4853417,29128\u00a0\u00a0\u00a0\u00a0College graduate35,2704331,11350\u00a0\u00a0\u00a0\u00a0Post graduate9,9711210,68117\u00a0\u00a0\u00a0\u00a0Missing1,60721,0102Marital status\u00a0\u00a0\u00a0\u00a0Not married25,2353116,90527\u00a0\u00a0\u00a0\u00a0Married54,1816744,59871\u00a0\u00a0\u00a0\u00a0Missing1,80421,2952Current BMI\u00a0\u00a0\u00a0\u00a0<202,64036,47910\u00a0\u00a0\u00a0\u00a020\u201322.499,8481216,57026\u00a0\u00a0\u00a0\u00a022.5\u201324.9915,4801916,06026\u00a0\u00a0\u00a0\u00a025\u201327.4914,266189,81216\u00a0\u00a0\u00a0\u00a027.5\u201329.9912,689166,35310\u00a0\u00a0\u00a0\u00a030\u201334.9915,197195,3268\u00a0\u00a0\u00a0\u00a035+11,100142,1984Family history breast cancer\u00a0\u00a0\u00a0\u00a0No55,5916841,23466\u00a0\u00a0\u00a0\u00a0Yes25,6293221,56434Age at menarche\u00a0\u00a0\u00a0\u00a0<116,13083,0945\u00a0\u00a0\u00a0\u00a01114,028179,32815\u00a0\u00a0\u00a0\u00a01220,9282615,83325\u00a0\u00a0\u00a0\u00a01322,0242718,28129\u00a0\u00a0\u00a0\u00a0149,430128,53414\u00a0\u00a0\u00a0\u00a015+7,41396,83011\u00a0\u00a0\u00a0\u00a0Missing1,26728981Age at first birtha\u00a0\u00a0\u00a0\u00a0<2015,537218,55415\u00a0\u00a0\u00a0\u00a020\u20132430,5744118,67434\u00a0\u00a0\u00a0\u00a025\u20132916,6942214,27626\u00a0\u00a0\u00a0\u00a030\u2013346,03886,70512\u00a0\u00a0\u00a0\u00a035+1,97232,4784\u00a0\u00a0\u00a0\u00a0Missing4,52665,0269Parity\u00a0\u00a0\u00a0\u00a005,87987,08511\u00a0\u00a0\u00a0\u00a017,02397,66512\u00a0\u00a0\u00a0\u00a0219,7912418,26229\u00a0\u00a0\u00a0\u00a0317,6522213,03221\u00a0\u00a0\u00a0\u00a0411,723146,90511\u00a0\u00a0\u00a0\u00a05+13,795175,8969\u00a0\u00a0\u00a0\u00a0Missing5,35773,9536Menopausal status\u00a0\u00a0\u00a0\u00a0Premenopausal29,1323637,55460\u00a0\u00a0\u00a0\u00a0Postmenopausal46,8745821,89535\u00a0\u00a0\u00a0\u00a0Missing5,21463,3495HT useb\u00a0\u00a0\u00a0\u00a0No32,8296312,86751\u00a0\u00a0\u00a0\u00a0Yes16,3383110,72242\u00a0\u00a0\u00a0\u00a0Missing2,92161,6557aAge at first birth in parous womenbA small proportion of women (5.5%) who reported whether they used HT did not give their menopausal status\nResults\nIn all women, scattered density (45%) was most frequently recorded, followed by heterogeneous density (34%), fatty breasts (12%), and extremely dense breasts (10%). The distribution of factors by breast density, classified as not dense versus dense, is shown in Table\u00a01. In general, based on the cutpoints shown, there was a tendency for younger women, those with higher education, and married women to have denser breasts. Women with lower BMI also had greater breast density, consistent with our use of BI-RADS categories, which assess the proportion of fibroglandular tissue (versus fat) in the breast. Women with a family history of breast cancer, later menarcheal age, later age at first birth, and low parity had a tendency toward higher breast density. High breast density was more common in premenopausal than in postmenopausal women, and in women currently using HT compared to nonusers.\nAge-stratified analyses, adjusted for age and BMI, showed a weak, positive influence of family history of breast cancer across all age groups assessed (Table\u00a02). A small positive association between age at menarche and breast density appeared to vary by age, with weaker effects in the older and youngest age groups. There were no clear age-related patterns for either the positive effect of age at first birth or the inverse effect of parity or menopausal status. Current HT use, compared to nonuse, was inversely associated with breast density in the younger age groups, and positively associated in women of age 50 or more. In women of age 70 or more, those using HT, compared to nonusers, had twice the odds of having dense breasts.\nTable\u00a02Odds ratios (OR) and 95% confidence intervals (CI) for the association with breast density according to age groupCharacteristicaAge group<4040\u20134950\u20135960\u20136970+n\u00a0=\u00a016,739n\u00a0=\u00a054,924n\u00a0=\u00a035,134n\u00a0=\u00a020,593n\u00a0=\u00a016,628OR (95% CI)OR (95% CI)OR (95% CI)OR (95% CI)OR (95% CI)Family history breast cancer\u00a0\u00a0\u00a0\u00a0NoReferenceReferenceReferenceReferenceReference\u00a0\u00a0\u00a0\u00a0Yes1.06 (0.99, 1.13)1.10 (1.06, 1.14) 1.07 (1.02, 1.13) 1.10 (1.03, 1.18) 1.11 (1.02, 1.20) Age at menarche\u00a0\u00a0\u00a0\u00a0Overall OR (95% CI)b1.04 (1.01, 1.06)1.06 (1.05, 1.08)1.03 (1.02, 1.05)1.01 (0.99, 1.03)1.01 (0.98, 1.03)\u00a0\u00a0\u00a0\u00a0p for trendb0.002<0.00010.00010.310.70\u00a0\u00a0\u00a0\u00a0<11ReferenceReferenceReferenceReferenceReference\u00a0\u00a0\u00a0\u00a0110.99 (0.85, 1.18)1.14 (1.04, 1.24)1.28 (1.15, 1.42)1.06 (0.90, 1.24)1.11 (0.89, 1.38)\u00a0\u00a0\u00a0\u00a0121.04 (0.89, 1.21)1.15 (1.06, 1.25)1.26 (1.15, 1.39)1.12 (0.96, 1.30)1.18 (0.96, 1.45)\u00a0\u00a0\u00a0\u00a0131.15 (0.98, 1.35)1.25 (1.16, 1.36)1.27 (1.15, 1.40)1.20 (1.03, 1.39)1.00 (0.82, 1.23)\u00a0\u00a0\u00a0\u00a0141.21 (1.02, 1.44)1.34 (1.22, 1.46)1.37 (1.23, 1.53)1.23 (1.05, 1.46)1.22 (0.99, 1.51)\u00a0\u00a0\u00a0\u00a015+1.29 (1.08, 1.55)1.45 (1.32, 1.60)1.36 (1.21, 1.52)1.05 (0.89, 1.25)1.15 (0.92, 1.43)Age at first birthc\u00a0\u00a0\u00a0\u00a0Overall OR (95% CI)d1.08 (1.06, 1.10)1.05 (1.04, 1.06)1.07 (1.06, 1.08)1.07 (1.05, 1.09)1.06 (1.05, 1.08)\u00a0\u00a0\u00a0\u00a0p for trendd<0.0001<0.0001<0.0001<0.0001<0.0001\u00a0\u00a0\u00a0\u00a0<20ReferenceReferenceReferenceReferenceReference\u00a0\u00a0\u00a0\u00a020\u2013241.06 (0.95, 1.19)1.14 (1.08, 1.21)1.21 (1.14, 1.29)1.23 (1.12, 1.34)1.07 (0.93, 1.24)\u00a0\u00a0\u00a0\u00a025\u2013291.09 (0.98, 1.22)1.19 (1.12, 1.26)1.40 (1.30, 1.51)1.51 (1.36, 1.68)1.28 (1.11, 1.49)\u00a0\u00a0\u00a0\u00a030\u2013341.35 (1.18, 1.53)1.23 (1.15, 1.36)1.58 (1.42, 1.76)1.64 (1.39, 1.95)1.42 (1.18, 1.70)\u00a0\u00a0\u00a0\u00a035+1.67 (1.30, 2.16)1.48 (1.35, 1.63)1.85 (1.58, 2.17)1.90 (1.44, 2.49)1.47 (1.14, 1.91)Parity\u00a0\u00a0\u00a0\u00a0Overall OR (95% CI)d0.86 (0.84, 0.88)0.91 (0.90, 0.93)0.90 (0.86, 0.91)0.89 (0.87, 0.90)0.87 (0.85, 0.89)\u00a0\u00a0\u00a0\u00a0p for trendd<0.0001<0.0001<0.0001<0.0001<0.0001\u00a0\u00a0\u00a0\u00a00ReferenceReferenceReferenceReferenceReference\u00a0\u00a0\u00a0\u00a010.74 (0.65, 0.86)0.90 (0.83, 0.87)0.87 (0.79, 0.97)0.87 (0.73, 1.05)0.88 (0.73, 1.05)\u00a0\u00a0\u00a0\u00a020.58 (0.52, 0.66)0.73 (0.69, 0.78)0.77 (0.71, 0.85)0.77 (0.67, 0.89)0.66 (0.57, 0.78)\u00a0\u00a0\u00a0\u00a030.48 (0.42, 0.55)0.69 (0.65, 0.74)0.67 (0.61, 0.73)0.65 (0.57, 0.76)0.59 (0.51, 0.69)\u00a0\u00a0\u00a0\u00a040.49 (0.42, 0.57)0.61 (0.57, 0.66)0.61 (0.55, 0.67)0.55 (0.48, 0.64)0.49 (0.42, 0.58)\u00a0\u00a0\u00a0\u00a05+0.38 (0.33, 0.45)0.60 (0.55, 0.66)0.52 (0.47, 0.58)0.49 (0.42, 0.56)0.39 (0.33, 0.46)Menopausal status\u00a0\u00a0\u00a0\u00a0PremenopausalReferenceReferenceReference\u00a0\u00a0\u00a0\u00a0Postmenopausal0.69 (0.60 , 0.79 )0.71 ( 0.68, 0.75)0.71 (0.67, 0.75)N\/AN\/AHT use\u00a0\u00a0\u00a0\u00a0NoReferenceReferenceReferenceReferenceReference\u00a0\u00a0\u00a0\u00a0Yes0.69 (0.57, 0.84)0.80 (0.75, 0.86)1.39 (1.32, 1.46)1.82 (1.70, 1.95)2.02 (1.84, 2.22)aAdjusted for age and current BMI as continuous variablesbBased on the cutpoints showncAmong parous womendBased on the continuous form of the variable\nAnalyses stratified on BMI suggested that the influence of some factors was less evident in women of BMI \u226530 (high BMI) than in those of BMI <30 (low BMI) (Table\u00a03). In particular, the positive influence of age at menarche and age at first birth, and the inverse influence of parity were least apparent in the highest BMI group. The data suggested an inverse effect of being postmenopausal, relative to premenopausal, which decreased consistently across the BMI groupings, but the change in OR from the lowest to the highest BMI group was slight. Although the influence of current HT use fluctuated somewhat over BMI groups, there were no obvious patterns.\nTable\u00a03Odds ratios (OR) and 95% confidence intervals (CI) for the association with breast density according to BMI groupCharacteristicaCurrent BMI <22.522.5\u201325.4925.5\u201327.4927.5\u201329.99\u226530n\u00a0=\u00a035,547n\u00a0=\u00a037,153n\u00a0=\u00a018,744n\u00a0=\u00a018,775n\u00a0=\u00a033,799OR (95% CI)OR (95% CI)OR (95% CI)OR (95% CI)OR (95% CI)Family history of breast cancer\u00a0\u00a0\u00a0\u00a0NoReferenceReferenceReferenceReferenceReference\u00a0\u00a0\u00a0\u00a0Yes1.09 (1.03, 1.14)1.03 (0.99, 1.08) 1.08 (1.01, 1.15) 1.15 (1.08, 1.23) 1.09 (1.03, 1.15) Age at menarche\u00a0\u00a0\u00a0\u00a0Overall OR (95% CI)b1.04 (1.03, 1.06)1.05 (1.03, 1.06)1.04 (1.02, 1.06)1.03 (1.01, 1.05)1.01 (0.99, 1.03)\u00a0\u00a0\u00a0\u00a0p for trendb<0.0010.0010.0010.0040.25\u00a0\u00a0\u00a0\u00a0<11ReferenceReferenceReferenceReferenceReference\u00a0\u00a0\u00a0\u00a0111.25 (1.09, 1.43)1.17 (1.05, 1.31)1.13 (0.98, 1.31)1.20 (1.05, 1.38)1.10 (0.99, 1.22)\u00a0\u00a0\u00a0\u00a0121.36 (1.20, 1.55)1.26 (1.13, 1.40)1.10 (0.96, 1.27)1.16 (1.01, 1.32)1.09 (0.98, 1.21)\u00a0\u00a0\u00a0\u00a0131.43 (1.26, 1.63)1.33 (1.20, 1.48)1.16 (1.01, 1.33)1.21 (1.06, 1.38)1.06 (0.94, 1.16)\u00a0\u00a0\u00a0\u00a0141.54 (1.34, 1.75)1.44 (1.28, 1.61)1.25 (1.08, 1.45)1.19 (1.02, 1.38)1.16 (1.02, 1.31)\u00a0\u00a0\u00a0\u00a015+1.52 (1.32, 1.75)1.43 (1.27, 1.61)1.26 (1.07, 1.48)1.33 (1.13, 1.56)1.06 (0.93, 1.21)Age at first birthc\u00a0\u00a0\u00a0\u00a0Overall OR (95% CI)d1.08 (1.07, 1.09)1.07 (1.06, 1.08)1.07 (1.06, 1.09)1.06 (1.04, 1.07)1.03 (1.02, 1.04)\u00a0\u00a0\u00a0\u00a0p for trendd<0.0001<0.0001<0.0001<0.0001<0.0001\u00a0\u00a0\u00a0\u00a0<20ReferenceReferenceReferenceReferenceReference\u00a0\u00a0\u00a0\u00a020\u2013241.21 (1.13, 1.31)1.20 (1.13, 1.28)1.15 (1.05, 1.26)1.13 (1.03, 1.23)1.04 (0.96, 1.11)\u00a0\u00a0\u00a0\u00a025\u2013291.39 (1.29, 1.51)1.34 (1.25, 1.44)1.21 (1.10, 1.34)1.31 (1.19, 1.45)1.10 (1.01, 1.19)\u00a0\u00a0\u00a0\u00a030\u2013341.45 (1.32, 1.60)1.55 (1.42, 1.70)1.40 (1.23, 1.59)1.42 (1.25, 1.63)1.11 (0.99, 1.24)\u00a0\u00a0\u00a0\u00a035+1.79 (1.56, 2.06)1.84 (1.61, 2.09)1.88 (1.56, 2.28)1.62 (1.33, 1.97)1.29 (1.08, 1.53)Parity\u00a0\u00a0\u00a0\u00a0Overall OR (95% CI)d0.86 (0.85, 0.87)0.88 (0.87, 0.89)0.90 (0.88, 0.92)0.91 (0.89, 0.93)0.93 (0.92, 0.95)\u00a0\u00a0\u00a0\u00a0p for trendd<0.0001<0.0001<0.0001<0.0001<0.0001\u00a0\u00a0\u00a0\u00a00ReferenceReferenceReferenceReferenceReference\u00a0\u00a0\u00a0\u00a010.75 (0.67, 0.84)0.80 (0.73, 0.89)0.93 (0.81, 1.08)1.13 (0.98, 1.30)0.95 (0.85, 1.07)\u00a0\u00a0\u00a0\u00a020.61 (0.55, 0.66)0.67 (0.62, 0.73)0.74 (0.66, 0.84)0.90 (0.79, 1.02)0.85 (0.78, 0.94)\u00a0\u00a0\u00a0\u00a030.52 (0.48, 0.58)0.58 (0.53, 0.63)0.68 (0.60, 0.77)0.80 (0.71, 0.91)0.81 (0.73, 0.89)\u00a0\u00a0\u00a0\u00a040.44 (0.39, 0.48)0.52 (0.47, 0.57)0.60 (0.52, 0.69)0.71 (0.62, 0.81)0.75 (0.68, 0.84)\u00a0\u00a0\u00a0\u00a05+0.37 (0.34, 0.41)0.44 (0.40, 0.48)0.52 (0.45, 0.60)0.64 (0.56, 0.74)0.66 (0.59, 0.74)Menopausal status\u00a0\u00a0\u00a0\u00a0PremenopausalReferenceReferenceReferenceReferenceReference\u00a0\u00a0\u00a0\u00a0Postmenopausal0.67 (0.63, 0.72)0.68 (0.64, 0.72)0.71 (0.65, 0.77)0.73 (0.67, 0.79)0.75 (0.70, 0.80)HT use\u00a0\u00a0\u00a0\u00a0NoReferenceReferenceReferenceReferenceReference\u00a0\u00a0\u00a0\u00a0Yes1.23 (1.15, 1.31)1.33 (1.26, 1.42)1.34 (1.23, 1.46)1.41 (1.30, 1.55)1.34 (1.25, 1.45)aAdjusted for age and current BMI as continuous variablesbBased on the cutpoints showncAmong parous womendBased on the continuous form of the variable\nAnalyses stratified by menopausal status revealed largely similar results for most variables, but the relationship between age at first birth and breast density was stronger in the postmenopausal women (Table\u00a04).\nTable\u00a04Odds ratios (OR) and 95% confidence intervals (CI) for the association with breast density according to menopausal statusCharacteristicaMenopausal status PremenopausalPostmenopausaln\u00a0=\u00a066,686n\u00a0=\u00a068,769OR (95% CI)OR (95% CI)Family history of breast cancer\u00a0\u00a0\u00a0\u00a0NoReferenceReference\u00a0\u00a0\u00a0\u00a0Yes1.09 (1.05, 1.13)1.08 (1.04, 1.12) Age at first birth\u00a0\u00a0\u00a0\u00a0Overall OR (95% CI) 1.05 (1.04, 1.06)1.03 (1.02, 1.04)\u00a0\u00a0\u00a0\u00a0p for trend<0.0001<0.0001\u00a0\u00a0\u00a0\u00a0<11ReferenceReference\u00a0\u00a0\u00a0\u00a0111.12 (1.03, 1.21)1.15 (1.06, 1.25)\u00a0\u00a0\u00a0\u00a0121.12 (1.04, 1.22)1.20 (1.12, 1.30)\u00a0\u00a0\u00a0\u00a0131.22 (1.13, 1.32)1.21 (1.12, 1.31)\u00a0\u00a0\u00a0\u00a0141.31 (1.21, 1.43)1.29 (1.18, 1.40)\u00a0\u00a0\u00a0\u00a0\u2265151.40 (1.28, 1.53)1.25 (1.15, 1.36)Age at first birthb\u00a0\u00a0\u00a0\u00a0Overall OR (95% CI)c1.05 (1.05, 1.06)1.07 (1.06, 1.08)\u00a0\u00a0\u00a0\u00a0p for trendc<0.0001<0.0001\u00a0\u00a0\u00a0\u00a0<20ReferenceReference\u00a0\u00a0\u00a0\u00a020\u2013241.07 (1.01, 1.13)1.21 (1.15, 1.27)\u00a0\u00a0\u00a0\u00a025\u2013291.11 (1.05, 1.17)1.41 (1.33, 1.49)\u00a0\u00a0\u00a0\u00a030\u2013341.21 (1.13, 1.29)1.57 (1.45, 1.71)\u00a0\u00a0\u00a0\u00a0\u2265351.48 (1.35, 1.62)1.77 (1.57, 2.00)Parity\u00a0\u00a0\u00a0\u00a0Overall OR (95% CI)c0.90 (0.87, 0.91)0.88 (0.88, 0.89)\u00a0\u00a0\u00a0\u00a0p for trendc<0.0001<0.0001\u00a0\u00a0\u00a0\u00a00ReferenceReference\u00a0\u00a0\u00a0\u00a010.87 (0.81, 0.93)0.86 (0.79, 0.93)\u00a0\u00a0\u00a0\u00a020.70 (0.66, 0.74)0.72 (0.68, 0.77)\u00a0\u00a0\u00a0\u00a030.63 (0.60, 0.68)0.62 (0.58, 0.67)\u00a0\u00a0\u00a0\u00a040.57 (0.53, 0.62)0.55 (0.51, 0.59)\u00a0\u00a0\u00a0\u00a0\u226550.52 (0.49, 0.57)0.46 (0.43, 0.50)HT use\u00a0\u00a0\u00a0\u00a0NoNAReference\u00a0\u00a0\u00a0\u00a0YesNA1.47 (1.41, 1.52)aAdjusted for age and current BMI as continuous variablesbAmong parous womencBased on the continuous form of the variable\nWe assessed risk factors and potential interactions in a multivariable model (Table\u00a05). Only one factor, family history of breast cancer, which showed a weak but significant positive effect (OR\u00a01.09; 95% CI\u00a01.05\u20131.14) on breast density, was not involved in an interaction. The possible interaction involving age and age at menarche, suggested by the age-stratified analyses, was not statistically significant (p\u00a0=\u00a00.10). A statistically significant interaction was found between current HT use and age (p\u00a0<\u00a00.0001). Interactions were also present between BMI and age at menarche (p\u00a0=\u00a00.04), age at first birth (p\u00a0<\u00a00.0001), and parity (p\u00a0=\u00a00.01). We also noted a significant interaction between menopausal status and age at first birth (p\u00a0=\u00a00.004), but the coefficients were inconsistent across categories of age at first birth. When the interaction between menopausal status and age at first birth was omitted from the multivariable model, results for the remaining terms were essentially unchanged. A possible interaction involving age, menopausal status, and HT was not significant (p\u00a0=\u00a00.10).\nTable\u00a05Betas (\u03b2) and standard errors (SE) for the association between factors and breast density in all women and in subgroupsaCharacteristicAll women n\u00a0=\u00a0144,018Women without breast cancer n\u00a0=\u00a0136,283\u03b2 (SE)\u03b2 (SE)Intercept4.82 (0.17)4.88 (0.17)Age\u22120.04 (0.001)\u22120.04 (0.001)Current BMI\u22120.12 (0.003)\u22120.12 (0.003)Family history of breast cancer0.09 (0.02)0.09 (0.02)BMI <30\u22120.53 (0.11)\u22120.53 (0.11)Postmenopausal\u22120.43 (0.09)\u22120.43 (0.09)HT use\u22121.22 (0.11)\u22121.25 (0.11)Age at menarche\u00a0\u00a0\u00a0\u00a015+\u22120.01 (0.10)\u22120.03 (0.11)\u00a0\u00a0\u00a0\u00a0140.04 (0.10)0.01 (0.10)\u00a0\u00a0\u00a0\u00a013\u22120.01 (0.08)\u22120.03 (0.08)\u00a0\u00a0\u00a0\u00a0120.08 (0.08)0.06 (0.08)\u00a0\u00a0\u00a0\u00a0110.04 (0.08)0.02 (0.08)BMI <30 * Age at menarche\u00a0\u00a0\u00a0\u00a015+0.30 (0.12)0.32 (0.12)\u00a0\u00a0\u00a0\u00a0140.24 (0.11)0.28 (0.11)\u00a0\u00a0\u00a0\u00a0130.22 (0.09)0.24 (0.10)\u00a0\u00a0\u00a0\u00a012 0.12 (0.09)0.15 (0.10)\u00a0\u00a0\u00a0\u00a0110.11 (0.10)0.14 (0.10)Age at first birth\u00a0\u00a0\u00a0\u00a0Nulliparous0.38 (0.16)0.13 (0.17)\u00a0\u00a0\u00a0\u00a035+0.19 (0.22)0.25 (0.23)\u00a0\u00a0\u00a0\u00a030\u201334\u22120.19 (0.15)\u22120.18 (0.15)\u00a0\u00a0\u00a0\u00a025\u201329\u22120.11 (0.12)\u22120.12 (0.12)\u00a0\u00a0\u00a0\u00a020\u2013240.14 (0.11)0.13 (0.11)BMI <30 * Age at first birth\u00a0\u00a0\u00a0\u00a0Nulliparous0.57 (0.10)0.59 (0.11)\u00a0\u00a0\u00a0\u00a035+0.41 (0.18)0.40 (0.18)\u00a0\u00a0\u00a0\u00a030\u2013340.31 (0.11)0.32 (0.12)\u00a0\u00a0\u00a0\u00a025\u2013290.12 (0.08)0.15 (0.08)\u00a0\u00a0\u00a0\u00a020\u2013240.06 (0.06)0.06 (0.06)Postmenopausal * Age at first birth\u00a0\u00a0\u00a0\u00a0Nulliparous0.09 (0.15)0.10 (0.15)\u00a0\u00a0\u00a0\u00a035+\u22120.08 (0.19)\u22120.14 (0.19)\u00a0\u00a0\u00a0\u00a030\u2013340.27 (0.13)0.26 (0.13)\u00a0\u00a0\u00a0\u00a025\u2013290.25 (0.11)0.23 (0.11)\u00a0\u00a0\u00a0\u00a020\u201324\u22120.05 (0.10)\u22120.04 (0.11)Paritya\u00a0\u00a0\u00a0\u00a010.29 (0.09)0.28 (0.09)\u00a0\u00a0\u00a0\u00a020.22 (0.07)0.23 (0.07)\u00a0\u00a0\u00a0\u00a030.14 (0.07)0.17 (0.07)\u00a0\u00a0\u00a0\u00a040.09 (0.07)0.11 (0.08)BMI <30 * Parityb\u00a0\u00a0\u00a0\u00a010.30 (0.10)0.30 (0.10)\u00a0\u00a0\u00a0\u00a020.22 (0.07)0.20 (0.08)\u00a0\u00a0\u00a0\u00a030.18 (0.08)0.15 (0.08)\u00a0\u00a0\u00a0\u00a040.09 (0.08)0.07 (0.09)Age * HT use0.03 (0.002)0.03 (0.002)aBased on a multivariable model containing all terms shown in the tablebThe nulliparous parameter and its interaction with BMI were set to 0, since these variables were linear combinations of other variables\nWe repeated the analyses in the subgroup of women who did not have a personal history of breast cancer, and found similar results (Table\u00a05). The findings were also comparable when the analyses were confined to parous women, or to those with a screening mammogram (data not shown).\nDiscussion\nEvidence accumulating for nearly 30\u00a0years supports the association between breast density and breast cancer [1\u20135]. Although the notion remains controversial, breast density may be a biomarker of risk [25]. In addition to its influence on breast cancer risk, breast density reduces the accuracy of screening mammography [11, 12, 26, 27], particularly in younger women [9] who tend to have denser breasts [28]. Perhaps as a direct consequence of reduced screening accuracy, breast density is associated with increased risk of interval breast cancers [11], with an adverse impact on breast cancer prognosis.\nMost studies of breast cancer risk have shown an inverse effect of age at menarche [13\u201315, 29], but previous studies of the relationship between age at menarche and breast density have produced inconsistent results. A positive association was seen in two studies [16, 18], including a large HMO population of nearly 30,000 women in Seattle [16]. Studies of breast cancer family members [17], Singaporean women [20], and Hispanic women [19] found no association between menarcheal age and breast density. Findings from the HMO-based study suggested the positive effect of age at menarche was stronger in the youngest and oldest age groups [16], whereas a study of nearly 5,000 women in Guernsey found significant positive effects only in postmenopausal women [6]. In contrast, our age-stratified analyses suggested weaker effects in the oldest age groups, although the interaction involving age and age at menarche was not statistically significant. Also in this study, analyses stratified on BMI suggested that age at menarche was positively associated with breast density in most BMI groups, but the association was tenuous in women with high BMI, and the interaction involving age at menarche and high BMI was statistically significant. A positive association between age at menarche and breast density, even if confined to women with lower BMI, seems paradoxical, given the usual inverse association between age at menarche and breast cancer and the strong positive association between breast density and breast cancer risk.\nConsistent with some [16\u201318, 20, 30, 31], but not all [6, 19] previous efforts, our findings show that age at first birth and parity are generally associated with breast density in a pattern resembling known associations with breast cancer risk. However, our stratified analyses indicate that the influence of the reproductive variables is less pronounced in women with high BMI, and our modeling results confirmed interactions involving BMI and both variables. These findings, along with our findings for age at menarche, are consistent with the possibility that hormonal or reproductive events are less influential in heavier women, whose circulating hormone levels may be influenced by conversion in peripheral adipose tissue. We also noted a stronger positive influence of age at first birth in postmenopausal women, although a previous study of Native American women found stronger effects in premenopausal women [30]. Our sample was large, and the multivariable results were inconsistent across categories of age at first birth; thus, it is possible the interaction between menopausal status and age at first birth was due to statistical artifact rather than true effect modification.\nOnly one variable, family history of breast cancer, was not involved in interactions with age, BMI, or menopausal status. Although previous studies have not shown an effect of family history on breast density [3, 6, 19], this is likely due to limited power to detect a weak association. The modest inverse effect of menopausal status has been noted previously [19, 20, 30]. Most previous reports, although not all [20], found a positive association between use of HT and breast density [16, 17, 19, 30, 32, 33], resembling the well-known association between use of these hormones and breast cancer risk. The large size of our study allowed an assessment of HT use in young postmenopausal women, and these analyses showed a modest but significant inverse effect in postmenopausal women less than 50\u00a0years of age, a phenomenon that has no clear explanation. In women of age 50 or more, the positive effect of HT on density increased with age, perhaps reflecting a corresponding decrease in density in untreated women in the same age group. Consistent with our findings, at least two previous studies of breast density showed an increasing effect of HT use when examined over increasing age groups [16, 32]. A prospective study of breast cancer risk also noted stronger HT effects in older women [37], although this is not always seen [36]. The age-related increase observed in our study could potentially reflect a longer duration of HT use, but at least two studies have shown that most of the increase in breast density occurs soon after HT initiation [33, 34], and duration of use was not associated with increased breast density in the HMO study [16]. In contrast, the positive influence of HT on breast cancer risk is usually observed for current\/recent and long-term use [35\u201340]. While speculative, it is possible that sustained breast density associated with long term HT use mediates the relationship between HT and breast cancer risk. Finally, our data did not indicate a stronger effect of HT on breast density in leaner woman, but a few studies [38\u201340], including a collaborative analysis of 51 studies [38], suggested a stronger association between HT and breast cancer risk in leaner women.\nAlthough the type of HT used (estrogen alone or estrogen combined with progesterone) was not assessed in our study, a possible role of progesterone is suggested by reports that breast density is greater during the luteal phase of the menstrual cycle [41\u201343]. In addition, at least two studies have found substantially greater changes in parenchymal patterns in women initiating use of a combined estrogen plus progesterone hormone regimen, as opposed to single agent estrogen [33, 34]. Results from the Women\u2019s Health Initiative randomized clinical trials of postmenopausal hormones also indicate that the increased risk of breast cancer is due to the combined regimen [44] rather than single agent estrogen [45].\nStrengths of our study include the large size of our sample, allowing analyses stratified by relatively refined age and BMI groups, which has not been possible in most previous studies, and good representation of the underlying population. Epidemiologic data were obtained on the time of the mammographic visit, ensuring updated information, and importantly, a high level of correspondence between use of hormone replacement therapy and the assessment of breast density. Limitations of our study include 11% of women for whom BMI is missing, reliance of self-report for BMI, and a lack of information regarding the type and duration of HT use. Our definition of family history of breast cancer included second degree relatives, which may have attenuated the effect of this variable. Also, the BI-RADS scores are qualitative, as opposed to digital quantification of density, and were applied by community radiologists, who despite being trained to use this system, may apply it differently. Nevertheless, our general findings in terms of the direction of effects for reproductive factors and HT use were compatible with those of most previous studies.\nIn conclusion, our results, based on the largest study to date, confirm earlier findings that most established breast cancer risk factors behave similarly in relation to breast density, consistent with the notion that breast density mediates breast cancer risk. However, our data indicated an inverse effect of HT in younger women, and a positive influence in older women, which has not been reported previously. We also noted effect modification by BMI, in which the positive effects of age at menarche and age at first birth, and the inverse effects of parity were less apparent in heavier women. Further investigation, including biological studies, may elucidate the complex interrelationships of hormones, BMI, breast density, and breast cancer and potentially offer opportunities for breast cancer prevention.","keyphrases":["hormone replacement therapy","reproductive history","mammographic breast density"],"prmu":["P","R","R"]}
{"id":"Graefes_Arch_Clin_Exp_Ophthalmol-4-1-2367393","title":"Posterior capsulorhexis combined with optic buttonholing: an alternative to standard in-the-bag implantation of sharp-edged intraocular lenses? A critical analysis of 1000 consecutive cases\n","text":"Background Current after-cataract prevention relies on optimizing the natural barrier effect of the optic rim against lens epithelial cell (LEC) migration. However, deficiencies in circumferential capsular bag closure caused by the intraocular lens (IOL) haptic or delayed secondary re-division of the fused capsules by Soemmering\u00b4s ring formation lead to primary or secondary barrier failure. Consequently, surprisingly high posterior laser capsulotomy rates have been reported long-term, even with optimal capsular surgery and the most widespread hydrophobic acrylic IOLs, considered to be the most advanced. Intraoperative removal of the central posterior capsule has been shown to be effective in further reducing LEC immigration. However, efficacy has turned out to be limited because of the propensity of LECs to use the posterior optic surface as an alternative scaffold.\nBackground\nThe current concept of after-cataract prevention (\u201cposterior capsule opacification\u201d, or \u201cPCO\u201d) is based on fully exploiting the natural barrier effect of the optic rim against lens epithelial cell (LEC) migration. This barrier effect is inherent to any optic rim, even when rounded. However, it can be optimized by the following measures: (1) by providing a circumferential overlap of the optic by the anterior capsular leaf, (2) by sharpening the posterior optic edge, and (3) by enhancing the collagenous sealing of both capsular leaves along the optic rim [10]. The latter is achieved by using fibrosis-inducing optic materials, and by preserving the integrity of the LEC layer as the substrate of fibrosis. The importance of fibrotic sealing is mirrored by two facts: (1) intraocular lenses (IOLs) made of materials with a lower fibrogenetic potential, such as acrylics, undergo a sharp increase in after-cataract score after 3\u20135\u00a0years [27], while with those made from materials with a higher fibrogenetic potential, such as silicones, it remains fairly constant, and (2) LEC abrasion by anterior capsule polishing significantly increases regeneratory after-cataract formation and the need for Nd: YAG laser capsulotomy (YAG-LCT) [11]. Implementation of all three above-mentioned parameters into modern cataract surgery has led to a drastical reduction of after-cataract and YAG-LCT rates.\nHowever, utilization of the edge barrier effect has two major shortcomings: (1) overlap of the optic by the anterior capsule leaf induces fibrotic whitening and shrinkage of the latter, thereby reducing the size of the free optic zone (this is especially true if the rhexis opening is small or decentered, be it because of inadequate dimensioning or due to subsequent contraction), and (2) the barrier function can be lacking from the very beginning, or wear off over the years, even when all the three above-mentioned requirements are fully met.\nThe barrier cannot build up when fusion of both capsular leaves and consecutive bending of the posterior capsule at the posterior optic edge is not initiated. Such \u201cprimary barrier failure\u201d can be limited to sectors or comprise the whole circumference of the optic. By nature, capsular fusion is impeded at the haptic-optic junction. While with looped haptics the barrier at the junction may remain fully preserved, broad-based haptics, including those used with the currently popular one-piece IOLs, inevitably disrupt the barrier at the optic insertion [24]. As a result, a gateway is opened up for LECs migrating from the germinative equatorial zone inwards. Deficiencies of capsular bag fusion may also arise independent of the haptic junction. When the reason is not evident, these have been termed \u201cidiopathic primary barrier failure\u201d. Most often, however, it is caused by ovalization of the capsular bag by overly long and rigid haptics, which impedes capsular fusion along the longitudinal IOL axis.\nEven when primary capsular fusion has been complete, the barrier may eventually fail. As Soemmering\u00b4s ring formation sets in, the two capsular leaves are increasingly redivided from the capsular equator inwards. If collagenous sealing of the capsule leaves along the optic rim is insufficient, these are increasingly forced apart, and the bend at the posterior optic edge is finally annihilated. Dormant LECs may thus become reactivated [15] and access the retrolental space. This phenomenon has been termed \u201csecondary barrier failure\u201d.\nThese barrier failures are the reason why in the long run YAG-LCT is still required in a considerable proportion of cases, even with optimized IOL designs and appropriate surgery. For the Acrysof 3-piece IOL, the reported YAG-LCT rate at 5\u00a0years ranges between 16% [2] and 27% [own data in publication]. After 10\u00a0years, a cumulative YAG-LCT rate of 42% was found (L. Vock, R. Menapace R. Long-term YAG laser capsulotomy and after-cataract rates with the sharp edge Acrysof and round edge PhacoFlex intraocular lenses: 10\u00a0year results. Abstract XXVth Congress of the ESCRS, 8.\u221212.9.2007, Stockholm). Thereby it must be kept in mind that the YAG-LCT rate only reflects those cases where central pearl formation is significant enough to interfere with vision. When also considering those eyes where LEC invasion remains limited to the periphery of the retrooptical capsule, the rate of barrier failure is significantly higher.\nTherefore, the potential for avoiding after-cataract formation by optimizing the edge barrier effect is limited, even when all relevant parameters are fully exploited. In addition, even in those cases where the sharp edge forms a permanent circumferential barrier, visibility of the peripheral retina may be significantly reduced by pearl formation in the intercapsular space outside of the optic rim.\nThe sharp edge as such also has the severe disadvantage of immanently causing dysphotopsia. Negative dysphotopsia is especially annoying for the patient. In the consultation section of the April 2005 issue of the Journal of Cataract and Refractive Surgery, dysphotopsia has been classified the \u201cproblem number one of modern cataract surgery\u201d [16]. Interestingly, this problem is not limited to acrylic IOLs with double square-edged (truncated) optics, but is also experienced with frosted edges, and even with rounded anterior edges. Also, it has been found with both acrylics and silicones [3].\nRationale\nThe still significant percentage of after-cataract formation requiring YAG-LCT in the longer run, and the problem of dysphotopsia, have fostered the search for better alternatives. Thereby, a new approach was required, since the concept of optimizing the migrational barrier effect of the optic edge is largely exploited.\nAs one option, intraoperative removal of the central posterior capsule by performing a primary posterior continuous curvilinear capsulorhexis (PPCCC) seemed promising, since the posterior capsule serves as a scaffold for centrally migrating LECs. As such, it is not an alternative, but much more supplements the current sharp edge concept by providing a \u201csecond barrier of defence\u201d when the optic edge barrier is overcome by migrating LECs.\nHowever, even though it has been shown to be safe [26, 4], the efficacy of adding a PPCCC is also limited. Firstly, the residual peripheral capsule can still be invaded and opacified by LECs which reduces the free optic zone. Secondly, similar to the anterior LEC ongrowth inside the ACCC rim often observed with acrylics [1], equatorial LECs can also grow over the PPCCC rim unto the free posterior optic surface, as they may utilize the latter as an alternative scaffold. Incidence and severity vary depending upon surface tension, wetability, and contact angle of the optic material, as well as the individual biological responsiveness, and is more pronounced in eyes with a defective blood\u2013aqueous barrier [25]. We performed a randomized prospective intraindividual comparison study including 29 patients with bilateral PPCCC, whereby one eye received a silicone, the other a hydrophilic acrylic IOL. Since round-edged optic IOLs were used at that time, this made the full impact of a PPCCC more obvious. After 2\u00a0years, partial reclosure of the PPCCC opening due to peripheral LEC ongrowth was found in 55% of the acrylic, and in 28% of the silicone IOL eyes. Total reclosure of the PCCC was observed in two eyes of the acrylic and in one eye of the silicone IOL group [5] (Fig.\u00a01). When evaluated with an objective grading system, after-cataract formation within the PPCCC opening exceeded 2 and 3 out of 10 possible grades for the silicone and acrylic IOL respectively.\nFig.\u00a01Primary posterior capsulorhexis decreases YAG laser capsulotomy rate by removing scaffold for LEC migration; however, residual capsule may still opacify, and LECs may alternatively grow upon optic surface\nThus, a PPCCC constitutes a useful additive surgical measure, but does not fully prevent after-cataract formation.\nOne promising alternative concept is combining a PPCCC with posterior buttoning-in of the optic (\u201cposterior optic buttonholing\u201d, or POBH), a technique which has been reserved for pediatric cataract surgery [6]. By doing so, the posterior capsule ends up lying on top of the optic. Instead of hindering the LECs from migrating, these are diverted towards the anterior optic surface, thus bypassing the optic edge. By doing so, migrating LECs, by principle, can no longer access the retrolental space.\nIn order to elucidate the suitability of the posterior optic buttonholing (POBH) technique as a routine procedure for senile cataracts, an extensive prospective study comprising a large number of eyes was initiated. In several substudies, a prospective randomized bilateral design was used to elucidate the risk profile compared to the standard in-the-bag implantation of IOLs in detail.\nFor this purpose, the surgical technique of POBH was revised and optimized to be then systematically investigated. From September 2004 to June 2007, over 1000 eyes underwent this POBH procedure. Patients were randomly taken from the waiting list. All prospective comparison studies were approved by the Medical University of Vienna Ethics Committee.\nTechnique\nThe surgical technique comprises an additional step of sophisticated, but perfectly controlled capsular and viscosurgery. After performing a standard 5\u20136\u00a0mm anterior continuous curvilinear capsulorhexis (ACCC), and after removing the lens contents with phacoemulsification and meticulously cleaning the capsular bag, the posterior capsule is flattened and the capsular fornix collapsed by pushing the iris and anterior capsule backwards with a low-viscosity cohesive ocular viscoelastic device (OVD, preferably Healon\u00ae). Thereby, the pupil is additionally widened. Due to this pupil-dilating effect, all eyes with a pupil size larger than 4\u00a0mm under full pharmacological dilatation were amenable to the procedure. After incising the center of the posterior capsule with a 30-gauge hypodermic needle (Sterican\u00ae manufactured by B. Braun, Melsungen, or Fine-Ject\u00ae by Henke-Sass-Wolf, Tuttlingen, Germany), a small amount of OVD is injected through the capsular opening. Then, the edge of the incised capsule is taken up by Utrata forceps and the incision extended peripherally to create a well-centered 4\u20135\u00a0mm PPCCC opening. When after the first pass one quadrant of the PPCCC has been completed, OVD is again injected in order to separate the underlying anterior hyaloid surface from the posterior capsule. After completion of the PPCCC and removal of the flap, OVD is again injected, this time in order to ensure full circumferential separation of the anterior hyaloid membrane from the residual posterior capsule up to the very periphery. Adequate viscoseparation of capsule and hyaloid is considered crucial to allow safe buttoning-in of the optic, and will be addressed in more detail in the Discussion Section below. Then, an open-loop IOL is implanted through an injector and fixated in the capsular bag fornix. When both loops are placed in the bag, the optic is buttoned-in into the PPCCC opening by gently pressing it down. As the elastic posterior capsule thereby wraps around the optic periphery in between the haptic junctions, the distended capsule and the optic form a mechanically stable and watertight diaphragm (Figs.\u00a02 and 3). Finally, the OVD is aspirated from the anterior chamber, the globe is tonisized, and the paracenteses are hydrated. The cataract incision is left unsutured. The surgery is performed under topical plus optional intracameral anesthesia (Lidocaine 4% plus Lidocaine unpreserved 1%) to ensure bright and stable retroillumination during the PPCCC manoeuvre. The HOYA AF-1 with a 6-mm optic is preferably used due to its particular haptic design (continuous optic-haptic transition: Fig.\u00a04; \u201chopper loop\u201d). Should the PPCCC result to be larger than the optic, as may occur in eyes with lax zonules or a large capsular bag, a 6.5-mm optic IOL should be resorted to, in order to ensure sealing of the diaphragm by sufficient circumferential capsular overlap. Additional implantation of a capsular tension ring facilitates estimation and performance of an appropriately sized and centered PPCCC in these eyes [9].\nFig.\u00a02Schematic of interrelationship between capsule leaves and IOL. Loops residing in capsular fornix, optic buttoned-in in PPCCC. Posterior capsule lying on top of optic (crescent-shaped blue areas); this precludes optic contact and thus fibrosis of the anterior capsule (red lines: rim of ACCC). If the ACCC is smaller than the optic (bold line), anterior capsule fibrosis limited to area adjacent to the haptic junction (red area); if larger than the optic (hatched line), no fibrosis also in this areaFig.\u00a03Retroillumination photography of buttoned-in IOLs (HOYA AF-1): note perfect autocentration of optic and run of posterior capsule rimFig.\u00a04HOYA AF-1-type IOL preferred because of continuous haptic\u2013optic junction and \u201chopper loop\u201d haptic. POBH causes optic to position (by approximately 1\u00a0mm) more posteriorly (compared to standard in-the-bag placement), thereby stabilizing the vitreous body\nApproximately 150 eyes underwent additional anterior capsule polishing with a special LEC aspiration curette [13] (Fig.\u00a05), the fibrosis-preventing efficacy of which had been substantiated in an earlier clinical study [19]. In this subgroup of eyes, the impact and potential advantages of this additional treatment was evaluated. Polishing was performed after completion of cortex aspiration and meticulous peeling of any residual lens fibers adhering to the peripheral posterior capsule.\nFig.\u00a05\u201cAspiration curette\u201d for effective and safe debridement of anterior LEC layer additionally reduces capsular fibrosis\nResults\nSurgical complications In a preliminary evaluation of the first 376 consecutive cases performed during the first year (Menapace R. Routine posterior optic buttonholing for after-cataract prevention: The better alternative to the sharp edge optic? Breaking News Session, XXIIIth Congress of the ESCRS, 10.\u221214.9.2005, Lisbon) no case of cystoid macular edema (CME), and only one case of retinal detachment (RD) were encountered. Since the latter occurred only 4\u00a0months after surgery in a highly myopic eye with an intact anterior hyaloid surface, the causative role of POBH is questionable. The RD emerged from a group of small round holes in the superotemporal quadrant, and was repaired by cryopexy and gas injection. In only three cases could buttoning-in not be performed as planned because of a too small or decentered PPCCC. However, even with a suboptimally centered PPCCC the optic always perfectly centered permanently. During surgery, there was no case of vitreous prolapse into the anterior chamber, and therefore no need for an anterior vitrectomy. Postoperatively, four eyes presented with a minor, and two eyes with a major vitreous entrapment without apparent vitreous traction. These cases happened at a time when the surgical procedure was still being elaborated, and the importance of performing a complete circumferential viscodissection of anterior hyaloid and posterior capsule prior to the optic buttoning-in manoeuvre to preclude vitreous incarceration was not yet fully recognized. One of the two patients with major entrapment underwent bimanual anterior vitrectomy in topical anesthesia through the pre-existing paracentesis openings while the optic was temporarily desenclavated. The other patient declined the proposed reintervention. No CME or RD ensued in any of these eyes in the 3-year follow-up period. The technique, results, and complications of the first 500 consecutive cases have been extensively published in a peer-reviewed journal [12]. In a later sub-series which investigated the effect of creating an ACCC opening larger than the optic diameter to further reduce anterior capsule fibrosis especially at the haptic\u2013optic junction, two other cases of delayed vitreous herniation into the anterior chamber occurred when a relatively large but still overlapping PPCCC was secondarily distended by posterior capsule contraction due to fibrosis emerging from the edge of the ACCC rim outside the optic, thus exposing the optic edge and creating a gap in the capsular diaphragm along the optic rim (Fig.\u00a06). Consequently, due to the saccadeous eye movements, mobile vitreous incrementally crept into the anterior chamber. Using bimanual vitrectomy, with the infusion inserted through one of the pre-existing paracentesis openings and the vitrectome through a pars plana sclerotomy, the floppy vitreous strand was retracted into the vitreous cavity and excised. Except for these few cases, no further cases of vitreous entrapment or herniation were encountered in the second 500 consecutive cases. In two other cases of the above-mentioned sub-series, spontaneous desenclavation of the optic without vitreous presentation was noted the day after surgery due to a too largely dimensioned PPCCC. There was no case of endophthalmitis in the 1000 cases performed up to June 2007. The results of the first 150 cases\u2014which may represent the learning curve\u2014and the subsequent 850 cases are summarized in Table\u00a01.\nFig.\u00a06Retraction of scarcely overlapping PPCCC rim from optic rim due to fibrosis emerging from ACCC larger than optic allows for delayed vitreous herniationTable\u00a01ComplicationsResultsFirst 150 cases:\u00a0\u2022 vitreous presenting in AC during surgery: none\u00a0\u2022 vitreous entrapment detected postoperatively\u00a0\u00a0\u2022 minor: four eyes\u00a0\u00a0\u2022 major: two eyes (\u2192 translimbal AVE in one eye)\u00a0\u2022 CME \/ RD: noneCases # 151 to >1000:\u00a0\u2022 oversized PPCCC with incomplete diaphragm: four eyes\u00a0\u00a0\u2022 secondary desenclavation : two cases\u00a0\u00a0\u2022 delayed vitreous herniation: two cases (\u2192 single-port ppVE)\u00a0\u2022 retinal detachement: one eye\u00a0\u00a0\u2022 highly myopic 58\u00a0yr-old male, AL>26.5\u00a0mm, 4\u00a0months postop: Cryo+gas\u00a0\u2022 CME: none\u00a0\u2022 endophthalmitis: none\nAfter-cataract preventive effect After a maximum follow-up exceeding 3\u00a0years, there was no case of retrolental after-cataract formation. The optic inside the capsular edges remained completely clear in all cases (Figs.\u00a07right and 8). When the anterior capsule was left unpolished, the posterior capsule overlying the optic periphery was often covered with a thin layer of translucent regeneratory LECs that formed into delicate pearls. These, however, did not relevantly compromise visualization of the peripheral retina. In some cases, localized Soemmering\u00b4s ring formation was observed (Fig.\u00a08left). Additional anterior capsule polishing significantly reduced regeneratory LEC proliferation on the residual posterior capsule, thereby optimizing visibility of the peripheral retina (Fig.\u00a08right). Fibrotic after-cataract formation depended upon whether anterior polishing had been performed or not. If so, no fibrosis was observed at all. If not, fibrosis of the anterior capsule was essentially restricted to the area adjacent to the haptic junction, where the undercrossing posterior capsule allowed an anterior capsule smaller than the optic diameter to establish direct contact with the optic (Fig.\u00a09 and 10). From there, limited encroachment of the fibrosis unto the neighboring capsular areas was often noted. Typically, some amount of fibrosis was observed to have spread out along both the anterior and posterior capsulorhexis edges. In the areas between the haptic junctions where the posterior capsule overlapped the optic and had been sandwiched between the latter and the anterior capsule, however, both capsules remained essentially clear and transparent. If the anterior rhexis was larger than the optic, no fibrosis formed, also adjacent to the haptic junctions, even without capsular polishing due to the lack of contact to the optic. However, fibrosis extending from the ACCC edge unto the posterior capsule retracted the latter from the optic edge in two cases with scarce optic overlap, resulting in gaping of the capsular diaphragm and delayed vitreous herniation as reported above.\nFig.\u00a07Right. Two\u00a0years after POBH: optic within capsule edges completely clear, posterior capsule overlying optic shows thin layer of regeneratory LEC proliferation with scarce small pearls. Left. Contralateral eye with in-the-bag placed IOL: regeneratory LEC proliferation on posterior capsule behind optic with abundant large pearlsFig.\u00a08Right. Two\u00a0years after POBH with polishing: significantly lower amount of regeneratory LEC proliferation\u2014compared to contralateral eye (left) having undergone POBH without polishing Fig.\u00a09Posterior capsule sandwiched between anterior capsule and optic precludes direct contact which catalyses anterior LEC transdifferentiation leading to fibrosis. Residual fibrosis essentially confined to haptic\u2013optic junctionFig.\u00a010Schematic close-up view highlighting capsule\u2013IOL interplay at haptic junction. Undercrossing of PPCCC edge allows for localized anterior capsule contact with optic, resulting in fibrosis adjacent to haptic junction which may spread out along rhexis rim\nDetailed risk profile Several substudies were initiated to exactly determine potential risks compared to the standard in-the-bag implantation technique. All were prospective randomized intraindividual comparison studies. There was no statistically significant difference in the early postoperative pressure course, either with or without the use of pressure-lowering drops [21]. This was also true for the inflammatory response as measured with a laser-flare-cell-meter, even after inadvertent puncturing of the anterior hyaloid surface [20]. Regarding the postoperative axial shift of the IOL optic and its final position, there was a significant difference. While during the first 4\u00a0weeks the bag-fixated optics generally moved forward to finally settle more anteriorly, the buttoned-in optics immediately stabilized and did not move thereafter. This resulted in a difference of mean anterior chamber depth of approximately 1\u00a0mm. Accordingly, there was a significant mean myopic shift after bag-fixation, while refraction remained unchanged after buttoning-in of the optic [22]. Though no clinically apparent CME was reported, subclinical postoperative changes had to be ruled out. This was done by investigating the thickness and morphology of the central retina using a last-generation optical coherence tomography (OCT) device. In a series of 50 bilateral cases, no difference could be found in any of these parameters (Menapace R, Stifter E. Macular integrity after cataract surgery with combined primary posterior capsulorhexis and posterior optic buttonholing as evaluated by optical coherence tomography. Abstract XXVth Congress of the ESCRS, 8.\u221212.9.2007, Stockholm), [23]. Another ongoing study is proving the clinically obvious lack of optic decentration and tilt even within a suboptimally centered PPCCC.\nDiscussion\nThe advantages, possible downsides, and specific features of the technique are addressed in the following:\nThe advantages of PPCCC fixation of the optic as compared to the standard in-the-bag fixation were found to be:\nEradication of after-cataract formation behind the optic by diverting migrating LECs to bypass the optic rim towards the anterior optic surface. When the optic is buttoned-in, the posterior capsule leaf is relocated to lie on top of the anterior optic surface (Figs.\u00a02, 3 and 8). By principle, migrating LECs can no longer access the retrolental space. At the optic junctions where the posterior capsule leaf undercrosses the haptic base, the high pressure exerted by the distended posterior capsule ring blocks LEC migration beneath the junctions.Significant reduction of capsular fibrosis without polishing. Myofibroblastic transdifferentiation is the substrate of capsular whitening and shrinkage. It mainly affects the anterior capsule, and is the cause for numerous complications including rhexis contraction (\u201crhexis phimosis\u201d) and rhexis retraction with secondary extrusion of the optic out of the bag (\u201canterior optic buttonholing\u201d), with possible decentration and tilt [10]. Direct contact between the anterior capsule and the optic is the trigger mechanism. The optic material then catalyses the myofibroblastic transdifferentiation of the anterior LECs on the back side of the anterior capsule, with consecutive capsule contraction and collagen deposition [8]. Buttoning-in causes the posterior capsule to be sandwiched between the anterior capsule and the optic, avoiding direct contact between the two. Consequently, LEC transdifferentiation of the anterior LEC layer is not initiated (\u201cno contact - no fibrosis\u201d). Only along the capsulorhexis edges some residual fibrosis may be seen. The usually seen extensive fibrosis of the whole anterior LEC layer remains restricted to the area of direct optic contact inside the optic junction.Reduced anterior optic ongrowth without polishing. After standard in-the-bag fixation, ongrowth of LECs unto the free optic zone is regularly observed with acrylates to a variable extent [1]. Usually transient, it may also be permanent. Though mostly limited to the periphery, it may in cases cover the whole free optic zone. With POBH this complication is abolished, since the crescent-shaped projection of posterior capsule forms a buffer zone between the anterior rhexis edge and the free optic. As with fibrosis, the optic ongrowth is restricted to the areas of direct anterior capsule contact to optic inside the haptic-optic junction.Eradication of residual fibrosis and ongrowth with additional polishing. Additional polishing of the anterior capsule specifically avoids fibrosis or ongrowth inside the haptic-optic junction, and fully excludes residual fibrosis in the areas where both capsules overlap.Capsular polishing without negative impact of retrolental regenerate formation. With bag-fixated IOLs, the strength and permanence of the barrier effect at the optic edge is largely dependent upon fibrosis [10]. Polishing, though effectively reducing fibrosis, therefore results in a significant increase of the YAG-LCT rate due to weakening collagenous edge sealing [11]. Since with POBH the migrating LECs are redirected to the anterior optic surface instead of being halted at the optic rim, the after-cataract preventive effect is completely independent of fibrosis. Polishing, therefore, does not promote retrolental LEC ingrowth when combined with POBH.Autocentration of optic. Even with a suboptimally centered PPCCC, the optic will automatically center within the buttonhole and align with the anatomical axis of the eye (Fig.\u00a011), since the centering force is exerted by the loops which reside in the capsular bag fornix. As opposed to standard capsular bag fixation, centration of the optic is also independent of the size and centration of the ACCC.Forgivingness of undersized PPCCC. Due to the extreme stretchability and elasticity of the posterior capsule, POBH will still be feasible with a PPCCC opening smaller than 4\u00a0mm in diameter (Fig.\u00a012). If the PPCC opening should happen to be too small to allow for buttonholing, the PPCCC can be either secondarily enlarged (similar to an ACCC), or the optic is simply placed in the bag without being buttoned in. In fact, this only happened in the early series in cases with a poorly dilating pupil.Immediate stability of capsular diaphragm and implant. Due to the lack of forward movement of the optic as seen after in-the bag fixation due to the loss of haptic memory and the distension of the capsule diaphragm as the capsules seal and fibrose, no myopic refractive shift occurs. While delayed by up to 4\u00a0weeks after bag-fixation depending upon the haptic design and material, final refraction is thus instantly attained, allowing prescription of final glasses 1\u00a0week after surgery, when the patient is seen by the referring ophthalmologist.Posterior positioning of the IOL optic by about 1\u00a0mm compared to bag-fixation significantly increases the interspace between the iris and optic. This avoids chronic iris chafing when a sulcus-fixated add-on IOL is secondarily implanted in order to correct for an erroneous IOL power selection, or to add astigmatic correction or multifocality.Fig.\u00a011Bag-fixated haptics compensate for decentration of PPCCC openingFig.\u00a012Exquisite stetchability and elasticity of posterior capsule compensates for undersizing of PPCCC opening\nTwo advantages of POBH deserve special mentioning: \n1. After-cataract prevention is entirely independent of the optic edge design. Thus, the design of the optic rim can be optimized in order to minimize reflectivity and thus dysphotopsia [16] without compromising the after-cataract performance of the IOL.2. After-cataract prevention is entirely independent of the optic material. Thus, the choice of optic material can be solely driven by achieving optimum biocompatibility, leading to minimum cell and bacteria adhesion. This may bring about a renaissance of the round-edged optics. Also, efforts in improving IOL materials may solely concentrate on optical performance, biostability and biocompatibility irrespective of their LEC inhibiting properties.\nAs opposed to PPCCC with bag-fixation of the optic, POBH creates a stable and watertight diaphragm. Vitreous prolapse or entanglement will not occur should the anterior chamber flatten after retracting the aspiration\u2013infusion tip following removal of residual OVD from the anterior chamber.\nThe POBH technique as such is safe and highly effective with regard to after-cataract prevention. It is not only feasible, but highly recommended to perform it under topical anesthesia in order to guarantee bright and stable retroillumination when performing the PPCCC. No special or extra instruments or implants are required.\nInterestingly, no case of endophthalmitis occurred in the whole series, despite the fact that no intracameral or postoperative antibiotic prophylaxis had been performed. Though this series is not large enough to draw a meaningful conclusion, it may be attributed to the fact that buttonholing avoids seclusion of bacteria within the retrolental space. Other than with bag-fixation, the optic and capsule surface are fully exposed to the aqueous humor circulation from both sides. In case of an endophthalmitis, the vitreous cavity may be easily accessed for translimbal tapping and bimanual vitrectomy after temporary desenclavation of the IOL optic.\nFour crucial surgical steps deserve in-depth discussion:\nSizing of the ACCC. Apart from capsular polishing, formation of residual fibrosis, in particular adjacent to the haptic-optic junction, can also be avoided by making the ACCC larger than the optic diameter (Fig.\u00a013right). However, combining a standard 5\u00a0mm ACCC with anterior capsule polishing is definitely recommended over creating an overly large ACCC,because of the following downsides of the latter. Appropriate dimensioning of the PPCCC is rendered more difficult since the ACCC can no longer be utilized as a ruler, making unintentional under- or oversizing more likely. Should a PPCCC result to be much larger than the optic, alternative buttoning-in of the IOL optic through the ACCC opening can then no longer be resorted to. Also, a posterior capsule that only slightly overlaps the optic may be pulled off its edge as fibrosis of the anterior capsule sets in. Delayed gaping of the capsule-IOL diaphragm along the optic rim, with possible consecutive vitreous herniation, may result, as has happened in two cases. Therefore, a 5-mm ACCC combined with anterior capsule polishing is definitely preferable over creating an oversized ACCC.Viscodissection of the central hyaloid-capsular interspace (Berger\u00b4s space and Wieger\u00b4s ligament).In the young eye, the anterior hyaloid surface is separated from the posterior capsule by an interspace with two concentric compartments: Berger\u00b4s space in the center, and Girard\u00b4s space in the periphery. These two compartments are separated by a concentric circular ring along which both membranes are attached to each other, the Wieger\u00b4s ligament, which has a diameter of 8\u20139\u00a0mm and a width of 1\u20132\u00a0mm (Fig.\u00a014). In practice, a great anatomical variability is experienced while performing a PPCCC in adult or senile eyes. In some eyes, attachment along Wieger\u00b4s ligament is no longer present, with the two united compartments thus forming one commun patent interspace. In others, there is hardly any interspace present, and viscoseparation of the strongly adhering membranes may be difficult to perform. This may be very difficult or not at all anticipated using the slitlamp with a cataractous lens. Therefore, utmost care must be used when centrally incising the posterior capsule, and inadvertent puncturing of the anterior hyaloid membrane still cannot be fully excluded. If in such a case larger quantities of OVD were injected through the capsular opening immediately after puncturing, the OVD might find its way through the punctured hyaloid surface and spread behind it within the vitreous body. This is excluded by performing part of the PPCCC beforehand. Only then, OVD is injected into Berger\u00b4s space to ensure that separation of the anterior hyaloid surface from the capsule has been attained. Viscodissection is then continued until Wieger\u00b4s ligament, if still adherent to the capsule, is severed and Girard\u00b4s space peripheral to it is accessed.Sizing and centration of the PPCCC.The posterior capsule being extremely stretchable and elastic, buttoning-in will also be feasible when a PPCCC turns out to be smaller than 4\u00a0mm. Also, the forces of the haptics will center the optic unto the anatomical axis within a suboptimally centered PPCCC. Therefore, though a 5\u00a0mm diameter and perfect centration should always be aimed at, deviation from this target will be tolerated. However, with a too large PPCCC that evades the optic rim, even when only along part of optic circumference, the IOL must be buttonholed through the ACCC with the loops either placed in the sulcus or capsular bag equator in order to avoid delayed vitreous prolapse. Therefore, the ACCC should be well-centered and no larger than 5\u20136\u00a0mm in diameter, to preserve the option of anterior rhexis fixation of the optic. Apart from providing an alternative for safe IOL fixation, an appropriately dimensioned ACCC also serves as a ruler for adequate sizing of the PPCCC.Sizing of the PPCCC may be difficult in eyes with a large capsular bag or lax zonules. This is heralded by wrinkles in the posterior capsule when flattened by OVD, and by traction folds when trying to create a central puncture or when pulling on the capsule edge when starting out with the PPCC. In such a case, implantation of a capsular tension ring remedies the problem as it puts the posterior capsule on radial stretch, creating uniform vector forces along the whole circumference. Such a ring can still be safely implanted when the PPCCC has already been started. However, bimanual tangential insertion should be preferred over injector implantation, and a smaller ring with a low resilience should be chosen. This minimizes capsular stress and distortion of the anterior segment [9], which could potentially lead to extension of the PPCCC or protrusion of the vitreous surface.Viscodissection of the peripheral hyaloid-capsular interspace (Girard\u00b4s space).Before implanting the IOL, the peripheral interspace between posterior capsule and anterior hyaloid must be circumferentially dissected with OVD in preparation of optic buttonholing. If not appropriately performed, the hyaloid surface may be shoveled up and damaged by the rim of the optic.Selection of IOL design. Though 1-piece IOLs with broad loops have been successfully used for the procedure [12], 3-piece IOLs with slim loops are definitely preferable. Of these, the HOYA AF-1 is most appropriate, since its continuous transition between haptic and optic allows the rim of the PPCCC to smoothly slide along it while the IOL is buttoned-in and centers itself within the PPCCC opening (Fig.\u00a015)Fig.\u00a013Schematic detailing capsular interplay with ACCC smaller (left) and larger than optic (right). Creating an ACCC larger than the optic precludes contact and thus fibrosis at the haptic\u2013optic junction (green circle), but may retract a scarcely overlapping PCCC and thus expose optic rimFig.\u00a014Schematic depicting interrelationship between posterior capsule and anterior hyaloid (Courtesy of Dr. T. Miyoshi). In practice, Wieger\u00b4s ligament may be completely detached; conversely, hyaloid may be closely adhering to capsule over extended areasFig.\u00a015Continuous haptic-optic transitions allow PPCCC rim to smoothly slide along IOL circumference as optic is buttoned-in and centered within PPCCC by loops residing in capsular bag fornix\nAre there any drawbacks of the PCCC plus POBH concept long-term with regard to delayed after-cataract formation or retinal complications?\nDelayed after-cataract formation. In principle, equatorial LECs might access the retrolental space beneath the haptic junction where the PCCC rim under-crosses the haptic base. This has been observed in some cases, but was always minimal and limited to the very periphery. The explanation may be that the stretched PPCCC rim firmly adheres to the haptic base, and LEC migration is very much limited, due to the narrow door for potential invasion and the lack of capsular as a scaffold for migration should the barrier fail. Moreover, because of the lack of collagenous sealing between the capsular leaves LECs might theoretically invade the exposed posterior capsule and form pearls that accumulate at the anterior capsular rim or protrude into the anterior chamber, as is seen along the edges of YAG-LCT openings. This, however, has also not been observed to a clinically significant extent. LEC ongrowth unto the exposed posterior capsule, if at all present, was always flat with no or only hinted pearl formation not exceeding the PPCCC rim. Both observations have been recently confirmed by a pertinent publication on the eyes of children who had undergone a similar procedure 5 to 12\u00a0years earlier [7], which may be explained by the proliferation-inhibiting effect of the aqueous. Considering the fact that children\u2019s eyes exhibit a much higher proliferative potential, long-term results in adults may be even better.Retinal complications. An increased incidence of CME and RD rate has been a concern. An earlier report did not find an increased incidence with PPCCC alone [26]. In our series of over 1000 POBH cases performed so far, not a single case of clinically significant CME was reported, and our OCT study revealed no difference in macular thickness or morphology.\nCME is provoked by the dissipation of cytokines released in the anterior segment, and by vitreous traction exerted by the anterior displacement of the vitreous body following exchange of the natural lens volume by a thin artificial lens. In these regards, POBH has certain advantages over standard in-the-bag IOL implantation. Firstly, as opposed to a sole PPCCC, optic buttoning-in creates a watertight diaphragm blocking the posterior dissipation of cytokines. In addition, viscodissection of the posterior capsule and anterior hyaloid creates a contiguous cushion of OVD behind the capsule\u2013IOL diaphragm including the zonular region which precludes access of cytokines to the posterior segment until it is eventually resorbed. The more posterior positioning of the buttonholed optic together with the OVD cushion behind it, and the lack of subsequent anterior movement of the optic, prevent both immediate and delayed anterior displacement of the vitreous body. Severing the attachments between the posterior capsule and the hyaloid, namely Wieger\u00b4s ligament, may help to avoid traction on the vitreous when the chamber happens to flatten during OVD aspiration, or thereafter.\nRegarding RD, one case was encountered 4\u00a0months after surgery in a young male with high axial myopia. For standard cataract and IOL surgery, a recent study reported a 5-year RD incidence of 0.7% and 21%, depending on the absence or presence of lattice degenerations [18]. In the 5\u00a0years following standard cataract surgery, such posterior vitreous detachment occurs in about four out of five eyes with the vitreous still attached. This is triggered by the anterior positioning of a bag-placed optic. Though our follow-up with POBH surgery is shorter than 5\u00a0years, the excessively low RD rate of only 0.1% overall seems to indicate a significantly lower risk. This may explained as follows: as an IOL optic buttoned-in into a PCCC is positioned 1\u00a0mm more posteriorly compared to bag-fixation [22] (Fig.\u00a04bottom), it inherently stabilizes the vitreous, and thus may avoid or delay posterior vitreous detachment. Severing of all adhesions between the posterior capsule and the anterior hyaloid and creating a cushion of OVD behind the capsule-IOL diaphragm, which is only slowly resorbed, may serve as an additional protective factor. A longterm study, including the incidence of vitreous and retinal detachment following POBH, is required to establish possible advantages of this technique over standard in-the-bag implantation of IOLs.\nIf all the precautions detailed above are carefully met, PPCCC with POBH is a very controlled and safe procedure which carries the potential of becoming a routine procedure. In any case, apart from pediatric cases, it may be considered in following clinical situations: pseudoexfoliation syndrome because of the pronounced tendency of capsular shrinkage; high myopia because of the significantly greater inclination to develop after-cataract; and peripheral retinal pathologies requiring controls and treatment (myopia, diabetes) because of the unimpeded insight for diagnosis and therapy.\nTwo potential future applications may be mentioned separately:\nToric IOLs. Overly long plate and z-shaped open-loop haptics have been used to provide for rotational stability. However, these cannot completely avoid rotation and may interfere with capsular bag closure and thus after-cataract performance. Posterior rhexis fixation makes postoperative optic rotation impossible without the need for special haptics (Fig.\u00a016).Accommodative shift IOLs. Currently marketed passive-shift IOLs have been designed to move anteriorly within the capsular bag upon accommodative effort [14].\nThe fact is that any bag-fixated IOL is immobilized due to fibrotic shrinkage. Instead of moving the optic, movement of the whole IOL-capsule complex is therefore more promising. However, fibrotic distension of the zonules would again obviate axial movement. POBH drastically reduces capsular fibrosis, as the sandwiched posterior capsule precludes the contact between the anterior capsule and the optic as the prerequisite for LEC transdifferentiation. Additional polishing of the anterior capsule, with special regard to the areas adjacent to the haptic\u2013optic junctions, has been shown to keep the capsule fully transparent and possibly elastic enough to allow axial movement of the optic under ciliary muscle contraction. This may fulfil the conditions of sufficient passive axial mobility, as postulated by Preussner, to allow for clinically relevant shift-induced accommodation when embedded in his magnet-driven concept [17] (Figure\u00a017). Instead of implementing the magnets into a special CTR as suggested by Preussner, these could be directly mounted to the optic periphery. A study investigating passive mobility under pharmacological stimulation with cycloplegics and pilocarpine is ongoing.\nFig.\u00a016Rhexis-fixation precludes postoperative IOL rotation in an oversized bag and delayed optic rotation by haptic compression arising from capsular bag shrinkageFig.\u00a017Repelling magnets located in the optic periphery (open square at 3\/9 o\u00b4clock) and under the superior\/inferior rectus muscle insertions. Upon ciliary muscle contraction\/zonular relaxation, the optic is pushed forward, increasing the power of the IOL optic within the eye\nTable\u00a02 summarizes current and potential future indications.\nTable\u00a02IndicationsSummary of indications[Pediatric cataracts]PEX-SyndromaHigh myopia (reduced barrier effect)Peripheral retinal disease: axial myopia, diabetes (improved insight for diagnosis & therapy)Multifocal IOLs (patients more susceptible to capsular opacification)\u2192ToricIOLs\u2192Accommodativelens system\nIn conclusion, primary posterior capsulorhexis combined with posterior optic buttonholing is a well-controlled, safe, and highly effective procedure with a steep learning curve, thus carrying the potential of becoming a routine alternative to standard in-the-bag implantation of intraocular lenses. As opposed to standard in-the-bag placement, its effectiveness is completely independent of optic material and optic edge design. This technique is meant for the skilled and dedicated surgeon, and must be carefully approached. It should be reserved to large pupil eyes. Should the pupil come down during the surgery, one may switch to standard in-the-bag surgery, optionally combined with a small PPCCC. Thorough viscodissection of anterior hyaloid and posterior capsule is crucial. Additional anterior capsule polishing is highly recommended, as it abolishes any residual fibrosis without any risks or downsides. Though long-term results in children's eyes support sustained efficacy and lack of complications, 5-year results should be awaited until the technique may be considered or recommended as a routine alternative to standard in-the-bag fixation of the IOL. For the time being, it is recommended with pseudoexfoliation syndrome, high axial myopia, peripheral retinal disease, multifocal IOLs, and toric IOLs when made available.","keyphrases":["optic buttonholing","posterior capsule opacification","fibrosis","regeneratory after-cataract","reclosure","primary posterior capsulorhexis","cystoid macular edema","retinal detachment","vitreous entrapment","toric iols","iop","flare","axial optic shift","optic centration"],"prmu":["P","P","P","P","P","P","P","P","P","P","U","U","R","R"]}
{"id":"Psychopharmacologia-4-1-2244695","title":"Selective PDE inhibitors rolipram and sildenafil improve object retrieval performance in adult cynomolgus macaques\n","text":"Rationale Selective phosphodiesterase (PDE) inhibitors improve the formation of hippocampus-dependent memories in several rodent models of cognition. However, studies evaluating the effects of PDE inhibition on prefrontal cortex-dependent cognition and in monkeys are rare.\nIntroduction\nSecond messenger cyclic nucleotides, i.e., cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), have been strongly implicated in processes of synaptic long-term potentiation (LTP; Frey et al. 1993; Zhuo et al. 1994) and have cognition-enhancing properties (Bernabeu et al. 1996; Blokland et al. 2006; Prickaerts et al. 2002a; Prickaerts et al. 2005). cAMP and cGMP are hydrolyzed by phosphodiesterase (PDE) enzymes, and inhibitors of PDEs (PDE-Is) produce cognition-enhancing effects in animal models of cognition. In this field, research has focused on PDE4 and PDE5 inhibitors (Blokland et al. 2006; Rose et al. 2005; Rutten et al. 2006) and more recently PDE2 (Boess et al. 2004; Rutten et al. 2007b) and PDE10 (Rodefer et al. 2005) inhibitors. For example, inhibition of PDE2, PDE4, and PDE5 can improve memory in the object recognition task in rodents (Boess et al. 2004; Prickaerts et al. 2004; Rutten et al. 2007b).\nMoreover, the cAMP-selective PDE4-I rolipram is an effective performance enhancer in the passive avoidance task (Egawa et al. 1997; Imanishi et al. 1997), fear-conditioning test (Barad et al. 1998), and radial arm maze (Zhang and O'Donnell 2000). The cGMP-selective PDE5-I sildenafil has been shown to enhance performance in the object recognition task, in the T-maze, and spatial plus maze (Devan et al. 2006; Devan et al. 2004; Patil et al. 2006; Prickaerts et al. 2004). In addition, sildenafil improved object recognition memory in mice (Rutten et al. 2005), and another PDE5-I, zaprinast, improved performance in a passive avoidance task in neonatal chicks (Campbell and Edwards 2006). Taken together, these results confirm the beneficial effects of PDE inhibition on cognition involving the hippocampus.\nThe present study assesses the effects of PDE inhibition on object retrieval (OR) performance, a task of prefrontal cognition, in monkeys. OR (also known as the detour reaching task) involves attention, response inhibition, and planning (i.e., executive function; Diamond et al. 1989). Because the prefrontal cortex is not well represented in rodents (especially the dorsolateral prefrontal cortex) and considerable debate exists on whether the rodent prefrontal cortex subserves the same behavioral functions as the primate\/human prefrontal cortex (Brown and Bowman 2002; Uylings et al. 2003), tests of prefrontal functioning (i.e., executive function) are preferentially carried out in monkeys. Successful completion of the OR task requires various hypothetical steps. An animal must appreciate the contradiction between visual and tactile input, ignore the incorrect input, remember the previous incorrect approach, and formulate a novel approach to obtain the reward, which acts against the innate tendency of perseveration toward sustained sensory stimulation (Lipina and Colombo 2001). Direct support for the involvement of the frontal cortex in OR comes from studies in which lesions of the frontal cortex impaired, while lesions of the hippocampus did not impair, performance in the task (Diamond et al. 1989; Wilkinson et al. 1997). Furthermore, deficits in OR tasks have been documented in monkeys after treatment with methylphenyl tetrahydropyridine (Schneider and Roeltgen 1993; Taylor et al. 1990a; Taylor et al. 1990b), phencyclidine (Jentsch et al. 2000; Jentsch et al. 1999a; Jentsch et al. 1999b), and after excitotoxic lesioning with quinolinic acid of the striatum (Roitberg et al. 2002). The effect of PDE-Is in the OR task have, to our knowledge, never been evaluated before in unimpaired monkeys.\nAs mentioned above, improvement in hippocampus-dependent memory tasks in rodents is well established through PDE inhibition. However, no evidence exists on the effects of rolipram or sildenafil on prefrontal cortex-dependent tests of executive function. As the PDE4 enzyme is omnipresent throughout the brain and PDE5 is expressed in the hippocampus, cortex, and cerebellum (van Staveren et al. 2004), we hypothesized that OR performance would be improved by the PDE4 and PDE5 inhibitors rolipram and sildenafil, respectively.\nMaterials and methods\nSubjects\nFourteen adult (age 5\u201312\u00a0years; weights 6\u20139\u00a0kg) male cynomolgus macaques (Macaca fascicularis) were housed in same-sex pairs in a colony room maintained at 21\u2009\u00b1\u20092\u00b0C, 40\u2009\u00b1\u200910% humidity, and on a normal 12-h light\/dark cycle (lights on at 7:00\u00a0a.m.). Food (Purina High Protein no. 5045) and water were available ad libitum. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Roche Palo Alto and were in accordance with National Institutes of Health guidelines.\nObject retrieval task\nThe OR task has been previously described by Diamond et al. (1989). Briefly, this task requires a monkey to reach into a clear acrylic box (dimensions\u2009=\u20095\u2009\u00d7\u20095\u2009\u00d7\u20095\u00a0cm) with one open side, to retrieve a food treat (cubes of apple or pear, 1\u20132\u00a0cm2). The box was positioned in front of the monkey and outside of the home cage, with the open side facing left, right, or toward the monkey. Food treats were placed on the outer edge, inner edge, or deep within the box. A test session consisted of 17 trials with nine \u201ceasy\u201d food retrievals (i.e., placement of the food reward on the inner or outer edges of the box or when the opening is toward the monkey) and eight \u201cdifficult\u201d food retrievals (i.e., placement of the food reward deep within the box and the open side facing left or right, see Fig.\u00a01). The order of presentation never varied (Table\u00a01), there were no contingencies for incorrect reaches (i.e., monkeys typically acquired the treat after the incorrect reach) or dropped treats, and trials were terminated if there were no reaches within 3\u00a0min. The box was cleaned diligently between trials to minimize cues that could influence the task and subsequent task performance. After an initial period (1\u00a0week) to acclimatize the monkeys to the apparatus and procedure, tests were conducted twice a week.\nFig.\u00a01A schematic overview of possible trials in the object retrieval (detour-reaching) task. The transparent box containing a food reward is depicted form the monkey\u2019s point of view. The top five orientations depict easy trials, and the bottom two orientations depict difficult trialsTable\u00a01Primate object retrieval: trial orderTrial numberDescriptionLevel1LOS\u2014line of sightEasy2LOS\u2014line of sightEasy3RO\u2014right outsideEasy4RD\u2014right deepDifficult5RO\u2014right outsideEasy6RI\u2014right insideEasy7RD\u2014right deepDifficult8LO\u2014left outsideEasy9LD\u2014left deepDifficult10LO\u2014left outsideEasy11li\u2014left insideEasy12ld\u2014left deepDifficult13ld\u2014left deepDifficult14rd\u2014right deepDifficult15ld\u2014left deepDifficult16rd\u2014right deepDifficult17los\u2014line of sightEasy\nDrugs\nRolipram (0.003, 0.01, 0.03, and 0.1\u00a0mg\/kg; Sigma Chemicals, St Louis, MO) and sildenafil (0.3, 1, and 3\u00a0mg\/kg; Sequoia Research Products Limited, Pangbourne, UK) were prepared fresh daily in a suspension of 10% cremaphore\/90% saline and administered 30 or 60\u00a0min before testing, respectively. Drugs were administered via intramuscular injection (i.m.) in a volume of 0.1\u00a0ml\/kg. Compounds were typically administered on Tuesday and Friday each week. Weekly test sessions were comprised of a vehicle session and a drug test session. Drug administration and behavioral measurement were completed blind, and each drug\u2013dose combination was tested once. Drug doses were administered in a pseudorandom order.\nData analysis\nThe mean percent correct first reaches for easy and difficult food retrievals were analyzed with a one-factor (Dose) repeated-measures analysis of variance. When significant effects were demonstrated, a post-hoc Dunnett analysis comparing dose conditions to vehicle performance was performed. For all tests, the significance level was 0.05 (two-tailed).\nResults\nDuring vehicle sessions, monkeys readily reached for the food treats, successfully acquiring the treat on the first reach 100\u2009\u00b1\u20090% during easy trials and 52\u2009\u00b1\u20093% during difficult trials. Over the course of the experiment (2\u00a0months), there were no changes in vehicle performance.\nThe PDE4 inhibitor rolipram (0.003\u20130.1\u00a0mg\/kg, i.m.) dose-dependently increased correct first reaches during difficult trials (F[3, 55]\u2009=\u200913.5, p\u2009<\u20090.05), reaching significance at 0.01\u00a0mg\/kg (Fig.\u00a02a). At the highest dose tested without side effects (0.03\u00a0mg\/kg), the percentage correct first reaches were increased approximately 20 to 71\u2009\u00b1\u20093%. Note that at 0.1\u00a0mg\/kg (data not shown), emesis was observed in all but two monkeys; these results were therefore excluded from analysis.\nFig.\u00a02The effects of PDE-I on object retrieval (OR) performance (mean values and SEM) a The effects of the PDE4 inhibitor rolipram and the PDE5 inhibitor sildenafil (b) on the percentage correct first reaches on difficult trials in the OR task. Asterisks indicate significant differences from baseline (P\u2009<\u20090.05)\nThe PDE5 inhibitor sildenafil (0.3\u20133\u00a0mg\/kg, i.m.) dose-dependently increased correct first reaches during difficult trials (F[3, 50]\u2009=\u20098.6, p\u2009<\u20090.05), reaching significance at 1\u00a0mg\/kg (Fig.\u00a02b). At the highest dose tested (3\u00a0mg\/kg), correct reaches were increased approximately 20 to 73\u2009\u00b1\u20093%. No side effects were observed.\nNeither rolipram nor sildenafil altered performance during the easy trials, with the exception of the high dose of rolipram (0.1\u00a0mg\/kg), in which monkeys failed to perform the task (data not shown) because of emetic side effects.\nDiscussion\nThe present study demonstrates performance-enhancing effects of two selective PDE-Is on OR performance in monkeys. OR is subserved by the prefrontal cortex and\/or fronto-striatal pathways, within which modulation of dopamine and acetylcholine transmission are involved in attention, response inhibition (i.e., executive function), and working memory in rodents and primates (Jentsch et al. 2000; Lipina and Colombo 2001; Palfi et al. 1996; Ramos et al. 2003; Wilkinson et al. 1997). To our knowledge, this is the first study to investigate the effects of PDE5 inhibition on executive function in monkeys. Of note, two previous studies have investigated the effects of sildenafil on cognition-related variables in humans. In these studies, sildenafil was shown to enhance simple reaction times and to some extent enhancement of focused attention (Grass et al. 2001; Schultheiss et al. 2001). However, these findings have not been further described in the literature. The effects of the PDE4 inhibitor rolipram on executive function have yet to be assessed in humans. Thus, the current results complement and extend to the cognition-enhancing effects of PDE inhibition.\nPrevious studies have repeatedly shown that PDE-Is can have cognition-enhancing effects, mainly in hippocampus-dependent memory tasks, in rodents. For example, the PDE4 inhibitor rolipram improved long-term memory in the object recognition task, in passive avoidance learning, and fear conditioning (Barad et al. 1998; Rutten et al. 2006; Zhang et al. 2005). In addition, rolipram had performance-enhancing effects in a prefrontal cortex-dependent working memory task, i.e., delayed alternation, in young rats and young monkeys (Ramos et al. 2003). Results from the present study corroborate the cognition-enhancing effects of low-dose (0.01\u00a0mg\/kg) rolipram treatment in a prefrontal cortex-dependent task, although the present OR task does not involve working memory but requires attention and response inhibition (i.e., executive function; Diamond et al. 1989). Furthermore, in aged mice, rolipram ameliorated the age-related deficits in the passive avoidance task, a test of hippocampus-dependent memory. In contrast, rolipram impaired prefrontal cortex-dependent working memory performance in aged rodents and aged monkeys. With advancing age, opposite profiles between the function of protein kinase A (PKA) in the hippocampus and prefrontal cortex were suggested to explain these results; that is, the prefrontal cortex showed indices of increased PKA activity, while the hippocampus exhibited evidence of decreased PKA activity (Ramos et al. 2003). Although in the present study, cognition-enhancing effects on executive function were observed after rolipram treatment in young monkeys, the possible cognition-impairing effects of rolipram on prefrontal cortex-dependent tests in aged monkeys should be further investigated.\nCompared to PDE4 inhibition, the cognition-enhancing effects of PDE5 inhibition have not been studied as extensively. However, a growing number of studies have shown cognition-enhancing effects of PDE5-Is in multiple tests and in multiple species. PDE5-Is improved cognitive performance in object recognition and inhibitory and passive avoidance tasks (Baratti and Boccia 1999; Prickaerts et al. 2005; Prickaerts et al. 2004; Prickaerts et al. 2002b; Rutten et al. 2005; Shafiei et al. 2006; Singh and Parle 2003). In addition, the inhibition of PDE5 reversed memory deficits induced by scopolamine, diabetic neuropathy, or nitric oxide (NO) synthase inhibitors in rats (Devan et al. 2006; Devan et al. 2004; Patil et al. 2004; Prickaerts et al. 1997). To our knowledge, no literature exists on the possible cognition-enhancing effects of PDE5 inhibitors in nonhuman primates. Furthermore, the effects of PDE5 inhibition on prefrontal cortex-depending cognition, i.e., working memory and executive function, are unknown.\nThe underlying mechanisms of PDE-Is and cognition enhancement are still elusive, but several possible pathways have been described. Possible mechanisms of action for rolipram and sildenafil are based on the proposed underlying signaling pathways of LTP. Both cAMP and cGMP have been strongly implicated in hippocampal LTP (Frey et al. 1993). Activation of LTP-related signaling pathways of cAMP\/PKA\/cAMP response element-binding protein (CREB) and cGMP\/protein kinase G (PKG)\/CREB have been implicated as the underlying mechanisms for the cognition-enhancing effects of PDE4 and PDE5 inhibitors (Bernabeu et al. 1996; Blokland et al. 2006; Lu and Hawkins 2002; Prickaerts et al. 2002a; Rutten et al. 2007b). A very recent study suggested another pathway through which cGMP can influence cognitive processes. This study showed that the hippocampal NO\/cGMP pathway directly stimulates the postsynaptic cAMP\/PKA\/CREB pathway (Matsumoto et al. 2006). Alternatively, cGMP has also been found to maintain LTP via a presynaptic cGMP\/PKG pathway (Zhuo et al. 1994). However, the present study involves prefrontal cortex-dependent behavior, and it remains to be proven that the above mentioned hippocampal LTP mechanisms of synaptical strengthening are also applicable in the prefrontal cortex. Of note, the effects of rolipram on prefrontal cortex performance were explained via activation of the postsynaptic PKA pathway (Ramos et al. 2003). Thus, both cGMP and cAMP can activate cellular cascades resulting in CREB phosphorylation, which could induce long-lasting changes in neuronal signaling and may thereby explain the performance enhancement observed in the present study.\nApart from LTP-related mechanisms of action, the effects of rolipram and sildenafil on prefrontal functioning in the OR may be explained by their stimulatory effect on neurotransmission. Because rolipram is independent of a specific receptor system, it is suggested that rolipram may ameliorate physiological abnormalities that occur secondary to alterations in dopaminergic, glutamatergic, serotonergic, and\/or adrenergic transmission (Maxwell et al. 2004; Rutten et al. 2007a). Elevated cAMP levels are thought to excite noradrenergic and dopaminergic neurotransmitter systems (Schoffelmeer et al. 1985), thus enhancing their availability, hence also enhancing synaptic transmission. Similar to rolipram, sildenafil is a drug that is not linked to specific neurotransmitter systems. Modulatory activity of sildenafil was suggested on central dopaminergic pathways (Ferrari et al. 2002). Dopaminergic dysfunction in prefrontal cortex may subserve at least a component of the impaired OR\/detour performance in deficit models (Jentsch et al. 1999a). Moreover, serotonin depletions of the prefrontal cortex in the common marmoset result in deficits in the acquisition of the OR task (Walker et al. 2006). Hence, activation of cGMP or cAMP may improve dopaminergic turnover and prefrontal functioning in the OR task. Thus far, it remains unclear which of these abovementioned mechanisms are involved in the prefrontal cortex and how they explain cognition enhancement by inhibition of PDEs in the OR task.\nTo summarize, previous research demonstrated the cognition- and largely memory-enhancing effects of PDE4 and PDE5 inhibition in rodents. The results from the current study extend these cognition-enhancing effects of PDE-Is in rodents to nonhuman primates. More specifically, rolipram and sildenafil improve OR performance, thus demonstrating that PDE4 and PDE5 inhibition enhance performance in a cognitive domain involving executive function (and attention) mediated by the prefrontal cortex.","keyphrases":["rolipram","sildenafil","object retrieval","phosphodiesterase","cognition","monkey","pde4","pde5","attention","executive function","primate","cyclic amp","cyclic gmp"],"prmu":["P","P","P","P","P","P","P","P","P","P","P","M","M"]}
{"id":"Behav_Genet-4-1-2257997","title":"Speed, Variability, and Timing of Motor Output in ADHD: Which Measures are Useful for Endophenotypic Research?\n","text":"Attention-Deficit\/Hyperactivity Disorder (ADHD) shares a genetic basis with motor coordination problems and probably motor timing problems. In line with this, comparable problems in motor timing should be observed in first degree relatives and might, therefore, form a suitable endophenotypic candidate. This hypothesis was investigated in 238 ADHD-families (545 children) and 147 control-families (271 children). A motor timing task was administered, in which children had to produce a 1,000 ms interval. In addition to this task, two basic motor tasks were administered to examine speed and variability of motor output, when no timing component was required. Results indicated that variability in motor timing is a useful endophenotypic candidate: It was clearly associated with ADHD, it was also present in non-affected siblings, and it correlated within families. Accuracy (under- versus over-production) in motor timing appeared less useful: Even though accuracy was associated with ADHD (probands and affected siblings had a tendency to under-produce the 1,000 ms interval compared to controls), non-affected siblings did not differ from controls and sibling correlations were only marginally significant. Slow and variable motor output without timing component also appears present in ADHD, but not in non-affected siblings, suggesting these deficits not to be related to a familial vulnerability for ADHD. Deficits in motor timing could not be explained by deficits already present in basic motor output without a timing component. This suggests abnormalities in motor timing were predominantly related to deficient motor timing processes and not to general deficient motor functioning. The finding that deficits in motor timing run in ADHD-families suggests this to be a fruitful domain for further exploration in relation to the genetic underpinnings of ADHD.\nIntroduction\nIt has become apparent from twin- and adoption studies that the development of the Attention-Deficit\/Hyperactivity Disorder (ADHD) (American Psychiatric Association 1994) is strongly genetically based (Faraone et\u00a0al. 2005; Willcutt, in press). Overall heritability estimates exceed 0.70 (Faraone and Doyle 2000; Smalley 1997) and are fairly constant across studies conducted worldwide (Faraone et\u00a0al. 2005). Research aimed at the molecular genetic basis of the disorder (genotype) has had success in identifying some susceptibility genes by using information from behaviorally observable symptoms (phenotype) (Faraone et\u00a0al. 2005). However, the current knowledge about the genetic basis of the disorder is still limited and the causal pathway(s) leading from genotype to phenotype have yet to be revealed.\nBoth issues have been the aims of investigation in endophenotypic research. Endophenotypes are defined as heritable, vulnerability traits that mark a risk for the development of the disorder (Almasy and Blangero 2001; Castellanos and Tannock 2002; Doyle et\u00a0al. 2005; Gottesman and Gould 2003; Skuse 2001; Waldman 2005; Zobel and Maier 2004). They are conceptualized as forming an intermediate link between the genotype and phenotype and are presumably genetically less complex compared to phenotypic symptoms (Gottesman and Gould 2003; Waldman 2005). Because of these characteristics, it is thought that, compared to phenotypic symptoms, endophenotypes are more suitable for detecting disease genes and for unraveling the modes of actions of these disease genes.\nSeveral criteria have been proposed to discriminate an endophenotype from other biological markers that are not causally involved in the disorder but are merely associated with the disorder (Durston et\u00a0al. 2004; Gottesman and Gould 2003). Although these criteria do not appear to be universally agreed upon, several key criteria have emerged from the literature (Almasy and Blangero 2001; Castellanos and Tannock 2002; Doyle et\u00a0al. 2005; Gottesman and Gould 2003; Skuse 2001; Waldman 2005; Zobel and Maier 2004). First, an endophenotype should co-occur with the disorder, although given the heterogeneity of ADHD, it is unlikely that a single endophenotype will occur in all patients with ADHD (Doyle et\u00a0al. 2005). Second, non-affected relatives should also exhibit the endophenotype to some extent, indicating that the endophenotype contributes to a familial susceptibility for the disorder. Because non-affected relatives share, on average, 50% of their genes with the affected family member, it is theorized that they also carry some of the susceptibility genes of ADHD which translate into subtle abnormalities in the endophenotype (Gottesman and Gould 2003; Waldman 2005). Third, the endophenotype should show familial resemblance, reflected by significant sibling correlations for the endophenotypic measure. In addition to these criteria, several other important characteristics of an endophenotype have been put forward, such as reliability of measurement, stability over time (i.e. expressed regardless whether or not the disorder is currently manifested), and acting as a mediator and\/or moderator between genes and disorder (Doyle et\u00a0al. 2005; Waldman 2005). The focus of our study lies on the first three key criteria of an endophenotype.\nPossible endophenotypic candidates might be found in the deficits in motor output associated with ADHD. Fine motor coordination problems as well as gross motor coordination problems are frequently observed in co-occurrence with ADHD (Carte et\u00a0al. 1996; Korkman and Pesonen 1994; Marcotte and Stern 1997; Piek et\u00a0al. 1999; Pitcher et\u00a0al. 2003; Whitmont and Clark 1996). Because of this highly frequent co-occurrence, it is feasible that motor problems might be genetically related to the risk for developing ADHD (Gillberg 2003), which has indeed been reported recently (Martin et\u00a0al. 2006). This might make motor measures useful endophenotypic candidates, which was underlined by the findings of motor control difficulties in non-affected siblings of children with ADHD (Rommelse et\u00a0al. 2007a; Slaats-Willemse et\u00a0al. 2005).\nA specific aspect of motor output that might serve as a candidate endophenotype is temporal organization (Castellanos and Tannock 2002; Waldman et\u00a0al. 2006). Temporal organization of motor output refers to the timing of movements (i.e. motor timing) and seems to be predominantly mediated by the cerebellum and basal ganglia and their reciprocal connections with the cerebral cortex (Handy et\u00a0al. 2003; Harrington et\u00a0al. 1998; Ivry 1996; Nenadic et\u00a0al. 2003). The timing of motor output is hypothesized to consist of two components: a clock component, which reflects central time keeping operations, and a motor delay component, which reflects random variability due to response implementation processes (Harrington et\u00a0al. 1998; Keele et\u00a0al. 1985; Wing and Kristofferson 1973). Children with ADHD seem to be predominantly impaired in the clock component, as evidenced by paradigms specifically assessing timing operations independent of motor operations, such as duration discrimination tasks (Keele et\u00a0al. 1985; see for review Toplak et\u00a0al. 2006). The motor component is possibly best assessed using simple reaction time tasks and free Tapping tasks in which timing is minimized. Some have found no impairments in children with ADHD (Kalff et\u00a0al. 2003; Seidman et\u00a0al. 1997, 2000), others have (Kalff et\u00a0al. 2005). By combining both components, one can assess timing that is predominantly related to motor output, which reflects the interplay between timing and motor skills. Studies using these motor timing tasks have, however, not revealed consistent results of impairments in children with ADHD, possibly due to inconsistencies in methodological approaches across studies (Toplak et\u00a0al. 2006). However, greater variability in motor output when a timing component is required is a frequently reported finding in children with ADHD (Leth-Steensen et\u00a0al. 2000; Pitcher et\u00a0al. 2002; Rubia et\u00a0al. 1999, 2003; Toplak et\u00a0al. 2006; Van Meel et\u00a0al. 2005). None of these studies, however, used a measure of basic motor speed\/variability (without requirements regarding timing\/rhythm) in addition to the motor timing measure. Therefore, it remains unclear whether the variability in motor timing is primarily due to variability of motor processes, or due to variability of timing processes or both. Here, we administered two simple motor tasks in addition to the motor timing task, in order to investigate whether deficits were specifically related to the timing of motor output or whether deficits were also observed in tasks that do not require timing of motor output.\nThe current study is, to our knowledge, the first to examine motor timing abilities in not only children with ADHD, but also in their siblings, in order to investigate whether motor timing measures might be suitable endophenotypic candidates. Previous studies on related topics have shown that non-affected siblings of children with ADHD have comparable problems in time reproduction skills as their affected siblings (Rommelse et\u00a0al. 2007b) and that variability in reaction time shows familial overlap with ADHD (Andreou et\u00a0al. 2007), giving support to the hypothesis that motor timing measures may be suitable endophenotypic candidates. It was expected that (1) children with ADHD would be impaired on motor timing measures and not (or to a lesser extent) on motor measures without a timing component, indicating an association between ADHD and motor timing deficits. Furthermore, we expected (2) to find similar findings in the non-affected siblings of the children with ADHD, suggesting motor timing deficits are not merely associated with the disorder. Last, we expected (3) to find correlations between siblings indicating familial resemblance on motor timing measures.\nMethod\nParticipants\nFamilies with at least one child with the combined subtype of ADHD (proband) and at least one additional sibling (regardless of possible ADHD-status) were recruited in order to participate in the Dutch part of the International Multicenter ADHD Genes study (IMAGE). The IMAGE project is an international collaborative study that aims to identify genes that increase the risk for ADHD using QTL linkage and association strategies (Brookes et\u00a0al. 2006). Additional control families were recruited from primary and high schools from the same geographical regions as the participating ADHD-families. Controls and their first degree relatives had no formal or suspected ADHD diagnosis. A total of 238 ADHD-families and 147 control-families fulfilled inclusion and exclusion criteria. Within the ADHD-families, 238 probands (all with combined subtype of ADHD), 112 affected siblings (64 with combined subtype, 28 with inattentive subtype and 20 with hyperactive-impulsive subtype of ADHD) and 195 non-affected siblings participated. Control-families consisted of 271 children. For 51 control children, no additional control sibling could be recruited for the study (see for an overview Tables\u00a01, 2).\nTable\u00a01Distribution of family sizesNumber of siblings within a familyADHDControlFamilies (n)Individuals (n)Families (n)Individuals (n)100515121773547214435315920604832416Total238545147271Note: ADHD\u00a0=\u00a0Attention-Deficit\/Hyperactivity DisorderTable\u00a02Distribution of affected and non-affected siblings within ADHD-familiesTotal number of children within a familyDiagnostic statusADHD-families (n)Proband (n)Affected siblings (n)Non-affected siblings (n)211\u2013621\u20131115312\u20137111241\u2013222413\u20132121111241\u201331Total238Note: ADHD\u00a0=\u00a0Attention-Deficit\/Hyperactivity Disorder\nAll children were between the ages of 5 and 19\u00a0years and were of European Caucasian descent. Participants were excluded, if they had an IQ\u00a0<\u00a070, a diagnosis of autism, epilepsy, brain disorders or known genetic disorders, such as Down syndrome or Fragile-X-syndrome.\nWithin an ADHD-family, both proband and siblings were similarly screened using the standard procedures of the IMAGE project described by Brookes et\u00a0al. (2006). Briefly, screening questionnaires (parent and teacher Conners\u2019 long version rating scales [Conners 1996] and parent and teacher Strengths and Difficulties Questionnaires [SDQ, Goodman 1997]), were used to identify children with ADHD symptoms. T-scores\u00a0\u2265\u00a063 on the Conners\u2019-N-scale (DSM-IV total symptom score) and scores\u00a0>\u00a090th percentile on the SDQ-hyperactivity scale were considered as clinical. For all children within a family scoring clinically on any of the questionnaires, a semi-structured, standardized, investigator-based interview was administered separately for each child: the Parental Account of Children\u2019s Symptoms (PACS; Taylor 1986). The PACS covers DSM-IV symptoms of ADHD, conduct disorder, oppositional defiant disorder, anxiety, mood, and other internalizing disorders. The section on autistic behaviour traits was administered, if a clinical score (raw score\u00a0\u2265\u00a015) was obtained on the Social Communication Questionnaire (SCQ; Berument et\u00a0al. 1999). A standardised algorithm was applied to the PACS and parent rated Conners\u2019 to derive each of the 18 DSM-IV ADHD items, providing operational definitions for each behavioural symptom. These were combined with items that were scored 2 (\u2018pretty much true\u2019) or 3 (\u2018very much true\u2019) in the teacher rated Conners\u2019 ADHD subscale to generate the total number of hyperactive-impulsive and inattentive symptoms of the DSM-IV. Situational pervasiveness was defined as at least one symptom occurring within two or more different situations as indicated by the parents in the PACS interview as well as the presence of at least one symptom scoring 2 or 3 on the ADHD subscale as indicated by teachers on the Conners\u2019. Siblings were regarded as non-affected, if they obtained scores in the non-clinical range on both the parent and teacher questionnaires (Conners\u2019-N-scale: T-score\u00a0\u2264\u00a062, SDQ\u00a0<\u00a090th percentile). No PACS interview was administered concerning non-affected siblings.\nThe Conners\u2019 long version for both parents and teachers was completed for control children. Control children had to obtain non-clinical scores on both the parent and teacher version (Conners\u2019-N-scale: T-score\u00a0\u2264\u00a062). Table\u00a03 provides the characteristics of the four groups.\nTable\u00a03Sample characteristicsProbandsAffected siblingsNon-affected siblingsNormal controlsF3,812Contrastsn\u00a0=\u00a0238n\u00a0=\u00a0112n\u00a0=\u00a0195n\u00a0=\u00a0271MSDMSDMSDMSDAge in years12.02.512.03.411.53.611.63.2ns% Right handed91.187.589.285.5nsa% Male84.556.345.140.6113.9*a1\u00a0>\u00a02,3,42\u00a0=\u00a03 & 2\u00a0>\u00a043\u00a0=\u00a04Estimated full scale IQ97.913.0100.710.6103.810.9106.010.223.5*1\u00a0=\u00a02 & 1\u00a0<\u00a03\u00a0=\u00a042\u00a0=\u00a03 & 2\u00a0<\u00a043\u00a0=\u00a04Conners\u2019 parent DSM-IV\u00a0\u00a0\u00a0\u00a0Inattentive71.18.466.011.647.97.046.54.8585.4*1\u00a0>\u00a02\u00a0>\u00a03\u00a0=\u00a04\u00a0\u00a0\u00a0\u00a0Hyperactive-impulsive79.19.267.813.649.06.947.35.1767.3*1\u00a0>\u00a02\u00a0>\u00a03\u00a0=\u00a04\u00a0\u00a0\u00a0\u00a0Total76.98.668.311.648.26.846.54.5875.7*1\u00a0>\u00a02\u00a0>\u00a03\u00a0=\u00a04Conners\u2019 teacher DSM-IV\u00a0\u00a0\u00a0\u00a0Inattentive66.09.161.710.248.36.046.44.6386.3*1\u00a0>\u00a02\u00a0>\u00a03\u00a0=\u00a04\u00a0\u00a0\u00a0\u00a0Hyperactive-impulsive70.210.763.513.348.36.547.25.0378.1*1\u00a0>\u00a02\u00a0>\u00a03\u00a0=\u00a04\u00a0\u00a0\u00a0\u00a0Total69.89.863.811.448.35.846.44.5485.8*1\u00a0>\u00a02\u00a0>\u00a03\u00a0=\u00a04ADHD diagnosis\u00a0\u00a0\u00a0\u00a0Inattentive\u201328\u2013\u2013\u00a0\u00a0\u00a0\u00a0Hyperactive-impulsive\u201320\u2013\u2013\u00a0\u00a0\u00a0\u00a0Combined23864\u2013\u2013Note: 1\u00a0=\u00a0Probands; 2\u00a0=\u00a0Affected siblings; 3\u00a0=\u00a0Non-affected siblings; 4\u00a0=\u00a0Normal controlsADHD\u00a0=\u00a0Attention-Deficit\/Hyperactivity Disorder; DSM-IV\u00a0=\u00a0Diagnostic and Statistical Manual for Mental Disorders (4th edition)*\u00a0P\u00a0<\u00a00.001; a\u00a0\u03c72; Contrasts based on p-values of 0.05\nMeasures\nMotor Timing Task\nThis task was designed to measure the accuracy and variability of motor timing (Van Meel et\u00a0al. 2005). Subjects were instructed to press a button with their preferred index finger when they thought a 1-second time interval had elapsed. The start of the interval was announced by a tone. After the subject\u2019s response, visual feedback concerning the accuracy of the response was presented on the screen, indicating whether the response was correct, too short or too long. A response was regarded as correct, if it fell between the lower and upper boundary set by a dynamic tracking algorithm. Boundaries were set at 500 to 1,500\u00a0ms at the beginning of the task. If the response fell within these boundaries, the boundaries of the subsequent trial were narrowed by 100\u00a0ms. Likewise, the boundaries of the subsequent trial were widened with 100\u00a0ms, if the response on the previous trial fell outside the boundaries.\nThe practice session consisted of 20 trials, the experimental session of 80 trials. Both sessions were preceded by presenting 10\u00a0times a cartoon figure for exactly 1\u00a0s on the screen to demonstrate the duration of 1\u00a0s (Van Meel et\u00a0al. 2005). Dependent measures were accuracy (median of productions in ms, which reflects under- versus over-production) and variability (SD of productions in ms).\nBaseline Speed task\nThis task was designed to measure the speed and variability of motor output in response to an external cue and comparable to a simple reaction time task (De Sonneville 1999). Subjects were required to press a key as quickly as possible, when a fixation cross in the centre of a computer screen changed into a white square. Immediately following the response, the white square changed back into the fixation cross. The time interval between a response and the emergence of the next white square varied randomly between 500 and 2,500\u00a0ms in order to prevent anticipation strategies.\nA practice session (10 trials) and an experimental session (32 trials) were administered for both hands separately. The task was first practised and executed with the index finger of the non-preferred hand, thereafter practised and executed with the index finger of the preferred hand. Dependent measures were the speed (mean reaction time in ms) and variability (SD of reaction times in ms) of responses.\nTapping task\nThis task measured the speed and variability of self-generated motor output (without internal or external cues) (De Sonneville 1999). Subjects were required to tap as frequently as possible within an interval of 18\u00a0s. The beginning and end of the interval were announced by a tone. During tapping, the number of taps was continuously counted and displayed on the screen.\nA practice session (5\u00a0s) and an experimental session (18\u00a0s) were administered for both hands separately. The task was first practised and executed with the index finger of the non-preferred hand, thereafter practised and executed with the index finger of the preferred hand. Dependent measures were speed (mean intertap interval in ms) and variability (SD of intertap intervals in ms) of motor output.\nIntelligence\nFull-scale IQ was estimated by four subtests of the WISC-III (Wechsler 2002) or WAIS-III (Wechsler 2000) (depending on the child\u2019s age): Vocabulary, Similarities, Block Design and Picture Completion. These subtests are known to correlate between .90\u2013.95 with the Full-scale IQ (Groth-Marnat 1997).\nProcedure\nTesting of ADHD children and their siblings took place at the VU University Amsterdam or at the Radboud University Nijmegen Medical Centre and was conducted simultaneously for children within a family. Psychostimulants were discontinued for at least 48\u00a0h before testing took place (Pelham et\u00a0al. 1999). Participants that took other medication than stimulants to suppress their symptoms of ADHD were also off medication during testing. The medication of these children was gradually decreased in line with standard procedures to allow for sufficient wash-out. Children were motivated with small breaks. At the end of the session, a gift worth approximately \u20ac 4, was given. Control children were tested in a similar way in a quiet room at their school. The study had medical-ethical approval.\nAnalyses\nThe percentage of missing data was less than 5% for each of the dependent variables. Missing data were replaced by using the Estimation Maximization procedure (Tabachnick and Fidell 2001). None of the dependent variables was normally distributed. Therefore, variables were successfully normalized by applying a Van der Waerden transformation (Statistical Package for the Social Sciences [SPSS] version 14). The Van der Waerden transformation transforms raw scores into z-scores corresponding to the estimated cumulative proportion of the distribution corresponding to a particular rank. It is defined by the formula r\/(w\u00a0+\u00a01), in which w is the sum of the case weights and r is the rank, ranging from 1 to w (Lehmann 1975). Cases are given different weights by means of simulated replication. The value of the new standardized variable equals the sum of case weights (SPSS version 14). This transformation has two important advantages: It handles the (extreme) influence outliers may have on the data, by ranking them as (very) high or low within the normal distribution, and the comparison between the variables was facilitated since the variables were all depicted on the same scale. Homogeneity of variance was tested by calculating Fmax (ratio of the largest cell variance to the smallest). Since sample sizes were relatively equal (i.e. within a ratio of 4 to 1 or less), an Fmax of 10 and lower was acceptable (Tabachnick and Fidell 2001). For all six normalized variables, the ratio was well within acceptable limits (all below 1.37). Alpha was set at .01 for all tests. Following Cohen\u2019s guidelines (Cohen 1988), effect sizes were defined in terms of the percentage of explained variance: 1, 9 and 25% were used to define small, medium, and large effects. These figures translate into \u03b72-values of 0.01, 0.06 and 0.14.\nLinear mixed models were used for the analyses. The linear mixed model expands the general linear model so that the data are permitted to exhibit correlated variability. This model allows for the investigation of group differences while correcting for the non-independency of data (i.e. more than one child participated per family, which resulted in related measurements within groups and between groups). In first instance, we tested the main effects of possible confounders (hand, gender, IQ, and age) on performance on the three different tasks. This was done within the control group to avoid dependency with the factor group. Thereafter, interactions between group and the confounders were examined to investigate whether effects of possible confounders were comparable across groups. In second instance, we investigated whether group differences existed for each task measure. Group was used as factor (four groups: proband, affected sibling, non-affected sibling, and control), age (linear and\/or curvilinear) as covariate(s), and family as random effect to account for within family correlation. Pairwise comparisons were used to compare groups and it was analyzed whether a linear trend was present in polynomial group contrasts. It was expected that probands and affected siblings performed worse than controls (mainly on the motor timing measures but not or to a lesser extent on the other motor measures), indicating an association between motor timing deficits and ADHD. It was also expected that the non-affected siblings performed worse than controls and formed an intermediate group in between their affected siblings and controls, suggesting motor timing deficits were related to a familial susceptibility to the disorder. The Conners\u2019 Total ADHD scale (averaged across parents and teachers) was used as an additional covariate in the analyses to rule out that possible deficits in the non-affected siblings group could be attributed to sub-clinical ADHD symptoms in this group. Correlations (with 95% confidence intervals) were calculated between siblings to test the familial resemblance of the motor measures (Statistical Analysis for Genetic Epidemiology [S.A.G.E] 5.3.1, 2007).\nResults\nTesting of possible confounders\nMain effects of hand, gender, IQ, and age\nWe tested for the effects of hand, gender, IQ, and age within the control group to avoid dependency with the factor group. No significant effect of hand was found on the Baseline Speed task (F(1, 271.0)\u00a0=\u00a00.05, P\u00a0=\u00a0.83, \u03b7p2\u00a0<\u00a0.01), but there was a significant effect on the Tapping task (F(1, 271.0)\u00a0=\u00a05.99, P\u00a0=\u00a0.02, \u03b7p2\u00a0=\u00a0.02). Control children were faster and less variable, when performing the Tapping task with their right hand than their left hand, likely reflecting an effect of hand dominance. The Motor Timing task was only performed with the preferred hand. Gender had no effect on the Motor Timing task and Baseline Speed task (F(1, 241.5)\u00a0=\u00a00.03, P\u00a0=\u00a0.87, \u03b7p2\u00a0<\u00a0.01 and F(1, 229.5)\u00a0=\u00a00.36, P\u00a0=\u00a0.55, \u03b7p2\u00a0<\u00a0.01, respectively), but had an effect on the Tapping task (F(1, 225.1)\u00a0=\u00a014.91, P\u00a0<\u00a0.001, \u03b7p2\u00a0=\u00a0.05). Control boys were faster and less variable in their tapping performance than control girls. No effect of IQ was found on the Motor Timing task, Baseline Speed task, or Tapping task (F(1, 258.4)\u00a0=\u00a00.20, P\u00a0=\u00a0.66, \u03b7p2\u00a0<\u00a0.01, F(1, 254.8)\u00a0=\u00a00.06, P\u00a0=\u00a0.81, \u03b7p2\u00a0<\u00a0.01 and F(1, 259.9)\u00a0=\u00a00.95, P\u00a0=\u00a0.33, \u03b7p2\u00a0<\u00a0.01, respectively). In order to assess whether age could be best modeled in linear and\/or curvilinear terms, we first analyzed the linear effect of age and then in a second model analyzed the quadratic effect of age, while keeping the linear term for age in the model. In this manner, the incremental contribution of the curvilinear relation with age over and above the linear relation was tested. Age (in linear terms) had a strong effect on the Motor Timing task, Baseline Speed task, and Tapping task (F(1, 252.7)\u00a0=\u00a026.65, P\u00a0<\u00a0.001, \u03b7p2\u00a0=\u00a0.22, F(1, 239.1)\u00a0=\u00a0292.38, P\u00a0<\u00a0.001, \u03b7p2\u00a0=\u00a0.57, and F(1, 244.5)\u00a0=\u00a0278.31, P\u00a0<\u00a0.001, \u03b7p2\u00a0=\u00a0.57, respectively). Older control children were more accurate, faster and less variable in their motor output. Results indicated that when both age terms were implemented in the model, no curvilinear effect of age was present on the Motor Timing task (F(1, 259.3)\u00a0=\u00a01.34, P\u00a0=\u00a0.25), but there was on the Baseline Speed task and Tapping task (F(1, 257.8)\u00a0=\u00a020.59, P\u00a0<\u00a0.001 and F(1, 258.3)\u00a0=\u00a012.47, P\u00a0<\u00a0.001, respectively). No significant interactions were present between gender and age, whether age was modelled in linear or curvilinear terms.\nInteractions between group and possible confounders\nGroup did not interact with hand on the Baseline Speed task (F(3, 816.0)\u00a0=\u00a01.14, P\u00a0=\u00a0.33, \u03b7p2\u00a0<\u00a0.01), but did interact marginally significantly with hand on the Tapping task (F(3, 816.0)\u00a0=\u00a02.73, P\u00a0=\u00a0.04, \u03b7p2\u00a0=\u00a0.01). Group did not interact with gender on the Motor Timing, Baseline Speed, or Tapping tasks (F(3, 762.9)\u00a0=\u00a01.96, P\u00a0=\u00a0.12, \u03b7p2\u00a0<\u00a0.01, F(3, 743.3)\u00a0=\u00a00.52, P\u00a0=\u00a0.67, \u03b7p2\u00a0<\u00a0.01, and F(3, 751.8)\u00a0=\u00a00.53, P\u00a0=\u00a0.66, \u03b7p2\u00a0<\u00a0.01, respectively) nor with IQ (F(3, 730.1)\u00a0=\u00a02.01, P\u00a0=\u00a0.11, \u03b7p2\u00a0=\u00a0.01, F(3, 714.3)\u00a0=\u00a01.28, P\u00a0=\u00a0.28, \u03b7p2\u00a0=\u00a0.01, and F(3, 727.2)\u00a0=\u00a02.11, P\u00a0=\u00a0.09, \u03b7p2\u00a0=\u00a0.01, respectively), nor with linear age (F(3, 732.0)\u00a0=\u00a01.67, P\u00a0=\u00a0.17, \u03b7p2\u00a0=\u00a0.01, F(3, 731.1)\u00a0=\u00a00.88, P\u00a0=\u00a0.45, \u03b7p2\u00a0<\u00a0.01, and F(3, 755.2)\u00a0=\u00a01.36, P\u00a0=\u00a0.26, \u03b7p2\u00a0=\u00a0.01, respectively), nor with curvilinear age (F(3, 727.1)\u00a0=\u00a01.61, P\u00a0=\u00a0.19, \u03b7p2\u00a0<\u00a0.01, F(3, 733.2)\u00a0=\u00a00.51, P\u00a0=\u00a0.67, \u03b7p2\u00a0<\u00a0.01, and F(3, 754.4)\u00a0=\u00a01.21, P\u00a0=\u00a0.31, \u03b7p2\u00a0<\u00a0.01, respectively).\nBased on the results of these analyses, it was decided to average the measures across hands to simplify results, since no group differences were found for the percentage of right- and left-handed (Table\u00a03) and since there was only a marginal significant interaction of small effect between group and hand for one of the tasks (Tapping). Furthermore, not included as covariates were IQ (had no effect on motor performance and did not interact with group) and gender (had only a small effect on one of the tasks and even in the opposite direction i.e. boys performing better than girls, and gender did not interact with group). Both the linear and curvilinear effects of age were included as covariates in the analyses for the Baseline Speed task and Tapping task. Only the linear effect of age was included as covariate in the analyses for the Motor Timing task. Raw means and SDs are presented in Table\u00a04.\nTable\u00a04Means and standard deviations of the motor measures in msDependent variableProbandAffected siblingNon-affected siblingControl\u03b7p2ContrastsMSDMSDMSDMSDMotor timing\u00a0\u00a0\u00a0\u00a0Accuracy98199997961,0071141,020100.031\u00a0=\u00a02\u00a0>\u00a03\u00a0=\u00a04\u00a0\u00a0\u00a0\u00a0Variability389265375265344260295218.101\u00a0=\u00a02\u00a0>\u00a03\u00a0>\u00a04Baseline Speed\u00a0\u00a0\u00a0\u00a0Speed35575356853538135178.021\u00a0=\u00a02\u00a0>\u00a03\u00a0=\u00a04\u00a0\u00a0\u00a0\u00a0Variability13285131871238211775.031\u00a0=\u00a02\u00a0>\u00a03\u00a0=\u00a04Tapping\u00a0\u00a0\u00a0\u00a0Speed23939248482565124948.01ns\u00a0\u00a0\u00a0\u00a0Variability4620482345204418.01nsNote: 1\u00a0=\u00a0Probands; 2\u00a0=\u00a0Affected Siblings; 3\u00a0=\u00a0Non-Affected Siblings; 4\u00a0=\u00a0Controls. ns\u00a0=\u00a0not significantScores were averaged across hands for the Baseline Speed and TappingOutliers (|z|\u00a0>\u00a03) were removed\nEndophenotypic analyses\nMotor Timing task\nA significant small effect of group was found for accuracy (F(3, 532.9)\u00a0=\u00a07.21, P\u00a0<\u00a0.001, \u03b7p2\u00a0=\u00a0.03). Pairwise comparisons indicated that probands and affected siblings did not differ from each other (P\u00a0=\u00a0.21). Both differed significantly from controls (P\u00a0<\u00a0.001 and P\u00a0=\u00a0.02, respectively): probands and (to a lesser extent) affected siblings tended to under-produce the 1,000\u00a0ms interval (M\u00a0=\u00a0981\u00a0ms and M\u00a0=\u00a0997\u00a0ms, respectively) compared to controls (M\u00a0=\u00a01,020\u00a0ms). A tendency to under reproduce appeared to be associated with ADHD, but was not convincingly related to a familial predisposition for the disorder, since non-affected siblings did not show this tendency: they differed significantly from probands (P\u00a0<\u00a0.001), marginally significantly from affected siblings (P\u00a0=\u00a0.02) but not from controls (P\u00a0=\u00a0.08) (see Fig.\u00a01). Using the Conners\u2019 Total ADHD score as covariate did not change the difference between non-affected siblings and controls (P\u00a0=\u00a0.04). However, a polynomial group contrast indicated a linear trend to be present (Contrast Estimate [CE]\u00a0=\u00a00.29, P\u00a0<\u00a0.001), suggesting probands performed most abnormal, followed by affected siblings, then non-affected siblings and then controls. Siblings marginally significantly resembled each other (r\u00a0=\u00a0.11 confidence interval [CI\u00a0=\u00a0.02\u2013.19], P\u00a0=\u00a0.02).\nFig.\u00a01Accuracy and variability of motor timing (adjusted for the linear effect of age) in probands, affected siblings, non-affected siblings and control children. Error bars represent 1 standard error from the mean\nGroups also differed with respect to the variability of motor timing (medium effect) (F(3, 524.6)\u00a0=\u00a025.12, P\u00a0<\u00a0.001, \u03b7p2\u00a0=\u00a0.10). Pairwise comparisons indicated that probands and affected siblings were equally variable (P\u00a0=\u00a0.45) and both were more variable than controls (both P\u00a0<\u00a0.001), suggesting ADHD and variability in motor timing were associated. Moreover, variability in motor timing appeared related to a familial predisposition for the disorder, since non-affected siblings formed an intermediate group: They significantly differed from probands, affected siblings, and controls (P\u00a0<\u00a0.001, P\u00a0=\u00a0.009 and P\u00a0<\u00a0.001, respectively). Using the Conners\u2019 Total ADHD score as covariate did not change the difference between non-affected siblings and controls (P\u00a0=\u00a0.001). A linear group contrast was present (CE\u00a0=\u00a0\u22120.49, P\u00a0<\u00a0.001). A significant familial resemblance was found for variability (r\u00a0=\u00a0.29 [CI\u00a0=\u00a0.20\u2013.38], P\u00a0<\u00a0.001). These findings lend support for variability of motor timing as endophenotype, though the accuracy of motor timing appears only to be associated with ADHD and not conclusively related to a familial susceptibility for the disorder (Fig.\u00a01).\nBaseline Speed task\nA small but significant effect of group was found for speed (F(3, 537.2)\u00a0=\u00a06.92, P\u00a0<\u00a0.001, \u03b7p2\u00a0=\u00a0.02). Pairwise comparisons revealed that probands and affected siblings did not differ from each other (P\u00a0=\u00a0.82) and both were slower than controls (P\u00a0<\u00a0.001 and P\u00a0=\u00a0.007, respectively), indicating a relationship between speed and ADHD. It appeared that speed was not related to a familial vulnerability for ADHD, since non-affected siblings differed from probands and affected siblings (P\u00a0<\u00a0.001 and P\u00a0=\u00a0.01, respectively), but not from controls (P\u00a0=\u00a0.84). Using the Conners\u2019 Total ADHD score as covariate did not change the difference between non-affected siblings and controls (P\u00a0=\u00a0.99). Nevertheless, polynomial group contrasts indicated a linear trend (CE\u00a0=\u00a0\u22120.14, P\u00a0=\u00a0.001), suggesting probands to perform most abnormal, followed by affected siblings, then non-affected siblings, and then controls. A significant correlation between siblings was found (r\u00a0=\u00a0.29 [CI\u00a0=\u00a0.20\u2013.38], P\u00a0<\u00a0.001).\nGroups differed somewhat (small effect size) in the variability on the Baseline Speed task (F(3, 555.1)\u00a0=\u00a06.96, P\u00a0<\u00a0.001, \u03b7p2\u00a0=\u00a0.03). Pairwise comparisons indicated that probands and affected siblings were equally variable (P\u00a0=\u00a0.74) and both groups were more variable than normal controls (P\u00a0<\u00a0.001 and P\u00a0=\u00a0.003, respectively), signaling an association between ADHD and variability in self generated motor output. Again, non-affected siblings did differ from probands (P\u00a0=\u00a0.003) and affected siblings (P\u00a0=\u00a0.04), but not from controls (P\u00a0=\u00a0.38). Using the Conners\u2019 Total ADHD score as covariate did not change the difference between non-affected siblings and controls (P\u00a0=\u00a0.41). Nevertheless, a linear group contrast was present (CE\u00a0=\u00a0\u22120.22, P\u00a0<\u00a0.001). Variability correlated between siblings (r\u00a0=\u00a0.16 [CI\u00a0=\u00a0.07\u2013.25], P\u00a0<\u00a0.001). These findings suggest slow and variable motor output in response to an external cue is associated with ADHD, but probably not related to a familial vulnerability for ADHD, since motor output of non-affected siblings resembles that of normal controls more than that of their affected siblings (Fig.\u00a02).\nFig.\u00a02Speed and variability of Baseline Speed (adjusted for the linear and curvilinear effects age) in probands, affected siblings, non-affected siblings and control children. Error bars represent 1 standard error from the mean. Scores were averaged across hands\nTapping Task\nNo significant effect of group was found for speed (F(3, 547.4)\u00a0=\u00a01.88, P\u00a0=\u00a0.13, \u03b7p2\u00a0=\u00a0.01) or variability (F(3, 553.6)\u00a0=\u00a01.95, P\u00a0=\u00a0.12, \u03b7p2\u00a0=\u00a0.01). No significant linear trend was present in polynomial group contrasts for speed or variability (CE\u00a0=\u00a00.10, P\u00a0=\u00a0.04 and CE\u00a0=\u00a0\u22120.12, P\u00a0=\u00a0.03, respectively). Siblings did resemble each other in the speed and variability of tapping (speed: r\u00a0=\u00a0.27 [CI\u00a0=\u00a0.18\u2013.36], P\u00a0<\u00a0.001; variability: r\u00a0=\u00a0.18 [CI\u00a0=\u00a0.09\u2013.27], P\u00a0<\u00a0.001). These findings indicated that speed and variability in self generated motor output were not familially associated with ADHD (Fig.\u00a03).\nFig.\u00a03Speed and variability of tapping (adjusted for the linear and curvilinear effects of age) in probands, affected siblings, non-affected siblings and control children. Error bars represent 1 standard error from the mean. Scores were averaged across hands\nSince there were group differences for speed and variability of externally cued motor output (Baseline Speed task), the issue was raised whether the deficits found on the Motor Timing task were primarily related to these group differences in basic motor output. Therefore, analyses were undertaken whereby the speed on the Baseline Speed task was used as an additional covariate in the analyses on accuracy on the Motor Timing task. The variability on the Baseline Speed task was used as additional covariate for the analyses on variability on the Motor Timing task.\nThe effect of group on accuracy on the Motor Timing task remained significant after accounting for speed on the Baseline Speed task (F(3, 532.4)\u00a0=\u00a07.51, P\u00a0<\u00a0.001, \u03b7p2\u00a0=\u00a0.03). The unadjusted means of the raw (unstandardized) data of the accuracy on the Motor Timing task for probands, affected siblings, non-affected siblings, and controls were: 981, 997, 1,007, and 1,020\u00a0ms, respectively. The adjusted means after covarying for speed on the Baseline Speed task were: 979, 998, 997, and 1,018\u00a0ms, respectively.\nThe same was true for variability of motor timing: the medium effect of group remained significant after accounting for variability on the Baseline Speed task (F(3, 528.2)\u00a0=\u00a021.72, P\u00a0<\u00a0.001, \u03b7p2\u00a0=\u00a0.09). The unadjusted means of the raw (unstandardized) data of the variability on the Motor Timing task for probands, affected siblings, non-affected siblings, and controls were: 389, 375, 344, and 295\u00a0ms, respectively. The adjusted menas after covarying for the variability on the Baseline Speed task were: 438, 428, 424, and 316\u00a0ms, respectively. Group contrasts for accuracy and variability as reported above also remained unchanged. These findings suggest that the deficits found on the Motor Timing task can not be explained by the deficits found in basic motor output.\nDiscussion\nThere was investigated whether accuracy and variability of motor timing were viable endophenotypic candidates as reflected by poor performance on these measures in children with ADHD (i.e. indicating an association between the deficits and the disorder), reflected by poor performance in non-affected siblings in between their affected siblings and controls (i.e. suggesting a relation between the deficits and a familial susceptibility for the disorder), and reflected by sibling correlations (i.e. signalling familial resemblance for deficits). We administered two motor tasks in addition to a motor timing task, in order to investigate whether deficits were specifically related to the timing of motor output or whether deficits were also observed in tasks requiring motor output without any timing demands.\nProbands and affected siblings were dissociated from controls with respect to accuracy of motor timing. Both groups tended to under-produce the 1,000\u00a0ms compared to control children (who tended to over-produce the interval). This finding has been reported previously using exactly the same task (Van Meel et\u00a0al. 2005) and is comparable to some other studies documenting on under estimation\/(re)production in patients with ADHD compared to controls (see for review Toplak et\u00a0al. 2006). These findings suggest a relation between under reproduction (possibly reflecting a somewhat speeded internal clock and\/or impulsivity) and ADHD. The findings were less convincing with respect to non-affected siblings: despite a linear trend in group contrasts, their accuracy of motor timing was more like controls than that of affected siblings. Furthermore, correlations between siblings on accuracy were also modest suggesting familial resemblance for accuracy was present but not strongly. Therefore, accuracy of motor timing seems not to be a strong endophenotypic candidate.\nVariability of motor timing, however, convincingly met all characteristics of an endophenotype as investigated in our study: Probands and affected siblings were clearly more variable in their motor output than controls, non-affected siblings also differed significantly from controls and had variability scores in between their affected siblings and controls, and greater variability in motor timing was evidently familial. Greater variability in motor timing in children with ADHD compared to controls concurs with previous studies (Pitcher et\u00a0al. 2002; Rubia et\u00a0al. 1999, 2003; Toplak et\u00a0al. 2006; Van Meel et\u00a0al. 2005) and suggests variability in motor timing is characteristic of ADHD. Our study adds important knowledge to this topic, showing that non-affected siblings portray a similar type of variability in their motor timing and that siblings resemble each other in the variability of motor timing. These findings suggest the variability in motor timing is not only associated with the disorder, but is related to familial vulnerability for ADHD, which may make it a useful tool in future studies aimed at unraveling the genetic underpinnings of ADHD.\nAll in all, variability of motor timing may form a fruitful endophenotypic candidate. However, group differences were also present on motor measures that did not require a timing component, suggesting motor deficits not to be specifically related to timing but to be more generalized. Probands and affected siblings were significantly slower and more variable than controls concerning motor output in response to an external cue (Baseline Speed). These findings of slow and variable responding are in line with a study using the same task in young children at risk for ADHD (Kalff et\u00a0al. 2005) and suggests slow and variable responding are characteristic of ADHD. However, in contrast to variability of motor timing, non-affected siblings did not differ from controls with respect to speed and variability of externally cued motor output. So, despite the finding that slow and variable motor output is associated with ADHD, it does not appear to be convincingly associated with a familial vulnerability for the disorder.\nGiven that poor motor output was observed in probands and affected siblings (Baseline Speed), it was surprising that no such abnormalities were found in self-generated motor output (Tapping). Probands and affected siblings had a normal speed and variability in self generated motor output. The discrepancy in results between both tasks may lie in the suggestion that Baseline Speed may have required some form of cognitive processing (i.e. registering a stimulus and responding to it), whereas Tapping only required executing a motor action. The normal performance of children with ADHD on the Tapping task is in line with some studies (Seidman et\u00a0al. 1997, 2000), but in contrast with others (Toplak et\u00a0al. 2006). This might be explained by the important difference in timing requirements necessary in the Tapping task used here and in some previous studies. Here, no timing was required to execute the Tapping task. The instruction was simply to press the button as often as possible within a certain time interval. However, in other studies the child was required to modify his\/her tapping rate to be in synchrony with the stimulus and maintain the rhythm in the absence of the stimulus (Rubia et\u00a0al. 1999; Toplak et\u00a0al. 2006). These tasks load differently on timing processes. Our findings suggest that self-generated motor output does not form a viable area of endophenotypic research, even though speed and variability of self-generated motor output correlate within families.\nSince group differences were not only present on measures of motor timing, but also on measures of motor output (Baseline Speed), it was investigated whether motor timing impairments may be due to deficits in basic motor output. This appeared not to be the case. Even when speed and variability of basic motor output were used as covariates in the analyses on motor timing, group differences for motor timing remained. These findings suggest abnormalities in the accuracy and variability of motor timing are relatively independent of general deficits in basic motor output (Keele et\u00a0al. 1985) and suggest abnormalities in motor timing are predominantly related to timing operations and not to motor functioning. Since motor timing appears predominantly regulated by the cerebellum, basal ganglia and their reciprocal connections with the cerebral cortex (Handy et\u00a0al. 2003; Harrington et\u00a0al. 1998; Ivry 1996; Nenadic et\u00a0al. 2003), subcortical regions in addition to cortical regions might be important in the etiology of ADHD (Halperin and Schulz 2006).\nLimitations\nWe did not administer the PACS interview for nonaffected siblings. This might have resulted in undetected ADHD cases in the nonaffected sibling group, which in turn might explain the deficits of this group. However, we do not believe this to be the case, because (1) all siblings were thoroughly screened and, if they scored clinically on any of the screening questionnaires, the PACS interview was administered, and (2) even when symptom severity was used as a covariate, group differences between nonaffected siblings and controls remained significant. Furthermore, including measures aimed at isolating aspects of timing performance, such as time estimation and reproduction paradigms, in addition to the measure of time production would have enhanced the comprehensiveness of our findings with respect to the internal clock. Some previous studies using time estimation and reproduction tasks have reported that children with ADHD performed abnormally (see for review Toplak et\u00a0al. 2006). We documented previously on time reproduction deficits present in both children with ADHD as well as their non-affected siblings (Rommelse et\u00a0al. 2007b), suggesting familial deficits in timing in ADHD generalize across timing paradigms and extent beyond motor timing as reported in the current study.\nConclusions\nVariability in motor timing appears a useful endophenotypic candidate: It is clearly associated with ADHD, it is also present in non-affected siblings, and it correlates within families. Accuracy (under- versus over production) in motor timing appears less useful: even though accuracy is associated with ADHD (probands and affected siblings have a tendency to under-produce compared to controls), non-affected siblings did not exhibit this tendency and sibling correlations were only marginally significant. There were group differences in motor speed and variability (Baseline Speed task): probands and affected siblings were slower and more variable in their motor output as response to an external cue. Even though siblings resembled each other in their speed and variability, non-affected siblings performed more like controls. These findings suggest that speed and variability of externally cued motor output are associated with having ADHD, but probably not related to a familial vulnerability for the disorder. Interestingly, the speed and variability in self-generated motor output (Tapping) is normal in probands and affected siblings, making it unsuitable to unravel underlying vulnerabilities leading up to ADHD. Deficits in motor timing cannot be explained by deficits already present in basic motor output without a timing component (Baseline Speed), suggesting abnormalities in motor timing are predominantly related to deficient timing operations but not to deficient motor functioning. The finding that deficits in motor timing run in ADHD-families suggests this to be a fruitful domain for further exploration in relation to the genetic underpinnings of ADHD.","keyphrases":["adhd","endophenotype","motor timing","siblings","motor speed","motor variability"],"prmu":["P","P","P","P","P","R"]}
{"id":"Biotechnol_Lett-3-1-2045120","title":"Solute transport in orthorhombic lysozyme crystals: a molecular simulation study\n","text":"Long-time equilibrium molecular dynamics simulations were performed to study the passage of a substrate, l-arabinose, through nanopores of orthorhombic hen egg white lysozyme crystals. Cross-linked protein crystals (CLPC), as novel biological nanoporous media, consist of an extensive regular matrix of chiral solvent-filled nanopores via which ions and solutes, e.g. sugars and amino acids, travel in and out. We studied the diffusive motion of arabinose inside protein channels. The computed diffusion coefficients within the crystal were orders of magnitudes lower relative to the diffusion coefficient of the solute in water. This study is valuable for understanding the nature of solute\u2013protein interactions and transport phenomena in CLPCs and provides an understanding of biocatalytic and bioseparation processes using CLPC.\nIntroduction\nProtein crystals are conventionally thought as important elements in structure determination of biomolecules as well as in protein purification (Johnson and Philips 1965; Blundell et\u00a0al. 2003; Haring and Schnrier 1991). They contain pores that range from approx. 0.3\u00a0nm to 10\u00a0nm and occupy 25\u201375% of the crystal volume (Bishop and Richards 1968). Their porosity is comparable to that of inorganic porous catalysts and sorbents, such as zeolites and silica-gel (Bishop and Richards 1968; Vilenchik et\u00a0al. 1998; Margolin and Navia 2001). More robust cross-linked protein crystals (CLPC) have been applied successfully as extremely stable biocatalysts (Vilenchik et\u00a0al. 1998) and as selective (chiral) separation media (Margolin and Navia 2001). The complex crystal structure of the protein contains many functional active sites. Moreover, it resists environmental degradation once it is cross-linked (Vilenchik et\u00a0al. 1998; Margolin and Navia 2001).\nUnderstanding the nature of transport of solutes in CLEC is relevant to many biotechnological processes (Margolin and Navia 2001; Margolin and Vilenchik 1996; Margolin et al. 2000; Vuolanto et\u00a0al. 2002, 2004; Jokela et\u00a0al. 2002). Properties of intracrystalline water molecules and ions, and their transport through the crystal, are essential to many of these applications. Apart from their practical use, protein crystals also provide a unique model to study transport phenomena in protein channels in cell membranes since the structure of water-filled pores in the crystals is determined to atomic resolution by X-ray diffraction (Morozova et\u00a0al. 1996). There are effects of both the solute on protein and protein on the solute (Nagendra et\u00a0al. 1998). The latter effect is more pronounced in protein crystals, where proteins make up the lining of small pores and water and ions affect the passage of substances through such pores (Nagendra et\u00a0al. 1998; Eisenberg and Kauzmann 1969). Several studies have focused on the experimental determination of the solute and water transport in protein crystals (Bishop and Richards 1968; Morozov et\u00a0al 1995; Velev et\u00a0al. 2000; Cvetkovic et\u00a0al. 2005a, b; O\u2019Hara et al. 1995). Recent studies on diffusion in protein crystals still leave open questions concerning the mobility of solutes and protein\u2013solute interactions near proteins that constitute the pore walls (Cvetkovic et\u00a0al. 2005a, b). In addition to advanced experimental techniques, versatile computational tools are generally needed to correlate reactivity of the protein and transport of solute with nanoporosity of the enzyme crystals at atomistic level (Malek et\u00a0al. 2004, 2005).\nMolecular dynamics simulations with explicit representation of molecules and ions should, in principle, provide realistic information about the diffusive motion of water, individual solute molecules and ions at atomic resolution. These simulations, however, are only practical at longer time and length scales. Due to heterogeneity of the protein surface, it is necessary to treat transport of molecules at the protein\u2013water interface as a local property (Bon et\u00a0al. 1999). There have been a number of such studies to determine how the water molecules on the protein surface are perturbed from the bulk (Bizzari and Cannistraro 1996; Bizzari et\u00a0al. 1996). To our knowledge, there have been no such studies on dynamical motion of small solute molecules in a protein crystal. The mobility of small dipolar molecules, such as sugars and amino acids, in the vicinity and far from the protein surface in a confined biological channel, exhibits different dynamical and structural behavior from that in a free protein in solution (Velev et\u00a0al. 2000; Cvetkovic et\u00a0al. 2005a, b; O\u2019Hara et al. 1995; Malek et\u00a0al. 2004, 2005).\nIn the crystalline form, proteins are in a highly ordered three-dimensional structure, where the protein molecules strongly bind to each other with specific intermolecular interactions. Protein crystals are particularly interesting materials for chiral separations (Margolin and Navia 2001; Margolin and Vilenchik 1996; Margolin et al. 2000; Vuolanto et\u00a0al. 2002, 2004; Jokela et\u00a0al. 2002). The crystal framework poses an asymmetric environment and is made of l-amino acids. Enzymes in crystals have also specific binding sites for the substrates and cofactors. In addition, protein crystals are nanoporous materials. Based on these properties, many CLPCs have recently been used in chiral separation of racemic mixtures (Vuolanto et\u00a0al. 2004; Jokela et\u00a0al. 2002). These novel insights are valuable for biotechnological applications to devise highly selective biocatalysts, biosensors and bioseparators.\nHere, we use orthorhombic lysozyme as a simple model enzyme to study diffusion of a chiral substrate through pore network of the enzyme crystal. Its well-known and extremely stable molecular structure makes it a good choice for computational studies. Moreover, lysozyme belongs to a large class of enzymes known as glycosidases. These are extremely efficient glycosidic hydrolysis catalysts. Biocatalysis inside the protein lattice has advanced features of product separation and catalyst recovery. Once the catalytic reaction occurs in lysozyme crystals, understanding the transport of products (sugars) through the lattice becomes an important issue. In short, the simple and well-known structure of lysozyme as model protein, the catalytic activity of lysozyme for hydrolyzing the glycosidic bond, the practical potential of protein crystals as biochemical porous media for chiral separation and as a model for biological channels, in general, were our first motivations for choosing a simulation model based on transport of sugar substrate into orthorhombic lysozyme lattice. Here we provide, for the first time, long-time MD simulations of diffusion of l-arabinose in orthorhombic hen egg white lysozyme crystals. Fundamental questions are; do solute molecules move by translation and\/or rotation? How solute\u2013protein interactions influence transport of a chiral solute through the protein crystal? What is the detailed nature of the diffusive motion of solute molecules in a lysozyme crystal and what are the length scales and the time scales (dynamics) of all those events.\nComputational methodology\nWe use MD simulations to examine diffusion of l-arabinose (Ara) in a fully hydrated orthorhombic lysozyme crystal. Ara is an aldopentose (Fig.\u00a01) that has been extensively used as a substrate to probe diffusion properties of protein channels (Nikaido and Rosenberg 1981). Lysozyme consists of 129 amino acids with 1001 non-hydrogen atoms. The crystal structures of lysozyme, entry 1AKI (Artymiuk et\u00a0al. 1982), is taken from the Brookhaven Protein Database and used as a starting point. Hydrogen atoms attached to aliphatic carbon atoms are incorporated with the latter, but the remaining 342 hydrogen atoms are treated explicitly, leading to 1,343 (pseudo-) atoms in total. The simulations are done at pH\u00a07. The amino acids Glu and Asp are taken to be deprotonated while Lys, Arg and His residues are protonated (Artymiuk et\u00a0al. 1982; Stocker et\u00a0al. 2000). This leads to +8 electron charges per protein molecule. Chloride ions are then added for electroneutrality. In an orthorhombic crystal, four protein molecules related by the crystallographic symmetry P212121 are placed in the unit cell with a\u00a0=\u00a05.9062\u00a0nm, b\u00a0=\u00a06.8451\u00a0nm, and c\u00a0=\u00a03.0517\u00a0nm. Repeating the unit cell along crystallographic axes generates the pore network (Fig.\u00a02). Channels and cavities within the orthorhombic lysozyme lattices are determined by using a procedure explained elsewhere (Malek et\u00a0al. 2004), based on HOLE and CHANNEL algorithms (Smart et\u00a0al. 1996; Kisljuk et\u00a0al. 1994). The radius of a pore is determined at any given distance along the pore axis by calculating the maximum size for a spherical probe to still fit in the pore without overlap with the van der Waals radii of atoms in the pore wall (Kisljuk et\u00a0al. 1994).\nFig.\u00a01Ball-and-stick model of l-arabinose. CHn groups are represented by united atomsFig.\u00a02Computer generated images of an orthorhombic lysozyme lattice, visualized along z- crystallographic axis. The solvent channels are shown in grey. A single unit cell is labelled by square and its sphere representation is shown in (b). Red lines represent water molecules. The surface representation in (a) was computed from the electron density calculations using the crystal structure data (1AKI) available in the Brookhaven protein data bank. Hydrophilic, hydrophobic and polar regions are shown in blue, red and green respectively\nSimulations are carried out using a fully atomistic 1\u00a0\u00d7\u00a01\u00a0\u00d7\u00a05 lysozyme lattice (a\u00a0=\u00a05.9062\u00a0nm, b\u00a0=\u00a06.8451\u00a0nm, and c\u00a0=\u00a015.2585\u00a0nm). The single unit cell in Fig.\u00a02b is repeated five times along the z-axis, so that a long pore is constructed. We start from an initial configuration by random placing arabinose inside the lysozyme channel. The system contains 20 lysozyme molecules, 21 arabinoses, 6,239 crystallographic and non-crystallographic water molecules and 160 chloride ions, leading to 46,031 atoms in total. After that the system was equilibrated for 500\u00a0ps using harmonic position restraints (1000\u00a0kJ\u00a0mol\u22121\u00a0nm\u22122). Production runs were performed for another 26\u00a0ns, the last 25\u00a0ns of which was used for the analysis. Simulations are performed using GROMOS96 force field (van Gunsteren et\u00a0al. 1990, 1996). In our force field, interactions between atoms are divided into non-bonded interactions, between any pair of atoms that are within a given cut-off radius, and bonded interactions between atoms connected by chemical bonds. In case of the non-bonded interactions (electrostatic and van der Waals), a partial charge and parameters for repulsion and attraction are assigned to each atom. The bonded interaction consists of bond, angle and dihedral terms. Bond and angle bending are given by simple harmonic potentials. The torsional rotational potential for the dihedral angle is a periodic function with a 3-fold barrier. The partial charges and interaction parameters for all species are taken from GROMOS96 database (van Gunsteren et\u00a0al. 1990).\nIn our MD simulation, a cut-off of 1.4\u00a0nm is used for Van der Waals interactions, while a cut-off of 1\u00a0nm and PME with a grid spacing of 0.12\u00a0nm and fourth order interpolation are used for electrostatics interactions (Morozova et\u00a0al. 1996). During the simulations, the potential energy and the total energy are monitored in order to check if the system is in equilibrium. MD simulations are performed in a canonical (NVT) ensemble at 300\u00a0K. The temperature (300\u00a0K) is controlled by the Berendsen weak-coupling algorithm, separately for protein, Ara and solvent plus ions with a time constant of 0.1\u00a0ps. During the simulations, polar hydrogen atoms are treated as dummy atoms with an increased mass of 4\u00a0Da. This allowed the integration time step to be 5\u00a0fs. Simulations are done with the GROMACS package (Lindahl et\u00a0al. 2001; Berendsen et\u00a0al. 1995; http:\/\/www.gromacs.org). Visualization is done by using the VMD v1.8.3 (Humphrey et\u00a0al. 1996) commercial package.\nResults and discussions\nFigure\u00a02a shows an instantaneous configuration of the atomic model of the orthorhombic lysozyme lattice, constructed of 3\u00a0\u00d7\u00a03\u00a0\u00d7\u00a03\u00a0unit cells, whereas a single unit cell is framed in Fig.\u00a02b. In each unit cell, there is a main pore lies along the z-axis and there are no remarkable pores in the other directions (Malek et\u00a0al. 2004; Geremia et\u00a0al. 2005; Falkner et\u00a0al. 2005). Figure\u00a03a visualizes part of the main pores along the z-axis. The average pore radius is about 0.88\u00a0\u00b1\u00a00.02\u00a0nm. Figure\u00a03b shows the pore radius as a function of the pore axis. The profiles show that there are constricted zones inside each pore. The pore radius slowly decreases from over 0.95\u00a0nm to slightly less than 0.82\u00a0nm at its narrowest point. The charged residues Lys1, Lys13, Lys33, Lys96, Lys97, Lys116, Arg14, Arg21, Arg114, Asp87, and Asp119 belonging to the lysozyme molecules lie in the pore space. The motion of these residues may affect the pore shape and pore size during the transport of Ara. Our analysis shows that the effect of the protein fluctuations on pore shape is negligible, particularly for solutes of sizes much smaller than the pore diameter, such as Ara.\nFig.\u00a03(a) Visualized pore along z-axis in a 1\u00a0\u00d7\u00a01\u00a0\u00d7\u00a05 lysozyme lattice. (b) Pore radius profile along the pore axis\nThe averaged root mean square fluctuation (RMSF) of C-\u03b1 of each residue, calculated from the distance fluctuation matrix of a 25\u00a0ns trajectory in crystalline orthorhombic lysozyme is shown in Fig.\u00a04a. The root mean square fluctuations from the X-ray structure are averaged over 20 proteins. The fluctuation results (Fig.\u00a04a) are consistent with previous works on fluctuations and correlations in lysozyme crystals (H\u00e9ry et\u00a0al. 1997, 1998). The largest fluctuations are for the \u03b1-helix loops. The \u03b2-strand residues show a low mobility with values on the order of 0.1\u00a0nm. \u03b2-Strands exhibit a high degree of rigidity with moderate correlations, while only parts of \u03b1-helices are rigid or correlated (H\u00e9ry et\u00a0al. 1998). Although the protein atoms fluctuate slightly during the simulation, these are too small to cause significant changes in pore size or in diffusivity. Figure\u00a04b shows that the RMSF patterns are similar for all twenty lysozyme molecules. The residues Gly117, Gly71 and Asp101 show fluctuations above 0.10\u00a0nm. Among these, Gly117 displays the maximum fluctuations ranging from 0.15\u00a0nm to 0.25\u00a0nm. Most of the charged residues accessible from the pore space show fluctuations around 0.05\u00a0nm. The latter confirms that the pore shape and pore size are not affected by protein fluctuations during 25\u00a0ns MD simulation.\nFig.\u00a04(a) A typical root mean square fluctuation of the C-\u03b1 atoms of each residue of one of the 20 lysozyme molecules in the simulation box with respect to their average position. (b) RMSD of each resides of all lysozyme molecules. The curves are on the same scale. The full lines are shifted by 0.2\u00a0nm in the vertical direction\nIn order to study the motion of solutes inside orthorhombic lysozyme crystals, we take into account that proteins affect the dynamics of solute molecules. MD simulations provide information to the dynamic picture of the solute motion in the vicinity of protein molecules. The self-diffusion coefficient D, which has been widely used in both spectroscopic experiments and MD simulations, is a suitable parameter characterizing the dynamical behavior of solute in the water filled protein channels in protein crystals. The diffusion coefficient D, is related to the slope of the mean square displacement (MSD) of water molecules by the Einstein relation, which in d dimension is: where \nis the MSD of solute molecules during the time \u0394t, averaged over the ensemble of molecules in a d dimensional space, from the moment t0 that their motion is equilibrated in the pore space. Use of the Einstein equation for the determination of D requires a linear dependence of the MSD with time. For the determination of the solute diffusivity, the MSD\u2013t linear relation is usually fulfilled on time scales longer than a few to hundred nanoseconds (Bizzari and Cannistraro 1996).\nIn order to study diffusion of a solute (Ara) inside pore space of an orthorhombic lysozyme crystal, we have performed extensive MD simulations of Ara transport in a fully hydrated lysozyme crystal. Protein atoms are subject to move according to the equation of motions. Ara molecules are randomly incorporated inside the main pore along z-axis (Fig.\u00a03a). The dynamical motion of solute molecules was sampled each 10\u00a0ps during 25\u00a0ns simulation.\nFigure\u00a05a shows the displacement of nine Ara molecules along the z-axis as a function of time. The location of the solute molecules is sufficiently randomized during the simulation time, so that the diffusion properties do not depend on the arbitrary initial placement of the Ara molecules. The motion of Ara molecules displays many jumps, with little motion between jumps. A few Ara molecules travel within the pore space with displacement in the order of 5\u201310\u00a0nm, while a few others remain around the same axial (z) position in the pore region, moving rather in xy plane. Overall, solute molecules move freely, some travel all the way within the pore network, a few remain in the pore and some go deep into the pore and return after some time. At some points along the trajectory, a group of two or three Ara molecules establish hydrogen bonding and perform a collective motion as a united body. The latter is particularly observed in longer simulation times thereby bringing an artefact in to the diffusion analysis of single Ara molecules. To calculate the self-diffusion (D) of solute molecules in the pore, the Einstein relationship (Eq. 1) is used.\nFig.\u00a05(a) The displacement of nine arabinose molecules along the z-axis through the pore region of lysozyme crystal shown in Fig.\u00a02a. Different colours represent different Ara molecules. (b) Mean square displacement (MSD) of Ara molecules versus time\nFigure\u00a04b shows Mean square displacement values (MSD) values versus time for Ara molecules diffusing in the pore network of lysozyme crystal. Only trajectories of individual Ara molecules are included in calculation of MSD and therefore the collective motion of solute molecules is not included in Fig.\u00a05b. Overall, the log-log behavior of MSD vs. time (for 5\u00a0ns\u00a0<\u00a0t\u00a0<\u00a020) is linear with a slope close to one. This shows that in fully hydrated pores of the lattice, the Einstein relation, Eq. 1, can describe the diffusion of Ara. The diffusion coefficient of Ara in pores along z-axis is calculated from the intercept of the log\u2013log plot, that is (8.51\u00a0\u00b1\u00a00.064)\u00a0\u00d7\u00a010\u221213\u00a0m2\u00a0s\u22121. Recently, we have performed combined dynamic Monte-Carlo and Brownian dynamic (MC\u2013BD) simulations to study electrostatic and steric confinement effects on the mobility of spherical probes in orthorhombic and tetragonal lysosyme crystals (Malek et\u00a0al. 2004). The diffusion coefficient of a probe of size comparable to that of the hydrated Ara (0.86\u00a0\u00b1\u00a00.01\u00a0nm), calculated by MC\u2013BD simulations is (8.85\u00a0\u00b1\u00a00.05)\u00a0\u00d7\u00a010\u221213\u00a0m2\u00a0s\u22121, which is in good agreement with the MD prediction. This indicates that size exclusion is the main reason for uncharged solute transport in orthorhombic lysozyme crystals. The diffusion of solutes within lysozyme crystals has also been investigated using different experimental techniques (Velev et\u00a0al. 2000; Cvetkovic et\u00a0al. 2005a, b; O\u2019Hara et al. 1995). Velev et\u00a0al. (2000) studied the diffusion of surfactants in lysozyme crystals using fluorescent probes by means of quantitative fluorescence microscopy. The diffusion coefficients obtained range from 2 to 30\u00a0\u00d7\u00a010\u221214\u00a0m2\u00a0s\u22121.\nBy using 3-D laser scanning confocal microscopy visualizations, Cvetkovic et\u00a0al. (2005a, b) investigated detailed diffusion coefficients of some solutes, fluorescein and rhodamine B, into tetragonal, orthorhombic, and triclinic lysozyme crystals. The results showed that the transport of solute molecules depends on the chemical nature of solutes (e.g., hydrophobic vs. hydrophilic), charge and size of solutes and pore network within protein crystal. The diffusion of fluorescein, with an average diameter of 0.69\u00a0\u00b1\u00a00.02\u00a0nm, in orthorhombic lysozyme crystals was highly anisotropic and the diffusion coefficient was calculated as approx. 7.0\u00a0\u00b1\u00a00.5\u00a0\u00d7\u00a010\u221213\u00a0m2\u00a0s\u22121.\nThe diffusion coefficient of Ara calculated from our MD simulation is in reasonable agreement to these experiments. The diffusion rate is 4\u20135 orders of magnitude slower than that in free water (\u223c7.73\u00a0\u00d7\u00a010\u221210\u00a0m2\u00a0s\u22121 at 298\u00a0K) (Nikaido and Rosenberg 1981; Mogi et\u00a0al. 2007). The deviation is most likely as a result of single-point interactions between solute and side chains of the XI, which are usually characterized by hydrogen bindings and dipole-dipole interactions. Our preliminary analysis based on hydrogen bonds, number of contacts and minimum distance between Ara molecules and residues on the pore wall showed that the solute molecules spend considerable time in the vicinity of active site residues Lys96, Lys97, Lys116, Arg14, Arg21, and Asp87. This indicates that the orientation and translational-rotational motion of Ara play an important role in its transport through channels in orthorhombic lysozyme crystals.\nThese results suggest that in lysozyme lattice, and in contrast to the free lysozyme in solution, the interactions between dipolar solute and protein are mostly promoted by a large electric field on the residues far from the active site cleft (which are Asp52, Asp53, Glu35, and Asp99). Many of the latter charged amino acids that act as adsorption\/desorption sites for the solute are, in fact, buried, while the residues Lys96, Lys97, Lys116, Arg14, Arg21, and Asp87 are more easily accessible. Considerable experimental and computational evidence supports several aspects of the mechanism that has been proposed for the catalytic activity of the lysozyme in solution (Dao-Pin et\u00a0al. 1989; Bottoni et\u00a0al. 2005). In a manner similar to that proposed for free lysozyme, our simulation results can be validated based on finding the most favorable interaction of the substrate molecules in the mixture with the catalytic active centers of the enzyme molecules in lattice. Such qualitative agreement with experimental data corroborates predictive capabilities of our model. Moreover, our study highlights the importance of the protein\u2013solute interactions on the transport in protein crystals.\nDespite its simplicity and obvious limitations, this computational study provides insight into the main features of solute transport in protein crystals. Our study allows relating transport properties of the nanopores in protein crystals to solvent and ion motion as well as to protein fluctuations. Although our findings are in good qualitative and quantitative agreement with existing experimental data, more experimental studies are still needed by which we can compare our data directly. These results in combination with experimental information provide vital insights for understanding biocatalytic and chiral separation processes in CLPC.\nConclusion\nIn the present study, we performed MD simulations to study the motion of an aldopentose molecule (l-arabinose) in nanopores of a fully hydrated orthorhombic lysozyme crystal. The electrostatic and steric interaction inside the crystal channel significantly influences the diffusion of solute. No clear adsorption site in the crystal was detected in the simulation, most likely due to the low solute concentration. However, our analysis show that the solute molecules spend considerable time in the vicinity of active site residues Lys96, Lys97, Lys116, Arg14, Arg21, and Asp87. According to our results, the average mobility of solute molecules in lysozyme crystal channels is reduced orders of magnitude compared to that in free water. This is why crystalline proteins are easily accessible to solute molecules that are smaller than the necks of the crystal channels. The results are of practical interest to evaluate the time necessary to impregnate the crystals with ligands, water and solutes and provide valuable insights of solute\u2013protein interactions during solute transport in nanopores of protein crystals.","keyphrases":["lysozyme","molecular dynamics","l-arabinose","protein crystal","diffusion"],"prmu":["P","P","P","P","P"]}
{"id":"J_Mol_Med-4-1-2359832","title":"Analysis of human MDM4 variants in papillary thyroid carcinomas reveals new potential markers of cancer properties\n","text":"A wild-type (wt) p53 gene characterizes thyroid tumors, except for the rare anaplastic histotype. Because p53 inactivation is a prerequisite for tumor development, alterations of p53 regulators represent an alternative way to impair p53 function. Indeed, murine double minute 2 (MDM2), the main p53 negative regulator, is overexpressed in many tumor histotypes including those of the thyroid. A new p53 regulator, MDM4 (a.k.a. MDMX or HDMX) an analog of MDM2, represents a new oncogene although its impact on tumor properties remains largely unexplored. We estimated levels of MDM2, MDM4, and its variants, MDM4-S (originally HDMX-S) and MDM4-211 (originally HDMX211), in a group of 57 papillary thyroid carcinomas (PTC), characterized by wt tumor protein 53, in comparison to matched contra-lateral lobe normal tissue. Further, we evaluated the association between expression levels of these genes and the histopathological features of tumors. Quantitative real-time polymerase chain reaction revealed a highly significant downregulation of MDM4 mRNA in tumor tissue compared to control tissue (P < 0.0001), a finding confirmed by western blot on a subset of 20 tissue pairs. Moreover, the tumor-to-normal ratio of MDM4 levels for each individual was significantly lower in late tumor stages, suggesting a specific downregulation of MDM4 expression with tumor progression. In comparison, MDM2 messenger RNA (mRNA) and protein levels were frequently upregulated with no correlation with MDM4 levels. Lastly, we frequently detected overexpression of MDM4-S mRNA and presence of the aberrant form, MDM4-211 in this tumor group. These findings indicate that MDM4 alterations are a frequent event in PTC. It is worthy to note that the significant downregulation of full-length MDM4 in PTC reveals a novel status of this factor in human cancer that counsels careful evaluation of its role in human tumorigenesis and of its potential as therapeutic target.\nIntroduction\nThe murine double minute (MDM) family members are key regulators of levels and activity of the oncosuppressor p53 [1]. MDM2, acting as specific E3 ubiquitin ligase and as transcriptional repressor, is the best known among them. MDM2 overexpression has been observed in many human tumors characterized by tumor protein 53 (TP53) wild-type (wt) status, supporting the model of multiple means of p53 inactivation in tumor cells [2]. In recent years, another member of the MDM family, MDM4 (also named MDMX) has come into the limelight and its function in the inactivation of p53 has been revealed by molecular and genetic approaches [1, 3]. In particular, MDM4 can negatively regulate p53-mediated cell cycle arrest, a role that distinguishes its function from that of MDM2, more related to the suppression of p53-mediated apoptotic response [1, 3\u20135]. However, others have reported on the antiproliferative and proapoptotic effect of MDM4 in the presence of wild-type TP53 under conditions such as stress [6, 7]. Studies aimed at characterizing the human MDM4 (also named HDMX) status in human tumors have shown amplification of its locus in the presence of wild-type TP53 in breast cancers, glioblastoma, retinoblastoma, and soft-tissue sarcomas [8\u201311], confirming that it may contribute to p53 inactivation during tumorigenesis. The impact of human MDM4 on tumor properties as well as its behavior during tumor progression however still poorly explored.\nIn addition to the full-length (fl) protein, different splice variants of MDM4 have been described, two of which detected in human tumors. A short form named MDM4-S (a.k.a. HDMX-S) was identified first [12]. This variant derives from an alternative splicing that produces a truncated protein, containing essentially the p53-binding domain. It is a more potent inhibitor of p53 transcriptional activity than wt MDM4 and although present in both normal and tumor tissues, it is overexpressed specifically in tumor samples [11, 12]. Recently, our group has characterized another MDM4-splicing form, HDMX211 (hereafter named MDM4-211), in a thyroid tumor cell line and in some lung cancers [13]. This form derives from an aberrant splicing and produces a protein containing essentially the RING finger COOH-terminus, where the MDM2 binding site resides. As a consequence, this variant is able to stabilize MDM2 protein levels, thus contributing to inactivation of p53. These data, while underlying the complexity of MDM4 expression in human tumors, strongly encourage studies on the comparative analysis of this protein and its derivative forms in human cancer.\nThyroid tumors represent over 90% of all endocrine cancers and are characterized by different genetic alterations, among which TP53 mutations are confined quite exclusively to the infrequent anaplastic and poorly differentiated histotype [14]. According to current models of human carcinogenesis that consider inactivation of the oncosuppressor p53, a common feature of almost all tumors, MDM2 amplification and\/or overexpression have been reported in thyroid tumors too [15\u201317]. However, the overall frequency of MDM2 overexpression is only about 30%, suggesting the existence of other pathways of p53 inactivation.\nThe aim of our study has been to analyze papillary thyroid carcinomas (PTC), the most frequent thyroid cancer, for the presence of MDM4 and its derivative forms to investigate alterations of these proteins in this tumor histotype and correlate them with histopathological features. In addition, we have analyzed MDM2 levels to assess the potential relationship between alterations of the two MDM family members. Our data have revealed the aberrant presence of variants MDM4-S and MDM4-211 messenger RNA (mRNA) in PTC as well as lack of correlation between MDM4 and MDM2 mRNA levels. Of note, levels of MDM4 mRNA were significantly downregulated in tumor samples in comparison to normal counterparts, and such downregulation appears significantly associated with tumor stage.\nMaterials and methods\nTissue samples and patients\nFifty-seven papillary thyroid carcinomas and 57 matched normal thyroid tissue samples from the contra-lateral lobe (CTRL) of 57 patients were studied. In addition, to confirm statistical analyses, 26 papillary thyroid carcinomas and three normal thyroid tissues from independent individuals were analyzed. All specimens were obtained from patients undergoing surgery at the University of Perugia from 1997 to 2007. Before the surgical procedure, all patients signed informed consent forms for collection of fresh thyroid samples for genetic studies. All specimens were sampled from the primary tumor at the time of surgery, snap frozen, and stored at \u221280\u00b0C until use. Tumors containing at least 70% of tumor cells based on the hematoxylin\u2013eosin staining were selected. All normal thyroid tissue from the contra-lateral lobe were histopathologically analyzed for the presence of tumor. Available medical records of the patients were consulted to gain information about the clinical features of the disease and, when possible, the tumor stage was defined according to the sixth edition of the \u201cAmerican Joint Committee on Cancer\u201d Cancer Staging Manual (American Joint Committee on Cancer 2002) based on pathological tumor\u2013node\u2013metastasis parameters and distinguishing patients in two groups: (1) patient age\u2009<\u200945\u00a0years (stage I and II), (2) patient age\u2009\u2265\u200945\u00a0years (stage I, II, III, IV).\nThe total of 83 papillary thyroid carcinomas included 55 of the classic variant, 20 of the follicular variant, five of other variants (tall cell, solid, diffuse sclerosing), and three showing the coexistence of PTC histology with areas of dedifferentiation. The mean period of follow-up was 45.8\u2009\u00b1\u200930.4\u00a0months.\nGenetic analysis of TP53 mutation, BRAF mutations and ret\/PTC rearrangements\nAnalysis of the TP53 gene status was performed as follows: exons 5, 6, 7, and 8 of p53 gene were sequentially amplified by polymerase chain reaction (PCR) assay with the use of AmpliTaq Gold (Applied Biosystems) and following primer sets: exon 5 sense: TTCCTCTTCCTACAGTACTC; exon 5 antisense: GCCCCAGCTGTTCAC; exon 6 sense: ACTGATTGCTCTTAG; exon 6 antisense: AGTTGCAAACCAGAC; exon 7 sense: AGTTGTGTTATCTCCTAG; exon 7 antisense: CAAGTGGCTCCTGAC; exon 8 sense: TCCTATCCTGAGTAG; exon 8 antisense: GTCCTGCTTGCTTAC. Purified PCR products were sequenced in both directions with the use of the BigDye terminator Cycle sequencing Kit (version 1.1, Applied Biosystems) and an ABI Genetic Analyzer (Model 3130, Applied Biosystems). Sequence data were analyzed by means of SeqScape software (version 2.1, Applied Biosystems) followed by manual review.\nSearch for mutations of BRAF was conducted by single-stranded conformational polymorphism (SSCP) screening of real-time (RT)-PCR products of exons 15, followed by sequencing, as previously described [18]. Screening for ret\/PTC 1 and ret\/PTC 3 rearrangements was performed by RT-PCR using primers spanning the breakpoints, as previously described [18].\nQuantitative real-time PCR\nTotal RNA was extracted with Trizol\u2122 (Invitrogen Corp., Carlsbad, CA, USA), according to the manufacturer\u2019s instructions. The expression of MDM2, MDM4, and MDM4-211 in tumors and matching normal thyroid samples was measured by quantitative real-time PCR (qRT-PCR), based on TaqMan methodology, using the ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA) according to Giglio et al. [13]. Briefly, custom-designed TaqMan primers and probes (Applied Biosystems) specific for MDM4, MDM2, and MDM4-211 were used. Our results are expressed as relative units of target mRNA, referred to a sample called calibrator, chosen to represent 1\u00d7 expression of the target gene. The calibrator used was the lowest value in the tissue collection under study. All analyzed samples express n-fold mRNA relative to the calibrator. Each sample mRNA was normalized relative to the \u03b2-actin mRNA. Predesigned TaqMan primers and probe (Applied Biosystems) for the housekeeping gene \u00df-actin were used. Each sample was treated with deoxyribonuclease (DNase) I amplification grade (Invitrogen) and tested before and after DNase treatment.\nComparative analysis of flMDM4 and MDM4-S was driven by qRT-PCR according to Bartel et al., [11] using specific probes and SYBR Master mix (Applied Biosystems) with evaluation of dissociation curves. A MDM4-S-to-flMDM4 ratio of >1.5 was considered indicative of overexpressed MDM4-S.\nWestern blot analysis\nTumor and normal tissue pairs whose material was sufficient for western blot analysis were selected. All samples were homogenized with Tissue Lyser (Qiagen) and western blots were performed according to Giglio et al. [13]. Briefly, samples were lysed in radio-immunoprecipitation assay buffer (50\u00a0mM Tris\u2013Cl pH 7.5, 150\u00a0mM NaCl, 1% Nonidet P-40, 0.5% Na desoxicholate, 0.1% sodium dodecyl sulfate (SDS), 1\u00a0mM ethylenediaminetetraacetic acid) supplemented with a cocktail of protease inhibitors (Boehringer). Whole lysates were boiled in SDS Laemnli sample buffer, resolved by SDS-polyacrylamide gel electrophoresis on precast 10% gels (Invitrogen) and subsequently transferred to polyvinylidene fluoride membranes (Millipore). After protein transfer, membranes were blocked for 30\u00a0min with Tris-buffered saline containing 0.1% Tween-20 and 5% nonfat dry milk and incubated with specific antibodies. The following primary antibodies were used: rabbit anti-MDM4 polyclonal antibody R1 (raised against full-length MDM4 protein) and (Bethyl Laboratories) mouse anti-MDM2 monoclonal antibody 2A10 and monoclonal antibody Ab-1 (oncogene), mouse anti-\u03b1-tubulin monoclonal antibody DM-1A (Sigma), and mouse \u03b1-actin monoclonal antibody AC-40 (Sigma). MDM4 was probed with both Bethyl and R1 antibodies, giving similar results although, in comparison, the polyclonal antibody R1 was more sensitive. The homemade R1 antibody has been raised using the entire MDM4 protein. Experiments of epitope mapping by using MDM4-deletion mutants have shown that R1 does not recognize the NH2-terminus of MDM4. It is therefore unable to recognize the MDM4-S form.\nStatistical analysis\nStatistic analysis was carried out using the Analyze-it software for Microsoft Excel (Analyze-it Software, Ltd.). Spearman rank correlations and Kendall rank correlation (for measure of linear association) were used to evaluate the correlation between not-normally-distributed variables. To compare groups, we used the Wilcoxon Signed-Ranks test for related samples (57 tumors vs. matched thyroid normal tissues) and Mann\u2013Whitney U test for independent samples (83 tumors vs. 60 normal thyroid samples). A probability of p\u2009<\u20090.05 was considered as statistically significant. Intraindividual variation was calculated by utilizing the T-to-N ratio that derives from the ratio of corrected tumor (mRNA or protein) levels to corrected normal tissue levels of MDM4 and MDM2.\nResults\nAnalysis of MDM4 and MDM2 levels in papillary thyroid tumors\nA group of 57 thyroid tumors samples and 57 matched normal thyroid tissues from the CTRL were analyzed. All tumors were of papillary histotype (PTC). The mean age of the studied population was 49.4\u2009\u00b1\u200916.81 (SD); the female percentage was 72%. The histopathological features of the 57 tumors are shown in Table\u00a01. Tumor stage was defined according to the sixth edition of the \u201cAmerican Joint Committee on Cancer.\u201d.Table\u00a01Histopathological features of the 57 Papillary Thyroid Tumors (PTC)\u00a0Yes, n (%)No, n (%)ND, n (%)BRAF mutation35 (61)20 (35)2 (3)Ret\/PTC rearrangement5 (9)50 (88)2 (3)TP53 mutation0 (0)57 (100)0Extra-thyroidal invasion15 (26)41 (72)1 (2)Multifocality24 (42)32 (56)1 (2)Nodal metastasis24 (42)33 (58)0Distant metastasis6 (10)35 (62)16 (28)Tumor stage of patients <45\u00a0years (n\u2009=\u200920)I15 (75)05 (25)II0 (0)15 (75)5 (25)Tumor stage of patients \u226545\u00a0years (n\u2009=\u200937)I8 (22)18 (48)11 (30)II2 (5)24 (65)11 (30)III3 (8)23 (62)11 (30)IV13 (35)13 (35)11 (30)HistologyClassic38 (67)19 (33)0Follicular13 (23)44 (77)0Other4 (7)53 (93)0Dedifferentiated2 (3)55 (97)0ND Not determined\nAll samples were screened for the presence of TP53 mutations (limitedly to exons 5\u20138 where 80% of mutations occur), BRAF mutations (by SSCP followed by sequence analysis), and for ret\/PTC 1 and ret\/PTC 3 rearrangements (by RT-PCR; Table\u00a02). MDM4 and flMDM2 mRNA levels were evaluated by qRT-PCR, using specific primers and probes. MDM4 primers were chosen in a region not present in the variant forms MDM4-S, HDMX-G [19] MDM4-211, XALT1, and XALT2 [20] to amplify specifically the fl product. Similarly, the chosen MDM2 primers do not amplify the MDM2-A, MDM2-B, MDM2-C, MDM2-D, and MDM2-E alternative spliced forms and most of the aberrantly spliced forms [21].\nTable\u00a02Tumor sample dataSampleM4M4 T\/NMDM4-S\/flM4MDM4-211MDM4-211 to flM4M2 T\/NM4 to M2TP53 statusBRAF mutRet\/PTC rearr.Patient statusaTumor stageMultifocalityN1M1Histologyb19.420.91.81.360.1411.3wt+\u221213\u2212\u2212\u22122218.961.35.21.42.5wt\u2212\u221244+++4329.341.60.49.030.311.53.5wt\u2212\u221211\u2212\u2212\u22122414.931.79.71.12wt\u2212+11+\u2212\u2212154.390.30.90.41.1wt\u2212\u221244\u2212++3611.160.41.30.51.9wt\u2212\u221244\u2212++3712.600.80.78.940.710.73.9wt\u2212\u221211++\u2212184.440.40.82.10.7wt+\u2212n.d.n.d.\u2212\u2212n.d.1915.511.40.61.41.6wt+\u221214++\u22121108.630.30.60.91.3wt\u2212\u2212n.d.n.d.+\u2212n.d.1115.960.21.20.90.7wt\u2212\u221224\u2212+\u22121128.110.51.110.9wt+\u221231++\u22121136.250.41.310.161.80.8wt+\u2212n.d.n.d.\u2212+n.d.2146.130.41.50.80.9wt+\u2212n.d.n.d.\u2212\u2212n.d.1158.340.31.60.61.2wt\u2212\u221211\u2212+\u22121163.820.21.41.230.320.80.5wt+\u221231\u2212\u2212\u221211718.8310.80.38.8wt+\u221224++\u22122186.321.20.71.11.2wt+\u221211\u2212\u2212\u22121193.610.21.82.60.1wt\u2212\u221211\u2212\u2212\u22122205.120.330.90.8wt+\u2212n.d.1+\u2212\u22121218.40.54.20.61.1wt\u2212+2n.d.+\u2212n.d.1225.10.3n.d.*10.6wt\u2212+13\u2212\u2212\u22121237.060.90.92.60.7wt+\u2212n.d.n.d.\u2212\u2212n.d.1248.850.60.111.4wt+\u221211+\u2212\u22121256.210.60.12.30.6wt\u2212\u2212n.d.n.d.+\u2212n.d.1269.6110.11.21.1wt+\u221211\u2212\u2212\u22122277.110.30.60.41.6wt+\u2212n.d.n.d.++n.d.1283.530.2n.d.0.21.4wt+\u221212\u2212\u2212\u22122295.760.42.20.71.2wt+\u2212n.d.n.d.+\u2212n.d.13010.7111.91.2wt+\u221211\u2212\u2212\u221213111.470.3n.d.0.80.9wt\u2212+31++\u221213214.421.5n.d.2.41.4wt+\u221211\u2212\u2212\u22121333.920.30.20.41.7wt+\u221234\u2212\u2212+13411.960.710.52.8wt\u2212\u221211\u2212\u2212\u22122353.690.11.81.170.320.40.7wt+\u221214++\u22122368.030.42.70.71.4wt+\u221211\u2212\u2212\u221213721.3320.40.85.6wt\u2212\u221211++\u22124385.560.44.71.60.6wt+\u221234++\u22122391.120.11.12.40wt\u2212\u221214\u2212+\u221224013.981.41.43.50.9wt+\u221211+\u2212\u22122415.840.33.610.6wt+\u221234\u2212++1427.360.31.31.830.250.90.8wt+\u221211\u2212\u2212\u22121434.770.31.20.80.8wt\u2212+3n.d.\u2212+n.d.4442.450.12.20.70.4wt+\u221213\u2212+\u22121455.120.30.70.80.7wt+\u221234\u2212++1463.10.32.11.60.2wt+\u221211\u2212\u2212\u221214710.271.12.419.60.1wt+\u221211++\u221224812.131.12.41.12.2wt+\u2212n.d.n.d.+\u2212n.d.1497.940.81.60.82.2wt\u2212\u221231++\u221215014.720.82.80.82.2wt+\u221234n.d.+\u221215115.671112.2wt+\u221212\u2212\u2212\u221215266.493.60.63.62.2wt+\u22121n.d.+\u2212n.d.15328.941.41.71.42.2wt+\u22121n.d.\u2212\u2212n.d.1543.720.40.32.80.5wt\u2212\u22121n.d.++n.d.4555.30.40.71.80.5wt+\u221211\u2212\u2212\u22121562.040.60.92.30.3wtn.d.n.d.1n.d.+\u2212n.d.1576.870.7n.d.1.40.8wtn.d.n.d.1n.d.\u2212\u2212n.d.1n.d. Not determined, N1 nodal metastasis, M1 distant metastasisa1, recovered; 2, recurrence occurred, 3,disease persistence, 4, expiredb1, classic variant; 2, follicular variant; 3, dedifferentiated variant; 4, other\nMDM4 and MDM2 mRNA levels in normal tissues followed a normal distribution according to Kurtosis analysis (Fig.\u00a01a,b). On the contrary, MDM4 and MDM2 values in tumor samples were not normally distributed (p\u2009<\u20090.0001; Fig.\u00a01c,d). Comparative analysis by Wilcoxon signed-ranks test for paired samples revealed a highly significant difference in the distribution of MDM4 values between tumor and CTRL groups (Wilcoxon\u2019s W\u2009\n=\u20091,385, p\u2009<\u20090.0001; Fig. 1e). Conversely, comparison of MDM2 values did not show significant difference (p\u2009\n=\u20090.61; Fig.\u00a01f), in spite of the presence of four upper outliers in the tumor sample set. There was no correlation between MDM4 and MDM2 mRNA tumor levels indicating that the alterations of the two transcripts are independent from one another (data not shown).\nFig.\u00a01a\u2013d Frequency histograms showing the distribution of the mRNA levels of the indicated genes in the indicated sample groups. Relative units of target mRNA are referred to a sample called calibrator, chosen to represent 1\u00d7 expression of the target gene. Each sample mRNA was normalized relative to the \u03b2-actin mRNA. The superimposed line shows the normal distribution curve. For each distribution, the mean (M)\u2009\u00b1\u2009standard deviation (SD) is shown. e, f Comparison of MDM4 and MDM2 levels, respectively, between control and tumor samples. Each plot shows graphically the central location and scatter\u2013dispersion of the values of each group: the line series shows parametric statistics (mean and confidence interval of mean) while the notched box and whiskers show nonparametric statistics (median, confidence interval of median, and interquartile range). Crosses and circles indicate possible outliers, between 1.5 and 3 interquartile range and over 3 interquartile range, respectively. p value was calculated according to Wilcoxon signed-ranks test\nTo confirm the decrease of MDM4 mRNA levels in PTC in comparison to CTRL, we analyzed these levels in an additional 26 independent PTC and three normal thyroid samples. Comparison of tumor and CTRL groups still resulted in a highly significant correlation (Mann\u2013Whitney for independent samples U\u2009\n=\u2009634, p\u2009<\u20090.0001) and confirmed previous results of a strong downregulation of MDM4 expression in tumor samples. Indeed, study of prediction by receive operator characteristics curves (ROC curves) indicated that the use of MDM4 mRNA levels as potential diagnostic test has a highly significant ability to discriminate between normal and tumor tissues (area under the ROC 0.84, p\u2009<\u20090.0001 Fig.\u00a02).\nFig.\u00a02Receive operator characteristics curves for MDM4 levels between normal and tumor samples, assessing variable MDM4 levels as diagnostic test. The plot shows sensitivity of the test against specificity. p value was 0.0001. For each decision threshold, the percentage of tumor cases correctly identified as such (true positives) against the percentage of normal cases incorrectly diagnosed as tumor (false positives) is shown\nTo ascertain whether the decrease of MDM4 mRNA in tumor samples results in decreased protein levels, we analyzed whole-cell extracts from 20 available pairs of tumors and CTRLs (Fig.\u00a03). In 16 out of 20 pairs, the tumor-to-CTRL ratio of densitometric values of the MDM4 protein levels correlated significantly with the observed ratio of the mRNA levels (Kendall tau\u2009=\u20090.7, p\u2009\n=\u20090.0002). Interestingly, western blot confirmed the decrease of MDM4 mRNA in tumor relative to normal tissue in seven samples, reinforcing previous observations. Notably, in three samples (44, 27, and 33), we observed an increase of MDM4 proteins in tumor compared to CTRL despite equal or even lower levels of corresponding tumor mRNA relative to CTRL, indicating MDM4 protein stabilization independent of transcription. Western blot analysis revealed also the presence of an additional species of about 58\u00a0kDa in some tumor samples (Fig.\u00a03, arrows). This band was recognized also by a mix of three monoclonal antibodies to MDM4 (6B1A, 11F4D, and 12G11G, data not shown), suggesting it may be a MDM4 variant. However, its size does not correspond to any of the MDM4 variant forms described so far.\nFig.\u00a03Western blot of WCE derived from 20 couples of tumor (T) and matching control (N). The values derive from the ratio of densitometric value of MDM4\u2013actin, both at the protein and mRNA levels. Asterisks mark tumor samples expressing MDM4-211 mRNA; section signs mark tumor samples showing increased levels of MDM2 protein with no increased levels of the corresponding mRNA. Arrows mark a lower weight protein of about 58\u00a0kDa, recognized by different \u03b1MDM4 antibodies\nFurther, western blot revealed frequent overexpression of the MDM2 protein in tumors that did not show overexpression of the mRNA (Fig.\u00a03, samples marked by section signs).\nAnalysis of intraindividual variation of MDM4 and MDM2 levels\nPrevious data were obtained by comparing tumors versus CTRLs. We then analyzed the intraindividual variation of MDM4 and MDM2 mRNA levels for each tumor (T) and its matched CTRL (N) by calculating the relative ratio, T to N. The range of MDM4 T-to-N ratios varied from 0.1 to 3.6 (Fig.\u00a04a and Table\u00a02). The majority of tumor samples showed a decrease of MDM4 levels compared to the corresponding CTRLs (29 out of 57 samples having a ratio of <0.5), confirming the previously observed decrease of tumor MDM4 expression at individual levels too. In other tumor histotypes, amplification of MDM4 gene and overexpression of the mRNA have been reported. In this PTC group, MDM4 overexpression (as defined by a T-to-N ratio of \u22655) was not detectable; in only two cases, the ratio of T to N showed a value of \u22652 (2 and 3.6, respectively). On the contrary, comparison of MDM2 mRNA levels showed tumor values twofold higher than matching CTRL in 11 samples, among which one had a ratio of >19, suggesting an amplification event (Fig.\u00a04b and Table\u00a02). Moreover, only six samples (10%) showed a ratio of <0.5, indicating that downregulation of MDM2 levels in PTC tumor is a rare event in comparison to that of MDM4. Notably, in two of these tumors, western blot analysis did not confirm the mRNA MDM2 decrease (Fig.\u00a03; samples 35, 27).\nFig.\u00a04a, b Frequency histograms showing the distribution of the T-to-N ratios derived from tumor\u2013CTRL levels of the indicated genes. The superimposed line shows the normal distribution curve. For each distribution, the mean\u2009\u00b1\u2009standard deviation is shown\nCorrelation of MDM4 and MDM2 with tumor histopathological features\nWe then investigated whether tumor MDM4 and MDM2 mRNA levels as well as the T-to-N ratio varied according to tumor properties by correlating them with the histopathological features of samples as reported in Table\u00a01. First, we examined their association with BRAF mutations and ret\/PTC rearrangements, two hallmarks of thyroid carcinogenesis. BRAF mutation were detected in 35 out of 57 patients, ret\/PTC rearrangements in 5 out of 57 (Table\u00a02). All BRAF mutations but one involved a heterozygous T\u2009>\u2009A transversion at nucleotide 1799, resulting in the prototypic valine-to-glutamic-acid substitution at position 600 (BRAFV600E). In one case, the in-frame insertion at position 1796 of an additional codon, coding for Valine, was detected (BRAFV599Ins). This was shown to be a \u201cgain-of-function\u201d mutation in all super imposable to BRAFV600E [22]. No association was found between MDM4 and MDM2 expression, BRAF mutations, and ret\/PTC rearrangements suggesting that these oncogenic hits are independent events. We then analyzed the correlation between MDM4 or MDM2 expression with the other tumor histopathological features. This revealed association of MDM4 with multifocality of tumors: specifically, tumors presenting as multifocal show higher values than nonmultifocal (Spearman rank correlation rs\u2009=\u20090.29, p\u2009\n=\u20090.032; Fig.\u00a05a, Table\u00a03). This association was not evident when we correlated multifocality with the MDM4 levels of matching normal tissues (p\u2009\n=\u20090.23), indicating that this tumor property is not attributable to a patient predisposition given by individual MDM4 expression levels but rather it is a specific feature of tumor cells.\nFig.\u00a05a Vertical box-whisker plots showing MDM4 levels between nonmultifocal (no) and multifocal (yes) tumors. Each plot shows graphically the central location and scatter\u2013dispersion of the values of each group: the line series shows parametric statistics (mean and confidence interval of mean) while the notched box and whiskers show nonparametric statistics (median, confidence interval of median, and interquartile range). Crosses and circles indicate possible outliers, between 1.5 and 3 interquartile range and over 3 interquartile range, respectively. p value was calculated according to Spearman test. b Vertical box-whisker plots showing tumor\u2013CTRL MDM4 mRNA levels in stage 1 compared to other stages of tumors. Each plot shows graphically the central location and scatter\u2013dispersion of the values of each group: the line series shows parametric statistics (mean and confidence interval of mean) while the notched box and whiskers show nonparametric statistics (median, confidence interval of median, and interquartile range). Crosses and circles indicate possible outliers, between 1.5 and 3 interquartile range and over 3 interquartile range, respectively. p value was calculated according to Spearman testTable\u00a03Summary of significant correlations of MDM4 and MDM4 T\u2013N values with histopathological features\u00a0MultifocalityTumor StagePresenceAbsenceIII\u2013IVn\u2009=\u200924n\u2009=\u200932n\u2009=\u200923n\u2009=\u200918MDM4 T\u2013NMean\u2009\u00b1\u2009SD0.9\u2009\u00b1\u20090.760.56\u2009\u00b1\u20090.430.81\u2009\u00b1\u20090.540.51\u2009\u00b1\u20090.43Median\u2009\u00b1\u2009IQR0.63\u2009\u00b1\u20090.830.38\u2009\u00b1\u20090.60.65\u2009\u00b1\u20090.810.3\u2009\u00b1\u20090.58MDM4Mean\u2009\u00b1SD12.4\u2009\u00b1\u200912.68.1\u2009\u00b1\u20096.410.28\u2009\u00b1\u20095.98.38\u2009\u00b1\u20095.9Median\u2009\u00b1\u2009IQR8.74\u2009\u00b1\u20098.76.19\u2009\u00b1\u20095.18.85\u2009\u00b1\u20095.445.7\u2009\u00b1\u20098.0\nWe also correlated MDM4 T-to-N ratio with tumor features. We observed an even stronger association between this parameter and multifocality (Spearman correlation coefficient rs\u2009=\u20090.32, p\u2009=\u20090.01, Table\u00a03), confirming previous observations. Further, we found a significant correlation of MDM4 T-to-N ratio with tumor stage (Spearman correlation coefficient, rs\u2009=\u20090.35 for n\u2009=\u200941, p\u2009=\u20090.02; Fig.\u00a05b); in particular, stage I tumors showed the highest ratio, indicating downregulation of MDM4 levels in late-stage tumors in comparison to early-stage ones (Table\u00a03).\nOn the contrary, MDM2 levels as well as MDM2 T-to-N ratio did not show correlation with any histopathological parameter.\nCharacterization of MDM4 variants in thyroid tumors\nWe have analyzed the presence of the MDM4 variant forms, MDM4-S and MDM4-211, in the same sample group. So far, these are the only variant forms of MDM4 detected in human tumors. MDM4-S is a splicing variant present both in normal and tumor tissues; however, in tumor cells, MDM4-S is overexpressed and, as a consequence, the ratio MDM4-S to flMDM4 is >1. At first, we performed comparative analysis of flMDM4 and MDM4-S by qRT-PCR in CTRL samples. In agreement with the literature, we detected both transcripts in all samples with a ratio MDM4-S to fl-MDM4 ranging from 0.09 to 0.79 with only two samples showing values of 1.14 and 1.05 (data not shown). On the basis of these results, we considered overexpression as defined by a ratio of \u22651.5 The same analysis in PTC detected MDM4-S in all but five samples; the resulting MDM4-S-to-flMDM4 ratio was \u22651.5 in 20 out of 57 of tumors (35%; Table\u00a02). The same percentage resulted from the analysis of the additional group of 26 tumors (9 out of 26), in agreement with other reports [11]. Statistical analysis did not reveal any significant association of MDM4-S-to-flMDM4 ratio with the features described in Table\u00a01.\nThe second variant form we investigated was MDM4-211 (HDMX211), previously isolated from a thyroid tumor cell line [13]. MDM4-211 mRNA was present in 7 out of 57 original tumor samples and in 8 out of 26 additional samples analyzed with an overall frequency of 18% (Table\u00a02 and data not shown). Its mRNA was not detected in any of the CRTL samples, thus confirming its specific presence in tumor tissue. In agreement with our previous reports [13], western blot analysis of available tumor and CTRL samples confirmed enhanced levels of the oncoprotein MDM2 in tumors expressing MDM4-211 (Fig.\u00a03, samples marked by *). In our tumor set, the presence of MDM4-211 did not correlate with any of the properties described in Table\u00a01 nor with the expression of the form MDM4-S, indicating that the presence of these two variants is an independent event.\nSome authors have hypothesized that the presence of MDM4 variants may correlate with lower level of flMDM4 [11]. We therefore assessed the presence and expression of MDM4-S and MDM4-211 in correlation with flMDM4 and with the MDM4 T-to-N ratio. We observed no significant correlation but rather a slight association between low levels of both MDM4-S and flMDM4 (p\u2009=\u20090.056).\nDiscussion\nIn this study, we have analyzed the expression of MDM4 and MDM2 mRNAs in a group of 57 papillary thyroid tumors in comparison to matching normal tissues. We observed that MDM4 levels are significantly lower in tumor samples than in control. Specifically, tumor samples expressing levels of MDM4 lower than matching control are very frequent (51%) and this decrease in expression appears to correlate significantly with higher tumor stages (stage I in comparison to the other), indicating that it occurs with tumor progression. These data led us to hypothesize that a robust presence of MDM4 might exert an antagonistic role in PTC evolution. To date, the majority of the literature has reported amplification of MDM4 in the presence of wt TP53 in different tumor histotypes [3], partly in contrast with our data. However, a different status of MDM4 in some cancer has also been reported. In a gene profiling analysis, MDM4 downregulation was observed in prostate tumors with poor prognosis in comparison to those with good prognosis in agreement with the hypothesis that MDM4 may be detrimental to tumor progression [23]. Moreover, two studies reported low levels of MDM4 protein in chronic myeloid leukemia tumor samples and in tumor cell lines [24, 25]. It would be interesting to know whether those cell lines have features resembling those of advanced-stage tumors. Some authors have suggested that low MDM4 expression levels may be correlated to the presence of a mutated TP53 [11, 25]. However, lack of detection of any of the most frequent mutation of TP53 gene in this PTC group does not support this hypothesis, at least in this type of cancer. Similarly, in the prestate tumor study TP53 was found to be wild type by sequencing (A. Farsetti, personal communication).\nThese data counteract the holistic view of MDM4 as a p53 inactivation tool and suggest that its function may be more complex than previously considered, at least in some tissues. Thus far, molecular studies indicating MDM4 functions different from negative regulation of 53 have provided evidence that MDM4 may display antiproliferative or proapoptotic activities, under stress conditions [6, 7]. Further, a recent work has shown for MDM4 a role in suppressing tumorigenesis through the control of bipolar mitosis [26]. Whether these mechanisms occur in PTC remain to be elucidated.\nIn the analysis of tumor MDM4 expression levels, we found that high levels of MDM4 are associated with multifocal tumors. Different hypothesis have been postulated to explain the origin of these tumors. Certainly, multifocality is related to the high proliferative potential of thyrocites. MDM4 has been reported as inhibitor of p53-mediated growth arrest [1]; thus, it might be hypothesized that the presence of MDM4 may predispose to multifocality and tumor development, while a decrease in its expression may confer an advantage to tumor progression. Interestingly, one study has documented a statistically significant association of MDM4 amplification with low-grade astrocytomas, thus supporting the hypothesis that high levels of MDM4 may represent an early advantageous event for tumor development [27]. Further, the association between MDM4 levels and tumor multifocality is of particular clinical relevance. Indeed, the possibility of early diagnosis of tumor multifocality might allow discrimination between partial or total thyroid gland ablation. The extension of this type of analysis to a broader set of samples, including benign lesions, should help to confirm these findings and to investigate potential applications of this molecule in clinical practice.\nIn addition, we have observed with high frequency the aberrant presence of MDM4 alternatively spliced forms, whose oncogenic activity has been assessed so far only in vitro. In our analysis, the aberrant presence of these variants did not correlate with any of the histopathological features. In a study with primary sarcomas, the MDM4-S-to-flMDM4 ratio correlated with enhanced cell proliferative potential and decreased patient survival [11]. The high incidence of patient survival in PTC (only three deaths in our group, Table\u00a02) does not allow to confirm this association. For MDM4-211, this is the first frequency report in a large group of tumor samples and indicates that its presence is appreciable in PTC (18%). Interestingly, these MDM4 variants forms show in vitro an even stronger inhibitory activity toward p53 than the full-length protein. Their presence may concur to modify the activity of flMDM4, making more complex the assessment of its function in tumor cells. It has to be mentioned that in many PTC samples we observed the presence of a MDM4 minor form whose nature is unclear, supporting the hypothesis that MDM4 variants may concur to MDM4 oncogenic functions.\nConversely, MDM2 mRNA levels were increased in majority of tumor samples. In particular, we observed with high frequency enhanced protein levels in tumor samples that do not show enhanced mRNA levels. This is in accord with previous reports and confirms that in thyroid tumors MDM2 overexpression takes place frequently by mechanisms other than enhanced transcription [28]. At least in some of these samples, the simultaneous presence of MDM4-211 variant may cause such upregulation. These data further confirm the general view of the antagonistic role of MDM2 toward the oncosuppressor p53. Finally, in this study, we did not find any correlation between MDM2 and MDM4 mRNA levels, suggesting that the alterations we observed occur independently from one another.\nOverall, our results reveal that MDM4 alterations, particularly the presence of its variant forms with p53-inhibitory properties, are present at high frequency in PTC confirming the general model of different ways of p53 inactivation in human tumors. However, our data represent the first statistical report of a strongly significant downregulation of flMDM4 expression in cancer and particularly in advanced-stage tumors, suggesting that this protein may play additional roles besides p53 inactivation at least in PTC. In view of the suggested strategies for abrogation of MDM4 in human tumors [1, 3], our findings counsel acquisition of more information on the molecular functions of this protein in different human tumors in order to carefully evaluate the application of such therapies.","keyphrases":["mdm4","mdm4 variants","thyroid carcinoma","p53","mdm2"],"prmu":["P","P","P","P","P"]}
{"id":"Histochem_Cell_Biol-3-1-1779629","title":"BAC constructs in transgenic reporter mouse lines control efficient and specific LacZ expression in hypertrophic chondrocytes under the complete Col10a1 promoter\n","text":"During endochondral ossification hypertrophic chondrocytes in the growth plate of fetal long bones, ribs and vertebrae play a key role in preparing growth plate cartilage for replacement by bone. In order to establish a reporter gene mouse to facilitate functional analysis of genes expressed in hypertrophic chondrocytes in this process, Col10a1- BAC reporter gene mouse lines were established expressing LacZ specifically in hypertrophic cartilage under the control of the complete Col10a1 gene. For this purpose, a bacterial artificial chromosome (BAC RP23-192A7) containing the entire murine Col10a1 gene together with 200 kb flanking sequences was modified by inserting a LacZ-Neo cassette into the second exon of Col10a1 by homologous recombination in E. coli. Transgenic mice containing between one and seven transgene copies were generated by injection of the purified BAC-Col10a1- lLacZ DNA. X-gal staining of newborns and embryos revealed strong and robust LacZ activity exclusively in hypertrophic cartilage of the fetal and neonatal skeleton of the transgenic offspring. This indicates that expression of the reporter gene in its proper genomic context in the BAC Col10a1 environment is independent of the integration site and reflects authentic Col10a1 expression in vivo. The Col10a1 specific BAC recombination vector described here will enable the specific analysis of effector gene functions in hypertrophic cartilage during skeletal development, endochondral ossification, and fracture callus healing.\nIntroduction\nCartilage\u2013bone transition by endochondral ossification is a highly complex process which determines not only longitudinal growth of long bones, ribs and vertebrae, but also plays a critical role in bone fracture callus healing, osteophyte formation and cartilage tissue engineering. Decisive steps in this process are proliferation, maturation and hypertrophy of chondrocytes in the growth plate which play a key role in mineralization, apoptosis and induction of resorption of hypertrophic cartilage by invading bone marrow. These events are regulated by synergistic action of several signaling pathways and transcriptions factors controlled by a multitude of growth factors and hormones.\nA major signaling system regulating chondrocyte proliferation and maturation is controlled by PTHrP, which is induced by Indian hedgehog, stimulates chondrocyte proliferation and delays maturation through the PTHrP receptor PTHR1 [for reviews see (Kronenberg et al. 1998; Vortkamp 2000)]. Also FGF factors stimulate proliferation and block maturation through FGF receptor 3, but their action in the growth plate is more complex with respect to the differential distribution of different FGFs and FGF receptors in the various zones of the growth plate (Ornitz and Marie 2002).\nThe role of BMPs during chondrocyte maturation and hypertrophy is somewhat controversially discussed: several studies show that BMP factors such as BMP-6 and BMP-7 stimulate hypertrophic differentiation of chondrocytes and promote collagen X expression, thus preparing growth plate cartilage for replacement by endochondral bone (Enomoto-Iwamoto et al. 1998; Volk et al. 1998; Grimsrud et al. 2001). On the other hand, it was reported that BMP-2 and BMP-4 overexpression in developing chick limbs caused delayed hypertrophy of chondrocytes (Duprez et al. 1996).\nThe fourth family regulating chondrocyte maturation are Wnt factors and the Wnt\/\u03b2-catenin signaling system (Dong et al. 2005), also acting in diverse directions. For example, Wnt 7a inhibits chondrogenesis and Wnt 5a impairs chondrocyte hypertrophy and type X collagen (Col10a1) expression in vitro (Daumer et al. 2004), but activation of the canonical \u03b2-catenin pathway by Wnt 8c and Wnt 9a induces chondrocytes maturation, hypertrophy and calcification, associated with upregulation of Col10a1 expression (Enomoto-Iwamoto et al. 2002; Dong et al. 2005, 2006; Hu et al. 2005).\nRecently increasing evidence points to complex crosstalks between these signaling pathways. For example, there is experimental evidence for a coordination between Indian hedgehog and BMP signaling in chondrocyte proliferation (Minina et al. 2002). Targets of the signaling pathways in the growth plate are transcription factors such as Sox9 responsible for chondrogenic differentiation (Ng et al. 1997; Lefebvre and de Crombrugghe 1998) and cbfa1\/runx2 inducing osteogenic differentiation (Karsenty et al. 1999; Ducy 2000) as well as chondrocyte maturation and Col10a1 expression (Kim et al. 1999; Enomoto et al. 2000; Zheng et al. 2003).\nThere is ample evidence in the literature that factors regulating chondrocyte maturation and hypertrophy may be also directly involved in the regulation of Col10a1 gene expression, e.g. PTHrP (Iwamoto et al. 1994; Ionescu et al. 2001; Riemer et al. 2002; Gebhard et al. 2004), Runx2 (Enomoto-Iwamoto et al. 2001; Leboy et al. 2001; Zheng et al. 2003), BMP2 (Volk et al. 1998; Grimsrud et al. 2001), or c-fos (Thomas et al. 2000; Riemer et al. 2002). Thus, in order to elucidate the exact role of the various signaling systems and involved factors regulating endochondral ossification, it would be useful to generate a reporter gene mouse in which the expression of LacZ is under the control of the complete Col10a1 gene regulatory sequences.\nPreviously we have reported on the generation of transgenic mouse lines in which LacZ expression is under the control of the 4.6\u00a0kb promoter of Col10a1 which includes a strong enhancer element (Gebhard et al. 2004) and runx2 binding sites (Zheng et al. 2003). We have shown that this promoter is sufficient to drive specific expression of LacZ reporter genes in hypertrophic chondrocytes of growth plate cartilage of long bones, ribs, vertebrae and sternal cartilage (Gebhard et al. 2004). Reporter gene expression was restricted to hypertrophic cartilage, but not all hypertrophic chondrocytes in the growth plate of 18.5-day embryos were found to be lacZ positive, and only weak lacZ expression was observed at early embryonic stages before day E17. Furthermore, not all transgenic founders containing the LacZ transgene showed lacZ activity in the growth plates, suggesting a high level of sensitivity to interfering activities of the genomic context of insertion or additional regulatory elements in the Col10a1 gene further upstream of the enhancer or downstream of the coding sequences.\nHere we report on the construction of a BAC reporter mouse expressing lacZ under the control of the Col10a1 gene. A LacZ-Neo cassette was inserted into the second exon of Col10a1 within the context of a 215\u00a0kb BAC using a phage-based homologous recombination system in E. coli (Yu et al. 2000; Lee et al. 2001). Transgenic mouse lines established with this modified BAC show specific LacZlacZ expression at high levels in hypertrophic zones of long bones, ribs, vertebrae, mandibles and sterna of transgenic mouse lines. No significant unspecific expression was detected in other chondrogenic or non-chondrogenic tissues except some transient, probably unspecific X-gal reaction in the prenatal epidermis and hair papillae.\nThe robust and specific expression of Col10a1-based BAC recombineering vectors in transgenic mice opens new and unique possibilities to study the role of growth factors and transcription factors in chondrocyte hypertrophy and endochondral ossification, and to define further cis-acting regions in the Col10a1 gene.\nMaterials and methods\nBAC clones\nMurine Col10a1 containing BAC clones were selected from the UCSC Bioinformatic databases (http:\/\/www.genome.ucsc.edu\/) (Kent et al. 2002); clone RP23-192A7 contained 211\u00a0kb of genomic DNA flanking the Col10a1 gene derived from the L129 mouse strain. RP23-192A7 (cloned in the pBACe3.6 vector) was obtained from the BACPAC Resources Center at Children\u2019s Hospital Oakland Research Institute, (CHORI). The presence of the full-length Col10a1 gene was verified by PCR amplification using both primers for detecting the enhancer at \u22124.6\u00a0kb (Enmcol10a1-3\u2032: ATT CTC GAG CTT TGG GAA GCA TGG TG ; Enmcol10a1-5\u2032: CAA GAA ATC TTT GGG AAA TGA ATG AAT G ) (Gebhard et al. 2004) , and for a region 2036 bp downstream of the poly A site of the Col10a1 gene (mcol10a1-psg-5\u2032: TCT ACC AGG AGG CCT CTC TTC AAT GTT AC; mcol10a1-psg-3\u2032:\nGGG AAT TCT TAC CTT AAA GTA GAT ACA TG (amplicon size 222\u00a0bp).\nConstruction of targeting vector placH+Col10a1-lacZ-frt-neo-frt\nThe 5\u2032 recombination site was generated by PCR amplification of a 129\u00a0bp fragment mapping to the 113\u00a0bp 3\u2032-end of intron 1 and 16\u00a0bp of the 5\u2032-UTR encoded in exon 2 by using BAC RP23-192A7 as template (see Fig.\u00a01). Terminal SalI and NcoI restriction sites were added by using primers Col10a1-5\u2032SalI (5\u2032-ACG CGT CGA CGA TAT CTC AGT CAT TTA AAA AAC CAT GA) and Col10a1-5\u2032NcoI (5\u2032-CAT GCC ATG GAT TTT CAG ATA GAT TCT GAA AAG CAG A. The product was digested with SalI and NcoI and cloned into the multiple cloning site 2 of plasmid placH (Zhou et al. 1995) kindly provided by Dr. V. Lefebvre, Cleveland) containing the lacZ gene and the 3\u2032-end of the murine protamine 1 gene with an intron and poly A signal resulting in clone placH+5\u2032COL10a1. Use of the NcoI site linked the lacZ reading frame to the start ATG of Col10a1.\nFig.\u00a01Generation of the BAC-Col10a1-lacZ-neo DNA for the transgenic expression of the lacZ reporter gene in hypertrophic cartilage. a Genomic structure of the murine Col10a1 gene, with exons (gray boxes) and introns. The sequences used for generation of the 5\u2032 and 3\u2032 homologous regions in exon 2 and intron 1 are shown in black. b The targeting vector placH- Col10a1-lacZ-frt-neo-frt contains Col10a1 derived 5\u2032 and 3\u2032 homology domains flanking a lacZ-frt-neo-frt cassette for homologous recombination in E. coli. The lacZ coding sequence was fused with the start ATG of the Col10a1 reading frame; shown is only the insert of the targeting vector, contained in the placH vector. c Partial map of the BAC-clone Col10a1-lacZ-frt-neo-frt clone after homologous recombination with the targeting vector placH-Col10a1-lacZ-frt-neo-frt\nThe 3\u2032 recombination site was generated by overlapping primers corresponding to a 57\u00a0bp sequence of exon 2. Additional HindIII sites were introduced at both ends as well as a XhoI site at the 3\u2032-end. The frt-neo-frt cassette of the pICGN21 vector (Yu et al. 2000; Lee et al. 2001); kindly provided by Dr. Copeland) harboring the neomycin phosphotransferase gene under the SV40 promoter, an HSV thymidine kinase and a poly A signal was amplified by PCR using primer FRTNeo5\u00b4HindIII (5\u2032-AGT CAA GCT TTA ACT GAT CGC GGC CAG CTT GAA GTT) (pIGCN21 sequence pos. 4664\u20134689, marked in bold) and the primer Col10a1-3\u2032HindIII (5\u2032-TAA AGC TTC TCG AGT TGT GTC TTG GGG CTA GCAAG TGG GCC CTT TAT GCC TGT GGG CGT TTG GTA CCG TTC TAT TCC AGA AGT AGT GAG GAG GCT TT) and plasmid pICGN21 as template (pICGN2 sequences pos. 6103 to 6123 marked in italics) and Col10a1 from exon 2 (82 to 138, underlined). The product was digested with HindIII and inserted into the multiple cloning site behind the mP1 gene of placH+5\u2032Col10a1. The resulting plasmid placH+Col10a1-lacZ-frt-neo-frt was verified to be correct by restriction digestion and sequencing for correct insertion and composition.\nHomologous recombination and detection of BAC transgene\nBAC RP23-192A7 was electroporated into the E. coli strain EL250 (DH10B[\u03bbcl857(cro-bioA)<>araC-PBADflpe]) kindly provided by Dr. Neal G. Copeland, NCI, Frederick, MD, USA, containing a defective \u03bb prophage which supplies essential functions (exo, bet, gam) to protect and enhance recombination of linear DNA (Lee et al. 2001).\nThe 5.1\u00a0kb recombination cassette was excised from plasmid placH+COL10a1-lacZ-frt-neo-frt by XhoI; complete linearization with XhoI was important to avoid amplification of the circular targeting vector in E. coli.\nFor homologous recombination 300\u00a0ng linear fragment was transformed by electroporation according to Dower (Dower et al. 1988) in 0.1\u00a0ml cuvettes containing 50\u00a0\u03bcl of ice-cold competent EL250 containing BAC RP23-192A7, using a Bio-Rad gene pulser set at 1,750\u00a0kV, 23\u00a0mF with a pulse controller set at 200\u00a0\u03a9. Electrocompetent bacteria were prepared as described in (Lee et al. 2001). A 15\u00a0min heat shock at 42\u00b0C was used for induction of the homologous recombination machinery according to (Yu et al. 2000; Lee et al. 2001). Transformed cells were incubated for 1.5\u00a0h at 32\u00b0C in 1\u00a0ml of LB and selected for chloramphenicol- and kanamycin-resistance. Resulting clones (RP23-192A7\u00a0+\u00a0lacZ-frt-neo-frt) were screened for homologous recombination by PCR detecting the 5\u2032 recombination event (amplicon 437\u00a0bp) using primers P1 (5\u2032-TTT AGA GCA TTA TTT CAA GGC AGT TTC CA) and P2 (5\u2032-CGG CAC CGC TTC TGG TGC CGG AAA CCA GGC), and the 3\u2032 recombination event (product 378\u00a0bp) using primers P3 (5\u2032-ACA GAA TAA AAC GCA CGG GTG TTG GGC GT) and P4 (5\u2032-ATC ATT CCG CTG TAC TAG CTC AAG CCA ATC). Direct sequencing of BAC DNA was performed with primers P1 and P4.\nBAC DNA purification and generation of transgenic mice\nBAC-Col10a1\u00a0-\u00a0lacZ-neo DNA from clone#11 was extracted from multiple E. coli EL250-BAC#11 minipreps by alkaline lysis (Sambrook et al. 2001), purified by potassium acetate precipitation, washed with ethanol and dissolved in Tris-EDTA (TE) buffer. For size and quality control, aliquots were subjected to pulsed field gel electrophoresis (PFGE) (Fig.\u00a02). For purification, 50\u00a0\u03bcg BAC DNA of clone#11(BAC-Col10a1-LacZ-neo) were dissolved in TE buffer and concentrated to 500\u00a0\u03bcl by vacuum centrifugation. The DNA was linearized overnight with PISceI enzyme (NEBiolabs) cleaving the unique site in the BACe3.6 vector part. For chromatographic purification a 5\u00a0ml plastic pipette was packed with Sepharose CL-4B (Pharmacia) equilibrated in microinjection buffer (5\u00a0mM Tris\/HCl, pH 7.4, 0.1\u00a0mM EDTA, 5\u00a0mM NaCl) (Zeilhofer et al. 2005), the linearized BAC#11-DNA was mixed with bromophenol blue and chromatographed. Half milliliter fractions were collected until the dye reached the column bottom, and BAC DNA concentration in each fraction was analyzed by PFGE using serial dilutions of BAC RP23-192A7 DNA as internal standard. The BAC DNA concentration of the main fraction was adjusted to 1.5\u00a0ng\/\u03bcl, and NaCl concentration was increased to 100\u00a0mM to stabilize BAC DNA. Fractions were stored at 4\u00b0C until microinjection. Injection into fertilized oocytes and generation of transgenic mice was performed by standard techniques using oocytes from FVB mice and FVB\/ FVBxC57BL6 F1 hybrids as described (Zeilhofer et al. 2005).\nFig.\u00a02Characterization of one of the BAC-Col10a1-LacZ-frt-neo-frt clones (clone#11). Digestion with PISceI (lane 3), NotI (lane 4), PmeI (lane 5) and NruI (lane 6) resulted in fragments of the expected sizes. Sizes are compared with 1\u00a0kb marker (lane 1), 1\u00a0kb extension (lane 2) and PFG marker (lane 7), and the lengths are indicated. For microinjection, BAC #11 DNA was linearized with PISceI, purified by gel chromatography on Sepharose CL4B equilibrated in microinjection buffer and analyzed and quantitated by pulsed field gel electrophoresis\nGenotyping of transgenic mouse lines\nTransgenic founder animals were selected by PCR of tail biopsies using primers pairs P1\/P2 and P3\/P4 (see above) detecting recombination events. Each sample was standardized by PCR using P1\/P4 primer pair which generates a 538 bp fragment from the wt Col10a1 allele. Founders derived from injections of F1 hybrids were crossed with C57BL6, whereas founders derived from FVB oocytes were crossed with FVB mice. For genotyping of newborn mice and embryos DNA was extracted from skin or placenta, and transgene DNA was identified by PCR as above.\nHistological analysis and in situ hybridization\nNewborns mice were anesthetized under CO2, dissected and subjected to X-gal staining, followed by either clearing with KOH fixation, embedding into paraffin or by cryotome sectioning as described (Zhou et al. 1995). Embryos at 12.5 to 16.5\u00a0day were left undissected and stained with X-gal. Paraffin and frozen sections were counterstained with eosin.\nIn situ hybridization for collagen X was performed as described previously (Aigner et al. 1992), but using a digoxigenin-labeled mouse Col10a1-specific anti-sense probe derived from the 3\u2032-end of the Col10a1 gene instead of 32P-labeled dCTP. Non-radioactive in situ hybridization was done as described elsewhere (Schmidl et al. 2006), using alkaline phosphatase labeled anti-digoxigenin antibodies and BM purple (Roche) as color substrate according to the manufacturers protocol.\nDetermination of transgene copy number by Southern hybridization\nFor quantitative analysis of transgene copy number, genomic DNA was isolated from skin of newborns or from placenta of embryos and cleaved with BamHI. For standardization 5\u00a0\u03bcg wild-type DNA were mixed with 0.37\u00a0ng up to 18.38\u00a0ng RP23-192A7\u00a0+\u00a0lacZ-frt-neo-frt DNA corresponding to 1 up to 50 copies of recombined BAC per lane. Samples for standardization and 5\u00a0\u03bcg of DNA from transgenic founders were cleaved with BamHI, separated on 1% agarose gels and blotted onto nylon membranes. Membranes were hybridized to with a 32P-labeled probe derived for from the pBACe3.6 vector, prepared by PCR using primers BAC11-5 (5\u2032-TTTAAACGTGGCCAATATGGA) and BAC11-6 (5\u2032-CGCGGATCCTCTCCCTAT). An 8\u00a0kb band was detected in transgenic samples and the intensity was measured in a FLA-3000 phosphoimager (Fujifilm) using the AIDA program (Advanced Image Data Analyzer, Raytest ).\nResults \nConstruction of a placH-Col10a1-lacZ-frt-neo-frt targeting vector and homologous recombination\nThe BAC clone RP23-192A7 selected from the CHORI BAC library RPCI23 contains the mouse Col10a1 gene (7.2\u00a0kb) with 171\u00a0kb of upstream and 33\u00a0kb of downstream genomic regions. For insertion of the lacZ reporter gene into the second exon of the Col10a1 gene by homologous recombination, a targeting vector was constructed containing the lacZ gene linked to a neomycin resistance cassette flanked by two frt sites. The targeting vector was flanked by a 129\u00a0bp 5\u2032-terminal homology arm overlapping parts of the 3\u2032-end of intron 1 and 14\u00a0bp of exon 2 including the start ATG for fusion with NcoI. The 3\u2032 recombination site mapped to a 57\u00a0bp sequence in the 3\u2032 part of exon 2 (Fig.\u00a01b). The vector construct was verified by restriction mapping, Southern blotting and sequencing. Initial attempts using shorter homology regions resulted in mostly incorrect recombination events.\nFor homologous recombination, the BAC clone RP23-192A7 was electroporated into the E. coli strain EL250, resulting in EL250\/RP23-192A7. The Col10a1-lacZ-frt-neo-frt insert was excised from the targeting vector with XhoI (Fig.\u00a01) and electroporated into electrocompetent EL250\/RP23-192A7. Chloramphenicol- and kanamycin-resistant BAC-positive clones were selected and tested by PCR for homologous recombination by using two primer pairs P1\/P2 and P3\/P4 (see Fig.\u00a01 and Materials and Methods) specific for the 5\u2032- and 3\u2032- recombination sites, respectively. To verify the integrity of the Col10a1 gene in its genomic context after homologous recombination, the presence of the enhancer at 4.6\u00a0kb upstream the transcription site and the 5\u2032-end of a pseudogene downstream of the 3\u2032 UTR sequences was verified by PCR with specific primers (see Fig.\u00a01 and Materials and Methods) and sequencing of the BAC DNA. Furthermore, intact BAC ends were confirmed by PCR, using primers derived from the pBACe3.6 vector and the genomic insert.\nTo minimize BAC degradation, DNA was prepared from BAC-Col10a1-LacZ-Neo clone #11 by alkaline lysis and ethanol precipitation, avoiding absorption to minicolumns and shear stress. Control of the BAC DNA by PFGE confirmed the expected size of 220\u00a0kb (Fig.\u00a02).\nGeneration of transgenic mice\nDNA from BAC-Col10a1-lacZ-neo (clone #11) was linearized with PISceI at the unique restriction site in the pBACe3.6 vector sequence, purified by molecular sieve chromatography, and injected into the pronuclei of fertilized oocytes of FVB mice and FVB\/C59Bl F1 hybrids. Both strains were by far superior in litter size and successful raising offspring as compared to C57\/Bl6 mice. Out of 70 newborn pups, 13 were found harboring the lacZ gene after PCR analysis of genomic DNA, using primer pairs P1\/P2 and P3\/P4 (see Fig.\u00a01). Southern blot analysis of genomic DNA (Fig.\u00a03a) as well as Real- time PCR analysis of genomic DNA using Col10a1 intron specific primers showed that the founders contained between one and seven transgene copies (Table\u00a01)\nFig.\u00a03a Analysis of BAC transgene copy numbers in 13 transgenic founders by Southern hybridization. Genomic DNA was isolated from skin of newborns, cleaved with BamHI, separated on 1% agarose gels and blotted onto nylon membranes. Membranes were hybridized with a 586\u00a0bp 32P-labeled probe derived from the pBACe3.6 vector which recognizes an 8\u00a0kb band in transgenic samples. Band intensities were quantitated in a phosphoimager. Copy numbers of transgene BAC DNA (Table\u00a01) were calculated on the basis of a standard curve established with serial dilutions of BAC DNA starting from 50 copies down to 1 copy of BAC RP23-.192A7, diluted in 5\u00a0\u00b5g of wt DNA. b Comparison of LacZ activity in the scapulae of three different BAC transgenic lines harboring different BAC copy numbers shows some correlation between staining intensity and BAC copy number. All specimen were from new born animals and stained under identical conditions with X-gal for 18\u00a0hTable\u00a01BAC transgene copy number in 13 founders, determined by Southern hybridization (see Materials and methods)Founder #Strain sex\u03b2-gal activityBAC copy no.800FVB, m+5986FVB, f+31501FVB, f+11504FVB, m+31508F1, fND21510F1, mND11515F1, fND41516F1, f+61520F1, m+71524F1, fND21527F1, mND11533FVB, mND11534FVB, m+4ND not determined\nSeven of the 13 independent founder lines were analyzed so far for LacZ activity by X-gal staining of newborns or embryos (all F1 generation). All offspring analyzed revealed strong LacZ activity after X-gal staining which was restricted to the hypertrophic zones in all growth plates of long bones, ribs, vertebrae, sternebrae and to hypertrophic cartilage in the skull in E14.5- and E16.5-day embryos and newborns (see Figs.\u00a04, 5, 6). The X-gal staining pattern was identical in all seven lines investigated, while staining intensity varied in relation to the BAC transgene copy number (Fig.\u00a03b). Under identical staining conditions, X-gal staining was strongest in founder #1520 (seven transgene copies), followed by #800 (five copies) and #986 (three copies) (Fig.\u00a03b).\nFig.\u00a04Detection of lacZ expression in BAC-Col10a1-lacZ-Neo transgenic embryos by X-gal staining at E14.5 (a), E16.5 (b) and P1 (e) of the F1 generation of founder lines #1534 (a), #1504 (b), and #1520 (c). X-gal staining reveals strongest expression in chondrocytes in the hypertrophic zones of the scapula (s), humerus (h), femur (f), clavicula (cl) and all other long bones, furthermore in the hypertrophic zone of the ribs and in cervical vertebrae (c1, c2), (p\u00a0pelvic bone). c, d In E16.5 embryos some X-gal staining was also seen in the epidermis of the tail (barrow) and in hair papillae of the forehead and snout, and at nostrils (n). This expression is restricted to some basal keratinocytes in the epidermis, here shown in a section of the snout (d). It disappears after birth and may be a result of unspecific \u03b2-gal activity. No LacZ activity was detected in non-hypertrophic chondrocytes or in non-chondrogenic tissues.Fig.\u00a05X-gal staining of transgenic newborn mice of BAC-Col10a1-lacZ-neo (a, b founder line #800, F1; c\u2013i founder line #1520, F1). All hypertrophic zones of the growth plates of long bones, ribs, and vertebrae exhibit strong LacZ activity. a Humerus, distal; b ulna, proximal; c forearm, d hand; e skull: C1 cervical vertebrae 1 (atlas); eb ecto-occipital bone; h\u00a0hyoid bone; m mandibular condyle; MC Meckels cartilage; mh\u00a0head of malleus; r head of ramus. f Thoracal vertebrae (left four) and cervical vertebrae (right three), view from ventral; g ribs; h spine, view form dorsal, i sternumFig.\u00a06Paraffin sections of transgenic newborns (a\u2013e) (founder line #1520, F1) and E16.5\u00a0day (g) (founder line #1534) embryos showing strong and specific LacZ activity in all hypertrophic chondrocytes, e.g. in the growth plate of the humerus (a) (distal), elbow joint of the radius (b), sternebrae (c), atlas (d), ribs (e) and spine (f). Comparison of the lacZ staining pattern in the elbow joint (h radius) with that observed after in situ hybridization with a Col10a1 probe (g) in a parallel section illustrates that LacZ is expressed in the BAC transgenic mice only in collagen X expressing hypertrophic chondrocytes\nSpecific expression of the BAC-Col10a1-LacZ-neo transgene in hypertrophic cartilage\nVarious stages of embryonic development were analyzed for expression of the BAC-Col10a1-lacZ-neo transgene by X-gal staining. In E14.5-day embryos, strong LacZ activity was seen in all ribs, the hypertrophic zones of scapula, humerus, radius and ulna in the forearm and femur, tibia and fibula of the hindleg, as well as in mandibles and cervical vertebrae (Fig.\u00a04a). At day 16.5, the extent of X-gal staining expanded in the diaphyseal region of scapula, humerus and in the ribs, corresponding to the expansion of the hypertrophic zones at this embryonic stage. (Fig.\u00a04b, e) Sectioning of X-gal-stained embryos revealed that the lacZ transgene expression in the skeleton occurred exclusively in the hypertrophic zone of growth plates and ribs (see Figs.\u00a05, 6), correlating with collagen X expression as seen by in situ hybridization (Fig.\u00a06g, h).\nAt embryonic stages E12.5 to 16.5 X-gal staining also occurred in some regions of the epidermis (Fig.\u00a04b, arrow, Fig.\u00a04c, d), mainly in the papillae of the forehead in the snout (Fig.\u00a04c), and in the skin of the tail, hindlegs, forelegs, and shoulder It was restricted to the basal layer of keratinocytes (Fig.\u00a04d) and seen at E12.5\u00a0day before the first appearance of hypertrophic chondrocytes in cartilage, but was not detected after birth. It seemed unspecific as no \u03b11(X) mRNA signals were seen in the epidermis or hair papillae at any stage of development by in situ hybridization (N. Adam and K. von der Mark, unpublished data).\nTransgenic newborn F1 offspring of all seven founders analyzed so far revealed an even and strong LacZ activity in all growth plates of long bones (Figs.\u00a04e, 5a, b), ribs (Figs.\u00a04e, 5g), vertebrae (Figs.\u00a04e, 5f, h), and sternebrae (Figs.\u00a04e, 5i). In the skull, LacZ activity was seen in mandibular condyles, in hyoid cartilage, heads of malleus and ramus, ectooccipital and basisphenoid bone anlagen, cervical vertebrae and other ossifying cartilage anlagen (Fig.\u00a05e). The high intensity of X-gal staining allowed the visualization of discrete zones of Col10a1 expression in hypertrophic cartilage, for example the inner and outer aspects of the thoracal and cervical vertebrae (Fig.\u00a05f, h).\nAnalysis of the X-gal staining pattern in paraffin and frozen sections of transgenic embryos and newborn mice confirmed that the LacZ activity was restricted to hypertrophic chondrocytes (Fig.\u00a06), shown here for the humerus (a), radius (b), sternum (c), atlas (d), and ribs (e, f). The X-gal staining pattern correlated well with the expression pattern of \u03b11(X) collagen mRNA seen by in situ hybridization analysis of a similar section (Fig.\u00a06g, h). Neither non-hypertrophic chondrocytes nor any non-chondrocytic cell types revealed detectable levels of LacZ-activity in newborn transgenic animals. After prolonged staining, a non-specific reaction appeared in osteoclasts of long bones in transgenic and wildtype animals owing to endogenous galactosidase activity. Regardless of the transgene copy number, the LacZ activity in the offspring of all founders was seen persistently in all hypertrophic chondrocytes of the developing skeleton.\nDiscussion\nIn a previous report on the generation of Col10a1-specific reporter gene mouse lines the expression of lacZ under the control of a 4.6\u00a0kb mouse Col10a1 promoter region was restricted to hypertrophic cartilage, indicating that the -4.6\u00a0kb mouse promoter including a 500\u00a0bp enhancer element located at the 5\u2032-end of this region is sufficient for tissue specific expression (Gebhard et al. 2004). Also in another transgenic mouse expressing LacZ under a \u22124.0\u00a0kb proximal Col10a1 promoter lacking the enhancer, lacZ expression was restricted to hypertrophic cartilage, although mostly to the lower hypertrophic zone (Zheng et al. 2003). The Col10a1 enhancer has been originally described in the human COL10A1 gene; it was located between \u22121.9 and \u22122.4\u00a0kb by in vitro transfection studies in primary hypertrophic chondrocytes with various reporter gene constructs (Thomas et al. 1995; Beier et al. 1997; Chambers et al. 2002; Riemer et al. 2002). This enhancer is active in hypertrophic, but not in resting zone chondrocytes. Further upstream, a silencer element between \u22122.4 and \u22122.8\u00a0kb suppressed transcription in non-chondrogenic cells (Beier et al. 1997). The enhancer element is highly conserved in mammals with 78\u201385% sequence identity between human, bovine and murine Col10a1 genes (Gebhard et al. 2004) and contains a conserved AP-1 site which is essential for high transcription of enhancer- reporter genes in hypertrophic chondrocytes (Gebhard et al. 2004). In the murine gene, the enhancer is located between \u22124.1 and \u22124.6\u00a0kb due to insertion of two virus-related sequences in the promoter (Gebhard et al. 2004). Further cis-regulatory elements in the 4.6\u00a0kb promoter include an AP-1 site in the proximal promoter (Harada et al. 1996) and several runx2 bindings sites (OSE elements), (Zheng et al. 2003).\nYet, although lacZ transgene expression in the 4.6\u00a0kb Col10a1\u2013LacZ reporter mice was specific and restricted to hypertrophic chondrocytes, not all hypertrophic chondrocytes in the growth plates of 18.5-day embryos were found to be LacZ positive, and only weak LacZ expression was observed at early embryonic stages before day E17 (Gebhard et al. 2004). Furthermore, not all transgenic founders containing the lacZ transgene were stained with X-gal in the growth plates, suggesting positional effects of additional regulatory elements in the Col10a1 gene further upstream of the enhancer or downstream of the coding sequences. It may also indicate a high level of sensitivity to interfering activities of the genomic context of insertion. It is possible that the \u22124.6\u00a0kb promoter including the first intron contains essential silencing elements required for suppressing Col10a1 expression in non-hypertrophic cartilage and in non-chondrogenic tissues, but possibly lacks additional regulatory elements beyond the enhancer region.\nIn contrast, in the Col10a1-BAC transgenic lines presented here, all hypertrophic chondrocytes showed strong and even LacZ expression in all zones of endochondral ossification in embryonic and postnatal transgenic animals. The LacZ reaction of hypertrophic cartilage became visible already after 3\u20134\u00a0h of X-gal staining, while under the control of the 4.6\u00a0kb Col10a1 promoter significant LacZ activity in transgenic mice did not become visible before 18\u00a0h staining time (Gebhard et al. 2004).\nRecently, evidence is accumulating that in many genes further tissue-specific enhancers are located in large distances from the start site of transcription. For example, regulatory sequences were identified 20\u201330\u00a0kb upstream of the transcription start site of the Myo D gene (Goldhamer et al. 1992) or the Ubx gene in Drosophila (Irvine et al. 1991), up to 270\u00a0kb downstream in the mouse BMP5 gene (DiLeone et al. 2000) or between 290K\u00a0kb upstream and 450\u00a0kb downstream of the Sox9 gene (Bagheri-Fam et al. 2006). Thus we cannot exclude that additional tissue-specific regulatory elements may be present in the Col10a1 gene, either upstream in the region between \u2212170\u00a0kb and the enhancer at \u22124.6\u00a0kb, or downstream of the 3\u2032-end of the Col10a1 gene. The BAC transgenic reporter lines established here would facilitate the identification of such elements by generating further mutants using the homologous recombination technique (DiLeone et al. 2000).\nReasons for the high reporter gene expression levels in the BAC-Col10a1-lacZ-neo reporter lines in comparison to the 4.6\u00a0kb Col10a1-lacZ mice are for one part the high copy number of BAC transgenes, and for the other part the favorable und undisturbed conditions for transgene expression in the BAC environment. The X-gal staining intensity in founder line #1520 harboring seven BAC transgene copies was clearly higher than that of founder line #986 harboring only three BAC copies. But in addition, the use of targeted BAC recombination permits the expression of genes in their genomic context, containing most relevant regulatory regions in the correct orientation and distance, in contrast to conventional transgenic mice generated by injection of short reporter genes. This strategy is less sensitive to unpredictable influences by regions surrounding the random integration site (Mortlock et al. 2003). The expression pattern of LacZ, however, was very similar in all seven BAC lines analyzed so far, apparently independent of the integration sites and the copy number of the BAC transgene.\nMost E14.5- to E16.5-day BAC-Col10a1-lacZ-neo transgenic embryos showed some X-gal staining in hair follicles of the forehead and snout, and of the epidermis of the tail, legs and shoulder, which disappears after birth. In the X-gal positive spots, staining was restricted to the basal layer of keratinocytes. Since these cells do not express type X collagen, the effect may be due to endogenous galactosidase activity, or a result of LacZ activation by other, Col10a1-unrelated regulatory elements located in the BAC clone.\nIn addition to Col10a1, the BAC clone RP23-192A7 contains coding regions of three additional genes Tspyl-1, Tspyl1-4 and Nt5dc1. Whether these genes are expressed under their endogenous promoters in the BAC transgenic mice is currently investigated. If they are expressed, they could affect the phenotype of the established transgenic mouse lines, but no phenotypic alteration in skeletal elements nor in other tissues have been observed in transgenic embryos, newborn or adults as compared to wildtype littermates.\nThe high intensity of X-gal staining under the control of the BAC-Col10a1 promoter allowed the visualization of discrete zones of Col10a1 expression in hypertrophic cartilage of anatomical structures not investigated so far in such detail and completeness, for example the skull or the inner and outer aspects of the thoracal and cervical vertebrae. The data presented in this study illustrate the superior potential of reporter gene expression analysis in combination with BAC recombineering technique as compared to conventional in situ hybridization analysis.\nThe robust and specific expression of the lacZ reporter gene under the control of BAC-Col10a1 environment furthermore shows that the placH+Col10a1-lacZ-frt-neo-frt targeting vector may be an extremely versatile vector for the targeted overexpression of other genes in hypertrophic chondrocytes of growth plates with high efficiency and specificity. Thus, the Col10a1-based BAC vector may be a useful tool to analyze the role of effector genes in skeletal development, endochondral ossification, and fracture callus formation.","keyphrases":["bac","hypertrophic cartilage","homologous recombination","collagen x","transgenic mouse"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_J_Nucl_Med_Mol_Imaging-3-1-1998881","title":"ApoSense: a novel technology for functional molecular imaging of cell death in models of acute renal tubular necrosis\n","text":"Purpose Acute renal tubular necrosis (ATN), a common cause of acute renal failure, is a dynamic, rapidly evolving clinical condition associated with apoptotic and necrotic tubular cell death. Its early identification is critical, but current detection methods relying upon clinical assessment, such as kidney biopsy and functional assays, are insufficient. We have developed a family of small molecule compounds, ApoSense, that is capable, upon systemic administration, of selectively targeting and accumulating within apoptotic\/necrotic cells and is suitable for attachment of different markers for clinical imaging. The purpose of this study was to test the applicability of these molecules as a diagnostic imaging agent for the detection of renal tubular cell injury following renal ischemia.\nIntroduction\nAcute renal failure (ARF) that results from ischemic or toxic insults to the kidney is usually referred to clinically as acute tubular necrosis (ATN), where tubular damage and altered glomerular hemodynamics may coexist or even lead to each other [1\u20133]. In some cases of either human ARF [4, 5] or experimental models of renal injury [6], a lack of correlation between histological evidence of injury and renal function has been found, and the relative contribution of functional versus structural changes to the evolving renal dysfunction has not been unequivocally substantiated. Since ATN is a dynamic clinical condition, rapidly evolving from the incipient to the established phase, its early identification is critical. However, the various currently available methods of clinical and laboratory assessment are insufficient to differentiate between ischemic ARF, resulting in acute tubular dysfunction secondary to cell injury, and prerenal azotemia, characterized by decreased glomerular filtration but no epithelial cell injury. The inability to distinguish between these situations leads to long disputes over the pathogenesis, diagnosis, and management of evolving renal failure. Renal biopsy provides limited data because of the small sample size and the very focal pattern of tubular injury [2, 7]. In addition and most importantly, it is impractical in the human syndrome. Functional data in early phases are also of little help, because prerenal azotemia becomes an important consideration at that time, and only the evolution of the clinical situation really defines the nature of the problem [8, 9]. The utilization of specific urinary biomarkers of tubular injury, such as KIM-1, is not yet considered valid or sufficiently sensitive to establish ATN [10, 11]. Moreover, these tests remain qualitative rather than quantitative markers of injury. Since therapeutic interventions might differ at various points in the evolution of ATN, advanced techniques are required to noninvasively follow the dynamic status of tubular injury. Furthermore, a technology that enables imaging endpoints, instead of time-consuming dissection and detection of renal parenchymal cellular damage, would be highly valuable.\nMultiple lines of evidence suggest that both apoptotic and necrotic cell death are important pathogenic mechanisms in acute renal failure [12, 13], where renal parenchymal cells are the primary target of a broad spectrum of inciting factors, ranging from ischemic and nephrotoxic agents to endotoxemia. Apoptotic cell death is a feature of both ischemia\u2013reflow injury [14] and radiocontrast-induced acute renal failure [15], and is a documented event in transplant biopsy as well [16]. The role of apoptosis in sepsis syndrome has not been adequately explored, but there is rapidly developing evidence to suggest that increased apoptotic processes may play a determining role in the outcome of sepsis syndrome [17, 18]. Although apoptosis is recognized as playing a role in these conditions, there is a lack of sensitive markers to monitor programmed cell death in association with disease progression or regression.\nRecently, we developed a family of small molecule compounds, ApoSense, capable of discriminating between vital and apoptotic cells. ApoSense molecules are small non-peptidic fluorescent compounds that are capable of selective targeting, binding, uptake, and accumulation within apoptotic and\/or necrotic cell cytoplasm, from the early stages of the death process. Our preliminary results from different animal models associated with apoptosis showed that the recognition of damaged cells by ApoSense is universal, irrespective of the cell type or the apoptotic\/necrotic trigger. Indeed, examined in multiple animal models, ApoSense was proven to be a highly sensitive and specific sensor for in vivo detection of neoplastic, hepatic, myocardial, atherosclerotic, and neuronal apoptotic and necrotic cell death (in preparation). In the present study the potential of both fluorescent and radiolabeled derivatives of one of the ApoSense compounds, didansyl cystine (DDC), was explored as a diagnostic imaging agent for detection of renal tubular injury in three different in vivo animal models of ATN: renal warm ischemia\/reperfusion, radiocontrast-induced distal tubular necrosis, and cecal ligature\/perforation-induced sepsis. These different models illustrate the applicability of ApoSense as a diagnostic imaging agent enabling real-time accurate detection of renal parenchymal damage of various types, and evaluation of its distribution and magnitude.\nMaterials and methods\nMale Sprague-Dawley rats weighing 180\u2013250\u00a0g and 8- to 10-week-old C57Bl mice (Harlan Laboratories, Jerusalem, Israel) were used for these experiments. All experimental protocols were performed according to the Guiding Principles for Research Involving Animals, and approved by the local Animal Care Committee; the experiments described in this manuscript also comply with the current laws of Israel.\nApoSense synthesis and radiolabeling\nThe ApoSense compound used in the present study was N,N\u2032-didansyl-L-cystine (DDC). Both its inherent fluorescent characteristics and its radiolabeled derivative (labeled with 3H radionuclide) were used for identification and quantification of apoptotic cells.\nFor DDC synthesis, dansyl chloride (675\u00a0mg) and cystine (240\u00a0mg) were dissolved in water\/acetone solution with potassium carbonate (550\u00a0mg), and after 1.5\u00a0h at room temperature, the aqueous mixture was extracted with ethyl acetate. The crude DDC was dissolved in 5% sodium carbonate, washed with ether, and reacidified to pH 3 to afford a yellow precipitate. The product was analyzed using 1H-NMR.\nLabeling of DDC with 3H was performed by consecutive addition of 0.16\u00a0mg cystine (0.65\u00a0\u03bcmol), 0.57\u00a0mg sodium carbonate (5.3\u00a0\u03bcmol), and 37\u00a0MBq (1\u00a0mCi) of 3H-dansyl chloride (Vitrax) at a concentration of 1.4\u00d710\u22124\u00a0mmol. The product was purified using a Bond-Elut C-18 cartridge eluted with 25% acetonitrile in 0.1% TFA in water, then lyophilized and dissolved in 1\u00a0ml of NaPPi buffer (0.1\u00a0M, pH=7.4). Radiochemical purity was assessed by thin-layer chromatography.\nEvaluation of ApoSense binding to apoptotic cells in vitro\nHuman adult T-cell leukemia Jurkat cells (clone E6-1) and cervical carcinoma HeLa cells (CCL-2.2), were purchased from ATCC (Rockville, MD, USA) and grown in Dulbecco\u2019s modified Eagle\u2019s medium with 10% fetal calf serum at 37\u00b0C in 5% CO2. For induction of apoptosis in the Jurkat cell sample, 1\u00d7106 cells\/ml were treated with IgM anti-Fas (CD95) antibody, clone CH11 (Medical and Biological laboratories, Japan) at a concentration of 0.1\u00a0\u03bcg\/ml for 120\u2013180\u00a0min. In additional studies, before incubation with anti-Fas antibody, Jurkat cells were subjected to 50\u00a0\u03bcM of caspase inhibitor Z-VAD-FMK in 0.05% DMSO (Enzyme System Products, Dublin, CA). For apoptosis induction in HeLa cells, growing cells were treated with staurosporin (STS; 250\u00a0nM; Sigma) for 18\u00a0h. At the end of the incubation period, cells were harvested by trypsinization (Trypsin-EDTA, Bet-Haemek, Israel) and collected by centrifugation.\nFor assessment of apoptosis, both fluorescence microscopy and flow cytometry were employed. Trypsinized HeLa cells were incubated with DDC (50\u00a0\u03bcM) and annexin V-FITC (Phosphatidyl Serine Detection Kit, IQP-116F) for 20\u00a0min and the staining pattern was evaluated under a fluorescence microscope (BX51TF; Olympus Optical.Co., Ltd., UK) equipped with relevant filters. For DDC staining, excitation at 365 and emission at 420 (band pass) nm was employed, whereas for FITC detection, excitation was at 488\u00a0nm and emission at 530\u00a0nm. Apoptotic Jurkat cells were detected by flow cytometry after double staining with annexin V-FITC and PI or with DDC and PI. Briefly, 30\u00a0\u03bcl of cell suspension (1\u00d7106 cells\/ml) was diluted in 300\u00a0\u03bcl Hepes buffer (10\u00a0mM Hepes and 140\u00a0mM NaCl, pH 7.4) containing annexin V-FITC\/PI or 50\u00a0\u03bcM of DDC\/PI. Following 40\u00a0min of incubation at room temperature, the cells were subjected to analysis using FACS Vantage VE (BD Biosciences, San Jose, CA) and CellQuest software. A total of 10,000 events were collected for each sample.\nEvaluation of ApoSense uptake into apoptotic\/necrotic cells in vivo\nThe detection of renal parenchymal cell death by ApoSense was assessed in three animal models of ATN, generally classified under ischemic and septic etiologies [19\u201321].\nRat renal ischemia\u2013reperfusion (I\/R) model Operative procedures were performed in rats under general anesthesia induced by the combination of ketamine, 80\u00a0mg\/kg, and xylazine, 10\u00a0mg\/kg, administered intraperitoneally. Renal ischemia was induced by unilateral left renal artery clamping, using a small nontraumatic vascular clamp, for 45\u00a0min [12]. Reperfusion was initiated by removal of the clamp. The period of renal reperfusion was 24\u00a0h. Preliminary experiments revealed no difference between the I\/R rat contralateral kidney and sham-operated normal rat kidneys with regard to cell death of renal tubular epithelium after 24\u00a0h. Thus, in the present study, the contralateral, untreated kidney from the same animal served as the sham-operated control kidney. During the course of reperfusion, animals were injected intravenously with 20\u00a0mg\/kg of DDC and 4\u00a0h later both kidneys were excised, frozen in liquid nitrogen, and stored at \u221270\u00b0C until use. For microscopic analysis and fluorescence imaging, 5-\u03bcm-thick cryo-sections of both kidneys were evaluated either by fluorescence microscopy or were stained with hematoxylin and eosin (H&E) for light microscopy analysis of apoptotic and\/or necrotic lesion distribution. Furthermore, the presence of apoptotic cell nuclei was confirmed by apoptosis detection kit staining (ApopTag Fluorescein Kit, Intergen Company, Purchase, NY) using the TUNEL (terminal deoxynucleotidyl transferase-mediated uridine triphosphate nick end labeling) method. Additional ex vivo whole organ fluorescence imaging was carried out on freshly excised kidneys analyzed under a Leica (Leica MZ FL III, Leica, Switzerland) stereomicroscope. DDC was visualized using a 360Ex and >420Em nm (NU) filter set. Emitted fluorescence was collected on a Leica DC 300F digital camera and images were processed and analyzed by Leica QWin Lite software. For quantitative analysis of the extent of renal DDC accumulation, previously frozen kidneys were thawed, weighed, and homogenized in buffer containing 50\u00a0mM Tris-HCl and 0.005% Triton X-100, pH 7.4 (1:7 w\/v), using Heidolph RZR 2020 homogenizer (Heidolph Instruments GmbH & Co. KG, Schwabach, Germany). Solubilized tissue extract samples were centrifuged twice at 13,800\u00a0rpm (20\u00a0min, 4\u00b0C) and the supernatant fractions were analyzed using a microplate reader (GENious Fl Reader, TECAN, Gr\u00f6dig, Austria) equipped with optical filters for fluorescence measurements. All samples were pipetted in triplicate onto black Ritter flat bottom microplates and read at 360\u00a0nm Ex and 535\u00a0nm Em. For the calibration curve, a dilution series of DDC were prepared in the same Tris-Triton buffer, supplemented with 0.05% kidney extract of control vehicle-injected animals. The same control animal samples were also used for background values. In all samples, DDC concentration (\u03bcg\/g tissue) and the percentage of the injected dose (% ID\/g) were calculated.Additional biodistribution studies were carried out to assess the spread of radiolabeled DDC into different organs. Twenty-four hours after unilateral renal ischemia\/reperfusion, rats were intravenously injected with 5\u201320\u00a0\u03bcg\/kg of the tritiated form of DDC (3H-DDC) [111\u2013370 kBq (3\u201310\u00a0\u03bcCi)\/animal]. Four hours later, both kidneys, liver, gut, and blood samples were collected, weighed, and submitted to lyses by SOLVABLE reagent (GNE9100, Packard Bioscience), according to the manufacturer\u2019s instructions, and sample radioactivity was determined using a beta counter (TRI-CARB 2100TR, liquid scintillation analyzer, Packard Bioscience). Both tissue uptake (% ID\/g) and the ratios of the radioactivity of the ischemic kidney (DPM\/g) to that of the contralateral kidney and the other non-target tissues were calculated.\nRat model of radiocontrast-induced distal tubular necrosis (DTN) The selectivity of DDC uptake in injured renal tissues was evaluated in this rat model, characterized by selective medullary hypoxic tubular damage. Nephropathy was induced by the combined administration of indomethacin (Sigma Chemical Co.), 10\u00a0mg\/kg, i.v., N-\u03c9-nitro-L-arginine methyl ester (L-NAME, Sigma Chemical Co.), 10\u00a0mg\/kg, i.v., and the radiocontrast agent sodium iothalamate 80% (Angio-Conray, Mallinckrodt Inc), 6\u00a0ml\/kg, i.a., as previously described [22]. The study was performed on six rats, with an additional two rats injected with vehicles serving as control (these two control animals studied are representative of at least 29 control rats tested in our previous experiments). Twenty-four hours after insult, animals were intravenously injected with 20\u00a0mg\/kg DDC and 2\u00a0h later they were sacrificed. The left kidney was removed and snap-frozen for fluorescence analysis (and for H&E and TUNEL staining, as detailed above), whereas the right kidney, used for morphological evaluation, was in vivo fixed with glutaraldehyde perfusion through the abdominal aorta, as described elsewhere [22, 23]. Perfusion-fixed kidneys were immersed in buffered 2% OsO4, dehydrated, and embedded in an Araldite-EM bed 812 mixture. Large sections were cut perpendicular to the renal capsule, containing cortex and medulla, including fornix and papillae. One-micrometer sections were stained with methylene blue and analyzed in a blinded fashion (i.e., without knowing the fluorescence results) for morphological alterations. As previously detailed [23], tubular necrosis was determined separately for S3 proximal tubules in the outer stripe and medullary rays, and for medullary thick ascending limbs (mTALs) in the outer, mid, and inner zones of the inner stripe of the outer medulla. The extent of damage was expressed as the percentage of necrotic tubules out of total tubules counted. Papillary necrosis was semiquantitatively assessed using a score of 0\u20133.\nSepsis model in the mouse The sensitivity of cell death recognition by DDC was tested in the cecal ligature and perforation (CLP) model in mice, where rare, focal tubular cell damage is hardly detected by regular morphological evaluation [24, 25]. The cecum of six anesthetized mice was isolated, ligated distal to the ileocecal valve, and punctured twice with a 26-gauge needle. An additional four sham-treated animals were laparotomized, but the cecum was neither ligated nor punctured. Twenty-four hours later, mice were injected i.v. with DDC, and 2\u00a0h afterwards, kidneys were removed and subjected to fluorescence microscopy analysis as detailed above.\nStatistical analysis\nData reported are mean\u00b1s.e.m. Student\u2019s t test was used to assess the significance of differences between two groups. Significance was accepted at p<0.05\nResults\nBinding of ApoSense to apoptotic cells in vitro\nConcomitant confocal imaging of HeLa cells undergoing apoptosis, double stained with DDC and annexin V, revealed that DDC accumulates within the cytoplasm of the apoptotic cell while annexin V is attached to the external apoptotic cell membrane (Fig.\u00a01). Accumulation of ApoSense within apoptotic cells can contribute to amplification of the signal obtained and consequently to the high signal-to-noise ratio. Previous studies have shown that through an interaction with the death receptor CD95 induces apoptosis by formation of a signaling complex at the cell membrane and subsequent caspase-8 and caspase-3 activation [26]. Therefore, the specificity of DDC uptake was tested in apoptotic Jurkat cells that were co-treated with caspase inhibitor and analyzed by flow cytometry. As shown in Fig.\u00a02, in viable, non-apoptotic cells (green line), no uptake of DDC was detected, except in a small population of spontaneously dying cells representing late events. Following 150\u00a0min of incubation with anti-Fas, most cells accumulated DDC and created a new peak of cells (red line) with a higher fluorescence intensity (the geometric mean fluorescence intensity value, GMFI, was 120.18) while the peak of late apoptotic cells remained unchanged. In the presence of the caspase inhibitor Z-VAD-FMK, cells lost the ability to bind DDC (black line), while their viability remained preserved, suggesting that accumulation of DDC compound is dependent on the apoptotic process itself. Upon addition of the caspase inhibitor, a marked shift towards a lower fluorescence intensity was noted, and the final intensity values were almost the same as the control cell values (GMFI 46.38 and 46.02, respectively), suggesting maximal inhibition. Fig.\u00a01Concomitant confocal imaging of HeLa cell undergoing apoptosis by DDC (green fluorescence) and annexin V (red fluorescence). DDC accumulates within the cytoplasm of the apoptotic cell while annexin V is attached to the external membraneFig.\u00a02Binding of DDC to apoptotic cells is caspase dependent. Jurkat cells were treated with CD95 in the absence (red line) or presence (black line) of the pan caspase inhibitor z-VAD-fmk for 150\u00a0min. Following incubation, cells were centrifuged and incubated in the presence of DDC for 40\u00a0min before FACS analysis. This histogram charts the number of cells counted (y-axis) and the fluorescence intensity (x-axis). The green line represents the control non-apoptotic sample\nApoSense uptake into ischemia\/reperfusion (I\/R) injured kidneys\nEvaluation of DDC accumulation into injured tubular cells of an ischemic rat kidney was performed on the basis of its intrinsic fluorescent properties. Either ex vivo whole kidney fluorescent imaging or cryo-section fluorescent analysis was employed. As seen in Fig.\u00a03, DDC accumulated in the injured (a) but not in the contralateral intact kidney (b), thereby exhibiting substantial capability for imaging apoptosis and\/or necrosis in vivo. Microscopic analysis of corresponding kidney sections revealed intense cytoplasmatic staining of tubular epithelial cells (Fig.\u00a03c,d) located at the corticomedullary junction. No uptake of DDC into viable renal parenchyma was detected. The extent of DDC accumulation in these cells was quantitatively determined in homogenized whole kidney extracts from five rats, using microplate reader fluorescence measurement. As shown in Fig.\u00a03e, the average DDC uptake into the ischemic kidney (expressed as %ID\/g) was 6.73\u00b11.34 fold higher (p=0.0076) than that in the intact control kidney (0.41\u00b10.11 and 0.06\u00b10.02, respectively). H&E-stained histological sections of kidneys from I\/R-treated rats revealed multiple apoptotic as well as necrotic cells situated along the tubule epithelium or in the tubule lumen (Fig.\u00a04). A spectrum of changes characteristic of tubular cell apoptosis, including shrinkage of renal tubular cells, cytoplasmic acidophilia, chromatin dense condensation and margination, nuclear fragmentation, and pyknosis, is shown in Fig.\u00a04a, left. In addition, as previously described [27\u201330], a large percentage of tubules in this region showed substantial tubular dilatation, epithelial necrosis, dissolving of tubular cells, luminal necrotic debris, and cast formation (Fig.\u00a04b, left). The presence of apoptotic\/necrotic cells was also defined by intensive green fluorescence of TUNEL-positive nuclei (Fig.\u00a04c, left). As shown in Fig.\u00a04a\u2013c, right panel, the accumulation of DDC within the damaged areas in ischemic kidneys corresponds completely, even at the single cell level, to the injured areas detected by H&E and TUNEL staining. However, it seems that in some areas, accumulation of DDC in apoptotic cell cytoplasm occurred earlier than DNA destruction and thereby cells were not stained by TUNEL but merely by DDC (Fig.\u00a04c, arrow). These results may indicate that DDC has the ability to identify early apoptotic cells and that use of TUNEL alone may not reveal the full spectrum of cell death stages. Fig.\u00a03DDC uptake into renal damaged areas. a,b Ex vivo images of the left ischemic (a) and the right sham-operated (b) kidney derived from a single I\/R rat (\u00d77). Whole organs were viewed under a stereomicroscope using a NU filter set without light flux, 4\u00a0h after DDC administration. c,d Histological sections of kidneys (c, \u00d7100; d, \u00d7600) revealing intense uptake along the tubule epithelium or in the tubule lumen. e A quantitative estimate of DDC uptake into I\/R ischemic and intact (sham) kidney from five rats, normalized to total injection dose for each gram of tissue. Values are means\u00b1s.e.m.Fig.\u00a04Morphological evidence for renal tubular cell apoptosis\/necrosis following ischemia\u2013reflow. a Tubular cells (H&E staining) are seen with changes typical of apoptosis (left). In an adjacent 5-\u03bcm section (right), these cells take up DDC. \u00d71,000. b Necrotic tubular cells in tubular lumina (H&E staining; left) and corresponding DDC fluorescence of the same injured cells (right). \u00d7400. c Co-localization of DCC fluorescence (right) with TUNEL-positive nuclear staining (left). Some cells are likely in the early stages of apoptosis and are TUNEL negative, but take up DDC (arrow). \u00d7200\nBiodistribution studies of 3H-DDC were performed in seven additional rats following the I\/R procedure. The accumulation of radioactivity in the blood, gut, liver and kidney at 4\u00a0h after intravenous administration is shown in Fig.\u00a05. Uptake in the ischemic kidney was significantly higher than that in the intact contralateral one(p<0.05) and also higher than liver, gut and blood uptake values (p<0.01). The average uptake values at 4\u00a0h, rendered as % ID\/g, were 1.20\u00b10.13 for the ischemic kidney, 0.58\u00b10.12 for the intact kidney, 0.41\u00b10.07 for the liver , 0.42\u00b10.09 for the gut, and 0.28\u00b10.01 for blood. The corresponding mean ischemic kidney-to-tissue ratios were 2.1\u00b10.5, 2.9\u00b10.5, 2.8\u00b10.7, and 4.3\u00b10.6, respectively. These results indicate that at 4\u00a0h following administration, DDC remained accumulated in the target organ, while being cleared from non-target ones. Fig.\u00a05Biodistribution of radiolabeled 3H-DDC after renal I\/R treatment. Uptake of 3H-DDC (% injected dose per gram corrected for background) in target and non-target tissues, determined 18\u201324\u00a0h after ischemia and 4\u00a0h after 3H-DDC administration, is shown. Data are presented as mean\u00b1s.e.m., n=7. Asterisks denote significantly higher accumulation of 3H-DDC in ischemic kidney compared with other organs (tissues): *p<0.05, **p<0.01\nRadiocontrast-induced distal tubular necrosis\nHeyman et al. [23] reported previously that animals subjected to the radiocontrast model of DTN developed varying degrees of renal dysfunction and structural damage, but the presence, distribution, and extent of tubular injury on an individual basis could not be predicted from the magnitude of kidney dysfunction. In the present study, as well as in previously published studies [22, 23, 31], renal morphology analysis disclosed a wide-ranging extent of medullary (namely mTALs and S3 segments in the outer medulla and medullary rays) and papillary damage, with a poor overall correlation between structural damage and functional deterioration (data not shown). By contrast, as shown in Figs.\u00a06 and 7, DDC distribution pattern and degree of deposition closely mirrored the morphological findings in the contralateral perfused kidney, suggesting that ApoSense uptake may reflect the level and degree of renal damage more closely than do functional tests. Results from both fluorescence and morphological analysis disclosed the characteristic striped pattern of injury (Fig.\u00a06a,b), where tubules adjacent to the vasa recta are preserved while those distant from vascular bundles are injured (Fig.\u00a07a). Homing of DDC was primarily restricted to injured regions within the outer medulla (Fig.\u00a06c). No DDC uptake at a specified region in the absence of morphological damage was seen, as demonstrated in Fig.\u00a07b, where necrosis of the papillary tip in one of the kidneys was clearly morphologically demarcated and DDC deposition was restricted to precisely the same injured area. Uptake occurred preferentially in apoptotic\/necrotic lesions, as confirmed by co-localization with TUNEL staining (Fig.\u00a06d). Thus, DDC was found to be a specific molecular marker for tubular injury in various segments of the kidney. Fig.\u00a06Renal macroscopic and microscopic findings in rats subjected to the ARF protocol (indomethacin, L-NAME and iothalamate) and subsequently injected with DDC. a Macroscopic view of the characteristic striped pattern of severe hypoxic outer medullary damage in an in vivo fixed kidney. b The in vivo microscopic distribution of DDC in the contralateral kidney correlates closely with the distribution of the striped pattern of outer medullary necrosis in the ipsilateral one (\u00d730). c Details (\u00d7100) of the cross-section, showing fluorescent imaging of DDC uptake in injured areas of both the inner strip and the forniceal region of the outer medulla. Examination of contiguous 5-\u03bcm sections revealed that the accumulation of DDC in damaged areas (d, upper panel) matched the infarct areas stained by TUNEL (d, lower panel) (\u00d7200). OM outer medulla,IM inner medulla, SP secondary pyramidFig.\u00a07Characteristic morphological findings in rats subjected to the ARF protocol and injected with DDC. Cryo-sections from the left kidney were used for DDC fluorescence analysis, whereas the in vivo fixed right kidney was used for light microscopy morphological examination of methylene blue-stained slides. Analyses of both kidneys were performed in a blinded fashion. An example is shown of the striped pattern reflecting the damage gradient, where tubules adjacent to the vasa recta (VR) are preserved (red stars) while those distant from vasa recta are injured (black stars) (a, left panel, \u00d7100). Note the excellent co-localization of morphologically damaged tubules with the DDC fluorescence (a, right panel, \u00d7100) in the contralateral kidney. DDC fluorescence was selectively identified in all morphologically injured regions, including papillary tip structures (b: left panel \u00d740, right panel \u00d7100)\nCecal ligature\/perforation-induced sepsis\nDDC was evaluated as a potential in vivo marker for renal tubular cell apoptosis in another animal model of ATN, in which sepsis-associated renal dysfunction was induced by cecal ligation and puncture. Morphological alterations of the kidney due to sepsis can hardly be identified using routine histological techniques [32, 33], and experimental data regarding the apoptotic cells of kidneys affected by sepsis are therefore almost non-existent. Indeed, in all septic mice we found only rare focal renal tubular cell injury, seen either in the outer medulla or in the cortex (Fig.\u00a08a). As seen in Fig.\u00a08b, focal renal tubular cell injury is apparently identified by DDC fluorescence, but is not clearly traced using H&E histopathological analysis; this demonstrates the high sensitivity of DDC in the detection of such renal damage. TUNEL staining confirmed the presumption that renal cells stained by DDC are indeed dying cells (Fig.\u00a08c). Fig.\u00a08Focal uptake of DDC in kidneys of six mice following sepsis caused by cecal ligature and perforation (CLP). Apoptotic cells, imaged by DDC, were shown either in the outer medulla (a, left) or in the cortex (a, right) of individual mice (\u00d7100). Most apoptotic cells appeared to be tubular epithelial cells (b, left, arrows). An evident correlation was demonstrated between DDC fluorescence (b, right and c, right) and apoptotic cells detected on H&E (b, left) or TUNEL (c, left) stained slides (\u00d7200)\nDiscussion\nAs a result of recent progress in medicine, biology, and physics, the field of diagnostic imaging is shifting from conventional anatomical imaging to the sphere of functional and molecular imaging, with the aim of imaging biological processes related to both health and disease. The implementation of the new ApoSense technology for clinical imaging of apoptosis would represent a major addition to this emerging field, allowing non-invasive imaging of cell death processes in vivo as a means to both the diagnosis of disease and the monitoring of treatment efficacy in a broad spectrum of disease states.\nIn the present study, we have reported the successful application of ApoSense technology as a diagnostic imaging tool for the detection of apoptotic and\/or necrotic cell death. ApoSense technology may be particularly useful in ARF, where current clinical and physiological assessment and complementary imaging tests are insufficient to discriminate between true renal parenchymal damage and prerenal azotemia (in which kidney filtration function is drastically reduced even though the internal structures of the kidney are intact). An inherent fluorescent property of one of the ApoSense family of compounds, DDC, was utilized for detection of tubular cell death in three animal models of ATN. The results obtained are in complete agreement with in vitro findings of DDC-specific binding to apoptotic cells (Figs.\u00a01, 2), and thereby strongly suggest the ability of DDC to image renal tubular cell apoptosis in vivo.\nThe three in vivo experimental models of ATN used in the present study complement each other regarding the applicability of ApoSense technology in the detection of ATN. The results obtained using the I\/R model showed the qualitatively remarkable capability of ApoSense in targeting of injured tissues and ex vivo imaging of apoptosis (Fig.\u00a03). Additionally, the model provides quantitative analysis of the extent of renal tubular cell damage based on the extent of DDC accumulation in these cells. It was shown that the uptake of DDC in the ischemic kidney was 6.73-fold higher than in the non-ischemic one. Moreover, the specificity of targeting of the injury by ApoSense was demonstrated by its localization to regions of apoptotic\/necrotic cell death, detected by H&E and TUNEL staining (Fig.\u00a04). Furthermore, it was observed that some TUNEL-negative cells with a normal overall nuclear morphology were distinctively stained by ApoSense (Fig.\u00a04c), conceivably representing cells in the initial phases of apoptosis. These results possibly indicate that ApoSense technology can provide a tool which will directly assess for early stages of programmed cell death in the ischemic kidney, before membrane vesicle formation and DNA degradation, particularly as measured by the TUNEL method [34].\nThe radiocontrast model of distal tubular necrosis provides a good example of specific targeting by ApoSense. Indeed, the presence of DDC was restricted to regions (medullary outer and inner stripe; papillary tip) that corresponded closely to the injured zones in the contralateral, perfusion-fixed kidney (Figs.\u00a06, 7). Methylene blue-stained thin sections from those in vivo fixed kidneys enabled precise evaluation of the renal morphology and accurate identification of tubular cell types at various stages of evolving structural damage. The complete correlation between DDC fluorescence and histological evidence of damaged areas, mainly made up of apoptotic and necrotic cells, is indicative of the specificity of DDC uptake. Thus, while the degree of renal dysfunction served as an inaccurate indicator of tubular injury [4, 5], ApoSense uptake closely predicted the existence of ATN, with an absolute specificity for involved tubular segments.\nOur findings derived from the mouse sepsis model further illustrated the sensitivity of ApoSense in the detection of rare focal tubular cell injury that is not well defined by routine H&E staining (Fig.\u00a08). It has to be reiterated that data regarding the morphology of sepsis-related ATN are nearly non-existent, mainly because such rare, focal tubular cell damage can hardly be detected by regular morphological evaluation [35, 36].\nThe inherent fluorescent properties of the ApoSense molecule DDC have been used in the present study to evaluate its specific binding to apoptotic cells. On the other hand, the structure of ApoSense molecules is modular, allowing for versatile attachment of various clinical imaging markers and, therefore, enabling their use as non-invasive imaging agents. In the current study, a 3H-labeled DDC molecule was used for biodistribution and quantitative uptake studies. Nevertheless, autoradiography with radiolabeled DDC was not performed in this series of experiments and this may be the main limitation of the present study. Moreover, additional studies based on preparation of ApoSense as a PET imaging agent (following its conjugation with 18F) are necessary before the introduction of the technology in the form of diagnostic imaging tests. These studies are currently in progress.\nOverall, the results presented here are promising and illustrate the high sensitivity and specificity of ApoSense in the assessment of tubular cell damage in multiple experimental templates that may have clinically relevant applications. For instance, ApoSense technology may be implemented for the in vivo assessment of the degree to which renal parenchymal cell apoptosis is contributing to renal failure in sepsis and for the differentiation of evolving ATN from renal hemodynamic alterations; as a result it can enable therapy to be tailored to each patient\u2019s needs. The ability of radiolabeled derivatives of ApoSense to visualize both apoptosis and necrosis, non-invasively and in real-time, may prove useful in reducing the need for routine biopsy. Furthermore, in clinical practice, ApoSense imaging may provide diagnostic information superior to that obtained by means of biopsy, especially as regards the dynamic nature and focal distribution of the disorder; furthermore, it does not suffer from the risks and limitations associated with tissue sampling.\nImaging sciences have grown tremendously during recent decades, and many non-invasive techniques for the detection and monitoring of renal hemodynamics, such as advanced ultrasonography techniques, magnetic resonance imaging, and nuclear and X-ray computed tomography [37\u201339], have become essential clinical tools. Similarly, non-invasive technologies and imaging probes that would enable the detection of evolving renal parenchymal apoptotic and necrotic injury are critically needed. During the past few years, the best-characterized apoptosis marker, annexin V, a calcium-dependent phospholipid binding protein [40, 41], has been extensively investigated in multiple animal models as well as in clinical trials [42]. However, annexin V has a strong affinity for intact renal cortex, which has been attributed to the intrinsic lipid profile of the kidney, where a high concentration of phosphatidylserine exists [43, 44]. The high accumulation of annexin V in the kidneys precludes its use for the study of apoptosis in these organs or in their immediate vicinity [45, 46]. In addition, the biological rationale for engineering DDC, a small non-peptide molecule with a molecular weight of 700\u00a0Da (as compared with the 36-kDa annexin V protein), is that such small molecules could benefit from less immunogenicity and the favorable biodistribution kinetics that small compounds in general exhibit over bigger proteins.\nIn summary, this study demonstrates that ApoSense, once successfully radiolabeled with an imaging moiety, may provide a sensitive means for real-time diagnosis and\/or monitoring of the extent of renal damage in ATN, utilizing selective targeting, binding, uptake, and accumulation within renal tubular apoptotic and\/or necrotic cells. Moreover, as a non-invasive method, ApoSense imaging would allow longitudinal studies in a single individual, rendering important information on the optimal timing and dosing of drugs and on the efficacy of therapeutic interventions.","keyphrases":["apoptosis imaging","cell death detection","renal disease","renal uptake","small animal imaging"],"prmu":["R","R","R","R","R"]}
{"id":"Psychopharmacologia-3-1-2048539","title":"Acute and constitutive increases in central serotonin levels reduce social play behaviour in peri-adolescent rats\n","text":"Rationale Serotonin is an important modulator of social behaviour. Individual differences in serotonergic signalling are considered to be a marker of personality that is stable throughout lifetime. While a large body of evidence indicates that central serotonin levels are inversely related to aggression and sexual behaviour in adult rats, the relationship between serotonin and social behaviour during peri-adolescence has hardly been explored.\nThere is a large body of literature that describes the association between serotonin (5-hydroxytryptamine, 5-HT) signalling, social status, aggression and sexual behaviour in humans and animals. For instance, cerebrospinal fluid (CSF) 5-hydroxyindoleacetic acid (5-HIAA; the major metabolite of 5-HT) levels are inversely related to aggression in humans, primates and rodents (Higley and Linnoila 1997; Fairbanks et al. 2001). Selective serotonin reuptake inhibitors (SSRIs), which are well-known anti-depressants, reduce impulsive aggression as well as sexual behaviour in humans (New et al. 2004; Olivier et al. 2006). Furthermore, depending on the social structure of the community, serotonergic drugs are able to interchange the dominant and subordinate status of community members (Edwards and Kravitz 1997; Larson and Summers 2001). It has been proposed that individual differences in 5-HT neurotransmission are an important neural underpinning of personality (Serretti et al. 2006), because central 5-HT levels are relatively stable throughout lifetime (Higley and Linnoila 1997). Studies showing that polymorphisms in 5-HT-related genes are linked to impulsive aggression in humans (Ferrari et al. 2005; Haberstick et al. 2006; Popova 2006) support the idea that the serotonergic modulation of social behaviour is heritable (Higley and Linnoila 1997).\nAn important form of social behaviour is social play behaviour, which is the earliest form of non-mother-directed social behaviour in young mammals. Social play behaviour consists of behaviours found in adult sexual, affiliative and aggressive encounters (Bolles and Woods 1964; Baenninger 1967; Poole and Fish 1975; Meaney and Stewart 1981). However, during social play, these behaviours are displayed in an exaggerated and\/or out-of-context fashion (Poole and Fish 1975). In rats, a bout of social play behaviour starts with one rat soliciting another animal, by attempting to nose or rub the nape of its neck. If the animal that is solicited upon rotates to its dorsal surface, \u2018pinning\u2019 is the result, i.e. one animal lying with its dorsal surface on the floor with the other animal standing over it. From this position, the supine animal can initiate another bout of play, by trying to gain access to the other animal\u2019s neck. Thus, during social play, pinning, which is considered to be the most obvious posture in social play behaviour in rats, is not an endpoint but rather functions as a releaser of a prolonged play bout (Poole and Fish 1975; Pellis and Pellis 1987; Pellis 1988). The animal that is pounced upon can also respond by evading or by turning around to face the other animal. In the latter situation, a brief period of boxing\/wrestling may follow, in which the animals try to push each other away. If the solicited animal evades, the other animal may start to chase it, thus making another attempt to launch a play bout (see Vanderschuren et al. 1995a, for a detailed analysis of the temporal structure of social play behaviour in rats). It is thought that social play sub-serves the facilitation of social and cognitive development because play deprivation, i.e. social isolation during 2\u00a0weeks in peri-adolescence when social play is most abundant (i.e. from postnatal day\u00a021 until postnatal day\u00a035), leads to behavioural disturbances, most prominently in the social domain (e.g. Hol et al. 1999; Van den Berg et al. 1999). These behavioural changes are more selective than those of isolation rearing, which entails continuous isolation from weaning throughout further lifetime. This not only affects social behaviour (Ferdman et al. 2007) but also induces behavioural disturbances related to exploratory behaviour (Heidbreder et al. 2000), cognition (Heidbreder et al. 2000; Dalley et al. 2002; Bianchi et al. 2006) and reward sensitivity (Howes et al. 2000), suggesting that social play plays a rather selective role in the development of social skills.\nThere is evidence indicating that 5-HT modulates social play in non-human primates. For instance, low CSF 5-HIAA levels are associated with increased solitary play (Maestripieri et al. 2006; Higley and Linnoila 1997), although these findings are difficult to interpret in terms of central 5-HT levels. The neurobiology of social play has been widely investigated in rats (Vanderschuren et al. 1997; Siviy 1998), but surprisingly, little attention has been paid to the role of the serotonergic system in rat social play itself. While treatment with para-chloro-phenylalanine or a low tryptophan diet, resulting in a decrease in central 5-HT levels, did not affect social play, fenfluramine and the SSRI fluoxetine, agents that increase extra-neuronal 5-HT levels, inhibited play (Panksepp et al. 1987; Knutson et al. 1996). Further, the 5-HT receptor agonist quipazine reduced social play (Normansell and Panksepp 1985). These data suggest that there is an inverse relationship between 5-HT signalling and social play. Furthermore, the findings that 5-HT modulates social behaviour in peri-adolescent animals indicates that 5-HT affects social behaviour throughout development, consistent with the proposed trait-like relationship between 5-HT and social behaviour.\nThe serotonin transporter (SERT) critically regulates extracellular 5-HT levels by its re-uptake into pre-synaptic terminals (Lesch et al. 1996), and SERT knockout models would therefore be very useful to test the hypothesis that central 5-HT levels and social play behaviour are inversely related in a trait-like manner. SERT knockout mice are available (e.g. Bengel et al. 1998), which display reduced aggression (Holmes et al. 2002a) and social interaction (Kalueff et al. 2007). However, because social play behaviour in mice is much less developed than in rats (Poole and Fish 1975; Pellis and Pasztor 1999), mice are not the preferred rodent species to study age-specific patterns of social behaviour during development. As social play behaviour in rats has been well described (Bolles and Woods 1964; Baenninger 1967; Poole and Fish 1975; Meaney and Stewart 1981; Pellis and Pellis 1987; Pellis 1988; Vanderschuren et al. 1997), we measured social play in the recently generated SERT knockout rat (Smits et al. 2006), which displays a ninefold increase in extra-neuronal 5-HT levels (Homberg et al. 2007). In line with the findings that increases in 5-HT levels reduce aggressive and sexual behaviour, we have observed that both aggressive and sexual behaviour (Homberg et al., unpublished observations) are reduced in the SERT knockout rat. The constitutive absence of the SERT in mice causes changes in the functioning of 5-HT receptors (Fabre et al. 2000; Bouali et al. 2003), so that changes in behaviour may be the result of adaptations that have occurred during development. Therefore, we also investigated social play behaviour in wild-type rats after acute treatment with compounds that increase central 5-HT levels, i.e. the SERT-blocker fluoxetine and 3,4-methylenedioxymethamphetamine (MDMA or \u201cecstasy\u201d), which causes 5-HT release by reversing 5-HT transport.\nMaterials and methods\nSubjects\nAll experiments were conducted with the approval of the animal ethics committees of the VU Medical Center, Amsterdam, The Netherlands, and the University Medical Center Utrecht, The Netherlands.\nBoth commercially supplied and SERT knockout rats were used in this study. Male Wistar rats (Harlan, Horst, the Netherlands) arrived at the age of 3\u00a0weeks in the animal facility. SERT knockout rats, which have been generated by ethylnitrosourea-induced mutagenesis in a Wistar background (for detailed description, see Smits et al. 2006), were bred by crosses between outcrossed (six generations) SERT+\/\u2212 rats. At the age of 2\u00a0weeks, ear cuts were taken under anaesthesia and used for genotyping. At the age of 3\u00a0weeks, male SERT\u2212\/\u2212 and SERT+\/+ rats (littermates served as controls) were transported from the breeding room to the experimental room. The animals were socially housed (four rats per type IV macrolon cage) under a normal day\/light cycle (lights off at 7\u00a0p.m.) at controlled room temperature (21\u2009\u00b1\u20092\u00b0C) and relative humidity of 60\u2009\u00b1\u200915%. Food and water were available ad libitum. Three days after arrival, the rats were weighed and handled daily until testing.\nBehavioural procedure\nTesting was performed as described previously (Vanderschuren et al. 1995b). Rats, aged 28\u201335\u00a0days, were tested in an acrylic plastic cage (40\u2009\u00d7\u200940\u2009\u00d7\u200960\u00a0cm [l\u2009\u00d7\u2009w\u2009\u00d7\u2009h]) with approximately 2\u00a0cm of wood shavings covering the floor. The test cage was illuminated by a 25-W red light bulb mounted 60\u00a0cm above the test cage. Background noise, produced by a radio, was used to minimize the influence of extraneous sounds.\nTwo days preceding the test, the animals were habituated to the test cage during 10\u00a0min. The animals of a test pair did not differ more than 10\u00a0g in body weight and had no previous common social experience. On the test day, test pairs were isolated for 3.5\u00a0h before the test to induce a half-maximal increase in the amount of social play behaviour (Niesink and Van Ree 1989). Thirty minutes before the test, pairs of animals in group 1 were treated with 0 or 1\u00a0mg\/kg fluoxetine (subcutaneous [s.c.]), animals in group 2 were pre-treated with 0 or 10\u00a0mg\/kg fluoxetine and group 3 received 0, 0.5, 2 or 5\u00a0mg\/kg MDMA (s.c.). Pairs of similarly treated rats were tested for 15\u00a0min in the test cage and the test order was randomized across treatments. Behaviour of the animals was recorded on video tape, and analysis from the video tape recordings was performed afterwards. Coding of the drug solutions ensured that both during experimentation and analysis, the experimenter was unaware of the treatment of the animals. Using Observer 4.0 (Noldus Information Technology B.V., Wageningen, The Netherlands), frequencies and durations of the following behaviours was scored\u2014pinning: one of the animals lying with its dorsal surface on the floor of the test cage with the other animal standing over it; pouncing: play soliciting by nosing the partner\u2019s nape; boxing\/wrestling: facing each other in vertical position and struggling using the forepaws; following\/chasing: moving in the direction of or pursuing the test partner, who moves away; social grooming and social exploration: sniffing or licking any body part of the test partner. Behaviour was assessed per pair of animals. Animals were used only once.\nDrugs\nFluoxetine was purchased from Sigma-Aldrich (Schnelldorf, Germany), and MDMA was purchased from O.P.G. (Utrecht, The Netherlands). Both drugs were freshly dissolved in saline on the day of the experiment and injected s.c. in the flank in a volume of 2\u00a0ml\/kg.\nStatistical analyses\nData were analysed using one-way analysis of variance (ANOVA), followed by Student\u2013Newman\u2013Keuls post-hoc tests where appropriate. The level of probability for statistically significant effects was set at p\u2009<\u20090.05 (n.s.\u2009=\u2009non-significant).\nResults\nSERT\u2212\/\u2212 pairs of rats, as compared to SERT+\/+ pairs, showed a strong reduction in pinning frequency (Fig.\u00a01a; F(2, 13)\u2009=\u20095.78, p\u2009<\u20090.05), pouncing frequency (F(2, 13)\u2009=\u20097.08, p\u2009<\u20090.05) and boxing\/wresting frequency (F(2, 11)\u2009=\u20099.91, p\u2009<\u20090.01). The duration of following\/chasing was significantly increased in SERT\u2212\/\u2212 rats (Fig.\u00a01b; F(2, 13)\u2009=\u20096.5, p\u2009<\u20090.05), while social grooming and social exploration were not affected by deletion of the SERT gene (social exploration: F(2, 12)\u2009=\u20090.22, n.s.; social grooming: F(2, 12)\u2009=\u20090.76, n.s.).\nFig.\u00a01Social play behaviour in peri-adolescent male SERT\u2212\/\u2212 and SERT+\/+ rats. The data represent mean\u2009\u00b1\u2009SEM of the number of pins, pounces and boxing\/wresting episodes (a) and mean\u2009\u00b1\u2009SEM of the duration of social exploration and following\/chasing (b) during a 15\u00a0min test. Asterisk, p\u2009<\u20090.05 SERT\u2212\/\u2212 vs SERT+\/+\nAs illustrated in Fig.\u00a02a, 1\u00a0mg\/kg fluoxetine did not affect pinning (F(2, 13)\u2009=\u20091.33, n.s.), pouncing (F(2, 13)\u2009=\u20092.56, n.s.) and boxing\/wrestling (F(2, 13)\u2009=\u20091.75, n.s.). One-way ANOVA did not also reveal effects of 1\u00a0mg\/kg fluoxetine on social exploration (F(2, 13)\u2009=\u20090.55, n.s.), following\/chasing (F(2, 13)\u2009=\u20090.69, n.s.) or social grooming (F(2, 13)\u2009=\u20090.00, n.s.; Fig.\u00a02b). A higher dose of fluoxetine, 10\u00a0mg\/kg, on the other hand, significantly reduced pinning (F(2, 14)\u2009=\u200915.86, p\u2009<\u20090.005), pouncing (F(2, 14)\u2009=\u200936.18, p\u2009<\u20090.0001), boxing\/wrestling (F(2, 14)\u2009=\u200923.82, p\u2009<\u20090.0005; Fig.\u00a02c) and following\/chasing (F(2, 14)\u2009=\u200920.17, p\u2009<\u20090.001) but not social exploration (F(2, 14)\u2009=\u20093.03, n.s.) and social grooming (F(2, 14)\u2009=\u20090.03, n.s.; Fig.\u00a02d).\nFig.\u00a02Effect of fluoxetine on social play behaviour in peri-adolescent male wild-type rats. Thirty minutes before the 15-min test, the animals were treated with 1 (a, b) or 10\u00a0mg\/kg (c, d) fluoxetine (s.c.). The data represent mean\u2009\u00b1\u2009SEM of the number of pins, pounces and boxing\/wresting episodes (a, c) and mean\u2009\u00b1\u2009SEM of the duration of social exploration, following\/chasing and social grooming (b, d). Asterisk, p\u2009<\u20090.05 vs saline\nMDMA was tested in three different doses: 0.5, 2.0 and 5.0\u00a0mg\/kg. One-way ANOVA indicated that MDMA treatment dose-dependently suppressed pinning (Fig.\u00a03a; F(2, 30)\u2009=\u200928.8, p\u2009<\u20090.0001, post-hoc testing: all MDMA groups vs saline p\u2009<\u20090.05), pouncing (F(2, 29)\u2009=\u200931.77, p\u2009<\u20090.0001, post-hoc testing: all MDMA groups vs saline p\u2009<\u20090.05) and boxing (F(2, 29)\u2009=\u200922.07, p\u2009<\u20090.0001, post-hoc testing: all MDMA groups vs saline p\u2009<\u20090.05). In addition, MDMA decreased following\/chasing (Fig.\u00a03b; F(2, 29)\u2009=\u20098.81, p\u2009<\u20090.0005, post-hoc testing: all MDMA groups vs saline p\u2009<\u20090.05). Social exploration (F(2, 30)\u2009=\u20096.41, p\u2009<\u20090.005, post-hoc testing: 2 and 5\u00a0mg\/kg MDMA vs saline p\u2009<\u20090.05) and social grooming (F(2, 28)\u2009=\u200924.89, p\u2009<\u20090.0001, post-hoc testing: 2 and 5\u00a0mg\/kg MDMA groups vs saline p\u2009<\u20090.05; Fig.\u00a03b) were also attenuated by MDMA treatment.\nFig.\u00a03Effect of MDMA on social play behaviour in peri-adolescent male wild-type rats. Thirty minutes before the 15-min test, the animals were treated with 0, 0.5, 2 or 5\u00a0mg\/kg MDMA (s.c.). The data represent mean\u2009\u00b1\u2009SEM of the number of pins, pounces and boxing\/wresting episodes (a) and mean\u2009\u00b1 SEM of the duration of social exploration, following\/chasing and social grooming (b). Asterisk, p\u2009<\u20090.05 vs saline; double cross, p\u2009<\u20090.05 vs 0.5\u00a0mg\/kg MDMA\nDiscussion\nIn this study, we show that increasing extra-neuronal 5-HT levels, either constitutively by deletion of the SERT gene or transiently by pharmacological manipulation (i.e. treatment with fluoxetine or MDMA), decreases social play behaviour in peri-adolescent rats. These effects were specific for playful social behaviours, i.e. pinning, pouncing and boxing\/wrestling. Mixed effects were observed for following\/chasing, social grooming and social exploration. The similarities between the genetic and pharmacological manipulations of the serotonergic system suggest that reduced social play behaviour in SERT\u2212\/\u2212 rats is the result of increased extracellular 5-HT levels (Homberg et al. 2007).\nFluoxetine and MDMA are well known to increase central 5-HT levels, but they affect dopaminergic neurotransmission as well, raising the possibility that reduced social play is due to an effect on the dopaminergic system. Fluoxetine, at 10\u00a0mg\/kg as used in the present study, increases dopamine release in the frontal cortex (Tanda et al. 1994). MDMA is a potent releaser of 5-HT, but it releases dopamine as well, albeit at higher concentrations (for reviews, see White et al. 1996; Green et al. 2003). Although psychostimulant drugs that increase central dopaminergic neurotransmission have been found to suppress social play behaviour (e.g. Beatty et al. 1982; for review, see Vanderschuren et al. 1997), these effects are most likely mediated through non-dopaminergic mechanisms because the selective dopamine reuptake inhibitor GBR-12909 and the dopamine receptor agonist apomorphine did not suppress play (Vanderschuren et al. 2005). Moreover, the pre-synaptic functioning of dopaminergic, as well as noradrenergic neurons, was not altered in SERT\u2212\/\u2212 rats (Homberg et al. 2007). Therefore, the present findings are most likely the result of altered function of the serotonergic system, rather than the dopaminergic system.\nIndirect 5-HT agonists such as MDMA and parachloroamphetamine enhance locomotor activity (Geyer 1996), but the doses required to enhance locomotor activity in peri-adolescent rats are higher than those that suppress play in the present study (see \u00c5berg et al. 2007). Fluoxetine does not alter locomotor activity (Homberg et al. 2004), and locomotor activity does not differ between SERT+\/+ and SERT\u2212\/\u2212 rats under both a novel and habituated conditions (Homberg et al., unpublished observations). In the present study, following\/chasing, the social parameter most closely related to locomotor activity, was increased in SERT\u2212\/\u2212 rats and decreased by fluoxetine and MDMA. Thus, there is no parallel in the effects of fluoxetine, MDMA and SERT knockout on locomotor activity and following\/chasing. This indicates that the reduction in social play found in SERT\u2212\/\u2212 rats and after treatment with fluoxetine and MDMA is not likely to be secondary to effects on locomotor activity.\nAn alternative explanation of the present findings is that the decrease in social play behaviour is due to increased anxiety. Although the animals were habituated to the test cage and tested under red light conditions, reluctance to contact an unfamiliar conspecific may have interfered with our findings. SERT\u2212\/\u2212 mice display anxiety-related symptoms and reduced social interaction (Holmes et al. 2002b; Kalueff et al. 2007), MDMA and fluoxetine have anxiogenic effects in a variety of tests (Lin et al. 1999; Morley and McGregor 2000; Drapier et al. 2007) and the SSRI citalopram reduces social interaction (Dekeyne et al. 2000). MDMA has also been reported to decrease social investigation and aggression and to increase non-social exploration, which was interpreted as attempts to escape from the test arena, i.e. an anxiogenic-like effect of MDMA (Maldonado and Navarro 2001). On the other hand, pro-social effects of MDMA have also been reported (Morley and McGregor 2000). SERT\u2212\/\u2212 rats spent more time on following and chasing than SERT+\/+ rats. This suggests that while social play was reduced, SERT\u2212\/\u2212 rats were still interested in their playing partner, which argues against social anxiety as an explanation of reduced play behaviour in SERT\u2212\/\u2212 rats. Moreover, social exploratory behaviour, which is the parameter used in the social interaction test of anxiety (File and Seth 2003), was not affected in SERT\u2212\/\u2212 and in fluoxetine-treated rats, whereas MDMA only mildly reduced social exploration at doses that completely eliminated social play. Thus, the observed effects were relatively selective for social play, and increases in extra-neuronal 5-HT levels do not induce a generalized aversion to contact an unfamiliar rat.\nLong-term SSRI treatment is associated with increased extra-neuronal 5-HT levels and several compensatory adaptations (Dawson et al. 2002) that are strikingly similar to those seen in SERT\u2212\/\u2212 rats, like desensitization of pre- and post-synaptic 5-HT1A receptors (Homberg et al., unpublished observations). Chronic SSRI treatment decreases aggressive (Fuller 1997) and sexual behaviour (Olivier et al. 2006) in rats, phenotypes we also observed in SERT\u2212\/\u2212 rats (Homberg et al., unpublished observations). Because acute fluoxetine and MDMA treatment cause an immediate increase in extra-neuronal 5-HT levels without compensatory adaptations, it is likely that reduced social play in SERT\u2212\/\u2212 rats is due to the increased extra-neuronal 5-HT levels, rather than compensatory adaptations. Ansorge et al. (2004) showed that early chronic SSRI treatment in wild-type mice induces anxiety- and depression-like phenotypes that resembles those of SERT\u2212\/\u2212 mice. It may follow that SERT\u2212\/\u2212 rats behave as being chronically treated with SSRIs, but given that acute and chronic SSRI treatment induce opposite effects (Silva and Brandao 2000) and that the effects of anti-depressants on the immature developing brain may differ as opposed to the effects on the mature brain of adults (Ansorge et al. 2004; Taravosh-Lahn et al. 2006), further research is required to support this suggestion. Anti-depressants are frequently prescribed to children with major depressive disorder and anxiety (Cheung et al. 2006), while there is only sparse knowledge of the effects of SSRIs on the developing brain (Spear and Brake 1983; Ansorge et al. 2004). Because social behaviour is reduced in peri-adolescent and adult SERT\u2212\/\u2212 rats, it is likely that changes in 5-HT homeostasis found at adulthood in SERT\u2212\/\u2212 rats (Homberg et al. 2007) reflect the situation in the peri-adolescent SERT\u2212\/\u2212 brain. We thus propose that insight into the function of the serotonergic system in the SERT\u2212\/\u2212 (peri-adolescent) brain may be helpful to gain insight into the effects of SSRIs on the developing brain.\n5-HT is considered to be an important marker of personality traits such as harm avoidance (Gerra et al. 2000), social dominance (Edwards and Kravitz 1997; Larson and Summers 2001), social attachment (Beech and Mitchell 2005) and social impulsivity (Fairbanks et al. 2001), which are relatively stable within individuals. These traits may be the result of changes in SERT function because a polymorphism in the SERT gene of humans and non-human primates, which decreases SERT expression and function (Lesch et al. 1996), has quite consistently been associated with childhood (Beitchman et al. 2006; Haberstick et al. 2006) and adult aggression (Sakai et al. 2006) and childhood (Nobile et al. 2004) and adult depression (Caspi et al. 2003). Furthermore, this polymorphism has been associated with reduced free play behaviour in monkeys (Bethea et al. 2004). Anti-social behaviour in children has been suggested to be related to developmental abnormalities in the serotonergic system, and altered social behaviour during childhood likely extends into adulthood (Van Goozen and Fairchild 2006). Thus, the observations that SERT\u2212\/\u2212 rats display reduced social play behaviour, reduced aggression and reduced sexual behaviour is likely to be the result of constitutive disruptions in 5-HT function. On the other hand, because social play sub-serves the development of an adequate social repertoire (Vanderschuren et al. 1997; Hol et al. 1999; Van den Berg et al. 1999), the reduction in social play in SERT\u2212\/\u2212 rats could also itself cause changes in adult social behaviour, independent of 5-HT function. Whichever possibility holds true, we think that because of their lifelong altered 5-HT homeostasis, the SERT knockout rat model may contribute to our understanding of the role of 5-HT during the development of social behaviour.","keyphrases":["serotonin","social play","peri-adolescence","fluoxetine","knockout rat","mdma"],"prmu":["P","P","P","P","P","P"]}
{"id":"J_Mol_Med-3-1-2121656","title":"Harnessing hypoxic adaptation to prevent, treat, and repair stroke\n","text":"The brain demands oxygen and glucose to fulfill its roles as the master regulator of body functions as diverse as bladder control and creative thinking. Chemical and electrical transmission in the nervous system is rapidly disrupted in stroke as a result of hypoxia and hypoglycemia. Despite being highly evolved in its architecture, the human brain appears to utilize phylogenetically conserved homeostatic strategies to combat hypoxia and ischemia. Specifically, several converging lines of inquiry have demonstrated that the transcription factor hypoxia-inducible factor-1 (HIF1-1) mediates the activation of a large cassette of genes involved in adaptation to hypoxia in surviving neurons after stroke. Accordingly, pharmacological or molecular approaches that engage hypoxic adaptation at the point of one of its sensors (e.g., inhibition of HIF prolyl 4 hydroxylases) leads to profound sparing of brain tissue and enhanced recovery of function. In this review, we discuss the potential mechanisms that could subserve protective and restorative effects of augmenting hypoxic adaptation in the brain. The strategy appears to involve HIF-dependent and HIF-independent pathways and more than 70 genes and proteins activated transcriptionally and post-transcriptionally that can act at cellular, local, and system levels to compensate for oxygen insufficiency. The breadth and depth of this homeostatic program offers a hopeful alternative to the current pessimism towards stroke therapeutics.\nStroke is defined as injury to the brain accruing from a vascular etiology. Strikingly, it has emerged as the third leading cause of death and the leading cause of disability in the USA. Accordingly, the estimated financial costs of stroke are more than 50 billion dollars a year in the USA alone. These financial costs do not begin to tell the story of the personal suffering that amasses from the silent epidemic of stroke disability\u2014over 5 million Americans face the challenges of handicaps from stroke each day. The recognition of stroke as a leading age-associated public health issue has led the government and the pharmaceutical industry to expend enormous resources on developing interventions in the form of drugs that minimize brain damage associated with stroke. Despite their promise, these efforts have been disappointing and have left a nearly indelible sense of frustration on the biomedical research community [1]. In this chapter, we will discuss the pathophysiology of stroke with particular attention to one of its primary mediators, hypoxia, and attempt to revive a sense of optimism and enthusiasm for stroke therapeutics moving forward.\nIschemia is a process in which perfusion to tissue is critically reduced creating a deficit in necessary brain fuels. The brain is highly vulnerable to ischemia because the eloquent functions it is assigned to carry out, in the pluralistic society of organ functions, depend integrally on energy\u2014specifically adenosine triphosphate (ATP). The brain comprises only 2% of body weight, but it utilizes nearly 20% of cardiac output to achieve its supply of essential nutrients including oxygen and glucose. Abnormal central nervous system (CNS) symptoms begin to manifest at 40\u201350% of normal resting hemispheric cerebral blood flow resulting in slowing of the EEG, attenuation of evoked potentials, and reduction in the membrane potential in individual cortical neurons [2]. The resting membrane potential must be maintained to allow proper neuronal function, including synaptic activity and axonal conduction. Once the neuronal membrane potential begins to dissipate, neuronal function ceases. It is important to note that ATP levels at this point may be normal or only slightly reduced [3]. The reason for this inordinate sensitivity is not entirely clear but is likely related to the Km of neurotransmitter systems for ATP or their exquisite sensitivity to tissue acidosis [4]. Gross deterioration and damage requires even more severe reductions in blood flow to 20\u201330% of normal resting hemispheric cerebral blood flow (Fig.\u00a01). With this level of ischemia, a deterioration of ionic membrane gradients ensues, and the tissue begins to accumulate hydrogen ions (acidosis) [5]. Changes in ionic fluxes likely accrue directly from a reduction of intracellular ATP, a failure of the Na+\/K+ATPase activity, and increases in intracellular Na+ and extracellular K+. The failure of ionic homeostasis reflects loss of activity of multiple transporters that normally maintain the electrochemical gradients necessary for normal neuronal signal transduction [6, 7]. Among the ions deregulated, intracellular calcium appears to play a critical role in further ion dyshomeostasis via the calmodulin-dependent activation of neuronal nitric oxide synthase (nNOS) [8\u201310]. Increased nNOS activity leads to increase in the ambient levels of neuronal nitric oxide (NO) [11]. Changes in NO coupled with ischemia-associated increases in cytosolic and mitochondrially derived superoxide, combine to form toxic peroxynitrite. Peroxynitrite can trigger cell death pathways via DNA damage, poly(ADP-ribose)polymerase (PARP) activation, liberation of apoptosis-inducing factor (AIF) from the mitochondria, and activation of death signaling pathways leading to apoptosis [12, 13]; alternatively, non-selective cation channels such as TRPM2 and TRPM7 can be activated to ensure sustained calcium overload and death [14]. Extracellular acidosis is also postulated to activate acid sensing ion channels, which also contribute to destruction of the normal ionic environment [15]. As one can appreciate from a partial description of the sequence of events after stroke that occur in the neuron alone, targeting a single molecule in the complex parallel and serial pathways of acute hypoxia-ischemia will not maintain neuronal survival (Fig.\u00a02). It is also unlikely to enhance the ability of energy-thirsty neurons to carry out their sophisticated roles in maintaining posture, movement, sequencing language, or making critical executive decisions. Therefore, how might we move forward? One of the most important advances in the treatment of complex medical problems has been the discovery that multimodal therapies can greatly enhance therapeutic efficiency. Treatment of cancer, HIV infection, and tuberculosis with multimodal therapies yields results that are not obtained with the application of single therapeutic agents.\nFig.\u00a01Neuronal hypoxia leads to calcium overload and production of free radicals. Stroke is associated with a decrease in cerebral blood flow to the brain. The consequent loss of metabolic fuels leads to failure of sodium pumps leading to an intracellular accumulation of sodium and calcium, depolarization, and activation of voltage sensitive and ligand gated [N-methyl-d-aspartate (NMDA)] ion channels. Increased in calcium in microdomains near the NMDA receptor leads to activation of neuronal nitric oxide synthase. Global dysregulation of calcium in the neuron leads to mitochondrial overload and superoxide production. Nitric oxide and superoxide combine to form peroxynitrite. Peroxynitrite can damage DNA leading to PARP activation and consumption of NAD+. It can also activate TRPM2\/7 channels leading to further calcium dysregulation. Hypoxia is sensed by decreased activity of HIF prolyl 4 hydroxylases that can lead to activation genetic responses capable of compensating for the sentinel metabolic stress (decreased cerebral blood flow). Decrease HIF PHD activity can also prevent death via HIF-independent pathways. Acidosis and ROS can also combine with HIF regulated prodeath proteins to trigger cell deathFig.\u00a02Contribution of neurons and astrocytes in mediating excitotoxic neuronal death. 1 Loss of ATP in ischemia leads to inhibition of the Na+\/K+ATPase and subsequent collapse of normal ionic gradients. 2 In turn, neuronal membrane depolarization activates voltage sensitive Ca2+ channels, which increase intracellular Ca2+ and stimulate vesicular glutamate release. Severe loss of ionic gradients found in certain ischemic regions may also lead to the reversal of 3 neuronal specific and 4 astrocyte specific glutamate transporters, which in the reverse mode act to release glutamate into the extracellular space. 5 Cell swelling in cerebral ischemia, which is mainly localized to astrocytes, likely activates swelling sensitive anion channels, referred to as volume regulated anion channels (VRACs). VRACs, which are permeable to organic osmolytes, contribute to glutamate release predominantly in the ischemic penumbra. 6 Glutamate regulated NMDA receptors (NMDA-R) are activated by (1) extracellular glutamate and (2) release of Mg2+ from its pore after membrane depolarization (in part due to activation of glutamate regulated AMPA receptors, not shown). 7 NMDA-Rs are permeable to Ca2+ and as such, overabundant NMDA-R activation leads to an intracellular Ca2+ overload. This increase in intracellular Ca2+ then contributes to neuronal death via several mechanisms\nAs stroke does not represent a single homogeneous category of injury, it is also a poor candidate for a single approach to treatment [16]. Challenges involved in promoting recovery from stroke involve reducing the extent of damage that occurs in acute injury. Moreover, many different kinds of damage are found in individuals with stroke ranging from necrotic, apoptotic, or parthanatotic death of neurons [17, 18], demyelination of otherwise functional axons [19], and transection of axons and subsequent loss of critical neuronal populations [20]. Acute injury itself is extremely complex, including waves of cell death, inflammatory responses, edema, and scarring. Indeed, experimental studies have continued to demonstrate that interventions that target single aspects of the complex cascade, including blocking ion gradients, scavenging free radicals, or enhancing growth factors on their own are insufficient to overcome the considerable barriers to protection against hypoxia and ischemia in acute stroke [21].\nInstructive data from the experimental paradigm of ischemic preconditioning has pointed the way towards novel strategies that can address the heterogeneity and complexity inherent in stroke pathophysiology [22]. Animals subjected to a non-lethal exposure to hypoxia are found to be more resistant to a host of subsequent lethal stresses, including cerebral ischemia. The mechanism by which a sublethal exposure to hypoxia can render the brain resistant to cerebral ischemia and a host of other insults is a topic of active investigation and debate. However, one point appears irrefutable: The tolerance that develops after a short duration of hypoxia involves not only the activation or inactivation of pre-existing proteins but also de novo gene expression [23, 24]. These transcriptional and post-transcriptional mechanisms reflect a cassette of genes and proteins that work collectively at the cellular, local, and systemic levels to compensate for a discrepancy in oxygen supply and demand. The findings suggest that under conditions where adaptive homeostatic mechanisms are appropriately engaged, damage to the brain can be substantially lessened or even prevented (Fig.\u00a03). Stroke is, almost by definition, a failure of homeostasis. Accordingly, identification of small molecules that augment endogenous adaptive strategies provides a mechanism to tilt the balance away from cell damage and death and toward cell survival and repair (Fig.\u00a03).\nFig.\u00a03Drugs that augment endogenous homeostatic mechanisms will more effectively neutralize the heterogeneity inherent in stroke pathophysiology. As these pathways are already used by the body, their activation can occur with decreased threat of toxicity. The term \u201chomeostasis\u201d was coined by Walter Canon in the early twentieth century. It refers to the innate tendency of organisms to mobilize adaptive responses physiological and pathological perturbations that ultimately return the system to a set point that is consistent with survival. a The experimental paradigm of \u201cischemic preconditioning\u201d has shown that a short, sublethal exposure to hypoxia, or hypoxia-ischemia induces homeostatic responses that make the organism \u201cimmune\u201d or \u201ctolerant\u201d to a lethal ischemic insult. Mechanistic studies have revealed that tolerance is the consequence of activation of pre-existing proteins and de novo gene expression. b According to this model, stroke can be conceptualized as a failure of homeostasis. Consequently, neurons die and the brain is permanently damaged. c By extension, small molecules that engage homeostatic mechanisms designed to alleviate hypoxia\/ischemia early or enhance their activation should tip the balance away from cell death and toward survival and repair. Such small molecules are currently being developed and represent a new generation of stroke therapies\nExamination of adaptive responses to hypoxia in the central nervous system has affirmed that the family of transcriptional regulators known as the hypoxia-inducible factors are central players [25\u201327]. HIF-1 was purified and cloned as a result of a search for proteins that regulate the expression of genes involved in hypoxic adaptation, such as erythropoietin, vascular endothelial growth factor, and glycolytic enzymes [27]. HIF is a heterodimeric transcriptional activator composed of an inducible HIF-1\u03b1 subunit and a constitutively expressed HIF-1\u03b2 subunit [28, 29]. HIF-1\u03b1 stability is regulated via the activity of a class of oxygen, 2-oxoglutarate, and iron dependent enzymes known as the HIF prolyl-4 hydroxylases (HIF PHDs, Fig.\u00a04) [30]. As intracellular oxygen levels drop below a critical threshold, these enzymes fail to hydroxylate HIF-1\u03b1. As hydroxylation is required for the recruitment of the constitutively active E3 Ubiquitin Ligase, Von Hippel Lindau protein, HIF-1\u03b1 becomes stabilized. HIF-1\u03b1 can partner with its constitutively expressed but induced partner HIF-1\u03b2 and translocate to the nucleus to regulate the expression of a host of genes involved in hypoxic adaptation [31]. Consistent with this model, several groups have shown that HIF-1\u03b1 immunoreactivity increases in areas of the cortex that become hypoxic due to stroke [25, 32] An unanswered question has been whether HIF is also upregulated in areas connected to but remote from the site of ischemia. Indeed, neurons projecting to an area of infarction are at risk for cell death due to a loss of trophic support from their damaged targets. Moreover, neurons projecting from an area of damage are at risk for cell death due to a loss of trophic excitatory input from their targets. An elegant recent study in non-human primates demonstrated a dramatic increase in neuronal immunoreactivity for HIF-1\u03b1 and one of its target genes in the infarct and peri-infarct region [33, 34]. Indeed, the neuronal immunoreactivity for HIF-1\u03b1 increased from less than 5% to nearly 90% in both regions. Interestingly, areas remote to the area of hypoxia and ischemia also experience increased HIF and vascular endothelial growth factor (VEGF) levels but to a quantitatively much smaller extent. Together, the published rodent and primate histochemical studies support the hypothesis that increased HIF protein levels resulting from direct hypoxia and non-hypoxic mediators such as IGF-1 are a marker for surviving and regenerating neurons after ischemia [32].\nFig.\u00a04HIF prolyl 4 hydroxylases sense hypoxia and transduce a critical insufficiency in oxygen in the brain into transcriptional and post-transcriptional signal changes that mediate protection and repair. Hypoxia regulates the activity of HIF PHDs via direct or indirect mechanisms; production of peroxide via reduction in mitochondrial ATP production and electron transport chain (ETC) impairment (1, 2); accumulation of the tricarboxylic cycle (TCA) intermediates succinate and fumarate (3); or direct inhibition of the activity of PHDs due to lack of oxygen (4). Accumulation of hydrogen peroxide, succinate or fumarate can inhibits the activity of PHDs by competing with 2-oxoglutarate or by oxidizing the active site iron (5). Among its numerous downstream effects, inhibition of HIF PHD activity leads to stabilization of HIF-1\u03b1. Stabilized HIF-1\u03b1 dimerizes with HIF-1\u03b2 in the nucleus and increases gene transcription (6)\nPharmacological and molecular studies have provided additional support for the notion that stabilization of HIF-1 after ischemia is associated with enhanced survival of neurons. Small molecule hypoxia mimics, deferoxamine, and cobalt chloride, were found to stabilize HIF-1\u03b1 levels, increase DNA binding to a cognate hypoxia response element, and increase the expression of HIF target genes in vitro cultured neurons (Zaman et al. [31]) and in vivo in the intact brain (Fig.\u00a04; [35]). Pretreatment or post-treatment with desferrioxamine or cobalt chloride resulted in reduced cell loss in models of focal or global ischemia in vitro and in vivo [25, 31, 36\u201339]. Subsequent studies have confirmed that hypoxia, iron chelation, or cobalt chloride appear to confer protective effects on neurons via their ability to inhibit HIF PHDs [35]. Emerging data indicate that inhibition of each of the three of the HIF PHD isoforms (1\u20133) may lead to cell survival in the nervous system. Of note, inhibition of each isoform may enhance survival via distinct but mutually supportive pathways.\nOur studies demonstrate that pharmacological inhibition of PHDs, in vitro, leads to inhibition of oxidative stress-induced death, an established mediator of neural injury and death in stroke [35]. More recent studies indicate that molecular suppression of HIF PHD 1 alone can mimic this effect (Siddiq et al., unpublished observations). Interestingly, while HIF-1, HIF-2, and cAMP response element-binding protein are stabilized by PHD inhibition in neurons or astrocytes, the molecular deletion or inhibition of each of these three transcription factors fails to abrogate the protective effects of PHD inhibition suggesting that other, as yet unidentified, pathways are important in protection (Siddiq et al., unpublished observations). Studies are underway to clarify whether molecular deletion of PHD1 selectively in the CNS confers resistance to stroke in a HIF-independent manner.\nIn contrast to PHD1, PHD2 appears to be the isoform most important for tagging HIF for degradation. Deletion of PHD2 but not PHD1 or PHD3 results in increased HIF and VEGF protein and consequent angiogenesis in multiple organs including the brain [40]. Underscoring PHD2\u2019s important role in HIF signaling, the expression level of this isoform is significantly higher as compared to its brethren [41]. Ischemia-induced PHD2 inhibition stabilizes HIF and enhances expression of genes that mediate cellular (e.g., glycolytic enzymes), local (vascular endothelial growth factor), and systemic (erythropoietin, Epo) adaptive responses to hypoxia or hypoxia-ischemia [42]. While Epo is best known as a hematopoietic growth factor that can enhance oxygen carrying capacity to tissue, it also has organ-autonomous roles in the nervous system. Epo is produced in astrocytes in response to hypoxia or ischemia and mediates a number of responses critical to stroke prevention and recovery [43, 44]. Epo can inhibit neuronal death due to excitotoxicity or growth factor deprivation [45\u201349]. It can also stimulate the proliferation of neural progenitors in the germinal zones of the brain and enhance their migration to sites of injury [50, 51]. The concomitant HIF-dependent increase in VEGF expression provides, among other things, the appropriate angiogenic niche for neural progenitors to survive [52]. New neurons could mediate recovery responses via paracrine effects or due to direct participation in functional circuits. A recent study affirmed the requisite role for HIF transcription factors in mediating some of the salutary effects of low molecular weight PHD inhibitors given after cerebral ischemia [53]. Available evidence suggests that these inhibitors are likely targeting PHD2 to induce HIF and its gene targets in the CNS.\nInhibition of the third isoform of PHDs (PHD3) by hypoxia or hypoxia mimetics has also been linked to neuronal survival [54]. Freeman and colleagues first identified PHD3 (then known as SM-20) as a message and protein that is highly upregulated in sympathetic neurons after growth factor deprivation. Subsequent studies have shown that pharmacological or molecular deletion of HIF PHD3 prevents apoptosis associated with trophic loss in neurons and that the protection is HIF independent [55]. As target (post-synaptic) derived trophic factors appear to be lost after stroke and result in cell death remote from the infarct site, HIF PHD3 inhibition may be a rational strategy for maintaining the viability of these neurons in evolving or stable stroke.\nFrom the above discussion, a model begins to emerge whereby inhibition of HIF PHD1, 2, or 3 by hypoxia or consequences of hypoxia can mediate HIF-dependent and HIF-independent compensatory responses via distinct but clearly reinforcing mechanisms. How does one reconcile this model with observations from several laboratories, including our own, that HIF PHD inhibition or HIF activation leads to the upregulation of prodeath, Bcl-2 family proteins such as Puma, Bnip3, and NIX [56]? Moreover, constitutive HIF activation has been associated with potentiation and suppression of death [56]. These findings appear to make perfect sense if one considers apoptosis as an adaptive response to stress. After stroke, oxygen may fall below a critical level rendering the prolyl hydroxylases inactive. Accordingly, HIF is stabilized and it translocates to the nucleus to upregulate genes involved in preventing and executing death. The anthropomorphized cell can be visualized stepping to the edge of a steep cliff in response to a potentially lethal stress (Fig.\u00a05). If adequate compensation for hypoxia occurs, prodeath proteins are not activated and the cell survives as a functional component of a complex neural network\u2014the cell moves away from the cliff to survival promoting, terra firma. By contrast, if oxidative stress, hypoxia, and acidosis dominate, indicating that compensatory mechanisms have failed, then proapoptotic proteins such as BNIP3 undergo a conformational change, insertion into the mitochondrial membrane and activation of permeabilization transition and release of apoptotic effectors [57\u201359]. In the latter scenario, the cell jumps off the cliff to preserve limiting resources for its neighbors. The model suggests that state changes in the cell (e.g., redox and pH) that follow the initial ischemic insult will determine the fate of the tissue and potential for recovery (Fig.\u00a05). Of note, low molecular weight or peptide inhibitors of the PHDs, antioxidants, and a small interfering RNA to BNIP3 prevent the prodeath effects of HIF (Amino et al., personal communication). These findings suggest that engaging the adaptive response at an upstream point where stress is detected by the cell (the stress sensor) is going to be more effective in stemming cell loss and facilitating repair than at the point of the transcription factor (HIF). It is our hypothesis that engaging the adaptive response at an upstream point in the pathway results in post-transcriptional changes essential for the homeostatic response. These changes in concert with transcriptional changes optimally alleviate the discrepancy between oxygen supply and demand.\nFig.\u00a05Adaptation to hypoxia-cell fate and beyond. Expression of HIF in neurons leads to the constitutive expression of proteins associated with cell death (BNIP3, NIX, and PUMA) and cell survival (VEGF, glycolytic enzymes, Epo, and p21 waf1\/cip1). Similar prodeath gene expression is found in neurons exposed to hypoxia or hypoxia mimetics despite the absence of cell death. It appears the oxygen \u201cstarved\u201d neurons have stepped to the edge of the cliff. If during the ensuing hours to days the neuron becomes acidotic or oxidized, then prodeath proteins such as BNIP3 undergo a conformational change, insertion into the mitochondrial membrane, release of apoptotic effectors, and death. By contrast, if the survival genes are effective in neutralizing the hypoxic stress (e.g., no acidosis or oxidative stress), then the death genes never get activated. Our studies indicate that antioxidants, short interfering RNAs to BNIP3 or inhibitors of the HIF prolyl 4 hydroxylases tip the balance toward survival (away from the cliff)\nThe model has some clear predictions. First, low molecular weight global inhibitors of the HIF prolyl 4 hydroxylases will be more effective at preventing injury and repairing damage after stroke than selective isoform inhibitors. These inhibitors will engage HIF-dependent and HIF-independent pathways at cellular, local, and systemic levels and ultimately alleviate the discrepancy in nutrient supply and demand. They can also (via mechanisms that are only beginning to be defined) divert HIF away from its tendencies as a prodeath transcription factor. The ability of single \u201cdrugs\u201d to target an oligopoly of proteins (HIF PHD1\u20133) to affect a concerted program of neuroprotection involving more than 70 genes and larger number of proteins suggests a strategy for overcoming the heterogeneity inherent in stroke pathophysiology in the short term. While a significant amount of work needs to be done to adequately assess the viability of this strategy for human therapeutics, the notion of augmenting endogenous adaptive programs via HIF PHDs to thwart disease continues to gain currency.","keyphrases":["stroke","brain","hypoxia","hif","therapeutics","hif prolyl hydroxylase"],"prmu":["P","P","P","P","P","R"]}
{"id":"Environ_Health_Perspect-114-2-1367850","title":"Prenatal Methylmercury Exposure and Developmental Outcomes: Review of the Evidence and Discussion of Future Directions\n","text":"I conducted a review of the published literature to assess the strength of the evidence for an association between prenatal exposure to methylmercury (MeHg) and subsequent child development. I identified 12 studies on this subject published since 1980. Of these, 3 were longitudinal studies\u20142 conducted in the Seychelle Islands, and 1 in the Faroe Islands. Nine were cross-sectional studies conducted in different countries where seafood, a source of MeHg, constituted a major part of the diet. The ages of the children studied ranged from 2 weeks to 12 years. The results of the longitudinal studies were contradictory. Researchers in the Faroe Islands identified an association between MeHg exposure and developmental effects, whereas those in the Seychelle Islands identified no such association. This inconsistency was mirrored in the results of the cross-sectional studies where there were some positive and some negative findings. It was concluded that it was not possible from currently available data to determine whether there is an association between prenatal MeHg exposure and adverse developmental effects in children. In advance of future research, consideration should be given to resolving the uncertainties surrounding exposure assessment and outcome measurement, as both elements varied between studies. It was suggested that questions of exposure assessment would benefit from the application of an expert review process. Outcome assessment would benefit from the development of theoretically based measures of specific aspects of cognitive functioning to replace the relatively crude measures of attainment and IQ currently employed in most studies. This would assist in the development of classic longitudinal studies by allowing repeated assessment over the full age range and providing data that are more readily interpretable and comparable between studies.\nOrganic compounds of mercury have a variety of industrial uses, and most of the data relating to the neurotoxic effects of mercury exposure have been derived from occupational populations (Chipman et al. 1995). However, in the 1950s a potential risk to the wider community was highlighted when large numbers of people living in the Minamata Bay area of Kyushu Island, Japan, developed symptoms of mercury poisoning. This well-documented incident was traced to a factory using mercuric chloride as a catalyst in the production of vinyl chloride and discharging effluent into the sea (Harada 1995). In aquatic environments, mercury is methylated by the action of common bacteria and methylmercury (MeHg), then passes up the food chain and becomes concentrated in fish and sea mammals. The heavy dependence of the Minamata inhabitants on a fish diet meant that they were subject to high levels of MeHg exposure. Both marine and freshwater fish routinely contain low levels of MeHg as a result of mercury leaching from the earth\u2019s crust. Cases of frank poisoning have not been recorded in fish-eating populations other than those in Minamata, where special circumstances prevailed. However, the Minamata findings raised questions about whether less severe neurologic effects might occur at lower levels of exposure in populations whose diets were rich in seafood.\nGiven the high level of fetal abnormalities observed in Minamata (Harada 1995), particular concern focused on the potentially hazardous effects of prenatal exposure. This concern increased after an outbreak of mercury poisoning in Iraq in 1971\u20131972, when large numbers of people consumed bread made from grain treated with a mercury-based fungicide. Subsequent neurologic examination of 80 children born to mothers who had eaten the affected bread over a 2- to 3-month period suggested an increase in neurologic abnormalities (Amin-Zaki et al. 1974). Animal and other data have indicated that the developing fetus is more vulnerable both to exposure to neuro-toxicants such as heavy metals and to the effects of such exposure (Hanson 1997). Any adverse effects are likely to be structurally and functionally different from those seen in the exposed adult. Thus, in addition to the obvious concern for the health of those with diets high in seafood, this issue has wider public health implications in terms of the general advisability of consuming seafood during pregnancy.\nThe systematic investigation of potential developmental effects in children after prenatal exposure to MeHg began in the 1980s. Since that time two major longitudinal programs of work and a number of smaller cross-sectional studies have been conducted. These investigations assessed levels of prenatal exposure to MeHg in seafood-eating populations and examined subsequent developmental outcomes in children of varying ages. However, despite considerable research effort over a period of 20 years, there remains a lack of consensus on the central issue of whether exposure to MeHg derived from the routine diets of pregnant women presents a risk to their unborn children. This article contributes to the current debate on this subject with a brief description of the currently available data, some possible explanations for the inconclusive results that have so far emerged, and suggestions for potential ways forward in this contentious area of research.\nCurrent Evidence\nA search of relevant databases [Medline (http:\/\/omni.ac.uk\/medline), Toxfile (http:\/\/www.dialog.com), Embase (http:\/\/www.embase.com), Cancerlit (http:\/\/cancerweb.ncl.ac.uk\/cancernet\/cancerlit\/), Biosis Previews (http:\/\/www.biosis.org.uk), SciSearch (http:\/\/www.bids.ac.uk), and the Web of Science (http:\/\/wok.mimas.ac.uk)], using keywords such as \u201cmethylmercury,\u201d \u201cprenatal exposure,\u201d \u201cchild development,\u201d and \u201cneurobehavior,\u201d revealed that 12 investigations have been published thus far on this subject, all of which have been conducted since 1980.These investigations have used either neurologic examinations, developmental rating scales, or psychological tests to evaluate postnatal neurologic effects in children prenatally exposed to MeHg. Nine of the studies were cross-sectional (Table 1), in the sense that a sample of children was tested on one occasion and associations between test results and a measure of prenatal exposure to MeHg were examined. (Cordier et al. 2002; Grandjean et al. 1999b; Kjellstrom 1991; Marsh et al. 1995b; McKeown-Eyssen et al. 1983; Murata et al. 1999b; Steuerwald et al. 2000; Stewert et al. 2003; Weihe et al. 2002). Of these cross-sectional studies, however, one consisted of a form of follow-up, in that children were tested at 6\u20137 years of age and divided into comparison groups on the basis of exposure data collected from their mothers soon after birth (Crump et al. 1998; Kjellstrom 1991). In addition the study of Stewert et al. (2003) comprised a cross-sectional element of a larger longitudinal investigation concerned primarily with the adverse effects of prenatal exposure to polychlorinated biphenyls (PCBs). During the course of this investigation, possible interactions between PCB and MeHg exposure as determinants of adverse health effects were also investigated. Sample sizes in the cross-sectional studies ranged from 43 to 351 and response rates from 64 to 99% (Table 1).\nThree studies were longitudinal in design (Table 2) and followed children from birth, at intervals, for several years. Of these, the Faroe Islands study (Budtz-Jorgensen et al. 1999, 2000, 2002; Grandjean et al. 1992, 1995, 1997, 1998, 1999a, 2001a, 2001b, 2002a, 2002b, 2003; Murata et al. 1999a, 2002a, 2002b; Weihe et al. 1996) commenced in 1986 with a cohort of newborn infants who were subsequently tested at ages 12 months, 7 years, and 14 years. Two further longitudinal studies were carried out in the Seychelle Islands. The first commenced in 1987 with a sample of newborn infants who were followed up at age 5 weeks, 66 months, and 108 months (Cernichiari et al. 1995a; Davidson et al. 2000; Marsh et al. 1995a; Myers et al. 1995a, 1995b; Shamlaye et al. 1995). This was considered a pilot study by the authors, although a large amount of data was collected of a good standard and was therefore reported. A subsequent study commenced in the Seychelle Islands in 1989 with a sample of newborns who were followed up at ages 6.5 months, 19 months, 29 months, 66 months, and 108 months (Axtell et al. 1998, 2000; Cernichiari et al. 1995b; Cox et al. 1999; Crump et al. 2000; Davidson et al. 1995a, 1995b, 1998, 1999b, 2001; Myers et al. 1995c, 1995d, 1997, 2000, 2003; Palumbo et al. 2000). Sample sizes in the longitudinal studies were larger than those in the cross-sectional studies (Tables 1 and 2), although there was inevitable attrition over the periods of longitudinal investigation (Table 2).\nExposure assessment.\nWith the exception of the longitudinal Faroe Islands study and the study of Steuerwald et al. (2000), all studies used maternal hair as the vehicle for measuring prenatal exposure. In the case of the two longitudinal Seychelle Islands studies and six of the cross-sectional studies (Kjellstrom 1991; Marsh et al. 1995b; McKeown-Eyssen et al. 1983; Steuerwald et al. 2000; Stewert et al. 2003; Weihe et al. 2002), hair samples were taken from the mothers at parturition. However, three of the cross-sectional studies used hair samples taken from the mother and\/or the child at the time the child was tested (Cordier et al. 2002; Grandjean et al. 1999b; Murata et al. 1999b). In the study by Grandjean et al. (1999b), for example, exposure was assessed by reference to MeHg concentrations in maternal hair samples and, where this was not available, by reference to concentrations in the child\u2019s hair at the time of testing. Dietary habits and social circumstances had changed little in the community during the previous years. For this reason maternal hair samples were judged likely to be representative of parturition samples. In addition child and maternal hair concentrations were highly correlated. Thus, where it was necessary to use child hair samples, these were also regarded as representing an adequate proxy measure for maternal exposure at parturition The longitudinal Faroe Islands study and the cross-sectional study of Steuerwald et al. (2000) used samples of cord blood as well as maternal hair to assess levels of prenatal exposure. All studies reported details of quality control measures for the analysis of samples. However, the specific form of mercury included in the measurement was not always clear, and a number of different exposure metrics were employed (Tables 1 and 2).\nOutcome measurements.\nA variety of outcome measures were used that included neurologic examination, developmental rating scales, neuropsychological tests, and attainment tests. Although this variety was accounted for partly by the differing ages of the children, necessitating different forms of assessment, variation also occurred between studies in terms of the tests used for children of the same age group. All studies used tests or rating scales that were established, published assessment tools. With the exception of the Seychelle Islands studies, information on testing procedures provided in the published reports was rather limited and therefore difficult to evaluate. All studies reported control of some potential effect modifiers such as socioeconomic status, ethnicity, and parental IQ, but the particular factors selected for inclusion varied between studies. All studies used multivariate analysis techniques.\nThree of the 12 studies, namely, the longitudinal Faroe Islands study and the studies of Kjellstrom (1991) and Grandjean et al. (1999b), reported a statistically significant relationship between prenatal exposure to MeHg and at least one developmental outcome. In the case of the Faroe Islands study, significant associations were observed for the group as a whole, at 7 years of age, between MeHg in cord blood and some, but not all, of the outcome measures on six neurobehavioral tests. Many of these associations were of borderline statistical significance. However, a dose\u2013response relationship between cord blood MeHg and some test outcomes was demonstrated in a subgroup with the highest MeHg levels. Initial analysis in the Kjellstrom study (1991) did not indicate any significant associations between test outcomes and MeHg. However, exclusion of one outlier whose mother had particularly high MeHg levels resulted in the emergence of associations between MeHg and six test outcomes (Crump et al. 2000; Kjellstrom 1991). In the study by Grandjean et al. (1999b), an association between MeHg exposure was observed for three test outcomes. No discernible pattern was evident in these results in terms of effects on particular tests. Three further studies reported some positive associations between MeHg exposure and developmental outcomes, although all qualify their conclusions and express some reservations about the strength of their findings. In two of these studies, children were under 3 years of age, and assessments were in the form of neurologic examination. Steuerwald et al. (2000) reported that examination of children at 2 weeks of age showed that those with higher prenatal exposures had slightly lower overall neurologic scores, although there was no discernible pattern of suboptimal findings. Similarly, McKeown-Eyssen et al. (1983) carried out neurologic examinations on Cree Indian children between 12 and 30 months of age and found an association between MeHg exposure and the prevalence of abnormal muscle tone reflexes in males only. The authors note the mildness of the abnormality, which they consider to be of doubtful clinical significance. Cordier et al. (2002), in a study of children 9 to 12 years of age, found an association between scores on one test and MeHg exposure, but this did not appear in separate analysis of the highest exposure subgroup. Moreover, results from one test showed a positive association with MeHg exposure. This type of counterintuitive result also occurred in a study of children 7\u201312 years of age (Weihe et al. 2002) and in the main Seychelle Islands study when children were assessed at 66 months (Davidson et al. 1998). Neither of these studies or the first Seychelle Islands study report associations between other test results and MeHg exposure. Similarly, the results of Marsh et al. (1995b) and Murata et al. (1999b) with children between 6 and 7 years of age indicated no associations between psychological test scores and MeHg exposure. Murata et al. (1999b), however, demonstrated an effect of MeHg exposure on two neurophysiologic measures. Finally, the study of Stewert et al. (2003) of younger children 38 and 54 months of age reported an interactive effect of MeHg and PCB exposure as an incidental finding that emerged in the course of a study to investigate the adverse effects of PCB exposure. A finding of this nature, although interesting, should perhaps be regarded as speculative only and requiring further investigation.\nOur relatively brief review of the literature highlights the current uncertainty in this field of enquiry. The two major longitudinal investigations report contradictory findings, and a number of cross-sectional studies have similarly produced inconsistent results. This data set has already been subjected to much examination [National Research Council (NRC) 2000] and further analysis in an attempt to derive appropriate environmental exposure limits for pre-natal MeHg (Office of Environmental Health 1999; Rice et al. 2003). The generally assumed superiority of longitudinal over cross-sectional designs has tended to focus most attention on the studies conducted in the Faroes and the Seychelle Islands, which are of high methodologic quality (Jacobson 2001). However, it should be noted that, given the complex and challenging nature of this type of research, the majority of the cross-sectional studies are also of relatively good quality and would normally be taken into account in a systematic review. The results of these cross-sectional studies in fact mirror the general inconsistency evident in the results of the longitudinal investigations. One is therefore drawn to the conclusion that it is not possible from currently available data to determine whether prenatal exposure to MeHg, at levels routinely experienced by populations whose diets are rich in seafood, results in adverse effects on the nervous system of the developing child.\nDiscussion\nA primary objective of the review process is the derivation of conclusions from the available data to guide future policy. In this case, however, the development of such a policy would appear to be hindered by the existence of directly contradictory results obtained from research of equal quality. As noted above, the data from the studies contained in this review have already been the subject of extensive evaluation (NRC 2000; Rice et al. 2003) and continue to excite controversy that is so far unresolved (Davidson et al. 1999a; Grandjean and White 1999; Stern and Gochfeld 1999). A secondary objective of the review process, the identification of data gaps in the literature, appears inappropriate in circumstances where so much research has been carried out to date. Although the contribution of existing published research is unquestioned, it may be time to concede that there is little further that can be drawn from these data or, one suspects, from repeated studies of a similar type. Experience from other fields (Spurgeon 2002) suggests that further cross-sectional studies employing similar neurobehavioral outcomes will serve only to increase rather than reduce the uncertainty surrounding this issue. In the remainder of this article, therefore, I discuss some of the possible reasons for the inconsistency in the existing data and indicate some areas where alternative approaches might be required to achieve some progress in this field.\nThe common objective of the investigations reviewed above was to establish whether there is an association between prenatal exposure to MeHg and developmental effects. Although the various studies had many elements in common, perhaps the most noticeable feature of the studies as a group was the variation in the methods used to assess the two basic elements of the association, namely, the exposure and the effect. It is not surprising that research using different combinations of biological and psychological measures produces inconsistent results. The debate surrounding each of these elements, although undoubtedly complex, merits resolution in advance of any further research.\nExposure.\nIn terms of the most appropriate biological marker of prenatal exposure, opinion is divided between maternal hair and cord blood as the biological sample of choice. Studies that have attempted to define the relationship between different biological indices have produced inconsistent and somewhat wide-ranging results, and conversion from one set of values to another appears to involve a number of questionable assumptions (Office of Environmental Health 1999). Other difficulties in the interpretation of the data set arise as a result of the use of different units of measurement and a lack of clarity in some studies about whether the measure is of organic, inorganic, or total mercury concentration. Thus, there is continuing uncertainty about the association between elements of the diet and concentrations in child hair, maternal hair, cord blood, and maternal blood, as well as uncertainty about the strength of any relationship between each of these elements and the relationship between each and the actual exposure of the fetus. Elements of the debate about hair versus blood samples must be linked to a large number of other unanswered questions surrounding prenatal exposure measurement. These relate particularly to the relative importance of exposure at different periods of gestation, the relationship between these and average exposures, and the importance of peak exposures. The development of the central nervous system is time related and unidirectional. The inhibition of one stage of development tends to cause alterations to subsequent processes, with limited capacity for compensation for cell loss (Annau and Eccles 1986; Trask and Kosofsky 2000). Both the dose and timing of any environmental insult are important in terms of the specific nature of any adverse effects. How far do our current methods of prenatal exposure assessment reflect the need to take this into account?\nThe present enthusiasm for evidence-based policy and practice appears to offer an ideal opportunity to address these types of questions, either through the medium of an expert workshop or that of a written systematic review. The important issues in either process include a) definition of the important questions to be addressed to achieve valid and reliable assessment of prenatal exposure, b) identification of available data that could be used to answer these questions, and c) identification of new research required to fill any identified data gaps. In advance of some consensus on these issues, further research is likely to provoke more controversy rather than lead to any resolution of the current uncertainty.\nOutcomes.\nThe outcomes used in these studies were predominantly psychological tests. Use of such tests in environmental and occupational health research, which began in the early 1980s, has always been controversial, and the apparent inconsistencies in the data produced has provoked much debate in both environmental and occupational health research (Koller et al. 2004; Levy et al. 2004). Results relating to prenatal MeHg exposure represent a particular example of a wider problem and highlight a number of questions related to the more general field of neurobehavioral toxicology.\nSpecifically, two main areas are of concern. The first, and perhaps the more straightforward, relates to the control of variables that either represent potential confounders or may act as modifiers of the effects under investigation (Spurgeon and Gamberale 1997). They are perhaps best considered under the broad headings of situational variables (physical testing conditions and test procedures), tester variables (reliability of the examiners), and subject variables (individual characteristics such as age, gender, and socioeconomic group). In all epidemiologic research involving psychological testing, the list of these variables is potentially very long, and researchers appear divided about which to include. In research on MeHg, the majority of studies consider important subject characteristics such as age, ethnic and socioeconomic group, and aspects of parental lifestyle. However, for a number of other variables (e.g., aspects of the caregiving environment), inclusion is patchy. For many of these variables, useful literature is available on their effects on children\u2019s abilities or on test performance, and it may be possible to reach an evidence-based consensus on their inclusion or exclusion. For other, mainly procedural factors, data appear relatively scarce. A systematic review that encompasses other areas of psychology, for example, that pertaining to human\u2013computer interaction, might reveal relevant information. For example, how much does the size of the screen affect performance on a computer-administered test? How much does the physical location of testing (home, laboratory, hospital) affect test performance? Existing data on the effects of time of day (Smith 1992), for example, indicate that in epidemiologic studies this factor should always be controlled. Intuitively it would seem appropriate that the physical testing situation and procedures should be standardized for all subjects as far as is practically possible, regardless of whether firm evidence exists about the influence of heating, lighting, noise control, or the arrangement of furniture. Less well-researched aspects of the test situation can be explored usefully within the researchers\u2019 data. Is there, for example, a significant difference between test scores obtained at the beginning and at the end of the week or at different times of the year?\nThe effects of the tester, particularly where tests are not computer-administered, may be important, not only because of different interactions with different subjects but also because of the examiner\u2019s variable moods, motivation, levels of fatigue, and tendency to introduce systematic errors into the testing procedure. It cannot be assumed that confining testing to one examiner or using examiners who have undergone a single period of training removes tester variation. In terms of reliability, it may be advantageous to employ more than one tester in some circumstances. Measures such as the videotaping of testing procedures, double scoring, and examination of the test data for trends related to some of these factors have all been used to account for or eliminate this potential source of variation (Harvey et al. 1988). Similarly some estimation, albeit a subjective rating, of the child\u2019s level of co-operation with the testing procedure is important to include. Potentially this is a major source of variation in test performance rarely alluded to in published reports. Ideally, tests should also include parallel forms or practice trials to ensure that maximal performance level is recorded for each subject.\nAll except one of the studies in the field of research under discussion here present detailed accounts of quality control procedures in relation to MeHg assessment. It is relatively rare to find equally detailed discussion of procedures for outcome assessment. This is a situation that occurs frequently in neurobehavioral investigations. Lack of reference to quality control does not necessarily imply that control was limited but may suggest something about the attitude of researchers toward its importance. The implications for further research are 2-fold. First, systematic work is needed on the effects of factors considered likely to affect test performance, including both a review of the available data and, if necessary, further investigative work. Second, consensus must be reached on good practice such as that available in some other areas of toxicology, notably animal experimentation. Although this consensus may exist at an informal level in the field, the considerable methodologic variations between different neurobehavioral studies suggest that many aspects are currently opinion based rather than evidence based.\nA second and fundamental issue in terms of outcome measures relates to the types of tests used and, by implication, the interpretation of the results they provide and the comparability of these between studies. The tests employed in the studies described above are mainly tests of intellectual functioning. However, those used in different studies, and sometimes within the same study, derive from a number of separate traditions of intellectual assessment, each of which was developed for a different purpose and different client group. Although each has some advantages, none were developed specifically for neurotoxicity research and none is entirely appropriate for this type of application.\nAttainment tests are attractive in the sense that they offer the opportunity to benchmark the performance of children in basic skills such as literacy and numeracy against that of their peers. However, such tests tend to reflect the use of abilities rather than the underlying abilities themselves. Given the range of social and educational factors interacting with the ability to produce attainment, this effectively introduces additional variables into the equation (Gadzella et al. 1989).\nIn contrast, the neuropsychological approach characterized, for example, by tests such as the Trailmaking test used in the Seychelle Islands study or the Bender Gestalt test used in the Faroes Islands study was developed to provide detailed evaluation of patients with suspected damage to the brain. Such damage might have resulted from head injury or other insult or from a degenerative disease of the nervous system. In these circumstances the purpose of assessment is to provide detailed information about the nature of the problem in functional terms and thus provide a basis for rehabilitation and progress monitoring. Assessment in a clinical setting tends to be a flexible process that draws as much on the qualitative aspects of the interaction between psychologist and client as it does on the numerical test scores. The clinician is interested in the patient as an individual and reaches a professional judgment on the basis of a number of sources of information. There is a risk that tests of this nature lose much of their value when applied in a routine fashion to large groups of people. Many who work in the field of clinical neuropsychology appear to be deeply uneasy about the transfer of these techniques to an epidemiologic setting (British Psychological Society 2001). Particular concern arises when tests designed for administration by a psychologist are adapted for computer presentation. Researchers with a neuropsychological background tend to adopt a clinical approach by administering a very large battery of tests to cover all aspects of functioning (Davidson et al. 1995a; Grandjean et al. 1997; Kjellstrom 1991). In an epidemiologic setting this can be inappropriate, resulting in multiple comparisons and the possibility of chance findings. Moreover, it often leads to confusion from a psychological point of view, where the results appear as a collection of apparently unconnected findings with no discernible meaningful pattern. Where studies use the same tests, it is common for significant associations to appear in both studies but in relation to different outcomes (Grandjean et al. 1997, 1999b). Finally, there are questions about the ability of tests designed for more severely affected patient groups to detect relatively subtle effects in community samples (Spurgeon 1996; Stollery 1985, 1990).\nTests derived from a psychometric tradition are concerned with the assessment of intelligence quotients (IQ) in the general population and were originally developed to describe normal distributions of cognitive functioning. The Wechsler Scale (Wechsler 1991) represents the most widely used test battery in this respect. Developmental scales for very young children fit within this tradition, replacing formal testing where this is impractical, although it should be noted that maternal reports of developmental milestones are subject to numerous sources of error such as inaccurate recall, differing definitions of certain behaviors, and presentational bias (Axelson and Rylander 1984).\nThe measurement of IQ is a reassuringly familiar concept supported by a wealth of normative data and experience built up over many years. Unfortunately, IQ tests were originally developed within a theoretical framework of cognitive functioning that prevailed more than a half-century ago. Such tests reflected a contemporary need to categorize individuals on a quantitative scale to predict future performance, an approach now considered somewhat crude and simplistic. Although such tests maintain their predictive validity in some settings (Neisser et al. 1996), they are relatively blunt instruments that combine a number of different abilities within each test (Lezak 1988). This aspect limits the information that can be derived from the assessment and makes interpretation difficult when conflicting results emerge from different studies. When placed in the context of more recent theoretical developments in cognitive psychology, established IQ tests do not provide results that can be easily linked to current models of cognitive processes.\nA primary objective of neurobehavioral research is the detection of subtle effects on cognitive functioning in community samples after neurotoxicant exposure. For epidemiologic purposes, tests should be quick and easy to administer. The results should be interpretable at group level and comparable between different studies. Given these criteria, none of the tests currently in use appear to be entirely fit for this purpose. Speed and ease of administration do not represent major challenges in an age of advanced information technology. However, improvements in interpretability and comparability are more complex issues likely to require a radical change of approach. In recent years a number of authors have pointed to the overemphasis on empiricism in this field and the lack of a strong theoretical underpinning for the assessment tools employed (Stephens and Barker 1998; Stollery 1990, 1996; Williamson 1990). The development of tests grounded in well-established cognitive theory would allow results to be discussed in terms of the specific aspects of cognitive processing under investigation rather than simply by reference to broad and largely uninformative categories of effect such as \u201cmemory\u201d or \u201cattention.\u201d Modern approaches to the study of memory processes, for example, have long distinguished between several elements that contribute to the final outcome (initial registration of information, encoding, transfer to long-term store, loss of information by decay or interference, and use of cues for retrieval) (Baddeley 1987). Each may be differentially susceptible to neurotoxic insult, but effects on one specific process cannot be uncovered by most current tests that provide a simple global outcome score. Moreover, overall scores may mask specific effects where subjects employ compensatory strategies among different processes to achieve maximum performance. The development of tests, for both children and adults, based on techniques currently available to separate and measure these specific processes would provide much more useful information about the nature and size of any observed effect. This type of approach would ultimately pave the way for much greater comparability between the results of different investigations and for the development of comparable assessment techniques for children at different ages during longitudinal investigations. Despite much international effort during the last 25 years, agreement on a universally approved set of tests has proved elusive (World Health Oganization 1989). At the same time, the pursuit of the goal to achieve comparability over time and between studies has tended to inhibit the development of new techniques. It seems unlikely that consensus on appropriate assessment tools will be achieved in advance of a consensus on the theoretical basis for those tools. Fortunately, much of the information required for these new developments is readily available in the existing cognitive, experimental, and developmental psychology literature.\nConclusion\nReviews of the data relating to the developmental effects of prenatal MeHg exposure have highlighted the inconsistency of the currently available evidence. The size and nature of the risk to children that is associated with seafood consumption by their mothers remains uncertain and a source of considerable controversy. It has been argued here that the present uncertainty derives from the variation between studies in the methods used to measure both the exposure and the effect. Each element would merit further consideration in advance of any future research in this field. Although consensus is required on the appropriate biological marker of exposure, there is also a particular need to address issues of both procedure and content in psychological assessment. Discussion of these issues, particularly those relating to psychological tests, may have implications that go well beyond the immediate needs of this field of inquiry. Investigation of the effects of MeHg provides one particular example of the difficulties in data interpretation that occur repeatedly in neurobehavioral studies and threaten to undermine confidence in this methodology. The increasing international anxiety about potential adverse effects of low-level neurotoxicant exposure in the environment underlines the importance of addressing these concerns, as psychological methods currently represent one of the main tools of research in this field.","keyphrases":["methylmercury","developmental outcomes","prenatal exposure","neurobehavioral tests"],"prmu":["P","P","P","P"]}
{"id":"J_Gastrointest_Surg-3-1-1852381","title":"Successful Internalization of a Chronic Biliary Cutaneous Fistula After Liver Transplantation: Deepithelializing the Fistula Tract\n","text":"Biliary cutaneous fistulas are uncommon sequelae after biliary surgery and can be a source of significant morbidity. We describe a liver recipient who developed a biliary cutaneous fistula secondary to hepatic artery thrombosis; this subsequently drained for over 7 years. Through a novel approach, using the transabdominal fistula tract as a conduit, the fistula skin opening was deepithelialized and anastomosed to a jejunal loop, internally draining the tract. For over 7 years postoperatively, this internal drainage procedure has continued to function effectively. This approach may have value in internalizing longstanding biliary cutaneous fistulas in well-selected patients in whom there is no existing biliary ductal system or the existing system anatomically does not lend itself to restoration of functional internal drainage through conventional approaches.\nIntroduction\nBiliary cutaneous fistulas are uncommon sequelae after biliary surgery and hepatic trauma. Persistent biliary fistulas can be associated with local skin morbidity, malabsorption of fat soluble vitamins, steatorrhea, impaired wound healing, and sepsis as a result of fistula tract obstruction.1 We describe a rare scenario in which a liver transplant recipient developed a biliary cutaneous fistula secondary to hepatic artery thrombosis. Through a novel approach, the fistula skin opening was deepithelialized and, using this transabdominal fistula tract as a conduit, anastomosed to a jejunal loop, thereby internally draining the fistula. This operative procedure, which has provided a successful long-term result, is discussed.\nMaterials and Methods\nCase Report\nA 60-year-old man of Chaldean descent underwent orthotopic liver transplant for cirrhosis secondary to chronic alcohol use. His early postoperative course was complicated by hepatic artery thrombosis with necrosis of the extrahepatic biliary system. Because of sepsis and family issues, retransplantation was not a realistic option. Surprisingly, his graft remained viable with good metabolic function despite the absence of normal extrahepatic drainage. For 7\u00a0years subsequent to transplantation, his biliary drainage was managed with a U-tube connected to a bulb drain exiting the skin in the right subcostal region (Fig.\u00a01). As a result, he suffered from severe malnutrition, pain, and skin breakdown. The persistent external drainage markedly affected his lifestyle, as the continuously draining bile required dressing changes over the exit site two to four times per day. The U-tube, frequently becoming obstructed with biliary sludge, required bimonthly replacement. Preoperative fistulogram (Fig.\u00a02) demonstrated the continuity of the cutaneous opening with a large channel through the mid portion of the liver, but with no evidence of a true ductal system.\nFigure\u00a01(a) Longstanding (7-year-old) biliary cutaneous fistula draining through right upper quadrant scar. Functional U-tube in place with copious biliary drainage. (b) Close-up of U-tube exit site.Figure\u00a02Preoperative fistulogram in which contrast was injected through the existing U-tube. The cutaneous opening at the upper left side of the frame is in continuity with the large contrast-filled channel that enters the substance of the liver. Note the complete absence of an extrahepatic biliary system.\nSurgical Procedure\nWith the patient under general anesthesia, the abdominal cavity was entered through a right subcostal incision along the scar of the previous transplant incision. The incision was fashioned so as to preserve a 1\" diameter button of full-thickness skin surrounding the opening to the fistula tract (Fig.\u00a03). Extensive adhesions between the fascia, liver surface, and small intestine were carefully lysed. The fistula tract was dissected retrogradely from surrounding subcutaneous tissue and muscle, preserving a rectus fascial ring. This created a conduit that could be anastomosed to small intestine. Dissection of the tract was stopped proximally at the level of the liver surface to preserve its integrity and vascularity. The fistula opening was lowered into the abdominal cavity. A 60-cm long Roux-en-Y jejunal limb was constructed 50\u00a0cm distal to the ligament of Treitz using stapled technique.\nFigure\u00a03(a) At operation, the U-tube was prepped into the surgical field. (b) Dissection of the epithelialized chronic biliary cutaneous fistula tract. In entering the abdomen through the previous right subcostal incision, a 1\" diameter button of full-thickness skin containing the fistula tract was preserved.\nA 4-mm-wide skin edge was sharply deepithelialized circumferentially around the dissected fistula opening. This conduit was then anastomosed to the Roux limb using two layers of running 4-0 Prolene suture (Fig.\u00a04). The inner layer approximated the deepithelialized skin edge of the fistula tract to full-thickness jejunum. The outer layer approximated the conduit\u2019s scar tissue and the preserved rectus fascial ring to seromuscular jejunum. An omental wrap was fashioned around the anastomosis. The anastomosis was performed over an 8-French feeding tube, which was exteriorized through a previous U-tube exit site and connected to a bile bag.\nFigure\u00a04(a) Anastomosis of Roux-en-Y jejunal limb to deepithelialized fistula tract. (b) Illustration depicting the anastomosis.\nThe abdominal cavity was irrigated and closed in the standard fashion. The patient tolerated the 3-h procedure well. Blood loss was minimal. He was discharged on postoperative day\u00a08.\nResult\nThis patient has now been followed for 7\u00a0years since the described procedure and is doing well without external drainage of bile. The condition of his periincisional skin improved dramatically shortly after the procedure. At this time his liver function tests and nutritional parameters are normal and his graft function remains adequate on a low dose immunosuppressive regimen.\nDiscussion\nChronic biliary cutaneous fistulas are challenging to manage from many standpoints: metabolic, nutritional, hygienic, and quality of life. The patient\u2019s being a liver transplant recipient, added further complexity. First, malignant change was reported in chronic biliary cutaneous fistulas 2; this possibility is of real concern in light of this patient\u2019s immunosuppressed state. Second, this patient had severe malabsorption and resulting difficulty in maintaining acceptable cyclosporin blood levels. Finally, the ongoing presence of his U-tube poses the threats of ascending cholangitis, hemobilia, intrahepatic biloma, and biliary-venous fistula, which are all well-documented complications that have been associated with U-tubes.3 The decision for operative intervention was made out of these concerns and for progressive difficulty in maintaining adequate external biliary drainage. Late retransplantation was considered; however, the patient\u2019s graft function had been excellent over the 7\u00a0years subsequent to his transplant, and the patient and his family refused to consider it.\nIn both the posttraumatic and postbiliary surgery settings, existing literature advocates initial nonsurgical management of biliary cutaneous fistulas, as a significant number will close either spontaneously or with nonsurgical intervention.1,4,5 In the series by Zer, four of seven biliary fistulas sealed spontaneously.5 Endoscopic approaches to reducing intrabiliary pressure and thereby encouraging drainage along a path of least resistance include endobiliary stenting, sphincterotomy, and nasobiliary drainage.1,4 Finally, selective biliary embolization, percutaneous transhepatic catheter drainage, sclerosis with tetracycline, and oral nitrates were also described to promote closure.4,6\u20139\nOperative procedures for refractory biliary cutaneous fistula were dictated by the anatomic site of biliary tract disruption. Roux-en-Y hepaticojejunostomy, in which a jejunal loop is sutured directly to the liver capsule, was employed in the context of intrahepatic biliary injury after trauma.11,10,4 Reports in which an actual fistula tract is used as a conduit and sutured to a loop of small bowel, however, are rare. Smith described the anastomosis of a jejunal loop to a divided fistulous tract arising from the lateral surface of the liver in a patient with a penetrating injury to the upper abdomen.12 That same author described the internal drainage of a biliary cutaneous fistula secondary to blunt liver trauma into the gallbladder.12 In both cases the fistula tract was divided and then the proximal end of the divided tract was anastamosed to an intestinal loop over a tube.\nDeepithelialization of the skin opening and its direct usage in an anastomosis has not, to our knowledge, been previously described. Furthermore, this procedure is unique in that the internalization was done in the case of a functioning liver after hepatic artery thrombosis. The existing anatomy that had been established over 7\u00a0years of fistulous drainage dictated the type of procedure that was performed. In contrast to fistulas that arise after biliary tract surgery (for example, after cholecystectomy with common bile duct exploration), there was no remnant of a previous biliary ductal system. The preoperative fistulogram revealed flow of contrast from the skin directly to the liver surface with no evidence of an extrahepatic ductal system. The fistula therefore provided the only source of biliary drainage for the entire liver.\nAn alternative surgical option would have been hepaticojejunostomy, in which the jejunum would have been sutured to a fibrous ring at the origin of the fistula tract from the liver surface. This, however, would have required more extensive dissection with possible disruption of collateral vasculature to the previously ischemic liver and to the tract itself, as well as dissection of the transverse colon, which in part bordered the tract. At operation, the superficial portion of the fistula tract was well established and appeared well vascularized. The potentially harmful deep dissection necessary for a hepaticojejunostomy was therefore avoided.\nWe emphasize the decision to have fashioned a tongue of omentum over the anastomosis. By nature, the tissue comprising a fistula tract is probably somewhat tenuous. This patient\u2019s immunosuppressed and malnourished state compound the risk of anastomotic breakdown. We feel that the well-vascularized, adherent omental tongue was an important aspect of the operation in reinforcing the anastomosis.\nAs of 7\u00a0years of follow-up, the patient has not developed signs of biliary obstruction. His immunosuppressant levels are easier to maintain and he does not have evidence of malabsorption. It is unlikely that he will develop cicatricial narrowing of the tract, as it had remained well epithelialized for the 7\u00a0years before this operation. There is a theoretical concern about formation of squamous cell carcinoma at the anastomotic site, which we continue to keep in mind during long-term follow-up.\nConclusion\nIn summary, through a novel approach this patient\u2019s biliary cutaneous fistula was internalized, which has eliminated his associated skin morbidity and malabsorption. Furthermore, his well-functioning graft was salvaged, thus saving the cost of retransplantation and sparing an additional liver for another patient. Internalization of a long-standing biliary cutaneous fistula through deepithelializing the skin opening and using the tract as a conduit is technically feasible. We believe it to be a valid approach in the patient in whom there is no existing biliary ductal system or the existing system anatomically does not lend itself to restoration of functional internal biliary drainage by conventional approaches.","keyphrases":["liver transplantation","biliary fistula"],"prmu":["P","P"]}
{"id":"J_Gastrointest_Surg-3-1-1852388","title":"Middle Segment Pancreatectomy: A Useful Tool in the Management of Pancreatic Neoplasms\n","text":"Small, benign, or low-grade malignant tumors located in the neck of the pancreas are usually treated with enucleation. However, if enucleation is too risky because of possible damage of the main pancreatic duct, standard pancreatic resections are performed. Such operations can lead to impaired long-term exocrine\u2013endocrine function. Middle segment pancreatectomy consists of a limited resection of the midportion of the pancreas and can be performed in selected patients affected by tumors of the pancreatic neck. Middle segment pancreatectomy is a safe and feasible procedure for treating tumors of the pancreatic neck; in experienced hands it is associated with no mortality but with high morbidity, even if the rate of \u201cclinical\u201d pancreatic fistula is about 20%. Moreover, it allows a surgeon to preserve pancreatic parenchyma and consequently long-term endocrine and exocrine pancreatic function.\nIntroduction\nWhereas neoplastic lesion located in the pancreatic head or body-tail are usually resected by pancreaticoduodenectomy or distal pancreatectomy, tumors in the neck represent a real challenge for a surgeon. In these cases, standard or extended pancreatectomies performed for benign or borderline cases can determine the loss of a great amount of glandular tissue, significantly increasing the risk of diabetes, impaired exocrine function, and splenic loss.1\u20136\nEnucleation would be an adequate alternative for small, benign, and low-grade malignant tumors, such as endocrine and cystic neoplasms of the pancreas. Unfortunately this conservative procedure cannot be always applicable. When the neoplastic lesion measures up to 2\u00a0cm or more, or it is encased within the pancreatic gland, enucleation is associated with a high risk of Wirsung\u2019s duct damage; moreover in the case of tumors with uncertain biological behavior this approach should be avoided because of the risk of tumor recurrence1\u20135.\nLetton and Wilson7 reported for the first time in the English literature in 1959 two cases of traumatic midpancreatic transection followed by a reconstruction with a Roux-en-Y jejunal loop anastomized to the distal part of the gland. Dagradi and Serio,8 from our own Department of Surgery, were the first in 1984 to propose middle pancreatectomy with an \u201concological\u201d indication, treating a pancreatic insulinoma. Subsequently, other authors reported cases of resection of the middle pancreas, of varying extent, using various terms such as \u201ccentral pancreatectomy,\u201d \u201cmiddle segment pancreatectomy,\u201d \u201csegmental pancreatectomy,\u201d and \u201cintermediate pancreatectomy.\u201d9\u201313 The underlying indications for surgery ranged from chronic pancreatitis to benign, uncertain behavior, or low-grade malignant exocrine and endocrine neoplasms1\u201319. Different techniques were adopted for gastrointestinal reconstruction, including jejunal anastomosis of both the proximal and distal stump, or of only the distal stump, with pancreaticoduodenal or pancreaticogastric anastomosis.1\u201321\nSurgical Technique\nThe abdomen is entered through a midline incision. The gastrocolic ligament is opened, preserving the gastroepiploic vessels, and the pancreatic gland is exposed. The posterior peritoneum along the superior and inferior margin of the pancreas is incised. The superior mesenteric vein and the portal vein must be identified and their surfaces cleared below the gland. The plane between the superior mesenteric and portal vein should be teased apart. The splenic artery and vein are dissected free and separated from the gland. Some venous tributaries to the portomesenteric axis and some minor collaterals of the splenic artery can be ligated. Then, the posterior surface of the pancreatic neck is isolated from the portomesenteric axis and a ribbon is passed behind the gland to elevate it. Sutures are placed along the superior and inferior margins to indicate where the proximal and distal transection should be performed and to ligate those vessels running along the margins. The segment of the pancreas with the tumor is subsequently transected through a knife or a stapler to the left and to the right of the lesion. The cephalic stump is sutured with interrupted stitches after elective ligation of the Wirsung\u2019s duct or by means of a stapler. A small stent is placed in the main pancreatic duct while performing pancreojejunostomy or pancreogastrostomy; the stent can be left in place, even if in our experience we have never done it. Two closed-system suction drains are used to drain the cephalic stump of the gland and the pancreaticojejunostomy\/pancreaticogastrostomy.\nDiscussion\nIt has been shown that standard pancreatic resections are nowadays associated with low mortality and morbidity if performed in high-volume centers by experienced surgeons.22\u201324 It is remarkable that this type of surgery can lead to long-term complications, such as diabetes, exocrine insufficiency, and late postsplenectomy infection25,26.\nThe incidence of postoperative exocrine and endocrine impairment is not predictable in patients with apparently \u201cnormal pancreas.\u201d Factors such as fibrosis of the remnant, Wirsung\u2019s duct obstruction, preexisting chronic pancreatitis, benign or malignant disease, and subclinical diabetes may play a role as \u201crisk factors.\u201d1\u20133 After standard left-sided resection there is an increased incidence of endocrine impairment and onset of diabetes reported from 17 to 85% of patients; it is reasonable that the extent of the resection is strictly related to the incidence of endocrine\u2013exocrine long-term insufficiency27\u201331.\nFor all these reasons, more conservative surgical techniques have been advocated for small, benign, or low-grade malignant tumors located in the neck of the gland, aimed for sparing, as much as possible, pancreatic parenchyma. Whenever neoplastic lesions are not small and superficial enough to be simply enucleated, middle segment pancreatectomy should be considerated.1\u20136\nMiddle segment pancreatectomy accounts for only 3% of the pancreatic resections performed at our institution and about 100 cases have been reported in the English literature1\u201321,32\u201334: this means careful selection of patients. In fact, the small number of patients who underwent this type of operation is related to different factors: specific localizations of the neoplasm, well-selected indications (benign or low-grade malignant tumors), and a distal pancreatic stump of at least 5\u00a0cm in length.\nSome authors1,3 have reported that this operation can be performed only in the case of small tumors (<5\u00a0cm in diameter); in our experience, although the mean diameter of the resected lesions was 27.4\u00a0mm, we have safely performed middle segment resection for tumor measuring more than 5\u00a0cm, harboring from the anterior face of the pancreas.\nMiddle segment pancreatectomy was also occasionally used for malignant disease: two islet cell carcinomas, one vipoma who subsequently developed hepatic metastases, one cystadenocarcinoma, and one carcinoma in situ2,3,10\u201312.\nIn the past we have also performed this operation for malignant tumor but we had pancreatic recurrence of the tumor in two patients (one affected by metastasis and one by intraductal papillary mucinous neoplasms [IPMN] with in situ carcinoma); moreover, two patients with adenoma and borderline main duct IPMN had a tumor recurrence in the pancreatic gland. Thus, we believe that in patients affected by primary or metastatic malignant tumor, a standard resection would be more appropriate. Moreover, middle pancreatectomy in our experience should also be avoided in patients affected by IPMN, especially main duct type because of their potential malignity and the possibility to have different degrees of dysplasia along the Wirsung\u2019s duct in the same patients.\nThe surgeon must be sure to achieve tumor-free proximal and distal resection margins after performing middle segment pancreatectomy and, for this reason, frozen section examination is mandatory.\nMiddle segment pancreatectomy is a meticulous procedure. There is the possibility of leaks from both the closed cut edge of the head and the pancreaticojejunostomy, considering that in most patients we are dealing with a normal soft pancreatic texture with a small Wirsung\u2019s duct. Thus, not only great care must be taken in selecting the patients who will benefit from this operation, but also an experienced pancreatic surgeon working in a high-volume center is required for performing the procedure.1\u20134,6,32,33\nMedian pancreatectomy is reported to be associated with no mortality but with a high postoperative morbidity, above all consisting of pancreatic fistula.6 In our experience the \u201cclinical\u201d pancreatic fistula rates after pancreaticoduodenectomy and left pancreatectomy are 10 and 20%, respectively.23,35\u201337 Between January 1990 and December 2005 61 patients underwent middle segment pancreatectomy at our institution. The incidence of pancreatic fistula\u2014according to the International Study Group on Pancreatic Fistula definition22\u2014was 51%. It is remarkable that most patients complained of Grade A fistula, which is a \u201cbiochemical\u201d fistula without any clinical impact, whereas 13 patients (21%) developed a grade B or C fistula, which required prolonged in-hospital stay. In almost all patients the conservative management was successful; no one underwent reoperation and in four cases intraabdominal collections were treated with ultrasound-guided drainage. The mortality rate was zero.\nThe risk of developing a pancreatic fistula must be taken into account in the preoperative decision making; we believe that this risk is acceptable when the procedure is performed in a high-volume center and for patients with a long-life expectancy, such as young or middle-aged people affected by benign or low-grade tumors.\nThe most important advantage of middle segment pancreatectomy is the good endocrine and exocrine long-term function.1\u20136,10 Iacono et al.1 in a series of 13 patients demonstrated that postoperative oral glucose tolerance, pancreaticolauryl and fecal fat excretion were normal in all cases and they studied six patients pre- and postoperatively with oral glucose tolerance test showing no significant differences before and after surgery. Moreover, Sperti et al.3 showed, in a review of the literature, no case of impaired endocrine function in 59 evaluable patients whereas exocrine function was reported to be normal in 56 out of 59.\nAnother advantage of this procedure is the possibility to preserve the spleen, preventing the risk of postsplenectomy sepsis and hematologic disorders, which is low but exists in an adult.38,39\nIn conclusion, middle segment pancreatectomy is a safe and technically feasible surgical approach for removing pancreatic neck tumors in well-selected patients; in experienced hands it is associated with no mortality but with high morbidity. Most of the complications do no require reoperation or prolonged in-hospital stay and can be successfully managed conservatively. Moreover, it allows the surgeon to preserve pancreatic parenchyma and consequently long-term endocrine and exocrine pancreatic function.","keyphrases":["middle segment pancreatectomy","pancreatic resection","pancreatic function","pancreatic tumors","surgical complications"],"prmu":["P","P","P","R","R"]}
{"id":"Ann_Surg_Oncol-4-1-2277445","title":"Metinel Node\u2014The First Lymph Node Draining a Metastasis\u2014Contains Tumor-Reactive Lymphocytes\n","text":"Background We previously identified tumor-reactive lymphocytes in the first lymph nodes that drain the primary tumor. In this study, we performed lymphatic mapping to investigate the possibility of finding the first lymph nodes that drain metastases, and of learning whether these lymph nodes contained tumor-reactive lymphocytes suitable for adoptive immunotherapy.\nThe global cancer registry Globocan shows 10.9\u00a0million new incident cases and 6.7\u00a0million deaths worldwide due to cancer in 2002.1 A total of 24.6\u00a0million persons are estimated to be alive with cancer (within 3\u00a0years of diagnosis). Leading causes of death from cancer, in order, are those of the lung (1.18\u00a0million), stomach (700,000), and liver (598,000). Most of these deaths were caused by metastatic disease, and they verify the fact that surgery and oncological therapy do not cure most patients with disseminated cancer. The incidence of cancer will continue to increase, especially in developing countries, largely due to smoking, infections, and lifestyles resembling those of people living in industrialized countries.1,2 Thus, intensified efforts are warranted, both to prevent cancer and to find new modalities of treating patients with metastatic cancer.\nTumor cells spread by lymphogenous and hematogenous routes into the systemic circulation to disseminate the disease. The importance of tumor lymphangiogenesis has lately been proven in studies where high levels in colorectal cancer specimens of lymphangiogenic peptides, and vascular endothelial growth factor (VEGF)-C and\/or VEGF-D promote tumor lymphangiogenesis and metastasis.3 The sentinel node concept implies that the lymphatic drainage from a primary tumor first drains to a certain locoregional lymph node specific for each individual and that its tumor status is representative for the entire lymphatic field. The concept is established in staging for breast cancer4 and malignant melanoma5 and has also been evaluated in several other types of malignancies, such as colon cancer,6 ovarian cancer,7 pancreatic cancer,8 and urinary bladder cancer.9 The technique has been used as a diagnostic tool to improve staging and to tailor the extent of regional lymph node dissections. Probably most solid tumors disseminate first through the lymphatics and thereafter enter the systemic circulation via lymphovenous shunts present in the first draining lymph nodes.10\u201312 Approximately half of the lymphatic fluid entering a lymph node continues directly into the systemic circulation; therefore, lymphogenic spreading of the tumor cells may be mandatory for hematogenic dissemination.3,13\nHowever, to our knowledge, only one study has previously evaluated lymphatic mapping in metastases where liver metastases were investigated.14 Because it has been proven that both metastases and primary tumors have the capacity to metastasize15\u201317 and induce lymphangiogenesis,18 we assume that lymphatic drainage from metastases may have an equal importance in metastases as the sentinel node concept in many solid primary tumors for correct staging. We have previously experienced surprises in lymphatic mapping within patients with colon cancer6 and urinary bladder cancer9 regarding primary tumor lymphatic drainage. We believe that a similar technique may be of use in surgery for liver metastases and other solid metastatic tumors for correct staging, and thereby for giving the patients the most appropriate therapy.\nWe previously showed that a sentinel node derived from a primary tumor contains specific tumor-reactive lymphocytes that have immunological reactivity toward the tumor in patients with colon cancer19 and in patients with urinary bladder cancer.20 Through the injection of Patent blue dye or radioactive tracer subserosally during surgery around the colonic tumor or transurethrally around the urinary bladder tumor, the sentinel nodes were found. A biopsy was performed, and the nodes\u2019 immunological role and function were studied. After preparation, the lymphocytes were identified, isolated, and proliferated in vitro by stimulation of interleukins (ILs) and tumor antigen. Lymphocytes from sentinel nodes proliferated dose dependently and secreted interferon (IFN)-\u03b3 on stimulation with tumor homogenate.\nThe aim of the present study was to investigate lymphatic drainage from metastases and identify first draining lymph nodes. We also analyzed tumor-reactive immunological properties in lymphocytes derived from these lymph nodes.\nMATERIALS AND METHODS\nPatients\nNineteen patients (9 men and 10 women, average age 51.5\u00a0years) with metastases from solid tumors were included in the study. Patient characteristics are shown in Table\u00a01. The operations were performed during the period of November 2003 to January 2007. The study was approved by the local ethical committee, and each patient provided informed consent.\nTable\u00a01.Patient characteristicsaPatientAge (y)SexPrimary tumorOrigin metinel nodeTracerNumber of metinel nodesMetinel node pos\/neg for metastasesSuccessful expansionAliveFollow-up time (mo)148MColonIntra-abdominal local recurrencePB33 negYesYes38254MRectumLiver metastasisPB22 negYesYes38350MMal melanomaSubcutaneous metastasisPB22 posNoNo\u2013477FColonLiver metastasisPB33 negNoNo\u2013574MColonLiver metastasisPB21 posYesNo\u20131 neg666MColonLiver metastasisPB1MINoYes29751FOvarian cancerLiver metastasisPB21 posYesbNo\u20131 neg864FOvarian cancerGroin lymph nodePB\/RT55 posNoNo\u2013959FPancreatic cancerIntra-abdominal local recurrencePB4MINoNo\u20131039FMal melanomaGroin lymph nodePB\/RT42 negNoYes111147MMal melanomaTruncal metastasisRT12 posNoNo\u20131245FBreast cancerCervical lymph nodePB\/RT22 negYesYes51333FTongue cancerTruncal lymph nodeRT3MINoYes61464FColonIntra-abdominal local recurrencePB21 posYesYes41 neg1541FBreast cancerLiver metastasisPB22 negNoYes41639MMal melanomaCervical lymph nodePB\/RT22 negNoYes21720MCholangiocarcinomaLiver metastasisPB33 negYesNo\u20131865MColonLiver metastasisPB31 posYesYes12 neg1942FLeiomyosarcomaLiver metastasisPB44 posYesYes1pos, positive; neg, negative; Mal, malignant; PB, Patent blue dye; MI, missing information; RT, radioactive tracer (4\u00a0\u00d7\u00a010\u201315\u00a0MBq Tc-nanokolloid).a Data regarding the expansions for patients 1, 2, 5, and 6 also exist in unpublished data.b Patient died on the projected day of transfusion.\nThe lymph nodes that drained metastases were identified by injecting approximately 1\u00a0mL Patent blue dye (PB) (Guerbet, Paris) subserosally or subcutaneously in four places around one of the metastasis or in the tissues close to the local recurrences with a 27-gauge needle. After the injection of PB, the first draining lymph nodes turned blue within 3 to 10\u00a0minutes and were regarded as metastases-draining lymph nodes. In our previous work, a 10-minute cutoff was sufficient to identify sentinel nodes to primary tumors. We have experienced that during this period of time the first draining lymph nodes turn blue. Waiting longer means that a second echelon of draining lymph nodes may be colored blue, which does not represent the first draining lymph nodes. We arbitrarily applied the same 10-minute limit when detecting lymph nodes that drain metastases. These lymph nodes were either immediately removed or marked with sutures.\nThe sources of the liver metastases in our study were from colorectal cancer in five patients and in one patient each from ovarian cancer, breast cancer, cholangiocarcinoma, and leiomyosarcoma. Four patients had subcutaneous lymph node metastases from disseminated malignant melanoma, and one patient each had metastases from ovarian cancer, breast cancer, and squamous cell carcinoma of the tongue. Two patients had intra-abdominal local recurrences after surgery for colon cancer, and one patient had an intra-abdominal local recurrence after surgery for pancreatic cancer.\nBecause of these liver metastases, five patients underwent partial right-sided liver resections (patients 4, 5, 6, 15, and 17), and three patients underwent partial left-sided liver resection (patients 2, 18, and 19). The patient with metastases resulting from ovarian cancer (patient 7) had several large, bulky, cystic tumors, and surgeons performed an almost complete right-sided liver resection. In four cases, surgery was performed for solitary colorectal liver metastases (patients 2, 4, 5 and 6), and in the remaining five cases, tumor-reducing surgery was performed.\nIn six cases, a preoperative lymphoscintigraphy was performed by a subcutaneous injection of 4\u00a0\u00d7\u00a010 to 15\u00a0MBq Tc-nanokolloid radioactive tracer in quadrants around the metastasis. The lymphoscintigraphies were performed to plan the surgical procedure by localizing the lymph node or nodes draining the metastasis. The position of the metastasis-draining lymph node or nodes was marked on the skin with a felt-tip pen. When the lymphoscintigraphy was not performed on the day of surgery, a new peritumoral injection of radioactive tracer was made at start of surgery. The draining nodes were intraoperatively identified by the use of a handheld gamma detection tube. In four of six cases, a simultaneous intraoperative injection of PB was performed.\nPreparation of Specimens\nLymph nodes draining metastases and nondraining lymph nodes (for analytical purposes) were surgically removed and immediately taken to the laboratory on ice. In addition, one 5-mm slice of the whole metastasis (including the invasive margin) was also dissected and sent for analysis. Frequently, most of the lymph node or nodes were used for the expansion procedure. The remaining specimens underwent routine histopathological examinations with hematoxylin and eosin staining. At the laboratory, slices <1\u00a0mm were cut from the central and peripheral part of the lymph nodes for analysis by flow cytometry (FACS) and proliferation analyses. The metastasis was also analyzed by FACS, and a preparation of the whole tumor (homogenate) was used.\nImmunological Evaluation\nSingle cell suspensions were made from the lymph nodes and metastases by gentle pressure with a loose-fitting glass homogenizer as previously described.19 Briefly, 1\u00a0\u00d7\u00a0106 cells were washed in phosphate-buffered saline containing 2% fetal calf serum and .05% NaN3, then stained with fluorophore conjugated antibodies toward the cell surface markers CD4, CD8, CD19, and CD56 (Becton Dickinson). Thereafter the cells were investigated with a FACSCalibur (Becton Dickinson).\nFor cell cultures, single cell suspensions of metastasis-draining lymph node\u2013derived cells were resuspended in RPMI 1640 proliferation media containing 10% human AB serum (Sigma), 1% penicillin-streptomycin (Sigma), and 1% glutamine (Sigma). For preparation of the antigen source, the metastasis was homogenized by an Ultra-turrax homogenizer in 5 volumes (w\/v) of 2\u00a0\u00d7\u00a0 phosphate-buffered saline, followed by 5\u00a0minutes\u2019 denaturation at 95\u00b0C. This tumor homogenate was then used for in vitro activation and clonal expansion of lymphocytes. The metastasis-draining lymph node\u2013derived lymphocytes were finally dispersed into cell culture flasks, at 2\u00a0\u00d7\u00a0106 cells\/mL of proliferation media, supplemented with 100 IU of IL-2 (Proleukin, Chiron) per milliliter of culture every 3 to 4\u00a0days. Tumor homogenate was diluted to 1:10 and 1:100 and added to the cell cultures after 3 to 4\u00a0days. After 1 to 3\u00a0weeks, the cell cultures were restimulated by irradiated peripheral blood mononuclear cells from the patient. These cells were collected by Ficoll-Hypaque centrifugation19 and used as antigen-presenting cells for restimulation together with further addition of tumor homogenate. To verify clonal expansion of T lymphocytes, V\u03b2 repertoire analyses were performed in a few cases. To test for functionality, single cell suspensions were investigated for Th1 and Th2 cytokine production, IFN-\u03b3, and IL-4.\nAfter an average period of 4\u00a0weeks, expanded cells were counted, evaluated by FACS, and investigated for the exclusion of malignant cells and bacteria before they were considered ready for transfusion. Cells were washed three times in .9% saline solution (Natriumklorid Baxter Viaflo 9\u00a0mg\/mL, Baxter) containing 2% human serum albumin (Baxter) and prepared in a sterile erythrocyte transfusion bag at 4\u00a0\u00d7\u00a0106 cells\/mL.\nThe transfusions took place at the surgical ward as an intravenous transfusion over the course of 1\u00a0hour. Our criterion for adoptive transfer is a transfusion of lymphocytes to a patient, regardless of the number of transferred cells. In our previous studies (M.K., unpublished data) of adoptive transfer originating from sentinel nodes, on average, 71\u00a0million clonally expanded autologous tumor-reactive lymphocytes were transfused to each patient, and in that pilot study, the disease of all 16 patients responded to therapy. We do not know the optimal number of cells to be transfused for the best or maximum immune response, but according to our experience, 39.5\u00a0million transfused cells (the average number of transfused cells in the metinel node study) may be sufficient. The explanation may be that the CD4 cells proliferate and promote division of effector T cells and memory T cells when they are stimulated by their antigen (which is derived from the tumor). The cell expansion continues in the patient.\nRESULTS\nMetinel Nodes\nLymph nodes draining liver metastases, intra-abdominal recurrences, and subcutaneous metastases were identified in all 19 cases, with an average number of 2.6 nodes found per patient (range, 1\u20135) (Table\u00a01). We denoted these metastasis-draining lymph nodes metinel nodes. Seventeen (40%) of the analyzed metinel nodes were positive for metastatic disease. In 4 (25%) of 16 patients where data were available, all metinel nodes were positive for metastatic disease (range, 2\u20135), and in 8 (50%) of 16 patients, all metinel nodes (range, 2\u20133) were negative for metastatic disease.\nTracer was injected around liver metastases (n\u00a0=\u00a09), intra-abdominal recurrences (n\u00a0=\u00a03), or close to subcutaneous metastases (n\u00a0=\u00a07). Draining metinel nodes appeared visually blue (Fig.\u00a01A) within 3 to 10\u00a0minutes after the injection of PB or were found to be disease positive by lymphoscintigraphy (Fig.\u00a01B). No complications due to the lymph node mapping procedure were observed. The method seems valid for detecting the draining metinel node regardless of tumor type because the following were identified by this method: colorectal cancer (n\u00a0=\u00a07), malignant melanoma (n\u00a0=\u00a04), ovarian cancer (n\u00a0=\u00a02), breast cancer (n\u00a0=\u00a02), pancreatic cancer (n\u00a0=\u00a01), squamous cell cancer (n\u00a0=\u00a01), cholangiocarcinoma (n\u00a0=\u00a01), and leiomyosarcoma (n\u00a0=\u00a01). Thus, we conclude that lymph nodes draining liver metastases or local recurrences can readily be identified by this simple, safe, and quick procedure.\nFig.\u00a01.(A) After intraoperative injection of Patent blue dye around liver metastasis in patient 18, the metinel node in the hepatoduodenal ligament is blue. (B) Preoperative percutaneous injection of 4\u00a0\u00d7\u00a015\u00a0MBq Tc-nanokolloid around ovarian cancer groin local recurrence (patient 8) demonstrates a medially and distally situated metinel node after 10\u00a0minutes.\nIn all cases of surgery for liver metastases, lymph nodes draining the area of the metastases were found within the liver hilum or hepatoduodenal ligament. The average number of metinel nodes from liver metastases were 2.4, and 7 (32%) of 22 of the analyzed metinel nodes were positive for metastatic disease. Two patients underwent surgery to treat intra-abdominal local recurrences from colon cancer (patients 1 and 14); resections of the recurrence and reresection of the bowel en bloc were performed. One patient with pancreatic cancer (patient 9) underwent tumor-reducing surgery to treat a local intra-abdominal recurrence after a previous Whipple operation.\nFour patients underwent surgery, with lymph node biopsies performed, to treat subcutaneous metastases from disseminated malignant melanoma (patients 3, 10, 11, and 16), and one patient each underwent surgery to treat subcutaneous metastases from disseminated breast cancer (patient 12), groin lymph node metastasis due to ovarian cancer (patient 8), and subcutaneous metastases related to squamous cellular carcinoma of the tongue (patient 13) (Table\u00a01).\nImmunology\nWe previously demonstrated the presence of tumor-reactive T lymphocytes in primary tumor-draining sentinel nodes from patients with colon19 and urinary bladder cancers.20 Here, we investigated whether signs of immune recognition also occurred in metinel nodes draining metastases. Single cell suspensions were collected from identified metinel nodes and investigated by flow cytometry for the presence of CD4+, CD8+ T lymphocytes, CD19+ B lymphocytes, and CD56+ natural killer (NK) cells (Table\u00a02). Most of the metinel nodes were predominated by CD4+ T helper cells, with an average CD4+\/CD8+ ratio of 2.3 (range, .1\u20136.6). However, five patients (patients 3, 6, 11, 12, and 19) displayed an increase in the fraction of CD8+ cytotoxic T cells. Two of these patients had malignant melanoma. The metinel nodes contained an average of 9.5% (range, .1\u201355) CD19+ B lymphocytes and an average of 2.1% (range, 0\u201318.9) CD56+ NK cells.\nTable\u00a02.Immunology of single cell suspensions collected from metinel nodesPatientTime in culture (d)No. of cells at start (millions)No. of cells at end (millions)Ratio of cells start\/endStart (% gated), ratio CD4\/CD8 and CD19\/CD56End, ratio CD4\/CD8 and CD19\/CD56Ratio CD4\/CD8 startRatio CD4\/CD8 end13110080.824\/11 and 55\/\u221224\/16 and \u2212\/\u22121.81.52334240.9551\/15 and 13\/\u221224 and .12\/\u22123.41.5328\u2013\u2013\u20138\/39 and 1\/\u2212\u2013.2\u201342065.4\u2013\u201337\/9 and \u2212\/\u2212\u20134.1\u2013543130\u20131.347\/7.1 and 28\/1.373\/22 and .09\/.176.63.3665\u2013\u2013.2\/1.6 and .4\/.4\u2013.125\u2013765.7.0216.4\/6.5 and 24.2\/1.6\u20132.5\u201385242.6\u2013\u201315.9\/7.6 and 9\/3.4\u20132.1\u201391512\u2013\u2013\u2013\u2013\u2013\u20131074166.85.0340\/7 and .5\/.3\u20135.7\u201311352091409.312.8\/16.3 and 1.9\/0\u2013.8\u201312312852.0116.4\/31.8 and .1\/0\u2013.5\u201313243\u2013\u2013\u2013\u2013\u2013\u20131432301.05\u2013\u2013\u2013\u2013154836\u2013\u20130\/.1 and 0\/0\u20130\u2013164520\u2013\u201328.25\/6.95 and 1.15\/0\u20134.1\u20131720894.045.8\/2.6 and 4.2\/.234.2\/30.4 and 7.6\/20.12.21.11829674.069.7\/3.8 and 1.3\/.1\u20132.6\u2013194311151.3612.7\/15.8 and 3\/18.985\/3 and .9\/8.4.828.3\nCD4+ cells are crucial for initializing both the cellular and humoral immune response toward, for example, tumors. CD4+ cells recognize antigens expressed on major histocompatibility complex II and have the capacity to catalyze the immune response in B cells, Antigen presenting cells (APCs), and CD8+ cells. CD4+ cells have, compared with CD8+ cells, a long-term capacity of acting both as effector T cells and as memory cells. CD8+ cells are mainly effector cells and will act during a few weeks, but further recruitment of CD+8 cells is maintained by CD4+ cells. The expansion procedure aims to increase the number of CD4+ cells, and the CD4+\/CD8+ ratio can be regarded as a measurement of how well this can be achieved. The average ratio of CD4+\/CD8+ cells at the start of the procedure was 2.6 for patients who underwent successful expansions and 2.1 for patients who underwent unsuccessful expansions. Consequently, the CD4+\/CD8+ ratios were 24% higher for patients with successful expansions.\nTo test for recognition of tumor antigen, the number of metinel node\u2013acquired lymphocytes from nine patients (patients 1, 2, 5, 7, 12, 14, 17, 18, and 19) were increased successfully by the addition of low dose IL-2 and tumor homogenate. An average of 97\u00a0\u00d7\u00a0106 cells (range, 5\u2013285\u00a0\u00d7\u00a0106) were collected, and the cells were expanded on average for 32\u00a0days before transfusion. At the time of transfusion, a mean of 39.5\u00a0\u00d7\u00a0106 tumor-reactive T cells (range, .7\u2013170\u00a0\u00d7\u00a0106) were returned (Table\u00a02). In patient 7, cells were expanded and prepared for transfusion, but the patient suddenly died without receiving any cells. No side effects from transfusion of expanded T lymphocytes were seen, and the patients were discharged from the hospital on the same day of the transfusion. The expansions were successful in five of seven patients who had colorectal primary tumor; one of two in patients with ovarian cancer and breast cancer; and one patient each with cholangiocarcinoma and leiomyosarcoma. The following expansions failed in all patients: malignant melanoma (n\u00a0=\u00a04), pancreatic cancer (n\u00a0=\u00a01), and squamous cellular cancer (n\u00a0=\u00a01). An analysis of the CD4 V\u03b2 repertoire from patient 11 was investigated by flow cytometry of metinel node\u2013acquired lymphocytes (Fig.\u00a02) and revealed an increase and clonal expansion of V\u03b2 families 7.1, 13.2, and 20. In addition, analysis of the V\u03b2 repertoire in two independently cultured metinel nodes derived from one breast cancer metastasis (patient 12) (data not shown) demonstrated clonal expansion after 12\u00a0days\u2019 short-term culture of the same T cell receptor families in both nodes (V\u03b2 4, V\u03b2 9, V\u03b2 20, and V\u03b2 21.3). We conclude that cultures of metinel node\u2013acquired lymphocytes expand in the presence of autologous tumor extract and IL-2.\nFig.\u00a02.In patient 13, the T cell receptor V\u03b2 repertoire was investigated by flow cytometry in two sentinel (nodes at the day of operation and after in vitro cell culture (43\u00a0days). Clonal expansion of V\u03b2 families 7.1, 13.2, and 20 were detected in the CD4+ T cell population.\nCells from patient 8, stimulated at the end of the expansion, demonstrated a high production of IFN-\u03b3 of >1000\u00a0pg\/mL (Fig.\u00a03) and no detectable IL-4 (data not shown), indicating a Th1 response. In an additional patient (patient 5), the stimulated IFN-\u03b3 secretion was 155\u00a0pg\/mL while only a low IL-4 production of 30\u00a0pg\/mL was found, again demonstrating a Th1-predominant response pattern.\nFig.\u00a03.Amount of interferon gamma secreted into the supernatant measured by enzyme-linked immunosorbent assay. Data presented are for patient 7 after 35\u00a0days of in vitro cell culture.\nAccording to more detailed analyses of some cases, we found that some T cells expressed Fox P3, indicating that these cells in fact were T regulatory cells.\nDISCUSSION\nWe show that it is possible to find the first lymph nodes draining various types of metastases by lymphatic mapping by means of dye or radioactive tracers. We have named these metastasis-draining lymph nodes \u201cmetinel nodes\u201d to show both the analogy and difference to the well-known term \u201csentinel node,\u201d which means the first node to receive lymphatic drainage from a primary tumor. In 19 studied cases, we demonstrated three different ways of identifying these nodes by injection of tracers subserosally around liver or intra-abdominal metastases, close to subcutaneous metastases, or into local lymph node metastases.\nThe fact that it is possible to expand the metinel node\u2013derived lymphocytes via in vitro expansions during several weeks of time, together with our analysis that they contain lymphocytes that show clonal expansion toward tumor antigens and produce high levels of IFN-\u03b3 in the expansions, are evidence that they contain tumor-reactive lymphocytes. These cells are mainly T-helper 1 cells that have developed an immunological response toward the metastatic cells. We suggest that these T cells may be used in future trials of adoptive cellular therapy of disseminated solid cancer.\nStudies have demonstrated that tumor growth beyond a certain size required angiogenesis21 and that the amount of neovascularization peritumorally was a predictor of metastatic disease.22 Far less is known about the specific factors regulating the lymphangiogenesis around a tumor and its possible importance, much as a result of the difficulty of discriminating between lymphatics and blood vessels. However, during recent years, the molecular pathways signaling for lymphangiogenesis have been described, and these studies have revealed that lymphangiogenesis is a major component of the metastatic process.\nThe lymphatic capillaries consist of thin-walled, low-pressure vessels that drain the interstitium. When the surrounding interstitial fluid pressure increases, the walls of the lymphatics are stretched and open to passage of cells or fluids. The lymphatic capillaries are joined into major lymphatic collecting vessels and filtered through at least one lymph node before entering the venous circulation.23 After entering the node, approximately half of the lymphatic fluid continues directly into the blood before the rest drains into an efferent lymphatic vessel.24\nA malignant tumor causes a peritumoral increase of lymphatic vessels mediated through VEGF. Studies have shown that high levels of the cytokine cascade of VEGF-C and VEGF-D promote the tumoral lymphangiogenesis and that inhibition of their joint receptor, VEGFR-3, suppresses the effects. Kawakami et al.25 showed that high levels of VEGF-C and its receptor, VEGFR-3, correlated with increased lymph node metastases and lymphatic invasion in human colorectal cancer. White et al.26 concluded that patients operated on for colon cancer had an overexpression of VEGF-D, but not of its receptor VEGFR-3, which correlated with a decrease in disease-free interval and overall survival. A study based on 110 patients with breast cancer showed that lymph vessels were present more often in metastatic axillary lymph nodes (85%) than in nonmetastatic lymph nodes (25%),18 and that intranodal and perinodal lymphatic endothelial cell proliferation fractions were higher in metastatically involved lymph nodes. Taken together, all these findings support lymphangiogenesis in metastases.\nThus, a tumor causes an upregulation of lymphangiogenic factors that leads to an increase in peritumoral lymphatic vessels in both tumors and metastases. It is likely that these newly developed lymph vessels connect to the preexisting lymphatic vessels and lymph nodes. Shed tumor cells may invade these newly formed vessels, and the dissemination of tumor cells is facilitated. The present study shows that this process can be visualized by lymphatic mapping, and the first draining lymph node from the metastasis, the metinel node, may be identified.\nAnimal studies showed that metastases had the capacity to metastasize. Mice inoculated with pulmonary venous blood from mice having pulmonary metastases developed disseminated cancer disease after injection into the tail vein, showing that pulmonary metastases shed viable circulating cells.16 Similarly, Hoover and Ketcham15 demonstrated that mice who had undergone amputation of their primary tumors and now only had pulmonary metastases experienced spread of the tumor cells specifically into the lungs after parabiosis.\nIn surgery for colorectal liver metastases, it is not standard to perform a biopsy of perihepatic lymph nodes. Nakamura et al.27 reported a 4-year survival of 40% in node-positive patients, but the study cohort was small. Most studies identify few 5-year survivors after liver resection for colorectal liver metastases involving positive perihepatic lymph nodes.28 These lymph node metastases are unlikely to be of lymphatic origin from the primary colonic tumor; instead, they may be explained by hematogenous spread to the liver followed by lymphogenic spreading from the liver metastases to the perihepatic lymph nodes.29 Correct perihepatic lymphatic staging is beyond dispute, not only because the increased morbidity in patients having perihepatic lymph node metastases, but also because of the improvements in systemic therapies. In the nine cases of resected liver metastases in our study, all metinel nodes were found among perihepatic nodes within the hepatoduodenal ligament or liver hilum. Most of the liver is drained by deep hepatic lymphatics, the upper inner part of the liver drains to nodes around the end of inferior caval vein, and the lower inner part drains to hepatic nodes through the liver hilum.30 The superficial part of the liver lymphatic drainage is divided into two main directions; the posterior surface drains into pericaval nodes, and the inferior, anterior, and superior surface drains into hepatic nodes through the liver hilum. In all cases of resected liver metastases, the injections of PB were performed subserosally on the inferior, anterior, or superior surface of the liver. Our identified perihepatic metinel nodes were located corresponding to the superficial lymphatic drainage of the liver.\nDespite the prognostic importance of these perihepatic lymph nodes, we have only found one article in the literature describing lymphatic mapping originating from liver metastases. Kane et al.14 demonstrated in a study of 11 patients with colorectal liver metastases that it was possible to perform lymphatic mapping by the intraoperative injection of isosulfan blue dye in a peritumoral and subcapsular manner. The basic premise was that the occurrence of extrahepatic metastases within the normal lymphatic drainage of the liver was considered to be remetastases from the colorectal metastases. They found blue-colored nodes in seven patients (54%), all within the liver hilum, falciform ligament, or gallbladder fossa. In three of these patients, the nodes were not detected by the surgeon before the mapping procedure. Like our results, they concluded that hepatic lymphatic mapping was safe, rapid, and simple technique to perform in humans.\nThe importance of lymphatic mapping in liver surgery for metastases was underlined by Grobmyer et al.,31 who concluded that routine lymph node sampling had a low yield in patients without suspected perihepatic disease by computed tomographic scan or positron emission tomography, or at surgical exploration. Further, another study demonstrated that microscopic perihepatic positive lymph node disease is associated with the same poor prognosis as in macroscopic positive disease and suggests, therefore, that lymph node dissection should be systematic.32\nLymphoscintigraphy has been used to find sentinel nodes from primary tumors,33 but to our knowledge, it has not been reported as a tool to locate lymph nodes draining subcutaneous metastases. The radioactive tracer facilitated the intraoperative identification of the metinel nodes by a gamma probe used as a complement to PB. In those cases, the lymphoscintigraphy was made days before surgery, and new injections of radioactive tracers were provided on the day of surgery. One to three metinel nodes were detected in our six patients.\nIn the late 1950s, the immunosurveillance hypothesis was introduced34 supporting the fact that tumor cells possessed new antigenic potentials and provoked an immunological reaction that could eradicate a tumor completely. In the last decade, the hypothesis has been proven in mice and humans. The role of the immune system in cancer diseases is demonstrated by immunodeficient patients having both an overrepresentation of virus-induced malignancies and an increased probability of developing cancer of nonviral cause. Cardiac transplant recipients have, for example, been shown to have a 25-fold higher prevalence of lung tumors than the general population.35 Much data also supports the fact that both the cellular and the humoral immune systems develop immune responses against tumors. Bevacizumab, a monoclonal antibody against VEGF, functions as an inhibitor of angiogenesis, inhibits growth of human tumor cells in mice, and prolongs the overall median survival in human metastatic colorectal cancer.36 The presence of tumor-reactive lymphocytes in sentinel nodes19,20 is an example of the function of the cellular immune system and a positive prognostic factor for survival in colorectal and ovarian cancer.37\u201339\nIn our earlier study, we made a single cell suspension of the lymphocytes from sentinel nodes. They were expanded in vitro, stimulated by tumor homogenate, IL-2, and antigen-presenting cells. In the present expansions, we proved it was possible to find tumor-reactive lymphocytes in metinel nodes and to perform adoptive immunotherapy based on them. The preliminary results seems promising, with total or partial regression of some liver and lung metastases (M.K., unpublished data). In this study, the expansions were performed in exactly the same way as in our previous studies, except that the source of the lymphocytes was derived from lymph nodes draining metastases\u2014that is, metinel nodes. The fact that we succeeded in nine cases in proliferating the lymphocytes via in vitro expansions over more than a month is evidence for the presence of tumor-reactive lymphocytes. Because the expansions were continuously stimulated with autologous tumor homogenate and antigen-presenting cells together with IL, only lymphocytes that were a priori stimulated by antigens derived from the tumor could proliferate and survive for such a long time. Figure\u00a02 demonstrates the V\u03b2 repertoire in CD4-positive cells in two metinel nodes in patient 11, thus showing which types of T-helper cell receptors were found at the end of expansion. The figure shows clonal expansion of V\u03b2 7.1, 13.2, and 20, demonstrating the presence of tumor-reactive lymphocytes toward three different types of tumor antigens.\nThese collective facts point out the presence of tumor-reactive type 1 T-helper lymphocytes in metinel nodes. T helper lymphocytes play a central role in initiating the adoptive immune defense. In 9 of 19 patients, the cells survived until the time for transfusion, and 8 patients underwent intravenous adoptive autologous T cell transfusion. Approximately half of all patients who underwent the expansion procedure died as a result of a combination of too few cells at the start and the initial presence of immunosuppressive factors that not could be overcome. Six of eight patients who received transfusions are still alive, although the follow-up time varies (Table\u00a01). Our results are encouraging because the prognosis in these type of cases is usually poor.\nIn this study, we have shown that it is possible to find the draining lymph nodes from different types of metastases. We named these nodes \u201cmetinel nodes,\u201d and they contain tumor-reactive lymphocytes, preferentially T-helper cells with Th1 response. We have also demonstrated that it is possible to proliferate these cells in vitro and, if the expansion procedure is successful, to use the cells for adoptive immunotherapy.","keyphrases":["metastases","lymphatic mapping","immunotherapy","sentinel node","solid tumors"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_Arch_Otorhinolaryngol-3-1-2099162","title":"Tonsillar metastasis of oesophageal adenocarcinoma\n","text":"In the literature less than 100 cases of metastatic carcinoma of the palatine tonsil have been reported. Tonsillar metastasis of adenocarcinoma of the oesophagus has not been reported previously. We report a case of a 57-year-old male with a primary adenocarcinoma of the distal esophagus with a metastasis in the right palatine tonsil. Metastatic tumour involving the palatine tonsil is rare. The route of dissemination remains unclear. Hypothetically the dissemination of tumour cells could be lymphogenic or secondary by transportation due to vomiting or at the time of endoscopy, but most likely represents haematogenous spread.\nIntroduction\nThe vast majority of cancers in the head and neck region are of epithelial origin. Lymphoma is the next most common malignancy in this area. Metastases in the head and neck other than lymph node metastases are rare.\nThe occurrence of tonsillar metastases from non-haematological malignant neoplasms is very rare [10]. We present a patient with metastasis in the right tonsil of a primary adenocarcinoma of the distal oesophagus.\nCase report\nA 57-year-old man was referred to our department of otolaryngology and head and neck surgery, because of dysphagia, pain and swelling of the right palatine tonsil. He also complained of frequent vomiting. Five months previously, an adenocarcinoma of the distal oesophagus with lymph node and liver metastases had been diagnosed. He received chemotherapy with combined capecitabine and oxaliplatin, which was stopped after two cycles because of disease progression. On physical examination an enlarged and ulcerating right palatine tonsil was seen (Fig.\u00a01). A suspicious ipsilateral cervical lymph node of 2\u00a0\u00d7\u00a04\u00a0cm was found at level II. A biopsy of the tonsillar tumour was performed and revealed intestinal-type adenocarcinoma. The histopathological picture corresponded with that of the oesophageal tumour (Fig.\u00a02). Tonsillectomy was not performed because of severe clotting disorders attributed to liver metastases. The patient died 4\u00a0weeks after his first visit to our department.\nFig.\u00a01Enlarged right palatine tonsil with ulcerationFig.\u00a02Biopsy right tonsil: adenocarcinoma (intestinal type)\nDiscussion\nSecond primary tumours relatively often involve the head and neck region. These tumours develop in head and neck cancer patients due to their common factors in carcinogenesis, e.g., tobacco, alcohol and, probably, genetic susceptibility [4]. However, this common aetiology applies for squamous cell carcinoma and not for adenocarcinoma. Simultaneous primary adenocarcinomas are not frequently found. In our patient the corresponding histopathology indicates a common origin, suggesting metastatic disease.\nPrimary oesophageal adenocarcinoma is a relatively uncommon tumour in western countries [9]. The pattern of metastasis of oesophageal adenocarcinoma can be lymphogenic, haematogenic and by transportation of tumour cells by regurgitation or endoscopes [2, 10]. Lymphogenic spread of distal oesophageal adenocarcinoma generally involves the regional nodes in the lower posterior mediastinum, the paracardiac region, along the lesser curvature and the left gastric artery [6]. In case of extensive lymphogenic spread, lymph-node metastases can be found in the tracheal bifurcation region, the proximal mediastinum, coeliac axis and supra-clavicular region [6]. Lymphogenic spread of adenocarcinoma of the distal oesophagus and the gastro-oesophageal junction is present in 70% of the patients at the time of diagnosis of the primary tumour [9].\nHaematogenous dissemination occurs at a later stage and is less frequent. Liver and lung are the most common organs involved [6].\nMetastatic tumour involving the palatine tonsil is distinctly rare. These metastases represent only 0.8% of all tonsillar tumours [5]. Bilateral metastases of gastric carcinoma to the tonsils have been described [7]. Less than 100 cases of metastatic carcinoma of the palatine tonsil have been reported [3, 7]. The most frequent tumours metastasizing to the tonsil are melanoma, lung carcinoma, breast carcinoma and renal cell carcinoma [3]. Eleven cases were associated with gastric adenocarcinoma [2, 7, 8]. Tonsillar metastasis of adenocarcinoma of the esophagus has not been reported previously. The occurrence of a tonsillar metastasis as the first manifestation of an occult neoplasm is very unusual [8]. In the majority of cases the occurrence of a tonsillar metastasis becomes manifest after the diagnosis of the primary tumour and is generally part of a widespread systemic disease [2]. Therefore, mean survival time for patients with tonsillar metastases is short, with a mean of 9\u00a0months, irrespective of the histological type of primary tumour [3].\nIn this case there are four hypothetical pathways for metastatic spread to the tonsils: Haematogenous dissemination of tumour from an esophageal carcinoma may occur via the portal circulation, the liver, the pulmonary circulation and systemic arterial vessels, thus reaching the palatine tonsil. A second haematogenous route may be through the paravertebral plexus of Batson, bypassing the lungs [1]. Retrograde flow via this valveless plexus occurs during elevated intra-abdominal and intrathoracic pressure, as is brought about by straining, coughing or as in this case, vomiting [1]. A third route of metastasis may be through the thoracic duct followed by retrograde cervical lymphatic spread to the tonsil [3]. This route of dissemination is very rare and is mostly seen in cases of seminomas [3]. Finally, some authors believe the possibility of metastasis by direct translumenal implantation, which might be favoured by a previous lesion in the mucosa of the tonsil [2]. The tumour cells would be transported secondary to regurgitation from the oesophagus [8], or as in this case possibly because of frequent vomiting, or at the time of endoscopy [2].\nIn conclusion, when finding a tumour of the tonsil a metastasis from a distant primary malignancy should be considered, although this is very rare. The route of dissemination eventually remains unclear.","keyphrases":["tonsil","metastasis","adenocarcinoma","oesophagus","head and neck"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_J_Health_Econ-_-_-1388078","title":"Defining the \u201cHealth Benefit Basket\u201d in nine European countries\n","text":"This article identifies and analyses a framework for \u201chealth baskets,\u201d the taxonomy of benefit catalogues for curative services, and the criteria for the in- or exclusion of benefits in nine EU member states (Denmark, England, France, Germany, Hungary, Italy, The Netherlands, Poland and Spain). Focusing on services of curative care, it is found that the explicitness of benefit catalogues varies largely between the countries. In the absence of explicitly defined benefit catalogues, in- and outpatient remuneration schemes have the character of benefit catalogues. The criteria for the in- or exclusion into benefit catalogues are often not transparent and (cost-)effectiveness is applied only for certain sectors. An EU-wide harmonization of benefit baskets does not seem realistic in the short or medium term as the variation in criteria and the taxonomies of benefit catalogues are large but not insurmountable. There may be scope for a European core basket.\nThe Kohll\/Decker judgments of the European Court of Justice and more recently the Geraets-Smits\/Peerbooms cases, have demonstrated that health services can no longer be regarded as operating in isolation from other EU member states [1]. Increasingly there are flows of patients from one member state to another, sometimes due to individual choice, sometimes induced by sickness funds or even by health ministries. Such movements have the potential to stimulate competition between health care systems of the member states. However, the average volume of imported health care services in the EU has been rather small, with approx. \u20ac1.99 per person as yet (1998) [2]. One major reason for this low volume of cross country flows is the lack of accurate information as the basis for competitive behavior. Actors in one health care system, for example, sickness funds, do not have sufficient information on benefit catalogues and prices for benefits in other health care systems in order to induce in- or exports of health services. So far research has mainly focused on health services for selected indications or on the comprehensiveness of services, but information on the benefit catalogues, their taxonomy, and inclusion criteria in each country is widely lacking [3, 4, 5, 6, 7].\nAs one result of the project \u201cHealth Benefits and Service Costs in Europe\u2013Health BASKET\u201d funded by the European Commission, this article identifies and analyses the framework for health baskets, the taxonomy of benefit catalogues for curative services, and the criteria for in- or exclusion of benefits in nine EU member states (Denmark, England, France, Germany, Hungary, Italy, The Netherlands, Poland and Spain). It focuses on services of curative care, although an in-depth analysis of all other health sectors is also available as part of the project results (Reports of the EU Health BASKET project on health benefit baskets in each of the nine countries are available at: http:\/\/www.ehma.org\/projects\/healthbasket.asp). One of the main hypotheses of the study is that in the absence of explicit benefit catalogues, inpatient, and outpatient remuneration schemes have the character of (less explicit) benefit catalogues. After introducing the terminology and methodology of the analysis, different regulatory frameworks for the definition of the overall benefit basket in each country are compared and analyzed. Finally the taxonomy and the inclusion criteria of benefit catalogues and their substitutes for curative services are compared and analyzed.\nMethodology\nIn general we differentiate between the terms \u201cbenefit basket\u201d (also \u201cbenefit package\u201d) and \u201cbenefit catalogues.\u201d The benefit basket refers to the totality of services, activities, and goods covered by publicly funded statutory\/mandatory insurance schemes (social health insurance, SHI) or by National Health Services (NHS). In contrast, we define benefit catalogues as the document(s) in which the different components of the benefit basket are stated in detail, i.e., which enumerate the services, activities, and goods in a more detailed way, listing single interventions (i.e., specific technologies). Thus a benefit basket may be further defined by one or more benefit catalogues.\nBasically, the coverage of a given population for health services can be characterized in three dimensions: \u201cbreadth\u201d as the extent of covered population, \u201cdepth\u201d as the number and character of covered services, and \u201cheight\u201d as the extent to which costs of the defined services are covered by prepaid financial resources as opposed to cost-sharing requirements. Services or goods which are not covered at all (i.e., with a \u201ccopayment\u201d of 100%) are not considered to be part of the benefit basket. Figure\u00a01 summarizes the three dimensions of a benefit basket. This article mainly focuses on the coverage of services (depth of benefit coverage) [8].Fig.\u00a01 Three dimensions of coverage\nAn open questionnaire was developed to explore the different benefit baskets and their underlying benefit catalogues of the participating nine countries, and this served as guide in compiling standardized country reports. To scan the different health systems in search for existing benefit catalogues we followed the framework of functional categories of \u201chealth care services and goods\u201d proposed by the OECD in its \u201cSystem of Health Accounts\u201d (Table\u00a01) [9]. As an initial step the researchers in each country were asked to identify the decision-making processes on the benefit basket as a whole in their country. As a second step they were asked to provide a detailed description of the existing benefit catalogues for each functional category, the actors involved in decision making, and the decision criteria.Table\u00a01 Framework of health care functional categories, system of health accountsHC.1 Services of curative care\u00a0\u00a0HC.1.1 In-patient curative care\u00a0\u00a0HC.1.2 Day cases of curative care\u00a0\u00a0HC.1.3 Out-patient care\u00a0\u00a0\u00a0\u00a0HC.1.3.1 Basic medical and diagnostic services (primary health care)\u00a0\u00a0\u00a0\u00a0HC.1.3.2 Out-patient dental care\u00a0\u00a0\u00a0\u00a0HC.1.3.3 All other specialized care\u00a0\u00a0\u00a0\u00a0HC.1.3.9 All other out-patient curative care\u00a0\u00a0HC.1.4 Services of curative home careHC.2 Services of rehabilitative care\u00a0\u00a0HC.2.1 In-patient rehabilitative care\u00a0\u00a0HC.2.2 Day cases of rehabilitative care\u00a0\u00a0HC.2.3 Out-patient rehabilitative care\u00a0\u00a0HC.2.4 Services of rehabilitative home careHC.3 Services of long-term nursing care\u00a0\u00a0HC.3.1 In-patient long-term nursing care\u00a0\u00a0HC.3.2 Day cases of long-term nursing care\u00a0\u00a0HC.3.3 Long-term nursing care at homeHC.4 Ancillary services to health care\u00a0\u00a0HC.4.1 Clinical laboratory\u00a0\u00a0HC.4.2 Diagnostic imaging\u00a0\u00a0HC.4.3 Patient transport and emergency rescue\u00a0\u00a0HC.4.9 All other miscellaneous servicesHC.5 Medical goods dispensed to out-patients\u00a0\u00a0HC.5.1 Pharmaceuticals and other medical non-durables\u00a0\u00a0\u00a0\u00a0HC.5.1.1 Prescription medicines\u00a0\u00a0\u00a0\u00a0HC.5.1.2 Over-the-counter medicines\u00a0\u00a0HC.5.2 Therapeutic appliances and other medical durables\u00a0\u00a0\u00a0\u00a0HC.5.2.1 Glasses and vision products\u00a0\u00a0\u00a0\u00a0HC.5.2.2 Orthopedic appliances and other prosthetics\u00a0\u00a0\u00a0\u00a0HC.5.2.3 Hearing aids\u00a0\u00a0\u00a0\u00a0HC.5.2.4 Medico-technical devices\u00a0\u00a0\u00a0\u00a0HC.5.2.9 All other miscellaneous medical durablesHC.6 Prevention and public health services\u00a0\u00a0HC.6.1 Maternal and child health; family planning and counseling\u00a0\u00a0HC.6.2 School health services\u00a0\u00a0HC.6.3 Prevention of communicable diseases\u00a0\u00a0HC.6.4 Prevention of non-communicable diseases\u00a0\u00a0HC.6.5 Occupational health care\u00a0\u00a0HC.6.9 All other miscellaneous public health services\nEstablishing and shaping health baskets\nA generic pattern of establishing and shaping health baskets is found in most of the countries consisting of two levels. At the higher level, legislation passed by the national parliaments (sometimes even rooted in the country\u2019s constitution, e.g., Poland [10]) establishes the general framework by enumerating the areas of health care, often similar to the OECD health care categories, included in the benefit basket. At the lower level, the benefit basket is further shaped by specifying certain procedures provided within each area of health care as part of the benefit catalogues. These catalogues contain recommendations as well as explicit in- or exclusions of services. The extent to and the way in which this shaping actually takes place varies considerably from country to country and within each country from sector to sector of health care. There are several ways used to establish benefit catalogues such as legislations passed by central or regional parliaments, decrees issued by national or regional governments, directives issued by self-governing bodies or by national and\/or local authorities, and other documents considered as \u201cquasilaws\u201d (rules without legal character, e.g., clinical guidelines).\nIn all countries studied a general definition of a benefit basket could be identified at the higher level. Depending on the organization of the system (NHS or SHI), the logic underlying the general definition of the benefit basket differs. In NHS countries the definition of a benefit basket refers to the specification of the duties and obligations of the purchasing organization (regional health authorities), and in SHI countries the issue of the benefit basket is more related to the specification of entitlements of the insured persons. However, it cannot be said that either NHS or SHI countries generally define their benefits more explicitly than the other.\nIn most NHS countries a coherent legislation contains a list of the areas of care to be provided by the respective NHS, including \u201cregional health services\u201d as part of the benefit basket of regions in decentralized NHS systems, for example, Italy and Spain. Denmark represents an exception from this as the legislation consists of separate acts concerning the categories of hospital, primary and long-term care, and pharmaceuticals [11]. The level of explicitness varies considerably from country to country. The vaguest definition of a benefit basket may be that of the NHS Foundation Act (1946) in England and related posterior documents, where the Secretary of State for Health is legally required to provide services \u201cto such extent as he considers necessary to meet all reasonable requirements\u201d [12]. The responsibility for making available general practitioner, dental, ophthalmic, and pharmaceutical services lay until 2003 with health authorities and since then with Primary Care Trusts. In contrast, the framework of the Italian and Spanish benefit baskets, each of which was established in recently implemented legal documents, is structured in more detail [13, 14]. Common to all four, however, is the differentiation between hospital care and primary care, including specialist outpatient services, as well as preventive or health promotion services.\nThe level of explicitness is not only heterogeneous across different countries, but also within the same country. For instance, in Spain some areas of health care may be further shaped by mentioning specific services or in some cases even specific procedures or technologies being in- or excluded from the benefit basket. Thus the definition of the benefit basket within NHS countries does not always follow a systematic approach of going into further detail. It rather addresses shortcomings perceived by decision makers of each health care system, not necessarily being based on evidence. Concerning the origin of more detailed benefit catalogues some similarities are observed across countries with similar organization of the health care system or with similar organizational demands (i.e., the need to reimburse providers in free practice). In the two countries with regionalized NHS, Italy and Spain, the motivation for defining a detailed benefit catalogue is to be seen in the autonomous status of regions. The national benefit basket can be regarded as a minimum basket of health services that must be provided by the regional health authorities. Therefore the regions in both countries are free to offer additional services not included in the national benefit basket.\nHealth baskets in SHI countries stem from two different roots. On the one hand, SHI countries formulate the basket as an entitlement for persons insured under the respective statutory health insurance schemes. The main reason for this lies in the fact that SHI schemes have evolved from fragmented voluntary\/statutory health insurance schemes not covering the whole population and only covering certain services, for example, sickness benefits. Statutory health insurance in Germany as well as in The Netherlands does still not cover the whole population [15, 16]. Therefore in SHI countries the health basket is also used to indicate the boundaries between those insured under the statutory health insurance and those insured under other schemes. The second root are fee catalogues which were originally more prevalent in SHI than in NHS countries due to the frequently used fee-for-service reimbursement system, at least in ambulatory specialist care.\nAnother common characteristic of decision making on health baskets in most SHI countries is the role of the self-governing institutions. Within a general framework stipulated by laws, self-governing institutions (e.g., sickness funds, physician associations) specify the rules to explicit benefit catalogues or remuneration schemes with the character of benefit catalogues, limiting the scope of service provision.\nAs observed in NHS countries, the level of explicitness also varies considerably among SHI countries. Poland has by far the most explicit benefit basket, which is even rooted in the constitution. Different legal acts define benefit catalogues specifying detailed procedures or even technologies being provided. At the other extreme, Germany probably has the vaguest legal definition of the benefit basket among the SHI countries, with the Social Code Book as a general framework. For instance, inpatient services in Germany are limited to only a certain extent by the diagnosis-related group (DRG) reimbursement system because they can be provided unless they are explicitly excluded by directives [17]. There is a trend in all SHI countries towards more explicit benefit baskets. In The Netherlands a system of diagnosis-procedure combinations has been introduced in 2005 defining procedures provided in inpatient care and by specialists in outpatient care [18]. In France a similar list is currently being prepared, while Germany and Hungary recently updated their outpatient remuneration schemes specifying certain procedures that are reimbursed by the statutory health insurance [17, 19].\nDefinition of benefit catalogues for curative services\nBenefit catalogues for inpatient care\nAs in other sectors of the health care system, the provided inpatient services can either be listed as procedures being part of an explicit benefit catalogue or indirectly determined by grouping systems that serve remuneration purposes, for example, DRGs. France, Poland, and Spain have defined explicit benefit catalogues, grouped according to medical specialties, for inpatient services listing detailed procedures serving as positive lists [10, 14, 20]. While there are clear decision criteria for the inclusion of benefits in the benefit catalogue in France and Spain, no transparent criteria are applied in Poland. In all other countries DRG and other grouping systems serve as a tool for estimating resource consumption supporting budget assignations or providing the basis for remuneration. In general, they classify a single episode of care according to main diagnoses, comorbidities and main surgical interventions into one of a limited number of groups. Thus each classified episode is assumed to require more or less homogeneous resource consumption, independently of whether exactly the same items (e.g., drugs, diagnostics) are used. As, finally, monetary values are attached to the different groups, the use of drugs, diagnostics etc. is confined to the given monetary scope of each group.\nIn our study four countries have introduced DRG systems: Italy [13], Germany [17], Hungary [19], and Denmark [11]. England and The Netherlands have DRG-like grouping systems called, respectively, health care resource groups and Diagnose Behandeling Combinaties. The main features of the DRG and other grouping systems are very similar (Table\u00a02) [12, 18]. In each country the groups have been developed from data collected on resource consumption, clustering in homogeneous resource consumption groups. The observed variability in the number of classified groups in each system may be explained by the different criteria used to classify them (Table\u00a02). Another possible explanation is the creation of additional groups in certain countries to increase the scope for the use of new innovative technologies (devices, procedures, and even drugs theoretically) which are considered to be worth the promotion (i.e., because of higher efficacy). This has been the case in Italy, where the regional health authority of Lombardy added three new DRGs to its system in order to specifically consider the use of drug-eluting stents and to encourage its utilisation [13].Table\u00a02 Inpatient benefit catalogues or substitutesCountryName of taxonomy, year of introductionApplied geographical areaTaxonomy (and grouping criteria)Actors involved in decision makingCriteria for in-\/ exclusion of benefitsDenmarkDRG system (Nordic-DRG); 1998National25 MDC (anatomical, etiological, other) with 589 DRG, special category for chemotherapy and radiotherapy. Grouping criteria: main diagnosis, procedures, sex, age, cause of dischargeLegislation at the national level (law, general framework)Need, budgetMinistry for the Interior and Health (approval)National Board of Health (DRG catalogue)County level (budgeting, hospital plan)Clinicians (priority setting in hospital)FranceCommon Classification of Medical Procedures (CCAM); 2005NationalCCAM lists reimbursable and excluded medical procedures thus being a positive and negative list; Grouping criteria: anatomic classification, medical specialtiesNational level (law, general framework)Effectiveness, safetyMinistry of Health (approval)National Union of Health Insurance Funds (in- and exclusion of services)High Health Authority (advisory body on in- and exclusion of services)GermanyG-DRG system (based on AR-DRG 4.1); stepwise 2003\u20132009National25 MDC (anatomical\/ etiological\/ other) with 876 DRG, 71 extra remunerations for special services (in 2005). Grouping criteria: main diagnosis, procedures, age, comorbidity, cause of dischargeLegislation at the national level (law, general framework)Services can be provided as long as they are not explicitly excluded. Adequate, expedient and cost-effectiveMinistry of Health (approval)Federal Joint Committee (exclusion of benefits)Institute for Hospital Reimbursement with assistance of the Committee of on HospitalPayment (DRG catalogue)Clinicians (priority setting in hospital)HungaryDRG system (US-DRG); 1993National26 MDC (anatomical, etiological, other) with 786 DRG. Grouping criteria: main diagnosis, procedures, age, comorbidityLegislation at the national level (law, general framework, budgeting)Costs, effectivenessMinistry of Welfare, Health division (DRG catalogue)National Health Insurance FundAdministration, especially (prepares decisions)Clinicians (priority setting in hospitals)ItalyDRG system (HCFA no. 10); 1995National reference list with regional differences23 MDC (anatomical, etiological, other) with 489 to 506 DRG, exceptional DRGs (e.g. liver and bone marrow transplantation). Grouping criteria: main diagnosis, procedures, age, comorbidity, cause of dischargeLegislation at the national level (law, general framework)Effectiveness, costsCentral level (national DRG catalogue)Regional level (redefines DRG catalogue, sets tariffs)Clinicians (priority setting in hospitals)The NetherlandsDiagnose Behandeling Combinaties (DBC) (DRG-like system); Jan. 2005National111,527 procedures regarding diagnosis and therapy (DBCs) are combined to 641 product groups. DBCs are on three different lists determining the status for tariff negotiations or excluding DBCs from the benefit package. Grouping criteria: medical specialty, product groupLegislation at the national level (law, general framework)Costs, effectiveness, Ministry of Health (decrees)DBC Maintenance Organization (DBC system)Clinicians (priority setting in hospitals)PolandGovernmental decrees and catalogue of benefitsNationalCatalogue lists all services covered under social health insurance scheme; services are linked to the respective regulation\/law. Grouping criteria: area of care (e.g., hospital care), medical specialtyLegislation at the national level (law, general framework)\/Ministry of Health (regulations decrees)National Health Fund (catalogue)SpainRoyal Decree 63\/1995 \/ Law 16\/2003 on cohesion and qualtiy of the National Health SystemNational with regional differencesServices are listed explicitly in decree. In some cases services are restricted to specific patient groups. Grouping criteria: area of care, medical specialtyLegislation at the national level (law, general framework)Safety, efficacy, efficiencyFederal Government (decree)Inter-territorial Council and Council of the State (inclusion of new benefits)Clinicians (provision of services relating to entitlements defined by decree)EnglandHealth care resource group (DRG-like system); stepwise 2004\u20132009NationalIn April 2004 there were only 48 HRGs in use. Grouping criteria: diagnosis, complexity, procedureLegislation at the national level (law, general framework)Costs, budgetMinistry of Health (catalogue)Primary Care Trusts (negotiate with providers on quantity and tariffs)\nThe fact that specific procedures and technologies drive the development of DRG systems confirms our hypothesis that they serve as some kind of benefit catalogues. Technologies specifically mentioned in certain groups may not be used if less resource consuming alternatives are available. In this way a less specific DRG system may act as a hidden negative list of technologies which de facto are not available for beneficiaries of publicly financed care since the monetary value assigned to certain groups does not cover the actual resource consumption associated with its use. Furthermore, in most applied grouping systems certain groups (e.g., in DRG systems so-called surgical DRGs) are even defined by specific procedures or specific technologies, such as drug-eluting stents. These groups can therefore be considered as a kind of explicitly defined benefits, similar to a positive list. Thus it can be assumed that grouping systems are applied as substitutes for benefit catalogues, containing incentives to limit the provision of benefits for those services not being explicitly mentioned.\nBenefit catalogues for outpatient care\nIn the outpatient sector benefit catalogues are, again, often replaced by grouping systems serving remuneration purposes. Although they generally seem to be more explicit than the inpatient catalogues, the explicitness varies even more than in the inpatient sector (Table\u00a03). These different degrees of explicitness are due mainly to the applied remuneration schemes in each country. If physicians receive fixed budgets or capitations, the benefit \u201ccatalogue\u201d (i.e., the procedures that they can offer) is indirectly restricted by the amount of money allocated to them. Therefore in these countries the benefit package for outpatient care is regulated rather implicitly through decrees issued by national or regional health authorities describing the obligation of physicians to provide those benefits that are considered necessary. Examples of these kind of implicit benefit catalogues are the \u201cHealth Insurance Treatment and Services Decree\u201d for care provided by general practitioners in The Netherlands and the \u201cGeneral Medical Services Contract\u201d in England. These decrees do not mention specific procedures, although in the case of The Netherlands, the general practitioners\u2019 association, the Landelijke Huisartsen Vereniging, defined a basic general practitioner benefit package in the 1980s [21].Table\u00a03 Outpatient benefit catalogues or substitutesCountryName of taxonomyApplied geographical areaTaxonomy (and grouping criteria)Actors involved in decision makingCriteria for in-\/exclusion of benefitsBenefits, procedures explicitly excludedDenmarkHealth Care Reimbursement Scheme Fee ScheduleNationalServices are grouped according to medical specialty and for GPs additionally in basic, supplementary, laboratory and miscellaneous services. Each service has an item number. It is referred to the respective legislation decree specifying the benefit, certain goods, procedures or in rare cases indicationsNational level (law, general framework)NeedAlternative careMinistry for the Interior and Health (approval)Counties (budgeting, health plan)Health care Reimbursement Negotiating Committee and health professional associations (negotiate catalogue)FranceCommon Classification of Medical Procedures (CCAM)NationalLists all medical procedures reimbursable and excluded. Grouping criteria: anatomic classification, medical specialtiesNational level (law, general framework)Effectiveness, safetySpa treatments; cosmetic surgeryMinistry of Health (approval)National Union of Health Insurance Funds (in- and exclusion of services)High Health Authority (advisory body on in- and exclusion of services)GermanySHI-EBM, SHI-BEMA, SHI-BEL-IINationalServices are grouped according to the medical specialty allowed to provide the service. Each service is assigned a numeric code in accordance with the subjection of the catalogueNational level (law, general framework)Diagnostic and therapeutic expedience, medical necessity and cost-effectivenessOrthopedic services after the age of 18 yearsFederal Joint Committee (approval of new benefits)Valuation Committee (negotiates EBM)Dental Valuation Committee (negotiates BEMA, BEL-II)HungaryGovernmental decrees and reimbursement cataloguesNationalSimilar services are listed in groups. Governmental decrees relate to different areas of care (e.g., dental care, specialist services). Items in reimbursement catalogues are listed with the respective ICPM code and a point valueLegislation at the national level (law, general framework, budgeting)Costs, effectiveness\u2013Ministry of Welfare (decrees, approval)National Health Insurance FundAdministration, especially (prepares decisions)Payment Codes Updating Committee (reimbursement catalogues)ItalyNational contract for primary care; decree on specialist outpatient servicesNational benefit package, regions include additional servicesContract for primary care describes obligations of GP. Individual services are not further itemized. Decree on specialist outpatient services lists services in three sections: available, availability restricted to specific indications, excludedGovernment at national level (sets decree, negotiates contract)Effectiveness, costsNonconventional treatments (e.g., acupuncture, phytotherapy); vaccination for traveling purposesRepresentatives of GPs (negotiate contract)Ministry of Health (transfers contract into law)Government at regional level (negotiates additional contracts)The NetherlandsHealth Insurance (Treatment and Services) Decree; Diagnose Behandeling Combinaties (DBC; DRG-like system); Jan. 2005NationalGP services are regulated in generic terms only by decree, DBC catalogue (111,527 DBCs) combine information on diagnosis and treatment for medical specialists. DBCs are on three different lists determining the status for tariff negotiations or excluding DBCs from the benefit package. Grouping criteria: medical specialty, product groupLegislation at the national level (law, general framework)Costs, effectiveness\u2013Ministry of Health (decrees)DBC-Maintenance Organization (DBC-System)Physicians (priority setting)PolandGovernmental decrees and catalogue of benefitsNationalCatalogue lists all services covered under social health insurance scheme. Services are linked to the respective regulation\/law. Grouping criteria: area of care, medical specialtyLegislation at the national level (law, general framework)\u2013Vaccination; acupuncture, unless part of chronic pain managementMinistry of Health (regulations)National Health Fund (catalogue)SpainRoyal Decree 63\/1995National with regional differencesServices are listed explicitly in decree. In some cases, services are restricted to specific patient groups. Decree lists services in 5 areas of care (e.g., primary care, specialized care, pharmaceutical care) which are further subdivided.Legislation at the national level (law, general framework)Safety, efficacy, efficiencyCosmetic surgery (transplantation of hair and nails); sex changeFederal Government (decree)Inter-territorial Council and Council of the State (inclusion of new benefits)Clinicians (provision of services relating to entitlements defined by decree)EnglandNational Service Framework NationalHealth Resource Groups are linked to procedures. Currently only 48 HRGs are in use. Guidelines recommend services to be used on certain indicationsLegislator at national level (law, general framework)Need, effectivenessCosmetic dental treatmentsGeneral Medical Services ContractNational, with possible variation at PCT-levelNHS Confederation and General Practitioners Committee (negotiate contract)Need, costsClinical GuidelinesNationalPrimary Care Trusts (PCT) (negotiate additional contracts)Need, costs, effectivenessNICE (clinical guidelines)\nIn contrast, the countries remunerating providers on the basis of fee-for-service schemes need detailed lists of procedures or at least of service complexes (aggregated multiple procedures) to be able to negotiate on price and\/or volumes. These lists can therefore be interpreted as substitutes for benefit catalogues, as physicians are usually reimbursed only for those items listed. The explicitness of these lists differs largely. Some countries issue detailed lists of all procedures to be performed by physicians (e.g. the \u201cCommon Classification of Medical Procedures\u201d in France [20]) while other countries list service complexes making physicians responsible for the priority setting within such a service complex (e.g., SHI-EBM or SHI-BEMA in Germany [17] and the Health Care Reimbursement Scheme Fee Schedule in Denmark [11]).\nInterestingly, taxonomy and structure are very similar in all countries. For example, in Denmark, France, Germany, Hungary, and The Netherlands services are grouped according to medical specialty. Certain outpatient benefits are also linked to indications or special patient groups in Poland and Spain [10, 14]. The high degree of explicitness regarding the definition of the benefit package is also underlined by the diverse lists of excluded services, common in all countries. Exclusion practices vary from issuing negative lists (e.g., Spain, Poland) and directives of self-governmental institutions with the character of negative lists (e.g., Germany) to excluded services within the framework of the national law (e.g., England). However, most countries exclude similar benefits such as cosmetic surgery (if not closely linked to certain treatments), vaccination for nonstandard diseases (e.g., for traveling purposes), and certain nonconventional treatments (e.g., acupuncture). Benefits of dental care are either restricted to specific treatment methods or age groups (e.g., Germany).\nDiscussion\nThe analysis of benefits defined in the countries under study reveals that there is a clear trend towards a more explicit definition of benefit baskets and their benefit catalogues. Those countries which recently introduced new health care legislations, such as Italy, Poland, and Spain, have more explicitly defined benefit catalogues. Other countries with older health care legislations, for example, the UK\u2019s English-NHS Foundation Act (1946) and Germany\u2019s Social Code Book (1988) have rather implicitly defined benefit catalogues, but increasingly work with negative lists, based on evidence provided by independent institutions such as the Englisch NICE and the German Institute for Quality and Efficiency (IQWiG) [12, 17]. Apart from negative lists remuneration schemes, for example, DRGs and procedures catalogues used for grouping are more and more used as benefit catalogues. These developments indicate that all included countries move towards a more explicit definition of benefit catalogues. Explicitly defined benefit catalogues, however, require clear and transparent decision criteria for the in- or exclusion of benefits.\nMost countries officially state that (cost)-effectiveness is an important decision criteria. However, further inquiries often demonstrate that there is no rational process of reviewing the available evidence on specific procedures or technologies [22]. In reality the decision-making process is rather guided by lobbying activities of certain actors in the system. Especially those countries with very explicit benefit baskets, e.g. Poland, often lack transparency of decision criteria [10]. In contrast to this, countries with rather implicitly defined benefit baskets, such as England and Germany, define very transparent criteria for benefit exclusion, although lists with excluded services are minor compared to explicitly oriented countries. In addition, criteria such as cost-effectiveness and even effectiveness are often restricted to one or few sectors of the health care system, for example, pharmaceuticals or medical devices, and are not generalizable to all products or services [23]. In general the transparency of decision criteria must be improved in all countries in order to achieve accountability for all actors of the health care systems as well as consumers.\nThis contribution, as well as the overall EU Health BASKET project, provides useful information for health care providers and industrial companies willing to invest in EU countries but do not have the necessary information on benefit baskets and their underlying decision-making processes. However, to improve the environment for investments and to provide confidence for foreign investors, public documents should be regularly prepared by each country giving a transparent overview of the health baskets and the decision-making criteria.\nThe information provided will be beneficial especially to decision makers at all levels of health policy enabling them to compare different approaches of benefit definitions in order to develop their own position. The need for benchmarking will grow in line with the further development of cross boarder flows and the establishment of coherent benchmark criteria as part of the \u201cOpen Method of Coordination\u201d initiated by EU policy makers [24, 25]. However, the project results also demonstrate that a harmonization of health baskets of EU countries, which in the view of certain decision makers could be the final stage after identifying best practice in benchmarking, is not realistic in the short or medium term since the definitions of benefit baskets vary substantially. Additionally, as shown in the cases of Italy and Spain, especially in NHS countries there is rather a trend towards more decentralization of decision making on benefits, delegating to regions the autonomy to offer certain benefits in addition to nationally defined health baskets [26, 27]. On the other hand, this could also mean that in future a minimum basket of health benefits may be defined by all countries on the national level, which could be harmonized on the EU level at a certain stage due to systems\u2019 competition as a result of increased cross-border flows. Beyond this minimum basket, there could be regional variations reflecting differences in wealth and of preferences.","keyphrases":["health services","health benefit plans","health priorities","national health programs","insurance benefits"],"prmu":["P","R","R","M","R"]}
{"id":"J_Fluoresc-3-1-1915606","title":"Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red\n","text":"The fluorescent dye Nile red was used as a probe for the sensitive detection of large, denatured aggregates of the model protein \u03b2-galactosidase (E. coli) in solution. Aggregates were formed by irreversible heat denaturation of \u03b2-galactosidase below and above the protein\u2019s unfolding temperature of 57.4\u00b0C, and the presence of aggregates in heated solutions was confirmed by static light scattering. Interaction of Nile red with \u03b2-galactosidase aggregates led to a shift of the emission maximum (\u03bbmax) from 660 to 611 nm, and to an increase of fluorescence intensity. Time-resolved fluorescence and fluorescence correlation spectroscopy (FCS) measurements showed that Nile red detected large aggregates with hydrodynamic radii around 130 nm. By steady-state fluorescence measurements, it was possible to detect 1 nM of denatured and aggregated \u03b2-galactosidase in solution. The comparison with size exclusion chromatography (SEC) showed that native \u03b2-galactosidase and small aggregates thereof had no substantial effect on the fluorescence of Nile red. Large aggregates were not detected by SEC, because they were excluded from the column. The results with \u03b2-galactosidase demonstrate the potential of Nile red for developing complementary analytical methods that overcome the size limitations of SEC, and can detect the formation of large protein aggregates at early stages.\nIntroduction\nAmong the biological macromolecules, proteins have become an important source of active substances for medical purposes. Insulin and growth hormone are two prominent examples of the many proteins used in medicine nowadays [1]. Many protein formulations can cause immune reactions in patients, and the presence of protein aggregates, even in minute amounts, has been identified as an important factor leading to the breaking of immune tolerance [2, 3]. Large protein aggregates are regarded as particularly immunogenic [4], and for this reason, analytical procedures for their sensitive and selective detection are important. The most widely used technique for analyzing aggregates in protein formulations is size exclusion chromatography (SEC) [2]. However, large protein aggregates of more than about 5 million Da are out of the separation range of SEC and can even be excluded from separation columns. Moreover, protein-column interactions may cause aberrant elution leading to erroneous interpretations. Field flow fractionation and analytical ultracentrifugation have a greater capacity than SEC for detecting high molecular weight protein aggregates [5, 6], but until now, these techniques are not readily available in every laboratory. Analysis by light scattering techniques [7] and Fourier transform infrared spectroscopy (FTIR) [8\u201310] is experimentally easy in the sense that these techniques do not rely on physical separation steps. However, the former is prone to artifacts when aggregates are present at low concentrations, and the latter is not very sensitive. Fluorescence spectroscopy with probes covalently labeled to proteins has been used for the direct detection of aggregated species [11], but this approach suffers from the problem that the modified protein may exhibit an altered aggregation behavior. Methods without protein labeling are rarer, but environment sensitive fluorescent dyes can be useful for detecting aggregated structures. For example, Congo red is used for the detection and identification of amyloid fibrils [12, 13]. Another potentially interesting dye is Nile red, which is established for the detection of hydrophobic areas on the surfaces of proteins [14\u201317]. Recently, this dye has been used for the detection of aggregates of an IgG1 recombinant humanized monoclonal antibody, as well as human calcitonin fibrils by fluorescence microscopy [18].\nThe fluorescence of Nile red is strongly dependent on the polarity of its environment. Decreasing polarity leads to a substantial blue shift of the absorption and emission maxima, as well as a marked increase of quantum yield and fluorescence lifetime. This has been related to the large dipole moment of the molecules in the excited state and the existence of a non-emissive twisted intramolecular charge transfer (TICT) state, which is more likely to be formed in polar than apolar environments [19, 20]. It has been proposed that the TICT state is non-emissive because of fast, non-radiative transition to the triplet state [19]. Nile red is a hydrophobic molecule with low solubility in water of \u223c1\u00a0\u03bcM [21]. Therefore, it has the tendency to partition into hydrophobic phases [22].\n\u03b2-Galactosidase from Escherichia coli is used as a model protein in the present study. This tetrameric enzyme (MW\u00a0=\u00a0465\u00a0kDa) consists of four equal subunits. It contains four catalytic sites for the hydrolysis of lactose or other \u03b2-galactosides to monosaccharides. For maximal enzymatic activity, the presence of Mg2+ or, alternatively, Mn2+ is required [23, 24]. It has been shown that Mg2+ increases the unfolding temperature of \u03b2-galactosidase in aqueous solution. In the presence of Mg2+, heat induced unfolding is irreversible. It coincides with activity loss and leads to the formation of denatured monomers, dimers, and aggregates [25].\nIn this study, steady-state and time-resolved fluorescence spectroscopy experiments with Nile red are used for the sensitive detection of large \u03b2-galactosidase aggregates, present in solution at concentrations in the low nanomolar range. Denaturation and aggregation of native protein is induced by incubating aqueous solutions below and above the experimentally determined unfolding temperature of 57.4\u00b0C. The presence of aggregates in heat-treated solutions is confirmed by light scattering. Time-resolved fluorescence anisotropy and fluorescence correlation spectroscopy (FCS) are used for analyzing the size of the aggregates detected by Nile red. The results obtained with Nile red are compared with results from SEC analysis.\nMaterials and methods\nMaterials\nFreeze-dried \u03b2-Galactosidase from Escherichia coli (protein content \u223c80%) was purchased from Fluka (Buchs, Switzerland) and stored at \u221220\u00b0C. Phosphate buffer (pH 7.4) containing 55\u00a0mM sodium-phosphate and 10\u00a0mM MgCl2 was prepared with reverse osmosis water, and rendered isotonic by the addition of NaCl. Upon preparation, buffer solution was filtered through a 0.2\u00a0\u03bcm cellulose acetate filter (Schleicher and Schuell, Dassel, Germany), and stored at 4\u00b0C for at most 4 weeks. 0.2\u00a0\u03bcm HPLC filters were obtained from Alltech (Deerfield, IL). Nile red (9-diethylamino-5H-benzo[\u03b1]phenoxazine-5-one) was obtained from Fluka. A stock solution containing 0.25\u00a0mM Nile red was prepared with water-free dimethylsulfoxide (DMSO) and stored at \u221220\u00b0C.\nMethods\nPreparation of \u03b2-galactosidase solution\nA fresh solution of \u03b2-galactosidase in phosphate buffer was prepared before each experiment. The protein was dissolved in the buffer and subsequently, the solution was filtered through a 0.2\u00a0\u03bcm HPLC filter. Protein concentration after filtration was determined by measuring absorption at 280\u00a0nm, assuming an absorption coefficient of 2.09\u00a0cm2\/mg [26]. Reproducibly, a protein concentration close to 0.10\u00a0\u03bcM was found after filtration.\nFor incubation at elevated temperatures, 1\u00a0ml aliquots of \u03b2-galactosidase solution containing 0.10\u00a0\u03bcM protein were filled into 1.5\u00a0ml polypropylene Eppendorf tubes. They were heated for a defined period of time without shaking using an Eppendorf Thermomixer (Eppendorf, Hamburg, Germany), and then cooled at room temperature.\nSteady-state fluorescence measurements\nInstrument and measurement settings\nSteady-state fluorescence measurements were performed with a Fluorolog FL3-21 spectrofluorometer (Jobin Yvon \u2013 Horiba, Edison, NJ), equipped with a short-arc xenon lamp. The slit openings of the excitation and emission monochromators were set to a bandwidth of 3\u00a0nm. The integration time was 0.05\u00a0s, and the signals were corrected for lamp intensity fluctuations by a simultaneously recorded reference signal. Each sample was measured 3\u20135 times, and the average spectrum was calculated. The sample temperature was controlled by a water bath with a temperature sensor connected to the sample holder. Samples were measured in quartz cuvettes (Hellma GmbH, Muellheim, Germany).\nIntrinsic tryptophan fluorescence\nMeasurements of intrinsic tryptophan fluorescence of \u03b2-galactosidase were performed by exciting 0.10\u00a0\u03bcM protein solutions at 298\u00a0nm and scanning emission between 310 and 450\u00a0nm. To establish a protein denaturation curve, the sample temperature was increased from 25 to 70\u00b0C in one degree steps. At every temperature, the sample was equilibrated for 5\u00a0min and then measured.\nNile red fluorescence\nBefore every measurement, a fresh solution of 25\u00a0\u03bcM dye in phosphate buffer was prepared by dilution of Nile red stock solution. This solution was immediately added to heated 0.10\u00a0\u03bcM \u03b2-galactosidase solution, non-heated 0.10\u00a0\u03bcM \u03b2-galactosidase solution or phosphate buffer in a cuvette, and mixed well. The final concentration of Nile red was 18.75\u00a0nM, well below the solubility limit of Nile red in water of 1\u00a0\u03bcM [21]. Nile red fluorescence was measured by exciting at 550\u00a0nm and scanning emission between 565 and 750\u00a0nm. All measurements were performed at 25\u00b0C. Fluorescence spectra were measured immediately after addition of Nile red. However, Nile red emission in water remained unchanged for at least 25\u00a0min upon sample preparation.\nTime-resolved fluorescence measurements\nInstrument and measurement settings\nTime-resolved fluorescence measurements were performed by time-correlated single photon counting, as described in detail elsewhere [27]. Excitation source was a mode-locked titanium:sapphire laser (model Verdi V10, Coherent Inc., Santa Clara, CA), pumped by a CW diode-pumped, frequency doubled Nd:YVO4 laser (model Mira 900-D in fs mode, Coherent Inc., Santa Clara, CA), and tuned at 980\u00a0nm. An excitation wavelength of 490\u00a0nm was obtained by frequency doubling, using a second harmonic generation system (model 5-050 Ultrafast Harmonic Generation System, Inrad Inc., Northvale, NJ). The repetition rate of the excitation pulses was 3.8\u00a0MHz. The emission filters were a Schott 3\u00a0mm OG 530\u00a0nm cut-off filter (Schott AG, Mainz, Germany), combined with a Schott IL 611.6 nm (\u0394\u03bb=11.0\u00a0nm) interference filter.\nMeasurements were performed at 25\u00b0C, and consisted of repeated 10\u00a0s sequences of measuring parallel and perpendicularly polarized fluorescence emission until a maximum peak content of at least 50,000 counts in the data files was reached. Samples of \u03b2-galactosidase solution without Nile red were measured for background correction. To minimize background luminescence, all solutions, including buffer, were prepared with fluorescence spectroscopy grade water (Fluka, Buchs, Switzerland). For the performance of a deconvolution procedure in data analysis, the dynamic instrumental response of the experimental setup was recorded using the fast and single-exponential fluorescence decay of the reference compound erythrosine B in water.\nData analysis was performed using the \u201cTRFA Data Processing Package\u201d of the Scientific Software Technologies Center (Belarusian State University, Minsk, Belarus) [28]. The decay of fluorescence intensity with time, I(t), could be described by an exponential model with three independent fluorescence lifetimes (\u03c4i) [29, 30]:\nwhere \u03b1i are the pre-exponential factors, which were normalized. I(t) itself was calculated from the measured parallel and perpendicular polarized fluorescence emission, III(t) and I\u22a5(t):\nG in the above equation represents the g-factor, which had a value of 1 for the used experimental setup.\nThe course of fluorescence anisotropy with time r(t) was calculated from the measured intensities III(t) and I\u22a5(t):\nFluorescence correlation spectroscopy measurements\nInstrument and measurement settings\nFluorescence correlation spectroscopy measurements were performed with a system composed of a krypton-argon laser and an MRC1024 confocal laser-scanning microscope (Biorad, Hercules, CA), a TE300D inverted microscope (Nikon, Tokyo, Japan) with a water immersion objective lens (Plan Apo 60\u00d7, NA 1.2, collar rim correction, Nikon), and ALV-5000\/E avalanche photodiode detectors (ALV GmbH, Langen, Germany). The 568 nm line of the krypton-argon laser was used for excitation of Nile red samples. Fluorescence emission above 585 nm was detected.\nSamples were prepared by mixing heated 0.10\u00a0\u03bcM \u03b2-galactosidase solution, non-heated 0.10\u00a0\u03bcM \u03b2-galactosidase solution or phosphate buffer with diluted Nile red stock solution in polypropylene Eppendorf tubes. Heated \u03b2-galactosidase solution was mixed with Nile red in the same tubes that were used for heating. The final concentration of Nile red in the samples was 18.75\u00a0nM. One hundred\u00a0\u03bcl sample was transferred into a well of a 96 glass bottom well plate (Bio-one, Greiner, Frickenhausen, Germany). For measurements, the focal volume was positioned 100\u00a0\u03bcm above the bottom of the well. Raw data, i.e. fluctuations of fluorescence intensity with time, were collected during 30\u00a0s. The laser power was minimized to prevent photobleaching during this period. Every sample was measured ten times, and ten experimental autocorrelation functions were subsequently obtained from the raw data.\nAnalysis of the experimental autocorrelation functions was performed by non-linear regression, based on a theoretical expression that describes the decay of an autocorrelation function, G(\u03c4), in consequence of diffusion and fast, non-diffusive processes, such as excitation of fluorescent molecules to the triplet state [31\u201333]:\nHere, Ei is the relative emission rate of species i, and \u2329Ni\u232a the average number of molecules of species i in the focal volume. Mi(\u03c4) is the mobility term for free, 3-dimensional diffusion:\nwhere \u03c4D,i is the diffusion time of fluorescent species i. The coefficients  and z0 are defined, respectively, as the distances from and along the optical axis at which the excitation light intensity has dropped by 1\/e2. These coefficients were determined from calibration of the focal volume with rhodamine green of known diffusion coefficient (2.8\u00d710\u22126cm2\u00a0s\u22121).\nThe coefficient F in Eq. (4) represents the fraction of molecules involved in a non-diffusive process, and \u03c4f is the decay time of this process. It is justified to assume the existence of a non-diffusive decay process, because the transition of excited state Nile red molecules to a non-emissive TICT state and to the triplet state has been proposed [19]. G(\u221e) is the value of the autocorrelation function at long time intervals (\u03c4).\nTo reduce the number of fitting parameters, Eq. (4) was simplified. For one diffusing species, it can readily be written as:\nThe assumption of two or more diffusing species gives:\nwhere\nBefore data analysis, the experimental autocorrelation functions were truncated at very short time intervals below 0.001\u00a0ms and at very long time intervals above 600\u20131000\u00a0ms. The autocorrelation functions were then normalized [34]:\nwhere G(0) is the start value of the autocorrelation function and G(\u221e) is its value at very long time intervals. G(0) and G(\u221e) were estimated individually for every experimental autocorrelation function by non-linear regression, using Eqs. (6) and (7) [34]. Normalization eliminated possible differences in experimental autocorrelation functions that were related to systematic errors, such as adsorption of dye molecules to the surfaces of the well plate during experiments.\nThe above described normalization changes Eq. (6) to:\nHere, Gnorm(0) is the start value of the normalized autocorrelation function, which is expected to be one. The value at long time intervals, accounted for by Gnorm(\u221e), is expected to be zero. Equation (7) changes to:\nwhere\nEquation (12) shows that fi,norm represents an intensity-weighted fraction of diffusing species i. Equations (10) and (11) were used for analyzing the normalized, experimental autocorrelation functions by non-linear regression using the Levenberg-Marquardt algorithm. The calculations were performed with IGOR Pro V.4.04 (WaveMetrics, Inc., Lake Oswego, OR). When applicable, up to ten autocorrelation functions obtained from the same sample were concatenated into one data set and analyzed by a global analysis procedure contained in the software. The standard deviation (\u03c3) of experimental autocorrelation functions, estimated from the measurement repeats, was used as the weighting factor during fitting [34].\nSolutions with a heterogeneous distribution of particle sizes showed very complex decays of the experimental autocorrelation functions. These could not be adequately fit by the discrete multi-exponential model using a small number of diffusion times, and increasing the number of diffusion times above three in the model resulted in substantially increased error margins of the determined parameters. For such complex data, calculation of the distribution of diffusion times was performed by the maximum entropy method (MEM) using the MEMFCS software [35].\nTranslation of diffusion time (\u03c4D) to a diffusion coefficient (D) was done by the following relationship:\nCalculation of the hydrodynamic radius (Rh) was then possible using the Stokes-Einstein equation:\nLight scattering\nDetection of \u03b2-galactosidase aggregates in solution was done with static light scattering measurements using a fluorometer (Fluorolog FL3-21, Jobin Yvon\u2013Horiba, Edison, NJ). The excitation and emission wavelengths were set to 400\u00a0nm, and measurements were performed at 25\u00b0C.\nFurthermore, the hydrodynamic radius of native \u03b2-galactosidase was determined by dynamic light scattering measurements of 0.10\u00a0\u03bcM protein solutions in phosphate buffer. Experiments were performed at 25\u00b0C with a CGS-3 goniometer system and an LSE-5003 correlator (ALV, Langen, Germany). The average hydrodynamic radius (Rh) of the \u03b2-galactosidase molecules was determined using the method of cumulants.\nSize exclusion chromatography (SEC)\nSEC measurements were performed using an Alliance 2695 system with a type 2487 dual wavelength detector and a type 2475 fluorescence detector (Waters, Milford, MA). A Superose 6 column with a separation range of 5\u00a0\u00d7\u00a0103 to 5\u00a0\u00d7\u00a0106\u00a0Da, and an exclusion limit of 4\u00a0\u00d7\u00a0107\u00a0Da (GE Healthcare Europe GmBH, Roosendaal, The Netherlands) was used. Phosphate buffered saline (pH 7.4) was used as an eluent, and the injection volume was 40\u00a0\u03bcl. Detection was by UV absorption at 210\u00a0nm or by fluorescence emission (\u03bbex 280\u00a0nm and \u03bbem 340\u00a0nm for intrinsic protein fluorescence; \u03bbex 550\u00a0nm and \u03bbem 611\u00a0nm for Nile red fluorescence). Before injection, samples were centrifuged at 10000 RPM for 5\u00a0min. A molecular weight calibration curve using chymotrypsin (25\u00a0kDa), human serum albumin (66\u00a0kDa and 132\u00a0kDa dimer), and thyroglobulin (670\u00a0kDa) as molecular weight standards was established.\nResults and discussion\nHeat denaturation of \u03b2-galactosidase\nThe temperature induced denaturation of \u03b2-galactosidase was followed by measuring intrinsic tryptophan fluorescence emission of this protein (not shown). Denaturation started between 50 and 55\u00b0C and was completed between 60 and 65\u00b0C. An unfolding temperature of 57.4\u00b0C was obtained by fitting the denaturation curve with a model for a two-state unfolding process [36]. This is in good agreement with DSC experiments and activity assays performed under comparable conditions [25].\nHeating \u03b2-galactosidase solutions above the unfolding temperature leads to irreversible aggregation of protein molecules [25]. Accordingly, a 0.10\u00a0\u03bcM \u03b2-galactosidase solution incubated at 62\u00b0C for 5\u00a0min was heavily aggregated and remained so upon cooling back to room temperature, as detected by light scattering (not shown). The aggregates formed under these conditions were too large to be detected by SEC. This is shown in Fig. 1a, which depicts SEC chromatograms of a non-heated \u03b2-galactosidase solution and of a \u03b2-galactosidase solutions that was heated at 62\u00b0C for 5\u00a0min. The former consists of peaks representing native, tetrameric \u03b2-galactosidase, oligomers of two and three \u03b2-galactosidase molecules, and even some aggregates of higher molecular weight. The latter consists only of a peak representing native, tetrameric \u03b2-galactosidase. However, the area under the curve of this peak is reduced to about 10% of the area under the curve of the corresponding peak of non-heated \u03b2-galactosidase solution. The apparent loss of protein was caused by the formation of large aggregates that were excluded from the SEC column.Fig. 1(a) SEC chromatograms of non-heated 0.10\u00a0\u03bcM \u03b2-galactosidase solution (solid line) and \u03b2-galactosidase solution incubated at 62\u00b0C for 5\u00a0min (dotted line). The peaks represent native (i.e. tetrameric) \u03b2-galactosidase (1) and oligomers of two (2) and three (3) \u03b2-galactosidase molecules. (b) Plot of the increase of scattered light intensity at 400\u00a0nm against incubation time of 0.10\u00a0\u03bcM \u03b2-galactosidase solution at 49\u00b0C. Data points are averages of 5\u201310 individual samples, and the error bars indicate \u00b11 SD. (c) SEC chromatograms of non-heated 0.10\u00a0\u03bcM \u03b2-galactosidase solution and solutions heated at 49\u00b0C for 15 and 60\u00a0min. The peaks represent native, tetrameric \u03b2-galactosidase (1) and oligomers of two (2) and three (3) \u03b2-galactosidase molecules. The insert shows that after 60\u00a0min incubation, high-order aggregates of \u03b2-galactosidase appeared, with a retention time of 29\u00a0min. There was no detectable difference between non-heated solutions and solutions heated at 49\u00b0C for 15\u00a0min\nStatic light scattering measurements using a fluorometer indicated that heating \u03b2-galactosidase solutions at 49\u00b0C also led to the formation of aggregates. Figure 1b shows the intensity of scattered light of 0.10\u00a0\u03bcM \u03b2-galactosidase solutions heated for different times between 15\u00a0min and 6\u00a0h. The increase of scattered light intensity indicates the formation of protein aggregates. This is clearly an effect of heating, because scattered light intensity did not increase upon storage at 25\u00b0C (not shown). Figure 1b also shows that a maximum of scattered light intensity was reached after about 120\u00a0min of incubation. This is possibly related to the sedimentation of very large aggregates that had been formed after long incubation times, which would also explain the increasing error margins of the long time-points.Fig. 2(a) Fluorescence emission spectra of Nile red added to phosphate buffer (broken line; \u03bbmax\u00a0=\u00a0662\u00a0nm), non-heated 0.10\u00a0\u03bcM \u03b2-galactosidase solution (dotted line; \u03bbmax\u00a0=\u00a0659\u00a0nm), and 0.1\u00a0\u03bcM \u03b2-galactosidase solution that was previously heated at 62\u00b0C for 5\u00a0min (solid line; \u03bbmax\u00a0=\u00a0611\u00a0nm). Excitation wavelength was 550\u00a0nm. (b) Fit of two Voigt-shaped peaks (broken lines) to an emission spectrum (circles) of Nile red added to \u03b2-galactosidase solution that was heated at 49\u00b0C for 360\u00a0min. The solid black line is the fitted spectrum, and the residuals are indicated. The positions of the fitted peaks are 605.0\u00a0\u00b1\u00a02.2\u00a0nm and 658.3\u00a0\u00b1\u00a01.4\u00a0nm. (c) Plot of Nile red fluorescence intensity at 611\u00a0nm against incubation time at 49\u00b0C of 0.10\u00a0\u03bcM \u03b2-galactosidase solution. Data points are averages of 5 individual samples, and the error bars indicate \u00b11 SD\nThe SEC chromatograms in Fig. 1c show the formation of large \u03b2-galactosidase aggregates by heating solutions at 49\u00b0C. Non-heated solution mainly contained the native, tetrameric \u03b2-galactosidase, along with some dimer, trimer, and even higher order aggregates. Heating for 15\u00a0min at 49\u00b0C did not lead to a change of the SEC chromatogram. However, after 60\u00a0min incubation an additional peak with a retention time of 29\u00a0min appeared, representing large \u03b2-galactosidase aggregates. The area of this peak was 1.5% of the total area under the curve of the protein and increased with increasing incubation time at 49\u00b0C (not shown). The chromatograms shown in Fig. 1c were obtained by measuring absorbance at 210\u00a0nm. The use of an on-line fluorescence detector system (\u03bbex 280\u00a0nm and \u03bbem 340\u00a0nm) led to corresponding results, i.e. aggregation was only detected in \u03b2-galactosidase solutions that were heated at 49\u00b0C for at least 60\u00a0min.\nNile red steady-state fluorescence\nFigure 2a depicts the emission spectra of Nile red in buffer, in non-heated 0.10\u00a0\u03bcM \u03b2-galactosidase solution, and in 0.10\u00a0\u03bcM \u03b2-galactosidase solution that was previously heated at 62\u00b0C for 5\u00a0min. The emission of Nile red in the presence of non-heated protein was almost identical to the dye\u2019s emission in buffer. This indicates that the surface area of native \u03b2-galactosidase is highly polar, and that possible apolar sites are not accessible to Nile red. Storage of unheated \u03b2-galactosidase solutions containing Nile red for up to 25\u00a0min at room temperature did not lead to a change of the dyes fluorescence, showing that the presence of Nile red does not lead to a change of \u03b2-galactosidase surface hydrophobicity over time.\nInteraction of Nile red with heat-denatured \u03b2-galactosidase resulted in a shift of emission maximum from 660 to 611\u00a0nm and in a substantial increase in fluorescence intensity (Fig. 2a). This is typically observed when Nile red molecules bind to hydrophobic, apolar sites on the surface of a protein [14], and shows that irreversible denaturation and aggregation by heating above the unfolding temperature has increased the surface hydrophobicity of \u03b2-galactosidase.\nUpon 15\u00a0min incubation of 0.10\u00a0\u03bcM \u03b2-galactosidase solution at 49\u00b0C, the emission spectrum of Nile red reproducibly showed an increased intensity below 640\u00a0nm. Upon longer incubation between 1 and 6\u00a0h, a shoulder between 605 and 615\u00a0nm emerged. Figure 2b shows a two-peak fit of an emission spectrum obtained with Nile red added to \u03b2-galactosidase solution that was previously heated at 49\u00b0C for 6\u00a0h. In Fig. 2b, the \u03bbmax of the main peak is 658\u00a0nm, whereas the peak responsible for the shoulder is positioned at 605\u00a0nm. Emission at 658\u00a0nm originates from free Nile red molecules, and emission at 605\u00a0nm from Nile red interacting with denatured \u03b2-galactosidase. The latter is comparable with the \u03bbmax of Nile red in \u03b2-galactosidase solution that had been heated above the unfolding temperature (Fig. 2a). This indicates that the surface polarity of protein molecules denatured at different temperatures is similar. The fluorescence of Nile red did not depend on the time that protein solutions were kept at room temperature upon heating at 49\u00b0C (not shown), which indicates that irreversibly denatured \u03b2-galactosidase species were detected.\nThe effect of increasing incubation time at 49\u00b0C on Nile red fluorescence intensity is shown in Fig. 2c. An observation wavelength of 611\u00a0nm was chosen, which corresponds to the average emission wavelength of Nile red interacting with irreversibly denatured \u03b2-galactosidase. The profile of Fig. 2c is similar to that of Fig. 1b, suggesting that Nile red interacted with aggregated \u03b2-galactosidase. A clear decrease of Nile red emission at 611\u00a0nm upon centrifugation at 10000 RPM (not shown) supported this notion. However, it was not possible to confirm this by SEC, neither by adding Nile red to the samples before injection and performing on-line fluorescence detection (\u03bbex 550\u00a0nm and \u03bbem 611\u00a0nm), nor by staining fractions from SEC runs with Nile red and performing off-line fluorescence detection. For this reason, time-resolved fluorescence spectroscopy and FCS were used to further investigate the interaction between Nile red and aggregated \u03b2-galactosidase.Fig. 3Fluorescence intensity decay, I(t), of Nile red in 0.10\u00a0\u03bcM \u03b2-galactosidase solution that was heated at 49\u00b0C for 60\u00a0min. The fitted decay curve and the weighted residuals are shown. Fitting gave three lifetimes (\u03c41\u00a0=\u00a00.11, \u03c42\u00a0=\u00a01.11, and \u03c43\u00a0=\u00a04.36\u00a0ns) and three (normalized) pre-exponential factors (\u03b11,norm\u00a0=\u00a00.40, \u03b12,norm\u00a0=\u00a00.24, and \u03b13,norm\u00a0=\u00a00.36). The value of \u03c72 was 1.056Table 1Lifetime analysisSample\u03c72 totala\u2329\u03c4\u232abbuffer1.0390.20non-heated1.0110.4215\u201349\u00b0C1.0621.3060\u201349\u00b0C1.0561.89120\u201349\u00b0C1.0642.48240\u201349\u00b0C1.0002.105\u201362\u00b0C1.0321.50a\u03c72 obtained with Eq. (1) using three fluorescence lifetimes.b\nTime-resolved fluorescence spectroscopy with Nile red\nFigure 3 shows the analysis of the fluorescence intensity decay, I(t), of Nile red in 0.10\u00a0\u03bcM \u03b2-galactosidase solution that was heated at 49\u00b0C for 60\u00a0min. The data points could be adequately described using Eq. (1) with three fluorescent lifetimes, as can be seen from the regular distribution of the residuals around zero and the proximity of \u03c72 to unity. The use of three lifetimes was necessary for the analysis of all intensity decays, and it was not possible to relate particular lifetimes to the free and the protein-bound state of Nile red. In Table 1, the results are summarized by listing \u2329\u03c4\u232a values, which are proportional to steady state intensities. In agreement with the increase of Nile red steady state intensity at 611\u00a0nm, heating of \u03b2-galactosidase solution at 49\u00b0C for 15\u00a0min led to a clear increase of \u2329\u03c4\u232a as compared to Nile red in buffer or non-heated protein solution. The tendency of \u2329\u03c4\u232a to further increase with increasing heating time at 49\u00b0C corresponds nicely to the increase of fluorescence intensity upon prolonged heating shown in Fig. 2c. Also with \u03b2-galactosidase solution that was heated at 62\u00b0C for 5\u00a0min, \u2329\u03c4\u232a was substantially increased as compared to non-heated protein solution. However, it was lower than expected from the steady-state fluorescence intensity at 611\u00a0nm (Fig. 2a). Possible reasons for this difference are the variation between individual experiments with solutions containing highly aggregated protein and, since there is a lot of light scattering in such samples, different sensitivities of the steady-state and time-resolved instrumentation for scattered light.\nThe course of fluorescence anisotropy with time, r(t), of Nile red in 0.10\u00a0\u03bcM \u03b2-galactosidase solution that was heated at 49\u00b0C for 15, 60, 120, and 240\u00a0min was measured (not shown). With all samples, anisotropy decreased at short times, then increased again, and finally remained at an almost constant value. The practically constant value of anisotropy at long times indicated rotational diffusion that was too slow to be detected by Nile red fluorescence. Therefore, although indicating that Nile red was in fact bound to large particles such as aggregates of \u03b2-galactosidase, it was not possible to estimate the size of these particles by time-resolved fluorescence anisotropy.\nFluorescence correlation spectroscopy (FCS) measurements with Nile red\nFigure 4a depicts an overlay of ten normalized, experimental autocorrelation functions that were obtained with Nile red in buffer. These data were analyzed simultaneously using the one-component model (Eq. (10)). The fitted curve included in Fig. 4a, the regular distribution of the weighted residuals around zero, and the proximity of \u03c72 to unity show that Eq. (10) was appropriate for describing the experimental data. The determined decay time for non-diffusive process (\u03c4f) was 0.022\u00a0ms, the fraction of dye molecules involved in this process (F) was 0.45, and the determined diffusion time (\u03c4D,1) was 0.309\u00a0ms (Table 2). A diffusion time of Nile red around 0.3\u00a0ms is in agreement with reported values [37]. It corresponds to a diffusion coefficient of 2.9\u00a0\u00d7\u00a010\u22126\u00a0cm2\u00a0s\u22121, which is reasonable for a molecule with a molecular weight of 318\u00a0Da. Therefore, \u03c4D,1 can be associated with the diffusion of free dye molecules. Gnorm(0) deviated slightly from the expected value of unity (Table 2). Forcing it to unity increased \u03c72 slightly, but had no effect on the determined parameters \u03c4D,1, \u03c4f, and F. Also Gnorm(\u221e) deviated somewhat from the expected value of zero (Table 2). This is related to the background subtraction described in the materials and methods section. Leaving out background subtraction did not lead to different values of the coefficients \u03c4D,1, \u03c4f, and F. Measurement of Nile red in non-heated 0.10\u00a0\u03bcM \u03b2-galactosidase solution gave the same parameters as Nile red in buffer (Table 2), confirming that there was no substantial interaction between the dye and non-heated protein.Table 2Coefficients from autocorrelation function analysisSample withNile redModel\u03c4D,1a (ms)f1,norma\u03c4D,2a (ms)f2,norma\u03c4fa (ms)FaGnorm(0)aGnorm(\u221e)aPhosphate bufferOne-Component0.309\u00b10.008\u2013\u2013\u20130.022\u00b10.0020.451\u00b10.0100.920\u00b10.016\u22120.0049\u00b10.00031.03\u03b2-Galactosidase non-heatedOne-Component0.291\u00b10.009\u2013\u2013\u20130.018\u00b10.0020.434\u00b10.0150.915\u00b10.021\u22120.0047\u00b10.00041.08\u03b2-Galactosidase 62\u00b0C\/5\u00a0minTwo-Component0.287\u00b10.0170.335\u00b10.00545.7\u00b11.020.690\u00b10.0070.019\u00b10.0010.246\u00b10.008\u2013\u22120.0368\u00b10.00211.44aEstimates \u00b11 SD.Fig. 4Analysis of FCS data. (a) Simultaneous fit of 10 normalized autocorrelation functions obtained with Nile red in buffer. Analysis was performed by non-linear regression using Eq. (10). The fitted curve (black) and the weighted residuals for one of the normalized autocorrelation functions are shown. Determined parameters (value\u00b11 SD) were F=0.451\u00b10.010, \u03c4f=0.022\u00b10.002 ms, \u03c4D,1=0.309\u00b10.008 ms, Gnorm(0)=0.920\u00b10.016, and Gnorm(\u221e)=\u22120.0049\u00b10.0003. The value of  was 1.03. (b) Simultaneous fit of 9 normalized autocorrelation functions obtained with Nile red in 0.10 \u03bcM \u03b2-galactosidase solution that was previously heated at 62\u00b0C for 5 min. Analysis was performed by non-linear regression using Eq. (11) for two diffusing species. The fitted curve (black) and the weighted residuals for one of the normalized autocorrelation functions are shown. Determined parameters (value \u00b11 SD) were F=0.246\u00b10.008, \u03c4f=0.019\u00b10.001 ms, f1,norm=0.335\u00b10.005, \u03c4D,1=0.287\u00b10.017 ms, f2,norm=0.690\u00b10.007, \u03c4D,2=45.70\u00b11.02 ms, and Gnorm(\u221e)=\u22120.037\u00b10.0021. The value of  was 1.44Fig. 5Analysis of FCS data. (a) MEM analysis of a single autocorrelation function obtained with Nile red in 0.10\u00a0\u03bcM \u03b2-galactosidase solution that was previously heated at 62\u00b0C for 5\u00a0min. The non-weighted residuals are shown. (b) Distribution of diffusion times (\u03c4D) calculated by MEM analysisFig. 6Raw data of two FCS measurements obtained with 0.10\u00a0\u03bcM \u03b2-galactosidase solution that was heated at 62\u00b0C for 5\u00a0min (upper curve) and at 49\u00b0C for 15\u00a0min\nAn overlay of nine normalized, experimental autocorrelation functions, obtained with Nile red in 0.10\u00a0\u03bcM \u03b2-galactosidase solution that was previously heated at 62\u00b0C for 5\u00a0min, is shown in Fig. 4b. There is an additional decay of the autocorrelation functions between 20 and 200\u00a0ms, which was not observed with Nile red in buffer. Data analysis was therefore performed using a two-component model, i.e. Eq. (11) assuming two diffusing species. The fitted line and the weighted residuals in Fig. 4b show that up to time intervals between 1 and 10\u00a0ms, the data could be well described by the two component model. Within this range, the decay of the autocorrelation functions is governed by \u03c4f and \u03c4D,1. The determined values were 0.019 and 0.287\u00a0ms, respectively (Table 2), which is in good agreement with the values found for Nile red in buffer. This shows that also in the heated \u03b2-galactosidase solution, not all Nile red was bound to denatured protein. The fraction of dye molecules (F) involved in non-diffusive process was 0.25, which is substantially lower than for Nile red in buffer (Table 2). A possible explanation for this is that in solutions with large amounts of denatured protein, less Nile red molecules undergo transition to the TICT and triplet state, because they can interact with hydrophobic surfaces [19]. The second diffusion time (\u03c4D,2) of 46 ms (Table 2) governed the decay of autocorrelation functions between 20 and 200\u00a0ms. It corresponds to a diffusion coefficient (D) of 1.9\u00a0\u00d7\u00a010\u22128\u00a0cm2\u00a0s\u22121 and a hydrodynamic radius (Rh) of 130\u00a0nm, which deviates substantially from the values of native \u03b2-galactosidase (D\u00a0=\u00a03.1\u00a0\u00d7\u00a010\u22127\u00a0cm2\u00a0s\u22121; Rh\u00a0=\u00a08\u00a0nm). This shows that incubation at 62\u00b0C for 5\u00a0min had led to very large, denatured aggregates of \u03b2-galactosidase, which could be detected by Nile red. However, the two-component model (Eq. (11)) was not able to fully describe the very complex autocorrelation function decays shown in Fig. 4b, as reflected by the residuals and the relatively high \u03c72 value of 1.44 (Table 2). For this reason, the distribution of diffusion times was determined by the maximum entropy method (MEM) [35]. Such an analysis is shown in Fig. 5a for an individual experimental autocorrelation function. The distribution of diffusion times (Fig. 5b) shows 3 peaks, centered at 0.02, 0.36, and 39\u00a0ms. The 0.02 and 0.36\u00a0ms peaks correspond to \u03c4f and \u03c4D,1 determined with Eq. (11). The peak around 39\u00a0ms is very broad and ranges from 5 to 150\u00a0ms, which corresponds to hydrodynamic radii between 14 and 420\u00a0nm. The peak maximum of 39\u00a0ms corresponds to an Rh of 109\u00a0nm, in good agreement with \u03c4D,2 obtained with Eq. (11). This is well above the hydrodynamic radius of small products of \u03b2-galactosidase denaturation, i.e. denatured monomers, dimers, and tetramers [25], and confirms that interaction with large \u03b2-galactosidase aggregates was the reason for the change of Nile red fluorescence.\nNile red also detected large aggregates in 0.10\u00a0\u03bcM \u03b2-galactosidase solutions that had been incubated at 49\u00b0C. As for protein samples heated at 62\u00b0C, these aggregates were detectable by an additional decrease of FCS autocorrelation functions at long times. However, aggregate concentration in samples heated at 49\u00b0C was much lower than in samples heated at 62\u00b0C. This is shown in Fig. 6, which compares the raw FCS data obtained with Nile red in \u03b2-galactosidase solutions heated for 5\u00a0min at 62\u00b0C and for 15\u00a0min at 49\u00b0C. The former is full of high-intensity peaks that were caused by large aggregates with bound Nile red crossing the focal volume. The latter contains only one high-intensity peak, representing a single large aggregate crossing the focal volume. As a consequence of the low aggregate concentration in samples heated at 49\u00b0C, only 3 out of 100 measurements contained high-intensity peaks. In spite of their rare occurrence, these peaks were not artifacts. No high-intensity peaks were detected in a total of 150 control experiments with Nile red in buffer and in non-heated \u03b2-galactosidase solution.\nThe combined results of steady-state fluorescence spectroscopy, time-resolved fluorescence spectroscopy, and FCS show that Nile red fluorescence gave complementary information to SEC about the aggregation of \u03b2-galactosidase. Native protein molecules and small oligomers could be detected by SEC (Fig. 1a and c), but did not substantially affect Nile red fluorescence. It is possible that both the surface hydrophobicity and the surface area of these protein species were not sufficient for an effective interaction with the dye. However, Nile red was able to interact with large, denatured aggregates of \u03b2-galactosidase and could therefore be used as a probe for their specific detection. Although some higher order aggregates could be detected by SEC upon heating at 49\u00b0C (Fig. 1c), Nile red fluorescence was sensitive to the presence of even larger aggregates, which were missed by SEC (Fig. 1a).\nThe interaction of Nile red with large aggregates of \u03b2-galactosidase makes it very difficult to use the SEC results for estimating the sensitivity of the Nile red method. Figure 1c shows that some large aggregates were first detected by SEC in protein samples that had been heated at 49\u00b0C for 60\u00a0min. However, the inability of Nile red to stain these aggregates fractionated from SEC runs (see results above) and the possible exclusion from the SEC column of even larger protein aggregates, which are detectable by Nile red, render the use of SEC as a reference method questionable. Furthermore, Nile red fluorescence was clearly more sensitive than SEC, as it already detected irreversible changes in samples that were heated at 49\u00b0C for 15\u00a0min. Therefore, to estimate the sensitivity of Nile red, non-heated \u03b2-galactosidase solutions were doped with defined amounts of \u03b2-galactosidase solution that had been irreversibly denatured and aggregated by heating at 62\u00b0C for 5\u00a0min. The lower limit of detection by Nile red steady-state fluorescence was 1% (v\/v) of heated protein solution, corresponding to 1\u00a0nM denatured and aggregated \u03b2-galactosidase. The fluorescence intensity of this solution at 611\u00a0nm corresponded to that of \u03b2-galactosidase solution that had been heated at 49\u00b0C for 15\u00a0min (Fig. 2c). This comparison is justified, because nearly all \u03b2-galactosidase molecules had formed large aggregates upon heating at 62\u00b0C for 5\u00a0min (Fig. 1a), and because the fluorescence intensity of the detected aggregates did not depend on the incubation temperature (Fig. 6), which means that Nile red was equally sensitive to aggregates formed by heating at 49 and 62\u00b0C.\nConclusions\nUsing the model protein \u03b2-galactosidase, it was shown that the presence small amounts of large, denatured protein aggregates in solution can be detected by Nile red fluorescence. Aggregates detected by Nile red had hydrodynamic radii around 130\u00a0nm with a broad size distribution. Native protein and small aggregates thereof had no substantial effect on Nile red fluorescence intensity. By steady-state fluorescence measurements, it was possible to detect 1\u00a0nM denatured and highly aggregated \u03b2-galactosidase in solution.\nThe spectroscopic detection of protein aggregates by Nile red is potentially useful for formulation screening or quality control of protein pharmaceutics. The presence of even minute fractions of aggregates in protein formulations needs to be avoided, because this can cause immune reactions in patients. Since large aggregates are particularly potent for breaking immune tolerance, their analytical detection is very important. SEC, which is the standard method for studying protein aggregation, is not reliable for detecting large aggregates, because they may be excluded from the separation column. In this work we showed that an analytical detection method with Nile red is a possible approach to overcome this shortcoming of SEC. After establishing the method with time-resolved fluorescence spectroscopy and FCS, as was done for \u03b2-galactosidase, steady-state fluorescence measurements, which can be performed in most labs, may enable an experimentally simple and sensitive detection of large protein aggregates.","keyphrases":["protein aggregation","nile red","fluorescence correlation spectroscopy","size exclusion chromatography","fluorescence spectroscopy"],"prmu":["P","P","P","P","P"]}
{"id":"Diabetologia-3-1-1914285","title":"Absence of an adipogenic effect of rosiglitazone on mature 3T3-L1 adipocytes: increase of lipid catabolism and reduction of adipokine expression\n","text":"Aims\/hypothesis The thiazolidinedione (TZD) rosiglitazone is a peroxisome proliferator-activated receptor-\u03b3 agonist that induces adipocyte differentiation and, hence, lipid accumulation. This is in apparent contrast to the long-term glucose-lowering, insulin-sensitising effect of rosiglitazone. We tested whether the action of rosiglitazone involves specific effects on mature adipocytes, which are different from those on preadipocytes.\nIntroduction\nRosiglitazone (BRL-49653), a thiazolidinedione (TZD) drug, is used in the treatment of type 2 diabetes mellitus [1]. It has been proposed that the glucose-lowering action of TZDs is mainly mediated by activating peroxisome proliferator-activated receptor (PPAR)-\u03b3 (PPARG) [2, 3]. PPARG is most abundantly produced in adipose tissue, suggesting that this is the primary site of action of TZDs [4]. Proposed mechanisms underlying the adipocyte-mediated glucose-lowering action of TZDs are the trapping of fatty acids inside adipocytes and away from muscle, the so-called \u2018fatty acid steal\u2019 hypothesis and the altering adipokine release [1].\nPPARG is a critical transcription factor in adipogenesis [5]. Its production increases strongly during adipocyte differentiation [6]. By activating PPARG, rosiglitazone promotes adipocyte differentiation in vitro [7, 8]. An increase in the number of small adipocytes and fat mass by TZDs in both animal models and human subjects, suggests that adipocyte differentiation also occurs in vivo [9, 10]. Compared with preadipocytes, adipocytes have a much higher capacity for fatty acid uptake and lipid storage and are potentially more insulin-sensitive due to higher numbers of glucose transporters and insulin receptors [6]. By increasing the number of adipocytes, TZDs can lower circulating NEFA and improve lipid exposure of peripheral tissues, thus improving whole-body insulin sensitivity [1].\nThis hypothesis is clearly attractive, but other effects of TZDs may occur. It has been pointed out that the increased number of small adipocytes caused by TZD treatment is not only a result of the appearance of new adipocytes, but also due to the shrinkage and\/or disappearance of existing mature adipocytes [10]. TZD-induced body weight gain in vivo is used as a marker of increased adiposity. However, there is evidence that this may also be due to fluid retention [11]. In fact, the fat mass may be unaffected or even decreased. In ob\/ob mouse, whole body weight was increased, but the fat pad weight was not changed after rosiglitazone treatment [12]. In type 2 diabetes patients, the visceral fat area was found to be decreased [13]. \u2018Fatty acid steal\u2019 therefore is not just a matter of increased lipid storage.\nThe maturation of adipocytes is positively correlated with the production of adipokines. Several of these profoundly influence insulin sensitivity and glucose metabolism, e.g. leptin, adiponectin and resistin [14]. However, it has been observed that TZDs decrease the circulating level of individual adipokines such as leptin [15], resistin [16] and plasminogen activator inhibitor-1 [17], and also of cytokines [18]. A decreased production of these adipokines by TZDs seems to conflict with the adipogenic properties of these drugs.\nThe above discrepancy could be due to different effects on preadipocytes and mature adipocytes in adipose tissue. It is clear that TZDs stimulate preadipocytes into the process of differentiation. However, their effect on mature adipocytes is less clear. In vivo studies do not allow us to distinguish between the effects on these two cell types. Therefore, to study the effects of TZDs on mature adipocytes, we used in vitro differentiated mature 3T3-L1 adipocytes. In addition to the assessment of lipid accumulation and adipokine secretion, we used transcriptomics to obtain insight into the effects of rosiglitazone. Although high insulin and high glucose levels mimic the situation in vivo when rosiglitazone is administered, we also included low insulin and low glucose levels for comparison.\nMaterials and methods\nCell culture and sample preparation Murine 3T3-L1 fibroblasts (CL-173; American Type Culture Collection, Manassas, VA, US) were cultured and differentiated to adipocytes in vitro as described [19]. On day 8, differentiated cells, grown in DMEM\/F-12 (1:1) supplemented with 10% fetal calf serum (Perbio Science, Erembodegem, Belgium), were treated with 0.5\u00a0\u03bcmol\/l rosiglitazone maleate (GlaxoSmithKline, Worthing, UK) with low (4.5\u00a0mmol\/l) glucose and (fetal calf serum level) insulin (Rosi), or with high (15.75\u00a0mmol\/l) glucose and 1\u00a0\u03bcmol\/l insulin (RosiIG). In parallel, control cells were cultured in the same medium with low (basal) or high glucose and insulin (IG). Each condition was created in quadruplicate. On day 10, two replications were collected separately for total RNA isolation using Trizol as described [19]. Cells of the other two replications were washed and incubated with the corresponding serum-free medium, supplemented with 2.2\u00a0mg\/l transferrin and 10.6\u00a0nmol\/l sodium selenite for 6\u00a0h. The number of cells was counted and culture medium proteins were collected as described [20]. The duplicated protein samples were pooled for later analysis. Three independent experiments were performed.\nOil red O staining An optimised Oil Red O staining method [21] was adapted for 3T3-L1 adipocytes cultured in a six-well plate. We added an additional quick wash with 70% ethanol after fixing and washing with water. After staining and washing, the neutral lipid-bound pigment was dissolved in DMSO and absorbance at 540\u00a0nm was measured. The staining for 3T3-L1 preadipocytes was used as background to correct for unspecific binding. The obtained absorbance values were further corrected for cell numbers measured in replicated wells. Five independent experiments were performed in duplicate, but in the first two experiments Basal and Rosi conditions were not measured.\nGlycerol assay The culture media of cells that had been treated for 48\u00a0h were collected per condition, frozen in liquid N2, then stored at \u221280\u00b0C until analysis. The concentration of glycerol in the medium was measured by a quantitative enzymatic assay kit (Sigma, St Louis, MO, USA), according to the manufacturer\u2019s instructions. The concentrations were corrected for cell numbers measured in replicated wells in parallel.\nMicroarray The mouse 10K_A oligo set (MWG, Ebersberg, Germany) and additional oligonucleotides of genes encoding secretory proteins and proteins involved in energy metabolism were printed in-house and further annotated as described [19]. Five total RNA samples of three independent experiments per condition were used for hybridisation against reference RNA, also as described [19].\nMicroarray data analysis The data were analysed as described [19]. Briefly, spots with an average intensity, over all arrays, of lower than twofold above average background were removed from further analysis, then the intensities of the remaining 4,019 spots were normalised against reference. Fold-change calculations, Student\u2019s t tests and correlation test (Pearson) were performed in Excel (Microsoft). Fold-change equals ratio in the case of increase and equals \u22121\/ratio in the case of decrease. Cut-off criteria for differential expression was set at fold-change >1.3 and p\u2009<\u20090.05. For genes with replicated spots, the average value of individual spots was used.Data were further analysed using Ingenuity Pathways Analysis (Ingenuity Systems, http:\/\/www.ingenuity.com). The program recognised 4,005 of 4,019 spot entries, and thus 3,718 genes. Differentially expressed genes that associated with a canonical pathway in the Ingenuity Pathways Knowledge Base were considered for evaluation. The significance of the association between the data set and the canonical pathway was measured in two ways: (1) a ratio of the number of genes from the data set that map to the pathway divided by the total number of genes that map to the canonical pathway is displayed; (2) Fischer\u2019s exact test was used to calculate a p value determining the probability that the association between the genes in the dataset and the canonical pathway is explained by chance alone. A pathway with a significance <0.05 was taken as significantly regulated.Quantitative real-time RT-PCR All six total RNA samples per treatment were used for quantitative real-time RT-PCR (Q-PCR) of 22 genes (primer information in Electronic supplementary material [ESM] Table\u00a01). Q-PCR was performed as described [19]. Ribosomal protein S15 (Rps15) mRNA was used as reference.\nOne- and two-dimensional gel electrophoresis Protein samples were analysed by one-dimensional (1D) and two-dimensional (2D) gel electrophoresis, corrected for cell numbers, as described [19]. Brefeldin-A-treated adipocyte medium protein sample [20] was loaded on the same 1D gel, to verify protein secretion. Differentially expressed secreted protein bands were excised and identified by matrix-assisted laser desorption ionisation-time of flight mass spectrometry [22].\nWestern blotting The secretion level of adiponectin was analysed by 1D western blotting as described [20].\nResults\nEffect of rosiglitazone on the lipid content of mature adipocytes To assess the effect of rosiglitazone on lipid accumulation in mature adipocytes, we measured the lipid content of differentiated 3T3-L1 adipocytes. Visual inspection by microscopy showed no change in morphology and fat cell percentage in the population of 3T3-L1 adipocytes after 2\u00a0days of treatment with RosiIG or Rosi (0.5\u00a0\u03bcmol\/l rosiglitazone at either high or low insulin\/glucose levels, respectively), as compared with untreated adipocytes. Nevertheless, the lipid content, as detected by Oil Red O staining, was 10% lower (p\u2009<\u20090.001, n\u2009=\u20095) in RosiIG-treated cells, while no difference was observed in Rosi-treated cells (Fig.\u00a01).\nFig.\u00a01Oil Red O staining (ORO) for lipid content in 3T3-L1 adipocytes. Data are expressed as mean\u00b1SD. n\u2009=\u20095 for high glucose and insulin (control condition) (IG) and rosiglitazone with high insulin and glucose condition (RosiIG); n\u2009=\u20093 for basal and rosiglitazone with low insulin and glucose condition (Rosi). Blank bars: absorbance, relative value; filled bars: absorbance, relative value corrected for cell numbers. ** p\u2009<\u20090.01 compared with IG condition before cell number correction (t test), *** p\u2009<\u20090.001 compared with IG condition after cell number correction (t test)\nEffect of rosiglitazone on gene expression To reveal the possible mechanisms for the decreased lipid content of rosiglitazone-treated 3T3-L1 adipocytes, we used DNA microarray to profile the gene expression. The complete analysis set was imported into Ingenuity Pathway Analysis for canonical pathway analysis. The significantly changed pathways were involved in metabolism, but not in signalling, such as PPAR signalling or insulin receptor signalling (Fig.\u00a02). The expression of Pparg itself was downregulated 1.8- and 2.2-fold (p\u2009<\u20090.005) by Rosi and RosiIG treatment, respectively. Notably, pathways related to energy metabolism were among those that were most changed. Taking the high redundancy among these pathways in the Ingenuity database into account, we checked the individual pathways for redundancy. This showed that oxidative phosphorylation, fatty acid metabolism, glycolysis and gluconeogenesis, the pentose phosphate pathway, the citric acid (TCA) cycle, glycerolipid metabolism, glutathione metabolism, steroid (androgen, oestrogen and C21 steroids) metabolism and prostaglandin metabolism are independent pathways, while for the others it cannot be ruled out that they were identified as significant just because they contain genes overlapping with other pathways.\nFig.\u00a02Regulated canonical pathways by rosiglitazone. Microarray data analysed by Ingenuity Pathway Analysis. Blank bars, with low insulin\/glucose; filled bars, with high insulin\/glucose. Bold line, thresholdFor these independently regulated pathways, we determined the direction of change by examining the individual genes in the pathways. The regulation of gene expression of rate-limiting or otherwise representative enzymes is presented in Table\u00a01. High insulin\/glucose attenuated the effect of rosiglitazone on some genes of complex I and II of the electron transport chain. However, high insulin\/glucose treatment itself did not induce a change in the expression of these genes from the basal state. In general, rosiglitazone produced dominant effects, as indicated by the Pearson product moment correlation coefficient of 0.875 for the fold changes of the complete analysis set of Rosi\/Basal and RosiIG\/IG. The changes in the pathways indicate that rosiglitazone enhanced both energy metabolism at the TCA cycle and oxidative phosphorylation. The generated energy may be dispensed with the help of upregulated uncoupling protein 2 (Ucp2). Enhanced energy metabolism is mainly supported by upregulation of the pathways involved in uptake and activation of fatty acids and in beta-oxidation in both mitochondria and peroxisomes. In contrast, de novo fatty acid synthesis was downregulated, as indicated by fatty acid synthase (Fasn) and stearoyl-coenzyme A desaturase 1 (Scd1). The downregulation of triacylglycerol synthesis enzymes and upregulation of adipocyte lipase (also known as patatin-like phospholipase domain containing 2) together imply a decreased lipogenesis in mature adipocytes (Table\u00a01). \nTable\u00a01Key enzymes of metabolism pathways regulated by rosiglitazonePathwayChangeGene symbolGene IDNameRosi\/BasalRosiIG\/IGFold-changep valueFold- changep valueTCA cycleUpIdh3a67834Isocitrate dehydrogenase 3 (NAD+) alpha2.530.0002.550.000Cs12974Citrate synthase1.950.0001.470.000Oxidative  phosphorylationUpNdufa568202NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 51.720.0091.200.235Sdhd66925Succinate dehydrogenase complex, subunit D1.830.0011.510.039Uqcrc122273Ubiquinol-cytochrome c reductase core protein I1.470.0071.330.017Cox7a112865Cytochrome c oxidase subunit VIIa polypeptide 11.780.0021.550.003Atp5h71679ATP synthase, H+ transporting, mitochondrial  F0 complex, subunit d1.710.0061.490.005Fatty acid uptake  and transportUpCd3612491CD36 antigen, fatty acid translocase2.540.0002.270.006Fabp411770Fatty acid bind protein 4, adipocyte1.640.0881.570.142Fatty acid  activationUpAcsl114081Acyl-CoA synthetase long-chain family member 12.500.0001.970.000Fatty acid beta  oxidationUpCrat12908Carnitine acetyltransferase1.450.0061.470.007Slc25a2057279Solute carrier family 25 (mitochondrial carnitine\/acylcarnitine translocase), member 201.630.0031.220.092Acox111430Acyl-coenzyme A oxidase 1, palmitoyl2.700.0002.540.000Acaa1113868Acetyl-coenzyme A acyltransferase 1 (peroxisomal)1.670.0021.290.017Acaa252538Acetyl-coenzyme A acyltransferase 2 (mitochondrial)2.700.0001.940.000Fatty acid  synthesisDownFasn14104Fatty acid synthase\u22121.520.000\u22121.550.001Scd120249Stearoyl-CoA desaturase 1\u22123.650,000\u22124.310.000Triacylglycerol  synthesisDownPpap2b67916Phosphatidic acid phosphatase type 2B\u22121.380.001\u22121.480.000Dgat267800Diacylglycerol O-acyltransferase 2\u22121.310.003\u22121.370.006Lipid droplet  formationUp\/downAdfp11520Adipose differentiation related protein5.970.0006.600.000Cav112389Caveolin, caveolae protein 1\u22122.200.000\u22122.300.001Glycerolipid lysisUpPnpla266853Patatin-like phospholipase domain  containing 21.520.0061.670.007Glycerol metabolismUpGk14933Glycerol kinase1.860.0071.880.003Gpd114555Glycerol-3-phosphate dehydrogenase 1 (soluble)2.070.0091.680.032Energy expenditureUpUcp222228Uncoupling protein 2 (mitochondrial,  proton carrier)1.780.0011.500.003Glutathione  metabolismUp\/downMgst366447Microsomal glutathione S-transferase 32.110.0011.880.001Gsta414860Glutathione S-transferase A41.360.046\u22121.010.924Gstm114862Glutathione S-transferase M1\u22121.880.000\u22122.010.010Gstt114871Glutathione S-transferase theta 1\u22121.380.060\u22121.510.044Gstz114874Glutathione transferase zeta 1\u22121.440.001\u22121.570.015Prostaglandin  metabolismUp\/downCbr3109857Carbonyl reductase 33.040.0003.960.000Ptges296979Prostaglandin E synthase 21.450.0121.610.000Ptgis19223Prostaglandin I2 (prostacyclin) synthase\u22121.710.002\u22121.410.004Steroids  metabolismUp\/downHsd17b715490Hydroxysteroid 17-beta dehydrogenase 71.630.0031.730.002Nsdhl18194NAD(P) dependent steroid dehydrogenase-like1.510.0391.370.017Hsd11b115483Hydroxysteroid 11-beta dehydrogenase 1\u22122.730.001\u22122.050.004GlyconeogenesisUpFbp214120Fructose-1,6-bisphosphatase 22.920.0032.820.014Glycogen synthesisUpGyg27357Glycogenin1.520.0011.340.008Pyruvate  metabolismUpPdha118597Pyruvate dehydrogenase E1 alpha 11.380.0011.330.001Pdhb68263Pyruvate dehydrogenase (lipoamide) beta1.590.0051.610.004Pentose phosphateUpH6pd100198Hexose-6-phosphate dehydrogenase (glucose  1-dehydrogenase)1.600.0001.470.001Taldo121531Transaldolase 11.570.0011.330.036Leu catabolismDownIvd56357Isovaleryl coenzyme A dehydrogenase\u22121.420.004\u22121.720.000Mccc172039Methylcrotonoyl-coenzyme A carboxylase 1  (alpha)\u22121.260.026\u22121.350.031Mean value of microarray data (n\u2009=\u20095)Rosi\/basal: the effect with low insulin\/glucose; RosiIG\/IG: the effect with high insulin\/glucoseAccompanying fatty acid catabolism, glycerol activation by glycerol kinase and oxidation by glycerol phosphate dehydrogenase to dihydroxyacetone phosphate were upregulated, feeding glycolysis and gluconeogenesis. The enzymes catalysing glycolysis\/gluconeogenesis were all upregulated, possibly in favour of gluconeogenesis. In addition, the upregulation of glycogenin, a gene priming glycogen synthesis, implies an increase of glycogen synthesis.Most genes in the amino acid metabolism pathways that were regulated also participate in fatty acid metabolism or in glycolysis. Therefore, we did not take amino acid metabolism pathways as independent rosiglitazone-regulated pathways. However, two key enzymes that catalyse the catabolism of leucine (Table\u00a01) were downregulated. This implies that rosiglitazone saves amino acids from being used as fuel in energy metabolism.Glutathione metabolism was significantly changed by rosiglitazone treatment. Two glutathione S-transferases that detoxify lipid peroxides, microsomal glutathione S-transferase 3 and glutathione S-transferase A4, were upregulated, while other glutathione S-transferases were downregulated by rosiglitazone. This is in line with the upregulation of fatty acid oxidation by rosiglitazone.Microsomal glutathione S-transferase 3 is involved not only in glutathione metabolism, but also in eicosanoid metabolism, which is also regulated by rosiglitazone. The expression of genes encoding enzymes involved in prostaglandin metabolism implies that the conversion from prostaglandin H2 to E2, F2 and D2 may be enhanced, while conversion to I2 may be reduced.Cholesterol biosynthesis and steroid hormone metabolism were also affected by rosiglitazone. Expression of some steroid dehydrogenases were upregulated, while hydroxysteroid 11-beta dehydrogenase 1 was downregulated (Table\u00a01). The latter has been reported as one of the beneficial effects of TZDs, leading to a decrease in the stress hormone cortisol [23].\nEffect of rosiglitazone on the expression of genes encoding adipocyte-secreted proteins To assess whether protein secretion by mature adipocytes is altered by rosiglitazone, we checked genes encoding adipocyte-secreted proteins. The list was based on literature [24\u201327] and our previous work [20]. In total, 61 genes were present on the array. Il1b, Il6, Il10 and leptin showed very low signals (less than twofold of background) and were excluded from the analysis. Among the remaining 57 genes, only apolipoprotein E (Apoe) was upregulated by Rosi treatment, and more than 40% of the genes were significantly downregulated with both Rosi and RosiIG treatment (Table\u00a02). \nTable\u00a02Effect of rosiglitazone on the expression of genes encoding adipocyte-secreted proteinsFunctionGene symbolGene IDNameRosi\/BasalRosiIG\/IGFold-changep valueFold-changep valueLipid  metabolismAdn11537Complement factor D (adipsin)\u22123.550.000\u22124.230.000C312266Complement component 3\u22122.250.000\u22122.680.001Lpl16956Lipoprotein lipase1.120.166\u22121.020.627Apoe11816Apolipoprotein E1.340.0261.180.145Insulin  sensitivityRetn57264Resistin\u22123.070.000\u22123.170.001Igf116000Insulin-like growth factor 1\u22121.550.012\u22121.580.001Pbef159027Pre-B-cell colony-enhancing factor 1\u22121.240.050\u22121.280.085Adipoq11450Adiponectin\u22121.090.462\u22121.250.108InflammationHp15439Haptoglobin\u22122.930.000\u22124.420.000Ptx319288Pentaxin-related gene\u22121.680.001\u22121.580.000Tgfb321809Transforming growth factor, beta 3\u22121.580.008\u22121.440.056B2m12010Beta-2 microglobulin\u22121.540.018\u22121.640.006Ccl220296Chemokine (C-C motif) ligand 2 (previously known as monocyte chemoattractant protein-1)\u22121.540.012\u22121.480.023Saa320210Serum amyloid A 3\u22121.490.047\u22121.380.001Cxcl1220315Chemokine (C-X-C motif) ligand 12\u22121.450.006\u22121.420.046Il6ra16194Interleukin 6 receptor, alpha\u22121.160.128\u22121.150.037Tgfb121803Transforming growth factor, beta 1\u22121.150.1631.020.914Tgfb221808Transforming growth factor, beta 2\u22121.150.306\u22121.070.584Il1816173Interleukin 18\u22121.150.224\u22121.010.937D17Wsu104e28106DNA segment, Chr 17, Wayne State University 104,  expressed (previously known as IL25)\u22121.030.794\u22121.160.088Lgals116852Lectin, galactose binding, soluble 11.050.7741.080.590Lcn216819Lipocalin 21.110.2281.200.179Mif17319Macrophage migration inhibitory factor1.180.0761.170.137Vascular  functionAdm11535Adrenomedullin\u22121.410.003\u22121.500.000Agt11606Angiotensinogen\u22121.220.161\u22121.150.150Serpinf120317Serine (or cysteine) peptidase inhibitor, clade F, member 1  (previously known as pigment epithelium derived factor)\u22121.150.237\u22121.250.187Vegfa22339Vascular endothelial growth factor A\u22121.030.724\u22121.080.644Apln30878Apelin1.010.9691.020.790Serpine118787Serine (or cysteine) peptidase inhibitor, clade E, member 1  (previously known as plasminogen activator inhibitor-1)\u22121.010.8971.270.013AntioxidantSod320657Superoxide dismutase 3, extracellular\u22121.090.500\u22121.390.036Mt117748Metallothionein 1\u22121.110.577\u22121.120.417Extracellular  matrix  componentNid218074Nidogen 2\u22121.640.002\u22121.960.003Sparc20692Secreted acidic cysteine rich glycoprotein\u22121.630.000\u22121.560.000Col5a212832Procollagen, type V, alpha 2\u22121.620.000\u22121.620.003Col5a112831Procollagen, type V, alpha 1\u22121.590.003\u22121.480.013Col1a212843Procollagen, type I, alpha 2\u22121.640.020\u22121.400.002Col1a112842Procollagen, type I, alpha 1\u22121.600.065\u22121.380.020Col3a112825Procollagen, type III, alpha 1\u22121.520.024\u22121.590.002Col4a512830Procollagen, type IV, alpha 5\u22121.440.034\u22121.370.019Col6a212834Procollagen, type VI, alpha 2\u22121.320.011\u22121.190.108Col6a112833Procollagen, type VI, alpha 1\u22121.310.1171.050.740Col5a353867Procollagen, type V, alpha 3\u22121.200.046\u22121.290.076Col4a112826Procollagen, type IV, alpha 1\u22121.160.114\u22121.160.069Col4a212827Procollagen, type IV, alpha 2\u22121.160.153\u22121.290.003Nid118073Nidogen 1\u22121.040.7231.040.767Col6a312835Procollagen, type VI, alpha 3\u22121.030.690\u22121.010.960Extracellular  matrix  processingMmp317392Matrix metalloproteinase 3\u22122.010.003\u22122.020.013Lox16948Lysyl oxidase\u22121.870.005\u22122.170.002Timp221858Tissue inhibitor of metalloproteinase 2\u22121.810.002\u22121.920.000Mmp217390Matrix metalloproteinase 2\u22121.440.054\u22121.300.100Timp121857Tissue inhibitor of metalloproteinase 1\u22121.320.095\u22121.300.055Pcolce18542Procollagen C-endopeptidase enhancer protein\u22121.310.029\u22121.480.002Mmp1958223Matrix metalloproteinase 19\u22121.010.848\u22121.080.347Mmp1117385Matrix metalloproteinase 111.050.860\u22121.120.413Timp4110595Tissue inhibitor of metalloproteinase 41.060.6721.250.055MiscellaneousGal14419Galanin\u22123.040.000\u22122.890.000Gsn227753Gelsolin\u22121.630.000\u22121.520.000Ppic19038Peptidylprolyl isomerase C\u22121.270.049\u22121.380.001Mean value of microarray data (n\u2009=\u20095)Rosi\/basal: the effect with low insulin\/glucose; RosiIG\/IG: the effect with high insulin\/glucoseInterestingly, the general downregulation of adipokines was also observed for other genes encoding secreted proteins. We used Gene Ontology to cluster the expressed genes on the microarray into secreted protein encoding genes and intracellular\/unknown location protein-encoding genes (additional gene expression information is available at www.foodbioactives.nl). In the first cluster, more genes were significantly downregulated (Fig.\u00a03a and c). Of 477 genes, 24.9 and 25.8% were significantly downregulated, while 9.6 and 6.7% were upregulated by Rosi and RosiIG, respectively. Of the upregulated genes, most encode multi-location proteins, which can also reside in the nucleus, mitochondria and\/or membranes. If these genes are omitted from the analysis, only seven genes were upregulated (1.5%). In the cellular protein cluster, by contrast, the majority of the regulated genes were upregulated (Fig.\u00a03b and d). Of 3,382 genes, 10.3 and 10.1% were significantly upregulated, while 5.1 and 5.0% were downregulated by Rosi and RosiIG treatment, respectively.\nFig.\u00a03Regulation pattern by rosiglitazone of genes encoding extracellular proteins and of genes encoding intracellular proteins or proteins of unknown location. a, c: extracellular pattern; b, d: intracellular pattern. a, b: rosiglitazone with low insulin\/glucose; c, d: rosiglitazone with high insulin\/glucoseTo complement array data, we used 2D and 1D gel electrophoresis and western blotting to examine the effect of rosiglitazone on secretion. With regard to complement C3, a 30% reduction of secretion by Rosi or RosiIG treatment was observed on the 1D gel (Fig.\u00a04). This is less than the change observed at the gene expression level. Nine spots on the 2D gel were identified as adipsin. We assessed the changes for individual spots, as well as for all spots in total. In both cases, the decrease effected by rosiglitazone at the gene expression level was similar to the decrease found at the secreted protein level with RosiIG treatment. However, this decrease at the protein level was less prominent with Rosi treatment (Fig.\u00a05). The secretion of adiponectin was also analysed by western blotting. A slight increase with rosiglitazone was detected (Fig.\u00a06), although there was no change at the gene expression level.\nFig.\u00a04The effect of rosiglitazone on the secretion of complement C3 as measured by 1D gel analysis. The marked bands (arrows) were identified as C3. One representative image is shown. A sample treated by Brefeldin A (+BFA) was used to verify secretionFig.\u00a05The effect of rosiglitazone on the secretion of adipsin as measured by 2D gel analysis. A section of one representative 2D gel image is shown for an individual adipsin spot (squares) from a low glucose and insulin control condition (Basal), b rosiglitazone with low insulin and glucose condition (Rosi), c high glucose and insulin control condition (IG) and d rosiglitazone with high insulin and glucose condition (RosiIG). e Total quantity of adipsin in the culture medium of 3T3-L1 adipocytes expressed in arbitrary units (AU)Fig.\u00a06The effect of rosiglitazone on the secretion of adiponectin as measured by western blotting. Data are expressed as mean \u00b1 SD (n\u2009=\u20093) and one representative western blot image is shown. *p\u2009<\u20090.05 compared with Basal condition (t test); **p\u2009<\u20090.01 compared with IG condition (t test). Basal, low glucose and insulin control condition; Rosi, rosiglitazone with low insulin and glucose condition; IG, high glucose and insulin control condition; RosiIG, rosiglitazone with high insulin and glucose condition\nGeneral confirmation of the microarray data by quantitative real-time RT-PCR analysis To confirm our microarray data, we checked the expression of 22 genes that encode secreted proteins or are related to lipid metabolism, by Q-PCR, using Rps15 as reference. Rps15 was chosen because of its constant expression behaviour on the arrays. A gene was selected as target gene because either itself or its family member showed changed expression based on microarray data. The comparison showed a strong correlation between array data and Q-PCR data (ESM Fig.\u00a01), with Pearson correlation coefficients of 0.987 and 0.986 for Rosi\/basal and RosiIG\/IG, respectively.\nThe effect of rosiglitazone on lipolysis We assessed the glycerol level in the culture medium as a measure of the lipolysis of adipocytes. RosiIG treatment significantly increased the release of glycerol from 3T3-L1 mature adipocytes, whereas glycerol release was significantly decreased by Rosi treatment (Fig.\u00a07).\nFig.\u00a07The effect of rosiglitazone on the culture medium glycerol levels. Data are expressed as mean\u00b1SD (n\u2009=\u20093). *p\u2009<\u20090.05 compared with IG condition (t test); **p\u2009<\u20090.01 compared with Basal condition (t test). Basal, low glucose and insulin control condition; Rosi, rosiglitazone with low insulin and glucose condition; IG, high glucose and insulin control condition; RosiIG, rosiglitazone with high insulin and glucose condition\nDiscussion\nAs a PPARG agonist, rosiglitazone stimulates adipogenesis, which is accompanied by an increasing lipid content of the differentiating cells. However, our results showed that rosiglitazone does not induce an increase of the lipid content, but decreases it in mature adipocytes at high insulin\/glucose level. The pathway analysis based on transcriptome data points out that an altered energy metabolism may underlie this phenomenon.\nAlthough the level of insulin\/glucose has some influence, the effect of rosiglitazone on the transcriptome was clearly dominant. The most significantly regulated pathways by rosiglitazone in 3T3-L1 adipocytes were fatty acid oxidation, the TCA cycle and oxidative phosphorylation. Besides an upregulation of fatty acid catabolism pathways, our data indicate that rosiglitazone encourages adipocytes to utilise fatty acids for energy production and spares glucose and amino acids by enhancing glycogen synthesis and inhibiting amino acid catabolism pathways. This is in line with the upregulation of Ucp2 and the downregulation of Scd1 expression. The increase in the level of UCP2 is correlated with a shift in substrate utilisation in favour of lipids as the dominant metabolic fuel [28]. SCD1 is involved in fatty acid synthesis, but it has also been shown that SCD1 deficiency activates metabolic pathways that promote beta-oxidation and decrease lipogenesis [29].\nOur conclusions regarding energy metabolism are consistent with recent studies. Mitochondrial changes have been shown in response to TZD treatment in 3T3-L1 adipocytes [30], human adipocytes in vitro [31] and mouse adipocytes in vivo [12]. These studies show that TZDs, including rosiglitazone, upregulate fatty acid oxidation by increasing mitochondrial protein and gene expression, but not in a trans-differentiation manner [12, 30]. This has been confirmed by direct measurement of labelled palmitic acid oxidation [12] or indirect calorimetric calculation [32]. We also found that among the genes involved in protein biosynthesis, more than 80% of those upregulated by rosiglitazone were genes encoding mitochondrial ribosomal proteins. This is consistent with an increase of mitochondrial biogenesis and remodelling [12]. We speculate that the maintenance of increased mitochondrial mass and function utilises (part of) the energy that the cell no longer needs for lipid synthesis under rosiglitazone treatment.\nA decrease in lipogenesis pathways accompanies the increased fatty acid catabolism pathways caused by rosiglitazone in mature adipocytes, as shown by decreased expression of genes encoding triacylglycerol synthesis enzymes and the genes encoding lipase. The downregulation of Fasn by TZD has also been observed in brown adipocytes [33]. Caveolin-1 (CAV1) is an important protein for efficient lipid droplet formation [34]. The downregulation of its gene expression supports a decrease of lipid accumulation. The upregulation of adipocyte differentiation related protein gene (Adfp), which encodes a lipid droplet-associated protein, seems to conflict with the decreased lipogenesis observed by us (Table\u00a01). However, in mature 3T3-L1 adipocytes, ADFP is spatially associated with lipid droplets subjected to lipolysis and is suggested to play a role in lipolysis [35]. Furthermore, the protein level of ADFP in mature adipocytes is not determined by the mRNA expression, but by the amount of micro-lipid droplets [35]. Therefore the increase in Adfp expression might also be interpreted as being in agreement with upregulated lipolysis.\nThe pentose phosphate pathway provides NADPH to be used in reductive biosynthesis, including fatty acid biosynthesis [36]. Rosiglitazone enhances the pentose phosphate pathway, but inhibits fatty acid biosynthesis, while stimulating other NADPH-dependent pathways, such as glutathione detoxification, cholesterol and steroid biosynthesis, and prostaglandin metabolism (carbonyl reductase 3). The excess of NADPH may be a driving force behind the upregulation of these pathways by rosiglitazone.\nThe present study indicates that in mature 3T3-L1 adipocytes rosiglitazone enhances fatty acid catabolism, increases lipolysis and reduces lipogenesis pathways. This may lead to a net decrease of lipid content of the cells. Indeed, we observed increased lipolysis expressed in terms of an increased glycerol level in the medium and a decreased lipid content of the cells under RosiIG treatment. This is in line with the increased lipolysis effected by rosiglitazone in a recent study on rat white adipose tissue [37]. We did not observe increased lipolysis under Rosi treatment, where in fact the glycerol level was decreased rather than increased. The difference implies that the insulin\/glucose level alters the effect of rosiglitazone. It is likely that at low insulin\/glucose level, the \u2018starved\u2019 adipocytes use glycerol as an energy source, rather than releasing it into the medium. However, it should be noted that low insulin\/glucose is not a realistic situation in vivo when rosiglitazone is administered. The decreased lipid content is consistent with the physiological effects of TZDs in both animal models [10, 38] and in humans [9], where TZDs increase the number of small adipocytes. In adipose tissue treated with TZDs, mature adipocytes can become smaller by actively burning fat, in parallel to the emerging of small immature adipocytes due to preadipocyte differentiation. With rosiglitazone, mature adipocytes take fatty acids for burning rather than for storage. This still fits the \u2018fatty acid steal\u2019 hypothesis [1] and can explain part of the glucose-lowering effects of rosiglitazone.\nRosiglitazone affects the function of mature adipocytes not only with regard to fat metabolism, but also with regard to adipokine secretion. Despite the limitations of the present study, namely that a mid-size array was used and that the expression changes of several genes could not be detected due to sensitivity, our data at both the mRNA level and the protein level confirmed that rosiglitazone represses adipokine expression, except adiponectin and Apoe. Since the majority of adipokines are negatively correlated with insulin sensitivity [39], the general repression may correct the disturbance of insulin sensitivity caused by obesity. Furthermore, our transcriptome analysis suggests that rosiglitazone induces a general decrease in expression of genes encoding secreted proteins. This implies a general mechanism, in which energy redistribution might be involved.\nWe observed that repression at the secretion level was less significant than at the gene expression level. One explanation is that the effect on protein lags behind the effect on mRNA. Another reason might be the influence of insulin. We have recently studied the role of insulin in 3T3-L1 adipocyte secretion [19]. We observed that at a low insulin level, the secretion of proteins is already suppressed. Consequently, the inhibitory effect of rosiglitazone on secretion is less prominent in the low insulin condition.\nIn contrast to most adipokines, adiponectin is reversely correlated with obesity, and its circulating level is upregulated by TZDs [40]. A functional PPAR-responsive element (PPRE) in the adiponectin promoter could be the reason for the stimulation [41]. We observed an upregulation only at the secretion level, but not at gene expression level. This is in line with the conclusion from another study, that although plasma levels of adiponectin were upregulated by TZDs, there was no significant effect on adiponectin gene expression [16].\nRosiglitazone is a potent PPARG agonist. It seems logical to assume that all these effects are mediated through regulation of PPARG. Indeed, we observed that rosiglitazone induced a significant decrease of Pparg expression in mature 3T3-L1 adipocytes. This is in contrast to an overwhelming increase during adipogenesis, but it is in line with several reports [42\u201344]. With respect to PPARG downstream effects, we found here that two PPRE-bearing genes, Cav1 and Scd1, were downregulated, and two other genes, adiponectin and lipoprotein lipase, remained unchanged, while most PPRE-bearing genes [45], such as CD36 antigen, fatty acid binding protein 4, adipocyte and acyl-coenzyme A oxidase 1, palmitoyl, were upregulated by rosiglitazone (Tables\u00a01 and 2). The effect of TZDs on Scd1 in mature adipocytes is opposite to that in preadipocytes [46], and the effect on Cav1 is in contrast to the reports in other cell types [47, 48]. It has been known that PPARG acts with different transcriptional partners, co-repressors and\/or co-activators as a complex, to activate or repress the expression of target genes [49, 50]. Possibly, the PPARG complex is modulated by rosiglitazone in mature adipocytes in a manner that is different from that occurring in preadipocytes and other cell types.\nIn conclusion, we have shown that rosiglitazone, in contrast to the stimulation of adipogenesis in preadipocytes, reduces adiposity of mature adipocytes by increasing fatty acid catabolism pathways. In addition, rosiglitazone represses the expression and secretion of adipokines of mature adipocytes. Our findings suggest that the change of adiposity as seen in vivo [13] may reflect a shift in balance between the effects of TZDs on preadipocytes and those on mature adipocytes, while the changes in circulating adipokine levels primarily result from an effect on mature adipocytes. These aspects all contribute to the therapeutic effects of TZDs.\nElectronic Supplementary Material\nBelow is the link to the electronic supplementary material \nTable 1\nQ-PCR primer sequences (DOC 26\u00a0kb)\nFig. 1\nComparison between microarray and quantitative real time RT-PCR analysis. The mean of the fold-change is shown. n\u2009=\u20095 for array n\u2009=\u20096 for RT-PCR. The lines at 1.3 and \u22121.3 fold changes indicate thresholds of differentially expression (DOC 51\u00a0kb)","keyphrases":["adipokine","thiazolidinediones","mature adipocytes","adipose tissue","secretion","microarray","energy metabolism","lipogenesis","lipolysis"],"prmu":["P","P","P","P","P","P","P","P","P"]}
{"id":"Ann_Biomed_Eng-2-2-1705476","title":"Autonomous Reovirus Strain Classification Using Filament-Coupled Antibodies\n","text":"We previously described a filament-based antibody recognition assay (FARA) that generates ELISA-like sandwich structures immobilized on a filament. FARA allows the coupling of antibodies to precise locations along a filament, on-line fluorescence detection of captured pathogen, and feedback-directed filament motion. These properties suggest that this approach might be useful as an automated means to rapidly classify unknown pathogens. In this report, we describe validation of the novel decision tree aspect of this technology using mammalian reovirus. Based on available antibodies, we developed a decision tree algorithm to detect virus with increasing specificity at each level of the tree. Using three strains of reovirus and a bacteriophage control, our system correctly classified the reovirus strains at a concentration of 2 \u00d7 1012 virions ml\u22121 and M13K07 phage at 3 \u00d7 1011 virions ml\u22121. Classification of reovirus strain type 3 Dearing (T3D) required three levels of testing: general reovirus classification in level 1, serotype 3 classification in level 2, and final T3D strain classification in level 3. Strain T3SA + also required three levels of testing before a final classification was returned in level 3. Classification of strain type 1 Lang (T1L) required two levels of testing. M13K07 phage detection required only one level of testing for classification. These results indicate that automated pathogen classification using FARA is feasible. Furthermore, the simplicity of the design could be exploited for development of more complex sub-classification networks with additional levels and branches.\nIntroduction\nIdentification of specific pathogens is essential for the selection of pathogen-specific treatments, minimizing the spread of infection, and monitoring for long-term complications. Currently, specific pathogen identification is achieved through available RT-PCR and antibody-based strategies. In the clinic, these tests are usually applied consecutively to evaluate for the presence of the most likely to least likely pathogen based on patient findings. An automated classification strategy that is less dependent on clinical knowledge but achieves rapid accurate identification of a single pathogen from among a group of possible pathogens is currently unavailable.\nOur previously described filament-based antibody recognition assay (FARA) employs antibodies immobilized at known locations along a filament to detect specific pathogens.9,10 The filament is pulled through a series of small reaction chambers, and pathogens, if present, are captured by filament-bound antibodies. Detection of pathogen binding is achieved by using a fluorescently labeled second antibody specific for the pathogen.\nFARA was first reported using immobilized anti-M13K07 antibody to detect M13K07 phage.10 This virus and antibody pair provided a well-characterized test system to demonstrate the feasibility of a filament-based, pathogen-detection platform. However, in this first generation approach, the filaments were removed from the system for fluorescence scanning. Subsequent improvements to FARA include an integrated fluorescence detector and a feedback algorithm to control filament position.9 The integrated detector enables adaptive pathogen detection in which regions of interest along the filament can be reincubated in the appropriate reaction chambers to increase filament fluorescence when the initial signal is low.\nIn this report, we describe a small-scale test of the use of a simple classification tree together with feedback-controlled FARA to identify four viruses. The feedback feature of online FARA is used to guide the selection of subsequent tests within the classification tree. Unnecessary tests are not performed, and each subsequent test becomes more specific for a single pathogen. These results establish FARA as a robust platform for classification of diverse pathogens.\nMaterials and Methods\nCells and Viruses\nMurine L929 (L) cells were cultured in suspension in Joklik\u2019s modified Eagle\u2019s medium supplemented to contain 5% fetal bovine serum, 2\u00a0mM l-glutamine, 100\u00a0U\u00a0mL\u22121 penicillin, 100\u00a0\u03bcg\u00a0mL\u22121 streptomycin, and 0.25\u00a0g\u00a0mL\u22121 amphotericin-B. Reovirus strains T1L and T3D are laboratory stocks. T3SA\u00a0+\u00a0is a monoreassortant virus isolated from L cells co-infected with T1L and T3C44MA.1 T3SA\u00a0+\u00a0contains the S1 gene segment from the type 3 parental strain and all other gene segments from T1L.1 Reovirus particles were purified as previously described.1,3,6 L cells were inoculated with second-passage L-cell lysate stocks of twice plaque-purified reovirus at a multiplicity of infection of 10 plaque-forming units per cell. Virus was purified from infected cells by freon extraction and CsCl-gradient centrifugation. Purified M13K07 virus was obtained from the Vanderbilt Molecular Recognition Core.\nAntibodies\nMouse monoclonal antibodies 4F2, 5C6, 8H6, and 9BG5 specific for reovirus proteins2,12,13 (Table\u00a01) were purified from mouse hybridoma supernatants using Protein A column chromatography. Anti-M13K07 monoclonal antibody was purchased from Amersham Biosciences (Piscataway, NJ).\nTable\u00a01.Antibody specificity.AntibodyProtein specificityReovirus strainT1LT3DT3SA+5C6T1 \u03c31+\u2212\u22129BG5T3 \u03c31\u2212++8H6T1 and T3\u00a0\u03bc1+++4F2T3D \u03c33\u2212+\u2212\nAntibodies 4F2 and 8H6 were used for fluorescence detection of virus (step 4, Table\u00a02) and were labeled with Alexa Fluor 555 or Alexa Fluor 647 (AF555 or AF647, Molecular Probes, Eugene, OR), respectively, according to the manufacturer\u2019s instructions (Molecular Probes). Labeled antibodies were purified using PD-10 size-exclusion chromatography (Amersham Biosciences). Antibody concentration and number of fluores per labeled antibody were determined by using absorbance measurements at 280\u00a0nm and the peak absorbance wavelength of each label. Aliquots of both labeled and unlabeled antibodies were stored at \u221220\u00b0C, and working solutions were kept at 4\u00b0C. Final antibody concentration was adjusted immediately prior to experiments.\nTable\u00a02.The five reaction chambers in FARA processingChamberDescriptionSolutionIncubation timeChamber ID \/ Volume (mm \/ \u03bcl)1Block\/Wash FilamentPBS-Ta15\u00a0min2\/2352Virus IncubationUnknown Virus40\u00a0min1\/60 3WashPBS-T1\u00a0min2\/2354Detecting Antibody IncubationFluorescently Labeled Detecting Antibody5\u00a0min1\/60 5WashPBS-T1\u00a0min2\/235aTween-20 (0.1%) was added to PBS as a blocking agent.\nFilament Preparation\nCapture antibodies were passively adsorbed to the filament surface in groups of three by placing the filament across the concave teeth of a PhastGel sample applicator (Amersham Biosciences) (Fig.\u00a01). Three capture-antibody regions, corresponding to the three levels of testing, were prepared along each filament using three applicator combs glued end to end. Antibody solution pipetted onto the filament without the comb spread unimpeded along the filament. Surface tension within the teeth of the comb overcame this tendency and produced a small, distinct circumferential band of immobilized antibody. Antibody was spotted in a volume of 0.75\u00a0\u03bcL and allowed to passively adsorb to the filament for 45\u00a0min in a humidified box. Following incubation at 25\u00b0C, filaments were rinsed in phosphate buffered saline with 0.1% Tween 20 (PBS-T) and threaded through the reaction chambers for virus detection experiments. Preliminary experiments were performed to determine the optimal concentration of each capture antibody.\nFigure\u00a01.Schematic of a device for adsorption of capture antibodies to the filament. The filament is placed within the concave teeth of a PhastGel applicator to localize the spotted antibody to a small circumferential band around the filament. \nRed fingernail polish (Poisonberry, Noxel Corporartion, Hunt Valley, MO) was used as a visible and fluorescent fiducial marker to identify the leading and trailing edges of capture-antibody regions during experiments and during laser scanning. The polish was applied by pipette between the teeth of the comb flanking the antibody region. A simple bar code system was used to identify each of the three capture-antibody regions. The first and second regions were preceded by a single fiducial marker, and the third region was preceded by two fiducial markers.\nMicro-reaction Chambers\nGlass microreaction chambers were fashioned from 0.25\u00a0inch stock tubing into 75\u00a0mm lengths. The ends were flared outward to facilitate movement of the filament through the chambers. Interior diameters of the chambers were 1 or 2\u00a0mm depending on the required reaction volume (Table\u00a02). Chambers were carefully positioned in a straight line on the top of a horizontal aluminum stage using machined aluminum mounts.9 Fine adjustment of chamber position was achieved by using oblong mounting holes on each mount. PBS-T was used in all wash chambers and for virus and labeled antibody solutions. Reovirus was used at a concentration of 2\u00a0\u00d7\u00a01012\u00a0virions\u00a0mL\u22121, and phage M13K07 was used at a concentration of 3\u00a0\u00d7\u00a01011\u00a0virions\u00a0mL\u22121. Detecting antibodies were present in the detecting-antibody chamber at a concentration of approximately 40\u00a0\u03bcg\u00a0mL\u22121 for each antibody in the three antibody mixture. Solutions were added to appropriate chambers at the initiation of experiments and used for all three levels of testing (Table\u00a02). If testing proceeded to level three, fluid loss from the chambers was monitored and replenished if necessary.\nFilament Control\nMovement of the filament and, therefore, the antibody bands through the chambers, was achieved by using a rotary stage to wind or unwind the filament around a spindle. A small weight was attached to the opposite end of the filament to maintain a constant tension. Filament positioning to within several microns was achieved by using a rotary stage encoder from Yaskawa Instruments (Waukegan, IL) and a custom control algorithm written as a LabView Virtual Instrument (VI) (National Instruments, Austin, TX). Parameters including filament speed and residence times within chambers were controlled by using the LabView software interface.\nBetween chambers, the speed of the filament was 1\u00a0cm\u00a0sec\u22121. Within each chamber, the capture-antibody region undergoing processing was oscillated 2.5\u00a0cm back and forth at a speed of 1\u00a0cm sec\u22121 to increase interactions between immobilized molecular structures attached to the filament and the molecular species in solution. Within the detector, filament speed was 4\u00a0cm\u00a0sec\u22121.\nLasers and PMTs\nFilament fluorescence was measured by passing the filament between two diode lasers. The two laser excitation sources were attached to either side of a detection chamber (Fig.\u00a02). Laser 1 (638\u00a0nm, 25\u00a0mW diode laser; Coherent, Santa Clara, CA) was used to excite the antibody tag AF647. Laser 2 (532\u00a0nm, 20\u00a0mW diode-pumped, solid state laser; B&W Tek, Inc., Newark, DE) was used to excite the antibody tag AF555. The effective power of laser 1 was reduced to approximately 5\u00a0mW by using a polarizer and an excitation slit. Laser 2 was not attenuated. Two Hamamatsu R928 photomultiplier tubes (PMT) were attached to the top and bottom of the sample chamber and powered by 850 and 800\u00a0V signals for the AF647 and AF555 channels, respectively. Current from the PMT was converted to voltage by using transimpedance amplifiers that amplified the signal by a factor of 106 for AF647 and 105 for AF555. Voltage was sampled at a rate of 800 samples\u00a0sec\u22121 by using a digital acquisition board from National Instruments (DaqPAD 6020E). Fluorescence signal was acquired as a function of filament position by using LabView. Following fluorescence scanning for most experiments, filament regions were cut and scanned again in a microarray flatbed scanner (GenePix 4000B, Axon Instruments, Union City, CA).\nFigure\u00a02.Schematic of lasers and photomultipliers (PMT) to detect online fluorescence of the filament. The filament is moved (arrow) through a rectangular sample chamber with a laser excitation source attached to either side. Photomultiplier tubes are attached to the top and bottom of the chamber.\nFilters\nFilter sets were placed in the light path between the sample chamber and the photomultipliers (Fig.\u00a02). Long-pass filters with cutoffs at 685\u00a0nm (Chroma, Rockingham, VT) and 665\u00a0nm (Melles Griot, Rochester, NY) were combined to reduce reflected laser light from the AF647 laser. For the AF555 channel, two long-pass filters (570\u00a0nm cutoff, Melles Griot) were combined with a bandpass filter centered at 565\u00a0nm (30\u00a0nm bandwidth, Chroma) to reduce reflected light.\nClassification Algorithm\nThe LabView program coordinated all filament movement, filament scanning, and feedback control. Elements of the feedback control are described by the nodes of the decision tree used to classify the viruses shown in Fig.\u00a03. The fluorescent fiducial markers on either side of the capture-antibody region produced characteristically sharp emission peaks. Since the physical location of the immobilized antibody positions between the markers was known, experimental conclusions were based on the distance of the first detected peak from the initial fluorescent marker.\nFigure\u00a03.Decision-tree algorithm to classify virus strains. The filament control program enters the decision tree from the top at level 1 and, based on the type of virus found at each level, follows different branches of the decision tree. When reovirus is detected at level one, the filament is moved forward to the next antibody region for level 2 testing. If a serotype 3 reovirus strain is detected (T3D or T3SA+\u00a0) at level 2, testing continues to level 3 to distinguish between these strains. \nThe filament-control program was designed to find each peak, calculate its location along the filament, and make a decision about additional tests. Spatially localized fluorescence from the filament was measured as a 0\u201314\u00a0V signal from the transimpedance amplifier. Voltage data from the scan were used as input data by a peak-detection function in LabView. The LabView peak detector fit a quadratic polynomial to sequential sets of points depending on a width parameter entered by the user. Data were then compared to a threshold parameter, also entered by the user, to identify peaks. A binary decision to stop or continue testing was based on the location and number of peaks found. Peak-detection threshold parameters were defined as 0.3\u00a0V (approximately three times background) with a width of 30 data points, which corresponded to a width slightly larger than the physical width of the comb tooth.\nA common set of parameters was used in all experiments based on previous work with M13K07.10 A filament region containing three capture antibodies and two fiducial markers was incubated within each chamber for the times shown in Table\u00a02. Each of the capture-antibody regions contained a PBS negative control in the first position. In level one the second position was a mixture of 9BG5 and 5C6 (0.25\u00a0mg\u00a0mL\u22121, 0.2\u00a0mg\u00a0mL\u22121). The third position was anti-M13K07 (0.5\u00a0mg\u00a0mL\u22121). In levels two and three, the negative control position was followed by 9BG5 (mg\u00a0mL\u22121) in the second position and 5C6 (0.2\u00a0mg\u00a0mL\u22121) in the third position.\nThe logic encoded in the decision tree shown in Fig.\u00a03 was followed. In level 1, if fluorescence was detected from the anti-M13K07 position of the first region of the filament, a classification of M13K07 virus was made, and no further testing was conducted. If fluorescence was detected in the 9BG5\/5C6 region, a classification of reovirus was made and the filament was advanced to the appropriate region for level 2 testing, where a more specific test for reovirus was performed. In level 2 testing, fluorescence from the 5C6 region indicated a serotype 1 reovirus, which in this scheme corresponded to reovirus T1L. A classification of T1L represented an endpoint in the decision tree, and testing was discontinued. Fluorescence from the 9BG5 region indicated the presence of a serotype 3 reovirus, and the program advanced the filament for further subtyping in level 3. Level 3 testing used AF555 conjugated 4F2 antibody as the detecting antibody and a second detection channel with a green excitation laser. If fluorescence was found in the 9BG5 region using this channel, a classification of reovirus T3D was made and testing along that branch ended. If no fluorescence was detected from the second channel for that region, the program ended with a classification of reovirus T3SA+\u00a0.\nIf no peaks were found between the fiducial markers, a message was generated indicating that no virus was found. Similarly, if a peak was detected in the negative control region of the filament, a warning message was generated. Although theoretically possible, this situation did not arise during testing.\nResults\nFARA utilizes a polyester filament with circumferential bands of immobilized antibody that is passed through a series of five glass micro-reaction chambers that are similar to the five major steps of an ELISA (Table\u00a02). In the first reaction chamber of the FARA approach employed in this study, capture antibody coupled to the filament was rehydrated. In this chamber the filament also was blocked to prevent non-specific binding of virus. In the second chamber, the immobilized capture antibody was incubated with virus solution. In this chamber, if virus in solution bound to antibody coupled to the filament, then the virus became coupled to the filament via this interaction. In the third chamber, non-specifically bound virus was removed by washing before incubation with a fluorescently labeled detecting antibody in the fourth chamber. A final wash in the fifth chamber removed non-specifically bound detecting antibody. The capture antibody region was then passed through an integrated fluorescence detector.\nIn these experiments, the test virus was classified with greater specificity at each level of a decision tree (Fig.\u00a03). Representative fluorescence signals in volts obtained during testing for phage M13K07 and reoviruses T1L, T3D, and T3SA+\u00a0are shown in Fig.\u00a04. Labels for the PBS control position and the antibodies in each capture antibody position are shown adjacent to the filament. The specificity of each test antibody for its corresponding virus was high, with little or no cross-reactivity. No signal was detected in the PBS negative-control position.\nFigure\u00a04.Fluorescence intensity in volts as a function of filament position for M13K07 phage (A) and reoviruses T1L (B), T3D (C), and T3SA+\u00a0(D). The immobilized capture antibody positions on the filament are labeled within each panel. Captured virus was detected by a fluorescently labeled second antibody (anti-M13K07 [red], 8H6 [red], or 4F2 [green]). Corresponding images of the scanned filaments are shown at the top of each graph. Each panel shows a representative experiment of three performed. \nDetection of M13K07 was achieved by level 1 testing. Strong fluorescence was observed in the anti-M13K07 position but not in the 9BG5\/5C6 or PBS positions (Fig.\u00a04a). This finding indicated capture of the phage M13K07 by the anti-M13K07 capture antibody. For detection of M13K07, AF647 conjugated anti-M13K07 was used. Because a classification of M13K07 was made, the other two capture regions of the filament were not evaluated.\nDetection of the reovirus strains employed in this study required analysis beyond level 1. Level 1 testing indicated the presence of a reovirus for all three strains with a peak in the second position corresponding to virus capture by the 9BG5\/5C6 antibody mixture. In level 2 testing, strain T1L was detected in the 5C6 antibody position but not in the 9BG5 or PBS positions (Fig.\u00a04b). In this case, the capture and detecting antibodies differed. Because a reovirus T1L classification was made, the third capture region of the filament was not evaluated.\nFor reovirus T3D, level 1 testing indicated the presence of a reovirus with a peak in the second position corresponding to virus capture by 9BG5\/5C6 (data not shown). Level 2 testing showed a distinct peak in the 9BG5 antibody position (Fig.\u00a04c, red curve). In this case, 8H6 antibody labeled with AF647 was the detecting antibody indicating a type 3 reovirus. Level 3 testing showed a distinct peak in the 9BG5 antibody position (Fig.\u00a04c, green curve). In this case, 4F2 antibody labeled with AF555 was the detecting antibody. Neither curve showed cross reactivity of reovirus T3D with the immobilized 5C6 region or the negative-control PBS region.\nFor reovirus T3SA+\u00a0, level 1 testing indicated the presence of a reovirus with a peak in the second position corresponding to virus capture by 9BG5\/5C6 (data not shown). Level 2 testing yielded a distinct peak in the 9BG5 antibody position (Fig.\u00a04d, red curve), like the result obtained with T3D. However, level 3 testing with 4F2 did not yield a positive signal in the 9BG5 antibody position, indicating that the captured virus was not T3D. No detectable cross reactivity was found in the 5C6 antibody region or the negative-control PBS region. Thus, the virus was classified as T3SA+.\nDiscussion\nIn this study, we demonstrated the feasibility of a FARA pathogen-classification approach using a well-characterized virus system. Three reovirus strains and one phage were successfully classified by performing sequential antibody-binding assays directed by the decision tree. The structure of the decision tree was based on published characteristics of each virus strain and the known specificity of each antibody. Because the capture antibodies were arrayed at known locations along the filament, and the fluorescence of the detection antibodies was measured as a function of filament location, an increase in filament fluorescence at a particular location indicated antibody interaction with the virus. For each virus strain tested, the expected fluorescence peaks were observed, and the correct classification was made. Moreover, successful transit of all decision tree branches was demonstrated.\nThe biochemical components of FARA are similar to those used in standard ELISAs. Both assays create a dual-antibody sandwich that results in similar detection limits for M13K07 for FARA and ELISA.10 One of the virus-specific antibodies acts as the capture antibody, and the second acts as the detecting antibody. The main difference in the biochemistry of the two assays is that, in the current implementation of FARA, enzyme amplification is not utilized. Like ELISA, FARA utilizes antibodies adsorbed to the surface of a substrate to capture virus from solution. The capacity of bound antibody to retain its antigen-binding activity is essential to the success of both assays. Each of the immobilized test antibodies in our system retained functionality and bound its corresponding virus (Fig.\u00a04). The absence of peaks in the negative-control positions and in positions occupied by antibodies not specific to the test virus demonstrates minimal cross reactivity in the FARA platform employed in this study.\nWe chose reovirus as a test system to show clinical relevance and to avoid potential safety concerns in the laboratory. Reovirus has been used as a model to study mechanisms of viral pathogenesis in mice.14 Although it is a human pathogen, it is rarely associated with human disease.11 Many reovirus field-isolate strains have been characterized,4,7,8 and a broad array of antibodies are available that recognize reovirus with varying specificity.12\nWe found that not every antibody tested was suitable for use as an immobilized capture antibody. Neither antibody 8H6 nor 4F2 bound virus when immobilized on the filament, even after virus incubation time was increased to greater than 100\u00a0min (data not shown). It is possible that these antibodies undergo conformational changes when passively adsorbed to a solid substrate, rendering them inactive. Alternatively, these antibodies may be incapable of antigen binding when immobilized as a consequence of steric hindrance. Antibodies 8H6 and 4F2 recognize the \u03bc1 and \u03c33 proteins, respectively,12 which form the bulk of the viral outer capsid.5 It is possible that extension of the viral attachment protein \u03c31 may shield the \u03bc1 and \u03c33 proteins from binding to the 8H6 and 4F2 antibodies on the filament surface. This conclusion also is consistent with our observation that 9BG5 and 5C6 were both excellent capture antibodies, since they recognize the \u03c31 protein, which extends farthest from the viral capsid. The failure of the 8H6 and 4F2 antibodies to function as effective capture antibodies complicated our virus detection scheme. Although 8H6 is not serotype-specific and should bind all reovirus strains, we could not use this antibody as the immobilized capture antibody for level 1 testing. Antibodies 5C6 and 9BG5 are specific for serotype 1 and serotype 3 reovirus strains, respectively;12 therefore, we immobilized a mixture of these antibodies for detection of all reovirus isolates in level 1. Although 4F2 recognizes most type 3 strains, T3SA+\u00a0is a reassortant virus that contains a \u03c33-encoding gene segment from T1L.1 As a result, 4F2 does not recognize T3SA+\u00a0and is specific only for T3D in our scheme. Since 4F2 could not be used as a capture antibody in level 3, we incorporated a fluorescently labeled 4F2 antibody as a detecting antibody in solution.\nAlthough the nature of the biochemical interactions is similar in FARA and ELISA, the capture antibody employed in a standard ELISA is static, and solutions are changed in the well-plate in a fixed sequence. In FARA, the capture antibody is attached to a mobile substrate, and solutions are changed by positioning the filament in different solution chambers. As we have shown previously, this gives FARA a sensitivity limit similar to ELISA on the order of 107 virus particles.10 A major advantage of FARA is that it is dynamic and allows modification of processing in response to results.9 Virus incubation time with the capture antibody can be reduced to as low as one minute depending on the virus concentration and antibody affinity, reducing the overall assay duration. On the other hand, the incubation time can be increased in order to increase sensitivity.9 Moreover, because the filament is mobile, the capture antibody is brought to the virus solution, thus enabling use of capture antibodies with different specificities to interrogate a single aliquot of virus solution.\nAttachment of the capture antibody to a mobile filament in FARA makes accurate positioning of the filament and correct identification of the capture antibody regions essential for virus strain classification. These parameters are particularly important in multilevel testing of the type used in our study. A simple bar code system based on fluorescent fiducial markers was used to identify the leading edge of each of the capture antibody regions and position the filament in the reaction chambers. The pattern of the fluorescent marks also was used to identify the level of testing. Since our simple test system involved three levels, and testing always began in level 1, only searches for patterns containing one or two fluorescent marks corresponding to levels two and three were required. However, this simple approach allows for the incorporation of more complex bar codes using additional spots or patterns of spots.\nThis study demonstrates the feasibility of an automated system for diagnosing specific virus strains. Although the scheme we report is a relatively simple implementation of this system, more complex designs are possible. For example, each antibody capture region could contain additional antibodies. In addition, there is essentially no limit to the overall length of the filament, which would allow the incorporation of many additional testing regions. The sensitivity of FARA could also be increased by the use of quantum-dot labeled detection antibodies as well as a more sensitive fluorescence detection system. The most important requirement is the availability of a wide range of antibodies to the specific pathogens of interest. Such a strategy may have applications for detecting specific pathogens from complex mixtures. This approach would have both clinical and environmental applications.","keyphrases":["fluorescence detection","pathogen detection","virus detection","virus classification"],"prmu":["P","P","P","R"]}
{"id":"Diabetologia-3-1-2063563","title":"Impaired glucagon-like peptide-1-induced insulin secretion in carriers of transcription factor 7-like 2 (TCF7L2) gene polymorphisms\n","text":"Aims\/hypothesis Polymorphisms in the transcription factor 7-like 2 (TCF7L2) gene are associated with type 2 diabetes and reduced insulin secretion. The transcription factor TCF7L2 is an essential factor for glucagon-like peptide-1 (GLP-1) secretion from intestinal L cells. We studied whether a defect in the enteroinsular axis contributes to impaired insulin secretion in carriers of TCF7L2 polymorphisms.\nIntroduction\nGenome-wide scans for diabetes susceptibility genes have been performed in various populations. In early 2006, it was reported that variants in the transcription factor 7-like 2 (TCF7L2) gene were strongly associated with an increased risk of type 2 diabetes in an Icelandic, a Danish and a US population [1]. The estimated population attributable risk of type 2 diabetes of this variation lies between 10 and 25%, which is in the range of the peroxisome proliferator-activated receptor \u03b3 gene (PPAR\u03b3) Pro12Ala (25% [2]) and the potassium inwardly-rectifying channel, subfamily J, member 11 gene (KCNJ11) Glu23Lys (15% [3]) polymorphisms.\nIn the initial report of Grant et al. [1], five single nucleotide polymorphisms (SNPs) within introns 3 and 4 of the TCF7L2 gene (rs12255372, rs7903146, rs7901695, rs11196205, rs7895340) were identified to associate with an increased risk of type 2 diabetes. This finding initiated a series of reports in various populations confirming the effect of these polymorphisms within the TCF7L2 gene on the type 2 diabetes risk [4\u201312]. Recently, the T allele of rs7903146 was identified as the variant that most strongly determines the risk of type 2 diabetes [13]. In carriers of the risk alleles for SNPs within TCF7L2 several studies additionally showed an impaired insulin secretion estimated from an OGTT or IVGTT [6, 7, 9, 10]. These studies indicate that progressive loss of insulin secretion might be the essential component of the phenotype which predisposes carriers of the TCF7L2 variant to develop type 2 diabetes. The pathogenic mechanism of the impaired insulin secretion due to polymorphisms in the TCF7L2 gene is not yet clear. It could be the consequence of a reduction in beta cell mass or could reflect a dysfunction of pancreatic beta cells. Furthermore, a defect in incretin-induced stimulus secretion coupling could mediate a reduction of insulin secretion in carriers of the TCF7L2 polymorphisms. The latter hypothesis is supported by the molecular mode of action of the transcription factor TCF7L2. TCF7L2 is an essential component of the wingless-type MMTV integration site family, member 1 (WNT) signalling pathway, which is crucial for the regulation of the glucagon gene (GCG) expression and the secretion of its product GLP-1 by the intestinal endocrine L cells [14]. Therefore, an alteration in the WNT signalling pathway through polymorphisms in the TCF7L2 gene might result in an altered GLP-1 response, which in turn could lead to a lower postprandial insulin secretion.\nTo test this hypothesis we analysed the associations of the above-described TCF7L2 polymorphisms with basal GLP-1 secretion and glucose-induced GLP-1 response during an OGTT in non-diabetic participants. We further studied the influence of the TCF7L2 SNPs on insulin secretion kinetics to i.v. administered glucose during an IVGTT and a hyperglycaemic clamp. In addition, we particularly investigated the influence on GLP-1-induced insulin secretion using a combined hyperglycaemic clamp with additional GLP-1 infusion and arginine bolus [15].\nMethods\nParticipants We studied 1,110 non-diabetic participants by an OGTT (Table\u00a01). Secretion patterns of GLP-1 were obtained in a subgroup of 155 participants. The anthropometric characteristics of this subgroup are shown in Table\u00a02. All 1,110 participants were also genotyped for the following five SNPs in the TCF7L2 gene: rs7903146, rs12255372, rs7901695, rs11196205 and rs7895340.\nTable\u00a01Anthropometric and metabolic data from all 1,110 participants who underwent an OGTT\u00a0rs7903146p value (ANOVA)p value (CC vs XT)GenotypeC\/CC\/TT\/Tn54647490NGT\/IGT461\/85386\/8863\/270.0070.03Sex (female\/male)346\/200315\/15950\/400.130.64Age (years)39\u2009\u00b1\u2009140\u2009\u00b1\u2009141\u2009\u00b1\u200910.390.67BMI (kg\/m2)29.5\u2009\u00b1\u20090.428.9\u2009\u00b1\u20090.428.2\u2009\u00b1\u20091.00.510.24Plasma glucose (mmol\/l)\u00a0Fasting 5.1\u2009\u00b1\u20090.025.1\u2009\u00b1\u20090.025.2\u2009\u00b1\u20090.070.250.39\u00a02\u00a0h 6.1\u2009\u00b1\u20090.076.2\u2009\u00b1\u20090.076.7\u2009\u00b1\u20090.20.060.17Plasma insulin (pmol\/l)\u00a0Fasting62.0\u2009\u00b1\u20092.153.4\u2009\u00b1\u20091.749.8\u2009\u00b1\u20093.10.0040.001\u00a02\u00a0h442\u2009\u00b1\u200919356\u2009\u00b1\u200917372\u2009\u00b1\u2009340.120.04Insulin sensitivityOGTT (arbitrary units)16.8\u2009\u00b1\u20090.518.2\u2009\u00b1\u20090.517.8\u2009\u00b1\u20091.20.02\/0.02a0.005\/0.006aInsulin secretionOGTT (pmol\/mmol)319\u2009\u00b1\u20095301\u2009\u00b1\u20095292\u2009\u00b1\u2009100.003\/0.04b0.0009\/0.02bInsulin\/glucose ratio (pmol\/mmol)143\u2009\u00b1\u20095127\u2009\u00b1\u20095124\u2009\u00b1\u2009130.003\/0.03b0.001\/0.01bData are means\u2009\u00b1\u2009SEMp values were obtained using \u03c72 test or ANOVAAnalysis for rs12255372 and rs7901695 also revealed a significant difference in insulin secretion (p\u2009=\u20090.003 and p\u2009=\u20090.05), whereas for rs7895340 and rs11196205 no significant differences in insulin secretion were detected (p\u2009=\u20090.53 and 0.45)ap values are derived from multivariate linear regression models: adjusted for age, sex and BMIbp values are derived from multivariate linear regression models: adjusted for age, sex, BMI and insulin sensitivityTable\u00a02Anthropometric and metabolic data of the subgroup of 155 participants with additional measurements of GLP-1\u00a0rs7903146p value (ANOVA)p value (CC vs XT)GenotypeC\/CC\/TT\/Tn736715NGT\/IGT64\/948\/1910\/50.030.009Sex (female\/male)50\/2342\/258\/70.500.33Age (years)46\u2009\u00b1\u2009147\u2009\u00b1\u2009147\u2009\u00b1\u200930.990.96BMI (kg\/m2)29.8\u2009\u00b1\u20090.730.3\u2009\u00b1\u20090.727.1\u2009\u00b1\u20091.10.150.88Plasma glucose (mmol\/l)\u00a0Fasting5.2\u2009\u00b1\u20090.055.3\u2009\u00b1\u20090.075.6\u2009\u00b1\u20090.20.170.21\u00a02\u00a0h6.5\u2009\u00b1\u20090.17.0\u2009\u00b1\u20090.27.2\u2009\u00b1\u20090.50.220.10Insulin sensitivityOGTT (arbitrary units) 12.8\u2009\u00b1\u20090.713.6\u2009\u00b1\u20091.015.7\u2009\u00b1\u20092.60.74\/0.92a0.78\/0.75aInsulin secretionOGTT (pmol\/mmol)304\u2009\u00b1\u200912287\u2009\u00b1\u200913235\u2009\u00b1\u2009190.01\/0.02b0.02\/0.01bGLP-1 (pmol\/l)\u00a00\u00a0min 16.1\u2009\u00b1\u20090.917.3\u2009\u00b1\u20091.317.3\u2009\u00b1\u20092.00.910.88\u00a030\u00a0min34.1\u2009\u00b1\u20092.138.8\u2009\u00b1\u20094.038.1\u2009\u00b1\u20093.50.450.36\u00a0120\u00a0min28.9\u2009\u00b1\u20091.529.0\u2009\u00b1\u20091.728.9\u2009\u00b1\u20092.40.870.88\u00a0Fold increase 0\u201330\u00a0min2.5\u2009\u00b1\u20090.22.7\u2009\u00b1\u20090.32.4\u2009\u00b1\u20090.40.770.84Data are means\u2009\u00b1\u2009SEMp values were obtained using \u03c72 test or ANOVAAnalysis for rs7901695 also revealed a significant difference in insulin secretion (p\u2009=\u20090.02), whereas for rs7895340 and rs11196205 no significant differences in insulin secretion were detected (p\u2009=\u20090.73 and 0.76)ap values were derived from multivariate linear regression models: adjusted for age, sex and BMIbp values were derived from multivariate linear regression models: adjusted for age, sex, BMI and insulin sensitivityA subset of 210 participants were studied by an IVGTT combined with a euglycaemic\u2013hyperinsulinaemic clamp to determine insulin secretion capacity and insulin sensitivity in one test [16] (Table\u00a03).\nTable\u00a03Anthropometric and metabolic data of 210 participants who underwent a combined IVGTT and hyperinsulinaemic\u2013euglycaemic clamp\u00a0rs7903146p value (CC vs XT)GenotypeC\/CX\/Tn97113NGT\/IGT80\/1781\/320.06Sex (female\/male)59\/3864\/490.58Age (years)44\u2009\u00b1\u2009145\u2009\u00b1\u200910.29BMI (kg\/m2)29.0\u2009\u00b1\u20090.528.8\u2009\u00b1\u20090.50.82Insulin sensitivityclamp (\u03bcmol kg\u22121 min\u22121 [pmol\/l]\u22121)0.07\u2009\u00b1\u20090.010.07\u2009\u00b1\u20090.010.77Insulin secretion indexa\u00a0IVGTT C-peptide secretion (pmol\/l)7,904\u2009\u00b1\u20093067,679\u2009\u00b1\u20093230.40\u00a0IVGTT insulin secretion (pmol\/l)2,059\u2009\u00b1\u20091311,924\u2009\u00b1\u20091290.25\u00a0OGTT insulin secretion (pmol\/mmol)314\u2009\u00b1\u20098292\u2009\u00b1\u200980.04Data are means\u2009\u00b1\u2009SEMp values were obtained using \u03c72 test or unpaired t test or multivariate linear regression modelsNone of the four other SNPs in TCF7L2 (rs12255372, rs7901695, rs11196205 and rs7895340) affected significantly insulin secretion in the IVGTT (all p\u2009>\u20090.2)aAdjusted for age, sex and BMI, and insulin sensitivityAdditionally, 233 participants were studied by a hyperglycaemic clamp [17]. The participants were selected from three independent studies in the Netherlands and Germany. The German and the Dutch cohort from Utrecht consisted of NGT and IGT participants, the Dutch cohort from Hoorn consisted of IGT participants only (Table\u00a04). All participants in our studies underwent a standard OGTT to define their glucose tolerance status. Details of the study groups have been described previously [17\u201319]. In the German subgroup (n\u2009=\u200973), the hyperglycaemic clamp was continued with an additional GLP-1 and arginine administration [15, 20\u201322].\nTable\u00a04Anthropometric and metabolic data of 233 participants who underwent a hyperglycaemic clamp and of 73 participants who underwent a modified hyperglycaemic clamp with additional GLP-1 and arginine administration\u00a0GermanyThe Netherlands (Utrecht)The Netherlands (Hoorn)rs7903146p valuers7903146p valuers7903146p valueGenotypeC\/CX\/TC\/CX\/TC\/CX\/TSex (female\/male)15\/2024\/140.085\/3122\/11<0.0117\/1130\/330.27NGT\/IGT30\/527\/110.1331\/526\/70.530\/280\/66\u2013Age (years)38\u2009\u00b1\u2009240\u2009\u00b1\u200920.6647\u2009\u00b1\u2009147\u2009\u00b1\u200910.9456\u2009\u00b1\u2009158\u2009\u00b1\u200910.43BMI (kg\/m2)26.1\u2009\u00b1\u20090.925.1\u2009\u00b1\u20090.60.4125.6\u2009\u00b1\u20090.726.2\u2009\u00b1\u20090.70.4929.2\u2009\u00b1\u20090.827.9\u2009\u00b1\u20090.40.13Plasma glucose (mmol\/l)\u00a0Fasting5.1\u2009\u00b1\u20090.15.0\u2009\u00b1\u20090.10.724.6\u2009\u00b1\u20090.14.8\u2009\u00b1\u20090.10.186.6\u2009\u00b1\u20090.16.6\u2009\u00b1\u20090.10.75\u00a02\u00a0h glucose5.9\u2009\u00b1\u20090.36.5\u2009\u00b1\u20090.30.215.7\u2009\u00b1\u20090.35.6\u2009\u00b1\u20090.30.749.5\u2009\u00b1\u20090.19.5\u2009\u00b1\u20090.10.84Insulin sensitivity (\u03bcmol kg\u22121 min\u22121 [pmol\/l]\u22121)0.15\u2009\u00b1\u20090.020.15\u2009\u00b1\u20090.010.900.17\u2009\u00b1\u20090.020.18\u2009\u00b1\u20090.020.600.11\u2009\u00b1\u20090.010.14\u2009\u00b1\u20090.010.21Insulin secretion index (pmol\/l)a\u00a0First phase (sum 0\u201310\u00a0min)1,067\u2009\u00b1\u2009158815\u2009\u00b1\u20091050.18963\u2009\u00b1\u200975891\u2009\u00b1\u2009960.76699\u2009\u00b1\u200970679\u2009\u00b1\u2009720.68\u00a0Second phase308\u2009\u00b1\u200947291\u2009\u00b1\u2009410.80389\u2009\u00b1\u200942376\u2009\u00b1\u2009470.71304\u2009\u00b1\u200945303\u2009\u00b1\u2009380.18\u00a0First phase GLP-12,049\u2009\u00b1\u20092581,466\u2009\u00b1\u20091750.03n.a.n.a.n.a.n.a.\u00a0Second phase GLP-14,567\u2009\u00b1\u20095682,881\u2009\u00b1\u20093410.006n.a.n.a.n.a.n.a.\u00a0Arginine2,680\u2009\u00b1\u20092262,252\u2009\u00b1\u20091810.40n.a.n.a.n.a.n.a.Data are means\u00b1SEp values were obtained using \u03c72 test, unpaired t test or multivariate linear regression modelsCarriers of the risk allele for SNP rs12255372 showed reduced first phase (p\u2009=\u20090.02) and second phase (p\u2009=\u20090.005) GLP-1-induced insulin secretionCarriers of the risk allele for SNP rs7901695 showed reduced second phase (p\u2009=\u20090.02) GLP-1-induced insulin secretionn.a. Not availableaAdjusted for age, sex and BMI, and insulin sensitivityThe participants were not related to each other. Informed written consent for all studies was obtained from all participants, and the local ethics committee approved the protocols.\nGenotyping Detection of the TCF7L2 polymorphisms described by Grant et al. [1] was done using the TaqMan assay (Applied Biosystems, Forster City, CA, USA) in the German population. The TaqMan genotyping reaction was amplified on a GeneAmp PCR system 7000, and fluorescence was detected on an ABI PRISM 7000 sequence detector (Applied Biosystems). As a quality standard, we randomly included six positive (two homozygous wild-type allele carriers, two heterozygous and two homozygous risk allele carriers) and two negative (all components excluding DNA) sequenced controls in each TaqMan reader plate. Because all controls were correctly identified, we assumed that the genotyping error rate of this method did not exceed 0.3% [23].In the Dutch population, only rs7903146 was determined using an ABI PRISM 7900HT sequence detector (Applied Biosystems).\nOGTT At 08:00 hours, participants ingested a solution containing 75\u00a0g glucose. Venous blood samples were obtained at 0, 30, 60, 90 and 120\u00a0min for determination of plasma glucose, insulin and C-peptide concentrations and 0, 30 and 120\u00a0min for determination of GLP-1 concentrations. The participants did not take any medication known to affect glucose tolerance or insulin sensitivity. Tests were performed after an overnight fast of 12\u00a0h.\nCombined IVGTT and hyperinsulinaemic\u2013euglycaemic clamp After an overnight fast and after baseline samples had been obtained, 0.3\u00a0g\/kg bodyweight of a 20% (vol.\/vol.) glucose solution was given at time 0. Blood samples for the measurement of plasma glucose, plasma insulin and C-peptide were obtained at 2, 4, 6, 8, 10, 20, 30, 40, 50 and 60\u00a0min. After 60\u00a0min, a priming dose of insulin was given followed by an infusion (40\u00a0mU\/m2) of short-acting human insulin for 120\u00a0min. A variable infusion of 20% glucose was started to maintain the plasma glucose concentration at 5.5\u00a0mmol\/l. Blood samples for the measurement of plasma glucose were obtained at 5\u00a0min intervals throughout the clamp.\nHyperglycaemic clamp Hyperglycaemic clamps were performed at 10\u00a0mmol\/l in all participants. The Dutch NGT and IGT participants underwent a 3\u00a0h clamp. In the German NGT participants, the clamp lasted for 2\u00a0h followed by the GLP-1 and arginine stimulation (see below). After an overnight fast, the participants received an i.v. glucose bolus to acutely raise glucose levels to 10\u00a0mmol\/l. Plasma glucose levels were measured at the appropriate intervals to maintain a constant plasma glucose during the clamp. Blood samples for insulin were drawn at 2.5\u00a0min intervals during the first 10\u00a0min of the clamp and at 10\u201320\u00a0min intervals during the remainder. Exact details of the clamping procedures in the different study groups have been described previously [15, 18, 19].\nCombined hyperglycaemic clamp This hyperglycaemic clamp combined with GLP-1 and arginine administration was performed as previously described [15, 20\u201322]. After 120\u00a0min of hyperglycaemic clamp at 10\u00a0mmol\/l, a bolus of GLP-1 (0.6\u00a0pmol\/kg) was given (human GLP-1(7-36)amide; Poly Peptide, Wolfenb\u00fcttel, Germany) followed by a continuous GLP-1 infusion (1.5\u00a0pmol kg\u22121 min\u22121) during the next 80\u00a0min. At 180\u00a0min, a bolus of 5\u00a0g arginine hydrochloride (Pharmacia & Upjohn, Erlangen, Germany) was injected over 45\u00a0s while the GLP-1 infusion was continued. Blood for the measurement of glucose, insulin, proinsulin and C-peptide was obtained during the time-points shown in Fig.\u00a01. This clamp allows measurement of different aspects of stimulus secretion coupling: first and second phases of glucose-induced insulin secretion, GLP-1-induced insulin secretion, and the response to additional arginine administration.\nFig.\u00a01Associations between the genotypes of rs7903146 polymorphism in the TCF7L2 gene with insulin secretion during a hyperglycaemic clamp in 73 German participants. Open circles CC; closed circles CT and TT. AIR Acute insulin response. Arrow Administration of 5\u00a0g arginine. The p values show the differences for first and second phases of glucose-induced insulin secretion, first and second phases of GLP-1-induced insulin secretion and acute insulin secretory response to arginine (AIR) (for calculation see \u2018Methods\u2019; insulin secretion is adjusted for insulin sensitivity)\nAnalytical procedures Plasma glucose was determined as previously described [14, 17, 20, 21]. GLP-1 was determined during the OGTT at baseline, 30 and 120\u00a0min. GLP-1 immunoreactivity was determined using an RIA specific for the C-terminus of the peptide [24]. Samples were stored at \u221280\u00b0C. The assay measures the sum of the intact peptide plus the primary metabolite, GLP-1(9\u201336)amide using the polyclonal anti-GLP-1 antibody 89390 and synthetic GLP-1(7\u201336)amide as standard.Plasma insulin and C-peptide concentrations in the German cohort were measured by a microparticle enzyme immunoassay (Abbott, Wiesbaden, Germany) and an RIA (Byk-Santec, Dietzenbach, Germany). In the participants from the Netherlands, insulin was measured using an in-house competitive RIA employing a polyclonal anti-insulin antibody (Caris 46), 125I-labelled insulin (IM 166; Amersham, Roosdaal, the Netherlands) as a tracer and regular insulin (Humulin; Eli Lilly, Indianapolis, IN, USA) as a standard.\nCalculations Insulin secretion in the OGTT was assessed by calculating the AUC for C-peptide divided by the AUC for glucose (AUCCP\/AUCGlc). AUCs were determined by the trapezoidal method. Furthermore, insulin secretion was calculated as insulin:glucose ratio by dividing (insulin at 30\u00a0min\u2013insulin at 0\u00a0min) by (glucose at 30\u00a0min\u2013glucose at 0\u00a0min). Insulin sensitivity during the OGTT was estimated from glucose and insulin values as proposed by Matsuda and DeFronzo [25].Insulin secretion during the IVGTT was calculated as the sum of C-peptide levels during the first 10\u00a0min after glucose administration. Insulin sensitivity during the hyperinsulinaemic\u2013euglycaemic clamp was calculated by dividing the average glucose infusion rate during the last 40\u00a0min of the clamp by the average plasma insulin concentration during the same time interval.Insulin secretion during the hyperglycaemic clamp was calculated using insulin levels determined during the clamp. The first phase of insulin secretion was defined as the sum of the insulin levels during the first 10\u00a0min of the clamp. The second phase of insulin secretion was defined as the mean of the insulin values during the last 40\u00a0min (80\u2013120\u00a0min, NGT group, Germany) or the last 30\u00a0min (150\u2013180\u00a0min, NGT and IGT groups, the Netherlands) of the clamp.In the combined hyperglycaemic clamp with GLP-1 and arginine administration, first-phase GLP-1-induced insulin secretion was defined as the mean of the 125 and 130\u00a0min insulin levels, second-phase GLP-induced insulin secretion (plateau) was defined as the mean of the 160\u2013180\u00a0min insulin levels. The acute insulin response to arginine was calculated as the mean of 182.5 and 185\u00a0min minus 180\u00a0min insulin levels [21].The insulin sensitivity index was determined by relating the glucose infusion rate to the plasma insulin concentration during the last 40\u00a0min (NGT, Germany) or 30\u00a0min of the clamp.\nStatistical analysis Data are given as means\u2009\u00b1\u2009SEM. Non-normally distributed parameters were logarithmically transformed to approximate linearity for statistical comparison. Distribution was tested for normality using the Shapiro\u2013Wilk W test. Differences in anthropometrics and metabolic characteristics between genotypes were tested using ANOVA for the three genotype groups. The secretion indices were compared using multivariate regression models. In these models the trait was the dependent variable whereas age, sex, insulin sensitivity and genotype were the independent variables. Due to the relatively low number of participants who were homozygous for the rare alleles in the hyperglycaemic clamp group, a dominant model was used. A p value of <0.05 was considered to be statistically significant. The statistical software package JMP (SAS Institute, Cary, NC, USA) was used.\nResults\nGenetic variants in the TCF7L2 gene The SNPs described in the paper of Grant et al. [1] had an allelic frequency of the minor allele of 30% (rs7903146), 28% (rs12255372), 31% (rs7901695), 46% (rs11196205) and 45% (rs7895340) in our population. These polymorphisms were all in Hardy\u2013Weinberg equilibrium and in linkage disequilibrium (Electronic supplementary material Table 1).\nOGTT: glucose tolerance, insulin secretion, insulin sensitivity and GLP-1 levels The percentage of participants with IGT was significantly higher in carriers of the risk allele for rs7903146 (p\u2009=\u20090.007), rs12255372 (p\u2009=\u20090.02) and rs7901695 (p\u2009=\u20090.02).Insulin secretion assessed as AUCCP\/AUCGlc during the OGTT was significantly reduced in participants with the risk alleles for rs7903146 and rs12255372 in the additive and dominant model both unadjusted and adjusted for relevant covariates. Participants with the risk allele for rs7901695 had a significantly decreased insulin secretion in the additive model (p\u2009=\u20090.02) (Table\u00a01).Insulin sensitivity, estimated by the index of Matsuda and DeFronzo [25], was significantly higher in participants carrying the risk alleles for rs7903146 and rs1255372 in TCF7L2 (Table\u00a01).In a subgroup of 155 participants we measured GLP-1 concentrations at baseline, at 30\u00a0min and 120\u00a0min after oral ingestion of 75\u00a0g glucose. The anthropometric characteristics are shown in (Table\u00a01). Neither basal GLP-1 plasma levels nor GLP-1 levels during the OGTT significantly differed between the genotypes in any of the above-described SNPs in the TCF7L2 gene (Table\u00a02).\nCombined IVGTT and hyperinsulinaemic\u2013euglycaemic clamp: glucose-induced insulin secretion and insulin sensitivity C-peptide and insulin values during the IVGTT were not significantly different between the genotypes in any of the above-described SNPs in the TCF7L2 gene. Table\u00a03 shows the results for rs7903146 in the additive and dominant model adjusted for relevant covariates. Including the glucose tolerance status as an additional co-variate in the model did not affect the results. Interestingly, a lower insulin secretion in this subgroup of participants could be demonstrated when insulin secretion was calculated from the OGTT as described above (Table\u00a03). Insulin sensitivity measured with the clamp technique was not affected by any of the genotypes (all p\u2009>\u20090.4).\nHyperglycaemic clamp: glucose-, GLP-1- and arginine-induced insulin secretion and insulin sensitivity First- and second-phase insulin secretion during the hyperglycaemic clamp were not significantly different between carriers and non-carriers of the risk allele for rs7903146 (Table\u00a04) in any of the three populations from the Netherlands and from Germany. The other four SNPs in TCF7L2 which were described above were determined in the German population only and were not associated with glucose-induced insulin secretion (data not shown) during the hyperglycaemic clamp.The first phase of GLP-1-induced insulin secretion was significantly reduced in carriers of the risk alleles for rs7903146 (p\u2009=\u20090.03) and rs12255372 (p\u2009=\u20090.02). In addition, we found significant differences in the second phase of GLP-1-induced insulin secretion for rs7903146 (p\u2009=\u20090.006), rs12255372 (p\u2009=\u20090.005) and rs7901695 (p\u2009=\u20090.02) with carriers of the risk alleles having a significantly lower second phase of GLP-1-induced insulin secretion. Table\u00a04 and Fig.\u00a01 show the results for SNP rs7903146.In contrast to GLP-1-induced insulin secretion, arginine-induced insulin secretion was not affected by any of the analysed SNPs in TCF7L2 (Table\u00a04).\nDiscussion\nThe variants in TCF7L2 described by Grant et al. [1] that mediate an increased risk of type 2 diabetes have been found to be associated with reduced insulin secretion [6, 7, 9, 10]. We could confirm this reduced insulin secretion in our study in a group of 1,110 German non-diabetic participants using C-peptide levels during an OGTT. Unexpectedly, the i.v. application of glucose during an IVGTT did not affect insulin secretion in carriers of the risk alleles for SNPs in TCF7L2. The same results were obtained using i.v. glucose challenge during a hyperglycaemic clamp in subgroups of the German population and a population from the Netherlands. The observed different results between an orally and i.v. administered glucose challenge generate the hypothesis that incretin-induced insulin secretion is affected by the variants in the TCF7L2 gene. Possible mechanisms explaining these findings include impaired incretin production or incretin signalling.\nTo address this issue we first measured basal GLP-1 concentrations and GLP-1 response during an OGTT. None of the tested variants that have been shown in the literature to exert a reproducible effect on type 2 diabetes were associated with basal GLP-1 levels or with GLP-1 response during the OGTT.\nFurthermore, we combined the hyperglycaemic clamp with additional GLP-1 infusion to measure GLP-1-induced insulin secretion. Both acute (so called first phase) and prolonged (so called second phase) GLP-1-induced insulin secretion during the hyperglycaemic clamp were significantly impaired in carriers of the risk alleles in TCF7L2. These data suggest that participants carrying the risk alleles have an impaired insulin secretion due to an impairment of the GLP-1 signalling chain.\nThe putative molecular mechanism for this finding is not clear. As the transcription factor TCF7L2 plays an essential role in the WNT signalling pathway [14], which on the one hand is required for a normal development of the pancreas and the pancreatic islets [26] and on the other hand is crucial for the regulation of GLP-1 expression and secretion in intestinal L cells [14]. In theory, polymorphisms in the TCF7L2 gene might therefore through alteration of the WNT signalling pathway directly affect beta cell growth, beta cell differentiation and beta cell function. Furthermore insulin secretion might also be affected indirectly via the enteroinsular axis either through an impaired overall GLP-1 secretion or through a defective or dysfunctional GLP-1-induced insulin secretion. An impaired or dysfunctional GLP-1 effect might result first in a reduced postprandial insulin secretion and second, might influence stimulation of beta cell growth and beta cell differentiation.\nOur data show that basal and glucose-induced GLP-1 secretion during the OGTT were not affected by any of the analysed SNPs, whereas the first and second phases of GLP-1-induced insulin secretion were significantly reduced in carriers of the risk alleles in three of the five above-described SNPs in the TCF7L2 gene, among them rs7903146, which is most strongly associated in the literature with type 2 diabetes [1, 6, 9, 12, 13]. The finding that basal and glucose-stimulated GLP-1 secretion were not influenced by the TCF7L2 polymorphisms indicates that a reduced GLP-1 secretion is not likely to explain the impaired insulin secretion in carriers of the risk genotypes, although it can not fully exclude an effect of these SNPs on GLP-1 levels. First, by measuring total GLP-1 levels, we may have missed a subtle defect in GLP-1 secretion, which may have been detected by measuring the active form of GLP-1. Second, systemic GLP-1 levels may not adequately reflect the level of the active hormone acting in the gut wall on the autonomic nervous system [27]. Third, impaired TCF7L2 activity might tissue-specifically reduce the GLP-1 levels in the brain, which are believed as well to be important for insulin secretion [28].\nWhile we have no evidence for reduced GLP-1 secretion we have strong evidence for an impaired GLP-1-induced insulin secretion in carriers of the above-described polymorphisms in TCF7L2. The data show a reduced efficiency of GLP-1 to stimulate insulin secretion in pancreatic beta cells. Alterations of the GLP-1 signalling pathway in beta cells might thereby play a role. Recently the TCF7L2 gene was also identified in human pancreas [14]. Therefore variations in this transcription factor could specifically impair the transcription of genes involved in the incretin signalling chain, resulting in resistance of the pancreatic beta cells to GLP-1.\nIn contrast to the observed reduction in the first and second phases of GLP-1-induced insulin secretion, the arginine-induced insulin secretion was not significantly affected by the analysed SNPs in the TCF7L2 gene. The arginine bolus in the combined hyperglycaemic clamp produces a maximal challenge for the secretory capacity of the beta cell and can be considered as a surrogate for beta cell mass [15, 20]. The SNPs do not affect this maximal insulin secretion, suggesting that the variants in TCF7L2 do not influence beta cell mass, at least in the prediabetic state. In addition, impaired beta cell function may also include the efficiency of the conversion from proinsulin to insulin [21]. However, there was no evidence for this abnormality related to the variants in the TCF7L2 gene during the hyperglycaemic clamp (data not shown).\nWe also found that insulin sensitivity calculated from the OGTT using an established index [25] was significantly increased in participants carrying the risk alleles for all analysed SNPs in the TCF7L2 gene. This was also described in the study of Florez et al. [9]. When we measured insulin sensitivity with the gold standard, the euglycaemic\u2013hyperinsulinaemic clamp and the hyperglycaemic\u2013hyperinsulinaemic clamp, we found no association of the risk alleles with insulin sensitivity. Taken together, our data suggest that the variants in the TCF7L2 gene do not have a strong effect on insulin sensitivity.\nIn summary, our data show that variations in the TCF7L2 gene are associated with impaired GLP-1-induced insulin secretion. This might be consistent with a state of relative incretin resistance. Increasing the incretin levels by pharmacological tools may thus be a logical therapy to overcome impaired insulin secretion in carriers of the TCF7L2 variants.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nESM 1\nLinkage disequilibrium statistics (D\u2032, r2) among the five SNPs reported by Grant et al. [1] in the German population (n=1,110) (PDF 17 KB).","keyphrases":["insulin secretion","tcf7l2","polymorphism","glp-1"],"prmu":["P","P","P","P"]}
{"id":"Plant_Mol_Biol-4-1-2295253","title":"Interaction between sugar and abscisic acid signalling during early seedling development in Arabidopsis\n","text":"Sugars regulate important processes and affect the expression of many genes in plants. Characterization of Arabidopsis (Arabidopsis thaliana) mutants with altered sugar sensitivity revealed the function of abscisic acid (ABA) signalling in sugar responses. However, the exact interaction between sugar signalling and ABA is obscure. Therefore ABA deficient plants with constitutive ABI4 expression (aba2-1\/35S::ABI4) were generated. Enhanced ABI4 expression did not rescue the glucose insensitive (gin) phenotype of aba2 seedlings indicating that other ABA regulated factors are essential as well. Interestingly, both glucose and ABA treatment of Arabidopsis seeds trigger a post-germination seedling developmental arrest. The glucose-arrested seedlings had a drought tolerant phenotype and showed glucose-induced expression of ABSCISIC ACID INSENSITIVE3 (ABI3), ABI5 and LATE EMBRYOGENESIS ABUNDANT (LEA) genes reminiscent of ABA signalling during early seedling development. ABI3 is a key regulator of the ABA-induced arrest and it is shown here that ABI3 functions in glucose signalling as well. Multiple abi3 alleles have a glucose insensitive (gin) phenotype comparable to that of other known gin mutants. Importantly, glucose-regulated gene expression is disturbed in the abi3 background. Moreover, abi3 was insensitive to sugars during germination and showed sugar insensitive (sis) and sucrose uncoupled (sun) phenotypes. Mutant analysis further identified the ABA response pathway genes ENHANCED RESPONSE TO ABA1 (ERA1) and ABI2 as intermediates in glucose signalling. Hence, three previously unidentified sugar signalling genes have been identified, showing that ABA and glucose signalling overlap to a larger extend than originally thought.\nIntroduction\nPhotosynthesis provides plants with sugars that play a central role in the plant life cycle as energy sources, storage molecules, structural components or intermediates for the synthesis of other organic molecules. Next to these metabolic functions, sugars act as signalling molecules with hormone-like properties. Both hexoses and disaccharides are able to induce signalling via different pathways (Jang and Sheen 1997; Loreti et\u00a0al. 2001; Rolland et\u00a0al. 2006). In plants glucose has been shown to affect many processes, including germination, early seedling growth, flowering and senescence (Gibson 2000, 2005; Smeekens 2000; Rolland et\u00a0al. 2006). Moreover, glucose feeding of Arabidopsis seedlings affected the expression of many genes as shown by micro array studies (Price et\u00a0al. 2004; Villadsen and Smith 2004; Li et\u00a0al. 2006). Sugar-induced signal transduction has been shown to control gene expression via diverse mechanisms that include transcription, translation, and modification of mRNA and protein stability (Rolland et\u00a0al. 2006).\nGenetic analysis showed that sugar signalling in plants is closely associated with plant hormone biosynthesis and signalling, in particular with that of abscisic acid (ABA, for review see Finkelstein and Gibson 2001; Gazzarini and McCourt 2001; Le\u00f3n and Sheen 2003; Rook et\u00a0al. 2006; Dekkers and Smeekens 2007). Four screens for sugar response mutants i.e. sucrose uncoupled (sun), impaired sucrose induction (isi), glucose insensitive (gin) and sugar insensitive (sis) identified ABA deficient mutants (i.e. aba2\/isi4\/gin1\/sis4 and aba3\/gin5) and ABA insensitive4 (abi4\/sun6\/isi3\/gin6\/sis5) as sugar insensitive (Arenas-Huertero et\u00a0al. 2000; Huijser et\u00a0al. 2000; Laby et\u00a0al. 2000; Rook et\u00a0al. 2001). ABI4 encodes an AP2 domain containing transcription factor that binds a CE1-like element present in many ABA and sugar regulated promoters (Finkelstein et\u00a0al. 1998; Niu et\u00a0al. 2002; Acevedo-Hernandez et\u00a0al. 2005). These observations linked sugar regulation to ABA signalling. However, there are many more examples of co-regulation of sugar and ABA in plants. The expression of many genes is co-regulated by sugar and ABA. A study of Li et\u00a0al. (2006) showed that 14% of the ABA upregulated genes are induced in response to glucose as well, in total nearly 100 genes. These included genes in stress responses, carbohydrate and nitrogen metabolism and signal transduction. Another group of nearly 40 genes was repressed by both ABA and glucose. In addition the authors identified a group of genes that showed a synergistic upregulation when treated with both ABA and glucose, including the ApL3 (ADP pyrophosphorylase large subunit) gene, which is involved in starch biosynthesis. An earlier study showed that ABA was not able to induce the ApL3 gene but that ABA in combination with sugar boosted transcription levels in comparison to sugar treatment alone (Rook et\u00a0al. 2001). Interestingly, Akihiro et\u00a0al. (2005) found a similar ABA\/sugar interaction for the regulation of ApL3 expression and starch biosynthesis in rice (Oryza sativa). There are many other examples of processes and genes that are regulated by sugar and ABA. E.g. the ABA, stress and ripening-induced (ASR) protein in grape (Vitis vinifera) that binds to the sugar response boxes in the promoter of the monosaccharide transporter VvHT1. ASR mRNA expression is responsive to sugar and is modulated by ABA (Cakir et\u00a0al. 2003). Both sugar and ABA signals also regulate the maize (Zea mays) invertase gene, Ivr2 (Trouverie et\u00a0al. 2004). Thus ABA\/sugar interactions were reported for a multitude of processes and in different plant species indicating that these interactions are physiologically relevant. Therefore, a thorough understanding of these sugar and ABA interactions is of importance.\nAn important strategy for the isolation of sugar response mutants makes use of the effect of exogenous supplied sugars on early seedling development in Arabidopsis Although the use of high sugar concentrations (>300\u00a0mM) possibly are above the physiological range, it provided an efficient screening method for mutants (reviewed by Rognoni et\u00a0al. 2007). Such elevated sugar concentrations arrest early seedling development, which is characterized by the absence of cotyledon greening and leaf formation. The difference in phenotype between arrested and non-arrested seedlings was exploited to isolate sugar insensitive mutants that showed proper seedling development on high glucose (gin) and sucrose (sis) media. In addition to the aforementioned role of ABA biosynthesis and signalling such sugar response mutants revealed a role for HEXOKINASE1 (HXK1\/GIN2) and ethylene signalling in the GIN response pathway (Zhou et\u00a0al. 1998; Gibson et\u00a0al. 2001; Cheng et\u00a0al. 2002; Moore et\u00a0al. 2003). ABA deficiency caused by either mutations in ABA1, ABA2 or ABA3 (Koornneef et\u00a0al. 1998), resulted in a clear gin phenotype but analysis of ABA insensitive mutants resulted in more ambiguous phenotypes. In addition to abi4, only abi5 and abi8 showed a sugar insensitive phenotype, while for abi1-1, abi2-1 and abi3-1 no or only a weak phenotype was reported (Arenas-Huertero et\u00a0al. 2000; Huijser et\u00a0al. 2000; Laby et\u00a0al. 2000; Brocard et\u00a0al. 2002; Brocard-Gifford et\u00a0al. 2004). Nevertheless, a role for ABI3 in sugar signalling during early seedling development was hinted too by later studies. E.g. ABI3 over expression lines were sugar hypersensitive (Finkelstein et\u00a0al. 2002; Zeng and Kermode 2004) and abi3 mutants were insensitive to glucose in combination with ABA (Nambara et\u00a0al. 2002). This insensitivity appeared to be allele specific and the assay used was different from other sugar sensitivity assays. However, in a more recent report WT glucose sensitivity during early seedling development for the abi3 mutant was observed (Yuan and Wysocka-Diller 2006). Thus it is unclear how and to what extend sugar and ABA signalling overlap.\nABA by itself blocked post-germination early seedling development, in addition to the role of ABA in the glucose-induced seedling arrest (Lopez-Molina et\u00a0al. 2001). Similar to glucose-arrested seedlings, ABA-arrested seedlings lacked greening and leaf formation. The ABA-induced seedling arrest is dependent on ABI3 and ABI5 activity and both abi3 and abi5 mutant do not arrest early seedling growth in response to ABA (Lopez-Molina et\u00a0al. 2002). This ABA sensitivity is confined to a limited time window after the start of germination (Lopez-Molina et\u00a0al. 2001; Lopez-Molina et\u00a0al. 2002) Adding ABA after this sensitive phase to seedlings no longer affects vegetative development. Similarly, the glucose-induced seedling arrest could only be triggered in a similar limited time frame after sowing (Gibson et\u00a0al. 2001).\nHere a study of the role of ABA biosynthesis and ABA signalling in sugar response pathways is presented. In particular this study was focussed on the glucose-induced early seedling developmental arrest (the GIN pathway). Analysis of transgenic ABA deficient lines with ectopic ABI4 expression (aba2-1\/35S::ABI4) suggested that additional ABA-controlled factors are necessary to arrest early seedling development in response to glucose. ABI3 expression and protein stability are controlled by ABA and ABI3 is a key component in the ABA mediated pathway that blocks early development (Lopez-Molina et\u00a0al. 2002; Zhang et\u00a0al. 2005). Therefore, we investigated the role of ABI3 in the GIN pathway. ABI3 encodes a B3 domain transcription factor that contains four conserved domains, one acidic activation domain (A1) and three basic domains (B1, B2 and B3, Giraudat et\u00a0al. 1992; Finkelstein et\u00a0al. 2002). ABI3 directs seed specific gene expression and is required for induction of desiccation tolerance and seed dormancy and represses genes related to seed germination (Ooms et\u00a0al. 1993; Hoecker et\u00a0al. 1995; Nambara et\u00a0al. 2000; Suzuki et\u00a0al. 2001; Finkelstein et\u00a0al. 2002). Other functions for ABI3 include regulation of flowering, resource allocation, lateral root growth in response to auxin and serve as a signal transduction component downstream of PHYB signalling in seeds (Robinson and Hill 1999; Suzuki et\u00a0al. 2001; Brady et\u00a0al. 2003; Mazzella et\u00a0al. 2005). Here we show that multiple abi3 mutants display a gin phenotype. The glucose insensitive phenotype observed for abi3 mutants was comparable to that of other known gin mutants like aba1, abi4 and abi5. Moreover, abi3 mutants showed a reduced expression level of glucose-regulated genes. In addition, physiological and molecular experiments led to the conclusion that the glucose-arrested seedlings mimic ABA-arrested seedlings. This was supported by the observations that two other ABA response mutants, i.e. era1 and abi2-1, showed altered glucose responsiveness as well. The identification of ABI3 and two other ABA response loci as components in GIN signalling suggests that glucose and ABA signalling share more components than originally anticipated.\nMaterial and methods\nPlant material\nArabidopsis plants were grown in a climate chamber at 22\u00b0C with 70% humidity and a 16\u00a0h\/8\u00a0h light\/dark cycle (Sylvania GRO-LUX fluorescent lamps; Technische Unie, Utrecht) for seed production. WT and mutant seeds that were compared in all experiments presented were produced together and stored under identical conditions. Seeds were dry-stored in paper bags at room temperature for at least a month before use in our experiments. The transgenic aba2-1\/35S::ABI4 lines were constructed as follows. ABI4 cDNA sequence was PCR amplified and cloned in pDONR vector and sequenced. Using Gateway cloning ABI4 was cloned behind the Cauliflower Mosaic Virus 35S promoter in the pGD625 vector. The pGD625 vector containing 35S::ABI4 was introduced in Agrobacterium which were used to transform aba2-1 plants by the floral dip method (Clough and Bent 1998).\nGermination assays\nAll germination assays were performed on 0.5\u00a0MS: half-strength Murashige and Skoog medium (pH 5.8), including vitamins, solidified with 0.8% plant agar (Duchefa, Haarlem, The Netherlands). Before plating, seeds were surface-sterilized in 20% (v\/v) commercial bleach (Glorix) for 15\u201320\u00a0min and rinsed four to five times with sterile water. After a three days stratification period at 4\u00b0C in the dark, plates (with or without sugars present as indicated) were incubated in the growth chamber (22\u00b0C and 16\u00a0h\/8\u00a0h light\/dark cycle). To investigate the effect of stratification on sugar-free (0.5\u00a0MS) media seeds were transferred to sugar media after this stratification period. Control experiments showed that transferring of seeds did not affect seed germination. Germination defined as radicle emergence from the seed coat, was scored daily for 3\u20138\u00a0days. Experiments were performed in duplo, each plate containing 50\u2013100\u00a0seeds, and every experiment was repeated one to three times.\nSugar response assays\nFor gin assay experiments seeds were sterilized, sown on 0.5\u00a0MS media and stratified for three days at 4\u00b0C in the dark. Hereafter seeds were collected and sown on 0.5\u00a0MS media supplemented with 1% sucrose (control) or on control media with the indicated amount of sugar unless otherwise indicated. Different seed batches show slightly different responses and therefore both 7% and 8% glucose media were used. Plates were incubated in the growth chamber (22\u00b0C and 16\u00a0h\/8\u00a0h light\/dark cycle) for up to two weeks. Seedlings were scored gin when green cotyledons emerged. To determine the isi phenotype of WT Col, aba2-1, L6 and L10 the seeds were stratified first for three days on sugar-free 0.5\u00a0MS media. After stratification the seeds were sown on 100\u00a0mM sucrose media. After four days plant material was harvested and ApL3 expression levels determined. For the sun phenotype analysis seeds were sterilized, sown on 0.5\u00a0MS and stratified for three days at 4\u00b0C in darkness. After stratification seeds were transferred to plates containing 0.5\u00a0MS or 0.5\u00a0MS with the indicated amount of sugar. Seeds were light-treated for at least one hour before they were incubated in the dark (wrapped in three layers of aluminium foil) at 22\u00b0C. After two days plant material was harvested for PC expression analysis by qRT-PCR.\nGene expression analysis\nWT Ler-0, abi3-1 and abi3-5 seeds were stratified for three days and germinated on 0.5\u00a0MS media. Seed batches were germinated to approximately 70% (t\u00a0=\u00a00) before the start of the different treatments. At this stage all WT seedlings were developmentally arrested in response to elevated glucose concentrations and ABA treatment. For RNA isolation and qRT-PCR analysis plant material was ground using mortar and pestle in liquid nitrogen and additionally bead-beaten in a mikro-dismembrator S (B. Braun Biotech International, Germany). RNA was isolated according to Schuurmans et\u00a0al. (2003). Additionally, RNA was purified using RNeasy columns (Qiagen USA, Valencia, CA). RNA samples were DNase treated, checked for the absence of DNA by PCR and used for cDNA synthesis and qRT-PCR analysis as described before by van Dijken et\u00a0al. (2004). For expression analysis by qRT-PCR either a 5\u2032 FAM\/3\u2032 TAMRA labelled probe (with TaqMan\u00ae 2\u00d7 Universal PCR Master Mix, Applied Biosystems, Foster City, CA) or SYBR\u00aeGreen technology (Power SYBR\u00aeGreen PCR Master Mix, Applied Biosystems, Foster City, CA) was used. Relative quantitation of gene expression is based on the comparative CT method (User Bulletin No. 2: ABI PRISM 7700 Sequence Detection System, 1997) using the AtACTIN2 (ACT2) as reference gene. The EM1 primers were obtained for the CATMA database (http:\/\/www.catma.org, EM1 forward primer ID: 3a44750.5, EM1 reverse primer ID: 3a44750.3). The sequences of primers and probes that were used for gene expression analysis are listed in \nTable\u00a01. The qRT-PCRs were run on a ABI-prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA).\nTable\u00a01Primers and probes used for gene expression analysisGeneForward primerReverse primerProbeACT2gctgagagattcagatgcccaatgggagctgctggaatccacagtcttgttccagccctcgtttgtgcABI3cacagccagagttccttccttttgtggcatgggaccagactcttgaatctccaccgtcatggccacABI4cggtgggttcgagtctatcaacggatccagacccatagaacaacctcatccaccgccgttggttgaABI5ggaggtggcgttgggtttgggcttaacggtccaaccatcccatttgctgtccacccgctEM1agatgggacacaaaggaggagtgttggtgaactttgactcatcgEM6ggtacgggaggcaaaagcttttgcgtcccatctgctgataRAB18gagcaactccacaaggaaaggtagccaccagcatcatatcApL3cgagaagtgccggattgtaaaggaacgttggatgctgcattcccaagaaacatccgtgtgagattaccgPCtctttgaaggatttcggtgtcacatggccatcgcatttccaaaacgatcgaagctgctgttgccact\nResults\nTransgenic ABA deficient lines with constitutive expression of ABI4 are glucose insensitive\nScreens for sugar insensitive mutants revealed the importance of ABA and ABA signal transduction, in particular ABI4 (Arenas-Huertero et\u00a0al. 2000; Huijser et\u00a0al. 2000; Laby et\u00a0al. 2000; Rook et\u00a0al. 2001). To study the interaction between ABA biosynthesis and ABI4 in sugar signalling we generated transgenic lines with constitutive ABI4 expression driven by a CaMV 35S promoter in an ABA deficient (aba2-1) background. ABI4 expression in these transgenic aba2-1 plants is independent from plant sugar status. Two lines homozygous for the 35S::ABI4 construct were selected, i.e. lines L6 and L10. Both lines germinated normally but showed a stunted growth phenotype (Fig.\u00a01a) similar to what has been reported previously for severe ABI4 overexpression lines (S\u00f6derman et\u00a0al. 2000). Expression analysis on control media showed that ABI4 expression is high in L6 and L10 and exceeded glucose-induced ABI4 expression in developmentally arrested WT Col seedlings by approximately 5\u20138 fold (Fig.\u00a01b).\nFig.\u00a01Analysis of transgenic ABA deficient lines with constitutive ABI4 expression. Plant phenotypes of WT Col, aba2-1, and two transgenic lines with constitutive ABI4 expression in aba2-1 (L6 and L10) after 2,5\u00a0weeks of growth on soil (a). ABI4 expression was detected by qRT-PCR in Col, aba2-1, L6 and L10 seedlings grown for two weeks on control or 8% glucose media. Values are averages of two experiments \u00b1SD (b). Seedling phenotypes after two weeks of growth on control, 8% sorbitol (sorb), 8% glucose (glc) or 8% glucose\u00a0+\u00a00,1\u00a0\u03bcM ABA (glc\u00a0+\u00a0ABA). Similar results were obtained in three experiments (c). ApL3 expression was detected by qRT-PCR in Col, aba2-1, L6 and L10 seedlings grown for four days on control half-strength MS (con) or half-strength MS\u00a0+\u00a0100\u00a0mM sucrose (suc) media. Values are averages of three experiments \u00b1SD (d)\nThe gin phenotype of Col, aba2-1, L6 and L10 was tested by growing the lines for two weeks on control, 8% sorbitol and 8% glucose media. All lines showed greening on control (con) and sorbitol (sorb, osmotic control) media as expected (Fig.\u00a01c). On glucose the Col seedlings showed an arrested phenotype whereas aba2-1 showed a gin phenotype as reported before (Fig.\u00a01c, Le\u00f3n and Sheen 2003). Also ABI4 expression levels in glucose-arrested Col seedlings were enhanced whereas in aba2-1 ABI4 expression was very low. The L6 and L10 aba2-1\/35S::ABI4 lines have very high ABI4 levels but, importantly, display a gin phenotype (Fig.\u00a01c). The gin phenotype of aba2-1 and the transgenic lines could be reverted to WT by the addition of 100\u00a0nM ABA as was reported before for aba mutants (Fig.\u00a01c, Arenas-Huertero et\u00a0al. 2000). Thus, in an ABA deficient background ABI4 overexpression does not restore wild type sensitivity to glucose.\nSucrose feeding induces ApL3 gene expression in Arabidopsis seedlings. This sugar regulation of the ApL3 promoter has been used as basis for a screen for sugar response mutants. Rook et\u00a0al. (2001) reported on isi sugar response mutants that were unable to fully activate ApL3 expression in response to sucrose. The identification of aba2\/isi4 and abi4\/isi3 as mutants with an impaired sucrose induction phenotype revealed a role for ABA biosynthesis and signalling in control of ApL3 expression (Rook et\u00a0al. 2001). To study the sugar-induced ApL3 expression in WT Col, aba2-1, L6 and L10 stratified seeds were sown on 100\u00a0mM sucrose media. After four days plant material was harvested and ApL3 expression levels determined. Sucrose feeding resulted in an approximate six fold induction of ApL3 in Col seedlings (Fig.\u00a01d). The induction of ApL3 is 25% lower in the aba2-1 seedlings, which is in agreement with the isi phenotype of aba2\/isi4 (Rook et\u00a0al. 2001). In the L6 and L10 aba2-1\/35S::ABI4 lines the ApL3 expression level on sugar-free control media already exceeds that of sugar-treated WT seedlings. Sugar feeding of L6 and L10 seedlings further boosted ApL3 expression 7\u20139 fold higher than sugar-treated WT seedlings (Fig.\u00a01d).\nGlucose and ABA trigger a similar developmental early seedling arrest\nHigh sugar concentrations arrest early seedling development in Arabidopsis. After germination vegetative seedling growth is blocked, characterized by the absence of cotyledon greening and leaf formation. Equimolar concentrations of an osmotic control did not block development in this early seedling stage (Fig.\u00a02a; Zhou et\u00a0al. 1998; Laby et\u00a0al. 2000; Rognoni et\u00a0al. 2007). Previously, Lopez-Molina et\u00a0al. (2001) showed that treatment with ABA arrested this post-germination switch from embryonic to vegetative growth as well. ABA-arrested seedlings showed neither greening nor leaf formation similar to glucose-arrested seedlings (Fig.\u00a02a). In comparison to ABA-arrested seedlings, glucose-arrested seedlings were larger due to the longer hypocotyl and larger cotyledons (Fig.\u00a02b). Moreover, significant root growth was observed in glucose-arrested seedlings, which is absent in ABA-arrested seedlings (Fig.\u00a02a, b). Further, in glucose-arrested seedlings often a pink colouration was observed, indicative for anthocyanin accumulation that was absent in ABA-arrested seedlings (Fig.\u00a02a, b).\nFig.\u00a02Glucose and ABA signalling arrest early seedling development and induce a drought tolerant phenotype. Ler-0 seedling phenotypes are shown after growth for twelve days on control (con, half-strength MS\u00a0+\u00a01% sucrose), or on con medium supplemented with either 7% sorbitol (sorb), 7% glucose (glc) or 10\u00a0\u03bcM ABA (ABA) (a). A close-up image of a glc-arrested seedling (left) and an ABA-arrested seedling (right) is shown (b). Developmentally arrested seedlings have a drought tolerant phenotype. Seeds were grown on control (con) or on con media containing either 10\u00a0\u03bcM ABA (ABA) or 7% glucose (glc) for nine days. At day nine the seedlings were subjected to a drought treatment as indicated (c). Hereafter seedlings were allowed to recover on control media for five days after which survival was scored. The survival percentage following drought treatment is presented (c)\nABA-arrested seedlings possess an enhanced resistance to drought stress compared to non-arrested seedlings (Lopez-Molina et\u00a0al. 2001). A comparison was made between the drought resistance phenotype of glucose- and ABA-arrested seedlings. Control (non-arrested) seedlings did not survive a drought treatment of 2.5\u00a0h (Fig.\u00a02c). ABA-treated seedlings showed a near 100% survival rate after 6\u00a0h of drought, the longest treatment tested in our experiments. Glucose-arrested seedlings showed an intermediate drought resistance. After 2.5\u00a0h of drought stress, glucose-treated seedlings showed a nearly 90% survival rate and after 6\u00a0h of drought the survival rate was 50% compared to 0% survival observed for control seedlings. Thus, a clear drought resistant phenotype was observed for glucose-arrested seedlings.\nThe ABA-induced early seedling developmental arrest depends on functional ABI3 and ABI5 genes. abi3 and abi5 mutants did not block early seedling development in response to ABA. Moreover, WT seedlings exposed to ABA showed strongly enhanced ABI3 and ABI5 protein levels (Lopez-Molina et\u00a0al. 2001, 2002). Both genes encode transcriptional regulators important for seed development and LEA gene expression (Koornneef and Karssen 1994; Parcy et\u00a0al. 1994, Finkelstein and Lynch 2000; Carles et\u00a0al. 2002). Lopez-Molina et\u00a0al. (2002) showed that during the early seedling developmental arrest several LEA genes were re-induced as well. This re-induction of seed transcriptional regulators and LEA gene expression suggested that a late embryogenesis program is re-initiated by ABA treatment, inducing a drought tolerant phenotype in such arrested seedlings (Lopez-Molina et\u00a0al. 2002). It was investigated whether the same transcriptional regulators and LEA genes were expressed by glucose signalling during early seedling development. A role for ABI5 has been proposed in GIN signalling and ABI5 expression is sugar responsive (Arenas-Huertero et\u00a0al. 2000; Laby et\u00a0al. 2000; Brocard et\u00a0al. 2002; Arroyo et\u00a0al. 2003). ABI5 expression thus is a good positive control in this experiment. Gene expression upon glucose feeding was studied by germinating seeds on control medium until the batch reached 70% germination (time point zero, t\u00a0=\u00a00). At this time point a sample was collected for RNA isolation while the rest of the seeds were transferred to control, sorbitol, glucose or ABA containing media for an additional 24\u00a0h. After 24\u00a0h samples were collected for gene expression analysis. Using quantitative RT-PCR (qRT-PCR), the expression of ABI3, ABI5, AtEM1, AtEM6 and RAB18 was studied in response to the different treatments. Gene expression was studied in three independent experiments. In each experiment the gene expression level at t\u00a0=\u00a00 was set to one (Table\u00a02). Some variation in gene expression levels between the three experiments was observed, likely due to the fact that for each experiment independently generated seed batches were used. Generally, continued growth on control media after t\u00a0=\u00a00 resulted in a decreased expression of all five genes (Table\u00a02). In contrast, treatments with sorbitol, glucose and ABA enhanced the expression of all genes studied, except for the sorbitol treatment in the second experiment. In this particular experiment gene expression decreased on sorbitol, however, this decrease was less pronounced compared to the control treatment. ABA treatment resulted in the re-induction of ABI3, ABI5 and the LEA genes, which is in line with results reported by Lopez-Molina et\u00a0al. (2002). Also glucose exposure significantly induced the expression levels of all five genes although the expression levels are in general somewhat lower than in response to ABA. Thus ABI3 is upregulated by glucose as well, which indicated a possible role for ABI3 in glucose responsiveness. The observation that the expression levels of the genes studied are in general lower after sorbitol treatment compared to glucose treatment indicates that the glucose-induced expression is not an osmotic response.\nTable\u00a02Relative expression levels of ABI3, ABI5, EM1, EM6 and RAB18 after glucose treatmentGeneTreatmentExp. 1Exp. 2Exp. 3ABI3t\u00a0=\u00a00111con0.140.090.06sorb2.20.191.7glc9.04.57.9ABA178.36ABI5t\u00a0=\u00a00111con0.480.200.29sorb8.70.4112glc191319ABA404123EM1t\u00a0=\u00a00111con0.260.340.20sorb740.71164glc7972300ABA749742200EM6t\u00a0=\u00a00111con0.220.140.15sorb360.3878glc5140173ABA450132237RAB18t\u00a0=\u00a00111con0.160.120.00sorb770.461.7glc324416.2ABA618633315The relative expression levels of five genes were determined using quantitative RT-PCR. The gene expression levels were measured at t\u00a0=\u00a00 and after a 24\u00a0h treatment on control media (con, half-strength MS\u00a0+\u00a01% sucrose) and on con media supplemented with either 8% sorbitol (sorb), 8% glucose (glc) or 10\u00a0\u03bcM ABA (ABA) media. The expression level at t\u00a0=\u00a00 was set to 1. The experiment was performed three times and the results for each experiment are indicated\nExpression studies using seedlings grown for five days on control, sorbitol (7%) or glucose (7%) media confirmed the induction of these genes (Fig.\u00a03). In five days old glucose-arrested seedlings enhanced expression levels were detected for ABI3 and ABI5 as well as the LEA genes AtEM1, AtEM6 and RAB18. The induction of these genes by sorbitol was roughly five fold lower (Fig.\u00a03). After 72\u00a0h of growth on ABA-free media, seedlings lose the capacity to re-induce ABI3, ABI5 and LEA expression and to arrest early seedling development in response to ABA (Lopez-Molina et\u00a0al. 2001, 2002). To study whether the induction by glucose is restricted to a similar time frame seeds were germinated and grown for three days on half-strength MS and subsequently switched for two additional days to elevated sorbitol and glucose media. The re-induction by glucose of ABI3, ABI5, EM1, EM6 and RAB18 was largely lost (Fig.\u00a03, glc 72\u00a0h). The weaker induction by sorbitol is essentially lost after three days (Fig.\u00a03, sorb 72\u00a0h). Thus, the glucose-response that induced ABI3, ABI5 and LEA expression is confined to a similar time frame as observed for ABA. This is in agreement with the finding that glucose regulation of ABI4 and ABI5 expression was dependent on developmental stage (Arroyo et\u00a0al. 2003) and that sugar arrests early seedling development only in a small 2\u20133\u00a0day time frame after sowing (Gibson et\u00a0al. 2001).\nFig.\u00a03The glucose-regulated expression of ABI3, ABI5 and LEA genes is restricted to a limited time frame after the start of germination. The expression of EM1, EM6, RAB18, ABI3 and ABI5 genes was quantified by qRT-PCR after germination and growth for five days on different media. Ler-0 seeds were plated for five days on control media (con, half-strength MS\u00a0+\u00a01% sucrose), or on con medium supplemented with either 7% sorbitol (sorb) or 7% glucose (glc). The values are an average of three experiments and in each experiment the expression on glc was set to one. It was investigated whether this gene expression regulation is limited to a small time window after the start of germination as has been shown for ABA regulation (Lopez-Molina et\u00a0al. 2002). Therefore seeds were germinated on con media for three days and thereafter switched to either sorb (sorb 72\u00a0h) or glc media (glc 72\u00a0h)\nIn conclusion, the glucose-induced block of early seedling development mimics the ABA-induced arrest of vegetative development in young seedlings. Both glucose- and ABA-arrested seedlings have a drought resistant phenotype and show enhanced expression levels of seed expressed genes. The observation that ABI4 overexpression is unable to restore WT glucose sensitivity in the aba2-1 mutant shows that WT ABA biosynthesis is essential for the GIN response. Apparently, next to ABI4 additional factors which are under control of ABA are important for GIN signalling. Two candidates are ABI3 and ABI5. Both play a role in the ABA-induced early seedling developmental arrest and gene expression and protein stability of both transcription factors is affected by ABA (Lopez-Molina et\u00a0al. 2001; Lopez-Molina et\u00a0al. 2002; Lopez-Molina et\u00a0al. 2003; Zhang et\u00a0al. 2005; this study). A role for ABI5 in glucose signalling has been established; abi5-1 has a gin phenotype (although somewhat weaker compared to abi4) and ABI5 expression is regulated by sugar (Arenas-Huertero et\u00a0al. 2000; Laby et\u00a0al. 2000; Brocard et\u00a0al. 2002; Arroyo et\u00a0al. 2003; this study see \nTable 2). However, a role for ABI3 is controversial (for review Finkelstein and Gibson 2001; Leon and Sheen 2003; Gibson 2004; Rognoni et\u00a0al. 2007). Since ABI3 is important for the ABA-induced developmental arrest and is induced by glucose, our hypothesis is that ABI3 is involved in GIN signalling as well. Therefore, the gin phenotype and glucose-regulated gene expression were studied in abi3 mutants.\nabi3 mutants are glucose insensitive\nThe glucose insensitive phenotype of WT Ler-0 and the abi3 mutant was investigated by plating seeds of both genotypes on media with elevated glucose concentrations (6\u20138% glucose). Initially, two abi3 mutant alleles were tested, i.e. the weak abi3-1 allele (producing normal, drought tolerant seeds) and the strong abi3-5 allele (producing green, drought intolerant seeds, Ooms et\u00a0al. 1993). As expected WT Ler-0 early seedling development was arrested in response to high glucose concentrations. In contrast, approximately 60\u2013100% (differed between experiments) of both abi3-1 and abi3-5 seedlings showed greening on high glucose media, suggesting that abi3 is a gin mutant, (Fig.\u00a04a). All genotypes showed greening on control and osmotic control media (Fig.\u00a04a, control and 7% sorbitol, respectively). The gin phenotype was observed for abi3 mutants in several experiments using independently grown and harvested seed batches (data not shown). Next, the strength of the gin phenotype of abi3-1 was assessed in comparison to that of the ABA deficient mutant aba1-1, which is in the Ler-0 background as well. The aba1-1 mutant, like other ABA deficient mutants tested, was shown to possess a gin phenotype (Arenas-Huertero et\u00a0al. 2000; Huijser et\u00a0al. 2000; Laby et\u00a0al. 2000). A similar percentage of seedling greening was observed for aba1-1 and abi3-1 when grown on glucose media (Fig.\u00a04b) suggesting that both mutants possess a comparable gin phenotype.\nFig.\u00a04abi3 mutants have a gin phenotype. Representative Ler-0, abi3-1 and abi3-5 seedlings are shown that were grown for 12\u00a0days on control media (half-strength MS\u00a0+\u00a01% sucrose), or on control medium supplemented with either 7% sorbitol or 7% glucose (a). The gin phenotype of abi3-1 was compared to that of the ABA deficient mutant aba1-1. Seedlings were grown for 12\u00a0days on the indicated media before seed germination and seedling greening were scored (b)\nSix additional mutant alleles (abi3-8, abi3-9, abi3-10, abi3-11, abi3-12, and abi3-13) were obtained (a kind gift of Dr. E. Nambara) and tested for their gin phenotype. These abi3 mutants were tested along with two known gin mutants, abi4-3 and abi5-7 in the gin assay. All these mutants were retrieved from a screen for mutants insensitive to the unnatural (\u2212)-ABA (Nambara et\u00a0al. 2002). The ABA insensitivity of the mutants was tested by plating the mutants on half-strength MS media containing 1% sucrose\u00a0+\u00a010\u00a0\u03bcM ABA. The ABA insensitivity observed was in agreement with that reported by Nambara et\u00a0al. (2002) (see Table\u00a03). The abi3-8, abi3-9, abi3-10, abi4-3 and abi5-7 mutants had a strong ABA insensitivity, the abi3-12 and abi3-13 showed a weaker phenotype. We did observe 40% greening for abi3-13 on ABA media while originally no abi phenotype was observed for this allele (Nambara et\u00a0al. 2002). These mutants were isolated on (\u2212)-ABA and the (\u2212)-ABA insensitivity does not necessarily parallel (+)-ABA insensitivity (Nambara et\u00a0al. 2002). Next, these mutants were plated on control, sorbitol- and glucose-containing media, respectively. On 7% glucose media strong gin phenotypes were observed for abi4\/gin6 and abi5 mutants as shown by the high percentages of greening, in agreement with earlier reports (Arenas-Huertero et\u00a0al. 2000; Huijser et\u00a0al. 2000; Laby et\u00a0al. 2000). Again a strong gin phenotype was observed for abi3, in agreement with our observation using abi3-1 and abi3-5 mutants. Five out of the six additional abi3 mutants tested show a gin phenotype. For four abi3 mutants the gin phenotype was comparable to that of abi4-3 and abi5-7 (Table\u00a03). The strong ABA insensitive abi3 mutants are strong gin mutants as well whereas the mutants with a weaker phenotype (abi3-11 and abi3-12) show a weaker phenotype on glucose media. However, exceptionally abi3-13 shows a weak abi phenotype but a strong gin phenotype. The abi3-11 mutant lacks an ABI insensitive phenotype and did not show a gin phenotype either. In conclusion, gin phenotype analysis of eight abi3 alleles confirms the glucose insensitive phenotype for abi3. Moreover, the gin phenotype of abi3 is comparable to that of other known gin mutants like aba1, abi4 and abi5.\nTable\u00a03Glucose insensitive phenotype of six abi3 allelesGenotypeTreatmentcon7% sorb7% glc10\u00a0\u03bcM ABA3\u00a0\u03bcM ABAaWT Col10099200abi4-3999894100100abi5-799988996100abi3-81001009791100abi3-9100100100100100abi-310100100100100100abi3-11100100520abi3-1210096338350abi3-1310010098400Seeds of WT Col, abi4-3, abi5-7 and six abi3 mutants were stratified on half-strength MS and after stratification switched to control medium (con, 0.5\u00a0MS\u00a0+\u00a01% sucrose) or con medium containing 7% sorbitol, 7% glucose or 10\u00a0\u03bcM ABA. Values are the percentage of cotyledon greening scored after 11\u00a0days. The experiment was repeated once with similar resultsaThe ABA insensitivity scored for the different mutant alleles based on cotyledon greening by Nambara et\u00a0al. (2002)\nGlucose-induced expression of ABI4, ABI5 and RAB18 is reduced in abi3\nGlucose-induced early seedling developmental arrest is associated with expression of ABI3, ABI4, ABI5 and a subset of LEA genes (Arenas-Huertero et\u00a0al. 2000; Cheng et\u00a0al. 2002; Arroyo et\u00a0al. 2003; this study). The involvement of ABI3 in glucose-regulated gene expression was tested for three genes ABI4, ABI5 and RAB18 as a representative of the LEA genes. For expression analysis the weak abi3-1 and the strong abi3-5 mutants were used. Seedlings of all three genotypes were grown for 24\u00a0h on control media or on control media containing 8% glucose.\nBoth RAB18 and ABI5 expression were sensitive to glucose addition. In WT seedlings, glucose treatment induced these two genes on average 14\u00a0times in comparison to t\u00a0=\u00a00 (Fig.\u00a05a, c). However, on glucose the expression levels of both genes were much reduced in abi3-1 and abi3-5 compared to WT (Fig.\u00a05a, c). Glucose treatment did not result in a clear induction of ABI4, especially when compared to ABI5 and RAB18. Instead ABI4 expression was roughly maintained to the same level observed for t\u00a0=\u00a00 but was enhanced when compared to the control treatment which displayed a strong reduction of ABI4 expression (Fig.\u00a05b). ABI4 transcripts were 2\u20138 fold lower in the abi3 mutant backgrounds upon glucose treatment. Thus, the abi3 mutation did not allow full glucose-induced expression of RAB18, ABI4 and ABI5. Interestingly, the reduced expression is most pronounced in the strong abi3-5 mutant background.\nFig.\u00a05Altered glucose-regulated gene expression in abi3 mutants. Glucose-regulated gene expression was studied in WT Ler-0 and abi3-1 and abi3-5 mutants. Gene expression was quantified by qRT-PCR at t\u00a0=\u00a00, the time of glucose application and after 24\u00a0h on control medium (con, half-strength MS\u00a0+\u00a01% sucrose) or con medium supplemented with 8 % glucose. At the t\u00a0=\u00a00 time point approximately 70% of the seeds had germinated. The values indicate an average of three independent experiments (two experiments for abi3-5). The WT Ler-0 expression on 8% glucose was set to one in each experiment. The glucose-regulated expression in Ler-0 and the two abi3 mutants was studied for RAB18 (a), ABI4 (b) and ABI5 (c). The gene expression ratio of Ler-0 over abi3 was calculated because all three genes were already under expressed in abi3-1 and abi3-5 at t\u00a0=\u00a00. This ratio was calculated by dividing the average gene expression in Ler-0 by the expression in abi3 (for both mutants) at t\u00a0=\u00a00 and after 8% glucose treatment for all three genes (d)\nAlready at t\u00a0=\u00a00 reduced expression levels for all three genes were observed in the abi3 mutant backgrounds. This was expected since the ABI transcription factors were found to affect each others expression in seeds (S\u00f6derman et\u00a0al. 2000). Moreover, ABI3 is an important regulator of LEA gene expression (Parcy et\u00a0al. 1994; Kermode 2005). To rule out that this reduced expression is responsible for the phenotype observed after glucose treatment the gene expression ratios of WT over mutant were determined for t\u00a0=\u00a00 and after glucose feeding. For each gene the expression level of WT is divided by the expression level of each mutant. These values show the fold difference of expression between WT and both abi3 mutants (Fig.\u00a05d). E.g. the level of RAB18 expression is 9 fold higher in WT compared to abi3-1 at t\u00a0=\u00a00. After glucose treatment the fold difference for RAB18 expression was 38. Except for the ABI4 expression ratio of Ler-0 over abi3-1, all WT\/mutant expression ratios showed increased values after glucose exposure. This confirms that both mutants have a reduced ability to control glucose-regulated gene expression.\nOther sugar insensitive phenotypes of abi3\nThe sugar response phenotype of the abi3-1 mutant was investigated in the sis, sun and glucose-induced delay of germination assays. High sucrose concentrations block early seedling development similar to high glucose concentrations. High sucrose resistant mutants are known as sis mutants (Laby et\u00a0al. 2000). Several mutants affected in the same genes were retrieved from gin and sis screens, e.g. gin1\/sis4\/aba2, gin6\/sis5\/abi4 and gin4\/sis1\/ctr1. The sis phenotype of WT Ler-0, abi3-1 and abi3-5 was analysed by plating these genotypes on 13% sucrose media. Both abi3 mutants show a sucrose insensitive phenotype (Fig.\u00a06a, b) and can thus be considered as sis as well as gin mutants.\nFig.\u00a06The abi3-1 mutant shows additional sugar signalling defects. The sugar response phenotype of abi3-1 was assessed in three additional assays. The sugar insensitive (sis) phenotype was assessed by scoring the early seedling developmental phenotype (by seedling greening) after growth on elevated sucrose concentration. Representative Ler-0, abi3-1 and abi3-5 seedling phenotypes are shown after growth on 13% sucrose media for 13\u00a0days (a). The percentage of germination and seedling greening of Ler-0, abi3-1 and abi3-5 was determined after growth for 13\u00a0days on control (half-strength MS\u00a0+\u00a01% sucrose) or on control medium supplemented with either 7% sorbitol or 13% sucrose media (b). The sucrose uncoupled (sun) phenotype was assessed by measuring plastocyanin (PC) expression levels by qRT-PCR in two days old seedlings grown on sucrose containing media in darkness. Sucrose repressed the PC transcript level in dark-grown two days old WT seedlings in comparison to WT seedlings grown on sugar-free media, however in sun mutants PC repression by sucrose is much less pronounced (Dijkwel et\u00a0al. 1997). Indicated are the relative PC expression on sucrose containing media for WT PC-LUC and sun6\/abi4 (assayed on 2% sucrose) and Ler-0 and abi3-1 (assayed on 1% sucrose). Values indicate an average of at least three experiments. PC expression of two days old seedlings grown on sugar-free half-strength MS was set to 1 for each genotype in every experiment (c). Germination is delayed by glucose addition. abi3-1 was tested for glucose-induced delay of germination by plating seeds on half-strength MS (0.5\u00a0MS), 0.5\u00a0MS\u00a0+\u00a07% sorbitol (7% sorb) and 0.5\u00a0MS\u00a0+\u00a07% glucose (7% glc). Sugars were already present during stratification (solid lines). In addition, seeds were stratified on sugar-free 0.5\u00a0MS media and directly after stratification transferred to 0.5\u00a0MS\u00a0+\u00a07% sorbitol (MS\u00a0>\u00a07% sorb) and 0.5\u00a0MS\u00a0+\u00a07% glucose (MS\u00a0>\u00a07% glc, dashed lines). Seed germination after the different treatments was scored daily for eight days for Ler-0 (d) and abi3-1 (e)\nDijkwel et\u00a0al. (1997) previously reported on sugar insensitive mutants that have been isolated using the sun screen. Dark-grown seedlings transiently express photosynthesis genes, including plastocyanin (PC, Dijkwel et\u00a0al. 1996). This transient increase of photosynthesis gene expression is repressed on sucrose media. In sun mutants sucrose did not repress this transient PC gene expression in dark grown seedlings (Dijkwel et\u00a0al. 1997). sun6 is such a mutant which was shown to be allelic to abi4 (Huijser et\u00a0al. 2000). A possible sun phenotype of abi3-1 was investigated by quantifying PC mRNA levels in two days old, dark-grown seedlings using qRT-PCR. Elevated PC expression was observed in sucrose-treated seedlings of sun6\/abi4-3 compared to its WT PC-LUC parent confirming its sun phenotype (Fig.\u00a06c). Interestingly, the abi3-1 mutant showed enhanced PC expression as well and, therefore, abi3 has a sun phenotype (Fig.\u00a06c). abi4 mutants displayed a nine fold increased PC expression level in sugar-treated seedlings compared to WT. In the abi3 mutants the PC expression level is only two fold increased, indicating that abi3 is a weak sun mutant.\nSugars delay seed germination in Arabidopsis. ABA plays a role in this glucose-induced delay of germination (Ullah et\u00a0al. 2002; Price et\u00a0al. 2003; Dekkers et\u00a0al. 2004; Chen et\u00a0al. 2006). Despite the involvement of ABA, several abi mutants (abi1-1, abi2-1, abi4 and abi5) are normally sensitive to glucose during germination (Price et\u00a0al. 2003; Dekkers et\u00a0al. 2004). Sugar insensitive mutants like gin6\/abi4 and gin2\/hxk1 that are insensitive to the glucose-induced early seedling developmental arrest are sensitive to glucose during germination (defined as radicle emergence). This indicates that the glucose response during germination (which delays radicle protrusion) and early seedling growth (which arrests seedling development) are two distinct processes (Price et\u00a0al. 2003; Dekkers et\u00a0al. 2004). The, abi3-1 and abi3-5 alleles were plated on 2.5% glucose. The presence of 2.5% glucose clearly delayed germination of WT seeds. The osmotic control of 2.5% sorbitol only slightly affected germination compared to control media. Both abi3 mutants showed a clear resistance against glucose as displayed by their reduced germination delay (data not shown), which is in agreement with observations of Yuan and Wysocka-Diller (2006) who reported on the glucose insensitivity of abi3-1 during germination.\nIn our experiments seeds were sterilized, plated on sugar media, and after a three day stratification period moved to the growth chamber and germination was scored the following days. Interestingly, when WT seeds were stratified on sugar-free media (half-strength MS) and after stratification transferred to sugar-containing plates, the delay of germination by 2.5% glucose was completely lost (data not shown). Thus, stratification on sugar-free media suppressed the inhibitory effect of glucose on germination. This effect of stratification was further investigated using higher (7%) glucose and sorbitol concentrations. Ler-0 and abi3-1 seeds were plated either on control, sorbitol or glucose media, stratified for three days and moved to the growth chamber. Alternatively, seeds were stratified on sugar-free control media and shifted to media containing sorbitol or glucose, and then placed in the growth chamber. Continuous treatment with 7% glucose severely delayed germination of WT seeds but affected abi3-1 significantly less as expected (Fig.\u00a06d, e). Also in this experiment, stratification on sugar-free media strongly suppressed the germination response to glucose in WT Ler-0. Even germination of the abi3-1 mutant, which already showed a reduced sensitivity to glucose during germination could be improved by stratification on sugar-free media. The germination speed of the abi3-1 mutant after stratification on sugar-free media was similar to the osmotic control indicating that this combination completely suppressed the germination delay by glucose. The glucose response during germination was affected by stratification but not the osmotic response (sorbitol treatment). This was observed for WT Ler-0 as well as the abi3-1 mutant thereby differentiating glucose from osmotic signalling.\nabi2-1 and era1-2 mutant are glucose response mutants\nOur results on the physiological and molecular level indicate that glucose-arrested seedlings mimic ABA-arrested seedlings. The finding that ABI3 is important for glucose signalling in addition to ABI4 and ABI5 led us to test the glucose insensitive phenotype of two other ABA signalling mutants, i.e. abi2-1 and era1. Brady et\u00a0al. (2003) proposed a genetic pathway for ERA1 and the ABI1-ABI5 genes in seed ABA responsiveness based on double mutant analysis between the era1 mutant and the abi mutants. This analysis suggested that ABI1 and ABI2 act at or upstream of ERA1, while ERA1 acts at or upstream of ABI3, ABI4 and ABI5 (Brady et\u00a0al. 2003). ABI2 encodes a phosphatase 2C protein that acts as a negative regulator of ABA signalling and the abi2-1 mutation results in an ABA insensitive phenotype (Koornneef et\u00a0al. 1984; Leung et\u00a0al. 1997). WT Ler-0 and abi2-1 seeds were plated along with abi3-1 on control, 7% sorbitol and 7% glucose media. In addition to abi3, the abi2-1 mutant displayed a gin phenotype as well, although its phenotype is not as strong as that of abi3-1 (Fig.\u00a07a). On control and sorbitol media all seedlings showed greening.\nFig.\u00a07Other ABA signalling mutants have sugar signalling defects as well. Putative glucose signalling defects were determined for the ABA insensitive mutant abi2-1 and the ABA oversensitive mutant era1-2. Ler-0, abi2-1 and abi3-1 were grown on control media (half-strength MS\u00a0+\u00a01% sucrose), or on control medium supplemented with either 7% sorbitol or 7% glucose. After 12\u00a0days germination and seedling greening percentage were scored (a). To investigate whether era1-2 has a glucose oversensitive phenotype both WT Col and mutant were grown on a sugar concentration which did not arrest early development of WT Col. Therefore, seedling greening of Col and the era1-2 mutant was scored daily on half-strength MS (0.5\u00a0MS) and on 0.5\u00a0MS\u00a0+\u00a06% glucose. The seedling phenotype was scored for 11\u00a0days (b)\nThe ERA1 gene encodes a \u03b2 subunit of a protein farnesyl transferase (Cutler et\u00a0al. 1996) and acts as a negative regulator of ABA signalling. The era1-2 mutant has an ABA hypersensitive phenotype. If ERA1 exerts a similar function in glucose signalling, a glucose oversensitive (glo) phenotype is expected for the era1-2 mutant. Seeds of WT Col and era1-2 were plated on half-strength MS\u00a0+\u00a06% glucose media. This glucose concentration allowed seedling greening of the WT Col seedlings. Greening started for WT seedlings from day four onwards. However, this glucose concentration arrested early seedling development of era1-2 seedlings. Even after eleven days no greening was observed for era1-2 seedlings (Fig.\u00a07b), which indicates that era1-2 indeed has a glo phenotype. The era1-2 mutant has a more dormant phenotype compared to WT (Cutler et\u00a0al. 1996). Therefore, seed germination was synchronized as much as possible by using after-ripened seeds and employing a three day stratification period on sugar-free media. On control media (half-strength MS) germination was somewhat delayed but that did not affect cotyledon greening. On 6% glucose era1-2 germination reached similar germination levels as WT seeds despite that germination was 1\u20132 days delayed (data not shown). This slower germination of era1-2 does not explain the much larger difference observed for cotyledon greening on glucose media. Thus, the small difference in germination timing between era1-2 and WT is not responsible for the seedling greening phenotype.\nDiscussion\nGLUCOSE INSENSITIVE signalling depends on a functional ABI3 gene\nOur results show that ABI3 is an essential regulator of GIN signalling. This conclusion is based on three observations. Firstly, ABI3 transcript levels accumulate in response to glucose treatment. In our study seedlings were treated for a relatively short period of 24\u00a0h and showed a 5\u20139 fold enhanced ABI3 levels, while ABI3 expression levels dropped after 24\u00a0h growth on control media (Table\u00a02). The osmotic control conditions induced ABI3 expression at most two fold showing that the strong induction of ABI3 by glucose is not solely due to osmotic stress. Secondly, glucose-regulated gene expression was altered in the abi3 mutant background. Glucose enhanced the expression of LEA genes and two important regulators of GIN signalling ABI4 and ABI5. In WT, glucose treatment led to a 14 fold induction of RAB18 and ABI5, and a 1.5 fold induction of ABI4. The abi3 mutation caused 2\u201360 fold under expression of these glucose-regulated genes (Fig.\u00a05). Thirdly, we observed a gin phenotype for seven out of eight abi3 alleles in both Ler-0 and Col accessions. The majority of the abi3 alleles tested showed a gin phenotype that was comparable to well-known gin mutants like aba1, abi4 and abi5 (Fig.\u00a04 and Table\u00a03). This indicates that ABI3 is a key regulator of GIN signalling which is in agreement with previous reports showing that ABI3 overexpression (either AtABI3 or CnABI3) in Arabidopsis results in a glucose oversensitive phenotype (Finkelstein et\u00a0al. 2002; Zeng and Kermode 2004). Kermode (2005) distinguished at least three functions for ABI3 in seeds. (i) dormancy induction and maintenance (ii) induction of storage proteins and desiccation\/stress protectants and (iii) repression of post-germinative gene expression. The observations that abi3 mutants showed a disturbed glucose-regulated induction of RAB18 and a gin phenotype (lack of post-germinative gene repression) are in agreement with the functions defined for ABI3.\nAlthough a role for ABI3 in sugar signalling during early seedling development was hinted by ABI3 over expression lines (Finkelstein et\u00a0al. 2002; Zeng and Kermode 2004) and the observation that abi3 mutants were insensitive to glucose in combination with ABA (Nambara et\u00a0al. 2002) its role remained controversial. This because studies that investigated the gin phenotype of abi3-1 in the Ler-0 background observed at most a weak phenotype. Importantly however, Ler-0 is a sugar oversensitive accession compared to the Col accession (Laby et\u00a0al. 2000) and likely, the sugar insensitive phenotype of abi3-1 has been underestimated in comparison with mutants in genetic backgrounds that show more rapid greening. Further, the absence of a clear gin phenotype for abi3-1 in earlier studies could be due to the seed material used. We observed that freshly harvested seeds were particularly sensitive to sugars during germination (data not shown). Low sugar concentrations delay germination while higher levels resulted in severely reduced germination rates. Therefore, we routinely after-ripened seeds for at least a month before these were used in our sugar signalling experiments. Seed quality changes per batch produced, which affects germination and most likely responses to internal and external stimuli as well. Hence, WT and mutant seeds used in the sugar response assays were produced simultaneously under the same growth conditions and seeds were stored under identical conditions. Moreover, we routinely stratified the seeds on sugar-free half-strength MS media before the start of the gin assays, since this procedure dramatically improves germination rates following transfer to glucose-containing media (Fig.\u00a06d; Dekkers and Smeekens 2007). Under these conditions we were able to obtain clear gin phenotypes within 12\u201314\u00a0days for abi3 in the Ler-0 background.\nIn addition, this study identified both abi3-1 and abi3-5 as sis mutants (which are insensitive for the sucrose-induced early seedling developmental arrest, Laby et\u00a0al. 2000) as well. In addition, abi3 mutants are insensitive to the glucose-induced delay of germination (Yuan and Wysocka-Diller 2006; Fig.\u00a06d, e) and abi3 is a weak sun mutant. These observations imply a broader role for ABI3 in mediating sugar responsiveness. Interestingly, another B3 domain transcription factor, HSI2, was found to regulate the sugar-inducible sporamin promoter (Tsukagoshi et\u00a0al. 2005). HSI2 is an active transcriptional repressor. A mutation in this gene causes high sporamin promoter activity in both low and high sugar conditions.\nGlucose and ABA induce a similar early seedling arrest in Arabidopsis\nGlucose treatment of germinated seeds resulted in an enhanced expression of several transcriptional regulators of late seed maturation (ABI3, ABI4 and ABI5). GIN signalling depends on these glucose-induced factors as shown by mutant analysis (Le\u00f3n and Sheen 2003; this study). LEA genes (EM1, EM6 and RAB18) were also induced by glucose treatment. The ABI transcription factors are known to regulate the expression of LEA genes (Parcy et\u00a0al. 1994; Finkelstein et\u00a0al. 1998; Finkelstein and Lynch 2000; Carles et\u00a0al. 2002; Kermode 2005). LEA gene expression is correlated with drought and desiccation tolerance (Hoekstra et\u00a0al. 2001) and its enhanced expression is in agreement with the drought tolerant phenotype observed for glucose-arrested seedlings. Interestingly, the ABA response during the post-germination developmental arrest depends on the same ABI3, ABI4 and ABI5 transcription factors. ABA similarly induces ABI3, ABI5 and a subset a LEA genes. Lopez-Molina et\u00a0al. (2001; 2002) suggested that a late embryogenesis program was re-induced by ABA, which resulted in developmentally arrested, drought tolerant seedlings and that this may constitute an important checkpoint during germination and seedling development.\nThe developmentally arrested state could only be induced within a limited time frame and after 60\u201372\u00a0h ABA sensitivity was lost. Beyond this limited time window ABA did not induce ABI3 and ABI5 protein levels, nor, re-induce LEA gene transcription and did not block early seedling development. The sugar-induced seedling arrest is restricted to a similar time window (Gibson et\u00a0al. 2001). Following this time window glucose failed to re-induce ABI3, ABI5 and LEA genes. Thus far it is not clear whether this time frame specifies a sugar sensitive window. Since ABA insensitivity results in a gin phenotype the loss of ABA sensitivity beyond this time frame may account for the glucose insensitivity observed. In conclusion, both glucose and ABA signalling depend on the same set of genes, induce similar transcription factors and LEA genes, result both in drought tolerant phenotypes and are sensitive for a similar time window. This indicates that both trigger a similar seedling developmental arrest.\nInterestingly, the induction of desiccation tolerance in germinated radicles is limited to a small time window after germination as well. In germinated seeds with short radicles (<3\u00a0mm) desiccation tolerance can be induced while in seeds with longer radicles (>4\u00a0mm) this capacity is lost (Buitink et\u00a0al. 2003). Possibly, this phenomenon depends on the same genetic components and is sensitive in a similar time window observed for glucose- and ABA-induced post-germination developmental block. A possible role was suggested for sugar signalling in desiccation tolerance induction in barrel medic (Medicago truncatula) and cucumber (Cucumis sativa) radicles (Leprince et\u00a0al. 2004). Non-reducing di- and trisaccharides, e.g. sucrose, protect the structure of membranes and proteins by the formation of a glassy state (Koster 1991; Hoekstra et\u00a0al. 2001). E.g. elevated sucrose concentrations were measured in desiccation tolerant radicles (Koster and Leopold 1988; Bruggink and van der Toorn 1995). Our study suggests that such elevated sugar concentrations may serve a double role, by protecting cellular structures and by acting as a signal for the induction of protectants such as LEA protein.\nInteractions between sugar and ABA signalling\nThe early seedling developmental arrest phenotype has been extensively exploited to isolate mutants with sugar signalling defects like gin mutants (Rognoni et\u00a0al. 2007). The gin2\/hxk1 mutant revealed a role for HXK1 in glucose signalling. Such HXK1 signalling does not depend on its metabolic function (Moore et\u00a0al. 2003) and HXK1 was shown to interact with unusual partners (a vacualor H+-ATPase and a 19S proteasome subunit) to regulate glucose signalling (Cho et\u00a0al. 2006). Other gin mutants suggested a link between glucose signalling and plant hormone biosynthesis and signalling. The lack of ABA biosynthesis and signalling (gin1\/aba2, gin5\/aba3 and gin6\/abi4) resulted in glucose insensitivity. To study the interaction between ABA biosynthesis, ABI4 and sugar signalling we used ABA deficient lines with constitutive ABI4 expression (aba2-1\/35S::ABI4). These strong ABI4 overexpression lines affected ApL3 expression (ISI pathway) and showed a stunted growth phenotype similar to what has been reported previously for severe ABI4 overexpression lines (S\u00f6derman et\u00a0al. 2000), indicating that ABI4 is active in an ABA deficient background. Nevertheless, the gin phenotype of aba2-1 could not be restored by high ABI4 expression levels (Fig.\u00a01c). Thus, ABI4 is necessary but not sufficient for GIN signalling in an ABA deficient background, indicating that other ABA regulated factors are essential for proper GIN signalling as well. Both ISI and GIN signalling rely on ABA biosynthesis and ABI4 but surprisingly, ABI4 overexpression only affected the isi phenotype of aba2-1. Thus, gin and isi sugar signalling depend on the same components but this study shows that the relationship between sugar, ABA and ABI4 is different for both sugar signalling pathways.\nABA biosynthesis and ABA signalling are required for the sugar induced seedling arrest. Genetic studies showed that two transcription factors which are involved in ABA signalling, i.e. ABI4 and ABI5, function in the sugar-induced seedling arrest (Arenas-Huertero et\u00a0al. 2000; Huijser et\u00a0al. 2000; Laby et\u00a0al. 2000; Brocard et\u00a0al. 2002). Here we show that ABI3 is also essential for GIN signalling. Glucose induces ABA biosynthesis genes (Cheng et\u00a0al. 2002) and increased ABA levels were measured in developmentally arrested seedlings. ABA is able to induce ABI3 transcription (Table\u00a02) and affects ABI3 protein stability (Zhang et\u00a0al. 2005). Thus glucose may impinge on ABI3 via activation of ABA biosynthesis. ABI3 also functions in seed development and developmental timing (Rohde et\u00a0al. 1999, 2000). Severe abi3 mutant embryos remain green and are unable to acquire desiccation tolerance and dormancy during seed development (Ooms et\u00a0al. 1993). Such mature abi3 embryos resemble a developing seedling rather than a dormant embryo (Nambara et\u00a0al. 1995). The seedling developmental arrest as induced by ABA and glucose is sensitive to developmental signals as well. Arrest of seedling development by ABA and sugars only occurs within a two to three days time window after imbibition (Gibson et\u00a0al. 2001; Lopez-Molina et\u00a0al. 2001). Therefore, the phenotype of the severe abi3 mutants is probably not solely due to ABA insensitivity but to seed developmental defects and the heterochronic nature of this mutation as well.\nTwo additional ABA response mutants with altered glucose responsiveness, abi2 and era1, strengthen the link between glucose and ABA signalling. The abi2-1 mutant showed a gin phenotype that is weaker compared to abi3-1. Possibly additional seed developmental phenotypes of abi3 may explain the stronger phenotype of the abi3-1 mutant compared to abi2-1. However, the ABA insensitive phenotype of abi2-1 is somewhat weaker compared to abi3-1 (Koornneef et\u00a0al. 1984; Finkelstein and Somerville 1990), which could explain the weaker gin phenotype as well. The era1-2 mutant is hypersensitive to ABA and shows a glucose oversensitive phenotype. Interestingly, era3 another ABA hypersensitive mutant shows a similar glo phenotype. Era3 is allelic to ethylene insensitive2 (ein2, Ghassemian et\u00a0al. 2000) and also other ethylene related mutants show sugar signalling defects (Zhou et\u00a0al. 1998; Gibson et\u00a0al. 2001; Le\u00f3n and Sheen 2003).\nThe role of ABA and ABA signalling in sugar signalling is well established. Thus far, sugar signalling was thought to rely on only a part of the ABA signalling cascade. The analysis of aba2\/35S::ABI4 transgenic lines showed that ABA related factors other than ABI4 are involved in glucose signalling. Furthermore, the glucose-induced early seedling arrest mimics the ABA-induced seedling arrest. Our study indicates that all response loci tested that act in the genetic pathway regulating ABA responsiveness as proposed by Brady et\u00a0al. (2003) affect glucose signalling. These observations suggest that the genetic pathways regulating glucose and ABA signalling are much more closely linked than assumed thus far. Obviously, both pathways are different as well. Genetic analysis suggested that HXK1 acts upstream of ABA signalling in glucose signalling. Moreover, the gaolaozhuangren2 mutant has a gin phenotype but is not affected in ABA signalling (Chen et\u00a0al. 2004). Such mutants may provide insight in the mechanism by which glucose affects ABA signalling. Interestingly, glucose delays germination in a process that depends on ABI3 but that does not require ABI1, ABI2ABI4 and ABI5 (Gibson 2005; Yuan and Wysocka-Diller 2006; Fig.\u00a06d, e), showing that the tight interaction between glucose and ABA is not present in all sugar response pathways.","keyphrases":["abscisic acid","seedling development","sugar signalling","glucose insensitive","aba insensitive"],"prmu":["P","P","P","P","P"]}
{"id":"Ann_Biomed_Eng-2-2-1705502","title":"Single-Step 3-D Image Reconstruction in Magnetic Induction Tomography: Theoretical Limits of Spatial Resolution and Contrast to Noise Ratio\n","text":"Magnetic induction tomography (MIT) is a low-resolution imaging modality for reconstructing the changes of the complex conductivity in an object. MIT is based on determining the perturbation of an alternating magnetic field, which is coupled from several excitation coils to the object. The conductivity distribution is reconstructed from the corresponding voltage changes induced in several receiver coils. Potential medical applications comprise the continuous, non-invasive monitoring of tissue alterations which are reflected in the change of the conductivity, e.g. edema, ventilation disorders, wound healing and ischemic processes. MIT requires the solution of an ill-posed inverse eddy current problem. A linearized version of this problem was solved for 16 excitation coils and 32 receiver coils with a model of two spherical perturbations within a cylindrical phantom. The method was tested with simulated measurement data. Images were reconstructed with a regularized single-step Gauss\u2013Newton approach. Theoretical limits for spatial resolution and contrast\/noise ratio were calculated and compared with the empirical results from a Monte-Carlo study. The conductivity perturbations inside a homogeneous cylinder were localized for a SNR between 44 and 64 dB. The results prove the feasibility of difference imaging with MIT and give some quantitative data on the limitations of the method.\nIntroduction\nMagnetic induction tomography (MIT) is a non-invasive and contact-less imaging modality for reconstructing the changes \u0394\u03ba of the complex conductivity distribution \u03ba =\u00a0\u03c3\u00a0+\u00a0j\u03c9 \u025b0\u025br in a target object.8,13\u201316,22 MIT requires an array of excitation (EXC) and receiving coils. Each EXC couples an alternating magnetic field B0 to the object under investigation (see Fig.\u00a01). Changes \u0394\u03ba of the complex conductivity cause a field perturbation \u0394B due to the induction of eddy currents. The perturbation induces voltage changes \u0394V in the receiver coils. It is convenient to normalize \u0394V to V0, the voltage which is induced by the unperturbed field B0.Figure\u00a01.Schematic of a possible coil system for MIT with 16 excitation coils and 32 receiver coils.\nPrevious reviews of MIT have been given in8,22,32. The method has been developed for industrial process tomography already more than 10\u00a0years ago but is comparatively new in medical imaging. Potential medical applications usually aim at the characterization of biological tissues by means of their complex conductivity. The motivation for measuring the electrical properties is their characteristic dependence on the (patho-) physiological state of tissues, especially hydration and membrane disorders. Medical applications so far suggested are: imaging of limbs,2 imaging of the brain, e.g. for the monitoring of brain edema,14,16,24,26 measurement of human body composition,7 monitoring of wound healing.23\nIn contrast to electrical impedance tomography (EIT) MIT avoids the ill-defined electrode-skin interface due to its inherently contact-less operation.\nFigure\u00a01 shows a schematic MIT coil configuration with rectangular coils as receivers and a cylindrical object space. The solenoid excitation coils are distributed on two different rings in order to obtain a true 3-D-arrangement.\nThe reconstruction of the absolute conductivity in a target region \u03a9 requires the solution of a complex inverse eddy current problem. Let be\nthe discretized non-linear forward mapping of the conductivity vector\nto the vector of induced voltages y. y contains M\u00a0=\u00a0a\u00a0\u00d7 b entries, a being the number of EXC and b that of receiving coils. The corresponding inverse problem\nis ill-posed and usually underdetermined. Uniqueness of the solution for this inverse boundary value problem was established in21 provided the angular frequency \u03c9 of the AC field is not a resonant frequency. The generic approach for the solution of this type of non-linear problem is the application of an iterative scheme such as the regularized Gauss\u2013Newton approach, including an appropriate regularization scheme.\nTo the knowledge of the authors the full inverse problem of medical MIT in 3-D has not yet been solved satisfactorily, although some approximate solutions, especially for 2-D, have been presented.3,13,33 Some authors9,15 proposed the use of weighted back-projection, similar to EIT. In all published cases the back-projection is done along magnetic flux tubes between excitation and receiving coils, the weights being calculated for the case of conducting perturbations in the empty space. However, own observations17,27,29 suggest that the basic requirements for the applicability of this kind of back-projection are not fulfilled in realistic anatomical structures, so that a more appropriate inverse approach is necessary. This paper is dedicated to demonstrating the feasibility of the 3-D reconstruction of a spherical perturbation within a cylindrical conducting body by means of a regularized one-step Gauss\u2013Newton reconstructor. The conductivities were chosen in the physiological range of human tissues.\nMethods\nThe solution of (2) requires the target region to be discretized into N voxels. Within each voxel i the assigned component \u03bai of the vector of conductivity\nis assumed to be constant. A grid of tetrahedral finite elements of second order was employed. In a general setup\nis then found with the iterative scheme: Define the forward problem with an initial parameter vector\n.\nMeasure the data vector ym.Solve iteratively for the estimated true parameters\nwhereby\nmeans the estimated \u201ctrue\u201d parameter vector. R and \u03bb are a regularization matrix and a regularization parameter, respectively, which are required to stabilize the iteration.\nWhen applying Newton\u2019s method starting from an initial guess the parameter vector\nis updated by an increment pk in each iteration step k+1\nwith the update step\nwith\n. The Jacobian\n, also called sensitivity matrix, must be recalculated in each iteration step. This procedure is very time consuming, hence a complete iterative identification run requires significant computing power, the bottleneck being the solution of the forward model. However, in EIT it could be shown that in practice most features of the image can already be recognized very satisfactorily after the first iteration. This fact led to the development of the so-called Newton-one-step reconstructor (NOSER4). NOSER is especially appropriate for so-called dynamical imaging where only the change in the conductivity between two different states of the object under investigation (e.g. lung ventilation) are of interest. In this case the first Newton step corresponds to the solution of the linearized forward problem\nwhereby\nis the change of the conductivity between two states of the observed object, and \u0394ym is the corresponding change of the measured data. In the case of comparatively small changes the inversion of (6) according to (7) yields a fairly correct localization of the perturbed regions. We evaluated the feasibility of this kind of reconstruction in MIT by implementing a NOSER-approach according to (7) under consideration of four different regularization methods.\nCalculation of the Forward Solution and the Sensitivity Matrix\nThe forward mapping\nis given by Maxwell\u2019s equations for harmonic excitation:\nwith H: magnetic field intensity, B magnetic flux density, E electric field strength, J: current density, \u025b: dielectric constant,\u00a0\u03bc: magnetic permeability, \u03c3: real conductivity, \u03ba: complex conductivity, \u03c9: angular frequency. \u03a9 denotes the interior of the object under investigation.\nThis forward problem is solved with a previously published finite element program,12,17 which employs an Ar\u2013V, Ar \u2013 formulation with edge elements of second order for the reduced magnetic vector potential Ar and nodal elements of second order for the electric scalar potential V. Boundary conditions on the far boundary (normal component of B vanishes) were prescribed on a spherical surface with a radius sufficiently large such that a change of this radius by 50% resulted in a change of the induced voltages by less than 1 %.\nSpecial attention must be paid to the efficient calculation of the Jacobian\n. A mathematically rigorous treatment of this topic has been given in.31 In our implementation we exploited the integral formulation published by Mortarelli20, which is based on a physical mutual energy concept. With this approach the absolute sensitivity dy\/d for a certain pair of coils is calculated according to (9).\nwith\nA\u03d5, A\u03a8, V\u03d5 and V\u03c8 denote the total magnetic vector potential and the electric scalar potential in the region \u03a9 due to currents I\u03d5 and I\u03c8 in the excitation and receiver coils, respectively. The sensitivity matrix\nis then obtained by evaluating (9) for all individual elements and all coil pairs. The exact numerical implementation of (9) was described in detail in11. The calculation of the sensitivity map for one pair of coils requires only two forward solutions of the eddy current problem for generating L\u03d5 and L\u03a8 in (10).\nRegularization\nIn EIT the regularization matrix RTR most frequently used is either the identity matrix I or a discrete spatial derivative operator of first or second order. Such approaches have been discussed extensively in the literature, for a good review see e.g.10 Several regularization matrices can be regarded as simple smoothness criteria for the solution but they have also a more general statistical meaning in the framework of Bayesian estimation theory (see e.g.1). In the case of uncorrelated noise with equal variance for all measurement data the estimator in (7) is a maximum a posteriori (MAP) estimator with RTR being the inverse of the expected covariance matrix of the image E[ppT]. In that sense e.g. the neighbouring matrix accounts for the case that all image values are de-correlated at borders between homogeneous regions with different mean values.\nAccording to our own observations good results can be achieved with variance uniformization5 which imposes a special assumption of the prior distribution. The objective here is to uniformize the expected variance of the reconstructed conductivity changes over the region \u03a9, thus providing approximately equal image noise in the center as in the periphery. The algorithm has been described in detail in5 and requires singular value decomposition of G according to G\u00a0=\u00a0U\u03a3VT. Then the regularization term is expressed as \u03bb RTR\u00a0= VDVT, with D a diagonal matrix with the entries di whereby \u03c3i is the Ith singular value. c is a free scalar tuning parameter.\nAlternatively truncated singular value decomposition (TSVD) has been applied in EIT-reconstruction,19 hence this approach was also implemented for MIT. In this case the inverse solution becomes\nwhereby t denotes the truncation level of the original matrices V, \u03a3 and U, thus removing the contributions of singular values with index >\u00a0t.\nIn this paper the results obtained with four different regularization schemes were compared: RTR\u00a0=\u00a0I. Using the identity matrix is the most simple Tikhonov-regularization method, penalizing high values of the reconstructed conductivity changes. In the following this method will be abbreviated as \u2018IM\u2019.RTR\u00a0=\u00a0N with N the neighbouring matrix defined as:\nnn is the number of neighbouring elements for element i, whereby only elements with common facets are considered as neighbours. N is an approximation of the spatial derivative operator of second order. Due to the irregular structure of the grid this filter is not spatially invariant, nevertheless it gives good results and is common practice for this type of inverse problem. In the following this method will be abbreviated as \u2018NM\u2019. \u03bb RTR\u00a0=\u00a0VDVT according to the variance uniformization approach. In the following this method will be abbreviated as \u2018VU\u2019.TSVD while choosing the truncation level t such as to remove all singular values below an appropriately chosen threshold.\nMethods 1 and 2 require the regularization parameter to be chosen optimally while method 3 in addition implies the choice of the tuning parameter c. In practice it turns out that the value of c is not critical over a very wide range of values because the optimal \u03bb depends on c. That means that c can be fixed at a more or less arbitrary value if a method for the automatic determination of \u03bb is applied. In our case c was set to 0.1.\nThe regularization parameter \u03bb accounts for the degree of smoothness of the reconstructed image and determines the condition number of the term (GTG\u00a0+\u00a0\u03bbRTR) in (6). Several methods for the optimal choice of this parameter have been published in the past, the most well-known ones being L-curves, Generalized Cross-Validation and the Morozov-criterion.10 Because of its clear physical interpretation we choose the latter method. In this case the optimal \u03bb is the one where the estimated residuals Gp\u00a0\u2212\u00a0\u0394ym have the same variance as the measurement noise. The motivation for this criterion is that it is obviously meaningless to make the residuals lower than expected from the statistics of the data. This method provided always stable images independently of the regularization matrix and was considered as a good basis for a fair comparison between the different regularization methods.\nModeling Setup\nThe inverse solver was tested with the simple 3-D model comprising a cylindrical conductor with two spherical inhomogeneities placed with their centers at (x\u00a0=\u00a0\u00a0\u2212\u00a060, y\u00a0=\u00a00, z\u00a0=\u00a00) (mm) and (x\u00a0=\u00a0\u00a0\u2212\u00a030, y\u00a0=\u00a052 and z\u00a0=\u00a00) (mm) and the array of 16 excitation coils and 32 receiving coils shown in Fig.\u00a01. The exact geometry is illustrated in Fig.\u00a02. The cylinder had a radius and a height of 100\u00a0mm, the perturbing sphere had a radius of 20\u00a0mm. The solenoid excitation coils were modeled by cylindrical rings with an inner diameter of 60\u00a0mm, a thickness of 1\u00a0mm and a width of 21\u00a0mm. The exciters were placed in groups of 8 with their centers on two rings with radii of 125\u00a0mm in two transversal planes of the cylinder. The lower ring is rotated by 22.5 degrees versus the upper one in order to achieve a lower degree of symmetry. The square receiver coils with an edge length of 40\u00a0mm were placed with their centers on two symmetrically arranged parallel rings with a radius of 120\u00a0mm, each comprising 16 evenly spaced coils. The orientation of the windings were opposite in both rings so that the 16 vertical pairs can be combined to planar gradiometers as published previously26. The measured data were simulated in terms of induced voltage changes when changing the conductivity of the spherical perturbation from 0.2\u00a0S\/m (=\u00a0background conductivity, homogeneous cylinder) to 0.3\u00a0S\/m. The relative permittivity was kept constant at 80 in all compartments. The excitation frequency was 100\u00a0kHz.Figure\u00a02.Schematic of the simulation model. 16 excitation coils and 32 receiver coils are placed on two concentric rings around the tank, respectively. Two spherical perturbations are placed at the shown locations with (x\u00a0=\u00a0\u00a0\u2212\u00a060, y\u00a0=\u00a00, z\u00a0=\u00a00) and (x\u00a0=\u00a0\u00a0\u2212\u00a030, y\u00a0=\u00a052, z\u00a0=\u00a00). All measures are given in (mm).\nThis arrangement was chosen as the model system for our analysis because of two reasons: It represents a true 3-D-arrangement which delivers theoretically 512 independent measuring combinations, i.e. 512 data points for one image reconstruction.It is similar to our experimental system which employs 16 excitation sites in one plane and 14 planar gradiometers which are formed by connecting in counter-phase the coils in the upper and in the lower receiver plane.\nTwo different meshes A and B were used for the generation of an artificial dataset and for the reconstruction. Mesh A comprised 11000 elements within the cylinder and approximately 1200 in the spheres while mesh B comprised 17000 elements for the homogeneous cylinder without perturbation. In both cases the diameter of the surrounding spherical surface which approximated the far boundary was chosen as 1m, requiring approximately 30000 elements (mesh A) and 50000 elements (mesh B), respectively. Uncorrelated Gaussian noise was added to the voltage data in order to simulate the noise of the receiver channels. This type of noise, although common practice in simulations of this kind,, is not entirely valid for real situations. In addition the noise of the excitation coils is propagated to all receiver coils, thus resulting in a certain amount of correlated noise in all receiver channels. This phenomenon has been studied in detail for EIT,6 but should be disregarded here for simplicity.\nThe calculation of the complete sensitivity matrix required 48 forward solutions according to the Mortarelli-approach.\nTheoretical Limits of Resolution and Contrast\/Noise Ratio\nFor EIT the theoretical limits of image quality in terms of contrast\/noise ratio (CNR) and resolution have been studied carefully in30 while no such study exists for MIT. A similarly rigorous discussion for MIT is certainly beyond the scope of this paper. However, a simplified analysis on resolution and CNR was carried out for our linear reconstruction scheme so as to have a certain theoretical basis for interpreting our empirical results.\nThere is a fundamental limit for the resolution which depends on the amount of available information in the data. This information depends on the number of data points, i.e. the number of possible sensor\u2013detector-combinations and on the degree of independence between these data points. In the case of EIT and MIT the number of independent data points is usually much lower than the number of voxels, so that the system is under-determined. Moreover the different data are correlated to a certain degree, so that the effective rank is comparatively low. In EIT, e.g. 16 electrodes provide 104 independent data points so that the information is no more than 104 \u2018effective pixels\u2019. Including some a priori-information in the form of the regularization terms leads to a defined \u2018smearing\u2019 of this information over the imaging plane and provides the typical diffuse images known from EIT.\nWe characterized the resolution of MIT with the Raleigh criterion. Accordingly two point-shaped perturbations are still separable if their point spread functions (PSF) overlap in such a way that the peak of the first one coincides with the first zero crossing of the second one. In the case of a sinc-shaped PSF the lowest separable distance is equivalent to the 64%-width of the PSF. In contrast to e.g. X-ray CT in MIT the PSF depends on the location of the perturbation and on the geometry of the object under investigation. In this paper the object is the model cylinder which is also used for the numerical reconstruction examples and for the phantoms. The PSF is calculated by mapping the true parameter values p* to the reconstructed ones p via the reconstruction equation\nA means the expression\nA\u00a0=\u00a0(GTG\u00a0+\u00a0\u03bb RTR)\u22121GT for regularized methods 1\u20133 or A\u00a0=\u00a0Vt \u03a3t\u22121Ut for TSVD.\nThe ith column of M in Eq. 13 is then the shifted PSF for the ith voxel. The theoretical limit was approximated by applying TSVD with the full set of non-zero singular values, i.e.\nWe chose TSVD for the estimation of the theoretical limit because it requires the least explicit assumptions about the a-priori distribution of p. In MIT an additional difficulty is that, in general, the 64%-boundary of the three-dimensional PSF is not spherical and thus the resolution is anisotropic. In our simplified analysis we define as Raleigh-width the largest axis of the ellipsoid which best approximates the 64%-boundary.\nAs CNR at the point x we defined\nwhereby \u0394p is the reconstructed difference between perturbation and background and std(np) is the standard deviation of the image noise. We recognize that \u0394p is not a contrast in the classical sense. Usually contrast is the difference between p in the perturbation and background divided by the background value. However, as our method is a differential one, the background value of the image is always zero and the classical contrast definition is meaningless. Moreover the determinant for the detectability of a perturbation is the difference \u0394p rather than the contrast. The CNR depends on the size and location of the perturbation as well as on the noise level of the measured data. In contrast to resolution there is no theoretical lower limit in the case of noise-free data and a perfect reconstruction method.\nWe estimated std(np(x)) by calculating the Cramer-Rao lower bound of the covariance of the parameters. Given the covariance matrix X of the voltage changes \u0394ym the Cramer-Rao lower bound of the covariance matrix of the image noise vector np is\nFrom the diagonal elements of this matrix the expected lower bound of the coefficient of variation (CV) of the reconstructed conductivity in the perturbation can be calculated for any voxel. For the evaluation of Eq. 15 the reconstructed value \u0394p in voxel i follows from Eq. 13:\nwhereby P is the set of the indices of all voxels inside the true perturbation. This equation clearly shows that the CNR must decrease when the size of the perturbation shrinks. Assuming a small perturbation and an approximately constant PSF inside this region the CNR is approximately inversely proportional to the volume of the perturbation. The detectability limit can then be defined as the one where the CNR equals 1.\nThe shown approach is simple and allows the calculation of detectability limits for perturbations with different radii and locations but it is only valid in the linear case. Alternatively Monte-Carlo studies can be carried out for different perturbations varying in size and contrast. Such a study has been published previously for a spherical perturbation in the center of a brain model.18\nFor simplicity we assumed the noise to be Gaussian with zero mean, although in the general non-linear case this may not be entirely valid. For characterizing the noise level we defined as SNR the ratio max(|\u0394V|)\/std(V) whereby std(V) is the standard deviation of the noise voltage. \u0394V is the vector of voltage changes in all excitation\/sensor combinations when a test object is placed into the empty measurement system. In order to be independent on size and location of the perturbation we chose as the test object the homogeneous background cylinder of our model.\nResults\nThe PSF was evaluated at 20 equally spaced points along the x-axis between the center and the border of the cylinder. This set includes also the point (0.6R0,0, 0), i.e. the center of one of the two perturbations in our simulation model. To mitigate discretization errors the PSF was calculated by rotating the coordinate system 8\u00a0times about 45\u00b0 and averaging the data assuming radial symmetry of the true model. Then the normalized resolution was calculated from the PSF as the inverse of the ratio between the Raleigh-width and the cylinder radius. This normalized resolution can be interpreted as the number of points which can be resolved per cylinder radius. The resulting data were plotted in Fig.\u00a03 as a function of the normalized x-coordinate in the xy-plane. Curves are depicted for noise-free data (TSVD with truncation level 512) and TSVD with truncation levels corresponding to a SNR of 44, 50 and 64\u00a0dB, respectively. The truncation levels were chosen according to the Morozov-criterion and are listed in table\u00a01. The above three SNR levels were chosen because they correspond to the range in which our current measurement system operates.25Figure\u00a03.Dependence of the theoretical normalized resolution on the noise level. Curves are depicted for noise-free data (TSVD with truncation level 512) and TSVD with truncation levels corresponding to a SNR of 44, 50 and 64\u00a0dB, respectively.TABLE\u00a01.Tuning parameters for the regularization, chosen according to the Morozov-criterion.Regularization methodTSVD Truncation levelIM \u03bbNM \u03bbVU \u03bbSNR\u00a0=\u00a064\u00a0dB1822.40E\u00a0\u2212\u00a0202.40E\u00a0\u2212\u00a0202.35E\u00a0\u2212\u00a011SNR\u00a0=\u00a050\u00a0dB1008.40E\u00a0\u2212\u00a0198.80E\u00a0\u2212\u00a0193.20E\u00a0\u2212\u00a010SNR\u00a0=\u00a044\u00a0dB681.25E\u00a0\u2212\u00a0171.19E\u00a0\u2212\u00a0177.40E\u00a0\u2212\u00a010The parameter c for VU was always kept at 0.1.\nA clear increase of the resolution with the distance from the center is observable. The theoretical limit for a point-shaped perturbation in the xy-plane is 3.5 (corresponding to 2.9\u00a0cm separation) in the center and a maximum resolution of more than 6 (corresponding to 1.7\u00a0cm separation) at the periphery. At the locations of the test spheres the resolution is in the range of 4, i.e. 2.5\u00a0cm separation. The relative loss of resolution with the noise level is stronger in the center than in the periphery. At a SNR of 44\u00a0dB the resolution does not increase any more continuously but levels off above a normalized x position of about 0.6.\nIn analogy to EIT the PSF depends strongly on the location, showing the broadest distribution in the center of the cylinder. This is reflected by increasing resolution when moving from the center towards the periphery. Increasing the amount of regularization or decreasing the truncation level according to increasing measurement noise the PSF broadens and its center of gravity is shifted towards the borders of the cylinder. Moreover increasingly strong ringing in form of star-like patterns becomes observable close to the border (not shown explicitly in this paper).\nIn Fig.\u00a04 the four regularization methods are compared at a SNR of 50\u00a0dB. Except for few points TSVD, performs poorest which is in accordance with the assumption of least explicit a-priori information.Figure\u00a04.Theoretical normalized resolution for the four regularization methods at a SNR of 50\u00a0dB.\nFigure\u00a05a shows the CNR for TSVD as a function of the normalized x-coordinate and in dependence on the noise level while Fig.\u00a05b compares the four methods at a SNR of 50\u00a0dB. As expected the theoretical CNR depends strongly on the location of the perturbation increasing from values around 2 in the center up to about 60\u00a0at the cylinder border. At the location of the perturbing spheres the CNR drops from about 26 to 13 when decreasing the SNR from 64\u00a0dB to 44\u00a0dB. In the simulated images the CNR drops from 24 to 9, i.e. remains fairly in the same range. Figure\u00a05b reveals that IM and NM yield the highest CNR, followed by TSVD and VU, whereby VU is characterized by dramatically lower values. In the center VU yields a CNR around 2 which is already very close to the limit of detectability. When comparing the theoretical values with the reconstructed ones (see table 2) the reconstructions always produce a lower CNR than expected, the discrepancy being stronger at high noise levels.Figure\u00a05.Panel A: CNR for TSVD as a function of the normalized x-coordinate (relative to the cylinder radius) and in dependence on the noise level. Panel B: comparison of the four methods at a SNR of 50\u00a0dB.\nFigures\u00a06 and 7 show the reconstructed mean images from a Monte-Carlo study with 50 runs for each of the four methods and an SNR of 64 and 50\u00a0dB, respectively. Representative cross-sections in the xy-plane and in the xz-plane were selected. The respective regularization parameters are listed in Table\u00a01.Figure\u00a06.Mean images of the Monte-Carlo study. Reconstructed \u0394 \u03c3 (transversal and saggittal section through the origin) for the spherical perturbations with four different regularization matrices and a SNR of 64\u00a0dB.Figure\u00a07.Mean images of the Monte-Carlo study. Reconstructed \u0394 \u03c3 (transversal and saggittal section through the origin) for the spherical perturbations with four different regularization matrices and a SNR of 50\u00a0dB.\nIn all cases the two perturbations can be recognized as diffuse bright disks. The dotted circles in the figures delineate the original position of the perturbing spheres.\nA number of performance indices were calculated in order to quantitatively assess the results in Figs.\u00a06 and 7. They are summarized in Table\u00a02 and comprise:\nMean and CNR of the pixel values in the center of gravity of each reconstructed perturbation. These parameters quantify the correctness of the reconstructed values as well as their uncertainty. The theoretically expected values are listed for comparison. The center of gravity was chosen as evaluation point because the reconstructed perturbations deviate more or less from the spherical shape and show significant outward shift with increasing noise level.Radial outward shift of the spheres in the reconstructed image (fidelity of the location). This shift was determined by localizing the center of gravity for each spot within a wedge-shaped search region with a height of 2.6\u00a0times the sphere\u2019s radius and excluding the outermost 2\u00a0mm as well as the innermost 40\u00a0mm in radial direction from the center. The restriction of the search region to this volume prevented spurious contributions from outliers and negative image values far away from the real perturbing regions. Also for this parameter we present theoretical values as expected from the PSF.TABLE\u00a02.Summary of the performance indices defined in the text.SNR\u00a0=\u00a064\u00a0dBSNR\u00a0=\u00a050\u00a0dBSNR\u00a0=\u00a044\u00a0dBMethodPerformance indexSphere realSphere theoretBackgroundSphere realSphere theoretBackgroundSphere realSphere theoretBackgroundIMMean central0.0210.0160.000540.0060.00430.00350.001NM0.0220.0160.000560.0060.0040.000510.00320.0010.00035VU0.0240.0240.000550.010.00650.000430.00600.00360.00035TSVD0.0330.0250.000540.010.00740.000500.01100.0020.00055IMCNR42.345.016.123.01220.1NM41.144.016.522.510.719.3VU7.98.25.65.23.43.3TSVD23.925.813.718.49.113IMResolution2.842.602.53NM2.812.582.50VU2.942.462.58TSVD2.582.422.24IMNormalized outward shift*0.100.100.210.250.330.35NM0.100.100.210.250.330.35VU0.100.060.110.150.210.15TSVD0.100.100.180.210.350.33Performance measures for the comparison of the reconstruction methods.*Normalized to the cylinder radius.\nIn addition Table\u00a02 lists the theoretical resolution limits for all methods and noise levels at the position of the perturbations.\nWith 64\u00a0dB SNR noise the two spheres can be resolved comparatively easily with all four methods. With 50\u00a0dB SNR noise the resolution is theoretically still possible for all methods. In the reconstruction the resolution is already somewhat below the limit for IM and VU, the image values in the notch between the two peaks being around 71% of the peak values. TSVD and NM appear to separate the perturbations even worse although theoretically this should not be the case. At higher noise all algorithms tend to shift the objects towards the borders of the cylinder when looking at the parameter \u2018normalized outward shift\u2019 in Table\u00a02. Here again the VU performs best by producing the lowest shift. At 64\u00a0dB SNR the mean images (not shown here) are in general comparatively poor. IM and NM interestingly still allow a clear separation of two objects, but their localization is very poor, the outward shift being extremely large (see Table\u00a02). VU still provides a much better localization but only at the cost of CNR. TSVD failed to produce a clear image, an observation which was not expected from theory.\nDepending on the regularization method the central voxel value of the perturbation at (\u2212\u00a00.6R0,0,0) decreases from 0.02\u20130.03\u00a0S\/m to 0.002\u20130.006\u00a0S\/m, compared to the true value of 0.1\u00a0S\/m. This means that even under nearly ideal conditions (64\u00a0dB SNR) the conductivity changes are strongly underestimated. NM and TSVD yield nearly the same central voxel values as TSVD while VU produces much lower values.\nFigure\u00a08 shows single reconstructions for IM and VU at all three noise levels. Both methods allow a separation of the perturbations in all cases, but the poor CNR of VU implicates a very noisy image at 44\u00a0dB. The pronounced difference in outward shift is clearly visible at 44\u00a0dB, where VU still allows a fair localization while IM fails completely to reconstruct the perturbations at the right positions.Figure\u00a08.Comparison of single-shot images for VU and IM at the three different noise levels.\nIM and NM perform nearly identically, also their optimal regularization parameters are almost identical. VU yields, in general, larger values in the perturbed regions but also a larger STD.\nDiscussion\nThe results demonstrate the feasibility of image reconstruction from MIT-data with the same methods as suggested for EIT. This finding is not self-evident, as the sensitivity distribution is significantly different in EIT and MIT.27,29 In EIT the region of maximum sensitivity is located between the equipotential surfaces which meet the surface at the detection electrodes, i.e. within a tube-shaped region which connects injection and detection sites. As shown in27,29 in MIT the sensitivity is not concentrated within a field tube between excitation and receiver coil but increases with the distance from the tube axis, according to the increase of the eddy current density. This may be the main reason why the reconstructed solution tends to be displaced towards the nearest border of the cylindrical tank, especially at higher noise levels. An extreme case for this effect can be observed if the perturbation is placed exactly in the origin and if the senders and receivers are all in the same plane (image not shown due to space restrictions). Instead of the expected spot in the origin two widely separated spots appear on the cylinder axis close to the top and bottom of the cylinder, respectively. In fact such a coil arrangement cannot distinguish between an object in the center and two objects on the cylinder axis placed symmetrically with respect to the origin, because it is always possible to find two corresponding conductivitiy changes so that the field perturbations in the median plane are the same. Obviously, in this ambiguous situation, the algorithm favors the splitted solution according to the sensitivity distribution. A similar ambiguity occurs when using differential sensors, such as the gradiometers employed in our setup. For getting rid of such artifacts it is very important to use a less symmetric transceiver setup which provides enough spatial information in 3-D.\nThe theoretical resolution limit was calculated from the PSF as derived from the TSVD method. This limit depends on the chosen regularization method, the geometry of the object and on the location within the object. The respective dependences are shown in Fig.\u00a03 and Table\u00a02 for some selected positions inside a cylinder. TSVD was chosen for the calculation of the theoretical limits because it requires no explicit assumptions about any prior distribution of p. In this sense it contains less a-priori information than the other methods and thus describes the worst case, as confirmed by Fig.\u00a03 and Table\u00a02. The calculated PSF shows all basic features of the reconstructed images.\nThe PSF is a 3-D-distribution similar to a 3-D analog of the sinc function. This means that most of its energy is concentrated in a diffuse cloud around the considered point but that there exist three-dimensional \u2018side lobes\u2019 which decay with the distance and show some kind of \u2018periodicity\u2019. The \u2018bean\u2019-shaped artifacts which are visible in most top views of Figs.\u00a06 and 7 are typical features of the PSF as well as the \u2018star-artifact\u2019 in the TSVD-images. Therefore these ringing artifacts do not stem from inaccuracies of the reconstruction method or measurement errors, but, instead, are inherent in the PSF.\nThe resolution clearly also depends on the contrast in the presence of noise, because the contrast determines the SNR. Increased noise requires more regularization and hence leads to a broadening of the PSF-distributions. Figure\u00a03 shows the dependence of the resolution on the noise in the case of TSVD at one single contrast of 0.5 only. A more complete analysis similar to that given for EIT in30 should also show the dependence of the resolution on contrast, size and location of the perturbation at a given noise level. However, such a comprehensive analysis requires a separate paper and should not be given here.\nThe CNR depends strongly on the radius and, to a less extent, on the noise level. Obviously IM and NM produce very similar values, followed by TSVD. VU in general yields comparatively small CNR but higher central voxel values. For centrally placed spheres with 4\u00a0cm diameter VU yields CNRs close to the detection limit. When comparing the theoretical values with the reconstructed ones (see Table\u00a02) the reconstruction always produces a lower CNR than expected, the discrepancy being stronger at high noise levels. One surprising detail of Fig.\u00a05a is that at higher noise levels the CNR-curves cross the curve for 64\u00a0dB. This means that very close to the periphery noisier data yield higher CNR values than less noisy data. The reason for this counter-intuitive effect is not yet entirely clear but may be related to the strong outward shift of the PSF at higher noise. In those cases the evaluation of the CNR at the original position of the perturbation may not be appropriate any more and should be interpreted with caution.\nIn the case of weak perturbation we can assume that the CNR depends approximately linearly on the conductivity difference \u0394\u03c3. The dependence on the volume of the perturbation is, in general, more complicated because the PSF depends on the location and is therefore not constant throughout the whole perturbation. Only in the case of small spatial extension of the perturbation an approximately linear dependence on the volume can be assumed.\nThe low number of significant singular values even at comparatively low noise (64\u00a0dB SNR) suggests that, similar as in EIT, a significant amount of sensor combinations does not provide enough independent information. Intuitively one would expect this finding because there exist pairs of excitation\/receiving coils which nearly fulfill the reciprocity condition and hence reduce the amount of useful combinations to about half of the number of possible combinations, i.e. to 256 in our case.\nFurther investigations should determine the maximum \u2018useful\u2019 number of sensors in one plane, i.e. that number beyond which additional sensors do not increase the resolution significantly. Adding more sensors off-plane may add more 3-D-information and hence still provide improvement. This possibility should be studied in further research.\nWhen comparing the regularization schemes after application of the Morozov-criterion, the IM and the NM approach yield the smoothest visual appearance and the highest CNR. However, they also tend to displace the perturbations towards the border of the tank. The best localization is obtained with VU, probably because the imposed variance counteracts somewhat the lower sensitivity in the center of the object. However, VU yields also the lowest CNR, i.e. the less homogeneous images and more pronounced ghosts. The failure of TSVD at a SNR of 44\u00a0dB was not expected theoretically, although, in general, it produces the poorest theoretical resolution. In terms of separability of the two perturbations VU performs best, especially when also taking into account the correct localization.\nIn neither case, however, the single-step solution provides the correct values for \u0394\u03c3. Even at a SNR of 64\u00a0dB the reconstructed differences are too low by a factor of at least 5, thus demonstrating that the method yields the correct search direction but not the correct step size.\nThe highest mean voxel values are provided by VU and TSVD, the drop with the noise levels being lowest. However, on the other hand VU yields the highest standard deviations. Moreover VU tends to produce more pronounced \u2018ghost objects\u2019 in the homogeneous region than IM and NM. As expected from the PSF TSVD tends to produce \u2018star-artifacts\u2019 at the cylinder border, i.e. a periodic pattern with 16 peaks close to the centers of the receiving coils. This artifact gets worse at increasing noise level.\nFirst experiments with smaller models and at least 10 iterations with an iterative solver show that the solution converges towards the correct voxel values. Nevertheless the single-step method may be completely justified in cases where only qualitative changes are sought for or where proportions are to be reconstructed, e.g. in frequency differential spectroscopic imaging. Therefore the area of applicability of a single-step approach has to be analyzed carefully in future work.\nAt least for the shown examples MIT appears relatively robust against Gaussian measurement noise. A SNR of 64\u00a0dB allows for a stable and distinct solution. Even 44\u00a0dB allow the recognition of the two spheres when applying the correct regularization. This result is very important for the practical implementation because, due to technical reasons, MIT is expected to yield low SNR (around 50\u00a0dB) at frequencies as low as 100\u00a0kHz, which are interesting for the imaging of pathophysiological processes.28,26 However, our results have only been achieved with two single focal perturbations with a relatively large diameter of 20% of the background object. In a more advanced study the stability and the resolution of the images should be investigated for a series of perturbations with different diameter and spacing. For the monitoring of brain edema which usually do not split up in separate sub-regions our approach may be sufficiently stable. This hypothesis has to be tested both theoretically and empirically for centrally placed perturbations (worst case).\nAs to the detectability of spherical perturbations in a human brain simulation results in18 have shown that a sphere with a diameter of about 40\u00a0mm, a background conductivity of 0.1\u00a0S\/m a contrast of 2 yields a SNR of 24\u00a0dB at 100\u00a0kHz when applying 1 A to an excitation coil with 45 turns and a receiver coil with 1 turn. The assumed acquisition time was 200\u00a0ms, With our present technology single shot images are generated with an acquisition time of 20\u00a0ms, an excitation coil with five turns and a current up to 20\u00a0A. The receiver coils have 40 turns with otherwise unchanged geometry. This means an overall increase in SNR by 28\u00a0dB. Extrapolating the analysis given in,28 an improvement of the SNR by a factor of 5\u201310 is still technically possible, thus reaching 50\u201360\u00a0dB, which is obviously sufficient for producing fairly acceptable difference images.\nAnother open question is the influence of the mesh quality on the reconstruction results. We used a comparatively coarse non-uniform grid for the reconstruction. Therefore non-negligible numerical errors are to be expected which may explain the discrepancies between theoretically expected and the reconstructed values for CNR and radial displacement. Also the apparently somewhat worse spatial resolution in the reconstructed images than theoretically expected may be due to such numerical problems. The influence of the mesh and the optimization of mesh quality should be a major issue for further developments.\nThe results were obtained at a single frequency only. Future work should concentrate on the exploitation of the frequency dependence of the tissue conductivity and measurements at frequencies up to several MHz. A multi-frequency approach is expected to increase significantly the available information and thus the quality of the images. Possible applications may then in fact be the same as for EIT (lung function monitoring, lung edema monitoring) and hydration monitoring in the brain.","keyphrases":["magnetic induction tomography","regularization","inverse problem","passive electrical properties","conductivity imaging"],"prmu":["P","P","P","M","R"]}
{"id":"Eur_J_Pediatr-4-1-2413080","title":"Growth charts for Wolf-Hirschhorn syndrome (0\u20134 years of age)\n","text":"Wolf-Hirschhorn syndrome is characterized by severe growth and mental retardation, microcephaly, seizures and \u2018Greek helmet\u2019 facies, caused by partial deletion of the short arm of chromosome 4. Growth charts are given from 0\u20134 years of age, based on the study of 101 individuals. Use of these specific growth charts is recommended, because standard growth charts are inapplicable for patients with WHS.\nIntroduction\nWolf-Hirschhorn syndrome (WHS) is caused by partial deletion of the short arm of chromosome 4 (4p- syndrome) [3\u20135, 7]. Cardinal features of this rare syndrome are severe growth and mental retardation, microcephaly, seizures, \u201cGreek helmet\u201d facies and closure defects (Fig.\u00a01) [1, 2, 4, 5, 7]. Growth in children with Wolf-Hirschhorn syndrome differs from that of normal children [1]. Up to date, there are no Wolf-Hirschhorn specific growth charts available.\nFig.\u00a01A 7-year-old boy with Wolf-Hirschhorn syndrome\nMaterials and methods\nThe study is based on data from 1,057 examinations of 101 children with WHS, 35 males and 66 females, born between 1975 and 2002. Data from 32 children were collected from records of individuals with WHS in the Netherlands. Another set of data was obtained from 69 children with WHS from Great Britain, North America, Germany and Australia who were contacted through WHS support groups. There were no exclusion criteria except growth hormone treatment. Thus, all children regardless of complicating disease such as heart defects, renal disease or feeding problems, were included. The number of observations in the Dutch group of children was higher than in the group of children from other countries. No difference in parameters related to growth was observed between Dutch and non-Dutch patients. The data used for creation of the growth charts were age at examination, height (cm), weight (kg) and head circumference (cm). The growth charts cover the time period from birth until 4\u00a0years of age.\nThe data for each sex were divided into 30 different age groups, with 1-month intervals during the first 2 years of life, 3-month intervals during the 3rd and 4th years of life. Each child contributed only one single set of data for each age group. If data from more than one examination within an interval were available, the data from the first examination were used. The growth data were compared to standard growth charts for healthy children based upon the fourth Dutch growth study of 1997 [6]. As there are no growth charts for children with WHS elsewhere in the world, no comparison could be made with growth charts from children with WHS from other countries.\nData for weight and height were transformed into logarithms before statiscal analysis in order to obtain normal distributions. All growth charts are based on means and standard deviations. The software used was SPSS version 10 and Sigmaplot version 8.02 bij SPSS Inc.\nResults\nFigures\u00a02 and 3 present growth charts for height for boys and girls. Mean birth length was 43.0\u00a0cm in girls and 41.5\u00a0cm in boys, corresponding to \u22123 SDS on growth charts for healthy Dutch children. Mean height at 4\u00a0years was 87.2\u00a0cm for girls and 85.8\u00a0cm for boys, corresponding to \u22124 and \u22124.5 SDS, respectively.\nFig.\u00a02Growth chart for height for girlsFig.\u00a03Growth chart for height for boys\nFigures\u00a04 and 5 represent growth charts for weight for boys and girls. Mean birth weight was 1.9\u00a0kg for girls and 2.1\u00a0kg for boys, corresponding to \u22123 SDS on growth charts for healthy Dutch children. Mean weight at 4\u00a0years was 9.8\u00a0kg for girls and 9.7\u00a0kg for boys, corresponding to \u22124 SDS and \u22124.5 SDS respectively. Figure\u00a06 represents the growth chart for head circumference for boys and girls. Mean head circumference at birth was 30.5\u00a0cm for girls and boys, corresponding to \u22123.5 SDS for both sexes on normal growth charts. Mean head circumference at 4\u00a0years was 45.8\u00a0cm for girls and boys, corresponding to \u22123.5 SDS, respectively.\nFig.\u00a04Growth chart for weight for girlsFig.\u00a05Growth chart for weight for boysFig.\u00a06Growth chart for head circumference for boys and girls\nDiscussion\nGrowth is used in pediatrics as a marker of health and is an important tool in the medical care for children. Severe growth retardation is one of the cardinal features of WHS [1]. It might be caused by genetic factors, poor feeding status, inherited parental factors and associated diseases. Whether patients with WHS suffer from an additional endocrine disorder is not known and is beyond the scope of this study. Up to date there are no WHS-specific growth charts available in contrast to more common chromosomal disorders like Down\u2019s syndrome, Turner syndrome and Prader-Willi syndrome. Although the ideal manner in constructing growth charts would be a prospective, longitudinal study based on a large study group, this method cannot be implied for rare syndromes such as WHS. To increase the number of observations we combined the available longitudinal data with cross-sectional measurements. We have used data from all children with WHS, including those receiving tube-feeding. Several other associated diseases such as heart or renal disease theoretically could influence growth of the patients. Although 33% of children with WHS have congenital heart defects, these defects are in most cases not complex in nature [1]. Closure defects, hypotonia, gastro-eosphageal reflux, epilepsia, repeated infections and surgical procedures all can have a significant influence on growth. Not all patients with WHS have the same deletion size. Until now there has been no evidence of an influence of the deletion size on growth, but all studies have only involved small numbers of children. Deletion size can be expected to be of influence on growth, and therefore we tried not to include patients with a known microdeletion. However, this study inevitably includes patients with different deletion sizes, but the number of included children was too small to show an effect on growth. Based on the available data from the Dutch cohort, we could not identify a major co-morbidity risk factor aggravating growth retardation. Almost all children with WHS showed a marked intra-uterine growth retardation. Despite adequate feeding none of the children with WHS, demonstrated catch-up growth; all remained short with a profound microcephaly. The developed specific growth charts should be used for children with WHS from birth to 4\u00a0years of age for the estimation of their growth and health condition as standard growth charts are inapplicable in these patients. At present the authors are constructing growth charts for older children with WHS.","keyphrases":["growth","growth charts","wolf-hirschhorn","whs"],"prmu":["P","P","P","P"]}
{"id":"Pediatr_Nephrol-4-1-2335288","title":"Chronic kidney disease after liver, cardiac, lung, heart\u2013lung, and hematopoietic stem cell transplant\n","text":"Patient survival after cardiac, liver, and hematopoietic stem cell transplant (HSCT) is improving; however, this survival is limited by substantial pretransplant and treatment-related toxicities. A major cause of morbidity and mortality after transplant is chronic kidney disease (CKD). Although the majority of CKD after transplant is attributed to the use of calcineurin inhibitors, various other conditions such as thrombotic microangiopathy, nephrotic syndrome, and focal segmental glomerulosclerosis have been described. Though the immunosuppression used for each of the transplant types, cardiac, liver and HSCT is similar, the risk factors for developing CKD and the CKD severity described in patients after transplant vary. As the indications for transplant and the long-term survival improves for these children, so will the burden of CKD. Nephrologists should be involved early in the pretransplant workup of these patients. Transplant physicians and nephrologists will need to work together to identify those patients at risk of developing CKD early to prevent its development and progression to end-stage renal disease.\nIntroduction\nChronic kidney disease (CKD) is a frequent and increasingly recognized complication of solid-organ and hematopoietic stem cell transplantation (HSCT), increasing the complexity of patient management and impacting survival. The incidence of CKD after cardiac, liver, and HSCT varies from 7\u201386% in pediatric patients, partly due to the lack of a uniform definition of CKD after transplant (Table\u00a01). Up to 16% of transplant survivors will develop end-stage renal disease (ESRD) [1\u201319].\nTable\u00a01Percentage of patients with chronic kidney disease (CKD) and end-stage renal disease (ESRD) based on transplant typeTransplant typeCKDESRDReferenceLiver28\u201386%0\u20138%[1\u20135]Cardiac7\u201354%2%[6\u201310]Heart\u2013lung34%7\u201316%[11\u201313]Hematopoietic stem cell transplantation18\u201342%5\u20138%[14\u201319]\nThis review focuses on the epidemiology, risk factors, and outcomes of children who develop CKD after liver, cardiac, lung, heart\u2013lung, and stem cell transplant.\nLiver transplant\nEpidemiology\nThe majority of studies of renal disease after liver transplantation has been done in adults, and the cumulative incidence of CKD varies from 5% to 30% over 1\u20135\u00a0years after transplant [20]. In a retrospective study of 117 pediatric liver transplant patients who survived 3\u00a0years after transplant (median 7.6, range 3\u201314.6\u00a0years), the prevalence of CKD, defined as a glomerular filtration rate (GFR) <70\u00a0ml\/min per 1.73\u00a0m2 at last follow-up was 32% [1]. In a 10-year follow-up study of 12 children undergoing a liver transplant in France, mild to moderate CKD (mild = GFR 60\u201380\u00a0ml\/min per 1.73\u00a0m2; moderate = GFR 20\u201360\u00a0ml\/min per 1.73\u00a0m2) developed in six of seven patients. Early deterioration of renal function is usually seen within the first year following transplant, followed by a period of stabilization, then with progression on long-term follow-up [2, 3]. In contrast, in a study of 50 pediatric liver transplant patients in Poland, CKD stages 2 and 3 as defined by the National Kidney Foundation Kidney Disease Outcomes Quality Initiative (NKF\/KDOQI) (Table\u00a02) developed at 1\u00a0year after transplant and remained stable after 3\u00a0years of follow-up [4]. In this study, GFR was calculated using both diethylenetriamine pentaacetic acid (DTPA) plasma clearance and estimated using the Schwartz formula. Little correlation was found between the two methods with the Schwartz formula, consistently overestimating GFR [4]. Therefore, the prevalence of CKD is likely underestimated in this patient population if estimated measures of GFR or serum creatinine are used to define CKD.\nTable\u00a02National Kidney Foundation Kidney Disease Outcomes Quality Initiative definition of chronic kidney disease by stageStageDescriptioneGFR (ml\/min per 1.73\u00a0m2)1Kidney damage with normal or \u2191 GFR\u2265 902Kidney damage with mild \u2193 GFR60\u2013893Moderate \u2193 GFR30\u2013594Severe \u2193 GFR15\u2013295Kidney failure<15 or dialysiseGFR estimated glomerular filtration rate\nRisk factors for developing CKD\nMany patients will have renal disease at the time of liver transplant secondary to hepatorenal syndrome and\/or acute tubular necrosis. In one study, renal histology pretransplant revealed glomerulosclerosis and other mild glomerular changes with mesangial matrix expansion, capillary-wall changes, and mesangial immunoglobulin (Ig) G, IgA, and IgM in the majority of cases despite normal serum creatinines [21]. Ultrasonographic findings included nephromegaly and increased echogenicity, which improved in the majority of cases after transplant [22]. In pediatric patients, there can be renal involvement from the primary liver disease, as in patients with primary hyperoxaluria, autosomal recessive polycystic kidney disease, Alagille\u2019s syndrome, and tyrosinemia (Table\u00a03). Renal dysfunction in patients with these disorders often improves after liver transplant. However, the long-term affects of preexisting renal disease on developing CKD after orthotopic liver transplant has not been studied systematically in pediatric patients. In contrast, in adult liver transplant patients who develop acute renal failure during the peri- and postoperative periods, there is an increased risk for developing CKD after transplant [23] and progression to CKD stage 5 requiring dialysis [24].\nTable\u00a03Liver diseases associated with preexisting renal diseaseTyrosinemiaAutosomal recessive polycystic kidney diseaseAlagille\u2019s syndromePrimary hyperoxaluriaHepatitis B- and C-related glomerulonephritis\nOther identified risk factors (Table\u00a04) for CKD include GFR <70\u00a0ml\/min per 1.73\u00a0m2 at 1\u00a0year after transplant and cyclosporine use [1]. In this study, there was an inverse relationship between hypertension at 1\u00a0year after transplant and development of renal dysfunction. In addition, in adult patients, preexisting diabetes, pre- and postoperative renal failure, hypertension, age, female gender, and hepatitis C infection increased the risk of CKD and ESRD [20]. Though the majority of renal dysfunction after liver transplant is attributed to the use of cyclosporine and\/or tacrolimus, there have been reports of other types of pathology present leading to CKD. Studies have found preexisting renal disease such as focal segmental glomerulosclerosis (FSGS) and hepatitis-C-related injury including membranoproliferative glomerulonephritis, unresolved hepatorenal syndrome, and diabetic nephropathy on renal biopsy after liver transplant [25, 26].\nTable\u00a04Risk factors for developing chronic kidney disease by type of transplantRisk factor common to all transplant typesCalcineurin inhibitor useLiverGFR of <70\u00a0ml\/min per 1.73\u00a0m2 at 1\u00a0year after transplantPretransplant renal dysfunctionAcute renal failurePreexisting diabetesAgeFemale genderHepatitis CCardiacPretransplant dialysisHypertrophic cardiomyopathyAfrican American racePrevious transplantPretransplant diabetesExtracorporeal membrane oxygenation useHeart\u2013lungHypertension posttransplantElevated serum creatinine at 1\u00a0month posttransplantHematopoietic stem cell transplantAcute graft-versus-host disease grades II\u2013IVOlder ageTransplant from an unrelated donorAcute renal failureChronic graft vs. host diseaseTotal body irradiation\nCalcineurin inhibitors\nThe use of cyclosporine (CSA) and tacrolimus in managing liver transplant patients has greatly improved outcomes. However, the improvement in survival is associated with an increased development of CKD. The manifestations of CSA toxicity range from asymptomatic azotemia and proteinuria to fulminant multiorgan failure [27]. In addition to the commonly described striped fibrosis and arterial and vascular lesions seen in patients on these medications, thrombotic microangiopathy has also been described in as many as 50% of patients after liver transplant [25]. These lesions are typically characterized by mesangiolysis, thrombus formation within glomerular capillaries, and widening of the subendothelial spaces. The nephrotoxic effects of CSA correlate with drug serum levels and therapy duration. CSA is also known to cause arteriolar injury, glomerulosclerosis, and interstitial fibrosis, as well as diffuse expansion of the mesangial matrix [28, 29]. The hypothesized mechanisms behind this injury appears to be vasoconstriction secondary to an imbalance between the vasodilatory hormones such as prostaglandin E1 and vasoconstrictive ones such as thromboxane A2 [30]. Increased synthesis of transforming growth factor (TGF)-\u03b21 by calcineurin inhibitors also contributes to the development of CKD in patients after transplant, and genetic polymorphisms in the TGF-\u03b21 gene have been associated with the development of ESRD after cardiac transplant [31].\nReducing calcineurin inhibitor levels while adding mycophenolate mofetil (MMF) in patients with CKD 5 years after liver transplant resulted in renal function improvements as measured by serum creatinine and creatinine clearance after 24\u00a0months of combined therapy, with only minor changes noted in immune function in these patients. Decreases in microalbuminuria were also noted[32]. Some authors have suggested that thrice-daily dosing of cyclosporine leads to decreased nephrotoxicity and only mild histopathologic changes in the kidney after 3\u00a0years of follow-up [33]. The authors attribute their findings to more constant trough blood levels and lower, less toxic, peak levels as well as the use of a calcium-channel blockers to manage their patients\u2019 hypertension [33]. In a case series, three children who underwent combined liver and kidney transplant were switched to sirolimus because of prolonged renal failure requiring dialysis and acute calcineurin inhibitor toxicity on biopsy. All three demonstrated improved renal function with cessation of dialysis [34]. These studies suggest that decreasing calcineurin inhibitor exposure by adding MMF or eliminating long-term exposure by switching to sirolimus can have beneficial effects on renal function in children after liver transplantation.\nCardiac transplantation\nEpidemiology\nApproximately 350 pediatric cardiac transplants are performed annually in the USA [7]. In a 2006 report from the International Society for Heart and Lung Transplantation, the incidence of renal dysfunction (defined as an abnormal serum creatinine) was 10% at 5\u00a0years after cardiac transplant [6]. Sixty-three percent of patients had hypertension. After 8\u00a0years of follow-up, the percentage of patient with renal dysfunction did not change, but 2% of patients were on dialysis or had received a renal transplant. In reports from single centers, the incidence of CKD at approximately 10\u00a0years after cardiac transplant in pediatric patients ranged from 7% to 54%; however, only a small percentage of these patients progress to ESRD requiring dialysis and\/or renal transplantation [7\u201310]. Hypertension is a more common finding, occurring in approximately 70% of patients after transplant [7\u201310]. Some centers actually report an improvement in kidney function in the first year following cardiac transplant and stabilization thereafter [9]. In adult studies, there is an increased risk of mortality associated with development of CKD [35, 36].\nRisk factors for development of CKD after cardiac transplant\nA recent study of 2,032 pediatric cardiac transplant patients transplanted between 1990 and 1999 identified pretransplant dialysis, hypertrophic cardiomyopathy, African American race, and previous transplant as risk factors for developing CKD (defined in this study as a creatinine >2.5\u00a0mg\/dl) [37]. Additional risk factors for developing ESRD included pretransplant diabetes and intensive care unit stay or extracorporeal membrane oxygenation [37]. In the adult population, several risk factors for CKD development have been identified and include older age at transplant, pretransplant serum creatinine, preexisting diabetes, abnormal GFR at 1\u00a0year after transplant, hypertension after transplant, and cyclosporine immunosuppression within the first 6\u00a0months after transplant [12, 35, 36, 38, 39]. Risk factors for progression to ESRD include postoperative development of hypertension and proteinuria of >1\u00a0g\/24 h [13, 40].\nCalcineurin inhibitors\nThough the nephrotoxic effects of calcineurin inhibitors are well known, data in the pediatric cardiac transplant population are conflicting. After 18\u00a0months of triple immunosuppression including cyclosporine, patients\u2019 GFR remained stable, and renal biopsy specimens in four patients did not show signs of cyclosporine toxicity [41]. Minor abnormalities in tubular function resulting in hyperuricemia and hyperkalemia were reported. Similarly, in a study of adult cardiac transplant patients treated with cyclosporine for 5\u00a0years, GFR (measured by inulin clearance) remained stable at 66\u00a0ml\/min per 1.73\u00a0m2, as did tubular function. The only abnormalities noted were hypertension and the presence of microalbuminuria. However, other studies of pediatric cardiac transplant patients found an association between cyclosporine use within the first 2\u00a0months after transplant and decreases in GFR years after transplant [42]. High cyclosporine levels (>500\u00a0\u03bcg\/L) in the first 6\u00a0months after cardiac transplant were associated with developing ESRD at anytime after cardiac transplant [43]. In a pediatric study of 14 patients with progressive decline in renal function over 2\u20135\u00a0years, inulin clearance declined from 84\u00a0ml\/min per 1.73\u00a0m2 at 1\u00a0year to 49.8\u00a0ml\/min per 1.73\u00a0m2 at 5\u00a0years. Biopsies performed on 13 patients revealed chronic tubulointerstitial lesions of grade II associated with acute changes of vacuolization of the proximal tubules. Arteriolar lesions were also present, along with focal glomerular scarring and fibrosis. Lesion extent correlated with calcineurin therapy duration. After reduction in calcineurin inhibitor dose by 50% and change from azathioprine to MMF, a 67% improvement in GFR (77\u00a0ml\/min per 1.73\u00a0m2) was noted 1\u00a0year after the change. The authors suggest that this improvement may be related to a decrease in preglomerular vasoconstriction [44]. In a study of pediatric patients treated with tacrolimus after transplant, 41% of patients had elevations in serum creatinine of 1\u20132\u00a0mg\/dl approximately 2\u20133\u00a0years after transplant [45].\nHeart\u2013lung and lung transplantation\nEpidemiology\nThe cumulative incidence of CKD, defined as a doubling of serum creatinine, after lung or heart\u2013lung transplantation varies from 34% at 1\u00a0year after transplant to 53% by 5\u00a0years after transplant [11]. ESRD occurs in 7.3\u201316% of patients 5\u00a0years after transplant [11\u201313]. The majority of patients, 51%, developed CKD stage 3 by 1\u00a0year posttransplant [12].\nRisk factors for CKD after heart\u2013lung and lung transplant\nRisk factors identified for developing CKD in a study of 219 patients undergoing a lung or a heart\u2013lung transplant include cumulative periods of a diastolic blood pressure >90\u00a0mmHg and serum creatinine value at 1\u00a0month posttransplant [11]. In this same study, the authors found that tacrolimus use in the first 6\u00a0months posttransplant decreased the risk of CKD compared with those who received cyclosporine alone. Other studies found GFR at 1 month to be a predictor of later CKD [12]. Development of postoperative hypertension is associated with an increased risk of ESRD after lung and heart\u2013lung transplants [13].\nHematopoietic stem cell transplant\nEpidemiology\nThe cumulative incidence of CKD varies from 13% to 60% in adult studies [15\u201317] to as high as 62% in children [14]. CKD usually becomes apparent 6\u201312\u00a0months after HSCT, although it has been described as early as 2\u00a0months and as late as 10\u00a0years posttransplant. Though this section discusses injury that occurs after HSCT, baseline or pretransplant renal function can impact the results [46]. Therefore, baseline assessments not only of serum creatinine but also urinalyses and more formal estimations of GFR are warranted. Accurate assessment of baseline renal function can help guide later medication dosing.\nThere are three distinct clinical manifestations of renal disease that can occur in the HSCT patient: thrombotic microangiopathy (TMA), typically hemolytic uremic syndrome (HUS), graft-versus-host-disease (GVHD)-related CKD, and nephrotic syndrome (NS). TMA syndromes represent a spectrum of clinical diseases characterized by systemic or intrarenal platelet aggregation, thrombocytopenia, and microvascular fragmentation of erythrocytes. Platelet aggregation can result in ischemia and organ injury. When renal injury is predominant, a diagnosis of HUS is usually rendered, whereas the presence of extensive extrarenal manifestations leads to a diagnosis of thrombotic thrombocytopenic purpura (TTP) (reviewed in [47]).\nHistopathology of TMA after HSCT\nMicroscopic examination of kidney biopsy specimens from patients with TMA-associated CKD demonstrates mesangiolysis and loss of endothelial cells with expansion of the subendothelium and occlusion of capillary loops. On electron micrographs, there is extensive widening of the space between the glomerular basement membrane and the subendothelium, with amorphous deposits that are not immune complexes.\nRisk factors for HUS after HSCT\nThough no clear relationships have been found to date for the development of TMA after HSCT, a number of risk factors have been examined. In earlier studies, where HUS was the primary diagnosis, risk factors identified were total body irradiation (TBI) [14, 15, 48, 49] and calcineurin inhibitor use [15, 27, 50\u201354]. However, in more recent studies of TMA, acute graft-versus-host disease (GVHD) grades 2\u20134, older age, and transplant from an unrelated donor are the primary risk factors identified [55, 56]. Other investigators identified sinusoidal obstruction syndrome, matched unrelated donors or haploidentical donors, and lymphoid malignancy as significant predictors of TMA after HSCT in addition to the above risk factors [17, 57\u201359]. However, in children who develop HUS after HSCT, the presumptive risk factor in these studies was TBI used as part of the conditioning regimen. For example, Tarbell et al. studied 44 children (aged 3\u201315\u00a0years) with acute lymphocytic leukemia (ALL) or neuroblastoma (NB) who underwent HSCT [14]. Twenty-nine of these patients were alive and in remission 3\u00a0months after HSCT and were evaluated in the study. Eleven patients developed increases in blood urea nitrogen (BUN) and creatinine, and ten were anemic and thrombocytopenic with evidence of hemolysis on peripheral blood smear; they also had elevated lactate dehydrogenase (LDH) levels. In every patient except one, the hemolytic process resolved, yet renal insufficiency persisted. The pathologic findings of mesangiolysis with intraglomerular capillary aneurysm formation in conjunction with laboratory abnormalities support the diagnosis of HUS in these children. In another small study, Antignac et al. described seven children referred to their nephrology clinic with CKD approximately 5\u201310\u00a0months after TBI followed by HSCT [48]. All seven children had leukemia, and all received cyclophosphamide alone or with cytosine arabinoside and vepeside, in addition to single-dose TBI as part of their conditioning regimen. Three patients developed CKD without hypertension, and four developed HUS with severe hypertension and microangiopathic hemolytic anemia. Of these, two of the four had normalization of their renal function. Follow-up biopsies, however, showed extensive scarring of the renal parenchyma but almost complete resolution of the mesangiolysis. The glomeruli were globally sclerotic, ischemic, or demonstrated mesangial hypercellularity. Thus, there was evidence of persistent and progressive renal damage in these patients despite normalization of serum creatinine and urinalysis. The occurrence of two different clinical presentations, CKD without hypertension and HUS with hypertension, but similar pathology in these children supports the notion that this is a spectrum of disease rather than distinct pathophysiologic processes.\nGVHD-related CKD\nGVHD-related CKD in this patient population is usually defined as an elevated serum creatinine or an abnormal GFR 6\u201312\u00a0months after transplant. The incidence of GVHD-related CKD in children after HSCT varies from 11% to 41% [60\u201363]. In one recent study, the incidence of CKD (GFR <70\u00a0ml\/min per 1.73\u00a0m2) changed over time, with 41% of children having CKD at 1\u00a0year, 31% at 3\u00a0years, and only 11% 7\u00a0years after transplant [63]. In approximately 19% of patients, hematuria and proteinuria persisted up to 10\u00a0years after HSCT. Berg and Bolme followed 44 children with acute lymphocytic leukemia (ALL), acute myeloblastic leukemia (AML), and severe aplastic anemia (SAA) and found a significant decrease in GFR 1\u20132\u00a0years after HSCT when compared with their baseline GFR (ALL and AML groups) or with a healthy control group despite serum creatinines that remained within normal limits. An initial decrease in GFR was followed by stabilization up to 5\u00a0years posttransplant [64]. This study supports the contention that serum creatinine is not an accurate measure of kidney function. Serum creatinine level and related estimating equations, routinely used clinical measures to estimate kidney function, are dependent on muscle mass and are influenced by age, race, gender, and weight [65, 66]. Patients undergoing HSCT may have large fluctuations in their nutritional status, muscle mass, and weight that will influence GFR based on estimation equations or serum creatinine levels. Proximal tubular dysfunction has also been described in 14\u201345% of pediatric patients 1\u20132\u00a0years after HSCT, with initial injury to the proximal tubules being nonspecific as reflected by elevated urinary excretion of alpha-1 microglobulin and beta-N-acetylglucosaminidase (\u03b2-NAG) followed by more specific damage manifested by decreases in phosphate reabsorption [67].\nRisk factors for GVHD-related CKD\nThe risk factors for GVHD-related CKD in children are similar to those identified in adult studies. Kist-van Holthe et al. also retrospectively identified risk factors for developing both acute and chronic renal insufficiency in a cohort of 142 children undergoing transplant over a period of 5\u00a0years in the Netherlands [68]. All children received allogeneic transplants. Ninety-one children received radiation, and 82 of these 91 received TBI. Twenty-five children (18%) had CKD (defined as a GFR <85\u00a0ml\/min per 1.73\u00a0m2). These authors found no correlation between radiation dose used and renal insufficiency at 1\u00a0year. In a later study from the same group, only acute renal insufficiency predicted the later development of CKD in patients after HSCT [62]. These studies contradict others in the literature that found TBI to be associated with renal injury [60, 61, 69]. However, the doses used here (5\u20138 Gy in a single fraction) were much lower than described elsewhere. In a study of 92 pediatric HSCT patients by van Why et al., late renal insufficiency developed in 18 of 64 (28%) patients; in half of these patients, the renal disease persisted for 3\u00a0months to 3\u00a0years [60]. Amphotericin B use, cyclosporine, and TBI were associated with the later development of CKD. In a large retrospective review of 1,635 children and adults, risk factors for developing CKD after HSCT included acute renal failure and acute and chronic GVHD [17]. In this study, TBI was not associated with development of CKD.\nNephrotic syndrome (NS) after HSCT\nChronic GVHD may manifest itself in the kidney as NS with or without renal insufficiency (reviewed in [70]). Patients usually present with proteinuria, edema, and hypoalbuminemia. The majority of these case reports demonstrate membranous nephropathy (MN) with subepithelial deposits on biopsy; it is postulated that these deposits are antigen\/antibody complexes representing GVHD in the kidney. However, cases of minimal-change disease (MCD), which is thought to be a T-cell-mediated process, have also been described [70]. Comparisons between case reports of MN and MCD after HSCT found that MN occurs in 61% of cases compared with 22% of cases having MCD [71]. The majority of reported patients with MN were slightly older males with a history of acute and chronic GVHD. Both MCD and MN occur later after transplant, at 8 and 14\u00a0months, respectively, and tend to occur within 1\u20135\u00a0months of GVHD development and\/or the tapering of immunosuppression for their chronic GVHD. MN is more difficult to treat, with only 27% of patients reportedly achieving remission compared with 90% of patients with MCD [71]. Others have reported cases of diffuse proliferative glomerulonephritis, anti-nuclear-cytoplasmic-antibody (ANCA)-related glomerulonephritis, focal segmental glomerulosclerosis, and IgA nephropathy [72\u201376] occurring after HSCT. The development of each of these diseases seems to be associated with chronic GVHD and\/or immunosuppression tapering. Treatment with high-dose prednisone and\/or reinstitution of calcineurin inhibitors usually results in resolution of NS. Some physicians have used rituximab successfully in patients with NS after HSCT, typically in cases of MN [77].\nManagement of CKD after liver, cardiac, lung, and HSCT\nPatients who develop CKD after liver, cardiac, lung, and hematopoietic stem cell transplants are at increased risk of mortality [18, 26, 78]. Transplant physicians and nephrologists should work together starting from the time prior to transplant to monitor these patients closely. More accurate measures of kidney function are needed, and baseline and follow-up iohexol or iothalamate studies to measure GFR may be indicated to identify patients with underlying CKD prior to transplant and to allow early identification and intervention in patients with mild changes in GFR posttransplant. New markers to estimate GFR, such as cystatin C, may be more informative in certain patient populations than is serum creatinine. Cystatin C is a cysteine protease inhibitor expressed by all nucleated cells and is freely filtered by the glomerulus. Serum cystatin C correlates well with measured GFR and more accurately measures kidney function than does serum creatinine in the elderly, cancer patients, diabetics, and renal transplant recipients [79\u201383]. Normal ranges for cystatin C have been validated in children. The normal reference range for children older than 1\u00a0year is 0.7\u20131.38\u00a0mg\/dl [84]. An equation to estimate GFR based on cystatin C levels in children has also been created: log (GFR) = 1.962 + [1.123*log(1\/cystatin C)] [85]. Urinalyses including a microalbumin to creatinine ratio should also be part of the pretransplant workup, and urinalyses should be monitored closely following transplant.\nAnimal models of HSCT, and specifically of radiation-induced HUS, offer potential interventions for patients with HUS after HSCT. Angiotensin-converting enzyme inhibitors (ACEI) have been used in rodent models of HSCT-related renal injury. The use of captopril or enalapril at the time of TBI in these animals resulted in less azotemia, lower blood pressures, decreased proteinuria, and long-term preservation of renal function [86]. ACEI and angiotensin receptor blockers (ARBs) also help reduce inflammation and inflammatory markers and reduce circulating levels of TGF-\u03b21 in patients after transplant [87\u201390]. These agents have also been shown to slow CKD progression and decrease proteinuria in patients with renal disease from various causes [91, 92].\nHypertension management early after transplant is important to prevent CKD development and progression to ESRD in certain transplant populations. ARBs and ACEIs have been shown to be effective and safe in managing hypertension in cardiac transplant patients [93], and ACEIs have been shown to stabilize renal function over 2\u00a0years of follow-up [94]. These drugs should be considered as first-line agents to manage hypertension in patients after transplant. In addition, hyperlipidemia management may be important to prevent CKD after transplant. In a study of adult patients undergoing a cardiac transplant, statin use was associated with a decreased risk of development of CKD after transplant [78]. The difficult decision is when to intervene (Fig.\u00a01). Should patients be started on these medications prior to transplant, at the first signs of hypertension and microalbuminuria, or at some set time point after transplant to help protect their kidneys? These decisions need to be made on an individual basis and will vary based on the type of transplant. Frequent discussions between the transplant physician and nephrologist are required to optimize the management of patients with CKD after transplant.\nFig.\u00a01Timing of intervention. Bold numbers are estimated glomerular filtration rate based on the Kidney Disease Outcomes Quality Initiative (KDOQI) guidelines. Adapted with permission from [102]\nIn addition to the above medications, immunosuppression adjustment is important in managing the CKD that develops after transplant, further supporting the need for close collaboration between transplant physicians and nephrologists. In two reported series of lung transplant recipients, sirolimus was used to decrease and eventually stop calcineurin inhibitors in patients with CKD after transplant. Patients had a sustained improvement in kidney function as reflected by a decrease in serum creatinine for 1\u201312\u00a0months [95, 96]. However, prior to changes in immunosuppression, a kidney biopsy may be indicated to define the etiology of the kidney injury and better tailor therapy to prevent or slow disease progression.\nManagement of patients with ESRD after transplant include the use of peritoneal and\/or hemodialysis. Caring for these patients also involves management of complications associated with ESRD, which include anemia, bone disease, hypertension, and metabolic abnormalities. There have been case series of patients undergoing renal transplantation successfully after cardiac, liver or HSCT; renal transplantation is a viable option for patients with ESRD after transplant and can improve outcomes [19, 97\u2013101].\nQuestions\n(Answers appear following the reference list)\nWhich of the following are risk factors for developing CKD after cardiac, liver, and HSCT?\nCalcineurin inhibitorsTotal body irradiationAcute renal failureHypertensionAll of the following are manifestations of CKD seen in patients after HSCT except:\nNephrotic syndromeAcute glomerulonephritisThrombotic microangiopathyGVHD-related CKDWhat pretransplant factors increase the risk of CKD in pediatric cardiac and liver transplant patients?\nPreexisting renal diseaseRacePretransplant diabetesAll of the aboveManagement of pediatric patients who develop CKD after transplant should include all of the following except:\nReducing exposure to calcineurin inhibitorsACEI and\/or ARBCalcium-channel blockersStatin useWhat is the most common cause for developing TMA after transplant in patients receiving a heart, liver, or stem cell transplant?\nGraft-versus-host diseaseTotal body irradiationCalcineurin inhibitor useDiabetes\nAnswers:\na and c\nb\nd\nc\nc","keyphrases":["chronic kidney disease","hematopoietic stem cell transplant","calcineurin inhibitors","risk factors","epidemiology","liver transplant","cardiac transplant","lung transplant"],"prmu":["P","P","P","P","P","P","P","P"]}
{"id":"World_J_Urol-3-1-1913171","title":"The influence of thermo-chemotherapy on bladder tumours: an immunohistochemical analysis\n","text":"To study the influence of microwave induced thermo-chemotherapy on high-grade urothelial cell carcinomas. Five groups of each three patients were formed of whom initial biopsies and cystectomy samples were collected. Patients were treated 2 days prior to cystectomy with mitomycin-C (group 1), hyperthermia (group 2) or thermo-chemotherapy (group 3). Group 4 patients had been treated with a cycle of six thermo-chemotherapy treatments prior to cystectomy and group 5 patients served as control (no treatment). Tumour samples were stained with Haematoxylin and Eosin, monoclonal antibody Ki-67 and the monoclonal antibody p53. In six out of the nine patients treated with hyperthermia a decrease in proliferation activity in the tumour was found. Seven out of nine patients treated with hyperthermia showed a decrease in p53 activity. A decrease in proliferation activity and p53 activity illustrate the potential role of thermo-chemotherapy as a promising intravesical treatment.\nIntroduction\nBladder cancer is the seventh most common cancer worldwide, accounting for 3.2% of all cancers. In 2000 an estimated 260,000 new cases in men and 76,000 in women were found worldwide [5]. The highest incidence rates of bladder cancer in both sexes are observed in Europe, North America and Australia [12]. Bladder cancer incidence is still rising moderately in most developed countries. Of all these malignant bladder tumours, more than 90% are transitional cell carcinomas of which two third is superficial and one third is muscle invasive. The management of these two types of transitional cell carcinoma differs enormously. The management of superficial bladder cancer consists of transurethral resection in the first place, whereas the primary treatment for muscle invasive bladder cancer consists of cystectomy, a complete different approach.\nAfter transurethral resection of a superficial bladder tumour 30\u201385% of patients develop recurrences, despite the most accurate resection technique. This high risk of recurrences makes bladder cancer one of the most prevalent human tumours. To decrease the number of recurrences after transurethral resection, patients are treated subsequently with chemotherapeutic or immunotherapeutic agents. Immunotherapy, usually BCG treatment, is more effective than any intravesical chemotherapeutic agent, but has more serious and more frequent side effects. So, there is a need for more effective treatment options or treatment options with less frequent and less severe side effects. A new treatment option in patients with intermediate to high risk tumours is the combination of intravesical hyperthermia and intravesical chemotherapy [2, 6, 15].\nThe endocavitary location and ease of accessibility by the urethra makes thermo-chemotherapy a good therapeutic option for superficial bladder cancer. Local hyperthermia at temperatures of 40\u201344\u00b0C in combination with selected cytostatic agents in several tumours, including transitional cell carcinoma, results in a synergistic anti-tumour effect [10].\nIn this descriptive study the influence of different treatment modalities on bladder tumours (thermo-chemotherapy, chemotherapy and thermotherapy) was investigated by several immunohistochemical stainings.\nMethods\nSubjects\nThe study included 15 patients with high-grade transitional cell carcinoma of the bladder. All patients signed a patient informed consent and all these patients were facing cystectomy. The 15 patients were divided in five groups with each three patients. Group 1 patients were treated 2\u00a0days before cystectomy with an intravesical mitomycin-C (MMC) instillation. After the bladder was emptied, 20\u00a0mg MMC (Kyowa Hakko Kogyo Co., Tokyo, Japan) in 50\u00a0ml saline was instilled. In order to stabilize the MMC concentration in the bladder throughout the entire session, the bladder was emptied after 30\u00a0min and with urine diluted solution was replaced by a new solution containing 20\u00a0mg MMC. Group 2 patients were treated 2\u00a0days before cystectomy with sterile water and local hyperthermia. To standardize treatments the instillation was replaced after 30\u00a0min. The local microwave induced hyperthermia was delivered by the SB-TS 101 system as described by Colombo et\u00a0al. [3]. This system consists of a 915\u00a0MHz intravesical microwave applicator that delivers hyperthermia of the bladder walls via direct irradiation. The applicator is part of the specially designed 20F transurethral catheter. The catheter also contains five thermocouples. Two thermocouples measure the temperature in the prostatic urethral tract; the other three are spread out and pushed tangentially against the posterior and lateral walls of the bladder. To avoid urethral overheating, the solution is continuously pumped out of the bladder and re-instilled after being cooled. Hyperthermia was delivered within a temperature range of 41\u201342\u00b0C.\nGroup 3 patients were treated 2\u00a0days before cystectomy with two times 30\u00a0min intravesical MMC (20\u00a0mg in 50\u00a0ml sterile water) combined with local microwave induced hyperthermia delivered by the SB-TS 101 system. Group 4 patients were treated with a cycle of six thermo-chemotherapy treatments during the last 3\u00a0months. Finally, group 5 patients served as control group. These patients did not receive any intravesical instillations within 3\u00a0months prior to cystectomy.\nImmunohistochemistry\nAll tumour tissue samples were fixed in a 10% buffered formaldehyde solution. The specimens were embedded in paraffin blocks and sections of 4\u00a0\u03bcm were cut. All specimens were deparaffinized and stained with Haematoxylin and Eosin. A microscopic examination of the samples was performed and the extend of inflammation (1+ to 3+) haemorrhage (1+ to 3+) were semi quantitatively scored.\nKi-67, a nuclear protein present during phases G1, S, G2 and M of cycling cells, is accepted as a good indicator of cell proliferation [11]. Sections were deparaffinized and immersed in a 10\u00a0mM sodium citrate buffer (pH 6.0) in a microwave oven for two times 5\u00a0min to enhance antigen retrieval. After washing, the slides were incubated with 0.3% H2O2 in methanol to quench endogenous peroxidase activity. After incubation at room temperature for 2\u00a0h with the anti-Ki67 monoclonal antibody clone MIB-1 (BioGenex) diluted 1:30, a biotinylated antibody that recognises murine IgG (BioGenex) was applied for 20\u00a0min, followed by incubation in streptavidin-peroxidase complex (BioGenex) for 20\u00a0min. The peroxidase reaction was developed using 0.5\u00a0mg\/ml diaminobenzidine tetrahydrochloride (Sigma) in 0.01% H2O2. Haematoxylin was used as a light counter stain. The slides were dehydrated and mounted with a xylene-soluble mounting medium.\nThe mutated p53 tumour suppressor gene product can be detected immunohistochemically and is associated with a lower survival in patients with bladder cancer [7]. Sections were deparaffinized and immersed in a 10\u00a0mM sodium citrate buffer (pH 6.0) in a microwave oven for two times 5\u00a0min to enhance antigen retrieval. After cooling down, 0.6% H2O2 in a 40% methanol solution was used for 30\u00a0min to block endogenous peroxidase activity. Slides were pre-treated with 10% normal swine serum for 10\u00a0min to block non-specific staining followed by adding primary antibody DO-7 for 1\u00a0h at 20\u00b0C. After extensive rinsing, sections were incubated for 30\u00a0min with biotinylated swine anti mouse antibody (1:200 dilution) and then for 30\u00a0min with avidin-biotin complexes (1:50 dilution) at 20\u00b0C. Diaminobenzidine (DAB) staining were finally followed by haematoxylin nuclear counter staining. The slides were dehydrated and mounted with a xylene-soluble mounting medium.\nImmunoreactivity scoring\nThe screening of tumour tissue samples was performed by two independent investigators (CH and AH). The sections were screened for positive cells, defined as cells with nuclear staining. The amount of Ki67 or p53 staining is scored in percentages. The threshold for p53 \u201cpositivity\u201d is \u226520% positive staining. The areas with maximal immunohistochemical staining were used for scoring. In total, 300\u2013500 tumour cells were scored. In the visual estimation only definitely brown nuclei were recorded as positive. The results were expressed as percentage of immunoreactive tumour cell nuclei.\nResults\nFifteen patients (11 males, 4 females) signed a patient informed consent and participated in this study. Ages ranged from 42 to 75\u00a0years (mean 63.2\u00a0years). All patients underwent cystectomy for transitional cell carcinoma of the bladder. Eight patients had a de novo invasive bladder tumour, whereas seven patients had a history of high-risk superficial disease. Patient details are summarized in Table\u00a01.\nTable\u00a01Patient details containing number of previous occurrences, interval period between initial diagnosis and cystectomy in months, pathology data of biopsy and cystectomy (grading according to WHO 2002)GroupsPatient no\/age\/sexN previous occurrencesInterval biopsy\u2013cystectomy (in months)Histopathology initial biopsyHistopathology cystectomy1 (MMC)1\/75\/M02,0\u2265pT2aGIIIpT3aGIIIN22\/46\/F04,4pT2GIIpT3bGIIIN13\/67\/F01,2\u2265pT2GIII\u2265pT2GIII 2 (HT)4\/64\/F01,9pT1GIIIpT2aGIII5\/71\/M338,7pT1GIII\u00a0+\u00a0CISpT1GIII\u00a0+\u00a0CIS6\/42\/M10121,9pTaGIIpTaGII3 (MMC\u00a0+\u00a0HT)7\/55\/M02,6pT1GIII\u2265pT2GIII8\/67\/M01,9pT2GIIIpT3aGIIIN19\/48\/F13,3pT1GIIpT2bGIIIN14 (History of MMC\u00a0+\u00a0HT)10\/54\/M18107,5pTaGIIpTaGII11\/73\/M222,3CISpT4aGIIIN212\/75\/M1057,7pTaGIIpTaGII5 (Control)13\/71\/M9115,5CISpT2GIII14\/71\/M03,1pT2GIIIpT2GIII15\/71\/M03,3pT2GIIIpT2GIIIN1HT microwave induced hyperthermia\nThe initial biopsies and tumour tissues obtained with cystectomy were used for histopathological diagnosis and immunohistochemical analysis of Ki-67 and p53. The results from the different treatment groups are summarized in Table\u00a02. In one patient of the MMC group no residual tumour could be retrieved in the cystectomy specimen. The proliferation activity and p53 activity could not be scored in that patient.\nTable\u00a02The p53 and Ki67 immunoreactivity scorings in percentages of all patients divided in five different treatment groupsGroupsMaterialInflammationProliferation (%)P53 (%)Haemorrhage1 (MMC)\u00a0aBiopsy+++3020+Cystectomy+++7520++\u00a0bBiopsy++2075\u2212Cystectomy+++1575+\u00a0cBiopsy+++4075+++CystectomyNANANANA2 (HT)\u00a0aBiopsy+6080\u2212Cystectomy+++2040++\u00a0bBiopsy+3090\u2212Cystectomy++2040\u2212\u00a0cBiopsy+10-\u2212Cystectomy++10-+3 (MMC\u00a0+\u00a0HT)\u00a0aBiopsy+++40>75\u2212Cystectomy+++2575\u2212\u00a0bBiopsy+++>75>75+Cystectomy+++6015\u2212\u00a0cBiopsy+++30>75+Cystectomy+++1575++4 (History of MMC\u00a0+\u00a0HT)\u00a0aBiopsy+1075\u2212Cystectomy+20\u2212\u2212 \u00a0bBiopsy+++3025\u2212Cystectomy+++3025\u2212\u00a0cBiopsy++5020\u2212Cystectomy++2010+5 (Control)\u00a0aBiopsy+15>75\u2212Cystectomy+++40>75\u2212\u00a0bBiopsy++4020\u2212Cystectomy++4020\u2212\u00a0cBiopsy++3575+Cystectomy+++3575\u2212The extend of inflammation and haemorrhage is semi quantitatively scored (1+ to 3+)NA not applicable due to absence of residual tumour\nThe intensity of inflammation increased in three out of nine patients treated with hyperthermia. In three out of five patients from the MMC group and control group an increase was seen. The intensity of haemorrhage increased in four out of nine patients treated with hyperthermia, one patient showed a decrease and four patients did not show any difference. In the MMC group and in the control group two patients showed an increase, one patient a decrease and two patients did not show any difference.\nIn six out of the nine patients treated with hyperthermia a decrease in proliferation activity in the tumour tissue sample was found (Fig.\u00a01a). In the MMC group one patient showed a decrease and one showed an increase in proliferation activity. In the control group two out of three patients showed no changes, whereas in one patient an increase in proliferation activity was seen.\nFig.\u00a01The p53 and Ki67 positive staining in patient no. 4 (hyperthermia group). a1 shows 60% Ki67 positivity before treatment; a2 shows 20% Ki67 positivity after treatment; b1 shows 80% p53 positivity before treatment and b2 shows 40% p53 positivity after treatment\nAs regards p53, seven out of nine patients treated with hyperthermia showed a decrease in p53 activity (Fig.\u00a01b). In one patient no change was found. The control group and the group treated with MMC did not show any differences concerning p53.\nDiscussion\nThermo-chemotherapy has shown to be a promising method for treating several kinds of malignant tumours including superficial bladder cancer [15]. While hyperthermia is important in cancer therapy, it can also damage normal tissues adjacent to the tumour. Fajardo [4] reviewed the effects of hyperthermia from several studies on various tissues of mammals and humans. Hyperthermia with a maximum applied temperature of 44.5\u00b0C showed no gross or microscopic alterations of the bladder of dogs and rabbits. Subsequently, Rath-Wolfson et\u00a0al. [13] studied the effect of hyperthermia, with a maximum applied temperature of 46\u00b0C, and simultaneous MMC treatment in sheep. In this study thermo-chemotherapy showed no significant macroscopic or microscopic differences in the bladder wall as compared to a control group with untreated sheep.\nIn the current study the effect of the combination thermo-chemotherapy is compared with solely hyperthermia, solely chemotherapy and no treatment (control group) in human patients with high-grade urothelial cell carcinoma of the bladder. The system SB-TS 101 used to deliver local microwave induced hyperthermia makes it possible to dose the hyperthermia very precisely [1]. In this study hyperthermia was delivered within a temperature range of 41\u201342\u00b0C, the clinical situation. This makes comparison between the different groups more accurate.\nThe sample size of the group studied is small due to the fact that recruitment of patients was difficult. Patients had to agree on an extra treatment session only two days before cystectomy, which did not give them any benefit at all.\nThe degree of inflammation increased in the group treated with solely hyperthermia (N\u00a0=\u00a03), solely MMC (N\u00a0=\u00a01) and the control group (N\u00a0=\u00a02). In the group treated with thermo-chemotherapy 2\u00a0days before surgery the degree of inflammation was initially already maximal. In the group treated with a cycle of six thermo-chemotherapy treatments during the last 3\u00a0months the degree of inflammation apparently had returned to baseline again.\nAt least one patient per group (except the control group) showed an increase in the degree of haemorrhage. Patients from group 1 (solely MMC) showed the highest degree of haemorrhage.\nGroup 3 patients (treated with thermo-chemotherapy prior to cystectomy) show in one patient an increase, in one patient a decrease and in one patient a stable extend of haemorrhage. This is in line with the results from Rath-Wolfson et\u00a0al. [13]. Nevertheless, since solely MMC and solely hyperthermia gives an increase in haemorrhage, it would have been logical to find a higher extend of haemorrhage after the combination of both treatments. Possibly, this is not found due to the small sample size.\nThe inhibition of proliferation activity is one of the most important goals in cancer treatment. All groups treated with hyperthermia showed to a different extend a decrease in proliferation activity. Most interesting are the three patients treated with thermo-chemotherapy preceding transurethral resection. All three patients showed a decrease in proliferation activity. The control group on the other hand shows in two patients no difference and in one patient an increase in proliferation activity. In earlier in vitro studies using hyperthermia and bladder cancer cell lines, this decrease in cell proliferation due to hyperthermia combined with chemotherapeutic agents was already shown [14, 16]. However, in these in vitro studies solely hyperthermia did not cause a significant decrease in cell proliferation. Nevertheless, there is a difference between in vitro and in vivo studies. This in vivo study is the first one showing a decreased proliferation activity in humans treated with microwave-induced hyperthermia with or without MMC. The effect of hyperthermia was seen in six out of nine patients treated with hyperthermia preceding cystectomy.\nFurthermore, the group treated with solely MMC did not show a significant decrease in proliferation activity. This is probably due to the limited penetration properties of this intravesical used drug, especially after one single treatment.\np53 is known to be responsible for repair or apoptosis in response to DNA damage. The p53 activity, in other words the expression of mutant p53, decreased exclusively in patients treated with hyperthermia with or without MMC.\nPrevious experiments showed that the p53 pathway is heat sensitive and that the p53 protein is inactivated at temperatures above 41\u00b0C [8, 9]. When cells were heated to 42.5\u00b0C and returned to normal temperatures, a strong p53 response with an increase in protein levels was observed. In our study we found the opposite, but the time interval between treatment and cystectomy was with 2\u00a0days (group 3) and several months (group 4) significantly longer than the time interval used by Guan et\u00a0al. [8]. Furthermore the results of this study are based on a small number of patients, which makes statistic analysis unreliable. On the other hand, the current results suggest that thermo-chemotherapy could be an effective treatment in patients with a p53 tumour suppressor gene mutated tumour. In all, the results show several trends and encourage carrying out larger experimental studies.\nConclusion\nThe degree of inflammation and haemorrhage in bladder tumours did not increase in patients treated with hyperthermia. This, in combination with a decrease in proliferation activity and a decrease in p53 activity, implies that thermo-chemotherapy is a safe and promising treatment.","keyphrases":["chemotherapy","hyperthermia","ki-67","p53","superficial bladder cancer"],"prmu":["P","P","P","P","P"]}
{"id":"J_Gastrointest_Surg-3-1-1852393","title":"CD24 Expression is an Independent Prognostic Marker in Cholangiocarcinoma\n","text":"CD24 has been described as an adverse prognostic marker in several malignancies. This study evaluates CD24 expression in cholangiocarcinoma and correlates the findings with clinicopathologic data and patient survival. Between 1996 and 2002, 22 consecutive patients with cholangiocarcinoma were treated at our institution. Demographic data, SEER stage, pathologic data, treatment, expression of CD24, mitogen-activated protein kinase (MAPK), phosphorylated MAPK, and survival were analyzed. The majority of the tumors demonstrated CD24 (81.8%) and p-MAPK (87%) expression. A negative association was noted between the expression of CD24 and p-MAPK. Median survival for patients with low expression of CD24 was 36 months and high expression was 8 months. Median survival for patients who received chemotherapy with low CD24 expression was 163 months, and for seven patients with high CD24 expression, it was 17 months (p = 0.04). With the addition of radiation therapy, median survival for patients with low expression of CD24 was 52 months and high expression was 17 months (p = 0.08). On multivariate analysis, the use of chemotherapy (p = 0.0014, hazard ratio 0.069) and the CD24 overexpression (p = 0.02, hazard ratio 7.528) were predictive of survival. CD24 is commonly expressed in cholangiocarcinoma, and overexpression is predictive of poor survival and possibly of lack of response to chemotherapy and radiation therapy. These findings may improve selection of patients for the appropriate treatment modality and the development of CD24-targeted therapy.\nIntroduction\nMalignancies of the biliary tract are uncommon in the Western world. Two-thirds arise in the gallbladder and the remainder in the biliary tree or periampullary region. Cholangiocarcinoma or bile duct cancer is a rare but lethal malignancy with an incidence of 1\u20132 cases per 100,000 patients in the United States.1 Clinicopathologic factors predictive of survival include curative resection, tumor stage and grade, serum bilirubin level <10\u00a0mg\/dl, low CA19-9 level, hepatitis viral infection,2\u20134 lymphovascular or portal vein invasion,5 intrahepatic satellite lesions, inraductal papillary compvonent, tumor angiogenesis,6 and DNA ploidy.7 Reports of molecular markers predictive of survival in cholangiocarcinoma include cluster of differentiation CD24,8 MMP-2, TIMP-2,9 cholinesterase level,10 MUC-4,11 cyclin D1,12 VEGF-C,13 p27,14 p53, and p73.15\nRecently, CD24 has been described in a wide variety of malignancies and shown to be a prognostic marker in several solid tumors including colorectal, stomach, lung, prostate, ovarian, and breast.16\u201321 CD24 is a small, heavily glycosylated, mucin-like, cell-surface protein expressed in developing cells including pre-B cells, keratinocytes, and renal tubular epithelium.22\u201324 It functions as an alternative ligand of P-selectin, an adhesion receptor expressed on activated endothelial cells and platelets which can enhance the metastatic potential of CD24-expressing tumor cells.25\u201328 CD24 has apoptotic activity, and its cross-linking induces the sustained activation of p38 MAPK (mitogen-activated protein kinases)\u2014the magnitude of which may determine the survival or death of pre-B cells.29 An improved understanding of the molecular pathways involved in the pathogenesis and progression of cholangiocarcinoma will contribute to the development of targeted therapy.\nThis study correlates CD24 and MAPK expression with patient survival in cholangiocarcinoma with the objective of identifying a subset of patients who may benefit from targeted molecular therapy.\nPatients and Methods\nClinical Data\nAfter obtaining approval of the Institutional Review Board, a review of the tumor registry at Roswell Park Cancer Institute identified 31 consecutive patients with histologically proven cholangiocarcinoma between 1996 and 2002. Twenty-two patients had adequate tissue for further histopathologic studies and constitute the basis of this study. Medical records of these patients were reviewed for demographic data including age; gender; surveillance, epidemiology, and end results (SEER) stage at presentation; treatment; and survival from the time of diagnosis.\nImmunohistochemical Staining\nFor most of the patients, diagnosis was established by examination of conventional hematoxylin and eosin (H&E)-stained slides and, in the remainder diagnosis, was confirmed with ancillary techniques including immunohistochemistry and special histochemistry with mucin and PAS stains. Uniform tissue fixation techniques were used for all patients. For each patient, a representative block containing adequate neoplastic and nonneoplastic tissue was selected. Five-micrometer tissue sections from these blocks were placed on charged slides and dried in a 60\u00b0C oven for 1\u00a0h. Upon return to room temperature, the slides were deparaffinized in three changes of xylene and rehydrated using graded alcohols. Endogenous peroxidase was quenched with 3% aqueous H2O2 for 15\u00a0min and washed with phosphate buffered saline with 0.05% Tween-20 (PBS\/T). CD24 primary antibody was obtained from BD Biosciences (clone ML5) and used with the recommended incubation time and antigen retrieval procedures. The primary antibody used for MAPK was obtained from Cell Signaling and for phosphorylated\/activated MAPK from Santa Cruz. After a PBS\/T wash, 0.03% casein (in PBS\/T) was used as a block for 30\u00a0min followed by the application of the primary antibody to the slides for an hour or overnight. Another PBS\/T wash was followed by exposure to the biotinylated secondary antibody for 30\u00a0min. A third PBS\/T wash was followed by exposure to the streptavidin\u2013peroxidase complex for 30\u00a0min. A PBS\/T wash was followed by the application of the chromogen DAB (DAKO, Carpinteria CA, USA) for 5\u00a0min. The slides were then counterstained with hematoxylin, rinsed with water, dehydrated, and cleared, and a coverslip was placed. The use of biomarkers, related controls, and interpretation of results using the Histoscore system for quantification of results have been described previously by our group.30 Histoscore was defined as the product of the percentage of positive cells and the intensity of stain. The grade of positive staining depended upon the intensity of staining (0: no staining, 1: weak, 2: moderate, and 3: strong staining) and the percentage of cells stained. The final score was calculated as a sum of each stain intensity multiplied by the percentage of stained cells in the area of interest. For example: if tumor showed 50% weak, 30% moderate, and 20% strong staining, the score assigned was .\nHistological Grading\nThe cholangiocarcinoma tissue specimens were also stained by routine H&E stains. The specimens were graded based on the degree of tumor differentiation using the World Health Organization (WHO) system.\nStatistical Analysis\nAssociation between biomarker expressions in tumor tissue was investigated using the Kendall\u2019s tau. Biomarker expressions were classified as high and low based on whether their scores were above or below the median value, and survival between low and high expressions was compared using the log-rank test. The Kaplan\u2013Meier method was also used to estimate the survival curves and median survival. The Cox\u2019s proportional hazards survival analysis was used in the multivariate analysis of survival data to explain the effect of biomarker expressions together with other diagnostic parameters. Patient demographics including age, tumor grade, SEER stage, and treatment received were considered as possible parameters for explanatory variables in the model. All statistical tests were two-sided with statistical significance level at 5%.\nResults\nPatient Characteristics\nOf the 22 patients included in the study, 7 were males and 15 females. The median age was 66.5\u00a0years (range: 35\u201377). SEER staging was local in 1 (4.6%), regional in 14 (63.6%), and distant in 7 (31.8%) patients. Differentiation of the tumor was classified as grade 1 in 3 (13.6%), grade 2 in 9 (40.9%), and grade 3 in 10 (45.5%) patients according to the WHO classification. Treatment for cholangiocarcinoma included surgery only (n\u2009=\u20098), surgery and chemotherapy (n\u2009=\u20095), surgery and radiation (n\u2009=\u20091), chemotherapy only (n\u2009=\u20092), and all three treatment modalities (n\u2009=\u20095), and one patient did not receive any treatment.\nImmunohistochemical Staining\nNormal bile duct staining was used to set the score intensity. Most of the bile ducts were negative. Occasionally, they demonstrated weak focal and incomplete staining as seen in Fig.\u00a01. The cholangiocarcinoma cells were scored as 1+ when they demonstrated weak expression of CD24, 2+ for moderate expression, and 3+ for strong expression of CD24. Figure\u00a01 depicts cholangiocarcinoma positive for CD24 expression adjacent to normal biliary epithelium. The majority of the tumors demonstrated CD24 (81.8%) and p-MAPK (87%) expression. Immunohistochemical staining for these proteins was higher in malignant tissue in comparison to normal biliary epithelium. The pattern of staining was usually a combination of cytoplasmic and apical, and few specimens demonstrated the apical pattern only.\nFigure\u00a01Moderately differentiated cholangiocarcinoma with overexpression of CD24. Normal bile duct (right upper corner) demonstrated weak apical staining, whereas neoplastic cells had strong apical and cytoplasmic staining (20\u00d7).\nRelationship Between Biomarkers\nA negative association was suggested between the expression of CD24 and phosphorylated\/activated p-MAPK (Kendall\u2019s \u03c4\u2009=\u2009\u22120.32408, p\u2009=\u20090.0501).\nSurvival\nMedian survival was 36\u00a0months for nine patients with low expression of CD24 and 8\u00a0months for 13 patients with high expression of CD24 as shown in Fig.\u00a02. The median survival for five patients who received chemotherapy with low CD24 expression was 163\u00a0months, and for seven patients with high CD24 expression, it was 17\u00a0months (Fig.\u00a03, p\u2009=\u20090.04). Median survival for four patients treated with radiation in the presence of low CD24 expression was 52\u00a0months, and it was 17\u00a0months for two patients with overexpression of CD24 (Fig.\u00a04, p\u2009=\u20090.08). Overexpression of CD24 continued to affect survival adversely despite the overall improvement noted with the addition of radiation therapy. Multivariate analysis using the Cox\u2019s proportional hazards survival analysis demonstrated that overexpression of CD24 (p\u2009=\u20090.02, hazard ratio 7.528) and use of chemotherapy (p\u2009=\u20090.0014, hazard ratio 0.069) were predictive of survival (Table\u00a01). There was no significant association noted between survival and patient\u2019s age, sex, SEER stage, grade of the tumor, surgery, radiation therapy, or expression of MAPK.\nFigure\u00a02Kaplan\u2013Meier survival curve for patients of cholangiocarcinoma with low and high levels of CD24 expression (n\u2009=\u200922). p\u2009=\u20090.02. Low CD24 (\u2013\u2013\u2013\u2013\u2013\u2013\u2013), high CD24 (- - - - - - - - - - - -).Figure\u00a03Kaplan\u2013Meier survival curve for patients who received chemotherapy with low and high levels of CD24 expression (n\u2009=\u200912). p\u2009=\u20090.04. Low CD24 (\u2013\u2013\u2013\u2013\u2013\u2013\u2013), high CD24 (\u2013 - \u2013 - \u2013 - \u2013).Figure\u00a04Kaplan\u2013Meier survival curve for patients who received radiation therapy with low and high levels of CD24 expression (n\u2009=\u20096). p\u2009=\u20090.08. Low CD24 (\u2013\u2013\u2013\u2013\u2013\u2013\u2013), high CD24 (\u2013 - \u2013 - \u2013 - \u2013).Table\u00a01Prognostic Variables for Survival in 22 Patients with CholangiocarcinomaVariablenMedian survival (months)Survival (p value)Multivariate analysis (p value)Age<681117 (8\u201336)0.60>681111 (5\u201352)GenderMale712 (8\u2013163)0.52Female1517 (6\u201352)Grade1, 21215 (6\u201352)0.9931011 (8\u2013*)SEER stage1, 21511 (6\u201352)0.483728 (6\u2013163)ChemotherapyNo106 (4\u201313)0.00050.0014Yes1252 (17\u2013163)RadiationNo168 (6\u201328)0.12Yes644 (17\u2013*)SurgeryNo38 (0\u201328)0.17Yes1917 (7\u201352)MAPKLow1017 (8\u201336)0.68High1128 (5\u2013163)P-MAPKLow1013 (8\u201352)0.34High1136 (4\u2013163)CD24<120936 (13\u2013163)0.100.02>120138 (6\u201328)*The estimate was not provided because the upper limit of the survival curve had not reached a 50% failure rate.CD denotes cluster of differentiation.p-MAPK denotes phosphorylated form of mitogen-activated protein kinase.\nDiscussion\nThe physiologic function of CD24 is incompletely understood but it has been shown to increase tumor proliferation, cell adhesion, motility, invasion, and apoptosis.22\u201324,31 Selectins are cell adhesion molecules involved in the rolling adhesion of leukocytes to endothelial cells and platelets under the shear forces of circulation, and P-selectin expressed by thrombin-activated platelets and endothelial cells is a major ligand for CD24 on carcinoma cells.26,27 This suggests that CD24-expressing tumor cells can disseminate more readily due to their capacity to form thrombi with activated platelets or to adhere to endothelial cells. Friederichs et al.28 have demonstrated that the carbohydrate sialylLex abundantly expressed on human cancers is essential for CD24-mediated rolling of tumor cells on P-selectin, and in its absence, human adenocarcinoma cells failed to arrest and colonize the lungs. CD24, a metastasis-associated protein, has been recently identified as a downstream target of Ral signaling.32 Ral GTPases are important mediators of transformation, tumorigenesis, and cancer progression. Microarray by immunohistochemistry of a human bladder cancer identified CD24 as a novel Ral-regulated target and a prognostic biological marker.\nIn this study, 81.8% of patients with cholangiocarcinoma expressed CD24. Median survival for patients with overexpression of CD24 was significantly shorter, and the addition of chemotherapy improved survival. A negative association was noted between the expression of CD24 and p-MAPK. The use of chemotherapy in patients with low expression of CD24 was associated with a median survival of 163\u00a0months compared to 17\u00a0months in patients with a high CD24 expression (p\u2009=\u20090.04). The use of radiation therapy in patients with low expression of CD24 was also associated with an improved survival than with overexpression of CD24 although the data did not attain statistical significance possibly due to the small number of patients in this series.\nIt has been reported by Taguchi et al.29 that the cross-linking of CD24 induces apoptosis in Burkitt\u2019s lymphoma enhanced by a B-cell antigen receptor (BCR)-mediated signal. They observed that simultaneous cross-linking of pre-BCR clearly inhibited CD24-mediated apoptosis in pre-B cells. CD24 cross-linking also induces the sustained activation of p38 MAPK, and whether pre-B cells survive or die may be determined by the magnitude of MAPK activation. Consistent with these observations, the present study suggests an inverse association between CD24 and p-MAPK, and eventual cellular proliferation or apoptosis might be a consequence of the dominant effect in a complex interplay of opposing influences.33\nOur data indicate that high expression of CD24 remains an adverse prognosticator despite the use of additional therapy. Chemotherapy and radiation were noted to provide maximal survival benefit to low expressors of CD24 although the data for the use of radiation was statistically insignificant probably due to the small number of patients in this study. Furthermore, correlation between CD24 expression and radiation sensitivity has been noted to vary with the cell type as in human small cell lung cancer, and radiation doses required to induce apoptosis of CD24-negative human ALL (acute lymphoblastic leukemia) cells were higher than those required for CD24-positive cells, suggesting that lack of CD24 surface antigen expression is associated with intrinsic radiation resistance.34,35\nHypoxia is a characteristic feature of tumor cells due to the sustained proliferation which progressively results in an acidic, nutrient-deprived, and hypoxic tumor microenvironment. Tumor oxygenation has been identified as an independent prognostic variable for locoregional control and overall survival following definitive irradiation for squamous cell carcinoma of the head, neck, and uterine cervix.36,37 Recent reports have indicated decreased efficacy of chemotherapy under hypoxic conditions in several tumor types including pancreatic cancer and testicular tumors.38,39 Because treatment failure was a consequence of hypoxia, the authors recommend novel treatment strategies aimed at improving tumor oxygenation or enhancing the treatment sensitivity of hypoxic tumor cells. Aimed at identifying potential oxygen-dependent markers in vascular endothelial cells for therapeutic intervention in tumor angiogenesis, Scheurer et al.40 performed a broad-range transcriptomic analysis of selected extracellular matrix protein gene expression levels in human umbilical cord vein endothelial cells exposed in vitro to hypoxia for different time periods. They noted several genes transcriptionally upregulated including CD24 at late times of exposure to hypoxia, indicating that it was a useful marker of hypoxic activation in vascular endothelial cells. In the present series, low expressors of CD24 demonstrated greater survival benefit from chemotherapy and radiation than the high expressors, suggesting that its expression may be a marker for tumor hypoxia and response to therapy. This finding that shows that patients with low expression of CD24 may benefit from chemotherapy or radiation is of importance because it has been previously reported that adjuvant or palliative radiation had no effect on survival in patients with cholangiocarcinoma.41 However, the small number of patients in the present series limits interpretation of data suggesting that CD24 overexpression may be predictive of lack of response to radiation or chemotherapy.\nCD24 has been shown to be a prognostic marker for shortened survival and disease progression in several malignancies including colorectal, stomach, lung, prostate, ovarian, and breast cancers.16\u201321 Weichert et al. report that in colorectal cancer, only the subset of patients with exceptionally strong cytoplasmic CD24 staining comprising 10% of their study group demonstrated a markedly shortened mean survival of 31.5\u00a0months compared to 67.5\u00a0months for the remaining patients.16 They also reported that cytoplasmic CD24 staining pattern is prognostically more significant than the membranous pattern\u2014the biological significance of which was unclear. Su et al. noted a 51% expression of CD24 by immunohistochemistry in intrahepatic cholangiocarcinoma as compared to the 81.8% in the present series. They reported CD24 expression and tumor stage as independently predictive of survival on multivariate analysis and suggested membrane-bound CD24 protein as a potential target for immunotherapy.8\nIn conclusion, overexpression of the molecular marker CD24 in cholangiocarcinoma is predictive of poor survival. CD24 overexpressors demonstrated a lack of response to chemotherapy and possibly radiation therapy although these observations were limited by the small sample size. Additional properties of tumor proliferation, invasion, metastasis, and apoptosis make CD24 a potent target for specifically directed molecular therapy and its overexpression a potential criterion in the selection of patients for the appropriate conventional treatment modality.","keyphrases":["cd24 expression","cholangiocarcinoma","survival"],"prmu":["P","P","P"]}
{"id":"Knee_Surg_Sports_Traumatol_Arthrosc-4-1-2358931","title":"A cadaveric analysis of contact stress restoration after osteochondral transplantation of a cylindrical cartilage defect\n","text":"Osteochondral transplantation is a successful treatment for full-thickness cartilage defects, which without treatment would lead to early osteoarthritis. Restoration of surface congruency and stability of the reconstruction may be jeopardized by early mobilization. To investigate the biomechanical effectiveness of osteochondral transplantation, we performed a standardized osteochondral transplantation in eight intact human cadaver knees, using three cylindrical plugs on a full-thickness cartilage defect, bottomed on one condyle, unbottomed on the contralateral condyle. Surface pressure measurements with Tekscan pressure transducers were performed after five conditions. In the presence of a defect the border contact pressure of the articular cartilage defect significantly increased to 192% as compared to the initially intact joint surface. This was partially restored with osteochondral transplantation (mosaicplasty), as the rim stress subsequently decreased to 135% of the preoperative value. Following weight bearing motion two out of eight unbottomed mosaicplasties showed subsidence of the plugs according to Tekscan measurements. This study demonstrates that a three-plug mosaicplasty is effective in restoring the increased border contact pressure of a cartilage defect, which may postpone the development of early osteoarthritis. Unbottomed mosaicplasties may be more susceptible for subsidence below flush level after (unintended) weight bearing motion.\nIntroduction\nFull-thickness cartilage defects may lead to early osteoarthritis [6,13,14]. Osteochondral transplantation is a successful treatment for these defects, but the outcome is dependent on, among other parameters, stability and restoration of surface congruency [9,15,21]. In our previous study about the effect of perfect depth alignment of a transferred plug [8], we evaluated the difference in stability between bottomed plugs (donor plug length and recipient defect depth accurately matched) and unbottomed plugs (donor plug is left shorter than the recipient defect depth). That study demonstrated that bottoming plugs resulted in a significantly more stable situation than unbottoming plugs. Unbottomed plugs leave a cavity at the bottom of the defect and therefore rely exclusively on frictional forces. When performing only a single-plug transfer the whole plug is, in most cases, nicely surrounded by (subchondral) recipient bone, and stability is fairly good, bottomed or not. However, when performing osteochondral transplantation where more than one plug is needed, stability is probably lower because of gaps between the round plugs and their surrounding bone. Kordas et al. [10] reported in agreement with this that the push-in force below flush level for (unbottomed) multiple grafts was significantly lower compared to a single graft. Theoretically, width differences of the cartilage layer between trochlea (donor area) and condyl (defect area) might have an influence on restoration of surface congruency.\nUsually a patient is restricted in weight bearing after osteochondral transplantation. Rehabilitation periods of two weeks of non-weight bearing and an additional two to three weeks of partial weight bearing are reported in the literature [6]. This period facilitates good ingrowth of (subchondral) bone [11,15] and thus ensures the preservation of surface congruency as it was intended directly post-operative. Histological research has proven that after transplantation bone resorption takes place on the recipient site and on the graft surface [5,18]. This might be one reason for a gradual loss of the initial press-fit stability postoperatively and a cause of subsiding below flush level of the graft during follow-up. Accordingly, Whiteside et al. [20] reported a reduction in short-term load bearing capacity of a single-plug transplant one week after transplantation. Press-fit mechanisms provide stability up to 4\u00a0weeks [6]; subsequently, this is taken over by the integration of the graft and the recipient bone [11].\nThe objective of the present study was to investigate the biomechanical effectiveness of osteochondral transplantation. More specifically we assessed whether the treatment would decrease peak stresses at the boundaries of the original articular cartilage defect. Subsequently, we analysed if articular stresses were dependent on plug placement (bottomed versus unbottomed) and how this was affected by loads that represented (for instance unintended) early weight bearing.\nMaterials and methods\nMaterials\nEight intact human cadaver knees obtained from the anatomical department, from individuals 70\u201380\u00a0years old of unknown gender, were used. Exclusion criteria were severe arthrosis of donor or graft area and malignant processes within the knee. Specimens were freshly frozen and thawed at room temperature overnight before preparation and testing. The K-scan 4000 (Tekscan Inc., Boston, MA) (Fig.\u00a01) was used for measuring surface congruency according to a previously discussed protocol [4]. Before testing, each new Tekscan sensors was pre-loaded and calibrated, as suggested by the Tekscan manual and by Brimacombe et al. [2]. For this loading, a circular plastic plunger, that almost covered the entire width of the sensor, was placed underneath a small loading platform where weights could be placed on top. Each sensor was calibrated separately and calibration files were stored. A new sensor was used for each knee. The sensor measurements were presented as a 22 by 26\u00a0pixel matrix with square pixels. For the osteochondral transplantations, a disposable 8\u00a0mm Osteochondral Autograft Transfer System (OATS) was used (Arthrex, Somas, Sint Anthonis, The Netherlands).\nFig.\u00a01Tekscan K-4000 measuring device with two thin (0.089\u00a0mm) and flexible pressure sensors, which were able to conform to the anatomy of the medial and lateral knee compartments. The sensors consist of printed circuits divided into grids of load-sensing regions. Each sensor measured 28\u00a0\u00d7\u00a033\u00a0mm, with a total of 572\u00a0pixels with a surface per pixels of 1.27\u00a0mm2\nPreparations\nSkin, muscles, excess soft tissues, patella and the anterior part of the knee capsule were removed from each knee, whereas collateral ligaments and intra-articular structures were left intact. In this way the tibio-femoral joint area was uncompromised and yet was accessible from the anterior side. Dorsally two small \u2018windows\u2019 were created in the capsule, giving access to the posterior part of both condyles. The femur as well as the tibia and fibula were sectioned at \u223c18\u00a0cm from the joint space. The knee was then placed in a knee-testing device, which was used in earlier experiments [1,19] (Fig.\u00a02). This device provides five degrees of freedom of motion for the tibial side, whereas the femoral side has one degree of freedom and can additionally be manually displaced for flexing or extending movements of the knee (Fig.\u00a02). Weights could be attached to the femur side such that the force may be directed through the longitudinal axis of the femur. To simulate partial weight bearing, we applied 350\u00a0N as a tibio-femoral compressive force. Previous research at our institution has shown that a force of 171\u00a0N (unbottomed) and 384\u00a0N (bottomed) was necessary to push a single-plug-mosaicplasty below flush-level [8].\nFig.\u00a02a Knee-testing device. b Circular plastic cap for positioning of Tekscan sensor. c Tekscan sensor in situ. d Special mould for OATS. e Three-plug mosaicplasty placed flush in full-thickness osteochondral defect\nOperation and testing\nFirst, donor sites were marked with a permanent marker on the trochleae or the posterior region of the condyles. Three plugs were harvested with a length of \u223c15\u00a0mm (to be unbottomed) and three plugs of \u223c18\u00a0mm length (to be bottomed). After harvesting all six donor plugs, Tekscan sensors were placed inside the tibio-femoral joint space. After positioning the knee in approximately 45\u00b0 flexion, the sensors were guided alongside the cruciate ligaments (one on each side), and placed between each femoral condyle and tibial plateau. Subsequently, a small circular plastic cap (diameter 10\u00a0mm, height 1\u00a0mm) was pinned on both condyles, at the location where the mosaicplasty was to be performed (Fig.\u00a02). The compression of both markers on the sensors was clearly visible on the computer screen and assisted in positioning the sensors centrally over the area where the mosaicplasty would be executed. To facilitate repositioning of the sensors between different interventions, the sensors were fixated to the joint by four small metal \u2018anchors\u2019 on all four corners of the sensor that could be used to re-attach the sensors to the joint (Fig.\u00a02). In order to confirm validity of this method a test was performed where the sensor was repeatedly fixated and completely removed from the knee and the plastic marker was visualized. Reproducibility of the re-attachement of the sensor was confirmed by five independent measurements of the position of a circular cap, each after removing a re-attachment of the Tekscan sensor. Four out of five measurements showed exactly the same position of the cap within the Tekscan matrix, which proves a high accuracy of re-attachment.\nTo investigate the consequences of a cartilage defect and subsequent mosaicplasty on the stress distribution under various conditions, Tekscan recordings were performed under five different conditions: (A) preoperatively, intact condition, (B) cartilage defect without any reconstruction, (C) following mosaicplasty, (D) after mosaicplasty and subsequent non-weight bearing motion of the knee and (E) after mosaicplasty with weight bearing motion (E). All surface measurements were performed with the knee in 0\u00b0 extension, thereby ensuring loading of the (restored) defective sites, and an axial load of 350\u00a0N.\nThe \u2018preoperative\u2019 measurement (A) was with intact condyles. After removing the sensors, both on the medial and lateral condyle a standardized subchondral defect, 8\u00a0mm deep was created by a circular drill of 16\u00a0mm diameter, the center being at exactly the same location as the center of the plastic marker that was measured previously.\nThe \u2018defect\u2019 measurement (B) of the condyle surfaces was performed after creating the osteochondral defect. With the use of a special mould (Fig.\u00a02) and the OATS, these defects were prepared to receive three osteochondral plugs. When the mosaicplasty was to be performed as bottomed, three defects were created, approximately 15\u00a0mm deep and the bottoms of the defects were tamped. Following measuring of the depths of these defects, three of the 18-mm donor plugs were matched for these depths by carefully removing some subchondral bone with surgical bone-nibbling pliers according to clinical practice. Plugs were orientated such that the most congruent surface would be achieved. These plugs were tampered in place, until flush level of the cartilage surface with the recipient site was achieved (Fig.\u00a02). At the opposite condyle, which was to be unbottomed, three defects of approximately 20\u00a0mm depth were created, and the donor plugs of 15\u00a0mm were used. The difference of 5\u00a0mm in length between the defects was essential for the unbottomed plugs to be sure to have a cavity at the bottom of the defect and therefore rely exclusively on frictional forces.\nThe \u2018mosaicplasty\u2019 surface measurement (C) was executed after performing both mosaicplasties. The \u2018non-weight bearing motion\u2019 measurement (D) was performed following flexing and extending the knee 20 times, while no weight was attached to the test device. The axial load of 350\u00a0N was re-applied before the measurement was performed. The final \u2018weight bearing motion\u2019 measurement (E), was performed once the knee had been flexed and extended 20 times with 350\u00a0N of axial force applied to the knee while performing the flexion-extension movements.\nEvaluation of variables\nEach of the eight knees provided two paired standardized mosaicplasties of three plugs, one bottomed and one unbottomed. Thus, there were 16 mosaicplasties: eight bottomed and eight unbottomed. Three general groups were created for statistical evaluation: the whole group of all mosaicplasties (bottomed and un-bottomed combined), a group with only bottomed and one with only unbottomed mosaicplasties. The five measurements performed were: (A) preoperative, (B) defect, (C) mosaicplasty, (D) non-weight bearing motion, (E) and weight bearing motion.\nRelative to the reconstructed surface, we defined two specific regions of interest: the mosaicplasty area itself and the border of the reconstructed surface. The first region obviously quantified the amount of stress transferred at the mosaic site, whereas the second region was selected to quantify the amount of stress transferred to the border of the defect. The mosaicplasty area (region 1) was a circle with a diameter of 16\u00a0mm, for which the best fitting circle representing the standardized defect was obtained on the Tekscan sensor matrix (Fig.\u00a03). This circle was obtained by calculating the lowest surface pressure of a 16-mm diameter circle in the non-reconstructed (defect) case. The border region (region 2) was selected around the first region and had a width of 3\u00a0pixels (3.81\u00a0mm). To ensure that the two regions were clearly separated a transition zone with a width of 1\u00a0pixel was selected and has not included in the measurements (Fig.\u00a03). The total of the pixel values in regions 1 and 2, respectively, were used for further comparative evaluations. To allow for a direct comparison of the pressure values, the pressures on the border and mosaic areas were normalized to the total force on the sensor.\nFig.\u00a03a Typical example of Tekscan report on load distribution for a cartilage defect without any reconstruction. b The mosaicplasty area (region 1) is a circle consisted of square pixels with a diameter of 12\u00a0pixels. The border area (region 2) is a ring with a diameter of 3\u00a0pixels around the defect circle. A transition zone of 1\u00a0pixel around the mosaicplasty area is found between regions 1 and 2\nStatistics\nContact pressures were calculated at the boundaries of the cartilage defect (region 2) as well as for the defect\/mosaicplasty (region 1) itself. This was done for five different conditions as described before (A\u2013E). These values were expressed for three groups, namely bottomed, unbottomed and combined.\nDifferences were examined within and among the groups. For statistical analysis, we used SPSS 12.0 for Windows. The linear mixed model was used to evaluate the influence of bottoming or unbottoming the plugs, with Bonferroni correction. P\u00a0<\u00a00.05 were considered to be statistically significant.\nResults\nThe averaged contact-pressures at the border contact surface are shown in Fig.\u00a04. Overall, there were no significant differences between bottomed and unbottomed. All the data showed the same pattern for the three groups: in presence of a defect (B) the border contact surface pressure was significantly higher compared to the intact (preoperative) situation (A), performing a mosaicplasty made the border pressure decline to approach preoperative values (C), and after motion with and without weight the border contact pressure remained stable (C\u2013E).\nFig.\u00a04Graph showing the border contact pressure for three groups; bottomed (blue line), unbottomed (green line) and the whole group (grey line). In presence of a defect (b) the border contact surface pressure was significantly higher compared to the intact (preoperative) situation (a), performing a mosaicplasty made the border pressure decline to approach preoperative values (c), and after motion with and without weight the border contact pressure remained stable (c\u2013e). Standardized deviation values are presented in Table\u00a01\nThe pressure pattern of the mosaic contact surface was inverse to the border contact surface pressure (Fig.\u00a05): the mosaicplasty area had almost no contact pressure after creating a defect (B), in the presence of the mosaicplasty the contact surface pressure was regained to some extent (C vs. A) and the contact pressure remained relatively constant after non-weight bearing and weight bearing motions (C\u2013E).\nFig.\u00a05Graph showing the mosaicplasty contact pressure for three groups; bottomed (blue line), unbottomed (green line) and the whole group (grey line). The mosaicplasty area had almost complete lack of contact pressure after creating a defect (b), in presence of the mosaicplasty the contact surface pressure was regained (c vs. a) and the contact pressure remained stable after non-weight bearing and weight bearing motion (c\u2013e). Standardized deviation values are presented in Table\u00a01\nTwo out of eight unbottomed versus zero bottomed mosaicplasties showed a decreased mosaicplasty pressure and increased rim stress after weight bearing motion on individual Tekscan measurements, indicating that the mosaicplasty subsided below flush level. Five out of eight bottomed plugs were placed on the medial condyle and no significant differences in pressure or rim stress was found.\nStatistics\nMean pressure values measured are given in Table\u00a01.\nTable\u00a01Results of the surface contact pressure measurements of five conditionsMeasurementWhole group (n\u00a0=\u00a016)P-valueBottomed (n\u00a0=\u00a08)Unbottomed (n\u00a0=\u00a08)BorderA. Preoperative38.7 (7.1)a40.2 (8.3)37.1 (6.0)B. Defect74.3 (5.5)(B vs. A\u2013C\u2013D\u2013E) 0.00075.5 (3.9)73.2 (6.8)C. Mosaicplasty52.1 (9.3)(A vs. C) 0.00154.6 (7.5)49.7 (10.7)D. Motion without weight53.2 (9.8)(A vs. D) 0.00153.9 (8.4)52.4 (11.6)E. Motion with weight53.8 (9.6)(A vs. E) 0.00055.1 (9.0)52.5 (10.6)MosaicA. Preoperative35.7 (7.8)33.6 (9.3)37.9 (5.6)B. Defect4.0 (4.0)(B vs. A\u2013C\u2013D\u2013E) 0.0003.7 (3.8)4.3 (4.4)C. Mosaicplasty23.8 (8.2)(A vs. C) 0.00221.4 (7.4)26.2 (8.9)D. Motion without weight22.8 (8.7)(A vs. D) 0.00122.1 (8.1)23.5 (9.7)E. Motion with weight22.0 (9.1)(A vs. E) 0.00121.0 (8.4)23.0 (10.1)a Values are given as mean (SD), only significant P-values are shown\nEffect of defect (A\u2013B): Pressure redistribution occurred in the presence of a defect from the mosaic contact surface to the cartilage surrounding it. The border contact pressure increased to 192% (P\u00a0=\u00a00.000) compared to the preoperative border contact surface pressure, while the pressure on the defect contact surface decreased to 11% (P\u00a0=\u00a00.000).\nEffect of mosaicplasty (B\u2013C): The mean stress elevation around the defect was partially restored in the presence of a mosaicplasty, as the border contact pressure decreased by 30% compared to the defect measurement. This resulted in a border contact pressure of 135% compared to intact cartilage. The mosaic contact pressure was restored from 11 to 67% (P\u00a0=\u00a00.000).\nEffect of flexion-extension motion (C\u2013D\u2013E): The border contact pressure did not return to preoperative values after performing a mosaicplasty, and did not change after flexion and extension motions. The border contact pressure after non-weight bearing motion\u2019 and \u2018weight bearing motion\u2019 remained equal to the mosaicplasty measurement, 138 and 139% (P\u00a0=\u00a00.001 and 0.000, respectively) compared to intact, preoperative cartilage. For these measurements the mosaic contact pressures remained lower than those of intact cartilage, with pressures 64 and 62%, respectively (respectively P\u00a0=\u00a00.001 and 0.001).\nDiscussion\nIn this biomechanical human cadaver study we clearly demonstrated that an osteochondral cartilage defect severely affects the contact pressure on the remaining intact joint surface. Obviously, there are some additional limitations to our study. The study had only a limited number of knees. During testing osteoarthritic changes in bone were found in some of these elderly knees. Clearly this is different to the bone quality of the typical patient who is relatively young and active. Another limitation was that during the preparations the knee was positioned in such a way that both condyles would be loaded with approximately the same force, which may be different from in vivo loads. This might have had an influence on the absence of medial\/lateral differences. Contrary to other experiments we kept all collateral ligaments and intra-articular structures intact, which resembles the anatomical situation more closely and thus gives a greater translational value of pressure transfer. Clearly, no biological effects were taken into account such as the resorption of the plugs (thereby reducing the stability) or bony ingrowth (thereby enhancing stability). We also measured the contact patterns under static conditions with the knee in extension, whereas in reality shear forces at different flexion angles are also applied to the reconstruction. These limitations should be taken into account when interpreting the results.\nThe results showed that the average border contact pressure almost doubled (increase of 92%) compared to the pressure on an intact congruent joint surface. In the literature, increases in peak pressure between 10 and 30% were found in the presence of a defect with a diameter ranging from 1 to 7\u00a0mm [3,9]. Guettler et al. [4] claimed to have found no difference in rim contact pressure for defects below 10\u00a0mm diameter, but they found a 64% increase with respect to the healthy situation for all defects above 10\u00a0mm. These findings are supported with a recent finite element model, in which it was found that large defects (greater than 0.78\u00a0cm2) resulted in significantly increased border contact surface pressures, which may have clinical implications [16].\nThe relation between increased contact stresses on the joint surface and progressive degenerative changes of the cartilage is well recognized. Lefkoe et al. found a significant decrease in proteoglycan content in the cartilage sampled from the rim of 20-week-old defects [12]. Jackson et al. [7] introduced a \u2018zone of influence\u2019, as he found cartilage adjacent to the defect being affected, which may lead to early secondary gonarthrosis. Messner and Maletius [14] reported in a follow-up study radiographic joint space reduction in almost 50% of the patients with severe cartilage damage 14\u00a0years earlier. Linden et al. [13] found in a follow-up study of 33\u00a0years that 80% of the adults with osteochondritis dissecans developed secondary gonarthrosis. The gonarthrosis seemed to have its onset 10\u00a0years earlier (mean age 49 instead of 59\u00a0years) in life than primary gonarthrosis. Clearly, the increased potential of degenerative changes in the knee following an articular cartilage defect is of great clinical importance. Especially because this type of cartilage defect frequently occurs in the younger aged population.\nIn our study we were able to reduce the contact pressure at the boundaries of a large articular cartilage defect by 30% with a osteochondral transplantation. In a study by Raimondi et al. [17] a 16% reduction in peak pressure was found in the presence of a fibrin glue graft. One could question as to what extent this reduction lasts since fibrin glue degrades quite rapidly. Koh et al. [9] reported peak contact pressure reduced to normal when plugs were flush. However, they used only one plug, which does not correspond to clinical practice. It may be more difficult to obtain a smooth cartilage surface when using more plugs, which explains why we did not find a complete normalization of the contact stresses.\nAnother objective of this study was to investigate the stability of a mosaicplasty during motion (with or without weight). For the whole group (unbottomed and bottomed together), neither motion protocols significantly changed surface congruency; it remained equal to the mosaicplasty directly after surgery. This can be concluded from the fact that the pressure patterns did not change after weight bearing or non weight bearing motion, as the pressure pattern and rim stress remained equal. When comparing the bottomed and unbottomed groups, two unbottomed mosaicplasties showed a pressure pattern according to subsidence below flush level after weight bearing motion. Since the main focus of this study was to measure peak stresses on the articular surface, we did not assess the actual amount of subsidence. Although no significant differences in contact stresses could be detected between bottomed and unbottomed plugs by comparing the total groups; one can at least have some concern about potential subsidence of unbottomed plugs. Nevertheless, most unbottomed plugs remained stable, which is in agreement with a study of Pearce et al. [15], who reported that unbottomed multiple plug mosaicplasties that were placed flush, continued to stay flush after 3\u00a0months of weight bearing motion in sheep. However, human bone is much softer and therefore it may be more difficult to obtain a stable reconstruction with unbottomed plugs. In our previous study with human femora we measured the force required to push a single plug below flush level. For an unbottomed plug of 16\u00a0mm, 151\u00a0N had to be applied in comparison with 294\u00a0N for the bottomed plug [8]. For the comparison with the multiple plug mosaicplasty, Kordas et al. [10] reported that application of a mean force of 54\u00a0N was enough to push an unbottomed multiple plug mosaicplasty 3\u00a0mm below flush level; unfortunately, there was not a bottomed comparison in that study. The applied force of 350\u00a0N during the weight bearing motion in our study may have been too low, to destabilize the reconstruction. This load, which is relatively low, was chosen, as it would simulate partial weight-bearing motion during the direct postoperative rehabilitation period and not level walking. The latter is, according to our protocol, permitted after 2\u20134\u00a0weeks, which should allow for in-growth of the plugs [11,15].\nClinical relevance\nA cartilage defect results in increased stress levels at the articular cartilage boundaries of the defect. A multiple plug mosaicplasty has a positive effect in reducing these stress elevations, which will reduce the potential of cartilage degeneration, and thus may postpone secondary osteoarthritis. Post-operative non-weight bearing and weight bearing motions did not seem to influence the surface congruency of the mosaicplasty, although the unbottomed mosaicplasties showed a trend of subsidence below flush level after weight bearing motion. It appears feasible to allow limited weight bearing of the knee after osteochondral transplantation, especially when plugs have been bottomed.","keyphrases":["transplantation","cartilage","biomechanics","humans","pressure","weight-bearing","articular\/pathology","knee joint\/surgery","surface properties","autologous"],"prmu":["P","P","P","P","P","P","M","R","M","U"]}
{"id":"Knee_Surg_Sports_Traumatol_Arthrosc-3-1-1779626","title":"Membrane-seeded autologous chondrocytes: cell viability and characterization at surgery\n","text":"The implantation of chondrocytes, seeded on matrices such as hyaluronic acid or collagen membranes, is a method that is being widely used for the treatment of chondral defects. The aim of the present study was to evaluate the distribution, viability and phenotype expression of the cells seeded on a collagen membrane just at the time of the implantation. Twelve patients who were suffering from articular cartilage lesions were treated by the MACI\u00ae procedure. The residual part of each membrane was tested by colorimetric assay (MTT) and histochemical and ultrastructural analyses were carried out. In all of the samples a large number of viable cells, quite homogenously distributed, was detected. The cells expressed the markers of the differentiated hyaline chondrocytes. These data reassure in that the MACI procedure provides a suitable engineered tissue for cartilage repair, in line with the clinical evidences emerging in the literature.\nIntroduction\nAutologous chondrocyte implantation (ACI) is an established method for the treatment of chondral defects [6]. Some prospective studies have demonstrated subjective and objective improvement in joint function at 12\u00a0months after surgery [3, 13]. MRI, second-look surgery and biopsies have shown the formation of nearly normal cartilage in a good number of patients [13, 18, 19]. On the basis of these results, ACI may be considered one of the most effective treatments of articular cartilage lesions. However, this method is not devoid of some problems with regard to, for example, the wide surgical approach, the demanding technique, the presence of periosteal flap and the cell-holding in site [14, 16].\nIn order to overcome some of these hurdles, new tissue engineering techniques, widely used nowadays, have been developed using chondrocytes seeded on biological matrices such as hyaluronic acid [15] or collagen membranes [7] or atelocollagen gel [17]. Despite the wide diffusion of these methods, there are still some areas that would need better clarification. The presence of numerous, viable and well-differentiated cells at surgery is an essential requisite for the success of all these methods. It has been shown that autologous chondrocytes in suspension before implantation are able to maintain their differentiated phenotype and are capable of proliferating fairly well [9, 20]. On the other hand, little has been reported so far about cell characterization in membrane-seeded ACI techniques.\nThe aim of the present study was to evaluate the distribution, viability and phenotype expression of the cells seeded on a collagen membrane just at the moment of the implantation.\nMaterials and methods\nIn 2003, 12 consecutive patients, 8 males and 4 females, mean age of 34\u00a0years, suffering from cartilage lesions of the knee (10 cases) and the ankle (2 cases), underwent a collagenic scaffold-based ACI procedure (MACI\u00ae-Verigen, D). Autologous chondrocytes were isolated at the Verigen laboratories from cartilage slices obtained from non-bearing areas of the patients\u2019 joints during a preliminary arthroscopic surgery. Cells were propagated in monolayer cultures in autologous serum for 2 or 3\u00a0weeks, according to the cellular growth rate, and were passaged on average 3 times to obtain at least 10\u00a0\u00d7\u00a0106 cells. Cells were then seeded on 20\u00a0cm2 type I\/III collagen membrane of porcine origin. The cell-seeded membranes were implanted in cartilage defects by means of either arthroscopic or mini-open surgery, using fibrin glue to ensure adhesion. At each implantation, the residual part of the membrane was collected and tested for cell viability, and histochemical and ultrastructural analyses were also performed.\nCell viability analysis\nCell viability was evaluated by MTT (dimethylthiazol-diphenyltetrazol bromide; thiazolil blue) colorimetric assay. MTT (Sigma, Italy) is a water soluble tetrazolium salt that yields a yellowish solution when prepared in medium lacking in phenol red (RTMI 1640, Sigma). Dissolved MTT is converted to an insoluble purple formazan by cleavage of the tetrazolium ring by the active mitochondrial dehydrogenases of living cells. The MTT solution (5\u00a0mg MTT\/ml medium) was added to three samples (1\u00a0cm2) of each membrane, being assayed to equal 1\/10 of the original culture medium volume, and incubated for 3\u00a0h. The solution was then removed and acidic isopropanol (0.04\u20130.1\u00a0N HCl in absolute isopropanol) was added to solubilize the stain.\nThe results were evaluated by means of the spectrophotometric assay (570\u00a0nm), yielding absorbance as a function of viable cell number.\nHistochemical and immunohistochemical analysis\nThe samples were fixed by immersion in 4% para-formaldehyde in 0.1\u00a0M phosphate buffer, pH 7.4, at 4\u00b0C and then embedded in paraffin. Specimens were stained with safranin-O. For the immunohistochemistry, non-specific binding was blocked with 3% normal goat serum in a phosphate-buffered saline (PBS), pH 7.4, for 30\u00a0min at room temperature; slides were then incubated overnight with primary antibodies at 4\u00b0C. Sections were incubated with polyclonal antibodies anti S-100 protein (Dako, Italy), a cytoplasmatic marker of chondrocyte phenotype, diluted at 1:3,000, anti-collagen type I (Monosan, The Netherlands) and II (Calbiochem-Oncogene, CA, USA) at 1:150, and monoclonal antibodies anti chondroitin sulphate (chondroitin-S) (Sigma) at 1:200. Rabbit and mouse immunoglobulins, at the same dilutions as the primary antibodies, were used as controls. After three washes with Tris\u2013HCl (0.05\u00a0M, pH 7.6), revelation of the reactions was accomplished by DAKO LSAB\u00a0+\u00a0kit, HRP. Stainings were viewed and photographed with a Leica Microscope (Leica Cambridge Ltd., UK).\nUltrastructural analysis\nFor scanning electron microscopy (SEM), the membranes were fixed in 2% glutaraldeyde in 0.1\u00a0M cacodylate buffer (pH 7.4), post-fixed in 1% osmium tetroxide, dehydrated in increasing ethanol concentrations and then CPD-dried. They were mounted on stubs and gold-sputtered. Specimens were observed with a Philips 505 microscope.\nResults\nCell viability analysis\nThe presence of numerous viable cells was observed in all samples, with quite a homogeneous stain distribution, even if some of the areas revealed a greater concentration of converted dye (Fig.\u00a01).\nFig.\u00a01Macroscopic view of human chondrocytes seeded on type I, III collagen membrane after MTT incubation. The converted blue dye shows the presence of numerous viable cells with quite a homogeneous stain distribution\nConsiderable variability was found in mean cell numbers between the samples from different patients, in relation to the different amounts of cells obtained from cartilage slices and seeded on the membrane. Highest values were observed in samples from the younger patients (Fig.\u00a02, Table\u00a01).Fig.\u00a02The standard curve obtained from each membrane by MTT colorimetric assay (see also Table\u00a01 )Table\u00a01The table shows the mean viable cell number\/cm2 obtained from each membrane by MTT colorimetric assayCaseJointAgeSexSeeded cellsViable cells\/cm2 (mean\u00a0\u00b1\u00a0SD)1 Knee54M19\u00a0\u00d7\u00a0106(7.52\u00a0\u00b1\u00a02.79)\u00a0\u00d7\u00a01032 Knee22F17\u00a0\u00d7\u00a0106(12.94\u00a0\u00b1\u00a02.54)\u00a0\u00d7\u00a01033Knee50F15.4\u00a0\u00d7\u00a0106(17.82\u00a0\u00b1\u00a06.25)\u00a0\u00d7\u00a01034 Knee23M20.3\u00a0\u00d7\u00a0106(100.28\u00a0\u00b1\u00a022.14)\u00a0\u00d7\u00a01035Knee29F20.4\u00a0\u00d7\u00a0106(110.07\u00a0\u00b1\u00a039.18)\u00a0\u00d7\u00a01036Knee43M18.7\u00a0\u00d7\u00a0106(5.34\u00a0\u00b1\u00a03.25)\u00a0\u00d7\u00a01037Knee48M17.5\u00a0\u00d7\u00a0106(4.74\u00a0\u00b1\u00a02.37)\u00a0\u00d7\u00a01038Ankle42F18.2\u00a0\u00d7\u00a0106(33.13\u00a0\u00b1\u00a025.92)\u00a0\u00d7\u00a01039Knee19M21.6\u00a0\u00d7\u00a0106(99.54\u00a0\u00b1\u00a025)\u00a0\u00d7\u00a010310Knee35M20\u00a0\u00d7\u00a0106(103.81\u00a0\u00b1\u00a037.26)\u00a0\u00d7\u00a010311Ankle39M19\u00a0\u00d7\u00a0106(79.48\u00a0\u00b1\u00a062.57)\u00a0\u00d7\u00a010312Knee13M20.1\u00a0\u00d7\u00a0106(87.88\u00a0\u00b1\u00a055.17)\u00a0\u00d7\u00a0103 Patients\u2019 characteristics and the number of cells initially seeded on each membrane are also reported (see also Fig.\u00a02)\nHistochemical and immunohistochemical analysis\nIn all of the samples, chondrocytes that were grown on the membrane were arranged in multi-layered sheets, and sometimes invaded deeper into the matrix. The cells appeared to be quite well differentiated, although some had a flattened morphology. They slightly stained metachromatically for safranin O (Fig.\u00a03), and clearly immunoreacted with anti-S-100 protein (Fig.\u00a04), type II collagen (Fig.\u00a05) and chondroitin-S antibodies. Only a few cells stained for type I collagen.\nFig.\u00a03Human chondrocytes seeded on type I, III collagen membrane before the implantation. Numerous cells are located on the membrane and deeper in the matrix forming a multi-layer engineered tissue. (Safranin O stain, \u00d7200)Fig.\u00a04Human chondrocytes seeded on type I, III collagen membrane. A marked cytoplasmic immunoreaction for S-100 protein is evident (\u00d7400)Fig.\u00a05Human chondrocytes seeded on type I, III collagen membrane. Cells express a positive immunoreaction for type II collagen, while the membrane remains unstained (\u00d7400)\nUltrastructural analysis\nThe membranes presented a dual appearance: there was a smooth side with tightly packed fibres and a rougher side, a sparse layer, to which the cells were adhered. Numerous cells firmly adhered to the membrane and showed a round shaped morphology and a rough surface (Fig.\u00a06).\nFig.\u00a06Scanning electron micrograph of human chondrocytes seeded on type I, III collagen membrane. Numerous cells (arrows) firmly adhere to the membrane and show a round shaped morphology and a rough surface (\u00d77,500)\nDiscussion\nCell phenotype and proliferation analysis should be an essential step in the evaluation of all tissue-engineered products because the implantation of dedifferentiated or not-proliferating cells would not justify the therapeutic employment of these biotechnologies.\nThe chondrocyte culture may undergo a dedifferentiation process, consisting in a fibroblast-like morphology, a reduction of the type II collagen and aggregating proteoglycans synthesis, and an increase of type I collagen expression [1, 4]. Chondrocyte dedifferentiation is common in monolayer cultures, while a long phenotype maintenance is supported by the presence of a tri-dimensional matrix [1, 4, 11]. The chemical and ultrastructural characteristics of the matrix may have great influence on the behaviour of chondrocytes in culture. A Type I collagen membrane appears to be a matrix endowed with such properties which make that ideal and useful for cartilage tissue engineering [8, 10].\nMACI is a method for the treatment of articular cartilage defects, that employs autologous chondrocytes expanded in monolayer culture, suspended, and then seeded on type I\u2013III collagen membrane. In a previous study [9], we had observed that autologous chondrocytes in suspension before implantation were of \u201cgood cartilaginous quality\u201d and that they possessed an excellent proliferation capacity. Zheng et al. [20] confirmed that cells in ACI procedure maintained chondrocyte phenotype and showed a low apoptotic rate. To our knowledge, there is no previous reference to the analysis of the phenotype and the proliferation activity of human chondrocytes before MACI implantation. Data about cell number and viability, but not about cell phenotype and distribution are provided by Verigen\u2019s laboratory for each implant. It should, however, be remembered that it is quite possible that the presence of a membrane like the one found in the MACI technique could interfere with cell behaviour and modify phenotype expression.\nThe present study has demonstrated that the autologous chondrocytes that were analysed adhered to the membrane and had quite a homogenous distribution. These cells were viable in all the cases analysed, as shown by the spectrophotometric assay. The differences observed among patients in the number of cells seeded on the membrane and those found to be viable are probably due to several factors. In the first place, the amount of tissue collected from patients and sent to the laboratory for culture was variable. The cellularity of cartilage tissue and the proliferation capacity of chondrocytes in identical culture conditions are also quite variable between subjects, and this variability cannot merely be explained with age, but is more likely to stem from a number of characteristics that define tissue \u201cquality\u201d [5, 12].\nWe did not investigate whether during monolayer culture chondrocytes underwent dedifferentiation, however, other authors [20] demonstrated that cells grown in monolayer with the same culture conditions maintained a differentiated phenotype. Moreover, we felt it was important to establish the phenotypical expression of chondrocytes seeded on the I\/III collagen membrane, since those are the cells that are then implanted, independently of what has taken place in the previous steps of the procedure. The positive immunostaining for S-100 protein, chondroitin-S, type II, but not type I collagen, confirms that cells maintained the characteristics of differentiated hyaline chondrocytes at the moment they were implanted.\nThese data reassure that the MACI procedure can indeed provide a suitable engineered tissue for cartilage repair, in line with the mid-term clinical results described in the literature so far [2, 7]. Nevertheless, clinical and histological results of this method to attain stable clinical recovery and a hyaline-like cartilaginous scar are auspicated to substantiate these evidences in the long term.","keyphrases":["chondrocytes","cells","articular cartilage","tissue engineering","cultured","histocytochemistry"],"prmu":["P","P","P","P","P","U"]}
{"id":"Cancer_Causes_Control-2-2-1764867","title":"Wine and other alcohol consumption and risk of ovarian cancer in the California Teachers Study cohort\n","text":"Objective Whether alcohol consumption influences ovarian cancer risk is unclear. Therefore, we investigated the association between alcohol intake at various ages and risk of ovarian cancer.\nIntroduction\nConsistent evidence that moderate alcohol consumption increases the risk of breast cancer, presumably by elevating estrogen and androgen levels [1], suggests that alcohol intake might also influence the development of other hormone-related malignancies, such as ovarian cancer. From a public health standpoint, this hypothesis is attractive since alcohol consumption could represent a readily modifiable ovarian cancer risk factor, whereas other risk factors, particularly reproductive characteristics [2, 3], are generally less amenable to change. Findings from previous studies examining the association between alcohol and ovarian cancer risk have been discrepant, with published reports of mostly null [3\u201319], some positive [15, 16, 20, 21], and some inverse associations [14, 17, 22\u201325], including occasional variation in associations by type of alcohol and\/or subgroup of ovarian cancer cases. No systematic differences in findings are apparent between case\u2013control and cohort studies.\nAlthough ethanol itself is not a direct ovarian carcinogen, it could influence ovarian cancer risk through effects on steroid hormones, especially estrogens, which are believed to play a primary role in ovarian carcinogenesis [26]. In pre-menopausal women, moderate to high alcohol consumption is associated with elevated levels of total and bioavailable estrogens and androgens [27\u201329], reduced fertility [30, 31], and decreased menstrual cycle variability and length, resulting in increased cumulative estrogen exposure [32]. In post-menopausal women, moderate alcohol intake markedly increases circulating estrogen levels in hormone therapy (HT) users [33, 34], and it may elevate estrogen and androgen levels in women not using HT [35]. Other mechanisms for alcohol-related carcinogenesis include alteration of gonadotropin levels, promotion of DNA damage, impaired folate metabolism and DNA hypomethylation, inhibition of carcinogen detoxification or clearance, and increased metastatic potential of tumor cells [1].\nGiven the biologic plausibility of a role of alcohol intake in ovarian cancer etiology, we examined the association between alcohol consumption and risk of ovarian cancer in a prospective cohort in which baseline alcohol consumption was associated with increased breast cancer risk [36, 37]. With data on past and baseline intake of specific alcoholic beverages, as well as information on a variety of demographic and behavioral factors, we were also able to explore the importance of drinking patterns and potential effect modifiers of any association.\nMethods\nStudy population\nThe California Teachers Study (CTS) cohort includes 133,479 active and retired female public school teachers and administrators who were members of the California State Teachers Retirement System and returned a mailed questionnaire in 1995\u20131996 [38]. The questionnaire assessed a range of potential cancer risk factors including menstrual and reproductive history, personal and family medical history, physical activity, dietary intake during the previous year (using a food-frequency questionnaire and portion-size assessment [39\u201342]), alcohol and tobacco use, and other factors. All women provided written informed consent to participate in the study, and the study protocol was approved by the institutional review boards of all participating institutions.\nFor this analysis, we excluded women (in a hierarchical manner) as follows: those who 1.) lived outside of California at baseline (n = 8,867); 2.) did not provide adequate information on personal history of cancer (n = 662); 3.) consented to participate only in analyses of breast cancer (n = 18); 4.) reported having had ovarian cancer before baseline or were identified by the California Cancer Registry as having been previously diagnosed with ovarian cancer (n = 640); 5.) reported having had a bilateral oophorectomy before baseline (n = 14,422); 6.) were aged 85 years or older at baseline (n = 1,874); 7.) reported never having had a first menstrual period (n = 51); 8.) provided multiple invalid, inconsistent, or blank responses to the dietary questionnaire (n = 2,942); 9.) reported food consumption that was judged to be implausibly low (i.e., < 600 calories\/day) or high (i.e., > 5,000 calories\/day) (n = 1,565 and 69, respectively); or 10.) provided invalid, missing, or inconsistent data with respect to alcohol intake during the previous year (n = 5,094) or the earlier two periods evaluated (n = 6,904). Of the 90,371 remaining women included in this analysis, 227 were diagnosed with invasive epithelial ovarian cancer and 26 were diagnosed with borderline epithelial ovarian cancer (ICD-O-3 site C569, excluding non-epithelial ovarian cancer cases [morphology codes 8240\u20138245, 8590\u20138671, and 9060\u20139989] [43]) after joining the cohort and on or before 31 December 2003.\nAlcohol assessment\nParticipants reported average weekly consumption of beer, wine\/champagne, and cocktails\/liquor at ages 18\u201322 years, at ages 30\u201335 years, and in the year prior to baseline. Available response categories for average number of drinks per week were none, \u2264 3, 4\u201310, 11\u201317, 18\u201324, and \u2265 25. A typical drink was defined as one bottle, can, or glass of beer; one glass of wine, champagne, or wine cooler; or one cocktail, shot, or mixed drink of liquor. A single drink of beer, wine, or liquor was assumed to contain 13.2, 11.1, or 15.0 grams of alcohol, respectively. Based on these standards, daily intake of grams of alcohol from each type of drink was calculated for each woman during each time period. Alcohol intake in the cohort was reproducible (r = 0.87) and valid compared to multiple 24\u2013hour recalls (r = 0.74) (Pamela Horn-Ross, unpublished data).\nDaily consumption of alcohol from beer was categorized as 0, < 13.2, or \u2265 13.2 grams\/day; alcohol from wine was categorized as 0, < 11.1, or \u2265 11.1 grams\/day; alcohol from liquor was categorized as 0, < 15.0, or \u2265 15.0 grams\/day; and total alcohol was categorized as 0, < 10.0, 10.0 to < 20.0, or \u2265 20.0 grams\/day. These categories were defined based on the grams of alcohol per standard drink, and on findings from previous analyses of alcohol intake and breast cancer risk in the CTS [36, 37].\nBased on their drinking patterns in any two time periods (ages 18\u201322 years and baseline, ages 30\u201335 years and baseline, or ages 18\u201322 years and 30\u201335 years), women were categorized as having been non-drinkers, moderate drinkers (in the middle category or categories of intake), or heavy drinkers (in the highest category of intake) in both time periods; or decreasing or increasing drinkers if their intake changed between the earlier and the later time period. For each type of alcoholic drink and each time period, women also reported how many days per week they usually had at least one drink. Women were categorized as non-drinkers, as drinking alcohol on 1\u20134 days\/week, or as drinking alcohol on \u2265 5 days\/week [37].\nResidence-based measures\nBased on residential address at entry, women were geocoded to census block groups. To obtain a measure of each cohort member\u2019s relative socioeconomic status (SES), all 1990 census block groups in the state of California were ranked by three measures, according to deciles based on the statewide adult population: percentage of adults over age 25 years who had completed a college degree or higher; median family income; and percentage of adults employed in managerial\/professional occupations [44]. A summary SES metric was created by adding the scores (1 through 10) across these attributes; participants were then categorized into deciles or quartiles of the total score. Women were also categorized by median family income in their census block group, based on deciles in the cohort.\nResidential census block groups were categorized as rural, town, small city, metropolitan suburban, or metropolitan urban based on population size and density [44]. Women were also classified as residents of the Greater San Francisco Bay Area (Alameda, Contra Costa, Marin, Monterey, San Benito, San Francisco, San Mateo, Santa Clara, and Santa Cruz Counties); the Southern Coastal area (Orange, Los Angeles, and San Diego Counties); or the rest of California [45].\nFollow-up\nPerson-time was accrued from the date of completion of the baseline questionnaire until the date of first diagnosis with borderline or invasive ovarian cancer, relocation out of California, death, or 31 December 2003, whichever occurred earliest. Information on incident ovarian cancer and tumor characteristics was obtained through annual linkage of cohort members to the California Cancer Registry (CCR) based on full name, date of birth, address, and social security number, including manual review of possible matches. The CCR is the population-based cancer registry that covers the entire state of California, has agreements with 13 other states for case-sharing purposes, and maintains high-quality data standards as part of the National Cancer Institute\u2019s Surveillance, Epidemiology, and End Results program. Reporting of new cancer diagnoses to the CCR has been mandated by California state law since 1985, and coverage is estimated to be 99% complete [46], such that all members of the CTS cohort are effectively in active follow-up for cancer outcomes as long as they reside in California.\nDate and cause of death are ascertained through linkages with the California state mortality file and the national Social Security Administration death master file, as well as reports from relatives. Address changes are obtained through annual mailed newsletters, notifications by participants, and record linkages with the California Department of Motor Vehicles, the US Postal Service National Change of Address database, and other sources.\nStatistical analysis\nMultivariable Cox proportional hazards regression analysis was performed to evaluate the association between alcohol consumption and risk of ovarian cancer, using ages at the start and end of follow-up (in days) to define the time scale. Models were adjusted for race (White or non-White), total daily caloric intake (continuous), parity (0, 1\u20132, or \u2265 3 full-term pregnancies), use of oral contraceptives (never, < 5 years, or \u2265 5 years), average strenuous physical exercise ( < 0.5, 0.5\u20133.99, or \u2265 4 hours\/week during lifetime up to age 54 years), menopausal status\/use of HT (pre-menopausal, unknown menopausal status, or peri-\/post-menopausal and: never used HT, used combination estrogen-progestin HT, used a mixture of combination and estrogen-only (\u201cmixed\u201d) HT, used estrogen-only HT for \u2264 5 years, used estrogen-only HT for > 5 years, used estrogen-only HT for an unknown duration, or unknown HT use), and an interaction between menopausal status\/HT use and the time scale (because menopausal status\/HT use violated the proportionality assumption), and were stratified by age at baseline (in years). These potential confounders were chosen based on statistically significant (p-value \u2264 0.05) associations with risk of ovarian cancer, and on prior knowledge of ovarian cancer risk factors. Missing values were coded as dummy variables, none of which were found to be associated with ovarian cancer risk. We tested the assumption of proportional hazards for each alcohol variable using significance tests of interactions with the time scale and visual examination of scaled Schoenfeld residual plots [47], and found no violations of the proportionality assumption.\nHazard rate ratios, presented as relative risks (RR), and corresponding 95% confidence intervals (CI) were estimated for each type of alcoholic beverage, comparing categories of consumption to non-drinkers of that alcohol type as the reference group. Tests for linear trend across exposure categories were conducted using the median of each category coded as an ordinal variable. The median of the highest category of beer, wine, or liquor intake was equal to the lower boundary because most women in those categories reported consuming one drink per day.\nAnalyses were first performed for all eligible women, and then repeated with restriction to women who were peri- or post-menopausal at baseline. In addition, analyses were performed with restriction to cases of invasive ovarian cancer (excluding borderline cases) or serous ovarian cancer (ICD-O-3 morphology codes 8441\u20138462 and 9014 [43]; n= 114). Likelihood ratio tests were used to evaluate homogeneity of the estimated RR between strata of women, as well as significant differences in fit between models with and without additional covariates. Tests for non-linearity of trend were based on a likelihood ratio test comparing models with the exposure coded as an ordinal or a categorical variable [48]. Associations with wine drinking were evaluated using multivariate logistic regression, using covariates as defined above. All analyses were performed using SAS Version 9.1 (SAS Institute, Cary, NC).\nResults\nDistributions of demographic characteristics and ovarian cancer risk factors in the eligible study cohort are shown in Table 1. The median length of follow-up was 2,959 days (8.1 years) and the median age of participants at baseline was 50 years. As shown in Table 2, there was no overall difference in risk of ovarian cancer by total alcohol consumption in the year before baseline, at ages 30\u201335 years, or at ages 18\u201322 years. Similarly, consumption of alcohol from beer or liquor during any of the three time periods was not significantly associated with risk of ovarian cancer. In contrast, intake of alcohol from wine during the year before baseline was associated with statistically significantly elevated risk of ovarian cancer. After adjusting for alcohol intake from beer and liquor, as well as ovarian cancer risk factors, women who drank at least 11.1 grams per day of alcohol from wine\u2014the equivalent of one glass per day\u2014at baseline were at 57% higher risk of ovarian cancer, compared to women who did not drink wine (Ptrend = 0.01). Intake of alcohol from wine at ages 30\u201335 years or ages 18\u201322 years was not significantly associated with ovarian cancer risk, although the estimated RRs were consistent with those for baseline wine intake.\nTable 1Selected baseline characteristics of the California Teachers Study (CTS) cohort included in the present analysis (n= 90,371)Characteristicn(%)Age at baseline (years) <3510,456(11.6%) 35\u20134418,546(20.5%) 45\u20135428,275(31.3%) 55\u20136416,229(18.0%) 65\u20137411,496(12.7%) 75\u2013845,369(5.9%)Race\/ethnicity White78,468(86.8%) Non-white11,266(12.5%) Unknown637(0.7%)Parity (full-term pregnancies) None23,810(26.3%) 1\u2013243,674(48.3%) \u2265321,412(23.7%) Unknown1,475(1.6%)Oral contraceptive use (years) None26,643(29.5%) <527,608(30.5%) \u2265532,463(35.9%) Unknown3,657(4.0%)Lifetime strenuous physical activity (average hours\/week) <0.525,254(27.9%) 0.5\u20133.949,467(54.7%) \u22654.015,293(16.9%) Unknown357(0.4%)Menopausal status Pre-menopausal42,204(46.7%) Peri-menopausal2,202(2.4%) Post-menopausal39,744(44.0%) Unknown6,221(6.9%)Hormone therapy (HT) use (peri-\/post-menopausal women only) None12,968(30.9%) Combination estrogen + progestin HT14,235(33.9%) Estrogen-only HT, \u22645 years4,342(10.4%) Estrogen-only HT, >5 years4,899(11.7%) Estrogen-only HT, unknown duration332(0.8%) Mixed combination and estrogen-only HT4,776(11.4%) Unknown394(0.9%)Region of residence within Californiaa Greater San Francisco Bay Area18,444(20.4%) Southern Coastal\/Los Angeles Area35,388(39.2%) Other areas36,483(40.4%) Unknown56(0.1%)Type of residence Rural12,677(14.0%) Town3,136(3.5%) Small city16,135(17.9%) Metropolitan suburban48,278(53.4%) Metropolitan urban9,038(10.0%) Unknown1,107(1.2%)Statewide percentile of socioeconomic status in census block groupa \u226449th18,962(21.0%) 50\u201359th10,199(11.3%) 60\u201369th12,620(14.0%) 70\u201379th14,508(16.1%) 80\u201389th16,864(18.7%) 90\u201399th16,076(17.8%) Unknown1,142(1.3%)Smoking history Never60,868(67.4%) Former24,989(27.7%) Current4,428(4.9%) Unknown86(0.1%)Alcohol drinking in the year prior to baseline None31,024(34.3%) Beer only2,222(2.5%) Wine only19,365(21.4%) Liquor only2,868(3.2%) Beer and liquor only1,005(1.1%) Wine and beer\/liquor33,887(37.5%)a See Methods for definitionTable 2Relative risks (RRs) and 95% confidence intervals (CIs) for associations between intake of specific types of alcohol at various ages and risk of ovarian cancerAlcohol typeTime periodDaily intake (g\/day)Median (g\/day)Cases (n)RRa95% CIaRRb95% CIbTotal alcoholYear before baselineNone0.0771.00(reference)\u2014<10.04.5811.04(0.76, 1.42)\u201410.0\u2013 < 20.011.8721.47(1.06, 2.03)\u2014\u226520.028.2231.15(0.71, 1.84)\u2014Ptrend = 0.19Ages 30\u201335 yearscNone0.0671.00(reference)\u2014<10.07.31011.14(0.83, 1.56)\u201410.0\u2013 < 20.011.8471.08(0.74, 1.59)\u2014\u226520.029.7160.99(0.56, 1.71)\u2014Ptrend = 0.99Ages 18\u201322 yearsdNone0.01311.00(reference)\u2014<10.04.5620.76(0.55, 1.03)\u201410.0\u2013 < 20.011.8361.26(0.86, 1.84)\u2014\u226520.028.891.00(0.50, 1.99)\u2014Ptrend = 0.63BeerYear before baselineNone0.01991.00(reference)1.00(reference)<13.24.0510.96(0.70, 1.31)0.89(0.64, 1.24)\u226513.213.230.58(0.19, 1.84)0.54(0.17, 1.70)Ptrend = 0.40Ptrend = 0.22Ages 30\u201335 yearscNone0.01801.00(reference)1.00(reference)<13.24.0450.81(0.58, 1.13)0.75(0.53, 1.06)\u226513.213.260.72(0.32, 1.64)0.73(0.32, 1.69)Ptrend = 0.20Ptrend = 0.16Ages 18\u201322 yearsdNone0.01791.00(reference)1.00(reference)<13.24.0460.95(0.68, 1.32)0.93(0.65, 1.33)\u226513.213.2131.21(0.68, 2.16)1.30(0.70, 2.39)Ptrend = 0.67Ptrend = 0.57WineYear before baselineNone0.0911.00(reference)1.00(reference)<11.13.3991.08(0.81, 1.43)1.09(0.80, 1.50)\u226511.111.1631.50(1.08, 2.09)1.57(1.11, 2.22)Ptrend = 0.01ePtrend = 0.01eAges 30\u201335 yearscNone0.0901.00(reference)1.00(reference)<11.13.31121.19(0.90, 1.59)1.26(0.92, 1.71)\u226511.111.1291.21(0.78, 1.86)1.38(0.87, 2.19)Ptrend = 0.36Ptrend = 0.16Ages 18\u201322 yearsdNone0.01671.00(reference)1.00(reference)<11.13.3631.11(0.82, 1.50)1.17(0.84, 1.63)\u226511.111.181.42(0.69, 2.91)1.63(0.76, 3.50)Ptrend = 0.28Ptrend = 0.15LiquorYear before baselineNone0.01691.00(reference)1.00(reference)<15.04.5681.09(0.82, 1.45)1.06(0.78, 1.44)\u226515.015.0160.87(0.52, 1.47)0.82(0.48, 1.39)Ptrend = 0.80Ptrend = 0.56Ages 30\u201335 yearscNone0.01241.00(reference)1.00(reference)<15.04.5931.07(0.81, 1.41)1.04(0.77, 1.40)\u226515.015.0140.77(0.44, 1.35)0.75(0.42, 1.36)Ptrend = 0.52Ptrend = 0.44Ages 18\u201322 yearsdNone0.01641.00(reference)1.00(reference)<15.04.5690.99(0.74, 1.32)0.94(0.68, 1.29)\u226515.015.050.62(0.25, 1.52)0.49(0.19, 1.26)Ptrend = 0.39Ptrend = 0.17a Adjusted for race, total energy intake, parity, oral contraceptive use, strenuous exercise, and menopausal status\/hormone therapy use; stratified by age at baselineb Additionally adjusted for consumption of other alcohol types in the same time periodc Among women over age 35 years at baseline with non-missing data on alcohol consumption at ages 30\u201335 yearsd Among women over age 22 years at baseline with non-missing data on alcohol consumption at ages 18\u201322 yearsep for non-linearity of trend > 0.05\nIntake of specific alcohol types, beyond total alcohol, was associated with ovarian cancer risk, as assessed by comparing a multivariate model with beer, wine, and alcohol consumption in the year before baseline to a model with alcohol consumption alone (p = 0.05, 2 d.f.). Furthermore, the association of wine consumption with ovarian cancer risk differed from that of beer or liquor consumption, as assessed by comparing a multivariate model with beer\/liquor and wine consumption to a model with alcohol consumption (p = 0.02, 1 d.f.). Controlling for total alcohol intake did not attenuate the positive association between wine intake at any time period and risk of ovarian cancer (data not shown). Women who drank wine only in the year before baseline (median daily alcohol intake = 11.1 grams), relative to non-drinkers, had an ovarian cancer RR of 1.40 (95% CI 1.01\u20131.93). In contrast, risk did not vary between non-drinkers and women who drank beer or liquor only (median daily intake of alcohol = 4.5 grams; RR = 1.03 [95% CI 0.58\u20131.83]) or beer\/liquor and wine (median daily alcohol intake = 11.8 grams; RR = 1.08 [95% CI 0.79\u20131.48]). The multivariate RR among women who exclusively drank at least one glass of wine per day, compared to non-drinkers, was 1.70 (95% CI 1.10\u20132.62). After simultaneously adjusting for wine drinking during the year before baseline and at ages 18\u201322 years and 30\u201335 years, the RR associated with drinking at least one daily glass of wine at baseline was 1.33 (95% CI 0.91\u20131.96); at ages 30\u201335 years, RR = 1.01 (95% CI 0.61\u20131.68); and at ages 18\u201322 years, RR = 1.28 (95% CI 0.58\u20132.87).\nWomen who drank at least one glass per day of wine both in an earlier time period (18\u201322 years or 30\u201335 years) and at baseline were at significantly higher risk of ovarian cancer, compared to wine non-drinkers (Table 3). Results were similar when based on intake at ages 18\u201322 years and 30\u201335 years (data not shown). Women who were heavy wine drinkers in all three time periods (n= 5 cases) had over four times the risk of ovarian cancer relative to women who never drank wine in any time period (RR = 4.60 [95% CI = 1.76\u201312.01]), adjusting for beer and liquor drinking patterns. Marginally elevated risk of ovarian cancer was also observed among women who increased or decreased their wine intake between ages 18\u201322 years and either baseline or ages 30\u201335 years (latter data not shown). No patterns of total alcohol, beer, or liquor intake were significantly associated with ovarian cancer risk. Furthermore, ovarian cancer risk did not vary according to number of drinking days per week at baseline, whether for total alcohol, beer, wine, or liquor, although the risk of ovarian cancer rose slightly with increasing frequency of wine consumption at baseline (Ptrend = 0.11).\nTable 3Relative risks (RRs) and 95% confidence intervals (CIs) for associations between patterns of drinking specific types of alcohol and risk of ovarian cancerAlcohol typeTime periodDrinking patternCases (n)RRa95% CIaRRb95% CIbTotal alcoholAge 30\u201335 years and the year before baselinecNever491.00(reference)\u2014Steady moderate1181.25(0.89, 1.76)\u2014Decreasing291.06(0.67, 1.68)\u2014Increasing271.10(0.68, 1.77)\u2014Steady heavy81.32(0.62, 2.82)\u2014Age 18\u201322 years and the year before baselinedNever551.00(reference)\u2014Steady moderate701.07(0.74, 1.53)\u2014Decreasing241.06(0.65, 1.72)\u2014Increasing861.30(0.92, 1.84)\u2014Steady heavy31.87(0.58, 6.04)\u2014Year before baseline0 days\/week771.00(reference)\u20141 to 4 days\/week981.25(0.92, 1.69)\u20145 to 7 days\/week, \u2264 20 g\/day301.36(0.88, 2.08)\u20145 to 7 days\/week, > 20 g\/day181.14(0.68, 1.93)\u2014BeerAge 30\u201335 years and the year before baselinecNever1631.00(reference)1.00(reference)Steady moderate260.80(0.53, 1.21)0.73(0.47, 1.13)Decreasing250.94(0.62, 1.44)0.88(0.57, 1.38)Increasing171.08(0.65, 1.79)0.99(0.59, 1.66)Steady heavy0\u2014\u2014Age 18\u201322 years and the year before baselinedNever1591.00(reference)1.00(reference)Steady moderate241.06(0.68, 1.64)1.02(0.64, 1.62)Decreasing340.95(0.65, 1.38)0.92(0.61, 1.37)Increasing210.77(0.49, 1.22)0.71(0.44, 1.14)Steady heavy0\u2014\u2014Year before baselinee0 days\/week1991.00(reference)1.00(reference)1 to 4 days\/week400.98(0.70, 1.39)0.79(0.53, 1.16)5 to 7 days.week10.33(0.05, 2.37)0.36(0.05, 2.59)Ptrend = 0.30Ptrend = 0.14WineAge 30\u201335 years and the year before baselinecNever601.00(reference)1.00(reference)Steady moderate651.15(0.80, 1.64)1.19(0.80, 1.77)Decreasing341.25(0.82, 1.92)1.32(0.82, 2.11)Increasing531.25(0.86, 1.82)1.29(0.86, 1.93)Steady heavy191.54(0.91, 2.62)1.75(1.00, 3.04)Age 18\u201322 years and the year before baselinedNever711.00(reference)1.00(reference)Steady moderate290.98(0.63, 1.52)1.01(0.62, 1.63)Decreasing201.48(0.89, 2.45)1.73(1.00, 2.99)Increasing1131.29(0.95, 1.74)1.38(0.99, 1.92)Steady heavy52.47(0.99, 6.19)2.76(1.09, 7.00)Year before baselinee0 days\/week911.00(reference)1.00(reference)1 to 4 days\/week971.19(0.89, 1.60)1.23(0.89, 1.71)5 to 7 days.week371.34(0.91, 1.98)1.45(0.96, 2.20)Ptrend = 0.18Ptrend = 0.11LiquorAge 30\u201335 years and the year before baselinecNever1101.00(reference)1.00(reference)Steady moderate471.13(0.80, 1.60)1.12(0.76, 1.63)Decreasing501.04(0.74, 1.46)0.97(0.67, 1.41)Increasing211.11(0.69, 1.77)1.04(0.63, 1.70)Steady heavy30.49(0.15, 1.54)0.46(0.15, 1.49)Age 18\u201322 years and the year before baselinedNever1261.00(reference)1.00(reference)Steady moderate321.12(0.76, 1.66)1.10(0.71, 1.69)Decreasing350.84(0.57, 1.23)0.76(0.50, 1.15)Increasing440.98(0.69, 1.38)0.92(0.64, 1.33)Steady heavy10.80(0.11, 5.77)0.69(0.10, 5.04)Year before baselinee0 days\/week1691.00(reference)1.00(reference)1 to 4 days\/week571.13(0.83, 1.53)1.10(0.78, 1.55)5 to 7 days.week131.00(0.57, 1.78)0.73(0.35, 1.51)Ptrend = 0.92Ptrend = 0.44a Adjusted for race, total energy intake, parity, oral contraceptive use, strenuous exercise, and menopausal status\/hormone therapy use; stratified by age at baselineb Additionally adjusted for drinking patterns of other alcohol types across the same time periodsc Among women over age 35 years at baseline with non-missing data on alcohol consumption at ages 30\u201335 yearsd Among women over age 22 years at baseline with non-missing data on alcohol consumption at ages 18\u201322 yearse Excluding women with missing data on number of drinking days per week\nThe lack of an association with total alcohol, beer, or liquor intake, as well as the persistence of the association between wine intake and risk of ovarian cancer after adjustment for alcohol consumption, suggested that determinants of wine drinking, or ingredients of wine other than alcohol, were responsible for the observed positive association. After mutual adjustment, older age (up to ages 65\u201369 years), White race, higher total caloric intake, nulliparity, ever-use of oral contraceptives, more physical activity, ever-use of HT, higher SES or median family income, residence in the Greater San Francisco Bay Area, lower body mass index, ever-smoking of cigarettes, and higher intake of coffee and\/or tea were significantly associated with drinking at least one daily glass of wine, compared to none (data not shown). However, the positive association between wine consumption and risk of ovarian cancer remained statistically significant even after adjustment for these characteristics (data not shown).\nTo further explore the relationship between wine consumption and ovarian cancer risk, we stratified the association by various potential effect modifiers, including demographic characteristics and ovarian cancer risk factors (Table 4). There was no statistically significant heterogeneity in the association between intake of alcohol from wine at baseline and risk of ovarian cancer by any of the factors examined. Among peri-\/post-menopausal women, we observed no association with wine intake among those who had never used HT, nor among those who used combined estrogen-progestin HT, adjusting for duration of HT use. In contrast, women who used unopposed estrogen HT were at twice the risk of ovarian cancer if they drank one glass of wine per day at baseline, compared to wine non-drinkers. The association was especially strong among women who used estrogen-only HT for over 5 years (RR = 2.39 [95% CI 0.97\u20135.89], Ptrend = 0.02), whereas there was no such association among women who used combination HT for over 5 years (RR = 1.34 [95% CI 0.51\u20133.54], Ptrend = 0.29). We also found that the women in the highest quartile of SES statewide were at double the risk of ovarian cancer if they drank at least a glass per day of wine, compared to none, whereas there was no such association among women in the lower three quartiles of SES.\nTable 4Stratified relative risks (RRs) and 95% confidence intervals (CIs) for associations between wine intake in the year before baseline and risk of ovarian cancer within participant subgroupsCharacteristic (at baseline)Alcohol consumption from winePtrendPheterogeneity between subgroupsNone < 11.1 g\/day\u2265 11.1 g\/dayCasesRRaCasesRRa(95% CI)aCasesRRa(95% CI)aAge\u2264Median (50 years)231.00271.07(0.58, 1.99)121.43(0.67, 3.04)0.34>Median681.00721.10(0.76, 1.57)511.62(1.09, 2.39)0.010.95ParityNulliparous181.00261.34(0.69, 2.62)131.56(0.71, 3.40)0.31Parous711.00731.05(0.73, 1.50)481.57(1.06, 2.34)0.020.61Oral contraceptive useNever451.00391.00(0.62, 1.61)291.70(1.02, 2.82)0.03Ever221.00140.76(0.37, 1.58)141.78(0.85, 3.72)0.090.54Lifetime strenuous physical activity\u2264Median (1.4 hours\/week)611.00581.07(0.72, 1.59)401.68(1.09, 2.59)0.01>Median (1.4 hours\/week)301.00411.11(0.66, 1.86)231.39(0.77, 2.50)0.260.55Menopausal statusPre-menopausal211.00200.83(0.42, 1.65)101.24(0.55, 2.83)0.53Peri-\/Post-menopausal661.00721.16(0.80, 1.66)511.72(1.16, 2.55)0.010.86Hormone therapy (HT) useNoneb211.00221.27(0.64, 2.51)91.20(0.51, 2.78)0.73Combination estrogen + progestin HTb231.00180.69(0.35, 1.37)161.17(0.58, 2.34)0.45Estrogen-only HTb161.00191.27(0.62, 2.61)152.03(0.95, 4.35)0.060.37Region of residenceGreater Bay\/Southern Coastal regions451.00621.20(0.79, 1.84)381.69(1.06, 2.71)0.02Other California regions461.00370.94(0.59, 1.52)251.46(0.87, 2.48)0.120.41Type of residenceRural\/town\/small city351.00341.23(0.74, 2.06)231.77(1.01, 3.11)0.05Metropolitan suburban\/urban541.00651.04(0.70, 1.55)401.51(0.97, 2.37)0.050.77Statewide percentile of socioeconomic status in census block groupcLower 75% 571.00501.09(0.72, 1.67)241.35(0.81, 2.27)0.25Upper 25% 321.00491.16(0.72, 1.88)391.96(1.19, 3.24)0.0040.43Body mass index\u2264Median (23.5 kg\/m2)331.00461.31(0.81, 2.11)321.64(0.97, 2.76)0.07>Median531.00520.97(0.63, 1.49)271.48(0.89, 2.45)0.100.69Cigarette smoking statusNever661.00611.05(0.71, 1.56)361.77(1.13, 2.78)0.01Ever251.00381.19(0.69, 2.03)271.42(0.80, 2.50)0.240.57Dietary folate intake\u2264Median (307.1 \u03bcg\/day)d231.00210.73(0.37, 1.43)131.15(0.54, 2.44)0.55>Mediand211.00231.16(0.60, 2.24)101.17(0.51, 2.66)0.750.74Total folate intake\u2264Median (473.0 \u03bcg\/day)e431.00461.07(0.68, 1.70)251.34(0.78, 2.30)0.27>Mediane411.00481.20(0.77, 1.89)372.07(1.29, 3.35)0.0020.43a Adjusted for race, total energy intake, parity, oral contraceptive use, strenuous exercise, menopausal status\/hormone therapy use, and consumption of beer and liquor in the past year; stratified by age at baselineb Including peri-\/post-menopausal women onlyc See Methods for definitiond Excluding all multivitamin users (i.e., women consuming supplemental folate)e Excluding short-term multivitamin users (i.e., women consuming supplemental folate for < 2 years)\nIn secondary analyses, we examined the associations between alcohol consumption and risk of ovarian cancer among only women who were peri- or post-menopausal at baseline (46% of the study population, 75% of cases). In this group, we observed the same lack of a significant association with total alcohol, beer, or liquor intake, along with a significant positive association with baseline wine intake. Likewise, when we restricted the case population to invasive ovarian cancer (90% of cases) or to serous ovarian cancer (45% of cases), baseline intake of alcohol from wine, but not from other sources, was associated with significantly increased risk of ovarian cancer.\nDiscussion\nThe lack of association between overall alcohol consumption and risk of ovarian cancer in our study is consistent with most previous studies [3\u201318], including a pooled analysis of 10 prospective cohorts [19]. Alcohol consumption during ages 18\u201322 years, ages 30\u201335 years, or the year before baseline\u2014whether from beer, liquor, or all sources combined\u2014was unrelated to ovarian cancer development. Additionally, changes in beer, liquor, or total alcohol consumption over time, as well as frequency of drinking in the year before baseline, did not affect ovarian cancer risk in this cohort.\nIn contrast, average consumption of at least one glass per day of wine in the year before baseline was associated with elevated risk of ovarian cancer, while wine intake at ages 30\u201335 years or 18\u201322 years was associated with nonsignificantly increased risk. The positive association with baseline wine intake persisted after adjustment for total alcohol intake, suggesting that the apparent effect of wine on ovarian cancer risk was independent of alcohol content. The association was also unchanged by further adjustment for characteristics and behaviors associated with wine drinking in this cohort, and was not significantly modified by reproductive characteristics, demographic factors, or folate intake. However, there was a significant positive association between wine consumption and ovarian cancer risk among peri-\/post-menopausal women who used unopposed estrogen HT, whereas there was no such association among peri-\/post-menopausal women who did not use HT or used combined estrogen-progestin HT. Wine consumption was also associated with increased ovarian cancer risk among women of high SES, but not among women of relatively low SES.\nTo our knowledge, no other study of ovarian cancer has examined alcohol consumption at various ages or drinking patterns over time. A positive association between wine consumption and risk of ovarian cancer has previously been detected in two case\u2013control studies [15, 49], although both of those studies reported a positive association with total alcohol intake as well. In contrast, two case\u2013control studies [14, 24] and one cohort study (among women with high dietary folate intake) [17] found an inverse association between wine consumption and ovarian cancer risk. However, there was no association with wine (or total alcohol) consumption in other studies [13, 18, 19].\nFew previous studies have examined modification of the association between alcohol consumption and risk of ovarian cancer. A pooled analysis of 529,638 women, including 2,001 incident ovarian cancer cases, found no interaction between alcohol intake and oral contraceptive or HT use, parity, menopausal status, folate intake, BMI, or smoking [19]. In contrast, a case\u2013control study found that an inverse association between wine consumption and ovarian cancer risk was significantly stronger in women who were more highly educated, had never smoked, or had used oral contraceptives\u2014characteristics that describe the majority of CTS cohort members\u2014but found no heterogeneity by menopausal status, BMI, or HT use [24]. Unlike us, others reported an interaction with folate intake [16, 17, 25] or heterogeneity by histologic subtype of ovarian cancer [14, 15], although others did not [3, 12, 19, 24]. We lacked sufficient cases to perform detailed analyses of ovarian cancer histologic subtypes other than the most common serous type.\nThe restriction of the positive association between wine intake and ovarian cancer risk to peri-\/post-menopausal women taking estrogen-only HT suggests a biological mechanism involving estrogen. Likewise, the restriction of the association to women of high SES may reflect reproductive characteristics and\/or HT use favoring increased estrogen levels in higher-SES women. A high background level of circulating estrogen, compounded by an upsurge of estrogen resulting from alcohol consumption [1], may promote ovarian carcinogenesis. Furthermore, the lack of an interaction between wine intake and use of estrogen-progestin HT could be explained by a protective effect of progestin against ovarian cancer. Phytochemicals, such as resveratrol, in red wine have been considered as promising cancer preventive agents due to their anti-estrogenic, antioxidant, anti-proliferative, and other anti-carcinogenic effects [50, 51]. However, such phytochemicals have multifarious effects, including pro-estrogenic activity and possible genotoxicity [52\u201354]; thus, both the potentially beneficial and potentially harmful effects of phytochemicals on cancer development must be considered together. In the present analysis, we were not able to distinguish red from white wine intake.\nOur findings should be interpreted in light of some limitations. It is possible that the observed positive association between wine consumption and ovarian cancer risk was due to confounding by unmeasured characteristics beyond those for which we attempted to adjust, or by residual confounding or chance. Our efforts to fully evaluate effect modifiers or confounders of the association between wine consumption and ovarian cancer risk were partly hampered by the lack of heterogeneity in some characteristics among CTS participants. The proportion of women who drank beer or liquor was low, although the range of total alcohol intake was adequate to reveal a significant positive association with risk of breast cancer in an earlier analysis [37]. Even though reporting of alcohol intake in the past year using our questionnaire has been validated, we were not able to assess the reliability or validity of self-reported alcohol consumption at earlier ages, which may be difficult to recall, especially among older women. Misclassification of distant past alcohol intake may explain the absence of a significant association between wine drinking at earlier ages and ovarian cancer risk. Nevertheless, it is unlikely that any exposure misclassification differed systematically between ovarian cancer cases and non-cases, since all information was assessed prospectively.\nIn summary, given the lack of association between overall alcohol consumption and ovarian cancer risk in our study, further investigations are necessary to determine whether ingredients of wine, but not beer or liquor, foster ovarian cancer development; whether correlates of wine drinking not measured or imperfectly measured in our study population are associated with ovarian cancer risk; or if an association is largely due to the apparent interaction between wine consumption and unopposed estrogen HT use and\/or other characteristics of women of high SES. If the observed association between wine drinking and ovarian cancer risk is due to confounding, then there may exist an as-yet unidentified ovarian cancer risk factor that is also associated with wine drinking. On the other hand, if the interactions with estrogen HT use and SES are confirmed, there may be a biological basis for the increased risk of ovarian cancer among wine drinkers with high endogenous and exogenous estrogen levels. However, if alcohol consumption is indeed unrelated to ovarian cancer development but positively associated with the risk of breast cancer, then differences in the hormonal and non-hormonal triggers between these two malignancies may help us understand the carcinogenic effects of alcohol on hormonally responsive tissue. Further understanding of the complex relationships among steroid hormone levels, metabolism of alcohol and wine, and carcinogenesis will help clarify what role, if any, alcohol and wine play in the development of ovarian cancer.","keyphrases":["ovarian cancer","cohort studies","alcoholic beverages","women\u2019s health"],"prmu":["P","P","P","R"]}
{"id":"J_Urban_Health-2-2-1705540","title":"Methods to Recruit Hard-to-Reach Groups: Comparing Two Chain Referral Sampling Methods of Recruiting Injecting Drug Users Across Nine Studies in Russia and Estonia\n","text":"Evidence suggests rapid diffusion of injecting drug use and associated outbreaks of HIV among injecting drug users (IDUs) in the Russian Federation and Eastern Europe. There remains a need for research among non-treatment and community-recruited samples of IDUs to better estimate the dynamics of HIV transmission and to improve treatment and health services access. We compare two sampling methodologies \u201crespondent-driven sampling\u201d (RDS) and chain referral sampling using \u201cindigenous field workers\u201d (IFS) to investigate the relative effectiveness of RDS to reach more marginal and hard-to-reach groups and perhaps to include those with the riskiest behaviour around HIV transmission. We evaluate the relative efficiency of RDS to recruit a lower cost sample in comparison to IFS. We also provide a theoretical comparison of the two approaches. We draw upon nine community-recruited surveys of IDUs undertaken in the Russian Federation and Estonia between 2001 and 2005 that used either IFS or RDS. Sampling effects on the demographic composition and injecting risk behaviours of the samples generated are compared using multivariate analysis. Our findings suggest that RDS does not appear to recruit more marginalised sections of the IDU community nor those engaging in riskier injecting behaviours in comparison with IFS. RDS appears to have practical advantages over IFS in the implementation of fieldwork in terms of greater recruitment efficiency and safety of field workers, but at a greater cost. Further research is needed to assess how the practicalities of implementing RDS in the field compromises the requirements mandated by the theoretical guidelines of RDS for adjusting the sample estimates to obtain estimates of the wider IDU population.\nIntroduction\nEvidence suggests recent diffusion of injecting drug use and associated HIV infection in the Russian Federation since 1996.1,2 Approximately 60% of HIV case reports have been associated with injecting drug use,1,3 with recent estimates indicative of increased sexual HIV transmission.4 According to UNAIDS classifications, HIV in much of the Russian Federation and former Soviet Union is a concentrated epidemic, with prevalence consistently above 5% in a single risk group (i.e., IDUs) but less than 1% in the general population.5 Concentrated epidemics require targeted surveillance of the population group most at risk in order to track the spread within that group as well as potential transmission to others.\nSurveillance among IDUs is problematic, and there has been much discussion on the merits of different methods to recruit marginalized and hidden groups for these purposes.6\u20138 We know that surveying drug users in treatment settings misses an important segment of the drug using population. Evidence suggests that behaviours, characteristics and HIV prevalence amongst IDUs in treatment often systematically differ to IDUs not in treatment.9\u201313\nMany surveillance studies of IDUs conducted in the 1990s relied on non-probability sampling such as convenience, snowball sampling or chain referral sampling to recruit members of the target group.12,14 These methods work on the assumption that peers are better able to recruit members of a hidden population than researchers.15 Typically studies employed \u2018privileged access interviewers\u2019 or \u2018indigenous field workers\u2019 to recruit IDUs from community settings. Indigenous field workers are interviewers who are either current or former drug users or individuals who have experience working with drug users and have privileged access to IDU networks. Over the last 15\u00a0years, this has become the established sampling method for recruiting hidden populations of IDUs and sex workers both in the UK and internationally.16\u201321\nA refinement of the chain referral methodology called respondent-driven sampling (RDS), has recently been developed.22 RDS is inspired by the insight of \u201csmall world theory\u201d that suggests that every person is indirectly associated with every other person through approximately six intermediaries,23 and therefore that everyone in a defined population could be potentially reached through several waves of recruitment in a chain-referral sample.24 This implies that there is a probability greater than zero that everyone in that population will be sampled.\nThe unique selling point of RDS is that the collection of data on participants\u2019 social networks allow for adjustment for non-random recruitment. RDS uses social network data to make inferences about the wider target population from which the sample is drawn to provide proportional population estimates of characteristics and behaviours.24,25 In this paper, we do not attempt to test the statistical superiority of RDS in providing \u2018population\u2019 estimates over other sampling strategies but instead focus on RDS as a recruitment strategy examining the unadjusted RDS sample characteristics.\nThis paper compares two sampling methodologies, RDS and chain referral sampling using indigenous field workers (IFS), in terms of cost effectiveness, duration of fieldwork and effects on the demographic composition of the sample. First we offer a theoretical descriptive comparison of the two approaches.\nTheoretical Comparison of the Sampling Methods\nIndigenous Field Worker Sampling\nThe IFS recruitment method uses a standard chain referral approach. Indigenous field workers undergo training covering aims of the study, fieldwork protocols, ethics, informed consent, interview skills and safety procedures. Field workers (FWs) identify individuals known to them from IDU networks, recruit; and then interview them in community settings, separate from the rest of the research team. Eligible participants are given an incentive to take part and also asked to introduce their peers to the FW. The use of multiple site and network recruitment ensures a wide coverage of the population, providing as representative a sample as possible. There is some evidence that the use of FWs with direct access to IDU social networks facilitates recruitment and reduces masking (undersampling reclusive respondents), volunteer bias (oversampling cooperative respondents) and underreporting of socially undesirable behaviours.10,26\nRespondent-driven Sampling\nIn RDS, a fixed site or \u201cstore front\u201d is established where all interviewing takes place, providing the research team with greater control over the fieldwork. Unlike IFS and other chain referral samples, RDS uses a dual incentive system, a primary incentive for participating in the study and a secondary incentive for recruiting others into the study.22 Sampling begins with a set of initial subjects who serve as \u2018seeds\u2019 for an expanding chain of referrals, with respondents from each link in the chain or \u2018wave\u2019 referring respondents who form subsequent waves. Rather than being asked to identify their peers to interviewers, respondents inform their peers about the study and allow them to decide independently whether they want to participate or not. This theoretically reduces masking since recruiters are part of the target group with direct access to other IDUs, and it reduces volunteer bias since recruitees decide themselves whether to participate.\nInformation on the relationships between recruiters and recruited and their estimated network size is collected during the interview to allow for the calculation of selection probabilities.27 This information is used to assess homophily, the extent to which recruiters are likely to recruit individuals similar to themselves, and to weight the sample to compensate or control for differences in network size, homophily and recruitment success.24\nMaterials and Methods\nData Collection\nBetween 2001 and 2005, we undertook nine community surveys of IDUs in the Russian Federation and Estonia (Table\u00a01). Four studies used IFS to recruit IDUs, and five used RDS. All IDUs were recruited from community settings. Seven of the studies had an epidemiological focus and measured the prevalence of HIV, HCV and associated injecting and sexual risk behaviours in IDUs.13,28,29 Two of the studies collected data on the social and economic characteristics of IDUs.30 All studies collected some standardised indicators and defined current IDUs as individuals who injected drugs for non-medical purposes in the last 4\u00a0weeks.\nTable\u00a01.Research amongst hard to reach populations recruited via respondent-driven sampling (RDS) and indigenous field workers (IFS) in Estonia and the Russian Federation, 2001\u20132005\u00a0PlacePopulation  sizeDates of field work  (days)Average number  of interviews  conducted  per daySample  sizeAim of studyRecruitment  methodReference1Togliatti,Russia740,6361\/10\/2001\u201318\/10\/2001 (18)23426HIV prevalence  and risk behaviourIFS292Moscow, Russia13,251,40127\/09\/2003\u201321\/10\/2003 (27)18514HIV prevalence  and risk behaviourIFS133Volgograd, Russia\u223c1,012,00026\/09\/2003\u201323\/10\/2003 (21)21597HIV prevalence  and risk behaviourIFS134Barnaul, Russia\u223c750,00026\/09\/2003\u201330\/10\/2003 (21)24512HIV prevalence  and risk behaviourIFS135Volgograd, Russia\u223c1,012,00019\/8\/2004\u20137\/9\/2004 (20)20400Social and economic  studyRDS306Barnaul, Russia \u223c750,00026\/8\/2004\u201316\/9\/2004 (22)18400Social and economic  studyRDS307Togliatti, Russia740,63621\/05\/2004\u201309\/06\/2004 (20)24472HIV prevalence  and risk behaviourRDS348Tallinn, Estonia 396,37502\/05\/2005 (34)10350HIV prevalence  and risk behaviourRDS289Kohtla Jarve, Estonia46,34602\/05\/2005 (16)6100HIV prevalence  and risk behaviourRDS28\nFor each of the IFS studies, IDUs were recruited using a team of 10\u201312 FWs at each site. Settings included street locations and respondents\u2019 homes but excluded drug treatment centres and STI clinics. Volunteers and outreach workers at local harm reduction non-governmental organizations (NGOs) were employed as FWs, as well as two researchers at a local university in each site. In all IFS studies, two experienced supervisors from Moscow and a researcher from the UK provided technical expertise and management for all studies. Measures to ensure data quality and to minimise network bias included limiting the number of interviews per FW, random spot-checks in the field, and follow-up validation interviews with 10% of participants. Primary incentives included HIV prevention materials (including needles\/syringes), chocolates and cigarettes.\nIn each RDS study, recruitment was undertaken by a team of seven to eight FWs at each site. The interview team comprised trained research staff from a local university, two FWs recruited from local harm reduction NGOs, two to three trained research staff from a local university, and two supervisors from Moscow. A researcher from the UK was also present at the studies, with the exception of the two socio-economic studies in Volgograd and Barnaul, Russia. In each study, a pre-fieldwork focus group was held with outreach workers from the local harm reduction NGOs to obtain information about the drug scene and to identify seeds to begin recruitment. Respondents received the same primary incentives for participating in the RDS study as in the IFS study and also an additional secondary incentive for each respondent they recruited into the study.22,24\nIn all studies FWs recorded their observations on the drug scene, progress of the fieldwork and any difficulties arising from the research in detailed notes. These observations provide a useful additional comparison between the two sampling methods.\nDuration and Cost of Fieldwork\nWe compared the duration of fieldwork for the IFS and RDS methods by calculating the mean number of days of fieldwork for each method and the proportion of the sample recruited on each day as the studies progressed. Means were compared using t-tests.\nCosts were estimated for five of the seven surveys conducted in Russia and analysis focused on examining the cost effectiveness of recruiting a given sample for each of the sampling methodologies from a programmatic point of view as opposed to examining societal or health system costs. The IFS studies in Moscow, Barnaul and Volgograd were conducted in 2003 and the RDS studies in Togliatti, Barnaul and Volgograd were conducted in 2004. Costs were calculated as: (1) \u2018outside\u2019 costs including salary, accommodation and travel of field work consultants; (2) local salary costs of FWs and researchers; (3) recruitment costs including the packages of goods valued at 140 roubles and 300 roubles, respectively, for primary and secondary incentives; and (4) other costs including local transport, telephone calls and logistical costs of training FWs. For the RDS study the cost of the fixed site used for interviews is not included as an explicit cost, rather it is subsumed into the local salary costs since local staff contracted to undertake the work were employed from syringe exchange programmes. Costs are presented assuming that there are elements of fixed and variable costs at each sample size and that an extra 20 respondents will require keeping the entire survey team in the field for one extra day.\nDemographic and Injecting Risk Behaviours of Sample\nDemographic and injecting risk behaviours of IDUs recruited through IFS and RDS were compared in the two sites (Volgograd and Barnaul) where both survey methodologies were used to ensure a cleaner comparison between survey methods. Demographic and injecting characteristics were used as the outcome variables with recruitment method included as an independent variable. In the univariate analysis, chi-squared tests were used to compare outcomes for categorical variables and Bartlett\u2019s test for equal variance to compare continuous variables. For the multivariate analysis, logistic regression models were used to explore associations between explanatory variables and a binary outcome, multinomial logit models were used for categorical variables with multiple values and ordinary least squares for continuous variables. The multivariate analysis includes all common independent variables and a categorical variable indicating survey method used. This allows outcomes to be compared controlling for all independent variables and to identify impacts associated with only survey methodology. All statistical analyses were conducted using Stata 7 with significance set at 5% (Stata Corporation, College Station, Texas).\nResults\nThe Surveys\nA total of 3,771 IDUs were recruited into nine surveys across four cities in the Russian Federation and two cities in Estonia (Table\u00a01). A total of 2,049 (54%) participants were recruited through IFS and 1,722 (46%) through RDS. Only IDUs are included in the analyses we present here.\nDuration of Field Work\nThe mean (standard deviation) duration of fieldwork for IFS surveys was 23.8 (4.1)\u00a0days and for RDS 20.6 (0.9)\u00a0days (t\u2009 = \u200927.9, p\u2009<\u20090.001). Figure\u00a01 depicts the number of respondents recruited by each successive day of fieldwork by city and recruitment method. The RDS studies appear to follow a pattern of recruitment that we might expect: the number of respondents increases steadily as the number of waves increase and then declines towards the end of the study as completion of the target sample size approaches and respondents are asked to refer fewer contacts to the study. Kohtla Jarve, Estonia, appears to be an exception to this as recruitment peaks more sharply then abruptly finishes. The recruitment for the IFS studies does not appear to follow any set pattern across the cities. In Moscow the highest number of respondents in any 1\u00a0day occurs at the start of the study. In Barnaul, and to a less extent Volgograd, the number of participants recruited per day is more even across the duration of the study.\nFigure\u00a01Frequency of recruitment per day amongst studies of injecting drug users in Russia and Estonia (2001\u20132005), by city and recruitment method.\nSample Characteristics\nTable\u00a02 summarizes the characteristics of IDUs by recruitment method from the four surveys conducted in Volgograd and Barnaul. In both cities, RDS participants were younger, more likely to be male, to have attended higher education and to have official residency permits for the city. RDS participants were more likely to report injecting heroin in both sites and less likely to report injecting vint or mak1 than IFS participants. Frequency of injecting did not differ by recruitment method in either city. Regarding injecting risk behaviour, there was no difference between recruitment methods in the proportion of IDUs reporting injecting with a used needle\/syringe in the last 4\u00a0weeks in Volgograd but in Barnaul a higher proportion of RDS respondents reported this behaviour (21 vs. 15%, p\u2009 = \u20090.02). In Volgograd IFS respondents were more likely to report ever having injected with a used needle\/syringe than RDS respondents (61 vs. 40%, p\u2009 < \u20090.001). The opposite was found in Barnaul (53 vs. 63%, respectively, p\u2009 < \u20090.003). In both cities and with both methods, the main source of new needles\/syringes was pharmacies. A higher proportion of IFS respondents reported using needle\/syringe exchanges or treatment centres in both cities and in Barnaul a higher proportion of RDS respondents reported using a source other than needle\/syringe exchange (defined as friend, dealer, family or on the street) as their main source of needles\/syringes.\nTable\u00a02.Comparison of characteristics and injecting risk behaviours among injecting drug users in Volgograd and Barnaul (2003\u20132004) by recruitment method\u00a0CharacteristicBarnaulVolgogradIFRDSaIFRDSan\/total%\/mean  (SD)n\/total%\/mean  (SD)\u03c72P valuen\/total%\/mean  (SD)n\/total%\/mean  (SD)\u03c72P valueTotal\u2020 age (years)25.8 (8.2)24.8 (6.1)2.20.0324.5 (3.9)23.8 (5.4)2.30.022Male348\/50469%293\/36580%13.8<0.001388\/51276%317\/34692%35.3<0.001Attended higher education 67\/50413%136\/35339%73.1<0.001140\/50928%150\/33445%27.1<0.001Duration of injection (years)7.1 (6.9)6.7 (4.3)1.10.295.4 (3.4)6.1 (4.3)\u22122.60.009No official residency permit34\/4957%7\/3562%11.20.00119\/4714%8\/3382%1.90.174Injecting risk behaviours  in the last 4\u00a0weeksMain drug injected\u00a0\u00a0Heroin280\/50456%263\/35774%424\/50883%328\/34595%\u00a0\u00a0Vint135\/50427%38\/35711%37\/5087%7\/3452%\u00a0\u00a0Mak84\/50417%51\/35714%21\/5084%6\/3452%\u00a0\u00a0Other5\/5041%5\/3571%39<0.00126\/5085%4\/3451%27<0.001\u00a0Daily injection 100\/50020%69\/35619%0.050.82386\/51217%63\/34118%0.40.53\u00a0Injected with used  needles\/syringes 75\/50015%75\/35321%5.60.01863\/51012%37\/33611%0.350.55Main source of new  needles\/syringes\u00a0\u00a0Pharmacy466\/49894%325\/36589%463\/50891%327\/34196%\u00a0\u00a0Treatment21\/4984%12\/3653%33\/5086%6\/3412%\u00a0\u00a0Other**11\/4982%28\/3658%14.90.00112\/5082%8\/3412%10.50.005\u00a0Ever injected with used  needles\/syringes255\/48153%225\/35663%8.70.003301\/49361%137\/34540%37.1<0.001aThe RDS data are not adjusted for homophily and network effects.\u2020Numbers do not always add up to total because not all respondents answered each question.**Other refers to friends, family, found on the street, drug dealers.\nMultivariate Analysis\nTable\u00a03 summarizes the multivariate analysis for the categorical and continuous variables for Barnaul and Volgograd. After controlling for all independent variables, our findings indicate that RDS recruited a population 0.07\u00a0years younger in both cities. In both cities RDS participants had been injecting slightly longer than IFS participants. In Barnaul, RDS participants were less likely to report obtaining their new needles\/syringes from pharmacies (\u22125%) but there was no evidence to suggest a difference in Volgograd. In Volgograd RDS participants were less likely to report using needle\/syringe exchanges (\u22123%) but there was no difference in Barnaul. RDS participants in Barnaul were 4% more likely to report using another source for their new needles\/syringes (Table\u00a03).\nTable\u00a03.Effect of recruitment method on demographic indicators and injecting risk behaviours amongst injecting drug users in Barnaul and Volgograd (2003\u20132004)OutcomesRDSaType of analysisBarnaulVolgogradCoefficient95% CIStd. err.Coefficient95% CIStd. err.log AGE\u22120.07\u22120.1\u2013(\u22120.04)0.01\u22120.07\u2212.0.9\u2013(\u22120.05)0.01Ordinary least squaresYears of injecting0.1 0.04\u20130.20.040.20.1\u20130.20.04Negative binomial regressionIncrease in probability  of using pharmacies\u22125%\u22128\u2013(\u22121%)0.023%0.1%\u20135%)0.1Multinomial logitIncrease in probability  of using needle\/syringes  exchanges0.5%\u22121\u20133%0.01\u22123%\u22125%\u2013(\u22120.2%)0.01Multinomial logitIncrease in probability  of using another source  for needles\/syringes4%1\u20137%0.01n\/an\/an\/aMultinomial logitaThe RDS data are not adjusted for homophily and network effects.n\/a = not applicable.\nIn the logistic regression analysis (Table\u00a04), RDS was more likely to result in a higher proportion of male IDUs (Barnaul OR\u2009 = \u20092.0, Volgograd OR\u2009 = \u20093.8) and participants who had attended higher education (Barnaul OR\u2009 = \u20095.2, Volgograd OR\u2009 = \u20093.0). In Barnaul RDS participants were more likely to have official residency permits (OR\u2009 = \u20094.6) but not in Volgograd. In both cities, RDS participants were more likely to inject heroin over mak or vint than IFS participants (Barnaul OR\u2009 = \u20092.5 and Volgograd OR\u2009 = \u20093.4). In Volgograd RDS participants had almost twice the odds of reporting daily injection (OR\u2009 = \u20091.7) than IFS participants and had reduced odds of ever injecting with used needles\/syringes (OR\u2009 = \u20090.3). In Barnaul, RDS participants were more likely to report injecting with used needles\/syringes in the last 4\u00a0weeks and ever (OR\u2009 = \u20091.6 and OR\u2009 = \u20091.4, respectively).\nTable\u00a04.Effect of recruitment method on demographic indicators and injecting risk behaviours amongst injecting drug users in Barnaul and Volgograd (2003\u20132004)OutcomesRDSaType of analysisBarnaulVolgogradOdds ratio95% CIStd. err.Odds ratio95% CIStd. err.Male2.01.3\u20133.10.43.82.1\u20136.91.2Logistic regressionAttended higher education5.23.5\u20137.61.03.02.1\u20134.30.5Logistic regressionOfficial residency permit 4.61.8\u201312.12.31.40.5\u20134.30.8Logistic regression Injected heroin as primary drug in the last 4\u00a0weeks versus mak\/vint2.51.7\u20133.50.43.41.7\u20137.01.2Logistic regressionDaily injected0.80.5\u20131.20.21.71.04\u20132.60.4Logistic regression Injected with used needles\/syringes in the last 4\u00a0weeks1.61.0\u20132.10.41.00.6\u20131.80.3Logistic regression Ever injected with used needles\/syringes1.41.0\u20131.90.20.30.2\u20130.50.1Logistic regression Sold sex in the last 4\u00a0weeks1.40.6\u20133.00.51.80.6\u20135.41.0Logistic regressionaThe RDS data are not adjusted homophily and network effects.\nCosts\nThe total cost of conducting an IFS survey recruiting 400 respondents was estimated to be $14,651 (USD) but $16,100 for the RDS survey (Table\u00a05). This translates to $43 per respondent using RDS and $37 using the IFS method. Increasing the sample from 400 to 500 reduced the average cost per respondent by $1 for the RDS method and by $3 for the IFS method. Reducing the sample from 400 to 300 respondents increased the cost per respondent by $2 for the RDS method and $5 for the IFS method. These results are presented in Table\u00a05.\nTable\u00a05.Analysis of costs of recruiting IDUs and sex workers from five surveys in Moscow, Volgograd and Barnaul, Russian Federation (2003\u20132004) by recruitment method and sample size\u00a0Recruitment methodIndigenous field workersRespondent-driven samplingSample size300400500300400500Outside costsa\u221215.40$20.3011.40\u22120.18$19.620.18Local costsb\u221213.20$7.9310.40\u22120.25$5.500.25Recruitment of respondentsc\u221233.30$7.0020.00\u22120.25$9.950.25Other0.00$1.400.00\u22120.25$5.250.25Total\u221217.20$14,651.0012.60\u22120.21$16,1000.21Cost per respondent$42.00$37.00$34.00$43.00$41.00$40.00At the time of writing 28 rubles was equivalent to one US Dollar. The 2003 costs are adjusted for inflation that occurred between August 2003 and 2004 based on price indices taken from the Bank of Russia.aCosts are presented assuming that there are elements of fixed and variable costs in each sample size and that an extra 20 respondents will take one extra day necessitating employing the entire fieldwork team for that extra day.bIn order to protect the confidentiality of staff we report only the total amount of all salaries and fees paid to project staff.cThe costs for the RDS surveys are also based on providing a package of goods valued at 140 rubles for each survey participant and a secondary reward of 300 rubles for each participant recruited. Costs for the IFS surveys are based on providing a package of goods valued 140 rubles for each participant and no secondary reward.\nDiscussion\nOur findings suggest that RDS does appear to be a faster recruitment method and that there are significant differences in the demographic characteristics of IDUs recruited via RDS in comparison with those recruited via IFS. However, evidence from the two cities is conflicting with regard to whether RDS recruits IDUs who engage in riskier injecting practices.\nOne of the suggested benefits of RDS is its apparent ability to recruit the hardest to reach sections of hidden populations.22 We found some differences in measures of marginalization and risk behaviours between the two recruitment methods, RDS participants tend to be slightly younger and are less likely to use needle\/syringe exchange programs. Some evidence in Russia suggests that IDUs whose primary source of needles\/syringes is informal are at more risk of engaging in high-risk behaviours.31 However, IFS participants were less likely to have attended higher education and have official residency permits to live in the city and more likely to be female. Lack of residency permit is an indicator of marginalization as it will affect an individual\u2019s ability to use health services or obtain employment.32,33\nAs no consistent trend emerges from the analysis of the effect of recruitment method on sample characteristics, then choice of method might be made on the basis of methodology and cost.\nInclusion Criteria and Data Validity\nWith IFS the responsibility for selecting the right target group is placed with the FWs, and its success depends on establishing a trusting relationship between the researchers and the fieldwork team. With RDS, issues of trust are less important, as researchers undertake the interviews themselves. The problems of establishing whether respondents are genuine members of the target group remain. Although measures can be put in place which might reduce this from happening, such as using indigenous field workers to screen participants or recording biometric measurements to avoid the same respondent being interviewed twice, it is very difficult to measure to what extent fabricated data may enter a survey.\nA disadvantage of both methods is that study participants who are not members of the target group may lie about their membership in order to receive a reward. This was the case in two of the IFS sites where 9 and 14% of questionnaires were subsequently found to be fakes. This was discovered because strict validation processes had been set up and there was a good relationship between the FW supervisor and indigenous field workers. In the Togliatti RDS study 15 people were refused entry into the study, as they were suspected of not being current injectors. However these may be considered a minimum estimate as one cannot rule out that additional fabrication might have occurred and gone undetected. Having a modest primary and secondary incentive can minimize the chances that participants who are not members of the target population will be recruited.\nDetermining the best incentive size is difficult and has many implications for the study, especially for RDS studies where the secondary incentive is so crucial to recruitment success. The networks recruited through RDS are largely artificial, created as a result of the study and since their composition is dependent on the incentive, changes to the amount of incentive offered would change the composition of the network. This is illustrated with the case of sex workers in Eastern Europe, who have been found to be harder to recruit through RDS in part due to the small incentive and social network properties; this is discussed in more detail in a paper in this issue by Simic et al.\nAdjusting the RDS sample to obtain \u2018population\u2019 estimates depends on the ability to recruit a random population within a subject\u2019s social networks and a positive probability of recruiting everyone in that network. The possibility that the network is highly dependant on the incentive raises the question whether the latter condition obtains. This is particularly relevant when the definition of the population of study is fluid or artificially constructed by the research as with IDUs and sex workers. It should also be noted that the collection of information describing network characteristics which allows RDS analysis to produce \u2018population\u2019 estimates requires the respondent to recall detailed information on the composition of their network, including its size and each member\u2019s relationship with the recruiter. This process carries a large potential for error.\nPersonal Safety and Capacity Building\nThere are safety considerations that favour RDS as respondents attend a fixed site for an interview in which a minimum number of staff is always present. In the IFS method, interviewers may find themselves travelling to an area in which they are unfamiliar, and unintentionally put themselves in danger, especially if it becomes known that they are carrying financial rewards or gift packs.\nLimitations\nWhilst we have tried to limit confounding in our analysis by comparing RDS and IFS studies conducted in the same cities, the studies were conducted in different years, and the findings may be confounded by time. Time may be important in relation to behaviour, but is likely to be less important in relation to socio demographic characteristics of the target group. None of the studies were set up specifically with the aim of comparing sampling methodologies, and this limited the number of characteristics that could be compared between study methodologies. A study set up specifically with the aim of comparing the methodologies might produce different results and facilitate more detailed comparison of characteristics. Additionally the starting point for both the IFS and RDS studies in all sites was the local outreach team. This may have led to more similarities in the sample composition than would have occurred if seeds had been selected through other methods. However, according to the principles of RDS, the selection of seeds does not ultimately influence the composition of the sample, since after several waves of recruitment the sample should be independent of the non-randomly selected seeds.24\nConclusion\nThe HIV epidemic is driven by populations engaging in high-risk behaviours mixing with those engaging in lower risk behaviours. It is important to identify the parameters of risk behaviour in order to model these epidemics and to design appropriate interventions. If we assume that, after adjusting for network sizes and homophily, RDS is more successful at estimating risk behaviours across a more representative population than IFS, then it could lead to more effective modelling and prediction of such epidemics; however to date there is no evidence to suggest that this is the case. Our findings indicate that as a recruitment strategy, RDS is no better than IFS in identifying populations with highest risk behaviours. It does have practical advantages in terms of safety of the FW team, with faster recruitment at only additional costs. In the meantime, until the statistical superiority of RDS can be proven, a preferred approach may be to adopt the best aspects of both methodologies, depending on the resources available. A combination could include the use of coupons for recruitment, but also training indigenous field workers to work alongside researchers to undertake interviews, serving to increase their capacity in research skills whilst ensuring that the correct target group is being reached.","keyphrases":["injecting drug users","estonia","hiv","respondent-driven sampling","indigenous field workers","russia."],"prmu":["P","P","P","P","P","P"]}
{"id":"Skeletal_Radiol-3-1-2042032","title":"Are radiographs needed when MR imaging is performed for non-acute knee symptoms in patients younger than 45 years of age?\n","text":"Objective The objective was to determine the value of radiographs in young adults with non-acute knee symptoms who are scheduled for magnetic resonance imaging (MRI).\nIntroduction\nRadiographs in patients with acute knee problems that are obtained at, or close to, the time of trauma serve mainly to exclude fractures and most of these radiographs are normal. To reduce the total number, and especially the number of normal radiographs, clinical decision rules that identify patients with a high risk of fracture have been developed and validated [2\u20134]. In patients with non-acute knee symptoms radiographs are the mandatory minimal initial exam in patients with non-traumatic knee pain, according to ACR appropriateness criteria [5]. Magnetic resonance imaging (MRI) is frequently obtained if additional imaging is clinically needed to search for suspected intra-articular abnormalities. The value of radiographs relative to MRI in a heterogeneous group of patients was recently reported [6]. Radiographs were rated \u201cessential\u201d or \u201cvery important\u201d in 46\u201358% of cases and added \u201cnot needed information\u201d in 14\u201329% of cases. However, the value of radiographs relative to MRI in young adults presenting with non-acute knee symptoms has, to our knowledge, not been established. If in these patients radiographs could be omitted, or obtained only when needed when reading MRI, this could possibly lead to a significant reduction of the volume of knee radiographs with ensuing reduction in radiation dose and costs.\nThe purpose of our prospective study was to determine the value of radiographs in young patients with non-acute knee symptoms and suspected intra-articular pathology who are scheduled for MRI based on the results of a physical exam. We therefore determined the prevalence of osseous abnormalities visible on radiographs and MRI and the yield of MRI compared with that of radiographs. In addition, we measured the radiation dose of standard radiographs of the knee.\nMaterial and methods\nOver 3\u00a0years, 961 patients referred from general practitioners to the outpatient surgery or orthopedic surgery clinics of three hospitals (two general hospitals and one university hospital) because of non-acute knee symptoms agreed to participate in our prospective study. The symptoms had to have been present for at least 4\u00a0weeks. The institutional review board of each hospital approved the study. We obtained written informed consent from all patients. At study entry a standardized history, including inventory of traumatic events and other knee-related events such as morbidity and medical procedures was taken. Physical exam was performed by one of 15 (orthopedic) surgeons or by residents under their supervision. All the patients included underwent MRI and radiographs of the knee. Recent radiographs taken for the same complaints that were obtained prior to study inclusion were not repeated.\nWe included patients who met the following inclusion criteria: knee symptoms (pain, swelling of the joint, feeling of instability or giving way, history of locking) of at least 4\u00a0weeks\u2019 duration and age between 16 and 45\u00a0years. To obtain a homogenous study population without co-morbidity we did not include patients who had had previous surgery of the affected knee (including arthroscopy) or previous MRI, a history of rheumatoid arthritis, and clinical diagnosis of retropatellar chondromalacia, contra-indication for MRI, or an incomplete data set. One hundred and sixty-three patients (17.0%) were eventually excluded (Table\u00a01), leaving 798 patients as our study group. Of these excluded patients a substantial number should not have been included by the orthopedic surgeons in the first place according to our study protocol, for instance, because they were either too young, too old or had knee complaints of less than 4\u00a0weeks\u2019 duration.\nTable\u00a01Excluded patients. Of the 961 patients referred, 163 (17.0%) were excludedExclusion criterianSymptoms of less than 4\u00a0weeks\u2019 duration65Aged under 16\u00a0years1Aged over 45\u00a0years6Previous surgery8Retropatellar chondromalacia14Previous MRI1Radiographs unavailable or not obtained65MRI or arthroscopy prohibited2Immediate arthroscopy required30Criteria are not mutually exclusive.\nOne out of six musculoskeletal radiologists (all with at least 5\u00a0years\u2019 experience) evaluated the radiographs without knowledge of the MR images and recorded their findings on a Case Record Form (CRF).\nThe radiation dose of standard antero-posterior and lateral radiographs was determined in two of the three hospitals using flat ionization chambers. We used PCXMC software (STUK, Finland) to convert the measured radiation doses into effective doses.\nThe radiologist evaluated the radiographs for articular surface lesions, using the classification developed by Bohndorf [7]. Osteoarthritis was scored using the scoring system developed by Kellgren et al. [1]. In the analysis, grades 1 to 3 were considered as one group, with osteoarthritis being present.\nRadiographs were also evaluated for fractures [8\u201310], loose bodies, and incidental findings. A loose body was defined as a radiodense circumscriptive lesion located in the femorotibial, patellofemoral jointline or suprapatellar pouch. Incidental findings, such as non-ossifying fibromas and enchondromas were diagnosed using established criteria [11, 12]. On radiographs a lesion was considered a non-ossifying fibroma if it met the following criteria: asymptomatic, cortical metaphyseal location, and radiographically non-aggressive. A lesion was considered an enchondroma if it met the following criteria: central metaphyseal location, chondroid matrix, geographic pattern of destruction with or without sclerotic margin.\nWe performed the MR studies in all three hospitals on an identical 0.5-T system (Gyroscan T5, Philips Medical Systems, Best, the Netherlands), with the same software release using a dedicated transmit\u2013receive knee coil.\nThe standardized scanning protocol consisted of three sequences: a sagittal and a coronal dual spin-echo (SE) sequence and a sagittal T1-weighted 3D gradient-echo (GE) sequence with frequency selective fat suppression. The following parameters were identical for both SE sequences: field of view 140\u2013160\u00a0mm, echo time (TE) 20\/80\u00a0ms. The coronal dual SE had a repetition time (TR) of 2,100\u00a0ms, a 256\u2009\u00d7\u2009205 matrix and a slice thickness of 5\u00a0mm with a 0.5-mm interslice gap. The sagittal dual SE had a TR of 2,350\u00a0ms, a 256\u2009\u00d7\u2009179 matrix and a slice thickness of 4\u00a0mm with a 0.4-mm interslice gap. The parameters for the sagittal frequency selective fat-suppressed T1-weighted 3D GE were: TR 70\u00a0ms, TE 13\u00a0ms, 45\u00b0 flip angle, field of view 160\u00a0mm, 256\u2009\u00d7\u2009205 matrix, 4\u00a0mm slice thickness with a 2-mm overlap.\nThe total imaging time of the standard protocol (including the initial survey sequence) was 26\u00a0min.\nAfter reading the radiographs one out of six radiologists evaluated MR images and reported his findings on a CRF. In accordance with usual management the observer had access to the radiographs, but not to the reports, when evaluating MR images. The CRF was similar to the one used for radiographs [8\u201312], except that bone marrow edema was added as an item [13]. For articular surface lesions we again used the classification developed by Bohndorf [7]. For osteoarthritis we used the scoring system developed by Recht et al. [14]. For the purpose of this study, grades 1 to 4 were considered as one group, with osteoarthritis being present.\nTo overcome bias in the reading of MR studies, introduced by knowledge of the results of conventional radiographs, one radiologist (JLB) re-evaluated MR studies of patients with abnormalities on conventional radiographs with the exception of osteoarthritis of Kellgren [1] grades 1\u20132. We used the same CRF format. At this second evaluation, MR studies of patients with osseous abnormalities on conventional radiographs were mixed with age- and gender-matched MR studies of patients without abnormalities on conventional radiographs. The radiographs and the findings on the CRF were not available at that time.\nWe divided the completed CRFs over two groups: group A had no history of trauma, and group B had a history of trauma of more than 4\u00a0weeks before presentation at the hospital.\nWe used the Chi-squared test to identify significant differences between the yield of radiographic and MR diagnoses, and between the number of findings in group A and that in group B. To look for statistically significant differences between the yield of radiographs and MRI within groups A, B and the study population as a whole, we used McNemar\u2019s Chi-squared test.\nResults\nThe median age of the 798 patients included was 31\u00a0years (range 16\u201345\u00a0years), and 261 patients (32.7%) were female. Minimal and median duration of symptoms were 4 and 20\u00a0weeks respectively (range 4\u20131,490\u00a0weeks). Three hundred and thirty-two patients reported no history of trauma (group A), and 466 patients reported an old (more than 4\u00a0weeks prior to consultation) history of trauma (group B).\nMagnetic resonance imaging depicted arthroscopically treatable intra-articular pathology in 341 patients (42.7%). Medial and lateral meniscal tears were found in 225 (28.2%) and 111 patients (13.9%) respectively. The average radiation dose of supine antero-posterior and lateral radiographs was 0.2 and 0.3\u00a0mSv respectively. The median duration of the interval between radiographs and MRI was 8\u00a0days (95 percentile, 85\u00a0days). In 196 patients (24.6%) radiographs were obtained prior to inclusion in the study. These radiographs were not repeated since the knee complaints had not changed between the moment they were obtained and inclusion in this study.\nIn group A, radiographs showed 36 osseous abnormalities in 332 patients (10.8%, Table\u00a02). All these abnormalities, except 13 Kellgren [1] grade 1 osteoarthritis cases, were also depicted on MRI. There were 23 concordant radiographic and MRI diagnoses (Fig.\u00a03; Table\u00a02). One incidental finding (enchondroma) was demonstrated on radiographs and MRI. The majority of the 72 diagnoses made only with MRI were osteoarthritis and bone bruising (Table\u00a02). Most osseous lesions (osteoarthritis, articular surface lesions, loose bodies and bone marrow edema) with the exception of incidental findings (one case) and old fractures (not present in this group) were significantly more often diagnosed with MRI than with radiographs (Table\u00a02).\nTable\u00a02Osseous abnormalities detected on radiographs only, on radiographs and MRI, and on MRI only in 332 patients with non-acute non-traumatic knee complaintsOsseous abnormalityR (%)R and MR (%)MRI (%)Total (%)McNemar test, p valueOA13 (3.9)18 (5.4)39 (11.7)70 (21.1)0.00ASL0 (0)3 (0.9)7 (2.1)10 (3.0)0.02LB0 (0)1 (0.3)6 (1.8)7 (2.1)0.03Fractures0 (0)0 (0)0 (0)0 (0)NAIncidental finding0 (0)1 (0.3)0 (0.0)1 (0.3)1.00Bone marrow edema0 (0)0 (0)20 (6.0)20 (6.0)NA Total13 (3.9)23 (6.9)72 (21.7)108 (32.5)0.00OA: osteoarthritis grade 1 or higher according to Kellgren [1] on radiographs and according to Recht [14] on MRI ; ASL: articular surface lesion with or without disrupted cartilage; LB: loose body; R: abnormality detected with radiographs only; R and MR: abnormality detected with both radiograph and MRI; MRI: abnormality detected with MRI only; NA: not applicable. Categories are not mutually exclusive.\nIn group B, radiographs showed 40 osseous abnormalities in 466 patients (8.6%, Table\u00a03). All these abnormalities, except 15 Kellgren [1] grade 1 osteoarthritis cases, were also depicted on MRI. There were 25 concordant radiographic and MRI diagnoses (Figs.\u00a01, 4, 5 and 6; Table\u00a03). Four of the six incidental findings were diagnosed on radiographs and MRI (1 non-ossifying fibroma and 3 enchondromas, Fig.\u00a07). Two enchondromas were depicted on MRI only (Fig.\u00a08). The majority of the 192 diagnoses made only with MRI were bone marrow edema (Fig.\u00a02), osteoarthritis and articular surface lesions (Table\u00a03). These three diagnoses were significantly more often diagnosed with MRI than with radiographs (Table\u00a03). There was no significant difference between MRI and radiographs in the diagnosis of loose bodies, old fractures and incidental findings.\nTable\u00a03Osseous abnormalities detected on radiographs only, on radiographs and MRI, and on MRI only in 466 patients with non-acute traumatic knee complaintsOsseous abnormalityR (%)R and MR (%)MRI (%)Total (%)McNemar test, p valueOA15 (3.2)13 (2.8)65 (13.9)93 (20.0)0.00ASL0 (0)3 (0.6)22 (4.7)25 (5.4)0.00LB0 (0)1 (0.2)4 (0.9)5 (1.1)0.13Fractures0 (0)4 (0.9)3 (0.6)7 (1.5)0.25Incidental finding0 (0)4 (0.9)2 (0.4)6 (1.3)0.5Bone marrow edema0 (0)0 (0)96 (20.6)96 (20.6)NA Total15 (3.2)25 (5.4)192 (41.2)232 (49.8)0.00Categories are not mutually exclusive.Fig.\u00a01Severe osteoarthritis on both radiographs and MRI. a, b Lateral and antero-posterior radiographs showing deformation of the femoral condyles and tibia plateau with marked subchondral cyst formation and osteophyte formation in a 44-year-old female patient with a history of knee trauma (group B). c\u2013e Coronal proton density and T2-weighted spin-echo (SE) images and of the same lesion showing, in addition to the deformities and changes already noted on the radiographs, the osteochondral defect to a better extent. e Sagittal T2-weighted SE image showing the same features, but also focal full-thickness cartilage lossFig.\u00a02Three-month-old transchondral fracture on both radiographs and MRI. a, b Antero-posterior and lateral radiograph showing irregularity and deformation of the lateral femoral condyle consistent with a transchondral fracture of the lateral femoral condyle in a 40-year-old male with a history of knee trauma (group B). c\u2013d Sagittal T2-weighted SE images and T1-weighted 3D gradient-echo (GE) with fat suppression showing subtle contour deformity of the lateral femoral condyle with kissing bone bruises in the lateral femoral condyle and tibial plateau\nOsseous abnormalities were more frequently encountered in group B than in group A (p\u2009<\u20090.001). This difference is explained by a significant difference in the number of old fractures (p\u2009<\u20090.05, no old fractures were seen in group A), and patients with bone marrow edema (p\u2009<\u20090.001).\nBone marrow edema was demonstrated with MRI in 116 patients (14.5%, Table\u00a04); 96 of these were found in group B (82.8% of patients with bone marrow edema, Table\u00a03).\nTable\u00a04Osseous abnormalities detected on radiographs only, on radiographs and MRI, and on MRI only in 798 patients with non-acute knee complaintsOsseous abnormalityR (%)R and MRI (%)MRI (%)Total (%)McNemar test, p valueOA28 (3.5)31 (3.9)104 (13.0)163 (20.4)0.00ASL0 (0)6 (0.8)29 (3.6)35 (4.4)0.00LB0 (0)2 0.3)10 (1.3)12 (1.5)0.00Fractures0 (0)4 (0.53 (0.4)7 (0.9)0.25Incidental finding0 (0)5(0.6)2 (0.3)7 (0.9)0.5Bone marrow edema0 (0)0 (0)96 (12.0)96 (12.0)NATotal28 (3.5)48 (6.0)264 (33.1)340 (42.6)0.00Categories are not mutually exclusive.\nIn 6 (30%) of the 20 group A patients with bone marrow edema, it was either associated with intra-articular damage of non-osseous origin or it was an isolated finding. The associated osseous abnormalities in the other 14 patients were as follows: bone marrow edema was found in 4 of the 10 patients with articular surface lesions, in 1 of the 7 patients with a loose body, and in 9 of the 70 patients with osteoarthritis.\nIn 89 (92.7%) of the 96 group B patients with bone marrow edema, it was either associated with intra-articular damage of non-osseous origin or it was an isolated finding. The associated osseous abnormalities in the other 7 patients were as follows: bone marrow edema was found in 4 of the 25 patients with articular surface lesions (Fig.\u00a02), in 1 of the 5 patients with a loose body and in 2 of the 93 patients with osteoarthritis.\nIn our population we did not find any other osseous lesions like osteopenia or femoropatellar disease. Also, we did not find chondrocalcinosis on radiographs.\nSubsequently, all patients with radiographically detected abnormalities with the exception of Kellgren [1] grade 1 and 2 osteoarthritis were identified. MRI studies of the 17 patients with 18 radiographically detected osseous abnormalities were mixed with 17 gender- and age-matched MRI studies of patients without abnormalities on radiographs. There were 6 females in each group, and the mean age in each group was 33.1\u00a0years, SD 7.9\u00a0years). At revision, the reader (JLB) was blinded to all information and had no access to the radiographs. All 18 osseous abnormalities (1 caae of osteoarthritis Kellgren grade 3 [Fig.\u00a01], 6 articular surface lesions [Figs.\u00a02, 3], 2 loose bodies [Fig.\u00a04], 4 old healed fractures [Figs.\u00a05, 6] and 5 incidental findings [4 enchondromas and 1 non-ossifying fibroma, Figs.\u00a07, 8]) were all diagnosed on MRI.\nFig.\u00a03Twenty-six-month-old transchondral fracture on both radiographs and MRI. a Antero-posterior radiograph showing abnormal density, subtle irregularity and deformation of the medial femoral condyle in a 35-year-old male patient with no history of knee trauma (group A). b\u2013d Sagittal T1-weighted 3D GE with fat suppression and coronal proton density and T2-weighted SE images of this knee showing the transchondral fractureFig.\u00a04Loose body on both radiography and MRI. a Lateral radiograph showing a ventrally located loose body in the left femorotibial joint in an 18-year-old male professional skater with a history of knee trauma (group B). b\u2013c Sagittal T1-weighted 3D GE with fat suppression and coronal proton density SE images of the same patient, also showing the loose body that is ventrally located in the lateral compartment of the femorotibial joint. At subsequent arthroscopy this loose body was removedFig.\u00a05Eight-month-old healed fracture on both radiograph and MRI. a Lateral radiograph showing a healed fracture of the inferior patellar pole in an 18-year-old male patient with a history of knee trauma (group B). b Sagittal proton density SE image of the same patient, also delineating the healed fracture of the inferior pole of the patellaFig.\u00a06Two-month-old healing fracture on both radiograph and MRI. a Lateral radiograph showing a healing fracture of the left inferior patellar pole in a 40-year-old male patient with a history of knee trauma (group B). b, c Sagittal T1-weighted 3D GE with fat suppression and T2-weighted SE images of the same patient, also delineating the healing fracture of the inferior pole of the patella without cartilaginous irregularities. Around the fracture line bone marrow edema can be appreciatedFig.\u00a07Incidental finding depicted on both radiographs and MRI. a, b Lateral and antero-posterior radiographs showing a central, metaphyseally located lesion with chondroid matrix consistent with enchondroma in the femur in a 44-year-old female patient with a history of knee trauma (group B). c, d Sagittal proton density and T2-weighted SE images of the same lesionFig.\u00a08Incidental finding depicted on MRI only. a Lateral radiograph showing no femoral osseous lesion in a 40-year-old female patient with a history of knee trauma (group B). b, c Sagittal proton density and T2-weighted SE images showing a metaphyseal lesion consistent with an enchondroma\nDiscussion\nKey characteristics of our population such as age, gender, clinical suspicion of intra-articular abnormalities, intra-articular abnormalities detected with MRI, and history suggest that our conclusions can be applied to typical populations scheduled for MRI to analyze non-acute knee problems.\nThe yield of radiographs in patients with subacute or chronic knee symptoms who are otherwise healthy and who are scheduled for MRI based on the results of a physical exam suggesting the presence of intra-articular damage is only 9.5% (76 patients). The majority of these patients (77.6%) have osteoarthritis. We failed to confirm with MRI the radiographic diagnosis of Kellgren [1] grade 1 osteoarthritis in 28 patients, but none of the other abnormalities were diagnosed only with radiographs. This was confirmed in a second reading of MRI blinded to clinical and radiographic information. Radiographs do not, in the setting of this study, contribute to clinical decision-making or clinical management of the patient. Although the dose administered when obtaining radiographs of the knee is small, the size of the population with knee complaints make the reduction of an administered dose a relevant issue. Radiographs can therefore be omitted in this specific population when the decision to perform MRI is taken based on clinical assessment. The disadvantage of occasionally missing Kellgren [1] grade 1 osteoarthritis is limited when we realize that because of the lack of a gold standard these radiographic diagnoses may also be false-positive.\nTaljanovic et al. [6] rated radiographs as \u201cessential\u201d or \u201cvery important\u201d in 46\u201358% of cases and radiographs added \u201cnot needed information\u201d in 14\u201329% of cases. They conclude in their study that radiographs are an important, and sometimes essential initial, complementary study for reading musculoskeletal MR examinations. This apparently different conclusion can be explained by the differences in study populations. Taljanovic et al. [6] analyzed a large variety of diagnosis (trauma, infection, tumors, degenerative or miscellaneous) in all anatomical locations in patients of all ages. We only analyzed patients between 16 and 45\u00a0years of age with non-acute knee symptoms lasting at least 4\u00a0weeks, excluding amongst others patients with acute knee symptoms (i.e., less than 4\u00a0weeks), history of rheumatoid arthritis, and clinical diagnosis of retropatellar chondromalacia. Other major methodological differences are mainly related to this difference.\nThe concordance between radiographs and MRI is not high because of the large number of diagnoses (77.6%) made only with MRI. In groups A and B osteoarthritis, articular surface lesions, and bone marrow edema were significantly more often diagnosed with MRI than with radiographs. Loose bodies were significantly more frequently diagnosed with MRI in group A only. This higher yield of MRI is no surprise, since abnormalities such as articular surface lesions and bone marrow edema are known to be better, or even exclusively appreciated on MRI. These findings have, in addition to the intra-articular abnormalities detected with MRI, potential clinical significance in view of the patients\u2019 presenting symptoms.\nThe advantage and comprehensiveness of MRI is illustrated by its ability to demonstrate bone marrow edema that is associated with other abnormalities. In the patients with a history of old trauma, bone marrow edema was mainly (92.7%) associated with intra-articular damage. In the group with no history of trauma, however, bone marrow edema was mainly (70%) seen in association with osseous abnormalities, thus facilitating the diagnosis of articular surface lesions and osteoarthritis.\nThis prospective study has several limitations that are mainly related to the decision to follow usual care. This means that radiographs were available at the time of MRI. To overcome this limitation, one radiologist (JLB) re-evaluated MR studies of patients with abnormalities on conventional radiographs without knowledge of the radiographs, as described.\nAnother limitation is that we could not determine accuracy because of the absence of a reference standard. If we look at the discordant cases, there are no potentially false-negative MRI diagnoses (with the exception of Kellgren [1] grade 1 osteoarthritis), but the number of potential false-positive MRI diagnoses (abnormal finding on MRI in combination with a normal radiograph) is substantial, mainly because of the high prevalence of bone marrow edema.\nAnother possible limitation of this study is the selection bias introduced by including only patients between 16 and 45\u00a0years of age and excluding patients with co-morbidity. The study design was not tailored toward assessment of the value of conventional radiographs only. The overall goal of this multicenter study was to evaluate the efficacy of MRI compared with direct arthroscopy in patients with subacute knee complaints, based on clinical outcome. To this end we tried to obtain a homogenous study population without co-morbidity. This is most likely the reason we encountered only 1 patient with high-grade osteoarthritis (Kellgren [1] grade 3). However, because the majority of MR examinations performed in our institutions will be of patients in this age range without known co-morbidity, we do not think this selection bias will alter our overall conclusions.\nWe tried to mimic usual care as much as possible in this study. However, in our study MRI was performed within 2\u00a0weeks of inclusion in the study. Due to the limited availability of MRI compared with conventional radiographs in most hospitals, MRI often cannot be performed at such short notice. This can lead to a considerable waiting time for MR examinations and this may direct physicians to prior conventional radiographs to rule out gross pathology and reassure the patient. To prevent these unnecessary radiographs, the waiting time for MRI must be as short as possible. In the Netherlands waiting times for MRI in most institutions are down from several months a couple of years ago to several weeks nowadays.\nWe conclude that radiographs should not be obtained routinely when MRI is being performed in non-acute young patients with knee complaints because the number of clinically relevant lesions detected on radiographs is low. In addition, the small number of abnormalities found on radiographs is, with the exception of an occasional case of Kellgren [1] grade 1 osteoarthritis, also depicted on MRI. Reduction of costs and radiation dose is small in the individual patient, but, because of its size, substantial in the entire population with subacute and chronic complaints suspected to be knee injury.","keyphrases":["radiographs","knee","magnetic resonance imaging"],"prmu":["P","P","P"]}
{"id":"Int_Arch_Occup_Environ_Health-3-1-1915641","title":"Work-related physical and psychosocial risk factors for sick leave in patients with neck or upper extremity complaints\n","text":"Objectives To study work-related physical and psychosocial risk factors for sick leave among patients who have visited their general practitioner for neck or upper extremity complaints.\nIntroduction\nMany people suffer from work-related neck and upper extremity complaints. In a population-based study in the Netherlands, the 12-month prevalence of work-related neck and upper extremity symptoms was found to be 31% (Blatter and Bongers 1999). These complaints have an important impact on loss of time from work. Sick leave has enormous cost implications, considering costs to employers, insurers, health care providers, and patients themselves. In the Netherlands, 8% of the employed population reported lost days from work in the past year because of complaints at the neck, shoulder, arm, elbow, wrist or hand (Blatter et al. 2001). The results of a British national survey in 1995 showed that musculoskeletal disorders of the neck and upper extremity were responsible for the loss of 4.2 million working days in a 12-month period (Jones et al. 1998). Borghouts et al. (1999) estimated the total number of sick days due to neck pain in 1996 in the Netherlands to be 1.4 million, with a total cost of US$ 185.4 million.\nIt is well recognized that neck and upper extremity complaints have a multi-factorial aetiology. Several reviews have identified individual characteristics, and (work-related) physical and psychosocial factors as risk factors for the onset of neck and upper extremity complaints (Bernard 1997; Bongers et al. 2002a, b; Malchaire et al. 2001; Muggleton et al. 1999; van der Windt et al. 2000; Zakaria et al 2002). Furthermore, these factors have also been recognized as potential prognostic factors for chronic pain and disability in patients with neck or upper extremity complaints (Ari\u00ebns et al. 2000, 2001; Cole and Hudak 1996; Eriksen et al. 1999; Feuerstein et al. 2000). However, relatively little is known about the association of work-related factors with sick leave in patients with neck or upper extremity complaints. Risk factors for the onset of neck and upper extremity complaints may differ from risk factors for sick leave due to these complaints (IJzelenberg et al. 2004). Ekberg and Wildhagen (1996) studied the impact of physical workload, work organization, psychosocial conditions and individual characteristics on days of sick leave after rehabilitation in patients with neck and shoulder disorders. They found two work-related factors (i.e., work content and uncomfortable sitting) to be independently related to sick leave days. A prospective cohort study on work-related determinants of sick leave in employees without neck pain at baseline suggested that work-related neck flexion and rotation, sitting, decision authority and skill discretion were independently related to sick leave due to neck pain (Ari\u00ebns et al. 2002). In contrast to these findings, a recent cross-sectional study on risk factors for musculoskeletal sick leave showed that physical workload nor psychosocial workload were significantly related to sick leave due to neck or upper extremity pain (IJzelenberg et al. 2004). Hansson and Jensen (2004) reviewed the literature on the causes for back and neck disorders and found that heavy physical workload, bent or twisted working position and low work satisfaction increased the risk of short-term and long-term sick leave, and self-reported pain and functional impairments were associated with a high risk for long-term sick leave.\nExposure to work-related physical risk factors may lead to pain and disability, which in turn may lead to sick leave. Psychosocial exposures at work may lead to psychological distress that may cause physiologic changes in the neck and upper extremity (e.g., increased muscle tension (Hagg and Astrom 1997; Lundberg 2002; Rissen et al. 2000), causing pain and disability, which in turn may lead to sick leave. Furthermore, psychosocial exposures at work may modify the effect of physical workload on sick leave. The objective of our study was to determine the influence of work-related physical and psychosocial risk factors on sick leave due to neck or upper extremity complaints in patients who have visited their general practitioner (GP) for these complaints.\nMethods\nDesign and study population\nThis study is part of a large observational cohort study on musculoskeletal disorders conducted in 61 general practices (97 GPs) (van der Waal et al. 2003). Part of the GPs participated in the second Dutch National Survey of General Practice (NS2), carried out by the Netherlands Institute for Health Services Research in co-operation with the National Information Network of General Practice in 2001 (Schellevis et al. 2003). The Medical Ethics Committee of the VU University Medical Center approved our study. Written informed consent was obtained from all patients. Details of the study design are described elsewhere (van der Waal et al 2003).\nOver a period of 1\u00a0year the GPs recruited 727 patients with a new complaint or new episode of a complaint at the neck, shoulder, elbow, arm, wrist or hand. An episode was considered to be \u2018new\u2019 if patients had not visited their GP for the same complaint during the preceding 3\u00a0months. Patients were eligible for participation if they were 18\u00a0years or older and capable of filling in Dutch questionnaires. Patients were excluded from the study if the presented symptoms were presumably caused by a fracture, malignancy, prosthesis, amputation or congenital defect or if the patient was pregnant.\nAt baseline a self-report questionnaire was sent to collect data on a broad range of factors (including work-related factors) that may be predictive of sick leave. In total, 643 patients (88%) returned the baseline questionnaire, of whom five patients were excluded (two were too young; three were pregnant). For our study, patients who reported to have paid work for more than 8\u00a0h per week were included (8\u201316\u00a0h: n\u00a0=\u00a041; 17\u201324\u00a0h: n\u00a0=\u00a069; 25\u201336\u00a0h: n\u00a0=\u00a0101; >36\u00a0h: n\u00a0=\u00a0172). Outcome (sick leave due to neck and\/or upper extremity complaints) was assessed after 3\u00a0months using a self-report questionnaire.\nOutcome measure\nSick leave was measured at 3\u00a0months after baseline by the question: \u201chow long were you absent from work because of your complaint since the baseline questionnaire\u201d. Response categories were (1) no sick leave; (2) less than 1\u00a0week; (3) between 1 and 2\u00a0weeks; (4) between 2\u00a0weeks and 1\u00a0month; (5) between 1 and 3\u00a0months; (6) more than 3\u00a0months. Sick leave was dichotomised into no sick leave (response category 1) and sick leave (response categories 2\u20136).\nDeterminants\nAt baseline, work-related physical risk factors were assessed by a questionnaire consisting of two scales \u201cheavy physical workload\u201d [12 items; e.g., does your work involve (a) standing for long periods of time, (b) walking for long periods of time, (c) moving loads, (d) physical hard work] and \u201clong-lasting postures and repetitive movements\u201d [six items; e.g., does your work involve (a) making the same movement for long periods of time; (b) bending or twisting your neck often; (c) working in the same position for long periods of time; (d) doing repetitive tasks with arms, hands or fingers many times per minute) (Bot et al. 2004). Response options varied from \u201cseldom or never\u201d, \u201csometimes\u201d, \u201coften\u201d, and \u201c(almost) always\u201d. Scoring was done by simply adding up the response to each item, which produced a raw score from 0 to 36 for the first subscale, and 0 to 18 for the second subscale. The final scores were calculated by dividing the raw score by the maximum score, multiplied by 100, resulting in a final score ranging between 0 (no workload) and 100 (highest workload) for both subscales. The internal consistency and validity of this questionnaire were considered to be good in a population with upper extremity and lower extremity musculoskeletal disorders (Bot et al. 2004). Furthermore, two questions about \u201cprolonged sitting\u201d and \u201c working with visual display units (VDU) for long periods of time\u201d (yes\/no) were asked.\nWork-related psychosocial risk factors were assessed according to the Demands\u2013Control model of Karasek (1979). We used three subscales of the Job Content Questionnaire (JCQ): \u201cjob demands\u201d, \u201cdecision authority\u201d and \u201cco-worker support\u201d and one single item on \u201cjob security\u201d (Karasek et al. 1998). The hypothesis of the Demands\u2013Control model is that the most adverse reactions of psychological strain occur when demands are high and the worker\u2019s decision latitude is low (Karasek et al. 1998). Therefore, we decided to combine the subscales high job demands and low decision authority into an additional determinant \u201cjob strain\u201d. Both subscales were dichotomized by their median score and four categories were created: (1) high decision authority and low job demands (reference category); (2) high decision authority and high job demands; (3) low decision authority and low job demands; (4) low decision authority and high job demands.\nStatistical analysis\nDescriptive statistics (e.g., means and standard deviations) were used to present baseline characteristics of the study population. Differences between patients reporting sick leave and patients without sick leave were tested with the unpaired Student T-test (continuous variables) and the Chi square (\u03c72) test (categorical and dichotomous variables). In case a continuous variable was not normally distributed the Mann\u2013Whitney test was used.\nInstead of using logistic regression analysis, Cox regression models with equal survival time were used to investigate the association between work-related risk factors (determinants) and sick leave (outcome). Hazard ratios are more easy to interpret and provide a better estimate of relative risks than odds ratios (Barros and Hirakata 2003; Lee and Chia 1994; Skov et al. 1998; Thompson et al. 1998) Associations were expressed as hazard ratios (HR) and corresponding 95% confidence intervals (95% CI) per unit increase of the risk factor involved. For continuous factors the linearity of the relation between the risk factor and sick leave was examined. Factors that were found to be non-linearly related to sick leave were either dichotomized or divided into tertiles with equally sized groups (Table\u00a01). The \u201clow-category\u201d served as the reference category in all analyses.\nTable\u00a01Potential confounders of the association between work-related risk factors and sick leave in patients with neck or upper extremity complaintsPotential confoundersCategorizationIndividual factors\u00a0AgeContinuous (per year)\u00a0SexMale versus female\u00a0SmokingPresent versus previous smoker\u00a0EducationPrimary versus secondary; college\/university\u00a0Pain copinga: retreating (seven items)Continuous (scale 7\u201328)\u00a0Pain copinga: worrying (nine items)Continuous (scale 9\u201336)\u00a0Distress (six items)Continuous (scale 1\u20139)General health\u00a0ACSM position standcNorm not met versus norm met\u00a0Norm healthy activityNorm not met versus norm met\u00a0Perceived healthdContinuous (scale 1\u20135)\u00a0Vitalityd (four items)Continuous (scale 0\u2013100)\u00a0Quality of lifeContinuous (scale 1\u20135)Characteristics of the complaint\u00a0Kind of complaintLocalized versus generalized\u00a0Duration of the complaintCategorical (<1, 1\u20134\u00a0weeks; 1\u20136\u00a0, >6\u00a0months)\u00a0History of neck\/upper limb complaintsHistory versus no history\u00a0Musculoskeletal co-morbidityYes versus no\u00a0Other co-morbidityYes versus noWork-related psychosocial factors (%)\u00a0Decision authorityf (three items)Tertiles (low, medium, high)\u00a0Job demandsf (five items)Tertiles (low, medium, high)\u00a0Co-worker supportf (four items)Tertiles (low, medium, high)\u00a0Job-securityfLow security versus high security\u00a0Job as perceived cause of complaintYes versus noaPain Coping Inventory (Kraaimaat and Evers 2003; Kraaimaat et al. 1997)bFour Dimensional Symptom Questionnaire (Terluin 1998)cAmerican College of Sports Medicine Position Stand (American College of Sports Medicine Position Stand 1990)dMedical Outcomes Study 36-item Short Form Health Survey (Ware and Sherbourne 1992)ePhysical workload questionnaire (Bot et al. 2004)fJob Content Questionnaire (Karasek et al. 1998); %work-related psychosocial factors were considered as potential confounders in the relation between work-related physical factors and sick leave\nTo prevent multicollinearity we checked whether the psychosocial risk factors, the physical risk factors, and potential confounders were highly correlated (P\u00a0>\u00a00.5 or P\u00a0<\u00a0\u22120.5). The factor with the highest association with sick leave was retained and the other factor was removed from further analyses. This was the case for prolonged sitting and prolonged VDU work (P\u00a0=\u00a00.69), distress and worrying (P\u00a0=\u00a00.52); prolonged VDU-work and heavy physical work (P\u00a0=\u00a0\u22120.56), and prolonged sitting and heavy physical work (P\u00a0=\u00a0\u22120.65).\nWe performed the analyses in several stages. First, the unadjusted relationships between the work-related risk factors and sick leave were examined. Second, these relationships were adjusted for each of the potential confounders individually. Potential confounders of the relationship between work-related risk factors and sick leave included individual factors, general health, characteristics of the complaint, and other work-related risk factors (Table\u00a01). Factors were considered as potential confounders if they had been reported as predictive of sick leave or poor prognosis in previously published scientific papers. Only those factors that led to a considerable change in the regression coefficient of the risk factor (>10%) were considered as potential confounders in the multiple regression models. Next, a manual forward selection procedure was used to sequentially include potential confounders that induced the most change in the regression coefficient of the risk factor (i.e., in order of highest change of the regression coefficient). Confounders that changed the regression coefficient of sick leave by more than 10% were retained in the model. If the unadjusted relationship between the work-related risk factor and sick leave was not statistically significant (P\u00a0>\u00a00.05) and remained not significant after controlling for all potential confounders individually, the relationship was adjusted for sick leave at baseline and sex only.\nIndividual factors may interact in the relation between work-related physical and psychosocial risk factors and sick leave. We considered possible effect modification by sick leave at baseline, sex, coping style worrying (dichotomized) and musculoskeletal co-morbidity. Product terms of the potential effect modifier and the work-related risk factor were added to the model (e.g., sex\u00a0\u00d7\u00a0risk factor). Furthermore, possible effect modification by psychosocial risk factors was investigated in the relation between the four physical risk factors and sick leave. Factors significantly interacting with determinants in the model were retained in the models. In case of significant effect modification stratified analyses were carried out presenting the effect of the work-related risk factor for relevant subgroups of workers. All analyses were performed with the use of SPSS for Windows version 10.1 (SPSS Inc., Chicago, IL, USA).\nResults\nIn total, 342 of the 383 included patients (89%) completed the 3\u00a0months follow-up questionnaire. Fifty-eight of the patients were excluded, because data on sick leave at follow-up were missing. There were no significant differences between responders and dropouts by age, sex, and any of the determinants (P\u00a0>\u00a00.05). Baseline characteristics of patients reporting sick leave in the 3\u00a0months following baseline (n\u00a0=\u00a073) and patients without sick leave (n\u00a0=\u00a0252) are shown in Table\u00a02. The mean age of the total study population was 43\u00a0years (SD 10.3) and 54% was female. At baseline 80 patients reported sick leave in the 3\u00a0months preceding baseline due to their neck or upper extremity complaint. Half of these patients reported sick leave in the 3\u00a0months following baseline. With reference to physical and psychosocial risk factors, there was a statistically significant difference between patients reporting sick leave and patients without sick leave for decision authority, heavy physical work, static postures and repetitive movements, prolonged sitting and prolonged VDU-work.\nTable\u00a02Baseline characteristics and values of patients with neck or upper extremity complaints reporting sick leave (n\u00a0=\u00a073) and of patients reporting no sick leave (n\u00a0=\u00a0252) due to their complaints in the 3\u00a0months following baseline assessmentCharacteristicSick leaveaNo sick leaveaTotalIndividual factors\u00a0Age, years [mean (SD)]42.0 (11.5)43.6 (9.8)43.0 (10.3)\u00a0Sex (female)75%49%**54%\u00a0Sick leave in 3\u00a0months before baseline59%16%**25%\u00a0Education level\u00a0\u00a0Primary48%30%*34%\u00a0\u00a0Secondary40%49%48%\u00a0\u00a0College\/university12%21%18%\u00a0Number of hours work (h)\u00a0\u00a08\u20131611%12%11%\u00a0\u00a017\u20132418%25%20%\u00a0\u00a025\u20133626%30%27%\u00a0\u00a0>3645%33%42%Married\/living together63%83%*78%Smoking (now, ever)77%67%70%Pain coping: retreating [scale 7\u201328; median (IR)]11.0 (5.0)9.0 (4.8)**10.0 (4.5)Pain coping: worrying (scale 9\u201336; median (IR))18.0 (7.0)14.0 (5.0)**15.0 (6.0)Psychological distress (scale 0\u201312; median (IR))7.0 (5.0)4.0 (5.0)**5.0 (6.0)General health\u00a0Meeting ACSM position stand4%16%*13%\u00a0Meeting Norm Healthy Activity41%42%43%\u00a0Perceived health [scale 1\u20135; mean (SD)]3.1 (0.8)3.4 (0.9)*3.3 (0.8)\u00a0Quality of life [scale 1\u20135; mean (SD)]3.2 (0.7)3.5 (0.8)*3.4 (0.8)\u00a0Vitality [scale 0\u2013100; mean (SD)]54.3 (20.9)62.4 (17.2)**60.0 (18.5)Characteristics complaint\u00a0Localized complaint40%53%*49%\u00a0Complaint at both arms10%15%13%\u00a0Complaint at dominant arm50%44%47%Duration of current episode\u00a0<1\u00a0week5%7%7%\u00a01week\u20131\u00a0month36%32%33%\u00a01\u20136\u00a0months29%37%34%\u00a0>6\u00a0months30%24%26%History of elbow complaints51%43%45%Musculoskeletal co-morbidity56%47%50%Pain intensity [scale 0\u201310; mean, (SD)]6.0 (1.9)4.4 (2.1)**4.8 (2.2)Functional disability [scale 0\u2013100; median (IR)]36.3 (28.8)16.5 (21.3)**20.0 (25.3)Work-related factors\u00a0Heavy physical work [scale 0\u2013100; mean (SD)]36.1 (40.2)16.7 (36.1)**22.2 (42.8)\u00a0Static postures and repetitive movements (scale 0\u2013100)55.6 (47.2)44.4 (44.4)**44.4 (38.9)\u00a0Sitting for a long period of time22%48%**42%VDU-work for a long period of time21%36%*31%\u00a0Decision authority [scale 3\u201312; mean (SD)]9.0 (3.0)9.0 (3.0)*9.0 (3.0)\u00a0Job demands [scale 5\u201320; mean (SD)]13.4 (3.6)12.8 (2.9)12.9 (3.1)\u00a0Co-worker support [scale 4\u201316; mean (SD)]12.0 (1.0)12.0 (2.0)12.0 (2.0)\u00a0Job security78%85%83%\u00a0Job as perceived cause of complaint62%49%56%VDU visual display unit, SD standard deviation, IR interquartile range*\u00a0P\u00a0<\u00a00.05; **\u00a0P\u00a0<\u00a00.001 (independent T-test, Mann\u2013Whitney test, or \u03c72-test)avalues are percentages of patients unless indicated\nTable\u00a03 shows the effect of work-related physical and psychosocial risk factors on sick leave. After adjustment for confounders, a statistically significant effect was found for heavy physical work and prolonged sitting, but only in patients with a high score on pain coping strategy \u201cworrying\u201d. In patients who scored low on the pain coping subscale \u201cworrying\u201d the hazard ratio of sick leave was 1.05 (0.93\u20131.18, P\u00a0=\u00a00.46) per 10% increase in heavy physical work. In patients scoring high on \u201cworrying\u201d the relative risk of sick leave was 1.32 (95% CI 1.07\u20131.62) per 10% increase in heavy physical work. Patients who were sitting for long periods of time had a reduced risk of sick leave as compared to patients who did not spent a lot of time sitting, again only in patients who scored high on \u201cworrying\u201d (HR\u00a0=\u00a00.17, 95% CI 0.04\u20130.72). In patients who scored low on \u201cworrying\u201d, sitting for long periods of time did not have an effect on sick leave (P\u00a0=\u00a00.43). Sick leave at baseline, sex and musculoskeletal co-morbidity did not modify the relationship between the physical and psychosocial risk factors and sick leave. The psychosocial risk factors neither modified the relationship between heavy physical work and sick leave.\nTable\u00a03Relationship between work-related risk factors and sick leave in patients with neck or upper extremity complaints: results from the multiple Cox regression analysesDeterminantNCrude HR95% CIAdjusted HR95% CIHeavy physical work (per 10% increase)69\/3201.23[1.12; 1.36]*\u00a0For patients with low scores on worrying d21\/1401.05[0.93; 1.18]a\u00a0For patients with high scores on worryingd48\/1801.32[1.07; 1.62]a,*Static postures, repetitive movements (per 10% increase)69\/3201.16[1.06; 1.27]*1.04[0.94; 1.14]bSitting for long periods of time (vs. never\/now and then)16\/3220.40[0.23; 0.70]*\u00a0For patients with low scores on worryingd2\/1800.81[0.43; 1.52]a\u00a0For patients with high scores on worryingd14\/1420.17[0.04; 0.72]a,*VDU-work long periods of time (vs. never\/now and then)15\/3220.56[0.32; 1.00]*0.70[0.36; 1.36]cDecision authority\u00a0Low1.00\u00a0Medium0.80[0.47; 1.35]0.94[0.55; 1.59]a\u00a0High0.59[0.31; 1.13]0.87[0.45; 1.68]aJob demands\u00a0Low23\/1151.001.00\u00a0Medium20\/1110.90[0.49; 1.64]0.91[0.50; 1.66]a\u00a0High27\/951.42[0.81; 2.48]1.13[0.64; 1.97]aCo-worker support\u00a0Low16\/861.001.00\u00a0Medium31\/1401.19[0.65; 2.18]1.22[0.67; 2.23]a\u00a0High23\/881.40[0.74; 2.66]1.14[0.59; 2.17]aLow job security (vs high)70\/3191.43[0.78; 2.60]1.47[0.80; 2.69]aJob strain\u00a0High decision authority, low job demands10\/771.001.00\u00a0High decision authority, high job demands11\/521.63[0.69; 3.84]1.29[0.54; 3.07]b\u00a0Low decision authority, low job demands25\/1041.85[0.89; 3.85]1.30[0.62; 2.73]b\u00a0Low decision authority, high job demands\u00a024\/862.15[1.03; 4.49]*1.15[0.54; 2.47]bN Number of patients reporting sick leave\/total, HR hazard ratio*\u00a0P\u00a0<\u00a00.05aAdjusted for sick leave at baseline and sexbAdjusted for sick leave at baseline, sex and worryingcAdjusted for sick leave at baseline, sex, education level, quality of life, working in static postures and with repetitive movementsdEffect modification by worrying\nDiscussion\nWe studied a population of working employees who consulted their GP with a complaint at the neck or upper extremity. The results show that heavy physical work and prolonged sitting in patients that worry a lot predicted sick leave in the three successive months after baseline. Other physical and psychosocial risk factors appeared not to be related to sick leave after adjustment for confounding variables.\nIndividual factors and stress may have a modifying effect on the relation between work related factors and outcome in musculoskeletal complaints. The pain coping style \u201cworrying\u201d appeared to be an effect modifier in the relationship between heavy physical work and sick leave. An increase of 10% of heavy physical work in patients who worried a lot increased the probability on sick leave with 1.3. A passive coping style like worrying (e.g., \u201cfocus on pain all the time\u201d; \u201cI think the pain will get worse\u201d) is thought to generate a preoccupation with bodily symptoms, which may increase the perception of pain and disability (Covic et al. 2000; Evers et al. 2003) or generate a different appraisal of the work situation and musculoskeletal symptoms (Bongers et al. 2002b), which in turn may lead to sick leave. Sitting for long periods of time reduced the risk of sick leave, only in patients who scored high on the pain subscale \u201cworrying\u201d In the study of Ari\u00ebns et al. (2002) sitting was also related to a reduced risk of sick leave. However, uncomfortable sitting has been found to increase the number of days on sick leave due to neck and shoulder disorders (Ekberg and Wildhagen 1996). Sitting was highly negatively correlated with heavy physical work. Perhaps, people who sit for long periods of time at their work do not experience heavy physical work, and thus the absence of heavy physical work results in a reduced risk. This may explain why sitting had only a protective effect in patients that worry a lot.\nIn the unadjusted analyses, all the physical risk factors and two of the psychosocial risk factors (i.e., decision authority and job strain) had a statistically significant effect on sick leave. It is partly due to the underlying distribution of the exposure which physical factors are most strongly associated with sick leave in the multivariate analyses. Adjustment for sick leave at baseline had the strongest effect on the presented associations, diminishing the strength of the relations between work-related risk factors and sick leave. Sick leave may have been the result of exposure to these risk factors, but once patients were on sick leave work-related risk factors no longer seem to have an impact on continued sick leave. Half of the patients who reported sick leave within the 3\u00a0months before baseline also reported sick leave in the 3\u00a0months following baseline. The effect of sick leave in the months preceding baseline apparently outweighed the impact of work-related risk factors in the analyses. The proportion of patients reporting sickness during the 3\u00a0months follow-up was too small to be able to exclude patients reporting sick leave at baseline. Cox regression produces large estimates of the standard errors, which results in conservative estimates of the confidence intervals (Skov et al. 1998). Therefore, it may be possible that relevant associations did not reach statistical significance.\nSick leave data were collected by a self-reported questionnaire. Sick leave data collected by an objective method (i.e., sick leave records) may be more accurate than self-reported sick leave. However, in large epidemiological studies the use of questionnaires is more feasible and can be a source of reliable data on sick leave. Burdorf et al. (1996) investigated the reliability of self-reported sick leave with company records as reference. A good agreement was found between prevalence, frequency and duration of self reported sick leave and company records in patients with back pain. In our study we only examined the prevalence of sick leave and consider the reported sick leave data to be fairly reliable.\nOur study population differs from an occupational cohort, consisting of workers from a wide variety of occupational settings who had visited the GP due to neck or upper extremity complaints. This makes our results more widely generalisable than a selective sample of workers from a specific company or industry. However, due to the fact that our population already had symptoms at baseline we may not be able to discriminate between factors that are a consequence of sick leave at baseline and factors that may increase the risk of sick leave. For example, patients may worry more about their pain problem because they are no longer able to work, or worrying about their pain problem may be the reason for sick leave after their visit to the doctor.\nIn the Netherlands the GP is often confronted with patients who are on sick leave due to musculoskeletal complaints, as patients who seek medical care usually first consult their GP. The GP acts as a gatekeeper in the health care system. At the time of our study, referrals to the second or third level of care could, in principle, only be made by the GP. This is comparable to, for instance, the British and the Canadian health care systems. As a result of their position in the Dutch health care system, GPs could play an important role in the prevention of aggravation of complaints. In this study the copying style \u201cworrying\u201d seemed to be an important factor in patients who have jobs involving heavy physical work. Reassuring patients might help to reduce aggravation of complaints, which might improve the chances of return to work in this group of patients with neck or upper extremity complaints. It may be interesting to investigate whether early intervention aimed at promoting particular coping styles can prevent or reduce sick leave in patients with neck or upper extremity complaints in a primary care setting.\nIn conclusion, heavy physical work increased the risk of sick leave and prolonged sitting reduced the risk of sick leave in a subgroup of patients who worry much. Other work-related physical risk factors and work-related psychosocial risk factors were not significantly related to sick leave. Additional large longitudinal studies of sufficiently large size among employees with neck or upper extremity complaints are needed to confirm our results.","keyphrases":["sick leave","neck","upper extremity","workload","cohort study"],"prmu":["P","P","P","P","P"]}
{"id":"Curr_Eye_Res-1-1-2430176","title":"Branch Retinal Vein Occlusion: Pathogenesis, Visual Prognosis, and Treatment Modalities\n","text":"In branch retinal vein occlusion (BRVO), abnormal arteriovenous crossing with vein compression, degenerative changes of the vessel wall and abnormal hematological factors constitute the primary mechanism of vessel occlusion. In general, BRVO has a good prognosis: 50\u201360% of eyes are reported to have a final visual acuity (VA) of 20\/40 or better even without treatment. One important prognostic factor for final VA appears to be the initial VA. Grid laser photocoagulation is an established treatment for macular edema in a particular group of patients with BRVO, while promising results for this condition are shown by intravitreal application of steroids or new vascular endothelial growth factor inhibitors. Vitrectomy with or without arteriovenous sheathotomy combined with removal of the internal limiting membrane may improve vision in eyes with macular edema which are unresponsive to or ineligible for laser treatment.\nBACKGROUND\nMethod of Literature Search\nEligible studies were identified through a comprehensive literature search of electronic databases (Medline, 1966\u2013September 2007 and Science Direct, all years). Additional articles were selected from review of the reference lists of the articles generated from the above search. The following keywords and combinations of these words were used in compiling the search: branch retinal vein occlusion, retinal circulatory disorders, pathogenesis, hematological disorders, risk factors, therapy methods, visual prognosis. In total, 150 of these were used for this mini-review.\nEpidemiology, Classification\nRetinal vein occlusion (RVO) is the second most common retinal vascular disorder after diabetic retinopathy and is a significant cause of visual handicap. Its prevalence has been shown to vary from 0.7% to 1.6%.1,2 In a population-based study,3 an overall incidence of symptomatic RVO was found in 0.21% of patients aged 40 or older. Hayreh et al.4 investigated the demographic characteristics of various types of RVO in 1108 patients (1229 eyes). In this study, a male:female ratio of 1.2:1 was noted in a group of patients with RVO. Of the two main types of RVO, central retinal vein occlusion (CRVO) and branch retinal vein occlusion (BRVO), the latter is more common. A further group is hemi-vein occlusion, a distinct clinical entity presenting as occlusion of only one trunk of the central retinal vein in the area of the anterior part of the optic nerve.4 Hayreh et al.4 postulated that its pathogenesis is closely related to CRVO.\nThe first case of BRVO was reported by Leber in 1877.5 Some studies showed a higher proportion of BRVO patients older than 65 at the onset of the disease compared to CRVO,4,6 but others found no significance of age in the distribution of CRVO and BRVO.7,8 BRVO is divided into two distinct entities: major BRVO, when one of the major branch retinal veins is occluded, and macular BRVO, when one of the macular venules is occluded. In 66% of eyes with BRVO, there is occlusion of the major branch in the superotemporal quadrant followed by 22\u201343% of eyes with occlusion of the major branch in the inferotemporal quadrant.9 Owing to absent subjective BRVO symptoms in nasal quadrants, the diagnosis of occlusion in this localization is mostly accidental and therefore rare.10 Very often BRVO in nasal quadrants is diagnosed only when its complication as bleeding from neovascularizations into the vitreous cavity occurs. The cumulative probability of developing a second episode of occlusion in the other eye within 4 years is about 7% in patients with BRVO.4\nPathogenesis\nThe pathogenesis of RVO is multifactorial while BRVO may be due to a combination of three primary mechanisms: compression of the vein at the arteriovenous (A\/V) crossing, degenerative changes of the vessel wall, and abnormal hematological factors. In the following sections these factors are discussed.\nArteriovenous Crossing\nKoyanagi in 192811 first reported the association between BRVO and A\/V crossing, and now it is established that mechanical narrowing of the venous lumen at these intersections plays a role in the pathogenesis of BRVO. Anatomic features of A\/V crossings and secondary effects of arteriolar sclerosis may explain the apparent vulnerability of the crossing site to venous occlusion. In the majority of A\/V crossings, the thin-walled vein lies between the more rigid thick-walled artery and the highly cellular retina. The sharing by artery and vein of the common adventitial sheath and the narrowing of the venous lumen that normally occurs at the A\/V crossing provide the setting for BRVO.12 The risk of occlusion may be accentuated when arteriolar sclerosis results in increased rigidity of the crossing artery. Duker and Brown13 provided further support for a mechanical basis of BRVO development when they examined the relative anatomic position of the crossing artery and vein at the site of occlusion in 26 eyes with BRVO. They found in all 26 eyes the artery anterior to the vein (towards the vitreous cavity). Zhao et al.12 evaluated the anatomic position of the crossing vessels in 106 eyes with BRVO and found the artery anterior to the vein at the obstructed site in 99% of affected eyes. However, other mentioned risk factors must play a role, too, because in approximately 60% of normal A\/V crossings without BRVO the artery lies anterior to vein.12\nDegenerative Changes of Vessel Wall\nA number of studies have investigated the histological changes of vessel wall at the A\/V crossing.14,15 An investigation by Jefferies et al.14 showed that the expected venous compression at the crossing in histological view does not exist. He described the bending of the vein into the nerve fiber layer at this point without its compression. Histological investigation of the venous lumen at the A\/V crossing in patients with a number of months to several years duration of BRVO showed organized thrombus with varied extent of recanalization in this part. Seitz15 described the clinical histological correlation in one eye with BRVO of a few hours after onset. There was no blood thrombus obliterating the venous lumen at the A\/V crossing and even the fundoscopic examination showed strong dilated and tortuous vein distal to the crossing. In the area of the A\/V crossing, alteration of the endothelium and intima media was present. Seitz suggests that the trophic changes of venous endothelium and intima media, as they follow the compression from overlaying artery, is the root of the pathogenesis of BRVO.15 The formation of the thrombus follows as a secondary process. The findings of Frangieh et al.16 support this hypothesis; 90% of the patients in their study had evidence of intima media layer hypertrophy, and all had evidence of intravenous thrombosis.\nSystemic hypertension, diabetes mellitus, atherosclerosis, and smoking are reported to be more common in patients with RVO.1,2,10 Sclerosis of the retinal artery which is associated with these systemic disorders may result in further compression of the vein, when the increased rigidity of arterial wall and contraction of the adventitial sheath shared by artery and vein occur. Mechanical obstruction of the vein through the rigid artery in the A\/V crossing may result in turbulent blood flow producing damage to venous endothelium and intima media and the sequence of events leading to occlusion of the vein.12,14 The turbulent blood flow was confirmed by Christoffersen and Larsen in an investigation which analyzed the fluorescein angiograms of 250 patients with BRVO.17\nHematological Disorders\nSome studies have revealed an association between BRVO and hyperviscosity due to high hemotocrit.18,19 Higher blood viscosity increases under conditions of low blood flow and erythrocyte aggregation.18 Viscosity is mainly dependent upon the hematocrit (the greater the number of erythrocytes, the larger they aggregate) and plasma fibrinogen (required for aggregation to occur).20 Another discussed hematological disorder in the pathogenesis of BRVO is dysregulation of the thrombosis-fibrinolysis balance.21 The coagulation cascade including different blood factors results in the production of thrombin which converts circulating fibrinogen to fibrin. The coagulation sequence is held in check and inhibited by specific anticoagulants including protein C, protein S, and antithrombin. Table 1 shows the major disorders studied in patients with RVO. The results of published studies, however, are inconsistent, and the role of coagulation factors in the development of RVO remains unclear.\nTABLE 1\nMost discussed coagulation and anticoagulation disorders in the etiology of BRVO\nResistance to activated protein C (especially factor V Leiden mutation)\nProtein C or protein S deficiency\nDeficiency of antithrombin III\nGenetic mutation in the prothrombin (factor II) gene\nAnti-phospholipid antibodies\nHyperhomocysteinemia\nResistance to Activated Protein C and Deficiency of Protein C or Protein S\nProtein C is serine proteinase whose activated form is a potent inhibitor of coagulation factors V and VIII.22 Factors V and VIII are a part of the coagulation cascade leading to conversion of fibrinogen to fibrin. Patients with protein C deficiency frequently manifest superficial and deep venous thrombosis and pulmonary embolism. Protein S and phospholipids are co-factors in the inactivation of factors V and VIII by activated protein C.22 An absolute deficiency of protein C or S is relatively rare. Tekeli23 and several other authors have reported normal levels in patients with RVO.24\u201326 The concept of resistance to activated protein C (so-called APC resistance) was first introduced by Dahlb\u00e4ck et al. in 1993.27 APC resistance was subsequently shown to be a risk factor for venous thrombosis.28 More than 90% of patients with APC resistance have been shown to have a single point mutation in factor V gene.29 This mutation hinders the degradation of factor V normally occurring through protein C. Several investigators have reported an increased frequency of APC resistance in a cohort of patients with RVO,30\u201334 but this association has not been confirmed in other studies.35,36 Moreover, some results are inconclusive due to small patient samples or the lack of control groups. The meta-analysis of Janssen et al.21 showed the odds ratios for factor V Leiden mutation in patients with RVO 1.5 (95% CI 0.8\u20133.2). Despite the evidence of the significance of Leiden mutation, the effect of this hematological disorder in the etiology of RVO is only marginal.21\nDeficiency of Antithrombin and Mutation in the Prothrombin Gene\nIn recent studies of patients with RVO, no significant association with a deficiency of antithrombin or with prothrombin mutation was found.21,26,34,37\u201340\nAnti-Phospholipid Antibodies and Hyperhomocysteinemia\nAntiphospholipid antibodies (APA) consist of a heterogeneous group of immunoglobulins, mainly anti-cardiolipin antibodies (ACA) and lupus anticoagulants (LA). Circulating APA leads to a hypercoagulable state and recurrent thrombosis through thrombocyte activation and inhibition of the natural anticoagulant pathways by binding of membrane phospholipids. Both the presence of LA and increased level of ACA are associated with a 3- to 10-fold increased risk of venous thrombosis.41\nAn elevated level of the amino acid, homocysteine is now generally accepted to be a risk factor for systemic vascular disease.42 Homocysteine appears to have a deleterious effect on vascular endothelium and may induce increased platelet aggregation and thrombosis. Levels of homocysteine may be increased by dietary habits, prescription medicines, or enzymatic mutations affecting homocysteine metabolism.43 The results of meta-analyses confirm total homocysteine to be an independent risk factor for RVO.21,44 Loewenstein et al.45 investigated the prevalence of genetic mutation in the enzyme methylentetrahydrofolate reductase (MTHFR) whose impaired activity may lead to hyperhomocysteinemia. The prevalence of this mutation was significantly higher in patients with RVO compared with the incidence of MTHFR in a control population. However, these results were not confirmed in other studies.46 The meta-analysis of Cahill et al.44 showed an association between retinal vascular occlusion and hyperhomocysteinemia but not with the mutation in the gene for MTHFR.\nPathogenesis of Macular Edema in BRVO\nThe development of macular edema (ME) followed by BRVO has been hypothesized to be caused by fluid flux from vessels to tissue according to Starling's law,47,48 which is based on the breakdown of the blood-retinal barrier (BRB) as a result of damage to the tight junctions of capillary endothelial cells,49 vitreoretinal adhesion,50 and secretion into the vitreous of vasopermeability factors produced in the retina.51,52 Observations by Noma et al.52 suggest that in patients with BRVO, vascular occlusion induces the expression of vascular endothelial growth factor (VEGF) and Interleukin-6 (IL-6), resulting in BRB breakdown and increased vascular permeability. Thus, VEGF and IL-6 may contribute to the development and progression of vasogenic ME in BRVO. ME is closely associated with retinal hypoxia, and the degree of hypoxia in the center of the macula corresponds to the decrease in visual acuity (VA). If marked hypoxia persists, irreversible structural changes in the macula occur, and the disturbed VA is almost always lasting. It is generally known that ME and intraretinal hemorrhage occurring in BRVO usually disappear within 6 to 12 months.53 In these cases, collateral systems often develop. The main purpose of the treatment is to decrease the duration of edema to prevent photoreceptor damage, if no spontaneous improvement occurs.\nClinical Signs and Diagnosis\nIn general, diagnosis of BRVO is not a problem owing to its classical features. Major BRVO can be asymptomatic or with visual blurring usually involving the sector of visual field corresponding to the area of the retina involved. In macular BRVO, there is always a central visual disturbance with normal peripheral vision. Acute BRVO presents characteristic clinical features with flame-shaped, dot and blot hemorrhage, soft and hard exudates, retinal edema, and dilated, tortuous vein in a segmental distribution. Signs of old occlusion are vascular sheathing and venous collaterals. The diagnosis is based on clinical examination under slit lamp and fundoscopy in artificial mydriasis. VA is of great importance for future visual prognosis. BRVO often leads to retinal non-perfusion zones in the occlusion area. Fluorescein angiography is particularly useful in determining the extent of ME and ischemia, although the ischemic areas are often obscured by the presence of intraretinal hemorrhage. Retinal neovascularization occurs in 36% of eyes with an area of non-perfusion greater than 5 disc diameter.54\nRVO is associated with an increase in vascular causes of death (both cerebral and cardiac) in large prospective follow-up studies.55 In all patients with RVO, the systemic risk factors (hypertension, diabetes mellitus, blood lipid disorders) should be investigated and managed by appropriate specialists.\nNatural Course and Visual Prognosis\nThe visual outcome following the natural course of BRVO is well documented.56\u201358 In general, BRVO has a good prognosis: 50\u201360% of eyes have been reported to have a final VA of 20\/40 or better even without any treatment.56\u201359 The natural course of BRVO is determined by the site and degree of occlusion, the integrity of arterial perfusion to the affected sector, and the efficiency of the developing collateral circulation.59 Chronic ME and bleeding into the vitreous from neovascularizations account most frequently for a poor final VA.54,58,60 Retinal neovascularization and persistent ME develop in 25% and 60% of eyes, respectively.57,61 Gutman et al.60 found that in the natural course of BRVO, only 14% of eyes with chronic ME retained a VA of 20\/40 or better, while 86% had a final VA of 20\/50 or worse. He concluded that chronic ME has a poor prognosis in terms of final VA.60 Schilling et al.62 observed a worse visual prognosis in cases of ischemic ME compared to perfused ME. However, findings by Finkelstein63 showed that 91% of 23 eyes with macular ischemia recovered vision within one year with a VA of 20\/40 or better. The conflicting reports and small number of studied eyes make it difficult to reach definitive conclusions on visual prognosis in patients with BRVO.\nVA is a very sensitive indicator of the oxygen situation in the macula. For this reason, pre-treatment VA may be an important prognostic factor. Six studies analyzing the relation between initial and final VA were found.53,56,58,64\u201366 Five were used in an analysis of the data of eyes with unsatisfactory final VA (20\/200 or worse) in relation to initial VA (Table 2). There were 2 groups; the first consisted of eyes with an initial VA of 20\/50 or better and the second group of eyes with an initial VA of 20\/200 or worse. In the second group were found a considerably higher percentage of eyes with a final VA of 20\/200 or worse, regardless whether the eyes had undergone laser treatment or not. Since there were differently divided subgroups for final VA, the study of Subramanian et al.65 was not included in our analysis. Magargal et al.58 investigated the visual prognosis in 246 eyes with BRVO divided into two groups: with and without laser treatment. The obtained analysis illustrates that in the group of eyes with an initial VA 20\/50 or better, no eye (not receiving laser treatment) and only 13% eyes (had undergone laser treatment) had a final VA of 20\/200 or worse, whereas in the group of eyes with an initial VA 20\/200 or worse, 83% of eyes (not receiving laser treatment) and 50% of eyes (had undergone laser treatment) had this unsatisfactory final VA. In an analogous way, the data for the final VA 20\/50 or better in relation to the initial VA were analyzed (Table 3). We can see that in the group of eyes with an initial VA 20\/50 or better, 89% of eyes (not receiving laser treatment), and 75% of eyes (had undergone laser treatment) retained this good VA, whereas in the group of eyes with an initial VA 20\/200 or worse, only 14% of eyes (not receiving laser treatment) and only 22% of eyes (had undergone laser treatment) had a final VA 20\/50 or better.58 Similar data are reported in the other studies (Tables 2 and 3). A chi-squared test with Yates correction was used to analyze the data. In 4 cases, in Table 2, and in 5 cases, in Table 3, respectively, the results were statistically significant (p < 0.05). Our analysis shows that in eyes with an initial VA 20\/50 or better, the visual prognosis is good even without treatment. It could also be concluded that the cases of BRVO with an initial VA of 20\/200 or worse have a statistically significantly poorer visual prognosis than those with an initial VA of 20\/50 or better. Subramanian et al.65 showed that in patients with BRVO who underwent laser treatment of ME, the level of preoperative VA can be a useful predictor of visual outcome.\nTABLE 2\nFinal visual acuity of 20\/200 or worse in relation to initial visual acuity. Chi-squared test with Yates correction (p < 0.05)\nInitial visual acuity 20\/50 or better\nInitial visual acuity 20\/200 or worse\nChi-squared test p < 0.05\nNatural course\u2014without laser treatment\nGutman56\n5% (1\/20)\n50% (6\/12)\nSignificant\nMagargal58\n0% (0\/35)\n83% (24\/29)\nSignificant\nHad undergone laser treatment\nWetzig53\n25% (2\/8)\n67% (10\/15)\nNo\nJalkh64\n0% (0\/9)\n33% (3\/12)\nNo\nMagargal58\n13% (5\/40)\n50% (32\/64)\nSignificant\nLang66\n8% (1\/13)\n50% (8\/16)\nSignificant\nTABLE 3\nFinal visual acuity 20\/50 or better in relation to initial visual acuity. Chi-squared test with Yates correction (p < 0.05)\nInitial visual acuity 20\/50 or better\nInitial visual acuity 20\/200 or worse\nChi-squared test p < 0.05\nNatural course\u2014without laser treatment\nGutman56\n90% (18\/20)\n33% (4\/12)\nSignificant\nMagargal58\n89% (31\/35)\n14% (4\/29)\nSignificant\nHad undergone laser treatment\nWetzig53\n63% (5\/8)\n20% (3\/15)\nNo\nJalkh64\n56% (5\/9)\n9% (1\/12)\nSignificant\nMagargal58\n75% (30\/40)\n22% (14\/64)\nSignificant\nLang66\n77% (10\/13)\n13% (2\/16)\nSignificant\nTreatment\nCurrent treatment options focus on the sequelae of the occluded venous branch, such as ME, retinal neovascularization, vitreous hemorrhage, and traction retinal detachment. There have been a number of treatment modalities advocated for the management of BRVO (Table 4). Many studies that examine interventions for BRVO suffer from methodological limitations, including insufficient power resulting from small sample sizes, short follow-up periods, absence of a control group or inappropriate control group (absence of placebo or best practice intervention as control groups), and lack of distinction between clinical entities. A number of such investigations have therefore produced conflicting data. Hence, the results of randomized clinical trials are the most important. The complex pathogenesis of this disease requires investigation and treatment of all risk factors (hypertension, diabetes mellitus, blood lipid disorders, hematological disorders).\nTABLE 4\nTreatment modalities for BRVO\nAnti-aggregative therapy and firbrinolysis\nIsovolemic hemodilution\nLaser treatment\nIntravitreal and periocular application of steroids\nIntravitreal injection of VEGF inhibitors\nSheathotomy and vitrectomy\nAnti-Aggregative Therapy and Fibrinolysis\nSystemic treatment with oral acetylsalicylic acid, subcutaneous heparin, or intravenous thrombolysis have not been shown to be effective treatments for CRVO, while for BRVO no randomized clinical trials have been published as of the date of this review. Thrombolysis using administration of tissue plasminogen activator intravitreally or directly into the retinal vein (mostly upper temporal branch close to the optic disc) has been demonstrated to improve VA in patients with CRVO,67 but the results were based only on noncomparative interventional series. There is no general current acceptance of this treatment.\nHoutsmuller et al.,68 in a double-blind study, examined the platelet aggregation inhibiting effect of ticlopidine in 54 patients with BRVO less than 3 weeks from the onset of symptoms. Compared with placebo therapy a significant improvement in VA was observed with ticlopidine therapy for six months. In the treated group, 69% of patients experienced an improvement in VA, whereas 52% of placebo group reported improvement.\nTroxerutin has been suggested to inhibit erythrocyte and platelet aggregation and to improve erythrocyte deformability, thus reducing blood viscosity and the retinal microcirculation.69 A double-blind randomized study of 26 patients with BRVO compared troxerutin with placebo.69 At 4 months follow-up, more of the patients receiving troxerutin treatment had a mean VA of 20\/40 or better than the control group, although this difference was not found to be statistically significant. After 4 months, all patients were treated with troxerutin for 2 years. At the completion of this follow-up period for those patients initially treated with troxerutin, a significant improvement in VA and improvement of ME was demonstrated. The limitation of this study is that there is no separation in the analysis of results for patients with BRVO and CRVO who were included in the study, too.\nBoth studies mentioned that investigated the medical treatment of BRVO are limited by a small sample size and short follow-up period (6 and 4 months).\nIsovolaemic Hemodilution\nChen et al.20 demonstrated positive results for isovolemic hemodilution given up to 3 months after the on-set of the symptoms of BRVO in patients with a hematocrit of 35% or more. In this randomized controlled study, 18 patients were treated for 6 weeks with venesection and volume replacement using hydroxyethylstarch and compared to 16 untreated control patients. After a one year follow-up, the final VAs were 20\/40 and 20\/80 for treated and untreated patients, respectively (p = 0.03). Patients with ME and a VA 20\/40 or worse underwent 3 months after including into the study macular grid laser photocoagulation (MLG). Sector photocoagulation was applied if ocular neovascularization developed or if, at 3 months, the fluorescein angiogram showed an area of capillary non-perfusion greater than 5 disc areas. 28% of the hemodiluted patients required MLG compared to 44% of the control group; this difference was not statistically significant (p = 0.2). Sector photocoagulation was required by 50% of both groups of patients.20\nHydroxyethylstarch has a capacity to expand the plasma volume by up to 172% of the volume infused and has a duration of action of approximately 36 hours.70 It is non-antigenic and has a low incidence of allergic reactions.71 Poupard et al.72 randomized 25 patients to either hemodilution with dextran for 21 days (n = 10), hemodilution combined with heparin for 21 days (n = 10), or heparin treatment for 21 days followed by anti-vitamin K drugs for a further 30 days (n = 5). The study showed that, for those receiving heparin followed by anti-vitamin K drugs, mean VA remained unchanged to baseline values by 60 days. For those treated with hemodilution and heparin, a statistically significant increase in VA was found by 60 days. For those treated with hemodilution alone, a significant improvement in VA was found by day 14. In a randomized study by Hansen et al.73 of 35 patients with BRVO, 18 patients were treated by hemodilution for a period of 5 to 6 weeks (targeted hematocrit 30\u201335%). A control group of 17 patients were only observed. At follow-up 12 months later, 25 patients had completed the therapy. Seven of the 13 who received hemodilution demonstrated a VA increase of 2 lines or more compared with none of the 12 patients who did not receive hemodilution (p < 0.005). Reported complications of hemodilution include headache, exertional dyspnea, tiredness, deep vein thrombosis, and hypotension. The treatment was noted to be generally well-tolerated even in elderly patients.20,73,74\nThe use of hemodilution to treat BRVO is currently not generally accepted. Interpretation of the above-mentioned studies is difficult because most of them incorporated other treatments in combination with the hemodilution. Further prospective randomized trials with adequate controls and sufficient follow-up are required for any definitive conclusions and recommendations.\nArteriovenous Crossing Sheathotomy and Vitrectomy\nOsterloh and Charles75 first reported improvement in VA in patients with BRVO after treatment using the technique of surgical sheathotomy. The principle steps of this procedure are a pars plana vitrectomy followed by separation of the retinal artery from the vein by creating an incision in the adventitial sheath adjacent to the A\/V crossing and then separation of the adhesions. Several studies have shown significantly better functional outcomes in patients treated by sheathotomy compared to controls (Table 5).75\u201396 Reported complications are few but include cataract, hemorrhage, retinal tears, postoperative gliosis, and retinal detachment.75\u201396 Garcia-Arumi76 described a combination of A\/V sheathotomy and injection of thrombolytic into the occluded vein which resulted in thrombus release in 28% cases and significant correlation with early surgery and better final VA. The role of the sheathotomy alone in visual improvement is insufficiently clear. Some authors suggest that vitrectomy is the most important part of the sheathotomy surgery, leading to reduction of ME.77,80,96 Yamamoto et al.77 compared the effect of sheathotomy combined with vitrectomy to the effect of vitrectomy alone and found no advantage of sheathotomy. Eyes with pre-existing posterior vitreous detachment were not studied. For this reason, the benefit of vitrectomy of these eyes is unknown. Surgical detachment of posterior hyaloid could be more important than the sheathotomy itself .78 The vitreous is postulated to have a role in the pathogenesis of neovascularization and ME, which may complicate BRVO and its removal may help in the management of these sight threatening complications.78 Vitrectomy and removal of the posterior hyaloid with peeling of the internal limiting membrane (ILM) appears to improve oxygenation of the retina, which may lead to visual improvement.97,98 Peeling of the ILM improves the surgical outcome during A\/V adventitial sheathotomy, too.84 To date, no randomized clinical trials on the surgical treatment of BRVO have been published. Any evidence supporting these procedures is based on non-ramdomized case series only.\nTABLE 5\nSummary of studies evaluating the treatment of macular edema in BRVO by sheathotomy (VA = visual acuity, ME = macular edema, ILM = internal limiting membrane)\nAuthor\nStudy type\nPatients\nFollow-up (mean)\nOutcomes\nComments\nOsterloh and Charles75\nCase report.\n1 eye.\n8 months.\nVA improved from 20\/200 to 20\/25.\nFirst report of sheathotomy.\nGarcia-Arumi et al.76\nProspective interventional nonrandomized study.\n40 eyes\u2014all underwent vitrectomy, sheathotomy and injection of 25 mg of tissue plasminogen activator into occluded vein.\n13 months.\nThrombus release in 11 eyes (27.5%)\u2014correlated with early surgery. VA increased from 20\/100 to 20\/40 (p = 0.016).\nYamamoto et al.77\nRetrospective interventional comparative case series.\n20 eyes\u2014sheathotomy 16 control eyes (posterior vitreous detachment via vitrectomy).\n12 months.\nVA: significantly better in both groups (p = 0.008 and p = 0.001, respectively). VA and foveal thickness were not significantly different between the groups.\nCharbonnel et al.78\nProspective nonrandomized, interventional case series.\n13 eyes\u2014sheathotomy.\n7 months.\nImprovement in VA \u2265 2 ETDRS lines in 9 eyes (69%).\nAbsence of previous posterior vitreous detachment correlated with improvement in VA.\nSohn et al.79\nRetrospective interventional case series.\n22 eyes\u2014sheathotomy + ILM peeling in all eyes.\n3 months.\nImprovement in VA (log MAR) from 0.79 \u00b1 0.29 to 0.57 \u00b1 0.33 (p < 0.01).\nAll eyes pretreated with grid laser or triamcinolone.\nKumagai et al.80\nProspective, randomized, comparative, interventional study.\nGroup 1: 18 eyes\u2014sheathotomy. Group 2: 18 controls (vitrectomy without sheathotomy).\n31 months.\nVA (log MAR) in group 1: 0.52 \u2192 0.08. In group 2: 0.53 \u2192 0.014. Differences between group 1 and 2 was not significant.\nAvci et al.81\nRetrospective interventional comparative case series.\n11 eyes\u2014sheathotomy. 10 control eyes\u2014grid laser photocoagulation.\n9 months.\nVA (log MAR): sheathotomy: 0.84 \u2192 0.36. Grid laser: 1.06 \u2192 0.82. Difference was significant.\nHorio et al.82\nInterventional case series.\n7 eyes.\n6 months.\nSignificant improvement in retinal blood flow (p < 0.01) and reduced macular thickness (p = 0.03).\nLakhanpal et al.83\nRetrospective interventional case series.\n12 eyes.\n49.9 weeks.\nVA (logMAR) improved from 1.00 \u00b1 0.32 to 0.56 \u00b1 0.28 (p = 0.0003).\n25-gauge transvitreal limited arteriovenous crossing manipulation without vitrectomy.\nMester et al.84\nProspective interventional nonrandomized case-control study.\n43 eyes\u2014sheathotomy. 16 eyes additionally + ILM peeling. 25 control eyes.\n6 weeks.\n26 patients (60%) gained \u2265 2 lines of VA. Better result in patients with ILM peeling. ME and intraretinal hemorrhage resorbed in all patients.\nAll patients had isovolaemic hemodilution for 10 days.\nOpremcak et al.85\nProspective interventional case series.\n15 eyes.\n6.5 years.\nSnellen VA improved in 10 patients (67%) by an average of 4 lines vision (range 1\u20139 lines). In 3 patients resolution of ME but no improvement of VA.\nRetinal vascular bleeding in 2 patients.\nAsensio Sanchez et al.86\nProspective interventional nonrandomized study.\n13 eyes\u2014sheathotomy, 5 eyes underwent additionally ILM peeling.\n12 months.\nVA improved in 12 patients (92%). Better results in patients with ILM peeling.\nLerche et al.87\nProspective nonrandomized intervention case series.\n12 eyes \u2013 sheathotomy.\n3 months.\nVA (logMAR) improved from 0.74 to 0.56.\nMason et al.88\nProspective, nonrandomized, comparative interventional study with concurrent control group.\n20 eyes\u2014sheathotomy. 20 control eyes (10 of them without intervention and another 10 underwent grid laser).\n14 months (sheathotomy). 19 months (controls).\nVA improvement: Sheathotomy: from 20\/250 to 20\/63. Controls: from 20\/180 to 20\/125 (p = 0.02). 45% of the surgical group had final VA \u2265 20\/40 compared with 15% of the controls.\nData only for whole control group together.\nCahil et al.89\nRetrospective non-controlled case series.\n27 eyes\u2014sheathotomy.\n12 months.\nResolution of ME in 8 (29.6%) patients, reduction in 14 (51.8%) and persistence in 5 (18.5.%).\nBecquet et al.90\nProspective nonrandomized interventional case series.\n6 eyes (sheathotomy + ILM peeling. 6 controls (ILM peeling only).\n6 months.\nSignificant improvement of VA in both groups. No difference in VA or foveolar thickness between the groups (p = 0.5; p = 0.6 respectively).\nMartinez-Soroa et al.91\nRetrospective interventional case series.\n17 eyes\u2014sheathotomy.\n6 months.\nImprovement in VA from 0.26 to 0.4. 53% patients improved \u2265 4 lines (Snellen).\nLe Rouic92\nRetrospective interventional case series.\n3 eyes\u2014sheathotomy.\n10 months.\nNo improvement in VA observed.\nAll patients with initial VA < 20\/40.\nDotrelova et al.93\nRetrospective interventional case series.\n3 eyes\u2014sheathotomy.\n12 months.\nVA improved in 2 patients to 20\/40, in 1 patient stabilized (20\/180).\nShah et al.94\nRetrospective interventional case series.\n5 eyes\u2014sheathotomy.\n6.5 years.\nVA preoperative in all patients \u2264 20\/200. Improved in 4 eyes from 20\/30 to 20\/70. 1 eye with counting fingers remained unchanged.\nCrafoord et al.95\nRetrospective interventional case series.\n12 eyes\u2014sheathotomy.\n20 months.\nVA improved in 9 eyes (75%), in 1 eye (8.3%) remained unchanged and deteriored in 2 eyes (16.7%).\n2 patients received additionally 25 mg triamcinolone acetonide at the end of the surgery.\nHan et al.96\nRetrospective interventional case series.\n20 eyes\u2014pars plana vitrectomy and dissection of the arteriovenous crossing without separation of the vessels.\n10.5 months.\nIn 16 eyes (80%) improved VA \u2265 2 lines. Mean improvement of VA (logMAR) was = 0.44 \u00b1 0.14 (p = 0.016).\nSteroids\nIntravitreal Corticosteroids\nIn several nonrandomized comparative studies, intravitreal corticosteroids were successfully used for the treatment of BRVO. Currently published randomized studies are very rare and limited by virtue of evaluating patients with ME of different etiology, making comparisons difficult. In various studied doses from 4 to 25 mg, triamcinolone acetonide (TA) has been reported to be effective99\u2013117 (Table 6). In a randomized, interventional, three-arm clinical trial, Avitable et al.99 compared the results of treating diabetic patients and a small group of BVRO patients with cystoid ME by TA and MLG. From a total of 63 patients, 22 were treated by TA (4 mg), 21 underwent MLG, and in 20 patients these methods were combined (TA + MLG). The greatest improvement in VA was found in patients treated by TA combined with MLG. VA (log MAR) in this group increased significantly from 0.83 at baseline to 0.20 at the end of follow-up 9 months later (p = 0.003). In patients treated by TA, VA improved significantly, from 0.82 at baseline to 0.23 at 9 months after injection (p = 0.04). VA in the group of patients treated by MLG remained the same. The results of this study are limited, however, owing to the different ME etiologies in evaluated patients; only 6 patients had ME secondary to BRVO. Oh et al.100 used a retrospective interventional case series to compare VA after single TA injection (4 mg) in 10 patients with mean duration of ME \u2264 3 months after onset of BRVO versus 10 patients with ME > 3 months after onset. In patients with a disease duration \u22643 months, VA significantly improved from baseline over 6 months of follow-up. However in those with a duration of >3 months, improved VA, though apparent at one month, was not maintained at 3 or 6 months after TA injection. This study is limited by its retrospective design and short follow-up period. Ozkiris et al.101 evaluated the effect of TA injection on persistent ME in BRVO that failed to respond to previous laser photocoagulation. During a mean follow-up time of 6.2 months, best corrected VA (log MAR) improved significantly (p < 0.001) from 1.01 at baseline to 0.55 at one month after the injection. VA after 3 months was 0.56, and at the end of follow-up was 0.62. The authors concluded that intravitreal application of TA may be helpful in patients who do not respond to laser photocoagulation. However, in published studies, the resulting reduced macular thickness and improved VA, is only temporary and requires repeated treatment. One to four times re-application has been reported. Cekic et al.103 performed a retrospective chart review of 13 patients who underwent intravitreal injections with 4 mg TA. Six eyes received a single injection. Repeated injections were performed in 1 eye twice, 4 eyes three times, and 2 eyes four times. During a mean follow-up of 13 months, central foveal thickness decreased by more than 50%. Final VA improved in 7 eyes (range 2\u20136 Snellen lines), remained the same in 4 eyes (range 0\u20131 Snellen lines), and worsened in 2 eyes (range 1\u20134 Snellen lines) compared to baseline. Retinal thickness decreased in all cases, while vision improved in most cases. One of the most common side effects of TA was steroid-induced elevation of intraocular pressure.118 Other complications were infectious endophthalmitis, post-injection steroid-induced cataract, and retinal detachment.119,120 Reported risk of infectious endophthalmitis per injection range was from 0.1% to 1.6%.120 The most recent report by Bhavsar et al.121 found in two large studies-Diabetic Retinopathy Clinical Research Network (DRCR.net) and SCORE (Standard Care versus Corticosteroid for Retinal Vein Occlusion), an endophthalmitis prevalence of 0.05% (one case in the 2009 injections).\nTABLE 6\nSummary of studies evaluating the treatment of macular edema in BRVO by intravitreal application of triamcinolone acetonide (TA = triamcinolone acetonide, VA = visual acuity, ME = macular edema, MLG = macular laser grid photocoagulation)\nAuthor\nStudy type\nPatients\nFollow-up (mean)\nOutcomes\nComments\nAvitabile et al.99\nRandomized interventional, parallel, three-arm clinical trial.\nIntravitreal TA (4mg): 22 eyes. MLG: 21 eyes. TA+MLG: 20 eyes.\n9 months.\nTA group: VA improved from 0.82 to 0.23 log MAR (p = 0.04). MLG-group: VA unchanged. TA+MLG group, VA improved from 0.83 to 0.20. logMAR (p = 0.003).\nDifferent etiology of ME, only 6 eyes with BRVO.\nOh et al.100\nRetrospective interventional comparative case series.\n20 eyes with ME (4 mg TA) Disease duration: 10 eyes \u2264 3 months; 10 eyes > 3 months.\n6 months.\nGroup \u2264 3 months: VA (logMAR) improved from 1.07to 0.63 in 1 month (p = 0.012) and to 0.34 in 6 months (p = 0.005).\nOzkiris et al.101\nRetrospective, non-controlled case series.\n19 treated eyes (8 mg TA).\n6.2 months.\nVA (logMAR) improved from 1.01 \u00b1 0.16 to 0.62 \u00b1 0.22. VA improved in 17 eyes and remained unchanged in 2 eyes.\nJonas et al.102\nProspective nonrandomized comparative study.\n10 treated eyes (20 mg TA). 20 untreated controls.\nTA patients: 10.1 months. Controls: 6 months.\nTA patients: VA increased from 0.27 \u00b1 0.11 to 0.45 \u00b1 0.27 (p = 0.02). Controls: VA decreased significantly (p = 0.007).\nVA increased higher in non-ischemic group. Significant increase of intraocular pressure in treated group.\nCekic et al.103\nRetrospective non-controlled case series.\n13 eyes (4 mg TA).\n13 months.\nVA: improved in 7 eyes, remained the same in 4 eyes, worsened in 2 eyes. Foveolar thickness decreased in 56% of patients (p < 0.001).\nVA improvement significantly correlated with patient age (p = 0.026).\nLee et al.104\nRetrospective, non-controlled case series.\n6 eyes (4 mg TA).\n149.5 days.\nImprovement in VA \u2265 2 lines in 5 eyes (83.3%). VA from 20\/166 to final 20\/106.\n3 eyes treated with re-application of TA.\nOzkiris et al.105\nRetrospective interventional comparative case series.\n15 eyes (8 mg TA). 19 eyes MLG.\n6.3 months.\nVA (logMAR) improved in TA group from 0.98 to 0.24 and in MLG group from 1.02 to 0.5 (in both groups p < 0.001). Improvement in TA group was significantly higher than in MLG (p < 0.001).\nYepremyan et al.106\nRetrospective, non-controlled case series.\n12 eyes (4 mg TA).\n15.3 months.\nVA improved >3 lines in 50% of eyes after 1 month and in 42% of eyes at last follow up.\n8 eyes developed recurrent ME at an average of 5.5 months after initial TA injection.\nCheng et al.107\nProspective nonrandomized interventional comparative study.\n16 eyes (4 mg TA). 11 controls (without TA).\n103 days in TA-group. 94.5 days in controls.\nVA (logMAR). In TA-group: improvement from 0.77 \u00b1 0.43 to 0.44 \u00b1 0.43 (p < 0.001). No significant change of VA in controls.\nSignificant reduction of ME in TA-group (P < 0.001).\nChen et al.108\nCase report.\n1 eye (4 mg TA).\n3 months.\nImprovement in VA from counting fingers to 20\/80.\nEye with macular ischaemia.\nChen et al.109\nProspective interventional non-controlled case series.\n18 eyes (4 mg TA).\nAll patients completed 9 months, 12 eyes completed 12 months.\nVA (logMAR) improved from 0.81 \u00b1 0.36 to 0.65 \u00b1 0.3 (p = 0.03) after 1 months, no significant difference in VA after 3, 6, 9 and 12 months.\nEye with macular ischaemia. All eyes with macular ischaemia.\nTsujikawa et al.110\nProspective interventional non-controlled case series.\n17 eyes (vitrectomy + 10 mg TA intravitreal), 12 eyes of them with recurrent ME received sub-tenon 20 mg TA.\n12.1 months.\n82% of eyes rapid resolution of ME within 2 months (p = 0.041). 12 eyes (70.5%) received sub-tenon TA because of recurrent ME. Final VA (logMAR) improved from 0.74 \u00b1 0.40 to 0.40 \u00b1 0.34 (p = 0.010).\n14 eyes with vitrectomy underwent additional phacoemulsification with lens implantation.\nKaracorlu et al.111\nProspective interventional non-controlled case series.\n8 eyes (4 mg TA). All eyes with serous macular detachment.\n6 months.\nAfter TA regression of ME and serous macular detachment in all eyes. After 6 months recurrence in 2 eyes (25%) re-treatment occurred. Final VA improved in 7 eyes (87.5%).\nKrepler et al.112\nProspective interventional non-controlled case series.\n9 eyes (4 mg TA).\n6 months.\nSignificant improvement in reading VA only after 1 month (p = 0.02). No significant improvement in VA for distance. No significant reduction in macular thickness.\n5 eyes non-ischaemic BRVO. 4 eyes ischaemic BRVO.\nDegenring et al.113\nCase report.\n2 patients: 1 eye BRVO 1 eye CRVO (25 mg TA).\n5 weeks.\nPatient with BRVO improved VA from 0.25 to 0.5. Patient with CRVO from 0.4 to 0.5.\nWakabayshi et al.114\nProspective interventional non-controlled case series.\n5 eyes with CRVO. 11 eyes with BRVO. All eyes received sub-tenon injection of 20 mg TA.\n7 months.\n8 eyes (50%) improved VA and 2 eyes (12.5%) had worsening of VA at the time of final examination. Reduction of ME >30% of initial thickness in 13 eyes (81.3%). Because of recurrent ME \u2013 in 7 eyes repeated sub-tenon application of TA.\n1 eye with BRVO pretreated with laser photocoagulation because of retinal ischemia.\nSalinas-Alaman et al.115\nRetrospective interventional case series.\n5 eyes (4 mg TA).\n6 months\nImprovement of VA in 4 eyes. 1 eye underwent re-injection after 3 months because of recurrent ME.\nHirano et al.116\nRetrospective interventional comparative case-control study.\n8 eyes TA-injected group (simultaneous intravitreal and sub-tenon TA injection). 7 eyes vitrectomy with TA group (treated by vitrectomy and intravitreal or sub-tenon TA).\n12 months.\nVA improved significantly from baseline in both the TA-injected (p = 0.0069) and vitrectomy with TA groups (p = 0.0145). There was no significant difference in VA and macular thickness between the two groups.\nKuppermann et al.117\nRandomized interventional clinical trial.\n105 (20 with venous occlusion) eyes in each group: I. 700 \u03bcg Posurdex II. 350 \u03bcg Posurdex III. controls.\n3 months.\nImprovement of VA of \u226510 lines (ETDRS) 35% eyes in group 700 \u03bcg Posurdex, 24% in 350 \u03bcg Posurdex and 13% in control group (p < 0.001 versus 700 \u03bcg group; p = 0.04 versus 350 \u03bcg group).\nPreliminary report 60 eyes with retinal venous occlusion include eyes with CRVO and BRVO.\nMost published studies on intravitreal TA for BRVO, however, suffer from two serious flaws: either the designs are not randomized or they often do not clearly differentiate between nonischemic types and ischemic types of occlusion. To compare the effectiveness and safety of standard care versus TA injection in the treatment of ME in patients with CRVO and BRVO, the multicenter randomized study SCORE is ongoing (https:\/\/web.emmes.com\/study\/score). In each of the two disease areas, 630 participants will be randomized in a 1:1:1 ratio to one of three groups: standard care, intravitreal 4 mg of TA, or 1 mg of TA. The follow-up is planned for 3 years. The results are not published as yet. Biodegradable intravitreal implants may allow steroid delivery over a more sustained period, permitting longer duration of action. A multicenter randomized clinical trial which evaluates implantation of dexamethasone 350 \u03bcg or 700 \u03bcg (Posurdex) versus observation (no therapy) for ME secondary to a variety of retinal disorders (including BRVO) has been reported.117 The preliminary 90-day results of all 315 evaluated patients showed that an improvement in VA of 10 letters or more (in ETDRS) was achieved by a greater proportion of patients treated with dexamethasone 700 \u03bcg (35%) or 350 \u03bcg (24%), than untreated patients (13%; p < 0.001 versus 700 \u03bcg group; p = 0.04 versus 350 \u03bcg group). The results were similar for patients with diabetic retinopathy, retinal vein occlusion, or uveitis or Irvine-Gass syndrome. In total, 60 patients with BRVO were randomized 1:1:1 to receive 350 \u03bcg or 700 \u03bcg dexamethasone or observation (no therapy). In the case of RVO, the effect of the treatment was evaluated only in a common group (CRVO and BRVO patients together): an improvement in VA of 10 letters or more was achieved in 15% of untreated patients versus 31% of patients treated with dexamethasone 700 \u03bcg. The number of patients with an increase in intraocular pressure of more than 10 mmHg from baseline anytime during the study was 12% for 350 \u03bcg, 17% for 700 \u03bcg, and 3% for the untreated controls.117\nPeriocular Application of Triamcinolone Acetonide\nKawaji et al.122 evaluated in 20 patients the effectiveness and safety of trans-tenon retrobulbar injection of 40 mg of TA for ME associated with BRVO after vitrectomy. Improvement in VA was seen in 14 (70%) eyes. Hayashi et al.123 compared in a randomized clinical trial, the short-term effect of intravitreal versus retrobulbar injection of TA for the treatment of ME caused by BRVO. Sixty patients received either a single intravitreal injection (4 mg) or repeated retrobulbar injections (40 mg, three times) of TA. The first injection in the retrobulbar group was given approximately one week after focal laser photocoagulation. Foveal thickness, macular volume, and improvement in VA were significantly better after intravitreal injection than after repeated retrobulbar injections. The need for re-injections was significantly greater in the retrobulbar group than in the intravitreal group.\nIntravitreal Injection of VEGF Inhibitors\nVEGF inhibitors are a treatment option for ME associated with RVO that target the disease at the causal molecular level. Randomized studies evaluating the results of treatment of all available VEGF inhibitors (bevacizumab, ranibizumab, and pegaptanib) are ongoing. Case reports, small retrospective or prospective non-controlled studies of VEGF inhibitors in the treatment of ME and retinal neovascularizations secondary to BRVO, have been published.124\u2013140\nRosenfeld et al.124 first reported improved VA and reduced ME measured by optical coherent tomography (OCT) following intravitreal injection of bevacizumab for recurrent ME secondary to CRVO in an eye previously treated by intravitreal TA injection. In a short-term study, Iturralde et al.125 treated 16 eyes of CRVO with ME that had failed intravitreal corticosteroid therapy, and nearly every eye showed some anatomic or VA improvement following bevacizumab injection. In various reports, doses from 1.25 to 2.5 mg bevacizumab have been intravitreally administrated.125\u2013134 The most recently published studies evaluated the results in a group of patients with BRVO combined with patients with CRVO. In all of these studies, bevacizumab injection improved VA and reduced macular thickness measured by OCT within the first 3 to 9 weeks. Few studies are available for BRVO patients alone.126,127 Rabena et al.126 reported a significantly increased VA and reduced macular thickness after treatment with 1.25 mg bevacizumab in a retrospective study of 27 patients with BRVO. Recurrent ME was observed in 6 (22%) patients an average of 2.1 months after the initial injection. These patients were reinjected and all showed moderate to complete reduction in ME. The limitations of this retrospective study are short follow-up and lack of control group. Additionally, most of the eyes in the study were previously treated and thus failed standard treatment, and perhaps represent a group unlikely to benefit from any treatment. All published reports provide evidence that this treatment is well tolerated. The most common adverse events were conjunctival hyperemia and subconjunctival hemorrhage at the injection site. However, the duration of reduced ME after bevacizumab administration is currently unknown. Frequent repeated injections are required to prevent a rebound effect with no clearly defined endpoint.128\nCampochiaro et al.129 presented preliminary results of a randomized study in the treatment of BRVO with intravitreal injection of ranibizumab at the 2007 Annual Meeting of Association on Research and Vision in Ophthalmology (ARVO). Patients with ME due to CRVO or BRVO were randomized 1:1 to receive 3 monthly injections of 0.5 or 0.3 mg of ranibizumab. Interim results without regard to treatment assignment, which is unknown, showed that 12 randomized patients with BRVO gained an improvement in VA (in ETDRS) from 21 to 37 letters and a reduction in ME from 508 to 208 \u03bcm after 3 months of treatment. The endpoint results are expected to clarify any differences between the treatment groups. Another indication for anti-VEGF drugs are retinal neovascularizations, rubeosis iridis, and neovascular glaucoma. Rapid regression of neovascularizations and compensation of intraocular pressure have been described in several studies.136\u2013139 Intracameral application of bevacizumab as successful treatment of rubeosis iridis and neovascular glaucoma has also been reported.140\nProspective, controlled studies are mandatory to develop standardized treatment protocols that allow safe and effective application of anti-VEGF drugs.\nLaser Treatment\nLaser treatment is an established method for use in patients with BRVO. A large number of publications concerning the role of photocoagulation in the management of BRVO have appeared. Various laser techniques can be used: macula grid photocoagulation and the method of arterial crimping for treatment of ME, and peripheral scatter photocoagulation for treatment of retinal and\/or disc neovascularization.\nMacular Grid Laser Photocoagulation\nThe Branch vein occlusion study group remains the largest randomized prospective trial that has evaluated the efficacy of grid-pattern laser photocoagulation for the treatment of ME in BRVO.141 In this study, only eyes with recent BRVO, perfused ME, resolved foveal hemorrhage, VA 20\/40 or worse, and no other ocular comorbidities were included. After a 3-year follow-up period, 65% of treated eyes gained improvement of 2 or more lines from baseline, as opposed to 37% of untreated eyes. The number of eyes that lost 2 or more lines was not significantly different in the two groups.141 Parodi et al. published two randomized controlled studies, in which no significant benefit of MLG on VA was found.142,143 MLG is recommended as an effective treatment to reduce the ME in BRVO after a period of 3 to 6 months after onset and following absorption of the majority of hemorrhage if VA is 20\/40 or worse.61,62,141 If the fluorescein angiogram reveals macular nonperfusion, laser therapy is not warranted.141 Subramanian et al.65 recommended laser treatment in patients with poor VA (20\/200 or worse) secondary to ME due to BRVO, before more aggressive approaches (as intravitreal TA). Argon MLG is usually used for this purpose. However, diode laser (810 nm) and krypton red laser (647 nm) also can be used.63,141,144\nScatter Photocoagulation\nThe randomized controlled study by Branch vein occlusion study group54 reported that peripheral scatter laser photocoagulation significantly reduced the development of retinal neovascularization and vitreous hemorrhage. This study also demonstrated that, if all eyes with large retinal nonperfusion were treated, 64% of these patients would never develop neovascularization. If only the eyes that develop neovascularization were treated, the events of vitreous hemorrhage would decrease from 61% to 29%. Since loss in the lower part of the visual field can produce marked disability and BRVO involving the superior retina is common, a significant worsening of visual fields with laser treatment becomes a very important, clinically relevant finding.145 Therefore, waiting is generally advocated until neovascularization actually develops before scatter photocoagulation is considered.54\nArteriolar Constriction\nAn alternative type of laser treatment involves arteriolar constriction (called also \u201ccrimping technique\u201d) and may be considered in order to reduce the inflow into the affected area if the ME is excessive. This procedure was first described by L'Esperance146 in 1975. It may lead to a decrease in arterial pressure in the occluded region resulting in better drainage of the ME due to reduced blood inflow. The technique is employed by placing coagulations at approximate intervals of \u00bd disc diameter (using the green beam of argon laser) through the afferent arteriole in the region of venous blockage. In 1984 Jalkh et al.64 proposed their own modification of this method and published the results obtained in 41 eyes. In this study, arterial constriction was applied in the treatment of the chronic stage of BRVO. Rehak et al. published several studies describing the modified arteriolar constriction in patients with BRVO.147\u2013149 This technique consists of the application of coalescent coagulation spots through the afferent arteriole that supports the occluded venous region. 83% of patients treated by this method within the first 2 months after the onset of occlusion achieved a final VA 20\/40 or better.149 In a study by Erdol and Akyol,150 the improvement in VA was higher in a group of patients receiving the MLG combined with arteriolar constriction than in a group treated by MLG only. However, the difference in the resolution of ME between the groups was not statistically significant. The authors suggest that arteriolar constriction in addition to grid pattern laser photocoagulation is more effective for resolving ME in patients with BRVO.\nCONCLUSIONS\nThe pathogenesis of BRVO is multifactorial. Its resulting visual loss is due primarily to ME, macular nonperfusion, and retinal neovascularization. A large number of treatments have been advocated in its management. Unfortunately, almost all of these lack sufficient evidence for their effectiveness. Randomized prospective trials are essential. The only one established treatment for ME is macular grid photocoagulation in patients with BRVO longer than 3 months and a VA of 20\/40 or worse. Additionally, the initial VA may play a crucial role in the prognosis of BRVO and determinates the final VA.","keyphrases":["branch retinal vein occlusion","pathogenesis","visual prognosis","treatment","risk factors"],"prmu":["P","P","P","P","P"]}
{"id":"Curr_Genet-4-1-2413079","title":"A codon-optimized luciferase from Gaussia princeps facilitates the in vivo monitoring of gene expression in the model alga Chlamydomonas reinhardtii\n","text":"The unicellular green alga Chlamydomonas reinhardtii has emerged as a superb model species in plant biology. Although the alga is easily transformable, the low efficiency of transgene expression from the Chlamydomonas nuclear genome has severely hampered functional genomics research. For example, poor transgene expression is held responsible for the lack of sensitive reporter genes to monitor gene expression in vivo, analyze subcellular protein localization or study protein\u2013protein interactions. Here, we have tested the luciferase from the marine copepod Gaussia princeps (G-Luc) for its suitability as a sensitive bioluminescent reporter of gene expression in Chlamydomonas. We show that a Gaussia luciferase gene variant, engineered to match the codon usage in the Chlamydomonas nuclear genome, serves as a highly sensitive reporter of gene expression from both constitutive and inducible algal promoters. Its bioluminescence signal intensity greatly surpasses previously developed reporters for Chlamydomonas nuclear gene expression and reaches values high enough for utilizing the reporter as a tool to monitor responses to environmental stresses in vivo and to conduct high-throughput screenings for signaling mutants in Chlamydomonas.\nIntroduction\nThe unicellular green alga Chlamydomonas reinhardtii has become an invaluable model organism for plant biology (Harris 2001; Gutman and Niyogi 2004; Pr\u00f6schold et al. 2005). It represents one of the simplest photosynthetic eukaryotes, can be easily grown at large scale either photoautotrophically, mixotrophically or heterotrophically, and can be propagated sexually or asexually. Moreover, Chlamydomonas combines a powerful genetics with the availability of unique genetic and genomic resources: all three genomes are fully sequenced (nuclear, plastid, and mitochondrial; Merchant et al. 2007), large mutant collections have been established, and all three genomes are amenable to genetic manipulation by transformation (Hippler et al. 1998; Remacle et al. 2006). One of the few drawbacks of Chlamydomonas is that it has been notoriously difficult to express transgenes to reasonably high levels from the nuclear genome. Use of specialized promoters (Schroda et al. 2000; Fischer and Rochaix 2001) and adjustment of the transgene\u2019s codon usage to that of the highly GC-rich nuclear genome of the alga (Fuhrmann et al. 1999, 2004) helped in some cases, but no general solution to the problem has been found to date. This is highly unfortunate, because a number of valuable tools available in higher plants currently cannot be used routinely in Chlamydomonas. These include all applications of in vivo reporters of gene expression, such as, promoter-YFP\/GFP fusions for gene expression analyses and subcellular localization studies, as well as fluorescence resonance energy transfer (FRET) and biomolecular fluorescence complementation (BiFC) for monitoring protein\u2013protein interactions.\nRecently, codon-optimized reporter genes have been developed for nuclear and chloroplast expression in C. reinhardtii. Two such genes have been developed for nuclear expression: a gfp gene encoding the green fluorescent protein from the jellyfish Aequorea victoria (Fuhrmann et al. 1999) and a luciferase gene (R-Luc) from the sea pansy Renilla reniformis (Fuhrmann et al. 2004). Synthetic luciferase and gfp genes were also designed for chloroplast transformation and successfully used to measure plastid gene expression (Minko et al. 1999; Mayfield and Schultz 2004; Barnes et al. 2005). However, the use of these reporter genes for nuclear transformation is still far from being routine. While both reporters allowed detection or quantitation of the expression of some (fusion) genes (Fuhrmann et al. 1999; Shao et al. 2007), their generally low sensitivity has precluded the universal use of these reporters.\nTo overcome these limitations, we have explored a recently discovered new luciferase for its suitability as a more sensitive reporter of gene expression in Chlamydomonas. The luciferase from the copepod marine organism Gaussia princeps represents one of the smallest and brightest bioluminescent proteins known to date (Tannous et al. 2005; Remy and Michnick 2006). In an ATP-independent reaction, it catalyzes the oxidation of the substrate coelenterazine resulting in light emission at a wavelength of 480\u00a0nm. The Gaussia luciferase gene (G-Luc) has been successfully used as a reporter of gene expression in mammalian cells (Tannous et al. 2005) and could also be split and used for the detection of protein\u2013protein interactions by protein fragment complementation assays (Remy and Michnick 2006). It encodes a non-toxic, monomeric protein of only 185 amino acids and this small size may make it particularly suitable for expression in recalcitrant species.\nWe report here the development of the luciferase gene from G. princeps as a sensitive reporter gene for the in vivo monitoring of gene expression in Chlamydomonas. A codon-optimized version of the gene fused to Chlamydomonas expression signals generated more than 7-fold higher bioluminescence activity than the R-Luc gene from R. reniformis. Furthermore, G-Luc displayed drastically higher signal intensity than R-Luc in luminescence imaging (about 40-fold), thus facilitating the in vivo monitoring of responses to environmental stress stimuli in C. reinhardtii.\nMaterials and methods\nAlgal strains and culture conditions\nChlamydomonas reinhardtii strain 325 (CW15,mt+, arg7\u20138) was used in this study (kindly provided by Dr. Christoph F. Beck, University of Freiburg, Germany). Cultures were grown mixotrophically in Tris\u2013acetate phosphate (TAP) medium (Harris 1989) on a rotary shaker at 23\u00b0C under continuous irradiation with white light (55\u00a0\u03bcE\u00a0m\u22122\u00a0s\u22121). The TAP medium was supplemented with 100\u00a0mg\u00a0l\u22121 of arginine when required.\nNucleic acid manipulations\nThe coding region of the G. princeps luciferase gene (G-Luc) was synthesized de novo according to the nuclear codon usage of C. reinhardtii (GenScript, Piscataway, NJ). The synthetic G-Luc gene (GenBank accession number EU372000) was ligated as NdeI\/EcoRI fragment into the similarly digested PsaD expression cassette (Fischer and Rochaix 2001). Analogously, a codon-optimized R. reniformis luciferase gene (R-Luc; Fuhrmann et al. 2004) was cloned as NdeI\/EcoRI fragment into the PsaD cassette. For inducible expression, the G-Luc coding region was excised by digestion with HincII and BamHI and inserted into an inducible expression cassette driven by the Hsp70A promoter (Shao et al. 2007; Fig.\u00a01b).Fig.\u00a01Codon usage optimization of the G. princes luciferase and construction of expression cassettes for the transformation of Chlamydomonas reinhardtii. a Adaptation of the G-Luc gene to the codon usage in the nuclear genome of Chlamydomonas reinhardtii. The relative frequencies of the individual codons of the native G-Luc gene (GenBank accession number AY015993) in the Chlamydomonas nuclear genome are indicated by grey bars with the most frequently used triplet in Chlamydomonas set to 100%. The synthetic G-Luc gene (diamonds) was optimized by changing all codons to the most frequently used ones in Chlamydomonas reinhardtii. bG-Luc and R-Luc expression cassettes. To comparatively assess constitutive expression levels, the G-Luc (dark grey box) and R-Luc (hatched box) coding regions were inserted into the PsaD expression cassette (PsaD promoter and 5\u2032 UTR shown as open box; Fischer and Rochaix 2001). Both the R-Luc (Fuhrmann et al. 2004) and the G-Luc genes are optimized with regard to the codon usage in the Chlamydomonas nuclear genome. For inducible expression, the Hsp70A promoter (from position \u221223 to \u2212285 with respect to the translation initiation codon of Hsp70A) was fused to a gene fragment containing the 5\u2032 UTR and the first three exons of the Hsp70B gene (Shao et al. 2007). The positions of the three heat shock elements (HSE, black boxes) within the promoter region are indicated. Light grey bars represent exons, introns are depicted as broken lines. The third exon of Hsp70B was fused to the coding region of R-Luc and G-Luc, respectively. The 3\u2032 UTR of the reporter gene cassettes is derived from RBCS2, a nuclear gene for the small subunit of Rubisco (Shao et al. 2007)\nNuclear transformation of Chlamydomonas\nChlamydomonas nuclear co-transformation was carried out using the glass bead method (Kindle 1990). Plasmid DNA used for transformation was purified by PEG precipitation. Prior to transformation, plasmid pCB412 containing the C. reinhardtiiARG7 gene as selectable marker was linearized by digestion with EcoRI; all Luc constructs were linearized with ScaI. Co-transformation and selection for arginine prototrophy were employed to introduce the Luc constructs into the Chlamydomonas nuclear genome. Arginine prototrophic clones were selected on TAP medium. Transformants harboring the Luc constructs were identified by luciferase assays.\nBioluminescence assays\nCoelenterazine (P.J.K. GmbH; Kleinblittersdorf, Germany), the substrate for G-Luc and R-Luc, was dissolved in ethanol (1\u00a0mM stock solution). To assay luciferase activity, C.\u00a0reinhardtii cultures were grown in liquid TAP medium under constant illumination (55\u00a0\u03bcE\u00a0m\u22122\u00a0s\u22121) to a final cell density of 3\u20136\u00a0\u00d7\u00a0106\u00a0cells\u00a0ml\u22121. After sampling, cells were spun down, resuspended in the same volume of sample buffer [1.5\u00a0mM Tris\u2013HCl (pH\u00a07.8), 1\u00a0mM EDTA], and frozen at \u221220\u00b0C for at least 20\u00a0min. After thawing, 20\u00a0\u03bcl samples were transferred to 96-well, white microtiter plates and 125\u00a0\u03bcl of the assay buffer [0.1\u00a0M K2HPO4 (pH\u00a07.6), 0.5\u00a0M NaCl, 1\u00a0mM EDTA] was added to each well. Following incubation at room temperature for 15\u00a0min in the dark, bioluminescence was assayed using a luminometer (MicroBeta TriLux; PerkinElmer) by auto-injecting the substrate (coelenterazine 0.01\u00a0mM; 50\u00a0\u03bcl per well). The luminescence units are presented as luminescence counts per second (LCPS). The background was normalized by measuring wells containing only buffer or buffer with cells lacking the Luc gene. Inducible expression of the Luc fusion genes was normalized to the chlorophyll content of the cultures (Porra et al. 1989). The induction factor was calculated by comparison with untreated cells.\nFor in vivo luminescence imaging, C.\u00a0reinhardtii cultures were grown in TAP medium under constant illumination (55\u00a0\u03bcE\u00a0m\u22122\u00a0s\u22121) to a final density of 1\u20132\u00a0\u00d7\u00a0106\u00a0cells\u00a0ml\u22121. A volume of 3\u00a0\u03bcl cell suspension were spotted onto TAP agar plates and incubated under constant illumination (55\u00a0\u03bcE m\u22122\u00a0s\u22121) for 4\u00a0days. Luminescence of the cells was visualized in the presence of the substrate (coelenterazine 0.05\u00a0mM) using an ultra sensitive Photon Counting Camera (C2400-30H; Hamamatsu). Samples imaged in the absence of the substrate served as control. The luminescence images were acquired and processed with the HPD-LIS software (Hamamatsu) using an integration time of 20\u00a0min and linear signal intensity.\nRNA gel blot analyses\nTotal cellular RNA was extracted according to published protocols (von Gromoff et al. 1989). RNA samples (15\u00a0\u03bcg total RNA) were electrophoresed in formaldehyde-containing 1% agarose gels and blotted onto Hybond XL membranes (GE Healthcare). To produce a hybridization probe for detection of G-Luc transcripts, the coding region of the gene was excised from a plasmid clone. A Hsp70A-specific probe was prepared from a plasmid clone originally described as hsp70-2 (von Gromoff et al. 1989). The probes were purified by agarose gel electrophoresis following extraction of the DNA fragments of interest from excised gel slices using the Nucleospin Extract II kit (Macherey-Nagel, D\u00fcren, Germany) and then radiolabeled with 32P-dCTP using the MegaPrime kit (GE Healthcare). Hybridizations were performed at 65\u00b0C in Church buffer (Church and Gilbert 1984).\nResults\nDesign of the Gaussia luciferase as reporter gene for Chlamydomonas\nBioluminescent proteins are widely used as reporter genes to measure gene expression, determine subcellular protein localization, and study protein\u2013protein interactions. Luciferases are nontoxic, bioluminescent reporter proteins suitable to monitor gene expression quantitatively. Unfortunately, the previously constructed Chlamydomonas-specific Renilla luciferase (Fuhrmann et al. 2004) suffers from low sensitivity, presumably due to low expression levels and\/or low protein stability. As in mammalian cells, the luciferase from the marine copepod G. princeps proved to be a much more sensitive reporter than firefly and Renilla luciferases (Tannous et al. 2005); we set out to test this luciferase as a reporter gene in C. reinhardtii.\nPrevious work had shown that, in Chlamydomonas, adaptation of the codon usage of trangenes significantly improves expression levels (Fuhrmann et al. 1999, 2004). As the codon usage in the native luciferase from G. princeps deviated strongly from that in nuclear genes of C. reinhardtii, we adjusted all codons to the most preferred triplets in Chlamydomonas (according to the codon usage table for Chlamydomonas: http:\/\/www.kazusa.org.jp\/codon; Fig.\u00a01a). Following this codon optimization in silico, the gene was resynthesized and will be subsequently referred to as G-Luc standing for Gaussia luciferase gene (GenBank accession number EU372000). In all subsequent experiments, G-Luc was compared side-by-side to the Renilla luciferases (Fuhrmann et al. 2004), referred to as R-Luc.\nBoth luciferase genes were cloned into two different expression cassettes: (1) the PsaD cassette (Fischer and Rochaix 2001), whose promoter is constitutively active at least under photosynthetic conditions and (2) an inducible expression cassette driven by the Hsp70A promoter fused to the 5\u2032 region of the Hsp70B gene (Shao et al. 2007; Fig.\u00a01b). This heat shock gene promoter was shown previously to positively respond to a variety of inducing signals, including heat stress, light, retrograde signals from the plastid, and reactive oxygen species (von Gromoff et al. 1989; Kropat et al. 1997; Kropat and Beck 1998; Schroda et al. 2000; Shao et al. 2007). All gene constructs were introduced into arginine-auxotroph Chlamydomonas cells by glass bead-mediated co-transformation followed by selection for arginine prototrophy.\nExpression and heat inducibility of the Gaussia luciferase reporter\nTo compare the sensitivity of the Chlamydomonas-specific G-Luc as a reporter gene with that of the previously designed R-Luc, 24 transgenic clones from each construct with the constitutive PsaD promoter were randomly chosen and the six best-expressing ones were assayed for their luciferase activities. While only three out of six best R-Luc clones had significant luciferase activity, all six G-Luc clones showed high activity (Fig.\u00a02a). Moreover, when the activities were compared quantitatively, the G-Luc clones displayed, on average, more than 7-fold higher bioluminescence signal intensity than the R-Luc clones (Fig.\u00a02a), indicating that the new G-Luc is more sensitive and more efficient than previously established reporter genes for Chlamydomonas. The very high luciferase activities measured indicate that G-Luc will also be suitable for reporting expression from promoters that are considerably weaker than the PsaD promoter.Fig.\u00a02Comparison of G-Luc and R-Luc activity in transgenic Chlamydomonas reinhardtii strains. a Expression of G-Luc and R-Luc under the control of the constitutive PsaD promoter. For each construct, bioluminescence assays with six independent transformants were performed using a luminescence counter. The bars represent the mean of three independent experiments. The standard deviation is indicated. b Heat induction of the G-Luc and R-Luc reporters under the control of the Hsp70A promoter. Expression of luciferase in transformants harboring the PHsp70A-Luc constructs was induced by a temperature shift from 23 to 40\u00b0C for 1\u00a0h. After a 1\u00a0h recovery phase at room temperature, luciferase activity was assayed and the induction factors were calculated by comparison with untreated samples. Four arbitrarily chosen transformants were assayed for each luciferase construct. The bars represent the mean of three independent experiments, the standard deviation is indicated. The basal expression levels of the transformants under non-inducing conditions were 105, 5, 133 and 2\u00a0LCPS\u00a0\u03bcg\u22121 chlorophyll for the four R-Luc clones and 4, 5, 37 and 18\u00a0LCPS\u00a0\u03bcg\u22121 chlorophyll for the four G-Luc clones\nNext we wanted to compare the two luciferase genes when expressed under the control of the inducible Hsp70A promoter. To this end, inducibility was determined by measuring luciferase activities in the uninduced and induced states for the four best-expressing co-transformants from each construct (identified among 32 randomly picked clones). Transgene expression was induced by shifting the growth temperature of the algal culture from 23 to 40\u00b0C for 1\u00a0h. While background expression under non-inducing conditions was comparably low in R-Luc and G-Luc transformants, the G-Luc transformants showed much higher bioluminescence under inducing conditions (on average more than 7-fold; Fig.\u00a02b). This confirms the higher sensitivity of the G-Luc reporter for another expression cassette (Hsp70A promoter\u00a0+\u00a0RbcS terminator) and, moreover, indicates that G-Luc can be used as a highly sensitive reporter gene for measuring inducible gene expression in C. reinhardtii.\nLuciferase imaging and assessment of protein stability\nOne of the most powerful applications of luminescent reporter proteins is their use in genetic screens for mutants in cellular signal transduction pathways. This usually requires detection of the reporter gene activity by imaging techniques to facilitate high-throughput screening of mutagenized organisms. Unfortunately, due to the lack of sufficiently sensitive reporters, this has not been possible in Chlamydomonas to date. We, therefore, were interested in testing whether the sensitivity of our new G-Luc reporter gene was sufficiently high to allow visualization of gene expression by luciferase imaging.\nTo this end, we assayed luciferase activity from both the constitutive and the inducible expression constructs in vivo using a photon-counting camera. Even the best-expressing clone with R-Luc controlled by the PsaD expression cassette (Fig.\u00a02a) did not show enough luminescence to be detectable by luciferase imaging (Fig.\u00a03a). In contrast, G-Luc activity was sufficiently strong to be readily detectable (Fig.\u00a03a). Similar results were obtained for inducible expression from the Hsp70A promoter. While R-Luc activity was barely above the detection limit, inducibility of G-Luc expression was detected with high sensitivity (Fig.\u00a03b).Fig.\u00a03In vivo assay of Gaussia luciferase and Renilla luciferase activities in Chlamydomonas reinhardtii by visualizing luminescence with a photon-counting camera. Left panels show photographs of the algal colonies prior to luminescence imaging, right panels show the luminescence images. The clones with the highest luciferase expression levels in Fig.\u00a02 were used. a For detection of luciferase activity in living algae, wild-type cells (WT) and transformants harboring the PpsaD R-Luc or PpsaD G-Luc constructs (strains R1 and G9 from Fig.\u00a02a) were spotted in three replicas onto agar-solidified medium and grown under normal light conditions (55\u00a0\u03bcE\u00a0m\u22122\u00a0s\u22121) for 4\u00a0days. The ratio of luciferase signal intensities of PpsaD G-Luc:PpsaD R-Luc was approximately 40 (40.4\u00a0\u00b1\u00a02.5); the signal intensity of PpsaD R-Luc was only slightly above background (PpsaD R-Luc: WT\u00a0=\u00a03.4\u00a0\u00b1\u00a01.5). b Luminescence of the PHsp70A-Luc transformants induced by heat shock. The cultures were shifted from 23 to 40\u00b0C for 1\u00a0h. After recovery at room temperature for 1\u00a0h, the luminescence image was taken with a photon-counting camera. Luminescence intensities are color-coded with the maximum set to 1.3\u00a0\u00d7\u00a0105\nDifferent possible explanations can account for the much better performance of G-Luc compared to R-Luc: higher expression rates, higher enzymatic activity or higher stability of the Gaussia enzyme. To distinguish between these possibilities, we performed stability assays by measuring luciferase activities in dependence on the temperature. To this end, algal cultures were subjected to 30\u00a0min of high temperature incubation followed by a 30\u00a0min recovery phase at room temperature prior to measurement of luciferase activity. If the Gaussia enzyme were indeed more stable than the Renilla enzyme, its activity should decline less sharply with temperature. This was indeed the case (Fig.\u00a04a): while the Renilla luciferase suffered a strong temperature-dependent decline in activity, the Gaussia enzyme was much less affected, suggesting that higher enzyme stability contributes substantially to the superior performance of G-Luc.Fig.\u00a04Monitoring the heat stress response in Chlamydomonas strains expressing luciferase constructs driven by the heat-inducible Hsp70A promoter. a Assessment of the thermostability of the Gaussia and Renilla luciferases. PpsaD-Luc transformants were subjected to 30\u00a0min of high temperature treatment as indicated followed by a 30\u00a0min recovery phase at room temperature. Luciferase activities were measured with a luminescence counter. Note the high temperature sensitivity of the Renilla enzyme with all activity being lost at temperatures above 46\u00b0C. In contrast, the Gaussia enzyme appears to be much more stable, facilitating its use as a sensitive reporter of heat stress-induced gene expression. b Visualization of the heat stress response with G-Luc. For luciferase imaging, wild-type cells (WT) and transformants harboring PHsp70A R-Luc, PHsp70A G-Luc or P\u2206HSEG-Luc (G-Luc fusion to a Hsp70A promoter lacking the HSE region; Shao et al. 2007) were spotted in three replicas on agar plates, grown under normal light conditions (55\u00a0\u03bcE\u00a0m\u22122\u00a0s\u22121) for 4\u00a0days, photographed (left picture), and then shifted from 23 to 47\u00b0C for 15\u00a0min. After 3.5\u00a0h at room temperature (RT), the luminescence of the transformants upon substrate addition was recorded with a photon-counting camera (right picture). c Analysis of the kinetics of luciferase induction in a PHsp70A G-Luc transformant exposed to heat stress. Colonies of the PHsp70A G-Luc strain growing on a TAP agar plate were exposed to 40 or 47\u00b0C for 15\u00a0min. After 1, 3 or 5\u00a0h recovery at RT, the luminescence of the colonies was visualized using a photon-counting camera. d Comparison of the induction of G-Luc expression by heat stress with induction of the endogenous Hsp70A gene at the mRNA level. Expression from the Hsp70A promoter was induced by a 15-min incubation at 40 or 47\u00b0C, and then followed over time by incubation at room temperature (RT) for the time spans indicated. The induction kinetics of luciferase expression from the Hsp70A promoter parallels that of the endogenous Hsp70A gene\nTo explore the heat inducibility of G-Luc under the control of the Hsp70A promoter in somewhat greater detail, we sought to identify optimum experimental conditions for conducting genetic screens for signaling mutants. We, therefore, tested different combinations of temperatures of the heat shock and recovery times and also included a control construct, in which the heat-shock elements (HSE) were deleted from the Hsp70A promoter (Shao et al. 2007). As expected, this deletion completely abolished heat inducibility under all conditions tested (Fig.\u00a04b and data not shown). Efficient heat induction of the G-Luc reporter was achieved in a wide temperature range, from 40 to 47\u00b0C (cp. Figs.\u00a03b, 4b). However, heat shock at higher temperatures required longer recovery times at room temperature before luciferase activity could be visualized by imaging. Whereas following heat shock at 40\u00b0C, maximum bioluminescence was measured after 1\u00a0h recovery, a recovery phase of 3\u00a0h was required to obtain similarly high bioluminescence after a heat shock at 47\u00b0C (Fig.\u00a04c).\nNext we wanted to confirm that heat induction of luciferase activity parallels G-Luc mRNA accumulation. This was clearly the case upon both induction at 40\u00b0C and induction at 47\u00b0C (Fig.\u00a04d). At both temperatures, mRNA levels peaked at about the same time as enzyme activities (cp. Fig.\u00a04c, d). Moreover, the kinetics of G-Luc mRNA accumulation correlated, by and large, with heat induction of the endogenous Hsp70A gene (Fig.\u00a04d), ultimately confirming that the luciferase reporter faithfully mirrors promoter activity.\nHaving established that G-Luc expression can be readily monitored by luminescence imaging, we finally wanted to provide a quantitative assessment of the superior performance of the G-Luc reporter by direct luciferase imaging of primary transformants. To this end, Petri dishes with transformed Chlamydomonas colonies were exposed to the substrate and analyzed by luminescence imaging (Fig.\u00a05). While transformation with G-Luc produced a high number of brightly luminescing colonies, R-Luc luminescence was much lower and barely detectable (Fig.\u00a05). These data ultimately confirm the much higher sensitivity of the G-Luc reporter and its suitability for luminescence imaging.Fig.\u00a05In vivo assay of Gaussia luciferase and Renilla luciferase activities in primary Chlamydomonas transformants by visualizing luminescence with a photon-counting camera. Co-transformation experiments were conducted using identical amounts of linearized DNA (500\u00a0ng Luc-containing plasmid\u00a0+\u00a0100\u00a0ng ARG7-containing plasmid) and identical amounts of algal cells. Upper panels show photographs of the algal colonies prior to luminescence imaging, lower panels show the luminescence images. Luminescence intensities are color-coded with the maximum set to 1.3\u00a0\u00d7\u00a0105. a Luminescence of PpsaD-Luc transformants. The ratio of total-plate luminescence of PpsaD G-Luc:PpsaD R-Luc was approximately 36. b Luminescence of PHsp70A-Luc transformants induced by heat shock. The cultures were shifted from 23 to 40\u00b0C for 1\u00a0h. After recovery at room temperature for 1\u00a0h, the luminescence image was taken. The ratio of total-plate luminescence of PHsp70A G-Luc:PHsp70A R-Luc was approximately 16. Note that a large fraction of the non-luminescing colonies is not co-transformed (i.e., harbors the selectable marker gene but not the luciferase reporter)\nDiscussion\nIn this work, we have established the luciferase from the marine copepod G. princeps as a novel and highly sensitive bioluminescent reporter in the model alga C. reinhardtii. The G-Luc reporter outperforms previously developed reporter genes for Chlamydomonas, improves the monitoring of gene expression and, most importantly, represents the first nuclear reporter gene that is sufficiently sensitive to facilitate in vivo imaging in Chlamydomonas. This expands the toolbox available for Chlamydomonas genetics and cell biology and will make possible experimental approaches that theretofore could not be taken in Chlamydomonas. First and foremost, the possibility to conduct large-scale mutant screens by bioluminescence imaging of live algal colonies will facilitate powerful genetic strategies for the isolation of novel components of all those signal transduction cascades that modify gene expression by targeting specific promoters. For example, the inducible promoter used in this study (Hsp70A) is the target of several distinct signal transduction pathways in response to heat, retrograde signals from the chloroplast, and reactive oxygen species (Shao et al. 2007). Mutagenesis of our algal strains expressing G-Luc from this promoter (Fig.\u00a04b, c), followed by selection for mutants incapable of inducing the luciferase gene in response to a specific stress stimulus, should allow the genetic dissection of the underlying signal transduction pathways. Our preliminary results indicate that reactive oxygen species, which are much weaker inducers of the promoter than heat stress (Shao et al. 2007), induce G-Luc expression sufficiently strongly to facilitate such a screen in a microtiter plate format, although screening on agar plates will require further optimization and improvement of the assay sensitivity.\nIn mammalian cells, a split version of the Gaussia luciferase was successfully used for the detection of protein\u2013protein interactions in vivo by protein fragment complementation assays (Remy and Michnick 2006). As currently no method is available to identify protein\u2013protein interactions in Chlamydomonas cells, the development of screens for protein interaction partners seems to be a particularly promising future application of the G-Luc reporter.\nIt should be noted that thus far, the G-luc reporter gene has been tested only in cell wall-deficient Chlamydomonas strains, which are easily transformable. It remains to be tested whether substrate uptake or luminescence imaging are influenced by the more rigid walls present in strains with wild type-like cell wall structure.\nAlthough the Chlamydomonas-specific G-Luc described here provides a workable reporter of gene expression that is significantly more sensitive than previously established reporter genes, our data indicate that the superior performance of the Gaussia luciferase gene is not due to its better expression in C. reinhardtii, but rather due to its very high enzyme stability. Most probably, the adaptation of other widely used reporter genes to C. reinhardtii (like the genes for the fluorescent proteins GFP and YFP) will require a general solution to the transgene expression problem in Chlamydomonas. This could be achieved by either developing novel expression tools or generating dedicated expression strains in which the suspected epigenetic transgene silencing mechanism is inactivated.","keyphrases":["luciferase","gaussia princeps","chlamydomonas reinhardtii","reporter gene","bioluminescence","heat-inducible expression"],"prmu":["P","P","P","P","P","R"]}
{"id":"Osteoporos_Int-3-1-1820756","title":"Bone loss in elderly men: increased endosteal bone loss and stable periosteal apposition. The prospective MINOS study\n","text":"Introduction Longitudinal studies on the age-related bone loss in men concerns the decrease in areal bone mineral density (aBMD), which can be qualified as \u201capparent bone loss\u201d because it does not reflect the change in bone mineral content (BMC). Loss of BMC can be referred to as \u201cnet bone loss\u201d because it does not take into account the morphological basis of the bone loss (decreased periosteal apposition; endosteal bone loss, i.e. bone loss on the trabecular, endocortical and intracortical surfaces). The aim of this study was to assess age-related apparent net and endosteal bone loss as well as their morphological basis and age-related changes during a prospective follow-up in a large cohort of elderly men.\nIntroduction\nOsteoporosis in elderly men is a major problem of public health. Fracture incidence increases exponentially with age, thus, total number of fragility fractures in men increases rapidly, mainly due to the increasing life expectancy [1]. Currently, 25\u201330% of fragility fractures occur in men [1, 2]. Moreover, postfracture morbidity and mortality are higher in men than in women [3, 4].\nBone loss is defined usually as the rate of decrease in areal bone mineral density (aBMD) measured by dual X-ray absorptiometry (DXA) [5\u20137]. However, aBMD of a given region of interest (ROI) is determined by its bone mineral content (BMC) and by its projected area. For tubular bones, ROI length is kept constant, and the projected area depends on bone width. Bone width increases with ageing due to the periosteal apposition [8, 9]. Thus, if the amount of bone deposited on the periosteal surface and that resorbed on the endosteal surfaces (i.e. trabecular, endocortical and intracortical) are equal, BMC remains constant, but aBMD decreases because bone width and its projected area have increased. Therefore, it is more appropriate to refer the decrease in aBMD assessed longitudinally as \u201capparent bone loss\u201d.\nChanges in BMC of a given ROI are determined not only by the quantity of bone resorbed on the endosteal surfaces but also by the quantity of bone deposited on the periosteal surface. Thus, change in BMC does not reflect the quantity of bone really lost on the endosteal surfaces and, therefore, the decrease in BMC is better qualified as \u201cnet bone loss\u201d. Estimation of bone gain due to periosteal apposition would allow assessment of the quantity of bone really lost on the endosteal surfaces. This quantity of bone could be referred as to \u201cendosteal bone loss\u201d.\nIn elderly men, apparent bone loss accelerates with age [5\u20137]. However, the morphological basis underlying this acceleration has not been studied, and it is unclear whether it is related to faster outward displacement of a constant amount of bone, to higher endosteal bone loss and\/or to a lower periosteal apposition. Moreover, only a few studies have compared the rate of apparent bone loss at different sites of measurement in elderly men [10\u201312], although it appears that the morphological basis underlying apprent bone loss may vary according to ROI [13].\nTherefore, the aim of this study was to assess age-related apparent net and endosteal bone loss as well as their morphological basis and the age-related changes during a long-term prospective follow-up in a large cohort of elderly men (the MINOS study).\nSubjects and methods\nCohort\nThe MINOS study is a prospective study of osteoporosis and its determinants in men, which was initiated in 1995 [14]. It is the result of a collaboration between the National Institute of Health and Medical Research (INSERM) and Soci\u00e9t\u00e9 de Secours Mini\u00e8re de Bourgogne (SSBM) in Montceau les Mines, a town located 130\u00a0km northwest of Lyon in the Department (District) of Sa\u00f4ne et Loire. The town has a population of 21,000 inhabitants, including 7,150 men > 19\u00a0years if age. SSMB is one of the largest health insurance companies in this town. The study was performed in accord with the Helsinki Declaration of 1975 as revised in 1983. The MINOS cohort consists of 1,040 men aged 19\u201385 recruited between 1995 and 1998. All men responded to an epidemiological questionnaire covering demographic and behavioural information as well as detailed medical history. Men aged 19\u201350 were examined once, whereas 790 men aged 50\u201385 were followed up prospectively for 90\u00a0months. Every 18\u00a0months, they were invited to attend the follow-up examination composed of an epidemiological questionnaire and DXA measurement. Radiograph of the spine were performed at baseline, 36 and 90\u00a0months.\nMeasurements\naBMD and BMC were measured at the lumbar spine (L2\u2013L4), hip and whole body using pencil-beam DXA (QDR 1500, Hologic Inc., Waltham, MA, USA) and at the distal nondominant forearm using single energy X-ray absorptiometry (Osteometer, DTX-100, Denmark). The OsteoDyne Hip Positioner System (HPS) was used to minimise hip positioning error. ROI of the femoral neck was positioned perpendicularly to the axis of the femoral neck to cover its narrowest part. When necessary, the femoral neck edges were adjusted manually. The QDR 1500 device was calibrated daily using a lumbar spine phantom, yielding a CV for aBMD of 0.33 %. Twice a month, the Hologic hip phantom was measured, yielding a long-term CV of 0.94 % for femoral neck aBMD and 1.05 % for the femoral neck projected area. Also twice a month, a human lumbar spine embedded in methyl methacrylate was measured. Its long-term CV was 1.07 % for BMC of L2\u2013L4, 1.07 % for the projected area of L2\u2013L4, and 0.62 % for aBMD of L2\u2013L4. At the distal forearm, the distal site includes 20\u00a0mm of radius situated proximally to the site where the spacing between the medial edge of the radius and the lateral edge of the ulna is 8\u00a0mm. Scans with evident error of positioning were excluded. The densitometer was calibrated daily using a calibration standard for DTX 100; its long-term CV was 0.47 % for aBMD and 0.15 % for the projected area.\nDimensions of the vertebral body of the third lumbar vertebra (L3) were measured on the anteroposterior and lateral radiographs of the lumbar spine performed at baseline and at 90\u00a0months. The cross-sectional area of L3 was calculated based on the anteroposterior and frontal diameters measured in the narrowest site of the vertebral body. Osteoarthritis of the lumbar spine was assessed, as described previously [14].\nThe external diameter of bone was calculated as the projected area of ROI divided by its length. The rate of age-related periosteal expansion was calculated as the average annual increase in the external diameter (femoral neck, distal radius and distal ulna), projected area (total hip) or cross-sectional area (L3). Age-related periosteal apposition (\u0394BMCPA) was estimated as the mass of bone deposited on the outer surface of bone since the first measurement (Fig.\u00a01). The volume of the ellipsoid cylinder was calculated assuming that the short axis was 0.75 of the long axis (external diameter). Then, the mass of the deposited bone was calculated as the product of the cylinder volume and the volumetric density of bone mineral (1.15\u00a0g\/cm3) [15]. We assumed that during the follow-up, BMC was determined by baseline BMC, bone mass deposited on the outer surface (\u0394BMCPA) and endosteal bone loss (\u0394BMCEBL). Thus, \u0394BMCEBL can be calculated using BMC at baseline and during the follow-up and the calculated value of \u0394BMCPA. The concept of endosteal bone loss does not make any assumption as to its underlying morphological basis (cortical thinning, increased cortical porosity, trabecular bone loss) nor on the proportion of cortical to trabecular bone. It only reflects the loss of bone mineral \u201cinside\u201d bone, which is represented in Fig.\u00a01 by the change of the colour from black to grey.\nFig.\u00a01Calculation of bone mass deposited by periosteal apposition (\u0394BMCPA) and of endosteal bone loss (\u0394BMCEBL) between baseline (t0) and follow-up (t1). Long semi-axes of the ellipse at baseline (R0) and during the followup (R1) are equal to half the external diameter of the region of interest (ROI). Bone mass deposited by periosteal apposition (\u0394BMCPA) is the product of the ellipsoid volume and the vBMD of cortical bone (d\u2009=\u20091.15\u00a0g\/cm3). Coefficient 0.75 reflects bone flattening in the anteroposterior projection in comparison with its diameter in the frontal projection. Endosteal bone loss (\u0394BMCEBL) is presented graphically as the brighter colour of the entire cross-sectional area of bone (black at baseline and light grey at follow-up) in order to indicate that the \u201cendosteal bone loss\u201d makes no distinction as to the morphological basis (cortical thninning, increase in cortical porosity, trabecular bone loss)\nStatistical methods\nAll calculations were performed by using SAS version 8.2 software (SAS Institute Inc., Cary, NC, USA). Correlation between continuous variables was assessed by Pearson\u2019s simple correlation coefficient. Individual slopes were calculated by using simple linear regression. Comparisons of the individual slopes between age groups were performed by analysis of variance (ANOVA) and adjusted for multiple comparisons by Dunnett-Hsu test. Participants attended two to six exams; those who had few measurements, e.g. two, had them over different periods of follow-up (18\u201390\u00a0months). Individual slopes were calculated by using two to six points distributed over 18\u201390\u00a0months, which could influence accuracy of the calculation. We adjusted for the duration of follow-up or the number of measurements to check whether duration of follow-up and number of measurements influenced the results. Each of them entered significant in the majority of models, although they influenced the results only to a limited degree. We present data adjusted for the duration of follow-up because this variable attained higher level of significance in the models and contributed more to the final model.\nResults\nCharacteristics of the investigated cohort\nSixty-five men who abandoned the study after the first examination were, at baseline, older and had lower physical activity but higher prevalence of diabetes mellitus, rheumatoid arthritis and parkinsonism than the 725 men who participated in the follow-up (Table\u00a01). They had lower aBMD and BMC at all sites of measurement except for the lumbar spine. The difference between the groups became weaker after the adjustment for age; however, it remained significant for certain sites. \nTable\u00a01Comparison of 725 men participating in the prospective study and 65 men lost to follow-up after recruitmentParameterFollow-up (n\u2009=\u2009725)No follow-up (n\u2009=\u200965)p value*p value**Age (years)65\u2009\u00b1\u2009770\u2009\u00b1\u20098< 0.0001Body weight (kg)80\u2009\u00b1\u20091379\u2009\u00b1\u2009150.29Body height (cm)169\u2009\u00b1\u20096168\u2009\u00b1\u200970.21BMI (kg\/m2)27.98\u2009\u00b1\u20093.6427.75\u2009\u00b1\u20094.510.63Fat mass (kg)22.02\u2009\u00b1\u20097.4823.23\u2009\u00b1\u20099.110.28Lean mass (kg)54.54\u2009\u00b1\u20096.6652.16\u2009\u00b1\u20097.56< 0.01NSTobacco smoking (%)11.811.60.98Physical activity (h\/week)21.8\u2009\u00b1\u200912.717.2\u2009\u00b1\u200911.3< 0.005NSPrevalent fractures (%)13.819.70.18Diabetes (%)6.515.7< 0.005Rhumatoid arthritis (%)1.45.7< 0.01Parkinsonism (%)1.55.7< 0.02Lumbar spine BMD (g\/cm2)1.031\u2009\u00b1\u20090.1841.052\u2009\u00b1\u20090.2130.21Femoral neck BMD (g\/cm2)0.845\u2009\u00b1\u20090.1210.803\u2009\u00b1\u20090.127< 0.01NSFemoral neck BMC (g)5.111\u2009\u00b1\u20090.8494.883\u2009\u00b1\u20090.934< 0.04NSFemoral neck width (cm)4.082\u2009\u00b1\u20090.3164.123\u2009\u00b1\u20090.3560.31Trochanter BMD (g\/cm2)0.740\u2009\u00b1\u20090.1090.691\u2009\u00b1\u20090.121< 0.001< 0.03Total hip BMD (g\/cm2)0.966\u2009\u00b1\u20090.1270.910\u2009\u00b1\u20090.157< 0.001NSWhole-body BMC (g)2706.6\u2009\u00b1\u2009410.42550.3\u2009\u00b1\u2009472.7< 0.005NSWhole-body BMD (g\/cm2)1.210\u2009\u00b1\u20090.1081.167\u2009\u00b1\u20090.121< 0.003< 0.05Distal forearm BMD0.524\u2009\u00b1\u20090.0650.483\u2009\u00b1\u20090.070< 0.0001< 0.01Ultradistal radius BMD0.430\u2009\u00b1\u20090.0640.399\u2009\u00b1\u20090.072< 0.001< 0.05Radius BMD (g\/cm2)0.556\u2009\u00b1\u20090.0680.513\u2009\u00b1\u20090.075< 0.0001< 0.01Radius BMC (g)2.743\u2009\u00b1\u20090.4032.527\u2009\u00b1\u20090.422< 0.0001< 0.01Radius width (cm)2.471\u2009\u00b1\u20090.2072.468\u2009\u00b1\u20090.2160.94Ulna BMD0.476\u2009\u00b1\u20090.0660.438\u2009\u00b1\u20090.070< 0.0001< 0.01Ulna BMC (g)1.502\u2009\u00b1\u20090.2441.401\u2009\u00b1\u20090.246< 0.0001< 0.01Ulna width (cm)1.659\u2009\u00b1\u20090.1361.684\u2009\u00b1\u20090.1520.16BMD bone mineral density, BMC bone mineral content, NS not significantp* difference between the groupsp** age-adjusted difference between the groups\nCharacteristics of bone loss\nBMC and aBMD of the lumbar spine increased significantly (Table\u00a02) faster in men with severe arthritis (5.95\u2009\u00b1\u200916.15 vs 1.74\u2009\u00b1\u200910.96\u00a0mg\/cm2\/year; p\u2009<\u20090.0001). At the femoral neck, aBMD decreased, whereas BMC increased. For other sites of measurement (hip, whole body, distal forearm), both aBMD and BMC decreased. The fractional apparent and net bone loss (percentage of initial value) were fastest at the distal forearm and slowest at the whole body. All the above absolute and fractional changes in aBMD and BMC were significantly different from 0 for all ROIs. \nTable\u00a02Average rate of apparent bone loss [change in areal bone mineral density (aBMD)], net bone loss [change in bone mineral content (BMC)] and of periosteal expansion (increase in bone width or area) as well as the simple correlation coefficients of these variables with age in 725 men aged 50\u201385 at baseline followed up prospectively for 90\u00a0months (the prospective MINOS study)Site of measurementYearly changeCorrelation with ageBone mineral density(mg\/cm2\/year)(%\/year)rp\u00a0Lumbar spine4.205\u2009\u00b1\u200914.210.495\u2009\u00b1\u20092.910\u22120.0690.07\u00a0Femoral neck\u22122.463\u2009\u00b1\u20098.305\u22120.282\u2009\u00b1\u20091.019\u22120.154< 0.0001\u00a0Trochanter\u22121.963\u2009\u00b1\u20098.031\u22120.276\u2009\u00b1\u20091.123\u22120.196< 0.0001\u00a0Total hip\u22124.714\u2009\u00b1\u20098.451\u22120.496\u2009\u00b1\u20090.930\u22120.213< 0.0001\u00a0Whole body\u22122.081\u2009\u00b1\u20098.712\u22120.177\u2009\u00b1\u20090.723\u22120.0450.22\u00a0Distal forearm\u22122.937\u2009\u00b1\u20094.178\u22120.580\u2009\u00b1\u20090.870\u22120.202< 0.0001\u00a0Distal radius\u22122.986\u2009\u00b1\u20095.340\u22120.561\u2009\u00b1\u20091.041\u22120.180< 0.0001\u00a0Distal ulna\u22123.353\u2009\u00b1\u20095.131\u22120.730\u2009\u00b1\u20091.206\u22120.119< 0.002\u00a0Ultradistal radius\u22121.823\u2009\u00b1\u20095.334\u22120.426\u2009\u00b1\u20091.285\u22120.128< 0.001Bone mineral content(mg\/year) (%\/year) \u22120.0410.27\u00a0L3118.17\u2009\u00b1\u2009552.900.658\u2009\u00b1\u20092.954\u00a0Femoral neck12.67\u2009\u00b1\u200950.380.263\u2009\u00b1\u20091.010\u22120.0510.18\u00a0Total hip\u2212226.1\u2009\u00b1\u20090.687\u22120.504\u2009\u00b1\u20091.551\u22120.120< 0.002\u00a0Whole body\u22127565.9\u2009\u00b1\u200922500.9\u22120.294\u2009\u00b1\u20090.877\u22120.192< 0.0001\u00a0Distal radius\u221211.26\u2009\u00b1\u200925.90\u22120.426\u2009\u00b1\u20090.978\u22120.179< 0.0001\u00a0Distal ulna\u22128.64\u2009\u00b1\u200916.82\u22120.559\u2009\u00b1\u20091.164\u22120.123< 0.001Bone size\u00a0L3 cross-section (mm2\/year)35.64\u2009\u00b1\u2009225.400.167\u2009\u00b1\u20091.184\u22120.0560.10\u00a0Femoral neck (\u03bcm\/year)133.1\u2009\u00b1\u2009217.20.321\u2009\u00b1\u20090.5030.0700.06\u00a0Total hip (mm2\/year)4.82\u2009\u00b1\u200942.230.112\u2009\u00b1\u20090.9150.0070.85\u00a0Distal radius (\u03bcm\/year)60.48\u2009\u00b1\u2009362.660.257\u2009\u00b1\u20091.4970.0340.36\u00a0Distal ulna (\u03bcm\/year)58.27\u2009\u00b1\u2009156.610.355\u2009\u00b1\u20090.9370.0130.73\nExternal diameter of the femoral neck, distal radius and distal ulna as well as the cross-sectional area of L3 and the projected area of total hip increased significantly during the follow-up. Fractional increase in bone size varied from 0.17% to 0.36% per year across the sites.\nCharacteristics of the rate of bone loss according to age at baseline\nAt the hip and distal forearm, rate of apparent bone loss (change in aBMD) was negatively correlated with age, indicating and age-related acceleration of apparent bone loss (Table\u00a02). After the age of 70, the apparent bone loss was two (for distal forearm) to 23 (for trochanter) times faster than in men aged younger than 60 at baseline (Fig.\u00a02). A similar trend was found when apparent bone loss was expressed as the percentage of the baseline value of aBMD.\nFig.\u00a02Comparison of the absolute values of apparent bone loss [change in areal bone mineral density (aBMD)] according to age group (black bars 50\u201360\u00a0years, pointed bars 61\u201370\u00a0years, white bars > 70\u00a0years at baseline). The slopes are significantly different from 0 (p\u2009<\u20090.005\u20130.0001) for all regions of interest (ROIs) and for all age groups, except for the trochanter in the youngest group\nNet bone loss (change in BMC) also accelerated with age at the total hip, whole body and bones of distal forearm. In men aged older than 70, net bone loss was two to four times as fast in comparison with men aged younger than 60 at baseline, both for the absolute values of net bone loss and for data expressed as percentage of baseline BMC (Fig.\u00a03).\nFig.\u00a03Comparison of the absolute values of net bone loss [(change in bone mineral content (BMC)] according to age group (black bars 50\u201360\u00a0years, pointed bars 61\u201370\u00a0years, white bars >70\u00a0years old). The slopes are different from 0 (p\u2009<\u20090.005\u20130.0001) for all regions of interest (ROIs) and for all age groups\nIn 183 men aged 50\u201360, rates of apparent and net bone loss were significantly different from 0 for all ROIs, except the trochanter. In 387 men aged 61\u201370 and in 155 men aged older than 70 at baseline, rates of apparent and net bone loss were significantly different from 0 for all ROIs. The rate of periosteal expansion (expressed as the change in the projected area of bone) was significantly different from 0 for all ROIs and for all three age groups. However, it did not vary across age groups for any site of measurement. Similarly, at distal forearm bones, periosteal apposition estimated as bone mass deposited annually did not change across the age groups (Fig.\u00a04). By contrast, estimated endosteal bone loss was about twice as high after the age of 70 compared with men aged younger than 60 at baseline.\nFig.\u00a04Upper panel: comparison of absolute values of annual rates of increase in external diameter at the distal radius and ulna according to age group. Lower panel: comparison of absolute values of rates of deposition of bone mass by periosteal apposition (positive hatched bars) and rate of endosteal bone loss (negative pointed bars) at the distal radius and ulna according to age group. The slopes are significantly different from 0 (<\u20090.005\u20130.0001) for periosteal apposition and for endosteal bone loss for both bones and for all age groups. Please note, in the lower panel, scales are different for radius and ulna\nDiscussion\nIn men, aBMD increased at the spine and decreased at the hip, distal forearm and whole body. At these sites, apparent bone loss (change in aBMD) was associated with net bone loss (change in BMC) except for the femoral neck. Apparent and net bone loss accelerated with age, whereas periosteal expansion rate remained stable. At the distal radius and ulna, endosteal bone loss accelerated with age, whereas the rate of periosteal expansion remained stable. Apparent bone loss at the hip and distal forearm was associated with a parallel net bone loss. At these ROIs, endosteal bone loss exceeded the periosteal apposition, and apparent bone loss was determined by outward displacement of lower quantity of bone mineral. Longitudinally assessed age-related apparent bone loss at the hip and distal forearm has been described in several cohorts of Caucasian men [5\u20137, 10, 16], but their morphological basis has not been investigated. In elderly men, apparent bone loss was associated with net bone loss at the intertrochanter ROI but with a gain in BMC at the total hip and femoral neck (narrow neck ROI) [13]. We found the same trend for the femoral neck but not for the total hip. This discrepancy is surprising because our cohort is younger. As the net bone loss accelerates with age, faster net bone loss (and not bone gain) could be expected in the older British cohort.\nWe confirm longitudinal and cross-sectional data on the gain in aBMD of lumbar spine in elderly men [7, 12, 14, 16\u201318]. Increase in the cross-sectional area of L3 at the midheight of the vertebral body is determined mainly by the periosteal expansion. This part is less involved in arthritis. Measurements performed on the X-rays allow for exclusion of the pathological calcifications. We do not speculate on the morphological basis underlying the increase in spine aBMD. Vertebral BMC and aBMD in the posteroanterior projection are determined by changes in the vertebral body and the posterior arch. Posterior arch and its processes are built mainly of cortical bone. As their periosteal surface is relatively high, its age-related gain of BMC due to periosteal apposition may be considerable. At the vertebral body, changes in BMC are determined by loss of trabecular bone inside and by the increase in BMC due to the periosteal apposition and the aggravation of arthritis. At the hip and distal forearm, both aBMD and BMC decreased, in agreement with previous studies [6, 16, 17]. Both apparent and net bone loss accelerated with ageing; by contrast, periosteal apposition remained constant. Thus, age-related acceleration of the apparent and net bone loss is determined by the acceleration of bone loss on endosteal surfaces. Apparent and net bone loss were significant in all age groups.\nIn women, menopause results in the acceleration of endocortical resorption and deceleration of periosteal apposition [19]. Thus, after menopause, periosteal apposition continues, as reported previously by Ahlborg et al. [20]. However, it does not increase sufficiently to offset endocortical bone loss. On the contrary, it decreases, which may contribute to the deterioration of bone strength and increased fracture risk. We postulated that postmenopausal osteoporosis is a disease of failed adaptation of bone [19]. However, in men also, age-related increase in endosteal bone loss is not accompanied by an increase in periosteal apposition that would offset the possible loss of strength. Periosteal apposition does not adapt to the endosteal bone loss and remains stable. This imbalance may result in a decrease of bone strength. Thus, in men also, osteoporosis seems to be a disease of failed adaptation.\nOur data are consistent with the cross-sectional data obtained in elderly men by using the high-resolution peripheral quantitative computerised tomography (hr-pQCT) showing the increase in bone size at the axial and peripheral skeleton, as well as endosteal bone loss characterised by decrease in the trabecular volumetric BMD (vBMD) determined mainly by the decrease in the trabecular number, increase in total marrow area and cortical thinning, indicating endocortical resorption, and decrease in cortical vBMD [21, 22]. The decrease in cortical vBMD is determined mainly by the increase in the midcortical and subendocortical porosity, whereas subperiosteal porosity and real volumetric density of the mineral of cortical bone remain relatively stable during ageing [15, 23].\nOur data help in understanding the difference in the age-related increase in fracture incidence. Young men have bigger bones than women, even after adjustment for age, weight and height [24, 25]. Then, aBMD and BMC decrease. Age-related acceleration of apparent and net bone loss is consistent with the exponential increase in fracture incidence. However, in contrast to women, periosteal apposition rate remains stable in men and may partly offset the endosteal bone loss and associated loss of bone strength. In elderly men, bones are larger than in women (because they were larger in youth and age-related periosteal apposition was greater), net bone loss is less (because periosteal apposition continues to deposit bone on the outer surface) and loss of bone strength starts later (because continuing periosteal expansion compensates partly for the loss of bone strength due to endosteal bone loss). These data are consistent with epidemiological observations that in men, fracture incidence increases later and, for a given age, is lower than in women [1, 26, 27].\nOur study has limitations. Montceau les Mines is a small town, and its inhabitants may not be representative of the French population. The response rate for the invitation was 23%. Men who abandoned the study after the first examination were older and sicker, although they represent only 8% of the initial cohort. Men who were followed up may have been healthier than the general population, especially in the oldest group. However, this difference would have underestimated the age-related bone loss and its age-related acceleration. A number of men had lower number of DXA scans because they did not attend examinations regularly or abandoned the study before the end of the follow-up. A low number of DXA values and shorter follow-up could influence the accuracy of estimation of slopes. However, adjustment for the follow-up duration or the number of scans did not influence the results.\nDXA presents limitations in the evaluation of bone width. In very old men, subperiosteal bone mass can be low and not recognised by the edge-detection system. This artefact can underestimate the bone width in elderly men and the age-related increase in bone width mainly in the cross-sectional studies (where the age range is large) but less so in the longitudinal study (where the follow-up period is shorter). The projected area of femoral neck may be overestimated because of calcifications in fibrous tissue. The measured radius site is established by the device. According to the individual anatomy, this site may be more distal (larger and more trabecular) or more proximal (narrower and cortical). Again, this artefact may introduce a bias mainly in cross-sectional studies.\nCalculation of endosteal bone loss is indirect and based on the assumptions such as uniform bone flattening, constant subperiosteal bone vBMD and proportional periosteal expansion in all axes. By contrast, the advantage of this concept is that we do not make any assumption on the morphological basis underlying the endosteal bone loss (cortical thinning or trabecular bone loss, proportion of cortical to trabecular bone, similar or different rates of trabecular and cortical bone loss, etc.). Finally, our calculation of endosteal bone loss was carried out for the predominantly cortical sites and, although globally consistent with the cross-sectional data obtained by hr-pQCT, may not necessarily apply for the predominantly trabecular sites.\nIn conclusion, in a large cohort of elderly men, age-related apparent bone loss (aBMD) at the hip, distal forearm and whole body was determined by the net bone loss (BMC), except for the femoral neck. Apparent and net bone loss accelerated with age, whereas the periosteal expansion rate (widening of ROI) remained constant. At the distal forearm, age-related acceleration of the apparent bone loss was determined by the higher endosteal bone loss, whereas the periosteal apposition rate (estimated mass of deposited bone) remained constant.","keyphrases":["bone loss","men","periosteal apposition","osteoporosis"],"prmu":["P","P","P","P"]}
{"id":"Eur_J_Pediatr-4-1-2292481","title":"Symptomatic asymmetry in the first six months of life: differential diagnosis\n","text":"Asymmetry in infancy is a clinical condition with a wide variation in appearances (shape, posture, and movement), etiology, localization, and severity. The prevalence of an asymmetric positional preference is 12% of all newborns during the first six months of life. The asymmetry is either idiopathic or symptomatic. Pediatricians and physiotherapists have to distinguish symptomatic asymmetry (SA) from idiopathic asymmetry (IA) when examining young infants with a positional preference to determine the prognosis and the intervention strategy. The majority of cases will be idiopathic, but the initial presentation of a positional preference might be a symptom of a more serious underlying disorder. The purpose of this review is to synthesize the current information on the incidence of SA, as well as the possible causes and the accompanying signs that differentiate SA from IA. This review presents an overview of the nine most prevalent disorders in infants in their first six months of life leading to SA. We have discovered that the literature does not provide a comprehensive analysis of the incidence, characteristics, signs, and symptoms of SA. Knowledge of the presented clues is important in the clinical decision making with regard to young infants with asymmetry. We recommend to design a valid and useful screening instrument.\nIntroduction\nThe objective of this descriptive review is to determine and classify the possible causes of asymmetry seen in young infants who have an asymmetric head and\/or body posture, as well as to present an overview of the nine most prevalent disorders in infants in the first six months of life leading to the diagnosis of symptomatic asymmetry (SA). Asymmetric infants form an increasing and complicated group of children seen by professionals from various clinical specialties, such as well baby clinic physicians, pediatricians, pediatric physiotherapists, orthopedic surgeons, and plastic surgeons [4, 6, 19, 37, 50, 61]. Asymmetry in infancy is a mostly benign symptom, but in this early phase of life, the differential diagnostics are extensive. The background of the professional influences the way in which associated clinical problems are evaluated. A screening instrument would be helpful. The first step in this process is to synthesize the current information in the literature about differential diagnostics.\nTwelve percent of all Dutch newborns develop a positional preference in the first few months of life, different from the physiological asymmetry [6]. A \u201cpositional preference\u201d is defined as a condition in which the infant\u2019s head is turned toward one side most of the time and active movement to the other side is restricted [4, 6]. About 25% of these infants (approximately 5,000 a year in the Netherlands) are referred to pediatric physical therapists [6]. Asymmetry in infancy is a clinical condition with a wide variation in appearances (shape, posture, and movement), etiology, localization, and severity. From the referred infants, the asymmetry is either idiopathic or symptomatic, and originates ante- and\/or postpartum [37, 48, 50, 51, 61]. In case of an idiopathic asymmetry (IA), the etiology is uncertain; environmental factors play a major role in the development of the asymmetry [6, 34, 35, 48, 62]. In SA, an underlying disorder, disease, or dysfunction causes the asymmetry. The majority of cases will be idiopathic, but an initial presentation of positional preference might be a symptom of a serious underlying problem. In the last decade, many studies on the appearances of IA have been published. If the focus in diagnostics and pattern recognition is on IA, there is a chance that an SA will be missed [3, 20]. When examining young infants with a positional preference, differentiating SA from IA is necessary to determine the prognosis and to choose appropriate intervention strategies.\nThis review will address the following question: which diagnoses, incidence rates, signs, and symptoms are described in the literature and are thought to cause a symptomatic asymmetrical posture or movement pattern in infants during their first six months of life?\nMethods\nSearch strategy\nThis review is based on a comprehensive literature search on SA. The following strategy is used: peer-reviewed literature on this topic in journals with a science citation index was searched, as well as clinical textbooks from the various clinical specialties. Computerized bibliographic databases were searched (PubMed, Pedro, Cinahl, and Cochrane Controlled Trials Register), and related papers and their references. General keywords used were: asymmetry, plagiocephaly, torticollis, posture, scoliosis, (differential) diagnosis, and screening. The search then focused on specific diagnoses that might cause asymmetry in infancy condition with the keywords: etiology, tumors, disorders (related to) vision, hearing, central nervous, or musculoskeletal system, obstetric complications, brachial plexus palsy\/lesion, clinical syndromes (Grisel, Sandifer), congenital anomalies and syndromes, gastroesophageal reflux, developmental dysplasia of the hip, paroxysmal torticollis, (birth) trauma, and clavicle fracture. Finally, we focused on the incidence and prevalence. The search was limited to citations that included: \u201call infants, birth\u201323\u00a0months,\u201d had an abstract, were written in English, and the search terms were in the title or abstract. The year of publication was not restricted. When more papers on the same subject were found, the most current studies were chosen. Only diagnoses that could be observed in infants in the first six months of life were included. Unique case reports and innocuous abnormalities that require no specific treatment were not included.\nResults\nThe prevalence and\/or incidence of the various medical diagnoses leading to SA was not always documented. Some disorders had no consistency in their reported incidence rates. The majority of children with a positional preference or asymmetry during the first six months of life are diagnosed with an IA [4, 6, 34, 35, 37, 48, 50, 51]. Table\u00a01 shows a selection of the most frequently detected disorders causing an SA.\nTable\u00a01Disorders related to symptomatic asymmetry (SA) from the literature searchDisorders with known incidenceIncidence\/1,0001. Developmental dysplasia of the hip (DDH)402. Perinatal fracture of the clavicle353. Congenital muscular torticollis (CMT)204. Obstetric brachial plexus palsy45. Central nervous system disorders26. Craniosynostosis\/lambdoid suture0.03Remaining groups of disorders7. Congenital abnormalities or malformations\u00a0Musculoskeletal\u00a0Chromosomal8. Sensory systems\u00a0Ocular disorders\u00a0Hearing disorders9. Acquired asymmetry postpartum in one of the remaining systems (non musculoskeletal)\nIn the last decade, discussion on positional preference leading to deformational plagiocephaly (DP) has increased substantially. A relatively high number of hits found during a search in January 2008 within PubMed resulted in the following: asymmetry (811), plagiocephaly (206), torticollis (225), posture (405), and scoliosis (623). However, when combined with \u201cdifferential diagnosis\u201d or \u201cscreening,\u201d the result decreased to less than 20 each. Differential diagnosis from craniosynostosis was often described [24, 33, 36, 51]. The main designs were retrospective or prospective descriptive studies and reviews. In diagnoses with a low incidence, the studies were predominantly case reports.\nAll diagnoses were classified according to the International Classification of Diseases [1]. The results of the literature search are presented below, starting with the diagnosis with the highest incidence rate.\nDevelopmental dysplasia of the hip\nDevelopmental dysplasia of the hip (DDH) has a high rate of co-morbidity with congenital muscular torticollis (8\u201320%) [11] and, to a lesser extent, with postural torticollis or scoliosis [50]. The reported incidence rate in the Netherlands ranged from 3 to 4% of all newborns [4, 11, 48], with 80% being unilateral [5, 56]. The clinical signs which are described include asymmetry in hip abduction and leg length and\/or asymmetrical skin folds in the inguinal and upper thigh region. The strong association with other asymmetries warrants a thorough screening on the signs of developmental dysplasia of the hip in infants with an asymmetry.\nPerinatal fracture of the clavicle\nA fracture of the clavicle during birth may induce a positional preference in the first weeks of life and, as such, may cause an asymmetry. A perinatal fracture can be an option in the differential diagnostics of asymmetry during the first weeks of life. A co-incidence with other perinatal injuries (like brachial plexus injury) was described by Perlow et al. [49]. The obstetric brachial plexus lesion is described separately. The incidence varies between 0.1 and 3.5% [28, 40, 49, 54], and these fractures usually consolidate within 3\u00a0weeks without complications. The clinical presentation can be asymptomatic. When symptomatic, signs include: decreased or absent movement and pain, or tenderness on movement of the arm on the affected side and palpable irregularity along the clavicle [54].\nCongenital muscular torticollis\nCongenital muscular torticollis (CMT) is frequently described in the literature. Unilateral fibrosis or thickening of and tightness in the sternocleidomastoid muscle can cause a characteristic posture of the head and restricted neck movements. The etiology of the pseudo-tumor or mass is unclear [10, 11, 19, 58]. A compartment syndrome due to intra-uterine malposition is the most frequently mentioned etiological hypothesis [10, 12, 19, 39]. An association with birth trauma and breech presentation is mentioned, but the evidence is weak, since CMT is also seen in infants born via a cesarean section [11, 32]. A pseudo-tumor can be palpated in the second or third week after birth. Incidence rates of CMT vary between 0.3 and 2% [10\u201312, 19, 58]. Ultrasound screening soon after birth has indicated that the incidence rate could be 3.9%. This screening method tends to be especially sensitive in detecting occult cases of fibrosis [10].\nFibroids of the uterus and other intrauterine tumors are described as a possible etiology to an atrophy of the sternocleidomastoid muscle of the child [18]. This phenomenon can cause a unilateral muscular dysfunction and a strong imbalance between both muscles. The distinction between CMT and postural torticollis is not always clear [15]. A CMT is primarily a condition with a structural fibroid shortening of the sternocleidomastoid muscle, visible and palpable in the first weeks of life, as opposed to a postural torticollis that occurs secondary to a positional preference and a DP [6, 37, 48, 51, 61].\nObstetric brachial plexus palsy\nPalsy of the brachial plexus during delivery is caused by traction or compression of the plexus during labor. In most cases, the upper brachial plexus is affected; in 15% of the patients, hand function is also impaired. The described incidence is 0.1\u20130.4% [31, 49, 52]. The extent of the neural damage becomes evident during the first six months of life [52], although in severe cases, the inactivity of the extremity is observed from birth onwards. Timely recognition of severe cases is important, since neurosurgical intervention can enhance future capacities. Between 20 and 25% of the infants experience persistent functional impairments [31].\nCentral nervous system disorders\nCerebral palsy (CP) syndromes, in particular, spastic unilateral CP, are neurological disorders that can cause asymmetry [3, 7]. Serious disorders of the central nervous system are generally easy to recognize, but a CP may also be discrete with subtle features. Early diagnosis, before the age of six months, might be difficult [45, 57]. The neurodevelopmental (motor) behavior is an important issue in early recognition: persistent infantile reflexes and abnormal muscle tone, motor delay, abnormal spontaneous movement patterns (especially \u201cgeneral movements\u201d), and poor postural control are more or less predictors of CP [25, 45, 46]. In a review on the epidemiology of CP, the world-wide prevalence of all types of CP is estimated at 0.2% [43].\nHypotonia and developmental delay were also mentioned as causes for developing positional preference and DP. A neurological disorder might be the underlying problem, but it is not always diagnosed at this young age [3, 7, 51].\nCraniosynostosis\nCraniosynostosis, the premature fusion of one or more cranial sutures, is most frequently described in relation to asymmetry and plagiocephaly, possibly as a result of the over-referral of infants with deformational non-synostotic DP to craniofacial or plastic surgery clinics. Primary craniosynostosis is either simple or compound and part of a genetic syndrome [36]. Premature closing of one lambdoid suture is the most frequently mentioned differential diagnosis of DP [24, 33, 36]. The incidence of this single suture craniosynostosis is rare (1\u20133 cases to 100,000 newborns) [37, 42] and can be clinically differentiated from DP by four major signs: from the vertex view, a trapezoid head shape can be observed, a palpable unilateral ridge, bulging of the unilateral mastoid, and an asymmetric skull base with tilt to the ipsilateral side [24, 33, 42]. The impact of the premature closure of cranial sutures in complex craniosynostosis is impressive: strong progressive deformation of the skull, risk of increasing intracranial pressure, and developmental problems. When evident, timely surgical intervention is warranted [24, 33, 36, 50, 51].\nThe following three categories are groups of disorders. Clear incidence rates could not be found.\nCongenital abnormalities or malformations\nMusculoskeletal congenital malformations must be considered in an asymmetry that is present immediately after birth [3, 7]. Well-known malformations are those of the spine, such as a Klippel-Feil syndrome, hemi-vertebrae, and a hemi-atlas [22, 38, 64]. Exceptional phenomena are hypoplasia or aplasia of the face, neck, or trunk muscles [2]. Patients may show defects in other systems as well, such as syndactyly, deafness, or a congenital heart disease. The co-incidence of defects may be an important sign of a syndrome. An asymmetric development or posture can be an associated finding in a variety of syndromes and abnormalities. These features are often present immediately after birth, but will not always be discovered until a second stage [64]. Local abnormalities, such as a vascular ring (around the trachea) or tracheomalacia, are occasionally an indirect cause of an asymmetric posture [60].\nDisorders in sensory systems\nIn the screening of infants with asymmetry, eye movement and\/or vision and hearing disorders must be considered. Infants with congenital nystagmus and restrictive or paralytic strabismus may use anomalous head positions to maximize visual function [26, 47, 63]. No clear incidence rates were found. A predictive factor for an ocular origin of torticollis is the family history of ocular problems, in particular, congenital nystagmus. The ocular pathology may be subtle. In case of doubt, infants must be referred to an ophthalmologist [63].\nTheoretically, a unilateral hearing disorder can induce a positional preference in young infants. In the literature search, no match was found for hearing loss and torticollis, except in syndromes such as Klippel-Feil or Moebius. A connection between ear malformation and hearing loss is mentioned [23].\nAcquired asymmetry, non-musculoskeletal\nA number of disorders in systems other than the musculoskeletal system can cause a postpartum asymmetry, but the asymmetry is not the only symptom. The disorders have in common that their symptoms are not stable and occur some time after birth. The signs and symptoms can be seen as so-called \u201cred flags\u201d and require immediate medical evaluation. It may be secondary to a trauma [27] or to inflammatory conditions, such as pharyngitis [8, 13, 29, 55]. Grisel syndrome (a non-traumatic atlanto-axial rotatory subluxation following infections of the upper respiratory tract) is often described, but never under the age of six months [7, 19, 29]. Another cause can be related to the cardio-respiratory or the digestive system, such as Sandifer syndrome (fluctuating asymmetry with abnormal body movements and contortions of the neck, associated with gastroesophageal reflux) [9, 17, 21]. The most alarming causes of asymmetry are related to neurological syndromes, such as syringomyelia, epilepsy, high intracranial pressure, postencephalitic syndromes, or life-threatening tumors of the central nervous system [3, 7, 12, 19, 20, 30]. These disorders are mainly described in case studies, without proven incidence rates. The signs and symptoms of these non-musculoskeletal causes are described in Table\u00a02.\nTable\u00a02Signs and symptoms of acquired symptomatic asymmetry disorders with a low incidenceSigns and symptomsHints for disordersGeneral historyHeavy painRetro-pharyngeal abscess [13]Vomiting\/drowsinessIncreased intracranial pressure [30]Lethargy\/irritabilityTumor [12, 30], intracranial injury [27]TraumaIntracranial injury [27]Seizures\/convulsionsEpilepsy; increased intracranial pressure; Sandifer syndrome [17]Acute onsetInfection, abscess [13]; Grisel syndrome (>6\u00a0months) [29, 55]Stridor, dyspneaVascular ring [60]RefluxSandifer syndrome; pathological gastroesophageal reflux [17]FeverInfection, abscess [13]Specific examinationSunset phenomenonIncreased intracranial pressureBulging anterior fontanelIncreased intracranial pressure, intracranial injury [27]Abnormal courseIncreasing head tiltInfection [13]; tumor [12, 30]Recurrent episodesBenign paroxysmal torticollis [9, 21]\nDiscussion\nAsymmetry in infancy is a condition with a high prevalence in infants in the first six months of life. In the majority of cases, the origin is idiopathic and is often related to environmental factors [6, 15, 34, 35, 37, 62]. This review addresses the possible causes, incidence rates, and symptoms of symptomatic asymmetries due to an underlying disorder, dysfunction, or disease.\nNot all of the incidence rates could be found, while some inconsistencies were observed in the current literature. The disorders with a high prevalence are well described in epidemiologic studies. The rarer diseases were, most of the time, documented in case reports without incidence rates. The incidence rates mentioned in the studies are inconsistent because of different opinions regarding the operationalization and assessment of the SA. Frequently, psychometric properties of instruments and concepts have not been described or evaluated. Variations in incidence rates (e.g. CMT) are inevitable, considering the variety in inclusion criteria and diagnostic tests used in the studies. The sequence in estimated incidence rates, as proposed in Table\u00a01, is open to debate.\nA clear description of signs and symptoms was not always presented in the literature. The variety in the etiology of asymmetry is considerable. The level of evidence of the included studies varies. Literature of more than 10\u00a0years ago mainly described underlying causes of SA, in particular, non-muscular torticollis [3, 7]. They still turned out to be useful in establishing criteria for differential diagnostic screening and are widely cited in current studies. However, an update regarding new developments in studies on infant asymmetry is needed. The exponential increase of plagiocephaly in the last decade, related to the recommendations to put babies on their back to sleep, is reflected in the objectives of recent studies [4, 6, 37, 41]. They mainly focused on IA and its predispositions, with little attention to SA. A number of recent papers described features to distinguish craniosynostosis from DP. Although craniosynostosis has a very low incidence, craniofacial clinics are deluged with infants with DP [14, 15, 37]. One of the positive effects of this situation is that authors from this background described useful clinical diagnostic criteria for craniosynostosis.\nvan Vlimmeren et al. [61] stated in their review on diagnostic strategies, that asymmetry in infancy is a diagnosis with a large spectrum of features and a multifactorial etiology without consensus on definition, nomenclature, or classification. In the present review, a classification by virtue of etiology is proposed. The dichotomy, symptomatic versus idiopathic, is often used in medicine [16, 44, 53, 59] and fits well with this health problem, since a large number of children have unexplained asymmetry.\nAlthough flow diagrams for diagnostic strategies are presented in some reviews [3, 7, 15, 19, 61], clear clinical diagnostic criteria that could be used were not mentioned. The criteria found in the present review might be considered in a future study. An expert validation, such as a Delphi study with clinical experts, could be a next step towards establishing clinical diagnostic criteria as warning flags in young infants with IA or SA.\nThis review presents an overview of the most common disorders underlying SA in infants less than six months of age. We have discovered that the literature does not provide a comprehensive analysis of the incidence, characteristics, signs, and symptoms of SA. Knowledge of the presented clues is important in the clinical decision making with regard to young infants with asymmetry. The endpoint of this review may be a starting document for the creation of a protocol, but it needs additional studies in order for it to become a valid and useful screening instrument.","keyphrases":["symptomatic asymmetry","differential diagnosis","etiology","infant","screening","plagiocephaly","torticollis"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"J_Urban_Health-2-2-1705478","title":"Assessment of Respondent Driven Sampling for Recruiting Female Sex Workers in Two Vietnamese Cities: Reaching the Unseen Sex Worker\n","text":"Respondent driven sampling (RDS) is a relatively new method to sample hard-to-reach populations. Until this study, female sex workers (FSWs) in Vietnam were sampled using a variety of methods, including time location sampling (TLS), which may not access the more hidden types of FSWs. This paper presents an analysis from an HIV biological and behavioral surveillance survey to assess the feasibility and effectiveness of RDS to sample FSWs, to determine if RDS can reach otherwise inaccessible FSWs in Vietnam and to compare RDS findings of HIV risk factors with a theoretical TLS. Through face-to-face interviews with FSWs in Ho Chi Minh City (HCMC) and Hai Phong (HP), data were collected about the venues where they most often solicit their clients. These data were used to create three variables to assess whether FSWs solicit their clients in locations that are visible, semi-visible and non-visible. For this analysis, the visible group simulates a sample captured using TLS. Survey results in HIV prevalence and related risk factors and service utilization, adjusted for sampling methodology, were compared across each of the three FSW visibility groups to assess potential bias in TLS relative to RDS. The number of self-reported visible FSWs (HCMC: n=311; HP: n=162) was much larger than those of the semi-visible (HCMC: n=65; HP: n=43) and non-visible (HCMC: n=37; HP: n=10) FSWs in HCMC and HP. Non-visible FSWs in both cities were just as likely as visible and semi-visible FSWs to be HIV positive (HCMC: visible 14.5%, semi-visible 13.8%, non-visible 13.5%, p value = 0.982; HP: visible 35.2%, semi-visible 30.2%, non-visible 30.0%, p value = 0.801), to practice behaviors that put them at risk for contracting and transmitting HIV (injecting drug use\u2014HCMC: visible 13.8%, semi-visible 12.3%, non-visible 5.4%, p value = 0.347; HP: visible 38.9%, semi-visible 23.3%, non-visible 30.0%, p value = 0.378, to have no condom use in the past month \u2014HCMC only: visible 52.7%, semi-visible 63.1%, non-visible 48.6%, p value = 0.249) and to have symptoms of a sexually transmitted infection (STI) in the past year (HCMC: visible 16.1%, semi-visible 12.3%, non-visible 16.2%, p value = 0.742; HP: visible 13.6%, semi-visible 18.6%, non-visible 20.0%, p value = 0.640). There was a difference found among the visible, semi-visible and non-visible groups in HP for no past month condom use (visible 53.1%, semi-visible 79.1%, non-visible 60.0%, p value = 0.009). This study found that RDS was successful at recruiting hidden types of FSWs in Vietnam. Past reports of FSWs in Vietnam have assessed the more visible FSWs as being the most vulnerable and at risk for HIV. Although the number of visible FSWs is much higher than those of the semi and non-visible groups, this study found that the non-visible FSWs are very vulnerable to HIV infection. If prevention programs are targeting and responding to those who are most likely to be assessed (e.g., more visible types of FSWs) then this analysis indicates that a significant proportion of the FSW population at risk for HIV may not be receiving optimal HIV information and services.\nIntroduction and Background\nVietnam is in the midst of an HIV epidemic concentrated among injection drug users (IDUs) and, increasingly, female sex workers (FSWs). In recent years, the epidemic in Vietnam appears to be moving rapidly from IDU populations to FSWs through sexual contact with injecting male clients and the adoption of injection drug use behaviors by FSWs. Sexual transmission of HIV from FSWs to their non-injecting clients, who in turn have non-commercial sex partners, greatly increases the probability that HIV will spread to the general population. In 2002, the Department of Social Evils in the Ministry of Labor, Invalids, and Social Affairs estimated that 130,000 women work as sex workers in Vietnam (other estimates put the number much higher).1\u20133\nTo understand and respond to HIV transmission patterns in Vietnam, numerous surveys have measured HIV prevalence and related risk factors among FSWs. However, the lack of sampling frames, relative rarity of this population, and their stigmatized and\/or illegal behaviors, hinder investigators from obtaining representative samples of FSWs. Further, it is important to recognize the heterogeneity of the sex worker population. Broadly, FSW can be dichotomized into direct and indirect sex workers. Indirect sex workers typically have a primary job or source of income other than sex work, though that job may lead to contacts with potential clients (e.g., bar maids or masseuses). Direct sex workers typically do not have another job. Direct sex workers can be further broken down into street-based, brothel-based, or gatekeeper-based. The latter may involve a formal \u201cpimp\u201d\/agent relationship or informal referrals from hotel concierges or taxi drivers. Use of the Internet to find clients is increasing also.\nThere is a concerted effort by the Government of Vietnam to reduce prostitution by placing FSWs in administrative detention in government rehabilitation centers where they can spend anywhere from 1\u00a0month to several years. Fear of detention leads FSWs to exercise greater caution and more subtlety on the streets. Recently, FSWs have become even more difficult to reach because the Vietnamese government is imposing penalties (fines, jail sentences and possible closure) on the owners of entertainment establishments where sex is allegedly sold.4 As a result, FSWs will now wait off-site for an establishment owner or some other gatekeeper to contact them at a client\u2019s request.\nFSWs are also hard to reach because they are highly mobile and move regularly from bar to bar, between districts and towns, and within and to other provinces.5\u20139 Some FSWs are difficult to locate because they have primary jobs and engage in sex work part-time. For instance, women from rural areas may travel to urban areas to sell agricultural goods and only sell sex as a way to earn extra income.5 Higher paid FSWs, such as those who solicit clients from scooters, the Internet, through agents and over the telephone, have the financial capacity to remain hidden.5,10\nPast efforts to survey FSWs in Vietnam, including annual sentinel HIV surveillance, have relied on convenience samples from rehabilitation centers and street-based FSWs. These samples comprise the type of FSWs who are reportedly the most vulnerable, and therefore most at risk, and visible, (i.e., visible enough to be captured by the police or found by public health staff) and do not represent the entire sex work community.5 Further efforts to assess HIV related risk factors among FSWs have used other non-probability sampling methods, such as institutional sampling, targeted and chain referral sampling.11\u201316 FSWs in Vietnam have also been sampled using a probability sampling method called time location sampling (TLS), also known as time\u2013space or venue\u2013day\u2013time sampling.17,18 However, TLS can only be representative of those FSWs who are easily located at visible sites, thereby missing potentially important information from other types of FSWs.\nThe Vietnamese Ministry of Health, with technical assistance from Centers for Disease Control and Prevention\u2014Global AIDS Program, conducted a survey using a relatively new sampling method to assess HIV prevalence, risk factors and service utilization among FSWs. The goal of the survey was to provide baseline information before increasing numbers of HIV prevention programs targeting these populations were initiated. This sampling method, known as respondent driven sampling (RDS), is a variant of chain-referral sampling that utilizes an incentive for being interviewed and another incentive for recruiting peers (other FSWs) to be interviewed.19\u201322 Sampling begins by selecting a small number of non-randomly selected members of the target population, known as seeds, to take part in the study. Seeds initiate the chain referral by recruiting a fixed number of peers who, in turn, recruit other peers. The process continues until the target sample size is attained. Links among peers are recorded using anonymous identification numbers.\nAlthough RDS is relatively new to public health researchers, it is starting to be used widely in place of other well known types of sampling methods for hard to reach populations. Researchers are considering the advantages of using RDS in place of TLS, the only other probability method for hard to reach populations, to conduct future HIV biological and behavioral surveillance surveys. TLS has been used to sample FSWs who gather and are accessible at public locations (or venues) that can be listed in a sampling frame.23\u201325 TLS entails identifying days and times when the target population gathers at specific locations (e.g., brothels, city blocks, bars), constructing a sampling frame of time and location units, randomly selecting these units and systematically intercepting consenting members of the target population for interviews.24\u201326 When implemented correctly, TLS allows researchers to construct a sample with known probabilities, make statistical inferences to the larger population of venue visitors, and theorize about the introduction of biases that may limit generalization of results to the target population.23\nTwo TLS surveys of FSWs in Vietnam have been reported. In 2000 Family Health International (FHI) conducted a behavioral survey with karaoke (n=2,302) and street-based (n=1,300) FSWs in five Vietnamese cities using TLS.18 During 2003, the Vietnamese National Institute of Hygiene and Epidemiology collected data from karaoke (n=1,367) and street-based (n=976) FSWs from five provinces.17\nAlthough TLS can produce a probability sample by ensuring that time\u2013location units are enumerated and selected randomly, it allows researchers to reach only those segments of the population who are visible and identifiable. Depending on the population of interest, this may introduce a bias by differential sampling of the portion of the population which is most at-risk. TLS can be expected to miss those segments that are less visible (e.g., FSWs who solicit clients through agents, by telephone or Internet). The \u201cvisible\u201d portion of a population does not represent the whole population any more than an institution or clinic-based population does; \u201chidden\u201d population members have a zero probability of selection, reducing representativeness of the sample.\nThe objectives of this paper are to present findings from a survey on HIV prevalence, risk behaviors and service utilization to (1) assess the effectiveness of RDS to sample FSWs in Vietnam; and (2) to compare RDS findings with theoretical TLS. This is especially important given that evaluations of RDS used for HIV surveillance have only come from IDUs and males who have sex with males populations in the United States.20,22,27\u201329 Until this study, RDS has not been evaluated in Asia among FSWs. This methodology warrants investigation to assess its ability to sample hard to reach populations in international settings.\nMaterials and Methods\nData to evaluate RDS were collected from FSWs in Ho Chi Minh City (HCMC) and Hai Phong (HP), Vietnam. Eligible participants were over the age of 18 and had exchanged sex for money in the previous month. FSWs were recruited by their peers (other FSWs) and provided a small monetary incentive for completing a questionnaire and providing a saliva sample to test for HIV (US \\$3 in HP and US \\$4 in HCMC). FSWs were provided another incentive for recruiting up to three other FSWs to participate (approximately US \\$1 in HP and US \\$1.50 in HCMC for each eligible recruit). Data were gathered from FSWs in HP from April to June and in HCMC from May to July, 2004. Recruitment began with 20 FSWs, non-randomly selected by survey staff, in each city. Outreach workers selected these initial recruits, or \u201cseeds,\u201d based on where they most often solicited their clients. A majority of the seeds were from the visible groups, street-based and karaoke-based FSWs. A few seeds from hotels and guesthouses were recruited.\nOnce seeds consented, they completed a one-hour face-to-face interview with a trained Vietnamese interviewer. The interview instrument consisted of questions about the participant\u2019s demographic background, current and past drug use, sexual behaviors, HIV knowledge, risk perceptions and service utilization, and about where they usually solicit their clients. After the interview, survey staff provided participants with three coupons with which to recruit up to three other FSWs who were co-workers, friends, acquaintances or family members. Each coupon contained a unique identification number and provided basic information about the study and the addresses of the interview sites. Respondent\/recruiters were asked to return to collect their incentives for successful recruits. A brief follow-up questionnaire was administered during this visit to ascertain the recruiter\u2019s relationship to their recruits and the number and type of refusals they received. This permitted the assessment of participation bias due to refusals or no-shows.\nOraquick\u2122 HIV antibody rapid test kits for oral fluids were used for HIV testing. No one refused to provide an oral sample. Participants did not learn their test results at the interview site but were provided a voucher to receive a free HIV test with counseling at a nearby voluntary counseling and testing (VCT) center.\nFor the assessment of bias in a theoretical TLS-drawn sample in comparison to an actual RDS-drawn sample, respondents were classified as accessible, i.e., visible to TLS, if they reached their clients in a public venue. Categories were established from the response to the question, \u201cWhere do you usually reach your clients?\u201d Responses were aggregated into three groups: visible (street, park, karaoke bar); semi-visible (brothel, guesthouse, hotel); non-visible (agent, telephone, Internet). Our assumption is that the visible group could potentially be captured by TLS. The semi visible group may or may not be captured with TLS, and the non-visible group would be missed with TLS.1\nStatistical Analysis\nAdjusted proportions and 95% confidence intervals (ci) for FSWs\u2019 level of visibility were analyzed using the RDS analysis tool (RDSAT).30 RDSAT adjusts proportion estimates for participants\u2019 network sizes and recruitment patterns.20,31 RDSAT adjustments use principles of social network theory to account for biases found in conventional chain referral sampling.31,32\nChi-square proportions and p-values for comparisons of HIV risk factors and service utilization of RDS and theoretical TLS were analyzed using SPSS 11.0. Five of the 20 non-randomly selected seeds in the HP sample, and four seeds in the HCMC sample were not used in the final analysis since they did not recruit any peers. It is not known why these seeds did not recruit anyone. The sample size was calculated based on 15% increase in use and knowledge of CDC sponsored VCT HIV services and outreach. We estimated low baseline knowledge and use of services (no actual data available) with high HIV prevalence. HP sample size was 200 based on higher knowledge from existing health education programs. The sample size in HCMC was 400, assuming lower baseline knowledge. Design effect was set at 2.\nThis study was reviewed by the Associated Director for Science of the National Center for HIV, STD and TB prevention at CDC who classified this as a surveillance activity. The protocol for this study was reviewed and approved by the LIFE-GAP Office of the Vietnam Ministry of Health.\nFindings\nA total of 628 individuals, including seeds, were interviewed in fixed interview sites in HCMC (n=413) and HP (n=215). The majority of the FSWs in the HCMC (54.6%) and HP (51.3%) sample were under the age of 29\u00a0years old. More than 50% of FSWs in HCMC and HP had no occupation other than selling sex.\nDuring the follow-up visits to collect recruitment incentives, most participants reported giving out all of their coupons. According to the 174 recruiting participants in HCMC and 90 in HP, each of whom received three coupons, a total of 75 (15.3%) individuals in HCMC and 24 (10%) in HP refused to accept a coupon; 147 recruiters in HCMC and 83 in HP reported that they were successful in distributing their coupons and that no one to whom they tried to give a coupon rejected one; so refusals were clustered among a handful of recruiters, 27 in HCMC and 7 in HP. According to recruiters, most refusers did so because of \u201cfear of identification,\u201d (57% in HCMC; 75% in HP), others \u201cfelt uncomfortable\u201d (27% in HCMC; 8.3% in HP), still others thought they were \u201ctoo busy\u201d (10.7% in HCMC), others thought the \u201cincentive not worth the time\u201d (17% HP) and a small number of FSWs in HCMC responded that they were \u201cnot interested.\u201d\nOverall, the recruited FSWs were diverse with respect to where they solicited their clients. Figure\u00a01 depicts the diversity of sex work types among the links within a single recruitment chain of FSWs in HCMC (n=59 recruits). In this example, the seed recruited three FSWs who were not street-based: two based in guesthouses and one based in karaoke.2 The selected karaoke-based FSW recruited two other karaoke-based and one guesthouse-based FSW. Near the outer waves of this recruitment chain, there are some more hidden types of FSWs such as agent-based and telephone-based FSWs.\nFigure\u00a01RDS recruitment network for one seed for type of female sex worker in HCMC, Vietnam.\nThe distribution by visibility was visible (HCMC, n=311; HP, n=161), semi-visible (HCMC, n=65; HP, n=43) and non-visible (HCMC, n=37; HP, n=12). Figure\u00a02 shows that the largest adjusted proportions of FSWs in both HCMC and HP reached by RDS were visible [HCMC, 73.7%; 95% ci(67.0%, 79.5%) and HP, 75.9%; ci(67.1%, 85.3%)]. RDS reached a small proportion of non-visible [HCMC, 9.3%; ci(6.0%, 13.1%) and HP, 9.7%; ci(1.3%, 18.2%)] and semi-visible FSWs [HCMC, 16.9%; ci(11.5%, 23.4%) and HP, 14.2%; ci(9.1%, 21.2%)], who theoretically could have been missed by TLS.\nFigure\u00a02Distribution of visible, semi-visible and non-visible FSWs in HCMC and Hai Phong, Vietnam; adjusted proportions (number). Visible FSWs are assessed to be the most visible of the FSWs in Vietnam; semi-visible FSWs are assessed as not always being easily identifiable; and non-visible FSWs are assessed as the most hidden types of FSWs in Vietnam.\nHIV Risk Factors\nThere were minor differences among FSW visibility groups with regard to HIV status and related risk behaviors (see Table\u00a01).\nTable\u00a01HIV prevalence, risk factors and drug use among visible, semi-visible and non-visible FSWs in Ho Chi Minh City and Hai Phong; unadjusted proportions (number), p-value\u00a0Ho Chi Minh City (n=413)Hai Phong(n=215)Visible % (n)Semi-visible % (n)Non-visible % (n)p-valueVisible % (n)Semi-visible % (n)Non-visible % (n)p-valueHIV positive status14.5 (45)13.8 (9)13.5 (5)0.98235.2 (57)30.2 (13)30.0 (3)0.801No condom use with a  partner in the past month52.7 (164)63.1 (41)48.6 (18)0.24953.1 (86)79.1 (34)60.0 (6)0.009Ever used non-injection  illicit drugs20.9 (65)18.5 (12)29.7 (11)0.38546.9 (76)37.2 (16)60.0 (6)0.338Ever injected illicit drugs13.8 (43)12.3 (8)5.4 (2)0.34738.9 (63)23.3 (10)30.0 (3)0.378Symptoms of a sexually  transmitted  infection in past year16.1 (50)12.3 (8)16.2 (6)0.74213.6 (22)18.6 (8)20.0 (2)0.640\nHIV positive test status did not differ by visibility group in HCMC or HP. In HCMC and HP, a higher proportion of visible FSWs tested positive for HIV (14.5 and 35.2%, respectively) than semi-visible (13.8%, 30.2%) and non-visible (13.5 and 30.0%) FSWs. The only significant difference among the groups for any variable was in HP, for \u201cno past month condom use with a partner\u201d (p value = 0.009).\nVisibility groups in both HCMC and HP did not differ with respect to reported use of non-injection illicit drugs ever and injected illicit drugs ever or reporting a sexually transmitted infection (STI) during the previous year.\nService Utilization\nContact with a peer educator (PE), source of HIV information and HIV testing did not vary significantly by visibility group in either city. Among all three visibility groups in both cities, fewer than 50% had ever made contact with a PE. More than 50% of FSWs in both cities (with the exception of semi-visible FSWs in HP) reported receiving their HIV information from television, radio, newspapers or magazines. More HCMC FSWs reported receiving HIV information from friends than from health workers and PEs; this pattern was reversed for HP FSWs (Table\u00a02).\nTable\u00a02HIV service utilization among visible, semi-visible and non-visible FSWs in Ho Chi Minh City and Hai Phong; unadjusted proportions (number), p-value\u00a0Ho Chi Minh City (n = 413)Hai Phong (n=215)Visible % (n)Semi-visible % (n)Non-visible % (n)p-valueVisible % (n)Semi-visible % (n)Non-visible % (n)p-valueEver met with a peer educator33.8 (105)49.2 (32)40.5 (15)0.31031.5 (51)27.9 (12)40.0 (4)0.720Most common source  of HIV information0.0880.106\u00a0TV, radio, newspaper  or magazine56.1 (171)70.8 (46)52.9 (18)57.5 (92)45.2 (19)70.0 (7)\u00a0Health worker\/peer educator17.0 (52)9.2 (6)20.6 (7)26.3 (42)38.1 (16)30.0 (3)\u00a0Friends20.7 (63)13.8 (9)26.5 (9)11.3 (18)16.7 (7)0\u00a0Other6.2 (19)6.2 (4)05.0 (8)00Ever had an HIV test45.3 (141)46.2 (30)40.5 (15)0.84134.0 (55)27.9 (12)50.0 (5)0.398Type of facility providing  last HIV test0.9760.285\u00a0Public clinic35.6 (51)43.4 (13)33.3 (5)46.3 (25)16.7 (2)40.0 (2)\u00a0Hospital43.7 (62)36.7 (11)33.3 (5)29.6 (16)25.0 (3)40.0 (2)\u00a0Private clinic4.2 (6)3.3 (1)6.7 (1)1.9 (2)16.7 (2)0\u00a0VCT center6.3 (9)6.7 (2)13.3 (2)9.3 (5)16.7 (2)2.0 (2)\u00a0Rehab center9.9 (14)10.0 (3)13.3 (2)13.0 (7)25.0 (3)0\nFifty percent or fewer FSWs in each visibility group reported ever having an HIV test. Among those HCMC and HP FSWs who ever had an HIV test, the majority received their test at either a public clinic (district or community public health center or STI clinic) or hospital, with the exception of HP semi-visible FSWs who reported receiving their HIV test from either a hospital or a drug user\/sex worker rehabilitation center. Twice as many non-visible (13.3%), in comparison to visible and semi-visible (6.3% each) FSWs in HCMC, reported receiving their HIV test at a VCT center.\nDiscussion\nThe analysis of RDS coverage demonstrates that this method may provide modestly better estimates for FSW populations than TLS because it reaches individuals ostensibly inaccessible by TLS. The level of risk and HIV prevalence in the less visible groups of FSWs was similar to the semi-visible and visible FSWs. This would indicate that less visible FSWs merit reasonable efforts to assure that these groups are targeted within all interventions for FSWs. The differences might have differed if two groups emerged rather than three. However, the semi-visible group could not be clearly dichotomized.\nThe three visibility groups in HCMC and HP did not differ significantly, which may contradict previous reports that higher paid, indirect FSWs, in general, have less HIV risk than direct FSWs.5,11,33 High proportions of respondents among all visibility groups in HCMC and HP reported inconsistent condom use with a sexual partner in the past month, possibly indicating that all three groups of FSWs are not being appropriately educated about using condoms or that they cannot easily access them. Other possible barriers to increased condom use might include poor negotiating skills with their clients or a false perception that a well-dressed client with an occupation may not be HIV-infected. Understanding the perceptions, negotiation skills and the reasons why some FSWs use condoms and others do not is essential to addressing the low proportion of condom use among FSWs in these cities.\nThe high proportion of non-visible FSWs in HCMC and HP who ever met with a PE indicates that the more hidden FSWs may be receiving more exposure to HIV information than previously believed. HCMC and HP FSWs reported similar sources for HIV information, indicating that PEs may be equally disseminated among the visibility groups. Furthermore the high proportion of non-visible FSWs who ever had an HIV test and their access to HIV testing at public clinics, hospitals and VCT centers indicate that this group has some awareness of and interest in their risk. It would be useful to gather qualitative data to determine how they obtained information and services.\nAnother key finding was the nature of social networks formed by FSWs in Vietnam. Networks were dense and diverse enough to provide a large sample in a short time that crossed the various types, direct and indirect, of FSWs working in the respective cities. These network properties are essential to the successful use of RDS. RDS had an added advantage over TLS by providing some measure of selection bias by questioning respondents on the characteristics of peers who demurred. TLS cannot determine reasons for not using a venue to solicit clients, only for respondents\u2019 refusal to speak with interviewers.\nThere were limitations to our study. Categories of visibility were based on the authors\u2019 perceptions of the sex work industry in the respective cities. We did not conduct extensive fieldwork so there was limited knowledge about the types of FSWs in Vietnam. There was too little formal research about characteristics of hotel and guesthouse-based FSWs to confidently dichotomize them as visible or non-visible. With a better understanding of these types of FSWs, reclassification and reanalysis of the data could better demonstrate the capacity of RDS to reach hidden FSWs. Furthermore, the question \u201cWhere do you usually reach your clients?\u201d does not signify where a FSW always reaches her clients. The findings here may have non-differentially misclassified some respondents.\nCurrently RDS data analysis is limited to estimates of proportions. Stratified analysis of the differences in risk factors among visibility groups might yield more information when RDSAT is updated.\nThe best scenario for testing whether RDS captures more hidden elements of the FSW population would be to conduct nearly contemporaneous surveys using TLS and RDS in the same population. Using RDS data to simulate TLS for comparisons of the two sampling methods is subject to any biases unique to RDS that might not be found in TLS.\nAlthough non-visible FSWs make up a small proportion of the FSW sample in HCMC, their high HIV prevalence and associated risk factors is alarming, especially when past efforts to sample FSWs, using TLS and convenience sampling, and sentinel surveillance in STI clinics, are likely to have missed these groups. Missing the most inaccessible portions of the FSW population could result in a biased understanding of HIV sero-prevalence and risk factors and, consequently, could lead to inefficient planning for HIV prevention and intervention programs for the entire FSW population. Recurrent round-ups of FSWs drive FSWs off the street, making them increasingly difficult to find in public venues. If current prevention programs are targeting and responding to those who are most likely to be assessed (more visible FSWs) then it is possible that an important segment of the FSW population at risk for HIV is not receiving optimal HIV information and services. However, this study found that segments were accessing services at about the same low rates, indicating a need to increase service utilization among all groups of FSWs.\nBased on these findings, we recommend using RDS for future surveys of FSWs in Vietnam to prevent the omission of the less accessible members of this population.","keyphrases":["sex workers","vietnam","hiv","respondent-driven sampling"],"prmu":["P","P","P","M"]}
{"id":"Purinergic_Signal-4-2-2377319","title":"Purinergic signaling microenvironments: An introduction\n","text":"The common theme of this introductory article and the minireviews that follow in this special issue is the concept of microenvironments within tissues and surrounding cells that would be ideal signaling venues for a biologically active purinergic ligand. Collectively, the editors\/authors and the other contributing authors agree that nucleotides and nucleosides would be most potent within a confined system. A talented cadre of purinergics has been solicited to discuss purinergic signaling in his or her favorite microenvironment within a given organ or tissue. We are gratified by the large number of original articles that also have successfully navigated the peer review process and are part of this special issue. These concepts are not simply purinergic, but the idea of maximal potency in a tissue microenvironment and surrounding specialized cells within a tissue pertains to any autacoid or paracrine agonist.\nIntroduction\nAdenosine 5\u2032 triphosphate (ATP) and adenosine are extracellular autocrine and paracrine signaling molecules. The most potent effects of ATP and adenosine are likely limited to extracellular microenvironments within tissues or immediately surrounding a cell or group of cells. These extracellular microenvironments may have a limited area of diffusion, have a small volume, have an ideal ionic composition and pH, represent an unstirred layer near a membrane surface, represent a trapped interstitium, or any and all of the above. If P1 or P2 nucleotide receptors are present on a membrane that is exposed to released nucleotide or nucleoside in this microenvironment, these ligands can engage P1 G-protein-coupled receptors, P2Y G-protein-coupled receptors or P2X receptor channels and affect the function of the cell on which it binds. Besides ATP and adenosine, other forms of nucleotides and nucleosides may also be active extracellular signaling ligands and may have unappreciated receptors. Inosine, thymidine, and even cyclic nucleotides are released by and may have effects on mammalian cells. The self-aggregation signal for the slime mold Dictyostelium discoideum, is cyclic adenosine monophosphate (AMP). Receptors for cyclic nucleotides have only been defined until now in the lower biological systems. Receptors for cyclic nucleotides as well as inosine or thymidine may also exist on mammalian cells and receive this autocrine signal within a microenvironment.\nIdeal microenvironments for purinergic signaling\nFor simplicity, we focus on ATP and its metabolites. Many different microenvironments have been studied in great detail and are described and included below. One classical microenvironment that has emerged in importance in the cystic fibrosis (CF) research community is in an open system but is a stirred liquid layer trapped between the luminal membrane of the airways epithelium and the humidified air that fills the lung and airways. The airway surface liquid [ASL, also termed airway surface fluid (ASF), the periciliary liquid layer (PCL), and the epithelial lining fluid (ELF)] bathes motile respiratory cilia [1\u20134]. The humidified air above this layer and the ciliated apical or luminal membrane beneath this layer help close this microenvironment. Submucosal gland secretions may also contribute importantly to its composition. Ric Boucher and his many coworkers have presented many recent seminal contributions on the purinergic signaling that may occur in the ASL\u2014among its many other properties [3, 4]. There are similar microenvironments of this type that are also discussed in review articles within this special issue. Daniel Marcus and colleagues and Claire Mitchell and coworkers discuss liquid microenvironments in the ear and the eye, respectively [5, 6], which provide a medium for purinergic signaling.\nA second classical microenvironment is a closed and open system that is compromised of a secretory gland or acinus and a collecting or draining duct. Iva Novak and colleagues have done seminal work in the pancreas in this regard [7, 8]. Geoff Burnstock has often discussed the rich purinergic signaling that occurs in tubes or ducts [9, 10]. In this microenvironment, glandular and acinar secretions may convey the purinergic ligands so that they may act in an autocrine manner at the acinus or in a paracrine manner within the duct. However, it is an open system in that these secretions eventually exit this tissue environment. The airway submucosal gland beneath the airway surface epithelium may be one such example that conveys purinergic signals to the surface where they may drive ciliary beat and other functions. The pancreatic acinus and duct, the sweat gland and duct, and the bile secretory apparatus and the bile duct are all important examples.\nA third microenvironment is a closed system in which the purinergic ligands concentrate within a small volume and within a tissue. Here, the purinergic ligands bind and activate receptors until they are either degraded or reabsorbed by the cells that secreted them or by neighboring cells. The endolymph of the ear and the humors of the eye are ideal examples of such closed systems [11, 12]. Marcus and colleagues and Mitchell and coworkers discuss these systems in minireviews alluded to above [5, 6]. Original articles and reviews in this issue and future issues of Purinergic Signalling speak to such systems in pancreatic islets and in the many renal tubules that comprise the nephron of the kidney [13]. In polycystic kidney and liver disease, tubules remodel into abnormal fluid-filled cysts. Multiple laboratories have argued that the fluid-filled lumen of the encapsulated cyst is a rich yet pathophysiological microenvironment for purinergic signaling [14\u201320]. The minireviews that follow this introductory article focus on specific microenvironments, cells and tissues that create and\/or come in contact with such microenvironments, and all possible modes of purinergic signaling within these microenvironments.\nAn ideal microenvironment paradigm for extracellular purinergic signaling: the synapse\nMany reviews in the past [21\u201323] and in the initial issues of this journal have documented well the roles of the P2X receptor channel at the synapse between neurons. Indeed, P2Y receptors may play roles in synaptic transmission as well. This is analogous to the interplay between cholinergic and muscarinic receptor systems in cholinergic neurons and synapses in many different nuclei and regions of the brain. In fact, the purinergic field was born from the classical studies of extracellular ATP on the heart by Drury and Szent-Gyorgyi [24], in the definition of purinergic nerves in the enteric nervous system by Burnstock and colleagues [25], and on the release and effects of purinergic ligands such as ATP on skeletal muscle contractility by Tom Forrester (see \u201cHistorical Perspective\u201d by T. Forrester in this issue, [26]). The authors lead this series of minireviews with the idea that the synapse is the ideal and, perhaps, the first or founding microenvironment for autocrine and paracrine purinergic biology.\nThe synapse is designed to have a limited diffusion distance between pre- and postsynaptic membranes where the principal ligand or neurotransmitter is reabsorbed by the presynapse or degraded in the synapse. For the sake of simplicity, our narrative here focused first on a purinergic synapse where released ATP acts as the principal ligand to evoke an action potential in the postsynaptic membrane. ATP as the lead neurotransmitter is released in response to an action potential in the presynaptic bulb that opens voltage-dependent calcium (Ca2+) channels. This voltage-dependent Ca2+ entry signal triggers the fusion or exocytosis of ATP-filled synaptic vesicles so that labile ATP is secreted into the synaptic cleft. Extracellular ATP-gated P2X receptor channels on the postsynaptic membrane have a large extracellular domain poised to bind that ATP immediately upon release within a synaptic microenvironment where there is a short diffusion distance and a small volume. In other words, the synapse is an ideal microenvironment for paracrine purinergic signaling. In this paradigm, the ATP is likely degraded to adenosine diphosphate (ADP), 5\u2032 AMP and adenosine, where adenosine may be recycled back into the presynapse by nucleoside transporters only to be a substrate to synthesize new ATP for metabolism or neurotransmission.\nSeveral additional functions for P2X receptor channels have been proposed in neurotransmission driven by other neurotransmitters. For example, at the glutamate synapse, it is well known that ATP is released as a cotransmitter. A classical example of this is the hair-cell sound transduction process, where glutamate and ATP are coreleased to stimulate auditory afferents [11]. How may ATP affect responses to glutamate at glutamate, N-methyl-D-aspartate (NMDA), and kainate receptor channels in the postsynaptic membrane and\/or at the presynapse in terms of release of ligand? Many possible cotransmitter effects have also been entertained and studied. We do not wish to debate the relative roles of P2X receptor channels in neurotransmission. The possible explanations of ATP effects at an excitatory glutamatergic synapse are given in simplistic terms. We only wish to show the synapse as a lead microenvironment to illustrate how and why local ATP signaling may be important.\nFirst, the presence of a postsynaptic P2X receptor channel or channels would potentiate the excitation of glutamate postsynapse. Not only do NMDA, kainate, and glutamate itself potentiate Ca2+ and sodium (Na+) entry as Ca2+ permeable nonselective cation channels, but extracellular ATP-gated P2X receptor channels open a separate pathway for Ca2+ and Na+. Flow of Ca2+ and Na+ through parallel pathways driven by these cotransmitters would lead to a much quicker development of a postsynaptic potential. Moreover, with Ca2+ entering with Na+, it is likely that the action potential may be more long-lived and possibly more robust (i.e., having a shoulder or plateau due to Ca2+ entry beyond the Na+-driven spike). Second, ATP could potentiate glutamate release at the presynaptic neuron by potentiating Ca2+ entry driven by the preceding action potential. A more robust Ca2+ entry would cause more or secondary glutamate release and would keep the postsynaptic neuron firing. As for acetylcholine, ATP also has G-protein-coupled receptors, the P2Y receptors, which could modulate presynaptic and\/or postsynaptic neuron function. Third, could metabolites of ATP limit activation of the postsynaptic and presynaptic neuron? One does not wish for the postsynaptic neuron to continue firing ad infinitum. One must have a limiting or negative feedback signal. Could this signal be adenosine? Not only does adenosine need to be recycled back into the neuron as a substrate for new ATP synthesis, but adenosine may also bind to P1 G-protein-coupled receptors to have limiting effects or neuronal function. Of course, ATP release and signaling at an inhibitory synapse driven by \u03b3-aminobutyric acid (GABA) or glycine would have different and modulating effects on inhibitory synapse. We do not discuss or illustrate these possible mechanisms because that is not the intent of this introductory minireview. Synaptic purinergic neurotransmission has been reviewed by Burnstock and others in many seminal reviews [9, 10, 21\u201323].\nConclusions and future directions\nThe founding concepts of purinergic signaling within tissues and tissue microenvironments were found in the heart, the enteric system (later to be duplicated in urinary bladder afferents, nociceptive neurons at the dorsal root ganglia of the spinal cord, etc.), and skeletal muscle by Forrester and coworkers [26]. This special issue of Purinergic Signalling focusing on the \u201cPhysiology of Nucleotide Release\u201d is dedicated to these and other pioneers who would not give up the fight. All purinergics would agree that we owe the purinergic pioneers a great debt. Through their efforts, establishment of this journal, and the increasing volume of research in purinergic signaling, it is now accepted that ATP and its metabolites have two principal biological roles\u2014as an extracellular signaling ligand and as an intracellular fuel.","keyphrases":["signaling","nucleotides","nucleosides","adenosine","atp"],"prmu":["P","P","P","P","P"]}
{"id":"Matern_Child_Health_J-2-2-1592154","title":"Women with Depression\u2014\u201cYou Can't Tell by Looking\u201d\u2122\n","text":"Introduction\nPerinatal depression, a mood disorder that occurs during pregnancy or the first year postpartum, affects 10\u201315% of women and up to 28% of women living in poverty. A previous history of depression, either preconceptionally or during the interconception period, is the strongest predictor that a woman will suffer from perinatal depression. Perinatal depression can adversely affect not only the woman, but also her fetus or infant, and has been linked with prematurity, difficulties with maternal-infant bonding and infant behavior problems later in life. If a woman has a known history of depression, she can be counseled preconceptionally about her increased risk of experiencing a subsequent episode during the perinatal years and thus take an active role in recognizing signs and symptoms of depression and accessing treatment as early as possible should an episode occur. The earlier treatment is sought for perinatal depression, the greater the chance for improved outcomes.\nBackground\nDepression is currently the leading cause of disease-related disability among women worldwide [1]. Depression can cause moodiness, irritability, changes in sleep patterns, sexual interest or appetite, and an inability to carry on with daily activities. In the most severe cases depression can cause thoughts of harming others or oneself or suicide. Prevalence rates of perinatal depression, which occurs during pregnancy or at some point within the first postpartum year, are similar to those in the general population, affecting 10\u201315% of women and up to 28% of women living in poverty [2, 3]. Some studies suggest that a woman's risk of becoming depressed during the early postpartum period is approximately three times that of women in the general population [4]. Depression may affect how a woman is able to relate and respond to her infant and if untreated, may lead to thoughts of harming the infant or infanticide. In addition, depression can have significant effects on prenatal care-seeking behavior [5], substance abuse [6], and infant\/child mental health [7\u20139] and nutrition [10]. Depression has also been associated with low birthweight [11] and premature delivery [12].\nApproximately 50% of women with perinatal depression remain undiagnosed when they are screened based on clinical observations alone [13]. Perhaps the strongest argument for routine screening of all pregnant and postpartum women is the fact that you can't tell simply by looking that a woman is depressed. However, there are some women who may be at increased risk for perinatal depression, including women living in poverty, those in abusive relationships, and women with a prior history of depression [14]. In fact, it is thought that having a history of depression is the strongest predictor that a woman will suffer from an episode of major depression during pregnancy or the postpartum period [15, 16]. Women with other mental disorders are at risk as well. For example, women with bipolar disorder also have an increased risk of experiencing postpartum depression, and some studies suggest an increased risk for a much more serious illness, postpartum psychosis [17]. Due to the predictive value of a history of depression, it is vital that women with a history of depression be counseled about the relationship between pregnancy and depression prior to conception.\nOne method to incorporate depression screening & counseling into preconception care\nCounseling about depression should become a routine part of preconception care. Health care providers who have contact with women during the preconception period can assist women in becoming aware of how pregnancy and depression can affect one another. By controlling depression prior to conception, just as one would with an illness such as diabetes or epilepsy, a woman's chances for healthy perinatal outcomes may improve [18].\nThe Wisconsin Association for Perinatal Care (WAPC) is a 35-year-old statewide, non-profit organization dedicated to the improvement of healthy outcomes for women, infants and their families. Since 1989, WAPC has been working toward infusing depression screening and counseling into preconception care by encouraging health care providers to ask women and their partners about their histories with depression and other mental illnesses prior to conception. One tool that has been developed by WAPC is an information sheet entitled Women with Depression that answers four basic questions: How does depression affect pregnancy? How does pregnancy affect depression? How could medications for depression affect the pregnancy? What can you do before pregnancy?\nCase examples\nThe following are two case examples illustrating the impact of depression on pregnancy and the positive effects of being educated prior to a subsequent depressive episode. Although these women were not educated about perinatal depression prior to their first pregnancies, these examples illustrate the importance of depression counseling in the interconception period as well. These cases demonstrate that addressing the questions in Women with Depression can provide an anticipatory contextual framework on which to develop a plan of care.\nCase example #1\nSara began experiencing symptoms of depression six months after giving birth to her first child. She had no prior history of depression and had received no counseling prior to conception about the symptoms and presentation of perinatal depression. Sara waited approximately six months before seeking help, but encountered numerous barriers to accessing care. She did eventually get into treatment and attended 15 sessions of interpersonal therapy.\nThrough both her therapy and additional self-education, Sara became quite informed about the effects and consequences of postpartum depression. Approximately six months after giving birth to her second child, Sara began experiencing depression again. This time, Sara sought treatment within one week of the presentation of symptoms. In addition to being able to recognize the signs of depression earlier, as reported by both her and her husband, Sara also remarked on how much easier it was to access treatment, as she knew how to \u201cnavigate the system.\u201d She knew what type of therapy worked for her and was able to resolve her depression in only seven sessions.\nCase example #2\nMelanie had a history of anxiety and depression prior to conception, but was never diagnosed or treated. Melanie experienced extreme continuous sleep disturbances almost immediately after the birth of her first child. When this problem had not resolved itself within two months, Melanie sought treatment. She was initially reticent about taking medications because she was breastfeeding her infant, but at nine months postpartum, Melanie began a regimen of paroxetine, which finally resolved the depression.\nAfter Melanie's second birth, she felt slight anxiety about the possibility of suffering from postpartum depression again, but this time had a plan of action in place. When she began having trouble sleeping at two months postpartum, she immediately contacted her therapist and began treatment. She was once again hesitant about taking medication, but at nine months postpartum, began taking paroxetine on the recommendation of her care provider. Although she waited to begin medications with the second depressive episode, Melanie reported having \u201ca kind of comfort knowing I was on paroxetine before and it didn't hurt my baby.\u201d\nBenefits of preconception counseling for women with a history of depression\nThe benefits of counseling women about depression prior to conception are numerous. By treating depression as a chronic condition and by actively managing depression prior to conception, a woman's chances of being prepared for a depressive episode during the perinatal period greatly increase. In a recent meta-analysis of psychosocial and psychological interventions to reduce the risk of postpartum depression there was no statistically significant effect on the prevention of postpartum depression [19]. In the absence of preventive treatment, then, understanding postpartum depression could allow families to anticipate possible consequences of maternal depression so they could take steps to help assure healthy outcomes [20]. By fully disclosing the increased risk of depression if she chooses to become pregnant, a woman can make informed decisions about planning for a family [21]. If the woman is taking antidepressant medication, she can discuss the impact of those medications on her future pregnancy and breastfeeding. By working with her health care provider, steps can be taken to change the medication or dose if necessary.\nOne of the most devastating effects of perinatal depression is that it occurs at a time when most women feel they should be experiencing joy. By educating women prior to pregnancy or birth about the high prevalence of perinatal depression, they may be better prepared for an episode, should it occur. Women with a history of depression especially should have a plan of action in place in the event of perinatal depression.\nWomen who have suffered from depression once may be more likely to recognize the signs and symptoms of a subsequent episode whether or not the first episode occurred preconceptionally or postpartum. This may be especially true if they are counseled about the possibility of a subsequent episode occurring. Women are not the only ones who can be counseled about the interaction between pregnancy and depression. Loved ones often do not know how to help women suffering from perinatal depression and cannot understand why the woman is depressed. By counseling significant others and family members, they, too, can become better prepared and in addition, can help recognize signs of severe depression and assist with accessing treatment.\nSeeking treatment may present fewer challenges to women who have been successfully treated and counseled about depression as well. Women with a history of depression can be counseled about seeking treatment at the earliest possible sign of depression, rather than waiting, as so often happens in primary episodes. The earlier treatment can be sought, the better the outcomes [22]. Stigma may be reduced as well if women are counseled ahead of time, making them more open to seeking treatment and more comfortable with their treatment decisions.\nConclusions\nTestimonials from affected women suggest that counseling women with a history of depression leads to improved outcomes should they suffer from a subsequent episode during the perinatal period. Women may be more likely to recognize the signs of depression, more likely to seek treatment earlier and will know what treatment options work for them. Due to an earlier recognition of depression, treatment may have a shorter duration as well. In addition, by counseling women about depression prior to conception, they and their families will have time to develop a plan of action should a depressive episode occur and will be equipped to make informed decisions about family planning. Tools such as Women with Depression may be beneficial in both educating consumers and assisting providers with consultations about depression prior to pregnancy or during the interconception period.\nThe major challenge currently lies in convincing health care providers to address women's mental health and to screen and counsel for depression during the preconception period. Intervention strategies using medication or counseling have not been significantly effective in preventing postpartum depression. Anticipating the potential interplay between depression and pregnancy may be an intervention that can lead to better mental health and perinatal outcomes.\nFuture research should address the content and timing of preconception counseling, including what information is needed and when and how it should be disseminated. Treatment algorithms and strategies could then be developed for practitioners and families to help prepare for the postpartum period. Finally, well-controlled intervention studies utilizing the materials developed could focus on maternal mental health, perinatal, and infant outcomes to answer questions about efficacy and effectiveness.","keyphrases":["depression","perinatal","mood disorder","preconception"],"prmu":["P","P","P","P"]}
{"id":"Addiction-1-_-2253702","title":"Social cognitive determinants of ecstasy use to target in evidence-based interventions: a meta-analytical review\n","text":"Aims The health hazards and prevalence of ecstasy use have been documented in two decades of research, but no review reporting on potentially modifiable antecedents of use is available. The aim of this study was to integrate systematically research identifying cognitive correlates of ecstasy use. Such research has the potential to identify targets for evidence-based interventions designed to discourage use.\nINTRODUCTION\nEcstasy use is potentially damaging to health [1\u20133] yet prevalent [4,5]. Legislative changes have not been effective in discouraging ecstasy use, and the development of theory-based behavioural interventions is warranted because these have been successful in generating behaviour change in other areas [6,7].\nBehaviour change interventions are more likely to be effective if they target modifiable antecedents of the target behaviour. For example, if expected positive outcomes of a target behaviour differentiate between those who do and do not engage in that behaviour, it is prudent to target outcome expectancies in behaviour change interventions [8]. Which potentially modifiable cognitive antecedents of ecstasy use should interventions target? Unfortunately, although ecstasy was synthesized in 1912 [9], and ecstasy use has been studied for 20 years (e.g. [10,11]), there is no systematic review of this research identifying potentially modifiable cognitive antecedents of use. We aimed to summarize research to date, synthesizing quantitatively all published, quantitative studies of psychological determinants of ecstasy use among young people living in western society.\nMETHOD\nThe search strategy comprised three iterative steps. First, the databases PsycINFO, MedLine and ERIC (Education Resources Information Center) were searched using several combinations of keywords (see Appendix I). The results of the final query were then scanned manually for relevant entries by examining the paper titles and abstracts (see Appendix II). Four inclusion criteria were used. Firstly, a study should investigate the target population of young recreative ecstasy users in western society, as factors influencing behaviour can be population-specific [12\u201314]. Secondly, a study should measure one or more potentially modifiable determinants of ecstasy use-related behaviour; that is, an antecedent that could be influenced potentially by health promotional interventions (excluding for example demographics, personality, etc.); for a list, see chapter 7 of Bartholomew et al. [8]. Thirdly, the study should measure either actual behaviour or intention. Finally, it should assess quantitatively the relationship between determinants and behaviour or intention. Publications selected by this process were examined in detail. Second, reference lists of these papers were scanned for relevant publications (the ancestry approach). Third, texts citing the relevant papers were located using the Web of Science database (the descendancy approach).\nPotentially modifiable determinants were extracted from the studies using a recommended published list [8], on the basis of which two authors selected determinants from each paper and perfect agreement was observed. Associations between determinants and behaviour or intention across studies were integrated by converting all statistics to the correlation coefficient r. These coefficients were then transformed to Fisher's Z and weighed by sample size \u22123 (cf. [15]). The mean Fisher's Z was then transformed back to the correlation coefficient r+. When a study tested a variable several times (e.g. frequency of use and intensity of use), the resulting effect sizes were averaged before being included in the calculations.\nRESULTS\nThe search yielded 367 hits, from which 15 publications were included (see Appendix II). Many excluded studies had a biological focus (e.g. [3]), examined determinants not feasibly changed by health promoting interventions (e.g. [16]) or used qualitative methods (e.g. [17]). All included publications studied the behaviour \u2018using ecstasy\u2019 (or the intention to use); none examined determinants of trying out ecstasy, ceasing use, changing use patterns or applying harm reduction practices. Of these 15 publications, six were discarded after thorough examination, as they were then discovered to yield no quantitative information on the relevance of potentially modifiable determinants of behaviour in the target population ([18\u201323]; see also Appendix II). Application of the ancestry approach yielded no additional publications (365 citations scanned), but the descendancy approach yielded one additional publication (of 85 unique citing publications; [24]). Of the final set of 10 publications [24\u201333], one publication described two studies ([26]; 26a and 26b refer to studies 1 and 2, respectively). Table 1 describes the 11 included studies, listing the sample details, the extracted potentially modifiable antecedents and how they were measured in the original studies.\nTable 1\nQuantitative studies into the determinants of using ecstasy and measures used.\nNo.\nSample details\nn\nAge\n% \u2640\nRelevant variables\nI\nR\n[24]\nUK, before July 2002 Polydrug users\n364\n19\n44%\nNegative mood function scale\n3\n5\nSocial function scale\n5\n5\nNegative effects\n4\n5\nOther functions (9 functions)\n1\n5\nExtent of peer use\n1\n4\nPartner\/best friend use\n1\n2\nIntensity of use\n1\n\u2013\n[25]\nUK, before March 1998 Alcohol and drug users\n100\n19\n45%\nMood function scale\n3\n5\nSocial\/contextual function scale\n5\n5\nNegative effects\/events scale\n3\n5\nExtent of peer use\n1\n5\nIntention\n1\n7\nIntensity of use\n1\n\u2013\nFrequency of use\n1\n\u2013\n[26a]\nUK, March 1992 Students\n186\n19\u201325\n58%\nAttitudes\n6\n7\nSubjective norms\n1\n7\nPerceived behavioural control\n6\n7\nBehavioural beliefs (17)\n2\n19\nNormative beliefs (5)\n2\n19\nControl beliefs (8)\n2\n19\nIntention to use ecstasy\n4\n7\n[26b]*\nUK, mid-1996 Club members\nt1: 203 t2: 123\n23\n41%\nAttitudes\n8\n7\nNormative influences\n13\n7\nPerceived behavioural control\n1\n7\nSelf-efficacy\n1\n7\nBehavioural beliefs\n13\n7\nControl beliefs\n6\n7\nIntention\n3\n7\nBehaviour (longitudinal)\n4\n\u2013\n[27]\nNetherlands, 2000, 2001 Party attendants\n844\n22\n33%\nNegative outcome expectancies\n11\n2\nEnhancement outcome expectancies\n3\n2\nEuphoria outcome expectancies\n4\n2\nSex outcome expectancies\n4\n2\nDancing outcome expectancies\n3\n2\nInsight outcome expectancies\n4\n2\nCommunication outcome expectancies\n4\n2\nWhether ecstasy was currently used\n1\n2\n[28]\n*\nUK t1: October 1994\u20131995 t2: May1995\u20131996 Students\nt1: 461 t2: 136\n19\u201322 19\u201322\n55% 65%\nAttitude\n2\n7\nInjunctive norms\n6\n7\nPerceived behavioural control\n8\n7\nDescriptive norms\n2\n6\nMoral norm\n1\n7\nIntention\n1\n8\nBehaviour (longitudinal)\n1\n8\n[29]\nUK, before 2003 College students\n657\n19\n55%\nFrequency of past use\n1\n7\nIntentions to use\n1\n9\nNormative influence (friends\u2019 use)\n1\n6\nBeliefs about ecstasy use (7 beliefs)\n1\n5\n[30]\n*\nUK\nt1: 84 t2: 32\n20\n74%\nAttitude\n10\n8\nSubjective norm\n2\n8\nPerceived behavioural control over obtaining ecstasy\n3\n8\nPerceived behavioural control over taking ecstasy\n4\n8\nIntention\n5\n8\nHabit\n2\n8\nSpecific attitudinal beliefs (13 beliefs)\n1\n5\nBehaviour (longitudinal)\n1\n2\n[31]\nthe Netherlands, 2001\u20132002\n490\n22\n34%\nEnergy motives\n4\n5\nEuphoria motives\n3\n5\nSelf-insight motives\n2\n5\nSociability\/flirtatiousness motives\n8\n5\nSexiness motives\n4\n5\nCoping motives\n3\n5\nConformism motives\n4\n5\nPerceived positive effects\n24\n2\nPerceived negative effects\n11\n2\nPerceived friends\u2019 use\n1\n5\nFrequency of ecstasy use\n1\n5\n[32]\nUK\n200\n21\n66%\nAttitude\n5\n?\nSubjective norm\n5\n5\nPerceived behavioural control over obtaining ecstasy\n3\n7\nPerceived behavioural control over taking ecstasy\n11\n?\nIntention\n6\n7\nHabit\n2\n7\n[33]\nUSA Club rave attendees\n70\n20\n47%\nRisk associated with using ecstasy once or twice\n1\n4\nRisk associated with using ecstasy regularly\n1\n4\nHarmful short-term physical effects\n1\n4\nHarmful long-term physical effects\n1\n4\nHarmful short-term psychological effects\n1\n4\nHarmful long-term psychological effects\n1\n4\nPositive physical effects\n1\n4\nPositive psychological effects\n1\n4\nEcstasy use within the past 12 months\n1\n2\nNo. = number in reference list, I = number of items used to measure variable, R = number of scale points on response scale of each item.\n*\nLongitudinal design.\nAll 11 studies can be viewed as tests of two theoretical frameworks which are applied frequently in drug use research [34]. Six studies [26a,26b,28\u201330,32] tested the theory of planned behaviour (TPB; [35]). The TPB proposes that the most proximal cognitive determinant of behaviour is intention which, in turn, is predicted by attitude (i.e. evaluation of probable consequences of that behaviour), subjective norm (i.e. perception of others' approval of the behaviour) and perceived behavioural control (PBC; i.e. perception of control based on perception of skills and external obstacles\/facilitators). Each of these constructs is based on underlying beliefs. Several extensions of the TPB have been proposed [36]; for example, personal norm (personal moral evaluation of the behaviour), descriptive norm (perception of others' performance of the behaviour [37]), habit [38] and anticipated regret (the regret one experiences when prospectively imagining having\u2014or not having\u2014performed a behaviour [39,40]).\nFive studies [24,25,27,31,33] tested expectancy models (e.g. [41]), which propose that behaviour is determined by expectations people have of the behaviours' consequences. Two studies [24,25] assessed how often participants used ecstasy for particular reasons (e.g. \u2018in the past year, how often have you used ecstasy to help you to let go of inhibitions?\u2019), implying germane expectations (such as \u2018taking ecstasy helps me let go of inhibitions\u2019). Three studies [27,31,33] used more traditional measures (e.g. \u2018I have experienced\/would expect that ecstasy makes it easier to communicate\u2019). Note that these expectations are viewed as underlying beliefs in the TPB, particularly in relation to the structure of attitudes [12,35].\nIn both the TPB and outcome expectancy models, higher-level constructs, such as attitudes, are based on lower-level beliefs. Most studies tested models involving this hierarchical cognitive structure. For the purposes of this review, higher-level constructs are referred to as \u2018compound constructs\u2019 and lower-order cognitions as \u2018expectancies\u2019 (e.g. beliefs about probable consequences of ecstasy use). Details of the particular theoretical models explored in the original studies are not provided here.\nOnly bivariate analyses were synthesized because multivariate analyses were incommensurable over studies as they tested different models. [In order to resolve this by conducting uniform regression analyses, all authors were asked to provide matrices of covariance. However, due to lost data sets, missing data and non-response, not enough data could be retrieved to render this feasible. The authors are grateful to M. Conner and T. ter Bogt, who did supply additional data.] It is worth noting none the less that in the two TPB-based studiesin which ecstasy use was regressed onto compound constructs, the average (weighed) R2 was 0.51 [26b,28], and in the five TPB-based studies in which intention to use was regressed onto compound constructs the average R2 was 0.67 [26a,26b,28,30,32]. The average R2 for the three expectancy studies in which ecstasy use was regressed onto expectancies was 0.35 [24,27,31], and in the expectancy study where intention to use was regressed onto expectancies was 0.64 [25]. In three prospective studies ecstasy use was found to be related strongly to prior intention to use with an average (weighed) r of 0.71 [26b,28,30].\nTable 2 shows the strength of association between predictor variables and measures of ecstasy use and intention to use. The strongest predictor of intention and use was the TPB-specified attitude measure. Attitudes are thought to be based on more specific expectancies [12,35] and these are shown separately in Table 2, grouped into \u2018positive\u2019 and \u2018negative\u2019 expectancies. In addition to these perceived advantages and disadvantages of ecstasy use, normative measures have been used, especially subjective and descriptive norms, as well as perceived behavioural control over ecstasy use. Variables proposed as extensions to the TPB have been categorized as \u2018miscellaneous\u2019.\nTable 2\nEffect sizes of predictors of ecstasy use and intention to use.\nAssociation with behaviour\nAssociation with intention\nVariable type\nCompound or expectancy\nk\nr+\nk\nr+\nAttitude\nAttitude [C]\n5\n0.53 (L)\n5\n0.63 (L)\nPositive expectancies\nSocial function [C]\n4\n0.09\n2\n0.30 (M)\nProduces a positive mood state\n3\n0.12 (S)\n2\n0.28 (M)\nMood function [C]\n4\n0.20 (S)\n1\n0.41 (L)\nHelps lose weight\n2\n0.09\n1\n0.08\nEnhances sex\n3\n0.11 (S)\nHelps stay awake\n3\n0.08\nHelps keep fit\n1\n0.14 (S)\n1\n0.22 (S)\nEnhances insight\/openness\n2\n0.18 (S)\nHelps to relax\/helps coping\n2\n0.08\nProduces excitement\n1\n0.31 (M)\nProduces intoxication\n1\n0.19 (S)\nEases after-effects\n1\n0.15 (S)\nImproves other drugs' effects\n1\n0.11 (S)\nIs conforming to peers\n1\n0.05\nHelps work\n1\n0.05\nNegative expectancies\nShort-term negative effects [C]\n4\n\u22120.47 (L)\nProduces mood swings\n1\n\u22120.34 (M)\n2\n\u22120.31 (M)\nLeads on to more frequent use\n1\n\u22120.30 (M)\n2\n\u22120.39 (L)\nProduces mental side-effects\n1\n\u22120.24 (M)\n2\n\u22120.40 (L)\nLeads on to \u2018worse\u2019 drugs\n1\n\u22120.23 (S)\n2\n\u22120.31 (M)\nProduces physical side-effects\n1\n\u22120.18 (S)\n2\n\u22120.39 (L)\nMakes one unhealthy\n1\n\u22120.18 (S)\n2\n\u22120.30 (M)\nProduces depression\n1\n\u22120.18 (S)\n2\n\u22120.28 (M)\nProduces lethargy\n1\n\u22120.05\n2\n\u22120.20 (S)\nProduces addiction\n1\n\u22120.32 (M)\n1\n\u22120.12 (S)\nLeads to death\n1\n\u22120.30 (M)\n1\n\u22120.32 (M)\nProduces paranoia\n1\n\u22120.27 (M)\n1\n\u22120.26 (M)\nMakes one feel run down\n1\n\u22120.17 (S)\n1\n\u22120.25 (M)\nSubjective injunctive norm\nSubjective norms (approval) [C]\n5\n0.39 (L)\n5\n0.52 (L)\nBest friends' approval\n1\n0.36 (M)\n1\n0.25 (M)\nPartners approval\n1\n0.15 (S)\n1\n0.30 (M)\nParents' approval\n1\n0.10 (S)\n1\n0.01\nHealth experts' approval\n1\n0.09\n1\n0.00\nOther ecstasy users' approval\n1\n0.07\n1\n0.08\nSubjective descriptive norm\nSubjective norms (descriptive) [C]\n1\n0.52 (L)\n1\n0.63 (L)\nPerceived peer use\n2\n0.39 (L)\n1\n0.43 (L)\nPerceived use in close relatives\n2\n0.21 (S)\nPerceived best friend\/partner use\n1\n0.37 (L)\nPerceived behavioural control\nPBC [C]\n3\n0.33 (M)\n3\n0.52 (L)\nPBC (over obtaining ecstasy)\n2\n0.20 (M)\n2\n0.25 (M)\nPBC (over taking ecstasy)\n2\n0.01\n2\n\u22120.03\nBeing with friends who use\n1\n0.45 (L)\n2\n0.62 (L)\nGoing out dancing\n1\n0.41 (L)\n2\n0.67 (L)\nBeing offered ecstasy\n1\n0.40 (L)\n2\n0.59 (L)\nEcstasy being available\n1\n0.40 (L)\n2\n0.57 (L)\nCheap ecstasy\n1\n0.19 (M)\n2\n0.54 (L)\nHaving alcohol\n1\n\u22120.02\n2\n0.10 (S)\nPBC (over not taking ecstasy)\n1\n\u22120.06\n1\n0.07\nNeeding to lose weight\n1\n\u22120.05\n1\n0.00\nNeeding to exercise\n1\n\u22120.01\n1\n0.05\nMiscellaneous\nHabit [C]\n2\n0.45 (L)\n2\n0.46 (L)\nMoral norm [C]\n2\n\u22120.28 (M)\n2\n\u22120.31 (M)\nDenial of negative consequences\n1\n0.17 (S)\n1\n0.18 (S)\nAnticipated regret\n1\n\u22120.11 (S)\n1\n\u22120.22 (S)\n[C] = compound construct, k = no. of samples, r+ = weighed average correlation, (S) = small, (M) = medium, (L) = large effect size magnitude according to Cohen [42]. PBC: perceived behavioural control.\nTable 2 is based on the Pearson correlations reported in five studies [24,25,28,30,32] and correlation matrices received from the authors of two publications [26,31]. One study [27] reported t-values, which were converted into effect size correlations using r = \u221a[t2\/(t2 + df)]. The results from two studies [29,33] could not be converted and will be provided later. Only associations found in at least two samples, of magnitudes corresponding to a medium (Cohen's d = 0.5 [42], r = 0.24) or large effect size (d = 0.8 and r = 0.37), are considered.\nAll significant associations were in the expected direction and some large effect sizes were observed, suggesting that both the theory of planned behaviour and the expectancy approach can help to explain ecstasy use and intentions to use. In the TPB studies, intention and behaviour are associated most strongly with TPB's attitude, with a large effect size (r+ = 0.53 with behaviour, r+ = 0.63 with intention). Specifically, this compound construct appears to be associated mainly with positive outcomes regarding mood control and social facilitation, and negative expectations regarding short-term negative effects, escalating use and physical and mental side effects.\nNormative influences are also important covariates of use and intention to use with stronger associations observed for descriptive (r+ = 0.52 with behaviour, r+ = 0.63 with intention) than injunctive norms (r+ = 0.39 with behaviour, r+ = 0.52 with intention). The strongest effect sizes for expectancies underpinning these compound constructs were observed for perceived ecstasy use by peers and approval of use by one's best friend and partner. The results also indicate that whether parents, health experts and \u2018other ecstasy users\u2019 approve is inconsequential.\nPerceived behavioural control was related to use with a medium (near large) effect size (r+ = 0.33) and also related strongly to intention (r+ = 0.52). Underlying beliefs showed a medium effect regarding control over obtaining ecstasy and large effects of control in relation to being with friends who use, going out dancing, being offered ecstasy and ecstasy being available. Two proposed extensions to TPB appear relevant: habit, with a large effect size (r+ = 0.45 with behaviour, r+ = 0.46 with intention) and moral norm, with a medium effect size (r+ = \u22120.28 with behaviour, r+ = \u22120.31 with intention).\nTwo studies reported results using statistics that could not be transformed to an effect size measure. One study [29] conducted a polynomial logistic regression predicting user group (six levels: rejectors, at-risk non-users, ex-users, and light, moderate and heavy users; for details, see [29]), testing whether a number of variables contributed significantly to model fit when predicting user group. Another study [33] conducted t-tests on beliefs (also predicting user group, with two levels: non-user and user), but did not report t-values, exact P-values, or variance information. The variables tested in these studies are shown in Table 3. Effect sizes were estimated (conservatively) on the basis of sample size and significance.\nTable 3\nSignificant and non-significant predictors of user group (user group had six levels in study [29] and two levels in study [33]).\nVariable\nStudy\nSignificance\nAssociation\nEffect size\nPerceived use by friends\n[29]\n< 0.001\nPositive\nM\nAnticipated regret (\u2018use would induce guilt\u2019)\n[29]\n< 0.001\nNegative\nM\nIs hard to resist\n[29]\n< 0.001\nPositive\nM\nMoral norm (\u2018ecstasy use is immoral\u2019)\n[29]\n< 0.05\nNegative\nS\nPerceived availability of ecstasy\n[29]\n< 0.05\nPositive\nS\nIs bad for one's physical health\n[29]\nNone\n\u2013\n\u2013\nIs bad for one's mental health\n[29]\nNone\n\u2013\n\u2013\nSubjective norm (approval)\n[29]\nNone\n\u2013\n\u2013\nHarmful long-term physical effects\n[33]\n< 0.01\nNegative\nM\nRisk associated with using regularly\n[33]\n< 0.01\nNegative\nM\nRisk association with using once or twice\n[33]\n< 0.05\nNegative\nM\nHarmful long-term psychological effects\n[33]\n< 0.05\nNegative\nM\nHarmful short-term physical effects\n[33]\nNone\n\u2013\n\u2013\nHarmful short-term psychological effects\n[33]\nNone\n\u2013\n\u2013\nPositive physical effects\n[33]\nNone\n\u2013\n\u2013\nPositive psychological effects\n[33]\nNone\n\u2013\n\u2013\nS = small, M = medium, effect size magnitude according to Cohen [42].\nThese additional results confirm the relevance of descriptive norm, negative expectancies (particularly long-term effects) and perceived control, and add anticipated regret, with at least a medium effect size, to the list.\nDISCUSSION\nSynthesis of the included studies shows the main predictors of intention to use and actual ecstasy use to be attitude (specifically positive outcomes regarding mood control and social facilitation and negative outcomes regarding escalating use and physical and mental side-effects); subjective and descriptive norms regarding one's friends, partner and peers; perceived control regarding obtaining ecstasy and control in relation to being with friends who use, going out dancing, being offered ecstasy and ecstasy being available; and habit, moral norm and anticipated regret. In addition to lending support to both the TPB and expectancy models, these findings show that some expectancies underlying attitude are irrelevant (e.g. ecstasy enhances sex), as are norms relating to some social referents (e.g. parents), and perceived control regarding some ecstasy-related behaviours (e.g. taking ecstasy).\nAs it is now clear which determinants best predict intention to use and ecstasy use according to the research so far, these determinants seem advisable intervention targets. However, not all determinants are equally easy to modify. As attitude encompasses several specific expectancies, it may be easier to target these more concrete expectancies than the abstract attitude construct. Also, not all expectancies are influenced equally easily. Because most users have experienced positive mood shifts, it may be difficult to develop persuasive messages that undermine this expectancy among users [43]. Negative variables are also associated strongly, and changed more easily. However, simply presenting information about negative outcomes (\u2018fear appeals\u2019) has been shown not to work, or even work inversely, unless a number of critical conditions are met, such as efficacy enhancement (see [6,44]; also see [45]). Similarly, it may be difficult to change a subjective norm if it reflects reality. As ecstasy use is social [46] and most users take ecstasy at a dance event [47] where ecstasy use is high (in the Netherlands, about two-thirds of the visitors use ecstasy; [48]), it might be hard to reduce descriptive norms, especially if participants' friends use ecstasy. Similarly, it may be challenging to establish a disapproving norm (see [49]).\nSimilarly, it may be difficult to reduce perceived control over obtaining ecstasy among users, as they probably have repeatedly performed this behaviour successfully. However, the large effects of specific control beliefs suggest that users who wish to stop may well be aided by adopting a stimulus control strategy avoiding the social contexts of use. In addition, enhancing refusal skills would influence perceived behavioural control, while also diminishing the effect of undesirable subjective norms (by decreasing users' motivation to comply, see [35]). Thus, among those with intentions not to use, refusal skills training and stimulus control are recommended [50]. Finally, habit and moral norm are useful predictors but may be difficult to change ([8], but also see [51]). Although anticipated regret has a smaller association with use and intention to use, it can be changed more easily (e.g. [39]). A list of methods and strategies to change these determinants, and the theoretical parameters prerequisite to success, is provided in chapter 7 of Bartholemew et al. [8].\nThis review is limited mainly by the fact that only bivariate associations could be synthesized and by two consequences of the paucity of research into ecstasy use thus far. First, the small number of studies into determinants of ecstasy use limit the robustness of the current findings. Second, many theories and models have not yet been studied and are therefore not included in this review. Only social cognitive theories have been tested; no studies have investigated, for example, the predictive value of implicit cognitions. However, recent research implies that implicit processes may be changeable [52]. Moreover, within social cognitive research, recently developed constructs such as consideration of future consequences [53] have not yet been applied to ecstasy use, but may aid in intervention development.\nRegarding the theories that have been studied, in order to gain a more comprehensive understanding of motives for ecstasy use future research should combine the two theoretical perspectives studied thus far, so that relative overlap can be determined. It would be interesting to see whether, and to what degree, particular expectancies account for the predictive utility of the TPB attitude measures. Also, the list of expectancies that has been studied so far may omit a number of consequences (such as \u2018suicide Tuesday\u2019, a term for a period following use when low serotonin levels can induce depressive feelings).\nAnother important gap in the literature concerns behaviours other than \u2018using ecstasy\u2019. Not only can the determinants of trying ecstasy out, starting use, ceasing use and maintaining cessation differ from the determinants of using ecstasy [12], little is known about the determinants of harm reduction practices, such as having one's ecstasy tested by a test service, ensuring sufficient hydration and maintaining a low body temperature (although studies such as [54] are a step in the right direction). Intervening to promote these behaviours could prove to be more beneficial to the health of party visitors, given the difficulty of intervening on most variables determining ecstasy use.\nIn conclusion, this review suggests that there is sufficient evidence to guide intervention development so that evidence-based practice is established. These interventions could then be evaluated to test the utility of particular theoretical frameworks. The priorities for interventions should be negative expectancies, perceived behavioural control and anticipated regret. Tailored interventions can offer refusal skills training and strategies to avoid risky situations to participants not intending to use ecstasy.\nAppendix I\nSearch terms used in PsycINFO (equivalent terms used in corresponding fields in MedLine and ERIC) [query in words, as corresponding to \u2018concepts\u2019 column, in brackets].No.ConceptOperationalizationFields1Language(English) or (Dutch)Language2Publication type(journal*) or (peer-reviewed-journal)Publication type3Publication date> 1980Publication year4Ecstasy(clubdrug*) or (club near drug*) or (dance near drug*) or (dancedrug*) or (party near drug*) or (partydrug*) or (xtc) or (mdma) or (methylenedioxymethamphetamine) or (\u20183,4-methylenedioxymethamphetamine\u2019) or (ecstasy)Title, abstract, keywords5Theoretical(theor*) or (attitud*) or (motivat* near functio*) or (mode*) or (norm*) or (perceived near control) or (pbc) or ('social cognitive\u2019) or (self adj efficacy) or (stages near change) or (perceived adj (harm or risk or functions)) or (functional) or (outcome adj (expectancies or expectations)) or (sct) or (tpb) or (patter*) or (psychosoc*) or (health adj belief adj model) or (hbm)Title, abstract, keywords6Determinants(determin*) or (facto*) or (variabl*) or (parameter*) or (reason*) or (caus*) or (motiv*) or (incentive*) or (correlat*) or (antecedent*) or (character*)Title, abstract, keywords7Initiation(start*) or (commenc*) or (originat*) or (onset) or (initiat*) or (instigat*) or ((use) not (user)) or (using) or (usage) or (establish*)Title, abstract, keywords8Maintenance(maint*) or (sustain*) or (continu*) or (uphold*) or (persist*) or (further*) or (prolong*)Title, abstract, keywords9Cessation(end*) or (stop*) or (discontinu*) or (terminat*) or (ceas*) or (cessat*) or (abstain*) or (abstin*) or (quit*) or (remiss*) or (resolut*) or (recover*)Title, abstract, keywords10Harm reduction(harm or risk or damage or casualt*) and (reduc* or manag* or limit* or minimi*)Title, abstract, keywords11Excluded((treatment not (\u2018not in treatment\u2019 or \u2018non-treatment\u2019 or \u2018non- treatment\u2019 or \u2018no treatment\u2019)) or rat or rats or mouse or mice or animal or monkey* or pigeon* or spectro* or cardio* or seroton* or dopamin* or neurotransm* or receptor* or psychiatr* or psychopath* or cell* or diagnos*)Anywhere12Inclusion#1 and #2 and #3 [Language and Publication Type and Publication Date]\u201313Behaviour#7 or #8 or #9 or #10 [Initiation or Maintenance or Cessation or Harm reduction]\u201314Empirical*#6 near #13 [Determinants near Behaviour]\u201315Final query*#12 and #4 near (#5 or #14) not #11 [Inclusion and Ecstasy near (Theoretical or Empirical) not Excluded]\u2013When executed, the query consisted of one command; therefore the use of the \u2018near\u2019-operator was valid here.\nAppendix II\nSearch procedure, number of resulting hits, and results of each step.\nStep\nActivity\nNumber of resulting publications\n1\nInput of query at 20 August 2007 in PsycINFO (162), MedLine (194) and ERIC (11)\n367\n2\nRemoval of duplicate records (83)\n284\n3\nRemoval of records about publications that (entries were removed in this order):\n3.1\nstudied biological variables (e.g. sequelae of ecstasy use; 75)\n209\n3.2\ndid not study ecstasy use or a related behaviour (such as trying out ecstasy, ceasing use, changing use patterns, or applying harm reduction practices; 32)\n177\n3.3\nstudied variables that cannot be changed using a health promotion intervention (e.g. sex, ethnicity or religion; 68)\n109\n3.4\nstudied ecstasy use as an independent variable in a multivariate or longitudinal analysis (22)\n87\n3.5\ndid not employ quantitative methods (e.g. qualitative studies; 30)\n57\n3.6\ndid not study behaviour or cognitions (25)\n32\n3.7\nstudied a \u2018non-normal\u2019 subpopulation or gathering data from samples inseparably encompassing these subpopulations (e.g. dependent participants, patients or delinquents)*, studying generic drug categories (e.g. \u2018hard drugs\u2019)\u2020, or not explicitly stating which drugs were studied (14)\n18\n3.8\nwere not published in a peer-reviewed journal (3)\n15\n*\ninclusion of these subpopulations would restrict generalization of the results to the target population of the current study (i.e. the average adolescent; see [12,55]).\n\u2020\nthis demand of drug specificity is necessary because previous research has shown that beliefs about drugs can vary between different drugs [56], rendering aggregation questionable.","keyphrases":["determinants","ecstasy","review","expectancies","drugs","theory of planned behaviour"],"prmu":["P","P","P","P","P","P"]}
{"id":"Eur_J_Pediatr-3-1-2151775","title":"Incidence of late vitamin K deficiency bleeding in newborns in the Netherlands in 2005: evaluation of the current guideline\n","text":"Vitamin K prophylaxis is recommended to prevent the hazard of haemorrhage caused by vitamin K deficiency in newborns. The present Dutch guideline recommends 1 mg of vitamin K1 orally at birth, followed by a daily dose of 25 \u03bcg of vitamin K1 from 1 to 13 weeks of age for breastfed infants. Since the introduction of this prophylaxis, the incidence of vitamin K deficiency bleeding (VKDB) has decreased; however, late VKDB is still reported. From 1 January to 31 December 2005, a nationwide active surveillance was performed by the Netherlands Paediatric Surveillance Unit (NSCK) to study the current incidence and aetiology of late VKDB in infants. Six cases could be validated as late VKDB: all were breastfed, one fatal idiopathic intracranial haemorrhage at the age of 5 weeks and five bleedings secondary to an underlying cholestatic liver disease between the age of 3 and 7 weeks. The total incidence of late VKDB and idiopathic late VKDB was calculated to be 3.2 (95% CI: 1.2\u20136.9) and 0.5 (95% CI: 0\u20132.9) per 100,000 live births, respectively. With the current Dutch guideline, idiopathic late VKDB is rare but late VKDB secondary to cholestasis still occurs in breastfed infants. Doubling the daily dose of vitamin K1 to 50 \u03bcg, as is comparable to formula-feeding, may possibly prevent VKDB in this group. Further research, however, is needed to prove this hypothesis.\nIntroduction\nVitamin K deficiency can cause severe haemorrhage in the newborn. The term \u201chaemorrhagic disease of the newborn\u201d (HDN) was coined by Charles Townsend in 1894 to describe an acquired bleeding disorder in the newborn due to vitamin K deficiency [26]. Nowadays, the term HDN is replaced by vitamin K deficiency bleeding (VKDB), as neonatal bleeding is often not due to vitamin K deficiency and VKDB often occurs after the 4-week neonatal period [23].\nVKDB can be classified according to the time of presentation after birth into early (0\u201324\u00a0h), classical (1\u20137\u00a0days) and late (2\u201312\u00a0weeks) VKDB. Late VKDB occurs primarily in exclusively breastfed infants who have received no or inadequate vitamin K prophylaxis. In addition, intestinal malabsorption defects (cholestatic jaundice, cystic fibrosis, \u03b1-1-antitrypsin deficiency etc.) are a well known frequent cause of late VKDB [13]. In some children, \u201cwarning bleeds\u201d such as mild bruises, nose bleeds or umbilical oozing are the first manifestation of VKDB. In about 50% of the cases in late VKDB, intracranial haemorrhage occurs, accounting for death and serious neurological and mental handicaps [22]. The efficacy of neonatal vitamin K prophylaxis (oral or parenteral) in the prevention of classic VKDB is firmly established [16]. It has been the standard of care since the Committee on Nutrition of the American Academy of Paediatrics recommended to administer vitamin K prophylaxis to all newborn infants in 1961 [1]. Since then, many countries adopted this recommendation, although controversies concerning the best dose, route and frequency of administration still exist.\nIn 1990, the Dutch Paediatric Association recommended the administration of a single oral dose of 1\u00a0mg of vitamin K1 at birth to all healthy infants. Parenteral administration of vitamin K1 at birth was recommended for newborns especially at risk (preterm babies, babies born with instrumental help or caesarean section, asphyxia or if maternal medication was interfering with coagulation). For infants who are largely or fully breastfed, a daily dose of 25\u00a0\u03bcg of vitamin K1 orally was recommended from 1 to 13\u00a0weeks of age. In case of additional risk factors like a known malabsorption, this dose should be increased to 50\u00a0\u03bcg per day [29].\nAfter the introduction of this guideline, the incidence of late VKDB, which was studied by a nationwide active surveillance performed by the Netherlands Paediatric Surveillance Unit (NSCK) in 1992 to 1994, decreased from about 7.0 to 1.1\/100,000 live births [2]. However, still, some cases of late VKDB have been reported in the Netherlands, merely due to an underlying cholestatic liver disease [7, 10, 11].\nTherefore, a one-year surveillance was started again to study the current incidence and the aetiology of late VKDB in the Netherlands. Discussion will rise about the efficacy of the current guideline in order to completely prevent late VKDB.\nMethods\nFrom 1 January to 31 December 2005, an active surveillance programme for VKDB was in operation in the Netherlands, using methods developed by the British Paediatric Association Surveillance Unit [8]. Surveillance was based on monthly report cards sent to paediatricians responsible for in- and outpatient care in all hospitals by the NSCK. A \u201cnothing to report\u201d option was included. The return rate of these report cards was 94.7% in 2004 [17].\nThe paediatricians were asked to report all infants in whom bleeding may have resulted from VKDB. The reported cases were validated with a questionnaire asking for information about the infant, type of feeding, clinical presentation, dose of vitamin K prophylaxis, route of vitamin K prophylaxis, associated diseases and laboratory data. For all reported cases, we asked for an anonymous hospital discharge letter to verify the diagnosis of late-onset VKDB.\nThe case definition of late-onset VKDB was as internationally defined: any infant between 8\u00a0days to 26\u00a0weeks of age, with spontaneous bruising, bleeding or intracranial haemorrhage associated with a grossly prolonged prothrombin assay, not due to an inherited coagulopathy or disseminated intravascular coagulation [23].\nConfirmed VKDB was diagnosed when the prothrombin assay results were grossly abnormal compared with the standards for age: international normalized ratio (INR) \u22654 control value, prothrombin time (PT) \u22654 control value and at least one of the following was present: \nPlatelet count normal or raised and normal fibrinogenProthrombin assay returned to normal after vitamin K administrationConcentration of proteins induced by vitamin K absence (PIVKA) exceeded normal controls\nPIVKA is a sensitive indicator of vitamin K status, allowing the detection of subclinical deficiency states which would not be detected by conventional coagulation assays [20].\nIdiopathic cases were defined as cases in whom no factor predisposing to vitamin K deficiency was identified. Secondary cases were those in whom an underlying condition such as cholestasis or other causes of malabsorption of vitamin K could be identified.\nVitamin K prophylaxis was considered to be \u201cas recommended\u201d if the Dutch guideline was followed at the time of bleeding: 1\u00a0mg of vitamin K1 (oral, intravenous, intramuscular) directly after birth, followed by a daily dose of 25\u00a0\u03bcg of vitamin K1 from 1 to 13\u00a0weeks of age for all breastfed babies.\nThe Konakion mixed micellar preparate (Phytomenadion 10\u00a0mg\/ml, Hoffmann-La Roche, Basel, Switzerland), which can be used for both oral, intramuscular and intravenous administration, an oral preparate called Davitamon K (marketed by Chefaro, Rotterdam, the Netherlands, 25\u00a0\u03bcg\/5gtt) and an oral preparate, Phytomenadion, which is made by the Dutch pharmacist Mixtura (Phytomenadion 10\u00a0mg\/ml FNA) were the only vitamin K preparations licensed in the Netherlands.\nThe 95% confidence intervals (CI) were calculated according to a binomial distribution and the chi-squared test was used for the comparison of incidences between different periods, with a value of P\u2009<\u20090.05 being considered as significant.\nResults\nThe response rate to the surveillance amounted to 93.4% and the response to our questionnaires was 100%. In total, seven cases of late VKDB were reported: four reports from academic paediatric centres and three from general district hospitals. A hospital discharge letter was obtained in six cases. In all cases, the diagnosis could be verified by the completed questionnaire or the additional discharge letter.\nOne case did not fulfil the criteria of the case definition because there were no clinical signs of bleeding, only biochemical clotting disorders due to a vitamin K deficiency.\nSix cases fulfilled the criteria of the case definition and could be validated as late VKDB. The surveillance data are shown in Table\u00a01. The mean age of presentation was 5\u00a0weeks and 2\u00a0days, and all of the cases were exclusively breastfed. In all cases, the PT time or INR was at least four times greater than normal and vitamin K deficiency was confirmed by a documented PT or INR normalisation after the administration of vitamin K. Five of the cases also received fresh frozen plasma. \nTable\u00a01Confirmed cases of late vitamin K deficiency bleeding (VKDB) in the Netherlands from 1 January to 31 December 2005Patient; sex; age (weeks)PresentationFeedingVitamin K prophylaxisProthrombin assay (after vitamin K administration) Additional morbidityOutcome of bleedingA; female; 5\u2009+\u20091\/7Intracranial bleedingBreastfeedingBirth: 1\u00a0mg p.o.\u219225\u00a0\u03bcg\/dayPT>120\u00a0s (13.3); APTT>120\u00a0s (37)NoDiedB; female; 6\u2009+\u20096\/7Intracranial bleedingBreastfeedingBirth: 1\u00a0mg p.o.\u219225\u00a0\u03bcg\/dayINR>8 (INR<2); APTT>240\u00a0s (49)Biliary atresiaHemipareses rightC; male; 4\u2009+\u20094\/7Intracranial bleedingBreastfeedingBirth: 1\u00a0mg p.o.\u219225\u00a0\u03bcg\/dayPT>90\u00a0s (30); APTT>120\u00a0s (38); Vitamin K: 0.1\u00a0nmol\/lBiliary atresiaHemipareses rightD; male; 5\u2009+\u20092\/7Nasal bleedingBreastfeedingBirth: 1\u00a0mg p.o.\u219225\u00a0\u03bcg\/dayPT>75\u00a0s (normal); APTT: 118\u00a0s (normal)Biliary atresiaNo sequelaeE; male; 6\u2009+\u20092\/7Nasal bleedingBreastfeedingBirth: 1\u00a0mg p.o.\u219225\u00a0\u03bcg\/dayPT>165\u00a0s (normal); APTT>240\u00a0s (normal)Biliary atresiaNo sequelaeF; male; 3\u2009+\u20096\/7Nasal bleedingBreastfeedingBirth: 1\u00a0mg p.o.\u219225\u00a0\u03bcg\/dayPT>71\u00a0s (12.7); APTT: 128\u00a0s (39.1)CholestasisNo sequelae\nConcerning the cause of VKDB, there was one case of idiopathic VKDB, who presented with signs of bleeding at the age of 5\u00a0weeks. She was breastfed and died due to the consequence of an intracranial bleeding. She received exactly the recommended prophylaxis. Unfortunately, no post-mortem obduction was performed but, biochemically, there were no signs of cholestasis. Five out of six cases could be validated as secondary late VKDB. The age of presentation in this group was between 3 and 7\u00a0weeks of age. All of these bleedings were due to an underlying cholestasis which was diagnosed after the first presentation of bleeding. In two cases, an intracranial bleeding was the first presenting sign. Four cases were diagnosed as having a bile duct atresia. All infants had been exclusively breastfed and all had received exactly the recommended prophylaxis.\nIn the study period, the number of live births amounted to 187,910 [21]. Therefore, the total incidence of late VKDB and idiopathic late VKDB was calculated to be 3.2 (95% CI: 1.2\u20136.9) and 0.5 (95% CI: 0\u20132.9) per 100,000 live births, respectively.\nDiscussion\nData from the United Kingdom, Germany and Switzerland prove that intramuscular vitamin K1 prophylaxis (1\u00a0mg) is highly effective in the prevention of late VKDB. This is supported by data from Australia, which demonstrate a higher incidence of late VKDB in babies who received oral vitamin K compared to those with parenteral vitamin K at birth. However, most of these failures of oral vitamin K were in cases reported with an underlying cholestatic liver disease [3, 12, 14, 25, 32, 33].\nIn 1992, Golding et al. [5] reported an increased risk of developing childhood cancer after parenteral vitamin K1 prophylaxis. Recent studies, however, show that solid tumours are no commoner in children who had parenteral vitamin K. Although no convincing evidence between parenteral vitamin K and childhood leukaemia was found in these studies, it cannot be fully excluded that there is no risk [4, 18].\nSince the report of Golding et al., there has been an increasing trend towards oral vitamin K administration and many oral prophylaxis regimes have been developed.\nIn 1997, we summarised the results of surveillance studies of over 2 million infants in four different countries using different multiple oral dose schedules and parenteral administration of vitamin K. A daily low oral dose of 25\u00a0\u03bcg of vitamin K1 following an initial oral dose of 1\u00a0mg vitamin K1 directly after birth for exclusively breastfed infants, as was used in the Netherlands since 1990, seemed to be as effective as parenteral vitamin K1 prophylaxis [3].\nThe present surveillance study shows that the current Dutch guideline still seems quite effective in preventing idiopathic late VKDB, but it is insufficient against secondary late VKDB. In our surveillance, the incidence of all late VKDB and idiopathic late VKDB was 3.2 (95% CI: 1.2\u20136.9) and 0.5 (95% CI: 0\u20132.9) per 100,000 live births, respectively. The incidence of the latter is comparable to 0.5 (95% CI: 0.1\u20131.6) per 100,000 live births reported in our surveillance study performed by the same Netherlands Paediatric Surveillance Unit (NSCK) in 1992\u20131994 [2]. The incidence of late VKDB secondary to an underlying cholestatic liver disease is, however, significantly higher (P\u2009<\u20090.05); 2.7 (95% CI: 0.9\u20136.2) compared to 0.7 (95% CI: 0.1\u20132.0) in 1992\u20131994. A higher incidence of cholestatic liver diseases in general or a delayed recognition of symptoms can be explanations for this difference. Unfortunately, data about the incidence or recognition of cholestatic liver diseases in the Netherlands are not available.\nIn this surveillance, there were no reports of late VKDB after parenteral administration of vitamin K1. This can be explained by the assumption of the higher efficacy of parenteral vitamin K1 or the low use of intramuscular and intravenous vitamin K1 these days.\nSince 1994, a new preparate, Konakion mixed micellar, has been available. This mixed micellar formulation is prepared with the phospholipids lecithin and the bile salt glycocholic acid, and it was presumed that this preparate would reduce the incidence of late VKDB due to a better absorption of oral vitamin K. However, a recent randomised controlled trial shows that the mixed micellar vitamin K gives unreliable and still severely impaired intestinal absorption in infants with cholestasis [15]. Furthermore, von Kries et al. [31] reported no significant reduction in the incidence of late VKDB in healthy infants and infants with an underlying cholestatic liver disease with this preparate compared to the use of the original Konakion (Cremophor EL) and Kanavit (Polysorbat 80).\nIn view of the fact that vitamin K has limited tissue reserves and is rapidly catabolised, with 60\u201370% of a single dose being excreted via the urine and bile in about 3\u00a0days, it seems reasonable that repeated doses rather than a higher single dose is more effective in preventing late VKDB [15].\nAn important study from Denmark has established that 1\u00a0mg of oral vitamin K1 given weekly during the first 3 months of life, following an initial oral dose of 2\u00a0mg of vitamin K1 directly after birth, is as effective as 1\u00a0mg of vitamin K1 given intramuscularly at birth [9]. Therefore, we hypothesise that the dosage of 25 \u03bcg of vitamin K1 daily is not enough to prevent the secondary cases of late VKDB, as we encountered in this study.\nIn most co-operative studies, the upper age limit for inclusion was set arbitrarily at the end of 12\u00a0weeks of age [32]. In our study, we included infants presenting with VKDB up to the age of 26\u00a0weeks of age, as suggested by Tripp et al. [27]. However, no infant between 12 and 26\u00a0weeks of age was reported.\nThe mean age of all infants with late VKDB was 5\u00a0weeks, which is comparable to other reports [22].\nIn our study, the boy:girl ratio was 2.5:1. Why late VKDB occurs more in boys than girls is unknown, but this is consistent with other European reports [22, 24].\nUnfortunately, this study has some limitations. First, this surveillance was based on one data source only: the Netherlands Paediatric Surveillance Unit. In spite of the high return rate of the report cards (93.4%), it is plausible that dramatic conditions, such as intracranial bleedings, are more likely to be reported, as also noted by von Kries et al. [31]. For this reason, our data could give an under-reporting of the true incidence of late VKDB in the Netherlands. However, the fact that 50% of late VKDB cases were intracranial bleedings is comparable to other, larger reports [3, 13, 14, 19, 22, 30, 31, 33].\nSecondly, the surveillance lasted only 1\u00a0year. In all probability, the confidence interval would be smaller when the length of the surveillance was prolonged. Third, the proportion of infants that is breastfed is unknown, and, additionally, we lack data about compliance to the prophylaxis. It is likely, however, that less than 100% of the eligible cases received the recommended prophylaxis. Prophylaxis failure, therefore, is likely to be underestimated due to a greater denominator than the true numbers exposed.\nWe would suggest to double the daily dose of vitamin K1 to 50\u00a0\u03bcg for all breastfed babies from 1 to 13\u00a0weeks of age to prevent bleedings in infants with an underlying cholestatic liver disease as well. This amount is comparable to the ingested dose of vitamin K1 by formula-feeding [6]. Formula is supplemented with vitamin K1 to a minimal concentration of 50\u00a0\u03bcg\/l. VKDB are not reported in formula-fed babies and there is no evidence that they are at an increased risk of childhood cancer [28]. Studies with such a concentration of vitamin K1 have never been performed and further research is necessary to study the efficacy of this prophylaxis in infants with underlying cholestatic liver disease. Due to the low incidence of late VKDB, it would be difficult to perform a randomised trial of 25\u00a0\u03bcg versus 50\u00a0\u03bcg of vitamin K1 daily. Alternatively, a new surveillance for 1 or 2\u00a0years after implementing 50\u00a0\u03bcg of vitamin K1 as the prophylaxis could give an indication of the efficacy of this dose.\nFurthermore, an earlier recognition of symptoms associated with cholestatic liver disease and immediate investigation and treatment of \u201cwarning bleeds\u201d helps to prevent the severe consequences of secondary late VKDB.\nIn conclusion, with the current guideline of vitamin K prophylaxis in the Netherlands, idiopathic late VKDB is rare, but late VKDB secondary to an underlying cholestatic liver disease still occurs. We suggest to double the daily maintenance dose of vitamin K1 to 50\u00a0\u03bcg per day for all breastfed infants from 1 to 13\u00a0weeks of age in order to prevent bleedings in children with an underlying cholestatic liver disease as well. Further research, however, is recommended.","keyphrases":["vitamin k","vitamin k deficiency","newborn","prophylaxis","infant"],"prmu":["P","P","P","P","P"]}
{"id":"Pediatr_Nephrol-3-1-2064946","title":"Molecular mechanism of edema formation in nephrotic syndrome: therapeutic implications\n","text":"Sodium retention and edema are common features of nephrotic syndrome that are classically attributed to hypovolemia and activation of the renin\u2013angiotensin\u2013aldosterone system. However, numbers of clinical and experimental findings argue against this underfill theory. In this review we analyze data from the literature in both nephrotic patients and experimental models of nephrotic syndrome that converge to demonstrate that sodium retention is not related to the renin\u2013angiotensin\u2013aldosterone status and that fluid leakage from capillary to the interstitium does not result from an imbalance of Starling forces, but from changes of the intrinsic properties of the capillary endothelial filtration barrier. We also discuss how most recent findings on the cellular and molecular mechanisms of sodium retention has allowed the development of an efficient treatment of edema in nephrotic patients.\nIntroduction\nInterstitial edema is a common clinical feature of nephrotic syndrome (NS). It is often massive (up to 30% of body weight) and constitutes a functional constraint, owing to locomotive restriction and eyelid shutting. Expansion of the interstitial compartment volume results from the combination of abnormal renal sodium retention and alterations of fluid transfer across capillary walls.\nRenal retention of sodium in nephrotic syndrome\nSite of sodium retention Most of our current knowledge on the site and mechanism of renal sodium retention in NS comes from experimental models of the disease, in particular the puromycin aminonucleoside (PAN) rat model. Following a single injection of PAN, rats develop massive proteinuria within 4\u20135\u00a0days and sodium retention within 2\u20133\u00a0days. Sodium excretion resumes after 9\u201310\u00a0days, while proteinuria lasts for 2\u20133\u00a0weeks [1, 2]. Unilateral NS can be induced by the injection of PAN into one of the renal arteries [3]. Early in vivo micropuncture studies in rats with unilateral PAN nephrosis demonstrated that sodium retention in the nephrotic kidney originates beyond the last nephron segment accessible to micropuncture, i.e., between the late distal convoluted tubule and the tip of the collecting duct [3]. This part of the nephron encompasses the connecting tubule and the cortical and outer medullary collecting ducts, which reabsorb sodium and are the major sites of the adjustment of sodium balance under the homeostatic control of aldosterone, and the inner medullary collecting duct, which is able to secrete an overload of sodium in response to atrial natriuretic peptide [4]. Methods allowing in vitro analysis of isolated sub-segments of the distal nephron demonstrated a marked stimulation of sodium reabsorption in the cortical collecting duct (CCD) of PAN nephrotic rats [1, 5]. This stimulation of sodium transport likely extends upstream to the connecting duct but not downstream to the outer medullary collecting duct. In addition, in NS, the inner medullary collecting duct becomes insensitive to the natriuretic action of atrial natriuretic peptide [6\u20138], thereby preventing any compensation of sodium retention in upstream nephron segments.\nCellular mechanism of sodium retention in the collecting duct In vitro microperfusion of isolated CCDs confirmed that the rate of sodium reabsorption and the trans-epithelial voltage are negligible in CCDs from control rats, whereas both are high in CCDs from PAN nephrotic rats [5]. CCDs are made of principal and intercalated cells, which account for sodium and water reabsorption and potassium secretion for principal cells, and proton, bicarbonate and likely chloride transport for intercalated cells [4]. In principal cells, sodium reabsorption proceeds along a two-step mechanism (Fig.\u00a01): Na,K-ATPase, exclusively present in the basolateral membrane, energizes the active extrusion of sodium at the expense of ATP hydrolysis. The electrochemical gradient for sodium generated by this primary process drives passive entry of sodium through the apical membrane, which contains selective sodium channels (epithelial sodium channels, ENaCs). Both Na,K-ATPase and ENaCs are targets for multiple, and often coordinated, regulations in CCDs [4].Increased sodium reabsorption along the CCDs of PAN nephrotic rats, as well as sodium retention and edema formation, are associated with stimulation of both basolateral Na,K-ATPase [1, 9, 10] and apical ENaC [11\u201313] (Fig.\u00a01), but the latter is dispensable (see below). Stimulation of Na,KATPase is fully accounted for by transcriptional induction of its \u03b1 and \u03b2 subunits and targeting of newly synthesized pumps to the basolateral membrane of principal cells [14]. In contrast, stimulation of ENaC mainly results from the targeting of a pre-existing pool of latent intracellular channels to the apical membrane of principal cells and slightly from transcriptional induction of the \u03b1 and \u03b2 subunits [11\u201313]. The patch-clamp technique shows that the intrinsic properties of ENaC (open probability and unitary conductance) are not altered in the CCD of nephrotic animals [13].\nFig.\u00a01Cellular mechanism of sodium reabsorption in principal cells of collecting ducts from normal rats and nephrotic rats. Sodium reabsorption in principal cells proceeds along a two-step mechanism that includes active extrusion of intracellular sodium ions by the basolateral Na,K-ATPase and passive apical entry of sodium via the amiloride-sensitive epithelial sodium channel (ENaC). In CCDs from normal rats (top panels), most ENaCs are sequestered in the intracellular compartment of principal cells (left panel), and basolateral expression of Na,K-ATPase in collecting ducts (asterisk) principal cells is very weak, in comparison with that in thick ascending limbs (T) and even proximal tubules (P) (right panel). Accordingly, the rate of sodium reabsorption is very low. In CCDs from PAN nephrotic rats (bottom panels), ENaC is expressed at the apical border of principal cells (left panel), and expression of basolateral Na,K-ATPase is drastically increased in collecting ducts (asterisk). Polarized increases in expression of ENaC and Na,K-ATPase in principal cells account for increased sodium reabsorption in CCDs. In both normal and nephrotic rats expression of Na,K-ATPase is undetectable in the glomerulus (G); in CCDs, unlabeled cells for both ENaC and Na,K-ATPase are intercalated cells (redrawn from [13, 14])\nAldosterone and activation of mineralo-corticoid receptors are not involved in sodium retention in nephrotic syndrome Historically, it is acknowledged that sodium retention and edema formation result from hypovolemia-induced stimulation of the renin\u2013angiotensin\u2013aldosterone system. Hypovolemia is supposed to proceed as follows: proteinuria induces hypoalbuminemia and reduces plasma oncotic pressure, which generates an imbalance of Starling\u2019s forces across capillary walls, leading to interstitial leakage of fluid and decreased efficient volume [15]. Although this mechanism is consistent with the renal site of sodium retention, with the activation of ENaC and the induction of Na,K-ATPase in the collecting duct, and with the hyperaldosteronemia observed in PAN nephrotic rats [13], a large body of clinical evidence argues against this theory.\nAnalbuminemic patients display no sodium retention and no, or only modest, edema, despite low plasma oncotic pressure [16].In children with steroid-sensitive minimal change disease, natriuresis resumes at the same time as proteinuria stops, before normalization of albuminemia [17].Blood volume is not correlated with plasma oncotic pressure in nephrotic patients [18].Among children with relapse idiopathic nephrotic syndrome, (a) 21% display hypertension but only 4% exhibit collapse [19], and (b) only 1% display low blood volume, whereas 17% present hypervolemia [20].Intravenous injection of albumin induces volume expansion but promotes only mild natriuresis (reviewed in [21]).Blockade of mineralo-corticoid receptor or inhibition of angiotensin-converting enzyme has no effect on natriuresis in most patients [22, 23].The lack of a role for hyperaldosteronemia in sodium retention in NS was directly demonstrated in PAN nephrotic rats. In this model it is possible to blunt PAN-induced hyperaldosteronemia through bilateral adrenalectomy and corticosteroid replacement through implanted mini-pumps delivering constant physiological level of aldosterone and glucocorticoids. Administration of PAN to these corticosteroid-clamped rats reduces sodium excretion, establishes sodium balance and promotes ascites formation with similar time course and intensity as in adrenal-intact rats [11, 13].These findings also exclude a possible role in sodium retention of promiscuous activation of the mineralo-corticoid receptor (MR) by glucocorticoid brought about by decreased 11\u03b2-hydroxysteroid dehydrogenase type\u00a02 (11\u03b2-HSD2). Indeed, use of dexamethasone instead of corticosterone for glucocorticoid replacement prevents MR activation, even in the presence of 11\u03b2-HSD2 inhibition. The lack of involvement of MR activation in sodium retention in nephrotic syndrome is consistent with the fact that nephrotic syndrome does not induce potassium secretion.\nNone of the known factors that control sodium reabsorption in the collecting duct accounts for sodium retention in PAN-nephrotic rats Since aldosterone, the major factor that controls sodium reabsorption in collecting ducts, is not involved in sodium retention, several hormones or paracrine factors that modulate this process have been considered as putative candidates:\nVasopressin (AVP), through activation of its V2 receptors coupled to the cAMP pathway, increases sodium reabsorption in the collecting duct synergistically with aldosterone [24, 25].Angiotensin\u00a0II (AII) increases sodium reabsorption directly through activation of AT1 receptors coupled to phospholipase\u00a0C, independently of the induction of aldosterone release [26].Insulin-like growth factor I (IGF-1) used in the treatment of insulin resistance in type\u00a02 diabetes can induce sodium retention and edema [27], likely through its stimulatory action on sodium transport along the collecting duct [28].In mouse collecting duct cells, tumor necrosis factor\u00a0alpha (TNF\u03b1) increases membrane expression of Na,K-ATPase [29], and administration of etanercept, a TNF\u03b1 receptor antagonist, to diabetic rats induces natriuresis [30].More recently, the thiazolidinedione agonists of peroxisome proliferator-activated receptor \u03b3 (PPAR\u03b3) were reported to induce sodium retention [31] through a stimulatory effect on the collecting duct [32].Quite unexpectedly, inhibition of nitric oxide synthase promotes sodium excretion in cirrhotic rats with ascites [33].Except for AII, all maneuvers aimed at blocking these different pathways had no effect on sodium retention in nephrotic syndrome (Fig.\u00a02). Only the AT1 blocker irbesartan was able to improve sodium excretion, although not up to normal level. However, this effect was limited to the initial sodium retention observed 2\u20134\u00a0days after PAN administration and did not alter the main effect observed at days\u00a05 and 6. This suggests that AII, independently of aldosterone, may be involved in the early retention of sodium observed after the peak of sodium excretion at day\u00a01.\nFig.\u00a02Profile of sodium excretion in PAN nephrotic rats. Daily urinary sodium excretion, expressed as a function of urinary creatinine excretion, following administration of puromycin aminonucleoside (150\u00a0mg\/kg body wt, intravenously) in normal rats (dotted lines) or genetically modified or pharmacologically treated rats (solid lines). Arrows or grey boxes show the time of treatment. Brattleboro rats genetically lack vasopressin secretion. JB1, an inhibitor of IGF-1 receptors, was continuously administered via subcutaneous mini-pumps at a dose of 12\u00a0\u03bcg\/100\u00a0g body wt per day, starting on day\u00a03. The antagonist of AT1 receptor, irbesartan, was administered per os at a dose of 2 mg\/100g body wt per day. Etanercept, a chimeric antibody directed against TNF receptor, was administered twice (days \u22121 and 2) at a dose of 0.2\u00a0mg\/100g body wt. The inhibitor of nitric oxide synthase, L-NAME, was administered twice daily by gavage (0.5\u00a0mg\/100\u00a0g body wt per 12\u00a0h) throughout the study. The antagonist of PPAR\u03b3, SR202, was given per os at a dose of 20\u00a0mg\/100\u00a0g body wt. All controls were treated in parallel with the vehicle. Values are means \u00b1 SE from 4\u20135 ratsAs a matter of fact, sodium retention in PAN nephrosis unlikely results from the presence of an abnormal blood concentration of any factor, since only the treated kidney displays increased sodium reabsorption in unilateral PAN-nephrotic rats. Rather, alterations in collecting duct function may be accounted for either by a direct effect of PAN on CCD or by the abnormal presence of an unidentified stimulatory factor in the luminal fluid that reaches the distal nephron. The presence of this factor may result from its abnormal filtration secondarily to the alteration of the glomerular filtration barrier (e.g., a high molecular weight protein), or from its generation in the proximal tubule secondarily to alteration of protein\/peptide handling.\nNa,K-ATPase is the primary target of sodium retention in PAN nephrosis Analysis of corticosteroid-clamped rats not only demonstrated that hyperaldosteronemia is not involved in the etiology of edemas in NS, but it also provided insights into the primary molecular target of sodium retention in the CCD. Indeed, in CCDs from corticosteroid-clamped PAN rats, Na,K-ATPase is induced as in adrenal-intact nephrotic rats [13]. In contrast, targeting of ENaC to the apical membrane is fully blunted [11, 13], and the residual channels expressed at the apical membrane are drastically inhibited, as their open probability is reduced >10-fold [13].Thus, induction of Na,K-ATPase is primarily responsible for sodium retention in PAN nephrotic syndrome. This conclusion likely applies to all experimental models of nephrotic syndrome and, possibly, to the disease in humans, since there is a significant reverse correlation between Na,K-ATPase activity in collecting duct and urinary sodium excretion during the phase of sodium retention in rats with nephrotic syndrome induced by PAN, Adriamycin or mercury chloride [1].\nEdema formation in nephrotic syndrome\nMost sodium-retaining states are associated with high blood pressure but not with development of edema or ascites. Edema formation in NS results from the asymmetry of extracellular volume expansion brought about by sodium retention: the vascular volume is not, or only slightly, modified, whereas water and solutes accumulate in the interstitium. Distribution of fluid between vascular and interstitium compartments is governed by fluid exchanges across the capillary wall and by lymphatic draining. Capillary filtration capacity is increased almost twofold in nephrotic patients [34]. This fluid leakage is governed by several parameters according to Starling\u2019s law:\nwhere Jv is the trans-capillary flux of fluid, Lp the hydraulic conductivity of capillaries, S the exchange surface, Pc and Pi the hydrostatic pressure of the capillary and interstitium, respectively, \u03c3 the reflection coefficient of proteins across the capillary wall, and \u03a0c and \u03a0i the oncotic pressure in the capillaries and interstitium, respectively.\nUnexpectedly, data from the literature indicate that the increase in Jv in NS is not accounted for by change in the oncotic pressure gradients, as intuitively thought, but by changes in the intrinsic properties of the capillary walls that govern their hydraulic conductance and reflexion coefficient for proteins. This means that the capillary wall is a direct target in NS, to the same right as the glomerular filtration barrier.\nThe trans-capillary gradient of oncotic pressure (\u03a0c\u2013\u03a0i) is unchanged in NS The lack of edema and ascites in analbuminemic rats and patients [16, 35] has questioned the importance of low plasma oncotic pressure in the genesis of edema in NS. As a matter of fact, the trans-capillary gradient of oncotic pressure is unchanged in analbuminemic rats, owing to a parallel decrease in plasma and interstitium oncotic pressure [35]. Experiments in dogs, in which plasma oncotic pressure was progressively decreased by 50% through plasmapheresis, showed that the interstitium oncotic pressure decreased in parallel and that the trans-capillary gradient remained unaffected. Extracellular volume increased transiently during the phase of variation of plasma oncotic pressure, in response to hyperaldosteronemia and renal sodium retention, but returned to normal level during the period of stabilized low plasma oncotic pressure [36].Essentially similar observations were made in humans. During NS, it was also observed that the oncotic pressure of the interstitium decreases in parallel with that of the vascular compartment, so that the trans-capillary gradient is only slightly reduced [37]. In addition, diuretic treatments [37] or extracorporeal ultrafiltration [38] allow the withdrawal of significant amounts of edema without significant change in the trans-capillary gradient of oncotic pressure.Thus, decrease in plasma oncotic pressure in animal models as well as in nephrotic patients does not alter significantly the trans-capillary gradient of oncotic pressure and is neither a determinant parameter in the genesis of edema nor a resistance factor to edema withdrawal.\nThe trans-capillary gradient of hydrostatic pressure (Pc\u2013Pi) is unchanged in NS Capillary pressure is unchanged in nephrotic patients [34]. Because soft tissues display an almost infinite compliance, their interstitium pressure increases by only 2\u00a0mmHg with their fluid filling [39]. In nephrotic patients interstitial pressure in edematous and non-edematous sectors differs by <4\u00a0mmHg [40]. Thus, it can be admitted that the trans-capillary gradient of hydrostatic pressure is not significantly altered in soft edematous tissues during NS.\nThe capillary hydraulic conductivity Lp is increased in NS The threshold of venous pressure triggering fluid transfer across capillaries is significantly reduced in NS [34], suggesting that endothelial Lp is decreased. The main determinants of the hydraulic conductivity of capillaries are the occlusive junctions (constituted by occludin, claudins and proteins ZO) and adhesive junctions (made of cadherins, catenins and actinin) between endothelial cells. These proteins are direct targets for intracellular signaling cascades, in particular protein kinase\u00a0C (PKC), which phosphorylates occludin [41] and alters the endothelial permeability [42]. In NS this pathway may be activated through two mechanisms. Firstly, hypoalbuminemia increases Lp via an increase in intracellular calcium [43]. Secondly, high plasma levels of TNF\u03b1 observed in patients with minimal change disease [44] activate PKC and increase Lp [45].\nThe reflexion coefficient of proteins (\u03c3) is increased in NS Increased coefficient of reflexion of macromolecules in NS is evidenced by the higher rate of leakage of technetium-labeled albumin towards the interstitium in nephrotic patients than in controls [46]. Because this change in reflexion coefficient is observed in patients with NS of different origin, it is likely not accounted for by the circulating permeability factor of lymphocyte origin responsible for glomerular hyperfiltration in minimal change disease. Increased \u03c3 was also reported in blood\u2013peritoneal barrier permeability in PAN-nephrotic rats [47].\nTherapeutic implications\nNephrotic edema results from the combination of renal sodium retention and increased capillary permeability. Although treatment of either of these two alterations would prevent edema, treatment of capillary permeability alone would lead to hypertension. Thus, treatments of edema must primarily target renal sodium retention.\nDifferent therapeutic strategies are needed to prevent edema formation in children with chronic proteinuria and to treat massive edema. In the first case it is sufficient to prevent sodium accumulation, either through limiting dietary sodium uptake or by natriuretic drugs that target the collecting duct, e.g., amiloride. In clinical practice the daily uptake of sodium is usually limited to 0.5\u00a0mmol\/kg. In PAN-nephrotic rats administration of amiloride starting before the onset of sodium retention fully prevents sodium retention and formation of ascites [5].\nMore classically, clinicians are confronted with patients who already display massive edema. Treatment aims both at limiting further sodium retention and at promoting the excretion of the mass of sodium and water sequestered in edema. While amiloride is well suited to block further sodium retention, it is inefficient in promoting massive sodium excretion, because, under normal conditions, sodium reabsorption along the collecting duct is quantitatively low. Recourse to more potent diuretics, such as loop diuretics, is restricted by the functional resistance of nephrotic patients to the natriuretic effect of furosemide. Several explanations have been proposed to account for this resistance, but none has been confirmed:\nThe pharmacokinetics of furosemide urinary elimination is not significantly altered in nephrotic children [48]Binding of furosemide to albumin in the tubular fluid does not account for furosemide resistance, since, despite controversial data [49, 50], inhibitors of furosemide\u2013albumin binding do not improve sodium excretion significantly.The intrinsic sensitivity of the Na\/K\/2Cl transporter of the loop of Henle, the molecular target of furosemide, is not altered in nephrotic rats [5].\nBased on our present knowledge of the site and cellular mechanism of sodium retention in NS, another explanation of furosemide resistance, and a therapeutic strategy to circumvent it, can be proposed. In non-nephrotic patients, furosemide decreases sodium reabsorption along the thick ascending limb of the loop of Henle, which increases sodium delivery to the distal nephron. Because the sodium reabsorption capacity of the distal nephron is rather limited, only a small fraction of the overload of sodium is reabsorbed, and the major fraction is excreted in the urine, accounting for the natriuretic effect of loop diuretics. Note that following long-term treatment with furosemide, there are adaptations of the distal nephron which increase its sodium reabsorption capacity and, thereby, reduce the natriuretic effect of furosemide. Because nephrotic patients display a huge sodium reabsorption capacity along their connecting and cortical collecting tubules, most of the overload of sodium brought about by furosemide-induced inhibition of transport in the thick ascending limb is reabsorbed, thereby blunting the natriuretic effect. Thus, the apparent resistance of nephrotic patients to loop diuretics can be circumvented by the inhibition of distal sodium reabsorption with amiloride. As a matter of fact, co-administration of furosemide and amiloride to nephrotic children increases urinary sodium excretion, induces a negative sodium balance and promotes complete edema withdrawal within 1 week [21].\nConclusion\nWhatever their etiology, nephrotic syndromes are always associated with renal retention of sodium. Renal sodium retention results from enhanced sodium reabsorption along the connecting and cortical collecting ducts and from blunted responsiveness of medullary collecting ducts to the natriuretic response to atrial natriuretic peptide. Induction of de novo synthesis of Na,K-ATPase is the primary effector of increased sodium reabsorption. It is not accounted for by any circulating factor, in particular aldosterone, known to stimulate sodium reabsorption along the distal nephron. New research strategies will be required to identify the unknown regulatory pathway that is dysregulated in NS.\nSodium retention in NS does not lead to high blood pressure but leads to an asymmetric expansion of the interstitium, while the vascular volume remains unchanged in most patients. This asymmetry of extracellular volume expansion is accounted for by changes in the intrinsic properties of the endothelial capillary barriers, i.e., an increase in its hydraulic conductivity and permeability to proteins, rather than to an imbalance of Starling\u2019s forces.\nThus, the pathophysiology of nephrotic syndrome relies on at least three disorders: a major alteration of the glomerular filtration barrier responsible for proteinuria and hypoalbuminemia, an induction of distal nephron Na,K-ATPase responsible for sodium retention, and alterations in the capillary permeability accounting for the asymmetry of volume expansion. Although causal relationships between these three events have not been formally established, it is assumed that the glomerular defect engenders both the tubular and the capillary alterations. This conclusion is based on the facts that: (a) whatever its origin, alteration of the glomerular filtration barrier always leads to sodium retention and edema formation, i.e., to the tubular and capillary defects, and (b) loss of function mutations of nephrin, which is expressed in the glomerular slit diaphragm but in neither the collecting duct nor capillary endothelial cells, is sufficient to promote proteinuria, sodium retention and edema [51]. Curiously, in PAN-induced nephrosis, sodium retention precedes proteinuria, suggesting that it is not secondary to the glomerular dysfunction. It should be stressed, however, that microproteinuria may appear sooner and concomitantly with sodium retention.","keyphrases":["sodium retention","aldosterone","collecting duct","na,k-atpase","epithelial sodium channels (enacs)","diuretics","capillary hydraulic conductivity"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Anal_Bioanal_Chem-3-1-1797063","title":"The effect of the chemical, biological, and physical environment on quorum sensing in structured microbial communities\n","text":"As researchers attempt to study quorum sensing in relevant clinical or environmental settings, it is apparent that many factors have the potential to affect signaling. These factors span a range of physical, chemical, and biological variables that can impact signal production, stability and distribution. Optimizing experimental systems to natural or clinical environments may be crucial for defining when and where quorum sensing occurs. These points are illustrated in our case study of S. aureus signaling in biofilms, where signal stability may be affected by the host environment. The basic signaling schemes have been worked out at the molecular level for a few of the major quorum-sensing systems. As these studies continue to refine our understanding of these mechanisms, an emerging challenge is to identify if and when the local environment can affect signaling.\nIntroduction\nAs illustrated by many articles in this special issue, the diversity of mechanisms bacteria use to communicate with one another is remarkable. Quorum sensing is widespread among different bacterial species, emphasizing the importance of coordinating behavior as a group.\nWhen considering the relevant context of quorum sensing, it is clear that the environment can be profoundly important. Many bacteria exist in spatially structured, multi-species communities, such as biofilms on submerged surfaces or flocs in the water column of aquatic environments [1, 2]. Within these communities bacteria may achieve the high cell numbers capable of generating the local signal concentrations required to produce a quorum sensing response. Quorum sensing undoubtedly occurs in situations where bacteria are growing planktonically, for example Vibrio harveyi quorum sensing-controlled bioluminescence in the ocean, thought to produce the \u201cmilky seas\u201d phenomenon [3, 4]. An argument could, however, be made that quorum sensing is more likely to occur in structured communities. If the reader accepts this point, further considerations lead to some interesting questions for example: \nAre certain signals better suited for signaling in different types of environments?What are the important parameters influencing signaling in different environments?\nTake, as an example, the well-studied, environmentally ubiquitous Pseudomonas aeruginosa. This bacterium has been isolated from a range of soil and aquatic samples [5\u20137]. The nature of signaling in these environments could be quite different, because signal production and diffusion dynamics may change.\nThis review will discuss factors that may affect signaling in structured microbial communities. In addition, quorum sensing in Staphylococcus aureus, a peptide-based signaling system, will be briefly reviewed and its role in biofilm formation will be discussed.\nFactors affecting signal diffusion, stability, and distribution\nThe traditional way of studying quorum sensing in the laboratory is in shaken liquid-batch cultures. The quorum sensing response occurs at a specific point in the growth curve, coinciding with a threshold concentration of signal. Volume is fixed in a culture flask and signal molecules produced by bacteria usually increase in concentration with time (unless they are degraded). Batch cultures represent a closed system in which only gaseous exchange occurs. Most structured communities are in open systems in which exchange occurs with the surrounding liquid [1]. In an open, flowing system, signals may be \u201cwashed\u201d away by the overlying fluid. In open systems the concentration of signal molecules within structured communities is primarily a function of: \nsignal-production rate;the degradation rate or half-life of the signal;the diffusion properties of the signal; andthe external hydrodynamic or mass-transfer conditions.\nThe prevailing environmental conditions and resident biology are, to different degrees, important for each of these processes.\nThe effects of signal and environmental chemistry on quorum sensing\nSome of the best-studied quorum-sensing signals include the acyl-homoserine lactones (AHL) used by many Gram-negative bacterial species, small peptides used by some Gram-positive species, and the furans of the phylogenetically widely spread AI-2 signaling (reviews are available elsewhere [8\u201310]). Signal chemistry varies widely in different quorum-sensing systems and is important for determining its stability and diffusion characteristics in different environments.\nAll AHL signals have a characteristic homoserine lactone moiety. The homoserine lactone is derived from S-adenosylmethionine, one of the substrates for AHL synthesis [11\u201313]. Although stable at neutral and slightly acidic pH, the lactone ring is subject to chemical hydrolysis under basic conditions [14, 15]. The resulting product (e.g. butyryl-homoserine lactone would become butyryl-homoserine) lacks biological signaling activity and is capable of spontaneously re-lactonizing when the pH is lowered. The formula describing the relationship between pH and the lactone ring stability is 1\/(1\u00d7107\u00d7[OH\u2212]) [14], which gives the half life in days. For example, at pH 7 the homoserine lactone ring would be stable for hours whereas at pH 8.5 the stability would be of the order of minutes (Fig.\u00a01). In many environments this might be insignificant. In some alkaline environments, however, like the photosynthetic mats at Octopus Springs in Yellowstone National Park, the pH is high enough to potentially affect signal gradients. Kaufmann et al. recently demonstrated that in addition to lactone hydrolysis, the 3-oxo-C12 AHL of P. aeruginosa is capable of spontaneously undergoing a Claisen-like condensation reaction in aqueous environments, forming a tetramic acid product, 3-hydroxydecylidene 5-(2-hydroxyethyl) pyrrolidine-2,4-dione (Fig.\u00a02) [16]. This compound was shown to have significant antimicrobial activity against other species and a high affinity for iron. It was proposed this iron-binding activity constituted an alternative means of acquiring iron by P. aeruginosa.\nFig.\u00a01Predicted AHL half-lives in different alkaline environmentsFig.\u00a02Structures of quorum-sensing signals and their derivatives. Letter designations for the Gram-positive peptide signals indicate amino acids. For the L. lactis signal nisin, the structural abbreviations were: Bu, dehydrobutirine with a lanthionine bridge; Ha, dehydroalanine; Hb, dehydrobutirine\nStructured communities can create highly heterogeneous localized niches where chemistry can vary drastically over very small distances. For example, consumption of dissolved oxygen by laboratory biofilm cells can result in completely anaerobic regions within the biofilm only 100\u00a0\u03bcm from the fully oxygen saturated overlying liquid [17]. Similarly, pH can vary dramatically, as demonstrated by pH microelectrodes positioned in biofilms formed from the dental pathogen Streptococcus mutans. On addition of the growth substrate sucrose, the pH dropped from 7 outside the biofilm to 4.6 approximately 100\u00a0\u03bcm deep within the biofilm (Gieseke A, Nguyen D, von Ohle C, Stoodley P; unpublished results). Clearly, if the half-life of signaling molecules is a function of local chemistry it is reasonable to expect that degradation rates will vary in the community.\nThe acyl side-chains of AHLs range in length from 4 to 18 carbon atoms [18, 19]. The substitution chemistry of the acyl side-chain is also highly variable. Although most AHLs have a ketone group at the C1 position, some AHLs also have either a ketone or hydroxyl group at the C3 position. In addition, the acyl side-chain can vary in saturation. Although most AHLs have fully saturated acyl side-chains, some, for example the 3-hydroxy,7-cis-tetradecanoyl homoserine lactone of Rhizobium leguminosarum, are unsaturated [19, 20]. All these features of AHL acyl side-chain chemistry, in particular acyl chain length, affect the relative hydrophobicity of the signal. Pearson et al. observed this for P. aeruginosa, which produces two major AHL signals, a C4 (i.e. four-carbon side-chain) and 3-oxo-C12 AHL (Fig.\u00a02) [21]. They found that the C4 signal diffused freely across the cell membrane whereas the 3-oxo-C12 signal partitioned to the cell membrane, where its extrusion was aided by the activity of the MexAB-OprM efflux pump.\nThe hydrophobicity of the local environment could have a large effect on AHL diffusion properties. For example, several AHL-utilizing bacterial species are also capable of producing highly hydrophobic, secreted polysaccharides (e.g. the pel\/psl polysaccharides of P. aeruginosa) [22]. In a structured community, such as a biofilm, secreted polysaccharides can serve as the scaffolding holding the community together [23]. Under such conditions these polysaccharides might also act as an AHL-sequestering matrix. Charlton et al. provided evidence of this, indicating that the 3-oxo-C12 signal of P. aeruginosa partitioned into the extracellular biofilm matrix [24]. Perhaps the C4 signal has reduced reactivity with the biofilm matrix, making it a more effective signal in a biofilm and explaining why it is produced in greater amounts than 3-oxo-C12 by P. aeruginosa growing as a biofilm [25].\nAnother factor affecting AHL stability in the environment is biologically-driven signal degradation. Emphasizing this point, Wang et al. demonstrated that radiolabled AHLs fed to soil microbial communities were almost instantly mineralized [26]! Several bacterial species have the ability to use AHLs as carbon and nitrogen sources. These include species that produce their own AHLs and those known not to make them. Why some organisms both make and break down their own AHLs is a mystery. AHL degradation occurs as a result of two primary types of enzymatic activity, lactonases, which break the homoserine lactone ring, and acylases, which cleave the amide bond linking the acyl side-chain to the homoserine lactone ring [27\u201331]. Biological AHL degradation is a key consideration in multi-species environments. Leadbetter proposed the concept of \u201cinsulation\u201d in which AHL-degrading organisms could prevent AHLs produced by bacteria spatially fixed in one location from reaching other bacteria of the same species [26].\nEnvironmental chemistry also plays a key role in AI-2 signaling. The paradigm for this type of quorum sensing is the luxS system of Vibrio harveyi [8]. One of the key steps in AI-2 signal synthesis is the LuxS-catalyzed conversion of S-ribosyl homocysteine to homocysteine and 4,5-dihydroxy-2,3-pentanedione (DPD) [32]. DPD is believed to be the key precursor to all AI-2 signals. DPD undergoes non-enzymatic cyclization and a number of spontaneous, reversible rearrangements to form the active signal-a furanosyl borate diester and other furans [33, 34]. The AI-2 signal for Salmonella typhimurium was recently solved and found to be, (2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran, a molecule related to, but structurally distinct from, the V. harveyi AI-2 (Fig.\u00a02) [35]. Spontaneous extracellular chemical rearrangements of S. typhimurium AI-2 can produce the V. harveyi AI-2 signal; the reverse is also true [35]. Current thinking is that the local chemical environment can affect the ratio of the different furanosyl esters. In particular, the presence of borate may shift the equilibrium toward formation of the V. harveyi AI-2 [35].\nThe chemical structures of the Gram-positive quorum-sensing peptides vary greatly in the number of residues and the types of modification (Fig.\u00a02). Because of the post-translational modifications on these peptides and their inability to diffuse across membranes, their biosynthetic pathways are more complex than those of the AHLs and dedicated signal-export systems are usually required. Many also induce their own biosynthesis, and are thus frequently termed \u201cautoinducing peptides\u201d. Several signals are simple linear peptides, for example the 17-residue competence-stimulating peptide (CSP) which regulates competence in Streptococcus pneumoniae [36]. The corresponding linear competence pheromone in Bacillus subtilis, called ComX, is more unusual in that an internal tryptophan residue has been cyclized and isoprenylated with a geranyl moiety [37], a modification that is catalyzed by ComQ [38].\nPerhaps the largest and most complex peptide signals are the lantibiotics, which also have antimicrobial activity [39]. These molecules, for example nisin produced by Lactococcus lactis, are known to positively regulate their own biosynthesis in a density-dependent manner. They also have extensive post-translation modifications, including dehydrated residues, such as dehydroalanine and dehydrobutyrine, and thioether bridges called lanthionines (Fig.\u00a02 depicts the base structure of the lantibiotic, nisin, of L. lactis). An emerging class of peptide signals are the cyclic lactones and thiolactones [40]. The first of this class to be discovered was the type I autoinducing peptide (AIP) of Staphylococcus aureus, an eight-residue thiolactone-containing peptide with the C-terminus constrained through linkage with a cysteine side-chain [41]. Since the discovery of AIPs, related signals have been identified in Enterococcus faecalis [42], Listeria monocytogenes [43], and other Staphylococci [44].\nEnvironmental conditions can have a significant effect on peptide signal stability. The linear peptides are likely to have short half-lives as they are rapidly metabolized by the action of secreted proteases. Indeed, this issue has led some to question their role as a means of communication, raising the possibility they serve as a general strategy for a single cell to time regulatory events [45]. In contrast, the lantibiotics have multiple lanthionine bridges that block the activity of many proteases [46], greatly improving the longevity of these molecules. Like AHLs, lantibiotics are pH-sensitive, with solubility and stability dropping rapidly at higher pH; those with dehydrated residues are also reactive with thiols [47]. Because of these chemical constraints, the lantibiotic nisin has a half-life of only 0.9\u00a0h in mouse serum [48], the pH of which is approximately 7.3. For L. lactis in the environment, however, growth and production of nisin are optimum at lower pH, at which stability is greater, facilitating autoinduction. In a similar fashion, the thiolactone-containing peptides are resistant to proteolysis and sensitive to high pH and reactive thiols (chemistry of the S. aureus AIP is discussed in more detail below), and some have increased sensitivity to oxidative damage [49]. Although it remains to be demonstrated, protease resistance may be one of the most significant environmental constraints faced by bacteria using peptide communication signals and extensive post-translational modifications could have evolved to overcome this problem.\nThe effect of the hydrodynamic environment on quorum sensing\nMass transfer is the physical process by which molecules are transported in a system, and it has the potential to affect cell-to-cell signaling in many ways. Delivery of nutrients to the active biomass in the structured community is one example. This could affect the relative metabolic activity of the community, which in turn has the potential to affect signal-production rates. Mass transfer is affected by the hydrodynamics of the bulk fluid and the geometry of the structured community. These two factors affect each other, because a microbial community both shapes, and is shaped by, its external environment.\nIf liquid flow is a feature of the environment, it will wash signal away, diluting its concentration within the community. In these circumstances the system can be divided into three zones (Fig.\u00a03). First, the biomass of the community which is producing signaling molecules, second, the viscous sublayer (\u03b4) located at the biomass-bulk fluid interface, and, third, the well mixed turbulent core of the overlying fluid. In laminar flow there is no turbulent core and \u03b4 essentially extends to either the center of the channel if flow is in a closed conduit (e.g. pipe or catheter) or to the fluid surface if flow is in an open channel (e.g. river). Using this simplified system we have constructed a one-dimensional model based on a flat biofilm to illustrate how different values of \u03b4 may result in different concentration profiles while the thickness of the biofilm, the cell density in the biofilm, and the rate of signal-production in the biofilm remain constant. The model is based on coupling the diffusion of signal through the viscous sublayer with the simultaneous diffusion and production of signal that occurs within the biofilm itself. In the fluid outside the biofilm, the concentration of signal changes linearly: \nwhere Cz is the signal concentration (mg L\u22121) at depth z, C0 is the signal concentration outside the viscous sublayer (0\u00a0mg L\u22121), k0 is the signal production rate (2.96\u00d710\u221217\u00a0mg cell\u22121 s\u22121), Xb is the active cell density in the biofilm (1\u00d71010\u00a0CFU cm\u22123), Lf is the biofilm thickness (50\u00a0\u03bcm), \u03b4 is the thickness of the viscous sublayer (0, 25, or 50\u00a0\u03bcm), Daq is the diffusion coefficient of the signal in water (4.9\u00d710\u22126\u00a0cm2 s\u22121), and \u03b4z is the proportional depth within \u03b4 (\u03bcm). Within the biofilm, the concentration of the signal changes quadratically with depth: \nwhere De is the effective diffusion coefficient of signal in the biofilm (1.23\u00d710\u22126\u00a0cm2 s\u22121) and Lz is the proportional depth within the biofilm (\u03bcm).\nFig.\u00a03Schematic diagram illustrating how external flow may affect the concentration of a cell signal produced by biofilm cells. The biofilm is 50\u00a0\u03bcm thick (so there is no oxygen limitation and we can assume equal signal production throughout the depth of the biofilm) and the relative concentration profiles are for three flow conditions: (a) Very high shear, negligible diffusion boundary layer (\u03b4). (b) Lower flow with a diffusion boundary layer of 25\u00a0\u03bcm (\u03b4B). (c) Lower flow, again, with a diffusion boundary layer of 50\u00a0\u03bc (\u03b4C). The model is steady-state Fickian diffusion for flat-slab geometry with a uniform density of bacteria producing signals at a constant rate with no degradation. There is no convection within the biofilm and no flux at the substratum. The concentration of signal in the turbulent core of the flow is zero. The effective diffusion coefficient in the biofilm is assumed to be 0.25 times that of the bulk fluid based on the molecular weight of 3-oxo-C12 AHL [50]. For simplification the viscous sublayer is shown as the diffusion boundary layer. In reality the transition (the \u201cbuffer region\u201d of the boundary layer) from the viscous sublayer to the turbulent core is not a sharp line, as depicted, but gradual. If we assume an inducing 3-oxo-C12 AHL concentration of 3\u00a0mg L\u22121 (10\u00a0\u03bcmol), depicted by the vertical dashed line, biofilm \u201cA\u201d is not induced at all, in biofilm \u201cB\u201d the bottom 50% is induced, and in biofilm \u201cC\u201d the bottom 75% is induced, as shown by the horizontal dashed lines where the inducing concentration intersects the concentration profile\nWhere known we used data as best we could, based on the signal 3-oxo-C12 AHL, unknown model data were hypothetical. All data were based on those used for illustrative purposes by Stewart [50].\nThese considerations led us to hypothesize that external flow conditions can affect quorum sensing in many ways. For example, in a somewhat closed, static system (e.g. a biofilm in a mud puddle or in the well of a microtitre plate), signals produced by a large, biofilm cell aggregate may induce a quorum-sensing response in neighboring bacteria that are not part of the aggregate. In contrast, in an open system subject to convective flow (e.g. a biofilm in a river, or the channel of a flow cell biofilm reactor), signals might be continuously removed from the system. Under these conditions mass transfer may prevent signals produced by a large biofilm cell aggregate from inducing cells in the vicinity of the aggregate. Experimental results on interspecies communications in dental plaque biofilms by Egland et al. [51] support this idea, and suggest that diffusible signals are adapted to function over short distances. At higher flows the higher flux of signal molecules from structured communities may reduce signal concentration in the interior or core of the community. A subsequent consequence is that more biomass would be necessary to produce an inducing concentration of signal.\nFew studies have directly addressed the effect of hydrodynamics on quorum sensing in structured communities. Purevdorj et al. [52] found that at relatively high flow rates flow velocity was a stronger determinant of P. aeruginosa biofilm structure than quorum-sensing-required functions. Yarwood et al. [54] grew wild type (WT) and accessory gene regulator (agr) mutant biofilms of S. aureus by batch culture under static conditions, batch culture on spinning disks, and in flow cells. The QS mutation had the greatest effect under static conditions, resulting in an increase in biofilm formation, consistent with the study of Vuong et al. [55], who hypothesized that agr was a repressor of biofilm formation. Interestingly, there was no difference between WT and agr (quorum sensing) mutant biofilms when grown in flow cells. Care must, however, be taken in correlating growth system with phenotype, because media type and concentration were also varied in this study. Nevertheless, Fux et al. [56] obtained similar results for flow cell-grown biofilms. After 24\u00a0h the WT S. aureus biofilms had less biomass than a TRAP mutant but were significantly rougher. After 4\u00a0days, however, there was little difference between the biofilms as measured by COMSTAT confocal image-analysis software [57]. In the same study the opposite trend, as measured by viable plate counts, was observed for \u201cfilter colony biofilms\u201d [58] grown under static conditions on nutrient agar. The WT biofilms contained an order of magnitude more cells than the TRAP mutant. Interestingly, a regulatory network mathematical model has predicted that at intermediate concentrations of 3-oxo-C12, bacteria can suddenly switch between induced and noninduced states [59]. This effect may help explain experimental observation of different biofilm phenotypes under what are assumed to be similar or even replicate growth conditions.\nQuorum sensing in the gas phase with volatile signals\nQuorum sensing is usually discussed and studied in the context of aqueous environments. Some environmental conditions, however, for example those found in soil, may not always be ideal for signaling in the liquid phase. Soil is subject to wetting and drying cycles, and even somewhat dry soils are teeming with metabolically active bacteria [60, 61]. Under these conditions it may be advantageous to some bacterial species to engage in quorum sensing. Local cell numbers may be quite high, but not linked to one another through the liquid phase. The production of signals able to act through the gas phase would circumvent these issues. There is precedence for volatile signaling in the microbial world. An interesting example is the signaling molecule, 3-OH palmitic acid methyl ester (3-OH PAME) of Ralstonia solanacearum (Fig.\u00a02) [62]. The signal of this quorum-sensing system can act in the gas phase. This plant pathogen regulates the production of secreted polysaccharides (an important virulence factor) by use of 3-OH PAME [63, 64], the synthesis of which is catalyzed by the phcB gene product [63]. When 3-OH PAME concentrations reach \u223c5\u00a0nmol L\u22121, 3-OH PAME-regulated genes are induced, presumably because of interaction of the signal with the membrane-associated PhcS sensor kinase. Although homologs of phcB are not found on many of the microbial genomes yet sequenced, signaling in the gas phase may be one of the next important frontiers in quorum sensing.\nA case study of signaling in structured communities-Quorum-sensing in Staphylococcus aureus biofilms\nContinuing with the themes of this review, a case study will be presented with a discussion on environmental, chemical, and biological factors discussed above that have the potential to affect quorum-sensing in S. aureus biofilms.\nS. aureus is a human commensal that resides in a non-pathogenic state in the nasal airways. When there is a breach in the host defenses enabling access, S. aureus can convert to a pathogenic state and secrete an impressive array of toxins, hemolysins, and degratory enzymes [40], causing damage to host tissues. This lifestyle switch is mediated, in a cell-density-dependent manner, by the action of the AIP molecule. Early in the growth phase surface adhesins and antigens are produced, and when AIP reaches a critical concentration the quorum-sensing cascade is activated and these surface proteins are down-regulated and invasive factors are secreted. Microarray studies have revealed that 104 genes are up-regulated and 34 are down-regulated by the action of this quorum-sensing molecule [65]; this represents almost five percent of the genome. Intriguingly, there are four specific classes of AIP molecule that approximately correlate with the type of disease caused by the producing S. aureus strain. In a fascinating mechanism of bacterial cross-talk these different classes of AIP cross-inhibit quorum-sensing in other S. aureus groups and other staphylococcal species [41]; this may serve to isolate specific sub-populations for cooperative action.\nThe locus responsible for the quorum-sensing regulation in S. aureus is the accessory gene regulator or Agr locus and is known to contain two divergent transcripts, named RNAII and RNAIII [40]. The RNAII transcript is an operon of four genes, agrBDCA, that encode factors required to synthesize AIP and activate the regulatory cascade (Fig.\u00a04). The biosynthetic pathway leading to functional AIP is not clear, but it is known that AgrD is the peptide precursor of AIP and AgrB is a membrane protease involved in its processing [66]. AgrC and AgrA form a typical two-component regulatory pair, and the binding of AIP to a surface receptor on AgrC activates this phosphoryl-transfer cascade [67]. When phosphorylated, AgrA is known to bind and induce expression of the RNAIII transcript [68], which encodes a regulatory RNA molecule that acts as the primary effector of S. aureus quorum-sensing [69]. AgrA also induces expression of the Agr proteins through the RNAII transcript, triggering the autoinduction phenomenon.\nFig.\u00a04Schematic diagram of quorum-sensing systems of S. aureus. The gene locus for the agr system is shown in black and contains two divergent transcripts, RNAII and RNAIII, driven by the P2 and P3 promoters, respectively. The RNAII transcript encodes the agrBDCA operon, which encodes the signal, processing, and detection components for quorum-sensing in S. aureus (see text). The RNAIII transcript is a regulatory RNA that up-regulates and down-regulates all genes related to quorum-sensing. This transcript also encodes the amphipathic peptide \u03b4-hemolysin. RAP and TRAP are part of a second quorum-sensing system thought to be a precursor of the agr system. RAP is secreted and induces TRAP phosphorylation, which in turn induces expression of the RNAII transcript. RIP is a heptapeptide known to inhibit the ability of RAP to induce TRAP phosphorylation\nA second quorum-sensing cascade is thought to serve as a precursor to the Agr system [70], setting the stage for AIP regulation. As S. aureus cells multiply, the RNAIII-activating protein (RAP) is secreted and accumulates outside the cell, and at a threshold concentration, RAP triggers the phosphorylation of the cytoplasmic protein TRAP [71], which induces expression of the RNAII transcript (Fig.\u00a03). Intriguingly, a linear heptapeptide called RIP is known to block the activity of RAP, enabling small-molecule control over S. aureus pathogenicity [72].\nChemistry of the S. aureus AIP signal\nThe reasons for evolving a cyclic thiolactone structure as a quorum-sensing signal are still not clear. The thioester bond and the presence of other labile amino acids reduce stability, because of the potential for oxidative damage, base-catalyzed hydrolysis, and thioester exchange. Indeed, the methionine residue in AIP is rapidly oxidized in vitro to a methionyl sulfoxide, converting the signal to an inactive byproduct [49]. Similarly, oxidants produced in vivo by the phagocyte NADPH oxidase are known to accelerate this inactivation [73]. Despite these issues, the AIP lifetime in host tissues is reported to be 3\u00a0h [74], which is more than adequate for regulation, bearing in mind that S. aureus doubles every 60\u00a0min in the host [75].\nStructure-function studies have shown that the thiolactone cannot be replaced with a more stabile lactone, suggesting that the signal receptor might require acylation to activate the cascade [76], although a lactam substitution does activate at high concentrations [49]. As discussed above, constraining the peptide will improve metabolic stability to proteases [77], by protecting the C-terminus and impeding access to endoproteases, which probably increases the half-life compared with that of the linear AIP counterpart. Clearly, further study is necessary to determine the physiological benefit of this thiolactone structure.\nThe relationship between S. aureus quorum-sensing and biofilm communities\nBiofilm formation is increasingly being recognized as an important virulence factor in S. aureus pathogenesis. Several biofilm-associated diseases, including osteomyelitis [78], endocarditis [79], medical device infections [80], and potentially even skin infections [81], have much clinical relevance. The formation of S. aureus biofilms progresses in a similar fashion to the Gram-negative biofilms, with attachment followed by development into a highly-structured cell community. Several attachment factors, for example the microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) and the surface-attached Atl protein, have been implicated in this initial stage of biofilm formation (reviewed elsewhere [82, 83]). Secretion of a polysaccharide adhesin is thought to be critical for development of a structured biofilm [84], although some biofilm-forming S. aureus strains cannot produce this polymer [85]. Unlike some well-studied Gram-negative bacterial species, S. aureus are non-motile, which leads to significantly reduced biofilm architecture in flowing systems. The significance of this difference is not clear, because simple alterations of growth conditions for P. aeruginosa are known to eliminate higher-ordered biofilm structure [86].\nBecause the development of a robust P. aeruginosa biofilm under some conditions requires an active quorum-sensing system [53], one might assume the behavior of S. aureus is similar; the opposite seems to be true, however, because inactivation of the Agr system tends to enhance attachment [87]. Agr is known to down-regulate surface attachment factors, providing an explanation of this observation. The Agr cascade also up-regulates both the secretion of proteases that can degrade these attachment factors and the secretion of amphipathic peptides (phenol-soluble modulins) that facilitate detachment [83, 88], suggesting quorum-sensing may play a role in biofilm turnover. Despite these intriguing observations, deciphering the literature on this topic has been challenging, because of non-uniform biofilm culturing methods and strain-to-strain differences [89]. To emphasize this point, enhanced attachment by Agr mutants in static biofilm systems is not observed in flowing biofilm reactor systems [87, 90].\nAn elegant study by Yarwood et al. addressed some of the discrepancies in the literature and suggested an alterative role for S. aureus quorum-sensing in biofilms [87]. Under static conditions attachment of Agr mutants was markedly better than that of wild-type; as shear force increased with increased flow rate, however, this advantage was lost and under some experimental conditions became a disadvantage. As in P. aeruginosa biofilm studies, the method and type of experiment seemed to dictate the requirement for the Agr system. By following a quorum-sensing promoter in a flow cell biofilm, Yarwood et al. observed that only patches of surface cells activated the Agr cascade. Surprisingly, these cells detached from the biofilm in periodic waves, suggesting an alternative role for the Agr system in the detachment and recycling phase of biofilm development.\nIn a biofilm infection of the host it seems probable that S. aureus will be subjected to increased flow and\/or shear force, in addition to many other factors, which will affect the quorum-sensing phenomenon. Increased flow will probably dictate the local AIP concentration, potentially deactivating quorum-sensing by washing away the signal, whereas increased shear force could also perform this function, while also accelerating cell detachment. We can only speculate on how this interplay will affect S. aureus in the context of an infection, because studies in this area are limited, but given the medical importance of these biofilms, it warrants further exploration.\nComplicating these biofilm studies are the frequent occurrence of quorum-sensing negative isolates in clinical models. In one study, 36% of S. epidermidis isolates from joint prostheses infections were Agr mutants [80]; these mutants can also be isolated from chronic infections or through extended passage in vitro [91, 92]. It has been proposed that the metabolic burden of the Agr cascade leads to the mutations in this locus [40], with parallels to the in vitro conversion of mucoid P. aeruginosa strains to non-mucoid [93]. The frequent occurrence of Agr mutants suggests it may be advantageous for some portion of an S. aureus population to dispense with the quorum-sensing system. By inactivating Agr, a more heterogeneous, robust biofilm is likely to form, with attachment of the mutants improved by the higher level of surface factor expression. Although it remains to be investigated, this heterogeneity could mirror findings for P. aeruginosa biofilms, in which variants arise at a greater frequency than in planktonic cultures [94].\nAs studies on S. aureus progress, a plausible picture of the role of quorum-sensing in biofilm development is beginning to emerge. As S. aureus attaches and develops an initial biofilm, heterogeneity arising as a result of generation of Agr mutants may lead to a more robust structure with improved adherence properties. Communication between cells will be dictated by the secretion and sensing of AIP molecules, which will enable cross-activation or inhibition of quorum-sensing, depending on the staphylococcal subspecies present in close proximity. When a quorum is reached, a portion of the biofilm will slough off by down-regulation of adherence factors, which could potentially be dictated by the action of secreted proteases or amphipathic peptides. The detached S. aureus cells will have an activated quorum-sensing system that leads to the secretion of numerous invasive factors, enabling spread through the host tissues and the development of an infection. This model is in direct contrast to the well-studied P. aeruginosa paradigm, for which quorum sensing-regulated functions are important for maintaining the structural integrity of the biofilm. Clearly more studies are required to confirm or alter this hypothetical view of the S. aureus lifecycle, but as this model is refined, it may play a significant role in the development of treatment for staphylococcal diseases.","keyphrases":["quorum sensing","biofilm","clinical\/biomedical analysis","cell systems\/single cell analysis","biosensors"],"prmu":["P","P","M","M","U"]}
{"id":"Dev_Biol-2-1-2279743","title":"Redundancy and evolution of GATA factor requirements in development of the myocardium\n","text":"The transcription factors, GATA4, 5 and 6, recognize the same DNA sequence and are all expressed in the developing myocardium. However, knockout studies in the mouse have indicated that none of them are absolutely required for the specification of the myocardium. Here we present evidence for redundancy in this family for the first time. Using morpholinos in both Xenopus and zebrafish embryos, we show that GATA4 knockdown, for example, only affects cardiac marker expression in the absence of either GATA5 or GATA6. A similar situation pertains for GATA5 in Xenopus whereas, in zebrafish, GATA5 (faust) plays a major role in driving the myocardial programme. This requirement for GATA5 in zebrafish is for induction of the myocardium, in contrast to the GATA6 requirement in both species, which is for differentiation. This early role for GATA5 in zebrafish correlates with its earlier expression and with an earlier requirement for BMP signalling, suggesting that a mutual maintenance loop for GATA, BMP and Nkx expression is the evolutionarily conserved entity.\nIntroduction\nThe GATA factors are zinc finger transcriptional activators that bind to the consensus DNA sequence (A\/T)GATA(A\/G). They have been identified throughout eukaryotes and been shown to play critical roles in both haematopoiesis and cardiogenesis in vertebrates and Drosophila (Fossett and Schulz, 2001; Nemer and Nemer, 2001). Of the six evolutionarily conserved GATA genes in vertebrates, GATA4, 5 and 6 are expressed in the heart as it develops.\nLoss and gain of function studies in P19 embryonal carcinoma cells indicated a requirement for GATA4 in the differentiation of cardiac restricted cells to beating cardiomyocytes (Grepin et al., 1997, 1995). In addition, overexpression of GATA4 in Xenopus embryos and explants resulted in expression of cardiac differentiation markers and in some cases spontaneously beating tissue (Jiang and Evans, 1996; Latinkic et al., 2003). However, in the GATA4 null mouse, normal amounts of myocardial tissue appeared to be formed (Holtzinger and Evans, 2005; Kuo et al., 1997; Molkentin et al., 1997; Narita et al., 1996). Thus, even though cardia bifida and defects in looping morphogenesis were observed in the null mouse embryos, specification of the myocardium appeared to take place normally. A suggested explanation for this was the elevated expression of GATA6 (Holtzinger and Evans, 2005; Kuo et al., 1997; Molkentin et al., 1997; Narita et al., 1996; Pu et al., 2004). Consistent with this proposed redundancy of function within the family, GATA5 and 6 are also active in the P19 cell line and Xenopus explant assays described above. Thus, it appears that each of these three GATA family members possesses the capability of inducing cardiac differentiation in gain-of-function assays, however demonstration that they exhibit such redundancy in vivo awaits combinatorial loss-of-function assays.\nThe GATA5 null mouse shows no cardiac phenotype, however it may not be a true knockout due to the potential formation of a truncated protein containing the DNA binding domain (Nemer and Nemer, 2002). In the zebrafish, a critical role for GATA5 in specification of the myocardium has been demonstrated by loss and gain of function assays (Reiter et al., 1999). The fausttm236 mutant shows a severe reduction in expression of cardiac markers and injection of GATA5 RNA induces ectopic expression of the same markers. However, GATA4 expression in the zebrafish fausttm23 mutant is significantly reduced and overexpression of GATA5 results in ectopic expression of GATA4. Thus, these studies raise the possibility that the GATA5 knockdown phenotype is due to the combined loss of GATA4 and 5.\nThe GATA6 null mouse is an embryonic lethal due to an extra-embryonic defect and chimeras have indicated that GATA6 is not required for specification of the myocardium (Kabrun et al., 1997; Koutsourakis et al., 1999; Kuo et al., 1997; Molkentin, 2000; Molkentin et al., 1997; Morrisey et al., 1997). However, we have presented evidence that GATA6 is required for the maintenance and differentiation of cardiac progenitors in zebrafish and Xenopus embryos (Peterkin et al., 2003). The likely resolution of these apparently contradictory data is that the major consequence of lost GATA6 function is non-cell autonomous and can therefore be rescued by surrounding wild type cells in mouse chimeras. The likely non-cell autonomous target for GATA6 is BMP (Peterkin et al., 2003). However, this requirement for GATA6 is for differentiation of cardiac progenitors and not for their initial specification.\nIn this study we use antisense morpholinos in Xenopus and zebrafish embryos to deplete combinations of GATA4, 5 and 6 for the first time. This has allowed us to provide the first experimental support for redundancy in vivo. In addition, we show that the strong dependence of zebrafish on GATA5 is not mirrored in Xenopus, where this GATA factor plays only a redundant role, like GATA4 in both species. The requirement for GATA5 in the zebrafish is for the induction of the myocardial programme, whereas in Xenopus, GATA activity is only required for differentiation. We propose that the primary function for GATA factors in development of the myocardium is in creating a sub-circuit of the regulatory network, involving another critical transcription factor, Nkx, and a crucial signalling pathway, BMP. This mutually supportive sub-network is evolutionarily stable, even though where the network is initiated appears to be more flexible.\nMaterials and methods\nIn situ hybridisation of whole-mounted and sectioned embryos\nXenopus and zebrafish were maintained and embryos were raised and staged using standard conditions (Nieuwkoop and Faber, 1967; Westerfield, 1993). In situ hybridisations on whole-mounted and sectioned embryos were carried out as previously described (Ciau-Uitz et al., 2000; Jowett, 2001; Walmsley et al., 1994). All RNA probes used were labelled with digoxigenin (DIG) except for MyoD and Krox20 which were used in double in situ hybridisations and labelled with fluorescein. Detection of the antibody\u2013alkaline phosphatase was done using BM purple (Roche) or Fast red (Sigma). After in situ hybridisation, embryos were re-fixed in 4% paraformaldehyde, zebrafish embryos were transferred into 75% glycerol to be photographed. Cryostat sections were performed after in situ hybridisation, embryos were fixed as above and washed in 30% sucrose. Embryos were transferred into embedding chambers in O.C.T Compound (Tissue-Tek) and 30\u00a0\u03bcm sections were cut on a Leica CM3050S.\nMorpholino (MO) injection\nThe GATA4\/5\/6 antisense morpholinos were designed and manufactured by Genetools. Morpholino sequences: Xenopus GATA4 MO 5\u2032ctggcaactcaatccacaaaatcca3\u2032 (data shown here), a second morpholino designed against the same pseudo-allele as described by Afouda et al. (2005) (data not shown, 5\u2032agctatactctgatacatcctgatc3\u2032), and a third GATA4 MO designed to block both pseudo-alleles (a kind gift from Todd Evans) (data not shown) gave the same results. Zebrafish GATA4 MO 5\u2032gccatcgttacaccttgatacatat3\u2032 or a second splice morpholino as described by Holtzinger and Evans (2005). For Xenopus GATA5 MO 5\u2032gctacaaacctcacagctcc3\u2032 see Afouda et al. (2005). Zebrafish splice GATA5 MO 5\u2032tgttaagatttttacctatactgga3\u2032. For Xenopus and zebrafish GATA6 MOs see Peterkin et al. (2003). MOs were diluted in deionised water and injected as described (Peterkin et al., 2003). For zebrafish, 25\u00a0ng GATA4 MO, 25\u00a0ng GATA5 MO and\/or 5\u00a0ng GATA6 MO were injected into single-cell embryos individually and in combination. For Xenopus embryos, a total of 20\u00a0ng of GATA4 MO or GATA5 MO and 10\u00a0ng of GATA6 MO were injected individually or in combination.\nResults\nGATA6 is the only essential GATA activity in Xenopus myocardium\nWe have reported previously that GATA6 is required for the maintenance and maturation of cardiomyocytes in Xenopus (Peterkin et al., 2003). The phenotypes consequential upon depletion of GATA4 or 5 in Xenopus, however, have not been previously reported. In the case of GATA5, the need to know is made greater because of its major contribution in zebrafish, and the inability to determine if this is a general requirement in vertebrates by comparison with the mouse knockout, because the reported mutation in the mouse appears not to be a null (Nemer et al., 1999). Therefore, before examining depletion of combinations of GATA factors, we examined the individual loss of GATA4 and 5 in comparison to the already known GATA6 phenotype.\nThe design and quality control of MOs against Xenopus GATA4, 5 and 6 have been reported previously (Peterkin et al., 2003; Afouda et al., 2005). For GATA4, as well as the MO reported previously, two other MOs, one against both pseudo-alleles (Todd Evans, personal communication), were tested and gave the same results. For GATA5 and 6, MOs were designed to target both pseudo-alleles of the Xenopus laevis genes. The optimal amount of each MO injected was determined by titration to ensure that the maximum dose without non-specific effects was used. The extent of knockdown by these ATG MOs was determined by co-injection of tagged reporter RNAs followed by Western blotting (Afouda et al., 2005; Peterkin et al., 2003). Very little residual protein was detected in each case.\nWhen MOs against individual GATA factors were injected separately into the presumptive heart field, the dorsolateral marginal zone, of 4-cell Xenopus embryos, cardia bifida was induced in each case (Fig. 1A, visualised by staining the cells for expression of Myosin Light Chain 2 (MLC)). Cardia bifida has been reported previously for the GATA4 knockout mouse (Kuo et al., 1997; Molkentin et al., 1997), the GATA5 mutant zebrafish, faust (Reiter et al., 1999), and GATA6 morphant Xenopus and zebrafish embryos (Peterkin et al., 2003), but this is the first direct comparison in a single species showing that all three GATA factors are required for the timely migration of cardiac precursors to the midline for fusion of the heart tube. This is in contrast to requirements in the differentiation of the myocardium (see below), where only one member of the family is essential. It seems likely that the requirement for GATA4, 5 and 6 in midline migration of myocardial precursors is actually in the underlying endoderm, where they are all expressed and which has been shown to be essential in mouse and zebrafish for heart tube fusion (Afouda et al., 2005; Alexander et al., 1999; Molkentin et al., 1997; Narita et al., 1997; Reiter et al., 1999; Weber et al., 2000).\nTo determine the effects of the GATA MOs on programming of the myocardial cells, as opposed to their morphological movements, the levels of expression of the transcription factors, Nkx2.5 and Tbx5, and of the contractile machinery genes, cardiac actin (CA), and MLC, were monitored by whole mount in situ hybridisation. In contrast to the GATA6 MO, which causes a profound reduction in the expression of these genes (Peterkin et al., 2003) (Fig. 1D), GATA4 and GATA5 MOs had minimal effects (Figs. 1B, C; for all three MOs and for each marker n was 30\u201350). Despite the cardia bifida at tailbud stages (stages 28\u201332) (Fig. 1A), the gross morphology of the hearts at later stages (stage 43) in GATA4 and GATA5 morphants looked similar to those in wild type embryos, i.e. the cardia bifida was only transient (data not shown). In contrast, as previously described (Peterkin et al., 2003), little or no cardiac tissue was observed in embryos depleted of GATA6 (data not shown). Thus, it would appear that, apart from the transient bifida, the loss of GATA4 or GATA5 has little effect on cardiogenesis in Xenopus. To ensure that the GATA4 and 5 morpholinos were properly functional, they were injected vegetally at the single-cell stage, and were shown to reduce the expression of Sox17\u03b1 during gastrulation (data not shown) (Afouda et al., 2005). Furthermore, the gut of GATA5 morphants failed to coil properly, as previously reported (Afouda et al., 2005). In addition, for these and several of the combination experiments described below, all three GATA4 MOs gave the same results. We therefore conclude that for development of the myocardium in Xenopus embryos, GATA6 is the only essential GATA factor.\nGATA factor redundancy in Xenopus myocardium\nOn the basis of slightly increased expression of GATA6 in GATA4 knockout mice, redundant roles for the GATA factors in the myocardium have been suggested (Kuo et al., 1997; Molkentin et al., 1997; Narita et al., 1996; Watt et al., 2004). In Xenopus, expression of neither GATA5 nor GATA6 was significantly increased in GATA4 MO injected embryos (data not shown). Similarly, in GATA5 and GATA6 MO injected embryos: in neither case was an increase in expression of the other two GATA factors observed (data not shown). However, redundancy does not necessarily depend on an increase in expression of the redundant family member: continued expression could suffice, and that is what we see in all three cases. Therefore to formally test redundancy within the GATA family, we injected combinations of MOs into the presumptive heart field of 4-cell Xenopus embryos, and monitored MLC and Nkx2.5 expression by whole mount in situ hybridisation (Fig. 2). Embryos were classified as unaffected (wild type, +), mildly (\u2212) or strongly (\u2212\u00a0\u2212) down regulated, or displaying no expression at all (\u2212\u00a0\u2212\u00a0\u2212) (Fig. 2A). Numbers of embryos in each category were scored and the results displayed in histograms (n\u00a0=\u00a031\u201394) (Figs. 2B, C). The greater effects of the GATA6 MO are immediately apparent, with clear increases in the affected categories at the expense of the wild type category compared to both control embryos and also to GATA4 or GATA5 MO injected embryos.\nWhen combinations of two MOs were injected, evidence for redundancy was revealed (Figs. 2B, C). Despite having little effect on their own, both GATA4 and GATA5 MOs made the phenotype of GATA6 MO injected embryos more severe when injected with it. Furthermore, the phenotype observed when GATA4 and 5 MOs were injected in combination was significantly worse than either alone, suggesting that the minimal phenotype for the single injections relied on the continued presence of the other GATA factor. When all three MOs were injected together, the phenotype was the most extreme of all with the vast majority of embryos having no expression of MLC at all. We therefore conclude that, while GATA6 is the only individually essential player in driving the myocardial programme in Xenopus, the other two GATA factors are responsible for the residual expression of cardiac genes. Furthermore, in the absence of GATA6, their roles are increased. This is evident from their significantly greater effects on embryo phenotypes when combined with GATA6 MO compared to on their own.\nGATA activity is required for differentiation but not induction of the myocardium in Xenopus\nWe have shown previously that GATA6 is required for the maintenance\/maturation of the myocardium rather than its induction in both Xenopus and zebrafish embryos (Peterkin et al., 2003). As expected, based on the absence of a late phenotype, embryos injected with GATA4 or GATA5 MOs had no effect on early Nkx2.5 expression, as seen for GATA6 MO (n\u00a0=\u00a060, 72, and 89, respectively) (Fig. 3A). In order to determine if the lack of an early effect, even for GATA6 which has a strong late phenotype, was the result of redundancy within the GATA family, we examined Nkx2.5 expression at neurula stages in embryos injected with all three MOs (n\u00a0=\u00a0102) (Fig. 3A). Expression was unaffected, as seen with each of the MOs on their own. Although Nkx2.5 is also expressed in the underlying endoderm at this time, we showed by examining sections that the signal in the cardiac mesoderm is unaffected (Fig. 3B, territory delineated by dashed lines). Furthermore, a similar result was obtained for Nkx2.3 (n\u00a0=\u00a055) (Fig. 3C), which is not expressed or is very weak in the endoderm at this time (Fig. 3D). The expression of eHAND was also unaffected at this stage (Fig. 3E, territory delineated by dashed lines). We therefore conclude that, despite their earlier expression, GATA factors are not required for induction of the myocardial programme in Xenopus, as seen by the continued expression of the other early regulators, Nkx2.5, Nkx2.3 and eHAND, and their own continued expression, but rather for its maintenance\/maturation.\nGATA4 is not essential for induction or differentiation of zebrafish myocardium\nGATA5 (faust) mutant zebrafish have profound defects in the myocardium, displaying reduced expression of several myocardial genes (Reiter et al., 1999). In addition, GATA6 has been shown to be required for maintenance\/maturation of the myocardial programme in zebrafish as seen in Xenopus (Peterkin et al., 2003). In order to determine the relative effects of these two GATA factors, and to determine the contribution of GATA4, we separately injected into zebrafish embryos MOs against each of these GATA factors. The GATA4 MO was shown to specifically block translation of a co-injected GATA4 RNA and not GATA5 or GATA6 RNAs (Supplementary Figs. 1A, B, C). The GATA5 MO was designed to block splicing between exons 1 and 2 of the GATA5 gene, which was confirmed in injected embryos by RT\u2013PCR (Supplementary Figs. 1D, E). This splice blocking morpholino was designed upstream of the exons encoding the zinc fingers to prevent any protein produced binding DNA. However, the creation of a dominant negative GATA5 via splicing from an upstream cryptic site is formally possible (see Supplementary Fig. 1D) but the ability of GATA4 and 6 morpholinos to enhance the cardiac phenotype in combinations (see below) makes this unlikely. Furthermore, the GATA5 morphant heart phenotype was indistinguishable from that seen in the faust mutant, both in single and combination experiments (Supplementary Fig. 1F and see below). The GATA6 MO has been reported previously (Peterkin et al., 2003).\nThe effects of the three MOs injected separately into zebrafish embryos were determined by monitoring expression of the transcription factor, nkx2.5, and the contractile machinery genes, ventricular myosin heavy chain (vmhc) and cardiac myosin light chain 2 (cmlc2) (Fig. 4). GATA5 and 6 MOs induced cardia bifida as described previously for the faust mutant and the GATA6 MO (Peterkin et al., 2003; Reiter et al., 1999). In contrast, in GATA4 MO injected embryos, the myocardial cells appeared to have migrated and fused normally at the midline. We therefore conclude that, in zebrafish, only GATA5 and 6 are required for the proper migration of cardiac precursors. In contrast to mice and Xenopus, GATA4 appears to be uninvolved in this process.\nThe previously reported effects on myocardial gene expression of GATA5 or GATA6 knockdown (Peterkin et al., 2003; Reiter et al., 1999) were immediately evident in these MO injected embryos (Fig. 4). GATA5 MO injection led to substantially reduced expression of nkx2.5 (19\/19), vmhc (42\/43) and cmlc2 (40\/41), as seen for the faust mutant. GATA6 MO injection also resulted in reduced expression of these markers (6\/6, 60\/60 and 42\/44) but to a lesser extent. In contrast, GATA4 MO injection had little or no effect on cardiac marker gene expression levels (n\u00a0=\u00a028, 69 and 53). Spatially the expression of the markers in the GATA4 morphants looks altered compared with the controls due to defects in late cardiac morphogenesis, consistent with those described by Holtzinger and Evans (2005). We therefore conclude that for laying down the myocardial programme in zebrafish, GATA5 has the greatest effect with a significant contribution from GATA6. In contrast, GATA4 makes little or no contribution, at least to the expression of the markers tested.\nGATA factor redundancy in zebrafish myocardium\nTo determine if, as in Xenopus, there is redundancy within the GATA family in zebrafish, we injected the MOs in combinations (Fig. 4). Morphant embryos were classified into three types, unaffected (type +), down regulated (type \u2212) or absent (type \u2212\u2212). Both the GATA5 and the GATA6 MO phenotypes were made worse by the co-injection of the GATA4 MO (Figs. 4B, D and F), as seen in Xenopus, and despite the fact that the GATA4 MO had little or no effect when injected on its own (n\u00a0=\u00a018\u201339). We therefore conclude that a significant proportion of the residual cardiac gene expression in GATA5 or GATA6 MO injected embryos is driven by GATA4, even though the consequences of its loss in the presence of GATA5 or GATA6 are minimal. Thus, redundancy within the GATA family is apparent in the zebrafish myocardium as in Xenopus.\nThe level of residual cardiac marker expression in the GATA4 and 5 MO combination or the GATA4 and 6 MO combination at 26\u00a0hpf was very low (Fig. 4). The level for the GATA5 and GATA6 MO combination was undetectable with 100% of the embryos losing expression, suggesting that, while GATA4 can cover for the absence of either GATA5 or GATA6, it cannot cover for the absence of both, which seems unlikely. We therefore monitored the expression of GATA4 in flat-mounted (Fig. 5A) MO injected 10-somite embryos to determine if it was still expressed (Fig. 5C). We found that GATA5 MO on its own caused a reduction in GATA4 expression (22\/36 embryos), and residual expression was removed completely by the addition of the GATA6 MO (n\u00a0=\u00a035) (Fig. 5C). The GATA4 expression seen in GATA4 morphants reflects the use of a translation-blocking morpholino rather than a splice-blocker. In the same experiment the expression of nkx2.5 was affected in the same way as already described (Fig. 5B). We therefore conclude that the complete absence of cardiac marker expression in GATA5 plus GATA6 MO injected embryos results from the simultaneous absence of GATA4 expression. Thus, as seen for Xenopus embryos, the absence of all three GATA factors completely abolished cardiac marker expression.\nGATA activity is required for induction of the myocardial programme in zebrafish\nWe have shown that GATA activity is only required for the maintenance\/maturation of the myocardial programme in Xenopus. While we have shown that the GATA6 requirement in zebrafish is also late (Peterkin et al., 2003), nkx2.5 expression at 6 somites has been shown to be affected in zebrafish faust mutants (Reiter et al., 1999), suggesting that an additional difference between the species might be the timing of requirements for GATA activity. We therefore tested this earlier requirement with more markers and to determine if it is subject to redundancy. Firstly, we examined nkx2.5 expression in MO injected embryos at 5 somites when it is first expressed (Fig. 6A). For the GATA5 MO, we found a major reduction in expression totally consistent with the reductions seen later and with those reported for the faust mutant (Reiter et al., 1999). We also observed very little effect for the GATA4 or 6 MOs on their own, but both made the GATA5 MO phenotype more severe, consistent with their back-up roles being active at this early stage. Similar observations were made for GATA4 and hrt expression in 5-somite embryos and for tbx5 expression in 10-somite embryos (tbx5 expression is first detected at \u223c\u00a07 somites) (Figs. 6B, C, D). In contrast, nkx2.7 expression was unaffected even by triple knockdown (Fig. 6E). We therefore conclude that, for the markers studied and in contrast to Xenopus, establishing the full early myocardial programme in zebrafish depends on GATA activity. The continued presence of cells expressing nkx2.7 suggested that apoptosis had not yet occurred, and this was confirmed by TUNEL and acridine orange assays (data not shown). Furthermore, re-specification to more anterior or more posterior mesodermal fates was not observed, as judged by the domains of expression of anterior lateral plate and pronephric markers (data not shown). We therefore conclude that in the absence of GATA activity, the cells remain undifferentiated at least up to the 10-somite stage.\nDiscussion\nRedundancy\nGATA4, 5 and 6 are an example of a gene family co-expressed in a specific tissue, in this case the myocardium. Although some differences in their binding site preferences have been detected (Sakai et al., 1998), all three bind to canonical GATA sites with high affinity. Because of this and the relatively mild phenotypes generated in loss of function experiments, they have been suggested to act redundantly (Jiang et al., 1998; Kuo et al., 1997; Molkentin et al., 1997; Narita et al., 1996; Watt et al., 2004). Here for the first time we present evidence in support of this with respect to laying down the genetic programme of the myocardium. The redundancy is particularly striking for GATA4, whose individual loss has essentially no effect on induction or maturation of the myocardium in either zebrafish or Xenopus, in contrast to assumptions of its importance in much of the literature. For this member of the family, its contribution is only revealed in the absence of GATA5 or 6, thereby constituting a formal demonstration of redundancy. Similar demonstrations are evident for both Xenopus GATA5 and in early heart induction for zebrafish GATA6, where they are not the essential players. These redundant GATA activities thus most likely account for the residual expression of cardiac markers in the absence of the essential GATA factor. Indeed little change was observed in expression of the remaining GATA factor in double morphant embryos compared to wild type siblings in either zebrafish or Xenopus (data not shown). The one exception was GATA5 and 6 double morphant zebrafish embryos where the complete loss of GATA4 was used to effect a triple knockout (Fig. 5C).\nGATA factors are an ancient family and in vertebrates have existed with three family members in the heart at least since fish (Patient and McGhee, 2002). Thus, the redundancy reported here would appear to be evolutionarily very stable. Maynard Smith and colleagues have developed simple genetic models to analyse selection pressures on redundant genes and have concluded that evolutionary stability can be achieved if the two (or more) genes perform the same function, but with slightly different efficacies, as seen here (Nowak et al., 1997). The less efficient family member comes into its own when paired with a mutant form of the more efficient family member. Another evolutionarily stable model can be achieved where two (or more) genes perform more than one function: the redundancy occurring only with respect to one specific function. GATA4, 5 and 6 have an ever-growing list of functions in other tissues, so this scenario is more than adequately satisfied as well (Afouda et al., 2005; Capo-Chichi et al., 2005; Ketola et al., 2004; Molkentin, 2000; Yang et al., 2002). The evolutionary stability of this model depends on random mutations being more likely to render the genes inactive for all functions rather than just for one of their functions. Finally, yet another model suggests that redundancy should be more common in genes displaying specific spatio-temporal expression patterns during development, as is the case for GATA4, 5 and 6. For this model, the developmental error rates applicable to these genes need to be higher than their germ line mutation rates: a requirement that is currently unknown.\nThe primary GATA factor\nAn unexpected finding was that the member of the family whose loss has the biggest effect differs between Xenopus and zebrafish. For single knock downs, GATA6 has the strongest effect on myocardial gene expression in Xenopus whereas GATA5 does so in zebrafish. Although at first glance this might suggest a switch in roles for GATA5 and 6, a consideration of the timing of their actions suggests an alternative view. The action of GATA6 in Xenopus is after the initial expression of other early markers such as Nkx2.5, suggesting a role in differentiation of the myocardium (Peterkin et al., 2003). GATA6 knockdown in zebrafish has a very similar effect. Thus, in both organisms, knockdown of GATA6 leaves early marker expression initially intact but decaying with time, whereas when GATA5 was knocked down in zebrafish, expression of Nkx2.5 and other early markers was compromised from the outset (Reiter et al., 1999; this study). The difference between the two organisms therefore can be characterised as the gain or loss of an early function for GATA5. The early role for GATA activity in zebrafish appears not to be masked by redundancy in Xenopus because even triple knockdown of GATA4, 5 and 6 leaves early expression of myocardial markers intact.\nThe role of GATA5 in myocardial induction in mouse and chick embryos is currently unclear. Although in P19 embryonal carcinoma cells induced to differentiate into cardiomyocytes, GATA5 up-regulation occurs after Nkx2.5, precluding an early function during induction (Alexandrovich et al., 2006), the mouse knockout of GATA5 retained the capacity to synthesise a truncated form of the protein containing both zinc fingers, which would likely have significant activity, preventing a definitive conclusion (Nemer and Nemer, 2002). Likewise, the attempts to date to knock down GATA5 activity in the chick were only partial and, in addition, attempted after induction of the myocardium (Jiang et al., 1998). It is therefore not yet clear if the early role for GATA5 has been acquired by zebrafish or lost by Xenopus. In Drosophila, the GATA factor, pannier, is required both upstream and downstream of the Nkx2.5 homologue, tinman (Gajewski et al., 2001; Klinedinst and Bodmer, 2003). In the nematode, the GATA factors, Med1 and 2, are expressed in the mesendodermal precursor to the mesoderm giving rise to part of the pharynx, an organ that has homologies to the heart, and upstream of the Nkx2.5 homologue, ceh22, suggesting that an early role for GATA factors may be ancestral (Broitman-Maduro et al., 2006; Maduro et al., 2001; Rodaway and Patient, 2001). Mesendodermal expression is seen for both GATA5 and GATA6 in zebrafish, while in Xenopus, mesendodermal expression is seen for GATA4 and 6 (Fletcher et al., in press; Rodaway et al., 1999; J. Broadbent, A. Gibson and R. Patient, unpublished observations). Thus, the early role for GATA5 in zebrafish may reflect its early expression in the lineage of cells leading to the myocardium whereas, in Xenopus, early expression of GATA5 is restricted to the endoderm, appearing in the cardiac mesoderm at a later stage (Weber et al., 2000). That GATA6 is also expressed early in this lineage in both species, and GATA4 likewise in Xenopus, and yet neither plays a role in induction of the myocardium, suggests that GATA5, at least in zebrafish, is alone in containing the requisite amino acid sequence for this function. All three GATA factors recognise the same DNA sequence therefore, in the absence of known binding site preferences for GATA5, activities specific to GATA5 are likely to include protein\u2013protein interactions. Thus, GATA5 may be more suited to the necessary interactions in the early mesendoderm than either GATA4 or 6.\nFeedback loops and timing\nGenetic regulatory networks (GRNs) consist of functionally linked regulatory genes encoding transcription factors and their controlling extra-cellular signals (Davidson et al., 2002). They contain motifs, or recurring wiring patterns, which occur with frequencies far greater than in randomised networks (Lee et al., 2002; Milo et al., 2002; Shen-Orr et al., 2002). One such motif is a feedback loop. Mathematical modelling of positive feedback loops indicate that they promote the persistence of signals and have the potential to store information, such that, for example, signalling can readily flip the system from one state to another (Bhalla and Iyengar, 1999). The observation in Drosophila that pannier is both upstream and downstream of tinman, raises the possibility that the establishment of a mutual regulatory loop for these two key regulators is critical evolutionarily (Gajewski et al., 2001; Klinedinst and Bodmer, 2003). Evidence for a similar feedback loop exists in mice, where a cardiac GATA gene has been shown to be Nkx dependent and vice versa (Brewer et al., 2005; Davis et al., 2000; Lien et al., 1999; Molkentin et al., 2000). Davidson and Erwin have recently proposed that regulatory motifs of this type are evolutionarily stable components of GRNs, which they called kernels (Davidson and Erwin, 2006). They highlight a heart-field specification kernel, which is conserved from Drosophila to vertebrates. Strikingly the Nkx2.5\/tinman GATA\/pannier feedback loop is central to this kernel. Our work supports this hypothesis and further suggests that the establishment of this kernel is more critical in evolution than where the loop is initiated. Thus, GATA activity is required to initiate Nkx2.5 expression in zebrafish but not in Xenopus, nevertheless both establish the loop (Fig. 7A).\nInterestingly, as seen for GATA activity, the requirement for BMP signalling differs between zebrafish and Xenopus. Thus, in zebrafish, BMP signalling is required to initiate expression of cardiac markers including GATA5 (Reiter et al., 2001), whereas in Xenopus, it is only required for their maintenance (Walters et al., 2001). In view of the links between BMP and GATA factors in several different tissues, including the myocardium, it seems likely that the early requirement for GATA activity in zebrafish is linked to the early requirement for BMP, and likewise the later requirement in Xenopus (Fig. 7B) (Peterkin, 2003). The cascade of events in Drosophila predicts that the Drosophila BMP signal, Decapentaplegic (Dpp), activates tinman, and they then act in concert to initiate the expression of pannier. Subsequently tinman and pannier maintain each other's expression, whilst pannier (in the ectoderm) maintains Dpp expression. Dpp signalling feeds back to maintain expression of tinman and pannier thus completing the loop (Fig. 7C; for review see Sorrentino et al., 2005). Thus, in summary, the data imply that the initiating factor and the direction in which the loop flows are not important. Ultimately it is the establishment of the loop that is essential and failure to do so leads to the loss of differentiated myocardium.","keyphrases":["redundancy","evolution","xenopus","zebrafish","bmp","nkx","heart","feedback loops","gene networks"],"prmu":["P","P","P","P","P","P","P","P","R"]}
{"id":"Bioinformation-2-1-2139994","title":"Hypo, hype and \u2018hyp\u2019 human proteins\n","text":"Genes with unknown function are called orphan genes while their transcripts and peptides are called hypothetical proteins. There are many genes and their associated proteins that remain uncharacterized in the human genome. A database of human hypothetical proteins with ascribed functions could be helpful for biologists to search for potential proteins of interest. In recent years, the rapid completion of genome sequences has created essential information to link genes to gene products. In order to better explain functions for un-annotated proteins we designed BioinformaTRICKS (an open source project) and used it to develop a database called HYPO.\nBackground\nAs the amount of genome sequence data now available is enormous with more than 750 genomes being either finished or in progress, a biologist is thrown into using \nseveral databases with increasing attention to find any novel genes or proteins or function. However, various analyses based on sequence, structure, function and \u201cOmic\u201d \ndata have revealed different annotation criteria leading to different sets of predicted genes. However, more than 50% of proteins in the proteome zone remain un-annotated \nand un-identified for function (Table 1 in supplementary material). The human genome contains many different regulatory sequences that have roles in controlling gene expression. \nThe protein-coding sequences is only less than 1.5 percent of the genome and then rest remains as non-coding inter and intra-genic regions with undetermined role. \n[1]\nThe annotation of various human chromosomes is well supported by computational predictions where there is no similarity to known proteins or EST sequences. The genes that \nhave unknown function called as orphan genes code for proteins annotated as \u201chypothetical proteins\u201d. Hence, there is a need to begin constructing and analyzing protein families \nclustered as \u201chypothetical proteins\u201d with an aim to elucidate function and protein subunit interactions.\nAfter several drafts of Human Genome Project, there are many proteins that remain to be characterized. Tools have been developed to utilize evolutionary relationships \ntowards understanding uncharacterized proteins despite the need to generate functional interaction networks. [2,3] \nIn particular, these approaches are being used to annotate functions for hypothetical proteins. Although several databases explore protein functions through data-mining, \nthere is a requirement to list all hypothetical proteins. There are reports that address the problem of orphan genes. [4] \nHowever, there is no adequate information to necessitate function of genes that cannot be based on homology alone, except connected to other known gene family. \nThere are several hypothetical proteins such as the KIAA that have remained hype for some time now. [5] Systems biology \nintegrated with protein-protein interaction (PPI) studies could identify the role of these unknown proteins (Figure 1a). Systems biology is a science of constructing networks of \ngenes and proteins thereby providing a framework for predicting models. [6] The proteins connected through the networks \ncould perhaps throw light on the plausible function of the hypothetical proteins through the bona fide interactions they are involved with. \nIn the context of PPI networks, we could consider if a model is to be developed from the network or a network is to be generated with an already established model. Precisely, \nthe putative function of a protein could be better known from a PPI network to develop a model from it. We show here an example in finding the putative function of a hypothetical \nprotein (figure 1b, NP_438169 - B3BP Homo sapiens Nedd4-binding protein 2) using a PPI network. The nearest interacting partners of the protein B3BP were mapped using the STRING \nthat could show the probable function of the query [7]. Information on \u201cknown\u201d or \u201cunknown\u201d protein-protein interactions is \nstill mostly limited but integrating tools such as these could generalize a way to find the function of hypothetical proteins. \nWhile we started mining the proteins, it seemed that there are a few hypothetical proteins that have amino acid residues HYP (histidine, tyrosine and proline) in succession. \nThese might have been long-established through the mutations that are introduced into the proteins at one or more predicted non-essential residues. \nWhile a few KIAA proteins are conserved and have been known to be identified as novel [8], functional analysis of the \nproteins encoded by these KIAA cDNAs could be established from our database of hypothetical proteins. [5] A \u201cconservative \namino acid substitution\u201d is the one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. The families of amino acid residues having \nsimilar side chains are distinct and include conserved amino acids such as histidine (aromatic\/basic side chains), tyrosine (polar side chain) and proline (non polar side chains). \nOn the other hand, these might have appeared during annotation through the mutations introduced randomly along all or part of a coding sequence. Our database has over 6362 hypothetical \nproteins that could be searched for different functions.\nObservations and challenges\nWe observe in tandem that few hypothetical proteins present on different chromosomal loci are known to have the same putative function. Categorizing several approaches beyond \ntraditional sequence similarity that utilize tremendously large amounts of data that is available for computational prediction of functions is the need of the hour these days. \nHaving said this, one could use a subset of proteins that match from several of the experimental approaches and be used as a predictor to circumvent the use of wet laboratory \nexperiments in the near future.\nAnother concern now is specifically on hypothetical protein domains in intrinsically disordered proteins (IDPs). [7] With \nProtein Data Bank (PDB), not accepting theoretical structures, there is an emphasis for experimental approach by researchers to determine the structure and functional relationship \nof a protein. Nevertheless, carefully considering the functional annotation methods as discussed above could definitely devise selection for proteins that could be experimented. \nThis could be an interesting approach to pursue further.\nYet another issue to be noted is the appraisal to understand if any of the hypothetical proteins have proper functional annotations\u2019 been attributed to sequence: structure: \nfunction relationship in case of ordered proteins while sequence: un-structure: function in case of intrinsically disordered proteins. In conclusion, the current methods could \nplay an important role in establishing functions for proteins annotated as hypothetical in the genome.\nNote\nThe title of the article contains hypo abbreviated for hypothetical proteins.\nSupplementary material\nData 1","keyphrases":["hypothetical proteins","database","networks","mining"],"prmu":["P","P","P","P"]}
{"id":"Cancer_Immunol_Immunother-3-1-1914259","title":"No evidence for circulating HuD-specific CD8+ T cells in patients with paraneoplastic neurological syndromes and Hu antibodies\n","text":"Aim In paraneoplastic neurological syndromes (PNS) associated with small cell lung cancer (SCLC) and Hu antibodies (Hu-PNS), Hu antigens expressed by the tumour hypothetically trigger an immune response that also reacts with Hu antigens in the nervous system, resulting in tumour suppression and neuronal damage. To gain more insight into the hypothesized CD8+ T cell-mediated immune pathogenesis of these syndromes, we searched for circulating HuD-specific CD8+ T cells in a large cohort of Hu-PNS patients and controls.\nIntroduction \nParaneoplastic neurological syndromes (PNS) are considered as naturally occurring, successful anti-tumour immune responses in humans [6]. However, this tumour immunity goes along with autoaggression against the nervous system, resulting in severe neurological dysfunction [9]. The mechanisms responsible for the anti-tumour response and neuronal damage are poorly understood. Antigens expressed by the tumour that are normally restricted to neurons (so-called onconeuronal antigens) hypothetically trigger an immune response that cross-reacts with the same antigens in the nervous system [7]. One of the most frequently involved tumours is small cell lung cancer (SCLC) and approximately 50% of patients with PNS and SCLC have high-titre Hu antibodies (Hu-PNS). Hu antibodies are directed against a family of neuron specific, mRNA binding proteins, of which HuD is the best-documented member. Consistent expression of HuD in all SCLC suggests that HuD plays a central role in triggering the immune response [15, 17].\nHigh titres of Hu antibodies in serum and cerebrospinal fluid suggested a pathogenic role for these antibodies, that could, however, never be proven in animal models [3, 22]. Furthermore, expression of Hu antigens is exclusively intracellular, and it is therefore difficult to understand how such antibodies could target tumours or neurons [5]. In addition, pathological examination of PNS neuronal tissue demonstrated localized inflammatory cell infiltrates, containing B cells, CD4+ and CD8+ T cells, in the proximity of overt neuronal cell damage [2, 13, 18, 25]. The presence of an oligoclonal CD8+ T cell infiltrate in nervous tissues and tumours of Hu-PNS patients further suggests an immunopathogenic role for such cells [18, 25].\nSome authors report the presence of HuD peptide-specific CD8+ T cells in the blood of these patients, but also in that of apparently healthy controls (HC) [19, 20, 24]. In addition, the presence of circulating HuD-specific CD4+ T cells has been suggested [1].\nHere, we have investigated the presence of circulating HuD-specific T cells in a large cohort of Hu-PNS patients and controls. Despite a multifaceted approach mainly geared towards the detection of HuD-reactive CD8+ T cells and, to a somewhat lesser extent, CD4+ T cells, no such cells were detected in the blood of Hu-PNS patients and controls.\nMaterials and methods\nPatients\nForty-three patients with high-titred Hu antibodies and a definite clinical diagnosis of PNS [12], 31 Hu antibody negative SCLC patients without neurological symptoms or signs (SCLC) and 54 apparently HC were tested. The Erasmus MC Institutional Review Board approved the study and all individuals provided written informed consent. The individuals\u2019 class I HLA alleles were typed by standard diagnostic PCR at the two-digit resolution level. Patient characteristics are shown in the Table\u00a01. Anti-Hu IgG titres were determined as described previously [21] and in Hu-PNS the dependence in activities of daily living was scored using the modified Rankin scale [21]. Twenty-five HC were male and 29 female, their median age was 46\u00a0years (range 17\u201389) and all were Hu antibody negative. No HC had received previous chemotherapy or immunosuppressive treatment and 30 HC (56%) were CMV-seropositive.\nTable\u00a01Patient characteristics at the time of study entryHu-PNS SCLC N4331Age (median, range)64 (4\u201381)a61 (40\u201383)Gender (M\/F)15\/2821\/10Hu-Ab titre (median, range)12,800 (400\u2013204,800)NegativeCMV serostatus (pos\/neg)29\/1417\/14Paraneoplastic neurological syndromeNA\u00a0PSN27\u00a0PEM5\u00a0PCD4\u00a0PLE\/ BE3\u00a0Pseudo-obstruction2\u00a0Motor neuron disease2Tumour\u00a0No tumour8b0\u00a0SCLC3131\u00a0\u00a0Limited2817\u00a0\u00a0Extended314\u00a0NSCLC20\u00a0Prostate10\u00a0Neuroblastoma10Prior treatment\u00a0None2727\u00a0Chemotherapy\u00a0\u00b1\u00a0immunosuppression164Neurological symptomsNA\u00a0Interval onset symptoms (study entry)5\u00a0months (2\u201315)c\u00a0Interval onset symptoms (diagnosis)4\u00a0months (1\u201312)c\u00a0Progressive at study entryd34 (79%)Modified Rankin scoreNA\u00a0MRS\u00a0=\u00a027\u00a0MRS\u00a0=\u00a0322\u00a0MRS\u00a0=\u00a0410\u00a0MRS\u00a0=\u00a054Hu-Ab Hu antibody, CMV cytomegalovirus, pos positive, neg negative, NA not applicable, PSN paraneoplastic sensory neuronopathy, PEM paraneoplastic encephalomyelitis, PCD paraneoplastic cerebellar degeneration, PLE paraneoplastic limbic encephalitis, BE brainstem encephalitis, SCLC small cell lung cancer, NSCLC non-small cell lung cancer, MRS modified Rankin scorea One Hu-PNS patient was a 4-year-old boy with an underlying neuroblastoma. The remaining Hu-PNS patients were aged between 49 and 81\u00a0yearsb No tumour mass visible on CT-scan or FDG-PET scanc Median (ranges) of intervals are shownd Progression of neurological symptoms was defined by the increase of at least one point on the modified Rankin scale during 2\u00a0months prior to study entry\nReagents\nNinety-three HuD protein-spanning synthetic peptides, 15-mers with 11 amino acids overlap, were pooled to constitute the HuD peptide mix (HuDmix) and smaller peptide pools [Jerini Peptide Technologies (JPT), Berlin, Germany]. For interferon-\u03b3 enzyme-linked immunosorbent spot-forming (IFN-\u03b3 ELISPOT) assays and the construction of HLA class-I multimers, HuD-derived 9- and 10-mers were selected based on previous studies [19, 20]. The phycoerythrin (PE)-labeled multimers and corresponding peptides used were: HLA-A*0101-147ELEQLFSQY155, HLA-A*0101-245RLDNLLNMAY254, HLA-A*0201-86SLGYGFVNYI95, HLA-A*0201-248NLLNMAYGV256, HLA-A*0201-315QLFGPFGAV323, HLA-A*0201-362RLGDRVLQV370, and HLA-A*2402-154QYGRIITSRI163 (ProImmune, Oxford, UK). As positive and negative controls, HLA-A*0201-495NLVPMVATV503 [CMV phosphoprotein-65 (CMV-pp65); Beckman Coulter, San Diego, CA] and HLA-A*0201 presenting an irrelevant peptide (ProImmune), respectively, were included.\nTo measure general T-cell responsiveness, we used phorbol myristate acetate (PMA) plus ionomycin, or phytohemagglutinin (PHA). A peptide pool containing 15-mers spanning CMV-pp65 (JPT) was used as positive- and negative antigen-specific control in CMV seropositive and seronegative individuals, respectively.\nCytokine flow cytometry\nPeripheral blood mononuclear cells (PBMC) were isolated within 12\u00a0h after venipuncture and stimulated in duplicate as described elsewhere [14]. Briefly, 2\u00a0\u00d7\u00a0106 PBMC were incubated at 37\u00b0C in a CO2 incubator for 18\u00a0h with 1\u00a0\u03bcg\/ml HuDmix, 1\u00a0\u03bcg\/ml CMV-pp65, 1\u00a0\u03bcg\/ml ionomycin plus 25\u00a0ng\/ml PMA, or without antigen. After 2\u00a0h of stimulation, brefeldin A was added to one of the duplicate tubes allowing for intracellular accumulation of cytokines in activated T cells. Brefeldin A was not added to the second tube to allow detection of secreted cytokines in the supernatant. In some individuals, additional stimulation was performed in the presence of co-stimulatory monoclonal antibodies (mAb) directed against CD28 and CD49d (BD Biosciences, San Jose, CA). Stimulated PBMC were stained and analysed using anti-CD3 conjugated with peridinyl chlorophyllin (PerCP), anti-CD8 conjugated with allophycocyanin (APC), anti-interferon (IFN)-\u03b3 conjugated with fluorescein isothiocyanate (FITC), anti-tumour necrosis factor (TNF)-\u03b1 conjugated with PE, or appropriate isotype control mAb (all from BD Biosciences) [14]. CD4+ T cells were defined as CD3+, 8\u2212. Positive responses were defined by (1) percentage of cytokine-positive CD4+ or CD8+ T cells >2 times the negative control (i.e., no antigen) and (2) \u22650.1% of the total number of CD4+ or CD8+ T cells, each after subtraction of isotype control results.\nDetection of secreted cytokines\nThe secretion levels of IFN-\u03b3, TNF-\u03b1, interleukin (IL)-2, IL-4, IL-5 and IL-10 were measured in supernatants using a cytometric bead array (BD Biosciences). Based on CMV data (not shown), a positive result was defined as cytokine concentration >2 times the background (no antigen) and a minimum level of 50\u00a0pg\/ml.\nIFN-\u03b3 ELISPOT\nPBMC (2\u00a0\u00d7\u00a0105\/well) were pre-stimulated in duplicate in 96-well plates with culture medium containing 3\u00a0\u03bcg\/ml HuD 9-mers, HuDmix, CMV-pp65, PHA, or no antigen for 1.5\u00a0h at 37\u00b0C and 5% CO2 [10]. The PBMC were subsequently transferred to anti-IFN-\u03b3-coated ELISPOT plates (Nalge Nunc, Rochester, NY) for a further 18-h incubation. The ELISPOT assay was performed using standard protocols and automated reading (AELVIS GmbH, Hanover, Germany) [10]. The mean number of spot-forming cells (SFC) in duplicate wells was used as assay outcome. Positive results were defined by numbers of SFC\/well >3 times background (no antigen) and a minimum of 15 SFC\/well.\nDetection of HuD-specific CD8+ T cells using Class-I HLA multimers\nThawed PBMC were stained as described previously [11]. Following acquisition of 1\u00a0\u00d7\u00a0105 viable (i.e., 7-amino-actinomycin-D [7AAD] negative) CD8+ T cells, a positive result required a percentage of \u22650.1% of viable CD8+ T cells binding the HuD multimer and a brightly staining HuD multimer-binding CD8+ T-cell population that did not overlap with the dimly staining irrelevant multimer-binding T-cell population.\nResults\nCytokine flow cytometry of PBMC after stimulation with HuD-derived peptides\nStimulation with PMA and ionomycin induced IFN-\u03b3 production in both CD4+ and CD8+ T cells of all individuals (not shown). Whilst all CMV-seropositive individuals specifically responded to CMV-pp65, no HuD-specific T-cell reactivity was observed in any of the Hu-PNS patients, SCLC patients or HC (Fig.\u00a01a, b). Similar results were obtained when intracellular TNF-\u03b1 (not shown) or secreted cytokines (Fig.\u00a01c) were measured. The use of co-stimulatory antibodies in combination with HuDmix did not result in the detection of HuD-specific T-cell reactivity (not shown).\nFig.\u00a01Cytokine production in response to HuDmix and HLA-matched HuD 9-mers. Proportions of CD8+ (panel a) and CD4+ (panel b) T cells expressing intracellular IFN-\u03b3 after stimulation with HuDmix in Hu-PNS patients, SCLC and healthy controls. Each dot represents the result observed in a single individual (panel c). After stimulation of PBMC with HuDmix and control antigens, the indicated cytokines were measured in assay supernatants. The results are shown for Hu-PNS patients only. Horizontal lines indicate median values of each group (panel d). ELISPOT assay showing the number of IFN-\u03b3 SFC after stimulation of 2\u00a0\u00d7\u00a0105 PBMC with HuDmix or HuD 9-mers. Each dot represents the mean result of duplicates for each stimulus in each individual (panels a\u2013d). Responses to CMV antigens are shown for CMV seropositive individuals only, as they were consistently negative in CMV seronegative individuals (not shown). The numbers of individuals tested are given in between brackets. NEG negative control (incubation without antigen), HuDmix HuD protein-spanning peptide pool, PP65 CMV pp-65 protein-spanning peptide pool, IL interleukin TNF tumour necrosis factor, IFN interferon, SFC spot-forming cell, PHA phytohemagglutinin. ELE, RLD, SLG, NLL, QLF, RLG and QYG designate individual HuD-based peptides\nIFN\u03b3-ELISPOT assay on PBMC stimulated with HuD-derived 9-mers\nAll individuals responded to PHA as determined by IFN\u03b3-ELISPOT assay. In addition, all CMV-seropositive individuals responded to CMV-pp65. However, no T cell reactivity towards the previously described class-I HLA-binding HuD peptides [20] was detected in individuals with the appropriate HLA types in any of the study groups (Fig.\u00a01d).\nAnalysis of HuD-specific CD8+ T cells using Class-I HLA multimers\nFinally, we investigated the presence of HuD-specific CD8+ T cells in PBMC using Class-I HLA multimers containing HuD-derived peptides. Whilst CMV-seropositive individuals with the appropriate HLA types showed distinct populations of pp65 multimer-binding CD8+ T cells, no HuD-specific CD8+ T cells were observed in individuals with the appropriate HLA types in any of the study groups (Fig.\u00a02).\nFig.\u00a02Analysis of HuD peptide-loaded, Class-I HLA multimer-binding CD8+ T cells. PBMC from a CMV-seropositive Hu-PNS patient were stained with HLA-A*0201\u00a0multimers loaded with the HuD peptide SLGYGFVNYI (panel a), an irrelevant peptide (panel b) or with the CMV-pp65 peptide NLVPMVATV (panel c). All data shown are obtained after selection of T cells (i.e., CD3+, low sideward scatter signals) [12]. Binding of Class-I HLA multimers (panels a\u2013c, horizontal axes) was analysed in relation to CD8 expression. In this example, the proportion of SLGYGFVNYI multimer-binding CD8+ T cells (0.36%) was similar to that of irrelevant multimer-binding CD8+ T cells (0.46%); binding resulted in low-intensity fluorescence signals only. In contrast, 2.46% of CD8+ T cells bound the NLVPMVATV multimer resulting in high-intensity fluorescence signals from most CD8+ T cells\nDiscussion\nWe set out to detect HuD-specific T cells in the blood of Hu-PNS patients as they are postulated to play a pivotal role in the immunopathology of this disease. Although we applied three different approaches we could not detect circulating HuD-specific T cells.\nFirst, to induce cytokine responses in T cells, we used 15-mer protein-spanning peptide pools that have the advantage of covering the full protein sequence and of eliciting both CD8+ and CD4+ T-cell responses [14], as demonstrated by the CD8+ and CD4+ CMV-pp65-specific T-cell responses. However, no CD8+ or CD4+ HuD-specific T-cell responses were observed. These results are at variance with the detection of HuD-specific CD4+ T-cell proliferative responses in PBMC of Hu-PNS patients by Benyahia et\u00a0al. [1]. This discrepancy may be explained by differences in read-out (i.e., 3-day lymphocyte proliferation in Benyahia\u2019s study [1] vs. overnight cytokine production in ours) and the use of recombinant HuD protein [1] versus a protein-spanning 15-mer peptide pool (this manuscript).\nWe then studied responses to HLA class-I binding HuD peptides that were previously selected [20]. Using the same experimental setup and HuD 9-mer peptides, Rousseau et\u00a0al. [20] reported HuD-specific T-cell reactivity in 7\/10 Hu-PNS patients and in 3\/10 HC [20]. In that small study a positive response was defined as an experimental value \u22652 times above background. With that criterion, 3 PNS, 2 SCLC and 4 HC would have been classified as HuD T-cell responders in our study. However, using that cut-off we would also have detected T-cell reactivity in individuals whose Class-I HLA molecules did not have the appropriate binding motifs (data not shown). Therefore, we used more stringent cut-off levels resulting in a negative outcome. As most patients in both studies had progressive neurological disease and were tested shortly after start of symptoms and prior to therapy, differences between the study populations do not explain this discrepancy.\nFinally, we could not detect HuD-specific circulating CD8+ T cells using Class-I HLA multimers with the same fine specificities as defined by Rousseau et\u00a0al. [20].\nThe absence of detectable circulating HuD-specific CD8+ T cells may not be surprising. In a PCR-based study, Plonquet et\u00a0al. [18] detected the same T-cell clone in neoplastic and nervous tissues, but not in blood. This finding suggests that T cells involved in the pathogenesis of Hu-PNS circulate in concentrations below detection level. An immune response taking place in the central nervous system parenchyma may deplete the circulating pool of CD8+ T cells with that specificity [23]. Furthermore, vaccination studies in melanoma patients demonstrate that clinically effective anti-tumour immune responses may occur despite low levels of melanoma-specific cytotoxic T cells, i.e., below the detection limit of multimer-based assays [4, 16].\nIn conclusion, we were unable to detect HuD-specific T cells in a large cohort of Hu-PNS patients and controls. However, two of our three assays were designed for the detection of CD8+ T cells only. The IgG1 isotype predominance of serum Hu antibodies in Hu-PNS indicates a T-helper response to the Hu antigen [13]. Therefore, further studies are warranted that focus on the detection of circulating HuD-specific CD4+ T cells. In this context, regulatory CD4+ T cells\u2014which down regulate immune responses towards auto-antigens and tumour-antigens\u2014are of interest. Although the numbers of regulatory T cells are increased in cancer patients [26] and in PNS patients [8] (de Beukelaar et al. unpublished data) their (possibly impaired) function in PNS remains to be studied. Finally, examination of the antigen-specificity of T cells in affected tissues may shed further light on the role of HuD-specific T cells in the pathogenesis of Hu-PNS.","keyphrases":["hud","paraneoplastic","sclc","anti-tumour immunity","t lymphocyte","hla class i multimer"],"prmu":["P","P","P","P","R","R"]}
{"id":"Rev_Endocr_Metab_Disord-3-1-1894828","title":"Noonan syndrome and related disorders: Alterations in growth and puberty\n","text":"Noonan syndrome is a relatively common multiple malformation syndrome with characteristic facies, short stature and congenital heart disease, most commonly pulmonary stenosis (Noonan, Clin Pediatr, 33:548\u2013555, 1994). Recently, a mutation in the PTPN11 gene (Tartaglia, Mehler, Goldberg, Zampino, Brunner, Kremer et al., Nat Genet, 29:465\u2013468, 2001) was found to be present in about 50% of individuals with Noonan syndrome. The phenotype noted in Noonan syndrome is also found in a number of other syndromes which include LEOPARD (Gorlin, Anderson, Blaw, Am J Dis Child, 17:652\u2013662, 1969), Cardio-facio-cutaneous syndrome (Reynolds, Neri, Hermann, Blumberg, Coldwell, Miles et al., Am J Med Genet, 28:413\u2013427, 1986) and Costello syndrome (Hennekam, Am J Med Genet, 117C(1):42\u201348, 2003). All three of these syndromes share similar cardiac defects and all have postnatal short stature. Very recently, HRAS mutations (Aoki, Niihori, Kawame, Kurosawa, Ohashi, Tanaka et al., Nat Genet, 37:1038\u20131040, 2005) have been found in the Costello syndrome and germline mutations in KRAS and BRAF genes (Rodriguez-Viciana, Tetsu, Tidyman, Estep, Conger, Santa Cruz et al., Nat Genet,2006; Niihori, Aoki, Narumi, Neri, Cave, Verloes et al., Nat Genet, 38:294\u2013296, 2006) in the Cardio-facio-cutaneous syndrome. Phenotypic overlap between these genetic disorders can now be explained since each is caused by germline mutations that are major components of the RAS-MAPK pathway. This pathway plays an important role in growth factor and cytokine signaling as well as cancer pathogenesis.\nIntroduction\nNoonan Syndrome (NS) has been recognized for about 40\u00a0years [1] but the genetic cause was not found until 2003 when mutations in the PTPN11 gene were reported [2]. Shortly afterwards LEOPARD syndrome (LS), a rare alleic variant of NS was found to have specific mutations in the PTPN11 gene [25]. Cardio-facio-cutaneous (CFC) syndrome and Costello syndrome (CS), both rare syndromes, share significant phenotypic overlap with NS especially in infancy. The common features of facial dysmorphism, short stature and similar cardiac findings in all four conditions suggest a similar underlying pathogenesis. In 2005 [6], mutations in the HRAS oncogene were found to be the cause of CS and in 2006 mutations in KRAS, BRAF, MEK1 and MEK2 genes [7, 8] were identified in CFC. All of these germline mutations are components of the RAS-MAPK pathway which plays an important role in growth factors and cytokine signaling. In this review, the clinical findings of the four syndromes will be reviewed. NS will be discussed in more detail since it is common and more clinical studies are available.\nNoonan syndrome\nNS is one of the more common non-chromosomal syndromes seen in children with congenital heart disease with an estimated incidence of 1 in 1,500 [1]. Although there is wide phenotypic variation in NS, distinctive facial features include hypertelorism, down-slanting palpebral fissures, a high arched palate, low set posteriorly rotated ears, malar hypoplasia, ptosis and often a short neck. The phenotype changes significantly with time. In the newborn, there is excessive nuchal skin which is the result of prenatal cystic hygroma. During infancy, the head is relatively large, the eyes are often prominent and round, there is a high nasal bridge which may be flat, and the neck appears short. At 3 to 4\u00a0years of age, the body becomes more stocky and the chest more prominent. The chest deformities often become significant. In later childhood, the facial appearance begins to show coarse features and becomes more triangular as the chin lengthens. The eyes become less prominent and the ptosis may be more apparent. In the teenager and young adult, as the neck lengthens webbing may become more apparent, the facial features are more triangular and become sharper, the nose has a pinched root and a thin high bridge. An older adult has prominent nasal labial folds, a high anterior hairline and the skin often appears rather transparent and wrinkled.\nIn most, the prenatal history is unremarkable but polyhydramnios is frequent. Height and weight are within normal limits at birth but height begins to drop off within a few months and over 70% of patients with NS have significantly short stature. Some patients with NS have significant feeding difficulties with resulting failure-to-thrive and require tube feedings. Although this may contribute to the poor gain in weight, short stature occurs equally in children who have no feeding problems in infancy. Other important findings include a chest deformity which may be in the form of a pectus carinatum or pectus excavatum, apparent widely spaced nipples and a relatively broad chest. Scoliosis and kyphosis occur in about 15% of patients. Muscle hypotonia is frequent and may account for some of the motor delay. Significant mental retardation is uncommon but some degree of learning disability is frequent and may require special help in school. Eye findings, especially strabismus and refractive errors are common and an occasional patient will have a coloboma. All children with NS should have a complete eye examination. Since conductive hearing loss is rather frequent, children should have a hearing evaluation. Over half of the males with NS have either one or both testes undescended and delay in puberty is common for both males and females.\nEasy bruising is common in NS and a variety of bleeding problems have been reported [9]. These include deficiency of Factor XI, Von Willebrand\u2019s disease, thrombocytopenia and platelet function defects. In addition, low levels of Factor VIII and XII have also been noted. Hepatosplenomegaly, usually unexplained, is present in about 25% of patients particularly in infancy. Lymphatic abnormalities occur in less than 20% of patients but may present serious problems. Over 80% of patients with NS have a cardiac finding. Pulmonary stenosis is, by far, the most common but nearly every cardiac lesion has been reported. In addition, hypertrophic cardiomyopathy may occur.\nIt was recognized early on that NS could be transmitted in an autosomal dominant manner. In 1994, Jamieson et al [10] was able to map the gene for NS to the distal part of chromosome 12q. Not all families with NS studied showed this linkage suggesting that more than one gene was likely to be involved in the etiology. In 2002, Tartaglia [2] found a mutation in the PTPN11 gene to be present in about 50% of patients with NS. This gene regulates the production of a protein called SHP-2 which is essential in several intracellular signal transduction pathways and controls a number of developmental processes including cardiac semilunar valvulogenesis. The protein is expressed throughout the body and it is an important player in cellular response to growth factors, hormones, cytokines and cell adhesion molecules. The PTPN11 mutations in NS are clustered in interacting portions of the N-SH2 in PTTP domains. This mutation results in a gain of function for SHP-2.\nChildren with NS often present to the endocrinologists because of the short stature, delayed puberty or undescended testes in males. Although height and weight are usually in the normal range at birth, height drops off within the first few months. In general, there is at least a 2-year delay between bone age and chronological age. Continued growth may occur until the early 20s. In both males and females, there is a delay in puberty. Females seem to possess normal fertility. Males, as expected due to undescended testes, appear to have decreased fertility but male transmission is well described and not uncommon.\nThe cause of short stature in NS is really not clear. After pharmacological stimuli, growth hormone secretion was usually normal in patients studied but a small number showed a subnormal response. Others have shown a neurosecretory growth hormone dysfunction to be present in some patients but this did not appear to have any effect on the response to growth hormone [11]. There have been previous studies looking at the IgF-1 levels which have been below normal for the majority reported. A considerable number of children have undergone treatment with human biosynthetic growth hormone. The majority of studies have shown similar results. There is a significant increase in growth velocity in the first and second year of growth hormone treatment [12\u201316]. The velocity in growth tends to diminish in succeeding years. Several authors claim that the predicted adult height has been increased in patients treated with growth hormone but there have been no real controlled studies to document the long-term effect of growth hormone on adult height. In those studies where bone age as well as actual height were measured, the increase in bone age was equal or slightly greater than the overall increase in height. If the bone age acceleration exceeds the acceleration in height, it is even possible that growth hormone therapy could actually decrease the adult height. Since there was little data available regarding adult height in NS, we recently [17] published a study of 73 adults who were all over 21\u00a0years of age at the time of their last measurement. Thirty-one percent of males and 32% of females achieved an adult height that was at the tenth percentile or greater which would be considered within a normal range. The remainder all fell below the tenth percentile but importantly 38% of males and 54.5% of females had an adult height below the third percentile. Adult height was unrelated to the presence or severity of cardiac disease and none of the adults achieving a normal height had been treated with growth hormone. Dr. Municchi et al [16] suggests that long-term serial height measurement over years comparing treated and untreated patients is needed to clearly show the benefit of growth hormone therapy on final adult height. There is no question that adult height can be achieved at an earlier age with the use of growth hormone.\nWith the discovery of the PTPN11 mutation, it is now possible to study patients with NS positive for a mutation and compare them with NS patients who do not carry a mutation of the PTPN11 gene. The large cohort reported by Ranke [18] suggested that weight and length were normal in 119 newborns with NS. With the availability of genetic testing, several recent studies have shown that mean birth length for NS patients with a mutation is slightly below normal and is less than that of the non-mutated NS newborns [19, 20]. In a study by Zenker [20], 88% of PTPN11 positive mutated children older than 3\u00a0years of age had a height less than two standard deviations and were significantly shorter than non-mutated children. All recent studies [21] suggest that there is a more severe mechanism acting on growth retardation in NS patients carrying a PTPN11 mutation. A recent study from France [19] evaluated 35 patients with NS, 20 of whom had a PTPN11 mutation. There was a trend to a shorter birth length in mutated versus non-mutated newborns and small for gestational age tended to be more frequent in mutated versus non-mutated patients. By 6\u00a0years of age, patients with mutations were significantly shorter than patients without mutations. The results of hormonal studies showed a normal growth hormone secretion after pharmacological stimuli and a low serum IgF-1 and ALS concentrations which is in contrast with a normal IgFBP-3 level. Since the PTPN11 gene has a negative effect on intracellular signaling downstream from several growth factor receptors, a growth hormone post receptor signaling resistance could represent the mechanism of stunted growth in NS. They felt that the lower growth response to growth hormone treatment observed in mutated versus non-mutated patients suggests some degree of resistance to growth hormone. Another recent study by Binder et al [22] showed a similar pattern. Data from these two studies would be in favor of growth hormone resistance by a late post receptor signaling defect specific for IgF-1 and ALS that does not effect IgFBP-3 stimulation. Changes in height during the first 2\u00a0years of growth hormone therapy in the pre-pubertal group show catch-up growth was less pronounced in patients with a mutation compared to those without a mutation. Fortunately, in all the studies using growth hormone, no adverse results have been observed. Many patients have undergone serial echocardiograms and none have shown an increase in their left ventricular mass index during growth hormone treatment [23]. These recent studies suggesting that there may be a primary IgF-1 deficiency in NS has stimulated and been incorporated into a phase II clinical study which will investigate the use of IPLEXtm (mecasermin rinfabate) (rDNA origin) which will be given by injection to treat growth failure due to insulin-like growth factor IgF-1 deficiency. Although we still do not understand how the mutation in the PTPN11 gene affects SHP-2, the result apparently is a disruption in the growth hormone IgF-1 axis and IgF-1 deficiency which could be the cause of growth failure.\nLEOPARD syndrome (LS)\nGorlin [3], in 1969, introduced the acronym LEOPARD (LS) to describe a rare syndrome that shares many features similar to NS. These include autosomal dominant inheritance, similar facial dysmorphism and similar cardiac defects with an overabundance of hypertrophic cardiomyopathy compared to pulmonary stenosis. The characteristic cutaneous finding of lentigines is the main distinguishing characteristic. In addition, unilateral or bilateral hearing loss is frequent.\nSarkozy et al [24, 25] recently reported clinical and molecular studies in a consecutive study of 30 patients with LS and found mutations in the PTPN11 gene in 27 of the 30 patients studied. Mutations in LS have all occurred in exons 7, 12 and 13 while the more typical NS have the great majority of mutations occur in exons 3, 8 and 13. It is of interest that the mutations in patients with LS show a loss of function rather than gain a function as is found in the more typical NS patients.\nZenker [20] noted a specific mutation T468M in exon 12 which was also reported by Sarkozy in seven of their patients with LS to have less adverse effects on body growth. Only two of the ten patients with the T468M mutation have short stature.A cardiac abnormality was present in 71% of the patients with LS, with 80% of those showing hypertrophic cardiomyopathy. Pulmonary stenosis was present in two patients and a partial AV canal in another.\nCardio-facio-cutaneous syndrome (CFC)\nIt is often difficult to distinguish an infant with CFC from NS although, with time, the phenotype becomes more distinctive. Patients with CFC [4] have a high forehead, a relatively large head and bitemporal constriction. Like NS, they have a downward slant of the palpebral fissures, posteriorly rotated ears and a flat nasal bridge. The hair is usually sparse, curly and friable and absent eyebrows are frequent. The skin changes are variable but include keratosis pilaris with patchy hyperkeratosis [26]. In time, the phenotype for CFC becomes more distinctive and less Noonan-like. These patients are significantly delayed in both motor and mental skills and, like NS, they are hypotonic, often have failure-to-thrive with frequent gastrointestinal complaints and often require tube feedings. Cardiovascular abnormalities are similar to NS although hypertrophic cardiomyopathy is more common than in the typical NS patient. Although these children appear to be of normal height and weight at birth, they soon fail-to-thrive and short stature is found in 78% [27]. Bone age is significantly delayed and osteopenia is occasionally observed.\nVery recently, mutations in four separate genes have been found to be associated with CFC [3, 4]. These four genes include BRAF, KRAS, MEK1 and MEK2. All of these genes belong to the same RAS-ERK pathway that regulates cell differentiation, proliferation and apoptosis. It is likely that the mechanism causing short stature in NS may be similar to that causing short stature in CFC. This is a very rare disease and there is little information regarding endocrine studies in patients with CFC.\nCostello syndrome\nCostello Syndrome (CS) [28] is a rare condition with a distinctive facial appearance which may be difficult to distinguish from NS and CFC in infancy. Although height and weight are normal or above normal at birth, severe growth retardation is the rule postnatally. Like NS, they have a large head, short wide nose and short neck. Unlike NS or CFC, these patients usually have thick and relatively prominent lips and tongue. They also have loose skin on the hands and feet and deep palmar and plantar creases. There is significant mental and motor delay. Cardiovascular abnormalities are found in about 60% and they are remarkably similar to that found in NS and CFC. Pulmonary stenosis, atrial septal defect and hypertrophic cardiomyopathy are the most common lesions [29]. Of particular interest is the high incidence of cardiac arrhythmias noted particularly in infancy [30] that is not characteristic of either CFC or NS.\nRecently Aoki et al [4] reported mutations in HRAS a proto-oncogene to cause CS. Gripp et al [31] confirmed this finding and reported 33 of 40 patients with a clinical diagnosis of CS to have a HRAS mutation. All the mutations were de novo. Patients with CS are at a significantly increased risk for the development of malignancy, particularly rhabdomyosarcoma, neuroblastoma, ganglioneuroblastoma, and transitional cell carcinoma of the bladder. Although few studies of growth hormone have been carried out in Costello patients, the findings are very similar to that of NS. Response to growth hormone treatment has been variable. Kerr et al [32] have suggested that in CS, hormone treatment may be harmful because of the propensity to tumor formation and the presence of hypertrophic cardiomyopathy. He described two patients, one who had mild left ventricular hypertrophy with normal function before growth hormone was started. After 3\u00a0months of treatment, the cardiomyopathy progressed with significant left ventricular outflow tract obstruction with a gradient of 60\u00a0mmHg. He was treated with Propranolol. There was no further progression of the cardiomyopathy in spite of continuation of growth hormone. In the second case, growth hormone was started at 12\u00a0months and continued until age 26\u00a0months when a large pelvic mass was discovered which was proven to be an embryonal rhabdomyosarcoma. Growth hormone was stopped after the tumor was recognized. In spite of extensive chemotherapy, the patient died. Since CS is associated with both subaortic hypertrophic cardiomyopathy and tumors, the role of growth hormone in these two patients is unknown. At the present time, it is unclear whether growth hormone is beneficial or harmful in CS.\nConclusion\nNS, LS, CFC and CS all have significant phenotypic overlap. Although they may be difficult to distinguish early on in life, in time they can usually be distinguished clinically. Recent studies show that each of these syndromes is caused by a germline mutation in a key component of the highly conserved RAS, RAF-ERK-MAP kinase cascade which is better known for its roles in growth factor and cytokines signaling in cancer pathogenesis. We still do not understand how these specific mutations cause the disease and why there are such distinct phenotypic differences in mutations within the same signaling pathway. Tartaglia et al [33] recently provided evidence that specificity in amino acid substitution is relevant to the functional deregulation of SHP-2 and disease pathogenesis. They showed NS mutations have less potency for promoting SHP-2 gain of function than do leukemia-associated mutations and that Y279C and T468M amino acid substitutions noted in LS engender a loss of SHP-2 catalytic activity. It is not surprising that a mutation in the HRAS gene associated with CS has an increased incidence of tumors since these mutations are identical to the human tumor associated mutations. So far, CFC syndrome has not been associated with malignancy but the number of reported cases is still quite small with long-term follow-up limited. For the endocrinologist, it is interesting that all of the syndromes have a high incidence of short stature. LS, on the other hand, with loss of function appears to have a lower incidence of short stature but the higher incidence of hypertrophic cardiomyopathy. It will be of interest to see how effective IPLEXtm will be in treating the growth failure in NS and perhaps these related syndromes as well. Since the growth failure starts very soon after birth and may indeed start before birth, it is certainly possible that treatment may be necessary very early in life to achieve anywhere near a normal growth stature.","keyphrases":["noonan syndrome","short stature","cardio-facio-cutaneous syndrome","costello syndrome","leopard syndrome","growth hormone"],"prmu":["P","P","P","P","P","P"]}
{"id":"Eur_J_Clin_Pharmacol-3-1-1914304","title":"Hypothermia following antipsychotic drug use\n","text":"Objective Hypothermia is an adverse drug reaction (ADR) of antipsychotic drug (APD) use. Risk factors for hypothermia in ADP users are unknown. We studied which risk factors for hypothermia can be identified based on case reports.\nIntroduction\nAntipsychotic drugs (APDs) can influence thermoregulation. Even before its psychotropic properties were clear in the early 1950s, the first manufactured APD, chloropromazine, was used to suppress compensatory responses to body cooling in surgery (artificial hibernation) [1].\nThe hypothermic effects of APDs seem less well known than the hyperthermic effects (e.g., malignant neuroleptic syndrome). Besides occasional case reports, little emphasis has been placed in scientific literature on hypothermia as adverse drug reaction (ADR). To our knowledge, a review of all reported and published cases of antipsychotic associated hypothermia has not yet been published.\nMethods\nThe WHO international database for Adverse Drug Reactions was searched for reports of hypothermia and APD use (ATC-code N05A, with exclusion of lithium). The data in the database are collected from 77 countries participating in the WHO Program for International Drug Monitoring. This database comprises more than 3.5 million case reports, to which around 50,000 new reports are added quarterly. The relationship between the APD and hypothermia is evaluated by calculating the Reporting Odds Ratios (RORs) and 95% confidence intervals (95% CI) in a case\/non-case design. The ROR compares the frequency of the reported ADR for a certain drug with the frequency of reports of that adverse drug reaction for all other drugs in the database. Reports concerning hypothermia were considered as cases, all other reports as non-cases. Index reports included all reports on an APD (ATC code beginning with N05A, with exclusion of lithium), all other reports were controls. When the number of reports of hypothermia in association with the APD is high and the number of reports of hypothermia in association with other drugs is low, ROR will be high. This also happens when the number of reports of other ADRs in association with the APD is low and the number of reports of other ADRs in association with other drugs is low. Since the vast majority of cases in the WHO database do not contain any details on indication of drug use, start and\/or end dates and outcome, these factors cannot be analyzed with data from the WHO database. To get more information regarding characteristics of patients developing hypothermia during APD use, we performed a literature search in Medline and Embase for case reports with search terms \u201c(antipsychotic OR neuroleptic) AND (hypothermia OR body temperature regulation), with no selection on date or language. From these articles, we searched the references for missing articles. Two reviewers judged all case reports. All relevant case reports were studied for patient, drug and environmental characteristics.\nResults\nIn the WHO database, in January 2007, 480 reports were registered of patients developing hypothermia during the use of APDs. Characteristics of these reports are presented in Table\u00a01. In the same period, 524 reports of hyperthermia associated with antipsychotic drug use were registered. Based on the reports, no specific pharmacological subgroup can be associated with an increased risk for hypothermia. Atypical antipsychotics are responsible for 55% of the reports, but this is mainly attributable to risperidone. Risperidone alone was responsible for 27% of all reports. A remarkable high association is found for pipamperone (ROR:24.62; 95% CI 13.16\u201346.07), an antipsychotic drug mainly used in Europe.\nTable 1Antipsychotic drugs and hypothermia: reports from the WHO databaseAntipsychotic drugNumber of reports for hypothermiaNumber of reports for any ADR with this drugReported OR (CI)a for ADRs with \u22653 reportsTioxanthenes Zuclopenthixol13109415.88 (9.18\u201327.47) Flupenthixol616774.73 (2.12\u201310.55) Chlorprothixen450210.58 (3.95\u201328.31) Tiotixene39044.38 (1.41\u201313.62) Clopenthixol2293-Phenothiazines Thioridazine2344366.90 (4.57\u201310.41) Chlorpromazine1661823.43 (2.09\u20135.60) Levomepromazine1123486.21 (3.43\u201311.24) Cyamemazine911979.99 (5.18\u201319.27) Periciazine843025,00 (12.41\u201350.36) Pipothiazine319720.36 (6.50\u201363.71) Fluphenazine326561.49 (0.48\u20134.62) Trifluoperazine318952.09 (0.67\u20136.48) Perphenazine21522- Prochlorperazine24451- Promazine2307- Mesoridazine1215-Butyrofenones Haloperidol32105436.21 (3.43\u201311.24) Pipamperone1054624.62 (13.16\u201346.07) Droperidol21178- Benperidol1146-Benzamides Tiapride559611.14 (4.62\u201326.89) Sulpiride418282.89 (1.08\u20137.70) Amisulpiride11514- Sultopride172-Others Loxapine49285.70 (2.13\u201315.23) Pimozide36286.31 (2.03\u201319.65) Zotepine2260- Prothipendyl193- Penfluridol158-Atypical Risperidone129184319.65 (8.09\u201311.52) Clozapine68442552.05 (1.61\u20132.61) Olanzapine44160903.65 (2.71\u20134.91) Quetiapine2153745.19 (3.38\u20137.98) Aripiprazole1145663.18 (1.76\u20135.76) Ziprasidone829633.57 (1.78\u20137.15)aROR= (a\/c) \/ (b\/d); (a = no. of reports of adverse drug reaction with suspected drug; b = no. of reports of adverse drug reaction in total database; c = no. of reports regarding the suspected drug in database; d = total no. of reports in database). ROR is only calculated for APDs with three or more reports\nThe literature search resulted in 32 articles containing 43 case reports (December 2006) [3\u201334] from which characteristics are summarized in Table\u00a02. Hypothermia following antipsychotic drug use is not associated with a specific age group. Reported ages vary from 0 to 90\u00a0years. In most cases, hypothermia is detected shortly following the start or dose increase of an antipsychotic drug. Most patients suffered from schizophrenia.\nTable\u00a02Characteristics of cases with hypothermia following antipsychotic drug use in literature (43 case reports, 46 episodes)CharacteristicsDataMale41%Age: mean (SD)49 (23.0)Range 0\u201390\u00a0yearsReported body temperature: mean (SD)32,6\u00b0C (2.7)Range 20.0\u201336.1\u00b0CDiagnosis known (n\u2009=\u200935)Schizophrenia51%Mental retardation11%\u00a0Bipolar disorder11%Dementia11%Drug change Start or dose increase80%\u00a0No change16%Interval drug change detection hypothermia\u00a0<2\u00a0days57%\u00a02\u20137\u00a0days16%Outcome death4%\u00a0ICU admission24%Hospitalization (incl. prolonged)69%\nDiscussion\nHypothermia in patients using an APD is a serious, unpredictable, type B adverse event frequently leading to hospital and ICU admission and sometimes even to death. Some authors have even suggested that a substantial proportion of unexplained deaths should be attributed to antipsychotic-induced hypothermia [3, 35]. No single sufficient cause for hypothermia can be found in case reports. First, drug-receptor profile may play a role. Serotonin is associated with thermoregulation and APDs with a stronger affinity for the 5-HT2a receptor than for the D2-receptor (pipamperone, the atypical APDs) seem to be associated with hypothermia. The high association for relatively new drugs, like the atypical APDs, can partially be explained by reporting bias (reporting incidence for adverse drug effects is higher for new drugs and tends to decline in time; the so-called Weber-effect), but the high number of reports for risperidone should keep clinicians alert. Blocking alpha2-adrenergic receptors (e.g., chloropromazine, risperidone, clozapine, thioridazine) may also increase the hypothermic effect, by inhibiting peripheral responses to cooling (vasoconstriction and shivering). Next to this receptor profile, many patient-bound factors must be considered. Patients with pre-existing brain damage may be more susceptible to hypothermic effects. The pre-optic anterior hypothalamic region regulates body temperature. Animal studies show that lesions of this region give a hypothermic response following administration of an APD [36, 37]. In patients with multiple sclerosis, hypothermia is also associated with thalamic lesions [38]. Studies in schizophrenic patients show that core temperature decreases following the administration of APDs [39, 40]. Our search shows a predominance of case reports for schizophrenic patients and little for other frequent APD user groups like demented or delirious elderly. In schizophrenia, thermal regulation is altered. This may be explained by changes in neurotensine levels in schizophrenia. Neurotensine (NT) is one of the most important thermoregulatory peptides that also plays a role in the antipsychotic actions of APDs. In schizophrenic patients, NT concentration in cerebro-spinal fluid (CSF) is low and will be normalized following antipsychotic drug use [41]. The hypothermic reaction is also dependent on ambient temperature. In animal studies, APD administration at ambient temperatures below 22\u00b0C led to hypothermia, whereas APD administration in a room temperature of 29\u00b0C gave no thermal response and at 32\u00b0C an increase in rectal temperature [41, 42]. Normally, a cold environment will result in behavior aimed at protection against the cold (taking extra blankets or clothes). APDs, however, will induce apathy and indifference, resulting in unawareness of developing hypothermia. Since some case reports also mention the co-existence of infections at the time of development of hypothermia, this may also play a role in the dysregulation of thermal homeostasis.\nThere is an ongoing discussion concerning the value of case reports and spontaneous reports in the field of drug safety. There can be no discussion that pharmacodiligence requires prospective studies, access to regulatory filings for controlled and monitored use of drugs, and some sense of relative potency for comparison between different drug entities. However, large cohorts are needed for the detection and analysis of type B reactions. Since these data are lacking, case and spontaneous reports by alert clinicians must be analyzed [43]. The outcome of such an analysis cannot be seen as solid evidence, but can help to get more insight in the adverse drug reaction (ADR). The use of measures like ROR and disproportionality can help to detect drugs with an increased association with an ADR. A statistically significant ROR may be indicative of a higher risk for that particular event during the use of a specific medication, but is never conclusive for the actual existence of a causal relation [2].\nThe results of this study should alert physicians of the risk of hypothermia in psychiatric patients using APDs. There seems to be no direct relation between stable drug dose and the ADR; the period shortly after starting the APD or dose increase seems to be the high risk period. Often, drug changes are indicated by behavioral problems also leading to separation or isolation of the patient. In the case of separation, patients will be dressed lightly and, even at normal room temperature, can cool down easily. In these cases, the patient\u2019s body temperature should be monitored daily (with a thermometer that can measure low body temperatures). Also, every change in behaviour or co-morbidity (e.g., infections) should be a warning sign to look for hypothermia.","keyphrases":["hypothermia","thermoregulation","schizophrenia","adverse drug effect","antipsychotic agents"],"prmu":["P","P","P","P","M"]}
{"id":"Virchows_Arch-3-1-1888718","title":"Frequency and diagnostic patterns of lymphomas in liver biopsies with respect to the WHO classification\n","text":"The recent World Health Organization (WHO) classification of hematopoietic and lymphoid tissue tumors represents the first worldwide consensus classification of these malignancies. However, the applicability of this classification to a representative number of hepatic lymphomas in liver biopsy specimens has not yet been investigated. The frequency and infiltration pattern of a series of 205 liver biopsies with lymphoma manifestations was analyzed with the aid of immunohistochemical and molecular pathological analyses. Diffuse large B-cell lymphoma (DLBCL) was by far the most frequent entity, comprising 45% of the cases analyzed. Using a previously published immunohistochemical algorithm, 35% of 80 DLBCL were assigned to a germinal center B-cell-like (GCB) and 65% to a non-GCB group. Most B-cell lymphoma entities involving the liver revealed a characteristic infiltration pattern. Diagnostically challenging entities were T-cell-rich B-cell lymphomas, anaplastic large cell lymphomas and peripheral T-cell lymphomas, which frequently required additional molecular clonality assessment. Overall, the percentage of T-cell lymphomas in the liver (12%) was higher as compared to other extranodal sites except for the skin and the small intestine. This study provides relevant data on the distribution of hepatic lymphomas and demonstrates the applicability of the WHO classification proposing a diagnostic algorithm for liver biopsies.\nIntroduction\nThe liver is most commonly involved in non-Hodgkin lymphomas (NHL) next to lymph nodes, spleen, and bone marrow. In the vast majority of cases hepatic involvement reflects secondary dissemination in advanced disease [15, 18] rather than a primary site according to the definition of Caccamo and coworkers [5]. Histopathological analysis of liver biopsy may be required in patients with an established diagnosis of malignant lymphoma to differentiate lymphoma manifestation from other causes of hepatic dysfunction or to clarify elevated transaminases such as toxic damage because of chemotherapy or other medications. On the other hand, a biopsy may reveal a previously unknown lymphoma during the exploration of a solid hepatic mass or after measuring elevated liver enzyme serology [12]. Occasionally, a malignant lymphoma can be detectable in the setting of other liver diseases such as chronic hepatitis B [20, 22], chronic hepatitis C [2, 3, 7, 21, 25], or primary biliary cirrhosis (PBC) [24].\nThe goals of the present study were to test the feasibility of lymphoma subtyping according to the World Health Organisation (WHO) classification of tumors of hematopoietic and lymphoid tissues [16] in liver biopsy specimens and to describe the frequencies by which the different entities occur. For this purpose, a series of 205 liver biopsies with a diagnosis of malignant lymphoma were analyzed retrospectively with a specific focus on the histopathology, especially infiltration patterns, which may ultimately allow the use of diagnostic algorithms for subtyping of lymphomas in liver biopsies. This is an important addition in relation to previous studies, which have mainly described the frequency of liver involvement in autopsy material of patients with leukemia or lymphoma [8, 13, 26, 28, 29] differentiating only between low-grade and high-grade NHL [26] or having performed a categorization according to the outdated Kiel classification [29].\nOverall, this study represents the largest series on hepatic lymphomas to date and due to its restriction to bioptic material reflects the primary diagnostic situation.\nMaterials and methods\nAll cases of hepatic lymphoma involvement diagnosed by liver biopsy during the years 1994\u20132003 were retrieved from the archives of the Institute of Pathology, Campus Benjamin Franklin, Charit\u00e9-University Medicine Berlin and the Institute of Pathology, University of Cologne, Germany. The series comprised a total of 205 cases with 135 cases from Berlin and 70 cases from Cologne. All cases were reevaluated and reclassified independently by three pathologists (C.L., T.L., H.S.) according to the WHO classification [16]. For cases in which the diagnosis was not unanimous, a final consensus diagnosis was reached after further immunophenotyping, molecular analyses, and final consultation. In six cases initially considered \u201csuspicious\u201d for lymphoma, a final diagnosis of lymphoma was made after reevaluation and additional analyses. Nine liver biopsies with insufficient material for additional immunohistochemical or molecular analyses were excluded beforehand, as well as five cases where T-cell receptor (TCR) polymerase chain reaction (PCR) had revealed a polyclonal or oligoclonal rearrangement pattern without a reproducible dominant PCR product.\nOverall, 32\/205 (16%) cases were reclassified, including eight \u201clow-grade\u201d [5\u00d7 B-CLL, 2\u00d7 follicular lymphomas (FL), 1\u00d7 marginal zone lymphoma (MZL)] and 20 \u201chigh-grade\u201d [19\u00d7 DLBCL, 1\u00d7 Burkitt lymphoma (BL)] B-cell NHL that were assigned to a specific WHO lymphoma category as well as four cases with a change in the final diagnosis [BL, classical Hodgkin lymphoma (cHL), T-cell-rich B-cell lymphoma (TCRBCL), lymphoplasmacytic lymphoma (LPL) to DLBCL, anaplastic large cell lymphoma (ALCL), peripheral T-cell lymphoma (pTCL) and B-CLL, respectively].\nThe analyses of the infiltration pattern were based on the assessment of the hepatic architecture. Three main patterns were distinguished: Infiltrates for which an exclusive or predominant association to portal tracts was evident were recorded as portal infiltrates. The second pattern consisted of lymphoma infiltrates which showed a coherent growth pattern thus predominantly resulting in the replacement of the acinar structures. These infiltrates were designated as a nodular growth pattern. Finally, infiltrates that showed prominent intrasinusoidal dispersion of the lymphoma cells were recorded as a sinusoidal growth pattern. Additionally, the density of the infiltrate was semiquantitatively assessed. The presence of scattered neoplastic cells in a background rich in reactive bystander cells (e.g., nonneoplastic T-cells and\/or macrophages in T-cell rich B-cell lymphoma) was defined as a loose infiltration pattern, whereas the appearance of coherently appearing neoplastic B- or T-cells was referred to as a dense infiltrate.\nAccording to clinical information available, in 76\/205 (37%) of the cases the diagnosis of a lymphoma had been previously established from extrahepatic biopsies or peripheral blood indicating secondary involvement of the liver. In four of these cases with known low-grade lymphoma (2\u00d7 B-CLL, 2\u00d7 FL) liver biopsy revealed transformation to a high-grade lymphoma (DLBCL). In the remaining cases, the biopsy obtained from the liver represented the site of primary diagnosis. In 26 (13%) patients, additional biopsies from extrahepatic sites were available (13 bone marrow biopsies, 10 lymph node biopsies, and three spleen biopsies). Furthermore, in a few cases clinical data regarding a potential predisposing condition for a primary hepatic lymphoma was available: chronic hepatitis C was reported in four patients (3\u00d7 DLBCL, 1\u00d7 B-CLL), HIV infection was present in three patients (2\u00d7 DLBCL, 1\u00d7 BL), and one marginal zone lymphoma occurred in a patient after liver transplantation.\nThe frequency of the various lymphoma entities diagnosed in liver biopsy specimen was compared to their frequency and distribution in other extranodal sites (cases from the Consultation and Reference Center for Lymph Node Pathology and Hematopathology, Berlin).\nImmunohistochemistry\nFor immunostaining, 4\u00a0\u03bcm thick sections were cut, deparaffinized, and subjected to heat-induced epitope retrieval before incubation with antibodies. For this purpose, sections were immersed either in sodium citrate buffer at pH\u00a06.0 or, alternatively, in ethylenediamintetraacetic acid (EDTA) at pH\u00a08.0 and heated in a high-pressure cooker. After cooking, the slides were rinsed in running water, washed with Tris-buffered saline, pH\u00a07.4, and incubated with the respective primary antibodies. All primary antibodies employed are listed in Table\u00a01. With the exception of immunoglobulin detection, in which the streptavidin\u2013biotin peroxidase method was applied [14], labeling was carried out using the alkaline\u2013phosphatase\/anti-alkaline\u2013phosphatase complex method [6]. Alkaline phosphatase was developed using Fast Red as the chromogen, whereas peroxidase was visualized with diaminobenzidine chromogen as the substrate. \nTable\u00a01Antibodies used in this studyAntibodyCloneAntigen retrievalDilutionSourceALK1ALK1Citrate1:20DakoBCL2124Citrate1:25DakoBCL6594Citrate1:25DakoCD2AB75Citrate1:50NovocastraCD3F7.2.38Citrate1:100DakoCD41F6Citrate1:25NovocastraCD54C7Citrate1:25NovocastraCD7CD7-272EDTA1:50NovocastraCD8C8\/144BCitrate1:100DakoCD1056C6Citrate1:25NovocastraCD15C3D1Citrate1:20DakoCD20L26Citrate1:50DakoCD211F8Protease1:50DakoCD231B12Citrate1:20NovocastraCD27137B4EDTA1:100NovocastraCD30BerH2Citrate1:50DakoCD43DF-T1Citrate1:50DakoCD68PG-M1Citrate1:50DakoCD79aJCB117Citrate1:100DakoCD138B-B4Citrate1:10SerotecCyclin D1P2D11F11Citrate1:50NovocastraEBV-LMPCS1-4Citrate1:100Dako\u03b1-heavychainRabbit polyclonalCitrate1:40 000Dako\u03b3-heavy chainRabbit polyclonalCitrate1:30 000Dako\u03b4-heavy chainRabbit polyclonalCitrate1:2000Dako\u03bc-heavy chainRabbit polyclonalCitrate1:2000DakoKi-67MIB-1Citrate1:2000Dako\u03ba-light chainRabbit polyclonalCitrate1:100 000Dako\u03bb-light chainHP6054Citrate1:16 000DakoMUM1\/IRF4MUM1pCitrate1:20Generously provided by Prof. B. Falini, Perugia, ItalyPax-524Citrate1:10Transduction LaboratoriesTIA-12G9Citrate1:500Coulter\nMolecular pathology analyses\nAdditional molecular pathology analyses were selectively performed for those cases in which immunoprofiling alone was not sufficient to establish a definite diagnosis of a malignant lymphoma.\nFor this purpose, DNA was extracted after dewaxing 20-\u03bcm-thick paraffin sections with QIAEX (Qiagen, Hilden, Germany) according to the manufacturer\u2019s recommendations. TCR-gamma rearrangements were analyzed using four different primer combinations (JGT1\/2, JGT3, BioMed-2 Set A and Set B). The primer combinations JGT1\/2 und JGT3 suitable to amplify the most frequent TCR-gamma rearrangements were used in a two-step seminested PCR. The BioMed-2 primer sets A and B able to detect all possible TCR-gamma rearrangements were performed as single round PCRs. The detailed cycling conditions (50 rounds of amplification for BioMed-2 set A and set B; two-time 35 cycles for primer combinations JGT1\/2 and JGT3) for all PCRs have been described in detail elsewhere [9, 27].\nAmplification of rearranged IgH genes was independently performed at least twice per case employing three different framework primer sets (BioMed-2 FR1, FR2, and FR3) separately in conjunction with a JH primer (JH22). PCR conditions consisted of 50 cycles of denaturation (95\u00b0C, 15\u00a0s), primer annealing (60\u00b0C, 40\u00a0s), and elongation (72\u00b0C, 45\u00a0s), and the reaction mixture contained 1.5\u00a0mM MgCl2, 0.8\u00a0mM deoxyribonucleotide triphosphates (dNTPs), 70\u00a0pmol VH primers, 30\u00a0pmol JH22 primer, and 2\u00a0U of AmpliTaq Gold polymerase (Applied Biosystems, Weiterstadt, Germany).\nResults\nCharacteristics of hepatic lymphomas\nAlmost 90% (182\/205) of the hepatic lymphomas belonged to the group of NHL, whereas only 23 of the 205 cases were cHL. Regarding NHL, 86% (157\/182) were of B-cell and 14% (25\/182) of T-cell origin. Overall, diffuse large B-cell lymphoma (DLBCL) was the most common type [51% (93\/182) of all B-NHL analyzed] with TCRBCL\u2014a morphologic variant of DLBCL\u2014comprising 14% (13\/93) of all DLBCL. According to the algorithm of Hans et al. [11] a predominance of DLBCL of non-GCB (germinal center B-cell-like) type (52\/80; 65%) compared to the DLBCL of GCB type (28\/80; 35%) was found (Fig.\u00a01a\u2013d,f). Only 4 out of 80 cases (5%) corresponded to the group of CD5-positive DLBCL (Fig.\u00a01e).\nFig.\u00a01Nodular pattern: DLBCL of GCB group positive for CD10 (a) and BCL6 (b) and negative for IRF4\/MUM1 (few scattered plasma cells serve as positive intrinsic control; c). CD5 positive DLBCL of ABC type negative for CD10 (positive bile canaliculi serve as intrinsic control; d), positive for CD5 (e) and IRF4\/MUM1 (f). TCRBCL with less than 10% large CD20 positive neoplastic B-cells (g) associated with numerous CD3 positive T-cells (h) and CD68 positive histiocytes (i)\nIn all lymphoma subtypes, men were affected more often, except for FL, which occurred with equal frequencies in both sexes. The male predominance was most striking in lymphomas of T-cell origin, in which ALCL and hepatosplenic T-cell lymphoma (HSTCL) were encountered exclusively in men. Additional molecular analyses were essential to confirm the suspected diagnosis of a lymphoma in 5 out of 19 T-cell lymphomas (26%) and 3 out of 116 (3%) B-NHLs (Fig.\u00a02).\nFig.\u00a02Size fragment analyses (GeneScan) of the amplificates after TCR-gamma PCR (primer combination JGT1\/2). Fluorescence-labelled PCR products (blue lines) were separated on capillary electrophoresis system (Applied Biosystems, Model 310A) in parallel to a size standard (red line). The dominant PCR product (arrow) indicating the presence of a clonal T-cell population was reproducibly detectable in addition to a moderate oligo-\/polyclonal T-cell background\nThe relative frequencies of the lymphoma subtypes, the available patient data and the infiltration patterns are summarized in Table\u00a02. \nTable\u00a02Frequency, age distribution and pattern of infiltration in the different lymphoma entities presenting in the liver (Berlin\/Cologne 1994\u20132003)\u00a0BerlinCologneBerlin + Cologne, n (%)Age (range)Age (mean)Male\/femalePatternDensity of infiltrateSinusoidalPortalNodularDenseLooseDiffuse large B-cell lymphoma (DLBCL)a522880(39)16\u2013876450\/3061972755T-cell rich B-cell lymphoma (TCRBCL)9413(6)35\u201385599\/40115013B-cell chronic lymphocytic leukemia (B-CLL)131326(13)44\u2013826616\/107252251Classical Hodgkin lymphoma (cHL)101323(11)21\u2013915414\/912110320Follicular lymphoma (FL)11314(7)38\u201383627\/70114140Marginal zone lymphoma (MZL)617(3)49\u201380706\/127061Leukemic Plasmacytoma (PL)426(3)61\u201388704\/241251Burkitt lymphoma (BL)5\u20135(2)30\u201370473\/200550Mantle cell lymphoma (MCL)3\u20133(1)60\u201366632\/113130B-lymphoblastic leukaemia (B-ALL)2\u20132(1)18\u201328221\/120020Hairy cell leukaemia (HCL)1\u2013142\u20130\/110010Peripheral T-cell lymphoma, unspecified (pTCL)13518(9)38\u2013846214\/4668117Anaplastic large cell lymphoma (ALCL)b5\u20135(2)24\u201376585\/033223Hepatosplenic T-cell lymphoma (HSTCL)112(1)38\u201351452\/020020Total13570205aIncluding centroblastic (n\u2009=\u200969), immunoblastic (n\u2009=\u20093) and anaplastic variant (n\u2009=\u20098)bIncluding one anaplastic large cell lymphoma kinase (ALK) protein positive case\nInfiltration patterns of the various lymphoma types\nMost of the cases showed the predominance of a certain growth pattern, although some cases exhibited more than one growth pattern. Almost all DLBCL and BL showed a nodular infiltration pattern (91%) composed of a dense infiltrate (94%; Table\u00a03), except for the TCRBCL variant, which was located predominantly within the portal tracts (85%) and exhibited the characteristic scattered infiltrate of fewer than 10% large neoplastic B-cells admixed with many reactive T-cells and histiocytes (Fig.\u00a01g\u2013i). Lymphomas showing a dense portal infiltration pattern were chronic lymphocytic leukemia\/small lymphocytic lymphoma (96%; Fig.\u00a03a\u2013c), FL (79%; Fig.\u00a03d\u2013f), MZL (100%; Fig.\u00a03g\u2013i), and mantle cell lymphoma (100%). In contrast, in precursor B lymphoblastic leukemia\/lymphoblastic lymphoma (B-ALL\/B-LBL; Fig.\u00a04a\u2013c), HSTCL (Fig.\u00a04d\u2013f), leukemic plasmacytoma (Fig.\u00a04g\u2013i), and hairy cell leukemia, a sinusoidal infiltration pattern was observed in the majority of cases. The cHL displayed a predominantly portal infiltration pattern (Fig.\u00a05a\u2013c). Peripheral T-cell lymphoma and ALCL showed a spectrum of portal, sinusoidal, nodular, or mixed patterns with a varying density of infiltration, and thus did not allow for pattern-based selection of additional analyses (Fig.\u00a05d\u2013f). The infiltration patterns, differential diagnoses, and discriminative immunohistochemical markers are summarized in Table\u00a03. \nTable\u00a03Infiltration pattern, differential diagnosis and characteristic immunohistochemical markersFig.\u00a03Portal pattern: chronic lymphocytic leukemia (B-CLL) with a predominantly portal infiltrate of small lymphocytes (a) with expression of CD23 (b) and CD5 (c). FL (d) with expression of CD10 (e) and BCL2 (f). Marginal zone B-cell lymphoma with portal involvement (g), expression of memory B-cell marker CD27 (h) and a lymphoepithelial lesion of a CK7 positive bile duct (i)Fig.\u00a04Sinusoidal pattern: precursor B lymphoblastic lymphoma (B-LBL) with a sinusoidal infiltrate of small blasts (a) with cytoplasmic expression of CD79a (b) and nuclear expression of terminal deoxynucleotidyl transferase (TdT; c). HSTCL with a sinusoidal infiltrate of monotonous neoplastic cells (d) with expression of CD3 (e) and the cytotoxic granule associated protein TIA-1 (f). Peripheral blood involvement (plasma cell leukemia) in a plasmacytoma with a sinusoidal infiltrate of plasma cells (g) with expression of CD138 (h) and IgG (i)Fig.\u00a05Classical Hodgkin lymphoma vs. anaplastic large cell lymphoma: cHL with portal infiltrates of Hodgkin and Reed-Sternberg HRS cells (arrows) in a background rich in eosinophils (a). The HRS cells strongly express CD30 (b) and the EBV encoded latent membrane protein 1 (LMP1) (c). Anaplastic large cell lymphoma (ALCL) with portal infiltrates of pleomorphic large cells resembling HRS cells (arrows) (d) and strong positivity for CD30 (e) and the cytotoxic molecule perforin (f)\nComparison of hepatic lymphomas with other extranodal lymphomas\nThe percentage of T-cell lymphomas was considerably higher in the liver (12%, 25\/205) when compared to other extranodal sites (5%, 165\/3252) except for the skin (50%, mainly mycosis fungoides\/S\u00e9zary syndrome) and small intestine (34%, mainly enteropathy-type T-cell lymphomas).\nThe high proportion of DLBCL in lymphomas of the liver (45%) was only surpassed by the small intestine (64%), the brain (83%), and the testis (97%). Of note, TCRBCL was extremely rare in other extranodal sites (0,3%) with only a few cases occurring in the bone marrow and spleen. Interestingly, one case fulfilled the criteria of TCRBCL in the liver, and an additional lymph node biopsy showed the picture of a typical DLBCL, whereas in another case, both liver and spleen biopsy showed the characteristic features of TCRBCL. The frequencies of other extranodal lymphomas compared to hepatic lymphomas are summarized in Table\u00a04. \nTable\u00a04Frequency of extranodal lymphoma at different sites (Berlin 1994\u20132003)\u00a0Brain (n\u2009=\u200931)Testis (n\u2009=\u200961)Lung (n\u2009=\u200979)Bone marrow (n\u2009=\u20091996)Stomach (n\u2009=\u2009855)Small intestine (n\u2009=\u200971)Large intestine (n\u2009=\u2009102)Spleen (n\u2009=\u2009128)Skin (n\u2009=\u2009427)Liver (n\u2009=\u2009135)DLBCL255722662383032217852TCRBCL\u2013\u2013\u20137\u2013\u2013\u20133\u20139FL1\u201321681633209111B-CLL1\u2013265612\u2013410713PL3\u2013\u2013473\u2013\u2013\u2013124MZL\u2013\u2013401757741218*126MCL\u2013\u2013310777411963LPL\u2013\u20133122\u2013\u20132135\u2013HCL\u2013\u2013\u2013112\u2013\u2013\u20136\u20131B-ALL\u20131\u201378\u2013\u2013\u20131\u20132BL\u20131\u20134\u201332\u201325cHL\u2013\u2013352\u2013\u2013\u201331010B-NHL total (%)30\/31 (97%)59\/61 (97%)75\/79 (95%)1862\/1996 (93%)850\/855 (99%)47\/71 (66%)96\/102 (94%)115\/128 (90%)213\/427 (50%)116\/135 (86%)pTCL1\u2013393466129413NK\/T\u20131\u2013\u2013\u20132\u2013\u20132\u2013T-ALL\u20131\u201329\u2013\u2013\u2013\u20139\u2013ALCL\u2013\u20131816\u2013\u2013265Enteropathy-type TCL\u2013\u2013\u2013\u2013\u201310\u2013\u2013\u2013\u2013HSTCL\u2013\u2013\u20135\u2013\u2013\u20131\u20131MF\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u201383\u2013T-NHL total (%)1\/31 (3%)2\/61 (3%)4\/79 (5%)134\/1996 (7%)5\/855 (1%)24\/71 (34%)6\/102 (6%)13\/128 (10%)214\/427 (50%)19\/135 (14%)*Including nine cases of splenic marginal zone lymphoma (SMZL)\nDiscussion\nTo the best of our knowledge, this study represents the largest series focusing on the histopathology of hepatic lymphoma involvement and the first application of the WHO classification of tumors of hematopoietic and lymphoid tissues for this purpose [16]. Our data demonstrate the feasibility of in vivo lymphoma subtyping in liver biopsy with the aid of infiltration pattern analysis, the selection of differentiating immunophenotypic markers and additional molecular methods.\nMany B-cell-derived lymphomas involving the liver revealed a characteristic infiltration pattern, which facilitated the use of a restricted panel of immunohistochemical markers to reach a final diagnosis. This approach is critical in those liver biopsies in which a lymphoma diagnosis has previously not been established, as accurate subtyping of lymphomas is fundamental for the initiation of adequate treatment.\nDiffuse large B-cell lymphomas (DLBCL) accounted for 51% of all B-NHL cases. DLBCL was recently subdivided into different prognostic groups designated GCB, activated B-cell-like (ABC or non-GCB) and Type 3, according to their gene expression profiles [1]. The GCB group is described to have a better survival than the ABC group and the heterogeneous Type 3 group. Hans et al. [11] have proposed a simple immunohistochemical algorithm to assign cases to a GCB and a non-GCB group. In the present study, the frequency of either group in 80 liver biopsies with DLBCL was investigated, excluding the T-cell\/histiocyte rich variant (n\u2009=\u200913). DLBCL of non-GCB type (65%) were more frequent in the liver than the GCB type (35%), whereas these two groups are described in other sites at about equal frequency [23]. As liver involvement with malignant lymphomas occurs secondary in advanced disease [15, 18], this observation may reflect the biological behavior of ABC-group DLBCLs through the selection of more aggressive cases by liver biopsy.\nApproximately 10% of the de novo DLBCL express CD5 [4] and may have a poor outcome [31]. They tend to be associated with extranodal sites, especially bone marrow and spleen [19]. In the present series of hepatic lymphoma involvement, the number of CD5 positive DLBCL (4\/80 or 5%) was not increased.\nThe TCRBCL, a variant of DLBCL in which the majority of cells are nonneoplastic T-cells and histiocytes, are often misdiagnosed as a reactive inflammatory condition or as a T-cell lymphoma infiltration of the liver [8, 17]. TCRBCL is relatively rare in lymph node biopsies [(23\/976) or 2% of the DLBCL in the Berlin Reference Centre for Lymph Node Pathology from 1994\u20132003]. However, in the present study, 13\/93 (14%) of the DLBCL in the liver were found to fulfill the WHO criteria for TCRBCL thus confirming previous data from Dargent and coworkers in their series of 62 liver specimens [8]. Additionally, one case with TCRBCL characteristics in the liver showed the typical morphology of a conventional DLBCL in an additional lymph node biopsy. This finding indicates that the composition of the reactive coinfiltrate may be influenced by the tissue-specific microenvironment. Therefore, the specific environment of a certain organ may influence the intensity of the T-cell response and subsequently explain the higher frequency of the TCRBCL variant of DLBLC presenting in the liver [10]. Limitations were encountered in the grading of follicular lymphoma and the subtyping of the cHL, which is generally not possible with sufficient certainty in needle biopsies.\nIn contrast to B-cell lymphomas, T-cell lymphomas infiltrating the liver generally lack a typical infiltration pattern, making their diagnosis more challenging. In particular, ALCL may mimic cHL due to the possible resemblance of Hodgkin and Reed-Sternberg cells and expression of CD30. Thus, additional markers against pan-T and pan-B cell antigens (including pax-5), CD15, ALK-1, cytotoxic molecules, e.g., TIA-1, granzyme B, and perforin) are required when confronted with this differential diagnosis (Fig.\u00a05).\nSimilarly, the distinction between a T-cell lymphoma and a drug-induced or viral hepatitis (e.g., due to Epstein-Barr virus) can be very difficult in biopsies containing an increased number of sinusoidal T-cells. Therefore, additional clonality analyses by TCR or IgH PCR were necessary in 5 out of 19 (26%) T-cell lymphomas compared to only 3 out of 116 (3%) B-cell lymphomas to establish a diagnosis.\nThe reason for the relatively high frequency of liver involvement by T-cell lymphomas is not known. As T-cells play an important role in the regulation of the hepatic immune responses, especially in chronic viral and autoimmune hepatitis [30], it is tempting to speculate whether this may influence the relatively high incidence of T-cell lymphomas in the liver compared to other extranodal sites. In the present study, 25 of the 205 (12%) cases were found to represent lymphomas derived from T-cells, whereas lymphomas of the testis, brain, lung, and stomach were B-cell lymphomas in nearly all instances (Table 4).\nIn summary, the present study demonstrates the feasibility of subtyping lymphoma infiltrates in liver biopsies according to the WHO classification. The large number of 205 cases provides reliable information regarding the relative frequencies of the different lymphoma entities encountered in the liver and demonstrates the usefulness of infiltration pattern analysis for diagnostic purposes.","keyphrases":["lymphoma","liver","pcr","immunohistochemistry","differential diagnosis"],"prmu":["P","P","P","P","P"]}
{"id":"Int_Urogynecol_J_Pelvic_Floor_Dysfunct-3-1-2062491","title":"Is there an association between depressive and urinary symptoms during and after pregnancy?\n","text":"Depressive symptoms and urinary symptoms are both highly prevalent in pregnancy. In the general population, an association is reported between urinary symptoms and depressive symptoms. The association of depressive and urinary symptoms has not yet been assessed in pregnancy. In this study, we assessed (1) the prevalence of depressive symptoms, over-active bladder (OAB) syndrome, urge urinary incontinence (UUI) and stress urinary incontinence (SUI) during and after pregnancy using the Center for Epidemiologic Studies Depression Scale (CES-D) and the Urogenital Distress Inventory (UDI) and (2) the association of depressive symptoms with urinary incontinence and over-active bladder syndrome during and after pregnancy, controlling for confounding socioeconomic, psychosocial, behavioural and biomedical factors in a cohort of healthy nulliparous women. Our data show a significant increase in prevalence of depressive symptoms, UUI, SUI and OAB during pregnancy and a significant reduction in prevalence of depressive symptoms, SUI and OAB after childbirth. UUI prevalence did not significantly decrease after childbirth. In univariate analysis, urinary incontinence and the OAB syndrome were significantly associated with a CES-D score indicative of a possible clinical depression at 36 weeks gestation. However, after adjusting for possible confounding factors, only the OAB syndrome remained significantly associated (OR 4.4 [1.8\u201310.5]). No association was found between depressive and urinary symptoms at 1 year post-partum. Only OAB was independently associated with depressive symptoms during pregnancy. Possible explanations for this association are discussed.\nIntroduction\nThe lifetime risk of depressive symptoms in women (5.9\u201321.3%) is about twice that in men and symptoms often start in the childbearing years [1\u20133]. Prevalence rates of 10\u201325% during pregnancy [4\u20139] and of 6\u201316% post-partum are reported [5, 6, 8, 10\u201312]. Prevalence rates vary because of the diversity of populations and diagnostic methodology. Most researchers found higher rates during pregnancy than after childbirth [5, 6, 8]. Urogenital symptoms are also more prevalent in pregnancy than in the general population. Prevalence rates of 9\u201350% have been reported for urinary incontinence (UI) during first pregnancy and 34\u201395% for frequency and urgency symptoms (over-active bladder [OAB] syndrome) [13, 14]. Strong associations are found between depressive symptoms, urinary incontinence and over-active bladder syndrome in non-pregnant women [15\u201319]. Whilst the relationship of depressive symptoms and urinary symptoms has not yet been explored in pregnancy, several factors have been found to be associated with depressive symptoms in a general, pregnant and post-partum population. These factors include biomedical factors such as obesity, age, chronic pain (like back pain), previous depressive symptoms [12, 20, 21], behavioural factors like excessive use of alcohol, smoking, lack of leisure time physical exercise and socioeconomic factors like unemployment and low job satisfaction [5, 7, 22]. Psychosocial factors, such as poorer social support, stressful life events and personality features have also been found related to depressive symptoms [11, 22\u201325].\nThe aim of this study was to (1) analyse the prevalence of depressive symptoms and urinary symptoms during and after pregnancy and (2) assess the association of urinary symptoms with depressive symptoms, controlling for psychosocial, behavioural, socioeconomic and biomedical factors during and after pregnancy.\nMaterials and methods\nStudy population\nBetween January 2002 and July 2003, 1,366 nulliparous pregnant women from 10 urban midwifery practices in the center of The Netherlands were approached to take part in a prospective longitudinal cohort study assessing pelvic floor problems, sexuality and back pain during first pregnancy until 1\u00a0year after delivery. All nulliparous pregnant women received information about the study from the midwives. After 1\u00a0week, the women were approached by phone and asked if they wanted to participate in the study. Inclusion criteria were a singleton low risk pregnancy and sufficient knowledge of the Dutch language.\nOne hundred and twenty-two women were excluded due to having a twin pregnancy (n\u2009=\u20092), miscarriage (n\u2009=\u200913) or insufficient knowledge of the Dutch language (107). Thus, 1,244 women met the inclusion criteria. Of these, 672 (54%) decided to participate in the study. The most common reasons for refusal were lack of time and the intensity and intrusiveness of the questions. The present study is a separate analysis of data collected from the larger study. The Medical Ethics Committee of the University Medical Center Utrecht approved the study. All participants signed an informed consent form.\nData collection\nTo assess the prevalence rates at different points in time of depressive symptoms, stress urinary incontinence (SUI), urge urinary incontinence (UUI) and over-active bladder (OAB) syndrome, we used data obtained from all respondents of questionnaires sent at 12 and 36\u00a0weeks gestation and 3 and 12\u00a0months post-partum. In addition, we analysed data obtained from the questionnaires sent at 36\u00a0weeks gestation and 12\u00a0months after delivery to assess the possible association between urinary symptoms and depression. We did so because the prevalence of urinary symptoms peak in the third trimester and because pelvic floor symptoms, occurring in pregnancy and persisting 1\u00a0year after delivery, may be associated with depression at this time [26].\nDepressive symptoms were investigated using the Center for Epidemiologic Studies Depression Scale (CES-D). This scale is developed for use in non-psychiatric populations and gives an impression of depressive symptoms [27, 28]. The total score ranges from 0 to 60; a higher score corresponds with more depressive symptoms. A cut-off score of 16 is frequently used as an indication of a possible clinical depression. We refer in this study to women who scored B16 on the CES-D as having depressive symptoms.\nUrinary symptoms were assessed with the Urogenital Distress Inventory (UDI) [29, 30]. The UDI is a validated, standardised questionnaire, translated in Dutch. This questionnaire consists of 19 questions about urogenital symptoms and the experienced discomfort of these symptoms. We looked specifically at self-reported urge and stress urinary incontinence and over-active bladder syndrome. Although the UDI also consists of questions on prolapse, obstructive micturition and pain and heaviness in the pelvic area, these symptoms have not been linked to depression in current literature. Therefore, these questions were not used in the present study. We followed the definitions of the International Continence Society (ICS) [31]. Urge incontinence was determined by a positive answer to the question: \u201cDo you experience urine leakage related to the feeling of urgency?\u201d Stress incontinence was determined by a positive answer to the question: \u201cDo you experience urine leakage related to physical activity, coughing or sneezing?\u201d Over-active bladder syndrome was determined when both of the following questions were answered positively: \u201cDo you experience frequent urination?\u201d and \u201cDo you experience a strong feeling of urgency to empty your bladder?\u201d\nValidated questionnaires were used to assess potential confounding variables. The Dutch Personality Questionnaire (DPQ) contains 133 statements, which are divided into 7 domains: inadequacy, social inadequacy, rigidity, hostility, egoism, dominance and self-esteem [32]. The higher the score, the more these characteristics are part of the subject\u2019s personality. Personality traits are stable and this questionnaire was completed at 24\u00a0weeks gestation for logistic reasons. The Maudsley Marital Questionnaire (MMQ) was used to measure the subjective emotional and sexual relationship of the woman with her partner [33]. The MMQ consists of 15 questions, of which 10 concern emotional aspects (range 0\u201380) and 5 concern sexual aspects (range 0\u201340) of the relationship. The higher the score, the worse this specific aspect of the relationship is perceived. In addition, the questionnaires addressed biomedical and socioeconomic variables.\nBiomedical factors included length and weight at 36\u00a0weeks gestation and 1\u00a0year after delivery, age at time of delivery, the presence of a chronic illness or back pain and the use of medication. Data on mode of delivery and pregnancy-related complications (hypertensive disorders, premature delivery, growth retardation and vaginal blood loss) were obtained from the case files of obstetricians and midwifes. Data on length and weight were transformed into a body mass index (BMI\u2009=\u2009weight\/(height)2). Socioeconomic factors consisted of level of education, marital state, employment and job satisfaction. For practical reasons, the education level was dichotomised into high school or less and more than high school. Marital state was dichotomised into married and unmarried\/divorced. Behavioural factors we studied were whether participants smoked, used alcohol or were involved in leisure time physical activity in early pregnancy. In addition, women were able to add comments to the standard questions.\nStatistical analyses\nChanges in prevalence of depressive symptoms, UUI, SUI and OAB between 6 and 12\u00a0weeks gestation and between 36\u00a0weeks gestation and 3 and 12\u00a0months after delivery was determined using the McNemar tests. Possible variables associated with depressive symptoms, including urinary symptoms, were analysed in women that responded to the questionnaire at 36\u00a0weeks gestation and 1\u00a0year after delivery. Women with and without CES-D B16 were compared in univariate analyses using the independent samples t-test for continuous variables and chi-squared test for categorical variables. The relationship between depressive symptoms (CES-D _16) (dependent variable), urinary symptoms and potential confounders was assessed using multivariate logistic regression analysis (stepwise forward method) to assess whether urinary symptoms remained associated with depressive symptoms.\nOnly significantly associated variables in univariate analyses (p\u2009<\u20090.05) were put in the model. Odds ratios (Exp (B)) for continuous variables were calculated when appropriate. The Hosmer and Lemeshow test was used to establish the goodness of fit of the model. A p value of >0.05 indicates that the model provides a valid representation of data. All analyses were performed with the SPSS for Windows 11.5.\nResults\nOf the 672 women who started in the study at 12\u00a0weeks gestation, 642 (95%) women responded to the personality questionnaire sent at 24\u00a0weeks gestation. The questionnaires at 36\u00a0weeks gestation and 3 and 12\u00a0months after delivery were answered by 511 (76%), 503 (75%) and 509 (76%) women, respectively. The average age at delivery was 30.0\u00a0years (standard deviation (SD) 3.6\u00a0years). Average BMI at 36\u00a0weeks gestation was 27.9\u00a0kg\/m2 (SD 4.1\u00a0kg\/m2). The percentage of women whose education stopped after high school was 52.7%. None of the women were breast-feeding 1\u00a0year after delivery. The percentage of women who did not have a spontaneous vaginal delivery was 33.9%. Pregnancy-related complications were reported in 18.3%. In our series, urinary tract infection was not mentioned by any of the women.\nThe prevalence rates of depressive symptoms, UUI, SUI and OAB at different points in time during and after pregnancy are shown in Table\u00a01. The increase in prevalence during pregnancy (from 12 to 36\u00a0weeks gestation) and decrease in prevalence after pregnancy (from 36\u00a0weeks gestation to 12\u00a0months after delivery) of all symptoms, except urge urinary incontinence, was statistically significant in the McNemar tests (p\u2009<\u20090.05). The increase in prevalence of urge incontinence during pregnancy was significant; however, the seemingly decrease after childbirth was not significant. Factors significantly associated with depressive symptoms at 36\u00a0weeks gestation in univariate analyses and in multivariate logistic regression model are shown in Table\u00a02. Many factors were significantly associated in univariate analysis; however, in logistic regression only (social) inadequacy (personality traits), worse sexual relationship with the partner, not being involved in leisure time physical activity, pregnancy-related complications and OAB syndrome remained independently associated with depressive symptoms. The Hosmer and Lemeshow test provided a p value of 0.214, which indicates a proper goodness of fit for this model. Explained variance in this model (R2) is 40.9%. At 1\u00a0year post-partum, no significant association was found between depressive and urinary symptoms in the chi-squared tests; therefore, no logistic regression analysis was performed.\nTable\u00a01Incidence of depressive symptoms (CES-D\u2009\u2265\u200916), incontinence and over-active bladder syndrome\u00a012\u00a0weeks gestation (%)36\u00a0weeks gestation (%)3\u00a0months post-partum (%)12\u00a0months post-partum (%)Depressive symptoms18.220.716.712.2Urge incontinence7.319.116.115.6Stress incontinence20.142.226.534.3Over-active bladder syndrome54.260.17.814.4Table\u00a02Univariate and multivariate associated factors with depressive symptoms at 36\u00a0weeks gestation\u00a0CES-D\u2009<\u200916 (n\u2009=\u2009405)CES-D\u2009\u2265\u200916 (n\u2009=\u2009106)Crude OR (95% CI)Adjusted OR (95% CI)UDIUrge incontinence17.4%26.7%1.73 (1.05\u20132.86)Stress incontinence39.2%52.4%1.71 (1.11\u20132.63)Over-active bladder syndrome57.0%73.3%2.08 (1.29\u20133.34)4.40 (1.84\u201310.48)MMQEmotionality6.62 (6.21)13.74 (11.33)1.10 (1.07\u20131.13)Sexuality11.52 (6.95)16.53 (8.12)1.09 (1.06\u20131.13)1.12 (1.06\u20131.17)NPVInadequacy8.08 (5.09)14.50 (7.79)1.17 (1.13\u20131.22)1.21 (1.13\u20131.30)Social inadequacy6.89 (6.19)9.84 (6.96)1.07 (1.04\u20131.10)1.08 (1.01\u20131.16)Rigidity24.65 (6.81)26.47 (5.91)1.04 (1.01\u20131.08)Hostility12.36 (5.36)17.12 (6.71)1.14 (1.10\u20131.19)Egoism9.03 (4.23)10.56 (4.90)1.08 (1.03\u20131.13)Self-esteem30.30 (4.83)27.09 (4.56)0.88 (0.84\u20130.92)BMI (kg\/m2)27.59 (3.65)29.22 (5.24)1.10 (1.04\u20131.15)Age (years)30.50 (3.67)29.66 (4.02)0.94 (0.89\u20131.00)Education high school\/below46.9%63.2%1.95 (1.25\u20133.02)Unemployed4.5%12.3%3.00 (1.42\u20136.34)Low job satisfaction5.2%15.1%3.23 (1.57\u20136.68)Smoking7.1%16.2%2.53 (1.33\u20134.83)Use of alcohol16.0%6.7%0.38 (0.17\u20130.85)No leisure time physical activity43.2%65.1%2.46 (1.57\u20133.83)2.83 (1.35\u20135.92)Back pain51.9%67.0%1.90 (1.21\u20132.98)Pregnancy related complications8.8%20.8%2.71 (1.38\u20135.34)3.22 (1.12\u20138.87)Shown are significantly associated factors at the p\u2009<\u20090.05 level with depressive symptoms. Non-significantly associated factors are left out in this table.CES-D: Center for Epidemiologic Studies Depression Scale, UDI: Urogenital Distress Inventory, MMQ: Maudsley Marital Questionnaire, DPQ: Dutch Personality Questionnaire, BMI: body mass index, OR: odds ratio, 95% CI: 95% confidence interval.\nDiscussion\nIn our study, we set out to analyse the prevalence of depressive and urinary symptoms during and after pregnancy and to assess the possible association of urinary symptoms with depression, whilst controlling for psychosocial, behavioural, socioeconomic and biomedical factors. We showed a significant increase in prevalence of depressive symptoms, UUI, SUI and OAB during pregnancy and a significant reduction in the prevalence of depressive symptoms, SUI and OAB after childbirth (p\u2009<\u20090.05). The prevalence of UUI did not significantly decrease after childbirth. In univariate analysis, UUI, SUI and the OAB syndrome was significantly associated with a CES-D score of B16 at 36\u00a0weeks gestation. However, after adjusting for possible confounding factors, only the OAB syndrome remained significantly associated. At 1\u00a0year after delivery, no association was found between urinary incontinence, OAB syndrome and depressive symptoms.\nIn non-pregnant women, an association is reported between depression and urinary incontinence, especially urge incontinence, and over-active bladder syndrome with and without urge incontinence [15\u201319]. These studies mainly used univariate statistics and did not account for potential confounding factors. Our univariate analysis also showed that urinary incontinence and over-active bladder syndrome were significantly associated with depressive symptoms. However, in multivariate analysis, we did not find an association between depressive symptoms and stress or urge urinary incontinence during or after pregnancy. On the other hand, we did find an independent significant relation between depressive symptoms and over-active bladder syndrome at 36\u00a0weeks gestation, though not 1\u00a0year after delivery. In a research in a non-pregnant population, several explanations are suggested. Firstly, urinary incontinence as a chronic disorder may lead to depressive symptoms. The finding that depression at 1\u00a0year after delivery was not associated with urinary symptoms does not support this explanation. Secondly, it is suggested that psychological factors might influence urgency and detrusor instability [34]. Thirdly, a mutual pathologic origin of urinary incontinence and depression is proposed: both are suggested to be caused by the reduction of serotonin [15, 35]. This suggestion is at the least dubious because serotonergic depletion as the suggested aetiology in depression has been seriously questioned [36]. Furthermore, the therapeutic effect of selective serotonin re-uptake inhibitors (SSRIs) in the treatment of depression is only slightly better than placebo [37].\nIn a pregnant population, several other explanations can be considered. First, during the third trimester of pregnancy, the increase in cardiac output, increasing size of the uterus with compression of the bladder and sleep disturbances may all contribute to an increased voiding frequency. Secondly, the definition used for over-active bladder syndrome (combination of urgency and frequency) has been developed for use in a non-pregnant population. Because the majority of women will have frequency and urgency symptoms as part of their normal third trimester pregnancy, it is questionable if the diagnosis of OAB in pregnancy has the same meaning and impact as OAB in non-pregnant women. The strength of this study is that we used a prospective, longitudinal design with validated questionnaires in a large number of healthy nulliparous women. By measuring not only urogenital symptoms and depressive symptoms, but also psychosocial, behavioural, socioeconomic and biomedical factors, we were able to perform solid multivariate analysis techniques on the associations between bladder symptoms and depression.\nOur study has also some potential drawbacks that need to be discussed. First, because of the observational, epidemiological design of the study, all information gathered through the questionnaires is subjective. We used a symptom-based definition of urinary symptoms recommended by the ICS [31]. Although we could rule out, to a certain extend, urinary tract infection as potential confounding factor for urinary symptoms from the records of the midwife or gynecologist, we were unable to confirm the reported symptoms by means of a clinical diagnosis. However, the sensitivity of clinical tests like the stress test and also urodynamic investigation in relation to urogenital symptoms is still under debate [38, 39]. Our data have to be interpreted from a symptom-based point of view, which is most often the best there is to get out of epidemiological studies. It would be worthwhile to test our findings in a clinical setting, although the use of urodynamics during pregnancy in healthy women may turn out to be a difficult ethical question. Secondly, we had a moderate participation rate. Due to the quantity and intimate nature of the questions on sexuality and pelvic floor discomfort, the response rate was 54%. However, the prevalence rates of pelvic floor problems and back pain found in this cohort are in concordance with prevalence rates in other studies and the obstetrical outcome (mode of delivery, birth weight, etc.) of the study population was similar to that of comparable women registered in The Netherlands Perinatal Registry 2001 [13, 40, 41]. In addition, the percentage of women scoring above the cut-off score of 16 on the CES-D is similar to that found in a large American study among pregnant women [7]. Finally, the associations we found between psychosocial, behavioural, socioeconomic and biomedical factors at 36\u00a0weeks gestation have all been reported before in ante-natal and post-natal study populations [5, 7, 21, 22, 42\u201345]. So, the chance that our results are biased by the moderate participation rate is low. A third limitation is that we did not have information on urogenital and depressive symptoms before pregnancy. We decided not to ask about previous symptoms because of the risk of recall bias. Longstanding pre-pregnancy depressive disorders or urogenital symptoms may affect the prevalence rate recorded in pregnancy, but it is questionable if it would affect the associations we studied. Our main conclusion that there is no association between UUI, SUI and depressive symptoms during and after pregnancy was reached after multivariate adjustment in which many factors were accounted for. Finally, the prevalence of depressive symptoms was measured with the CES-D and diagnosis of depression could therefore differ from diagnosis obtained through interview following the DSM-IV criteria. However, the CES-D is a widely used questionnaire with adequate sensitivity and specificity [46], gives a good indication of depressive symptoms and is very suitable for large cohort studies.\nConclusion\nWe found significantly higher rates of depressive symptoms, SUI and OAB syndrome during pregnancy than after childbirth. After controlling for other associated factors, we found an independent association between depressive symptoms and the OAB syndrome in pregnancy but not with urinary incontinence. This association between OAB syndrome and depressive symptoms is lost after pregnancy, so it is likely that pregnancy-related factors have confounded this association. In general practice, this information on the natural course of the OAB syndrome, and its lack in causing major depressive symptoms after childbirth, can be used to counsel women who are confronted with these problems during pregnancy.","keyphrases":["depression","pregnancy","urge urinary incontinence","stress urinary incontinence","ces-d","over-active bladder syndrome"],"prmu":["P","P","P","P","P","P"]}
{"id":"Matern_Child_Health_J-4-1-2335294","title":"Preconception Brief: Occupational\/Environmental Exposures\n","text":"In the last decade, more than half of U.S. children were born to working mothers and 65% of working men and women were of reproductive age. In 2004 more than 28 million women age 18\u201344 were employed full time. This implies the need for clinicians to possess an awareness about the impact of work on the health of their patients and their future offspring. Most chemicals in the workplace have not been evaluated for reproductive toxicity, and where exposure limits do exist, they were generally not designed to mitigate reproductive risk. Therefore, many toxicants with unambiguous reproductive and developmental effects are still in regular commercial or therapeutic use and thus present exposure potential to workers. Examples of these include heavy metals, (lead, cadmium), organic solvents (glycol ethers, percholoroethylene), pesticides and herbicides (ethylene dibromide) and sterilants, anesthetic gases and anti-cancer drugs used in healthcare. Surprisingly, many of these reproductive toxicants are well represented in traditional employment sectors of women, such as healthcare and cosmetology. Environmental exposures also figure prominently in evaluating a woman\u2019s health risk and that to a pregnancy. Food and water quality and pesticide and solvent usage are increasingly topics raised by women and men contemplating pregnancy. The microenvironment of a woman, such as her choices of hobbies and leisure time activities also come into play. Caregivers must be aware of their patients\u2019 potential environmental and workplace exposures and weigh any risk of exposure in the context of the time-dependent window of reproductive susceptibility. This will allow informed decision-making about the need for changes in behavior, diet, hobbies or the need for added protections on the job or alternative duty assignment. Examples of such environmental and occupational history elements will be presented together with counseling strategies for the clinician.\nIntroduction\nThe influence of environmental exposures on the general status of health has been increasingly acknowledged for numerous disease outcomes. The connection between air pollution and acute respiratory disease, for example and, more recently, the observation linking poor indoor air quality to increases in the incidence of childhood asthma has been widely publicized. Such epidemiologic observations are often reported in the media and, when combined with the growing public interest in a clean and healthy environment, have translated into an increasingly sophisticated patient population that expects healthcare providers to be conversant with the environmental contribution to disease risk. Indeed, the lay public often has shown more interest in\u2013 and occasionally knowledge of\u2013 the relationship between health and the environment than has the medical community.\nMedical educators and the Institute of Medicine (IOM) both have addressed this dilemma by promoting enhanced medical student and physician training in environmental medicine in order to develop \u2018environmentally literate\u2019 physicians. In this brief, the term \u2018environmental medicine\u2019 will refer to \u201cdiagnosing and caring for people exposed to chemical and physical hazards in their homes, communities and workplaces through such media as contaminated soil, water and air\u201d [1, 2]. This definition was taken from a 1992\nIOM Committee report on Curriculum Development in Environmental Medicine. The preconception clinical visit already includes environmental history queries regarding smoking and alcohol use, [3] but must be enlarged to address the broader concept of environmental exposures occurring in the woman\u2019s home, community and workplace.\nSources of environmental exposure\nOrganizing the environmental exposure history can be facilitated by using the definition referred to above and by eliciting information on aspects of the patient\u2019s larger environment; this includes the specific community locale (workplace and her home environment) and infrastructure which relates to air quality and soil or water pollution, all of which the patient deals with on a regular basis.\nCommunity\nSurprisingly, a clinician would likely not know if a given patient in his\/her practice was living in an area near by an environmentally polluted location, such as a National Priority Listed (NPL) waste site or a local toxic waste dump. In large metropolitan areas, many such sites are present and unknown to most citizens. Although clinically significant exposure to a toxic hazard present on the site would be unlikely to threaten the wider community, a risk may exist for some residents living very close to the site, possibly allowing for soil or drinking water contamination. In specific instances often covered by the news media, patients would likely be able to report knowledge of living in the vicinity of such a place. Such a \u2018self-report\u2019 response could be elicited by simply asking the question, \u201cDo you live near or have contact with a waste site?\u201d Probably the most potentially important exposure opportunity is via contaminated drinking water. Here the biggest risk is usually from a small private water source such as a well, not subject to municipal water treatment standards and testing. According to the EPA approximately 15% of the U.S. population has a private drinking water source.\nWorkplace\nAn intimate relationship exists between occupational and environmental health, because often the source of environmental contamination is a former (or present day) work site. The workplace is an important \u2018special case\u2019 in environmental history taking, because populations exposed at work tend to be exposed at higher concentrations than the larger community, regardless whether contamination is via air, water or soil. Thus, though the working population is smaller than the public at large, workers are exposed at higher \u2018doses\u2019. It is thus sometimes observed that workers are the \u2018canaries\u2019 or \u2018sentinels\u2019, exhibiting first the health effects which might be expected in a wider community exposure from an environmental pathway.\nIt is fair to say that the principal exposure opportunity to an environmental reproductive or developmental toxicant in patients will be from their work place. Many toxicants with unambiguous reproductive and developmental effects are still in regular commercial or therapeutic use, and thus pose a continued potential risk in the occupational environment. Several employment sectors where such toxicants commonly are found employ women workers primarily and include: laboratory and clinical medicine, printing and dry cleaning [4] though this is clearly not always the case and the gender mix varies by sector. (See Table\u00a01). Whereas some of these chemical toxicants are regulated by public health agencies, the majority of chemicals considered for regulation are not evaluated for reproductive endpoints [5] but rather, for other toxic effects. This gap in the regulatory safety net\u00a0allows reproductive toxicants to be encountered in both environmental and work settings by men and women.Table 1Employment sectors and associated reproductive\/develop-mental toxicantsSectorToxicantExamplesAgriculturePesticides\/HerbicidesEthylene DibromideManufacturingOrganic SolventsGlycol ethers, lead,Heavy MetalsCadmiumDry CleaningSolventsPerchloroethylenePrintingSolvents\/inksPharmaceutical Compounding\/ManufactureHazardous DrugsAntineoplastics, hormones, immunologic modifiersHealth CareBiologicsRubella, CMV, Hepatitis virusPhysical AgentsIonizing Radiation\/HeatChemicalsAntineoplastics\/Hazardous Drugs Anesthetic Gases SterilantsPhysical ExertionLifting\/Prolonged Standing Shift WorkNote. Ref: Summarized from GAO, 1988; Stellman, 1994.\nHome\nHere, one must consider the exposure opportunities posed by the woman\u2019s residential environment, such as those involved with household tasks, those related to her pursuit of hobbies and those related to her \u2018micro-environment,\u2019 including diet.\nDiet\nBeyond the current recommendations related to healthy eating, the uses of non-prescription herbal or alternative medicines or supplements are crucial parts of the medical history for women planning a pregnancy. Also, some ethnic home remedies must be queried for, as some contain hazardous contaminants such as lead and mercury [6]. There are also several dietary cautions, which should be reviewed with the woman contemplating pregnancy. One is not new and relates to the warning regarding exposure to food borne listeria infections. Although listeriosis may cause only mild flu-like symptoms in the pregnant mother, serious outcomes in the fetus may result. These include premature delivery, stillbirth or neonatal infection [7\u20138]. General advice relies upon avoidance of soft cheese such as feta, Brie, Camembert, blue-veined and Mexican-style cheeses and unpasteurized milk or milk products [9]. Strict adherence to food preparation safety, such as vigilant washing of raw fruits and vegetables, avoidance of undercooked and raw meat and careful separation of stored raw food from uncooked meats serves to enhance safety for the entire family as most food borne illness is preventable.\nConsumer advisory on methylmercury in fish\nIn 2004, the Food and Drug Administration (FDA) and the Environmental Protection Agency (EPA) issued a joint consumer advisory on methylmercury in fish and shellfish. The warning specifically targets women who may become pregnant, are pregnant or are nursing mothers, and also includes young children. The document emphasizes the health benefits of fish in the diet generally as a good source of protein, and also warns of several types of fish which contain comparatively higher concentrations of mercury and should be avoided. These include: shark, swordfish, King mackerel and Tilefish. Other types of fish may be consumed in up to two meals (6 oz each) per week, including: shrimp, canned light tuna (but not albacore, which has more mercury), salmon, pollock and catfish. The Advisory also warns about local fish advisories, which are generally posted for specific populations which supplement their diets with locally, caught fish. Appropriate discussion of relevant advisories should take place in the preconception visit and is especially important for non-meat eating patients whose total dietary intake of fish may be relatively greater than is the case for meat consumers. Also of note, the U.S. Department of Health\u2019s Women\u2019s Infants, and Children Program (WIC) sometimes gives canned tuna as a diet supplement. This practice should be weighed in light of the above concerns, however, at least in terms of the relative amount of tuna in a weekly diet. The complete consumer advisory can be found at: www.epa.gov\/ost\/fish.\nHobbies and home-based work\nThe hobbies of concern generally would include those involving similar types of chemical toxicants discussed in the occupational section above, including heavy metals (lead, cadmium, arsenic) and solvents (paints (other than latex based), furniture stripper, metal cleaners etc.) Hobbies to be discouraged include: painting, ceramics, stained-glass window making (lead solder), furniture re-finishing and the like. Leisure activities to be avoided include use of saunas and hot tubs.\nA number of home-based work activities, which resulted in contamination of the home and family members, have been documented in the literature. Although uncommon, they deserve mention here. At one end of the spectrum most involve the use of metals, either in metal parts reclamation (lead batteries being melted down) or jewelry making (heating and soldering) [10]. At the other, the home environment may be unwittingly contaminated by family members who bring work contaminants home on their shoes or dirty work clothes. If the work clothes are laundered at home, there not only is opportunity for exposure to the launderer but also other household members. For this reason, some OSHA standards require changing rooms so work clothes will not secondarily expose family members, in the case of lead and asbestos work, for example [11].\nHousehold exposures\nPesticides, herbicides and rodenticides are likely the most common chemical toxicants in the average home. Certainly, a pregnant woman or one actively attempting to conceive, should not apply any of these [12]. Because the concept of \u2018second hand\u2019 exposure as described above applies here also, care needs be taken so that the chemicals are not \u2018tracked\u2019 into or throughout the house, no matter who performs the chemical applications. Of far greater importance on a population basis is second hand smoke in the home and\/or workplace. Numerous polycyclic aromatic hydrocarbons present in cigarette smoke are carcinogenic and have been shown to affect reproductive outcomes in animal studies. They cross and affect the placenta, and enter breast milk [13]. Laundering of contaminated work clothes is also a concern, as discussed above. A hiatus from caring for the family cat and litter box should be mentioned due to the toxoplasmosis risk.\nFig. 1Example checklist for initial qualitative evaluation of reproductive hazards, (modified from Grajewski, 2005)Fig. 2Example of checklist for initial quantitative evaluation of reproductive hazards, (modified from Grajewski, 2005)\nMany non-latex paints are solvent based and contain small amounts of metals to enhance wear, and as preservatives. This is especially true of exterior paint, such as that used on porches or building exteriors or even in the interiors of older buildings. Rehabbing older homes, which often involve paint stripping, either with a heat gun or a chemical stripper is particularly hazardous. Inhalation is a very efficient means of producing a clinically significant exposure to lead which was commonly used years ago in interiors of homes. Many commercial paint strippers contain methylene chloride (dichloromethane), which metabolizes to carbon monoxide, and is particularly toxic to the fetus.\nGuidance for clinician\nCertainly, awareness that occupational and environmental hazards encountered by patients may play a clinically significant role in a pregnancy outcome is the first step in effective patient management. Therefore, enlarging the standard health history form, completed by the patient on or before the first visit to obtain a more detailed environmental history is a necessary first step and is also often very informative. Whereas a number of preconception checklists exist, only some of which address fish consumption or residence near a waste site, most poorly capture, or even fail to query about occupation, despite this being the likely greatest \u2018environmental\u2019 risk the patient faces.\nA \u2018qualitative\u2019 evaluation of a patient\u2019s potential exposure to reproductive and developmental toxicants can be obtained with a screening questionnaire as seen in Fig.\u00a01. This involves examining occupation by industry sector of employment and then by chemical, physical and biological agents of concern. If a reproductive hazard is present in the patient\u2019s work environment, an initial quantitative assessment can be made regarding the exposure intensity (Fig.\u00a02). However, at this point, the clinician may want to consult an occupational medicine colleague to elicit a more detailed history and assist in the preconception recommendations.\nPreconception management strategies, should be based on the occupational history and include a decision on the safety of continued employment during the preconception period and pregnancy. If the patient works with bona fide reproductive and development toxicants, continued employment and under what circumstances must be determined. This may involve identifying additional protections, such as a respirator, gloves or other personal protective apparel or equipment that may make the job safer for the patient. There are some jobs, however, where it is recommended by professional organizations as well as governmental safety and health agencies, that pregnant persons, or those actively trying to conceive be provided with alternative duty [14]. A good example here is nurses who handle cancer chemotherapy [15]. However, a related issue involves crafting elements of an existing job which the pregnant patient, such as the oncology nurse may safely continue, such as patient education, telephone triage and the like. This arrangement is termed \u2018alternative duty\u2019. Also involved in preconception counseling is the potential impact on the quality of breast milk if the mother is planning to breastfeed. The majority of organic solvents and pharmaceuticals absorbed by the mother in the workplace invariably make their way into milk. Thus, both work during pregnancy and return to work while breastfeeding need to be considered.\nWe have thus far focused on the working patient\u2019s current job, which is generally considered the most influential on pregnancy outcome, as most reproductive toxicants are thought to act via an acute toxicity mechanism during the three to four months prior to conception and pregnancy [16\u201318]. However, there are also several examples of remote past exposure that are important to elicit, though likely less commonly encountered. An example includes the patient who has high bone lead stores from remote past (childhood) exposure that can be mobilized during pregnancy and expose the fetus [19\u201320]. Medical management at the preconception visit would include a blood lead level so this can be tracked during pregnancy. Also, maternal stores of fat-soluble organic chemicals, such as dioxins and PCBs may also expose the fetus [21, 22]. Positive responses to exposure to occupational or environmental agents by the preconception patient may require consultation. The Association of Occupational and Environmental Clinics can supply referrals to clinicians in various locales nationwide at (www.aoec.org).\nConclusion\nThe preconception office clinic visit presents a strategic opportunity to minimize the environmental and occupational sources of reproductive risk facing the preconception patient. This requires the two-way exchange of information between the patient and her clinician, clarifying misunderstandings and implementing reasonable strategies to minimize exposures from the wider community, in the workplace and at home. Taking an environmental and occupational history and tailoring recommendations based on that, enlarges the likelihood that preventable, adverse pregnancy outcomes can be avoided.","keyphrases":["environmental exposure","reproductive","reproductive toxicants"],"prmu":["P","P","P"]}
{"id":"Cancer_Immunol_Immunother-3-1-2150627","title":"The CIMT-monitoring panel: a two-step approach to harmonize the enumeration of antigen-specific CD8+ T lymphocytes by structural and functional assays\n","text":"The interpretation of the results obtained from immunomonitoring of clinical trials is a difficult task due to the variety of methods and protocols available to detect vaccine-specific T-cell responses. This heterogeneity as well as the lack of standards has led to significant scepticism towards published results. In February 2005, a working group was therefore founded under the aegis of the Association for Immunotherapy of Cancer (\u201cCIMT\u201d) in order to compare techniques and protocols applied for the enumeration of antigen-specific T-cell responses. Here we present the results from two consecutive phases of an international inter-laboratory testing project referred to as the \u201cCIMT monitoring panel\u201d. A total of 13 centers from six European countries participated in the study in which pre-tested PBMC samples, synthetic peptides and PE-conjugated HLA-tetramers were prepared centrally and distributed to participants. All were asked to determine the number of antigen-specific T-cells in each sample using tetramer staining and one functional assay. The results of the first testing round revealed that the total number of cells analyzed was the most important determinant for the sensitive detection of antigen-specific CD8+ T-cells by tetramer staining. Analysis by ELISPOT was influenced by a combination of cell number and a resting phase after thawing of peripheral blood mononuclear cells. Therefore, the experiments were repeated in a second phase but now the participants were asked to change their protocols according to the new guidelines distilled from the results of the first phase. The recommendations improved the number of antigen-specific T-cell responses that were detected and decreased the variability between the laboratories. We conclude that a two-step approach in inter-laboratory testing allows the identification of distinct variables that influence the sensitivity of different T-cell assays and to formally show that a defined correction to the protocols successfully increases the sensitivity and reduces the inter-center variability. Such \u201ctwo-step\u201d inter-laboratory projects could define rational bases for accepted international guidelines and thereby lead to the harmonization of the techniques used for immune monitoring.\nIntroduction\nIn the last two decades, more than 200 clinical trials of different anti-tumor vaccines aiming to induce tumor-specific immunity in cancer patients have been described [1]. Most of these trials primarily assessed safety and immunogenicity while reporting partial or complete clinical responses in a minority of patients [2, 3]. Despite the fact that the low fraction of clinical responders still precludes the establishment of a direct correlation between clinical efficacy and T-cell reactivity, it has become clear from animal models and clinical observations that naturally-occurring or vaccine-induced CD8+ or CD4+ T-cells play an important role in the control and regression of tumors [4\u20139]. Therefore, the number of subjects that mount a vaccine-induced T-cell response as well as the strength of a detected T-cell response represent important surrogate markers for vaccine efficacy. The enzyme-linked immunospot (ELISPOT) assay [10, 11], staining with HLA-peptide multimers [12] and intracellular cytokine staining (ICS) [13, 14] are technologies used commonly for the monitoring of antigen-specific immune responses. For these three assays, a huge variety of different protocols are available worldwide. This heterogeneity, together with the fact that the sensitivity of the individual protocols can vary significantly, makes a comparison of the results obtained in different trials a difficult task. Moreover, an increasing number of new technologies are constantly being introduced to the field, which makes interpretations even more complex [15\u201325].\nCurrent data and opinion support the use of a functional assay like the ELISPOT or ICS in combination with a phenotyping assay like HLA-multimers [26, 27], but recognized international standards for all these methodologies are still lacking.\nThe main aim of the \u201cCIMT monitoring panel\u201d is to harmonize and optimize the monitoring of antigen-specific T-cells among the participating laboratories, based on objective rationales with respect to the testing procedure, the analysis and the interpretation of results. Important requirements for an immunological test are sensitivity, applicability to large amounts of clinical material and feasibility at reasonable cost. The results generated by the tests should be reproducible and sensitive, independently of the place where they have been performed. After the first meeting of the working group, a series of inter-laboratory testing projects was initiated, in which individual laboratories could compare their performance, express their needs and exchange experience in order to improve their local assays. Here we report the results of the first two phases of the CIMT monitoring panel, with 13 participating centers from six European countries.\nMaterials and methods\nPreparation and screening of PBMC samples\nBuffy coats from HLA-typed healthy volunteers were kindly provided from the Blood Bank of the University Mainz. HCMV sero-status was known. PBMC were isolated by Ficoll density gradient separation (Pharmacia, Uppsala, Sweden), washed two times in RPMI 1640 (GIBCO BRL, Grand Island, NY, USA) containing 10\u00a0mM Hepes buffer, l-arginine (116\u00a0mg\/ml), l-glutamine (216\u00a0mg\/ml), penicillin (10\u00a0IU\/ml), streptomycin (100\u00a0mg\/ml) and 10% FCS (GIBCO BRL), counted and frozen at 10 to 20\u00a0\u00d7\u00a0106 cells per cryovial in 1\u00a0ml of FCS 90%\u00a0+\u00a0DMSO 10% at \u221280\u00b0C in freezing-boxes filled with iso-propanol. After 20\u00a0h, all cryovials were transferred to liquid nitrogen and stored until distribution to the participating laboratories.\nPre-screening and selection of the PBMC donors for influenza- and CMV- T-cell reactivities were performed by a central lab using the IFN\u03b3 ELISPOT assay following a local protocol as described previously [38]. Five donors were selected for the first phase of the panel and eight for the second phase. One HLA-A*0201-negative donor was included in each phase (negative control), all other samples were HLA-A*0201-positive.\nSynthetic peptides and HLA-tetramers\nPeptides were synthesized using standard Fmoc chemistry, dissolved at 10\u00a0mg\/ml in DMSO, aliquotted and stored at \u221280\u00b0C. The purity was checked by reverse-phase HPLC and was found to be >80%. Two known HLA-A*0201 T-cell epitopes were used: influenza MP 58\u201366 GILGFVFTL and HCMV pp65 495\u2013503 NLVPMVATV (http:\/\/www.syfpeithi.de). Biotinylated recombinant HLA-A*0201 monomers folded with the influenza MP 58\u201366 or the HMCV pp65 peptides were produced essentially as described, purified by gel filtration and stored as aliquots at \u221280\u00b0C [12]. Fluorescent multimers were obtained by incubation with streptavidin-PE (Molecular Probes, Leiden, The Netherlands), then frozen as aliquots after addition of 0.5% BSA and 16% glycerol. HLA-concentrations of influenza-tetramer and HCMV-tetramers were 700 and 350\u00a0\u03bcg\/ml, respectively. Both tetramers were checked by HPLC and\/or validated by staining of a specific CD8+ T-cell line (Influenza) or PBMC from HLA-A2-negative and HLA-A2-positive CMV seronegative donors (CMV). Such tetramers are stable at 4\u00b0C for at least 1\u00a0month (personal observation) and participants were asked to perform all tests within this time period.\nParticipating centers\nTwelve centers from five European countries participated in the first phase of the monitoring panel. As one of the investigators moved to another institution during the study a 13th center from a 6th European country was added to the group in the second phase of the panel. Participation in the panel was open to all interested laboratories with a focus on T-cell monitoring, independently of membership in the Association for Immunotherapy of Cancer.\nReagent distribution and assay guidelines\nCoded PBMC samples, synthetic peptides and HLA-A*0201 tetramers were shipped on dry ice to the participants. Additionally, guidelines for the two T-cell assays were distributed for each phase:\nPhase I\/2005. A protocol for tetramer staining was included. Briefly, 1\u00a0\u00d7\u00a0106 PBMC per test were transferred directly after thawing into one well of a 96 well u-bottom plate and washed in FACS buffer consisting of PBS, 2% FCS, 2\u00a0mM EDTA, 0.02% azide. Incubation with 5\u00a0\u03bcg\/ml HLA-tetramer was then performed in FACS buffer with 50% FCS for 30\u00a0min at room temperature in the dark. After one wash in FACS buffer, mAb for T-cell staining were added for 20\u00a0min at 4\u00b0C. Finally, cells were washed twice before fixing in FACS buffer containing 1% formaldehyde solution. Three mAb combinations were proposed, CD8 alone, CD3 plus CD8, or CD4 plus CD8. Each lab could choose here the antibody clones, fluorescent dye and concentrations used. Stainings were performed in duplicate.\nFor the functional assays, synthetic peptides were diluted at 1\u00a0mg\/ml in PBS as a stock solution. Concentrations in further tests were 1\u201310\u00a0\u03bcg\/ml, left to the choice of the participants. There were no recommendation which functional test should be performed, so that each group could choose the test either routinely used, or to be implemented for its own needs. In this first phase, 11\/12 laboratories chose the IFN\u03b3 ELISPOT assay, one lab (Z10) a FACS-based intracellular IFN\u03b3 staining and one lab performed both assays (Z7). Spot counting was performed locally.\nPhase II\/2006. Following the results obtained in the first testing phase, requirements were introduced and participants were asked to apply exactly these new criteria (two for the tetramer staining, and four for the ELISPOT, see \u201cResults\u201d section). The assay guidelines were modified accordingly. However, in order to reduce the variability in the FACS analysis of the 13 laboratories, a figure showing exemplary dot-plots, settings of gates and quadrants, and statistical analysis was provided. All laboratories were now required to perform an IFN\u03b3 ELISPOT as the functional test, with a fixed peptide concentration of 1\u00a0\u03bcg\/ml. Participants were encouraged to use a distributed model protocol but were allowed to use their local protocol, provided that they applied the four new requirements introduced in the second phase.\nCollection and analysis of results\nAfter performing the required tests in each phase, participants returned a completed report form containing all relevant information. Number of cells recovered after thawing was included to assess viability after transport. For the tetramer staining experiments, mAb clone, manufacturer, amount, cytometer type and number of lymphocytes and\/or CD8+ cells analyzed were noted. Results were expressed as percentage of tetramer-positive cells among CD8+, CD3+CD8+, or CD4\u2212 lymphocytes, depending on which mAb combination was used for the staining. Additionally, FACS dot-plots containing all gates, quadrants and deduced statistical analysis were collected. For the functional test, medium and thawing procedure (e.g. addition of DNAse, of a resting phase, etc.) had to be described, as well as the number of cells per test, the antibodies used (clone, manufacturer, concentration), the final peptide concentration and the incubation times. For the ELISPOT assay, the type of plate, the enzymatic visualization system and the spot reader were also noted. Absolute spot numbers were given by each participant, and filter plates were kept for possible second analysis.\nAll results from both phases were collected and centrally analyzed. For the tetramer stainings, the number of lymphocytes, number of CD8+ T-cells and frequencies of tetramer-positive cells were calculated on the basis of the stainings and statistics provided by the participants. Apart from these calculated frequencies, a \u201cvisual evaluation\u201d was necessary (see \u201cResults\u201d). For the ELISPOT, analysis was performed based on the spot numbers reported by the participants, followed by a student t test. Results were accepted as positive reaction only when the numbers of antigen-specific spots exceeded the number of spots in the background wells by atleast a factor two. The coefficients of variation (CV) were calculated for all results (CV\u00a0=\u00a0SD\/mean\u00a0\u00d7\u00a0100) and are shown in supplementary Tables S1a, b.\nThe raw data from both panel phases will be provided to interested readers upon request.\nResults\nPhase I\/2005 of the interlaboratory testing project\u2014general aspects\nCoded PBMC samples from four HLA-A*0201-positive and one HLA-A*0201-negative healthy donor (D1\u2013D5) were included in this first testing phase. The thawing procedure for PBMC samples in the test centers was not standardized and the recovery of viable cells varied greatly between 45 and 102% (mean 73%) in the 12 labs. However, the number of cells recovered was in all cases sufficient to perform the required analyses. When all the data from the tetramer staining and functional tests were combined it became clear that subjects D1 and D5 had responded to the HLA-A*0201 restricted CMV-derived peptide, consistent with their CMV seropositive-status, and that subjects D1, D2, D3, and D5 had responded to influenza. In total, each laboratory should in theory have been able to measure six positive (2\u00d7 CMV and 4\u00d7 influenza) responses.\nDetection of antigen-specific T-cells by tetramer staining and IFN\u03b3 ELISPOT\nThe protocol required that all PBMC samples should be analyzed by the 12 participants for the presence of HLA-A*0201-restricted CMV-specific and influenza-specific CD8+ T-cells using centrally-prepared tetramers. The indicated frequencies of antigen-specific CD8+ cells generally represent the mean of two separate stainings with CD3 Ab\/CD8 Ab\/tetramer, except for centers Z1 (CD8\/tetramer), Z7 (CD3\/CD4\/CD8\/tetramer), Z5 and Z10 (one staining CD3\/CD8\/tetramer and one staining CD3\/CD4\/tetramer) and are based on the analysis and dot plots provided by each participant. As illustrated in Fig.\u00a01, the absolute numbers of tetramer-positive T-cells were influenced by the individual decision of where to set the gates and quadrant markers for the analysis. For example, the inclusion of the subset of T lymphocytes expressing CD8 at a low density influenced the number of CD8+ and consequently the frequency of tetramer+ cells. Moreover, non-specific binding of the tetramer (as seen on the CD8-negative subset) also varied between the different laboratories. For these reasons, not only the frequencies, but also the appearance of the tetramer-positive populations was carefully examined. Two parameters were chosen for validation of \u201cpositive\u201d results: (1) a clustered, but not diffuse, tetramer binding-population, and (2) strong intensity of tetramer staining, especially marked for the CMV-tetramer-binding population (Fig.\u00a01). Table\u00a01 shows: (I) the minimum, mean and maximum frequencies of antigen-specific CD8+ T-cells, (II) the results obtained from the individual centers Z1\u2013Z12, and (III) the number and percentage of centers that detected a response. The high frequencies of CMV-specific CD8+ T-cells in donors D1 and D5 were readily detected by all participants (mean of 1 per 141 CD8\u00a0\u00b1\u00a0113 in D1 and mean of 1 per 80 CD8\u00a0\u00b1\u00a024 in D5, respectively). For influenza-specific CD8+ T-cells, the results were more variable. Influenza-tetramer+ cells in donor D3 were detected by all participants with a mean frequency of one cell in 1014 CD8+ T-cells\u00a0\u00b1\u00a0355. In Donor D5, 11 of 12 laboratories detected a mean of one tetramer binding cell per 1106 CD8+ T-cells\u00a0\u00b1\u00a0508. Influenza-specific cells were less numerous in healthy subjects D1 and D2 and were only detected by five and eight laboratories, respectively. No false positive reactivity was reported by any of the participants.\nFig.\u00a01Example of tetramer staining results as provided by four selected participating centers Z5, Z12, Z8 and Z1. All stainings were performed on donor D1 from phase I\/2005 who showed reactivity with both of the tested tetramers. Cells were gated either on the lymphocyte population (Z1), or the subsets of CD3+CD8+ (Z5) or CD3+ (Z8, Z12), according to the Ab combination used by each lab. The upper panel shows results for tests with the CMV-tetramer, the lower panel shows results for tests with the influenza-tetramer. In all dot-plots, the tetramer staining is displayed on the y-axis and anti-CD8-staining on the x-axis. Number of counted CD8+ T-cells and percentage of tetramer-positive cells among the CD8 subset are indicatedTable\u00a01Overview of the tetramer results from phase I\/2005 of the CIMT monitoring panelD1 CMVD1 FluD2 FluD3 FluD5 CMVD5 FluMin44820,0006,6671,8181061,786(I)Mean1418,0951,9091,014801,106Max353,77452659526588Z11293,774635870106758(II)Z2112\u2013\u20131,818701,770Z3n.d\u2013\u2013701n.d\u2013Z497\u2013\u20138541061,342Z5161\u2013\u201390988606Z61396,8961,3161,00091651Z744820,0006,6671,266821,724Z81005,12854559587877Z9n.d\u20131,3161,274n.d1,786Z1094\u20132,5971,360861,439Z11994,6751,66787758588Z1235\u201352664526625Detected by10\/105\/128\/1212\/1210\/1011\/12(III)Detected %100426710010092(I) Minimum, mean and maximum frequencies of antigen-specific T-cells(II) Results obtained from the individual centers Z1\u2013Z12. All frequencies are indicated as 1 per x counted CD8+ T-cells(III) Number and percentage of centers which detected a given reactivity in donors D1, D2, D3 and D5\nEleven laboratories analyzed the five PBMC samples for the presence of HLA-A*0201-restricted CMV-specific and influenza-specific IFN\u03b3-producing T-cells by ELISPOT assay. Only one group (Z10) used an intracellular cytokine staining as a functional test (data not shown because no comparison with other groups possible). Table\u00a02 shows (I) the minimum, mean and maximum frequencies of antigen-specific cells, (II) the results obtained from the individual centers Z1\u2013Z12, and (III) the number and percentage of centers that detected each reactivity. As described in the \u201cMaterials and methods\u201d, results of spot-forming cells per seeded PBMC were accepted as a positive reaction only when passing statistical testing and when the number of antigen-specific spots exceeded the number of spots in the background wells by at least a factor of two. IFN\u03b3-producing cells reactive against CMV were detected by 10 of the 11 laboratories in donor D1 (mean reactivity was 1 per 1,855 PBMC\u00a0\u00b1\u00a0825) but only by 8 of 11 in donor D5 (mean reactivity was 1 per 4,405 PBMC\u00a0\u00b1\u00a03,762). The influenza-specific T-cells present in subject D3 were detected by six laboratories, while the responses in the healthy subjects with markedly lower numbers of peripheral specific T-cells (D1, D2 and D5) were detected by three laboratories only.\nTable\u00a02Overview of the IFN\u03b3 ELISPOT results from phase I\/2005 of the CIMT monitoring panelD1 CMVD1 FluD2 FluD3 FluD5 CMVD5 FluMin3,06162,50055,55533,33311,42850,000(I)Mean1,85538,14143,58917,5474,40530,811Max88810,25630,7698,5711,03914,705Z11,006\u2013\u20138,571\u2013\u2013(II)Z22,439\u2013\u2013\u20132,816\u2013Z31,29562,500\u201322,7271,41227,727Z41,312\u2013\u201310,9091,980\u2013Z5\u2013\u2013\u2013\u2013\u2013\u2013Z6233*\u2013\u20131,960*253*\u2013Z71,89510,25644,44433,33311,42850,000Z83,061\u2013\u2013\u20136,896\u2013Z988841,66655,55512,1951,03914,705Z10NDNDNDNDNDNDZ111,769\u201330,769\u2013\u2013\u2013Z123,030\u2013\u2013\u20135,263\u2013Detected by10\/113\/113\/116\/118\/113\/11(III)Detected %912727557327(I) Minimum, mean and maximum frequencies of antigen-specific cells(II) Results obtained from the individual centers Z1\u2013Z12. All frequencies are indicated as 1 per x seeded PBMC except for Z6* where it is indicated as 1 per x seeded CD8+ T-cellsResults from Z6 were not included for calculation of the mean frequency of antigen-specific T-cells in D1, D3 and D5(III) Number and percentage of centers that detected a given response in donors D1, D2, D3 and D5ND not determined\nSubgroup analysis reveals that the number of CD8+ T-lymphocytes analyzed affects the sensitivity of the tetramer staining\nAlthough the tetramer stainings were performed with centrally prepared reagents following set guidelines, centers were left free to select several parameters according to their own protocols, and this could have influenced the test results (see \u201cMaterials and methods\u201d). Most of the participants used monoclonal antibodies specific for CD3 and CD8 to co-stain the cells. There were no obvious differences in the performance of the centers depending on which antibody clones, antibody combinations or cytometer were used (data not shown).\nThere was a high degree of variability in the number of CD8+ cells which were analyzed per staining, ranging from only 0.5\u00a0\u00d7\u00a0104 to about 19\u00a0\u00d7\u00a0104 (inter-center variation). In addition, a non-negligible intra-center variation was observed for the number of counted CD8+. We therefore analyzed each individual staining independently of the center that performed it and focused on the number of CD8+ T-cells that had been counted. For the six different antigen-specific populations detectable, a total of 68 tests was performed by the group (see Table\u00a01). Overall, antigen-specific T-cell reactivities were reported in 82% of the tests (56\/68, mean of duplicate stainings). When less than 30,000 CD8+ T-cells were counted, only 70% of all responses were found. In contrast, 89% of all responses were manifest when more than 30,000 CD8+ T-cells were counted (Fig.\u00a02a). When antigen-specific T-cells were present at high frequency, the number of cells counted did not influence the result, because CMV-specific T-cells from donors D1 and D5 were detected irrespective of the number of CD8+ T-cells in the test. However, for the influenza-specific cells, positivity was registered in only 75% of all tests performed (36 of 48 tests). Strikingly, we observed a marked difference for the results derived from those tests involving less than 30,000 CD8+ T-cells (56% success in detection) as compared to tests performed with more than 30,000 CD8+ T-cells (84%).\nFig.\u00a02a Subgroup analysis of tetramer results from phase I\/2005. Bars indicate the percentage of positives that could be detected by tetramer staining. The first group of bars shows the results for all of the six detectable positives, the second group shows results from stainings with the CMV-tetramer and the third group of columns shows results from stainings with the influenza-tetramer. The open bars in each group represent all tests performed, grey bars represent results obtained in tests that were performed on more than 3\u00a0\u00d7\u00a0104 CD8+ T-cells and black bars represent results obtained in tests that were performed on less than 3\u00a0\u00d7\u00a0104 CD8+ T-cells. The boxes within each bar indicate the fraction of tests with a positive result. The asterisk indicates a P-value\u00a0<\u00a00.05 by Chi-square analysis. b Subgroup analysis of ELISPOT results from phase I\/2005. The bars indicate the percentage of positive reactivities detected by IFN\u03b3 ELISPOT assays. The open bar shows the percentage of all reactivities detected by all 11 centers that performed the ELISPOT assay as the functional test. Criteria for division of centers into two subgroups were based on the following requirements: do not use allo-APC (first subgroup analysis), use a resting time (second subgroup analysis) or use equal or more than 400,000 PBMC per well (third subgroup analysis). Grey bars always represent centers that were in conformity with the indicated minimum requirement, black bars show results from centers that did not fulfil that requirement. The boxes within each column indicate the fraction of centers in each category. The asterisks indicate a P-value\u00a0<\u00a00.05 in Chi-square analysis\nIn conclusion, the ability to detect antigen-specific T-cell reactivities by tetramer staining was mainly affected by the number of CD8+ T-cells stained and analyzed, especially when the antigen-specific T-cells were present at low or moderate frequencies. We therefore modified our guidelines for the tetramer assay and recommended staining at least 1\u00a0\u00d7\u00a0106 PBMC and analyzing all cells in the tube. In addition, we provided an example of how optimal cell gates and dot-plot quadrants could be selected.\nELISPOT assays are heterogeneous and require standardization\nThe ELISPOT analyses were performed according to 11 more or less different protocols. The most discernible differences that were observed in these protocols concerned (1) the different types of multi-screen plates, (2) the serum origin, (3) the use of duplicates, triplicates or quadruplicates, (4) the use of allogeneic APC, (5) the inclusion of a resting phase after thawing the PBMC, (6) the number of PBMC per well, (7) the type of antibodies used, (8) the type of spot-reader, and the (9) enzyme and substrate for staining of the spots. Each center also used a different plate protocol (distribution of the wells, number of replicates, control tests).\nThe influence of each of these parameters on the number of positive responses was studied by further analysis in which the laboratories were divided into two subgroups. As a result, several criteria were identified which could help to improve the sensitivity and comparability of detection.\nAll data sets (duplicates, triplicates or quadruplicates) were first analyzed by Student t test for unpaired samples (\u201cMaterials and methods\u201d). In our panel, one center used quadruplicates, nine centers used triplicates and one center performed the ELISPOT analysis in duplicates. Due to the variety in the replicates, responses measured by duplicate wells failed to pass the Student t test more often as compared to triplicates.\nOverall, the 11 centers were able to detect 50% of all possible reactivities in this panel phase (Table\u00a02; Fig.\u00a02b). In a subgroup of three laboratories (Z5, Z6 and Z8), an allogeneic APC population (T2 or K562-A*0201 cells) was added for binding and presentation of the synthetic peptides. The three centers that used allo-APC detected only 28% of all responses, while the other centers detected 58% of all responses.\nIn five laboratories (Z3, Z4, Z7, Z8, and Z9) PBMC were thawed, and then incubated in culture medium at 37\u00b0C. After this resting phase of 2\u201320\u00a0h, living cells were washed, counted and seeded into ELISPOT plates. Laboratories using a resting phase detected 73% of the positive reactivities (22 out of 30 potentially positive tests). No significant difference in the ability to detect antigen-specific T cells was found using shorter or longer resting-times. In contrast, the laboratories that did not use a resting procedure detected only 30% of all positives (Fig.\u00a02b).\nFinally, the number of cells seeded per well differed considerably between all participants and ranged from 1 to 6\u00a0\u00d7\u00a0105 PBMC. We divided the laboratories arbitrarily into two groups, those using either more than 4\u00a0\u00d7\u00a0105 PBMC (Z4, Z7, Z8, and Z9) or less than 4\u00a0\u00d7\u00a0105 PBMC (Z1, Z2, Z3, Z11 and Z12). The first group detected 71% of all positive samples, whereas the second group was able to detect only 43% of all positives (Fig.\u00a02b). Centers Z5 and Z6 used a defined number of separated CD8+ T-cells in the ELISPOT and were therefore not included in this subgroup analysis.\nNone other of the nine depicted protocol variables had any obvious impact on the detection of specific T-cells. As a conclusion from these results, four minimum requirements were formulated for the ELISPOT protocol: (1) perform triplicates for each test antigen (2) do not use allo-APC (3) add a resting time to increase the proportion of living cells seeded and (4) use a minimum number of 4\u00a0\u00d7\u00a0105 PBMC per well.\nPhase II\/2006 of the interlaboratory testing project\u2014general aspects\nTo formally prove that the requirements formulated for tetramer staining and ELISPOT analysis increase the ability of the participants to detect antigen-specific CD8+ T-cells and reduce the inter-center variability, we decided to repeat the analysis in a second phase of the panel, with the same participants (phase II\/2006). In this round, all groups were asked to follow our modified guidelines for the tetramer- and the ELISPOT-assays.\nAgain, all PBMC samples were prepared and pre-tested in one central lab and peptide antigens and PE-conjugated tetramers were also provided from one source. As one investigator had meanwhile moved to another lab, we added a 13th center to the group. PBMC from seven selected healthy HLA-A*0201-positive donors and 1 HLA-A*0201-negative donor (D3) were required to be analyzed for the presence of HLA-A*0201-restricted CMV-specific T cells and for influenza-specific T-cells. The mean number of recovered cells after thawing was sufficient to perform the tests. When all the data were combined, it became clear that subjects D2, D5 and D8 possessed CMV-specific CD8+ T-cell subsets, and D1, D2, D4, D6 and D7 possessed influenza-specific CD8+ T-cells. Therefore, each laboratory could theoretically have measured eight positives (3\u00d7 CMV and 5\u00d7 Influenza) in this second phase.\nAnalysis of CD8+ T-cell tetramer binding using the new guidelines\nIn the second phase, a total of 104 tests were performed to detect the eight possible tetramer reactivities. Following the modified guidelines for tetramer staining, the mean number of CD8+ T-cells that were counted in each separate test increased markedly (+36%): a mean of about 49,000 CD8+ cells were analyzed in the phase I (n\u00a0=\u00a068 tests) and a mean of 67,000 CD8+ T-cells in phase II (n\u00a0=\u00a0104 tests). The number of cells per test ranged from 12,000 to 467,000 CD8+. In 81% (84 of 104) of the tests >30,000 CD8+ were counted (compared to 66% of all relevant tests in the first phase). Table\u00a03 shows (I) the minimum, mean and maximum frequencies of antigen-specific T-cells, (II) the results obtained from the individual centers Z1\u2013Z13, and (III) the number and percentage of centers that detected each T-cell specificity. Donors D2, D5 and D8 showed very strong reactivities with the CMV-tetramer, with mean frequencies of 1\/45 CD8+ T-cells, 1\/37 CD8+ T-cells, and 1\/19 CD8+ T-cells, respectively. All 13 laboratories were able to detect these populations (Table\u00a03). All but one center detected the influenza-specific cells present at high frequencies in donors D6 (1\/1116 CD8+ T-cells) and D7 (1\/347 CD8+ T-cells). Donors D1, D2 and D4 possessed fewer specific cells (1\/3,739, 1\/3,573 and 1\/5,278 CD8+ T-cells) which were found by 12, 9 and 9 centers, respectively. Three laboratories also reported influenza tetramer-binding CD8+ cells in D5 or D8. According to the results of the other centers as well as from the ELISPOT (see below), these stainings were considered as false positive (not shown). One center (Z13) was not able to detect any of the influenza-specific CD8+ T-cell reactivities. Finally, no tetramer+ cells were described in the HLA-A*0201-negative donor (D3).\nTable\u00a03Overview of tetramer results from phase II\/2006 of the CIMT monitoring panelD1 FluD2 CMVD2 FluD4 FluD5 CMVD6 FluD7 FluD8 CMVMin7,1437710,00010,000603,33386942(I)Medium3,739453,5735,278371,11634719Max1,538281,2502,500305712028Z13,333 453,3335,0003976929410(II)Z24,000303,3335,000301,10025021Z34,00047\u20135,0004558827020Z41,538712,8576,6663376926342Z56,666542,500\u2013431,42837724Z65,00077\u201310,000601,66686927Z72,857473,3336,6663883329022Z82,000353,3333,333316662448Z93,3333110,0003,333323,33362520Z103,333282,222\u20133095220213Z111,666361,2502,5003257121515Z127,41337\u2013\u2013357142608Z13\u201353\u2013\u201334\u2013\u201320Detected by12\/1313\/139\/139\/1313\/1312\/1312\/1313\/13(III)Detected %9210069691009292100(I) Minimum, mean and maximum frequencies of antigen-specific T-cells(II) Results obtained from the individual centers Z1\u2013Z13. All frequencies are indicated as 1 per x counted CD8+ T-cells(III) Number and percentage of centers that detected each of the eight possible responses\nAnalysis of CD8+ T-cell responses by ELISPOT following the introduction of a set of four rules\nIn this second phase, all laboratories performed ELISPOT analysis following local protocols, all of which conformed to the newly introduced minimum requirements. Table\u00a04 shows (I) the minimum, mean and maximum frequencies of antigen-specific cells, (II) the results obtained from the individual centers Z1\u2013Z13, and (III) the number and percentage of centers that detected the response. High frequency T-cell responses against CMV could readily be detected by all 13 centers in donors D5 and D8 and by 12 of 13 in donor D2. Failure of center Z4 to detect the CMV reactivity in donor D2 was due to a very high background of the medium control. The number of spots representing IFN\u03b3-producing cells after influenza-peptide stimulation was generally lower, and consequently, the influenza-specific T-cell responses in subjects D1, D2, D4 and D6 were detected by fewer laboratories (four centers for D1, three centers for D2, two centers for D4 and ten centers for D6). The high numbers of influenza-specific T-cells present in D7 were detected by all 13 laboratories (Table\u00a04).\nTable\u00a04Overview of IFN\u03b3 ELISPOT results from phase II\/2006 of the CIMT monitoring panelD1 FluD2 CMVD2 FluD4 FluD5 CMVD6 FluD7 FluD8 CMVMin44,1181,79133,80358,8241,74548,38714,7201,698(I)Medium28,8231,08816,39549,96099914,2654,6691,023Max10,3453961,23141,0963914,4581,706269Z144,118596\u2013\u201359648,38714,720318(II)Z2\u20131,732\u2013\u20131,7454,7392,2221,698Z3\u20131,333\u2013\u20131,117\u20132,9821,292Z4\u2013\u2013\u2013\u2013774\u20132,273447Z5\u2013997\u2013\u20131,15717,6473,8271,209Z610,3451,03114,151\u20131,006\u20131,7061,005Z7\u20131,317\u2013\u20131,06110,0006,0001,661Z832,258396\u201358,8243914,4583,623269Z928,571966\u201341,09691214,4238,9551,081Z10\u20138471,231\u20138067,4263,052696Z11\u20131,04433,803\u20131,08720,3393,6701,273Z12\u20131,791\u2013\u20131,38710,6193,0571,583Z13\u20131,008\u2013\u20139494,6154,615770Detected by4\/1312\/133\/132\/1313\/1310\/1313\/1313\/13(III)Detected %3192231510077100100(I) Minimum, mean and maximum frequencies of antigen-specific cells(II) Results obtained from the individual centers Z1\u2013Z13. All frequencies are indicated as 1 per x seeded PBMC(III) Number and percentage of centers that detected each of the eight possible responses\nComparison of the results obtained in both phases\nWhen the mean frequencies of all T-cell responses in both testing rounds were compared, it became clear that there was a difference in the distribution of reactivities (Fig.\u00a03). In the tetramer assay, the mean T-cell frequency of the six possible positives in the first phase was 1 per 2,083 CD8+ T-cells. This value was 1 per 1,769 CD8+ T-cells for the eight possible positives in the second phase. Similarly, the mean T-cell frequency of the responses detected in IFN\u03b3 ELISPOT was 1 per 22,369 PBMC for Phase I\/2005 but 1 per 14,653 PBMC for Phase II\/2006. To allow a comparison of the overall performance in both phases of the panel, we therefore decided to define theoretical thresholds for high, moderate and low T-cell responses and then to compare data of the participating laboratories within these groups.\nFig.\u00a03Distribution of antigen-specific T-cell frequencies in the two testing phases as obtained by tetramer staining (a) and IFN\u03b3 ELISPOT assays (b). The figure shows the six reactivities (filled circle) and the calculated mean of all reactivities from phase I\/2005 (filled line) as well as the eight reactivities (open circle) and calculated mean of all reactivities from phase II\/2006 (open line). The frequency of antigen-specific T-cells is indicated on the y-axis as 1 per x counted CD8+ T-cells for the tetramer test and as 1 per x seeded PBMC for the ELISPOT assay\nIn order to define such thresholds for low, medium and high T-cell responses, we first displayed the probability of detecting each of the 14 different reactivities as a value in a coordinate system and inserted a trendline. For both the tetramer assay and the ELISPOT assay, we observed a clear correlation between the frequencies of antigen-specific T-cells and the number of participating centers that were able to detect these populations. We then calculated the theoretical frequencies at which 90% (y\u00a0=\u00a090) and 50% (y\u00a0=\u00a050) of all participants could detect a given response (Fig.\u00a04a, b) and used these two thresholds to divide all reactivities into three distinct classes of T-cell responses (\u201chigh\u201d, \u201cmoderate\u201d and \u201clow\u201d).\nFig.\u00a04Probability of detecting a reactivity by a tetramer staining, or b IFN\u03b3 ELISPOT assay. A trendline was inserted on the basis of results from all 14 reactivities from both phases of the panel. The figure shows the six reactivities from phase I\/2005 (filled squares) and the eight reactivities from phase II\/2006 (open squares). The frequency of antigen-specific T-cells is shown on the x-axis in 1 per x counted CD8+ T-cells for the tetramer assay (a) or 1 per x seeded PBMC for the ELISPOT assay (b). X-values for y\u00a0=\u00a090% and y\u00a0=\u00a050% are indicated by the broken lines\nFor the tetramer assay, T-cell frequencies exceeding 1 per 1,200 CD8+ T-cells were therefore classified as \u201chigh\u201d, whereas frequencies of less than 1 per 7,650 CD8+ were classified as \u201clow\u201d (Fig.\u00a04a). Following the same rules for the ELISPOT assay, T-cell responses of at least one IFN\u03b3 spot per 2,850 PBMC can be considered as \u201chigh\u201d and T-cell responses of less than one spot per 19,000 PBMC as \u201clow\u201d (Fig.\u00a04b).\nWith these calculated assay-specific thresholds for high, moderate and low T-cell responses, we compared the results obtained in the two phases. For the tetramer assay, the ability to detect high frequency T-cells (>1 per 1,200 CD8+) did not differ in the two phases, and was not influenced by the number of CD8+ analyzed, as previously seen for each of the two phases separately (Fig.\u00a05a). However, for moderate and low T-cell frequencies, we found that they could be successfully detected in only 54% of cases in the first phase but this improved to 77% in the second phase. Moreover, here, the number of cells counted did have an impact on the ability to detect low frequency T-cells. In the first phase, only 14% were detected when less than 30,000 CD8+ were counted, as compared to 71% when more than 30,000 CD8+ T-cells were counted. The same trend was observed in phase II\/2006, but in this case 40% of assays with less than 30,000 CD8+ successfully detected the moderate to low T-cell frequencies compared to 83% counting more than 30,000 CD8+ (Fig.\u00a05a).\nFig.\u00a05a Percentage of reactivities actually detected by tetramer staining. The first two groups of bars show the detection rate for the nine high reactivities (>1 per 1,200 CD8+ T-cells) in phase I\/2005 and phase II\/2006. The next two groups of bars show the detection rate for five moderate to low reactivities (<1 per 1,200 CD8+) in phase I\/2005 (third group) or phase II\/2006 (fourth group). The open bars represent all tests performed, grey bars represent results obtained in tests that were performed on more than 3\u00a0\u00d7\u00a0104 CD8+ T-cells and filled bars represent results obtained in tests that were performed on less than 3\u00a0\u00d7\u00a0104 CD8+ T-cells. b Percentage of reactivities detected in IFN\u03b3 ELISPOT assays. The first two groups of bars show the rate of detection of the four high reactivities (>1 per 2,850 PBMC in phase I\/2005 and phase II\/2006. The next two groups of columns show the rate of detection for the ten moderate to low reactivities (<1 per 2,850 PBMC) in phase I\/2005 and phase II\/2006. The open bars represent the performances of all centers in the respective panel phase, grey bars represent results obtained from the five centers that already fulfilled at least three of the four minimum criteria in phase I\/2005 and filled bars represent results obtained from centers that fulfilled less than three of the four minimum criteria in phase I\/2005\nWe then analyzed the capacity of the laboratories to measure either high T-cell responses (>1 per 2,850 PBMC) or low to moderate T-cell responses (<1 per 2,850 PBMC) in the ELISPOT assay. This analysis was performed for two defined subgroups of participants. The first subgroup included those five centers (Z3, Z4, Z7, Z8 and Z9) that already fulfilled three or four of the requirements in the first phase of the panel. These five centers did not have to introduce any change or at least no major changes to their protocol for the repetition of the experiments in phase II. The second subgroup included the new center Z13 (led by a colleague that had been in a laboratory that only fulfilled one of four requirements in phase I) and all others that had fulfilled only one or two of the four requirements in the first phase. All laboratories in this second group had to introduce marked changes to their locally established protocols. Similar to the tetramer analysis, the new requirements were not necessary to detect antigen-specific responses among the category of high T-cell frequencies in either the first or second phases (Fig.\u00a05b). However, applying the set of rules defined in phase I markedly improved the capacity of centers to detect the low to moderate T-cell responses. The first subgroup detected a total of 68% of the low to moderate reactivities in phase I, whereas the second subgroup detected only 20% (Fig.\u00a05b). After harmonization of the protocols, both subgroups performed equally well. In addition, the inter-group variability in detecting positive responses was reduced in phase II (percentage of detected responses ranged from 38 to 88% with a mean of 67\u00a0\u00b1\u00a016%) as compared to phase I (percentage of detected responses ranged from 0 to 100% with a mean of 55\u00a0\u00b1\u00a033%).\nExperience does not equal performance\nAmong the 13 centers that had participated in phase II, tetramer stainings had been performed for 1\u20138\u00a0years. Similarly, the experience in the ELISPOT technology varied between 1 and 10\u00a0years. For both techniques, we could not find any correlation between the years of experience and the ability to detect T-cell responses, not even among the subgroups of moderate or low T-cell responses (not shown).\nDiscussion\nWhenever new techniques are introduced to the scientific community, they are first only available to a small group of expert laboratories. If these assays are robust and applicable for specific research or routine applications, they spread to the international community. In general, the \u201coriginal\u201d protocol then undergoes several adaptations in order to meet specific needs. On the one hand, changes can be beneficial and result in the improvement of protocols. On the other, this evolutionary process leads to employment of many different protocol variants, limiting comparison of the study results obtained by different laboratories. Thus, standardization approaches should be omitted during the initial development but are absolutely required when assays have become firmly established. In recent years, several activities aiming at the harmonization of techniques used to monitor the presence of antigen-specific T-cells have been initiated for ELISPOT [28\u201331], tetramer staining [32] and ICS [33\u201336]. While these studies showed the feasibility, general applicability and the diversity of performance among participants, they were not designed to either systematically investigate the influence of distinct protocol variables nor to test whether changes to these parameters can lead to a global improvement of the group. The CIMT monitoring panel is the first initiative that has now introduced the two-step approach proposing a strategy where technical variables that influence the performance of a defined assay are first systematically identified (\u201cfirst step\u201d) followed by a new testing phase where resultant protocol changes are validated under controlled conditions within the same group of investigators (\u201csecond step\u201d).\nAs soon as a number of protocol variables that might have influenced the sensitivity and the quality of the tests were identified in the first phase of our study, it was decided to validate this finding in a second phase. Because this two-step approach was not initially foreseen, the second phase was performed with PBMC samples obtained from different donors than those used in the first round. The distribution and the frequencies of detectable T-cell responses directed against the chosen model antigens were different in the first and second group of donors (Fig.\u00a03) precluding a direct comparison of the results obtained in both phases of the panel. To circumvent this problem, two assay-specific frequency thresholds were introduced that allowed us to distinguish classes of T-cell responses (low, moderate and high) (Fig.\u00a04a, b). Clearly, high-frequency T-cell responses were detected irrespective of the protocol used and as such did not allow the identification of factors that exert a strong influence on the sensitivity and variability of the protocols used. Relevant parameters could only be detected when the comparison was focused on the detection of T-cells that are present at low to moderate frequencies in PBMC. This finding should be taken into account when selecting model antigens for use in monitoring panels [37], in particular by laboratories that are interested in the detection of peripheral tumor-specific T-cells, which are often present at low frequencies, even after vaccination.\nAlthough our experiments do not specifically address the question of detection limits for the ELISPOT and tetramer assays, we could detect a high variability in the sensitivity of protocols used by the different participants. The majority of labs (y\u00a0=\u00a090%) is able to detect responses with a frequency above 1 per 1,200 CD8+ T cells in the tetramer assay or responses with a frequency above 1 per 2,859 PBMC in the ELISPOT. Note that some of the centers could reliably detect a response with a frequency of about 1 per 8,000 CD8+ T cells in the tetramer assay and about 1 per 40,000 PBMC in the ELISPOT assay. These low frequencies are in the range of that is commonly reported as the detection limit for internally validated protocols for both technologies [39, 40, own unpublished observations]. Another important task of standardization efforts should be to decrease the variation of results obtained in a group of several laboratories down to the stable and low values (15\u201330%) that can be reproducibly found within single labs. In order to quantify the variation of results among laboratories we calculated the coefficient of variation for all 14 reactivities of the two panel phases. The CVs were determined on the base of centers that were able to detect the respective T cell response and the results are shown in supplementary Tables S1a, b. As expected, the CVs we found in our inter-laboratory testing project were higher than those reported from intra-center analysis [39, 40].\nIn the ELISPOT assay, the background spot numbers obtained by the different participants varied greatly, but we were unable to correlate this finding to a distinct variable. Since the spontaneous cytokine secretion impacts significantly on the sensitivity of this assay, factors that especially influence the non-specific spot production, possibly the medium type or serum source, will need to be systematically analyzed in a separate study.\nThe main conclusions from our study have been drawn on the basis of subgroup analyses. Although the CIMT panel in general (13 centers in this initial action), and consequently the subgroups formed during the analysis were rather small, we could already identify statistically significant differences in the ability to detect positive responses. We concluded that the number of counted CD8+ T-cells is the most influential crucial factor for the tetramer assay and that the combination of a resting-time and a high number of PBMC leads to increased sensitivity in the ELISPOT assay. This suggests that the impact of the identified technical variables on the quality of the assays is high. In order to identify those protocol variables that lead to more subtle differences, a larger group of participants would be needed.\nIn addition to the systematic identification of variables that correlate with sensitivity\/insensitivity of various assays, inter-laboratory testing projects also allow the rapid evaluation of individual performance among a group. Interestingly, the finding that experienced laboratories did not perform better than laboratories which recently applied these techniques strongly suggests that non-optimal protocols, once established in a lab, can commonly be maintained for several years. Periodic comparison of local protocols with those of other centers is recommended. Even if a new staff member uses an established protocol, it is recommended to have them participate in inter-laboratory testing\/teaching exercise. Regular participation in multi-center comparisons could thereby help to optimize and validate participants\u2019 performance over time and to maintain sensitive protocols or minimal standards. This is of great importance when material from expensive clinical trials has to be analyzed.\nAll data from the CMV-serology, from the pre-testing experiments and from the results generated by the participating laboratories in ELISPOT and tetramer staining were taken together for each donor in order to qualitatively validate the presence of CMV- and influenza-specific T-cells. To estimate the quantity, i.e. the frequency of specific T-cells in each donor, we calculated the average of all qualitatively positive results, as well as the standard deviations. This procedure constitutes only an approximation of the real number of antigen-specific cells present in a given sample, and cannot be taken as a method for determining absolute T-cell frequencies. Cell samples that contain pre-defined numbers of antigen-specific T-cells (e.g. spiked T-cell clones), especially tumor-reactive T-cells, are not easily available for use in multi-center comparisons, although such standard samples are urgently needed. We see this as one major bottle-neck for the optimization and standardization of immunomonitoring techniques. Methods to generate such standard samples for broader use will therefore be elucidated with high priority in the near future for the next phases of this international collaboration. Another big challenge will be to define accepted rules for the settings of the equipment used in these analyses (flow cytometer or ELISPOT reader) in order to uniformly process and analyze the raw data. Ten from eleven laboratories that performed the ELISPOT assay in the first phase used an ELISPOT reader for spot counting. It is known that spot counts between centers can differ significantly and this may be explained by the use of different reading machines, different settings for the same type of machine or by the experience of the operator. Within this group, four different commercially available reading systems were used (supplementary Table\u00a0S2). We were not able to identify differences between the types of ELISPOT readers. A new ELISPOT panel phase is currently in preparation, that will specifically focus on the performances of different ELISPOT readers and try to introduce tools to control inter center variation. In addition, none of the participant reported on the use of live\/dead cell discrimination on thawed PBMC samples for the FACS-based experiments. Whether the combination of staining with Ab\/HLA-tetramers and vital dyes or with a resting phase is beneficial for increasing the sensitivity of the tetramer staining assay could be addressed in future testing actions.\nResults from a proficiency panel of 36 laboratories from nine different countries in which the ELISPOT assay was validated are now also being reported [41]. This initiative, conducted under the aegis of the Cancer Vaccine Consortium (CVC), was mainly designed to offer an external validation to the participating laboratories but the in depth analysis of the obtained data sets lead to similar findings and recommendations as the CIMT monitoring panel. It confirmed that a resting phase of cells prior to addition to the ELISPOT plates is advantageous and should therefore be generally recommended. Furthermore, a long year experience in a technology did not guarantee for a sensitive test and failure to detect specific T cell responses concentrated on the weak responses. The fact that two independent initiatives come to similar findings is surely notable and shows the necessity to carry on running proficiency panels.\nLast but not least, we would like to stress that even the best guidelines and protocols alone cannot guarantee good performance. Monitoring of antigen-specific T-cell responses requires skills as well as experience. Participation in immunomonitoring panels cannot compensate for the need to constantly educate and train staff and to develop specific expertise for covering individual needs. Nevertheless, we strongly believe that by organizing further two-step inter-laboratory testing projects, the CIMT monitoring panel will be able to improve the sensitivity of the assays used for immunomonitoring as well as to actively participate in the harmonization of these assays, which is required to enable the comparison of immunotherapeutic trials performed in different centers.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nESM (PPT 82\u00a0kB)","keyphrases":["standardization","tetramer","elispot","interlaboratory testing"],"prmu":["P","P","P","P"]}
{"id":"Histochem_Cell_Biol-3-1-2137945","title":"Physiological importance of RNA and protein mobility in the cell nucleus\n","text":"Trafficking of proteins and RNAs is essential for cellular function and homeostasis. While it has long been appreciated that proteins and RNAs move within cells, only recently has it become possible to visualize trafficking events in vivo. Analysis of protein and RNA motion within the cell nucleus have been particularly intriguing as they have revealed an unanticipated degree of dynamics within the organelle. These methods have revealed that the intranuclear trafficking occurs largely by energy-independent mechanisms and is driven by diffusion. RNA molecules and non-DNA binding proteins undergo constrained diffusion, largely limited by the spatial constraint imposed by chromatin, and chromatin binding proteins move by a stop-and-go mechanism where their free diffusion is interrupted by random association with the chromatin fiber. The ability and mode of motion of proteins and RNAs has implications for how they find nuclear targets on chromatin and in nuclear subcompartments and how macromolecular complexes are assembled in vivo. Most importantly, the dynamic nature of proteins and RNAs is emerging as a means to control physiological cellular responses and pathways.\nIntroduction\nIt is an irony of science that the discovery of fundamental principles is often delayed. A case in point is the cell biological investigation of the cell nucleus. The nucleus is arguably the functionally most prominent cellular organelle as it houses the vast majority of the genetic information and is the exclusive site of essential processes including transcription, RNA processing, replication and DNA repair. Yet, our understanding of how the nucleus is organized and how nuclear architecture contributes to cellular function is only now emerging and still lags behind that of most other cellular compartments. Fortunately, pioneering studies over the past decade have uncovered several of the fundamental principles which determine nuclear architecture and function (Lanctot et al. 2007; Misteli 2001; Spector 2003). These studies have revealed the presence of distinct structural elements, particularly lamin proteins and possibly short actin filaments, within the nucleus and they have led to the identification of a large number of distinct nuclear subcompartments in which specific nuclear functions, such as the synthesis and processing of ribosomal RNA in the nucleolus, occur (Burke and Stewart 2006; Gruenbaum et al. 2005; Handwerger and Gall 2006; Hernandez-Verdun 2006; Lamond and Sleeman 2003) (Fig.\u00a01a). In addition, it has become clear that the genome is non-randomly organized within the spatial confines of the nucleus with specific chromosomes and genes preferentially localizing to specific sites (Lanctot et al. 2007; Meaburn and Misteli 2007) (Fig.\u00a01b, c). These observations paint a picture of a highly complex and heterogeneous cell nucleus. They also indicate that some properties of the nucleus are counterintuitive. For example, despite the requirement to accommodate \u223c2\u00a0m of DNA within the cell nucleus of a diameter of typically \u223c10\u00a0\u03bcm, chromatin is estimated to only occupy about 15% of the nuclear volume.\nFig.\u00a01Nuclear architecture and function. The mammalian cell nucleus contains a large number of non-random and structural features. a Nuclear lamins (green) form a network of architectural elements at the periphery. This nuclear lamina is implicated in mechanical support of the nucleus, organization of chromatin (red\/blue) in the nuclear interior, as a platform for signaling events and in the sequestration of nuclear proteins. b, c Genomes are non-randomly organized within the nucleus. b Each chromosome occupies a distinct chromosome territory (red chromosome 14; green chromosome 17 in human breast epithelial cells). c Pericentromeric heterochromatin regions (white) from multiple chromosomes often cluster into chromocenters. Bar 6.3\u00a0\u03bcm. Image courtesy: a Gianluca Pegoraro, b Karen Meaburn, c Manjari Mazumdar, all National Cancer Institute, NIH\nA key question in understanding nuclear architecture is how protein and RNAs move within the nucleus. This is a key issue if we are to gain a full appreciation of what the nuclear environment is like. More importantly, the mode of nuclear motion of molecules has physiological implications as it affects how proteins find their target sequences in the genome, how RNAs are exported from the nucleus upon transcription and how proteins are sequestered within the nucleus for regulatory purposes. Some initial insights into these issues are summarized in broad terms here.\nThe motion of RNA and proteins in the cell nucleus\nWe have long known that proteins and RNAs must be able to move within the cell nucleus. Messenger RNAs traverse the nucleus and are promptly exported from the nucleus after their synthesis and complete processing. Newly synthesized proteins on the other hand are imported into the nucleus and rapidly disperse throughout its volume, implying effective intranuclear trafficking. Furthermore, proteins rapidly relocalize within the nucleus upon experimental or physiological changes in conditions. For example, inhibition of protein synthesis leads to rapid and dramatic relocalization of a large number of nucleolar proteins (Andersen et al. 2005), and pre-mRNA splicing factors swiftly respond to transcriptional activation and inhibition (Jim\u00e9nez-Garc\u00eda and Spector 1993; Misteli et al. 1997). How proteins and RNAs move within the nucleus and how they find their targets, however, was unknown for long. It was not clear whether movements are energy dependent, whether they occur by directed transport or what the speed of intranuclear trafficking is. These fundamental questions have recently been answered (Misteli 2001).\nThe investigation of intranuclear protein and RNA dynamics was made possible by the development of techniques to visualize and measure the motion of these molecules by time-lapse microscopy. Tracking of proteins by photobleaching methods revealed a surprising degree and speed of intranuclear trafficking. The analysis of tracers such as GFP or dextran which do not undergo specific interactions with DNA or other proteins indicated that the nuclear environment is permissive for rapid diffusional motion (G\u00f6risch et al. 2005; Misteli 2001; Seksek et al. 1997). The diffusion of molecules within the nucleus is only limited by the steric constraints imposed by chromatin and nuclear bodies (G\u00f6risch et al. 2005; Misteli 2001; Seksek et al. 1997). The typically measured diffusion coefficients of 10\u2013100\u00a0\u03bcm2\u00a0s\u22121 are similar to those observed in the cytoplasm and are only \u223c5 times less than that in solution. As expected for diffusion, this rapid motion is energy-independent and non-directional.\nRNA motion within the nucleus is similarly rapid and non-directional. Several methodological approaches involving fluorescently labeled, microinjected, engineered or endogenous RNAs demonstrate that ribosomal RNAs as well as polyA-RNAs move freely in a non-directional manner with a diffusion coefficient of 0.03\u20130.1\u00a0\u03bcm2\u00a0s\u22121 within the nucleus (Ritland-Politz et al. 2006; Shav-Tal et al. 2004). A diffusion coefficient of this magnitude is sufficient to ensure transport of an RNA particle from deep within the nucleus to the cytoplasm within a few minutes, consistent with biochemical observations on kinetics of RNA maturation and transport. Thus energy-independent, diffusion-based movement of RNA particles alone can account for the observed kinetics of RNA export, and no active mechanisms are required to ensure rapid export. An impressive demonstration of the non-directional motion of mRNA comes from studies in which a nascent RNA is visualized at its site of transcription and its export to the cytoplasm measured in living cells (Ritland-Politz et al. 2006; Shav-Tal et al. 2004). These studies show that RNAs synthesized from genes positioned in proximity to the nuclear envelope diffuse away from their site of synthesis in all directions rather than follow a direct path to the nearest nuclear pore. This observation powerfully demonstrates the non-directed, diffusion-based motion of RNAs in the nucleus.\nTrafficking as a means of targeting\nOne of the conceptually most challenging problems in cell biology is the question of how molecules find their specific targets within a cell or within an organelle. This problem is particularly complex in the cell nucleus where transcriptional regulators need to find their specific target genes amongst the myriad of potential binding sites within the genome. Somewhat counter-intuitively, the non-directional, but rapid, motion of proteins within the cell nucleus provides a means to ensure targeting of proteins to specific genome locations (Misteli 2001). The power of targeting by random diffusion within the nucleus is best illustrated when considering how a transcription factor finds its target genes but it also applies to targeting of proteins to nuclear compartments or any other nuclear site.\nProteins a priori do not know where their targets are and we do know of any directed transport systems, such as a molecular motor-based mechanism, that would bring a factor to a specific gene or a specific location within the nucleus. Thus, the only way for a transcription factor to find its target is to scan the genome. The intrinsic ability of proteins to rapidly move within the nucleus by diffusion-based mechanisms permits such genome scanning (Misteli 2001). This occurs by a transcription factor freely diffusing within the nucleoplasm until it interacts by chance with chromatin. The molecule will now probe whether the sequence it has encountered is a specific binding site such as in the promoter of one of its target genes. If it is, the transcription factor will be captured and stably associated with its specific target site. If the sequence is not a binding site, the molecule, after a short interaction, will dissociate from chromatin and continue its diffusional journey through the nucleus.\nIt might appear at first glance that such a stop-and-go model for genome scanning would be insufficiently effective. However, the observed dynamic properties of transcription factors are entirely consistent with this model. We know that the residence time of most transcription factors even on specific DNA binding sites is in the order of a few seconds and that their interaction with non-specific sites is even faster, most likely in the order of tens of milliseconds (Gorski et al. 2006; Houtsmuller et al. 1999; McNally et al. 2000; Phair et al. 2004; Sprague et al. 2006). Assuming these time scales for binding, one can calculate that it takes a single transcription factor molecule only a few minutes to search the entire genome space. Considering that most transcription factors exist in several thousand copies and have multiple target genes, their random diffusional motion is entirely sufficient to ensure a steady supply at their target genes. This assumption is further supported by direct measurement of transcription factor flux on an endogenous rRNA promoter demonstrating the collision of several hundred molecules per second (Dundr et al. 2002). Since proteins similarly move by diffusional motion within the cytoplasm, it stands to reason that the random scanning is also a key mechanism of protein targeting in the cytoplasm and represents a universal mechanism for how proteins find their targets.\nA key feature, and a requirement, in a genome-scanning model of targeting is that the interactions of proteins with chromatin are transient. This has been confirmed by photobleaching methods on a large number of DNA binding proteins (Gorski et al. 2006; Phair et al. 2004; Sprague et al. 2006). The transient nature of protein\u2013chromatin interactions is important for three reasons. First, it allows proteins to maintain a high rate of motion and thus allows faster scanning. Were protein\u2013chromatin interactions static, they would get stuck at non-specific or incorrect binding sites which would slow down their overall motion. Second, the short-life of protein\u2013chromatin interactions continuously makes available binding sites which can then be scanned by diffusing transcription factors. If proteins interacted for extended periods of time on chromatin, non-specific or improper binding would block access of the correct factors. Third, the dynamic dissociation allows for change. Were protein complexes permanently bound to their target sites, changes in transcriptional activity such as in response to physiological stimuli could only occur after active removal of the bound complex, presumably by dedicated and specialized molecular machinery. In contrast, in a dynamic binding model the natural flux of proteins provides a window of opportunity for association of a distinct regulator each time a bound protein or complex dissociates as part of its normal binding cycle.\nProtein dynamics as the key for formation of nuclear compartments\nA hallmark of the mammalian cell nucleus is the presence of distinct subnuclear compartments and domains in which particular functions occur (Handwerger and Gall 2006; Hernandez-Verdun 2006) (Fig.\u00a02). The prototypical nuclear compartment is the nucleolus, a distinct intranuclear compartment in which ribosomal RNAs are synthesized and partially processed. Other prominent nuclear domains include splicing factor compartments which serve as storage and assembly sites for spliceosomal components, and the Cajal bodies which are possibly involved in maturation of small nuclear RNPs. The structure of intranuclear bodies is not determined by a membrane, and the principles underlying their biogenesis are extremely poorly understood. The recently revealed dynamic properties of proteins in nuclear compartments give a hint as to the principles involved in subnuclear compartment assembly.\nFig.\u00a02Intranuclear compartments. The mammalian cell nucleus contains a larger number of distinct intranuclear compartments. The nucleolus is the site of ribosomal RNA synthesis and is a prototypical nuclear body. The complex organization of the nucleolus is revealed by multi-color staining of distinct nucleolar components. RPA43 RNA polymerase I transcription factor, Fibrillarin snRNP component, B23 rRNA processing factor. Image courtesy of Miroslav Dundr, Rosalind Franklin University of Medicine and Science, Chicago\nThe key property in understanding the formation of nuclear compartment is the remarkable fact that the association of proteins with their compartments is highly dynamic (Dundr et al. 2004; Kruhlak et al. 2000; Phair and Misteli 2000). Typical residence times of proteins within a compartment are in the seconds range. In practical terms the dynamic behavior of nuclear body proteins means that they undergo repeated rapid cycles of association and dissociation between the nuclear body and the nucleoplasm. As a consequence, a nuclear body is in perpetual flux and its structure is determined by the ratio of on-rate versus off-rate of its proteins.\nGiven these observations, the simplest scenario to explain the formation of nuclear subdomains is a self-organization model in which the collective sum of all interactions amongst proteins in a body establishes and determines its structure (Carrero et al. 2006; Misteli 2007). In this model, nuclear bodies are essentially dynamic protein aggregates which form as a consequence of multiple, transient interactions amongst a large number of proteins. This is consistent with the lack of defining intranuclear membranes and with the absence of any known dedicated structural elements within intranuclear bodies. While dynamic observations on nucleoli, splicing factor compartments and Cajal bodies support this view, this model has not been rigorously tested experimentally. A key prediction, and a way to experimentally address this fundamental question, is that it should be possible to create nuclear bodies de novo and that any nuclear body component should be able to nucleate the formation of a body. Experimental systems to test these predictions are now available, and we are eagerly awaiting the results of these key experiments.\nThe importance of protein and RNA dynamics in regulatory events\nThe observation of dynamic properties of proteins in the cell nucleus of living cells has suggested that dynamic trafficking is an intrinsic property of proteins and RNAs. The dynamic behavior of proteins and RNAs clearly contributes to their proper function. However, is dynamic trafficking important for physiological regulation? Several observations demonstrate that dynamic trafficking of both proteins and RNA indeed can have regulatory function by several means.\nOf particular importance as a regulatory mechanism is intranuclear sequestration (Fig.\u00a03). Accumulation of a protein at a particular nuclear site or within a nuclear compartment can serve to either increase the local concentration of a factor at that site or to reduce its abundance in the rest of the nucleus. Several proteins are now known to accumulate, in particular nuclear locations in response to physiological cues and their sequestration is thought to contribute to their cellular function.\nFig.\u00a03Intranuclear retention and sequestration as a regulatory means. a Retention of proteins within intranuclear subcompartments regulates their nuclear concentration and their rate of nuclear export. b Sequestration of proteins at the nuclear periphery, particularly via interaction with the lamina (blue) controls their nucleoplasmic concentration and their availability at target genes. Sequestration can act both as a negative or a positive regulatory mechanism. Sequestration of an activator leads to repression, sequestration of a repression leads to activation. c Retention of partially or fully processed RNA within intranuclear compartments modulates RNA export rate\nPrototypical example of sequestration as a regulatory mechanism is the accumulation of the ubiquitin ligases MDM2 and the von Hippel-Lindau tumor suppressor protein VHL in the nucleolus (Mekhail et al. 2005, 2007). These two proteins are responsible for the ubiquitination of the p53 tumor suppressor and the hypoxia-induced factor HIF, respectively. Modification of these two targets leads to their nuclear export and degradation in the cytoplasm. Since ubiquitination is a major regulatory mechanism in their function, the concentration of the ubiquitin ligase in the nucleus significantly determines their fate. Both MDM2 and VHL diffuse freely within the nucleus. However, in response to physiological cues, both MDM2 and VHL can become sequestered in the nucleolus, thus reducing their nucleoplasmic concentration and the extent of ubiquitination of their targets (Mekhail et al. 2005, 2007) (Fig.\u00a01a). Sequestration of these proteins appears to occur by increased retention of diffusing molecules within the nucleolus. How this retention is regulated is currently unclear (Mekhail et al. 2005, 2007). The possibility that retention is a highly controlled mechanism comes from observations on nucleostemin, a nucleolar protein implicated in stem cell maintenance and cancer proliferation (Meng et al. 2007; Tsai and McKay 2005). The retention of nucleostemin in the nucleolus is controlled by a GTP\u2013GDP binding cycle which might be directly or indirectly linked to signaling pathways. In addition to nucleostemin the localization of several additional GTP-binding domain containing nucleolar proteins seems to be controlled in a similar fashion (Meng et al. 2007; Tsai and McKay 2005).\nIntranuclear sequestration can also be a means of negative regulation. The basic helix\u2013loop\u2013helix transcription factor Hand1 is expressed in trophoblast stem cells and is required for their differentiation along several lineages (Martindill et al. 2007). Hand1 is sequestered in the nucleolus and upon stimulation during differentiation is rapidly released and moves into the nucleoplasm where it presumably acts on target genes. Hand1 is retained in the nucleolus by its interaction with I-mfa, and this interaction is sensitive to phosphorylation of Hand1. Upon phosphorylation Hand1 dissociates from I-mfa and is released from the nucleolus. Interestingly, both the sequestration of Hand1 in the nucleolus and its release are functionally important. Premature release of Hand1 triggers differentiation, whereas the presence of Hand1 is also required to maintain the stem-cell potential of trophoblast stem cells. Thus nucleolar sequestration of this key cell fate regulator serves a dual regulatory role (Martindill et al. 2007).\nMost examples of intranuclear sequestration involve the nucleolus. However, the nuclear lamina has recently also emerged as a major site for transcription factor sequestration (Heessen and Fornerod 2007) (Fig.\u00a01b). Prominent transcription factors which associate with the nuclear periphery either via interaction with the lamins or with the inner nuclear membrane proteins include c-fos, Oct-1 and Rb (Imai et al. 1997; Ivorra et al. 2006; Johnson et al. 2004). It is tempting to speculate that the association of these factors with the periphery reduces the intranuclear concentration and thus their availability at target genes (Heessen and Fornerod 2007). A physiological role for such peripheral sequestration is most strongly suggested by observations on the pro-proliferation factor c-fos which associates with the lamina in quiescent cells but is released upon entry of cells into the cell cycle correlating with activation of some of its target genes (Ivorra et al. 2006). Similarly, release of Oct-1 from the lamina correlates with activation of collagenase, one of its prime target genes (Imai et al. 1997). The sequestration of transcription factors at the nuclear periphery is a potentially powerful and simple mechanism of transcriptional regulation. It will be important to determine how widespread this mechanism is and how precisely the association of transcription factors with the lamina is controlled.\nDynamic trafficking of RNA is similarly used as a regulatory mechanism (Prasanth and Spector 2007). In particular, it appears that RNA retention can act as a quality control mechanism for RNA export. For one, incompletely spliced or processed RNAs are retained at their sites of transcription and not released into the nucleoplasm for export. In addition, pools of stable polyadenylated RNAs are enriched in the nuclear splicing factor compartments, although their function remains unclear (Huang et al. 1994). But retention is also emerging as a mechanism for regulation of specific RNAs. A non-coding RNA transcribed from the mouse Cat2 gene encoding a cationic amino acid transporter is retained in the nucleus via a dedicated 3\u2032 end containing extensive repeat sequences (Prasanth et al. 2005). These sequences are responsible for accumulation of the RNA in a nuclear compartment identified as paraspeckles (Fig.\u00a01c). This retention serves a physiological function because upon stress the Cat2 RNA is cleaved, released from paraspeckles and rapidly exported into the cytoplasm for translation, thus ensuring a rapid physiological stress response. Although the Cat2 RNA is to-date the best characterized example of such retention, a similar mechanism is likely at work for the migration-stimulating factor (MSF) mRNA whose 3\u2032 end resembles that of Cat2 and is retained within the nucleus (Kay et al. 2005). Under conditions of MSF secretion the RNA appears to be released from the nucleus and rapidly translated. A further candidate for control via nuclear retention is the neuron-specific gomafu RNA which is retained in intranuclear compartments. Interestingly, gomafu does not seem to encode for any protein and might thus be a non-coding regulatory RNA which is retained in the nucleus (Sone et al. 2007).\nConclusions\nThe past few years have seen a dramatic change in how we view the cell nucleus. We have come to appreciate the presence of distinct structural elements within the nucleus, the presence of a multitude of intranuclear bodies and the fact that genomes are non-randomly organized within the nuclear space. One of the most consequential findings has been the realization that just about every aspect of nuclear organization is highly dynamic. Both proteins and RNAs move rapidly within the nucleus, and they only transiently interact with chromatin and nuclear bodies. It is now clear that the dynamic nature of nuclear components is a fundamental property and has implications for how molecules are targeted to their final destinations and for how intranuclear compartments form. Most importantly, it is becoming clear that the dynamic properties of nuclear proteins are critical for various mechanisms of physiological regulation, particularly via sequestration and retention of proteins and RNA. It is likely that these recently discovered examples of regulation by modulation of dynamic interactions within the cell nucleus are only the proverbial tip of the iceberg, and it stands to reason that we should consider the contribution of dynamic interactions and trafficking in any nuclear event we investigate in the future.","keyphrases":["rna","dynamics","diffusion","nuclear architecture"],"prmu":["P","P","P","P"]}
{"id":"Mod_Rheumatol-4-1-2279153","title":"Retrospective clinical study on the notable efficacy and related factors of infliximab therapy in a rheumatoid arthritis management group in Japan: one-year clinical outcomes (RECONFIRM-2)\n","text":"Biologics targeting TNF have brought about a paradigm shift in the treatment of rheumatoid arthritis (RA) and infliximab, anti-TNF-\u03b1 chimeric monoclonal antibody, was marketed in 2003 in Japan. We previously reported on the RECONFIRM study, a retrospective clinical study on the efficacy of infliximab therapy in a RA management group in Japan, where we evaluated the clinical response after 22 weeks of the therapy in 258 patients. The study reported here was aimed at reconfirming the clinical efficacy of the infliximab therapy and demographic factors related to the efficacy over a 54-week study period in 410 RA patients in the same study group. Infliximab was infused according to the domestically approved method, and the clinical response was evaluated following 54 weeks of infliximab therapy using the European League Against Rheumatism (EULAR) response criteria. Disease activity was assessed by DAS28-CRP (Disease Activity Score including a 28-joint count\/C-reactive protein). Infliximab was discontinued in 24.4% of the 410 patients at 54 weeks and 9.3% and 8.1% discontinued the therapy due to adverse events and inefficiency, respectively. Average DAS28-CRP decreased from 5.5 at week 0 to 3.1 at week 54 after the therapy. Patients in remission and those showing low-, moderate-, and high-disease activity changed from 0.0, 1.0, 9.0 and 90.0%, respectively, at the start of the study to 27.6, 11.7, 34.4 and 26.3%, respectively, at week 54. Younger age, RF-negativity and low scores of DAS28-CRP showed significant correlations with remission at week 54. EULAR response criteria\u2014good, moderate, and no response to infliximab\u2014were 37.0, 41.7 and 21.2%, respectively. In conclusion, we reconfirmed the clinical efficacy of infliximab and demographic factors related to the efficacy over a 54-week study period in 410 Japanese patients with RA using DAS28-CRP and EULAR response criteria.\nIntroduction\nRheumatoid arthritis (RA) is a chronic, systemic inflammatory disease that causes significant morbidity and mortality. RA patients should be started with DMARDs as early as possible, and among multiple DMARDs methotrexate (MTX) is considered the anchor drug and should be used first of all. However, even the use of MTX often fails to control disease activity and to prevent structural damage, and so more effective treatment strategies are needed. TNF-\u03b1 plays a pivotal role in the pathological processes of RA through the accumulation of inflammatory cells and the self-perpetuation of inflammation, leading to cartilage and bone destruction. The combinational use of biologics targeting TNF-\u03b1 and MTX has revolutionized the treatment of RA, producing significant improvements in clinical, radiographic, and functional outcomes that were not previously observed [1\u20135].\nInfliximab, anti-TNF-\u03b1 chimeric monoclonal antibody, has been marketed since July 2003 in Japan and currently provides high efficacy and potential adverse events. Although global evidence of the efficacy and safety of infliximab has accumulated, including the ATTRACT study, ASPIRE study and many others [6\u201310], there is no well-established firm evidence of the efficacy of this agent in Japan. Only the RECONFIRM study, a retrospective clinical study on the notable efficacy and related factors of infliximab therapy in a RA management group in Japan, has reported clinical evidence on its efficacy and safety; this study was performed in several major rheumatology centers in Japan [11]. However, the RECONFIRM study evaluated the clinical response following only 22\u00a0weeks of infliximab therapy in 258 patients.\nIn this RECONFIRM-2 study, we assessed the clinical efficacy and safety of infliximab and MTX over a 54-week study period when used in 410 RA patients in the same group, in order to reconfirm not only its clinical efficacy but also demographic factors related to the efficacy.\nPatients and methods\nData and information on RA patients that fulfilled the diagnostic criteria of the American College of Rheumatology (ACR) were collected from three major rheumatology centers in Japan, including the First Department of Internal Medicine, School of Medicine, University of Occupational and Environmental Health Japan, Kitakyushu; the Division of Rheumatology and Clinical Immunology, Department of Internal Medicine, Saitama Medical Center, Saitama Medical University, Saitama; and the Institute of Rheumatology, Tokyo Women\u2019s Medical University. All patients that received infliximab treatment in each institution by December 2005 were registered with this retrospective study. Demographic data, including disease duration and concomitant therapy, were collected from medical charts. The following parameters were evaluated before and at 54\u00a0weeks after the initial infliximab infusion: tender joint count (TJC) 28, swollen joint count (SJC) 28, patient\u2019s assessment of pain on a visual analog scale (patient\u2019s pain VAS), patient\u2019s global assessment of disease activity (patient\u2019s global VAS), physician\u2019s global assessment of disease activity (physician\u2019s global VAS), and C-reactive protein (CRP).\nInfliximab therapy\nInfliximab was infused to patients at zero, two, and six\u00a0weeks and thereafter every eight\u00a0weeks at a dose of 3\u00a0mg\/kg according to the drug labeling and the guidelines of the Infliximab Study Group in the Ministry of Health, Welfare and Labor in Japan [12]. Concomitant use of MTX was instituted in all cases, although the dose of MTX was determined by each attending physician.\nTherapeutic response\nDisease activity was assessed by Disease Activity Score, including a 28-joint count (DAS28)-CRP that was calculated according to the authorized formula (http:\/\/www.das-score.nl\/). The value of DAS28-CRP is reported to be less than the original DAS28 using the erythrocyte sedimentation rate (ESR), and we used a threshold of 4.1 instead of the original 5.1 as the cut-off for high activity and 2.7 instead of 3.2 as the cut-off for low activity. Thus, we defined a value of DAS28-CRP >4.1 as high activity, 2.7\u20134.1 as moderate activity, <2.7 as low activity, with <2.3 being defined as remission [9]. The response to infliximab therapy at 22\u00a0weeks was evaluated by the European League Against Arthritis (EULAR) response criteria using 4.1 and 2.7 as the thresholds for the high and low disease activities, respectively [13]. Secondary insufficiency to infliximab was defined as patients with good or moderate EULAR response at week 22, and whose DAS28 increased to >0.6 or >1.2 from week 22 to 54.\nDiscontinued subjects\nThe continuation rate of infliximab therapy was calculated by all causes of discontinuation. Cumulative hazards and associated factors were analyzed in relation to three types of discontinuation: adverse events, inefficacy, and remission.\nStatistical analysis\nBaseline characteristics of patients are summarized in Table\u00a01 using the mean, standard deviation, median, 25%, and 75% values for continuous variables. The continuation rate of infliximab therapy was calculated using the Kaplan\u2013Meier estimator in pooled samples of three institutes. The discontinuation-cause specific hazards were estimated using the Nelson\u2013Aalen estimator. Next, risk factors for the discontinuations were evaluated using the stratified proportional hazards model in order to adjust for differences among institutions. The clinical efficacies of infliximab were evaluated using a multivariate logistic regression to adjust for institutional effects. In this analysis, the last observed DAS28-CRP values were used for discontinued patients. All multivariate analyses were conducted using the variables gender, age, duration of disease, positive\/negative rheumatoid factor (RF), concomitant MTX dose, concomitant prednisolone (PSL) dose, and DAS28-CRP at baseline. All reported P-values are two-sided and are not adjusted for multiple testing. The significance level was set at a P value of <0.05.\nTable\u00a01Baseline characteristics of patients in three institutions of rheumatology in JapanMeanSDMin25%Median75%MaxFemale (%)87.6\u2013\u2013\u2013\u2013\u2013\u2013Age53.112.71946556280Duration9.48.8036.61354Stage3.01.012344Class2.20.512224RF positive (%)87.6\u2013\u2013\u2013\u2013\u2013\u2013RF (titer)213331139962412980MTX dose7.82.0068820PSL dose3.83.7004522.5CRP3.32.801.182.74.713.7TJC2810.57.30591528SJC2810.66.106101428GH63.121.9049.36680100DAS28-CRP5.51.11.94.85.66.38.0MTX methotrexate, PSL prednisolone, RF rheumatoid factor, CRP C-reactive protein, GH general health, TJC tender joint count, SJC swollen joint count, DAS disease activity score\nResults\nBaseline demographic and clinical characteristics \nThe baseline demographic and clinical characteristics of 410 patients receiving infliximab therapy are summarized in Table\u00a01. Patients had active disease at baseline as evidenced by high mean counts of tender and swollen joints, CRP levels, and the DAS28. Age, sex, and disease duration were similar among these three institutes, while the %user and dose of MTX or PSL were divergent and the disease activities as assessed by TJC, SJC, GH, and DAS28 was also different.\nContinuation of infliximab therapy\nInfliximab was discontinued in 100 cases (24.4%) among 410 patients during a 54-week period and the survival rate for infliximab use was comparable among three institutes by week 54 after the treatment according to Kaplan\u2013Meier analysis (Fig.\u00a01a). Cumulative hazards of the discontinuation during the 54-week infliximab therapy were different among the causes of discontinuation; discontinuation due to adverse events, inefficiency, remission and other causes (such as change of hospitals\/clinics and economic reasons), were 0.093, 0.081, 0.007 and 0.063, respectively (Fig.\u00a01b). Although the cause of the discontinuation was similar among three institutes, adverse events were higher in Center 1 than in the other two institutes, and remission and other causes including economic problems of the patient were greater in Center 3 than in the other two (data not shown). In 100 patients who terminated infliximab treatment, stratified Cox regression was performed to analyze factors associated with the discontinuation of the infusion. Male, older age and RF-negativity were significantly associated with the discontinuation of infliximab due to adverse reactions, whereas there was no significant factor responsible for the discontinuation due to maintained remission or a lack of efficacy (Table\u00a02).\nFig.\u00a01Continuation of the infliximab therapy in RA patients for 54 weeks. a Survival rate of RA patients treated with infliximab (n\u00a0=\u00a0410, total and three institutes) during the 54-week therapy. b Cumulative hazards of the discontinuation of infliximab therapy by week 54 of the treatmentTable\u00a02Results from a Cox regression analysis performed to examine the factors related to the discontinuation of infliximab therapyVariableAE (n\u00a0=\u00a038)Inefficacy (n\u00a0=\u00a033)Remission (n\u00a0=\u00a03)Coef.HRCLCUPCoef.HRCLCUPCoef.HRCLCUPAge0.0371.0381.0061.0700.020\u22120.0140.9870.9571.0170.38\u22120.0210.9800.8881.0810.68Gender\u22120.9400.3910.1770.8630.0200.1601.1730.3523.9100.79\u2013\u2013\u2013\u2013\u2013RA duration0.0181.0180.9831.0540.3200.0051.0050.9641.0460.83\u22120.1140.8930.6221.2800.54RF (\u00b1)\u22120.9490.3870.1710.8780.0231.5454.6860.63534.5730.13\u22121.1500.3170.0234.2910.39MTX dose0.0961.1010.9201.3180.290\u22120.1420.8670.7091.0600.160.3391.4030.7992.4650.24PSL dose\u22120.0660.9360.8481.0330.1900.0031.0030.9081.1090.95\u22120.6360.5290.1871.5000.23DAS (0 week)0.2051.2270.8901.6920.2100.1131.1200.8151.5370.49\u22120.4740.6230.1642.3670.49Coef. coefficient, HR hazard ratio,   CL 95% confidence lower limit of HR, CU 95% confidence upper limit of HR\nEfficacy of infliximab therapy\nThe average DAS28-CRP before starting infliximab was 5.5\u00a0\u00b1\u00a01.1, and this decreased to 3.4\u00a0\u00b1\u00a01.2, 3.2\u00a0\u00b1\u00a01.4, 3.1\u00a0\u00b1\u00a01.3 at weeks\u00a02, 22 and 54, respectively, after the infliximab therapy (Fig.\u00a02). Disease characteristics at baseline and after 2, 22 and 54\u00a0weeks of the inflilximab therapy were as follows: tender joints count (10.5, 4.2, 3.2 and 2.9), swollen joints count (10.6, 4.2, 2.7 and 2.3), GH (63, 34, 33 and 33\u00a0mm), and CRP (3.3, 1.0, 1.5 and 1.1\u00a0mg\/dl) (Fig.\u00a03). Before starting infliximab, the proportions of patients showing low, moderate, and high disease activity were 1.0, 9.0 and 90.0%, respectively. At week 22, patients in remission (defined as DAS28-CRP <2.3) and those showing low (<2.7), moderate (2.7\u20134.1), and high disease activity (>4.1) had changed to 27.8, 12.0, 32.4 and 27.8%, respectively, and at week 54 patients in remission and those showing low, moderate, and high disease activity were 27.6, 11.7, 34.4 and 26.3%, respectively (Fig.\u00a04). Thus, approximately 27\u201328% of the patients satisfied the remission criteria at week\u00a022 and still remained at week\u00a054 after the infliximab therapy. Also, when the responses were evaluated by the EULAR response criteria, the proportions for good, moderate, and no response to infliximab as measured by DAS28-CRP were 37.3, 43.7 and 19.0%, respectively, at week\u00a022 and 37.0, 41.7 and 21.2%, respectively, at week\u00a054 (Fig.\u00a05).\nFig.\u00a02Longitudinal analysis of DAS28 values during the 54-week study of patients using infliximab. Line in the box represents the median and the upper and lower ends of the box show the 25th and 75th percentiles of the populationFig.\u00a03Longitudinal analysis of a SJC28, b TJC28, c CRP, and d GH values during the 54-week study of patients using infliximab. Line in the box represents the median, and the upper and lower ends of the box show the 25th and 75th percentiles of the populationFig.\u00a04Changes in DAS28 values during the 54-week study of patients using infliximab. The ratios of patients who demonstrated high disease activity (defined as DAS28-CRP >4.1), moderate activity (2.7\u20134.1), low activity (<2.7) and remission (<2.3) at each observation point during the 54-week study are shownFig.\u00a05The response to infliximab therapy during the 54-week study. The ratios of patients whose responses were evaluated by the European League Against Arthritis (EULAR) response criteria are shown\nDemographic factors related to the clinical efficacy of infliximab therapy\nIn order to clarify demographic factors related to the clinical efficacy of infliximab therapy, we performed a multivariate analysis adjust for institutional differences. Younger age, RF-negativity and lower levels of DAS28-CRP were significantly associated with the clinical remission induced by infliximab therapy at week\u00a054, whereas gender, duration of the disease, dose of MTX and dose of PSL did not show a significant association (Table\u00a03). On the other hand, most of the demographic factors, except for older age, did not affect secondary insufficiency from week\u00a022 to 54 after the infliximab therapy according to the logistic regression analysis (Table\u00a04).\nTable\u00a03Results from the logistic regression analysis used to examine the factors related to clinical remission at weeks 22 and 54 induced by infliximab therapyVariablesWeek 22 (n\u00a0=\u00a0113)Week 54 (n\u00a0=\u00a0112)Coef.ORSECLCUPCoef.ORSECLCUPIntercept1.5304.6171.0340.60835.0670.1391.7705.8681.0100.81142.4450.080Center 2 vs. Center 10.1741.1900.3590.5882.4050.6290.5641.7580.3500.8863.4880.107Center 3 vs. Center 11.0722.9220.3241.5495.5100.0010.9892.6890.3251.4235.0820.002Age\u22120.0140.9860.0100.9671.0060.168\u22120.0240.9760.0100.9570.9950.014Gender0.5631.7550.3920.8133.7870.1520.1541.1660.3700.5652.4090.677RA duration\u22120.0070.9930.0150.9641.0230.6400.0101.0100.0140.9821.0390.491RF (\u00b1)\u22120.4130.6620.3390.3401.2870.224\u22120.7420.4760.3290.2500.9080.024MTX dose0.0421.0430.0680.9131.1930.5350.0051.0050.0660.8841.1430.936PSL dose\u22120.0630.9390.0350.8761.0050.070\u22120.0580.9440.0340.8821.0090.092DAS28-CRP\u22120.4640.6290.1160.5010.7890.000\u22120.2760.7590.1120.6090.9450.014Coef. coefficient HR, OR odds ratio, SE standard error, CL 95% confidence lower limit of OR, CU 95% confidence upper limit of ORTable\u00a04Results from the logistic regression analysis used to examine the factors related to the secondary inefficiency from week 22 to 54 during infliximab therapyVariablesWeek 54\u201322 >0.6 (n\u00a0=\u00a085)Week 54\u201322 >1.2 (n\u00a0=\u00a047)Coef.ORSECLCUPCoef.ORSECLCUP(Intercept)\u22121.4610.2321.0790.0281.9230.176\u22124.3310.0131.4390.0010.2210.003Center 2 vs. Center 1\u22120.0080.9920.3500.4991.9700.9810.0741.0770.4400.4552.5490.866Center 3 vs. Center 10.0711.0730.3330.5592.0600.8310.0541.0560.4260.4582.4330.899Age0.0191.0190.0110.9981.0420.0790.0321.0320.0141.0041.0620.028Gender\u22120.2830.7530.3580.3731.5200.4290.2501.2840.5110.4713.5000.625RA duration\u22120.0290.9720.0170.9411.0040.083\u22120.0200.9800.0200.9421.0190.313RF (\u00b1)\u22120.2330.7920.3660.3871.6220.5240.1641.1780.5120.4323.2120.749MTX dose\u22120.0010.9990.0690.8731.1440.9900.0391.0400.0860.8791.2300.649PSL dose\u22120.0150.9850.0360.9181.0570.6820.0191.0190.0450.9341.1120.676DAS28-CRP (0 week)\u22120.0330.9670.1180.7671.2190.779\u22120.0110.9890.1480.7391.3230.941Coef. coefficient HR, OR odds ratio, SE standard error, CL 95% confidence lower limit of OR, CU 95% confidence upper limit of OR\nDiscussion\nThe RECONFIRM-2 study was designed to fully evaluate the effect of infliximab used in combination with MTX on the clinical results during a 54-week study period in DMARD-resistant RA patients of a RA management group in Japan. The safety profile of infliximab therapy for a total of 5,000 cases was investigated in Japan using an all-case registered post-marketing surveillance system, and the entire profile of adverse events related to infliximab therapy was clearly identified [14]. The assessment of efficacy, however, was based only on the physician\u2019s general evaluation and not on quantitative measures such as EULAR criteria. In this report, therefore, efficacy data based on DAS28-CRP and EULAR improvement criteria were intensively assessed. The average DAS28-CRP before starting infliximab was 5.5, it decreased to 3.2 at week 22 and it remained steady at 3.1 by week 54 after the infliximab therapy. At week 22 after the therapy, about 28% and 40% of patients satisfied the remission and low disease activity criteria, respectively, and these effects remained (28 and 39%, respectively) by week 54. Also, about 37% of the patients exhibited good response according to EULAR criteria continuously from week 22 to  54. These results reconfirmed that the clinical efficacy of infliximab at week 22 was maintained until week 54 in most of the patients treated with infliximab. Thus, appropriate treatment with infliximab and MTX in this study group could minimize secondary insufficiency of the therapy for at least one\u00a0year.\nOn the other hand, the discontinuation of infliximab was observed in approximately 24% of the patients during the 54-week period. This rate of discontinuation of the therapy was comparable to that obtained in other countries: 26.7% in the Anti-Tumor Necrosis Factor Trial in Rheumatoid Arthritis with Concomitant Therapy (ATTRACT) international study carried out in the USA and the EU, 34.6% in a German domestic study and 26% in a French group study during the first one-year period [7, 15, 16]. Cumulative hazards differed among causes of discontinuation; discontinuations due to adverse events, inefficiency, remission and other causes (including changes of hospital\/clinic and economic problems) were 0.093, 0.081, 0.007 and 0.063, respectively. Male gender, older age and RF-negativity were significantly correlated with discontinuation of infliximab due to adverse reactions, including infusion reactions (N\u00a0=\u00a07), toxicoderma (4), bacterial pneumonia (3), pneumocystis jirovecii pneumonia (3), but more than two-thirds of the withdrawals due to adverse events were observed within the first 14 weeks after the therapy.\nIn this study, to clarify how predisposing factors from the demographic characteristics of RA patients were related to the clinical efficacy of infliximab therapy, a multivariate analysis using a logistic regression was performed. In this study, multiple variables including sex, age, duration of disease, stage, class, positive\/negative RF, concomitant MTX dose, concomitant PSL dose, and initial levels of CRP, TJC, SJC and GH were assessed. It is worth noting that, among multiple variables, younger age, RF-negativity and lower levels of DAS28-CRP at the baseline were significantly correlated with the clinical remission induced by infliximab therapy at week 54. These results imply that the timely use of MTX and infliximab can be strongly recommended for younger RA patients that show RF-negativity in order to efficiently achieve clinical remission. On the other hand, male gender, older age and RF-negativity were significantly correlated with discontinuation of infliximab due to adverse reactions, whereas there was no significant factor responsible for discontinuation as a result of inefficacy, and only older age affected secondary insufficiency from week 22 to 54 after the infliximab therapy. Although it is intriguing that male gender predisposes for discontinuation as a result of adverse events, these results provide the first information that can be used to facilitate the more efficacious use of infliximab and MTX in the daily practice of rheumatologists.\nTaken together, this REOCNFIRM-2 study reconfirms the clinical efficacy of treatment with infliximab and MTX in Japanese RA patients using the DAS28-CRP and EULAR response criteria. Among 410 patients with active RA, approximately 28% and 39% of the patients satisfied the remission and low disease activity criteria, and good response according to the EULAR criteria was achieved in 37% of the patients treated with infliximab plus MTX during the 54-week study period. The clinical efficacy of infliximab was maintained from week 22 to 54 after the treatment, and secondary insufficiency after week 22 was marginal after the appropriate treatment in this study group. Several demographic factors, including male gender, RF-negativity and lower scores of DAS28-CRP, were significant predisposing factors for remission. The promising effectiveness of infliximab at improving measures of disease activity in RA patients has led to this therapy becoming one of the key advances in the management of RA. Thus, this study is important because it provides obvious and invaluable evidence concerning the efficacy of the combinational use of infliximab and MTX, and can guide the real clinical use of infliximab in the future.","keyphrases":["infliximab","rheumatoid arthritis","eular response","retrospective study"],"prmu":["P","P","P","P"]}
{"id":"Eur_J_Epidemiol-3-1-2039783","title":"From evidence based bioethics to evidence based social policies\n","text":"In this issue, Norwegian authors demonstrate that causes of early expulsion out the workforce are rooted in childhood. They reconstruct individual biographies in administrative databases linked by an unique national identification number, looking forward 15 years in early adulthood and looking back 20 years till birth with close to negligible loss to follow up. Evidence based bioethics suggest that it is better to live in a country that allows reconstructing biographies in administrative databases then in countries that forbid access by restrictive legislation based on privacy considerations. The benefits of gained knowledge from existing and accessible information are tangible, particularly for the weak and the poor, while the harms of theoretical privacy invasion have not yet materialised. The study shows once again that disadvantage runs in families. Low parental education, parental disability and unstable marital unions predict early disability pensions and premature expulsion out gainful employment. The effect of low parental education is mediated by low education of the index person. However, in a feast of descriptive studies of socio-economic causes of ill health we still face a famine of evaluative intervention studies. An evidence based social policy should be based on effective interventions that are able to break the vicious circles of disability handed down from generation to generation.\nNatural selection is for biology what the human life course is for epidemiology: an overarching framework needed to understand the occurrence of disease in the biography of the individual person [1]. The life course of human beings as a history of health and disease starts long before conception, in the genes and life course of their parents. The odds at facing a successful life are entirely different if conception started with the rape of a young teenager by an HIV positive warrior in a horrifying African civil war or as the consequence of the deep desire to raise a child among a healthy and wealthy European loving couple. To expect a life in good health, healthy parents have to provide their offspring with good genes, food, shelter, love, an upbringing and an education.\nHowever, a general problem of human life course epidemiology is that the human life course is a lot longer than the scientific career of epidemiologists. While there is a scope for ambitious programmes of prospective research spanning multiple generations, such programmes will nevertheless run into questions not addressed by the original design. In this issue, the Norwegian study of Gravseth et\u00a0al. show how existing data from administrative databases can be put to good use to understand disabling processes originating in youth [2].\nEvidence based bioethics\nAs in other Scandinavian countries, several databases (here the medical birth registry, benefit and income registries in the National Insurance Administration, the education register of Statistics Norway, and the Central Population Register) can be linked by a unique national identification numbers of the child and the parents. In many European countries, such linkage is considered a breach of personal autonomy through invasion of privacy. Bioethical theory is very concerned with the potential of abuse of knowledge, but seems to neglect the potential of harm by lack of available but inaccessible knowledge [3]. Gravseth et\u00a0al. shows how linked administrative databases can yield important insights in the human life course, supporting policy decisions protecting the poor and the weak. Bioethical theory mistrusts human nature, suggesting that the risk of abuse of private information from linked administrative databases is high. This has never been confirmed by empirical evidence. Bioethics should consider the possibility that untested theories supported by unproved assumptions may look good in theory, but in practice waste healthy life. Medicine knows a large array of such theories. Although critical of parenting experts, we too put our first babies to sleep on their front, likely following advice that started by the celebrated parenting expert, Dr Spock. In 1970, a statistically significantly increased risk of cot death for front sleeping compared with back sleeping (pooled odds ratio 2.93; 95% confidence interval 1.15, 7.47) could already be identified from the then available studies [4]. Preferring the cool theory of babies developing their brain and body better by looking up to spot their mother over the neglected harsh evidence has caused the death of 50,000 babies worldwide, this after 1970 only [4]. Where empirical investigation is possible, bioethical theory needs corroboration by observable evidence [5]. If evidence of harms and benefits is available, as with the long-standing practice of data linkage in the Scandinavian countries, observable evidence of benefits should have precedence over theoretical considerations based on unproved assumptions.\nThe use of administrative databases\nThe use of administrative databases can be very tantalising: they carry a lot of information, but what one wants is not available or not reliable [6]. However, as in all study designs, its\u2019 use depends on one\u2019s aims. Flagging persons in a population register and being informed by a mortality register of their death will obtain survival figures at low costs. Death is one of those few health states even epidemiologists do not argue about. Follow up is always close to 100%.\nAn important principle of administrative databases is that they don\u2019t follow patients but money. As soon as money is at stake, administrative registers become highly reliable to count processes amenable to financing, and loss to follow-up is close to negligible. Eligibility for benefits may change over periods, but the fact of being reimbursed is coded extremely well. One of the strong characteristics of administrative databases is their completeness, even over very long periods. In the article of Gravseth et\u00a0al., early disability pension registered in 1988\u20132003 is a highly reliable proxy of definitive expulsion from paid work. Looking back to the medical birth registry in 1967\u20131976 and a limited number of parental characteristics between ages 0 and 18 of the child, the authors have explored the consequences of a limited numbers of determinants in youth for later expulsion out of work.\nShaping the life course\nLosing the capability to do paid work between the ages 20 and 35 is a highly relevant health outcome, as defined by Sen\u2019s capability approach [7]. Being unable to work at young adult age is a heavy burden of disability. In terms of attributable risks, education is by far the most important determinant, explaining 57% of the risk of early disability. Here, the administrative data show their inherent weakness: they contain little information of the process leading to low levels of education, be it personal factors related to the health of the child or social factors related to its\u2019 family and living situation. However, the stratified analysis suggests that low education of the child finds it roots in low education of parents. It is worrying that even in a country as Norway, with one of the highest human development indices in the world, disadvantage as expressed by low education of parents and parental disability is still handed down from generation to generation. Last but not least, the relative importance of maternal marital status shows the importance of stable marital unions to raise healthy children. With low European fertility and postponement of first pregnancy after the age of 30, policy makers are tempted to try to push women into earlier childbirth. This policy may be ill advised, if it leads to more children of less well educated younger mothers in less stable unions.\nMoving to evidence based policy\nGravseth et\u00a0al. show, by using linked administrative databases, that causes of early disability in young adulthood are rooted in childhood and in parental disadvantage. A consequence is that interventions targeting the adults at risk will likely add more to the problems than to the solutions. The horns of the dilemma to give or to withhold a disability pension are terribly sharp. An early disability pension is a social death sentence, crippling the individual by removing incentives to seek work and declaring him or her definitively incurable. But those who are effectively crippled won\u2019t be helped by the misery of repeated frustrations in a labour market in which they cannot compete. Studies carefully designed to identify determinants of irreversible disability are needed, inclusive randomised controlled experiments evaluating pension decisions as important health care interventions.\nI opened this editorial by making the strong comparison between the little African girl, pregnant after being raped and the highly educated European women in her 30s. However, even in Norway, disadvantage is still a disease that runs in families. We have now a descriptive feast of the lifelong health consequences of social disadvantage and low education. However, the evidence supporting effective interventions is remaining an evaluative famine [8]. Successful interventions need to target the vicious circles of disadvantage that are handed down from generation to generation. We need theories and models that identify potentially effective interventions and study designs able to collect the evidence that supports the effectiveness. The transgenerational aspects of life course epidemiology may be a hard nut to crack in evaluative research. But as good health starts with a good education, good education will always be a worthy proxy.","keyphrases":["databases","disability","pensions","epidemiology","norway","risk factors","educational status","socioeconomic factors"],"prmu":["P","P","P","P","P","R","R","M"]}
{"id":"Diabetologia-3-1-1914282","title":"The effect of moderate alcohol consumption on adiponectin oligomers and muscle oxidative capacity: a human intervention study\n","text":"Aims\/hypothesis The aim of this study was to investigate whether moderate alcohol consumption increases plasma high molecular weight (HMW) adiponectin and\/or muscle oxidative capacity.\nIntroduction\nModerate alcohol consumption is associated with a decreased risk of type 2 diabetes compared with abstention [1], which could be explained by improved insulin sensitivity [2]. The underlying mechanism for this is not clear, but several pathways may be involved. First, moderate alcohol consumption increases plasma adiponectin concentrations, which could precede changes in insulin sensitivity [2]. Adiponectin improves insulin sensitivity by increasing muscle fat oxidation and\/or decreasing intramyocellular triacylglycerols (IMTGs) [3]. Adiponectin is present in plasma as a trimer, hexamer or high molecular weight (HMW) form, the latter possibly being the most relevant in the aetiology of insulin resistance [4]. Second, moderate alcohol consumption acutely affects energy expenditure, diet-induced thermogenesis, lipolysis and lipid oxidation [5]. These changes may result from acetate production from ethanol, which is converted to acetyl-CoA mainly in peripheral tissue such as muscle [5]. Such acute changes could cumulatively affect oxidative capacity and insulin sensitivity. This is the first human study to investigate whether chronic moderate alcohol consumption affects plasma adiponectin oligomers and muscle oxidative capacity.\nMaterials and methods\nIn a randomised, controlled, crossover trial, conducted at TNO Quality of Life, 19 healthy, moderate-drinking, lean (BMI 18\u201325\u00a0kg\/m2, n\u2009=\u200911) and overweight (BMI \u226527\u00a0kg\/m2, n\u2009=\u20098) men (aged 18\u201340\u00a0years), consumed 100\u00a0ml whisky (32\u00a0g alcohol per day; Famous Grouse Scotch Whisky, 40% vol, Perth, Scotland) or mineral water (Spa Reine, Spa, Belgium) daily for 4\u00a0weeks. For the last 7\u00a0days of each treatment period the diet was fully controlled, and treatments were separated by a 2\u00a0day wash-out period. This sample size was sufficient to detect \u223c15% difference of \u03b2-3-hydroxyacyl-CoA dehydrogenase (\u03b2-HAD) and citrate synthase activity in this crossover trial and correlation coefficients of 0.55 or higher with 80% power and accepting an alpha of 0.05. At the end of each treatment period, muscle biopsies from the vastus lateralis muscle were taken to assess \u03b2-HAD, cytochrome c oxidase and citrate synthase activity [6], IMTG [7] and succinate dehydrogenase activity [8]. Fasting blood samples were collected and an OGTT was performed to calculate whole-body insulin sensitivity. Subjects gave written informed consent and the University Medical Center Utrecht Medical Ethics Committee approved the protocol. The study was conducted according to the Declaration of Helsinki (2000) and the International Conference on Harmonisation Guidelines for Good Clinical Practice. Data were analysed using the SAS statistical software package (SAS\/STAT version 8, SAS Institute, Cary, NC, USA). Treatment effects were assessed by ANOVA, using general linear modelling, with BMI, period, treatment and treatment order included in the model. Two-sided p values below 0.05 were considered statistically significant.\nResults\nCompliance to treatments was good as assessed by measurements of urinary ethyl glucuronide every 5\u00a0days, showing one negative sample during the whisky-drinking and no positive samples during the water-drinking period. Another indication was the 11% increase (p\u2009<\u20090.001) of serum HDL-cholesterol after whisky compared with water consumption. Table\u00a01 shows results on insulin sensitivity, adiponectin and muscle enzyme activities. Insulin sensitivity was not changed after moderate alcohol consumption, but plasma adiponectin concentrations increased from 7.9\u2009\u00b1\u20090.2\u00a0mg\/l after water to 9.0\u2009\u00b1\u20090.2\u00a0mg\/l (means\u00b1SEM) after whisky consumption. Moderate alcohol consumption tended to increase HMW and medium molecular weight (MMW) adiponectin, but not low molecular weight (LMW) adiponectin. Differences in insulin sensitivity and adiponectin and its oligomers tended to be more pronounced among lean than overweight men (Table\u00a02). Muscle oxidative capacity measured by \u03b2-HAD, cytochrome c oxidase and citrate synthase activity did not differ between treatments. A borderline significant interaction between treatment and BMI was observed for cytochrome c oxidase (p\u2009=\u20090.072) and citrate synthase (p\u2009=\u20090.102) activity. Moderate alcohol consumption tended to increase cytochrome c oxidase (+23.7%; 95% CI \u22123.9 to +51.2%, p\u2009=\u20090.08) and citrate synthase (+24.7%; 95% CI \u22127.7 to +57.2%, p\u2009=\u20090.11) activity among lean, but not overweight men (Table\u00a02). Succinate dehydrogenase activity in mixed muscle fibres decreased (p\u2009=\u20090.03) by 15% after moderate alcohol consumption. IMTGs did not differ, despite a 15\u201320% difference between both treatments. These results were not different for lean and overweight men (Table\u00a02). After whisky consumption, HMW correlated positively with activities of citrate synthase (r\u2009=\u20090.64, p\u2009=\u20090.004), cytochrome c oxidase (r\u2009=\u20090.59, p\u2009=\u20090.009) and \u03b2-HAD (r\u2009=\u20090.46, p\u2009=\u20090.056). Activities of citrate synthase (r\u2009=\u20090.44, p\u2009=\u20090.07), cytochrome c oxidase (r\u2009=\u20090.32, p\u2009=\u20090.20) and \u03b2-HAD (r\u2009=\u20090.44, p\u2009=\u20090.07) also tended to correlate with MMW adiponectin, but not LMW adiponectin. A similar pattern was observed for correlations of adiponectin oligomers with HDL-cholesterol (HMW: r\u2009=\u20090.55, p\u2009=\u20090.014; MMW: r\u2009=\u20090.55, p\u2009=\u20090.014; LMW: r\u2009=\u20090.21, p\u2009=\u20090.38).\nTable\u00a01Insulin sensitivity index, adiponectin oligomer concentrations, HbA1c and enzyme activities after 4\u00a0weeks of consumption of whisky or mineral water in 19 lean or overweight men\u00a0Mineral waterWhisky% Changep valueInsulin sensitivity indexa10.6\u2009\u00b1\u20091.39.6\u2009\u00b1\u20091.3\u22129.40.61Adiponectin (mg\/l)7.9\u2009\u00b1\u20090.29.0\u2009\u00b1\u20090.212.50.0008\u00a0HMW0.7\u2009\u00b1\u20090.11.1\u2009\u00b1\u20090.157.1 0.074\u00a0MMW4.0\u2009\u00b1\u20090.24.5\u2009\u00b1\u20090.212.50.065\u00a0LMW3.2\u2009\u00b1\u20090.33.5\u2009\u00b1\u20090.39.40.442HbA1c (%)4.9\u2009\u00b1\u20090.024.8\u2009\u00b1\u20090.02\u22122.00.023Muscle \u03b2-HAD (mol\/\u03bcg protein)4.22\u2009\u00b1\u20090.484.37\u2009\u00b1\u20090.493.60.827Muscle citrate synthase (mol\/\u03bcg protein)0.66\u2009\u00b1\u20090.050.75\u2009\u00b1\u20090.0513.60.262Muscle cytochrome c oxidase (mol\/\u03bcg protein)1.48\u2009\u00b1\u20090.121.67\u2009\u00b1\u20090.1212.80.262Intramyocellular triacylglycerols (AU)\u00a0Type 10.027\u2009\u00b1\u20090.0040.022\u2009\u00b1\u20090.004\u221218.50.339\u00a0Type 20.010\u2009\u00b1\u20090.0020.008\u2009\u00b1\u20090.002\u221220.00.489\u00a0Mixed0.019\u2009\u00b1\u20090.0030.016\u2009\u00b1\u20090.002\u221215.8 0.429Succinate dehydrogenase activity (AU)\u00a0Type 10.084\u2009\u00b1\u20090.0050.075\u2009\u00b1\u20090.004\u221210.70.122\u00a0Type 20.057\u2009\u00b1\u20090.0030.052\u2009\u00b1\u20090.003\u22128.80.221\u00a0Mixed0.073\u2009\u00b1\u20090.0030.062\u2009\u00b1\u20090.003\u221215.10.028Data are presented as means\u00b1SEM or %AU Arbitrary unitsaInsulin sensitivity according to Matsuda and DeFronzo [15]Table\u00a02Insulin sensitivity index, adiponectin oligomer concentrations, HbA1c and enzyme activities (means\u00b1SEM) after 4\u00a0weeks of consumption of whisky or water in lean and overweight subjects\u00a0Lean groupOverweight groupWaterWhisky% Changep valueWaterWhisky% Changep valueInsulin sensitivity indexa13.3\u2009\u00b1\u20091.913.0\u2009\u00b1\u20091.9\u22122.30.937.8\u2009\u00b1\u20091.64.6\u2009\u00b1\u20091.6\u221241.0 0.21Adiponectin (mg\/l)8.1\u2009\u00b1\u20090.39.4\u2009\u00b1\u20090.316.10.017.7\u2009\u00b1\u20090.28.5\u2009\u00b1\u20090.210.40.08\u00a0HMW0.8\u2009\u00b1\u20091.21.2\u2009\u00b1\u20090.250.00.120.8\u2009\u00b1\u20090.10.8\u2009\u00b1\u20090.100.62\u00a0MMW4.2\u2009\u00b1\u20090.34.8\u2009\u00b1\u20090.314.30.133.9\u2009\u00b1\u20090.24.1\u2009\u00b1\u20090.25.10.52\u00a0LMW3.2\u2009\u00b1\u20090.43.4\u2009\u00b1\u20090.46.30.693.1\u2009\u00b1\u20090.43.5\u2009\u00b1\u20090.412.90.50HbA1c (%)4.9\u2009\u00b1\u20090.024.8\u2009\u00b1\u20090.02\u22122.00.044.9\u2009\u00b1\u20090.024.9\u2009\u00b1\u20090.0200.53Muscle \u03b2-HAD (mol\/\u03bcg protein)4.67\u2009\u00b1\u20090.505.10\u2009\u00b1\u20090.459.20.553.46\u2009\u00b1\u20090.903.69\u2009\u00b1\u20091.016.70.87Muscle citrate synthase (mol\/\u03bcg protein)0.73\u2009\u00b1\u20090.080.92\u2009\u00b1\u20090.0726.00.110.60\u2009\u00b1\u20090.050.54\u2009\u00b1\u20090.06\u221210.00.51Muscle cytochrome c oxidase (mol\/\u03bcg protein)1.72\u2009\u00b1\u20090.152.12\u2009\u00b1\u20090.1423.30.081.33\u2009\u00b1\u20090.131.13\u2009\u00b1\u20090.15\u221215.00.36Intramyocellular triacylglycerols (AU)\u00a0Type 10.022\u2009\u00b1\u20090.0060.016\u2009\u00b1\u20090.005\u221227.30.500.032\u2009\u00b1\u20090.0060.028\u2009\u00b1\u20090.006\u221212.50.61\u00a0Type 20.006\u2009\u00b1\u20090.0020.008\u2009\u00b1\u20090.00233.30.410.013\u2009\u00b1\u20090.0030.007\u2009\u00b1\u20090.003\u221246.20.14\u00a0Mixed0.014\u2009\u00b1\u20090.0040.013\u2009\u00b1\u20090.003\u22127.10.860.024\u2009\u00b1\u20090.0040.019\u2009\u00b1\u20090.004\u221220.80.40Succinate dehydrogenase activity (AU)\u00a0Type 10.086\u2009\u00b1\u20090.0050.080\u2009\u00b1\u20090.005\u22127.00.400.084\u2009\u00b1\u20090.0090.069\u2009\u00b1\u20090.008\u221217.90.29\u00a0Type 20.060\u2009\u00b1\u20090.0050.055\u2009\u00b1\u20090.005\u22128.30.500.054\u2009\u00b1\u20090.0050.048\u2009\u00b1\u20090.004\u221211.10.34\u00a0Mixed0.076\u2009\u00b1\u20090.0050.066\u2009\u00b1\u20090.005\u221213.20.160.069\u2009\u00b1\u20090.0050.057\u2009\u00b1\u20090.004\u221217.40.15Data are presented as means\u00b1SEM or %AU Arbitrary unitsaInsulin sensitivity according to Matsuda and DeFronzo [15]\nDiscussion\nThis study showed that moderate alcohol consumption increases adiponectin concentrations, consistent with previous reports [2]. We have now observed that the alcohol-induced increase of adiponectin may be oligomer specific. Moderate alcohol consumption increased particularly HMW adiponectin, MMW adiponectin to a lesser extent, but not LMW adiponectin. These results are in line with those of Bobbert et al. [9] showing a similar pattern of changes in adiponectin oligomers after moderate weight reduction. Studies with thiazolidinedione or rigorous weight reduction show similar, but more pronounced results [10]. HDL-cholesterol concentrations correlated particularly with HMW adiponectin, as previously shown [9]. We have now also observed that HMW and MMW adiponectin are correlated with markers of muscle oxidative capacity, in line with reports of increased fat oxidation after adiponectin infusion [3] and a recent study showing that adiponectin increases muscle oxidative capacity [11]. Our results tend to confirm these findings and show that these relationships may be specific to HMW adiponectin. Altogether, this could provide an underlying mechanism for the proposed importance of HMW adiponectin in the aetiology of insulin resistance [4].\nDespite this, the alcohol-induced increase of adiponectin and specifically HMW adiponectin did not affect muscle oxidative capacity, IMTG content and insulin sensitivity. As some subtle differences were present, we cannot completely rule out the hypothesis that changes in IMTG content and\/or oxidative capacity could occur. Although it did not reach significance, we observed a 15\u201320% lower IMTG content after whisky than water consumption, which is of similar magnitude to that observed for a weight loss and physical activity intervention [12]. Furthermore, moderate alcohol consumption tended to increase muscle citrate synthase activity among lean men, but succinate dehydrogenase activity declined after moderate alcohol consumption in mixed muscle fibres. All citric acid cycle and respiratory chain enzymes are thought to change in parallel to perturbation [13]. Our findings with citrate synthase and succinate dehydrogenase, however, are contradictory to this notion. This could simply be due to the measurement of ex vivo oxidative capacity or to chance. Alternatively, moderate alcohol consumption could differentially affect various enzymes in oxidative pathways such as the citric acid or glyoxylate cycle as suggested by Kokavec and Crowe [14]. Because acetyl-CoA from ethanol oxidation is generated independently from activation of pyruvate dehydrogenase complex, it may not affect the citric acid cycle. Alcohol may instead affect the glyoxylate cycle that bypasses part of the citric acid cycle, including succinate dehydrogenase [13, 14].\nStrengths of this study are its randomised, controlled crossover design. We assessed compliance to study treatments several times throughout the study and observed no significant deviations. It is therefore unlikely that our results are confounded by diet or lifestyle. Our study was, however, limited by a slightly small sample size for certain contrasts such as analyses in subgroups of lean and overweight men or of IMTG content. Therefore these results may be somewhat preliminary and need to be confirmed with larger sample sizes. Second, because insulin sensitivity was not the primary endpoint of this study, an OGTT was used to assess insulin sensitivity. However, we used an insulin sensitivity index that correlates well with the gold standard, the hyperinsulinaemic\u2013euglycaemic clamp technique [15]. In addition, our results on insulin sensitivity do not differ from our previous studies using the clamp technique [2]. We therefore believe that using this insulin sensitivity index has not influenced our results to a large extent.\nIn conclusion, this study shows that 4\u00a0weeks of daily moderate alcohol consumption increases adiponectin concentrations and particularly HMW adiponectin concentrations, but does not affect insulin sensitivity and oxidative capacity. Concentrations of HMW and MMW adiponectin were positively associated with muscle oxidative capacity.","keyphrases":["moderate alcohol consumption","adiponectin","muscle oxidative capacity","insulin sensitivity","intramyocellular triacylglycerols","high molecular weight adiponectin"],"prmu":["P","P","P","P","P","R"]}
{"id":"Appl_Microbiol_Biotechnol-4-1-2243256","title":"Discovery and characterization of a putrescine oxidase from Rhodococcus erythropolis NCIMB 11540\n","text":"A gene encoding a putrescine oxidase (PuORh, EC 1.4.3.10) was identified from the genome of Rhodococcus erythropolis NCIMB 11540. The gene was cloned in the pBAD vector and overexpressed at high levels in Escherichia coli. The purified enzyme was shown to be a soluble dimeric flavoprotein consisting of subunits of 50 kDa and contains non-covalently bound flavin adenine dinucleotide as a cofactor. From all substrates, the highest catalytic efficiency was found with putrescine (KM = 8.2 \u03bcM, kcat = 26 s\u22121). PuORh accepts longer polyamines, while short diamines and monoamines strongly inhibit activity. PuORh is a reasonably thermostable enzyme with t1\/2 at 50\u00b0C of 2 h. Based on the crystal structure of human monoamine oxidase B, we constructed a model structure of PuORh, which hinted to a crucial role of Glu324 for substrate binding. Mutation of this residue resulted in a drastic drop (five orders of magnitude) in catalytic efficiency. Interestingly, the mutant enzyme showed activity with monoamines, which are not accepted by wt-PuORh.\nIntroduction\nFlavoprotein oxidases catalyze the oxidation of a wide range of compounds, while at the same time they reduce oxygen to hydrogen peroxide. They are valuable biocatalysts for the oxidative activation of biomolecules, as they usually selectively oxidize their substrate at a specific position, leaving other positions unaffected. Due to their ability to use molecular oxygen as electron acceptor, no expensive coenzymes like NAD(P)H are needed. This makes oxidases inexpensive and rather straightforward in usage compared to other redox enzymes.\nBy far, the best studied oxidase is glucose oxidase from Aspergillus niger. This enzyme has been applied for decades, mostly for diagnostic applications (Wilson and Turner 1992). Another example is pyranose oxidase (glucose-2-oxidase) from Peniophora gigantea, which oxidizes specifically the C2 position of glucose and has been applied for the chemoenzymatic synthesis of rare sugars and sugar-based synthons (Giffhorn et al. 2000). Human monoamine oxidases (MAO-A and MAO-B) are important for the oxidation of neurotransmitters (Abell and Kwan 2001). An engineered mutant of the homologous A. niger enzyme (MAO-N) has been applied in an elegant biocatalytic deracemization process to obtain enantiomerically pure chiral amines (Carr et al. 2005; Dunsmore et al. 2006).\nSo far, the most described oxidases are from eukaryotic origin, and heterologous expression of the recombinant protein in Escherichia coli for large scale enzyme production can be problematic. In a search for novel bacterial oxidases, we looked in sequenced genomes and identified genes encoding novel oxidases. By this, we discovered oxidases primarily acting on alditols (Heuts et al. 2007) and phenolic compounds (Jin et al. 2007). These oxidases, both from actinomycetes, could be highly overexpressed in E. coli. Another way of searching for novel bacterial oxidases is to screen unsequenced bacterial genome libraries directly for genes encoding enzyme activity. As actinomycetes appear to be rich in oxidases, we have chosen to explore these organisms for relevant oxidases. Using a plate-based screening protocol for oxidase activity (Alexeeva et al. 2002), we screened a genomic DNA library of the actinomycete Rhodococcus erythropolis NCIMB 11540 to find novel oxidases acting on polyols and\/or amines.\nIn this paper, we describe the discovery and characterization of a novel flavin-containing oxidase primarily acting on putrescine. This putrescine oxidase (PuORh) shares 67% sequence identity with the enzyme from Micrococcus rubens (PuOMr; Ishizuka et al. 1993). We show that PuORh is highly overexpressed in E. coli and can be easily purified. Using a structural model, we were able to identify a glutamate residue (Glu324) that is crucial for substrate binding, and we could alter the substrate specificity by protein engineering.\nMaterials and methods\nChemicals\nRestriction enzymes were from Roche and New England Biolabs. One-shot electrocompetent E. coli TOP10 cells and the TOPO TA Cloning Kit were purchased from Invitrogen. Plasmid isolation was performed using the Qiagen DNA purification kit. Oligonucleotides were purchased from Sigma. Nitrocellulose filters (Protran BA85 132\u00a0mm, 0.45\u00a0\u03bcm pore size) were from Schleicher and Schuell BioScience, Dassel (Germany). Deprenyl and rasagiline were a kind gift from Prof. A. Mattevi (University of Pavia, Italy). All other chemicals were of analytical grade. The genomic DNA library from R. erythropolis NCIMB 11540 was provided by DSM (Geleen, The Netherlands). Constructs were sequenced at GATC Biotech (Kostanz, Germany).\nPlate-based screening method for oxidase activity\nA gene library of R. erythropolis NCIMB 11540 in pZErO-2 was screened for oxidases using the plate-based oxidase activity screening method adapted from the group of Turner (Alexeeva et al. 2002). This gene library was constructed by partial digestion of genomic DNA from R. erythropolis NCIMB 11540 by Sau3A1. Fragments of 4\u201310\u00a0kb were isolated, ligated in pZErO-2 (cut with BamHI), and followed by transformation of E. coli DH10B. Colonies were collected from plate, stored as glycerol stock, and plasmids were isolated. The total amount of plasmids contained a mean insert size of 6.0\u00a0kb and 1% of self-ligated vector molecules.\nElectrocompetent E. coli TOP10 cells were transformed with the gene library, and the transformed cells were diluted in Luria\u2013Bertani (LB) medium to obtain single colonies on plate. The diluted cell suspensions were plated on nitrocellulose filters. The filters were placed on top of LB agar containing 0.05\u00a0mg\/ml kanamycin and incubated for 48\u00a0h at 30\u00b0C. Subsequently, the nitrocellulose filters were transferred to empty petri dishes and stored at \u221220\u00b0C to partially lyse the cells. Each filter was submersed with 50\u00a0ml 50\u00a0mM sodium phosphate buffer pH\u00a07.5 containing 1% (w\/v) agarose, 2\u00a0U\/ml Horseradish peroxidase (HRP) and a mixture of 1\u00a0mM 4-chloro-1-naphthol, 100\u00a0\u03bcM cholesterol, 10\u00a0mM xylitol, 10\u00a0mM sarcosine, 10\u00a0mM l-alanine, 10\u00a0mM d-glucose, 10\u00a0mM d-galactose, 10\u00a0mM lactose, and 10\u00a0mM d-glucosamine. Plates were incubated at room temperature and regularly checked by visual inspection for color formation in and around colonies. Positives were picked and cultivated overnight in 5\u00a0ml liquid LB medium and subjected to another screening cycle to assure that color formation was caused by single clones.\nSequence analysis\nTo identify unique positive clones, plasmids were isolated and subjected to restriction analysis by EcoRI. Inserts were sequenced and open reading frames (ORFs) were identified using the ORF Finder tool of the National Center for Biotechnology Information (NCBI) (http:\/\/www.ncbi.nlm.nih.gov\/gorf\/gorf.html). A BLAST search (blastp) was performed with the protein sequence of PuORh using the BLAST function of the NCBI website (http:\/\/www.ncbi.nlm.nih.gov\/BLAST).\nCloning and expression of the gene encoding PuORh\nTo overexpress the novel putrescine oxidase, the corresponding gene (puoRh) was amplified from the fragment of genomic DNA of R. erythropolis using the following primers: puoRh_fw: 5\u2032-GCTCCATATGCCTACTCTCCAGAGAGATG (NdeI site shown in italics) and puoRh_rv: 5\u2032-GCTCAAGCTTTCAGGCCTTGCTGCGGGCG (HindIII site shown in italics). The amplified gene was purified from gel and ligated between the NdeI and HindIII restriction sites of the pBAD\/Myc-His vector (Invitrogen). The plasmid carrying the puoRh gene (pBADpuoRh) was transformed to CaCl2-competent E. coli TOP10 cells and spread on LB agar plates containing 50\u00a0\u03bcg\/ml of ampicillin. E. coli TOP10 cells containing pBADpuoRh were tested for overexpression of the protein at 17, 30, and 37\u00b0C and at arabinose concentrations of 0, 0.00002, 0.0002, 0.002, 0.02, and 0.2% (w\/v). Cell extracts and cell-free extracts of E. coli TOP10 containing pBADpuoRh were analyzed on a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel to find the best conditions for overexpression of soluble PuORh.\nEnzyme purification\nTo obtain purified protein, E. coli TOP10 containing pBADpuoRh was cultivated for 24\u00a0h at 30\u00b0C in 1\u00a0l of terrific broth medium containing 50\u00a0\u03bcg\/ml ampicillin and 0.02% (w\/v) arabinose. Cells were harvested by centrifugation for 15\u00a0min at 6,000\u00a0rpm at 4\u00b0C. The supernatant was discarded, and the pellet was suspended in 40\u00a0ml of 50\u00a0mM Tris\u2013HCl pH\u00a07.5 and sonicated for 10\u00a0min to break the cells. The cell extract was centrifuged for 30\u00a0min at 15,000\u00a0rpm at 4\u00b0C to remove the broken cells and obtain cell-free extract. From this cell-free extract PuORh was purified using a Q-Sepharose anion exchange column. Unbound protein fractions were washed from the column with 50\u00a0mM Tris\u2013HCl buffer pH\u00a07.5. PuORh was eluted from the column with a 50\u00a0mM Tris\u2013HCl buffer pH\u00a07.5 by increasing the KCl concentration. Eluted fractions containing PuORh were combined, concentrated using an Amicon filter, and desalted using a HiPrep 26\/10 Desalting Column (Amersham Biosciences).\nEnzyme activity assay and determination of steady-state kinetic parameters\nPuORh activity was measured at 25\u00b0C using a peroxidase-coupled assay containing 2,2\u2032-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) as the chromogenic substrate (Childs and Bardsley 1975). The H2O2 formed by PuORh can be coupled to the Horseradish-peroxidase-mediated oxidation of ABTS resulting in the formation of a green product that can be measured at 420\u00a0nm (\u025b420\u2009=\u200935.7\u00a0mM\u22121 cm\u22121). The standard assay mixture with ABTS (HRP-ABTS assay) contained 50\u00a0\u03bcM putrescine, 50\u00a0mM Tris\u2013HCl buffer, pH\u00a08.0, 100\u00a0\u03bcM ABTS, 5\u00a0U of Horseradish peroxidase, 10\u00a0\u03bcl of enzyme dilution (appropriately diluted), and a fixed amount of substrate. The reaction was started with adding the enzyme solution. Alternatively, 0.1\u00a0mM 4-aminoantipyrine (AAP) and 1.0\u00a0mM 3,5-dichloro-2-hydroxybenzenesulfonic acid (DCHBS) were used as chromogenic substrates for HRP (Federico et al. 1997; HRP-AAP\/DCHBS assay). The oxidation of these substrates results in the formation of a purple product, which can be measured at 515\u00a0nm (\u025b515\u2009=\u200926\u00a0mM\u22121 cm\u22121). For the determination of kinetic parameters, PuORh activity was measured at different substrate concentrations at 25\u00b0C using the HRP-ABTS enzyme assay. The kinetic parameters of the mutant enzymes (PuORh Glu324Ala and PuORh Glu324Leu) for putrescine were determined by measuring directly the H2O2 production at 240\u00a0nm (\u025b420\u2009=\u200943.6\u00a0M\u22121 cm\u22121). Data were fitted with Origin 7.0 using the Michaelis\u2013Menten equation for enzyme kinetics.\nInfluence of temperature and pH on enzyme activity and stability\nTo determine the optimal temperature for PuORh activity, the oxidase activity was measured at temperatures between 20 and 60\u00b0C. Before adding the enzyme, the assay mixture was equilibrated for 20\u00a0min to assure the right temperature. The influence of temperature on the enzyme stability was determined by incubating 1-ml portions of the enzyme at 4, 30, 37, and 50\u00b0C. Twenty-microliter samples were taken, placed on ice after which activity was measured. In both experiments, the activity was determined with the HRP-AAP\/DCHBS activity assay using 50\u00a0\u03bcM putrescine and 0.01\u00a0\u03bcM PuORh.\nThe pH optimum for putrescine oxidase was determined by measuring the activity at different pH values at 25\u00b0C. The following buffers were used: 50\u00a0mM Pipes buffer (pH\u00a06.4\u20137.2), 50\u00a0mM Tris\u2013HCl (pH\u00a07.4\u20138.8), and 50\u00a0mM Ches buffer (pH\u00a08.7\u20139.6). The enzyme activity was measured using the HRP-AAP\/DCHBS assay.\nInhibition experiments\nRasagiline, deprenyl, and cyclopropylamine were tested to probe whether they were able to inhibit PuORh by the formation of a covalent adduct with the flavin adenine dinucleotide (FAD) cofactor, as is the case for monoamine oxidase B (Binda et al. 2005). For 900\u00a0\u03bcl of a 20\u00a0\u03bcM PuORh solution, an absorbance scan was recorded from 650\u2013300\u00a0nm. After the addition of 100\u00a0\u03bcl of 1\u00a0mM inhibitor (final inhibitor concentration, 100\u00a0\u03bcM), absorbance spectra were recorded regularly in time to see if addition of the inhibitor would cause an alteration of the typical FAD spectrum of PuORh. For cyclopropylamine, also a final inhibitor concentration of 1\u00a0mM was tested as described above.\nButylamine, aminoethanol, ethylenediamine, 1,3-diaminopropane, and (2-aminoethyl)-trimethylammonium were tested as competitive inhibitors of PuORh. For this, the Michaelis constant (KM) of PuORh for putrescine was determined for three different concentrations for each of these amino compounds with the HRP-ABTS assay. Inhibition constants (KI) for each compound were calculated with the following formula:\nWhere [I] represents the inhibitor concentration, KM,inhibitor the apparent Michaelis constant in presence of the inhibitor and the KI the inhibition constant.\nAnalytical methods\nAll absorbance spectra were recorded in 50\u00a0mM Tris\u2013HCl pH\u00a08.0 at 25\u00b0C on a Perkin Elmer Lambda Bio40 spectrophotometer. From a cuvet containing 5\u00a0\u03bcM PuORh all oxygen was removed by flushing with argon and an absorbance spectrum was recorded from 650 to 300\u00a0nm. After adding 50\u00a0\u03bcM putrescine, another spectrum was recorded for the reduced enzyme. Reoxidation was monitored by collecting spectra in time after the cuvet was exposed to air. A spectrum of the unfolded enzyme was recorded by adding 0.1% SDS and heating for 5\u00a0min at 80\u00b0C.\nModeling and mutant construction\nBased on the structure of MAO-B in complex with rasagiline (PDB\/1S2Q; Binda et al. 2004b), a model of PuORh was made using the CHPmodels 2.0 Server. Mutants were constructed by Quick Change PCR. PuORh Glu324Ala was made by a substitution of the codon GAG for GCG using the following primers: puoRhGlu324Ala_fw, 5\u2032-CGAGGTAGTGCAGGCGGTGTACGACAACACC, and puoRhGlu324Ala_rv, 5\u2032-GGTGTT GTCGTACACCGCCTGCACTACCTCG (mutated codon underlined). For construction of PuORh Glu324, the codon GAG was replaced by CTG using the following primers: puoRhGlu324Leu_fw, 5\u2032-CGAGGTAGTGCAGCTGGTGTACGACAACACC, and puoRhGlu324Leu_rv, 5\u2032- GGTGTTGTCGTACACCAGCTGCACTACCTCG (mutated codon underlined). Both mutants were expressed in E. coli TOP 10 and cultivated and purified in the same way as wild-type PuORh.\nNucleotide sequence accession number\nThe nucleotide sequence of the PuORh encoding gene (puoRh) has been submitted to GenBank under accession number EU240877.\nResults\nIdentification of a novel putrescine oxidase\nTo identify novel oxidases acting on alcohols and\/or amines, a genomic library from the actinomycete R. erythropolis NCIMB 11540 was screened using a plate-based oxidase assay. Positive clones, expressing oxidase activity, could be identified by their purple color formation. From 55,000 transformants, 5 were observed to produce a purple color around the colony. Retransformation to E. coli TOP10 cells and repeated screening on plate resulted in three positive clones, which showed oxidase activity. Restriction analysis with EcoRI and PvuI showed a different insert for each clone indicating that these were independent clones. Sequencing of one of these clones revealed an inserted fragment of genomic DNA of 5,311\u00a0bp. On this fragment, an ORF was identified which shares 67% sequence identity (at the amino acid level) with putrescine oxidase (EC 1.4.3.10) from M. rubens, a FAD-containing amine oxidase that catalyzes the oxidative deamination of putrescine (Ishizuka et al. 1993). When we tested the cell extract of the clone containing the above-mentioned insert of 5,311\u00a0bp, we found that it was indeed active with putrescine. The other two positive clones probably contained the same gene, as for both clones by using the primers puoRh_fw and puoRh_rv a PCR product was seen on gel.\nGene cloning, overexpression, and protein purification\nFor overexpression, the PuORh encoding gene was cloned into the pBAD\/Myc-His A vector behind the araBAD promotor (without His-tag), and the plasmid carrying the puo gene was transformed to E. coli TOP10. Several temperatures (17, 30, and 37\u00b0C) and inducing conditions (0\u20130.2% L-(+)-arabinose) were tested to find the optimal conditions for overexpression. The gene was well expressed at arabinose concentrations of 0.02 and 0.2% as judged by a dominant protein band of \u00b154\u00a0kDa on SDS-PAGE. At 17 and 30\u00b0C, the protein was mainly present in the soluble fractions while at 37\u00b0C the unsolube fractions contained most of the overexpressed protein. The overexpressed protein did not show any fluorescence under UV-light upon SDS-PAGE, which is an indication that this protein does not contain a covalently histidyl-bound flavin cofactor (Fraaije et al. 1997).\nPuORh was purified from a 1-l culture containing 0.02% arabinose that was grown for 24\u00a0h at 30\u00b0C. Due to the high level of overexpression (200\u00a0mg), PuORh could be easily purified in one-step by anion exchange chromotography. SDS-PAGE analysis of the purified protein revealed that, besides the major protein band present at around \u223c54\u00a0kDa, there is a minor band corresponding to a protein mass which is approximately 3\u00a0kDa larger (Fig.\u00a01, lane A). It was concluded that this must be the product of the same gene with a His-tag extension, as this minor protein band could be removed by an additional purification step using a Ni-agarose column. (Fig.\u00a01, lane B). Although in the plasmid pBADpuoRh the stop codon (TGA) was properly introduced, it is known that in some cases, this stop codon can be translated (MacBeath and Kast 1998). With the constructed plasmid, this would indeed result in a 3\u00a0kDa larger protein.\nFig.\u00a01SDS-PAGE gel of purified recombinant PuORh. Lane A Purified PuORh. Lane B Purified PuORh after removal of His-tagged PuORh. Lane C Low molecular weight marker\nSpectral and molecular properties of PuORh\nPuORh shows a typical flavoprotein spectrum with absorbance maxima at 377 and 459\u00a0nm (Fig.\u00a02). In the presence of 5% trichloroacetic acid, PuORh precipitated and was visible as a white pellet after centrifugation. The supernatant contained a yellow color, and its absorbance spectrum with maxima at 375 and 450\u00a0nm was typical for that of free FAD. This shows that the FAD cofactor in PuORh, like in PuOMr, is not covalently bound. At room temperature, the addition of 0.1% SDS did not unfold PuORh, indicating this flavoprotein is rather robust. When 50\u00a0\u03bcM putrescine was mixed with PuORh under anaerobic conditions, the FAD cofactor was completely reduced (Fig.\u00a02, dotted line). Heating the same sample for 5\u00a0min at 80\u00b0C completely unfolded the enzyme as can be seen from the resulting free FAD UV\/VIS spectrum (Fig.\u00a02, dashed line). The reduced enzyme could be rapidly and fully reoxidized by addition of oxygen, indicating that the enzyme is a true oxidase. From the difference in absorbance between protein-bound FAD and free FAD, the molar extinction coefficient for PuORh (\u025b459) was calculated, 11.0\u00a0mM\u22121 cm\u22121. Based on the protein concentration as determined by Wadell\u2019s method (Wolf 1983) and the FAD concentration (A459), a protein\/FAD ratio of 0.5 was calculated. This is consistent with what has been described earlier for PuOMr (Desa 1972). Incubation of PuORh with additional FAD did not yield an increase of FAD incorporation or oxidase activity. Gel filtration experiments revealed that PuORh is mainly present as a dimer of \u00b1100\u00a0kDa. This corresponds well with the theoretical monomeric mass of 49,375\u00a0Da.\nFig.\u00a02Spectral properties of PuORh. Absorbance spectra of oxidized native (solid line) and unfolded (dashed line) PuORh are shown. After addition of 50\u00a0\u03bcM putrescine under anaerobic conditions, the native enzyme is fully reduced (dotted line)\nTemperature and pH dependence of activity and stability\nTo investigate the effect of temperature on enzyme activity and stability, both were determined at different temperatures. The optimal temperature for enzyme activity was found to be 30\u00b0C. Temperature stability experiments revealed that PuORh is a reasonable thermostable enzyme. At 50\u00b0C, half of the activity was lost after 2\u00a0h, while at 37\u00b0C, such a degree of inactivation was reached only after 1\u00a0day. PuORh has a rather sharp pH optimum for activity with putrescine with a peak around pH\u00a08. Below pH\u00a06.4, no significant activity can be detected.\nSubstrate specificity\nSeveral alcohols, amino alcohols, and amines were tested as substrate, and if they turned out to be converted by PuORh, the steady-state kinetic parameters were determined at pH\u00a08.0. Enzyme activity was found with several aliphatic diamines, amino alcohols, and polyamines. From Table\u00a01, it is clear that putrescine is by far the best substrate. With aromatic amines and amino alcohols, no activity was observed. An increase in carbon chain length of the diamine from 4 to 6 C-atoms resulted in a more than 1,000-fold drop in catalytic efficiency (kcat\/KM). For diamines containing 3 or 2 C-atoms and for n-butylamine, no activity could be detected. These amines were found to strongly inhibit the enzyme (Table\u00a02). Apparently, they bind efficiently but are not converted. Clearly, a minimum of two amino groups is required to be a substrate for PuORh, and these amino groups should be preferably 4 C-atoms apart. Polyamines like spermine and spermidine were also converted by PuORh but are poor substrates. Compared to putrescine, 4-amino-1-butanol is also a poor substrate, while 1,4-butanediol is not accepted at all. This again indicates that two amino groups are essential for efficient catalysis, and it confirms that PuORh is a true amine oxidase. Inhibition studies showed that aminoethanol is a very effective competitive inhibitor of PuORh (KI\u2009=\u20091.8\u00a0\u03bcM). Apparently, this amino alcohol binds very strongly in the active site, while it cannot be oxidized. When we tested 2-hydroxyputrescine, which contains an aminoethanol moiety, we found that this compound is readily oxidized. l-Ornithine was found to be a very poor substrate. Apparently, the presence of a negatively charged and relative bulky carboxylic acid group at the C1 position prevents efficient amine oxidation. Taken together, it can be concluded that PuORh has a narrow substrate specificity and is very selective for putrescine.\nTable\u00a01Steady-state kinetic parameters of PuORh at pH 8.0.ND Not determinedaValues of putrescine oxidase from Micrococcus rubens (Okada et al. 1980)Table\u00a02Inhibition constants of competitive inhibitors of PuORhaValues of putrescine oxidase from Micrococcus rubens (Swain and Desa 1976)\nModel structure of PuORh reveals a key role for Glu324 in substrate binding\nPuORh shares 32% sequence identity with human monoamine oxidase B (MAO-B), which is a 59\u00a0kDa FAD-containing protein involved in the oxidation of neurotransmitters and other arylalkylamines like benzylamine and phenylethylamine (Shih et al. 1999). Based on the structure of MAO-B (Binda et al. 2004b), it was possible to construct a model for PuORh. The C-terminal membrane anchor of MAO-B is absent in the resulting model structure of PuORh. This could explain why PuORh is well expressed in the cytosol as a soluble protein in contrast to MAO-B, which is membrane associated (Binda et al. 2004a). In the model, the active site of the PuORh exhibits a similar architecture as that of MAO-B (Fig.\u00a03). The catalytically important \u201caromatic cage,\u201d present in flavin-containing amine oxidases (Li et al. 2006), is formed by His432 and Tyr395. The other two key amino acid residues, Lys296 and Trp385, involved in non-covalent FAD binding (Binda et al. 2002b) can also be found in the model of PuORh. However, there are also some striking differences. In MAO-B, FAD is covalently attached to Cys397 at the C8\u03b1 position of the flavin. PuORh has an alanine residue at this position (Ala394), which precludes covalent binding of FAD. Furthermore, the active site of PuORh seems to be narrower than that of MAO-B due to the presence of Trp60 and Met173 in PuORh (Tyr60 and Cys172 in MAO-B). The model of PuORh also shows a glutamic acid residue (Glu324) pointing towards the N5 of the FAD, while MAO-B contains a tyrosine at this position (Tyr326). The presence of such a negatively charged amino acid in the active site has already been predicted for PuOMr several decades ago by Swain and Desa. They suggested the presence of a negatively charged carboxyl group in the active site, as PuOMr was found to be irreversibly inactivated by carbodiimides (Swain and Desa 1976). The presence and position of Glu324 can well explain the difference in substrate specificity between MAO-B and PuORh. PuORh prefers short aliphatic diamines, while MAO-B is active with a range of aromatic monoamines. Probably, Glu324 is involved in binding the protonated amino group of the diamine substrate, while the other amino group is positioned in the \u201caromatic cage\u201d near the N5 of the flavin. Such a mode of binding for putrescine is consistent with our PuORh model. To confirm this hypothesis, we mutated Glu324 into an alanine (PuORh Glu324Ala) and a leucine residue (PuORh Glu324Leu) and found that the catalytic efficiency for putrescine drops, respectively, 100,000 and 200,000-fold (Table\u00a03).\nFig.\u00a03Active site residues in the structure of MAO-B and in the model of PuORh. In MAO-B, the inhibitor rasagiline is covalently bound to the flavin. In the model of PuORh, the substrate putrescine is modeled in the active siteTable\u00a03Steady-state kinetic parameters for putrescine of wild-type PuORh and of the mutants Glu324Ala and Glu324Leu at pH\u00a09.0.\u00a0Km (\u03bcM)kcat (s\u22121)kcat\/Km (s\u22121 mM\u22121)PuORh WT3.5\u2009\u00b1\u20090.620.7\u2009\u00b1\u20091.15900PuORh Glu324Ala110,000\u2009\u00b1\u200920,0006.1\u2009\u00b1\u20090.40.05PuORh Glu324Leu110,000\u2009\u00b1\u200920,0003.2\u2009\u00b1\u20090.20.03\nThe distance between Glu324 and the N5 of the isoalloxazine ring of the flavin cofactor (9\u00a0\u00c5) may explain the specificity for the specific chain length of the substrate. To be a substrate, a minimal carbon chain length of 4 C-atoms is required. Shorter diamines can bind to Glu324 but cannot reach the flavin and therefore are strong competitive inhibitors of PuORh (Table\u00a02). In case of longer diamines or polyamines, the position of the amino group that is oxidized is less favorable compared to the case of putrescine resulting in a decrease in catalytic efficiency. Aliphatic monoamines like n-butylamine will bind to Glu324 via the amine moiety but cannot be oxidized and therefore are also inhibitors of PuORh. Initial substrate screening using 96-wells plates for the mutants PuORh Glu324Ala and PuORh Glu324Leu revealed that both showed some activity with aromatic amines like phenyl-1-butylamine and with aliphatic monoamines (e.g., kcat\/KM for n-butylamine\u2009=\u20090.001\u00a0s\u22121 mM\u22121 at pH\u00a09.0 for PuORh Glu324Leu). Unfortunately, accurate determination of the kinetic parameters of the mutant enzymes is not straightforward due to their low substrate affinity, which requires the use of high concentrations of amines, causing interference with the peroxidase-based assay.\nDiscussion\nIn an attempt to discover novel bacterial oxidases with biocatalytic potential, a putrescine oxidase from R. erythropolis NCIMB 11540 was identified. This putrescine oxidase was discovered by using a plate-based screening method for oxidase activity, which has been used before to screen for improved mutants during directed evolution experiments (Alexeeva et al. 2002). Our study shows that this method is also valuable for the discovery of novel bacterial oxidases of unsequenced genomes. Although the physiological substrate of the discovered putrescine oxidase, 1,4-diaminobutane, was not present during screening, clones expressing this oxidase could still be detected based on their oxidase activity. As polyamines like putrescine, spermine, and spermidine are present in most living cells, including E. coli (Tabor and Tabor 1984), this resulted in the formation of hydrogen peroxide and detection of positive clones.\nThe physiological role of putrescine oxidase in R. erythropolis is probably related to polyamine degradation (Large 1992), which is supported by the presence of a neighboring gene encoding a putative aldehyde dehydrogenase on the sequenced DNA fragment (data not shown). PuORh displays 67% sequence identity with PuOMr, which is the only bacterial putrescine oxidase that has been characterized so far. A BLAST search with the amino acid sequence of PuORh resulted in a high number of homologous (putative) flavin-containing amine oxidases, which are widely distributed among most kingdoms of life. The seven closest homologs (sequence identity >65%) can all be found in actinomycetes. These sequences all contain the active site glutamate (Glu324), involved in substrate binding, the alanine residue (Ala394), which excludes covalent FAD binding, and the residues, which limit the size of the active site cavity (Trp60 and Met173). Most likely, they represent a clade of orthologous putrescine oxidases, which are not active with monoamines, within the family of (putative) flavin-containing amine oxidases (Supplementary information, ESM 1).\nPuORh shares some properties with PuOMr. Both enzymes are soluble dimeric proteins of approximately 100\u00a0kDa. Interestingly, both contain only 1\u00a0mol of non-covalently bound FAD per mole of dimer, which is rather unique among flavoproteins. The substrate specificity of PuORh is very narrow, like PuOMr, the enzyme is very specific for putrescine. Polyamines are also accepted, but aliphatic monoamines are not converted at all. In general, PuORh appears to have a higher affinity for its substrates than PuOMr (Km for putrescine\u2009=\u20098.2 vs 38\u00a0\u03bcM).\nThe narrow substrate specificity of PuOMr for di- and polyamines was already rationalized by the suggestion of the presence of an \u2018anionic point\u2019 in the active site. This anionic point, likely due to a carboxylate function, binds one positively charged amino group of the substrate, while another amino group is oxidized (Swain and Desa 1976; Okada et al. 1979). Based on sequence alignment between PuORh and PuOMr and a structural model of PuORh, we can now identify Glu344 as the anionic point in PuOMr. The presence of a carboxylate function as a manner to bind a positively charged amino group is not restricted to putrescine oxidase. In polyamine oxidase (PAO), a negatively charged \u2018carboxylate ring\u2019 can be found at one side of its substrate tunnel to guide polyamine substrate molecules into the active site (Binda et al. 1999). In addition, in PAO, two glutamate residues (Glu62 and Glu170) are present in the active site opposite to the N5 of the flavin. However, these residues have been suggested to be protonated (Binda et al. 1999). Moreover, in PAO, Glu62 and Glu170 are in close proximity to a substrate secondary amine group, which will be oxidized, while in PuORh, Glu324 interacts with the (primary) amine group, which will not be oxidized. The amine group to be oxidized in PuORh is placed between His432 and Tyr395, near the N5 of the isoalloxazine ring. This structural feature resembles the \u201caromatic cage\u201d found in other flavin-containing amine oxidases, like MAO-A (De Colibus et al. 2005), MAO-B (Binda et al. 2002a), and PAO (Binda et al. 1999). For MAO-B, this \u201caromatic cage\u201d has been shown to play a steric role in substrate binding and in flavin accessibility and helps to increase the substrate amine nucleophilicity (Li et al. 2006).\nPolyamines like putrescine, cadaverine, spermine, and spermidine are related to cell growth and differentiation processes. Increased levels in body fluids and tissue occur in cancer patients, and therefore, polyamines represent important cancer markers (Casero and Marton 2007). Polyamines are also used to monitor food freshness, as an increased level can be found in spoiled food due to amino acid degradation (Bard\u00f3cz 1995). Due to efficient heterologous expression and its catalytic properties, PuORh may develop as a valuable diagnostic enzyme for the detection of low amounts of putrescine and polyamines. Moreover, based on the structural model, enzyme redesign may also allow creation of mutants that can be used for synthetic purposes.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nESM\u00a01\nUnrooted phylogenetic tree representation of PuORh homologs, which exhibit over 30% sequence identity (at the amino acid level) throughout the whole sequence. In gray, the clade of putrescine oxidases. MAO-B Human monoamine oxidase B; PuORh putrescine oxidase from Rhodococcus erythropolis NCIMB 11540; PuOMr putrescine oxidase from Micrococcus rubens. (DOC 36\u00a0kb)","keyphrases":["putrescine oxidase","flavin","amine","activity screening"],"prmu":["P","P","P","P"]}
{"id":"Breast_Cancer_Res_Treat-3-1-2137941","title":"The spectrum of ATM missense variants and their contribution to contralateral breast cancer\n","text":"Heterozygous carriers of ATM mutations are at increased risk of breast cancer. In this case-control study, we evaluated the significance of germline ATM missense variants to the risk of contralateral breast cancer (CBC). We have determined the spectrum and frequency of ATM missense variants in 443 breast cancer patients diagnosed before age 50, including 247 patients who subsequently developed CBC. Twenty-one per cent of the women with unilateral breast cancer and 17% of the women with CBC had at least one ATM germline missense variant, indicating no significant difference in variant frequency between these two groups. We have found that carriers of an ATM missense mutation, who were treated with radiotherapy for the first breast tumour, developed their second tumour on average in a 92-month interval compared to a 136-month mean interval for those CBC patients who neither received RT nor carried a germline variant, (p = 0.029). Our results indicate that the presence of ATM variants does not have a major impact on the overall risk of CBC. However, the combination of RT and (certain) ATM missense variants seems to accelerate tumour development.\nIntroduction\nHomozygous or compound heterozygous germline mutations in the ATM gene cause the autosomal recessive disorder ataxia-telangiectasia (A-T). This progressive neurological childhood disease is characterized by cerebellar degeneration, immunological defects, extreme sensitivity for ionising radiation and increased risk for cancers, particularly lymphomas [1]. ATM mutations identified in A-T families can be classified in three categories; truncating mutations, mutations that lead to some expression of mutant protein that lacks kinase activity and missense mutations with reduced kinase activity (http:\/\/chromium.liacs.nk\/lovd\/).\nHeterozygous pathogenic ATM mutation carriers, \u223c0.5\u20131% of the general population, do not display the symptoms observed in A-T patients. Several epidemiological studies have consistently shown elevated rates of breast cancer among female blood relatives of patients with A-T [2, 3]. Thompson et\u00a0al. have shown that the overall relative risk in carriers was 2.23 [95% confidence interval (CI) 1.16\u20134.28] compared to the general population and 4.95 (95% CI 1.9\u201312.9) in those younger that age 50. A large review showed that ATM mutations are more frequent in breast cancer patients selected on the basis of a family history of breast cancer than in unselected patients [4]. Besides pathogenic ATM mutations, a large number of ATM variants (common polymorphisms and unclassified variants) have been described, which were found in cancer patients as well as in the general population. It has been hypothesized that the cancer risk among ATM heterozygotes might be related to mutation type, suggesting that particularly missense mutations are associated with an increased risk [5, 6]. However, two recent studies by Thompson et\u00a0al. and Renwick et\u00a0al. showed that pathogenic ATM mutations that cause A-T are breast cancer susceptibility alleles [2, 7]. This argues against the hypothesis that missense rather that truncating are associated with breast cancer.\nWomen with breast cancer have in general a three to fourfold increased risk of developing a new primary cancer in the opposite breast [8]. The contralateral breast cancer (CBC) risk might be explained by the same genetic and hormonal factors that caused the first breast cancer. Treatment related factors, e.g. radiotherapy for primary breast cancer, may also contribute to the development of cancer in the contralateral breast [9] (our own data, manuscript under review).\nTo evaluate whether germline ATM missense variants are significantly associated with CBC risk (results regarding ATM truncating mutations are reported elsewhere) and whether treatment modifies this risk, we conducted a case-control study in which we assessed the ATM missense mutation spectrum and frequency in women who developed their first breast cancer before age 50, with and without a second primary breast cancer.\nMethods\nPatients\nThe consecutive breast cancer patients included in this study were all selected from the hospital tumour registries of The Netherlands Cancer Institute, Amsterdam (NKI-AVL) or The Dr. Daniel den Hoed Cancer Center\/Erasmus Medical Center, Rotterdam (DDHK). Of all patients that were invited to participate we achieved an 80% response rate. The breast cancer patients were included if their (first) breast cancer was diagnosed before age 50 (n\u00a0=\u00a0443). For CBC we required an interval of at least 1\u00a0year (n\u00a0=\u00a0247). The unilateral breast cancer patients (UBC) patients all had to be disease-free (of a second breast cancer) for at least 5\u00a0years. The first 57 CBC patients were individually age-matched (1:3) to UBC controls. All patients had invasive breast carcinoma and were treated with surgery. Of the CBC patients 169 did and 78 did not receive radiotherapy treatment for their primary breast tumour. Average age at diagnosis for the first breast cancer in the RT group was 41.2\/41.3\u00a0years (mean\/median) and the non-exposed group 42.0\/43.2\u00a0years (mean\/median). Detailed treatment data, disease and patient characteristics were obtained from medical records and risk factor questionnaires (data not shown) [10, 11]. Patients were asked to donate a 20\u00a0ml blood sample or permission for use of paraffin-embedded tissue blocks and patients gave their written informed consent for mutation analysis. This study received approval of the Medical Ethical Committees of NKI-AVL and DDHK.\nGenomic DNA isolation\nGenomic DNA was either isolated from peripheral blood lymphocytes with DNAzol (Invitrogen, Breda, The Netherlands) methods according to the manufacturer\u2019s instructions, or from three 10-\u03bcm paraffin embedded normal tissue slides according to standard protocols [12]. For histopathological examination we used a hematoxylin-eosin stained slide.\nMutation analysis\nThe complete ATM Open Reading Frame (ORF) was analysed, each exon (exon 4-65) and all intron-exon boundaries were screened for germline mutations using Denaturing Gradient Gel Electrophoresis (DGGE) identifying \u223c90% of all ATM mutations and polymorphisms (details from the author upon request). All aberrations were confirmed with genomic sequence analysis, performed using the ABI PRISM BigDyeTerminator Cycle Sequencing Ready Reaction Kit Version 3.1 (Applied Biosystems, Nieuwerkerk a\/d yssel, The Netherlands). Sequencing products were analysed with the ABI PRISM 3700 DNA Analyzer and corresponding software.\nStatistical analysis\nStatistical analyses were performed using standard methods for analysis of case-control studies [13]. We compared the mutation frequency between UBC and CBC and between CBC cases previously treated with RT and cases not-treated with RT. Odds ratios (ORs) and 95% CI were calculated to evaluate the association between mutation carriers status and breast cancer risk. We have used the Mann\u2013Whitney test to determine whether the difference between the intervals between the two breast cancers of the CBC patients was significant. All analyses were performed using SPSS 12.0 (SPSS Inc., Chicago, IL, USA).\nResults and discussion\nATM germline mutations\nIn the present study, we have used the DGGE method to screen the complete ATM ORF to obtain insight in the ATM missense mutation spectrum in (contralateral) breast cancer patients. With DGGE we were able to confirm all the previously identified truncating mutations. A subset of the CBC patients described in this study had been screened in the past for ATM truncating mutations with the Protein Truncating Test, revealing seven ATM truncating mutations (including a non-sense mutation and small insertions and deletions; generating stop codons within a previously functional protein coding sequence causing premature termination of translation of the protein) [10]. Among all 443-breast cancer patients that were tested in this study with DGGE we detected a large number of ATM silent mutations (presumed neutral polymorphisms, data not shown and excluded from all analyses) and missense mutations (causing an amino acid substitution in the coded protein, most common ones; i.e. D1853N, not included in further analysis).\nATM missense mutation spectrum\nIn our study cohort we have detected 35 distinct ATM missense variants and 6 distinct truncating mutations. Several of the detected missense variants have been reported in the ATM database as being detected in A-T patients\/or as polymorphisms (Table\u00a01). None of the missense variants identified in this study are known as pathogenic A-T causing missense mutations. Seventeen of the missense variants have not been reported previously. Eleven missense variants were exclusively found in the CBC group and 10 exclusively in the UBC group. Whether this distinction in the spectrum indicates an association between particular variants and bilateral breast cancer risk cannot be concluded from the small numbers obtained in this study population. The ATM protein has several functional domains and the identified missense variants are located throughout the ORF. Potential functional implications of the newly identified unclassified variants remain to be established.\nTable 1ATM missense variant and truncating mutation spectrum in contralateral and unilateral breast cancer patientsMissense variantsAmino acid changeCBC n\u00a0=\u00a0247UBC n\u00a0=\u00a0190Databasea or literature37C>TR13C1[10]146C>GS49C55database162T>CY54H21 [4], [14]378A>TD126E1database1009C>TR337C1Novel1132A>GS377G1Novel1229T>GV410A21 [4]1810C>TP604S1database2119T>CS707P78database2276G>A S759NNovel2336T>CM779T1Novel2414G>AR805Q2Novel2572T>CF858L43database2650C>TP884S12650C>TP884S1Novel2614C>TP872S [15]3161C>GP1054R813database3925G>AA1309T11 [16]4138C>TH1380Y1database4258C>TL1420F54database4324T>CY1442H2Novel4362A>CK1454N1database4477C>GL1493V1Novel4664T>AL1555H1Novel4722G>TL1574F1Novel5044G>TD1682Y1database5071A>CS1691R22database5557G>AbD1853N3549database5558A>TD1853V31database5741A>GD1914GNovel6067G>AG2023R1database6820G>AA2274T1database6919C>TL2307F1 [14]7446G>AM2482I1Novel7874A>GD2625G1Novel8659C>GH2887D1NovelTruncating mutationsIVS10-6T>G419X12database [10], [17]1563delAG521X1database1660delA554X1NovelIVS14\u00a0+\u00a02T>Gdel 601-6331database2572insTF858X1Novel3115A>TR1039X1Novela\u00a0http:\/\/chromium.liacs.nk\/lovd\/b\u00a0Not included in frequency analysis\nDespite the fact that ATM plays a role in breast cancer risk, the role of most distinct ATM missense variants remains unclear. Some studies tried to predict the relevance of each particular mutation on basis of co-segregation with breast cancer in families, the location in a functional domain or interference with the splicing machinery. Only a few studies present functional analysis that are necessary to assess the biological impact of unidentified variants found frequently in ATM [18\u201320].\nATM missense mutations and contralateral breast cancer\nTwenty-one per cent of the patients carried at least one ATM germline variant (missense and truncating; Table\u00a02). Among the patients with CBC (n\u00a0=\u00a0247) we identified in total 55 ATM variants in 45 individuals (18%); 51 missense variants and 4 truncating mutations (Table\u00a02). Eight CBC patients had multiple ATM missense variants and 2 patients carried both a missense and a truncating ATM mutation. In the women with UBC (n\u00a0=\u00a0196) we identified 52 ATM variants in 46 individuals (23%); 48 missense and 4 truncating mutations. Three UBC patients carried double missense and 3 patients both a truncating and a missense variant. Although it is known from the literature that ATM missense variants might be involved in breast cancer pathogenesis, the identified 17% missense variant carriers among the CBC patients compared to the 21% missense variants among the UBC patients indicate that there is not a significantly increased risk for bilateral breast cancer among ATM missense variant carriers, OR 0.77 (95% CI 0.48\u20131.24).\nTable\u00a02ATM variant frequencies in all breast cancer patients diagnosed under age 50 and according to uni- or contralateral breast cancerBreast cancer patients withAll patients n\u00a0=\u00a0443CBC n\u00a0=\u00a0247UBC n\u00a0=\u00a0196Total ATM variantsa55: 51 missense and 4 truncating52: 48 missense and 4 truncatingAt least one ATM variant91 (21%)45 (18%)46 (23%)At least one ATM missense variant85 (19%)43 (17%)42 (21%)Only one ATM truncating mutations321One truncating and one missense variant523Double missense variants1183a\u00a0Not included are the most common and silent variants\nAssociation with radiation treatment\nWomen at high risk for developing breast cancer may respond differently to radiation exposures associated with screening and treatment, than the general population. Candidate-genes like ATM are implicated in maintenance of genome integrity. Their involvements in breast cancer susceptibility as well as their role in DNA-damage repair signalling make them excellent candidates for a role in radiation-induced breast cancer [21]. Recently, we showed that women with a pathogenic germline mutation in a DNA repair pathway gene (e.g. BRCA1, BRCA2, CHEK2 and ATM) have an over 2-fold increased risk of developing radiation-associated breast cancer (manuscript under review). Therefore, we now investigated whether exposure to ionising radiation had a greater biological impact on certain ATM genotypes than on others.\nWe did not detect a significantly increased risk of developing radiation-associated CBC among missense mutation carriers. Among those 169 CBC patients who had developed a second primary breast tumour following radiotherapy for their first breast tumour we identified 19.5% ATM missense variants carriers compared to 13% among those CBC patients who did not receive RT, the OR from this case-only analysis is 1.65 [95% CI (0.77\u20133.55) p\u00a0=\u00a00.2]. Furthermore, we have observed that 21% of the UBC patients, who received RT but did not develop a CBC carried an ATM missense variant, compared to 19.5% of the CBC patients that received RT for their first tumour [OR 0.86 (95% CI 0.52\u20131.43)]. These results suggest that RT is not a strong risk factor for the development of CBC among carriers of those ATM missense variants.\nIt has however been shown that particular alterations in the ATM gene are associated with increased radiation sensitivity [22\u201324]. Gutierrez-Enriquez et\u00a0al. showed that lymfoblastoid cell lines carrying the ATM variant 3161G (linked to 2572C) was associated with increased in\u00a0vitro chromosomal radio-sensitivity, perhaps by interfering with ATM function in a dominant-negative manner [22]. We found this particular variant allele (3161G\/2572C) exclusively in our CBC group exposed to radiotherapy (four times) and not in the non-RT-exposed CBC group. This finding supports the hypothesis that particular ATM variants might play a differential role in radiation response. Although a subset of the missense variants was only detected in the RT exposed subpopulation, individual numbers were probably too small to detect a significant effect of particular mutations associated with treatment.\nWe observed that CBC patients with an ATM missense variant had an mean interval between the first and second breast tumour of \u223c101\u00a0months, compared to 122\u00a0months for non-carriers CBC patients (p\u00a0=\u00a00.085). Interestingly, the combination of radiation treatment and a missense variant resulted in an even shorter mean interval of a 92\u00a0months in the CBC patients compared to a 136-month interval for CBC patients who neither received RT nor carried a germline variant (p\u00a0=\u00a00.029). These data suggest that carrier-ship of an ATM missense variant may accelerate the development of a second tumour and decreases the age at onset of the second breast tumour, especially in case of exposure to RT.\nThe suggestion of a shorter induction period of RT-associated breast cancer in patients, who carry an ATM missense mutation, while the proportion of patients with missense variants was similar in CBC and UBC cases, might be attributable to a different spectrum of mutations in those patients who developed CBC. A big challenge in such a study remains to assess which particular missense mutations have an impact on ATM function. Large association studies, as performed by the Breast Cancer Association Consortium (coordinated by Doug Easton and Paul Pharoah, Cambridge), and functional studies are clearly necessary to determine the importance of particular variants and their contribution to the breast cancer risk.","keyphrases":["atm","missense variants","breast cancer","radiotherapy"],"prmu":["P","P","P","P"]}
{"id":"Eur_J_Epidemiol-4-1-2270357","title":"Data splitting as a countermeasure against hypothesis fishing: with a case study of predictors for low back pain\n","text":"There is growing concern in the scientific community that many published scientific findings may represent spurious patterns that are not reproducible in independent data sets. A reason for this is that significance levels or confidence intervals are often applied to secondary variables or sub-samples within the trial, in addition to the primary hypotheses (multiple hypotheses). This problem is likely to be extensive for population-based surveys, in which epidemiological hypotheses are derived after seeing the data set (hypothesis fishing). We recommend a data-splitting procedure to counteract this methodological problem, in which one part of the data set is used for identifying hypotheses, and the other is used for hypothesis testing. The procedure is similar to two-stage analysis of microarray data. We illustrate the process using a real data set related to predictors of low back pain at 14-year follow-up in a population initially free of low back pain. \u201cWidespreadness\u201d of pain (pain reported in several other places than the low back) was a statistically significant predictor, while smoking was not, despite its strong association with low back pain in the first half of the data set. We argue that the application of data splitting, in which an independent party handles the data set, will achieve for epidemiological surveys what pre-registration has done for clinical studies.\nIntroduction\nThe concept of statistical significance may be the single-most important mathematical invention for applied science. Its use has become so widespread and commonplace that many non-mathematical readers may not be actively aware of its true meaning. To briefly review, the statement \u201cX is correlated with Y at significance level alpha\u201d signifies, \u201cIf no true correlation between X and Y exists, the probability of obtaining the observed correlation is less than alpha.\u201d The P-value of a test is therefore a measure of surprise; the smaller the P-value, the greater the surprise. Standard practice has been to set alpha at 0.05, which literally allows for a 5% chance of erroneously reporting a significant finding (Type I error). One cannot interpret the P-value as a probability of having made a Type I error, so 5% significance does not imply that the conclusion is correct with a 95% probability. Such statements are meaningful only in a Bayesian context where one assigns a priori probabilities to hypotheses. The present article addresses non-Bayesian (frequentist) analysis, which is by far the most common in epidemiology.\nIoannidis [1] purports that most scientific findings are likely to be false, despite being reported as statistically significant. One of his arguments, which we support, is that the pressure to publish creates an incentive for researchers to simultaneously address a large number of hypotheses and selectively report only \u201csignificant\u201d results. This conduct is labelled hypothesis fishing or data dredging. (In older papers the term data mining has also been used, but the meaning of this has shifted toward discovery of valid patterns in databases.) Also, the investigator may run different statistical tests (e.g. parametric and non-parametric tests) for any given hypothesis, which is a less recognized form of hypothesis fishing.\nIn an epidemiological and health services setting, complex data sets based on statistical surveys are commonplace, in which hundreds of variables are collected from thousands of people. Data collection for a survey requires enormous effort, with both individual and collective demands on the researchers and respondents. From a purely economic point of view, it appears logical to \u201cturn the data set upside down,\u201d searching for anything of interest buried in it. Therein lies the temptation to launch a large-scale fishing expedition for all potentially interesting hypotheses. Similar to real-life commercial ocean fishing, which requires adjusting the mesh size of the nets upward, researchers investigating multiple hypotheses need to adjust the level of statistical significance down in order to preserve the meaning of statistical significance. The general rule of thumb is to divide alpha by the number of hypotheses, referred to as a Bonferroni correction [2]. However, in accordance with Ioannidis [1], we contend this method is rarely applied. The limited use of Bonferroni correction may result from uncertainty surrounding the exact number of hypotheses \u201cfished for,\u201d but some researchers may also be motivated to keep the \u201ccatch.\u201d In epidemiological research, upwards of 100 possible hypotheses are common, and setting the alpha at 0.0005 eliminates much of the fun in a fishing expedition. As a consequence, researchers may be tempted to discuss only a handful of \u201csignificant\u201d results, failing to mention the 95 that proved non-significant.\nHypothesis fishing renders P-values almost completely meaningless, and we consider it to be a serious problem for epidemiological survey analysis. The present paper recommends a very simple countermeasure against hypothesis fishing, based on data splitting. The procedure is similar to two-stage analysis of microarray data. We argue that if our method could be applied for survey data in general, it could do for epidemiological studies what pre-experiment registration of RCTs has done for medical experiments.\nThe rest of the article is laid out as follows: First we review some mathematical methods that might be considered useful for counteracting hypothesis fishing. Then we explain our method in detail, and compare it to two-stage analysis of microarray data, followed by a case study where the method is applied to an analysis of low back pain. The article ends with discussion and conclusion.\nMathematical remedies (that fail to solve the problem)\nIf one considers hypotheses fishing to be a mathematical problem, it is reasonable to look for mathematical solutions. There is a large mathematical literature that relates to model building and multiple hypotheses, and we will only try to point out the main themes.\nThe simplest way of handling the problem of multiple hypotheses is to reduce the significance level through Bonferroni correction. It is also possible to use so-called false discovery rate (FDR) [3], which is a way of controlling the expected percentage of rejected null-hypotheses (discoveries) that are falsely rejected. If all the null-hypotheses are true, FDR is equivalent to Bonferroni correction, but otherwise it is less strict. As we mentioned in the introduction, it can be hard to keep track of the number of hypotheses one is really addressing, which makes the use of Bonferroni or FDR cumbersome.\nComplex mathematical methods exist that adjust for the effect of model selection on inference (for example, see Madigan and Raftery [4]). In order to utilize these methods, however, one must describe the model-building strategy in a formal mathematical way. Faraway [5] has analysed various data splitting approaches similar to the one we apply, and compares them to mathematical inference adjustment methods. Based on his simulation study, he concluded that data splitting costs more in reduced accuracy than it gains in \u201chonesty\u201d. This conclusion is to be expected, because adjustment methods utilize information that the splitting methods disregard. In our present setting, however, accurate information on modelling procedure is not available, so adjustment methods are not an option.\nThe task of choosing a set of hypotheses is\u2014at least superficially\u2014related to the problem of choosing the set of predictors to include in a statistical model for the data. An overview of this field is given by Hastie et al.  [6], and we only mention a few key concepts. An obvious goal in statistical modelling of a given set of data is to develop a model that fits the data well, and a model will always fit better when the set of predictors is increased. However, if one includes too many predictors, the model is prone to imitating random properties of the data set, which are not present in the underlying sampling distribution of the data. Therefore, one needs ways to make trade-offs between model fit and model size. This can be done directly through various information criteria, such as Akaike\u2019s Information Criterion (AIC), the Bayesian Information Criterion (BIC), or the Divergence Information Criterion (DIC). A different approach called cross validation is to split the data set in two parts, using one part for estimating model parameters, and the other for evaluating model fit. A problem with this method is that only half of the data is used for each task. A clever remedy is to leave one data point out at the time, estimate the model from the remaining data points, and measure model fit by taking the average fit on the points left out (leave-one-out cross validation).\nWe mention these methods mainly to point out that they are not very relevant for controlling hypotheses fishing. The use of information criteria or cross validation helps in trading model fit for model size, but does not produce adjustments for the total number of variables. The cross validation procedure of splitting the data set, using one part to estimate model parameters and the other for validation is deceptively similar to the method we apply. But the purpose of validating a model\u2019s predictions is entirely different from our purpose of conducting sound hypothesis testing.\nIn addition to the inherent difficulties related to mathematical ways of dealing with hypothesis fishing, we do not see the problem as mainly a mathematical one. Some researchers may not be aware of the fact that P-values lose their meaning when the hypotheses are chosen from a large pool of undocumented ones. Others may be vaguely aware of the problem, but choose not to address it unless reviewers demand it. Reviewers, on the other hand, may not feel inclined to insist on purity beyond what they have done in their own scientific work.\nThe solution: data splitting\nWe recommend splitting the data set randomly into two sections (Parts 1 and 2). This allows the investigators to identify hypotheses in Part 1 of the data set, while remaining blind to Part 2 until the hypotheses are specified. True hypothesis testing is then performed using only Part 2 of the data. At this point there is no second-guessing. If the alpha-level is set to 0.05, and the P-value in Part 2 is 0.051, the result is by definition not significant, even if it received a P-value of 0.001 in Part 1. In such cases, there will be a temptation to \u201cmake a compromise\u201d by computing the average of the P-values from Parts 1 and 2, but this is not allowed. Because the data in Part 1 was used to construct the hypotheses, it is tainted, and cannot take any part in the hypotheses testing.\nThis procedure is strict with respect to identifying statistical significance. Once a hypothesis is supported, however, the entire data set is used for estimating the effect size. Thus, the purpose is to ensure the proper use of the term statistical significance. Once a significant finding is established, though, it is preferable to obtain the most accurate parameter estimates possible.\nModels: hypothesis variables, and confounders\nEpidemiological hypotheses are usually formulated within the framework of a model. Assume the hypothesis is that eating mushrooms increases the risk of cancer. To test this hypothesis, one would build a model with predictors like age, gender, smoking status, as well as mushroom habits, in order to control for these confounding factors. (If old people eat more mushrooms, excluding age from the model would give an incorrect positive association between mushroom eating and cancer.)\nFrom a purely computational point of view, no differences exist between predictors associated with hypotheses and confounders, yet the semantics are very different. The confounders are included only as a means of estimating the causal link between the cause (mushrooms) and its hypothesized effect (cancer).\nResearchers often use P-values as a road map (in addition to literature reviews and general medical knowledge), when deciding which variables should be included as confounders. A common piece of advice is to include confounders demonstrating an association at a P-value below 0.1 or 0.2. Despite this rationale for including a confounding factor, no similar demands are made for inclusion of the confounder in Part 2. The chosen set of confounders provides the framework within which the hypothesis is defined, and it is not the framework that is being tested. In the mushroom example, the hypothesis is that mushroom eating is associated with cancer when controlling for age, gender, and smoking, not that controlling for each of these factors is necessary.\nSize of Part 1 and Part 2\nWhen deciding upon the relative size of Parts 1 and 2, a trade-off exists between the need to identify hypotheses by exploration in Part 1, and the need to achieve statistical significance in Part 2. An even split may be reasonable in cases where the need for exploration is high, particularly if the data set is large, so that half of the data set is sufficient to achieve statistical significance for stronger effects. In cases where greater domain knowledge is available based on the existing literature, a smaller Part 1 is reasonable, especially when the sample size is small.\nMultiple hypotheses\nIt is possible to investigate multiple hypotheses within our splitting regime, using Bonferroni corrections. Assume, in the mushroom-cancer example, that the analysis of Part 1 also provided strong support for the hypothesis that eating bananas protects against cancer. One might then choose to include both mushroom habits and banana habits as hypothesis variables, and consequently divide alpha by 2 (the number of hypotheses). If either mushroom or banana habit fails the significance test in Part 2, it will still be in the model, as a confounder.\nIt might be the case that both banana and mushroom habits get P-values that fall between alpha\/2 and alpha. In this situation, both hypotheses would have passed the significance test individually, but the choice to include two hypotheses resulted in failure of both variables to reach significance. Many non-statisticians would argue that scientific procedures and statistical analysis should be objective, with conclusions based on \u2018hard facts\u2019, independent of arbitrary choices of hypotheses. Unfortunately, this is not the case if we wish to claim statistical significance.\nThe mushroom-banana example is a clear case in which investigators should reduce the alpha-level to account for multiple hypotheses. At the other extreme, if independent research groups investigating different research questions based on independent data sets, their combined effort is obviously not a case of \u2018multiple hypothesis testing\u2019. A grey area exists with partially overlapping data sets, hypotheses, and research groups, often making it difficult to decide whether Bonferroni corrections are called for. A pragmatic solution may be to view a published article as a unit, and apply Bonferroni corrections within each one.\nRelation to two-stage analysis in genetics\nReaders who are familiar with microarray analysis will recognize that our data splitting method is similar to two-stage analysis, as it is routinely performed in genetics [7].\nThere are a few differences, however. In a microarray context the set of possible hypotheses is given by the number of genes, and the FDR method is normally used to limit the number of incorrect findings. Rather than primarily counteracting hypothesis fishing, microarray two-stage analysis is usually motivated by cost effectiveness: By screening out promising candidates first, and then evaluating them, researchers can make a higher number of valuable discoveries for each monetary unit spent. In a microarray setting the procedure is also likely to be more automatic, as interesting genes are filtered out in two more or less mechanical steps of analysis. In our epidemiological application, on the other hand, there will be a man-in-the-loop, as the researcher builds a model with hypothesis variables and confounders based on a combination of his domain knowledge and Part 1 of the data.\nCase study of low back pain in the Ullensaker study\nStudy sample and setting\nData material consisted of adults enrolled in an epidemiological survey for musculoskeletal pain (MSP) in the Ullensaker municipality, 40\u00a0km northeast of Oslo in Norway. In 1990, 4050 inhabitants born in 1918\u20131920, 1928\u20131930, 1938\u20131940, 1948\u20131950, 1958\u20131960 and 1968\u20131970 (age 20\u201370\u00a0years) were sent a postal questionnaire about MSP. Of these people, 67% responded. Individuals who reported low back pain (LBP) during the past year (1990) were excluded from this material (N\u00a0=\u00a01439), such that the original sample consisted of 1283 participants who were free of LBP in 1990. In 2004, a 14-year follow-up was conducted. A total of 763 participants (59%) responded and formed the present study sample. These 763 participants were randomly divided in two samples with n\u00a0=\u00a0369 (Part 1) and n\u00a0=\u00a0394 (Part 2), respectively.\nOutcome measures \nTo identify respondents with LBP, we used the answer to the question, \u201cDuring the past year, have you experienced pain or discomfort in your lower back?\u201d. This item was based on the Standardised Nordic Pain Questionnaire [8], which is a self-report questionnaire frequently used in Scandinavian epidemiological studies.\nIndependent variables (potential risk factors)\nIn 1990, the survey questionnaire contained a number of socio-demographic and health-related factors, which could be included as risk factors in the present study. Socio-demographic variables were gender, age, marital status, and work status. Health-related variables were body mass index (BMI), smoking status, number of MSP sites other than the low back, duration of previous MSP, use of medication due to MSP, having been examined by a health care provider due to MSP during the last year, comorbidity, family history of musculoskeletal problems, emotional distress, leisure physical activity, participation in competitive sports, sleeping problems, and self-perceived health.\nModel and hypotheses\nA logistic regression model was developed based on Part 1 of the data set and medical expertise. The number of pain sites and smoking status were included as independent variables. Smoking status was dichotomised as smoking and non-smoking. Number of pain sites was operationalized using participant responses on the Nordic Pain Questionnaire [8]. Specifically, respondents reported whether they had experienced any pain or discomfort from the following 10 areas during the previous year: head, neck, shoulder, elbow, hand\/wrist, upper back, low back, hip, knee and ankle\/foot (responses were \u201cyes\/no\u201d). The total number of pain sites was computed and categorized into the following four categories: no pain sites, 1 or 2 sites, 3 or 4 sites, and 5 or more pain sites.\nWe also included age, which was categorized into values corresponding to the six birth cohorts: 1918\u20131920, 1928\u20131930, 1938\u20131940, 1948\u20131950, 1958\u20131960, and 1968\u20131970. In addition, gender and marital status (dichotomised into married\/partnership versus living alone) were included. Results of the logistic regression model are presented in Table\u00a01.\nWe hypothesized that smoking would be positively associated with LBP. Therefore, a 1-tailed hypothesis test was conducted. It was also hypothesized that individual pain sites would be positively associated with LBP. To limit the number of hypotheses, though, we hypothesized that the total number of pain sites would affect LBP probability, rather than run analyses for each level of the variable.\nHypotheses testing\nThe significance level alpha was set to the usual value of 0.05. With two hypotheses, the critical P-value becomes 0.025. Results for Part 2 of the data set are illustrated in Table\u00a02. The P-value of the pain sites variable (0.015) was below the critical value of 0.025, and therefore it is concluded that the number of pain sites was significantly associated with LBP at the 14-year follow-up.\nTable\u00a01Parameter estimates from Part 1, controlling for age, gender, and marital statusPredictorOR estimate 95% CI for ORP-valueNumber of pain sitesa0.0121 or 2 pain sites2.292(1.248\u20134.208)0.0073 or 4 pain sites2.690(1.406\u20135.147)0.0035 or more pain sites2.944(1.193\u20137.262)0.019Smoking2.079(1.285\u20133.363)0.003aThe reference category for number of pain sites was no pain sitesTable\u00a02Parameter estimates from Part 2, controlling for age, gender, and marital statusPredictorOR estimate 95% CI for ORP-valueNumber of pain sitesa0.0151 or 2 pain sites1.328(0.793\u20132.224)0.2813 or 4 pain sites1.598(0.857\u20132.979)0.1415 or more pain sites3.941(1.700\u20139.136)0.001Smoking0.993(0.627\u20131.571)0.487*aThe reference category for number of pain sites was no pain sites*1-sided P-value\nSmoking status received a 1-sided P-value of 0.487, which exceeds the limit of 0.025 by a large margin, and this variable is thus deemed non-significant. This result may seem surprising, given the variable\u2019s strong association with the dependent variable in Part 1 of the data set (2-sided P-value\u00a0=\u00a00.003). This illustrates the dangers of hypothesis fishing: Our analysis suggests that the smoking status variable may only have been a \u201clucky winner\u201d in the \u201cP-value lottery\u201d of Part 1.\nParameter estimates\nHaving concluded that number of pain sites has a significant effect on low back pain at follow up, we estimated the magnitude of the effect from the full data set (Table\u00a03). Because the hypothesis test failed to give significance for smoking status, it is included only as a confounder together with age, gender, and marital status. The 2-sided P-value for smoking status was 0.040, so researchers following a hypothesis fishing procedure would probably have reported it as a statistically significant predictor.\nTable\u00a03Parameter estimates of the hypotheses variable number of pain sites, from the complete data set controlling for age, gender, marital status and smoking statusPredictorOR estimate 95% CI for ORP-valueNumber of pain sitesa0.0001 or 2 pain sites1.637(1.116\u20132.400)0.0123 or 4 pain sites1.983(1.285\u20133.061)0.0025 or more pain sites3.346(1.846\u20136.067)0.000aThe reference category for number of pain sites was no pain sites\nDiscussion\nOur study was designed to illustrate a simple and straightforward data splitting method to counteract hypothesis fishing in large-scale epidemiological surveys. This method involves splitting the data set, where the first half is used to identify hypotheses, while the remaining data is used to test the hypotheses. The data splitting procedure was illustrated using data material collected for a population-based health survey administered in Norway in 1990 and 2004. Results demonstrated that the number of pain sites (\u201cwidespreadness\u201d of pain) was significantly associated with LBP following a 14-year follow-up. Smoking status was a strong predictor of LBP in Part 1 of the data set (hypothesis identification), but did not achieve significance in Part 2 (hypothesis testing). Therefore, this finding was dismissed as non-significant in our study. For the full data set, the P-value for smoking status was 0.040, so the traditional way of analysing epidemiological data would have given a different conclusion.\nIn this study, the investigators had free access to the entire data set prior to data splitting and during model development. However, any temptation to \u201cpeak\u201d at the material was successfully avoided, as indicated by the discrepant results for smoking status. This indicates that the data splitting procedure can indeed function properly in the absence of strict external control of the data. Nevertheless, we recommend that the data set be handled by an independent party, so that researchers can document claims that only Part 1 of the data set was used for model and hypothesis development.\nIdeally, the establishment of an independent international body is recommended to manage splitting of survey data. A fixed date for releasing data for Part 2 would be agreed upon, so that only those hypotheses specified prior to the release date would undergo a true significance test. Although several challenges and practical issues would inevitably need resolution (i.e., data collection, confidentiality, release of data), such an organization should be feasible and acceptable to the scientific community.\nConclusions\nResults demonstrated that the number of musculoskeletal pain sites significantly predicts low back pain at a 14-year follow-up, when controlling for age, gender, marital status, and smoking. The application of the data splitting method in our study indicates its potential as an effective and useful method to counteract hypothesis fishing in population surveys. In our opinion, systematic data splitting administered by an independent party would accomplish for statistical surveys what pre-registration has already done for clinical trials.","keyphrases":["data splitting","hypothesis fishing","low back pain","two-stage analysis","data dredging"],"prmu":["P","P","P","P","P"]}
{"id":"Neurochem_Res-3-1-1832151","title":"The Role of Presenilin and its Interacting Proteins in the Biogenesis of Alzheimer\u2019s Beta Amyloid\n","text":"The biogenesis and accumulation of the beta amyloid protein (A\u03b2) is a key event in the cascade of oxidative and inflammatory processes that characterises Alzheimer\u2019s disease. The presenilins and its interacting proteins play a pivotal role in the generation of A\u03b2 from the amyloid precursor protein (APP). In particular, three proteins (nicastrin, aph-1 and pen-2) interact with presenilins to form a large multi-subunit enzymatic complex (\u03b3-secretase) that cleaves APP to generate A\u03b2. Reconstitution studies in yeast and insect cells have provided strong evidence that these four proteins are the major components of the \u03b3-secretase enzyme. Current research is directed at elucidating the roles that each of these protein play in the function of this enzyme. In addition, a number of presenilin interacting proteins that are not components of \u03b3-secretase play important roles in modulating A\u03b2 production. This review will discuss the components of the \u03b3-secretase complex and the role of presenilin interacting proteins on \u03b3-secretase activity.\nIntroduction: the generation of beta amyloid by presenilin and its binding proteins\nAlzheimer\u2019s disease (AD) is a complex, progressive neurodegenerative disorder that is neuropathologically characterised by extensive neuronal loss and the presence of neurofibrillary tangles and senile plaques. While the majority of AD cases are sporadic, \u223c5% of AD cases are familial (FAD) with mutations in three genes, amyloid precursor protein, presenilin 1 (PS1) and presenilin 2 (PS2) accounting for the majority of cases. A major feature of both sporadic and familial forms of AD, is the accumulation and deposition of a small peptide referred to as beta amyloid (A\u03b2) within brain tissue of AD sufferers. The mechanisms that underlie the disease processes are poorly understood. However, the accumulation of A\u03b2 is thought to play a pivotal role in neuronal loss or dysfunction through a cascade of events that include the generation of free radicals, mitochondrial oxidative damage and inflammatory processes (reviewed in Refs. [1, 2]). The primary event that results in the abnormal accumulation of A\u03b2 is thought to be the dysregulated proteolytic processing of its parent molecule, the amyloid precursor protein (APP).\nThe APP molecule is a transmembrane glycoprotein that is proteolytically processed by two competing pathways, the non-amyloidogenic and amyloidogenic (A\u03b2 forming) pathways. How these pathways are regulated remains unclear. However, there are many factors including diet, hormonal status, and genetic mutations that influence the processing of APP to generate A\u03b2 (reviewed in Refs. [1, 3]). Three major secretases are postulated to be involved in the proteolytic cleavage of APP. These include \u03b1-secretase (of which the metalloproteases ADAM17\/TACE and ADAM 10 are likely candidates), beta APP cleaving enzyme (BACE, formally known as \u03b2-secretase) and the \u03b3-secretase. The \u03b1-secretase cleaves within the A\u03b2 domain of APP thus precluding the formation A\u03b2 and generating non-amyloidogenic fragments and a secreted form of APP (\u03b1-APPs). In the amyloidogenic pathway (Fig.\u00a01), BACE cleaves near the N-terminus of the A\u03b2 domain on the APP molecule, liberating another soluble form of APP, \u03b2-APPs and a C-terminal fragment (C99) containing the whole A\u03b2 domain. The final step in the amyloidogenic pathway is the intramembranous cleavage of the C99 fragment by \u03b3-secretase, to liberate the A\u03b2 peptide (reviewed in Ref. [1]). Identification of multiple cleavage sites within the APP molecule has suggested the presence of other secretases, namely \u03b5- and \u03b6-secretase; that generate the APP intracellular domain (AICD) and a longer fragment of A\u03b2 (A\u03b246), respectively [4\u20137]. Although different enzymes may exists, the same \u03b3-secretase complex that generates A\u03b2 may also generate AICD and A\u03b246 as these products are inhibited by \u03b3-secretase inhibitors [4\u20137] indicating that this enzyme has multiple cleavage sites. However, over-expression and mutagenesis studies have shown that the production of A\u03b2 and AICD may be independent catalytic events [8, 9].\nFig\u00a01Formation of the \u03b3-secretase complex and A\u03b2 generation. A schematic representation of the proposed formation of the \u03b3-secretase complex and the enzymatic activity resulting in A\u03b2 generation. Evidence has been provided to show that PS, nicastrin (NCT), aph-1, and pen-2 are the only components of the \u03b3-secretase complex. Nicastrin and aph-1 are involved in the early formation of the complex, interacting with each other and forming a dimeric sub-complex. The presenilin holoprotein is incorporated into the aph-1: NCT sub-complex, nicastrin undergoes post-translational modifications and the complex is transported to the cell surface (or other A\u03b2 generating compartments, i.e. TGN) as a trimeric complex. Pen-2 is incorporated into the complex and presenilin is cleaved forming the active \u03b3-secretase complex. Following cleavage of APP by BACE to liberate secreted APP, the APP-C99 fragment left embedded within the membrane undergoes regulated intramembranous processing (RIP) by the \u03b3-secretase complex, liberating A\u03b21\u201340 or A\u03b21\u201342 (Modified from Ref. [165])\nThe \u03b3-secretase enzyme is thought to be an aspartyl protease that has the unusual ability to regulate intramembrane proteolysis (RIP) for a growing list of type 1 integral membrane proteins which include, APP, APP like proteins (APLPs), E-Cadherin, CD44, lipoprotein receptor related protein (LRP), Notch, sterol regulatory element-binding protein (SREBP), interferon response element (IRE1) and activated transcription factor 6 (ATF-6) (for recent review see [10]). The mechanism of RIP and \u03b3-secretase activity is unknown. However a transient hydrophilic environment for catalysis within the lipid membrane must be created. Furthermore, the enzyme (or an enzyme domain) must have the ability to bend and unwind the \u03b1-helical substrates, exposing their amide bonds to hydrolysis. It is therefore conceivable that the \u03b3-secretase enzyme is an integral protein of the lipid bi-layer and contains a number of proteins that may have different functions within an enzyme complex. The exact conformation or molecular architecture of the \u03b3-secretase enzyme remains unknown. However, large molecular mass complexes of \u223c250\u00a0kDa [11, 12], \u223c500\u00a0kDa [13\u201317] and \u223c2,000\u00a0kDa [13, 18] have been identified.\nOver the last few years biochemical and genetic approaches have identified four components of the \u03b3-secretase complex, presenilins, nicastrin, anterior pharynx defective (aph-1) and presenilin enhancer 2 (pen-2). Over-expression and expression knockdown studies have provided strong evidence that these proteins are essential for \u03b3-secretase activity [15, 19\u201324]. Subsequent reconstitution studies in non-mammalian cells have provided evidence that these are the only components responsible for \u03b3-secretase catalytic activity [25\u201327]. However the active component and the active catalytic site of this enzymatic complex are yet to be identified.\nThe roles of presenilin and its binding partners within the \u03b3-secretase complex in A\u03b2 generation \nPresenilin 1: the catalytic component of the \u03b3-secretase enzyme\nPresenilin 1 (PS1) is thought to be the most critical component of the \u03b3-secretase complex as evidence indicates that it is the catalytic component of this proteolytic enzyme. The finding that mutations in PS1 account for the majority of inherited early onset forms of AD and result in the overproduction of the highly amyloidogenic A\u03b242 [28\u201330] provided the initial evidence that PS1 facilitates \u03b3-secretase activity. Subsequent studies showed that PS1 ablation or mutagenesis of two highly conserved aspartate residues within transmembrane domains 6 and 7 of PS1 results in a reduction in A\u03b2 levels [31\u201334]. Furthermore, aspartyl protease inhibitors and transition state analogue inhibitors which are designed to target the active site of the protease, all reduce A\u03b240 and A\u03b242 levels and have been shown to affinity label and bind to PS1 [35\u201337]. In addition, physical interactions between PS1 and the APP-C99 fragment have been identified [38\u201340]. The studies outlined above provide substantial evidence to indicate that PS1 is the catalytic subunit of \u03b3-secretase. However, this transmembrane protein is not typical of a conventional aspartyl protease, and lacks the typical D(T\/S)G motif for an aspartyl protease active site (recently reviewed in [41]. Aspartyl proteases require the presence of two aspartate residues for the hydrolysis of the peptide bond. Although PS1 lacks the typical D(T\/S)G aspartyl protease active site it does contain the two aspartyl residues (D257 and D385) which are either critical for the active site on the \u03b3-secretase complex or constitute the active site [42]. Other proteases that contain atypical aspartyl protease motifs and have weak homology with PS1 include the bacterial membrane embedded aspartyl protease, type 4 prepilin protease and the signal peptidases, which are all transmembrane proteins. These proteins share the unusual aspartyl motif GxGD and perform intramembranous cleavage of type I membrane proteins (reviewed in [43]). The topology and atypical characteristics of these proteins make them an unprecedented class of protease and as is the case for PS1, would require interaction with of other proteins within the complex.\nThe interaction between the components of the \u03b3-secretase complex is not sufficient to create a macromolecular enzyme complex such as \u03b3-secretase (i.e. total mass of all the components is \u223c200\u00a0kDa). Thus, dimer or trimer formation must occur within the complex. To date the only member of the complex that is known to undergo this process is PS1. The full length PS1 protein is rapidly endoproteolytically cleaved within its characteristic large hydrophilic loop into amino- and carboxy-terminal fragments (NTF\/CTF) of \u223c27 and \u223c17\u00a0kDa, respectively [44, 45]. These fragments are thought to interact with each other to form the catalytic component of the \u03b3-secretase enzyme since in the absence of full length PS1, co-expressed NTF and CTF were able to generate \u03b3-secretase activity in presenilin deficient mammalian cells [46, 47]. The stoichiometry and the nature of the interaction between these fragments remain unclear. It has been shown by many studies that the NTF:CTF form a heterodimer in mammalian cells [12, 48\u201351] leading to suggestions that this heterodimer is the active \u03b3-secretase [52]. However, Cervantes and colleagues [53] provided evidence that the presenilin fragments can form a tetramer by identifying heterodimers as well as NTF and CTF homodimers in yeast. Evidence for heterodimer and NTF homodimer (but not CTF homodimer) formation has been provided by photo-affinity labelled cross-linking studies [54]. This formation provides a core of aspartyl residues required for aspartyl protease activity. However, it has yet to be established whether the hypothetical \u201ccore\u201d is formed between fragments from one PS1 molecule or multiple molecules within the complex. Further, if PS1 is the \u201ccatalytic core\u201d of the \u03b3-secretase complex the roles of the other interacting proteins need to be determined.\nNicastrin: the substrate docking site and scaffolding for building the \u03b3-secretase complex\nNicastrin was discovered to be part of the \u03b3-secretase complex when it was found to co-immunoprecipitate with PS1 [55]. Nicastrin is a type I transmembrane protein with a 670 amino acid long hydrophilic N-terminal domain, a transmembrane domain, and a relatively short cytoplasmic C-terminus of twenty amino acid residues [55]. The protein undergoes glycosylation and sialylation within the secretory pathway to yield a mature \u223c150\u00a0kDa protein, the largest component of the \u03b3-secretase complex [14], [56\u201358]. Nicastrin has been shown to be a critical component of the \u03b3-secretase complex as inhibition of nicastrin function in vitro and in vivo results in a complete loss of APP and Notch cleavage [14, 55, 59\u201361]. Within this complex, nicastrin appears to be involved in the early formation of the complex, particularly in the formation of the first sub-complex between itself and aph-1 (Fig.\u00a01). Recent evidence suggests that nicastrin is critical for the correct assembly of the \u03b3-secretase complex within the endoplasmic reticulum and the intracellular trafficking of the complex to the cell surface [62, 63]. Furthermore, nicastrin is essential for the interaction between the complex and APP-C99 and thus may act as a receptor for \u03b3-secretase substrates [63, 64]. Therefore, although nicastrin is a critical component, evidence to date suggests that it is unlikely to be a catalytic component of the \u03b3-secretase complex. Instead, nicastrin acts as a scaffold for the building of the active complex, and a possible substrate docking site.\nAnterior pharynx defective 1 (aph-1): scaffolding partner for nicastrin and may have possible catalytic activity\nThe aph-1 protein is a \u223c30\u00a0kDa, 308 amino acid long 7-transmembrane protein [65] existing in two homologous forms, located on chromosome 1 and chromosome 15 (aph-1a & aph-1b respectively; [19]). In addition, as the result of alternate splicing, a long and short isoform of aph-1a have also been reported, with the short isoform more abundantly expressed in most tissues [21]. Although the exact function of these isoforms remains unknown, multiple \u03b3-secretase complexes containing aph-1 isoforms are thought to exist [66, 67]. The aph-1a isoform appears to be incorporated into active \u03b3-secretase complexes. Deletion of aph-1a causes a reduction in A\u03b2 generation [66] and is the major isoform present in \u03b3-secretase complexes during embryonic development [67]. The \u03b3-secretase complexes incorporating aph-1b are thought to have a redundant function as aph-1b has been found not to be involved in the regulation of other \u03b3-secretase components or \u03b3-secretase activity [66].\nThe aph-1 protein forms a stable and intermediate \u223c140\u00a0kDa complex with Nicastrin, before binding PS1. Aph-1 has been shown to interact with immature and mature forms of presenilins and nicastrin [21]. The GXXXG motif located within transmembrane 4 of aph-1 has been shown to be important in this interaction [68], [69]. The aph-1: nicastrin sub-complex appears to play a role in the stabilisation and assembly of PS1 in the \u03b3-secretase complex (Fig\u00a01; [24, 70, 68]). However, the exact role of aph-1 in the \u03b3-secretase complex remains unknown. Expression studies have shown that removal of aph-1 destabilises the presenilin holoprotein and the addition of aph-1 leads to the accumulation of the holoprotein [11, 19\u201324, 71]. Thus together with nicastrin, aph-1 is thought to contribute the initial formation and trafficking of the \u03b3-secretase complex and thereby provide a scaffold for the proteolytic processing of PS1 and the formation of the active site.\nAph-1 is critical for the activity of the fully constructed \u03b3-secretase complex. Evidence has been provided that as well as binding to immature components of \u03b3-secretase in early stages of complex formation, aph-1 also interacts with the mature forms of PS1, nicastrin and pen-2 [68, 72]. Furthermore, recent evidence has been provided to suggest that this interaction occurs on the cell surface where it also binds the \u03b3-secretase substrate, Notch and facilitates its cleavage [72]. These studies suggest that apart from the scaffolding function it shares with nicastrin, aph-1 has another function within the fully constructed, active \u03b3-secretase complex. Structural and functional similarities between aph-1 and other proteases that possess the ability for intramembranous cleavage (such as rhomboid, [68, 73] suggests that this transmembrane protein may have an enzymatic function within the complex. This postulated novel function for aph-1 needs to be validated by future studies.\nPresenilin Enhancer 2 (pen-2): the elusive presenilinase or \u201cmolecular clamp\u201d that holds the complex together?\nThe pen-2 protein is a \u223c12\u00a0kDa, 101 amino acid long protein containing two transmembrane domains and its N- and C-terminals facing the lumen of the endoplasmic reticulum [74, 75]. Pen-2 has been shown to interact with Nicastrin, aph-1, and also PS1. Without this interaction pen-2 has been shown to be degraded, possibly by the proteasome [76, 77]. Ablation of pen-2 results in significantly reduced PS1 endoproteolysis and A\u03b2 production, suggesting that pen-2 is essential for both presenilinase and \u03b3-secretase activity [15, 19, 22\u201324]. Likewise, combinational expression studies have shown that when pen-2 is co-expressed with the other components of the \u03b3-secretase complex in a step-wise manner, endoproteolysis of the PS1 holoprotein was enhanced [11, 22, 24\u201327, 71]. A trimeric sub-complex between presenilin, nicastrin and aph-1 has been identified in cells lacking pen-2 [24, 70] suggesting that pen-2 may be the final protein incorporated into the \u03b3-secretase complex (Fig.\u00a01). Recent evidence has suggested that pen-2 is incorporated into the \u03b3-secretase complex through an interaction with presenilins. The \u201cDYLSF\u201d domain of pen-2 and a \u201cNF\u201d motif on transmembrane 4 of PS1 have been shown to be critical for the interaction between these proteins [78\u201380]. The cleavage of PS1 occurs within the hydrophilic loop and is distal from the interaction site on transmembrane 4, suggesting that another domain of pen-2 is responsible for the cleavage of PS1 or pen-2 may present PS1 to the elusive \u201cpresenilinase\u201d which may be another member of the \u03b3-secretase complex. The C-terminal end of pen-2 has been shown to be important for \u03b3-secretase activity since altering the length of this region by addition or deletion of residues has been shown to reduce A\u03b240 and 42 generation without altering the binding of pen-2 to the complex [78] suggesting that pen-2 may have an alternative function within the complex. One suggestion put forward by Hasegawa and colleagues [78] is that the C-terminus of pen-2 may be a linker\/space molecule that maintains spatial interactions between proteins within the complex. However, recent evidence using pen-2 C-terminal loss of function mutations suggest that the C-terminus acts as a \u201cmolecular clamp\u201d holding together the presenilin fragments and the whole \u03b3-secretase complex [81]. If this is the case then PEN-2 is an integral part of the catalytic process holding the complex together whilst the \u03b3-secretase products are generated.\nThe newest member of the complex: TMP21, a modulator of \u03b3-secretase activity\nRecently another member of the \u03b3-secretase complex was immuno-purified from wild-type PS1 and PS2 blastocyte derived cells [82]. The type 1 transmembrane protein termed TMP21 is a member of the p24 cargo-family which may have a signalling role in the sorting and transport of proteins from the endoplasmic reticulum to the Golgi [83, 84]. It was shown by Chen and colleagues [82] that TMP21 is a member of the complex as it was isolated in a high molecular weight presenilin complex, interact with all of the known components of the \u03b3-secretase complex, co-localised with the complex components in the ER, Golgi and cell surface and destabilised from the complex in the absence of the presenilins and pen-2. Although the over-expression of TMP21 did not alter \u03b3-secretase activity, its suppression resulted in and increase in A\u03b240 and A\u03b242. However, suppression of TMP21 did not alter the production of AICD, notch cleavage to generate NICD or cleavage of E-cadherin to generate CICD. These results are consistent with the notion that \u03b3- and \u03b5-secretase cleavage activities are independently regulated and indicate a role for TMP21 in modulating \u03b3-secretase activity to generate A\u03b2. This role for TMP21 appears to be independent of its role in protein transport since the suppression of both TMP21 and p24a (a member of the p24 cargo family that interacts with TMP21) does not result in additional increases in A\u03b2 production to that observed following the suppression of TMP21 only [82]. This finding led the authors to postulate that there are two pools of TMP21, a major pool that is stabilised by, p24a and has no role in A\u03b2 production and a minor pool that modulates A\u03b2 production, independent of p24a. Although, further investigation is required to determine the precise mechanism of action, it appears that TMP21 may function to regulate intramembrane proteolysis controlling \u03b3-secretase activity and thus preventing the over-production of A\u03b2. This modulator role for TMP21 is important to consider when elucidating mechanisms on how \u03b3-secretase activity is altered (for example by presenilin mutations) resulting in enhanced A\u03b240 and A\u03b242 production.\nPresenilin 2 (PS2): capable of forming a fully functional \u03b3-secretase complex\nThe presenilin proteins share an overall homology of 67% with the highest similarities in the predicted transmembrane domains. Fewer similarities are found at the N-terminus and in the central region of the hydrophilic loop [85, 86]. Although there are some structural similarities between PS1 and PS2 several lines of evidence suggest that these proteins may have distinct functions. Mice lacking PS1 die before birth and have severe skeletal and brain deformities, whilst those lacking PS2 only develop a mild pulmonary fibrosis and haemorrhage with age [33, 87\u201390]. Compared to neuronal cultures isolated from PS1 ablated mice, those isolated from PS2 knockout mice exhibit higher A\u03b2 production and are less sensitive to \u03b3-secretase inhibitors [89, 91]. Lai and colleagues [91] also provided evidence to suggest that distinct PS1 containing and PS2 containing complexes exist [91]. If this is the case, evidence to date suggests that PS2 containing complexes have different functions and have less \u03b3-secretase processing power than PS1 containing complexes. However, recent in vitro evidence has suggested that chimeras of PS1 and PS2 fragments retain \u03b3-secretase activity [92] suggesting that active complexes containing both PS1 and PS2 could occur. However, this is unlikely to occur in vivo as PS2 and PS1 transgenic mice have differential effects on \u03b3-secretase activity [93]. In this study mice lacking PS1 were crossbred with transgenic mice containing wild type or mutant PS2\/PS1. Compared to mice harbouring wild-type PS1, those harbouring wild-type PS2 had a \u223c4-fold reduction in A\u03b240 and \u223c2-fold reduction A\u03b242 brain levels. In addition, the authors reported that although mutations in PS1 lead to higher brain levels of A\u03b240 and A\u03b242 than PS2 mutations, those mice harbouring PS2 mutations had higher brain levels of A\u03b242, whilst A\u03b240 brain levels remain unchanged [93]. Taken together these studies suggest that PS1 containing complexes predominate in generating A\u03b240 and A\u03b242. However, the finding that PS2 is involved in A\u03b242 (but not A\u03b240) generation suggests that two A\u03b2 generating \u03b3-secretase complexes (\u03b3-40 secretase and \u03b3-42 secretase) exist, with \u03b3-42 secretase complexes containing both PS1 and PS2.\nThe roles of presenilin binding proteins not incorporated into the active \u03b3-secretase complex\nIn addition to A\u03b2 generation, the presenilins have been implicated in a variety of intracellular processes including membrane trafficking, Notch signalling, neuronal plasticity, cell adhesion, regulation of calcium homeostasis, the unfolded protein response, and apoptosis. Therefore it is not surprising that the presenilins have a growing list of binding partners (see Table\u00a01). Though the presenilin binding proteins that are incorporated into the \u03b3-secretase complex are critical for the activity of this enzyme, those that are not integral components have equally important roles in modulating A\u03b2 production. A number of non-complex presenilin binding proteins have been shown to alter \u03b3-secretase activity (see Table\u00a01). However, only a few have had supporting evidence that their direct interaction with the presenilins modulate \u03b3-secretase activity. These are described below and shown in Fig.\u00a02.\nTable\u00a01Presenilin interacting proteinsInteracting proteinProposed functionInteraction with presenilins shown to modulate A\u03b2 production ReferenceComplex componentsAPH-1Component of the \u03b3-secretase complex; role for initial formation of the complex, or a proteolytic roleYes[19, 65, 67, 123].NicastrinComponent of \u03b3-secretase complex; possible role as a receptor for APP-C99Yes[14, 55\u201358].PEN-2Component of the \u03b3-secretase complex, role in proteolytic cleavage of the presenilins Yes [19, 24, 70, 76, 77]. TMP21Recently identified component of the \u03b3-secretase complex, role in modulating A\u03b2 productionYes[82]\u03b3-secretase substratesAPP-C99Substrate for the \u03b3-secretase complex: precursor to A\u03b2 generationYes [38\u201340]E\/N cadherin\u03b3-secretase substrate; role in Cell-Cell AdhesionNo[124]IRE1\u03b3-secretase substrate; role in unfolded protein responseNo[125, 126]LRP\u03b3-secretase substrate; Lipid metabolismYes [119]Notch1 \u03b3-secretase substrate; precursor to NICD generationYes [112]Interacting binding proteinsABP-280, Filamin homolog1Actin binding proteinNo[127]Bcl-2 Anti-apoptotic moleculeNo[128]Bcl-XLAnti-apoptotic moleculeNo[129]CalmyrinPossible calcium-myrstol switchNo[130]CALP- calsenilin like proteinNovel member of the calsenilin\/KChIp protein familyNo[131]CalsenilinCalcium binding proteinYes[94, 96\u201399, 101, 102]CLIP-170Linking membrane organelles to microtubules No[132]DRALLIM-domain containing proteinNo[133]FHL2Role in PI3K\/Akt activationNo[134]FKBP38Role in mitochondria mediated apoptosis No[135]GoSignalling moleculeNo[136]GSK3\u03b2Wnt signalling, serine threonine protein kinase No[137]HC5\/ZETASubunits of the catalytic 20S proteasomeYes[138]Met1Putative methyltransferaseNo[139]Modifier of cell adhesion protein (MOCA)Regulates proteasomal activity on APPYes[140]Omi\/HtrA2Serine protease, proapoptoticNo[141]PAGNeuronal proliferation proteinNo[142]PAMP and PARLMetalloproteases No[143]PKASerine\/Theronine protein kinase; \u03b2-catenin phosphorylationNo[144]PlakoglobinRole in b-catenin\/Tcf-4 activityNo[145]PLD1Phospholipid-modifying enzymeYes [146]PSAPPDZ like proteinNo[147]QM\/Jif1Negative regulator of c-JunNo[148]Rab proteinsVesicle mediated protein traffickingYes[149, 150]RyR2Cardiac ryanodine receptor No[151]SEL-10Ubiquitination of proteinsYes[152]SorcinCalcium binding regulator of ryanodine receptorNo[153]Syntaxin 1ASynaptic plasma-membrane proteinNo[154]Syntaxin 5ER-Golgi vesicular transportYes[155, 156]TauMicrotubule binding proteinNo[137]TelencephalinNeuron specific adhesion moleculeNo[40]TPIPTetratricopeptide repeat-containing proteinNo[157]UbiquilinUbiquitin domain-containing proteinNo[158\u2013160]X11 family of proteinsCytoplasmic adaptor proteinsYes[110, 111]\u03b2-catenin\/\u03b4-catenin (NPARP)Wnt signalling. Cell adhesionNo[161\u2013163] \u03bc-CalpainCalcium-dependent thiol proteaseNo[164]Fig.\u00a02The postulated roles for presenilin interacting proteins not incorporated into the \u03b3-secretase complex in A\u03b2 generation. A schematic representation of the postulated roles of presenilin interacting proteins in (A) promoting or (B) attenuating \u03b3-secretase activity and A\u03b2 generation. (A) Together with the formation of the \u03b3-secretase complex, calsenilin have been shown to interact with the presenilins and promote A\u03b2 generation when over-expressed in mammalian cells. The role of calsenilin in A\u03b2 generation remains unclear but maybe involved in the presentation of the substrate to the \u03b3-secretase complex. (B) The presenilin interacting protein, X11 and other \u03b3-secretase substrates that compete for enzyme activity such as Notch fragments (i.e. NEXT) have all been shown to reduce A\u03b2 generation when over-expressed in mammalian cells. X11 may reduce A\u03b2 production by binding to PS and APP preventing PS mediated cleavage of APP or its presentation to the \u03b3-secretase complex \nCalsenilin\nCalsenilin (also called DREAM or kChIP3) is a calcium binding protein, that was first identified through yeast two hybrid screening using the PS2 C-terminus as the bait protein [94]. The interaction between calsenilin and the presenilins appear to have a role in APP processing. It was initially demonstrated that calsenilin can form a complex with PS1 and PS2 and regulate presenilin fragment formation in mammalian cells [95]. This study also showed that calsenilin may facilitate presenilin mediated apoptosis, since calsenilin was shown to preferentially interact with the C-terminal fragment of PS2 that results from caspase cleavage of the presenilin holoprotein [96]. Expression studies in vivo (Xenopus) and in vitro (mammalian cells) have shown that the interaction between presenilin and calsenilin may be involved in ER calcium release and apoptosis [97, 98]. Over-expressing calsenilin in neuronal cells resulted in cell death which was enhanced by the co-expression of wild-type or PS1 mutations [99]. These studies have suggested that the interaction between calsenilin and presenilins may function in apoptotic pathways. However, several lines of evidence suggest that calsenilin may also be implicated in APP processing and A\u03b2 generation. Initial in vitro studies showed that over-expression of calsenilin in mouse neuroblastoma cells resulted in a gene dose dependent accumulation of A\u03b242 [100]. Using a \u03b3-secretase luciferase based assay and a cell free system established by Li and colleagues [18] to characterise the \u03b3-secretase complex in purified cell membranes, Jo and colleagues [101] recently reported that the over-expression of calsenilin resulted in an increase in \u03b3-secretase activity. This calsenilin\/presenilin, mediated activity was markedly reduced using a well-characterised \u03b3-secretase inhibitor (L-685, 458). Furthermore, compared to wild-type mice, brain A\u03b240 and A\u03b242 levels were reduced in calsenilin ablated mice [102]. Together these studies would suggest that calsenilin has a second function, to facilitate \u03b3-secretase activity.\nX11\/Mint proteins\nA family of cytoplasmic adaptor proteins called X11 (also known as munc-18-interacting proteins\u2014mints) have also shown to have a direct role in APP processing. In particular, several studies have reported that X11\u03b1 and X11\u03b2 stabilise APP and inhibit the secretion of A\u03b2 and APP from neuronal cells [103\u2013106]. The molecular mechanisms by which the X11s influence APP processing and A\u03b2 production are not clear. However, this effect appears to be modulated by a direct interaction between X11 and APP. The interaction between the phosphotyrosine binding (PTB) domain within X-11 and the YENPTY motif within APP appears to modulate A\u03b2 generation, since introduction of point mutations within these domains enhanced A\u03b2 generation [103, 106\u2013109]. The binding of X11 appears to stabilise APP, leading to its intracellular accumulation [103, 106\u2013109]. It is conceivable that the X11\u2013APP interaction may inhibit secretase accessibility to APP resulting in impaired A\u03b2 secretion. The X11 proteins have multiple binding domains and have been shown to interact with the PS1 C-terminal fragment through their PDZ domain [110]. The authors also showed an interaction between PS1 and APP in the presence of X11 that was otherwise ablated in the absence of X11. This suggests that an interaction between APP and PS1 is mediated through X11. A subsequent study has reported that X11\u03b2 and alcadein (and X11\u03b2-binding protein) expression prevents the binding of PS1 to APP [111]. The significance of the results from these interaction studies remains unclear. However, they do suggest that X11 proteins may act as inhibitory molecules preventing PS1 mediated proteolytic processing of APP and thus attenuating A\u03b2 production.\nCompeting substrates\nSome presenilin binding proteins reduce the likelihood of A\u03b2 generation by competing for the catalytic activity offered by \u03b3-secretase. One example is Notch1 which undergoes cleavage by \u03b3-secretase to liberate an intracellular domain (NICD) that translocates to the nucleus where it co-activates transcription of genes involved in several developmental pathways [112\u2013115]. The first evidence of substrate competition came from one study that treated neurons with the Noch1 ligand, delta, and found a dose dependent reduction in A\u03b2 levels [116]. In contrast, the over-expression of APP in these neurons resulted in a reduction in Notch signalling [116]. Further evidence for substrate competition was provided by Kimberley and colleagues [117] where APP and Notch based substrates directly competed for \u03b3-secretase activity in a cell free assay. Similar results were achieved in a subsequent study where cells expressing Notch showed a reduction in A\u03b2 production [118]. Additional evidence for substrate competition has been provided recently for another substrate of \u03b3-secretase, the cytoplasmic tail of the low-density lipoprotein receptor\u2014related protein (LRP), which has also been shown to interact with PS1 [119]. The co- expression of the LRP C-terminal fragment and APP in CHO cells has been shown to result in a reduction in secreted levels of A\u03b240 and A\u03b242 [119].\nTaken together, these studies not only suggest substrate competition but also the presence of a single \u03b3-secretase. However, the presence of multiple \u03b3-secretase complexes cannot be ruled out. As discussed above there is evidence in the literature for the formation of \u03b3-secretase complexes with distinct functions. All of the studies described above are undertaken in conditions where there is a large amount of substrate available which could down-regulate the activity of one \u03b3-secretase for another. Whether substrate competition occurs under physiological conditions and if there are certain conditions that promote a higher expression of one substrate over another remains to be determined.\nConcluding comments\nIt is well established that together with presenilin, its binding proteins, nicastrin, aph-1 and pen-2 are the only proteins that constitute the \u03b3-secretase complex responsible for generating A\u03b2 from APP. The function(s) of each \u03b3-secretase component within the complex is gradually becoming apparent. However, there are many facets of \u03b3-secretase activity that are yet to be identified such as the domain responsible for actual catalytic activity; the mechanisms of enzymatic activity within the lipid bi-layer; and the existence of single or multiple \u03b3-secretase complexes. Further investigation using appropriate protein expression models and reconstitution studies such as those used to validate the protein components of \u03b3-secretase complex is required to provide some insight into the processes that underlie the catalytic activity of this enzyme. Indeed, reconstitution of the \u03b3-secretase complex in insect cells and purification of this complex from the cells have provided researchers with the first 3D images of the structure of this intricate enzyme [120, 121]. Identification of the specific catalytic domain(s) within the complex would provide a site that could be a potential target to develop agents that specifically modulate A\u03b2 levels without altering the other known (and unknown) activities of the \u03b3-secretase enzyme. Recent insight into this notion was provided by the finding that certain compounds selectively block the cleavage of APP but not Notch [122]. However, it is important to note that other presenilin interacting proteins not incorporated into the complex and the pathways they are involved in play important roles in APP processing and A\u03b2 generation. These interactions assist in many facets of \u03b3-secretase activity, including modulating substrate\/enzyme availability, and thus are important to consider when developing appropriate therapeutic strategies.","keyphrases":["presenilin","interacting proteins","beta amyloid","alzheimer\u2019s disease","amyloid precursor protein","gamma secretase"],"prmu":["P","P","P","P","P","M"]}
{"id":"Biochim_Biophys_Acta-2-1-2396231","title":"Differential regulation of Kr\u00fcppel-like factor family transcription factor expression in neonatal rat cardiac myocytes: Effects of endothelin-1, oxidative stress and cytokines\n","text":"Kr\u00fcppel-like transcription factors (Klfs) modulate fundamental cell processes. Cardiac myocytes are terminally-differentiated, but hypertrophy in response to stimuli such as endothelin-1. H2O2 or cytokines promote myocyte apoptosis. Microarray studies of neonatal rat myocytes identified several Klfs as endothelin-1-responsive genes. We used quantitative PCR for further analysis of Klf expression in neonatal rat myocytes. In response to endothelin-1, Klf2 mRNA expression was rapidly increased (\u223c 9-fold; 15\u201330 min) with later increases in expression of Klf4 and Klf6 (\u223c 5-fold; 30\u201360 min). All were regulated as immediate early genes (cycloheximide did not inhibit the increases in expression). Klf5 expression was increased at 1\u20132 h (\u223c 13-fold) as a second phase response (cycloheximide inhibited the increase). These increases were transient and attenuated by U0126. H2O2 increased expression of Klf2, Klf4 and Klf6, but interleukin-1\u03b2 or tumor necrosis factor \u03b1 downregulated Klf2 expression with no effect on Klf4 or Klf6. Of the Klfs which repress transcription, endothelin-1 rapidly downregulated expression of Klf3, Klf11 and Klf15. The dynamic regulation of expression of multiple Klf family members in cardiac myocytes suggests that, as a family, they are actively involved in regulating phenotypic responses (hypertrophy and apoptosis) to extracellular stimuli.\n1\nIntroduction\nC2H2 zinc finger transcription factors possess two cysteine and two histidine residues which co-ordinate Zn2+ within each finger to form a conserved DNA-binding structure. Sp1-like proteins and Kr\u00fcppel-like factors (Klfs) each have three C2H2 zinc fingers at their C-termini, and Klfs are distinguished by a highly conserved interfinger space sequence [1]. The seventeen mammalian Klfs which have been identified are now designated Klf1-17 [2,3], though many have alternative names. Several were named according to the tissues in which they were originally shown to be enriched (e.g. Klf1 is erythroid Eklf, Klf2 is lung Lklf, Klf4 is gut Gklf, Klf5 is intestinal Iklf and Klf15 is kidney Kklf [2]). Other Klfs are widely expressed including Klf3 (basic Bklf), Klf6 and Klf7 (ubiquitous Uklf). Klf9 was identified as a basal transcription element binding (BTEB) protein, with Klf5 and Klf13 as homologues (BTEB2 and BTEB3, respectively). Klf10 and Klf11 were identified as early genes induced by transforming growth factor \u03b2 and were named TIEG and TIEG2, respectively.\nKlfs bind to consensus GC-rich or CACCC sequences through the three C-terminal zinc fingers [1]. The frequency of such sequences in gene promoters, coupled with the high sequence identity between Klf proteins in their DNA-binding domains, raises questions relating to the specificity of any Klf for a particular promoter and potential functional redundancy. Nevertheless, Klfs can be clustered according to whether they act primarily as transcriptional repressors and\/or activators, and the domain structure of the N-terminal regulatory regions. Klf3, Klf8 and Klf12 recruit C-terminal binding proteins (CtBPs) to repress transcription, whereas Klf9, Klf10, Klf11, Klf13 and Klf16 repress transcription through interaction with mSIN3a [1,4]. Klf15 also represses transcription, though the mechanism is not clear. Klf2, Klf4, Klf5, Klf6, and Klf7 are largely transcriptional activators [1], but may suppress gene expression in specific circumstances (e.g. Klf5 negatively regulates expression of Klf4 [5]). In addition to interactions with other proteins, post-translational modifications (e.g. phosphorylation or acetylation) regulate the transactivating activities of different Klfs [1,6]. Globally, Klfs regulate fundamental cellular responses such as growth, apoptosis, angiogenesis and proliferation. For example, Klf5 overexpression is associated with cell proliferation, whereas Klf2, Klf4 and Klf6 are more consistently implicated in cell cycle arrest [7\u20139]. Klf4 and Klf5 may both promote apoptosis induced by oxidative stress though the mechanism is unclear [9].\nMammalian cardiac myocytes (the contractile cells of the heart) become terminally-differentiated shortly after birth. Subsequent growth of the heart results from an increase in size of individual cardiac myocytes and, in the adult, myocytes may hypertrophy in order to maintain or increase cardiac output. This is associated with physiological and morphological changes (increases in cell size and myofibrillogenesis) and changes in gene expression including increased expression of immediate early genes (IEGs), and re-expression of genes normally expressed in early development [10]. Stimuli such as endothelin-1 (ET-1), which activate Gq protein-coupled receptors are particularly implicated in cardiac myocyte hypertrophy, and promote both the physiological\/morphological changes and the changes in gene expression associated with the response [11]. In contrast, oxidative stresses or pro-inflammatory cytokines may induce cardiac myocyte apoptosis [12]. Many studies have examined the intracellular signalling pathways which are activated by various stimuli in cardiac myocytes, and these are presumed to lead to changes in gene and protein expression to promote hypertrophy or to facilitate myocyte death [13].\nMany Klfs are expressed in adult hearts to a degree although the cell types in which they are expressed cannot be ascertained. For example, Klf2 is associated with endothelial cells [14,15], and cardiac endothelial cells could account for expression of Klf2 in whole heart extracts [16]. As reviewed by Haldar et al. [17], Klf13 and Klf15 are the only Klfs which have so far been shown to play a role in cardiac myocytes. Klf13 is highly expressed in adult hearts [18] and is required for normal cardiac development in Xenopus\n[19]. Klf15 is highly expressed in adult hearts and in cardiac myocytes [20,21]. It appears to be anti-hypertrophic since it is downregulated during hypertrophy, and overexpression of Klf15 in cardiac myocytes suppresses the morphological changes and changes in gene expression induced by hypertrophic stimuli [21]. Our microarray studies highlight the dynamic changes in expression of many genes (including Klfs) induced by H2O2 (an example of oxidative stress) or ET-1 in cardiac myocytes over 4\u00a0h [22\u201325]. Here, we explore further the regulation of Klf family members in cardiac myocytes in response to ET-1, H2O2 and pro-inflammatory cytokines [interleukin 1\u03b2 (IL-1\u03b2) and tumour necrosis factor \u03b1 (TNF\u03b1)]. The dynamic regulation of expression of multiple Klf family members suggests that, as a family, they are actively involved in regulating phenotypic responses of cells to extracellular stimuli.\n2\nMaterials and methods\n2.1\nPrimary culture of neonatal rat cardiac myocytes and preparation of polysomes\nMyocytes were dissociated from the ventricles of 2- to 4-day-old Sprague\u2011Dawley rat hearts by an adaptation of the method of Iwaki et al. [26] as previously described [27]. Cells were plated at 4\u00a0\u00d7\u00a0106 cells\/60\u00a0mm Primaria culture dish (BD Biosciences) for 18\u00a0h in 15% (v\/v) foetal calf serum, then serum was withdrawn for 24\u00a0h before experimentation. Myocytes were exposed to ET-1 (100 nM), H2O2 (0.2\u00a0mM), IL-1\u03b2 (100\u00a0ng\/ml) or TNF\u03b1 (10\u00a0ng\/ml) with or without pre-treatment (10\u00a0min) with inhibitors (10\u00a0\u03bcM U0126; 50\u00a0\u03bcM LY294002; 5\u00a0\u03bcM SB203580). Cardiac myocyte polysomes were prepared by sucrose density centrifugation (0.8\u20131.6\u00a0M sucrose gradients; 5\u00a0ml) as previously described [25]. Fractions were collected by upward displacement (fraction 1 from the top of the gradient, 0.8\u00a0M sucrose; fraction 12 from the bottom of the gradient, 1.6\u00a0M sucrose) whilst monitoring absorbance at 254\u00a0nm.\n2.2\nRNA preparation and quantitative PCR (qPCR)\nTotal RNA and polysome RNA (fractions 6\u201311 of the sucrose gradients) were extracted and cDNA synthesized using reverse transcription as previously described [22,25]. qPCR was performed using a Real\u2011Time PCR System (Model 7500, Applied Biosystems) as described [25] using primers designed to amplify mRNA sequences across an intron (Table 1). qPCR analysis of glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was performed as a control and the relative quantification protocol was used. PCR conditions were 50\u00a0\u00b0C for 2\u00a0min, 95\u00a0\u00b0C for 10\u00a0min (Jump-Start Taq polymerase activation step), followed by 40\u00a0cycles of 95\u00a0\u00b0C for 15\u00a0s and 59\u00a0\u00b0C for 60\u00a0s. Following qPCR, dissociation curve analysis was routinely performed to check for aberrant amplification products (e.g. primer\u2011dimers).\n2.3\nWestern blotting\nCardiac myocyte nuclear extracts were prepared and Western blotting performed essentially as described [28]. Extracts from 1.8\u00a0\u00d7\u00a0106 cells were analysed with separation of proteins on 10% (v\/v) polyacrylamide gels. Nitrocellulose blots were probed with Klf6 rabbit polyclonal antibodies (Santa Cruz Biotechnology Inc.; Klf6(R-173), sc-7158, 1\/500 dilution). Bands were detected and analysed by scanning densitometry as described [29].\n3\nResults\n3.1\nET-1 regulates expression of multiple klf family members in cardiac myocytes\nOur microarray studies (with Affymetrix rat genome 230 2.0 arrays) of the effects of ET-1 on cardiac myocyte gene expression over 4\u00a0h [25] identified Klfs as a family of transcription factors which appeared to be dynamically regulated at the mRNA level. Mining the data specifically for Klfs, we identified rapid and transient increases in expression of Klf2, Klf4, Klf5, Klf6, Klf9 and Klf10, with concomitant downregulation of Klf3, Klf11 and Klf15 (Table 2). There was no statistically significant change in expression of Klf13 or Klf16, although Klf13 exhibited a small (non-significant; one-way ANOVA with Tukey post-test) increase in expression over 2\u2013 4\u00a0h. Klf1 (probeset 1382033_at), Klf7 (probesets 1380363_at, 1377618_at, 1384497_at) and Klf12 (probeset 1385545_at) were not consistently called \u201cpresent\u201d with low fluorescence values and are thus expressed at minimal levels. We could not identify Klf8, Klf14 (Sp6) or Klf17 on the arrays.\nUsing qPCR, we examined more carefully the effects of 100 nM ET-1 on Klf mRNA expression. Klf2 mRNA expression was significantly increased within 15\u00a0min, with maximal expression (\u223c\u00a09-fold relative to controls) at 30\u00a0min (Fig. 1A). Klf4 and Klf6 mRNAs were also upregulated (Fig. 1, B and C), though the relative stimulation was less (\u223c\u00a05-fold relative to controls) and the response was delayed relative to Klf2 (maximal expression at 0.5\u20131\u00a0h). Klf5 mRNA was upregulated from 1\u00a0h, with maximal expression at 1.5\u00a0h (\u223c\u00a013-fold relative to controls) (Fig. 1D). For these Klfs, the increase in expression was transient, with expression of Klf2 returning to baseline within 90\u00a0min and expression of Klf4, Klf5 and Klf6 declining to basal levels within 4\u00a0h. The increases in expression of Klf9 and Klf10 were relatively small (maximal expression < 2-fold at 1.5\u20132\u00a0h) although levels remained elevated over 4\u20136\u00a0h (Fig. 1, E and F). Consistent with our microarray data, Klf3, Klf11 and Klf15 were downregulated in response to ET-1. Whereas the decrease in expression of Klf3 was transient with minimal expression at \u223c\u00a030\u00a0min (Fig. 1G), the decrease in expression of Klf11 and Klf15 was sustained over at least 6\u00a0h (Fig. 1, H and I). It is notable that the Klfs which were downregulated and those with only a small increase in expression are largely associated with transcriptional repression, whereas the Klfs with the greatest increases in expression (Klf2, Klf4, Klf5 and Klf6) are mostly associated with transcriptional activation [1,4,6]. Further studies focused on this latter group.\n3.2\nRegulation of Klf2, Klf4, Klf5 and Klf6 by ET-1\nTo determine whether Klf2, Klf4, Klf5 and Klf6 mRNAs were upregulated as IEGs (i.e. the increase in expression is regulated by pre-existing transcription factors and does not require synthesis of new proteins), we examined the effects of 20\u00a0\u03bcM cycloheximide (to inhibit protein synthesis). Cycloheximide alone increased the expression of Klf2, Klf4 and Klf6 mRNAs (Fig. 2A; probably due to activation of stress-activated signalling pathways [30,31]), but did not inhibit the increase in expression induced by ET-1. However, the increase in expression of Klf5 by ET-1 was inhibited by cycloheximide (Fig. 2B), indicating that de novo protein synthesis is required for upregulation of Klf5 mRNA. Thus, Klf2, Klf4 and Klf6 are regulated as IEGs in response to ET-1, whereas Klf5 is a second phase gene.\nET-1 potently activates the extracellular signal-regulated kinases 1\/2 (ERK1\/2) in cardiac myocytes [32]. We used 10\u00a0\u03bcM U0126 (a selective inhibitor of the ERK1\/2\/5 cascades [33\u201335], though ERK5 is not significantly activated by ET-1 [24]) to determine whether the increase in expression of Klf2, Klf4, Klf5 and Klf6 requires ERK1\/2 signalling. U0126 minimally affected basal expression of each Klf, but partially inhibited the ET-1-induced increase in expression of Klf2 (56\u00a0\u00b1\u00a04% inhibition at 30\u00a0min; mean\u00a0\u00b1\u00a0SEM, n\u00a0=\u00a05), Klf4 (43\u00a0\u00b1\u00a09% inhibition at 1\u00a0h; n\u00a0=\u00a04), Klf5 (72\u00a0\u00b1\u00a08% inhibition at 1\u00a0h; n\u00a0=\u00a04) and Klf6 (45\u00a0\u00b1\u00a08% inhibition at 1\u00a0h; n\u00a0=\u00a04) (Fig. 3A). These data suggest that ERK1\/2 signalling is required, at least in part, for the upregulation of Klf2, Klf4, Klf5 and Klf6 mRNAs by ET-1.\nSignalling through phosphoinositide 3\u2032 kinase (PI3K) may increase expression of Klf2 [36], and p38-MAPK is activated by ET-1 in cardiac myocytes [37], so we examined the effects of 50\u00a0\u03bcM LY294002 or 5\u00a0\u03bcM SB203580 (selective inhibitors of PI3K and p38-MAPK, respectively [34]). Surprisingly, LY294002 increased basal expression of Klf2 and Klf4 in cardiac myocytes and promoted further the increase in expression induced by ET-1 (Fig. 3B), indicating that PI3K signalling is not required for the upregulation induced by ET-1 and suggesting that basal PI3K signalling negatively regulates Klf2\/Klf4 expression. LY294002 did not affect Klf6 expression, but attenuated the increase in expression of Klf5 by ET-1. Since basal PI3K signalling is required for protein synthesis in cardiac myocytes [38], the effect of LY294002 on the increase in Klf5 induced by ET-1 (which requires de novo protein synthesis; Fig. 2B) may be a reflection of this. SB203580 had a minimal effect on the basal expression of Klf2, Klf4, Klf5 or Klf6, or on the increase in expression induced by ET-1 (Fig. 3B). It is unlikely that p38-MAPK signalling plays a significant role in the response.\n3.3\nTranslation-state analysis of Klf2, Klf4, Klf5 and Klf6 mRNA expression\nGiven the increasing evidence for translational regulation of specific mRNAs [39,40], we performed translation-state analysis of Klf2, Klf4, Klf5 or Klf6 mRNAs. We used qPCR to compare the relative levels of expression in the total RNA pool and in RNAs associated with cardiac myocyte polysomes (prepared by sucrose density centrifugation; Fig. 4, A and B). Klf2, Klf4 and Klf6 mRNAs were marginally enriched in cardiac myocyte polysomes in unstimulated cells and, following stimulation with ET-1 (1\u00a0h), the relative increase in the polysomes was at least as great as in the total RNA pool (Fig. 4, C\u2013E). Klf2 was increased 5.6-fold in the polysomes (4.8-fold in total RNA), Klf4 was increased 6.4-fold (5.0-fold in total RNA) and Klf6 was increased 4.6-fold (4.8-fold in total RNA). Klf5 mRNA was marginally decreased in the polysomes relative to the total pool, but the increase in expression induced by ET-1 in the polysomes (4.3-fold) was slightly higher than in the total pool (3.9-fold) (Fig. 4F). We therefore expect all these Klfs to be efficiently translated into protein. Consistent with this, Klf6 protein (detected as bands of \u223c\u00a038\u201340\u00a0kDa on Western blots) was increased in cardiac myocytes exposed to ET-1, with maximal expression at \u223c\u00a01\u00a0h (Fig. 4G). It could be argued that early genes should be efficiently translated and this may be a property of all such genes. However, Irs2 was upregulated as an IEG by ET-1 (no inhibition by cycloheximide, Fig. 2A), but the mRNA was not enriched in the polysomes and the increase in the polysomes was significantly less than that in the total RNA pool (Fig. 4H). Similarly, for interleukin 1 receptor-like 1 (Il1rl1), a second phase gene (upregulation was inhibited by cycloheximide; Fig. 2B), the increase in expression in the polysomes was significantly less than in the total RNA pool (Fig. 4I). It is therefore notable that Klf2, Klf4, Klf5 and Klf6 mRNAs are efficiently recruited to the polysomes for translation into protein.\n3.4\nRegulation of Klf2, Klf4 and Klf6 by H2O2 and pro-inflammatory cytokines\nWe considered whether other stimuli also regulate expression of Klf2, Klf4 or Klf6 mRNAs. H2O2 (0.2\u00a0mM) promotes cardiac myocyte apoptosis [41], but also stimulates ERK1\/2 (and other pathways) [42], and stimulated a transient increase in expression of Klf2 (Fig. 5A). However, the response was less than that of ET-1 (\u223c\u00a03.6-fold) and was delayed (maximal increase at 1\u00a0h) (Fig. 1A). Consistent with our previous report [23], 0.2\u00a0mM H2O2 upregulated Klf4 and Klf6 mRNAs with maximal expression (5.0-fold) from 1\u00a0h and sustained expression over at least 6\u00a0h (Fig. 5, B and C). We have observed a similar delay in induction of other IEGs by H2O2 compared with ET-1 (e.g. Fos family members, unpublished data), which may reflect the delayed time course for activation of ERK1\/2 signalling (maximal activation of ERK1\/2 by ET-1 is at \u223c\u00a05\u00a0min [43]; maximal activation by H2O2 is at 15\u201330\u00a0min [42]). Consistent with a role for ERK1\/2 signalling, U0126 inhibited the increase in expression of Klf2, Klf4 and Klf6 by H2O2 (Fig. 5D). As with ET-1, SB203580 did not affect the response to H2O2 (Fig. 5D), whereas LY294002 enhanced the response (Fig. 5E). In endothelial cells, Klf2 is downregulated by TNF\u03b1 [15]. IL-1\u03b2 or TNF\u03b1 profoundly and rapidly (from \u223c\u00a01\u00a0h) downregulated Klf2 mRNA expression, a response which was sustained over at least 6\u00a0h (Fig. 6, A and B). Neither cytokine significantly affected the expression of Klf4 or Klf6 (data not shown). The decrease in expression of Klf2 in response to IL-1\u03b2 was not affected by either SB203580 or U0126, although LY294002 (which itself increased expression of Klf2 mRNA, Fig. 3B) restored the baseline levels of expression (Fig. 6C).\n4\nDiscussion\n4.1\nRegulation of gene expression by Klf family members\nMany studies of individual Klfs relate to specific cells\/tissues in which they are highly expressed or for which the phenotype of a transgenic mouse suggests they may play some role. Thus, most studies of Klf2 focus on its regulation in lung tissue, endothelial cells or T cells, whereas many studies of Klf4 are in gut, endothelial cells or smooth muscle cells, and Klf5 is studied largely in gut or smooth muscle cells. Although there are indications that Klfs are transcriptionally regulated in these various cells (e.g. Klf2 expression is increased by shear stress in endothelial cells [14]), and there is an indication of interplay between Klf family members (e.g. Klf4 and Klf5 appear to operate in an antagonistic manner to regulate cell cycle progression [8]), few studies consider the regulation and function of the Klfs as a family. Our data demonstrate substantial and dynamic regulation of nine Klfs by ET-1 in a single cell type (Fig. 1) with at least three regulated as IEGs (Fig. 2A), suggesting that Klfs potentially play a much more significant role in the phenotypic responses of more cell types than has so far been considered.\nKlfs are defined by the high degree of homology in their C-terminal DNA-binding domains [1,6]. It is probably not surprising, therefore, that all bind to similar consensus DNA sequences or, with 17 family members, that such elements are relatively common in gene promoters. The Klfs form two principal groups of transcriptional repressors (acting through CtBPs or mSin3a) plus a group which are primarily transcriptional activators, and it could be argued that the different Klf groups all do bind to similar sequences but the effect depends on whether the Klf is an activator or repressor and, if the latter, the mode of action. An additional factor could be that different cells express different Klfs and interactions with other cell-specific transcription factors may result in differential gene expression. In cardiac myocytes, we detected expression of at least 11 Klfs (Table 2), with regulation of several family members from each of the activator\/repressor groups by ET-1 (Fig. 1), suggesting that (at least in this system) neither tissue specificity nor classification according to global function is responsible for functional differences between individual Klfs. Since there is evidence for functional specificity in other cells [44,45], minor variations in primary and secondary structure may be sufficient for individual Klfs to have differing affinities for precise sequences in various gene promoters. Expression of any individual gene may therefore reflect the balance of Klfs expressed at any particular time in relation to the precise DNA-binding sites which can be accessed by them.\n4.2\nRegulation of expression of Klf2, Klf4, Klf5 and Klf6\nOf the Klfs which were upregulated by ET-1, Klf2 mRNA expression increased very rapidly and transiently (Fig. 1A), and it was regulated as an IEG (Fig. 2A). Partial inhibition of the response by U0126 (Fig. 3A) suggests that ERK1\/2 signalling is required to some degree. ERK1\/2 signalling was also required for upregulation of Klf2 by H2O2 (Fig. 5D). In contrast to endothelial cells subjected to shear stress [36], the increase in Klf2 expression by LY294002 and enhanced response to ET-1 in the presence of LY294002 (Fig. 3B) indicated that PI3K signalling negatively regulates Klf2 expression in cardiac myocytes. ERK5 phosphorylation of the transcription factor MEF2 promotes Klf2 expression in other cells [46], but ERK5 is not significantly activated in cardiac myocytes by ET-1 [24], ERK1\/2 do not efficiently activate MEF2 [47] and inhibition of p38-MAPKs (which also phosphorylate MEF2 [47]) by SB203580 had no effect on Klf2 expression induced by ET-1 (Fig. 3B), suggesting that MEF2 is not the principal factor involved in this context. Of the transcription factors associated with the Klf2 promoter identified by Ahmed and Lingrel [48], the most probable candidate for regulating Klf2 expression by ET-1 in our study is CREB. CREB is rapidly (within 5\u201310\u00a0min) phosphorylated in cardiac myocytes exposed to ET-1, a response which is inhibited by U0126 [49].\nLike Klf2, the increases in expression of Klf4 and Klf6 (Fig. 1, B and C) were inhibited by U0126, (Figs. 3A and 5D), and both were regulated as IEGs (Fig. 2A). Our data are consistent with other systems in which Klf4 is regulated as an IEG by 15-deoxy-\u039412,14 prostaglandin J2\n[50] or platelet-derived growth factor [51], and Klf6 is regulated as an IEG by phorbol esters or serum [52,53]. However, for either Klf4 or Klf6, the potential transcription factors or mRNA stabilisation factors which promote the increase in expression are not known. The increase in expression of Klf5 induced by ET-1 was inhibited by cycloheximide (Fig. 2B), so the principal factors promoting its expression must be newly-synthesized proteins, and the response was inhibited by U0126 (Fig. 3A), implicating ERK1\/2 signalling in the response. These data are consistent with other studies in which phorbol esters or fibroblast growth factor increase Klf5 expression in smooth muscle cells in an ERK1\/2-dependent manner [54] and, in fibroblasts, the increase in expression induced by phorbol esters is inhibited by cycloheximide [55].\nStudies of mRNA expression raise the question of whether or not changes in mRNA expression equate to changes in protein expression. In cardiac myocytes treated with ET-1, translation-state analysis indicated that mRNAs for Klf2, Klf4, Klf5 and Klf6 were increased to a similar or greater extent in the polysomes than in the total RNA pool (Fig. 4). Since ET-1 also increases the global rate of protein synthesis in cardiac myocytes [56] (illustrated by the increase in A254 profile of polysome fractions 6\u201311 with concomitant decrease of monosome fractions 2\u20134; Fig. 4A), overall, we would predict that the rate of synthesis of each of the Klf proteins should be increased at least in proportion to the mRNA. Consistent with this, we detected a significant, transient increase in expression of Klf6 protein following stimulation with ET-1 (Fig. 4G). Although cellular stresses such as oxidative stress can generally inhibit translation, 0.2\u00a0mM H2O2 does not have a significant effect on global protein synthesis over 4\u00a0h, and our microarray data indicate that Klf2, Klf4, and Klf6 are increased to a similar extent in the polysomes as in total RNA (data not shown).\n4.3\nRegulating Klf-dependent gene expression in cardiac myocytes\nDownregulation of Klf15 is associated with the hypertrophic response of cardiac myocytes and may be required for hypertrophy to develop [21]. However, our data suggest that the situation regarding Klf-mediated regulation of gene expression in cardiac myocytes is more complex. In cardiac myocytes exposed to ET-1, we detected a rapid decrease in expression of mRNAs for three established repressors of gene expression (Klf3, Klf11 and Klf15; Fig. 1, G, H and I), with simultaneous rapid increases in expression of four Klfs more commonly associated with transcriptional activation (Klf2, Klf4, Klf5 and Klf6; Fig. 1, A\u2013D). In response to ET-1, therefore, there appears to be an immediate overall switch towards increasing Klf-directed gene expression. However, the increases in expression of Klf2, Klf4, Klf5 and Klf6, and decrease in expression of Klf3 were transient, with a delayed increase in expression of Klf9 and Klf10, suggesting that the balance of Klf-regulated gene expression continues to change. A similar effect was detected with H2O2, with an early increase in expression of Klf2, Klf4, Klf5 and Klf6 and simultaneous decrease in expression of Klf3, Klf11 and Klf15 (Fig. 5 and [23]). In contrast, Il-1\u03b2 or TNF\u03b1 promoted downregulation of Klf2 expression (Fig. 6) with little effect on Klf4 or Klf6. Clearly, the genes which each of the Klfs regulates in cardiac myocytes remain to be established.","keyphrases":["kr\u00fcppel-like factors","cardiac myocytes","endothelin-1","cytokines","immediate early genes","gene expression"],"prmu":["P","P","P","P","P","P"]}
{"id":"Histochem_Cell_Biol-4-1-2248605","title":"Imaging and imagination: understanding the endo-lysosomal system\n","text":"Lysosomes are specialized compartments for the degradation of endocytosed and intracellular material and essential regulators of cellular homeostasis. The importance of lysosomes is illustrated by the rapidly growing number of human disorders related to a defect in lysosomal functioning. Here, we review current insights in the mechanisms of lysosome biogenesis and protein sorting within the endo-lysosomal system. We present increasing evidence for the existence of parallel pathways for the delivery of newly synthesized lysosomal proteins directly from the trans-Golgi network (TGN) to the endo-lysosomal system. These pathways are either dependent or independent of mannose 6-phosphate receptors and likely involve multiple exits for lysosomal proteins from the TGN. In addition, we discuss the different endosomal intermediates and subdomains that are involved in sorting of endocytosed cargo. Throughout our review, we highlight some examples in the literature showing how imaging, especially electron microscopy, has made major contributions to our understanding of the endo-lysosomal system today.\nIntroduction\nLysosomes are the terminal degradative compartments of the endocytic pathway. They receive extracellular components via endocytosis and intracellular material via autophagy, as well as via the biosynthetic pathway (Eskelinen 2005; Klionsky 2007; Luzio et al. 2007; Sachse et al. 2002a). By modulating the degradation of a multitude of proteins, lysosomes are key organelles in the regulation of cellular homeostasis. Moreover, these catabolic functions are complemented by a group of cell type-specific lysosome-related organelles, which store and\/or secrete key factors involved in a diversity of functions, such as blood clotting, antigen presentation, pigmentation and alveolar surface tension regulation (dell\u2019Angelica et al. 2000a; Raposo et al. 2007; Weaver et al. 2002). Because of their many functions, defects in lysosome functioning can have devastating consequences, as is illustrated by the rapidly growing number of human disorders displaying a primary or secondary defect in the lysosomal system. To date, lysosomal aberrations have been found in the \u2018classical\u2019 lysosomal storage disorders, as well as in muscular and neurological disorders, numerous types of cancer, immune-deficiency diseases and pigmentation-bleeding disorders (Futerman and van Meer 2004; Kroemer and J\u00e4\u00e4ttel\u00e4 2005; Mohamed and Sloane 2006; Nixon 2005; Saftig et al. 2001).\nUnraveling the intrinsic complexity of the endo-lysosomal system is a major challenge for cell biologists. Here, we will review some of the current insights in the mechanisms involved in lysosome biogenesis and functioning. Imaging of the endo-lysosomal system combined with imaginative visions has been instrumental for furthering the conceptual understanding of the endo-lysosomal system. Therefore, on the special occasion of the 50th anniversary of the Journal of Histochemistry and Cell Biology, we will also highlight some examples in the literature showing how imaging, especially electron microscopy (EM), has contributed to our understanding of the endo-lysosomal system today.\nThe discovery of the lysosome\nThe famous story of the discovery of the lysosome reads as a compelling detective novel (Bainton 1981; de Duve 2005). A major question that was faced by the cell biological society of the fifties was how cells could exert proteolytic activity without being digested by autolysis. The first indications on the existence of lysosomes came from studies designed to answer an entirely different question-i.e. to clarify the action of insulin on the liver-in which acid phosphatase activity (nowadays known as a lysosomal activity) measurements were used as a control. A combination of experiments from the labs of Christian de Duve and Albert Claude led to the intriguing observation that acid phosphatase in fresh fractions, prepared by a relatively gentle protocol, showed astonishingly low activity levels. By contrast, samples that were obtained by a crude fractionation protocol or stored for some days before assaying showed elevated levels of phosphatase activity. When this same hide-and-seek strategy was found for four additional hydrolytic enzymes, all with an acidic pH optimum and the same sedimentation characteristics as acid phosphatase, de Duve concluded that these enzymes must be contained together in particles surrounded by a membrane \u2018to prevent the enzymes getting out and the substrate getting in\u2019. The term lysosome, for lytic particle or digestive body, was introduced (de Duve et al. 1955).\nNovikoff, together with de Duve, provided the first EM pictures of these liver fractions that in addition to numerous mitochondria also showed an enrichment of electron dense bodies (Fig.\u00a01). These were tentatively recognized as the \u2018pericanalicular dense bodies\u2019 previously found in intact liver cells (Novikoff et al. 1956). In these same years, Gomori had developed a protocol for acid phosphatase cytochemistry (Gomori 1952), which was subsequently optimized for EM by Novikoff, Holt, Barka and Anderson (Barka and Anderson 1962; Holt 1959; Novikoff 1961). The circle was closed when the acid phosphatase staining was applied to the ultrastructural level, thereby providing the direct evidence that acid phosphatase activity localized to the dense bodies of the liver parenchyma cells (Essner and Novikoff 1961) (Fig.\u00a02), as well as similar bodies in various other cell types.\nFig.\u00a01Reproduction of the first electron micrograph ever that indicated the existence of lysosomes (arrows), as originally published in 1956 by Novikoff and colleagues. The sample represents a liver fraction that also contains mitochondria (MT) and endoplasmic reticulum (ER). Reproduced from Novikoff et al. (1956)Fig.\u00a02Reproduction from Essner and Novikoff (1961). This picture shows for the first time at the ultrastructural level that acid phosphatase activity is localized in lysosomes (LY) of liver cells. BC bile canaliculus, ER endoplasmic reticulum, EX extraneous precipitate, MI microvilli, V vacuole\nAnother major clue was provided by Werner Straus. He found that \u2018droplets\u2019 of unknown function within the proximal tubules of the kidney contained reabsorbed (i.e. endocytosed) material as well as acid phosphatase activity and other lysosomal hydrolases (Straus 1954, 1956). This observation provided the first link between lysosomal digestion and endocytic uptake of extracellular material. Hence, the concept was born of the lysosome as a membrane-bound organelle that contains acid hydrolases involved in the digestion of substances that enter the cell via endocytosis (de Duve 1959). It explained that cells digest without autolysis by setting apart the degradative enzymes together with their endocytosed substrates in a membrane-bound compartment. The answer to the riddle was found.\nThe endocytic pathway in a nutshell\nLysosomes receive extracellular substances for degradation via endocytosis: the invagination and pinching-off of membrane-bound vesicles from the plasma membrane (Fig.\u00a03a). There are at least five highly controlled different entries into the cell, of which the \u2018classical\u2019 clathrin-mediated pathway is best-documented. However, the importance of non-clathrin-mediated pathways is becoming increasingly evident (Conner and Schmid 2003; Kirkham and Parton 2005; Mayor and Pagano 2007; Nichols 2003). Clathrin-mediated endocytosis begins with the sorting of receptors into a clathrin-coated pit at the plasma membrane, which then buds off to form a transport vesicle. These vesicles fuse with the first intracellular sorting station, the early endosome (Fig.\u00a03a). Early endosomes have a mildly acidic pH that triggers the dissociation of some ligands from their receptors. Emptied receptors enter membrane tubules that emerge from the endosomal vacuole, resulting in transport via the recycling endosome back to the plasma membrane or, alternatively, the trans-Golgi network (TGN) (Fig.\u00a03a). Receptors that remain ligand-bound are sequestered into small 50\u201380\u00a0nm diameter intraluminal vesicles (ILVs) that bud from the endosomal-limiting membrane into the endosomal lumen. Through a series of such vesicle fusion and fission events involving protein sorting, early endosomes gradually transform into late endosomes, a process called endosomal maturation. The formation of ILVs can continue up to the late endosome, which is why during endosomal maturation the number of these internal vesicles increases and late endosomes are often referred to as multivesicular bodies (MVBs, Fig.\u00a03a). Degradation of cargo starts in late endosomes\/MVBs and continues in the lysosome. Late endosomes\/MVBs develop into lysosomes by multiple fusion and fission cycles (Futter et al. 1996; Luzio et al. 2007). After degradation of the lysosomal content, transporter molecules translocate sugars, amino acids and lipids across the limiting lysosomal membranes into the cytosol where they function as building blocks in the synthesis of new products.\nFig.\u00a03Sorting and transport within the biosynthetic and endo-lysosomal pathways. a Schematic representation of the interplay between the biosynthetic pathway and endo-lysosomal system depicting the main endosomal compartments that are discussed in the text. b Detailed overview of the various sorting steps at the early endosome. At the endosomal vacuole or sorting endosome, receptor\u2013ligand complexes destined for degradation accumulate in the bilayered clathrin coat (1), upon which they are sorted into intraluminal vesicles (ILV) that form by inward budding of the vacuolar limiting membrane. Recycling cargo by default enters the membrane tubules of the tubular sorting endosome (2) from which it can recycle back to the plasma membrane via the recycling endosome, or is actively sorted towards late endosomes\/lysosomes (AP-3 pathway for lysosomal membrane proteins) or TGN (AP-1 pathway). A third exit from the vacuolar endosome is provided by the endosome-to-TGN carriers (3) that mediate SNX1-dependent recycling of MPRs and sortilin to the TGN. c From the TGN, multiple pathways arise that mediate transport to the endo-lysosomal system and plasma membrane. It is still an open question to what extent these pathways use different or overlapping molecular machinery and transport carriers. AP adaptor protein complex, ER endoplasmic reticulum, M6P mannose 6-phosphate, MPR mannose 6-phosphate receptor, SAP sphingolipid activator protein, SNX1 sorting nexin 1, TGNtrans-Golgi network\nDefining early and late endosomes\nWhen studying the endo-lysosomal system, one is forced to continuously pose the question how to discriminate between the distinct endosomal intermediates. The distinction \u2018early\u2019 and \u2018late\u2019 endosomes often has too limited discriminating power to describe the dynamic continuum of the endo-lysosomal pathway. In a recent EM study (Mari et al. 2007), a novel method to distinguish early and late endosomes was introduced by combining morphological, kinetic and molecular criteria. Early endosomes are by definition accessible to internalized transferrin (Tf) that recycles to the plasma membrane. As a result, late endosomes contain only marginal levels of Tf. With this in mind, the number of ILVs within the endosomal vacuoles was correlated with the occurrence of internalized Tf. Tf-positive early endosomal vacuoles on average contained one to eight ILVs, whereas endosomal vacuoles with nine or more ILVs were mostly devoid of Tf and therefore designated as late endosomes. A similar analysis with various other established early endosomal marker proteins corroborated and extended this definition. For example, the early endosomal antigen 1 (EEA1) appeared to be connected with only the very early maturation stages of early endosomes (one to five ILVs), whereas Rab11 appeared only on the later maturation stages of early endosomes (four ILVs or more). The early endosomal markers Rab4 and Hrs were more evenly distributed. Although the interpretation of this study is currently limited to the cell line in which the analysis was performed, i.e. the human hepatoma cell line HepG2, these observations illustrate that distinct stages of endosomes can be better monitored with increasing means of analysis.\nTGN-to-endosome transport pathways\nTo fulfill their degradative function, lysosomes must also receive lysosomal proteins. These are synthesized in the endoplasmic reticulum and Golgi complex, after which they enter the TGN (Fig.\u00a03a). From the TGN, multiple pathways to the endosomal system arise, mediating delivery of essential components to various endosomal intermediates. Thus, lysosomes receive their components via both the endocytic pathway and biosynthetic pathway.\nTwo classes of proteins together are essential for lysosomal functioning: the soluble lysosomal hydrolases and lysosomal membrane proteins. Each lysosomal hydrolase targets specific substrates for degradation and presently more than 50 different types are known. The heavily glycosylated lysosomal membrane proteins encompass a group of proteins with various functions. The major lysosomal membrane proteins (Marsh et al. 1987) are the lysosome-associated membrane proteins (LAMP)-1 and -2, the lysosomal integral membrane protein (LIMP)-2 and the tetraspanin CD63 [originally identified as platelet-activating glycoprotein (Pltgp40) and also often referred to as melanoma-associated antigen ME491, LIMP-1 or LAMP-3]. Initially, lysosomal membrane proteins were thought to mainly play a role in lysosomal stability and integrity, but currently new functions emerge involving chaperone-mediated autophagy and macroautophagy (reviewed by Eskelinen et al. 2003). Thus, the proper targeting of lysosomal proteins from the TGN to the endo-lysosomal system is an essential process in the biogenesis and maintenance of lysosomes. The best-known pathway for TGN-to-endosome transport is the delivery of soluble lysosomal hydrolases by the 300\u00a0kD cation-independent (CI) and 46\u00a0kD cation-dependent (CD) mannose 6-phosphate receptors (MPR) (Kornfeld and Mellman 1989; von Figura 1991). However, as will be outlined in the next paragraphs, increasing evidence indicates the existence of multiple additional or alternative pathways from the TGN to endosomes.\nMPR-dependent transport of soluble lysosomal hydrolases\nBoth the CI and CD-MPR are present in almost all mammalian cell types. The lumenal domain of the CI-MPR is composed of 15 repetitive units, with two mannose 6-phosphate (M6P)-binding sites, whereas the CD-MPR has a lumenal domain of 159 amino acids, resembling one such unit, and has a single M6P-binding site. The M6P-binding sites recognize M6P-moieties, which are specifically present on the precursor forms of newly synthesized lysosomal hydrolases. Both receptors predominantly function as non-covalently bound homodimers. A specific role for each MPR in the targeting of lysosomal hydrolases has not been established, but distinct lysosomal hydrolases can exhibit different affinities for CI or CD-MPR (Qian et al. 2008; Sleat and Lobel 1997). CI-MPR can endocytose extracellular lysosomal hydrolases, whereas under physiological conditions CD-MPR is not involved in endocytosis. CD-MPR shows enhanced ligand binding in the presence of divalent cations (Hoflack and Kornfeld 1985a, b). Interestingly, the CI-MPR also has several other functions; it regulates the levels of circulating insulin-like growth factor II by binding it at the plasma membrane for delivery to lysosomes. Furthermore, it facilitates the activation of the precursor of transforming growth factor-\u03b21, which is a growth inhibitor, and also binds retinoic acid, thereby mediating retinoic acid-induced apoptosis and growth inhibition, further supporting the idea that CI-MPR could be a tumor suppressor (reviewed by Ghosh et al. 2003a).\nTogether, the two MPRs mediate the targeting of the huge majority of newly synthesized lysosomal hydrolases from the TGN to the endo-lysosomal system (Fig.\u00a03c) (Hoflack and Kornfeld 1985a, b; Sahagian et al. 1981). In steady state, both CD and CI-MPR have been localized by immuno-EM to the TGN, plasma membrane, endosomes and associated vesicles (Bleekemolen et al. 1988; Geuze et al. 1984, 1985; Griffiths et al. 1988; Klumperman et al. 1993, 1998). Sorting of MPRs at the TGN was long thought to predominantly depend on binding to the heterotetrameric adaptor-protein complex (AP)-1, which also mediates the recruitment of clathrin (Fig.\u00a04). The discovery of the Golgi-localized, \u03b3-ear-containing, Arf-binding family of proteins (GGA) as clathrin adaptors changed this view (Boman et al. 2000; dell\u2019Angelica et al. 2000b; Hirst et al. 2000; Poussu et al. 2000). GGAs are monomeric proteins and three different forms are known in mammals, GGA1, GGA2 and GGA3. GGAs and AP-1 might function in parallel to generate distinct MPR-containing vesicles at the TGN, thereby allowing delivery to different endosomal compartments. Alternatively or additionally, GGAs may facilitate entry of MPR into clathrin-coated vesicles by interacting with AP-1. A major clue in favor of the latter hypothesis was provided by immuno-EM showing that GGAs and AP-1 colocalize in the same clathrin-coated TGN buds (Doray et al. 2002; Puertollano et al. 2003). Furthermore, the three GGAs were found to interact with each other and also to bind directly with their hinge domain to the \u03b3-ear domain of AP-1. Depletion of each GGA resulted in decreased levels of the other GGAs, their redistribution to the cytosol and impaired sorting of CI-MPR and the lysosomal hydrolase cathepsin D (Doray et al. 2002; Ghosh et al. 2003b). However, these findings do not rule out the possibility that different adaptor proteins can also act separately to a certain extent. Interestingly, it is not precisely known where the MPRs deliver their bound ligands. A small percentage of the MPRs travels to the cell surface, from where they are internalized by clathrin-mediated endocytosis and routed to the early endosomes. However, the majority takes a direct intracellular pathway to the endo-lysosomal system, and it is generally believed that both early and late endosomes can be recipients for TGN-derived MPR. The involvement of multiple adaptors could provide a means to establish these distinct pathways.\nFig.\u00a04Immuno-EM of a human hepatoma cell (HepG2) showing the occurrence of CIMPR (represented by 10\u00a0nm gold particles) in AP-1 (represented by 15\u00a0nm gold particles) coated TGN membranes (arrowheads). CI-MPR and AP-1 positive membranes also show the characteristic cytoplasmic dense coat indicative for the presence of clathrin. The TGN area is enclosed by distinct golgi stacks (G). Bar, 200 nm\nMPR-independent transport of soluble lysosomal hydrolases\nIn addition to the well-characterized MPR pathway, several additional transport pathways for lysosomal hydrolases exist (Fig.\u00a03c). For example, the lysosomal hydrolases lysosomal acid phosphatase and \u03b2-glucocerebrosidase do not acquire M6P residues and their transport is not depending on MPRs (Aerts et al. 1988; Waheed et al. 1988).\nLysosomal acid phosphatase is a type I transmembrane protein that follows the constitutive secretory pathway to the plasma membrane and reaches the lysosome via endocytosis. In lysosomes, the transmembrane precursor is processed into a soluble form, but both proteins are enzymatically active (Waheed et al. 1988). Lysosomal acid phosphatase contains the tyrosine-based motif YRHV in its cytosolic domain, which is required for endocytosis, but does not mediate direct TGN-to-endosome targeting (Oberm\u00fcller et al. 2002; Peters et al. 1990; Pohlmann et al. 1988).\n\u03b2-Glucocerebrosidase has no membrane spanning domain (Erickson et al. 1985). After synthesis, it is glycosylated upon which it becomes membrane-associated (Rijnboutt et al. 1991). The machinery required for proper transport of \u03b2-glucocerebrosidase was long unknown. Recently, Paul Saftig and colleagues unexpectedly identified the lysosomal membrane protein LIMP-2 as a specific receptor for \u03b2-glucocerebrosidase. LIMP-2 binds \u03b2-glucocerebrosidase in a pH-dependent manner, enabling association in the ER and guiding it all the way to the lysosome, where the complex likely dissociates because of the acidic pH (Reczek et al. 2007). Whether LIMP-2 recycles between endosomes and TGN is not known. This study for the first time implies a lysosomal membrane protein in the transport of a lysosomal hydrolase. It will be very interesting to establish whether LIMP-2 acts as a more general binding partner for lysosomal hydrolases. In addition, characterization of the molecular machinery required for transport will provide further insight into this TGN-to-endosome transport pathway.\nLysosomal hydrolases that are normally modified with a M6P residue can also traffic in an MPR-independent way to the lysosomes (Fig.\u00a03c). This became apparent by comparing different cell types from patients with I-cell disease, also known as Mucolipidosis type II. In this disease, soluble lysosomal hydrolases do not acquire M6P groups due to a deficient activity of the enzyme GlcNAc-phosphotransferase (Hasilik et al. 1981; Reitman et al. 1981). As a consequence, the lysosomal hydrolases follow the default constitutive pathway to the plasma membrane (Fig.\u00a03c) and are thus secreted. However, in cells of non-mesenchymal origin, most lysosomal hydrolases are still targeted correctly to the lysosome (Fig.\u00a05) (Little et al. 1987; Owada and Neufeld 1982; Waheed et al. 1982), suggesting the existence of alternative pathways. The same phenomenon was observed in a very recently established mouse model system for I-cell disease, achieved by GlcNAc-phosphotransferase knockout (Gelfman et al. 2007). Furthermore, in mice that are deficient of both MPRs, the intracellular activities of several lysosomal hydrolases are comparable to the control situation (Dittmer et al. 1999). These findings indicate that lysosomal hydrolases can follow a pathway to the lysosome that is not mediated by MPRs.\nFig. 5\nImmuno-EM of a B lymphoblast derived from a patient with I-cell disease showing a lysosome (L) positively labeled for the lysosomal hydrolase cathepsin D (represented by 10\u00a0nm gold particles). This picture indicates that although the MPR pathway is impaired in these cells, lysosomal enzymes can still reach lysosomes. Bar 200 nm\nThe mechanisms and pathways of MPR-independent transport are still poorly defined. Recent studies, however, have implicated a role for the multiligand receptor sortilin as an alternative receptor for a subset of lysosomal proteins, called sphingolipid activator proteins (SAPs), which are non-enzymatic cofactors required for the degradation of glycosphingolipids. Two SAPs are known; the precursor protein prosaposin, which renders saposin A, B, C and D after proteolysis, and GM2 activator protein (AP), an essential cofactor for \u03b2-hexosaminidase A. Studies from Lefrancois and colleagues have shown that sortilin interacts with both prosaposin and GM2AP, which is independent of M6P tags, whereas depletion of sortilin induced their secretion (Lefrancois et al. 2003). It remains to be established, however, whether sortilin is the exclusive receptor for SAPs, or whether MPR has an additional role in their transport. Interestingly, sortilin, together with SorLA and SorCS1\u20133, forms a protein family sharing homology to the lumenal domain of the yeast vacuolar sorting protein Vps10p, which directs carboxypeptidase Y to the vacuole (Marcusson et al. 1994). Immuno-localization studies positioned sortilin predominantly in endosomes and the TGN (Mari et al. 2007), where it colocalized with CI-MPR in AP-1 and GGA3 positive clathrin-coated transport carriers, indicating that sortilin and MPRs travel via the same carriers to the endo-lysosomal system. Currently, the functions of the SorCS-subgroup are unknown, but since SorCS3 is predominantly localized at the plasma membrane it is unlikely to be involved in TGN-to-endosome trafficking (Westergaard et al. 2005). Whether sortilin or possibly SorLA could act as alternative receptors for lysosomal hydrolases is still unclear.\nTGN-to-lysosome transport of lysosomal membrane proteins\nLysosomal membrane proteins that exit the TGN can be transported to lysosomes following either a direct or indirect pathway, involving passage over the plasma membrane. Virtually nothing is known about the carriers and molecular machinery that mediate the direct route of lysosomal membrane proteins to the lysosomes. Moreover, the contribution of this pathway remains debated (Carlsson and Fukuda 1992; Janvier and Bonifacino 2005). Important for the sorting of lysosomal membrane proteins to lysosomes are the tyrosine and dileucine-based consensus motifs within their cytosolic tails. LAMP-1, LAMP-2 and CD63 (LIMP-1) all bear a YXX\u00d8 motif, with an essential tyrosine residue, hydrophobic XX residues and the \u00d8 residue being a bulky hydrophobic amino acid (reviewed by Bonifacino and Traub 2003). The YXX\u00d8 motif is required for endocytosis at the plasma membrane, but has also been implicated in the direct targeting from TGN to lysosomes. For TGN sorting, however, additional characteristics seem to be required; a glycine residue preceding the critical tyrosine, the XX residues being acidic and the entire motif positioned six to nine residues from the transmembrane domain.\nLIMP-2, the recently identified receptor for \u03b2-glucocerebrosidase, has a dileucine-based sorting motif DXXLL, also referred to as acidic cluster-dileucine motif, which has also been implicated in both internalization and TGN-to-lysosome targeting. The YXX\u00d8 motifs are recognized by the \u03bc subunits of AP-1, AP-2, AP-3 and AP-4 and the DXXLL motif by the VHS domain of the GGAs (reviewed by Bonifacino and Traub 2003). The role of adaptor proteins in TGN sorting of lysosomal membrane proteins remains to be established. Segregation of LAMP-1 into AP-1 positive vesicles was observed in a biochemical study and also confirmed by immuno-EM, indicating exit from the TGN in clathrin-coated vesicles (H\u00f6ning et al. 1996; Hunziker and Geuze 1996). However, sorting of LAMP-1 is not strictly dependent on AP-1. In mice lacking the \u03bc1A subunit, which impairs AP-1 binding and clathrin assembly at the TGN, there was no significant increase in plasma membrane levels of LAMP-1 (Meyer et al. 2000), whereas the protein was still found in lysosomes. Depletion of clathrin in HeLa cells did result in increased levels of LAMPs at the cell surface, but not a complete redistribution of LAMPs to the plasma membrane (Janvier and Bonifacino 2005), indicating the existence of a direct TGN-to-lysosome pathway independent of clathrin (Karlsson and Carlsson 1998). Interestingly, depletion of AP-1 in HeLa cells, like in mice, again had only modest effects on LAMP transport over the surface (Janvier and Bonifacino 2005). Together, these data indicate that in addition to AP-1 and clathrin, other factors might be involved in direct TGN-to-lysosome transport of LAMPs.\nThe presence of a clathrin-independent TGN-to-endosome pathway was also indicated by studies on cells that present antigens, like B lymphocytes, macrophages and dendritic cells, in the context of major histocompatibility complex (MHC) class II molecules (Fig.\u00a03c). The newly synthesized MHC II molecules are transported from the TGN either via the plasma membrane or directly to so-called MHC II loading compartments (MIICs) that are lysosome-related organelles representing early and late endosomes in these cells (Kleijmeer et al. 1997). For proper transport of the integral membrane protein MHC II from TGN to MIICs, association with the type 2 transmembrane protein, invariant chain (Cresswell 1996), is important. Invariant chain contains [DE]XXX[LI]-type signals in its C-terminus, but also the cytoplasmic tail of class II was found to contain endosomal sorting information (Bakke and Nordeng 1999; Bonifacino and Traub 2003). The TGN-to-endosome pathway of MHC II\/invariant chain was studied in detail in B lymphoblasts by a combined biochemical and immuno-EM approach (Glickman et al. 1996). This revealed that MHC II, together with cathepsin D, in both normal and I-cell disease B lymphoblasts entered TGN exit domains that did not contain AP-1 or clathrin and were devoid of CD-MPR. Further studies are required to establish the molecular machinery of this pathway.\nInterestingly, a recent study linked the TGN exit of lysosomal membrane proteins to GGA3, the same component involved in the exit of MPRs. GGAs are monoubiquitinated at their GAT (GGA and TOM) domain, which supports the idea that ubiquitin is a universal sorting signal, acting at various intracellular sites. The lysosomal-associated protein transmembrane-5 (LAPTM5), which is specifically expressed in hematopoietic cells, associates in the TGN with both the ubiquitin ligase Nedd4 and GGA3 (Pak et al. 2006). This association is required for efficient targeting of LAPTM5 to lysosomes and indicates that ubiquitination of GGA3 is possibly accomplished by Nedd4. The finding that GGAs can be involved in TGN-to-lysosome transport of a lysosomal membrane protein might explain the mild effect of AP-1 knockdown. Clearly, further studies are required to clarify this direct TGN-to-endosome transport pathway of lysosomal membrane proteins.\nThe early endosome: from CURL to tubular sorting endosome\nThe combined data on TGN-to-endosome transport of lysosomal hydrolases and lysosomal membrane proteins suggest the existence of multiple TGN exits for direct delivery to the endosomal system (Fig.\u00a03c). There may be several reasons for such different pathways. For example, they allow custom tailored regulation of the delivery of distinct types of cargoes and provide a back-up system in case a given pathway is no longer available (such as in I-cell disease). The existence of alternative TGN-to-endosome pathways would also allow different cargoes to enter the endo-lysosomal system at distinct stages, which may coincide with their function and mode of sorting. In addition to understanding TGN exits, it is therefore equally important to define the recipient endo-lysosomal intermediates in lysosome biogenesis. With the refining of EM techniques, our understanding of the endo-lysosomal system has equally evolved. The technique that we now know as \u2018classical\u2019 EM, i.e. thin sections of plastic-embedded samples of chemically fixed cells, allowed our first acquaintance with the fascinating complexity of the cell\u2019s inner world. Immunocytochemistry added another dimension by allowing the localization of enzymatic activities to specific compartments (as illustrated for acid phosphatase). The next step was the development of immunocytochemical procedures, which greatly increased the repertoire of proteins that can be visualized in the cell.\nOne of the first published examples of immuno-EM also marked a major leap in our understanding of the endo-lysosomal system; i.e. the identification of the early endosome as sorting device. In 1983, a novel immuno-EM technique was introduced by Hans Geuze and Jan Slot, in which differentially sized gold particles were used to simultaneously localize two distinct proteins. By applying this technique to visualize a ligand-asialoglycoprotein (ASGP), which is endocytosed by liver cells for transport to lysosomes-as well as its receptor (ASGPR, which recycles after endocytosis), they for the first time visualized a protein-sorting event. The ASGPR was found in tubular membranes that lacked ASGP and were connected to vacuoles that were enriched for ASGP but depleted of ASGPR (Geuze et al. 1983) (Fig.\u00a06, left panel). The ASGPR-containing tubules were implicated in the recycling of the ASGPR to the plasma membrane, and the ASGP-containing vacuoles were defined as \u2018precursor compartments en route to the lysosome\u2019. The entire compartment was called \u2018CURL\u2019: \u2018compartment of uncoupling receptor and ligand\u2019.\nFig.\u00a06From CURL to tubular sorting endosome. Left panel reproduction of the first electron micrograph using double-immunogold labeling, showing the uncoupling of a ligand (asialoglycoprotein (ASGP), indicated by 5\u00a0nm gold particles) from its receptor (indicated by 15\u00a0nm gold particles) in early endosomes, at that time called CURL (compartment for uncoupling receptor and ligand). The ASGP receptor is found in tubules implicated in its recycling, whereas ASGP en route to lysosomes remains in the vacuole. Originally published by Geuze et al. (1983). Right panel, reproduction from Peden et al. (2004), showing by triple labeling that the adaptor protein AP-3 (15 nm gold particles), the lysosomal membrane protein LAMP-1 (10 nm gold particles) and the recycling ASGP receptor (5 nm gold particles) co-localize in the tubules emanating from endosomal vacuoles. This work gave rise to the concept of the tubular sorting endosome\nOver the years, it became clear that CURL was involved in the sorting of multiple types of proteins and the modern structural equivalent of CURL is the early endosome. Indeed, the early endosome is the major cellular site, where cargo destined for recycling is sorted from cargo en route to late endosomes and lysosomes. To indicate this, early endosomes are often subdivided into the globular \u2018vacuolar or sorting\u2019 endosome (Fig.\u00a03b), whereas the associated tubules were indicated as \u2018recycling tubules\u2019 or \u2018recycling endosomes\u2019. However, recent studies provided accumulating evidence that early endosome-associated tubules are more than just transport intermediates for recycling cargo; they are also actively involved in sorting (Fig.\u00a03b).\nWith the increasing number of antibodies against distinct types of cargoes and by optimizing immuno-EM techniques (Slot and Geuze 2007), it became possible to localize less abundant cargoes. This revealed that at steady state conditions minor amounts of LAMP-1 and LAMP-2 are found in the early endosome-associated tubules (Fig.\u00a06, right panel). The importance of this localization became apparent from studies on the pigmentation-bleeding disorder Hermansky-Pudlak-syndrome-2 (HPS-2). Cells from HPS-2 patients lack a functional AP-3 adaptor complex and show an increased transport of lysosomal membrane proteins over the plasma membrane (dell\u2019Angelica et al. 1999). A combination of biochemical and immuno-EM approaches revealed that AP-3 mediates the exit of lysosomal membrane proteins from the early endosome-associated tubules, most likely for transport to late endosomes or lysosomes (dell\u2019Angelica et al. 1998; Peden et al. 2004) (Fig.\u00a03b). Impairment of AP-3 function results in an increased cycling of lysosomal membrane proteins between early endosomes and plasma membrane (Peden et al. 2004), which explains the increased levels of lysosomal membrane proteins at the plasma membrane of HPS-2 cells. Importantly, early endosomes can be reached by lysosomal membrane proteins via the plasma membrane, but possibly also via a direct pathway from the TGN. This may explain why in the literature AP-3 has been implicated in the direct as well as indirect pathway to lysosomes (Ihrke et al. 2004; Rous et al. 2002). In melanocytes, AP-3 also mediates a pathway from early endosomes to melanosomes, which are lysosome-related organelles (Theos et al. 2005).\nImportantly, by multiple double- and triple immunogold labellings, it was shown that a particular early endosome-associated tubule could contain the recycling proteins Tf and ASGPR, the CD and CI-MPR, and LAMP-1 and LAMP-2. In addition, one single tubule could display multiple budding profiles that were positive for either the adaptor complex AP-1 or AP-3 (Peden et al. 2004). AP-1 on endosomal tubules has been implicated in the retrograde transport of the CD-MPR and of the Shiga toxin B subunit to the TGN (Meyer et al. 2000), as well as recycling of internalized Tf (Mallard et al. 1998; van Dam and Stoorvogel 2002). The presence of distinct types of recycling proteins and the association of multiple adaptor complexes indicated that the early endosome-associated tubules mediate sorting of cargo proteins to distinct destinations in the cell, i.e. plasma membrane, TGN and late endosomes or lysosomes. Therefore, these AP-3 positive compartments were introduced as tubular sorting endosomes (Figs.\u00a03b and 6, right panel) (Peden et al. 2004). A recent review of Bonifacino and Rojas extends this concept (indicated here as \u2018tubular endosomal network\u2019), by ascribing all protein machinery currently known to be involved in endosome-to-TGN transport to distinct exits of the tubular sorting endosome (Bonifacino and Rojas 2006).\nTogether, the available data launch the concept of a two-step rocket. In the first step, at the vacuolar domain of early endosomes, proteins destined for late endosomes-lysosomes are sorted from recycling proteins that accumulate in the attached tubules. Then, in the second step, taking place in the tubular sorting endosome, recycling proteins are sorted out to distinct destinations in the cell. Within this model, the recycling endosomes are one type of transport intermediate that evolve from the tubular sorting endosomes, mediating the recycling of cargo to the plasma membrane (Fig.\u00a03b).\nMultiple sorting events at the early endosomal limiting membrane\nDespite the relatively simple and attractive concept of the two-step rocket, sorting at the early endosomes displays another level of complexity; recycling carriers not only evolve from tubular sorting endosomes, but also from endosomal vacuoles. A recent study showed the formation of so-called endosome-to-TGN carriers (ETCs) from the limiting membrane of early endosomal vacuoles (Fig.\u00a03b) (Mari et al. 2007). These ETCs are so far specifically involved in the endosome-to-TGN recycling of lysosomal protein receptors, i.e. the CD- and CI-MPRs and sortilin. Importantly, ETCs constitute an entirely different population of recycling carriers than those emanating from the tubular sorting endosome. With the exception of the MPRs, ETCs are devoid of any of the cargo or machinery proteins found in the tubular sorting endosome. The associated machinery proteins of ETCs are SNX1, SNX2 (Carlton et al. 2004, 2005; Mari et al. 2007) and hVps26p (Arighi et al. 2004; Mari, personal communication). ETCs also differ from tubular sorting endosomes by morphology. By 3D-tomographic analyses, ETCs were identified as non-branched, short tubules and vesicles, not organized in a reticulum.\nApart from segregating proteins into tubular sorting endosomes and ETCs, the limiting membrane of early endosomal vacuoles is involved in a third protein sorting event; i.e. segregation of proteins into a characteristic bilayered coated area that is involved in protein sorting into ILVs (Fig.\u00a03b, see also below) (Bonifacino and Rojas 2006; Peden et al. 2004; Raiborg et al. 2002; Raposo et al. 2001; Sachse et al. 2002b). Thus, proteins present in early endosomal vacuoles may be sorted to at least three types of specialized sorting domains: tubular sorting endosomes, ETC or the bilayered coat. The formation of two types of exits from the non-coated regions of the early endosomal vacuoles predicts the existence of complementary molecular machineries that specifically recruit cargo to either of these recycling compartments (i.e. ETC or tubular sorting endosome). Future investigations are needed to further unveil the components of these machineries.\nSorting to late endosomes and lysosomes \nThe default pathway for molecules entering early endosomes is recycling back to the plasma membrane, a pathway that starts with the segregation of cargo into the tubular extensions of the early endosomes (Draye et al. 1988; Yamashiro et al. 1984). Endocytosed cargo from the plasma membrane that is not destined for recycling requires active sorting to late endosomes\/lysosomes, a process mostly mediated by inclusion into ILVs (Gruenberg and Stenmark 2004; Katzmann et al. 2002; Sachse et al. 2002b).\nProtein sorting into ILVs is critical for growth factors and their receptors, such as growth hormone (GH) (Sachse et al. 2002b) and epidermal growth factor (EGF) (Raiborg et al. 2003; Urbe et al. 2003). Sorting into ILVs segregates these ligand\u2013receptor complexes from the cytoplasm, thereby terminating signaling. ILV sorting requires ubiquitination and involves among others the cytoplasmic proteins Hrs and STAM (Bache et al. 2003b; Raiborg et al. 2001, 2002; Urbe et al. 2003). By immuno-EM, it was shown that at the limiting membrane of the early endosomal vacuole the GHR and EGFR are concentrated in flat, bilayered coated areas with a fuzzy outer layer of clathrin and a dense inner layer of unknown protein composition. These bilayered coated domains were enriched in Hrs (Sachse et al. 2002b). These findings led to the proposition of the \u2018sorting-by-retention\u2019 model for endosomal sorting (Sachse et al. 2002b). This model describes that receptor\u2013ligand complexes prior to their incorporation into ILVs are retained and concentrated in the bilayered coated areas of endosomal vacuoles, whereas recycling proteins pass through the vacuolar-limiting membrane and follow the bulk flow route towards recycling endosomes. A recent study showed that the recruitment of clathrin is responsible for concentrating Hrs in these endosomal microdomains, reinforcing an active role for clathrin in protein retention (Raiborg et al. 2006).\nDownstream of Hrs, the targeting of ubiquitinated receptors into ILVs requires the \u2018endosomal sorting complexes required for transport\u2019 (ESCRT)-1, -2 and -3 in concert with Alix\/AIP-4 and the AAA-ATPase (hVps4). hVps4 is required for disassembly and recycling of ESCRT complexes (Babst 2005; Bache et al. 2003a; Katzmann et al. 2003) and interestingly, also for the disassembly of the bilayered coat (Sachse et al. 2004). Impairment of hVps4p function gives rise to an elongated coat and decreased formation of ILVs, indicating that coat disassembly is required for the inward budding process to proceed (Sachse et al. 2004). Indeed, 3D-electron tomography of early endosomes showed that inward budding of ILVs is mostly seen at the edges of the coats (Murk et al. 2003). Notably, the sorting-by-retention model proposes a novel role for clathrin in addition to its role in the formation of transport vesicles, i.e. defining endosomal subdomains involved in protein retention.\nFuture developments\nWith the increasing number of molecular markers of endosomes and lysosomes, there is a growing awareness that distinct populations of endosomes and perhaps even lysosomes, with different functions, can coexist within one cell (B\u00f6kel et al. 2006; Lakadamyali et al. 2006; M\u00f6bius et al. 2003; White et al. 2006). For example, ILVs enriched in the lipid lysobisphosphatidic acid (LBPA) are found in distinct MVBs from those enriched in cholesterol (M\u00f6bius et al. 2003). Another example is illustrated in Fig.\u00a07 showing three seemingly identical late endosomes, of which only two are reached by endocytosed BSA-gold after 3\u00a0h of uptake. One explanation is that cargo drives the formation of specific endocytic compartments. For example, addition of EGF to cells results in the specific increase of the type of MVBs that do not contain LBPA (White et al. 2006). Formation of cargo-dependent carriers can start at the plasma membrane. The recent identification of clathrin-independent endocytosis, as well as the discovery of cargo specific adaptors that lead to clathrin-coated pit formation-either in conjunction with or independent of AP-2-illustrates that alternative endocytic pathways can arise from the plasma membrane. If and how these pathways converge at the endosomal level is presently unclear (Benmerah and Lamaze 2007). EM is an indispensable tool to search for cargo-specific endosomal compartments, since by fluorescence microscopy only the labelled compartment is visible and a nearby compartment negative for the cargo under study will remain unseen.\nFig.\u00a07Electron micrograph illustrating the heterogeneity of late endosomes (LE). HepG2 cells were allowed to internalize bovine serum albumin (BSA) conjugated to 5\u00a0nm gold particles (abundant labeling) for 3\u00a0h and processed for immuno-EM of cathepsin D (10 nm gold particles, scarce labeling). Three seemingly identical late endosomes containing multiple ILVs and partially degraded material are shown, two of which are heavily loaded with BSA-gold, whereas the upper one is devoid of the endocytic tracer. G Golgi stacks; Bar 200 nm\nAnother point of interest is that not all cargo might be recruited to ILVs by the same mechanism. Both in yeast (Bilodeau et al. 2002; Epple et al. 2003) and mammalian cells (Hislop et al. 2004), examples of cargoes are described that do not require direct ubiquitination. In case of the melanosomal protein Pmel17, which is also targeted to ILVs independently of ubiquitin and Hrs, two N-terminal lumenal subdomains seem necessary and sufficient for efficient transport (Theos et al. 2006). Possibly, these non-ubiquitinated cargoes associate with others for their association with the ESCRT machinery, but perhaps they use an alternative mechanism. Interesting in this respect is that LBPA can induce ILV formation in liposomes, in the absence of protein, suggesting that lipid-driven and ESCRT-dependent pathways may coexist (Matsuo et al. 2004). Also the lysosomal targeting of LAMP-1 and LAMP-2 remains an interesting open question, since these proteins are predominantly located on the limiting membranes of late endosomes and lysosomes, and incorporation into ILVs seems therefore not involved. By a more detailed characterization of the distinct endosomal maturation stages (Mari et al. 2007), it will be feasible to obtain a more accurate definition of the stage in which a specific cargo enters or exits the endo-lysosomal system. One of the questions that is currently addressed in our lab is whether lysosomal membrane proteins can travel directly to late endosomes or lysosomes, thereby bypassing the need of ESCRT sorting machinery.\nFinally, with the development of live cell imaging methods and correlative live cell imaging EM, a unique tool is provided to integrate dynamic parameters with ultrastructural protein localization studies, which opens a whole new field of analysis.","keyphrases":["imaging","lysosomes","protein sorting","trans-golgi network","mannose 6-phosphate receptor","endosomes","electron microscopy","lysosomal membrane proteins"],"prmu":["P","P","P","P","P","P","P","P"]}
{"id":"Pediatr_Radiol-4-1-2367394","title":"Imaging findings in noncraniofacial childhood rhabdomyosarcoma\n","text":"Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood. This paper is focuses on imaging for diagnosis, staging, and follow-up of noncraniofacial RMS.\nIntroduction\nRhabdomyosarcoma (RMS) is the most common paediatric soft-tissue sarcoma and constitutes 3\u20135% of all malignancies in childhood [1]. In children, the age-standardized annual incidence rate for RMS is between 4 and 7 per million [2]. This review focuses on the imaging of all RMS occurring outside the head and neck region, which comprise 40% of RMS; around 15% are genitourinary (GU) nonbladder prostate tumours (GU-NBP, i.e. paratesticular, vaginal and uterine tumours), 10% are bladder prostate tumours (BP), 15% occur in the limbs, and 20% occur in other sites (i.e. thoracic or abdominal tumours).\nClinical symptoms vary widely, based on the location of the primary tumour, but in general presenting symptoms often are indolent, with nonspecific or minimal symptoms, at the start mimicking innocent general paediatric diseases. Often the duration or progression of symptoms alerts the physician to the presence of a malignant tumour. Site-specific symptoms in GU-NBP tumours are a paratesticular mass in boys that may be painful or not, while girls may present with a grape-like (botryoid) vaginal extrusion of mucosanguineous tissue or micturition problems. BP tumours may present with urinary retention, haematuria, constipation or an abdominal mass. Limb tumours often present with a painless swelling and\/or with enlarged regional lymph nodes, while presentation of tumours in other locations may vary from biliary obstruction in tumours of the biliary tract, to painless masses, all depending on their primary site.\nPatients with metastatic disease present with more general symptoms of fatigue, weight loss, and low blood counts. As soon as a RMS is suspected an extensive work-up must be performed to get a histological diagnosis and correct staging of the tumour. Imaging of the primary site with US, MRI or CT is required in all patients at the start of the diagnostic work-up [3]. Once the diagnosis has been confirmed histologically, the most frequently involved metastatic sites will be investigated; CT of the lungs, imaging of regional lymph nodes, and a technetium bone scan are recommended for every patient. Furthermore, bone marrow aspirates and trephines should be performed in all patients, while examination of the cerebral spinal fluid is only required in patients with paraspinal and parameningeal locations. As soon as the work-up has been completed, the patient can be stratified to receive treatment according to the child\u2019s risk group, based on the six significant prognostic factors for localized RMS that came out of a retrospective European analysis (European Paediatric Soft-tissue Sarcoma Study Group, EpSSG); histology, postsurgical status according to the Intergroup Rhabdomyosarcoma Study Group (IRSG), tumour site, node involvement, tumour size, and patient age (Table\u00a01).\nTable\u00a01EpSSG prognostic factors [28]\u00a0FavourableUnfavourableHistologyEmbryonalAlveolarIRSG statusHigher grades more unfavourableTumour siteHead and neck non-parameningealAll other sitesOrbitalGenitourinary \u2013 nonbladder\/prostateNode involvementN0N1Tumour size (cm)\u22645>5Age (years)<10\u226510\nPathology\nRMS is a fast-growing, primitive, high-grade, malignant mesenchymal tumour. Depending on their degree of differentiation, the tumour cells manifest features that more or less can be found in the cells of skeletal muscle. These features, essential for the diagnosis, are the presence of myofibrils and cross striations (on light and electron microscopy) and\/or positive immunohistochemical staining for markers of muscle differentiation such as desmin and myoD1. For detailed descriptions of the histopathological aspects of RMS, reference should be made to Weiss and Goldblum [4] and Fletcher et al. [5].\nBased on morphology, RMS is traditionally subdivided into embryonal, alveolar and pleomorphic. Pleomorphic RMS, in contrast to embryonal and alveolar RMS, almost exclusively occurs in adults (median age sixth decade), and is therefore not discussed further. Embryonal RMS is the most common type (60\u201370% of all RMS). The cells show a close resemblance to various stages in the embryogenesis of normal skeletal muscle (Fig.\u00a01). Subtypes are botryoid RMS and spindle-cell RMS (Fig.\u00a02). When arising in the submucosa, embryonal RMS may present as a fast-growing exophytic, polypoid mass. This macroscopic variant is called botryoid RMS (grape-like) and, due to its growth pattern (primary exophytic and not invasive), has a better prognosis.\nFig.\u00a01Embryonal RMS: small cells and primitive spindle-shaped cells resembling the first stages of developing normal skeletal muscle (H&E, original magnification 10\u00d720)Fig.\u00a02Embryonal RMS, spindle-cell type: closely packed spindle cells arranged in perpendicular crossing fascicles giving a leiomyosarcoma-like appearance (H&E, original magnification 10\u00d720)\nSpindle-cell RMS comprising about 4% of all RMS, seems to behave less aggressively and is most often encountered in a paratesticular location (about 30% of all paratesticular RMS) and the head and neck [6, 7]. Histologically spindle cell RMS is characterized by parallel orientation of spindle cells with an eosinophilic, fibrillary cytoplasm and an elongated, hyperchromatic nucleus. The cells are arranged in broad fascicles (fibro-\/leiomyosarcoma-like) or in short, interdigitating fascicles (storiform) and whirls with, in contrast to the broad fasciculated variant (leiomyomatous type), an abundant, partly hyalinized collagenous matrix.\nAlveolar RMS is composed of ill-defined, dense aggregates of poorly differentiated round or oval tumour cells that frequently show loss of cohesion (Fig.\u00a03). This loss of cohesion and the presence of thin fibrous septa result in an alveolar pattern. In cases where the tumour cells do not show loss of cohesion, the term \u2018solid alveolar RMS\u2019 is used. Alveolar RMS represents about 20% of all RMS and has two specific translocations with specific fusion transcripts that can be detected by RT-PCR. The majority (about 55%) show a t(2;13)(q35;q14) translocation with the corresponding fusion transcript PAX3-FKHR [8]. In about 22% a t(1;13)(p36;q14) translocation is found with fusion transcript PAX7-FKHR. In rare cases, RMS with a more or less alveolar pattern lacks these translocations.\nFig.\u00a03Alveolar RMS: cluster of primitive cells with loss of cellular cohesion and bordered by dense fibrous septa, resulting in an alveolar pattern (H&E, original magnification 10\u00d720)\nBoth embryonal and alveolar RMS may show rhabdoid tumour-like features and anaplasia (focal or diffuse) [9, 10]. More recently, under the heading sclerosing RMS, a variant with hyalin sclerosis has been described (Fig.\u00a04) [11]. It is unclear whether this is a distinct subtype.\nFig.\u00a04Sclerosing RMS: small cells, primitive spindle-shaped cells and eosinophilic rhabdomyoblasts in a background of hyaline sclerosis (H&E, original magnification 10\u00d720)\nIn 1995, the IRSG and the International Society of Paediatric Oncology (SIOP) suggested a prognostically more significant classification (Table\u00a02) [12].\nTable\u00a02RMS: IRSG and SIOP classification, extended with new variants [12]GroupPrognosisSubtypeIBetter prognosisEmbryonal RMS, botryoid typeEmbryonal RMS, spindle-cell typeIIIntermediate prognosisEmbryonal RMS (remaining)IIIWorse prognosisAlveolar RMSIVUnclear prognosisRMS with rhabdoid featuresEmbryonal RMS with diffuse anaplasiaSclerosing RMS\nImaging findings\nRadiography\nAs RMS is a soft-tissue tumour, conventional radiology plays an insignificant role in its diagnosis. Localized bony erosion adjacent to the primary site is a recognized complication; this area may be hot on a 99mTc-MDP bone scan in the absence of metastatic disease in the skeleton. In contrast to the initial diagnostic work-up, where for the assessment of pulmonary metastases CT is mandatory, AP and lateral chest radiographs are used in the follow-up period.\nUltrasonography\nUS is often the first imaging modality used in children with soft-tissue masses because it is readily available, has high resolution, and can easily assess extent and vascularity of a mass. One should not forget that most soft-tissue lesions are benign, can readily be diagnosed with US, and do not need further diagnostic work-up or even treatment.\nOn US, RMS in general shows as a well-defined, slightly hypoechoic inhomogeneous mass that can show significantly increased flow (Fig.\u00a05). In the particular case of paratesticular RMS, US is the imaging modality of choice, although CT of the abdomen is also necessary to evaluate for retroperitoneal lymphadenopathy. In all other RMS locations additional imaging using CT or MRI is mandatory.\nFig.\u00a05A 6-year-old boy with a mass in the left scrotum. US image shows an ill-defined heterogeneous mass surrounding the testis (open arrow). The mass shows increased flow (solid arrow). Histopathology: embryonal RMS\nUS is also of use in image-guided biopsies. Recently Sebire and Roebuck [13] systematically reviewed the pathological diagnosis of paediatric tumours from image-guided needle-core biopsies. They concluded that image-guided biopsy material was sufficient to come to a diagnosis in 94% (95%; CI 92\u201396%) of patients. Complications needing treatment, mostly haemorrhage requiring transfusion, were reported in only 1% of patients. For image-guided biopsies the material obtained should be transported fresh to the pathology department. Fixation should not be performed as this precludes further cytogenetic studies.\nCT\nIn order to assess pulmonary metastases from RMS, CT of the chest is a mandatory examination. However, assessment of pulmonary involvement can sometimes be difficult. Although criteria such as number and size of lesions, morphology (noncalcified, round and well-defined) and location (inferior lobes, subpleural spaces, branching vessels) have been applied in adult patients, none has shown 100% specificity. According to the EpSSG guidelines for evaluating chest CT the following criteria with respect to the diagnosis of pulmonary metastases should be applied: one pulmonary or one pleural nodule over 10\u00a0mm in diameter, two or more well-defined nodules of 5\u201310\u00a0mm or five or more lesions >5\u00a0mm [3]. This comes with the assumption that there is no other medical explanation for these lesions. When there is a high suspicion that a small lesion is metastatic, and appears to be the only site of metastatic disease, biopsy may be performed. In the EpSSG protocol, lung biopsy is not recommended.\nWith the introduction of multidetector CT (MDCT) the advantage of MRI of being a multiplanar imaging modality has been overtaken, and by virtue of its underlying physics, CT is superior to MRI in detecting osseous changes. The drawback of CT in children is the use of ionizing radiation. Since the seminal papers of Paterson et al. [14] and Brenner et al. [15], we should be aware of the theoretical risk of CT-induced cancer fatalities and take this into consideration especially in the work-up of children with cancer, as they have already proven their tendency to present with a malignancy early in life [14, 15].\nMRI\nWith its superior ability to depict soft-tissue changes, MRI is the primary imaging modality in RMS [16]. Although imaging protocols should be tailored for each individual patient, they should at least consist of axial T1-W and T2-W images (for anatomic detail and assessment of neurovascular structures), T1-W images perpendicular to the axial plane, and imaging after gadolinium administration. It is important that at least two series should be identical, one before and one after contrast agent administration, to be able to discern enhancement. Contrast-enhanced series are mandatory and ideally be performed with fat saturation.\nThe imaging characteristics of RMS are relatively nonspecific. Like most soft-tissue tumours they have intermediate signal intensity on T1-W images (Fig.\u00a06). On T2-W images they tend to be of intermediate-to-high signal intensity. If the tumour contains a high number of septa it may have a lobular shape. RMS in general show strong enhancement on postcontrast imaging (Fig.\u00a06). In very rare instances the tumour may show a predominantly cystic appearance (Fig.\u00a07). Dynamic series are useful in order to assess tumour vascularity, and to differentiate between postchemotherapy\/surgery residual disease and fibrosis.\nFig.\u00a06A 13-year-old girl who noticed a small lump near the anus. a T1-W MR image shows a well-defined pararectal lesion (arrow). b After intravenous gadolinium administration the lesion shows homogeneous enhancement (arrow) Histopathology: alveolar RMSFig.\u00a07An 8-year-old girl with haemolysis, fever and a mass underneath the scapula. a Coronal STIR image shows a lesion with mixed signal intensity (open arrow) and multiple enlarged lymph nodes in the neck (solid arrow). b Axial T2-W image shows multiple cystic lesions with fluid-fluid levels (open arrow). Histopathology: stage IV embryonal RMS\nFor the surgeon, in order to plan surgery, it is of importance to describe the compartment in which the RMS is located [17]. Vascular involvement is considered to be absent if there is a normal tissue plane visible between the tumour and the vessel, or if the tumour has a less then a 180\u00b0 circumferential relationship. If the tumour surrounds the vessel for more than 180\u00b0, it is considered to be encased.\nTwo studies have addressed the use of whole-body MRI (WB-MRI) in paediatric oncology [18, 19]. In the first study with various malignant tumours WB-MRI had a superior positive predictive value for skeletal metastases compared to bone scintigraphy (94 vs. 76%, respectively), and also a significantly higher sensitivity (99 vs. 26%, respectively) [18]. In the second study, WB-MRI had a higher sensitivity (82%) than skeletal scintigraphy (71%) for the detection of bone marrow metastases, but a lower sensitivity than FDG-PET (90%) [19]. The authors of both studies concluded that WB-MRI can replace bone scintigraphy. One advantage of this approach would be the implementation of a one-stop-shop approach to childhood RMS. A drawback of MRI in young children is, however, the need for general anaesthesia.\nBone scintigraphy\nIn the current EpSSG protocols, bone scintigraphy is mandatory as part of the work-up in patients with RMS. The finding of an isolated hot spot on the bone scan should be evaluated with conventional radiography or MRI.\nCogswell et al. [20] reported a retrospective series of 40, primarily adult, patients with RMS and found bone metastases in 18%. Bone scintigraphy in their study had a sensitivity of 70% and specificity 95% in the detection of metastatic disease. In contrast to this, in a Dutch study of 109 patients with soft-tissue sarcoma, bone metastases were found in only 8 patients (7%) [21]. However, of these eight patients, six reported bone pain and all had other sites of metastatic disease. The authors conclude that the yield of routine bone scintigraphy is low and that it should be reserved for symptomatic or high-risk patients only.\nPositron emission tomography-CT\nIn PET-CT studies fluorine-18 fluorodeoxyglucose (18F-FDG), a radiolabelled glucose analogue, is used [22]. As 18F-FDG is a glucose analogue, it shows uptake in metabolically active cells, which most malignant tumour cells are. The combination of PET with CT, without moving the relative position of the patient, yields a higher diagnostic accuracy than PET alone (Fig.\u00a08). In general, the CT scan will be low-dose CT scan only meant to identify anatomical structures. However, as the CT scanners in modern PET-CT systems are of high diagnostic quality, it is also possible to combine a diagnostic CT scan, e.g. for the depiction of pulmonary metastases, with a PET scan.\nFig.\u00a08A 19-year-old boy with a history of treated metastatic RMS presented with low back pain. The PET-CT image shows intense 18F-FDG uptake in the spinal canal (open arrow). Physiological excretion of the radiopharmaceutical via the kidneys is visible (solid arrows). Histopathology: embryonal RMS\nThe literature on the use of PET-CT in children with RMS is limited to several case reports or small studies [23, 24]. Although in some cases PET-CT has been shown to be of benefit in individual patients, larger prospective studies are needed.\nStaging and follow-up\nStaging of RMS is of importance for the individual patient as it gives an indication of prognosis, and thus treatment stratification. From a broader perspective staging makes compiling data on larger patient groups for research purposes possible, enabling evaluation of the outcome of different treatment regimens.\nThe main staging system is the postsurgical staging system developed by the IRSG. This is currently used by study groups both in the USA and now in Europe also (Table\u00a03). The IRSG was formed in 1972 and consisted of surgeons, pathologists, oncologists, and radiation oncologists. The absence of radiologists is striking, and paediatric radiologists are still infrequently involved in development of paediatric oncology study protocols, although in the EpSSG RMS 2005, paediatric radiologists were involved in the development of the protocol. For staging regional nodes it is important to be familiar with the regional node stations. Lymph node involvement has a negative impact on prognosis, as has been shown in the SIOP Malignant Mesenchymal Tumor 89 trial [25]. Overall 5-year survival was 60% for N1 patients versus 73% in N0 patients (P\u2009=\u20090.03). Distant lymph node involvement upgrades a patient to stage IV disease (Fig.\u00a07), having an adverse impact on prognosis: overall 5-year survival becomes 24% [26].\nTable\u00a03IRSG classificationStageCharacteristicsILocalized disease completely resected (regional nodes not involved)A: Tumour confined to muscle or organ of originB: Tumour infiltrating outside organ of (muscle of) originIILocalized or regional disease with total resection of gross tumourA: Primary tumour grossly resected, with microscopic residual disease (negative findings in local nodes)B: Primary tumour and positive nodes completely resectedC: Primary tumour and positive nodes resected, with evidence of microscopic residual diseaseIIIIncomplete resection of tumour or biopsy, with gross residual diseaseIVDistant metastatic disease present at diagnosis\nTable\u00a04 lists the regional node stations by primary tumour site. Oval-shape lymph nodes and a short axis <1\u00a0cm are considered to be normal [3]. If the node shows peripheral enhancement or is round with a short axis of 1.5\u20132\u00a0cm then the node should be considered probably invaded by tumour. Besides surgical resection or needle-core biopsies, lymph node involvement can also be assessed using fine-needle aspiration (FNA). Klijanienko et al. [27] reported a review of the use of FNA in 180 tumours; 176 (97.8%) were either diagnosed accurately or as round-cell sarcoma.\nTable\u00a04Regional node stations by primary tumour site. Disease with involvement of other lymph nodes than those specified in the table should be classified as stage IVAnatomical siteNode stationExtremityLower extremityInguinal, femoral, popliteal nodes (rare)Upper extremityAxillary, brachial, epitrochlear, and infraclavicular nodesGenitourinaryBladder, prostate, cervix, uterus, paratesticularPelvic, retroperitoneal nodes at renal artery level or belowVaginaRetroperitoneal, pelvic nodes at or below common iliac inguinal nodes\u00a0VulvaInguinal nodesThoracicIntrathoracicInternal mammary, mediastinal nodesRetroperitoneum\/pelvisPelvic, retroperitoneal nodesTrunkAbdominal wallInguinal, femoral nodesChest wallAxillary, internal mammary, and infraclavicular nodesOtherBiliaryLiver hilar nodes\u00a0Perianal\/perinealInguinal, pelvic nodes (may be bilateral)\nOnly in patients with intraspinal or suspected meningeal extension (on imaging or clinical assessment) does the EpSSG RMS protocol state that craniospinal MR should also be performed. In the current EpSSG RMS 2005 protocol, risk stratification is based on six criteria that have emerged from the analyses of previous European studies: histology (embryonal vs. alveolar), postsurgical stage (IRSG), tumour site, node stage, tumour size and patient age (Table\u00a01) [28].\nDuring follow-up, tumour size is an important parameter in assessing tumour response. In the current EpSSG protocol complete remission is defined as disappearance of tumour both clinically and on imaging. Minor response is defined as >33% reduction in volume after three courses of chemotherapy; if not reached the patient is eligible for second-line chemotherapy. In recording tumour response the EpSSG uses volumetric evaluation; additionally the presence or absence of a posttherapeutic residue should be mentioned in the radiology report [28].\nIn studies in adults the use of Response Evaluation Criteria In Solid Tumours (RECIST) has been advocated by the European Organization for Research and Treatment of Cancer (EORTC), National Cancer Institute of Canada Clinical Trials Group and the National Cancer Institute (NCI) of the United States [29]. With respect to implementation in children, RECIST have been a matter of debate [30]. Recently RECIST were retrospectively applied to ten consecutive children with cancer [31]. The authors concluded that tumour size was underestimated and that in disseminated disease many lesions were either calcified or too small to measure and, therefore, that RECIST are not readily applicable in paediatric oncology. Currently the EpSSG has incorporated RECIST to be used alongside the volumetric measurements in their latest protocol in order to prospectively assess the validity of RECIST in a large patient population with a single tumour type.\nTumour relapse in patients most commonly presents with locoregional disease (51%) compared to distant relapse (41%) [32] (Fig.\u00a09). In a retrospective case-based study the use of PET-CT was advocated; this, however, needs to be evaluated in larger prospective studies [33].\nFig.\u00a09Two years after initial diagnosis the patient shown in Fig.\u00a06 presented at the outpatient clinic complaining of back pain. a Coronal STIR image of the pelvis shows discrete increased signal intensity in the left ischium (open arrow). b Subsequently acquired PET-CT image confirms the presence of recurrent disease in the same location (open arrow). Note excretion of tracer into the urinary bladder (solid arrow). c PET-CT image also shows a second lesion in the thoracic spine (open arrow). Additional rib and pleural metastases were also visible (not visible on this image)\nTumour locations\nGenitourinary\nApproximately 25% of all RMS are GU RMS [34, 35]. As mentioned above, GU RMS can simply be subdivided into two subgroups based on different prognosis and subsequent treatment strategy, GU bladder\/prostate (GU-BP) being an unfavourable location (Fig.\u00a010). Tumours at other GU non-bladder\/prostate (GU-NBP) sites, such as a paratesticular location (testes, epididymis and spermatic cord; Fig.\u00a05), vagina or uterus (Fig.\u00a011), have a favourable prognosis, and thus require less-intensive treatment [36\u201348]. There is a caveat to MRI of the bladder in RMS: after intravenous contrast medium administration, layering of contrast medium can occur making it difficult to appreciate bladder wall enhancement [49]. T2-W sequences can be particularly useful in this setting to assess bladder wall thickening. Additional cystoscopy is often warranted [50]. At the end of treatment, some residual soft-tissue thickening may persist, and on MRI it is impossible to decide whether this is residual scarring or tumour; in these cases endoscopic biopsy is mandatory.\nFig.\u00a010A 3-year-old boy with RMS of the prostate. The sagittal T1-W contrast-enhanced MR image shows the lesion invading the bladder wall. A transurethral catheter has been inserted (open arrow). Histopathology: botryoid RMSFig.\u00a011A 2-year-old girl presented with a mass in the vagina. a Axial T1-W contrast-enhanced image shows the mass with heterogeneous enhancement. The tumour has both solid (asterisk) and fluid (open arrow) compartments. b Sagittal T2-W MR image shows the mass with mixed signal intensity. The bladder is displaced anteriorly and the uterus cannot be visualized. Histopathology: embryonal RMS\nExtremities\nRMS of the extremities (Figs.\u00a012 and 13) is almost always of alveolar histology, tends to occur in older children and young adults, is often present with positive regional lymph nodes, and has a propensity to metastasize to unusual sites; these negative prognostic factors contribute to the relatively poor prognosis of RMS in this location [51, 52]. In approximately 12% of patients, nodal involvement is seen on imaging; however, when nodal dissection is performed the rate of nodal involvement increases to almost 50% [53]. This discrepancy between imaging findings and nodal dissection might be reduced by using high-quality state-of-the-art US. In current treatment protocols systematic biopsy of regional nodes is advocated, even if the nodes are not palpable or enlarged on imaging; sentinel node procedures are recommended whenever feasible, although the value of upgrading a patient to a higher risk group based merely on a positive sentinel node has not been studied yet. As in all tumours of the extremities, preoperative imaging plays a vital role in the depiction of the relationship between neurovascular bundles and the tumour.\nFig.\u00a012A 4-year-old girl presenting with a mass in the left lower leg. a Axial T1-W contrast-enhanced MR image shows an ill-defined mass circumferential to the fibula. Note the cortical thinning (open arrow) of the fibula. b Sagittal PD-weighted image shows diffuse bone marrow metastases. Histopathology: embryonal RMSFig.\u00a013A 2-month-old boy with a mass in the third ray of the left foot. T1-W MR image shows a discrete lesion (open arrow) of intermediate signal intensity. Histopathology: embryonal RMS\nOther\nChest wall\nChest wall RMS (Fig.\u00a014) is a relatively rare finding with a reported incidence of 3.7% in the IRSG II and IRSG III studies [54]. Most of the reports of chest wall involvement are either case reports or small series [55\u201357]. In a retrospective analysis of 15 patients, Saenz et al. [57] found a 5-year survival of 67%.\nFig.\u00a014A 4-year-old boy presenting with a mass on the right chest wall. a US image shows a heterogeneous mass in the pectoralis major muscle (the pectoralis minor is not involved; asterisk). b T1-W MR image of the chest shows a mass of intermediate signal intensity. c After intravenous contrast medium administration the lesion shows homogeneous enhancement. Histopathology: alveolar RMS\nPulmonary\nThere is an ongoing debate and controversy whether congenital cystic anomalies predispose children to intralesional development of RMS [58\u201362]. It has been reported that pleuropulmonary blastoma (PPB) has been mistaken for or classified as RMS arising in congenital cystic adenomatoid malformation (CCAM) on a number of occasions [63]. Despite the fact that the exact incidence in CCAM is unknown, it has prompted paediatric surgeons to resect even small pulmonary cystic lesions (Fig.\u00a015) [64, 65].\nFig.\u00a015A 3-year-old boy with dyspnoea. Chest CT image shows displacement of the trachea (open arrow) and oesophagus (solid arrow) to the right due to a large mass (asterisk) with accompanying pleural effusion. Histopathology: embryonal RMS\nBiliary tree\nRMS is the most common tumour of the biliary tree in childhood, although it only accounts for approximately 0.04% of all childhood tumours [66]. The initial diagnosis will, in most patients, be made on US, which may show a solid or cystic mass situated in the liver hilum, and intrahepatic bile duct dilatation (Fig.\u00a016). MRI is mandatory for presurgical evaluation, where magnetic resonance cholangiopancreatography (MRCP) can depict the biliary tree (Fig.\u00a016). In many patients, however, endoscopic retrograde cholangiopancreatography (ERCP) will have to be performed in order to depict intraductal irregularities (Fig.\u00a016). Biliary tree RMS is a tumour that does not necessarily need to be fully resected in order to achieve long-term survival, as long as adequate radiotherapy is added [67]. Intraperitoneal metastases, which can also be found on follow-up, should also be born in mind.\nFig.\u00a016An 8-year-old boy presented with abdominal pain and jaundice. a US image shows a central process in the liver hilum (open arrow) and dilatation of the intrahepatic bile ducts (solid arrow). b T2-W MR image shows a circumscribed lesion with increased signal intensity (open arrow). c MRCP image shows intrahepatic bile duct dilatation. Note that the right and left duct systems do not communicate (open arrow). d ERCP image (ERCP performed in order to insert a stent in the common bile duct). Histopathology: embryonal RMS\nOther locations\nIn extremely rare instances RMS can be found in other organs such as the heart, the diaphragm (Fig.\u00a017), the omentum, the urachus and the digestive tract [68\u201376].\nFig.\u00a017A 4-year-old boy was shown to have a right-sided pleural effusion on a chest radiograph. Balanced FFE sagittal MR image shows a mass (open arrow) arising from the diaphragm (courtesy of S.G.F. Robben, Academic Hospital Maastricht, The Netherlands)\nCongenital\nRMS has been reported to occur as a congenital tumour (Fig.\u00a018) [77\u201381]. In congenital alveolar RMS the prognosis is reported to be extremely poor, despite otherwise adequate treatment [79]. Orbach et al. [82] reported the SIOP data on soft-tissue sarcoma in the first year of life. In their study population of 16 newborns, with a follow-up of 1.8\u201310.0\u00a0years, 3 out of 5 newborns with RMS survived. It has been noted that in congenital RMS the disease may be metastatic at the time of birth, with metastases described in a number of organs and in the placenta [81].\nFig.\u00a018A 4-day-old girl born with a lump on the left foot. Antenatal ultrasonography at 20\u00a0weeks showed no abnormalities. a T1-W MR image shows a large inhomogeneous mass arising from the left foot. b Abdominal US image shows popliteal and inguinal nodal invasion, and hepatic and pancreatic metastases (open arrow). Due to the poor prognosis, no therapy was given, and the child died several weeks later. Histopathology: poorly differentiated soft-tissue sarcoma without distinct translocations\nAdult patients\nEvery once in a while paediatric radiologists and paediatric oncologists receive a request for help in the management of an adult patient (Fig.\u00a019). RMS, although seen as a soft-tissue tumour of childhood, can also occur later in life [83\u201386]. Compared to childhood RMS, adult RMS has a poor outcome. In a large retrospective study of 171 patients 5-year overall survival was only 40% [85]. However, the patients in this series treated according to the guidelines for treatment of childhood RMS showed survival figures comparable to those seen in children. This suggests that treatment of adult RMS should be based on paediatric protocols tailored to adults, to increase survival in this age group. In the Academic Medical Centre Amsterdam we have a working group on childhood tumours in (often young) adults that specifically deals with this challenging population. This working group consists of medical oncologists, paediatric oncologists, radiation oncologists, (orthopaedic) surgeons and a paediatric radiologist. Imaging features will in general not be of help, as the pretest likelihood of RMS in an adolescent or adult is extremely low.\nFig.\u00a019A 45-year-old man with a mass in the thigh. T1-W contrast-enhanced MR image shows a heterogeneous circumscribed mass in the vastus lateralis muscle of the right leg. Histopathology: alveolar RMS\nDifferential diagnosis\nGiven the wide variety of locations in which RMS can be found it is difficult to give a concise list of differential diagnoses. The site of the primary lesion determines the differential diagnosis. Keeping location out of the equation there are, however, certain tumours, such as haemangiomas\/vascular malformations (Fig.\u00a020), adult-type soft-tissue sarcomas, peripheral neuroectodermal tumours (PNET), infantile fibrosarcoma, aggressive fibromatosis, desmoplastic small round-cell tumours and rhabdoid tumours, and other more even rarer soft-tissue tumours such as nonosseous Ewing sarcoma (Fig.\u00a021), that should be kept in mind when performing US or reading CT or MRI studies of soft-tissue tumours in childhood.\nFig.\u00a020A 1-year-old girl with a mass on the left buttock. a Duplex US image shows a highly vascularized, well-delineated heterogeneous mass that was initially thought be a haemangioma. b Coronal STIR image shows a circumscribed solid lesion that invades the pelvis via the greater sciatic foramen (open arrow). c After initial resection, with incomplete margins, tumour recurrence was seen. MR image 2.6\u00a0years after initial diagnosis shows progression of disease extending to the abdominal wall (open arrow). Histopathology: alveolar RMSFig.\u00a021A 22-month-old boy with a mass in the right groin. MRI shows a heterogeneous lesion adjacent to the gracilis muscle (open arrow). Histopathology: extraosseous Ewing sarcoma\nTreatment and prognosis\nTreatment of RMS requires a multidisciplinary approach, where chemotherapy, surgery and radiotherapy (RT) each has its own specific role.\nChemotherapy\nBefore the introduction of chemotherapy only 25% of patients with RMS survived, despite adequate local therapy. This indirectly indicates that the vast majority of patients had at least minimal disseminated disease at diagnosis, whereas, with state-of-the-art imaging techniques and bone marrow investigations, only 15% of patients with RMS present with overt metastatic disease. Besides treating minimal disseminated disease, chemotherapy enables local therapy to be more conservative. In most patients, surgery at diagnosis consists of biopsy only. Chemotherapy is given to all patients; it reduces tumour size and extension and often allows a delay in surgery. The tumour is more likely to be completely resected and mutilating surgery avoided. Most international protocols use vincristine and dactinomycin as standard drugs. The choice of alkylating agent differs between Europe (ifosfamide) and North America (cyclophosphamide). These drugs have proved to be equally effective, but differences in toxicity exist: ifosfamide is more nephrotoxic, whereas cyclophosphamide more gonadotoxic [87]. The addition of other antineoplastic agents to vincristine, dactinomycin and alkylators in the treatment of RMS has been investigated, but no significant improvement in outcome has been found [88].\nSurgery\nIn children surgery often starts during the diagnostic phase with biopsy for histological studies, although here interventional radiology plays an increasing role. Excisional biopsy is not advocated except for paratesticular tumours. Most patients end up with postsurgical stage IRS group III. Surgery is generally delayed until after tumour reduction by chemotherapy. The surgical treatment of RMS is site-specific, but the current paradigm is complete wide excision of the primary tumour with a margin of uninvolved tissue whenever possible. Debulking and mutilating procedures should be avoided.\nRadiotherapy\nThe philosophies underlying the treatment strategies in North America and Western Europe have differed in the past. North American protocols involved aggressive surgery and routine RT, except for those tumours that were microscopically radically resected at diagnosis, followed by prolonged chemotherapy regimens for up to 2 years. The SIOP-MMT (International Society for Paediatric Oncology\u2013Malignant Mesenchymal Tumour) group advocated the use of chemotherapy and surgery in order to achieve complete remission in as many patients as possible in order to avoid RT in these often very young patients. RT often has devastating effects in growing children, potentially leading to significant cosmetic and functional problems. Radiation tolerance of growing bone is \u226420\u00a0Gy, but the radiation dose from the treatment of RMS ranges from 36 to 50.4\u00a0Gy. For certain sites this policy worked out well (e.g. orbital RMS), and in many patients RT could be avoided [89]. At most other sites, relapse rates were high, and salvage rates after relapse were low. The treatment protocols in Germany (Co-operative Weichteilsarcom Studie, CWS) and Italy (Italian Cooperative Soft Tissue sarcoma Group, ICG) were between that in North America and that of the SIOP-MMT group. In 2005, the former SIOP-MMT, CWS and ICG joined to form the EpSSG. As a result of extensive cooperation between the European and the North American groups, the similarities in treatment strategies now outnumber the differences [90]. Although RT is still deemed essential in many patients to achieve cure, the possibility of radiation-induced second malignant neoplasms must be kept in mind [91, 92].\nEuropean approach\nToday more than 70% of nonmetastatic RMS are cured, but survivors may suffer from sequelae [93\u201398]. Therefore, the SIOP-MMT group has tried in their RMS75, MMT84, MMT89 and MMT95 studies to avoid mutilating surgery and RT [93, 99, 100]. Only children \u22653\u00a0years of age with parameningeal RMS at high risk of meningeal extension and children not achieving complete remission after chemotherapy and surgery were irradiated after intensive chemotherapy. Of all survivors, 49% were treated without significant local therapy [93]. Locoregional relapse occurred in 34%. This has lead to an event-free survival (EFS) of 57% and an overall survival (OS) of 71% [93]. Alveolar histology was associated with a significantly higher risk of relapse and a much higher risk of metastases.\nNorth American approach\nIn the studies performed by the IRSG the need for RT is based on surgical radicality, localization, and size of the primary tumour. Only completely resected tumours (IRS group I) with a favourable location (stage I; Table\u00a05), and small tumours (\u22645\u00a0cm) at unfavourable locations (stage II) did not receive RT in the IRS-IV study [87]. Grossly resected tumours with microscopic residual disease (IRS group II), and incompletely resected tumours or tumours with gross residual disease after biopsy (IRS group III) were irradiated [87, 88, 101, 102]. With this approach in the IRS-IV study an EFS of 78% and an OS of 84% were obtained. Between the IRSG and SIOP-MMT group a significant difference in OS in alveolar RMS was seen (71% vs. 38%) [90]. The salvage rate after relapse of an alveolar RMS was low, and therefore in the current European trial, RT is mandatory for all patients with alveolar histology.\nTable\u00a05Pediatric Oncology Group and Children\u2019s Cancer Group for the Study of Rhabdomyosarcoma classification [28]StageSitePrimary tumouraSizebRegional nodescDistant metastasisdIOrbitT1 or T2a or bN0, N1 or NXM0Head and neck (excluding parameningeal)Genitourinary, nonbladder nonprostateIIBladder and prostateT1 or T2aN0 or NXM0ExtremityCranial parameningealOther (including trunk, retroperitoneum, etc)IIIBladder and prostateT1 or T2aN1M0ExtremityCranial parameningealT1 or T2bN0, N1 or NXM0Other (including trunk, retroperitoneum, etc)IVAll sitesT1 or T2a or bN0 or N1M1aT1 tumour limited to original muscle or organ, T2 tumour has extension or fixation to the surrounding tissue.ba tumour equal to or less then 5\u00a0cm in greatest dimension, b tumour larger than 5\u00a0cm in greatest dimension.cN0 no clinical involvement of regional nodes, N1 clinical involvement of regional nodes, NX status of regional nodes unknown.dM0 no distant metastasis, M1 distant metastasis.\nAs described above, European and American approaches, although historically different, have converged based on the results of successive international trials and extensive cooperation between the European and North American groups [90]. As approaches now are very similar, in this review the current EpSSG approach for the different clinical groups is discussed.\nCurrent EpSSG approach\nIRS group I In the IRS-I study, a randomized trial in clinical IRS group I patients, no difference was seen in survival between patients treated with chemotherapy and those treated with chemotherapy and RT [101]. A subsequent retrospective study based on the subsequent IRS-I to IRS-III trials confirmed that for embryonal RMS although there was a small difference in failure-free survival (FFS) with and without RT, OS was not significantly different [103]. On the other hand, for alveolar RMS and undifferentiated RMS, 10-year FFS was 73% vs. 44%, and OS 82% vs. 52% for patients treated with and without RT, respectively [103]. Therefore the current EpSSG study recommends RT in non-embryonal RMS only.\nIRS group II Evaluation of the role of RT in IRS group II patients in subsequent Cooperative Soft Tissue Sarcoma studies (CWS 81, 86, 91 and 96) showed that for embryonal RMS, EFS was significantly different for patients treated with and without RT [104]. However, OS at 5\u00a0years was not significantly different (84% vs. 77%). For patients with tumours of unfavourable histology (independent of site and size), EFS and OS were significantly better when RT was part of the treatment [104]. Therefore, in IRS group II patients, RT is recommended. It is compulsory in patients with high-risk features, but may be omitted in patients with favourable histology in whom RT may be considered too toxic when considering patient age and site of the tumour.\nIRS group III In patients with IRS group III tumours, RT is the only available therapy in patients who cannot receive a secondary complete resection. Patients who do receive a delayed complete resection benefit from additional RT. In the CWS trials 81\u201396 5-year EFS was 77% and 58% for those treated with and without additional RT, respectively [28]. OS, however, was not significantly different between the two groups: 84% and 79%, respectively. RT is, therefore, usually indicated except in patients with a favourable site and histology.\nPrognosis\nAs malignant tumours in childhood are rare diseases, most children with cancer have been included in international treatment protocols. This way survival for localized disease (85% of patients) has improved from 25% in the early 1970s to 75% in the most recent international trials [101, 105\u2013107]. Although results for localized RMS have improved dramatically in the past decades, patients with disseminated disease still have a dismal prognosis, with a 5-year OS of 24% [26].\nA way to reduce long-term sequelae of RT may be by using brachytherapy, especially in girls with genital tract RMS and patients with bladder-prostate, extremity, and head and neck RMS [100, 108\u2013111]. Other options are the use of modulated RT (IMRT) and proton therapy [112, 113].\nThe postoperative patient\nImage interpretation and management of the patient after surgery and often RT are challenges (Fig.\u00a022). Most important is proper knowledge of the surgical procedure and\/or the radiation field. The following postoperative changes can be encountered in children treated for RMS:\nHaematomaOedemaSoft-tissue infection\/abscessCalcificationForeign bodiesMuscle flaps\/fat padsDistorted anatomyRadiation effect Figure\u00a023 shows a possible decision tree to manage postoperative findings on follow-up imaging.\nFig.\u00a022A 6-year-old boy with a history of treated bladder RMS. At surgical resection the urethra was damaged leading to a persistent urinoma in, after RT, nonvital tissue. MRI image after treatment shows a mass (asterisk) between the urethra (open arrow) and the rectum (solid arrow). This mass is a vascularized gracilis muscle flap used to repair the defect. Without proper knowledge of the surgical history of the patient, this might have been interpreted as tumour recurrence. Histopathology: embryonal RMSFig.\u00a023Flow chart for posttreatment lesions found on MRI. SI signal intensity, CE contrast-enhanced [114]\nConclusion\nIn this review we have discussed the findings of RMS outside the craniofacial region. The treatment of RMS requires a multidisciplinary approach, in which paediatric oncologists, radiologists, paediatric surgeons, pathologists and radiation oncologists all play a vital role. Although they are the most common soft-tissue tumour of childhood, these still rare tumours should be evaluated and treated in specialized centres.","keyphrases":["imaging","rhabdomyosarcoma","children"],"prmu":["P","P","P"]}
{"id":"Obes_Surg-4-1-2367390","title":"Delayed Intrathoracic Gastric Perforation After Obesity Surgery: A Severe Complication\n","text":"We describe a case of a patient with an intrathoracic gastric perforation, 6 months after she underwent a gastric banding procedure for the treatment of morbid obesity. After an urgent laparotomy during which the stomach was replaced and oversewn, she recovered uneventfully. The possible mechanism of this severe complication is discussed.\nIntroduction\nGastric banding is a frequently performed operation for patients with morbid obesity. Complications have been described extensively. We describe a rare and severe complication of an intrathoracic stomach in a patient with gastric banding, 6\u00a0months after the initial procedure, and its treatment.\nCase Report\nA 37-year-old woman was admitted to our emergency department with acute onset of abdominal pain, vomiting, and dyspnea. Six months earlier, she had undergone laparoscopic adjustable gastric banding (Lap-Band\u00ae)for severe obesity. Physical examination now showed an anxious, dyspneic woman with a respiratory rate of 45 per minute. There was a tachycardia (140\/minute) and blood pressure difference between the left (60\/40\u00a0mmHg) and the right arm (110\/70\u00a0mmHg). Auscultation of the chest revealed diminished breathing sounds on the left side. Chest x-ray showed an intrathoracic position of the stomach through a paraesophageal hernia with a shift of the mediastinum to the right (Fig.\u00a01). A small pneumothorax was seen.\nFig.\u00a01Intrathoracic position of the stomach with a shift of the mediastinum to the right\nAfter a nasogastric tube was inserted, the patient was intubated because of respiratory insufficiency. A computed tomography (CT) scan of the chest showed a complete atelectasis of the left lung caused by an intrathoracic stomach and a left-sided pneumothorax (Fig.\u00a02). The condition of the patient worsened and she was taken to the operating room for urgent laparotomy.\nFig.\u00a02Complete atelectasis of the left lung caused by an intrathoracic stomach and a left-sided pneumothorax\nDuring laparotomy an ischemic gastric perforation was identified at the orifice of the paraesophageal hernia, with spill of gastric content into the abdomen and thoracic cavity. The intrathoracic stomach was reduced into the abdomen. The gastric perforation was oversewn. The    Lap-band was removed, and a Nissen fundoplication was performed. At last, a thoracic drain was placed to treat the pneumothorax.\nThe patient was treated with intravenous antibiotics (metronidazole and cefuroxime) for 5\u00a0days. After 2\u00a0days, she was able to leave the Intensive Care Unit. Her postoperative course was uneventful. Thirteen days after surgery, she left the hospital.\nDiscussion\nGastric perforation after gastric banding has been described earlier [1]. In all reported cases, the perforation was caused by erosion of the band through the stomach with an incidence of 0.3\u20133% of the patients. Furthermore, herniation of the stomach into the thoracic cavity is a well-known entity. It has been described as a late complication after Nissen fundoplication, even in combination with intrathoracic perforation [2\u20134].\nWe describe the first case in which both perforation and herniation have occurred simultaneously after obesity surgery and even not as an immediate consequence of the operation. In our case, the gastric band was not the primary cause of the perforation. Most probably, the stomach had moved into the thoracic cavity through a preexistent paraesophageal hernia. Because of air-trapping distal to the band accompanied by having a copious meal, the intrathoracic stomach distended, rotated, and perforated at the orifice of the hernia. The acute onset of pain can be explained by the perforation and the dyspnea appears to be caused by mediastinal shift as well as the pressure atelectasis of the left lung.\nRetrospectively, our patient was known to have a paraesophageal hernia before she underwent gastric banding. The possible severe complications of this combination should be considered. A Nissen fundoplication before or in the same operation could be an option for this subgroup of obese patients.","keyphrases":["gastric perforation","complication","gastric banding","morbid obesity","bariatric surgery"],"prmu":["P","P","P","P","M"]}
{"id":"J_Biol_Inorg_Chem-3-1-2099461","title":"A steady-state and pre-steady-state kinetics study of the tungstoenzyme formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus\n","text":"Formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus is a homotetrameric protein with one tungstodipterin and one [4Fe\u20134S] cubane per 69-kDa subunit. The enzyme kinetics have been studied under steady-state conditions at 80 \u00b0C and pre-steady state conditions at 50 \u00b0C, in the latter case via monitoring of the relatively weak (\u03b5 \u2248 2 mM\u22121 cm\u22121) optical spectrum of the tungsten cofactor. The steady-state data are consistent with a substrate substituted-enzyme mechanism for three substrates (formaldehyde plus two ferredoxin molecules). The KM value for free formaldehyde (21 \u03bcM) with ferredoxin as an electron acceptor is approximately 3 times lower than the value measured when benzyl viologen is used as an acceptor. The KM of ferredoxin (14 \u03bcM) is an order of magnitude less than previously reported values. An explanation for this discrepancy may be the fact that high concentrations of substrate are inhibitory and denaturing to the enzyme. Pre-steady-state difference spectra reveal peak shifts and a lack of isosbestic points, an indication that several processes happen in the first seconds of the reaction. Two fast processes (kobs1 = 4.7 s\u22121, kobs2 = 1.9 s\u22121) are interpreted as oxidation of the substrate followed by rearrangement of the active site. Alternatively, these processes could be the entry\/binding of the substrate followed by its oxidation. The release of the product and the electron shuffling over the tungsten and iron\u2013sulfur center in the absence of an external electron acceptor are slower (kobs3 = 6.10 \u00d7 10\u22122 s\u22121, kobs4 = 2.18 \u00d7 10\u22122 s\u22121). On the basis of these results in combination with results from previous electron paramagnetic resonance studies an activation route plus catalytic redox cycle is proposed.\nIntroduction\nTungstoenzymes and molybdoenzymes typically catalyze the generic n\u00a0=\u00a02 reaction: R\u00a0+\u00a0H2O\u00a0\u2194\u00a0RO\u00a0+\u00a02[H], where R is either an oxoanion (e.g., sulfite) or a relatively small organic metabolite (e.g., an aldehyde). On the basis of structural (cofactor and protein) and functional properties molybdoenzymes are classified in three families: the sulfite oxidase family, the xanthine oxidase family, and the dimethyl sulfoxide reductase (DMSOR) family [1]. The latter also encompasses a limited number of tungsten-containing formate dehydrogenases. The majority of the tungstoenzymes, however, cannot be classified in any of these three families; they form a separate family of aldehyde oxidoreductases (AORs) together with a few apparently less common AOR molybdoenzyme members [2]. The mechanism of action of molybdoenzymes has been extensively studied for several decades, especially for xanthine oxidase [3]. In contrast, attempts to understand the reaction mechanism of tungstoenzymes, in particular for the members of the AOR family, are more recent and of limited extent, although the available structural information on tungstoenzymes and that on molybdoenzymes are comparable. The present study leads to the first proposal of a complete catalytic cycle of the AOR family member formaldehyde oxidoreductase (FOR).\nThe cofactor common to the enzymes of all four families consists of a single Mo(VI\/IV) or W(VI\/IV) ion coordinated by the dithioleno sulfurs of one or two three-ring pterin ligands sometimes with an additional nucleotide attached through a phosphoester bond [4]. All tungsten systems appear to be nonnucleotide dipterin systems with an additional magnesium phosphate coordination between the pterins [5, 6]. The coordination number of the Mo or W is 4\u20137; extra ligands are provided by an amino acid side chain and\/or by O,S of a small, nonprotein ligand. The vast majority of the Mo\/W enzymes have additional prosthetic groups for interaction with a second substrate (e.g., NADH) and\/or for electron transfer with a protein natural redox partner (e.g., ferredoxin). These heme, flavin, and\/or iron\u2013sulfur prosthetic groups are strongly colored to the extent that optical monitoring of the relatively weakly colored (see below) metallopterin has thus far not been tried for mechanistic studies of these complex enzymes. Electron paramagnetic resonance (EPR) spectroscopy is not a competitive alternative, because the only EPR-detectable oxidation state, Mo(V) or W(V), does not appear to be a competent intermediate in the primary, n\u00a0=\u00a02 reaction.\nA small group of molybdoenzymes, exemplified by DMSOR, are exceptional because these proteins contain no other prosthetic groups in addition to the active-site molybdopterin. Furthermore, substitution of W for Mo in Rhodobacter capsulatus DMSOR affords an active enzyme with an unmodified 3D structure. The Mo(VI) and W(VI) metallopterin UV\u2013vis spectra of this enzyme have been reported to exhibit several broad peaks in the visible and near-UV region with extinction coefficients of the order of \u03b5\u00a0\u2248\u00a02\u00a0mM\u22121\u00a0cm\u22121 [7], and the molybdopterin absorption has been used as monitor in a pre-steady-state kinetics study [8].\nFollowing the DMSOR, the next step up in optical complexity is found in the AOR family of tungstoenzymes, which all contain\u2014in addition to the tungstopterin active site\u2014a single [4Fe\u20134S](2+;1+) cluster for electron transfer. The visible spectrum of the iron\u2013sulfur cubane consists of a single broad line at approximately 400\u2013430\u00a0nm, which is not only a simpler pattern to that found for [2Fe\u20132S] clusters (e.g., in the xanthine oxidase family of molybdoenzymes), but it also has a significantly smaller extinction coefficient than flavin or heme prosthetic groups, and so chances are more favorable for monitoring the weaker absorptions from the metallopterin cofactor in these complex enzymes. The homotetrameric (4\u00a0\u00d7\u00a069\u00a0kDa) Pyrococcus furiosus FOR is a member of this family, and we describe here a study of this enzyme as the first example of pre-steady-state kinetics research of a complex, group-6 metalloenzyme using the color of the active center as the monitor. Our choice of FOR was also indicated by the availability of a 3D structure, which was the basis of an initial proposal of part of the enzyme\u2019s working mechanism [6], and was further stimulated by the unusual behavior of the enzyme\u2019s substrate in aqueous solution [9, 10].\nMaterials and methods\nP. furiosus (DSM 3638) was grown at 90\u00a0\u00b0C under anaerobic conditions with starch as a carbon source, as previously described [11]. Cells were broken by osmotic shock, diluting with 5\u00a0vol 30\u00a0mM tris(hydroxymethyl)aminomethane (Tris)\/HCl, pH 8.0, containing, 0.1\u00a0mg\u00a0ml\u22121 DNase I, 0.1\u00a0mg\u00a0ml\u22121 RNase, and 1\u00a0mM cysteine. A cell-free extract was obtained as the supernatant after 30\u00a0min centrifugation at 15,000g.\n\u201cHigh-activity\u201d FOR was purified as reported previously [9, 10]. Samples were anaerobically purified in 20\u00a0mM Tris\/HCl buffer, pH 8.0. An extra column of (diethylamino)ethyl (2.0\u00a0cm\u00a0\u00d7\u00a010\u00a0cm) was equilibrated with 20\u00a0mM piperazine\/diethanesulfonic acid buffer, pH 6.4. A 100-ml gradient was used from 0 to 0.5\u00a0M NaCl. Ferredoxin was purified as reported previously [12]. Samples were anaerobically purified in 20\u00a0mM Tris\/HCl buffer, pH 8.0. Protein concentration was determined using the bicinchoninic acid method using bovine serum albumin as the standard. Subunit molecular weight and degree of purity were determined with sodium dodecyl sulfate polyacrylamide gel electrophoresis using a Phast system (GE Healthcare).\nFOR activity was routinely assayed at 80\u00a0\u00b0C, under anaerobic conditions, with formaldehyde, or glutardialdehyde, as the substrate and 3\u00a0mM benzyl viologen as the electron acceptor in 50\u00a0mM 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (Epps) buffer, pH 8.4.\nSteady-state kinetics of FOR were determined by activity measurements in triplicate in an optical assay with different concentrations of formaldehyde (0.5, 1, 2, 5, 10, 25, 50\u00a0mM) as a substrate. Benzyl viologen (\u03b5600\u00a0=\u00a010.6\u00a0mM\u22121\u00a0cm\u22121 [13]) or ferredoxin (\u03b5400\u00a0=\u00a017.0\u00a0mM\u22121\u00a0cm\u22121 [14]) were used as an electron acceptor at various concentrations in 50\u00a0mM Epps buffer, pH 8.4, and \u00a080\u00a0\u00b0C, where one unit is defined as 2\u00a0\u03bcmol electron acceptor reduced per minute. The concentrations of benzyl viologen and ferredoxin were, respectively, 50\u20133,000 and 10\u2013200\u00a0\u03bcM, and the ferredoxin concentration is per dimer [15].\nThe UV\u2013vis spectrum was recorded with a Hewlett-Packard 8452A diode-array spectrophotometer. The optimal excitation wavelength for fluorescence measurements was obtained using a Shimadzu RF-5001PC spectrofluorophotometer. Pre-steady-state studies were carried out under anaerobiosis in 50\u00a0mM Epps buffer, pH 8.4, at 50\u00a0\u00b0C with formaldehyde (25\u00a0mM) and formaldehyde-d2 (25\u00a0mM) as a substrate using an SX.18\u00a0MV stopped-flow apparatus equipped with a sequential mixing capability and a diode-array rapid-scan detection system (Applied Photophysics). The UV\u2013vis data were obtained by recording 400 spectra at different time scales (16, 65 and 250\u00a0s). A spectrum obtained 2\u00a0ms after mixing was taken as the \u201coxidized\u201d reference spectrum. Fluorescence data were obtained by exciting the samples at 280\u00a0nm, and the change in maximum fluorescence emission was measured at 340\u00a0nm.\nResults\nSteady-state kinetics\nSteady-state kinetics studies at 80\u00a0\u00b0C were performed on the combinations FOR\/formaldehyde\/benzyl viologen and FOR\/formaldehyde\/ferredoxin. FOR catalyzes a two-electron transfer from formaldehyde to the one-electron acceptor benzyl viologen or ferredoxin. This results in a reaction with three substrates; one formaldehyde molecule and two benzyl viologen or two ferredoxin molecules. The possible mechanisms for this reaction are so-called enzyme-substitution mechanisms: the triple-transfer mechanism or the concerted-substitution mechanism [16].\nTriple-transfer mechanism: \nConcerted-substitution mechanism: The equation for the reaction rate can be simplified, when substrates B and C (ferredoxin or viologen) are the same. The results have the same form as equations for the reaction rate of an enzyme with two substrates. The triple-transfer mechanism will transform into the substituted-enzyme mechanism for two substrates and the concerted-substitution mechanism will transform into the ternary-complex mechanism for two substrates [16]. The apparent KM values for the second and third substrates are identical, and the measured KM is twice the real KM of the second or third substrate [16, 17]. To predict whether the systems function according to a ternary-complex mechanism or a substituted-enzyme mechanism, one substrate was varied, while the other substrate concentration was held constant.\nSubstrate inhibition of one of the substrates is described by Eq.\u00a03, where KsiB is the constant that defines the strength of the inhibition of substrate B with concentration b: Primary plots of the concentration of the inhibitor divided by the reaction rate (b\/v) versus the concentration of the inhibitor (b) are parabolic and intersect at a common point on the b\/v axis. The primary plots of the concentration of the noninhibiting substrate divided by the reaction rate (a\/v) versus the concentration of the noninhibiting substrate (a) are linear and intersect at a positive value of the concentration of noninhibiting substrate [16]. A more complex situation occurs when both substrates show substrate inhibition. At low concentrations of both substrates the shapes of both curves are parabolic. Substrate inhibition by one substrate at low concentrations of the other provides strong positive evidence that the substituted-enzyme mechanism applies [16].\nThe plots of a\/v versus a and b\/v versus b, with substrate A is formaldehyde and B is benzyl viologen or ferredoxin, are presented in Figs.\u00a01 and 2. All intersection points are situated within experimental error at positive values of the substrates. The parabolic shape of all the figures indicates, as stated above, substrate inhibition by both substrates. All four graphs in Figs.\u00a01 and 2 are consistent with a substrate substituted-enzyme mechanism. Fig.\u00a01Primary plots for Pyrococcus furiosus formaldehyde oxidoreductase with formaldehyde as a substrate and benzyl viologen as an electron acceptor. Formaldehyde was used in concentrations of 1, 2, 5, 10, 25, and 50\u00a0mM. Benzyl viologen was used at concentrations of 50, 100, 500, 1,000, 2,000, and 3,000\u00a0\u03bcM. Each data point is the average value from three measurementsFig.\u00a02Primary plots for P. furiosus formaldehyde oxidoreductase with formaldehyde as a an substrate and ferredoxin as electron acceptor. Formaldehyde was used at concentrations of 0.5, 2, 5, 10, 25, and 50\u00a0mM. Ferredoxin was used at concentrations of 10, 20, 40, 80, 100, and 200\u00a0\u03bcM. Each data point is the average value from three measurements\nOne-substrate Michaelis\u2013Menten kinetics was used to fit the data and to calculate KMapp and Vmaxapp at the different concentrations of substrate and acceptor. To calculate the KM and the Vmax of the different substrates the KMapp and vapp data were fitted using the equations for the apparent values of the Michaelis\u2013Menten parameters for the substituted-enzyme mechanism [16]: with equivalent expressions for substrate A.\nThe KM and Vmax values of formaldehyde measured with excess benzyl viologen as an electron acceptor (Table\u00a01) are in agreement with previous measurements [10, 18]. The KM value for formaldehyde (3.8\u00a0mM) with ferredoxin as an electron acceptor is approximately 3\u00a0times less than when benzyl viologen is used as the acceptor. The conversion rate of formaldehyde with ferredoxin as the electron acceptor is 6\u00a0times less than when benzyl viologen is used as the acceptor. The values for KM are unusually high for a physiological substrate. We have previously provided experimental evidence indicating that only \u201cfree\u201d formaldehyde (i.e., the nonhydrated form which is 0.2% at 80\u00a0\u00b0C) is a substrate for FOR [10]. The corrected KM values for free formaldehyde with acceptor benzyl viologen or ferredoxin are, respectively, 71 and 21\u00a0\u03bcM. Note that Vmax decreases by a factor of 4 when the temperature is lowered from 80 to 20\u00a0\u00b0C, and that KM for free formaldehyde is temperature-independent [10].\nTable\u00a01\nCatalytic properties of formaldehyde oxidoreductase determined at 80\u00a0\u00b0C with formaldehyde as the substrate and ferredoxin or benzyl viologen as the electron acceptor\nElectron acceptor\nBenzyl viologen\nFerredoxin\nKM total formaldehyde (mM)\n13\u00a0\u00b1\u00a02\n3.8\u00a0\u00b1\u00a00.5\nKM free formaldehyde (\u03bcM) \n71\u00a0\u00b1\u00a010\n21\u00a0\u00b1\u00a03\nVmax formaldehyde (s\u22121)\n28\u00a0\u00b1\u00a01a\n4.5\u00a0\u00b1\u00a00.5\nKM electron acceptor (\u03bcM)\n73\u00a0\u00b1\u00a019\n14\u00a0\u00b1\u00a07\nVmax electron acceptor (s\u22121)\n32\u00a0\u00b1\u00a02\n6.2\u00a0\u00b1\u00a03.2\nKM glutardialdehyde (mM)\n12\u00a0\u00b1\u00a02\nVmax glutardialdehyde (s\u22121)\n13\u00a0\u00b1\u00a01\nThe ratio of activities at 80\u00a0\u00b0C compared with 50\u00a0\u00b0C is 2.8 under standard conditions (see \u201cMaterials and methods\u201d) with 50\u00a0mM total formaldehyde\naIn our previous work [10] Vmax\u00a0=\u00a054\u00a0s\u22121 was reported on a per tungsten atom basis; this is equal to 28\u00a0s\u22121 on a per protein molecule basis\nVarying the formaldehyde concentration gives the KM and Vmax values for the electron acceptors. The KM value of benzyl viologen of 73\u00a0\u03bcM is comparable to values found in literature for other enzymes that react with benzyl viologen as an electron acceptor. The KM value of ferredoxin (14\u00a0\u03bcM) is, however, uncharacteristically low: previously reported literature values for FOR and AOR are 100 and 200\u00a0\u03bcM, respectively [9, 19]. An explanation for this discrepancy may be found in the fact that high concentrations of substrate, up to 50\u00a0mM of total formaldehyde for FOR, have been used in previous studies to determine KM of ferredoxin. In the present work concentrations from 0 to 10\u00a0mM were used to determine KMapp and vapp. When activity was measured with high concentrations of formaldehyde (i.e., resulting in significant substrate inhibition) the apparent KM of ferredoxin increased to approach values found in the literature (data not shown).\nIn a previous study alternative substrates for FOR were reported with KM values of the order of a few millimoles per liter. The lowest KM value was found for glutardialdehyde (KM\u00a0=\u00a00.8\u00a0mM) with benzyl viologen as an electron acceptor [9]. We have been unable to reproduce these results. In our hands the KM for glutardialdehyde was determined to be 12\u00a0mM, i.e., approximately the same as the apparent KM for total formaldehyde.\nPre-steady-state kinetics\nPre-steady-state kinetics studies were performed on FOR (20\u00a0\u03bcM) and formaldehyde at 50\u00a0\u00b0C. Formaldehyde was used at a concentration of 25\u00a0mM (50\u00a0\u03bcM free formaldehyde). UV\u2013vis spectra were recorded at 40- and at 160-ms intervals. At least two spectra per time point were averaged for noise reduction. In the spectrum of oxidized FOR the broad feature of the [4Fe\u20134S]2+ cluster dominates. However, close inspection reveals weak (\u03b5max\u00a0\u2248\u00a01\u20132\u00a0mM\u22121\u00a0cm\u22121) absorptions at higher wavelengths similar to those reported for the single-cofactor DMSOR [20]. After subtraction of the spectrum at t\u00a0=\u00a02\u00a0ms, difference spectra were obtained (Fig.\u00a03), at 0.1, 0.5, 1, 2, 5, 10, and 16\u00a0s, which reveal peak shifts and a lack of isosbestic points. Fig.\u00a03Difference in absorbance versus wavelength measured in pre-steady-state experiments with formaldehyde as a substrate at times 0.1, 0.5, 1, and 2\u00a0s (a) and 2, 5, 10, and 16\u00a0s (b). The reference spectrum was taken at t\u00a0=\u00a02\u00a0ms. c UV\u2013vis spectra of the oxidized and the substrate-reduced form (after 65-s incubation time) of P. furiosus formaldehyde oxidoreductase\nFrom the difference spectra five different wavelengths were selected: 335, 390, 435, 595, and 695\u00a0nm. At 595 and 695\u00a0nm, approximately corresponding absorption bands can be found in the spectrum of oxidized DMSOR from R. capsulatus [8]. Amplitude versus time traces of the 40 and 160\u00a0ms spectra at the different wavelengths were combined. After 65\u00a0s the time traces for all the wavelengths essentially leveled off (Fig.\u00a04). Data points were taken up to 250\u00a0s but no further significant changes were found (not shown). Fig.\u00a04Stopped-flow UV\u2013vis traces obtained for the reduction of P. furiosus formaldehyde oxidoreductase by formaldehyde at 335, 390, 435, 595, and 695\u00a0nm\nThe major changes at the different wavelengths occur in the first seconds. Three phases starting at different times can be identified in Fig.\u00a05a. The first phase starts at or before t\u00a0\u2248\u00a020\u00a0ms; the second and the third phases start at approximately 100 and 300\u00a0ms, respectively. Changes in the amplitude versus time traces at 390 and 435\u00a0nm start at or before 20\u00a0ms; the trace of the wavelength of 595\u00a0nm follows the trend of the 335\u00a0nm trace, with the difference that all phases start approximately 200\u00a0ms later. All data at the different wavelengths were globally simulated with a four-phase exponential least-squares fit (Table\u00a02). Fig.\u00a05Stopped-flow UV\u2013vis traces plus fits on a logarithmic time scale obtained for the reduction of P. furiosus formaldehyde oxidoreductase by a formaldehyde or b deuterated formaldehyde at 335, 390, 435, 595, and 695\u00a0nm. c Stopped-flow fluorescence trace plus fit obtained for the reduction of P. furiosus formaldehyde oxidoreductase by formaldehyde at an excitation wavelength of 295\u00a0nm and an emission wavelength of 340\u00a0nm. d Stopped-flow UV\u2013vis traces plus fits obtained for the binding of formate by P. furiosus formaldehyde oxidoreductase at 335, 390, 435, 595, and 695\u00a0nmTable\u00a02Rate constants from pre-steady-state kinetics of formaldehyde oxidoreductase plus formaldehyde or deuterated formaldehyde or formate at 50\u00a0\u00b0CUV\u2013visFluorescenceCH2OCD2OFormateCH2Ok1 (s\u22121)4.71.120.514.7k2 (s\u22121)1.90.942.00\u00a0\u00d7\u00a010\u221221.9k3 (s\u22121)6.10\u00a0\u00d7\u00a010\u221226.07\u00a0\u00d7\u00a010\u221226.10\u00a0\u00d7\u00a010\u22122k4 (s\u22121)2.18\u00a0\u00d7\u00a010\u221222.26\u00a0\u00d7\u00a010\u22122\nA previous steady-state kinetics study on the substrate kinetic isotope effect of FOR showed a significant primary isotope effect for C\u2013H\/D bond breaking. The specific activity of FOR dropped by a factor of 3 when deuterated formaldehyde was used as a substrate [21]. In the present work the pre-steady-state kinetics of FOR with formaldehyde-d2 was determined in the same fashion as for the experiments with formaldehyde. The amplitude versus time traces at selected wavelengths of FOR incubated with formaldehyde-d2 resemble the traces of the different wavelengths when formaldehyde was used as the substrate; the phases are similar, only the duration and the onset of the changes of states are different. Again the data were fitted with a four-phase exponential fit (Fig.\u00a05b; Table\u00a02).\nTryptophan residues are excellent probes for protein conformational changes. In FOR a single tryptophan (Trp441) can be found in the active site [6]. A fluorescence stopped-flow experiment was performed using 280\u00a0nm as the excitation wavelength and 340\u00a0nm as the emission wavelength. The concentrations of FOR and formaldehyde used were the same as in the previous UV\u2013vis stopped-flow experiments. Traces were measured on different time scales: 1, 10, and 100\u00a0s, and were combined (Fig.\u00a05c). The data can be fitted to a three-phase exponential model. The noise levels however are high. The fluorescence data were fitted simultaneously with the UV\u2013vis data of Fig.\u00a05a, affording a consistent set of parameters (Table\u00a02).\nFormaldehyde is oxidized to formate by FOR with reduction of the W(VI) center. We checked whether FOR can oxidize formate to carbon dioxide. Activity measurements in an optical assay were done at 80\u00a0\u00b0C using formate as the substrate and methyl viologen as the electron acceptor. FOR was unable to oxidize formate. Pre-steady-state binding experiments with oxidized FOR using formate as the nonreducible substrate were done at the same time intervals as for the experiments with formaldehyde (Fig.\u00a05d). Although formate is not converted to carbon dioxide by FOR, absorption changes are found in the time traces at the different wavelengths. The first phase starts at t\u00a0\u2248\u00a020\u00a0ms; the second and the third phases start at approximately 100 and 300\u00a0ms, respectively. The time trace at 335\u00a0nm resembles the trace obtained with formaldehyde. The time traces at 390 and 435\u00a0nm are not similar to each other in contrast to when formaldehyde was used as the substrate. The time traces at 595 and 695\u00a0nm are essentially unaffected by the formate. All data at the different wavelengths were globally fitted with a two-phase exponential least-squares model (Table\u00a02).\nDiscussion\nSteady-state kinetics\nAn initial proposal for part of the working mechanism of P. furiosus FOR was deduced by Hu et al. [6] from the enzyme\u2019s 3D structure: two electrons are transferred from the substrate to the tungsten center. The pterin acts as a noninnocent ligand and transfers the electrons via the N8 atom one by one through Cys491 to the iron\u2013sulfur cluster of FOR. The iron\u2013sulfur cluster of ferredoxin accepts an electron through its Asp14 [6]. The electron is then transferred from the iron\u2013sulfur cluster of ferredoxin to a membrane-bound hydrogenase [22]. After reduction of the iron\u2013sulfur cluster of ferredoxin, the electron transfer comes to a halt. Only one electron at a time can pass through this route. Two ferredoxin molecules are necessary to transport both electrons; alternatively, a ferredoxin dimer [15] can twice in a row bind to FOR and accept an electron. This means that a two-substrate mechanism is insufficient to describe this three-substrate system. The second and the third substrates are both ferredoxin. This results in steady-state Michaelis\u2013Menten equations that have the same form as equations for the reaction rate of an enzyme with two substrates; however, the measured KM for ferredoxin will be twice the \u201creal\u201d KM.\nThe triple-transfer mechanism for three substrates with substrates two and three (ferredoxin) the same is consistent with the experiments.\nBoth substrates, either formaldehyde and ferredoxin or formaldehyde and benzyl viologen, inhibit FOR. The KM values of 20\u201370\u00a0\u03bcM for free formaldehyde are in agreement with our previous studies [10]. When a nonphysiological electron acceptor such as benzyl viologen is used then the KM for formaldehyde is 3\u20134\u00a0times higher. It is possible that when ferredoxin binds to FOR the enzyme experiences a slight conformational change, resulting in a lower KM for formaldehyde.\nIn these studies a KM of 14\u00a0\u03bcM was determined for ferredoxin, which is 5\u00a0times lower than the KM of benzyl viologen. The high KM reported in the literature for ferredoxin was determined with high concentrations of formaldehyde. An explanation for this high value is that at high concentrations (50\u00a0mM) formaldehyde inhibits and partially denatures FOR.\nPre-steady-state kinetics\nPre-steady-state kinetics studies were performed at 50\u00a0\u00b0C. This relatively low temperature was dictated by technical limitations of the stopped-flow apparatus. FOR activity at 50\u00a0\u00b0C is 2.8\u00a0times less than at 80\u00a0\u00b0C, which affords an increased time resolution of enzyme intermediates.\nDifference spectra from the pre-steady-state data revealed peak shifts and a lack of isosbestic points. These features are an indication that several processes are happening in the first seconds of the reaction. The visible spectrum of the iron\u2013sulfur cubane consists of a single broad line at approximately 400\u2013430\u00a0nm; the relatively weakly colored metallopterin is found at different wavelengths.\nA minimal first-order exponential fit model was used to fit the pre-steady-state data. The UV\u2013vis data could be fitted globally with a three-phase exponential fit. Two fast processes and one slow process were found. The fluorescence data were initially modeled with a two-phase exponential fit, resulting in one fast and one slow process. The fast and slow processes deduced from the fluorescence and from the UV\u2013vis data were clearly different. A four-phase exponential model afforded a significantly improved fit to the UV-data (two fast and two slow processes), and the fluorescence data were fitted more accurately with a three-phase exponential model (two fast processes and one slow process). The complete set of fluorescence and UV\u2013vis data obtained with the same enzyme and substrate were then fitted in a global analysis with a three-phase and a four-phase exponential model, respectively. The same four-phase model was used to fit the pre-steady-state data obtained using formaldehyde-d2 as the substrate. The fit is comparable with the fit of formaldehyde but the rate constants of the first and second phases are lower, as expected from the steady-state studies. The first phase is 4\u00a0times slower (k1\u00a0=\u00a01.12\u00a0s\u22121) than with formaldehyde as the substrate. The rate constant of the second phase (k2\u00a0=\u00a00.94\u00a0s\u22121) is lower than when formaldehyde is used as the substrate, consistent with a previously reported steady-state kinetics experiment [21]. The rates for the first two phases with CD2O as the substrate are not significantly different within experimental error. The rate constant for intramolecular electron shuffling appears to be unaffected by the use of formaldehyde-d2.\nResiduals of the individual fits plotted versus time were essentially randomly distributed around the x-axis, thus attesting to the quality of the global fit. Several intermediate species are formed during the first seconds of the reaction. These results are in contrast with previous studies on molybdenum\u2013DMSOR, where in stopped-flow experiments clear isosbestic points were found, and analysis of the time course produced a simple exponential, indicating a simple concerted two-electron reduction of the enzyme with no intermediate species [23].\nTwo alternative interpretations can be envisioned for the three phases in pre-steady-state kinetics (Fig.\u00a06). Fig.\u00a06Proposed alternative models for the pre-steady-state phases of fully oxidized P. furiosus formaldehyde oxidoreductase reacting with formaldehyde. Rate constants with capital letters as a subscript involve binding, oxidizing the substrate, releasing the product, and rearranging electrons between the tungsten center and the iron\u2013sulfur cluster. Alternatively, rate constants with numbered subscripts involve oxidation of substrate, a conformational change leading to weakened product binding, release of product, and electron rearrangement\nFOR is represented in Fig.\u00a06 as a rectangle with two redox groups, a tungstopterin (W) with oxidation states 6+, 5+, or 4+, and a [4Fe\u20134S] cubane cluster with oxidation states 2+ or 1+. On the left of W is an empty binding pocket which can be occupied by substrate (A), by product (P), or by ready-to-leave deactivated product (P*). Occupation by A means that the enzyme has acquired a molecule of substrate in the previous reaction. Similarly, a W capped by a double-bonded O means that the enzyme has previously acquired an oxygen (protonation undefined) from the solvent. The protein is initially in the fully oxidized form; the tungsten center is 6+ and has an oxygen atom bound that was previously acquired from a water molecule, and the iron\u2013sulfur cluster is 2+.\nIn the first model the initial step (kA\u00a0=\u00a04.7\u00a0s\u22121) observed in the optical monitoring is taken to be the overall binding process of formaldehyde to the enzyme. No changes to the formal oxidation state of the tungsten center and iron\u2013sulfur cluster are assumed to occur in this stage. Subsequently, in what appears to be a single event, the oxygen atom is transferred, the substrate is oxidized to the product (kB\u00a0=\u00a01.9\u00a0s\u22121), and the tungsten center receives two electrons from the substrate and is reduced to 4+. The product then leaves the protein (kC\u00a0=\u00a06.10\u00a0\u00d7\u00a010\u22122\u00a0s\u22121). In this step there are no changes in the oxidation states of the tungsten center and the iron\u2013sulfur cluster. The active site of FOR has been proposed by Hu et al. [6] to be closed off from the solvent by a lid provided by the protein itself. It is possible that the substrate, free formaldehyde, is so small that it will not experience significant resistance in reaching the active site. The bigger and charged product may have more difficulty getting free of the enzyme, resulting in a low rate. The protein will then, in the absence of an external electron acceptor, redistribute (to a minor extent; see below) its electrons over the tungsten center and the iron\u2013sulfur cluster (kD\u00a0=\u00a02.18\u00a0\u00d7\u00a010\u22122\u00a0s\u22121). The tungsten center will become 5+ and the iron\u2013sulfur cluster 1+, consistent with previously determined redox properties for \u201chigh-activity\u201d enzyme [10]. An isotope effect of 4.2 is found for the rate constant of the first step (Table\u00a01), which seems to be unrealistically large for a noncovalent binding event, and would rather seem to point toward an event involving the breaking and making of covalent chemical bonds. Therefore, the following would appear to be a rather more likely interpretation of the stopped-flow data.\nAlternatively, if the entry and binding of the substrate does not lead to a detectable change in the optical absorption, then the first step (k1\u00a0=\u00a04.7\u00a0s\u22121) can be the two-electron reduction of the tungsten. In this model the second step (k2\u00a0=\u00a01.9\u00a0s\u22121) is a conformational change leading to an \u201copen\u201d state with decreased affinity for the product formed. The third step (k3\u00a0=\u00a06.10\u00a0\u00d7\u00a010\u22122\u00a0s\u22121) then is the ready release of this now weakly bound formate. The pre-steady-state phase ends with the two-electron-reduced intermediate which, in the absence of an external electron acceptor, slowly, and only partially converts, by electron shuffling, to the EPR-detectable, paramagnetic side product. Previous EPR studies on FOR revealed this slow electron shuffling at 20\u201360\u00a0\u00b0C between the tungsten center and the iron\u2013sulfur cluster upon reduction with formaldehyde or with dithionite [10]. Only after minutes the W(V) signal appeared in the EPR measurements. This observation is in qualitative agreement with the low rate constants found here for the decay of the fourth state.\nThe pre-steady-state data from the experiments with formate and FOR were fitted with a two-phase exponential model: there is one fast process and one slow process. Formate is not oxidized or reduced by FOR; therefore, the fast process (kobs1\u00a0=\u00a00.51\u00a0s\u22121) could be the entry and the binding of formate to the active site of FOR. As in the FOR\/formaldehyde system the slow process (kobs2\u00a0=\u00a02.00\u00a0\u00d7\u00a010\u22122\u00a0s\u22121) is probably a nonphysiological process: the protein reaches a dead end.\nExcluding the very slow electron redistribution step in the absence of external electron acceptor (the last step in Fig.\u00a06) a complete cycle could now be written in which the enzyme shuttles between fully oxidized, one-electron-reduced, and two-electron-reduced, by inclusion of the reoxidation steps of the two-electron-reduced intermediate through serial reactions with two molecules of oxidized ferredoxin. However, such a scheme would be inconsistent with steady-state turnover rates, because the rate constant (k3 or kC) of the product-release step (the penultimate step in Fig.\u00a06) is more than an order of magnitude less than kcat. On the other hand, it is conceivable, that the one-electron-reduced intermediate reacts with a second substrate molecule to become three-electron-reduced (with respect to the resting enzyme). This would lead to the cycle presented in Fig.\u00a07 in which the enzyme shuttles between one-electron-reduced, two-electron-reduced, and three-electron-reduced. Fig.\u00a07Proposed model for the catalytic redox cycle of P. furiosus formaldehyde oxidoreductase. A is the substrate formaldehyde; P is the product formate. The cube-enclosing circle represents the natural redox partner, ferredoxin. Pre-steady-state kinetics measures either the steps with rate constants kA through kD or, alternatively, those with constants k1 through k4. The fully oxidized enzyme is reductively activated by the reaction with formaldehyde to enter a redox cycle in which the enzyme shuttles between one--electron-reduced, two--electron-reduced, and three-electron-reduced states. See the text for additional explanation\nUnder this model the steps observed in the pre-steady-state experiments would actually constitute an activation process. Note that once the steady state of this cycle has been reached, the enzyme reacts sequentially with one formaldehyde and two ferredoxin molecules consistent with the \u201cA+2B\u201d Michaelis\u2013Menten steady-state analysis.\nThe proposed reaction cycle of Fig.\u00a07 is based on a combination of steady state-kinetics, pre-steady-state kinetics, and EPR studies in the absence of an electron acceptor and available structural data. Future pre-steady-state kinetics studies in the presence of excess natural electron acceptor ferredoxin may put the model to a rigorous, be it experimentally challenging, test.","keyphrases":["pre-steady-state kinetics","steady-state kinetics","pyrococcus furiosus","tungsten","formaldehyde oxidoreductase"],"prmu":["P","P","P","P","P"]}
{"id":"Ann_Hematol-4-1-2275303","title":"Practical recommendations on the use of lenalidomide in the management of myelodysplastic syndromes\n","text":"Lenalidomide, an oral immunomodulatory agent, has received approval in the USA from the Food and Drug Administration (FDA) for the management of myelodysplastic syndromes (MDS) classified by the International Prognostic Scoring System (IPSS) as low risk or intermediate-1 risk and with a deletion 5q (del(5q)) cytogenetic abnormality. Although some patients with del(5q) have a relatively good prognosis, all del(5q) patients will become transfusion-dependent at some point during the course of their disease. The results of two clinical trials in more than 160 patients with MDS have demonstrated clear therapeutic benefits of lenalidomide, with >60% of patients achieving independence from transfusion during therapy, irrespective of age, prior therapy, sex, or disease-risk assessment. The recommendations presented in this review will aid the safe administration of lenalidomide for the treatment of patients with low-risk or intermediate-1-risk MDS and a del(5q) cytogenetic abnormality, and they will help physicians avoid unnecessary dose reduction or interruption, thus assuring the best efficacy for patients.\nIntroduction\nMyelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis and leading to peripheral cytopenias and a genetic instability with enhanced risk of disease transformation to acute myeloid leukemia (AML).\nIn the general population, MDS affects approximately five in every 100,000 individuals and is regarded as a relatively rare disease [1]. However, its incidence is highest in people aged >70 years, and in this age group, MDS is the most frequently occurring malignant hematological disorder: twice as frequent as AML, more common than chronic lymphocytic leukemia, and more common than all other malignant lymphomas combined [2]. All patients diagnosed with MDS will eventually die from their disease, and in about 30%, the disease will progress to AML [3].\nBetween 40% and 60% of patients with primary MDS and about 90% of patients with secondary MDS have chromosomal abnormalities at the time of diagnosis [4]. Chromosomal abnormalities may occur as a single abnormality or as part of a complex karyotype; disease severity increases with the number of abnormalities. The most common chromosomal abnormalities are deletions of chromosome 5 and chromosome 7 and trisomy of chromosome 8 [5]. In a subgroup of patients who have an isolated deletion 5q (del(5q)), termed 5q-syndrome, the clinical profile is distinct [6]. Typically, this clinical profile includes macrocytic anemia, a normal to increased platelet count, mild leukopenia, hypolobulated megakaryocytes in the bone marrow, a medullary blast count <5%, and an isolated del(5q) abnormality including a common deleted region between 5q31 and 5q33 [4, 7]. Consistent with the observation that disease severity is related to the number of chromosomal abnormalities, patients with del(5q) plus additional cytogenetic abnormalities have a worse prognosis than those with an isolated del(5q) abnormality [3, 5].\nOn the basis of the results of an international phase II trial in 148 patients [8], the oral immunomodulatory, antiangiogenic, and antineoplastic agent lenalidomide (Revlimid\u00ae; Celgene Corporation, NJ, USA) was approved in December 2005 by the US Food and Drug Administration (FDA) for the treatment of patients with transfusion-dependent International Prognostic Scoring System (IPSS) classified low-risk to intermediate-1-risk MDS with a del(5q) cytogenetic abnormality, with or without additional cytogenetic abnormalities. The first 46 patients recruited received lenalidomide 10mg\/day for 21days every 28-day cycle, for up to 24weeks. After a protocol amendment, the remaining 102 patients received lenalidomide 10mg\/day on a continuous dosing schedule [8]. Overall, 67% of patients who were initially transfusion-dependent achieved transfusion independence, and another 9% achieved a decrease (\u226550%) in the total number of transfusions required during lenalidomide therapy [8]. Forty-five percent of the 85 evaluable patients in this study achieved a complete cytogenetic response, irrespective of chromosomal complexity [8].\nIn January 2007, an international group of MDS specialists met to discuss the practical management of lenalidomide in patients with transfusion-dependent, IPSS-classified low-risk or intermediate-1-risk MDS and a del(5q) cytogenetic abnormality. The recommendations presented here are based on the clinical data derived from the current literature [8, 9], from the known clinical profile of lenalidomide in other disorders, and the clinical experience of the panel members in the practical use of this agent. Although lenalidomide has been used in IPSS-classified intermediate-2-risk and high-risk patients and in patients without an associated del(5q) abnormality, the recommendations presented here focus on the current approved indication of the drug in the USA.\nSelection of patients for lenalidomide therapy\nThe FDA approved lenalidomide for patients with transfusion-dependent anemia due to low- or intermediate-1-risk MDS associated with a del(5q) cytogenetic abnormality, with or without additional cytogenetic abnormalities. This is an important limitation, as patients with a complex karyotype and del(5q) are eligible for treatment only if they have no more than one cytopenia (i.e., anemia, neutropenia, or thrombocytopenia) and their medullary bone marrow blast count is <5%. Furthermore, patients with a bone marrow blast count up to 10% receive a score of 0.5 points in the IPSS classification (Table\u00a01) [3]. Therefore, in order to remain in the IPSS intermediate-1-risk category, one additional chromosomal abnormality (not involving chromosome 7) or two or more peripheral cytopenias are allowed, but not both. Although teratogenicity has not been reported with lenalidomide, as it is an analogue of thalidomide, women of childbearing potential should have two negative pregnancy tests performed within 14days prior to lenalidomide intake.\nTable\u00a01International prognostic scoring system for myelodysplastic syndromes: survival and evolution of acute myeloid leukemia\u00a0Score valuePrognostic variable00.51.01.52.0Bone marrow blasts, %<55\u201310\u201311\u20132021\u201330KaryotypeaGoodIntermediatePoor\u00a0\u00a0Cytopeniasb0\/12\/3\u00a0\u00a0\u00a0Scores for risk groups are as follows: Low, 0; Int-1, 0.5\u20131.0; Int-2, 1.5\u20132.0; and High, \u22652.5aGood: normal, \u2212Y, isolated del(5q), isolated del(20q); poor: \u22653 abnormalities or chromosome 7 anomalies; intermediate: other abnormalitiesbNeutrophils <1800\u2009\u00d7\u2009106\/l, hemoglobin <10 g\/dl, platelets <100\u2009\u00d7\u2009109\/lTable reproduced with permission from [3] \u00a9 The American Society of Hematology\nLenalidomide therapy is effective regardless of prior erythropoietin [8, 9] or prior thalidomide therapy [9], and those previous therapies should not be regarded as contraindications for its use, although concurrent therapy with erythropoietin is not recommended, owing to concerns regarding venous thromboembolism.\nClinical data have shown that the frequency of response to lenalidomide is similar across all age groups evaluated [8]. Importantly, the overall adverse-event burden did not increase in older patients, but serious adverse events were more frequent in patients aged over 65years compared with younger patients (54% vs. 33%, respectively) [10].\nAlthough the impact of renal impairment on lenalidomide efficacy or toxicity in patients with MDS has not been evaluated, the risk of toxicity is expected to be greater in patients with impaired renal function because lenalidomide is renally excreted [11]. In patients with known renal impairment, the expert panel support the dosing schedule proposed by Chen et al., which is based on creatinine clearance (Table\u00a02) [11]. As advancing age is associated with declining renal function, it is recommended that patients older than 65years who are prescribed lenalidomide are monitored carefully throughout their treatment (Table\u00a03).\nTable\u00a02Recommended dose adjustments for patients with impaired renal functionaRenal impairmentDoseMild10\u00a0mg (full dose) every 24\u00a0h\u00a0(80\u2009>\u2009CLcr\u2009\u2265\u200950\u00a0ml\/min)Moderate5\u00a0mg every 24\u00a0h\u00a0(30\u2009\u2264\u2009CLcr\u2009<\u200950\u00a0ml\/min)Severe5\u00a0mg every 48\u00a0h\u00a0(CLcr\u2009<\u200930\u00a0ml\/min, not requiring dialysis)End-stage renal disease5\u00a0mg three times a week after each dialysis\u00a0(CLcr\u2009<\u200930\u00a0ml\/min, requiring dialysis)aRecommendations based on a pharmacokinetic study by Chen et al. [11]CLcr creatinine clearanceTable adapted from [11] \u00a9 2007 SAGE Publications. Adapted by permission of SAGE Publications, Inc.Table\u00a03Recommendations for laboratory monitoring during treatmentFunctionTestRecommendation(s)Kidney functionCreatinineEvery 4\u00a0weeks in patients aged 65\u00a0years and olderBloodFBCWeekly monitoring of full blood count mandatory for the first 2\u00a0months (it may be continued for 5\u00a0months).Biweekly or monthly monitoring should be considered thereafter, depending on hematological status.If treatment is interrupted in patients who had a previous episode of neutropenia or thrombocytopenia while on lenalidomide treatment, the same monitoring guidelines apply at re-initiationThyroid functionTSH, T4Monitor every month during the course of treatmentIn case of loss of response during lenalidomide treatmentGonadal functionTestosteroneIn case of loss of response during lenalidomide treatmentDigoxinDigoxinIn patients concomitantly taking Digoxin, the plasma level should be monitored periodicallyPregnancy testUrine testDay \u221214 and day 0 at initiation of therapy, monthly thereafter (in women of childbearing potential)Bone marrowBM aspiration and cytogenetic testing; Trephine biopsy optionalAt commencement of therapy.In case of loss of response to rule out progressive disease or cytogenetic evolutionBM Bone marrow, FBC full blood count, T4 thyroxine, TSH thyroid-stimulating hormone\nLikewise, the safety, efficacy, and pharmacokinetics of lenalidomide therapy have not been fully investigated in patients with evidence of hepatic impairment, or those with frank hepatic dysfunction. Clinical trials conducted to date have excluded patients with inadequate hepatic function, with individual trials using different serum transaminase measures as exclusion criteria; most trials excluded patients with serum transaminase levels greater than three times the upper limit of normal.\nPharmacokinetic studies have revealed that the co-administration of lenalidomide with digoxin may increase the maximal digoxin concentration, although the area under the concentration\u2013time curve remained unchanged [10]. Importantly, lenalidomide does not interact with the cytochrome P450 system, and so, can be confidently co-administered with other medications metabolized by this route (Celgene data on file) [10]. This latter finding is particularly relevant for older patients, who may be more likely to be receiving concomitant medications for co-morbidities. The expert panel concurred with the current prescribing information for lenalidomide and recommended that patients taking digoxin should have their digoxin plasma levels monitored periodically (Table\u00a03).\nTreatment with lenalidomide in del(5q) MDS\nThe time-to-response during lenalidomide therapy was evaluated in the larger of the two pivotal clinical trials [8]. For most patients, the median time to initiation of the transfusion-independent period was 4.6weeks, but responses have been noted to take up to 12months [8]. Therefore, the expert panel recommends a treatment duration of at least 4months in order to obtain an initial response (Table\u00a04). Patients were followed for a median of 104weeks, but as more than half of them remained transfusion-free at this assessment point, the median duration of transfusion independence could not be calculated. Encouragingly, among the pooled patients with MDS with a del(5q) abnormality from the two studies [8, 9], transfusion independence and major erythroid response were maintained for a median duration of at least 2years [12]. Even though there are anecdotal reports of long-lasting erythroid remissions in some patients with hematological complete response who discontinued lenalidomide, the panel recommends continuation of lenalidomide treatment in responders for as long as it is tolerated. In patients where the dose has initially been reduced for mild adverse events, dose escalations may be considered (Table\u00a04). In responders with high ferritin levels due to long transfusion histories who achieve normal hemoglobin levels, lenalidomide treatment should be continued and therapeutic phlebotomy be considered to reduce the secondary iron overload. In responders who discontinue due to adverse events, the panel recommends checking for response continuation before initiating another treatment (Table\u00a04). Preliminary data show that prolonged treatment with lenalidomide does not increase the risk of transition to acute myeloid leukemia [13]. Of note, there are reports of patients acquiring additional cytogenetic abnormalities while on lenalidomide [8], but at this time the relevance of this phenomenon is not entirely clear. Until there is data to the contrary, patients with continuous hematological response should remain on the drug. Patients losing their response and developing a complex karyotype should be approached as they would be in the absence of lenalidomide, and alternative therapies, including transplantation, should be considered.\nTable\u00a04Recommendations for treatment duration\u00a0Recommendation(s)Initial treatmentTreatment should be continued for at least 4\u00a0months in order to obtain an initial responsePatients who\u00a0Have a complete hematological responseContinue lenalidomide therapy for as long as it continues to be well tolerated to avoid relapse (both erythroid and cytogenetic)\u00a0Have a partial responseContinue lenalidomide therapy, and consider an escalation of the lenalidomide dose to a maximum of 10 mg per day, if tolerable\u00a0Discontinue treatment because of adverse eventsPatients should not begin another therapy immediately; it is recommended to wait 8\u201312\u00a0weeks to determine whether the response continues\nTolerability of lenalidomide in del(5q) MDS\nLenalidomide has been shown to be well tolerated in the two clinical trials reported to date [8, 9]. The most commonly reported adverse events of grade 3 or 4 severity were neutropenia (55%), thrombocytopenia (44%), anemia (7%), leukopenia (6%), rash (6%), diarrhea (3%), pruritus (3%), pneumonia (3%), and fatigue (3%) [8].\nManagement of hematological adverse events\nHematological adverse events, including neutropenia and thrombocytopenia, were the most common adverse events with lenalidomide therapy and the most frequent reasons for dose adjustment [9]. As such, thrombocytopenia and neutropenia should be expected to occur in most patients\u2014in fact, they appear to be associated with a higher likelihood of a response to therapy [14]. In general, regular monitoring of blood cell counts is recommended (Table\u00a03).\nThe majority of hematological adverse events (62%) occurred early in the course of treatment (within the first 8weeks) [8]. Grade 3 or 4 neutropenia was reported in 55% of patients [8]. Monitoring and, in some cases, additional treatment with granulocyte colony-stimulating factor (G-CSF) is recommended (Tables\u00a03 and 5, and Fig.\u00a01). Neutropenic sepsis was the only reported cause of death (occurring in three patients) that was regarded as possibly related to the study drug [8], therefore, patients should receive clear guidance on how to react in the event of febrile neutropenia (Table\u00a05). Grade 3 or 4 thrombocytopenia was observed in 44% of patients [8]. In some thrombocytopenic patients, interruption of lenalidomide treatment may be necessary (Table\u00a05 and Fig.\u00a01). Lenalidomide has not been tested in patients with a baseline neutrophil count <500 \u00d7 106\/l or baseline platelet count <50 \u00d7 109\/l.\nFig.\u00a01Recommendations for the management of a neutropenia and b thrombocytopenia. aNo consensus was reached, recommendation is based on the opinion of some of the panel members G-CSF granulocyte colony-stimulating factorTable\u00a05Recommendations for the management of hematological adverse events\u00a0Recommendation(s)NeutropeniaFor treatment recommendations for neutropenia see Fig. 1Febrile neutropeniaProvide patients with clear guidance on how to react in the event of febrile neutropenia (patient education, specialized hematological care at all times, and broad-spectrum antibiotics within 3\u00a0h of fever onset)ThrombocytopeniaFor treatment recommendations for thrombocytopenia see Fig. 1VTEVTE prophylaxis is not generally recommended in patients with MDS. Combining lenalidomide with erythropoietin is also not recommended.If erythropoietin is used, be aware of a potentially increased risk of VTE. Patients should be informed about the risk of VTE and monitored for symptomsIf VTE does occur, interrupt lenalidomide treatment, treat the VTE, and carefully re-introduce lenalidomide once stable anticoagulation has been establishedPolycythemiaLenalidomide should be continued and phlebotomy considered, depending on ferritin levels. Although polycythemia is usually transient, treatment interruption may be necessary if additional risk factors for VTE are presentMDS myelodysplastic syndromes, VTE venous thromboembolism\nIn the opinion of the panel, patients presenting with a neutrophil count <1,000 \u00d7 106\/l prior to the start of lenalidomide therapy can still be treated with lenalidomide if G-CSF is co-administered. When neutropenia occurs during lenalidomide therapy, interruption of the lenalidomide treatment schedule might be necessary (Table\u00a05 and Fig.\u00a01). When thrombocytopenia (platelet count <25 \u00d7 109\/l) is diagnosed at presentation, based on the rationale that once a response is achieved thrombocytopenia is likely to resolve, some experts would consider administering lenalidomide with platelet support (Table\u00a05 and Fig.\u00a01). However, other experts advise against the use of lenalidomide in patients with a platelet count <50 \u00d7 109\/l because there is a risk of sustained deterioration of thrombocytopenia, despite an erythroid response. In some patients responding to lenalidomide, polycythemia may occur [8]; if appropriate, therapeutic phlebotomy or interruption of lenalidomide therapy may be considered (Table\u00a05).\nGrade 3 or 4 venous thromboembolism (VTE) was observed in 3% of patients [8]. Generally, the risk of VTE is increased if there is a history of superficial vein thrombosis, or previous VTE [15, 16]. It is unknown if this applies to patients treated with lenalidomide as well. Nevertheless, these patients should be carefully monitored. In patients with a history of previous VTE, low-molecular-weight heparin should be used to prevent recurrent thrombosis. Although aspirin is effective in the prevention of thrombosis in multiple myeloma, aspirin cannot be recommended alongside lenalidomide as lenalidomide frequently leads to grade 3 or 4 thrombocytopenia in patients with MDS with del(5q). There are insufficient data on low-dose coumadin treatment for the prophylaxis of VTE, and therefore the panel did not recommend its use. Of particular relevance to patients with MDS, VTE risk is increased by concomitant erythropoietin use [17]. Since the incidence of VTE in patients with lenalidomide-treated MDS is generally low [8], prophylaxis in patients without antecedents of VTE is not recommended. If VTE does occur, it should be treated according to standard protocols. Treatment with lenalidomide should be interrupted until stable anticoagulation is achieved and then carefully reintroduced (Table\u00a05). Once VTE has occurred during lenalidomide therapy, patients must remain on anticoagulation therapy as lenalidomide treatment continues.\nManagement of non-hematological adverse events\nUnlike thalidomide, lenalidomide does not lead to dose-dependent peripheral neuropathy or somnolence [18], but other non-hematological adverse events (NHAEs) may occur. The most common NHAEs (all grades) associated with lenalidomide therapy are diarrhea (49%), pruritus (42%), rash (36%), and fatigue (31%) [10]. The most commonly reported grade \u22653 NHAEs were rash (6%), fatigue (3%), diarrhea (3%), and pruritus (3%) [8]. In general, should NHAEs arise during lenalidomide therapy, alternative causes should be ruled out and symptomatic treatment initiated (Table\u00a06). Dry skin and pruritus occur regularly, and itching of the scalp is a characteristic adverse event during the first few weeks of therapy. These NHAEs are usually self-limiting and seldom need treatment. Unselective antihistamines (e.g., clemastine) are usually helpful. In severe cases, a short course of systemic corticosteroids (10mg of prednisone or equivalent), or local steroid application may be helpful. Rash often resolves spontaneously with time [19], and the panel do not recommend discontinuation of lenalidomide treatment should rash arise; however, in patients with severe or persistent rash, the temporary interruption of lenalidomide may be necessary (Table\u00a06).\nTable\u00a06Recommendations for management of non-hematological adverse events (NHAEs)NHAERecommendation(s)RashUsually resolves within 2\u20133\u00a0weeks, no interruption of lenalidomide treatment neededIf required treat with unselective antihistamines (e.g. clemastine), topical steroids, or a short course (14\u00a0days) of oral 10\u00a0mg prednisoneIf rash is severe or persists after treatment, lenalidomide should be interrupted until rash resolves. In the experience of the panel, lenalidomide can be restarted thereafter without recurrence of rashDiarrheaTreat symptomatically after ruling out other underlying causesHypothyroidismIn case of hypothyroidism, thyroid replacement therapy must be initiatedOther NHAEsTreat symptomatically after ruling out other underlying causes such as anemia or autoimmune disorders\nDiarrhea is a frequent problem and may impact on a patient\u2019s quality of life. Patients with known lactose intolerance should add lactase to their diet, as lenalidomide capsules contain small amounts of lactose. Diverse symptomatic therapies have been reported to be effective in those patients, including, but not limited to, loperamide, pipaverium bromide, uzara root extract, and tinctura opii. Muscle cramps have been reported by a number of patients after a variable time of lenalidomide intake. Magnesium dietary supplements may be tried, but are usually of limited value. Quinine sulphate (up to 200mg thrice-daily) works well, however, physicians should check for other cytochrome-P450-interacting drugs (e.g., sotalol, terfenadine, astemizole, voriconazole, erythromycin, rifampicin, and cisapride) that might prolong QT time in these patients. Patients who experience fatigue will be likely to benefit from counselling [20], and in cases of severe fatigue, dose reduction may be needed. Hypothyroidism has been reported in approximately 7% of patients [10]. It is almost exclusively of autoimmune cause. Patients should be screened for hypothyroidism every other month during the course of treatment (Table\u00a03). If hypothyroidism is confirmed, hormone replacement therapy is indicated (Table\u00a06). Patients losing their response to lenalidomide during treatment may have developed either hypothyroidism or hypogonadism, and should be screened for both conditions.\nConclusions\nThe approval of lenalidomide for the treatment of MDS patients with low-risk or intermediate-1-risk disease with a deletion of chromosome 5q represents a significant step forward for this underserved group of patients. The key goal of the strategies presented here is to avoid unnecessary dose delays and reductions, and to maximize probability of response. Patient selection is straightforward, as efficacy does not appear to be influenced appreciably by relevant prognostic factors including age, prior erythropoietin therapy, sex, French\u2013American\u2013British type, or IPSS score. Treatment should be continued for a minimum of 4months to ensure treatment response. Neutropenia and thrombocytopenia are the most common adverse events during lenalidomide therapy but can be managed by the introduction of G-CSF and dose interruption. Other side-effects are generally manageable, and a range of simple (prophylactic) interventions are recommended to address the more common and more serious associated adverse events.","keyphrases":["lenalidomide","myelodysplastic syndromes","mds","treatment guidelines"],"prmu":["P","P","P","R"]}
{"id":"Environ_Manage-2-2-1705511","title":"Perceived Conflicts Between Pastoralism and Conservation of the Kiang Equus kiang in the Ladakh Trans-Himalaya, India\n","text":"An emerging conflict with Trans-Himalayan pastoral communities in Ladakh\u2019s Changthang Plateau threatens the conservation prospects of the kiang (Equus kiang) in India. It is locally believed that Changthang\u2019s rangelands are overstocked with kiang, resulting in forage competition with livestock. Here, we provide a review and preliminary data on the causes of this conflict. Erosion of people\u2019s tolerance of the kiang can be attributed to factors such as the loss of traditional pastures during an Indo-Chinese war fought in 1962, immigration of refugees from Tibet, doubling of the livestock population in about 20 years, and increasing commercialization of cashmere (pashmina) production. The perception of kiang overstocking appears misplaced, because our range-wide density estimate of 0.24 kiang km\u22122 (\u00b1 0.44, 95% CL) is comparable to kiang densities reported from Tibet. A catastrophic decline during the war and subsequent recovery of the kiang population apparently led to the overstocking perception in Ladakh. In the Hanle Valley, an important area for the kiang, its density was higher (0.56 km\u22122) although even here, we estimated the total forage consumed by kiang to be only 3\u20134% compared to 96\u201397% consumed by the large livestock population (78 km\u22122). Our analysis nevertheless suggests that at a localized scale, some herders do face serious forage competition from kiang in key areas such as moist sedge meadows, and thus management strategies also need to be devised at this scale. In-depth socioeconomic surveys are needed to understand the full extent of the conflicts, and herder-centered participatory resolution needs to be facilitated to ensure that a sustainable solution for livelihoods and kiang conservation is achieved.\nOne of the seven equid species in the world, the kiang Equus kiang, occurs in parts of China and India, with small populations also reported from Pakistan and Nepal. Unlike the Asian wild ass E. hemionus, of which the population has declined drastically over the last century, kiang continues to have a wide distribution with fairly large populations (Schaller 1998). Within India, Ladakh (approximately 75\u00b0 50\u2032 to 75\u00b0 80\u2032 E; 32\u00b0 30\u2032 N to 32\u00b0 37\u2032 N) remains a stronghold for the kiang (Fox and others 1991; Shah 1996), where local Buddhist communities have been fairly tolerant of the species, and large herds of kiang can be relatively easily seen in eastern Ladakh. Kiang is classed as a Least Concern species under the IUCN Red List category; however the Western Kiang, E. k. kiang, which occurs in Ladakh, is classed as \u201cdata deficient\u201d (Shah 2002).\nYet, with growing integration of the local economy of Ladakh with better developed cash markets, pastoral communities are fast losing their tolerance towards the kiang and it is increasingly seen as a competitor of livestock (Fox and others 1991). Ironically, its relatively large population and conspicuousness are becoming concerns for the continued conservation of the species. Although the species is not persecuted (except for driving them away from pastures by herders on horseback) in Ladakh largely due to the Buddhist beliefs of the local pastoral communities, this perceived conflict is currently one of the most serious issues being faced by the local district administration and the wildlife department, with vehement demands for compensation. Indeed, a decreasing tolerance for the species may lead in future to a worsening willingness to preserve it. There is a belief among the administration that Ladakh\u2019s rangelands are presently overstocked by kiang, and that they are degrading the pastures (Anon 2003). Over the last decade, as a measure to protect forage from kiang and make it available to livestock, the local government and some nongovernmental organizations have started fencing the most productive pastures, the sedge meadows near rivers and streams, thereby increasing forage availability for livestock and reducing the available habitat for the kiang (Richard 1999; Bhatnagar and Wangchuk 2001).\nThe purpose of this article is to document this intensifying conflict between pastoralism and conservation of the kiang in Ladakh. The kiang subspecies in question is categorized as \u201cdata-deficient\u201d by the IUCN (Moehlman 2002). We examine the primary ecological, socioeconomic, and political aspects of kiang\u2013human conflict, and summarize the results of our rangewide surveys of the kiang population in Ladakh. We also discuss the growing linkages between a traditional way of life and the global cashmere market, and their consequences for conservation of the kiang.\nMethods\nStudy Area\nAdministratively, the Ladakh region in the state of Jammu and Kashmir (India) is divided into two districts: the western Kargil district and the eastern Leh district. Leh district is a high-altitude cold desert spread over approximately 45,000 km2. This rain-shadow region is semiarid to arid, with winter temperatures dropping to below \u221225\u00b0C. Western and central Leh are mostly rugged, relatively lower in elevation, and inhabited by agropastoralists. The eastern part of the district, called Changthang, on the other hand, comprises high plateaus and rolling hills (usually >4000 m) interspersed with lake and river basins that have moist patches of relatively dense graminoid vegetation. Most of the remaining arid region is dominated by medium to sparse steppe vegetation. Changthang covers approximately 20,000 km2 and is inhabited by the nomadic Changpa community and Tibetan refugees, both of whom primarily rear goat and sheep. According to the human census in 2001 conducted by the government, the population of the Leh district was 117,637, with the majority (85%) being in the western region. The population is sparse in the Changthang (13,444) with a density of approximately 0.7 km\u22122. Ladakh is home to a diverse assemblage of wild carnivores, ungulates, and birds (Pfister 2004).\nWithin Ladakh, the kiang occurs in the eastern Changthang part of the Leh district. Our survey covered most of this region, from the Pangong Tso in the North, along the eastern border with Tibet, to Hanle in the south (Figure\u00a01). We divided the entire region into five blocks based on their location and overall similarity in terms of topography and vegetation, and surveyed each block (Table\u00a01). Our repeated counts of kiang were conducted in the Hanle Valley. This valley (79\u00b0 0\u2032 45\u2033 E, 32\u00b0 37\u2032 29\u2033 N; approximately 2800 km2) extends from the Zanskar mountains in the south to the Indus Valley in the north into which the Hanle River drains at Loma. The higher reaches are primarily rolling mountains and plateaus, while the mid and lower portion is a wide valley fringed by a high range exceeding 5000 m on the east, and a lower, more gradual ridge on the west.\nFig.\u00a01The five blocks surveyed for kiang in eastern Ladakh during 2000 to 2003. The estimated \u201cviewscape\u201d is based on modeling for areas visible within 3 km on either side of the road (see text for details). Distribution of kiang in Ladakh is also shown (based on Chundawat and Qureshi 1999). We also surveyed some additional adjacent areas with similar habitat.Table\u00a01Density of kiang in the surveyed blocks of eastern Ladakh, IndiaSurvey blockTransect length (km)Area (km2)No. KiangKiang density (km\u22122)Loma-Demchok (Indus)822882490.86Hanle Valley66347780.23Chushul-Loma63299190.06Parma Valley7436090.03Pongong Tso80359100.03Total36516533650.24 (95% CL \u00b1 0.44)\nData Collection and Analysis\nThrough archival research and field observations, we documented the intensifying human\u2013kiang conflict in Ladakh. We describe the perceptions of the Ladakhi administration regarding this conflict, and the efforts they have undertaken to address it. Through archival research, we also examine recent land-use changes in eastern Ladakh, describe the growing pashmina or cashmere industry, and its fallouts for the kiang\u2013human conflict. Literature was also surveyed for obtaining information on the past status and distribution of the kiang. We also obtained data on livestock populations from the Sheep Husbandry Departments in Nyoma and Leh, to estimate the livestock density in eastern Ladakh. We had informal conversations with over 50 herders to learn about their perceptions regarding conflicts.\nOur data on the kiang population in Ladakh come from rangewide surveys conducted in the year 2000. The study area was surveyed from a slow-moving vehicle (traveling at 10\u201312 km\/hour) and areas on either side were periodically scanned using an 8\u00d7 binocular and\/or 20\u00d7 spotting scope. On every sighting, animals were counted and classified, and habitat variables such as topographic feature, elevation, slope, aspect, dominant plant species, and approximate plant cover were recorded. Classification of kiang by sex was not possible in most instances due to the similarity between the sexes, especially when seen from a distance. A total distance of 365 km was covered in the Changthang, traveling on an average 60\u201370 km per day. We also covered approximately 50 km on foot in the upper catchment of the Hanle Valley. Subsequently, in the spring of 2001, 2003, and 2004, we conducted repeated vehicle counts of kiang along an approximately 100-km stretch in the Hanle Valley, which had emerged as an area with moderate to high kiang density during the 2000 survey.\nDensity for kiang and livestock was calculated by estimating the area covered in each segment using Geographical Information System (GIS) tools. The area surveyed along the roads was estimated by creating a \u201cviewscape\u201d in ArcInfo, which effectively created polygons of all area visible from the road. The base map used for this estimation was the National Imagery and Mapping Agency\u2019s Vector Map Level 0 (Digital Chart of the World) Edition: 5. This was integrated with the Digital Elevation Model (resolution 90 m or 3 arc seconds) Shuttle Radar Topography Mission (SRTM) data from the Space Shuttle mission (for the year 2000) obtained from ftp:\/\/e0mss21u.ecs.nasa.gov\/srtm\/ to generate maps of the study area with numerous layers of information that included elevation, aspect, and drainage. After creating the viewscape, we used a cutoff distance of 3 km on either side of the road, within which all visible areas were calculated. This was generally our maximum sighting distance for the kiang (most Rangefinders do not work at this distance). We estimated the extent of the surveyed areas (through viewscapes) for both the rangewide surveys as well as the region of our repeated kiang counts in Hanle Valley.\nAs a preliminary exercise to understand the extent of forage consumption by kiang vis-\u00e0-vis livestock, we estimated total daily forage consumption by kiang and livestock through established relationships between body mass and daily food intake (as percent of body mass) among herbivores (Foose 1982). In the absence of population structure data for both kiang and livestock, we used adult body masses (averaged between sexes) from Mishra and others (2002). We used the established relationships for foregut fermenters (goat, sheep, cattle, yak, cattle-yak hybrids) and hindgut fermenters (kiang, horses, donkeys), as appropriate. Forage quality in the highly seasonal cold desert steppe of Ladakh is expected to vary strongly between seasons, with high-protein, low-fiber forage being available in summer, and low-protein, high-fiber forage in winter. We therefore estimated forage consumption by each species separately for winter and summer, using the relationships for low-protein, high-fiber grass hay diet for winter (4.5\u20137.4% crude protein and 65\u201370% cell wall) and high-protein, low-fiber legume hay diet for summer (17.4\u201322.2% crude protein and 31\u201356% cell wall) (Foose 1982). For hindgut fermenters, daily forage intake as a percent of body mass (W) in summer was estimated as 13.8W\u22120.315, and 6.95W\u22120.236 in winter. Similarly, for the ruminants (goat, sheep, cow, dzo\/dzomo, yak), it was estimated as 7.31W\u22120.231 in summer and 4.04 W\u22120.184 in winter (Foose 1982).\nResults and Discussion\nGenesis of the Conflict: Kiang Population Changes in Recent History\nKiang inhabits the open plains and rolling mountains of the Changthang plateau in eastern Ladakh at altitudes between 4000 m and 5500 m. It has a total estimated range of approximately 7400 km2 in Ladakh (Chundawat and Qureshi 1999; Figure\u00a01). The species was common in Ladakh during the earlier part of the 20th century (Stockley 1936), but is believed to have declined substantially with the buildup of armed forces in Ladakh during the war between India and China in 1962 (Fox and others 1991). There are no population estimates available for the period before or immediately following this war, but it is believed that the population had recovered by the 1980s. The only published estimate of their population comes from the late 1980s when approximately 1500 animals were estimated to occur in Ladakh (Fox and others 1991; Shah 2002).\nIt thus appears that for at least a decade after the war, the kiang population remained relatively small in Ladakh. The genesis of the present conflict can be traced back to the early 1980s, by which time their population had apparently recovered (Fox and others 1994). The prevalent belief in Ladakh during that time, and one that continues until today, is that the kiang population has drastically increased in eastern Ladakh because many animals have moved from Tibet to the Indian side of the border to escape persecution (Fox and others 1991; Anon. 2003). Even in the 1980s, there were complaints by herders regarding the extent of forage consumption by kiang and consequent competition with livestock, and demands that the government should \u201cdrive the kiang back into Tibet\u201d (Fox and others 1991).\nIntensification of the Conflict: Changing Face of Pastoralism in Ladakh\nThe Tibetan plateau was inhabited by nomadic hunting people 30,000 years ago, and nomadic pastoralists have been herding livestock on the plateau for at least two millennia (Schaller 1998). The nomads, who became Buddhists presumably in the last millennium, live in tents (ribos) in seasonal camps. In the past, the few nomadic groups inhabiting eastern Ladakh belonging to the Changpa community traveled distances exceeding 100 km during their seasonal migrations (Phuntsok 2000; Hagalia 2004). This has recently changed, and most seasonal movements are now restricted to within 40\u201350 km (Hagalia 2004).\nAfter the 1960s, there has been a steady increase in the population of the Changpa and their livestock, presumably facilitated by access to better health care, provisions, and subsidies, both from the government as well as the army (Chaudhuri 2000; Phuntsok 2001; Bhatnagar and Wangchuk 2001). Supplemental forage provided by the government in the event of severe winters now offsets most of the starvation-related livestock mortality, which in the past was perhaps an important factor limiting the livestock population. The 1962 war between India and China also led to the curtailment of movement of the Changpa herders from eastern Ladakh into parts of Tibet, considerably reducing the availability of grazing land. Many of their pastures such as Skagzung, the primary winter pasture of the Rupshu Changpas (Figure\u00a01), became unavailable, effectively escalating the stocking density in eastern Ladakh. Furthermore, a large number of Tibetan refugees have moved into eastern Ladakh with their livestock herds since the late 1950s, thus contributing to a substantial increase in the number of herding families and the livestock population (Bhatnagar and Wangchuk, 2001; Hagalia 2004). As per the government records, the livestock population in Ladakh (including Changthang) has doubled in about two decades, from approximately 212,500 in 1977 to 487,000 in 1999 (Richard 1999; Bhatnagar and Wangchuk 2001). In 1999, Changthang constituted 44% of Ladakh\u2019s livestock population, most of which (94%) were sheep and goats.\nOne of the most important livestock products from the region is the pashmina or cashmere, a fine luxury fiber derived from the underwool of the local Changra goat. Traditionally, more than 95% of cashmere from Ladakh has been used for barter with or sale to traders from the neighboring Kashmir region under a treaty with the erstwhile kingdom of Kashmir dating from 1684 (Rizvi 1999). Over the last two decades, however, cashmere production in Ladakh has been promoted by the government in a substantial way. This has included setting up of goat breeding farms, efforts at improving yield, and provisioning of veterinary services to nomads that has brought down the kid mortality rates to 2\u20133% from an estimated 30\u201340% (Jina 1995). However, the efforts at increasing the per animal cashmere yield have apparently had very limited success (Jina 1995), suggesting that increasing cashmere production necessitates an increase in the goat population. More recently, with part support from the central government of India, the International Fund for Agricultural Development and the UNDP, the Ladakh administration has set up a cashmere de-hairing plant, expected to result in considerable added value to the cashmere from Ladakh (Joshi and Morup 2003; Mital 2004). Presently, Ladakh contributes merely 30,000 kg (0.37%) to the global cashmere production and the administration is eager to increase Ladakh\u2019s contribution in global trade.\nThe recent intensification of human\u2013kiang conflict needs to be viewed against these socioeconomic and political developments. The human and livestock populations in kiang habitat have significantly increased in the last four decades due to factors ranging from better healthcare to the influx of refugees. Furthermore, promotion of production and value of cashmere has meant that livestock rearing is fast becoming a cash-based enterprise that increasingly caters directly to the lucrative global cashmere market. Herders are realizing the importance of every bit of pastureland in maximizing cashmere production and see kiang as a direct threat to pasture production. Against this background, it is perhaps not surprising that people are fast losing their tolerance of the kiang.\nThe Ecological Context: Current Kiang Population and Relationships with Livestock\nIn our rangewide survey of the kiang, we surveyed an estimated area of 1653 km2, which is 22% of the total range of the kiang in Ladakh (7400 km2; Chundawat and Qureshi 1999). We recorded a total of 181 distinct kiang groups totaling 365 animals. The average density in the surveyed blocks was 0.24 (95% CL \u00b1 0.44) kiang km\u22122, with the highest abundance along the upper Indus from Loma to Demchok followed by the Hanle Valley (Table\u00a01, Figure\u00a01). With an overall livestock population of 210,000 (as per unpublished records from the Nyoma office of the Sheep Husbandry Department (2004)) in the Nyoma and Durbuk blocks that comprise the Changthang region of Ladakh, the livestock density in the region was 11 km\u22122. Thus, in terms of density, kiang represents only 2% of the total herbivore population (kiang and livestock together) in the Changthang, with the bulk being contributed by goats (57%).\nThe group sizes of kiang observed during the rangewide survey varied from 1 to 74. The mean group size was 2.8 (95% CL \u00b1 0.8), with most of the sightings (55%) being of solitary animals. Most kiang were seen in open wide valleys (79%, n = 202), followed by stony slopes (19%) and very rarely in broken areas (2%). There were no sightings on slopes steeper than 30\u00b0. We observed kiang between elevations of 4100 and 5500 m, with most sightings between 4200 and 4300 m. Most kiang groups (73%) were seen on sedge (Carex spp. and Kobresia spp.) and grass (Stipa spp.) meadows in the valley bottom, which were occasionally interspersed with bushes of Caragana sp. These meadows appear to be critical habitat for kiang during summer and winter as also reported by Schaller (1998), but are limited in extent to narrow discontinuous strips in moist areas along valleys. The remaining sightings were in areas that had sparse to dense Artemisia steppe (25%; with Tanacitum sp. and Sedum sp. being the other dominant plants) and high altitude forb meadows (2%; Saussurea spp., Salsola sp., and Polygonum sp. as the dominant plants).\nAt a landscape level covering entire eastern Ladakh, of the total daily forage consumption by large herbivores, we estimated that kiang consume 10\u201311% and livestock 89\u201390% (Table\u00a02). Among livestock, goats were estimated to consume the maximum forage (41% to 43% of the total forage consumed), followed by sheep (26% to 27%).\nTable\u00a02Herbivore density and estimated forage consumption by kiang vis-\u00e0-vis livestock in ChangthangSpeciesBody weight(kg)Density(animals km\u22122)Total forage consumption in summer (kg km\u22122)Percent forage consumption in summerTotal forage consumption in winter (kg km\u22122)Percent forage consumption in winterKiang2750.241.6101.211Livestock\u00a0\u00a0Donkey900.040.110.11\u00a0\u00a0Horse2480.150.950.76\u00a0\u00a0Cattle1910.160.740.54\u00a0\u00a0Yak2980.261.591.110\u00a0\u00a0Yak cattle hybrids2270.010.03<10.02<1\u00a0\u00a0Sheep343.984.4272.926\u00a0\u00a0Goat336.446.9434.541Livestock total112111.0314.5909.789Total11.2716.0810.96Body weights are taken from Mishra (2001) and have been averaged between sexes. Note that this will result in an overestimate of the offtake by kiang because we assumed that all kiang were adult. Livestock density was calculated only for adult animals.\nOur subsequent estimate of the kiang population in the Hanle Valley (the region between Rongo and Kalangtartar) based on repeated counts between 2001 and 2004 yielded a density of 0.56 kiang\/km2 (95% CL \u00b1 0.08). We estimated that on an average, kiang remove only 3% to 4% of the total forage consumed in the Hanle Valley, with the greater part being consumed by goats (45% to 47%) followed by yaks (20% to 21%) and sheep (20%) (Table\u00a03).\nTable\u00a03Herbivore density and estimated forage consumption by kiang vis-\u00e0-vis livestock in the Hanle ValleySpeciesBody weight (kg)Density(animals km\u22122)Total forage consumption in summer (kg km\u22122)Percent forage consumption in summerTotal forage consumption in winter (kg km\u22122)Percent forage consumption in winterKiang2750.563.632.84Livestock\u00a0\u00a0Donkey9000000\u00a0\u00a0Horse2481.579.587.49\u00a0\u00a0Cattle1910.662.721.92\u00a0\u00a0Yak2983.9122.82016.521\u00a0\u00a0Yak cattle hybrids22700000\u00a0\u00a0Sheep3421.5223.72015.519\u00a0\u00a0Goat3350.6954.54735.545Livestock total112178.34113.29776.796Total78.9032.479.6\nSynthesis of the Conflict: Is It Real or Perceived?\nMost wars in the last five decades have resulted in detrimental effects on wildlife (Blom 2000; Dudley and others 2002; Mishra and Fitzherbert 2004), and the war between India and China in 1962 was no exception, particularly from the viewpoint of kiang conservation. Our review suggests that the kiang population in Ladakh declined drastically in the years during and after the war between India and China in the 1960s, possibly due to hunting, land mines, and disturbances associated with the war. Thus, for two decades during and after the war, a generation of herders presumably saw and became used to a very low density of kiang in Ladakh\u2019s rangelands. Not surprisingly, the genesis of kiang\u2013human conflict in Ladakh can be traced back to the 1980s, the period by which the kiang population had recovered. It seems that the recovery of the kiang from a very low density to the current estimated density of 0.24\/km2 has led to the perception to a generation of nomads that Ladakh\u2019s rangelands are now overstocked with kiang. Available estimates from three regions in Tibet place the density of kiang between 0.15 and 0.39 kiang km\u22122 (Schaller 1998), suggesting that the kiang density in Ladakh is comparable with those in Tibet. Furthermore, our results show that the share of forage consumed by the kiang vis-\u00e0-vis livestock is relatively small (10\u201311% in the whole of eastern Ladakh, and 3\u20134% in Hanle Valley). Thus, at a landscape level, the perception of the overabundance of kiang in Ladakh, and of kiang compromising cashmere production, is clearly unfounded.\nNevertheless, there is variation in kiang density locally (0.03 to 0.86 kiang km\u22122 recorded in our surveys). Kiang occasionally congregate in large herds and can together remove considerable forage from the low-productivity Trans-Himalayan pastures. Using the data in Table\u00a02, a group of 70 kiang (the largest group seen during the survey was 74) can be estimated to potentially consume 199\u2013254 kg dry forage in a single day. The peak standing biomass in sedge meadows, which seem to be important kiang habitats, has been estimated in the adjoining region of Spiti at 857 kg ha\u22121 (Mishra 2001). These habitats seem critical for both kiang and livestock, but cover a small fraction of the landscape, with an estimated 150 km2 in the entire approximately 20,000 km2 of Changthang. Illustratively, this means that in a single day a large group of 70 kiang can potentially consume more than a quarter of the standing biomass from 1 ha of a key resource area. This consumption is also very similar to the estimated daily forage consumption by a herd of 200 goats (approximately 215 kg). Because of the long winters (November to April) and short summers (July\u2013August), the potential for regrowth during summer is low, aggravating the impact. Considering the potential of large kiang herds to remove significant amounts of forage, together with the fact that grazing areas have been reduced and the livestock population in Ladakh more than doubled in the last 30 years, it is not difficult to see that at a localized scale of the valued sedge meadows, some herders must be facing serious forage competition from the kiang. Given the intensifying commercialization of cashmere production in Ladakh and its integration with global markets, this localized forage competition is, perhaps understandably, no longer tolerated, and the conflict needs to be addressed immediately. However, it must be borne in mind that the problem seems to be evident at local scales, and will need to be addressed at that scale.\nIn the absence of any scientific understanding of the conflict so far, or efforts to document it, this local conflict has already begun to take on a serious political dimension. The politicians and the district administration in Ladakh are blaming the kiang for compromising cashmere production, and the wildlife department is being looked upon to provide a solution. The only on-ground reaction of the district administration has been to fence large productive sedge and grass meadows from the kiang. In the Hanle Valley alone, an estimated 120 ha of meadows have been fenced already. Three fourths of our total kiang sightings during the rangewide surveys were in such meadows, suggesting that this is an important habitat for the species, and continued fencing of this habitat may cause a decline in the kiang population of Ladakh.\nHow can this conflict be managed? Although our review of the recent history of political and socioeconomic developments in kiang habitat and our estimates of density and forage consumption of kiang and livestock bring to light interesting dimensions of the kiang\u2013human conflict, the current level of understanding of the kiang\u2019s ecology and its relationships with livestock is still very preliminary. Intensive research needs to be promoted to assist in better management of the species, and its conflicts with humans. Monitoring programs need to assess kiang and livestock density, as well as the land use changes in kiang habitat. Our article shows that the problem of kiang\u2013human conflict is locally concentrated in the key resource areas that contain the sedge meadows, and management strategies need to be devised at that local scale. Surveys are needed to identify all high-conflict areas, and adequate compensatory mechanisms need to be worked out for herders who face genuine forage competition from the kiang. It needs to be examined whether the fact that herders share the forage with kiang could actually be used for adding value to the cashmere produced in Ladakh. The kiang is a striking and conspicuous wild animal that may provide a potential for ecotourism. Finally, after taking up the above interventions, it is important that fences on the sedge meadows to exclude kiang be removed and the practice of fencing discontinued because they lock out key resource areas from the kiangs\u2019 range. Without access to key resource areas, kiang and other wild herbivores, such as the Tibetan gazelle, Procapra picticaudata, may face severe population declines in Ladakh.","keyphrases":["conflicts","pastoralism","equus kiang","ladakh","livestock","nomads"],"prmu":["P","P","P","P","P","P"]}
{"id":"Semin_Immunopathol-3-1-2071951","title":"Complement in glomerular injury\n","text":"In recent years, research into the role of complement in the immunopathogenesis of renal disease has broadened our understanding of the fragile balance between the protective and harmful functions of the complement system. Interventions into the complement system in various models of immune-mediated renal disease have resulted in both favourable and unfavourable effects and will allow us to precisely define the level of the complement cascade at which a therapeutic intervention will result in an optimal effect. The discovery of mutations of complement regulatory molecules has established a role of complement in the haemolytic uremic syndrome and membranoproliferative glomerulonephritis, and genotyping for mutations of the complement system are already leaving the research laboratory and have entered clinical practice. These clinical discoveries have resulted in the creation of relevant animal models which may provide crucial information for the development of highly specific therapeutic agents. Research into the role of complement in proteinuria has helped to understand pathways of inflammation which ultimately lead to renal failure irrespective of the underlying renal disease and is of major importance for the majority of renal patients. Complement science is a highly exciting area of translational research and hopefully will result in meaningful therapeutic advances in the near future.\nIntroduction\nThe complement system involves approximately 30 plasma- and membrane-bound proteins (reviewed in [1, 2]). These proteins play an important role in anti-microbial defence and the clearance of immune complexes and apoptotic and necrotic cells. The role of complement is not restricted to the innate immune system but includes important functions in the regulation of the adaptive immune response. Although complement undoubtedly contributes to tissue damage in numerous forms of glomerulonephritis, we will also see that complement protects against immune-mediated tissue damage in a number of settings.\nIn the following, we will first give an introduction to the pathways of complement activation and their regulation. This will be followed by a review of glomerular renal diseases in which complement plays a prominent role. The role of complement in non-glomerular renal disease entities such as transplantation and ischaemia\/reperfusion damage falls outside the scope of this study and is reviewed in references [3] and [4].\nOverview of the complement pathways\nThe complement system consists of three different pathways that all converge in the activation of the central complement molecule C3 (Fig.\u00a01). Sufficient activation of C3 will then lead to the formation of the membrane attack complex.\nFig.\u00a01Schematic overview of the three pathways of complement activation\nThe first component in the activation of the classical pathway is C1. Binding of at least two bindings sites of C1q to antigen-bound IgG or IgM, acute phase proteins such as CRP or dead cells leads to conformational changes that result in the activation of the associated serine proteases C1r and C1s. Activated C1s cleaves C4 into C4a and C4b. C4b then covalently binds to nearby structures. The bound C4b then binds C2 whereupon the smaller C2b fragment is cleaved off, resulting in the formation of the C4bC2a complex, which is the classical pathway C3 convertase.\nThe lectin pathway is related to the classical pathway and uses the same C3 convertase, C4bC2a. The initiation molecules of the lectin pathway, mannose-binding lectin (MBL) and the ficolins, recognise carbohydrate ligands present on a wide range of microorganisms in a pattern-like fashion. The interaction of MBL with its ligand leads to the activation of the MBL-associated serine proteases (MASP-1, MASP-2 and MASP-3). MASP-2 then cleaves C4 and subsequently C2 leading to the formation of the C3 convertase which is identical to the classical route C3 convertase, C4bC2a.\nMBL consists of up to six trimeric subunits which are arranged in a structure similar to C1q. Frequently occurring single nucleotide polymorphisms within exon 1 of the MBL-2 gene and polymorphisms of the promoter region explain the up to 1,000-fold inter-individual variation in MBL plasma levels.\nThe activation of the alternative route of complement depends on spontaneous activation of the C3 molecule by hydrolysis of the internal thioester bond of C3. This results in the formation of the C3b-like molecule C3(H2O). Hydrolysed C3 then binds to factor B. This interaction renders factor B susceptible to cleavage by factor D resulting in the release of the Ba fragment and the formation of the C3 convertase C3(H2O)Bb. This initial convertase constantly cleaves C3 at a low rate generating C3b. This constant low rate generation of C3b is referred to as the \u201ctick over\u201d of the alternative pathway. The generated C3b can interact with factor B to form the more active alternative pathway C3 convertase C3bBb.\nThe majority of the generated C3b is rapidly inactivated by circulating factor I together with its co-factors, factor H (fH) and membrane co-factor protein (MCP). However, if C3b binds to an activator surface (e.g. a bacterial wall or damaged tissue), the molecule is protected against inactivation and further amplification of the alternative route will occur. Properdin is an important positive regulator of the alternative route. Binding of properdin leads to stabilisation of the labile C3 convertase and promotes the assembly of a complement-activating lattice by further binding factor B and C3b molecules [5\u20137].\nThe early activation steps of the classical, lectin and alternative route of complement activation converge in a common terminal pathway. The addition of a further C3b molecule to the C3 convertase complex leads to the formation of C3bBbC3b in the case of the alternative pathway and to the formation of C4bC2aC3b in the case of both the classical and lectin pathways. These C5 convertases then initiate the assembly of the membrane attack complex by cleavage of C5 to C5a and C5b. C5a can then function as a potent anaphylotoxin. The newly formed C5b forms a tri-molecular complex by binding C6 and C7. After inserting into a cell membrane, this complex binds C8 and multiple C9 molecules. This results in the completion of the pore-forming membrane attack complex (C5b-9). This complex can lead to cell lysis and, in the absence of complete lysis, to cell activation.\nRegulation of complement activation\nThe complement system consists of numerous regulatory molecules that protect the host from uncontrolled tissue destruction and activation by the complement system. Recently, defective complement regulation has been shown to play an important role in the pathogenesis of some forms of the haemolytic uremic syndrome (HUS) and membranoproliferative glomerulonephritis (MPGN). The role of complement in these diseases will be discussed in more detail below.\nC1-inhibitor is a powerful inhibitor of the classical pathway of complement activation. It binds to activated C1r and C1s and causes dissociation of these inactivated enzymes from C1q. Recent data shows that C1 inhibitor also inhibits the activation of the lectin pathway by inactivation of MASP-2 and, at higher concentrations, fluid phase activation of the alternative pathway [8].\nFactor I is a circulating serine protease that proteolytically degrades C3b and C4b in the presence of the co-factors fH and C4-binding protein (C4bp). Next to its function as a co-factor, fH also inhibits activation of the alternative pathway by binding to C3b and displacing Bb from the C3 convertase complex. Similarly, C4bp regulates activation of the classical and lectin pathway by displacing C2a from C4b. Both fH and C4bp promote the degradation of the C3 and C5 convertases of the respective pathways.\nCell-membrane-bound inhibitors of complement activation also contribute to the defence against inappropriate tissue damage by homologous complement. Decay-accelerating factor (CD55) exerts its effect early in the complement cascade by inhibiting the activation of C3 by preventing the formation and accelerating the decay of both the alternative and classical pathway C3 and C5 convertases. Membrane co-factor protein (MCP, CD46) serves as a co-factor for the cleavage of C3b and C4b by factor I. CD59 interacts with the final section of the complement activation pathway by inhibiting the formation of C5b-9.\nComplement receptor 1 (CD35, CR1) also functions as a complement regulator by accelerating the decay of the C3 convertases. A functionally intact soluble form of CR1 can be detected in plasma [9]. Recently, a new complement receptor, the human complement receptor of the immunoglobulin superfamily (CRIg), has been described [10]. CRIg is present on macrophages in both humans and mice and plays a role in pathogen clearance. It binds to C3b and selectively inhibits the C3 and C5 convertases of the alternative pathway. A recombinant soluble form of CRIg suppressed inflammation in two murine models of arthritis [11].\nImmune-complex-mediated glomerulonephritis\nImmune complex glomerulonephritis is a good example for the dual role of the complement system. Immune complexes can either be deposited in the glomerulus by passive deposition from the circulation or by in situ formation via binding of antibody to local antigens. Alternatively, local formation of immune complexes may occur when a circulation antigen is recognised by antibodies after deposition in the glomerulus (planted antigen). Subepithelial complement deposition as found in membranous nephropathy leads to a non-inflammatory complement-mediated damage because the anaphylotoxins produced during the local activation do not reach circulating leucocytes. Subendothelial deposition of complement factors is associated with a brisk inflammatory response because the produced anaphylotoxins easily come into contact with circulating cells. Subendothelial immune complex deposition is typical of proliferative lupus nephritis.\nVarious studies have underscored the role of complement in immune-complex-mediated glomerulonephritis. Complement depletion by treating rats with aggregated human IgG resulted in a marked decrease of neutrophil influx and renal damage in a nephrotoxic serum model of acute glomerulonephritis [12]. Later studies demonstrating a beneficial effect of complement depletion in the non-inflammatory Heymann nephritis model of membranous nephropathy showed that complement-mediated damage is not dependent on the influx of inflammatory cells [13].\nThe contribution of terminal pathway of complement-mediated injury was established in various glomerulonephritis models in C6-depleted or C6-deficient rats. Renal damage is ameliorated in both the anti-Thy-1 and the passive Heymann nephritis models in the absence of C6 [14, 15]. As a follow-up to these findings, soluble CR1 was successfully used to treat the disease in both models [16]. A recent paper pointed towards an important role of the alternative pathway of complement activation in mouse models of type I and type II cryoglobulinemia [17]. The glomerular influx of neutrophils was significantly less in mice deficient for C3, factor B and C5, whereas C1q deficiency had no protective effect, suggesting involvement of the alternative pathway or lectin pathway.\nIt is interesting to note that many of the complement-deficient models of renal disease show spontaneous or worsened renal disease. This is compatible with the observation that the complement system plays an important role in the clearance of immune complexes from the circulation and in the solubilisation of deposited immune complexes. Immune complexes are rapidly opsonised with C4b and C3b. These complement components mediate the binding of the immune complexes to CR1 on erythrocytes. The complexes are then stripped off the erythrocytes when they pass through the liver or spleen. Thus, CR1-mediated clearance plays an important role in the handling of immune complexes and in keeping soluble immune complexes away from the endothelial surface thereby preventing vascular injury.\nNumerous animal models of immune-complex-mediated renal disease demonstrate a protective role of complement. C3 deficiency did not protect mice from the formation of immune complexes and proteinuria in a planted antigen model of immune complex nephritis [18]. This can be explained by the role of Fc-receptor-mediated activation of either resident or infiltrating cells at the site of injury. In the complete absence of complement activation, deposition of immune complexes can still lead to renal damage via Fc-receptor-mediated cell activation [19, 20].\nAn interesting recent study demonstrated that factor-D-deficient mice spontaneously develop immune complex glomerulonephritis with mesangial deposition of IgM and C3 [21]. Apparently, amplification of C3 activation is necessary for the processing or dissociation of IgM-containing immune complexes in the kidney.\nThe important role of the complement system in immune complex clearance is underscored by the finding that humans with complement deficiency are prone to immune-complex-mediated disease. Systemic lupus erythematosus (SLE) is a highly relevant example for this dual role of the complement system as will be discussed in the next section.\nRole of complement in lupus nephritis\nThe degree of peripheral complement consumption and the heavy glomerular deposition of complement in SLE nephritis point towards an important role of complement in lupus nephritis. Various approaches at inhibiting complement activation have been successful in treating experimental lupus nephritis. Wang et al. prevented the development of glomerulonephritis with an anti-C5 antibody in lupus-prone NZB\/W mice. Both treatments with the soluble rodent complement inhibitor rCrry and transgenic expression of this CR1-like molecule limited renal damage in the MLR\/lpr mouse model of SLE [22, 23].\nHowever, in humans, deficiencies of the early complement proteins C1q, C2 and C4 are associated with an increased risk of developing SLE [24]. Similarly, mice with C1q or C4 deficiency develop an autoimmune disease which resembles SLE [25\u201327].\nNext to the loss of clearance of immune complexes, complement deficiency may also lead to auto-immune phenomena due to defective clearance of apoptotic cells. C1q binds to apoptotic cells [28] and C1q-deficient mice are impaired in their capacity to clear these cells [29]. Defective clearance of this rich source of auto-antigens may contribute to the emergence of auto-immunity in patients with SLE.\nTaken together, it seems that, in lupus, the early components of the classical pathway of complement activation are beneficial due to their role in the clearance of immune complexes and apoptotic cells. Probably, the damage caused by Fc-receptor-mediated mechanisms in the presence of an increased deposition of immune complexes overrides the benefit of complement inhibition in these models. However, the inhibition of complement activation downstream of C3 may be a promising therapeutic approach.\nLupus nephritis is strongly associated with the presence of anti-C1q antibodies. These antibodies are present in 30\u201340% of SLE patients [30] and correlate with active lupus nephritis with a sensitivity of 87 to 97% and a specificity of 92% [31, 32]. Antibodies against MBL are also present in SLE, but no association with disease activity was detected [33]. The strong association of anti-C1q antibodies with active lupus nephritis suggests a pathogenic role of these antibodies. To study this question, our group has generated homologous mouse anti-mouse C1q antibodies. The administration of these antibodies to healthy mice resulted in deposition of C1q in the glomeruli together with an influx of granulocytes. However, this was not accompanied by a reduction of renal function or significant proteinuria [34]. When mice were pre-treated with a sub-nephritogenic dose of rabbit anti-C1q antibodies, the subsequent administration of mouse anti-C1q antibodies resulted in an increased deposition of immunoglobulin. The use of mice deficient for C3, C4 or for all three Fc gamma receptors showed that both complement and Fc-gamma-receptor-mediated damage is involved in this model.\nIgA nephropathy\nMesangial IgA deposition is the hallmark of IgA nephropathy. Early studies on complement deposition in IgA nephropathy reported co-deposition of C3 and properdin [35]. Because C1q and C4 are usually not detected in kidneys with IgA nephropathy, this complement deposition was thought to be caused by alternative pathway activation. Mesangial deposition of C5b-9 is also present and underscores the possible pathogenic importance of complement activation in this setting [36]. More recent studies suggest a role of the lectin pathway of complement activation in IgA nephropathy. Co-deposition of IgA and MBL has been described by several authors in both IgA nephropathy [37\u201339] and the related entity Henoch Sch\u00f6nlein purpura [40]. Hisano et al. found a relation between the presence of MBL deposition and the severity of the disease [41].\nThe discovery of MBL-binding properties of IgA has provided a mechanistic explanation for the link between IgA and MBL deposition [42]. MBL binds to polymeric IgA in a calcium-dependent fashion via its lectin domain, suggesting an interaction with carbohydrates exposed on the IgA molecule. The binding of MBL to IgA results in complement deposition and offers an explanation for the complement deposition found in IgA nephropathy.\nA recent publication underscored the potential clinical importance of MBL deposition in IgA nephropathy [43]. About 75% of the 60 biopsies in this study were negative for MBL and C4d, indicating that the C3 and C5b-9 deposition in these patients is most probably caused by the alternative pathway. The 25% of biopsies in which glomerular MBL deposition was found were positive for C4d in the absence of Clq, indicating complement activation via the lectin pathway. MBL deposition in the glomerulus was associated with both clinical and histological markers of more severe renal damage including more severe proteinuria, renal failure, extracapillary proliferation, glomerular sclerosis and interstitial fibrosis. The MASP-associated lectin, L-ficolin, was also present in these biopsies and may contribute to the activation of the lectin pathway next to MBL.\nMembranoproliferative glomerulonephritis\nComplement research has greatly contributed to the understanding of the pathophysiology of type II membranoproliferative glomerulonephritis (dense deposit disease) [44]. MPGN type II is characterised by deposits within the glomerular basement membrane together with staining for C3 along the glomerular basement membrane. In contrast to MPGN type I, deposition of immunoglobulins is usually not detected. The glomerular complement deposition is usually accompanied by decreased circulating C3 levels and alternative route activity. More than 80% of patients with MPGN type II are positive for serum C3-nephritic factor (C3NeF) [45].\nC3NeF is an antibody directed against the alternative pathway C3 convertase. The binding of C3NeF to C3bBb prolongs the half-life of the C3 convertase by slowing down the dissociation of Bb form C3b [46]. One of the mechanisms by which C3NeF increases the half-life of C3bBb is by inhibition of fH-mediated inactivation of the convertase [47]. Consistent with complement activation by stabilisation of alternative pathway convertase activity, serum complement profiles of patients with MPGN II show predominant depletion of C3 with no consumption of C1q and C4. The glomeruli of affected kidneys show marked deposition of C3 along the glomerular capillary walls without deposition of C1q, C4 or immunoglobulins. MPGN II is also associated with acquired partial lipodystrophy. This entity is also associated with the presence of C3NeF and marked C3 depletion. In vitro data shows that C3NeF is capable of inducing alternative-pathway-mediated damage on adipocytes [48]\nThe role of a deregulated alternative pathway in MPGN type II was highlighted by the discovery of FH mutations in both humans [49] and pigs with MPGN type II. Mice with a targeted deletion of FH have significantly reduced levels of C3 and consistently develop MPGN with deposition of C3 in the capillary walls [50]. If fH-deficient mice are also deficient for factor B, they cannot activate the alternative pathway and no renal disease develops.\nA chronic serum sickness model of immune complex disease demonstrated increased deposition if IgG immune complexes with increased C3 deposition in fH-deficient mice compared to wild-type mice. The fH-deficient mice developed diffuse proliferative glomerulonephritis, while the wild-type mice were protected against glomerular pathology. These findings indicate a role of fH in processing immune complexes and protecting the glomerulus against immune-complex-mediated disease.\nPatients with MPGN type II develop ocular lesions which are similar to the drusen that are found in patients with age-related macular degeneration (AMD). The finding of a close association of AMD with factor H mutations suggests that complement is also involved in the pathogenesis of this visually disabling disease [51\u201354].\nA recent study into the role of the terminal complement pathway in MPGN has pointed towards the potential of therapeutic inhibition of the complement system in MPGN [55]. Mice deficient for both fH and C5 developed less severe glomerulonephritis with better renal function, lower mortality and reduced glomerular cellularity in comparison to fH-deficient mice with normal C5. It is interesting to note that C5-deficient mice were not protected against proteinuria, suggesting that the glomerular C3 activation is sufficient to disrupt the glomerular permselectivity. The induction of heterologous nephrotoxic serum nephritis in fH-deficient mice resulted in markedly increased renal damage when compared to wild-type mice. The renal damage was clearly reduced in fH-deficient mice lacking C5 whereas C6-deficient mice were not protected. These findings demonstrated that the complement-mediated renal damage depended on the formation of the anaphylotoxin C5a but not on the formation of C5b-9. The role of C5 was supported by the reduction of proteinuria and glomerular neutrophil accumulation after treatment with an antibody against C5.\nThese observations suggest that anti-C5 treatment could serve as a treatment option in MPGN type II.\nComplement and the atypical haemolytic uremic syndrome\nThe haemolytic uremic syndrome is characterised by microangiopathic haemolytic anemia, consumptive thrombocytopaenia and the formation of microvascular thrombi. The vascular damage is particularly severe in the kidney and can lead to acute renal failure. Most cases of HUS are associated with diarrhoea and are caused by the verotoxin-producing Escherichia coli strain O157:H7. The less common form that is not associated with diarrhoea is referred to as atypical HUS (aHUS). Especially in children, the outcome and prognosis of diarrhoea-associated HUS is good, whereas atypical HUS is associated with substantial chronic renal failure and mortality.\nThe familial occurrence of aHUS [56] and the occasional finding of complement consumption [57] and deposition [58] in familial aHUS suggested a hereditary defect in alternative pathway activation or control. Importantly low levels of C3 persisted in patients with familial HUS after remission of the disease and low C3 levels were also detected in unaffected relatives. The discovery of fH mutations in families with aHUS confirmed this hypothesis [59, 60]. Until now, more than more than 100 fH mutations have been described [61]. These can be searched in an interactive HUS database (http:\/\/www.FH-HUS.org). The fH mutation frequency is 38% in familial forms of aHUS and 20% in sporadic forms [62]. In contrast to the fH mutations in patients with MPGN type II, patients with aHUS are usually heterozygous for the fH mutation. The penetrance is around 60%. Patients usually have normal levels of circulating fH protein, but reduced C3 levels are found in about 50% of the aHUS cases with a fH mutation [62]. Also in contrast with the mutations associated with MPGN, the fH mutations in patients with aHUS are located in the C-terminal region which is important for binding to cellular surfaces [63\u201365] via an interaction with C3b deposited in the surface of these cells [66]. Because mutated fH cannot bind to surface-bound C3b, circulating factor B can associate with the C3b and C3 convertase is formed, leading to unopposed complement activation on the endothelium.\nThe creation of a transgenic mouse that lacks the exons encoding for the C-terminal region of fH that is responsible for the binding to cellular surfaces has resulted in an aHUS model that is highly similar to the human disease [67]. These mice have a preserved capacity to regulate fluid phase complement activation and did not develop glomerulonephritis. The absence of systemic complement depletion in the presence of defective endothelial protection against complement attack led to a typical picture of HUS including the formation of glomerular microthrombi, fragmentocytes in peripheral blood and thrombocytopenia.\nNext to fH mutations, other mutations in complement regulatory proteins have been discovered in patients with aHUS. MCP mutations are found in approximately 14% of the patients with aHUS [62] and until now 43 mutations have been reported [61]. The course of the disease is milder in patients with MCP mutations, and plasma therapy does not seem to contribute to the outcome.\nFactor I mutations are quite rare and are found in about 4.5% of the patients with aHUS. More recently, two factor B mutations have been discovered [68]. One of these mutations increases the affinity of factor B for C3b, while the other increases the half-life of C3bBb. Both mutations result in an increased activity of the alternative pathway.\nTaken together, the clinical and experimental findings clearly point towards an important role of complement regulation in the pathogenesis of aHUS. However, until now, mutations of complement-regulatory proteins are only found in about 50% of the affected patients and family members of affected patients can share the mutations without manifesting aHUS. It seems that both additional predisposing factors and triggering circumstances, e.g. infections, are necessary to initiate the full-blown microangiopathy of aHUS.\nNevertheless, it is clinically useful to screen patients with aHUS for the known mutations because the findings may influence the prognosis and therapeutic decisions. Although evidence is lacking, patients with fH deficiency are usually treated with plasma therapy [69]. On the other hand, MCP is a membrane-bound protein and there is no rationale for plasma substitution in patients with MCP mutations and aHUS. As mentioned above, retrospective data do not indicate that plasma therapy results in improved outcomes in these patients [62]. However, patients with MCP mutations do well after kidney transplantation [70] as functional MCP is present on the endothelium of the transplanted kidney whereas patients with fH mutations have a very high rate of disease recurrence and graft loss after kidney transplantation [71]. Preemptive plasma therapy may be an option in these patients, but data are lacking to support this approach. Others have attempted combined liver and kidney transplantation in children with fH-associated HUS, but liver transplantation is associated with greatly increased risks when compared with kidney transplantation alone, and this procedure has not been uniformly successful until now [72\u201374].\nComplement and progressive renal damage\nIndependent of the underlying renal disease, proteinuria is associated with tubulointerstitial fibrosis and a progressive loss of renal function [75\u201377]. A number of mechanisms by which proteinuria may cause renal function loss have been suggested. These include oxidative damage induced by transferrin, lysosomal rupture caused by overload of the resorptive capacity for urinary proteins and pro-inflammatory effects of albumin-bound free fatty acids [78\u201381].\nFor many years, complement deposition along the brush border has been noted in proteinuric renal disease [82]. C5b-9 is found in urine from patients with various proteinuric renal diseases including diabetic nephropathy [83]. The C5b-9 in the urine is very probably generated within the tubulular lumen due to an intrinsic complement-activating property of the tubular cells [84, 85]. The exact mechanism of tubular complement activation is not understood, but tubular ammonia production [86] and a low expression of complement regulatory proteins on the apical cell surface [87] are thought to contribute.\nThe insertion of sublytic amounts of C5b-9 in the cell membrane of tubular cells leads to the production of pro-inflammatory cytokines [88, 89] and collagen. In vivo evidence for the role of complement in proteinuria-mediated renal damage has been derived from studies in C6-deficient rats. Rats with an inherited C6 deficiency were protected against tubulointerstitial damage in both the remnant kidney model [90] and the puromycin model of proteinuric renal disease [91]. A therapeutic intervention with either the murine complement inhibitor Crry or CD59 targeted to the renal tubulus resulted in improved renal function and less interstitial damage when compared with untreated animals [92].\nConclusions\nIncreasing knowledge about the complement system has taught us about both the protective and harmful roles of complement in renal disease. In the course of this review, it has repeatedly become clear that complement inhibition early on in both the classical and alternative pathways is associated with the risk of increased deposition of immune complexes and the resulting damage may outweigh the benefit. On the other hand, it seems that complement inhibition distal of the formation of the C3 convertases is safe and offers more promising therapeutic options for renal diseases for which no satisfying treatment has been established until now.\nIndependently of these promising therapeutic prospects, complement has become an invaluable tool in the diagnosis and monitoring of renal disease and results of complement studies have a strong impact on day-to-day decision making in the care of our patients with renal disease.","keyphrases":["complement","glomerulonephritis","immune complexes","hemolytic uremic syndrome"],"prmu":["P","P","P","M"]}
{"id":"J_Chem_Ecol-3-1-2039843","title":"Flower vs. Leaf Feeding by Pieris brassicae: Glucosinolate-Rich Flower Tissues are Preferred and Sustain Higher Growth Rate\n","text":"Interactions between butterflies and caterpillars in the genus Pieris and plants in the family Brassicaceae are among the best explored in the field of insect\u2013plant biology. However, we report here for the first time that Pieris brassicae, commonly assumed to be a typical folivore, actually prefers to feed on flowers of three Brassica nigra genotypes rather than on their leaves. First- and second-instar caterpillars were observed to feed primarily on leaves, whereas late second and early third instars migrated via the small leaves of the flower branches to the flower buds and flowers. Once flower feeding began, no further leaf feeding was observed. We investigated growth rates of caterpillars having access exclusively to either leaves of flowering plants or flowers. In addition, we analyzed glucosinolate concentrations in leaves and flowers. Late-second- and early-third-instar P. brassicae caterpillars moved upward into the inflorescences of B. nigra and fed on buds and flowers until the end of the final (fifth) instar, after which they entered into the wandering stage, leaving the plant in search of a pupation site. Flower feeding sustained a significantly higher growth rate than leaf feeding. Flowers contained levels of glucosinolates up to five times higher than those of leaves. Five glucosinolates were identified: the aliphatic sinigrin, the aromatic phenyethylglucosinolate, and three indole glucosinolates: glucobrassicin, 4-methoxyglucobrassicin, and 4-hydroxyglucobrassicin. Tissue type and genotype were the most important factors affecting levels of identified glucosinolates. Sinigrin was by far the most abundant compound in all three genotypes. Sinigrin, 4-hydroxyglucobrassicin, and phenylethylglucosinolate were present at significantly higher levels in flowers than in leaves. In response to caterpillar feeding, sinigrin levels in both leaves and flowers were significantly higher than in undamaged plants, whereas 4-hydroxyglucobrassicin leaf levels were lower. Our results show that feeding on flower tissues, containing higher concentrations of glucosinolates, provides P. brassicae with a nutritional benefit in terms of higher growth rate. This preference appears to be in contrast to published negative effects of volatile glucosinolate breakdown products on the closely related Pieris rapae.\nIntroduction\nMost plants are not simply homogeneous resources to herbivorous insects but produce discrete organs with different functions. In addition to the vegetative leaf tissues, plants produce reproductive organs (flowers, seeds) that may exhibit significant differences in levels of both nutrients and defensive secondary metabolites. Specialized guilds of herbivores are known to exploit specific plant structures (Schoonhoven et al. 2005). Flowers and seeds may be attacked by insects that rarely feed on leaf tissues, and vice versa. Other herbivores exhibit more plasticity in food selection and will readily feed on all available parts of the plant. Even the latter herbivore types display some preference for a specific plant structure, presumably as a means of obtaining optimal nutrient intake, limiting competition, or obtaining enemy-free space.\nThe cabbage white butterflies Pieris brassicae L., Pieris rapae L., and Pieris napi L. are specialized on the plant family Brassicaceae. Interactions between these butterflies and their host plants have been explored in detail, and they have acquired the status of a model system in the field of insect\u2013plant biology (Feltwell 1982; Chew and Renwick 1995; Renwick 2002). Glucosinolates, characteristic of the Brassicaceae, play a crucial role as chemical mediators of these interactions. Adult females of P. brassicae and P. rapae, as well as their larvae, exploit glucosinolates as token stimuli during selection of host plants for oviposition and feeding (Renwick et al. 1992; van Loon et al. 1992; Moyes et al. 2000; Schoonhoven and van Loon 2002). The majority of Pieris\u2013Brassica interaction studies has focused on cultivated forms of Brassica oleracea L., which are biennials. Other wild crucifers, such as the annual black mustard, Brassica nigra L. (Koch), are reported to be host plants for P. brassicae in Europe as well (Harvey et al. 2003). We studied within-plant feeding site location in detail on three genotypes of B. nigra. We included the analysis of leaf and flower glucosinolates to assess whether a chemical basis for selection of either plant organ in terms of quality or quantity of these compounds could be determined. In addition, we investigated whether growth rates differed between caterpillars feeding on either leaves or flowers to assess whether the observed preference behavior would confer a nutritional benefit.\nMethods and Materials\nPlants Seeds of B. nigra var. abyssinica A. Braun from three early flowering accessions were obtained from the Centre for Genetic Resources (CGN, Wageningen, The Netherlands). The accessions were: A, CGN06619 (feral population, collected in 1975 from the Peloponesus, Greece); B, Junius CGN06618 (advanced cultivar, collected in 1977 in Germany); and C, Giebra CGN06620 (advanced cultivar, donated in 1965 by the Botanical Garden of the Justus Liebig University, Giessen, Germany). Sowing was done on April 7, 2004, in a greenhouse at 22\u2009\u00b1\u20092\u00b0C and 60\u2009\u00b1\u20095% RH. Two weeks later, plants were individually transplanted into 1.5-l pots and transferred to another greenhouse compartment on April 30, 2004. This compartment was kept at 22\u2009\u00b1\u20092\u00b0C, 60% RH, natural light supplemented by additional illumination by 500-W SON-T lamps (Philips, Eindhoven, The Netherlands) for 16\u00a0hr per day. Flower bud formation and flower opening were recorded daily to determine developmental stage (Harper and Berkenkamp 1975).\nInsects Pieris brassicae caterpillars were obtained from a laboratory strain established in 2004 and reared on Brussels sprouts (B. oleracea var. gemmifera cv Cyrus) plants grown in a climatized greenhouse, under the same conditions as given above for B. nigra plants. The colony was maintained in a climatized room at 22\u2009\u00b1\u20091\u00b0C, RH 40\u2009\u00b1\u20095%, and a photoperiod of L16:D8.\nCaterpillar Position on the Plant On the first day of an experiment, newly hatched P. brassicae larvae were collected from the laboratory culture and taken to the greenhouse. Three caterpillars were placed on the first true leaf below the lowest flowering branch of a plant in growth stage 4.1 (Harper and Berkenkamp 1975). Total numbers of plants used from each accession were A, 8; B, 7; and C, 9. Caterpillar position on the plants was scored each morning in four categories: on a true leaf, on a small leaf in the inflorescence, on a flower, and on the stem. Caterpillar instar stage was recorded daily until the fifth instar was reached. In the late fifth instar, caterpillars that had entered the wandering phase left the plants.\nCaterpillar Growth on Leaves and Flowers Three neonate caterpillars were inoculated on each of 20 plants of accession A, on a mature leaf just below the inflorescence. The inoculation took place when plants had just started to flower (growth stage 4.1). Just after caterpillars had molted to the third instar, they were transferred to the lowest branches of the inflorescence on half of the plants. We applied a specially constructed water barrier to prevent caterpillars from migrating from leaves to the inflorescence or vice versa, while not influencing the microclimate around the plant (Fig.\u00a01). Fresh body weights of larvae were determined to the nearest milligram on a Mettler electronic balance at three time points separated by 3-d intervals.\nFig.\u00a01Schematic drawing (top and lateral views) and dimensions of water barrier applied around the stem of B. nigra plants to prevent movement of caterpillars from leaves to flowers and vice versa. A circular water container was manufactured of plastic. The middle circle consisted of a central platform, preventing drowning of caterpillars feeding on the inflorescence in the rare events of falling down or downward migration. In the center of the platform, a hole allowed the main stem to grow through. The platform was put in place around the main stem just below the branch carrying the first flower buds. The circular opening between the central hole and the stem was blocked with soft foam (not drawn)\nPreparation of Plant Samples for Glucosinolate Analysis Ten seedlings of each of the three accessions were individually transplanted to 1 1\/2-l pots. Plants were maintained at 22\u2009\u00b1\u20092\u00b0C, RH 60\u2009\u00b1\u20095%, during the 16-hr photophase (8 Philips SON-T Agro 400-W sodium lamps) and 8-hr scotophase.At day\u00a047 after sowing, leaf and flower samples were taken from all 10 plants of each accession. After sampling, five plants of each accession were designated untreated (controls). Three groups of 10 newly hatched P. brassicae caterpillars were placed on three leaves of the five treated plants. As soon as the caterpillars had migrated to the flowers, which took place late in the second or early in the third instar (days\u00a05\u20136), leaves damaged by the caterpillars were sampled. Leaves of a similar age were sampled from the control plants and intact flowers were sampled from both control and treated plants.The flower and leaf samples were kept in paper envelopes and stored in a \u221220\u00b0C freezer before and after freeze-drying. Samples were freeze-dried in the envelopes for 2\u00a0d (Labconco Freedry system). After freeze-drying, the envelopes were sealed in plastic and sent to the laboratory of NA in Denmark for chemical analysis.\nDetermination of Glucosinolate Content Freeze-dried samples were weighed accurately into 10-ml centrifuge tubes (excluding the petiole part of leaves). For samples below 300\u00a0mg, the entire sample [27\u2013257\u00a0mg dry weight (DW)] was used to avoid errors associated with subsampling, but from a few samples above 300\u00a0mg, a representative subsample was taken. Each sample was extracted 3\u00d7 in 70% boiling aq. methanol with benzylglucosinolate as internal standard. The combined extracts were adjusted to 10.00\u00a0ml, of which an amount was applied to an anion exchange column (Agerbirk et al. 2001a). To keep within the linear range of the additional sample preparation steps, the amount of crude extract applied depended on the weight of the sample extracted as follows (DW extracted\/volume applied): 25\u201375\u00a0mg\/8\u00a0ml, 75\u2013150\u00a0mg\/4\u00a0ml, and 150\u2013300\u00a0mg\/2\u00a0ml. Glucosinolates were converted to desulfoglucosinolates (Agerbirk et al. 2007), which were eluted and quantified by high-performance liquid chromatography (HPLC) (Agerbirk et al. 2001a) and identified by comparison of retention times and diode array UV spectra with those of authentic standards (Agerbirk et al. 2001b). A number of unidentified trace peaks with areas typically less than 0.2% of the desulfosinigrin area were ignored. Generally accepted relative response factors were used for calculation of glucosinolate levels based on the HPLC peak areas (at 229\u00a0nm, band width 8\u00a0nm) of the desulfoderivatives (Wathelet et al. 2004). The recoveries of internal standard in the three extraction series were [mean (SD)] 99% (6%), 97% (13%), and 98% (4%), as compared to HPLC peak areas obtained after application of the internal standard only to DEAE columns in parallel control experiments. Preliminary experiments without the addition of internal standard (comprising both leaf and flower samples from plants both with and without caterpillars feeding) confirmed the absence of benzylglucosinolate as an endogenous glucosinolate in the material, in accordance with previous reports (Feeny and Rosenberry 1982; Sang et al. 1984), allowing the use of this glucosinolate as internal standard. The identities of all desulfoglucosinolate peaks were confirmed by liquid chromatograph mass spectrometry (LC\u2013MS) of representative samples with an Agilent 1100 Series LC (Agilent Technologies, Waldbronn, Germany) coupled to a Bruker Esquire 3000+ ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany). An XTerra MS C18 column (Waters, Milford, MA, USA; 3.5\u00a0\u03bcM, 2.1\u2009\u00d7\u2009100\u00a0mm) was used at a flow rate of 0.2\u00a0ml min\u22121. The mobile phases were as follows: A, 0.1% (v\/v) HCOOH and 50\u00a0\u03bcM NaCl; B, 0.1% (v\/v) HCOOH and 80% (v\/v) MeCN. The gradient program was 0 to 4\u00a0min, isocratic 2% (v\/v) B; 4 to 10\u00a0min, linear gradient 2 to 8% B; 10 to 30\u00a0min, linear gradient 8% to 50% (v\/v) B; 30 to 35\u00a0min, linear gradient 50 to 100% (v\/v) B; 35 to 40\u00a0min, isocratic 100% B. The mass spectrometer was run in electrospray mode, observing positive ions. Mass spectral data were treated with the native DataAnalysis software.The extraction procedure was somewhat simplified compared to the standard protocol (Agerbirk et al. 2001a, originally optimized for seed extraction), as variable sample weights were accepted, and mechanical homogenization during extraction was avoided. These simplifications were justified by results of initial control experiments: A number of both leaf and flower samples of varying weights (up to 400\u00a0mg) were first subjected to the extraction described above. Then, the residue was further extracted for another three times, but this time with mechanical homogenization. In all cases, low relative amounts of glucosinolates (1\u201316% compared to the first extract) were detected in the extract obtained with mechanical homogenization (with a tendency for increasing percentage with increasing sample weight). Proportional amounts of internal standard were similarly recovered in all the extracts obtained with mechanical homogenization, showing that the internal standard had already been completely mixed with endogenous analytes during the extraction without homogenization. Hence, it was concluded that the ratio of internal standard to endogenous glucosinolates extracted without mechanical homogenization was representative of their initial concentrations.\nStatistical Analyses Proportion of days caterpillars spent on different positions on the plant was treated as binomial data. Distributions over the four positions were compared by using the Kolmogorov\u2013Smirnoff test (GenStat release 8.11; Anonymous 2005). Caterpillar weights were analyzed by analysis of variance. The amount (\u03bcmol\/g DW) of each glucosinolate detected in the leaf and flower samples were analyzed with a generalized linear model after a logarithmic transformation. The effect of time (before and during caterpillar feeding), treatment (undamaged and damaged by caterpillars), plant tissue (leaf and flower), and genotype (accessions) and all possible two- and three-way interactions were investigated (GenStat release 8.11).\nResults\nPosition on the Plant First and second instars fed primarily on leaves, whereas third instars migrated via the small leaves of the flower branches to the flower buds and flowers. Once flower feeding began, no further leaf feeding was observed (Fig.\u00a02). The distribution of caterpillars over the four positions was statistically similar for the three genotypes (Kolmogorov\u2013Smirnoff test, P\u2009>\u20090.05).\nFig.\u00a02Frequency distribution of caterpillar position on B. nigra plants observed daily over a period of 12\u201313\u00a0d. On day\u00a01, neonates were introduced on a true leaf just below the inflorescence. Four organ positions were distinguished: on a true leaf, on a small leaf in the inflorescence, on a flower, or on a stem in the inflorescence. Percentages are averaged based on three caterpillars per plant for eight, seven, and nine plants of accessions A, B, and C, respectivelyCaterpillars fed in discrete meals two to three times each hour, interspersed with phases without feeding. On a leaf, after termination of a meal, they moved away from the freshly damaged wound, after which they rested most of the time elsewhere on the leaf until they moved back to the feeding site. By contrast, in the inflorescence, during a single meal, caterpillars moved from flower to flower along flowering branches. A fifth-instar caterpillar consumed on average 135\u2009\u00b1\u200921 buds and flowers. When they were not feeding, P. brassicae larvae rested on stems immediately adjacent to the flowers.\nCaterpillar Growth Caterpillars feeding on flowers of accession A since molting to the third instar had significantly higher fresh body weights on each of the three measuring points (mid-L4 and early and mid-L5 and late L5; Fig.\u00a03) than those that were confined to leaves (P\u2009<\u20090.01, generalized linear model).\nFig.\u00a03Fresh body mass of P. brassicae on either flowers (circles) or leaves (squares) of B. nigra in mid-fourth, mid-fifth, and late-fifth instar developing on B. nigra, accession A. Mean and SEM are plotted for 30 caterpillars\nGlucosinolate Levels Five glucosinolates were identified: the aliphatic allylglucosinolate (sinigrin), which occurred in large amounts, and low levels of four aromatic glucosinolates including 2-phenylethylglucosinolate and the three indole glucosinolates indol-3-ylmethylglucosinolate (glucobrassicin), 4-methoxyglucobrassicin, and 4-hydroxyglucobrassicin. Despite the minor levels of the identified aromatic glucosinolates, they were quantified and included in the statistical analyses because of their well established chemosensory influences on Pieris species (Schoonhoven and van Loon 2002). No other significant peaks were detected. 2-Phenylethylglucosinolate was either below the detection limit in leaves (accession C) or present at very low levels (range 0.001\u20130.013\u00a0\u03bcmol\/ g DW). We carefully distinguished the peak with the exact retention time of (desulfo) 2-phenylethylglucosinolate from an unidentified trace peak that was more intense in leaf chromatograms (estimated levels 0.03\u20130.35\u00a0\u03bcmol\/g DW in leaves, 0.01\u20130.08\u00a0\u03bcmol\/g DW in flowers), that had a retention time 0.7\u00a0min longer than that of (desulfo) 2-phenylethylglucosinolate, and that did not coelute with the authentic standard of 2-phenylethylglucosinolate after spiking.Genotype and tissue type were the most important factors affecting the levels of identified glucosinolates, and these factors showed a significant interaction (Table\u00a01). No significant interactions were found between treatment and tissue (P\u2009>\u20090.05 for all glucosinolates). Glucosinolate concentrations have been graphically depicted for accession A in Fig.\u00a04. Sinigrin was by far the most abundant compound in all three genotypes, with levels at least 44 times higher (range 44\u2013305) in leaves and at least 87 times higher (range 87\u2013447) in flowers of any of the three genotypes and treatments than the second most abundant compound, 4-hydroxyglucobrassicin. Sinigrin, 4-hydroxyglucobrassicin, and phenylethylglucosinolate were present at significantly higher levels in flowers than in leaves. Sinigrin levels were ca. fivefold higher in flowers than in leaves. In response to caterpillar feeding, sinigrin levels in leaves were higher than in undamaged plants (P\u2009<\u20090.05; Table\u00a01). In flowers of accession A (Fig.\u00a04), there was a similar tendency for induction of sinigrin by caterpillar feeding, but such induction was not seen with flowers from the other two genotypes (results not shown). Levels of other glucosinolates showed moderate variations that were not affected by the experimental variables (Table\u00a01, Fig.\u00a04 and results not shown). There was no systematic induction of any indole glucosinolate or of total indole glucosinolates in response to caterpillar feeding (results not shown).\nTable\u00a01Results from generalized linear model analysis on log-transformed data of glucosinolate concentrations in two tissue types (leaves and flowers) for three genotypes of B. nigraFactorSinigrinPEGB4OHGB4MeOGBTimensnsns***nsTissue******ns***nsTreatment*nsns*nsGenotype************nsTissue \u00d7 genotype*****ns*nsTreatment means presence or absence of third-instar caterpillars feeding on flowers during 4\u00a0d. Effect of the factor \u201ctime\u201d refers to changes over 4\u00a0d in intact plantsPE = phenyethylglucosinolate; GB = glucobrassicin; 4OHGB = 4-hydroxyglucobrassicin; 4MeOGB = 4-methoxyglucobrassicin*P\u2009<\u20090.05; **P\u2009<\u20090.01; ***P\u2009<\u20090.001Fig.\u00a04Concentrations of five glucosinolates (mean + SEM) in leaves and flowers of 10 plants of accession A. For both leaves and flowers, the initial concentration just before treatment, the concentration in leaves or flowers of plants damaged by feeding caterpillars during 4\u00a0d, and the concentration determined in leaves or flowers of intact plants 4\u00a0d after caterpillars were introduced on the treated plants\nDiscussion\nOur data show that P. brassicae third, fourth, and fifth instars preferentially feed on B. nigra flowers. Preference was absolute: although leaf material was available in surplus, caterpillars fed exclusively on flowers. Historically, P. brassicae has been commonly considered a folivore (Feltwell 1982), with its impact on plants examined primarily from this perspective. To the best of our knowledge, this is the first detailed study showing that P. brassicae larvae preferentially feed on the buds and flowers of its host plant. The status of P. brassicae as an agricultural pest has invariably resulted in a focus on cultivated, biennial forms of B. oleracea in studies of its host plant relationships. We also observed flower and silique feeding under field conditions (Smallegange et al., unpublished results).\nThe impact of folivores on plants has both direct and indirect effects. Directly, these herbivores reduce the leaf area available for photosynthesis, thereby decreasing the plants\u2019 ability to garner resources for defense and reproduction (Marquis 1984; Mothershead and Marquis 2000; Schoonhoven et al. 2005). Indirectly, leaf herbivory can delay flowering (Meyer and Root 1993; Strauss et al. 1996) or can alter developing flowers both physically (Strauss et al. 1996; Lehtil\u00e4 and Strauss 1999; Mothershead and Marquis 2000) and chemically (Lohman et al. 1996; Karban and Baldwin 1997). This may cause a reduction in plant attractiveness to pollinators or in the efficiency of pollen delivery, resulting in decreased pollination and, subsequently, reduction of seed set and overall fitness of the plant. Florivory, feeding on flowers, has received less attention than folivory or feeding on other vegetative plant parts despite its common occurrence and potentially high impact on plant fitness (recently reviewed by McCall and Irwin 2006). Feeding on flowers causes damage to structures essential for plant reproductive output (Juenger and Bergelson 1997; Adler et al. 2001). Indirectly, petal damage leads to a reduction in pollinator visitation that not only reduces reproductive success but also affects male fitness (via pollen removal) of the plant as well (Karban and Strauss 1993; Krupnick and Weis 1998; Adler et al. 2001). However, the compensatory abilities of plants can partially overcome the impact of floral herbivory (Hendrix 1988; Krupnick and Weis 1998). Removal of reproductive organs as a preferred mode of feeding has evolutionary consequences, as this type of attack is likely to exert a stronger selection pressure than leaf feeding, especially in annual species like B. nigra. Although many plant species accumulate higher concentrations of defensive secondary metabolites in reproductive organs than in vegetative organs (Schoonhoven et al. 2005), in B. nigra, these higher levels are not sufficient to deter the specialist feeder P. brassicae. A putative barrier to florivory by generalist herbivores lies in the higher quantities of secondary plant compounds that are typically found in these organs relative to the levels reported for leaves (Rask et al. 2000; Fahey et al. 2001; Brown et al. 2003; Strauss et al. 2004; this study). The location of high concentrations of defensive secondary plant compounds, including glucosinolates, in reproductive structures is consistent with the optimal distribution of chemical defenses predicted by plant defense theory (Zangerl and Bazzaz 1992; van Dam et al. 1996; Wallace and Eigenbrode 2002; Strauss et al. 2004). Myrosinases, \u03b2-thioglucosidase enzymes that convert glucosinolates into products such as isothiocyanates and nitriles, which are volatile in the case of, e.g., sinigrin and 2-phenylethylglucosinolate, occur in different forms in flowers and leaves (Rask et al. 2000). By using headspace techniques, isothiocyanates and nitriles have been reported from mechanically macerated bud samples of several Brassica species (Tollsten and Bergstr\u00f6m 1988). Phenylacetonitrile was detected in headspace samples of intact B. rapa L. flowers (Omura et al. 1999). Benzylcyanide was released in significantly higher amounts from Brassica napus L. in midflowering than in bud stage (J\u00f6nsson et al. 2005). High levels of isothiocyanates are potentially toxic to both specialist and generalist herbivorous insects (e.g., Li et al. 2000; Agrawal and Kurashige 2003), while even low levels may be detected by specialized sensory receptors and assist specialized herbivores in host plant finding (Renwick et al. 2006).\nThe growth rate we observed for caterpillars feeding on flowers was remarkable. The feeding site preference of L3\u2013L5 caterpillars presents a case of within-plant selective foraging, the adaptive value of which might be the ingestion of higher nutritional content in flowers, known to act as nutrient sinks, compared to leaves. Nutrient acquisition during the larval stage can affect pupal survival, longevity, and fecundity of Lepidoptera (Feltwell 1982; Van der Meijden et al. 1984; Metspalu et al. 2003). For example, female pupae of Tyria jacobaeae were significantly larger when caterpillars were reared on leaves and inflorescences of Senecio jacobaea plants than when reared on leaves only. This is advantageous because large pupae have a higher eclosion success, and it may explain why T. jacobaeae females prefer flowering plants for oviposition (Vrieling and de Boer 1999).\nFaster growth has often been interpreted as a mortality-reducing factor. It is assumed that by reducing the amount of time that larvae are early instars, when they are most vulnerable to attack by natural enemies, they will experience higher survival (the \u2018slow-growth\u2013high-mortality hypothesis\u2019 sensu Clancy and Price 1987; Benrey and Denno 1997). This has been experimentally confirmed under field conditions for the interaction between P. rapae feeding on B. oleracea and its predators and parasitoids such as Cladophora glomerata (Loader and Damman 1991; Benrey and Denno 1997). However, for other tritrophic interactions, faster growth was associated with higher mortality due to natural enemies (e.g., Clancy and Price 1987). Biere et al. (2002) found that larvae of the moth Hadena bicruris exhibit a strong preference for seeds of their food plant, Silena latifolia, over leaf tissues. Herbivore development proceeded more rapidly on seeds, and the larvae were less susceptible to parasitism from their main endoparasitoid, Microplitis tristis.\nHerbivorous insects specialized on glucosinolate-containing plants typically avoid the formation of toxic isothiocyanates by employing specialized detoxifying mechanisms. In the case of P. brassicae, this is accomplished by a nitrile specifier protein (NSP) in the gut that changes the products of the myrosinase-catalysed hydrolysis of glucosinolates from isothiocyanates to relatively harmless nitriles (Wittstock et al. 2004), which may be further metabolized before excretion depending on side chain structure (Agerbirk et al. 2006; 2007). Shortly before this mechanism was reported, it was discovered that allyl isothiocyanate, the volatile hydrolysis product of sinigrin, is toxic to the specialist feeder P. rapae when feeding on an artificial diet (Agrawal and Kurashige 2003). Assuming a 20% DW in the artificial diet as used by Agrawal and Kurashige (2003), the maximum concentration to which P. rapae caterpillars were exposed in their study was 8.5\u00a0\u03bcmol\/g DW. This dose caused significant negative effects on growth rate and inhibited development. In contrast, sinigrin levels in B. nigra flowers were more than 10 times higher (Fig.\u00a04) and flowers sustained higher growth rates of P. brassicae than leaves, which had average levels of sinigrin two times higher than the corresponding amount of allylisothiocyanate as applied in the artificial diet. Assuming that isothiocyanates were produced in significant amounts during ingestion of glucosinolate-containing foliage by P. rapae, an involvement of the glucosinolate\u2013myrosinase system in defense against this specialist species was suggested. Brassica nigra plants that had been induced by previous P. rapae feeding damage had a 27% increase in sinigrin concentration, but also 43% higher trichome density (Traw and Dawson 2002). Our data confirm the reported induction of sinigrin in leaves upon feeding, but also demonstrate that the magnitude of the induction is minor relative to the difference between leaves and flowers. With the elucidation of the NSP-based mechanism for detoxification of the glucosinolate\u2013myrosinase system, the suggested involvement of the glucosinolate\u2013myrosinase system and defense to specialists now needs to be re-evaluated. Formation of isothiocyanates is to be expected only if the myrosinase activity in the plant is so high that available NSP cannot completely direct the reaction towards nitriles, combined with glucosinolate levels sufficiently high to produce toxic levels of isothiocyanates. The selective foraging on flowers by P. brassicae documented here demonstrates that the caterpillars are able to detoxify even high levels of sinigrin, such as those present in B. nigra flower tissue. Not only high glucosinolate levels but also high myrosinase activity would be needed to exert toxic effects on Pieris caterpillars, as has been experimentally demonstrated for another glucosinolate-adapted herbivorous insect, Plutella xylostella (Li et al. 2000). Glucosinolates occur in cells of all organs of Brassicaceous plants, whereas myrosinases can be found in scattered glucosinolate-free cells. It remains to be examined whether flower buds of different age, flowers, and siliques of B. nigra have cells that contain myrosinases. In the related B. napus L., expression of the MYR1-gene coding for particular myrosinase isoforms has been demonstrated to occur in all organs investigated, including petals (Falk et al. 1992), although myrosinase was absent in very young flower buds in this species (Andr\u00e9asson et al. 2001). Myrosinase gene promoters fused to beta-glucuronidase as a reporter showed expression of myrosinase in idioblast myrosin cells in immature and mature seeds and phloem myrosin cells of B. napus (Thangstad et al. 2004). Data on headspace composition of inflorescences actually fed upon by Pieris spp. are not available. Follow-up studies on the types and quantities of volatile breakdown products emitted by herbivore-damaged flowers are required to quantify myrosinase activity levels in flowers. If these levels are low, the better performance associated with flower feeding might be reconciled with a role of the glucosinolate\u2013myrosinase system in defense against the specialist P. brassicae. However, a likely and simple explanation of the better performance associated with flower-feeding may be that the caterpillars are able to efficiently detoxify the glucosinolate\u2013myrosinase defense system in both organs, and that flowers, major nutrient sinks on a plant, are more nutritious than leaves. The fact that early instars did not migrate to flowers may be an adaptation to factors other than nutrient content: leaves are the site of oviposition, are present earlier in the season than flowers, and may provide better refuge from natural enemies for small instars. We have never observed P. brassicae or P. rapae ovipositing on flowers or flowering stems.\nNeither P. rapae nor P. brassicae sequester glucosinolates for their own defense against natural enemies (M\u00fcller et al. 2003), in contrast to what had been published earlier by Aplin et al. (1975). The intensely green, clear regurgitant these caterpillars instantly, and typically, produce when attacked may contain highly concentrated glucosinolate hydrolysis products (nitriles) stored in the foregut. Presence of concentrated glucosinolate hydrolysis products may explain the obvious repellent effect of regurgitant to natural enemies such as Cotesia wasps and predators (Brodeur et al. 1996). Consequently, caterpillars may prefer flower tissues that contain high levels of these compounds, thus boosting their defense against both specialist and generalist natural enemies (Soler et al. 2005). Behavioral experiments with parasitoids and predators known to act as common natural enemies of P. brassicae (Feltwell 1982) are required to test this hypothesis.","keyphrases":["brassica nigra","nutrition","herbivory","myrosinase","isothiocyanates","lepidoptera","pieridae"],"prmu":["P","P","P","P","P","P","U"]}
{"id":"Hum_Reprod-1-1-2387218","title":"Cell identity in the preimplantation mammalian embryo: an epigenetic perspective from the mouse\n","text":"The early preimplantation mouse embryo is a unique system where it is possible to explore the foundations of totipotency and differentiation. Following fertilization, a single cell, the zygote, will give rise to all tissues of the organism. The first signs of differentiation in the embryo are evident at the blastocyst stage with the formation of the trophectoderm, a differentiated tissue that envelopes the inner cell mass. The question of when and how the cells start to be different from each other in the embryo is central to developmental biology: as cell fate decisions are undertaken, loss of totipotency comes about. Although the blastomeres of the preimplantation embryo are totipotent, as the embryo develops some differences appear to develop between them which are, at least partially, related to the epigenetic information of each of these cells. The hypothesis of epigenetic asymmetries acting as driver for lineage allocation is presented. Although there are now some indications that epigenetic mechanisms are involved in cell fate determination, much work is needed to discover how such mechanisms are set in play upon fertilization and how they are transmitted through cell division. These considerations are further discussed in the context of preimplantation genetic diagnosis: does it matter to the embryo which cell is used for genetic diagnosis? The exquisite complexity and richness of chromatin-regulated events in the early embryo will certainly be the subject of exciting research in the future.\nEmbryonic development starts from a single cell, the zygote. In this cell, the two gametes convey and contribute information to start a new developmental programme. The formation of the newly fertilized zygote constitutes therefore the climax of totipotency because of the resulting zygote\u2019s inherent ability to produce all cell types in a new organism.\nIn the mammalian embryo, the first differentiative event occurs as inner cells form upon cell division at the 8-cell stage. As a result of this division, an \u2018inner\u2019 and an \u2018outer\u2019 population of cells can be distinguished in the 16-cell stage embryo, which will respectively occupy different positions in the morula (Johnson and Ziomek, 1981). Much work has been done in trying to understand how cell polarity develops in the inner and outer cells, but I will not deal with this topic here and instead will refer the reader to an excellent review published elsewhere (Johnson and McConnell, 2004). The inner cells will develop into the inner cell mass (ICM) and the outer layer of cells will differentiate into the trophectoderm (Tarkowski and Wroblewska, 1967; Ziomek and Johnson, 1982). Morphologically, however, the first overt signs of differentiation are evident only at the blastocyst stage, with the formation of the trophectoderm, which is the first differentiated tissue to form as an epithelial layer that envelops the ICM (Fig.\u00a01A). The latter, in contrast to the trophectoderm, retains its pluripotent character and the ability to self renew. While the ICM will give rise to the embryo proper, the trophectoderm will give rise to the extraembryonic tissues that will support development of the embryo during gestation. The ICM will also give rise to yet another extraembryonic tissue, the primitive endoderm, which is first visible as a cuboidal layer of cells lining the blastocoelic cavity on the fourth day of development. The trophectoderm and the ICM each display molecular identity, which is reflected in part by the expression of specific genes that are, for the ICM, involved in its specification and the maintenance of pluripotency (such as Nanog and Oct4) or, for the trophectoderm, that are required for its differentiation (such as Cdx2) (Palmieri et al., 1994; Nichols et al., 1998; Chambers et al., 2003; Mitsui et al., 2003; Strumpf et al., 2005). The bifurcation of these two lineages is complete at the late blastocyst stage. At this stage, cells from the ICM do no longer have the potential to form trophectoderm derivates in vivo upon transplantation (Rossant and Lis, 1979), indicating that these cells have lost their totipotency and that lineage allocation has definitely occurred.\nFigure\u00a01:\nCell lineages of the mammalian blastocyst and epigenetic marking\n(A) Representation of the lineages in the mammalian blastocyst on the third day of gestation (E3.5). The blastocyst is composed of two distinct populations of cells: the trophectoderm (red) and the inner cell mass (ICM, green), which display molecular identity and epigenetic asymmetries. The embryonic-abembryonic regions (dotted line) of the blastocyst are determined by the position of the ICM, which lies within the embryonic region of the blastocyst. (B) Diagram illustrating some of the epigenetic marks. The DNA wrapped around the nucleosome (beige cylinders) is shown as light blue. The DNA is subject to DNA methylation, which constitutes one of the main epigenetic players. The core histones (beige) that form the nucleosome can be covalently modified (by acetylation, methylation, phosphorylation), particularly on their N-terminal tails. Each of these marks can have an effect on how the information contained in the DNA is read by modulating downstream events such as transcriptional activation or repression. For example, histone methylation (Me) can have a positive effect on transcription (green) or a repressive one (red). The marks can be present in different combinations and may change during the cell cycle. (C) Model for epigenetic marking and lineage allocation. In this model, an epigenetic mark would be laid down in a given cell during development. There could be other epigenetic event(s) that reinforce and\/or are influenced by the first marking event. Cumulatively, this could result in determination of the fate of that cell towards a lineage in the blastocyst. The lineage specific marks could stabilize such cell identity and might be necessary for further differentiation. Alternatively, the acquisition of these epigenetic marks could be the result of cell fate determination. One should also consider that not only the nature of the mark would be important, but also the different regions of the chromatin that would be affected by such marks. Because the cells in the preimplantation embryo are totipotent and because the chromatin will still need to be dynamically remodelled during subsequent development, flexibility should be an important component of epigenetic mechanisms taking place during early development. As cell fate decisions are taken, a concomitant loss of totipotency takes place\nInvestigations during recent years have recreated an interest in whether the blastomeres of the mammalian embryo are truly alike throughout preimplantation development before the first differentiative division mentioned above. In other words, whether they acquire a \u2018fate\u2019 or whether they start to differ from each other prior to their spatial \u2018inner\/outer\u2019 allocation within the embryo upon the formation of the morula. Of course, if this were to be the case, the big challenge would be to ascribe molecular mechanisms to these processes.\nThe question of when and how the cells start to be different from each other is not a trivial one. In particular, because as the first cell fate decisions are undertaken, a concomitant loss of totipotency occurs. The developmental time window when this first cell fate decision occurs comprises a number of epigenetic events (Morgan et al., 2005; Surani et al., 2007). These events include the reprogramming of the parental chromatin. Whether such epigenetic events are the cause or the consequence of reprogramming remains an exciting open question, but it is probably a combination of the two. Moreover, the two lineages of the blastocyst exhibit some epigenetic asymmetries.\nThis mini-review is divided in two parts, the first one will deal with the main epigenetic mechanisms that are known to occur during mammalian preimplantation development. The second one will give an overview on data obtained through experimental embryology manipulations and lineage tracing observations to study cell fate in the early mouse embryo.\nEpigenetic mechanisms in the preimplantation embryo\nIn general terms, epigenetic mechanisms include DNA methylation, covalent modification of histones, chromatin remodelling and histone replacement through incorporation of the so-called histone variants (Fig. 1B). Histone marks have emerged as one of the main players involved in epigenetic mechanisms (Kouzarides, 2007). Histone modifications can be highly dynamic, or have a function in epigenetic memory. Although it is still unclear whether they are the actors of the epigenetic information or the epigenetic information itself, it is evident that covalent modifications of histones are essential components of the epigenome.\nHistones can be modified by a number of enzymes that mediate methylation, acetylation, phosphorylation, ubiquitynation and ADP-ribosylation of specific amino acid residues (reviewed in Kouzarides, 2007). By and large, the highest density of modifications so far described occurs in histone H3, particularly on its tail. The effects of these modifications on the chromatin and on cellular processes are very diverse, and a modification of the same residue can even have opposite effects depending on the type of modification. For example, trimethylation of H3K9 is considered as a repressive mark, whereas acetylation of the same lysine has a positive effect on transcription (Bannister and Kouzarides, 2005). Likewise, methylation of arginine residues can have a positive effect on transcription (Chen et al., 1999) or a repressive effect (Pal et al., 2004), depending both on the targeted residue and on whether the methylation is symmetric or asymmetric. For some of the modified residues, there is a very clear view of the outcome of an eventual modification: H3K9me3 creates a specific docking site for the heterochromatin protein 1 (HP1), which subsequently recruits the H3K9 methyltransferase Su(var)3-9 and reinforces an autoregulatory loop for heterochromatin formation and maintenance (Bannister et al., 2001; Lachner et al., 2001; Nakayama et al., 2001).\nThe levels of regulation of epigenetic events in the preimplantation mouse embryo are multiple. They include the regulation of the subcellular localization of DNA methyltransferase activity, highlighted by the cytoplasmic retention of Dnmt1o (Carlson et al., 1992); the exclusion of a particular histone modification from the chromatin, which is exemplified by the lack of detection of H3K9me3 in the paternal pronucleus after fertilization resulting in an asymmetry of histone marks between the two pronuclei (Arney et al., 2002; Santos et al., 2005), the differential incorporation of chaperons and histone variants in the parental chromatin (van der Heijden et al., 2005; Torres-Padilla et al., 2006) and the acquisition of highly specific histone variants in the gametes (Clarke et al., 1992; Tanaka et al., 2001; Govin et al., 2007). Further, the maternal and paternal pronuclei exhibit different patterns of global DNA methylation: while the paternal pronucleus is rapidly demethylated\u2014presumably through an active mechanism\u2014right after fertilization, the maternal pronucleus is only passively demethylated through the subsequent rounds of replication and cell division that follow the first mitosis of the embryo (Mayer et al., 2000). Moreover, while the centromeric and pericentric paternal chromatin remain DNA methylated, the maternal DNA loses methylation in such regions (Rougier et al., 1998).\nThe changes in the levels of DNA methylation as development proceeds in the preimplantation embryo are dynamic. Global levels of DNA methylation have been analysed by immunofluorescence, bisulphate sequencing and restriction digestion (Rougier et al., 1998; Mayer et al., 2000; Santos et al., 2002; Aranyi and Paldi, 2006). Bisulphate sequencing and restriction digestion have also been used to analyse the methylation status of repeat sequences (such as L1 and IAP repeats) and some single-copy sequences (such as actin) (Howlett and Reik, 1991; Oswald et al., 2000). These studies have revealed that although global levels of DNA methylation decrease until the blastocyst stage, changes in DNA methylation do not occur to the same extent on all genes. Remarkably, imprinted genes (such as H19) and some repeat sequences (such as IAPs) do not undergo demethylation (Tremblay et al., 1997).\nDuring early stages of development, decisions involved in cell fate determination and pluripotency have to be assumed. These processes require the chromatin to be dynamically remodelled to ensure its plasticity. This implies that the mechanisms involved in regulation of chromatin structure need to ensure stability across generations and cell division, but they also need to be flexible (Reik, 2007). The double nature of a covalent modification either on histones and\/or on the DNA as dynamic (because in principle it can be added and removed) and at the same time its potential ability to propagate a memory, fits well with these aforementioned needs. Moreover, in keeping with the importance of epigenetic mechanisms during early development, the possibility for an epigenetic mark(s) underlying these phenomena appears very attractive.\nConcerning the epigenetic asymmetries of the ICM and the trophectoderm, the ICM displays, in global terms, higher levels of DNA methylation compared with the trophectoderm (Dean et al., 2001; Santos et al., 2002). Specific histone marks such as trimethylation of lysines 9 and 27 of histone H3 (H3K9me3 and H3K27me3, respectively) are enriched in the ICM compared with the trophectoderm (Erhardt et al., 2003). Likewise, the trophectoderm retains an imprinted form of X inactivation, where the paternal X chromosome is silenced (Heard and Disteche, 2006). This is in contrast to the ICM, where there is reactivation of the inactive X chromosome and a subsequent round of inactivation occurs at random in which either the maternal or the paternal chromosome is inactivated (Mak et al., 2004; Okamoto et al., 2004).\nThe aforementioned epigenetic asymmetries of the two lineages of the blastocyst are evident once lineage allocation has taken place and might reinforce their molecular identity. However, epigenetic asymmetry could also act as a driver for lineage allocation, in which case, the former would precede the latter (Fig. 1C). This constitutes a fascinating current working hypothesis.\nDevelopment of cell identity in the mouse embryo\nFrom experimental embryology, we have learnt from pioneer experiments performed in the 50\u2019s that after mechanical separation of the blastomeres of a 2-cell stage embryo and transfer into foster mothers, each of these two cells gives rise to an adult mouse (Tarkowski, 1959). This indicates that mouse embryos are very flexible in what people have referred to as developmental potential. Derivation of twins from mouse blastomeres at later stages of development (e.g. 4-cell stage or later) has not been possible. Although this has been linked to the low number of cells present in the resulting embryos and hence their inability to form an ICM with a normal cell number, rather than to their developmental potential or identity (Tarkowski and Wroblewska, 1967; Rossant, 1976). Indeed, when random single 4- and 8-cell stage blastomeres are aggregated with \u2018carrier\u2019 blastomeres, their progeny is able to contribute to all the tissues of the embryo (Kelly, 1977; Tarkowski et al., 2005), and in this sense, the blastomeres were considered to be totipotent. So, what are these carrier cells providing? Is it a simple matter of cell number? and\/or of an appropriate environment? It is also possible however that some subtle intrinsic differences of these blastomeres might be masked by the limitations of the outcome of transplantation procedures, given that the results of these studies are very often far from 100% and the transplantation efficiency is never absolute. Whether this is solely related to technical difficulties linked to these challenging manipulations or to an intrinsic property of specific blastomeres of the embryo is impossible to ascertain.\nDespite a controversial viewpoint on whether there is any polarity in the early mouse embryo or not, most reports coincide with the interpretation that a blastomere at the 2-cell stage contributes to both the ICM and the trophectoderm (reviewed in Edwards and Beard, 1997; Zernicka-Goetz, 2006). The suggestion of a given blastomere at the 2-cell stage having a \u2018preferential\u2019 fate towards either of the blastocyst lineage is not resolved and some researchers have suggested that a slight, but distinct difference in the fate of 2-cell stage blastomeres might be disturbed by experimental manipulations (Alarcon and Marikawa, 2005; Hiiragi et al., 2006). The role of extrinsic factors to the embryo, such as the shape of the zona pellucida, in axis specification of the blastocyst is also a matter of controversy (Gardner, 2007; Kurotaki et al., 2007). However, there are some indications that a bias for a blastomere to contribute to a given region of the embryo in the blastocyst could already exist at the 4-cell stage (Fujimori et al., 2003; Piotrowska-Nitsche and Zernicka-Goetz, 2005). These conclusions are mostly based on lineage tracing experiments of labelled blastomeres, and their degree of invasiveness is debatable.\nSome groups have used the plane of division in relation to the animal\u2013vegetal axis of the embryo as a sort of guideline to distinguish and characterize blastomeres according to their cleavage plane. By convention, the animal pole is demarcated by the position of the second polar body (which is extruded after resumption of meiosis II upon fertilization) and hence the vegetal pole lies on the opposite side (Fig. 2A). The division from the 2- to the 4-cell stage would segregate for the first time the \u2018animal\u2019 and the \u2018vegetal\u2019 components of the zygote if it occurs equatorially, that is, perpendicular to the animal\u2013vegetal axis of the conceptus (Gardner, 2002). Thus, whereas a cell that derives from a meridional division (parallel to the animal\u2013vegetal axis) inherits both components, an equatorial division gives rise to an \u2018animal\u2019 and a \u2018vegetal\u2019 blastomere (Fig. 2B). By looking into the plane of division and the order at which this division occurs from the 2- to the 4-cell stage and subsequent lineage tracing, a subgroup of embryos was identified where it is possible, with a relatively high probability, to predict the future position of the blastomeres in the blastocyst (Piotrowska-Nitsche et al., 2005). This group of embryos are referred to as ME embryos (for Meridional\u2013Equatorial, reflecting the type of cleavage plane and order of division that generated them (Fig. 2B) (Piotrowska-Nitsche et al., 2005; Piotrowska-Nitsche and Zernicka-Goetz, 2005). Although the ME type embryos constitute only a small part (20%) of a complete litter, they provide a very nice system where it is possible to explore the foundations of differentiation in the embryo. Indeed, the \u2018vegetal\u2019 blastomere would most often populate the abembryonic region of the blastocyst, which contains mainly mural trophectoderm.\nFigure 2:\nBlastomere division planes according to the animal-vegetal axis in the embryo\n(A) The animal\u2013vegetal (A\u2013V) axis of the preimplantation embryo is demarcated, by convention, by the position of the second polar body, which marks the animal pole. The opposite side to the site of extrusion of the polar body is, by default, the vegetal pole of the embryo. (B) The division pattern from 2-to- 4-cell stage of a typical embryo that undergoes one meridional (M) and one equatorial (E) division (ME embryo) is represented. The cleavage plane is depicted by a red dashed line (embryo on the left). The blastomere that divides earlier is represented on the left. A Meridional division has a cleavage plane that is parallel to the A\u2013V axis of the embryo and hence gives rise to two cells containing both \u2018animal\u2019 and \u2018vegetal\u2019 components (two cells with pink and yellow motifs on the embryo depicted on the right). In contrast, when a 2-cell stage blastomere divides equatorially, a segregation of the \u2018animal\u2019 and \u2018vegetal\u2019 cytoplasm occurs and follows derivation of an \u2018animal\u2019 (pink) and a \u2018vegetal\u2019 (yellow) blastomere\nIn looking for epigenetic marks that could be involved in an eventual cell fate decision of the blastomeres of these ME embryos, it was found that the \u2018vegetal\u2019 blastomere displays the lowest levels of dimethylated arginine 26 of H3 (H3R26me2). If H3R26me2 participates in lineage allocation, one might predict that modulating the levels of histone arginine methylation, would have an effect over cell fate. Overexpression of the histone methyltransferase that methylates this residue on H3, PRMT4\/CARM1, into individual blastomeres not only induced upregulation of Nanog and Sox2, but also resulted in an almost complete allocation of these blastomeres into the ICM compartment (Torres-Padilla et al., 2007).\nIt is interesting to note that in the mouse, the differences described in histone H3 arginine methylation appear at the 4-cell stage (Torres-Padilla et al., 2007), that is. as early as one cell cycle after the major wave of embryonic genome activation occurs (Schultz, 2002; Hamatani et al., 2004), which suggests that these events might be, at least in part, linked to the transcriptional programme of the embryo. The developmental stage at which genome activation occurs in other mammalian species varies considerably: it takes place at the 1-to- 2-cell stage in mice, the 4-to- 8-cell stage in cows and humans, and the 8-to- 16-cell stage in sheep and rabbits (Schultz and Heyner, 1992). Would this anticipate a different timing for an eventual \u2018cell fate path\u2019 for other species? Normal fertile adults can be derived from single blastomeres from 2-, 4- and 8-cell stage embryos in the rabbit, sheep and cattle (Moore et al., 1968; Willadsen, 1981; Willadsen and Polge, 1981). Thus, these species indeed support blastomere isolation and further development at later stages than the mouse does.\nTransplantation of isolated 4-cell blastomeres into morula stage embryos has demonstrated that the blastomeres at the 4-cell stage are totipotent (Kelly, 1977). In this context, it is important to note that aggregating the \u2018vegetal\u2019 cell from ME embryos to form chimeric embryos, showed that this cell is able to contribute to all tissues in the embryo. However, aggregating the same blastomere with other \u2018vegetal\u2019 blastomeres from ME embryos exclusively, results in a failure to proceed through development (Piotrowska-Nitsche et al., 2005). Thus, the environment where the blastomeres develop is crucial for the success of the embryo throughout development, and in a \u2018normal\u2019 situation, where the embryo has not been perturbed and a given cell develops in its niche, some differences appear to develop, which are, at least partially, related to the epigenetic information of each of them (Torres-Padilla et al., 2007). If epigenetic asymmetries of the early embryo are related to lineage allocation, it is still uncertain whether they are a cause or a consequence for lineage choice. Also, it remains unknown whether such epigenetic asymmetries would affect only particular regions of the genome. For example, whether genes involved in specification of the ICM such as Sox2 and Nanog would all be targeted by the same epigenetic marks in the same blastomere or whether such marks would vary among genes and\/or among blastomeres. Further, are \u2018inner\u2019 and \u2018outer\u2019 cells at the 16-cell stage distinguishable in terms of their chromatin landscapes?\nThe experiments showing that blastomeres are able to respond to the overexpression of a histone modifier and change their fate, indicate that these cells have not yet acquired a \u2018fix\u2019 destiny, but that they can still be responsive to some kind of signals. These experiments have an important impact on showing that manipulating the epigenetic information can affect cell fate in the preimplantation embryo, in line with the importance of epigenetic mechanisms being crucial for early development. Moreover, these results do illustrate that such cells can still be flexible and accommodate themselves after a perturbing event (in this case, overexpression of a histone methyltransferase and the downstream effects on the information that is. imparted through specific histone modifications).\nThese studies have originated some interest from the part of the medical community, particularly, in the context of preimplantation genetic diagnosis (PGD) (Goldman, 2007). Does it matter to the embryo which cell is used for genetic diagnosis? Might the death of one of these cells have an effect on subsequent development? This question is equally valid on the impact of cell loss upon cryopreservation (Cohen et al., 2007). Although these are very delicate questions with very likely no easy answer, from the perspective of the mouse embryo, at least four things are to be considered. A tendency for a blastomere of some 4-cell stage embryos to contribute to a given region of the embryo has been documented. Second, the blastomeres in the 4-cell stage show clear differences not only in the levels of histone methylation, but also in their transcriptional activiy when they develop without being perturbed. However (third), the cells undergo a redirection of cell fate when a histone methyltransferase is overexpressed, indicating that they can readapt. Finally, the environment in which cells develop seems to be crucial for completing development and somehow the remaining cells in the embryo could compensate provided they are somehow different from each other. It is also important to note that a 4-cell stage mouse embryo might correspond to a very different developmental stage than a 4-cell embryo in other mammalian species, as illustrated by the differences in the onset of genome activation between them. Indeed, PGD is most often performed at the 8-cell stage and some reports document a better rate of development when 6-to- 9-cell stage embryos are diagnosed, as opposed to 3-to- 4-cell stage embryos (Wang et al., 2007). Moreover, the effects of in vitro fertilization procedures and culture on embryonic development are also extremely important, as they have been shown to alter epigenetic information in the mouse (Li et al., 2005).\nAs a final consideration, I would like to leave the reader with an open perspective of some ongoing questions in the field. Although there are strong indications that epigenetic mechanisms are involved in cell fate determination, we are still far from establishing a direct link between an epigenetic mark(s) and the derivation of a particular cell lineage in the embryo. Much work is still to be done to determine how these mechanisms are set in play upon fertilization and how they are transmitted during subsequent cleavage stages. Also, what other epigenetic marks contribute to the inheritability of cell fate decisions? How do these marks relate to and influence each other? Are different lineage-specific genes marked by a different combination(s) of epigenetic marks? Do the marking of these genes occur at different stages of development? It is also tempting to expand these notions into the stem cell field and question whether these mechanisms would also underlie the intrinsic self renewal ability of adult stem cells and their potential to differentiate into other cell types. The exquisite complexity and richness of chromatin-regulated events in the early embryo will certainly be the subject of exciting research in the future.\nFunding\nThe author acknowledges support from the PNRRE\/INSERM.","keyphrases":["epigenetics","mouse embryo","cell fate","pluripotency","histone methylation"],"prmu":["P","P","P","P","P"]}
{"id":"J_Autism_Dev_Disord-4-1-2175022","title":"Brief Report: Adults with Mild Autism Spectrum Disorders (ASD): Scores on the Autism Spectrum Quotient (AQ) and Comorbid Psychopathology\n","text":"While knowledge about symptom presentation of adults with mild ASD, including comorbid psychopathology, is limited, referral of adults with suspected mild PDD is increasing. We report on pilot research investigating whether patients diagnosed with mild ASD (n = 15) and patients who were not diagnosed with ASD (n = 21) differed in terms of (a) AQ scores and (b) Axis I and II disorders, assessed by the SCAN and the IPDE. Additionally, AQ scores were compared with those from non-ASD patients referred to a general outpatient clinic (n = 369). The results showed very few differences between ASD patients and non-ASD patients. Self-report may not differentiate mild ASD patients from non-ASD patients and Axis I and II disorders seem equally prevalent among these two groups.\nIntroduction\nAlthough there is consensus (APA, 1994) about DSM IV criteria for children and adolescents with Autistic Disorder and Asperger\u2019s disorder, the largest category, PDDNOS, remains difficult to classify. The latter is even more true for adult patients since the DSM-IV criteria are not formulated with adult age in mind (Gillberg, 1998; Tamtam, 1991; Vermeulen, 2002). The Autism Diagnostic Interview \u2013 Revised (ADI-R, Lord, Rutter, & Le Couteur, 1994) and the Autism Diagnostic Observation Schedule-Generic (ADOS-G; Lord et al., 2000) are validated only for children and adolescents. Follow-up studies of adolescents and adults with autism indicate that a slow decrease in symptoms across time occurs in a small group, particularly the less severely affected patients (Seltzer, Shattuck, Abedutto, & Greenberg, 2004). However, there is a lack of knowledge about adults with mild ASD, i.e., Asperger\u2019s Disorder and PDDNOS.\nIn light of this limited knowledge on symptom presentation in adults, the present paper reports on a pilot research-project that was done with two aims: (a) to test the Autism Spectrum Quotient (AQ; Baron-Cohen, Wheelwright, Skinner, Martin, & Clubley, 2001) as an instrument for screening adult non-mentally retarded patients for ASD. We hypothesised that scores would be higher for an ASD group than for two non-ASD comparison groups; (b) there is \u201ca clinical feeling\u201d that mild ASD symptoms often mix with other disorders (e.g., Obsessive Compulsive Disorder or Schizoid Personality Disorder). Therefore, in patients referred to an ASD expertise-centre, prevalences of Axis I and II comorbid disorders were estimated to test the hypothesis that certain diagnoses are more prevalent among ASD patients than non-ASD patients.\nMethod\nParticipants\nTwo groups of patients were investigated; the first group (n\u00a0=\u00a036) consisted of patients referred to the \u201cAutism Team North Netherlands\u201d (ATN). The ATN is the ASD outpatient center of expertise for the three Northern provinces of the Netherlands (1.5\u00a0million inhabitants). On a yearly basis, about 80 new adult patients are referred for diagnostic evaluation and (advice for) treatment. Since the ATN is a \u201cthird line\u201d center of expertise, all patients were referred by psychiatrists and psychologists, working in the field of adult (but not forensic) psychiatry. Only patients with parents who were able to give the required information were included in the present study. They were 18\u00a0years or older, with an estimated IQ higher than 80. Over a span of 10\u00a0months from February 2003 through October 2003, 60 eligible patients were asked to participate, of which 35 patients and their parents consented. The patients were randomly assigned to two experienced (child) psychiatrists (CK or EH) for diagnostic assessment.\nThe second group (n\u00a0=\u00a0369) consisted of patients from the same region, who were referred in this same period to the general outpatient psychiatric clinic of the University Medical Centre Groningen. This group from the general outpatient clinic (GOC) enabled us to compare the scores on the AQ screening instrument.\nInstruments\nThe clinical standardized diagnostic protocol that was used in the intake of ATN-referred patients consisted of a semi-structured interview (taken from at least one of the parents), and observations from the ADI-R, ADOS-G, as well as clinical experience. Actual ASD symptoms were translated in the interview as much as possible to the adult-world situation. Questions about the past were used to reconstruct the developmental history of the patient as adequately as possible. The information derived from the diagnostic protocol was subsequently used to score the DSM-IV criteria on ASD. Intelligence was assessed by using a short version of the \u201cGroninger Intelligentie Test\u201d (GIT, Luteijn, 1966).\nFor classification of PDDNOS, the minimal amount of positive DSM-IV criteria was two, of which at least one derived from the social interaction domain. For Asperger\u2019s Disorder at least two items on the social domain and at least one item on the stereotypic domain needed to be present with no early delay of language. For High Functioning Autism at least six items were needed to be present, with at least two items on the social domain and at least one positive item on each of the other two domains, with onset of abnormal functioning before the age of three years.\nThe reliability of the diagnostic protocol was tested by assessing a series of seven patients simultaneously by two experienced (child) psychiatrists (CK and EH). These independent classifications differed only on one patient (PDDNOS versus non-ASD). The seven patients were not included in the present study, which started after having determined diagnostic reliability.\nThe AQ was translated in Dutch and was named AQ-D (Dutch). This self-report screening questionnaire has 50 items, which score on 5 domains of behavior: social skill, attention switching, attention to detail, communication, and imagination. Each domain has a maximum score of 10 (for details on this instrument, see Baron-Cohen et\u00a0al., 2001) The AQ-D was completed by the patient before the clinical procedure started. The investigators were blind to the outcome of AQ-D scores. Internal consistency reliability estimates (Cronbach\u2019s \u03b1) for AQ subscales were acceptable for the ASD-group but somewhat on the lower side for the non-ASD groups. Averaged across the five subscales Cronbach\u2019s \u03b1 was .67 for the ASD group, .62 for the non-ASD group referred to the ATN, and .59 for the GOC group. For the total scale internal consistency reliability was acceptable in all three groups: i.e., .85, .82, and .79, respectively.\nPresent and life-time comorbidity were assessed by using the Schedules for Clinical Assessment in Neuropsychiatry (SCAN-2.1, Giel & Nienhuis, 1996; World Health Organisation, 1992) and the International Personality Disorder Examination (IPDE, Loranger et\u00a0al., 1994). IQ, SCAN and IPDE assessments were done by a psychologist (JB) who was blind to the outcome of the ASD psychiatric diagnosis. JB was formally trained by the Groningen WHO Training Center for administering the SCAN.\nStatistical Analysis\nChi-square tests and analyses of variance were conducted to analyze possible differences between the groups.\nResults\nFifteen of the 36 patients were diagnosed with ASD. In this ASD-group, 10 patients classified as PDDNOS, 4 patients as Asperger\u2019s Disorder and 1 patient as High Functioning Autism. The number of positive DSM-IV items ranged from 2 to 8, with an average score of 3.8.\nTable\u00a01 shows gender, age, and IQ of ASD and non-ASD patients. Also included in this table are gender and age of the GOC patients. The significant differences between the groups were (a) the younger age of the ASD patients relative to the other patients groups; (b) the younger age of the non-ASD group referred to the ATN relative to the GOC patients; and (c) the predominance of males in the ATN-referred patients compared with the GOC patients.\nTable\u00a01Characteristics of participantsDiagnosisASD (n\u00a0=\u00a015)No ASD (n\u00a0=\u00a021)General outpatient clinic (n\u00a0=\u00a0369)Male\/femaleam\u00a0=\u00a012, f\u00a0=\u00a03m\u00a0=\u00a018, f\u00a0=\u00a03m\u00a0=\u00a0180, f\u00a0=\u00a0189 Average ageb (range, sd)22\u00a0years (18\u201324, 5)27\u00a0years (18\u201355, 9)35\u00a0years (14\u201373, 11)Mean IQ (sd)104 (10)105 (12)a\u00a0Difference between GOC group and non-ASD group (chi-square\u00a0=\u00a014.2, df\u00a0=\u00a01, p\u00a0<\u00a00.0001); difference between GOC group and ASD group (chi-square\u00a0=\u00a05.8, df\u00a0=\u00a01, p\u00a0<\u00a00.05); no difference between ASD group and non-ASD groupb\u00a0GOC group differs from non-ASD group (p\u00a0<\u00a00.01) and from ASD group (p\u00a0<\u00a00.0001); no difference between ASD group and non-ASD group\nTable\u00a02 shows AQ-D sub-domain and total scores for the three groups. Generally, there were no significant differences between the three groups. The one significant difference was on the sub-domain Communication between the ASD group and the general outpatient group. Further, within the ASD group, average AQ-D scores varied with the severity of ASD: High Functioning Autism: 31 (n\u00a0=\u00a01); Asperger\u2019s Disorder: 24 (n\u00a0=\u00a04), and PDDNOS: 21 (n\u00a0=\u00a010). In light of the small sample sizes the latter differences were not tested for statistical significance.\nTable\u00a02Mean scores AQ-D (sd) and ANOVA tests for differences in mean scoresASD (n\u00a0=\u00a015)No ASD (n\u00a0=\u00a021)General outpatient clinic (n\u00a0=\u00a0369)Social skill5.0 (2.8)4.2 (2.6)4.1 (2.6)Attention switching5.1 (2.6)5.3 (2.4) 5.3 (2.3) Attention to detail3.5 (2.3)4.4 (2.2)4.2 (2.3)Communication*4.5 (2.1)3.5 (2.2)2.8 (1.8)Imagination4.4 (2.4)4.3 (2.0)3.5 (1.9)Total22.5 (8.4)21.8 (7.6)19.9 (7.0)*\u00a0Significant difference between ASD group and GOC group at p\u00a0<\u00a00.01\nTable\u00a03 reports Axis I diagnoses in ASD and non-ASD patients as assessed by the SCAN. With one exception, there were no differences in either Axis I or Axis II diagnoses between the two groups. This held for both actual and lifetime (not reported) Axis I diagnoses. The one exception was that more patients were diagnosed with actual psychotic disorder NOS in the non-ASD group than in the ASD group. When we aggregated separate categories into more broad diagnostic categories, i.e., anxiety disorders, psychotic disorders, and mood disorders, there were no differences between the two groups in past-month or lifetime diagnoses either, except for actual psychotic disorders (results not shown).\nTable\u00a03SCAN-derived actual (past month) DSM IV Axis I diagnosesASD (N\u00a0=\u00a015)n (%)No ASD (N\u00a0=\u00a021)n (%)No disorder7 (47)7 (33)Mood disorder with psychotic symptoms2 (13)\u2013Mood disorder without psychotic symptoms2 (13)3 (14)Substance abuse 3 (20)2 (10)Sleep disorders4 (27)6 (29)Psychotic disorder NOS\u20134 (19)*Schizophrenia\u2013\u2013Social Phobia3 (20)4 (19)Panic attacks\/Agoraphobia2 (13)1 (5)Other anxiety disorders1 (7)\u2013Obsessive compulsive disorders1 (7)1 (5)Other disorders1 (7)2 (10)*\u00a0Significant difference between ASD group and no ASD group at p\u00a0<\u00a00.05\nTable\u00a04 reports Axis II diagnoses as assessed by the IPDE. ASD and non-ASD patients did not differ on individual Axis 2 diagnoses. Also, when we tested whether ASD and non-ASD groups differed with regard to the total number of patients with any complete, partial, or either of these two, Axis 2 disorders (bottom two rows, Table\u00a04), we found no difference between the two groups.\nTable\u00a04IPDE derived Axis II diagnoses by ASD statusASD (N\u00a0=\u00a015)No ASD (N\u00a0=\u00a021)Completen (%)Partialn (%)Completen (%)Partialn (%)Parano\u00efd\u2013\u2013\u20131 (5)Schizo\u00efd 1 (7)2 (13)1 (5)1 (5)Schizotypical\u20131 (7)\u2013\u2013Antisocial\u20131 (7)\u2013\u2013Borderline 1 (7)\u2013\u20131 (5)Avoidant 1 (7)1 (7)2 (10)\u2013Obsessive compulsive\u2013\u20133 (14)1 (5)Personality disorder NOS1 (7)2 (13)3 (14)1 (5)Any personality disorder3 (20)6 (40)8 (38)3 (14)Any personality disorder partial or complete7 (47)10 (48)\nDiscussion\nThus far, publications about screening instruments for adult ASD use the Autism-Spectrum Quotient (AQ; Baron-Cohen et\u00a0al., 2001) or the Autism Spectrum Disorder in Adults Screening Questionnaire (ASDASQ; Nylander & Gillberg, 2001). With the help of the latter instrument, Nylander & Gillberg (2001) and Chang et\u00a0al. (2003) estimated a 1.4 and 0.6 % ASD prevalence in a general psychiatry outpatient clinic for adults. The AQ was investigated by Baron-Cohen in 2001 in Asperger\u2019s Disorder or High Functioning Autism patients and compared with several subgroups in the general population, and was tested again in 2005 at the national diagnostic clinic for Asperger\u2019s Disorder (Woodbury-Smith, Robinson, Wheelwright, & Baron-Cohen, 2005). The AQ differentiated the ASD patients adequately from healthy controls, and ASD patients from non-ASD patients.\nThe present study tried to replicate the findings of Baron-Cohen. However, our results showed that the AQ-D did not differentiate between an ASD group (predominantly PDDNOS) and two non-ASD patient groups. Only for the communication domain, the ASD patients had higher scores relative to the general outpatient group. It is of interest to mention here that this domain was also one of the two domains that differentiated parents of autistic patients from healthy controls (Bishop et\u00a0al., 2004). The most probable explanation of this discrepant outcome is the fact that the Dutch patients were less severely affected than the British patients. The ASD patients but also the non-ASD patients in the Baron-Cohen papers had higher total mean scores on the AQ (for ASD: 35.8 and 35.6 and for non-ASD: 26.2) than our patients (ASD: 22.5, non-ASD and general outpatients: 21.8 and 19.9). By comparison, British, non-psychiatric, control groups typically have total scores of around 16\u201317 (Baron-Cohen et\u00a0al., 2001). Interestingly, the mean score on the recent Japanese version of the AQ was 29.4 in ASD patients and 22.2 in non-psychiatric controls (Kurita, Koyoma, & Osada, 2005). The Japanese authors, like we, reasoned that part of the Japanese patients may have been less severely affected relative to the British patients. They also argued that it is possible that autistic related behavior as assessed by the AQ is more prevalent in the Japanese population than in the British. In the absence of the actual diagnoses of the non-ASD patients in both the British and one of the Dutch samples, it remains difficult to pinpoint why scores between the British and Dutch non-ASD patients (26.2 and 21.8\/19.9, respectively) differ. The absence of differences between scores of the AQ-D in the present study suggests that there might be \u201cASD-like\u201d symptoms, as felt by the patients themselves, present in the non-ASD patients in the ATN-referred group and in the patients of the general outpatient clinic, and revealed by self-report. Indeed, the individuals in the non-ASD group were referred to the ATN for the possible presence of an ASD diagnosis, so ASD-like symptoms must have been voiced by these patients even if these could not be diagnosed as such on the basis of the standardized diagnostic protocol. In sum, although there are still many open questions, for example about differences in scores cross-culturally, the present data suggest that self-report questionnaires are not adequate for differentiating less severe ASD patients from other patient groups. Another study is needed to investigate the merits of an alternative approach to this problem; i.e., a parents\/caregivers questionnaire as a screening tool for adult ASD.\nOur second aim was to look into possible differences in comorbidity of Axis I and Axis II DSM IV classifications in ASD and non-ASD groups. This because of the notion that mild ASD symptoms often mix with symptoms of other disorders such as Obsessive Compulsive Disorder or Schizoid Personality Disorder. There is a scarcity of research on this topic. For example, (comorbid) diagnoses of the British patients were not reported in the paper of Woodbury-Smith et\u00a0al. (2005). For the Dutch, ATN-referred group, however, we took a systematic approach to diagnosing DSM IV Axis I and II disorders. The results indicated that, except for Psychotic Disorder NOS which was diagnosed in roughly 20% of the non-ASD group and not in the ASD group, there were no significant differences in the pattern of diagnoses between the ASD and non-ASD patients. Possibly, the difference in Psychotic Disorder partly accounts for the referral to the ATN and for the \u201cASD-like\u201d symptoms in these patients as discussed in the above paragraph. However, the general picture indicated by our data is that clinical accounts of a relatively high prevalence and\/or a specific profile of comorbid disorders in the ASD group relative to the non-ASD group could not be confirmed.\nThere are important limitations to this study, among which is the small number of patients in the ATN-referred group. This major drawback precludes any definite conclusions. Of note is further that the ASD patients in this investigation belonged to the less-severe side of the spectrum. This makes it difficult to compare results with the investigations by Baron-Cohen and collegues. Clinical experience indicates that ASD patients, compared to non-ASD patients, benefit more from treatment by structuring, long-term repetitive treatment, and adaptations of the environment. This may hold even more when comorbid conditions are present. Since the prevalence of patients with less severe ASD is relatively high compared to the more severe ASD categories, efficient and valid screening of this group and charting possible comorbidity is consequently an even more important issue. The present study provided a modest contribution in improving our knowledge in this direction; clearly, much more research is needed.","keyphrases":["autism spectrum quotient","comorbidity","pddnos","personality disorders","adult autism spectrum disorders"],"prmu":["P","P","P","P","R"]}
{"id":"Int_J_Biochem_Cell_Biol-1-5-1906734","title":"Cytosolic and ER J-domains of mammalian and parasitic origin can functionally interact with DnaK\n","text":"Both prokaryotic and eukaryotic cells contain multiple heat shock protein 40 (Hsp40) and heat shock protein 70 (Hsp70) proteins, which cooperate as molecular chaperones to ensure fidelity at all stages of protein biogenesis. The Hsp40 signature domain, the J-domain, is required for binding of an Hsp40 to a partner Hsp70, and may also play a role in the specificity of the association. Through the creation of chimeric Hsp40 proteins by the replacement of the J-domain of a prokaryotic Hsp40 (DnaJ), we have tested the functional equivalence of J-domains from a number of divergent Hsp40s of mammalian and parasitic origin (malarial Pfj1 and Pfj4, trypanosomal Tcj3, human ERj3, ERj5, and Hsj1, and murine ERj1). An in vivo functional assay was used to test the functionality of the chimeric proteins on the basis of their ability to reverse the thermosensitivity of a dnaJ cbpA mutant Escherichia coli strain (OD259). The Hsp40 chimeras containing J-domains originating from soluble (cytosolic or endoplasmic reticulum (ER)-lumenal) Hsp40s were able to reverse the thermosensitivity of E. coli OD259. In all cases, modified derivatives of these chimeric proteins containing an His to Gln substitution in the HPD motif of the J-domain were unable to reverse the thermosensitivity of E. coli OD259. This suggested that these J-domains exerted their in vivo functionality through a specific interaction with E. coli Hsp70, DnaK. Interestingly, a Hsp40 chimera containing the J-domain of ERj1, an integral membrane-bound ER Hsp40, was unable to reverse the thermosensitivity of E. coli OD259, suggesting that this J-domain was unable to functionally interact with DnaK. Substitutions of conserved amino acid residues and motifs were made in all four helices (I\u2013IV) and the loop regions of the J-domains, and the modified chimeric Hsp40s were tested for functionality using the in vivo assay. Substitution of a highly conserved basic residue in helix II of the J-domain was found to disrupt in vivo functionality for all the J-domains tested. We propose that helix II and the HPD motif of the J-domain represent the fundamental elements of a binding surface required for the interaction of Hsp40s with Hsp70s, and that this surface has been conserved in mammalian, parasitic and bacterial systems.\n1\nIntroduction\nWhile many classes of molecular chaperones exist, members of the heat shock protein 40 (Hsp40) and heat shock protein 70 (Hsp70) families form chaperone pairs that are amongst the most ubiquitous (Fink, 1999). The diverse cellular processes involving these chaperones include the correct folding of nascent polypeptide chains, prevention of protein denaturation and misfolding during cellular stress, degradation of proteins, protein translocation, and quaternary assembly\/disassembly (Hennessy, Nicoll, Zimmermann, Cheetham, & Blatch, 2005b).\nThe major molecular chaperone, Hsp70, consists of an N-terminal ATPase domain and a C-terminal substrate-binding domain. The affinity of Hsp70 for protein client is modulated by ATP binding and hydrolysis. In the ATP bound state, affinity of the substrate-binding domain for the client protein is low and exchange rates are high. Hydrolysis of ATP to ADP results in high affinity for the substrate and low exchange rates, effectively locking the substrate into the binding pocket (Schmid, Baici, Gehring, & Christen, 1994). This integral step in assisted protein folding is directly modulated by the binding of Hsp40 proteins (Cheetham, Jackson, & Anderton, 1994; Liberek, Marszalek, Ang, Georgopoulos, & Zylicz, 1991). Furthermore, there is evidence that some Hsp40 proteins bind client protein first and then target client to Hsp70 (Han & Christen, 2003; Suh, Lu, & Gross, 1999).\nHsp40s are defined by the presence of an approximately 70 amino acid region known as the J-domain, which is essential for interaction with Hsp70. The J-domain is a highly conserved \u03b1-helical structure that interacts with the Hsp70 ATPase domain and possibly also with the Hsp70 substrate-binding domain (Auger & Roudier, 1997; Hennessy et al., 2005b; Suh et al., 1999). Hsp40s are divided into three groups based on their possession of domains in addition to the J-domain (Cheetham & Caplan, 1998). Type I Hsp40s contain four primary domains: an N-terminal J-domain, a glycine\/phenylalanine (GF)-rich region, a zinc finger domain and a C-terminal domain. Type I Hsp40s have been shown to bind protein substrates at their C-terminal domain and to have independent chaperone activity by inhibiting denaturation and aggregation (Langer et al., 1992; R\u00fcdiger, Schneider-Mergener, & Bukau, 2001). Type II Hsp40s contain an N-terminal J-domain, a GF-rich region and a C-terminal domain. Type III Hsp40s contain the J-domain, and this may occur at any position within the protein. Other than the J-domain, the type III Hsp40s are highly divergent in size, sequence and structure and tend to serve highly specialized functions. Specific Hsp40-Hsp70 partnerships have been identified that are dedicated to the correct folding of distinct subsets of client proteins. We and others have proposed that the J-domain makes important contributions to the affinity and specificity of binding of a specific Hsp40 protein to its partner Hsp70 (Garimella et al., 2006; Hennessy et al., 2005b).\nThe structures of the J-domain from six Hsp40 and Hsp40-like proteins have been published: E. coli DnaJ (Huang, Flanagan, & Prestegard, 1998; Pellecchia, Szyperski, Wall, Georgopoulos, & W\u00fcthrich, 1996); human HDJ1 (Qian, Patel, Hartl, & McColl, 1996); E. coli Hsc20 (Cupp-Vickery & Vickery, 2000); the large T antigen from murine polyomavirus (Berjanskii et al., 2000); the large T antigen from SV40 in conjunction with the retinoblastoma tumour suppressor (Kim, Ahn, & Cho, 2001); and bovine auxilin (Gruschus, Greene, Eisenberg, & Ferretti, 2004; Jiang et al., 2003). The J-domain structures reveal the presence of four \u03b1-helices (helices I\u2013IV) and a loop region between helices II and III. Helix I is usually seen as a short helix in type I Hsp40s. However, although there are a number of highly conserved hydrophobic residues in helix I, the tertiary structure of helix I, as seen from X-ray and NMR studies, becomes divergent in types II and III Hsp40s. The helices II and III are structurally conserved in all known J-domains, in particular helix II which bears an overall positive charge and is thought to interact with the negatively charged underside of the ATPase domain of Hsp70. Of particular importance is the HPD tripeptide that resides in the transhelix loop between helices II and III. Alteration of these residues always results in loss of functional interaction between Hsp40 and Hsp70 (Genevaux, Wawrzynow, Zylicz, Georgopoulos, & Kelley, 2001; Laufen et al., 1999; Mayer, Laufen, Paal, McCarty, & Bukau, 1999; Tsai & Douglas, 1996; Wittung-Stafshede, Guidry, Horne, & Landry, 2003). Apart from the HPD motif, the other amino acids on the J-domain of Hsp40 proteins that are involved in the binding to a partner Hsp70 are less precisely defined. However, as a result of our work (Hennessy, Cheetham, Dirr, & Blatch, 2000; Hennessy, Boshoff, & Blatch, 2005a) and that of other researchers (Garimella et al., 2006; Genevaux, Schwager, Georgopoulos, & Kelley, 2002; Genevaux et al., 2003; Lu & Cyr, 1998; Suh et al., 1999), other residues and regions outside the HPD motif, especially helices II, III and IV, are gradually being implicated in the general binding and specificity of interaction of Hsp40 proteins with Hsp70 proteins.\nThe high resolution structure of the E. coli DnaJ J-domain suggested that J-domain stabilization occurred through a buried core of hydrophobic residues, primarily Ile9, Leu10, Val12, Ile21, Ala53 and Leu57 (Hill, Flanagan, & Prestegard, 1995; Pellecchia et al., 1996; Szyperski, Pellecchia, Wall, Georgopoulos, & Wuthrich, 1994). Amino acid sequence alignment analysis of over 200 type I Hsp40s showed that Tyr7, Ala53 and Leu57 were conserved in over 98% of all sequences, and that Leu10 was absolutely conserved in all sequences (Hennessy et al., 2000). Tyr7 projects upward in the E. coli DnaJ J-domain tertiary structure and potentially makes contacts with residues of helices II, III and IV. Leu10 projects toward the helix II\u2013helix III inter-helical space, potentially interacting with residues of helices II and III. Therefore, Tyr7 and Leu10 may be critical in ensuring the stability of the helix-loop structure of helix II\u2013helix III for presentation to Hsp70 (Hennessy et al., 2005a). Arg26 has been shown to be critical for J-domain function in E. coli DnaJ and Agrobacterium tumefaciens DnaJ (Agt DnaJ), and has been proposed to be part of a network of residues on helix II (and possibly helix III) that form an Hsp70-binding site on the J-domain (Genevaux et al., 2002; Hennessy et al., 2005a,b). In addition, Genevaux et al. (2002) described Tyr25 of E. coli DnaJ as a candidate catalytic residue that potentially comprises part of this binding site. His33 of the HPD motif in the loop region has been extensively documented as being a critical residue in regulation of Hsp70 ATPase stimulation, and substitution of this residue results in loss of functional interaction between Hsp40 and Hsp70 (Genevaux et al., 2002; Kelley & Georgopoulos, 1997; Laufen et al., 1999; Mayer et al., 1999; Tsai and Douglas, 1996). Genevaux et al. (2002) revealed that a glycine substitution at Arg36 (Lys36 in Agt DnaJ) resulted in loss of J-domain function and suggested that the critical cluster for J-domain function is not only the HPD tripeptide but could also include Arg36 and Asn37 to form the critical HPD-R\/K-N pentapeptide. Helix III appears to contain a so-called KFK motif (Hennessy et al., 2000), and a number of amino acid substitutions have been conducted on this motif. In particular, the F47A substitution in the J-domain of E. coli DnaJ resulted in loss of in vivo function (Genevaux et al., 2002), suggesting this was an essential amino acid for J-domain structure and function, potentially by forming interactions with His33 of the HPD motif. Interestingly, an F47L substitution in Agt DnaJ had no detectable effect on its in vivo function, suggesting that the Leu residue could sufficiently contribute to the contacts originally made by the Phe residue (Hennessy et al., 2005a). The highly conserved Leu57 of helix III projects into the J-domain interior and is likely to be a key residue in holding helices II and III together. Furthermore, experimental evidence has suggested that Leu57 was essential for J-domain function (Hennessy et al., 2005a). While residues of helices II and III and the loop region linking the two helices are crucial to J-domain function (Pellecchia et al., 1996), it has been proposed that the residues of helix IV are not essential to the co-chaperone function of DnaJ (Genevaux et al., 2002). However, recent studies involving the J-domain of Agt DnaJ identified residues on helix IV that were important for its in vivo function, suggesting a structural or functional role of this helix in other DnaJ homologues, potentially in the enhancement of the affinity or specificity of interactions with Hsp70 (Hennessy et al., 2005a,b). Asp59 and Arg63 are part of a conserved charged cluster of residues in helix IV, with Arg63 being part of the conserved QKRAA motif on helix IV of the J-domain of DnaJ. Data from studies on E. coli DnaJ (Suh et al., 1999) and Agt DnaJ (Hennessy et al., 2005a) suggested that substitutions of Asp59 and Arg63 partially disrupted the structure and function of these proteins, and recently it has been suggested that helix IV may contribute to the specificity of J-domains for their Hsp70 partners (Garimella et al., 2006; Hennessy et al., 2005b).\nHsp40 proteins are not completely interchangeable with respect to their interaction with distinct Hsp70s. The cytosolic Hsp70s, Hsc70, yeast Ssa1 and E. coli DnaK, are not interchangeable with BiP with respect to protein translocation into the endoplasmic reticulum (Brodsky, Hamamoto, Feldheim, & Schekman, 1993; Wiech, Buchner, Zimmermann, Zimmermann, & Jakob, 1993). Furthermore, E. coli DnaJ is capable of stimulating the ATPase activity of mammalian Hsc70, whereas mammalian Hdj1 is incapable of stimulating the ATPase activity of DnaK (Minami, H\u00f6hfeld, Ohtsuka, & Hartl, 1996). Hence, the J-domain may contain sequence and structural features that mediate the specificity of binding between Hsp40s and partner Hsp70s, and a number of J-domain swapping experiments have been conducted to establish the elements of specificity (reviewed in Hennessy et al., 2005b). In general, J-domains appear to be interchangeable when they are derived from Hsp40 proteins that interact with functionally equivalent or homologous Hsp70 proteins, or are involved in similar cellular processes (Deloche, Kelley, & Georgopoulos, 1997; Genevaux et al., 2001). However, J-domains appear to be less interchangeable when derived from Hsp40 proteins that are involved in very different cellular processes; for example, interchanging J-domains of type I Hsp40s with those of membrane-bound Hsp40s (Schlenstedt, Harris, Risse, Lill, & Silver, 1995) or viral Hsp40-like proteins such as the T antigen (Kelley and Georgopoulos, 1997; Sullivan et al., 2000). This suggests that two broad classes of J-domains may have evolved; those J-domains that have evolved to specifically interact with Hsp70s involved in assisted protein folding, and those J-domains that have evolved to specifically interact with Hsp70s involved in more specialized cellular processes. To date no systematic analysis has been conducted on the interchangeability of J-domains between all the types I, II and III Hsp40-like proteins from any one cell type, compartment or system.\nIn this study we have conducted domain swapping of the J-domains from a selection of divergent Hsp40s of mammalian and parasitic origin (malarial Pfj1 and Pfj4, trypanosomal Tcj3, human ERj3, ERj5, and Hsj1, and murine ERj1) in an attempt to identify system specific and common factors in Hsp40\u2013Hsp70 interactions. The similarities and differences in the structure and function of Hsp40s of parasites and their hosts have yet to be determined, and therefore from this broader perspective we were interested in a comparative analysis of the J-domains of parasitic and human origin. An in vivo functional assay was used to assess the ability of the J-domains to substitute for the J-domain of a prokaryotic type I Hsp40. Furthermore, the functional importance of specific residues was addressed through single amino acid substitution analysis. The data suggested that cytosolic and ER J-domains of mammalian and parasitic origin can interact with DnaK using a common mechanism, and that a fundamental binding surface appears to be conserved in J-domains of Hsp40s of mammalian, parasitic and bacterial origin.\n2\nMaterials and methods\n2.1\nMaterials\nE. coli OD259 (MC4100 araD139 \u0394ara714 \u0394cbpA::kan dnaJ::Tn10-42) was kindly provided by Dr. Olivier Deloche (University of Geneva, Switzerland). The pGEX-4T-ERj1, pGEX-4T-ERj3 and pGEX-4T-ERj5 plasmids encode mouse ERj1, human ERj3 and human ERj5. The pET23b-Tcj3 construct encoding Tcj3 has been described (Edkins, Ludewig, & Blatch, 2004), while the pCMV-Tag3a(Hsj1a) construct encodes Hsj1a. Mutagenesis was performed using the QuikChange site directed mutagenesis kit (Stratagene, USA) as per the manufacturer's instructions. Mutagenesis and PCR primers were synthesised by IDT (USA) and Inqaba Biotec (SA).\n2.2\nCreation of the Agt DnaJ chimera proteins\nThe pQE30-derived pRJ30 vector containing the Agt DnaJ coding sequence (Hennessy et al., 2005a) served as a base vector for all domain swapping and subsequent mutagenesis. A silent mutation encoding a BstBI restriction site was introduced directly downstream of the J-domain at residue Phe74 of Agt DnaJ to produce vector pRJ-B (Fig. 1A). Removal of the coding region of the Agt DnaJ J-domain and insertion of the respective coding regions of the J-domains under investigation into the Agt DnaJ coding region backbone was achieved through use of the BstBI restriction site and a BamHI restriction site immediately upstream of the start of Agt DnaJ coding region (Fig. 1A).\nE. coli codon optimized versions of the Plasmodium falciparum Pfj1 J-domain (Fig. 1B) and full length Pfj4 (Nicoll et al., 2006) were synthesized using Polymerase Chain Reaction (PCR) assembly (Stemmer, Crameri, Ha, Brennan, & Heyneker, 1995). The gene design process was also utilized to introduce the BamHI and BstBI sites to allow subsequent domain swapping. Subcloning of the coding regions for the Pfj1 (residues 60\u2013128) and Pfj4 (residues 1\u201379) J-domains into the pRJ-B vector resulted in the creation of the expression constructs encoding the chimera Pfj1-J-Agt-DnaJ and Pfj4-J-Agt-DnaJ, respectively. The coding region for the Tcj3 (residues 1\u201374) J-domain was amplified by PCR from pET23b-Tcj3 with BamHI and BstBI restriction sites to allow subcloning into pRJ-B. Insertion of the coding region for the Tcj3 J-domain into the pRJ-B vector resulted in the creation of the expression construct encoding the Tcj3-J-Agt-DnaJ chimera. Similarly, the coding regions for the J-domains of Hsj1a (residues 1\u201372), ERj1 (residues 56\u2013128), ERj3 (residues 23\u201397) and ERj5 (residues 35\u2013100) were PCR amplified from pCMV-Tag3a (Hsj1a), pGEX-4T-ERj1, pGEX-4T-ERj3 and pGEX-4T-ERj5, respectively, and subcloned into pRJ-B to give expression constructs encoding Hsj1-J-Agt-DnaJ, ERj1-J-Agt-DnaJ, ERj3-J-Agt-DnaJ and ERj5-J-Agt-DnaJ. All mutations were produced by the whole-plasmid linear amplification approach using complementary oligonucleotides (QuikChange site directed mutagenesis kit, Stratagene, USA). Primers were designed so as to introduce or eliminate a restriction endonuclease site to facilitate the identification of successful mutants by restriction analysis. Mutations were confirmed by subsequent DNA sequencing.\n2.3\nIn vivo complementation assays\nComplementation assays were performed in the thermosensitive E. coli dnaJ cbpA strain, OD259 (Deloche et al., 1997; Kelley and Georgopoulos, 1997). Agt DnaJ has been shown to functionally replace CbpA and DnaJ in E. coli OD259 at 40\u00a0\u00b0C similarly to E. coli DnaJ (Hennessy et al., 2005a). Thus E. coli OD259 cells exogenously producing Agt DnaJ from a pQE30-based plasmid served as the positive control for the functional in vivo assays. Substitution of His33 of the HPD motif of Agt DnaJ is known to abolish interactions of the protein with DnaK (Hennessy et al., 2005a), therefore E. coli OD259 cells exogenously producing Agt DnaJ-H33Q from a pQE30-based plasmid served as the negative control.\nPlasmids were transformed into competent E. coli OD259, and single colonies were used to inoculate 5\u00a0ml yeast-tryptone (YT) broth containing 100\u00a0\u03bcg\/ml ampicillin for plasmid selection and 50\u00a0\u03bcg\/ml kanamycin for strain selection. The cultures were grown overnight at 30\u00a0\u00b0C, before being diluted 1:100 with YT broth containing 100\u00a0\u03bcg\/ml ampicillin and 50\u00a0\u03bcg\/ml kanamycin, and grown further at 30\u00a0\u00b0C until an A600 of approximately 2.0 was reached. Cultures were diluted to an A600 of 0.3 and serial dilutions were performed to a final dilution of 1\u00a0\u00d7\u00a010\u22128. Aliquots (3\u00a0\u03bcl) of each of these dilutions were spotted onto agar plates containing 50\u00a0\u03bcM IPTG. Plates were grown at 30\u00a0\u00b0C, 40\u00a0\u00b0C and 42\u00a0\u00b0C, respectively, to determine the ability of Agt DnaJ, Agt DnaJ-H33Q and the chimera proteins to reverse the thermosensitivity of E. coli OD259.\n2.4\nWestern analysis for the detection of chimeras\nWestern analysis was performed on whole cell lysates of E. coli OD259 and its transformants producing His6-tagged Agt DnaJ and chimera proteins. Proteins were resolved on a 12% (acrylamide, w\/v) sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to nitrocellulose membrane. Proteins of interest were detected using a mouse anti-His antibody (Amersham, UK) and horseradish-peroxidase-conjugated anti-mouse secondary antibody (Amersham, UK) using chemiluminescence-based detection (ECL Western blotting kit, Amersham, UK). Images were captured using a Chemidoc chemiluminescence imaging system (Biorad, USA).\n2.5\nBinding studies with ERj1-J and BiP\nThe pGEX-4T-1-based construct containing the coding region for the J-domain of murine ERj1 (originally called Mtj1; Brightman, Blatch, & Zetter, 1995) fused downstream of the coding region for glutathione S-transferase (GST) has been described previously (Dudek et al., 2002). The heterologous overproduction and purification of the GST-ERj1-J fusion protein was carried out as described previously for other GST-J-domain fusion proteins (Tyedmers et al., 2000). Hamster BiP was generously provided by Dr. Martin Jung (Universit\u00e4t des Saarlandes, Germany). For the ERj1-BiP pull-down binding assays, purified GST-ERj1-J and its mutant derivatives were buffer exchanged into phosphate buffered saline (PBS) at 4\u00a0\u00b0C so as to remove GSH from the purified protein. A sample (80\u00a0\u03bcl) of a 50% slurry of GSH-Sepharose beads was equilibrated in PBS (200\u00a0\u03bcl). Sufficient GST-ERj1-J was added to the GSH-Sepharose suspension to give 0.5\u00a0\u03bcM final concentration. The suspension was incubated for 1\u00a0h at 4\u00a0\u00b0C to allow binding of GST-ERj1-J to the GSH-beads, before washing twice with PBS (300\u00a0\u03bcl). For the binding reaction, the bead-bound GST-ERj1-J was reconstituted in 200\u00a0\u03bcl of PBS, with and without ATP (2\u00a0mM), and with BiP (0.5\u00a0\u03bcM), and binding allowed to occur for 1\u00a0h at 4\u00a0\u00b0C. The beads were washed twice with PBS (200\u00a0\u03bcl), and the proteins eluted from the beads by treatment with SDS-PAGE sample treatment buffer (40\u00a0\u03bcl), and analysed by SDS-PAGE.\nSurface plasmon resonance spectroscopy was carried out in a BIAlite upgrade system. Monoclonal goat anti-GST-antibodies (BIACORE, Uppsala, Sweden) were immobilized on a sensor chip CM5 (BIACORE, Uppsala, Sweden) by amine coupling according to the manufacturer's protocol. The sensor chip was equilibrated with running buffer (phosphate buffered saline containing 3\u00a0mM KCl, 1\u00a0mM MgCl2, 0.1% Tween 20 and 2\u00a0mM ATP). GST was bound to the immobilized antibodies in the reference cell, while the GST-ERj1-J fusion proteins were immobilized separately in the measuring cell (400 response units; flow rate 5\u00a0\u03bcl\/min). Subsequently, solutions containing increasing concentrations of purified BiP (0.25\u20132\u00a0\u03bcM) were passed over the chip in the presence of ATP (flow rate 20\u00a0\u03bcl\/min). Each BiP application was followed by the application of running buffer until baseline was reached. The analysis of the data were carried out using the BIAevaluation software version 2.2.4.\n3\nResults\n3.1\nBioinformatic analysis of the J-domain and the identification of structurally and functionally important residues\nThe J-domain sequences analyzed in this work covered a wide range of diverse Hsp40 types (Fig. 2). Agt DnaJ, a prokaryotic type I Hsp40 that has 57% identity to the E. coli DnaJ, was previously shown to be able to reverse the thermosensitivity of a dnaJ cbpA mutant E. coli strain (OD259), suggesting that it was capable of functionally replacing DnaJ and CbpA (Hennessy et al., 2005a). Furthermore, Agt DnaJ-H33Q was unable to reverse the thermosensitivity of E. coli OD259, suggesting that Agt DnaJ reversed the thermosensitivity of E. coli OD259 through J-domain-based regulation of the chaperone activity of E. coli DnaK (Hennessy et al., 2005a). Due to the consistency and reproducibility of the results produced in functional in vivo complementation assays of E. coli OD259 producing Agt DnaJ, this protein was chosen as the type I Hsp40 backbone molecule for the creation of chimeras. It should be noted that J-domains chimeras were created using the E. coli DnaJ backbone, and the same complementation trends were observed as for the Agt DnaJ chimeras; however, the results were not as reproducible (data not shown). The lack of reproducibility of complementation assays using the E. coli DnaJ chimeras could possibly be related to the toxic effects that have been observed for the overproduction of E. coli DnaJ (Pr\u00f6ls et al., 2001). Tcj3 is a type I Hsp40 from Trypanosoma cruzi, the protozoan causative agent of Chagas\u2019 disease (Edkins et al., 2004), while Pfj1 is a type I Hsp40 and Pfj4 is a type II Hsp40 identified from the protozoan malarial parasite P. falciparum (Watanabe, 1997). While no experimental evidence exists for the subcellular localization of Tcj3, the knowledge-based subcellular localization program PSORTII (Nakai & Kanehisa, 1992) predicted a cytosolic localization. Pfj1 has a potential mitochondrial import signal (RRKVCS; Watanabe, 1997), while Pfj4 was predicted to have a nuclear localization signal by PSORTII. HSJ1 is a human Hsp40 that is preferentially expressed in neuronal cells (Cheetham, Brion, & Anderton, 1992). Two forms of HSJ1 are found in vivo, both of which contain an identical J-domain. One form is localized to the cytosolic face of the ER (HSJ1b) while the other is cytosolic and nuclear (HSJ1a) (Chapple & Cheetham, 2003). ERj1 (also called Mtj1) is a mouse type III Hsp40 that has been shown to be enriched in microsomal and nuclear fractions of murine cells (Brightman et al., 1995). In particular it has been shown to be present in the endoplasmic reticulum (ER) in close association with active ribosomes (Dudek et al., 2002, 2005), and to interact with the ER Hsp70, BiP (Chevalier, Rhee, Elguindi, & Blond, 2000; Dudek et al., 2002). ERj3 (also called HEDJ) is a soluble Type I\/II Hsp40 that has a Cys-rich region instead of a typical Cys-repeat region, and been shown to be present in the ER lumen and to functionally interact with BiP (Bies et al., 1999, 2004; Yu & Haslam, 2005). ERj5 (also called JPDI) is a type III Hsp40 that contains thioredoxin motifs characteristic of a protein disulfide isomerase (Cunnea et al., 2003; Hosoda, Kimata, Tsuru, & Konho, 2003). ERj5 is located in the ER lumen and has been shown to interact with BiP in an ATP-dependent manner (Cunnea et al., 2003).\nThe sequences of the J-domains used in this study were aligned, phylogenetically analysed, and key conserved residues identified and highlighted on the three-dimensional structure of the E. coli DnaJ J-domain (Fig. 2). We derived a J-domain consensus sequence for the highly conserved sequences, and also identified the positions of highly conserved charged residues (Fig. 2A). The numbering used for residues in the ensuing text will be the J-domain consensus numbering which is equivalent to the E. coli DnaJ J-domain numbering (Fig. 2A). All of the J-domain sequences exhibited conservation of most of the residues previously identified to be conserved from a study of over 200 J-domains (Hennessy et al., 2000). Phylogenetic tree analysis (Fig. 2B) revealed the relative relatedness of the J-domains of the prokaryotic Hsp40s (Agt DnaJ and E. coli DnaJ), certain parasitic Hsp40s (Pfj1 and Tcj3) and certain mammalian Hsp40s (HSJ1 and ERj3; ERj1 and ERj5). The J-domain of Pfj4 appeared to be relatively divergent from all the other J-domains. In this study, the amino acid residues targeted for substitution were chosen based on their conservation (Fig. 2A), predicted orientation in three dimensions (Fig. 2C), and predicted structural and functional roles as determined from previous studies on DnaJ from our group and others (Genevaux et al., 2002; Hennessy et al., 2005a).\n3.2\nCharacterization of the chimeras\nEach of the Hsp40 J-domain chimera-encoding plasmid constructs were transformed into E. coli OD259, and the ability of the chimeras to reverse the thermosensitivity of this strain was assessed by comparing growth at 30\u00a0\u00b0C and 40\u00a0\u00b0C, respectively. For the E. coli OD259 cells producing Agt DnaJ (positive control), growth at 30\u00a0\u00b0C occurred up to the highest dilution (10\u22128), indicating that Agt DnaJ was not toxic to the cells. Furthermore, the growth profile observed at 40\u00a0\u00b0C was similar to that observed at 30\u00a0\u00b0C, suggesting that Agt DnaJ was able to reverse the thermosensitivity of E. coli OD259 (Fig. 3). This result was consistent with our previously published data that demonstrated Agt DnaJ was able to replace DnaJ and CbpA in E. coli OD259 and reverse the thermosensitivity of this strain (Hennessy et al., 2005a). As expected, E. coli OD259 producing Agt DnaJ-H33Q (negative control), was able to grow at 30\u00a0\u00b0C, but was unable to grow at 40\u00a0\u00b0C. E. coli OD259 transformants producing the chimeras HSJ1-J-Agt-DnaJ, Pfj1-J-Agt-DnaJ, Pfj4-J-Agt-DnaJ, Tcj3-J-Agt-DnaJ, ERj3-J-Agt-DnaJ and ERj5-J-Agt-DnaJ were all able to grow at 30\u00a0\u00b0C, and reversed the thermosensitivity of this strain at 40\u00a0\u00b0C similar to the control (Fig. 3). Furthermore, the H33Q mutant version of all these chimeras disrupted their in vivo functionality (Table 1 and data not shown). These data indicated that each of the J-domains were able to functionally replace the J-domain of Agt DnaJ in this prokaryotic system, and most likely exerted their in vivo function through a functional interaction with E. coli DnaK. The only chimera that was unable to reverse the thermosensitivity of E. coli OD259 was the ERj1-J-Agt-DnaJ. Western analysis to determine the protein production levels of the various chimeras in E. coli OD259, demonstrated that the chimeric proteins were produced and detectable in all the transformed strains (Fig. 3). This indicated that the inability of the ERj1-J-Agt-DnaJ chimera to reverse the thermosensitivity of E. coli OD259 was due to a lack of functionality rather than an absence of protein.\n3.3\nPfj1, Pfj4 and Hsj1 J-domain mutants\nThe ability of this wide range of J-domains to functionally substitute for the Agt DnaJ J-domain in the E. coli system suggested that an underlying commonality existed in their mode of interaction with the E. coli DnaK. Therefore, a comparative analysis was conducted of the functional effects of the substitution of certain conserved amino acids of the J-domains of these proteins (Table 1).\nProduction of the helix I mutant protein Pfj4-J-Agt-DnaJ-Y7A in E. coli OD259 partially reversed its thermosensitivity indicating this protein was partially functional in this assay, while the Pfj4-J-Agt-DnaJ-L10A was unable to reverse the thermosensitivity of this strain indicating it was non-functional in this assay (Table 1). Similar results were observed for the equivalent helix I mutant proteins of HSJ1-J-Agt-DnaJ. The helix II mutant protein Pfj4-J-Agt-DnaJ-R26A was partially functional (Table 1), while the HSJ1-J-Agt-DnaJ-R26A, Pfj1-J-Agt-DnaJ-R26A, and Pfj1-J-Agt-DnaJ-F25A mutant proteins were all non-functional (Table 1).\nAs discussed in the previous section, the H33Q substitution in the loop HPD motif was found to disrupt the functionality of all the chimeras. By contrast, substitution of the basic residue of the so-called HPD-R\/K-N pentapeptide in Pfj4-J-Agt-DnaJ-K36A, did not affect its functionality in the in vivo assay (Table 1).\nThe so called KFK motif of helix III is replaced by a KMA motif in the J-domain of Pfj1. Substitution of Met47 with Phe47 (the KMA to KFA mutation) resulted in a mutant Pfj1-J-Agt-DnaJ protein that was only partially functional in the in vivo assay (Table 1). Further substitution of the KMA motif of the Pfj1 J-domain to produce KMK and KFK, resulted in mutant Pfj1-J-Agt-DnaJ proteins that were fully functional. The RFK motif on helix III of the J-domain of Pfj4 was modified to RAK, and the resultant mutant protein Pfj4-J-Agt-DnaJ-F47A was found to be non-functional. Furthermore, substitution of the conserved helix III L57 residue in Pfj4-J-Agt-DnaJ-L57A resulted in a non-functional mutant protein (Table 1).\nThe roles of key conserved residues (e.g. D59) and motifs (e.g. the \u201cQKRAA\u201d motif) in helix IV were also investigated in this study (Table 1). The equivalent helix IV mutant proteins Pfj4-J-Agt-DnaJ-D59A and Hsj1-J-Agt-DnaJ-D59A were both found to be functional. The Pfj4-J-Agt-DnaJ-R63A mutant protein (Arg63 of the KRRRK motif of Helix IV in Pfj4, corresponding to the QKRAA motif of Agt DnaJ) was found to be functional, while in contrast the Pfj1-J-Agt-DnaJ-K63A mutant protein (substitution of Lys63 of the KKKEF motif of Helix IV in Pfj1) was only partially functional. Mutation of the Pfj1 KKKEF motif to the more conserved QKRAA motif produced a functional Pfj1-J-Agt-DnaJ mutant protein.\nProduction of all the mutant chimeric proteins was detected by Western analysis, suggesting that the inability of certain mutant proteins to reverse thermosensitivity was the result of a lack of protein functionality rather than a lack of protein production (Table 1). As has been previously observed (Hennessy et al., 2005a), levels of protein production in E. coli OD259 often vary between mutants and do not necessarily correlate with levels of functional recovery.\n3.4\nERj1 J-domain\nSince it was not possible to characterize the ERj1 J-domain any further through an in vivo assay, an in vitro analysis was conducted. Using a GST-ERj1-J fusion protein and mutant derivatives, GST-ERj1-J-R26A and GST-ERj1-JH33Q, affinity pull-down assays were conducted to evaluate the interaction of the J-domains with BiP in the presence and absence of ATP (Fig. 4A). While GST-ERj1-J functionally interacted with BiP in an ATP-dependent manner, GST-ERj1-J-H33Q had no significant interaction with BiP since the levels of BiP detected in the pull-down assay were equivalent in the presence and absence of ATP. The GST-ERj1-J-R26A protein exhibited an ATP-dependent interaction with BiP, but at reduced levels compared to GST-ERj1-J. The interaction of GST-ERj1-J-R26A was further assessed using surface plasmon resonance spectroscopy (SPR; Fig. 4B). Using a range of BiP concentrations (0.25\u20132.0\u00a0\u03bcM), the apparent affinity of GST-ERj1-J-R26A for BiP was found to be reduced by 50% compared to the affinity of GST-ERj1-J for BiP (Fig. 4B). The results of the in vitro study on GST-ERj1-J-R26A were consistent with the results of the in vivo assays on mutant J-domain chimeras, where we have found that substitution of the equivalent residue at position 26 on helix II of other J-domains disrupted their functionality in the in vivo assay (J-domains of Pfj1, Pfj4 and HSJ1).\n4\nDiscussion\nApart from the J-domain of the integral-membrane-bound ER Hsp40, ERj1, our findings suggested that all the J-domains tested exerted their functionality in the in vivo assay through a specific interaction with E. coli Hsp70, DnaK. Therefore, we propose that cytosolic and ER J-domains of mammalian and parasitic origin can interact with DnaK using a common mechanism. Furthermore, we found that substitution of a basic residue at position 26 of the helix II of the J-domain compromised functionality in all the J-domains in which this mutation was investigated (J-domains of Pfj1, Pfj4, HSJ1 and ERj1). Similar results have been found for E. coli DnaJ (Genevaux et al., 2002) and Agt DnaJ (Hennessy et al., 2005a,b). Therefore, we propose that this basic residue of helix II together with the HPD motif of the loop region are important elements of a fundamental binding surface required for J-domain-based Hsp40-Hsp70 interaction. This fundamental binding surface appears to be conserved in J-domains of Hsp40s of mammalian, parasitic and bacterial origin.\n4.1\nJ-domain interchangeability\nThe number of different Hsp40 proteins in any organism generally outweighs the number of different Hsp70 proteins identified; for example, there are 6 Hsp40s and 3 Hsp70s in E. coli (Hennessy et al., 2005a), while there are 43 Hsp40s and 6 Hsp70s in P. falciparum (Matambo, Odunuga, Boshoff, & Blatch, 2004; Sargeant et al., 2006). This suggests the Hsp70 protein is the more promiscuous member of the Hsp40\/Hsp70 pair and that the Hsp40 protein provides the specificity to this chaperone partnership. The J-domain may provide some of the molecular determinants of this specificity (Garimella et al., 2006; Hennessy et al., 2005a,b). In this study we have analyzed the J-domains from a number of diverse Hsp40s to assess their ability to functionally replace the J-domain of an exogenously produced Agt DnaJ in the prokaryotic E. coli OD259 in vivo complementation system. Interestingly, while Pfj1, Pfj4 and Tcj3 were all of parasitic origin and HSJ1, ERj3, and ERj5 were of mammalian origin, the J-domains of each protein were able to functionally replace the J-domain of the prokaryotic Agt DnaJ J-domain. In contrast, the J-domain of ERj1, the only integral membrane-bound Hsp40 tested, was unable to substitute for the J-domain of Agt DnaJ in the in vivo complementation assay. Two other membrane-bound ER Hsp40s, ERj2 and ERj4, have been investigated previously by others. Using a yeast complementation assay, Schlenstedt et al. (1995) found that the J-domains from the yeast Hsp40s Sis1p (cytosolic) and Mdj1p (mitochondrial lumen) were unable to functionally replace the J-domain of the integral membrane-bound Hsp40, Sec63p (ERj2 homologue). By contrast, the J-domain of the membrane-associated ERj4 (also called Mdg1) was shown to functionally replace the J-domain of E. coli DnaJ in an E. coli complementation assay (Pr\u00f6ls et al., 2001). Membrane-bound Hsp40s have also been investigated in prokaryotic systems. Kluck et al. (2002) showed that the J-domain of a membrane-bound E. coli Hsp40, DjLC, could not replace that of cytosolic E. coli DnaJ. This result may reflect that fact that DjLC was shown to interact with a specialized E. coli Hsp70 called HscC, which did not interact with E. coli DnaJ (Kluck et al., 2002). The lack of interaction of the J-domain of ERj1 with DnaK in the in vivo complementation assay, suggested that it may have reduced affinity for Hsp70s other than its partner BiP. It is well established that ERj1 can functionally interact with BiP, and that this association is important for the role of ERj1 in protein translocation into the ER (Chevalier et al., 2000; Dudek et al., 2002). It has also been shown that the ERj1 J-domain could stimulate E. coli DnaK ATPase activity in vitro, however, only at levels greater than that required for similar stimulation by E. coli DnaJ (Chevalier et al., 2000). This finding suggests that the ERj1 J-domain has low affinity and specificity for DnaK, and is consistent with our findings. It would be worthwhile conducting a gain-of-function analysis on the ERj1 J-domain by changing certain divergent residues of the ERj1 J-domain to those found in other J-domains (e.g. Phe9 to Ile or Leu), and evaluating which residues promote a functional interaction with DnaK using both in vitro and in vivo assays.\n4.2\nTargeted mutagenesis\n4.2.1\nHelix I\nPublished J-domain structures have revealed that while helix IV is relatively mobile, helix I is relatively fixed in position (Berjanskii et al., 2000; Cupp-Vickery & Vickery, 1997, 2000; Huang et al., 1998; Pellecchia et al., 1996; Qian et al., 1996). The side-chains of the helix I residues Tyr7 and Leu10 may be involved in core contacts with helices I, II and III thereby stabilizing the J-domain scaffold for interactions with Hsp70 (Berjanskii et al., 2000). Substitution of these residues to Ala resulted in partial and complete reduction of J-domain functionality, respectively, in both the Pfj4-J-Agt-DnaJ and HSJ1-J-Agt-DnaJ chimeras. This was consistent with the findings of Hennessy et al. (2005a,b) where a lack of functionality was shown for Y7A and L10A mutant Agt DnaJ proteins in the same in vivo functional assay. These substitutions most likely destabilized the J-domain, resulting in a destabilized mutant protein with compromised functionality. Therefore, while these data suggest that Y7 and L10 of helix I have primarily a structural role, they do not exclude the possibility that these residues have an indirect functional role ensuring that the J-domain is correctly orientated for interactions with Hsp70 (Hennessy et al., 2005a).\n4.2.2\nHelix II and the loop\nBased on structural data from the J-domain of polyomavirus T antigen (Berjanskii et al., 2000), residues Tyr25 and Lys26 of the positively charged helix II of the J-domain of DnaJ were predicted to form part of the interaction surface that contacts the ATPase domain of DnaK. When substituted, these residues were found to disrupt in vivo functionality of E. coli DnaJ and Agt DnaJ (Genevaux et al., 2002; Hennessy et al., 2005a,b). Our findings were consistent with these data, in that the R26A mutations in the J-domains of Pfj1-J-Agt-DnaJ, Pfj4-J-Agt-DnaJ, HSJ1-Agt-DnaJ and GST-ERj1-J, and the Y25A mutation of the J-domain of Pfj1-J-Agt-DnaJ J-domain disrupted functionality. Lys36 (equivalent to Arg36 in E .coli DnaJ) is part of the functionally critical pentapeptide (HPD-R\/KN) predicted by Genevaux et al. (2002). While it has been shown that a R36G mutation in E. coli DnaJ resulted in loss of function (Genevaux et al., 2002), we have demonstrated that a K36A mutation in Pfj4-J-Agt-DnaJ had no effect on its in vivo functionality. This suggested that while Arg36 appeared to be a structurally and\/or functionally critical residue in E. coli DnaJ, it did not appear to be critical in all Hsp40s.\n4.2.3\nHelix III\nPhe47 of the KFK motif has been predicted to be important in J-domain function and was previously the only residue in close proximity to His33 that abolished Hsp40 function when mutated (Genevaux et al., 2002). Since Phe47 protrudes into the inter-helical space of helices II and III, it is tempting to propose that it has a largely structural role in maintaining the orientation of the J-domain and particularly the loop region. Indeed, it has been suggested that Phe47 may sterically constrain the movement of the helix II\u2013helix III inter-helical loop (Genevaux et al., 2002); however, its conserved nature and proximity to His33 also suggest a potential mechanistic role in Hsp40\u2013Hsp70 interactions (Hennessy et al., 2000; Landry, 2003). Here we have demonstrated that the mutation F47A in the Pfj4-J-Agt-DnaJ chimera completely abolished functionality of the chimera, as is seen for the equivalent mutation in E. coli DnaJ, yeast Ydj1 and an Hdj1 E. coli DnaJ chimera (Genevaux et al., 2002; Hennessy et al., 2005a,b; Johnson & Craig, 2000). While the function of Phe47 is unclear, these findings demonstrate that this residue is necessary for the functioning of certain diverse Hsp40s. Interestingly, the F47L substitution in Agt DnaJ had no adverse effect on its in vivo functionality (Hennessy et al., 2005a,b), suggesting that the minimum requirement for functionality at this position was the presence of a large hydrophobic residue. This was further supported by our data in which the M47F substitution of the KMA motif of the Pfj1-J-Agt-DnaJ chimera preserved functionality of the mutant protein, albeit at reduced levels. This was similarly the case when the neighbouring hydrophobic residue Ala48 was mutated to the more frequently encountered conserved residue Lys48, and when the double mutation M47F\/A48K (producing a KFK motif) was performed on the Pfj1-J-Agt-DnaJ chimera. Leu57 is predicted to be a key residue involved in maintaining the structural integrity of helices II and III, since its side chain projects directly into the helix II\u2013helix III cleft (Hennessy et al., 2005a,b). The L57A mutation of the Pfj4-J-Agt-DnaJ chimera, and the L57S mutation of Agt DnaJ (Hennessy et al., 2005a,b) abolished in vivo functionality of these Hsp40 proteins, suggesting that Leu57 was indeed required for the structure and function of the J-domain.\n4.2.4\nHelix IV\nResidue Asp59 is predicted to be of structural importance in the J-domain due to its location on the border of helices III and IV. Structural data suggests that helix IV is mobile, and its exact functional location is unclear. Hennessy et al. (2005a,b) showed that a D59A mutation of Agt DnaJ abolished its in vivo functionality; however since the mutant protein was undetectable, the inability of the protein to function in vivo may be attributed to a lack of protein production. Conversely, Genevaux et al. (2002) demonstrated that a T58A\/D59A double mutation in E. coli DnaJ had no effect on its in vivo functionality. Our data showing that the Pfj4-J-Agt-DnaJ-D59A and Hsj1-J-Agt-DnaJ-D59A chimeras were functional in the in vivo assay, were consistent with the E. coli DnaJ data (Genevaux et al., 2002). Arg63 is part of the conserved QKRAA motif of helix IV that has been proposed to play a role in enhancing the affinity or specificity of interactions with Hsp70 (Garimella et al., 2006; Hennessy et al., 2005a,b). In this study, Pfj1-J-Agt-DnaJ-K63A was fully functional in the in vivo assay, and Pfj4-J-Agt-DnaJ-R63A was partially functional. While this result suggested that a basic residue at position 63 was not absolutely critical for J-domain-based interactions with Hsp70, it was consistent with its proposed role in the affinity or specificity of interaction.\n4.3\nConclusion and future perspectives\nThis study has analysed cytosolic and ER Hsp40s of mammalian and parasitic origin, and found that certain key features of the J-domain appear to be fundamental to the function of all the J-domains studied, and perhaps to the function of J-domains in general. Interestingly, the differences appear to be subtle (e.g. the effects of the Y7A and R26A substitutions on the function of the J-domains of Hsj1 versus Pfj4), and may reflect slight differences in affinity or specificity of these J-domains for DnaK. These differences need to be probed further using quantitative in vitro assays, and incorporating an analysis of the less-conserved J-domain residues shown by NMR analysis to occur at J-domain-DnaK\/Hsp70 binding interfaces. Furthermore, the possibility that there are specialized features unique to the J-domains of integral-membrane-bound Hsp40s needs to be investigated more extensively.","keyphrases":["dnaj","hsj1","malarial hsp40s","trypanosomal hsp40s","endoplasmic reticulum hsp40s"],"prmu":["P","P","R","R","R"]}
{"id":"Arch_Sex_Behav-3-1-1914258","title":"Sexual Decision-Making in HIV-Positive Men Who Have Sex with Men: How Moral Concerns and Sexual Motives Guide Intended Condom Use with Steady and Casual Sex Partners\n","text":"Determinants of intended condom use with steady and casual sex partners were examined among Dutch HIV-positive men who have sex with men (MSM) (N = 296). Given the proposition that safer sex behavior among HIV-positive people is a form of prosocial behavior, the present study extended the general framework of the Theory of Planned Behavior with Schwartz\u2019s norm-activation theory and tested the assumption that personal norms would mediate the effects of other psychosocial factors on intended condom use for anal sex. In addition, it was hypothesized that, depending on the context in which sex occurs, specific motives for unprotected anal sex may have a negative influence on intended condom use and, as such, undermine a prosocial tendency to practice safer sex. Therefore, we also investigated the influence of sexual motives for unprotected anal sex on intended condom use with steady and casual sex partners. Results indicated that the Theory of Planned Behavior adequately predicted condom use intentions (for casual sex partners and steady sex partners, the explained variance was 52% and 53%, respectively). However, our proposed model of sexual decision-making significantly improved the prediction of behavioral intentions. For steady and casual sex partners, the assumption of the mediating role of personal norms on condom use intention was confirmed empirically. Additionally, sexual motives for unprotected anal sex exerted, as expected, a direct, negative effect on condom use intention with casual sex partners. The implications of the findings for future research and the development of HIV-prevention programs for HIV-positive MSM are discussed.\nIntroduction\nStudies on the effects of HIV counseling and testing show that most individuals who are tested HIV-positive respond by reducing their sexual risk behavior (Weinhardt, Carey, Johnson, & Bickman, 1999). Nevertheless, it has been found that approximately one third of HIV-positive men who have sex with men (MSM) engaged in unprotected anal sex in the last two to three months (for a review, see Kalichman, 2000). Moreover, increased prevalence rates of gonorrhea and syphilis have been reported among HIV-positive and HIV-negative MSM in several western regions (e.g., Anon, 2002; Macdonald et\u00a0al., 2004; Van de Laar & Op de Coul, 2003). These findings suggest a rise in unprotected anal sex among HIV-negative and HIV-positive MSM. Given that at least a subgroup of HIV-positive MSM, for various reasons, engage in risky sexual behavior, an understanding of why this occurs remains a matter of significant public healthy concern.\nStudies have been conducted that examined possible antecedents of (un)safe sexual behavior in HIV-positive MSM (Crepaz & Marks, 2002). One of the factors that has been identified as promoting sexual behavior change is perception of risk, i.e., awareness that unprotected sex increases the risk for HIV infection and STDs. Accordingly, studies have indicated that HIV-positive MSM who believe that HIV-superinfection and other STDs may have negative consequences for their own health are more likely to use condoms for anal sex (Colfax et\u00a0al., 2004; McConnell, Grant, & Greenwood, 2002). However, other studies suggest that a person\u2019s own risk of HIV-superinfection and STDs is not a key concern. Of great importance is the finding that safer sex in HIV-positive MSM is primarily motivated by concerns about the risks for the other and that concerns about one\u2019s own risks are subordinate (Keogh, Weatherburn, & Stephens, 1999; Van Kesteren, Hospers, Kok, & Van Empelen, 2005).\nThe notion that HIV-positive MSM engage in safer sex because of concerns about the welfare of sex partners can be characterized as a form of prosocial behavior. Prosocial behavior \u201crepresents a broad category of acts\u2026that are defined as generally beneficial to other people\u201d (Penner, Dovidio, Piliavin, & Schoeder, 2005, p. 366). If HIV-positive MSM indeed engage in safer sex to benefit others, or more specifically, are motivated to prevent harm to others, it is of particular importance to understand why this is the case and to determine which factors promote such behavior. The literature offers several explanations to account for prosocial behavior. One explanation for a wide range of moral and prosocial behaviors, which has received considerable attention, is the so-called personal standards approach (e.g., Manstead, 2000). This approach emphasizes how internalized, self-reinforced standards, such as altruism, can promote prosocial behavior as people strive to maintain a positive self-image or achieve their ideals (Penner et\u00a0al., 2005).\nIndeed, some qualitative studies suggest that internalized values or personal standards play an important role in the practice of safer sexual behavior. For example, Nimmons and Folkman (1999) showed that HIV-positive MSM described moral and altruistic values in regard to the need to practice safer sex. A study by Wolitski, Bailey, O\u2019Leary, G\u00f3mez, and Parsons (2003) found that many HIV-positive MSM perceived that they had a particular responsibility for protecting their partners and that this perception influenced safer sexual decision-making. Participants cited altruism and self-imposed standards as the motivations underlying their beliefs about personal responsibility. Similarly, Van Kesteren et\u00a0al. (2005) found that a greater concern about potential HIV transmission to sex partners was related to enhanced feelings of personal responsibility for safer sex.\nWhile these studies have contributed greatly to a preliminary understanding of the role of intrinsic motivation in safer sexual behavior, the factors that may motivate HIV-positive MSM to adopt safer sex practices for the protection of others warrants further examination. This study represents such an attempt, examining social-psychological factors of condom use in the context of steady and casual sex. For this reason, we combined the Theory of Planned Behavior (Ajzen, 1991), a well-established framework used to understand behavior in a broad range of contexts, with the norm-activation theory (Schwartz, 1977; Schwartz & Howard, 1981), a framework specifically developed to understand prosocial behavior. In spite of the supposed importance of prosocial motivation in safer sexual behavior, it is possible that other motivations (e.g., sexual motives) for unprotected anal sex may have a negative influence on intended condom use in certain circumstances (Cooper, Shapiro, & Powers, 1998). Therefore, for both steady and casual sex partners, we examined the extent to which sexual motives for unprotected anal sex negatively affect intended condom use.\nTheory of Planned Behavior\nThe Theory of Planned Behavior (TPB) (Ajzen, 1991) is one of the most important social psychological theories for predicting and understanding behavior. The TPB posits that the most proximal determinant of whether or not a person performs a behavior is his intention to do so. According to the theory, behavioral intention is determined by attitude, subjective norm, and perceived behavioral control. Attitude is the person\u2019s overall evaluation of the advantages and disadvantages of a particular behavior whereas subjective norms are determined by the perceived social pressure to perform the behavior. Perceived behavioral control is the person\u2019s conviction about whether or not the required skills and resources to perform the behavior are at his disposal and is closely related to Bandura\u2019s (1986) concept of self-efficacy. The TPB has received significant support from research on the adoption of many health-related behaviors (Godin & Kok, 1996), including safer sex behavior (Albarrac\u00edn, Fishbein, Johnson, & Muellerleile, 2001; Rye, Fisher, & Fisher, 2001; Sheeran, Abraham, & Orbell, 1999).\nEssentially, the TPB stresses the importance of cognitive, information processing mechanisms in explaining behavior. However, not every act of safer sex is necessarily the result of deliberate thought processes. Of particular interest are the growing number of studies that suggest that HIV-positive MSM are motivated by concerns about the effect that their sexual behavior may have on others; that is, that they feel personally responsible for protecting their partners from HIV. Although we agree that the TPB provides a valuable framework for predicting safer sex practices, we argue that this perspective is too narrow and may not fully account for feelings of personal responsibility that may promote safer sexual behavior. More specifically, we believe that safer sexual behavior in HIV-positive MSM is based on concerns that go beyond a purely rational weighing of personal advantages and disadvantages, i.e., that safer sex is simply the right thing to do. One theory that may help explain why HIV-positive MSM are motivated to adopt safer sexual practices for the protection of others is Schwartz\u2019 (1977) norm-activation theory.\nNorm-Activation Theory\nThe norm-activation theory (Schwartz, 1977; Schwartz & Howard, 1981) seeks to predict and understand prosocial or altruistic behavior. According to norm-activation theory, personal norms are the immediate determinant of behavior. Personal norms are considered as strong intrinsic motivators, as they trigger an individual\u2019s internal value system and are tied to one\u2019s self-concept. In the case of sexual behavior, these aspects reflect the feeling of moral obligation to practice safer sex because of concern about the welfare of sex partners. That personal norms may play an important role in safer sexual behavior in HIV-positive MSM was shown in a study by Godin, Savard, Kok, Fortin, and Boyer (1996). Their study showed that personal norms, in addition to perceived behavioral control, are important predictors of intended condom use for anal sex.\nThe important relationship between personal norms and condom use intention underscores the assumption that safer sexual behavior is influenced by feelings of moral obligation. However, Schwartz argued that there will be no moral motivation unless: (1) an individual is aware of the specific action that is needed and the consequences of action or inaction to oneself and others (awareness of need); (2) an individual ascribes responsibility to himself for the consequences of the action or inaction (ascription of responsibility); (3) a person identifies actions that might be effective in handling the specific situation (efficacy); and (4) a person believes that he is capable of performing the required actions or behavior (ability). As such, we expect that, in predicting safer sex, awareness of need and ascription of responsibility may be important determinants of behavioral intention to use condoms for anal sex with steady and casual sex partners. Note that in this context, efficacy and ability are not differentiated because both concepts seem to be covered by the theoretical paradigm of the TPB (i.e., attitude and self-efficacy, respectively).\nSexual motives for unprotected anal sex\nNotwithstanding our argument that personal norms play a central role in sexual decision-making, we expect that, depending on the social context in which sex occurs, strong motives to engage in unprotected sex may conflict with one\u2019s personal norm for safer sex. For instance, Cooper et\u00a0al. (1998) have demonstrated that sexual behaviors, whether risky or safe, may serve a range of psychological functions that have little to do with health protection and disease avoidance. Indeed, the study by Van Kesteren et\u00a0al. (2005) indicated that the extent to which HIV-positive MSM acted in accordance with their feelings of personal responsibility to practice safer sex depended, in part, on their sexual motives for engaging in unprotected anal sex. Typically, HIV-positive MSM reported having engaged in unprotected anal sex to express emotions related to love or because they were \u201csexually turned-on\u201d by their sex partners. Thus, sexual motives appear to have a negative influence on intended condom use, which may undermine a prosocial motivation to practice safer sex. However, it can be argued that a conflict such as this may play a role more in casual sex encounters than in steady sexual relationships, as it is likely that the benefits of unprotected sex are more salient in the context of casual sex (i.e., when one does not necessarily know one\u2019s sex partner and is less likely to be confronted with the potential adverse longer term social consequences of having engaged in unprotected sex) than with steady partners (i.e., when one is emotionally involved with a partner and committed to an ongoing relationship) (Flowers, Marriott, & Hart, 2000; Van Kesteren et\u00a0al., 2005). It has been shown that when people perceive increased benefits of a specific behavior (unsafe sex), it leads to a more favorable affective impression, resulting in lower judgments of risk (so-called affect-heuristic; Finucane, Alhakami, Slovic, & Johnson, 2000). Therefore, we expect that sexual motives may have a negative impact on intended condom use, particularly in casual sex encounters.\nProposed model of sexual decision-making\nSafer sex in HIV-positive MSM can be seen as a prosocial behavior, making it vital to integrate variables specific to the TPB with variables specific to the norm-activation theory. In accordance with the TPB, we propose a model (see Fig. 1) in which intention is the most proximal determinant of behavior. However, in line with the norm-activation theory, we assume that the decision to use condoms for anal sex largely depends on one\u2019s intrinsic motivation to engage in safer sex. Accordingly, we expect personal norms to be the most direct determinant of intention, thereby mediating the effects of the other determinants specific to the TPB and the norm-activation theory. Following Cooper et\u00a0al. (1998), we further assume that sexual motives for unprotected anal sex may have a direct and negative impact on intended condom use and, as such, may compete with a prosocial tendency to engage in safer sex. However, due to the social context in which sex occurs, we hypothesize that sexual motives will contribute to the explanation of intended condom use with casual sex partners, whereas this is unlikely to be the case in the context of steady sex partners.Fig. 1Proposed model of sexual decision-making\nMethod\nParticipants\nData were obtained by means of a self-administered questionnaire between July 2002 and April 2003. Participants were recruited through AIDS consultants working in 15 Dutch hospitals. Participants were asked to fill out the questionnaire at home and return the questionnaires directly to the research institution by means of a stamped, pre-addressed envelope. Consequently, no information was available about the response rate per hospital. Participation was on a voluntary and anonymous basis. A pilot study indicated that completion of the questionnaire took about 30 to 45\u00a0min. Participants received no compensation for filling out the questionnaires. Approval for the study was obtained from the Ethics Committee of Maastricht University Hospital.\nParticipants who met the following criteria were included in the study: (1) tested positive for HIV-antibodies; (2) identified themselves as homosexual or bisexual; (3) aged between 20 and 65 years; and (4) were sufficiently fluent in Dutch to complete the questionnaire. A total of 1,050 questionnaires were distributed, of which 296 were completed and returned (response rate\u00a0=\u00a028.2%).\nTable 1 summarizes the demographic characteristics of the participants. Participants were predominantly Dutch nationals, were mainly from the Western part of the Netherlands, including Amsterdam, and ranged in age from 25 to 59 years, with a mean age of 42.1 years. Most participants had a medium or high level of education and were employed full-time or part-time. Most participants identified themselves as exclusively homosexual. More than half of the men had known themselves to be HIV-positive for 6 years or less. The majority of men was aware of their CD4 counts and viral load and indicated that they were currently on some form of HIV antiretroviral therapy.Table 1Sociodemographic characteristics of the sample (maximum N\u00a0=\u00a0296)CharacteristicsN%Nationality\u2003Dutch25185.7\u2003Surinam\/Antillean\u200351.7\u2003Other3712.6Geographic area of the Netherlands\u2003Amsterdam7325.3\u2003West (excluding Amsterdam)11038.0\u2003South6723.2\u2003Northeast3913.5Age\u200325\u2013355518.9\u200336\u20134513646.7\u200346\u2013558228.2\u2003>56186.2Education\u2003Higher vocational education or university12443.1\u2003Secondary vocational training or high school12041.7\u2003Primary school or basic vocational training3411.8\u2003Other103.5Employment status\u2003Full-time11743.5\u2003Part-time8029.7\u2003Unemployed7226.8Self-identification\u2003Homosexual24482.4\u2003More homosexual than heterosexual3812.8\u2003Bisexual\u200382.7\u2003More heterosexual than bisexual\u200341.4\u2003Other\u200320.7Years knowing HIV-positive status\u2003<2 yrs5920.1\u20032\u20136 yrs11639.6\u20037\u201311 yrs7124.2\u200312\u201316 yrs3712.6\u2003>16 yrs103.4Self-reported CD4+\u2003<2003311.1\u2003200\u201350012040.5\u2003>5009231.1\u2003Don\u2019t know5117.2Self-reported viral load\u2003Detectable [median=10.000, range 50\u20132.080.0007225.9\u2003Undetectable17663.3\u2003Don\u2019t know3010.8Antiviral treatment\u2003No5920.1\u2003Yes23479.9\nMeasures\nThe questionnaire was based on earlier studies (Hogeweg & Hospers, 2000; van Kesteren et\u00a0al., 2005) and consisted of several questions on determinants for explaining condom use behavior: attitudinal beliefs, subjective norms, self-efficacy, personal norms, awareness of consequences, ascription of responsibility, sexual motives, intention and demographic variables. Separate scales were used for condom use with steady and casual sex partners for all determinants. The questionnaire was pre-tested among a group of 12 HIV-positive MSM for comprehension and completeness.\nAttitudinal beliefs were measured through 16 items drawn from interviews with HIV-positive MSM (van Kesteren et\u00a0al., 2005) and from an earlier quantitative study among MSM (Hogeweg & Hospers, 2000). Four items were used to assess response efficacy (e.g., \u201cBy using condoms when having anal intercourse, I protect my steady partner\/casual sex partners against the AIDS virus\/HIV-superinfection\u201d). In addition, seven possible pros (e.g., \u201cBy using condoms during anal sex with my steady partner\/casual sex partners, I worry less about his\/their well-being\u201d), and five possible cons of condom use behavior (e.g., \u201cUsing condoms for anal sex with my steady partner\/casual sex partners reminds me of HIV\/AIDS\u201d) were included in the questionnaire. Items were indexed on 5-point scales (1\u00a0=\u00a0totally disagree, 5\u00a0=\u00a0totally agree), and had an alpha of .81 for steady and .76 for casual sex partners.\nSubjective norms were measured through the use of three items on 5-point scales. Two items assessed the perception of participants of the opinions of other important people on whether or not they should use condoms when having anal intercourse (1\u00a0=\u00a0certainly not, 5\u00a0=\u00a0certainly). Following Schwartz (1973), one item assessed perceived social sanctions regarding not using condoms during anal intercourse and was expressed as follows: \u201cHow do you think important others would react if you told them that you don\u2019t use condoms when having anal intercourse with your steady partner\/casual sex partners?\u201d (1\u00a0=\u00a0very approving, 5\u00a0=\u00a0very disapproving). Alpha for steady and casual sex partners was. 76 and .63, respectively.\nSelf-efficacy was measured with seven items based on van Kesteren et\u00a0al. (2005) and Hogeweg and Hospers (2000) (e.g., \u201cSuppose you want to use condoms when having anal intercourse, will you be able to discuss condom use with your steady partner\/casual sex partners?\u201d). Items were measured on 5-point scales (1\u00a0=\u00a0certainly not, 5\u00a0=\u00a0certainly), and had an alpha of .89 for steady and .87 for casual sex partners.\nPersonal norms were measured by means of three items on 5-point scales (Godin et\u00a0al., 1996; Parker, West, Stradling, & Manstead, 1995), such as: \u201cAs a matter of principle, I use condoms every time I have anal intercourse with my steady partner\/casual partners.\u201d Items were rated on 5-point scales (1\u00a0=\u00a0totally disagree, 5\u00a0=\u00a0totally agree) and had an alpha of .96 and .90 for steady and casual sex partners, respectively.\nBoth awareness of consequences for self and awareness of consequences for others were assessed separately for condom use with steady and casual sex partners by a single item on a 5-point (1\u00a0=\u00a0totally agree, 5\u00a0=\u00a0totally disagree) scale. Awareness of consequences for self measured the participant\u2019s view about whether the consequences for his health were negligible when not using condoms during anal sex. Awareness of consequences for others measured the participant\u2019s views about whether the consequences for the health of his partner were negligible when not using condoms during anal sex.\nAscription of responsibility to self was measured by means of four items on 5-point scales concerning responsibility for condom use behavior and one item concerning transmission of HIV. For example: \u201cHow responsible are you yourself for negotiating condom use?\u201d (1\u00a0=\u00a0not at all, 5\u00a0=\u00a0completely). Alpha for steady and casual sex partners was .91 and .89, respectively.\nSexual motives for unprotected anal sex were measured using seven affective states on 7-point scales (1\u00a0=\u00a0considerably less, 7\u00a0=\u00a0considerably more), such as lust, love, excitement, and feeling good. Following Nelissen, Dijker, and De Vries (in press), participants were asked to indicate the extent to which they would feel a particular emotion when engaging in unprotected anal sex with steady (alpha\u00a0=\u00a0.89) and casual sex partners (alpha\u00a0=\u00a0.85).\nIntention to use condoms when having anal intercourse was measure by the following three items on 5-point scales: \u201cI intend to use a condom when having anal intercourse with my steady partner\/casual partners\u201d; \u201cI will try to use a condom when\u2009\u2026\u201d; and \u201cI plan to use a condom when\u2009\u2026\u201d (1\u00a0=\u00a0certainly not, 5\u00a0=\u00a0certainly). Alpha for both steady and casual sex partners was .93.\nWith respect to sexual risk behavior, participants were asked whether or not they had engaged in receptive and insertive oral sex with ejaculation and in receptive and insertive anal sex in the past six months (separate for steady and casual sex partners). For each sexual behavior they had engaged in, the frequency of condom use (1\u00a0=\u00a0never, 5\u00a0=\u00a0always) was assessed.\nResults\nSexual behavior\nTable 2 shows that about 60% of the participants reported a male steady partner in the preceding six months. Among these, about one third (30.3%) reported that they had not had oral or anal sex with their steady partner. Among those who had had oral sex with ejaculation with their steady partners (23.6%), only one person reported that he consistently had used a condom. Among those who had had anal sex with their steady sex partners (51.1%), about half (48.4%) reported that they had not used condoms consistently. Approximately 72% of the participants reported male casual sex partners in the preceding six months. When oral intercourse was considered, none of the men who had engaged in oral sex with ejaculation (31.8%) reported consistent condom use. When anal sex was considered, about half (45.6%) of the participants who had engaged in anal intercourse with casual sex partners (74.8%) had not consistently used condoms.Table 2Descriptives of sexual behavior and disclosure in casual sex encounters of the sample HIV-positive MSM (N\u00a0=\u00a0296)N%Steady partner(s) in the preceding\u2003\u20036 months17860.1\u2003Anal or oral sex with steady sex\u2003\u2003partner124 (178)69.7\u2003Oral intercourse with ejaculation\u2003\u2003with steady sex partner42 (178)23.6\u2003Unprotected oral intercourse\u2003\u2003with steady sex partner41 (42)97.6\u2003Anal intercourse with steady sex\u2003\u2003partner91 (178)51.1\u2003Unprotected anal intercourse with\u2003\u2003steady sex partner44 (91)48.4Casual partner(s) in the preceding\u2003\u20036 months21472.3\u2003Anal or oral sex with casual sex\u2003\u2003partners167 (214)78.0\u2003Oral intercourse with ejaculation\u2003\u2003with casual sex partners68 (214)31.8\u2003Unprotected oral intercourse\u2003\u2003with casual sex partners68 (68)100.0\u2003Anal intercourse with casual sex\u2003\u2003partners160 (214)74.8\u2003Unprotected anal intercourse with\u2003\u2003casual sex partner73 (160)45.6Disclosure of HIV status in casual\u2003\u2003sex encounters\u2003Has never\/rarely informed\u2003\u2003casual sex partners about\u2003\u2003HIV-positive status177 (214)82.8\u2003Was never\/rarely informed about\u2003\u2003HIV-status of casual sex partners189 (214)88.3\nAmong men who had anal sex with their steady sex partner, unprotected sex occurred more often within the context of HIV-seroconcordant relationships than within HIV-serodiscordant relationships: 23.3% (7 out of 30) of the men with an HIV-positive partner reported consistent condom use in the preceding six months compared to 61.0% (25 out of 41) of the men with an HIV-negative partner and 73.7% (14 out of 19) of the men with a partner whose HIV status was unknown, \u03c72(2)\u00a0=\u00a014.7, p\u00a0<\u00a0.01.\nAlthough no data were available on the HIV-status of casual sex partners, participants were asked two questions regarding disclosure of HIV-status. The majority of the participants (82.8%) reported that they never or rarely had informed casual sex partners about their HIV-status in the preceding six months. Similarly, most participants (88.3%) reported that casual sex partners had never or rarely informed them of their HIV-status (see Table 2). Thus, accurate knowledge of each others HIV-status appears to be minimal, which makes the use of effective partner selection strategies (i.e., \u201cserosorting\u201d) as an explanation for the occurrence of unprotected sex within casual sex encounters less likely.\nDescriptive statistics and correlations\nMeans, SDs, and correlations for the determinants of condom use for anal sex are provided in Table 3. A missing value analysis was conducted for independent variables relating to condom use with steady and casual sex partners. Participants with missing values exceeding 10% of all items were excluded from further data analyses. For participants with less than 10% missing values, the item score was replaced with the mean of the remaining participants for the respective item. As a result, analyses of intention to use condoms were conducted among 149 out of 178 participants who reported a steady sex partner and 188 out of 214 participants who reported casual sex partners in the preceding six months. Of the participants who were included in the analyses with steady and casual sex partners, there were 22 and 24, respectively, who had one or more missing items (range 1\u20134). In both samples, however, the majority was missing only one of the questionnaire items.Table 3Means and correlations for determinants of condom use with steady (N\u00a0=\u00a0149; below diagonal) and casual sex partners (N\u00a0=\u00a0188; above diagonal)ScaleRange(1)(2)(3)(4)(5)(6)(7)(8)(9)Intention (1)1\u20135\u2013.56**.57**.67**.83**.36**.25**.64**\u2212.56**Attitudinal beliefs (2)1\u20135.60**\u2013.49**.48**.57**.28**.18*.53**\u2212.55**Subjective norms (3)1\u20135.77**.57**\u2013.45**.62**.17*.07.45**\u2212.43**Self-efficacy (4)1\u20135.74**.62**.69**\u2013.62**.36**.24**.56**\u2212.49**Personal norms (5)1\u20135.88**.64**.80**.74**\u2013.32**.22**.64**\u2212.53**Awareness consequences self (6)1\u20135.23*.30**.21*.26**.22**\u2013.72**.26**\u2212.29**Awareness consequences others (7)1\u20135.39**.35**.38**.37**.41**.53**\u2013.23**\u2212.23**Ascription of responsibility (8)1\u20135.60**.60**.51**.71**.60**.18*.35**\u2013\u2212.49**Sexual motives (9)1\u20137\u2212.57**\u2212.62**\u2212.57**\u2212.59**\u2212.59**\u2212.16*\u2212.31**\u2212.47**\u2013Steady\u2003\nM4.13.54.34.24.13.34.04.23.5\u2003\nSD1.30.71.00.91.41.61.40.71.6Casual\u2003\nM4.33.84.34.24.33.84.24.33.4\u2003\nSD1.10.60.60.81.11.21.20.81.4*p\u00a0<\u00a0.05.**p\u00a0<\u00a0.01.\nThe descriptive statistics indicates that, in general, participants\u2019 intentions to use condoms during anal sex with steady and casual sex partners were high. In addition, the means of most of the other determinants were on the positive side of the scale. The exception was for sexual motives for unprotected anal sex, where participants scored negative with regard to both steady and casual sex partners. Consistent with our proposed model (see Fig. 1), attitudinal beliefs, subjective norms, self-efficacy, personal norms awareness of consequences for self and others, and ascription of responsibility to self were all positively correlated with behavioral intention, with the exception of sexual motives for unprotected anal sex. As expected, the correlation between sexual motives for unprotected anal sex and intended condom use was negative for both steady and casual sex partners.\nPredictors of intended condom use\nBecause intention for both steady and casual sex partners was positively skewed (more than 50% of the sample scored 5 on a 5-point scale), intended condom use with steady and casual sex partners was dichotomized on its median into high versus low intention. To test our proposed model (see Fig. 1), two hierarchical logistic regression analyses were conducted with intended condom use with steady and casual sex partners as the outcome variables. Personal characteristics and partner variables were controlled in both analyses and were entered first. In order to examine the relative contribution of the TPB variables (i.e., attitudinal beliefs, subjective norms and self-efficacy), these variables were entered in the second step. Awareness of consequences for self and others and ascription of responsibility were entered in the third step, followed by sexual motives for unprotected anal sex in the fourth step. Personal norms were entered in the final step of the regression analyses. In addition, we examined the mediating role of personal norms following the standard procedure specified by Baron and Kenny (1986).\nPrior to analyses, collinearity between all variables in the regression analyses was checked by computing the Variance Inflation Factor (VIF) of each variable. Values above 10 were regarded as an indication of near-collinearity (Kleinbaum, Kupper, Muller, & Nizam, 1998). With respect to steady and casual sex partner variables, all VIFs were <5 and <3, respectively. Additionally, initial regression analyses were conducted to minimize the number of potential personal characteristics and partner variables used in the logistic regression. Only those personal characteristics and partner variables making significant contributions to prediction of intended condom use with steady or casual sex partners were included in subsequent analyses.\nSteady sex partners\nFirst, a regression of intended condom use with steady sex partners on personal characteristics (age, education, antiviral treatment, and years knowing HIV-positive status) and partner variables (steady partner only versus casual partners as well and HIV-negative or unknown status partner versus HIV-positive status) showed education level and partner HIV status to be the only significant predictors. Therefore, these variables were entered in the first step of the subsequent regression analysis, yielding an explained variance of 13% (see Table 4). When the TPB variables were entered in the second step, a further 40% (p\u00a0<.001) of variance was explained, with self-efficacy as a significant predictor. Subjective norms were borderline significant (p\u00a0=\u00a0.05). As shown in Table 4, there was no significant increase in variance explained with the addition of awareness of consequences for self and others and ascription of responsibility in Step 3, or with sexual motives for unprotected anal sex in Step 4. At this stage of the analysis, self-efficacy significantly contributed to the prediction of intention, whereas a marginally significant effect was found for subjective norms (p\u00a0<.10). When personal norms were entered in the final step of the analysis, a further 9% (p\u00a0<.001) of variance was explained. In this model, the effect of personal norms was significant; however, the effects of subjective norms and self-efficacy dropped below significance, which suggested that the effects of subjective norms and self-efficacy were mediated through personal norms.\nTesting this suggestion required that three conditions be met (see Baron & Kenny, 1986). The first condition-that subjective norms and self-efficacy be related to intended condom use-was assessed in the above analysis (see Step 4, Table 4). The second condition requires that the mediator (i.e., personal norms) predict intended condom use and that subjective norms and self-efficacy have weaker effects when controlling for personal norms. This was also confirmed in the above analysis (see Step 5, Table 4). The third condition requires that subjective norms and self-efficacy significantly predict the mediator. To test this condition, personal norms were regressed on subjective norms and self-efficacy, while controlling for educational level and partner HIV status. Results revealed that subjective norms (OR\u00a0=\u00a04.6, p\u00a0<.001) and self-efficacy (OR\u00a0=\u00a016.3, p\u00a0<.001) were significantly related to personal norms. Thus, there was evidence that personal norms mediated the effects of self-efficacy on intended condom use. In addition, there was evidence that personal norms mediated the tentative influence of subjective norms.\nCasual sex partners\nInitially, a regression of condom use intention with casual sex partners on personal characteristics (age, education, antiviral treatment, and years knowing HIV-positive status) and partner variables (casual partners only versus steady partners as well) showed antiviral therapy to be the only statistically significant predictor. Therefore, only this variable was subsequently entered in Step 1 of the logistic regression analysis, yielding an explained variance of 6% (see Table 5). When the TPB variables were entered in the second step, a further 46% (p\u00a0<.001) of the variance was explained. Attitudinal beliefs, subjective norms and self-efficacy significantly predicted intention, whereas a marginally significant effect was found for antiviral therapy. The entry of awareness of consequences for self and others and ascription of responsibility in Step 3 led to a further 8% (p\u00a0<.001) of explained variance. Step 4 also accounted for a significant increase of variance explained with 3% (p\u00a0<.01). In this step of the analysis, antiviral therapy, self-efficacy, ascription of responsibility and sexual motives significantly contributed to the prediction of intention, whereas a marginally significant effect was found for subjective norms (p\u00a0<.10). When personal norms were entered in the final step, a further 5% (p\u00a0<.001) of variance was explained. As shown in Table 5, antiviral therapy and personal norms positively and sexual motives negatively predicted behavioral intention. Furthermore, the effects of subjective norms, self-efficacy and ascription of responsibility became non-significant, which suggested that these variables were mediated through personal norms.\nTo test this notion, we again assessed the three conditions specified by Baron and Kenny (1986). First, the analysis above revealed that self-efficacy and ascription of responsibility significantly predicted intended condom use, whereas a marginally significant effect was found for subjective norms (see Step 4, Table 5). Second, the above analysis showed that adding personal norms gave a significant regression coefficient for personal norms, and non-significant regression coefficients for subjective norms, self-efficacy, and ascription of responsibility (see Step 5, Table 5). Finally, in an analysis predicting personal norms while controlling for antiviral therapy, subjective norms (OR\u00a0=\u00a02.7, p\u00a0<.05), self-efficacy (OR\u00a0=\u00a03.0, p\u00a0<.01), and ascription of responsibility (OR\u00a0=\u00a010.5, p\u00a0<.001) emerged as significant predictors. Taken together, these results provided evidence that personal norms were a mediator of the self-efficacy-intention and ascription of responsibility-intention relationships. In addition, evidence was found that personal norms mediated the tentative influence of subjective norms.Table 4Logistic regression analyses predicting high intention (0\u00a0=\u00a0no, 1\u00a0=\u00a0yes) to use condoms with steady sex partners (N\u00a0=\u00a0149)Model 1Model 2Model 3Model 4Model 5BOR95% CIBOR95% CIBOR95% CIBOR95% CIBOR95% CIStep 1\u2003Education level.251.3*1.1\u20131.6.111.1.86\u20131.5.071.1.81\u20131.4.071.1.81\u20131.4.01\u20031.0.74\u20131.4\u2003Partner HIV statusa\u22121.2.30**.14\u2013.64\u2212.43\u2212.65.24\u20131.8\u2212.40.67.24\u20131.9\u2212.40.67.23\u20131.9\u2212.35.70.22\u20132.3Step 2\u2003Attitudinal beliefs.491.6.67\u20134.0.351.4.55\u20133.7.371.5.53\u20134.01.1.37\u20133.1\u2003Subjective norms.672.01.0\u20133.8.611.8.92\u20133.7.621.9.92\u20133.7.07.64.20\u20132.1\u2003Self-efficacy1.75.4***2.1\u201313.71.64.9**1.7\u201313.61.64.9**1.7\u201314.3\u2212.442.7.82\u20138.9Step 3\u2003ACSb\u2212.02.98.71\u20131.4\u2212.02.98.70\u20131.4\u2212.01.99.70\u20131.4\u2003ACOc.241.3.88\u20131.8.241.3.88\u20131.8.221.3.85\u20131.8\u2003ARd.291.3.50\u20133.5.281.3.50\u20133.5.211.2.43\u20133.5Step 4\u2003Sexual motives.021.0.70\u20131.5\u2212.041.0.65\u20131.4Step 5\u2003Personal norms2.410.8**2.0\u201357.5\u2003Nagelkerke R2.13.53.54.54.62\u2003Model \u03c7215.874.577.177.192.1Note. At each step, variables were included in the equation simultaneously.aVariable coding: 0\u00a0=\u00a0HIV-negative or unknown, 1\u00a0=\u00a0HIV-positive.bAwareness consequences self.cAwareness consequences others.dAscription of responsibility.*p\u00a0<.05.**p\u00a0<.01.***p\u00a0<.001 (two-tailed).Table 5Logistic regression analyses predicting high intention (0\u00a0=\u00a0no, 1\u00a0=\u00a0yes) to use condoms with casual sex partners (N\u00a0=\u00a0188)Model 1Model 2Model 3Model 4Model 5BOR95% CIBOR95% CIBOR95% CIBOR95% CIBOR95% CIStep 1\u2003Antiviral treatmenta.992.7**1.4\u20135.2.742.1.88\u20135.01.12.9*1.1\u20137.31.13.1*1.2\u20138.41.23.3*1.2\u20139.2Step 2\u2003Attitudinal beliefs1.23.2**1.4\u20137.5.842.3.92\u20135.8.321.4.50\u20133.8.251.3.45\u20133.6\u2003Subjective norms1.12.9**1.3\u20136.5.902.51.0\u20136.1.862.4.91\u20136.1.391.5.50\u20134.4\u2003Self-efficacy1.54.2***2.3\u20137.91.12.9**1.5\u20135.9.862.4*1.1\u20135.0.531.7.76\u20133.8Step 3\u2003ACSb.241.3.85\u20131.9.251.3.85\u20132.0.131.1.72\u20131.8\u2003ACOc.341.4.89\u20132.2.341.4.88\u20132.2.341.4.86\u20132.3\u2003ARd1.13.0*1.2\u20137.51.13.0*1.1\u20137.9.641.9.65\u20135.5Step 4\u2003Sexual motives\u2212.61.54**.35\u20138.3\u2212.57.57*.36\u2013.89Step 5\u2003Personal norms1.33.8**1.6\u20138.9\u2003Nagelkerke R2.06.52.60.63.68\u2003Model \u03c728.991.4111.8120.5132.3Note. At each step, variables were included in the equation simultaneously.aVariable coding: 0\u00a0=\u00a0no, 1\u00a0=\u00a0yes.bAwareness consequences self.cAwareness consequences others.dAscription of responsibility.*p\u00a0<.05.**p\u00a0<.01.***p\u00a0<.001 (two-tailed).\nDiscussion\nThe present study showed that the majority of HIV-positive MSM reported engaging in safer sexual behavior either though abstinence or consistent condom use for anal sex. Nonetheless, a high number of men who had had anal sex reported that they had not used condoms consistently with steady and casual sex partners. Results further indicated that HIV-positive MSM were more likely to engage in unprotected anal intercourse in the context of casual sex encounters than in steady sexual relationships (overall 34.1% and 24.7%, respectively). This finding was consistent with patterns of sexual risk behavior observed in other populations of HIV-positive MSM (cf. Crepaz & Marks, 2002).\nIn this study, we tested a model of factors that influence intended condom use for anal sex with steady and casual sex partners. The results showed that, compared with the TPB (Ajzen, 1991), our proposed model of sexual decision-making led to a significant increase of explained variance from 53% to 62% for steady sex partners and from 52% to 68% for casual sex partners. Furthermore, evidence was obtained for the mediating role of personal norms on intended condom use with both steady and casual sex partners, suggesting that moral issues play an important role. However, the study also showed that sexual motives may undermine the influence of personal norms on intended condom use in the context of casual sex, with the likelihood of unprotected sexual intercourse. Taken together, the results revealed strong support for our model of sexual decision-making.\nIn this study, the finding that personal norms appeared to exert a strong direct effect on intention to use condoms with both steady and casual partners was consistent with results of earlier studies that suggest that safer sex in HIV-positive MSM is largely determined by moral concerns or feelings of personal responsibility (e.g., Godin et\u00a0al., 1996). Moreover, this finding lends further indirect support to the proposition that condom use behavior among HIV-positive MSM is a form of prosocial behavior (e.g., Kok, 1999; Nimmons, 1998). For steady partners, personal norms were identified as the most proximal determinant of intended condom use, whereas an indirect effect was found for self-efficacy on intended condom use through personal norms. Furthermore, some evidence that personal norms mediate the tentative effect of subjective norms on intended condom use with steady sex partners was found. Similar results were found for intended condom use with casual sex partners. However, we also found an additional indirect effect of ascription of responsibility on intended condom use through personal norms. These findings are important because they underscore the need to take into account the role of social expectations, self-efficacy expectations, and attributions about responsibility for condom use in promoting feelings of moral obligation-a process that has not been identified in previous research on sexual risk behavior in HIV-positive MSM.\nFurthermore, in the case of casual sex partners, condom use intention was not only directly related to personal norms, but was also negatively influenced by sexual motives for unprotected anal sex. Thus, the findings from this study suggest that the need to engage in unprotected anal sex for physical and emotional satisfaction may supercede the prosocial motivation for safer sex. The fact that sexual motives contributed toward explaining intended condom use in casual sex encounters but not in steady relationships does not imply that sexual motives are not important in the context of steady sex. More likely, there are some differences between steady and casual sex that encourage HIV-positive MSM to suppress sexual motives within the context of steady relationships but not within casual sex encounters. As discussed previously, a possible explanation may lie in the so-called affect-heuristic. In the context of casual sex, the benefits of unprotected sex may be more salient, thus resulting in a decreased assessment of risk. It is worth noting that the affect-heuristic is especially likely to color judgments under time pressure, which may play a more important role in the context of casual sex (Finucane et\u00a0al., 2000). The \u201cCoolidge effect\u201d may also explain why sexual motives play a role in the casual sexual context, but not in steady sexual relationships. The Coolidge effect refers to enhanced sexual arousal that is felt when sexual stimuli and partners are novel (Gregoire, 1999).1 A final explanation may be the greater ambivalence that HIV-positive MSM experience in casual sex encounters as opposed to steady relationships. Greater ambivalence is likely to result in a reduction in both behavioral intention and actual behavior. In addition, people who experience ambivalence are more easily persuaded (Conner & Armitage, 2000). It may be that pointing out the potential for casual sex partners to become steady partners in the future could increase the perceived benefits of safer sex, thus facilitating prosocial motivation to engage in protected sex. Indeed, the study by van Kesteren et\u00a0al. (2005) suggests that such a mechanism exists. Further, the results of this study demonstrated that the use of antiviral therapy was associated with a greater intention to use condoms with casual sex partners. To date, inconclusive evidence exists regarding the association between the use of antiviral therapy and unprotected sex (for a meta-analysis, see Crepaz, Hart, & Marks, 2004).\nSome study considerations and limitations and implications for practice should be mentioned. First, as Ajzen and Fishbein (1970) held, it may be argued that the important role of personal norms as a predictor of intention can be attributed to a certain amount of overlap between the measures of personal norms and intention. In this study, high correlations were found between personal norms and intention. On theoretical grounds, however, it seems imperative to distinguish personal norms from intentions. As Manstead (2000) argued, holding the belief that something is morally \u201cright\u201d or \u201cwrong\u201d is not the same as the perceived likelihood of performing certain behaviors. Consequently, an individual may feel a personal obligation to act in a certain way, but intend to behave in a way that is contradictory to his personal norm because the personal or social advantages of acting inconsistently with his personal norm outweigh the personal or social advantages of acting consistently with the norm. Second, it should be noted that the data were cross-sectional, which hampers conclusions about causality. Due to the cross-sectional design, we did not specifically address the relationship between the psychosocial factors and behavior. It should be noted, however, that tentative support for our model was found in an additional analysis when intention was examined as a mediator of the personal norms-behavior relationship, both for steady and casual sex partners. Yet, longitudinal studies are needed to determine causative relationships between the measured constructs. A third limitation of the present study concerns the use of a single-item approach to measure awareness of consequences, as single-item measures are known to be less reliable. This issue merits attention in future research. Finally, the sample used in this study warrants some concern. More than 70% of those who received questionnaires did not respond. This raises the question as to whether these participants were predominantly more aware of the need to practice safer sex and, as such, are not a representative sample of Dutch HIV-positive MSM. On the other hand, Swanborn (2002) found that 30% is a common response rate for this type of research in the Netherlands. It is possible that the sensitivity of the subject and the fact that the participants suffer from a chronic disease may create barriers to participation. Moreover, substantial efforts were made to recruit HIV-positive MSM from throughout the Netherlands. Although this approach did not ensure that the sample was representative, it did enhance its diversity and therefore strengthens the external validity of the study results.\nThe findings reported in this study carry several implications for HIV-prevention efforts to promote safer sexual behavior among HIV-positive MSM. When addressing condom use behavior with steady and casual sex partners, it seems particularly crucial to enhance feelings of moral obligation to use condoms for anal sex. Feelings of moral obligation could be addressed by inducing HIV-positive MSM to reflect on their own personal risk and personal standards for safer sex (Kalichman, 1998). The findings of this study further suggest that desirable changes in subjective norms and self-efficacy expectations might lead to corresponding changes in personal norms and intentions (e.g., Deci & Ryan, 2000; van Empelen, Kok, Jansen, & Hoebe, 2001; van Empelen, Schaalma, Kok, & Jansen, 2001). Therefore, preventive interventions should focus on helping HIV-positive MSM to cope with an unsupportive social environment and to encourage them to build the skills and confidence required for communicating and negotiating condom use. For the promotion of condom use with casual sex partners, personal norms may be further increased by urging HIV-positive MSM to accept responsibility for safer sexual behavior. Moreover, especially within casual sex encounters, it is imperative to help HIV-positive MSM to deal with feelings that may conflict with the goal of safer sex. Possible methods that can help HIV-positive MSM to identify and control high risk situations are, for example, action planning and coping planning (Sniehotta, Schwarzer, Scholz, & Sch\u00fcz, 2005). Action plans and coping plans are detailed plans of what a person needs to do when a specific situation occurs. When such situation arise, it is likely to function as a cue for the execution of those precise plans of implementation. Additionally, stimulating HIV-positive MSM to imagine how they would feel if they were in their sex partners shoes (i.e., perspective taking) may evoke a mixture of egoistic and altruistic motivation and, as such, lead to more protected sex (Batson, Early, & Salvarani, 1997). That such an approach is feasible has been shown in a study by van Kesteren, Kok, Hospers, Schippers, and De Wildt (2006), in which the above-mentioned methods have been integrated in an intervention to promote sexual health in HIV-positive MSM.","keyphrases":["hiv-positive","men who have sex with men","sexual motives","personal norms","sexual risk behavior"],"prmu":["P","P","P","P","P"]}
{"id":"Osteoporos_Int-3-1-1766477","title":"The prevalence of vertebral fracture amongst patients presenting with non-vertebral fractures\n","text":"Introduction Despite vertebral fracture being a significant risk factor for further fracture, vertebral fractures are often unrecognised. A study was therefore conducted to determine the proportion of patients presenting with a non-vertebral fracture who also have an unrecognised vertebral fracture.\nIntroduction\nOsteoporosis may be defined as a \u2018skeletal disorder characterized by compromised bone strength predisposing a person to an increased risk of fracture\u2019 [1]. Overall, osteoporotic or low trauma fractures are common - it has been estimated that one in two women and one in five men aged over 50\u00a0years will sustain at least one low trauma fracture in their lifetime [2].\nThe fracture sites which most commonly come to clinical attention include the distal radius (Colles\u2019), proximal femur (hip), humerus and ankle. In the Fracture Liaison Service covering Glasgow, 82% of presenting fractures were at these sites [3].\nThe spine is another key fracture site; however, it has been estimated that only 30% of vertebral fractures receive clinical attention - which means that the majority of patients with vertebral fractures remain undetected [4]. It appears that only those patients with the most severe vertebral fractures come to clinical attention\u2014it is likely that this is due to higher levels of back pain and disability [4].\nThe European Prospective Osteoporosis Study (EPOS) assessed vertebral fracture incidence in 3,174 men (mean age 63.1\u00a0years) and 3,614 women (mean age 62.2\u00a0years) over a mean of 3.8\u00a0years. The age-standardised incidence of morphometric fracture was 10.7\/1,000 person years in women and 5.7\/1,000 person years in men [6]. Vertebral fracture incidence increased markedly with age in both men and women. Indeed, the incidence in women aged 50\u201354 was 3.6\/1,000 person years, rising to 29.3\/1,000 person years in women aged 75\u201379 [5].\nLateral dorsal and lumbar X-rays were used in the European Vertebral Osteoporosis Study (EVOS) population to identify vertebral fracture over an 8-year period. The investigators found an incidence rate of 9.85\/1,000 person years compared with 3.06\/1,000 person years for hip fracture, 4.28\/1,000 person years for Colles\u2019 fracture and 4.76\/1,000 person years for other fractures [6].\nLow trauma fractures have substantial consequences. Although hip fracture has the greatest mortality and morbidity, vertebral fracture is also associated with significant mortality and morbidity. In one study, patients with vertebral deformity had a substantially higher risk of death compared to patients without vertebral deformity, regardless of age, sex and BMD (mortality rate 30.3% versus 10.7% over a 14-year follow-up). The risk of death was highest in those patients with a vertebral deformity who had a subsequent symptomatic fracture [HR 9.0 (3.1\u201326.0)] [7]. In terms of morbidity, around 70% of patients with symptomatic vertebral fracture complain of difficulty in standing and 65% of difficulty in bending, and 41% complain of constant pain [8].\nIt is well-established that the presence of one low trauma fracture significantly increases the risk of a further fracture [9\u201311]. In a systematic review carried out by Klotzbuecher et al. [9], women with a single pre-existing vertebral fracture, identified in eight studies at baseline by vertebral morphometry and in another seven as clinical (symptomatic) fractures, were 4.4-times more likely to have another vertebral fracture than women without vertebral fracture. Prior vertebral fracture also predicted non-vertebral fracture: in women with vertebral fracture, the risk of hip fracture was increased by 2.3-times, of Colles\u2019 by 1.4-times and of any non-vertebral fracture by 1.8-times. Fracture risk increased even further with each additional vertebral fracture; for example, the odds of a new vertebral fracture in women with five or more prior vertebral fractures were 35-times greater than for women without prior vertebral fracture.\nGiven that vertebral fracture is a significant risk factor for further fracture, and that vertebral fractures often go undiagnosed, this study set out to determine the proportion of patients attending a Fracture Liaison Service following a non-vertebral fracture who also have an unrecognised vertebral fracture.\nThe standard method to assess vertebral fracture is radiography of the thoraco-lumbar spine [12]. However, there is no gold standard for the definition of osteoporotic vertebral fracture. A number of methods have been developed for interpretation of spinal X-rays, including the Genant semi-quantitative method [13], which has been used as a surrogate \u2018gold standard\u2019 in a number of key osteoporosis studies. This approach is more objective and reproducible than other qualitative methods [12]. Vertebral morphometry using dual-energy X-ray absorptiometry (DXA) also known as morphometric X-ray absorptiometry (MXA) is a fast, low-radiation technique which produces images that are of sufficient quality to be used to diagnose the presence of vertebral deformity consistent with fracture [14].\nMethods\nThe Fracture Liaison Service in South Glasgow has been previously described [3]. This service assumes responsibility for all patients who present with a clinical low trauma fracture and ensures that appropriate assessment, diagnosis and treatment recommendations are made to prevent further fracture. A low trauma fracture is defined as one sustained from a standing height or less, and not occurring as a result of a road traffic accident. Patients with skull or facial fractures are not routinely offered assessment for osteoporosis [3]. All patients aged 50\u00a0years or over identified by an Osteoporosis Specialist Nurse have a DXA scan carried out if they are likely to be candidates for bisphosphonate therapy. It should be noted that patients aged over 70\u00a0years presenting to this service after hip fracture do not have a DXA scan carried out, since most of these patients have osteoporosis and require therapy.\nAll patients who presented with a low trauma fracture and who underwent DXA evaluation with vertebral morphometry from DV5\/6 to LV4\/5 were included in this prospective study. All scans were carried out on a Lunar Prodigy densitometer using the Dual Energy Vertebral Assessment (DVA) morphometry imaging software (version 6.3). Vertebral fractures were identified by direct visualisation using the Genant semi-quantitative grading scale [17]. All scans were reviewed by and vertebral deformities reported by the same physician (SJG). Although Genant\u2019s semi-quantitative grading is based on analysis of X-rays and is not validated for MXA, it was used in this study to assess vertebral status on MXA since it provides a practical and pragmatic option.\nStatistical analysis\nBaseline differences that potentially explained observed differences in morphometric vertebral fracture rates (sex, age, body mass index, T-score and prior fracture history) were clustered into pre-defined categories: \nSex: male\/femaleAge: 50\u201364, 65\u201374, 75+Body mass index:\u00a0\u00a0\u226418\u00a0kg\/m2, 19\u201324, 25\u201329, \u226530\u00a0kg\/m2; also compared by quartilesT-score: \u2265\u22121, \u22122.4 to \u22121.1,\u00a0\u2264\u22122.5Prior fracture history: yes\/no\nContingency tables for each set of categories were prepared and chi-squared values calculated to test for overall interaction. Yates continuity correction was used where 2\u00d72 tables resulted. Where the overall result was significant, individual pairwise comparisons and t-testing were then carried out.\nResults\nData was collected during the routine work of the FLS between September 2004 and March 2005. The data was analysed between July 2005 and July 2006.\nDemographics\nData were available for 337 patients presenting with low trauma fracture; 77.4% (n=261) were female. Of all patients, 10.4% were aged 50\u201364\u00a0years, 53.2% were aged 65\u201374\u00a0years and 36.2% were aged 75\u00a0years or over. Mean body mass index (BMI) was 24\u00a0kg\/m2 (SD 32); 6% (n=20) had a BMI \u226418\u00a0kg\/m2, 33% (n=112) between 19\u201324\u00a0kg\/m2, 35% (n=118) between 25\u201329\u00a0kg\/m2 and 26% (n=87) \u226530\u00a0kg\/m2 According to WHO definitions, 35.0% of patients had normal lumbar spine BMD (T-score \u22121 or above), 37.4% were osteopenic (T-score \u22121.1 to \u22122.4) and 27.6% were osteoporotic (T-score \u22122.5 or lower).\nHumerus (n=103, 31%), radius\u2013ulna (n=90, 27%) and hand\/foot (n=53,16%) were the most common presenting fractures (see Fig.\u00a01). Other fracture sites included hip (n=16, 5%), clavicle (n=11, 3%), rib (n=7, 2%), tibia-fibula (n=8, 2%), pelvis\u2013pubic rami (n=7, 2%) and olecranon, patella, scapula, sternum and shaft of femur (n=7, 2%).\nFig.\u00a01Presenting fracture and prevalence of vertebral deformity for each type of fracture. *Other includes olecranon, patella, scapula, sternum and shaft of femur\nThe presenting fracture was the first non-vertebral, low trauma fracture to come to clinical attention in 72% of patients (n=241), the second in 25% (n=85), the third in 1% (n=5) and the fourth in 2% (n=6).\nInformation regarding risk factors for fracture\u2014including prior history of fracture, early menopause, BMI, history of smoking or excess alcohol, thyrotoxicosis, history of maternal hip fracture, family history of osteoporosis, steroid use, rheumatoid disease, history of recurrent falls and partial gastrectomy\u2014was also collected. Of all patients, 63% had one fracture risk factor, 23% had two risk factors, 11% had three risk factors and 3% had more than three risk factors. Risk fractures for fracture were assessed after patients had presented to the FLS with a non-vertebral fracture; therefore, every patient had a prior history of fracture.\nVertebral deformity consistent with fracture\nThe overall prevalence of vertebral deformity established by MXA was 25%. The prevalence of vertebral deformity by type of presenting fracture is shown in Fig.\u00a01. Patients with a hip fracture were significantly more likely to have a vertebral deformity identified on MXA than were patients who presented with a non-hip fracture (p=0.009).\nOverall, 55.4% of patients with vertebral deformities had deformities in the thoracic spine and 26.4% had lumbar spine deformities, whilst 18.2% had deformities in both regions. Of the patients with vertebral deformities identified by MXA, 45% had deformities of more than one vertebra. Multiple vertebral deformity prevalence varied considerably according to the site of the presenting fracture (see Fig.\u00a02).\nFig.\u00a02Presenting fracture and prevalence of multiple (two or more) vertebral deformities\nOf the patients with vertebral deformities, 3 (3.6%) had unreadable scans for grading. Of the 80 remaining patients, 58 (72.5%) had Grade 2 or Grade 3 deformities. Thirty-two (55.2%) of the patients with vertebral deformity of Grade 2 and Grade 3 had multiple vertebral fractures. Of the sub-group of patients with vertebral deformity of Grade 2 and 3, 56.9% had deformities in the thoracic spine, 22.4% in the lumbar spine and 20.7% in both regions. There was no significant difference between the vertebral deformity population and the sub-population of patients with Grade 2 or 3 deformities, in terms of the proportion of patients with multiple vertebral deformities or in the site of deformity.\nTable\u00a01 shows the prevalence of vertebral deformity and multiple vertebral deformities by lumbar spine T-scores. Patients whose lumbar spine T-scores were consistent within the osteoporosis range (T-score \u22642.5) were significantly (p<0.05) more likely to have vertebral deformities (42%) than patients whose T-scores were in the osteopenic or normal ranges (20% and 16% respectively). They were also significantly more likely to have multiple vertebral deformities than those with a normal T-score (54% versus 14%, p<0.001). \nTable\u00a01Impact of lumbar spine T-score on prevalence of vertebral deformityLumbar spine T-scoreTotalVertebral deformityMultiple vertebral deformitiesFracture Grade 2 or 3n%n%%an%%bn%%aOsteoporotic (T-score \u2264\u22122.5).93283947.041.9205451.33255.234.4Osteopenic (T-score \u22122.4 \u2212 \u22121.1).126372530.119.8123248.01627.612.7Normal118351922.916.151426.31017.28.5Totals337100831003710058100Vertebral deformity: Chi-squared=13.471, p=0.0012Multiple vertebral deformities: Chi-squared=16.304, p=0.0003a% within each T-score group with vertebral deformityb% of vertebral deformity group with multiple deformities within each T-score group\nPatients with lower BMI (\u226418\u00a0kg\/m2) were significantly more likely to have vertebral deformity than patients with the highest BMI (\u226530\u00a0kg\/m2), (40% versus 17%, p<0.0001) (Table\u00a02). A very similar picture was seen when BMI was divided into quartiles: 45% of patients in the lowest quartile had a vertebral deformity, compared with 18% of those in the highest quartile (p<0.0001). The mean BMI for patients with and without vertebral deformity did not differ significantly (24.35\u00b14.78\u00a0kg\/m2 vs 25.22\u00b13.90\u00a0kg\/m2), nor did the mean BMI of patients with multiple vertebral deformities (23.70\u00b14.78\u00a0kg\/m2). \nTable\u00a02Impact of BMI on prevalence of vertebral deformity, n=336BMITotalVertebral deformityMultiple vertebral deformitiesFracture Grade 2 or 3n%n%%n%%n%%Underweight <18206.089.640.0513.562.5813.840.0Normal 19\u20132411133.03947.035.12054.151.32441.421.625\u20132911835.12226.518.6616.227.31729.314.430+8725.91416.916.1616.242.9915.510.3Totals336100831003710058100Vertebral deformity: Chi-squared=16.572, p=0.0009Multiple vertebral deformities: Chi-squared=15.288, p=0.0016\nPatients with a history of one or more non-vertebral fractures prior to the fracture they presented with were significantly more likely to also have vertebral deformity(ies) (Table\u00a03), (43% versus 20%, p=0.008). \nTable\u00a03Impact of prior fracture history on prevalence of vertebral deformity\u00a0TotalVertebral deformityMultiple vertebral deformitiesFracture Grade 2 or 3n%n%%n%%n%%024171.54959.020.32464.949.03560.314.51 or more9628.53441.042.71335.138.22339.724.0Totals337100831003710058100Vertebral deformity: Chi-squared=7.131, p=0.0078Multiple vertebral deformities: Chi-squared=0.237, p=0.63 (ns)\nTable\u00a01 also includes analysis of the sub-group of patients with vertebral deformities of Grade 2 and Grade 3 \nPatients with an osteoporotic T-score were significantly more likely to have vertebral deformities than those with a normal or osteopenic T-score (34% vs 12.7% [osteopenic] and 8.5% [normal] p<0.0001)Patients with BMI<18\u00a0kg\/m2 were significantly more likely to have vertebral deformities than those with BMI of over \u226530\u00a0kg\/m2 (40% vs 10.3%, p<0.0001)Patients with a history of one or more prior non-vertebral fractures were more likely to have vertebral deformity although this did not reach significance (24% vs 15%, p=0.055)\nOlder patients were slightly (although not statistically significantly) more likely to have a vertebral deformity: 30% of patients aged over 75 had a vertebral deformity, compared with 23% and 22% of patients aged 50\u201364\u00a0years and 65\u201374\u00a0years respectively. Gender did not appear to affect the likelihood of identifying a vertebral deformity (25% of women and 24% of men), although women were somewhat more likely to have multiple vertebral deformities (45% vs 40%, p=ns).\nDiscussion\nOne-quarter of patients presenting to our Fracture Liaison Service with a non-vertebral fracture had a previously undiagnosed vertebral deformity, of which almost one-half (45%) were multiple vertebral deformities. This is similar to the prevalence seen in previous studies using radiographic or MXA detection in women with and without non-vertebral fracture. Sub-analysis of patients by grade of deformity revealed that almost three-quarters of the patients had vertebral deformities of Grade 2 and Grade 3. These are more likely to be vertebral fractures, rather than vertebral deformities of non-osteoporosis aetiology, which might be more likely where Grade 1 deformities are present.\nIn the Study of Osteoporotic Fractures (SOF), a prospective study of almost 10,000 women, the prevalence of vertebral fracture was 20% (42% of which were multiple) in women aged over 65\u00a0years who did not have a non-vertebral fracture [11]. In another study of 482 women aged over 65\u00a0years without a non-vertebral fracture, the prevalence of vertebral fracture was 18.3% [15]. In a smaller study of 58 women with Colles\u2019 fracture, vertebral fractures were identified by MXA in 19% of patients [16].\nIn this present study, patients presenting with hip fracture, spine T-score \u2264\u22122.5, low BMI (either in the lowest quartile or \u226418\u00a0kg\/m2) or with more than one prior non-vertebral fracture were all significantly more likely to have evidence of a prevalent vertebral deformity. However, we found that 16% of patients with normal BMD at the lumbar spine had a prevalent vertebral deformity (26% of which were multiple). This is comparable to that seen in an earlier study conducted in women aged over 65\u00a0years without a non-vertebral fracture and with normal BMD at the spine, which revealed a prevalence of vertebral fracture of 18.7% [15].\nIn the UK, current treatment decisions for patients with low trauma fracture are based on fracture status and BMD as determined by DXA [17, 18]. In England, patients aged under 75 receive treatment if they have a fracture and osteoporosis is confirmed by DXA scanning [17]. In Scotland, patients with vertebral fracture and BMD outside the osteoporotic range, as defined by the WHO classification, do not routinely receive treatment unless they have two or more vertebral fractures, irrespective of BMD, or one vertebral fracture and an osteopenic BMD score [18]. Identification of those patients who already have an (often unrecognised) prior vertebral fracture is particularly important, as they are at high risk of further fracture by virtue of their prior vertebral fracture. Although these patients might not fulfil the criteria for therapy on the basis of their (non-vertebral) fracture history and BMD, they might be candidates for therapy if their underlying positive vertebral fracture status was known.\nIn this study, five of the 118 patients with normal BMD (4.2%) had two or more previously undiagnosed vertebral deformities plus a new non-vertebral fracture, and 25 of the 126 patients with osteopenic BMD (19.8%) had one or more previously undiagnosed vertebral deformities plus a non-vertebral fracture. These patients would not have been candidates for anti-resorptive treatment if MXA had not been performed and their vertebral deformities identified. Therefore, one in 20 patients with a normal T-score and almost one in five patients with an osteopenic T-score would experience a change in management by virtue of their underlying vertebral deformity or deformities.\nOverall, 30 (8.9%) of the patients in this study would be eligible for treatment by virtue of their newly identified vertebral deformity., which means that 11 patients who present with a non-vertebral fracture would need to undergo vertebral morphometry in order to identify one patient who ought to be managed differently. This \u2018Number Needed to Screen\u2019 gives an indication of the number of patients for whom treatment recommendations would change after the identification of vertebral fracture.\nThese data also provide the first attempt to quantify the overlap between patient populations with non-vertebral and vertebral fracture. Estimates suggest that 1.28 million post-menopausal women in the UK have experienced a prior non-vertebral fracture [19] and 1.3 million have at least one prior vertebral fracture [20]. In this study, 72% of patients presented with their first non-vertebral fracture, and vertebral morphometry subsequently identified vertebral deformities in one in five (21%) of these patients. Nearly one-half of vertebral deformities were multiple (48%). It is highly likely that these vertebral fractures had occurred prior to the first non-vertebral fracture, suggesting that a substantial number of patients will have experienced a vertebral fracture prior to any non-vertebral fracture event.\nOur results support the recommendation to perform vertebral morphometry in any patient who is referred for DXA testing after experiencing a non-vertebral fracture. Treatment decisions will then better reflect any given patient\u2019s future absolute fracture risk. The 'Number Needed to Screen' if MXA is used in this way would be seven to identify one patient with vertebral deformity, and 14 to identify one patient with two or more vertebral deformities.\nRoutinely performing lateral vertebral morphometry does raise a number of issues. Firstly, there is an increase in radiation exposure for the patient. A standard AP spine and hip DXA scan is associated with a radiation exposure [entrance surface dose (ESD)] of around 0.037\u00a0mGy, and results in an effective dose of less than 2\u00a0\u03bcSv (microsieverts). The ESD associated with lateral vertebral morphometry is 0.083\u00a0mGy [21],corresponding to an estimated effective dose of about 2.5\u00a0\u03bcSv. These are relatively small doses compared to the UK natural background radiation level of approximately 2.5\u00a0mSv. The small carcinogenic potential associated with these scans has to be balanced against both the prognostic importance (in terms of future fracture risk) that is associated with the identification of unrecognised vertebral deformities and also the much higher radiation exposure risk associated with plain spine radiology (effective dose \u223c0.6\u00a0mSv).\nLateral vertebral morphometry does have limitations; for example, it is less reliable than conventional lateral spine X-rays at the upper thoracic spine and in Grade 1 deformities.\nA recent study [22] comparing MXA with lateral spine X-rays found that vertebral morphometry using MXA allowed diagnosis of vertebral fracture in the lumbar and mid thoracic spine, where vertebral fractures are most common. MXA was less reliable at the upper thoracic spine, which is also an established limitation of X-rays.\nAn earlier study [23] compared identification of vertebral fracture on MXA using the Genant semi-quantitative method using MXA with conventional X-rays in 80 post-menopausal women. Of the 1,027 potentially evaluable vertebrae, 81% were adequately visualised on MXA to allow grading. Prevalent vertebral fracture was identified in 40 vertebral bodies using X-ray; of these, 28 (70%) were correctly identified on MXA, 17\/18 (95%) Grade 2 or 3 and 11\/22 (50%) of Grade 1 fractures. Of the non-fractured vertebrae, 96.2% were correctly classified as normal by MXA.\nAlthough the Genant semi-quantative method is not validated for MXA, it has been used in other studies [22, 23], and was used in this study as a pragmatic option. To carry out quantitative analysis on each scan would have been time-consuming and impractical.\nGiven that the sample population had already had at least one non-vertebral fracture (28% had more than one prior fracture), were drawn from the FLS population, and that almost three-quarters had Grade 2 or 3 vertebral deformity, it seems reasonable to expect the vertebral deformity to be due to osteoporosis, rather than other diagnoses such as degenerative change or Scheurmann\u2019s disease.\nThe sensitivity of detection of a vertebral fracture is lower both where there is significant spine scoliosis and also where there is marked disc-space osteoarthritic change. In these settings, proceeding to plain spine radiology would be recommended [24]. Plain spine X-rays were not carried out in this study. Furthermore, in keeping with our local protocol, bone densitometry was not carried out in older patients (over the age of 70) after hip fracture. Both of these features may mean that the overall prevalence of vertebral deformity in this population has been underestimated.\nIn conclusion, undiagnosed vertebral deformity is highly prevalent in patients aged over 50\u00a0years who present with a new non-vertebral fracture. Given that prior vertebral fracture significantly increases the risk of further fracture and because newer diagnostic techniques make identification of vertebral deformity consistent with fracture relatively straightforward with minimal radiation exposure, we recommend that all patients undergoing DXA should also undergo vertebral morphometry to identify prior vertebral deformities consistent with fractures.","keyphrases":["vertebral fracture","osteoporosis","low trauma fracture","vertebral morphometry","number needed to screen"],"prmu":["P","P","P","P","P"]}
{"id":"Int_J_Colorectal_Dis-4-1-2386753","title":"Fibrin glue and transanal rectal advancement flap for high transsphincteric perianal fistulas; is there any advantage?\n","text":"Backgrounds and aim In recent decades, fibrin glue has appeared as an alternative treatment for high perianal fistulas. Early results seemed promising, with high success rates being reported. However, with increasing follow-up, the enthusiasm was tempered because of disappointing results. The aim of this retrospective study was to assess the additional value of fibrin glue in combination with transanal advancement flap, compared to advancement flap alone, for the treatment of high transsphincteric fistulas of cryptoglandular origin.\nIntroduction\nPerianal fistulas of cryptoglandular origin cause considerable discomfort and arise from infections in anal glands lying in the intersphincteric space [1]. To delineate the fistula tract, magnetic resonance imaging (MRI) and anal endosonography are nowadays readily available and are increasingly used for fistula imaging [2]. The essence of surgical treatment of perianal fistulas is to eradicate the fistula tract and at the same time preserve continence. Low fistulas, where the fistula tract is submucosal, intersphincteric or located in the lower third of the external anal sphincter can be treated by fistulotomy with low recurrence rates and relatively little impact on continence [3]. In patients with high perianal fistulas, the fistula tract is located in the upper two-thirds of the external sphincter. Fistulotomy performed on high fistulas results in loss of sphincter function in a considerable number of patients due to the interference of the external sphincter complex [4, 5]. There are various alternative surgical options for high fistulas, namely, rectal advancement, fibrin glue, and seton drainage.\nIn 2006, Johnson et al. [6] reported a new biologic anal fistula plug to treat high transsphincteric perianal fistulas. The anal fistula plug is biologic absorbable and consists of lyophilized porcine intestinal submucosa. In their series of 46 patients treated with the anal fistula plug, a success rate of 83% was achieved at a median follow-up of 12 months [7]. After this publication, several authors have reported their experience with the anal fistula plug, resulting in success rates ranging from 41\u201388% [8\u201310].\nCurrently, the transanal rectal advancement flap (AF) remains the \u201cgold standard\u201d in the treatment of high transsphincteric perianal fistulas of cryptoglandular origin. The rationale behind the AF is that the open internal opening is the cause of the persisting fistula tract. By advancing the tissue over the internal opening, it would be impossible for fecal material to be forced into the fistula tract during defecation. However, recurrence rates of the advancement flap found in literature vary considerably and extend up to 63% [11\u201315]. Roughly, one out of every four patients requires multiple surgical interventions to close the fistula tract successfully.\nIn recent decades, fibrin glue has appeared as an alternative treatment for high perianal fistulas. As a result of the obliteration of the fistula tract and the closure of the internal opening, the fistula might heal. Early results seemed promising, with high success rates being reported. However, with increasing follow-up, the enthusiasm was tempered because of disappointing results [16\u201320]. Recently, Zmora et al. [21] conducted a retrospective study including 37 patients with high perianal fistulas. In a subset of 13 patients with fistulas of various etiologies, the advancement flap was used in addition to the fibrin glue installation. The results showed a recurrence rate of 46%.\nThe aim of this study was to assess the additional value of fibrin glue to the transanal rectal advancement flap in a well-defined group of patients with high transsphincteric fistulas of cryptoglandular origin. Patients with previous fistula surgery are a surgically more challenging group as the result of scar tissue and sometimes anal stenosis. Therefore, patients were matched for the presence of a history of fistula surgery.\nMaterials and methods\nPatient characteristics Between January 1995 and January 2006, a consecutive series of patients were treated by AF. Only patients with high transsphincteric perianal fistulas of cryptoglandular origin were analyzed. High perianal fistulas were defined as patients with fistulas running through the upper two thirds of the external sphincter complex, which is confined by the puborectal sling and the end of the anal canal. Patients in whom the internal fistula opening was not detectable and patients with perianal fistulas as a result of Crohn\u2019s disease, HIV, and other causes were excluded. Patients over 18years of age were included. A consecutive series of patients were operated on with fibrin glue (Tissucol Duo\u00ae, Baxter International) in addition to the AF in an attempt to decrease the recurrence rate of the AF. This series of patients were treated on between February 2003 and January 2006. Patients were matched for previous surgery and divided into two groups; one group with previous fistula surgery and the other without. Preoperatively, no routine imaging was performed. Only in selected case when the fistula was complex and\/or recurrent, MRI or anal endosonography was used to outline the fistula tract. In The Netherlands and Belgium, non-experimental clinical case series of patients treated with a CE-approved device do not require approval of the local Medical Ethics Commission. A subset of patients from this series were also included in a study where the aim was to assess the long-term functional outcome and identify risk factors for the development of recurrence in patients surgically treated for cryptoglandular fistulas [22].\nSurgical technique On the day of surgery, an enema was administered to the patient to clean the proctum. All procedures were performed under general or locoregional anesthesia in the lithotomy position, and broad spectrum antibiotics were administered perioperatively. Subsequently, the internal opening was located by probing the external opening. During surgery, the amount of sphincter involved was judged by palpation of the puborectal sling and the inferior edge of the external sphincter complex. In cases where the internal opening was not found by probing, hydrogen peroxide was injected to locate the internal opening. In case of active sepsis, a seton was placed for a period of at least 3 months insuring adequate drainage. In the group that was operated with the AF, the internal opening was excised followed by mobilization of the mucosa, submucosa, and a small amount of muscular fibers from the internal sphincter complex. The rectal flap was mobilized to sufficiently cover the internal opening with overlap. Hemostasis was performed to prevent a hematoma under the flap. The base of the advancement flap was kept wide enough to ensure adequate circulation in the flap. The internal opening was not closed before advancing the flap over the internal opening. This was followed by suturing the flap in the distal anal canal with Vicryl 2\/0 after the fistula tract had been curetted. In the AF + G group, the identical procedure was carried out. In addition to the advancement procedure, fibrin glue was installed retrogradely in the fistula tract after the AF was completed and the fistula tract had been curetted. Installation of the fibrin glue was performed via the external opening, under direct vision of the advancement flap to prevent flap dislocation and subsequent failure. No specific postoperative instructions were given to the patients.\nData collection Chart review was performed on age, gender, tertiary referral, previous fistula surgery, smoking habits, complications, and fistula recurrence. All patients visited the outpatient\u2019s clinics on a regular basis (every 2\u20134 weeks) until full closure of the fistula tract was achieved. The fistula was considered closed if the external opening was closed and no discharge or pain was experienced; otherwise, it was considered as a persistent or recurrent fistula. No routine postoperative imaging or proctoscopy was performed to confirm the closure of the fistula tract. The data was collected retrospectively and the outcome was compared between groups.\nStatistical analysis Data are presented as median values with ranges, unless otherwise specified. Categorical data are presented as frequencies or percentages. Differences between groups were tested using Mann\u2013Whitney U test for continuous data. Chi-square test or Fisher\u2019s exact test were used when appropriate to compare groups in case of categorical or dichotomous variables. All reported p values are two-sided. A p value of 5% or less was considered as statistical significant. Statistical analysis was done using the SPSS v.12.0 package (SPSS, Chicago, IL, USA).\nResults\nIn the study period, a total of 127 patients were operated for high perianal fistulas. Inflammatory bowel disease (n = 30), HIV (n = 12), or no internal opening found during surgery (n = 5) were the reason of exclusion in 47 patients. In total, 80 patients were analyzed in this comparative study. Of these, 54 patients were treated with the AF and 26 patients underwent AF combined with the installation of fibrin glue. Furthermore, patients were matched for a history of fistula surgery. Patient characteristics for both groups are shown in Tables\u00a01 and 2. The groups were comparable for patients\u2019 characteristics as sex, age, smoking, seton drainage, and number of tertiary referrals.\nTable\u00a01Characteristics of patients with high anorectal fistula without previous fistula surgeryVariableAF (n\u2009=\u200932)aAF\u2009+\u2009G (n\u2009=\u20099)bp valueM\/F (n)18:146:30.711Age (median, in years)42 (21\u201367)41 (29\u201355)0.653Tertiary referral26 (81%)7 (78%)1.000Smoking43%71%0.232Seton drainage18 (56%)6 (67%)0.711aRectal advancement groupbRectal advancement group with fibrin glueTable\u00a02Characteristics of patients with high anorectal fistulas with previous fistula surgeryVariableAF (n\u2009=\u200922)aAF\u2009+\u2009G (n\u2009=\u200917)bp valueM\/F (n)18:411:60.282Age (median, in years)43 (22\u201362)47 (35\u201372)0.136Tertiary referral15 (68%)8 (47%)0.209Smoking53%50%1.000Seton drainage10 (46%)12 (71%)0.193aRectal advancement groupbRectal advancement group with fibrin glue\nClinical outcome All patients were operated in day case setting. There were no intraoperative complications. In two patients out of the AF group, a postoperative complication was encountered, consisting of a minor bleeding (n = 1) and a bradycardia for which the patient was observed overnight (n = 1, patient with cardial history). In the AF + G group, there were no postoperative complications recorded. The minimal follow-up after surgery was 13 months with a median of 67 months (range, 13\u2013127). There were no patients lost to follow-up. The overall recurrence rate was 26% (n = 21; Table\u00a03.). In 17% of the patients, the fistula persisted in the AF group compared to 46% in the AF + G group (p = 0.05). In the matched group without previous fistula surgery, the result was significantly worse for the AF + G group compare to the AF group (p = 0.014). The recurrence rates were 56% (n = 5) and 13% (n = 4), respectively. In the group with a history of fistula surgery, the recurrence rate was 23% (n = 5) compared to 41% (n = 7) in the AF and the AF + G group, respectively (p = 0.216).\nTable\u00a03Recurrence rates for the matched group analysisGroupAFaAF\u2009+\u2009Gbp valueOverall (n\u2009=\u200980)9\/54 (17%)12\/26 (46%)0.050No previous fistula surgery (n\u2009=\u200941)4\/32 (13%)5\/9 (56%)0.014Previous fistula surgery (n\u2009=\u200939)5\/22 (23%)7\/17 (41%)0.216aRectal advancement groupbRectal advancement group with fibrin glue\nDiscussion\nHigh perianal fistulas remain a surgical challenge. There are various treatment options for treating high transsphincteric fistulas, e.g., the rectal and anodermal advancement flap, loose and cutting seton, fibrin glue, and potentially the newly developed anal fistula plug [3]. However, the results from these therapies vary. Transanal rectal advancement flap is nowadays the treatment of choice because of its sphincter-saving approach. Due to the low recurrence rate of only 30%, which leaves a lot of room for improvement [11, 14], fibrin glue has been widely studied. Fibrin glue was developed to obliterate the fistula tract by stimulating fibroblasts, which leads to permanent closure of the fistula tract. Unfortunately, the long-term results were not as good as expected [19, 20]. In the present series, an attempt was made to decrease the recurrence rate of the surgical treatment of high transsphincteric perianal fistulas of cryptoglandular origin by combining the two methods, i.e., fibrin glue and the rectal advancement flap in a consecutive series of patients. Overall, although not significant, a clear trend was found consisting of a worse outcome for patients from the AF + G group. The recurrence rate was 46% compared to 17% in the AF + G and AF group, respectively. In the group without a history of fistula surgery, patients in the AF + G group did significantly worse than the AF group. In the group with a history of fistula surgery, no significantly different recurrence rates were found.\nIn 2003, Zmora et al. [21] described a small retrospective series of 13 patients with perianal fistulas of different origins treated with fibrin glue in combination with the AF [14]. In their series, a recurrence rate of 46% was found after a mean follow-up of 12.1 months. The group contained patients with fistulas of cryptoglandular origin and fistulas associated with Crohn\u2019s disease or surgical trauma. In addition, two patients with rectovaginal fistulas were included. More recently, Ellis and Clark [23] reported on a series of 58 patients randomized into advancement flap repair alone or advancement flap repair combined with fibrin glue. Selected were patients with perianal fistulas where the fistula tract comprised more than 30 to 50% of the sphincter complex. Furthermore, patients were included when the fistula was located anteriorly in women or when the patient had a history of incontinence. In two thirds of the patients, the mucosal advancement flap was used and the remaining patients were treated by anodermal advancement flap. The recurrence rate was significantly higher in the group where fibrin glue was combined with the advancement flap compared to the group treated only by advancement (46% vs 20%). Two techniques, i.e., mucosal advancement flap and anodermal advancement flap, were used. Furthermore, no information was provided on the distribution of causes of the fistulas in both groups making the results difficult to interpret.\nThe effectiveness of the AF is the result of the closure of the internal opening [24]. The reason why addition of fibrin glue fails to decrease the recurrence rate and even seems to worsen the result is still unclear. After AF, the fistula tract acts as a drainage canal for any remaining sepsis, with the external opening left open. With the installation of the fibrin glue, a temporary closure of the fistula tract is theoretically achieved. After a few weeks, when the clot resolves, the fibroblasts activated by the matrix should provide collagen syntheses for a definitive closure of the tract [25]. A possible explanation to why the AF + G group does worse is that the closure of the fistula tract with the fibrin glue leads to a situation where insufficient drainage from the primary and eventual secondary fistula tracts occurs.\nSentovich reported on a prospective series of 48 patients (75% of cryptoglandular origin) treated with fibrin glue [20]. In their technique of using the fibrin glue, the procedure was combined with closure of the internal opening with only a figure eight suture without an advancement flap. After a median follow-up of 22 months, a recurrence rate of 31% was found. Surprisingly, the patients with longer fistula tracts did significantly worse than those with short fistula tracts. Loungnarath et al. [26] reported on 39 patients with perianal fistulas treated with fibrin glue. The overall recurrence rate was 69%. In 6 of the 39 patients, the internal opening was closed using a figure eight suture to avoid clot extrusion because of high pressure from the anal canal during defecation. Four of these patients had a recurrence (33%).\nThis retrospective study, in contrast to earlier studies [21, 23], assessed the additional value of fibrin glue to the transanal rectal advancement flap of only patients with high transsphincteric fistulas of cryptoglandular origin with a long follow-up. The rectal advancement flap combined with fibrin glue installation was associated with a significantly higher recurrence rate, compared to the advancement flap treatment alone, in patients without previous fistula surgery. This observation must be interpreted carefully because of the small sample size of the AF + G group. As the costs of the fibrin glue are considerable and the therapeutic effect very doubtful, it cannot be recommended routinely in the adjunct of transanal rectal advancement flap treating high perianal fistulas. The rectal advancement flap remains the treatment of choice for high transsphincteric perianal fistulas of cryptoglandular origin until novel methods like the anal fistula plug are studied sufficiently in randomized trials.","keyphrases":["fibrin glue","advancement","recurrence","surgery","rectal fistula"],"prmu":["P","P","P","P","R"]}
{"id":"J_Mol_Med-4-1-2374880","title":"Prorenin anno 2008\n","text":"For many years, prorenin has been considered to be nothing more than the inactive precursor of renin. Yet, its elevated levels in diabetic subjects with microvascular complications and its extrarenal production at various sites in the body suggest otherwise. This review discusses the origin, regulation, and enzymatic activity of prorenin, its role during renin inhibition, and the angiotensin-dependent and angiotensin-independent consequences of its binding to the recently discovered (pro)renin receptor. The review ends with the concept that prorenin rather than renin determines tissue angiotensin generation.\nIntroduction\nDespite nearly 40\u00a0years of research on prorenin, the renin precursor is still the least well-understood component of the renin\u2013angiotensin system (RAS). Initially, it was thought to have no function at all. Yet, it circulates in human plasma in excess to renin, sometimes at concentrations that are 100 times higher, and in plasma of anephric subjects, prorenin, but not renin, is still present [1, 2]. This suggests that prorenin, in contrast with renin, is also of extrarenal origin. Prorenin is particularly elevated in diabetic subjects with microvascular complications [3]. Moreover, the renal vasodilator response to captopril in diabetic subjects correlated better with plasma prorenin rather than with plasma renin [4]. Thus, (circulating) prorenin may have a function after all. Possibly, it is prorenin (and not renin) which is responsible for tissue angiotensin generation. Obviously, this would require local prorenin\u2013renin conversion, for which no evidence exists [5]. In support of this concept, however, transgenic rodents with (inducible) prorenin expression in the liver display increased cardiac angiotensin (Ang) I levels, cardiac hypertrophy, and\/or vascular damage [6, 7].\nOrigin and regulation of prorenin\nThe juxtaglomerular epithelioid cells, located in the walls of renal afferent arterioles, are the main source of renin in the body. Renin is synthesized as preprorenin. Preprorenin is converted to prorenin upon insertion into the endoplasmatic reticulum. The majority (75%) of prorenin is secreted constitutively, while the remainder is targeted to dense core secretory granules. In these granules, an acidic pH is created to optimize the activity of the proteases (cathepsin B, prohormone convertases) that cleave off the prosegment to yield renin. Prorenin and renin levels are highly correlated but do not alter in parallel under all circumstances [1]. Acute stimuli of renin will not affect prorenin levels, whereas chronic stimuli (like a decrease in Ang II) increase both renin and prorenin. This suggests that renin is stored as active enzyme and is released immediately upon stimulation of the juxtaglomerular apparatus. Prorenin is released constitutively, and no acute responses occur. Chronic stimulation causes more prorenin to be converted to renin, leading to an increased renin\/prorenin ratio in plasma. However, some exceptions to this rule exist. A well-known example is, as mentioned above, diabetes mellitus complicated by retinopathy and nephropathy [3]. Pregnant women also have high plasma prorenin levels, derived from the ovaries [8]. The function of this prorenin is unknown, as is the function of prorenin in amniotic fluid, in which prorenin was discovered. The reproductive organs, together with the adrenal, eye, and submandibular gland, are sites of extrarenal renin gene expression [9]. For reasons that are not understood, these tissues predominantly, if not exclusively, synthesize and release prorenin.\nEnzymatic activity of prorenin?\nA 43-amino-acid N-terminal propeptide explains the absence of enzymatic activity of prorenin. This propeptide covers the enzymatic cleft and obstructs access of angiotensinogen to the active site of renin. Prorenin can be activated in two ways: proteolytic or nonproteolytic [10]. Proteolytic activation is irreversible: it involves actual removal of the propeptide. Nonproteolytic activation of prorenin is reversible. It can best be imagined as an unfolding of the propeptide from the enzymatic cleft. Nonproteolytic activation can be induced by exposure to low pH (pH\u2009=\u20093.3) or cold (4\u00b0C) [10]. Nonproteolytically activated prorenin is enzymatically active and can be recognized by monoclonal antibodies that are specific for the active site. Kinetic studies of the nonproteolytic activation process have indicated that an equilibrium exists between the closed (inactive) and open (active) forms of prorenin. The inactivation step is highly temperature dependent and occurs very rapidly at neutral pH and 37\u00b0C. Consequently, under physiological conditions, <2% of prorenin is in the open and active form, i.e., displays enzymatic activity, and >98% is closed and inactive.\nProrenin receptor(s)?\nThe beneficial effects of RAS blockers are due, at least in part, to blockade of the generation or action of Ang II at tissue sites [11]. In tissues that are believed not to express the renin gene, like the heart and vascular wall, such angiotensin generation depends on renin\/prorenin taken up from the circulation. Simple diffusion cannot explain the relatively high renin levels in these organs, and thus a receptor-mediated mechanism may exist. Two candidates currently have been proposed: the mannose 6-phosphate\/insulin-like growth factor II receptor (M6P\/IGF2R) [12\u201314] and the (pro)renin receptor [15]. The M6P\/IGF2R nonselectively binds M6P-containing proteins like renin and prorenin. However, such binding did not result in angiotensin generation, and it is now believed that the M6P\/IGF2R is a clearance receptor for renin\/prorenin [16]. This leaves the (pro)renin receptor as the most promising candidate for tissue uptake of circulating renin\/prorenin. This receptor, a 350-amino-acid protein with a single transmembrane domain, binds prorenin with higher affinity than renin [17] and, unlike the M6P\/IGF2R, does not internalize these proteins. Interestingly, binding to the receptor allowed prorenin to become catalytically active without proteolytic cleavage of the prosegment [15, 17]. Apparently, therefore, binding induces a conformational change in the prorenin molecule, similar to the change occurring after exposure to cold or low pH.\nAngiotensin-independent effects of prorenin?\nAfter the discovery of the receptor, (pro)renin receptor antagonists were designed based on the idea that the prosegment contains a \u2018handle region\u2019 which binds to the receptor [18]. These (peptidic) antagonists (also known as \u2018handle region peptides\u2019, HRP) mimic the handle region and thus may bind to the receptor instead of prorenin. In support of this concept, HRP infusion normalized the elevated renal angiotensin content in diabetic rats [18] and simultaneously prevented the development of diabetic nephropathy.\nSurprisingly, identical effects occurred in diabetic angiotensin II type 1A receptor-deficient mice [19]. Since such mice no longer display the normal (constrictor) response to Ang II [20], the effect of the (pro)renin receptor antagonist in these mice cannot be due to suppression of local angiotensin generation. Thus, prorenin may also exert direct angiotensin-independent effects, possibly via the above described (pro)renin receptor (Fig.\u00a01). Indeed, prorenin (and renin) induced p42\/p44 mitogen-activated protein kinase (MAPK) activation and transforming growth factor-\u03b21 release in mesangial cells [15, 21], and these effects did not occur following deletion of the receptor with siRNA [21]. Moreover, in cardiomyocytes, prorenin concentration dependently activated p38 MAPK and phosphorylated heat shock protein 27 [22].\nFig.\u00a01Model depicting prorenin activation by the (pro)renin receptor (P)RR, allowing prorenin to generate angiotensin I from angiotensinogen. In addition, prorenin binding to the receptor results in effects (intracellular signaling) that are independent of angiotensin generation. HRP is assumed to block both prorenin activation and the direct prorenin-induced effects (see text for further explanation)\nOverexpression of the human (pro)renin receptor in rats resulted in elevated blood pressure, increased plasma aldosterone, and\/or glomerulosclerosis [23, 24]. Since such overexpression was not accompanied by changes in renin or Ang II, angiotensin-independent effects of the receptor may underlie this phenotype. HRP prevented the development of glomerulosclerosis in (pro)renin receptor transgenic rats [25]. Yet, transgenic rats with inducible hepatic prorenin expression (resulting in a >200-fold rise in plasma prorenin) did not develop glomerulosclerosis [26], although such animals did develop hypertension. Moreover, HRP blocked neither prorenin binding to cells overexpressing the human (pro)renin receptor [17] nor prorenin-induced signaling in U937 monocytes [27]. Thus, it is uncertain to what degree the beneficial in vivo effects of HRP are solely due to prorenin blockade. Such effects are unlikely to involve renin, since HRP will not block renin\u2013(pro)renin receptor interaction.\nProrenin and renin inhibition\nProrenin levels will rise during renin inhibition, as they do during any type of RAS blockade. Renin inhibitors affect the equilibrium between the open and closed forms of prorenin, because such drugs (due to their high affinity for the active site) prevent inactivation [10]. Therefore, renin inhibitors, like low pH, cold, and the (pro)renin receptor, are capable of nonproteolytically \u2018activating\u2019 prorenin although, of course, due to the presence of the renin inhibitor, this open prorenin cannot display enzymatic activity. Open prorenin will however be recognized by the active site-directed antibodies applied in renin immunoradiometric assays, thus leading to an overestimation of the renin rise during renin inhibition [28].\nTheoretically, the elevated (pro)renin levels during renin inhibition might result in (pro)renin receptor activation. In vitro studies suggest that renin inhibitors do not interfere with this process. The consequence of such overstimulation is unknown. However, since such a rise also occurs during other types of RAS blockade, its detrimental effects, if present, should have been known by now. A possible explanation comes from the work of Schefe et al. [29], who showed that, on activation of the receptor, the transcription factor promyelocytic zinc finger is translocated to the nucleus and represses transcription of the (pro)renin receptor itself, thus creating a short negative feedback loop. In other words, high (pro)renin levels, as occurring during RAS blockade, will suppress (pro)renin receptor expression, thereby preventing excessive receptor activation. Furthermore, the members of a family with a mutated renin allele, resulting in high plasma prorenin levels, were phenotypically normal [30].\nConclusion\nAfter many years, it now seems that a function for prorenin has been found. The \u2018inactive\u2019 renin precursor gains Ang I-generating activity by binding to a receptor, without undergoing proteolytic cleavage. This mechanism explains how prorenin might contribute to tissue angiotensin generation, even when no prorenin\u2013renin conversion occurs outside the kidney. Renin inhibitors will bind to such open activated prorenin, to the same degree as they bind to renin, and may thus be the ideal tools to block tissue angiotensin generation. In vitro studies suggest that prorenin also acts as an agonist of the (pro)renin receptor, inducing intracellular signaling pathways in an angiotensin-independent manner. However, given the contradictory data obtained with the (pro)renin receptor blocker HRP, more work is needed to verify the in vivo importance of such prorenin-induced (pro)renin receptor activation.","keyphrases":["renin","diabetes","(pro)renin receptor","receptor","tissue angiotensin","signaling","renin inhibitor","renin angiotensin system"],"prmu":["P","P","P","P","P","P","P","R"]}
{"id":"Dysphagia-3-1-1914227","title":"Viscosity Is Not a Parameter of Postdeglutitive Pharyngeal Residue: Quantification and Analysis with Scintigraphy\n","text":"The aim of this study was to explore the influence of viscosity on pharyngeal residue in normal healthy volunteers. Scintigraphy was used to measure pharyngeal residue in 11 healthy volunteers after swallowing three different substances (age = 20.2\u201348.3 years). The first substance was a 10-ml solution of tap water with 0.5% xanthan with a viscosity of 4500 mPa s, comparable to a yogurt drink. The second and third substances were a 0.75% xanthan and a 1.00% xanthan solution, with viscosities of 10,500 and 21,000 mPa s, comparable to low-fat yogurt and 3% fat yogurt, respectively. Tap water was used as the control substance. Mean pharyngeal residue after swallowing tap water was 2.3% (SD = 1.2) of the initial volume in the oral cavity. Pharyngeal residue after swallowing 0.5% xanthan solution was 1.8% (SD = 0.8), after swallowing 0.75% xanthan solution 2.6% (SD = 2.2), and after swallowing 1.00% xanthan solution 2.8% (SD = 1.7). No significant correlation between increase of viscosity and pharyngeal residue was found. In healthy persons viscosity does not seem to be a significant parameter for pharyngeal residue for boluses with viscosities ranging from tap water to solutions having a viscosity comparable to 3% fat yogurt.\nThe residual amount of food or beverage in the pharynx after swallowing (pharyngeal residue) is an important parameter in diagnostic procedures in the treatment of swallowing disorders. In common practice of management of swallowing disorders, the amount of pharyngeal residue is determined either by videofluoroscopy or by transnasal flexible endoscopy (FEES). With the help of logistic regression, Perlman et al. [1] predicted the chance of aspiration (food or liquid that gets into the lungs) based on the amount of pharyngeal residue. An odds ratio of 1.4 for aspiration was found for patients with pharyngeal residue. In this study 330 videofluoroscopies of dysphagic patients were analyzed and the amount of pharyngeal residue was rated on a four-point scale (absent, mild, moderate, severe). The study also showed that a patient with severe pharyngeal residue has an increased risk of aspiration (odds ratio of 4.0) and underlined the importance of quantifying pharyngeal residues. Dejaeger et al. [2] analyzed videofluoroscopies of 25 healthy elderly and clearly described localization of pharyngeal residue; unfortunately, they did not attempt to quantify the amount of residue.\nFew attempts have been made to quantify pharyngeal residue in normal and dysphagic populations, mainly because of the limited availability of appropriate diagnostic tools. Videofluoroscopy or FEES has been used to explore physiologic parameters of swallowing [3,4]. One of the limitations of the use of these techniques is the lack of reliable interpretation of physiologic parameters. Stoeckli et al. [5] analyzed the inter- and intrajudge reproducibility of the interpretation of videofluoroscopy and suggested that the agreement for assessing pharyngeal parameters is minimal to moderate (\u03ba = 0.01\u20130.56).\nScintigraphy allows quantitative measurement by using a technetium-99m (99mTc)-labeled colloid. 99mTc colloid is used in routine clinical practice for investigating gastrointestinal motility disorders of the esophagus and stomach [6,7]. The scintigraphic technique allows quantitative assessment because the amount of radioactivity (radioactive counts) can be measured with a gamma camera. Scintigraphy in dysphagia mainly is used for detection of aspiration in young children because the radiation doses are less than those of videofluoroscopy [8]. It is used less frequently in adults, although recently it has been described as a useful and reliable diagnostic tool in adult patients with oropharyngeal dysphagia [9]. Only three studies have been performed with scintigraphy to examine pharyngeal parameters of normal swallowing physiology: Cook et al. [10] used scintigraphy to study the influence of age on swallowing efficiency. Hamlet et al. [11] reported on the differences in bolus transit times and oropharyngeal residues in healthy volunteers, while Shaw et al. [12] studied the influence of bolus volume on pharyngeal residue. All these studies examined different parameters of swallowing and found different outcomes. The aim of this study was to explore the influence of viscosity on pharyngeal residue in normal healthy volunteers, where our hypothesis was that swallowing a more viscous product would result in more pharyngeal residue in healthy volunteers.\nMethods\nVolunteers\nEleven healthy volunteers (9 female, 2 male, mean age = 29.1 years, standard deviation [SD] = 8.9), recruited by public advertisement, were invited to participate. All volunteers reported no history of swallowing problems. All volunteers gave written informed consent to participate in the study, which was approved by the Medical Ethics Committee of the Academic Medical Center of the University of Amsterdam.\nBolus Characteristics\nBefore the examination, three different substances with three different viscosities were prepared in a research laboratory. All viscosities were measured with a viscosity meter (Brookfield LVT Viscometer, model DV-II+) after 24 h of storage in a refrigerator at a constant temperature of 4\u00b0C. Measurements were performed with spindle No. 63 at a velocity of 3 rpm and viscosities were recorded after 30 s. The substances consisted of tap water thickened with different percentages of xanthan, a polysaccharide commonly used in the food industry to thicken fluids. The first substance was a solution of tap water with 0.5% xanthan resulting in a substance with a viscosity of 4500 mPa s, comparable to a yogurt drink. The second and third substances were tap water with 0.75% and 1.00% xanthan, resulting in viscosities of 10,500 and 21,000 mPa s, comparable to low-fat yogurt and 3% fat yogurt, respectively. As reference, tap water with a viscosity of 1 mPa s (per definition) was used. For all solutions 200 ml was labeled with 260 MBq of 99mTc colloid (Hepatate, GE Health, Eindhoven, The Netherlands) dissolved in 1 ml 0.9% saline solution and prepared in such a way that for 10 ml of each solution the radiation dose was 13 MBq. After labeling, 44 syringes (4 per volunteer) were used to collect 10 ml of each of the four solutions (tap water, 0.5%, 0.75%, and 1.00% xanthan). The three xanthan solutions, stored for more than 24 h in a refrigerator at a stable temperature of 4\u00b0C, were mixed with the 99mTc colloid 1 h before the study.\nData Collection\nFor this study a gamma camera (Orbiter, Siemens Medical Systems, Germany) was fitted with a low-energy all-purpose collimator. A dynamic acquisition using 0.25-s frames was performed for 30 s. Data were acquired and processed on a Hermes workstation (Nuclear Diagnostics, Sweden). Volunteers were asked to sit in front of the gamma camera and hold their right cheek against the collimator. The oral cavity was placed in the middle of the field-of-view. When the volunteer was placed in the right position, 10 ml of one of the solutions was emptied in the mouth of the volunteer with a syringe. Volunteers were instructed to hold the bolus in their mouth and swallow on command. They were specifically instructed to swallow the whole bolus in one swallow and to breath through the mouth to inhibit a second swallow. Three cobalt markers were placed on the skin of the volunteers using a pen pointer: one on the mastoid bone, one on the hyoid bone, and one underneath the thyroid cartilage. The exact placement for the marker was found by palpation of the head and neck.\nFirst, the volunteers were asked to swallow the water with 99mTc colloid. After recording the swallow for 30 s, the volunteers were asked to rinse their mouth with unlabeled tap water and to swallow 200 ml unlabeled tap water to wash away any residue from the previous recording. The second recording was done with 10 ml of the 0.5% xanthan solution, the third with the 0.75% xanthan, and the last with the 1.00% xanthan. Between recordings the volunteers were instructed to rinse their mouth and swallow 200 ml unlabeled tap water to clear any possible residue. Each volunteer swallowed a consistency labeled with a dose of 13 MBq four times, for a total of 52 MBq, which is equal to a radiation dose of less than 1 milliSievert (mSv). According to the ICRP62 guidelines, this is a minor radiation dose (category IIa).\nAfter data collection the results were stored and processed on a Hermes workstation. Based on the markers, two regions of interest (ROI) were defined: the oral cavity (ROI-1), bordered by a straight line between the hyoid marker and the mastoid marker, and the pharynx (ROI-2), bordered by a straight line between the hyoid marker and the mastoid marker and a horizontal line from the thyroid marker, as described by previous studies [10,11]. To estimate background activity during the examinations, a third ROI with a comparable surface area to ROI-2 was defined to the right of the field of view. Figure\u00a01 shows the three ROIs. Collected data were transferred to a spreadsheet file (Excel, Microsoft Corp., Redmond, WA), which showed the number of counts within the specific ROI at each frame (0.25 s) of the total acquisition. For our analysis the average count of 8 frames (2 s) before swallowing was used to determine the average number of counts in the oral cavity. After swallowing the average count of 4 s (16 frames) was used to determine the average amount of pharyngeal residue, expressed in counts. Background activity was estimated by a measurement of 4 s; this was subtracted from the average count in the oral cavity and the pharyngeal residue. Hereafter, the number of pharyngeal counts was divided by the number of counts in the oral cavity, thus providing a percentage of pharyngeal residue.\nFig.\u00a01.Scintigraphic evaluation with three regions of interest (ROI). 1= oral cavity, 2 = pharynx, 3 = background radiation, a = mastoid marker, b = hyoid marker, 3 = thyroid marker.\nStatistical Analysis\nThe calculated percentages of pharyngeal residue were transferred to SPSS v12.0 (SPSS Inc., Chicago, IL) for further analysis. Distribution of the percentages was formally tested with the Kolmogorov\u2013Smirnoff test for normality and was not found to be normally distributed. To explore differences in median percentages of pharyngeal residues of the three xanthan solutions to the reference (tap water) on a group level, a nonparametric (Friedman) test was used. All statistical tests were two-tailed and differences were evaluated at the 5% level of significance.\nResults\nThe mean pharyngeal residue after swallowing tap water was 2.3% (SD = 1.2; range = 1.1%\u20134.5%) of the initial volume in the oral cavity. The percentage of pharyngeal residue after swallowing the 0.5% xanthan solution was 1.8% (SD = 0.8; range = 0.9%\u20132.8%), after swallowing the 0.75% xanthan solution the residue was 2.6% (SD = 2.2; range = 0.7%\u20138.1%), and after swallowing the 1.00% xanthan solution the pharyngeal residue was 2.8% (SD = 1.7; range = 1.2%\u20136.4%).\nMedian percentages of pharyngeal residue were analyzed. Figure\u00a02 shows by boxplot the distribution of residue for the four different viscosities in the 11 nondysphagic volunteers. No significant difference in the amount of residue between the four groups was found (\u03c72 = 0.927, df = 3, p = 0.819).Fig.\u00a02.Distribution of percentages residue on group level.\nAlthough the sample size was limited, post hoc analysis showed that this study had a power of 0.96 (n = 11 with an effect size of 0.47 based on a SD and correlation between measurements occasions of 1.1 and 0.7, respectively). Based on these results, a power calculation showed that in future research (with a power of 0.80 and alpha of 0.05) at least 137 healthy volunteers would be needed to detect any possible difference in the amount of pharyngeal residue between the four groups (tap water, 0.5%, 0.75%, and 1.00% xanthan) (Table\u00a01).\nTable\u00a01.Pharyngeal residue in percentages with 10ml bolusesNo.Water0.5% Xanthan0.75% Xanthan1.00% Xanthan11.11.20.71.221.81.21.01.731.30.78.13.441.41.51.71.354.12.32.66.462.91.51.51.271.20.91.22.482.42.55.35.493.12.52.42.4102.02.52.12.7114.52.82.12.4Mean2.3 (SD \u00b1 1.2)a1.8 (SD \u00b1 0.8)a2.6 (SD \u00b1 2.2)a2.8 (SD \u00b1 1.7)aaNo significant difference (p > 0.05).\nDiscussion\nOur study attempted to investigate the correlations between viscosity and pharyngeal residue in healthy volunteers; we did not find evidence for a positive correlation between an increase of viscosity and an increase in pharyngeal residue.\nBecause Shaw et al. [12] established that bolus volume does not influence pharyngeal residue, allowing extrapolation of our findings to other volumes, only one bolus volume was used in this study. It might be suggested that our volunteers drank 200 ml tap water after the first labeled bolus and thus the mucosa of the pharynx was relatively dry before the first estimation of pharyngeal residue. Because the larger proportion of our volunteers had drunk coffee, tea, or water in the last hour before the study, differences in dryness of the pharyngeal mucosa is not considered a confounder.\nOnly one bolus per viscosity was used, therefore no data on intrasubject differences could be provided. If multiple boluses per viscosity had been used in this study, the radiation exposure for our volunteers would have been much higher. Because none of the volunteers had swallowing disorders and all were used to swallowing tap water and yogurt, any adaptation in swallowing during the study to the circumstances during data collection or intrasubject differences can be considered negligible.\nRaut et al. [13] examined with manometry the influence of increased viscosity on pharyngeal pressures. It was concluded that an increased bolus viscosity led to increased amplitude of the bolus wave and clearing contractions within the pharynx. The increase in contraction of the pharyngeal muscles supports our findings of equivalent percentages of residue for the different viscosities.\nHamlet et al. [11] reported on the differences in bolus transit times and oropharyngeal residues in healthy volunteers. In that study 20 healthy volunteers (age = 39\u201365 years) swallowed tap water and a thickened substance labeled with 99mTc. The mean pharyngeal residue was found to be 4.0% (standard error [SE] = 0.5) for tap water and 10.5% (SE = 0.1) for a substance with a viscosity of 1070 mPa s.\nDifferences in the Hamlet et al. study and our study might be explained by temperature influences and different approaches to measure viscosity. In the Hamlet study [11] the bolus temperature was set at 25\u00b0C, whereas in our study syringes were stored in a chilled room. Therefore, temperature is not considered a confounding factor in our study. Also, in the Hamlet study [11] a Brookfield Viscometer type RVT was used, which is more suitable for measuring the viscosity of low-viscosity products like milk. In our study we used a Brookfield Viscometer type LVT, which is more suitable for high-viscosity substances, making an adequate comparison between measured viscosities of the two studies not possible.\nLiterature suggests that body positioning, bolus temperature, and taste affect swallowing [3,4,14]. In our study the volunteers had to rotate their heads to the right side to obtain adequate measurements, following the protocol used in the Hamlet study [11]. This might have reduced the amount of pharyngeal residue because the bolus passes the pharynx mainly on one side and the contact area between bolus and pharyngeal wall is decreased. It is known that in some dysphagic patients (i.e., patients with unilateral pharyngeal weaknesses) rotating the head decreases the amount of residue, but the effects of head rotation on healthy individuals has not been investigated. Looking at the wide distribution of percentages of residues in volunteers (Table\u00a01) and the slightly lower percentage (2.3%) found in our study compared with that of Shaw [12] (2.5%), where measurements were made in a right lateral position, one might conclude that the influence of head rotation in our study cannot be seen as a confounding factor on the outcome.\nConclusion\nIn 11 healthy volunteers, mean pharyngeal residue after swallowing tap water was 2.3% (SD = 1.2) of the initial volume in the oral cavity. The percentage of pharyngeal residue after swallowing 0.5% xanthan solution was 1.8% (SD = 0.8), after swallowing 0.75% xanthan solution the residue was 2.6% (SD = 2.2), and after swallowing 1.00% xanthan solution the residue was 2.8% (SD = 1.7). Our data do not show a significant increase in pharyngeal residue in healthy volunteers as a result of an increasing viscosity of viscous solutions, ranging from tap water to full-fat yogurt. Although some trends may be seen, (confounding) factors other than viscosity may play a role in the build up of pharyngeal residue.","keyphrases":["physiology","deglutition","radionuclide imaging","deglutition disorders"],"prmu":["P","U","U","M"]}
{"id":"Planta-4-1-2270920","title":"Subcellular concentrations of sugar alcohols and sugars in relation to phloem translocation in Plantago major, Plantago maritima, Prunus persica, and Apium graveolens\n","text":"Sugar and sugar alcohol concentrations were analyzed in subcellular compartments of mesophyll cells, in the apoplast, and in the phloem sap of leaves of Plantago major (common plantain), Plantago maritima (sea plantain), Prunus persica (peach) and Apium graveolens (celery). In addition to sucrose, common plantain, sea plantain, and peach also translocated substantial amounts of sorbitol, whereas celery translocated mannitol as well. Sucrose was always present in vacuole and cytosol of mesophyll cells, whereas sorbitol and mannitol were found in vacuole, stroma, and cytosol in all cases except for sea plantain. The concentration of sorbitol, mannitol and sucrose in phloem sap was 2- to 40-fold higher than that in the cytosol of mesophyll cells. Apoplastic carbohydrate concentrations in all species tested were in the low millimolar range versus high millimolar concentrations in symplastic compartments. Therefore, the concentration ratios between the apoplast and the phloem were very strong, ranging between 20- to 100-fold for sorbitol and mannitol, and between 200- and 2000-fold for sucrose. The woody species, peach, showed the smallest concentration ratios between the cytosol of mesophyll cells and the phloem as well as between the apoplast and the phloem, suggesting a mixture of apoplastic and symplastic phloem loading, in contrast to the herbal plant species (common plantain, sea plantain, celery) which likely exhibit an active loading mode for sorbitol and mannitol as well as sucrose from the apoplast into the phloem.\nIntroduction\nIn order to understand carbon partitioning, the regulation of photosynthesis, phloem loading, or sink\u2013source interactions, it is important to know how photoassimilates are distributed between the subcellular compartments of mesophyll cells of source leaves. Much of the information concerning subcellular metabolite concentrations is derived from studies of species such as spinach, barley, tobacco, and potato. In such plant species carbohydrates assimilated from CO2 are exported into the phloem exclusively in the form of sucrose (Riens et al. 1991; Winter et al. 1992; Lohaus et al. 1998). However, it has been shown that in several plant species, sugar alcohols like mannitol, sorbitol or dulcitol are also translocated in addition to sucrose (Zimmermann and Ziegler 1975). The advantage of transporting sucrose and sugar alcohols is that these compounds are highly soluble and chemically inert. In addition to functioning as a transport form for reduced carbon, sugar alcohols have been also shown to serve as storage for reduced carbon, as compatible solute synthesized in response to abiotic or biotic stress, or as osmoprotectants. It has been estimated that up to 30% of the carbon fixed by plants on earth may be present in the form of polyols (Bieleski 1982).\nMannitol is the most-widely distributed sugar alcohol and has been reported in more than 100 species of vascular plants, including most species of the Apiaceae (celery, parsley, and carrot), Oleaceae (olive, privet), Rubiaceae (coffee), and Scrophulariaceae (snapdragon) (Zimmermann and Ziegler 1975; Bieleski 1982). In higher plants, mannitol is synthesized by the action of an NADPH-dependent mannose-6-phosphate reductase that catalyzes the conversion of mannose-6-phosphate to mannitol-1-phosphate, followed by dephosphorylation by a phosphatase (Rumpho et al. 1983). The activity of mannose-6-phosphate reductase was only observed in mature leaves. In different plant species, mannitol synthesis occurs simultaneously with either sucrose synthesis, as in celery (Rumpho et al. 1983), or with raffinose oligosaccharide synthesis, as in Oleaceae (Zimmermann and Ziegler 1975). To date, there are only few reports on the intracellular distribution of mannitol (Keller and Matile 1989; Moore et al. 1997) and these studies do not include measurements of phloem concentrations of mannitol.\nAnother sugar alcohol present in higher plants is sorbitol. Sorbitol is a major photoassimilate translocated in addition to sucrose in the phloem of woody Rosaceae, including all members of the economically important genera Malus (apple), Pyrus (pear) and Prunus (stone fruits such as peach, cherry, plum and apricot; Zimmermann and Ziegler 1975; Bieleski 1982; Moing et al. 1997). Sorbitol is synthesized in source organs from glucose-6-phosphate by the activity of aldose-6-P-reductase (Negm and Loescher 1981) converting glucose-6-phosphate to sorbitol-6-phosphate. Sorbitol-6-phosphate is further converted to sorbitol by a specific phosphatase. Sinks have little or no capacity to synthesize sugar alcohols like mannitol or sorbitol (Loescher and Everard 1996). To our knowledge, there is only one report on the intracellular distribution of sorbitol in leaf cells (Moore et al. 1997) and with the exception of peach (Moing et al. 1997) there are no data on polyol concentrations in the phloem sap of sugar\u2013alcohol-translocating species.\nIn addition to mannitol and sorbitol, most plant species also contain the cyclitol myo-inositol (Zimmermann and Ziegler 1975) which is also produced from glucose-6-phosphate (Loewus and Loewus 1983). Myo-inositol and its derivatives are involved in membrane formation, cell wall synthesis, seed germination, and hormone responses as well as stress responses (Loewus and Loewus 1983). As an integral part of galactinol, myo-inositol also acts as a carrier of activated galactose that is transferred to sucrose, yielding raffinose and its polymeric derivates of higher order.\nKnowledge of concentrations of sugar alcohols and sugars in subcellular compartments of leaf cells and in the phloem is important to understand possible modes of phloem loading. Therefore, we examined the subcellular concentrations of sorbitol, mannitol, myo-inositol, hexoses and sucrose in peach (P. persica, Rosaceae), celery (A. graveolens, Apiaceae), common plantain (P. major, Plantaginaceae), and sea plantain (P. maritima, Plantaginaceae) as well as the corresponding concentrations in the apoplast and in the phloem sap. Celery and common plantain were selected because much of what is known about the transport of mannitol and sorbitol has come from studies of these plant species, and more recently, the relevant transporters for sucrose as well as for sugar alcohols were cloned from these species. In addition, sea plantain and peach were selected because both species also translocate sorbitol but the former belongs to the group of halophytes and the latter to the group of woody plants. In this manner, different groups of sorbitol-translocating plants are included in the comparison. The sugar alcohols and sugars listed above are the main soluble carbohydrates in these plants. Based on the subcellular distribution, we calculated the concentration ratios of soluble carbohydrates between the different subcellular compartments as well as in compartments which are involved in phloem loading. In combination with results from morphological studies and data about sucrose and polyol transporters available in the literature the results are compared to predictions based on models of symplastic and apoplastic phloem loading that are currently discussed in the field.\nMaterial and methods\nMaterials\nPlants of A. graveolens var dulce cv Vert d\u00b4Elne (celery; supplied by R. Lemoine, University of Poitiers cedex, France), P. major (common plantain; supplied by N. Sauer, University of Erlangen, Germany) and P. maritima (sea plantain; supplied by N. Sauer, University of Erlangen, Germany) were grown in 2-l pots in compost soil in a green-house with supplemental illumination at a 15\u00a0h photoperiod and a 23\/18\u00b0C day\u2013night thermoperiod. Two-month-old plants were taken for the experiments. P. persica (peach), cv. Red Haven (Jenssen, G\u00f6ttingen, Germany) was grown in 5-l pots in compost soil in a green house. Three-year-old plants were used for the experiments. Leaf samples were harvested in May and June.\nNon-aqueous fractionation of leaf tissue\nLeaves were cut from the plants after 9\u00a0h of the light period. The middle rib and larger veins were removed, and the samples were ground to a fine powder in liquid nitrogen in a precooled mortar. The leaf tissue was lyophilized at \u221225\u00b0C. The dry leaf powder was suspended in 20\u00a0ml of heptane:tetrachloroethylene mixture (density 1.27\u00a0g\u00a0ml\u22121, the solvents were dried and stored over molecule sieve beads from Merck, Darmstadt, Germany) and ultrasonicated with 5-s pulses and 5-s breaks for a total sonication period of 95\u00a0s (Cell Disrupter B15, Bronson Sonifer). The suspension was then poured through a polyester monolen sieve with a pore size <40\u00a0\u03bcm, diluted 3-fold with heptane, and centrifuged for 10\u00a0min at 2,200g (Centrifuge 5810R, Eppendorf). The clear supernatant was discarded and the sediment resuspended in 3\u00a0ml of a heptane:tetrachloroethylene mixture (density 1.27\u00a0g\u00a0ml\u22121). Two 200\u00a0\u03bcl aliquots were withdrawn for the determination of enzyme activity and carbohydrates in the unfractionated material. Two milliliter of the remaining material were added to an exponential heptane-tetrachlorethylene gradient with a density difference between 1.27 and 1.48\u00a0g\u00a0ml\u22121 for common plantain and sea plantain, and between 1.27 and 1.45\u00a0g\u00a0ml\u22121 for peach and celery. The bottom of the centrifugation tube contained a 2\u00a0ml cushion of a heptane:tetrachlrethylene mixture with the highest density (1.58\u00a0g\u00a0ml\u22121). After centrifugation (25,000g, 2.5\u00a0h) six or seven fractions were collected from the centrifuge tube content. Each of these fractions was divided into two portions, one for assay of marker enzymes and the other for assay of carbohydrates. NADP-glycerine aldehyde phosphatase, PEP-carboxylase and \u03b1-mannosidase were taken as marker enzymes for stroma, cytosol and vacuole. Chloroplastic material (including stroma) appeared in the lower region of the gradient, the cytosolic compartment was found to be enriched in the middle region of the gradient, whereas vacuolar material was mainly found in the fraction of highest density. Chloroform methanol extracts were prepared from the aliquots for the determination of the carbohydrate concentrations (see next section: \u201cExtraction of sugars and sugar alcohols from non-aqueous fractions\u201d).\nFor the evaluation of the subcellular distribution of sugars and sugar alcohols between the stromal, cytosolic, and vacuolar compartment, a calculation procedure according to Riens et al. (1991) was used. This calculation method is based on the assumption that the metabolites are confined to the three compartments as indicated by the corresponding marker enzymes. The evaluation is done by a computer program testing all possible cases for the distribution of a certain metabolite between the three compartments, using increments of 1%; for example: (1) vacuole 100%, stroma 0%, and cytosol 0%; (2) vacuole 99%, stroma 1%, and cytosol 0%; (3) vacuole 99%, stroma 0%, and cytosol 1%. There are 5,151 possibilities for the distribution of a metabolite between the three compartments and the program uses possibility yields to calculate the best fit (agreement) with the experimental results. To avoid the results being falsified by analytical errors, the calculations are based on mean values obtained from measurements of at least five independent density gradient fractionations.\nExtraction of sugars and sugar alcohols from non-aqueous fractions\nChloroform:methanol extracts were prepared to extract sugars and sugar alcohols from samples after non-aqueous fraction. The aliquots (see above) were dried and 5\u00a0ml chloroform:methanol (3:7, v\/v) was added to the powder. The samples were homogenized until completely thawed and kept on ice for 30\u00a0min. The homogenate was then extracted twice with 3\u00a0ml water. The aqueous phases were combined and evaporated in a rotary evaporator. The dried residue was dissolved in 0.7\u00a0ml ultrapure water (Millipore), syringe-filtrated (0.45\u00a0\u03bcm cellulose-acetate; Schleicher and Schuell, Dassel, Germany) and stored at \u221280\u00b0C.\nCollection of sieve tube sap\nSieve tube sap was obtained from severed stylets of the green-peach aphid, Myzus persicae (Sulz.). About 10 aphids were caged for about 5\u00a0h on the leaf. Their stylets were cut by a laser beam (Lohaus et al. 1995). The exuding phloem sap (10\u2013500\u00a0nl) was collected in micro-capillaries (total volume 0.5\u00a0\u03bcl) and the volume of the exudate was determined by measuring the length occupied by the solution. Evaporation of the phloem sap was prevented by bringing the front edge of the capillary in close contact with the leaf surface and surrounding the end with a plastic cap. The humidity around the capillary was about 80%. Under these conditions no evaporation from reference capillaries was detectable. The samples were ejected into 50\u00a0\u03bcl of distilled sterile water and stored at \u221280\u00b0C.\nExtraction of apoplastic washing fluids from leaves\nLeaves were cut 7\u20138\u00a0h after the onset of illumination and carefully washed with deionized water. Leaves were placed into a syringe filled with 40\u00a0ml deionized water and infiltrated by pulling the plunger, producing a reduced pressure of approximately 20\u00a0kPa. Thereafter, intact leaves were blotted dry, positioned with the xylem wound up in a 10\u00a0ml vessel and centrifuged immediately at 200g, for 4\u00a0min at 4\u00b0C.\nDue to the fact that infiltration of the apoplastic air space leads to a dilution of the apoplastic fluid, the solute concentrations in the apoplastic washing fluid were corrected by the ratio volume of the infiltration solution (which corresponds to the volume of the apoplastic gas space Vgas) to the volume of the apoplastic water space (Vwater). The ion concentration in the apoplast was calculated by multiplying the ion concentration in the apoplastic washing fluid by the dilution factor (=(Vwater\u00a0+\u00a0Vgas )\/Vwater). The mean dilution factor of several C3 herbal dicot plant species is about six (Lohaus et al. 2001). Therefore, the concentrations of the apoplastic fluid were diluted about 6-fold by the infiltration procedure in common plantain, sea plantain and celery. The dilution factor in peach was only 2.7 because the apoplastic water space was 17%, expressed as percentage of total leaf water content (Moing et al. 1997) which corresponds to about 113\u00a0\u03bcl\u00a0g\u22121 leaf water space (Table\u00a03), and the apoplastic gas space was 190\u00a0\u03bcl (g FW)\u22121 (Table\u00a03).\nCellular contamination of apoplastic washing fluids was quantified by comparing the activity of malate dehydrogenase in the apoplastic wash fluid with that in leaf extracts (Lohaus et al. 2001). According to this test, cellular cross-contamination was always below 0.1%.\nMetabolite analysis\nSugars and sugar alcohols in the samples after non-aqueous fractionation, apoplastic washing fluids, and phloem sap were analyzed by HPLC. An ion exchange column (CarboMA10; Dionex Corp, Sunnyvale, CA, USA) was eluted isocratically with 600\u00a0mM NaOH (0.4\u00a0ml\u00a0min\u22121) buffer for 60\u00a0min. Sugars were detected by a pulse amperometric detector with a gold electrode (ESA, Model 5200, Coulochem II, Bedford, MA, USA). Pulse setting was at 50, 700 and \u2212800\u00a0mV for 500, 540 and 540\u00a0ms, respectively. Sugar standards were measured daily, and plant samples were diluted sufficiently to provide signals within the linear range of the detector response (50\u2013500\u00a0\u03bcM). The metabolite amount was calculated from the peak area using Peaknet 5.1 software (Dionex, Idstein, Germany).\nElectron microscopy and determination of subcellular volumes\nSource leaves were cut into pieces of approximately 2\u00a0mm2 and prefixed in 5% glutaraldehyde (v\/v), 5 CaCl2, and 50\u00a0mM sodium-cacodylate buffer, pH 7.0. After 3\u00a0h at 4\u00b0C, the fixed slices were washed four times for 15\u00a0min each in cold 50\u00a0mM sodium-cacodylate buffer, pH 7.0, fixed in 2% (w\/v) osmium tetraoxide in cacodylate buffer and 0.8% (w\/v) K4Fe(CN)6 for 3\u00a0h at 4\u00b0C. The samples were washed five times in distilled water and stained with 5% uranyl acetate. Samples were dehydrated by subsequent incubation in higher concentrations of acetone [30, 50, 70, 90 and 100% (w\/v)]. The samples were then incubated in an acetone\/epoxyresin mix (Spurr 1969) with increasing concentrations of epoxyresin [33, 66 and 100% (w\/v)] for 1\u00a0h per concentration, and overnight and 4\u00a0h at 100% (w\/v). Sample blocks were than trimmed with a razor and sectioned in an ultra-microtome (Leica Microsystems, Wetzlar, Germany) with a diamond knife. Slices of approximately 60\u00a0nm were placed onto copper grids (Plano, Marburg, Germany), stained with uranylacetate and 0.3% lead citrate in NaOH and observed in a transmission electron microscope (EM 400, Philips, Eindhoven, NL).\nElectron micrographs of these sections were used for the evaluation of subcellular volumes according the principle of Delesse (1847): \u201cthe areal density of profiles on sections is an unbiased estimate of the volume density of structures\u201d (Weibel and Bolender 1973; Winter et al. 1993; Farr\u00e9 et al. 2001). About 50 electron micrographs were taken from 2 independent fixation procedures and from each 6 different leaves. Low magnification and high magnification (>x1,000) were used to determine the areas of most compartments. The cross-sectional areas of subcellular compartments were quantified using analysis software (NIH image, public domain software, developed at US National Institutes of Health, available at http:\/\/rsb.info.nih.gov\/nih-image\/).\nThe relative volumes (percent of total) were converted to absolute volumes per unit mass by taking into account the water content per mass fresh weight.\nResults\nSubcellular distributions of sugars and sugar alcohols in leaf cells\nSubcellular distributions of sugar alcohols, mono- and di-saccharides were measured by non-aqueous fractionation and HPLC in common plantain, sea plantain, peach and celery. The chosen method is based on the comparison of metabolite and marker enzyme distributions using a three-compartment calculation program (Riens et al. 1991). The method gives highly reproducible results for metabolites which are exclusively located (e.g., hexoses that are predominantly located in the vacuole). A higher variation is found for metabolites located in more than one compartment. The variability is greatest when the proportion found in a particular compartment is low. Farr\u00e9 et al. (2001) estimated that the limit of detection of a compound in a particular compartment is around 5% of the total amount in the tissue.\nHPLC analysis of gradient fractions of common plantain, sea plantain, and peach showed that sucrose, hexoses (glucose and fructose), myo-inositol, and sorbitol were present in leaf cells in these three plants (Fig.\u00a01a\u2013c). Celery contains mannitol instead of sorbitol (Fig.\u00a01d). In all four species the subcellular distribution of sorbitol and mannitol, respectively, was very different from those of hexoses and sucrose (Fig.\u00a01a\u2013d). Sorbitol (in common plantain and peach) and mannitol (in celery) were located in all three compartments, but predominantly in the vacuole (48\u201373%), followed by the stroma (19\u201327%) and the cytosol (8\u201325%). The distribution in sea plantain was different, and sorbitol was found mainly in the vacuole (88%) with only small portions in the stroma and in the cytosol. In contrast, in all four plants glucose and fructose were confined entirely to vacuoles (96\u201398%), and in common plantain and sea plantain sucrose was mainly distributed among the vacuolar and the cytosolic compartments (Fig.\u00a01a, b). In peach and celery, a greater proportion of sucrose was found in the vacuolar compartment and a smaller part in the cytosolic compartment (Fig.\u00a01c, d). The cyclitol, myo-inositol, was mostly present in the stroma of common plantain and sea plantain. In peach and celery the proportion was high in the stroma as well as in the vacuole, while the proportion found in the cytosol was always the lowest (Fig.\u00a01c, d).\nFig.\u00a01Percentage distribution of sugars and sugar alcohols among the vacuolar, stromal and cytosolic compartments of leaf cells from a common plantain, b sea plantain, c peach, and d celery. Data represent mean values\u00a0\u00b1\u00a0SD from five to six independent fractionations\nSubcellular volumes\nThe further conversion of the percentage distribution of sugars and sugar alcohols into concentrations requires an estimation of the volumes of the subcellular compartments of mesophyll cells. Leaves contain different tissues but in dicots about 75% of the aqueous volume of the leaf is occupied by mesophyll cells (Winter et al. 1993, 1994; Leidreiter et al. 1995). Table\u00a01 shows the relative proportions of the vacuolar, chloroplastic and cytoplastic (sum of cytosol, peroxisomes, mitochondria, and nucleus) compartments of TEM micrographs of mesophyll cells. In all plant species analyzed the most voluminous compartment was the vacuole, occupying 68\u201385% of the total cell, followed by the chloroplasts (10\u201321%) and the cytoplasm (5\u201312%). The proportion of the vacuole was lowest in the woody plant peach whereas in common plantain and sea plantain mesophyll cells were dominated by the vacuole (82\u201385%). Accordingly, the relative proportions of chloroplasts and cytoplasm were smallest in these plants. The relative proportion of the combined volumes of mitochondria, peroxisomes, and nuclei of the total cytoplasmic volume has been estimated to be about 20% (Winter et al. 1993, 1994) and the proportion of the stroma volume of the total chloroplast volume about 50% (Winter et al. 1993, 1994). From the relative proportions mentioned above, the data from Winter et al. (1993, 1994) and the average water contents of leaf cells (Table\u00a02) the volumes of the vacuolar, stroma, and cytosolic compartments can be estimated (Table\u00a02).\nTable\u00a01Relative volumes (%) of the subcellular compartments within the total volume of mesophyll cells from common plantain, sea plantain, peach and celeryVacuole %Chloroplast %Cytoplasm %Common plantain82\u00a0\u00b1\u00a0512\u00a0\u00b1\u00a046\u00a0\u00b1\u00a01Sea plantain85\u00a0\u00b1\u00a0510\u00a0\u00b1\u00a045\u00a0\u00b1\u00a02Peach68\u00a0\u00b1\u00a0721\u00a0\u00b1\u00a0611\u00a0\u00b1\u00a04Celery72\u00a0\u00b1\u00a0516\u00a0\u00b1\u00a0412\u00a0\u00b1\u00a03Data were obtained from TEM micrographes (n\u00a0=\u00a037\u201342). Cytoplasm is defined as cytosol, peroxisomes, nucleus and mitochondria. Mean values\u00a0\u00b1\u00a0SDTable\u00a02Dry weight (n\u00a0=\u00a08), water and gas space (n\u00a0=\u00a012) of leaves and the volumes of subcellulare compartments of mesophyll cells from common plantain, sea plantain, peach and celeryCommon plantainSea plantainPeachCeleryDry weight (mg\u00a0g\u22121 FW)147\u00a0\u00b1\u00a09124\u00a0\u00b1\u00a015336\u00a0\u00b1\u00a030140\u00a0\u00b1\u00a08Water space (\u03bcl\u00a0g\u22121 FW)853\u00a0\u00b1\u00a09876\u00a0\u00b1\u00a015664\u00a0\u00b1\u00a030860\u00a0\u00b1\u00a08Gas space (\u03bcl\u00a0g\u22121 FW)226\u00a0\u00b1\u00a048222\u00a0\u00b1\u00a021190\u00a0\u00b1\u00a044213\u00a0\u00b1\u00a033Volume vacuole (\u03bcl\u00a0g\u22121 FW)701743452860Volume stroma (\u03bcl\u00a0g\u22121 FW)50437068Volume cytosol (\u03bcl\u00a0g\u22121 FW)42385882Mean values\u00a0\u00b1\u00a0SD\nSubcellular sugar and sugar alcohol concentrations\nFrom the data on leaf carbohydrate contents, the subcelluar volumes, and the distribution of metabolites between the subcellular compartments the corresponding metabolite concentrations in the subcellular compartments were calculated. Sorbitol or mannitol, respectively, made up more than twothird of the total leaf content of soluble carbohydrates in all the plants (Table\u00a03). The concentrations of sorbitol and mannitol, respectively, were high in all compartments of peach and celery (Table\u00a03). Most of the sorbitol and mannitol were located in the vacuole (Fig.\u00a01a\u2013d) but due to the smaller volume cytosolic concentrations were calculated to be similar to those in the vacuole, and the highest level was found in the stroma (Table\u00a03). The sorbitol concentration in the cytosol of sea plantain was lower (13\u00a0mM) than in the vacuole (87\u00a0mM) whereas in common plantain the vacuole showed the lowest sorbitol concentration (15\u00a0mM).\nTable\u00a03Whole leaf contents as well as sugar and sugar alcohol concentrations in the vacuolar, stromal and cytosolic compartments of leaf cells from common plantain, sea plantain, peach, and celeryWhole leaf content \u03bcmol (g FW)\u22121Concentration (mM)VacuoleStroma CytosolCommon plantain\u00a0Sorbitol22\u00a0\u00b1\u00a03.115\u00a0\u00b1\u00a02.3118\u00a0\u00b1\u00a034133\u00a0\u00b1\u00a048\u00a0myo-Inositol0.7\u00a0\u00b1\u00a00.20.1\u00a0\u00b1\u00a00.18.8\u00a0\u00b1\u00a01.36.1\u00a0\u00b1\u00a00.7\u00a0Glucose and fructose2.4\u00a0\u00b1\u00a01.13.2\u00a0\u00b1\u00a00.21.4\u00a0\u00b1\u00a02.00.6\u00a0\u00b1\u00a00.7\u00a0Sucrose1.2\u00a0\u00b1\u00a00.10.9\u00a0\u00b1\u00a00.20.1\u00a0\u00b1\u00a00.212\u00a0\u00b1\u00a02.5Sea plantain\u00a0Sorbitol73\u00a0\u00b1\u00a09.187\u00a0\u00b1\u00a08.0201\u00a0\u00b1\u00a014013\u00a0\u00b1\u00a08.4\u00a0myo-Inositol0.8\u00a0\u00b1\u00a00.20.2\u00a0\u00b1\u00a00.115\u00a0\u00b1\u00a00.40\u00a0\u00b1\u00a00\u00a0Glucose and fructose11\u00a0\u00b1\u00a02.515\u00a0\u00b1\u00a00.50.2\u00a0\u00b1\u00a00.50\u00a0\u00b1\u00a00\u00a0Sucrose1.4\u00a0\u00b1\u00a00.20.8\u00a0\u00b1\u00a00.30.1\u00a0\u00b1\u00a00.120\u00a0\u00b1\u00a04.9Peach\u00a0Sorbitol148\u00a0\u00b1\u00a00.1220\u00a0\u00b1\u00a015461\u00a0\u00b1\u00a0165290\u00a0\u00b1\u00a062\u00a0myo-Inositol1.5\u00a0\u00b1\u00a00.11.5\u00a0\u00b1\u00a00.48.5\u00a0\u00b1\u00a03.64.1\u00a0\u00b1\u00a03.3\u00a0Glucose and fructose30\u00a0\u00b1\u00a06.165\u00a0\u00b1\u00a015.0\u00a0\u00b1\u00a05.05.0\u00a0\u00b1\u00a05.0\u00a0Sucrose38\u00a0\u00b1\u00a02.164\u00a0\u00b1\u00a04.539\u00a0\u00b1\u00a026106\u00a0\u00b1\u00a015Celery\u00a0Mannitol108\u00a0\u00b1\u00a012127\u00a0\u00b1\u00a07.0294\u00a0\u00b1\u00a030100\u00a0\u00b1\u00a039\u00a0myo-Inositol1.4\u00a0\u00b1\u00a00.11.1\u00a0\u00b1\u00a00.39.0\u00a0\u00b1\u00a01.91.7\u00a0\u00b1\u00a00.9\u00a0Glucose and fructose17\u00a0\u00b1\u00a02.028\u00a0\u00b1\u00a00.32.5\u00a0\u00b1\u00a01.72.9\u00a0\u00b1\u00a01.2\u00a0Sucrose35\u00a0\u00b1\u00a04.345\u00a0\u00b1\u00a03.35.2\u00a0\u00b1\u00a03.286\u00a0\u00b1\u00a028Mean values\u00a0\u00b1\u00a0SD\nIn each plant species sucrose was mainly concentrated in the cytosol, followed by the vacuole and was lowest in the stroma, however, concentrations varied widely between the species (cytosol 12\u2013106\u00a0mM, vacuole 0.8\u201364\u00a0mM, and stroma 0.1\u201339\u00a0mM; Table\u00a04). These differences between the plant species reflect the differences in the total leaf contents (Table\u00a03). The sucrose content in both plantain species was much lower than in peach and celery.\nTable\u00a04Carbohydrate concentrations in the cytosol of mesophyll cells of leaves as well as in the apoplast and in the phloem sap from common plantain, sea plantain, peach and celeryConcentration (mM) RatioRatioCytosolApoplast Phloem sapPhl\/apoPhl\/cytCommon plantain\u00a0Sorbitol133\u00a0\u00b1\u00a0485.5\u00a0\u00b1\u00a01.2422\u00a0\u00b1\u00a0129773.2\u00a0Sucrose12\u00a0\u00b1\u00a02.50.3\u00a0\u00b1\u00a00.1645\u00a0\u00b1\u00a02252,15040\u00a0Sorbitol\/sucrose11180.7Sea plantain\u00a0Sorbitol13\u00a0\u00b1\u00a08.47.9\u00a0\u00b1\u00a02.5315\u00a0\u00b1\u00a01054024\u00a0Sucrose20\u00a0\u00b1\u00a04.90.3\u00a0\u00b1\u00a00.1355\u00a0\u00b1\u00a01121,18318\u00a0Sorbitol\/sucrose0.7260.9Peach\u00a0Sorbitol290\u00a0\u00b1\u00a06224.4\u00a0\u00b1\u00a09.5582\u00a0\u00b1\u00a090242.0\u00a0Sucrose106\u00a0\u00b1\u00a0161.2\u00a0\u00b1\u00a00.6207\u00a0\u00b1\u00a0511732.0\u00a0Sorbitol\/sucrose2.7202.8Celery\u00a0Mannitol100\u00a0\u00b1\u00a0396.7\u00a0\u00b1\u00a03.3732\u00a0\u00b1\u00a01311097.3\u00a0Sucrose86\u00a0\u00b1\u00a0281.2\u00a0\u00b1\u00a00.9389\u00a0\u00b1\u00a0843244.5\u00a0Mannitol\/sucrose1.25.61.9Mean values of n\u00a0=\u00a03\u20139 independent measurements are shown. Mean values\u00a0\u00b1\u00a0SD\nOf the compartments analyzed the highest concentration of glucose and fructose was consistently found in the vacuole. As the evaluation of the subcellular fraction was performed in increments of 1%, glucose and fructose concentrations found in the cytosol or stroma represent an upper limit of 1% for these compounds.\nThe concentration of myo-inositol was highest in the stroma, with intermediate values in the cytosol and lowest in the vacuole (Table\u00a03). Myo-inositol, sorbitol and mannitol, respectively, made up between 90 and 99% of the total soluble carbohydrates in the stroma of the plant species.\nApoplastic and phloem sap concentrations\nCarbohydrates found in the phloem sap of the four plant species investigated were sucrose and mannitol or sorbitol (Table\u00a04). Concentrations were high in all cases, ranging between 200 and 700\u00a0mM. In the two Plantaginaceae sucrose concentrations, measured directly, were slightly higher than sorbitol concentrations, whereas in peach and celery the sorbitol and mannitol concentrations, respectively, were 3- and 2-fold higher than the sucrose concentration. Therefore, the ratios of sugar alcohol to sucrose concentrations for the four plants differ between 0.7 in common plantain and 2.8 in peach. No or only traces of glucose and fructose were found in the phloem, and the concentration of myo-inositol was in the low millimolar range (data not shown).\nSorbitol and mannitol, respectively, dominated the apoplast of all four plants, while sucrose was present at concentrations at or below 1\u00a0mM (Table\u00a04). Nevertheless, the polyol as well as the sucrose concentrations in the apoplast were much lower than in phloem sap (Table\u00a04). The concentration differences between the apoplast and the phloem were about 100-fold for mannitol, between 20- and 80-fold for sorbitol and between 170- and 2,200-fold for sucrose.\nDiscussion\nDespite the central importance of the compartmentation of sugar alcohols in plant cells in relation to phloem transport, up to now the sugar alcohol distribution in subcellular compartments has been the subject of very few studies. We have used non-aqueous fractionation in addition to HPLC to study subcellular sugar alcohol distributions in common plantain, sea plantain, peach, and celery which translocate large amounts of sorbitol or mannitol within the phloem. It should be pointed out that the non-aqueous fractionation technique was developed for determination of subcellular concentrations of metabolites that are exclusively located in the mesophyll (Gerhardt and Heldt 1984). For other metabolites such as sugars, which are also present in tissues other than the mesophyll, the method tends to somewhat overestimate concentrations in the mesophyll cell compartments, because the amounts of metabolites in other tissues are assumed to be negligible. However, Voitsekhovskaja et al. (2006) have shown that concentrations of hexoses and sucrose in mesophyll cells measured either by single-cell technique or by non-aqueous fractionation were similar. A possible explanation is that e.g., the dense phloem tissue remains intact and will be removed from the other tissue during fractionation process (either in the sieve or in the bottom of the gradient; see chapter \u201cNon-aqueous fractionation of leaf tissue\u201d).\nSorbitol and mannitol concentrations are high in stroma and cytosol despite of a large vacuolar polyol depot\nThe main proportion of sorbitol and mannitol was found in the vacuolar compartment (48\u201387%; Fig.\u00a01a\u2013d) whereas in most species analyzed the highest concentrations were found in the stroma (Table\u00a03). High concentrations of polyols in the stroma require either polyol-uptake or polyol-synthesis inside this compartment. Unfortunately, little is known about the intracellular distribution of the enzymes involved in sorbitol synthesis. Experiments with apple (Yamaki 1981) support a predominantly chloroplastic location for sorbitol-6-phosphate dehydrogenase. On the other hand, the intracellular immunocytochemical localization data for mannose-6-phosphate reductase, the key enzyme for mannitol synthesis, show that this enzyme is primarily cytosolic (Loescher and Everard 1996). So far no polyol transporter has been demonstrated to be localized in the envelopes of chloroplasts. The function of high concentrations of polyols in the stroma could be e.g., for osmoregulation because the concentration of several other metabolites or ions in this compartment is much lower than in the cytosol and in the vacuole (Table\u00a03; Winter et al. 1993; Lohaus et al. 1998; Voitsekhovskaja et al. 2006).\nOf the plants analyzed which produce sorbitol, only in sea plantain was the sorbitol concentration in the vacuole of mesophyll cells higher by about 7-fold than that in the cytosol (Table\u00a03). The only previous report about subcellular distribution of sorbitol was of snapdragon (Moore et al. 1997) where up to 100% of sorbitol was found in the vacuole. However, the total leaf content of sorbitol was much lower in snapdragon than in plants in our study. A higher sorbitol concentration in the vacuole than in the cytosol, as in sea plantain, demonstrates that there is an active transport of sorbitol into the vacuole, because most likely sorbitol is either produced in the cytosol or in other cell compartments. So far no sorbitol transporter has been demonstrated to be localized in the tonoplast of mesophyll cells.\nThe high sorbitol concentration in the vacuoles of sea plantain indicates that sorbitol may have more functions in that species than just serving as a transport-form for carbon. Sea plantain is a more salt tolerant plant species than common plantain (Ahmad et al. 1979). In the study presented here no salt stress was given but the plants have taken up sodium from the soil used for plant culture. As a result the sodium content in common plantain leaves was 4.3\u00a0\u00b1\u00a01.1\u00a0\u03bcmol (g FW)\u22121, whereas sea plantain contained 36\u00a0\u00b1\u00a06.5\u00a0\u03bcmol (g FW)\u22121 which corresponds to the higher sorbitol content in the leaves in sea plantain (Table\u00a03). Stoop et al. (1996) have discussed that mannitol may accumulate in the vacuole of salt-tolerant plant species for redistribution to the cytosol in response to stress, and recently Pommerrenig et al. (2007) demonstrated increased sorbitol contents in leaf cells of common plantain in response to salt treatment.\nIn leaves of celery three quarters of mannitol was located in the vacuole (Fig.\u00a01d). In A. barclaiana (Voitsekhovskaja et al. 2006), parsley and snapdragon (Moore et al. 1997) the vacuole, stroma and the cytosol contained each onethird of leaf mannitol, but the total leaf content of mannitol was much lower in these three plants than in celery. Probably the higher leaf content of mannitol requires the deposition of a greater portion of mannitol in the vacuole. Despite the high proportion of mannitol in the vacuole, cytosolic and stromal mannitol concentrations were calculated to be similar to be higher than those in the vacuole (Fig.\u00a01d and Table\u00a03). Mannitol is synthesized in the cytosol of leaf cells (Rumpho et al. 1983) and similar concentrations of mannitol in the vacuole and the cytosol indicate that diffusion of mannitol between both compartments could be responsible for the pattern observed. On the other hand, mannitol is transiently stored in the vacuoles of parenchyma cells of fleshy petioles (Greutert et al. 1998). Similarly, Keller and Matile (1989) estimated a 3-fold higher mannitol concentration in the vacuole than in the cytosol of petiole cells, suggesting that an active uptake system of mannitol is present in the tonoplast. The vacuolar mannitol concentration higher in storage petioles than in source leaves may be a reflection of the different functions of these two organs in celery.\nA large pool of myo-inositol is located in the stroma\nA great portion of myo-inositol is localized in the stroma (Fig.\u00a01a\u2013d) which corresponds to earlier findings (Voitsekhovskaja et al. 2006; Moore et al. 1997). The high proportion of myo-inositol in the stroma also resulted in the highest myo-inositol concentration in this compartment (8\u201315\u00a0mM, Table\u00a03), compared to other compartments. This stromal pool probably originates from its synthesis by the stromal isoform of the myo-inositol synthesizing enzyme, myo-inositolphosphate synthase (Adhikari et al. 1987). In the halophyte, Mesembryanthemum crystallinum, myo-inositol synthesis is increased in response to salt stress (Ishitani et al. 1996). Leaves of sea plantain contained a 8-fold higher salt content than those of common plantain (see above). Despite this difference, the content of myo-inositol was very similar in both plants in contrast to the sorbitol content (Table\u00a03). Therefore, it seems for plantain that the sorbitol concentration is correlated with the sodium concentration rather than the myo-inositol concentration, even under low sodium conditions.\nThe subcellular distribution of hexoses and sucrose is similar in plant leaves with and without sugar alcohols\nHexoses are often confined to the vacuole (Riens et al. 1991; Winter et al. 1992). Figure\u00a01a\u2013d shows that these findings are also true for the four species analyzed here: concentrations of glucose and fructose were always higher in the vacuole than in the cytosol or in the stroma (Table\u00a03). Both facilitated diffusion and active transport have been described for vacuolar uptake of glucose. Facilitated diffusion has been discussed for the transport of glucose across the tonoplast in celery (Daie 1987). Heineke et al. (1994) suggested that leaf vacuoles may contain transporters for the active uptake of hexoses. However, Martinoia et al. (2000) noted that accumulation of hexoses within the vacuole is not a proof for energized hexose uptake, because the vacuole contains acid invertases, allowing for enzymatic production of hexoses on the vacuoles.\nBetween 50 and 80% of the sucrose detected in the four species analyzed were located in the vacuole (Fig.\u00a01a\u2013d). However, due to the smaller volume of the cytosol relative to that of the vacuolar compartment, cytosolic sucrose concentrations were calculated to be similar to or even higher than those in the vacuole (Table\u00a03). A similar distribution has also been reported for leaves of other species (Riens et al. 1991; Winter et al. 1992). The lowest vacuolar sucrose concentration was found in the two Plantaginaceae and may be related to the overall low sucrose content in the leaves of these two species (Table\u00a03). Recently Endler et al. (2006) have localized sucrose transporters in the tonoplast of barley (HvSUT2) and Arabidopsis (AtSUT4) and proposed that sucrose transporters are involved in vacuolar transport.\nConcentration ratios between the cytosol of mesophyll cells and the phloem were similar for sucrose and sugar alcohols\nSugar alcohols and sucrose are the major compounds translocated in the four species analyzed. The phloem concentrations of sorbitol and mannitol (Table\u00a04) were in the same range as that of sucrose (300\u2013700 versus 200\u2013700\u00a0mM) although the ratio of sorbitol, or mannitol, to sucrose varied between 0.7 and 2.8. High concentrations of sugar alcohols in the phloem indicate that a high proportion of assimilated carbon is exported in this way. Moing et al. (1997) estimated that sorbitol accounts for 60\u201390% of the carbon exported from the leaf in peach and in celery, while mannitol was shown to account for 10\u201360% (Daie 1986).\nIn the four plants the sucrose concentration was always higher in the phloem sap than in the cytosol of mesophyll cells although the concentration ratio of sucrose between the cytosol of mesophyll cells and the phloem varied between 2- and 40-fold (Table\u00a04). The different ratios arose from variable cytosolic sucrose concentrations rather than by different phloem concentrations in the four plants (Table\u00a04). With the exception of common plantain the ratios of both, sorbitol and mannitol, between the phloem and the cytosol of mesophyll cells were similar to the corresponding ratios of sucrose. In common plantain the overall sucrose concentration was relatively low which resulted in a higher ratio for sucrose than for sorbitol.\nConcentration ratios between the apoplast and the phloem for sucrose and sugar alcohols with relation to the phloem loading mode\nGamalei (1989) has shown that the members of Plantaginaceae display a virtually complete symplastic isolation of the sieve element-companion cell complex, as indicated by a very low plasmodesmal abundance between phloem companion cells and bundle-sheath cells. Sucrose and sorbitol were present in the apoplast of both Plantaginaceae, however, the concentrations were much lower than in phloem sap (Table\u00a04). Therefore, the loading of both sucrose and sorbitol into the phloem must be energized. This is consistent with the observation that sucrose transporters (PmSUC1, PmSUC2, and PmSUC3; Gahrtz et al. 1994; Barth et al. 2003) as well as polyol transporters (PmPLT1 and PmPLT2; Ramsperger-Gleixner et al. 2004) were cloned from P. major. The function of the putative transporter proteins in phloem loading was corroborated by their specific localization in companion cells of source leaf phloem (PmSUC2, Stadler et al. 1995; PmPLT1 and PmPLT2 Ramsperger-Gleixner et al. 2004). The Km value of PmSUC2 for sucrose was about 1\u00a0mM (Gahrtz et al. 1994) and the Km value of PmPLT1 for sorbitol was about 12\u00a0mM (Ramsperger-Gleixner et al. 2004), which are in the same range as the apoplastic sucrose and sorbitol concentrations (Table\u00a04). No data of transporters exists for sea plantain, but the concentration gradients for sucrose as well as those for sorbitol strongly suggest active transport systems for both carbohydrates. Together, the data support apoplastic transfer of sucrose and sorbitol in the members of Plantaginaceae.\nPathway and mechanism of phloem loading in peach are still a matter of debate (Moing et al. 1997). The phloem structures of Rosaceae trees were shown to be of the intermediate-type (moderate numbers of plasmodesmal connections between mesophyll and companion cells; Gamalei 1989). Therefore, the mechanism of phloem loading in Prunus species could be symplastic, apoplastic or mixed. In apoplastic loaders, sucrose or sorbitol destined for phloem transport has to be released from the mesophyll cells into the apoplast, whereas in symplastic loaders, sugars and sugar alcohols synthesized in the mesophyll and destined for the phloem transport, are expected to stay in the symplast. Thus, it could be expected that the percentage of these carbohydrates in the cytosol of mesophyll cells in putative symplastic loaders would be much higher than in apoplastic loaders. Sorbitol concentrations were high in the phloem sap of peach (Table\u00a04, Moing et al. 1997) and in addition to sucrose sorbitol is in general a major photoassimilate translocated in the phloem of woody Rosaceae (Bieleski 1982). The concentration ratios for sucrose and sorbitol between the cytosol of mesophyll cells and the phloem were about 2-fold for each carbohydrate (Table\u00a04). These are the lowest ratios among the species analyzed and much lower than those determined for apoplastic phloem loaders (ratio of sucrose 5\u201330; Lohaus et al. 1995; Lohaus and M\u00f6llers 2000). A 2-fold concentration ratio for sucrose corresponds to the gradient found in Alonsoa meridionalis, a Scrophulariaceae with a symplastic phloem loading mode (Knop et al. 2001). A lower concentration ratio is obviously easier to balance or even reverse, depending on the metabolic situation, than a steeper ratio. Therefore, the possibility of phloem loading of sucrose and sorbitol by simple diffusion cannot be ruled out. Turgeon and Medville (1998) were unable to detect accumulation of sucrose against the concentration gradient in minor veins of willow leaves, representing another woody species.\nOn the other hand, leaf infiltration with p-chloromercuribenzenesulfonic acid (PCMBS) was found to inhibit sugar phloem transport in peach and Moing et al. (1992) further concluded from this result that apoplastic phloem loading predominates in peach. Unfortunately up to now no sucrose or sorbitol transporters were cloned from peach as they had from other Rosaceae (Prunus cerasus, Gao et al. 2003; Malus domestica, Watari et al. 2004). Watari et al. (2004) reported the existence of three sorbitol transporters in apple source leaves with apparent Km values for sorbitol between 0.7 and 3.2\u00a0mM. The apoplastic sorbitol concentration in apple leaves was not detected but the corresponding concentration in peach was about 25-fold higher (Table\u00a04).\nSucrose and sorbitol uptake in plasma membrane vesicles from peach leaves exhibited saturated kinetics (Marquat et al. 1997). These results suggest that uptake of sorbitol and sucrose is carrier mediated. Active absorption of sucrose was completely inhibited by PCMBS, contrary to the absorption of sorbitol (Marquat et al. 1997). The activities of sorbitol transporters from Rosaceae heterologously expressed in yeast were also not or only slightly inhibited by PCMBS (Gao et al. 2003; Watari et al. 2004). Ramsperger-Gleixner et al. (2004) have shown that sorbitol transport by PmPLT1, but not that driven by PmPLT2, is inhibited by PCMBS. Therefore, PCMBS sensitivity of sorbitol transport can vary and the insensitivity of transport processes against PCMBS conclusively does not rule out that carriers are involved.\nUnfortunately, the type of phloem loading in peach is still an open question; the available data are consistent with either an apoplastic or a symplastic mode of loading or, more likely, a combination of both.\nCelery is the only plant species analyzed here which translocates mannitol in addition to sucrose. The concentration ratio of sucrose between the apoplast and the phloem sap was about 300-fold and the ratio for mannitol was about 100-fold (Table\u00a04). Studies of metabolite uptake into plasma membrane vesicles from phloem tissues of celery petioles by Salmon et al. (1995) showed mannitol- as well as sucrose-proton-co-transport, indicating that in this species mannitol and sucrose transporters may be involved in phloem loading. Noiraud et al. (2000) have cloned two sucrose transporters from celery (AgSUT1, AgSUT2). AgSUT1 is expressed in mature leaves and phloem of petioles. Noiraud et al. (2001) have also cloned a cDNA of a mannitol transporter (AgMaT1) of celery which is not sensitive to PCMBS. The expression profile for AgMaT1 in source leaves and phloem was in agreement with a role in phloem loading of mannitol in celery. The AgMaT1 protein expressed in yeast cells exhibited a Km value for mannitol uptake of about 0.3\u00a0mM (Noiraud et al. 2001). The apoplastic mannitol concentration was 25-fold higher (Table\u00a04). In summary, celery shows all characteristics of a typical apoplastic phloem loader.\nThe comparison of common plantain, sea plantain, peach, and celery indicates that different modes of phloem loading are employed by different plant families, which may in part be related to the respective ecophysiological requirements.","keyphrases":["phloem","plantago","prunus","apium","sorbitol","mannitol"],"prmu":["P","P","P","P","P","P"]}
{"id":"Evid_Based_Complement_Alternat_Med-2-4-1297501","title":"Complementary and Alternative Medicine for Chronic Prostatitis\/Chronic Pelvic Pain Syndrome\n","text":"To discuss challenges concerning treatment for chronic prostatitis\/chronic pelvic pain syndrome (CP\/CPPS) and review complementary and alternative medical (CAM) therapies being evaluated for this condition, we performed a comprehensive search of articles published from 1990\u20132005 using the PubMed, Medline databases. Data from the articles were abstracted and pooled by subject. Keywords cross-searched with CP\/CPPS included: complementary, alternative, integrative, therapies, interventions, nutrition, antioxidants, herbs, supplements, biofeedback and acupuncture. Listed articles with no abstracts were not included. Various CAM therapies for CP\/CPPS exist including biofeedback, acupuncture, hyperthermia and electrostimulation. Additionally, a variety of in vitro and in vivo studies testing herbal and nutritional supplements were found. Saw palmetto, cernilton and quercetin were the most frequently tested supplements for CP\/CPPS. Although many CAM therapies demonstrate positive preliminary observations as prospective treatments for CP\/CPPS, further exploratory studies including more randomized, controlled trials are necessary for significant validation as treatment options for this complex disorder.\nIntroduction and Characterization of Prostatitis\nChronic prostatitis\/chronic pelvic pain syndrome (CP\/CPPS) is of significant interest in urology and accounts for up to 2 million office visits per year (1). Currently, there are multiple approaches to the management of CP\/CPPS depending on the classification of the related symptoms. However, there are no absolute findings or laboratory tests employed and diagnosis is often one of exclusion.\nIn 1995, the National Institutes of Health established an International Prostatitis Collaborative Network in order to construct a new classification of prostatitis syndromes and better define chronic prostatitis (2,3). The categories are now documented as follows:\nCategory 1. Acute bacterial prostatitisCategory 2. Chronic bacterial prostatitisCategory 3. Chronic prostatitis\/chronic pelvic pain syndrome\nA. InflammatoryB. Non-inflammatoryCategory 4. Asymptomatic inflammatory prostatitis.\nWhile most patients report a primary subjective symptom of local pain and\/or dysuria, the clinical presentation of acute versus chronic prostatitis varies. Laboratory cultures are standardly employed to detect bacterial involvement and this testing along with other diagnostic criteria determines each classification. Men with Category 1, acute prostatitis, frequently present with dysuria, fever, malaise, myalgia (non-specific) and positive culture analysis that often reveals coliform bacteria. This imparts to standard antibiotic treatment with good prognosis for recovery and minimal recurrence.\nPatients with Category 2, chronic bacterial prostatitis, present with similar symptoms as those with acute prostatitis. However, the frequency of symptoms (duration >3 months), recurrent urinary tract infections and additional diagnostic tests including analysis of lower urinary tract cultures contribute to its diagnosis as Category 2 prostatitis (2).\nMen with Category 4, asymptomatic inflammatory prostatitis, do not present with subjective symptoms. This diagnosis is often discovered via laboratory findings such as the positive presence of white blood cells in prostatic secretions or in prostate tissue during routine evaluation for other disorders (1).\nComparative to the total number of prostatitis cases reported, the majority of representative cases are Category 3, CP\/CPPS (1,4). This diagnosis is usually one of exclusion, as bacterial etiology acute or chronic is ruled out. Other exclusion criteria include urogenital cancer, urethral stricture and neurologic disease affecting the bladder. However, the patient may still present with polyuria, dysuria, generalized myalgia or specific pelvic pain, urethral discharge, voiding dysfunction, sexual dysfunction and negative impact on quality-of-life (QOL). The presentation of this symptom set is now termed Category 3, CP\/CPPS. Categories 3A and 3B are further differentiated by the presence or absence of inflammatory blood cells in prostatic secretions and seminal fluid, respectively (4,5). Table 1 further outlines the characteristics and treatment options of Category 3 prostatitis.\nDue to the complexity in diagnosing CP\/CPPS, the National Institutes of Diabetes and Digestive and Kidney Diseases funded the Chronic Prostatitis Collaborative Research Network (CPCRN) in 1995 (2,3). This network was fundamental in the construction and validation of the National Institutes of Health Chronic Prostatitis Symptom Index (NIH-CPSI), which was implemented in 1999 (3). The index has become a valid measure that quantifies the qualitative experience of men with CP\/CPPS and addresses three different aspects of CP\/CPPS: pain, function and QOL (3,6).\nThe formation of the CPCRN and the advent of the NIH-CPSI have better characterized diagnosis and treatment for CP\/CPPS, but challenges still exist. While standard treatment options including anti-inflammatory agents, analgesics and alpha-blockers are often prescribed, impact on QOL is another factor often overlooked in the treatment and management of CP\/CPPS (1,4,5,7). The focus on QOL, anecdotal data, epidemiological studies and the increased popularity and validation of herbal, complementary and alternative medicine (CAM) has led to exploration of the utility of CAM therapies as treatments for CP\/CPPS (8). CAM therapies including biofeedback, acupuncture, heat therapy, electrostimulation, herbal and nutritional supplements will be discussed below.\nCAM Background\nCAM, as defined by the National Center for Complementary and Alternative Medicine (NCCAM), is a group of diverse medical and health care systems, practices and products that are not presently considered part of conventional medicine. Conventional medicine is further defined as medicine as practiced by holders of MD (medical doctor) or DO (doctor of osteopathy) degrees (9). Though the list of what therapies or practices considered to be CAM changes continually, the pool of both practitioners of CAM modalities and patients utilizing CAM services continues to grow within the United States and globally (9).\nThe inclusion of CAM practices in urology is also being implemented in the clinic. Many groups such as the Committee of Complementary and Alternative Medicine within the American Urological Association (AUA) recognize the integration of non-conventional therapies into urological clinical practice. Additionally, both public demand for CAM therapies and their testing and validation within health science research centers has increased (8,10,11). It has been suggested that many urological conditions possessing subjective and QOL components such as in benign prostatic hyperplasia (BPH), chronic prostatitis, voiding, erectile dysfunction and cancer prevention and survivorship might be particularly amenable to CAM treatment strategies (10). This review will focus on current CAM therapies found in the literature for CP\/CPPS.\nCAM Therapies for CP\/CPPS\nBiofeedback\nBiofeedback therapy is considered a mind\u2013body technique that utilizes a monitoring machine to assist people in controlling bodily functions such as heart rate, blood pressure and muscle tension. This therapy has been studied for its efficacy in urological conditions such as incontinence, prolapse, pediatric voiding dysfunction and CP\/CPPS (12\u201317). The hypothesis of biofeedback's mechanism of action in treating CP\/CPPS is based on the principle that maximum muscle contraction prompts maximum muscle relaxation. This mechanism addresses the chronic pain aspect of CP\/CPPS and focuses on muscular reeducation, which may ultimately provide symptom relief (13\u201315).\nTwo studies testing the value of biofeedback therapy for CP\/CPPS yielded positive results. The first study assessed 62 patients who were refractory to conventional therapy (such as antibiotics and\/or alpha-blockers) for greater than half a year. These patients were treated utilizing the Urostym Biofeedback equipment five times a week for 2 weeks with a stimulus intensity of 15\u201323 mA and duration of 20 min. The NIH-CPSI index noted a significant overall reduction in score (P < 0.01) and no side effects were reported during the trial (13).\nA second pilot study evaluated biofeedback therapy in 19 men with pelvic floor tension and CP\/CPPS. These results demonstrated significant improvement in pain scores as measured by the AUA symptom index (P = 0.001). While this study focused on testing the effect of biofeedback therapy in treating the symptoms associated with CP\/CPPS, it also implicated the presence of pelvic floor tension contributing to pain and the paramount importance of muscular reeducation for its treatment (15). These initial, positive biofeedback studies may warrant larger randomized clinical trials to confirm safety and efficacy as well as explore the mechanism of action of biofeedback therapy.\nAcupuncture\nAcupuncture is a traditional Chinese method of medical treatment involving the insertion of fine, single-use, sterile needles in acupoints according to a system of channels and meridians that was developed by early practitioners of Traditional Chinese Medicine (TCM) over 2000 years ago. The needles are stimulated by manual manipulation, electrical stimulation or heat (18). Currently, acupuncture is often used with TCM and it is a recognized health profession with strict licensure and regulatory status in 40 states (19). Common applications include acupuncture as a complementary therapy for cancer patients undergoing chemotherapy or radiotherapy, for conditions involving pain such as migraines and back pain, and for relieving the impact of stress among patients with chronic conditions. The precise physiological mechanism of action of acupuncture is unknown but a variety of hypotheses exist. For example, acupuncture analgesia is thought to be mediated by central nervous system (CNS) mechanisms of pain control via the release of specific neurotransmitters, such as endorphins (20\u201323). Additionally, there are significant data which suggest that acupuncture treatment can decrease inflammation and relieve pain (24,25).\nData suggesting the ability of acupuncture treatment to decrease pain, positively impact QOL and potentially modulate inflammation and\/or affect the CNS has suggested it as potential therapeutic option for men with CP\/CPPS. While a number of studies listed in other journals test the utility of acupuncture treatment for CP\/CPPS (26) only two medline listed pilot studies are shown testing acupuncture treatment in patients with CP\/CPPS.\nThe first study examined whether acupuncture improved pain, voiding symptoms and QOL in 12 men with CP\/CPPS. This study reported a significant decrease in total NIH-CPSI pain, urinary and QOL scores (P < 0.05) over 6 weeks of treatment and an average 33 weeks of follow-up (27). The mechanism of action addressed in this paper suggests a neuropathic model of CP\/CPPS and the hypothesis that acupuncture, if considered a neuromodulatory therapy, may provide a therapeutic option for men with CP\/CPPS (27).\nA second study tested acupuncture treatment for CP\/CPPS patients with intrapelvic venous congestion. This study of 10 patients receiving 5 weeks of acupuncture treatment also reported a significant decease in NIH-CPSI pain and QOL scores (P < 0.05, P < 0.01). While the study reported no serious adverse events, the mechanism of action was not addressed (28). The promising clinical outcome of both studies testing acupuncture for CP\/CPPS implies that larger studies are required to confirm the utility of acupuncture in this patient population.\nHigh Frequency Electrostimulation\nOnly one study in English was found utilizing electrostimulation for chronic prostatitis. This study tested a new, high frequency, urethral\u2013anal prototype stimulation device in men with CP\/CPPS twice weekly for 5 weeks. The results demonstrated a significant decrease in the NIH-CPSI (P = 0.0002) with no urethral, anal complaints or other side effects (29). The authors suggest that due to the positive results, simple technology and ability to be self-administered, this new device may have utility in the treatment of CP\/CPPS. However, further studies and standardization of the electrostimulation device are essential.\nHyperthermia\nAnecdotal evidence and a few clinical trials have suggested heat therapy or hyperthermia as a treatment option for men with CP\/CPPS. Its mechanism of action is based on the application of heat to the prostate to relieve pain. An excellent review by Zeitlin (30) discusses the lack of literature concerning hyperthermia and CP\/CPPS. The review notes a variety of pitfalls in hyperthermia research including variation and lack of standardization of treatment. These concerns are applicable to both the type of heat utilized such as interstitial heat or microwaves and variation in its application, either transrectally or transurethrally. The review also suggests that the hyperthermia instruments used were not validated and outcome measures were subjective. However, the review implies that utilization of a quantitative assessment tool, applied statistics and greater documentation of therapy type may offer hyperthermia a better opportunity to be evaluated as a potential therapy for CP\/CPPS (30).\nWe also discovered the paucity of literature described by Zeitlin and only three listed clinical trials utilizing hyperthermia. The first study analyzed a group of 45 men with chronic abacterial prostatitis or prostadynia who underwent 6 weekly, 1 h sessions of local deep microwave hyperthermia (42.5 +\/\u22120.5\u00b0C) to the prostate. Although the authors report encouraging results in the decrease of pain, these subjective patient assessments were not quantified by the NIH-CPSI or other index for CP\/CPPS (31).\nA second abstract discussed a randomized, sham-controlled comparative study utilizing transrectal microwave hyperthermia in 80 men with CP\/CPPS. While this abstract noted a 75% symptomatic improvement in the treatment group, the study was available as an abstract only with no statistical significance or descriptive methodology reported (32). A third study also tested transrectal microwave hyperthermia for both chronic non-infectious and infectious prostatitis. While the study design incorporated obtaining measurement of prostatic secretions, uroflowmetry and transrectal color Dopplerographic mapping, the results from the study were not abstracted as the article was in Russian (33).\nHerbal and Nutritional Supplements\nHerbal and nutritional supplement therapies have been most widely investigated for their utility in CP\/CPPS and other prostate conditions such as BPH (34\u201340). While many formulations have been cited for their use in a wide variety of urological conditions, saw palmetto, pollen extract and quercetin were the supplements found with specific application to CP\/CPPS. While most herbal and nutritional supplements contain a wide variety of synergistic ingredients upon compositional analysis, some of the active components such as phytosterols or antioxidants are listed in Fig. 1.\nSaw palmetto\nSaw palmetto (Serenoa repens) garnered much attention in urology based on a great deal of anecdotal evidence regarding its prostate specific properties. It is widely used in many Asian, African and European countries and compositional analysis of the berry of S. repens exhibits sterols and free fatty acids as its major constituents (41). Initial studies suggested that the efficacy of saw palmetto may be similar to that of the pharmaceutical enzyme inhibitors such as finasteride. This preliminary data prompted the exploration of mechanism, utility and efficacy of saw palmetto in in vitro analysis and in clinical trial settings. While a number of trials have examined saw palmetto use for symptoms related to BPH (34\u201339), only a few have focused on it specifically for CP\/CPPS (42\u201344).\nThe first study compared the safety and efficacy of saw palmetto berry supplement versus finasteride in men with Category 3, CP\/CPPS. This prospective, open label 1 year study randomized 64 men to the saw palmetto or finasteride group, respectively. After 1 year of treatment, the NIH-CPSI score decreased from 23.9 to 18.1 in the finasteride group (P < 0.003) and from 24.7 to 24.6 in the saw palmetto group (P = 0.41). While significance was only achieved in the finasteride treatment arm, it was notable that at the end of the trial 41 and 66% of participants opted to continue the therapies of saw palmetto and finasteride, respectively, regardless of achieved statistical significance (42).\nThe second clinical trial from China examined the effects of prostadyn sabale capsules containing saw palmetto berry in patients with CP\/CPPS. While 125 men reported positive outcome and the NIH-CPSI was used as a primary end point, the article is only available in Chinese and the manufacturer, active constituents of the capsules and statistical significance was not reported (43).\nA third multicenter study testing a saw palmetto abstract called Permixon analyzed the response of Permixon therapy in 61 patients with Category 3B prostatitis. While 65% of the Permixon group reported improvement based on the Patients Subjective Global Assessment (SGA), the total NIH-CPSI and the pain, voiding and QOL\/impact domains of the NIH-CPSI, statistical significance was not reported (44). Additionally, prostate volume was unchanged in both the treatment and control groups. While this multicenter study suggests that Permixon may provide clinical benefit for CP\/CPPS 3B, the dosages and components of the Permixon product were not listed in the abstract.\nThe assessments of saw palmetto studies for CP\/CPPS are far fewer than those for BPH. However, marked and continued progress in molecular studies, increased mechanistic data and more clinical trials in CP\/CPPS are warranted to ascertain the utility and reproducibility of saw palmetto use in men with chronic prostatitis.\nPollen Extract: Cernilton\nPollen extract is traditionally collected from the flowers of various plant types and it contains carbohydrates, fat, protein, vitamins and minerals (41). The particular pollen extract named cernilton has been suggested to benefit a variety of urological conditions. Anecdotal evidence and references from traditional herbal texts have implicated cernilton's potent anti-inflammatory properties and potential in treating symptomatic relief of urinary pain and dysfunction often present in both CP\/CPPS (41) and BPH (45,46). In vitro studies demonstrate a variety of experimentation on this particular extract including histopathological analysis of its effect on cell proliferation, apoptosis, serum cytokines and testosterone (47,48). The literature also lists quite a few clinical trials on pollen extract; however, five are in the Japanese language and one in the German language (45,48\u201352). While many of these studies report the positive activity of pollen extract and suggest its usefulness for CP\/CPPS, data from these studies were not abstracted due to unavailability and translation of the articles.\nOne available study testing pollen extract reported a 78% favorable response of men with chronic prostatitis taking Cernilton\u00ae pollen extract at a dosage of 1 tablet TID for 6 months (53). While this study reported favorable results, the study was published in 1993 and similar subsequent larger phase clinical trials are not evident to further elucidate the possible utility of pollen extract in men with CP\/CPPS.\nQuercetin\nQuercetin is known chemically as a mixture of 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one and 3,3\u2032,4\u2032,5,7-pentahydroxy flavone. It belongs to a group of polyphenolic substances known as flavonoids and is a member of the class of flavonoids called flavonols. It is commonly found in the plant kingdom in the rinds and barks of certain foods such as onions, grapes and green tea (41). Since quercetin is thought to have antioxidant, anti-inflammatory, antiviral, immunomodulatory, anticancer, gastroprotective and antiallergy activities it has been studied for a variety of conditions (54\u201356).\nOne prospective, randomized, double-blind, placebo-controlled trial was performed to test the action of this bioflavonoid in men with CP\/CPPS. This placebo-based study assessed 30 men with CP\/CPPS to receive the bioflavonoid quercetin, 500 mg twice daily or the placebo pill for 1 month. Significant change in the NIH-CPSI score was observed in the quercetin (P = 0.003) versus the placebo group, who had an insignificant mean improvement in the NIH-CPSI score (57). While this was the only clinical trial found testing quercetin for CP\/CPPS, the positive outcome supports the need for further study including cost analysis of quercitin therapy in this patient population.\nOther Herbal Agents for Prostatitis\nFinally, a wide variety of herbal compounds not previously discussed were found during this review, many of which are commonly used in the TCM herbal material medica. Some of the compounds are Nan mi qing capsules containing Rheum palmatum and Rx. astragalus memberanaceus, Ye Ju Hua Shuan, an herbal suppository of Flos Chrysanthemi Indici and a variety of other formulae or capsules of which the ingredients were not listed (58\u201362).\nInitial in vitro studies examining some of the herbal compounds effects on markers of inflammation such as thromboxanes (TBX2) and 6-keto-PGF1-\u03b1 are promising as the link between chronic inflammation in CP\/CPPS is still under investigation (58). While the preliminary reports of additional herbal compounds are encouraging, a number of difficulties exist in the extraction of this data. Namely, the articles are in the Chinese or other language and data including the ingredients, constituents and other practices including good manufacturing are unable to be assessed. While this conundrum exists for many herbal agents despite their historical use in a variety of traditional herbal medicine systems, testing of safety and utility are still necessary. However, the variety of herbal agents available offers a large source to draw from and the possibility that other herbal products might be beneficial in the treatment of CP\/CPPS.\nConclusions\nA summary of the reviewed literature is outlined in Table 2 and a variety of CAM modalities tested in patients with CP\/CPPS include practitioner-based therapies such as biofeedback, hyperthermia, acupuncture and electrostimulation, and biological-based therapies including herbal and nutritional supplements. As the challenges in treating this complex and chronic disorder remain, further evidence of efficacious CAM treatment options for men with CP\/CPPS is needed. Thus far, promising data on the function and efficacy of certain CAM therapies suggest their potential as treatment options for this patient population. Further exploratory studies including more in vitro studies of herbal products, mechanistic data, cost analysis and randomized, controlled trials will assist in validation of certain CAM therapies as permanent treatment options for men with CP\/CPPS (63,64).","keyphrases":["alternative medicine","chronic prostatitis","herbs","supplements","biofeedback","acupuncture","hyperthermia","complementary medicine"],"prmu":["P","P","P","P","P","P","P","R"]}
{"id":"Dig_Dis_Sci-4-1-2413116","title":"Abuse in Women and Men with and without Functional Gastrointestinal Disorders\n","text":"We aimed to investigate the history of abuse in childhood and adulthood and health-related quality of life (HRQL) in women and men with FGID in the general adult population. A cross-sectional study in a random population sample (n = 1,537, 20\u201387 years) living in \u00d6sthammar municipality, Sweden, in 1995 was performed. Persons with FGID (n = 141) and a group of abdominal symptom-free controls (SSF, n = 97) were selected by means of a validated questionnaire assessing gastrointestinal symptoms (the ASQ). Abuse, anxiety and depression (the HADS) and HRQL (the PGWB) were measured. Women with FGID had a higher risk of having a history of some kind of abuse, as compared with the SSF controls (45% vs.16%, OR = 2.0, 95% CI: 1.01\u20133.9; SSF = 1), in contrast to men (29% vs. 24% n.s.). Women with a history of abuse and FGID had reduced HRQL 91 (95% CI 85\u201397) as compared with women without abuse history 100 (95% CI 96\u2013104, P = 0.01, \u201chealthy\u201d = 102\u2013105 on PGWB). Childhood emotional abuse was a predictor for consulting with OR = 4.20 (95% CI: 1.12\u201315.7.7). Thus, previous abuse is common in women with FGID and must be considered by the physician for diagnosis and treatment of the disorder.\nIntroduction\nMaltreatment, threats or violence from another person, often an intimate partner, is psychosocially traumatic and a growing problem in society. During the last decade, 1997\u20132006, the number of assaults reported to the police in Sweden increased by 40%, to 1,079\/100,000 inhabitants, and the number of sexual offences increased by 57% to 137\/100,000, of which rapes increased by 142% to 46\/100,000. The latter high percentage is partly attributable to a change in the Swedish legislation that lowered the threshold at which sexual offence is classified as rape. For children (0\u201315\u00a0years) the number of assaults reported to the police has increased by 64% to 97\/100,000 and the number of rapes has increased during the last decade by 300% to 12\/100,000 [1].\nAbout half of the women in Canada and Sweden have been described as having experienced at least one incident of violence by a man since the age of 16 [2, 3], and in a Japanese study three quarters of the women said they had experienced sexual, physical or emotional intimate partner violence [4]. A population survey in Los Angeles reported a childhood (lifetime for age\u00a0<16\u00a0years) sexual assault prevalence of 6.8% in women and 3.8% in men [5], and 10.5% in adults [6].\nFunctional gastrointestinal disorders (FGIDs) are a group of digestive diseases with a chronic or recurrent course in the absence of organic illness likely to explain the symptoms [7]. The most frequent FGIDs are irritable bowel syndrome (IBS), with an average estimated prevalence of 12%, and functional dyspepsia with a prevalence of 25% in the general adult population, but with a considerable proportion of persons with overlapping symptoms [8]. Most studies on IBS report more sufferers among women than men [9]. The difference in prevalence of dyspepsia between women and men is inconsistent; some studies report a higher prevalence of symptoms of dyspepsia in women than in men [10, 11], whereas others do not find any sex difference [12, 13]. Although FGID is common, only about 50% of people who have symptoms of dyspepsia ever seek medical attention for it [14, 15], while the consulting figures for IBS are somewhat higher [15]. FGIDs constitute about 5% of all consultations in primary care [16], and only a minority is referred to secondary care [17, 18].\nNevertheless, FGID accounts for up to 50% of all gastroenterology consultations [19].\nThe pathogenesis of FGID is at present often explained using the biopsychosocial model in which biologic and psychosocial factors participate in the origin of symptoms [20].\nAn overrepresentation of a history of past abuse has been reported in both patients and non-patients with IBS and among non-patients with functional dyspepsia [21, 22]. However, investigating abuse is difficult among patients with abdominal distress, since sexual and physical abuses are seldom reported to doctors by their patients [23].\nOur aims were to investigate the occurrence of a history of sexual, physical or emotional abuse experienced in childhood or later in life among women and men with FGID in the general population and the possible association with consultation rate, as compared with subjects free from FGID, controlling for age, sex, education and psychological distress.\nMethods\nSetting and sampling\nA population sample (n\u00a0=\u00a01,537) drawn from the National Swedish Population Registry in 1995 involved men and women 20\u201387\u00a0years of age, born on day 3, 12 or 24 of each month living in \u00d6sthammar municipality. The sampling method is equivalent to random sampling since there is no reason to believe that date of birth is associated with the variables measured. The subjects were sent a validated postal questionnaire, called \u201cThe Abdominal Symptom Questionnaire\u201d (ASQ) [24]. The responders (n\u00a0=\u00a0911) in the population sample were classified according to their responses in the ASQ as having either functional gastrointestinal disorder (FGID n\u00a0=\u00a0244), i.e., functional dyspepsia or irritable bowel syndrome, but not predominant symptoms on gastroesophageal reflux, or being strictly symptom free (SSF n\u00a0=\u00a0219), i.e., having reported no gastrointestinal (GI) symptoms and also no GI symptoms reported by subjects who also participated in two previous population studies in 1988\u20131989 [25].\nIn 1996, the FGID (n\u00a0=\u00a0244) and SSF (n\u00a0=\u00a0219) sample groups were invited by mail to an appointment at one of their six local health centres. About 187 (77%) with FGID and 156 (71%) SSF accepted the invitation. At the health centre they filled in the ASQ once again together with other questionnaires about health-related quality of life (HRQL) and their abuse history. A nurse assisted only when needed. With the same criteria for FGID applied to this ASQ response, 141 with FGID (IBS and dyspepsia: 99; only dyspepsia: 40; only IBS: 2) and 97 SSF remained, and thus constituted the study groups. Thus the FGID symptomatics were classified as FGID twice, in 1996 and 1995, and the symptom-free SSF group up to four times in 1996, 1995, 1989 and 1988. This sampling procedure assured persistent symptom status in the two study groups, as has been presented earlier [26] and in Fig.\u00a01.\nFig.\u00a01Formation and sampling procedure for the study groups from the population of \u00d6sthammar\nQuestionnaires\nThe Abdominal Symptom Questionnaire (ASQ) has previously been validated and found reliable and reproducible [27\u201329]. The definition of FGID used in this study is dyspepsia, IBS or both, as taken from the ASQ. Solitary heartburn and\/or regurgitations were symptoms not included in our definition of dyspepsia, which is in agreement with the current ROME III definition [30]. In the Abuse Questionnaire, the questions about sexual abuse were those initially developed for the National Population Survey of Canada [2] from survey questionnaires that fulfill reliability criteria [31]. The questions were translated into Swedish and back-translated into English for validation. Questions were written separately for childhood \u226413\u00a0years and adulthood (>13), including six categories of abuse: sexual, physical and emotional, each as a child or as an adult [23].\nThe Psychological General Well-Being (PGWB) index is a health-related quality of life instrument including 22 items divided into six domains: anxiety, depressed mood, positive well-being, self-control, general health and vitality. Items are scored on a six-grade Likert scale, with higher scores indicating better HRQL. The total score with a high responsiveness and validity for dyspepsia [32] was used in this study. HRQL values varying between 102 and 105 have been observed in a normal healthy population [33]. The total score was dichotomized at the median with a cut-off point 107\/108 at the upper 95% CI for functional dyspepsia. Consequently, a good HRQL was considered to be a total score of 108 or more, which was reached by 117 subjects (score 108\u2013132; mean 117.5, SD\u00a0=\u00a06.3), while a poor HRQL was reached by 120 subjects (score 49\u2013107; mean 90.8, SD\u00a0=\u00a012.6).\nThe Hospital Anxiety and Depression Scale (HADS) is a validated, reliable instrument with subscales for measuring anxiety and depression [34\u201336]. The questionnaire has seven items, graded 0\u20133, with possible ranges of 0\u201321 for each subscale (total minimum score of 0, total maximum score of 42). A score of 7 or less on each subscale (out of a maximum of 21) denotes a non-case, 8\u201310 a doubtful case, and 11 or more a definite case of anxiety or depression. The cut-off point 10\/11 was set for identifying sufferers in this study.\nAll participants per definition completed the ASQ, 231 (97.1%) the Abuse Questionnaire, 237 (99.6%) the PGWB questionnaire and 217 (91.1%) the Hospital Anxiety and Depression Questionnaire.\nEducational background was registered at five levels and dichotomized, with low including elementary, comprehensive, secondary level and high upper secondary, university level. Data of previous consultations for GI symptoms and educational background were taken from questions added to the 1995 ASQ.\nThis study was approved by the Ethics Committee of the Medical Faculty, Uppsala University, on 5 June  1996.\nStatistical analyses\nA Spearman rank correlation test was performed for the variables anxiety, depression and quality of life. A multiple logistic regression analysis was performed with FGID\/SSF and consulters\/non-consulters as outcome variables, each of the abuse variables as exposure variables and the possible confounding factors: age (\u221240\/40- years), sex (female\/male), education level, anxiety (1-HADS subscale >10, 0-HADS subscale <11), depression (1-HADS subscale >10, 0-HADS subscale <11) and HRQL score (1-PGWB >108, 0-PGWB \u2264108) as explanatory variables. In order to adjust for the influence of explanatory variables the variables were added one by one with a forward stepwise multiple logistic regression technique [37], and if the variable affected the odds ratios of the outcome variables less than \u00b110% and with P\u00a0>\u00a00.10, the variable was eliminated from the model. The Hosmer\u2013Lemeshow goodness-of-fit test was performed for each model, and model improvements were tested with the likelihood-ratio test. Tests for interactions were completed between the abuse variables and age, sex and HRQL. No significant interactions were found. STATA version 9.2 statistical package [38] was used for the analyses.\nResults\nDemography and sexual, physical and emotional abuse\nThe distribution of age, sex, education, GI consultation, anxiety, depression, HRQL and sexual, physical and emotional abuse for persons with FGID and SSF is presented in Table\u00a01. Forty-one percent of those with FGID had experienced some kind of abuse, as compared with 20% (P\u00a0<\u00a00.01) among SSF controls. Impaired anxiety and HRQL were significantly more common among subjects exposed to prior abuse, while depression was not.\nTable\u00a01Distribution of age, sex, education, previous GI consultation and abuse for women and men with functional gastrointestinal disorder (FGID) and strictly GI symptom free (SSF) and previous consulters\/non-consulters for FGIDFGID (n\u00a0=\u00a0141) n (%)\u00a0SSF (n\u00a0=\u00a097) n (%)Statistic PConsulters GI (n\u00a0=\u00a099) n (%)Non-consulters GI (n\u00a0=\u00a039) n (%)Statistic PMean age years (SD)b45.7 (14.3)52.4 (15.4)0.000746.7 (13.4)41.4 (14.2)0.041Femalea93 (66.0)51 (52.6)0.038 66 (66.7)26 (66.7)01Completed high schoola66 (47.1)28 (29.5)0.00743 (43.4)23 (60.0)0.100Consulted for GI evera99 (71.7)8 (8.3)<0.001Not relevantNot relevantPsychological general well-beingb96.8114.6<0.000196.896.60.963Anxietya13 (10.0)0 (0.0)0.0025 (7.5)8 (13.1)0.507Depressiona5 (4.8)1 (1.7)0.3193 (5.4)2 (4.3)0.942Childhood abuseChildhood sexual abusea19 (13.9)3 (3.1)0.00616 (16.5)3 (7.9)0.196Childhood physical abusea15 (11.0)3 (3.2)0.02914 (14.4)1 (2.6)0.050Childhood emotional abusea37 (27.4)15 (16.1)0.04632 (33.7)5 (13.2)0.017Any childhood abusea43 (31.4)18 (18.8)0.03137 (38.1)6 (15.8)0.012Adulthood abuseAdult sexual abusea17 (12.4)2 (2.1)0.00514 (14.4)3 (7.9)0.303Adult physical abusea3 (2.2)1 (1.1)0.5133 (3.1)0 (0.0)0.273Adult emotional abusea32 (23.7)5 (5.4)<0.00124 (25.3)8 (21.1)0.608Any adulthood abusea40 (29.2)8 (8.3)<0.00130 (30.9)10 (26.3)0.598Any childhood, not adulthood, abusea16 (14.6)11 (12.4)0.65415 (20.0)1 (3.0)0.022Any adulthood, not childhood, abusea13 (11.8)1 (1.1)0.0038 (10.7)5 (15.2)0.509Any abuse (child or adult)Sexual abusea30 (21.9)4 (4.2)<0.00125 (25.8)5 (13.2)0.113Physical abusea17 (12.4)3 (3.2)0.01416 (16.5)1 (2.6)0.029Emotional abusea48 (35.6)15 (16.1)0.00139 (41.1)9 (23.7)0.060Any abusea56 (40.9)19 (19.8)0.00145 (46.4)11 (29.0)0.064aPearson chi2-test,  b\u00a0Student\u2019s t-test\nWomen\nThere were 144 women, 93 with FGID and 51 SSF in the study. Almost half of the women, 42 out of 93 (45%) with FGID, had a history of abuse, in contrast to the 8 out of 51 (16%, P\u00a0<\u00a00.01) SSF controls. Women with FGID had a higher prevalence than SSF women for sexual, physical and emotional abuse in childhood and for sexual and emotional abuse in adulthood, as presented in Table\u00a02.\nTable\u00a02The number of people and the proportion of individuals (%) with a history of sexual, physical and emotional abuse in women and men with a functional gastrointestinal disorder (FGID) and strictly GI symptom free (SSF). The total number of FGID\/SSF for both childhood and adulthood abuse in women was 93\/51 and 48\/46 in menChildhood abuse number FGID\/SSF n(%) PAdulthood abuse number FGID\/SSF n(%) PSexualPhysicalEmotionalAnySexualPhysicalEmotionalAnyWomen18(19)\/3(6) 0.02711(12)\/1(2) 0.04826(28)\/5(10) 0.02132(34)\/7(14) 0.00614(15)\/2(4) 0.0442(2)\/0(0) 0.40324(26)\/2(4) 0.00131(33)\/4(8) <0.001Men1(1)\/0(0) 1.004(8)\/2(4) 0.87611(23)\/10(22) 0.93711(23)\/11(24) 1.003(6)\/0(0) 0.3311(2)\/1(2) 1.008(17)\/3(7) 0.3439(19)\/4(9) 0.382P value computed with Fisher\u2019s exact two-sided test\nOut of the 50 women with a history of abuse, 24 (48%, FGID n\u00a0=\u00a021, SSF n\u00a0=\u00a03, P\u00a0<\u00a00.01) had experienced sexual, physical or emotional abuse both in childhood and as adults. In the FGID group, 42 (46%) women reported abuse in childhood and\/or adulthood, as compared with 8 (16%, P\u00a0<\u00a00.01) in the SSF group. Moreover, in the FGID group, 32 (35%) women had a history of childhood abuse as compared with 7 (14%, P\u00a0<\u00a00.01) in the SSF group.\nMen\nIn contrast to the women, the men had no significant difference for a history of abuse: 14 out of 48 (29%) men with FGID versus 11 out of 46 (24%, P\u00a0=\u00a00.69) SSF men. Similarly, no statistically significant difference was found for a history of sexual, physical or emotional abuse in the subgroups childhood and adulthood for men; see Table\u00a02.\nAnxiety and depression\nWomen and men with FGID significantly more often reported anxiety (P\u00a0<\u00a00.01), consultations for GI problems (P\u00a0=\u00a00.02), childhood abuse (P\u00a0<\u00a00.01) and adulthood abuse (P\u00a0<\u00a00.01) than SSF women and men. In contrast, depression had no significant univariate association with FGID, GI consulting, childhood abuse or adulthood abuse. FGID (OR\u00a0=\u00a02.1, 95% CI: 1.1\u20134.0) and anxiety (OR\u00a0=\u00a010.7, 95% CI: 2.3\u201351) were the only remaining independent predictors for abuse when adjusted for age, sex, education and depression when tested with the multiple logistic regression technique.\nQuality of life\nSubjects with FGID had a significant reduction in HRQL, as measured by the PGWB, with a mean value of 97 (95% CI: 94\u201399), as compared with SSF controls, who scored 115 (95% CI: 112\u2013116). There was no significant difference in HRQL between women and men either for the FGID subjects (women 96; 95% CI: 92\u201399; men 99; 95% CI: 95\u2013102) or for the SSF group (women 114; 95% CI: 110\u2013117; men 115; 95% CI: 112\u2013119). Women with a history of some kind of abuse and FGID had significantly reduced HRQL, with a mean value of 91 (95% CI: 85\u201397) as compared with a mean value of 100 (95% CI: 96\u2013104) for women without abuse history. Similarly, men with a history of some kind of abuse and FGID had significantly reduced quality of life, with a mean value of 90 (95% CI: 82\u201399) as compared with a mean value of 102 (95% CI: 98\u2013105) for men without abuse history.\nThe HRQL for FGID consulters and non-consulters was the same; both groups had a mean value of 97 with 95% CI 93\u2013100 and 92\u2013102, respectively. However, FGID consulters with a history of abuse had significantly lower HRQL, with a mean value of 92 (95% CI: 86\u201397), than FGID consulters without a history of abuse with a mean value of 100 (95% CI: 97\u2013104, P\u00a0<\u00a00.05). There was a significant negative correlation between HRQL and anxiety [r\u00a0=\u00a0\u22120.61 (P\u00a0<\u00a00.01)] and between HRQL and depression [r\u00a0=\u00a0\u22120.54 (P\u00a0<\u00a00.01)].\nMultivariate risk modeling\nPersons with FGID had higher odds ratio 2.2 (95% CI: 1.1\u20134.4) of a history of some kind of abuse (in childhood or adulthood, sexual, physical or emotional) as compared with the SSF controls adjusted for age, sex and HRQL (main model, adding the variable anxiety or education did not improve the model). Some kind of abuse in adulthood had odds ratio 2.8 (95% CI: 1.1\u20137.1) with the same strategy. Emotional abuse in adulthood had the highest (and only significant) odds ratio 3.1 (95% CI: 1.0\u20139.4), while sexual abuse did not reach significance. In childhood, physical abuse seems to have the highest odds ratio 2.9 (95% CI: 0.7\u201312) for future FGID, although significance was not reached in this study (data not shown).\nWith the main model, women had significantly high odds ratios for FGID: from some kind of abuse 3.13 (95% CI: 1.21\u20138.10), emotional abuse 3.66 (95% CI: 1.22\u201311.0), physical abuse 5.07 (95% CI: 0.55\u201347.1) and sexual abuse 3.03 (95% CI: 0.89\u201310.3); the latter two types of abuse did not reach significance, as presented in Table\u00a03. There were significant high odds ratios in women with the crude model for FGID from all types of abuse in childhood and adulthood, except adult physical abuse (probably owing to the small number of observations). Similarly, with the main model, there were high odds ratios in women, but they did not reach significance, see Table\u00a03.\nTable\u00a03Odds ratio and 95% confidence interval (CI) for having a functional gastrointestinal disorder (FGID) (logistic regression) in different groups of abuse, women and men, in crude model and main model adjusted for age and HRQLVariableCrude modelMain model (adjusted for age and HRQL)WomenMenWomenMenChildhood abuseNo childhood sexual abuse11Childhood sexual abuse3.95 (1.10\u201314.1)*2.44 (0.60\u201310.0)*No childhood physical abuse1111Childhood physical abuse 6.88 (0.86\u201354.9)1.95 (0.34\u201311.2)4.93 (0.53\u201346.3)1.61 (0.21\u201312.3)No childhood emotional abuse1111Childhood emotional abuse 3.71 (1.32\u201310.4)1.00 (0.38\u20132.68)2.71 (0.87\u20138.4)1.14 (0.37\u20133.55)No childhood abuse1111Any childhood abuse3.41 (1.38\u20138.43)0.94 (0.36\u20132.47)2.50 (0.91\u20136.85)1.10 (0.36\u20133.38)Adulthood abuseNo adult sexual abuse111Adult sexual abuse4.45 (0.97\u201320.5)*3.20 (0.60\u201316.9)1.51 (0.53\u20134.29)No adult physical abuse11Adult physical abuse *0.93 (0.06\u201315.4)*0.84 (0.03\u201325.3)No adult emotional abuse1111Adult emotional abuse 8.86 (2.00\u201339.3)2.74 (0.67\u201311.1)3.42 (0.70\u201316.9)2.78 (0.55\u201314.0)No adulthood abuse1111Any adulthood abuse6.07 (2.00\u201318.4)2.43 (0.69\u20138.57)3.10 (0.92\u201310.3)2.41 (0.55\u201310.5)Any abuse (child or adult)No sexual abuse111Sexual abuse3.57 (1.34\u20139.5)*3.03 (0.89\u201310.3)1.49 (0.52\u20134.24)No physical abuse1111Physical abuse7.59 (0.96\u201360.2)2.5 (0.46\u201313.6)5.07 (0.55\u201347.1)1.84 (0.26\u201313.0)No emotional abuse1111Emotional abuse5.83 (2.11\u201316.1)1.26 (0.48\u20133.28)3.66 (1.22\u201311.0)1.31 (0.43\u20133.95)No abuse1111Any abuse4.61 (1.95\u201310.9)1.31 (0.52\u20133.32)3.13 (1.21\u20138.10)1.32 (0.45\u20133.90)1\u00a0=\u00a0Reference, *\u00a0too few observations\nIn contrast, men did not have the same elevation of odds ratio for FGID from any type of abuse as compared to women. The highest odds ratio in men for FGID was for some kind of adulthood abuse and was 2.41 (95% CI: 0.55\u201310.5), while adult emotional abuse was 2.78 (95% CI: 0.55\u201314.0), but neither were significant; see Table\u00a03.\nConsulters and non-consulters with FGID\nConsulters with FGID more often (46% of them) had a history of abuse as compared with non-consulters (29%, P\u00a0=\u00a00.06), although it did not reach significance as shown in Table\u00a01. Some kind of abuse in childhood was more prevalent: 38% in consulters versus 16% in non-consulters (P\u00a0=\u00a00.01) and correspondingly for childhood emotional abuse 34% and 13% (P\u00a0=\u00a00.02) and for childhood physical abuse 14% and 3% (P\u00a0=\u00a00.05). Moreover, there was a significantly higher prevalence of physical abuse in childhood or adulthood in consulters, 17%, versus non-consulters, 3%, P\u00a0=\u00a00.03. Female consulters with a history of some kind of abuse had an odds ratio of 2.47 (95% CI: 0.92\u20136.7), and with a history of childhood emotional abuse this was significant, 4.20 (95% CI: 1.12\u201315.7.7), in contrast to male consulters, with an odds ratio of 1.66 (95% CI: 0.37\u20137.50) for some kind of abuse and 2.10 (95% CI: 0.37\u201312.0) for childhood emotional abuse.\nDiscussion\nWomen, but not men, with longstanding FGID often have a history of abuse. Women with FGID reported past sexual, physical or emotional abuse in 45% of the cases, as compared with 16% for women without FGID, 29% in men with FGID and 25% in men without FGID. In this study emotional and sexual abuse are the most common type of threats to women\u2019s health related to FGID. This study also shows that childhood emotional abuse is a predictor for consulting for GI problems. Moreover, longstanding FGID is associated with a significantly reduced HRQL, and a history of abuse further reduces the HRQL in women and men. The findings reveal that a history of abuse is an important psycho-social factor linked to FGID in women. In contrast, the association in men is less clear, although men with a history of some kind of abuse and FGID had significantly reduced HRQL as compared with men with FGID without any history of abuse.\nOur study does not support the idea that consulters with FGID generally have a poorer HRQL than non-consulters, but a history of abuse had a negative effect on HRQL for consulters with FGID.\nMost studies that investigate FGID use a technique that implies questioning the subjects of the experiment at a single point in time. One of the strengths of this study is the repetitive sampling procedure, which makes it possible to compare individuals with longstanding FGID with persistently symptom-free individuals in the same population. We consider that our findings can be generalized to the general population, as the study groups were sampled from a well-defined and thoroughly investigated population [24, 26]. The individuals answered the abuse questionnaire in a quiet, safe environment, in reality anonymously, with the support of a nurse only when needed, which was an advantage since patients tend to underreport abuse when asked face to face by a doctor [23].\nThe abuse questionnaire has not been thoroughly validated in a Swedish population, but the questions are straightforward and the questionnaire was translated into Swedish and back-translated into English. The definition of functional dyspepsia and IBS used is not exactly the same as the now recommended Rome III criteria [39]. The definitions of dyspepsia and IBS used in this study were those used in the original study from 1988 [25], before current recommendations were available. We opted to retain our original study definition since our IBS definition was in accordance with the Rome III criteria and our definition of dyspepsia was more restricted in terms of symptoms, but wider in terms of abdominal location. We consider the overall prevalence of FGID in this study and the concordance on an individual level to be applicable within the current definition [39], and thus we consider our results conclusive.\nPrevious studies in the field have reported that about half the patients referred to a gastroenterology clinic had a history of sexual or physical abuse [23, 40]. Moreover, 71% of women who reported domestic violence to the police had FGID according to Perona et\u00a0al. [41]. In the literature, a history of childhood or adult abuse characterizes patients who present with a variety of functional symptoms apart from FGID [42]: headaches [43], pelvic pain [44], panic disorders [45], non-epileptic attack disorders [46] and back pain [47]. Psychological factors such as anxiety [48], as well as physiological factors such as enhanced visceral sensitivity, all contributed to a poor health outcome [42]. Only a few studies have focused on abuse and FGID in the general population, where Koloski et\u00a0al. [22] found abuse to be significantly associated with IBS and\/or functional dyspepsia, but less important when psychosocial factors were controlled for, a finding confirmed by others [49, 50].\nThe same research team [51] found that past sexual, physical and emotional abuse was not a significant predictor for seeking health care, in contrast to our finding that a history of childhood emotional abuse was associated with consulting, and anxiety and FGID were independent predictors for previous abuse. It is clear from our study that many subjects with traumatic memories and uncomfortable symptoms never seek health care and that when they do it is important to take a thorough medical history.\nThere is a bi-directional communication between the gut and the brain through the autonomic nervous system and the hypothalamic\u2013pituitary\u2013adrenal axis [51] that can mediate an emotional trauma such as previous abuse. Common links suggested are neuroticism [49], generally diminished pain threshold [52] and current depression [53]. There is a higher incidence of childhood abuse reported for women than for men in this study as in others [5]. This could explain the significant relation of consulting and childhood emotional abuse for women, but not for men found in this study, suggesting that FGID in some patients might be a sequel of previous abuse. The treatments available to date for FGIDs are of limited value. Creed et\u00a0al. [54] have reported that psychological treatment and antidepressants are effective in patients with IBS and a history of sexual abuse, which stresses the importance of discovering past abuse in patients with FGID. Future studies must focus on how the knowledge of a patient\u2019s previous exposure to different kinds of abuse and the effects of different kind of psychotherapy can improve the treatment for at least a subset of FGID sufferers.\nConclusions\nWe conclude that women with longstanding FGID in many cases have a history of physical, emotional or sexual abuse in childhood or adulthood, which is associated with a poor HRQL and increased health care seeking. This is important for physicians to consider when diagnosing and treating FGID in women.","keyphrases":["functional gastrointestinal disorders","irritable bowel syndrome","abused women","child abuse"],"prmu":["P","P","R","R"]}
{"id":"Eur_Spine_J-2-2-1602182","title":"A rare case of complete C2\u2013C3 dislocation with mild neurological symptoms\n","text":"The authors report a rare case of complete C2\u2013C3 dislocation with unexpectedly mild neurological symptoms in a 57 year old man involved in a motor vehicle accident, who had previously undergone posterior laminectomy from C3 through C7. A retrospective chart analysis and a thorough radiographic review were performed. X-rays and CT of the cervical spine demonstrated a complete dislocation at the C2\u2013C3 level. Computed tomographic angiography revealed disruption of both vertebral arteries; however, blood flow was evident in the basilar artery. After radiologically guided placement in cervical traction with tongs that reduced the subluxation by approximately 50% the patient had spontaneous eye opening and was able to follow commands. A two-stage 360o stabilization and fusion was performed and the patient was finally discharged 24 days after admission with his neurological status essentially unchanged. In conclusion, our patient presented with surprisingly mild neurological symptoms. The previously performed laminectomy could have both predisposed to injury as well as protected his spinal cord from potentially fatal trauma.\nIntroduction\nSubluxation of the cervical spine in adults usually occurs at its lower segments (C4\u2013C7) [12, 14]; 50% of anterior subluxation of the neck has been reported to involve the highly mobile C5\u2013C6 junction [11, 14]. In the pediatric population (up to 9\u00a0years old), the upper cervical spine is more susceptible to injury due to the anatomical differences in the developing spine [3]. Subluxation at the C2\u2013C3 level is a particularly uncommon injury. We report a case of complete dislocation at the C2\u2013C3 level in a patient involved in a motor vehicle accident who presented with surprisingly mild neurological symptoms.\nCase report\nA 57\u00a0year old African American man under the influence of alcohol and benzodiazepines was involved in a severe motor vehicle accident including one fatality. The patient was wearing a seat belt. He was transferred to our clinic via ambulance from a community hospital, fully immobilized with a hard cervical collar on, while the spinal cord injury solumedrol protocol had been initiated. On arrival to our Emergency Department, the patient had a Glascow Coma Scale (GCS) score of 11 and was thrashing all extremities. He responded to pain. He was intubated and thorough radiologic evaluation was undertaken.\nThe CT of the brain revealed a slender tentorial subdural hematoma not requiring surgical evacuation. X-rays and CT of the cervical spine revealed a complete dislocation at the C2\u2013C3 level with acute angulation between the two cervical vertebral segments (Fig.\u00a01). Additionally, the imaging studies demonstrated multi-level degenerative spondylosis, anterior bridging osteophytes from C3 through C7 consistent with Forestier\u2019s disease and extensive previously performed posterior laminectomy from C3 to C7. Laminectomy was performed, according to the patient, because of underlying degenerative disease. Computed tomographic angiography (CTA) revealed disruption of the vertebral arteries bilaterally at the level of C2\u2013C3 (Fig.\u00a02). However, flow was identified in the basilar artery indicating a patent posterior circle of Willis providing back flow to the basilar artery and the posterior circulation. No MRI could be obtained due to the presence of a previously implanted cardiac pacemaker.\nFig.\u00a01Plain lateral X-rays of the cervical spine demonstrating a complete C2\u2013C3 dislocation upon the patient\u2019s admissionFig.\u00a02Computed tomographic angiography obtained at patient\u2019s admission demonstrating bilateral complete disruption of vertebral arteries\nThe patient was placed in cervical traction with tongs under radiologic guidance for verifying alignment. The weight applied was gradually increased up to twelve pounds; at that point, fluoroscopic imaging demonstrated a significant improvement in the angulation of the two separate segments and a reduction of the degree of subluxation. However, a subluxation of approximately 50% of the cervical spine vertebral body length was still present (Fig.\u00a03). Upon reversal of sedation, the patient had spontaneous eye opening and was able to follow commands. His upper and lower extremities\u2019 muscle strength was 3\/5. The treatment plan for this patient consisted of a two stage 360o stabilization and fusion.\nFig.\u00a03Plain lateral X-rays of the cervical spine on the same patient obtained after applying external traction via tongs, demonstrating sub-optimal realignment of his cervical spine\nShortly after his arrival to our hospital, the patient started to experience respiratory difficulties, leading the authors to the decision to delay surgery until the respiratory condition of the patient was stabilized. Two days after his admission, anterior cervical discectomy and fusion (ACDF) with the standard Smith-Robinson technique and bone allograft was performed at the C2\u2013C3 level. A 24\u00a0mm Synthes (Paoli, PA, USA) statue plate was utilized after appropriate bending in order to contour the curvature of the cervical spine. Intraoperatively, disruption of the anterior longitudinal ligament at the C2\u2013C3 level as well as fracture of the anterior osteophytic bony bridges, were evident. The vertebral endplates of the vertebral bodies at that level were severely degenerated. At the conclusion of the case, adequate reduction, satisfactory alignment and stabilization of the anterior cervical spine were accomplished. A halo vest was applied post-operatively for additional stabilization, necessary for the aggressive physical therapy that the patient\u2019s respiratory condition demanded. Post-operative X-rays demonstrated reduction of the dislocation of C2 with respect to C3 and anatomic alignment of the structures of the cervical spine.\nThe patient\u2019s neurological condition remained unchanged during the following days. Respiratory distress was however noted, and a few days later, the patient was diagnosed with pneumonia. Appropriate treatment with antibiotics was initiated and it was decided to delay the second, posterior surgical approach until the patient\u2019s respiratory condition was stabilized and his pneumonia resolved. Eighteen days after the initial operation, a second, posterior approach was employed. Under general endotracheal anesthesia, through a midline skin incision, a posterior cervical fusion and instrumentation (Synthes, Paoli, PA, USA) with pedicle screws at C2 level (20\u00a0mm long and 4\u00a0mm in diameter) and lateral mass screws (12\u00a0mm in length and 3.5\u00a0mm in diameter) at C3 level was undertaken. After optimal position of the screws was fluoroscopically verified, a 40\u00a0mm rod was used, which was attached and secured on a C2 and a C3 screw. Allograft bone chips were then appropriately placed (Fig.\u00a04). The patient was placed back in the halo post-operatively due to his age and some concerns regarding osteoporosis. The halo vest was kept for four weeks after the second operation. He was finally discharged from the hospital to a rehabilitation clinic 24\u00a0days after admission. His neurologic status was essentially unchanged, with a muscle strength 3\/5 in the upper and lower extremities.\nFig.\u00a04Post-operative lateral X-rays on the same patient after completion of anterior and posterior fusion\nThe patient was followed at a different institution. At the last follow-up visit, 12\u00a0months postoperatively, his neurological status showed improvement with muscle strength of 4\/5 in the upper and lower extremities.(Fig.\u00a05a, b)\nFig.\u00a05a Post-operative CT (3D reformatted midsagittal image) of the cervical spine demonstrating adequate anatomical alignment at the C2\u2013C3 level. b Post-operative CT (3D reformatted midsagittal image) of the cervical spine demonstrating adequate anatomical alignment at the C2\u2013C3 level\nDiscussion\nInjury of the cervical spine is a potentially fatal and debilitating incident due to the risk of damage of the cervical spinal cord. Although the degree of subluxation does not necessarily correlate with clinical symptoms and neurological signs, one would agree that the patient described above presented with unusually mild symptomatology in regards to the complete dislocation of the cervical vertebrae of the C2\u2013C3 level, evident in his radiological examination.\nIn reviewing the possible parameters related to this patient\u2019s favorable outcome, attention should be drawn to the posterior cervical laminectomy previously performed on our patient due to underlying degenerative disease. It is quite likely that the lack of dorsal spinal elements of the C3 level, provided for adequate space for the posterior dislocation of the spinal cord to occur without sustaining the anticipated injury due to compression. This must have been of great importance both during the injury itself, as well as, after the dislocation had been established.\nInterestingly, Fountas et al. [5] noted in their study using postmyelogram computed tomographic measurements, that the cervical spinal cord might be smaller than considered. Specifically, they found that the diameter of the cervical spinal cord was 15\u201320% smaller than has been reported by autopsy studies. A smaller cervical spinal cord would also be less susceptible to injury due to compression. Although this is an interesting point that should perhaps be taken into consideration when referring to compression of the cervical spinal cord, its role in our case of a complete dislocation of the cervical spine could not have been, by itself, determinant without the history of previous laminectomy.\nOn the other hand, the removal of the posterior arc of the vertebral bone may have predisposed for the development of such a complete dislocation. Hansen-Schwartz et al. [7] observed that 26% of patients developed static subluxation at the cervical spine 7.7\u00b10.6\u00a0years after cervical laminectomy. Cusick et al [2], in a cadaveric study examining the biomechanical responses of the cervical spine to laminectomy, concluded that multilevel cervical laminectomy induces increases in total column flexibility, allowing additional motion in the flexion-extension plane. This observation may well describe the mechanism of injury in our case in which extension of C2\u2013C3 induced a posterior dislocation and was not limited to a severe sprain. Likewise, Hong-Wan et al. [8] noted in their biomechanical study a significant increase in intersegmental motions following cervical laminectomy. Furthermore, Fields et al. [4] observed in their randomized study comparing the effects of laminectomy and laminoplasty in the rabbit, that laminectomized animals had poorer clinical outcome at 3\u00a0months post-operatively, associated with statistically significant angular deformity. Similarly, Baisden et al. [1], using a goat model, concluded that laminoplasty is superior to laminectomy in maintaining sagittal cervical alignment and preventing spinal deformities. The removal of bony and ligamentous structures of the posterior cervical spine might alter the biomechanics of the vertebral column and predispose to instability. The involvement of the C2\u2013C3 level in our patient which coincides with the transition from non-laminectomized to laminectomized segments raises further suspicion regarding the putative role of laminectomy in the mechanism of injury in our case. Finally, the presence of osteophytes in the anterior cervical column in our patient (Forestier\u2019s disease) could predispose to injury; more severe neurologic deficits have been described in patients with diffuse idiopathic skeletal hyperostosis even following minor trauma [15].\nIt is interesting to note that our patient had no signs of posterior fossa infarction despite the fact that both of his vertebral arteries were disrupted at the C2\u2013C3 level. We assume that this is because of the presence of fetal circulation in this patient providing back flow to the basilar artery as this was demonstrated in his CTA, thus maintaining adequate perfusion of the posterior fossa [9].\nTreatment of subaxial cervical spinal injury remains controversial. Both the anterior and posterior procedures have serious advantages and disadvantages [6, 10, 13]. In our case, we decided to perform a combined anterior and posterior surgical procedure, since both the anterior and posterior longitudinal ligaments were disrupted and we felt that a circumferential approach would sufficiently restore the lost stability. Furthermore, the presence of disc fragments compromising the spinal canal could not be ruled out, due to the inability to obtain an MRI in our patient. A posterior approach, thus, without anterior stabilization was considered to harbor an increased risk of spinal cord compromise. It needs to be emphasized that further multiinstitutional prospective clinical studies are required in order to establish guidelines for the management of subaxial cervical spine injury.\nConclusion\nIn conclusion, we have reported a rare case of complete C2\u2013C3 dislocation with unexpectedly mild neurological symptoms in a patient who had previously undergone a C3\u2013C7 laminectomy. The previously performed laminectomy might have both predisposed and protected the patient\u2019s spinal cord from severe injury.","keyphrases":["dislocation","fusion","upper cervical","anteroposterior stabilization","post-laminectomy"],"prmu":["P","P","P","M","U"]}
{"id":"Eur_J_Health_Econ-_-_-1388084","title":"The \u201cEssential Levels of Care\u201d in Italy: when being explicit serves the devolution of powers\n","text":"The definition of an explicit health benefit package in Italy has gained importance because of devolution of powers from the national level to the regions. The set of services to be guaranteed by the public sector are defined at national level, while regions are accountable for their provision. This contribution discusses the entitlements and the decision criteria adopted by Italian policy-making bodies. Entitlements to services are clearly defined for few sectors (mainly outpatient specialist care); for hospital care the benefit catalogue is vague. The definition of the health benefit package in Italy is an essential element of the relationship between the central government and the regions. It is argued that adequate monitoring systems and accountability procedures are still needed to make the essential levels of care an effective pivotal element of the Italian National Health Service.\nItaly has a National Health Service based on principles of universalism and comprehensiveness [1, 2]. Established in 1978, the Italian National Health Service (INHS) absorbs 76.4% of total health care expenditure and it is financed by general taxation (OECD health data, 2005). As a consequence most care is provided free of charge at point of delivery, although user charges apply for outpatient services and procedures and, to a lesser extent, on pharmaceuticals [3].\nA major critical feature of the INHS rests on the distribution of powers between the central government and the regions. Mainly created to reverse the concentration of state power occurring during the Fascist regime, the 20 regions are the emerging institutional tier of the country as they benefit from a substantial, although controversial, devolution of powers from the State. They have almost full control over approx. 200 Local Health Units and 100 Independent INHS Hospitals and they are expected to cover their deficit [4]. The Italian Constitution, revised in 2001, reserves to the central government the exclusive power to set the so-called \u201cessential levels of care\u201d (Livelli Essenziali di Assistenza, LEAs), which must be guaranteed to all residents. Regions have virtually exclusive powers over regulation, organization, administration, and funding of publicly financed healthcare.\nThe Italian Constitution of 1948 specifies the citizen\u2019s right to health. This constitutional guarantee is expressed in very general terms. Article 32 of the Constitution says that \u201cThe Republic protects health as a fundamental right of the individual and as a concern of collectivity and guarantees free care to the indigent.\u201d The principle of a package of benefits available to all citizens irrespective of age, social condition, or income was stated later, in the law introducing the INHS in 1978. The expression \u201clevels of care\u201d was mentioned for the first time with the objective to guarantee equal health care coverage throughout the country: \u201cthe State is to set objectives for eliminating geographical differences in social and health care conditions\u201d (Art. 2) and \u201cand to determine levels of care to be guaranteed to all citizens\u201d (Art. 3). The same legislation also introduced another major feature of the INHS: the patient\u2019s right to choose \u201cprovider and place of treatment.\u201d\nAlthough the 1978 reform listed the areas in which treatments are to be delivered under the INHS, it did not define the benefits to be included and excluded in detail. The concept of levels of care gained prominence in the legislation reforming the INHS in 1992\u20131993. The delegation of new powers to regions was coupled with tighter accountability systems. On the provision side the regions must deliver uniform levels of care, while on the funding side regions are mandated to cover any deficit required to provide the LEAs and to use their own resources to provide services above those guaranteed by national laws.\nThe reform laid out a new logical framework, but it took time to establish the LEAs. Significant progress was made in the late 1990s with the approval of the National Health Plan 1998\u20132000 and a new reform approved in 1999. These emphasized the importance of the principle of equality in the access of care and introduced LEA system; they also clearly stated the criteria that should inform their definition: human dignity, effectiveness, appropriateness, and efficiency.\nThe LEAs notion was at risk of remaining an abstract concept as political costs and implementation difficulties of clarifying and limiting INHS coverage were paramount. However, substantial progress in the definitions of LEAs was necessary to make sense of the overall strategy of redistribution of powers between the central government and the regions. This progress was made with the agreement between the regions and the central government on 8 August 2001 which was followed by a governmental decree (the LEA decree). At present this decree is the pivotal element of the Italian health benefit catalogue (Fig.\u00a01). It defines the main areas of healthcare services to be guaranteed by the INHS (positive list), those completely excluded by public coverage (negative list), and those partially covered (only available for specific clinical conditions).Fig.\u00a01 Decision making on benefits in Italy\nThe positive list is based on the recognition and systematization of current legislation (other decrees, laws, guidelines, etc.), i.e., it includes all the services that the INHS is actually providing categorized in three macrolevels of care: (a) public health services, (b) community care, and (c) hospital care (Fig.\u00a02). Table\u00a01 displays the main elements of LEA decree and of the other benefit-defining laws and decrees currently in force in Italy. The present contribution focuses on curative services.Fig.\u00a02 DPCM, 29 November 2001. Definition of national standards of care (LEAs). MoH Ministry of HealthTable\u00a01 Benefit-defining laws\/decrees and catalogues for curative care services in Italy (LEAs DPCM, 29 November 2001, \u201cDefinition of Essential Levels of Care\u201d; Specialist DM, 22 July 1996, \u201cSpecialist outpatient services\u201d;\nNPF National Pharmaceutical Formulary; Prostheses DM\u00a0332\/1999: \u201cProsthetic devices, tariffs and provision modalities\u201d; Rehab Guidelines on Rehabilitative Care adopted on May 7th 1998;\nPrimary National Contract for Primary Care)LEAsSpecialistNPFaProsthesesaRehabaPrimaryInpatient curative care+\u2013\u2013\u2013\u2013\u2013Day cases curative care+\u2013\u2013\u2013\u2013\u2013Primary care+\u2013\u2013\u2013\u2013+Outpatient dental care+\u2013\u2013\u2013\u2013\u2013Specialist outpatient care++\u2013\u2013\u2013\u2013Alternative medicine+\u2013\u2013\u2013\u2013\u2013Rehabilitative care+\u2013\u2013\u2013+\u2013Long-term nursing care+\u2013\u2013\u2013\u2013\u2013Clinical laboratory\u2013+\u2013\u2013\u2013Emergency rescue+\u2013\u2013\u2013\u2013\u2013Pharmaceuticals+\u2013+\u2013\u2013\u2013Therapeutic devices+\u2013\u2013+\u2013\u2013Prevention and public health services+\u2013\u2013\u2013\u2013\u2013Legal statusLegislative decreeMinistry DecreeAgency administrative actLegislative decreeLawPresidential decreeDecision makerPermanent State-Regions Conference; MoHMinistry of HealthNational Drug AgencyMinistry of HealthMinistry of healthMinistry of health; Trade-Unions of GPsOriginal purposeEntitlementsFee-schedulePositive listFee-scheduleGuidelinesEntitlementsPositive\/negative definition of benefitsP and NPPPPPDegree of explicitnessb233311\/2If itemized: goods, procedures only; linked to indicationsVaries by area of careProcedures, sometimes linked to indicationsGoods, for certain categories specific indications (prescription notes)GoodsNoPartiallyUpdatingForeseenNo at national level; regular at regional levelRegularly (annually)No (foreseen)NoEvery 2\u20133\u00a0yearsCriteria\u2013\u2013\u2013\u2013\u2013\u2013\u00a0\u00a0Need+\u2013\u2013+\u2013+\u00a0\u00a0Costs++++\u2013\u2013\u00a0\u00a0Effectiveness\u2013++\u2013\u2013+\u00a0\u00a0Cost-effectiveness\u2013\u2013\u2013\u2013\u2013\u2013\u00a0\u00a0Budget+\u2013\u2013\u2013\u2013+\u00a0\u00a0Appropriateness+\u2013\u2013\u2013++aThese sections (noncurative care services) are not discussed in detail in the present contributionb1, \u201call necessary\u201d; 2, areas of care; 3, items\nThe decree also defined a system for monitoring LEA implementation across the country. Responsibility for this was assigned to a special technical body established in April 2002 and composed of representatives of the Ministry of Health, the Treasury, and the regional governments. The main objective of the commission is to \u201cmonitor and evaluate the actual provision of services included in the LEAs and their costs.\u201d\nIn 2004 a new technical body (the National LEA Commission) was established to update LEAs on the basis of scientific, technological, and economic evidence (Ministry decree of 25 February 2005). The Commission is set up of 14 members: 6 experts of healthcare management, planning, and organizational sciences are nominated by the Ministry of Health, 7 are regional representatives, and one is appointed by the Treasury.\nServices of curative care in the health benefit basket\nInpatient curative care\nTraditionally the services to be provided in hospital settings were never explicitly defined by the INHS. It has been rather implicitly recognized that all types of services deemed to be appropriately delivered at hospital level must be available to citizens. The LEA decree defines seven chapters as broad categories of services to be delivered in the hospital: (a) emergency services, (b) ordinary admissions (including rehabilitative and long-term inpatient care), (c) day hospital, (d) day surgery, (e) curative home-care, (f) collection, diffusion, control of blood-components and transfusion services, and (g) organ and tissue transplantation services. In addition, it is explicitly recognized that some benefits are available in hospitals although not included in the positive list of outpatient services provided under public coverage (e.g., certain pharmaceuticals or diagnostic tests).\nWhile the detailed spectrum of services to be provided by the hospitals is not explicitly defined, national and regional fee-schedules regulate their funding. Since January 1995 Italian hospitals have been financed mainly according to nationally predetermined rates based on classification into diagnosis-related groups (DRGs) [4, 5, 6]. Regions are free to modify the rates according to their own standards but must take the national rate as the maximum level [1]. To a certain extent DRG lists may be interpreted as the catalogue of hospital services and benefits covered by the INHS. This interpretation, however, should be done with some caution. DRGs are divided in two major groups: surgical and medical. The interventions to which surgical DRGs refer are expected to be offered and funded. Therefore surgical DRGs define a sort of list of services available to patients. For medical DRGs the situation is different as the classification includes all possible diagnoses, including those for which hospital admissions may not been appropriate. Therefore medical DRGs do not define a list of services to be guaranteed but rather economic constraints according to which providers act. Thus, although implicitly, the tariff values assigned to DRGs influence the specific content of the services provided in each diagnosis category.\nIn Italy as well as in other countries, determining tariffs for specific diagnosis and treatments is seen as detrimental to the adoption of innovative technologies. Fixed and outdated tariffs may discourage the adoption of new expensive technologies and may force hospitals to look for alternative source of funding, often resulting in wide disparities of their availability to citizen. Regional authorities may react to the deficiencies in national policy with a variety of measures as it is illustrated in the case of Drug Eluting Stents (DES). Since their introduction into the European market in 2002, DES have been gradually implemented in clinical practice in Italy, mainly by hospitals in the north of the country. Several position papers and recommendations have been provided by scientific societies (e.g., Italian Group for Heamodynamics Studies, GISE), study groups, local committees (e.g., Emilia-Romagna Cardiology-Cardiosurgery Committee) in order to guide the adoption of DES. Early updating of reimbursement policies has been advocated by Italian cardiologists, hospital administrators, and patients to allow this technology to be economically sustainable by the hospitals [7]. Faced with continuously growing clinical and economic evidence, some regional authorities started to adopt different policy measures [8]. In 2002 the Lombardy Region revolutionized its DRG classification by creating three new DRGs to cover stent reimbursement and to encourage utilization. Other regions (Emilia Romagna, Lazio, Marche, Puglia) allowed for particular DRG tariff increase while the Campania Region established a special regional fund for DES reimbursement. In addition, Emilia-Romagna and recently Sicily established a regional registry to monitor the rate of adoption of the new technology and its effectiveness in real-life conditions. The final aim is to identify the target populations for which the new technology would be most beneficial. Most of the regions still have not updated in any way their reimbursement policies, thus limiting to certain extent the diffusion of the device on their territory.\nThe example on DES illustrates the existence of wide variations across the regions concerning how to fund innovative technologies. Similar situations may be found in other sectors of health care. However, the issue is not that simple when we consider that one of the fundamental rights of Italian citizens is the \u201cfreedom of choice of provider and place of care\u201d [9], making regions responsible for the cost of treatment provided to their residents in other regions.\nDay cases of curative care\nServices available in day hospital regime are defined as \u201cdiagnostic, rehabilitation, and curative care, delivered as alternative to ordinary hospitalization, when the services to be provided require, due to their nature or complexity of provision, medical or\/and nurse continuous assistance, not available in the outpatient (ambulatory) setting\u201d (Presidential decree, 20 October 1992). Regarding inpatient care services available in day hospital are not explicitly defined, but rather there are specific criteria for the service to be considered appropriately delivered in this regime. Guidelines are defined at national level and they refer mostly to organizational aspects of services\u2019 provision rather than their specific types (i.e., number of beds assigned for day hospital services must be at least 10% of the total).\nThe situation is very similar as regards day surgery care. National guidelines provide a list of possible interventions that may be performed in day care setting, as an alternative to normal hospital care. The list includes about 780 interventions, itemized by service delivered and grouped according to the organ or system of organs they refer to (e.g., respiratory system interventions, cardiovascular system interventions)\nOutpatient curative care: primary care\nAll patients in Italy are registered with a general practitioner (GP) or a pediatrician who is in charge for providing most primary care, referring to specialists, and prescribing diagnostic interventions and drugs. The citizen\u2019s can freely choose his or her own GP, given the limit of maximum number of enrolled patients. Primary care services provided by GPs are outlined broadly in the National Contract for General Practitioners, which is the most important document regulating various aspect of primary care. The National Contract is a result of negotiations between the government and representatives of general practitioners organized in various trade unions. Once reached, the content of the National Contract is legislated through a Decree approved by the Ministry of Health (i.e., the agreement is a binding by law). Regions are autonomous in establishing further agreements (Accordi Integrativi Regionali) aimed mainly at identifying the most appropriate organizational arrangements for the provision of services set at national level. The regional agreements may define additional services to be provided in primary care. The categories of services that primary care physicians are obliged to provide under the National Contract are defined broadly as: (a) essential services: acute and chronic disease management, in line with best practice indications and in agreement with the patient; (b) health promotion activities; (c) patient management within programmed and integrative domiciliary care coordinated with providers of specialist and rehabilitative care services; and (d) community services defined on the basis of regional agreements. The National Contract also encourages various forms of integration between primary care physicians and district services such as social and home care. Additionally, the National Contract obliges Local Health Units to guarantee continuity of care, i.e., primary care services 24\u00a0h a day, 7\u00a0days a week. Organizational arrangements are decided at regional level.\nOutpatient dental care\nPublic coverage of dental care services has always been a debated issue in the Italian NHS. Current legislation explicitly excludes almost all types of dental services from the nationally defined benefit package. Some limited care is available to special groups of patients defined according to age and to specific clinical conditions. This explicit and rather broad exclusion of dental care services at national level had an impact on regional health policies. Numerous regions have adopted measures to guarantee some types of dental care services to citizens. An example of regionally defined benefit catalogue for dental care services is that in Veneto region, defined by Regional decree 2227\/02. The regional decree defines the list of services available free of charge or subject to copayment for special patient categories. The list of services is available under set conditions only for the residents of Veneto Region, while nonresidents are fully charged. Beneficiaries are identified according to age, income, employment status, and presence of specific disease criteria: (a) dental health care in developmental age (0\u201316\u00a0years): preventive, diagnostic, and curative services for patients under the age of 16\u00a0years, orthodontic care for patients under the age of 12, nonsurgical treatment of paradental pathology for patients under age 16; (b) dental and prosthetic care to very low-income residents (<\u20ac 8,500 per year) affected by chronic (e.g., cardiac insufficiency, psychosis) and rare (e.g., metabolic diseases, immunodeficiency) conditions; and (c) specialist curative care (excluding prosthetics) for anthalgic emergency cases caused by infections of caries and of paradental pathologies of traumatic events.\nSpecialized outpatient care\nSpecialized ambulatory services, including specialist visits, diagnostic, and curative interventions, are provided either by LHUs or by accredited public and private facilities. Patients are allowed to access specialist care only after approval by their general practitioner, who is responsible for the referral. Once the general practitioner has authorized the visit or the procedure, the patient is free to choose any provider among those accredited by the NHS anywhere in Italy. A list of outpatient services, including diagnostic procedures, specialist visits and laboratory tests was drawn up in 1996, and its original purpose was to define reimbursement fees of providers. Since then the national list of services has not been updated. However, regional authorities have often revised their fee schedules. The main criteria including services are effectiveness (based on solid scientific evidence) and costs. The benefits are classified in three different sections: (a) specialist outpatient care (including clinical laboratory and diagnostic imaging) provided under INHS coverage (a positive list of services, explicitly defined and enumerated, mainly without specific link to clinical conditions); (b) specialist services available only for specific indications (positive list of services limited to special patient categories); and (c) specialist outpatient care not covered by the INHS (negative list).\nRegions are free to deliver additional services for which they are financially responsible. These services should be marked separately in the fee schedule and added to the list in accordance with the coding system in place. The positive list of specialist outpatient services is itemized by service delivered. The items (approx. 2,000) are grouped into 16 categories on the basis of system of organs the intervention refers to (e.g., respiratory system interventions). Each category is further divided according to the specific organ (e.g., trachea and larynges interventions). Finally, each subcategory contains a list of specific services (e.g., laryngoscope). Some services in the positive list are limited to special settings (e.g., where special equipments are available). Services that are available only to special patients categories (i.e., limited for specific clinical conditions) include about 20 items, mainly laboratory and diagnostic examinations that are very costly (e.g., positron emission tomography) or in some way controversial (palliative pain treatments).\nAll other outpatient curative care\nNumerous services of physiotherapy are excluded from the national benefit package. Many regions, however, have approved their inclusion in the regional benefits so to generate substantial variability across the country. Lombardy, for example, includes all services listed on the national negative list while Veneto and Friuli Venezia Giulia authorize \u201cwater rehabilitation.\u201d Almost all regions provide anthalgic electrotherapy, ultrasound therapy, mesotherapy, and laser therapy. The national benefit package explicitly excludes all types of alternative and complementary medicine, leaving it to the regions to decide whether to provide some of these services to their citizens. Only 4 of 21 the regions have invested in this category of services: (a) acupuncture is available in Piedmont, Valle d\u2019Aosta, Umbria, and Tuscany; (b) homeopathy is available in Valle d\u2019Aosta (limited to specific clinical conditions); and (c) chiropractic services are available in Valle d\u2019Aosta only for spinal cord pathologies. Spa treatment is available for a limited number of pathologies, identified as those for which thermal treatment may provide actual benefits (based on scientific evidence). The list of pathologies is explicitly defined in a Ministry decree (1994) and includes: rheumatic diseases (e.g., osteoarthrosis and other degenerative forms, extrajoint rheumatisms), respiratory diseases (e.g., chronic pulmonary diseases), and dermatological diseases (e.g., psoriasis).\nDiscussion\nAlmost 40\u00a0year since its inception, the Italian NHS has an explicit system of national services guaranteed to all its citizens. The concept of providing a limited set of services under the INHS is now well established. Entitlements to services in particular areas (mainly outpatient care) are now clearly defined, and some services (e.g., dental and thermal care) are explicitly excluded. In the area of hospital care entitlements remain broad and general, although a strong reference to appropriateness criteria and the use of DRGs contribute to make benefits more explicit in this setting as well.\nAs in other countries, in Italy a clear definition of the benefits provided by the statutory system is thought to be beneficial for several reasons: it can contribute to a better allocation of resources, help to reassure patients about their rights and responsibilities, and facilitate the development of supplementary insurance [9]. In addition, the definition of the health benefit package in Italy is also an essential element in the relationship between the central government and the regions. The system of LEAs is the means to keep management and policy powers at regional level while assuring national guarantees. In this sense the basic package is primarily a policy devise to keep regions accountable to national standards.\nA constitutional reform aimed at a new redistribution of powers between the central and regional authorities is presently under parliamentary discussion. This specifies that powers on health matters are exclusively in the hands of regions, provided that national principles are respected. If this reform is approved, the basic package would gain even more importance. Therefore we foresee the need for substantial investments to further specify the content of the package and, more importantly, to develop adequate monitoring systems and accountability procedures.\nIn our opinion, two issues are particularly critical in this respect. First, the coherence between benefits and resources made available requires an adequate governance system. At present Italy does not have a higher Chamber (it may be introduced in the Constitution reform under discussion) in which regions are represented, and where negotiations between them and the central government can take place within an appropriate institutional framework. At present, the devolution process lacks adequate rules to govern negotiations and conflicts. The risk of institutional conflicts, endless negotiations, legal disputes, and lack of coordination is very real and hitherto affects mainly the health care sector. It should be clear that without adequate governance mechanisms conflicts between the two central and regional authorities and between the regions themselves may result in further acceleration of the fragmentation of the INHS. The second issue concerns an adequate infrastructure to sustain the national government as guarantor of health care rights. Without an appropriate information system and new jurisdictional powers the national tier cannot ensure its guarantor role. To implement effective national guarantees the central government needs to develop the benefit package, to implement an effective monitoring system, and to design appropriate rules to force regions to act adequately. On the other hand, these conditions are also needed to ensure that resources available to the regions are compatible with the cost of the provision of services included in the benefit package [10].\nIt has been suggested that LEAs serve two main policy goals: equality and cost-containment [11]. An explicit definition of the guarantees provided Italians is a major mechanism to promote equality in the access of care, while limiting coverage may be a strong tool to contain costs. In theory the two objectives are compatible as explicitness of coverage can coexist with a different extent of the benefit package. In practice, however, LEAs may results in an overall system to defend the basic principle of the INHS from the risk of poor funding. Should radical policies aimed at reducing the ambitious goals of the INHS prevail, LEAs may become the main pillar of the system.\nA few reflections can be made about the criteria adopted in Italy to build the benefit package. Effectiveness (in most cases efficacy), as confirmed by scientific evidence, is the dominant criterion in defining the package. Need criteria have been also used; ailments for minor conditions (cough, sore throat, minor headache), cosmetic surgery, and ritual circumcisions) are excluded on the basis of various interpretations of the need criteria. In general, clinical and organizational appropriateness is promoted as well. As the former requires that treatments and procedures are applied only to patients with particular clinical conditions, the latter tries to assure that patients are treated in the most adequate (and often cheapest) setting. Overall the use of appropriateness criteria suggests that benefit catalogues should be made of detailed lists of services for particular clinical conditions rather than simple lists of services.","keyphrases":["italy","health services","health benefit plans","health priorities","national health programs"],"prmu":["P","P","R","M","R"]}
{"id":"Clin_Rheumatol-3-1-1847465","title":"Measuring quality of life in rheumatic conditions\n","text":"Musculoskeletal disorders often have associated pain, functional impairment and work disability, and, not surprisingly, are the most common reasons for utilizing healthcare resources. Rheumatoid arthritis (RA) and fibromyalgia (FM) are causes of musculoskeletal pain and disability. Research indicates that there is a widespread impact of RA and FM on physical, psychological and social factors in affected individuals, and thus, outcome measures that encompass multiple aspects of quality of life are needed. Generic measures of quality of life identify associations between physical conditions and mental health and highlight the need to address psychological functioning to ultimately improve the individuals\u2019 quality of life.\nMusculoskeletal disorders often have associated pain, functional impairment and work disability, and, not surprisingly, are the most common reasons for utilizing healthcare resources [1, 2]. Rheumatoid arthritis (RA) and fibromyalgia (FM) are among the most common causes of musculoskeletal pain and disability. They are estimated in community studies to affect 0.5\u20131 and 2\u20136% of the population, respectively [3\u20135]. These two conditions differ in a number of ways: In particular, RA has a clear pathophysiology with a range of clinical measures being able to indicate its presence, whereas the biological basis for FM is less defined with no \u201cgold standard\u201d test available. Despite this difference, these two rheumatic conditions share a range of associated outcomes. These include not only pain and fatigue but also difficulties with activities of daily living, ranging from basic and intermediate functions through to more complex tasks such as paid employment and social roles, e.g. child rearing [2, 6]. Further, RA and FM are associated with poor psychological functioning with 20 to 60% reporting depressive symptoms [7\u20139]. It is clear that there is a widespread impact of RA and FM on physical, psychological and social factors in affected individuals, and thus, outcome measures that encompass multiple aspects of quality of life are needed.\nIn this volume, Birtane et al. [10] study the use of a generic measure of physical and psychological functioning in patients with RA and FM and thus provide insights on the quality of life outcomes in these disorders. Generic and self-report measures of quality of life provide a wealth of information regarding the experience of individuals with rheumatic conditions that medical and observational measures cannot capture [11]. One such measure is the Medical Outcomes Survey 36 Items (SF-36) [12], which is a self-report multidimensional measure of quality of life that has been used extensively in RA and FM research [13, 14]. The SF-36 includes subscales across eight dimensions of health and well-being, including physical functioning, role physical, role emotional, bodily pain, general health, vitality, social functioning and mental health. Scores for all subscales are expressed on a scale of 0 to 100, with a higher score indicating a better state of health. It has good reliability and validity for both clinical and healthy community-based samples [15].\nA generic measure of quality of life can offer a number of insights into the impact of rheumatic conditions on everyday functioning. The SF-36 provides the opportunity for interesting comparisons of the quality of life between healthy individuals and those with rheumatic conditions and other chronic physical conditions. Studies indicate that those with RA and FM, almost invariably, have reduced or compromised quality of life compared to healthy age-matched controls. Both RA and FM have a similar impact on psychological and physical functioning and social roles as other chronic conditions that contribute to the global burden of diseases, such as heart disease, chronic obstructive pulmonary disease and diabetes [10, 16, 17]. These findings have implications on health policy and allocation of funding for both healthcare and research. For instance, musculoskeletal conditions affect more than 17% of the community and 60% of working-aged adults, with projections indicating that arthritis will affect up to 20% of people by 2020 [18]. The prevalence, cost and burden of musculoskeletal disorders indicate that policymakers need to address the impact on economies, healthcare systems and society at large. For instance, if a public health intervention in 2005 enabled the onset of arthritis to be delayed by 10\u00a0years, the age-related incidence rates would be reduced by 11.1% before 2020 [18]. The burden of musculoskeletal disorders and issues of impaired quality of life have become an international health priority with initiatives such as the Bone and Joint Decade launched by the World Health Organization (WHO).\nBirtane\u2019s use of a generic measure of quality of life highlights the fact that FM, a condition with unclear pathophysiology, is associated with impaired quality of life to a similar extent as RA. This is in keeping with the recent models of quality of life or disability that place less emphasis on disease processes, pathophysiology and\/or structural damage. Previous models of outcomes for rheumatic conditions have a strong biomedical focus with many models of disability or quality of life having an implicit assumption that there is a linear relationship between disease processes and disability. Attempting to use such models of disability by reading from left to right has obvious shortcomings because there are many instances such as FM that do not have a fully identifiable disease process [19]. Indeed, focusing on pathophysiology and functional ability in a limited context without consideration of the individual\u2019s everyday tasks and roles sheds little light on the quality of life [20].\nNewer approaches to examining disability acknowledge the roles of demographic, physiological, psychological, social and environmental factors acting as buffers and exacerbators of poor outcomes for chronic physical conditions [2]. The International Classification of Functioning (ICF) promoted by the WHO incorporates a broader biopsychosocial framework of disability, health and health-related states [21]. Indeed, the ICF takes a neutral stand with regard to aetiology and allows researchers to arrive at causal inferences using appropriate scientific methods. The ICF attempts to dislodge assumptions that the body level dysfunctioning, classified as impairments, is the fundamental dimension of disability and quality of life [21\u201323]. In other words, the quality of life in both RA and FM is not contingent on aetiology. The presence of a disability implies a cause, but the cause may not be sufficient to explain the resulting quality of life [21, 24, 25]. Measures such as the SF-36 provide the building blocks to create models of quality of life and study the different perspectives of this process across a range of chronic physical conditions as well as healthy individuals. Within this framework, using generic measures of quality of life can offer opportunities to frame research and interventions that appropriately target the quality of life of individuals with musculoskeletal disorders.\nUsing this approach, Birtane et al. [10] identified in patients with RA and FM that the incidence of poor physical and psychological functioning is almost invariably greater than for healthy controls. Additionally, higher depression levels are evident in patients with FM compared to those with RA. It is not surprising that FM is associated with the worst psychological functioning, given that depression is often evident in individuals with FM [26]. These observations are consistent with previous findings, which indicate that significant depressive symptoms are reported by up to 60% of individuals with chronic physical conditions. Addressing poor psychological functioning, especially depression, in musculoskeletal conditions is critical due to its impact on the condition itself. Reciprocal relationships can occur between depression and pain and disability leading to a cycle of poor mental and physical health. Depression is related to poor treatment compliance and increased morbidity and mortality in rheumatic conditions [27]. Measures such as the SF-36 clearly identify the associations between chronic physical conditions and mental health and further highlight the importance of addressing psychological functioning in both research and interventions to ultimately improve the individuals\u2019 quality of life.\nRA and FM are of course not mutually exclusive. The pathophysiology of each disorder is different, and many patients with RA also have FM, as do many other patients with a variety of chronic rheumatic diseases. The substrate of FM lies in the pain system, which can be functionally activated by distress, a common feature of chronic painful and disabling disorders. How much of the FM mechanism contributes to the low quality of life of RA? Further exploration of the mechanisms causing FM, with its resultant highly significant effect on the quality of life, will have a great impact on the management of not only FM itself but also RA and related musculoskeletal conditions.","keyphrases":["quality of life","disability","rheumatoid arthritis","fibromyalgia"],"prmu":["P","P","P","P"]}
{"id":"Plant_Cell_Rep-4-1-2413084","title":"Arabidopsis R2R3-MYB transcription factor AtMYB60 functions as a transcriptional repressor of anthocyanin biosynthesis in lettuce (Lactuca sativa)\n","text":"The MYB transcription factors play important roles in the regulation of many secondary metabolites at the transcriptional level. We evaluated the possible roles of the Arabidopsis R2R3-MYB transcription factors in flavonoid biosynthesis because they are induced by UV-B irradiation but their associated phenotypes are largely unexplored. We isolated their genes by RACE-PCR, and performed transgenic approach and metabolite analyses in lettuce (Lactuca sativa). We found that one member of this protein family, AtMYB60, inhibits anthocyanin biosynthesis in the lettuce plant. Wild-type lettuce normally accumulates anthocyanin, predominantly cyanidin and traces of delphinidin, and develops a red pigmentation. However, the production and accumulation of anthocyanin pigments in AtMYB60-overexpressing lettuce was inhibited. Using RT-PCR analysis, we also identified the complete absence or reduction of dihydroflavonol 4-reductase (DFR) transcripts in AtMYB60- overexpressing lettuce (AtMYB60-117 and AtMYB60-112 lines). The correlation between the overexpression of AtMYB60 and the inhibition of anthocyanin accumulation suggests that the transcription factorAtMYB60 controls anthocyanin biosynthesis in the lettuce leaf. Clarification of the roles of the AtMYB60 transcription factor will facilitate further studies and provide genetic tools to better understand the regulation in plants of the genes controlled by the MYB-type transcription factors. Furthermore, the characterization of AtMYB60 has implications for the development of new varieties of lettuce and other commercially important plants with metabolic engineering approaches.\nIntroduction\nFlavonoid metabolism is unique to plants and produces a great number and variety of compounds including flavones, flavonols, anthocyanins, and proanthocyanins (Winkel-Shirley 2001; Park et al. 2004). These molecules play important roles in many fundamental processes in plants, such as the pigmentation of flowers and fruits, UV resistance, pollen fertility, responses to wounding and fungal attacks, and environmental stress responses (Shirley et al. 1995; Weisshaar and Jenkins 1998; Harborne and Williams 2000; Dixon and Piava 1995; Moyano et al. 1996; Pietta 2000; Cominelli et al. 2005). The regulation of the anthocyanin pigments in plants is particularly important in metabolic engineering applications because these compounds act as visual signals that attract the insects and animals required for pollination and seed dispersal (Holton and Cornish 1995).\nThe metabolic pathways involved in anthocyanin biosynthesis are well established, and the central pathways in this process are highly conserved in plants (Shih et al. 2006). Moreover, studies in model plants, such as Arabidopsis and maize, have facilitated a sound understanding of the genes involved and the assembly of the relevant enzyme complexes (Winkel-Shirley 2001). Two groups of genes are required for anthocyanin biosynthesis: structural genes that encode the enzymes that directly participate in the biosynthetic reactions, and genes for transcription factors that regulate the expression of these structural genes and the accumulation of flavonoid metabolites. Transcription factors can act as activators or repressors of gene expression, and mediate either increases or decreases in transcription through sequence-specific DNA binding and protein-protein interactions (Broun 2004).\nMYB proteins have been identified in a large number of eukaryotes, including fungi, plants and vertebrates (Ohi et al. 1994; Martin and Paz-Ares 1997; Slamon et al. 1986), and have roles in a wide array of cellular processes. These include the regulation of secondary metabolism, signal transduction, cell division, and responses to plant diseases and various forms of stress (UV-B light, cold and drought) (Larkin et al. 1994; Borevitz et al. 2000; Stracke et al. 2001; Vailleau et al. 2002; Cominelli et al. 2005). The R2R3-MYB-related proteins activate the transcription of structural genes that function in different branches of phenylpropanoid metabolism (Martin and Paz-Ares 1997). The promoters of these structural genes each contain potential MYB protein recognition motifs and also bind with the basic helix-loop-helix (bHLH) domain and conserved WD40 repeat proteins (Park et al. 2007a, b; Martin and Paz-Ares 1997; Winkel-Shirley 2001).\nThe classification of MYB transcription factors is based on the strong conservation of imperfect repeats in the MYB DNA-binding domain of the member proteins (Martin and Paz-Ares 1997). In contrast to animals (R1R2R3), the MYB domain transcription factors found in plants are characterized by the R2R3-type MYB domain, comprising a DNA-binding domain and an activation\/repression domain. Moreover, the balance between activators and repressors in this transcription factor family in plants may provide extra flexibility in terms of transcriptional control (Jin et al. 2000). There have been 126 R2R3 MYB genes identified in Arabidopsis thaliana, all of which exhibit strong homology within their N-terminal MYB DNA-binding domains and can be divided into 24 subgroups on the basis of their sequences (Stracke et al. 2001; Riechmann and Ratcliffe 2000; Kranz et al. 1998). To determine the biological roles of the MYB-related transcription factors in the production of phenylpropanoids, we isolated six R2R3-MYB proteins from Arabidopsis based on available information indicating that these genes are induced by environmental stress (UV-B irradiation) (Kranz et al. 1998). However, they do not have an informative phenotype, because of the structural and functional redundancy among these factors (Bouche and Bouchez 2006). Furthermore, although there have been other studies of anthocyanin accumulation in lettuce, one of the most popular and commercially important vegetables in the world, this process has not been characterized before at the level of transcriptional regulation. The regulation of anthocyanin accumulation in plants is also a key issue in our understanding of the regulation of leaf color. The identification of the factors that exert this control will provide valuable tools to moderate the extent and distribution of anthocyanin-derived pigmentation in plant tissues.\nIn this study, we overexpressed the AtMYB60 gene in lettuce plants (\u201cJinjachuckmyun\u201d cultivar) that are highly pigmented with a red color throughout their leaf tissues. Because the loss of these pigments is easily detected, the activity and repression of the MYB-type transcription factors that affect these pathways can be investigated. In the leaves of lettuce, coordinated transcriptional regulation controls virtually each step of the anthocyanin biosynthetic pathway (Park et al. 2007a). We compared the expression of the major anthocyanin biosynthetic genes and the AtMYB60 gene by RT-PCR. We show that this transcription factor is functionally active in repressing anthocyanin accumulation and can thus generate green lettuce leaves. We also show, by functionally repressing the accumulation of this compound, that AtMYB60 plays a significant role in controlling anthocyanin biosynthesis via the inhibition of a key target gene encoding dihydroflavonol reductase (DFR). Thus, we report here for the first time that anthocyanin biosynthesis and the transcription of the DFR gene are repressed through the overexpression of Arabidopsis R2R3-MYB transcription factors in transgenic lettuce plants. We also discuss the function of transcriptional regulators in the control of the expression of the structural protein that are required for anthocyanin biosynthesis in lettuce.\nMaterials and methods\nPlant materials and growth condition\nTo investigate the effects of exogenous AtMYB proteins on anthocyanin biosynthesis in lettuce leaves at the molecular level, Jinjachuckmyun plants, supplied by Syngenta (Seoul, Korea), were used. The plants were grown at the National Institute of Agricultural Biotechnology in Suwon, South Korea. Fresh lettuce leaves were plucked from the plants, frozen immediately in liquid nitrogen and stored at \u221280\u00b0C until required.\nIsolation of AtMYB cDNAs\nTo isolate the AtMYB genes, cDNAs were isolated from UV-B-irradiated Arabidopsis plants using the BD SMART RACE cDNA Amplification Kit (Clontech, USA) and then used as templates for PCR cloning. The following gene-specific primers were used for RACE-PCR: AtMYB4 (forward: 5\u2032-GTATGGGAAGGTCACCGTGCTGTGAGAAA-3\u2032; reverse: 5\u2032-TATTATTTCATCTCCAAGCTTCGAAAGCC-3\u2032), AtMYB29 (forward: 5\u2032-AAATGTCAAGAAAGCCATGTTGTGTGGGA-3\u2032; reverse: 5\u2032-GATCATATGAAGTTCTTGTCGTCATAATC-3\u2032), AtMYB30 (forward: 5\u2032-TAATGGTGAGGCCTCCTTGTTGTGACAAA-3\u2032; reverse: 5\u2032-CTTCAGAAGAAATTAGTGTTTTCATCCAA-3\u2032), AtMYB34 (forward: 5\u2032-AGATGGTGAGGACACCATGTTGCAAAGAA-3\u2032; reverse: 5\u2032-CGTCAGACAAAGACTCCAACCATATTGTC-3\u2032), AtMYB51 (forward: 5\u2032-GAATGGTGCGGACACCGTGTTGCAAAGCC-3\u2032; reverse: 5\u2032-ACTCATCCAAAATAGTTATCAATTTCGTC-3\u2032) and AtMYB60 (forward: 5\u2032-AGATGGGTAGGCCTCCATGCTGTGACAAG-3\u2032; reverse: 5\u2032-AATTAAAGCATATTAGAGAGCTCCATCAA-3\u2032). The resulting PCR products were then cloned into the pGEM-T Easy vector (Promega, USA) and sequenced on both strands.\nVector construction\nAtMYB coding regions were cloned into an expression vector containing the CaMV 35S promoter and the nopaline synthase 3\u2032 terminator. The resulting constructs were then introduced into the binary vector 22103 (a derivative of pCAMBIA3301; Cambia, Australia). The complete coding sequence of each AtMYB cDNA was amplified with a specific forward primer designed to introduce an MluI restriction site and a reverse primer designed to introduce an XbaI restriction site to allow subcloning, as follows: AtMYB4 (forward: 5\u2032-ATACGCGTATGGGAAGGTCACCGTGCTGTG-3\u2032; reverse: 5\u2032-TATCT AGATTATTTCATCTCCAAGCTTCGA-3\u2032), AtMYB29 (forward: 5\u2032-ATACGCGTATGTCAAGAAAGCCATGTTGTG-3\u2032; reverse: 5\u2032-TATCTAGATCATATGAAGTTCTTGTCGTCA-3\u2032), AtMYB30 (forward: 5\u2032-ATACGCGTATGGTGAGGCCTCCTTGTTGTG-3\u2032; reverse: 5\u2032-TATCTAGATCAGAAGAAATTAGTGTTTTCA-3\u2032), AtMYB34 (forward: 5\u2032- ATACGCGTATGGTGAGGACACCATGTTGCA-3\u2032; reverse: 5\u2032-TATCTAGATCAGACAAAGACTCCAACCATA-3\u2032), AtMYB51 (forward: 5\u2032-ATACGCGTATGGTGCGGACACCGTGTTGCA-3\u2032; reverse:5\u2032-TATCTAGATCATCCAAAATAGTTATCAATT-3\u2032), AtMYB60 (forward: 5\u2032-ATACGCGTATGGGTAGGCCTCCATGCTGTG-3\u2032; reverse: 5\u2032-TATCTAGATTAAAGCATATTAGAGAGCTCC-3\u2032). After PCR and digestion, the MluI\/XbaI fragments were cloned into the binary vector 22103 between CaMV 35S promoter and the nopaline synthase poly(A) addition site, creating the binary vector 22103-AtMYB plasmids (Fig.\u00a02). These constructs were then introduced into the Agrobacteriumtumefaciens EHA105 strain using the freeze\u2013thaw method.\nGeneration of AtMYB transgenic lettuce\nTransgenic lettuce plants were generated using the Agrobacterium-mediated transformation of cotyledons. Lettuce seeds of the Jinjachuckmyun cultivar were supplied by Syngenta (Korea) and were surface sterilized in 25% (v\/v) bleach for 7\u00a0min, washed (three changes) in sterile water, and placed on phytogel-solidified (0.2%, w\/v) MS medium at pH\u00a05.8. The seeds were germinated and maintained at 25\u00b0C (16\u00a0h photoperiod, 200\u00a0\u03bcmol\u00a0m\u22122\u00a0s\u22121, daylight fluorescent tubes). The cotyledons were excised from the 4-day-old lettuce seedling and inoculated with freshly grown A. tumefaciens. The inoculated cotyledons were then co-cultivated on MS salt medium solidified with 0.2% purified phytogel (Sigma, USA), for 2\u00a0days at 24\u00b0C in the dark. The cotyledon explants were then transferred to shoot initiation medium supplemented with 400\u00a0mg\u00a0l\u22121 carbenicillin (Duchefa, the Netherlands). After a washing step, these explants were cultured (ten per plate) on selection medium (MS salt 4.43\u00a0g\u00a0l\u22121, sucrose 30\u00a0g\u00a0l\u22121, phytogel 2\u00a0g\u00a0l\u22121, kinetin 0.5\u00a0mg\u00a0l\u22121, carbenicillin 400\u00a0mg\u00a0l\u22121, phosphinothricin 1.0\u00a0mg\u00a0l\u22121 and cysteine 10\u00a0mg\u00a0l\u22121) and subcultured every 2\u00a0weeks on the same medium. Shoots that regenerated from explants on medium containing phosphinothricin were rooted in rooting medium (MS salt 4.43\u00a0g\u00a0l\u22121, sucrose 30\u00a0g\u00a0l\u22121, phytogel 2\u00a0g\u00a0l\u22121 and phosphinothricin 1.0\u00a0mg\u00a0l\u22121), before transfer to the greenhouse, where they were allowed to self-pollinate and to set seed. The seeds were harvested and stored at 4\u00b0C.\nAnthocyanin extraction and HPLC analysis\nAnthocyanin in the lettuce plants was quantified by HPLC at a detection wavelength of 510\u00a0nm, comparing the sample retention times and peaks with those of known standards. The relative quantification of each phenolic compound samples was expressed as the equivalent quantity of purified standards for each treatment.\nAbout 0.2\u00a0g of ground and freeze-dried leaves was accurately weighed and extracted with 1\u00a0ml of a solution of methanol:water (80:20, v\/v) at 80\u00b0C for 1\u00a0h, then sonicated for 20\u00a0min. The extracted solution was filtered into a 50-ml volumetric flask, and the flask and filter were rinsed with a solution of methanol:water (80:20, v\/v). The filtrate was then made up to the required volume with the same solvent. Approximately 1\u00a0mL of the sample solution was passed through a 0.4\u00a0\u03bcm filter before analysis by HPLC. A Shimadzu HPLC system with 10AD dual pumps was used, with an RP18 (LiChrospher, 250\u00a0mm\u00a0\u00d7\u00a04\u00a0mm\u00a0\u00d7\u00a05\u00a0\u03bcm) column. The HPLC parameters were as follows: column temperature 30\u00b0C, solvent A\u00a0=\u00a00.1% trifluoroacetic acid in water, solvent B\u00a0=\u00a098% acetonitrile with 0.1% trifluoroacetic acid, solvent gradient, 0\u00a0min\u00a0=\u00a00% B, 3\u00a0min\u00a0=\u00a06% B 12\u00a0min\u00a0=\u00a018% B, 25\u00a0min\u00a0=\u00a025% B, 35\u00a0min\u00a0=\u00a0100% B, 40\u00a0min\u00a0=\u00a0100% B. The flow rate was 0.35\u00a0mL\u00a0min\u22121. The peaks were classified as either cyanidin or delphinidin derivatives by UV spectral analysis.\nRT-PCR analysis\nTotal RNA from the transgenic and wild-type lettuce leaves was extracted using RNeasy Plant Mini Kit (Qiagen, USA), according to the manufacturer\u2019s instructions. To estimate the transcript levels of the exogenous AtMYB4 and AtMYB60 genes in lettuce, we used the primers 5\u2032-ATGGGAAGATCGCCTTGTTGTGAA-3\u2032 (F) and 5\u2032-TCATTTCATCTCTAAGCTTCTGTAGTCCAAAA-3\u2032 (R) for AtMYB4 and 5\u2032-ATGGGGAGGCCTCCTTGTTGTG-3\u2032 (F) and 5\u2032-TCAATTATCGAAAAAATTAGGGTTTTCATCA-3\u2032 (R) for AtMYB60. The mRNA expression patterns of the CHS, F3H, DFR and UFGT genes were analyzed by RT-PCR with the All-in-one RT\/PCR Premix (SuperBio P7003, Suwon, Korea). The amplification of rRNA was used as the internal control. Total RNA (100\u00a0ng) in a volume of 20\u00a0\u03bcL containing 20\u00a0mM Tris\u2013HCl (pH 7.9), 100\u00a0mM KCl, 0.1\u00a0mM EDTA, 1\u00a0mM DTT, M-MLV reverse transcriptase (RNaseH Minus), Super Taq Plus DNA polymerase and 10\u00a0pmol of each gene-specific amplification primer was used for RT-PCR, according to the manufacturer\u2019s instructions. Gene-specific primers for CHS (forward: 5\u2032-GGTTTGCTCTGAGATTACAGCGGTTACC-3\u2032, reverse: 5\u2032-TCCTTGAGACCAAGCTTGAGCTCCACCT-3\u2032), F3H (forward: 5\u2032-GAGATCTTATCAGAGGCAATGGGCCTTG-3\u2032, reverse: 5\u2032-ACAACGGCCCGATGGTCTGCGTTCTTAA-3\u2032), DFR (forward: 5\u2032-GGTCTATGACGAGTCTCATTGGAGCGAT-3\u2032, reverse: 5\u2032-CTATCAATTGCTCCTTTGAACATCTCCT-3\u2032), UFGT (forward: 5\u2032-TGGAGAAGCGGGTTAGACAGTTGATGGA-3\u2032, reverse: 5\u2032-TATAGCTACCATGATTCAACCAACTTCG-3\u2032), rRNA (forward: 5\u2032-TACGGCACTGAAGGTGCCAAGCTCGTG-3\u2032, reverse: 5\u2032-CATCCTCTTGGCAGTCTTGGCGTAGGG-3\u2032) and bar (forward 5\u2032-GCCGCAGGAACCGCAGGAGT-3\u2032, reverse: 5\u2032-AGCCCGATGACAGCGACCAC-3\u2032) were used to confirm the expression levels of these genes in the lettuce plants. Reverse transcription of the total RNA was carried out at 50\u00b0C for 30\u00a0min, and the reaction was inactivated at 96\u00b0C for 3\u00a0min. The amplification conditions comprised 35 cycles of denaturation at 94\u00b0C for 30\u00a0s, annealing at 60\u00b0C for 30\u00a0s, extension at 72\u00b0C for 2\u00a0min, and a final extension at 72\u00b0C for 10\u00a0min. PCR was performed using a GeneAmp PCR system 9700 Cycler (Perkin-Elmer, MA, USA). The resultant RT-PCR products were resolved on a 1% agarose gel, stained with ethidium bromide, and photographed. The amplified products of the CHS, F3H, DFR, UFGT, rRNA and bar transcripts were 398, 319, 534, 153, 386 and 267\u00a0bp, respectively.\nResults\nCloning of MYB-type transcription factor genes from Arabidopsis\nTo isolate and characterize the MYB-type transcription factors in Arabidopsis (AtMYB) that play a role in phenylpropanoid metabolism, primers were designed to amplify the entire coding regions of the AtMYB genes that are induced by environmental stress (UV-B irradiation) (Meissner et al. 1999). Previous studies of Arabidopsis have suggested that the R2R3-MYB genes of this plant have important functions in the regulation of secondary metabolism, disease resistance, and hormonal responses (Kranz et al. 1998). We isolated six Arabidopsis R2R3-MYB transcription factor genes based on available information concerning the AtMYB genes induced by UV-B irradiation (Kranz et al. 1998). These were AtMYB4 (AF062860, AT4G38620), AtMYB29 (AF062872, At5G07690), AtMYB30 (AF062873, AT3G28910), AtMYB34 (U66462, AT5G60890), AtMYB51 (AF062887, AT1G18570) and AtMYB60 (AF062895, AT1G08810), with apparent full-length cDNAs of 849, 1011, 972, 885, 1059 and 843\u00a0bp, respectively, encoding proteins of 283, 337, 324, 295, 353 and 281 amino acids, respectively (Fig.\u00a01). The amino termini of these proteins contain the R2R3 repeats responsible for their binding to target DNA sequences and which are highly conserved among the R2R3-MYB proteins.\nFig.\u00a01Multiple alignment of AtMYB proteins. Amino acids identical in all six proteins are marked in black, amino acids found in four proteins are marked in gray. The line above indicates the R2 and R3 MYB repeats. GenBank accession numbers for the sequences: AtMYB4 (AF062860), AtMYB29 (AF062872), AtMYB30 (AF062873), AtMYB34 (U66462), AtMYB51 (AF062887) and AtMYB60 (AF062895)\nExpression of AtMYB genes in lettuce plants\nTo examine the effects of the six isolated AtMYB genes on leaf color changes and on the anthocyanin composition of lettuce, cotyledons of the cultivar \u201cJinjachuckmyun\u201d were transformed using the Agrobacterium tumefaciens strain EHA105 with the binary vector 22103-AtMYB (a derivative of pCAMBIA3301; Fig.\u00a02), carrying these genes and a barstar selection marker. Shoots were regenerated from the callus of phosphinothricin-resistant transformatants. To confirm the introduction and expression of the exogenous AtMYB genes in the Jinjachuckmyun cultivars, total RNA was extracted from the leaves of six-week-old wild-type and transformed lettuce plants (T1 generation) and analyzed by RT-PCR.\nFig.\u00a02Schematic diagram of the T-DNA region of the 22103-AtMYB binary vector used in the transformation of lettuce cultivars. LB left border, RB right border, P35S Cauliflower mosaic virus 35S RNA promoter, PolyA Cauliflower mosaic virus 3\u2032UTR terminator, Tnos nopaline synthase terminator\nTransgenic plants were confirmed by their resistance to a barstar spray and by subsequent RT-PCR with specific primers for six AtMYB genes. Because the T1 plants of the AtMYB4, AtMYB29, AtMYB30, AtMYB34 and AtMYB51 transformants were similar in leaf color and in the anthocyanin accumulation in their leaves, the AtMYB4 transformant (AtMYB4-101) and the AtMYB60 transformant (AtMYB60-117 and AtMYB60-112) were selected for further RT-PCR analysis to detect the expression of the structural genes that are involved in anthocyanin biosynthesis (Fig.\u00a03). RT-PCR analysis was performed using six-week-old lettuce leaves of wild-type and transgenic lettuce plants. The wild-type lettuce plants had no integrated bar gene whereas each of the transgenic plants expressed bar mRNA. Analysis of the exogenously expressed AtMYB4 and AtMYB60 genes showed that AtMYB4 was specifically expressed in AtMYB4-101 and AtMYB60 in AtMYB60-117 and AtMYB60-112. Ribosomal RNA levels were used as a normalization control.\nFig.\u00a03RT-PCR analysis confirming the transcription of the AtMYB4, AtMYB60 and Bar genes in the indicated transgenic lettuce lines and not in the wild-type (WT) plants. rRNA was used as the control\nPlant morphology in AtMYB-transformed lettuce\nIn our previous study, we demonstrated that the wild-type lettuce leaf accumulates anthocyanin, with cyanidin and delphinidin representing the major and minor components, respectively (Park et al. 2007a). To further characterize the functions of the AtMYB genes in anthocyanin biosynthesis, we compared the morphologies of transgenic lettuce plants overexpressing these genes with that of wild-type lettuce.\nMore than 20 independent transgenic plants, selected with barstar spray and subsequently confirmed by PCR analysis (data not shown), were obtained with each A. tumefaciens strain. There was a large variation in the leaf color phenotypes of these transgenic lines. The AtMYB4-101 line showed normal anthocyanin accumulation and a red coloration phenotype, similar to that of the wild-type, but the AtMYB60-117 line showed different levels and patterns of anthocyanin accumulation. Under standard growth conditions, transgenic lettuce plants of the T1 generation that overexpressed AtMYB60 under the control of the cauliflower mosaic virus (CaMV) 35S promoter showed an inhibition (AtMYB60-117) of anthocyanin accumulation (green leaves), compared with that of wild-type plants (red leaves) (Fig.\u00a04a, b). Apart from this inhibition of anthocyanin biosynthesis, no other morphological or developmental abnormalities were detected in this line under standard growth conditions. These results confirm that the exogenous AtMYB60 protein was responsible for the repressed anthocyanin phenotype.\nFig.\u00a04Morphological analysis showing the inhibition of anthocyanin biosynthesis in lettuce leaves, indicated by the lack of red pigmentation, following the introduction of the Arabidopsis MYB gene, AtMYB60: a young (20\u00a0days after sowing) wild-type (WT) lettuce and the AtMYB60 transgenic lettuce line, AtMYB60-117; b morphologies of both the WT and AtMYB-overexpressing lettuce plants at a later stage of growth. Both the AtMYB4-101 and AtMYB60-117 transgenic plants are shown and reveal that AtMYB4 does not inhibit anthocyanin production\nHigh-performance liquid chromatography (HPLC) analysis of AtMYB60 transgenic lettuce shows that accumulation of anthocyanin compounds is inhibited\nTo confirm the identity of the anthocyanins that are synthesized in lettuce after transformation with AtMYB genes, lettuce leaf tissues were harvested and analyzed by HPLC for the presence of soluble anthocyanins. The results confirmed the predominance of cyanidin in the wild-type lettuce leaves, with traces of delphinidin also evident, as we previously described (Park et al. 2007a). The cyanidin and delphinidin peaks were detected in the AtMYB4-overexpressing lettuce (AtMYB4-101) and wild-type lettuce. The AtMYB29-, AtMYB30-, AtMYB34- and AtMYB51-overexpressing lettuce plants that showed a similar red-colored phenotype also produced corresponding cyanidin and delphinidin peaks on HPLC analysis (data not shown). However, clear differences were evident in the patterns of anthocyanin accumulation in the AtMYB60-overexpressing lettuce plants (Fig.\u00a05). With HPLC, we found that the cyanidin (red pigment) peak was absent or sharply reduced in AtMYB60-117 and AtMYB60-112. The delphinidin (blue pigment) peak was absent in every AtMYB60-overpxpressing lines. Hence, the anthocyanin derivatives detected in lettuce leaves by HPLC can be correlated with the morphologies of the plants and with the inhibition of anthocyanin biosynthesis by the AtMYB60 protein.\nFig.\u00a05HPLC analysis of methanolic extracts from both wild-type control (WT) and AtMYB transgenic lettuce leaves (AtMYB4-101, AtMYB60-117 and AtMYB60-112). HPLC chromatograms were recorded at 510\u00a0nm. Peak C corresponds to cyanidin and D represents delphinidin. Each chromatogram was generated using the same quantity of injected sample\nRNA expression profiles of anthocyanin biosynthetic enzymes suggest their coordinated regulation in AtMYB60 transgenic lettuce plants\nBased on the correlation between the plant color morphology and our HPLC analysis of the anthocyanins, we undertook to verify the effects of the AtMYB proteins on the expression of the structural genes encoding the enzymes responsible for anthocyanin biosynthesis. As shown in Figs.\u00a04 and 6, a dramatic leaf color change was evident in the AtMYB60-117 (green leaf) and -112 (mosaic phenotype) lines, in which anthocyanin accumulation is inhibited, and these plants showed a strong green leaf color phenotype. The enzymes that act in the anthocyanin biosynthetic pathway have been well characterized in lettuce in our previous study (Park et al. 2007a), in which we also analyzed the expression of the structural genes encoding the relevant enzymes. Following environmental stimuli such as UV-B irradiation, the expression of chalcone synthase (CHS), flavanone 3-hydroxylase (F3H) and dihydroflavonol 4-reductase (DFR) is positively induced, and the accumulation of anthocyanin is increased in lettuce. We also performed RT-PCR analysis using gene-specific primers in that study to analyze the expression of these structural genes. They were identified from young UV-B-irradiated lettuce leaves and designated as CHS (CV700441), F3H (CV700152), DFR (CV700105) and UFGT (CV700246) (Park et al. 2007a). The transcript levels of these four anthocyanin biosynthetic genes were compared between the wild-type, AtMYB4- and AtMYB60-overexpressing lettuce plants in the present study.\nFig.\u00a06RT-PCR analysis of the genes involved in anthocyanin biosynthesis in the leaves of wild-type and AtMYB4- and AtMYB60-overexpressing lettuce plants. The transcripts of four biosynthetic genes involved in general anthocyanin metabolism (CHS, F3H, DFR and UFGT) were analyzed by RT-PCR in the AtMYB4-101 and AtMYB60-117 transgenic lettuce lines and were compared with the wild-type expression patterns. rRNA was used as the quantitative control\nTotal RNA extracts were prepared from the leaves of six-week-old lettuce plants of wild-type, AtMYB4-101 and AtMYB60-117 lines and were analyzed by RT-PCR for the expression of the previously determined structural genes that are involved in the major biochemical pathways of anthocyanin biosynthesis (Fig.\u00a06b). No reduction in the expression of any of these genes was observed in wild-type or AtMYB4-101 lines, both of which exhibited red coloration in their leaves. Moreover, the mRNA levels of the CHS, F3H and UFGT genes, which catalyze the synthesis of chalcones, flavanones and anthocyanin, respectively, were unchanged in any of the plants. In contrast, the mRNA levels of DFR, the production of which catalyzes the conversion of dihydroquercetin and dihydrokaempferol to leucocyanidin and leucopelargonidin (of the leucoanthocyanidins), respectively, was inhibited in the AtMYB60-117 line, which exhibits green-colored leaves with no red pigmentation.\nDiscussion\nTo isolate the MYB-type transcription factor genes involved in phenylpropanoid metabolism, the transgenic expression of six AtMYB genes was analyzed in lettuce. To date, a large number of transcription factors containing DNA-binding domains that are similar to those of the MYB proteins have been identified across the eukaryotic kingdom (Kranz et al. 1998). Several MYB transcription factors are known to regulate phenylpropanoid metabolism, and the quantities of the various end-products of the associated pathways that accumulate in specific cells, tissues and organs are thought to respresent the balanced expression of several interacting MYB proteins (Tamagnone et al. 1998; Espley et al. 2007). Recently, biochemical studies have also suggested that the MYB-type transcription factors are involved in regulating the branches of the phenylpropanoid metabolic networks in higher plants. Multiple MYBs are also thought to regulate the expression of the structural proteins that drive several downstream pathways of flavonoid metabolism (Moyano et al. 1996). A previous study has also shown that MYB proteins can regulate flavonoid biosynthesis via the transcriptional regulation of the gene encoding CHS, an enzyme that catalyzes the first committed step in flavonoid biosynthesis (Feldbrugge et al. 1997).\nThe lettuce cultivar \u201cJinjachuckmyun\u201d, which we used in our current transformation experiments, shows anthocyanin pigment accumulation in its leaves during development. We have shown in a recent study that environmental stresses upregulate the expression of the genes that encode anthocyanin biosynthetic enzymes in this same lettuce variety, as demonstrated by their responsiveness to UV-B irradiation (Park et al. 2007a).\nThe lettuce has merit as a model plant system to further characterize the functions of those gene products in anthocyanin metabolism, because of its leaf color is red under field conditions and green under chamber condition when exposed to UV-B irradiation. The findings described in this study further validate the use of lettuce as a model plant because it offers experimental versatility in terms of its genetic regulation of the inhibition of leaf pigmentation. The function of the MYB proteins as activators (green to red in a growth chamber) or repressors (red to green in the field) of anthocyanin biosynthesis can be characterized directly from a phenotypic analysis of the lettuce leaf color. This system has potential applications as a research tool in a number of areas of plant molecular biology.\nWe show in our present experiments that the anthocyanin accumulation in leaves is strongly inhibited in AtMYB60 transgenic plants compared with that in wild-type plants (Fig.\u00a04a, b). These results suggest that AtMYB60 functions as a repressor of anthocyanin biosynthesis. Based upon our current findings of the inhibition of anthocyanin biosynthesis and our metabolite analysis of AtMYB60-overexpressing lettuce plants compared with the wild-type, we speculate that the synthesis of anthocyanin is principally controlled by the AtMYB60 transcription factor. The production of anthocyanin is inhibited in the AtMYB60-117 and AtMYB60-112 lines, and this is likely to underlie the green leaf phenotype of these plants. Hence, this characterization of the function of AtMYB60 represents a crucial step in furthering our understanding of the molecular regulation of anthocyanin biosynthesis and the transcription of related genes in lettuce and other plants. It is noteworthy in this context that MYB-related proteins generally act as transcriptional activators (Foos et al. 1994).\nThe R2R3-MYB-related proteins have so far been shown to activate the transcription of structural proteins that act in different branches of the phenylpropanoid metabolism machinery (Martin and Paz-Ares 1997) or that play roles in the plant response to stress signals (Cominelli et al. 2005). Anthocyanin expression in maize is dependent on the presence of both the MYB and MYC types of regulatory factors. It is of some interest, therefore, that in this study, the expression of AtMYB60 alone, under the control of the CaMV 35S promoter, was sufficient to repress anthocyanin accumulation in a red lettuce variety.\nWe also investigated the effects of AtMYB60 on the expression of the different structural proteins involved in anthocyanin production, which we have identified previously in lettuce. Our results reveal that this MYB protein represses the expression of the DFR gene in the AtMYB-117 and -112 lines. Significantly, DFR is a crucial structural protein that facilitates anthocyanin pigmentation in lettuce and that no such reduction in the expression of this gene was found in the wild-type or AtMYB4-101 lines that also develop red-colored leaves. The DFR protein represents an important branching point within the anthocyanin biosynthetic pathway and catalyzes the commitment step in this process, which is the formation of leucoanthocyanidins from the substrate dihydroflavonol. This indicates that AtMYB60 specifically inhibits the flux of flavonoid intermediates toward the production of leucoanthocyanins by repressing the transcription of DFR, with a resulting green leaf phenotype (Fig.\u00a07).\nFig.\u00a07Schematic representation of the relationship between AtMYB60 expression and the structural proteins that play roles in anthocyanin biosynthesis\nIt is interesting to note that the transcriptional response to UV-B irradiation that operates through AtMYB60 focuses on the regulation of DFR expression and anthocyanin accumulation. Recently there has been some debate about the identity of the phenylpropanoids involved in the response to UV-B, which may provide the most effective components of sunscreen formulations. MYB transcription factors that are known to regulate the transcription of genes in the phenylpropanoid biosynthetic pathway have been studied previously (Jin and Martin 1999). The overexpression of PAP1 in Arabidopsis results in the up-regulation of the genes encoding phenylalanine ammonia-lyase (PAL), CHS and DFR (Borevitz et al. 2000; Tohge et al. 2005). Moreover, mutations in the CHS, DFR and LDOX genes reduce the pigmentation of plants (Shirley et al. 1995; Abrahams et al. 2003), suggesting that these proteins are required for the synthesis of both anthocyanidin and proanthocyanidin. Because MYB-binding sites are present in the promoter regions of these flavonoid biosynthetic genes (Nesi et al. 2001; Debeaujon et al. 2003), it is possible that the AtMYB60 transcription factor might also specifically and directly target them. It is also possible that additional target genes that are related to phenylpropanoid metabolism can be negatively regulated by AtMYB60 when it is overexpressed in lettuce.\nThe effects of AtMYB60 suggest that this MYB protein acts as a direct transcriptional regulator by binding to the MYB motifs common to the promoters of many structural genes in the same metabolic pathway, thus resulting in the downregulation of this pathway. The precise determination of this regulatory system at the molecular level remains to be achieved. Recently, Cominelli et al. (2005) reported that AtMYB60 is involved in the plant response to stress and its expression is negatively modulated during conditions of drought (Cominelli et al. 2005). A null mutation in AtMYB60 results in a constitutive reduction in stomatal openings and in decreased wilting under water-stress conditions. Therefore, we hypothesize that AtMYB60 has multiple functions in the regulation of anthocyanin biosynthesis and in the plant responses to different environmental conditions, including drought and exposure to UV-B light.\nAtMYB60 is the first MYB protein identified that functions as a transcriptional repressor of the lettuce DFR gene in anthocyanin biosynthesis, and thus extends the known roles of the R2R3-MYB proteins in secondary metabolism. Evidence from our transgenic lettuce experiments suggests that the direct repression exerted by AtMYB60 protein produces phenotypic effects in transgenic plants. The engineering of the anthocyanin biosynthetic pathway is potentially of great commercial significance. Because the AtMYB60 transcription factor should act effectively in most plant species, it can potentially be used to control anthocyanin biosynthesis in agriculturally and industrially important plants. This method of modifying the anthocyanin content in plant tissues also opens up new avenues to engineering improvements in commercial crops by altering metabolic pathways via the regulation of different structural genes in these pathways.","keyphrases":["arabidopsis","r2r3-myb transcription factor","repressor","lactuca sativa"],"prmu":["P","P","P","P"]}
{"id":"Cancer_Immunol_Immunother-3-1-2150634","title":"Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC\/SVI)\n","text":"The Cancer Vaccine Consortium of the Sabin Vaccine Institute (CVC\/SVI) is conducting an ongoing large-scale immune monitoring harmonization program through its members and affiliated associations. This effort was brought to life as an external validation program by conducting an international Elispot proficiency panel with 36 laboratories in 2005, and was followed by a second panel with 29 participating laboratories in 2006 allowing for application of learnings from the first panel. Critical protocol choices, as well as standardization and validation practices among laboratories were assessed through detailed surveys. Although panel participants had to follow general guidelines in order to allow comparison of results, each laboratory was able to use its own protocols, materials and reagents. The second panel recorded an overall significantly improved performance, as measured by the ability to detect all predefined responses correctly. Protocol choices and laboratory practices, which can have a dramatic effect on the overall assay outcome, were identified and lead to the following recommendations: (A) Establish a laboratory SOP for Elispot testing procedures including (A1) a counting method for apoptotic cells for determining adequate cell dilution for plating, and (A2) overnight rest of cells prior to plating and incubation, (B) Use only pre-tested serum optimized for low background: high signal ratio, (C) Establish a laboratory SOP for plate reading including (C1) human auditing during the reading process and (C2) adequate adjustments for technical artifacts, and (D) Only allow trained personnel, which is certified per laboratory SOPs to conduct assays. Recommendations described under (A) were found to make a statistically significant difference in assay performance, while the remaining recommendations are based on practical experiences confirmed by the panel results, which could not be statistically tested. These results provide initial harmonization guidelines to optimize Elispot assay performance to the immunotherapy community. Further optimization is in process with ongoing panels.\nIntroduction\nElispot is a widely used assay for immune monitoring purposes [6, 17, 25, 39]. Measuring immune responses has been accepted as an important endpoint in early clinical trial settings in order to prioritize further vaccine or other immunotherapy development [11, 22, 24, 26, 38]. Despite the overwhelming use of various immune assays for exactly that purpose, reported results are often met with skepticism, caused mainly by two reasons: (1) high variability among results from the same laboratories and\/or among different laboratories, and (2) the lack of demand to report standardization, validation and training strategies as well as assay acceptance criteria by the laboratories conducting immune testing. This is surprising since the reporting of results for other clinical endpoints, e.g., side effects, has to follow strict guidelines and definitions.\nThe Clinical Laboratory Standard Institute (CLSI) clearly phrased the demand for internal validation of immune assays as well as external validation in proficiency panels [28]. Proficiency panels are a common strategy for clinical laboratories to prove their ability to perform clinical tests at a level that permits patient testing. Defined as a program in which multiple specimens are periodically sent to laboratories for analysis, and in which each laboratory\u2019s results are compared with those of the other laboratories and\/or with an assigned value [27], proficiency panels serve various purposes:To provide regulatory and sponsoring agencies with confidence that reported data are generated following necessary standards and rigor that supports product licensure.To provide an external validation tool for individual labs.To provide proof to patients and volunteers that necessary measures have been taken to allow successful study participation [3].Some reports about comparison of Elispot performances among laboratories have been published [5, 32, 33]. A two-step approach to assay harmonization is being described by the C-IMT monitoring panel in this issue [2]. The earliest report, a four-center comparative trial in 2000, showed that most participants were able to correctly detect low frequency responses or absence of response against specific peptides in PBMCs from six donors, following their own protocol [33]. Another four-center comparison was conducted among members of the Elispot standardization group of the ANRS in France [32]. This study demonstrated overall good qualitative and quantitative agreement in Elispot results in the participating labs when testing HIV negative and positive donors for reactivity against a variety of peptides. All labs used their own protocol, but shared a high level of Elispot experience. An important program was launched by the NIAID for 11 international laboratories participating in HIV-1 clinical trials [5]. The panel demonstrated good concordance in qualitative detection of specific immune responses in previously defined and tested donors, but also notable inter-laboratory and intra-sample variability in spot counts, cell recovery and viability. This observation was met with strict standardization strategies across all panel members, and the panel was repeated twice with all laboratories following a standardized protocol. Variability was decreased under these conditions, but not abrogated (Cox, personal communication). Justification for the strict standardization approach among participating HIV vaccine laboratories can be found in the nature of HIV vaccine testing programs. Immune monitoring for these vaccine trials is performed at many sites simultaneously. Further, results from different trials need to be comparable in order to identify most suitable vaccine candidates. Importantly, the experimental Elispot setup is similar or identical in most laboratories, where PBMCs are tested against a variety of peptides [25, 31].\nIn contrast, immune monitoring approaches in the cancer vaccine field are more heterogeneous, based on the vast variety of vaccine design, type of cancer, and availability of antigen presenting cells. Standardization of the entire Elispot protocol across laboratories is therefore not feasible. We set out to devise a strategy to identify issues and deficiencies in current Elispot practices, and to identify common sources of assay variability within and between laboratories, with the extended goal of standardizing the identified factors in an assay harmonization effort across laboratories.\nIn 2005, the Cancer CVC\/SVI initiated an Elispot proficiency panel program to achieve this goal. In addition to offering an external validation program, the CVC addressed the need for such strategy by comparing assay performance across the field, identifying critical protocol choices and gaining an overview of training and validation practices among participating laboratories.\nFor this program, predefined PBMCs from four donors with different ranges of reactivity against two peptide pools were sent to participants for Elispot testing. Laboratories had to further provide cell recovery and viability data, as well as respond to surveys describing their protocol choices and training and validation status.\nIn response to the survey results, the CVC\/SVI established requirements for laboratories to participate in future proficiency panels, which included the existence of a Standard Operating Procedure (SOP) prior to joining the program. Further, individualized assay performance assessment was offered to all laboratories, together with suggestions for implementation of protocol optimization steps.\nThe results of the second Elispot proficiency panel conducted a year later demonstrated a significant improvement. The results and survey data allowed the identification of critical protocol choices for a successful assay performance (Fig.\u00a01).\nFig.\u00a01Initial guidelines for harmonization of the Elispot assay to optimize assay performance and reproducibility derived from two international proficiency panels, based on their findings and trends observed\nMaterials and methods\nParticipants and organizational setup\nAll participants were members of the Cancer Vaccine Consortium or its affiliated institutions, the Ludwig Institute for Cancer Research (LICR) and the Association for Immunotherapy of Cancer (C-IMT). Laboratories were located in ten countries (Australia, Belgium, Canada, Germany, Italy, Japan, France, Switzerland, UK, and USA). Each laboratory received an individual lab ID number. Panel leadership was provided by a scientific leader experienced in Elispot, in collaboration with the CVC Executive office. SeraCare BioServices, Gaithersburg, MD, served as central laboratory, providing cells, pretesting and shipping services, as well as logistical services like blinding of panelists. IDs were not revealed to panel leader, CVC or statistician during the panel.\nThirty-six laboratories including the central lab participated in panel 1, 29 including the central lab in panel 2. Twenty-three laboratories participated in both panels. Six new panelists were added to the second testing round. Thirteen dropped out after the first panel. Main reasons for drop out were switch of assay priorities and not meeting criteria for panel participation. Various groups stated that one time participation fulfilled their need for external validation.\nPBMCs and peptides\nPBMCs from healthy donors were obtained from a commercial donor bank, manufactured and processed under GMP conditions using established Standard Operating Procedures at SeraCare. PBMCs were frozen using a rate controlled freezer and transferred to the vapor phase of liquid nitrogen. A three lot validation study was performed [20] in which the following was validated: cell separation procedure, freezing media, dispensing effect on function, freezing procedure, and shipping on dry ice. It was demonstrated that functionality and viability were maintained throughout the procedure. In addition, Elispot values from fresh and frozen PBMCs, shipped on dry ice, were nearly equivalent.\nEach vial of PBMCs contained enough cells to ensure a recovery of 10\u00a0million cells or more under Seracare\u2019s SOP.\nPBMCs were pretested at the central laboratory for reactivity against the CEF [7] and CMV pp65 peptide pools [23]. The CEF peptide pool was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. It contains 32 8-11mers known to elicit CD8-restricted responses, and serves as a widely used control in IFN\u03b3 Elispot assays [7]. The CMV pp65 peptide pool was a generous gift of the NIAID and Becton Dickenson. This pool consists of 135 15mers overlapping by 11 amino acids, and has been shown to elicit CD8- and CD4-restricted responses [23]. PBMCs were selected for no, low, medium and strong responses against both peptide pools, and repeatedly Elispot-tested at Seracare in order to confirm responder status. Response definition was set arbitrarily for the spot number\/well (200,000 PBMC): no responder: average median\/panel 1\u00a0+\u00a02\u00a0=\u00a01 (CMV and CEF); low responder: average median\/panel 1\u00a0+\u00a02\u00a0=\u00a018 (CMV) and 40 (CEF); medium responder: average median\/panel 1\u00a0+\u00a02\u00a0=\u00a098 (CMV) and 127 (CEF); and high responder: average median\/panel 1\u00a0+\u00a02\u00a0=\u00a0396 (CMV) and 398 (CEF).\nPeptide pools were resuspended in DMSO and further diluted with PBS to a final concentration of 20\u00a0\u03bcg\/ml. Aliquots of 150\u00a0\u03bcl of peptide pool were prepared for final shipment to participants. Corresponding PBS\/DMSO aliquots for medium controls were also prepared. Participants were blinded to the content of these vials, which were labeled as \u201cReagent 1, 2 or 3\u201d.\nAll cells and reagents sent to participants in both panels were obtained from the same batches.\nCells and reagent vials were shipped to all participants for overnight delivery on sufficient dry ice for 48\u00a0h. Shipping was performed by Seracare under their existing SOPs.\nElispot\nParticipants received a plate layout template and instructions for a direct IFN\u03b3 Elispot assay which had to be performed in one Elispot plate. Each donor was tested in six replicates against three reagents (medium, CEF and CMV peptide pool). Further, 24 wells were tested for the occurrence of false positive spots by the addition of T cell medium only. About 200,000 PBMC\/well were tested against 1\u00a0\u03bcg\/ml peptide pool or the equivalent amount of PBS\/DMSO. All other protocol choices were left to the participants, including choices about: Elispot plate, antibodies, spot development, use of DNAse, resting of cells, T cell serum, cell counting and spot counting method. All plates were reevaluated at ZellNet Consulting (Fort Lee, NJ) with a KS Elispot system (Carl Zeiss, Thornwood, NY), software KS Elispot 4.7 (panel 1) and KS Elispot 4.9 (panel 2) in blinded fashion. Each well in each plate was audited.\nSince the focus of this study was on assay performance and protocol evaluation and not on the definition of a positive response, we prospectively defined the ability to detect a response (independent of magnitude) by using the following \u201cempirical\u201d method: the antigen-specific spot counts per 2\u00a0\u00d7\u00a0105 PBMCs had to be >10, and at least 3\u00d7 as high as the background reactivity. Similar approaches have been described elsewhere [5, 8].\nStatistical analysis\nThe following parameters were calculated for the overall panel and the individual participant\u2019s performance, using either lab-specific counts or central recounts: the mean, standard deviation, and coefficient of variation (CV), the median, minimum, and maximum spot counts for each donor and reagent and the media only wells. Box plots were used to illustrate the distribution of spot counts across the panel per given test condition. Further, individual results were represented as box plots comparing lab counts with recounts, central lab results and overall panel results. For panel 2, results from repeating laboratories were also compared in box plot format for each donor and condition. The Fisher\u2019s Exact test was used to compare the proportions of laboratories which missed to detect responses in each panel. For the comparison of recovery and viability, the Student\u2019s t test was applied.\nResults\nFeasibility\nIn the first proficiency panel, shipping and Elispot testing among 36 laboratories from 9 countries were conducted without delays. The success of this panel demonstrates the feasibility of such large international studies, the biggest of such format as of today, under the described organizational setup. The second panel with 29 participating laboratories from 6 countries followed the approach of panel 1. However, customs delays of dry ice shipments to some international sites required repeated shipments of cells and antigens to these destinations. Based on this experience, the use of dryshippers with liquid nitrogen is being implemented for international destinations in the third CVC panel round in 2007.\nRecovery and viability of PBMCs in panel 1 and 2\nParticipants were asked to record recovery and viability of cells immediately after thawing (Table\u00a01). The mean and median for both parameters were almost identical in both panels, and for all four donors (mean cell recovery between 11.5 and 13.3\u00a0million cells and mean viability between 85 and 91%). Only a small percentage of laboratories recovered less than 8\u00a0million cells (13% in panel 1 and 14% in panel 2). The percentage of groups reporting less than 70% viability was 7% for both panels.\nTable\u00a01Cell recovery and viability in both proficiency panelsDonoraMean P1\/P2bMedian P1\/P2Minimum P1\/P2Maximum P1\/P2Cell recovery in 106 cells112.5\/11.512.0\/12.06.8\/1.026.4\/18.8213.3\/12.313.2\/12.65.5\/0.328.4\/22.4313.1\/12.813.6\/12.86.7\/1.725.1\/24.3412.8\/12.411.9\/12.95.6\/6.234.4\/22.7Viability (%)188\/8589\/8948\/58100\/98287\/8889\/9057\/67100\/98391\/8793\/9054\/69100\/100486\/8990\/9243\/75100\/98aPBMCs from same donors and batches were used in both panelsbP1\/P2 refer to data from panel 1 (P1) and panel 2 (P2)\nInterestingly, only 4\/10 laboratories in panel 1, and 4\/7 laboratories in panel 2 with recoveries below 8\u00a0million cells were from international locations. Similar, only 1\/5 laboratories in panel 1 and 2\/5 in panel 2 reporting viabilities less than 70% belonged to international sites. This clearly demonstrates that location for dry ice shipment had no effect on overall cell recovery and viability.\nWe also investigated whether low (<8\u00a0million) or very high (>20\u00a0million) cell recovery had an influence on spot counts, assuming that these were potential erroneous cell counts, leading to too low or too high cell dilutions, respectively, what in turn would lead to too high (in case of underestimating cell number) or too low (in case of overestimating cell number) spots counts. However, except for a few sporadic incidents, there was no correlation between cell counts and spot counts (data not shown) in either direction. Only one laboratory with low recovery and low viability was found to have peptide pool-specific spot counts for all donors much below the panel median.\nThe use of an automated cell counter (Guava) did not reveal any trend in recovery for this group (recovery ranged from 6.7 to 25.1\u00a0million cells), nor a difference compared to the overall panel recovery data. In contrast, the mean cell viability per donor measured by Guava counter users was significantly lower than the overall cell viability in users of a hemocytometer (P\u00a0<\u00a00.01, see table in Fig.\u00a05).\nElispot results in panel 1\nAll 36 laboratories completed testing of all 4 donors against medium, CEF and CMV peptide pool. Spot appearance and size as well as occurrence of artifacts differed dramatically among laboratories (not shown). Four outlier laboratories were identified, which detected less than half of the responses correctly. In all four cases, detected responses were well below the panel median, and often, there was high background reactivity (up to 270 spots\/well) in medium controls. No obvious protocol choices could be identified which could have been responsible for the suboptimal performance. One out of the four laboratories had little experience at the date of the panel. Another group reported a less experienced scientist performing the assay. A third outlier repeated the assay, and was able to perform adequately. No feed back was available from the fourth group.\nThirty-two out of 36 labs were able to detect medium and strong responders as well as the non-responder. However, almost 50% of participants were challenged with the detection of the low responder. The responses measured for that particular donor (mean spot counts\/well for CEF\u00a0=\u00a042 and for CMV\u00a0=\u00a022) has been chosen for illustration purposes. Figure\u00a02 demonstrates the distribution of responses measured including intra-laboratory variability for this donor against all three reagents and across both panels.\nFig.\u00a02Laboratory spot counts and variability for low responder across panel participants. Panel 1 left column, panel 2 right column. The reagent tested is indicated. Lab-specific spot counts per 200,000 cells are depicted as box plots with the box presenting the interquartile range, the triangle the mean and the horizontal line the median. Maximum and minimum spot counts are illustrated through the upper and lower mark. The horizontal line across a graph demonstrates the overall panel median. The central laboratory performed the assay under two different conditions in panel 1. Results from both experiments are presented; therefore 37 laboratories for panel 1. Laboratory ID numbers do not correlate in both panels. In panel 1, laboratory #18 reported spot counts for the medium control as high as 270 per well (mean 81, median 34.5). For proper illustration of all other panel data, these data were omitted from the graph. Intra-laboratory variability and variability among participants as well as reactivity against medium are representative for all responder PBMCs tested\nIn panel 1, 17 labs did not detect the response against the CMV peptide pool. In one case, the response was missed due to high reactivity against medium. Three other labs missed to detect the response due to inaccurate spot counting, specifically in the medium control wells. Artifacts were erroneously included in counts (Fig.\u00a03). Reevaluation of those plates revealed that those labs had spot counts indicating a response against CMV.\nFig.\u00a03Elispot assay results can be confounded by plate evaluation accuracy. The table demonstrates spot counts for PBMC\/medium control wells with many artifacts from three different laboratories (Lab X, Y, Z). Respective well images are shown below each column for that specific group. Differences in lab-specific spot counts (\u201cown\u201d) and counts from reevaluation in an independent laboratory (\u201ccentral\u201d) including resulting variability measures are presented in the table\nA similar scenario was found for response detection against the CEF peptide pool. Thirteen laboratories missed to detect the response, one of which due to high reactivity against medium, and one due to inaccurate spot counting.\nAn interesting observation was that 23 groups reported false positive spots in a range of 1\u201326\u00a0spots\/well. Reevaluation revealed that the actual number of groups with false positive spots was lower (12), and the false positive spot number range per well fell between 0 and 8.\nSurvey results about protocol\nDuring the first panel, participants had to provide information about their protocol choices: plates, potential prewetting of PVDF, antibodies, enzyme, substrate, use of DNAse during PBMC thawing, resting of cells, serum used, cell counting, and plate reader. There was a wide range of protocol choices across the panel participants. The most common choices for the parameters listed above were as follows: use of PVDF plates (64%) prewetted with Ethanol (52%), coated with Mabtech antibodies (67%) at 0.5\u20130.75\u00a0\u03bcg\/well (33%); spot development with HRP (53%) from Vector Laboratories (33%) using AEC (44%) from Sigma (42%); no use of DNAse when thawing cells (83%), and no resting period for cells prior to the assay (53%); use of human serum (64%); cell counting with trypan blue exclusion using a hemocytometer (78%); and plate evaluation with a Zeiss reader (36%).\nThere were some clear trends for international sites with preferred use of the AP\/BCIP\/NBT development system (83 via 29% in the US), the use of nitrocellulose plates (75 via 21% in the US), and the use of Mabtech antibodies (83 via 58% in the US). On the other hand, 7\/8 laboratories using an automated cell counter were located in the US.\nWe stratified our panel participants into groups consisting of: (1) outliers (failed to detect more than 50% of responses correctly), (2) labs which missed weak response detection only, and (3) labs with correct qualitative response detection in all four donors. We then checked for specific parameters that could be potentially responsible for the groups\u2019 panel performance. We were not able to detect any parameter that seemed to be responsible for missed response detection. In contrast, laboratories with the same overall protocol choices (except serum) could have very different assay performance, whereas laboratories with different protocol choices could have almost identical spot counts (Fig.\u00a04). Reasons for the failure to detect protocol choices which significantly proved the advantage or disadvantage of their use can be found in (1) the study design with open protocol choices allowing participants to follow their own SOP, (2) the overall wide range in choices of reagents and materials (e.g., 7 different sources for the coating antibody with 10 different total antibody amounts reportedly used, 10 different enzyme sources, 7 different Elispot reader systems, cell resting times between 0.5 and 20\u00a0h, serum choices unique for each laboratory etc.), and (3) the small sample sizes resulting from this dispersion of protocol choices. These widely spread individual protocol choices among participants did not allow the aggregation of data for an adequately powered statistical analysis.\nFig.\u00a04Demonstration of examples of concordance of spot counts among three laboratories (Lab I\u2013III) using different Elispot protocols (a, high responder), or of disagreement of spot counts for two laboratories (Lab IV\u2013V) using almost identical protocol choices (b, medium responder). Lab-specific spot counts per 200,000 cells are depicted as box plots with the box presenting the interquartile range, the triangle the mean and the horizontal line the median. Maximum and minimum spot counts are illustrated through the upper and lower mark. The triangle refers to the overall panel mean for that specific condition. Results of Lab I\u2013III are shown in a, and results of IV\u2013V in b. The table contains reference to the figure above, and information about specific protocol choices\nOne trend observed was that the users of an automated cell counter (7 labs used a Guava cell counter) had an overall better assay performance (Fig.\u00a05), with no outliers in this group and only two laboratories who missed to detect the weak responders (29 vs. 52% in non-Guava users).\nFig.\u00a05Distribution of mean spot counts (upper two graphs) and cell viability (table) among the users of a Guava automated cell counter (a) and users of a hemocytometer (b). Spot counts per well (200,000 cells) are represented on a logarithmic scale. D1\u20134 refer to the donor tested with D1 being the strong, D2 the low, D3 the medium and D4 the non-responder. The tested reagent is indicated (med medium). The mean viability per donor reported by Guava users and users of other cell counting methods (one lab used an automated cell counter from Beckman Coulter, all others used a hemocytometer with trypan blue exclusion) is presented in the table below the figure\nAlmost half of the participants (47%) introduced a resting period for cells before adding them to the Elispot plate. The time frame varied from 0.5 to 20\u00a0h. A valuable side-by-side comparison was performed by the central laboratory, in which two tests were run in parallel using the lab\u2019s SOP with the exception that their standard resting period of 20\u00a0h was replaced by a shorter resting period of 2\u00a0h in the second test. The results are summarized in Fig.\u00a06, demonstrating a significant increase in spot numbers for all peptide pool-specific responders when cells were rested for 20\u00a0h, without an increase in background reactivity (P\u00a0<\u00a00.05 for the weak responder against CMV; for all other antigen-specific responses in all donors P\u00a0<\u00a00.01).\nFig.\u00a06Comparison of the effect of 2\u00a0h (checkered bars) and 20\u00a0h (solid bars) resting periods for cells after thawing, before adding to the assay. D1\u20134 CEF\/CMV refer to the specific donor and peptide pool tested. Background reactivity for all donors and testing conditions was between 0 and 5 spots (not shown). The standard error is shown. *\u00a0Indicates a statistical significant difference of P\u00a0<\u00a00.01 in spot counts between 2 and 20\u00a0h resting periods for a given donor and reagent; # indicates in statistical difference with P\u00a0<\u00a00.05 (Student\u2019s t test)\nSurvey results about validation and training practices\nDuring the lively discussion of the results of panel 1 and its protocol survey at the Annual CVC meeting in Alexandria in November 2005, it was suggested that the level of experience, standardization and validation of participating laboratories might have been the cause for the variability and performances observed. In response, we conducted a survey among panelists, in which 30 laboratories participated. As expected, the experience and Elispot usage varied significantly. Some laboratories had the Elispot assay established less than one year before panel testing, whereas others used the assay for more than 10\u00a0years. The experience of the actual performer of the panel assay also varied widely.\nInterestingly, even though 2\/3 of participants reported to have specific training guidelines for new Elispot performers in place, more than 50% never or rarely checked on the scientist\u2019s performance after the initial training.\nAlmost all laboratories indicated that they use an SOP that had been at least partially qualified and\/or validated. Validation tools and strategies varied widely. Only 12 groups monitored variability, whereas 23 reported the use of external controls of some kind (e.g., T cell lines, predefined PBMC, parallel tetramer testing).\nThirteen groups were found to have some kind of criteria implemented for assay acceptance. Among the 20 different criteria reported, not one was described by more than one lab.\nMirroring these survey results, 20 laboratories believed that they need to implement more validation steps. All except one group expressed their strong interest in published guidelines for validation and training strategies for Elispot.\nElispot results in panel 2\nBased on the experience from the first panel, acceptance criteria for participation in the program were redefined. Only laboratories with established SOPs were accepted. The second panel was repeated with identical experimental setup as the first panel, and with the same batches of PBMCs, peptides and control reagents. Twenty-nine laboratories participated including the central lab. This time, there were no outlier performers identified. The number of groups that did not detect the weak responder dropped dramatically (P\u00a0<\u00a00.01) from 47% (17\/36 labs) in panel 1 to 14% (4\/29) in panel 2 (Fig.\u00a02). The overall panel median for the CMV-response in the weak responder increased from 14 spots in panel 1 to 21 spots in panel 2, and for the CEF response from 30 to 51, respectively.\nOf the 23 labs repeating the panel, 8 had changed their protocol before panel 2, 3 of which as an immediate response to results from the first panel. One outlier lab from panel 1 participated in panel 2, and improved its performance by detecting all responses correctly as per reevaluation counts. Only their lab-specific evaluation did not detect the low CMV responder. Overall, 4\/23 panel-repeating labs (17%) did not detect the low CMV-responder, 3 of which also did not detect this response in panel 1. About 10\/23 groups missed the low CMV responder in panel 1, but 7 of these laboratories were able to detect it in panel 2. Only one repeater detected the low CMV response in panel 1, but not panel 2. This is a clear performance improvement for that group (47% missed this response in panel 1), and highlights the usefulness of multiple participation in panel testing as an external training program.\nWe ran an in-depth analysis of the results and previous survey responses, where available, from participants who missed the weak responder, as well as from laboratories with marginal detection of response, including personal communication. We were able to narrow down the possible sources for these performances. Two laboratories missed responses due to inaccurate evaluation, during which they either included artifacts into spot counts or simply did not count the majority of true spots, as central reevaluation revealed. One laboratory did not follow the assay guidelines. The majority of laboratories, however, followed common protocol choices, but had either very low response detection across all donors and antigens, or detected very high background reactivity in some or all donors. This pattern pointed to serum as the possible cause for suppressed reactivity or non-specific stimulation. Three of these laboratories shared with the CVC that retesting their serum choice indicated that they had worked with a suboptimal serum during the panel; and that they now successfully introduced a different serum\/medium to their protocol with improved spot counts. Serum choices included human AB serum, FCS, FBS, and various serum-free media. There was no difference in assay performance detectable between these groups.\nDiscussion\nThe CVC conducted two large international Elispot proficiency panels in which participants tested four batches of predefined donor PBMC for CEF and CMV peptide pool reactivity. Common guidelines like number of cells per well and amount of antigen had to be followed, in order to allow result comparison and reduce variability due to the known influence of cell numbers plated [2, 14]. A surprising finding from the first panel was that almost half of the participants were not able to detect the weak responder. The initial protocol survey did not allow the detection of common sources for this sub-optimal performance. In some cases, laboratories with identical protocol choices performed very differently, whereas others with distinct protocols had almost identical spot counts (Fig.\u00a04). This observation supports the premise that many common Elispot materials and reagents (e.g., plates, antibodies, spot development reagents) perform equally or similarly well; and that there are other factors which influence the outcome of the Elispot assay.\nVarious laboratories missed the response detection by inaccurately evaluating their Elispot plate, independent of the reader used, as reevaluation revealed (Fig.\u00a03). This observation is in contrast to results from the first NIAID proficiency panel, which describes good agreement in spot counts from participants and experienced, independent centers [5]. Operator-dependent variability in Elispot evaluation results is a known phenomenon [15]. Despite the availability of high resolution readers and software features for automated spot gating and other potentially helpful options, it is essential to employ well trained operators for spot counting, to audit all plates, and to implement changes of reading parameter in cases when well and spot appearances differ from the overall assay, typically for technical reasons. For that, SOPs used for plate evaluation might require revision. The use of available certification and training services can be helpful.\nCell counting is a protocol step known for introducing variability. Because of the wide range in PBMC recovery, we investigated whether cell counts could have been potentially erroneous, which would lead to wrong final cell dilutions, and can therefore lead to too high or low spot counts. Despite large differences in cell recovery (from 5.5 to 34.4\u00a0million cells per vial in panel 1), we did not find a correlation between cell recovery and spot counts. Very low (<8\u00a0million) and very high (>20\u00a0million) cell counts were consistently found for all donors among the same few laboratories. Both, hemocytometers and automated counters were used in those laboratories. Various automated cell counters have been introduced to the market, which do not only offer automated spot counting features, but also the discrimination of apoptotic, viable and dead cells. The use of such systems can potentially decrease variability in cell counts, and most importantly increase the accuracy of viable cell counts [4, 21]. Seven laboratories in panel 1 used the Guava counter, which allows the discrimination of apoptotic cells. Even though our panel did not reveal a difference in cell counts between Guava and hemocytometer users, it could be demonstrated that the overall cell viability reported by Guava users was significantly lower. This could likely be attributed to the ability to discriminate apoptotic cells with this method. Smith et al. [35] recently reported the usefulness of apoptosis acceptance criteria that allowed the separation of PBMC samples by their ability to respond to an antigenic stimulus or not.\nThe introduction of a resting period for thawed cells is known to be advantageous since apoptotic cells die during the resting period, and final dilutions for the assay are based on a more homogenous population of viable cells [16, 18]. In contrast, the addition of a mixture of viable and apoptotic cells, which are prone to die during assay incubation time, directly after thawing leads to lower spot counts. During panel 1, the central laboratory performed a side-by-side comparison of the influence of a 2 and 20\u00a0h resting period on final spot counts, and demonstrated that a 20\u00a0h resting period yielded significantly higher counts (Fig.\u00a06). Proficiency testing results from the C-IMT also support the introduction of a cell resting period for Elispot assays [2]. Even though almost half of participants in panel 1 let cells rest before addition to the plate, there was no clear correlation to the magnitude of peptide-specific spot counts. This might have been due to the variation of resting times between 0.5 and 20\u00a0h, and other protocol variables, which included the actual resting protocol. Factors like serum and serum concentration, cell concentration, and actual storage condition (e.g., tissue culture flasks or plates can lead to cell adherence and therefore loss of professional antigen-presenting cells) are known to influence the success of cell resting.\nThere are no scientific studies published about the effect of serum choices on immune assay results, one of the best known \u201csecrets\u201d in immunology [16]. It is critical to choose serum that supports low background reactivity, but strong signals. The leading choice in this panel to use human AB serum reflects the historic preference for human immune assays. Each serum batch, however, is unique in its ability to support optimal assay resolution, and may potentially contain mitogenic or immune suppressive factors. There was some anecdotal evidence that the serum choice among our panelists was responsible for suboptimal performance. Interestingly, six laboratories preferred to work with serum-free medium. None of these groups observed high background reactivity, but two failed to detect the weak responder.\nA survey conducted among participating laboratories shed light on validation and training practices. The wide experience range of participating laboratories, in combination with various levels and approaches to validation and training, correlated with the overall high variability in panel results. In response, new criteria for panel participation were introduced for panel 2. The outlier lab number decreased from 4 to 0 in the new panel, however 3\/4 outliers from panel 1 did not participate in panel 2. The percentage of laboratories that did not detect the weak responder decreased significantly from 47 to 14% (P\u00a0<\u00a00.01). This improvement might have been partially due to the stricter participant selection in panel 2. However, 7\/10 labs repeating the panel improved their performance by correctly detecting the low CMV responder in panel 2, while missed in panel 1. A striking finding was that 2\/3 of all laboratories stated that they believed they needed to implement more validation. And all but one group expressed their wish for published guidelines for Elispot assay validation and training.\nThe outcomes from these two proficiency panels first provide Initial Elispot Harmonization Guidelines for Optimizing Assay Performance (Fig.\u00a01) that can fulfill this need and may provide\u2014if implemented widely\u2014the grounds for substantial improvement of assay utility for research applications and development of immune therapies. This can be implemented in accordance with assay recommendations made for cancer immunotherapy clinical trials by the Cancer Vaccine Clinical Trial Working Group [12]. Further optimization is aimed for through ongoing proficiency panel work conducted by the CVC.\nValidation of assays is now a requirement for all endpoint parameters in clinical trials [30]. There are an increasing number of publications available describing validated Elispot assays [1, 19, 25, 34]. These papers contain valuable scientific information, but only limited referral to FDA regulations. The FDA guidelines for validation of analytical procedures [9] describe validation as the process of determining the suitability of a given methodology for providing useful analytical data, which consists of analyzing or verifying the eight or nine assay parameters as described in the US pharmacopeia or the ICH guidelines [13, 37]. Only few publications address validation of bioassays and Elispot in FDA terms [10, 16, 29, 36]. And even these few publications give only limited advice on how to validate the Elispot assay in a given laboratory setting, not to mention specific training guidelines.\nFurthermore, acceptance criteria for assay performance were only used by a limited number of laboratories, and each criterion was unique for the laboratory that used it.\nThese observations should be a wake-up call for the immune monitoring community, which does not only include the cancer vaccine field, but also the infectious disease and autoimmunity field and others. General assay practices for the detection of antigen-specific T cells are comparable across all fields. The CVC as part of the Sabin Vaccine Institute is intending to develop and tighten collaborations with groups from other research and vaccine development areas. Published documents with specific criteria for Elispot assay validation, assay acceptance criteria and training guidelines will be most valuable for the immune monitoring field, and are now being established as CVC guidelines as a result of the described studies. Continuous external validation programs need to be a part of these efforts in order to check upon the success of inter-laboratory harmonization including assay optimization, standardization and validation as well as of laboratory-specific implementation of guidelines and protocol recommendations. These efforts are essential to establish the Elispot assay and other immune assays as standard monitoring tools for clinical trials.","keyphrases":["harmonization","elispot","proficiency panel","immune monitoring","validation"],"prmu":["P","P","P","P","P"]}
{"id":"Histochem_Cell_Biol-4-1-2228376","title":"Topology of molecular machines of the endoplasmic reticulum: a compilation of proteomics and cytological data\n","text":"The endoplasmic reticulum (ER) is a key organelle of the secretion pathway involved in the synthesis of both proteins and lipids destined for multiple sites within and without the cell. The ER functions to both co- and post-translationally modify newly synthesized proteins and lipids and sort them for housekeeping within the ER and for transport to their sites of function away from the ER. In addition, the ER is involved in the metabolism and degradation of specific xenobiotics and endogenous biosynthetic products. A variety of proteomics studies have been reported on different subcompartments of the ER providing an ER protein dictionary with new data being made available on many protein complexes of relevance to the biology of the ER including the ribosome, the translocon, coatomer proteins, cytoskeletal proteins, folding proteins, the antigen-processing machinery, signaling proteins and proteins involved in membrane traffic. This review examines proteomics and cytological data in support of the presence of specific molecular machines at specific sites or subcompartments of the ER.\nIntroduction\nThe ER is a dynamic organelle essential for cell life. The main subdomains of the ER include the nuclear envelope (NE), rough ER (rER), and transitional ER (tER), each with its own characteristic structure and function. The NE forms a shell around the nucleus. The shell typically consists of a flattened saccule closely applied to nuclear chromatin and showing multiple discontinuities or nuclear pores (Fig.\u00a01a). The rER is continuous with the NE and consists mostly of stacked, flattened saccules. Each saccule is limited by membranes with attached ribosomal particles (Fig.\u00a01b). The tER is composed of two distinct but continuous membrane domains: a rough domain characterized by the presence of attached ribosomes and a smooth domain (sER), giving rise to membrane buds and tubules (Fig.\u00a01c). Clusters of vesicles, and tubules are often observed closely associated with the tER and represent the intermediate compartment, also called ER-Golgi Intermediate Compartment (ERGIC), vesiculo-tubular clusters (VTCs) or pre-Golgi intermediates (Bannykh et al. 1996; Hauri et al. 2000; Saraste and Kuismanen 1992; Fan et al. 2003). In certain cell types (such as steroid-synthesizing cells or hepatocytes) the smooth ER is extensive and consists of a network of interconnecting tubules limited by smooth membranes devoid of attached ribosomes. The tubules are linked by tripartite junctions and limit cytoplasmic regions often devoid of large organelles (Fig.\u00a01d).\nFig.\u00a01Different subcompartments of the endoplamic reticulum. The ER is composed of continuous but distinct subdomains. a The nuclear envelope (NE) is shown with nuclear pores and ribosomal particles attached to the outer membrane. b The rough ER (rER) is continuous with the NE and consists of stacked flattened saccules, whose limiting membranes have numerous attached ribosomal particles. c Transitional ER (tER) is composed of a rER subdomain continuous with the rER and a smooth ER (sER) subdomain consisting of buds and tubules devoid of associated ribosomes (arrowhead points to a coated bud). d In some cells (e.g., steroid secreting cells and hepatocytes) the sER is composed of a large network of interconnecting tubules showing tripartite junctions (arrows) and fenestrations. Micrograph in C is courtesy of Christian Zuber and Jurgen Roth\nThe diversity of ER structures parallels its many functions. The numerous ribosomes associated with the flattened saccules of the rER indicate its high capacity for protein synthesis, translocation, and folding. The two domains of the tER indicate an additional function within this compartment: that of the formation of tubules and vesicles allowing cargo exit and transport to the Golgi Apparatus, and the tubules of the sER, which are continuous with the rER indicate an increased ER volume thus providing enhanced capacity for calcium storage, drug handling, detoxification, and lipid and steroid synthesis (reviewed in Baumann and Walz 2001; Shibata et al. 2006). Based on these considerations, the subcompartments of the ER are predicted to have a presence and enrichment of very specific proteins. Such predictions can be analyzed by proteomics analysis of each ER subcompartment.\nA number of proteomics studies were done on the subcompartments of the ER providing an ER protein dictionary with new data being made available on many molecular protein complexes of relevance to the biology of the ER. As summarized in Table\u00a01, proteomics studies were done on the NE, total ER microsomes (mixture of rough and smooth ER membranes), ribosomes, purified rough microsomes, purified smooth microsomes, and the ERGIC compartment. Because of the paucity of proteomics data on pure outer nuclear envelope membrane, we have restricted comment on studies of this ER compartment to that within Table\u00a01. Subcompartments of the ER were isolated by subcellular fractionation using centrifugation and various sucrose gradients and some were isolated by immuno-isolation. The degree of purity of these fractions was analyzed by morphology and\/or morphometry and in many cases biochemically by measuring the level of enrichment of expected proteins (markers) compared to the starting material (Table\u00a01). Identifying the proteins present in these ER subfractions is an important step towards better understanding the specialized functions of the ER subcompartments and molecular mechanisms governing these functions.\nTable\u00a01Assessment of published endoplasmic reticulum proteomics datasetsOrganelleTissue\/cellsSpeciesER purificationMass spectrometryProteins detectedReferenceNENeuroblastoma N2a cellsMouseNE preparation (Triton-X-100 treatment)2D-BAC gels, MALDI MS148Dreger et al. (2001)NELiverMouseSubstractive proteomics (NE fraction-MM fraction)MudPIT, LCQ-Deca ion-trap MS, Tandem MS566Schirmer et al. (2003)NELiverRatNuclear pore complex fraction (enriched in nucleoporins) 1D gels, MALDI-QqTOF MS, Tandem MS 94Cronshaw et al. (2002)NESaccharomyces cerevisiaeYeastPurified nuclear pore complexHPLC, 1D gels, MALDI-TOF MS, Tandem MS174Rout et al. (2000)ERaLiverRatbMembrane proteins1D and 2D gels, MALDI-TOF MS68 (1D)39 (2D)Galeva and Altermann (2002)ERaLiverHamstercTenfold enrichmentj (calnexin marker, IB)2D gels, MALDI-Q-TOF MS39Morand et al. (2005)ERdLiverMousePCP-fraction co-sedimenting with calnexinLC, linear ion-trap Fourier transform MS, Tandem MS 229Foster et al. (2006)RERSaccharomyces cerevisiaeYeastPurified ribosomesMultidimensional LC, LCQ ion-trap MS, Tandem MS95Link et al. (1999)RERLiverMouse75% RMe, luminal proteins 2D gels, MALDI-TOF MS, Tandem MS141Knoblach et al. (2003)RERPancreasDogRibosome-associated membrane proteinBlue Native gels, LCQ ion- trap MS, Tandem MS30Shibatani et al. (2005)RER LiverRat4.0-fold enrichment j (G6Pase enzyme assay) 77% RMe, f1D gels, LC, QTOF-2 MS, Tandem MS787Gilchrist et al. (2006)SER LiverRat4.5-fold enrichment j (G6Pase enzyme assay) 58% SMe, 39% RMe, g1D gels, LC, QTOF-2 MS, Tandem MS998Gilchrist et al. (2006)ERGIChHepG2 cellsiHuman110-fold enrichmentj (ERGIC-53 marker, IB)1D gels, LC, Tandem MS24Breuza et al. (2004)ER-Golgi derived vesiclesdLiverMousePCP-fraction co-sedimenting with p115LC, linear ion-trap Fourier transform MS, Tandem MS220Foster et al. (2006)BAC 16-benzyldimethyl-n-hexadecyl ammonium chloride, ERGIC ER-golgi intermediate compartment, G6Pase glucose-6-phosphatase, HPLC high pressure liquid chromatography, IB immuno-blot, LC liquid chromatography, MALDI matrix-assisted laser desorbtion ionization, MM microsomal membrane, MS mass spectrometry, MudPIT Multidimensional protein identification technology, PCP protein correlation profiling, RM rough microsomes, SM smooth microsomes, TOF time of flightaTotal microsomes (containing both rough and smooth ER membrane derivatives)bUntreated and phenobarbital treated ratscModel of insulin resistance and metabolic dislipidemia fructose-fed animaldFractions obtained by rate-zonal centrifugation of the postnuclear supernatanteMorphometric characterization by electron microscopyfPresence of \u226511 ribosomal particles\/vesiclegPresence of 1\u20134 ribosomal particles\/vesiclehImmuno-affinity purified ERGIC membranesiCells were treated with brefeldinA to accumulate cycling proteins in the ERGICjEnrichment over homogenate\nProtein synthesis and secretion\nThe ER is a key organelle of the secretion pathway involved in the synthesis of both proteins and lipids destined for multiple sites within and without the cell.\nRibosomal proteins\nBecause ribosomes define the rough domain of the ER, studies of the proteins of purified ribosomes are relevant to the proteome of the rough ER. Link et al. (1999) reported on the proteome of ribosomes purified from Saccharomyces cerevisiae. Multidimensional chromatography and tandem MS were used to identify 70 of the 78 predicted ribosomal proteins in yeast. The YMR116p protein (homologue of human RACK1 an intracellular receptor for activated protein kinase C) was found to be associated with the 40S ribosomal subunit implicating this protein in translation in PKC-mediated signal transduction. RACK1 is now known to be a bona fide ribosomal protein and to play an important role in regulating eukaryotic translation (Nilsson et al. 2004). Of the proteomics studies carried out so far on mammalian liver ER subcompartments, Gilchrist et al. (2006) have identified most of the ribosomal proteins in purified rough microsomes from rat liver including 33 proteins of the 40S ribosomal subunit and 45 proteins of the 60S ribosomal subunit. This number is close to the 84 proteins isolated from rat ribosomal particles (Wool et al. 1995). The study by Gilchrist et al. (2006) revealed that the concentration of ribosomal proteins was greatest in high-density rough microsomes (HDM) when compared with the ribosomal protein concentration in a smooth microsomal fraction containing low-density rough microsomes (LDM). This is consistent with previous morphometric studies showing more ribosomal particles associated with more vesicles in the HDM fraction compared to that in the LDM fraction from normal rat liver (Gilchrist et al. 2006; Lavoie et al. 1996). Foster et al. (2006) identified a comparable number of ribosomal proteins in ER fractions enriched with the marker proteins calnexin and p115.\nProteins involved in RNA metabolism\nRough microsomal derivatives of the ER are expected to contain a variety of proteins involved in RNA metabolism because they contain and translate associated messenger RNA (Lerner and Nicchitta 2006). A variety of proteins known to be involved in the metabolism of mRNA and in translation of mRNA have been detected in ER membrane derivatives by mass spectrometry (Foster et al. 2006; Gilchrist et al. 2006). These include heterogeneous nuclear ribonucleoproteins (hnRNPs, hnRNP A1, hnRNP A2\/B1, hnRNP D, and hnRNP K), Poly(A)-binding protein 1 and many proteins that are involved in translation (eEF-1A, eEF-2, several subunits of eIF-3, and eIF-5A). Heterogeneous nuclear ribonucleoproteins are involved in mRNA metabolism both inside the nucleus and in the cytoplasm. In the cytoplasm hnRNPs are known to regulate mRNA localization, mRNA translation, and mRNA turnover (Dreyfuss et al. 2002; Shyu and Wilkinson 2000). Since hnRNP family proteins are known to accompany RNA from the gene through nuclear pores and into polysomes (Visa et al. 1996) the hnRNP family proteins detected in ER maybe associated with membrane-bound polysomes and modulate translation of specific proteins relevant to protein synthesis.\nProteins mediating targeting, co-translational translocation, and processing of nascent polypeptide chains\nSecretory and transmembrane proteins are synthesized on polysomes that are attached to the rough domain of the ER (Palade 1975). Ribosomes are bound to the ER membrane and are associated with a molecular complex involved in protein translocation (Gilmore 1993; Rapoport et al. 1996). Sec61p made up of the three subunits alpha, beta, and gamma comprises the main protein-conducting channel, whereas signal-recognition particle receptor, signal peptidase, oligosaccharyltransferase, TRAM, TRAP, p180, p34, Sec63, and BIP comprise associated protein complexes that assist in the signal sequence-mediated targeting, co-translational translocation, and processing of nascent polypeptide chains (Johnson and van Waes 1999; Nilsson et al. 2003; Osborne et al. 2005). As would be expected because of the continuity between rough and smooth ER membrane compartments and because of the capacity of translocon components to diffuse laterally along the ER membrane (Nikonov et al. 2002) translocon components were detected in microsomal-derivatives of both rough and smooth ER (Gilchrist et al. 2006). However, despite such membrane continuity polysomes are retained concentrated in the rough domain of the ER. Cytoskeletal elements (i.e., microtubules) known to interact with specific receptors of the ER (i.e., CLIMP-63) might play a role in mechanisms that restrict the lateral mobility of membrane-bound polysomes (Farah et al. 2005; Vedrenne et al. 2005; Nikonov et al. 2007).\nProteins involved in glycosylation and the calnexin cycle\nDuring the process of protein translocation, the asparagine residues in Asn-x-Ser\/Thr motifs on the nascent polypeptide chain are glycosylated in the lumen of the ER by a multisubunit membrane protein complex called oligosaccharyltransferase (OST). Although several subunits (mainly the ribophorins I and II) of OST have previously been identified in proteomics studies of ER subfractions the most complete analysis of these was reported by Shibatani et al. (2005) who carried out proteomics analysis of isolated ribosome-associated membrane protein from canine rough microsomes. These investigators identified five known subunits of OST (STT3-A, ribophorin I, ribophorin II, OST48, and DAD1) and two previously uncharacterized proteins that co-purified with these subunits DC2 and KCP2. Oligosaccharyltransferase transfers preassembled glucose3-mannose9-N-acetylglucosamine2 core oligosaccharides from the ER membrane lipid donor dolichol pyrophosphate to nascent polypeptide chains. Glycosylation facilitates protein folding by increasing the solubility of yet-unstructured nascent chains and allows nascent chains to enter the folding sensor system by recruiting the lectin chaperones calnexin and calreticulin (Ellgaard et al. 1999). Access to the calnexin\/calreticulin system requires modification of the N-glycans by the enzymes glucosidase I (GI), glucosidase II (GII), UDPglucose: glycoprotein glucosyltransferase (UGT1), ER-mannosidase 1 and EDEMs (ER degradation enhancing-mannosidase-like protein) (reviewed by Ruddock and Molinari 2006). Polypeptide released from this sensor system fulfils quality control requirements and can exit ER and transport to their final destination. However, misfolded polypeptides are retrotranslocated into the cytosol and degraded (described in \u201cUbiquitin metabolizing enzymes\u201d). A number of enzymes involved in N-glycan modification have been reported in association with ER subfractions including GI (Gilchrist et al. 2006), GII (Knoblach et al. 2003; Foster et al. 2006; Gilchrist et al. 2006) and UGT1 (Gilchrist et al. 2006) following analysis by mass spectrometry. In addition to the expected detection in the rough microsomes (Knoblach et al. 2003; Gilchrist et al. 2006), these enzymes have also been detected in smooth microsomes (Gilchrist et al. 2006). This is consistent with the localization of GII and UGT in smooth ER and also ERGIC by immuno-electron microscopy (reviewed by Roth et al. 2002). These data are also consistent with the detection of chaperones calnexin and calreticulin, in rough and smooth microsomes from rat liver (Gilchrist et al. 2006) as well as the presence the ERAD machinery in smooth microsomes (Gilchrist et al. 2006). Thus, glycoprotein quality control not only takes place in the rER but also may occur to a significant extent in the sER.\nBiosynthetic cargo\nMany hepatic secretory proteins were detected by proteomics analyses within ER fractions from normal liver. These include albumin, serotransferrin, apolipoproteins A, B, and E, Complement C3, alpha-2u-globulin, and transthyretin (Foster et al. 2006; Galeva and Altermann 2002; Gilchrist et al. 2006; Knoblach et al. 2003). The association of these proteins with the endoplasmic reticulum is assumed to be biogenetic. The proteins are thought to be in the lumen of the ER en route for secretion via the early secretion apparatus. Consistent with this suggestion quantitative proteomics revealed two to fivefold higher concentrations of these proteins in a Golgi fraction when compared with that in rough and smooth ER fractions from the same tissue (Gilchrist et al. 2006). Although transmembrane proteins destined for transport out of the ER to the Golgi Apparatus and to the plasma membrane are expected to be part of the biosynthetic cargo of the ER such proteins maybe either too low in abundance for detection by mass spectrometry or they could be considered as membrane contaminants of the ER preparations. Confirmation of the latter suggestion would require double labeling for specific membrane markers using imunocytochemistry of the subcellular fractions used for proteomics analysis.\nProteins involved in cargo exit and membrane traffic\nInteractions between membrane derivatives of the ER and the Golgi Apparatus are well known and this includes formation of ER exit sites to permit transport of newly synthesized protein out of the ER towards the Golgi (Palade 1975) and the formation of the ER-Golgi intermediate compartment to permit membrane recycling between the ER and the Golgi (Lee et al. 2004). Proteomics studies of different fractions of the ER have detected a number of proteins involved in membrane traffic. Coat proteins of the COPI and COPII protein complexes were detected in smooth microsomes (Gilchrist et al. 2006) and in ER\/Golgi\/vesicles (Foster et al. 2006). The smooth microsomes used in the studies of Gilchrist et al. (2006) are considered derivatives of the tER based on previous molecular, histochemical and morphological characterizations (Lavoie et al. 1996, 1999; Roy et al. 2000). As would be expected for proteins that recycle between the ER and the cis Golgi P24 family members (p24A, gp25L2, and TMP21) ERGIC-53, Rab1a, Sec22b, and the KDEL receptor were detected in smooth microsomes (Gilchrist et al. 2006) in ERGIC (Breuza et al. 2004) and in ER\/Golgi-derived vesicles marked by the cis Golgi marker p115 (Foster et al. 2006). The tER plays a key role in the formation of cargo exit sites and since the extent and amount of cargo exit sites may vary between different cell types (Fan et al. 2003; Bannykh et al. 1996) the relative content of cargo and membrane traffic proteins is expected to vary in proteomics studies using membrane-derivatives of the tER from different cell types.\nER chaperones\nThe ER functions to both co- and post-translationally modify newly synthesized proteins and lipids and sort them for housekeeping within the ER and for transport to their sites of function away from the ER (Palade 1975). A variety of proteins both membrane-bound and luminal are involved in the control of maturation of nascent proteins and lipids and include proteins involved in folding, in the regulation of calcium homeostasis, in oligomerization, and glycosylation (Hebert and Molinari 2007). Many such proteins have been detected by proteomics analysis of fractions representing different domains of the ER. Knoblach et al. (2003) have concentrated their efforts on the analysis of luminal proteins in purified rough microsomes from mouse liver and have detected BiP, GRP94, lectin-like chaperones (calnexin, calreticulin), peptidylprolyl isomerases, thiol disulphide oxidoreductases (PDI, P5 (CaBP1), ERp72, ERp57, ERp44, ERp29, and ERp46). Quantitative proteomics revealed these same chaperones to be in similar concentrations in rough and smooth microsomes from rat liver (Gilchrist et al. 2006). Detergent extraction of the rough and smooth microsomes after salt wash revealed the presence of the ER chaperones in the aqueous phase of the detergent extracts, thus confirming the luminal localization for all these proteins (Gilchrist et al. 2006).\nThe subunits of the chaperonin containing TCP-1 were detected associated with ER subcellular fractions (Foster et al. 2006; Gilchrist et al. 2006). Although tubulin and actin are well-known substrates for chaperonin t-complex proteins, new substrates are being defined and include proteins involved in cell cycle events (Liu et al. 2005). A role for chaperonin containing TCP-1 in protecting proteolytic intermediates in the MHC class I antigen-processing pathway has been described (Kunisawa and Shastri 2003). Whether this chaperone complex participates in MHC class I antigen processing at the cytosolic surface of a specific ER subcompartment remains to be confirmed.\nCalcium-handling proteins\nThe ER plays an important role in Ca2+-homeostasis. This organelle has been described as a heterogeneous compartment with respect to the distribution of its Ca2+-handling proteins including the Ca2+-binding proteins, the Ca2+-pumps, and the Ca2+-release channels (Papp et al. 2003). Whereas the quantitative proteomics analysis of Gilchrist et al. (2006) revealed the Ca2+-pump SERCA2 and the Ca2+-release channel inositol 1,4,5-trisphosphate (InsP3) receptor to be enriched in smooth microsomes most Ca2+-binding proteins including calreticulin, glucose-regulated protein 78 and 94 (Grp78\/BiP and Grp94), and protein disulfide isomerase, were similarly distributed between rough and smooth microsomes. Electron energy loss imaging analysis confirmed the heterogeneity of calcium distribution in ER of PC12 cells but was unable to distinguish distributions with respect to rough and smooth ER domains (Pezzati et al. 1997). The relative capacity for calcium storage and release of the rough and the smooth domains of the ER may vary between different cell types and the volume of these two subcompartments may influence this capacity.\nEnzymes of lipid and glucose metabolism\nThe endoplasmic reticulum plays an important role in lipid biosynthesis and in glucose metabolism. Because of the large amount of sER in steroid-producing cells, lipid biosynthesis has often been attributed to the smooth domain of the ER. Indeed enzymes involved in lipid biosynthesis have been localized to the sER in such cells (Frederiks et al. 2007). However, smooth microsomes purified from the steroid-producing adrenocortical cells revealed not only enzymes involved in lipid synthesis but surprising high levels of translocation apparatus, and oligosaccharyltransferase complex proteins (Black et al. 2005). The implication of this finding is that lipogenic enzymes and other proteins involved in lipid metabolism should be easily recognized in both sER and rER domains. Lipogenic enzymes (ex. fatty acid synthase, acetyl-CoA carboxylase 1, and ATP citrate lyase) and lipid transfer proteins (ex. microsomal triacylglycerol transfer protein) indeed have been detected by proteomics analysis in both rough microsomal (Foster et al. 2006; Gilchrist et al. 2006; Knoblach et al. 2003) and smooth microsomal (Gilchrist et al. 2006) fractions. In terms of the distribution of proteins involved in lipid metabolism the data suggests a lack of distinction between sER and rER membrane domains. This suggestion is consistent with the previous morphological demonstration of lipid droplet formation in association with rER domains in situ (Robenek et al. 2006) and the demonstration of the formation of lipid droplets using ribosome-depleted rough microsomes in a cell-free reconstitution system (Paiement et al. 1994).\nA number of proteins involved in the pentose-phosphate pathway (PPP) have been reported in association with ER subfractions including transketolase (Foster et al. 2006; Gilchrist et al. 2006), glucose-6-phosphate dehydrogenase (G6PD) (Gilchrist et al. 2006) and transaldolase (Gilchrist et al. 2006) following analysis by mass spectrometry. The detection of the transketolase and G6PD in the ER by mass spectrometry is consistent with previous electron microscope cytochemical studies showing these proteins in association with the ER in situ (Boren et al. 2006; Frederiks and Vreeling-Sindelarova 2001). Thus PPP enzymes may play a role in glucose metabolism at the level of the ER in specific cell types.\nProteins of detoxification and drug protein targets of the ER\nThe xenobiotic metabolism pathway of the ER includes functioning cytochrome p450 family (CYPs) proteins, UDP- glucuronosyltransferases, and carboxylesterases. Extensive biochemical and cytochemical data exist showing enrichment of such proteins in ER (reviewed in Seliskar and Rozman 2007) but little data differentiates between the rER and sER subcompartments. In the quantitative proteomics study of Gilchrist et al. (2006) over 30 different cytochrome P450s were detected as well as nine carboxylesterases and over 10 different glucuronosyltransferases and most of these proteins were in similar concentrations in rough and smooth microsomes. Continuity between the rough and smooth ER domains may endow specific ER proteins with the capacity to diffuse freely along the membranes (Nikonov et al. 2002) and within the lumen (Snapp et al. 2006), thus explaining equal distribution of enzymes involved in xenobiotic metabolism in rER and sER domains.\nEndoplasmic reticulum proteins of detoxification and drug protein targets are now being examined quantitatively by proteomics in diseased states and under controlled drug treatments. Proteomics studies of liver microsomes under controlled drug treatments have revealed differential expression of specific ER proteins. Cytochromes P450 2B1, and 2B2, protein disulfide isomerase A3 and A6, and 78-kDa glucose-regulated protein were differentially expressed following phenobarbitol treatment (Galeva and Altermann 2002). p450 family proteins CYP1A2, \u22122A4\/5, \u22122B10, \u22122B20, \u22122C29, \u22122C37, \u22122C38, \u22123A11, and \u221239A1 were up-regulated, and CYP2C40, \u22122E1, \u22123A41, and \u221227A1 were down-regulated by treatment with 1,4-bis-2-(3,5-dichloropyridyloxybenzene) (Lane et al. 2007).\nUbiquitin metabolizing enzymes\nEndoplasmic reticulum-associated degradation (ERAD) is a regulated process whereby misfolded and unassembled proteins are recognized and retained in the ER by the quality-control apparatus and subsequently extracted, polyubiquitinated, and finally degraded in the cytoplasm by the multi-subunit 26S proteasome complex (reviewed by Meusser et al. 2005 and Romisch 2005). Ubiquitin and ubiquitin-activating enzymes were shown by electron microscope immuno-gold labeling to be associated with the post-ER\/pre-Golgi network consisting of anastomosing tubulated and fenestrated membranes (Raposo et al. 1995). More recently misfolded protein was detected in an ER subdomain and in enlarged pre-Golgi intermediates by electron microscope immuno-gold labeling (Zuber et al. 2004). The quantitative proteomics analysis of Gilchrist et al. (2006) revealed proteasome subunits as well as ubiquitin metabolizing enzymes only in smooth microsomes. This data is consistent with a previous report showing that proteasomes were associated with the smooth endoplasmic reticulum and that they were practically absent from the rough endoplasmic reticulum (Palmer et al. 1996). Smooth microsomes used in the study of Gilchrist et al. (2006) may correspond to the compartment identified and described by Raposo et al. (1995) and Zuber et al. (2004). The smooth ER could be an important subcellular site for proteasome-dependent degradation of misfolded protein?\nProteins involved in antigen processing\nMajor histocompatibility complex (MHC) class I molecules present short, perfectly cleaved peptides on the cell surface for immune surveillance by T lymphocytes (Hammer et al. 2007). The intracellular processes that take part in creating the antigen-presenting machinery of the plasma membrane occur at the level of the ER and involve protein degradation, membrane translocation, and protein folding (reviewed by Elliott 2006 and Hammer et al. 2007). Protein degradation is carried out on the cytosolic side of the ER by the proteasome and the enzyme tripeptidyl peptidase II. The group II chaperonin TRiC (TCP-1 ring complex) is thought to chaperone the peptide between proteases and TAP the peptide transporter of the ER. TAP is composed of two transmembrane proteins, TAP1 and TAP2, and participates in the translocation of peptide to the lumen of the ER in an ATP-dependent step. On the luminal side of the ER the peptide is further processed by ERAAP, the ER aminopeptidase associated with antigen processing. The multicomponent MHC class I peptide-loading complex (PLC) includes MHC class I molecules, beta 2 microglobulin, the chaperone calreticulin, the oxidoreductases ERp57, and protein disulfide isomerase, the class I-specific accessory molecule tapasin and the peptide transporter TAP. This complex ensures the establishment of proper conformation of MHC class I molecules for peptide loading in the ER. A number of the components of the PLC have been reported in proteomics studies of ER fractions (Foster et al. 2006; Gilchrist et al. 2006). The study of Gilchrist et al. (2006) revealed higher amounts of beta 2 microglobulin, tapasin, and ERAAP in smooth microsomes compared to rough microsomes. Electron microscope immuno-gold labeling has previously revealed Class I molecules and beta 2 microglobulin in association with the post-ER\/pre-Golgi network consisting of anastomosing tubulated and fenestrated membranes (Raposo et al. 1995). Whether the transitional zone of the ER, which contains the smooth ER is a major site for antigen processing and peptide loading onto the PLC remains to be confirmed.\nEndoplasmic reticulum proteins were previously detected in purified phagosomes using proteomics analysis (Garin et al. 2001). This observation prompted consideration that the ER maybe involved in phagocytosis and indeed electron microscope cytochemical, electron microscope immuno-gold labeling, and biochemical data were obtained to testify to this possibility (Gagnon et al. 2002). Endoplasmic reticulum involvement in phagocytosis has been confirmed independently by other laboratories (Ackerman et al. 2006; Becker et al. 2005) and the findings have led to additional proteomics studies and the important conclusions explaining antigen cross-presentation (Guermonprez et al. 2003; Houde et al. 2003). Therefore even though organelle proteomics studies may often suggest the presence of organelle contaminants using intuitive reasoning and corollary experiments as was done by Michel Desjardins and colleagues, in the case of ER proteins associated with phagosomes such studies may also provide information leading to new paradigms.\nCytoskeletal proteins\nThe ER interacts with the cytoskeleton to maintain its position within sedentary cells and to establish new intracellular positions in motile cells. The ER contains a receptor for specific interaction with microtubules (Farah et al. 2005; Klopfenstein et al. 1998) and can move along both microtubules and actin cables (Allan and Vale 1994; Kachar and Reese 1988; Terasaki and Reese 1994). Based on these reports one might expect proteomics studies of the ER to reveal the presence of associated cytoskeletal proteins. Indeed, protein components of microtubules but not of microfilaments have previously been detected in association with ER subfractions from mammalian liver (Foster et al. 2006; Gilchrist et al. 2006). In addition a number of molecular motors including protein members of the myosin, kinesin, and dynein protein families have been detected in association with these same membrane fractions. Subcellular membrane movements are directed by molecular motors including myosin, dynein, and kinesin family molecular motors (Mallik and Gross 2004). Other cytoskeletal proteins, which have previously been detected in ER fractions include filamin A and B, the actin-binding proteins profilin 1, and transgelin 2.\nFilamin A and B were detected in smooth ER fractions (Gilchrist et al. 2006). Filamin A has been implicated in intracellular traffic of several cell-surface receptors and was observed to bind one of these at the level of the endoplasmic reticulum (Feng et al. 2005; Liu et al. 1997). Therefore filamin A may bind yet unidentified nascent receptors in the ER in hepatocytes and promote traffic of these molecules to the cell surface.\nThe actin-binding proteins profilin 1 and transgelin 2 were detected in the ER fractions. Higher amounts of these proteins were found in association with smooth microsomes compared with that in rough microsomes (Gilchrist et al. 2006). Profilin 1 is able to bind membrane phosphoinositides and thus affect cell signaling and has been implicated in membrane traffic (Witke 2004), thus it maybe involved in ER membrane traffic in hepatocytes. Transgelin 2 mRNA was reported overexpressed in hepatocellular carcinoma and this protein has been proposed as a potential diagnostic marker for this disease (Shi et al. 2005). The role of transgelin 2 at the level of the ER remains to be determined.\nCytoskeleton-associated protein 4\/CLIMP-63 was reported predominantly in rough microsomes of rat liver by the studies of Gilchrist et al. 2006. CLIMP-63 serves as a specific ER- microtubule receptor (Klopfenstein et al. 1998) and was shown to link the ER to microtubules via the neuronal microtubule-associated protein 2 (MAP-2) in the dendritic compartment of neurons (Farah et al. 2005). Interestingly, the non-neuronal microtubule-associated protein 4 (MAP-4), which has an identical microtubule-binding domain to MAP-2 was also detected in association with rough microsomes (Gilchrist et al. 2006). Whether MAP-4 and CLIMP-63 make a link between the rER and microtubules thus immobilizing rER structure in hepatocytes remains to be confirmed.\nReticulons constitute a family of proteins implicated in the tubular structure of the smooth ER (Voeltz et al. 2006). Reticulon 3 was detected by mass spectrometry in smooth microsomes of rat liver by Gilchrist et al. (2006) and over-expression of this protein in Hela cells revealed production of fluorescent tubules, which co-distributed along microtubules as revealed by double-labeling immunofluorescence (Wakana et al. 2005). As proposed by Shibata et al. (2006) reticulons promote long unbranched ER tubules. Endoplasmic reticulum tubules have previously been shown to be highly dynamic structures capable of moving along tracks of microtubules (Waterman-Storer and Salmon 1998). Perhaps the tubular structure of the ER favors ER\/motor\/microtubule interaction? However if reticulons promote long unbranched ER tubules, how do they affect tritubular jonctions as observed in the large inter-connecting networks of tubules in sER in steroid cells or in hepatocytes?\nProteins involved in signaling\nThe ER is involved in many signaling pathways including the unfolded protein response (UPR) (Bernales et al. 2006), apoptosis (Chae et al. 2004; Nakajima et al. 2004), ubiquitination and proteasome degradation (Kostova and Wolf 2003) and some of the involved signaling molecules have been shown to associate in a transient manner with ER membranes. This includes Ras protein, which is involved in cell signaling (Sobering et al. 2004), the AAA ATPase p97, which is involved in ER assembly (Lavoie et al. 2000) and in proteasome degradation (Ye et al. 2003) and BAX, which is involved in apoptosis (Zong et al. 2003). Excluding proteins involved in ubiquitination and proteasome degradation (see above), few signaling proteins have yet to be detected in proteomics studies of ER fractions. This is true for a number of regulatory proteins such as SREBP-1, which regulates lipogenic enzymes (Bengoechea-Alonso and Ericsson 2007), Ire-1 which regulates the UPR (Bernales et al. 2006) and Bax, which is involved in apoptosis (Hetz et al. 2006). These signaling proteins probably represent low abundance proteins of the ER and require up-regulation following physiological activation to be detected by existing mass spectrometry techniques.\nUnder conditions of accumulation of misfolded and unassembled proteins, the UPR acts to modify the ER by up-regulating chaperones so as to increase its folding capacity. Morand et al. (2005) used quantitative proteomics analysis and revealed a number of chaperones (GRP94 and PDI) to be up-regulated in ER microsomes from the liver of a fructose-fed, insulin-resistant hamster model and up-regulation was attributed to ER stress. Ras protein and p97 have been reported in association with ER fractions (Gilchrist et al. 2006; Galeva and Altermann 2002). Association of Ras with the ER is consistent with previous reports of the association of Ras with membranes of the secretory pathway including ER (Zheng et al. 2007). Association of p97 with ER is consistent with its role in ER assembly (Lavoie et al. 2000) and proteasome degradation (Ye et al. 2003).\nPhosphoproteins associated with the ER\nThe ER is a membrane-bound compartment that functions by interacting with cytoplasmic and luminal-soluble proteins. Among the soluble proteins that interact with the ER membrane in a transient manner are signaling proteins. Reversible phosphorylation has been shown to control protein interaction at the ER of several signaling proteins including valosin-containing protein\/p97 (Lavoie et al. 2000) BCL-2 (Lin et al. 2006) and Ire1 (Bernales et al. 2006). In addition, reversible phosphorylation has been shown to play a role in the control of ER structure. For example, phosphorylation of CLIMP-63 was shown to affect ER structure during interphase (Vedrenne et al. 2005) and phosphorylation of p47, a cofactor of p97, was shown to control ER structure at mitosis (Kano et al. 2005). Since the ER controls key metabolic events including events crucial to cell survival, it is expected that new phosphoproteins are yet to be identified, especially in relations to control of cell cycle events.\nA number of proteins have been detected in ER fractions, which have previously been shown to be phosphoproteins. Phosphoproteins were identified in transformed cells by analysis of tyrosine-phosphorylated peptides immunoprecipitated using anti-phosphotyrosine antibodies (Rush et al. 2005). A significant number of these previously identified tyrosine-phosphorylated proteins have in fact been detected in association with ER membranes by proteomics analysis (Foster et al. 2006; Gilchrist et al. 2006). Many of these proteins are not conventional ER proteins but rather are better known as cytosolic proteins; these include proteins involved in mRNA metabolism (PABP1, YB-1, ELAV-like 1, eEF-1-alpha), proteins involved in phospholipid binding (annexin I, annexin II), and proteins involved in de novo lipid synthesis (ATP citrate lyase). Some of these proteins are substrates of key signaling pathways including the Akt-signaling pathway (ex. ATP citrate lyase, Berwick et al. 2002) and some have been shown to have a phosphorylation status which has been associated with subcellular trafficking (Hibino et al. 2006). Thus proteins previously defined as tyrosine-phosphorylated proteins were observed in ER membranes by proteomics analysis and these may undergo cycles of tyrosine phosphorylation and dephosphorylation promoting specific ER-related functions in cells, which are yet to be better defined.\nSerine\/threonine protein phosphorylation can also modify subcellular protein localization. Phosphorylation of the disc\u2019s large tumor suppressor protein controls its membrane localization (Massimi et al. 2006). CLIMP-63 is serine phosphorylated during mitosis and regulates ER-microtubule binding (Vedrenne et al. 2005).\nConclusions and future perspectives\nFrom available proteomics data parts of which are supported by electron microscope protein localization studies some tentative conclusions can be drawn about the relative segregation of proteins and molecular machines in the subcompartments of the ER. CLIMP-63 and ribosomes are enriched in rER. Reticulon, enzymes involved in ubiquitination, the proteasome, some cytoskeletal proteins, proteins involved in antigen processing, and coat proteins are enriched in sER. In contrast, proteins that appear equally distributed between rER and sER include proteins of the translocon, biosynthetic cargo, chaperones, proteins of detoxification, and proteins involved in lipid and glucose metabolism.\nBecause proteomics of ER subcompartments is carried out using subcellular fractions, there are limitations that have to be considered when trying to interpret the results of the protein analysis. For example, the relative purity of the fractions will vary and proteins of fragments of contaminating organelles will be present in the fraction and be identified in the analysis. Moreover molecular dynamics at the cytosolic and luminal surfaces of the ER have to be taken into consideration. The composition of the ER is subject to change based on molecular interactions occurring on both cytosolic and luminal sides of the ER membrane. On the cytosolic side of the ER interaction with the cytoskeleton (to change the location, shape or size of the ER) or interaction with signaling proteins (to activate specific signaling pathways, e.g., apoptosis) leads to transient associations between the ER and cytosolic proteins. On the luminal side of the ER interactions between newly synthesized proteins and chaperones can vary under specific conditions (for example during ER stress) and will affect the overall molecular composition. Up-regulation of proteins involved in detoxification may occur under exposure to toxic chemicals and affect ER composition. Thus depending on the cell physiology protein associations with the ER may vary. Dynamics of protein interactions in organelles are often controlled by posttranslational modifications including phosphorylation. Understanding such modifications is key to understanding site-specific protein function.\nProteomics studies of the subcompartments of the ER have lead to insights into the function of the different compartments of this organelle and new paradigms. However data obtained using proteomics analysis should be complimented by cytological techniques to confirm the localization of the proteins in the ER subcompartments and molecular biology should be used to modulate protein expression to examine the function. The combination of these approaches not only will yield new information about the proteins but they will also expand knowledge on the protein families to which they belong and\/or protein complexes of which they are part of.","keyphrases":["proteomics","cytology","protein complexes","smooth endoplasmic reticulum","rough endoplasmic reticulum","protein localization","transitional endoplasmic reticulum","molecular topology"],"prmu":["P","P","P","P","P","P","R","R"]}
{"id":"Matern_Child_Health_J-2-2-1592147","title":"Treatment of Tobacco Use in Preconception Care\n","text":"The preconceptional period provides an important opportunity to actively assess and treat tobacco use and to assist parents-to-be in quitting permanently to avoid smoking-related pregnancy and long-term health consequences.\nThe use of tobacco prior to conception is associated with male impotence, conception delay, and primary as well as secondary infertility. During pregnancy, tobacco use increases the risk for spontaneous abortion, ectopic pregnancy, low birth weight, SIDS, premature birth, and other fetal and maternal complications [1]. Each year, almost one-half million babies in the United States are born to mothers who report smoking during pregnancy [2]. For smokers, tobacco use continues to be the leading cause of morbidity and mortality in the United States, resulting in approximately 440,000 deaths per year [3].\nWomen smokers are at increased risk for cancer, cardiovascular disease, and pulmonary disease. Since 1987, lung cancer has outpaced breast cancer as the leading cause of cancer death among women, and exposure to environmental tobacco smoke (ETS) is a cause of lung cancer and coronary heart disease among lifetime nonsmokers. Moreover, infants born to women who are exposed to ETS during pregnancy may also have increased health risks, including small decrements in birth weight and slightly increased risk for intrauterine growth retardation, compared with infants born to women who were not exposed [1].\nDespite all that is known about the devastating health consequences of smoking, 20% of U.S. women smoked cigarettes in 2003 [4] and 11.4% of women giving birth reported smoking during their pregnancy [5]. It is encouraging that 70% of smokers want to quit, and about two-fifths quit for at least a day every year [4]. To avoid early pregnancy complications, women who smoke should be counseled to quit before they become pregnant. In addition, smokers who are partners of mothers-to-be should also be included in any treatment plans given the strong association with partner smoking and relapse [6] as well as concerns about ETS exposure.\nTreatment for tobacco dependence is safe, effective, available, and affordable [7]. The 2000 Public Health Service document, Treating Tobacco Use and Dependence: A Clinical Practice Guideline [7], as well as the 2001 Centers for Disease Control and Prevention publication, Guide to Community Preventive Services [8], both provide strong evidence-based guidelines for treating smokers using a comprehensive system that includes the 5A's (a brief 5-step counseling approach recommended by the 2000 PHS clinical practice guideline for all smokers). These documents also provide details of how reminder systems, telephone quitlines, decreased co-pays and costs of treatment, mass media campaigns, smoke free environments and increased prices for tobacco products support the cessation process [7, 8].\nAt the clinical level, every clinician providing preconception care should implement a tobacco treatment system based on the 5 A's and beginning with a systematic way to assess tobacco use. This evidence-based counseling approach includes asking every patient about tobacco use and, if they smoke, advising them to quit, assessing willingness to quit, assisting them in quitting, and arranging follow-up. Assisting may include the offer of pharmacotherapies or additional counseling, each of which doubles the quit rates in non-pregnant adult populations [7]. The effectiveness of counseling increases as the intensity (number and length of sessions) increases. At present, seven first-line FDA-approved medications are available: bupropion varenicline and five nicotine replacement therapies (gum, patch, lozenge, inhaler, and nasal spray) [7]. Because the safety and efficacy of pharmacotherapy during pregnancy has yet to be established, the preconception period provides an excellent interval before pregnancy to offer these effective adjuncts to quitting.\nOnce abstinent, patients should be followed to help avoid relapse. For those smokers who are not willing to quit in the near future, the PHS clinical practice guidelines recommend the 5 R's (relevance, risks, rewards, roadblocks, repetition) to enhance patients\u2019 motivation to quit smoking [7].\nClinical systems that provide tobacco-dependence treatment should also include a reminder system to alert providers to advise smokers in their practice [8]. This system might be an electronic medical record alert or simply a stamp or sticker on the patient's chart to indicate tobacco status - current, former, or never. The PHS guideline also recommends using the 305.1 ICD-10 code for tobacco dependence for billing purposes [7]. This code also may be used by health-care systems to monitor and improve the provision of effective treatment.\nState quitlines provide an effective vehicle for telephone counseling and can be accessed by calling 1-800-QUIT NOW, a national portal number to refer tobacco users directly to their state quit line based on their area code. A small number of states also provide over-the-counter medications to eligible populations in conjunction with telephone counseling. Another group of states (e.g., Maine, Massachusetts, Oklahoma, Oregon, and Wisconsin) encourages providers to fax referrals (with patient consent) to the state quitline, which in turn proactively calls the smoker to begin counseling. Most quitlines send a \u201cquit kit\u201d to each caller.\nWith effective treatment, quit rates can be as high as 25%\u201330% in the general population [9]. Because many women spontaneously quit upon learning about their pregnancy\u2014ranging from 11% to 28% in publicly insured pregnant smokers to 40% to 65% in privately insured pregnant smokers [10], it is reasonable to expect that effective preconception tobacco-dependence treatment could exceed these rates, particularly if partners who smoke are treated as well. Placing these cost-effective treatment systems into preconception practice should not be insurmountable, given the evidence, the affordability, the lessons learned from other clinical tobacco-dependence systems, and the availability of resources.\nAvailable Resources for learning more about tobacco dependence treatment:Treating Tobacco Use and Dependence. JAMA June, 28, 2000 (Summary article)Treating Tobacco Use and Dependence (full document). Available at www.surgeongeneral.gov\/tobacco.Agency for Health Care Research and Quality, patient and provider materials. Available at www.ahrq.gov.The Guide to Community Preventive Services. Available at www.thecommunityguide.org .Telephone Quitlines: A Resource for Development, Implementation, and Evaluation. Available at www.cdc.gov\/tobacco.Smoke-Free Families provider, patient, and system materials. Available at www.smokefreefamilies.org.Environmental Protection Agency, smokefree home ban materials. Available at www.epa.gov .March of Dimes at www.marchofdimes.com.Nicotine and Tobacco Research Journal supplement: Helping Pregnant Women Quit Smoking: Progress and Future Directions. Available at http:\/\/www.ntrjournal.org\/pregnancy.html.","keyphrases":["preconception care","pregnancy","smoking","keywords"],"prmu":["P","P","P","U"]}
{"id":"Intensive_Care_Med-3-1-1915615","title":"Complete recovery from an unusual cause of coma\n","text":"Case\nA\u00a051-year-old man was admitted to the emergency room because of progressive lethargy, slurred speech, weakness of the right arm and low-grade fever. Physical examination showed no additional neurological deficits. He had a\u00a0history of a\u00a0tick bite 6 months previously which had been treated with antibiotics. Apart from leukocytosis (10.5\u202f\u00d7\u202f109\/l) and raised C-reactive protein (62\u202fmg\/l), additional blood examination was normal. Cerebral contrast-enhanced computed tomography (CT) revealed no abnormalities. Cerebrospinal fluid (CSF) examination showed an elevated cell count of 66\u202f\u00d7\u202f106 cells\/l (of which 97% were lymphocytes), slightly elevated protein concentration of 1.03\u202fg\/l, a\u00a0normal glucose level of 3.7\u202fmmol\/l and an elevated pressure of 29\u202fcmH2O.\nCase discussion\nThe onset of progressive lethargy over a\u00a0period of days, slurred speech and the weakness indicates a\u00a0lesion of the central nervous system. Given the progression of lethargy, the fever and the absence of acute onset, an infectious origin seemed more likely. This was suggested by the CSF examination, showing an elevated cell count, especially lymphocytes. The differential diagnosis consisted of (viral) meningo-encephalitis \u2013 most likely caused by enteroviruses, arboviruses or herpesviruses \u2013 neuroborelliosis and vasculitis. Antibiotics and antiviral drugs were started.\nCase\nWithin 2 days, the patient's condition deteriorated into a\u00a0deep coma requiring mechanical ventilation. Initial treatment consisted of acyclovir and ceftriaxone, anticipating a\u00a0possible neuroborelliosis and herpes simplex encephalitis. However, repeated blood and CSF cultures were normal, making an infective origin less likely. Serological tests for Lyme disease and repeated polymerase chain reaction (PCR) testing for herpes simplex virus (HSV) were negative. Systemic vasculitis was ruled out. The patient remained in an areactive coma without focal signs and abnormal brainstem reflexes. An electro-encephalogram (EEG) after 1 week showed non-specific diffuse slow activity. Three weeks after admission cerebral gadolinium-enhanced magnetic resonance imaging (MRI) was performed, showing numerous hyperintense lesions, on T2-weighted images, of the brainstem, the left cerebellar hemisphere, the basal ganglia on both sides and in periventricular locations (Fig.\u202f1a\u2013c).\nFig.\u00a01a\u2013c T2-weighted MRI of the brain, demonstrating hyperintense lesions of a brainstem and left cerebellar peduncle, b basal ganglia and c left periventricular region. c\u2013e T2-weighted MRI of the brain 2 months after therapy, demonstrating a\u00a0decrease in white matter lesions\nCase discussion\nOn ICU admission the main medical problems were (1) respiratory insufficiency requiring mechanical ventilation, (2) progressive loss of consciousness and (3) the unknown origin of the coma. Maintenance of an adequate airway and concomitant aspiration pneumonia necessitated mechanical ventilation. There are numerous possible causes of progressive loss of consciousness, including cerebrovascular accidents, cerebral infections (viral, bacterial, parasites, tuberculosis, Lyme disease), neoplastic and auto-immune diseases (sarcoidosis, vasculitis, SLE). Despite the elevated white cell count and the presence of protein in the CSF, no infectious origin was determined. Screening for HIV and coagulation disorders was negative. Repeated blood and CSF cultures were negative, and there was no response after 14 days of ceftriaxone and acyclovir. Nevertheless CSF analysis after treatment was interpreted cautiously. Many viruses responsible for meningo-encephalitis cannot be identified using common tests. MRI showed multiple hyperintense brain lesions, especially of the brainstem. Because of the massive involvement of the brainstem and persistence of coma for 2 weeks, the prognosis was considered poor.\nCase\nAcute disseminated encephalomyelitis (ADEM) could not be ruled out, so treatment was continued. Additionally, treatment with intravenous dexamethasone (4\u202fmg four times a\u00a0day) was started. Upon treatment with corticosteroids for 4 weeks the patient made a\u00a0slow but full recovery within 2\u00a0months. After 2 months cerebral gadolinium-enhanced MRI was repeated, showing substantial amelioration of the intracerebral lesions (Fig.\u202f1d\u2013f). One year after treatment the patient still functions well, without relapse.\nCase discussion\nElevated pressure, white cell count and protein level in the CSF in combination with the extensive white matter lesions confirmed the diagnosis of ADEM. ADEM is a\u00a0rare cause of prolonged coma with complete recovery under specific treatment, and intensivists should be aware of this disease. Withdrawal or withholding of care in patients with prolonged coma can hardly be discussed as long as the cause of the coma remains unknown and the prognosis cannot be accurately established. Treatment with intravenous corticosteroids resulted in a\u00a0full recovery. The absence of a\u00a0previous infection is a\u00a0peculiar aspect of our case.\nComments\nThis case illustrates the remarkable outcome after a\u00a0long period of coma. Good recovery from a\u00a0coma is observed in only 10% of reported cases and depends on the aetiology, the accompanying clinical signs, and the depth and duration of the coma\u00a0[1]. ADEM is a\u00a0rare monophasic illness that is thought to develop from antigenic mimicry, with antibodies having cross-reactivity to host epitopes in the nervous system. The disorder typically occurs following vaccination or a\u00a0viral prodrome and is predominantly seen in children and in Japan, where viral encephalitis is more common\u00a0[2, 3, 4, 5]. A\u00a0preceding infection is reported in 50\u201375% of cases\u00a0[3, 4, 5]. The estimated incidence is 0.8 per 100,000 population per year\u00a0[3]. Some cases of ADEM among adults and the elderly have been reported, but the incidence is estimated to be considerably lower. The diagnosis of ADEM is reached on clinical grounds, evidence of white matter lesions on MRI and exclusion of other causes. These classic features were present in our case. However, diagnosis of ADEM may be difficult. Diagnostic overlap with multiple sclerosis (MS) may lead to underestimation of the prevalence [4, 5]. Some 0\u201333% of children and 35% of adults initially diagnosed with ADEM will eventually develop MS\u00a0[4, 5, 6]. Therefore this diagnosis should be considered, especially in adults. Follow-up by repeated MRI, at intervals not shorter than 6 months, may anticipate this development\u00a0[7]. The differential diagnosis consists of meningo-encephalitis, meningitis and cerebral involvement in auto-immune diseases\u00a0[8]. In severe cases, ADEM may lead to coma. There is no standard treatment for ADEM. It is a\u00a0rare disease, and no formal clinical trials of any therapeutic agent have been published. Thus the management of the disease rests on strategies that have appropriate effects on the plausible disease mechanisms. Present treatments rely on immunosuppression and immunomodulation.\nIntravenous corticosteroids (preferably methylprednisolone) is the first choice of treatment and usually leads to full recovery. Plasma exchange and intravenous immunoglobulin should be considered by deterioration on corticosteroids\u00a0[9, 10].\nIn conclusion, treatment of coma should be continued when the cause of the coma is unclear, even if there are numerous lesions in the brain. ADEM should be considered in comatose patients with raised CSF pressure, protein and lymphocytes, negative CSF cultures, and multiple white matter lesions on cerebral MRI. ADEM is predominantly seen in children, but also occurs in adults. Recovery may last weeks.","keyphrases":["coma","acute disseminated encephalomyelitis"],"prmu":["P","P"]}
{"id":"J_Med_Internet_Res-4-2-1761933","title":"Where Are They Now? A Case Study of Health-related Web Site Attrition\n","text":"Background. When considering health-related Web sites, issues of quality generally focus on Web content. Little concern has been given to attrition of Web sites or the \"fleeting\" nature of health information on the World Wide Web. Since Web sites may be available for an uncertain period of time, a Web page may not be a sound reference.\nIntroduction\n\"We've all heard that a million monkeys banging on a million typewriters will eventually reproduce the entire works of Shakespeare. Now, thanks to the Internet, we know this is not true\" [1].\nRobert Wilensky\nThe results of a recent major national survey found that about 110 million people in the U.S. \u2014 over half of the adult population \u2014 may be seeking health information online [2]. This compares with 54 million in 1998, 69 million in 1999 and 97 million in 2001. And according to the American Medical Association, on any given day, more people go online for medical advice than actually visit health professionals [3].\nWhen considering health-related Web sites, issues of quality generally focus on Web content: how to find relevant information, and how to assess the credibility of the publisher as well as the accuracy and reliability of a document retrieved [4]. Little concern has been given to attrition of Web sites or the \"fleeting\" nature of health information on the Web.\nScientist and scholar Sir Isaac Newton once said, \"If I have seen farther than others, it is because I was standing on the shoulders of giants\" [5]. Much, if not all, scholarship is based on relation to previous work, and when new scholarly work is produced, it is important that detailed and accurate information on sources consulted are cited. To facilitate referencing, scholarly works have been routinely collected and preserved in print by libraries and database producers [6,7]. But in terms of cataloging, storage and retrieval as it relates to the Web, the status quo does not apply. With the advent of the Web, libraries must now consider Web site information that may be created, change, move, expire and disappear; with no record of the information being preserved. Few libraries made the practice of collecting copies of Web pages [8].\nSince Web sites may be available for an uncertain period of time, a Web page may not be a sound reference. If a Web page or link disappears, chances are almost nonexistent of locating the reference at a later time. As a safeguard, it has been recommended that individuals keep a personal copy of Web pages as evidence that the information existed [9].\nTo address the issue of attrition, a defined set of health-related Web sites was examined at two separate time intervals.\nMethods\nIn an earlier study, a systematic survey was conducted to determine the validity of health claims on the World Wide Web for the herbal remedy Opuntia [10]. From December 1998 to May 1999, 184 Web sites were collected from which health claims were identified. Web sites were retrieved utilizing multiple search engines, and the Uniform Resource Locator (URL) for each Web site was recorded.\nIn this study, to determine the degree of attrition, each of the 184 Web sites obtained and recorded from the previous study were revisited at a later period of time. During May 2002, the previously recorded URL for each Web site was entered into the address field of the browser Netscape Navigator (version 4.7, Netscape Communication Corporation, Mountain View, California.) It was documented whether the original Web site could not be found, moved to a different URL location, or the URL and site location was found unchanged from the original search. For A Web site whose URL remained unchanged, it was also noted whether the Web site had maintained currency, (i.e. updated) since the original posting.\nSince it is conceivable that inaccessibility of Web sites may be due to temporary server problems, another attempt was made to access the sites at different periods of time. For each \"HTTP Error 404\" or similar message obtained from the initial URL checks, an attempt to access these sites was made during June 2002 on various days and times of day in the manner described above.\nResults\nResults indicate that when each URL address from the original set of 184 Web sites was re-entered into the address field of the browser, 108 (59%) of the sites could not be found, 31 (17%) had moved to a new URL address, and 45 (24%) of the sites could be found from the original URLs obtained in the previous study. Of the Web sites that moved to a new URL address, only 7 sites provided a link from the original URL to redirect the viewer to the new location. Of the Web sites still in existence, 17 (38%) provided update information from the original posting. The information is summarized in Table 1.\nTable 1\nAttrition of Health-related Web Sites for a Three-year Period **\nWeb Site Sponsor (No. of Sites)\nNot Found\nMoved To New URL\nURL Redirected\nURL to Site as Original\nMaintenance Update Provided\nHerbal Vendor (74)\n46\n14\n1\n14\n7\nFood\/Recipes Products (7)\n5\n1\n0\n1\n0\nEducational Institution(24)\n12\n1\n0\n11\n6\nGovernment Institution (3)\n1\n2\n1\n0\n1\nHistorical Essay (8)\n1\n1\n1\n6\n0\nTravel and Tourism (5)\n1\n2\n0\n2\n1\nMessage Board (15)\n15\n0\n0\n0\n0\nReference Guide (16)\n8\n6\n3\n2\n1\nPrint Media* (24)\n17\n2\n1\n5\n1\nExpert (7)\n2\n2\n0\n3\n0\nDoomsday Group (1)\n0\n0\n0\n1\n0\nTotals (184)\n108 (59%)\n31(17%)\n7(4%)\n45 (24%)\n17 (38%)\n* Includes book excerpts, newspaper and magazine articles, newsletters, a calendar reprint and a radio broadcast transcript\n** Original Web site addresses and content are available on the World Wide Web at http:\/\/ismo.ama.ttuhsc.edu\/users\/~veronin\/WebOpuntia.pdf\nIn this study, attrition is defined as the unavailability of a Web site when known to be previously accessible based on a known URL address. This did not include sites that were redirected to a new URL.\nApproximately three years after initial posting, over two-thirds of the health-related Web sites reviewed could not be found or had moved with no forwarding URL, and about one-third of the remaining sites maintained currency of information. It appears that links are terminated as Web sites are moved or removed, or as servers close down. This supports the notion that it is difficult, if not impossible, to locate information that was previously found on the Web, and if a reference to an item is provided, there is no guarantee that viewers will be able to find the site at a later date.\nIn this study, a comprehensive data set of Web sites on a specific health-related topic was obtained, and attrition was examined. Obviously an example from a single health-related topic is limited in what conclusions should be drawn. These findings cannot be generalized to other medical topics. But this raises the question that other health-related sites on the World Wide Web may vary in their degree of attrition, and warrants further research into methods of dealing with attrition with other medical topics.\nDiscussion\nThe average life of a Web page is about 77 days [11]. The perceived value of the Web lies in the immediate accessibility to a seemingly endless pool of information with no central controlling authority. This also makes the Web difficult to maintain. According to Chris Sherman, Associate Editor of SearchEngineWatch.com, (http:\/\/searchenginewatch.com),as automatic maintenance, most search engines remove missing URLs from their index when they recrawl and find that the pages are gone [12]. A different problem arises, though, when an organization has gone out of business but its site still exists. This is a much more difficult problem to handle, and to date, no search engine exists to locate or remove these sites.\nEnhancements in Web technologies hope to improve the problem of attrition. A prime example is the Internet Archive.\nThe Internet Archive\nThe Internet Archive (http:\/\/www.archive.org) is a digitallibrary of Web pages created with the lofty goal of cataloging all of the past and present publicly available material on the World Wide Web [11]. Accessible to the public for free, it contains more than 100 terabytes of data and is growing by 10 to 12 terabytes a month. Since 1996, the Internet Archive has been storing Web pages, including graphics files, from publicly accessible Web sites. A feature implemented October 2001 known as the \"Wayback Machine\" allows users to go back and view earlier versions of current Web sites or of Web sites that no longer exist.\nThe Wayback Machine serves as a source to find Web pages when the page or host cannot be located [11]. When a user encounters a \"File Not Found\" or similar message on the Web, the Wayback Machine can be accessed to find a facsimile of the Web page.\nThough a significant accomplishment towards recovering lost Web pages, the Wayback Machine has limitations. It is not searchable by keywords or text in the manner of a general search engine. The user must know the precise URL of a particular Web page or site to access the Archive. Having entered a URL address, the viewer is presented with a list of dates that designates when a particular page was archived. Also, though the Internet Archive contains more than 100 terabytes of data, much is still missing. For example, it does not contain the older gopher content and other non-Web files prior to 1996, and a relatively small number of pages exists from 1996, with content increasing to recent times.\nIssues of Quality and Content\nThe question may arise as to whether a relationship exists between Web site quality and attrition. Are poor quality sites more likely to disappear in time than sites of higher quality?\nA consensus has yet to be reached as to the properties a Web site needs to have to be considered \"high quality.\" Wilson states that \"quality remains an inherently subjective assessment, which depends on the type of information needed, the type of information searched for, and the particular qualities and prejudices of the consumer\" [13]. Yet many organizations and individuals have identified standards of quality that should be applied to the Web [14]. A practical approach for assessment has been described by Risk that provides benchmarks of quality [15]. It includes assessing a site for information that is accurate, current, has a clear source, is referenced, has disclaimers and cautions if appropriate, clear, clean and pleasing design features and a well-defined purpose. These criteria were applied to the original sites in this study by this author to examine whether attrition may be influenced by quality. If a site possessed at least 5 of these attributes, it was considered \"high\" quality, 3 to 4 attributes, it was considered \"moderate,\" and 2 or less it was considered \"poor\" quality. The results are summarized in Table 2.\nIt appears that although the high quality sites make up only a small portion of the total number of sites retrieved (15%), half of the original high quality sites (14 of 28) could be located from the original URL or were redirected to a new URL. Conversely, only 10 of the 73 poor quality sites were accessible from the original URL entry, and only one poor quality site was redirected to another URL from the original site. This suggests that Web sites of higher quality may be less subject to attrition than those of poorer quality, and warrants further research on the relationship between Web site quality and attrition with other medical topics.\nConsidering subject matter and attrition, it may be that certain topics (such as herbal remedies) can have periods of enthusiasm by the public then wane \u2014 which may be the case with these sites. Perhaps information on more mainstream topics (such as health risks and smoking) is less vulnerable to attrition.\nTable 2\nQuality of Health-related Web Sites and Attrition\nWeb Site Quality* (No. of Sites)\nNot Found\nMoved To New URL\nURL Redirected\nURL to Site as Original\nHigh (28)\n10\n7\n3\n11\nModerate (83)\n38\n21\n3\n24\nPoor (73)\n60\n3\n1\n10\nTotals (184)\n108 (59%)\n31 (17%)\n7(4%)\n45 (24%)\n* Quality assessed by author based on attributes described by Risk [15]: High = 5 or more, Moderate = 3 to 4, Poor = 2 or less\nFuture Considerations\nIt has yet to be determined with certainty the forces that influence the survival of Web sites. With the complex and dynamic nature of information flow on the Web, is there a form of \"natural selection\" at work in health Web site survival? If attrition is not related to the site's quality or subject matter, perhaps those with strong commercial backing may survive with greatest frequency. At this point we can only speculate what will endure.\nIn some instances, Web site attrition may be desirable. A common complaint against search engines is that they return too many pages, and that many of the pages have low relevance to the query [16]. The most efficient search engines index only a fraction of the total number of documents on the Web, [17] and if sites of poorer quality go away, ideally this should help retrieval of documents of higher relevance to the user.\nMost quality issues with the Web focus on consumers, [18] however, a recent major poll revealed that physicians are using the Internet to increase their medical knowledge and improve the care they provide to patients [19]. Medical information can change rapidly with continuing breakthroughs and advances in medical knowledge. Availability of information through the Web would facilitate access to the most up-to-date information on current medical topics and scientific discoveries. Future research that is directed toward making sure Web site viewers always know the site will be accessible at a later time will enhance the Web as a valuable medical information resource.","keyphrases":["health-related web sites","attrition","world wide web","internet archive","web site quality"],"prmu":["P","P","P","P","P"]}
{"id":"J_Behav_Health_Serv_Res-4-1-2214828","title":"Healthcare Utilization of Individuals with Opiate Use Disorders: An Analysis of Integrated Medicaid and State Mental Health\/Substance Abuse Agency Data\n","text":"Data from the Substance Abuse and Mental Health Services Administration\u2019s Integrated Database (IDB) were used to examine the service use patterns of individuals with possible opiate use disorders in Washington State. Results indicate that regardless of Medicaid enrollment status, individuals who received mental health (MH) or substance abuse (SA) services only through state agencies received no inpatient substance abuse service. Furthermore, when compared with individuals who received at least one MH\/SA service through Medicaid, those who received services only through the state agencies were less likely to have received any MH services and were more likely to have received residential SA services. This analysis highlights the importance of using integrated client data in providing a more comprehensive understanding of services to inform policy and raises significant questions about how regulatory requirements affecting different funding mechanisms might drive settings of care in ways not related to the care needed.\nIntroduction\nThe use of, abuse of, and dependence on opiates is a major public health concern.1,2 Although only about 0.1% of the U.S. population reported past-year heroin use in 1998,3 heroin and other opiate use accounted for about 30% of total spending for illicit drug use treatment and almost 18% of spending on drug-related crime.4 Psychological and physical health problems were also common among heroin and other opiate users in the mid- to late-1990s.5 As recently as 2003, heroin accounted for 23% of the mentions of substances used among emergency room patients and 41% of drug-related deaths recorded by medical examiners or coroners.6 The social costs of opiate abuse are not limited to illegal drugs. A recent study suggests that in 2001, the social cost of prescription opioid abuse in the United States was $8.6 billion.7\nDespite the overwhelming societal costs associated with opiate use disorders, few studies have investigated the substance abuse (SA) or mental health (MH) treatment service patterns of individuals suffering from opiate abuse or dependence; rather, most studies focus almost exclusively on those in methadone maintenance treatment.8\u201310 These studies suggest that the co-occurrence of MH problems and other illicit drug dependence is quite high among individuals with an opiate use disorder.11\u201313 Yet, despite the increased incidence of MH conditions, individuals with a substance use disorder may not receive adequate levels of MH care beyond that directly related to their substance use disorder.10,11,14,15 Given these findings and the major public health concerns caused by opiate abuse and dependence, the lack of information about the broader MH\/SA treatment service patterns of individuals with an opiate use disorder is a critical gap in the knowledge base informing policies affecting this population.\nThis study used data from the Substance Abuse and Mental Health Services Administration\u2019s (SAMHSA) Integrated Database (IDB) on individuals in Washington State with an indication of an opiate use disorder to determine what MH\/SA services they received and through which auspice (i.e., Medicaid or state agency) they received them. Findings from this study offer two contributions to the current literature. First, the IDB presents a unique opportunity to study the behavioral health care utilization of individuals with opiate use disorders because it contains service use data linked at the client level from Washington Medicaid and from the Washington State Department of Social and Health Services\u2019 (DSHS) Mental Health Division (MHD) and Division of Alcohol and Substance Abuse (DASA). The combination of Medicaid and DSHS data represented in the IDB provides a more comprehensive picture of service use patterns than might be obtained from studies that focus on only one data source. Second, the late 1990s is often characterized as a time of a heroin epidemic in the United States.16 Therefore, examining the MH\/SA treatment service use patterns of individuals with opiate use disorders during this period can provide an especially relevant baseline for today\u2019s policy makers as they attempt to address issues surrounding the emergence of new opiates.\nData and Methods\nThis paper uses IDB data on Washington State from three full calendar years (1996\u20131998). For a more detailed description of the IDB, see Coffey et al.,17 and for a detailed description of the methods used to link IDB service records across state organizations, see Whalen et al.18 In addition to service use information, the IDB contains information on patient demographics (e.g., age, gender, race\/ethnicity), Medicaid enrollment status, MH\/SA diagnosis and service codes, and limited provider information.\nStudy population\nThe study population for this analysis consists of individuals who have at least one record in the IDB indicating an opiate use disorder. An opiate use disorder could be indicated by one or more of the following: a diagnosis (either primary or secondary) of an opiate use disorder, a provider type indicative of opiate treatment, a service or procedure code indicative of opiate treatment (including methadone maintenance therapy but excluding methadone used for pain management), or a report of an opioid as the drug of choice. Opiate use disorder diagnoses were defined using the International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM). ICD-9-CM codes of 304.0 or 305.5, including any subclassifications, were used to identify opiate dependence and abuse, respectively. State-specific provider codes were used to identify opiate treatment providers. Both standard [e.g., Healthcare Common Procedure Coding System (HCPCS)] and local procedure codes were used to identify opiate treatment services. Finally, drug-of-choice information was available from self-reports obtained by state agencies during client intake. In some cases, the drug-of-choice information was obtained more than 1\u00a0year before or after any SA or MH service provision, but these cases represent less than 3% of the sample.\nPreliminary analyses revealed that very few agency service records include diagnosis codes, but most Medicaid records do. Further investigation revealed that this is not an issue of Medicaid requiring a diagnosis for eligibility, but rather an issue of the data systems themselves. Agency services do not require a diagnosis code for reimbursement, so agency data systems do not track diagnosis. Conversely, Medicaid services often require a diagnosis for reimbursement, so Medicaid data systems track diagnosis. Because the presence or absence of a diagnosis is almost completely confounded by the use or nonuse of Medicaid services, information on service use differences associated with an opiate use disorder diagnosis is not presented.\nClient classification\nOne of the primary objectives of this study was to examine the extent to which the MH\/SA service use patterns of individuals with an opiate use disorder varied based on whether their MH\/SA services were tracked by Medicaid or a state MH\/SA agency. Importantly, service tracking may, or may not, be associated with full or partial financial coverage. Accordingly, for this study, individuals were classified based on the data source (Medicaid or state MH\/SA agency) from which their IDB MH\/SA service records were obtained. Based on this information, individuals were classified into three categories:\nAny Medicaid service: individuals who have at least one Medicaid MH\/SA service record, regardless of whether they have MH\/SA state agency service recordsAgency services only with Medicaid enrollment: individuals who have at least one\u00a0MH\/SA state agency service record and were Medicaid-enrolled at some point between their first and last observed MH\/SA service record but have no record of receiving an MH\/SA service through MedicaidAgency services only without Medicaid enrollment: individuals who have at least one\u00a0MH\/SA state agency service record and were not Medicaid-enrolled at any time between their first and last observed MH\/SA service record\nBecause the IDB integrates information from state Medicaid and state MH\/SA agency data sources, overlapping records may occur if Medicaid reimburses a bill, but the state agency provides the service. To avoid overstating utilization rates, only one service date was counted for cases in which the same client, service type (MH or SA), modality\/setting of service [i.e., inpatient (IP), residential\/long-term care, or outpatient (OP)], and service date were reported on both the Medicaid and state agency databases. Individuals with these types of records were classified as any Medicaid service. Importantly, although individuals in the any Medicaid service group may have received any number, or even the majority, of their services through the state agency, preliminary analyses indicated that individuals who received both Medicaid and agency services were more similar to those who received only Medicaid services than they were to those who received only agency services. For this reason, individuals receiving any Medicaid service were combined into a single category.\nIn addition to the client categorization described above, the standard IDB client classification was also used. The IDB client classification was used to identify individuals who received services for only MH conditions (MH-only), only SA conditions (SA-only), or co-occurring conditions (both MH and SA) during the study period. The IDB classifies individuals as having co-occurring conditions if they had any of the following within the 3-year study period: (1) both a primary MH and a SA diagnosis, (2) a primary MH and a secondary SA diagnosis, or (3) a primary SA and a secondary MH diagnosis. Individuals classified as having co-occurring conditions did not necessarily have MH and SA conditions concurrently. A client with an MH record at the beginning of the study period and an SA record at the end of the study period, for example, is classified by the IDB as having co-occurring conditions. In the absence of diagnosis information, MH-only and SA-only classifications were assigned based on the type of service received during the study period (see Coffey et al.17 and Bray et al.19 for a detailed definition of the primary MH\/SA diagnosis category).\nThe IDB client classification does not incorporate information on secondary diagnoses unless a primary MH\/SA diagnosis is also present, nor does it incorporate drug-of-choice information from state agency intake records. Because both of these pieces of information were used to identify individuals with an opiate use disorder for this study, it is possible for individuals with an opiate use disorder to be classified as MH-only or having received no MH\/SA service based on the IDB client classification.\nService classification\nStandard IDB service type classifications were used to classify the MH\/SA services received by individuals with an opiate use disorder. The IDB MH\/SA service type classifies MH\/SA service records as either MH or SA and within MH\/SA as IP, residential, or OP. Many inpatient programs are part of psychiatric or general hospitals and generally use a medical model of substance disorders in which intensive medication and counseling are provided over a relatively short period of time.20 Residential programs typically are provided in a free-standing, designated residential treatment facility. Residential treatment is usually of longer duration than IP treatment and relies less on medical professionals. Residential treatment provides organized services by designated treatment personnel who provide a planned regimen of care in a 24-h setting and is intended to serve clients who need a safe and stable living environment to develop sufficient recovery skills.21 For a complete description of the criteria used to classify services, see Coffey et al.17 The vast majority of individuals with a possible opiate use disorder have at least 1\u00a0SA service record, but relatively few have MH service records; therefore, the IP, residential, and OP subclassifications were examined for SA service records but not for MH service records.\nService encounters\nAn individual\u2019s total number of service encounter dates was defined as the count of unique dates within the MH\/SA service window on which the individual had a record with at least one service of a given service category (MH, IP SA, residential SA, or OP SA). Within a single IP or residential stay, each daily service encounter date was counted separately. Using standard IDB definitions for the full SA population, this same information is presented for the broader SA population, excluding individuals with an indication of an opiate use disorder.\nMethods\nTo characterize the level of contact individuals with an opiate use disorder have with the public treatment system, the analysis examined four key domains: (1) the proportion of clients using services, (2) the median length of the service window (i.e., the length of time between an individual\u2019s first and last MH\/SA service), (3) the number of days of Medicaid enrollment within the service window, and (4) the number of unique MH\/SA encounter dates within the service window.\nRegression analyses were conducted to assess whether the service use patterns of individuals with an opiate use disorder differed significantly across the client classification categories after controlling for differences in the length of the MH\/SA service window, the length of Medicaid enrollment, and demographics across the client categories. First, logistic regression models of the following form were estimated:\nwhere SERVi is a series of indicators for receipt of services of a given type. For the regression analyses, any MH services and OP and residential SA services were considered. Regression analyses for IP SA services were not conducted because those services were received solely by the any Medicaid service group. DEMOGi is a set of demographic characteristics (i.e., gender, age, and race\/ethnicity); GROUPi is a vector of variables representing the data source categories of any Medicaid service (the referent), agency services only with Medicaid enrollment, and agency services only without Medicaid enrollment; WINDOWi is the MH\/SA service window length in days; and ENROLLi is the months of Medicaid enrollment (including 0\u00a0months). The \u03b2s are vectors of coefficients to be estimated. Variables reflecting the primary diagnosis categories are not included because much of the information used to classify individuals as MH-only, SA-only, or co-occurring was also used to classify services as MH or SA.\nNext, regressions of the following form were run on days of service, conditional on service use:\nwhere DAYSi is a set of variables reflecting the unique days of care for each of the same types of care, and all other terms are as previously defined. Consistent with the recommendations of Manning and Mullahy,22 generalized linear model (GLM) estimation with a log link and a gamma distribution on the natural scale was used rather than ordinary least squares (OLS) on the log scale.\nResults\nTable\u00a01 presents the demographic characteristics of the individuals identified as having an opiate use disorder in the IDB. For a point of comparison, Table\u00a01 also presents the same information for the general IDB SA population, as defined in Coffey et al.,17 but excluding individuals with an opiate use disorder. Individuals with an opiate use disorder accounted for approximately 16% of the population with any SA disorder. In terms of demographics, when compared with individuals with a non-opiate SA disorder, individuals with an opiate use disorder are less likely to be male and more likely to be adults (as opposed to youth or elderly), but only minor differences in the distribution of clients across racial\/ethnic categories are observed. Individuals with an opiate use disorder are less likely to be in the SA-only IDB client classification category. This is largely by construction because as discussed earlier, the current paper uses more inclusive data to identify individuals with a possible opiate use disorder than is used by the IDB client classification. Individuals with an opiate use disorder were more likely to have IP and residential SA services than the general SA population, but less likely to have any OP SA service. Individuals with an opiate use disorder were also more likely to have received any MH service compared to the general SA population. They also had longer MH\/SA service windows, indicating longer periods of contact with the public MH\/SA treatment system, and were more likely to be in the any Medicaid service and the agency services only with Medicaid enrollment groups.\nTable\u00a01Characteristics of individuals with a possible opiate use disorder compared with the non-opiate SA populationPopulation characteristicNon-opiate SA populationOpiate usersAll users (N)83,79315,652Percentage of total SA population (%)84.315.7Gender (%)\u00a0Male63.256.8\u00a0Female36.843.2\u00a0Unknown0.00.0Age (%)\u00a0Youth (0\u201317)19.93.1\u00a0Adult (18\u201364)78.896.5\u00a0Elderly (65+)1.30.4\u00a0Unknown0.00.0Race\/ethnicity (%)\u00a0White70.077.6\u00a0Black9.010.6\u00a0Hispanic8.54.3\u00a0Native American7.84.5\u00a0Other3.62.6\u00a0Unknown1.00.3IDB client category (%)\u00a0SA-only68.859.9\u00a0Co-occurring (MH+SA)31.234.7\u00a0MH-only0.01.9\u00a0Neither MH nor SA0.03.4MH\/SA service type (%)\u00a0SA services (%)\u00a0\u00a0Any setting94.994.6\u00a0\u00a0Inpatient3.06.2\u00a0\u00a0Residential31.957.7\u00a0\u00a0Outpatient80.971.4\u00a0\u00a0No SA services5.15.4\u00a0\u00a0Any MH service31.236.5\u00a0MH\/SA service window length\u00a0\u00a025th percentile (days)38.078.0\u00a0\u00a0Median (days)168.0348.0\u00a0\u00a075th percentile (days)446.0770.0\u00a0MH\/SA service data source (%)\u00a0\u00a0Any Medicaid46.051.0\u00a0\u00a0Agency services only with Medicaid enrollment17.322.7\u00a0\u00a0Agency services only without Medicaid enrollment36.826.3SA Substance abuse, MH mental health\nFigure\u00a01 presents the median MH\/SA service window and length of Medicaid enrollment across individuals with a possible opiate use disorder in each of the data source categories. The any Medicaid service group has the longest median MH\/SA service window and the most median days of Medicaid enrollment within that window, followed by the agency services only with Medicaid enrollment group and then the agency services only without Medicaid enrollment group. Because the analysis only examines Medicaid enrollment within the MH\/SA service window, the length of Medicaid enrollment is always less than the total service window length. The total Medicaid enrollment of an individual may be greater than that reported here, but by definition of the service window, no MH\/SA service use occurred during days of Medicaid enrollment not captured by this measure. Importantly, the any Medicaid service group may have received substantial services from MH or SA state agencies, because individuals in this group are categorized as any Medicaid service only because they received at least one Medicaid service.\nFigure\u00a01Median MH\/SA service window length and days of medicaid enrollment among individuals with a possible opiate use disorder by data source category\nFigures\u00a02 and 3 present the probability of service use and the median days of service conditional on service utilization for each of the data source categories. IP SA care was not included in Figures\u00a02 and 3 because only individuals in the any Medicaid service group had IP SA utilization (the conditional median days of IP SA care for that group were 6). Figure\u00a02 suggests that the two agency services only groups are more likely to receive residential SA services and are less likely to receive OP SA services or MH services. Conditional on receipt of services, Figure\u00a03 suggests that the agency services only with Medicaid enrollment group received the most days of residential SA service, the agency services only without Medicaid enrollment group received the most days of OP SA care, and the any Medicaid services group received the most days of MH care.\nFigure\u00a02Percentage of individuals with a possible opiate use disorder using MH\/SA services by service type and data source categoryFigure\u00a03Median days of care conditional on service use among individuals with a possible opiate use disorder by service type and data source category\nTable\u00a02 presents results from the logistic regressions, including logit coefficients, their standard errors, and associated odds ratios (ORs) for the data source grouping variables. For all other covariates, only the estimated logit coefficients and their standard errors are presented. Table\u00a02 shows that even after controlling for the longer service window and more months of Medicaid enrollment, the pattern observed in Figure\u00a02 still holds. Compared with individuals in the any Medicaid service group, individuals in the agency services only with Medicaid enrollment and the agency services only without Medicaid enrollment groups had significantly higher odds of receiving at least residential SA service (OR\u2009=\u20092.941 and OR\u2009=\u20091.376, respectively) and lower odds of receiving any OP SA (OR\u2009=\u20090.311 and OR\u2009=\u20090.162, respectively) or any MH service (OR\u2009=\u20090.217 and OR\u2009=\u20090.110, respectively). Briefly examining the results for the control variables in the regression, females are significantly less likely to receive residential SA services. The racial\/ethnic categories that are statistically significant suggest that racial\/ethnic minorities are less likely than non-Hispanic whites to receive services of any type. Age categories reflecting individuals both younger and older than the referent of 36 to 40 were also, in general, less likely to receive services of any type when the associated coefficients were statistically significant. The exception is that individuals aged 21 to 35 with an opiate use disorder were more likely to receive residential SA services. Results with regard to demographic characteristics, especially race\/ethnicity, should not be interpreted as evidence of disparities, however. Rather, they indicate the differential representation of demographic groups across the data and highlight the importance of controlling for those factors when assessing statistical significance. Unexpectedly, months of Medicaid enrollment are positively associated with a greater likelihood of service use only for MH services. For both types of SA care examined, longer Medicaid enrollment was negatively associated with the probability of service use, although the relationship was not significant for residential care.\nTable\u00a02Logistic regression results for the probability of service useParametersProbability of residential SA service useProbability of OP SA service useProbability of any MH service useAgency services only with Medicaid enrollment1.079***\u22121.167***\u22121.526***Standard error0.0500.0610.052Odds ratio2.9410.3110.217Agency services only without Medicaid enrollment0.319***\u22121.819***\u22122.210***Standard error0.0520.0690.072Odds ratio1.3760.1620.110Control variablesIntercept0.446***1.091\u22120.274***Standard error0.0590.0780.065Female\u22120.519***0.344\u22120.064Standard error0.0360.0460.042Race\/ethnicity (referent is non-Hispanic White)Black\u22120.269***0.371\u22120.493***Standard error0.0570.0750.069Hispanic\u22120.0810.036\u22120.402***Standard error0.0860.0970.116Native American0.342***\u22120.067\u22120.221**Standard error0.0870.1010.101Other\u22120.357***0.229\u22120.169Standard error0.1100.1450.128Unknown\u22121.656***\u22121.122**0.863*Standard error0.5070.4410.459Age (referent is 36 to 40)0 to 17\u22120.384***0.765\u22120.058Standard error0.1030.1460.12418 to 20\u22120.032\u22120.037\u22120.126Standard error0.1120.1320.13421 to 250.361***\u22120.0980.097Standard error0.0750.0860.08526 to 300.279***\u22120.0600.027Standard error0.0630.0760.07231 to 350.175***0.0340.093Standard error0.0580.0720.06641 to 45\u22120.205***\u22120.047\u22120.116*Standard error0.0550.0690.06546 to 50\u22120.465***\u22120.010\u22120.268***Standard error0.0650.0820.07851 to 55\u22120.537***0.001\u22120.425***Standard error0.0990.1290.12156 to 60\u22121.096***0.443**\u22120.718***Standard error0.1590.2200.18361 to 64\u22120.656***\u22120.713**\u22120.729**Standard error0.2410.2970.28865 plus\u22121.417***\u22120.285\u22120.675**Standard error0.3010.3440.322Service window length (days)0.0000.0030.001***Standard error0.00010.00010.0001Medicaid enrollment (months)\u22120.005\u22120.017**0.053***Standard error0.0040.0070.005IP Inpatient, SA substance abuse, OP outpatient, MH mental health*p\u2009<\u20090.10**p\u2009<\u20090.05***p\u2009<\u20090.01\nTable\u00a03 presents the regression results for days of care, conditional on having at least 1\u00a0day of service of that type of care. Because the natural logarithm of days of care is the dependent variable, the coefficient estimates, standard errors, and associated percentage changes for the data source categories are presented. For all other variables, only the estimated coefficients and their standard errors are presented. For residential SA care, the agency services only with Medicaid enrollment group is significantly associated with more days of residential SA care (28% increase), while the agency services only without Medicaid enrollment group is significantly associated with fewer days of care (56% decrease). Both agency services only groups are significantly and positively related to OP SA days of care: an estimated 18% increase for the agency services only with Medicaid enrollment group and an estimated 60% increase for the agency services only without Medicaid enrollment group. Finally, the agency services only without Medicaid enrollment group is associated with significantly more days of MH care than the any Medicaid service group (56% increase). Unlike in Table\u00a02, no consistent pattern emerges with regard to the demographic control variables included in the linear regression. The length of the MH\/SA service window is positively and significantly associated with days of care. Months of Medicaid enrollment are negatively and significantly associated with days of residential and OP SA care, but positively and significantly associated with days of MH care.\nTable\u00a03Linear Regression Results for Unique Health Care Service Dates\u00a0ParametersUnique dates of residential SA serviceUnique dates of OP SA serviceUnique dates of MH serviceAgency services only with Medicaid enrollment0.248***0.169***\u22120.009Standard error0.0300.0450.063Percentage change0.2820.185\u22120.009Agency services only without Medicaid enrollment\u22120.818***0.472***0.448***Standard error0.0380.0770.094Percentage change\u22120.5590.6040.564Control variablesIntercept3.310***2.552***2.337***Standard error0.0420.0650.067Female\u22120.0170.322***\u22120.045Standard error0.0250.0390.041Race\/ethnicity (referent is non-Hispanic White)Black\u22120.0070.121**\u22120.010Standard error0.0400.0610.074Hispanic0.208***0.013\u22120.153Standard error0.0570.1060.133Native American0.254***\u22120.039\u22120.700***Standard error0.0520.0840.106Other\u22120.057\u22120.0160.211*Standard error0.0790.1230.128Unknown\u22121.268***0.000\u22121.340***Standard error0.4830.0000.443Age (referent is 36 to 40)0 to 170.549***0.374***0.224*Standard error0.0760.1120.12318 to 200.193***0.1350.366**Standard error0.0740.1230.14321 to 25\u22120.0280.188***0.062Standard error0.0450.0720.08526 to 300.0030.174***0.071Standard error0.0390.0610.07131 to 35\u22120.0260.0390.040Standard error0.0370.0570.06541 to 45\u22120.0310.137**0.115*Standard error0.0370.0580.06446 to 500.0370.1150.309***Standard error0.0460.0750.07851 to 550.0400.317***\u22120.080Standard error0.0730.1220.12356 to 60\u22120.229*0.264\u22120.037Standard error0.1340.2100.19661 to 64\u22120.618***0.283\u22120.093Standard error0.1900.3570.30865 plus0.701***\u22120.7000.116Standard error0.2711.3210.353Service window length (days)0.001***0.003***0.002***Standard error0.0000.0000.000Medicaid enrollment (months)\u22120.019***\u22120.024***0.013***Standard error0.0030.0040.003IP Inpatient, SA substance abuse, OP outpatient, MH mental health*p\u2009<\u20090.10**p\u2009<\u20090.05***p\u2009<\u20090.01\nImplications for Behavioral Health\nThis analysis examined the service use patterns of individuals with an indication of an opiate use disorder using IDB data from Washington for the period 1996 through 1998. Among individuals with opiate use disorders, the receipt of at least one MH\/SA service through Medicaid appears to be positively associated with IP SA service use in that only individuals who met this condition had any record of an IP SA service. The receipt of at least one\u00a0MH\/SA service through Medicaid was also positively associated with OP SA service and with MH service use among individuals with opiate use disorders. The use of only state MH\/SA agency services, on the other hand, was positively associated with the use of residential SA services.\nThe findings suggest that the regulatory restrictions faced by state agencies and Medicaid may drive observed patterns of care for individuals with opiate use disorders. Specifically, the finding that agency-only MH\/SA service use is associated with higher rates of residential service use and with no IP SA service use is likely tied to regulatory differences between Medicaid and the state agencies in Washington. For example, SA block grant funds, which are a key source of funding for both DASA and MHD, could not be used for IP hospital care during the period covered by this analysis. This restriction most likely induced state agencies to route patients for whom outpatient care is insufficient to residential care. Similarly, the Medicaid Institutions of Mental Disorders exclusion prohibits payment for psychiatric services received by adults in residential care facilities with more than 16 beds and so may induce providers to route more severe patients to IP care rather than to residential care. This explanation suggests that using administrative data to track service use patterns may be misleading because the collection of service data is driven by regulatory environments and billing systems that may not capture the actual intensity of care given.\nThe results are subject to several limitations. First, although the IDB represents one of the most comprehensive cross-system databases used to examine this critical issue to date, it does not capture all possible services that could be used by individuals with possible opiate use disorders in Washington. Other possible sources include self-pay, private insurance, and the Department of Veterans Affairs (VA) system, among others. Another important limitation common to all administrative databases is that the actual treatment need of the population studied is unknown, so definitive statements cannot be made about the appropriateness of the services received. Furthermore, because of the limited time frame of the IDB, data are unavailable for individuals who used MH\/SA services either before 1996 or after 1998. As a result, it is possible that this study has not captured the full service use history of some individuals who appear in the treatment system briefly at the beginning or end of the study period. A final limitation of this study is that information on prescription drug use is not available in the agency service records contained in the IDB and is therefore not considered in this study.\nDespite these limitations, the analysis of service use patterns of individuals with possible opiate use disorders during the late 1990s offers key implications for today\u2019s policy makers as they attempt to address issues surrounding the emergence of new opiates. First and foremost, if policy makers are trying to track the service use of a small but important segment of the overall SA population, using integrated data is a necessity. When funding for treatment services is cut in an effort to contain costs, it is important to determine if those services have simply been shifted to other state programs, reflecting little net cost savings for states overall. The present study combines state MH\/SA agency data with state Medicaid data, but including additional data sources (e.g., VA or criminal justice system) would provide an even more complete picture of service use patterns. Second, the utility of administrative data for analyses such as these is often limited by the influence of the regulatory environment, clinical practice patterns, and the institutional history of the data systems used. Given that state and federal policy makers increasingly rely on administrative data to assess the performance of the treatment system, the results clearly highlight the need to better track service provision. SAMHSA\u2019s IDB, therefore, represents the vanguard of a new, expansive, cross-agency philosophy regarding administrative data sources and serves as a model for new and more comprehensive data integration efforts.","keyphrases":["opiate use disorder","medicaid","state mh\/sa agencies","mh\/sa service utilization"],"prmu":["P","P","P","R"]}
{"id":"Virchows_Arch-3-1-2063564","title":"Pancreatic intraductal papillary-mucinous neoplasms: a new and evolving entity\n","text":"For a long time, intraductal tumors of the pancreas were neglected because they were misdiagnosed as mucinous cystadenocarcinoma, ordinary ductal adenocarcinoma, or chronic pancreatitis. Only in recent years have they been recognized as clinical and pathological entities. Most common are the intraductal papillary-mucinous neoplasms. Although they show an adenoma-carcinoma sequence, they have proved to have a more favorable prognosis than ductal adenocarcinoma, when resected in a preinvasive state. Recently, it has become clear that they constitute a heterogeneous group with at least four subtypes. Their stratification reveals that the various intraductal papillary-mucinous neoplasm subtypes have different biological properties with different prognostic implications.\nHistorical notes and a rising incidence\nThe pancreatic tumors that are characterized by an intraductal origin and growth pattern include intraductal papillary-mucinous neoplasms [3, 24, 41], intraductal tubular carcinomas [22, 45], intraductal tubular adenomas of the pyloric type [6], and intraductal acinar cell carcinomas [15]. Most common are the intraductal papillary-mucinous neoplasms (IPMNs). They are mucin-producing epithelial tumors that usually show a papillary architecture and are associated with dilatation of the ducts. Before 1990, these tumors were thought to be rare. They first started to be recognized approximately 20\u00a0years ago [31, 36, 39], when the many different names given to the tumor in the 1980s were replaced by the term IPMN [41]. This name was also introduced in the classification of exocrine pancreatic tumors propagated by the World Health Organization (WHO) [24] and the fascicles of the Armed Forces Institute of Pathology [44]. Since then, IPMNs have been reported with increasing frequency (Table\u00a01) and currently account for about 7% of clinically diagnosed pancreatic neoplasms and up to 16% of surgically resected pancreatic neoplasms and for almost 50% of pancreatic cysts found incidentally [8, 17]. When only the cystic tumors of the pancreas are considered, IPMNs take first place, with a frequency of 24% [26].\nTable\u00a01Incidence data on intraductal papillary-mucinous neoplasmsAuthorPeriod 1Number of incidencePeriod 2Number of incidenceSohn et al. [43]1987\u20132001582001\u2013200378Wada et al. [47]1988\u20132000632001\u2013200337Our series1981\u20132000552001\u2013200750\nThe new incidence data on IPMNs raise the question whether their increase in number is real or not. Of course, it is difficult to accept that IPMNs might have been overlooked in the past, not only clinically but also morphologically. There are good reasons, however, to believe that IPMNs did always exist and did not really increase in frequency. One reason is related to the rapid improvements in modern imaging techniques, which enable more precise recognition of cystic lesions, even if they are small and asymptomatic. Another is connected with the decreasing risk of pancreatic surgery. The most important fact, however, may be that until 1999, the distinction between IPMNs and mucinous cystic neoplasms (MCNs) was unclear, so that many IPMNs were classified as MCNs [50] or regarded as ductal adenocarcinomas or chronic pancreatitis.\nAdenoma-carcinoma sequence\nIn IPMNs, the normal ductal epithelium is replaced by mucin-producing columnar cells showing papillary proliferations and variable degrees of cellular atypia, even within an individual neoplasm. They are graded according to the most atypical area as IPMN with low grade dysplasia (adenoma), IPMN with moderate dysplasia (borderline), and IPMN with high grade dysplasia (carcinoma in situ). An invasive component may be found in 38\u201350% of the cases [7, 26, 40, 43]. Progression from adenoma to carcinoma is estimated to occur at about 5\u00a0years [43]. IPMNs therefore provide a model of neoplastic progression from a benign intraductal neoplasm through increasing grades of dysplasia to invasive carcinoma.\nPrognosis after resection\nBetween 80 and 90% of IPMNs are surgically resectable. For these IPMNs, a 5-year survival rate of 77\u2013100% was reported, provided the tumors did not have an invasive component (Table\u00a02). By contrast, IPMNs with an invasive component had a 3- to 5-year survival rate of only 36\u201346% [12, 16, 43] (Table\u00a02). Interestingly, the survival rate did not appear to be dependent on the grade of dysplasia in the IPMN if there was no invasive component [12, 16, 43, 47]. These data imply that the overall outcome of IPMNs therefore largely depends on the presence of an invasive component. If the tumor is already invasive, criteria for a poor outcome are lymph node involvement, vascular invasion, and bilirubin elevation [13]. A comparison of the prognosis of all patients with invasive IPMNs with that of patients with ductal adenocarcinoma reveals that patients with IPMNs survive longer than those with ductal adenocarcinomas [29, 40, 43].\nTable\u00a02Five year survival rate and recurrence in 349a intraductal papillary-mucinous neoplasms\u00a0NoninvasiveInvasive77\u2013100%36\u201346%No recurrence93\u201398.7%52\u201370%Recurrence1.3\u20137%30\u201348%Localup to 6%8\u201348%Distant (metastases)1%12%Both0%10\u201348%a[12, 43, 47]\nSeveral studies have reported recurrences after resection of noninvasive IPMNs, some of which revealed only moderate dysplasia [12, 43, 47, 48]. The recurrences were either local or metastatic (Table\u00a02). To explain the recurrences, particularly the local ones, it has to be assumed that either tumor tissue was overlooked at the pancreatic resection margin, or an invasive component remained undetected in the resected specimen, or there was multifocal disease. The last possibility has to be considered if the surgical margins were negative and the recurrence occurred in the pancreatic remnant. This has been observed only in a few cases [40]. When metastatic recurrences occur, it is most likely that they resulted from inadequate sampling that failed to detect an invasive component. Regarding the impact of a positive resection margin on IPMN recurrence, it is interesting to note that it has been reported that even IPMNs with positive margins did not recur during a median follow-up period varying from 19\u201340\u00a0months [13, 48]. The reason for this phenomenon might be that the growth of the remaining intraductal tumor tissue is so slow that clinical symptoms only appear after a follow-up period of 2 to 3\u00a0years. However, even if intraductal recurrences may take a long time to become clinically apparent, a positive margin in any IPMN case should lead to further tissue resection.\nHistological type and prognosis\nIn 1991, it was reported that the invasive component of IPMNs corresponded either to an ordinary ductal adenocarcinoma or, more frequently, to that of a mucinous (colloid) carcinoma [49]. This observation suggested that IPMNs form a group of heterogeneous neoplasms. A further argument for the heterogeneity of IPMNs was the detection of IPMNs in branch ducts rather than in the main duct, where most of the IPMNs are found. Finally, it was recognized that IPMNs differ in their histological and cytological features and in their mucin profile [3, 4, 18, 28, 32, 33]. Currently, four subtypes of IPMN can be distinguished: an intestinal type, a pancreatobiliary type, an oncocytic type, and a gastric type [18].\nThe most common type of IPMN is the intestinal type (Table\u00a03). It usually occurs in the main duct of the pancreatic head [9] and shows a villous growth pattern similar to that of villous adenoma in the colon. It also expresses MUC2 and CDX2 but not MUC1 (Fig.\u00a01a,b). When this IPMN type becomes invasive, the invasive component resembles mucinous (colloid) carcinoma [5, 28], a tumor of which at least 80% is composed of pools of extracellular mucin containing single cells or strands of neoplastic glandular epithelium or even a small component of signet ring cells. Patients with colloid carcinoma have a 55% 5-year survival rate after resection [5]. These tumors therefore seem to be much less aggressive than ordinary ductal adenocarcinomas.\nFig.\u00a01Histological subtypes of intraductal papillary-mucinous neoplasms of the pancreas and their usual mucin pattern. a, b IPMN of the intestinal type positive for MUC2; c, d IPMN of the pancreatobiliary type positive for MUC1; e, f IPMN of the oncocytic type showing scattered positivity for MUC2; g, h IPMN of the gastric type, positive for MUC5Table\u00a03Histopathological data on 105 intraductal papillary-mucinous neoplasms collected during a period of 26\u00a0yearsIPMNGastric (n\u2009=\u200927) (26%)Intestinal (n\u2009=\u200957) (54%)Pancreatobiliary (n\u2009=\u20097) (7%)Oncocytic (n\u2009=\u200914) (13%)Noninvasive (65%)n\u2009=\u200920 (74%)n\u2009=\u200935 (62%)n\u2009=\u20093 (43%)n\u2009=\u200910 (72%)Adenoma91215Borderline71821Carcinoma in situ4504Invasive (35%)n\u2009=\u20097 (26%)n\u2009=\u200922 (38%)n\u2009=\u20094 (57%)n\u2009=\u20094 (28%)\nThe pancreatobiliary type of IPMN is much rarer than the intestinal type IPMN (Table\u00a03). It shows complex arborizing papillae and expresses MUC1 only (Fig.\u00a01c,d). Its invasive component usually corresponds to a conventional ductal adenocarcinoma. The prognosis of this type of IPMN, if invasive, seems to be similar to that of ductal adenocarcinoma and therefore poorer than that of the intestinal type of IPMN [4].\nThe oncocytic type of IPMN (also called intraductal oncocytic papillary neoplasm [2]) shows the same complex papillae as the pancreatobiliary type, but the lining cells reveal strongly eosinophilic cytoplasm. In addition, there are often numerous goblet cells. The tumor cells express MUC1 and MUC2 inconsistently (Fig.\u00a01e,f). With fewer than 20 cases reported in the literature to date [2, 20, 34, 35, 37, 38, 42], the clinical and pathological behavior of this type is still unclear. Most of the cases (94%) were diagnosed as carcinoma, some of them with an invasive component or even distant metastases [37]. As the follow-up in this patient group is very short, no relevant data are available yet on survival and outcome.\nThe gastric type of IPMN exhibits papillary projections lined by epithelial cells resembling gastric foveolar cells and shows pyloric gland-like structures at the base of the papillae. These cells express MUC5 (Fig.\u00a01g,h), while MUC1, MUC2, or CDX2 positivity is only occasionally observed. The gastric type of IPMN corresponds to the branch duct type, which occurs in the periphery of the pancreatic parenchyma, most often in the uncinate process, where it usually presents as a multicystic lesion with cysts no larger than 3\u00a0cm [9]. The gastric type seems to be less aggressive, i.e., less invasive, than the other IPMN subtypes [10, 25, 33, 46] but may show severe cellular atypia in a few cases (up to 25%) [46] (Table\u00a03, Fig.\u00a02) The size of the lesion was unrelated to the grade of cellular atypia [46].\nFig.\u00a02IPMN of the gastric type showing severe cellular atypia and MUC5 positivity\nInterestingly, pancreatic intraepithelial neoplasia (PanIN)-like complexes are frequently observed next to gastric type IPMNs. This raises the question whether IPMNs of the gastric type are a focal accentuation of a diffuse disease rather than a localized lesion. They might therefore also be related to the small peripheral cystic changes described by Kimura et al. [21] in non-neoplastic pancreata or the patchy lobular fibrosis associated with PanIN-1B lesions described by Detlefsen et al. [14] (Fig.\u00a03). If this were the case, IPMNs of the gastric type would actually be large PanIN-1 lesions. An argument for this assumption is that both IPMNs of the gastric type and PanIN-1 lesions stain for MUC5 in the absence of MUC1 and MUC2 positivity. This assumption would also explain why it is difficult to distinguish PanIN lesions from some IPMNs [27] (Fig.\u00a04), despite a consensus definition of both lesions [19].\nFig.\u00a03PanIN-1 lesion associated with lobular fibrosisFig.\u00a04Small multicystic duct-associated lesion in the periphery of pancreatic tissue suggestive of an IPMN of the gastric type but difficult to distinguish from a large PanIN-1 lesion\nAlthough the malignant potential of IPMNs of the gastric type seems to be rather low, it has to be pointed out that the fibrocystic changes that have been described in pancreata removed from patients with a strong family history of pancreatic cancer [11, 30] are similar, if not identical, to IPMNs of the gastric type and their associated PanIN lesions. This implies that IPMNs of the gastric type\/PanIN-1 lesions are not innocuous lesions but have a malignant potential.\nSummary and perspectives\nThe significance of IPMNs among the pancreatic tumors has increased greatly in recent years because of their improved recognition, both clinically and histopathologically and their much better prognosis than ordinary ductal adenocarcinomas. Moreover, they appear to fall into four subtypes that have special biological properties with prognostic implications. Of particular interest in relation to the development of ductal adenocarcinomas is the fact that the so-called gastric type IPMNs seems to occur in pancreata from patients with a strong family history of pancreatic cancer. Furthermore, it is of interest that the pancreatic IPMNs have their counterparts in IPMNs of the biliary duct system, where the same subtypes may occur [1, 23, 51, 52]. The treatment of choice is resection, but future trials may reveal that the extent of resection could depend on the IPMN subtype.","keyphrases":["pancreas","outcome","intraductal papillary mucinous neoplasm"],"prmu":["P","P","R"]}
{"id":"Eur_J_Pediatr-4-1-2413082","title":"A 15-year-old girl with a large pericardial effusion\n","text":"Pericarditis is a rare manifestation of tuberculosis and can be fatal. We describe a 15-year-old girl admitted for a large pericardial effusion. Subxiphoid pericardial biopsy was performed. Biopsy samples were positive for M. tuberculosis DNA by PCR, which confirmed the diagnosis of tuberculous pericarditis.\nA 15-year-old girl was admitted with chest pain, progressive shortness of breath, fever, nonproductive cough, decreased appetite, weight loss, and fatigue. The patient was born in Angola, but had lived in the Netherlands for 14\u00a0years. Seven months before admission she had traveled to the Democratic Republic of Congo in Western Africa.\nHer temperature was 39.7\u00b0C, her pulse 90 beats\/min, respiratory rate 28 breaths\/min, and blood pressure 96\/58\u00a0mmHg. No enlarged lymph nodes were found. Soft cardiac tones and a friction rub were noticed. The liver was enlarged.\nLaboratory results showed normal white blood cells and platelets with a decreased hemoglobin (4.3\u00a0mmol\/l). The erythrocyte sedimentation rate was 60\u00a0mm\/h.\nA chest radiograph showed marked enlargement of the cardiac silhouette with no other abnormalities. Echocardiography revealed a large pericardial effusion containing exudative debris and fibrin strands (Fig.\u00a01). Extensive laboratory studies for auto-immune diseases and infections, including HIV, did not reveal the cause.\nFig.\u00a01Echocardiography showing a large pericardial effusion with exudative debris and fibrin strands. RV right ventricle, LV left ventricle, LA left atrium, PE pericardial effusion\nUnder general anesthesia the pericardial fluid was drained and a subxiphoid pericardial biopsy was performed. The PCR for M. tuberculosis DNA was positive on the pericardium, though not in the pericardial fluid. The patient was treated with four anti-tuberculous drugs (isoniazide, ethambutol, pyrazinamide, rifampicin) for 4\u00a0months and two anti-tuberculous drugs for an additional 4\u00a0months. Adjunctive treatment with predinisolone was tapered over a 12-week period. The patient completely recovered and did not develop any signs of constrictive pericarditis. She was followed up for more than 1\u00a0year and then discharged because of uneventful recovery.\nDiscussion\nPericarditis is a rare manifestation of tuberculosis that can be fatal and accounts for approximately 1\u20132% of all cases of tuberculosis [6]. Children in particular are at high risk of the development of extra-pulmonary disease [5]. Clinical manifestations of tuberculous pericarditis are nonspecific and cardiopulmonary complaints usually develop later. Accumulation of pericardial fluid may compromise venous return, leading to cardiac failure.\nEchocardiography plays a major role in confirming the presence of a pericardial effusion. In addition, several echocardiographic abnormalities including pericardial thickening, exudative coating and strands crossing the pericardial space are suggestive of tuberculous pericarditis. Thickened pericardium and fibrin strands are highly specific (94% and 88% respectively), and exudative coating has a high sensitivity (100%) in tuberculous pericarditis [4].\nThe diagnosis is confirmed with detection of Mycobacterium tuberculosis in either pericardial fluid or pericardial tissue. Pericardial fluid aspiration, however, has low diagnostic yield, both for PCR and culture, whereas pericardial biopsy provides the best chance of definitive diagnosis [2, 6]. A positive tuberculin skin test may increase the suspicion of tuberculous pericarditis, but a negative skin test does not exclude the diagnosis. A tuberculin skin test was not performed in this case because the girl had been immunized with BCG. There was no history of any family members being diagnosed with TBC.\nThe spread of tuberculosis to the pericardium, resulting in an effusion, occurs most often from the mediastinal lymph glands. However, pericarditis may also represent reactivation of disease in the absence of an apparent primary focus of infection [3].\nCurrent recommendations for treatment of tuberculous pericarditis include an initial four-drug regimen for a period of 8\u00a0weeks with a subsequent continuation phase of treatment for 4\u00a0months when the isolate is drug-susceptible [1]. Controversy exists with respect to the use of corticosteroids for tuberculous pericarditis. The American Thoracic Society\u2019s consensus statement on tuberculosis recommends adjunctive therapy with corticosteroids (1\u00a0mg\/kg of prednisolone with tapering over 11\u00a0weeks) for tuberculous pericarditis [1].","keyphrases":["pericarditis","tuberculosis","corticosteroids","subxiphoidal biopsy"],"prmu":["P","P","P","R"]}
{"id":"J_Headache_Pain-4-1-2276243","title":"The primary headaches: genetics, epigenetics and a behavioural genetic model\n","text":"The primary headaches, migraine with (MA) and without aura (MO) and cluster headache, all carry a substantial genetic liability. Familial hemiplegic migraine (FHM), an autosomal dominant mendelian disorder classified as a subtype of MA, is due to mutations in genes encoding neural channel subunits. MA\/MO are considered multifactorial genetic disorders, and FHM has been proposed as a model for migraine aetiology. However, a review of the genetic studies suggests that the FHM genes are not involved in the typical migraines and that FHM should be considered as a syndromic migraine rather than a subtype of MA. Adopting the concept of syndromic migraine could be useful in understanding migraine pathogenesis. We hypothesise that epigenetic mechanisms play an important role in headache pathogenesis. A behavioural model is proposed, whereby the primary headaches are construed as behaviours, not symptoms, evolutionarily conserved for their adaptive value and engendered out of a genetic repertoire by a network of pattern generators present in the brain and signalling homeostatic imbalance. This behavioural model could be incorporated into migraine genetic research.\nIntroduction\nThe genetics of the primary headaches scored recent scientific successes due to the unravelling of the genetics of FHM. Deciphering of the patho-physiological mechanisms of these common diseases promises to bring the much needed knowledge for pharmacological treatments and therapeutic interventions. There are however also problems and controversies, some not solved by the genetic studies performed to date. The following is a brief subjective review of the available evidence, suggesting a role for epigenetic mechanisms and ending with the proposal of a behavioural model of the primary headaches possibly useful for the genetic studies.\nPrimary and secondary headaches: symptoms, syndromes or diseases? Idiopathic and syndromic migraines\nHeadaches\/migraines are plagued by problems of definition: these terms describe symptoms (a feature which indicates a condition of disease, in particular one apparent to the patient, CED 2003), and at the same time, distinctive syndromes (a group of symptoms which consistently occur together) [1] or diseases (a disorder of structure or function in a human, animal, or plant, especially one that produces specific symptoms) [1] with recognisable diagnostic features, internationally defined [2]. The problems encountered with definitions become evident when dealing with primary or secondary headaches, and when considering idiopathic and syndromic migraines. Secondary headaches are those in which attacks occur due to a recognisable cause or disease, which itself represents the primary cause of the attacks. Syndromic migraines, contrasted with the idiopathic ones, are those in which attacks of migraine, clinically barely or not distinguishable from those occurring in the primary migraines, occur compounded with involvement of other systems. Syndromic migraines are often genetically determined. The concept of \u201csyndromic,\u201d potentially useful in the exploration of headache pathogenesis, has been applied to conditions such as deafness, visual loss and epilepsy, but has no place in the HCS classification that classifies headache attacks and not diseases, albeit distinguishing between primary and secondary headaches. These considerations may apply to the genetics of the primary headaches, since by adopting the HCS (2004), we consider symptoms, not diseases (much as if, studying the genetics of diabetes mellitus, we adopted a classification of the hyperglycemias).\nGenetic epidemiology of the typical migraines\nThe typical primary migraines (MO and MA) all have a substantial risk of familial recurrence. When estimating the population relative risk of migraine in specified groups of relatives (i.e. the ratio between the probability that a relative versus a random member of the population is affected), first-degree relatives of migraine without aura probands have 1.9 times the risk of MO and 1.4 times the risk of MA, whereas first-degree relatives of MA probands have nearly four times the risk of MA and no increased risk of MO [3]. Since a family aggregation is implied when the risk ratio exceeds one, this confirms the familial liability for the migraines, even though familiarity is not yet heredity. A further analysis showed however that spouses of MO probands have 1.4 times the risk of MO, and spouses of MA probands have no increased risk of MA [3], thus backing a hereditary liability for MO and especially MA. Twin studies concur with this increased familial liability. Concordance rates for migraine are consistently higher among monozygotic (MZ) than dizygotic (DZ) twins. In particular, heritability estimates were around 52% in female twin pairs raised together or apart since infancy. In MZ Danish twin pairs, liability to MO resulted from additive genetic effects (61%) and from individual-specific environmental effects (39%), while in MA, correlation in liability was 0.68 in MZ and 0.22 in DZ, with heritability estimated at 0.65. Therefore, twin studies reveal that approximately one-half of the variation in migraine is attributable to additive genes, while the remainder is caused by unshared rather than shared environmental factors between twins [4, 5]. Several studies have analysed pedigrees with migraine, segregation analysis being performed to discover the genetic transmission pattern. Studies at first envisioned migraine as a simple mendelian disorder, inherited according to monogenic rules of transmission. Various modes of inheritance, autosomal dominant with female preponderance, possibly sex determined; autosomal recessive with 70% penetrance; polygenic; maternal and X-linked transmissions have been proposed, or rejected [6, 7]. Finally, based on complex segregation analysis, a multifactorial inheritance was considered the most likely pattern even in high-risk families with MA [8]. A single gene was considered unlikely, but, notably, in some families, a mendelian or mitochondrial inheritance could not be excluded [3]. Currently, migraine is widely considered a complex disease with multifactorial inheritance. This type of inheritance applies to many complex\/quantitative traits, i.e. traits that vary continuously in a phenotypic range, and in which variation is quantitative, not qualitative. Examples of quantitative traits are height, body weight, etc. Such traits are influenced by multiple genes (each a quantitative trait locus QTL), each having a small quantitative effect and interacting with the environment. However, there is still no unequivocal evidence that migraine as a quantitative trait varies continuously in the general population, and moreover, genetic variation underlying a continuous character distribution can result from segregation at a single locus too. Therefore, considering migraine as a quantitative trait may still be unwarranted.\nThe primary headaches also display considerable comorbidity, rarely incorporated into genetic studies. MA is comorbid with hypomania, depression and anxiety, and MO with phobia, panic and major depression. Other comorbidities are stroke, dyslipoproteinemias, essential tremor, paroxysmal dyskinesia and epilepsy. Merikangas et al. in a longitudinal genetic epidemiology study found that migraine was associated with mood disorders and drew attention to the fact that age at onset of anxiety disorders preceded, while onset of affective disorders followed that of migraine, findings consistent with a syndromic relationship between migraine and anxiety\/depression [9].\nThese findings have been replicated, maternal depression being significantly associated with development of migraine in children [10]. Asthma, rhynitis and allergic bronchitis are also important comorbidities recurring in migraine families [11\u201313]. These comorbid clinical features should be properly incorporated in the genetic studies of the primary headaches.\nMendelian migraines? The genetics of FHM and their putative relationship with the typical migraines MA\/MO\nMigraines may be multifactorial, but mendelian migraines, i.e., migraines that conform to a mendelian type of genetic transmission, do exist. FHM is classified as a subtype of migraine with aura in the HCS (2004), and it conforms to an autosomal dominant pattern of hereditary transmission. Joutel et al. mapped FHM to chromosome 19, and in 1996 the first FHM gene, CACNL1A4, later termed CACNA1A, encoding the alpha1A subunit of the P\/Q neural calcium channel, was discovered, accounting for both FHM 1 and episodic ataxia type 2 (EA2) phenotypes [14, 15]. Spinocerebellar atrophy type 6 (SCA6) was added to the FHM and EA2 phenotypes in 1997 [16]. Thus, FHM 1, EA2 and SCA6 are all allelic channelopathies, with missense mutations mostly accounting for FHM, mutations disrupting the reading frame for EA2 and polyglutamine expansions in the COOH gene terminal for SCA6. The phenotypic spectrum of the CACNA1A mutations was at first believed to consist either of pure FHM or of FHM associated with cerebellar atrophy. Why some mutations originate pure FHM while others elicit progressive or intermittent cerebellar features remains unclear [17]. Ictal coma after trivial trauma and essential tremor were associated clinical features in families harbouring particular CACNA1A mutations. The phenotypic spectrum of the CACNA1A mutations has further expanded to include ataxia induced by fever or high temperature [18], childhood epilepsy [19,20] and status epilepticus [21], paroxysmal paranoid psychosis with anxiety [22], benign paroxysmal torticollis of infancy, considered a migraine equivalent [23], and even myasthenic syndrome [24], since CACNA1A is also expressed on presynaptic neuromuscular junction terminals where it modulates transmitter release [25] even in the absence of any morphological changes in the junction or muscle weakness [26].\nThe paroxysmal clinical features of migraine, ataxia and epilepsy, together with the consideration that CACNA1A specifies for a calcium channel and that in the tottering and leaner mouse with epilepsy and ataxia, similar mutations are found in the mouse homologue of the calcium channel alpha1A subunit gene, led to the proposal that migraine be considered a calcium channelopathy [27]. The concept of migraine as a brain channelopathy fits well with the phenomenon of spreading depression [28], implicated in migraine attack pathophysiology. It is now accepted by the scientific community as an explicative model for migraine. However, the available genetic evidence is controversial or negative (see below).\nFHM was soon proved to be genetically heterogeneous, some families linking to chromosome 1 [29,30], and a second gene, ATP1A2, encoding the alpha2 subunit of the Na\/K ATPase, was discovered in Italian families and accounting for a phenotype of pure FHM (FHM 2) [31]. New mutations were found in FHM 2 pedigrees [32], and soon the phenotypic spectrum of FHM 2, initially thought to be confined to pure FHM, broadened to include such features as coma, triggered by minor head trauma and angiography [33], recurrent comas [34] and epilepsy, namely benign familial infantile febrile convulsions [35]. Finally, cerebellar ataxia associated with epilepsy and mental retardation was described in an Italian FHM 2 family [36,37], findings later confirmed by Spadaro et al. and Vanmolkot et al. in other families [38, 39]. Phenotypes of alternating hemiplegia of childhood [40\u201342] and basilar migraine [43] described with ATP1A2 mutations further enlarged the clinical spectrum of FHM 2. Variability within the same family is notable, with FHM, cerebellar ataxia, recurrent paroxysmal dystonia and mental retardation all recurring together [42].\nLastly, mutations in the neuronal voltage-gated sodium channel SCN1A were reported by Dichgans et al. [44] to account for a phenotype of pure FHM (FHM 3), and there are still FHM families without mutations in any of the previously described genes, implying further genetic heterogeneity. Sporadic patients with HM, more common in clinical practice, also present problems, since mutations in the known FHM genes are only rarely encountered in this population [45].\nAn important corollary of the genetic discoveries obtained in the FHM was the proposal to consider FHM as a model for the typical migraines MO and MA [27]. This spurred the search for the involvement of FHM genes in MO\/MA. Up to now the effort has been largely unrewarding. This in our opinion is also due to the misclassification of FHM as a subtype of MA [2], whereas FHM represents a syndromic migraine (see below). Some evidence in favour of linkage of typical migraines to the FHM locus on chromosome 19 was initially offered by May et al. [46], Nyholt et al. [47] and Terwindt et al. [48]. However, early negative studies [49\u201351] were later substantiated by systematic screening investigations of the CACNA1A in families with MO and\/or MA [52\u201355], and to date, mutations in CACNA1A have never been demonstrated in kindreds without hemiplegic migraine, with or without aura. The same negative considerations apply to ATP1A2. Earlier evidence in favour of a role of the Chrlq3l locus or ATP1A2 gene in the typical migraines [56, 57] was superseded by negative findings and absent ATP1A2 mutations in typical migraine only pedigrees, even those displaying an apparently autosomal dominant mode of inheritance [58\u201361]. The FHM 3 SCN1A gene was discovered too recently for any conclusive study. Von Brevern et al. [62] however failed to find any CACNA1A, ATP1A2 or SCN1A mutations in patients with migrainous vertigo. Thus, there is no current evidence that the genes causing FHM represent major susceptibility loci for the typical migraines.\nDoes such negative genetic evidence imply that FHM is not a useful model for migraine etiology? Several reviews of migraine pathogenesis apply the FHM model of neural channelopathy to the typical migraines. While such models are not justified genetically, it may be contended that FHM is nonetheless helpful in elucidating the pathophysiology of the migraine attacks. This consideration however is likely to apply to several clinical conditions all characterised by headache attacks of the migraine type. Migraine-like attacks indeed are found not only in the typical migraines, but also in other conditions, diseases or syndromes, in which they occur together with symptoms and signs of multisystem nervous or extra-nervous involvement. These \u201csyndromic migraines\u201d thus display bona fide migraine headache attacks at some times in their clinical course, and most of them have a genetic basis (Table\u00a01). FHM is also characterised by multisystem neurologic involvement (migraine, hemiplegia, ictal recurrent comas, cerebellar atrophy, mental retardation, epilepsy, movement disorders, myasthenic syndrome, etc.), and therefore we make a plea for FHM to be considered more appropriately as a syndromic migraine and not a subtype of MA, as with the current HCS classification (2004).\nTable\u00a01A list of proposed (and provisional) syndromic migrainesSyndromic migrainesGenes (chromosome) involvedMigrainous features (references)MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes)MTTL1, MTTQ, MTTH, MTTK, MTTS1, MTND1, MTND5, MTND6, and MTTS2 (mtDNA)Most frequent symptom: episodic sudden headache with vomiting and convulsions [63, 64]CADASIL (cerebral arteriopathy, autosomal dominant, with subcortical infarcts and leukoencephalopathy)NOTCH 3 (19p13.2\u2013p13.1)MA in 22% [65]; migraine in 38% [66]HERNS (retinopathy, vascular, with cerebral and renal involvement and Raynaud and migraine phenomena)TREX1 (3p21.3\u2013p21.2)Migraine in 70% [67]CCM (familial cerebral cavernous malformations)KRYT 1 (7q11.2\u2013q21)Convulsions and migraine attacks [68, 69]\nLinkage and association studies in the typical migraines\nSeveral studies on the genetics of the typical migraines MO and MA applied genetic association, linkage and genome wide scanning methods. Most of these studies resulted in findings that either lack verification or are controversial. For MA, a genome wide scan on 50 multigenerational families in Finland identified a susceptibility locus on chromosome 4q24 [70]. Other loci for MA have been reported on chromosome 11q24 in Canadian families with an autosomal dominant transmission pattern [71], and on chromosome 15q11\u2013q13 to a genomic region containing genes encoding for GABA-A receptors in ten Italian families again displaying an autosomal dominant transmission pattern [72].\nFor MO, or for pedigrees with MO mixed with MA, susceptibility loci have been reported on chromosome 6p12.2\u2013p21.1 in Sweden [73], chromosome 5q21 [74], chromosome 14q21.2\u2013q22.3 in an Italian family with MO [75], chromosome Xq24\u201328 in two large Australian pedigrees [76] and chromosome 19p13.3\/2 to the insulin receptor gene INSR [77]. While many of such findings have still to be replicated, in some cases (the INSR gene) sequence studies have given negative results [78].\nOther studies have examined candidate genes, implying that a pathogenetic (and a priori) hypothesis was formulated beforehand. This may be risky, considering that the pathogenesis of the migraine headaches is still imperfectly understood. Candidate genes explored were the mitochondrial DNA (mtDNA), or genes involved in prothrombotic or cardiovascular disease, or in the metabolism of biologic amines such as dopamine or serotonin, or in a variety of other metabolic systems. Several of the studies applied to mtDNA genes have yielded negative results, even though in some families migraine was reported to segregate with the Leber mtDNA 14484 mutation [79], and mtDNA mutations and haplotypes (haplotype U) have been associated with juvenile migraine stroke [80, 81] and with cyclic vomiting, considered a migraine equivalent in the pediatric population [82\u201384]. Contrasting results for genes involved in prothrombotic\/cardiovascular risk, and for those involved in the metabolism of the biological amines serotonin and dopamine, or in several other metabolic pathways are summarised in Tables\u00a02, 3, 4, and 5.\nTable\u00a02Prothrombotic and cardiovascular risk genes and typical migraine geneticsProthrombotic\/vascular risk genes or mutations examinedPhenotypesLDL receptor (19p13.2)Associated with MO [87]; not associated [88]Factor V R\/Q 506 (Leiden mutation)Associated with MA [89]Not associated with migraine stroke [90]; not associated with MA\/MO [91]; not associated with juvenile MA [92]Factor II 20210 G\/ANot associated with MA\/MO [91]Not associated with migraine stroke [93]Factor XIII Val 34 LeuNot associated with migraine [94]Decanucleotide insertion\/deletion factor VII promoterNot associated with MA\/MO [91]Alloantigenic platelet systems HPA-1 and HPA-2Not associated with MA\/MO [91]Deficit of protein SAssociated with MA [89]Angiotensin converting enzyme (ACE)Allele D associated with MO and more frequent migraine attacks [95]Endothelial NO synthase inducible (NOS3; iNOS)Not associated with migraine [96, 97]Endothelin receptor A (ETA-231 A\/G) polymorphismAllele G protecting from migraine [98]MTHFR (methylene-tetra-hydrofolate reductase) C677T\/A1298CHomozygous mutation associated with MA [99], associated with MA [100]; risk for MA, modulated by thymidilate synthase gene [101]Table\u00a03Serotonin metabolism genes and typical migraine geneticsSerotonin metabolism genes examined Phenotypes5-HTSERT (17q11.2\u201312) Allelic association with MO (increase of allele STin2.12\u00a0+\u00a0decrease of allele STin2.10) and MA (same\u00a0+\u00a0increase of allele Stin2.9) [102]; 5HT-TLPR with MA [103]Allelic association with migraine (allele Stin2.10) [104]; borderline association with migraine [105]No association\/linkage with migraine [106, 107]5-HT2A (13q14\u201321)Allelic (allele C) association with migraine aura [108]No association with migraine [105, 106, 109, 110]5-HT1B (6q13)No association with migraine [106, 110, 111]5-HT1D (1p36.3\u201334.3)No association with migraine [106, 110, 111]5-HT2B (2q36.3\u2013q37.1)No association with migraine [106, 110, 111]5-HT2C (Xq22\u201325)No association with migraine [106, 110, 111]5-HT1B (6q13)No association with therapeutic response to triptans [112, 113]5-HT1F (3p12)No association with therapeutic response to triptans [112, 113]Table\u00a04Dopamine metabolism genes and typical migraine geneticsGenes examinedPhenotypesDopamine receptor 2 (DRD2)Allelic association (allele NcoI) with MA comorbid with anxiety\/depression [114]Allelic association (allele 1) with yawning\/nausea during attack of MO [115]No allelic association (allele NcoI) with MA [116]No allelic association with MO\/MA [107, 117, 118, 119]Dopamine receptors 1, 3, 4, 5 (DRD1, DRD3, DRD4, DRD5)No allelic association with migraine [115, 117, 120]Dopamine transporter (DAT)Association with chronic daily headache with drug abuse [86]COMT; MAO-A No association with migraine [117, 121]Dopamine-betahydroxylase (DBH)Association with migraine [107], especially males with MA [122]No association [123]Table\u00a05Other genes implicated in typical migraine geneticsGenes examinedPhenotypesAndrogen\/progesterone receptorsAndrogen receptor not associated; progesterone receptor associated with migraine [124]K channel KCNN3Allelic association (CAG repeats) with MO\/MA [125]Not associated (CAG repeats) [126]Cytotoxic T lymphocyte antigen 4 (CTLA-4) Not associated with migraine [127]HLA-DRB1Allelic association with MA [128]\nFinally, a few studies have focused on the genetics of the chronic headaches, a major social problem, since these chronic headaches are often associated with drug abuse and afflict a remarkable percentage of the general population. Chronic tension-type headache displays a substantial familial recurrence, with lifetime relative risk estimated at 3.87 for parents and 3.53 for children of probands; the risk is greater for females (3.35) than for males (2.59) [85]. A genetic association study of chronic headache with drug abuse versus the dopamine metabolism genes by Cevoli et al. [86] found that allele 4 of the exon III VNTR polymorphism of the dopamine receptor 4 gene DRD4 was associated with chronic daily headache, and allele 9 of the dopamine transporter gene SLC6A3 was more common in chronic daily headache associated with drug abuse than in episodic migraine.\nGenetics of tension-type headache\nRemarkably, apart from an epidemiological genetic study that demonstrated a familial aggregation for chronic tension-type headache [85], there are no other genetic studies of this common disorder.\nGenetics of cluster headache\nCluster headache (CH) has long been considered a sporadic disease. In recent decades, however, a familial recurrence has been appreciated, and the new HCS classification (2004) now states that CH may be transmitted as an autosomal dominant disease in about 5% of cases. Several CH cases have been reported among monozygotic twins and in family pedigrees [129\u2013137], and family studies indicate that I-degree relatives of CH probands carry a 5- to 18-fold, and II-degree relatives a 1- to 3-fold increased relative risk of the disease [138\u2013141]. CH has been considered a probable autosomal dominant disease with a penetrance of 0.3\u20130.34 in males and 0.17\u20130.21 in females [142]. The exact transmission pattern is however still debated [143, 144], and an autosomal recessive pattern has been advocated in certain families [137]. Several candidate genes have been analysed, in particular mtDNA mutations [145\u2013148], HLA antigens [149\u2013151] and CACNA1A polymorphisms [152, 153], usually with negative or controversial results. Other genes, such as the NO synthases NOS1, NOS2A and NOS3 [154], the elusive amine gene cluster [155], the CLOCK gene involved in the regulation of circadian rhythms [156,157] and the hemochromatosis gene [158], have been found not associated with CH. Recently, an association between CH and a polymorphism in the hypocretin receptor 2 gene HCRTR2 was reported by Rainero et al. [159], possibly accounting for the circadian recurrence of the CH attacks. Such an association, while confirmed by Sch\u00fcrks et al. [160], was rejected in a European multicentric study [161]. Recently, reports of CH associated with hemiparesis during the attacks suggested a relationship with FHM and ionic channelopathies [162]. Preliminary genic expression studies instead documented the activation of proinflammatory genes during the CH attack [163].\nGenetics and epigenetics\nEpigenetics is the study of the changes in DNA and DNA-binding proteins that, albeit altering the structure of chromatin, do not modify the nucleotide sequence of DNA. The remarkable feature here is that some of these modifications may be associated with heritable changes in gene function.\nCommonly held concepts of heredity indeed pit environmental influences (nurture) against genetic background (nature) as totally separate causative factors. Genetic advances themselves have however demonstrated that the hereditary transmission of biological changes not encoded in the DNA sequence and dictated by environmental influences is possible. This part of genetics, called epigenetics, has received little or no attention in the genetic studies of the primary headaches. It is the contention of the author however that future epigenetic studies will account for several hereditary features of the primary headaches, in particular their comorbidities.\nAll those (meiotic and mitotic) modifications in gene expression that are heritable but not encoded in the DNA sequence are defined as epigenetic. Molecular mechanisms implicated include (1) methylation of cytosine residues at C5 in dinucleotide CpG sites (localised especially in promoters of well over 40% of the genes and that, when methylated, cause silencing of the gene); (2) mechanisms of RNA interference, whereby microRNAs silence gene expression; (3) histone (DNA associated proteins) changes: activation or inactivation of genomic regions according to the \u201chistone code\u201d. All of these mechanisms result in the expression or silencing of genes, and underlie such phenomena as inactivation of the X chromosome and genomic imprinting. Several epigenetic diseases are already known that may be inherited through the somatic and the germinal line: fragile X syndrome, in which ATRX gene mutations modify the methylation pattern of ribosomial RNA and, by methylation of CGG expansions in the FMR1 gene, silence the gene; the Angelman and Prader-Willi, and Rett syndromes; also, many colonic cancers and leukemias. Important epigenetic differences that increase with age are found even between monochorial twins [164]. Notably, epigenetic modifications may increase with age and may also be prevented through interventions directed at DNA or histone methylation (with azanucleotides, antisense oligonucleotides, histone deacetylase). Even more remarkably, there is some evidence that lifestyles and even diet may play a role [165].\nEpigenetic models for the primary headaches?\nThere is consistent evidence that behavioural differences typical of specific inbred animal strains are the consequence of environmental influences acting especially during development rather than DNA changes. Mice strains with decreased environmental exploration behaviour (B6 strain) develop enhanced exploratory behaviour if nurtured in their first 3\u00a0months of life by BALB dams, a strain displaying intense exploratory behaviour; changes in behaviour appear to be linked to the type of maternal care, particularly licking of the pup by the mother, a behaviour demonstrated to affect the status of the endocrine stress system in mice [166]. Weaver et al. [167] showed how maternal care in the rat (licking and grooming the pup) modifies the methylation pattern of the promoter of the glucocorticoid receptor gene in the hippocampus; such epigenetic changes, evident from the first week of life, persist throughout the animal\u2019s life but are reversible upon treatment with histone deacetylase inhibitors or upon intracerebral administration of methionine (an intervention that modifies the methylation pattern) [168].\nStress plays a remarkable role in the development of the nervous system: removal of rat pups from the mother causes reduced neurogenesis in the adult hippocampus through steroid-dependent mechanisms [169], and alters serotonergic transporter densities and serotonergic 1A receptors in the rat brain [170]. Administration of steroids to the mother before delivery causes changes in behavioural patterns in juvenile rats [171], and maternal deprivation in the immediate post-natal period modifies locomotor and steroid release patterns in the adult rat [172]. Epigenetic mechanisms also seem relevant for the formation of memory traces [173] and more generally for cognitive development [174]. Epigenetic mechanisms have been hypothesised for psychiatric disorders [175, 176] and many complex and multifactorial diseases affecting the brain or the inflammatory and immune systems [177\u2013179].\nThere are still no studies of epigenetic mechanisms in the primary headaches. When considering, however, the important maternal influence in migraine genetics; the consistent and inherited co-morbidities especially for psychiatric and inflammatory-immune disorders; twin studies documenting that only about half of the variability is due to \u201cgenetic\u201d factors; it is possible to envision that epigenetic mechanisms, especially those acting during nervous system development in early infancy and childhood, play a role in the heritability and pathophysiology of the primary headaches. Preliminary studies have already analysed attachment styles in adult migraineurs [180, 181], and a prospective investigation demonstrated correlations between events suffered during pregnancy and early life, and quality of adult life 31\u201333\u00a0years later [182]. It is reasonable to suggest that early life factors and attachment styles between mother and child represent determinants of epigenetic changes relevant in migraine pathogenesis. Such early pre- and post-natal environmental behavioural factors could be usefully analysed to define endophenotypes of adaptive behaviour useful in the genetic studies of the primary headaches.\nFinal comments: a behavioural model of the primary headaches as fight-or-flight response and sickness behaviour to be incorporated into genetic research\nConsideration of epigenetic mechanisms may help in analysing behaviours during the headache attacks. Any genetic studies are ultimately dependent upon the definition of the phenomenon taken into consideration, and on how it is conceptualised. Therefore, studies have to rely upon conventional diagnostic criteria, in turn based on a priori interpretations. Most genetic studies have been performed within the frame of migraine interpreted simply as a \u201cpain\u201d trait with multifactorial inheritance. In a \u201charlequin\u201d model, several genetic factors, each one having a small specific weight, interact with environmental factors to determine the migraine attack. Such a model should however be better tailored to suit phenomena such as the migraine attack and the migraine diseases that are really behavioural \u201cprocesses\u201d with an intrinsic logic of their own [183], one that is consistent within attacks, within patients and within populations. There is a need to conceive of the primary headaches along more useful scientific lines. Consistently lacking in the genetic studies is for instance any consideration of migraine as a behavioural response to environmental and\/or endogenous triggers, a view that has scientific support [184] and that we recently revised to accomodate a Darwinian perspective [185]. According to our view, migraine and other primary headaches such as CH are behaviours, not symptoms, evolutionarily conserved for their adaptive value and engendered out of a genetic repertoire by networks of pattern generators present in the brain. These neural networks serve the homeostasis of the brain, with migraine pain considered a kind of visceral pain signalling homeostatic imbalance. The behavioural repertoire enacted during the migraine attack, complete with its full panoply of pain, cognitive, autonomic, motor, etc., symptoms and signs, is comparable to that defined as sickness behaviour and already known to develop in all mammals and other animals following challenge with infective and other pathogenic agents [186]. In contrast, behaviour during the CH attacks [187] resembles the fight-or-flight response of hypothalamic animals. These behaviours during the headache attacks really represent \u201chealing\u201d processes, and migraine may even be evolutionarily advantageous [188]. Thus, what is relevant in this new behavioural model is not the manifestations of the attack, but the factors triggering it, that, migraine being of the brain, must relate to still unknown disturbances of brain homeostasis [185]. Accordingly, it is these triggering factors rather than the manifestations during the attacks that may represent the features most relevant for a true dissection of the genetics of the primary headaches.","keyphrases":["genetics","epigenetics","migraine","cluster headache","tension-type headache"],"prmu":["P","P","P","P","P"]}
{"id":"Psychopharmacologia-3-1-1915617","title":"The PDE4 inhibitor rolipram reverses object memory impairment induced by acute tryptophan depletion in the rat\n","text":"Rationale The selective type IV phosphodiesterase inhibitor, rolipram, has been shown to improve long-term memory and can reverse the cholinergic deficit caused by scopolamine. However, the underlying mechanisms of action of rolipram remain obscure.\nIntroduction\nInhibition of phosphodiesterase type 4 (PDE4) leads to an increase in intracellular cyclic adenosine monophosphate (cAMP) availability (Silvestre et al. 1999). cAMP is an important second messenger molecule in the process of intracellular signal transduction mechanisms (Bailey et al. 1996). Several studies have shown that rolipram, a selective PDE4 inhibitor, improves cognitive performance in young rats (Blokland et al. 2006; Imanishi et al. 1997; Rose et al. 2005; Zhang et al. 2000, 2004) and ameliorates scopolamine-induced memory deficits (Egawa et al. 1997; Imanishi et al. 1997; Rutten et al. 2006; Zhang and O\u2019Donnell 2000).\nIn a previous study, we have shown that rolipram reversed a scopolamine-induced deficit in an object recognition task (ORT) in male Wistar rats (Rutten et al. 2006). It was suggested that these effects may be related to the effects of elevated cAMP levels on neurotransmitter release. There is also evidence that there is a link between PDE4 inhibition and serotonergic neurotransmission (Schoffelmeer et al. 1985; West and Galloway 1996). The adenylate cyclase activator forskolin, as well as 8-bromo-cAMP, can enhance the electrically evoked release of 3H-5-hydroxytryptamine (Schoffelmeer et al. 1985). Thus, elevating cAMP levels by a selective PDE4 inhibitor could exert a general facilitatory effect on 5-HT release. Based on these findings, we argued that rolipram may also reverse memory deficits induced by a lowered 5-HT neurotransmission.\nAcute tryptophan depletion (ATD) is a well-established model to assess the role of serotonin in cognitive and affective functioning (Booij et al. 2003). The neurotransmitter serotonin is synthesized from its amino acid precursor tryptophan (TRP), which is obtained from our food. Free TRP is transported into the brain across the blood\u2013brain barrier, but has to compete for entrance with five other large neutral amino acids (LNAAs: valine, leucine, isoleucine, phenylalanine, and tyrosine). The ratio of TRP and these other LNAAs (TRP\/\u03a3LNAA ratio) is thought to be a more sensitive index of brain TRP availability (Fernstrom 1981; Wurtman et al. 1980). As 5-HT is synthesized from TRP, through the rate-limiting enzyme TRP-hydroxylase, the availability of TRP in the brain determines the amount of central 5-HT (Wurtman et al. 1980). Due to its reversible and non-intrusive effects, the method of ATD can be repeatedly used in animals and humans.\nIn humans, acute tryptophan depletion has a negative effect on memory consolidation (Park et al. 1994; Riedel et al. 1999; Schmitt et al. 2000). Furthermore, memory impairments in the ORT have been observed in rats after ATD (Lieben et al. 2004b, 2005). Therefore, we hypothesized that rolipram could reverse an ATD-induced memory deficit in rats by elevating 5-HT release in the brain.\nIn addition to this aim of the study, we further examined the relation between peripheral TRP and memory performance. We investigated the effects of different pretreatment times of the TRP-depleted mixture on object recognition memory. These data should provide more experimental support for a relation between plasma TRP and, consequently, central 5-HT and memory performance.\nMaterials and methods\nAnimals\nAll experimental procedures were approved by the local ethical committee of the Maastricht University for animal experiments according to governmental guidelines. A total of 42 4-month-old male Wistar rats (Charles River, The Netherlands) were used (410\u2013450\u00a0g). Rats were randomly assigned to either the biochemistry (n\u2009=\u200918) or the behavior group (n\u2009=\u200924). In the biochemistry groups, the animals were randomly subdivided over three treatment groups, i.e., saline, TRP+, or TRP\u2212 (n\u2009=\u20096\/group). The animals were housed individually in standard type 3 Makrolon cages on sawdust bedding in an air-conditioned room (about 20\u00b0C). They were kept under a reversed 12\/12-h light\/dark cycle (lights on from 18:00 to 6:00\u00a0hours) and had free access to water. Rats were housed in the same room as where they were tested. A radio, which played softly, provided background noise in all rooms. All testing were done between 9:00 and 17:00\u00a0hours.\nDrugs and chemicals\nThe gelatin hydrolysate (Solugel C) was obtained from PB Gelatins (Tessenderlo, Belgium). Glucodry 210 was obtained from the Amylumgroup (Koog aan de Zaan, The Netherlands). l-Tryptophan and rolipram were obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands). Kaliumchloride (KCl), calciumchloride-dihydrate (CaCl2\u00b72H2O), and 5-sulfosalicylic acid dihydrate were purchased from Merck (Darmstadt, Germany).\nTreatment\nDuring a period of 2\u00a0weeks preceding the experiment, the rats were handled and habituated to oral injections with normal tap water (10\u00a0ml\/kg). On experimental days, the rats were not fed 14\u00a0h before treatment until the testing period was completed. This was done to minimize the availability of TRP from food, which would counteract the effects of the ATD treatment. The rats were orally treated with a protein\u2013carbohydrate mixture containing TRP (TRP+ group; in which 0.28% TRP of the total protein was added to the mixture) or one lacking TRP (TRP\u2212 group; no TRP added to the mixture), or with saline. The composition of the nutritional mixture is shown in Table\u00a01. \nTable\u00a01Composition of mixture and determination of the amino acids in gelatin-based protein (mol) MixtureComposition\u00a0Protein (Solugel) in 100\u00a0ml water100\u00a0g\u00a0Aspartic acid4.8\u00a0Glutamic acid10.3\u00a0Hydroxyproline11.4\u00a0Serine3.4\u00a0Glycine23.2\u00a0Histidine0.8\u00a0Arginine10.0\u00a0Threonine2.0\u00a0Alanine10.4\u00a0Proline12.0\u00a0Tyrosine0.4\u00a0Valine2.2\u00a0Methionine0.7\u00a0Isoleucine1.0\u00a0Leucine2.5\u00a0Hydroxylysine0.9\u00a0Phenylalanine1.1\u00a0Lysine2.9Carbohydrate (Glucodry 210) in 80\u00a0ml water50KCl0.094CaCl2\u00b72H2O2.32l-Tryptophan (TRP\u2212group)0l-Tryptophan (TRP+ group)0.28The composition of the mixture (g) used in this experiment is described in italic. The amino acid spectrum (%) of the Solugel protein was obtained from PB Gelatins, Tessenderlo, Belgium.\nIn every experiment, each dose contained Solugel C at 4.0\u00a0g\/kg and Glucodry at 2.0\u00a0g\/kg of the body weight and was given in a dose of 10\u00a0ml\/kg between 8:30 and 12:30\u00a0hours. Blood samples were taken at baseline (20\u00a0min before treatment) and 60, 180, and 360\u00a0min after the first treatment. For the behavioral studies in the object recognition task, the mixture was administered at different time points (see below).\nRolipram was freshly suspended in 5% ethanol, 1% tylose (methyl-cellulose), and 94% distilled water on every experimental day. The following doses of rolipram were tested: 0, 0.01, 0.03, or 0.1\u00a0mg\/kg always in combination with the TRP\u2212 mixture (3\u00a0h before the first trial, T1). Rolipram was always administered 30\u00a0min before T1 (i.p. injection, dose 2\u00a0ml\/kg).\nBiochemistry\nFor the determination of plasma amino acid levels, blood samples were taken at resting values and repeated at several points in time. Blood sampling was done via a tail-incision method (Fluttert et al. 2000). Promptly after collection of blood in sodium heparin tubes (Microvette\u00ae CB 300, Sarstedt, Germany), the samples were kept on ice. After centrifugation of the blood samples (at 4\u00b0C for 15\u00a0min at 3,000\u00d7g in a Hettich EBA 12 centrifuge), plasma was deproteinized with cups containing dry 5-sulfosalicylic acid (6\u00a0mg\/100\u00a0\u03bcl plasma), and the protein was spun down. Samples were frozen in liquid nitrogen and stored at \u221280\u00b0C. Before analysis, samples were thawed at 4\u00b0C, vortex-mixed vigorously, and centrifuged at 50,000\u00d7g in a Hereaus Model Biogufe Stratos for 10\u00a0min at 4\u00b0C. From the clear supernatant, 20\u00a0\u03bcl was mixed with 1,960\u00a0\u03bcl water and 20\u00a0\u03bcl norvaline and stored in the cooled (7\u00b0C) sample compartment until analysis.\nIn addition to total plasma TRP, the concentrations of several other amino acids were determined with a fully automated high-performance liquid chromatography system after precolumn derivatization with ophthaldialdehyde (OPA; Van Eijk et al. 1993). OPA amino acids derivates were quantified with fluorescence detection. The concentrations of the total plasma amino acids were expressed as micromole per liter.\nObject recognition memory\nThe object recognition test was performed as described elsewhere (Prickaerts et al. 2002).The apparatus consisted of a circular arena, 83\u00a0cm in diameter. Half of the 40-cm high wall was made of gray polyvinyl chloride, the other half of transparent polyvinyl chloride. A light bulb was switched on during testing only and provided a light intensity (20\u00a0lx) which was equal in the different parts of the apparatus. Two objects were placed in a symmetrical position about 10\u00a0cm away from the gray wall. We used four different sets of objects. The different objects were: (1) a cone consisting of a gray polyvinyl chloride base (maximal diameter 18\u00a0cm) with a collar on top made of brass (total height 16\u00a0cm), (2) a standard 1-l transparent glass bottle (diameter 10\u00a0cm, height 22\u00a0cm) filled with sand, (3) a massive metal cube (10.0\u2009\u00d7\u20095.0\u2009\u00d7\u20097.5\u00a0cm) with two holes (diameter 1.9\u00a0cm), and (4) a massive aluminum cube with a tapering top (13.0\u2009\u00d7\u20098.0\u2009\u00d7\u20098.0\u00a0cm). The objects could not be displaced by a rat.\nIn the first week, the animals were handled daily and were adapted to the procedure in 2\u00a0days; that is, they were allowed to explore the apparatus (without any objects) twice for 3\u00a0min each day. In the two following weeks, the rats were adapted to the testing and i.p. administration procedure by a saline injection (0.4\u00a0ml) 30\u00a0min before the first trial until they showed a stable discrimination performance, i.e., good object discrimination (a d2 value of about 0.30, see below) at a 1-h interval on two successive sessions. Subsequently, testing of the drugs began.\nA testing session comprised two trials. The duration of each trial was 3\u00a0min. During the first trial (T1) the apparatus contained two identical objects (samples). A rat was always placed in the apparatus facing the wall in the center of the transparent front segment. After the first exploration period, the rat was put back in its home cage. Subsequently, after a delay interval, the rat was put back in the apparatus for the second trial (T2), but now with two dissimilar objects, a familiar one (the sample) and a new one. The times spent exploring each object during T1 and T2 were recorded manually with a personal computer.\nExploration was defined as follows: directing the nose to the object at a distance of no more than 2\u00a0cm and\/or touching the object with the nose. Sitting on the object was not considered exploratory behavior. To avoid the presence of olfactory trails, the objects were always thoroughly cleaned with alcohol (70%). Moreover, each object was available in triplicate so neither of the two objects from the first trial had to be used as the familiar object in the second trial. In addition, all combinations and locations of objects were used in a balanced manner to reduce potential biases due to preferences for particular locations or objects.\nAs we expected ATD to impair memory performance, we needed a delay interval at which normal rats discriminate between the novel and familiar object. Therefore, we selected a delay interval of 1\u00a0h, as Wistar rats show good discrimination between the two objects after this interval (Rutten et al. 2006). Furthermore, we hypothesized that rolipram may reverse deficits induced by ATD in a 1-h delay ORT. In 1\u00a0week, three testing sessions were given, with a 48-h washout period in between. Tests were always conducted on Mondays, Wednesdays, and Fridays.\nIn the first behavioral experiment, we investigated the effects of administration with TRP+ or TRP\u2212 mixture at different time points (i.e., 30\u00a0min, 1, 3, or 6\u00a0h) before testing in the ORT. Each rat always participated in each condition, and the order of test conditions was decided at random.\nThe second behavioral experiment examined the effect of rolipram (30\u00a0min before T1) on object recognition performance in combination with the TRP\u2212 mixture at the most effective time point (i.e., 3\u00a0h before T1). Thus, five treatment conditions were tested, i.e., saline, vehicle and TRP\u2212, rolipram 0.01\u00a0mg\/kg and TRP\u2212, rolipram 0.03\u00a0mg\/kg and TRP\u2212, and rolipram 0.1\u00a0mg\/kg and TRP\u2212. Each rat was tested in each condition, and the order of treatment conditions was decided at random. The same animals (n\u2009=\u200924) were used for both behavioral experiments; thus, control conditions (TRP+) from experiment 1 and saline from experiment 2 can be compared to each other.\nStatistical analysis\nBiochemistry\nThe mean concentrations of plasma amino acids were determined for each treatment and time condition separately. The extent of reduction (expressed in absolute values and in percentage decline from resting values) was calculated for total plasma TRP concentrations and for the TRP\/\u03a3LNAA ratio. Extreme values, as determined by an extremity test (see Dixon 1959), were excluded from statistical analysis. Differences in plasma amino acid concentrations were analyzed with a general linear model, with main factor \u2018treatment\u2019 and repeated measure factor \u2018time\u2019. Separate one-way analyses of variance (ANOVA) were performed to analyze differences between treatment conditions per time point. A post-hoc Bonferroni test was used to further characterize the differences between treatment conditions. Differences were regarded as statistically significant if P\u2009<\u20090.05.\nBehavior\nThe basic measures were the times spent by rats exploring an object during T1 and T2. Table\u00a02 shows the measures involved in the object recognition task (Prickaerts et al. 1997). e1 and e2 are measures of the total exploration time of both objects during T1 and T2, respectively. d2 was considered as index measures of discrimination between the new and the familiar objects. d2 is a relative measure of discrimination which corrects the difference between exploring the old and the novel object for exploration activity (e2) and appears to be independent of the total exploration times (see \u015e\u0131k et al. 2003). In the object recognition task, results are not reliable if an animal explores both objects in the second trial for less than 5\u00a0s (\u015e\u0131k et al. 2003). Therefore, in every session, animals that explored less than 5\u00a0s in the second trial (i.e., e2\u2009<\u20095) were excluded from analysis. For all parameters, the effects of the different treatment, time, and interactions were analyzed with univariate ANOVA for the first experiment and one-way ANOVA for the second experiment. Significant effects were analyzed in more detail using the Bonferroni correction. To compare control conditions, i.e., the TRP+ (3\u00a0h before the first trial) condition of the first experiment with the saline condition of the second experiment, a t test was performed. Differences were regarded as statistically significant if P\u2009<\u20090.05. \nTable\u00a02Measures involved in the object recognition testExplorationDiscriminatione1 is the measure of the time spent in exploring both identical objects (a1 and a2) in T1; e2 is the measure of the time spent in exploring both the familiar (a) and new object (b) in T2; d2 is the measure of discrimination between the new and familiar objects.\nResults\nPlasma TRP values\nThe plasma TRP\/\u03a3LNAA ratio is shown in Fig.\u00a01. TRP\/\u03a3LNAA levels changed over the 6\u00a0h [F(3,60) = 4.92; P\u2009<\u20090.01]. There was a treatment effect for TRP\/\u03a3LNAA ratio [F(2,60) = 36.72, P\u2009<\u20090.01]. Post hoc analysis showed that the TRP\u2212 condition was significantly different than the TRP+ and the saline condition (Bonferroni, P\u2009<\u20090.05). In addition, the time \u00d7 treatment interaction was also significant for the TRP\/\u03a3LNAA ratio [F(6,60) = 7.80; P\u2009<\u20090.01]. Separate one-way ANOVA analyses for each treatment time point showed differences between treatment conditions at the 1-h time point [F(2,17) = 31.44; P\u2009<\u20090.01] and the 3-h time point [F(2,16) = 6.79; P\u2009<\u20090.01]. However, no differences between treatment conditions were observed at the baseline time point (\u221220\u00a0min) [F(2,15) = 2.03; n.s.] or the 6-h time point [F(2.17) = 3.33; n.s.] (see Fig.\u00a01). Furthermore, post hoc analysis showed that the TRP\/\u03a3LNAA ratio of the TRP-treated animals was lower than the TRP+- or saline-treated animals at the 1-h time point (Bonferroni; P\u2009<\u20090.01) and the 3-h time point (Bonferroni; P\u2009<\u20090.05).\nFig.\u00a01The effects of treatment time on the ratio plasma TRP\/\u03a3LNAA (mean values and SEM). Percentages indicate the difference from baseline levels in the TRP\u2212 conditions. Asterisks indicate significant differences from baseline (*P\u2009<\u20090.05; **P\u2009<\u20090.01)\nBehavioral experiment 1\nThe effects of ATD on ORT performance are shown in Fig.\u00a02 and Table\u00a03. There was a general treatment effect on ORT performance [F(1,165) = 36.44; P\u2009<\u20090.01]. In addition, a time effect was observed on ORT performance [F(3,165) \u2009=\u2009 2.86; P\u2009<\u20090.05]. The time \u00d7 treatment interaction was also significant for the memory performance in the ORT [F(3,165) = 4.47; P\u2009<\u20090.01]. Separate analysis per treatment condition showed that there was a significant time effect for the TRP\u2212 condition [F(3,87) = 5.41; P\u2009<\u20090.01] in memory performance, but not for the TRP+ condition [F(3,84) = 1.77; P\u2009=\u2009n.s.]. Post hoc analysis showed that object recognition performance was significantly impaired when the TRP\u2212 mixture was administered 3\u00a0h before the first trial.\nFig.\u00a02The effects of treatment time of TRP+ or TRP\u2212 on the performance in object recognition task. Asterisks indicate a significant impairment in object recognition performance (*P\u2009<\u20090.05)Table\u00a03Exploration times in the object recognition task at baseline and after ATD\u00a0T1\u201430\u00a0minT1\u20141\u00a0hT1\u20143\u00a0hT1\u20146\u00a0hTRP+e126.62 (2.09)32.30 (1.37)27.82 (2.06)29.00 (2.50)e233.64 (2.40)49.78 (3.11)*28.61 (2.39)28.37 (2.15)TRP\u2212e126.97 (2.11)30.39 (1.78)29.29 (1.66)24.98 (1.53)e239.26 (4.17)37.32 (2.40)30.06 (1.77)28.91 (2.69)*P\u2009<\u20090.05 (indicates significant differences to the baseline group)\nBehavioral experiment 2\nThe effects of ATD in combination with rolipram on object recognition performance are depicted in Fig.\u00a03 and Table\u00a04. A dose-dependent increase in discrimination performance was observed after treatment with rolipram [F(4,104) = 6.85; P\u2009<\u20090.01]. Post hoc analysis showed that a dose of 0.1\u00a0mg\/kg rolipram reversed the effects of TRP\u2212 treatment (P\u2009<\u20090.05). Rolipram treatment decreased exploration times in the first trial [F(4,106) = 16.24; P\u2009<\u20090.01], but had no effect on exploration times in the second trial [F(4,106) = 1.53; n.s.; see Table\u00a04). Post hoc analysis showed that for all of the rolipram doses the exploration in the first trial was lower than the saline control condition.\nFig.\u00a03The effects of rolipram treatment on ATD-induced deficits in the object recognition task. Asterisks indicate a significant difference in object recognition performance compared with the TRP\u2212 and vehicle condition (**P\u2009<\u20090.01). The 0.1-mg\/kg dose of rolipram reverses the ATD effectTable\u00a04Exploration times in the object recognition task after treatment with rolipram and TRP\u2212 mixture\u00a0SalineVehicle0.01\u00a0mg\/kg0.03\u00a0mg\/kg0.1\u00a0mg\/kgSalineTRP\u2212TRP\u2212TRP\u2212TRP\u2212e128.72 (2.52)26.18 (1.72)19.15 (1.66)*18.68 (1.35)*12.02 (1.53)*e228.73 (1.42)27.43 (1.80)21.68 (1.96)27.89 (2.63)27.29 (2.70)*P\u2009<\u20090.05 (indicates significant differences to the baseline group)\nA comparison between the d2 values of the control groups of the first and second experiment (TRP+ 3\u00a0h vs saline) showed that there was no difference between these two control groups [t(38) = 0.906; n.s.].\nDiscussion\nThe present study shows that acute tryptophan depletion results in reliable lowering of plasma TRP and the TRP\/\u03a3LNAA ratio. When rats were administered one dose (10\u00a0ml\/kg) of the TRP\u2212 mixture, depletion was maximal (48%) 1\u00a0h after the treatment. The present data provide strong evidence that blood TRP levels predict object recognition performance and that ATD has only temporary effects on TRP levels and memory performance. When injected 3\u00a0h before T1, object recognition was maximally impaired. This is in support of the results from the blood TRP values (see above). Thus, TRP levels in the blood seem to be related with behavioral performance in rats. As previous studies have shown (Lieben et al. 2004a) a clear relation between plasma TRP and central 5-HT, these data provide further support that ATD can be used as a serotonergic-deficit model in the object recognition task.\nIn the study by Lieben at al. (2004a,b), two doses of the TRP\u2212 mixture (10\u00a0ml\/kg) were administered, which resulted in a 70% drop of the TRP\/\u03a3LNAA ratio and in memory impairment in the ORT. In contrast, the present study showed that one dose of the TRP\u2212 mixture resulted in a 48% drop in the TRP\/\u03a3LNAA ratio, which was found to induce memory impairment in the ORT. These findings suggest that a 50% reduction in plasma TRP levels is sufficient to impair object memory in rats.\nIn the first behavioral experiment, no effects on exploration times in the ORT were observed, except from an increase in exploration time in the second trial of the TRP+ 1\u00a0h before T1 group. However, in the second experiment, a dose-dependent decrease in the exploration time of T1 was observed after treatment with rolipram. Previous work from our group has shown that as long as exploration in trial 1 is higher than 10\u00a0s, a reliable discrimination index (d2) can be calculated (\u015e\u0131k et al. 2003). Furthermore, as can be seen when exploration times are compared to d2 values, effects in exploration time are independent of effects in d2 values (\u015e\u0131k et al. 2003). Rolipram is known to have some sedative side effects (at higher dosages >0.1\u00a0mg\/kg) (Griebel et al. 1991; Silvestre et al. 1999); nevertheless, the low dosages used in the present study improved memory performance.\nRolipram has shown its pro-cognitive effects in several behavioral models. We have shown that rolipram treatment increases object memory in a time-dependent forgetting paradigm (Rutten et al. 2006). In addition, rolipram attenuated cholinergic deficits caused by scopolamine in several behavioral tasks (Imanishi et al. 1997; Rutten et al. 2006; Silvestre et al. 1999; Zhang and O\u2019Donnell 2000). When rolipram (0.1\u00a0mg\/kg) was administered in combination with the TRP\u2212 mixture (i.e., TRP\u2212 3\u00a0h before T1 and rolipram 30\u00a0min before T1), we found that it reversed the effects of ATD. These are the first data suggesting that rolipram can reverse a serotonergic-induced memory deficit in rats.\nOur previous study indicated that rolipram can reverse the cholinergic deficit caused by scopolamine in the ORT (Rutten et al. 2006). Remarkably, a similar dose of 0.1\u00a0mg\/kg rolipram was capable of reversing the serotonergic and cholinergic deficits in the object recognition task, which might be explained by a non-specific working mechanism of rolipram. Although the underlying mechanisms of action of the memory-enhancing effects of rolipram in these models remain to be determined, the present data could be explained via an enhanced cholinergic and serotonergic turnover by rolipram (Imanishi et al. 1997; Schoffelmeer et al. 1985).\nAlternatively, the effects of rolipram could also be explained in terms of only a cholinergic mechanism of action. Thus, previous studies have shown that the acetylcholinesterase inhibitor metrifonate ameliorates the effects of ATD as well and improves performance in scopolamine-deficit models of the ORT (Lieben et al. 2005). This suggests that the cholinergic and serotonergic systems act in a synergistic manner in memory performance (see Lieben et al. 2005 for a detailed discussion). Clearly, further research is needed to scrutinize the mechanism of action rolipram in the different animal models of memory.\nAnother mechanism of action of rolipram that could explain the present data is related to the intracellular signaling in long-term potentiation (LTP). In the hippocampus, rolipram has shown to improve LTP through activation of the cAMP\/PKA\/CREB pathway (Frey et al. 1993; Impey et al. 1996). Moreover, these mechanisms have been linked to behavioral improvement in several behavioral tasks (Barad et al. 1998; Bernabeu et al. 1997; Blokland et al. 2006; Rutten et al. 2006). Consequently, it could be argued that the present data could be explained by the effects of rolipram on the cAMP\/PKA\/CREB pathway. However, a recent study by our group showed that the administration time of rolipram was of critical relevance. Rolipram was only effective in the 24-h interval ORT when administered 3\u00a0h after T1 (Rutten et al. 2006), and the mechanism of late LTP was proposed to explain these effects. However, in the present study, a 1-h interval in the ORT was examined, in which no gene transcription or protein synthesis is required. Therefore, we assume that the cAMP\/PKA\/CREB pathway cannot explain these effects.\nA recent PET study used radioactive rolipram as a measure for PDE4 binding and as an indirect index of cAMP signaling (Lourenco et al. 2006). Acute elevation of synaptic neurotransmitter levels (noradrenaline, 5-HT, and histamine) resulted in elevated cAMP levels that in turn elevated (R)-[11C] rolipram binding to PDE4 (Lourenco et al. 2006). According to this rationale, elevation of cAMP levels through inhibition of PDE4 can reverse cAMP-mediated deficits in behavior that occur due to a shortage of 5-HT, ie, reversing the ATD-induced object recognition impairment. As rolipram non-selectively facilitates several neurotransmitter systems, a broad mechanism of action can be expected. This can be favorable because influencing multiple systems increases the likelihood that several aspects of behavior could be enhanced. This may explain previous reports that rolipram was effective in depression models (Bobon et al. 1988; Norman et al. 1992; Weishaar et al. 1985). On the downside, the broad mechanism of action increases the risks for negative side effects, e.g., emesis, sedation, and nausea.\nIn summary, the method of ATD can be used as a reliable method for inducing object memory impairment in rats. The selective PDE4 inhibitor rolipram was able to reverse this serotonergic deficit, which could be explained by different mechanisms of action. These data provide further support that PDE4 inhibition could be considered as a potential target to improve memory performance.","keyphrases":["pde4","rolipram","memory","tryptophan depletion","phosphodiesterase","cognition","object recognition","atd","cyclic amp"],"prmu":["P","P","P","P","P","P","P","P","M"]}
{"id":"Plant_Mol_Biol-3-1-1876254","title":"Tomato linalool synthase is induced in trichomes by jasmonic acid\n","text":"Tomato (Lycopersicon esculentum) plants emit a blend of volatile organic compounds, which mainly consists of terpenes. Upon herbivory or wounding, the emission of several terpenes increases. We have identified and characterized the first two tomato monoterpene synthases, LeMTS1 and LeMTS2. Although these proteins were highly homologous, recombinant LeMTS1 protein produced (R)-linalool from geranyl diphosphate (GPP) and (E)-nerolidol from farnesyl diphosphate (FPP), while recombinant LeMTS2 produced \u03b2-phellandrene, \u03b2-myrcene, and sabinene from GPP. In addition, these genes were expressed in different tissues: LeMTS1 was expressed in flowers, young leaves, stems, and petioles, while LeMTS2 was strongest expressed in stems and roots. LeMTS1 expression in leaves was induced by spider mite-infestation, wounding and jasmonic acid (JA)-treatment, while LeMTS2 did not respond to these stimuli. The expression of LeMTS1 in stems and petioles was predominantly detected in trichomes and could be induced by JA. Because JA treatment strongly induced emission of linalool and overexpression of LeMTS1 in tomato resulted in increased production of linalool, we propose that LeMTS1 is a genuine linalool synthase. Our results underline the importance of trichomes in JA-induced terpene emission in tomato.\nIntroduction\nPlants produce a wide variety of terpenoids that have primary functions as hormones (gibberellins, abscisic acid, brassinolide), sterols, pigments (carotenoids, phytol), and as parts of electron carrier moieties (ubiquinone, plastoquinone) (McGarvey and Croteau 1995). Most terpenoids, however, are secondary metabolites: over 20,000 different terpenoid structures from plants have been described (Sacchettini and Poulter 1997). Some terpenoids function in the direct defence strategy of plants as phytoalexins, which accumulate upon pathogen infection. Sesquiterpene phytoalexins (e.g., capsidiol) are characteristic for the Solanaceae (Chappell and Nable 1987; Egea et\u00a0al. 1996).\nVolatile monoterpenes and sesquiterpenes can function in the interaction of plants with other organisms. They are present in floral scents that attract pollinators (Knudsen et\u00a0al. 1993; Langenheim 1994) and are emitted by many plant species in response to herbivory by insects or spider mites. Induced terpene emission can attract predators or parasitoids of the herbivores, a mechanism commonly referred to as the indirect defense strategy of plants. This has been studied in, for instance, Arabidopsis (Arabidopsis thaliana) (Van Poecke et\u00a0al. 2001), lima bean (Phaseolus lunatus) (Dicke 1994; Takabayashi and Dicke 1996), tobacco (Nicotiana tabacum; N. attenuata), maize (Zea mays), and cotton (Gossypium hirsutum) (De Moraes et\u00a0al. 1998; Pare and Tumlinson 1999), and tomato (Kant et\u00a0al. 2004; Takabayashi et\u00a0al. 2000).\nThese volatile terpenes are produced by sesqui- and monoterpene synthases of which several are induced by herbivory. For instance, nerolidol synthase activity is induced in lima bean, cucumber (Cucumis sativus), and maize (Bouwmeester et\u00a0al. 1999; Degenhardt and Gershenzon 2000), whereas transcript levels of various other sesquiterpene synthases increase in, amongst others, maize (Shen et\u00a0al. 2000), cucumber (Mercke et\u00a0al. 2004), and wormwood (Artemisia annua) (Cai et\u00a0al. 2002). In Arabidopsis, caterpillars (Pieris rapae) induce the myrcene\/ocimene synthase AtTPS10 and the \u03b2-ocimene synthase AtTPS03, which coincides with increased myrcene emission (Van Poecke et\u00a0al. 2001). Moreover, a \u03b2-ocimene synthase (LjE\u03b2OS) has been identified from Lotus (Lotus japonicus), which is induced by spider mite-feeding, resulting in increased emission of \u03b2-ocimene (Arimura et\u00a0al. 2004b). However, artificial wounding or jasmonic acid (JA) treatment are often also sufficient to induce terpene synthases. The wound- and JA-induced synthesis of terpenes by coniferous plants is well described (Martin et\u00a0al. 2002; Miller et\u00a0al. 2005; Steele et\u00a0al. 1995), and these treatments also mimic herbivore-induced responses in several angiosperms (Arimura et\u00a0al. 2004a; Gomez et\u00a0al. 2005; Schnee et\u00a0al. 2002; Shen et\u00a0al. 2000).\nHere we describe the identification and characterization of the first two monoterpene synthases from a solanaceous species (tomato), LeMTS1 and LeMTS2. We provide evidence that these genes are differentially regulated and more importantly, that expression of linalool synthase LeMTS1 is restricted to trichomes and induced by JA.\nResults\nIdentification of tomato terpene synthases 1 and 2 (LeMTS1 and LeMTS2)\nIn order to identify tomato monoterpene synthases that are induced upon spider mite herbivory, we first queried tomato EST databases (www.tigr.org and www.ncbi.nlm.nih.gov\/BLAST\/) with known monoterpene synthase sequences from other plant species. Retrieved tomato EST sequences were used to design a primer specific for monoterpene synthases (Fig.\u00a01). We then used cDNA derived from spider mite-infested plants, to amplify a single 850\u00a0bp fragment with this primer and an oligo dT(18) primer. This fragment was subsequently used as a probe to screen available tomato cDNA libraries. Two different full-length cDNAs were identified; LeMTS1 (AY840091) was isolated from tomato leaves (sequence identical to the probe) and LeMTS2 (AY840092) from tomato roots and stems. The LeMTS1 and LeMTS2 open reading frames encode proteins of 609 and 590 amino acids (Fig.\u00a01), which have predicted masses of 70.8 and 68.6\u00a0kDa, respectively, and are 71% identical. LeMTS1 and LeMTS2 contain amino acid motifs conserved in angiosperm (mono)terpene synthases. These are, based on LeMTS1 amino acid positions: R(45)R(46)W(55), required for the initial diphosphate migration (Williams et\u00a0al. 1998); D(362)D(363)D(366), required for binding of a Mg2+ or Mn2+ ion, which is used as cofactor (Cane et\u00a0al. 1996; Starks et\u00a0al. 1997); D(505)T(509)E(513), required for binding of a second Mg2+ or Mn2+ cofactor; and D(586) in the active site cleft (Schwab et\u00a0al. 2001; Whittington et\u00a0al. 2002). LeMTS1 and LeMTS2 contain both putative (predicted with TargetP; Emanuelsson et\u00a0al. 2000) plastid targeting signal peptides, which should be processed in the region upstream of the conserved R(45)R(46) arginine pair.\nFig.\u00a01Alignment of deduced amino acid sequences of LeMTS1 and LeMTS2 with three terpene synthases. A ClustalW alignment is shown of Mentha aquatica linalool synthase, a monoterpene synthase (C10), MaLin (AAL99381); Lycopersicon esculentum monoterpene synthase 1, LeMTS1 (AY840091); L. esculentum monoterpene synthase 2, LeMTS2 (AY840092); Mentha longifolia limonene synthase, MlLim (AAD50304), and L. esculentum germacrene C synthase, a typical sesquiterpene synthase (C15), LeGerC (AAC39432). Amino acids are shaded dark if identical in at least three of the five sequences and light if identical in LeMTS1 and LeMTS2 only. Conserved amino acid motifs typical for terpene synthases are boxed: RRx8W (Williams et\u00a0al. 1998), DDxxD (Cane et\u00a0al. 1996; Starks et\u00a0al. 1997), Dx3Tx3E, and D (Schwab et\u00a0al. 2001; Whittington et\u00a0al. 2002). The presence of putative N-terminal plastid targeting peptides in the four monoterpene synthases and the primer used for amplifying the initial LeMTS fragment from cDNA of spider mite-infested leaves are indicated\nRemarkably, LeMTS1 contains an extra sequence stretch of 22 amino acids starting at N170 (Fig.\u00a01). This sequence is not found in any other terpene synthase, nor does it have homology to any protein in the NCBI database (www.ncbi.nlm.nih.gov\/BLAST\/). Its presence can also not be explained by alternative splicing since the flanks do not contain canonical splice sites. This extra internal element is not an artefact of the isolated cDNA since it was present in four independent cDNAs isolated from different organs (data not shown).\nLeMTS1 and LeMTS2 belong to the TPSb subfamily of terpene synthases\nLeMTS1 and LeMTS2 are more similar to typical monoterpene synthases from, e.g., mint and citrus than to a typical tomato sesquiterpene synthase (Fig.\u00a01). Based on protein sequence relatedness, LeMTS1 and LeMTS2 can be classified in the TPSb subfamily (Fig.\u00a02), a distinct group of angiosperm monoterpene synthases, according to the original classification proposed by Bohlmann et\u00a0al. (1997). So, unlike the snapdragon (Antirrhinum majus) monoterpene synthases from subfamily TPSg (Dudareva et\u00a0al. 2003), including an Arabidopsis and a strawberry terpene synthase, the monoterpene synthases from the solanaceous family do not seem to form a new subfamily.\nFig.\u00a02LeMTS1 and LeMTS2 belong to the TPSb subfamily of terpene synthases. The TPS family is divided into subfamilies according to the nomenclature of Bohlmann et\u00a0al. (1997). TPSa: angiosperm sesquiterpene synthases, TPSb: angiosperm monoterpene synthases, TPSc: angiosperm diterpene synthases, TPSd: gymnosperm mono-sesqui- and diterpene synthases, TPSe: a second group of angiosperm diterpene synthases, TPSf: a distinct group of (putative) linalool synthases mainly from Clarkia (Dudareva et\u00a0al. 1996) and TPSg: a recently identified group of monoterpene synthases mainly from Antirrhinum (Dudareva et\u00a0al. 2003). A limited amount of representative terpene synthases is shown. The bootstrapped phylogenetic tree was constructed using PAUP and TreeView software. A representative tree of 100 replicates is shown. Genbank protein accessions of terpene synthases shown are, top to bottom: TPSf: AAL24105, AAC49395; TPSe: BAC56714, AAC39443; TPSc: BAA84918, NP192187; TPSd: AAK83566, AAK39129, AAK39127, AAB71084; TPSg: NP176361, AAO42614, CAD57081; TPSa: AAU05951, AAK54279, AAC39432, AAG09949, Q40577; TPSb: NP189212, NP567511, AY840091, AY840092, AAC26018, AAV63791, AAL99381, AAD50304, AAG31435, AAM53943, AAM53945, AAF13356, AAK58723, CAC41012, AAS79351\nTerpene synthase activity of recombinant LeMTS1 and LeMTS2\nRecombinant LeMTS1 and LeMTS2 proteins were produced in Escherichia coli in order to characterize their enzymatic activities. The plastid targeting peptide was deleted up to one amino acid upstream of the RRX8W motif (Fig.\u00a01) to produce a His-tagged \u2018pseudo-mature\u2019 form of the proteins, since it has been reported that this can improve protein expression without affecting activity and product specificity of the enzymes (Bohlmann et\u00a0al. 1999; Williams et\u00a0al. 1998). The soluble protein fractions were assayed for terpene synthase activity and products were analyzed on a GC coupled to a Time Of Flight-MS. Using the monoterpene precursor geranyl diphosphate (GPP) as substrate, LeMTS1 generated only linalool whereas LeMTS2 generated several monoterpene products: \u03b2-phellandrene, \u03b2-myrcene, and sabinene (Fig.\u00a03a). Mass spectra and relative retention indices (Adams 2001) or authentic standards confirmed the identification of terpene reaction products. Extracts of E. coli cells expressing the empty vector control generated minor amounts of linalool (Fig.\u00a03a) and occasionally geraniol. Thermally induced autoconversion or solvolysis of GPP to linalool has been observed before (Crowell et\u00a0al. 2002; Jia et\u00a0al. 1999), as well as conversion to geraniol by aspecific hydrolase or phosphatase activity in protein extracts (Crowell et\u00a0al. 2002). However, LeMTS1 product levels are several hundred-fold higher than the background levels of the control (Fig.\u00a03a).\nFig.\u00a03Enzymatic activity of recombinant LeMTS1 and LeMTS2 proteins. (A) GC\/MS chromatograms of the LeMTS1 and LeMTS2 monoterpene products and the empty vector control with GPP as substrate. (B) GC\/MS chromatogram of the LeMTS1 sesquiterpene product and the empty vector control with FPP as substrate. The chromatograms show detector responses for the terpene-specific ion mass 93. Products were identified as linalool (1), sabinene (2), \u03b2-myrcene (3), \u03b2-phellandrene (4), and (E)-nerolidol (5). Representative chromatograms are shown from assays of at least two independent experiments\nEnantiomer separation on an enantiomer-selective column showed that LeMTS1 produced only (R)-linalool (data not shown). LeMTS1 was also able to utilize the sesquiterpene precursor farnesyl diphosphate (FPP) as substrate to produce (E)-nerolidol (Fig.\u00a03b), whereas LeMTS2 had no detectable sesquiterpene synthase activity. Both enzymes were unable to form terpene products from the diterpene precursor geranylgeranyl diphosphate.\nLeMTS1 and LeMTS2 are differentially expressed in various plant organs\nWe investigated the organ specificity of LeMTS1 and LeMTS2 expression in a mature (13\u00a0weeks old) tomato plant (Fig.\u00a04a). Transcripts of LeMTS1 were detected in young fruit, flower buds, petals, sepals, stems, petioles, and in young leaves. The expression pattern of LeMTS2 was very different: no transcripts were detected in petals, sepals, and leaves. However, LeMTS2 was expressed in roots.\nFig.\u00a04Spatial expression patterns of LeMTS1 and LeMTS2 in tomato. (A) Expression of LeMTS1 and LeMTS2 in different organs of an adult tomato plant. (B) Expression in trichomes or complementary tissue of petioles and stems. Expression was analyzed by RT-PCR. The data from one of two independent tissue sets that gave similar results is shown. RUB1 conjugating enzyme (RCE1, www.tigr.org: TC153679) was used as constitutive control. Ethidium bromide-stained agarose gels are shown\nFor plants that contain glandular trichomes, monoterpene production is considered to be localized exclusively in these organs (Gershenzon et\u00a0al. 1992; Iijima et\u00a0al. 2004; Kutchan 2005; Turner et\u00a0al. 1999; Turner and Croteau 2004). Tomato plants possess several types of trichomes including glandular trichomes that are present in high density on leaves, petioles, and stems and that contain terpenes (Maluf et\u00a0al. 2001; Snyder and Carter 1985). Therefore, we investigated the location of LeMTS1 and LeMTS2 expression in trichomes of petioles and stems. This showed that LeMTS1 is expressed in trichomes, whereas LeMTS2 is expressed in the complementary tissue (Fig.\u00a04b).\nLeMTS1 but not LeMTS2 is induced by spider mite feeding, artificial wounding, and jasmonic acid\nTo study whether LeMTS1 and LeMTS2 were induced upon spider mite feeding, changes in transcript levels of intact plants were analyzed by RT-PCR since LeMTS gene-expression was too low to determine by RNA gel-blot analysis. Expression of LeMTS1 was indeed induced in leaves by spider mite-feeding, but LeMTS2, which expression was very low in leaves, was not (Fig.\u00a05a). Spider mites damage plant tissue and induce JA- and SA-responsive genes such as a wound-induced proteinase inhibitor (WIPI-II) and a pathogenesis related protein (PRP6), respectively (Kant et\u00a0al. 2004). Therefore, we investigated the effects of artificial wounding and exogenous application of JA and SA (Fig.\u00a05a). Both artificial wounding and JA induced LeMTS1. In contrast, LeMTS2 was not induced by these treatments (Fig.\u00a05a). SA-treatment induced the SA-marker PRP6 but neither LeMTS1 nor LeMTS2 was increased. Induction of LeMTS1 and WIPI-II was validated by real-time Q-RT-PCR. This showed consistent and clear induction of both genes by JA, artificial wounding and spider mites, although there was large variation in the level of induction between independent experiments (Fig.\u00a05b). We routinely used the tomato cultivar Moneymaker for our experiments. However, in our earlier work (Ament et\u00a0al. 2004; Kant et\u00a0al. 2004) the cultivar Castlemart (CM) was used because it has the same genetic background as the defenseless1 (def-1) mutant (Howe et\u00a0al. 1996). Here, we show that spider mite- and JA-induced LeMTS1 expression occurred both in CM and Moneymaker (Fig.\u00a05b).\nFig.\u00a05Induction of LeMTS1 in tomato leaves in response to spider mites, wounding, and jasmonic acid (JA). (A) Gene expression analysis by semi-quantitative RT-PCR of LeMTS1 and LeMTS2 after various treatments, compared to the well known JA- and SA-marker genes WIPI-II (Graham et\u00a0al. 1985) and PRP6 (van Kan et\u00a0al. 1992), respectively. Treatments: JA, SA, and artificial wounding (W) after 24\u00a0h and spider mite-infestation (M) after 48\u00a0h. A representative result from three independent experiments is shown. RUB1 conjugating enzyme (RCE1) was used as constitutive control. Ethidium bromide-stained agarose gels are shown. (B) Validation of LeMTS1 and WIPI-II induction by real-time Q-RT-PCR. Two different tomato cultivars were used to verify induction by mites and JA. Values represent expression levels relative to controls, calculated after correction for expression of the control gene RCE1 (TC153679, www.tigr.org). Average values and standard errors are shown from three to five independent replicates\nTo investigate whether spider mite-induced expression of LeMTS1 was dependent on JA or SA we made use of the def-1 mutant, which is impaired in induced JA-accumulation (Howe et\u00a0al. 1996) and the SA-deficient, transgenic NahG tomato line (Brading et\u00a0al. 2000). In plants with the def-1 mutation, LeMTS1 was not induced by spider mites, whereas the expression could be induced by exogenous JA (Fig.\u00a06a). In contrast, the NahG plants showed higher and clearly induced LeMTS1 expression (Fig.\u00a06b). This shows that induction of LeMTS1 expression is dependent on JA but not on SA.\nFig.\u00a06Spider mite-induced LeMTS1 expression is dependent on jasmonic acid (JA). (A) Expression of LeMTS1 in wild type (wt) or JA deficient def-1 mutant plants infested with spider mites (M) or treated with JA for 2\u00a0days. (B) LeMTS1 expression in wild type and SA deficient NahG plants in response to 2\u00a0days of spider mite-feeding. Gene expression was analyzed by RT-PCR, a representative result from one out of two independent experiments is shown. RUB1 conjugating enzyme (RCE1) was used as constitutive control. Ethidium bromide-stained agarose gels are shown\nJA induces expression of LeMTS1 in trichomes\nSince basal LeMTS1 expression in stems and petioles seemed to be restricted to trichomes (Fig.\u00a04b), we addressed the question whether JA would induce expression of LeMTS1 solely in trichomes or also in other tissues. Indeed, JA-induced LeMTS1 expression was strongest in trichomes (Fig.\u00a07). Interestingly, WIPI-II expression showed the opposite pattern. WIPI-II expression is very low in trichomes and basal as well as strongly induced WIPI-II expression occurred mainly in the complementary stem tissues.\nFig.\u00a07LeMTS1 is induced in trichomes by JA. Expression analysis of LeMTS1 and WIPI-II by real-time Q-RT-PCR. Intact plants were treated with JA, and subsequently trichomes were separated from remaining tissues of stems. The results from four independent experiments are shown as mean values and standard errors. Expression levels are indicated as expression relative to the control gene RCE1\nLinalool accumulates in glandular trichomes and linalool emission is induced by wounding, jasmonic acid, and by ectopic LeMTS1 overexpression\nFigure\u00a04b showed that, in petioles and stems, LeMTS1 was expressed specifically in the trichomes. Extraction of terpenes from the same tissues of that experiment demonstrated that linalool was produced predominantly in trichomes (Fig.\u00a08). (E)-nerolidol and \u03b2-phellandrene also accumulated in trichomes. Conversely, the volatile benzenoid methyl-salicylate was not produced in trichomes and was also clearly detected in roots, in contrast to most terpenes.\nFig.\u00a08Linalool accumulation in trichomes. Relative levels of linalool, a selection of other terpenes and methyl salicylate in extracts of petioles and stems before and after removal of trichomes compared to total leaf and root extracts. Equal amounts of the different tissues were used (freshweight, 0.5\u00a0g), and trichome material was corrected for freshweight of the original petiole or stem. Averages of two measurements and min\/max are shown\nBecause LeMTS1 was induced by spider mite-infestation, wounding and JA, we investigated whether the emission of its in vitro products was induced concomitantly. Analysis of volatile production during 2\u00a0days of wounding or JA-treatment, conditions that induce LeMTS1 expression (Fig.\u00a05), revealed that plants emitted more linalool after both treatments (Fig.\u00a09a), while nerolidol emission was not induced.\nFig.\u00a09Increased linalool emission. (A) Linalool and (E)-nerolidol emission by intact tomato plants that were wounded (W) or treated with 1\u00a0mM jasmonic acid (JA) twice a day, for 2\u00a0days. The bar-graph shows the average (n\u00a0=\u00a03) emission over 2\u00a0days compared to the control and standard errors. Data (corrected for unequal variances) were analyzed by means of ANOVA followed by a Fishers-LSD post hoc-test. Linalool emission for both JA and wounding treatments was significantly increased compared to the control (P\u00a0<\u00a00.05), whereas (E)-nerolidol emission was not. (B) Linalool emission by a transgenic tomato line overexpressing LeMTS1. Headspaces were sampled from desiccators containing two plants each and compared to that of plants transformed with the empty vector. The graph expresses average values and standard deviations of four measurements. Emission of (E)-nerolidol, \u03b2-phellandrene, and methyl-salicylate did not significantly differ between plants. LeMTS1 overexpression in leaves was confirmed by RNA gel-blot analysis. The expression of RUB1 conjugating enzyme (RCE1) and ribosomal RNA levels (ethidium bromide-stained gel) are shown to indicate equal loading of the gel\nNext, we tried to overexpress the full length LeMTS1 cDNA (including the putative targeting peptide) in tomato plants, under control of the CaMV 35S promoter. Four transformation experiments yielded only two transformants of which one overexpressed LeMTS1. Compared to empty vector-transformed plants, LeMTS1 overexpressing plants emitted several hundred-fold more linalool into the plants headspace (Fig.\u00a09b), while emission of other terpenes, including nerolidol, did not differ significantly from control plants. This suggests that LeMTS1 functions in planta as a true linalool synthase.\nDiscussion\nMonoterpenes are abundantly emitted by tomato plants. For instance \u03b2-pinene, \u03b2-myrcene, 2-carene, \u03b2-phellandrene, limonene, and terpinolene are constitutively emitted, while linalool and \u03b2-ocimene emission is induced after spider mite feeding (Kant et\u00a0al. 2004). In this paper, we describe the first two monoterpene synthases from tomato, LeMTS1 and LeMTS2 and show that LeMTS1 is induced by JA in trichomes.\nIdentification of two tomato monoterpene synthases\nIn vitro assays with the recombinant LeMTS1 and LeMTS2 proteins show that LeMTS1 has both (R)-linalool synthase and (E)-nerolidol synthase activity, and that LeMTS2 is a \u03b2-phellandrene\/\u03b2-myrcene\/sabinene synthase (Fig.\u00a02a). LeMTS1 activity in planta seems restricted to linalool synthase activity since increased LeMTS1 expression by wounding, JA-treatment and ectopic overexpression led to increased linalool emission but did not affect nerolidol levels (Figs.\u00a05, 9).\nGenerally speaking, monoterpene synthases are plastid targeted and use GPP, while sesquiterpene synthases use FPP in the cytosol. There are however some exceptions to this rule. For instance, a cytosolic strawberry nerolidol synthase (FaNES1) has both (3S)(E)-nerolidol synthase and S-linalool synthase activity and is likely responsible for both linalool and nerolidol synthesis in planta (Aharoni et\u00a0al. 2004). Vice versa, when FaNES1 was targeted to the plastid in Arabidopsis it also produced both linalool and nerolidol (Aharoni et\u00a0al. 2003). The authors suggested that GPP and FPP substrate pools are not strictly separated between the cytosol and plastid, which has recently been supported by similar engineering experiments in tobacco (Wu et\u00a0al. 2006). These observations might be explainable by for instance cytosolic as well as plastidial targeting of GPP synthase (GPS) in Arabidopsis and Litospermum erythrorizon (Bouvier et\u00a0al. 2000; Sommer et\u00a0al. 1995). In addition, FPP might be transported from the cytosol into plastids (Lichtenthaler 1999) and finally, the prenyltransferases GPS, FPS or GGPS might generate multiple products. However, since induced LeMTS1 expression does not correlate with increased nerolidol emission but only with increased linalool emission (Figs.\u00a05, 9), we consider LeMTS1 to be a linalool synthase. Furthermore, we hypothesize that the plastidial FPP concentrations in tomato are negligible.\nDespite their high sequence homology (Fig.\u00a01), LeMTS1 and LeMTS2 have different biochemical activities. It is commonly found that different terpene synthases within one plant species often have higher sequence similarity to one another than to functionally related terpene synthases of other plant species. For example, \u03b2-pinene synthase from citrus (Citrus limon) is more homologous to the citrus \u03b3-terpinene synthase than to \u03b2-pinene synthase from wormwood (Fig.\u00a02). It is also known from work with other terpene synthases that product specificity can depend on only a few amino acids (Kollner et\u00a0al. 2004; Rising et\u00a0al. 2000; Yoshikuni et\u00a0al. 2006) and thus, product specificity of terpene synthases is not predictable on basis of protein sequence or specific amino acid residues. By comparing terpene synthase structures, attempts have been made to ascribe substrate binding and substrate modifications to certain residues. Modeling of the active sites of a Salvia (Salvia officinalis) sabinene synthase, a mint (Mentha citrata) limonene synthase and a mint linalool synthase led to the following observation (Crowell et\u00a0al. 2002): compared to the other two enzymes, the linalool synthase has a three amino acid deletion in the active site pocket (two amino acids after the conserved D591, see Fig.\u00a01), resulting in a more open structure of the so called J\/K loop and providing an easier access of water during substrate ionization. Water access results in the premature release of the intermediate monoterpenol carbo-cation, before cyclization to a monoterpene olefin (non-hydrated) can occur. The tomato linalool synthase LeMTS1 also has a deletion in the active site pocket after the conserved D586 (Fig.\u00a01). Although the deletion is eight instead of three amino acids, it indicates that LeMTS1 might utilize a similar structural mechanism to generate a monoterpene alcohol.\nLeMTS1 expression correlates with induced linalool emission\nThe induction of LeMTS1 expression correlated with the increased emission of linalool by tomato plants. Wounding and JA application induced LeMTS1 (Fig.\u00a05) as well as linalool emission (Fig.\u00a09a). Induction of LeMTS1 upon spider mite-feeding (Fig.\u00a05) coincided with linalool emission (Kant et\u00a0al. 2004) in the tomato cultivar CM. Moreover, overexpression of LeMTS1 resulted in enhanced production of linalool (Fig.\u00a09b) and no other volatile terpenes, providing evidence for in planta linalool synthase activity of LeMTS1. RNAi lines suppressing LeMTS1 expression in trichomes would provide additional evidence that LeMTS1 acts as a linalool synthase in planta.\nIt is generally known that successful overexpression of terpene synthases can be problematic (reviewed by Chappell 2004). Substrate limitation and product conversions can explain low-terpene production levels (Aharoni et\u00a0al. 2003; Lucker et\u00a0al. 2001; Ohara et\u00a0al. 2003). In addition, ectopic terpene synthase overexpression might lead to product toxicity (Aharoni et\u00a0al. 2006; Besumbes et\u00a0al. 2004) and selection for plant lines with low transgene expression or low terpene accumulation (Diemer et\u00a0al. 2001; Krasnyanski et\u00a0al. 1999). We managed to regenerate only one LeMTS1 overexpressing plant line after four successive, independent transformations. The empty vector and unrelated constructs gave at least 20-fold higher transformation frequencies. Similarly, LeMTS2 overexpression was unsuccessful, seven transgenic plant lines showed only expression of truncated transcripts (data not shown).\nLeMTS2 is expressed in roots, stems and petioles (Fig.\u00a04a). In roots, only minute amounts of the main LeMTS2 product \u03b2-phellandrene were detected, hardly visible in Fig.\u00a08 since levels are \u223c300-fold lower than in leaves. The role of monoterpenes in roots remains unclear, although they might play a role in the defense response of plants. The release of the monoterpene 1,8-cineole from Arabidopsis roots is induced upon bacterial pathogen infection (Steeghs et\u00a0al. 2004), although transcriptional regulation of the corresponding 1,8-cineole synthase (Chen et\u00a0al. 2004) has not been shown yet.\nAlthough LeMTS2 is hardly expressed in tomato leaves (Fig.\u00a04a), \u03b2-phellandrene and \u03b2-myrcene are constitutively emitted by tomato plants (Buttery et\u00a0al. 1987; Kant et\u00a0al. 2004). This suggests that the constitutive low expression of LeMTS2 might be sufficient to generate these monoterpenes. However, like the majority of volatile terpenes (data not shown), \u03b2-phellandrene is mostly located in trichomes (Fig.\u00a08) where LeMTS2 is not expressed (Fig.\u00a04b). This indicates that there are other \u03b2-phellandrene\/\u03b2-myrcene synthases in tomato that synthesize these terpenes.\nJA-induced LeMTS1 expression occurs in trichomes\nAlthough the presence of terpene synthases in trichomes has been well documented (Bertea et\u00a0al. 2006; Iijima et\u00a0al. 2004; Lange et\u00a0al. 2000), the regulation of their expression in trichomes remains obscure. Here we show that JA treatment of intact plants leads to higher transcript levels of LeMTS1 mainly in trichomes (Fig.\u00a07). In contrast, WIPI-II expression is induced by JA in the complementary tissues, indicating that there is a tissue-specific differential activation of JA-regulated genes. Basal expression of WIPI-II was very low in tomato trichomes, although constitutive expression of a WIPI-II in glandular trichomes was recently demonstrated in nightshade (Solanum americanum) (Liu et\u00a0al. 2006). It is unknown whether trichome-expressed WIPI-II plays a role in the defense against herbivores. Induction of WIPI-II in non-glandular tissue (mesophyll\/parenchyma) meets the expectation that it should be present in those cells that suffer from herbivory.\nPreviously, JA has been shown to increase trichome density on newly formed leaves of, for instance, Arabidopsis and tomato (Boughton et\u00a0al. 2005; Traw and Bergelson 2003). Also, JA might induce trichome-based defenses directly. The production of acylsugars on the leaf surface (probably in trichomes) of Datura wrightii plants increased without affecting trichome density (Hare and Walling 2006). More clearly, nornicotin production on the Nicotiana repanda plant surface increased twofold within 6\u00a0h of JA treatment (Laue et\u00a0al. 2000). Although it should be kept in mind that in the two above-mentioned studies, it was perhaps unjustly assumed that CHCl3 or CH2Cl2 extraction releases only trichome contents, it indicates that trichome-based secondary metabolite biosynthesis can be inducible. Results from our study show, for the first time, a specific transcriptional regulation of a gene involved in secondary metabolite biosynthesis in trichomes. Isolation of trichomes revealed that linalool synthase expression in tomato trichomes was induced more than twofold by JA after 24\u00a0h.\nIn this paper, we report the identification of the first two monoterpene synthases from a plant of the solanaceae family. As it has recently been described that increased volatile terpene production in transgenic plants can benefit the indirect defense (Kappers et\u00a0al. 2005; Schnee et\u00a0al. 2006), it will be interesting to investigate the biological effect of LeMTS1 overexpression in tomato. Furthermore, the role of LeMTS2-derived terpenes in roots and stems remains to be investigated. Finally, the specific induction of LeMTS1 in trichomes by JA can provide an excellent opportunity for identification of novel, JA-related promoter elements, and transcription factors.\nMaterials and methods\nIsolation of LeMTS1 and LeMTS2 cDNAs\nTomato plants, Lycopersicon esculentum cv. Moneymaker (3\u20134\u00a0weeks old) were infested with 150 spider mites (Tetranychus urticae) as described by Kant et\u00a0al. (2004). Leaves were collected and pooled after 2\u20135\u00a0days of infestation. RNA was isolated using Trizol (Invitrogen, Carlsbad, CA, USA); cDNA was made using SuperscriptII RNAse H- (Invitrogen). A LeMTS fragment was amplified from this cDNA using the primer 5\u2032-GATGACATTTATGATGTTTATGGC-3\u2032 in combination with an oligo dT(18) primer. The primer was designed based on putative tomato monoterpene synthases (www.tigr.org: TC168035, TC160168, BG131411, and cLED9K13) as such that it should not anneal to sesquiterpene synthase cDNAs. The 850\u00a0bp PCR product was cloned into pGEM-T easy (Promega, Madison, WI, USA) and sequenced using the ABI PRISM BigDye terminator kit (Applied Biosystems, Foster city, CA, USA). cDNA was synthesized from leaves of 3-week-old Moneymaker plants and from hypocotyls and roots of 11-week-old Moneymaker plants with a ZAP-cDNA synthesis kit, cloned into the Lambda-ACT vector (Elledge et\u00a0al. 1991) and packaged with a ZAP-cDNA Gigapack II gold cloning kit (Stratagene, La Jolla, CA, USA) 200,000 plaques of each library were screenend with the 850\u00a0bp RT-PCR product as probe, radioactively labeled by the ALL-IN-ONE Random prime labeling method (Sigma, Saint Louis, MI, USA). Filters were hybridized at 55\u00b0C in BLOTTO hybridization buffer (Sambrook and Russell 2001) and washed three times for 10\u00a0min in 1\u00a0\u00d7\u00a0 SSC, 0.1% SDS at 55\u00b0C. DNA from positive plaques was converted to pAct2 plasmids and their inserts were sequenced. LeMTS1 was obtained from the leaf cDNA library, LeMTS2 from the hypocotyl and root cDNA library. Sequences can be found in Genbank as AY840091 and AY840092, respectively. The presence of plastid targeting signals was predicted using PREDOTAR (http:\/\/genoplante-info.infobiogen.fr\/predotar\/predotar.html; Small et\u00a0al. 2004) and TargetP (http:\/\/www.cbs.dtu.dk\/services\/TargetP\/; Emanuelsson et\u00a0al. 2000).\nExpression of recombinant proteins in E. coli, enzyme assays and product analysis\nLeMTS1 and LeMTS2 were subcloned into the pET32-a expression vector (Novagen, San Diego, CA, USA) after removal of the plastid targeting signal sequences up to one amino acid upstream of the conserved arginine pair (Fig.\u00a01) by generating \u2018truncated\u2019 LeMTS1 and LeMTS2 PCR products with Pfu DNA polymerase (Stratagene). Forward primers contained NcoI- and reverse primers XhoI-restriction sites. LeMTS1 primers (5\u2032\u20133\u2032): GATCCATGGACACAAGGCGTTCAGGGAATTAC and GTACTCGAGCAAAGTAATAAAATGAAGCCTACG, LeMTS2 primers: GATCCATGGGTATCCGACGTTCAGGAAATTAC and GTACTCGAGAATAAAAGGTAATAATTCCTTGTC. pET32-LeMTS constructs were transformed into E. coli BL21(DE3) which were selected on Luria-Bertani (LB) plates containing 100\u00a0mg\/l ampicillin. A single colony was grown overnight at 37\u00b0C on a plate, and transferred to 100\u00a0ml of LB with ampicillin. Cultures were grown at 37\u00b0C for 30\u201360\u00a0min to A600 of 0.6\u20131.0 before addition of 1\u00a0mM isopropyl-1-thio-\u03b2-d-galactopyranoside and grown at 20\u00b0C for an additional 8\u00a0h. Cells were harvested by centrifugation and resuspended in 5\u00a0ml monoterpene synthase assay buffer containing 50\u00a0mM HEPES pH 7.5, 10% glycerol, 5\u00a0mM DTT, 2\u00a0mM MnCl2, 10\u00a0mM MgCl2, proteinase inhibitor cocktail Complete (Roche, Basel, Switzerland), and 1\u00a0mM ascorbic acid. Cells were disrupted by addition of 1\u00a0mg\/ml lysozym (Sigma), incubation on ice for 30\u00a0min and sonication. After centrifugation (10,000g) for 30\u00a0min at 4\u00b0C, 1\u00a0ml of the soluble enzyme fraction was assayed directly, or after purification of the His-tagged recombinant fusion protein on Ni-agarose beads (Novagen) for monoterpene, sesquiterpene or diterpene synthase activity using 10\u00a0\u03bcM of the substrates GPP, FPP or GGPP, respectively. The assay mix was incubated in a closed 20\u00a0ml vial at 30\u00b0C for 1\u00a0h under gentle shaking (150\u00a0rpm). Reaction products were sampled with a Solid Phase Micro Extraction fiber (SPME) for 10\u00a0min during which the vial was agitated and heated to 50\u00b0C. The SPME fiber was desorbed 1\u00a0min in an Optic injector port (ATAS GL Int., Zoeterwoude, The Netherlands) which was kept at 250\u00b0C. Compounds were separated on a DB-5 column (10\u00a0m\u00a0\u00d7\u00a0180\u00a0\u03bcm, 0.18\u00a0\u03bcm film thickness; Hewlett Packard, Palo Alto CA, USA) in an 6,890\u00a0N gas chromatograph (Agilent, Amstelveen, The Netherlands) with a temperature program set to 40\u00b0C for 1.5\u00a0min, ramp to 250\u00b0C at 30\u00b0C\/min and 250\u00b0C for an additional 2.5\u00a0min. Helium was used as carrier gas, the column flow was set to 3\u00a0ml\/min for 2\u00a0min and to 1.5\u00a0ml\/min thereafter. Mass spectra were generated with the ion source set to \u221270\u00a0V at 200\u00b0C and collected with a Time-of-Flight MS (Leco, Pegasus III, St. Joseph, MI, USA) at 1,671\u00a0V, with an acquisition rate of 20\u00a0scans\/s. Terpene products were identified using authentic standards and comparison of ion specra and relative retention times (Adams 2001). For separation of terpene enantiomers, a Cyclosil-B column (30\u00a0m\u00a0\u00d7\u00a0247\u00a0mm, 0.25\u00a0\u03bcm film thickness; Agilent) was used. The temperature program was isothermal at 115\u00b0C for 15\u00a0min, with a final ramp to 240\u00b0C at 120\u00b0C\/min and 240\u00b0C for 5\u00a0min. Column flow was 0.7\u00a0ml\/min.\nPlant treatments, plant headspace sampling, trichome isolation, and measurement of extracts\nFor determining tissue specific gene expression, a 13\u00a0weeks old, untreated tomato plant was dissected to obtain material of each of the described tissues. For separation of trichomes from petioles and stems, 4-week-old plants were used. Trichomes were collected on the bottom of a 50\u00a0ml tube after vortexing several N2(l)-frozen petiole or stem segments, standing in upright position. The trichome fraction was used directly, whereas the remaining cleaned petiole or stem segments were thoroughly brushed to remove all remaining trichome material. Wounding-, spider mite-, and hormone-treatments (for both volatile sampling and gene expression analysis) were done using 3\u20134-week-old tomato plants. Wounding was inflicted by squeezing leaflets several times with a hemostat; a total of 150 spider mites were put on 3\u20134 leaflets per plant for 3\u00a0days; JA and salicylic acid were applied to plants by spraying 1\u00a0mM solutions made with tap water containing 0.05% SilwetL-77. Wounding- and hormone-treatments were done (cumulative) at 0\u00a0h and at 18\u00a0h and leaves were sampled at 24\u00a0h. Headspace sampling of plants was performed as described by Kant et\u00a0al. (2004). Tenax sampling tubes were eluted with pentane\u00a0:\u00a0diethyl ether (4:1). The Eluted fractions were concentrated 20 times under a stream of N2 and 1\u00a0\u03bcl was injected into the GC injector port. For root-, leaf-, trichome-, stem- or petiole-extracts, 0.5\u00a0g tissue was used. The amount of trichome material was adjusted to be equivalent to the trichomes present on 0.5\u00a0g of the original stem or petiole. Tissues were ground in N2(l) and transferred to glass 20\u00a0ml vials containing 2\u00a0ml saturated CaCl2 (5\u00a0M) buffered in 100\u00a0mM sodium acetate (pH 4.5) which were capped immediately and kept at 5\u00b0C. Extracts were pre-incubated for 5\u00a0min at 60\u00b0C under agitation (500\u00a0rpm) and sampled for 10\u00a0min at 60\u00b0C on a 100\u00a0\u03bcM PDMS SPME fiber (Supelco, Zwijndrecht, The Netherlands). The SPME fiber was desorbed 1\u00a0min in the injector port of the GC\/MS, which was kept at 250\u00b0C. GC\/MS analysis was done as described earlier (Kant et\u00a0al. 2004).\nGene expression analysis by RT-PCR and real-time Q-RT-PCR\nFor semi-quantitative RT-PCR, the tomato RUB1 conjugating enzyme (RCE1) was used as constitutively expressed control gene (www.tigr.org: TC153679). Initial PCR was performed with RCE1 primers, product levels were compared and individual cDNA samples were diluted accordingly, to ensure equal template concentrations. PCRs with primers of genes of interest were subsequently performed for non-saturating number of amplification cycles. RCE1 primers were always included in each experiment. Used primers (5\u2032\u20133\u2032) for RCE1: forward (F) GATTCTCTCTCATCAATCAATTCG; reverse (R) GCATCCAAACTTTACAGACTCTC, WIPI-II: F: GACAAGGTACTAGTAATCAATTATCC; R: CACATAACACACAACTTTGATGCC, PRP6: F: TCAGTCCGACTAGGTTGTGG; R: TAGATAAGTGCTTGATGTGCC, LeMTS1: F: GATGACATTTATGATGTTTATGGC; R: GGCCATCTCGAGACTTGAGAGCGAATGCAACATTAG, LeMTS2: F: GATGACATTTATGATGTTTATGGC; R: GGGTAATAATTCCTTGTCTTATTTC. Expression differences were validated by varying the amount of PCR cycles.\nFor real-time Q-RT-PCR, total RNA was isolated using Trizol (Invitrogen) and DNA was subsequently removed with DNAse (Ambion, Huntingdon, UK). cDNA was synthesized from 5\u00a0\u03bcg RNA using SuperscriptII (Invitrogen) in 20\u00a0\u03bcl reaction volume that was diluted to 50\u00a0\u03bcl prior to using it for PCR. PCRs were performed in the ABI 7500 Real-Time PCR System (Applied Biosystems) using the Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen). About 20\u00a0\u03bcl PCR reactions contained 0.25\u00a0\u03bcM of each primer, 0.1\u00a0\u03bcl ROX reference dye, and 1\u00a0\u03bcl template. The cycling program was set to 2\u00a0min 50\u00b0C, 5\u00a0min 95\u00b0C, 40\u00a0cycles of 15\u00a0s at 95\u00b0C and 1\u00a0min 60\u00b0C, and a melting curve analysis. Primer pairs were tested for specificity and for linearity with a standard cDNA dilution curve. Primers used: LeRCE1 QF: 5\u2032-GATTCTCTCTCATCAATCAATTCG-3\u2032 QR: 5\u2032-GAACGTAAATGTGCCACCCATA-3\u2032, LeWIPI2 (K03291) QF: 5\u2032-GACAAGGTACTAGTAATCAATTATCC-3\u2032 QR: 5\u2032-GGGCATATCCCGAACCCAAGA-3\u2032, LeMTS1 (AY840091) QF: 5\u2032-TTTGGGGACATCTTCGGATGAA-3\u2032 QR: 5\u2032-CTACTCGAGTTACTTGAGAGCGAATGCAAC-3\u2032. Expression levels were normalized using RCE1 mRNA levels.\nGeneration of transgenic tomato plants\nThe complete LeMTS1 cDNA, including the putative signal peptide, was cloned into the EcoRI-digested and Klenow-blunted binary vector pGreen1K (Brandwagt et\u00a0al. 2002), resulting in a CaMV35S:MTS1:nos cassette. The construct was transferred to Agrobacterium tumefaciens strain EHA105 carrying the pSoup helper plasmid (Hellens et\u00a0al. 2000). Tomato plants cv. Moneymaker were transformed essentially as described by Vanroekel et\u00a0al. (1993). Heterozygous and homozygous LeMTS1 overexpressing plants were used to measure linalool production by sampling the headspace of intact plants (four sets of two plants for both LeMTS1 overexpressors and control plants).","keyphrases":["tomato","linalool","trichomes","jasmonic acid","terpenes","defence"],"prmu":["P","P","P","P","P","P"]}
{"id":"Eur_Arch_Otorhinolaryngol-4-1-2217619","title":"Morphology and function of Bast\u2019s valve: additional insight in its functioning using 3D-reconstruction\n","text":"The utriculo-endolymphatic valve was discovered by Bast in 1928. The function of Bast\u2019s valve is still unclear. By means of orthogonal-plane fluorescence optical sectioning (OPFOS) microscopy 3D-reconstructions of the valve and its surrounding region are depicted. The shape of the duct at the utricular side is that of a flattened funnel. In the direction of the endolymphatic duct and sac this funnel runs into a very narrow duct. The valve itself has a rigid \u2018arch-like\u2019 configuration. The opposing thin, one cell-layer thick, utricular membrane is highly compliant. We propose that opening and closure of the valve occurs through movement of the flexible base\/utricular membrane away from and toward the relatively rigid valve lip.\nHypothesis\nThe bottom or opposing wall of the uticulo-endolymphatic (Bast\u2019s) valve is the moving and thus functional part of the valve.\nBackground\nAt the utricular end the utricular duct has a valve-like opening. This structure was discovered by Bast [3], who called it the utriculo-endolymphatic valve. It is since then also called Bast\u2019s valve. The morphology of the valve has been researched in humans and several mammalians [1, 5, 10].\nThe function of Bast\u2019s valve is still unclear. Bast himself suggested the closure of the utricular duct as the main function [3, 4]. Bachor and Karmody [2] follow Bast\u2019s view, postulating that decreasing pressure in the whole endolymphatic system, secondary to collapse of the ductus reuniens, causes closure of the valve to prevent more loss of endolymph from the utricular system. Zechner [20] proposes that dysfunction of the valve causes endolymphatic hydrops.\nIn this paper orthogonal-plane fluorescence optical sectioning (OPFOS) microscopy [6, 15\u201317] was used to obtain detailed three-dimensional reconstructions of Bast\u2019s valve and the surrounding region in the intact guinea pig inner ear, as it is a technique capable of visualizing bone as well as soft tissue structures with high resolution, and is non-invasive.\nMaterial and methods\nSpecimen preparation\nTwo healthy female albino guinea pigs (Harlan Laboratories, UK), weighing 450\u00a0g, were used. Animal care and use were approved by the Experimental Animal Committee of Groningen University, protocol No. 2883, in accordance with the principles of the Declaration of Helsinki.\nThe animals were terminated by lethal administration of sodium pentobarbital. After decapitation the bullas were dissected and fixated in a 10% formalin solution, neutrally buffered. Then the bullas were rinsed in aqua-dest. Decalcification in ethylenediaminetetraacetic acid 10% solution (EDTA; Sigma, ED5SS, pH 7.4) took place at a temperature of 50\u00b0C in a microwave oven (T\/T MEGA microwave histoprocessor, Milestone) in eight sessions of 6\u00a0h. After decalcification the bullas were again rinsed with aqua-dest and dehydrated in a graded seven-step ethanol series (30%, 50%, 70%, 90%, 96%, 100%, and 100%).\nSpalteholz fluid, a 5:3 solution of methyl salicylate (Sigma, M-6752) and benzyl benzoate (Sigma, B-6630) [14], was thereafter used to achieve transparency of the specimen. The clearing process consisted of application of a succession of Spalteholz-ethanol solutions, 24\u00a0h each. The Spalteholz fluid fraction in the clearing session was 25%, 50%, 75%, 100%, 100%, respectively. Hereafter the specimen was dyed in a fluorescent dye bath of Rhodamine-B Isothiocyanate (RITC; Sigma, R-1755). RITC absorbs maximally at 570\u00a0nm and emits at 595\u00a0nm. The dye bath was prepared by dissolving 1.0\u00a0mg\/ml RITC into ethanol, followed by dilution in Spalteholz fluid to a final dye concentration of 5\u00a0\u00d7\u00a010\u22124\u00a0mg\/ml [15, 16] (Voie 2003). The specimen was dyed for four days.\nOPFOS imaging system\nA schematic diagram of the optical setup of our custom-made OPFOS imaging device is shown in Fig.\u00a01 [6]. Laser light, originating from a green frequency doubled Nd:YVO4 neodymium-laser (model DPGL-2050, Suwtech), with an emission wavelength of 532\u00a0nm and 52\u00a0mW maximal power, is expanded into a parallel beam of 28\u00a0mm diameter by a Galilean beam expander (BE, model 336, Spectra-Physics). The broadened and spatially filtered light falls onto a specially designed cylindrical lens (CL, custom-made by LiteTec Ltd., Essex, UK) of 30\u00a0\u00d7\u00a030\u00a0mm and 80\u00a0mm focal length, which focuses the light along a single dimension (Z), thus creating a hyperbolic light pattern along the X-axis. Near the center of this focus, we can locally approximate the light intensity profile as a sheet of light in the X\u2013Y plane, which will perform virtual slicing of a specimen object (O). In the plane where the object is sectioned by the sheet of light, the specimen emits fluorescence light, which is recorded by a CCD-camera (C, Firewire FO442BIC, Foculus) in the direction orthogonal to the light plane. In this way, a 2D image of a virtual section within the object is obtained. For the specimen to be compatible with the OPFOS method, the above described specimen preparation is necessary to make the object transparent, refraction index matched and fluorescent. In our setup the specimen is positioned inside a container with optical quality glass windows at the laser and CCD side, and filled with Spalteholz fluid, and can be positioned within the container by an object translation stage (OTS). Between recordings the object (and not the container) is moved along the Z-axis in small slicing steps with a high-precision DC-motor driven translation stage with position encoder (M112.1 High-Resolution Micro-Translation Stage with C862 Mercury II DC-Motor Controller, PI Polytec). A long working distance microscope objective lens (OL) with good numerical aperture (M Plan APO\u00a0\u00d7\u00a05, NA\u00a0=\u00a00.14, Mitutoyo) projects the fluorescence image on the CCD. A colour filter (Kenko R1 SR-60) placed before the objective lens blocks scattered laser light and transmits fluorescence light.\nFig.\u00a01Orthogonal-Plane Fluorescence Optional Sectioning (OPFOS) set-up. (BE: beam expander, FS: field stop, CL: cylindrical achromat, O: object, OTS: object translation stage, FTS: focusing translation stage, OL: objective lens with colour filter, CCD\u00a0=\u00a0charge coupled device camera)\nThe virtual slicing is performed by a \u2018plane\u2019 with 3\u00a0\u03bcm 1\/e\u00b2-thickness resolution in the center of the image and slightly thicker at the edges (less than 10\u00a0\u03bcm), with slicing steps of 2\u00a0\u03bcm, followed by storage in a personal computer. The two-dimensional stored images were processed with an IMOD (http:\/\/bio3d.colorado.edu\/imod) software package for 3D-reconstruction. Input of relevant contours in each 2D-image was manually performed with a writing tablet (Wacom Cintiq 15X).\nResults\nFigure\u00a02a shows an OPFOS image of a cross-section of part of the guinea pig inner ear. Figure\u00a02b is an enlargement of the part of Fig.\u00a02a inside the dashed box, in which Bast\u2019s valve and its surrounding structures are clearly visible. Saccule, endolymphatic sinus, utricule and endolymphatic duct are filled with endolymph. Walls of the endolymphatic space are on the right side of Fig.\u00a02b connected to bone (dense; non-compliant). The opposite walls are adjacent to perilymph (hypodense; compliant). Connective tissue (medium density; non-compliant) is present inside the lip of Bast\u2019s valve and between the upper side of the utricule and bone. Figure\u00a02c is another OPFOS image, showing Bast\u2019s valve in detail.\nFig.\u00a02(a) 2D OPFOS-image of the region around Bast\u2019s valve and the utricular duct, obtained with another OPFOS setup [7] than for (c) and following. The dashed box is shown in a larger magnification in (b), (b) Dashed box in (a) shown in a larger magnification, (c) 2D OPFOS-image of Bast\u2019s valve. The arrows point toward very narrow passages\nFigure\u00a03 shows a 3D-reconstruction of the entrance of Bast\u2019s valve as seen from inside the utricle. The proximal end of the valve shows a rather rigid \u2018arch-like\u2019 configuration. Figure\u00a04 shows a 3D-reconstruction of walls of Bast\u2019s valve and the utricular duct. The shape of the utricular duct at the utricular side is that of a flattened funnel. This funnel quickly runs into a very narrow duct.\nFig.\u00a033D-Reconstruction of Bast\u2019s valve as seen from inside the utriculeFig.\u00a043D-Reconstruction of walls of Bast\u2019s valve and the utricular duct. The shape of the utricular duct at the utricular side is that of a flattened funnel. This funnel runs into a very narrow duct\nFigure\u00a05 is a 3D-reconstruction of part of the outer walls of the utricle and utricular duct. It again shows the small caliber of the utricular duct in relation to the size of surrounding structures.\nFig.\u00a053D-Reconstruction of part of the outer walls of the utricle and utricular duct. Note the small caliber of the utricular duct in relation to the size of surrounding structures\nDiscussion\nIn the 3D OPFOS-reconstruction of the entrance of Bast\u2019s valve, shown in Fig.\u00a03, the lip of the valve appears as a relatively rigid structure. In a comparable scanning electron microscopy (SEM) image the entrance of the valve is visible as a narrow slit in the utricular wall [9]. The valve lip is filled with connective tissue (Fig.\u00a02c). This is even better visible in Fig.\u00a06, which is a light microscopy (LM) image. This figure also clearly shows that the bottom of the valve has a thickness of only one cell-layer, and is as result highly compliant.\nFig.\u00a06Light microscopy image of lip and bottom of Bast\u2019s valve\nSchuknecht and Belal [13] showed that the corium of the valve consists of fibroblasts and fibrocytes.\nBased on the study of 170 human temporal bones these authors propose that the valve is closed in normal ears and that its anatomical structure is ideally suited to permit the occasional egress of excessive accumulation of endolymph to be processed in the endolymphatic sac, while preventing an excessive loss of endolymph with the possible consequence of membrane distortions and interference with the motion mechanics of the vestibular sense organs. Opening of the valve, still according to Schuknecht and Belal [13], would then be accomplished by pressure displacement of the outer membraneous wall away from the more rigid inner valve lip.\nThe arrows in Fig.\u00a02c indicate positions were the valve appears to be closed. Also in Fig.\u00a06 valve lip and bottom touch.\nBast himself [5] was not sure about the valve being opened or closed in the normal situation. He writes: \u201cAs a matter of fact in many histological sections the epithelium of the valve is in contact with the opposing wall of the slit-like opening of the duct which suggests that such a closure may exist, at least at times, in the living ear.\u201d\nHe also was not certain about the mechanism that closes the valve [5].\nOn the one hand he supposed that a high perilymphatic pressure could close the valve by moving the opposing wall against the valve lip, as shown in Fig.\u00a07. On the other hand he proposed closing of the valve lip against the opposing duct wall in the case of a relatively greater intra-utricular pressure. In this case he supposes that it is not the tip of the valve that moves, because it is bulky, but that the valve lip rotates at its base, where it is made up of loose perilymphatic tissue.\nFig.\u00a07Possible mechanism of valve closure (black arrows) or valve opening (grey arrows)\nIn our opinion the latter mechanism is less likely, considering the observation that the valve opening in 3-D reconstruction is a rigid arch-like structure (Fig.\u00a03), and not a flap hinged at its base, that could close an underlying opening. The latter mechanism may be inspired by 2-D pictures, like Fig.\u00a02b, c and 6, where the valve tip appears as a flap-like structure.\nThe rigidity of the valve tip was also noticed by Scheerer and Hildmann [12], who also thought it to be improbable that this structure can function as a valve.\nThe walls of the endolymph filled spaces in the inner are highly compliant. As a result the pressure difference between endolymph and perilymph are negligible in the normal situation [19]. If the valve is open it seems, at first impression, easy to understand how an increase of perilymphatic pressure will close the valve by compression of its bottom, as is shown in Fig.\u00a07. However, an increasing perilymphatic pressure will also increase the endolymphatic pressure inside the utricule and force endolymph out through the slit-like opening and prevent closure of the valve [11, 18].\nAs can be seen in Figs.\u00a04 and 5 the utricular duct has a diameter in the order of 10\u00a0\u03bcm at its narrowest passage, preventing rapid flow of endolymph and possibly protecting the sensitive vestibular receptors against large fluid shifts in a short time.\nKonishi [8] found an open utriculo-endolymphatic valve in guinea pigs with an (experimental) endolymphatic hydrops. It is conceivable that an excess of endolymph will force the base of the valve opening away from the lip, as shown in Fig.\u00a07.\nIn future work, high-resolution OPFOS (HROPFOS) with slicing, a resolution of 2\u00a0\u03bcm [6] is feasible and may reveal even more detail.\nConclusion\nThe use of OPFOS imaging techniques and graphical 3D-reconstruction of the uticulo-endolymphatic (Bast\u2019s) valve and its surroundings has given some additional insight in its functioning. It is most likely that opening or closure of the valve occurs through movement of the flexible base away from or toward the relatively rigid valve lip.","keyphrases":["bast\u2019s valve","3d-reconstruction","utriculo-endolymphatic valve","opfos","endolymphatic system","imaging"],"prmu":["P","P","P","P","P","P"]}
{"id":"Eur_Radiol-3-1-1820761","title":"Imaging oxygenation of human tumours\n","text":"Tumour hypoxia represents a significant challenge to the curability of human tumours leading to treatment resistance and enhanced tumour progression. Tumour hypoxia can be detected by non-invasive and invasive techniques but the inter-relationships between these remains largely undefined. 18F-MISO and Cu-ATSM-PET, and BOLD-MRI are the lead contenders for human application based on their non-invasive nature, ease of use and robustness, measurement of hypoxia status, validity, ability to demonstrate heterogeneity and general availability, these techniques are the primary focus of this review. We discuss where developments are required for hypoxia imaging to become clinically useful and explore potential new uses for hypoxia imaging techniques including biological conformal radiotherapy.\nIntroduction\nThe suspicion that tumour hypoxia increased resistance to radiotherapy was first considered in the 1930\u2019s but it was not until 1955 that Tomlinson and Gray showed that chronic hypoxia occurred in human bronchial carcinomas with necrosis occurring approximately 150\u00a0\u03bcm from blood vessels [1], which is a little larger than the currently known diffusion distance of soluble oxygen in tissues (approximately 70\u00a0\u03bcm). Decades of research in radiation therapy then followed, much of which focused on attempts to circumvent hypoxia-mediated radio-resistance but these efforts were only moderately successful. Over the last decade, it has become evident that hypoxia changes the patterns of gene expression in several ways that alters the malignant potential of tumours, leading to more aggressive survival traits. As a result, hypoxic cancers are difficult to treat, particularly by radiation and photodynamic therapy [2], but also by cytotoxic chemotherapy. Attempts at circumventing the cure-limiting impact of hypoxia have included the use of hyperbaric oxygen and radiation sensitizer drugs but these have, in general, not proved widely advantageous. However, attempts to take advantage of the presence of tumour hypoxia, such as hypoxia-specific cytotoxins, are more promising. As hypoxia-directed therapies enter into clinical trials, it has become important to non-invasively assess for the presence of hypoxia and to be able to follow how it is modulated by new therapies. Hypoxia imaging may help select the most appropriate population that would benefit from novel hypoxia-directed therapies. In this review we describe the causes for and the effects of tumour hypoxia, as well as summarise the lead contenders for human tumour imaging. We also assess where developments are required for them to become clinically useful imaging tests and explore potential new uses for hypoxia imaging techniques including biologically-directed conformal radiotherapy.\nOverview of tumour hypoxia & its importance\nFor the majority of solid tumours hypoxia develops because of the inability of the vascular system to supply the growing tumour mass with adequate amounts of oxygen. Consequently, both low oxygen tensions and nutrient deprivation contribute to impaired tumour growth such that growth beyond 2\u00a0mm requires tumour neovascularisation. The major factors that play roles in the development of tumour cell hypoxia are the known abnormalities in structure and functioning of tumour microvessels [3], the increased diffusion distances between blood vessels (many of which may not even carry oxygenated red blood cells), the expanding tumour cell mass competing for oxygen and the reduced oxygen carrying capacity of blood due to disease- or treatment related anaemia. Thus, there are three distinct types of tumour hypoxia [4]: (1) Perfusion related (acute) hypoxia that results from inadequate blood flow in tumours that is generally the consequence of recognised structural and functional abnormalities of the tumour neovasculature. Such acute hypoxia is often transient, caused by temporary occlusions and temporary rises in interstitial pressure and can affect all cells right up to the vessel wall; (2) Diffusion related (chronic) hypoxia is caused by increased oxygen diffusion distances due to tumour expansion and affects cells greater than 70\u2013100\u00a0\u03bcm from the nearest capillary, depending on where tumour cells lie in relation to the arterial or venous end of a capillary; (3) Anaemic hypoxia, which relates to reduced O2-carrying capacity of the blood and may be tumour associated or treatment related.\nThe presence of hypoxia within human tumours before starting treatment has been observed in a variety of tumour types including squamous cell carcinomas, gliomas, adenocarcinomas (breast & pancreas) and in sarcomas. For example, in the normal cervix the pO2 is a median of 42\u00a0mmHg compared to a median of 10\u00a0mmHg in squamous carcinomas, and for cervix cancer the oxygenation status is independent of size, stage, histopathological type, and grade of malignancy [5]. Oxygen probes, that is, electrodes implanted directly into tumours to measure oxygen concentration by a polarographic technique [6\u20138] have shown (1) heterogeneity within and between the same tumour types of oxygen concentration and, (2) that hypoxia contributes to poor prognosis; pO2\u2009<\u200910\u00a0mmHg results in poor local tumour control, disease-free survival and overall survival in squamous carcinomas of the head and neck and of the cervix [9, 10].\nA large body of clinical evidence suggests that the hypoxia-mediated aggressive behavior of cancer cells and their resistance to therapy is orchestrated by the heterodimeric transcription factor, hypoxia inducible factor-1 alpha (HIF-1\u03b1), via a number of molecular events required for the adaptation of tumour cells to hypoxia (including unregulated glycolysis, angiogenesis and mutant p53) [11]. It is also important to realise that in some tumours including uterine lieomyomas, HIF expression is not always correlated with the presence of hypoxia suggesting that other factors including genetic events also contribute to activation of HIF, the most significant one of which is the loss of function of the Von Hippel-Lindau (VHL) tumour suppressor protein which results in constitutive activation of the HIF pathway. HIF-1 controls the expression of a variety of genes, the protein products of which play crucial roles in the acute and chronic adaptation of tumour cells to oxygen deficiency, including enhanced erythropoiesis & glycolysis, promotion of cell survival, inhibition of apoptosis, inhibition of cell differentiation, and angiogenesis. Thus, adaptive changes in the proteome and genome of neoplastic cells result in the emergence of more aggressive clones which are of cells that are more able to overcome nutrient deprivation or escape their hostile environments. Selection pressures by hypoxia and clonal expansion of the more aggressive cell types can result in exacerbations of regional hypoxia, further promoting the development of cell phenotypes that are treatment resistant (Fig.\u00a01). Given the central role of HIF-1 in hypoxia mediated aggressive behaviour of cancer cells and their resistance to therapy, HIF-1 has become a target for the development of anti-cancer drugs [12].\nFig.\u00a01Stylised diagram showing how hypoxia leads to therapy resistance and the development of an aggressive tumour cell phenotype. Figure adapted from [4]\nThere is debate about whether there is a critical intratumoural pO2 below which detrimental changes begin to occur that is common across cell types. This occurs because experiments performed in cell cultures may not be applicable to in-vivo environments and some of the literature variation can be attributed to the tumour cell type chosen for experiments and the demands of host tissues. With these caveats in mind, the critical pO2 tensions below which cellular functions progressively cease or anticancer treatments are impaired are approximately as follows [13]: Effectiveness of immunotherapy becomes impaired (30\u201335\u00a0mmHg); Photodynamic therapy (15\u201335\u00a0mmHg); Cell death on exposure to radiation (25\u201330\u00a0mmHg); Binding of hypoxia immunohistochemical markers (10\u201320\u00a0mmHg); Proteome changes (1\u201315\u00a0mmHg) and Genome changes (0.2\u20131\u00a0mmHg). The differences in these numbers are smaller than the similarities so that, from a practical perspective, for solid tissue tumors in vivo, a value of between 5\u201315\u00a0mmHg is a good number to remember because of its impact on therapy. This number is in contrast to ischaemic hypoxia in the myocardium or stroke where detrimental effects are experienced at higher O2 [14]. In all these instances the critical oxygen level in tissues reflects the drive to match delivery with metabolic demand.\nAs noted above, the presence of tumour hypoxia appears to impair the effectiveness of radiotherapy and radiosensitivity is progressively limited as tumour pO2 levels fall. Hypoxia-induced radioresistance is multifactorial with the presence of oxygen mediating DNA damage through the formation of oxygen free radicals which occurs after the interaction of radiation with intracellular water. The ratio of doses administered under well-oxygenated to hypoxic conditions needed to achieve the same biological effect (i.e., cell kill) is called the oxygen enhancement ratio (OER). For sparsely ionising radiations such as x- and gamma rays, the OER at therapeutic doses is between 2.5\u20133.5 [15]. That is, well oxygenated cells are about three times more sensitive to x- and gamma radiation than the same cells when they are hypoxic. Half maximal sensitivity to x- and gamma rays occurs at oxygen tensions of approximately 2\u20135\u00a0mmHg; above pO2 values of approximately 10\u201315\u00a0mmHg near maximal oxygen effects are seen. However, it should be recognised that sensitivity of cells to radiation is dependent on the phase of the cell cycle, with cells in the G1 phase having a lower OER (i.e., more radiosensitive) than cells in S-phase. As noted above, the oxygen effect is not the only mechanism for radioresistance in hypoxic tumour cells. Evidence is accumulating that the hypoxia-mediated proteomic and genomic changes may also contribute to radioresistance by increasing the levels of heat shock proteins (heat shock proteins (HSPs), are induced in response to environmental stresses like heat, cold and oxygen deprivation [16]) or by increasing the number of tumour cells that can resist apoptosis by mutating p53 (the slowing of cell division is dependent on a protein brake known as p53; the disruption of the functioning of this protein is associated with approximately 50\u201355% of human cancers).\nClinical imaging of hypoxia\nAs tumour hypoxia is an important biological characteristic and there is no good or easy clinical way to predict its presence, it has been suggested that imaging may be a good way of non-invasively selecting cancer patients who would benefit from treatments that overcome, circumvent or take advantage of the presence of hypoxia. Since tumour hypoxia is a key mechanism that leads to radioresistance, it has been repeatedly suggested that a hypoxia mapping technique could be integrated with conformal radiotherapy techniques to improve target delineation and dose delivery; this is discussed in more detail below. Imaging could also be used to document whether or not and the extent to which reoxygenation of tumours occurs during radiotherapy. Key requirements of any method that evaluates tumour hypoxia include non-invasive assessments that allow serial changes during treatment to be monitored and evaluation of heterogeneity between and within tumours.\nThere are a number of ways in which tissue oxygenation status can be assessed in vivo (both invasive and non-invasive) or in vitro using material from biopsy. Non-imaging methods of assessing for the presence of hypoxia in tissues include histological appearance, immunohistochemical staining for intrinsic markers of hypoxia (e.g., carbonic anhydrase IX (CA-IX) and hypoxia inducible factor-1 (HIF-1)) and for the binding of externally administered nitroimidazoles [17, 18].\nFrom an imaging perspective, an ideal test would: (1) distinguish normoxia \/ hypoxia \/anoxia\/necrosis, (2) distinguish between perfusion-related (acute) and diffusion-related (chronic) hypoxia if possible, (3) reflect cellular in preference to vascular pO2, (4) be applicable to any tumour site with complete loco-regional evaluation, (5) be simple to perform, non-toxic and allow repeated measurements, and (6) be sensitive at pO2 levels relevant to tumour therapy. Therefore, the challenge for hypoxia imaging is to measure low levels of tissue pO2 on a spatial scale similar to the O2-diffusion distance (70\u2013100\u00a0\u03bcm); a much smaller dimension than can be achieved with human imaging techniques. Currently available MRI and PET methods were compared at a National Institute of Health\/National Cancer Institute of the USA sponsored workshop in April 2004 and it was noted that only a few techniques have potential for in vivo assessment in humans particularly for repeated, sequential measurements. 18F-MISO and 60\/64Cu-ATSM PET, and BOLD-MRI are the lead contenders for human application based on their non-invasive nature, ease of use and robustness, measurement of hypoxia status, validity, ability to demonstrate heterogeneity and general availability (Table\u00a01). \nTable\u00a01Comparison of techniques for evaluating human tumour hypoxiaTechnique and key referencesInvasive investigationRequires injectionMeasuresClinically Validated in RTGeneral availability (1\u20135; poor-wide)Monitors changes in pO218F-MISO PET [19, 24, 51\u201353]NoYesHypoxia\u00b13No18F-AZA PET [54]NoYesHypoxiaNo2No18F-EF5 PET [55]NoYesHypoxia\u00b12No64Cu ATSM [25, 26, 28, 34, 35]NoYesHypoxia\u00b13NoBOLD-MRI [36]NoNo[dHb] in RBCsYes4YesPolorographic electrode [6\u20138]YesNopO2Yes2Yes\n18F-MISO\n[18F]Fluoromisonidazole, 3-fluoro-1-(2\u2032-nitro-1\u2032-imidazolyl)-2-propanol or (18F-MISO), is the prototype hypoxia imaging agent whose uptake is homogeneous in most normal tissues, reflecting its high partition coefficient that nears unity, and whose delivery to tumours is not limited by perfusion [19]. The initial distribution of 18F-MISO is flow dependent, as with any freely diffusible tracer, but local oxygen tension is the major determinant of its retention above normal background in tissues after 2 hours (Fig.\u00a02). 18F-MISO accumulates in tissues by binding to intracellular macromolecules when pO2\u2009<\u200910\u00a0mmHg. Retention within tissues is dependent on nitroreductase activity (that is, on reduction status of a NO2 group on the imidazole ring) (Fig.\u00a03) and accumulation in hypoxic tissues over a range of blood flows has been noted, including within the intestinal lumen where it is retained in anaerobes!\nFig.\u00a02The 18FDG-PET image (bottom left panel) shows increase uptake in both the oropharyngeal tumour (arrow) and in the left neck nodal metastasis (asterix). The 18F-MISO images (bottom right panel) were acquired in a dynamic mode and representative images after 1 minute, 30 minutes and 240 minutes are shown together with time-activity curves from the two regions of interest indicated in the FDG-PET image. The early distribution (1 minute) shows hyperperfusion in the region of the primary tumour and metastasis because of the high partition coefficient of 18F-MISO. After 2 hours, only the left neck nodal metastasis is shown to be hypoxicFig.\u00a03The structure of [18F]-fluoromisonidazole, 18F-MISO, and its mechanism of retention in hypoxic tissues. The partition coefficient of 18F-MISO is near unity so the molecule diffuses freely into all cells. Once 18F-MISO is in an environment where electron transport is occurring (viable tissues), the \u2013NO2 substituent (which has a high electron affinity) takes on an electron to form the radical anion reduction product. If O2 is also present, that electron is rapidly transferred to oxygen and 18F-MISO changes back to its original structure and can leave the cell. However, if a second electron from cellular metabolism reacts with the nitroimidazole to form the 2-electron reduction product, the molecule reacts non-discriminately with peptides and RNA within the cell and becomes trapped. Thus, retention of FMISO is inversely related to the intracellular partial pressure of O2 as shown in the lower left panel [45]. This mechanism is confirmed by the autoradiograph of a tumour spheroid (bottom right panel) with a radius of approximately 0.5\u00a0mm that shows no retention in the necrotic core or in the well-oxygenated outer sphere but intense uptake (white spots) in a donut like ring where cells are hypoxic\nHypoxia can be imaged with 18F-MISO PET in a procedure that is well-tolerated by the patients. Imaging requires 20\u201330\u00a0min and starts anywhere from 75 to 150\u00a0min after injection, making it similar to the bone scan with which most cancer patients are familiar. Useful and well-validated images can be achieved with a modest dose of radiation, typically 250\u00a0MBq. No arterial sampling or metabolite analysis is required and synthesis is achieved through relatively simple modifications of nucleophilic displacement \/ deprotection synthesis boxes such as are used for fluoro-deoxyglucose (18F-FDG). In the USA, F-MISO has investigational new drug (IND) authorisation from the Food and Drug Administration (FDA) as an investigational product for use in humans. Unlike Eppendorf pO2 histography, 18F-MISO is only sensitive to the presence of hypoxia in viable cells; 18F-MISO is not retained in necrosis because the electron transport chain that reduces the nitroimidazole to a bioreductive alkylating agent is no longer active (Fig.\u00a03). Limitations of 18F-MISO PET include the modest signal-to-noise ratio of raw 18F-MISO PET images but if a venous blood sample is acquired during the mid-course of the imaging procedure and used to calculate a Tumour:Blood (T\/B) ratio image, then normoxic uptake (T\/B\u2009<\u20091) can be electronically subtracted to increase image contrast. Several studies in a range of hypoxic tumours, stroke and hypoxic myocardium [14] have shown that a T\/B of >1.2 reliably identifies the presence of hypoxia. The presence of high normal liver uptake impairs complete assessment of liver lesions and urinary excretion interferes with imaging near the bladder.\n18F-MISO PET is able to monitor the changing hypoxia status of lung tumours during radiotherapy [20]. Studies in sarcoma [21] and head and neck cancer [22\u201324] have demonstrated a correlation of 18F-FMISO uptake with poor outcome to radiation and chemotherapy.\nCu-ATSM\nCu-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM) holds exceptional promise as an agent for delineating the extent of hypoxia within tumours with PET. Numerous pre-clinical studies have evaluated and validated its use for imaging of hypoxia in tumours and other tissues [25\u201332]. The mechanism of retention of the reagent in hypoxic tissues is largely attributed to the low oxygen tensions and the subsequent altered redox environment of hypoxic tumours (increased NADH levels) (Fig.\u00a04). Clinical studies, well-tolerated by patients, involved 60Cu-ATSM imaging sessions of about 60 minutes with analysis of 30\u201360 minute summed-images. This time frame not only yields excellent data with good image quality (Fig.\u00a04) in a very short time frame which opens up the opportunity with the shorter-lived 60Cu to perform multiple imaging sessions. A number of radioactive copper isotopes with longer half lives are available, e.g. 64Cu (t1\/2\u2009=\u200912.74\u00a0h) [33], enabling wide geographic distribution and the United States FDA recently approved an IND application for the study of 64Cu-ATSM for the imaging of hypoxia in human tumours.\nFig.\u00a04Retention mechanism of Cu(II)ATSM in hypoxic tissues. (a) Cu(II)ATSM is bioreduced (Cu(II) to Cu(I)) once entering the cell. The reduced intermediate species (likely to be [Cu-ATSM]-) is trapped within the cell because of its charge. This transient complex can then go through one of two competing pathways: reoxidation to the uncharged Cu(II) species (which can escape by diffusion), or proton-induced dissociation (which releases copper to be irreversibly sequestered by intracellular proteins). [Cu-ATSM]- favours the reoxidation route because it is easily oxidised but chemically more resistant to protonation. Copper from Cu(II) ATSM is trapped reversibly as [Cu-ATSM]- (if oxygen is absent), with the possibility of irreversible trapping by dissociation over a longer period. Cu(II)ATSM is thus hypoxia-selective. (b) High quality axial 60Cu-ATSM-PET image through the mid-upper thorax demonstrates heterogeneously increased uptake (arrow) within a known lung cancer in the aorto-pulmonary window and left suprahilar region. PET images representing summed data were obtained from 30 to 60 minutes after injection of 60Cu-ATSM\nIn human studies of lung [34] and cervix cervical cancers [35], encouraging evidence has emerged that 60Cu-ATSM can act as a prognostic indicator for response to therapy. In the prospective study of 14 humans with non-small cell lung cancer, a semi-quantitative analysis of the 60Cu-ATSM muscle-to-tumour ratio was able to discriminate those likely to respond to therapy from non-responders [34]. A similar study in 14 women with cervical cancer demonstrated a similar predictive value in the tumour response to therapy [35].\nBOLD-MRI\nBlood oxygenation level dependent (BOLD) and intrinsic susceptibility weighted MRI are interchangeable terms. As in any MR image, tissue contrast in BOLD images is affected by intrinsic tissue properties including spin-lattice and spin-spin relaxations. Additionally, BOLD MRI contrast is affected by blood flow and paramagnetic deoxyhaemoglobin within red blood cells (oxyhaemoglobin is not paramagnetic). Deoxyhaemoglobin increases the MR transverse relaxation rate (R2*) of water in blood and surrounding tissues thus BOLD-MRI is sensitive to pO2 within, and in tissues adjacent to perfused vessels [36]. Static tissue components include iron content (e.g. myoglobin found in muscle) and presence of fibrosis or ligamentous structures (e.g. in benign prostatic hyperplasia and the suspensory ligaments of the breasts) also affect the appearances of intrinsic susceptibility weighted images. In order to decouple the effects of flow from deoxyhaemoglobin and static components it is necessary to measure the T2* relaxation rate (R2*\u2009=\u20091\/ T2*) which can be done by using a multi-echo GRE sequence (Fig.\u00a05). Decoupling of flow from static effects on R2* images occurs because the flow component can be thought of as affecting individual T2* images of a multi-gradient echo sequence equally. It is important to remember that, although synthetic R2* images are free of the contribution of blood flow (that is, they mainly reflect deoxyhaemoglobin content and static tissue components), improving blood flow and vascular functioning will also increase tissue oxygenation, which can be seen by changes in R2* images.\nFig.\u00a05Data acquisition and quantification of BOLD-MRI in the prostate gland. Gradient recalled-echo MR images acquired at 1.5\u00a0T with a fixed repetition time and flip angle (TR ~100\u00a0msec; alpha 40 degrees) with lengthening echo-time (TE) are acquired through the prostate gland (bottom row of images). These images show increased susceptibility (T2*) effects with increasing TE. The rate of signal intensity decay in the dorsal aspect of the prostate is dependent on intrinsic T2* relaxation (local structure), deoxyhaemoglobin concentration [dHb] and local blood flow. Synthetic R2* (=1\/T2*) images are created by plotting the natural logarithm of the signal intensity against the TE (top left panel). R2* map (top right panel) reflects on the structure of tissues and local [dHb] but inflow effects are minimised; however, R2* maps retain sensitivity to pO2 changes caused by alterations in blood flow\nThe observations made in the previous paragraph imply two intuitive inferences. (1) BOLD-MRI images are more likely to reflect on acute (perfusion-related) tissue hypoxia which, as stated above, occurs because of transient occlusions of vessels, simply because hypoxic areas extend to the level of the blood vessels. In contradistinction, chronic hypoxia is less likely to be reflected by BOLD-MRI because the red blood cells in vessels are too distant from the area of hypoxia. (2) For BOLD-MRI to be able to inform on tissue oxygenation status, it is important for red blood cells to be delivered to the tissue in question. Human and xenograft studies have shown that tumour perfusion varies widely and that red blood cell perfusion is not simply related to the absence\/presence of vessels; plenty of tumour vessels maybe present but perfusion by red blood cells may not occur [37]. This observation probably explains in part why no direct correlations between baseline R2* and tissue pO2 have been observed (that is, R2* does NOT measure tissue pO2). So it is necessary to know or to determine the distribution of blood volume in tissue in order to be able to correctly interpret R2* images in order to infer oxygenation status. Thus, if a tissue is perfused but has a high baseline R2* in one area\/region compared to another area\/region in the same tissue (i.e. the statistic components are the same), then one can infer that the high R2* region is relatively more hypoxic; this hypothesis is supported by recent preclinical and clinical data [38, 39].\nAs stated above, the use of BOLD-MRI for assessment of tissue hypoxia is predicated on the assumption that the oxygenation of haemoglobin is proportional to blood arterial pO2 which is in equilibrium with oxygenation of surrounding tissues. Many studies have shown that changes in R2* in response to vasomodulation with Carbogen (95% CO2:5% O2) inhalation, for example, are temporally correlated with changes in tissue pO2. Tumours differ in their responses to carbogen inhalation with only 50\u201360% of human tumours showing changes in R2* [40, 41]. The reasons for these limited and heterogeneous responses are complex but undoubtedly include the fact that tumours have adapted to widely different perfusion and that, even when vessels are present, red blood cell transport along these vessels may not be effective as demonstrated by Robinson et al. [37]. Thus, hypoxic tumours with high blood volume (due to high microvessel density coupled with large vessels) will not only have raised baseline R2* values but are more likely to respond to Carbogen. This will be reflected by large changes in R2*; and it is these hypoxic tumours that show positive radiosensitisation with Carbogen. On the other hand, hypoxic tumours with low blood volume (due to lower microvessel density, or due to small vessels) will have lower baseline R2* values and are thus less likely to respond to Carbogen. In this situation, there will be negligible changes in R2* and such hypoxic tumours do not show radiosensitisation with Carbogen [42]. Readers should also note that the BOLD response to Carbogen is also dependent on the ability of the underlying maturity of the vasculature with mature vessels able to respond actively to vasoconstrictory and vasodilatory stimuli [43].\nThe primary advantages of BOLD-MRI are that there is no need to administer exogenous radioactive contrast material and images at high temporal and with high spatial resolution can be obtained and repeated as needed. It is possible to decouple the effects of flow and deoxyhaemoglobin which are seen in native BOLD images and so to demonstrate changes in oxygenation independent of changes in blood flow. Major limitations of BOLD-MRI include the fact that they do not measure tissue pO2 directly (either in blood or tissues because of a non-linear relationship of R2* and tissue pO2), the images obtained have low signal to noise ratio and clinical studies with Carbogen vasomodulation are technically challenging (approximately 25\u201335% of patient examinations fail due to respiratory distress caused by an increased respiratory drive induced by Carbogen [40, 41]. BOLD-MRI appears most sensitive to oxygen levels adjacent to perfused vessels (that is, perfusion related or acute hypoxia) and BOLD-MRI sensitivity to more distant diffusion related or chronic hypoxia is an unknown.\nHypoxia guided radiotherapy\nRecently introduced, technological improvements in radiotherapy delivery systems, including intensity-modulated radiotherapy (IMRT), have provided a means for shaping the dose distribution not only to the geometry of target volumes, and also to the differences in radiobiology across tumours [44]. Thus, it is now possible to define an additional \u201ctarget within the target\u201d as 3D pixel maps of the prescribed dose incorporating biological information derived from functional images; sometimes called dose painting by numbers [45, 46]. This allows treatment to the desired dose with escalation based on biologically relevant data, such as hypoxia which was discussed above, that is mechanistically related to therapeutic outcome. As an example of this, the spatial distribution of 60\/64Cu-ATSM uptake on PET was successfully fused with CT radiotherapy planning images to show a proof of concept. This theoretical treatment planning would deliver higher doses of radiation via intensity modulated radiotherapy techniques (IMRT) to the most hypoxic regions of head and neck tumours [47].\nIn the case of hypoxia, it is important to remember that if the tumour stays hypoxic throughout the course of a treatment, more (escalating) radiation alone may not be sufficient to overcome the hypoxia-induced resistance. Hence, from a therapeutic perspective it is additionally important to know when and if reoxygenation occurs after initial radiotherapy (either by a reduction of oxygen consumption by the arrest of proliferation or by death of well oxygenated cells [15] (Fig.\u00a06). However, data correlating treatment success with hypoxia and reoxygenation changes occurring during treatment are sparse; the extent and rapidity of reoxygenation is impossible to predict for individual tumours. The complexity of the processes leading to reoxygenation suggests the need for repeated imaging during the initial phase of treatment to determine the best time for dose adaptation. Koh et al. used 18F-MISO PET imaging to detect reoxygenation of some lung tumours after just a few treatments, whereas other patients exhibited no reduction in hypoxia even over the full course of radiotherapy [20]. Thus, dose escalation to target hypoxic areas in all patients at the beginning of the treatment could be a wasted effort because reoxygenation would change its distribution. Dose escalation is possibly best undertaken towards the end of treatment, at a time when hypoxic radioresistant cells are predominate.\nFig.\u00a06The re-oxygenation phenomenon. Tumours contain mixtures of aerated and hypoxic cells. Radiation is effective at eliminating well oxygenated cells because they are radiosensitive. Reoxygenation cause the preradiation pattern to return which can be eliminated by further radiation fractions but a progressive decrease in the tumour mass occurs after a series of fractions. Figure adapted from [15]\nRecently, Thorwarth et al. presented a study where dynamic (mapping of perfusion) and static (mapping of hypoxia) 18F-MISO scans were obtained in 15 patients with head-and-neck cancers who were subsequently treated with chemoradiation [48]. Their data suggested that subsequent treatment failure was related to both pretherapy hypoxia and concurrent poor perfusion. This result hints that reoxygenation did not occur as a consequence of the deficient vasculature of tumours [49]. Thus, it may be necessary not only to target hypoxic regions but also areas of poor blood flow. Dose escalation maps can be derived from dynamic 18F-MISO PET scans for targeting poor perfusion, while functional planning target volumes could be obtained from late static scans (that reflect hypoxia); both concepts lend themselves equally well to IMRT (Fig.\u00a07). As a caution, simulated treatment plans also suggest that some patients may require such high radiation doses that will lead to a high risk of late complications in the vicinity of the tumour bed, such as necrosis, rupture of blood vessels or excessive fibrosis.\nFig.\u00a07Dose escalation map superimposed on CT with Planning Target Volume (red). Isodose lines show conformality to hypoxic lymph node with a maximum dose increase by 20%. This is the same patient illustrated in Fig.\u00a02\nChallenges for hypoxia imaging techniques\nAs hypoxia imaging techniques move from academic research environments to routine clinical usage, it becomes important to recognise the unique challenges of clinical translation. For example, it is important that patient examination times are short to improve patient compliance particularly for repeated examinations. Thus, the need for doing dynamic scanning followed by several static scans over a prolonged period of time with PET techniques could prove a disincentive for patients. However, as noted above, not all patients undergoing hypoxia studies by PET need dynamic imaging although it may be useful in selected patients where the aim is to demonstrate both perfusion and hypoxia (for example to demonstrate reperfusion-reoxygenation). In-contradistinction, the interpretation of BOLD-MRI does require that the distribution of blood flow\/volume is known and this can be done in clinical studies using dynamic contrast enhanced MRI (DCE-MRI) [50]. Whatever the chosen technique for clinical translation, there needs to be standardisation of imaging procedures and analysis methods in order to allow techniques to become more completely validated, for use in clinical trials. Amongst other issues that require addressing when clinical trials are being designed include the need for quantification, test-retest variability and data collection in body parts where there is a large degree of physiological movement such as the lungs and liver.\nA practical question often asked is whether it is necessary to quantify imaging data to answer important clinical questions. Subjective assessments work well enough in the clinic; however it is important to realise that subjective criteria cannot be applied simply from one centre to another particularly, when different equipment and imaging routines and human observers are used. Quantification techniques aim to minimise errors that can result from the use of different equipment and imaging protocols. Quantification techniques also enable the derivation of parameters that are based on some understanding of physiological processes and so can provide insights into tumour biology, for example the simple T\/B and T\/M ratios described for the two PET procedures. Quantification techniques are preferred when serial imaging studies are anticipated, for example when evaluating response to novel anticancer therapeutics.\nThe reproducibility of the imaging technique should also be known in order to estimate the sample size required to evaluate therapy efficacy. Variation between measurements of the same quantity on the same individual can be caused either by measurement error or by physiological changes between measurements. Whilst it is possible (in theory) to reduce measurement error, physiological variation is inherent, and can cause difficulty in attempts to characterise disease or to monitor the effects of therapy. An estimate of measurement error enables us to decide whether a change in observation represents a real change. Data addressing the precision and measurement variability of hypoxia imaging techniques are urgently needed and should be an integral part of any prospective study that evaluates functional response to therapy to allow assessments of individual patients and group changes.\nIt is intuitive that analysis and presentation of imaging data needs to take into account the heterogeneity of tumour hypoxia. The presence of motion can invalidate functional parameter estimates particularly for pixel-by-pixel analyses and this is especially true for high spatial resolution techniques such as BOLD-MRI. Motion is averaged in PET imaging because imaging times are long. In that case, pixel-analyses of the data and the issues of heterogeneity assessment can become less meaningful. The first step in heterogeneity analysis includes ROI definition which should be performed independent on the hypoxia imaging being assessed. For BOLD-MRI this could be done by anatomic MRI images and for PET studies could include the CT component of CT-PET studies although some groups have used ROIs defined in 18FDG-PET images. However, ROIs defined on 18FDG-PET images are know to be prone to error as far as tumour boundaries are concerned and are highly dependent on the level of threshold chosen. Whole tumour ROIs yield outputs with good signal-to-noise ratio, but lack spatial resolution and are prone to partial volume averaging errors and thus are unable to evaluate tumour heterogeneity. Pixel mapping has the advantages of an improved appreciation of heterogeneity of hypoxia and the risk of missing important diagnostic information and of creating ROIs that contain more than one tissue type is reduced. An important advantage of pixel mapping is being able to spatially map tumour characteristics such as hypoxia, glucose metabolism and blood flow and to be able to probe the spatial correlations between different kinetic parameters, providing unique insights into tumour structure, function and response to treatment.\nConclusions\nTo summarise, tumour hypoxia is common and its effects represents a significant challenge to the curability of human tumours, leading to treatment resistance and enhanced tumour progression. Tumour hypoxia can be detected by non-invasive and invasive techniques but the inter-relationship between these techniques needs to be better defined; human validation of the utility of hypoxia imaging is sparse at best. Anti-hypoxia therapies exist in the clinic and more are on their way. Either they don\u2019t work very well or we don\u2019t know how to use them optimally. Hypoxia imaging may allow better definition of a sub-population of cancer patients that would benefit for novel anti-hypoxia directed therapies.","keyphrases":["hypoxia","bold-mri","radiotherapy","cancer","cu-atsm-pet","f-miso-pet","tumour resistance"],"prmu":["P","P","P","P","U","U","R"]}
{"id":"Surg_Endosc-3-1-2169269","title":"Comparison of three perioperative fluid regimes for laparoscopic donor nephrectomy\n","text":"Background Pneumoperitoneum (PP), as used for laparoscopic procedures, impairs stroke volume, renal blood flow, glomerular filtration rate and urine output. This study investigated whether perioperative fluid management can abolish these negative effects of PP on hemodynamics.\nLaparoscopic donor nephrectomy (LDN) has become the method of choice to procure kidneys from living donors, mainly because of the reduced procedure-related morbidity and faster convalescence period [1\u20133]. Despite the benefits to the donor, there are concerns over the transient deterioration of renal function in the recipient of the kidney procured by the laparoscopic technique, compared with open donor nephrectomy (ODN) [2, 4\u20136]. The exact mechanism of delayed graft function after LDN is not fully understood.\nPneumoperitoneum (PP) elevates intra-abdominal pressure (IAP), causing a decrease in renal blood flow (RBF) and glomerular filtration rate (GFR) resulting in oliguria [7\u201310]. In an animal model, London et al. have shown that PP resulted in a decrease in RBF during normal saline infusion, whereas RBF did not decrease if volume expansion was given [11]. From these results vigorous hydration up to 2 l\/h of crystalloids during LDN in patients has nowadays been advocated [1, 12\u201315].\nIn 52 patients, Bergman et al. [16] found, however, no difference in graft function after LDN between aggressive (>10ml\/kg\/h) and conservative (<10 ml\/kg\/h) intraoperative fluid management. Volume loading after establishment of PP is perhaps too late to counterbalance the collapsed venal system. Biancofiore et al. [17] studied the effect of volume loading on graft function with a crystalloid infusion starting the night before surgery. Early graft function did not differ between ODN and LDN, although the serum creatinine declined earlier, but not significantly, in those receiving kidneys from ODN procedure.\nFasting before operation and induction of anesthesia leads to relative hypovolemia and the goal is therefore to compensate this before PP is started. In this study, we compared three different fluid regimes in LDN patients, in which the effect of pre-hydration together with a bolus of colloids given just before induction of anesthesia and a second one just before inflation of PP on hemodynamics was of special interest.\nMethods\nPatients undergoing LDN from June 2001 to November 2001 (N\u00a0=\u00a021) were included in the study. The anesthetic procedure was performed according to a strict protocol for medication, ventilation and fluid regimen. In our hospital the donor patients are admitted the day before the operation, they are fasted during the night from 00:00 and operated on at 08.00 the next morning. Patients were randomized the day before operation by sealed envelopes by the responsible anesthetist to three different fluid regimens (Table\u00a01): in group 1 fluid administration was started at 22:00 the day before operation with 3 ml\/ideal body weight (IBW)\/h Ringers lactate (RL) until operation. Before induction of anesthesia, the patients received 6 ml\/IBW of colloid (6% HES 130\/0.4); thereafter 13 ml\/IBW\/h RL was started until nephrectomy, before installation of PP another bolus of 6 ml\/IBW colloid was given. Group 2 received overnight infusion in the same way as in group 1 with a bolus of 6 ml\/IBW colloid just before induction. During operation, an infusion was started with 13 ml\/IBW\/h RL and 2 ml\/IBW\/h of colloid was given for three hours. Group 3 was fasted from 00:00 on the day of operation and received only an infusion during operation with 13 ml\/IBW\/h RL and 4 ml\/IBW\/h of colloid for three hours. After nephrectomy the infusion protocol was adjusted, so that exactly six hours after start of operation all the patients had received in total 9 ml\/IBW\/h RL. Patients were fitted with anti-thrombosis stockings.\nTable\u00a01.Infusion protocols used in the three groupsGroupnml RL\/IBW\/h pre-hydrationaml RL\/IBW\/h preoperativebml HES\/IBW before inductioncml HES\/IBW before PPdml HES\/IBW\/h after PPeml HES\/ IBW total1731366122721363\u00a0\u00d7\u00a021237163\u00a0\u00d7\u00a0412IBW\u00a0=\u00a0ideal body weight n\u00a0=\u00a0number of patientsa Prehydration from 22:00 the day before laparoscopic donor nephrectomy, until operationb The amount of Ringer\u2019s lactate given during operation, until nephrectomyc Amount of 6% HES 130\/0.4 given before induction of anesthesiad Amount of 6% HES 130\/0.4 given before installation of pneumoperitoneume Amount of 6% HES 130\/0.4 given per hour after installation of pneumoperitoneumHES\u00a0=\u00a06% HES 130\/0.4\nInduction of anesthesia was performed with propofol (2 mg\/kg) after a bolus of sufentanil (0.3 \u03bcg\/kg). Muscle relaxation was achieved with rocuronium (0.8 mg\/kg) and monitored by train-of-four (TOF) guard, a bolus of rocuronium (0.3 mg\/kg) was given for three or more twitches. Anesthesia was maintained with propofol by continuous infusion (4\u201311 mg\/kg\/h), aiming at a bispectral index between 45 and 55 (BIS monitor; Aspect Medical Systems, Newton, MA, USA), and analgesia was achieved by continuous infusion of sufentanil (0.4 \u03bcg\/kg\/h) until nephrectomy. One hour after the start of operation 20 mg mannitol was given intravenously.\nAfter intubation all patients were ventilated in a pressure-controlled mode using a closed-loop ventilator (Physioflex\u00ae, Dr\u00e4ger, L\u00fcbeck, Germany) with the following initial settings: FiO2 of 0.4, positive end-expiratory pressure (PEEP) of 7 cm H2O and peak inspiratory pressure (PIP) of 22 cm H2O. Ventilation frequency was adjusted to keep PetCO2 between 4 and 5.5 kPa. After induction of anesthesia and before positioning of the patient, an esophageal Doppler probe (HemoSonicTM 100, Arrow International Inc., Reading, PA, USA) was positioned for measuring stroke volume (SV) and left ventricular ejection time, corrected for heart rate (LVETc) [18\u201320].\nAfter positioning the patient in full lateral nephrectomy position, PP was installed with an IAP of 12 mmHg, which was constantly maintained at this level. All operations were done by the same team of anesthesiologists and surgeons. The surgical techniques have been described in detail elsewhere [21].\nMean arterial pressure (MAP) and SV (available after induction of anesthesia) were monitored noninvasively every five minutes. Urine output was measured from 22:00 the day before until the introduction of PP (T0), and was then measured every hour up to six hours thereafter (T1\u20136). Blood samples of the donors were collected to determine creatinine levels the day before operation, after induction of anesthesia, six hours after installation of PP, two days, one month, and one year after operation. Creatinine clearance (CrCl) was determined using the Cockcroft-Gault formula [22].\nStatistical analysis\nData analysis was performed using SPSS for Windows (version 14.0, SPSS Inc., Chicago, USA). Data are presented as means with standard deviation (SD). Differences between the groups were analyzed using the independent t-test, depending on Levene\u2019s test, pooled or unpooled. Repeated measures with a general linear model from SPSS were used to assess significance for CrCl. A (two-sided) p-value\u00a0<\u00a00.05 was considered statistically significant.\nResults\nBaseline characteristics are shown in Table\u00a02 and were comparable between the three groups. After induction of anesthesia, SV was significantly higher in both pre-hydrated groups compared to the control group (Fig.\u00a01). After repositioning from supine to lateral, SV decreased significantly in the control group but not in groups 1 and 2 (Fig.\u00a01). After installation of PP, SV remained stable in group 1 but not in groups 2 and 3 (Fig.\u00a01).\nTable\u00a02.Demographic data on the three groups, mean (SD)Group 1Group 2Group 3Age (yrs)56 (11)53 (9)55 (12)Weight (kg)72 (9)82 (8)74 (7)IBW (kg)70 (8)78 (8)71 (6)Male\/female 2\/54\/33\/4Operation time (min)237 (37)251 (46)226 (31)IBW\u00a0=\u00a0ideal body weightFig.\u00a01.Stroke volume changes during laparoscopic donor nephrectomy, comparing three different fluid regimens: # symbol p\u00a0<\u00a00.05 for groups 1 and 2 versus control group 3; o symbol p\u00a0<\u00a00.05 for group 1 versus group 2; \u00d7 symbol p\u00a0<\u00a00.05 versus supine position. Data are mean\u00a0\u00b1\u00a0standard deviation.\nAfter induction, LVETc was higher in group 1 compared to the control group during the whole procedure and remained stable (Table\u00a03). In all groups MAP decreased after induction of anesthesia; in the control group MAP decreased significantly more compared to group 1 (p\u00a0=\u00a00.03). HR was comparable between the three groups (Table\u00a03).\nTable\u00a03.Data on hemodynamic parameters: heart rate (HR), mean arterial pressure (MAP) and left ventricular ejection time (LVETc), mean (SD). Columns correspond to seven times: before anesthesia (preop); after induction of anesthesia, supine position (supine); full lateral position (lateral); and mean measurement for the first, second, third and fourth 30 minutes after installation of pneumoperitoneum (PP30, PP60, PP90 and PP120, respectively)PreopSupineLateralPP30PP60PP90PP120HR\u00a0\u00a0Group 165 (6)65 (13)63 (20)60 (9)61 (8)60 (10)61 (9)\u00a0\u00a0Group 263 (11)60 (9)61 (14)62 (11)61 (9)63 (6)63 (8)\u00a0\u00a0Group 3 (control) 73 (9)69 (14)61 (6)65 (10)63 (6)60 (7)61 (6)MAP\u00a0\u00a0Group 1102 (11)85 (13)* #81 (19)*93 (14)96 (15)94 (17)98 (20)\u00a0\u00a0Group 2102 (14)73 (10)*81 (15)*107 (10)107 (18)104 (8)102 (12)\u00a0\u00a0Group 3 (control) 105 (12)70 (11)*79 (7)*103 (9)98 (11)95 (12)95 (15)LVETc\u00a0\u00a0Group 1336 (19)#339 (20)#355 (26)#345 (31)335 (30)336 (33)\u00a0\u00a0Group 2313 (29)308 (39)295 (58)\u00b0311 (20)\u00b0308 (27)300 (28)\u00a0\u00a0Group 3 (control) 294 (26)284 (31)292 (26)307 (18)309 (18)311 (25)* p\u00a0<\u00a00.05 versus preoperative values# p\u00a0<\u00a00.05 groups 1 and 2 versus control group\u00b0 p\u00a0<\u00a00.05 group 1 versus group 2\nUrine output, measured from the start of operation until the moment of kidney extraction, was 1.9 ml\/kg\/h (range 1.2\u20133.2) for group 1, 1.4 ml\/kg\/h (range 0.8\u20132.3) for group 2, and 1.1 ml\/kg\/h (range 0.6\u20131.6) for group 3. In controls, the urine production was significantly lower compared to group 1 (p\u00a0=\u00a00.01). CrCl decreased in the control group directly after PP, but not in the other groups (Table\u00a04). From two days postoperative, CrCl was comparable between the three study groups (Table\u00a04).\nTable\u00a04.Creatinine clearance [mean (SD)] in the three groups at six measurement points: one day before operation; just after induction of anesthesia; at 14:30, six hours after installation of pneumoperitoneum T6; two days after operation; one month after operation; and one year after operationGroup 1Group 2Group 3 (control)CrCl preop (ml\/min)89 (19)101 (25)102 (34)CrCl after induction (ml\/min)104 (18)109 (20)96 (31)CrCl after the operation (ml\/min)87 (17)94 (14)73 (23)\u00aaCrCl D2 (ml\/min)63 (10)*65 (11)*64 (19)*CrCl after 1 month (ml\/min)63 (12)*67 (11)*63 (24)*CrCl after 1 year (ml\/min)71 (13)71 (13)*66 (25)** p\u00a0<\u00a00.05 versus preoperative values\u00aa p\u00a0<\u00a00.05 difference between CrCl preop and CrCl after operation, control group versus groups 1 and 2\nDiscussion\nThis study showed that during LDN preoperative hydration together with a bolus of colloid given before induction of anesthesia and before installation of PP resulted in higher SV and higher urine output compared to a fluid regimen with only an intraoperative aggressive fluid infusion. The second group, which received no bolus of colloid before PP in contrast to group 1, showed a significant reduction in SV after installation of PP. In the control group, LVETc and urine output at the moment of kidney extraction showed significantly lower values compared to both pre-hydration groups. CrCl values six hours after the start of the operation was significantly reduced in the control group compared to preoperative values but not in the two pre-hydrated groups; this difference was reduced two days postoperatively.\nClinical studies yield conflicting data concerning the effect of LDN on recipient graft function compared to ODN. The largest study to date compared more than 5,000 kidney transplants from a database and found that LDN was associated with slower early graft function compared to ODN. However, renal function and graft survival at one year was similar between both groups. This was confirmed by retrospective analysis of 120 LDN and 100 ODN in our own institution in which serum creatinine in the recipients was significantly higher in the LDN group only in the first week after transplantation [4]. One very important discriminating factor between ODN and LDN is the pneumoperitoneum. From experimental studies it has become clear that PP decreases RBF and that the magnitude of this decrease is affected by the IAP used, the volume status, and positioning. To counterbalance the increased IAP, vigorous intravenous hydration during LDN is nowadays recommended in an attempt to optimize preload and promote diuresis, but randomized clinical data are missing. In a porcine model, Demyttenaere et al. [23] showed that the decrease in SV and renal cortical perfusion could be prevented by a simple hydration of 15 ml\/kg\/h saline combined with a bolus 20 ml\/kg saline, in accordance with the findings of London et al. [11]. This was also seen in the present study in which pre-hydration with a normal infusion with crystalloids during operation combined with a bolus of colloids just before PP did not decrease SV but improved diuresis. Besides PP, the kidney lateral decubitus position, which is an anti-Trendelenburg position, contributes to hemodynamic alterations by decreasing preload through the effect of gravity on venous return [24]. Yokoyama et al. [25] found no significant change in hemodynamic values after postural change of their patients from supine to lateral but a significant reduction in SV after postural change to kidney position; these patients received a fluid regime of 20 ml\/kg\/h of crystalloids. This was confirmed by our study in which the control group showed a significant reduction in SV after postural change from supine to kidney position whereas there was no reduction in the two pre-hydrated groups, which received a bolus of colloid just before induction (Fig.\u00a01).\nAfter pre-hydration with crystalloids we infused colloids to achieve optimal plasma expansion just before installation of PP [26]. In our hospital we use 6% HES 130\/0.4 for fluid expansion, because the rate for anaphylactic reactions is considerably lower than for gelatin products [27]. However, there are concerns that infusion of certain HES types may influence kidney function [28]. As long as adequate hydration using sufficient amounts of crystalloids are used, the latest generation of HES products (6% HES 130\/0.4) do not increase the risk for renal dysfunction even when used in large amounts [29, 30]. Lang et al. [31] even demonstrated that 6% HES 130\/0.4 improved tissue oxygenation during and after major surgical procedures compared with a crystalloid-based volume strategy.\nIn this study, we used the HemoSonicTM, a transoesophageal Doppler ultrasonography (TOD) device, to measure blood flow in the descending aorta. Several studies have confirmed good correlation with cardiac output measured by the thermodilution technique [18, 32]. It has been shown that the accuracy of the device is somewhat operator-dependent [20] and therefore the same two people did all the measurements with this device in the present study. Feldman et al. [33] used LVET to guide their fluid management in LDN patients. In the present study it was shown that LVETc was significantly lower in the control group that did not received pre-hydration and increased over time (Table\u00a02). It should, however, be taken into account that the blood flow with this device is measured in the descending aorta, which is around 70% of the total cardiac output. This could influence our measurements if redistribution of flow away from the descending aorta occurs because of elevated IAP and this is more pronounced in hypovolemic patients.\nSome other limitations of this study should be noted. In only four patients a MAG3 scan was performed, which provides the distribution of the function from the two kidneys of the donor. In the four measured patients the harvested kidney contributed 43\u201348% of the total kidney function, these four patients were divided over all three study groups. However because we do not have the data on the other patients, this could have biased our data on postoperative CrCl. Prehydration of the donor patients conform our protocol, started the night before operation, which contradicts fast-track surgery where kidney donor patients are admitted to hospital on the day of surgery. Also these patients can receive adequate pre-hydration, but further research should be done.\nIn this study we focused on intraoperative hemodynamic changes. Our data show that preoperative hydration together with a colloid bolus given before induction of anesthesia and before installation of PP resulted in higher SV and higher urine output during LDN, compared to controls that received only an aggressive intraoperative infusion. While under-hydration may contribute to renal dysfunction, perioperative fluid excess can also cause problems, such as pulmonary edema, ileus and increased risk of cardiopulmonary and wound healing complications, which might result in longer hospital stay [34]. However, there is a need to ensure adequate hydration status during PP without being overaggressive. First, our fluid regime will be tested in a large prospective study in order to prevent the negative effect of PP on early graft function in the recipient, and to study possible side-effects in the donor.","keyphrases":["pneumoperitoneum","transplantation","renal (kidneys)","anaesthesia","urbanization"],"prmu":["P","P","R","U","U"]}
{"id":"J_Occup_Rehabil-3-1-2039785","title":"Loss of Productivity Due to Neck\/Shoulder Symptoms and Hand\/Arm Symptoms: Results from the PROMO-Study\n","text":"Introduction The objective of the present study is to describe the extent of productivity loss among computer workers with neck\/shoulder symptoms and hand\/arm symptoms, and to examine associations between pain intensity, various physical and psychosocial factors and productivity loss in computer workers with neck\/shoulder and hand\/arm symptoms.\nIntroduction\nNeck\/shoulder and hand\/arm symptoms are a common problem in society, in particular among the working population. In the European Union, in 2000 and 2001, 23% of the working population reported work-related pain in neck or shoulders. The percentage reporting work-related muscular pain in the upper limbs was about 13% in the old member states and 20% in the new member states [1]. Although it is not clear to what extent work-related factors contribute to their origin, their impact on working life is huge. Neck\/shoulder and hand\/arm symptoms can interfere with activities at work, and can cause sickness absence and chronic occupational disability. In the Netherlands in 2001, incident cases of chronic disability for work due to neck and upper limb symptoms added up to 0.1% of the working population, and 6% of the total number of new disability benefits [2].\nBesides sickness absence and chronic disability, neck\/shoulder and hand\/arm symptoms could also lead to reduced work effectiveness. Many workers still go to work despite the feeling that, in the light of their health, they should have taken sick leave. This phenomenon is known as sickness presenteeism [3]. Although they are present at work, their productivity could be reduced due to functional limitations. The extent of productivity loss while present at work is uncertain, but it has been suggested that it accounts for the majority of lost productivity costs associated with chronic pain [4, 5]. Therefore, more knowledge is needed to estimate the magnitude of productivity loss associated with neck\/shoulder symptoms and hand\/arm symptoms.\nNot all musculoskeletal symptoms involve sickness absence or productivity loss. It would be interesting to know more about factors that might influence productivity loss in symptomatic workers. We are inclined to think that these factors might be similar to risk factors for the occurrence of symptoms or for sickness absence due to musculoskeletal symptoms. However, this is not necessarily true and these factors could easily diverge. Knowledge on both types of risk factors is important for primary, secondary and tertiary prevention. While knowledge on risk factors for the occurrence of symptoms is required to prevent them, knowledge on factors associated with productivity loss is needed in the process of retention, reintegration and rehabilitation of workers with symptoms.\nPrevious studies have shown that pain characteristics, like pain intensity or severity, were predictive factors for a poor prognosis of musculoskeletal symptoms [6\u20139]. Therefore, it seems plausible that high pain intensity will also have an impairing effect on productivity. Besides pain characteristics, physical, psychosocial and personal factors might affect productivity. In previous studies these factors were already identified as risk factors for the occurrence of symptoms [10\u201314]. However, as mentioned earlier, this does not necessarily mean they also affect productivity loss in symptomatic workers. The present study is different from studies examining risk factors for sickness absence, as productivity is a broader outcome measure. Moreover, the study population of this study consists of symptomatic workers, and distinguishes between symptomatic workers with and without productivity loss. Studies examining risk factors for sickness absence are usually held in a mixed study population and usually distinguish healthy and symptomatic workers from workers with sickness absence.\nThe objective of the present study is to describe the extent of productivity loss among computer workers with neck\/shoulder symptoms and hand\/arm symptoms, and to examine associations between pain intensity, various physical and psychosocial factors and productivity loss in computer workers with neck\/shoulder and hand\/arm symptoms.\nMethods\nStudy Population\nData were used from the baseline measurement of the PROMO-study: Prospective Research On Musculoskeletal Disorders in Office Workers [15]. The main purpose of this study was to determine risk factors for neck\/shoulder symptoms and hand\/arm symptoms among computer workers. The study design was approved by the Medical Ethics Committee of the VU University Medical Center.\nThe study included workers from five different companies. The five participating companies comprised an insurance company, a department of a university, a public transport company, a brewery, and a financial consultancy firm. Employees from these companies had administrative, professional or management jobs. Altogether approximately 9,000 employees were working in these companies. All employees were invited to participate in the study. To maximize the participation rate, various activities were arranged, varying from the dissemination of brochures to visits at the worksite. Almost 2,500 employees subscribed to the project and signed informed consent. They were requested by e-mail to fill out an electronic questionnaire, accessible via the Internet. Participants who did not want to fill out an electronic questionnaire could fill out a paper version. Out of these employees, 1,951 filled out the questionnaire at baseline, resulting in a response rate of 79% of the subscribed employees.\nIn the PROMO-study, productivity loss due to neck\/shoulder and hand\/arm symptoms was self-reported and only assessed in workers reporting symptoms. Therefore, analyses concerning associations between various factors and productivity were limited to respondents reporting work-related neck\/shoulder symptoms or hand\/arm symptoms during the past three months. This selection contained 654 computer workers.\nProductivity Loss\nProductivity loss was only assessed for those respondents that reported regular or prolonged neck\/shoulder symptoms or hand\/arm symptoms in the past three months. A dichotomous variable was constructed, based on the answers on the following questions: (1) \u2018Have your symptoms slowed down your work pace?\u2019; (2) \u2018Have your symptoms decreased your working hours?\u2019; or (3) \u2018Have your symptoms caused disability to work for one or more days?\u2019 These questions were adapted versions of items used in the Swedish questionnaire used in the epi-mouse study. In the epi-mouse study, questions on productivity loss were validated through interviews [16]. They were asked for neck\/shoulder symptoms and for hand\/arm symptoms separately and referred to the past three months. If one or more of these questions were answered affirmative, it was defined as productivity loss. It was assumed that respondents answering that their symptoms decreased their working hours (2), but did not cause disability for work (3), had not been on sick leave. Only if the question concerning disability for work was answered affirmatively, it was defined as sickness absence.\nNeck\/Shoulder Symptoms and Arm\/Hand Symptoms\nIn the questionnaire subjects were separately asked about symptoms in the neck\/shoulder region and in the arm\/hand region. The reason to separate these regions is the expected difference in relationship between symptoms and computer usage; computer usage seems to have more effect on arm\/hand symptoms than on neck\/shoulder symptoms [17]. In the present study the distinction between the regions was maintained, because the different relationship with computer usage might result in different effects on productivity loss.\nSubjects were asked to rate the occurrence of pain or discomfort in the neck\/shoulder region as well as in the hand\/arm region in the previous 3\u00a0months on a four-point scale: \u2018no, never\u2019; \u2018yes, sometimes\u2019; \u2018yes, regularly\u2019; \u2018yes, prolonged.\u2019 Subsequently, subjects were asked to estimate whether these symptoms were related to their work, which they could answer with \u2018yes, completely\u2019; \u2018yes, partly\u2019; \u2018possibly\u2019 or \u2018no.\u2019 Also, 8 possible specific causes of these symptoms were summed: sport injuries, accidents, skin diseases, a twist or sprain, a cut or burn, a congenital defect, rheumatic disorders and a slipped disc. Neck\/shoulder symptoms and arm\/hand symptoms were defined as regular or prolonged pain, completely, partly or possibly related to work, and not caused by any listed specific cause.\nIndependent Variables\nTo examine which factors are associated with productivity loss in computer workers with neck\/shoulder and hand\/arm symptoms, analyses were carried out with the following variables: pain intensity, physical activity in leisure time (with BMI as a possible confounder), working hours, mouse position, psychosocial load and overcommitment.\nIntensity of Symptoms\nThe intensity of symptoms was measured using Von Korff scales [18]. Respondents were asked to indicate the mean intensity of their symptoms in the past three months on a scale ranging from 0 (no pain) to 10 (worst pain ever). If they reported symptoms in both neck\/shoulder and hand\/arm region, the region with the highest score was used in the analyses.\nPhysical Activity and BMI\nConcerning physical activity, the respondents were asked about the number of days per week they usually performed activities of at least moderate intensity, and about the times per week they usually performed activities of vigorous intensity. One variable with three categories was constructed measuring physical activity. The categories were \u2018no significant physical activity,\u2019 \u2018moderate intensity physical activity, but no physical activity of vigorous intensity,\u2019 and \u2018vigorous intensity physical activity.\u2019 Moderate intensity physical activity was defined as performing physical activity causing increased breathing for at least 30\u00a0min per day with a frequency of at least 5\u00a0days per week [19, 20].Vigorous intensity physical acitivity was defined as performing physical activity causing sweating for at least 20\u00a0min per session with a frequency of at least 3\u00a0days per week [21, 22]. BMI was computed by body weight (kg) divided by square of height (m2). Data on weight and height were self-reported.\nWorking Hours\nRespondents were asked how many hours per week they worked according to their contract. A dichotomous variable was constructed that distinguished between full-time workers working 37\u201340\u00a0h and part-time workers working 4\u201336\u00a0h per week.\nMouse Position\nRespondents were asked about the position of their mouse while using it. This question was illustrated with five possible positions for respondents to select, and a category \u2018other position.\u2019 A dichotomous variable was constructed that distinguished between a mouse position close to the keyboard, and positions further away from the keyboard. This dichotomization corresponds to the dichotomization in previous research of Hagberg et\u00a0al., who defined nonoptimal computer mouse position as: \u201cThose who marked their computer mouse position outside a rectangle close to the operator in a workplace layout figure in the questionnaire\u201d [16].\nPsychosocial Load\nTo assess psychosocial load, two concepts were used, namely effort-reward imbalance and job satisfaction. For the first concept Siegrist\u2019s Effort-Reward Imbalance model (ERI) was used [23, 24]. The theory of this model is that a combination of high effort and low reward could lead to adverse health effects. Effort and reward were measured with the recommended scales of the ERI-model [25]. A validated Dutch version of the questionnaire was used [26] with scores varying from 1 (\u2018agree\u2019) to 5 (\u2018disagree, and I am very distressed\u2019). Many different methods have been used to construct a variable indicating ERI [27]. In this study a variable in four categories was constructed: no high effort and no low reward, high effort (but no low reward), low reward (but no high effort) and both high effort and low reward. High effort was assigned when a respondent reported to be distressed or very distressed about one or more of the 5 effort items. Low reward was assigned when a respondent reported to be distressed or very distressed about one or more of the 11 reward items. Cronbachs \u03b1 was 0.68 for the effort scale and 0.80 for the reward scale.\nTo assess job satisfaction respondents were asked how they enjoyed their work. To answer this question four categories were presented: \u2018never,\u2019 \u2018sometimes,\u2019 \u2018often,\u2019 and \u2018always.\u2019 This variable was dichotomized, resulting in a positive score on job satisfaction containing the responses \u2018often\u2019 or \u2018always,\u2019 and a negative score containing the responses \u2018never\u2019 or \u2018sometimes.\u2019\nPersonal Factor\u2014Overcommitment\nConcerning personal factors, the personality trait overcommitment was assessed. The concept \u201covercommitment\u201d specifies those cognitive, emotional and motivational components within the global concept of Type A behavior that are important in coping with work demands. Overcommitted workers may expose themselves more often to high demands at work, or they may exaggerate their efforts beyond what is formally needed [25]. Overcommitment was assessed with the short version of a standard questionnaire [23\u201325]. Subjects were asked if they strongly disagreed, disagreed, agreed or strongly agreed on 6 items (e.g., I get easily overwhelmed by time pressures at work). Scores were dichotomized (agree versus disagree) and the added scores on these items resulted in an overcommitment score ranging from 0 to 6. Cronbachs \u03b1 of the overcommitment scale was 0.74.\nAnalysis\nFirst, descriptive statistics were used to examine how often symptoms resulted in productivity loss, and to describe the components of productivity loss in terms of sickness absence and decreased performance at work. The association between productivity loss and several determinants was examined with logistic regression analyses using productivity loss as the dichotomous outcome variable. Univariate and multivariate analyses were carried out. In all multivariate analyses, age, gender, level of education, and intensity of symptoms were included as covariates. For the analysis with physical activity, BMI was checked for confounding and for the analysis with the psychosocial work characteristics, the other psychosocial work characteristics were checked for confounding. If their inclusion in the model did not result in a change of more than 10% in the effect estimate, these covariates were not considered as a confounder and not included in the final model.\nResults\nProductivity Loss\nFigure\u00a01 shows that neck\/shoulder symptoms were reported more frequently than hand\/arm symptoms. Of the total population 10% reported both symptoms. On average, in 26% of the cases reporting symptoms, productivity loss was involved. If both symptoms were reported, they were more often accompanied by productivity loss (36%).\nFig.\u00a01Distribution of neck\/shoulder and hand\/arm symptoms in a population of computer workers (n\u00a0=\u00a01,951), and the fraction involving productivity loss within workers reporting symptoms\nFigure\u00a02 shows what part of productivity loss was caused by sickness absence and what part was caused by a decreased performance at work. A decreased performance at work means decreased speed or decreased working hours, but no sickness absence. In 32% of all cases reporting productivity loss, this productivity loss was coming from sickness absence. Sickness absence occurred more frequently in workers reporting both symptoms (43%). Among workers reporting arm\/hand symptoms, and no neck\/shoulder symptoms, productivity loss was composed mainly of decreased productivity at work. Only 11% of these workers reporting productivity loss have been on sick leave during the last 3\u00a0months.\nFig.\u00a02Distribution of productivity loss due to neck\/shoulder symptoms, hand\/arm symptoms or both in a symptomatic population of computer workers (n\u00a0=\u00a0654) and the fraction involving sickness absence within symptomatic workers reporting productivity loss\nAssociations with Productivity Loss\nTable\u00a01 shows odds ratios resulting from the logistic regression analyses. A higher odds ratio represents a higher probability of productivity loss. Pain intensity and psychosocial load, i.e., high effort and low job satisfaction were associated with productivity loss in computer workers with neck\/shoulder or arm\/hand symptoms. In the analyses of the psychosocial work characteristics, the other psychosocial work characteristics were identified as confounders, while inclusion of physical activity in the model did not change the odds ratios for more than 10%.\nTable\u00a01Associations with productivity loss in a symptomatic population of computer workers (n\u00a0=\u00a0654)% (n)Crude OR (95%CI)Adjusted* OR (95% CI)R2N if item deletedFull model**.140Pain intensity\u00a0\u00a0\u00a0\u00a0Continuous measure (0\u201310)1.24 (1.13\u20131.37) 1.26 (1.12\u20131.41) .104Physical activity in leisure time\u00a0\u00a0\u00a0\u00a0No significant physical activity63 (408)1.001.00.131\u00a0\u00a0\u00a0\u00a0Moderate intensity (5\u00a0\u00d7\u00a030\u00a0min\/week)22 (141)0.73 (0.46\u20131.17)0.74 (0.43\u20131.27)\u00a0\u00a0\u00a0\u00a0Vigorous intensity (3\u00a0\u00d7\u00a020\u00a0min\/week)16 (99)1.48 (0.92\u20132.39)1.27 (0.70\u20132.27)Working hours\u00a0\u00a0\u00a0\u00a0Part-time (4\u201336\u00a0h)61 (399)1.001.00.135\u00a0\u00a0\u00a0\u00a0Full-time (37\u201340\u00a0h)39 (255)1.37 (0.96\u20131.96) 1.36 (0.87\u20132.11) Mouse position\u00a0\u00a0\u00a0\u00a0Close to the keyboard35 (229)1.001.00.134\u00a0\u00a0\u00a0\u00a0Other position65 (425)0.93 (0.64\u20131.34) 0.70 (0.45\u20131.08) Psychosocial loadEffort-Reward Imbalance\u00a0\u00a0\u00a0\u00a0No high effort, no low reward40 (258)1.001.00.115\u00a0\u00a0\u00a0\u00a0No high effort, low reward20 (130)1.81 (1.10\u20132.99)1.43 (0.76\u20132.67)\u00a0\u00a0\u00a0\u00a0High effort, no low reward18 (119)1.89 (1.14\u20133.16)2.26 (1.24\u20134.12)\u00a0\u00a0\u00a0\u00a0High effort, low reward21 (138)2.69 (1.68\u20134.32)1.95 (1.09\u20133.50)Job satisfaction\/task enjoyment\u00a0\u00a0\u00a0\u00a0Always21 (138)1.001.00.110\u00a0\u00a0\u00a0\u00a0Often65 (423)1.29 (0.80\u20132.08)1.62 (0.89\u20132.92)\u00a0\u00a0\u00a0\u00a0Never\/sometimes14 (93)3.10 (1.72\u20135.58)3.10 (1.44\u20136.67)Overcommitment\u00a0\u00a0\u00a0\u00a0Continuous measure (0\u20136)1.06 (0.96\u20131.16) 1.09 (0.97\u20131.22) .140Abbreviations: OR, odds ratio; CI, confidence interval; R2N: Nagelkerke R2 index of global model fit (0\u00a0=\u00a0lack of fit, 1\u00a0=\u00a0perfect fit)* Adjusted for gender, age, level of education and intensity of symptoms; the analyses with effort-reward imbalance were additionally adjusted for job satisfaction and the analyses with job satisfaction were additionally adjusted for effort-reward imbalance** The full model contained gender, age, level of education and all variables mentioned in the table\nPhysical activity in leisure time, working hours, mouse position and overcommitment were not associated with productivity loss in computer workers with neck\/shoulder symptoms or arm\/hand symptoms. Additional adjustment for BMI in the analyses with physical activity did not result in a change in odds ratio for more than 10%.\nDiscussion\nThe purpose of this study was to describe the extent of productivity loss among computer workers with neck\/shoulder symptoms and hand\/arm symptoms and to examine associations between various physical, psychosocial and personal factors and productivity. The results show that in 26% of all cases reporting regular or prolonged symptoms in the past three months, productivity loss was involved. Most productivity loss was found in workers reporting both neck\/shoulder symptoms and hand\/arm symptoms. Overall, about 32% of the productivity loss was coming from sickness absence. Sickness absence occurred more frequently in workers reporting both symptoms (43%) and considerably less frequent in workers reporting only hand\/arm symptoms (11%). Symptomatic workers reporting unfavorable psychosocial work characteristics reported more productivity loss.\nComparison with Previous Research\nThe study population of the present study consisted of office workers with neck\/shoulder or hand\/arm symptoms. So far, almost all studies using productivity loss as an outcome measure were studies in a mixed population, containing subjects with and without musculoskeletal symptoms. Moreover, most previous research on productivity loss concerned sickness absence, while in the present study a decreased productivity while working was also included. Therefore, the results of previous studies are hard to compare with the present study. In a mixed population, associations between productivity loss and potential risk factors partly reflect the risks for symptom occurrence. In the present study we wanted to examine which factors are related to decreased productivity once symptoms have occurred. These factors do not have to be similar. Nevertheless, we will mention the results of previous research in this section.\nIn the present study, physical activity in leisure time was not significantly associated with productivity loss in workers with neck\/shoulder or hand\/arm symptoms. We did not find other studies in a symptomatic working population. A few studies have examined the relation between physical activity and productivity loss in a general population. These studies often found a favorable effect of physical activity. A favorable effect was found of sporting activity on sick leave in general [28], and in particular sick leave due to musculoskeletal disorders [29, 30]. Also an association was found between higher levels of physical activity and job performance [31]. It seems that physical activity has a preventive effect and is positively associated with productivity, but does not affect productivity loss in workers already having symptoms.\nThe present study did not examine the association between physical load and productivity loss extensively. Our hypothesis was that computer workers with a high physical load (for example adverse ergonomic working conditions or sustained computer or mouse usage) might experience more productivity loss. Conversely, some studies in a general working population examining the effect of the improvement of ergonomic working conditions have shown beneficial effects on productivity [32, 33]. An observational study found a weak association between an adverse mouse position and productivity loss [16] in workers with musculoskeletal symptoms.\nThe present study could not confirm the relation between an adverse mouse position and productivity loss. It might be more interesting to examine the relation between productivity loss and duration of computer use, since studies show that duration is more consistently associated with musculoskeletal symptoms than posture [10, 17]. However, the design of the present study is not suitable to examine this relationship. The results of the analyses would produce a biased view, because in a population of computer workers, productivity loss means almost automatically less computer usage. Preliminary analyses confirmed this assumption: in the subpopulation of symptomatic computer workers, those reporting productivity loss also reported less computer usage, in particular mouse usage. Therefore, analyses examining the association between computer usage and productivity loss would result in a negative association, i.e., more computer usage, less productivity loss.\nHowever, we did examine the association with part-time versus full-time work, because this variable is probably not biased. Respondents were asked how many hours they worked according to their contract. The distinction might indicate a form of physical load, assuming that full-time workers are exposed to a higher physical load. Nevertheless, no relation was found between productivity loss and part-time\/full-time work. No previous studies were found examining this relationship.\nPsychosocial load, in this study defined as effort-reward imbalance and job satisfaction, was strongly associated with productivity loss. No studies were found that examined these psychosocial work characteristics in relation to productivity loss or sickness absence due to musculoskeletal symptoms. Also, no studies were found that examined the relation between psychosocial work characteristics in general and productivity loss, other than sickness absence. Finally, no studies were found that examined these relations in a symptomatic population.\nIn mixed populations, containing subjects with and without musculoskeletal symptoms, the relation between psychosocial load and sickness absence in general was demonstrated by several studies [34\u201336]. Few studies have examined the relation with sickness absence due to musculoskeletal symptoms, and their results were conflicting [37, 38].\nLimitations of the Study\nIn the present study, self-reported data were used to assess productivity loss. Productivity loss in this study contains two components: absenteeism and presenteeism. Self-reported data on absenteeism have been found to be reliable and valid when the recall periods are short [39]. However, measuring presenteeism is more complex. For most types of employment, there is no objective account of productivity with which to assess an employee\u2019s performance. In the present study computer usage was measured by a software program. Therefore, in this study population of computer workers it might be considered to use computer usage as a measure for productivity. We decided not to do so, because we assumed that differences in computer usage would be larger between jobs than between workers with and without productivity loss. For this reason, differences in computer usage would probably reflect different kinds of jobs instead of differences in productivity.\nThe lack of objective instruments to measure productivity also hampers the validation of self-reported measures. One study, using a rather similar productivity measure, compared self-reported productivity loss assessed with a questionnaire with interviews of 50 computer users who had reported musculoskeletal symptoms [16]. The results seem to indicate that productivity loss assessed in the questionnaire could be a slight underestimation.\nAnother limitation concerning the productivity measure is its dichotomy. No distinction could be made between a minimal and a major loss of productivity. It would be more accurate and it would provide more insight into the magnitude of the problem, if the precise size of the productivity loss was assessed. However, it is very hard for workers to estimate the size of their productivity loss. A recent review concluded that no study has shown that employees can accurately transform their perceived impairments into a quantitative measure [39]. Therefore, since objective instruments to measure productivity are lacking, we are forced to limit ourselves to a subjective dichotomous measure of productivity.\nAlthough in the PROMO-study follow-up measurements were available, we choose to use a cross-sectional design. The main reason is that data on productivity loss were only available for workers with symptoms. Since symptoms generally have an episodic nature the selection of workers with symptoms at baseline is not identical to the selection of workers with symptoms at follow-up. To select workers with data on productivity at baseline as well as at one or more of the follow-up measurements would mean a selection of workers with symptoms at all these measurements. Such a selection would result in a small study population of workers with chronic symptoms.\nIf we assume that the associations in this study are causal, still the direction of causality cannot be established due to the cross-sectional design. It could be that either psychosocial working conditions caused productivity loss, or productivity loss caused the reporting of adverse psychosocial working conditions, or both. Intervention studies focusing on the prevention of productivity loss among symptomatic workers might shed more light on the direction(s) of causality.\nImplications for Practice\nThe results of this study show that employers should be aware that the consequences of neck\/shoulder and hand\/arm symptoms are more extensive than the visible sickness absence due to these symptoms. Only one third of workers who experience productivity loss due to their symptoms actually take sick leave. For the other workers productivity loss expresses itself in decreased performance at work. The results concerning factors associated with productivity loss are more difficult to interpret, since there is still a lack of knowledge on how they relate. It seems that symptomatic workers perform better in favorable psychosocial working conditions. An advantageous psychosocial climate might prevent productivity loss in symptomatic workers.\nIn conclusion, most workers with neck\/shoulder symptoms or hand\/arm symptoms experience productivity loss from a decreased performance at work and not from sickness absence. Favorable psychosocial work characteristics might prevent productivity loss in symptomatic workers.","keyphrases":["productivity","computer workers","psychosocial factors","presenteeism","musculoskeletal symptoms"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_Spine_J-2-2-1602191","title":"Mycoplasma hominis deep wound infection after neuromuscular scoliosis surgery: the use of real-time polymerase chain reaction (PCR)\n","text":"Mycoplasma hominis is a commensal of the genitourinary tract. It mostly causes infections to associated structures of this system; however, occasionally it is a pathogen in nongenitourinary tract infections. Since, M. hominis strains require special growth conditions and cannot be Gram stained, they may be missed or delay diagnosis. This report describes a deep wound infection caused by M. hominis after neuromuscular scoliosis surgery; M. hominis was recovered by real-time polymerase chain reaction (PCR). An awareness of the role of M. hominis as an extragenital pathogen in musculoskeletal infections, especially in neuromuscular scoliosis, being a high-risk group for postoperative wound infection, it is necessary to identify this pathogen. Real-time PCR for postoperative deep wound infection, in patients with a history of genitourinary infections, decreases the delay in diagnosis and treatment. In these cases rapid real-time PCR on deep cultures should be considered.\nIntroduction\nDeep wound infections complicating spinal surgery are sources of major morbidity in neuromuscular scoliotic patients. Enterobacter, Enterococcus, Escherichia coli, Proteus and Staphylococci account for the majority of postoperative spinal wound infections in these patients [28]. Increasingly, previously uncommon pathogens are being identified in surgical infections. These organisms may be difficult to identify and\/or resistant to most of the broad-spectrum antibiotics used for preoperative prophylaxis and for the treatment of postoperative wound infections. Among these pathogens, Mycoplasma hominis has been recognized as a cause of postoperative wound infections [25, 27, 31]. M. hominis is a commensal bacterium in humans and is distinguished phenotypically from other bacteria by the minute size and lack of a cell wall. As a result, M. hominis cannot be Gram stained and is resistant to penicillin and other antibiotics that interfere with the cell wall metabolism. In addition, it is a fastidious slow-growing organism, which may not be readily identified by using routine culture protocols. Early diagnosis, however, is of utmost importance for adequate institution of appropriate antimicrobial therapy.\nTo our knowledge, only two cases of postoperative spinal wound infections due to M. hominis have previously been reported [18]. We describe a case report of a M. hominis postoperative deep wound infection in a patient with a progressive myelomeningocele scoliosis who underwent posterior scoliosis surgery with bone allograft. Real-time polymerase chain reaction (PCR) provided a fast and secure diagnosis, which prevented further complications.\nCase presentation\nAn 11-year-old girl with a complete paraplegia at level L2\u2013L3 caused by a myelomeningocele was admitted to our hospital for surgical correction of a progressive right convex scoliosis. Preoperative physical examination revealed a flexible right convex thoracic scoliosis with a left convex thoracolumbar curve. The unsupported sitting anteroposterior radiograph showed a right convex thoracic scoliosis of 55\u00b0and a left convex thoracolumbar curve of 42\u00b0with no pelvic obliquity. Her medical history revealed a shunted hydrocephalus with an Arnold Chiari type II malformation, chronic urinary tract infections, and an auto-augmentation of the bladder.\nThe patient was treated with single stage scoliosis correction involving posterior instrumentation (Xia\u2122 spinal system, Stryker Spine, Cestas, France) from T3 to L5. The spondylodesis was completed by applying allograft bone chips (Netherlands Bone Bank Foundation, Leiden, The Netherlands) over the laminae in the thoracolumbar region. In addition, Collagraft\u00ae (Neucoll, Campbell, CA, USA), a synthetic bone graft substitute composed of collagen and a composite mineral (hydroxyapatite and tricalcium phosphate), was applied posterolateral in the lumbosacral region.\nProphylactic antibiotics, cefazolin (cefalosporin, Kefzol\u00ae) 1,000\u00a0mg IV, were administered at the induction of anaesthesia, and as a second and third dose 8 and 16\u00a0h postoperatively, respectively [6]. Because of a chronic urinary tract infection including a positive culture with E. coli, cefradin (cefalosporin, Velosef\u00ae) 500\u00a0mg orally was continued for 9\u00a0days at 8-h intervals.\nThe postoperative course was uneventful. At day 8 the patient was discharged. At discharge the wound produced negligible clear fluid at the distal end without any signs of infection. However, at day 18 postoperatively, the patient developed fever (39\u00b0C) and was readmitted to our centre. On physical examination, an enlarged distal wound dehiscence was seen with increased fluid production. Infection parameters showed an increased C-reactive protein (CRP) (120\u00a0mg\/l; normal <10\u00a0mg\/l) and white blood cell (WBC) count (10.9\u00a0cells\u00d7109\/l; normal 4\u201310\u00a0cells\u00d7109\/l). A surgical intervention was performed including a thorough debridement of necrotic tissue and removal of the bone grafts. The instrumentation was left in place. According to the established guidelines, cultures were taken of various deep areas by fluid aspiration and from the applied bone graft [15, 28, 32]. After extensively irrigating with pulsatile lavage, the wound was closed leaving gentamicin collagen fleeces (Septocoll\u00ae, Biomed, Darmstadt, Germany) over the instrumentation. Adjuvant therapy with gentamicin (aminoglycoside, Garamycinl\u00ae) 210\u00a0mg IV and flucloxacillin (isoxazolylpenicillin, Floxapen\u00ae) 2,000\u00a0mg IV were initiated immediately after obtaining appropriate intraoperative cultures, and was continued postoperatively at 6-h intervals.\nIntraoperative cultures from both the fluid aspiration and the bone graft yielded Mycoplasma after 4\u00a0days incubation. Real-time PCR on the peri-operative biopsy material was only positive for Mycoplasma and the species was identified by 16S DNA amplification as M. hominis (Fig.\u00a01). Separately a Collagraft\u00ae sample of the same batch was tested for Mycoplasma, however, the culture as well as real-time PCR remained negative. Gentamicin and flucloxacillin were stopped, and doxycycline (tetracycline, Vibra-S\u00ae) 100\u00a0mg was administered orally at 12-h intervals and continued for 3\u00a0weeks. The patient was discharged 5\u00a0days after surgery.\nFig.\u00a01Agarose gel analysis of duplo PCR amplification of Mycoplasma DNA. M Molecular weight marker, Lanes 1 and 2 positive PCR amplification of clinical sample, lane 3 negative control, lane 4 positive control. Subsequent sequencing results revealed M. hominis\nWound healing and temperature were monitored at regular intervals at the outpatient clinic. After 3\u00a0months, the infection parameters were normalized and the wound was healed (Fig.\u00a02). Four years after surgery there were no signs of infection. Radiographs at final follow-up demonstrated no instrumentation failure and no loss of correction of the scoliotic deformity (Fig.\u00a03).Fig.\u00a02Clinical appearance 3\u00a0months after surgical intervention for deep wound infection, located at the distal end of the woundFig.\u00a03AP radiographs of the lumbar spine at the site of the deep wound infection immediately after surgical intervention and debridement of necrotic tissue including removal of the bone grafts (a) and at 4\u00a0years of follow-up (b)\nDiscussion\nMycoplasma hominis is a commensal bacterium in humans. The organism is commonly associated with infections of the genitourinary tract, particularly in females. Rates of colonization in the urogenital tract range from 21 to 54% among women and from 4 to 13% among men [20]. Reports on M. hominis infections outside the genitourinary tract are scarce. Most of them are case reports describing extragenital infections, such as septic arthritis [17, 21], septicemia [11, 12], prosthetic valve endocarditis [3, 10], postoperative wound infections [25, 31], peritonitis [13], neonatal encephalitis [30], meningitis [7, 9], brain abscess [33], thrombophlebitis [27], and mediastinitis [19]. Extragenital infections are mainly related to patients with immunosuppression [3, 7, 9, 17, 19, 21, 25, 27, 30, 33]. Osteomyelitis caused by M. hominis is predominantly reported in combination with hypogammaglobulinemia [5, 14, 16, 24]. In an extensive overview of nongenitourinary M. hominis infections, Madoff and Hooper [18] described only two cases of postoperative deep wound infections caused by M. hominis after orthopedic surgery. Both cases concerned with deep wound infections after scoliosis surgery, one of them with a history of pyelonefritis.\nPostoperative deep wound infections after scoliosis surgery are more common in neuromuscular patients than in patients with idiopathic scoliosis [28]. Risk factors associated with increased postoperative wound infection rates include a generalized decline in the immune status of neuromuscular patients, poor personal hygiene, and soiling of the wound. Sponseller et al. [28] reported 25 patients (12%) with a deep wound infection out of a series of 210 surgically treated patients with neuromuscular scoliosis in a 10-year retrospective study. From these 25 patients, 16 had a scoliosis related to myelomeningocele. Two risk factors were found to be significant: the degree of cognitive impairment and use of bone allograft. Furthermore, 52% of the infections were polymicrobial, which could point to contamination during or after surgery. However, the authors did not report any case of M. hominis wound infections in their series.\nAntibiotics are used routinely to prevent postoperative wound infection in patients undergoing spinal implant procedures. Currently, first or second-generation cephalosporin (e.g. cefazolin) are recommended as prophylaxis [6]. Prophylactic cefazolin was also routinely given in our case. Since, cephalosporins act on the cell wall of organisms in a manner similar to the penicillins, postoperative wound infection by M. hominis was not prevented. Antimicrobial treatment for M. hominis include protein-synthesis inhibitors such as tetracycline (e.g. doxycycline) [26] and doxycycline was administered accordingly. The postoperative deep wound infection resolved favorably after surgical debridement and appropriate antimicrobial treatment with doxycycline.\nThe source of M. hominis deep wound infection in our case is unclear. M. hominis is commonly associated with infections of the genitourinary tract [20] and patients with meningomyelocele are known to have a high incidence of urinary tract infections [8]. In addition, it has been shown that children with myelomeningocele have a high incidence of urological complications after surgical treatment of scoliosis [4]. Possibly, in our case the M. hominis could have been present in the urinary tract infection and spread hematogenous or by contamination, either at the time of surgery or secondarily through the wound. Other possible sources are the materials that were used during surgery. Obviously, the instrumentation and the applied Collagraft\u00ae, were sterilized. In addition, cultures and PCR on the same batch of the Collagraft\u00ae were negative. Bone allograft, used to induce and facilitate spinal fusion, could also have been the source of the infection. The fact that the bone allograft was positive for M. hominis, proved that it was infected before retrieval, however, not that it was the source of the infection itself. Bone allograft has not been reported as a carrier of M. hominis in the literature, however, it must be noted that the standard screening at the Netherlands Bone Bank Foundation does not include screening on M. hominis. Unfortunately, no other specimen of the donor could be retrieved for testing. As a result, haematogenous spread or contamination from a colonized urogenital tract or contamination by the bone allograft as source of the infection could not be excluded.\nIn the presented case, cultures taken during the reintervention proved to be positive for Mycoplasma. However, the diagnosis of a Mycoplasma infection was delayed due to the fastidious nature of mycoplasmas. To identify the Mycoplasma species, which in our case was cultured, 16S DNA amplification was used. Because isolation of M. hominis is difficult, time consuming, and not routinely done, a rapid specimen processing is required. Real-time PCR on deep cultures could provide a rapid alternative with a higher sensitivity and specificity than culture for the detection of M. hominis [1, 2].\nIn conclusion, M. hominis infections should be considered in postoperative deep wound infection after neuromuscular spinal surgery, especially in patients with genitourinary tract comorbidity. Since, M. hominis is not covered by routine prophylactic and therapeutic antibiotics, rapid real-time PCR is advised in these patients to initiate appropriate antibiotic treatment.","keyphrases":["mycoplasma hominis","wound infection","neuromuscular scoliosis","pcr"],"prmu":["P","P","P","P"]}
{"id":"J_Mol_Biol-1-5-1885968","title":"Global Changes in Local Protein Dynamics Reduce the Entropic Cost of Carbohydrate Binding in the Arabinose-binding Protein\n","text":"Protein dynamics make important but poorly understood contributions to molecular recognition phenomena. To address this, we measure changes in fast protein dynamics that accompany the interaction of the arabinose-binding protein (ABP) with its ligand, d-galactose, using NMR relaxation and molecular dynamics simulation. These two approaches present an entirely consistent view of the dynamic changes that occur in the protein backbone upon ligand binding. Increases in the amplitude of motions are observed throughout the protein, with the exception of a few residues in the binding site, which show restriction of dynamics. These counter-intuitive results imply that a localised binding event causes a global increase in the extent of protein dynamics on the pico- to nanosecond timescale. This global dynamic change constitutes a substantial favourable entropic contribution to the free energy of ligand binding. These results suggest that the structure and dynamics of ABP may be adapted to exploit dynamic changes to reduce the entropic costs of binding.\nIntroduction\nAn important goal of structural biology is to predict and manipulate the interactions between proteins and small-molecule ligands. To this end, extensive research efforts have been aimed at relating the known structure of protein\u2013ligand complexes to the thermodynamics of that interaction. The success of these attempts has been limited, in part because of their neglect of the role of protein and ligand conformational dynamics in determining ligand-binding thermodynamics.\nA common view of protein\u2013ligand interactions sees them arising from the hydrophobic effect (the entropically favourable exclusion of water from hydrophobic surfaces) together with shape complementarity between the protein and ligand. In this view, protein\u2013ligand interactions are expected to be driven by favourable changes in entropy. Recent results suggest closer scrutiny of this view is warranted, even for predominantly non-polar interactions.1,2 One can contrast this former view of protein\u2013ligand interactions with one that considers the restriction in conformational degrees of freedom, which must necessarily be entailed by binding. From this perspective, an unfavourable change in entropy is expected to characterise these interactions. This requires a larger, favourable enthalpic contribution, which may arise directly from the protein\u2013ligand interaction or from solvation effects.\nThe thermodynamic signature of protein\u2013ligand interactions in this second view is typical of protein\u2013carbohydrate interactions: entropic contributions to the interaction are typically large and unfavourable, and favourable enthalpic contributions drive the interaction. An example of such a system is the arabinose-binding protein (ABP). ABP is a member of the bacterial periplasmic binding protein family and serves as the initial component of the active transport system for the monosaccharides l-arabinose, d-fucose and d-galactose. Several members of this family, including ABP, have served as models for structural analysis of ligand-binding interactions,3,4 and have recently emerged as templates for design of novel ligand-binding proteins.5 ABP binds its ligands in the interface of two protein domains. The binding site displays an extensive network of H-bond and CH\u2013\u03c0 interactions characteristic of protein\u2013carbohydrate association. Both structural and thermodynamic aspects of this interaction have been studied extensively,4,6\u20139 yet there is no clear understanding of the basis of either the large favourable binding enthalpy or the unfavourable entropy change on binding.\nResults and Discussion\nEntropic cost of binding\nIt is evident that formation of a stable complex between a protein and a ligand will involve the loss of entropy associated with the free diffusion of one component with respect to the other. The magnitude of these unfavourable contributions to the protein\u2013ligand interaction may be only approximated; here, we take an estimate of the loss of ligand translational and rotational entropy from the work by Turnbull et al and Lundquist and Toone, which converge at approximately 25 kJ\/mol for the equivalent free energy penalty.10,11 We note that this estimate is consistent with a number of others in the literature, for systems ranging from small polar molecules to folded and unfolded proteins.12\u201315\nIt is generally assumed that the bound ligand will adopt a single conformation, optimised to the structure of the binding site, and thus will experience a loss of entropy reflecting the loss of conformational flexibility of the ligand in solution. Likewise, the protein-binding site might be expected to undergo a loss of entropy as it adopts only that subset of available conformations that are conducive to binding. This loss of internal conformational entropy of the ligand is challenging to assess, particularly for a flexible ligand such as galactose, which displays complex conformational behaviour in solution. Nonetheless, on the assumption that ligand degrees of freedom are substantially \u201cfrozen\u201d on binding, the entropic penalty arising from loss in degrees of freedom of the galactose hydroxyl rotors alone is likely to be \u223c\u00a030 kJ\/mol.11\nThe ABP binding site contains a significant number of tightly bound water molecules, which play a role in governing substrate specificity,9 and in maintaining the structure of the binding site. Examination of the structures of ABP in complex with arabinose (1ABE), fucose (1ABF) and galactose (5APB) reveals some 15 crystallographically resolved and structurally conserved water molecules within the binding site (within 10\u00a0\u00c5 of a ligand heavy atom and within 5\u00a0\u00c5 of heavy atoms of both protein domains; Figure 1). The entropic cost of confinement of water molecules has been estimated to be as much as 8 kJ\/mol per water molecule.16 Even if the cost of confinement of the water molecules in the binding site of ABP is much less than this maximal value, it is clear that the overall cost of constraining these water molecules in the binding site will be substantial.\nThe experimentally observed entropy of the ABP\u2013galactose interaction amounts to a T\u0394S\u00b0 of \u201361 kJ\/mol at 308 K.6 Together, the entropic costs above exceed significantly this experimental entropy change on binding. Similar discrepancies, in which measured entropies of association are more positive (i.e. more favourable) than might be expected from first principles, have been known for at least 50 years.17\u201319 Most often, the discrepancy has been attributed to solvation effects, such as the hydrophobic effect or desolvation of charged groups.17,18 It has been noted, however, that these discrepancies might be explained by the introduction of new degrees of freedom in the complex, which did not exist in the interacting partners before interaction.19 We investigate this latter possibility by examining the contribution of protein dynamics to the entropy of interaction of ABP with its ligands.\nAssignment of apoABP\nWe have reported the determination of near-complete backbone resonance assignments for ABP in complex with its ligand, d-galactose.20 Assignments for apoABP were determined from these results, using an approach that combined conventional triple-resonance assignment strategies and 1H-15N heteronuclear single quantum coherence (HSQC) titrations of ABP with the fast-exchanging ligand 1-deoxy-galactose. Approximately 80% of the expected backbone resonances of apoABP were assigned.\nFrom these assignments, a comparison was made of the chemical shifts of the backbone amide resonances of ABP in the apo state and in the complex (Figure 1). As expected, large chemical shift changes are seen in the binding site. In addition, large chemical shift changes are seen in the region linking the two domains of ABP, suggesting that ligand binding might be associated with a change in relative domain orientation in ABP. Domain reorientations on binding are observed in other members of the periplasmic-binding protein family, and have been proposed for ABP on the basis of the results of small-angle X-ray scattering and computational studies.21,22\nSmall chemical shift differences are seen at sites distal to the binding site and hinge region. These differences suggest that subtle changes in conformation or dynamics throughout the molecule occur on binding.\nDomain orientation of apoABP\nAnalysis of NMR relaxation for anisotropic molecules depends on knowledge of the molecular structure. It is therefore necessary to address the possibility of ligand-induced domain reorientation in ABP. We have done this using residual dipolar couplings (RDCs), which are sensitive to the average molecular orientation with respect to molecular alignment induced by a liquid crystalline solution.23\n1H-15N RDCs were measured for 127 backbone amides in regions of defined secondary structure of apoABP. The measured RDCs were initially compared to those predicted on the basis of the crystal structure of ABP in complex with galactose. Agreement was poor (RMSD\u00a0=\u00a013\u00a0Hz, R-factor\u00a0=\u00a065%), confirming that conformational change does indeed occur on ligand binding.\nThe extent of domain reorientation in ABP was determined using a structure calculation protocol in which the structure of each domain was minimised individually against the measured RDCs, followed by a simulated annealing procedure in which each domain was held rigid in its minimised conformation. This protocol was repeated 100 times with data generated by a Monte Carlo re-sampling of the experimental RDC data to assess the robustness of the protocol and the precision of the resulting structures. The agreement between the RDCs and the calculated structure is excellent, with an RMSD of 1.2\u00a0Hz and an R-factor of 6.0%. The quality of the structure is good, with 97% of residues in the core and allowed regions of the Ramachandran plot, and all residues showing correct peptide bond geometry. The backbone RMSD over the 100 structures derived from Monte Carlo re-sampling of the RDC data is 0.67\u00a0\u00c5.\nThe resulting ensemble of structures (Figure 2) showed apoABP to be \u201copened\u201d by a domain hinge motion of \u223c\u00a020\u00b0 with respect to the crystal structure of the ABP\u2013galactose complex. This represents a conformational change similar to those seen in other members of the periplasmic-binding protein family. In addition, small changes to individual domain structures are required for optimal agreement with RDCs. This supports the inference from chemical shift changes, that ligand binding has subtle conformational consequences throughout the protein.\nDynamics of ABP\nThe contribution of protein dynamics to the ABP\u2013galactose interaction was assessed by means of Lipari\u2013Szabo analysis of nuclear magnetic relaxation.24,25 This approach yields information on the extent and timescale of molecular motions occurring faster than the rotational diffusion of the protein. The Lipari\u2013Szabo formalism is relatively free of assumptions regarding the physical model describing the motion under investigation, requiring only that the dynamics be described by a Markov process and that the internal dynamics is uncorrelated with the global tumbling of the macromolecule. The results of the Lipari\u2013Szabo analysis, in the form of an order parameter, can be interpreted in terms of the conformational entropy associated with the measured motions by means of a specific motional model. It has been shown that for a wide range of models, the functional dependence of entropy on the order parameter is similar, suggesting that changes in order parameter can be related to entropy change in a model-independent fashion.26\nWe have measured relaxation data for 148 backbone amides of apoABP at three magnetic fields, and for 156 backbone amides of the ABP-galactose complex at two magnetic fields. The analysis of these data in terms of the Lipari\u2013Szabo formalism yields order parameters that measure the extent of angular motion of individual amide bond vectors (Figure 3(a) and (b)). In both apoABP and the complex we see almost exclusively the large order parameters typical of the relatively rigid backbone of globular proteins. Remarkably, order parameters for apoABP are generally larger than for the ABP\u2013galactose complex, indicating that pico- to nanosecond motions are more extensive in the complex than in the apo protein.\nOwing to the size of ABP and the associated spectral complexity, many residues have been excluded from the analysis, because spectral overlap precludes accurate measurement of relaxation rates. For this reason, no experimental data are available for residues involved directly in binding. Thus, our observations reflect changes in dynamics remote from the binding site, yet caused by the protein\u2013ligand interaction. Indeed, the observed changes are seen to be distributed throughout the protein, albeit with a slight bias towards the N domain (in the sense that larger dynamic changes are seen preferentially in this domain) (Figure 3(c)).\nTo confirm and further explore the basis of this result, we have performed molecular dynamics simulations for ABP in the apo state and in complex with galactose. By several measures, we see significant increases in backbone dynamics in the complex as compared with the apo protein. RMS deviations from the average structure for both the N and C domains are significantly larger for the ABP\u2013galactose complex than for apoABP. In addition, fluctuations of backbone heavy-atom positions across the trajectory are generally larger in the complex than in the apo protein (data not shown). Furthermore, these dynamic changes are seen to be more pronounced in the N domain than the C domain, consistent with the experimental observations.\nTo make a direct comparison between the experimentally observed order parameters and the simulations, backbone amide order parameters have been calculated from the molecular dynamics trajectory.27 We see good agreement between measured and calculated order parameters, albeit with a small tendency for the simulation to underestimate the experimental values (Figure 3(a) and (b)). Underestimation of measured order parameters by molecular dynamics simulations has been a common observation in previous comparisons between molecular dynamics simulation and NMR relaxation measurements, particularly in loops and other flexible regions.27\u201330 This small systematic discrepancy is of little importance in this instance, as we are interested principally in the difference in order parameter between the two states. Indeed, the changes in order parameter upon ligand binding reproduce the measured changes excellently, showing an approximately uniform decrease in order parameter upon binding across much of the protein (Figure 3(c)). The average change in order parameter (S2apoABP\u2013S2ABPgal) determined by the two methods is identical, at 0.038; the uncertainty on this average value, derived by propagation of experimental uncertainties in the individual order parameters, is 0.005. The correlation coefficient between the experimental and simulated order parameters is 0.83 for the complex protein and 0.47 for the apo-protein.\nAs well as validating our experimental results, the molecular dynamics simulations reveal details of dynamic changes in regions that could not be measured experimentally. Notably, the simulations reveal complex changes in dynamics in the binding site (Figure 4(b)). Several residues in the binding site display increases in flexibility upon binding, consistent with the trend seen throughout the rest of the molecule. Other binding site loops display a decrease in flexibility, more in keeping with the intuitive expectation that ligand binding will reduce the conformational freedom of binding site residues.\nA significant assumption entailed by the Lipari\u2013Szabo analysis performed here is that the rotational diffusion of the protein can be fully characterised by a single diffusive process uncorrelated with the internal motions under investigation.24 This assumption is generally robust for globular, single-domain proteins, but might be called into question in the case of ABP, where flexibility of the domain hinge may result in a complex interaction between local protein conformation and rotational diffusion.\nIn the case of the ABP\u2013galactose complex, we assume such flexibility to be insignificant, as the ligand binds in and stabilises the domain interface. No such assumption can be made for apo ABP, however. In an attempt to assess the influence of inter-domain flexibility in apoABP on our results, we have repeated the Lipari\u2013Szabo analysis, assuming each domain undergoes independent rotational diffusion. The expectation is that the apparent rotational diffusion of each domain should differ from that of the molecule as a whole if significant inter-domain flexibility is present. The best-fit diffusion tensors arising from the analysis of the two individual domains are very similar, but are slightly different from the diffusion tensor derived from the whole protein (Table 1). This suggests that inter-domain flexibility has only a minor influence on the measured relaxation rates. The orientation of the diffusion tensors for each domain is consistent with the RDC-derived structure of apo ABP, highlighting a consistency between the RDC and relaxation data that would not be expected if the relaxation data were influenced substantially by domain motions. Importantly, the Lipari\u2013Szabo dynamic parameters derived from this analysis are identical, within error, with those of the initial analysis.\nFurthermore, we note that inter-domain flexibility can, to a first approximation, be accounted for by the so-called extended Lipari\u2013Szabo treatment.25 Here, inter-domain flexibility is treated by the slow dynamic component (S2s, \u03c4s), while internal motions are treated by the fast dynamic component (S2f).31,32 Because the reported overall order parameter is the product of the fast and slow order parameters (S2LZ\u00a0=\u00a0S2f S2s), the effect of inter-domain flexibility (S2s\u00a0<\u00a01) will always be the overestimation of the extent of internal motions. As such, if domain flexibility in apoABP does influence our analysis, its effect will be the underestimation of the increase in internal dynamics upon ligand binding. In this context, it is of interest to note that there are more residues that are best treated by the extended Lipari\u2013Szabo model in apoABP than there are in the ABP\u2013galactose complex (Table 2).\nAdditional evidence that inter-domain motions have little impact on our overall results is obtained from the MD simulations. The contributions of rotational diffusion to the simulated dynamics are removed in the conventional way by alignment of the protein at each snapshot to a single reference structure. Alternatively, contributions of both rotational diffusion and inter-domain flexibility can be removed by separately aligning a single domain to a reference structure before analysis of the internal dynamics of that domain. There is no significant difference between these two approaches in terms of the calculated order parameters (data not shown). This reflects the small amplitude of the domain flexibility observable in the production phase of the apo ABP trajectory. On this basis, it is likely that domain flexibility is either very small or occurs on a timescale slower than is detectable in the 20\u00a0ns simulations analysed here. Given the anomalously low viscosity of the TIP3P water model used in these simulations, the rate of domain flexibility in the simulation is likely to significantly overestimate the in vitro rate. Thus, the failure to detect significant domain flexibility suggests that the relevant timescale is slower than the overall rotational diffusion of ABP (\u223c\u00a015\u00a0ns), and therefore is not expected to contribute to the measured relaxation data.\nA further caveat of the analysis of NMR relaxation data concerns its insensitivity to dynamics occurring on a timescale similar to or slower than the rate of rotational diffusion. Given this insensitivity, it is conceivable that the apparent increase in flexibility of ABP on ligand binding may in fact represent a shift in the dynamic timescale. This would imply that the dominant timescale for motions in apoABP is slower than \u223c\u00a015\u00a0ns, but that this shifts to a much faster timescale on ligand binding. There are two reasons to discount this interpretation of our data. Firstly, we consider it most likely that a timescale shift of the type considered here would result in a shift from predominantly simple Lipari\u2013Szabo models in the apo-protein to a preponderance of complex models in the ligand-bound form, as new observable motional modes are introduced by the change in timescale. In fact, precisely the opposite trend is observed (Table 2). Secondly, we note that there is no evidence of extensive slow timescale motions in our molecular dynamics studies. The presence of dynamics on this timescale would be reflected by inadequate sampling over the course of our 20\u00a0ns trajectories. In fact, using the convergence test proposed by Best et al.,29 the calculated order parameter is judged to have converged in all but nine residues in apo ABP, and in all but four residues in the ABP\u2013galactose complex. All residues for which dynamics are not converged are in flexible loops in ABP.\nTo understand the thermodynamic implications of the observed changes in dynamics, we exploit the relationship between Lipari\u2013Szabo order parameter (S2LZ) and conformational entropy (Sconf) derived by Yang and Kay.26 Of the 306 residues of ABP, we determined an entropy change associated with the measured change in pico- to nanosecond dynamics for 84 residues. Entropy change for the remaining residues could not be determined either because S2LZ could not be determined reliably for both the apo and ligand-bound states (203 residues), or because S2LZ\u00a0>\u00a00.95 in one or both states (19 residues). These data are plotted in Figure 5. The average entropy change per residue is 2.0(\u00b1\u00a00.3) J\/mol K. This amounts to an overall entropy change of 170(\u00b1\u00a030) J\/ mol K for the measured residues (T\u0394Sconf\u00a0=\u00a052(\u00b1\u00a09) kJ\/mol at 308 K). If it is assumed that this average entropy can be applied to all un-measured residues, and that dynamics changes for each residue are not correlated, the total entropy change from changes to pico- to nanosecond motion on ligand binding amounts to 610(\u00b1\u00a0120) J\/mol K (T\u0394Sconf\u00a0=\u00a0188(\u00b1\u00a037) kJ\/mol at 308 K). Clearly, this latter value amounts to an overestimate, as the assumption of un-correlated motion is not likely to hold for all residues of the protein. It is evident, however, that the entropy change associated with the change in pico- to nanosecond dynamics contributes significantly to the favourable free energy of binding, and in effect amounts to protein dynamics paying some of the unfavourable entropic cost of ligand binding.\nThe experimental data available for this system is limited to probes of backbone dynamics. Given the good agreement between experiment and the molecular dynamics simulations, it is perhaps reasonable to infer something of the side-chain dynamics from these simulations. We observe considerably more variability in side-chain order parameters than is evident for the backbone, consistent with findings in other proteins. Furthermore, there is much greater variability in the change in order parameter observed upon ligand binding. Despite this, the changes in side-chain dynamics are broadly similar to those seen for the backbone, with the majority of residues showing a small increase in flexibility on ligand binding. As fast side-chain dynamics are correlated with local backbone dynamics only weakly, this suggests an additional source of favourable entropy change accompanying binding. A number of residues around the binding site show larger changes in dynamics on binding, reflective of both increases and decreases in flexibility. These changes reflect similar heterogeneous dynamic changes seen in the protein backbone of the loops that comprise the binding site.\nOne particularly surprising aspect of these results is the dispersed and approximately uniform nature of the change in dynamics. This is unexpected, because fast motions in proteins, such as the pico- to nanosecond dynamics under study here, are almost exclusively local in character, with few if any correlations over distances longer than a few \u00e5ngstr\u00f6m units.30 One plausible explanation for the uniform nature of the observed effect is that it is an artefact arising from a systematic error in the determined rotational diffusion tensor for the apo and\/or holo protein. To exclude this possibility, we have repeated the analysis using the approach described by Schurr et al., which fits the relaxation data for each residue individually, with a so-called local \u03c4m term to account for rotational diffusion.33 In this analysis, the sets of dynamic parameters determined for each residue are statistically independent of one another, and are independent of any structural model for the protein. We find no significant difference between order parameters derived from this analysis and those obtained from the conventional analysis, where rotational diffusion is treated as a global parameter in the form of an anisotropic diffusion tensor. Furthermore, the local \u03c4m values determined in this analysis are fully consistent with the diffusion tensor determined conventionally.\nWe conclude that the results described here are robust with respect to the models of protein structure and rotational diffusion used in the analysis. These results therefore indicate a coordinated global change in local dynamics, initiated by the localised event of ligand binding. The physical basis for such a change is not clear, but it is not without precedent in the literature: several studies have identified favourable changes in pico- to nanosecond backbone dynamics on ligand binding to be similarly dispersed throughout the protein.34\u201336 Other studies have identified broadly dispersed changes in dynamics of contrasting sign, such that favourable changes in one part of a protein appear to compensate for unfavourable dynamic changes elsewhere in the protein.37,38\nIt has been appreciated for some time that protein dynamics might potentially mediate aspects of protein function including allosteric regulation39 and catalysis.40 Experimental evidence for such a functional role, principally from NMR spectroscopy, has been accumulating.41,42 These results raise the possibility that proteins may be adapted evolutionarily to exploit internal dynamic processes to functional ends. The results described here, together with previous observations,34\u201338 suggest that proteins may have evolved finely tuned networks of dispersed dynamic interactions that regulate the thermodynamics of protein\u2013ligand interactions. We are actively exploring the mechanism of this phenomenon in ABP, and the contribution of other degrees of freedom to the binding entropy, particularly ligand and protein side-chain dynamics.\nMaterials and Methods\nProtein expression and purification\nABP was expressed as essentially as described,6,20 except that the Escherichia coli host strain was BL21(DE3), and the growth medium was M-9 minimal medium, supplemented with BME vitamins (Sigma). For the production of 15N-labelled protein, 15NH4Cl was the sole nitrogen source, and for production of 50% 2H, 13C, 15N protein, the medium was 50% 2H2O and the carbon and nitrogen sources were [U-13C, 50% 2H]glucose and 15NH4Cl, respectively. Purification was as described.6,20\nNMR spectroscopy\nAll NMR samples contained approximately 1\u00a0mM ABP in 20\u00a0mM potassium phosphate (pH 7.0), 3\u00a0mM sodium azide, 0.1\u00a0mM EDTA, 10% 2H2O. For studies of the complex of ABP with galactose, the sample contained approximately 5\u00a0mM d-galactose. Except where noted, NMR experiments were performed at 308 K on Varian INOVA spectrometers operating at 500 MHz, 600 MHz or 750 MHz 1H frequency and equipped with room-temperature triple-resonance z-gradient probes. The transverse relaxation optimized spectroscopy (TROSY)-HNCA of apoABP was acquired at 600 MHz with 16 transients and 1024, 60 and 40 complex points and spectral windows of 7510, 2800 and 4000\u00a0Hz in the 1H, 15N, and 13C dimensions, respectively.\nRDCs were measured at 303 K and 750 MHz using 3.5% (w\/v) C5E12 alkyl-poly(ethylene glycol)\/hexanol (1:0.96 molar ratio)43 as an alignment medium. 1H-15N one-bond couplings in the presence and in the absence of the alignment medium were measured using the 1JNH modulated HSQC described by Tjandra et al.44 The dephasing delay was varied in 1\u00a0ms steps from 6\u00a0ms to 20\u00a0ms, with two points duplicated for error estimation. Each increment was acquired with 40 transients, 1024\u00a0\u00d7\u00a0128 complex points and spectral windows of 6500\u00a0\u00d7\u00a03200\u00a0Hz. Residues for which fewer than two zero-crossings could be clearly identified were excluded from further analysis. Uncertainty in the measured couplings was estimated using multiple fits using a bootstrap resampling procedure.45\nBackbone amide relaxation parameters (15N R1 and R2, and the 1H-15N heteronuclear NOE) were measured using the pulse sequences reported by Farrow et al.46 Typical relaxation delays were 10.7\u00a0ms, 53.3\u00a0ms, 107\u00a0ms, 213\u00a0ms, 426\u00a0ms, 640\u00a0ms, 906\u00a0ms, 1230\u00a0ms, 1600\u00a0ms and 2130\u00a0ms for R1 and 12.4\u00a0ms, 16.6\u00a0ms, 24.8\u00a0ms, 33.1\u00a0ms, 49.7\u00a0ms, 66.2\u00a0ms, 82.8\u00a0ms, 99.4\u00a0ms and 133\u00a0ms for R2. Duplicate measurements were made for at least two points in each series for estimation of experimental error. In the R2 experiment, dummy Carr\u2013Purcell\u2013Meiboom\u2013Gill sequences were applied before the recovery delay to compensate for sample heating caused by radio-frequency pulses, and all experimental series were acquired fully interleaved. Typically, 32 transients were acquired for R1 and R2 measurements, and 64 for the NOE, giving a total experimental time of approximately 60\u00a0h for the three experiments at each field. Measurements were made at 500 MHz, 600 MHz and 750 MHz for apo ABP, and at 500 MHz and 600 MHz for the ABP\u2013galactose complex.\nSpectral assignment\nResonance assignments for the ABP\u2013galactose complex have been determined.20 Many peaks in the 1H-15N HSQC show small chemical shift changes between the apo and complexed states, permitting partial apoABP assignments to be determined on the basis of those for the complex. For assignment of 1H-15N HSQC resonances that do move on binding ligand, a titration of ABP with 1-deoxygalactose was used. 1-deoxygalactose binds ABP with an affinity four orders of magnitude less than that of galactose, and is in the fast-exchange regime. Assignments for a number of resonances remained ambiguous at this stage, owing to substantial crowding of the 1H-15N HSQC spectrum. These remaining ambiguities were largely resolved by means of i to i\u20131 connectivities derived from an HNCA spectrum of apoABP.\nDomain orientation of apoABP\nABP is composed of two structural domains, the N domain (residues 1\u2013103 and 257\u2013277), and the C domain (residues 110\u2013253 and 286\u2013306). The remaining residues form a three-stranded linker between the domains, which retains some flexibility in the absence of ligand.21,22 The relative orientation of the two domains of apoABP was determined using 1H-15N RDCs. An initial estimate of the tensor describing molecular alignment of apoABP in 3.5% (v\/v) C5E12\/hexanol was obtained from the histogram of experimental RDCs.47 A starting structure was derived from the structure of ABP in complex with galactose (PDB ID 5ABP) by Powell minimisation of each domain individually against the measured RDCs.9 From this structure, domain orientation is determined by rigid-body\/torsion angle simulated annealing in which both domains are held rigid. All calculations were performed using the IVM module of Xplor-NIH.48 The annealing protocol comprised an initial 3 ps\/1000 steps of dynamics at 2000 K, after which the temperature was reduced to 25 K in 12.5 K steps, with 0.3 ps\/100 steps of dynamics performed at each temperature. Default Xplor-NIH parameters were used throughout, and force constants were ramped using default values. Square-well potentials were used for all RDC constraints.\nLipari\u2013Szabo analysis of relaxation data\nBackbone amide relaxation parameters were analysed in terms of the extended Lipari\u2013Szabo formalism,24,25 using the software relax.49,50 The 15N CSA was set at \u2212170\u00a0ppm and the effective N\u2013H bond length was 1.02\u00a0\u00c5. Initial estimates for the rotational diffusion tensor of both apoABP and the ABP\u2013galactose complex were obtained from the ratio of longitudinal and transverse relaxation rates.51 Residues that experience complex internal motion or Rex contribution were identified as described and excluded from this diffusion tensor estimation.52 For ABP\u2013galactose, an axially symmetric diffusion tensor produces an optimal fit, while the fit to the apo ABP data is improved significantly by the use of a fully anisotropic diffusion tensor. Similar results were obtained using data collected at each field strength.\nUsing these estimates, parameters of the extended model-free formalism were optimised for each residue individually, and the best parameter set identified by AIC model selection.49 All parameters, including the diffusion tensor, were then optimised. This process was repeated until the solution converged. The final optimised diffusion tensors are presented in Table 1, and the final dynamic parameter sets in Table 2.\nAdditional analyses were performed as described.33 Here, the contribution to relaxation due to overall rotational diffusion is treated as a mono-exponential component to the correlation function, with a time constant determined individually for each residue; the so-called local \u03c4m. In this analysis, the dynamic parameters and local \u03c4m for each residue are optimised individually, and the optimal parameter set selected by AIC.49 We did not include chemical exchange (Rex) contributions to R2 in this analysis, owing to the tendency for un-physically low local \u03c4m values to be compensated by artefactual Rex contributions and low order parameters, particularly in the case of the ABP\u2013galactose complex, where only two R2 values are available for each residue.\nChanges in conformational entropy associated with the observed changes in Lipari\u2013Szabo order parameters were determined using the relationship described by Yang and Kay.26 This relationship is essentially independent of motional model for S2LZ\u00a0\u2264\u00a00.95, so only residues consistent with this criterion in both apoABP and the complex have been included in considerations of entropy change. Uncertainty in the calculated entropy change deriving from experimental uncertainty in order parameters was determined by standard error propagation methods.\nMolecular dynamics\nThe simulations were carried out using AMBER 8,53 with the Cornell et al. force field.54 Initial coordinates of both holo and apo forms were based on the crystal structure of the ABP\u2013galactose complex (PDB code 5ABP).9 The structures were processed by the XleaP module of AMBER, and the hydrogen atoms were added to the system. The structure of \u03b2-d-galactopyranose was optimised ab initio using the Gaussian 98 program\u2020 with the HF\/6-31G* basis set, and RESP charges were generated and fitted.55 The ligand molecule was parameterised using GLYCAM force field.56 The models were subjected to short (1000 cycles) energy minimisation.\nProtein models were then immersed in periodic TIP3P water boxes. Approximately 5500 water molecules were added to each system. Simulations were carried out under NPT conditions at 300 K, using the particle mesh Ewald technique with 12\u00a0\u00c5 non-bonded cutoff and 2 fs time-step.57 SHAKE constraints with a tolerance of 10\u2212\u00a08 \u00c5 were applied to all hydrogen atoms during MD simulations to eliminate the fastest X-H vibrations and allow a longer simulation time-step. Translational and rotational centre-of-mass motion was removed every 10 ps. Equilibration started by 20 000 cycles of energy minimisation, with the atomic positions of the solute molecule restrained. It was followed by 100 ps of MD simulations, where the system was heated gently to 300 K and the constraints were released gradually (from 100 kcal\/(mol\u00b7\u00c52) applied initially). The further equilibration took 4.9\u00a0ns.\nThe production period took 20\u00a0ns for both systems. The coordinates were saved every 2 ps of MD simulation.\nGeneralised order parameters were calculated from the trajectory of individual back-bone amide bond vectors as:27where x, y and z are components of a unit vector along the amide bond, and angular brackets denote the time-average over the trajectory. Convergence of the dynamics of interest is tested using the approach described by Best et al.:29 a cumulative time function S2LZ(\u03c4) is defined using equation (1), with the time averages taken from t\u00a0=\u00a00 to t\u00a0=\u00a0\u03c4. This function was evaluated for 100 equally spaced time-points across the trajectory. The trajectory was deemed to have converged if the difference between the maximum and minimum values of this function over the final 50 time-points (i.e. the final 10\u00a0ns of the trajectory) was less than 0.05. Any residue judged not to have converged was excluded from the analysis.","keyphrases":["nmr relaxation","molecular dynamics","ligand binding","thermodynamics","periplasmic binding protein","abp, arabinose-binding protein","hsqc, heteronuclear single quantum coherence","rdc, residual dipolar coupling"],"prmu":["P","P","P","P","P","R","R","R"]}
{"id":"Extremophiles-4-1-2262144","title":"Metabolism of halophilic archaea\n","text":"In spite of their common hypersaline environment, halophilic archaea are surprisingly different in their nutritional demands and metabolic pathways. The metabolic diversity of halophilic archaea was investigated at the genomic level through systematic metabolic reconstruction and comparative analysis of four completely sequenced species: Halobacterium salinarum, Haloarcula marismortui, Haloquadratum walsbyi, and the haloalkaliphile Natronomonas pharaonis. The comparative study reveals different sets of enzyme genes amongst halophilic archaea, e.g. in glycerol degradation, pentose metabolism, and folate synthesis. The carefully assessed metabolic data represent a reliable resource for future system biology approaches as it also links to current experimental data on (halo)archaea from the literature.\nIntroduction\nExtremely halophilic archaea are a diverse group of euryarchaeota that inhabit hypersaline environments (3\u20135\u00a0M) such as salt lakes, salt ponds, and marine salterns. They are often referred to as \u201chalobacteria,\u201d named after the model organism Halobacterium salinarum, whose proton pump bacteriorhodopsin is one of the best-studied membrane proteins. Although haloarchaea share certain features in order to adapt to their extreme environment, i.e. acidic protein machineries, respiratory chains and rhodopsins, their metabolism is considerably different from each other. Although there are carbohydrate-utilizing species such as Haloferax mediterranei, Haloarcula marismortui, and Halococcus saccharolyticus, which catabolize hexoses (glucose, fructose), pentoses (arabinose, xylulose), sucrose, and lactose (Rawal et al. 1988; Altekar and Rangaswamy 1992; Johnsen et al. 2001; Johnsen and Schonheit 2004), other haloarchaea like H. salinarum are not capable of sugar degradation (Rawal et al. 1988). Instead, non-carbohydrate-utilizing species thrive on amino acids and typical compounds of hypersaline habitats. Haloferax volcanii, for example, is able to grow on glycerol and organic acids (Kauri et al. 1990) excreted by primary halophilic producers Dunaliella salina (Elevi Bardavid et al. 2006) and Microcoleus chthonoplastes (Zviagintseva et al. 1995), respectively. Haloarchaea differ not only in their catabolic pathways but also in their nutritional requirements. While simple growth media were described for Haloferax volcanii (Kauri et al. 1990) and Natronomonas pharaonis (Falb et al. 2005), H. salinarum exhibits complex nutritional demands. Growth of Halobacterium cells is often limited in synthetic media (Oesterhelt and Krippahl 1973; Grey and Fitt 1976), in spite of rich amino acid (at least 10 amino acids) and cofactor supplements (folate, biotin, thiamine).\nThe metabolic diversity of halophilic archaea has not yet been investigated at the genomic level by metabolic reconstruction and comparative analysis. The absence of enzyme genes for numerous metabolic reactions in archaeal genomes has limited reconstruction of metabolic pathways so far. However, many of these pathway gaps have been elucidated recently with the discovery of novel non-orthologous enzymes in archaea, e.g. for the de novo synthesis of mevalonate (Barkley et al. 2004; Grochowski et al. 2006b), purines (Graupner et al. 2002; Ownby et al. 2005), and cobamide (Woodson et al. 2003; Woodson and Escalante-Semerena 2004; Zayas et al. 2006). Archaea also employ novel enzymes and precursors for pentose formation (Grochowski et al. 2005) and aromatic amino acid synthesis (White 2004; Porat et al. 2006) circumventing absent enzymes of the classical pentose-phosphate pathway.\nFor this detailed review of haloarchaeal metabolism, metabolic pathways of halophilic archaea were systematically reconstructed. Currently, genome sequences of four diverse haloarchaeal species are available for comparative analysis (Table\u00a01), namely that of the model organism H. salinarum (Ng et al. 2000; Pfeiffer et al. 2008; http:\/\/www.halolex.mpg.de), the metabolic-versatile H. marismortui (Baliga et al. 2004), the haloalkaliphile N. pharaonis (Falb et al. 2005), and the square-shaped Haloquadratum walsbyi (Bolhuis et al. 2006). The presented metabolic data will be a valuable resource for future system biology approaches, as each metabolic reaction has been carefully assessed and linked to experimental data from the literature.\nTable\u00a01Overview of the currently sequenced haloarchaeaH. salinarum strains NRC-1\/R1aH. marismortuiH. walsbyiN. pharaonisGene identifierVNG\/OErrn, pNGHQNPSalt optimum [M]4\u20135\u00a0M4.5\u00a0M3.3\u00a0M3.5\u00a0M (pH 8.5)IsolationSalted fishDead Sea (Israel)Solar saltern (Spain)Soda lake (Egypt)Main research interestsRhodopsins, signal transductionVersatile nitrogen metabolismSquare-shaped cells, halomucinHaloalkaliphilicity, respiratory chainGenome size [Mb]2.61\/2.724.373.242.80# Plasmids2\/48 (incl. CHRII)12%GC chromosome68.062.447.963.4rRNA operons1321Fla genes (motility)YesYesNoYes# Transducer genes1821 (18)019# Rhodopsin genes (bop, hop, sop)4 (1,1,2)6 (3,1,2)3 (2,1,0)2 (0,1,1)aTwo strains of H. salinarum have been sequenced, strains NRC-1 (Ng et al. 2000) and R1 (Pfeiffer et al. 2008; http:\/\/www.halolex.mpg.de). These are virtually identical and differ only in their distribution of insertion elements and their plasmid arrangements. Unless mentioned otherwise, the two strains are not distinguished in this review, because they exhibit analogous sets of enzyme encoding genes. Gene identifiers of H. salinarum strain R1 (e.g. OE1001F) will be used throughout the text\nMaterials and methods\nMetabolic reconstruction procedure\nMetabolic pathways of H. salinarum were reconstructed in a two-step procedure. At first, relevant reactions that take part in a given metabolic pathway were chosen from the complete set of reference reactions downloaded from the KEGG ligand database (Kanehisa et al. 2004). In the second step, organism-specific metabolic pathways were reconstructed by flagging each reaction existent or non-existent in H. salinarum with a certain confidence value. The enzyme gene predicted to catalyze this reaction in H. salinarum was linked to the reaction. In case experimental data is available from the literature, a comment was linked to the reaction or enzyme gene, which includes the PubMed identifier of the external reference. Each reaction was manually assessed based on automatic enzyme assignments derived from various similarity searches (i.e. blast search with UniProt enzymes, COG assignments, Pfam search), genome data (http:\/\/www.halolex.mpg.de), and experimental literature data for H. salinarum. In case, a reaction or enzyme is confirmed by experiments but no genetic evidence was found in the genome (indicating novel yet unknown enzyme variants), the reaction was marked existent and the conflict was labeled. The metabolic data for H. salinarum given in Supplementary Material S1 was internally stored in a MySQL database and managed via a Web-interface.\nComparative analysis of the complete haloarchaeal gene sets\nFor comparison of haloarchaeal gene sets, protein databases (fasta format) of the four completely sequenced haloarchaeal genomes, H. salinarum strain R1, N. pharaonis, H. walsbyi, and H. marismortui, were blasted against each other and against the non-redundant (nr) database. For example, each predicted protein sequence of N. pharaonis (species 1) was searched against the H. salinarum database (species 2) and the nr database (all species), then the difference between the best blast score for the general database (nr) and for the related species H. salinarum was calculated. Listing N. pharaonis sequences by these differences in descending order detects genes present in N. pharaonis but absent in H. salinarum. In few cases, the N. pharaonis gene had a close homolog in the general database but only a more distant homolog in H. salinarum. Such a gene is considered present in H. salinarum when the blast hit is highly significant (E-value better than e\u221220) were considered. The differential blast analysis routine was applied to all combinations of the set of four haloarchaeal genomes. A list of genes present in only a subset of genomes was obtained and filtered for metabolic function (Supplementary Material S2).\nResults and discussion\nThe metabolism of H. salinarum was reconstructed by evaluating metabolic reactions of biochemical reference pathways from KEGG (Supplementary Material S1). Enzyme annotations from an automatic assignment routine and from curated genome data (http:\/\/www.halolex.mpg.de) were carefully assessed in order to avoid misassignments due to domain rearrangements (e.g. purine biosynthesis, Fig.\u00a02) or overannotation of the exact substrate specifity (e.g. 2-oxoacid dehydrogenase complex). Novel enzyme variants closing former pathway gaps in archaea as well as previously published experimental data of H. salinarum were also considered throughout the reconstruction process. Comparative analysis of the currently sequenced haloarchaea, H. marismortui, H. walsbyi, N. pharaonis, and H. salinarum, disclosed major metabolic differences, i.e. alternative enzymes and metabolic pathways employed by haloarchaea (Supplementary Material S2).\nIn the following, reconstructed metabolic pathways of halophilic archaea will be reviewed in detail with emphasis on different enzyme genes and metabolic pathways, which lead to the diverse catabolic and anabolic capabilities of halophilic archaea. In case an enzyme gene is present in all four haloarchaeal species, only the N. pharaonis identifier (e.g. NP1002A) will be exemplarily mentioned.\nGlycolytic pathways\nMajor differences in sugar degradation capabilities were identified amongst halophilic archaea that reflect the previous classification of halophilic archaea into carbohydrate- and non-carbohydrate utilizers (Fig.\u00a01, Supplementary Material S2). H. salinarum, H. marismortui, and H. walsbyi possess all required enzyme genes for the semi-phosphorylated Entner-Douderoff (ED) pathway previously described for carbohydrate-utilizing haloarchaea (Rawal et al. 1988; Danson and Hough 1992; Verhees et al. 2003). However, only H. marismortui and H. walsbyi encode a classical KDPG aldolase, which is the key enzyme of the pathway, in addition to the novel archaeal KD(P)G aldolase recently described for thermophilic archaea (Ahmed et al. 2005). Thus, operation of different ED pathway variants between H. salinarum and the other two species is indicated. It should further be noted that neither the ED pathway nor growth on glucose as the sole carbon source has been established for Halobacterium so far (Gochnauer and Kushner 1969; Rawal et al. 1988). N. pharaonis is likely to be incapable of glucose degradation, because it completely lacks all enzyme genes of the ED pathway.\nFig.\u00a01The central intermediary metabolism in halophilic archaea. The reaction arrows depict the reconstructed metabolism of the reference species H. salinarum (green reaction exists, red reaction absent). The four geometric symbols illustrate differences in enzyme gene sets between the four sequenced haloarchaea (square: H. marismortui, circle: H. walsbyi, diamond: N. pharaonis, triangle: H. salinarum, green gene exists, red gene absent). Reactions that have been investigated experimentally through NMR studies or enzyme activity tests in H. salinarum are highlighted by bold arrows (green reaction exists, red reaction absent). For some of the experimentally verified reactions, there are currently no genetic evidences in the H. salinarum genome (green arrows with red border). Vice versa, some reactions have been experimentally excluded, but probable enzyme genes are present in the H. salinarum genome (red arrows with green border). Compounds that have been identified through labeling studies are marked by asterisks. Proposed essential amino acids for H. salinarum are indicated (E). Compounds: AraHex\u2014d-arabino-3-hexulose-6P, Ery4P\u2014erythrose-4P, Frc\u2014fructose, GAP\u2014glyceraldehyde-3P, Glc\u2014glucose, Gluc\u2014gluconate, Glyn\u2014glycerone, Glyc\u2014glycerol, Glyox\u2014glyoxylate, Icit\u2014isocitrate, KDPG\u20142-dehydro-3-deoxy-6-phosphogluconate, Mal\u2014malate, OA\u2014oxalacetate, 2-OG\u20142-oxoglutarate, PEP\u2014phosphoenolpyruvate, PGA\u20143-phosphoglycerate, Pyr\u2014pyruvate, Rib5P\u2014ribose-5P, Ribul5P\u2014ribulose-5P, Suc\u2014succinate, Xyl5P\u2014xylulose-5P, Sed7P\u2014sedoheptulose 7-phosphate, AcCoA\u2014acetyl-CoA\nThe carbohydrate-utilizing H. marismortui has acquired a wide range of enzyme genes involved in the uptake and degradation for various sugars. Fructose and sucrose are likely degraded via the modified Embden-Meyerhof (EM) pathway, which has been described for haloarchaea (Altekar and Rangaswamy 1992). The pathway involves the key enzyme 1-phosphofructokinase (EC\u00a02.7.1.56, rrnAC0342) and sucrose 6-phosphate hydrolase (EC\u00a03.2.1.26, rrnAC1479). Haloarcula also contains probable gene clusters for maltose uptake (maltose ABC transporter, rrnAC2346-rrnAC2349) and metabolism (several \u03b1-glucosidases, e.g. rrnAC0224) as well as a gene cluster for D-xylulose oxidation (rrnAC3032\u2013rrnAC3039). The latter encodes the previously characterized D-xylulose dehydrogenase (rrnAC3034) (Johnsen and Schonheit 2004).\nEmbden-Meyerhof pathway and gluconeogenesis\nConsistent with the previous biochemical findings (Rawal et al. 1988; Altekar and Rangaswamy 1992), genes for 6-phosphofructokinase (EC 2.7.1.11), the key enzyme of the classical Embden-Meyerhof pathway, are absent in all four haloarchaeal genomes. Alternative archaeal types of 6-phosphofructokinases that depend on ADP (Thermococcus, Pyrococcus) or PP(i) (Thermoproteus) (Kengen et al. 1994; Selig et al. 1997) as co-substrates have not been found either. However, a 1-phosphofructokinase gene for fructose degradation via the haloarchaeal variant of the EM pathway (Altekar and Rangaswamy 1992; Rangaswamy and Altekar 1994a, b) is present in H. marismortui (rrnAC0342). Although the upper, hexose part of the classical EM pathway is missing in haloarchaea, the lower, triose part of the pathway that leads to pyruvate is likely to be functional in all species, as required enzyme genes are encoded in their genomes. In fact, several enzyme activities have already been proven in H. salinarum (Rawal et al. 1988). The final glycolysis step from phosphoenolpyruvate to pyruvate might be catalyzed by pyruvate kinase (EC 2.7.1.40, e.g. NP1746A) and pyruvate, water dikinase (EC 2.7.9.2, e.g. NP1196A) in haloarchaea. In a thermophilic archaeon, both enzymes participated in glycolysis but only deletion of the pyruvate, water dikinase gene completely omitted growth on sugars (Imanaka et al. 2006).\nComplete gene sets for the reverse EM pathway (gluconeogenesis) from pyruvate to phosphorylated glucose were identified in all four haloarchaeal genomes. Consistently, labeling experiments (Sonawat et al. 1990; Ghosh and Sonawat 1998) and enzyme activity studies (Rawal et al. 1988) have confirmed gluconeogenesis in H. salinarum, which is required to synthesize hexoses for membrane constituents. For example, glucose was found to be incorporated into different sugar moieties (glucose, mannose, galactose) of halobacterial glycolipids (Weik et al. 1998). Furthermore, saccharide units are attached to surface proteins of H. salinarum such as the S-layer protein and flagellins (Sumper 1987). Sugar moieties of lipids and proteins are likely synthesized via nucleotide sugars. In accordance with this, several nucleotide sugar enzymes such as UDP-glucose 4-epimerase (EC 5.1.3.2, e.g. NP4662A) and UDP-glucose 6-dehydrogenase (EC 1.1.1.22, e.g. NP2322A, NP4668A) are present in haloarchaeal genomes.\nEntner-Douderoff pathway\nVariants of the classical Entner-Douderoff pathway are common in the archaeal domain of life (Danson and Hough 1992). Halophilic archaea such as H. vallismortis (Altekar and Rangaswamy 1992) operate the semi-phosphorylated pathway, where glucose is converted to 2-dehydro-3-deoxygluconate (KDG). This intermediate is then phosphorylated to KDPG and subsequently split into pyruvate and glyceraldehyde 3-phosphate. In Sulfolobus and Thermoplasma, a non-phosphorylated ED pathway has been described (in addition to the semi-phophorylated pathway variant), where KDG is cleaved without prior phosphorylation by a novel bifunctional KD(P)G aldolase (same COG0329 as dihydrodipicolinate synthase) (Ahmed et al. 2005). Orthologs for all genes of the characterized Sulfolobus ED pathway cluster (Ahmed et al. 2005; Kim and Lee 2005) were found in H. salinarum, H. marismortui and H. walsbyi, namely genes encoding d-gluconate dehydratase (EC 4.2.1.39, gnaD, COG4948, e.g. OE1664R), KDG kinase (EC 2.7.1.45, kdgK, COG0524, e.g. OE1266R), and KD(P)G aldolase (kdgA, COG0329, e.g. OE1665R). The ED pathway genes, except kdgK, are located in one gene cluster in the H. salinarum genome, which additionally contains a glucose 1-dehydrogenase gene (EC 1.1.1.47, e.g. OE1669F) (Bonete et al. 1996) as well as two conserved genes (e.g. OE1668R, OE1672F), which are candidates for the yet missing gluconolactonase (EC 3.1.1.17). The non-carbohydrate-utilizing strain N. pharaonis lacks all enzyme genes for the ED pathway.\nIn addition to their archaeal KD(P)G aldolase (rrnAC0960, HQ2365A), H. marismortui (rrnAC3121), H. walsbyi (HQ1495A), and Haloferax alicantei (AAB40121, co-located with kdgK, AAB40122) also encode the canonical bacterial-type KDPG aldolase (EC 4.1.2.14, kdgA, COG0800), which is absent in Halobacterium, Sulfolobus, and Thermoplasma species. This suggests operation of different ED pathway variants in the two (halo)archaeal groups, and, probably, a \u2018reduced\u2019 semi-phosphorylated ED pathway in H. salinarum, which needs to be investigated in future. So far, experiments with glucose-grown cells of H. salinarum have shown the conversion from glucose to gluconate (Sonawat et al. 1990; Bhaumik and Sonawat 1999), but have not detected subsequent reactions from gluconate to pyruvate and glyceraldehyde 3-phosphate when applying indirect enzyme activity tests (Rawal et al. 1988).\nOxidative pentose phosphate pathway\nThrough the oxidative pentose phosphate pathway, phosphorylated glucose is oxidized to gluconate 6-phosphate (C6) and then converted to ribulose 5-phosphate (C5) by oxidative decarboxylation. In the archaeal domain of life, the oxidative PP pathway seems not to exist, because enzyme genes for this pathway are absent in archaeal genomes. However, haloarchaea show glucose 6-phosphate dehydrogenase activity (EC 1.1.1.49) in spite of lacking the respective enzyme gene (Aitken and Brown 1969) and encode orthologs of 6-phosphogluconate dehydrogenase (EC 1.1.1.44, COG1023, e.g. NP0286A). Thus, an operative, albeit modified oxidative PP pathway is indicated for haloarchaea.\nPentose metabolism\nIn bacteria, pentoses are synthesized via the non-oxidative part of the PP pathway, where fructose 6-phosphate (C6) and glyceraldehyde 3-phosphate (C3) are converted to ribulose 5-phosphate (C5) in a complex five-step pathway. The enzyme gene set for this pathway, consisting of transaldolase\u00a01 and 2 (EC 2.1.2.1), transketolase (EC\u00a02.2.1.1), and ribulose-phosphate 3-epimerase (EC 5.1.3.1), is absent in most archaea except Methanococcus jannaschii and Thermoplasma spp. (Soderberg 2005). Individual genes encoding ribose 5-phosphate isomerase (EC\u00a05.3.1.6, e.g. NP0786A) for the conversion of ribose 5-phosphate to ribulose 5-phosphate are present in archaeal genomes.\nAlthough Methanocaldococcus jannaschii encodes all enzyme genes for an operative non-oxidative PP pathway, relevant intermediates (i.e. erythrose 4-phosphate, xylose 5-phosphate, sedoheptulose 7-phosphate) are absent in its cells (Grochowski et al. 2005). Instead, ribulose 5-phosphate is exclusively produced through the ribulose monophosphate (RuMP) pathway in M. jannaschii. This pathway is commonly employed for formaldehyde fixation and detoxification in bacteria but operates in reverse mode in archaea and, thus, substitutes for the classical non-oxidative PP pathway (Orita et al. 2006; Grochowski et al. 2005). The RuMP pathway converts fructose 6-phosphate to d-arabino-3-hexulose 6-phosphate and then to formaldehyde and ribulose 5-phosphate involving 6-phospho-3-hexuloisomerase (COG0794) and 3-hexulose-6-phosphate synthase (COG0269). These enzymes are encoded in all archaea except Thermoplasma and haloarchaea, which presumably operate the non-oxidative and a modified oxidative PP pathway for pentose formation, respectively (Soderberg 2005).\nAbsence of the non-oxidative branch of the PP pathway in most archaea also results in the absence of the pathway intermediate erythrose 4-phosphate (C4), which is the precursor of aromatic amino acids in bacteria. While a group of archaea (e.g. Pyrococcus abyssi) encodes transketolase required for erythrose 4-phosphate synthesis, many archaea, amongst them haloarchaea, lack transketolase. These archaea operate an alternative pathway for aromatic amino acid synthesis starting from different precursors, 6-deoxy-5-ketofructose-1-phosphate (DKFP) and l-aspartate semialdehyde (see below) (White 2004).\nThe key enzyme of the reductive branch of the PP pathway for CO2 fixation is ribulose-bisphosphate carboxylase (EC 4.1.1.39, RuBisCO). RuBisCO activity was previously detected in haloarchaea such as H. mediterranei but not in H. salinarum (Rawal et al. 1988; Rajagopalan and Altekar 1994). The CO2 acceptor and substrate of RuBisCO, ribulose 1,5-bisphosphate is not directly synthesized from ribulose 5-phosphate in archaea, but from the purine precursor 5-phospho-d-ribosyl-1-pyrophosphate (PRPP) (Finn and Tabita 2004). The required gene for this conversion is present in all haloarchaea (e.g. NP5174A), while RuBisCO (NP2770A) is only encoded in the N. pharaonis genome. Recently, it has been shown that purine and pentose metabolism are connected in archaea, as archaeal type\u00a0III RuBisCO is involved in an AMP recycling pathway that is present in N. pharaonis (see below) (Sato et al. 2007). This novel pathway might be part of a cyclic CO2 fixation pathway in archaea consisting of (i) pentose formation (PRPP) from fructose 6-phosphate (e.g. via the RuMP pathway), (ii) conversion of PRPP and adenine to AMP by adenine phosphoribosyltransferase (EC\u00a02.4.2.7, e.g. NP1254A, NP1426A), (iii) AMP recycling releasing adenine and 3-phosphoglycerate (and involving the CO2 fixation step), and (iv) gluconeogenesis for the conversion of 3-phosphoglycerate back to fructose 6-phosphate (Sato et al. 2007).\nGlycerol metabolism\nIn many hypersaline habitats, glycerol is a highly abundant carbon source that is produced by the halotolerant green algae Dunaliella to protect itself from osmotic pressure (Borowitzka et al. 1977; Phadwal and Singh 2003). Haloarchaea are able to catabolize the abundant glycerol through phosphorylation to glycerol 3-phosphate and subsequent formation of dihydroxyacetone phosphate (DHAP) (Rawal et al. 1988; Nishihara et al. 1999). Consistently, haloarchaeal genomes encode glycerol kinase (EC 2.7.1.30, e.g. OE3762R), the multi-subunit glycerol 3-phosphate dehydrogenase (EC\u00a01.1.99.5, e.g. OE3763F-OE3765F) and a potential sn-glycerol-3-phosphate ABC transport system (e.g. OE5166F-OE5170F). The haloalkaliphile N. pharaonis lacks glycerol degrading enzymes, which might be due to its soda lake habitat, where Dunaliella is not a main primary producer. It should be noted that glycerol degradation and lipid formation occur through separate pathways in archaea, meaning that the intermediate glycerol 3-phosphate derived from glycerol catabolism is not used for the synthesis of the glycerophosphate backbone of archaeal lipids (see below) (Nishihara et al. 1999).\nIn H. salinarum, glycerol can also be converted to DHA by glycerol dehydrogenase (Rawal et al. 1988). For this reaction, a plasmid-encoded glycerol dehydrogenase (EC\u00a01.1.1.6, OE5160F) is employed, which has been characterized and structurally elucidated (Offermann 2003; Horn 2006). The produced DHA might be phosphorylated by DHA kinase (EC 2.7.1.29) and fed into the lower EM pathway. However, potential DHA kinases genes (HQ2672A, HQ2673A) are only encoded by H. walsbyi, where they probably depend on a cytosolic phosphoenolpyruvate-dependent phosphotransferase system (HQ2709A) (Bolhuis et al. 2006).\nPyruvate metabolism and tricarboxylic acid cycle\nPyruvate metabolism\nThe central metabolite pyruvate is converted to acetyl-CoA by pyruvate-ferredoxin oxidoreductase (EC 1.2.7.1, porAB, OE2623R\/OE2622R) (Kerscher and Oesterhelt 1981a, b) and subsequently fed into the tricarboxylic acid (TCA) cycle. NMR spectroscopy experiments for H. salinarum have shown that 90% of the flux is channelled into the TCA cycle via pyruvate-ferredoxin oxidoreductase (Ghosh and Sonawat 1998; Bhaumik and Sonawat 1994). The remaining 10% of the pyruvate is converted to the TCA intermediate oxaloacetate by pyruvate carboxylase (EC 6.4.1.1), in order to fill up the oxaloacetate pool of the TCA cycle, when its intermediates are drawn off for biosynthetic purposes (Ghosh and Sonawat 1998). However, haloarchaea do not encode archaeal-type pyruvate carboxylase (Mukhopadhyay et al. 2000), but biotin carboxylases that are more likely to be involved in fatty acid degradation (e.g. NP4250A\/NP4252A located within a fatty acid degradation cluster NP4230A\u2013NP4258A). H. marismortui and H. walsbyi encode a phosphoenolpyruvate carboxylase (EC 4.1.1.31, rrnAC0562, HQ3197A), which has been proposed to be involved with a novel cytosolic phosphotransferase system (pNG7387-pNG7391, HQ1667A, HQ2709A) (Bolhuis et al. 2006). During gluconeogenesis, phosphoenolpyruvate is synthesized from oxaloacetate through malic enzyme (EC 1.1.1.38\/39\/40, e.g. NP0132A, NP1772A) and pyruvate, water dikinase (EC 2.7.9.1, e.g. NP1196A) in haloarchaea. The former anaplerotic enzyme has been shown to be active in H. salinarum, while the anaplerotic reactions catalyzed by phosphoenolpyruvate carboxykinase (EC 4.1.1.32\/38\/49) and oxaloacetate decarboxylase (EC 4.1.1.3) are missing in haloarchaea (Bhaumik and Sonawat 1994; Ghosh and Sonawat 1998).\nUnder anaerobic conditions, it has been shown for H. salinarum that pyruvate is primarly converted to alanine, presumably by an aspartate transaminase, and to a larger extent to lactate and acetate (Bhaumik and Sonawat 1994; Ghosh and Sonawat 1998). In spite of proven lactate dehydrogenase activity in H. salinarum cell extracts (Bhaumik and Sonawat 1994), no clear lactate dehydrogenase homolog has been identified in haloarchaeal genomes. In M. jannaschii, it has been shown that lactate is produced from lactaldehyde, which might be derived from methylglyoxal (Grochowski et al. 2006a). The M. jannaschii lactaldehyde dehydrogenase is similar to several probable aldehyde dehydrogenases encoded in haloarchaea (e.g. NP1686A, NP3020A).\nTricarboxylic acid cycle\nAlthough TCA cycles are highly variable and often incomplete within the archaeal domain of life (Huynen et al. 1999), haloarchaeal genomes encode the complete set of enzymes. For H. salinarum, activity of all these enzymes has been proven (Aitken and Brown 1969; Hubbard and Miller 1972; Kerscher and Oesterhelt 1981a, b; Gradin et al. 1985) and an operative TCA cycle has been shown by NMR spectroscopy (Ghosh and Sonawat 1998).\nIn haloarchaea, pyruvate and 2-oxoglutarate are not converted by classical 2-oxoacid dehydrogenase complexes but by 2-oxoacid-ferredoxin oxidoreductases encoded by porAB and korAB genes, respectively (Kerscher and Oesterhelt 1981a, b). However, haloarchaeal and other archaeal genomes further contain gene clusters encoding all components of a 2-oxoacid dehydrogenase complex. In Thermoplasma acidophilum, it has recently been shown that the E1 component of the encoded 2-oxoacid dehydrogenase complex accepts branched-chain 2-oxoacids (Heath et al. 2004). Most likely, the haloarchaeal 2-oxoacid dehydrogenase complex is also involved in branched-chain amino acid degradation.\nWhen grown on acetate, Haloferax volcanii operates a glyoxylate bypass operon involving isocitrate lyase (EC 4.1.3.1) and a new type of malate synthase (EC 4.1.3.2) (Serrano and Bonete 2001). Homologs of both enzymes are present in the N. pharaonis (NP4432A, NP4430A) and H. walsbyi (HQ1720A, HQ3094A) genomes, while only a probable malate synthase is encoded in H. marismortui (rrnAC1965). Halobacterium lacks both glyoxylate cycle genes, although activity of both glyoxylate cycle enzymes has been demonstrated previously in this haloarchaeon (Aitken and Brown 1969).\nNucleotide metabolism\nDe novo synthesis of nucleotides\nThe complete gene sets that are required for de novo synthesis of purines from ribose 5-phosphate and for de novo synthesis of pyrimidines from carbamoylphosphate and ribose 5-phosphate are present in haloarchaeal genomes. Haloarchaea reveal an unusual domain fusion pattern of purine synthesis enzymes (Fig.\u00a02), because they contain a unique fusion of GAR and AICAR transformylases (EC 2.1.2.2\/EC 2.1.2.3, purN\/purH, e.g. NP1662A, OE1620R). Haloarchaea do not encode the novel AICAR transformylase (purP) (Ownby et al. 2005) present in most archaea, but encode the novel archaeal variant of IMP cyclohydrolase (purO, e.g. NP0732A, OE4329F) (Graupner et al. 2002). The four haloarchaeal strains show only few differences in their nucleotide metabolism, namely in the occurrence of pyrimidine kinases (EC 2.7.1.21: OE3159R, HQ1795, EC 2.7.1.48: OE2749F) (Supplementary Material S2).\nFig.\u00a02Domain rearrangement of enzymes involved in the de novo synthesis of purines. The pathway comprises 10 steps from PRPP to IMP. Fusions of enzyme genes are indicated by linked boxes. Non-orthologous enzymes are known for steps 3, 9, and 10 (unfilled boxes) but further archaeal enzymes for purine synthesis are unknown (question marks)\nArchaeal type III RuBisCO functions in AMP metabolism\nRibulose-bisphosphate carboxylase (EC 4.1.1.39, RuBisCO) is a key enzyme for CO2 fixation via the Calvin-Benson-Bassham cycle in plants. However, RuBisCO was found to be involved in bacterial methionine cleavage and in the AMP metabolism of archaea (Sato et al. 2007). The latter pathway recycles the intracellular pool of AMP produced by ADP-dependent (AMP-forming) sugar kinases, which are involved in glycolytic pathways of archaea (Kengen et al. 1994; Selig et al. 1997). N. pharaonis is the only haloarchaeal species encoding all enzyme genes for this novel AMP recycling pathway, i.e. AMP phosphorylase (NP3958A), ribose-1,5-biphosphate isomerase (NP3202A), and RuBisCO (NP2770A), while H. salinarum exhibits only the isomerase gene (OE3610R) (Sato et al. 2007). N. pharaonis and some other archaea do not possess ADP-dependent sugar kinases that would produce AMP though. Instead, AMP recycling might be part of a cyclic pathway for CO2 fixation as described earlier.\nLipid metabolism\nMembrane lipids of archaea consist of glycerol diether lipids with prenyl side chains instead of diacylglycerol esters. Specifically, membranes of H. salinarum contain core lipids with two phytanyl side chains (C20), and to lesser extents also other isoprenoid constituents such as squalenes (C30), phytoenes (C40), menaquinones (C40), and dolichol (C60) (Oesterhelt 1976; Lechner et al. 1985; Kushwaha et al. 1976) (Fig.\u00a03). Furthermore, H. salinarum synthesizes several carotenoids from prenyl precursors, preferentially bacterioruberins (C50) and photoactive retinal (C20) (Oesterhelt 1976; Oesterhelt and Stoeckenius 1973). Retinal is incorporated into bacteriorhodopsin and other retinal proteins, which are unique to haloarchaea within the archaeal domain of life. Although fatty acids are not part of archaeal membrane lipids, small amounts of fatty acids (C14, C16, C18) have been detected in membrane proteins of H. salinarum (Pugh and Kates, 1994). Other haloarchaeal species likely possess similar membrane constituents as H. salinarum because they have the same gene set for the de novo synthesis of isoprenoids. However, specific prenyl-based compounds might vary from species to species, as in the case of diether core lipids found in haloalkaliphiles, e.g. N. pharaonis (C20\u2013C20 and C20\u2013C25 prenyl chains) (Tindall et al. 1984).\nFig.\u00a03Biosynthesis of isoprenoids in halophilic archaea. The isoprenoid precursor IPP is synthesized via the mevalonate pathway as shown by labeling studies (green reaction exists, red reaction absent, bold experimental verification). Various isoprenoids detected in membranes of H. salinarum (listed in boxes) are synthesized by a series of condensation reactions with IPP, which is added in head\u2013tail (HT) or head\u2013head (HH) fashion, and through desaturase reactions ([2H]). Enzyme gene sets for isoprenoid synthesis differ only slightly between halorarchaea (square: H.\u00a0marismortui, circle: H.\u00a0walsbyi, diamond: N. pharaonis, triangle: H.\u00a0salinarum, green gene exists, red gene absent). Bacterial- (B) or archaeal-type (A) enzyme variants are indicated. Superscript \u201ca\u201d indicates C5-prenyl units are synthesized via the mevalonate pathway starting from two acetyl-CoA molecules and a still unknown C2-unit arising from amino acid degradation (Ekiel et al. 1986)\nPrenyl side chains of membrane lipids and other isoprenoids are derived via the mevalonate pathway in haloarchaea (Ekiel et al. 1986), while the glycerophosphate backbone of membrane lipids is formed from glycerol 1-phosphate. This membrane precursor is derived from the glycolytic intermediate DHAP via glycerol-1-phosphate dehydrogenase (EC\u00a01.1.1.261, e.g. NP4492A), which is ubiquitously found in archaea (Nishihara et al. 1999).\nDe novo synthesis of isoprenoids (mevalonate pathway)\nLike other archaea, halophiles synthesize activated C5-units [dimethylallyl and isopentenyl diphosphate (IPP)] for polycondensation of prenyl chains via the mevalonate pathway (Fig.\u00a03). A previous comparative analysis of the mevalonate pathway (Smit and Mushegian 2000) identified gaps for three pathway steps in archaea, namely the lack of bacterial-type phosphomevalonate kinase (EC 2.7.4.2, COG3890), diphosphomevalonate decarboxylase (EC\u00a04.1.1.33, COG3407), and IPP isomerase (EC 5.3.3.2, COG1443). Recently, an alternative type II IPP isomerase has been identified in archaea, which belongs to the same COG1304 as lactate dehydrogenase (Barkley et al. 2004). Furthermore, it has been suggested that mevalonate phosphate is first decarboxylated and then phosphorylated to synthesize IPP, while phosphorylation of mevalonate phosphate precedes the decarboxylation step in bacteria (Grochowski et al. 2006b). The archaeal-specific reactions involve a predicted phosphomevalonate decarboxylase (COG1355) and a characterized isopentenyl phosphate kinase (COG1608).\nInterestingly, haloarchaea may operate a chimeric mevalonate pathway (Fig.\u00a03). Like other archaea, they encode isopentenyl phosphate kinase (e.g. NP2852A) instead of a bacterial phosphomevalonate kinase. However, haloarchaea lack the proposed archaeal phosphomevalonate decarboxylase gene, and, instead, encode a bacterial-type diphospho-mevalonate decarboxylase (e.g. NP1580A). Thus, neither the classical bacterial nor the proposed archaeal mevalonate pathway is complete. For the last mevalonate pathway step, only N. pharaonis and H. salinarum encode an archaeal-type II IPP isomerase (e.g. NP0360A), while all four haloarchaea possess a bacterial-type IPP isomerase (e.g. NP4826A). Future investigations are needed in order to clarify whether archaeal and bacterial enzymes are employed simultaneously in haloarchaea. The acquired bacterial enzymes might possibly have a higher substrate specificity or turnover for covering increased isoprenoid demands for retinal and bacterioruberin biosynthesis.\nA functional mevalonate pathway has been verified for H. salinarum by labeling studies (Ekiel et al. 1986), which lead to the proposal of an unusual first step. Lipid labeling patterns indicated that mevalonate is not synthesized from three activated acetate precursors but rather from two acetyl-CoA molecules and an unknown C2-unit. The latter is not derived from acetate but from degraded amino acids such as lysine. C5-isoprenoid precursors derived via the mevalonate pathway are elongated to trans- and cis-polyprenyl chains in head-to-tail fashion by (E)- and (Z)-prenyltransferases, respectively (E: NP3696A, NP4556A, NP0604A, Z: NP4550A, NP4544A). Exact chain-specificity of prenyltransferase orthologs needs to be determined experimentally, but the biosynthesis of dolichol and menaquinones found in H. salinarum requires prenyltransferases with long-chain specifities. Potential enzymes for squalene and phytoene synthesis through head-to-head condensation are also encoded in haloarchaeal genomes.\nSynthesis of carotenoids and retinal\nFor carotene biosynthesis, phytoene is reduced to lycopene by phytoene desaturase that is encoded in haloarchaeal genomes (e.g. NP4764A, NP0204A). Lycopene is the branching point for the synthesis of \u03b2-carotene (C40) and bacterioruberins (C50) (Oesterhelt 1976). The reactions that lead to bacterioruberins have not yet been elucidated in detail, but lycopene cyclase activity (e.g. NP0652A) converting lycopene to \u03b2-carotene has been shown for H. salinarum (Peck et al. 2002). The derived \u03b2-carotene is cleaved by \u03b2-carotene mono-oxygenase into two retinal molecules (C20), which are incorporated in haloarchaeal rhodopsins. H. walsbyi encodes two cyanobacterial-like (HQ2381A, HQ2020A) and one plant-like \u03b2-carotene mono-oxygenase homologs (HQ3007A), and H. marismortui acquired a cyanobacterial-like \u03b2-carotene mono-oxygenase gene (pNG7272) on one of its plasmids (Bolhuis et al. 2006). H. salinarum and N. pharaonis lack \u03b2-carotene mono-oxygenase homologs and must therefore possess a still unknown non-orthologous enzyme for the oxidative cleavage of \u03b2-carotene. Previously, brp and blh have been shown to play a role in regulation or synthesis of retinal (Peck et al. 2001).\nFatty acid metabolism\nEven-numbered fatty acids like palmitic and stearic acid are part of membrane proteins but not of membrane lipids in archaea (Pugh and Kates 1994). For example, palmitic acid is associated with halorhodopsin as a free fatty acid in H.\u00a0salinarum (Kolbe et al. 2000). The origin of these fatty acids is unclear because archaea do not encode components of a fatty acid synthase complex. In methanogenic archaea, biosynthesis of fatty-acid-like compounds from 2-oxoglutarate by repeated 2-oxoacid chain elongation has been reported (White 1989).\nFor the degradation of activated fatty acids via the \u00df-oxidation pathway, gene candidates are present in haloarchaea and most other archaea. However, since chain-length specifity of these enzymes is currently unknown, fatty acid degradation might still be limited to short chain lengths (e.g. for derivatives of branched-chain amino acids). A series of genes were assigned for Natronomonas (37 genes) and Haloarcula (29 genes) for the repeated four-step reaction sequence of the \u00df-oxidation pathway indicating a versatile fatty-acid metabolism in these two species. In contrast, few \u00df-oxidation genes are present in H.\u00a0salinarum (12 genes) and H.\u00a0walsbyi (6 genes). In accordance to these findings, growth experiments showed that N.\u00a0pharaonis is able to grow on fatty acids of various lengths as carbon source (especially C14), while fatty acids seem not to be utilized by H.\u00a0salinarum, as growth is reduced or diminished by long-chain (C14\u2013C18) and medium-chain fatty acids (<C14), respectively (Konigsmaier 2006).\nHaloarchaea likely degrade odd-numbered fatty acids, because they possess probable propionyl-CoA carboxylase (EC 6.4.1.3, e.g. NP4250A\/NP4252A), methylmalonyl-CoA epimerase (EC 5.1.99.1, e.g. NP1228A) as well as methylmalonyl-CoA mutase (EC 5.4.99.2, e.g. NP1226A, NP2710A). Natronomonas further encodes enzymes that are probably involved in propionate catabolism (NP6212A, NP4432A, NP4820A).\nAmino acid synthesis\nWhile certain haloarchaea such as Haloarcula hispanica (Hochuli et al. 1999) do not require any amino acids for growth, H.\u00a0salinarum is grown in synthetic media with 10 to 15 amino acids (Oesterhelt and Krippahl 1973; Grey and Fitt 1976). Through metabolic pathway reconstruction and comparison, it can be proposed that H.\u00a0salinarum has indeed reduced capabilities to synthesize amino acids. Specifically, H.\u00a0salinarum lacks gene clusters for valine, leucine, isoleucine, lysine, and arginine (Supplementary Material S2). Furthermore, because of reduced folate biosynthesis, methionine biosynthesis via folate-dependent methionine synthase (EC 2.1.1.14) might be affected in H.\u00a0salinarum. The predicted set of essential amino acids fits well to the set of amino acids that can be sensed by H.\u00a0salinarum (Oren 2002) except for lysine, which is not an attractant signal, and for cysteine, which is sensed by BasT (Kokoeva et al. 2002) in spite of the fact that all enzyme genes for cysteine biosynthesis are present in its genome. The synthesis of several amino acids (glutamate, glutamine, proline, aspartate, asparagine, alanine, serine, phenylalanine, tryptophan, histidine) by H.\u00a0salinarum has been verified by NMR labeling studies (Ekiel et al. 1986; Bhaumik and Sonawat 1994; Ghosh and Sonawat 1998; Engelhard et al. 1989) (Fig.\u00a01).\nFor H. marismortui, N.\u00a0pharaonis, and H. walsbyi, complete independence from supplemented amino acids can be concluded from comparative analysis (Supplementary Material S2). De novo synthesis of all amino acids has already been proven for N. pharaonis by the development of a synthetic medium without amino acid supplements (Falb et al. 2005; Oberwinkler 2006). The three haloarchaea that do not require amino acids are also able to utilize several nitrogen sources because they posess gene sets required for urea conversion as well as for nitrate and ammonia assimilation (Falb et al. 2005) (Supplementary Material S2). Amino acid biosynthesis pathways of the three haloarchaea show only few differences, i.e. for proline, lysine, serine, and glycine synthesis (see below).\nGlutamate family (glutamate, glutamine, proline, arginine)\nThe biosynthesis of glutamate from the TCA cycle intermediate 2-oxoglutarate is a major metabolic conversion in haloarchaea as shown by NMR labeling studies in Halobacterium. Labeled pyruvate, alanine, acetate, and glycerol were mainly found to be incorporated into glutamate, and a considerable part of the flux through the TCA cycle was shown to be channelled to glutamate (Ghosh and Sonawat 1998; Ekiel et al. 1986). Three paralogous glutamate dehydrogenase genes were found in Natronomonas (NP1582A, NP1806A, NP6184A), Halobacterium (OE1270F, OE1943F, OE2728R), and Haloarcula (rrnAC0384, rrnAC0775, pNG7157), but only one gene in Haloquadratum (HQ1880A). The activity of two Halobacterium glutamate dehydrogenases with NADP+ (OE1943F) and NAD+ (OE1270F) cofactor specificity has been investigated in detail (Bonete et al. 1987, 1989, 1990; Perez-Pomares et al. 1999; Hayden et al. 2002). In N.\u00a0pharaonis, H.\u00a0walsbyi, and H.\u00a0marismortui, glutamate is also derived by glutamate synthase (e.g. NP1794A) from 2-oxoglutarate and glutamine, a reaction that is part of a proposed ammonia assimilation pathway (e.g. NP4224-NP4228A) (Falb et al. 2005). It should be noted that haloarchaeal cells amass glutamate in high concentrations as a compatible osmolyte (Kokoeva et al. 2002; Desmarais et al. 1997).\nGlutamine is derived from glutamate by glutamate-ammonia ligase (EC 6.3.1.2, e.g. NP4376A) which is present in all haloarchaea. Glutamate might further be converted to l-glutamate 5-semialdehyde and subsequently to proline. The required enzymes encoded by the proCBA gene cluster are only found in the genomes of Natronomonas (NP3974A-NP3978A) and Haloquadratum (HQ1844A-HQ1846A), but are absent in Halobacterium and Haloarcula. Biosynthesis of proline by H.\u00a0salinarum has been shown previously (Ghosh and Sonawat 1998) indicating an alternative pathway in the latter two haloarchaea. Proline might be synthesized via 1-pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.2) and proline dehydrogenase (EC\u00a01.5.99.8, putA). This notion is supported by the fact that the phylogenetic profiles of putA is complementary to that of the proABC cluster, i.e. putA is only encoded in H.\u00a0salinarum (OE3955F) and H.\u00a0marismortui (rrnAC2471), but is missing in N.\u00a0pharaonis and H. walsbyi. Proline might also be derived by the cyclisation of ornithine as shown for M. jannaschii (Graupner and White 2001). Homologs of ornithine cyclodeaminase (EC 4.3.1.12) are encoded in all haloarchaea (e.g. NP0448, NP3802A).\nGlutamate is also a precursor of ornithine, which is converted to arginine by urea cycle enzymes. The genomes of Natronomonas, Haloquadratum, and Haloarcula possess argXCBDEF clusters (e.g. NP5258A-NP5268A) containing all required enzymes (except the argA gene) for the de novo synthesis of ornithine as well as a probable transcription regulator ArgX. The complete gene set for arginine biosynthesis is absent in Halobacterium. Instead, halobacterial arginine requirements are covered by the uptake of external arginine via a verified arginine-ornithine antiporter (OE5204R) (J. Tittor, unpublished results) encoded next to the arginine deiminase pathway cluster on plasmid PHS3.\nAspartate family (aspartate, alanine, asparagine, threonine, methionine, lysine)\nAspartate is derived from the TCA cycle intermediate oxaloacetate by aspartate transaminase (EC 2.6.1.1) of which several paralogs are present in haloarchaeal genomes (e.g. NP0824A, NP1666A, NP4024A, and NP4410A). These seem to be also involved in pyruvate transamination to alanine since experiments showed that the transaminase, which is involved in alanine synthesis in Halobacterium, uses aspartate instead of glutamate as amino group donor (Bhaumik and Sonawat 1994). In agreement with this, no clear orthologs of glutamate-pyruvate transaminase (EC 2.6.1.2) have been found in haloarchaeal genomes.\nLabeling studies showed that aspartate is converted to asparagine inH. salinarum (Engelhard et al. 1989), and, consistently, all haloarchaeal genomes encode asparagine synthase (EC 6.3.5.4, e.g. NP2978A). In a pathway analogous to proline synthesis from glutamate, aspartate is likely to be converted to l-aspartate semialdehyde. This compound is not only a precursor of threonine, methionine, and lysine in archaea, but is also required for aromatic amino acid synthesis via a modified shikimate pathway (see below). Enzyme genes for threonine biosynthesis from l-aspartate semialdehyde via l-homoserine and O-phospho-l-homoserine are present in all haloarchaea (e.g. NP0302A, NP4524A, NP5280A). In M. jannaschii, O-phospho-l-homoserine is not only converted to threonine but also to homocysteine (White 2003). Additionally, haloarchaea might synthesize homocysteine from homoserine via O-acetyl-l-homoserine (but not via O-succinyl-l-homoserine). This pathway requires clustered metX and metY genes (e.g. NP0280A-NP0284A) in case of direct sulfhydrylation of O-acetyl-l-homoserine. Alternatively, O-acetyl-l-homoserine could be converted to cystathionine and subsequently to homocysteine by thiol-lyase\/-synthase (e.g. NP4746A). In the final step, methionine is likely derived from homocysteine by cobalamine-independent methionine synthase (EC 2.1.1.14, metE), of which two to three paralogs are found in haloarchaea (NP3670A\/NP3672A) except Halobacterium (OE2668R). Since methionine synthase depends on the availability of folate, reduced folate synthesis in H. salinarum might also affect its synthesis of methionine.\nThe diaminopimelate (DAP) pathway for the synthesis of lysine has already been proven for H.\u00a0hispanica (Hochuli et al. 1999). Enzymes for the DAP pathway are present in Natronomonas, Haloquadratum, and Haloarcula, i.e. the dapABD gene cluster (e.g. NP1490A-NP1494A, dapC is synonym with argD) as well as lysA (e.g. NP1646A) and argG genes (e.g. NP5252A). Gaps within the DAP pathway (dapE, dapF) occuring in Haloarcula and Natronomonas are likely to be bridged by still unknown non-orthologous enzymes. Halobacterium is not capable of lysine biosynthesis because it does not encode any of the dap genes.\nSerine family (serine, glycine, cysteine)\nSerine biosynthesis might occur via two different pathways. Within the phosphorylated pathway, glycerate 3-phosphate, a glycolytic intermediate, is oxidized to 3-phospho-hydroxy-pyruvate, which is further converted to phosphoserine and to serine by transaminase and phosphatase activity. Alternatively, non-phosphorylated glycerate is directly oxidized to hydroxypyruvate and then transaminated. All four haloarchaea possess genes for phosphoglycerate dehydrogenase (EC\u00a01.1.1.95, serA, e.g. NP0272A) and phosphoserine phosphatase (EC 3.1.3.3, serB, e.g. NP0274A) of the phosphorylated synthesis pathway. However, the enzyme gene for the intermediate pathway step, phosphoserine transaminase (EC 2.6.1.52, serC), is missing. Nevertheless, studies in Methanococcales revealed that broad-specificity class V aspartate transaminases (e.g. NP0884A, NP2578A) catalyze this reaction (Helgadottir et al. 2007). Apart from the phosphorylated serine synthesis pathways, Natronomonas, Haloquadratum, and Haloarcula might also operate the non-phosphorylated pathway because these species encode a probable hydroxypyruvate reductase (EC 1.1.1.81, e.g. NP1162A) for the initial step from glycerate to hydroxypyruvate. One of the class V aspartate transaminases might then catalyze the transaminase reaction from hydroxypyruvate to serine.\nGlycine is potentially derived from serine by glycine hydroxymethyltransferase (EC 2.1.2.1, e.g. NP2050A) in all haloarchaea. Halobacterium might additionally synthesize glycine from threonine employing threonine aldolase (EC\u00a04.1.2.5, OE4436R), which is unique to the archaeal domain of life. H. salinarum would thus be adapted to its reduced folate synthesis (see below) as the folate-dependent glycine hydroxymethyltransferase is circumvented. All haloarchaea except H.\u00a0walsbyi encode components of the glycine cleavage system (EC 1.4.4.2) which is encoded in a single gene cluster in Halobacterium (OE3274R-OE3278R). Future investigations might elucidate whether metabolic fluxes differ for the serine metabolism of Halobacterium and the other haloarchaea.\nSimilar to the conversion of homoserine to homocysteine via O-acetyl-l-homoserine, serine is likely to be converted in all halobacteria to O-acetyl-serine (cysE, e.g. NP4172A) and then to cysteine by the incorporation of sulfide (cysK, e.g. NP4748A). An alternative cysteine biosynthesis pathway employed by M.\u00a0jannaschii, which lacks cysE and cysK genes, starts from homocysteine and phosphoserine and leads via cystathionine to cysteine (White 2003). This pathway requires thiol-lyases and might be also operative in haloarchaea. Recently, also a tRNA-dependent synthesis pathway has been reported for M. jannaschii that leads from phosphoserine, an intermediate of the phosphorylated serine pathway, to Cys-tRNA (Sauerwald et al. 2005). Similar tRNA-dependent pathways have been suggested for glutamine and asparagine synthesis from glutamate and aspartate (Di Giulio 2003). For cysteine biosynthesis, sulfide needs to be assimilated from sulfate via adenylylsulfate, 3P-adenylylsulfate, and sulfite. Although archaea encode a gene for the small subunit (cysD) of the sulfate adenylyltransferase (EC 2.7.7.4, e.g. NP4570A), they lack the GTPase subunit (cysN). Genes for the two subsequent sulfate assimilation steps (cysC, cysH) are present in some archaea (e.g. Aeropyrum pernix) but are not present in any of the haloarchaeal genomes. Alternatively, thiosulfate might be converted to sulfite by thiosulfate sulfurtransferase [EC 2.8.1.1, e.g. NP3186A, NP4004A (N-terminus)] and subsequently to sulfide by one of the potential sulfite reductases [e.g. NP4004A (C-terminus), not in H.\u00a0salinarum].\nBiosynthesis of branched chain amino acids (valine, leucine, isoleucine)\nAll enzyme genes for the de novo synthesis of valine, leucine, and isoleucine are present in Natronomonas, Haloquadratum, and Haloarcula but not in Halobacterium. Threonine-ammonia lyase (EC 4.3.1.19, e.g. NP1076A), which is required to synthesize the isoleucine precursor 2-oxobutyrate, is encoded in all haloarchaea. The threonine pathway leading to isoleucine is employed by H.\u00a0hispanica (56% flux) but it is simultaneuosly operated together with the pyruvate pathway leading via citramalate to isoleucine (44% flux) (Hochuli et al. 1999). However, no sequences are yet available in public repositories for enzymes of this pathway, e.g. citramalate lyase (EC\u00a04.1.3.22). A third potential pathway from glutamate to isoleucine via methylaspartate (mesaconate pathway) is not employed by H.\u00a0hispanica (Hochuli et al. 1999). Mesaconate pathway genes (mamABC) were found in the genomes of H.\u00a0salinarum and H.\u00a0marismortui, however (see below). In Halobacterium, 2-oxobutyrate may further be derived from methionine by methionine gamma-lyase (EC\u00a04.4.1.11), a reaction that releases volatile methanthiol from the cells (Nordmann et al. 1994). The fact that Halobacterium encodes enzyme genes for 2-oxobutyrate synthesis but lacks other isoleucine synthesis enzymes implies that 2-oxobutyrate is a precursor for further metabolic pathways in haloarchaea.\nBiosynthesis of aromatic amino acids (phenylalanine, tyrosine, tryptophan, histidine)\nThe complete gene set for the classical shikimate pathway, where 3-dehydroquinate is derived from erythrose 4-phosphate (C4) and phosphoenolpyruvate (C3), is only present in Thermoplasma spp. and some other archaea (Soderberg 2005). In methanogenic archaea, 3-dehydroquinate is synthesized via a novel pathway starting from l-aspartate semialdehyde (C4) and 6-deoxy-5-ketofructose-1-phosphate (DKFP, C6) (White 2004). The DKFP pathway is also employed by haloarchaea as shown by the incorporation of labeled aspartate into the C11-position of tryptophan in H. salinarum (Engelhard et al. 1989). Furthermore, the two novel DKFP pathway enzymes for alternative 3-dehydroquinate synthesis, 2-amino-3,7-dideoxy-d-threo-hept-6-ulosonate synthase (COG1830 together with DhnA-type fructose bisphosphate aldolase; e.g. NP3160A) and 3-dehydroquinate synthase type II (COG1465, e.g. NP2238A) (Porat et al 2006) are encoded in haloarchaeal genomes.\nThe precursor of the modified shikimate pathway, DKFP, presumably derives from methylglyoxal rather than from nucleoside diphosphate sugars in archaea (White and Xu 2006). Methylglyoxal is not created by methylglyoxal synthase (EC\u00a04.2.3.3), which is absent in archaea, but from glyceraldehyde 3-phosphate using triosephosphate isomerase (EC 5.3.1.1, e.g. NP2182A, NP3716A). Another glycolytic enzyme, a multifunctional class I fructose-biphosphate aldolase (e.g. NP1594A) catalyzes the subsequent transaldolase reaction of methylglyoxal with fructose 1,6-diphosphate (or fructose 1-phosphate) to DKFP.\nThe remaining biosynthesis steps from 3-dehydroquinate to chorismate follow the classical shikimate pathway in haloarchaea. Enzyme genes for biosynthesis of tryptophan, tyrosine, and phenylalanine from chorismate are also present in haloarchaeal genomes. Consistent with this, de novo synthesis of phenylalanine and tryptophan has been proven for H. salinarum (Ekiel et al. 1986; Engelhard et al. 1989). A second synthesis pathway for aromatic amino acids in archaea has recently been described for M.\u00a0maripaludis (Porat et al. 2004), where aromatic amino acids are synthesized through the incorporation of exogenous aryl acids catalyzed by a ferredoxin-dependent indolepyruvate oxidoreductase (ior). This pathway is presumably not present in halophilic archaea, because their only two ferredoxin-dependent oxidoreductases have been already assigned otherwise (porAB, korAB) (Kerscher and Osterhelt 1981a, b).\nHistidine biosynthesis genes are partly clustered within haloarchaeal genomes. All enzymes for histidine biosynthesis starting from ribose 5-phosphate are encoded except for histidinol-phosphatase (EC 3.1.3.15). An unknown alternative enzyme is indicated for the missing pathway step, especially since histidine biosynthesis has already been proven experimentally in H.\u00a0salinarum (Ekiel et al. 1986).\nAmino acid degradation\nGlutamate and aspartate degradation\nGlutamate and aspartate are major carbon substrates for halophilic archaea that are fed into the TCA cycle and subsequently into the respiratory chain for ATP production. Several transaminases are encoded in haloarchaea which likely catalyze the conversion of aspartate, glutamate, and other amino acids to TCA cycle intermediates. In H.\u00a0salinarum and H.\u00a0marismortui, glutamate might further be degraded to mesaconate by methylaspartate mutase (EC\u00a05.4.99.1) and methylaspartate ammonia-lyase (EC 4.3.1.2). Genes for the mesaconate pathway (mamABC, OE4204F-OE4207F, rrn0684-rrn0687) are only encoded in the two haloarchaea and very few bacteria, i.e. two E. coli strains as well as a Clostridium and Treponema species. In thermophilic anaerobic bacteria, conversion of mesaconate to citramalate and subsequently to pyruvate and acetate has been proven (Plugge et al. 2001). However, the respective enzymes for the established reactions are not yet known. Mesaconate is also a potential precursor of isoleucine in haloarchaea, although not for the haloarchaeon H.\u00a0hispanica (Hochuli et al. 1999).\nArginine metabolism\nUrea cycle genes for the conversion of ornithine to arginine were found in all halophilic strains, but arginase (EC 3.5.3.1, rrnAC0383, rrnAC0453) converting arginine back to ornithine is only present in H. marismortui. Instead, H. salinarum metabolizes arginine to ornithine with concomitant ATP production via the arginine deiminase pathway (Hartmann et al. 1980). This pathway is rare amongst prokaryotes, and H.\u00a0salinarum is the only archaeon that has acquired an arginine deiminase gene cluster (arcRACB, OE5205R-OE5209R, plasmid PHS3) (Ruepp and Soppa 1996) encoding all required arginine deiminase pathway enzymes as well as a probable transcription regulator ArcR. The gene for an arginine-ornithine antiporter (OE5204R) (J. Tittor, unpublished results) is also located next to the arginine deiminase gene cluster.\nArchaea also utilize arginine for the biosynthesis of polyamines such as putrescine, spermidine, or spermine (Graham et al. 2002b), which seem to be required in archaea for nucleosome maintenance in high-temperature environments (Higashibata et al. 2000). Orthologs of a novel arginine decarboxylase (EC 4.1.1.19, e.g. NP4484A) (Graham et al. 2002b) and agmatinase (EC 3.5.3.11, e.g. NP3022A, NP4754A) for the conversion of arginine to putrescine are present in haloarchaeal genomes. For pathway steps leading from putrescine to spermidine, no enzyme genes are encoded in halophilic archaea.\nBranched-chain amino acid degradation\nFor amino acids with a more complex carbon scaffold, i.e. branched-chain and aromatic amino acids, separate biosynthesis and degradation pathways, regulated independently from each other, have evolved. Catabolism of valine, leucine, and isoleucine first involves a transaminase (e.g. NP5036A) for the conversion of branched-chain amino acids to the respective 2-oxoacids. The following decarboxylation is likely catalyzed by a previously unassigned 2-oxoacid dehydrogenase multienzyme complex (e.g. OE4113F-OE4116F), whose E1 component has recently been shown to accept branched-chain 2-oxoacids in T. acidophilum (Heath et al. 2004). The derived activated fatty acids are likely to be further degraded to acetyl-CoA and propionyl-CoA.\nAromatic amino acid degradation\nHistidine is likely to be degraded to glutamate via the urocanate pathway by H. salinarum and H.\u00a0marismortui encoding hutUGIH gene clusters (e.g. OE2734F\u2013OE2739F). The two haloarchaea (and A. pernix) also acquired the only archaeal tryptophanases (EC 4.1.99.1, OE4331R, rrnAC2439) and H. salinarum the only archaeal kynureninase (EC 3.7.1.3, OE2332F) for potential indole or anthranilate formation from tryptophan, respectively. Further, haloarchaea (except H.\u00a0walsbyi) encode orthologs (e.g. NP1194A) of the recently described l-tyrosine decarboxylase (EC 4.1.1.25) (Kezmarsky et al. 2005), which is required for synthesis of methanofuran in M.\u00a0jannaschii. Complete aromatic amino acid degradation pathways could not be reconstructed but probable aromatic compound dioxygenases (COG0346, e.g. NP2650A) are encoded in all haloarchaeal genomes.\nCofactor metabolism\nBacteria and archaea are commonly able to synthesize cofactors de novo. However, the relevant biosynthesis pathways are often not completely understood, i.e. early pathway steps leading to biotin and thiamine. In fact, many genes (e.g. thiI, moaA) which have been associated with coenzyme biosynthesis have not yet been assigned to specific metabolic reactions. Upon metabolic reconstruction, archaea reveal many gaps where enzyme genes are replaced by still unknown non-orthologous genes. Although some novel enzymes have been elucidated in recent years, e.g. novel classes of GTP cyclohydrolases (e.g. NP4142A, NP2514A) (Graham et al. 2002a; El Yacoubi et al. 2006; Grochowski et al. 2007), several pathway gaps in archaeal coenzyme metabolism remain to be filled. Taking these limitations into account, de novo synthesis pathways for common cofactors are likely in haloarchaea.\nThe compared haloarchaeal genomes show surprisingly different gene sets for cofactor synthesis pathways, and might have differing synthetic capabilities for coenzymes (Supplementary Material S2). N.\u00a0pharaonis has the most complete set of cofactor synthesis genes and has been shown to grow independently of cofactors (Falb et al. 2005). It is capable of synthesizing menaquinone, coenzyme\u00a0A, tetrahydrofolate (THF), molybdopterin, hemes, cobamide, flavins, nicotinamide derivates (NAD+\/NADP+), biotin, thiamine, and pyridoxal 5-phosphate. The latter is presumably synthesized by novel pyridoxal 5-phosphate synthesis enzymes (pdxS\/pdxT, e.g. NP4528A, NP0464A) like in B. subtilis (Raschle et al. 2005). While most enzymes for biotin synthesis are encoded in N.\u00a0pharaonis (bioA absent) and H.\u00a0marismortui (bioA and bioD absent), H.\u00a0salinarum and H.\u00a0walsbyi lack the complete biotin synthesis gene set. Thiamine biosynthesis pathways might also differ amongst haloarchaea because a couple of probable thiamine biosynthesis genes found in N. pharaonis are partly missing in other haloarchaea, i.e. thiM (NP4052A), thiE (NP4054A), and tenA (NP4080A, NP4082A). For the latter two enzymes genes, there are non-orthologous variants in haloarchaea (including N.\u00a0pharaonis), tenA is analogous to thiC (NP2210A) and thiN (NP5168A, NP0546A (fused to thiD)) to thiE (Morett et al. 2003). Finally, gene sets for folate metabolism differ greatly between various haloarchaeal strains (see below) leading to the reduced folate synthesis capability in H.\u00a0salinarum (Levin et al. 2004). Consistent with these findings, biotin, thiamine, and folate are supplied to the synthetic medium of Halobacterium (Oesterhelt and Krippahl 1973).\nThe utilization of several cofactors has been confirmed in haloarchaea such as the use of nicotinamide derivates by glutamate dehydrogenases (NAD+: OE1270F, NADP+: OE1943F) (Hayden et al. 2002), the association of coenzyme\u00a0A and ferredoxin to pyruvate- and 2-oxoglutarate ferredoxin oxidoreductases (OE1710R, OE1711R, OE2622R, OE2623R) (Kerscher and Oesterhelt 1981a, b) and the incorporation of flavin in dodecin (OE3073R) (Bieger et al. 2003). Respiratory chains of haloarchaea further involve menaquinone and certain hemes (Oesterhelt 1976; Sreeramulu et al. 1998; Scharf et al. 1997; Mattar and Engelhard 1997; Falb et al. 2005). Several copies of proteinaceous cofactors, e.g. ferredoxin, thioredoxin, halocyanin (Mattar et al. 1994), and Fe-S proteins, were also found in haloarchaeal genomes. These are likely used in various redox reactions.\nHeme and cobamide synthesis\nMenaquinone (men) and heme (hem) biosynthesis gene clusters are present in all haloarchaeal genomes. All heme synthesis genes for steps leading from glutamate to uroporphyrinogen\u00a0III have been found, but genes for successive modifications of the porphyrin system to protoheme (e.g. by hemE) are absent as in most other archaea (Supplementary Material S3). Thus, an alternative heme biosynthesis pathway via precorrin-2 as shown for M. barkeri seems likely (Buchenau et al. 2006). A novel archaeal variant of heme\u00a0A synthase (COG1612) (Lewin and Hederstedt 2006) for the conversion of heme O to heme A is present in haloarchaea (e.g. NP1770A, OE3306R).\nHalobacterium assimilates corrinoid precursors and cobamides through a high-affinity transport system, but is also able to synthesize cobamide (coenzyme B12) de novo (Woodson et al. 2003). Cobamide synthesis starting from uroporphyrinogen\u00a0III has mainly been studied in bacteria, e.g. in Pseudomonas denitrificans (cob genes) which uses an aerobic pathway and in Salmonella typhimurium (cbi\/cob genes) and Bacillus megaterium which employ an anaerobic pathway (Rodionov et al. 2003). The two pathway variants differ in their cobalt integration step occurring early in the anaerobic (S. typhimurium cbiK, B. megaterium cbiX) but late within the aerobic pathway (P.\u00a0denitrificans cobN). Haloarchaea encode both cobalt chelatases, the oxygen-dependent (cobN, e.g. NP1092A) and the oxygen-independent type (cbiX, e.g. NP1108A) (Supplementary Material\u00a0S3). Experimental data showed, however, that cobamides are synthesized under aerobic conditions in Halobacterium (Woodson et al. 2003). Haloarchaeal genomes encode most known cob genes but homologs of P. denitrificanscobG, cobF, cobK (S. typhimirium cobJ), and cobP (S. typhimirium cobU) are absent. This indicates alternative enzymes and reactions in archaeal cobalamin synthesis. For example, the bifunctional bacterial cobP enzyme gene (EC\u00a02.7.1.156\/2.7.7.62) is replaced by two novel genes cbiZ (e.g. NP5300A) and cobY (e.g. NP5304A) in archaea (Woodson et al. 2003; Woodson and Escalante-Semerena 2004). The novel archaeal variant of \u03b1-ribazole-5\u2032-phosphate phosphatase (EC\u00a03.1.3.71, cobZ, COG1267) (Zayas et al. 2006) for the synthesis of the cobamide \u03b1-ligand is not present in haloarchaea, however. Their genomes (except H. salinarum) contain the bacterial-type phosphatase gene (cobC in Salmonella, COG0406, e.g. NP1332A) encoded next to the hemCXD cluster (NP1326A\u2013NP1330A) in Natronomonas. The cobamide \u03b1-ligand is derived by cleavage of the ribityl tail from flavin mononucleotide cofactor by BluB (COG0778) (Campbell et al. 2006), of which distant homologs are present in haloarchaea (e.g. NP0176A).\nFolate synthesis\nFolic acid and its derivates are synthesized from p-aminobenzoate (PAB) and a pteridine intermediate (Fig.\u00a04, Supplementary Material S4). The latter is derived from GTP by a series of reactions involving folE, folB, and folK genes; all of them are missing in the archaeal domain of life except for H.\u00a0marismortui. This species has acquired an extensive folate metabolism gene cluster located on one of its plasmid, which contains canonical GTP cyclohydrolase I (EC 3.5.4.16, folE, pNG7382). Recently, a novel archaeal GTP cyclohydrolase (e.g. NP2514A) producing a cyclic phosphate intermediate has been reported in archaea (El Yacoubi et al. 2006; Grochowski et al. 2007), but novel enzyme genes replacing folB and folK remain to be elucidated. PAB is usually derived from chorismate by aminodeoxychorismate synthase (EC\u00a02.6.1.85, pabAB) and aminodeoxychorismate lyase (EC 4.1.3.38, pabC) which are clustered in haloarchaeal genomes (e.g. NP0798A-NP0802A). These enzymes have previously been annotated as copies of anthranilate synthase (same COG0147\/COG0512) and branched-chain amino acid aminotransferase (same COG0115), respectively. The pabA gene (OE1570F) of H.\u00a0salinarum strain R1 (but not of strain NRC-1) is disrupted by an insertion element, though, so that de novo synthesis of folate is likely omitted in this strain. Proposed haloarchaeal pab genes have not yet been characterized, and it should be noted that labeling studies for M.\u00a0maripaludis suggest the synthesis of PAB from 3-dehydroquinate and not from chorismate in this archaeon (Porat et al. 2006).\nFig.\u00a04Folate biosynthesis and metabolism in halophilic archaea. Sets of enzyme genes, which are involved in folate metabolism, differ considerably between haloarchaeal species (square: H. marismortui, circle: H. walsbyi, diamond: N. pharaonis, triangle: H. salinarum, hexagon: H. volcanii, green gene exists, red gene absent). For details see Supplementary Material S4\nFor folate synthesis, the pteridine intermediate and PAB are converted to 7,8-dihydropteroate and subsequently to 7,8-dihydrofolate by the gene products of folP and folC (Fig.\u00a04). Dihydrofolate is then reduced to 5,6,7,8-tetrahydrofolate by dihydrofolate reductase (EC 1.5.1.3, folA), which is encoded in all haloarchaea (e.g. NP2922A) except H.\u00a0salinarum. Tetrahydrofolate synthesis in H.\u00a0salinarum occurs via a novel alternative pathway, instead, where 7,8-dihydropteroate is reduced to 5,6,7,8-tetrahydropteroate by a flavin-binding Prd linker domain of the FolC-Prd-FolP fusion protein (e.g. OE1615R, NP1478A) (Levin et al. 2004, 2007). Tetrahydropteroate is subsequently converted to tetrahydrofolate by the FolC domain.\nThe canonical dihydrofolate reductase FolA is not only involved in de novo synthesis of folate but is also required for the regeneration of dihydrofolate produced by thymidylate synthase (EC\u00a02.1.1.45, thyA, NP2924A, HQ2456A). This enzyme is replaced by an alternative folate-independent enzyme encoded by thyX (OE2898R, rrnAC1121) in case a chromosomal folA gene is absent like in H.\u00a0salinarum and H.\u00a0marismortui (Supplementary Material S4). Tetrahydrofolate is likely metabolized to formyl- and methyl-THF in haloarchaea. However, only H. walsbyi and the H. marismortui plasmid pNG700 encode all required enzyme genes involved in folate coenzyme metabolism (e.g. HQ1768A, HQ2790A, HQ1756A).\nConclusions\nComparative analysis of enzyme gene sets of four halophilic archaea reveals analogous metabolic routes for the biosynthesis of nucleotides and prenyl-based lipids. Biosynthesis pathways for all proteinogenic amino acids are present for H. marismortui, H.\u00a0walsbyi, and N.\u00a0pharaonis, while H.\u00a0salinarum has no biosynthetic capabilities for five amino acids. The loss of amino acid synthesis genes and the acquisition of a catabolic gene clusters for arginine and glutamate fermentation in Halobacterium might have been driven by the constant availability of external amino acids under extreme salt conditions, where few other halophiles compete for nutrients. Several pathway variations were observed for folate-dependent enzymes such as the occurrence of different types of thymidylate synthases or the circumvention of glycine hydroxymethyltransferase by threonine aldolase. These differences might be due to varying gene sets for folate metabolism, in particular the replacement of the canonical dihydrofolate reductase by dihydropteroate reductase in H.\u00a0salinarum (Levin et al. 2004, 2007). While some haloarchaea may require thiamine and biotin supplements, all halophilic archaea possess the required enzyme genes for the de novo synthesis of cobamide, hemes, flavin and nicotinamide nucleotides, folate, coenzyme A, menaquinone, molybdopterin, pyridoxal 5-phosphate, and several proteinaceous cofactors like halocyanin.\nThe compared haloarchaeal species can best be distinguished by their catabolic and central intermediary pathways. While H.\u00a0marismortui has a versatile sugar and nitrogen metabolism, H.\u00a0walsbyi encodes a unique sugar phosphotransferase system and H.\u00a0salinarum is characterized by a variable energy metabolism. Its plasmids acquired arginine deiminase and cytochrome\u00a0d oxidase gene clusters (OE6185F\/OR6186F) as well as the gene for glycerol dehydrogenase. N.\u00a0pharaonis is not able to catabolize glycerol (that is not common in its alkaline environment) and is further incapable of sugar degradation. However, the haloalkaliphilic strain encodes an archaeal-type RuBisCO that is probably involved in a novel carbon dioxide-fixation pathway (Sato et al. 2007). It appears that halophilic archaea have adopted several strategies in order to adapt to the nutritional conditions of hypersaline environments, e.g. by gain\/loss of metabolic pathways, acquirement of plasmid-encoded enzymes, and utilization of secretion enzymes (Supplementary Material S2). Interestingly, haloarchaeal genomes often encode bacterial-type enzymes instead of (or alongside) non-orthologous archaeal variants, e.g. bacterial-type \u03b1-ribazole-5\u2032-phosphate phosphatase (cobC), folate reductase (folA), and \u03b2-carotene monooxygenases. Within the mevalonate pathway, two bacterial-type enzymes (IPP isomerase, diphosphomevalonate decarboxylase) exist, which might have been acquired to cover high isoprenoid demands of haloarchaea. Future studies might elucidate whether halophilic archaea employ different enzyme sets for the synthesis of their membrane lipids and carotenes, the latter of which are unique to the haloarchaeal branch of the archaeal domain.\nThe compiled data on haloarchaeal metabolism present a valuable resource for future system biology approaches (Gonzalez et al. 2008), because current knowledge of enzyme variants, domain rearrangements, and enzyme substrate specifities has been included throughout. Further, many of the predicted reactions and enzymes have been linked to available experimental data from enzyme activity tests and labeling studies conducted in the model species H.\u00a0salinarum. Some of these experimental results however disagree with genomic findings, e.g. for enzymes of the glyoxylate cycle (Aitken and Brown 1969) and the Entner-Douderoff pathway (Rawal et al. 1988) (Fig.\u00a01). These inconsistencies might be due to still unknown non-orthologous enzymes or may result from investigating different strains of Halobacterium, which have been renamed several times within the past decades. Although many pathway gaps in archaeal metabolic networks have been closed in recent years, novel enzyme and pathway variants still remain to be discovered within the archaeal domain of life. In haloarchaea, pathways for ribose 5-phosphate synthesis and for retinal formation need to be investigated. A future in-depth analysis of abundant non-orthologous gene displacements in halophilic and other archaea might further give interesting insights into the evolution of metabolic pathways.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nSupplementary Material S1 (DOC 1.71 MB)\nSupplementary Material S2 (DOC 116 KB)\nSupplementary Material S3 (DOC 95.5 KB)\nSupplementary Material S4 (DOC 66.5 KB)","keyphrases":["metabolism","archaea","metabolic pathways","halobacterium salinarum","enzymes","haloarchaea","pathway database","comparative genomics"],"prmu":["P","P","P","P","P","P","R","R"]}
{"id":"Pediatr_Nephrol-4-1-2275777","title":"The neurogenic bladder: medical treatment\n","text":"Neurogenic bladder sphincter dysfunction (NBSD) can cause severe and irreversible renal damage and bladder-wall destruction years before incontinence becomes an issue. Therefore, the first step in adequate management is to recognize early the bladder at risk for upper- and lower-tract deterioration and to start adequate medical treatment proactively. Clean intermittent catheterization combined with anticholinergics (oral or intravesical) is the standard therapy for NBSD. Early institution of such treatment can prevent both renal damage and secondary bladder-wall changes, thereby potentially improving long-term outcomes. In children with severe side effects or with insufficient suppression of detrusor overactivity despite maximal dosage of oral oxybutynin, intravesical instillation is an effective alternative. Intravesical instillation eliminates systemic side effects by reducing the first-pass metabolism and, compared with oral oxybutynin, intravesical oxybutynin is a more potent and long-acting detrusor suppressor. There is growing evidence that with early adequate treatment, kidneys are saved and normal bladder growth can be achieved in children so they will no longer need surgical bladder augmentation to achieve safe urinary continence in adolescence and adulthood.\nIntroduction\nNeurogenic bladder sphincter dysfunction (NBSD) can develop as a result of a lesion at any level in the nervous system, including the cerebral cortex, spinal cord, or peripheral nervous system. Neurologic conditions in children leading to neurogenic bladder dysfunction are predominantly congenital neural tube defects (including myelomeningocele, lipomeningocele, sacral agenesis, and occult lesions causing tethered cord). Acquired causes such as spinal cord tumors or trauma or sequelae of transverse myelitis are less frequent. Whereas from an etiologic standpoint neurogenic bladder dysfunction is a heterogeneous group, medical management will be similar irrespective of the underlying cause. The vast majority of knowledge about NBSD management comes from long-term experience with myelomeningocele (MMC), the most common neural tube defect.\nFollowing the institution of a general treatment policy with advances in neurosurgical and orthopedic treatments in previous decades, governing the associated NBSD has become crucial for improving quality of life and life expectancy in children with neural tube defects. In MMC patients, disordered innervation of the detrusor musculature and external sphincter adversely affects bladder function, which if untreated not only leads to incontinence but also will cause secondary damage and dysfunction of both the upper and lower urinary tracts. Key elements in optimal NBSD management are early diagnosis (including NBSD typology) and early (presymptomatic) institution of adequate medical treatment. There is indeed growing evidence that management decisions made during infancy, which prevent both renal damage and secondary bladder-wall changes, potentially impact long-term outcomes for renal function and safe urinary continence.\nAfter describing the pathophysiology of NBSD and its possible consequences, this paper focuses on diagnosis (including early identification of patients at risk), treatment goals, treatment tools, and practical management of NBSD.\nHistorical evolution\nThe management of NBSD in children has undergone major changes over the years. A first milestone was the introduction of clean intermittent catheterization (CIC) in 1972 [1]. CIC (combined with anticholinergics if required) has made \u201cconservative\u201d (medical) management a successful treatment option, with a good outcome for quality of life and kidney protection. Further important breakthroughs were the wider application of urodynamic testing in the evaluation of infants and young children with suspected NBSD [2\u20134] and the better pathophysiological understanding of the natural history of NBSD in patients with spina bifida. In spina bifida, the natural history of the urinary tract in untreated NBSD is one of progressive deterioration by the age of 3\u00a0years in up to 58% of patients [5]. Several reports have shown this deterioration to be directly related to increased intravesical pressure. In 1981, the bladder pressure at which urethral leakage occurred was found to be a useful predictor of unsafe bladder function [2]. The leak-point pressure, as it is now commonly referred to, has become accepted as one of the urodynamic parameters that allows clinicians to differentiate patients with relatively low or high risk for subsequent upper urinary tract deterioration. In 1984, detrusor external sphincter dyssynergia (DSD) was identified as an important factor leading to functional obstruction, and intravesical pressure was recognized as the pathophysiological mechanism of subsequent upper urinary tract deterioration [3]. Shortly thereafter, urodynamics in infants and children was shown to allow a functional classification of NBSD that correlated with clinical entities of incontinence and obstruction, an approach that has allowed the concept of individualized and presymptomatic therapy in high-risk patients [6].\nPathophysiology of the neurogenic bladder\nUnder normal conditions, the detrusor muscle, bladder neck, and striated external sphincter function as a synergistic unit for adequate storage and complete evacuation of urine. In healthy bladders, the change in bladder-filling pressure between empty and full is normally less than 10\u201315\u00a0cm H2O. Normal voiding pressures for males and females are from 50 to 80\u00a0cm H2O and from 40 to 65\u00a0cm H2O, respectively [7].\nIn patients with NBSD, disordered innervation of the detrusor musculature and external sphincter adversely affects bladder function. In recent years, it has become clear that children with this condition can be categorized into high- and low-risk groups for secondary damage from a neurogenic bladder based on intravesical pressure. When the detrusor (filling) pressure exceeds 40\u00a0cm H2O, glomerular filtration rate decreases and pyelocaliceal and ureteral drainage deteriorates, leading to obstructive hydronephrosis and\/or vesicoureteral reflux [2, 8\u201310]. Even in the absence of reflux or upper urinary tract dilatation, high intravesical pressure can impair drainage of urine into the bladder. Any pathophysiologic process that causes either intermittent or continuous elevation of bladder pressure above 40\u00a0cm H2O places the child at risk for upper urinary tract dysfunction, urinary tract infections, and ultimately renal failure. Intermittent elevation of bladder pressure may occur from detrusor hypertonia, hyperreflexia, or both. Hyperreflexia may cause intermittent elevation of bladder pressure, especially if the external sphincter acts reflexively and tightens rather than relaxes in an attempt to prevent micturition [detrusor sphincter dyssynergia (DSD)]. Over a long period of time, hyperreflexia with pressures greater than 40\u00a0cm H2O may result in detrusor decompensation (areflexia from myogenic failure) or in detrusor hypertrophy with associated sacculations and subsequent diverticula formation. These pathophysiologic changes affect the elastic and vesicoelastic properties of the bladder and also result in mechanical ureterovesical junction obstruction. Continuous elevation of bladder pressure above 40\u00a0cm H2O may occur from a hypertonic detrusor or a hypertrophic small-capacity bladder secondary to outflow obstruction [11]. Bladder outlet obstruction is caused by DSD, or by fibrosis of the external urethral sphincter secondary to partial or complete denervation [3, 12, 13]. Bladder outlet obstruction will lead to elevated (pathologic) voiding pressures, which will contribute to either detrusor decompensation or hypertrophy. Finally, recurrent urinary tract infections due to bladder residue may aggravate damage to the neurogenic bladder through processes of transmural inflammation and fibrosis. Together with high intravesical pressures and\/or vesicoureteral reflux, these lower urinary tract infections will lead to episodes of acute pyelonephritis and irreversible renal damage.\nManagement of the neurogenic bladder\nGeneral principles and treatment goals\nThe cornerstone of optimal NBSD management is early identification and characterization (typology) and the institution of proactive therapy. Crucial for long-term prognosis of patients with NBSD is the fact that the management must start before consequences of bladder dysfunction become apparent. From the outset, the goals of management are to prevent or minimize secondary damage to the upper urinary tracts and bladder from the primary neurogenic bladder dysfunction and to achieve safe social continence [14]. Thus, long before continence becomes an issue, starting from the first year of life, management is directed at creating a low-pressure reservoir and ensuring complete and safe bladder emptying.\nClean intermittent catheterization (CIC) or self-catheterization (CISC) in combination with anticholinergics (oxybutynin) is the standard therapy for children with neurogenic bladder dysfunction with detrusor hyperactivity and\/or DSD [11, 15, 16]. This treatment is also feasible and effective in developing countries, where untreated neuropathic bladder is an important cause of preventable chronic renal failure [17, 18]. CIC enables complete bladder emptying and thus avoids bladder residues and consequent risks for infections. In the high-risk bladder with DSD, CIC also allows bladder emptying before the occurrence of otherwise \u201cspontaneous\u201d high-pressure voiding, which is known to be detrimental for kidney function and drainage. Oxybutynin, a bladder smooth-muscle relaxant, is used to improve bladder dynamics through suppression of detrusor hypertonicity and hyperreflexia. By doing so, oxybutynin eliminates (high-pressure) uninhibited detrusor contractions (and thus urinary leakage) and prevents high-pressure bladder storage (due to detrusor hypertonicity or low bladder compliance) and high-pressure emptying (in case of DSD).\nEarly management, including diagnosis and identification of the high-risk bladder\nAt birth, the majority of patients with neurogenic bladder has normal upper urinary tracts. Without proper management, urinary tract infections and elevated bladder pressures with secondary bladder-wall changes may cause upper urinary tract deterioration within 3\u00a0years in up to 58% [5]. One third of children who develop impaired kidney drainage do so within the first year of life [19]. The specific abnormalities vary considerably and are not predicted by the level of the spinal cord defect. Furthermore, the dysfunctional pattern may be dynamic, influenced by spinal cord surgery, tethering, and denervation. In the initial baseline evaluation, clinical observations must be completed with urinalysis (microscopy and culture), renal\/bladder ultrasound, and cystourethrogram. These allow the experienced clinician to suspect the type of NBSD and to identify the high-risk subgroup. The next consideration is when to perform urodynamic studies.\nTwo different opinions exist in the literature on the use of urodynamic studies in the early evaluation and further follow-up. In one approach, urodynamic assessment has become an integral part of the initial evaluation and subsequent management, as it allows recognition of the different subtypes of NBSD (typology), proactive interventions, evaluation and guidance of therapy, and early detection of neurologic deterioration (such as symptomatic tethering of the spinal cord [20]). Advocates justify this approach of routine urodynamics to minimize the deleterious effects of high intravesical pressure by directly measuring it rather than indirectly suspecting it from the development of upper and lower urinary tract changes on serial radiologic imaging. Several studies have shown that early urodynamic evaluation of children with NBSD allows the prediction of which newborns are at risk for upper urinary tract deterioration. Urodynamic risk factors are low bladder compliance, intravesical pressure more than 40\u00a0cm H2O, and DSD [2, 3, 6, 21]. The alternative to urodynamic-based management is serial radiologic imaging to detect secondary evidence of high bladder pressure. Critics of newborn and early infancy urodynamics refer to a lack of standards for performance and interpretation, which might lead to unnecessary interventions [22, 23]. Those authors recommend careful history, physical examination, upper urinary tract imaging, and close follow-up during infancy and childhood, reserving urodynamic studies only for patients with evidence of urinary retention on physical examination, new-onset hydronephrosis or febrile urinary tract infection, or for evaluation to achieve continence. Proponents of this approach with selective urodynamics suggest that close monitoring with prompt intervention at first signs of deterioration is effective in protecting the upper urinary tracts (including preservation of nephrons and thus renal function in the long run). A remaining concern, however, could be that in this more expectant approach, high intravesical pressures may have already resulted in irreversible and avoidable damage to the bladder wall, resulting in small-capacity, low-compliance bladders later in life.\nAlthough many questions regarding optimal evaluation and management remain unanswered, the consensus on the need of close surveillance, especially in the first years of life, plus the possibility that proactive treatment may be better for the bladder in the very long term, emphasize the need for an integrated approach in which clinical observations, serial imaging, and urodynamics are the basis for early adequate treatment.\nUrodynamic studies: special considerations in children with NBSD\nIf properly performed, even with possible shortcomings in newborns and infancy, urodynamic studies allow direct diagnosis of NBSD and recognition of dysfunction subtypes. This functional classification allows adequate treatment for the different types and early proactive treatment for the bladder at risk [6].\nIt is important for the practitioner to understand the complexities involved in performing urodynamic studies in newborns, infants, and children. Urodynamic assessment can provide reproducible results in newborns and infants, but it requires attention to mechanical factors and filling rates. The younger the child, the higher the risk that mechanical factors (such as bladder-outlet obstruction by the catheter used for the investigation) may produce artificial information (elevated leak pressure or inability to void). It has also been shown that using a bladder infusion rate as close as possible to the natural filling rate is important for correct assessment of detrusor properties [24]. It is presumed that fast infusion rates overcome vesicoelastic detrusor properties, falsely indicating detrusor hypertonicity [11]. On the other hand, in children who have apparent low-pressure cystograms and who leak during filling (due to sphincter hypoactivity), detrusor hypertonia may be unrecognized [25]. In these children, it is important to perform a provocative study (including bladder neck occlusion with a balloon catheter) to identify unrecognized detrusor hyperactivity prior to bladder-neck surgery for treatment of incontinence. Electromyographic (EMG) evaluation of the external urethral sphincter is required to identify DSD. The use of concentric EMG needles is preferred, as it gives more reliable information than patch electrodes [11]. The combination of X-ray cystography with cystometrogram and sphincter EMG (video urodynamics) allows accurate evaluation of the link between intravesical pressure and vesicoureteral reflux and gives direct visual information of (dys)synergia between detrusor and sphincter mechanisms [26].\nClean intermittent catheterisation\nIn children with neurogenic bladder, CIC is the first-choice treatment to empty the bladder adequately (no residue, no infection) and safely (prior to high-pressure voiding), and it is a valuable tool for achieving continence. The wide variety of used materials and techniques for CIC does not seem to affect efficacy and safety as long as some basic principles are applied: proper education and training, clean and atraumatic application, and achievement of good patient compliance on a long-term basis. For education, training, and further guidance during follow-up, a dedicated continence nurse is invaluable. Patients and caregivers must understand what is wrong with the bladder\/sphincter and why CIC is proposed for treatment, and they have to learn how to catheterize properly. CIC has been successfully used by parents even in newborns and infants, becoming a part of their everyday routine [27]. Some authors prefer early institution of CIC in all infants with NBSD, given the fact that by the age of 3\u00a0years, CIC will be required in all for achieving continence, and given the difficulties of starting CIC at toddler age [28]. Such early institution of CIC seems to improve family compliance and their ability to assist the child in coping with their disease and with CIC [29]. CISC can be successfully taught to boys and girls who are motivated and who have developed the required dexterity, mostly around the age of 6\u00a0years. The required frequency of catheterization depends on several factors: fluid intake, bladder capacity, and bladder filling\/voiding pressures. In practice, it is recommended to catheterize six times a day in infants (linked with feeding time) and five times a day in school-aged children. Although reported incidences of CIC-related infection risks are variable, it is generally agreed that the risk is low as long as complete bladder emptying is achieved. Furthermore, reused supplies are not related to more urinary tract infections [30]. If symptomatic infections occur, these are mainly caused by incomplete bladder emptying, and CIC appliance by child or caregiver needs to be optimized. To prevent urethral strictures and false passage in boys, catheter lubrication and avoidance of forceful manipulation during catheter insertion are advocated. Nonreusable low-friction catheters are considered valuable in high-risk male patients with urethral false passage or very tense sphincters but are unnecessary in routine cases [31]. To maintain therapeutic compliance with CISC in adolescents, psychosocial support is often required. Neurogenic bowel dysfunction with constipation and fecal soiling can interfere with the institution of a successful CIC treatment. Retained stools may mechanically impair bladder filling, increase detrusor irritability, or contribute to urine retention. Stool incontinence increases the risk of bladder contamination and urinary tract infection. An effective bowel management program is therefore needed. Finally, given the high prevalence of latex allergy [32], in the spina bifida population, a strict latex-free approach is of extreme importance.\nPharmacologic treatment: anticholinergics\nOf the anticholinergic agents available, oxybutynin hydrochloride is most commonly used, and long-term experience supports its safety also in newborns and infants [33]. Oxybutynin is a tertiary amine with a well-documented therapeutic effect on detrusor hyperactivity, and its effectiveness is attributed to a combination of anticholinergic (M3-selective receptor subtype antagonism), antispasmodic, local anesthetic and calcium-channel-blocking activity [34]. Several studies have shown its efficacy for decreasing the filling pressure, increasing the capacity of the neurogenic bladder, and preserving renal function [35\u201337]. The usual dose regimen of oral oxybutynin is 0.3\u20130.6\u00a0mg\/kg per day in three doses.\nIn children with insufficient response or significant systemic side effects to oral oxybutynin, intravesical instillation of oxybutynin has been shown to be a highly efficacious, reliable, and well-tolerated therapy for children who would otherwise require surgical therapy [38\u201343]. Because a solution suitable for intravesical instillation was not available, crushed oxybutynin tablets were used in the earlier trials, with consequent problems of inconvenience and impracticability, and it was the belief of several authors that poor patient compliance could be resolved by an optimized drug preparation [40, 44]. It was subsequently shown that, indeed, eliminating the complex crushing preparation by child or parent makes intravesical oxybutynin therapy easy to use and acceptable for long-term therapy [41].\nThe mechanisms underlying the more potent and longer-acting detrusor-suppressive effects of intravesical oxybutyinin, as well as its better tolerability, have been investigated by several groups. It was demonstrated that a reduced first-pass metabolism of oxybutynin after intravesical instillation, resulting in a reduced generation of the N-desethyl metabolite, may explain the clinically relevant reduction of systemic side effects that characterizes intravesical compared with oral oxybutynin therapy [45]. In addition, these pharmacokinetic studies provided first evidence for a direct local rather than a systemic effect of intravesical oxybutynin on detrusor muscle [45]. Further evidence for a local effect of intravesically administered oxybutynin was provided by studies showing local (urothelial) accumulation, suppression of muscarinic receptor-mediated detrusor muscle contractions, and blocking of muscarinic receptors in bladder-afferent pathways [46, 47]. In most reports, intravesical oxybutynin is used in dosages between 0.3 and 0.6\u00a0mg\/kg per day in two or three doses. Given its better tolerability compared with oral treatment, if required, intravesical dosages can be further increased up to doses of 0.9\u00a0mg\/kg per day [43].\nTo date, the vast majority (\u223c 90%) of patients can be treated successfully with the gold standard treatment of oxybutynin (oral or intravesical) and CIC. Other bladder-relaxant drugs include propiverine (10\u201315\u00a0mg b.i.d. or t.i.d., adult dose), trospium (20\u00a0mg b.i.d., adult dose), extended-release oxybutynin, and tolterodine (children 0.25\u20131\u00a0mg b.i.d., adults 1\u20132\u00a0mg b.i.d.). The current experience with compounds other than oxybutynin is still limited in children with neurogenic bladder [48, 49]. Botulinum A toxin injections into the detrusor muscle have been shown to be a potentially valuable approach in the neurogenic overactive bladder [50]. Repeated botulinum A toxin injections (as an alternative for or an additive to anticholinergics) could be considered to postpone or avoid surgical procedures in the small minority of children not responding to standard therapy with CIC and anticholinergics [51]. However, further investigations are required, given remaining concerns about costs and long-term efficacy and safety of prolonged botulinum A toxin administration. Although some authors have advocated alfa-receptor stimulation of the bladder neck, no validated medical treatment is available to enhance the bladder outlet.\nMedical management of NBSD in clinical practice\nOptimal management involves first, early diagnosis, including recognition of high-risk subtypes, and second, proactive institution of adequate treatment. Early proactive treatment of high-pressure dyssynergic lower urinary tracts is important in the long term, not only to preserve renal function [52] but also to prevent poor bladder compliance and the subsequent need for bladder augmentation [35]. Urodynamic assessment is used in newborns and infants to come to a functional classification of NBSD, allowing presymptomatic interventions in the high-risk groups and individualized treatment planning according to the type of dysfunction [6, 29, 53].\nIn clinical practice, four major subtypes can be used to describe NBSD (Fig.\u00a01): sphincter overactivity combined with detrusor underactivity (type A) or overactivity (type B), and sphincter underactivity combined with detrusor underactivity (type C) or with detrusor overactivity (type D). The easiest type to treat is type A. This bladder type requires early treatment because of urine retention with high filling pressure and continuous leaking. Here, CIC alone is effective and sufficient and will make the bladder safe and infection free, and the patient will be dry in between (social continence). Good care to empty the bladder totally is most important to avoid bladder infections caused by residual urine. Dysfunctional type B will have high filling and high voiding pressures, being very unsafe from birth onward due to DSD. Here, the act of voiding has to be prevented. With oxybutynin, the overactive detrusor can be \u201cpharmacologically converted\u201d to an inactive reservoir (situation similar to type A), which has to be emptied with CIC. In type C, CIC reduces the degree of incontinence and offers much better control over urinary tract infections. To achieve continence, this type will at a later age need surgical intervention on the sphincter (e.g. sling operation). An important caveat here is that detrusor instability may emerge only after surgical improvement of outlet resistance. If this detrusor instability would remain unrecognized and untreated (with oxybutynin), bladder-outlet surgery would have converted a \u201cwet but safe\u201d into a \u201cdry but unsafe\u201d bladder. In the last dysfunctional subtype (type D), the bladder leaks due to detrusor instability and gradually becomes unsafe due to secondary bladder-wall changes with detrusor hypertrophy and loss of bladder compliance. Therefore, treatment consists of CIC combined with oxybutynin and, at a later age, bladder-outlet surgery.\nFig.\u00a01Classification of the neurogenic bladder, with four subtypes (a\u2013d) according to dysfunctional activities of sphincter and detrusor. For each subtype, clinical implications if untreated and principles of management are summarized\nOnce appropriate therapy has been initiated, adequate follow-up is required, with adjustments if needed (CIC frequency, medication dosing and administration route). Treatment efficacy can be assessed using clinical parameters (including CIC frequency and volume charts), urinalysis, renal and bladder ultrasound, X-ray cystography, and video urodynamics.\nAs long-term sequelae of insufficiently treated neurogenic bladders (renal scarring, noncompliant fibrotic bladder) already have their origin in the first years of life, the frequency of multidisciplinary follow-up visits must be age dependent (3\u00d7 yearly up to age 3\u00a0years, 2\u00d7 yearly in school-aged children, yearly in adults). Typically, urinalysis and ultrasound are performed at all visits, cystography to investigate unexpected upper urinary tract infections, and urodynamics periodically to verify that under treatment, the catheterized bladder volumes are age appropriate [54] and stored under safe pressure conditions (storage of expected bladder capacity at pressures below 30\u00a0cm H2O; see [55]).\nWith early instituted and optimal treatment, the large majority of patients can be adequately controlled without antireflux surgery or surgical bladder augmentation (Fig.\u00a02). Augmentation cystoplasty is limited to a small group of patients in whom medical treatment fails (persistence of high filling pressures). In patients with insufficient sphincter activity, continence achievement will require bladder-outlet surgery in addition to medical treatment. In female wheelchair users, surgical intervention to provide a continent stoma will facilitate self-catheterization.\nFig.\u00a02Suppression of detrusor hyperactivity with resolution of reflux by nonsurgical management. Illustrative patient with high-risk neurogenic bladder sphincter dysfunction (NBSD) (type B), urodynamically showing early unsafe filling pressures (A) with high-grade reflux (a) and urosepsis before treatment. Under clean intermittent catheterization (CIC) plus oxybutynin, the unsafe high-pressure bladder was converted into a safe low-pressure reservoir with good capacity and disappearance of the reflux at control cystography 3 months later (b). Severe systemic side effects, making continuation of oral oxybutynin impossible, disappeared after switching to intravesical oxybutynin. Further urodynamic evaluations (B: after first intravesical administration; C: after 4\u00a0months) documented adequate suppression of detrusor hyperactivity (modified from [40]). Long-term (currently 13\u00a0years) continuation of CIC and intravesical oxybutynin has resulted in a safe and adequate capacity bladder with social continence for the patient\nLong-term outcome evaluation and need for life-long follow-up\nLifelong follow-up with further periodic investigations of upper urinary tract changes, renal function, and bladder status is extremely important. There are two reasons why long-term outcome evaluation in adulthood and life-long patient follow-up are indispensable. First, for the individual patient, therapy is a life-long requisite, and verifying preservation of the patient\u2019s kidneys is only possible by repetitive assessment throughout adolescence and adulthood. Second, in general, detailed long-term follow-up data will show whether a treatment policy driven by long-term goals is sufficiently effective or requires further adaptations. The effectiveness of efforts preserving upper urinary tract function can only be judged by assessing the ultimate outcome once these patients have reached adolescence or adulthood [29]. In populations with NBSD, no consensus exists as to how renal status is ideally evaluated [56]. In clinical practice, upper urinary tract deterioration or protection is often monitored by radiographic images of hydronephrosis and vesicoureteral reflux. Modalities used to look at renal functions include nuclear imaging [dimercaptosuccinate acid (DMSA) renal scan], urinary concentrating ability, and glomerular filtration rate assessment. For the latter, creatinine (Cr) clearance can be used for patients who are socially continent; for others, inulin or Cr ethylenediaminetetraacetate (EDTA) clearance can be used. Which (combination) of these tests is best to evaluate renal function requires further investigation [56].\nConclusions\nMedical management with CIC and anticholinergics is effective in preserving renal function and providing safe urinary continence in more than 90% of patients with a neurogenic bladder. Early diagnosis and treatment institution, long before continence becomes an issue at toddler age, can prevent both renal damage and secondary bladder-wall changes, thereby improving long-term outcomes. Compared with oral oxybutynin, intravesical oxybutynin has more potent and longer-acting detrusor suppressive effects with good tolerance and should be used prior to considering surgical therapies. Therapeutic goals should no longer be restricted to prevention of secondary damage to both upper and lower urinary tracts. Instead, our goal should be to achieve normal renal and bladder growth at safe bladder pressure, with appliance-free continence.","keyphrases":["neurogenic bladder","intermittent catheterization","anticholinergics","urinary continence","urodynamics"],"prmu":["P","P","P","P","P"]}
{"id":"J_Mammary_Gland_Biol_Neoplasia-3-1-1820839","title":"Extracellular Proteolysis in Transgenic Mouse Models of Breast Cancer\n","text":"Growth and invasion of breast cancer require extracellular proteolysis in order to physically restructure the tissue microenvironment of the mammary gland. This pathological tissue remodeling process depends on a collaboration of epithelial and stromal cells. In fact, the majority of extracellular proteases are provided by stromal cells rather than cancer cells. This distinct expression pattern is seen in human breast cancers and also in transgenic mouse models of breast cancer. The similar expression patterns suggest that transgenic mouse models are ideally suited to study the role of extracellular proteases in cancer progression. Here we give a status report on protease intervention studies in transgenic models. These studies demonstrate that proteases are involved in all stages of breast cancer progression from carcinogenesis to metastasis. Transgenic models are now beginning to provide vital mechanistic insight that will allow us to combat breast cancer invasion and metastasis with new protease-targeted drugs.\nIntroduction\nBreast cancer progression is accompanied by increased expression of proteases that are capable of degrading the extracellular matrix (ECM) leading to cancer cell invasion and eventually to dissemination of cancer cells to other organs. The proteolytic capability of breast cancers is reminiscent of the inherent proteolytic capability of the healthy mammary gland. Extracellular proteases are necessary for the dynamic tissue remodeling that takes place during normal mammary gland development and pregnancy cycling (reviewed in [1]). During development, the invasive phenotype of terminal end buds is associated with a controlled proteolytic digestion of ECM in the fat pad, and side branching of the newly formed ducts requires a focal degradation of basement membrane. The proliferative response of the epithelium during pregnancy and lactation is accompanied by limited proteolytic activity. In contrast, the involution of the lactating gland to a resting gland after weaning restructures the entire gland and requires extensive degradation of ECM and basement membranes. Extracellular proteases are thus more abundant during involution than at any other stage during development and the pregnancy cycle. The similarity between the proteolytic repertoire of the normal gland and the proteolytic capability of mammary cancers is best illustrated by the fact that the cellular expression patterns of individual proteolytic components are very alike, especially when comparing involution and cancer [2]. In terms of extracellular proteolysis, breast cancer in many ways resembles a mammary gland in a state of continuous and uncontrolled tissue remodeling.\nMatrix Metalloproteases and Plasminogen Activators\nTwo families of extracellular proteases have been extensively studied in relation to mammary gland morphogenesis and cancer. These are the matrix metalloproteases (MMPs) and the group of serine proteases that govern the activation of plasminogen (Plg). Figure\u00a01 illustrates the connected extracellular proteolytic network of the MMPs and the Plg activation system (PA system).\nFigure\u00a01The PA and MMP systems in the mouse. Plasmin and MMPs are two major sources of extracellular proteolysis. Plg is converted to the active protease plasmin by one of three Plg activators, uPA, tPA, and pKal. The generation of plasmin is enhanced by the binding of uPA to the cell surface receptor uPAR, and by the binding of tPA to coagulated fibrin or to cell surface proteins (annexin II [86] and CKAP4 [87]). The conversion of pro-uPA to active uPA may be initiated by several proteases, depending on the tissue in question. Additional pro-uPA is then activated by plasmin in a positive feed-back loop. The PA system is tightly controlled by several serine protease inhibitors (serpins); the primary and most specific inhibitors are the Plg activator inhibitor PAI-1 that targets uPA and tPA, and the plasmin inhibitor \u03b12-antiplasmin (\u03b12AP). Major substrates of plasmin include fibrin, fibronectin, laminin, and other ECM proteins, latent TGF-\u03b2 and other growth factors, and pro-MMPs. The 22 different mouse MMPs are all extracellular, and are either soluble or membrane-anchored. Like the serine proteases of the PA system, the MMPs are also synthesized as inactive proforms that require proteolytic activation. Ten of the 22 pro-MMPs, including the membrane-anchored MMPs, have a furin cleavage site and may be activated by furin-like proprotein convertases before they are secreted. The remaining pro-MMPs are activated extracellularly, typically by plasmin or by other MMPs. The MMP system is counterbalanced by a group of four inhibitors, TIMPs, that have varying specificities for individual MMPs. In addition, the membrane-anchored MMP inhibitor RECK regulates a subgroup of MMPs including MMPs-2, -9, and -14 [88]. An excess of plasmin or MMP activity may ultimately be cleared by\u00a0\u03b12-macroglobulin (\u03b12M) which is a non-specific protease scavenger. The MMPs are collectively able to degrade any component of the ECM. Important substrates for the MMPs as a group include the native fibrillar collagens and all four major components of the basement membrane: collagen type IV, laminin, nidogen\/entactin, and heparan sulfate proteoglycans.\nThe liver-derived proenzyme Plg is found in high concentration throughout the body. In contrast, expression of the specific urokinase- and tissue-type Plg activators (uPA and tPA) is induced locally and thus determines where and when Plg is converted to the active protease plasmin [3, 4]. Plasmin, activated by uPA, is important for a number of tissue remodeling processes that, in addition to pregnancy cycling of the mammary gland [5], include wound healing [6] and embryo implantation [7]. In contrast, tPA is acknowledged to be primarily involved in thrombolysis and neurobiology [3, 4] but is also active during wound healing [8]. A newly identified Plg activator, plasma kallikrein (pKal), is involved in adipocyte differentiation in the involuting mammary gland [9] and in wound healing [8].\nThe MMPs constitute a large family of 22 extracellular metalloproteinases in mice (24 in humans) [10, 11] (an updated list of human and mouse proteases is available at http:\/\/web.uniovi.es\/degradome\/). MMPs are essential for a number of physiological tissue remodeling processes during ontogenesis and adult life, including mammary gland development [12, 13], bone development [14], wound healing [15], and embryo implantation [7]. During mammary gland development MMP-2 is involved in ductal elongation, and MMPs-2 and -3 are involved in ductal branching [13]. MMP-3 is also rate limiting for adipocyte differentiation during post-lactational involution [16]. The ductal tree is eventually fully formed in mice that are deficient in MMP-2, -3 or several other MMPs, indicating that the remaining MMPs are able to replace their activity in a functional overlap.\nSeveral members of the MMP and PA systems are involved in breast cancer progression. We will focus on the role played by these extracellular protease families during the various stages of breast cancer progression, from the initial hyperplastic lesion to disseminated disease, with particular emphasis on the insight gained from transgenic mouse models.\nMouse Models of Breast Cancer\nThe mouse mammary gland is ideally suited to study cancer progression for a number of reasons. First of all, there are many similarities between the human and mouse mammary glands (for detailed reviews see [17, 18]). Human and mouse mammary glands contain the same epithelial cell types: the ductal epithelial cells, luminal (milk secreting) epithelial cells, and the myoepithelial cells. They also contain adipose tissue and stroma, although the composition of the stroma differs between the human and mouse glands. One of the few differences between the species is that the epithelium in the mouse gland is surrounded by adipose tissue and a relatively sparse stroma, whereas the human mammary epithelium is encompassed by a specialized fibroblastoid stroma and is not in direct contact with adipocytes. The epithelial structure in the mature nulliparous mouse gland is also much less developed compared to the human gland and consists only of a branching but otherwise naked ductal tree. In contrast, the mature ductal tree in non-pregnant women contains groups of acini at the end of terminal ducts, named terminal ductal lobular units (TDLU). The mouse epithelium develops similar lobuloalveolar structures transiently during the estrus cycle in certain mouse strains [19] and always during early pregnancy, and in this way the resting human gland is structurally more similar to the pregnant mouse gland. In some transgenic breast cancer models, the earliest hyperplastic lesions may also resemble TDLU-like structures [20].\nA second ideal feature of the mouse mammary gland is that it is readily accessible for monitoring tumor growth and for other experimental procedures. This has lead to the development of numerous transplanted models, chemically induced models and transgenic models. We will limit our discussion to the transgenic and chemically induced models since there are inherent problems of transplanted models that may introduce bias. Depending on the study design, these obstacles can include: (1) a species barrier between proteolytic components supplied by the transplant and the host (as documented for human and mouse uPA and its receptor uPAR [21]); and (2) a cellular inconsistency with respect to protease expression in the sense that the majority of proteolytic components are supplied by stromal cells in human breast cancers while epithelial-based cancer cell lines often express the very same proteolytic components [22]. This is presumably due to genetic alterations that have occurred during in vitro culture. Furthermore, (3) when gene-deficient mice are used as hosts for syngeneic transplantation, it is possible that a tumor-suppressive phenotype in the protease-deficient host is due to an immune response directed at the protease-producing transplant.\nA third advantage of the mammary gland for cancer research is that there are more transgenic models of mammary cancer than for any other cancer form, primarily because relatively specific promoters are available for targeting oncogene expression to the mammary epithelium. The promoters most frequently used stem from the whey acidic protein (WAP) gene, and the mouse mammary tumor virus long terminal repeat (MMTV). The promoters are not equally active at all stages of mammary gland development and the pregnancy cycle [23]. The MMTV promoter is active during puberty but is greatly enhanced during pregnancy. The WAP promoter is primarily active from mid pregnancy and during lactation. Accordingly, cancer progression is accelerated by pregnancies in most transgenic models and some models only develop tumors after a number of pregnancies.\nFinally, the tumor histopathology of several transgenic models is very similar to human breast cancers [17]. The histopathology seen in a single mouse model is, however, relatively uniform and often characteristic of the inducing oncogene, and in this way no single model replicates the variability of tumor phenotypes seen among different breast cancer patients [17, 24]. This drawback is to be expected, considering the fact that transgenic models rely on a well-defined initiating oncogene in mice with a homogenous genetic background (inbred strains). In contrast, human breast cancers arise from combinations of multiple mutations in women with heterogenous genetic backgrounds. A notable similarity between mouse and human tumors is the development of a reactive stroma and the appearance of extracellular proteolysis in the tumor microenvironment. There are, however, also significant drawbacks to the use of transgenic models, such as the fact that they express oncogenes in the entire mammary epithelium (or at least a large part of it) often giving rise to multiple tumors and in some models a general hyperplasia of the entire gland. By contrast, human tumors derive from a single site, therefore resulting in a single tumor in the breast.\nA comprehensive list of transgenic breast cancer models is available at http:\/\/emice.nci.nih.gov\/ and has recently been reviewed [23]. Here we will focus on models that have been studied in relation to extracellular proteolysis. Table\u00a01 summarizes the studies that describe MMP or PA system intervention in transgenic and chemically induced breast cancer models. It is evident from Table\u00a01 that the role of proteases has almost exclusively been studied in models, in which one of the four oncoproteins ErbB-2 (neu) [25], Ras [26], Wnt1 [27], or the polyomavirus middle T antigen (PymT) [28] are induced by the MMTV promoter. ErbB-2 is often overexpressed in human breast cancers. Ras and the downstream effector proteins of Wnt1 and PymT are also frequently activated in human breast cancer, although mutation\/overexpression of Ras and Wnt1 are infrequent in breast cancer and there is no cellular counterpart of the PymT oncogene [23]. In all four transgenic models, tumor incidence is 100% or at least approaches 100% depending on the time allowed for observation [29\u201332]. Tumor incidence has only in a few cases [29, 32] been affected in protease intervention studies (Table\u00a01). Tumor onset in the ErbB-2, Ras, and Wnt1 models can be accelerated by multiple pregnancies [25, 30, 32] and mice used for experiments are frequently mated continuously, at least in the Ras and Wnt1 models [30, 32]. The PymT model is exceptionally fast even in non-pregnant mice, where the average tumor latency is approximately 1.5\u00a0months [31] compared to 4\u20137\u00a0months for multiparous mice in the three other models [25, 30, 32]. In all four models the tumors metastasize primarily to the lungs although the models differ in aggressiveness. The PymT model is the most aggressive and the Wnt1 model the least aggressive [31, 32]. A notable exception to the use of transgenic models in protease studies is the chemically induced mammary tumors that are induced by orally administered 7,12-dimethylbenzanthracene (DMBA) with or without hormone supplementation [33, 34]. The DMBA model is primarily a carcinogenesis model, since the tumors generally do not progress to form metastases [33, 34]. \nTable\u00a01PA system and MMP intervention studies in transgenic and chemically induced breast cancer models.Proteolytic interventionMouse br.ca. modelPhenotypic effect on mammary neoplasiaReferencetumor incidencetumor onsettumor growth ratefinal tumor sizelung metastasis incidencelung metastasis burdencomments and additional phenotypesPlg activation systemPlg nullMMTV-PymT\u2194\u2194 (\u2193 trend)\u2194\u2194\u2193\u2193reduced metastasis in 129:BSw:FVB mixed strain but not in FVB mice[44] and unpublished datauPA nullMMTV-PymT\u2194\u2194(\u2193 trend)\u2194\u2194\u2194\u2193+ reduced lymph node metastasis[31]PAI-1 nullMMTV-PymT\u2194\u2194\u2194\u2194\u2194\u2194 (\u2191 trend)tumor vascularization not affected[45]uPAR nullMMTV-PymT\u2194\u2194\u2194\u2194\u2194\u2194no phenotype in the FVB strainunpublished dataMMP protease family\u00a0MMP overexpressionMMTV-MMP-3 transgenenoneyes**tumors develop in the CD1 mouse strain but not in mixed B6:DBA mice[33, 61], see comment in [90]WAP-MMP-3 transgenenoneyes**tumors develop in the CD1 mouse strain but not in FVB mice[62], see comment in [90]MMTV-MMP-7 transgenenonenohyperplasia in multiparous mice but no tumors develop spontaneously[29]MMTV-MMP-14 transgenenoneyestumors develop spontaneously[63]MMTV-MMP-3 transgeneDMBA gavage\u2193\u2193opposite expectation[33]MMTV-MMP-3 transgeneDMBA gavage + MMTV-TGF\u03b1\u2194\u2194[33]MMTV-MMP-7 transgeneMMTV-neu\u2191\u2191\u2194\u2194[29]MMTV-MMP-7 transgeneApcMin + ENU\u2194[91]\u00a0MMP inhibitionMMP inhibitor-treatment (galardin\/GM6001)MMTV-PymT\u2193\u2193\u2193unpublished dataMMP-2 nullMMTV-PymT\u2194\u2194\u2194\u2194\u2193\u2193Fingleton and Matrisian, pers. comm.MMP-7 nullMMTV-PymT\u2194\u2194\u2194\u2194\u2194\u2194Fingleton and Matrisian, pers. comm.MMP-7 nullApcMin + ENU\u2193transient reduction in number of mammary tumors[91]MMP-9 nullMMTV-PymT\u2194\u2194\u2194\u2194\u2193\u2193reduced metastasis in mixed B6:FVB mouse strain but not in FVB miceFingleton and Matrisian, pers. comm.MMP-9 nullMMTV-neu\u2191(meeting report [67])MMP-11 nullMMTV-ras\u2194\u2193\u2193\u2191bidirectional effect[30]MMP-11 nullDMBA gavage\u2193\u2193ovarian and mammary carcinomas[66]MMP-13 nullMMTV-PymT\u2194\u2194\u2194\u2194\u2194\u2194unpublished dataWAP-TIMP-1 transgeneWAP-MMP-3\u2193reduced incidence of hyperplasias, no tumors develop[62]Albumin-TIMP-1 transgeneDMBA gavage + MPA\u2193early hyperplasia not affected[34]Albumin-TIMP-1 transgeneMMTV-PymT\u2194\u2193\u2193\u2194[34]MMTV-TIMP-1 transgeneDMBA gavage + MPA\u2194[34]MMTV-TIMP-1 transgeneMMTV-PymT\u2194\u2194\u2194[34]MMTV-TIMP-2 transgeneMMTV-Wnt1\u2193\u2193\u2193\u2193reduced mean vessel size in tumors[32]TIMP-2 gene therapyMMTV-neu\u2193\u2193[92]Abbreviations: ApcMin multiple intestinal neoplasia model in mice with a mutated Apc allele; B6, C57BL\/6J; BSw Black Swiss; DMBA 7,12-dimethylbenzanthracene; ENU N-ethyl-N-nitrosourea; MMP matrix metalloprotease; MMTV mouse mammary tumor virus long terminal repeat promoter; MPA medroxyprogesterone acetate; PAI-1 Plg activator inhibitor-1; Plg plasminogen; PymT polyomavirus middle T antigen; TIMP tissue inhibitor of metalloproteases; uPA urokinase plasminogen activator; uPAR uPA receptor; WAP whey acidic protein promoter.\nA feature of any transgenic model that is particularly relevant to extracellular proteolysis is the transition from pre-invasive (carcinoma in situ, CIS) to invasive breast carcinoma. Most human pre-invasive lesions are ductal CIS (DCIS), and in these lesions the carcinoma cells accumulate in the ducts confined by the myoepithelial cell layer and the basement membrane. Proteolytic degradation of the basement membrane leads to depolarization of the myoepithelial cells and offers the carcinoma cells access to the surrounding periductal stroma [35, 36]. In some human breast carcinomas the tumor suppressive myoepithelium surrounding an emerging tumor can persist for a relatively long period of time. In the MMTV-PymT model the late stage tumors resemble human invasive ductal breast cancer, but the myoepithelium is lost very early in tumor progression and it is therefore a poor model of the CIS stage ([20] and unpublished data). The lack of an intact myoepithelium, as seen in the MMTV-PymT model, does not necessarily make the neoplastic cells invasive. These lesions fall in the broad category of mammary intraepithelial neoplasia (MIN), which includes CIS-like lesions but is used more generally in mouse tumors to describe pre-malignant epithelial cells that have significant nuclear atypia and are surrounded by an intact basement membrane [37]. A few transgenic breast cancer models have been described to develop CIS lesions with morphologic similarities to human DCIS and later progress into invasive breast cancer. These include the C3(1)-SV40-T prostate cancer model that also develops mammary lesions [38], the WAP-SV40-T model [39], and the MMTV-neu models [17]. However, the CIS stage has not been the focus of protease intervention studies so far.\nProtease Expression in Transgenic Breast Cancer Models\nExtracellular proteases are generally absent in resting mammary glands but are present as the gland undergoes the pregnancy cycle. The levels of a number of extracellular proteolytic components are particularly upregulated during post-lactational mammary gland involution. These include uPA, tPA, MMPs-2, -3, -9, -11, and their corresponding inhibitors PAI-1 and TIMP-1 [5, 40\u201342]. The upregulated proteases and protease inhibitors are presumably required for the orderly restructuring of the lactating gland to a virgin-like state (discussed later). During breast cancer development, a similar proteolytic program is activated in the diseased gland and extracellular proteases are abundant in invasive breast cancers of both humans and mice. Figure\u00a02 gives a schematic comparison of the expression patterns of MMP and PA system components in human breast cancer and in the MMTV-PymT transgenic breast cancer model, which has been used to analyze the expression of several extracellular proteases. One study used laser microdissection to isolate cancer cells and stromal cells followed by quantitative RT-PCR to examine the expression level of MMPs and the PA system [43]. The study concluded that stromal tissue adjacent to cancer cells expresses higher levels of uPA, PAI-1 and MMPs-2, -3, -11, -13, and -14 than the cancer tissue. The predominantly stromal expression patterns of all these components in the MMTV-PymT model has been confirmed by in situ hybridization [31, 43\u201346]. Using immunohistochemistry, uPAR expression was found primarily in fibroblasts, endothelial cells, and in some macrophages in the MMTV-PymT model (unpublished data). In situ hybridization was used in another study on MMTV-PymT tumors to determine the expression pattern of four of the membrane-type MMPs: the transmembrane MMPs-14, -15, and -16 and the GPI-linked MMP-17 [46]. MMPs-14 and -16 were detected in the stroma whereas MMP-15 was the only protease found predominantly in the epithelium. MMP-17 expression was not observed in the MMTV-PymT tumors. The majority of these findings reflect the mRNA expression data for human breast cancer, since uPA [47], PAI-1 [48], and MMPs-2 [49], -3 [50], -11 [49], -13 [35], and -14 [51] all are found predominantly in the stroma (see suppl. data to review by Egeblad and Werb 2002 [10]). Immunoreactivity for uPAR is also primarily found in the stroma [52]. Figure\u00a03 provides two examples of this close correlation between human and transgenic mouse tumors: MMP-13 is focally expressed in stromal cells in patient material [35] and in MMTV-PymT transgenic tumors [43] (Fig.\u00a03c\u2013d). Similarly, the primary uPA inhibitor PAI-1 is expressed in stromal cells in MMTV-PymT mice [45] and in breast cancer patients [48] (Fig.\u00a03e\u2013f). The PAI-1-producing stromal cells were identified as myofibroblasts in the human samples. It is of note that this cell type is uncommon in the normal mouse and human glands but is abundant in tumors from both transgenic mice (unpublished data) and breast cancers patients [53].\nFigure\u00a02Expression of MMPs and PA components in breast tumors in humans and in MMTV-PymT transgenic mice. MMPs and PA components are expressed by cancer cells or stromal cells in breast tumors or are present as ubiquitous plasma-derived proteins. The predominant source of each mRNA\/protein is illustrated for human ductal breast cancer and for the MMTV-PymT model in mice. Only those proteases and related components that have been analyzed in the MMTV-PymT model are included in the comparison. Only mRNA analyses are included except for uPAR, which was analyzed by immunohistochemistry in both species. The \u201cNot detected\u201d category includes proteases that were sporadically expressed in the tumors. The data for the MMTV-PymT model: uPA [31]; PAI-1 [45]; uPAR, MMPs-7, -9, -10 (unpublished data); MMPs-2, -3, -11, -13, -14, uPA, PAI-1 [43]; MMPs-14, -15, -16, -17 [46]. The data for invasive ductal breast cancer: uPA [47]; PAI-1 [48]; uPAR [52]; MMP-2 [49]; MMP-3 [50], MMP-7 [89]; MMP-9 [72]; MMP-10 [89]; MMP-11 [49]; MMP-13 [35]; MMP-14 [51].Figure\u00a03Examples of protease expression in breast cancer from transgenic mice and humans. Expression of MMP-13 during early invasion in mouse C3(1)-SV40-T breast carcinoma (a) and in human ductal carcinoma in situ (DCIS) (b). MMP-13 is focally expressed in periductal fibroblast-like cells (arrows in a, a\u2019, and b\u2019) in areas with early cancer cell invasion (inv) and is not expressed in adjacent non-invasive areas with in situ carcinoma (CIS). Expression of MMP-13 in invasive MMTV-PymT carcinoma (c) and in human invasive ductal carcinoma (IDC) of the breast (d). Focal expression of MMP-13 is seen in fibroblast-like cells located in the stroma surrounding invasive cancer cells in both mouse and human breast cancers (arrows in c and d). Expression of PAI-1 in invasive MMTV-PymT carcinoma (e) and in human IDC of the breast (f). The expression of PAI-1 is seen in fibroblast-like cells located in the stroma surrounding invasive cancer cells in both the mouse and human breast cancers (arrows in e and f). All panels are in situ hybridization using 35S-labeled probes. Panels a, c\u2013f are counterstained with haematoxylin and eosin, whereas b is combined with pan-cytokeratin immunohistochemistry and (b\u2019) with CK14 immunohistochemistry for myoepithelial cells [35]. Bars: a,c, 200\u00a0\u03bcm; b, 100\u00a0\u03bcm; d\u2013f, 150\u00a0\u03bcm.\nRecently, the MMTV-Wnt1 model has also been used to study the expression levels of a number of MMPs during mammary tumor formation by RT-PCR and in situ hybridization [32]. MMPs-2, -3, -9, -13, and -14 displayed increased expression levels in hyperplastic glands and mammary tumors. MMPs-2, -3, and -9 were exclusively expressed in the stroma, while MMPs-13 and -14 were found predominantly in the cancer cells and to a lesser extent also in stromal cells. Again, most of these MMPs have been reported with similar expression patterns in human breast cancer, although MMPs-13 and -14 are predominantly stromal in ductal breast cancer [35, 51]. It is noteworthy that the MMTV-PymT and MMTV-Wnt1 models have broadly similar but not identical localization and expression of MMPs.\nThe initial expression of proteases in human breast cancer may be associated with the transition from DCIS to invasive ductal carcinoma. The transition to invasive cancer can be identified histopathologically in some DCIS lesions, particularly facilitated by immunohistochemical staining of myoepithelial cells and intraductal neoplastic cells. Expression studies of MMPs in human DCIS material have shown that MMP-13 is focally upregulated in microinvasive foci (Fig.\u00a03b) in contrast to MMPs-2, -11, and -14, which are all more broadly expressed in the samples [35]. All four of these MMPs are expressed in periductal (myo)fibroblasts, indicating an intense stromal involvement during early invasion. Brummer et al. found that TIMP-1 is also upregulated in areas of early invasion [54]. Mimicking the human situation, the C3(1)-SV40-T model develops DCIS-like lesions, in which MMP-13 is upregulated specifically in those DCIS-like structures that show signs of invasion (Fig.\u00a03a). In human samples, uPA and uPAR are also focally upregulated in areas of microinvasion together with MMP-13, suggesting that plasmin-directed proteolysis coincides with MMP activity in the transition to invasive cancer [36].\nThus, the extracellular proteases expressed in transgenic breast cancer models generally mirror those identified in human breast cancer both in terms of expression and localization. These findings suggest that transgenic models are useful tools for the study of protease involvement in breast cancer invasion and metastasis.\nFunctional Studies of Proteases in Breast Cancer Progression\nProteases in Post-lactational Mammary Gland Involution\nMammary gland involution following weaning can to some extent be regarded as a surrogate model for the role of proteases in breast cancer invasion due to the extensive tissue remodeling and extracellular proteolysis taking place. The first, reversible stage of involution is prematurely activated in Plg-deficient mice likely due to increased milk stasis during lactation [55]. However, the second, protease-dependent and irreversible stage of involution seems to be delayed in Plg-deficient mice [5]. Involution is eventually accomplished in Plg-deficient mice probably due to compensating action of other proteases including MMPs. In support of this hypothesis, implantation of TIMP-1 slow release pellets into involuting mammary glands delays alveolar regression [40], and likewise TIMP-3 null mice exhibit accelerated involution [56]. In contrast, transgenic overexpression of TIMP-1 in luminal cells did not provoke an involution phenotype [16, 57], which may be a cell-of-origin effect since endogenous TIMP-1 is produced by stromal cells during involution [40, 58]. Similarly, the accelerated involution observed in mice that overexpress MMP-3 in luminal cells [57, 59] could be an effect of overexpressing a stromal protein in luminal cells, and is not supported by evidence from MMP-3 null mice that do not show a change in mammary apoptosis [16, 57]. The fact that the PA system and the MMPs are required for the controlled tissue remodeling of the involuting mammary gland, suggests that it is the inherent proteolytic repertoire of the mammary gland that is reactivated in breast cancers.\nProteases in Hyperplasia and Carcinogenesis\nThe earliest stages of mammary carcinogenesis including the initial genetic alterations in the epithelial cells were previously considered to be independent of the surrounding stroma and extracellular proteolysis. It now seems that the structural integrity of the stroma is an essential factor in preventing carcinogenesis [60]. In fact, disruption of the ECM and cellular microenvironment caused by overexpression of MMPs-3, -7 or -14 in the mammary gland is sufficient to initiate mammary epithelial hyperplasia [29, 61\u201363]. Transgenic mice overexpressing MMP-3 or MMP-14 even develop tumors in the absence of a transgene-supplied oncogene, and a fraction of the tumors undergo malignant transformation [62, 63]. Similar transgenic overexpression of Plg activators has not been reported for the mammary gland, but a K14-uPA\/uPAR double transgene elicits extensive skin hyperplasia [64]. The MMP transgenes all direct MMP expression to the mammary epithelium, using either MMTV or WAP promoters. Only MMP-7 is expressed by normal mammary epithelium [65], while MMPs-3 and -14 are expressed by the mammary stroma during development and in the mature gland [46, 61]. Thus, tumor growth and malignancy have only been reported in cases where extracellular proteases were expressed by cells that would not normally produce them. Nevertheless, ectopically expressed proteases act as independent tumor promoters, presumably by compromising the growth-inhibitory role of the stroma, and eventually lead to genetic instability as if they were conventional oncogenes.\nIn light of the independent tumorigenic ability of some MMP transgenes, it is not surprising that tumor onset in transgenic breast cancer models is promoted by increased MMP activity and delayed by decreased MMP activity in some studies (Table\u00a01). Tumor onset is thus promoted by MMP-7 overexpression in the MMTV-neu model [29] and delayed by MMP-11 deficiency in the MMTV-ras model [30] or by TIMP-2 overexpression in the MMTV-Wnt1 model [32]. Similarly, the incidence of DMBA-induced mammary tumors is reduced in MMP-11-deficient [66] and in TIMP-1-overexpressing mice [34]. The results of two studies were inconsistent with the general finding: MMP-3 overexpression resulted in reduced rather than increased tumor incidence in the DMBA model [33], and MMP-9 deficiency led to increased tumorigenesis in the MMTV-neu model [67]. This suggests that some MMPs may have a protective function in mammary tumorigenesis, similar to the protective role of MMP-8 in chemical carcinogenesis of the skin [68].\nIn contrast to the documented influence of some of the MMPs on tumor initiation, the PA system (including genetic deficiency in Plg [44], uPA [31], PAI-1 [45], or uPAR [unpublished data]) does not affect tumor onset in the MMTV-PymT model. Neither does TIMP-1 overexpression [34] or genetic deficiencies of MMPs-2, -7, -9 (Fingleton and Matrisian, personal communication) or MMP-13 (unpublished data) in the MMTV-PymT model. At present it is difficult to determine whether these differences reflect the nature of the different proteases or perhaps that the very early tumor onset, which takes place during puberty in the MMTV-PymT model, is less sensitive to the proteolytic environment.\nProteases in Tumor Growth and Vascularization\nMammary hyperplasias and CIS lesions are generally supplied with oxygen and nutrients by the pre-existing vasculature of the mammary gland. Tumor expansion beyond a certain stage requires the formation of new blood vessels. Angiogenesis, the sprouting of new vessels from existing vasculature, depends on proteolytic dissection of the endothelial basement membrane and other ECM components. Endothelial cells penetrate a newly deposited fibrin-rich matrix and use it as a temporary scaffold for vessel structure [69]. Accordingly, the fibrinolytic capacity of the PA system is crucial for vascularization in certain experimental settings, and PAI-1 may play a dual role as a protease inhibitor and cell migration mediator [3]. In tumors from breast cancer patients, endothelial cells are a considerable source of uPA, PAI-1 and uPAR suggesting a possible role in vascularization [47, 48, 52]. This has not been experimentally confirmed in transgenic tumors thus far (Table\u00a01). In the MMTV-PymT model, tumors grow equally fast in the genetic absence or presence of Plg [44], uPA [31], PAI-1 [45] or uPAR (unpublished data), indicating that vascularization is not rate limiting for tumor growth in these cases. It is likely that functional redundancy with other proteolytic components compensates for the absence of a single component in the intact tumor. Even in PAI-1-deficient tumors, vascular density was not significantly different from wild type tumors [45]. It is also possible that vascularization of spontaneous tumors can be partially accomplished by endothelial precursor cells in a process termed vasculogenesis that may be independent of proteases or depend on a different subset of proteases [11].\nDeficiency of MMP-9 reduces tumor growth in transgenic models of skin or \u03b2-cell islet tumors, most likely due to a requirement for MMP-9 to release vascular endothelial growth factor (VEGF) from ECM deposits in order to activate the angiogenic response [70, 71]. In human breast tumors MMP-9 is expressed by a subset of inflammatory cells and vascular pericytes, which could suggest a similar role of MMP-9 in breast cancer vascularization [72]. However, the evidence is less clear in transgenic breast cancer models (Table\u00a01). Final tumor size in MMTV-PymT-transgenic mice is not significantly affected by single gene deficiencies of MMPs-2, -7, -9 (Fingleton and Matrisian, personal communication), or MMP-13 (unpublished data). Deficiency of a single MMP may be compensated by the presence of other MMPs. In contrast, simultaneous inhibition of a number of MMPs through a liver-specific TIMP-1 transgene significantly reduced final tumor size (by 42%) in MMTV-PymT mice [34]. Transgenic overexpression of TIMP-2 also caused a significant reduction in tumor growth rate and reduced the mean blood vessel size in MMTV-Wnt1 mammary tumors, providing direct evidence for an effect on vascularization [32]. MMP-11 may be important at this stage, since MMP-11-deficient mice develop significantly smaller tumors in the DMBA [66] and MMTV-ras [30] models. Surprisingly, a broad spectrum MMP inhibitor (galardin) only resulted in a 50% reduction in final tumor size in the MMTV-PymT model (unpublished data). It is possible that some MMPs promote while others inhibit tumor growth and vascularization, and the net effect of inhibiting a whole range of MMPs is rather limited. A protective role against tumor growth and vascularization has been observed in non-mammary models for MMP-3 [73] and MMP-19 [74].\nProteases in Cancer Metastasis\nIn order to successfully form metastases, cancer cells must enter the lymphatic system or the bloodstream, survive in circulation and arrest in the vasculature, extravasate and finally be able to proliferate in the new tissue. There are several metastasizing transgenic models, but the majority of models metastasize rather infrequently and only when the primary tumors are quite large. In addition to the MMTV-PymT [28] and MMTV-neu [25] models, very few other breast cancer models metastasize sufficiently to allow rigorous quantification (reviewed in [75]). Metastasis can be greatly accelerated by combining e.g. an MMTV-neu model with a second mammary-specific transgene for VEGF or transforming growth factor-\u03b21\u00a0(TGF\u03b21) [75\u201377], but these models require more elaborate breeding. Proteases are intimately associated with metastasis. A classic example is the osteolytic bone metastases that are so frequent in breast cancer patients. Breast cancer cells and the resident osteoclasts stimulate each other in a feed-back activation loop. Cancer cells stimulate MMP-dependent degradation of the collagen type I rich bone matrix by osteoclasts [78, 79]. As a consequence of bone lysis, latent growth factors are released from their deposits within the bone ECM, and are available to stimulate further cancer cell proliferation. Unfortunately, transgenic models generally metastasize to the lungs and in this way the models do not exactly mimic the organ preference of metastatic breast cancer in humans.\nThe PA system plays a unique role in metastasis at least in the MMTV-PymT model that metastasizes to the lungs [28] and lymph nodes [31] (Table\u00a01). uPA [31], PAI-1 [45], and uPAR (unpublished data) are all expressed both in the primary tumor and in the lung metastases. Metastasis is significantly reduced in both Plg-deficient and uPA-deficient mice, while these gene deficiencies have no significant effect on the primary MMTV-PymT tumors [31, 44]. Absence of the specific uPA inhibitor PAI-1 has a slight metastasis-promoting effect, but the difference is not significant perhaps due to the presence of alternative uPA inhibitors [45]. Deficiency of uPAR does not significantly affect metastasis in the inbred FVB strain (unpublished data).\nPlg and uPA may promote metastasis due to their fibrinolytic ability. Fibrin-deficient mice have not been studied in a transgenic model, but they are less susceptible to spontaneous metastasis from transplanted tumors [80] and lung colonization by tail vein-injected cells [81], suggesting that fibrin is important for sustained adhesion of circulating cancer cells in the target organ. Thus, metastasis may depend on an intact and balanced system of fibrin formation and fibrinolysis. Alternatively, Plg and uPA may promote metastasis by releasing growth factors from the ECM. Overexpression of relevant ECM-sequestered growth factors promotes breast cancer progression and metastasis in transgenic models [76, 77, 82, 83]. These growth factors include the possible plasmin substrates VEGF, TGF\u03b2, and FGF (fibroblast growth factor), as well as the possible uPA substrate HGF (hepatocyte growth factor).\nThere is also considerable evidence linking the MMPs to invasion and metastasis (reviewed in [10, 84]). In MMTV-PymT mice treated with the broad spectrum MMP inhibitor galardin, the average lung metastasis volume is reduced by approximately 99%, in the context of primary tumors that are only reduced by half (unpublished data). Metastasis incidence is also reduced in MMTV-PymT mice that overexpress TIMP-1 through a liver-specific transgene, but the reduction is less pronounced and is absent with a mammary gland-specific TIMP-1 transgene [34]. It will take a considerable effort to define which MMPs are responsible for the metastasis reduction observed with these inhibitors. MMP-9 deficiency results in reduced metastasis in the MMTV-PymT model, although this phenotype was mouse strain dependent (Fingleton and Matrisian, personal communication). Deficiency of MMP-2, which is abundantly expressed in the stroma of MMTV-PymT tumors [43], also results in reduced metastasis in the same model (Fingleton and Matrisian, personal communication). MMPs-2 and -9 have previously been associated with invasion and metastasis in a number of studies [84]. In contrast, there is no effect on metastasis in MMTV-PymT mice that are deficient in either MMP-7 (Fingleton and Matrisian, personal communication) or MMP-13 (unpublished data). Similarly, overexpression of MMP-7 in mammary glands has no effect on lung metastasis in the MMTV-neu model [29].\nMMP-11 may play an unexpected role in actually preventing metastasis. In the MMTV-ras model, MMP-11 deficiency results in a higher number of metastases despite a lower number and volume of primary tumors [30]. Apparently, the transition to invasive carcinoma is greatly accelerated in the MMP-11 null mice, indicative of a protective function of MMP-11. A similar protective role against metastasis has been reported in non-mammary models for MMP-3 [73] and MMP-12 [85]. Thus, while the general MMP inhibitor galardin has a large inhibitory effect on metastasis, several single MMP knock-out studies report no effect or even the opposite effect on metastasis in breast cancer models. This demonstrates the potential of MMP inhibitors in preventing spontaneous metastasis but also underlines the necessity of evaluating a second generation of MMP inhibitors for clinical trials that are more specific than previous compounds. Transgenic models with a well-characterized involvement of MMPs in metastasis could be a useful tool in testing such compounds.\nConclusion and Future Perspectives\nTransgenic breast cancer models are invaluable research tools because they recapitulate the entire process from the initial genetic events in normal cells to metastatic disease. Furthermore, the very similar expression patterns of extracellular proteases in human and mouse breast cancer samples suggest that transgenic mouse models are ideally suited to study the role of proteases in carcinogenesis and the progression to invasion and metastasis.\nWe have only begun to examine gene deficiencies and transgenic overexpression of the various PA and MMP components in transgenic models. The studies that have been performed so far suggest that no single MMP or PA system component is likely to be all-important for any stage in tumor progression. Practically all of the protease intervention studies report partial phenotypes such as delayed carcinogenesis, slower tumor growth or reduced metastasis, if they report any phenotype at all. The absence of any absolute phenotypes, such as prevention of metastasis, suggests that many of the proteases have mutually overlapping functions. The challenge for developing novel therapeutics in the future will be to identify those proteases that can be targeted simultaneously to obtain a synergistic tumor-suppressive effect.\nAnother area that has so far only been thoroughly examined in one or two transgenic models is the expression patterns of proteases. Using protease array data, it may be possible to draw some more detailed parallels between individual mouse models and certain histopathological subtypes of human breast cancers. It is very possible that the collective of transgenic breast cancer models available, rather than any single transgenic, will be needed to adequately model the heterogenous human disease. Another open question is whether certain proteases or inhibitors are up- or downregulated in response to certain gene deficiencies. This may provide vital information on functional overlap between individual proteases.\nTransgenic breast cancer mice may prove to be very reliable models not only for basic research but also for testing experimental anti-proteolytic therapeutics, due to the high degree of similarity with human breast cancer. However, the transgenic models also present some significant challenges. A key issue is that the experimental drug has to cross-react with the mouse version of the protease. Therefore, it may be necessary in some cases to develop two drugs in parallel, one for each species. Due to the extracellular nature of the PA and MMP systems, these represent excellent targets for therapeutic monoclonal antibodies. An elegant method for targeting extracellular proteases in transgenic mice is the development of monoclonal anti-mouse antibodies by immunizing protease-deficient mice with the target protease. In conclusion, transgenic breast cancer models will further the identification of extracellular proteases critical to cancer invasion and metastasis, and aid the development of new agents against the spread of cancer.","keyphrases":["plasminogen","upar","dcis","fibrinolysis","urokinase upa","matrix metalloprotease mmp"],"prmu":["P","P","P","P","R","R"]}
{"id":"J_Mol_Biol-1-5-1885970","title":"Crystal Structure of an Active Form of Human MMP-1\n","text":"The extracellular matrix is a dynamic environment that constantly undergoes remodelling and degradation during vital physiological processes such as angiogenesis, wound healing, and development. Unbalanced extracellular matrix breakdown is associated with many diseases such as arthritis, cancer and fibrosis. Interstitial collagen is degraded by matrix metalloproteinases with collagenolytic activity by MMP-1, MMP-8 and MMP-13, collectively known as the collagenases. Matrix metalloproteinase 1 (MMP-1) plays a pivotal role in degradation of interstitial collagen types I, II, and III. Here, we report the crystal structure of the active form of human MMP-1 at 2.67 \u00c5 resolution. This is the first MMP-1 structure that is free of inhibitor and a water molecule essential for peptide hydrolysis is observed coordinated with the active site zinc. Comparing this structure with the human proMMP-1 shows significant structural differences, mainly in the relative orientation of the hemopexin domain, between the pro form and active form of the human enzyme.\nIntroduction\nConnective tissue remodeling is a complex process involving a plethora of cytokines, growth factors, and turnover of extracellular matrix (ECM). The main enzymes that degrade ECM molecules are matrix metalloproteinases (MMPs), which are also known as matrixins. Under normal physiological conditions, MMP activity can be regulated at various stages: during transcription, proteolytic processing of their inactive pro forms, zymogens, as well as by inhibition of enzyme activity by endogenous inhibitors such as tissue inhibitors of metalloproteinases or TIMPs.1,2 These enzymes have a similar domain structure: an N-terminal signal sequence to target for secretion, a pro-peptide domain to maintain latency, a catalytic domain containing the catalytic zinc, a linker region, and a C-terminal four-bladed propeller structure called the hemopexin domain. Some of the MMPs have additional domains, e.g. the fibronectin repeats in gelatinases. These domains are important in substrate recognition and in inhibitor binding.3 The human MMP family to date comprises of about 23 enzymes that are classified based on their preferred substrate and cellular localisation: collagenases, gelatinases, stromelysins, elastase, membrane-type MMPs and so forth.4\nCollagenases (MMP-1, MMP-8 and MMP-13) are the key enzymes that are capable of cleaving interstitial fibrillar collagen. Apart from these enzymes MMP-2 (gelatinase A) and MMP-14 (MT1-MMP) are also able to initiate the breakdown of collagen fibrils.5,6 Interstitial collagens I, II and III are triple-helical proteins that are the essential structural components of all connective tissues such as the cartilage, bone, skin, tendons and ligaments. These extracellular glycoproteins provide scaffolding of the tissue and play an important role in cellular processes such as cell migration, proliferation and differentiation. Physiological collagenolysis is integral to several biological processes such as embryogenesis, tissue repair and remodeling, angiogenesis, organ morphogenesis and wound healing.7,9 The collagenases cleave the triple-helical collagen approximately three-quarters away from the N terminus of the substrate, resulting in three-quarters and one quarter length fragments that are unstable at body temperature and undergo denaturation, rendering them susceptible to other non-specific tissue proteinases. However, under aberrant circumstances degradation of collagen results in pathological conditions such as cancer, atherosclerosis, arthritis, aneurysm and fibrosis.8,10\nThe proteolytic activity of the enzyme resides in the catalytic domain but it requires the hemopexin domain in order to cleave the three chains of the triple-helical collagen.11 The crystal structure of porcine MMP-1,12 determined sometime ago, revealed details of the active site structure and the specificity pocket but this structure does not shed any light on how collagenolytic MMPs can cleave the triple-helical collagen. Recently, the X-ray structure of human proMMP-1 (MMP-1 zymogen) was elucidated.13 The structure revealed the interaction between the pro-peptide and the hemopexin domain of the enzyme, which results in a \u201cclosed\u201d conformation of the zymogen in contrast to the \u201copen\u201d conformation of catalytic domain of the active MMP-1.\nThe three-dimensional structure of the human MMP-1 (E200A), an active site mutant, reported here takes us a step closer towards a more complete understanding of the interaction of the collagenases with the triple-helical collagens. It reveals new features of the active protease and provides a platform for understanding the structural changes that accompany zymogen activation.\nResults\nOverall structure\nThe structure of human MMP-1 (E200A) was determined at 2.67\u00a0\u00c5 resolution with two monomers (chains A and B) in the asymmetric unit of the trigonal space group, P3221 (see Table 1 for crystallographic statistics). The overall domain structure of human MMP-1 is similar to that of the previously solved full-length enzymes (Figure 1). The structure comprises of the N-terminal catalytic domain, the linker region and the C-terminal hemopexin domain. The catalytic domain of one monomer contacts the hemopexin domain of the other monomer. Interestingly, the contact site used by the two monomers in the asymmetric unit to form the dimer is not the same as the dimerisation site observed in the proMMP-1 structure13 or that for the MMP-9 hemopexin domain.14 This indicates that the dimerisation mechanism is perhaps not a general rule of thumb for the hemopexin domains and is most probably a crystallisation artefact. Monomers A and B deviate from each other with an overall r.m.s. deviation of 0.87\u00a0\u00c5 (for 367 C\u03b1 atoms). The dimer is stabilised by eight hydrogen bonds and 54 van der Waals contacts (Table 2). These contacts are facilitated by a total of 33 residues (18 residues from monomer A and 15 residues from monomer B). Only nine amino acid residues are common to both the monomers: Pro104, Asp105, Leu106, Arg183, Trp184, Thr185, Val300, Phe301 and Gln335. Apart from the asymmetry seen in the residues that participate from each monomer at the dimer interface, we also observe a non-symmetric interaction pattern. However, it is believed that this dimer is not physiologically relevant, as we found that human MMP-1 is a monomer in solution (R.V. & H. N., unpublished results).\nCatalytic domain\nThe structure of MMP-1 catalytic domain is very similar to those of other MMPs. The metalloproteinase domain is about 160 amino acid residues in length with the catalytic zinc ion residing in the C-terminal segment of this domain. The catalytic fragment of the protease consists of three \u03b1-helices and a highly twisted five-stranded \u03b2-sheet. The active site zinc is bound in the sequence HELGHXXGXXH by the three His residues: His199, His203, His209 and a water molecule at the active site cleft (Figure 2). This is the first MMP-1 structure where a water molecule essential for peptide hydrolysis is observed at the active site because all the previous structures of the metalloproteinase domain of MMP-1 are in the inhibited state of the enzyme.\nAlso observed in this domain is the salt bridge between the ammonium group of the N-terminal, Phe81 and the carboxylate group of the side-chain of Asp232. The generation of the correct N terminus (Phe81) in the activation process of proMMP-1 is crucial for the enzyme to have full activity against collagen.15 If the N terminus is either longer or shorter the activity against collagen drops to 30\u201340%.15 Only with the correct N terminus the formation of the salt bridge is possible, and this stabilises the structure of the N terminus as was originally shown for MMP-8.16,17\nAlignment of the pro-enzyme (1SU3) structure with that of the mature enzyme (2CLT here) shows Phe81 to have moved some 17\u00a0\u00c5 from its original position in the zymogen form of collagenase-1 (Figure 3(a)). The structure of proMMP-3 (PDB code: 1SLM)18 was aligned as well to help calculate the displacement of Phe81. The first seven N-terminal residues were not observed in the proMMP-1 structure because of lack of proper visible electron density.13 This deviation gradually decreases and by residue Pro88 the two structures are in register and remain remarkably similar throughout the catalytic domain. There are only slight differences, if any, in the side-chain orientation of the N-terminal residues in the catalytic domain cleft (Figure 3(b)).\nThe structural zinc exhibits tetrahedral coordination facilitated by residues His149, Asp151, His164 and His177. The catalytic domain of MMP-1 also contains three calcium-binding sites. Either four or five liganding residues coordinate all the calcium ions, except one in the catalytic domain of monomer A (Table 3). One calcium ion packs the S-loop (between strands s3 and s4) against the liganding residues from strand s5. The second calcium ion of the catalytic domain is sandwiched between strand s3 and the loop containing strands s4 and s5. The third calcium site is determined by the presence of the critical residue, Asp105 that comes from the loop following strand s1. The other residues (Glu180 and Glu182) that provide coordination to this calcium ion come from the loop following strand s5. One interesting feature observed in this crystal structure by virtue of dimerisation is that residue Gln335 from monomer B contributes the fourth coordinating ligand for this calcium ion. This feature, however, is not replicated in the corresponding calcium-binding site in monomer B. Details of the metal site geometries can be found in Table 3.\nLinker region\nIn MMPs the catalytic domain is followed by a stretch of 15\u201365 amino acid residues referred to as the linker or the hinge region. This region is rich in proline residues and replacement of those with alanine drastically reduced the collagenolytic activity of MMP-8 (neutrophil collagenase),19 indicating that the presence of the correct linker structure is important for collagenolysis. This region in MMP-1 with 16 residues is well defined in the present structure unlike that in the full-length crystal structure of porcine collagenase. The conformation of the linker region is quite similar in both proMMP-113 and the present MMP-1 structures (Figure 4(a)). The residues of the linker region make extensive contacts (six hydrogen bonds and 73 van der Waals contacts; Table 4) with both the catalytic and the hemopexin domain of the enzyme. These interactions stabilise the domain arrangement in MMP-1, which is required for the concerted action of the two domains. Comparing the interactions of the linker region with the catalytic and the hemopexin domain in both the pro-enzyme and the active enzyme did not reveal many differences. Most of the interactions seen in proMMP-1 (seven hydrogen bonds and 71 van der Waals contacts) are still observed in the present structure, indicating that the activation of the enzyme does not result in structural changes and the overall structure of the areas around the linker region are mostly conserved. Interestingly though, the hydrogen-bonding interactions (Table 4) within this region are quite different between the two forms of the molecule. There are a total of seven hydrogen bonds within the linker region of active MMP-1 (Figure 4(b)) compared to the five observed in proMMP-1 (Figure 4(c)), with only two bonds common to both structures This difference in the number of hydrogen bonds is perhaps not of much significance when seen in the light of the extensive contacts this region makes with the other two domains.\nHemopexin-like domain\nThe hemopexin domain starts with Cys259 and forms a complete circle by joining to Cys447 in a disulphide bond that connects blade bI with blade bIV, giving this domain the characteristic four-bladed \u03b2-propeller structure. Each blade starts near the periphery with either the motif DAA or DAX, in which the Asp residues (Asp266, Asp359 and Asp408) coordinate the central calcium ion through their carbonyl oxygen atom. Glu310 provides the fourth coordination thus completing the acidic patch at the entrance of the central, solvent-accessible channel (Table 3). The side-chains of these residues form salt bridges to the neighbouring \u03b2-strands holding the entrance of the central channel together. Three water molecules are found trapped in the centre of this channel. These, however, are not involved in the coordination geometry of the calcium ion at the tunnel centre. Two of the water molecules are at positions corresponding to the sodium and chloride ion in the proMMP-1 structure.12 The water molecule corresponding to the sodium ion is at hydrogen-bonding distances to the carbonyl oxygen atom of Ile268, Ala312, Ala361 and Val410. The same cannot be said for the one corresponding to the chloride ion, which does not make any hydrogen bonds with the main-chain amides of the residues mentioned above. It is very likely that the presence of these ions is a consequence of the crystallisation conditions rather than a stability requirement for the hemopexin-like domain.\nComparison with the porcine MMP-1 and the human proMMP-1 structures\nFull-length human MMP-1, human proMMP-113 and porcine MMP-112 were superimposed on the basis of the catalytic domain of the three structures. Alignments were also done for just the hemopexin domain of the molecules. As expected both types of alignments gave the same outcome whereby the active form of the enzyme (human and porcine) were closer in their structure as opposed to the proMMP-1 structure. The average r.m.s. deviation over 367 C\u03b1 atoms of the porcine and human MMP-1 is \u223c1.4\u00a0\u00c5, whereas the r.m.s. deviation when aligning the full-length active MMP-1 with the proMMP-1 structure is 1.6\u00a0\u00c5. If, however, we take only the hemopexin-like domain into consideration, then the conformational differences that lie within this domain are brought to light (Figure 5(a)). The superimposed porcine and human MMP-1 hemopexin-like domains vary by 1.2\u00a0\u00c5 (maximum displacement of 5.2\u00a0\u00c5). This value increases to 1.7\u00a0\u00c5 when superimposing the hemopexin-like domains of the active human MMP-1 and the human proMMP-1. A considerable change in conformation is reflected in the residues that interact with the pro-domain. Most of these differences are by virtue of the Phe289-Tyr290-Pro291 region of the hemopexin-like domain. Table 5 shows these residues, their interactions and the shift in C\u03b1 position observed when the pro and active forms of human MMP-1 were superimposed. The largest movement of 16\u00a0\u00c5 is observed for the residue, Phe289 (Figure 5(b)). Such a perturbation in structural conformation is not evident in the catalytic domain of the enzyme (Table 5). It seems like Arg281 acts as a pivot around which the hemopexin-like domain undergoes displacement upon activation as suggested by Jozic et al.13\nOne region in the catalytic domain that generates interest is the region between Tyr218 and Tyr221. The shift in the C\u03b1 position between the active and the proMMP-1 is not as dramatic as that observed for the Phe289-Tyr290-Pro291 region but they are involved in several interactions with the pro domain around the cysteine-switch region. As a result when the pro-enzyme undergoes activation, this region of the catalytic domain opens wider and further exposes the crucial cis-configured Glu190-Tyr191 peptide in MMP-1.\nDiscussion\nReconciliation of structural findings: pro-collagenase versus active collagenase\nMMPs are multi-domain enzymes that consist of a pro domain, catalytic domain, a linker region and a hemopexin-like domain. Cleavage of the pro domain leads to a substantial rearrangement of the N-terminal residues 81\u201388 (Figure 3(a)). The activation process swings Phe81 towards the proteinase domain and terminates with a salt link between the amine of Phe81 and the carboxylate side-chain of Asp232. This interaction imparts several-fold greater enzymatic activity to MMP-1 than those with either an extended or a shorter N terminus, which lack the salt linkage.17\nPreviously an inhibitor-free structure of the catalytic domain of MMP-1 was reported.20 This structure was, however, unique in the sense that the N-terminal Leu-Thr-Glu-Gly (83\u201386) residues of one molecule occupied the active site of the other molecule thus forming an unnatural inhibited complex, which should not really be considered as an inhibitor-free structure. The present structure on the other hand stands unique in being elucidated in the absence of an inhibitor. This is the first unliganded structure of the full-length MMP-1 where a water molecule is seen at the active site providing the fourth ligand for the catalytic zinc in the tetrahedral coordination sphere (Figure 2). It must be mentioned here that the present structure is an active site mutant where the catalytic glutamate residue has been mutated to an alanine. It is likely that the scissile peptide bond and the catalytic glutamate (Glu200) flank the active site water molecule on either side. The favourable hydrogen bond between Glu200 and the water molecule would make it more nucleophilic. The strongly polarised carbonyl group of the scissile bond (by virtue of its interaction with the catalytic zinc) would face a properly oriented water molecule. An unstable transition state results in the transfer of one water proton to the leaving nitrogen (the amino group of the scissile bond) via the Glu200 carboxylate shuttle. This is followed by the cleavage of the peptide bond and concomitant shuttle of another water proton to the amino group. This hypothesis will, however, need to be substantiated with a transition state structure.\nThe linker region is considered to play an important role in collagenolysis,19,21 but its exact role is not clear. The structures of both proMMP-1 and active MMP-1 have revealed the residues of this region to be in close contact with the catalytic and the hemopexin-like domain. Comparative analysis of the extensively similar contacts made by the residues of the hinge region reveal that despite being highly exposed with no secondary structure, the conformation of the linker peptide is perhaps a signature of its sequence and does not depend on the crystal packing (Figure 4(a)). Mutagenesis studies of this region result in decreased collagenolytic activity of MMP-121 and MMP-8.19 This may be due to structural changes in this region affecting the interactions with the catalytic and hemopexin domains that are required for collagenolysis.22\nThe cleavage of the pro domain to form the active enzyme is accompanied by major conformational rearrangement in the residues that interact with the pro domain. Residues from both the catalytic domain and the hemopexin-like domain make interactions with the pro domain residues. The effects on the overall backbone structure of the catalytic domain is minimal; the effects on the hemopexin-like domain is, however, quite dramatic (Figure 5(a)). The Phe289-Tyr290-Pro291 region of the hemopexin-like domain undergoes the most significant conformational change (Figure 5(b)). In essence, the hemopexin-like domain undergoes a major displacement towards the catalytic domain, thus widening the cleft between the proteinase domain and the hemopexin-like domain on the active site face of the enzyme. This altered configuration makes the active site residues and the RWTNNFREY (residues 183\u2013191) interface, a segment critical for collagenolysis,23 more accessible for native collagen.\nImplications for collagenolysis\nDespite growing awareness of the importance of collagen-recognising determinants it is still unclear as to which part of the collagenase molecule makes the first contact with the triple-helical collagen. Several hypotheses have been suggested to try and explain the steps involved in collagenolysis (the most recent by Jozic et al.13). Docking studies were performed using a single-stranded, collagen \u03b11(I)-like 15mer peptide with the Gly-Ile cleavage site incorporated in an attempt to better understand the functioning of the active enzyme. The peptide was modelled on the known structure of one of the chains of the triple-helical collagen (PDB code: 1BKV).24 Several solutions were generated. We used localised interactions as our guide to help us pick one possible solution: (a) contact of the carbonyl group of the Gly-Ile bond of the peptide with the active site determinants, especially the catalytic zinc; and (b) contact with the critical cis-configured Glu190-Tyr191 peptide. This solution revealed the peptide to be aligned to the continuous bulge-edge strand (Gly160-Phe166) and the wall-forming segment (Pro219-Phe223) in an extended manner. This solution, however, upon energy minimisation moved outside of the coordination sphere of the catalytic zinc but it was still close enough to be polarised by the catalytic zinc. Since the peptide was only 15 amino acid residues long, we could not see any interactions with the C-terminal hemopexin-like domain. It has been proposed that the triple-helical collagen would run via the Glu190-Tyr191 cis-peptide exosite making extensive interactions with the residues of the hemopexin-like domain.22\nThe structure of the active form of human MMP-1 has provided us insights on the conformational changes that occur upon activation of the pro-enzyme and these are valuable clues towards understanding the mechanism of collagenolysis. A better understanding of the mechanism of collagen cleavage will assist towards design of inhibitors that would specifically interfere with collagenolysis without affecting (beneficial) the cleavage of other substrates.\nExperimental Procedures\nCloning, protein expression and protein purification\nThe catalytically inactive mutant proMMP-1 (E200A) was cloned, overproduced, refolded, and purified as described.22 A catalytic site mutant of this enzyme was chosen in order to prevent autocatalytic cleavage during crystallisation. The mutant zymogen was activated as described22 at a final concentration of 554\u00a0\u03bcM in the presence of 1:80 molar ratio of MMP-3 lacking the hemopexin-like domain (MMP-3\u0394C) and 1\u00a0mM 4-aminophenylmercuric acetate for 90\u00a0min at 37\u00a0\u00b0C. The activation mixture was directly applied to a Sephadex S-200 gel filtration column (diameter 26\u00a0mm, length 900\u00a0mm) in 50\u00a0mM Tris\u2013HCl (pH 7.5), 150\u00a0mM NaCl, 10\u00a0mM CaCl2, 0.02% (w\/v) sodium azide, to separate the \u201cactive\u201d form of MMP-1 (E200A) from MMP-3\u0394C, 4-aminophenylmercuric acetate, and remnants of the pro-peptide. Fractions containing MMP-1 (E200A) were pooled and concentrated using a Vivacell 250\u00a0ml with a 5\u00a0kDa cutoff membrane, followed by a Vivaspin 20 with a 5\u00a0kDa membrane (Vivascience). The protein was stored at room temperature as incubation at 4\u00a0\u00b0C resulted in precipitation.\nCrystallisation, data collection and processing\nMMP-1 (E200A) was crystallised using the hanging drop vapour-diffusion method. The protein (2\u00a0\u03bcl at a concentration of 21\u00a0mg\/ml) was mixed with 2\u00a0\u03bcl of the reservoir solution containing 0.1\u00a0M Tris (pH 7.5), 1.5\u00a0M ammonium formate and 10% (w\/v) polyethylene glycol (PEG) 8000. Crystals appeared and grew to their maximum size within two weeks at 16\u00a0\u00b0C. A cryoprotectant solution prepared by supplementing the reservoir with 25% (v\/v) glycerol enabled the crystals to be flash-frozen in liquid nitrogen.\nFlash-cooled MMP-1 (E200A) crystals were used to collect diffraction data to a resolution of 2.67\u00a0\u00c5 on PX 14.1 at the Synchrotron Radiation Source, Daresbury (UK). The data were processed and scaled using HKL2000.25 The crystals belong to the space group P3221, with cell dimensions of a\u00a0=\u00a0b\u00a0=\u00a0138.48\u00a0\u00c5 and c\u00a0=\u00a0110.05\u00a0\u00c5. There are two monomers in the asymmetric unit with solvent content of about 60%. Data reduction using the program TRUNCATE26 estimated an overall B-factor of 63.1\u00a0\u00c52\/Da from the Wilson plot. Details of the data processing statistics are presented in Table 1.\nStructure determination\nThe solution for the MMP-1 (E200A) structure was found using the program PHASER.26 Human proMMP-1 (PDB code: 1SU3)13 was used as the search model with the pro-peptide domain removed from the structure. Log-likelihood gain (LLG) of 161 for the first solution increased to an LLG value of 1091 when the second monomer in the asymmetric unit was located indicating the solutions were indeed the right ones.\nRefinement\nCrystallographic refinement was carried out using the program CNS27 at 2.67\u00a0\u00c5 resolution against 85.1% of the measured data. A test set of random reflections of 2.2% was excluded from the full data set for cross-validation purposes by calculating the free R-factor (Rfree) to monitor refinement trend.28 Initial round of refinement with the two monomers found by PHASER26 resulted in an Rcryst of 45.1% and an Rfree of 50.6%. Once the six N-terminal residues in both the monomers were built in and all the ions (two zinc ions and four calcium ions per monomer) were added and the model subjected to simulated annealing, the Rcryst and the Rfree dropped to 31% and 38%, respectively. Iterative cycles of refinement (energy minimisation, simulated annealing and individual temperature factor (B-factor) refinement) using CNS27 and model building with reference to 2Fo-Fc and Fo-Fc maps using the program Coot29 progressively improved the phases. In the final stages of refinement, water molecules with peaks greater than 3\u03c3 in the Fo-Fc maps and those within hydrogen bonding distances from appropriate atoms were incorporated into the structure.\nThe final refined structure at 2.67\u00a0\u00c5 resolution has an Rcryst of 22.3% and an Rfree of \u223c26%. Both monomers, chains A and B have the full complement of the amino acids: from Phe81 to Cys447. Residues 117, 356, 369, 385, 386 and 424 of chain A and residues 257, 272, 287, 424 and 446 of chain B have been modelled as alanine residues because of the lack of visible electron density beyond C\u03b2 atom. Analysis of the Ramachandran plot using the program PROCHECK26 indicated that \u223c81% of the residues are in the most favourable region of the \u03d5-\u03c6 plot and about 17% lie in the additional allowed region. The structure also consists of 198 water molecules. Details of the refinement statistics are given in Table 1.\nProtein Data Bank accession codes\nThe atomic coordinates and the structure factors of the human MMP-1 (E200A) have been deposited with the RCSB Protein Data Bank (accession codes 2CLT and R2CLTSF, respectively).","keyphrases":["collagen","matrix metalloproteinases","inhibitor-free","mmp, matrix metalloproteinase","ecm, extracellular matrix","fibroblast collagenase","x-ray crystallography"],"prmu":["P","P","P","R","R","M","M"]}
{"id":"Plant_Mol_Biol-4-1-2268718","title":"Comparative molecular biological analysis of membrane transport genes in organisms\n","text":"Comparative analyses of membrane transport genes revealed many differences in the features of transport homeostasis in eight diverse organisms, ranging from bacteria to animals and plants. In bacteria, membrane-transport systems depend mainly on single genes encoding proteins involved in an ATP-dependent pump and secondary transport proteins that use H+ as a co-transport molecule. Animals are especially divergent in their channel genes, and plants have larger numbers of P-type ATPase and secondary active transporters than do other organisms. The secondary transporter genes have diverged evolutionarily in both animals and plants for different co-transporter molecules. Animals use Na+ ions for the formation of concentration gradients across plasma membranes, dependent on secondary active transporters and on membrane voltages that in turn are dependent on ion transport regulation systems. Plants use H+ ions pooled in vacuoles and the apoplast to transport various substances; these proton gradients are also dependent on secondary active transporters. We also compared the numbers of membrane transporter genes in Arabidopsis and rice. Although many transporter genes are similar in these plants, Arabidopsis has a more diverse array of genes for multi-efflux transport and for response to stress signals, and rice has more secondary transporter genes for carbohydrate and nutrient transport.\nIntroduction\nCells maintain their biological activities by importing and exporting various substances. Provision of energy and nutrients and efflux of salts, biochemicals, and ions are necessary to maintaining biological activity in prokaryotic and eukaryotic cells. Environmental situations within cells differ among organisms: unicellular organisms cannot control the ion concentrations outside the cell, but multicellular eukaryotes (especially animals) can precisely regulate the ion concentrations of their cellular environments within micromolar ranges. Therefore, we can expect organisms to differ in gene number, structure, and function according to their biological abilities and environmental situations. Recent sequence analyses of entire genomes have made it possible to confirm the existence of homologous genes by computer data analysis. It is also possible to reveal the overall patterns of gene networks. In plants, complete genomic sequences are available for Arabidopsis and rice, but gene annotation programs are not yet sufficiently accurate to determine the function of all genes Instead, full-length cDNA data are useful for precise analysis of genes. Because transport activities are required at distinct levels in most tissues, we expected that the transcripts of most transmembrane transporters would be represented in full-length cDNA libraries from plants at various developmental stages, various plant tissues, and plants exposed to various treatments.\nWe searched for orthologs of known membrane transport genes by using the 35,180 full-length rice cDNA sequences (Rice Full-Length cDNA Consortium 2003; Satoh et al. 2007) and genomic sequence data from Arabidopsis (Arabidopsis Genome Initiative 2000) and japonica rice (Goff et al. 2002; International Rice Genome Sequencing Project 2005) and global functional gene annotation in Arabidopsis and rice (Munich Information Center for Protein Sequences (MIPS) data service (http:\/\/mips.gsf.de\/proj\/plant\/jsf\/) (Schoof et al. 2004; Karlowski et al. 2003)) (RAP-DB = Rice Annotation Project Data Base: http:\/\/rapdb.lab.nig.ac.jp\/ (Rice Annotation Project 2007, 2008)); the TIGR Rice Genome Annotation: http:\/\/www.tigr.org\/tdb\/e2k1\/osa1\/index.shtml (Ouyang et al. 2007). Transmembrane proteins have a hydrophobic structure, a pore-forming sequence, and molecule-binding sites. Because of these specific structural features, the identification of membrane transport orthologs is clear from computer calculations. Previous reports have characterized individual transporter protein families but have not extended to whole transport systems in general (Eng et al. 1998; Pao et al. 1999; M\u00e4ser et al. 2001; S\u00e1nchez-Fern\u00e1ndez et al. 2001). In a more general analysis of various organisms, the features of prokaryotes were contrasted with those of eukaryotes (Ren and Paulsen 2005). However, differences among eukaryotes\u2014especially animals and plants\u2014were not a focus of that report. We also searched for orthologs of membrane transport gene in various organisms database (The Human Gene Nomenclature Database Search Engine (http:\/\/www.genenames.org\/cgi-bin\/hgnc_search.pl) (Wain et al. 2004); Genomic comparison of membrane transport systems (TransportDB: http:\/\/www.membranetransport.org\/index.html) (Ren et al. 2004)), the functional genomics of plant transporters (PlantsT: http:\/\/plantst.genomics.purdue.edu\/ (Tchieu et al. 2003) and ARAMEMNON (http:\/\/aramemnon.botanik.uni-koeln.de\/) (Schwacke et al. 2003)). Here, we compare total membrane transport systems from diverse organisms and conclude that membrane transport genes exemplify evolutionary diversity of homeostatic systems. Evolutionary changes in gene families indicate the dynamics of alterations in biological systems and gene networks. Therefore, analysis of large categories of gene families may reveal many basic concepts of biological systems.\nGeneral comparisons of membrane transport genes\nMembrane transport protein carries various materials for homeostasis. There are many clear features of domains (ex. Transmembrane, pore-forming, ATP binding, molecular capture), and functional features in the transport proteins. According to the structure and its functional systems, the membrane transport proteins have divided to three categories-pump, channel and secondary transporter. The pump system is the slowest (1\u2013103\u00a0molecules\/s) but environmental-independent system which consume energy (mainly ATP) for transport. The channel is the most rapid (107\u2013108\u00a0molecules\/s) and non-energy consuming systems but its need concentration gradients previously (transport directions are only according to the gradients). The secondary transport system adapts co-transport molecules movement energy to carry molecules. Therefore, it needs co-transport molecules and the transport directions have depended on environmental conditions and the speeds (102\u2013104\u00a0molecules\/s) are the middle of pump and channel. We summarized all three categories (pump, channel and secondary transporter) of genes and compared the total numbers of membrane transport genes in Escherichia coli, Arabidopsis thaliana, Oryza sativa, Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens, Neurospora crassa, and Saccharomyces cerevisiae. The genome sizes among these organisms were diverse (4.6\u20133150 megabases), and the numbers of transmembrane genes ranged from 300 to 350 in E. coli, fungi, and yeast to about 1000\u20131200 in Arabidopsis and rice (Table\u00a02). This indicates that a minimum number of about 300 gene species may be required to retain cell homeostasis. The greater numbers of transmembrane transport genes in Arabidopsis and rice may indicate additional redundancy as well as the modification of genes for new roles (e.g. addition of new substances, adaptation of systems for regulating transport, divergence of stage- and tissue-specific material transport), specialization for the various tissues and cells of multicellular organisms, and increased complexity of cells, which in eukaryotes have many additional organelles.\nThe greater relative increase (plants versus bacterium, fungus, and yeast) in the numbers of membrane transport genes was less than has been reported for other gene categories, such as transcription factor genes and metabolic enzyme genes, in higher eukaryotes (Wray et\u00a0al. 2003). This may indicate that adaptations in membrane transport are critical for the survival of organisms during evolution. The total numbers of membrane transport genes in higher plants (Arabidopsis, about 1000; rice, 1200; Table\u00a02) are 1.2\u20132.0 times those in animals (fly, 600; nematode, 650; human, 750; Table\u00a02). These differences in numbers of transporter genes may be related to differences in the need for efflux and influx systems in restricted habitation environments. Because of their immobility and the simplicity of their uptake systems, plant cells have more opportunity to absorb inappropriate substances and greater amounts of substrates and to synthesize larger amounts of secondary products than do animals.\nIn accordance with their structures and mechanisms of action, transport proteins are classified into three classes: pump, channel, and secondary transporter (transporter). The composition ratios of these classes of protein were also compared (Table\u00a02, Fig.\u00a01). The numbers of pump genes in animals (72\u201382) were almost the same as in bacteria (70). The numbers of secondary transporter (animals, 350; bacteria, 230) and channel (animals, 160\u2013320; bacteria, 15) genes were increased in animals. In particular, vertebrates (humans) had more (322) channel gene species than plants (130\u2013180). We considered that this gene diversity in the development of channel systems was caused by the acquisition of a nervous system. The electrical transmission systems in nervous systems supplying organs (e.g. muscles, kidneys) need precisely controlled ion concentrations and the ability to make immediate changes in gradients. The development of active transport systems in animals allowed the regulation of rapid movements of the body and organs. Therefore, animals presumably acquired genes for the fastest transport-system channels. Plants also had more channel gene species than bacteria, although fewer than animals. Because plants do not transmit signals for quick movement of their organs, they do not need to regulate membrane voltages as precisely as animals. Additionally, signal-transmitting systems with ligand molecules (e.g. neurotransmitters) are not specific, unlike in animals. Therefore, the numbers of voltage-gated ion channels (VICs) and ligand-dependent channels were smaller in plants than in higher animals (Table\u00a01). On the other hand, higher plants had increased numbers of genes for pumps (170\u2013250) and secondary active transporters (660\u2013760). Plant cells have chloroplasts, which synthesize carbohydrates for many biological activities, including protein synthesis and functioning of the ATP-dependent pumps. Plants presumably use ATP-consuming systems more easily than animals, and the pumps transport the molecules that act as the driving forces of the secondary active transporters. Additionally, plant-specific organelles and vacuoles provide pools of ions and catabolite molecules. Co-transport molecules for secondary transport also are safely and stably stored in the vacuoles. Therefore, plants are presumably able to constantly supply co-transport molecules for secondary active transporters, independently of environmental conditions. The existence of vacuoles gives plant cells more self-sufficiency than animal cells and explains the evolution of membrane transport genes for individual cell homeostasis in plants. Therefore, pump and secondary transporter systems in plants are more divergent than in animals.\nFig.\u00a01Numbers of membrane transporter proteins of each class. Membrane transporter proteins were categorized into three classes (ATP-dependent [pump], channel, and secondary transporter) and compared among Escherichia coli K12-MG1655, Arabidopsis thaliana, Oryza sativa, Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens NCBI, Neurospora crassa 74-OR23-IVA, and Saccharomyces cerevisiae S228CTable\u00a01Comparative analysis of membrane transporter gene in many organismsGene familyE. coli K12A. thalianaO. sativaC. elegansD. melanogasterH. sapiens N. crassa 74S. cerevisiae MaterialPresent inEnergy-dependent (pump)ABC 671101534851473124VariousAllArsAB 00101111AnionAllF-ATPase (catalytic) 15621222H+, Na+AllH+-PPase 03900000H+P, a (vacuolar)IISP 03501329ProteinAllMPT 061411814818ProteinEukaryote (mitochondria)P-ATPase 446572219321916H+, Na+, Ca2+ etc.,AllChannelACC00000700Cation (Ca2+)A (neuron)Annexin 0810071310Ca2+EukaryoteBcl-2 000011200AnionA (mammal )Bestrophin 000214400Anion (Cl\u2212)A, P, F, B (G\u2212)CD2000000900Ca2+A (B-lymphocyte )ClC 3714631031Cl\u2212AllConnexin 000001800VariousA (vertebrate)CSC 02100000VariousP (chloroplast)CytB 01970101319H+AllE-ClC 04400400Cl\u2212A (mammal), P (distant homolog)ENaC0002025800Na+A (epithelial cell, brain)GIC 019179272000Metal (Ca2+, K+), GlutaminAllHsp70 017230141440Cation AllICC00000100Cl\u2212A (mammal)ICln 01210100Anion (Cl\u2212)A, P (distant homolog)Innexin 000228000VariousA (invertebrate)IRK-C000132200K+A, BLIC 00069234500VariousAMid100000001Cation (Ca2+)YMIP23838771114H2O, CO2, NH3AllMIT20000023Metal (Mg2+,Co2+, Ni2+)Y, a, BMscL 10000000VariousBMscS 68700000VariousP, BNSCC2 01111101CationA, Y, FO-ClC00001600Cl\u2212APCC 00005600Cation (Na+, K+ , Ca2+) APLM00000700AnionA (mammal)RIR-CaC 00053600Ca2+ATic11001200000VariousPTRP-CC 000572301Ca2+A, Y, UT00000200UreaA (vertebrate), B VIC 1351863319022K+, Na+ or Ca2+AllPhosphotransferase System (PTS)GPTS 60000000CarbohydrateBSSPTS 230000000CarbohydrateBSecondary TransporterAAA 02300000ATPPlant (chloroplast)AAAP 0437711151347Amino acid, AuxinA, P, F, YAAE10000000Aspartate, Alanine BAbgT10000000p-Aminobenzoyl-glutamate BACR3 00000011Arsenite, Antimonite Y, BAE 073421021Na+, HCO3\u2212 , H+, Cl\u2212 , H3BO3 (P, Y)A, P, YAEC 18800000AuxinP, Y, BAGCS 10000000Alanine, GlycineB, aAmt161362443NH3, CO2AllAPC2212141111141524Amino acid, PolyamineAllArsB 20005100Arsenite, Antimonite All (with distant homolog)BASS 15602501Organic acidAllBCCT 30000000Betaine\/Carnitine\/CholineB, aBenE 10000000BenzoateBCaCA 21223811884Ca2+AllCCC 01265911Na+, K+, Cl\u2212AllCDF 2811871085Zn2+ or Cd2+ etc.,AllCHR00000010SO42\u2212 , CrO42\u2212F, BCNT30022310NucleosideA, Y, BCPA1287115332Na+, H+AllCPA2 3321600121Na+, K+AllDAACS30062700Dicarboxylate, Amino acidA, BDASS 54543503VariousA, P, Y, BDcu 20000000C4-Dicarboxylate B (G\u2212)DcuC 20000000C4-Dicarboxylate B (G\u2212)DMT 161215715141869Various AllENT 08453411NucleosideA, P, YESS 10000000GlutamateBFBT 09800000Folate, BiopterinP, B, aFNT 40000011Formate, NitrateY, B, aGntP70000000CarbohydrateB (E. coli, Bacillus) GPH 69811520SugarA, P, F, a, BGUP 00100012GlycerolA, P, Y, F, BHAAAP 81000000Amino acidB, P (distant homolog)KDGT 100000002-Keto-3-DeoxygluconateBKUP 1132100010K+P, F, BLCT00002211CystineA, P, FLctP20000000Lactate, Glycolate B, aLIV-E10000000Amino acid (L,I,V)B, aLIVCS 10000000Amino acid (L,I,V)BLysE 10000000LysineBMC 052663445443434Various EukaryoteMET 00000300Nucleoside, etc.AMFS 70901451371448214185Various AllMOP8564800213Various (Drugs etc.,)AllMTC 02262311Various (Anionic substrate )EukaryoteNCS1 210000310Nucleobase, Thiamine P, F, Y, B, aNCS211121151410Nucleobase,etc.AllNhaA 10000000Na+,H+ProkaryoteNhaB10000000Na+, H+AllNhaD 02100000Na+(Li+), H+P, BNiCoT 00000010Ni2+, Co2+AllNramp171021223Metals (Fe2+, Zn2+ etc.,)AllNSS 00012211800Neurotransmitters etc.,AOAT 022381100VariousA, P, F, YOPT0153400043OligopeptideP, B, aOST 00000200Organic compound EukaryoteOxa114501101ProteinAll (with distant homolog)PiT21351211HPO42\u2212, SO42\u2212AllPnaS 10010200Inorganic phosphate A (mammal)POT 4506133421OligopeptideAllRFC 00033400Folate, TiamineARhtB 50000000Amino acidB, aRND 821244721Various AllSSS 4113191121VariousAllSulP 11115791144SO42\u2212AllTat 10100000ProteinB (G\u2212)TDT 14900021Tellurite, C4-Dicarboxylate All (with distant homolog)ThrE 10000002Threonine, Serine All (with distant homolog)TRAP-T 10000000VariousB, aTrk 21100022K+P, Y, BZIP 0131865253Zn2+, Fe2+AllUnclassifiedCtr1 00000001Dipicolinic Acid Y, BCtr2 05743222Cu2+AllFeoB 10000000Fe2+B, aFeT 00000001Fe2+, (Co2+, Cd2+)YFP 00000100Fe2+A (mammal)LPI 00000500ProteinAOFeT 10000012Fe2+,Fe3+Y, BPnuC 10000000Nicotinamide mononucleotideBPPI 00006630ProteinA, F, BPUP 115400000Peptide, Fatty acidP, BNote: Present in: A\u00a0=\u00a0animal, P =\u00a0plant, F\u00a0=\u00a0fungi, Y =\u00a0yeast, B\u00a0=\u00a0bacteria, a\u00a0=\u00a0archea, G\u2212\u00a0= gramm minus bacteria\nEnergy (ATP, pyrophosphate)-dependent (pump) system\nWe compared the ATP-dependent transport genes in this diverse set of organisms (Table\u00a01, Fig.\u00a02, and Supplemental data-2). The main roles of the ATP-dependent (pump) proteins are (1) to transport molecules in specific directions independently of the environmental situation; and (2) to transport ions to form a concentration gradient between the areas outside and inside the membrane (active transport). Because bacteria cannot control the concentrations of ions or metabolites outside the cell, their pumps work mainly to transport molecules. In E. coli, most of the ATP-dependent genes (93%) encode ATP-binding cassette (ABC) proteins, and some of them are reported to encode channel proteins (Fig.\u00a02, Table\u00a01) (Holland et\u00a0al. 2005). The ABC proteins transport various substances (e.g. ions, peptides, nucleosides, amino acids, carbohydrates, proteins) ATP-dependently in all organisms (Kolukisaoglu et\u00a0al. 2002; Garcia et\u00a0al. 2004). In eukaryotes, many functional units are present within one polypeptide, whereas many bacterial ABC subunits are encoded by individual genes. This results in an inverted relationship between prokaryotes (E. coli) (72) and primitive eukaryotes (yeast) (43). Many bacterial ABC proteins are located in the plasma membrane and serve as the main forces in energy-consuming transport for both import and export of substances. In higher eukaryotes, ABC proteins tend to export substances rather than function in import reactions.Fig.\u00a02Comparison of numbers of pump genes among various organisms. Pump gene numbers were compared among Escherichia coli K12-MG1655, Arabidopsis thaliana, Oryza sativa, Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens NCBI, Neurospora crassa 74-OR23-IVA, and Saccharomyces cerevisiae S228C. ABC: ATP-binding Cassette; ArsAB: Arsenite-antimonite Efflux; F-ATPase: H+ or Na+-translocating F-type, V-type and A-type ATPase; H+-Ppase: H+-translocating Pyrophosphatase; IISP: General Secretory Pathway (Sec); MPT: Mitochondrial Protein Translocase; P-ATPase: P-type ATPase\nStructural analyses revealed that ABC proteins could be classified as half size (homo- or heterodimer functional) or full size (monomer functional) in accordance with their construction. The number of genes encoding ABC proteins in animals is the same as, or slightly less than, in bacteria, most of them representing whole subunits of ABC proteins. In contrast, the number of ABC genes in plants is about twice that in animals and bacteria, primarily due to the encoding of half-size proteins (Table\u00a01, Fig.\u00a02, and Supplemental data-2) (Sanchez-Fernandez et\u00a0al. 2001; Sugiyama et\u00a0al. 2006). This increase in numbers of genes encoding half-size (e.g., WBC, ABC2) and full-size (PDR; pleiotropic drug resistance) proteins supposedly permits stage- and tissue-specific regulation of various substances (including plant-specific substances). Therefore, increases in the capacity of import and export transport systems by an increase in the number of half-size ABC proteins have allowed plants to fulfill their unique roles.\nIn the import and efflux of ions and the creation of ion gradients for secondary transport, P-type ATPases transport many species of ion (e.g. H+, Na+, K+, Ca2+) in both directions in the cell (Table\u00a01, Fig.\u00a02, and Supplementary data-2) (Axelsen and Palmgren 2001; Baxter et\u00a0al. 2003). Control of ion concentrations outside bacterial cells is not possible. Only a few of the bacterial genes involved in systems for the uptake or efflux of ions (uptake: K+, Mg2+; efflux: Ca2+, Ag+, Zn2+, Co2+, Pb2+, Ni2+, Cd2+; uptake or efflux, depending on the system: Cu2+) have been characterized, and each of the enzymes encoded comprises a distinct subfamily (Banci et\u00a0al. 2006).\nEukaryotes have diverged in terms of their transport substances and have also adapted genes for making the ion gradients for secondary transport. One of the most important ATPases in animals\u2014Na+\/K+ATPase\u2014does not exist in plants. Na+\/K+ATPase makes a Na+ ion gradient across the plasma membrane and forms the basis of membrane voltage and secondary active transport in animals. On the other hand, plants use \nH+ gradients for secondary transport, and more than 10 isoforms and vacuole-type H+ATPases are involved in stage- and tissue-specific control. This difference in adapted ion gradients reflects the ion concentrations in the cells and the demand for nutrient ions. A constant supply of Na+ ions is required to retain homeostasis of animal cells and rapid signal transmission systems (e.g. muscles, nerves). In contrast, plants use the more abundant H+ ion to make cation gradients and have special systems for transporting H+ ions into vacuolar pools. For transporting H+ into vacuoles, higher plants have a vacuole-type ATPase and an H+-translocating pyrophosphatase (H+-PPase) (Table\u00a01, Fig.\u00a02, and Supplementary data-2) that are comparable to bacterial ATPase and PPase, respectively (Sivula et\u00a0al. 1999; Sze et\u00a0al. 2002). Therefore, plants presumably adapted bacterial H+-ATPase systems to help in H+ ion concentration steps, whereas animals developed completely new systems to make Na+ ion gradients.\nIon channel systems\nIon channels are the \u201cgates\u201d in the membranes that open or close in response to signals such as mechanical or electrical stimulation and ligand binding. Therefore, ion channels are closely involved in determining whether or not ionic gradients are available. Multicellular organisms can control the ion concentrations in the tissues on both sides of the cell membrane, whereas monocellular organisms usually find it hard to control the ion gradient outside the cell. Thus, the use of ion channels becomes restrictive and unidirectional in these more primitive organisms. Unlike in eukaryotes, the channel system in prokaryotes is not well adapted to transport (Table\u00a01, Fig.\u00a03, and Supplemental data-3). Of the total number of genes encoding membrane transport proteins in prokaryotes, fewer than 5% are channel genes, and they mainly regulate osmotic homeostasis in the cell. Ions (e.g. Cl\u2212, K+, metal), water, and osmolytes are imported or exported by the channels in their restricted role. In contrast, animals can make various ion gradients precisely and, in particular, can develop channel systems very well.Fig.\u00a03Comparison of numbers of channel genes among various organisms. Channel gene numbers were compared among many organisms (E. coli K12-MG1655, A. thaliana, O. sativa, C. elegans, D. melanogaster, H. sapiens NCBI, N. crassa 74-OR23-IVA, and S. cerevisiae S228C). ACC: ATP-gated Cation Channel; Bcl-2: Bcl-2; Bestrophin: Anion Channel-forming Bestrophin; CD20: CD20 Ca2+ Channel; ClC: Chloride Channel; Connexin: Gap Junction-forming Connexin; CytB: gp91phox Phagocyte NADPH Oxidase-associated Cytochrome b558 (CytB) H+-channel; E-ClC: Epithelial Chloride Channel; EnaC: Epithelial Na+ Channel; GIC: Glutamate-gated Ion Channel; Hsp70: Cation Channel-forming Heat Shock Protein-70; ICC: Intracellular Chloride Channel; Icln: Nucleotide-sensitive Anion-selective Channel; Innexin: Gap Junction-forming Innexin; IRK-C: Inward Rectifier K+ Channel; LIC: Ligand-gated Ion Channel of Neurotransmitter Receptors; Mid1: Yeast Stretch-Activated, Cation-Selective Ca2+ Channel Mid1; MIP: Major Intrinsic Protein; MIT: CorA Metal Ion Transporter; MscL: Large Conductance Mechanosensitive Ion Channel; MscS: Small Conductance Mechanosensitive Ion Channel; NSCC2: Non-selective Cation Channel-2; O-ClC: Organellar Chloride Channel; PCC: Polycystin Cation Channel; PLM: Phospholemman; RIR-CaC; Ryanodine-Inositol 1,4,5-triphosphate Receptor Ca2+ Channel; Tic110: Chloroplast Envelope Anion Channel-forming Tic110; TRP-CC: Transient Receptor Potential Ca2+ Channel; UT: Urea Transporter; VIC: Voltage-gated Ion Channel\nCompared with other systems, the channel system is the fastest at transporting molecules without consuming energy (i.e. when it is not necessary to transport against electrical gradients). Therefore, animals (especially vertebrates) have adapted these systems to nerve and muscle signal transmission. Nervous-system-specific channels such as neurotransmitter-responsible channels (connexin, ENaC, innexin, LIC, RIR-CaC, TRP-CC) are found only in animals. The numbers of channels that are membrane-voltage-dependent for Ca2+, K+, and Na+ ion transport are dramatically increased in both vertebrates and invertebrates (Table\u00a01, Fig.\u00a03; Sheng et\u00a0al. 2000; Du et\u00a0al. 2002; Clapham 2003; Hua et\u00a0al. 2003; Miyazawa et\u00a0al. 2003). Because animals form Na+ and K+ gradients with the Na+\/K+ ATPase pump system, there is also an increase in the numbers of species of Na+ and K+ ion channels in animals. Thus, the total number of channel genes in humans (320) is about twice that in plants (130\u2013180) (Table\u00a02, Fig.\u00a03). This difference is supposedly related to the ecological and physiological specificity of plants. Sometimes constant acquisition of unevenly distributed resources is difficult for plants. Additionally, the ion concentrations in the vascular systems and intercellular spaces of plants, unlike those in animals, are difficult to control precisely. Therefore, some of the ion channels and membrane-voltage-dependent channels are not as divergent as in animals. Although plants lack neurotransmission systems and have fewer membrane-voltage-dependent systems that use Na+ and K+ ion channels, the channel genes for ion homeostasis and signal transduction (e.g. ClC, GIC) are as well developed as in animals, and the numbers of some channel genes (CytB and MscS) are in fact specifically increased in plants (Table\u00a01, Fig.\u00a03).Table\u00a02Comparison of the genome size, total and membrane transport gene numbers in many organismsE. coli K12A. thalianaO. sativaC. elegansD. melanogasterH. sapiens N. crassa 74S. cerevisiae Genome Size (Mb)4.61254309712031504013Total gene number4,290 26,000 37,000 20,621 13,489 30,000 10,082 5,804 Total Transporter Proteins3549841200654590754344300Transporters per Mb genome76.747.822.846.754.30.248.6325.38Transporters per whole gene0.0820.0380.0330.0330.0440.0250.0340.052ATP-dependent pumps72 (20%)173 (18%)249 (21%)72 (11%)72 (12%)82 (11%)53 (15%)43 (14%)Ion Channels15 (4%)131 (13%)178 (15%)229 (35%)158 (27%)322 (43%)12 (3%)24 (8%)Phosphotransferase Systems (PTS)30 (8%)0000000Secondary Transporters233 (66%)658 (67%)762 (63%)349 (53%)351 (59%)336 (44%)273 (79%)227 (75%)Unclassified3 (1%)22 (2%)11 (1%)4 (1%)9 (1%)14 (2%)6 (2%)6 (2%)\nComparison of protein structures indicates that the fundamental structures of ion channels (numbers of transmembrane domains, pore-forming helices) are common in many organisms, but local similarities in individual regions among organisms are low (especially in the N and C terminus fragments). Some of the genes (e.g. for shaker-type K+ channel, SKOR-type K+ channel) have low levels of similarity among the whole structures of plants and animals (Supplemental data-3).\nIn contrast, the major intrinsic protein (MIP) gene family, which encodes water-transport proteins, is specifically well developed in plants (Zardoya 2005). The numbers of these genes in tissues and at different stages are 3\u20135 times those in animals (38 vs. 7\u201311) (Table\u00a01, Fig.\u00a03) (Quigley et\u00a0al. 2002; Sakurai et\u00a0al. 2005). MIPs are abundant in the plasma membrane (15\u201320% of total membrane protein) and vacuoles (30\u201350% of total membrane protein) of plants; therefore, a high level of water transport is carried out at plant cell membranes. The importance of the acquisition of water has presumably resulted in the diversification and development of water channels in plants. Plants also use water pressure for regulation of movement (e.g. stomatal opening, leaf and petal angle changes) and for transmitting signals in various homeostatic functions. Therefore, plants have presumably developed signal transduction systems that rely on water molecules instead of the neurotransmitters and membrane voltage changes used in animals.\nStructural analyses indicate divergence in the level of conservation of MIP subfamilies (PIP, TIP, SIP, NIP, AQP, and GLP) (Supplemental data-3E). The plasma-membrane-intrinsic proteins (PIPs) are well conserved among organisms, but animal- (AQP) and plant-specific (SIP, NIP, TIP) genes have diverged, and closely related channel proteins have low levels of similarity within each organism (Supplementary data-3E). This also suggests a specific diversification of the water transport genes in plants. The structures of the channels in plants are simple compared with those in animals (Supplementary data-3). A decrease in the membrane spanning times and in the number of subunits of working systems can be detected in many channels (e.g. VIC [VDCC], CIC) (Supplementary data-3). Therefore, animals have developed channel systems especially for the exquisite control of ions, membrane voltages, and the signal transduction pathways of specific ligands (e.g. neurotransmitters).\nPhosphotransferase system (bacteria)\nBacteria have specific membrane transport systems (phosphotransferase systems) for carbohydrates (sugar) and phosphates (Tables\u00a01, 2) (Barabote and Saier 2005). These systems use phosphate phosphoenol pyruvate (PEP) as the energy source for phosphorylation and for transport of carbohydrates with the aid of enzyme complexes. Carbohydrates (e.g. glucose, fructose, mannitol, sorbitol) are transported against a concentration gradient, with concomitant phosphorylation. PEP is transferred via the soluble (and non-sugar-specific) enzymes EI and HPr to the enzyme complex EII. EII is made up of components A, B, and C, which, depending on the sugar specificity and bacterium involved, may be the domains of composite proteins; component\/domain C is a permease and is anchored to the cytoplasmic membrane. Because the amount of phosphorylation of the enzymes influences other regulatory mechanisms in the cells (e.g. catabolite repression, chemotaxis), the whole cell needs a multiple-component, complex enzymatic control system. Therefore, the larger and more \ncomplicated cells \nof animals and plants have not adapted this system and instead use ATP-dependent and secondary transporter systems to transport carbohydrates.\nSecondary transporter (transporter) system\nThe secondary transporter system works via the concentration gradient of co-transporter molecules. It is efficient to use a few abundant molecules as common co-transporter molecules for various transport substrates. Therefore, in accordance with the species of ion for which there is a gradient between the inside and outside of the cell, major co-transporter molecules were selected and secondary transporter systems developed for them. There are more than 100 species of gene families in the secondary transporter systems of all organisms, and various substances are transported. Compared with those in the pump and channel systems, the genes involved in the secondary transporter system are most divergent in plants and bacteria; in animals there are about 350 of these genes (44\u201359% of the total) (Tables\u00a01, 2, Fig.\u00a04).Fig.\u00a04Comparison of numbers of secondary transport genes among organisms. Secondary transporter gene numbers were compared among Escherichia coli K12-MG1655, Arabidopsis thaliana, Oryza sativa, Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens NCBI, Neurospora crassa 74-OR23-IVA, and Saccharomyces cerevisiae S228C. AAAP: Amino Acid\/Auxin Permease; AE: Anion Exchanger; AEC: Auxin Efflux Carrier; Amt: Ammonium or Ammonia Transporter; APC: Amino Acid-Polyamine-Organocation; BASS: Bile Acid:Na+ Symporter; BCCT: Betaine\/Carnitine\/Choline Transporter; BenE: Benzoate:H+ Symporter; CaCA: Ca2+:Cation Antiporter; CCC: Cation-Chloride Cotransporter; CDF: Cation Diffusion Facilitator; CNT: Concentrative Nucleoside Transporter; CPA1: Monovalent Cation:Proton Antiporter-1; CPA2: Monovalent Cation:Proton Antiporter-2; DAACS: Dicarboxylate\/Amino Acid:Cation (Na+ or H+) Symporter; DASS: Divalent Anion:Na+ Symporter; DcuC: C4-dicarboxylate Uptake C; DMT: Drug\/Metabolite Transporter; ENT: Equilibrative Nucleoside Transporter; FBT: Folate-Biopterin Transporter; GntP: Gluconate:H+ Symporter; GPH: Glycoside-Pentoside-Hexuronide (GPH):Cation Symporter; HAAAP: Hydroxy\/Aromatic Amino Acid Permease; KUP: K+ Uptake Permease; LCT: Lysosomal Cystine Transporter; MC: Mitochondrial Carrier; MET: 4 TMS Multidrug Endosomal Transporter; MFS: Major Facilitator Superfamily; MOP: Multidrug\/Oligosaccharidyl-lipid\/Polysaccharide Flippase Superfamily; NCS2: Nucleobase:Cation Symporter-2; Nramp: Metal Ion (Mn2+\u2013iron) Transporter; NSS: Neurotransmitter:Sodium Symporter; OAT: Organo Anion Transporter; OPT: Oligopeptide Transporter; PnaS: Phosphate:Na+ Symporter; POT: Proton-dependent Oligopeptide Transporter; RFC: Reduced Folate Carrier; RhtB: Resistance to Homoserine\/Threonine; RND: Resistance-Nodulation-Cell Division; SSS: Solute:Sodium Symporter; SulP: Sulfate Permease; TDT: Tellurite-resistance\/Dicarboxylate Transporter; Trk: K+ Transporter; ZIP: Zinc (Zn2+)\u2013Iron (Fe2+) Permease\nOrganism-specific gene families in bacteria include those encoding proteins (e.g. APC, HAAAP) that transport many species of amino acid or carbohydrate (GntP, Dcu), or other substances (BCCT, BenE) (Neidle et\u00a0al. 1991; Golby et\u00a0al. 1998; Saier et\u00a0al. 1999; Jack et\u00a0al. 2000; Samsonov et\u00a0al. 2002; Prakash et\u00a0al. 2003). Gene families in animals include genes for specific transport proteins (NSS, MET) that carry neurotransmitters and hydrophobic compounds (Beckman and Quick 1998; Hogue et\u00a0al. 1999; Yamashita et\u00a0al. 2005). Those in plants include genes with dramatically increased expression, encoding proteins (AAAP, CPA2, DMT, MFS, OPT, POT) that transport such substances as amino acids, cations, and carbohydrates (Fig.\u00a04) (Fischer et\u00a0al. 1998, 2002; Pao et\u00a0al. 1999; Saier et\u00a0al. 1999; Jack et\u00a0al. 2001; Koh et\u00a0al. 2002; Wipf et al. 2002). The greater number of secondary active transporters in plants is supposedly due to the existence of vacuoles. Secondary active transporters need an ion gradient across the membranes, and vacuoles can pool many substances at high concentrations and supply co-transporter molecules instantly at any time. Therefore, the secondary transporter systems easily control transport and have diverged more in plants than in animals.\nThe major facilitator superfamily (MFS) is the largest secondary transporter family in whole organisms; its members transport various substances (e.g. carbohydrates, phosphates, amino acids, cations) with Na+ or H+ ions (Pao et\u00a0al. 1999; Burckhardt and Wolff 2000; Lemoine 2000). There are many modifications of these superfamily genes among organisms. Bacteria have amino acid and drug efflux families, animals have neurotransmitter transport families, and plants have diverse carbohydrate-transport genes (Tables\u00a01, 2, Fig.\u00a04, and Supplementary data-4). The proteins have structures typical of secondary active transporters: 10\u201314 transmembrane domains, co-transporter molecules, and material-binding domains. Comparative analyses indicate that their fundamental structure\u2014membrane-spanning, pore-forming, and substrate-binding sites\u2014is conserved, but similarities between individual domains are generally low among organisms (Tables\u00a01, 2, Fig.\u00a04 and Supplementary data-4). The transporter proteins are encoded by single transcription units and work as monomers. Therefore, this transport system is easy to adapt to new purposes than are the higher pump and ion channel systems. Although the secondary transporter system can adapt to many kinds of concentration gradients, generally few ion species are used. Most of the secondary active transporters depend on two ions: Na+ and H+. In the whole organism, more than 80% of the genes depend on these ions as co-transporter molecules (Table\u00a03, Fig.\u00a05). However, in many cases each ion can be substituted: animals use Na+ ions as co-transporter molecules and bacteria and plants use H+ ions. This has caused animals to make a Na+ ion gradient with Na+\/K+ ATPase for membrane voltage and plants and bacteria to make H+ ion gradients with P-type ATPase and H+-PPase, respectively. The transporter genes of animals and plants have specifically adapted to Na+ and H+ co-transporter systems (Table\u00a03, Fig.\u00a05). Therefore, both animals and plants have developed transporter genes adapted to ion gradients.Table\u00a03Comparison of secondary transporter genesE. coli A. thalianaO. sativaC. elegansD. melanogasterH. sapiens N. crassa S. cerevisiae Present inMaterialCotransporterDirectionAAA 02300000P (chloroplast)ATPH+ in (same)AAAP0437711151347A, P, F, YAmino acid, AuxinH+ in (same)AAE 10000000Bl-Aspartate, l-Alanine each otherboth (anti)AbgT10000000Bp-Aminobenzoyl-glutamate H+ in (same)ACR300000011B, YArsenite, Antimonite ?outAE 073421021A, P, YNa+, HCO3\u2212, H+, Cr, H3BO3H+, Na+, Cl\u2212both (anti)AEC 18800024P, Y, BAuxinH+ out (anti)AGCS10000000a, BAlanine, GycineNa+, H+in (same)Amt 161362443AllNH3, CO2?bothAPC 2212141111141524AllAmino acid, PolyamineH+, Solutein (same)ArAE311900000P, Y, B, aArmate acid (Malate etc.,)?outArsB 20005100All (with distant homolog)Arsenite, Antimonite ?outBASS 15602501AllOrganic acidNa+in (same)BCCT 30000000B, aBetaine\/Carnitine\/CholineH+ in (same)BenE 10000000BBenzoateH+ in (same)CaCA 21223811884AllCa2+H+, Na+both (anti) CCC 01265911AllNa+, K+, Cl\u2212Na+, K+, Cl\u2212both (same)CDF2811871085AllDivalent cation (Zn2+, Cd2+) H+, K+out (antit)CHR 00000010F, BSO42\u2212 , CrO42\u2212H+both (anti)CNT30022310A, Y, BNucleosideH+, Na+in (same)CPA1 287115332AllNa+,H+H+, Na+, Cl\u2212both (anti)CPA2 3321600121AllNa+, K+H+ (or itself)out (anti)DAACS 30062700A, BDicarboxylate, Amino acidH+, Na+in (same)DASS54543503A, P, Y, B (G\u2212)Aminoacid, Sulfate, Phosphate etc.,H+, Na+in (same), both (anti)Dcu20000000B (G\u2212)C4-Dicarboxylate Dicarboxylateboth (anti)DcuC20000000B (G\u2212)C4-DicarboxylateH+, Dicarboxylatein (same\u00a0=\u00a0H+) , both (anti) DMT 161215715141869AllC3 Carbohydrate, Sugar, Nucleotide etc.,H+, Nucleotideout (anti)ENT08453411A, P, YNucleoside?bothESS 10000000BGlutamateNa+in (same)FBT 09800000P, a, BFolate, BiopterinH+ in (same)FNT 40000011Y, a, BFormate, NitrateH+ in (same)GntP 70000000B (E. coli, Bacillus)CarbohydrateH+ in (same)GPH69811520A, P, F, a, BSugarH+, Na+ , Li+in (same)GUP 00100012A, P, F, Y, BGlycerolH+ in (same)HAAAP 81000000B, P (distant homolog)Amino acidH+ in (same)KDGT 10000000B2-Keto-3-DeoxygluconateH+ in (same)KUP 1132100010P, F, BK+noneinLCT06302211A, P, FCystineH+ in (same)LctP 20000000a, BLactate, Glycolate H+ in (same)LIV-E10000000a, BAmino acid (L, I, V)H+ in (same)LIVCS10000000BAmino acid (L, I, V)H+, Na+in (same)LysE 10000000BLysineH+, OH\u2212out (OH\u2212\u00a0=\u00a0same; H+\u00a0=\u00a0anti)MC 052663445443434EukaryoteC3 Carbohydrate, Sugar, Nucleotide etc.,Variousboth (anti)MET00000300ANucleoside, Hydrophobic compound H+ both (anti)MFS 71921451341368114185AllC3 Carbohydrate, Sugar etc.,H+, Na+, Variousboth (H+, Na+= same; others\u00a0=\u00a0both)MOP (MATE) 8564800213AllVarious (Drugs, Polysaccharides etc.,)Na+, (H+)out (anti)MTC 02262311EukaryoteVarious (Anionic substrate )H+ in (same)NCS1210000310P, F, Y, a, BNucleobase, Thiamine H+ in (same)NCS2 10121151410AllNucleobase, (Ascorbate\u00a0=\u00a0(mouse only)) H+, (Na+= (Ascotbate))in (same)NhaA 10000000ProkaryoteNa+, H+Na+, H+both (anti)NhaB 10000000AllNa+, H+Na+, H+both (anti)NhaD 02100000P, BNa+, (Li+), H+Na+, (Li+), H+both (anti)NiCoT 02000010AllNi2+, Co2+Ni2+, Co2+both (anti)Nramp 171021223AllFe2+, Zn2+, Mn2+, Co2+, Ca2+, Cu2+, Ni2+, Pb2+etc.,H+ in (same)NSS 00012211800ANeurotransmitters, Amino acids, Osmolytes, Cl\u2212 etc.,Na+in (same)OAT 022681100A, P, F, YVariousAnion both (anti)OPT 0153400043P, B, aOligopeptideH+ in (same)OST00000200EukaryoteOrganic compound (mostly anions)each otherboth (anti)Oxa1 14501101All (with distant homolog)Proteineach otherboth (anti)PiT 21351211AllHPO42\u2212, SO42\u2212H+, Na+in (same)PNaS 10010200A, B (distant homolog)Inorganic phosphate Na+in (same)POT 4506133421AllOligopeptideH+ both (same)RFC00033400AFolate, TiamineH+, OH\u2212, anionboth (anti)RhtB50000000a, BAmino acidH+ out (anti)RND821244721AllHeavy metals, Drugs, Lipids etc.,H+ out (anti)SSS4113191121AllSugar, Amino acid, Organo cation, AnionNa+, (H+ can replace, but reduces affinity)in (same)SulP 11115791144AllSO42\u2212H+ in (same)Tat10100000B (G\u2212), P (distant homolog)ProteinnoneoutTDT 14900021All (with distant homolog)Tellurite, C4-Dicarboxylate H+ in (same)ThrE10000002All (with distant homolog)Threonine, Serine H+ both (anti).TRAP-T10000000a, BVariousH+ in (same)Trk 21100022P, Y, BK+H+ in (same)ZIP 0131865253AllZn2+, Fe2+noneinNote: Present in: A\u00a0=\u00a0animal, P\u00a0=\u00a0plant, F\u00a0=\u00a0fungi, Y\u00a0=\u00a0yeast, B\u00a0=\u00a0bacteria, a\u00a0=\u00a0archea, G\u2212\u00a0=\u00a0gramm minus bacteria; Direction: \u201cin\u201d , \u201cout\u201d, and \u201cboth\u201d indicate transport direction of materials through cell membrane, and \u201csame\u201d means material and cotranspoter move to the same dirction, \u201canti\u201d means the different directionsFig.\u00a05Comparison of co-transport molecules of secondary active transporters among various organisms. Numbers of co-transport molecules of secondary active transporters were compared among Escherichia coli K12-MG1655, Arabidopsis thaliana, Oryza sativa, Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens NCBI, Neurospora crassa 74-OR23-IVA, and Saccharomyces cerevisiae S228C\nIn animals, many molecules are transported with different co-transporter molecules. Examples are (1) molecules (e.g. neurotransmitters, organic anions, phosphates) transported with Na+ ion (NSS, BASS, CPA2, Pnas) (Beckman and Quick 1998; Saier et\u00a0al. 1999; Kramer et\u00a0al. 2001; Segawa et\u00a0al. 2002; Radchenko et\u00a0al. 2006); (2) molecules (e.g. amino acids, nucleosides) transported with H+ ions (e.g. AAAP, APC, DMT, LCT, MET) (Steiner et\u00a0al. 1994; Jack et\u00a0al. 2001; Zhai et\u00a0al. 2001; Gasol et\u00a0al. 2004); (3) molecules (e.g. anions, cations, sugars, nucleosides) transported with either Na+ or H+ ions (e.g. AE, CaCA, DAACS, GPH, CNT) (Reinders and Ward 2001; Ritzel et\u00a0al. 2001; Zhu et\u00a0al. 2003; Cai and Lytton 2004; Ryan et\u00a0al. 2004); and (4) others (e.g. CCC, OAT, RFC) transported with other molecules (Russell 2000; Flintoff et\u00a0al. 2003; Hagenbuch and Meier 2003).\nIn contrast, in plants, almost all substances (K+: KUP; N (): Amt; phosphate: PiT, Pht [MFS], and TPT [DMT]; : SulP; sucrose: GPH; sugar alcohol: SAT [MFS]; monosaccharide: MST [MFS]; amino acid: APC and AAAP; nucleosides: NCS1), including many cations, anions, metals, and drugs, are co-transported with H+ ions. Only BASS (organic acid transporter) and NhaD (Na+: H+ antiporter\u00a0=\u00a0Na+: efflux system of vacuole) use Na+ ions as co-transporter molecules for efflux of ions from the cytoplasm, so plants do not require extensive sodium uptake (Table\u00a03, Fig.\u00a05) (Smith et\u00a0al. 1995; Naderi and Saier 1996; Fu and Luan 1998; Nozaki et\u00a0al. 1998; Pao et\u00a0al. 1999; Daram et\u00a0al. 1999; de Koning and Diallinas 2000; Jack et\u00a0al. 2001; Khademi et\u00a0al. 2004). Accumulation of H+ ions makes solutions acidic and prevents many enzymatic and biochemical reactions, so plants pool H+ ions in vacuoles inside the cytoplasm, thereby establishing a stable ion gradient for transport energy. Plants make H+ gradients not only inside cells, but also outside cells by the H+ pump, and they absorb many substances by secondary transporter systems. This H+-adapting system presumably has led to diversification of H+ co-transport in secondary transporter systems in plants.\nComparative analysis of membrane transporter systems in Arabidopsis and rice\nWe also compared the numbers of membrane transporter genes in Arabidopsis and rice. The rice genome size (430\u00a0Mb) is more than three times that of Arabidopsis (125\u00a0Mb), but the total number of membrane transporter proteins (1200) is only 1.20 times that in Arabidopsis (1000) (Table\u00a02). The proportions of pump, channel, and secondary transporter proteins are almost the same, and the numbers of genes in many individual gene families are 1.1\u20131.3 times those in rice. However, Arabidopsis has more multi-efflux proteins and proteins involved in secondary active transport (CPA2, DMT, MOP, NCS2) and in channel-type signal transduction systems (CytB, GIC) (Fig.\u00a06). Arabidopsis is a wild plant that lives in diverse soil conditions and under environmental stress, whereas rice is a cultivated crop plant grown under more stable environmental conditions. Therefore, Arabidopsis has diverged more than the rice cultivar Nipponbare to form transporter systems involved in multi-efflux and stress response signaling. On the other hand, rice has more pump and secondary transporter genes (ABC, P-type ATPase, MFS, POT) for carbohydrate and nutrient transport systems (Fig.\u00a06). This divergence of carbohydrate transporters might have been influenced by artificial selection, during which individuals with larger seed size and numbers were chosen to be cultivated. Additionally, amino acid (AAAP), ammonia (Amt), sulfate (SulP), metal ion (ZIP), Ca2+ (CaCA), and K+ transport protein gene families (VIC, KUP) are specifically diverged in rice. This may have been caused by the need for nutrient supplements for rapid growth in subtropical plants. These differences may also result from dissimilarities between monocots and dicots. Although the cell structures are the same in both plants, there are many differences in the basic structure of tissues and organs. Differences in root and vascular bundle structure may be related to differences in uptake efficiency of nutrients. The levels of uptake and transport amounts of ions such as K+, Fe2+, and other nutrients differ between monocots and dicots; this might be related to differences in the numbers of membrane transport genes.\nFig.\u00a06Comparison of membrane transport genes in Arabidopsis and rice. Numbers of the membrane transporter gene families are compared in Arabidopsis and rice. ABC: ATP-binding Cassette; P-ATPase: P-type ATPase; CytB: gp91phox Phagocyte NADPH Oxidase-associated Cytochrome b558 (CytB) H+-channel; GIC: Glutamate-gated Ion Channel; MIP: Major Intrinsic Protein; VIC: Voltage-gated Ion Channel; AAAP: Amino Acid\/Auxin Permease; Amt: Ammonium or Ammonia Transporter; CaCA: Ca2+:Cation Antiporter; CPA2: Monovalent Cation:Proton Antiporter-2; DMT: Drug\/Metabolite Transporter; KUP: K+ Uptake Permease; MFS: Major Facilitator Superfamily; MOP: Multidrug\/Oligosaccharidyl-lipid\/Polysaccharide Flippase Superfamily; NCS2: Nucleobase:Cation Symporter-2; OPT: Oligopeptide Transporter; POT: Proton-dependent Oligopeptide Transporter; SulP: Sulfate Permease; ZIP: Zinc (Zn2+)\u2013Iron (Fe2+) Permease; PUP: Peptide Uptake or Activated Fatty Acid Export Permease\nComparison of the membrane transport genes in Arabidopsis and rice pointed to the directions of evolution of these plants in response to the selection pressures of their differing environmental situations. The level of conservation of gene families in Arabidopsis and rice varies, depending on the category. Rice membrane transporters were 72% orthologous with those of Arabidopsis; this is higher than the 50% ortholog found in transcription factors (Xiong et\u00a0al. 2005). The levels of expression of membrane transport genes are limited precisely within a small range, and these genes are critical for survival. This may be the reason why the membrane transport genes were less divergent than the transcription factors. Therefore, the specifically divergent gene families in Arabidopsis and rice are related to the indispensable and unique systems in each plant. Divergence of gene patterns might indicate differences between wild weeds and crop cultivars that have been selected for growth in specific and possibly new environments.\nComparison of the overall gene compositions of bacteria (E. coli), animals (H. sapiens), and plants\nIn the eight diverse organisms that we compared, the number of membrane transporter genes (300\u20131200) varied less than that of genes in other categories. The minimum number of genes indispensable to retaining cell membrane transport homeostasis thus seems to be 300\u2013350, as found in bacteria, yeasts, and fungi. Many of the additional newly diverged genes of higher animals and plants are channel transporter genes and secondary active transporters that facilitate adaptation to fluctuating concentration gradients present in their environments. Moreover, many newly acquired transporters are highly specifically stage- and tissue-regulated and transport special substrates such as neurotransmitters (ACC) in neural cells or carbohydrates (POT) in leaf tissue.\nComparison of the overall gene compositions of bacteria (E. coli), animals (H. sapiens), and plants (A. thaliana and O. sativa) reveals the strategies for osmotic pressure adjustment and the features of the substance-transport systems in each organism (Fig.\u00a07). Since a monad cannot control the ion environment of its external world and has no need to communicate with other cells, the role of its transporter proteins is restricted to the control of material transport into and out of the cell. Therefore, transport depends mainly on an energy-consuming system (pump) and an internal ion-gradient-dependent system (secondary transporter). Because the bacterium has a cell wall, osmotic pressure is opposed by cell wall pressure and there is no need to form a molecular concentration gradient to prevent excessive accumulation within the cell. Additionally, bacteria are small (1\u20135\u00a0\u03bcm, <1\/10 of the size of an animal cell) and do not have many of the organelles and membrane structures possessed by higher organisms. Therefore, transport of substances is simple and it is easy to control their concentrations in the cell. The genes encoding the ATP-dependent pump (ABC), phosphotransferase (PTS), small conductance mechanosensitive ion channel (MscS), and secondary active transporters (e.g. APC, DMT, MFS, NCS2, RND) with H+ or Na+ as co-transporter molecules have diverged in bacteria (Fig.\u00a07).\nFig.\u00a07Summaries of comparison of membrane transport genes in bacteria, animals, and plants. Bacteria-specific genes\u00a0=\u00a0blue; animal-specific genes\u00a0=\u00a0red; plant-specific genes\u00a0=\u00a0green; bacteria- and animal-specific genes\u00a0=\u00a0purple; bacteria- and plant-specific genes\u00a0=\u00a0brown; genes with divergent numbers in organisms\u00a0=\u00a0yellow. ABC: ATP-binding Cassette; ArsAB: Arsenite-Antimonite Efflux; F-ATPase: H+or Na+-translocating F-type, V-type and A-type ATPase; H+-Ppase: H+-translocating Pyrophosphatase; IISP: General Secretory Pathway (Sec); MPT: Mitochondrial Protein Translocase; P-ATPase: P-type ATPase; ACC: ATP-gated Cation Channel; Annexin: Annexin; Bcl-2: Bcl-2; Bestrophin: Anion Channel-forming Bestrophin; CD20: CD20 Ca2+ Channel; ClC: Chloride Channel; Connexin: Gap Junction-forming Connexin; CSC: Chloroplast Outer Envelope Solute Channel; CytB: gp91phox Phagocyte NADPH Oxidase-associated Cytochrome b558 (CytB) H+-channel; E-ClC: Epithelial Chloride Channel; EnaC: Epithelial Na+ Channel; GIC: Glutamate-gated Ion Channel; Hsp70: Cation Channel-forming Heat Shock Protein-70; ICC: Intracellular Chloride Channel; Ic ln: Nucleotide-sensitive Anion-selective Channel; Innexin: Gap Junction-forming Innexin; IRK-C: Inward Rectifier K+ Channel; LIC: Ligand-gated Ion Channel of Neurotransmitter Receptors; Mid1: Yeast Stretch-Activated, Cation-Selective Ca2+ Channel Mid1; MIP: Major Intrinsic Protein; MIT: CorA Metal Ion Transporter; MscL: Large Conductance Mechanosensitive Ion Channel; MscS: Small Conductance Mechanosensitive Ion Channel; NSCC2: Non-selective Cation Channel-2; O-ClC: Organellar Chloride Channel; PCC: Polycystin Cation Channel; PLM: Phospholemman; RIR-CaC; Ryanodine-Inositol 1,4,5-triphosphate Receptor Ca2+ Channel; Tic110: Chloroplast Envelope Anion Channel-forming Tic110; TRP-CC: Transient Receptor Potential Ca2+ Channel; UT: Urea Transporter; VIC: Voltage-gated Ion Channel; AAA: ATP:ADP Antiporter; AAAP: Amino Acid\/Auxin Permease; AAE: Aspartate:Alanine Exchanger; AbgT: p-Aminobenzoyl-glutamate Transporter; ACR3: Arsenical Resistance-3; AE: Anion Exchanger; AEC: Auxin Efflux Carrier; AGCS: Alanine or Glycine:Cation Symporter; Amt: Ammonium or Ammonia Transporter; APC: Amino Acid-Polyamine-Organocation; ArsB: Arsenite-Antimonite (ArsB) Efflux; BASS: Bile Acid:Na+ Symporter; BCCT: Betaine\/Carnitine\/Choline Transporter; BenE: Benzoate:H+ Symporter; CaCA: Ca2+:Cation Antiporter; CCC: Cation-Chloride Cotransporter; CDF: Cation Diffusion Facilitator; CHR: Chromate Ion Transporter; CNT: Concentrative Nucleoside Transporter; CPA1: Monovalent Cation:Proton Antiporter-1; CPA2: Monovalent Cation:Proton Antiporter-2; DAACS: Dicarboxylate\/Amino Acid:Cation (Na+ or H+) Symporter; DASS: Divalent Anion:Na+ Symporter; Dcu: C4-Dicarboxylate Uptake; DcuC: C4-dicarboxylate Uptake C; DMT: Drug\/Metabolite Transporter; ENT: Equilibrative Nucleoside Transporter; ESS: Glutamate:Na+ Symporter; FBT: Folate-Biopterin Transporter; FNT: Formate-Nitrite Transporter; GntP: Gluconate:H+ Symporter; GPH: Glycoside-Pentoside-Hexuronide (GPH):Cation Symporter; GUP: Glycerol Uptake; HAAAP: Hydroxy\/Aromatic Amino Acid Permease; KDGT: 2-Keto-3-Deoxygluconate Transporter; KUP: K+ Uptake Permease; LCT: Lysosomal Cystine Transporter; LctP: Lactate Permease; LIV-E: Branched Chain Amino Acid Exporter; LIVCS: Branched Chain Amino Acid:Cation Symporter; LysE: L-Lysine Exporter; MC: Mitochondrial Carrier; MET: 4 TMS Multidrug Endosomal Transporter; MFS: Major Facilitator Superfamily; MOP: Multidrug\/Oligosaccharidyl-lipid\/Polysaccharide Flippase Superfamily; MTC: Mitochondrial Tricarboxylate Carrier; NCS1: Nucleobase:Cation Symporter-1; NCS2: Nucleobase:Cation Symporter-2; NhaA: Na+:H+ Antiporter A; NhaB: Na+:H+ Antiporter B; NhaD: Na+:H+ Antiporter D; NiCoT: Ni2+\u2013Co2+ Transporter; Nramp: Metal Ion (Mn2+\u2013iron) Transporter; NSS: Neurotransmitter:Sodium Symporter; OAT: Organo Anion Transporter; OPT: Oligopeptide Transporter; OST: Organic Solute Transporter; Oxa1: Cytochrome Oxidase Biogenesis; PiT: Inorganic Phosphate Transporter; PnaS: Phosphate:Na+ Symporter; POT: Proton-dependent Oligopeptide Transporter; RFC: Reduced Folate Carrier; RhtB: Resistance to Homoserine\/Threonine; RND: Resistance-Nodulation-Cell Division; SSS: Solute:Sodium Symporter; SulP: Sulfate Permease; Tat: Twin Arginine Targeting; TDT: Tellurite-resistance\/Dicarboxylate Transporter; ThrE: Threonine\/Serine Exporter; TRAP-T: Tripartite ATP-independent Periplasmic Transporter; Trk: K+ Transporter; ZIP: Zinc (Zn2+)\u2013Iron (Fe2+) Permease; GPTS: general carbohydrate phosphotransferase; SSPTS: sugar specific phosphotransferase; Ctr1: Dipicolinic Acid Transporter; Ctr2: Copper Transporter; FeoB: Ferrous Iron Uptake; FeT: Low Affinity Fe2+ Transporter; FP: Ferroportin; LPI: Lysosomal Protein Import; OfeT: Iron\/Lead Transporter; PnuC: nicotinamide mononucleotide(NMN) uptake permease; PPI: Peroxisomal Protein Importer; PUP: Peptide Uptake or Activated Fatty Acid Export Permease\nIncreases in the size and complexity of cells with the emergence of organelles and an internal membrane structure arose in the process of evolution from prokaryote to eukaryote. The eukaryote\u2019s inclusion of the prokaryote\u2019s transport genes in the mitochondrion or chloroplast allowed the transport of homeostatic substrate to organelles, as well as other materials and the products of specific biosynthesis (tricarboxylic acid cycle, electron-transport chain, and photosynthesis). Some of the genes are indistinguishable between eukaryotic transcript and organelle transporter, but there are 50\u2013100 typical organelle transporters (e.g. MC, MCX, Tat) in eukaryotes, and they make a large group (7\u201312% of all transporters). Furthermore, with the development of multicellular eukaryotes there came a need for organisms to develop a system of transmission between cells. Animals and plants developed specific systems to solve the problem. Animals lost the cell wall structure and gained cellular mobility and flexibility, which also promoted integration of the whole body and specialization of tissues and cells. The loss of the cell wall also led to the need to control osmotic pressure against the plasma membrane. Animal cells regulate osmotic pressure by raising the Na+ concentration outside the plasma membrane by active transport with Na+\/K+ ATPase. This Na+ gradient is used not only for homeostasis of the osmotic pressure of the membrane, but also as an electrical potential to control the voltage-dependent channel system and for co-transport of symport or antiport molecules of secondary transportation. Animals then developed the channel system (e.g. Bcl-2, connexin, E-ClC, EnaC, ICC, Ic ln, LIC, PCC, PLM, RIR-CaC, VIC) for rapid transmission in nerve and muscle tissues, together with an Na+ gradient dependent on the secondary transport system (e.g. NSS, PnaS, SSS). This Na+ ion gradient adaptation system is a feature of the newly added transporters and has evolved with animal specificities, i.e. the need for rapid movement and adaptation to environmental stimulation.\nIn contrast to animals, plants have retained the cell wall instead of acquiring mobility. Because their cell structures and sizes became complicated and large, plants also evolved a means of controlling osmotic pressure; this means differed from the cell-wall-dependent system of bacteria. Because the plant cell controls osmotic pressure by the turgor pressure of the cell wall, it does not require a sodium or other ion gradient and lacks an Na+\/K+ ATPase for control of osmotic pressure against the plasma membrane. On the other hand, an increase in permeability to water is necessary for smooth transmission of water pressure in the large and complicated structures of the cell. Therefore, water channels are abundant in both the plasma membrane and the vacuoles (20\u201350% of membrane proteins), and the diversity of water channels (MIP) is also four to five times higher than that in bacteria and animals. Compared with circulation systems in animals, whole-plant circulation systems are simple and less controlled. Plant cells also need to be more self-complementary in their homeostasis. Additionally, plants have less choice in their environmental conditions (e.g. soil, weather, stress) and must have more transport systems for cell homeostasis than animals. This is why plant cells include vacuoles to pool many substances and bioactive compounds.\nBecause plants obtain energy through photosynthesis, there are more ATP-dependent (pump) transporters (ABC, P-type ATPase) in their cells than in animal cells. Unlike the case in animals, voltage-dependent systems and Na+-ion-dependent secondary transport systems have not developed in plants; systems that rely on H+ rather than Na+ have developed as part of plant secondary transporter systems. Because the increase in H+ brings acidification, plants maintain the neutrality of the cytoplasm by accumulating H+ in vacuoles, which are membrane-bound organelles separated from the other cytoplasmic contents. By having an internal ion pool, plant cells can control the ion gradient easily and safely. Therefore, secondary transport genes (e.g. CPA2, DMT, MFS, MOP, POT) that use H+ as a co-transporter molecule have diverged far in plants (Fig.\u00a07). The inclusion of pooling and energy-synthesizing organelles in the cell may be why plant cells (10\u2013100\u00a0\u03bcm) are larger than animal cells (10\u201330\u00a0\u03bcm).\nConclusions\nComparative analysis of membrane transporters among these eight diverse organisms indicates the type of cell homeostasis, as evidenced by the pattern of gene conservation and diversification. Evolutionary changes in gene families, in general, indicate the dynamics of alterations in biological systems and gene networks. Therefore, analysis of large categories of gene families may reveal many basic concepts of biological systems. In practice, analyses of the membrane transporter mechanism are useful in revealing changes in the absorption of molecules by, or their efflux from, cells and tissues. This information also is useful for examining changes in soil adaptability, nutritional demand, and stress tolerance in plants. It may also help to improve the harvest of crop cultivars or extend areas habitable by plant species. Gene networks are intricately related, and analysis of the whole genetic structure is needed to gain a full understanding of biological phenomena and systems of gene regulation. We are continuing to analyze whole categories of genes in an effort to develop an overview of total gene networks.\nElectronic supplementary material\n(DOC 21 kb)\n(XLS 189 kb)\n(PDF 151 kb)\n(PDF 192 kb)\n(PDF 165 kb)","keyphrases":["membrane transporter","oryza sativa","comparative molecular analysis"],"prmu":["P","P","R"]}
{"id":"Chiropr_Osteopat-13-_-1151650","title":"Is obesity a risk factor for low back pain? An example of using the evidence to answer a clinical question\n","text":"Background Obesity as a causal factor for low back pain has been controversial with no definitive answer to this date. The objective of this study was to determine whether obesity is associated with low back pain. In addition this paper aims to provide a step-by-step guide for chiropractors and osteopaths on how to ask and answer a clinical question using the literature.\nIntroduction\nObesity is a problem of epidemic proportion [1,2]. Despite record rates of non-physician supervised dieting and the availability of numerous weight loss programs, the problem is not abating [3]. Complicating this, is that most primary care physicians do not treat obesity, citing a lack of time, resources, insurance reimbursement, and knowledge of effective interventions as significant barriers [4]. Musculoskeletal disorders including low back pain (LBP) represent a considerable public health problem and a common diagnosis creating absenteeism and the need for disability pensions [5]. It is estimated that about 80% of the United States and Canadian population will experience LBP during adulthood [6]. Most low back pain is self-limiting and will ultimately resolve in two weeks (50% of those affected) to six weeks (90% of those affected), however it remains an intriguing clinical problem [6].\nIt is widely noted that the economic cost of obesity and its related disorders are staggering, with lifestyle related conditions such as diabetes mellitus and coronary heart disease placing a large economic burden on the health care system [1-4]. However, low back pain also has a significant socioeconomic impact. Cost estimates range from US$20 billion to $50 billion annually, with 10% of the patients accounting for 85 to 90% of the costs [6]. In Australia, Walker et al estimated the cost of low back pain in 2001 alone to be AUD$9.17 billion [7].\nOne question, which arises from the discussion concerning obesity, is whether obesity is a risk factor for low back pain. \"Buckwalter et al contended that a number of medical conditions including obesity, along with diabetes and hypertension, may influence the pathophysiology of diseases of the tendons and ligaments during the process of aging thus potentially leading to low back pain [8]. Along with low back pain, the conventional wisdom is that overweight persons are at risk of osteoarthritis in weight-bearing joints such as the knee, the hips, and feet [9].\nTo date, literature reviews have given conflicting views based on the available data and method of data retrieval. The purpose of this review is to establish, from recent research, if there is a causal link between obesity and the affliction of low back pain. A secondary purpose of this review is to present the concepts of evidence-based practice to aid the chiropractor or osteopath in looking for health-related evidence for their patients who present with obesity.\nMethods\nA MEDLINE search, from the National Library of Medicine, was used to ascertain pertinent articles between the years 1990 and 2004. The use of keywords \"obesity\", \"body mass index\", \"BMI\" and \"low back pain\" was used to obtain relevant studies. The references of papers retrieved were also reviewed, as were key texts and references.\nThis section is devoted to presenting the concepts of evidence-based practice (EBP) to demonstrate to the reader the discovery process for finding a possible link between obesity and low back pain. The EBP method used is shown in Table 1.\nTable 1\nSteps to asking the answerable question using EBP principles\nStep 1: Asking an answerable question\nStep 2: Selecting an evidence resource\nStep 3: Executing the search strategy\nStep 4: Examining the evidence summary\nStep 5: Application of the evidence\nThe first step in this process is \"asking an answerable question.\" In this paper we assume a patient has asked whether being overweight can cause low back pain. Construction of an appropriate answerable question would possibly be \"Does an increased BMI cause low back pain?\" \"Does being overweight create osteoarthritis?\" In this way questions can be constructed to allow the practitioner to effectively answer a clinical concern.\nOnce the answerable question has been constructed the next task is to find an adequate resource. Internet access to the U.S. National Library of Medicine's MEDLINE or PUBMED, these database systems are considered by many experts to be the most up to date data source on medically related topics. The next step is to determine keywords to place in the search engine. From the answerable question(s) it can be appreciated that the initial keywords will be \"low back pain\", \"BMI\" and \"osteoarthritis.\" This search constitutes the third step. In initiating the search, one should look for the search engines \"limits\" area. In this area can one designate an age group (ex: 45 to 60 years), date span of the literature search, (ex: 1998 to 2003), and to select either English language or articles in foreign languages.\nOnce the search has been completed, the articles, which may answer the question, are isolated and can be read. Step four involves collating the evidence to answer the question. In searches one may find answers that were not known to exist, and information that may challenge an already preconceived notion. The evidence summary should list the main author and year the paper was published. This is in order to retrieve the data should anyone wish to examine the source. As an example of an evidence summary see Table 2.\nTable 2\nRecent evidence: Obesity and low back pain (chronological order)\nAuthor, Year (Ref)\nN\nBMI\nLBP\nAssociation\nMelissas, 2003 [14]\n50\n>40\n58%\ndirect\nBener, 2003 [10]\n802\n(26.4 males\/ 27.8 females)\n56.1% males\n73.8% females\nmoderate\nTsuritani, 2002 [16]\n709\n--\n40.3%\nnone\nBowerman, 2001 [4]\n252\n--\n29.2%\nnone\nKostova, 2001 [12]\n898\n--\n--\nincreased risk\nBayramoglu, 2001 [15]\n25\n--\n--\ndirect\nMortimer, 2001 [13]\n475\n30 (43.6%)\n31\u201340 (28.8%)\n40+ (1.3%)\n--\nincreased risk\nHan, 1997 [11] 7018 women\n5887 men NR\n--\nfemales increased risk\nN = number; BMI = body mass index; LBP = low back pain; NR = not reported\nIt is at this time that the clinician is ready for the final step of applying the evidence. In our example clinical data from the experimental literature may or may not indicate that there is a link between overweight and low back pain.\nResults\nThe literature search into obesity and joint pain revealed several studies pertinent to the debate. Table 2 reveals an overview of the studies selected. Several studies [4,10-13] had large populations to draw from yet the data from these studies were not in agreement as to a cause or association. In fact, only two studies [14,15] found a direct association for obesity as a risk factor while two [4,16] studies found no association. Several of the studies reviewed were unable to clarify BMI to the satisfaction of the authors.\nDiscussion\nInterest in the association between obesity and low back pain has piqued researchers interest for many years. Intuitively, a burgeoning waistline and an increased lordotic lumbar spine led researchers to conclude that overweight people would be more prone to low back pain. Historically, Kellgren and Lawrence (1958) found that that the prevalence of disk degeneration with obesity was not significant [17]. However, it was not until the mid-1970's when several studies observed a possible association. Obesity was found to increase the prevalence of disk degeneration significantly in a study by Magora and Schwartz in 1976 [18]. Barton et al (1976), in a review of 144 cases, found that 70% of those who complained of low back pain had been classified as being overweight [19]. This basic research appeared to conclude what was already intuitively thought about low back pain and increased weight.\nBody mass index\nBefore an in-depth discussion of low back pain and obesity can ensue, the concept of Body Mass Index (BMI) needs to be discussed. BMI is a measure of fatness and is calculated by dividing the patient's weight in kilograms by height in metres squared kg\/M2 [20]. It is widely accepted, easily measured, and predicts morbidity and mortality in many populations [15]. Obesity is generally defined as a BMI of 30 kg\/m2 and higher [20,21]. Overweight is defined as a BMI between 25 and 30 kg\/m2 [19,20]. Overweight tends to be more common in men with obesity being more prevalent among women [21]. When body weight is increased 20% above average, mortality rises to 20% for men and 10% for women [22]. (Table 3) Overweight individuals demand more from their cardio-respiratory and musculoskeletal systems [22]. It is known that more than 50% of adult Americans have a BMI equal to or greater than 25 [23]. Although there are certain limitations to BMI i.e. large muscular athletes who are in good cardiovascular shape, the rationale behind these numbers is that, across large population groups, there is an increased prevalence of certain diseases in people with a BMI over 25, and a much greater risk of disease and death in those with a BMI over 30 [4]. Being overweight or obese substantially raises the risk of developing hypertension, coronary heart disease, type 2 diabetes, stroke, gallbladder disease, sleep apnea and other respiratory problems, prostate and colon cancers [4,23]. Yet, the evidence to date linking it to low back pain is not as clear cut as it is with the previously stated pathologies.\nTable 3\nClinically relevant differentiation between obesity and overweight\nOverweight\nObesity\nBMI of 25.0 to 29.9 kg\/m2\nBMI greater than 30 kg\/m2\nBMI calculation without benefit of BMI charts\nBody Mass Index (BMI) charts and hand held scales are available for individual clinician use. It is, however, unknown to what degree chiropractors or osteopaths use such tools. The following section is designed to aid the clinician with calculating BMI without benefit of chart or hand held scales.\nAs noted earlier, BMI is calculated as weight in kilograms divided by height in square metres [20,24]. This method is often too difficult to calculate for most people. A simpler method for those using the imperial system of measures is to take body weight in pounds \u00d7 703\/height in inches squared.\nFor example, a person weighing 150 pounds at 6 foot tall would correspond to a BMI of 20.3. TABLE 4\nTable 4\nCalculation of BMI\n150 \u00d7 703 = 105450 divided by 72 inches (6 foot) squared.\n105450 divided by 5184 (72 \u00d7 72) = 20.3 BMI.\nAdditional research findings\nLeboeuf-Yde concluded from a review of the literature that due to lack of evidence, body weight should be considered a possible weak risk indicator and suggested that there is insufficient data to assess if it is a true cause of LBP [25]. Kostova found that in men over 40, overweight, obesity and number of pack years of smoking, estimated by duration of smoking and daily cigarette consumption (more than 20 years and more than 20 cigarettes per day), increased the risk of developing back disorders [12].\nDespite these two studies, Garzillo et al and Leboeuf-Yde et al have given conflicting opinions [26,27]. Garzillo's review of the data revealed a possible association between obesity and low back pain only in the upper quintile of obesity, and no evidence of a temporal relationship between weight change and changes in low back pain [26]. Leboeuf-Yde concluded from a twin study that obesity is modestly positively associated with low back pain, in particular with chronic or recurrent low back pain [27].\nWhat appears to be a main concern in linking obesity as a causal factor for low back pain is the numerous variables encountered in these subjects. For example, it is hypothesized that overweight adult females may have negative self-concepts and body images compounded by chronic low back pain and obesity, these may be confounding factors [28]. Other variables such as less activity and\/or muscular weakness leading to obesity are also possible considerations.\nObesity and low back pain-related conditions\nNot only is there controversy in obesity and low back pain, but there exists conflicting views of obesity and low back pain-related conditions such as spondylosis, decreased physical activities and discal herniation. The studies demonstrating a positive association are many. O'Neil et al noted that increasing BMI is associated with more frequent findings of osteophytes (bone spurs) at both the thoracic and lumbar spines [29]. The correlation of osteophytes and increased BMI is highest at the thoracic level [29]. Biering-Strenson et al noted absolute weight and BMI are significantly higher in persons 60 years of age with spondylosis [30]. Both men and women with BMI of 30 kg\/m2 or higher were twice as likely to have difficulties in performing a range of basic daily physical activities [30]. Compared with women with BMI lower than 25 kg\/m2, those with BMI of 30 kg\/m2 or higher were 1.5 times more likely to have symptoms of intervertebral disk herniation [31].\nConversely, Luoma et al concluded that disc degeneration is not related to body height, overweight, smoking, or the frequency of physical activity [32]. In addition, studies by Riihimaki, Symmons, and Kang have shown no association between BMI and low back related problems [33-35].\nConfounding the data is that the mechanism by which excess body weight causes osteoarthritis is poorly understood [9]. It is believed that contributions from both local increased force across the joint and systemic factors play a role [9]. A discriminating factor between fit and unfit patients with back pain may be the fact that fit persons more frequently are still employed, and as such may be involved more in physical activity [36]. Table 5 indicates where the research currently exists for the link between low back pain and obesity along with obesity and osteoarthritis.\nTable 5\nBMI-related risk of osteoarthritis and low back pain\nIf your BMI is\nthen your risk based solely on BMI\n<25\nminimal\n25 \u2013 <27\nminimal\n27 \u2013 <30\nminimal\n30 \u2013 <35\nmoderate\n35 \u2013 <40\nmoderate\n>40\nmoderate to high\nWe conclude, based on the available evidence to date, that those individuals with a BMI of under 30 are at a minimal risk of developing low back pain while those persons whose BMI increases to over 30 are a moderate risk of developing low back pain. We also suggest, based on the findings of the Melissas study [14] of those patients who relieved their low back pain symptoms after obesity surgery, that patients with a BMI of greater than 40 are at a high risk of developing low back pain. Albeit controversial, Table 5 may lead to a further refinement of risk of osteoarthritis and low back pain based solely on BMI.\nLimitations of obesity as a risk factor for low back pain\nA significant difficulty in ascertaining cause and effect between obesity and low back pain is undoubtedly the term \"low back pain\" itself. Low back pain is a symptom not a diagnosis. A specific diagnosis, instead of the generalized form of \"low back pain\" may help separate out the association between LBP and obesity.\nThe Agency for Health Care Policy and Research (AHCPR) in their 1995 Acute Low Back Problems in Adults noted common diagnoses used to explain back problems [37] (Table 6). Given these possible diagnoses one can readily appreciate the dilemma in attempting to link obesity with its specificity in measurement to a broad symptom such as low back pain.\nTable 6\nCommon diagnoses used to explain back symptoms\nAnnular tear\nAdult spondylolysis\nMyofascitis\nFibromyalgia\nDisc syndrome\nStrain\nSpondylosis\nLumbar disc disease\nFacet syndrome\nDegenerative joint disease\nSprain\nSpinal OA\nDisc derangement\/disruption\nDislocation\n*Other potential causes of low back pain symptomology\nFailed Back Surgery Syndrome*\nOsteoporosis*\nUrinary tract infection*\nCompression fracture*\nSomato-visceral mimicry syndrome*\nOrganic pathology (tumor, rheumatoid, endometriosis, arthritic disorders)*\nLeg length inequity*\nSacro-iliac dysfunction*\nHip disorder*\n**Disagreement in research as cause of low back symptomology\nMorbid obesity?**\nOA = osteoarthritis\nAnother problem is the hypothesis that a person who suffers with continuing bouts of low back pain may be predisposed, due to inactivity or inability to exercise, to gain weight thus increasing their BMI. This hypothesis to our knowledge, has yet to be fully discussed and investigated.\nConclusion\nThe data for a link between obesity and low back pain appears to be controversial. Yet, this does not adequately address the appropriate therapeutic approach to the obese patient with low back pain. The studies chosen for this review fail to document a definitive causal link between obesity and low back pain. Further research and epidemiologic data is needed to continue the search for a definitive answer.\nCompeting interests\nThe author(s) declare that they have no competing interests.","keyphrases":["obesity","risk factor","low back pain","association","evidence-based practice"],"prmu":["P","P","P","P","P"]}
{"id":"Evid_Based_Complement_Alternat_Med-4-4-2176145","title":"A Review of Yoga Programs for Four Leading Risk Factors of Chronic Diseases\n","text":"Yoga, a form of physical activity, is rapidly gaining in popularity and has many health benefits. Yet healthcare providers have been slow to recognize yoga for its ability to improve health conditions, and few interventions have been developed that take full advantage of its benefits. The purpose of this article is to review published studies using yoga programs and to determine the effect of yoga interventions on common risk factors of chronic diseases (overweight, hypertension, high glucose level and high cholesterol). A systematic search yielded 32 articles published between 1980 and April 2007. The studies found that yoga interventions are generally effective in reducing body weight, blood pressure, glucose level and high cholesterol, but only a few studies examined long-term adherence. Additionally, not enough studies included diverse populations at high risk for diabetes and its related common health problems.\nChronic diseases such as heart disease, stroke and diabetes are leading causes of death in the US (1). Common conditions for these chronic diseases are overweight, high blood pressure (BP), high glucose and high cholesterol. These conditions are commonly associated with each other. According to a prospective cohort study focusing on men (2), there were positive relationships between body mass index (BMI) and hypertension incidence. Men with a higher BMI have a higher likelihood to have diabetes and high cholesterol. Another study with men and women showed that overweight is linked to type 2 diabetes (3). Even among patients newly diagnosed with type 2 diabetes, the Hypertension in Diabetes Study found that \u223c40% had hypertension, and that hypertension considerably increased mortality in people with type 2 diabetes (4). Another recent study found a 79% rate of hypertension in patients who had received outpatient care for type 2 diabetes for at least 2 years (5). Individuals with impaired glucose tolerance also are more likely to have risk factors for cardiovascular disease such as elevated levels of triglycerides and low-density lipoprotein (LDL) and low levels of high-density lipoprotein (HDL)(6).\nPromising in this regard is appropriate physical activity because it can reduce body weight, BP, glucose level and cholesterol (7,8). Yoga, a form of physical activity consisting of various postures (Asana) and breathing and meditation techniques (Pranayama) (9), has been shown to have therapeutic benefits for individuals with a wide range of health conditions, including hypertension (10) and diabetes (11). Yoga also appears to be effective in reducing stress (12) and improving exercise tolerance as it is related to cardiovascular response (13).\nThe number of people practicing yoga in the US increased significantly between 1997 and 2002 (14). According to the 2002 National Health Interview Survey (14), 5% of American adults practiced yoga in the month previous to the study. Yoga also is well received as a therapeutic intervention; for example, participants in a yoga intervention for insomnia found that it was easily learned and performed (15). Despite its popularity and positive physiologic effects, however, yoga has not been widely recognized in efforts to prevent and treat major chronic health conditions. The purpose of this article is to review published studies using yoga practice to determine the effects of yoga on common health problems, such as overweight, hypertension, high glucose level and high cholesterol.\nMethods\nArticles were retrieved from electronic databases (CINAHL, Ovid MEDLINE and PsychInfo) using yoga as a keyword. This initial retrieval, performed in April 2007, yielded 2349 articles, of which 861 have been published since 1980. A search within those 861 articles, using the keywords overweight, blood pressure, glucose and cholesterol, identified 56 research articles. After the author had read the full text of the 56 articles and identified those that met the purpose of the study, 24 articles were excluded, mainly because they described outcome variables and characteristics of yoga that were irrelevant. Poor quality was not a reason for exclusion, but studies were excluded if they focused only on meditation or relaxation (Pranayama) or if they were case studies. This process resulted in a final total of 32 articles to be reviewed.\nThe review comprised studies involving yoga interventions and using an experimental or quasi-experimental design. In addition, observational studies were included because they often supply important information beyond the results of clinical trials (16). Studies that used yoga as a control, not as an intervention, were included as long as they provided evidence of the effectiveness of yoga on variables of interest (overweight, BP, glucose and cholesterol).\nFindings\nOf the 32 articles reviewed, 12 described experimental studies, 18 described quasi-experimental studies and two described observational studies. Only 2 of the 12 true experimental studies (17,18) described the randomization method. Even though risk factors for chronic health conditions were used as keywords in the search strategy, many of the studies used healthy adult samples; only half of the 32 studies actually focused on subjects with diabetes (19\u201323), hypertension (10,11,18,24\u201327), or cardiovascular disease (28\u201331). Seven of the studies were conducted in the US; the others, in India and other countries.\nContent of Yoga Programs\nOther than duration and frequency, most articles did not describe the yoga sessions in detail. Only a few articles (17,20,22,31,32) provided details of the yoga sequences used. Some listed the names of postures and breathing techniques. Of the few articles that identified the type of yoga studied, the most common was Hatha yoga, a popular form of yoga in the Western world.\nThe Compendium of Physical Activities, (33) a coding scheme that classifies physical activity based on energy expenditure, does not define energy expenditure while performing various types of yoga. However, Hatha yoga is classified as a conditioning exercise, in the same category as stretching, which has a metabolic equivalent (MET) of 2.5 (1 MET equals the amount of energy used for resting). A recent study (34) found that when young adult women (19 to 40-years old) performed a 30-min session of Hatha yoga, the MET was 2.17. However, if the session was made more active using Sun Salutations, a flowing series of physical postures, the MET increased to 3.74 (SD = 0.70).\nFrequency and Duration of Yoga Sessions\nThe total dose of yoga training, which depends on both the duration and frequency of yoga sessions, also needs to be considered in evaluating and comparing yoga studies. The most common duration and frequency of yoga sessions in the studies were 30\u201360 min per session and sessions meeting daily for 4\u201310 weeks, but many studies used sessions meeting 2\u20133 times per week for 8\u201312 weeks. Some yoga programs met more frequently than others but for a shorter time span. For example, in a study by Damodaran et al. (24), persons with essential hypertension received 1 h of yoga training daily for 3 months, which is counted as 84 h of training, whereas Ray et al. (32) studied healthy young adults who received 1 h of yoga training three times a week for 10 months, for a total of 120 h.\nSome findings can be useful in trying to determine effective durations of yoga sessions. For example, in one study, 1 h daily yoga practice was associated with significant reductions in body weight and cholesterol levels after 4 weeks, and those significant effects lasted for 14 weeks (28). In a study of 20 patients with essential hypertension, daily 30-min sessions of yoga led to a decrease in BP by the fifth day (27).\nAdherence to the Yoga Program\nAny persistent benefits from yoga would rely on long-term adherence, which was examined in only a few of the reviewed studies. In some studies, after yoga training, subjects were asked to continue their programs with daily home practice (28,35). One of these (35) compared adherence to yoga practice after a 10-week yoga intervention between white and black American adults (mean age: 69 years for whites and 70 years for blacks). Although the dropout rate did not differ significantly (16% for whites, 22% for blacks), the black participants did not engage in yoga at home as frequently as whites. A different study (36) found greater compliance with subsequent home practice among participants in a yoga class that met three times per week than in those who attended a weekly yoga class (86% versus 65%, P < 0.05). However, found no difference in compliance with home practice between groups engaged in yoga or aerobic exercise (36).\nWeight Decreased\nAccording to a retrospective observational study of 15,550 adults aged 53\u201357 years (37), regular yoga practice for 4 or more years was significantly associated with weight loss by overweight participants. Several intervention studies (10,26,29,31,38\u201341) also showed that yoga practice was effective in reducing body weight. After 4-day residential yoga practice followed by 14 weeks of 1 h daily home practice, one study (28) found a significant loss in mean body weight from 72.26 to 70.48 kg among subjects with risk factors for coronary artery disease (CAD). Other studies found that yoga was associated with significant weight loss by subjects with CAD (29\u201331) and subjects without CAD (30). Manchanda et al. (29) showed a 7% loss of body weight among adult men with CAD after 1 year of yoga practice, and in a study by Schmidt and colleagues, healthy adults lost an average of 5.7 kg after 3 months of yoga practice (39). All overweight adults studied by Yogendra et al. reached a normal weight within 1 year after initiating yoga-based lifestyle modifications (31). However, this article did not show the effect size of this change.\nBlood Glucose Level Lowered\nOf the six studies that examined blood glucose, all found that yoga was effective in reducing blood glucose level (11,19\u201322,31). For example, after 4 months of yoga practice, fasting glucose fell significantly (from 144 to 119 mg dl\u22121, P < 0.005) in a group of adults with type 2 diabetes, (19). Another sample of 24 adults with type 2 diabetes had significantly decreased fasting glucose (from 190.1 to 141.5 mg dl\u22121, P < 0.001) after 40 consecutive days of yoga practice, (20). Adults with normal blood glucose levels (11) also had significantly lower glucose levels after 3\u20134 h of yoga practice for 8 days (P < 0.001).\nBP Decreased\nThis review found ample evidence that yoga was effective in reducing BP. In a group of low-income elderly people, effects on systolic BP did not differ between a yoga class and an aerobic exercise class, both held three times a week for 10 weeks (36). However, eight other studies found that yoga practice was effective in lowering BP in healthy samples, regardless of the type of yoga (13,32,38\u201344). Yoga practice also significantly improved BP among people with hypertension (10,11,18,24\u201327), cardiovascular disease (28\u201331) or type 2 diabetes (19\u201323). For example, in 13 patients, aged 41\u201360 years, with essential hypertension (25), BP dropped significantly during the third week of a 4-week yoga program (1 h per day, 6 days per week), and it fell further after the program. For example, systolic BP dropped from 141.7 to 127.9 mmHg by the third week and to 120.7 mmHg by the fourth week.\nCholesterol Level Improved\nThe practice of yoga was associated with significant decreases in cholesterol among subjects with cardiovascular disease (28,29), hypertension (11) or type 2 diabetes (19). One study (28) examined a regimen involving 4 days of a yoga program at a residential course, followed by 1 year of yoga practice at home. In both men with angina and asymptomatic participants with CAD risk factors, all lipid variables except HDL decreased beginning the fourth week of yoga practice (e.g. total cholesterol fell from 206.6 to 193.6 mg dl\u22121), and the level of total cholesterol continued falling to 176.06 mg dl\u22121 at 14 weeks. A study of subjects at risk for cardiovascular disease and diabetes (11) found significant improvements (P < 0.01) in total cholesterol, triglycerides, LDL, HDL and very-LDL (VLDL, defined as total cholesterol minus LDL minus HDL) after short-term intensive yoga practice (3\u20134 h per day for 8 days). Notably, for subjects whose baseline total cholesterol was 200 mg dl\u22121 or higher, the reduction in triglycerides (from 151.5 \u00b1 48.9 to 132.7 \u00b1 50.5 mg dl\u22121, P < 0.001) and VLDL (from 36.7 \u00b1 13.8 to 30.2 \u00b1 14.6 mg dl\u22121, P < 0.001) was significantly greater than in subjects with lower baseline total cholesterol (triglycerides falling from 113.6 \u00b1 46.5 to 110.5 \u00b1 38.1 mg dl\u22121, P > 0.05; VLDL from 23.7 \u00b1 12.8 to 23.2 \u00b1 12.5 mg dl\u22121, P > 0.05). Finally, a study of healthy adults over 40 years old found that 5 years of yoga practice reduced age-related deterioration in cardiovascular functions (45). Although the article describing this observational study did not detail the type of yoga performed nor the frequency or intensity of the yoga sessions, the data showed a long-term change indicating the effectiveness of yoga on cardiovascular functioning.\nFour studies used aerobic training as an intervention and yoga as a control (38,43,46,47). One of these, a study of healthy active people aged 65 years or older (38), found that aerobic exercise produced no significant reduction in weight or BP, whereas 4 weeks of yoga practice did lead to some reduction in weight or BP (for example, systolic BP decreased from 146 to 139 mmHg). DiPietro et al. (47) found no change in glucose and insulin responses in their yoga control group. However, the yoga control group was monitored to ensure that pulse rates did not exceed 90 beats per min during yoga practice (47). Therefore, this restriction should be considered in interpreting this result.\nDiscussion\nThe reviews showed that yoga had beneficial effects on body weight, BP, blood glucose level and cholesterol level (Fig. 1). Nonetheless, several shortcomings in research on this topic need to be addressed, in order for clinical programs to capitalize on these health benefits.\nFigure 1.The possible effects of yoga on behavioral, psychological and physiological measures are illustrated. This article focuses on current evidences for physiological changes.\nOf the articles describing interventions, only a few provided details regarding the yoga programs or the names and sequence of yoga postures. Asana and Pranayama provide different types of health benefits, and therapeutic yoga programs can involve various combinations of these two components. An optimal comparison of study results thus requires knowing the combination of Asana and Pranayama used. The sequence of yoga postures can be inferred from the list of posture names, but more straightforward information is essential to future studies seeking to replicate or generalize the results. A related issue that remains to be resolved is how to standardize yoga exercises for research purposes.\nThe optimal duration and intensity required to maximize the effectiveness of yoga need to be determined, as does the need for a booster to provide long-term effects. Because the MET of yoga is low, increasing the frequency may increase the benefits. However, these factors cannot be judged from the reviews studies; many articles did not clearly identify the dosage of the yoga program studied, and they focused on the short-term health benefits of yoga. Only a few studies included follow-up data beyond 6 months. It also remains to be determined whether more intensive training, that is, a greater dosage, improves the likelihood of adopting and maintaining active behavior in the long run.\nThe samples in the reviewed studies pose additional dilemmas. It was not a surprise that a large portion of the studies were conducted in India, where the philosophy and practice of yoga originated. However, this focus on one geographical region, where yoga is particularly ingrained in the culture, limits the generalizability of results. Few studies have addressed variables of interest specific to minorities in the US (Blacks, Hispanics and Asians), which are populations believed to be more vulnerable to type 2 diabetes (48,49) and physical inactivity (8).\nYoga has beneficial effects on various health conditions. A large portion of the reviewed studies analyzed the effects of yoga with healthy samples. There is evidence that yoga practice was more effective in lowering triglycerides among people with higher cholesterol than those with a lower cholesterol level (11). Therefore, it is important to consider participants\u2019 health conditions because there are possible differences in the effects of yoga by their health severity.\nConclusions\nAnalyses of yoga intervention should be designed and conducted to identify programs best suited for diverse populations and for specific populations with high-risk factors for chronic health conditions. Such studies could guide the development of more practical and effective interventions.","keyphrases":["yoga","risk factors","chronic disease","overweight","hypertension","glucose","cholesterol"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"J_Plant_Res-3-1-2039807","title":"Promiscuous, non-catalytic, tandem carbohydrate-binding modules modulate the cell-wall structure and development of transgenic tobacco (Nicotiana tabacum) plants\n","text":"We have compared heterologous expression of two types of carbohydrate binding module (CBM) in tobacco cell walls. These are the promiscuous CBM29 modules (a tandem CBM29-1-2 and its single derivative CBM29-2), derived from a non-catalytic protein1, NCP1, of the Piromyces equi cellulase\/hemicellulase complex, and the less promiscuous tandem CBM2b-1-2 from the Cellulomonas fimi xylanase 11A. CBM-labelling studies revealed that CBM29-1-2 binds indiscriminately to every tissue of the wild-type tobacco stem whereas binding of CBM2b-1-2 was restricted to vascular tissue. The promiscuous CBM29-1-2 had much more pronounced effects on transgenic tobacco plants than the less promiscuous CBM2b-1-2. Reduced stem elongation and prolonged juvenility, resulting in delayed flower development, were observed in transformants expressing CBM29-1-2 whereas such growth phenotypes were not observed for CBM2b-1-2 plants. Histological examination and electron microscopy revealed layers of collapsed cortical cells in the stems of CBM29-1-2 plants whereas cellular deformation in the stem cortical cells of CBM2b-1-2 transformants was less severe. Altered cell expansion was also observed in most parts of the CBM29-1-2 stem whereas for the CBM2b-1-2 stem this was observed in the xylem cells only. The cellulose content of the transgenic plants was not altered. These results support the hypothesis that CBMs can modify cell wall structure leading to modulation of wall loosening and plant growth.\nIntroduction\nPlant cell expansion depends on the interplay between internal driving forces and the controlled yielding of the cell wall. The ability of the wall to extend under this internal pressure is largely determined by wall loosening processes that modify the interaction of cellulose microfibrils with one another, and\/or with hemicelluloses (Darley et al. 2001). Likewise, organ growth is brought about by differential rates of wall loosening, which causes differential tissue growth and, consequently, tissue tension within the organ (Peters and Tomos 1996, 2000). Thus, progressive modification of the cellulose\u2013hemicellulose (mostly cellulose\u2013xyloglucan) networks is important in plant development.\nIn addition to their effect on plant growth, there are indications that interactions of hemicelluloses with cellulose can affect cellulose fibre properties. Using the Acetobacter model system, it was shown that cellulose\u2013glucomannan and cellulose\u2013galactomannan composites cause 57 and 32% loss of the crystallinity, respectively, of the native bacterial cellulose (Whitney et al. 1998). For cellulose\u2013xyloglucan composites 29% loss of cellulose crystallinity was observed (Whitney et al. 1999). It was also shown that the composites were less stiff, leading to a dramatic reduction in mechanical strength, for example 80% reduction in composites with xyloglucan (Whitney et al. 1999, 2000). There are also reports which indicate that fibre properties depend on the side-chain substitution of the hemicelluloses. Studies on Arabidopsis thaliana mutants with mutations of the MUR2 and MUR3 genes, which encode xyloglucan-specific fucosyl and galactosyl transferases, respectively, revealed that the tensile strength of the fibre was enhanced by galactosylation of the xyloglucan (Pena et al. 2004; Ryden et al. 2003).\nThere are indications that the interactions between mannan-based polysaccharides and cellulose can affect the structural properties of the cell wall (Carpita et al. 2001; Hosoo et al. 2002). An immuno-localisation study of A. thaliana revealed that mannans were present in all thickened cell walls of stems and leaves, including those of the xylem parenchyma and epidermis (Handford et al. 2003). Mannan transglycosylase was recently identified and characterized as cell wall enzyme acting on mannan-based plant polysaccharides in primary cell walls of higher plants (Schroder et al. 2004). This body of evidence thus supports an emerging idea that mannan-based polysaccharides in cell walls may have a role analogous to that of xyloglucans, introducing flexibility and forming a growth-restraining network with cellulose.\nProteins, for example expansins, have been shown to be involved in modifying cellulose\u2013hemicellulose interaction, thereby loosening the cell wall and ultimately regulating growth and developmental processes in which rearrangements in the cell wall are thought to be important (Cho and Cosgrove 2000; Choi et al. 2003; Pien et al. 2001; Zenoni et al. 2004). There is evidence that (heterologous) carbohydrate-binding modules (CBMs) could also affect these interactions (Levy et al. 2002; Shpigel et al. 1998), leading to altered structural morphology in transgenic plants (Kilburn et al. 2000; Quentin 2003; Shoseyov et al. 2001) or plant growth (Safra-Dassa et al. 2006). CBMs are non-catalytic polysaccharide-recognizing modules, appended to glycoside hydrolases, that often degrade water-insoluble polysaccharides and concentrate the enzymes on their target polysaccharides. CBMs are grouped into families based on amino acid sequence similarities; there are currently 47 families of CBMs in the database (http:\/\/www.afmb.cnrs-mrs.fr\/CAZY\/). The biochemical characteristics of members of most of the CBM families have been established, including those that target non-cell wall polysaccharides such as starch and glycogen (reviewed by Boraston et al. 2004). Although most CBMs are linked to the catalytic modules of carbohydrate-active enzymes, some non-catalytic carbohydrate-binding proteins are not appended to any catalytic module. Examples of such proteins include putative CBMs of the A. thaliana former X8 family, now CBM43 (GenBank accession no. AL161503) (Henrissat et al., 2001), with olive pollen allergen Ole e 10 (GenBank accession no. AY082335) being the first characterized member of this family (Barral et al. 2005). Another example is the non-catalytic protein (NCP1) (CBM29; GenBank accession no. AY026754), which is a component of the anaerobic fungus Piromyces equi cellulase\u2013hemicellulose complex (Freelove et al. 2001).\nBecause of the heterogeneous nature of the cell wall polysaccharides, several CBMs have evolved structures which enable them to recognize more than one wall polysaccharide (Boraston et al. 2004). Promiscuous recognition of carbohydrates by CBMs enables the associated enzyme (complex) to be more efficient in cell wall degradation. McCartney et al. (2006) showed that different xylan-specific CBM families can vary substantially in their binding to the cell wall. This indicates that the specificity of CBMs is so intricate that after expression in planta their effects on cell wall modification might be very local. To really affect wall architecture, it might be important to select CBMs that bind to different kinds of polysaccharide (promiscuous), so that the effect is not confined to one location or cell type. Most research activity relating to the potential use of CBMs for plant cell wall modification has been on cellulose-specific CBMs. In this investigation we have used different CBMs which not only recognise hemicellulose as opposed to CBM3 but are also to some extent promiscuous.\nThe CBM29 modules are an excellent model of promiscuity in protein\u2013carbohydrate recognition. The modules bind soluble glucomannan, galactomannan, \u03b2-glucan, and hydroxyethylcellulose (HEC), and insoluble forms of cellulose and mannan (Charnock et al. 2002; Freelove et al. 2001). For all the ligands tested two appended copies of CBM29 (CBM29-1-2) had affinity substantially higher than the additive value of the individual modules. This indicates the two CBMs act in synergy to bind all their target ligands, with CBM29-2 having more affinity than CBM29-1. The tandem CBM29-1-2 was, additionally, able to interact with xyloglucan and different forms of xylans, whereas the single CBM had little affinity for these polysaccharides (Freelove et al. 2001; McCartney et al. 2004). An ex-situ labelling study has revealed that the tandem CBM29-1-2 modules bind strongly to the cell walls of the maize coleoptile sheath and the enclosed developing leaves (McCartney et al. 2004). This is indicative of the great abundance of their interacting ligands in the primary cell walls of grasses. It was therefore of interest to investigate the heterologous expression of these promiscuous CBMs in plants, and, more importantly, to compare their effects with those of less promiscuous CBMs, whose characteristics are described below.\nSome ligand promiscuity is also observed for two consecutive family 2b CBMs (CBM2b-1 and CBM2b-2) from Cellulomonas fimi xylanase 11A. The tandem CBM2b-1-2 (note the underlining used throughout this report to distinguish it from CBM29-1-2) has very strong affinity for xylan and much lower affinity for insoluble acid-swollen cellulose (Bolam et al. 2001). It also has an 18 to 20-fold greater affinity for xylan than the individual modules.\nWe have expressed two CBM29 constructs, the tandem CBM29-1-2 and the single derivative CBM29-2, and the tandem CBM2b-1-2 construct in tobacco (all proteins targeted the cell wall) under the control of 35S cauliflower mosaic virus (CaMV) promoter. It is shown that cell wall structural alterations in transgenic plants expressing CBM29-1-2 are more severe than those in CBM2b-1-2 transformants. Our results indicate that promiscuous carbohydrate binding modules can modulate cell wall structure and the development of transgenic tobacco plants.\nMaterials and methods\nPreparation of constructs\nThree constructs were prepared for expression in tobacco plants, one for single CBM29-2, one for tandem CBM29-1-2, and one for tandem CBM2b-1-2. The CBM29 and CBM2b-1-2 constructs were prepared by amplifying their respective gene fragments from pET22b and pET28a, recombinant plasmid vectors used for cloning and expression in Escherichia coli (Bolam et al. 2001; Freelove et al. 2001). For the CBM29-1-2 fragment the polymerase chain reaction (PCR) was performed using primers that included BamHI and SmaI recognition sites (5\u2032- cgggatccgttagtgctacttactctgttgtttat-3\u2032 and 5\u2032-tcccccgggccttttaatttattgggtcaacgaaa-3\u2032; the BamHI and SmaI sites, respectively, are underlined). The three bases highlighted in bold type are the stop codon. The amplified 914-base-pair fragment of the tandem CBM29-1-2 was digested with BamHI and SmaI (Invitrogen, The Netherlands) and cloned into a similarly digested binary vector pGreen7k (Hellens et al. 2000). Similarly, amplification of the single CBM29-2 fragment were performed using primers with the same restriction sites as for the tandem CBM29-1-2 (5\u2032-cgggatcccgtaatgtcagagccacttacactgt-3\u2032 and 5\u2032-tcccccgggccttttaatttattgggtcaacgaaa-3\u2032; the BamHI and SmaI sites, respectively, are underlined). The amplified 559-base-pair fragment was digested with BamHI and SmaI (Invitrogen, The Netherlands) and cloned into pGreen7k binary vector. Similarly, amplification and cloning of the 635-base-pair CBM2b-1-2 fragment were performed using primers that included SmaI and EcoRI recognition sites at either end (5\u2032-cccgggtgacacgggcggaggcggcggt-3\u2032 and 5\u2032-ggaattctcagcccgtggcgca-3\u2032; the SmaI and EcoRI sites, respectively, are underlined). The cloning of the three fragments was in-frame with fusion peptides of two sequences, which were cloned upstream in the binary vector. The first sequence codes for a tobacco transit peptide for transporting a cellular glycoprotein NTP303 across the plasma membrane into the cell wall (Wittink et al. 2000) whereas the second sequence encodes a hexahistidine tag. The sequence of the transit peptide was obtained as a product of annealing two oligonucleotide primers, TP1 (5\u2032- agcttatgggaagtggtaaagtaacatttgtggctttgctactttgcctctccgtaggggtgatagctt-3\u2032) and TP2 (5\u2032-ctagaagctatcacccctacggagaggcaaagtagcaaagccacaaatgttactttaccacttcccata-3\u2032). The underlined nucleotides produced overhangs of HindIII and XbaI, respectively. This fragment was then cloned into the HindIII and XbaI cloning sites of the vector. Similarly, the sequence of the hexahistidine tag was obtained and ligated into XbaI and BamHI sites as annealing product of His1 (5\u2032-ctagaagaggatcgcatcaccatcaccatcacg-3\u2032) and His2 (5\u2032-gatccgtgatggtgatggtgatgcgatcctctt-3\u2032), with the underlined nucleotides producing overhangs of XbaI and BamHI, respectively. The purpose of using the hexahistidine tag was to facilitate affinity purification of the CBM29 proteins. The control construct did not contain any of the CBMs, the transit peptide, or the hexahistidine epitope tag. All constructs were sequenced to verify their integrity.\nImmunofluorescence detection of CBM29-1-2 and CBM2b-1-2 binding and microscopy\nWild-type tobacco plants (Nicotiana tabacum L.) were grown at 24\u00b0C under 16\u00a0h light and 8\u00a0h dark for 6\u20137\u00a0weeks. Regions of stem were excised and immediately fixed in PEM buffer (50\u00a0mmol\u00a0L\u22121 PIPES (piperazine-N,N\u2032-bis[2-ethanesulfonic acid]), 5\u00a0mmol\u00a0L\u22121 EGTA (ethylene glycol bis(\u03b2-aminoethyl ether)-N,N,N\u2032N\u2032-tetraacetic acid), and 5\u00a0mmol\u00a0L\u22121 MgSO4, pH\u00a06.9) containing 4% paraformaldehyde. Samples were then dehydrated in an ethanol series (30, 50, 70, 90, and 97%, each for 30\u00a0min at 4\u00b0C, ethanol and wax 1:1, 37\u00b0C, overnight) and embedded in Steedman\u2019s wax. Sections were cut to a thickness of 12\u00a0\u03bcm and collected on polylysine-coated microscope slides (BDH Laboratory Supplies, Dorset, UK), de-waxed, and re-hydrated through a reverse ethanol series into phosphate-buffered saline (PBS) (97% for 3\u00a0\u00d7\u00a010\u00a0min, 90%, 50% and water for 10\u00a0min each, and a final step of water 90\u00a0min). For CBM labelling, sections were incubated in milk protein\/PBS (to reduce any non-specific binding of proteins) and 5\u00a0\u03bcg\u00a0mL\u22121 CBM29-1-2 for 1.5\u00a0h. Immunofluorescence detection of binding of CBM29-1-2 and CBM2b-1-2 to sections and microscopy were performed as described elsewhere (McCartney et al. 2004).\nTobacco transformation and regeneration\nIn vitro leaf explants of N. tabacum cv. Samsun NN were used for Agrobacteriumtumefaciens-mediated transformation. Cloned binary vector pGreen7k was co-transformed with the helper plasmid pSoup (Hellens et al. 2000) into A.tumefaciens strain LBA4404 by electroporation. This was plated out on LB-agar plates containing kanamycin (100\u00a0\u03bcg\u00a0mL\u22121) and rifampicin (30\u00a0\u03bcg\u00a0mL\u22121), and incubated for three days at 28\u00b0C, to obtain single colonies. The integrity of the binary plasmids was tested by restriction analysis of plasmids isolated from A. tumefaciens cultures used for plant transformation. LB medium (20\u00a0mL) without selection was inoculated with a single colony and incubated overnight at 28\u00b0C. The grown culture (100\u00a0\u03bcL) was added to a Petri dish containing 10\u00a0mL Murashige\u2013Skoog (MS30). Leaf explants, without major veins and edges, from young seedlings were transferred upside-down in to the MS30 medium. Agrobacterium infection was performed in the dark for 2\u00a0days at 24\u201325\u00b0C. Two controls were used for the procedure, untransformed wild-type control and empty pGreen7k control without an insert. Leaf explants were washed in three changes of liquid washing medium of MS30 with 250\u00a0mg\u00a0L\u22121 carbenicillin. The washed explants were transferred upside-up to an MS30-phytagel plate containing 0.1\u00a0mg\u00a0L\u22121 \u03b1-naphthalene acetic acid (NAA), 1\u00a0mg\u00a0L\u22121 6-benzylaminopurine (BAP), 200\u00a0mg\u00a0L\u22121 kanamycin and 250\u00a0mg\u00a0L\u22121 carbenicillin, and incubated overnight in the dark at 28\u00b0C. Plants were transferred to a growth chamber (25\u00b0C), where they were gradually adapted to light and incubated for callus induction. After two weeks in culture, calli generated were transferred to shoot-inducing medium, MS20-phytagel, containing 0.2\u00a0mg\u00a0L\u22121 NAA, 2\u00a0mg\u00a0L\u22121 BAP, 200\u00a0mg\u00a0L\u22121 kanamycin, and 250\u00a0mg\u00a0L\u22121 carbenicillin. Well-formed shoots were harvested and transferred to root-inducing medium, MS15-phytagel plate containing 100\u00a0mg\u00a0L\u22121 kanamycin, 250\u00a0mg\u00a0L\u22121 carbenicillin, and 200\u00a0mg\u00a0L\u22121 vancomycin. Transformed plantlets were transferred to the greenhouse to generate mature plants. Between fifteen to eighteen antibiotic-resistant tobacco transformants were generated for each of the three CBM transgenes.\nRNA gel blot analysis of transgenic plants\nTotal RNA was isolated from 3 to 5\u00a0g transformed in-vitro shoots as described elsewhere (Kuipers et al. 1995). Aliquots of 20\u00a0\u03bcg per lane were separated on a 1% formaldehyde agarose gel and blotted onto a Hybond-N nylon membrane (Amersham) by vacuum transfer in 0.4\u00a0mol\u00a0L\u22121 sodium hydroxide. The membranes were hybridized with probes consisting of 60\u00a0ng [\u03b132P]dCTP-labelled restricted fragments of CBM29-1-2, CBM29-2, or CBM2b-1-2. The radioactive labelled blots were exposed to X-OMAT S and AR scientific imaging films (Kodak) at \u221280\u00b0C. The blots were re-hybridized, after stripping, with a 489\u00a0bp [\u03b132P]dCTP-labelled fragment of a tobacco 18S ribosomal RNA gene (GenBank accession no. AJ236016), as a control. The ribosomal probe was amplified from tobacco genomic DNA using oligonucleotides 5\u2032-gaaactgcgaatggctcatt-3\u2032and 5\u2032-attaccgcggctgctggc-3\u2032 for PCR amplification.\nProtein extraction\nProtein extracts were prepared from transformed tobacco stems using 250\u00a0mmol\u00a0L\u22121 sodium phosphate buffer (pH 6.0), 0.5 mol\u00a0L\u22121 NaCl, 5\u00a0mmol\u00a0L\u22121 EDTA, 0.1% (v\/v) Tween 20, protease inhibitor (Complete, Roche), 37.5\u00a0mg\u00a0mL\u22121 PVPP (adapted from Vincken et al. 1998). The protein samples were incubated for 2\u00a0h at 4\u00b0C with gentle shaking. After incubation the samples were centrifuged at 4,500g and 4\u00b0C for 20\u00a0min. The supernatant containing the soluble protein fraction was then collected. The protein samples were then concentrated with Centricon Plus-20 devices (Millipore) according to supplier\u2019s procedure. Protein concentrations were determined with an ESL protein assay (Roche) using BSA as standard.\nIncubation of RGS\u00b7(HIS)6-tagged size marker in protein extract of wild-type tobacco\nProtein extracts were prepared from wild-type tobacco as described above, either lacking or supplemented with 5\u00a0mmol\u00a0L\u22121 EDTA and protease inhibitor (Complete, Roche). RGS\u00b7(HIS)6-tagged size marker was incubated with the protein extracts as follows. A batch of RGS\u00b7(HIS)6-tagged size marker (Qiagen) was dissolved in 100\u00a0\u03bcL 50\u00a0mmol\u00a0L\u22121 Tris, pH 7.5, such that the final concentration of the individual proteins varied from 50 to 75\u00a0ng\u00a0\u03bcL\u22121. The epitope-tagged size marker (2\u00a0\u03bcL) was added to 18\u00a0\u03bcL tobacco extracts. The mixtures were incubated at room temperature for approximately 15\u00a0h. As a control, RGS\u00b7(HIS)6-tagged size marker was incubated in 50\u00a0mmol\u00a0L\u22121 Tris, pH 7.5.\nWestern blot analysis\nThe protein samples were separated with SDS-PAGE on 12% SDS-polyacrylamide gels (Mini-Protean II apparatus, Bio-Rad). The separated proteins were electroblotted on to a nitrocellulose membrane in 192\u00a0mmol\u00a0L\u22121 glycine, 25\u00a0mmol\u00a0L\u22121 Tris base, 0.1% (w\/v) SDS, 20% (v\/v) ethanol, pH 8.3. Blocking against non-specific binding was performed for 2\u00a0h with 3% (w\/v) BSA (Sigma) in TBS (10\u00a0mmol\u00a0L\u22121 Tris, 150\u00a0mmol\u00a0L\u22121 NaCl, pH 7.5). The membrane was then incubated with a 1:1000 dilution of the primary anti-RGS\u00b7HIS antibody (Qiagen) in TBS solution containing 3% (w\/v) BSA for 2\u00a0h. The membrane was washed twice in TBS-TT buffer (20\u00a0mmol\u00a0L\u22121 Tris base, 500\u00a0mmol\u00a0L\u22121 NaCl, pH 7.5, 0.05% (v\/v) Tween 20, 0.2% (v\/v) Triton X-100), and once in TBS buffer, for 10\u00a0min each. The membrane was then washed with 1:2000 dilution horseradish peroxidase (HRP) conjugated sheep anti-mouse antibody (Amersham) in TBS buffer containing 10% (w\/v) non-fat dried milk. The membrane was washed in TBS-TT buffer four times, 10\u00a0min each, and subsequently submerged in Supersignal ULTRA substrate working solution (Pierce), which is a mixture of equal parts of Ultra luminol\/enhancer solution and Ultra stable peroxide solution. Finally, the membrane was exposed to X-OMAT S Scientific Imaging films (Kodak) at room temperature.\nMonitoring the growth of the transformed plants\nTo monitor growth of the transformed plants with time, three replicates per line were grown in the greenhouse. The transformants were grown in two series, the CBM29s and CBM2b-1-2 series, with each series having its separate controls. Height measurements of the stems were taken weekly for nine weeks, starting from week two after transplant. The height measurements of six individual high expressers, each from transgenic lines containing CBM29-1-2 and CBM29-2 constructs, six transformed controls, and six untransformed controls from week 3 to week 9 were subjected to one-way analysis of variance (ANOVA), using an \u03b1 of 0.05. To test whether the differential stem elongation was significant, a Fisher\u2019s unprotected least significant difference test was used. The null hypothesis of no difference between plant heights was rejected at an error of 0.05.\nLight microscopy\nThree individual plants per transgenic tobacco line and three wild-type plants, as control, were used for microscopic examination. Three transgenic lines were used per construct (two high expressers and one low expresser). Stem samples were taken from the second internode from the top of the plant. Stem sections 1\u00a0mm-thick were fixed in 3% glutardialdehyde (Merck) and 3% paraformaldehyde (Merck) in 0.1\u00a0mol\u00a0L\u22121 phosphate buffer containing 0.1% Triton X-100 for 2\u00a0h. The samples were then washed and dehydrated in an ethanol series. After dehydration they were embedded in Technovit 7100 resin (USA) (Kuroiwa et al. 1990). Sections 4\u00a0\u03bcm-thick were stained with 0.1% toluidine blue (Aldrich) and examined under a bright field microscope.\nCryo-scanning electron microscopy and quantification of cell size and cell wall diameter\nOne high expresser from each of the CBM29s and CBM2b-1-2 transgenic lines and one individual plant each from their respective controls were used for cryo-SEM examination. The plants that were analysed were chosen on the basis of the results obtained from the extensive light microscopic analysis that showed a consistent pattern of alterations in high-expressing transgenic plants. Stem sampling was the same as for the light microscopy. Stem samples 6\u00a0mm-thick were mounted in a brass cylindrical sample holder with TBS tissue freezing medium (EMS, Washington, PA, USA). The frozen samples were placed in a sample holder in a cryo-ultra microtome (Reichert Ultracut E\/FC4D) and cut at a specimen temperature of 100\u00b0C. These samples were first planed with a glass knife, after which the surface was planed with a diamond knife (Histo no trough, 8\u00a0mm 45\u00b0C; Drukker International, The Netherlands). After planing, the samples were placed in a dedicated cryo-preparation chamber (CT 1500 HF, Oxford instruments, UK). All the samples in the cryo-preparation chamber were freeze dried for 3\u00a0min at \u221290\u00b0C and 8\u00a0\u00d7\u00a010\u22124\u00a0Pa, and subsequently sputtered with a 10-nm layer of Pt. The samples were cryo-transferred into the field emission scanning microscope (Jeol 6300F, Japan) on the sample stage at \u2212190\u00b0C. All images were recorded digitally (Orion, 6 E.L.I. sprl, Belgium) at a scan rate of 100\u00a0s (full frame) at the size of 2,528\u00a0\u00d7\u00a02,030, 8 bit. The images were optimized and resized for publication by use of Adobe Photoshop CS.\nTo quantify the micrographic images, files were opened with Image J software developed at the National Institute of Health, USA (http:\/\/www.rsb.info.nih.gov\/ij\/). The surface area of three hundred cortical and xylem cells was measured as shown by the insets in Figs.\u00a07a and 7b. One hundred cells per field of view were measured in three replicates. Similarly, cell wall thickness of 150 cells was quantified as shown by the inset in Fig.\u00a07c. Fifty cells per field of view were measured in three replicates. Standard deviations of three measurements were determined.\nIsolation of cell wall material and analysis of the cellulose content\nStem samples from one high expresser of the CBM29 and CBM2b-1-2 series, one pGreen7k vector control, and one wild-type control plant were ground to a fine powder in liquid nitrogen. For each isolation, 1\u00a0g of this stem material was extracted in a 50\u00a0mmol\u00a0L\u22121 Tris[HCl], pH 7.2 solution containing 1% SDS, for 3\u00a0h at room temperature (RT) with continuous shaking. The cell wall material CWM was spun down by centrifugation at 13,000\u00a0rpm for 15\u00a0min. Subsequently, the residue was washed with water, ethanol, and acetone, then air-dried. The different cell wall materials (10\u00a0mg) were hydrolysed in 1\u00a0mL 2\u00a0mol\u00a0L\u22121 TFA. The TFA-insoluble cellulose was spun down at 13,000\u00a0rpm for 15\u00a0min, and the pellet was suspended in 67% sulfuric acid. The suspension was heated and diluted appropriately to determine the cellulose content colorimetrically, using anthrone as colouring agent (Updegraff 1969). The acid hydrolysis was performed in quadruplicate.\nResults\nExpression of bacterial CBM29 and CBM2b-1-2 genes in tobacco\nTo detect expression of the introduced CBM genes in the tobacco plant, we performed Northern blot analysis with total RNA. Figure\u00a01 shows representatives of three classes of transcript expression (high, low, and none) of the three CBM modules as revealed by the Northern analysis. Ten of the CBM29-1-2 plants were classified as high expressers, five as low expressers, and two as none expressers. For the CBM29-2 plants, ten lines were classified as high expressers, seven as low expressers, and two as none expressers (note that none expressers may include plants with very low RNA expression, which could not be detected). For the CBM2b-1-2 plants, six each were classified as high and low expressers whereas four plants were classified as none expressers. Preliminary attempts at purifying CBM proteins using affinity purification with the hexahistidine tag were not successful (data not shown). Similarly, Western detection of a hexahistidine epitope-tagged fungal elicitor protein ECP2 that was infiltrated into the apoplast of leaves did not succeed in the tomato plants but succeeded in Arabidopsis thaliana (van Esse et al. 2006). The results indicated that the histidine tag was cleaved in the tomato plants but not in A. thaliana. These observations have since led to the idea that the hexahistidine epitope tags have cleavage sites recognized by proteases, which are specific for the cell walls of the solanaceous species.\nFig.\u00a01Transcript analysis of CBM29 and CBM2b-1-2 genes in transgenic tobacco leaves. A differential transcript expression pattern is shown in the upper panel with the representative of each class in the three transgenic lines CBM29-2 (a), CBM29-1-2 (b) and CBM2b-1-2 (c). Line 14 in aline 5 in both b and c, represent high expressers. Lines 9, 11 and 1 represent low expressers. Line 17 in a, line 6 in b and line 14 in c represent none expressers. The lower panel shows RNA blots for the 18S ribosomal RNA internal control for each of the series\nWe corroborated this notion by incubating a protein ladder, consisting of five different proteins of known sizes, each containing an N-terminal RGS\u00b7(HIS)6\u2013tag, in protein extracts of wild-type tobacco (Compier 2005). The tagged proteins of the size marker that were incubated in tobacco extract without EDTA and protease inhibitor were not detectable on a Western blot whereas the markers incubated in extract supplemented with EDTA and protease inhibitor were detected. This suggested that extracts from tobacco contained proteases that could remove the hexahistidine epitope tag.\nBecause grinding of the plant material and protein extraction were performed at 4\u00b0C, and the protein extraction buffer contained EDTA and protease inhibitor, removal of the RGS\u00b7(HIS)6-tag from the fusion proteins had probably occurred already in planta, most probably extracellularly. It should be noted that with the presence of the NTP303 signal peptide, the fusion proteins would be secreted into the apoplast. Cleavage of the RGS\u00b7(HIS)6-tag was, therefore, likely to occur after translocation into the extracellular space.\nCBM29-1-2 and CBM2b-1-2 have different binding patterns in the stem\nImmunofluorescence detection of CBM29-1-2 and CBM2b-1-2 binding to stems of the wild-type tobacco plant was performed order to determine, a priori, the particular tissues of the stem where the introduced CBM29-1-2 module would bind. Indirect immunofluorescence micrographs (Fig.\u00a02) revealed that CBM2b-1-2 binding is restricted to the secondary cell walls of stem vascular tissue\u2014xylem cells and phloem fibres (Fig.\u00a02a)\u2014whereas CBM29-1-2 indiscriminately binds to every tissue of the stem, from the epidermis through the cortex and the vascular tissue to the pith parenchyma in the innermost part (Fig.\u00a02b). In the control (no CBM) weak autofluorescence was observed in the xylem vessel cell walls (Fig.\u00a02c). This ex-situ evidence for unselective binding of the CBM29-1-2 to both the primary and the secondary cell walls of the mature stem of the tobacco plant thus complements earlier evidence for its binding to a predominantly primary-walled maize coleoptile (McCartney et al. 2004). It should also be noted that tobacco is a dicotyledonous plant whereas maize is monocotyledonous, and that there are many differences between the polysaccharide composition of the walls of these species. Both results thus confirm the binding promiscuity of the tandem CBM29-1-2, as previously characterized (Charnock et al. 2002; Freelove et al. 2001). We also conclude that the CBM2b-1-2 ligands are present in the vascular tissue of the stem.\nFig.\u00a02Micrographs, obtained by indirect immunofluorescence microscopy, showing binding of CBM2b-1-2 (a), CBM29-1-2 (b), and control (no CBM) (c) to transverse sections of wild-type tobacco stem. The CBM29-1-2 binds indiscriminately to every tissue of the stem, from the epidermis through the cortex and the vascular tissue to the pith parenchyma in the innermost part whereas CBM2b-1-2 binding is restricted to the vascular tissues. ct cortical parenchyma, xy xylem cells, ph pith. Arrow head indicates the epidermis. Arrow indicates the phloem fibres. Scale bar\u00a0=\u00a0100\u00a0\u03bcm\nReduced stem elongation and prolonged juvenility are observed for CBM29-1-2 transformants\nAn indication of reduced stem elongation in the transgenic plants expressing the tandem CBM29-1-2 was first apparent after the third week of transfer to the greenhouse. The different stem elongation of the tandem CBM29-1-2-expressing plants and the control plants became significant (P\u00a0<\u00a00.05) at weeks seven and eight, when other plants had reached their final height (Table\u00a01, Fig.\u00a03). The tandem CBM29-1-2-expressing plants continued to increase in height until the eleventh week, however, when they reached their final height, an average of 58\u00a0cm. There was, as a result, no significant difference between the heights of the tandem CBM29-1-2 lines at maturity and the heights of the single CBM29-2 lines, the pGreen vector control, and the wild-type control plants. There was no significant difference, at any time, between stem elongation of transgenic plants expressing CBM29-2 and the control plants.\nTable\u00a01Plant height (cm) of transgenic lines CBM 29-1-2, CBM 29-2, empty pGreen7k control, and wild-type controlPlant lineWeeks after transplant3456789CBM29-1-29.5\u00a0\u00b1\u00a01.316.1\u00a0\u00b1\u00a02.227.0\u00a0\u00b1\u00a03.837.7\u00a0\u00b1\u00a05.543.1\u00a0\u00b1\u00a05.3a50.5\u00a0\u00b1\u00a05.4a56.6\u00a0+\u00a06.7CBM29-29.3\u00a0\u00b1\u00a01.316.9\u00a0\u00b1\u00a01.927.5\u00a0\u00b1\u00a03.240.9\u00a0\u00b1\u00a05.050.7\u00a0\u00b1\u00a04.157.8\u00a0\u00b1\u00a04.163.8\u00a0+\u00a04.7PGreen7k9.5\u00a0\u00b1\u00a01.617.3\u00a0\u00b1\u00a02.330.1\u00a0\u00b1\u00a03.844.6\u00a0\u00b1\u00a05.257.6\u00a0\u00b1\u00a07.163.1\u00a0\u00b1\u00a05.865.4\u00a0+\u00a05.6WT Ctrl9.7\u00a0\u00b1\u00a01.317.1\u00a0\u00b1\u00a02.331.5\u00a0\u00b1\u00a03.750.6\u00a0\u00b1\u00a07.363.6\u00a0\u00b1\u00a07.168.4\u00a0\u00b1\u00a05.568.8\u00a0+\u00a05.6The values are means and standard deviations for six transgenic lines (high expressers) and six control plants. Each line has three individual plants (three replicates)aPlant height significantly different from that of controlsFig.\u00a03Reduced stem elongation in time, and delayed development of CBM 29-1-2 transgenic tobacco plants. The picture was taken at week eight\nIn addition to reduced stem elongation it was also observed that flower development was delayed in the tandem CBM29-1-2-expressing plants. We observed that at week eight, when most plants had already flowered (Fig.\u00a03) and set fruit (data not shown), the tandem CBM29-1-2-expressing plants were just developing flower buds (Fig.\u00a03). The flower buds eventually developed into normal flowers and also set fruits. Most plants, except the CBM29-1-2 plants, had also lost many leaves, because of faster ageing (Fig.\u00a03).\nThere was no morphological or developmental change in the transgenic plants expressing the tandem CBM2b-1-2 gene compared with the pGreen7k vector control. The average plant height of the tandem CBM2b-1-2 series at maturity was 71\u00a0cm, which was comparable with the 75\u00a0cm average height for their pGreen7k vector control plants. We also did not observe a particular trend in plant development with regard to stem elongation, leaf abscission, or flower formation.\nCellular alterations are observed in transgenic plants\nThe cellular basis for the CBM29-1-2 plant phenotypes was examined by histological examination of the transgenic tobacco stems. The examination revealed a prominent layer of collapsed cells in the cortex of the stems of the tandem CBM29-1-2-expressing plants (Figs.\u00a04a, 5a). We also observed that this morphological alteration is consistently more severe in the high expressers than in the low expressers. This alteration was also observed in the high expressers of the single CBM29-2, although it was much less severe (Fig.\u00a04c). For the CBM2b-1-2 stems, irregularly shaped cells were observed in the cortex of the high expressers (Fig.\u00a05c). The micrographs of both the empty vector control and untransformed wild-type control looked the same; hence the pGreen7k micrograph was taken to represent the controls (Fig.\u00a04b). Altered cell enlargement across the tissues in the CBM29s-expressing stems was also observed. This was particularly true for the tandem CBM29-1-2-expressing plants (compare, for example, Figs.\u00a05a and\u00a05d with 5b\/c and 5e\/f, respectively). The altered cell size phenotype is widespread in the high and low CBM29-1-2-expressing plants. This is not observed, however, in the single CBM29-2-expressing stems, in which only the high expressers had noticeably larger cells in the epidermal, cortex, and xylem tissues than those of the controls. Similarly, there was an indication of larger xylem cells in the outermost layer of the CBM2b-1-2 stems than in controls (compare Figs.\u00a05e and 5f). There was, however, no clear indication of altered enlargement of the stem pith parenchyma cells of the transgenic plants.\nFig.\u00a04Cross sections of transgenic and control stems, showing collapsed cortical cells and enlarged cortical and xylem cells of the CBM29-1-2 stems. Sections of CBM29-1-2 (a), empty Pgreen7k control (b), CBM29-2 stem (c), and CBM2b-1-2 (d) were stained with toluidine blue. The boxed area indicates tissues shown with higher magnification in Fig.\u00a05. ep epidermis, ct cortex, xy xylem, ph pithFig.\u00a05 Magnification (10\u00d7) of cross sections of CBM29-1-2 stem (a, d), control stem (b, e), and CBM2b-1-2 stem (c, f). Micrographs show collapsed cortical cells (a), enlarged cortical and xylem cells of the CBM29-1-2 stems (a, d), and irregularly shaped stem cortical cells of the CBM2b-1-2 stems (c). Asterisks indicate irregular cortical cells in the CBM2b-1-2 transformants; in the CBM29-1-2 transformants these cells are more apparent and are not highlighted. ep epidermis, ct cortex, xy xylem, pf phloem fibre\nCryo-scanning electron microscopy (cryo-SEM) of tobacco stems\nTo establish that the transgenic stems indeed had larger cells than those of the controls we made cryo-SEM examinations of stem samples. Figure\u00a06a shows the collapsed and enlarged cells of the cortex in the sections of transgenic line 5 of CBM29-1-2, as previously observed by light microscopic examination. The cortical cells of the control plant were smaller and the collapsed phenotype was not apparent (Fig.\u00a06b). The micrographs also showed that xylem cells were indeed larger in the CBM29s-expressing stems than in the controls (compare transgenic line 5, Fig.\u00a06c and the empty vector control, Fig.\u00a06d). The micrographs also indicated that the cell walls of the xylem cells were thinner in the CBM29s-expressing stems (Fig.\u00a06c) than in stems of controls (Fig.\u00a06d). Similar micrographs were obtained for the CBM2b-1-2 series, which confirmed that the radial surface area and cell wall diameter of their stem xylem cells were larger and thinner, respectively, than those of the controls (data not shown). These notable observations were later substantiated by quantification. Figure\u00a07a shows that almost 50% and 40% of the stem cortical cells of the wild-type and pGreen7k control plants, respectively, were grouped in the small class (50\u2013100\u00a0\u03bcm2), whereas fewer than 20% of the stem cortical cells of the CBM29s-expressing plants were grouped in the small class. An opposite trend was observed in the large class (150\u2013200\u00a0\u03bcm2)\u2014fewer than 20% of the cortical cells of the two control stems were grouped compared with more than 30 and 40% of the stems expressing the single CBM29-2 and the tandem CBM29-1-2, respectively. We thus infer that the cortical cells of the CBM29-2-expressing stems are larger than those of the control stems. Similar quantitative data were obtained from the xylem cells depicted in Fig.\u00a07b, which shows that only the xylem cells of the CBM29-1-2-expressing stems are larger than those of the control stems. There was no difference between the size of the xylem cells of the single CBM29-2 stems and those of the controls. Similarly, we quantified the thickness of the cell walls of the xylem cells of the CBM29s-expressing stems and the pGreen7k control stems as presented in Fig.\u00a07c. Approximately 45% of the cell walls of the xylem cells of the CBM29-1-2-expressing stem were grouped in the thin class (1.2\u20132\u00a0\u03bcm) whereas fewer than 10% of the stem xylem cell walls of the single CBM29-2, the pGreen7k control, and the wild-type control plants were grouped in this class. More than 60% of the xylem cell walls of the two control stems were grouped in the thick class (2.8\u20133.6\u00a0\u03bcm) whereas approximately 22 and 0% of the stem xylem cell walls of the single CBM29-2 and the tandem CBM29-1-2, respectively, were grouped in this class. We can also infer from this result that the thickness of the cell walls of the xylem cells of the CBM29-2-expressing stems (particularly of the tandem CBM29-1-2) are thinner than those of the pGreen7k and the wild-type control stems.\nFig.\u00a06Scanning electron micrographs of transgenic tobacco stems to show details of larger cells and thinner cell walls of the CBM 29-1-2 stems compared with the pGreen7k control. CBM29-1-2 (a) and (c), and empty pGreen7k control (b) and (d). a and b show the cortex tissue whereas c and d show the xylem tissue. pf phloem fibre, xy xylem, ct cortex. Arrow heads indicate collapsed layer of cortical cellsFig.\u00a07Cell size distribution in the cortex (a), cell size distribution in the xylem (b), and cell wall thickness of xylem cells (c) in the stems of transgenic and the wild-type tobacco plants. Insets show how the surface area and wall thickness measurements were taken. Surface areas of 300 cells were measured, 100 cells per field of view. Cell wall diameters of 150 cells were measured, 50 cells per field of view. Error bars represent standard deviation. Grey bars represent wild-type plants. Diagonal bars represent empty pGreen7k transformants. Open bars represent single CBM29-2 transformants. Horizontal brick bars represent double CBM29-1-2 transformants\nA similar trend was observed for the xylem cells of the CBM2b-1-2 stems, which were larger than those of the pGreen7k control plants (data not shown). Similarly, the cell walls of the xylem cells of the CBM2b-1-2-expressing stems were thinner than those of the pGreen7k control stems (data not shown).\nAnalysis of cellulose content\nA colorimetric assay was performed on samples of crystalline cellulose obtained from four separate acid hydrolyses to determine the cellulose content in the stem cell walls of the CBM29s and CBM2b-1-2 transformants and the control plants. There were no substantial differences between levels of cellulose in the stem cell walls of the transgenic tobacco lines expressing the single CBM29-2, the tandem CBM29-1-2, the tandem CBM2b-1-2, the empty vector control, and the wild-type control plants (data not shown). Hence, it can be concluded that use of these CBMs for cell wall modification did not interfere with the extent of cellulose biosynthesis.\nDiscussion\nIt has been suggested that networks of plant cell wall polysaccharides can affect the properties of cellulose fibres. We hypothesized that expression of promiscuous CBMs in tobacco plants might lead to modification of these networks, although they might also affect plant development in more general terms. In this study, expression of two promiscuous Piromyces CBM29-containing proteins was targeted at tobacco cell walls to provide baseline information on the possibility of using CBMs for in-planta modification of the properties of cellulose fibre for industrial applications. In parallel, we also targeted the expression of the less promiscuous tandem CBM2b-1-2 protein of the C. fimi xylanase 11A, with a view to comparing its effect with that of the more promiscuous CBM29-1-2.\nOur results reveal that the tandem CBM29-1-2 modified the tobacco cell wall structure with consequent altered cellular and organ morphology. The tandem CBM29-1-2 modules had more effect than the single CBM29-2. Expression of the tandem CBM29-1-2 modules in the tobacco cell wall affected plant development, as reflected by reduced stem elongation rate, enlarged xylem, and collapsed cortical cells. The stem phenotype of the CBM29-1-2-expressing plants was more of delayed development of the whole plant rather than mere elongation defects of the stem. We suggest that the delayed development (prolonged juvenility) was as a result of reduced rate of anabolism in the tandem CBM29-1-2-expressing plant. It could be that structural remodelling of the cell wall has affected the normal developmental cues that signal the cells to synthesize new cell wall materials. It is conceivable that when the cell walls are held loosely continuously as a result of the continuous presence of CBM29-1-2, the rate of wall extension will outpace biosynthetic rate. Consequently, excessive thinning of the cell walls may occur (Kutschera 1990), which may have led to the eventual collapse of, particularly, the primary walled cells (and not so much of the secondary-walled xylem cells). Thus, because of the extensive loosening of their cell walls and consequent altered expansion, the cortical cells may have yielded to tissue pressure within the stem. The altered cell enlargement, which led to the collapse of the cortical cells of the tandem CBM29-1-2-expressing stems, is proposed as the cellular basis for the reduced stem elongation. We have not noticed that the increased size of the xylem cells led to thicker stems. It is possible the gain in the size of the xylem cells is neutralized by the collapse of the cortical cells.\nThe explanation given above is based on the assumption that the CBMs do not bind to polymers until they have reached the wall. It should be noted, however, that by placing a signal peptide at the amino terminus of the CBMs we have targeted them through the secretory pathway, which includes the site of hemicellulose biosynthesis (the Golgi apparatus) (Denecke et al. 1990; Vitale and Denecke 1999). It is, therefore, possible the CBMs had already bound to hemicellulose before their deposition in the cell wall. As a result, the hemicellulose content, length, and\/or branching pattern might have changed; this, in turn, could affect their interaction with cellulose microfibrils, leading to structural alteration of the cell wall.\nThe expression of the CBM2b-1-2 modules in the tobacco cell wall altered the cellular morphology of the transgenic plants, as was apparent from the irregular shape of the cortical cells and altered cell enlargement of the xylem cells of the stems. The deformation of the cortical cells of the CBM2b-1-2 expressing plants is, however, less pronounced than that of the cortical cells of the CBM29-1-2 transformants. The more obvious phenotypic changes in the CBM29-1-2-expressing plants is consistent with the result of the immunolabelling study with the proteins, which revealed that CBM29-1-2 binds to polysaccharides that are integral to both primary and secondary cell wall structure. CBM2b-1-2, in contrast, binds only to xylan and non-crystalline cellulose. As xylan is mainly present in the secondary cell wall, it is not strange to see the effect of CBM2b-1-2 most pronounced in the secondary walled xylem. The finding that CBM2b-1-2 did not label the cortex (Fig.\u00a02a) suggests that the deformation of the cortical cells is an indirect effect of CBM2b-1-2. The cortical parenchyma has no secondary walls, suggesting that the walls are much weaker here; consequently, this tissue might collapse when challenged by excessive expansion of flanking tissues. The expression of the CBM2b-1-2 only had effect on cellular morphology and not on plant morphology whereas the expression of the CBM29-1-2 in tobacco led to altered structural morphology and altered development of the transgenic plants. The reduced stem elongation phenotype of the CBM29-1-2 tobacco plants is, however, in contrast with the report by Safra-Dassa et al. (2006) of enhanced growth of transgenic potato plants expressing a bacterial CBM3. These contrasting results might be because of the different specificities of the CBMs, because we have used double CBMs in our investigation as opposed to the single CBM they used. That the experiments were performed on different plant species may also have contributed to the different observations.\nThere are reports attributing cell wall phenotypes in mutant or transgenic plants to abnormal wall assembly in general, or depletion of the cellulose content in particular (Szyjanowicz et al. 2004; Taylor et al. 1999, 2000; Tsabary et al. 2003; Turner and Somerville 1997). In all these instances the plants had deformed walls in vascular cells and reduced growth as a result of cellulose depletion. Our results reveal that wall deformation (cortical cells) can also occur without a marked reduction in cellulose content.\nTo summarise, the results of our investigation suggest that expression of the promiscuous tandem CBM29-1-2 can alter cell morphology to a larger extent than that of more specific CBM CBM2b-1-2, although the actual effect is unpredictable. Use of CBMs for in-planta cell wall modification thus still needs further investigation.","keyphrases":["carbohydrate-binding modules","cell expansion","cell wall modification","cellulose\u2013hemicellulose networks","plant growth and development","promiscuous polysaccharide recognition"],"prmu":["P","P","P","R","R","R"]}
{"id":"Evid_Based_Complement_Alternat_Med-5-1-2249747","title":"The Treatment of Pulmonary Diseases and Respiratory-Related Conditions with Inhaled (Nebulized or Aerosolized) Glutathione\n","text":"Reduced glutathione or simply glutathione (\u03b3-glutamylcysteinylglycine; GSH) is found in the cytosol of most cells of the body. GSH in the epithelial lining fluid (ELF) of the lower respiratory tract is thought to be the first line of defense against oxidative stress. Inhalation (nebulized or aerosolized) is the only known method that increases GSH's levels in the ELF. A review of the literature was conducted to examine the clinical effectiveness of inhaled GSH as a treatment for various pulmonary diseases and respiratory-related conditions. This report also discusses clinical and theoretical indications for GSH inhalation, potential concerns with this treatment, its presumed mechanisms of action, optimal doses to be administered and other important details. Reasons for inhaled GSH's effectiveness include its role as a potent antioxidant, and possibly improved oxygenation and host defenses. Theoretical uses of this treatment include Farmer's lung, pre- and postexercise, multiple chemical sensitivity disorder and cigarette smoking. GSH inhalation should not be used as a treatment for primary lung cancer. Testing for sulfites in the urine is recommended prior to GSH inhalation. Minor side effects such as transient coughing and an unpleasant odor are common with this treatment. Major side effects such as bronchoconstriction have only occurred among asthma patients presumed to be sulfite-sensitive. The potential applications of inhaled GSH are numerous when one considers just how many pulmonary diseases and respiratory-related conditions are affected by deficient antioxidant status or an over production of oxidants, poor oxygenation and\/or impaired host defenses. More studies are clearly warranted.\nIntroduction\nReduced glutathione or simply glutathione (\u03b3-glutamylcysteinylglycine; GSH) is found in the cytosol of most cells of the body (1). It is a tripeptide consisting of glycine, cysteine and glutamate. GSH functions in several enzyme systems within the body that assist with the quenching of free radicals and the detoxification of fat-soluble compounds (Table 1) (2\u20135). It also plays a significant metabolic role in supporting many different biochemical processes (e.g. amino acid transport, deoxyribonucleic acid synthesis and immune system augmentation) considered to be important mediators of health status (6).\nTable 1.Enzyme systems involving glutathioneEnzyme systemFunctionGlutathione synthetaseGamma-glutamyl cycle.Riboflavin-containing glutathione reductaseCatalyzes the conversion of oxidized glutathione (glutathione disulfide; GSSG) to its reduced form.GSH transferase isoenzymesConjugation of GSH with fat-soluble substances for liver detoxification and the detoxification of environmental carcinogens, such as those found in tobacco smoke.Selenium-containing glutathione peroxidase (GPX)Protects cells from hydrogen peroxides and lipid hydroperoxides. If not neutralized, these peroxides will damage cellular membranes and other vital cellular components.Leukotriene C4 synthaseConjugation of leukotriene A4 with GSH, resulting in the generation of leukotrienes C4. Gamma-glutamyl transpeptidase then metabolizes leukotrienes C4 to leukotrienes D4.\nGlutathione in the epithelial lining fluid (ELF) of the lower respiratory tract is thought to be the first line of defense against oxidative stress (6). The ELF concentration of GSH is 140 times that of serum concentrations with a redox ratio of >\u20099\u2009:\u20091 (7). In fact, alternations in alveolar and lung GSH metabolism are widely recognized as a central feature among many inflammatory lung diseases (8\u201314). In healthy lungs, the oxidant burden is balanced by local antioxidant defenses. However, in lung diseases cellular damage and injury is mediated by an increased oxidant burden and\/or decreased antioxidant defenses.\nIn inflammatory lung diseases, supplementation with exogenous sources of GSH would be necessary to reduce the oxidant load and\/or correct for antioxidant deficiencies within the lungs. A few published clinical studies have shown the oral administration of GSH to be ineffective at increasing plasma levels when given to healthy subjects (15), or when used for the treatment of hepatic cirrhosis (16). If the oral administration of GSH cannot raise plasma levels in healthy and diseased patients, it is doubtful that this method of delivery would have any appreciable effects at increasing GSH concentrations within the lungs.\nIntravenous administration might be effective since it bypasses the gastrointestinal tract, immediately enters the blood stream, and presumably would saturate body tissues such as the lungs. Unfortunately, the results of a study did not show intravenous administration to be effective at increasing GSH levels within the ELF (17). When 600\u2009mg of GSH was delivered intravenously to sheep, the levels in the venous plasma, lung lymph and ELF increased only for a very brief period of time. However, when the same amount of GSH was delivered through inhalation (nebulized or aerosolized), the baseline GSH level in the ELF (45.7\u2009\u00b1\u200910\u2009\u03bcM) increased 7-fold at 30-min (337\u2009\u00b1\u200964\u2009\u03bcM), remained above the baseline level 1\u2009h later (P\u2009<\u20090.001) and returned toward baseline levels by 2\u2009h. Despite this short-term increase in GSH concentrations within the ELF, the inhalation method did not significantly increase the amount of GSH in the lung lymph, venous plasma and urine during the 2\u2009h study period. The authors of this report concluded that inhalation specifically increased GSH levels at the lung epithelial surface.\nGiven that inhalation is the only known method that increases GSH levels in the ELF for a significant duration, a review of the literature was conducted to examine the clinical effectiveness of inhaled GSH as a treatment for various pulmonary diseases and respiratory-related conditions. Only reports involving human subjects were included in the analysis. The clinical and theoretical indications for GSH inhalation were summarized and potential concerns with this treatment reported. Other pertinent details such as its presumed mechanisms of action and optimal doses to be administered were compiled and evaluated.\nMethods\nLiterature Search\nComputer searches were conducted of English and non-English language articles in the Biomedical Reference Collection (1984 to August 2006), CINAHL (1982 to August 2006), MEDLINE (1965 to August 2006) and Nursing and Allied Health Collection (1985 to August 2006) databases. Articles were searched with the key search terms \u2018Nebulized Glutathione,\u2019 and \u2018Glutathione\u2019 in combination with \u2018Aerosol\u2019 OR \u2018Inhalation.\u2019 These keywords were also searched with words related to pulmonary and\/or respiratory disease. To supplement the search, references of the articles found from the initial search were reviewed. Hand searching of relevant journals was also completed as part of the search.\nSelection of Articles\nTo be included in the final review, articles had to report on the use and administration of inhaled GSH for pulmonary diseases and respiratory-related conditions in human subjects. Only peer-reviewed articles were reviewed.\nQuality Assessment\nAn evidence grade was determined for each article. These evidence grades were adapted from the hierarchy of evidence developed by the Oxford Centre of Evidence Based Medicine (Table 2) (18).\nTable 2.Grades of evidenceASystematic reviews of randomized controlled trials and\/or randomized controlled trials with or without double-blind placebo control.BSystematic reviews of observational studies and\/or high-quality observational studies including cohort and case-control studies and\/or cohort \u2018outcomes\u2019 research and\/or nonrandomized controlled trials.CCase-series, case-reports, and\/or poor-quality cohort and case-control studies.DExpert opinion without explicit critical appraisal or based on physiology, bench research or \u2018first principles.\u2019\nResults\nA total of 12 reports were screened (9, 10, 17, 19\u201327). Only one report was excluded because it involved the use of inhaled GSH in sheep (17). In total, 11 articles were found to meet the inclusion criteria and were included in this review (9, 10, 19\u201327). Table 3 displays the characteristics of the studies included in this review.\nTable 3.Summary of articles demonstrating the effectiveness of inhaled glutathione for the treatment of pulmonary diseases and respiratory-related conditionsReferenceConditionNDosages of inhaled GSHOutcomeEvidence grade(21)AsthmaEight asthma patients [mean age, 29\u2009\u00b1\u20097 (standard deviation; SD) years]600\u2009mg once weekly for 3 monthsA subset of patients with clinically stable mild asthma experienced a bronchoconstrictor effect when treated with inhaled GSH.A: Randomized placebo-controlled trial(23)Chronic otitis media with effusion (chronic OME)30 patients (3\u201312 years of age; mean age, 5.8 years) and 30 controls (3\u201312 years of age; mean age, 6.1 years)600mg of GSH in 4\u2009ml of saline subdivided into five 2-min sessions by nasal aerosol every 3\u20134 waking h for 2 weeksGSH should be considered for the nonsurgical management of chronic OME.A: Randomized placebo-controlled trial(24)Cystic fibrosis (CF)Nine patients [mean age, 16.1\u2009\u00b1\u20091.44 (SD) years] received the S-nitrosoglutathione (GSNO) and 11 patients [mean age, 19.9\u2009\u00b1\u20093.45 (SD) years] received the phosphate- buffered saline (PBS) solution0.05\u2009ml\/kg of 10\u2009mM GSNOThe treatment group showed a modest improvement in oxygenation that was thought to be independent of the physiological effects of nitric oxide.A: Randomized placebo-controlled trial(27)CF19 patients (6\u201319 years of age) were randomized to treatment [mean age, 13.3\u2009\u00b1\u20094.1 (SD) years] or placebo groups [mean age, 12.9\u2009\u00b1\u20094.9 (SD) years]Total daily dose administered to the patients in the treatment group was 66\u2009mg\/kg of body weightGSH can improve clinical parameters in CF patients, and that effective treatment should include the correction of GSH deficiency.A: Randomized placebo-controlled trial(9)Idiopathic pulmonary fibrosis (IPF)10 patients with IPF [mean age, 46\u2009\u00b1\u20093 (SD) years] and 19 normal nonsmokers [mean age, 36\u2009\u00b1\u20093 (SD) years]600\u2009mg twice daily for 3 daysInhaled GSH might be beneficial among IPF patients by reversing the oxidant\u2013antioxidant imbalance.B: Nonrandomized controlled trial(19)Human immunodeficiency virus (HIV) seropositive individuals14 HIV seropositive individuals [mean age, 32\u2009\u00b1\u20092 (SD) years]600\u2009mg twice daily for 3 daysIt is a reasonable therapeutic strategy to augment the deficient GSH levels of the lower respiratory tracts of HIV seropositive individuals.B: Cohort \u2018outcomes\u2019 research(20)Chronic rhinitis13 patients with chronic rhinitis and 13 healthy subjects (4\u201315 years of age for all subjects; mean age, 8.2 years)600\u2009mg daily for 14 daysStatistically significant improvement in nasal obstruction, rhinorrhea and ear fullness.B: Nonrandomized controlled trial(10)CFSeven CF patients [mean age, 25\u2009\u00b1\u20091 (SD) years]600\u2009mg of GSH for 3 daysInhalation therapy with GSH does normalize the respiratory epithelial surface oxidant\u2013antioxidant balance in CF patients.B: Cohort \u2018outcomes\u2019 research(25)CF21 patients with CF (16\u201337 years of age for all subjects)300 or 450\u2009mg three times daily for 14 daysInhaled GSH can permeate the lower airways of the lungs and improve important parameters of lung function in CF patients despite not having any effect upon markers of oxidative injury.B: Cohort \u2018outcomes\u2019 research(26)CF17 patients with CF (18\u201329 years of age for all subjects; mean age, 24 years)450\u2009mg three times daily for 14 daysInhaled GSH did not affect the oxidative status of the patients who were tested, but it did favorably modulate their immune responses.B: Cohort \u2018outcomes\u2019 research(22)EmphysemaOne (95 year-old male)120\u2009mg of GSH in office, then 120\u2009mg twice daily for 3 days, and continuation of treatment (dose unknown) for 2 yearsWhen the patient returned for a follow-up visit, he no longer required the use of his wheelchair and oxygen. The striking results were unexpected and unlikely to be due to placebo alone.C: Case report\nDiscussion\nBased exclusively on the published evidence included in this review, inhaled GSH is potentially indicated for the following clinical conditions: cystic fibrosis (CF), chronic otitis media with effusion (OME), HIV seropositive individuals, idiopathic pulmonary fibrosis (IPF) and chronic rhinitis. These conditions were chosen since the published studies were of good quality, received A and B evidence grades, and their respective results demonstrated benefits from the use of GSH inhalation.\nInhaled GSH cannot be recommended as a potential treatment for emphysema since the quality of evidence is lacking at the present time. The emphysema case report had notable limitations since serial spirometry was not documented, and the placebo effect could not be ruled-out (22). However, this does not necessarily indicate that GSH inhalation would be of no benefit for emphysema patients. There is experimental and human data demonstrating a link between GSH, oxidant-derived damage and possible protection against the development of emphysema. An in vitro study demonstrated that GSH could retard the oxidant-mediated down-regulation of \u03b1-1-proteinase inhibitor activity in smokers\u2019 emphysema (28). This finding is important since one of the principal pathophysiological mechanisms of emphysema is the down-regulation of this enzyme by means of oxidative damage (29). Moreover, in a recent review of lung GSH and cigarette smoke-induced airway disease, increased GSH in the ELF of chronic smokers was presumed to be a protective adaptive mechanism against the development of chronic obstructive pulmonary disease (COPD) (30). Considering that not all chronic smokers go on to develop COPD, the authors in that review pointed out that genetic variations in the molecular mechanisms that regulate GSH metabolism might explain why some individuals are better protected against the development of COPD. It thus appears that emphysema patients are subjected to progressive tissue damage due, in part, to the consequences of GSH deficiency and\/or genetic variations in GSH metabolism. Since GSH inhalation would presumably offer both antioxidant protection and GSH replenishment, this method of treatment would potentially benefit emphysema patients.\nAsthma is another condition where inhaled GSH cannot be recommended since this treatment caused notable side effects (e.g. breathlessness, bronchoconstriction and cough) in the cited study (21). These side effects were linked primarily to the production of sulfites that occurred when GSH was in solution. GSH inhalation should continue to be explored as a potential treatment for asthma. None of the asthma patients in the study had their urine tested for sulfites. A positive test for sulfites would have eliminated these patients from entering the study. Accordingly, the results might have been much more favorable if patients without sulfite sensitivities were included.\nThis issue of asthma and sulfite sensitivities is an important one for clinicians to be mindful of. Sulfites are found in beer, wine, restaurant salad bars, seafood, potatoes, processed foods and many pharmaceuticals (31). Many asthma patients report being sensitive to sulfites. In an Australian study, \u223c30% of asthmatic patients reported being sensitive to sulfites in wine (32). A more recent and rigorous scientific study, however, demonstrated that asthma patients can tolerate varying amounts of sulfites in wines ranging from 20, 75 or 150 parts per million (ppm) (33). Only a small minority of patients in this study (4 of 24 self-reported wine-sensitive asthmatics) exhibited reactions when challenged with 300\u2009ppm of sulfites. One report indicated that 4\u20138% of asthmatics are sensitive to sulfites (34). Other reports have estimated the incidence of sulfite sensitivity to be around 5\u201311% (35,36). Even though the exact percentage of sulfite-sensitive asthmatics is difficult to ascertain, sulfite sensitivity is an important factor to assess when using or evaluating research done on inhaled GSH.\nFuture Research Directions\nThere are additional clinical conditions that might benefit from this type of treatment, but further studies are necessary. One such condition is Farmer's lung (FL), which is a hypersensitivity pneumonitis caused by the inhalation of thermophilic actinomycetes and spores of Aspergillus specie (11). A study was undertaken to investigate the effect of pulmonary GSH levels after hay exposure in patients with FL and in asymptomatic farmers (AF) (11). Fifteen symptomatic patients with FL [mean age, 42\u2009\u00b1\u20091 (SD) year] were compared with 10 AF [mean age, 43\u2009\u00b1\u20091 (SD) year] serving as the control group. All patients had baseline lung function testing and testing at various time intervals following hay exposures. The authors of this study concluded that FL and AF patients have characteristically different intrapulmonary levels of GSH, and that the pathogenesis of FL is likely related to GSH regulatory mechanisms. They also speculated that AF patients have a better ability to upregulate their pulmonary GSH levels, which would protect them against active disease. Clinical testing of inhaled GSH in patients with FL is warranted.\nThe administration of GSH inhalation before and\/or immediately following exercise is another potential application of this novel treatment. Exercise is a known inducer of oxidative stress leading to free radical production, which can encourage lipid peroxidation and tissue damage among individuals with deficient and\/or impaired antioxidant systems. As stated in the beginning of this report, selenium is a cofactor in the GPX enzyme that protects cells from hydrogen peroxides and lipid hydroperoxides. When under situations of oxidative stress, the GPX enzyme will markedly increase in the lungs as an antioxidant adaptive response (37). By supplying more GSH to the lung tissues, more of this enzyme might be available to help reduce the production of free radicals associated with exercise. Although these assumptions are very speculative, it does seem possible and even logical that GSH inhalation would benefit those who regularly exercise by increasing exercise tolerance, and by maintaining and\/or replenishing the antioxidant systems within the lungs.\nMultiple chemical sensitivity disorder (MCSD) is another condition that might be clinically responsive to this treatment. Patients with this disorder are known to have bronchial hyperreactivity and even exhibit asthma-like symptoms (38). Unlike asthma, MCSD is not associated with atopy and immunoglobulin E (IgE)-mediated allergic mechanisms (39). The prevailing theory explaining the cause of MCSD is a fusion between two separate theories\u2014the neural sensitization and nitric oxide\/peroxynitrite theories (40). This fusion theory, proposed by Pall, links long term potentiation of N-methyl-d-aspartate (NMDA) receptors at the synapses of nerve cells by glutamate and aspartate to an increased production of nitric oxide and its oxidant product, peroxynitrite (40,41). Treatment with antioxidants may improve symptoms of MCSD by reducing the peroxynitrite elevations and other biochemical dysfunctions that are associated with such elevations (40,41). Glutathione inhalation may be ideal since the primary route by which patients with MCSD get triggered is through smelling and breathing. Sulfite sensitivity would have to be considered since inhaled GSH could provoke adverse events. This treatment might be capable of providing antioxidant protection to both the upper and lower respiratory airways, which would theoretically help to reduce the symptoms of MCSD and the production of peroxynitrite. More research studies are necessary.\nTwo final conditions, cigarette smoking and lung cancer, are worth mentioning since they are intimately related to each other and are affected by GSH and its related enzymes. These conditions are influenced by the glutathione S-transferase (GST) group of enzymes that are found in significant quantities in the bronchioles and alveoli of the lungs (42), and in very high concentrations in the bronchial epithelium (43). Among smokers, a lack of the GST mu enzyme was thought to be associated with a greater risk of lung cancer, especially if there was a cancer and\/or lung cancer history among the relatives of the patients in this study (44). Since the GST mu enzyme detoxify carcinogens in tobacco, any deficiency of this enzyme was presumed to be associated with an increased risk of lung cancer. However, a more recent study pertaining to the GST group of enzymes found no such association (45). In this meta-analysis, polymorphisms in the GST genes had no associations or weakly positive associations with risk factors for lung cancer. Despite the need for more research, GSH inhalation might be beneficial for smokers to augment their GST enzymes, which would help facilitate the detoxification of carcinogens. Even though the best intervention for these patients would be smoking cessation, many patients lack the necessary willpower to quit. For these patients, regular GSH inhalation might reduce oxidants generated from cigarette smoke (\u223c1014 free radicals\/puff) (46), and the epithelial lung injury associated with smoking (47).\nFor lung cancer patients, the use of GSH inhalation is not recommended. Cancer cells use multiple mechanisms (e.g. altered transport of a drug, inhibition of drug-induced apoptosis and elevation of cellular GSH) to circumvent the cytotoxic effects of chemotherapeutic agents (48). Early research studies showed that GSH was able to reduce cytotoxicity to chemotherapeutic compounds by boosting the metabolism of drugs to less active compounds, or by the detoxification of free radicals (49,50). More recently, research has revealed that the levels of a specific GST enzyme increases among cancer cells with higher differentiation grades, and that these drug-resistant gene products are found in lung carcinomas at the time of surgical resection (51). There is also speculation that GSH might be capable of repairing drug-induced injury at the DNA level (48). A recent review article has described the involvement of glutathione in the detoxification or inactivation of platinum drugs\u2014the most commonly employed drugs for the treatment of advanced stage lung cancer patients (52). Based on this information, it would be unwise and illogical to use GSH inhalation while lung cancer patients are undergoing active chemotherapy treatment.\nMechanism of Action\nInhalation of GSH results in a mechanism of action confined to the upper airways and lungs (Fig. 1), and will not influence plasma levels to a significant degree. In the studies that measured both lung and plasma levels of GSH, the plasma levels remained essentially unchanged following GSH inhalation. Seven of the studies included in this review demonstrated that GSH inhalation exerts its effects upon the lower respiratory tract (9, 10, 19, 24\u201327). The upper respiratory tract also appears to benefit from GSH augmentation. Two studies involving patients with upper respiratory tract diseases showed clinical benefits from GSH inhalation treatment (20,23). The predominant mechanism responsible for GSH's therapeutic effects are probably related to its antioxidant properties that offer protection against oxidative injury, and\/or assist with the normalization of the oxidant\u2013antioxidant balance within the upper and lower respiratory tract. Even though the majority of these studies suggested that antioxidant protection was the principal reason for the favorable treatment responses, some of the studies were unable to demonstrate a change in markers of oxidation from this treatment. More data is necessary to confirm the precise nature of GSH's antioxidant properties within the upper and lower respiratory tract. Additional explanations for GSH's therapeutic effects might include an improvement in host defenses (e.g. increased cytotoxic lymphocytes), and better oxygenation (e.g. an increase in oxygen saturation). GSH inhalation produced clinically meaningful results in the majority of diseases that were studied. Specifically, GSH inhalation was shown to improve clinical markers of respiratory function that inevitably impact upon quality of life and disease progression. These improvements were the most important outcomes and features of this novel treatment.\nFigure 1.Inhaled GSH\u2019s mechanism of action. GSH, reduced glutathione; FEV1, forced expiratory volume in 1\u2009s; FVC, forced vital capacity.\nConsiderations Prior to Initiating GSH Inhalation\nThe urine should be tested for sulfite sensitivity. A special test strip can be dipped in the urine, and is known as the \u2018EM-Quant 10013 Sulfite Test.\u2019 It can be easily located through any search engine on the Internet (53). Even though instructions for sulfite testing have been published elsewhere (54), a brief description of the procedure is outlined below:\nA random (fresh) urine sample is suitable, but a first morning void may be preferable due to its higher concentration. Once the test strip is dipped in the urine (for 1\u2009s), the reaction zone changes color to indicate the concentration of sulfites present. After 30\u2009s, the color on the test strip is compared to a color scale on the bottle indicating the concentrations of sulfites in the urine (can detect 10, 40, 80, 180 and 400\u2009ppm of sulfites). The resultant concentration should be multiplied by a factor of 1.5 to provide the amount of free sulfites in mg\/l (ppm). The strip will not detect below 10\u2009ppm. The urine samples should be preservative free, and the urinary pH should also be tested with pH paper. If the urine pH is below 6, then the amount of sulfites might be underestimated by the test. In such cases, consider adding sodium acetate or sodium hydroxide to raise the pH to at least 7\u201310 (should not exceed a pH of 12), and then repeat with a new test strip.\nIf the urine test were positive for sulfites (normally they are absent), the use of inhaled GSH would be strictly contraindicated.\nMethod of Delivery, Recommended Daily Dosages and Side Effects\nWith a nebulizer, a solution of GSH is made into an aerosol and is delivered to the upper respiratory tract and the lungs through a mask that covers the nose and mouth, or is delivered directly into the lungs via a mouthpiece. Any compounding pharmacist would be able to prepare the solution of GSH at the desired concentrations. The typical dosages used in the studies cited in Table 3 were 600\u2009mg once daily, 600\u2009mg twice daily, 900\u2009mg daily, 1350\u2009mg daily or a daily dose of 66\u2009mg\/kg of body weight. Better results are more likely to be achieved with doses of at least 600\u2009mg or more each day. One of the studies used much larger doses (66\u2009mg\/kg of body weight) since the authors speculated that these would be necessary to replace half of the amount of GSH that is produced each day (e.g. a 150\u2009lb male synthesizes 10\u2009g daily and would need 5\u2009g as a replacement dose) (27). When patients are unresponsive to doses in the range of 600\u20131350\u2009mg per day, it might be suitable to try doses that would replace half the estimated amount of GSH that is synthesized each day. These gram doses might yield better clinical results.\nIn terms of side effects, GSH inhalation is very safe. Minor side effects such as mild coughing and an unpleasant odor were reported in some of the studies included in this review. These minor side effects, better described as mild nuisance problems, were not severe enough to cause any of the study participants to discontinue treatment with inhaled GSH. The only worrisome or potentially life-threatening side effect to note is bronchoconstriction, which would be more likely to occur among sulfite-sensitive asthma and MCSD patients. However, if proper precautions such as sulfite testing are done prior to treatment, this serious side effect should be avoidable.\nMonitoring the Clinical Response to Inhaled GSH\nFor pulmonary diseases or respiratory-related conditions, baseline pulmonary function testing with a spirometer or a simple peak flow meter is recommended prior to the first treatment. After a prescribed period of treatment time, pulmonary function tests should be repeated. This will help to establish if there are any clinical improvements from regular GSH inhalation.\nConclusions\nGSH inhalation is an effective treatment for a variety of pulmonary diseases and respiratory-related conditions. Even very serious and difficult-to-treat diseases (e.g., CF, IPF) yielded benefits from this novel treatment. GSH inhalation is very safe, and rarely causes major or life-threatening side effects. The potential applications are numerous when one considers just how many pulmonary diseases and respiratory-related conditions are affected by deficient antioxidant status, poor oxygenation and\/or impaired host defenses. More studies are clearly warranted.","keyphrases":["inhaled gsh","antioxidant","aerosolized glutathione (gsh)","nebulized gsh","reduced gsh"],"prmu":["P","P","R","R","R"]}
{"id":"Purinergic_Signal-4-1-2245999","title":"Internalization and desensitization of adenosine receptors\n","text":"Until now, more than 800 distinct G protein-coupled receptors (GPCRs) have been identified in the human genome. The four subtypes of the adenosine receptor (A1, A2A, A2B and A3 receptor) belong to this large family of GPCRs that represent the most widely targeted pharmacological protein class. Since adenosine receptors are widespread throughout the body and involved in a variety of physiological processes and diseases, there is great interest in understanding how the different subtypes are regulated, as a basis for designing therapeutic drugs that either avoid or make use of this regulation. The major GPCR regulatory pathway involves phosphorylation of activated receptors by G protein-coupled receptor kinases (GRKs), a process that is followed by binding of arrestin proteins. This prevents receptors from activating downstream heterotrimeric G protein pathways, but at the same time allows activation of arrestin-dependent signalling pathways. Upon agonist treatment, adenosine receptor subtypes are differently regulated. For instance, the A1Rs are not (readily) phosphorylated and internalize slowly, showing a typical half-life of several hours, whereas the A2AR and A2BR undergo much faster downregulation, usually shorter than 1 h. The A3R is subject to even faster downregulation, often a matter of minutes. The fast desensitization of the A3R after agonist exposure may be therapeutically equivalent to antagonist occupancy of the receptor. This review describes the process of desensitization and internalization of the different adenosine subtypes in cell systems, tissues and in vivo studies. In addition, molecular mechanisms involved in adenosine receptor desensitization are discussed.\nIntroduction\nAdenosine is an important neuromodulator involved in a variety of brain activities and it also serves many different functions in the periphery. This nucleoside, when extracellular, exerts its action via specific G protein-coupled receptors (GPCRs) of the P1 class, divided into four subtypes: A1R, A2AR, A2BR and A3R [1]. GPCRs consist of a single polypeptide, containing seven \u03b1-helices which are oriented perpendicular to the membrane. The N terminus is located at the extracellular side of the cell and often contains one or more glycosylation sites. The C terminus is located intracellularly and contains phosphorylation and palmitoylation sites, which are involved in regulation of receptor desensitization and internalization [2]. All adenosine receptors, with the exception of the A2AR, contain a palmitoylation site near the C terminus. The A2AR is the only subtype with an extraordinary long C terminus, 122 amino acids versus 36 amino acids in e.g. the A1R [3]. All the adenosine receptors are glycosylated on the second extracellular loop, although glycosylation does not appear to influence ligand binding. The third intracellular loop and\/or the C terminus are involved in coupling the adenosine receptors to G proteins. Phosphorylation of in particular intracellular loop 3 is involved in desensitization and internalization of adenosine receptors [4\u20136].\nAdenosine and analogues\nAdenosine, consisting of a purine ring connected to a ribose group, is the endogenous ligand for the adenosine receptors (Fig.\u00a01). Under normal conditions, adenosine is continuously formed extracellularly by dephosphorylation of ATP, ADP and\/or AMP to adenosine by NTPDases (ectonucleoside triphosphate diphosphohydrolases). However, the A3R can also be activated by inosine, a breakdown product from adenosine [5]. Most adenosine receptor agonists are analogues of adenosine, modified by N6, C2 and C8 substitutions at the adenine base, and C5\u2032 modifications of the ribose moiety [5, 6]. Antagonists lack the ribose group and usually possess a mono-, bi- or tricyclic core structure, e.g. caffe\u00efne, which contains a xanthine as basic structure (Fig.\u00a01). For extended reviews on high affinity agonists and antagonists for adenosine receptors and their structure-activity relationships, see Palmer and Stiles [1], Fredholm et al. [5], Jacobson and Gao [7], Beukers et al. [8], M\u00fcller [9] and Klotz [10].\nFig.\u00a01Chemical structures of the endogenous ligand adenosine and the antagonist caffeine\nOccurrence and physiological functions of adenosine receptors\nThe adenosine receptors are widespread throughout the body and exert many different functions both in the CNS and in the periphery.\nThe A1R is particularly prevalent in the central nervous system, with high levels in the cerebral cortex, hippocampus, cerebellum, thalamus, brain stem and spinal cord. Numerous peripheral tissues also express the A1R, including vas deferens, testis, white adipose tissue, stomach, spleen, pituitary, adrenal gland, heart, aorta, liver, eye and bladder. Low levels are found in the lung, kidney and small intestine [1, 5, 6]. The A1R is involved in cardiovascular effects (e.g. reducing heart rate), inhibition of lipolysis and stimulation of glucose uptake in white adipocytes and the modulation of neurotransmitter release in the CNS [1]. The A1R also plays a role in anxiety, hyperalgesia, bronchoconstriction and the glomerular filtration rate and renin release in the kidney [5, 6, 11].\nIn the CNS, the A2AR is highly expressed in the striatum and olfactory tubercle [1]. In the periphery, it is highly expressed in the spleen, thymus, leucocytes and blood platelets, and intermediate levels are found in the heart, lung and blood vessels [5, 6]. The A2AR is involved in the onset of vasodilation, inhibition of platelet aggregation, exploratory activity, aggressiveness and hypoalgesia [1]. In addition, A2AR plays a role in Parkinson\u2019s disease, Huntington\u2019s disease, Alzheimer\u2019s disease, ischaemia, attenuation of inflammation and neuroprotection, particularly in peripheral tissues [5, 6]. A2A receptor antagonists slow the neurodegeneration which occurs in Parkinson\u2019s and Huntington\u2019s disease and also prevent toxicity induced by beta-amyloid in the development of Alzheimer\u2019s disease [12\u201314].\nThe A2BR is widely expressed in the brain, but generally at very low levels. In the periphery, high levels of A2BR were detected in the caecum, large intestine and urinary bladder. Lower levels were observed in the lung, spinal cord, vas deferens, pituitary, adipose tissue, adrenal gland, kidney, liver and ovaries [5, 6]. Since there is a lack of specific agonists for the A2BR, little is known about the functional significance of this receptor. However, the A2BR plays a role in mediating vasodilation in a.o. the aorta, the renal artery and the coronary artery of different species. It is also involved in allergic and inflammatory disorders [5, 6].\nThe A3R is expressed in the CNS, but at relatively low levels, and only the hypothalamus and the thalamus have been reported to contain A3R [15]. The highest levels of adenosine A3R have been found in the lung and liver, and somewhat lower levels were found in the aorta [1]. In addition, the A3R was found in eosinophils, mast cells, testis, kidney, placenta, heart, spleen, uterus, bladder, jejunum, aorta, proximal colon and eye, although with pronounced differences in expression level between species [5, 6, 16]. The A3R has been implicated in mediating allergic responses, airway inflammation and apoptotic events; however, the latter is dependent on the cell type involved and\/or the type of activation [5, 16]. Furthermore, the A3R is involved in the control of the cell cycle and inhibition of tumour growth both in vitro and in vivo [6]. In fact, adenosine A3 receptors have been demonstrated to be more highly expressed in tumours than in healthy cells, suggesting a role for A3R as a tumour marker [17].\nSignal transduction of adenosine receptors\nG protein-coupling and second messengers\nHeterotrimeric G proteins are guanine-nucleotide regulatory protein complexes composed of \u03b1 and \u03b2\u03b3 subunits. They are responsible for transmitting signals from G protein-coupled receptors to effectors, e.g. adenylyl cyclase. Until now, 16 \u03b1, 5 \u03b2 and 14 \u03b3 isoforms have been reported [18]. G proteins are divided into several subclasses with a specific activity profile: Gs proteins stimulate adenylyl cyclase, Gi proteins inhibit adenylyl cyclase and stimulate GIRK channels, G0 proteins stimulate K+ ion channels, Gq\/11 proteins activate phospholipase C, G12 proteins activate Rho guanine-nucleotide exchange factors (GEFs) and the olfactory G protein, Golf, stimulates adenylyl cyclase. Upon receptor activation, both the \u03b1 subunit and the \u03b2\u03b3 subunit can signal, but to different effectors [19\u201321]. For a recent review on G proteins, see Milligan and Kostenis [18].\nThe A1R is usually coupled to a pertussis toxin-sensitive  protein, which mediates inhibition of adenylyl cyclase and regulates calcium and potassium channels [1, 3, 5, 6, 11]. Both the third intracellular loop and the C-terminal tail of the A1R are involved in  coupling [5]. In addition, it has been reported that under certain conditions the A1R couples to Gs to stimulate adenylyl cyclase, or to Gq\/11 to stimulate inositol phosphate production. Apparently, the specific activity state of the receptor or the nature of the agonist determine which G protein class is activated by the A1R [22, 23]. The A2AR in the periphery is coupled to cholera toxin-sensitive  proteins, which increase adenylyl cyclase activity upon receptor activation. The A2AR in the striatum is presumably coupled to Golf [5, 6]. The third intracellular loop, but not the C terminus of the A2AR, is involved in  coupling [5]. The A2BR is coupled to  proteins leading to stimulation of adenylyl cyclase upon receptor activation [6]. There is quite some evidence that A2BR can activate phospholipase C as well, via Gq\/11 proteins [5]. The A3R is coupled to pertussis toxin-sensitive  proteins, which mediate inhibition of adenylyl cyclase. In addition, A3R can stimulate phospholipase C via Gq\/11 proteins [5, 6]. For an extensive overview of adenosine receptor-G protein coupling, see Fredholm et al. [24].\nDesensitization and internalization\u2014general principles and players\nMechanisms to dampen GPCR signalling exist at every level in the cell. In this section attention will be paid to the underlying principles of desensitization and internalization and the protein partners involved in these processes.\nReceptor localization\nDepending on the localization signal, GPCRs in the plasma membrane can be targeted to lipid rafts1 or caveolae2. Different regions of GPCRs can influence not only the targeting to either lipid rafts or caveolae but may also enable an interaction of the receptor with constituents of these rafts and caveolae. For instance the extracellular part of the receptor might interact with GM1 gangliosides (glycosphingolipids) present in lipid rafts\/caveolae. In addition, the C-terminal fatty acid acylation or palmitoylation may also affect targeting of GPCRs to either lipid rafts or caveolae. Finally, transmembrane regions may interact with cholesterol in the lipid rafts resulting in a change in conformation of the \u03b1-helices. Since the conformation of \u03b1-helices depends on the activation state of GPCRs, it may well be that agonist binding to the receptor may affect its localization in lipid rafts by means of molecular transitions leading to receptor activation [25, 26].\nDesensitization\nDesensitization reduces receptor activity and plays a role in signal duration, intensity and quality. Desensitization is initiated by phosphorylation of serine and\/or threonine residues in the third intracellular loop and C terminus of the receptor. Two types of desensitization occur, heterologous and homologous desensitization, and both are the result of receptor phosphorylation. Heterologous desensitization is induced by phosphorylation of the receptor by protein kinase A or C\u2014sometimes even without agonist occupancy. On the other hand, homologous desensitization is specific for agonist-occupied receptors and consists in most cases of two steps. First, the receptor is phosphorylated by one of the G protein-coupled receptor kinases (GRKs 1-7); then it binds to \u03b2-arrestin, of which two subtypes exist, which exhibit high affinity for agonist-occupied, phosphorylated receptors. \u03b2-Arrestin serves to sterically inhibit G protein coupling, thereby terminating the G protein activation, and may also target the receptor to clathrin-coated pits3 for internalization (Fig.\u00a02) [19, 25, 27, 28].\nFig.\u00a02Homologous (a) and heterologous (b) desensitization and subsequent internalization of GPCRs.  subunit,  subunit, L ligand, GRK G protein-coupled receptor kinase, P phosphorylated amino acids, \u03b2-arr \u03b2-arrestin, AP2 adaptin, E effector, second messenger, PKA protein kinase A, PKC protein kinase C\nInternalization\nReceptor desensitization, initiated by phosphorylation of the receptor by different protein kinases (A or C) or GRKs, can be subsequently followed by receptor internalization. Upon phosphorylation, \u03b2-arrestin 1 or 2 is attracted to the receptor [31]. \u03b2-Arrestins not only interact with the phosphorylated receptor, but also bind to the heavy chain of clathrin, to the \u03b22-adaptin subunit of the clathrin adaptor protein AP2, and to phosphoinositides. These interactions direct the phosphorylated receptor to punctate clathrin-coated pits in the cell membrane, which are internalized by action of the GTPase dynamin. Upon internalization, receptors can either be rapidly recycled to the plasma membrane, targeted to larger endosomes and slowly recycled, or degraded in lysosomes. The final destination of the internalized receptors largely depends on the \u03b2-arrestin subtype (1 or 2) that is recruited by the receptor upon phosphorylation and the duration of \u03b2-arrestin binding. In this way, internalization may regulate receptor resensitization and contributes to a positive regulation of receptor signalling [19, 25, 31].\nInternalization pathways\nFrom internalization studies with several receptors, it appears that the internalization pathway is specific for receptor type, cell type, metabolic state of the cell, cell-specific factors etc. Receptor trafficking can be regulated in different ways (Fig.\u00a03): (a) the receptor resides mainly in lipid rafts\/caveolae and enters the cell via this pathway by default; (b) the receptor is in lipid rafts, but leaves these upon agonist binding to be internalized via clathrin-coated pits; (c) the receptor moves into lipid rafts upon agonist binding and is internalized via this pathway; (d) the receptor moves into lipid rafts after agonist binding to activate certain signalling events, but is eventually moved out of the lipid rafts to be internalized via clathrin-coated pits. Internalization can even be achieved via uncoated vesicles or by a combination of two or more of the aforementioned pathways. For example, \u03b21-AR is internalized via both lipid rafts and clathrin-coated pits. PKA phosphorylation directs \u03b21-AR to a clathrin-coated pit, whereas GRK phosphorylation directs the receptor to lipid raft-mediated internalization [19, 25, 26].\nFig.\u00a03Different internalization pathways, adapted from Chini and Parenti, 2004 [25]. Internalization via (a) lipid rafts\/caveolae, (b) upon agonist binding, the receptor moves to clathrin-coated pits to be internalized, (c) the receptor moves into lipid rafts upon agonist binding and is internalized, (d) the receptor moves into lipid rafts upon agonist binding to activate certain signalling events, but is eventually moved out to be internalized via clathrin-coated pits. Ligand (L, green triangle), clathrin-coated pits (dotted blue lines) and lipid rafts (solid pink lines) are indicated\nFunction of lipid rafts\/caveolae\nThe existence of lipid rafts\/caveolae serves different functions. First of all, lipid rafts act as \u2018stations\u2019 in which GPCRs accomplish specific signalling tasks by meeting a selected set of signalling molecules, e.g. G proteins and adenylyl cyclases. Another possible function of lipid rafts is the protection of receptors from rapid constitutive or agonist-induced internalization, thus allowing their coupling to specific signalling pathways. In addition, caveolin may regulate the constitutive activity of receptors [32]. Finally, the endocytic pathways that GPCRs choose may depend on cell-specific factors. Switching the internalization pathway from lipid rafts\/caveolae to clathrin-coated pits may alter the final receptor destination [19, 25, 26]. Research on adenosine receptors probably provided the first account of receptor internalization via caveolae and lipid rafts, as an alternative to the well-described \u03b2-arrestin pathway [33].\nG protein-coupled receptor kinases (GRKs)\nUpon agonist binding to GPCRs, not only G proteins are recruited to the activated receptor conformation, but also two other protein families, namely the G protein-coupled receptor kinases (GRKs) and the \u03b2-arrestins [31]. The GRK family consists of seven different genes. The GRKs have been divided into three protein subfamilies, based on sequence similarity. GRK1 and GRK7 belong together. The second subfamily consists of GRK2 and GRK3 since their membrane recruitment depends on interaction with G\u03b2\u03b3 subunits and phosphatidylinositol 4,5-bisphosphate. GRK2 and GRK3 are primarily responsible for agonist-dependent receptor phosphorylation, \u03b2-arrestin recruitment and functional uncoupling of the receptor. GRK4, GRK5 and GRK6 form the third subfamily and are constitutively associated with the membrane. GRK1 and GRK7 are expressed mainly in the retinal rods and cones, whereas GRK4 expression is limited to the cerebellum, testis and kidney. In contrast, GRK2, GRK3, GRK5 and GRK6 are widely expressed in mammalian tissues [31].\n\u03b2-Arrestins\n\u03b2-Arrestins belong to the arrestin family of which four members have been identified. Arrestin 1 and arrestin 4 are expressed in the visual system in retinal photoreceptor cells; arrestin 2 (also called \u03b2-arrestin 1) and arrestin 3 (also called \u03b2-arrestin 2) are more widely expressed and are involved in the regulation of nonvisual GPCRs. The \u03b2-arrestins were originally discovered as molecules that bind to and desensitize the activated and phosphorylated form of G protein-coupled receptors as described before [19, 25, 27, 28, 31]. The \u03b2-arrestin protein consists of an N and a C domain, which are almost entirely composed of antiparallel \u03b2 sheets, connected by a linker of 12 amino acids. A \u2018polar core\u2019 is embedded between those two domains, and its disruption by the phosphorylated C terminus of the activated receptor leads to conformational changes in the \u03b2-arrestin. The C tail of the \u03b2-arrestin is then released, exposing both the clathrin and the AP2 binding domains. Recently, it appeared that \u03b2-arrestins not only are involved in the desensitization of G protein-coupled receptors, but also act as signal transducers on their own. As a consequence, they emerge as multifunctional adaptor\/scaffold proteins which mediate cellular processes such as chemotaxis, apoptosis and metastasis besides receptor signalling and trafficking. For an extensive and recent review on the interactions of \u03b2-arrestins with other cellular proteins, see Lefkowitz et al. [34].\nGRKs and \u03b2-arrestins orchestrate GPCR activities at three different levels: (1) silencing: the functional uncoupling of the receptor from its G protein by a mechanism known as \u2018homologous desensitization\u2019, (2) trafficking, which involves receptor internalization, resensitization and\/or degradation, and (3) cross-signalling: activation or inhibition of intracellular signalling pathways, independent of heterotrimeric G proteins [31].\nDampening adenosine receptor signals\nIn this section we will discuss the evidence for and mechanisms of desensitization and internalization of the four adenosine receptor subtypes. For each receptor we will first summarize results from in vitro, ex vivo and in vivo studies. This will be followed by a more biochemical approach in which we will focus on the molecular mechanisms of adenosine receptor desensitization and internalization by paying attention to the role of accessory proteins, the influence of receptor mutations, etc. We will refer to earlier seminal publications, but mostly focus on more recent evidence for adenosine receptor desensitization and internalization.\nA1 receptor\nCellular and physiological studies\nThe early evidence for adenosine A1 receptor desensitization was largely obtained from primary cells, cell lines, tissues or tissue slices, and intact animals that were exposed to varying concentrations of adenosine receptor agonists (either A1-selective or not), often examined over several time periods.\nStiles and co-workers were among the first to study A1 receptor desensitization in detail (1991). Ramkumar et al. pretreated DDT1 MF-2 cells, a smooth muscle cell line expressing both A1 and A2A receptors, with R-phenylisopropyl adenosine (R-PIA) for up to 24\u00a0h, after which the adenylyl cyclase activity was reduced by approximately 50%. This was associated with a significant decrease in cell membrane-bound A1 receptors and a concomitant increase of receptor number in intracellular compartments. The authors also showed an increase in receptor phosphorylation, nicely paralleling the time course of adenylyl cyclase modulation [35]. In a later study, Nie et al. reported similar findings in the same cell line, although desensitization occurred at a somewhat faster pace (4\u00a0h) [36]. Interestingly, Palmer et al. were unable to demonstrate desensitization in CHO cells expressing the human A1R [37]. Klaasse et al., however, were able to show internalization of the human A1R tagged with a C-terminal yellow fluorescent protein (hA1YFP-R), stably expressed in CHO cells. Exposure of these cells for 16\u00a0h to 400\u00a0nM or 4\u00a0\u03bcM CPA resulted in 25 and 40% receptor internalization, respectively. Addition of 10\u00a0\u03bcM of the allosteric enhancer PD81,723 did not accelerate the internalization process, but lowered the threshold concentration at which internalization occurred. Under those conditions a small degree of internalization was observed already at a concentration of 40\u00a0nM CPA, and at 400\u00a0nM CPA, 59% of the receptors internalized [38].\nExposure of cerebellar granule cells, endogenously expressing A1R, to 100\u00a0nM R-PIA for 2\u201348\u00a0h led to a blunting of the inhibition of adenylyl cyclase. Along with this observation, a decrease of [3H]cyclohexyladenosine ([3H]CHA) binding to intact cerebellar granule cells and an increase of [3H]CHA binding in microsomes was detected. Simultaneously, a decrease in the steady-state level of  in plasma membrane and microsomes was observed. These findings point not only to homologous desensitization, but also to subsequent internalization of the A1R in the microsomal fraction of cerebellar granule cells upon long-term agonist exposure. However, no change in mRNA level was observed, suggesting that post-transcriptional regulation underlies receptor desensitization [39].\nIn hippocampal neurons, A1Rs are present on both presynaptic and postsynaptic terminals, as well as on the cell body and dendrites where they exert different actions. To address the question of whether desensitization was influenced by subcellular localization, neurons were exposed to the agonist 2-chloroadenosine (CADO, 20\u00a0\u03bcM) from 2 up to 96\u00a0h. It was found that upon agonist exposure in cultured hippocampal neurons, the presynaptic A1Rs desensitize less quickly (>12\u00a0h) than the postsynaptic A1Rs (2\u00a0h). In accordance, the recovery of desensitized presynaptic A1Rs also requires more time (48\u00a0h vs 8\u00a0h for postsynaptic A1R). Desensitization of the postsynaptic A1Rs apparently occurs at the level of the receptor, because the other elements of the signal transduction machinery appeared to be fully functional upon receptor desensitization. All in all, these results suggest that the extent and the kinetics of agonist-induced desensitization of A1Rs depend on the subcellular localization of the receptors [40].\nUsing a more intact preparation, Coelho et al. found that the density of A1R in rat hippocampal slices was decreased by 30% upon 60\u00a0min of imposed hypoxia. This desensitization could be mimicked by adding the A1R agonist CADO (10\u00a0\u03bcM) for 60\u00a0min and was prevented by adding the A1R antagonist DPCPX. These results suggest that hypoxia leads to an increase in extracellular adenosine levels, and a subsequent, quite rapid (<90\u00a0min) desensitization, possibly followed by subsequent internalization of the A1R in nerve terminals [41].\nRat in vivo studies also demonstrated A1R desensitization. In a study by Parsons and Stiles rats were chronically (6\u00a0days) infused with R-PIA. Examination of adipocyte membranes of both treated and control rats revealed a 40% lower inhibition of adenylyl cyclase in the treated animals, coinciding with a reduced number of A1Rs (68% agonist-occupied receptors remaining) as determined by radioligand binding [42]. Upon further study it was noticed that the effects on adenylyl cyclase were not A1R specific but were controlled at the level of adipocyte G proteins. Gi levels appeared downregulated, whereas the amount of Gs in the preparation was increased, although not at the level of their mRNA, suggesting a heterologous form of desensitization [43].\nChronic exposure of rats to R-PIA (6\u00a0days) also led to A1R desensitization in the brain, and subsequent reduced inhibition of adenylyl cyclase. This loss of response was accompanied by a significant decrease in both total numbers of A1R and  proteins in synaptic plasma membranes in the brain, paralleling the finding by Stiles and co-workers in adipocytes [44]. As a consequence, a significant increase of A1R was observed in microsomes and coated vesicles, which suggested a role for coated vesicles in the internalization of A1R. Similarly, chronic agonist exposure of rats to NECA (6\u00a0days) resulted in a significant decrease of A1R in the high-affinity state in the rat brain, however without changes in adenylyl cyclase activity or inhibition of the  proteins [45].\nMolecular mechanisms\nAs discussed in the general introduction to this review, receptor posttranslational modifications, receptor phosphorylation, recruitment of arrestins and the formation of clathrin-coated pits are elements of the molecular machinery of desensitization and internalization. In addition, other potential protein partners in the two processes have been studied for the various adenosine receptor subtypes, which will also be discussed.\nEffect of receptor posttranslational modifications on desensitization and\/or internalization\nGao et al. studied the effects of preventing palmitoylation of the A1R. It appeared that the Cys309Ala mutation, thus removing the palmitoylation site of the human A1R, had no effect on internalization [46]. These findings were later confirmed by Ferguson et al. [47].\nGRKs and arrestins\nA1R phosphorylation by GRKs has been subject to debate. Palmer et al. and Ferguson et al. failed to demonstrate GRK-2 phosphorylation of A1R; in the latter study it was found that nonphosphorylated A1R redistributes arrestin 3 from the cytoplasm into punctate clusters at the plasma membrane [37, 47]. Nie et al., however, reported that within 1\u00a0h of exposure of DDT1MF-2 cells to R-PIA, rapid translocation of GRKs was observed from the cytosol to the cytoplasm [36]. In a further biochemical approach with both purified receptor and GRK-2, a phosphorylated receptor was obtained that showed enhanced affinity for arrestins over G proteins. The same receptor kinase, upon overexpression in FRTL-5 cells, influenced A1R signalling, however, not via -mediated adenylyl cyclase but through -mediated MAP kinase activation [48]. It may be concluded that under physiological conditions A1R phosphorylation does not (readily) take place, which would be a rationale for the long time periods required for receptor internalization.\nOther protein partners\nThe ectoenzyme adenosine deaminase (ADA) regulates the extracellular adenosine concentration by converting excess adenosine into inosine. However, ADA also plays a role in the desensitization and internalization of A1R in smooth muscle DDT1MF-2 cells [49, 50] and LLC-PK1 epithelial cells [51], acting as a receptor activity-modifying protein (RAMP). Upon agonist exposure (100\u00a0nM R-PIA, 2\u00a0h), ADA and A1R formed complexes on the cell surface, clustered and internalized together to intracellular compartments. Such clustering of adenosine A1 receptors prior to internalization was also reported by Ciruela et al. and Saura et al. [49, 52]. The intracellular vesicles contained the lipid raft marker protein caveolin. Filipin, an agent that disrupts rafts or caveolae, inhibited A1R internalization. In contrast, acidic treatment disrupting clathrin-coated vesicles did not inhibit agonist-induced internalization of A1R. These results indicate that ADA and A1R form a stable complex in the cell membrane of LLC-PK1 cells, internalizing upon agonist exposure via lipid rafts, in a clathrin-independent pathway. Furthermore, a direct interaction of the C terminus of A1R with caveolin was demonstrated [50]. These and other data suggest that the mode of receptor compartmentalization in response to agonist stimulation may be governed by both receptor subtype and cell type [51].\nAnother accessory protein, the heat shock cognate protein 73 (hsc73), a member of the hsp70 family, was identified as a cytosolic component able to interact with the third intracellular loop of the A1R. The interaction between hsc73 (30\u00a0nM) and A1R led to a marked reduction in affinity of ligands for the A1R and also prevented activation of the G proteins, even more so than the addition of GTP analogues or GTP itself (100\u00a0\u03bcM). These effects were completely prevented by the addition of 25\u00a0nM ADA. A high percentage of A1R was coupled to hsc73 in cell lysate, according to immunoprecipitation experiments. A remarkable feature upon internalization of the receptor in DDT1MF-2 cells was found; A1Rs internalized via two different vesicle types, one in which A1R and hsc73 are colocalized and another in which hsc73 was absent. These observations open the possibility that signalling via GPCRs is regulated at least to some extent by heat shock proteins [53].\nReceptor-receptor interactions\nDunwiddie et al. found that activation of A3R with a selective A3R agonist resulted in subsequent heterologous desensitization of the A1R, as observed in electrophysiological experiments in the CA1 region of rat hippocampus. Similar results were obtained after A3R occupancy via a brief superfusion with a high concentration of adenosine [54].\nLopes et al. investigated how activated A2AR influenced A1R function and whether this interaction was modified in aged rats. In hippocampal and cortical nerve terminals, the A2AR agonist CGS 21680 (30\u00a0nM) was able to lower the binding affinity of the A1R-selective agonist CPA, and this was taken as proof for A1R desensitization. The effect was counteracted by the addition of the A2AR antagonist ZM 241385 (20\u00a0nM). This reduction in A1R function could only be detected in young adult rats (6\u00a0weeks), but not in old rats (24\u00a0months). The addition of a PKC inhibitor, chelerythrine (6\u00a0\u03bcM), also prevented the desensitization of A1R [55]. Similarly, Ciruela et al. [56] demonstrated that A1R-A2AR heteromers were able to modulate the glutamatergic neurotransmission in the rat striatum. The main biochemical characteristic of this heteromer is the ability of the agonist-occupied A2AR to reduce the agonist affinity of the A1R [56].\nBy binding to cannabinoid CB1 receptors in Purkinje fibers in the cerebellum, \u03949-tetrahydrocannabinol inhibits adenylyl cyclase and consequently motor coordination. Long-term \u03949-tetrahydrocannabinol treatment resulted in CB1R downregulation, desensitization of the  protein and desensitization of adenylyl cyclase. G protein activation by A1R, however, was unaffected. Surprisingly, heterologous attenuation of A1R-mediated inhibition of adenylyl cyclase was observed. These results indicate that long-term \u03949-tetrahydrocannabinol administration produces a disruption of inhibitory receptor control of cerebellar adenylyl cyclase and suggest a potential mechanism of cross-tolerance to the motor effects of cannabinoid and A1 agonists [57].\nD1R and A1R have been shown to form functionally interacting heteromeric complexes in engineered cell lines and in cortical neurons in culture. Pretreatment with an A1R agonist caused complex formation of both receptors. Combined pretreatment with selective agonists for both receptors (but not one agonist alone) substantially reduced cAMP accumulation induced via the D1R, indicative for this receptor\u2019s desensitization [58].\nA2A receptor\nCellular and physiological studies\nDDT1 MF-2 cells, expressing both A1R and A2AR, were exposed to an A2A-selective agonist, which resulted in a rapid loss (t1\/2\u2009=\u200945\u00a0min) of agonist-stimulated cAMP production in these cells. This receptor desensitization, however, did not involve a reduction in cell membrane receptor number or a change in ligand affinity [35].\nProlonged exposure of PC12 cells, expressing both A2AR and A2BR, to AR agonists led to a fast (30\u00a0min) and significant inhibition of A2AR stimulation by the A2AR-selective agonist CGS21680. This effect appeared to occur at the level of adenylyl cyclase, since no change was observed in receptor number or in CGS21680\u2019s affinity for the receptor. This conclusion was corroborated by the finding of reduced activation of adenylyl cyclase by forskolin. Longer agonist exposure (12\u201320\u00a0h) led to a reduction of  levels, whereas no changes occurred in the short-term protocol [59].\nNG108-15 neuroblastoma x glioma hybrid cells express both A2AR and A2BR [60, 61]. Treatment of the cells with the non-selective agonist NECA followed by its washout led to a rapid (t1\/2\u2009=\u200920\u00a0min) and pronounced reduction in cAMP production by the A2AR-selective agonist CGS21680 when compared to vehicle-treated cells.\nPalmer et al. [62] generated CHO cells solely expressing the recombinant (canine) A2AR and examined the desensitization process of this receptor. Cells exposed to NECA showed a rapid desensitization of A2AR-stimulated adenylyl cyclase activity with no obvious difference between pretreatment of 30\u00a0min or 24\u00a0h. This was associated with a slightly reduced affinity of the receptor for the A2AR-selective radiolabelled agonist [3H]CGS21680. Cell surface receptor numbers only diminished significantly (up to 40%) upon longer-term pretreatment (t1\/2\u2009=\u20098\u00a0h) [62].\nUsing a tissue preparation, Conti et al. investigated whether prolonged exposure of A2AR to the non-selective agonist NECA, or to the selective A2AR agonists CGS21680 and 2HE-NECA, influenced A2AR desensitization. The authors used the porcine coronary artery as a sensitive vascular model, expressing among others A2ARs. The arteries were first precontracted by adding 3\u00a0\u03bcM PGF\u03b1. NECA, CGS21680 and 2HE-NECA showed high affinities for the A2AR (EC50\u2019s of 72, 40 and 20\u00a0nM, respectively) inducing vasorelaxation. Next, coronary arteries were pretreated with 10\u00a0\u03bcM NECA for 30\u00a0min or 2\u00a0h. After a 2-h washout period, the functional response was assessed. It appeared that preincubation with NECA did not hamper the vasorelaxing effects of CGS21680 and 2HE-NECA. However, NECA response curves were shifted to the right after NECA pretreatment [63]. These results seem inconclusive, since NECA pretreatment might \u2018hit\u2019 other adenosine receptor subtypes.\nA2ARs, next to A1R, are present on presynaptic baroreceptor afferent terminals within the nucleus tractus solitarius (NTS) in the brain [64]. It was observed that these A2AR modulate 5-HT release as a mechanism of baroreflex control mediated by the NTS. Low concentrations of the non-selective agonist NECA (0.3\u20133\u00a0nM), briefly exposed (5\u00a0min) to NTS brain slices, induced the release of 5-HT caused by the activation of the A2AR present at presynaptic nerves. This effect could be blocked by the addition of the adenosine A2AR antagonist 8-(3-chlorostyryl)caffeine (CSC; 100\u00a0nM). Longer exposure of NECA (20\u00a0min) to the NTS slices resulted in inhibition of the 5-HT release, probably caused by quick desensitization of the A2AR (15\u00a0min) and subsequent involvement of the A1R [65]. These findings were corroborated in a similar study with CGS21680, an A2AR-selective agonist [66].\nThe effects on AR fate upon chronic agonist exposure were studied in vivo in rat brain [45]. After 6\u00a0days of NECA treatment, the adenylyl cyclase activity in synaptic plasma membranes was decreased, suggesting a desensitization of A2Rs, although the authors did not specify or study which A2R subtype was involved. Gs protein levels were decreased indicating Gs downregulation as the mechanism of desensitization.\nInterestingly, Rekik and Mustafa [67] showed that chronic antagonist treatment (3\u00a0days) of porcine coronary arteries with ZM241,385 led to a decreased agonist responsiveness. Although A2A receptor expression went up, it appeared that the levels of Gs had decreased, altogether leading to a functional desensitization of the relaxing response by e.g. CGS21680 [67]. It should be kept in mind that ZM241,385 is also a potent antagonist for the adenosine A2B receptor.\nMolecular mechanisms\nEffect of receptor C terminus on desensitization and\/or internalization\nTo investigate the importance of the (120 amino acid residues) long C terminus of the A2AR receptor in inducing desensitization and internalization, Palmer and Stiles introduced several mutations and deletions into the receptor tail. It appeared that deletion of the last 95 amino acids of the C terminus, containing 10 possible phosphorylation sites, did not have any effect on radioligand binding, adenylyl cyclase activity or desensitization kinetics compared to the wild-type A2AR. However, when two possible phosphorylation sites (Thr 298 and Ser 305) just upstream the 95 deleted amino acids were mutated to Ala, short-term (30\u00a0min) agonist-induced desensitization was attenuated, while the long-term (24\u00a0h) desensitization was not affected. Single mutations revealed that mutation of Thr 298 alone was sufficient to reduce receptor phosphorylation and agonist-induced short-term desensitization. This study also shows that short-term and long-term desensitization have distinct structural requirements and do not occur via the same mechanism [4].\nGRKs and arrestins\nWhich GRK isoforms are involved in the phosphorylation of the A2AR is not entirely clear yet. However, a putative role for GRK2 and\/or GRK5 has been suggested [4]. The role of GRK2 in agonist-induced phosphorylation and subsequent desensitization of A2AR and A2BR was thoroughly investigated by Kelly and co-workers. Wild-type GRK2 was stably expressed in NG108-15 cells, which endogenously express A2AR and A2BR. The acute stimulation of adenylyl cyclase by activation of A2AR was markedly reduced in NG108-15 cells overexpressing wild-type GRK2. This was probably caused by GRK2-dependent pre-desensitization of the A2AR by extracellular adenosine. This effect could be reversed by pretreating the cells 24\u00a0h with 0.5 unit\/ml ADA [68]. The same research group investigated the effect of a dominant-negative GRK2 mutant on the desensitization of the A2AR [60]. Stable transfection of a GRK2Lys220Arg mutant in NG108-15 cells reduced desensitization of the A2AR by 50% following a 30-min treatment with the adenosine agonist NECA.\nTumour necrosis factor (TNF)-\u03b1 treatment of human monocytoid THP-1 cells expressing the A2AR prevented desensitization of this receptor, occurring under control conditions upon pretreatment with CGS21680 or NECA [69]. It was discovered that the TNF-\u03b1 treatment prevented GRK2 translocation to and decreased GRK2 association with the plasma membranes of these cells as a consequence of the activation of a sphingomyelinase-dependent pathway.\nIn another study, Mundell and Kelly investigated the effect of inhibitors of receptor internalization on desensitization and resensitization of ARs in NG108-15 cells [70]. Before agonist exposure, cells were pretreated with hypertonic sucrose or concanavalin A (ConA), both inhibitors of internalization. This pretreatment did not affect the agonist-induced desensitization of the A2AR. However, the resensitization of the A2AR upon agonist removal was abolished in the presence of ConA or sucrose.\nArrestins 2 and 3 have been implicated in the downstream desensitization process. CGS21680 stimulation of a tagged A2AR transiently transfected in HEK293 cells induced the translocation of GFP-tagged arrestins 2 and 3 towards the plasma membrane. A dominant-negative arrestin 2 mutant inhibited agonist-induced internalization [71].\nOther protein partners\nThe long C terminus of the A2AR has been coined a \u2018coincidence detector\u2019 as it recognizes quite a number of other proteins such as \u03b1-actinin and ARNO, the Arf nucleotide site opener (for a review see Gsandtner and Freissmuth [72]). \u03b1-Actinin may play a role in receptor internalization, not unlikely for a protein involved in cytoskeletal arrangements [71], however ARNO does not [73].\nReceptor-receptor interactions\nThe A2AR colocalizes with D2 dopamine receptors in the basal ganglia, and their interaction has been documented on several occasions [74]. For instance, the protein-protein interaction between A2AR-D2R was confirmed in HEK293T cells. The most likely mode of interaction is that helix 5 and\/or helix 6 and the N-terminal portion of I3 in the D2R approach helix 4 and the C terminus of the A2AR [75]. Within the scope of this review fits the observation that activation of the D2 receptor actually sensitizes A2AR-mediated increases in cAMP production, in CHO cells expressing both receptors [76] as well as in CAD and NS20Y neuroblastoma cells in which the D2 receptor was expressed [77]. In SH-SY5Y neuroblastoma cells co-stimulation of A2AR and D2R accelerated D2R desensitization, probably by causing or enhancing D2R internalization [78]. Recently it was found that in these SH-SY5Y neuroblastoma cells the A2AR also formed heteromers with the endogenously expressed CB1R. The CB1R is negatively coupled to adenylyl cyclase and requires previous or simultaneous activation of A2AR to signal in these cells [79].\nA2B receptor\nCellular and physiological studies\nThe adenosine A2B receptor (A2BR) is endogenously expressed on most artificial cell lines, such as COS and HEK293 cells. The exception is formed by CHO cells that lack this adenosine receptor subtype.\nThe A2BR, like the other three adenosine receptor subtypes, is subject to agonist-induced desensitization. This was measured on the level of cAMP production by Peters et al. [80]. Pretreatment of COS7 cells endogenously expressing A2B receptors with the non-selective agonist NECA (1\u00a0\u03bcM) for 1 up to 17\u00a0h led to a significant reduction in cAMP production upon acute agonist challenge, already after 1\u00a0h of pretreatment. Maximal reduction of cAMP production was reached after 4\u00a0h of pretreatment. CHO cells stably expressing a 5\u2032 FLAG epitope-tagged A2B receptor showed the same result, albeit that maximal reduction of cAMP response was already achieved after 1\u00a0h of pretreatment. Also mouse 3T3-L1 cells and human HEK293 cells, endogenously expressing the A2BR, showed a decreased cAMP response after 2\u00a0h and 5.5\u00a0h of pretreatment with 1\u00a0\u03bcM NECA, respectively [80]. In NG108-15 cells, expressing both A2AR and A2BR, the rate of desensitization for both receptor subtypes appeared similar, with a half-life of 15\u201320\u00a0min. Pretreatment with NECA of these cells (0.1\u00a0mM, 30\u00a0min) reduced stimulation of adenylyl cyclase by A2BR by approximately 50% [60, 61].\nRat pheochromocytoma PC12 cells also endogenously express both A2AR and A2BR. Prolonged exposure (14\u00a0h) to 100\u00a0nM NECA or 1\u00a0\u03bcM CGS21680 significantly inhibited the cAMP response of the cells to subsequent stimulation with the A2A-selective agonist CGS21680. Although a 100-fold higher NECA concentration is needed to stimulate the A2BR compared to A2AR, Chern et al. found that the A2B receptor cAMP response was also desensitized upon desensitization of the A2A receptor. This observation can be explained by the fact that the  protein level and the adenylyl cyclase activity were diminished upon long-term desensitization of the A2AR by CGS21680 [59].\nThe domain in which the receptor resides is also important for desensitization events, according to Sitaraman et al. NECA (10\u00a0\u03bcM) added to either the apical or basolateral side of T84 intestinal cells resulted in desensitization of the A2BR on the corresponding side within 2\u20133\u00a0h. However, whereas applying NECA to the apical side of the membrane had no effect on the basolateral A2BR, basolateral NECA induced a complete desensitization of the apical receptor. Since receptor trafficking may play a role in this cross-desensitization process, this effect may contribute to the desensitization and subsequent downregulation of the A2BR [81].\nTrincavelli et al. observed a very rapid desensitization (t1\/2\u2009=\u20095\u00a0min) of A2BR in a human astrocytoma cell line. Both G protein coupling efficiency and cAMP production were diminished after pretreatment of the cells with NECA in the presence of an A2AR-selective antagonist [82].\nMundell et al. reported that acute exposure of human airway smooth muscle (ASM) cells to adenosine receptor agonists resulted in a rapid accumulation of cAMP, most probably via A2BR. Treatment with adenosine deaminase (ADA) suggested that ASM cells produce adenosine which feeds back on the cells\u2019 A2BRs, thereby regulating basal cAMP levels and inducing a small degree of A2BR desensitization. Chronic treatment with adenosine agonists had a dual effect; both A2BR desensitization and adenylyl cyclase sensitization (an increased responsiveness of adenylyl cyclase upon stimulation) were observed [83].\nHaynes et al. [84] studied A2BR desensitization in both pulmonary artery smooth muscle cells and a more integrated preparation, the isolated perfused lung. Pretreatment of the smooth muscle cells with NECA for 45\u00a0min abrogated the increase in cAMP response otherwise observed for both NECA and isoproterenol, suggesting heterologous desensitization. Indeed, experiments with cholera toxin showed that the desensitization took place at the level of the Gs protein-adenylyl cyclase complex. In the lung preparation NECA, when dosed acutely, caused a rapid vasodilation, which was not observed when the tissue was treated again with the same compound 45\u00a0min after the first dose [84].\nTaken together, these results suggest that desensitization of the A2B receptor is a robust phenomenon, rather independent from the cell type or tissue in which it is expressed.\nMolecular mechanisms\nEffects of receptor mutations on desensitization and\/or internalization\nMatharu et al. studied the regions of the A2BR which are responsible for the desensitization and internalization of the A2BR [85]. For this purpose they introduced point mutations or deletions in the C terminus of the A2BR. Deleting the final two amino acids (Leu330-stop) did not affect the internalization properties of the A2BR. The Phe328-stop and Gln325-stop mutants, however, were resistant to agonist-induced desensitization and internalization. GFP-tagged arrestin 2 did not translocate from the cytosol to the plasma membrane upon agonist stimulation of these two truncated receptors. From a single point mutation in the C terminus, Ser329Gly, it became clear that this serine residue is responsible for the rapid agonist-induced desensitization and internalization. Surprisingly, a further deletion mutant Ser326-stop was able to undergo rapid agonist-induced desensitization and internalization; however, arrestin 2 was not attracted to the plasma membrane upon agonist stimulation. It appeared that internalization of this truncated receptor occurred via an arrestin- and clathrin-independent pathway, probably via caveolin-mediated internalization since the internalization of Ser326-stop was dynamin dependent. The destination of this truncated receptor was also different: a compartment close to the plasma membrane, instead of early endosomes. Whether the receptor will undergo rapid agonist-induced desensitization and internalization or influence the rate, extent and mechanism of internalization depends on specific sites in the C terminus of the A2BR and can be influenced by their mutation or deletion.\nGRKs and arrestins\nThe A2BR, endogenously expressed in NG108-15 cells, is desensitized through phosphorylation by GRK2. This GRK2 effect was selective for A2AR and A2BR, since the other endogenously expressed receptors (secretin R and IP-prostanoid R) were not desensitized in this way [68]. In addition, stable transfection of a dominant negative GRK2(Lys220Arg) mutant in the NG108-15 cells reduced the NECA-induced (30\u00a0min, 100\u00a0\u03bcM) desensitization of the A2BR by 50% [60]. Peters et al. studied the role of phosphorylation by PKA and PKC rather than GRK2 in agonist-induced desensitization in COS-7 cells transiently expressing the 5\u2032-FLAG A2BR. They concluded that these second messenger-dependent kinases are not involved in A2BR phosphorylation [80]. A similar conclusion for A2BR phosphorylation in NG108-15 cells was reached by Mundell and Kelly [61]. Trincavelli et al. found that treatment of astrocytoma cells with TNF-\u03b1 increased the A2BR functional response, without changing receptor protein or mRNA levels. TNF-\u03b1 treatment appeared to markedly reduce agonist-dependent receptor phosphorylation and also attenuated agonist-mediated A2BR desensitization. These effects could be inhibited by the addition of the A2BR antagonist MRS 1706 [82]. These findings resemble data obtained with the A2AR (vide supra).\nMundell et al. found that within the arrestin family, both arrestin 2 and arrestin 3 are involved in the internalization process of the A2B receptor. Within 1\u00a0min of agonist exposure, both GFP-tagged arrestin isoforms were translocated to the plasma membrane. Longer agonist exposure (>10\u00a0min), however, revealed that only arrestin 2 colocalizes with the receptor in the early endosomes. One explanation for this may be a higher affinity of the A2BR for arrestin 2 compared to arrestin 3. Interestingly, arrestins are not only involved in the internalization of the A2B receptor, but also in its recycling. It appeared that the expression of stable arrestin anti-sense constructs, reducing the levels of endogenous arrestins, not only resulted in less internalization of the receptors, but also impaired the recycling process significantly. In contrast to the internalization process, arrestin 3 induced a significantly faster rate of A2BR recycling compared to arrestin 2 [86].\nPenn et al. also studied the behaviour of GFP-tagged arrestins, now in human airway smooth muscle cells. Arrestin 2 and arrestin 3 were able to reduce the cAMP production upon stimulation of endogenous A2BR by NECA and stimulation of \u03b22-AR by isoproterenol. In addition, a punctate clustering was observed in the membrane upon exposure to either NECA (100\u00a0\u03bcM) or isoproterenol (1\u00a0\u03bcM), indicating that arrestins play a role in receptor trafficking. On the contrary, signalling and trafficking of the prostaglandin E2-R, another endogenously expressed receptor, was not affected by arrestin 2 or arrestin 3 [27].\nA3 receptor\nAdenosine A3 receptors (A3R) evoke considerable interest as novel drug targets to address cerebral\/cardiac ischaemia, cancer, inflammation, asthma and chronic obstructive pulmonary disease. So far, potent and selective antagonists for the hA3R have been identified; the disadvantage however is that those antagonists show extremely low binding affinity for the rodent A3R, typically 1,000 times lower than in humans. Since rodent models are essential for the pharmacological evaluation of new therapeutic agents, this forms a serious drawback.\nIn addition, the adenosine A3 receptor (A3R) sequence holds a nuclear localization signal in its C-terminal tail [87]. This typical stretch of four amino acids (KKFK) in helix 8 may direct the receptor to the cell nucleus, not necessarily as a consequence of desensitization or internalization. This should be kept in mind when appreciating the studies described below.\nCellular and physiological studies\nRamkumar et al. studied the characteristics of rat A3R, endogenously expressed on RBL-2H3 mast cells. In one of their experiments the authors pretreated the cells with NECA and found a partial desensitization of the initial Ca2+ response to NECA [88]. Trincavelli et al. studied the relationship between agonist-induced desensitization, internalization and resensitization of hA3R in transfected CHO cells. Agonist-induced endocytosis of hA3R was investigated by immunogold electron microscopy of plasma membranes and intracellular vesicles and shown to occur with a half-life of 17\u00a0min. Subsequent removal of the agonist led to recycling of 90% of the receptor population to the cell surface, with a half-life of 35\u00a0min. Short-term exposure to agonist caused rapid desensitization, as assessed in cAMP assays. Removal of the agonist led to resensitization of the cAMP signal to 90% of the original signal within 120\u00a0min. Internalization did not affect signal transduction, as was demonstrated after blockade of internalization and recycling. Internalization occurred via clathrin-coated pits. These results show that hA3R undergoes agonist-induced endocytosis, which is not responsible for desensitization. Moreover, in the case of hA3R, receptor sequestration rather than desensitization seems to be the first step in a cycle of internalization, dephosphorylation and recycling to the plasma membrane [89]. A similar cell line was used by Palmer et al. In stably transfected CHO cells prolonged treatment with NECA (10\u00a0\u03bcM, 20\u00a0h) induced uncoupling of recombinant hA3R from Gi proteins and a functional desensitization. Upon this A3R desensitization, an approximately twofold increase in adenylyl cyclase activity was found in the presence or absence of forskolin. This sensitization of adenylyl cyclase activity was not caused by an altered level of inhibitory or stimulatory G protein expression. The occurrence of the sensitization was also not due to new protein synthesis, but probably to an increased coupling efficiency between Gs and adenylyl cyclase. Compared to control cells, long-term exposure of stably transfected CHO cells to NECA caused an increase in phosphorylation of the cAMP-responsive element binding protein upon addition of suboptimal concentrations of forskolin. The sensitization of adenylyl cyclase activity upon long-term treatment might provide a molecular basis for the observation that for several adenosine receptor-mediated events, acute agonist exposure produces opposite effects to those after chronic agonist treatment [90]. Ferguson et al. studied the fate of the rat A3R expressed in CHO cells. The receptors internalized rapidly after treatment with NECA or R-PIA over a time frame (t\u00bd\u2009=\u200910\u00a0min) that followed receptor phosphorylation (t\u00bd\u2009=\u20091\u00a0min) [91].\nThe desensitization, internalization and downregulation of hA3R was also investigated in human astrocytoma cells, natively expressing hA3R. Short-term (15\u00a0min) exposure of the cells to 100\u00a0nM Cl-IBMECA, an A3R-selective agonist, caused rapid receptor desensitization, subsequently followed by receptor internalization within 30\u00a0min. With the help of immunogold electron microscopy, the localization of the A3R was revealed. After 10\u00a0min of exposure, the A3R was found in smooth-surfaced pits and in uncoated vesicles in the cytoplasm. After 30\u00a0min of exposure, the A3R was found in vesicular endosomes. Upon removal of the agonist, resensitization of the A3R occurred within 120\u00a0min through receptor recycling to the cell surface. Long-term agonist exposure (1\u201324\u00a0h) resulted in a marked downregulation of A3R to 22\u2009\u00b1\u20093% of control after 24\u00a0h. In conclusion, multiple, temporally distinct and sequential processes are involved in the regulation of hA3R upon short- and long-term exposure to agonists [92].\nA3Rs were found to be highly expressed on murine B16-F10 melanoma cells [93]. The authors examined the association between A3R trafficking and receptor functionality and tumour growth inhibition upon activation with the A3R-selective agonist IB-MECA. Exposure to 10\u00a0nM IB-MECA (5\u00a0min) led to rapid internalization of A3R to the cytosol, upon which receptors were directed to the endosomes for recycling or to lysosomes for degradation. The addition of 100\u00a0nM MRS 1523 (5\u00a0min), an A3R antagonist, was able to counteract the internalization process as well as the modulation of the Wnt pathway leading to proliferation, thereby emphasizing the involvement of A3R in this process. When the melanoma cells were injected into nude mice, tumours rapidly developed. Tumour growth was significantly inhibited after administration of IB-MECA to the animals, paralleled by a decrease in A3R expression in tumour lesions. In hypoxic human A172 and U87MG glioblastoma cell lines it was found that adenosine upregulates the expression of hypoxia-inducible factor-1\u03b1 (HIF-1\u03b1) and vascular endothelial growth factor (VEGF) via the A3 receptor. HIF-1\u03b1 is a key regulator in the development of tumours. The addition of the A3-antagonist MRE 3008F20 inhibited the adenosine-induced HIF-1\u03b1 and VEGF accumulation in these hypoxic cells, thereby showing a putative role in inhibiting tumour growth [94].\nTo circumvent the species differences mentioned in the introduction to the A3R section, Yamano et al. generated A3R humanized (A3ARh\/h) mice in which the A3R was replaced by its human counterpart. The expression level of hA3R in the humanized (A3ARh\/h) mice was equal to the expression levels of A3R in wild-type mice. A3R agonists were able to elevate the intracellular Ca2+ concentration in bone marrow-derived mast cells from the humanized (A3ARh\/h) mice. However, the rate of hA3R internalization was markedly reduced compared with that of mA3R in these mast cells [95].\nMolecular mechanisms\nReceptor phosphorylation, GRKs and arrestins\nPalmer and Stiles as well as Ferguson et al. investigated which amino acid residues in the C terminus are responsible and crucial for the rapid desensitization of the A3R [96, 91]. A triple mutant (Thr307, Thr318 and Thr319 to Ala) exhibited dramatically reduced phosphorylation, desensitization and internalization of the rat A3AR. Individual mutation of each Thr residue showed that Thr318 and Thr319 were the most important sites for phosphorylation. In addition, phosphorylation of Thr318 was necessary to observe phosphorylation of Thr319, but not vice versa. Moreover, changing Thr318 for a negatively charged residue (Glu) was not sufficient to retain phosphorylation at Thr319. Mutating two predicted palmitoylation sites, Cys302,305 to Ala resulted in agonist-independent basal phosphorylation of the rat A3AR. Such findings strongly suggest that palmitoylation of these Cys residues is an important factor in controlling accessibility of the C terminus of the A3R in the process of recruiting GRKs. In fact, the palmitoylation sites are highly conserved between different species, e.g. rat, mouse, human, dog, sheep. Taking these results together, it appears that GRK-mediated phosphorylation of the A3R C terminus follows a sequential mechanism, with the receptor palmitoyl moieties in an important regulatory role, and phosphorylation of Thr318 being particularly crucial as an essential first step. In an earlier study Palmer et al. replaced the carboxyl terminus of the A1R by that of the A3R [37]. This chimaeric construct was able to undergo agonist-stimulated phosphorylation and functional desensitization, similar to A3R. It was also found that purified GRK2, GRK3 and GRK5 were all able to enhance agonist-dependent phosphorylation of A3R as well as the A1-A3 chimaera.\nTrincavelli et al. studied the involvement of extracellularly regulated kinases (ERK1 and 2), members of the mitogen-activated protein kinase (MAPK) family, in A3R phosphorylation. It was found that within 5\u00a0min of exposure to 10\u00a0\u03bcM NECA, ERK1 and 2 were already phosphorylated in CHO cells stably expressing hA3R. An inhibitor of MAPK activation (PD98059) also caused inhibition of A3R phosphorylation, desensitization and internalization, probably by preventing the membrane translocation of GRK2. These results indicate that the MAPK cascade is involved in A3R regulation by a feedback mechanism which controls GRK2 activity and probably involves direct receptor phosphorylation [97].\nThe rat basophilic leukaemia cell 2H3 cell line (RBL-2H3 cells) endogenously expresses equal levels of arrestin 2 and arrestin 3. Both arrestin isoforms also have a high and comparable affinity for clathrin, thereby promoting agonist-induced internalization. RBL-2H3 cells also endogenously express a high level of A3R, which, however, neither recruited arrestin 3 nor arrestin 2 upon stimulation with NECA. Also no changes in A3R distribution were observed. One explanation is that A3R follows an endocytic mechanism upon agonist stimulation that does not involve arrestin-mediated clathrin-coated pit internalization. Another possible explanation is that arrestin recruitment was below the limit of detection in the RBL-2H3 cells [98]. Ferguson et al. observed that upon phosphorylation of A3R by GRK arrestin 3 is redistributed into punctate vesicles both at the plasma membrane and within the cytoplasm. Nevertheless, these authors also noticed that there was no colocalization between A3R and arrestin 3 [47].\nConclusions\nThe adenosine receptor subtypes are differentially regulated when exposed to agonist treatment. A1Rs are not (readily) phosphorylated and slowly internalize with a typical half-life of several hours. This feature may cause less than average tachyphylaxis upon chronic agonist administration, for instance in type II diabetes. A2AR and A2BR show much faster downregulation with similar kinetics, usually < 1\u00a0h for short-term desensitization. Agonists for the A2A receptor, currently profiled in wound healing, may thus suffer from declining efficacy upon chronic administration, in contrast to those for the A1 receptor. The A3R undergoes even faster downregulation, often a matter of minutes. The latter receptor also holds a nuclear localization signal in its carboxy terminal tail, possibly obscuring true agonist-induced downregulation. The fast desensitization of the A3R after agonist exposure may be therapeutically equivalent to antagonist occupancy of the receptor.","keyphrases":["internalization","desensitization","adenosine receptors","phosphorylation","g protein-coupled receptor kinase","palmitoylation","lipid rafts","caveolae","\u03b2-arrestins"],"prmu":["P","P","P","P","P","P","P","P","P"]}
{"id":"Diabetologia-4-1-2170455","title":"Retinal haemodynamics in individuals with well-controlled type 1 diabetes\n","text":"Aims\/hypothesis Abnormalities in retinal haemodynamics have been reported in patients with type 1 diabetes in advance of clinical retinopathy. These abnormalities could therefore be useful as early markers or surrogate endpoints for studying the microangiopathy. Since the DCCT, the increased focus on good glycaemic control is changing the natural history of diabetic retinopathy. Based on this, the aim of this study was to investigate whether patients with type 1 diabetes treated entirely or mostly in the post-DCCT era and tested in the absence of confounding factors show retinal haemodynamic abnormalities.\nThe DCCT proved that good glycaemic control is a powerful means of preventing retinopathy or slowing its course in many patients [1]. It also proved that today\u2019s means of achieving good control are imperfect, and that some patients may still progress to sight-threatening stages of retinopathy. With the latter finding, the DCCT encouraged continuation of the quest for therapies able to pre-empt the effects of the residual hyperglycaemia; however, by increasing the prevalence of good glycaemic control it made any trial of such therapies extremely long and expensive. For this reason, there is heightened interest in early markers of retinopathy to be used as surrogates to test adjunct therapies.\nRetinal haemodynamics have been investigated extensively in the quest for early functional markers of diabetic retinopathy [2]; some of the changes detected appear to be a function of the duration of diabetes and the severity of retinopathy [2\u20134]. In patients with type 1 diabetes and absent or minimal retinopathy, several investigators using different techniques have observed decreased retinal blood speed [3\u20137]. These studies, performed in the early and mid-1990s, were in patients with rather poor glycaemic control by today\u2019s standards, yet the decreased retinal blood speed could not be attributed solely to elevated blood glucose levels at the time of measurement because hyperglycaemia actually increases blood speed [2, 3, 7]. We investigated whether patients with type 1 diabetes treated entirely or mostly in the post-DCCT era and tested in the absence of confounding factors show retinal haemodynamic abnormalities.\nMethods\nThe institutional review boards of the Schepens Eye Research Institute and Massachusetts General Hospital (MGH) approved this study. We recruited, from the MGH Diabetes Center and the greater Boston area, type 1 diabetic patients with the clinical characteristics of patients previously reported [3\u20137] to show decreased retinal blood speed: 18\u201345\u00a0years of age, 1\u201315\u00a0years of insulin dependence, and absent or minimal retinopathy. The non-diabetic participants, matched for age and sex, had no history of diabetes and had HbA1c levels lower than 6%. Exclusion criteria were pregnancy, smoking, systemic diseases other than type 1 diabetes, other retinal diseases, hypertension and renal insufficiency; also excluded were users of angiotensin-converting enzyme inhibitors, angiotensin receptor antagonists, vitamin E at doses greater than 400\u00a0IU\/day, aspirin or other antiplatelet drugs, and ibuprofen. After giving informed consent, all participants in the study underwent a complete eye examination. Diabetic individuals were excluded if they had diabetic retinopathy greater than level 20 in the Early Treatment Diabetic Retinopathy Study severity scale (microaneurysms only). HbA1c was measured by the MGH HbA1c laboratory; a level >10% was an exclusion criterion.\nThe retinal haemodynamic parameters were measured when capillary blood glucose levels were between 3.8 and 11.1\u00a0mmol\/l in order to avoid the confounding effects of acute hypo- or hyperglycaemia on retinal blood flow [7]. The investigator performing the laser Doppler measurements (G. T. Feke) was masked to the diabetes status of the participants. The study eye was chosen on the basis of vascular anatomy. The superior temporal retinal arteries are preferred because temporal arteries are larger than nasal arteries, and the lesions of diabetic retinopathy are more prevalent in the superior temporal than in the other quadrants. The arterial blood column diameter and centreline blood speed were measured using a Canon laser Doppler retinal blood flow instrument (CLBF 100; Canon, Tokyo, Japan) [8]. The blood flow rate in units of \u03bcl\/min is calculated automatically as the product of the cross-sectional area of the artery at the laser Doppler measurement site and the average blood speed, assuming a circular arterial cross-section [8].\nData are presented as means\u00b1SD. Parameters were compared between diabetic and control participants using the non-paired t test.\nResults\nTable\u00a01 summarises the characteristics of the study population. The diabetic and control groups differed only in the levels of HbA1c and blood glucose. The diabetic participants received insulin through multiple daily injections or an external pump. The mean HbA1c level was 7.5% and the range 5.2\u20139.3%, eight patients having HbA1c levels above 8%. The mean duration of type 1 diabetes was 8.8\u00a0years. Only three patients manifested retinopathy, two of them showing one microaneurysm and one several microaneurysms. Of note, no diabetic individual who sought participation in the study was excluded because of advanced retinopathy, and only one diabetic individual was not included because of HbA1c greater than 10% (10.8%).\nTable\u00a01Characteristics of the study population\u00a0Diabetic individuals (n\u2009=\u200933)Controls (n\u2009=\u200931)p valueAge (years)30\u2009\u00b1\u20097.030\u2009\u00b1\u20095.60.86Sex (% female)48580.44HbA1c (%)7.5\u2009\u00b1\u20091.25.0\u2009\u00b1\u20090.3<0.0001Diabetes duration (years)8.8\u2009\u00b1\u20094.6n\/an\/aBlood glucose level (mmol\/l)a7.7\u2009\u00b1\u20092.85.4\u2009\u00b1\u20090.8<0.0001Systolic blood pressure (mmHg)106\u2009\u00b1\u200911109\u2009\u00b1\u200970.27Diastolic blood pressure (mmHg)65\u2009\u00b1\u2009866\u2009\u00b1\u200970.47Retinopathyb (%)9n\/aData are means\u00b1SD or percentagesaMeasured immediately before the retinal haemodynamic studiesbAssessed using the Early Treatment Diabetic Retinopathy Study severity scale. Individuals with a score of greater than 20 were excluded from the studyn\/a, not applicable\nFigure\u00a01 presents the retinal haemodynamic measurements in 27 diabetic and 26 control participants in whom the measurements could be performed at the major superior temporal artery. Arterial diameter and blood speed were not significantly different between the diabetic patients and the controls (diameter: patients 116\u2009\u00b1\u200912\u00a0\u03bcm, controls 112\u2009\u00b1\u200910\u00a0\u03bcm, p\u2009=\u20090.18; blood speed: patients 32.9\u2009\u00b1\u20096.0\u00a0mm\/s, controls 34.6\u2009\u00b1\u20096.8\u00a0mm\/s, p\u2009=\u20090.34). Accordingly, retinal blood flow was the same in the two groups (patients 10.4\u2009\u00b1\u20092.4\u00a0\u03bcl\/min; controls 10.4\u2009\u00b1\u20093.2\u00a0\u03bcl\/min, p\u2009=\u20090.98). In the remaining six diabetic and five control participants there was early bifurcation of the artery, and one of the branches was used for the measurements; again, no differences were noted between the two groups. The sample size provided 80% power (\u03b1\u2009=\u20090.05) to detect differences as low as 0.75\u00a0SD between diabetic and control participants. This translates into differences of 7, 15 and 24% for retinal artery diameter, blood speed and blood flow, respectively.\nFig.\u00a01Retinal haemodynamic parameters in type 1 diabetic individuals and matched non-diabetic controls. The box plots present the data for the 27 diabetic and 26 control participants in whom the measurements could be performed at the major superior temporal artery. Each box plot shows the 10th, 25th, 50th (median), 75th and 90th percentiles of the indicated parameter. Values above the 90th and below the 10th percentile are plotted as points\nNone of the haemodynamic measurements showed a significant correlation with coincident blood glucose levels, HbA1c, age, diabetes duration, systolic or diastolic blood pressure in either the diabetic or the control participants (data not shown).\nDiscussion\nThe important finding of this study is that in young patients with relatively well-controlled type 1 diabetes and no complications the baseline retinal circulatory parameters are within the normal range, even after several years of diabetes. These results differ from those of studies completed before the publication and widespread application of the DCCT results. In these earlier studies [3\u20137] the average HbA1c levels were 8, 10 or 12% compared with 7.5% in our diabetic individuals. Although we did not observe a correlation of HbA1c with retinal blood speed or flow, most likely because of the narrow range of HbA1c values, it was previously shown that retinal blood flow decreased with increasing HbA1c in type 1 diabetic patients without retinopathy [4].\nThe relation between HbA1c and retinal circulatory parameters must be interpreted with the knowledge that acute elevations [2, 7] or coincident high levels [2, 3] of blood glucose increase retinal blood flow, by increasing blood speed. Thus, the decreased blood speed and blood flow found in type 1 patients in previous studies indicated the presence of chronic effects of hyperglycaemia on retinal vessels in advance of clinical retinopathy. Elevated plasma glucose levels at the time of haemodynamic measurements could actually have masked, to some extent, such chronic effects of hyperglycaemia, contributing to the inconsistency with which retinal circulatory abnormalities were detected in diabetic patients without retinopathy [2]. The design of our study, requiring relatively normal glucose levels prior to the laser Doppler measurements (Table\u00a01), limited such potential confounding effects, and the normal retinal haemodynamics that we recorded in diabetic participants should be informative of the status of retinal vessels.\nOur results are empowering for patients with type 1 diabetes and those who care for them, for two reasons. First, it is apparent that good glycaemic control is attainable in a non-clinical trial setting, and is sustainable over time. Second, such good control not only delays or prevents retinopathy, as shown by the DCCT, but also delays or prevents abnormalities in the retinal circulation that may be prodromes to the development of retinopathy. Normal retinal haemodynamics were also reported recently in patients with type 2 diabetes with no retinopathy and mean HbA1c of 7.2% [9] or 7.7% [2].\nFrom the standpoint of finding early markers of retinopathy, our observations indicate that measurements of retinal circulatory parameters in the unperturbed state may not be sufficiently sensitive in patients with absent or minimal retinopathy and well-controlled diabetes. Insofar as the HbA1c levels in such patients are often still greater than normal, the residual hyperglycaemia is likely to leave signs on the retinal vessels, but these may be uncovered only when a functional challenge is applied to the vessels. In response to hyperoxia, used as a stimulus to test vascular reactivity, patients with well-controlled type 2 diabetes showed normal retinal haemodynamics [9], but type 1 patients in another study did not [10]. Unfortunately, the latter study did not report levels of glycaemic control. The next step will be to compare in the same well-controlled type 1 diabetic individuals the behaviour of retinal haemodynamics at steady state and in response to a challenge. In this way we will begin to reconstruct systematically the history of retinal microangiopathy in the post-DCCT era.","keyphrases":["type 1 diabetes","diabetic retinopathy","retinal blood flow","retinal circulation"],"prmu":["P","P","P","P"]}
{"id":"Neurosurg_Rev-2-2-1705527","title":"Surgical management of dural arteriovenous fistulas with transosseous arterial feeders involving the jugular bulb\n","text":"Dural arteriovenous fistulas located in the vicinity of the jugular foramen are complex vascular malformations and belong to the most challenging skull base lesions to treat. The authors comprehensively analyze multiple features in a series of dural arteriovenous fistulas with transosseous arterial feeders involving the jugular bulb. Four patients who underwent surgery via the transcondylar approach to treat dural arteriovenous fistulas around the jugular foramen were retrospectively reviewed. Previously, endovascular treatment was attempted in all patients. The success of the surgical treatment was examined with postoperative angiography. Complete obliteration of the dural arteriovenous fistulas (DAVFs) was achieved in three patients, and significant flow reduction in one individual. All patients had a good postoperative outcome, and only one experienced mild hypoglossal nerve palsy. Despite extensive bone drilling, an occipitocervical fusion was necessary in only one patient with bilateral lesions. The use of an individually tailored transcondylar approach to treat dural arteriovenous fistulas at the region of the jugular foramen is most effective. This approach allows for complete obliteration of the connecting arterial feeders, and removal of bony structures containing pathological vessels.\nIntroduction\nTen to 15% of intracranial arteriovenous fistulas are dural lesions [27], and these are most commonly related to dural venous sinuses [7]. Only a few previous articles have reported the less common intraosseous dural arteriovenous fistulas occurring at the anterior or posterior skull base [8, 19, 22, 26, 30]. Although the pathogenesis of dural arteriovenous fistulas (DAVFs) is not yet well understood, angiogenic factors and local hypoxia might play the most important roles in angiogenesis of DAVFs [24, 36\u201339]. The known experience suggests that the natural history of DAVFs is sometimes progressive, and intervention aiming at closure of these fisulas should be considered in selected cases\u2014either surgically, endovascularly, or radiosurgically [6, 11, 15, 21]. In some circumstances, successful obliteration may not be possible by endovascular means, or patients may refuse the radiosurgical treatment. Direct surgery may be the best treatment option in such cases.\nSurgical treatment of the DAVFs at the jugular foramen region aims at removal of the bony parts that contain the pathological vessels, and physical interruption or obliteration of the arterialised leptomeningeal venous connection. In the patients we have treated so far, the fistula complex was located within or in the vicinity of the occipital condyle, rendering the suboccipital transcondylar approach as the best access route for surgical treatment. Several previous studies related to fistulas with transosseous arterial feeders dealt with endovascular embolization or with a similar surgical technique, but in many aspects different from ours [14, 20, 21, 26]. It was the aim of the present study to detail our surgical technique for the treatment of DAVFs in the region of the jugular foramen, and to retrospectively review the clinical outcome of four patients treated at our institute during a 5-year period.\nPatients and methods\nPatients\nBetween June 1999 and February 2004, four patients underwent surgery at the Department of Neurosurgery, Philipps University for removal of DAVFs involving the jugular foramen. Patients ranged in age from 58 to 72\u00a0years, with a mean of 67\u00a0years. There were three females and one male; one individual had bilateral lesions. Four lesions were located on the right, and one on the left side. All patients were symptomatic, the most common presenting symptom being pulsatile bruit. The clinical details are summarized in Table\u00a01. \nTable\u00a01Summary of clinical featuresCase no.Age\/sexPresentationLocationEmbolizationAngiographyAttempts and intervalOutcome1. WP58Y\/MPulsatile bruit, rt. hypoglossal nerve paresisRt. jugular process  and bulbYesFeeders: rt. ascending pharyngeal and occipital arteries of ECA2, 2\u00a0daysNo additional deficits; symptom free2. KW72Y\/FPulsatile bruit, swelling of rt. upper and lower eyelidsRt. jugular process  and bulbYesFeeders: Both ascending pharyngeal and rt. occipital arteries of ECA4, 5\u00a0daysNo deficits; symptoms improved3. SH70Y\/FPulsatile bruitDorsal rim of foramen magnum, both occipital condyles and jugular processesYesFeeders: Both internal maxillary and occipital arteries of ECA2, 1\u00a0monthNo deficits, symptom-free4. BE68Y\/FPulsatile bruitRt. Jugular tubercle and bulbYesFeeders: rt. ascending pharyngeal and occipital arteries of ECA2, 4\u00a0daysMild hypoglossal nerve palsy; symptom-freeAbbreviations: Y: years; M: male; F: female; Rt: right; ECA: external carotid artery; Attempts and interval: attempts of endovascular embolization, and timing between the last endovascular and surgical treatment\nAll patients underwent preoperative angiography and at least two attempts of endovascular embolization. For the occlusion of arterial feeders, Guglielmi electrolytically detachable platinum coils were used. The decision for surgical intervention was made after the endovascular approach proved unsuccessful in terms of complete obliteration, and the patient developed neurological deficits or intolerable symptoms of pulsatile bruit.\nCase histories\nCase 1 A 58-year-old male presented with a 1-year history of pulsatile tinnitus in the right ear. Neurological examination revealed a partial right-sided hypoglossal palsy. Superselective angiography disclosed multiple DAVFs between the distal right sigmoid sinus and transosseous arterial feeders from the right ascending pharyngeal and right occipital arteries. Although the endovascular intervention resulted in coil occlusion of the occipital artery, the DAVFs still persisted (Fig.\u00a01). Microsurgical intervention was performed due to intolerable pulsatile tinnitus, with the patient in the sitting position. Only minimal bony involvement around the jugular foramen was detectable on the CT scan (Fig.\u00a01).\nFig.\u00a01DAVFs with transosseous arterial feeders. Right external carotid angiogram, antero-posterior (AP) (a) and lateral (b) views, demonstrate arteriovenous fistulas (arrows) adjacent to the right internal jugular bulb. The fistulas are mainly fed by the right-sided ascending pharyngeal and occipital arteries. Symptoms were improved following transarterial embolization with coils (c), but the fistulas persisted. Intraoperative photograph with the patient in the sitting position showing the electrodes for lower cranial nerve monitoring (d). Postoperative angiograms (e and f) demonstrate cure of the DAVFs. The same patient\u2019s axial CT scan reveals the preoperative intraosseous lesion near the jugular process (g). Postoperative CT scan shows minimal bony removal (h)\nCase 2 This 72-year-old female had suffered from pulsatile tinnitus in the right ear for more than 18\u00a0years. During this period, the patient was angiographically diagnosed as having DAVFs in the region of the right jugular bulb. Four sessions of endovascular intervention were performed without success. Several months before admission, the patient experienced a clinical deterioration in the form of right periorbital swelling. In addition, there was excessive lacrimation of the right eye, and progressive numbness occurred around her right ear. On the CT scan, a bony erosion of the medial portion of the right occipital condyle and jugular tubercle became evident. A recent angiography disclosed the typical early filling of the right jugular bulb, and right sigmoid and transverse sinuses through intraosseous DAVFs mainly supplied by both ascending pharyngeal arteries and both occipital arteries (Fig.\u00a02). The patient subsequently underwent surgical intervention in the left park bench position (Fig.\u00a02).\nFig.\u00a02Lateral (a) and AP (b) preoperative angiograms of the right common carotid artery (CCA), demonstrating the right intraosseous DAVFs (arrows). Postoperative right CCA angiogram revealing the remaining fistula (c). On the CT scan (d) bony erosion of the medial portion of the right jugular tubercle becomes evident (white arrows). Selective injection of preoperative right occipital artery reveals intraosseous fistula (e). The patient is positioned in the left lateral park bench position (f)\nCase 3 A 70-year-old female presented with a 10-month history of bilateral pulsatile tinnitus predominant in the right ear. Neurological examination was irrelevant, and superselective angiography revealed numerous bilateral DAVFs in the foramen magnum region affecting both occipital condyles and jugular processes (Fig.\u00a03). The fistula drained into both jugular veins, respectively. Feeding arteries were branches from the internal maxillary and occipital arteries. After partial occlusion of feeders on the right side, the intensity of the tinnitus decreased but still persisted. The patient was severely affected by this symptom. Surgery was therefore carried out using a right transcondylar exposure, and post-operatively the patient reported further improvement, with minimal residual tinnitus. During a 6-month follow-up, the intensity of left pulsatile bruit increased. Additionally, the patient developed a movement-related neck pain which proved to be caused by a fracture of the right occipital condyle, with dislocation of a bony fragment and craniocervical instability (Fig.\u00a03). She underwent a second surgical procedure via a left transcondylar approach to treat the left DAVFs, followed by an occipitocervical fusion performed in the same surgical setting (Fig.\u00a04).\nFig.\u00a03Bilateral intraosseous DAVFs causing bilateral pulsatile tinnitus. Right lateral (a) and AP (b) external carotid angiograms, as well as right AP (c) common carotid angiogram demonstrate multiple arteriovenous shunts fed by the right internal maxillary and occipital arteries. MRI (d) and CT (e) studies show the abnormal vessels posterior to the right jugular tubercle and the enlarged jugular foramen with its surrounding bony erosion, respectively (arrows). The photograph shows the skin incision for the right transcondylar approach (f). Intraoperative screenshot of navigation, applied for localizing the exent of the arteriovenous fistulas (g)Fig.\u00a04Postoperative CT scan of the same patient shown in Fig.\u00a03 reveals a fracture of the right occipital condyle with dislocation of a bony fragment (a). Postoperative angiograms of the right external carotid artery (ECA), lateral (b) and AP (c) views showing no arteriovenous fistula after the surgical resection of right intraosseous DAVFs. Preoperative angiogram of the left ECA revealing arterial filling of the jugular vein and left-side DAVFs (d). The patient is placed in the prone position for the surgery of the left intraosseous DAVFs and the occipito-cervical fusion (e). Postoperative lateral plain radiogram (f) and a schematic drawing (g) demonstrating the instrumentation used for the occipitocervical fusion (g)\nCase 4 This 68-year-old female complained of progressive pulsatile right-sided tinnitus for 1\u00a0year. Neurological examination was irrelevant. An angiogram obtained at our institution furnished evidence of DAVFs at the right occipital condyle and jugular tubercle. The fistula was mainly fed by meningeal branches of the right occipital and ascending pharyngeal arteries. The patient underwent embolization of the right occipital artery, aimed at reducing the arterial supply. Due to persistent arteriovenous connection and impairment of the patient\u2019s quality of life, surgery was subsequently performed.\nSurgical technique\nAll surgical procedures were performed by the senior author (H.B.). The first aim of surgery was an adequate exposure of the dorsolateral suboccipital region, including the medial portion of the occipital condyle, the lateral rim of the foramen magnum, the arch of the atlas and the horizontal portion of the vertebral artery, as well as the medial mastoid and particularly the jugular process region (Fig.\u00a05). The patient was placed either in the sitting or in the lateral park bench position with the head flexed, turned to the ipsilateral and slightly tilted to the contralateral side. It was considered important to reduce the venous congestion by avoiding jugular-vein compression or by elevating the head when the lateral park bench position was chosen. Arterial feeders were occasionally encountered during muscular dissection in the course of the occipital artery in those patients in whom the artery has not been completely obliterated endovascularly before surgery.\nFig.\u00a05The particular arteriovenous fistulas are located within the occipital condyle and the jugular foramen region. These fistulas are fed by dural branches of both internal and external carotid arteries (upper). The drawing demonstrates the intraosseous fistulas, arterialized diploic veins and draining vein (lower)\nFirst, a small dorso-lateral suboccipital craniectomy was performed, which basically corresponds to a dorso-lateral enlargement of the foramen magnum. Bone drilling began in the supracondylar fossa, and was extended both medially towards the rim of the foramen magnum and laterally towards the mastoid. By gradually exposing the distal sigmoid sinus and the dura mater of the suboccipital and upper cervical region, transosseous arterial feeders were encountered, which occasionally caused a brisk arterial bleeding (Fig.\u00a06). To control this bony bleeding, drilling was temporarily performed using the diamond burrs without saline irrigation (Aesculap drills). Application of bone wax or bipolar coagulation to stop the bleeding was usually ineffective. In the patients with severe bony involvement, the consistency of the cancellous bone was remarkably altered. In these cases the bone marrow was packed with Surgicel and then slightly compressed with cottonoid to control the bleeding. In some cases, the arteriovenous fistulas involved the posterior condylar emissary vein located within\/around the posterior condylar canal (Fig.\u00a07). These bony portions were completely drilled away. While continuing drilling within the occipital condyle, the hypoglossal canal and the medial portion of the jugular bulb were gradually exposed. The medial wall of the jugular bulb, being the site of the arteriovenous fistulas, was coagulated to interrupt small arterial feeders of this area (Fig.\u00a06).\nFig.\u00a06Artistic illustration showing the transcondylar approach as applied to interrupt the connecting arterial feeders; the extent of the bony removal is judged according to the extent of the arteriovenous fistulas. Transosseous arterial feeders are encountered, which may occasionally cause a brisk arterial bleedingFig.\u00a07Schematic drawing of intraosseous dural arteriovenous fistulas in the right hypoglossal canal and in the vicinity of the jugular foramen region. The feeders are the dural branches of the ascending pharyngeal artery. These arterial feeders connect with the inferior petrosal sinus, the jugular bulb and the posterior condylar emissary vein. (1: right jugular bulb, 2: right posterior emissary vein, 3: right inferior petrosal sinus, 4: arterialized venous plexus within the right hypoglossal canal, 5: right ascending pharyngeal artery, a: right internal auditory canal, b: pars nervosa of the right jugular foramen, c: right hypoglossal canal, d: right occipital condyle, e: anterior rim of the foramen magnum)\nIntraosseous DAVFs were also encountered around the hypoglossal canal, with arteriovenous shunts flowing into the venous plexus surrounding the hypoglossal canal, and more anteriorly in the region of the inferior petrosal sinus. To reach this area, the bony resection was extended into a deeper region. Use of the intraoperative microdoppler probe gave a good impression of the arterialization of the jugular bulb. Thus, persistence of intraosseous arterial feeders indicated that bony resection had to be continued by drilling the jugular tubercle, the occipital condyle and the jugular process. The required extent of bone removal was also estimated from preoperative imaging, and was related to anatomical landmarks such as the hypoglossal canal, the posterior condylar emissary canal and the dural entrance of the vertebral artery. Drilling was stopped only when no more arterial feeders were encountered, indicating that all feeders had been interrupted. Normalization of blood flow within the jugular bulb could be documented by using the microdoppler probe. To adequately drill the dorsomedial portion of the occipital condyle, a small portion of the lateral atlantal mass had to be resected in some instances as well. In such cases, complete exposure of the horizontal portion of the vertebral artery was necessary. However, there was no need for complete C1 hemilaminectomy. The suboccipital dura mater was not opened, since an intradural inspection of the jugular foramen was unnecessary as there were no intradural arteriovenous shunts. After meticulous extradural hemostasis, wound closure was obtained in several layers.\nResults\nAll four patients underwent surgical obliteration of their DAVFs via the transcondylar approach. This access route provided a good exposure of the target site, and allowed occlusion of the intraosseous pathological vessels. There were no intra-operative complications, and no permanent major postoperative deficit occurred. Despite a certain blood loss during surgery, no patient required intra-operative or postoperative blood transfusion. Although an extensive resection of the involved occipital condyle, jugular process and jugular tubercle was carried out in all patients, an occipito-cervical fusion was necessary only in patient 3 with bilateral lesions. There were no additional neurological deficits, with the exception of patient 4, who experienced mild hypoglossal nerve palsy postoperatively.\nPostoperative angiography was obtained in each patient to confirm obliteration of the DAVFs. Three patients showed radiological cures, with no further treatment necessary. In patient 2, the DAVFs were significantly reduced surgically, but still persisted. Therefore, a second surgical intervention was proposed to the patient, who eventually refused. While preoperative complaints disappeared in three patients, they were clearly improved in one individual.\nDiscussion\nDefinition, terminology, pathology and symptoms\nDural arteriovenous fistulas [DAVFs] are defined as abnormal arteriovenous connections occurring within the leaflets of the dura mater, usually within or near the walls of a dural sinus [13]. Although the term \u201cdural arteriovenous malformations\u201d [DAVMs] has been used widely in the neuroscience literature, current nomenclatures include terms such as \u201cDAVFs\u201d and \u201cdural arteriovenous shunts\u201d, according to an accepted view that many of these lesions are acquired [7, 9, 10, 18]. Initially, DAVFs were described only in association with venous sinuses. Subsequently, they have been found in other locations such as the tentorium, the skull base and the intraorbital area as well [30, 31]. Piske and Lasjaunias hypothesized that DAVFs can develop wherever veins have their course [31]. This would explain why DAVFs can be located entirely within the bone, because emissary veins have transosseous courses. The particular group of dural arteriovenous fistulas located within the occipital condyle and jugular foramen region are intracavarial (intracranial) arteriovenous shunts fed by dural branches of both internal and external carotid arteries. Some authors have named this particular lesion \u201cintraosseous DAVMs\u201d [26] or \u201cintraossous DAVFs\u201d [22]. The fistulas are located within the bony structures surrounding the jugular bulb (the major draining vein), and may involve the adjacent draining veins [8, 26].\nDAVFs involving the transverse and sigmoid sinuses always cause a pulsatile tinnitus [28, 29]. It seems that this symptom is related to venous turbulence in contact with the petrous bone, or to the high shunt flow into the petrosal sinus [22]. This might explain why all our patients presented with bruit as the predominant symptom.\nImaging\nDAVFs are often invisible on CT and MR studies [17, 23]. Consequently, a normal contrast-enhanced CT or MR study does not exclude a DAVF. Contrary to the ordinary DAVFs, CT and MRI studies aid in giving the diagnosis of this particular DAVFs when the patient presents with pulsatile tinnitus due to demonstrable abnormal vessels in the soft tissue beneath the skull base [12, 32], or when bony erosion due to intraosseous abnormal vessels is present, as in the patients of our series. However, cerebral angiography remains the most important modality in evaluating DAVFs.\nGeneral management and endovascular intervention\nAccording to current opinion, no single treatment is ideal for the obliteration of DAVFs, and management requires an interdisciplinary team approach which must be individualized for each patient. Malik et al. reported two patients with intraosseous dural arteriovenous fistulas located at the foramen magnum. He postulated that the intraosseous nature was related to the large number of emissary veins in this region, and recommended the exploration of the adjacent bone until the entire fistula is exposed [26]. Our observations and intraoperative findings confirm this operative experience, that considerable but controllable hemorrhage can occur during bone removal.\nAs being less invasive than surgery, endovascular technique is given preference as the primary treatment. However, transvenous embolization or transarterial coil embolization of the ascending pharyngeal artery during treatment of hypoglossal DAVFs may sometimes be associated with complications [14, 22]. An alternative surgical therapy is warranted if the endovascular technique eventually fails, as it happened to all patients of this study. The reason for failure of the endovascular intervention is probably the great number of arterial feeders that traverse through the occipital condyle and jugular process to enter the jugular bulb. A transvenous embolization was not carried out in our patients, to avoid the risk of major venous occlusion. Although stereotactic radiosurgery either followed or not followed by transarterial particulate embolization of accessible external carotid feeding vessels became the primary mode of treatment at some institutions [6, 15, 25, 33], it may not always replace open surgery. Radiosurgery is expected to cause obliteration of DAVFs between 1 and 3\u00a0years after treatment [6], and it is not considered as a standard therapy for all types of DAVFs. Benign cranial DAVFs [6, 34], though, are a developing indication for radiosurgery [34]; stereotactic radiosurgery may be considered as an alternative treatment for this type of DAVFs that have failed other treatments [20].\nSurgical technique and avoidance of complications\nIn the majority of our previously treated patients, we have used the juxtacondylar\/transcondylar approach to expose various intradural craniospinal lesions [1\u20135, 16]. To treat DAVFs, this approach was slightly modified, and primarily applied with the purpose of interrupting the pathological arterial feeders. The extent of bony resection at the skull base was, therefore, individually tailored according to the extent of arteriovenous fistulas under intraoperative microdoppler guidance.\nThe use of this surgical procedure was most effective, as all patients in our study showed postoperative clinical improvement, and complete obliteration of the intraosseous DAVF in three patients. Only one patient (patient 3), whose angiogram revealed residual AV fistulas, experienced previously developed mild periorbital edema. We believe that this was due to residual pathological vessels anterior to the jugular bulb, an area very difficult to reach surgically. During the removal of pathological bone containing abnormal fistulas in this patient, the dilated thin wall of the jugular bulb was slightly injured. After packing this area of venous bleeding with muscle fascia, no space for further drilling was available. A second, slightly-modified surgical intervention was, therefore, proposed to the patient to obliterate the remaining fistulas, but she refused.\nPartial resection of the occipital condyle in DAVFs may be associated with the risk of injury to the vertebral artery and\/or jugular bulb, or damage to the hypoglossal nerve. Since the arteriovenous fistulas may be located within and around the hypoglossal canal as in patient 4 of our series, the rootlets of the nerve may be injured by surgical manipulation, even in the hands of a very experienced neurosurgeon. Preoperative imaging with thin-slice CT scans or image fusion technique (which was applied in the last three patients) [35] can provide valuable information about the local topographical anatomy. This information comprises the variable anatomical configuration of the jugular tubercle, the course of the canal containing the posterior condylar emissary vein, and the size and exact location of arteriovenous fistulas.\nUsually, the partial resection of the occipital condyle with opening of the atlanto-occipital joint does not create atlanto-occipital instability [2\u20135]. Only in the case of extensive bony drilling within the condyle, necessary due to the large number of transosseous pathological feeders, a partial condylar fracture may occur as it happened in patient 3 of this series. However, an occipito-cervical fusion was undertaken in this patient only because she harbored bilateral intraosseous DAVFs, and a second procedure on the opposite side was required that would have significantly destabilized the atlanto-occipital region.\nConclusion\nDural arteriovenous fistulas located around the jugular foramen are a less common type of DAVFs. The suboccipital transcondylar approach is considered the best access route when surgery of this particular pathology is intended. The amount of pathological bone that must be removed by drilling depends upon the extent of transosseous arterial feeders. Surgical obliteration of this complex vascular malformation is possible, at least in the cases in which the arterial feeders do not extend too far anterior to the jugular bulb. With this surgical technique, an atlanto-occipital instability may occur in very large or bilateral lesions, requiring occipito-cervical fusion.","keyphrases":["dural arteriovenous fistula","transosseous arterial feeder","jugular bulb","transcondylar approach","occipital condyle","intraosseous davfs"],"prmu":["P","P","P","P","P","P"]}
{"id":"Virchows_Arch-4-1-2233709","title":"Downregulated parafibromin expression is a promising marker for pathogenesis, invasion, metastasis and prognosis of gastric carcinomas\n","text":"Parafibromin is a protein encoded by the hyperparathyroidism 2 oncosuppressor gene and its downregulated expression is involved in pathogenesis of parathyroid carcinomas. To clarify the roles of parafibromin expression in tumourigenesis and progression of gastric carcinomas, it was examined by immunohistochemistry (IHC) on tissue microarray containing gastric carcinomas (n = 508), adenomas (n = 45) and gastritis (n = 49) with a comparison of its expression with clinicopathological parametres of carcinomas. Gastric carcinoma cell lines (MKN28, AGS, MKN45, KATO-III and HGC-27) were studied for parafibromin expression by IHC and western blot. Parafibromin expression was localised in the nucleus of gastric epithelial cells, adenoma, carcinoma cells and cell lines. Its expression was gradually decreased from gastritis to gastric carcinoma, through gastric adenomas (p < 0.05) and inversely correlated with tumour size, depth of invasion, lymphatic invasion, lymph node metastasis and Union Internationale Contre le Cancer (UICC) staging (p < 0.05) but not with sex or venous invasion (p > 0.05). Parafibromin was strongly expressed in older carcinoma patients compared with younger ones (p < 0.05). There was stronger positivity of parafibromin in intestinal-type than diffuse-type carcinomas (p < 0.05). Univariate analysis indicated cumulative survival rate of patients with positive parafibromin expression to be higher than without its expression (p < 0.05). Multivariate analysis showed that age, tumour size, depth of invasion, lymphatic invasion, lymph node metastasis, UICC staging and Lauren\u2019s classification but not sex, venous invasion or parafibromin expression were independent prognostic factors for carcinomas(p < 0.05). Downregulated parafibromin expression possibly contributed to pathogenesis, growth, invasion and metastasis of gastric carcinomas. It was considered as a promising marker to indicate the aggressive behaviours and prognosis of gastric carcinomas.\nIntroduction\nParafibromin is a protein encoded by the hyperparathyroidism 2 (HRPT2) oncosuppressor gene, whose mutation causes the hyperparathyroidism\u2013jaw tumour syndrome. The disease is an autosomal dominant disorder characterised by the occurrence of parathyroid adenoma or carcinoma, fibro-osseous jaw tumours of the mandible or maxilla and renal neoplastic and non-neoplastic abnormalities, such as Wilms\u2019 tumour, hamartoma or cystic renal disease [1, 16, 20]. HRPT2 gene is located in human chromosome 1q31.2, consists of 17 exons and spans 18.5\u00a0kb in the genome. It encodes a 2.7-kb transcript which is translated into a 531-amino-acid parafibromin protein with a molecular weight of 60\u00a0kd [3, 4, 23]. The 200-amino-acid C-terminal segment of parafibromin shares 32% identity and 54% homology with cell division cycle 73, a Saccharomyces cerevisiae protein forming the polymerase-associated factor 1 (Paf1) complex, which is associated with ribonucleic acid (RNA) polymerase II and involved in transcript site selection, transcriptional elongation, histone H2B ubiquitination, histone H3 methylation, poly (A) length control and coupling of transcriptional and posttranscriptional events [10, 17, 18, 24, 27]. Parafibromin overexpression was documented to inhibit colony formation and cellular proliferation and induce cell cycle arrest in the G1 phase, indicating that parafibromin has a critical role in cell growth [28]. Northern blot analysis showed HRPT2 expression in heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas [4]. Western blot study revealed parafibromin expression as a 60-kd band in the adrenal gland, heart, pancreas and kidney but 40-kd immunoreactive bands in the heart and skeletal muscle of human [25]. Immunohistochemically, higher expression of parafibromin was found widespread in glomerular mesangial cell, hepatocytes, cells of the base of gastric glands, renal cortex tubules and the pars intermedia of the hypophysis [17]. Subsequent investigations have revealed that mutations in HRPT2 are present in 66\u2013100% of sporadic parathyroid carcinomas [7, 20]. Hyperparathyroidism\u2013jaw-tumours-syndrome-related and sporadic parathyroid carcinomas are characterised by loss of parafibromin nuclear immunoreactivity [5, 22]. Selvarajan et al. [19] found that parafibromin expression was inversely linked to tumour size, pathologic stage and lymphovascular invasion of breast carcinomas using immunohistochemistry in a tissue microarray (TMA) study. These findings suggested the potential roles of parafibromin in pathogenesis and progression of malignancies.\nGastric carcinoma ranks as the world's second leading cause of cancer mortality behind lung cancer despite a sharp worldwide decline in both its incidence and mortality since the second half of the 20th century [9]. Tumourigenesis and progression of gastric carcinoma is a multistage process with the involvement of a multifactorial aetiology, which mainly results from gene\u2013environment interactions [32, 33]. Gastric carcinomas are classified into early and advanced ones on the basis of whether the carcinomas invade into the muscularis propria of the stomach [12]. In 1965, Lauren [13] classified gastric carcinomas into intestinal- and diffuse-type ones based on the morphological appearances. Intestinal-type carcinomas are characterised by cohesive carcinoma cells forming gland-like tubular structures with expanding or infiltrative growth pattern. However, the cell cohesion is less apparent or absent in diffuse-type carcinoma and cancer cells diffusely spread in the gastric wall [31]. Generally, there is a favorable prognosis for the patients with early or intestinal carcinoma compared with the other type. In our study, parafibromin expression was examined in gastric carcinoma, adenoma, gastritis and gastric carcinoma cell lines and compared with the clinicopathological parametres of carcinomas, as well as prognosis to explore the clinicopathological significance and molecular roles of parafibromin expression in stepwise development of gastric carcinoma.\nMaterials and methods\nSubjects\nGastric carcinomas (n\u2009=\u2009508) were collected from the surgical resection, adenoma (n\u2009=\u200945) from endoscopic biopsy or polypectomy and gastritis (n\u2009=\u200949) from the endoscopic biopsy in our affiliated hospital, Himi Citizen Hospital and Kouseiren Takanoka Hospital between 1993 and 2006. All carcinomas were adenocarcinomas and the adenoma group was free from non-neoplastic polyp types, leiomyomas and benign gastrointestinal stromal tumours. The patients with gastric carcinoma were 354 men and 154 women (29~91\u00a0years, mean\u2009=\u200965.4\u00a0years). Among them, 191 cases have carcinomas accompanied with lymph node metastasis. None of the patients underwent chemotherapy or radiotherapy before surgery. They all provided consent for use of tumour tissue for clinical research and our University Ethical Committee approved the research protocol. We followed up all patients by consulting their case documents or through telephone.\nPathology\nAll tissues were fixed in 4% neutralised formaldehyde, embedded in paraffin and incised into 4-\u03bcm sections. These sections were stained by haematoxylin and eosin (HE) to confirm their histological diagnosis and other microscopic characteristics. The staging for each gastric carcinoma was evaluated according to the Union Internationale Contre le Cancer (UICC) system for the extent of tumour spread [21]. Histological architecture of gastric carcinoma was expressed in terms of Lauren\u2019s [13, 31] classification. Furthermore, tumour size, depth of invasion, lymphatic and venous invasion were determined.\nTissue microarray\nRepresentative areas of solid tumours were identified in HE-stained sections of the selected tumour cases and a 2-mm-in-diametre tissue core per donor block was punched out and transferred to a recipient block with a maximum of 48 cores using a Tissue Microarrayer (AZUMAYA KIN-1, Japan). Four-micrometre-thick sections were consecutively incised from the recipient block and transferred to polylysine-coated glass slides. HE staining was performed on TMA for confirmation of tumour tissue.\nCell lines and culture\nGastric carcinoma cell lines come from the Japanese Physical and Chemical Institute, including MKN28 (well-differentiated adenocarcinoma), AGS (moderately differentiated adenocarcinoma), MNK45 (poorly differentiated adenocarcinoma), KATO-III (poorly differentiated adenocarcinoma) and HGC-27 (undifferentiated adenocarcinoma). They were maintained in Roswell Park Memorial Institute 1640 (MKN28, MKN45 and KATO-III), minimum essential (HGC-27) or Ham\u2019s F12 (AGS) medium supplemented with 10% foetal bovine serum, 100-units\/ml penicillin, and 100-\u03bcg\/ml streptomycin in a humidified atmosphere of 5% CO2 at 37\u00b0C. Total protein was prepared from all cells by cell disruption buffer according to Protein And RNA Isolation System manual (Arctiris Bioscience, USA). All cells were collected by centrifugation, rinsed with phosphate-buffered saline, fixed by 10% formalin and then embedded in paraffin as routinely processed.\nImmunohistochemistry\nConsecutive sections were deparaffinised with xylene, dehydrated with alcohol and subjected to antigen retrieval by irradiating in target retrieval solution citrate pH 6.0 (TRS, DAKO, Carpinteria, CA 93013, USA) for 15\u00a0min with microwave oven (Oriental Rotor Lmt. Co., Tokyo, Japan). Five percent bovine serum albumin was then applied for 1\u00a0min to prevent non-specific binding. The sections were incubated with mouse anti-parafibromin antibody (Clone 2H1, SC-33638, Santa Cruz, CA, USA; 1:40) for 15\u00a0min, then treated with the anti-mouse Envison-PO (DAKO, CA, USA) antibody for 15\u00a0min. Binding sites were visualised with 3, 3\u2032-diaminobenzidine with the 5-min reaction. All the incubations were performed in a microwave oven to allow intermittent irradiation as described previously [11]. After each treatment, the slides were washed with Tris-buffered saline with Tween 20 (TBST; 10\u00a0mM Tris-HCl, 150\u00a0mM NaCl, 0.1% Tween 20) three times for 1\u00a0min. After being counterstained with Mayer\u2019s haematoxylin, the sections were dehydrated, cleared and mounted. Omission of the primary antibody was used as a negative control.\nOne hundred cells were randomly selected and counted from five representative fields of each section blindly by three independent observers (Takano Y, Li XH and Zheng HC). The positive percentage of counted cells was graded semi-quantitatively according to a four-tier scoring system: negative (\u2212), 0~5%; weakly positive (+), 6~25%; moderately positive (++), 26~50%; and strongly positive (+++), 51~100%.\nWestern blot\nFifty-microgramme denatured protein was separated on an SDS-polyacrylamide gel (10% acrylamide) and transferred to Hybond membrane (Amersham, Germany), which was then blocked overnight in 5% milk in TBST. For immunoblotting, the membrane was incubated for 1\u00a0h with mouse anti-parafibromin antibody as described above. Then, it was rinsed by TBST and incubated with anti-mouse immunoglobulin G conjugated to horseradish peroxidase (DAKO, CA, USA, 1:1,000) for 1\u00a0h. Bands were visualised with X-ray film (Fujifilm, Japan) by ECL-Plus detection reagents (Amersham, Germany). After that, membrane was washed with WB Stripping Solution (pH 2\u20133, Nacalai, Tokyo, Japan) for 30\u00a0min and treated as described above except mouse anti-\u03b2-actin antibody (Sigma, MO, USA, 1:5,000) as an internal control.\nStatistical analysis\nStatistical evaluation was performed using Spearman correlation test to analyse the rank data. Kaplan\u2013Meier survival plots were generated and comparisons between survival curves were made with the log\u2013rank statistic. The Cox\u2019s proportional hazards model was employed for multivariate analysis. P\u2009<\u20090.05 was considered as statistically significant. SPSS 10.0 software was employed to analyse all data.\nResults\nParafibromin expression in gastric tumours and carcinoma cell lines\nAs shown in Fig.\u00a01, parafibromin was positively immunostained in the nucleus of MKN28, AGS, MKN45, KATO-III and HGC-27, and its expression level was consistent with the data of Western blot. Parafibromin was strongly expressed in the nucleus of gastric epithelial cells, adenomas and early carcinomas but not in given advanced carcinomas. Occasionally, it also appeared in stromal fibroblasts and lymphocytes but much weaker than epithelial cells or adenomas (Fig.\u00a02).Generally, the stromal lymphocytes and fibroblasts were negative in cases where the tumour was negative. Overall, parafibromin expression was detected respectively in all gastritis (100.0%), 36 out of 45 adenoma patients (80.0%) and 233 out of total 508 gastric carcinoma patients (45.9%). Statistically, gradually reduced expression of parafibromin was seen from gastritis to gastric carcinoma through gastric adenoma (p\u2009<\u20090.05, Table\u00a01). As summarised in Table 2, parafibromin expression was inversely correlated with tumour size, depth of invasion, lymphatic invasion, lymph node metastasis and UICC staging (p\u2009<\u20090.05) but not with sex or venous invasion (p\u2009>\u20090.05). Parafibromin was strongly expressed in older carcinoma patients compared with younger ones (p\u2009<\u20090.05). Intestinal-type carcinomas exhibited more frequent expression of parafibromin than diffuse-type ones (p\u2009<\u20090.05).\nFig.\u00a01Parafibromin expression in gastric carcinoma cell lines. a Parafibromin was positively immunostained in the nucleus of MKN28 (a), AGS (b), MKN45 (c), KATO-III (d) and HGC-27(e). b Cell lysate (50\u00a0\u00b5g) was loaded and probed with anti-human parafibromin antibody (60\u00a0kd) with \u03b2-actin (42\u00a0kd) as an internal control. Lane #1: MKN28; #2 AGS; #3 MKN45; #4 KATO-III; #5 HGC-27Fig.\u00a02Immunohistochemical staining of parafibromin in gastritis, adenoma and carcinoma. Note parafibromin positivity was strongly observed in the nucleus of gastric superficial epithelium (a), and adenoma (c) and early gastric carcinoma (b), occasionally weaker in the stromal fibroblasts and lymphocytes(a, c), but not in given advanced gastric carcinomas (d), indicating that the internal positive control (stromal cells) was negative adjacent to the negative staining carcinoma cells but positive adjacent to the positive epithelial cellsTable\u00a01Parafibromin expression in gastric tissue samplesGroupsNumberParafibromin expression\u2212++++++PR (%)Gastritis4901741100.0aGastric adenoma459582380.0bGastric carcinoma508275556011845.9PR Positive rateaCompared with gastric adenoma or carcinoma, p\u2009<\u20090.001bCompared with gastric carcinoma, p\u2009<\u20090.001Table\u00a02 Relationship between parafibromin expression and clinicopathological features of gastric carcinomasClinicopathological featuresNumberParafibromin expression\u2212++++++PR (%)Rsp valueAge (years)\u00a0\u00a0<6520912524243640.20.095<0.05\u00a0\u00a0\u22656529915031368249.8Sex\u00a0\u00a0Male35418837389146.90.054>0.05\u00a0\u00a0Female1548718222743.5Tumour size (cm)\u00a0\u00a0<426311626348555.9\u22120.237<0.001\u00a0\u00a0\u2265424515927263335.1Depth of invasion\u00a0\u00a0Tis\u2212126310230409161.2\u22120.344<0.001\u00a0\u00a0T2-424517325202729.4Lymphatic invasion\u00a0\u00a0\u221233115742458752.6\u22120.168<0.001\u00a0\u00a0+17711813153133.3Venous invasion\u00a0\u00a0\u2212443236495110746.7\u22120.051>0.05\u00a0\u00a0+6539691140.0Lymph node metastasis\u00a0\u00a0\u221231713838449756.5\u22120.285<0.001\u00a0\u00a0+19113717162128.3UICC staging\u00a0\u00a00\u2013I29212336419257.9\u22120.292<0.001\u00a0\u00a0II-IV21615219192629.6Lauren\u2019s classification\u00a0\u00a0Intestinal-type27310834389360.4\u22120.322<0.001\u00a0\u00a0Diffuse-type22515721222530.2PR Positive rate, Tis carcinoma in situ, T1 lamina propria and submucosa, T2 muscularis propria and subserosa, T3 exposure to serosa, T4 invasion into serosa, UICC Union Internationale Contre le Cancer\nUnivariate and multivariate survival analysis\nFollow-up information was available on 508 gastric carcinoma patients for periods ranging from 0.2\u00a0months to 12.2\u00a0years (median\u2009=\u200967.2\u00a0months). Figure 3 showed survival curves stratified according to parafibromin expression for gastric carcinomas. Univariate analysis using the Kaplan\u2013Meier method indicated cumulative survival rate of patients with weak, moderate or strong parafibromin expression to be obviously higher than without its expression (p\u2009<\u20090.05; Fig.\u00a03a). The significant difference disappeared if stratified according to the depth of invasion (Fig.\u00a03b, c). Multivariate analysis using Cox\u2019 s proportional hazard model indicated that age, tumour size, depth of invasion, lymphatic invasion, lymph node metastasis, UICC staging and Lauren\u2019s classification (p\u2009<\u20090.05) but not sex, venous invasion or parafibromin expression were independent prognostic factors for overall gastric carcinomas (p\u2009>\u20090.05; Table\u00a03).\nFig.\u00a03Correlation between parafibromin status and prognosis of the gastric carcinoma patients. Kaplan\u2013Meier curves for cumulative survival rate of patients with gastric carcinomas according to the parafibromin expression in overall (a), early (b, EGC) and advanced (c, AGC) gastric carcinomasTable\u00a03Multivariate analysis of clinicopathological variables for survival with gastric carcinomasNumberClinicopathological parametresRelative risk (95%CI)p valueAAge (\u226565\u00a0years)1.929 (1.357\u20132.743)<0.001BSex (female)1.463 (0.983\u20132.179)>0.05CTumour size (\u22654\u00a0cm)1.606 (1.013\u20132.547)<0.05DDepth of invasion (T2\u20134)6.530 (3.110\u201313.710)<0.001ELymphatic invasion (+)2.626 (1.516\u20133.374)<0.001FVenous invasion (+)0.959 (0.633\u20131.452)>0.05GLymph node metastasis (+)2.773 (1.525\u20135.043)<0.01HUICC staging (II-IV)0.294 (0.139\u20130.622)<0.01ILauren\u2019s classification (diffuse-type)1.796 (1.212\u20132.661)<0.01JParafibromin expression (+~+++)0.792 (0.529\u20131.187)>0.05CI Confidence interval, UICC= Union Internationale Contre le Cancer\nDiscussion\nHRPT2 has been isolated from complementary DNA libraries of parathyroid, kidney and bone tissue and encodes tumour suppressor protein parafibromin [4]. In the present study, the nuclear expression pattern was observed in the gastric epithelial cells, adenomas, adenocarcinomas and carcinoma cell lines consistent with previous reports in the gastric superficial mucosa, hepatocytes, kidney cortex tubules, adrenal gland, spleen lymphocytes, parathyroid tissue, adenoma and carcinomas, breast carcinoma [5, 8, 17, 19]. Although the result was in contrast with the paper of Porzionato et al. [17] possibly due to different incubation times of primary antibody, the great majority of immunohistochemical and cell transfection studies supported our observation of the nuclear staining [2, 5, 6, 8, 14, 19]. This study again demonstrates that parafibromin is nuclear and not cytoplasmic or nucleocytoplasmic in location as initially thought. The weaker expression of parafibromin in stromal cells than epithelial cells and adenoma might be due to the specificity of its cellular distribution as described previously [17]. It was found that the translocation of parafibromin to the nuclear compartment involved a function monopartite nuclear localisation signal at residues 136\u2013139 [2, 6]. Parafibromin is a component of Paf1 complex in the nucleus, where it plays a role in cell cycle regulation, histone methylation, lipid and nucleic acid metabolism [10, 17]. The distribution pattern of parafibromin protein in gastric epithelial cells or tumour cells demonstrated its biological function in the nucleus.\nStatistically, parafibromin expression was gradually reduced from gastritis to carcinoma through adenoma in line with parathyroid carcinogenesis, suggesting that downregulated parafibromin expression might contribute to the malignant transformation of gastric epithelial cells as an early event. The positive rate of parafibromin expression was reduced to 80% in gastric adenoma and reached about 46% of gastric adenocarcinoma, supporting the involvement of parafibromin in the gastric adenoma\u2013adenocarcinoma sequence. Actually, the adenoma can progress into and be incorporated with gastric well-differentiated carcinoma when it grows bigger and de novo carcinogenesis is well understood, especially in diffuse-type gastric carcinomas [34]. Higher parafibromin expression in adenoma and intestinal-type carcinoma indicated that decreased parafibromin expression might play an important role in de novo diffuse-type carcinogenesis but less in intestinal carcinogenic pathway.\nA body of evidences indicated that downregulation of tumour suppressor protein expression was due to genetic or epigenetic changes, like allelic loss, mutation, loss of heterozygosity (LOH), hypermethylation and microsatellite instability in malignancies [29, 30]. In the sporadic parathyroid carcinomas and hyperparathyroidism\u2013jaw tumours, LOH or mutation of HRPT2 might cause the loss and inactivation of parafibromin protein [20, 25, 26]. Furthermore, the reduced expression of parafibromin was found to closely link to the tumour size, depth of invasion, lymphatic or venous invasion and UICC staging in line with the observation in breast carcinomas [19], indicating the inhibitory effects of parafibromin on tumour growth, invasion, metastasis and progression of gastric carcinomas. Drosophila Hyrax and its human orthologue, parafibromin, are required for nuclear transduction of the Wnt\/Wg signal and bind directly to the C-terminal region of beta-catenin\u2013Armadillo, thereby controlling transcriptional initiation and elongation by RNA polymerase II [15]. Parafibromin overexpression can inhibit colony formation, anchorage-dependent cell growth and cellular proliferation and induce cell cycle arrest in the G1 phase [28]. These findings demonstrated that loss of parafibromin expression had impact on the pathogenesis and progression of malignancies by promoting cellular proliferation. Additionally, parafibromin was expressed with a higher incidence in intestinal-type gastric cancer, which is presumed to arise from preceding dysplastic lesions, than diffuse-type one, which evolves without any precedent dysplastic changes. It is also demonstrated that distinct parafibromin expression underlies the molecular mechanisms for the differentiation of intestinal- and diffuse-type carcinomas.\nUntil now, there is yet no paper describing the prognostic significance of parafibromin expression in malignancies. Here, for the first time, we analysed the relation of parafibromin expression with the survival rate of 508 patients with gastric carcinoma. The results revealed a close link between its loss and worse survival. If stratified according to the depth of invasion, the significant link disappeared, indicating that the relationship between parafibromin expression and prognosis depends on the depth of invasion. The multivariate analysis demonstrated that age, depth of invasion, lymphatic invasion, lymph node metastasis, UICC staging and Lauren\u2019s classification but not parafibromin expression, venous invasion or sex were independent prognostic factors for carcinomas. These findings suggested that parafibromin expression is a promising indicator for the favorable prognosis of gastric carcinoma patients, albeit not independent.\nIn the present study, a large number of gastric carcinoma cases were screened by TMA, which takes the advantages of high throughput, identical immunohistochemical conditions, and economy of samples, antibodies and time [33]. Although we used 2-mm-in-diametre needles, which are large enough to evaluate the morphological appearance and carefully selected representative regions with the reference of HE slides, it was difficult to avoid selection bias. Gill et al. [5] found that stronger parafibromin staining and more positive cells sometimes appeared at the edges of the parathyroid tumour than in the centre. This could be due to fixation methods, other processing issues or a biological phenomenon, for example tissue hypoxia in the centre of large tumours. Additionally, the collection of our samples (e.g. gastritis, adenoma and adenocarcinoma) respectively from the endoscopic biopsy, polypectomy, or surgical resection put forward their another possibility of selection bias because of different fixation and processing methods. Selvarajan et al. [19] found that parafibromin underexpression was correlated particularly with large tumour size which is in line with our finding. It was possible that weaker staining could be attributable to poor fixation properties in the centre of large tumours. Because tumour size is a key prognostic indicator, this artifact could explain the prognostic significance of parafibromin in gastric carcinomas. Therefore, it is a limitation of the present study not to separate the edge and centre of gastric carcinomas when establishing TMA. In the current study, the negatively staining carcinomas are associated with a negative internal control (stromal cells and lymphocytes) whereas the positive staining epithelium is adjacent to positive staining internal controls. Therefore, the negative staining of the carcinoma might be artificial, which should be considered as another limitation of the study. Our study might be mentioned as a preliminary experiment and the staining with original-size sections is an extensive work in the future using the gastric carcinoma samples, fixed and processed by the same approach.\nIn summary, downregulated parafibromin expression might play an important role in malignant transformation of gastric epithelial cells. Its reduced expression was closely related to growth, invasion, metastasis and worse prognosis of gastric carcinomas. Its expression could be employed to differentiate the intestinal- and diffuse-type carcinomas and underlay the molecular mechanism about the differentiation of both carcinomas. It was considered as a promising marker to indicate the pathobiological behaviours and prognosis of gastric carcinomas.","keyphrases":["parafibromin","pathogenesis","prognosis","gastric carcinoma","progression"],"prmu":["P","P","P","P","P"]}
{"id":"Health_Care_Anal-4-1-2226192","title":"The UNESCO Universal Declaration on Bioethics and Human Rights: Perspectives from Kenya and South Africa\n","text":"In October 2005, UNESCO (the United Nations Educational, Scientific and Cultural Organization) adopted the Universal Declaration on Bioethics and Human Rights. This was the culmination of nearly 2 years of deliberations and negotiations. As a non-binding instrument, the declaration must be incorporated by UNESCO\u2019s member states into their national laws, regulations or policies in order to take effect. Based on documentary evidence and data from interviews, this paper compares the declaration\u2019s universal principles with national bioethics guidelines and practice in Kenya and South Africa. It concentrates on areas of particular relevance to developing countries, such as protection of vulnerable persons and social responsibility. The comparison demonstrates the need for universal principles to be contextualised before they can be applied in a meaningful sense at national level. The paper also assesses the \u2018added value\u2019 of the declaration in terms of biomedical research ethics, given that there are already well-established international instruments on bioethics, namely the World Medical Association Declaration of Helsinki and the CIOMS (Council for International Organizations of Medical Sciences) guidelines on biomedical research. It may be that the added value lies as much in the follow-up capacity building activities being initiated by UNESCO as in the document itself.\nIntroduction\nIn October 2005,1 the United Nations Educational, Scientific and Cultural Organization (UNESCO) adopted the Universal Declaration on Bioethics and Human Rights. The preamble states: \u201cIt is necessary and timely for the international community to state universal principles that will provide a foundation for humanity\u2019s response to the ever-increasing dilemmas and controversies that science and technology present for humankind and the environment\u201d [20, p. 3]. As the declaration is non-binding, this universal foundation will be implemented at the level of the nation-state. The onus is on UNESCO\u2019s member states to incorporate the declaration\u2019s provisions into their national laws, regulations or policies. This paper examines the synergies between the declaration and bioethics regulation in two countries, Kenya and South Africa.\nBefore the UNESCO declaration was adopted, international instruments such as the World Medical Association (WMA) Declaration of Helsinki and the International Ethical Guidelines for Biomedical Research Involving Human Subjects of the Council for International Organizations of Medical Sciences (CIOMS) were already well-established in bioethics [5]. Indeed, the Helsinki declaration and the CIOMS guidelines are noted in the preamble of the UNESCO declaration [20]. These instruments have had a significant influence on the national bioethics policies of developing countries [1], including Kenya and South Africa.\nThe objectives of this paper are twofold. The first is to assess the translation of universal principles into national practice. The comparison between the UNESCO declaration and Kenyan and South African bioethics forms the basis for this analysis. The second is to locate the \u2018added value\u2019 of the declaration, particularly to developing countries, given the pre-existence of international level bioethics documents. Empirically based, the paper draws on documentary evidence and interviews conducted in Kenya and South Africa in 2005 and 2006.\nBackground\nThe empirical research for this paper was carried out as part of a larger doctoral project on the global governance of human genomic and biomedical research and in particular the part developing countries play in this. The theoretical framework for this project is provided by international relations, a sub-discipline of political science. While international relations theory is not referred to overtly in this paper\u2019s analytical sections, it provides the context for the understanding of universality contained therein. That is, universality is explored pragmatically, with regard to the relationship between broad principles negotiated at international level and their subsequent adaptation to national level policy and practice, rather than philosophically, in terms of universal versus pluralist moral reasoning.\n\u2018Bioethics\u2019\nWhat exactly is meant by \u2018bioethics\u2019 is notoriously difficult to determine. This is illustrated by the fact that the UNESCO declaration contains no clarification of the term. While a definition appeared in earlier drafts, the impossibility of reaching a consensus on wording necessitated its being left out of the final version [18]. For the purposes of this paper, bioethics is understood specifically in terms of the regulation of biomedical research. The UNESCO declaration itself has a wider remit, covering medical practice and protection of biodiversity and the environment as well as research ethics [20]. Since the overall doctoral project is concerned chiefly with human subjects research, however, these broader considerations will not be discussed here.\nMethodology and Limitations\nMuch of contemporary international relations theory concerns the roles of both state and non-state actors in global governance mechanisms. Reflecting this, the doctoral fieldwork consisted of 70 semi-structured interviews with a range of persons considered to be stakeholders in genomics and bioethics in France, the United Kingdom, Kenya and South Africa.2 The sample was chosen to reflect different societal perspectives on genomics and bioethics and thus consisted of those who formulate policy at international and national levels, those who must implement these policies in laboratories and ethics committees, those who claim to represent public concerns and those with commercial interests. The breakdown of sectors was as follows: policy-makers (20), scientists (17), ethicists3 (18), civil society representatives (12) and businesspersons (3).4 Of these 70 interviews, the data used in this paper draw on only 22 and come mostly from those with members of research ethics committees. Thus they are illustrative rather than representative.\nStudying a declaration as it evolves5 makes for exciting and contemporary research. It also carries limitations, however; in this instance, fieldwork may have been conducted too early to enable an assessment of the social and political impact of the UNESCO declaration in Kenya and South Africa. In the wider context of the doctoral research project, this limitation will be addressed by the inclusion of UNESCO\u2019s previous declarations on genomics and genetics, which have had longer to become established within national policy frameworks.6 It is worth noting that the research process became by default an awareness-raising exercise, in that many of the people interviewed were previously unaware of the bioethics declaration, or indeed its predecessors.\nPrevious Studies\nThe UNESCO declaration features in several publications. Developing World Bioethics devoted a whole issue to the draft text in September 2005. The articles were largely critical, questioning the content of the draft declaration, how it had been put together and whether UNESCO was the right body to be taking on such an endeavour [8]. Professor Henk ten Have, Director of the Division of Ethics of Science and Technology at UNESCO, responded that the journal\u2019s contributors were perhaps not au fait with how UN agencies work [14]. After the declaration was adopted, Herman Nys wrote an editorial for the European Journal of Health Law outlining its basic tenets and comparing it to the European Convention on Human Rights and Biomedicine. He emphasised the obligations of states to take on the declaration, despite its being legally non-binding [10]. Professor ten Have has himself written about the declaration, in the wider context of UNESCO\u2019s activities in ethics. In his article in the Kennedy Institute of Ethics Journal he described how these activities fall into three areas, namely the adoption of normative instruments such as the declaration and their subsequent implementation through national level capacity building and awareness raising [6].\nThere has been a growing literature in recent years on research ethics in developing countries. A few examples, taken from the British Medical Journal, the Bulletin of the World Health Organization and PloS Medicine, will serve to illustrate that contextualisation of international instruments at national level and capacity for ethical review have been among the major concerns raised. Sylvester Chima has suggested that international guidelines need to be interpreted legislatively at local and regional levels and has thus called on the African Union to pass binding directives that are nevertheless adaptable to the laws of each state [3]. Kass et\u00a0al., in a case study published in January 2007 reviewing the practices of research ethics committees in several African countries, including Kenya and South Africa, highlighted insufficient funding and training as the biggest challenges facing these committees and proposed that workshops be set up on how to apply ethical principles at local levels. They also called for more empirical investigation of ethics in African research [7]. Peter Singer and Solomon Benatar, in a 2001 article on the Helsinki declaration, contended that building capacity in research ethics will have far more impact on ethical standards than \u201crevisions of this or any other research ethics code,\u201d implying that declarations themselves are of limited use unless the capacity exists to implement them [15, p. 747). Zulfiqar Bhutta has also argued that strengthening local capacity in bioethics is key to promoting ethical health research in developing countries [1].\nThe UNESCO Declaration\nThe Universal Declaration on Bioethics and Human Rights was adopted \u201cby acclamation\u201d at the UNESCO General Conference on 19 October 2005 [20, p. 5). This was the end of a process that began with an invitation by the 2001 General Conference to the UNESCO International Bioethics Committee to report on the possibility of elaborating a universal instrument on bioethics. The Committee recommended that this instrument be declaratory in nature (that is, non-binding) and the drafting process was launched in January 2004 [17, 19]. Thus the actual negotiation period lasted under 2\u00a0years. The declaration is aimed primarily at states, but can also be implemented by \u201cindividuals, groups, communities, institutions and corporations, public and private\u201d where appropriate [20, p. 6]. It covers a wide range of bioethical principles, several of which had already become customary in bioethics and feature in documents such as the Helsinki declaration and the CIOMS guidelines (informed consent, for example). Some of the principles of particular relevance to developing countries will be elaborated further below.\nBioethics in Kenya and South Africa\nKenya and South Africa were chosen as fieldwork destinations because of their significant involvement in genomics and bioethics at local, national, regional and international levels. Both countries have recently adopted national guidelines on bioethics: in Kenya the 20047 National Council for Science and Technology (NCST) Guidelines for Ethical Conduct of Biomedical Research Involving Human Subjects in Kenya (\u2018human subjects guidelines\u2019) and the 2005 Ministry of Health (MoH) Kenya National Guidelines for Research and Development of HIV\/AIDS Vaccines (\u2018vaccines guidelines\u2019) and in South Africa the Department of Health (DoH) Ethics in Health Research: Principles, Structures and Processes, which were drawn up by members of both the Department and the Interim National Health Research Ethics Committee, appointed under the National Health Act of 2003 [13].\nBoth Kenya and South Africa decided that national bioethics guidelines were necessary partly in order to protect poor and marginalised people from being exploited by unscrupulous researchers [9, 13]. Among other texts, the guidelines draw on the Helsinki declaration, the CIOMS guidelines8 and several documents from the World Health Organization and the United States, but are tailored to their national contexts, with specific provisions addressing the vulnerabilities that may have enabled past abuses [9, 11, 13]. Participants from ethics committees cited a similar assortment of guidelines and regulations\u2014international, regional and national\u2014as influential, including some from Europe, the United Kingdom and Australia.\nSynergies between the UNESCO Declaration and Bioethics in Kenya and South Africa\nAs the UNESCO declaration is non-binding, if its principles are to be applied universally they will necessarily have to be reflected in national level documents and systems. This section compares the main tenets of the declaration that are of special relevance for developing countries with Kenyan and South African bioethics policy and practice. The comparison has two purposes. The first is to illustrate how internationally determined, universal principles might be implemented at national levels in developing countries. The second is to show to what extent these principles were already reflected in national systems, before the adoption of the declaration.\nCommunity Consent\u2014Article 6\nThe UNESCO declaration states that for a research project on a group or community, agreement from representatives may be sought, in addition to that of the individual participants [20]. Community Advisory Boards facilitate this in some areas of Kenya (interviews, K_06:2005 and K_25:2005) and dialogue with community members through such boards is a requirement for HIV\/AIDS vaccines research [11]. The South African guidelines stipulate community involvement and consultation for research involving \u2018collectivities\u2019, on issues such as ownership of data and distribution of benefits and harms [13].9\nVulnerability\u2014Article 8\nThis article holds that \u201cindividuals and groups of special vulnerability should be protected and the personal integrity of such individuals respected\u201d [20, p. 8]. The Kenyan human subjects guidelines give special instructions concerning research with underdeveloped communities, prisoners, married women in rural areas and pregnant or lactating women [9], while the vaccines guidelines state that the vulnerable and poor must be protected from exploitation [11]. The South African guidelines invite ethics committees to be \u201cespecially vigilant when considering research proposals involving vulnerable populations\u201d [13, Preamble] and contain detailed provisions for research involving pregnant women, foetuses, prisoners and vulnerable communities [13].\nCultural Diversity and Pluralism\u2014Article 9\nUnder the declaration these should be given \u201cdue regard\u201d [20, p. 8]. Again, the national guidelines contain specific examples of what this might entail. The Kenyan human subjects guidelines, in the context of gaining informed consent from married women in rural communities, remind researchers that each of Kenya\u2019s 42 tribes will have \u201cunique sociocultural backgrounds\u201d [9, p. 11], while the South African guidelines, in a section on indigenous medical systems, call on researchers to respect the cultures and traditional values of all communities [13]. The Kenyan vaccines guidelines are less detailed and, in a similar vein to the UNESCO declaration, simply require that research teams be sensitive to \u201csociocultural issues,\u201d without specifying what these issues might be [11, p. 30].\nSocial Responsibility\u2014Article 14\nThis article of the UNESCO declaration is considered particularly innovative [6]. It is perhaps not surprising, then, that it should prove more specific than any of the national guidelines as to how social responsibility could be implemented. Where the Kenyan and South African guidelines require research to be relevant to study populations and each country in general, by addressing either \u201chealth needs\u201d (Kenya) [9, pp. 13, 16] or \u201cbroad health and development needs\u201d (South Africa) [13, p. 3], the declaration states that scientific and technological progress should advance: access to healthcare and medicines; adequate nutrition and water; improved living conditions and environment; elimination of marginalisation and exclusion; and reductions in poverty and illiteracy [20].\nBenefit Sharing\u2014Article 15\nThe declaration is similarly specific with regard to the sharing of benefits, giving seven examples of what form this could take, including sustainable assistance to research participants and provision of new health products stemming from research [20]. It is the latter of these examples which features most prominently in the national guidelines. In Kenya, if research produces positive results, treatments should be available to participants [9]. Also, before HIV\/AIDS vaccine research can take place, the \u201cavailability, affordability and accessibility\u201d of its potential products must be considered [11, p. 30]. Research proposals in South Africa must indicate whether there is a reasonable likelihood that participants will benefit from the research and whether they will receive long-term therapy after the study [13]. More widely, research findings must \u201cbe translatable into mechanisms for improving the health status of South Africans\u201d [13, p. 3]. In practice, ethics committees in both countries try to assess the extent to which participants will benefit from a research project (interviews, K_25:2005 and SA_19:2006), although they do not always find this a straightforward endeavour (interviews, SA_10:2006 and SA_35:2006).\nTransnational Practices\u2014Article 21\nThis article calls on states to combat illicit trafficking of \u201corgans, tissues, samples, genetic resources and genetic-related materials\u201d [20, p. 10]. Transfer of tissues overseas has been a particular concern in both Kenya and South Africa, as reflected in their guidelines. The Kenyan vaccines guidelines aim to eliminate the unauthorised transfer of research materials and to this end contain a sample Biological Material Transfer Agreement [11]. Under chapter 810 of the South African National Health Act, an appendix to the DoH guidelines, the Health Minister may regulate \u201cthe importation and exportation of tissue, human cells, blood, blood products or gametes\u201d [12, pp. 70, 62]. Ethics committees in both countries take tissue transfer equally seriously, examining closely any research protocol that involves movement of samples across borders. Generally they prefer a project to train people to analyse data in-country (interviews, K_17:2005, K_21:2005, K_25:2005, SA_19:2006, SA_21:2006 and SA_30:2006).\nBioethics Education, Training and Information\u2014Article 23\nUnder this article states are invited to foster bioethics training and education \u201cat all levels\u201d and to encourage information dissemination on bioethics [20, p. 11]. Such training and education is perhaps more widespread in South Africa than Kenya, although the UNESCO National Commission was in the process of setting up a bioethics centre to serve the East Africa region at the time of interview (interviews, K_01:2005 and K_16:2005). Several South African universities run courses in ethics or bioethics and two training initiatives, IRENSA and SARETI, serve sub-Saharan Africa as a whole (including Kenya).11 All three set of guidelines advocate bioethics training [9, 11, 13].\nUniversal Principles in the National Context\nThe South African guidelines read:\nThe challenge to international research ethics is the development of universal rules for research at a time when health care is being delivered within very different health care systems and in a multicultural world in which people live under radically different economic conditions. [13, p. 7]This paper asks whether the UNESCO declaration has met this challenge. Although promulgated with the expressed purpose of stating universal principles, the declaration acknowledges that such cultural and economic differences exist, through its articles on community consent, cultural diversity, vulnerability, social responsibility and benefit sharing. The Kenyan and South African guidelines demonstrate how the declaration might be applied at state level, with their prescriptions concerning, for example, how researchers should engage with communities and which particular members of society should receive special attention as vulnerable persons. That states may need to adopt particular interpretations of the declaration\u2019s principles in order to realise them in national and local contexts is highlighted by ten Have in his paper on UNESCO\u2019s ethical activities. He describes the adoption of the declaration as only the \u201cstart of a long process of detailed elaboration and consequent application\u201d [6, p. 342] and in the context of the articles on consent and social responsibility states:As principles they are universally adopted, but in practice their application must be tailored in multiple ways to accommodate different types of research and health care, categories of patients and problems, and cultural settings and traditions. [6, pp. 342\u2013343]The need for any national implementation of the UNESCO declaration to be adequately contextualised is brought out in the interview data from Kenya and South Africa, as are differences in opinion as to the usefulness of international guidelines in general. In Kenya, an academic scientist and advisor to both the government and UNESCO stated that it would be necessary to work with those government officers responsible for effecting international documents in law, in order to \u201ctranslate it [the declaration] into what is happening locally\u201d (interview, K_13:2005). Similarly, the head of a research institution attested: Something which became more and more legalistic in the detail and binding but which ignored local realities would be unhelpful. Something which tried to establish clear, agreed principles, which had been widely consulted, not just between member states, but with the kind of communities that are affected, would be very useful. (Interview, K_07:2005)In South Africa, one ethics committee member thought that, given different cultural contexts and sensitivities, declarations such as the UNESCO one would have to be formulated as generally as possible to enable universal applicability (interview, SA_08:2006). Another was of the view that national and local ethics guidelines are \u201cthe things to follow,\u201d because what is applicable in one country may not be applicable in another (interview, SA_19:2006). Several saw at least some value in the UNESCO endeavour, but cautioned that universal principles must not be applied unthinkingly. Perhaps mirroring ten Have\u2019s prediction that elaboration of the declaration will be a long process, they described how working out how to apply such principles in different contexts is often the most challenging aspect of implementing international instruments (interviews, SA_10:2006, SA_17:2006 and SA_24:2006).\nAdded Value of the UNESCO Declaration\nThe UNESCO declaration is considered to be of added value because it is the first intergovernmental instrument on bioethics. Ten Have describes the commitment by governments to an agreed set of principles on bioethics as the \u201cinnovative dimension\u201d of the declaration [6, p. 342]. The Helsinki declaration and the CIOMS guidelines, by contrast, have been adopted by professional organisations (although CIOMS is in official relations with the World Health Organization). Where the former is officially directed at physicians or researchers, however, the latter, like the UNESCO declaration, are to be used in designing national policy on biomedical research ethics, particularly in developing countries [4, 21].\nIt may take time for the significance of the UNESCO declaration\u2019s governmental backing to filter through to those who practise research ethics. An anonymous reviewer of this paper described the declaration as the \u201ccommon denominator of global ethical thought,\u201d but for many stakeholders the Declaration of Helsinki and the CIOMS guidelines fulfil this role, their professional origins notwithstanding. The Helsinki declaration is generally considered the foremost document globally on medical research ethics [2, 5] and, with the CIOMS guidelines, forms the bedrock of research ethics in many developing countries [1]. In Kenya and South Africa, where policy-makers and ethics committees have looked to these two documents for guidance at the global level, several of those interviewed were unconvinced that the UNESCO declaration, which they saw as simply another international instrument on bioethics, was necessary. As the declaration was adopted only relatively recently, such perceptions may change as both its contents and its intergovernmental status become more widely established (particularly as it construes bioethics in broader terms than only medical research ethics).\nIf the UNESCO declaration is not to \u201cremain paperwork,\u201d as a non-binding instrument it must be effected by states [6, p. 343]. Kenya and South Africa already have national bioethics guidelines that complement the declaration to a large degree. They also have research ethics committees at institutional and national levels, or plans for constituting them. For those countries which have not already established bioethics systems, however, the UNESCO declaration could act as a catalyst to galvanise them into doing so. To this end, UNESCO\u2019s ethics programme supports states in building bioethics capacity, through various activities. The first is to construct a database with information on ethics experts, institutions, teaching programmes and policies around the world. The second is to promote bioethics education, through teacher training and curriculum development. The third is to support the setting up of ethics committees and their subsequent operations [6]. These activities were broadly welcomed among those interviewed in both countries, with the caveat that they should not overlap too far with the initiatives of other organisations. UNESCO is in fact piloting an ethics teacher training course in Kenya in July 2007 [16], where there are fewer bioethics courses available than in South Africa.\nOne area where ethics committees in Kenya and South Africa are in need of support is in the monitoring of research projects once they have been approved, as required by all three sets of national guidelines [9, 11, 13]. In practice, as found by Kass et\u00a0al., funding can be an issue. One prominent Kenyan ethics committee had only recently carried out its first on-the-spot inspection at the time when interview data were obtained in November 2005. Prevented from conducting these inspections more often by financial constraints, the committee generally relies on reports from investigators and word of mouth (interviews, K_17:2005 and K_25:2005). The South African DoH guidelines require at a minimum that ethics committees request annual reports from principal investigators and establish a complaints procedure [13]; members of two different ethics committees described such measures as \u201cpassive monitoring\u201d. As in Kenya, committees do not have the resources to carry out site visits (interviews, SA_10:2006 and SA_17:2006). The UNESCO declaration itself does not offer much by way of assistance, the only article that could possibly be taken to relate to post-approval monitoring stating, \u201cAppropriate assessment and adequate management of risk related to medicine, life sciences and associated technologies should be promoted\u201d [20, p. 10].12 Perhaps UNESCO\u2019s ethics programme could help with training in these areas or encourage better funding, however. As highlighted by Singer and Benatar, capacity building is likely to advance research ethics further than are reformulations of bioethical principles. It may be, then, that the added value of the declaration will prove to lie more in UNESCO\u2019s follow-up activities than in the document itself, at least in the medium term, the innovative sections on social responsibility and benefit sharing notwithstanding.\nConclusion\nThis paper has revealed something of a loose consensus, at least between UNESCO and those involved in bioethics in Kenya and South Africa, on two counts. With regard to universal principles, any attempt to implement them at national levels must be contextualised. Working out how to apply such principles in particular social and economic contexts is arguably as challenging as reaching agreement on how they should be constituted in the first place. With regard to the usefulness of the UNESCO declaration, the significance of its adoption as the first intergovernmental instrument on bioethics must be matched by action in the form of capacity building for it to be of added value in the realm of biomedical research ethics.\nThe scope of this paper has been limited to a primarily pragmatic analysis of how universal principles can be applied at national level. The paper has not commented on whether the value of the universality that UNESCO has aimed towards in terms of a foundation for humanity is compromised if these applications are very different. Deeper reflection on the nature of universality in this context would require the input of trained bioethicists and philosophers. This paper highlights, then, the need for a cross-disciplinary approach to the analysis of international bioethics instruments. The scope of the study that engendered this paper was necessarily limited by the time and resource constraints of doctoral research. Further research could explore whether the experiences of other African countries have been similar to those of Kenya and South Africa. Francophone and lusophone states, in particular, may have quite different bioethical traditions and thus have had very different experiences in implementing bioethics policies.","keyphrases":["unesco","kenya","south africa","universal principles","national bioethics","global bioethics"],"prmu":["P","P","P","P","P","R"]}
{"id":"J_Autism_Dev_Disord-4-1-2254472","title":"Brief Report: Eye Movements During Visual Search Tasks Indicate Enhanced Stimulus Discriminability in Subjects with PDD\n","text":"Subjects with PDD excel on certain visuo-spatial tasks, amongst which visual search tasks, and this has been attributed to enhanced perceptual discrimination. However, an alternative explanation is that subjects with PDD show a different, more effective search strategy. The present study aimed to test both hypotheses, by measuring eye movements during visual search tasks in high functioning adult men with PDD and a control group. Subjects with PDD were significantly faster than controls in these tasks, replicating earlier findings in children. Eye movement data showed that subjects with PDD made fewer eye movements than controls. No evidence was found for a different search strategy between the groups. The data indicate an enhanced ability to discriminate between stimulus elements in PDD.\nIntroduction\nWhile most diagnostic criteria of Pervasive Developmental Disorder (PDD) are characterised by impairments, mostly with respect to social and communicational abilities, subjects with PDD excel on certain visuo-spatial tasks that supposedly reflect superior processing of fine detail (see Dakin & Frith, 2005, for a review). An especially robust finding seems to be the behavior of subjects with PDD on visual search tasks (Plaisted, O\u2019Riordan, & Baron-Cohen, 1998; Plaisted, Saksida, Alcantara, & Weisblatt, 2003; O\u2019Riordan, Plaisted, Driver, & Baron-Cohen, 2001). In such tasks, subjects are required to detect a target in a display containing a variable number of distracters. If the task difficulty is increased by adding distracters, usually RTs increase. In studies on PDD, two search task versions that differed in difficulty have been used. In both tasks children with PDD showed shorter RTs, as compared with normally developing controls (Plaisted et\u00a0al., 1998; Plaisted et\u00a0al., 2003; O\u2019Riordan et\u00a0al., 2001). The reason for this superior performance is not clear, but has been related to enhanced ability to discriminate between stimulus elements in subjects with PDD (O\u2019Riordan & Plaisted, 2001), an explanation that has also been proposed for the superior performance of subjects with autism on other visuo-spatial tasks (Plaisted et\u00a0al., 2003, see also Bertone, Mottron, Jelenic, & Faubert, 2001).\nSo far, however, there have been no studies that have validated the claim for enhanced stimulus discrimination as an explanation for superior performance in search tasks in PDD more directly. It is well-known that stimulus discriminability affects eye movement parameters, especially number of fixations and fixation duration (Hooge & Erkelens, 1999). Therefore, studying eye movements during search tasks in subjects with PDD is a first step to gain more insight into the neurocognitive mechanisms of atypical visuo-spatial processing in this group. If increased stimulus discriminability is indeed the underlying factor for superior performance of subjects with PDD, it is expected that they need fewer and\/or shorter fixations to identify the target. Also, eye movement parameters can be used to test alternative hypotheses for shorter reaction times in search tasks in PDD. More specifically, it has been argued that (healthy) subjects in a visual search task have a tendency to move their eyes even though in some situations it would be a better strategy not to do so, since longer fixations allow better extraction of (peripheral) information (Hooge & Erkelens, 1999; see also Rayner, 1998). It is well possible that individuals with PDD use a different search strategy in which they show longer, but less, fixations than controls.\nThe first aim of the present study was to use eye movement parameters to test the two hypotheses described above for superior visual search in subjects with PDD. A second aim was to replicate the findings of Plaisted et\u00a0al. (1998) and O\u2019Riordan and Plaisted (2001) in high functioning adults with PDD. For these reasons we used the same search tasks as described in O\u2019Riordan et\u00a0al. (2001, second experiment) in a group of high functioning adults with PDD, matched on gender, IQ, and age to a control group.\nMethods\nParticipants\nTwo groups of young male adults participated, a group of eight high functioning individuals with PDD and a group of eight normal control individuals. The clinical subjects were recruited via the Department of Child and Adolescent Psychiatry at the Utrecht University Hospital. The study was described to the subjects and written informed consent was obtained. All subjects were administered the Wechsler Intelligence Scale, revised Dutch edition (WAIS). Diagnoses of either Autistic Disorder or Asperger Syndrome were made by a child psychiatrist and based on DSM-IV criteria. The parents of all subjects with PDD were administered the Autism Diagnostic Interview Revised (ADI-R) (Lord, Pickles, McLennan & Rutter, 1997; Lord, Rutter & LeCouteur, 1994). Due to technical problems, eye-movement data of one subject with PDD and one control subject were lost. The individual ADI scores of the remaining seven PDD subjects can be found in Table\u00a01. Unpaired t-tests revealed that there were no significant differences between the remaining seven PDD and seven control subjects with respect to either age, TIQ, VIQ, or PIQ (see Table\u00a02). None of the participants showed any visual or oculomotor pathology other than refraction anomalies. Subjects were allowed to wear their contact lenses or glasses.\nTable\u00a01ADI scores on the four domains for individual PDD subjectsADI scores individual subjectsSocial interaction (cutoff 10)Communication (cutoff 8)Stereotypy (cutoff 3)Age of onset (cutoff 1)12218202181390324163542616855242410362120437281752Table\u00a02Mean age and IQ characteristics of the control and PDD groupControlPDDAge21.222.1TIQ115.3121.9VIQ119.1124.0PIQ106.7114.0\nSet up and Data Analysis\nSubjects sat in front of a LaCie Blue Electron lll 22\u2032 Screen (0.394\u00a0m\u00a0\u00d7\u00a00.295\u00a0m, 1240\u00a0\u00d7\u00a01024\u00a0pixels at 85\u00a0Hz,) at a distance of 0.64\u00a0m. Stimuli were generated by a Apple PowerMac G4\/450 DP using a Matlab program based on routines taken from the Psychophysics Toolbox (Brainard, 1997; Pelli, 1997) and the EyeLink Toolbox (Cornelissen, Peters & Palmer, 2002).\nMovements of the left eye were measured at 250\u00a0Hz with the EyeLink 1 eye monitor. Head movements were prevented by the use of a chinrest. Data were stored on disk and were analysed off-line by a self-written Matlab program. The velocity signal of eye movements was searched for peak velocities above 20\u00b0\/s. Each peak (in the velocity signal) was considered a potential indicator of the presence of a saccade. The exact onset of the saccade was determined by going backward in time to the point where the velocity signal dropped below the average velocity plus two standard deviations during the stable fixation period before the saccade. The exact offset of the saccade was determined by going forward in time to the point where the velocity signal dropped below the average velocity plus two standard deviations during the stable fixation period after the saccade. This method was adopted from Van der Steen and Bruno (1995). This procedure was followed by rejection\/acceptation based on minimum saccade duration of 12\u00a0ms and minimum amplitude of 1\u00b0. When a saccade was removed, fixation time before and after this saccade and the duration of the saccade were added together.\nStimulus and Task\nTwo tasks were tested in separate sessions for each participant, with the order counterbalanced within participant groups. Both tasks contained tilted as well as vertical line elements. The tasks only differed in which of the two elements was target or distracter. Since an earlier study has shown a difference in task difficulty depending on target orientation (O\u2019Riordan et\u00a0al., 2001), the condition including a vertical target among tilted distracters will be referred to as the easy condition, and the condition with a tilted target (among vertical distracters) as the hard condition. Each task contained two crossed factors; set size (4, 16, or 25 items) and probe, target present or target absent. There were 30 trials at each unique combination of factors, yielding a session of 180 trials per task. The sequence of different tasks was randomised within each session. In each session, the participant was informed about the nature of the target (either vertical or tilted), but did not know when a target would be present or absent, or what the display size would be on any trial. The participants performed a binary choice RT task indicating whether a target was present or absent by a button press on each trial.\nProcedure\nThe order in which the two conditions were presented was counterbalanced. Prior to each task participants were given 10 practice trials. Participants were instructed to respond as quickly and accurate as possible by pressing the five key on the numeric pad when the target was present and the four key when the target was absent. Thereafter, the eye link camera was attached to the head and calibrated. On each trial the sequence of events was as follows: a fixation cross was presented. The participant had to fixate at the cross and press the space bar subsequently. Fixation of the fixation cross was used for on-line drift correction of the eye tracker. Then, the search display appeared and remained visible until the participant responded. After the response the next trial started.\nResults\nTo assure that a possible superior task performance of the individuals with PDD was not due to a difference in detection criteria (e.g., if speeded reaction time is accompanied by a decrease in number of correct answers), we performed an independent t-test on the error rates of both groups. No significant difference in error rate between the groups was found. One of the subjects in the PDD group showed a high number of errors and a deviant pattern in reaction time data, and was excluded from further analysis. The results described below are therefore based on six subjects with PDD, and seven controls.\nThe mean RT, first fixation times, remaining fixation times, and number of fixations data were analysed using a repeated measures ANOVA, with one between subjects factor of group (control or autistic) and three within subject factors, condition (condition 1: tilted target or condition 2: vertical target), probe (target present or target absent) and set size (4, 16, or 25 items). Analyses of the first and remaining fixations were done separately, because there is evidence that they belong to different distributions (Hooge & Erkelens, 1996). Unless otherwise stated, a significance level of p\u00a0<\u00a0.05 was adopted for all statistical comparisons. Only main effects or interactions with Group will be considered.\nRT Analysis\nThe analysis of variance revealed a main effect of Group, F(1,11)\u00a0=\u00a06.6, p\u00a0<\u00a00.05, reflecting the fact that individuals with PDD were significantly faster overall than the control individuals (737 vs. 1016\u00a0ms). A significant interaction Group\u00a0\u00d7\u00a0Display was found (F(2, 22)\u00a0= 3.8, p\u00a0<\u00a0.05), as well as an interaction of Group\u00a0\u00d7\u00a0Display\u00a0\u00d7\u00a0Probe (F(2, 22)\u00a0=\u00a04.7, p\u00a0<\u00a0.05). Partial analyses showed that the Groups difference was significant at all set sizes, but that the effects were largest for set size 25 (all p\u00a0<\u00a0.05) (resp., F(1,11) 5.7, 5.4, 6.8). The interaction Group\u00a0\u00d7\u00a0Probe was marginally significant for set size 25 only (F(1,11)\u00a0=\u00a04.0, p\u00a0=\u00a00.72), indicating that at this set size, the Groups difference was largest if the target was absent (See Fig.\u00a01).\nFig.\u00a01Means and standard errors of the reaction times for each group in trials with (left panel) and without a target (right panel), for the easy and hard condition\nEye Movement Analysis\nThe analysis of number of fixations revealed a significant effect of group (F(1,11)\u00a0=\u00a010.3, p\u00a0<\u00a0.01). The individuals with PDD made significantly less fixations than the control group (means, respectively, 1.3 and 2.1). In addition, there were significant interactions between Group\u00a0\u00d7\u00a0Probe, Group\u00a0\u00d7\u00a0Display and Group\u00a0\u00d7\u00a0Probe\u00a0\u00d7\u00a0Display. When tested per level of Display, subjects with PDD showed significantly fewer fixations for all display sizes (F(1,11)\u00a0=\u00a08.2, p\u00a0=\u00a0.015\/9.4, p\u00a0=\u00a0.011\/10.8, p\u00a0=\u00a0.007), but the effects were largest for the 25 set size. Interactions of Group\u00a0\u00d7\u00a0Probe effects were marginally significant for all set sizes (F(1,11)\u00a0=\u00a04.7, p\u00a0=\u00a0.052\/4.0, p\u00a0=\u00a0.072\/4.4, p\u00a0=\u00a0.06), indicating that group differences in number of fixations were largest in the target absent condition (see Fig.\u00a02).\nFig.\u00a02Means and standard errors of the number of fixations for each group in trials with (left panel) and without a target (right panel), for the easy and hard condition\nNo significant group differences were found for fixation times at either the first fixation or the remaining fixations.\nDiscussion\nThe present study was aimed to test two hypotheses on superior performance in visual search tasks in subjects with PDD. It has been suggested earlier that this superior performance reflects an enhanced ability to discriminate between stimulus elements (O\u2019Riordan et\u00a0al., 2001). We tested this hypothesis by measuring eye movements during search tasks in high functioning adult men with PDD and an age- and IQ-matched control group. In case of enhanced stimulus discriminability, shorter fixation times and\/or fewer fixations were expected in the PDD group, as compared to the control group. The alternative explanation, that subjects with PDD use more efficient search strategies, was also tested. A more efficient search strategy would be reflected in a longer fixation time.\nThe reaction time data showed that subjects with PDD were better than the control group for both easy and hard search tasks, especially in the largest set size and in trials in which the target was absent. These findings are in accordance with earlier studies on visual search in children with autism, indicating faster RT in the same type of tasks (O\u2019Riordan et\u00a0al., 2001; Plaisted et\u00a0al., 1998). This indicates that superior search performance in subjects with PDD is a robust finding that can be demonstrated in both children and adults.\nAnalyses of fixations during the search tasks showed that subjects with PDD made fewer fixations than controls. This difference in number of fixations was especially clear for the largest set size and for the condition in which the target was absent, analogous to the RT effects. In addition, it was noted that many subjects with PDD showed an absence of saccadic eye movements in target present trials, indicating that they were able to localize the target in a single glance. No differences between groups were found in fixation time. The absence of group differences in fixation time indicates that subjects with PDD followed the same search strategy as normal controls. However, the decreased number of fixations during the task in the PDD group is in agreement with the hypothesis of enhanced stimulus discriminability in subjects with PDD, as suggested by O\u2019Riordan and Plaisted (2001). The finding that the pattern of effects for fixation times was strongly similar to the RT effect provides additional evidence that superior performance in this task is indeed related to enhanced discriminatory abilities in PDD.\nThe question what the locus of this enhanced discriminability could be has been addressed by Cohen (1994), who tested a neural network model of processing in subjects with PDD. This model showed that an increase in processing units resulted in a strong ability to discriminate and learn stimulus patterns, along with a weak ability for generalization. Indeed, there is anatomical evidence for abnormal development of the cyto-architecture of the cerebral cortex of subjects with PDD, resulting in an increase in processing units. Recent post-mortem studies in subjects with PDD show an increased number of micro columns, albeit of reduced size (Casanova, Buxhoeveden, Switala, & Roy, 2002; Casanova et\u00a0al., 2006).\nConcluding, the measurement of eye movements during search tasks shows that the superior behavior of subjects with PDD in these tasks cannot be attributed to strategy difference, but point indeed to increased stimulus discriminability in this group.","keyphrases":["eye movement","search task","pdd","perception","eye fixation"],"prmu":["P","P","P","U","R"]}
{"id":"Mol_Biochem_Parasitol-1-5-2075530","title":"The role of metacaspase 1 in Plasmodium berghei development and apoptosis\n","text":"The malaria parasite encodes a wide range of proteases necessary to facilitate its many developmental transitions in vertebrate and insect hosts. Amongst these is a predicted cysteine protease structurally related to caspases, named Plasmodium metacaspase 1 (PxMC1). We have generated Plasmodium berghei parasites in which the PbMC1coding sequence is removed and replaced with a green fluorescent reporter gene to investigate the expression of PbMC1, its contribution to parasite development, and its involvement in previously reported apoptosis-like cell death of P. berghei ookinetes. Our results show that the pbmc1 gene is expressed in female gametocytes and all downstream mosquito stages including sporozoites, but not in asexual blood stages. We failed to detect an apparent loss-of-function phenotype, suggesting that PbMC1 constitutes a functionally redundant gene. We discuss these findings in the context of two other putative Plasmodium metacaspases that we describe here.\n1\nIntroduction\nMalaria remains the most important parasitic human disease responsible for millions of deaths each year. The life cycle of malaria parasites is complex involving multiple life stages and requiring two hosts: mosquito and human. The uptake of gametocytes by the mosquito leads to rapid gametogenesis and fertilisation. Ookinetes are formed in the mosquito midgut lumen, which cross the midgut wall and transform into oocysts. Each oocyst can produce thousands of sporozoites, which after release traverse the mosquito hemolymph and infect the salivary gland tissues before they can enter the human host via the mosquito bite. Following inoculation, sporozoites infect hepatocytes and develop into liver schizonts. Liver schizont produce merozoites, which upon release into the blood stream initiate the erythrocytic cycle, the prime cause of clinical symptoms in malaria patients.\nExisting malaria control strategies are hampered by increasing parasite resistance in the field to established antimalarial drugs, as well as by widespread insecticide resistance in the mosquito vectors. Effective antimalarial vaccines are only at the preliminary stages of development. There is thus a great need to develop novel strategies for malaria control. Proteases are attractive antimalarial targets because of their indispensable roles in parasite development and infectivity. A wide variety of proteases of malaria parasites have been described [1]. Amongst these are cysteine proteases belonging to the Peptidase_C14 family (clan CD) which include caspases. Caspases belong to a distinct class of cysteine proteases with a so called Caspase Hemoglobinase Fold (CHF) [2]. Recently, two new families of predicted CHF proteases closely related to the caspases were identified, named paracaspases and metacaspases [3].\nIn metazoa, caspases are centrally involved with the molecular machinery of programmed cell death (apoptosis), and are responsible for many of the biochemical and morphological changes that accompany it. They cleave a variety of proteins ultimately leading to the disintegration of the cell [4]. Metacaspases have thus far only been identified in organisms that do not possess classic caspase genes: plants, fungi and the parasitic protists Trypanosoma, Leishmania and Plasmodium, and there is some evidence linking metacaspases to apoptosis in these organisms [5\u201310]. In Plasmodium falciparum a metacaspase gene (GenBank accession CAD52669, here named PfMC1) has been described possessing consensus histidine and cysteine residues that typically form the catalytic dyad in this family of proteases [1,3], suggesting that this parasite species may possess a mechanism of programmed cell death. Indeed, apoptosis-like DNA fragmentation has been reported to occur in blood stage parasites of P. falciparum in response to chloroquine treatment [11]. In addition, ookinetes of Plasmodium berghei were reported to undergo an apoptosis-like mechanism of cell death resulting in very substantial parasite losses [12]. In this study we investigate the role of PbMC1 in the reported programmed cell death in Plasmodium by constructing PbMC1-deficient parasites. We discuss our findings in the context of two additional Plasmodium metacaspase-like proteins that we describe here.\n2\nMaterials and methods\n2.1\nParasite maintenance, culture and purification\nP. berghei ANKA (clone 2.34) parasites were maintained in female TO mice by mechanical blood passage and regular transmission in Anopheles stephensi, as described [13]. Ookinete cultures were set-up as described [12,13] both with and without prior removal of white blood cells by CF11 columns. In general, 21\u201324\u00a0h ookinete cultures were centrifuged at 1500\u00a0\u00d7\u00a0g for 5\u00a0min and the cell pellet incubated on ice in 20 volumes of 0.17\u00a0M NH4Cl to lyse the red blood cells. In some experiments ookinetes were further purified on Nycodenz cushens. Finally, parasites were concentrated by centrifugation at 1500\u00a0\u00d7\u00a0g for 5\u00a0min, resuspended in RPMI 1640 or phosphate buffered saline, and immediately analysed.\n2.2\nGeneration of PbMC1-KO parasites\nA 720\u00a0bp fragment was amplified by PCR from P. berghei genomic DNA with primers IF-MC3\u2032-F (TAAACCATTGGTCATACCAAAAAATAATCAAAAAAATAACCAA) and IF-MC3\u2032-R (CGGGCCGCTCTAGCATGACCAGGCTCAATAATTGAACA) and introduced into NdeI-digested pLP-DHFR2 via In-Fusion PCR cloning (BD Biosciences). The resulting plasmid, pLP-DHFR\/MC, contains a loxP-prokaryotic promotor cassette (BD Biosciences), followed by a modified Toxoplasma gondii dihydrofolate reductase (dhfr) gene cassette [14], followed by the 3\u2032UTR of pbmc1. A 680\u00a0bp fragment was amplified by PCR from P. berghei genomic DNA with primers pDNR-\u0394MC-F (ACGAAGTTATCAGTCGACGGTACCCCATCATAAAGCAAAAAGC) and pDNR-\u0394MC-R (ATGAGGGCCCCTAAGCTTATTTAACATAAATTTTGTCCATTT) and introduced into SalI\/HindIII-digested pDNR-EGFP via In-Fusion PCR cloning. The resulting plasmid, pDNR-\u0394MC\/EGFP, contains two loxP sites flanking the 5\u2032UTR plus first 11 codons of pbmc1 fused in-frame to the enhanced green fluorescent protein (egfp) gene, followed by the 3\u2032UTR of P. berghei dhfr. The pbmc1-specific insert contained within pDNR-\u0394MC\/EGFP was introduced into pLP-DHFR\/MC via Cre-loxP site-specific recombination (BD Biosciences) to give the transfection vector pLP-\u0394MC\/EGFP. Prior to performing transfections this plasmid was digested with KpnI and NotI to remove the vector backbone. Parasite transfection, pyrimethamine selection and dilution cloning were performed as previously described [15]. Genomic DNA extraction, and Southern blot were performed as previously described [14].\n2.3\nApoptosis assays\nPhosphatidylserine (PS) translocation was studied with fluorescein isothiocyanate (FITC)-conjugated Annexin V in conjunction with propidium iodide, by using the Annexin-FITC Apoptosis Detection Kit (Sigma\u2013Aldrich) according to manufacturer's instructions. DNA condensation in the nucleus was assessed using acridine orange staining as described [12]. DNA fragmentation was assessed by in situ terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labelling (TUNEL), by using the ApopTag\u00ae Fluorescein In Situ Apoptosis Detection Kit (Chemicon International) according to manufacturer's instructions. Caspase activation was assessed with sulphorhodamine-VAD-FMK inhibitor of caspases, by using the CaspaTag\u2122 Pan-Caspase In Situ Assay Kit (Chemicon International) according to manufacturer's instructions.\n3\nResults\n3.1\nDescription of Plasmodium metacaspases\nUsing BLAST searches of available Plasmodium genome sequences with the amino acid sequence of PfMC1 we identified orthologues in P. berghei and other Plasmodium species including P. chabaudi, P. yoelii, P. knowlesi, P. vivax and P. gallinaceum. Multiple alignment of the predicted PxMC1 proteins revealed conserved amino- and carboxy-terminal domains, separated by a region of variable length and sequence. These amino- and carboxy-terminal domains had, in Pfam and SMART searches, significant homologies with a C2 domain (a Ca2+-dependent membrane targeting module, Pfam identifier PF00168) and with the Peptidase_C14\/Caspase domain (Pfam identifier PF00656), respectively (Fig. 1A). We also identified two paralogues of PxMC1 (named PxMC2 and PxMC3) in all Plasmodium species, each containing a region of significant homology to the Peptidase_C14\/Caspase domain in Pfam and SMART searches. In contrast to the PxMC1 proteins however, sequence conservation in the much larger PxMC2 and PxMC3 proteins appeared to be limited to their C-terminal regions containing the putative caspase domains (Fig. 1A).\nMultiple alignment of the predicted caspase domains of PxMC1, PxMC2 and PxMC3 from P. berghei, P. falciparum and P. vivax, along with a metacaspase from yeast (YCA1) [6] shed further light on their structures (Fig. 1B). Like PfMC1, all PxMC1 orthologues possess histidine and cysteine residues predicted to form the catalytic dyad in this family of proteases. It is interesting to note that PbMC1 has the predicted catalytic cysteine located one residue upstream of the consensus position for this amino acid, with a proline in the consensus position (Fig. 1B). The same is true for PcMC1 and PyMC1 (data not shown). In contrast, PvMC1 has two adjacent cysteines, at the consensus position and the preceding residue (Fig. 1B). The same is found in PkMC1 and PgMC1 (data not shown). Catalytic dyad histidine and cysteine residues are absent in the consensus positions in the PxMC2 and PxMC3 proteins (Fig. 1B). However, PfMC3 and PvMC3 have a cysteine at the preceding amino acid position, similar to the situation in PxMC1 of rodent malaria species.\n3.2\nGeneration and molecular analyses of PbMC1 knockout parasites\nBecause PxMC1 proteins display the greatest sequence similarity with the consensus Peptidase_C14 domain and, of the three Plasmodium metacaspases described here, are therefore most likely to be active enzymes, we decided to investigate the expression of PbMC1 and its contribution to P. berghei parasite development. To this purpose we generated genetically modified parasites in which the PbMC1 coding sequence was removed and replaced with a reporter gene, enhanced green fluorescent protein (EGFP). In the strategy used, the 5\u2032UTR and 3\u2032UTR of the pbmc1 gene allow for double homologous recombination [15,16], introducing the EGFP coding sequence downstream of the first 11 codons of PbMC1 (coding for MSLQMDKIYVK), as well as inserting a modified T. gondii dihydrofolate reductase\/thymidylate synthase (tgdhfr\/ts) gene cassette conferring resistance to the antimalarial drug pyrimethamine into the pbmc1 locus (Fig. 2A).\nAfter transfection of purified P. berghei schizonts [15] we readily obtained pyrimethamine-resistant parasites. Diagnostic PCR amplification across the predicted integration sites showed that correct integration of the DHFR\/TS cassette into the PbMC1 locus had occurred (data not shown). This was confirmed by assessing the integrity of a clonal population of the genetically modified parasite (named PbMC1-KO) by Southern blot analysis of HincII-digested genomic DNA. Accordingly, a probe corresponding to the 5\u2032UTR plus PbMC1 coding region gave rise to expected bands of 4.5 and 2.1\u00a0kb in the parental wild-type (WT) parasites (Fig. 2B). In contrast, in the PbMC1-KO parasites expected bands of 4.5 and 0.9\u00a0kb were observed. The 0.9\u00a0kb fragment corresponds to 360\u00a0bp of the 5\u2032UTR of pbmc1, its first 11 codons, plus 490\u00a0bp of the egfp gene (Fig. 2B). The 4.5 and 0.9\u00a0kb bands are more weakly labelled than the 2.1\u00a0kb band, because a smaller portion of the probe anneals to these fragments (Fig. 2A). A probe corresponding to the tgdhfr\/ts gene gave rise to bands of 2.6 and 0.9\u00a0kb in the PbMC1-KO parasites, but no signal in the WT parasite sample as expected (Fig. 2B). These combined results are fully consistent with the structural removal of the PbMC1 coding sequence, and the integration of the reporter and selectable marker genes into the pbmc1 locus.\n3.3\nPbMC1 expression and loss-of-function phenotype\nPbMC1-KO parasites developed normally in mice and were morphologically indistinguishable from WT parasites in Giemsa-stained blood smears. Using UV microscopy EGFP fluorescence was readily observed in a subpopulation of parasitized erythrocytes. Upon gametocyte activation (by placing the infected blood in ookinete medium) the large majority (>90%) of fluorescent parasites emerged from the host cell (clearly visible by the rounding up of the parasite) by 15\u00a0min, typical of gametogenesis. The number of remaining intracellular fluorescent parasites was considerably lower than the percentage of trophozoites present in the sample as assessed by Giemsa staining. These are therefore likely to constitute immature gametocytes, or gametocytes that had failed to emerge. We were further able to distinguish between male and female gametocytes by observing exflagellation (i.e. the release of male gametes); this involved gametocytes that were not green. From these combined observations we conclude that the pbmc1 gene is expressed only in female gametocytes (Fig. 3A). As far as we could ascertain all females were positive and all males negative. Normal differentiation of gametocytes into ookinetes both in vitro and in vivo was observed. These ookinetes again displayed bright EGFP-based fluorescence under UV light (Fig. 3B). WT and PbMC1-KO parasite-infected A. stephensi mosquitoes developed comparable numbers of oocysts, indicating that the PbIMC1a-KO ookinetes are capable of normal midgut invasion and subsequent ookinete-to-oocyst transition on the hemocoel side of the midgut wall. Examination of oocysts on the midgut wall by UV microscopy displayed EGFP fluorescence in oocysts and the sporozoites contained within (Fig. 3C). Fluorescent sporozoites were observed in the mosquito salivary glands (Fig. 3D), which were infectious to mice upon infected mosquito bite. These combined results show that the pbmc1 gene is expressed in female gametocytes and all downstream mosquito stages including sporozoites, but not in the asexual blood stages. The apparently normal development of the PbMC1-KO parasite both in the vertebrate and insect hosts shows that pbmc1 is not an essential gene and may be functionally redundant.\n3.4\nApoptosis in ookinetes\nIt has been reported that, in P. berghei, a large proportion of ookinetes undergo cell death, displaying typical apoptosis-like features such as PS translocation, nuclear condensation and DNA fragmentation [12]. As caspases are well known to play a central role in apoptosis, we wanted to assess the effect of PbMC1 knockout on this process. Initially, we assessed the level of apoptosis-like cell death in WT ookinetes. Annexin V conjugated to FITC (staining green), which specifically binds to PS, was used to assess the proportion of ookinetes displaying PS translocation to the outer leaflet of the cell membrane. Propidium iodide (staining red) was used simultaneously to assess plasma membrane integrity (cell viability). Across 13 experiments conducted we observed an average 77% viable ookinetes (no staining), while an average 21% appeared dead (red only staining) (Fig. 4). The average proportion of ookinetes displaying PS translocation (green staining) was less than 3%. Only 0.5% of ookinetes examined showed PS translocation and were propidium iodide negative (green only staining) (Fig. 4). In addition to this assay, we used acridine orange staining to look for nuclear condensation, and TUNEL to assess DNA fragmentation in P. berghei ookinetes, as described [12]. We found no evidence for either of these processes occurring.\nGiven the low numbers of positive cells in the above assays, we decided to look for the activation of caspases, a key event in caspase-dependent apoptosis, by using a fluorochrome-labelled inhibitor of caspases (CapaTag). In mammalian cells, these labelled inhibitors are permeant and covalently bind to the active centres of caspases with a 1:1 stoichiometry. In this assay, WT ookinetes displayed an average 3.8\u00a0\u00b1\u00a00.5% of positive cells (two experiments) after 21\u00a0h of culture, which, at 24\u00a0h had gone up to an average 14\u00a0\u00b1\u00a09% (four experiments). Under the same conditions, PbMC1-KO ookinetes showed an average 2.6\u00a0\u00b1\u00a00.3% positive cells (two experiments) at 21\u00a0h, and 13\u00a0\u00b1\u00a06% (five experiments) at 24\u00a0h of culture. We also used a green fluorescent P. berghei parasite with an intact pbmc1 gene (PbGFPCON [17]), that displayed an average 3.8% and 9.0% of positive ookinetes at 21 and 24\u00a0h, respectively. Thus, knockout of PbMC1 does not appear to significantly affect the percentage of ookinetes that bind CaspaTag.\n4\nDiscussion\nIn this paper we describe the generation of P. berghei parasites in which the caspase-like protein PbMC1 is replaced by the fluorescent reporter EGFP, allowing us to study both its expression profile and its loss-of-function phenotype using a single genetically modified parasite line. Our EGFP reporter data show the pbmc1 gene to be transcribed, and probably translated, in female gametocytes and all downstream mosquito stages including sporozoites, but not in asexual blood stages. However, we cannot rule out that the pbmc1 gene may be subject to translational repression. There are many examples of genes that are transcribed in the female gametocyte, but which have a translational block and are not translated until later in development, as was recently published [18]. Interestingly, pbmc1 is not one of the genes identified by these authors, supporting a scenario of direct translation. Knockout of PbMC1 expression does not seem to adversely affect any stage of parasite development, at least in vivo in the mouse and mosquito, or in the culture systems used in this study. This was surprising because pxmc1 is a highly conserved single copy Plasmodium gene, and because PxMC1 is predicted to possess consensus catalytic dyad residues and thus is likely to possess caspase activity. Although the two paralogues, PxMC2 and PxMC3, do not share the typical consensus catalytic dyad residues of the Peptidase_C14\/Caspase family, it is possible that they constitute active zymogens by using alternate, functionally equivalent catalytic residues. Indeed, functional replacement between cysteine, serine or threonine residues has been reported for other cysteine and serine proteases [19\u201321]. If this is the case it is conceivable that one or both of the paralogues could carry out the role of PbMC1, hence leading to functional redundancy and resulting in an apparently normal mutant phenotype as observed. Interestingly, five metacaspase genes have been identified in Trypanosoma brucei, TbMCA1-TbMCA5, and two of these also lack a conserved histidine residue (replaced by a tyrosine, TbMCA1) and\/or cysteine residue (replaced by a serine, TbMCA1 and TbMCA4) (reviewed in [22]). Whether any of these molecules are active enzymes remains to be determined.\nIt has been previously shown in plants and fungi [6,9] that metacaspases can be involved in the regulation or the execution of programmed cell death. Because an apoptosis-like mechanism of cell death was reported to take place in ookinetes of P. berghei\n[12] we hypothesized that PbMC1 could be involved in this phenomenon. Of three typical apoptotic markers: PS translocation, nuclear condensation and DNA fragmentation, only PS translocation gave positive results in our hands. PS translocation does also occur when cells die from accidental cell death (oncosis), in which case membrane integrity is lost concomitantly. Accordingly, we cannot truly determine whether ookinetes that in our assay stained doubly positive for both Annexin V and propidium iodide were in the later stages of apoptosis, or whether they died from an unrelated death mechanism. The fact that a subset of ookinetes stained positive for Annexin V suggests that the low numbers of positive cells observed are not a result of a reduced PS content in the parasite plasma membrane. Overall, the results based on these three apoptotic markers indicate that apoptosis-like death in ookinetes, in our hands, is low.\nWhen we monitored for potential apoptotic ookinetes using CaspaTag, we obtained up to 14% positive cells. Surprisingly, no reduction of CaspaTag labelling was observed in PbMC1-KO parasites. Although PbMC1 is the most likely target of the CaspaTag inhibitor, it is possible that other targets of CaspaTag are present in ookinetes, which could mask any reduction in CaspaTag binding in the PbMC1-KO parasite. Clearly, the other two putative metacaspases are prime candidates. Indeed, P. falciparum transcriptome and proteome analyses indicate that PfMC2 is expressed in gametocytes and sporozoites [23\u201325]. Hence, its expression in other mosquito stages including ookinetes is likely. This scenario could also explain the normal development of the PbMC1 null mutant parasites by assuming a level of functional redundancy between the three Plasmodium metacaspases. Expression and knock out experiments with the other metacaspases are underway to test this hypothesis.\nCaspase activation is one of the earliest events in apoptotic mammalian cells. Hence, early apoptotic cells have activated caspases, but display no changes in plasma membrane integrity [26]. It was therefore notable that no EGFP-based fluorescence was observed in CaspaTag positive ookinetes neither in the PbMC1-KO nor PbGFPCON parasite lines, indicating that these cells had lost plasma membrane integrity before CaspaTag labelling occurred. This is consistent with results from Al-Olayan et al. [12] who reported that their caspase inhibitor-positive ookinetes were propidium iodide permeable. Moreover, we found that pre-treatment with the unlabelled inhibitor failed to reduce subsequent binding of CaspaTag (data not shown). In mammalian cells this has been reported to reduce CaspaTag binding by over 90% [26]. We should thus consider the possibility that these caspase inhibitors may have different specificities in Plasmodium than they do in mammalian cells.\nIt is difficult to explain why in our hands the number of potentially apoptotic ookinetes detected was far below the numbers previously reported [12]. Despite numerous attempts we were unable to find conditions that led to increased numbers of ookinetes displaying apoptotic markers. We cannot rule out that small differences in the experimental set-ups might explain the conflicting findings. Alternately, the level of apoptosis detected in P. berghei ookinetes may depend on multiple factors that are still to be defined, for instance the exact conditions under which the gametocytes are generated in the mouse host. A not dissimilar discrepancy also exists for the reported apoptosis-like DNA fragmentation that occurs in blood stage P. falciparum parasites in response to chloroquine treatment [11], an observation that was not corroborated in a more recent study [27].","keyphrases":["metacaspase","plasmodium berghei","apoptosis"],"prmu":["P","P","P"]}
{"id":"Atherosclerosis-2-1-2292239","title":"Early changes in arterial structure and function following statin initiation: Quantification by magnetic resonance imaging\n","text":"Effective LDL-cholesterol (LDL-C) reduction improves vascular function and can bring about regression of atherosclerosis. Alterations in endothelial function can occur rapidly, but changes in atherosclerosis are generally considered to occur more slowly. Vascular magnetic resonance imaging (MRI) is a powerful technique for accurate non-invasive assessment of central and peripheral arteries at multiple anatomical sites. We report the changes in atherosclerosis burden and arterial function in response to open label statin treatment, in 24 statin-na\u00efve newly diagnosed stable coronary artery disease patients. Patients underwent MRI before, and 3 and 12 months after commencing treatment. Mean LDL-C fell by 37% to 70.8 mg\/dL (P < 0.01). The plaque index (normalised vessel wall area) showed reductions in the aorta (2.3%, P < 0.05) and carotid (3.1%, P < 0.05) arteries at 3 months. Early reductions in atherosclerosis of aorta and carotid observed at 3 months were significantly correlated with later change at 12 months (R2 = 0.50, P < 0.001; R2 = 0.22, P < 0.05, respectively). Improvements in aortic distensibility and brachial endothelial function that were apparent after 3 months treatment were sustained at the 12-month time point.\n1\nIntroduction\nHMG CoA reductase inhibitors or \u2018statins\u2019 reduce cardiovascular events by approximately 25\u201330% in patients with stable atherosclerotic disease [1,2]. Previous studies of ultrasound carotid intima media thickness (CIMT) have suggested relatively slow regression of atherosclerosis after 1\u20132 years of high dose statin treatment [3,4]. More recently, intravascular ultrasound (IVUS) of the coronary arteries has demonstrated coronary plaque regression following intensive LDL-C reduction in over 300 patients treated with 40\u00a0mg rosuvastatin for 2 years [5]. Corti et al. [6] were among the first to use serial magnetic resonance imaging (MRI), to demonstrate reduction in carotid and aortic atherosclerosis in 18 patients in response to 12 months of statin treatment. Subsequently, the same group demonstrated that more effective lipid lowering, to LDL-C\u00a0<\u00a0100\u00a0mg\/dL, was associated with a larger decrease in plaque size at 12 months [7]. Similarly, Saam et al. [8] have recently identified statin treatment as an independent predictor of reduced annual rate of carotid atheroma progression, measured using MRI.\nIn the setting of acute coronary syndromes, early initiation of \u2018intensive\u2019 statin treatment can rapidly reduce cardiovascular risk within 4\u201316 weeks [9,10]. However, given the apparently slow changes in atheroma burden observed in previous studies [6], it has been hypothesised that the early clinical benefits of statins cannot reflect structural changes within arteries, but are due to \u2018pleiotropic\u2019 effects of statins such as improvement in endothelial function and reduction in inflammation and thrombosis [11]. Indeed, numerous studies have demonstrated rapid improvement in endothelial function in response to statin treatment [12\u201314]. However, there is also evidence that rapid changes in plaque size and composition might also be possible. Animal models of atherosclerosis indicate that potent correction of dyslipidaemia can result in prompt regression and favourable remodelling of plaques after only 9 weeks [15]. Furthermore, in humans, a recent study of treatment with intravenous apoAI-Milano induced modest but significant regression of coronary atherosclerosis after only 6 weeks [16].\nWe have previously shown that MRI can assess both central and peripheral vascular function, including measurements of arterial stiffness and endothelial function [17,18]. These parameters are of additional interest because they have been shown prospectively to predict cardiovascular risk [19,20]. By assessing both atherosclerosis and vascular function in the same patients we hoped to examine the extent to which changes in structural parameters might be anticipated by early functional changes. In this study, we used magnetic resonance imaging to evaluate, in vivo, the changes in structure and function in human aorta, carotid and brachial arteries at 0, 3 and 12 months in response to statin therapy. Notably, the mean post-treatment LDL-C achieved in this study reached the contemporary target of 70\u00a0mg\/dL [21].\n2\nMethods\n2.1\nStudy population\nNewly diagnosed patients (n\u00a0=\u00a032) with coronary artery disease were recruited from the Cardiology Department of a single tertiary centre. Diagnosis was based on a history of typical symptoms of angina together with an exercise ECG that showed ischaemic-type ST segment changes or a stenosis of \u226550% in at least one coronary artery at angiography. No patients had taken statins prior to study enrolment. No other cholesterol modifying agents were permitted, but no further restrictions were placed on concomitant cardiovascular medications. Patients with acute coronary syndromes or contra-indication to MRI were excluded. MRI was performed at baseline, 3 and 12 months. At each time point, venous blood samples were obtained after a 12-h fast. Statin treatment was withheld until immediately after the first MRI scan, when a statin was started according to the preference of the managing clinician. Decisions regarding cholesterol treatment and management of cardiac risk factors were taken by the responsible clinicians. Statin dose titration, whilst allowed, was not protocol-driven. The study protocol was approved by the local Research Ethics Committee and all subjects gave informed written consent. Of the 32 subjects initially recruited, 8 did not complete all three study time points: 1 patient died from an out of hospital cardiac arrest, 5 were unwilling to return for follow-up scans due to claustrophobia and 2 were lost to follow-up. The 8 patients who did not complete the study were not significantly different from the 24 who did in terms of either their risk factors (age, cholesterol, diabetes, hypertension and smoking) or MRI measures of atheroma or vascular function at baseline. The data reported below refer to the 24 patients, who completed the study protocol.\n2.2\nVascular MRI protocol\nImaging was performed on a 1.5\u00a0T magnetic resonance scanner (Siemens Sonata, Erlangen, Germany) as previously described [17,18]. In brief, aortic imaging was performed using a combination of a two-element array surface coil placed on the chest and spine coil array. For carotid artery imaging, a two-element array surface coil was used (Machnet BV, Eelde, Netherlands) and for brachial artery imaging a surface coil was attached at the right elbow. Brachial artery blood pressure was monitored using a blood pressure cuff on the left arm. For quantification of aortic wall area, ECG-gated double-inversion recovery (black-blood) fast spin echo images were acquired during breath-hold (Figs. 1b and 2b). Typical parameters were FOV 200\u00a0mm, TR 750\u00a0ms, TE 11\u00a0ms, in plane resolution 0.8\u00a0mm\u00a0\u00d7\u00a00.8\u00a0mm, slice thickness 5\u00a0mm. Using an oblique sagittal image of the aorta as a pilot, 11 serial axial images were acquired with 5\u00a0mm interslice gap to cover the entire descending thoracic aorta. The midpoint of the right pulmonary artery in cross section was used as the anatomical reference for the first slice in baseline and follow-up scans. For the carotid arteries axial ECG-gated T2 weighted, black blood images of the neck were acquired during free breathing (Fig. 2b). Sequence parameters: FOV 150\u00a0mm, TR 2 R\u2013R intervals, TE 81\u00a0ms, resolution 0.5\u00a0mm\u00a0\u00d7\u00a00.5\u00a0mm in plane, slice thickness 3\u00a0mm, no interslice gap. Nine images of the common carotid artery were acquired using the common carotid bifurcation as the anatomical reference position for baseline and follow-up scans. Care was taken to place aortic and carotid image slices perpendicular to the long axis of the vessel on the pilot images in order to limit partial volume effects. For analysis of aorta and carotid plaque images all identifying data were removed apart from a code number so that observers were blinded to both patient identity and study time point. The external vessel boundary and vessel lumen were contoured manually by one of two experienced observers (J.L., C.S.) using CMRtools software (Imperial College, London, UK). Vessel wall area was calculated from the difference between these two contours, and then normalised to external vessel area to yield a plaque index (PI), as previously described [22,23]. Plaque index for each patient was then expressed as the mean of all aortic or carotid slices. In keeping with previous studies of carotid atherosclerosis, a mean value was obtained for left and right carotid arteries combined [3,4]. A subset of images from four randomly selected patients (>100 images) were analysed by both observers to assess inter-observer variability.\nSteady state free precession (SSFP) acquisitions were used to determine aortic distensibility and brachial artery reactivity, as previously described [17,18]. Post processing of aortic and brachial images was performed using semi-automated edge detection methods developed with Matlab software (Mathworks Inc.) [24]. Maximum and minimum aortic cross-sectional areas over the cardiac cycle were measured, from which distensibility was calculated as the relative change in area divided by the pulse pressure. To assess brachial artery flow mediated dilatation (FMD), cross sectional images of the brachial artery were acquired at baseline and following release of a cuff inflated to 50\u00a0mmHg above systolic blood pressure on the forearm for 5\u00a0min. After 10\u00a0min, further brachial artery images were acquired following administration of 400\u00a0\u03bcg of sublingual glyceryl trinitrate to elicit maximal (endothelial-independent) dilatation. Maximum percentage change in cross sectional area was used to determine the response to each stimulus.\n2.3\nSerum and plasma assays\nCholesterol and lipoprotein assays were performed using a Cobas-Mira Analyser (ABX Diagnostics, Shefford, UK). Total cholesterol was assayed using the enzymatic CHOD-PAP method and triglycerides were assayed using the enzymatic GPO-PAP method. HDL-cholesterol was assayed using a homogenous second generation PEGME method (Roche Diagnostics, Burgess Hill, UK). Apolipoprotein AI (apoAI) and apolipoprotein B (apoB) were assayed using immunoturbidimetric methods, using reagents supplied by ABX Diagnostics. C-reactive protein was analysed using ELISA (MP Biomedicals, UK) according to the manufacturer's instructions.\n2.4\nStatistical analyses\nStatistical analysis was performed using SPSS 12.0 (SPSS Inc., Chicago IL). The Kolmogorov\u2013Smirnov test was used to assess whether data were normally distributed. Measurements at each time point were compared using repeated measures ANOVA for normally distributed data and Friedman analysis of variance by ranks for non-normally distributed data. Post-hoc analysis of paired time points was performed using a Bonferroni correction. Categorical data were analysed by the \u03c72 test. Data are presented as mean\u00a0\u00b1\u00a0standard deviation or median and interquartile range as appropriate. Statistical significance was assigned at P\u00a0<\u00a00.05.\n3\nResults\n3.1\nClinical and biochemical measures\nBaseline characteristics of the 24 patients who completed all three study time points are shown in Table 1. The most common statin dose used was simvastatin 40\u00a0mg daily (63% patients); other statin prescriptions are detailed in Table 1. Mean baseline total cholesterol was 187.9\u00a0mg\/dL (LDL-C 112.7\u00a0mg\/dL), however 3 months after commencing statins mean LDL-C was reduced by 37% to 70.8\u00a0mg\/dL (P\u00a0<\u00a00.01) with a corresponding 24% (P\u00a0<\u00a00.01) reduction in apoB. Mean LDL-C at 12 months (79.3\u00a0g\/dL) appeared slightly greater than at 3 months, but this was not statistically significant. Triglycerides and CRP did not change significantly at 3 or 12 months, though HDL cholesterol did show significant increase by 12 months \u2013 lipid and apolipoprotein data are shown in Table 2.\nCompared to baseline, at 3 months there were no significant changes in the proportion of patients taking either angiotensin converting enzyme inhibitors (12\/24 versus 8\/24: \u03c72\u00a0=\u00a00.77; P\u00a0=\u00a00.38) or beta adrenoceptor blockers (22\/24 versus 18\/24: \u03c72\u00a0=\u00a01.35; P\u00a0=\u00a00.25). By 12 months, the number of patients taking ACE inhibitors had increased significantly compared to baseline (17\/24 versus 8\/24: \u03c72\u00a0=\u00a05.3; P\u00a0<\u00a00.05), though this would not have influenced the study findings at the early time point of 3 months. Beta blocker usage was not changed at 12 months (19\/24 versus 18\/24: \u03c72\u00a0=\u00a00.11; P\u00a0=\u00a00.74). There was no significant change in either blood pressure or heart rate over the course of the study (Table 3).\n3.2\nAtherosclerosis regression\nAll 24 patients had aortic plaque images of sufficient quality for analysis. Four patients had carotid plaque images at one or more time points of insufficient quality for analysis; therefore they were excluded from statistical analysis. Images (total\u00a0>\u00a0100) from four randomly selected patients were analysed by both observers yielding inter-observer coefficients of variation for plaque index of 4.8% in the aorta, and 2.9% in the carotid. In both the aorta (Fig. 1a) and the carotid arteries (Fig. 2a), there were statistically significant reductions in plaque index after 3 months of statin treatment. Mean aortic plaque index decreased from 0.303\u00a0\u00b1\u00a00.024 at baseline to 0.296\u00a0\u00b1\u00a00.022 at 3 months (P\u00a0<\u00a00.05 versus baseline) and 0.288\u00a0\u00b1\u00a00.024 at 12 months (P\u00a0<\u00a00.01 versus baseline). Plaque regression in the aorta between 3 and 12 months was also significant (P\u00a0<\u00a00.05). In the carotid artery, plaque index fell from 0.446\u00a0\u00b1\u00a00.053 at baseline to 0.432\u00a0\u00b1\u00a00.046 at 3 months (P\u00a0<\u00a00.05 versus baseline) and 0.416\u00a0\u00b1\u00a00.032 at 12 months (P\u00a0<\u00a00.01 versus baseline). Plaque index for the carotid artery between 3 and 12 months did not show significant reduction (P\u00a0=\u00a00.09). The number of patients with early regression in the aorta at 3 months compared to baseline was 18\/24, similar to that at 12 months where 20\/24 showed regression (\u03c72\u00a0=\u00a00.126, P\u00a0=\u00a00.72) (Fig. 1c). The number of patients with measurable early regression at 3 months in the carotid was 14\/20, which was not significantly different to the 18\/20 observed at 12 months (\u03c72\u00a0=\u00a01.4, P\u00a0=\u00a00.24); see Fig. 2c. Furthermore within patients, the early (3 months) change in plaque index of aorta and carotid arteries showed significant correlation with the final change observed at 12 months (Figs. 1c and 2c).\nMean aortic lumen area was 431\u00a0\u00b1\u00a077\u00a0mm2 at baseline, 434\u00a0\u00b1\u00a077\u00a0mm2 at 3 months (P\u00a0=\u00a01.0 versus baseline) and 442\u00a0\u00b1\u00a085\u00a0mm2 at 12 months (P\u00a0=\u00a00.09 versus baseline). Mean lumen area in the carotid arteries was 44\u00a0\u00b1\u00a09\u00a0mm2 at baseline, 44\u00a0\u00b1\u00a08\u00a0mm2 at 3 months (P\u00a0=\u00a01.0 versus baseline) and 43\u00a0\u00b1\u00a08\u00a0mm2 at 12 months (P\u00a0=\u00a00.55 versus baseline).\n3.3\nPhysiological measures\nAfter 3 months of statin treatment, aortic distensibility increased by >20% at each of the three locations along its length. This effect was sustained but did not increase further at 12 months (Table 3). Flow-mediated dilatation of the brachial artery, a measure of endothelial function, also improved after 3 months by >30% (Table 3). Endothelial independent relaxation induced by GTN was not significantly changed after 3 months, but did show a significant increase by 12 months.\n3.4\nRelationship between variables\nWithin individual patients there was no correlation between MRI quantification of atheroma burden in the aorta and carotid at baseline. Although at a group level, endothelial function and aortic compliance improved and atheroma burden diminished, there was no correlation of these changes within individual patients. Furthermore, there were no significant associations between measures of vascular function or atheroma burden and any of: attained LDL-C; change in LDL-C, HDL-C, apoB, apoA-I, or CRP.\n4\nDiscussion\nIn this study, we have observed that regression of atherosclerosis in response to statin treatment can occur earlier than previously appreciated in both the aorta and carotid arteries. The robustness of this observation is enhanced by the finding that, within individual patients, regression at the early time point of 3 months was closely related to the magnitude and direction of change at 12 months. Patients also showed early and sustained improvement in aortic distensibility and in flow mediated vasodilatation of the brachial artery.\nThe magnitude of atheroma regression observed in our study after 3 months is consistent with previous longer-term studies of regression in response to statin treatment [6,7,25]. A lesser LDL-C reduction than that achieved in our study was not associated with early plaque regression after 6 months statin treatment [6]. Intensive LDL-C reduction has been reported to achieve greater regression, but this study did not include an early time point [7]. Thus, the early regression observed here is a new finding that accords with the analysis of Nissen et al. [5] in which atheroma regression, assessed by intravascular ultrasound in the coronary arteries, was predicted by attained mean LDL-C\u00a0<\u00a070\u00a0mg\/dL. A recent observational study reported overall carotid wall area progression by 2.2% per year [8], although individual cases of regression of up to 7.9% were reported. Importantly, the population in that study differed by the inclusion of older patients with a higher prevalence of hypertension and selected only patients with carotid plaques of \u226550% stenosis on a prior duplex scan.\nMean lumen size of the aorta and carotid appeared to show slight increase by 12 months, this did not reach statistical significance implying that regression was occurring predominantly by reversal of \u2018positive\u2019 vessel remodelling as suggested by Corti et al. [6]. There was significant change in plaque index between 3 and 12 months in the aorta, whilst the carotid artery showed a trend that did not reach significance (P\u00a0=\u00a00.09) over the same time period. This could reflect the smaller size of the carotid artery compared to the carotid, making a small change harder to detect. Alternatively the plaques in the aorta and carotid may differ in composition, with certain elements of the plaque likely to be more susceptible to removal than others. For instance, lowering plasma LDL-C could slow the rate of lipoprotein deposition in the arterial wall, allowing reverse cholesterol transport mechanisms to predominate, culminating in net regression [26]. Different effects on individual plaque components is suggested by MRI studies of advanced carotid atherosclerosis in which patients treated with aggressive lipid lowering therapy showed a reduction in size of lipid rich areas [27,28]. This study was not designed to examine plaque composition, though initiation of study to address this question has recently been reported [29]. New developments such as lipid selective contrast agents or higher field strength (3\u00a0T) imaging with improved resolution might further establish the degree to which plaque regression involves lipid removal [30,31].\nEarly clinical benefits of statins have also been ascribed to anti-inflammatory effects [32]. The degree of change in coronary atheroma volume measured using intravascular ultrasound has been related to the magnitude of change in both LDL and CRP [33]. However, in the present study, the early changes in plaque size occurred in the absence of significant change in CRP. Lack of measurable change in CRP probably reflects both the relatively small sample size, and the low baseline CRP level in this stable CAD population, comparable to the post treatment CRP levels in other studies [34]. We also found no relationship between the LDL-C attained and change in plaque size, which may again reflect the sample size and a clustering of LDL-C levels in the lower range precluding observation of a quantitative effect. However, the absence of changes in biochemical parameters despite plaque regression highlights the complementary role of MRI as an imaging biomarker.\nRapid improvements in endothelial vasomotor function have previously been demonstrated in patients with atherosclerosis within weeks [12,14] or even days [13] of starting statin treatment. Thus our finding of increased flow mediated dilatation at 3 months was not surprising. By contrast, timing of statin effects on central arterial stiffness is less established. Improvements in large artery stiffness have been demonstrated after a year of statin treatment [35,36], although a shorter study of 8 weeks pravastatin treatment in patients with familial hypercholesterolemia did not show any improvement [37]. Hypertensive patients without coronary artery disease treated with high dose atorvastatin (mean LDL of <70\u00a0mg\/dL) did show improvements in large artery stiffness after 3 months [38]. Our finding of rapid improvement in aortic stiffness was of similar magnitude and extends this potentially beneficial effect of statin treatment to a coronary artery disease population. We also observed that GTN mediated (endothelium independent) dilatation of the brachial artery was increased after 12 months statin treatment, as has been previously reported [39,40] and which might reflect downstream statin effects e.g. on smooth muscle cell sensitivity to nitric oxide.\nWe initially hypothesised that changes in arterial function might predict changes in structural parameters, but in this study we found no correlation on an individual patient basis between changes is structure and function. Endothelial function, aortic stiffness and atheroma burden all represent different aspects and stages of disease. Although at a population level all these parameters may change in a favourable direction in response to treatment, individual patients may show variable response in each according to their stage of disease. As a result, it is likely that measures of vascular structure and function will provide complementary insights into vascular disease [18].\nThe capability of MRI to perform non-invasive assessment of changes in the arterial wall using relatively small numbers of patients is highlighted by this and other studies [6,22]. Accurate and reproducible assessment of the vessel wall is key to detection of atherosclerosis progression or regression [41]. The rationale for this approach has been illustrated by previous carotid intima-media thickness or coronary IVUS studies [42\u201344]. An early appreciation of effects on both structure and function, as provided by MRI, could be used to guide selection of novel agents prior to investment in major Phase III trials [45]. MRI is an emerging technique and unlike ultrasound measures of IMT and FMD, has not yet been validated as a means to predict future events in large studies. However, recent data shows correlation of aortic plaque burden by MRI with clinical risk scores [46]. As our findings suggest MRI potentially offers additional value through the assessment of arterial structure in multiple locations and complementary measures of vascular function. Therefore, it seems increasingly likely MRI will play a key role in evaluation of new and existing therapies and may even become part of individual patient risk assessment.\n4.1\nStudy limitations\nA potential limitation of this study is the absence of a control group. However, given strong evidence of the benefits of early and intensive treatment with statins, a placebo controlled arm or even low intensity statin-treatment arm was not considered ethical. In common with several recent studies [5,6], we therefore conducted a longitudinal study of changes compared to baseline. As a result, it is not possible to exclude the possibility that the changes observed were due to an unknown factor, though in context, this is improbable. These patients were newly diagnosed with coronary artery disease, some of whom were taking vasoactive drugs such as ACE inhibitors and beta adrenoceptor blockers for hypertension prior to study enrolment. However, the increase in use of such medications between study baseline and the 3-month time point was small and not statistically significant and so the vascular changes observed over that period are not likely to be confounded by changes in these other medications. The 2\u20133% reductions in plaque index observed in this study at 3 months appear relatively modest when compared to decreases in lesion size of greater than 30% observed over a similar time frame in some animal studies. However, the interventions in animal models of atherosclerosis usually involve much more extreme changes in lipid levels than those achievable by statin treatment in humans. Thus, whilst statin treatment to achieve effective LDL-C reduction appears important for plaque regression, additional HDL-C based interventions in order to enhance reverse cholesterol transport may prove even more effective [26]. The patient population studied was overwhelmingly male and Caucasian therefore the findings may not be applicable across all population groups.\n5\nConclusions\nThis study shows that in a population of statin na\u00efve, clinically stable but otherwise unselected coronary artery disease patients, cholesterol reduction using statins to mean LDL-C of approximately 70\u00a0mg\/dL was associated with rapid regression of atheroma at 3 months. Early changes were highly correlated with changes after 12 months. These rapid structural changes were accompanied by early improvements in arterial stiffness and endothelial function that were sustained to 12 months. Use of multi-modal vascular MRI to detect early changes in atheroma and vascular function in small numbers of patients could prove to be an efficient strategy to screen novel anti-atherosclerotic agents.","keyphrases":["statin","magnetic resonance imaging","atherosclerosis","aorta","carotid"],"prmu":["P","P","P","P","P"]}
{"id":"Pediatr_Nephrol-3-1-1989764","title":"The nervous system and chronic kidney disease in children\n","text":"This paper provides a review of the literature on the nervous system involvement incurred by children and adolescents with chronic kidney disease (CKD), with a particular focus on neuropsychological functioning. In addition to an historical overview of earlier literature, published studies from the past 14 years that address both central and peripheral nervous system function in children with CKD are reviewed (1990\u20132003). These studies span work in neuroimaging, electrophysiology, and neuropsychology. A key focus for this review is on variables that might affect neurodevelopmental status in these children. The paper concludes with suggestions for achieving progress in the understanding of this complication of kidney disease in children.\nIntroduction\nAssociations between end-stage renal disease (ESRD) and neurocognitive dysfunction have been reported for over 25\u00a0years. Between 1977 and 1986, approximately 15 publications described dialysis in infants. In these early studies, approximately 65% of infants were reported to have developmental delay and 49% encephalopathy [1]. Between 1976 and 1984, the neurocognitive effects of aluminum-induced neurotoxicity secondary to then current treatments for chronic kidney disease (CKD) were reported [2, 3, 4]. These neurocognitive effects included seizures, speech disorders, dementia, and a slow electroencephalogram (EEG) pattern [2, 3]. These findings were important for the field in that they encouraged modifications to the treatment regimen for dialysis patients and highlighted a confounding factor in the relationship between CKD and neurocognitive integrity. By 1990 the use of aluminum was generally eliminated with improved dialysis water purification techniques and avoidance of aluminum-containing medications. In the early 1990s, erythropoietin was introduced into standard practice, resulting in improved anemia management for children and adults with ESRD and a diminution of anemia-related EEG and cognitive deficits reported in the adult ESRD population [5, 6, 7].\nThe purpose of this paper is to review the contemporary published literature that focuses on the neurological impact of CKD in children. Papers selected for this review have been published from 1990 to the present, and were selected to coincide with the improvements in treatment, including the diminution of aluminum exposure and availability of erythropoietin that occurred circa 1990. These studies span work in neuroimaging, electrophysiology, and neuropsychology to address central nervous system (CNS) function. We also present available studies addressing the integrity of the peripheral nervous system.\nCentral nervous system studies\nNeuroimaging\nImaging of the brain with computed tomography and magnetic resonance imaging (MRI) has been used to document the structural impact of ESRD in children. Contemporary estimates of the prevalence of cerebral atrophy are obtained from a report of 13 children with chronic renal failure from infancy. Brain atrophy was reported in 3 (23%) of these patients [8]. Publications with a focus on certain disease groups with a high risk for CNS disease, such as cystinosis, Lowe syndrome, and congenital nephrotic syndrome, estimate the prevalence of cerebral atrophy to range between 15% and 100% [9, 10, 11]. A recent study by Qvist et al. [11] described a cohort of 33 children, largely composed of children with ESRD from congenital nephrotic syndrome, tested an average of 6\u00a0years after renal transplant. MRI documented CNS infarcts or ischemic changes from both silent and clinically significant cerebrovascular events in 19 (58%). Although relevant for children with CKD, the frequency of brain structure abnormalities in these specific disorders may be greater than in a general pediatric CKD population with a variety of renal diseases.\nElectrophysiology findings\nThree reports of EEG findings in a cohort of children with CKD have been published since 1990. In one study focal and paroxysmal EEG abnormalities were observed in 12 (36%) of 33 children after renal transplant, with 5 (15%) receiving anticonvulsant therapy [11]. The second report of 14 patients documented unspecified EEG abnormalities in 6 (42%) [8]. This study also documented a normal auditory brain stem evoked response in 12 of 12 children tested [8]. Hurkx et al. [12] evaluated 22 children with chronic renal insufficiency (CRI) and dialysis dependence using somatosensory evoked potential of the right median nerve. They reported an increased interpeak latency (N13\u2013N20) suggesting a delayed thalamocortical conduction. No differences in CRI versus dialysis subgroups were found. Investigations of adults with ESRD demonstrate that these tools are sensitive to kidney disease-related effects and may prove useful in future pediatric studies [5, 6, 13].\nNeuropsychology findings\nTable\u00a01 provides a brief description of 11 published studies that have been conducted over the past 15\u00a0years, with follow-up discussion on each of the studies within the broad domains of cognition listed below. These studies have employed samples ranging from 9 to 62 infants, children, and adolescents with CKD. The subjects experienced different types of treatment for their CKD (e.g., hemodialysis, peritoneal dialysis, kidney transplant, conservative). The research design varied (e.g., longitudinal, pre-post transplant, control groups) and different kinds of assessment methodology were used (e.g., infant development batteries, IQ batteries, specific measures of cognition, experimental tasks). In this regard, drawing specific conclusions for neuropsychological findings will be tentative, at best, but should provide guidance to clinicians and researchers as to how the field should move forward within this assessment domain.Table\u00a01 Cognitive functioning in children and adolescents with end-stage renal disease (ESRD), 1990\u20132003 (CKD chronic kidney disease, SD standard deviation, CAPD continuous ambulatory peritoneal dialysis)Author\/dateSample featuresComparison groupFindingsDavis et al. 1990 [28]n=37Pre-post transplant designMental development20 Dialysis at pre-transplant evaluation\u00a0Improved from pre- (mean=77.0, range 50\u2013116) to post-transplant (mean=91.4, range 50\u2013117)17 Conservative at pre-transplant evaluationMean age at transplant=17.6\u00a0monthsMotor development\u00a0Improved from pre- (mean=68.7, range 50\u201386) to post-transplant (mean=85.6, range 65\u2013109)Cognition\u00a0No change from pre- (mean=92.0) to post-transplant (mean=90.4)Overall development worse with early onset of ESRDFennell et al. 1990 [22]n=56n=56 healthy childrenCognitionMean age=13.6\u00a0yearsTesting at 6-month intervals\u00a0Decreased verbal ability in CKDMean age of CKD onset=6.05\u00a0yearsVisual motor skills\u00a0Decreased in CKDModality:Memory and learning\u00a0Hemodialysis (7)\u00a0Decreased in CKD and loss of function over time\u00a0Peritoneal dialysis (12)Attention\u00a0Kidney transplant (10)\u00a0No differences between CKD and controls\u00a0Conservative (27)Lawry et al. 1994 [18]n=24Transplant compared with dialysis plus conservativeCognitionModality:\u00a0IQ: no differences between dialysis plus conservative (mean=92.91, SD=16.86) vs. transplant (mean=103.00, SD=11.97)\u00a0Dialysis (9)\u00a0Conservative (2)\u00a0Kidney transplant (13)\u00a0Correlation between age at onset of illness and IQ (r=0.49)Achievement\u00a0Dialysis plus conservative less than transplant in maths (P=0.020), reading (P=0.028), and written language (P=0.028)Elzouki et al. 1994 [8]N=15NoneDevelopmental screeningModality:\u00a03 of 15 with developmental delay\u00a0Dialysis (6)\u00a0Transplant (3)\u00a0Conservative (6)Hulstijn-Dirkmaat et al. 1995 [14]n=31NoneDevelopmental indexMean age=2.5\u00a0yearsTesting every 6\u00a0months\u00a0Conservative (mean=90.3, SD=14.3) greater than ESRD (mean=67.6, SD=17.3)Modality:\u00a0Dialysis (16)\u00a0Conservative (15)Verbal, perceptual performance, and quantitative scales\u00a0No change over timeMendley and Zelko 1999 [19]n=9Pre-post transplant designCognition (baseline only)\u00a0Full-scale IQ mean=91.6Mean age at pre-transplant testing=14.2\u00a0years\u00a0Verbal IQ mean=91.4\u00a0Performance IQ mean=95.1Mean age at post-transplant testing=15.8\u00a0yearsAttention\u00a0Improvement in sustained attention 1-year post-transplant (P=0.039)Pre-transplant modality:Executive functioning\u00a0Peritoneal dialysis (5)\u00a0Improvement in mental processing speed 1\u00a0year post transplant (P=0.008)\u00a0Hemodialysis (3)\u00a0Conservative (1)Mean age of onset of ESRD=11.9\u00a0yearsMemoryMean duration of ESRD prior to transplant=2.5\u00a0years\u00a0Improvement in working memory 1\u00a0year post transplant (P=0.016)Warady et al. 1999 [16]n=28 infantsLongitudinal designDevelopment, generalModality:\u00a06 of 28 children below the average range at 1\u00a0year of age\u00a0CAPD at \u22643\u00a0months of ageCognitionTransplant at mean age=2.1\u00b10.8\u00a0years\u00a0Verbal IQ: 5 of 18 children below the average range at \u22654\u00a0years of ageMean age at follow-up=7.8\u00b12.8\u00a0years (range 2.5\u201312.0\u00a0years)\u00a0Non-verbal IQ: 8 of 18 children below the average range at \u22654\u00a0years of age\u00a01 child within impaired range on both verbal and non-verbal IQLedermann et al. 2000 [15], Madden et al. 2003 [17]n=16 infants with ESRDLongitudinal designDevelopment, generalMean age at start of dialysis=0.38\u00a0years (range 0.02\u20131\u00a0year)\u00a02 of 8 school-aged children had general delaysDuration of dialysis=17.3\u00a0months (range=1\u201359\u00a0months)\u00a02 of 8 children <5\u00a0years of age had general delaysMean age at assessment=5.84 (range 1.58\u201312.00\u00a0years)Cognition\u00a0IQ: 67% in average range, and 20% in low-average range (mean=86.5, range 50\u2013102)\u00a0Lower IQ scores for children with co-morbid diagnoses (mean=67.0) than for those with ESRD alone (mean=94.2)Attention\u00a07 of 14 children with hyperactivity problemsSocial-behavioral\u00a06 of 14 children displaying conduct problemsBrouhard et al. 2000 [21]n=62n=62 siblingsCognitionMean age: 13.8\u00b10.4\u00a0years\u00a0IQ: ESRD less than siblingsModality: 26 dialysis, 36 transplant\u00a0No difference in dialysis vs. transplantAcademic achievement\u00a0ESRD less than controls for all measures of spelling, reading, and mathematics\u00a0No difference dialysis vs. transplantCorrelation between age of diagnosis and academic achievementCorrelation between parental education and academic achievementQvist et al. 2002 [11]n=33 transplant recipientsNoneCognitionMean age at assessment=8\u00a0years (range=7\u201312\u00a0years)\u00a0Verbal IQ: mean=87.5\u00a0Non-verbal IQ: mean=87.5IQ range\u00a0Low: 3\/33\u00a0Low-average: 14\/33\u00a0Average: 14\/33\u00a0Above average: 2\/33Neuropsychological battery\u00a0No overall group deficits with attention, language, memory, or visuospatial abilities when compared with normative populationNeuropsychological deficits\u00a0Attention: 8 of 33 children\u00a0Language: 2 of 33 children\u00a0Memory: 6 of 33 children\u00a0Visuospatial: 8 of 33 childrenMotor function\u00a0Hemiplegia: 3 of 33 children\u00a0Bilateral infarction: 1 of 33\u00a0Cerebral palsy: 1 of 33Auditory function\u00a02 of 33 children with moderate sensorineural hearing loss\nGeneral neurocognitive function\nIn infants and toddlers neurodevelopmental testing is conducted and analyzed in broad categories of overall development, mental development, and motor development. Hulstijn-Dirkmaat et al. [14] compared the general development in 15 toddlers receiving conservative therapy (CRI) with 16 dialysis-dependent children. The children with CRI had a better developmental index compared with the dialysis-dependent children (mean\u00b1standard deviation 90.3\u00b114.3 vs. 67.6\u00b117.3) [14]. Ledermann et al. [15] evaluated the long-term outcome of infants requiring peritoneal dialysis. In this study, 2 of 8 (25%) young children demonstrated developmental delay. Warady et al. [16] evaluated 28 infants at 1\u00a0year of age, all of whom were dialysis dependent. Of these 28 infants, 6 (21%) scored in the low-average or impaired range of general development. Based on these studies, approximately 20%\u201325% of children less than 5\u00a0years of age with CKD might be expected to show general developmental delays [8, 15, 16].\nFor older children, IQ is the typical measure of general cognitive function. In the sample of children studied with CKD, the distribution of IQ scores is shifted downward compared with the normal population, with low-average (IQ 80\u201389) and average (IQ 90\u2013109) range scores predominating [11, 14, 15, 16, 17, 18, 19, 20]. In a study of 62 children with ESRD, dialysis and transplant combined, Brouhard et al. [21] described a significantly lower IQ in the children with kidney disease compared with their sibling controls. In 19 children with a mean age of 6.6\u00b11.3\u00a0years who had ESRD from infancy, Warady et al. [16] reported a relatively intact IQ, with 15 of 19 (79%) in the average range. In this group, 13 of 18 (72%) achieved average verbal IQ scores, while only 10 (56%) scored in the average range in the nonverbal subtest [16]. When comparing transplanted patients with dialysis-dependent patients, variable results have been reported. Lawry et al. [18] compared 13 transplanted children with 11 dialysis-dependent children in a cross-sectional study and found a higher mean IQ in the transplant group (103.0\u00b111.97 vs. 92.9\u00b116.86). Conversely, Brouhard et al. [21] compared 36 transplant and 26 dialysis-dependent children in their cross-sectional study and found no significant difference between these patient groups. In aggregate it appears that general cognitive function is impaired in children with CKD, but the published literature is inadequate to fully characterize this effect with respect to CKD modality and differences between verbal and non-verbal IQ [11, 16, 19, 22].\nAttention and executive function\nThe findings in the pediatric CKD literature are mixed with respect to attention and executive function [11, 19, 22]. Fennell et al. [22] reported no differences in measures of sustained attention between children with all treatment modalities of CKD combined and matched controls. Conversely, Mendley and Zelko [19] documented longitudinal improvements in sustained attention (pre-transplant 2.19\u00b11.29 vs. post-transplant 2.95\u00b11.33, P=0.04) and mental processing speed (pre-transplant 2.28\u00b10.72 vs. post-transplant 1.64\u00b10.31\u00a0s, P=0.008) 1\u00a0year after transplant in 9 children. After renal transplant, Qvist et al. [11] reported no overall group deficits of attention in 33 children compared with the normative population [standard deviation score (SDS)=\u22120.2\u00b10.4], although 24% of the sample showed generalized attention deficits.\nLanguage\nThe prevalence of hearing loss among children with CKD is approximately 18%, and unrecognized or delayed diagnosis of hearing impairment may impede language development [11, 23]. Children with known hearing loss tend to be excluded from studies of cognitive function, and so their challenges are not likely to be fully reflected in the published literature [17]. Fennell et al. [22] documented deficits with verbal abstracting abilities in their matched control design for children with CKD. Qvist et al. [11] documented no overall group deficits of language compared with the normative population (SDS=\u22120.2\u00b10.4), with only 6% of children showing evidence of generalized language deficits. More research needs to be conducted before determining whether language abilities will prove to be an area of concern for children with CKD, particularly with respect to potential delays secondary to hearing impairment.\nVisuospatial abilities\nCompared with a matched control population, early studies by Fennell et al. [22] documented deficits in visual-motor abilities in a cohort of 56 children with CKD. Qvist et al. [11] documented no overall group deficits with visuospatial abilities in 33 transplant recipients when compared with the normative population (SDS=\u22120.5\u00b10.5); however, 24% of the cases did show generalized visuospatial deficits.\nMemory\nIn a heterogeneous sample of children with CKD, Fennell et al. [22] reported lower memory scores for children with CKD compared with controls. In addition, a general loss of memory function over time was observed in 26 children with all treatment modalities of CKD over a 12-month testing period [24]. After kidney transplantation, Qvist et al. [11] reported no overall group deficits with memory compared with the normative population (SDS=\u22120.4\u00b10.5), although 20% of the cases displayed generalized memory deficits. Conversely, Mendley and Zelko [19] documented longitudinal improvements in working memory in 9 children 1\u00a0year after transplant compared with the pre-transplant evaluation.\nAcademic achievement\nAcademic achievement in children with CKD is vulnerable to deficits in each cognitive domain noted above as well as an increased frequency of school absences. In a study by Lawry et al. [18], the combined dialysis and CRI group (n=11) with a mean chronological age of 14.9\u00b13.3\u00a0years achieved at an age equivalent of 13.8\u00b15.8 in mathematics, 15.6\u00b17.7 in reading, and 11.2\u00b17.2 in language. In comparison, the transplant group (n=13) with a mean chronological age of 13.8\u00b13.2\u00a0years, achieved at an age equivalent of 16.2\u00b17.8 in mathematics, 16.4\u00b17.7 in reading, and 16.2\u00b19.1 in language. This paper suggested that transplant recipients may have superior academic achievement compared with dialysis-dependent children [18]. Conversely, Brouhard et al. [21] compared the academic achievement of dialysis and transplant patients and normal sibling controls. They reported no difference in academic achievement between dialysis and transplant groups. However, the combined ESRD group of dialysis and transplant achieved below their sibling controls in spelling, arithmetic, and reading [21]. Observational studies of academic placement further indicate that regular education (with or without remedial tutoring) is used for 79%\u201394% of children with CKD, while 13%\u201315% receive special education services not related to hearing or visual impairments [11, 16]. This compares with national data in which approximately 10%\u201315% of students receive special education services [25]. Given the observed frequency of abnormalities across multiple areas of cognitive function, the frequency of special education services used by children with CKD appears low.\nPeripheral nervous system\nPeripheral neuropathy including diabetic neuropathy and dialysis-related amyloidosis has been reported in adults with ESRD [26, 27]. The frequency of peripheral neuropathy in children with CKD can only be grossly estimated based on the following study. Elzouki et al. [8] assessed nerve conduction velocity in 11 children with CKD and found 1 (9%) had diminished motor nerve conduction. Electromyograms performed in 9 of these patients revealed increased polyphagia of motor unit potentials in 4 (44%) [8]. At present, we are unable to find additional published studies examining peripheral neuropathies in children with CKD.\nKey variables affecting neurodevelopmental outcomes\nCKD is a complex disorder and, similar to other pediatric disorders, there are undoubtedly a variety of variables that contribute to the neurodevelopmental status of this population. Key variables presented here are postulates for the most part as the studies conducted since 1990 have not been adequately powered to assess multiple influential factors.\nThe duration of CKD and the age at onset of CKD may affect cognition. Two separate studies have reported a poorer developmental prognosis for children with earlier-onset ESRD [18, 28]. It has been postulated that this may be the result of insults to the CNS at particularly vulnerable periods in development or the result of continuous insults over the majority of postnatal life among some children with long-standing kidney disease. The congenital disorders presenting with kidney disease early in life may influence the perceived relationship between duration of CKD and cognition, as these disorders may have other associated CNS abnormalities, such as pulmonary hypoplasia and hypoxia associated with obstructive uropathies, or structural anomalies with Joubert syndrome and Smith-Lemli-Opitz syndrome [29]. Hulstijn-Dirkmaat et al. [14] identified the presence of co-morbid conditions as a risk factor for cognitive impairment based on mean IQ scored (CKD alone 86.7 vs. CKD with co-morbidity 61.4, P=0.001). Conversely, Fennell et al. [24] found no correlation between age at onset of ESRD and cognition. The available data suggest that the duration of CKD and the age at onset of CKD negatively impact on cognitive functioning, but it remains unclear how these variables contribute to the type and severity of cognitive dysfunction.\nHaving reached ESRD, the modality of renal replacement therapy may have an impact on neurodevelopment. Several studies have examined small groups of children either before and after renal transplant or as a cross-sectional study comparing renal replacement therapy modality groups [30, 31]. Fennell et al. [24] compared 10 transplant, 7 hemodialysis, and 12 peritoneal dialysis patients. Patients in both the transplant and peritoneal dialysis groups tended to have better attention and memory skills when compared with the performance of hemodialysis patients. Additional investigation is warranted to determine the differential effect of hemodialysis and peritoneal dialysis among children with ESRD with current dialysis adequacy standards [32, 33, 34]. From previous studies, a hypothesis can be formulated that motor skills, memory, attention, and mental processing may improve after renal transplantation [11, 16, 21]. Although existing literature suggests that a successful renal transplant may diminish the adverse developmental effects of ESRD, these same studies document the type and frequency of residual deficits in pediatric renal transplant recipients to be greater than in the normative American population [11, 16, 21].\nComplications of CKD such as anemia, hypertension, and malnutrition likely affect neurodevelopment. Anemia has been shown to slow the cognitive event related potential in adults with CKD (mean hematocrit=23.7%) and impair cognitive function among otherwise healthy children aged 6\u201311\u00a0years with hemoglobin levels less than 11.8\u00a0g\/dl [6, 35]. The optimal hemoglobin for cognitive function in children with CKD remains to be determined. The effects of hypertension on cognition may be related to the degree of blood pressure elevation, brain injury consequent to hypertensive or hypotensive episodes, and side effects of antihypertensive therapy. A recent study by Lande et al. [36] analyzed National Health and Nutrition Examination Survey III (NHANES III) data comparing 217 children with systolic blood pressure greater than or equal to the 90th percentile for age and gender with 4,860 normotensive children. The NHANES III study used subtests of the Wechsler Intelligence Scale for Children and reading and arithmetic subtests of a standardized academic achievement test. In this study, results suggested that hypertension was associated with lower scores in subtests representing memory, attention, and arithmetic. Furthermore, malnutrition in young infants without kidney or other chronic disease has been linked to impaired brain growth and developmental delay [37, 38, 39]. Significant effort has been applied to the nutritional support of children with CKD. However, in national registries of children with CKD, the mean weight z-score is 1.0\u20131.4 standard deviations below the American age-matched mean, and mean height z-scores are approximately 1.6 standard deviations below the American age-matched mean [40]. Anemia, hypertension, and malnutrition are likely key factors contributing to the cognitive deficits of children with CKD.\nChildren with CKD are at risk for additional disorders that may accompany congenital kidney disease. Prematurity occurs with a greater frequency in children with congenital kidney disease and is associated with a greater frequency of CNS injuries and later developmental deficits [11, 41]. Socioeconomic status and parental education have a known impact on academic achievement among all children [42]. Although not unique to children with CKD, the identification of poor socioeconomic status or limited parental education may trigger additional educational evaluation and or services for children with CKD in these families. School absences are increased in children with CKD due to the need for outpatient medical visits, hospitalizations, hemodialysis, and acute illness [20]. Finally, the CKD population is at excess risk for sensory deficits, including congenital and acquired hearing impairment (9%-18%) and visual impairment, although the latter has not been examined thoroughly [11, 43]. All of these factors may contribute to the neurodevelopmental dysfunction of children with kidney disease.\nConclusions\nEmergent findings appear to apply to three broad groups of children and adolescents: mild-to-moderate CKD, dialysis-dependent children, and transplant-dependent children. What is known about children with mild-to-moderate CKD is severely limited, with no focused studies addressing their neurodevelopmental needs. More is known about the pediatric dialysis population, with deficits in the areas of attention, language, visual-spatial abilities, and memory. However, the differentiation of problems that may relate to disease-specific variables, such as age at onset of kidney failure, anemia, and hypertension, remain relatively unknown and modestly examined at best. For the transplant-dependent group, cognitive deficits appear to persist, supporting the conclusion that the transplant does not result in complete neurocognitive \u201crecovery.\u201d\nThe low frequency of peripheral neuropathy in children with CKD observed clinically and reported in a single study may be attributed to the low prevalence of diseases with microvascular complications such as diabetes mellitus, advanced atherosclerosis, and dialysis-related amyloidosis [8]. This pediatric advantage may be lost as patients survive into young adulthood. It is unlikely that purely pediatric follow-up studies will be able to characterize this potential late-term complication.\nGiven the expected long-term patient survival of children with CKD, established guidelines for the provision of renal replacement therapy for children, and the data provided by studies among children with CKD, we believe that an organized, adequately powered approach toward the characterization of the neurodevelopmental impact of CKD is warranted [32, 33, 34, 44]. Ideally, this study or set of studies will include measures of neuroimaging, electrophysiology, and neuropsychology. Future research in this area should characterize potential risk factors, such as age at onset of disease, anemia management, and hypertension to advance our understanding of the magnitude of cognitive dysfunction and aid the identification of modifiable mediators of CNS deficits in children with CKD.","keyphrases":["neurodevelopment","cognition","renal failure"],"prmu":["P","P","P"]}
{"id":"Arch_Microbiol-4-1-2270922","title":"The influence of cultivation methods on Shewanella oneidensis physiology and proteome expression\n","text":"High-throughput analyses that are central to microbial systems biology and ecophysiology research benefit from highly homogeneous and physiologically well-defined cell cultures. While attention has focused on the technical variation associated with high-throughput technologies, biological variation introduced as a function of cell cultivation methods has been largely overlooked. This study evaluated the impact of cultivation methods, controlled batch or continuous culture in bioreactors versus shake flasks, on the reproducibility of global proteome measurements in Shewanellaoneidensis MR-1. Variability in dissolved oxygen concentration and consumption rate, metabolite profiles, and proteome was greater in shake flask than controlled batch or chemostat cultures. Proteins indicative of suboxic and anaerobic growth (e.g., fumarate reductase and decaheme c-type cytochromes) were more abundant in cells from shake flasks compared to bioreactor cultures, a finding consistent with data demonstrating that \u201caerobic\u201d flask cultures were O2 deficient due to poor mass transfer kinetics. The work described herein establishes the necessity of controlled cultivation for ensuring highly reproducible and homogenous microbial cultures. By decreasing cell to cell variability, higher quality samples will allow for the interpretive accuracy necessary for drawing conclusions relevant to microbial systems biology research.\nIntroduction\nThe cultivation of microorganisms has been performed for more than a century beginning with Louis Pasteur (1879), Robert Koch (1881) and R. J. Petri (1882) (Sedgwick 1916; Gest 1987), with ever increasingly sophisticated methods becoming available over time. These methods range from using undefined medium (e.g., boiled meat extracts) in shake flask cultures where growth rate and substrate utilization (Narang et al. 1997) as well as proteomic profiles (Valentine et al. 2005; Wunschel et al. 2005) can vary widely, to defined culturing media combined with state-of-the-science bioreactors (Nethe-Jaenchen and Thauer 1984; Vasconcelos et al. 1994; Elias et al. 2005).\nA considerable amount of physiological (Keltjens et al. 1990; Dolla et al. 2000; Elias et al. 2004) and high-throughput functional genomic data (Beliaev et al. 2002; Thompson et al. 2002; Wan et al. 2004) has been generated from microbial samples produced using shake flask batch cultures. Although results may be statistically defensible, the inherent short-comings of this cultivation technique are that essential parameters directly impacting organism physiology are poorly described and difficult to control. These parameters include nutrient availability, metabolite production, specific growth rate, and poor mixing leading to cultural\/environmental heterogeneity. Poor mass transfer kinetics can lead to unwanted limitations in dissolved gasses for use by the organism (such as oxygen or hydrogen) and the buildup of gaseous metabolic byproducts, such as CH4 and CO2 that can have secondary effects on cellular metabolism. For example, the accumulation of dissolved CO2 and the production of dissolved organic acids can significantly alter pH in poorly buffered systems (Ji et al. 1995; Narang et al. 1997; Mayville et al. 1999; Miller and Bassler 2001). Fluctuations of pH in bacterial cultures induce physiological responses in various organisms (Snoep et al. 1990; Blankenhorn et al. 1999; Stancik et al. 2002; Yohannes et al. 2004). Hence, the conditions of cultivation at the beginning of growth can be far different than those when the cells are harvested for analysis. Furthermore, the time of cell harvest during different \u201cphases of growth\u201d will also impact biochemical reaction rates (Wright and Holland 2003) and the complement of cellular proteins (Lipton et al. 2002; Wunschel et al. 2005). In actuality, the functional characteristics and relative abundance of proteins measured are derived from a heterogeneous cell population including cells in various stages of growth that are potentially expressing a range of metabolic states within relatively short time periods. We argue that the term \u201caerobic shake flask,\u201d therefore, is more an operational description than an accurate description of the physiological growth conditions.\nIn bioreactors, all relevant physiological parameters, including pH, dissolved oxygen (DO2), temperature, agitation, and the incoming gas flow rate and composition can be monitored and controlled. Because the nutrient medium can be of variable composition and added to the bioreactor at discreet rates, cells can be cultured with varying degrees of electron donor\/carbon source or electron acceptor limitation and over a range of growth rates. The former has important ramifications given the effects of these limitations on cell physiology, while being able to address the issue of different growth rates enables studies that more closely mimic the variable and potentially long generation times typical of many habitats including sedimentary and aquatic environments. A complete mass (fermentation) balance can also be obtained by measuring biogenic gases produced during the cultivation by using in-line mass spectrometry (MS) in addition to the byproducts of carbon and energy source metabolism and the amount of produced biomass. The utilization of such advanced technologies are allowing for unprecedented insights into the metabolic complexities of microbiological systems.\nControlled cultivation technologies, such as chemostats and turbidostats, minimize culture heterogeneity through continual and thorough agitation and by monitoring and controlling all culture parameters. This mixing substantially increases the mass transfer rates of both the influent gas stream and those gases resulting from microbial metabolism, thus creating a more homogenous gas profile throughout the culture (Pin et al. 2002) and reducing the gas effect on pH. Any change in pH that occurs during cell growth due to non-gaseous metabolic by-products is compensated for by monitoring probes and the addition of acid or base (typically HCl or NaOH) via computer controlled pumps. While there is a greater upfront capital investment for bioreactors, the investment is minor relative to the expense associated with high-throughput functional genomics techniques.\nSystems biology seeks to integrate high-throughput and comprehensive analytical techniques such as DNA and RNA microarrays, proteomics and protein interaction analyses, and metabolite measurements with computational biology (i.e., modeling) to describe the structure of the system and responses to individual perturbations. Such knowledge can be used, for example, to predict systems level responses to environmental changes\/perturbations. In our opinion, a key element of systems microbiology research is the use of controlled cultivation techniques to generate samples under well-defined conditions where variations in intra- as well as inter-culture variability, are minimized.\nThe work described herein was designed to quantitatively assess the relative suitability and limitations of various cultivation approaches for systems biology research. Cells cultivated in shake flasks, controlled batch bioreactors and chemostats were subjected to targeted as well as comprehensive proteomic analysis via two-dimensional gel electrophoresis (Beliaev et al. 2002; Abboud et al. 2005) and the MS based accurate mass and time (AMT) tag approach (Conrads et al. 2002; Smith et al. 2002), to determine the extent of biological variation. Recent reports have used controlled cultivation to determine the \u201cmetabolic flux\u201d with different carbon, electron donor or nitrogen sources (Daran-Lapujade et al. 2004; Kolkman et al. 2005, 2006; Adams et al. 2006). However, this is the first report documenting the metabolic differences with varied electron-acceptor between cultures. Finally, while others have utilized steady state cultures and cite advantages similar to those mentioned above, this is, to our knowledge, the first report that assesses and quantifies the effect of cultivation methods on microbial physiology and cell proteome.\nMaterials and methods\nMaterials\nAcetonitrile and methanol were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Urea, dithiothreitol (DTT), and CaCl2 were obtained from Sigma-Aldrich (St Louis, MO, USA) while thiourea, trifluoroacetic acid, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate and ammonium bicarbonate were obtained from Aldrich Chemical Company, Inc. (Milwaukee, WI, USA). Sequencing grade, modified trypsin was purchased from Promega (Madison, WI, USA) while ammonium formate was obtained from Fluka (St Louis, MO, USA). Water was purified using a Barnstead Nanopure Infinity water purification system (Dubuque, IA, USA).\nCell cultivation and harvesting\nShake flask cultures\nShewanella oneidensis MR-1 (ATCC 700550) were cultivated in a defined medium as described elsewhere (Elias et al. 2005) except that 18\u00a0mM lactate was used instead of 6\u00a0mM and the PIPES concentration was increased to 30\u00a0mM since there was no pH control. Shake flask cultures (0.15\u00a0l) were grown aerobically at 30\u00b0C with constant shaking at 150\u00a0rpm. Duplicate cultures were inoculated at 2-h intervals over 20\u00a0h. After an additional 4\u00a0h of incubation the optical density (OD; A600), pH and DO2 were recorded and culture samples for organic acid analysis were taken via syringe, filtered (0.22\u00a0\u03bcm) and stored at \u221280\u00b0C until analyzed. The cultures that had grown for 24\u00a0h were in the late log phase of growth and were then harvested as previously described (Elias et al. 2005). First, 100\u00a0ml of culture was centrifuged and the resulting pellet dehydrated for dry weight biomass measurement (MA100 moisture analyzer Sartorius, Inc.; http:\/\/www.sartorius.com\/). Then, the remaining cells were pelleted by centrifugation (27,000\u00d7g; 8\u00a0min; 4\u00b0C). The resulting pellets and approximately 2\u20133\u00a0ml of the supernatant were transferred to cryovials (1.0\u00a0ml) and centrifuged (9,000\u00d7g; 4\u00a0min; 4\u00b0C). After removal of the supernatant, the cryovials containing packed cell pellets were immediately stored at \u221280\u00b0C for later analysis by 2DE and AMT MS.\nControlled batch cultures\nCells were cultured in controlled batches using Bioflow model 110 reactors (New Brunswick Scientific, Edison, NJ, USA). For the direct proteome comparison, lactate was used at 18\u00a0mM as in the shake flasks but the PIPES concentration was lowered to 3\u00a0mM since pH control was present. Cells were grown with oxygen at 20% of dissolved saturation (DO2) using a combination of air and N2 gas while pH was constantly maintained at 7.00\u00a0\u00b1\u00a00.03 by the addition of 2\u00a0N HCl or 2\u00a0N NaOH. The temperature was a constant 30\u00b0C and the reactors were stirred at 400\u00a0rpm to minimize mass transfer limitations of O2 and CO2. Samples were taken for organic acid analysis as described above until the late log phase of growth at A600 \u223c0.6. At this point cells were harvested for the analyses as described above for the shake flasks.\nChemostat cultures\nSteady state cultures were attained using Bioflow model 3000 reactors (New Brunswick Scientific, Edison, NJ, USA) with the same medium and identical parameters and controls as described for the controlled batch cultures. Once the optical density (OD; A600) was approximately 0.6, fresh medium was pumped into the reactor at a dilution rate of 0.1\/h and a constant 3\u00a0l volume maintained. Culturing continued until a constant OD and acid addition rate (i.e., steady state) was attained. Samples for organic acids were collected periodically and the cells harvested as above.\nOrganic acid quantitation\nThe organic acids lactate, acetate, pyruvate, formate and fumarate were measured using isocratic flow (0.5\u00a0ml\/min) with both high performance liquid chromatography (HPLC) and ion chromatography (IC). The HPLC (Agilent 1100 series HPLC with a 300\u00a0\u00d7\u00a078\u00a0mm Rezex organic acid column (Phenomenex, Torrance, CA, USA); 60\u00b0C; 35\u00a0min run time) used a dilute acid mobile phase of 0.005\u00a0N H2SO4. Quantification was accomplished by injecting the sample (25\u00a0\u03bcl) and using either a diode array detector or a fixed wavelength detector at 210\u00a0nm (4\u00a0nm bandwidth). Samples were prepared by filtering 1\u00a0ml (0.2\u00a0\u03bcm) and acidifying with 10\u00a0\u03bcl of 2.5\u00a0N H2SO4. Lactate had a retention time of 18.8\u00a0min and detection limit of \u223c0.1\u00a0mM while pyruvate, acetate, and formate retention times were 13.4, 22.1, and 20.2\u00a0min, respectively with a detection limit each of \u223c0.2\u00a0mM. For IC (Dionex DX-500) analysis was performed for the above organic acids as previously described (Senko et al. 2002; Elias et al. 2004).\n2D-gel proteomic analysis\nFrozen cell pellets were mixed with 2 vols. of a solution containing 9\u00a0M urea, 2% 2-mercaptoethanol, 2% ampholytes (pH 8\u201310, BioRad), and 2% Nonidet P40 (a nonionic detergent). The cell lysate was then ultracentrifuged (435,000\u00d7g; 10\u00a0min) using a Beckman TL100 tabletop ultracentrifuge, and the soluble denatured proteins were recovered in supernatants from centrifugation. Protein concentrations were determined using a modification of the Bradford protein assay (Ramagli and Rodriguez 1985). Aliquots of sample containing 40\u00a0\u03bcg of protein were separated in the first dimension by isoelectric focusing using polyacrylamide gels containing 50% pH 5\u20137 with 50% pH 3\u201310 carrier ampholytes (Anderson and Anderson 1978a). After 14,000 V-h, the first-dimension gels were equilibrated with sodium dodecyl sulfate (SDS) and the proteins were separated by SDS-polyacrylamide gel electrophoresis as described (O\u2019Farrell 1975) using a linear gradient of 10\u201317% acrylamide (Anderson and Anderson 1978b). Proteins were then detected by staining with silver nitrate (Giometti et al. 1991). The 2DE images were digitized using an Eikonix1412 scanner interfaced with a VAX 4000-90 workstation. The images were then transferred to a PC, converted to TIFF format, and then processed for spot detection and pattern matching using the Progenesis software (Nonlinear USA). One 2DE image was used as a reference pattern for the experiment. All patterns in the experiment were matched to the reference pattern so that the protein spots were given identification numbers. Statistical analysis of the relative abundance of each matched protein spot across the data set was done using a two-tailed Student\u2019s t-test as previously described (Giometti and Taylor 1991). Proteins to be identified were cut from two to three replicate gels stained with Coomassie Blue R250 (approximately 200\u00a0\u03bcg of protein was loaded on each gel), and the proteins were digested in-gel with trypsin (Promega sequence-grade trypsin, 12.5\u00a0ng\/\u03bcg). The resulting peptides were eluted from the gel pieces by extracting three times, first with equal parts of 25\u00a0mM ammonium bicarbonate and acetonitrile, then twice with equal parts of 5% (v\/v) formic acid and acetonitrile. The eluted tryptic peptides were desalted and concentrated with a commercial ZipTip C18 pipette tip (Millipore). Peptide samples were then loaded onto a 365\u00a0\u00d7\u00a0100\u00a0\u03bcm fused silica capillary (FSC) column packed with 10\u00a0\u03bcm POROS 10 R2 packing material (PE Biosystem) at a length of 10\u201315\u00a0cm. Peptides were separated with a 30-min linear gradient of 0\u201360% solvent containing 80% acetonitrile and 0.5% acetic acid, and then entered into an LCQ ion trap mass spectrometer (Finnigan MAT). Tandem mass spectra were automatically collected under computer control during the 30-min LC\u2013MS runs. MS\/MS spectra were then directly subjected to SEQUEST database searches (Eng et al. 1994; Sadygov et al. 2002) by correlating experimental MS\/MS spectra to predicted protein sequences in the S. oneidensis MR-1 open reading frame database.\nMass spectrometry proteomic analysis\nCell lysis and tryptic digestion\nWhole cell lysis was achieved by bead beating while tryptic digestion was achieved with sequencing grade-modified trypsin as previously described (Elias et al. 2005) and digested for 5\u00a0h at 37\u00b0C using a 1:50 (w\/w) trypsin-to-protein ratio. Samples were quick frozen in liquid N2 and stored at \u221280\u00b0C until analyzed. Protein concentration was determined by the BCA assay kit (Pierce, Rockford, IL, USA).\nMass spectrometric analysis\nThe capillary LC system used was described previously (Elias et al. 2005) using 5,000 psi reversed-phase packed capillaries at \u223c1.8\u00a0\u03bcl\/min (150\u00a0\u03bcm i.d.\u00a0\u00d7\u00a0360\u00a0\u03bcm o.d.; Polymicro Technologies) (Shen et al. 2001, 2002) with two mobile phase solvents consisting of 0.2% acetic acid and 0.05% TFA in water (A) and 0.1% TFA in 90% acetonitrile\/10% water (B). The LTQ-FT data was processed using the PRISM data analysis system as described previously for LC-FTICR-MS data (Elias et al. 2005). Since the separation systems for both the LTQ-FT and the LCQ analyses were identical, peptide confirmation was based on both the calculated (from the mass tag database) and measured mass (from the FTICR analysis) of the peptide matching to within 6\u00a0ppm and the elution times matching to within 5%.\nAll samples were analyzed as previously described (Lipton et al. 2002; Elias et al. 2005) using an LTQ-FT (Thermo-Finnigan, MI, USA). The mass spectrometer measurements were analyzed with SEQUEST (Eng et al. 1994) using our current database as previously described (Elias et al. 2005). Mass spectra were acquired with approximately 105 resolution.\nThe peptide identifications were determined using the spatially localized confidence scoring (SLiC) algorithm (Norbeck et al. 2005) that incorporates a number of constraints and estimates the confidence of each peptide identification by yielding a score of 0\u20131. Recent work in our laboratory shows that a SLiC score of at least 0.7 is sufficiently rigorous (unpublished) and is used herein. Further, at least one high-confidence \u201cunique\u201d peptide (i.e., mapping to only one possible parent protein) and a total of two peptides was required for protein identification in each analysis.\nResults\nEffect of cultivation method on growth and metabolism\nDuplicate cultures were grown in; (1) shake flasks (uncontrolled batch) (2) controlled batch and (3) continuous flow reactors operating as steady state chemostats all with O2 as the sole terminal electron acceptor. Shake flask cultures exhibited an extended lag phase (approximately 13\u00a0h), which compared with the shorter lag phase ((5\u00a0h) in controlled batch cultures (Fig.\u00a01a,\u00a0b). The extended lag and slow growth rates ((0.15\/h) in shake flasks correlated with elevated DO2 concentration that gradually decreased from nearly 100% of air saturation to approximately 20% of air saturation (Fig.\u00a01a). Below 20% DO2 growth rates increased to approximately 0.275\/h, which is close to the growth rate observed for controlled batch culture operated at DOT 20% of air saturation (Fig.\u00a01a,\u00a0b). By definition, chemostat cultures operating at steady state were maintained at a constant growth rate (0.1\/h) and 20% DO2 throughout the course of the experiment (data not shown).\nFig.\u00a01A comparison of duplicate uncontrolled versus duplicate controlled (right) batch culturing of S. oneidensis MR-1. a The inconsistency in decreasing DO2 (dark filled diamond, open diamond) and growth (dark filled triangle, open triangle) in the uncontrolled cultures is apparent when compared to those for controlled batch cultures (b) using the same symbols. This variation is reflected in the (c, d) organic acid profiles. The lactate (dark filled diamond, open diamond), pyruvate (dark filled square, open square) and acetate (dark filled triangle, open triangle) all exhibited more similar profiles between culture replicates in the controlled batches\nDespite the similarity in growth rates between the duplicate flask cultures (Fig.\u00a01a), the decrease in DO2, i.e., the O2 consumption rates, were dissimilar. These differences correlated with variations in the organic acid concentration profiles in shake flask cultures (Fig.\u00a01c). Although the order of appearance and disappearance of the organic acids was the same between the flask culture replicates, the rate of lactate consumption varied significantly as did the transient concentrations of pyruvate and acetate. The differences in these profiles presumably reflect the metabolic variability between cultures at any given point along the growth curve, and may result from increased variability in the mass transfer kinetics in poorly mixed cultures compared to controlled batch.\nIn comparison, maintaining a constant DO2 concentration in the controlled batch cultures resulted in a shorter lag phase with nearly identical growth rates (Fig.\u00a01b). Cells in well-mixed controlled batch cultures were continuously exposed to a constant 20% DO2 concentration, as opposed to variable DO2 concentrations typical in \u201caerobic\u201d flask cultures that in fact become O2-limited as cell densities increase due to poor mass transfer rates. This resulted in much greater consistency in organic acid concentration profiles between the replicates (Fig.\u00a01d).\nRelating cultivation data to metabolic activity\nNot only does controlled cultivation result in more consistent metabolic activities within and between cultures as shown above, but the very nature of highly instrumented and controlled cultivation provides important characterization data that help to relate cellular metabolism with the environmental conditions experienced by the culture. In a controlled batch experiment designed to illustrate these relationships, cell growth (Fig.\u00a02a) was correlated with the DO2 consumption and the amount of acid needed to maintain a constant pH (Fig.\u00a02b), as well as the organic acid profiles (Fig.\u00a02c). Figure\u00a02a illustrates the increase in optical density measured during the course of the experiment. The solid circles indicate optical density of samples taken directly from the culture, while solid squares represent optical density measurements on the same samples after the addition of 10\u00a0\u03bcM Na-EDTA. This treatment destabilized cell aggregates (flocs) by chelating calcium, which can cross-link polysaccharides in extracellular polymeric substances. The ratio of flocculated:deflocculated cultures is termed the flocculation index (FCI) and is represented by the open triangles. Hence, flocculation indices above 1 indicate the presence of cell aggregates while values at or slightly below 1 indicate the absence of flocculated cells. The cultures lacked flocculated cells during the first 15\u00a0h of growth (Fig.\u00a01a). Acid addition (Fig.\u00a02b, red line) during this period directly tracked increases in optical density. Lactate served as the primary electron donor and carbon source, and decreased while pyruvate and acetate accumulated in the medium (Fig.\u00a02c).\nFig.\u00a02Alignment of the (a) growth, (b) controlled parameters and (c) organic acid profile for S. oneidensis MR-1 cells grown in controlled batch to indicate consequent shifts in metabolism. a The growth of flocculated (dark filled square; after EDTA addition), and non-flocculated cells (dark filled circle) were initially compared to profile the degree of flocculation (dark filled triangle) during growth. When coordinated with changes in (b) pH, DO2 and acid addition and (c) the appearance and exhaustion of the organic acids lactate (dark filled diamond), pyruvate (dark filled square), acetate (dark filled triangle), changes in metabolism can be identified. These include the DO2 spike (solid arrow) signaling lactate exhaustion with the concomitant increase in cell floculation as well as the decreased acid addition rate (broken arrow) showing major carbon source exhaustion and the concomitant disappearance of flocculation\nThe point at which lactate was completely consumed (18\u00a0h, solid gray arrow) corresponded with a transient spike in DO2 (Fig.\u00a02b, blue line). This peak resulted from a rapid decrease in O2 consumption rate and a delayed response of the control loop used by the reactor to maintain constant DO2 values. The coordinated decrease in respiration rate and the complete consumption of lactate as the primary electron donor also correlated with the onset of flocculation, as indicated by an increased FCI. The organic acid analyses clearly indicated that pyruvate assumed the role of primary electron donor with no apparent change in acetate concentration. Cell flocculation peaked at \u223c25\u00a0h, just as the last of detectable pyruvate was consumed. The subsequent decrease in the FCI was bimodal. The initial decrease in the FCI corresponded to a slow decline in alkalinity production as measured by decreasing acid addition to maintain pH. When the concentration of acetate fell to \u223c15\u00a0mM and the FCI entered its second phase and decreased sharply, acid addition increased dramatically. This was likely a result of both the decrease in microbial metabolism and the aeration of the bioreactor. During active growth, lactate will be mineralized to both CO2 and bicarbonate with the CO2 being stripped from the culture via aeration and bicarbonate modestly increasing the alkalinity of the culture. As the metabolic rate of the culture decreases due to nutrient limitation, CO2 and bicarbonate generation will also decrease, but the CO2 will continue to be removed from the system while increasing amounts of bicarbonate will form carbonic acid and CO2. This will result in a net loss of protons from the system, thus increasing the pH and cause the increased addition of acid to the culture.\nCorrelating protein expression with culture conditions\nSignificant differences were observed between the proteomes of cells from uncontrolled \u201caerobic\u201d flasks and controlled aerobic batch reactors when analyzed by 2DE and MS. In general, proteins predicted to be expressed in cells growing under low oxygen tensions or anaerobically [e.g., fumarate reductase (Myers and Myers 1992; Maier et al. 2003], OmpW, glutamine synthase, decaheme cytochrome c MtrA, MtrC, and OmcA (Beliaev et al. 2002, 2005; Fang et al. 2006) were exclusively expressed or detected with increased abundance in samples from shake flasks when compared to those from controlled batch and chemostat cultures. Fumarate reductase (spot 432, SO0970), an enzyme required for anaerobic growth with fumarate (Maier et al. 2003), was detected in high abundance in 2DE patterns of lysate proteins from cells grown in the flask cultures, but was not detected in samples from aerobic controlled batch reactors (Fig.\u00a03). Similarly, the outer membrane protein OmpW (spot 1945, SO1673), and a hypothetical protein (spot 1275, SO3549) were present in 2DE patterns of proteins from flask culture lysates, but not detected in aerobic controlled batch reactors. A significant increase in the number of peptides corresponding to OmpW and fumarate reductase (8.1\u00d7 and 6.3\u00d7 higher, respectively, in the flask samples compared to controlled batch samples) was observed in the MS-based AMT tag analyses of these lysates (Table\u00a01), confirming the 2DE results. It is presently unclear why OmpW and the hypothetical protein abundances increased in low DO2, but our laboratory has observed this trend in the former protein several times (unpublished). In addition, peptides representing the multiheme cytochromes MtrA, MtrC, and OmcA and outer membrane \u03b2-barrel protein MtrB, were observed in two- to fivefold higher abundance in the AMT tag analysis of samples from shake flasks versus those from controlled batch cultures. These observations strongly suggest the use of shake flask cultures to assess changes in protein expression patterns related to aerobic versus anaerobic metabolism could yield inaccurate results.\nFig.\u00a03Two-dimensional gel electrophoresis patterns of whole cell lysate proteins from S. oneidensis MR-1 grown in shake flasks or continuous batch cultures. The proteins indicated by spot number correspond to those identified as varying with statistical significance between the two different culture methods (see Table\u00a01). The gel images are oriented with the basic proteins to the right, acidic proteins to the left, high molecular weight toward the top and low molecular weight toward the bottom. Flask: shake flask cultures (two replicate experiments); CB, controlled batch cultures (two replicate experiments)Table\u00a01Differential protein detection between flask and controlled batch cultures as determined by 2DE and MSORFProtein nameSpot number2DEMSAvg flaskAvg CBP-valueFlask\/CB ratioAvg flaskAvg CBFlask\/CB ratioSO0970Fumarate reductase4324900ND\u2013\u2013106\u00a0\u00b1\u00a03417\u00a0\u00b1\u00a02b6.3SO4349Ketol-acid reductoisomerase636114831500.0030.422\u00a0\u00b1\u00a06139\u00a0\u00b1\u00a0260.2SO19312-Oxoglutarate dehydrogenase763495a9510.024a0.536\u00a0\u00b1\u00a0146\u00a0\u00b1\u00a040.8SO3237Flagellin999271112239.9E-052.2108\u00a0\u00b1\u00a03673\u00a0\u00b1\u00a0141.5\u2013ND104811961968.8E-046.1\u2013\u2013\u2013SO3549Hypothetical protein1275648ND\u2013\u2013\u2013\u2013\u2013SO3681Universal stress protein family138226007013.5E-063.751\u00a0\u00b1\u00a01213\u00a0\u00b1\u00a023.9\u2013ND1426313ND\u2013\u2013\u2013\u2013\u2013SO1673OmpW19452927ND\u2013\u201362\u00a0\u00b1\u00a0248\u00a0\u00b1\u00a018.1SO4410Glutamine synthetase, type I1946100117471.8E-040.658\u00a0\u00b1\u00a01263\u00a0\u00b1\u00a030.9SO1776MtrB7\u00a0\u00b1\u00a014\u00a0\u00b1\u00a012.1SO1777Decaheme cytochrome c MtrA8\u00a0\u00b1\u00a012\u00a0\u00b1\u00a005.5SO1778Decaheme cytochrome c MtrC36\u00a0\u00b1\u00a0618\u00a0\u00b1\u00a012.0SO1779Decaheme cytochrome c OmcA21\u00a0\u00b1\u00a0311\u00a0\u00b1\u00a011.9ORF open reading frame (www.ncbi.nih.gov\/), Protein name annotation for open reading frame (www.ncbi.nih.gov\/), Spot number spot number assigned by Progenesis software using images shown in Figs.\u00a03\u00a0and\u00a05, Avg flask average integrated spot density in 2DE images (3\u20134 images per sample) from shake flask samples, Avg CB average integrated spot density in 2DE images (3\u20134 images per sample) from continuous batch culture samples, P-valueP-value from two-tailed Student\u2019s t-test analysis of average integrated density values, Flask\/CB ratio ratio of average integrated density values for flask and continuous batch culture samplesaNote that the difference in spot 763 is a function of the variability in the protein between the two flask cultures. In Flask 1, the average integrated density was 1020 with a coefficient of variation (CV) of 12.5% whereas in Flask 2, the average integrated density was 316 with a CV of 31.6%. The 495 represents the averaging of integrated densities from all of the patterns from these two flasks together with a resulting CV of 60%bThe average MS abundance data\u00a0\u00b1\u00a0the standard deviation resulting from triplicate analysis of each biological sample\nChemostats provide enhanced experimental consistency\nChemostat cultures provide the highest level of control of any known cultivation technology (Monod 1950; Novick and Szilard 1950; Wimpenny 1985). To evaluate the reproducibility of chemostat cultures, duplicate bioreactors were operated in steady state with S. oneidensis MR-1 under 50% DO2 as above and the growth and carbon metabolism parameters were measured. Each of the aerobic cultures attained an initial maximum optical density of \u223c0.70 by 20\u00a0h and eventually achieved a steady state cell density at 0.53 (A600\u00a0nm) (Fig.\u00a04). The two reactors were highly similar in terms of growth and acid addition rates, as well as organic acid concentrations at the time of sampling of 100\u00a0h. Lactate, acetate, fumarate and succinate were all below detectable limits suggesting that the cultures were in fact carbon-limited during steady state growth. Pyruvate was present in both chemostatic cultures at 0.14\u00a0\u00b1\u00a00.02\u00a0mM. Dry weight biomass measurements confirmed the highly similar cell densities of 338.0\u00a0\u00b1\u00a04.3\u00a0mg\/l with only a 1.3% variance between the reactors. In comparison, the duplicate controlled batch and shake flask cultures used in this study yielded similar biomass amounts but varied by 2.3 and 17.6%, respectively. These results support our contention of greater biological variability among replicate flask cultures relative to bioreactor cultures.\nFig.\u00a04Shewanella oneidensis MR-1 cultured in duplicate bioreactors at 20% DO2. The same inoculum culture was used for both bioreactors. Cells were harvested from each reactor when the optical density and organic acid concentrations were similar. The average dry weight biomass was 338\u00a0\u00b1\u00a04.3\u00a0mg\/l\nCulture and proteome variability\nWhen the intercultural variability of the proteomes of cells from duplicate shake flasks, controlled batch bioreactors, and chemostat cultures was assessed using 2DE and MS, notable differences were identified. For 2DE analysis, digital images of the gels were processed for spot detection and pattern matching with one 2DE image as a reference pattern and all others matched to it to give the protein spots identification numbers. Statistical analysis of the relative abundance of each matched protein spot across the datasets employed a two-tailed Student\u2019s t-test as previously described (Giometti and Taylor 1991). Differences in 2DE spot patterns were observed between the replicate flask cultures (Fig.\u00a05a,\u00a0d), including one unidentified protein (spot 771; P\u00a0<\u00a00.004). In contrast, there was relatively little variability (P\u00a0<\u00a00.05) in 2DE proteome profiles from the replicate gels from either the controlled batch or chemostat cultures.\nFig.\u00a052DE protein patterns from biological replicates of the three growth methods. The within method replicates show few significant differences. The chemostat and controlled batch replicates showed no quantitative differences with a statistical significance of P\u00a0<\u00a00.05 whereas the flask culture replicates showed one quantitative difference (Spot 771) with a difference of P\u00a0<\u00a00.004. SS steady state culture, CB controlled batch culture. The gel images are oriented with the basic proteins to the right, acidic proteins to the left, high molecular weight toward the top and low molecular weight toward the bottom\nData from MS-proteome analysis was consistent with the 2DE results. Triplicate samples from duplicate cultures from shake flask, controlled batch, and chemostats were analyzed and Venn diagrams (Fig.\u00a06) were constructed to illustrate the similarities and differences between biological and analytical replicates based on the cultivation method. A comparison of protein detection consistency between the biological duplicates showed that the flask grown cells (Fig.\u00a05a) exhibit a higher degree of variability. Flask A averaged 972 proteins detected from triplicate analyses while flask B averaged 1,029 proteins with only 770 proteins (77%) in common. The consistency of detected proteins between biological replicates was higher in the controlled batch (84%, Fig.\u00a05c) and chemostat cultures (88%, Fig.\u00a05e). This was not due to a lack of consistency in the analytical replicates since protein detection consistency was between 88 and 89% for all three cultivation methods (Fig.\u00a05d\u2013f). Secondly, there was an overall decrease in the number of proteins detected from the flask grown (770) to the controlled batch (713) and to the chemostat (571) cultures. This can be explained by the presumed homogeneity of the culture increasing as the degree of control over pH and DO2 increased thus allowing the entire culture to experience similar environmental conditions.\nFig.\u00a06MS proteomic analysis of the same duplicate (left) shake flask, (middle) controlled batch and (right) chemostat grown cells that were analyzed by 2DE. The consistency in the proteins detected between biological duplicates from shake flask (a), controlled batch (c) and chemostat (e) cultures shows the increased similarity in proteomes with increased control over culturing parameters. This was not an effect of the instrumentation since analytical replicates from each of the shake flask (b), controlled batch (d) and chemostat (f) cultures consistently had (88% of the detected proteins in common. The values below each Venn diagram circle are the number of detected proteins for each biological (a, c, e) or analytical (b, d, f) replicate. The value in the overlap region is the number of proteins in common between the replicates with the corresponding percentage for ease of comparison. The percentage shown is the number of proteins in common divided by the average of the two replicates presented\nDiscussion\nThe advantages of controlled cultivation have been demonstrated for many different research applications in microbiology, including the determination of carbon, nitrogen or electron flow (Vasconcelos et al. 1994; Daran-Lapujade et al. 2004; Kolkman et al. 2005, 2006; Adams et al. 2006), kinetic constants for specific reactions (Nethe-Jaenchen and Thauer 1984), alternate roles for traditionally well-defined enzymes (Snoep et al. 1990), and whole cell protein fingerprinting (Wunschel et al. 2005). While there have been statements touting the advantages of controlled cultivation, an in depth investigation of the differences in metabolism between cultivation methods has not been performed. The closest exception to this reported that microarray results between three chemostatic cultures from three different laboratories were significantly more reproducible compared to a previous study that performed a similar comparison using shake flask cultures (Piper et al. 2002). However, the authors reported that there were differences in harvesting and other procedures which may well have skewed the findings. To this end, the experiments reported here were specifically directed toward determining whether or not controlled cultivation results in a more homogenous culture with regard to both targeted and comprehensive proteome profiles from three cultivation methods in our laboratory. Our results demonstrate that variations in DO2 concentration and consumption rates between replicate flask cultures in the late log phase of growth can impact metabolism. This was evidenced by the increased lag time in the uncontrolled flask cultures relative to the controlled cultivations. The data suggests that this may be an effect of oxygen toxicity when DO2 levels are above 20%. This has been recently tested in S. oneidensis MR-1 using sealed culturing vessels and levels above 21% headspace O2 were found to be inhibitory (McLean et al. 2007). Two possible explanations exist for this observation. It is possible that the extended lag and initial slow growth rate was the result of oxygen toxicity at DO2 values above 20%. However, the uncontrolled batch cultures contained 30\u00a0mM PIPES buffer to aid in the maintenance of pH during growth while the controlled batch and chemostatic cultures contained only 3\u00a0mM PIPES. Hence, there would have been a large difference in the ionic strength of the two media and so while DO2 toxicity may be the reason for the extended lag phase, other reasons cannot be ruled out. Separately, differences in acetate concentrations of the replicate shake flask cultures compared to controlled batch and chemostat cultures indicated that such differences may well have promoted additional biological variability.\nThe difference in acetate concentrations between replicate shake flask cultures compared to the consistent acetate concentrations in controlled batch and chemostat cultures has particular implications for S. oneidensis strain MR-1. MR-1 can use lactate and pyruvate as an electron donor during anaerobic growth, but not acetate (Lovley et al. 1989; Nealson and Myers 1992). Thus, variations in the concentration of acetate and the variable DO2 uptake rates in shake flask cultures will cause a temporal shift in carbon source usage and electron flux. Such shifts can be expected to result in variable metabolic, transcriptomic and proteomic profiles between independent shake flask cultures if only a small number (1\u20133) of samples are taken, since one culture may have exhausted a carbon source and consequently shifted metabolism and electron flow while the \u201cbiological replicate\u201d has not.\nGiven the general metabolic flexibility of bacteria including S. oneidensis MR-1 and their ability to respond rapidly to changes in environmental conditions, changes in the expressed proteins (proteome) would be expected as a result of exposing cultures to different environmental conditions. Through the use of comprehensive proteome measurements, the results presented here demonstrate that chemostat cultures provide the most consistent source of cellular material for biochemical measurements involving microbial cells with a minimum contribution to those measurements from biological variation. Two independent methods of proteome measurement, 2DE and AMT MS, both showed that flask cultures yielded protein samples with more culture-to-culture variability than observed in either controlled batch cultures or continuous chemostat cultures, primarily among proteins thought to be associated with anaerobic or microaerobic metabolism. A similar effect was found with respect to differential regulation of genes via microarrays when the results of two different investigations were compared from yeast cells, showing less discrepancy between chemostatic cultures (Daran-Lapujade et al. 2004).\nMany of the proteins present in higher abundance in the aerobic shake flask cultures relative to the controlled batch or chemostat cultures in this study have previously been reported to be up-regulated in anaerobic compared to aerobic cultures (Beliaev et al. 2002, 2005; Fang et al. 2006). MtrA,B,C and OmcA are involved in metal-reduction in anaerobic cultures supplied with metals as terminal electron acceptors (Beliaev and Saffarini 1998; Beliaev et al. 2001; Myers and Myers 2002; Pitts et al. 2003) while the outer membrane protein OmpW and fumarate reductase have both been observed to be significantly more abundant in anaerobic cultures supplied with iron or fumarate as electron acceptor than in aerobic cultures (Fang et al. 2006). Thus, the shake flask cultures in the experiments described here must have included cells experiencing a lack of oxygen due to the nutrient depletion that is an inherent complication of such cultures, resulting in a greater range of expressed proteins.\nAchieving a systems-level understanding of microorganisms, including the underlying metabolic and regulatory networks that control cell physiology and the ability to predict responses to perturbations, is indeed an ambitious goal. Systems biology takes full advantage of the state-of-the-science technology and genomic information, with the scientific as well as the practical benefits expected to be plentiful. Our results emphasize that functional genomics can greatly benefit from well-defined and homogeneous cell cultures provided by controlled cultivation techniques. The use of controlled cultivation techniques ensures that the large and comprehensive datasets used in systems biology research are derived from well-characterized and homogeneous biological samples, thus reducing the inherent variability that often complicates drawing biological conclusions and will allow for more accurate and robust identifications of metabolic and regulatory networks.","keyphrases":["shewanella","proteomics","systems biology","controlled cultivation"],"prmu":["P","P","P","P"]}
{"id":"Diabetologia-3-1-1914300","title":"Automated measurement of brain and white matter lesion volume in type 2 diabetes mellitus\n","text":"Aims\/hypothesis Type 2 diabetes mellitus has been associated with brain atrophy and cognitive decline, but the association with ischaemic white matter lesions is unclear. Previous neuroimaging studies have mainly used semiquantitative rating scales to measure atrophy and white matter lesions (WMLs). In this study we used an automated segmentation technique to investigate the association of type 2 diabetes, several diabetes-related risk factors and cognition with cerebral tissue and WML volumes.\nIntroduction\nDiabetes mellitus type 2 is associated with accelerated cognitive impairment and an increased incidence of dementia [1]. Recently, we have demonstrated that cognitive impairments in patients with type 2 diabetes are accompanied by brain atrophy and ischaemic white matter lesions (WMLs) [2]. Other neuroimaging studies have also demonstrated that type 2 diabetes is associated with a moderate degree of cerebral atrophy [3\u20135] and with an increased occurrence of cerebral infarcts [6]. These studies have used either semiquantitative rating scales [3, 4] or cerebrospinal fluid to brain ratios [2, 4] to assess atrophy. Quantitative assessment of volumes of grey matter, white matter, lateral ventricle, and cerebrospinal fluid in patients with type 2 diabetes has not been performed.\nThe association between WMLs and type 2 diabetes is debated. Although several studies found an association between type 2 diabetes and WML severity or progression [2, 7\u201313], others did not [4, 5, 14\u201318]. These inconsistencies are probably due to the use of different, mostly semiquantitative, WML grading methods, and differences in study design and population selection. Furthermore, even within the population of healthy elderly the variance in WML severity is large [19].\nThe present study aimed to determine quantitatively the effects of type 2 diabetes on cerebral tissue volumes and WML severity by using an accurate magnetic resonance (MR) imaging-based automated segmentation algorithm [20] in a large, representative, cross-sectional, population-based sample of patients with type 2 diabetes and control participants. A second aim was to study the association of several diabetes-related variables and cognition with cerebral tissue volumes and WML severity.\nSubjects and methods\nParticipants Participants were recruited between September 2002 and November 2004 as part of the Utrecht Diabetic Encephalopathy Study (UDES), a cross-sectional, population-based study on determinants of impaired cognition in type 2 diabetes [2]. The UDES study aimed to identify potential risk factors for cognitive impairment in type 2 diabetes. Therefore, patients were not selected for the presence or absence of diabetic complications, comorbid conditions (e.g. hypertension) or exposure to other risk factors (e.g. smoking). For inclusion in the present study, participants had to be 55\u201380\u00a0years of age, functionally independent, and Dutch-speaking. Type 2 diabetes patients (n\u2009=\u2009122; 56\u201380\u00a0years of age) were recruited through their general practitioner and had a diabetes duration of at least 1\u00a0year. Controls (n\u2009=\u200957, 55\u201378\u00a0years of age) were recruited among the spouses and acquaintances of the patients. Exclusion criteria for all participants were a psychiatric or neurological disorder unrelated to diabetes that could influence cognitive functioning, a history of alcohol or substance abuse, and dementia. Controls with a fasting blood glucose \u22657.0\u00a0mol\/l were also excluded. Twice as many patients as controls were included to increase the statistical power of within-group analyses in the type 2 diabetes group. The study was approved by the medical ethics committee of the University Medical Center Utrecht and each participant signed an informed consent form.In a standardised interview, participants were questioned about diabetes duration, height and weight, history of hypertension and smoking, level of education (seven categories, corresponding to years of education: <6, 6\u20137, 8, 9, 10\u201311, 12\u201318 and >18, respectively [21]), medication use and history of vascular disease. Furthermore, all participants measured their blood pressure at home at nine different time points during the day. These measurements were used to calculate the mean arterial pressure. Hypertension was defined as an average systolic blood pressure \u2265160\u00a0mmHg and\/or diastolic blood pressure \u226595\u00a0mmHg and\/or self-reported use of blood pressure-lowering drugs. BMI, fasting glucose and HbA1c were also determined. Hypercholesterolaemia was defined as a cholesterol:HDL-cholesterol ratio greater than 5 [22] or the use of cholesterol-lowering drugs. All subjects underwent a neuropsychological evaluation, including 11 different tests addressing the cognitive domains of visuo-construction, attention and executive function, information processing speed, memory and abstract reasoning [2]. For further analysis, the raw scores of the different tests were standardised into z-scores per domain. For the present study, these z-scores were averaged into one composite cognitive z-score.Brain MR images were acquired as part of the study. In 23 participants with type 2 diabetes and 11 control participants no MR images were available for automated analysis: in 14 cases (five controls, nine diabetic patients) an MR image had not been performed because of MR contraindications and in 20 cases (six controls, 14 diabetic patients) the scan could not be analysed automatically because of technical problems, such as failure to retrieve the digital MR images or incompleteness of the series of images, or because image quality did not allow automated processing. Data for 99 patients with type 2 diabetes and 46 controls were analysed. Age, sex and duration of diabetes for the excluded subjects were similar to those for the included subjects (control subjects, average age 64.6\u00a0years, five men and six women; diabetic subjects, average age 66.5\u00a0years, 13 men, ten women, average diabetes duration 8.7\u00a0years).\nMR imaging Brain MR images were acquired on a Philips Gyroscan ACS-NT 15 whole-body system operating at 1.5\u00a0T (Philips Medical Systems, Best, The Netherlands) with a standardised MR protocol (slice thickness 4\u00a0mm, 38 contiguous slices, 230\u2009\u00d7\u2009230\u00a0mm field of view, 256\u2009\u00d7\u2009256 scan matrix). Axial T1, inversion recovery (IR), T2, proton density (PD) and fluid attenuated inversion recovery (FLAIR) scans were made: T1, 234\/2\u00a0ms (repetition\/echo time); IR, 2919\/410\/22\u00a0ms (repetition\/inversion\/echo time); T2, 2200\/100\u00a0ms (repetition\/echo time); PD, 2200\/11\u00a0ms (repetition\/echo time); FLAIR, 6000\/2000\/100\u00a0ms (repetition\/inversion\/echo time).\nImage processing Preprocessing consisted of intrasubject registration of the five MR sequences and extraction of a brain mask. Registration was performed using an affine nine-parameter mutual information-based algorithm [23] with the FLAIR image as reference.Brain masks were constructed to exclude the skull, skin and background during classification. The masks were extracted by k-means clustering of the T1, IR, T2, PD and FLAIR images using eight clusters. The clusters containing cerebrospinal fluid and brain were combined. Holes in the mask were filled and appending structures, such as eyes, were removed using morphological operators. Dilation of the brain mask by three voxels ensured the inclusion of all cerebrospinal fluid. One mask had to be edited manually because appending structures were included. The final brain masks contained the whole brain including the cerebellum and brainstem.Segmentation of the MR IR and FLAIR images into white matter, cortical and subcortical grey matter, lateral ventricles, cerebrospinal fluid not including the lateral ventricles (CSF) and WML was executed fully automatically by means of a probabilistic k-nearest neighbour (KNN)-based classification algorithm [20]. The algorithm was trained on expert manual segmentations of ten subjects who had varying degrees of WML, who were similar in age to the participants of this study and scanned using the same protocol, but who did not participate in this study. Manual segmentation of grey matter and white matter was performed on MR IR images, because these provide optimal tissue contrast for the assessment of the grey matter\u2013white matter boundary, and manual segmentation of lateral ventricles, CSF and WML was performed on MR FLAIR images. All MR images were available to the expert for reference. Performance of the classification algorithm was validated previously using a leave-one-out procedure and resulted in similarity indexes of at least 0.808, indicating excellent agreement [20]. For each participant, classification produces five separate images of white matter, grey matter, lateral ventricles, CSF and WML probability per voxel. As an example, the result of the classification of the MR FLAIR and inversion recovery images of a diabetes patient is shown in Fig.\u00a01. White matter, grey matter, CSF and lateral ventricle volumes were calculated by summing over the probability image and multiplying with the voxel dimensions. WML volume calculation deviated slightly. A threshold of 0.5 was applied on the WML probability image and all unconnected voxels were removed. Then, WML volume was calculated by summation over the image and multiplication with the voxel dimensions. The intracranial volume was calculated as the sum of white matter, grey matter, lateral ventricles, CSF and WML volumes; total brain as white matter plus grey matter volume; and total CSF as CSF plus lateral ventricle volume. Volumes of automatically segmented tissues for the male control participants of the present study did not differ from the tissue volumes found for the manually segmented men (manually segmented men, n\u2009=\u20098; women, n\u2009=\u20092 [not compared with female controls]). Furthermore, all segmentations were carefully reviewed by one of the authors (C. Jongen). During this process, the author was blinded to the diabetes mellitus status of the participants. Twelve WML segmentations were manually edited to be sure that infarcted tissue was excluded and ten were edited because of artefacts in the FLAIR image.\nFig.\u00a01MR FLAIR (a) and inversion recovery image (b) of a diabetes patient with relatively severe WMLs. On the MR FLAIR image, the WMLs are clearly visible as white areas, whereas on the inversion recovery image the boundary between grey and white matter is much better defined. (c) The result of segmentation using the automated KNN-based algorithm. The colours indicate the different tissue classes: grey matter (yellow), white matter (dark blue), lateral ventricles (green), CSF (red) and WML (light blue)\nStatistical analysis Differences in demographic data and risk factors between control participants and those with type 2 diabetes were analysed using a univariate general linear model or logistic regression adjusting for age and sex. A univariate general linear model was used to analyse differences in tissue volume between type 2 diabetes and control participants adjusting for age, sex, intracranial volume and level of education. Additionally, separate analyses for men and women were performed, because cerebral tissue volumes are different among men and women. Kolmogorov\u2013Smirnov tests showed that WML volume was not normally distributed. Therefore, we used a natural log transformation of WML volumes in the analyses. Lateral ventricle volume distribution was slightly non-normal. Using natural log-transformed volumes gave similar results to using untransformed volumes. For ease of interpretation, results for untransformed volumes are reported. Additionally, the associations of age with tissue volumes were analysed. Furthermore, associations between the tissue volumes with the composite cognitive performance were analysed for type 2 diabetes and control participants separately, adjusting for age, sex, intracranial volume and level of education. Within the group of type 2 diabetes patients associations between MR volume measures and diabetes duration, HbA1c, hypertension, mean arterial pressure, history of macrovascular disease, and hypercholesterolaemia were determined, adjusting for age, sex, intracranial volume and level of education. All data were analysed using SPSS 12.0.1 (SPSS Inc., Chicago, IL, USA).\nResults\nPatient characteristics are shown in Table\u00a01. Table\u00a02 shows the brain tissue and WML volumes unadjusted for differences in age or intracranial volume. Table\u00a03 shows the results of the analysis of the tissue volume differences between controls and patients with type 2 diabetes. Diabetes patients had significantly smaller grey matter volume (estimated volume difference \u221221.8\u00a0ml; 95% CI \u221234.2, \u22129.4; p\u2009=\u20090.001; F\u2009=\u200912.091) and significantly larger lateral ventricle volume than controls (estimated volume difference 7.1\u00a0ml; 95% CI 2.3, 12.0; p\u2009=\u20090.004; F\u2009=\u20098.441). Total CSF volume was also larger in diabetes patients, but not significantly so (estimated volume difference 9.5\u00a0ml, 95% CI \u22120.4, 19.5; p\u2009=\u20090.060; F\u2009=\u20093.594). White matter volume was unaffected (estimated volume difference 2.8\u00a0ml, 95% CI \u22123.2, 8.8; NS; F\u2009=\u20090.844). WML volume was significantly larger in type 2 diabetes patients (56.5%; 95% CI 4.0, 135.8; p\u2009=\u20090.032; F\u2009=\u20094.684). The cumulative distribution of WML volume is shown in Fig.\u00a02. All participants had at least some WML. However, in type 2 diabetes patients a smaller proportion of the subjects had very small lesion volumes (<0.5\u00a0ml) (Pearson \u03c72 with continuity correction, p\u2009=\u20090.014). Such very small volumes mostly reflect pencil-thin lining and capping, which is often considered to be a normal finding in this age group [19]. The regression analyses were repeated for men and women separately (Table\u00a03). Although the direction of the effect of type 2 diabetes was similar in the two sexes, significant associations of type 2 diabetes with tissue volume were only found in women.\nTable\u00a01Demographics of participants and risk factors\u00a0Control participantsType 2 diabetes patientsMean difference (95% CI)aOdds ratio (95% CI)aMen\/women20\/2649\/50\u20131.3 (0.6, 2.6)Age (years)\u00a0 Men66.4 (6.3)65.9 (6.0)\u22120.5 (\u22122.8, 3.7)\u2013\u00a0 Women63.8 (5.0)65.9 (5.2)2.1 (\u22120.4, 4.5)\u2013Level of educationb4 (3\u20135)4 (3\u20135)\u2013\u2013Diabetes duration (years)\u20138.7 (6.1)\u2013\u2013HbA1c (%)5.5 (0.3)6.8 (1.2)1.4 (1.0, 1.7)***\u2013Use of insulin (%)\u201329.3\u2013\u2013Hypertension (%)c28.370.76.2 (2.8, 13.5)***Mean arterial pressure (mmHg)97.5 (10.6)102.6 (11.5)4.8 (0.9, 8.8)*\u2013History of macrovascular disease (%)4.327.3\u20138.3 (1.8, 37.0)**Hypercholesterolaemia (%)42.269.4\u20133.7 (1.7, 8.1)**Smoking ever (%)47.866.3\u20132.2 (1.0, 4.7)*BMI (kg\/m2)27.2 (4.4)28.0 (4.4)0.8 (\u22120.8, 2.4)\u2013Cognition (composite z-score)0.13 (0.45)\u22120.10 (0.63)\u22120.22 (\u22120.40, \u22120.05)*d\u2013Data in first two columns are mean (SD) except for level of education, which is given as median (interquartile range)*p\u2009<\u20090.05; **p\u2009<\u20090.01; ***p\u2009<\u20090.001aAdjusted for age and sexbSeven categories, corresponding to years of education: <6, 6\u20137, 8, 9, 10\u201311, 12\u201318 and >18, respectivelycAll controls with hypertension and 96% of diabetic patients with hypertension used antihypertensive drugsdAdjusted for age, sex and level of educationTable\u00a02Volumes of cerebral compartments (ml) unadjusted for age or intracranial volume\u00a0Control participantsType 2 diabetes patientsMenWomenMenWomenWhite matter688.0 (49.3)621.8 (46.9)687.2 (56.5)601.0 (56.5)Grey matter401.7 (47.8)404.4 (40.8)388.3 (48.8)360.1 (35.6)Total brain1,090 (81.0)1,026 (72.9)1,076 (91.0)961.1 (71.0)Lateral ventricle33.9 (18.7)22.1 (8.66)36.9 (17.4)29.8 (13.9)Lateral ventriclea30.1 (23.9\u201336.6)20.3 (16.6\u201328.0)33.8 (24.9\u201342.6)26.7 (20.6\u201335.3)CSF excluding lateral ventricles273.3 (27.1)222.1 (25.1)279.7 (37.8)229.4 (35.9)Total CSF307.2 (32.3)244.2 (26.3)316.5 (42.4)259.2 (41.2)WML3.89 (5.76)2.98 (4.70)3.66 (5.37)6.19 (14.2)WMLa1.81 (0.47\u20133.48)1.00 (0.48\u20133.20)2.16 (0.91\u20133.86)2.56 (0.86\u20134.46)Intracranial volume1,403 (90.7)1,275 (82.2)1,398 (104.2)1,228 (93.7)aLateral ventricle and WML volumes are median (interquartile range); other data are mean (SD)Between-group comparisons and statistical analyses are presented in Table\u00a03Table\u00a03Adjusted tissue volume differences between participants with type 2 diabetes and control participants\u00a0Estimated volume difference (ml)F valueAll\u00a0White matter2.8 (\u22123.2, 8.8)0.844\u00a0Grey matter\u221221.8 (\u221234.2, \u22129.4)**12.091\u00a0Total brain\u221219.0 (\u221229.9, \u22128.1)**11.863\u00a0Lateral ventricles7.1 (2.3, 12.0)**8.441\u00a0CSF not including lateral ventricles9.5 (\u22120.4, 19.5)3.594\u00a0Total CSF16.7 (6.8, 26.5)**11.247\u00a0LN WML0.45 (0.04, 0.86)a*4.684Men\u00a0White matter1.8 (\u22127.4, 11.0)0.157\u00a0Grey matter\u221214.5 (\u221233.6, 4.6)2.315\u00a0Total brain\u221212.7 (\u221230.6, 5.2)2.026\u00a0Lateral ventricles4.9 (\u22123.7, 13.5)1.283\u00a0CSF not including lateral ventricles6.9 (\u221210.2, 24.0)0.650\u00a0Total CSF11.8 (\u22124.9, 28.5)1.993\u00a0LN WML0.37 (\u22120.19, 0.93)b1.781Women\u00a0White matter7.0 (\u22121.5, 15.6)2.696\u00a0Grey matter\u221237.1 (\u221254.1, \u221220.1)***19.062\u00a0Total brain\u221230.1 (\u221244.9, 15.4)***16.695\u00a0Lateral ventricles9.0 (3.2, 14.8)**9.563\u00a0CSF not including lateral ventricles17.2 (4.9, 29.4)**7.802\u00a0Total CSF26.1 (13.6, 38.7)***17.215\u00a0LN WML0.54 (\u22120.11, 1.20)c2.745LN WML Natural log of WML volumeData are mean (95% CI) adjusted for age, sex, intracranial volume and level of education (data missing for one woman with type 2 diabetes)*p\u2009<\u20090.05; **p\u2009<\u20090.01; ***p\u2009<\u20090.001a56.5% (95% CI 4.0, 135.8)b44.9% (95% CI \u221217.0, 154.7)c72.4% (95% CI \u221210.6, 232.4)Fig.\u00a02Cumulative distribution of WML volume (control men, closed squares; men with type 2 diabetes, closed triangles; control women, open squares; women with type 2 diabetes, open triangles). Very small lesion volumes (<0.5\u00a0ml) were significantly more frequent among controls (p\u2009=\u20090.014) than participants with type 2 diabetes\nWithin the patients with type 2 diabetes, no significant associations between MR volumes and diabetes duration, hypertension, mean arterial pressure or hypercholesterolaemia were found. Higher HbA1c levels were associated with larger grey matter volume (6.479\u00a0ml per unit HbA1c (%); 95% CI 0.2, 12.8; p\u2009=\u20090.043; F\u2009=\u20094.203), but not with any other volume abnormalities. A history of macrovascular disease was associated with a larger total CSF volume (12.4\u00a0ml; 95% CI 2.6, 28.7; p\u2009=\u20090.020; F\u2009=\u20095.659) and a smaller total brain volume (\u221215.0\u00a0ml; 95% CI \u221229.8, \u22120.2; p\u2009=\u20090.047; F\u2009=\u20094.057). In the diabetic participants lower composite cognitive performance was associated with significantly smaller total brain volume (\u221215.4\u00a0ml per unit of the composite cognitive z-score; 95% CI \u221227.6, \u22123.1; p\u2009=\u20090.015; F\u2009=\u20096.199), larger WML volume (57.4%; 95% CI 2.2, 142.4; p\u2009=\u20090.040; F\u2009=\u20094.370) and non-significantly larger total CSF volume (11.0\u00a0ml; 95% CI \u22120.6, 22.1; p\u2009=\u20090.051; F\u2009=\u20093.911).\nIn the control group exploratory analyses showed an association between higher BMI and larger total CSF volume (2.0\u00a0ml; 95% CI 0.34, 3.6; p\u2009=\u20090.017; F\u2009=\u20096.287). No other significant associations were found within the control group.\nAcross the whole population, age was significantly associated with reduced grey matter and total brain volumes and larger lateral ventricle, CSF, total CSF and WML volumes (p\u2009<\u20090.001; F\u2009>\u200920.424; adjusted for diabetic status, sex, intracranial volume and level of education). Age was the most powerful predictor of WML and lateral ventricle volumes and it came second after intracranial volume for predicting grey matter, CSF, total brain and total CSF volumes. No significant interactions between age and type 2 diabetes were found (all p\u2009>\u20090.14). The interaction between age and sex showed a trend towards significance, men having more grey matter decrease (\u22121.8\u00a0ml\/year; 95% CI \u22123.9, 0.3; p\u2009=\u20090.092; F\u2009=\u20092.888) and women having more white matter decrease (\u22120.9\u00a0ml\/year; 95% CI \u22121.9, 0.1; p\u2009=\u20090.079; F\u2009=\u20093.134) with age (other volumes p\u2009>\u20090.26).\nDiscussion\nOur MR image-based segmentation paradigm showed that type 2 diabetes was significantly associated with smaller grey matter volumes and larger lateral ventricle and WML volumes, whereas white matter volume was not affected. A smaller grey matter volume suggests cortical atrophy, whereas a larger lateral ventricle volume may indicate subcortical atrophy. Separate analysis for men and women showed significantly smaller grey matter and total brain volumes and significantly larger lateral ventricle, CSF and total CSF volumes in female but not in male diabetic patients.\nThe apparent differential effects of diabetes on brain volumes in men and women were an unexpected finding. Previous studies on cognitive functioning or dementia in patients with diabetes have provided no clear indications that the effects of diabetes on the brain might be sex-specific [24], although it should be noted that the role of sex has not yet been studied systematically. In the general population, intracranial volume and relative and absolute grey matter and white matter volumes are known to differ between men and women [25]. Sex also influences the effects of ageing [26\u201328], men being more severely affected by age-related grey matter decrease than women [29, 30], which was also found in this study. WML volumes among male and female controls were similar, but in female type 2 diabetes patients we found significantly larger WML volumes than in male type 2 diabetes patients. In conditions other than diabetes, more severe WMLs in women than in men have been reported [15, 31\u201333], but similar WML severity in both sexes has also been reported [19]. Diabetic women had non-significantly smaller age-adjusted total intracranial volumes than control women. However, the expected effects of these intracranial volume differences on brain tissue volumes were smaller than the observed differences in effects on grey matter, CSF and lateral ventricle volumes. Age is a very important predictor of brain tissue and WML volumes and age effects may have confounded the effects of type 2 diabetes in men and women in this study. Hence, the implications of the effect of sex observed in the present study remain to be determined.\nNo statistically significant associations between MR image measures and hypertension or mean arterial pressure within the group of type 2 diabetes patients were found. Similar results were obtained with manual measurements of atrophy and with WML grading in the same study population [2], and in a study that assessed the relation between type 2 diabetes, blood pressure and temporal lobe atrophy [5]. In contrast, another study reported that hypertension was an important determinant of cortical atrophy in patients with type 2 diabetes [4]. We found an association between glycosylated haemoglobin and a slight increase in grey matter volume in type 2 diabetes patients. This counterintuitive finding may be due to chance. Other studies in subjects without diabetes have linked increased glycosylated haemoglobin with cerebral atrophy [34] and WMLs [35]. Cognitive impairment was associated with smaller total brain volume and larger WML volumes in type 2 diabetes patients. This is in line with observations from another recent study that reported an association between WMLs and subcortical brain atrophy and cognitive performance, in particular the speed of information processing [36]. This suggests that atrophy as well as WMLs have a negative impact on cognition.\nPrevious studies that analysed WML volume or progression in type 2 diabetes patients have reported inconsistent results [4, 5, 7\u201318]. Several methodological factors may have contributed to these inconsistencies. First, techniques to measure WML severity have varied widely, from noting lesion presence or absence [12] to volumetric measurements [13, 18]. The majority of studies applied semiquantitative ordinal grading scales. It has been shown that these scales can give variable results [37] and some scales are relatively insensitive to mild to moderate WMLs. Second, the examined populations and the study design varied considerably [38]. Only a small proportion of the studies had a true population-based setting, whereas the majority of studies were hospital-based, involving cohorts of patients with stroke, hypertension or other forms of cardiovascular disease. Furthermore, several studies involved fewer than 20 type 2 diabetes patients [8, 11\u201313, 18]. These small sample sizes may have reduced statistical power. The present study used volumetric measurements in a large population-based sample.\nThe combination of increased atrophy and increased WML volume indicates that type 2 diabetes is associated with mixed pathology in the brain. The association with vascular lesions, such as WML, is not surprising because diabetes is a well-known risk factor for cerebrovascular disease [39]. Indeed, previous imaging studies [38], as well as autopsy studies [40], have reported an increased occurrence of vascular lesions, in particular infarcts, in diabetic patients relative to controls. The mechanisms underlying accelerated brain atrophy in diabetic patients are less clear. Several processes related to glucose toxicity, abnormalities in cerebral insulin homeostasis, and microvascular abnormalities have been implicated [41], but their exact contributions to abnormalities in cerebral function and structure in diabetes still need to be elucidated.\nA limitation of our quantitative automated volumetric segmentation algorithm is slight overestimation of the CSF and slight underestimation of the lateral ventricle volume in subjects with markedly enlarged occipital horns. Some misclassification of interhemispheric CSF as lateral ventricle might also occur. However, the misclassified volumes are small compared with ventricle (about 2% and 6%) and CSF (about 0.3% and 0.4%) volumes and total CSF volumes are not affected. The artefacts were proportionally distributed across men and women and between type 2 diabetes patients and controls. Therefore, it is unlikely that these inaccuracies affected the analyses. Furthermore, all segmentations were inspected visually, so results will not have been affected by other segmentation errors.\nOur algorithm did not classify WMLs into deep and periventricular lesions. Such automated subclassification may provide valuable information; however, it is hard to achieve. WMLs tend to extend smoothly from the ventricular wall [42] and periventricular WMLs are often connected to deep WMLs on MR images. Therefore, defining meaningful boundary criteria that can be consistently and reproducibly applied automatically is difficult.\nOther published methods have used T1 and T2 [25], PD and T2 [26], T1 only [27], or T1, PD and T2 [30] MR images for segmentation. However, in a previous study it was shown that the use of IR and FLAIR images is highly preferable in brain tissue segmentation and that optimal performance of the automated KNN classification algorithm is achieved with this combination of images [20]. Furthermore, the learning data were segmented manually on the basis of IR and FLAIR images with the T1, T2 and PD images available to the expert for reference. Thus, our KNN classifier retrieves its learning data from expert segmentations based on all available information and segments using the optimal image combination for tissue classification.\nOur automated segmentation method offers major advantages over manual methods by enabling precise, objective and reproducible volumetric measurements of cerebral tissues in large numbers of patients. In future studies, this method might be used to quantify the progression of abnormalities on MR images in patients with type 2 diabetes, as well as in patients with other conditions. Such studies using quantitative MR data should focus not only on the differences between groups of patients and controls, but also on differences within a patient group to identify determinants of structural brain changes.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.","keyphrases":["brain","white matter lesion","diabetes mellitus type 2","image analysis","computer-assisted","leukoaraiosis","magnetic resonance imaging"],"prmu":["P","P","P","R","U","U","R"]}
{"id":"Cancer_Causes_Control-3-1-1914283","title":"Alcohol consumption, cigarette smoking, and endometrial cancer risk: results from the Netherlands Cohort Study\n","text":"Objective To examine the association between alcohol consumption, cigarette smoking, and endometrial cancer.\nIntroduction\nEndometrial cancer is the most frequently diagnosed gynecologic cancer in Europe [1]. The development of endometrial cancer has been related to exposure to estrogens unopposed by progestagens [2]. Many studies have shown a positive association between alcohol ingestion and estrogen levels in postmenopausal women. For instance, cross-sectional data from the European Prospective Investigation into Cancer and Nutrition (EPIC) suggested that elevated blood levels of estrone are observed with increasing alcohol consumption in postmenopausal women [3]. Thus, alcohol could be expected to increase endometrial cancer risk by elevating estrogen levels. An important determinant of estrogen levels in women is use of unopposed hormone replacement therapy (HRT). Accordingly, use of unopposed HRT is consistently associated with an increased risk of endometrial cancer [4\u20136]. Furthermore, it has been suggested that alcohol consumption increases estradiol levels in particular in postmenopausal women who are on HRT [7, 8]. However, previous studies have indicated that alcohol consumption is either weakly or not associated with a reduced risk of endometrial cancer and no significant interaction with use of HRT has been found [9]. Although several studies have analyzed the association between alcohol consumption and endometrial cancer risk [10\u201322], only few studies have examined the risk associated with various measures of alcohol consumption (e.g., amount and type of alcohol) including only one comprehensive prospective cohort study [18].\nIn contrast to alcohol, smoking has been hypothesized to exert anti-estrogenic effects [23] and to lower the risk of endometrial cancer in this way. Also, an effect modification by use of HRT seems reasonable [24]. Furthermore, it has been suggested that body mass index (BMI) and age at menopause might mediate part of the inverse association between smoking and endometrial cancer [24]. Earlier prospective studies [22, 25\u201329] have generally suggested that smoking is associated with a slight to moderate protection against endometrial cancer. However, to date, only the largest and most recent prospective cohort study has reported a significantly reduced risk in both current and past smokers [29]. Moreover, this large study explored the relationship between HRT and smoking and found no effect-modification, and also found that the association between smoking and endometrial cancer was not confounded by alcohol use [29]. The evidence from other epidemiological studies with regard to an effect-modification by use of HRT is ambiguous [24].\nAs many case\u2013control [24], but only a few cohort studies have reported on the association between smoking and endometrial cancer [22, 25\u201329], the International Agency for Research on Cancer concludes that prospective cohort studies, in which selection and recall bias are minimized, are scarce [30]. Only two cohort studies have explored the association between smoking and endometrial cancer comprehensively by examining the risk associated with all common quantitative smoking measures (e.g., smoking duration, time since cessation) [28, 29].\nSince only few prospective cohort studies have investigated the association between alcohol consumption, cigarette smoking, and endometrial cancer comprehensively, important features of this relationship are under-explored. Hence, we aim to provide additional evidence based on prospective data. Moreover, we intend to elucidate the hormonal mechanisms underlying endometrial carcinogenesis by investigating, first, whether BMI and age at menopause might act as intermediary variables in the association between smoking and endometrial cancer and by examining, second, whether there is evidence regarding a potential effect modification by HRT use.\nMaterials and methods\nThe Netherlands Cohort Study (NLCS) started in September 1986 when 62,573 women aged 55\u201369\u00a0years were enrolled in the cohort. Ethical approval was obtained from the ethics committee of the University Hospital Maastricht. All women were presumed to be postmenopausal. At baseline, data on dietary habits and other risk factors (such as alcohol consumption, smoking history, reproductive history, and anthropometry) were collected by means of a self-administered questionnaire. Data analysis was conducted according to the case\u2013cohort approach. In this approach, cases are derived from the cohort (providing numerator information for the incidence rates), while the accumulated person-years at risk of the cohort are estimated from a random sample from the cohort, i.e., the subcohort (providing denominator information for the incidence rates). Following this approach, a subcohort of 2,589 women was sampled after the baseline exposure measurement. The subcohort has been followed up biennially by mail for vital status information. The vital status of subcohort members, who did not respond was completed by contacting the municipal population registers. Incident cases occurring in the entire cohort were detected by annual record linkages to the Netherlands Cancer Registry and the nationwide network and registry of histopathology and cytopathology in the Netherlands (PALGA). Further details on the design of the study and methods of follow-up have been presented elsewhere [31, 32].\nThe present analysis is restricted to cancer incidence in the 11.3-year follow-up period from September 1986 to December 1997. The completeness of cancer follow up was estimated to be at least 96% [33], and no subcohort members were lost to follow up.\nThree hundred and twenty-seven incident, microscopically confirmed, invasive, primary endometrial carcinomas were detected after a follow-up period of 11.3\u00a0years. Cases were excluded from analysis if they had been diagnosed with non-epithelial tumors (n\u00a0=\u00a012), and if information on either alcohol consumption or cigarette smoking was incomplete (n\u00a0=\u00a035).\nWomen were eligible for the subcohort if they did not report at baseline that they had undergone hysterectomy. Application of this inclusion criterion yielded a subcohort of 2,229 members. Individuals were excluded from the analysis if they had been diagnosed with cancer other than skin cancer at baseline (n\u00a0=\u00a0151) and if information on either alcohol consumption or cigarette smoking was missing (n\u00a0=\u00a0177). After these exclusions, 280 cases and 1,901 subcohort members remained available for analysis.\nQuestionnaire data\nConsumption of alcoholic beverages during the year preceding the baseline interview was assessed by consumption frequency questions on beer, red wine, white wine, sherry, other fortified wine, liqueur, and liquor. Categories ranked from \u2018never\u2019 to \u20186\u20137\u00a0times per week\u2019 and information on the number of glasses per consumption day was also requested. Questionnaire data of all cases and subcohort members were key entered twice and processed in a manner blinded with regard to case\/subcohort status in order to minimize observer bias in the coding and interpretation of data. The questionnaire has been validated against a nine-day diet record [34, 35]. The Pearson correlation coefficient between the mean daily ethanol intake assessed by the questionnaire and that estimated by the nine-day record was 0.86 for all subjects and 0.78 for users of alcoholic beverages [34]. Respondents that reported to drink alcohol less than once per month were considered non-drinkers. Four items from the questionnaire (red wine, white wine, sherry, and liqueur) were combined into one single wine variable since these items were highly correlated and separate analysis would have resulted in small numbers of subjects within each stratum. Mean daily alcohol consumption was calculated using the Dutch food composition table [36]. Based on data from a pilot study, standard glasses were defined as follows: 200\u00a0ml for beer, 105\u00a0ml for wine, 80\u00a0ml for sherry, and 45\u00a0ml for both liqueur and liquor, corresponding to 8\u00a0g, 10\u00a0g, 11\u00a0g, 7\u00a0g, and 13\u00a0g of alcohol, respectively.\nSmoking was addressed at baseline by questions on age at first exposure to smoking, age at last exposure to smoking, smoking frequency, and smoking duration of cigarette, cigar, and pipe smokers. As the vast majority of smoking subcohort members was cigarette smokers, analyses were restricted to that particular group. Based on the questionnaire data, the following cigarette smoking variables were constructed: cigarette smoking status (never versus ever and never versus former or current), frequency (number of cigarettes per day), duration (years), age at first exposure (years), and time since cessation (years). Time since cessation was calculated as \u2018age at baseline\u2019 minus \u2018age at smoking cessation\u2019.\nConcerning the use of HRT, women were asked whether they had ever used HRT because of complaints related to the menopause. We can assume that all members of our cohort were postmenopausal in 1986, when the baseline questionnaires were completed, and that possible treatment with HRT took place prior to 1986 in most women. Based on information regarding HRT prescription in the Netherlands in the past [37\u201339], we assume that all HRT users enrolled in the NLCS were treated with unopposed oral estrogens.\nData analysis\nA variable was considered a confounder if (I) it was associated with endometrial cancer risk, if (II) it was associated with alcohol consumption or cigarette smoking, and if (III) age-adjusted hazard ratios changed by more than 10% after adjustment for the potentially confounding factor. Based on the literature [40] and previous analyses, we considered the following variables as potential confounders: age (continuous), age at menarche (continuous), use of oral contraceptives (ever versus never), duration of oral contraceptive use (continuous), age at first child birth (continuous), parity (continuous), age at menopause (continuous), use of postmenopausal hormones (ever versus never), duration of postmenopausal hormone use (continuous), non-occupational physical activity (categorized), BMI (continuous), height (continuous), energy intake (continuous), total fat intake (continuous), intake of saturated fat (continuous), intake of carbohydrates (continuous), intake of dietary fiber (continuous), intake of vegetables (continuous), intake of fruits (continuous), coffee consumption (yes versus no), education (categorized), diagnosis of hypertension (yes versus no), diagnosis of diabetes mellitus (yes versus no), family history of endometrial cancer (yes versus no), and if applicable: total alcohol consumption per day (continuous), type of alcoholic beverage (categorized), current smoking (yes versus no), number of cigarettes smoked per day (continuous), and duration of smoking (continuous).\nIncidence rate ratios (RR) and corresponding 95 percent confidence intervals (95% CI) for endometrial cancer were estimated in the age-adjusted and multivariate case\u2013cohort analyses with categorized and continuous alcohol and cigarette smoking variables, using the Cox proportional hazards model [41] processed with the Stata statistical software package [42]. Standard errors were estimated using the robust Hubert\u2013White sandwich estimator to account for additional variance introduced by sampling from the cohort. This method is equivalent to the variance\u2013covariance estimator by Barlow [43]. The proportional hazards assumption was tested using the scaled Schoenfeld residuals [44]. Tests for dose-response trends in risk of endometrial cancer were assessed by fitting ordinal exposure variables as continuous terms. Tests for interaction were performed by using the Wald test. Two-sided p values are reported throughout the paper.\nResults\nThe percentage of women reporting alcohol consumption was similar among cases and subcohort members (67.5% and 66.9%, respectively), as was the mean alcohol consumption per day among users in both groups (7.7\u00a0g with standard deviation (sd)\u00a0=\u00a010.8, and 8.5\u00a0g (sd\u00a0=\u00a010.4), respectively). Current smoking was less prevalent among cases than among subcohort members (15.4% vs. 21.8%), but the number of cigarettes smoked per day did not differ considerably between smokers in both groups (13.6 (sd\u00a0=\u00a08.4) and 13.2 (sd\u00a0=\u00a08.1), respectively).\nDrinkers reported a slightly higher age at menopause and a higher prevalence of both oral contraceptive use and current cigarette smoking than non-drinkers (see Table\u00a01). With regard to smoking status, former and current smokers were slightly leaner and had fewer children than never-smokers. On average, current smokers reported having reached menopause 1\u00a0year earlier than former-smokers and never-smokers. The prevalence of both oral contraceptive use and alcohol use was higher among smokers than among never-smokers. Also, average alcohol consumption was approximately twice as high among smokers as among never-smokers (see Table\u00a01).\nTable\u00a01Means (standard deviation) and distribution (n) of potential confounders according to alcohol consumption and cigarette smoking status among subcohort members, the Netherlands Cohort Study (1986\u20131995)\nCharacteristicUnitAlcohol consumption statusCigarette smoking statusNo (n\u00a0=\u00a0630)Yes (n\u00a0=\u00a01,271)Never (n\u00a0=\u00a01,100)Former (n\u00a0=\u00a0387)Current (n\u00a0=\u00a0414)Mean (sd)Mean (sd)Mean (sd)Mean (sd)Mean (sd)AgeYears61.8 (4.3)61.4 (4.3)62.0 (4.3)61.1 (4.4)60.7 (4.1)Body Mass Indexkg\/m225.4 (3.9)24.9 (3.4)25.3 (3.5)24.7 (3.3)24.6 (3.8)ParityNumber of children2.8 (2.4)2.7 (2.2)3.0 (2.4)2.5 (1.8)2.5 (2.1)Age at 1st child birthYears22.0 (11.2)21.9 (11.3)22.2 (11.2)22.3 (11.3)20.8 (11.3)Age at menopauseYears48.3 (4.8)49.2 (4.3)49.1 (4.4)49.2 (4.1)48.1 (4.8)Total energy intake (including alcohol)kcal1,628 (415)1,724 (386)1,694 (393)1,676 (406)1,705 (406)Alcohol consumption G\/day0 (0)8.5 (10.4)5.7 (7.8)10.4 (11.2)12.5 (12.8)CigarettesNo.\/day13.0 (8.8)11.1 (8.1)0 (0)9.8 (8.0)13.2 (8.1)n (%)an (%)an (%)an (%)an (%)aOral contraceptive useEver124 (20.0)336 (26.7)223 (20.6)127 (32.9)110 (26.8)Physical activity>30\u00a0min\/day425 (69.1)975 (77.8)796 (74.1)306 (79.5)298 (72.9)Diagnosis of hypertensionYes198 (31.4)351 (27.6)339 (30.8)107 (27.7)103 (24.9)Diagnosis of diabetesYes33 (5.2)40 (3.2)48 (4.4)13 (3.4)12 (2.9)Hormone replacement therapyEver62 (10.0)152 (12.1)107 (9.9)58 (15.1)49 (12.0)Family history of endometrial cancerYes20 (3.2)32 (2.5)23 (2.1)16 (4.1)13 (3.1)Alcohol usersYes\u2013\u2013663 (60.3)312 (80.6)296 (71.5)Currently smoking cigarettesYes118 (18.7)296 (23.3)\u2013\u2013\u2013a The percentage reported for some variables does sometimes not correspond with the numbers per smoker stratum since part of the information for these variables was missing\nBased on the literature and based on the methodological criteria specified above, we found the following confounders: age, BMI, parity, oral contraceptive use, non-occupational physical activity, hypertension, age at first child birth, and age at menopause. Alcohol consumption and cigarette smoking status were found to confound each other\u2019s association with endometrial cancer. We controlled for all these confounders in multivariate analyses. In additional analyses, we mutually controlled the age-adjusted risk estimates regarding qualitative smoking measures for the other smoking measures.\nThe multivariate risk estimates did not change substantially when oral contraceptive use (ever\/never) was replaced by duration of oral contraceptive use (data not shown). Accordingly, we considered it sufficient to control only for oral contraceptive use (ever\/never).\nTable\u00a02 shows the results for the association between alcohol consumption and endometrial cancer. The multivariate adjusted RR associated with alcohol consumption was 1.06 (95% CI\u00a0=\u00a00.78\u20131.43). The multivariate RRs of endometrial cancer for women who consumed up to 4, 5\u201314, 15\u201329, and 30 or more gram of alcohol per day versus non-drinkers were 1.09 (95% CI\u00a0=\u00a00.78\u20131.52), 0.95 (95% CI\u00a0=\u00a00.62\u20131.45), 0.94 (95% CI\u00a0=\u00a00.52\u20131.69), and 1.78 (95% CI\u00a0=\u00a00.88\u20133.60), respectively. No significant trend was observed (ptrend\u00a0=\u00a00.62).\nTable\u00a02Rate ratios of endometrial cancer according to baseline alcohol consumption in the Netherlands Cohort Study, 1986\u20131997Alcohol consumption (g\/ day)Age adjustedMultivariate adjustedCategorical medianCasesPerson-years in subcohortRR (95% CI)aCasesPerson-years in subcohortRR (95% CI)bTotal alcohol\u00a0\u00a0\u00a0\u00a0No0916,6411 (ref.)825,8371 (ref.)\u00a0\u00a0\u00a0\u00a0Yes4.018913,7461.01 (0.77\u20131.32)17212,1371.06 (0.78\u20131.43)\u00a0\u00a0\u00a0\u00a00.1\u201341.61147,5991.10 (0.82\u20131.48)1056,6431.09 (0.78\u20131.52)\u00a0\u00a0\u00a0\u00a05\u2013149.1473,6400.94 (0.65\u20131.37)393,2790.95 (0.62\u20131.45)\u00a0\u00a0\u00a0\u00a015\u20132920.9171,8220.69 (0.40\u20131.18)171,5750.94 (0.52\u20131.69)\u00a0\u00a0\u00a0\u00a0\u22653037.3116841.20 (0.62\u20132.34)116391.78 (0.88\u20133.60)\u00a0\u00a0\u00a0\u00a0p trend0.490.62Alcohol from wine\u00a0\u00a0\u00a0\u00a0Yes3.218213,0091.06 (0.81\u20131.37)16611,5071.13 (0.84\u20131.52)\u00a0\u00a0\u00a0\u00a00.1\u201341.51258,0721.17 (0.88\u20131.55)1127,1131.16 (0.84\u20131.59)\u00a0\u00a0\u00a0\u00a05\u2013148.9383,1460.91 (0.61\u20131.36)352,7841.07 (0.68\u20131.67)\u00a0\u00a0\u00a0\u00a0\u22651521.8191,7910.80 (0.48\u20131.35)191,6111.11 (0.64\u20131.93)\u00a0\u00a0\u00a0\u00a0p trend0.430.64Alcohol from beer\u00a0\u00a0\u00a0\u00a0Yes1.14291,8731.15 (0.76\u20131.74)261,6291.30 (0.82\u20132.07)Alcohol from liquor\u00a0\u00a0\u00a0\u00a0Yes3.7342,6480.93 (0.64\u20131.37)312,3491.11 (0.73\u20131.68)aRR\u00a0=\u00a0rate ratios; CI\u00a0=\u00a0confidence interval; n.a.\u00a0=\u00a0not applicablebRate ratios adjusted for age (years), body mass index (kg\/m2), parity (number of children), use of oral contraceptives (ever versus never), non-occupational physical activity (low, moderate, active, very active), hypertension (yes versus no), age at first child birth (years), age at menopause (years), and current cigarette smoking (yes versus no)\nThe multivariate RR of endometrial cancer for wine-consumers versus non-consumers was slightly, but non-significantly, elevated (RR\u00a0=\u00a01.13, 95% CI\u00a0=\u00a00.84\u20131.52). The RRs were also slightly, but non-significantly elevated when calculated according to different levels of wine consumption and no significant trend was observed (ptrend\u00a0=\u00a00.64). The multivariate RR of endometrial cancer associated with drinking beer equaled 1.30 (95% CI\u00a0=\u00a00.82\u20132.07) and the RR for drinking liquor was 1.11 (95% CI\u00a0=\u00a00.73\u20131.68).\nRegarding smoking, age-adjusted analysis revealed an inverse association between ever-smoking and endometrial cancer risk (RR\u00a0=\u00a00.70, 95% CI\u00a0=\u00a00.54\u20130.92, see Table\u00a03). We observed significant inverse trends of endometrial cancer risk with all quantitative smoking measures. However, these trends became non-significant when never-smokers were excluded. Some of these age-adjusted risk estimates changed substantially after additional adjustment for current smoking status, smoking frequency and smoking duration (see Table\u00a03).\nTable\u00a03Rate ratios of endometrial cancer according to baseline cigarette smoking features in the Netherlands Cohort Study, 1986\u20131997Cigarette smoking featuresCategorical medianAdjusted for ageAdjusted for age, current smoking status, frequency and duration of smokingAdjusted for all confoundersCasesPerson-years in subcohortRR (95% CI)aCasesPerson-years in subcohortRR (95% CI)bCasesPerson-years in subcohortRR (95% CI)cSmoking status\u00a0\u00a0\u00a0\u00a0Neverdn.a.18711,8721 (ref.)\u2013\u2013\u201316910,3301 (ref.)\u00a0\u00a0\u00a0\u00a0Ever smokers938,5160.70 (0.54\u20130.92)\u2013\u2013\u2013857,6440.71 (0.53\u20130.95)\u00a0\u00a0\u00a0\u00a0Neverd18711,8721 (ref.)\u2013\u2013\u201316910,3301 (ref.)\u00a0\u00a0\u00a0\u00a0Former smokersn.a504,1810.77 (0.55\u20131.07)\u2013\u2013\u2013473,7570.83 (0.58\u20131.20)\u00a0\u00a0\u00a0\u00a0Current smokersn.a434,3340.64 (0.45\u20130.91)\u2013\u2013\u2013383,8880.59 (0.40\u20130.88)\u00a0\u00a0\u00a0\u00a0p trend0.010.01Frequency (cigarettes\/day)\u00a0\u00a0\u00a0\u00a0Neverd018711,8721 (ref.)18711,8721 (ref.)16910,3301 (ref.)\u00a0\u00a0\u00a0\u00a00.1\u20139 4383,7190.65 (0.45\u20130.95)373,5690.86 (0.49\u20131.50)333,2231.07 (0.58\u20131.98)\u00a0\u00a0\u00a0\u00a010\u201319 12282,4380.74 (0.48\u20131.14)272,4151.02 (0.54\u20131.92)252,2141.28 (0.66\u20132.46)\u00a0\u00a0\u00a0\u00a020+20222,0140.70 (0.44\u20131.12)222,0141.03 (0.46\u20132.29)211,7981.31 (0.56\u20133.03)\u00a0\u00a0\u00a0\u00a0p trend0.040.750.43Duration (years)\u00a0\u00a0\u00a0\u00a0Neverd018711,8721 (ref.)18711,8721 (ref.)16910,3301.0 (ref.)\u00a0\u00a0\u00a0\u00a00.1\u201319 10.5232,0090.74 (0.47\u20131.17)221,9910.71 (0.42\u20131.18)211,8480.77 (0.43\u20131.39)\u00a0\u00a0\u00a0\u00a020\u20133930524,2740.79 (0.57\u20131.10)514,1200.80 (0.47\u20131.34)473,6900.89 (0.51\u20131.56)\u00a0\u00a0\u00a0\u00a040+411319700.42 (0.23\u20130.75131,8870.44 (0.19\u20131.02)111,6970.37 (0.15\u20130.90)\u00a0\u00a0\u00a0\u00a0p trend0.000.100.13Age at first exposure (years)\u00a0\u00a0\u00a0\u00a0Neverdn.a18711,8721 (ref.)18711,8721 (ref.)16910,3301 (ref.)\u00a0\u00a0\u00a0\u00a0<1917343,0980.71 (0.48\u20131.05)322,9470.78 (0.34\u20131.80)282,7240.91 (0.37\u20132.23)\u00a0\u00a0\u00a0\u00a019\u20132420212,5240.53 (0.33\u20130.86)202,3710.60 (0.29\u20131.27)202,1020.88 (0.38\u20132.02)\u00a0\u00a0\u00a0\u00a025+30312,6910.73 (0.49\u20131.09)312,5960.82 (0.47\u20131.46)282,3251.00 (0.53\u20131.87)\u00a0\u00a0\u00a0\u00a0p trend0.010.470.94Time since cessation (years)\u00a0\u00a0\u00a0\u00a0Neverdn.a18711,8721 (ref.)18711,8721 (ref.)16910,3301 (ref.)\u00a0\u00a0\u00a0\u00a020+e26.5201,1421.11 (0.68\u20131.84)171,0410.80 (0.41\u20131.57)179431.12 (0.52\u20132.41)\u00a0\u00a0\u00a0\u00a010\u201319e14141,4590.62 (0.35\u20131.09)141,4160.38 (0.14\u20131.03)131,2700.50 (0.17\u20131.45)\u00a0\u00a0\u00a0\u00a00.1\u20139e5131,3920.60 (0.33\u20131.08)131,3470.32 (0.10\u20131.06)121,2570.50 (0.12\u20132.00)\u00a0\u00a0\u00a0\u00a0p trend0.030.060.26aRR\u00a0=\u00a0rate ratios; CI\u00a0=\u00a0confidence interval; n.a.\u00a0=\u00a0not applicablebRRs were adjusted for age (years). In addition, frequency was adjusted for current smoking status (yes\/no) and duration (years); duration was adjusted for current smoking status and frequency (cigarettes\/day); age at first exposure was adjusted for current smoking status, frequency, and duration; time since cessation was adjusted for frequency and durationcRRs were adjusted as mentioned under footnote b and additionally for body mass index (kg\/m2), parity (number of children), use of oral contraceptives (ever versus never), non-occupational physical activity (low, moderate, active, very active), hypertension (yes versus no), age at first child birth (years), age at menopause (years), and alcohol consumption (gram\/ day)dNever smoked cigars, pipe, or cigaretteseEx-smokers only\nWhen we adjusted for all confounders, multivariate analysis showed a statistically significant 29% reduced risk of endometrial cancer for ever-smokers when contrasted with never-smokers. When considered separately, the risk reduction appeared to be stronger among current smokers (RR\u00a0=\u00a00.59, 95% CI\u00a0=\u00a00.40\u20130.88) than among former smokers (RR\u00a0=\u00a00.83, 95% CI\u00a0=\u00a00.58\u20131.20). Tests for trends were not significant for any of the quantitative smoking variables when these were adjusted for age and additional confounders.\nThe strongest reduction in risk, which could be observed in both univariate and multivariate models, was associated with a smoking history of 40 or more years compared with having never smoked (RR\u00a0=\u00a00.37, 95% CI\u00a0=\u00a00.15\u20130.90). Moreover, we observed a non-significant 50% reduction in risk in women that quit smoking either nine or less years ago or that quit 10\u201319\u00a0years ago. The data indicated no association between age at first smoking exposure and endometrial cancer.\nOmitting age at menopause and BMI from the multivariate models either separately or simultaneously did not cause meaningful changes in the corresponding estimates. No interactions in determining endometrial cancer risk could be observed between alcohol consumption and HRT use (p\u00a0=\u00a00.43), BMI (p\u00a0=\u00a00.38), age at menopause (p\u00a0=\u00a00.39), or current cigarette smoking (p\u00a0=\u00a00.83). When we stratified multivariate alcohol analyses according to smoking status, we observed a non-significantly lower risk of endometrial cancer among alcohol consumers that have ever smoked (RR\u00a0=\u00a00.91, 95% CI\u00a0=\u00a00.52\u20131.58) than among alcohol consumers that have never smoked (RR\u00a0=\u00a01.16, 95% CI\u00a0=\u00a00.81\u20131.67). Concerning smoking and HRT use, the interaction term was not statistically significant (p\u00a0=\u00a00.11). In this subset analysis, current smoking was associated with a reduced risk of endometrial cancer in women not using HRT (RR\u00a0=\u00a00.54, 95% CI\u00a0=\u00a00.35\u20130.84). In current smokers that did use HRT, the RR was 1.32 (95% CI\u00a0=\u00a00.57\u20133.04). Numbers were very small however: only 28 women were current smokers and used HRT and only eight women were current smokers and did not use HRT.\nDiscussion\nOur results do not suggest a meaningful association between alcohol consumption and endometrial cancer risk. Current smoking is associated with a reduced risk of endometrial cancer. This inverse relationship is neither mediated by BMI nor by age at menopause.\nRegarding the biological mechanism underlying endometrial carcinogenesis, the so-called \u201cunopposed estrogen hypothesis\u201d is widely accepted [2]. According to this hypothesis endometrial cancer develops when the endometrium is exposed to high levels of unopposed endogenous or exogenous estrogens for a long period of time. This exposure results in elevated mitotic proliferation of endometrial cells which, in turn, increases the risk of DNA replication errors and DNA mutations which can lead to endometrial cancer [2].\nAlthough female alcohol consumers could be expected to be at increased risk of endometrial cancer due to their hormonal profile, we have not detected significant associations between alcohol consumption and endometrial cancer risk. This finding is consistent with the vast majority of previous studies [13, 15\u201318]. However, in the EPIC study, significantly elevated blood estrone levels were found only in postmenopausal women who consumed more than 25\u00a0g of alcohol per day compared to non-drinkers [3]. Thus, possibly, a marked increase in estrone concentrations, and ultimately in endometrial cancer risk, can only be observed in women who consume more than moderate amounts. This notion might be supported by our data as we have observed an (non-significantly) elevated risk of endometrial cancer in women who reported to drink more than 30\u00a0g of alcohol per day. Based on literature reviews [7, 8], we hypothesized that we might find a positive association between alcohol consumption and endometrial cancer risk in particular among HRT users. However, our findings do not support the hypothesis of an effect-modification by HRT use; neither did most of the previous epidemiological studies [13, 17, 18].\nConcerning smoking, an anti-estrogenic effect has been suggested [23], which should lower the risk of endometrial cancer according to the unopposed estrogen hypothesis. Accordingly, our data indicated a significant risk reduction in current smokers, just like the Nurses\u2019 Health Study did, which has reported a RR of 0.72 (95% CI\u00a0=\u00a00.57\u20130.90) for current smokers [29]. In contrast, other cohort studies have observed non-significant associations [25, 26, 28]. We found that the inverse association between smoking and endometrial cancer was more pronounced among current smokers than among former smokers. These findings are in line with the evidence from several case\u2013control [17, 45\u201347] and two cohort studies [26, 28] and they suggest that the degree of protection might partly depend on the time since smoking cessation.\nAlthough not statistically significant, our prospective data support this notion, because we observed that endometrial cancer risk is possibly higher in women that quit smoking 20 or more years ago compared to the risk in women that quit 19\u00a0years ago or less. In the epidemiological literature, a few studies presented data on time since cessation [17, 28, 29, 48\u201350], but only one of them found a significantly lowered risk among women that quit smoking less than 10\u00a0years ago [48].\nConsidering the risk associated with duration of smoking, we have observed a reduction in risk with a long smoking history, just like previous studies [17, 29, 47, 48].\nWith regard to smoking intensity, we have found that smoking 20 or more cigarettes per day is associated with a non-significantly elevated risk of endometrial cancer. Though one needs to bear in mind that the corresponding confidence intervals were large, these point estimates are possibly not in line with the majority of previous studies, which generally indicated that a high smoking intensity (e.g., smoking 20 or more cigarettes per day) is associated with a decreased risk of endometrial cancer [17, 22, 28, 29, 45, 47, 48, 51]. This inconsistency could be explained by the lack of adjustment for other smoking variables in earlier studies: only one [29] out of six [22, 25\u201329] prospective cohort studies has adjusted its smoking frequency estimates for the potentially confounding effects of smoking duration. When we omitted smoking duration from our multivariate models, the RRs for smoking frequency also suggested an inverse association with endometrial cancer (data not shown).\nOur results do not indicate any important association between the age at starting smoking and endometrial cancer risk. Four previous studies [26, 28, 47, 49] found a (non-significant) risk reduction with young age at first exposure, that is, starting to smoke between age 15 [28] or age 20 or earlier [49], but none of these studies has reported a significant trend.\nIt has been hypothesized that smoking might lower the levels of estrogens partly by reducing the amount of fat tissue or by decreasing the age at menopause [24]. In our cohort, smokers were slightly leaner than non-smokers (24.6\u00a0kg\/m2 vs. 25.3\u00a0kg\/m2). Moreover, on average, current smokers appeared to have reached menopause 1\u00a0year earlier than never-smokers and former smokers (48.1\u00a0years vs. 49.1\u00a0years). However, in conclusion, our analyses indicated that BMI and age at menopause are no mediating factors.\nIn contrast to smoking, use of unopposed HRT increases endometrial cancer risk in postmenopausal women. Although an interaction between smoking and HRT use seems biologically plausible, the small numbers in our analysis did not allow to draw firm conclusions regarding a possible effect modification by HRT use. Moreover, we had no precise information on what type of HRT women in the NLCS have used. If we could have included such information in our analysis, results might differ according to type of HRT used.\nAnother potential drawback of our study might be misclassification of self-reported alcohol consumption and\/or self-reported cigarette smoking. However, these misclassifications might be non-differential owing to the prospective study design. Consequently, the risk estimates would probably be biased towards no effect. Moreover, the correlation between the alcohol consumption measured by the NLCS questionnaire and the measurement in a nine-day record was high due to the large variation in alcohol consumption [34].\nAn important strength of our study is that the exposures were assessed prior to the diagnosis of endometrial cancer. Therefore, our findings cannot be influenced by recall bias. Moreover, selection bias is unlikely as the follow-up of subcohort members and cases was almost complete [33, 52]. Another strength is the way alcohol consumption and cigarette smoking was assessed in the NLCS. The detailed assessment enabled us to evaluate associations between endometrial cancer and various measures of both exposures. Furthermore, we were able to control for confounding by the most important risk factors of endometrial cancer [40].\nTo sum up our major findings, we found that alcohol consumption is not associated with endometrial cancer. Current smoking was associated with a reduced risk of endometrial cancer in postmenopausal women. This association was probably not mediated by a decreased BMI or by an earlier age at menopause. Larger prospective studies with information on the type of HRT are needed in order to investigate possible effect modification by different types of HRT.\nPossibly, the incidence of endometrial cancer could be reduced if smoking was more common in female populations; however, such a reduction would be overshadowed by a dramatically increasing incidence of many other chronic diseases. Thus, individuals should still be encouraged to quit or not to start smoking.","keyphrases":["smoking","netherlands","cohort studies","alcohol drinking","endometrial neoplasms"],"prmu":["P","P","P","R","M"]}
{"id":"Med_Biol_Eng_Comput-4-1-2329731","title":"Decomposition cross-correlation for analysis of collagen matrix deformation by single smooth muscle cells\n","text":"Microvascular remodeling is known to depend on cellular interactions with matrix tissue. However, it is difficult to study the role of specific cells or matrix elements in an in vivo setting. The aim of this study is to develop an automated technique that can be employed to obtain and analyze local collagen matrix remodeling by single smooth muscle cells. We combined a motorized microscopic setup and time-lapse video microscopy with a new cross-correlation based image analysis algorithm to enable automated recording of cell-induced matrix reorganization. This method rendered 60\u201390 single cell studies per experiment, for which collagen deformation over time could be automatically derived. Thus, the current setup offers a tool to systematically study different components active in matrix remodeling.\nIntroduction\nThe extracellular matrix (ECM) provides a biophysical and biochemical environment for cell mechanical behavior. In turn, cellular interactions with the ECM resulting from adhesive, proteolytic and migratory activity govern continuous matrix reorganization. One such example occurs in eutrophic inward remodeling of small arteries [34]. Here, the existing collagen matrix is rearranged around a smaller diameter. Such inward remodeling is a hallmark of many hypertensive disorders and has been shown to have predictive value for cardiovascular events [9].\nIn vitro setups of cell-seeded matrix scaffolds allow studying the specific components that are active in vascular remodeling [22]. Especially, matrix reorganization can be monitored in the presence of specific cells, matrix elements, and blockers or markers of remodeling enzymes such as the matrix metalloproteases and transglutaminases [1, 2]. Several approaches using multi-cellular preparations have been developed. Thus, free-floating collagen gels can be seeded with contractile cells, and the remodeling is monitored from the dimensions of these gels [8, 13, 16, 20]. In the so-called culture force monitor, collagen gels mixed with cells are allowed to polymerize between a fixed plate and a force transducer. Contractile force development is measured during subsequent culture of this preparation [12, 14, 22, 36]. However, interpretation of data derived from these multi-cellular approaches is troubled by variability in cellular mechanical activity, lack of information on cell density, and synergistic contractile effects [30]. Therefore, microscopic observations on matrix reorganization by single cells may provide more detailed and fundamental information on the mechanisms of remodeling.\nSeveral groups have studied the effect of single cell activity on local deformation of matrices [5, 28]. The most frequently used substrates are silicone rubber [3, 6, 18, 21] and polyacrylamide sheets. Local deformation is then monitored by tracing the texture, sometimes facilitated by using fluorescent beads or imprinted micropatterns. Although these inert scaffolds provide a geometrical framework to study processes like cell locomotion, they do not resemble the physiological ECM. This has been solved in some cases by coating the polyacrylamide membrane with a thin layer of collagen [11, 17, 26, 27, 37]. However, remaining issues are the orientation of the collagen fibrils in the coating as compared to native collagen, and the inability of the cells to chemically modify (e.g. by cross-linking) the artificial sheets.\nA possible way to overcome these issues is the use of collagen-based matrices. Cell-induced deformation patterns of these matrices and the degree of reversibility of such deformation provide information on physical remodeling and the underlying biochemical processes. Deformations have been assessed by manually tracing beads or landmarks in consecutive images [6, 28, 30]. However, so far, only a few algorithms were developed for automatic detection of substrate deformations. In particular, very few of such algorithms allow tracing in the absence of embedded beads [23, 33, 35], and more stable algorithms are required.\nWhile single cell observations provide a more fundamental insight into matrix remodeling than macroscopic studies, a concern is efficiency. Time-lapsed video microscopy of single cells forms a labor-intensive and time-consuming experiment. Meaningful experiments require the comparison of large data sets, including different cell types, various matrix compositions, or the use of biochemical or molecular interventions. A fully automated, motorized microscopy setup is required that scans series of individual cells and their surrounding matrix. In addition, each individual cell should automatically be kept in focus during remodeling experiments, which take up 24\u00a0h or more. Finally, stable algorithms are needed for the automated analysis of geometrical reorganization with minimal user input.\nThe aim of this study is to develop an automated technique that can be employed to obtain and analyze local matrix remodeling by individual cells. The system that we present allows for monitoring of \u223c75 cells in parallel, using time-lapse video microscopy and computer-controlled stage positioning. In addition, we present and evaluate a new algorithm for automated detection of collagen matrix deformation around these cells.\nMethods\nCell culturing and collagen matrix preparation\nSmooth muscle cells, obtained from mesenteric small arteries, were cultured in Leibovitz medium with 10% (v\/v) heat-inactivated fetal calf serum. Cells from passages three to nine were used in experiments.\nMatrix constructs were produced from calf skin collagen (MP Biomedicals) at a concentration of 1\u00a0mg\/ml; pH was buffered by HEPES, and a mix of antibiotics (PSF and ciproxin) was added. Immediately after preparation at 4\u00b0C, the collagen mixture was poured into a 3.8\u00a0cm2 culture well and a 1.5-h polymerization period at 37\u00b0C was allowed. Then, SMCs were seeded at a concentration of about 1 cell per mm2 in the presence of 1\u00a0ml serum-free Leibovitz medium. The cells were maintained in an incubation chamber that was set to a temperature of 37\u00b0C throughout the experimental procedure. After a stabilization period of about 1\u00a0h, cell\u2013matrix interactions were monitored by microscopic imaging for a period of 24\u00a0h of spontaneous cell contraction.\nAutomated microscopic imaging\nIn order to enable simultaneous monitoring of cell-induced matrix remodeling at multiple locations, microscopy was combined with a motorized stage. Individual cells and their surrounding matrix were studied by phase-contrast microscopy (Olympus IMT-2 with 10\u00d7 objective and 2.5\u00d7 projection lens). Images were captured by a Qimaging Retiga SRV camera. The calibration factor for these images (1,392\u00a0\u00d7\u00a01,040\u00a0pixels) was 0.88\u00a0\u03bcm\/pixel. The microscopic field of view was set by a motorized stage, controlled by custom written software (Matlab 7.0 with Image Acquisition Toolbox 2.0). After manually determining and storing a set of x, y, z-coordinates for about 60\u201390 appropriate cells, these positions were tracked through time at a 15-min interval and images were captured by an automated procedure.\nDuring the time-lapsed image acquisition, samples were kept in focus by means of implementation into the acquisition software of one of the general auto-focus algorithms. Image contrast is optimal when a histogram of intensity values shows a broad distribution over all bins. This characteristic feature can be approximated by the standard deviation of pixel intensity values. For each image, contrast was enhanced by histogram equalization, and standard deviation was calculated. A normalized focus index (FI) was defined by dividing the standard deviation of the original image (SDoriginal) by the standard deviation of the contrast enhanced image (SDenhanced): For each cell, a series of images was captured at five different heights. The image was then defined to be in focus at the vertical level of maximal normalized focus. This height (z) was used as the central level when capturing the z-series in the next time step, thus allowing gradual vertical shift during the time-lapsed image acquisition. Finally, a stack of time-lapsed images was constructed for each x, y-position and analyzed off-line.\nGel dynamics analysis\nCell\u2013matrix interactions were quantified offline using in-house designed, automated image analysis software (Matlab 7.0 with Image Processing Toolbox 4.2). Matrix reorganization was assessed by calculation of the displacement field around a cell. This was achieved by performing a cross-correlation between each two successive images in an image stack; resolution of the displacement field was refined by correlation of subimages of decreasing size. This procedure is explained below and illustrated in Fig.\u00a01.\nFig.\u00a01Graphical representation of parameters used in matrix deformation analysis. The image shows a single SMC in the center, surrounded with a collagen matrix of relatively smooth texture. The white box (768\u00a0\u00d7\u00a0768\u00a0pixels) indicates the area for the most coarse correlation analysis. This correlation between two successive images was applied over an area as indicated by the yellow box (960\u00a0\u00d7\u00a0960\u00a0pixels). The green boxes show the more refined cross-correlation windows (stages 2\u20135). Table\u00a01 indicates the area expansion used for these correlation analyses\nFirst, gross displacement was defined at the point of maximal correlation between two parent images I\n1 (at t\u00a0=\u00a0t\n0) and I\n2 (at t\u00a0=\u00a0t\n0\u00a0+\u00a0\u0394t), using a correlation threshold of 0.5 and a maximum tested displacement of 11\u00a0pixels. Then, I\n1 was decomposed into four equal-sized square subimages, for which another cross-correlation was performed. The position of the correlation subwindow of I\n2 was refined using the displacement as calculated for the parent image, with a safety measure to prevent I\n2 crossing the borders of I\n1. This procedure of accurately positioning a correlation window not only reduced the possibility of an accidental cross-correlation match between a subimage of I\n1 and any random subimage of I\n2, but also drastically reduced calculation time. Resolution of the displacement field was refined to the fifth decomposition stage in our experiments (see Fig.\u00a01), using maximum tested displacements (n\ncc) as indicated in Table\u00a01. When insufficient correlation (r\u00a0<\u00a00.5) was found in any small area, displacement field from the next larger images were used. The calculated displacements were then assigned to the corresponding subimage centers. Subsequently, a continuous displacement field was obtained by bicubic interpolation. Finally, matrix compaction for each stack was quantified by tracking circular areas centered around the cell (see Fig.\u00a05).\nTable\u00a01Settings for matrix compaction analysis by decomposition cross-correlationDecomposition stageCross-correlation windowSearch area expansion (n\ncc)1768\u00a0\u00d7\u00a0768962384\u00a0\u00d7\u00a0384483192\u00a0\u00d7\u00a019224496\u00a0\u00d7\u00a09624\n5\n48\u00a0\u00d7\u00a048\n24\n624\u00a0\u00d7\u00a02412712\u00a0\u00d7\u00a0126Dimensions of the cross-correlation window I\n1 were reduced by 50% for each decomposition stage. The search area of I\n2 for correlation with I\n1 was obtained by increasing the cross-correlation window with n\ncc pixels on all image sides. The settings which were used for gel dynamics analysis and validation are given in bold\nValidation\nThe method described above (decomposition CC) was validated on several test series against a straightforward cross-correlation analysis (direct CC), with settings according to decomposition stage 1.\nThe first test case consisted of an image of a collagen-embedded cell, which was artificially resized by 3%, thereby simulating matrix compaction. Secondly, increasing amounts of white noise were added to the resized image in order to test the stability of both correlation methods. Relative dispersion (RD), which is defined as standard deviation divided by mean, was used as a noise level index. Finally, image resizing was followed by a horizontal translation of 60\u00a0pixels for a low and high noise example (see Table\u00a02).\nTable\u00a02Characteristics of validation images: CC was tested by addition of Gaussian white noise with mean 0.0 and increasing variance levels, in several cases the image was resized or translatedIndexScaling (%)Gaussian white noise varianceRD imageRD increase (%)RD noiseHorizontal shift (pixels)a10.00000.2500b0.970.00000.2510.50c0.970.00050.2604.10.1040d0.970.00100.2697.60.1460e0.970.00200.28514.10.2070f0.970.00300.30120.20.2530g0.970.00400.31626.30.2930h0.970.00500.32931.70.3270i0.970.00050.2594.10.10860j0.970.00300.30320.20.26560Relative dispersion (RD) in an image is defined as standard deviation divided by mean, RD increase is expressed as RD value compared to case \u201ca\u201d, RD noise is calculated as ratio of noise variance and image mean\nResults\nParallel recording of matrix compaction movies\nWe were able to record on average 66 movies on collagen compaction by single cells in parallel at a time resolution of 15\u00a0min (n\u00a0=\u00a010 experiments). Critical issues that limited this number were the need to avoid rapid acceleration and deceleration of the microscope stage, and the time-consuming autofocus algorithm. If cells were properly focused in the initialization stage, only a small fraction (<5%) ran out of focus during 24-h compaction experiments. Under the given incubation conditions, the majority of the cells adhered firmly to the collagen matrix and demonstrated little migratory activity. Typically, cell centroids remained within a 150\u00a0\u03bcm radius of their original xy position. z position decreased slowly in many cases, reflecting compaction of the matrix in the vertical direction (data not shown).\nValidation of the collagen compaction analysis\nAnalysis based on cross-correlation of images at a series of decomposition stages was compared with straightforward cross-correlation. This was performed on images simulating matrix compaction, subsequently followed by a challenge of increasing amounts of image noise and artificial translation.\nDecomposition CC was more time-consuming than direct CC: respectively, 84 and 54\u00a0s per image pair. This was due to a larger number of cross-correlations that have to be performed, and more complex data storage and lookup operations. For low noise levels (Table\u00a02, case c, d), both analysis methods render 100% correct displacement vectors (see Fig.\u00a02). When noise increased, the number of vectors in the smallest decomposition stage that had insufficient cross-correlation (r\u00a0<\u00a00.5) rose. However, this effect was smaller in the decomposition CC versus direct CC. As an example, the percentage correct displacement vectors at 20.2% noise (Table\u00a02, case f) was 77.7% in direct CC versus 89.5% in decomposition CC. Concurrently, the average displacement error at this noise level amounted to 2.1 and 0.7\u00a0pixels, respectively.\nFig.\u00a02Noise sensitivity of the cross-correlation methods. Top from left to right simulated displacement field of 3% compaction (Table\u00a02, case b), displacement field as predicted by direct CC, displacement field as predicted by decomposition CC; both cross-correlations were performed at a RD increase of 20.2% (Table\u00a02, case f); vectors were not scaled to absolute displacements, but to optimal visual illustration; red asterisks indicate vectors based on cross-correlation values below threshold (0.5) at the highest resolution. Middle the fraction of displacement vectors in the smallest decomposition stage that is calculated correctly (error in horizontal and vertical direction \u22641\u00a0pixel). This fraction decreased at higher noise levels (relative dispersion: Table\u00a02, case c\u2013h). Bottom the average error between calculated and simulated displacement vectors increased for higher noise levels (Table\u00a02, case c\u2013h). The decomposition method resulted in a slightly lower number of incorrect vectors, and a large decrease in displacement error\nFigure\u00a03 shows estimates for matrix compaction for these simulated deformations. The simulated compaction over a disc with 350\u00a0pixels radius was 6%. For noise levels up to 7.6% (Table\u00a02, case d), such compaction was indeed found by both correlation methods. At higher noise levels, direct CC underestimated compaction much more severely as compared to decomposition CC. As an example, after a noise increase of 20.2%, decomposition CC estimated a compaction value of 5.9% as compared to only 4.1% for direct CC.\nFig.\u00a03Calculated area at a radial distance of 350\u00a0pixels after a simulated 3% compaction. Increasing displacement errors, caused by higher noise levels, resulted in a mismatch between calculated area and simulated compacted area (94% of original). The decomposition method showed stable results at larger relative dispersion values (Table\u00a02, case c\u2013h)\nWhen an additional horizontal shift was imposed, both analysis methods correctly estimated compaction for 4.1% noise (Table\u00a02, case i). For higher noise levels, decomposition CC succeeded in a correct area assessment. In contrast, at 20.2% noise and 60\u00a0pixels translation (Table\u00a02, case j), direct CC predicted 1.2% rather than 6% compaction of the 350\u00a0pixels disc (see Fig.\u00a04). This effect was not due to more incorrect displacement vectors, but could be attributed to a rise in average displacement error up to 14.6\u00a0pixels.\nFig.\u00a04Sensitivity of the correlation methods to translation of the image. A horizontal displacement of 60\u00a0pixels was imposed after application of a 3% compaction and noise addition (4.1, respectively, 20.2% RD increase). Top left displacement field for high noise example (RD increase: 20.2%), as calculated by direct CC. Red asterisks indicate displacements where cross-correlation was below threshold (0.5); at these positions, a large mismatch between calculated compaction and simulated compaction occurs. Top right visual representation of matrix deformation at a radial distance of 270\u00a0pixels. Bottom calculated area remaining after a simulated compaction. At low noise levels (Table\u00a02, case i), a horizontal shift after image resizing did not affect the calculated area; for high noise (Table\u00a02, case j), failing correlation in the direct method results in a high offset in calculated area\nCollagen compaction by individual smooth muscle cells\nFigure\u00a05 shows an example of collagen matrix compaction by an individual smooth muscle cell. In this particular case, gross geometrical reorganization occurred in the first 6\u00a0h of the 22-h observation period. Compaction magnitude decreases with distance, and there was a time delay of around 1.5\u00a0h before compaction visible at a radius of 97\u00a0\u03bcm (110\u00a0pixels) became apparent at a distance of 308\u00a0\u03bcm (350\u00a0pixels).\nFig.\u00a05Typical example of collagen matrix compaction by individual smooth muscle cell as estimated by decomposition cross-correlation, obtained at a resolution of 1 frame per 15\u00a0min. Top left displacement field represented by vectors, refined in five decomposition stages. Vectors originating from a red asterisk were created at a larger correlation scale; for the smallest window, cross-correlation value at these positions was below threshold (0.5). Top right visual representation of matrix compaction at a radial distance of 350\u00a0pixels, as estimated from a series of displacement fields, showing an initial (yellow) circle and its deformed (green) state. Bottom estimation of compacted area after 22\u00a0h at distances of 110, 230 and 350\u00a0pixels (scaling: 0.88\u00a0\u03bcm\/pixel)\nDiscussion\nThis study aimed at developing an automated technique for obtaining and analyzing matrix remodeling by individual cells. Emphasis was put on construction of an automatic, reliable algorithm for assessment of a detailed matrix displacement field. Especially, refinement of a cross-correlation based image analysis with a decomposition scheme was investigated. While \u201cclassic\u201d direct CC sufficed for pairs of images with high correlation and low noise, this was no longer the case when substantial matrix remodeling occurred within the time frame between two consecutive images. This resulted in failing of CC at spots of high geometrical reorganization. However, when using decomposition CC, the gross displacements at these positions could be estimated by analysis of parent images with larger dimensions. This way, at a RD increase of 20.2% the average displacement error was lowered threefold in decomposition CC as compared to direct CC.\nThe method of refining displacement field accuracy with each decomposition step becomes progressively more important with larger displacements. Therefore we investigated the effect of a horizontal shift superimposed on a simulated compaction. Such shift occurred in our in vitro experiments when a group of neighboring cells pulled strongly on the matrix adjacent to a cell of interest. The shift interfered with direct CC because zero deformation was assumed when no proper correlation could be found. The result was an irregular displacement field (see Fig.\u00a04). When using decomposition CC, on the other hand, the gross displacement was already detected in the first decomposition stage. Displacement values were then adjusted when local compaction was detected in subsequent decomposition stages. Using this strategy, a lack of local correlation at the finest resolution resulted in only a minor displacement error, with an average of 0.9 as compared to 14.6\u00a0pixels for direct CC.\nBoth cross-correlation methods differ not only in stability of displacement field estimation, but in efficiency of calculation time as well. Direct CC requires a large search area, i.e. the subwindows of I\n2 have to extend considerably beyond the boundaries of their corresponding I\n1 subwindow in order to perform a meaningful correlation. On the other hand, with decomposition CC n\ncc can decrease with each stage (see Table\u00a01), since each subwindow of I\n2 is repositioned according to the preliminary displacement as calculated for its parent window. Due to the increased number of calculations and data manipulations that have to be performed, analysis time was still about 50% larger for decomposition CC at the settings as stated in Table\u00a01.\nWe chose a correlation threshold of 0.5 for both techniques, as well as cross-correlation windows as indicated in Table\u00a01. These values were empirically determined as an optimum in the trade-off between calculation time, false positive displacements and overlooking local deformations. Clearly, these choices depend on the contrast and texture of the images and the nature of the deformation, and will need to be optimized for specific future experiments.\nCell traction is frequently assessed by quantification of deformations in a flexible substratum. Both 2D and 3D approaches have been used, resulting in different cellular morphologies [19, 20, 29]. While the latter seems more physiological, interpretation of the 3D experiments is more complex [35]. 2D substrates offer more straightforward tools for analyzing mechanical behavior of single cells. These materials can be enriched with fluorescent microbeads to increase image contrast. In order to achieve a high resolution in the vicinity of cells, which can change their morphology rapidly, manual tracking of specific landmarks was employed in several studies [6, 28, 30]. However, this labor-intensive method inherently limits the number of cells under study. In several studies particle tracking was performed on individual microbeads [11]. In this case, resolution depends on particle distribution, which is typically far from homogeneous in collagen substrates [23].\nSeveral nested cross-correlation methods have been developed [35]. All of these algorithms are based on empirical determination of square size, distance for pattern search, and normalized CC threshold. In order to spatially limit the search region, a relative translational shift has been derived from image registration [23, 33]. Several schemes have been implemented to increase displacement field resolution in a step-by-step manner. In a single refinement cycle, Dembo and colleagues [26] decreased the subwindow size if significant displacement was observed. On the other hand, window size can be increased if correspondence failure occurs at high resolution [23]. The algorithm developed by Wang and coworkers [33] resembled our decomposition CC to a large extent. However, these authors aimed at construction of traction fields with a smooth nature by incorporation of filtering procedures at several stages.\nGeometrical matrix reorganization provides a qualitative index of the traction forces present in the underlying material. The actual forces can be derived from a displacement field series using material properties of the ECM construct. However, for collagen scaffolds these are highly heterogenous. Frequently, local stiffness is estimated by either microneedles [5, 10, 21, 30] or optical tweezers [4, 15, 32]. Tractions can then be calculated by application of stress\u2013strain relationships, including appropriate boundary conditions [7, 24, 25, 31, 37, 38]. However, this translation to quantitative traction forces lies beyond the scope of this article.\nIn conclusion, we presented integral methodology for the study of matrix remodeling. These techniques allow systematic screening of the role of matrix components such as collagen, elastin, fibronectin and laminin. Likewise, the function of stationary cells like smooth muscle cells, fibroblasts, and osteoblasts can be investigated. Our method can be applied under a wide variety of other experimental conditions. The single requirement for the image quality is a sufficiently high contrast in the material under investigation, without the neeed for laborious and potentially interfering micropatterning. Furthermore, our graphical user interface enables a flexible tuning of parameters such as number of decompositions, size of correlation window, search area and cross-correlation threshold. Using our motorized microscopy stage it is possible to patch individual images together in order to create one large field of view for the study of motile cells such as keratocytes. Finally, the method can be extended with fluorescence imaging of specific cell structures, cytokines, hormones and enzymes [15].","keyphrases":["cross-correlation","collagen","deformation","smooth muscle cell","matrix remodeling","microscopy","image analysis"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Osteoporos_Int-3-1-1894753","title":"Gender differences in the ratio between humerus width and length are established prior to puberty\n","text":"Summary On a sample of 1,317 children aged 9.9 years we developed a novel method of measuring humeral dimensions from total body dual-energy X-ray absorptiometry (DXA) scans and showed that gender differences in the ratio between humeral width and length are established prior to puberty.\nIntroduction\nTraditionally, the increased risk of osteoporotic fracture in women compared to men has been attributed to their low bone mass as a consequence of reduced peak bone mass acquisition and increased rates of bone loss following the menopause. However, more recent studies indicate that several other factors influence fracture risk independently of bone mass, such as skeletal geometry and the material properties of bone [1]. Furthermore there are gender differences in skeletal geometry that may contribute to the greater fracture risk in women compared to men. For example, long bone cross-sectional area is greater in men, which is thought to reflect higher rates of periosteal apposition from the time of puberty onwards [2]. One study of 68 girls and 59 boys aged 11.9\u00a0years, who underwent prospective peripheral quantitative computed tomography (pQCT) measurements, reported that periosteal growth was more rapid in pubertal boys compared to girls [3].\nSkeletal growth is coordinated to ensure that the ratio between different skeletal dimensions is maintained despite rapid changes in size [4]. Therefore, it is possible that the greater cross-sectional area in boys compared to girls is a reflection of their larger size. On the other hand, the ratio between periosteal growth and longitudinal bone growth may be different in boys compared to girls, leading to gender differences in bone shape. However, little is known of the inter-relationships between longitudinal and periosteal bone growth, since investigation of possible gender differences in skeletal geometry have generally been confined to analysis of cross-sectional area; in the absence of techniques capable of simultaneous measurement of long bone length, it has been difficult to accurately assess how the ratio between longitudinal and periosteal growth is affected by gender and other factors such as puberty.\nRecently, we developed a novel method for evaluating long bone geometry based on analysis of the humerus on total body dual-energy X-ray absorptiometry (DXA) scans. The humerus offers important advantages over other long bones in that its entire outline can readily be traced on total body DXA images, and its shape can be modelled as a cylinder with reasonable accuracy. To explore the utility of regional DXA analysis at the humerus, \u2018volumetric\u2019 bone density of the humerus was derived by dividing humeral bone mineral content (BMC) by estimated humeral cylindrical volume, and then analysed in relation to fracture risk. Interestingly, humeral volumetric bone density obtained in this way was indeed related to fracture risk, as analysed in a subgroup of 1,317 children from the Avon Longitudinal Study of Parents and Children (ALSPAC), in whom total body DXA scans were available at 9.9\u00a0years of age [5].\nIn the present study, we aimed to characterise the influence of gender and puberty on the ratio between longitudinal and periosteal growth, by exploiting this novel technique of humeral geometric analysis. In particular, we wished to determine whether, in analyses combining fracture and non-fracture controls from the study described above, gender differences exist in the ratio between width and length of the humerus, and if so whether these differences are established prior to puberty.\nMethods\nStudy population\nThe Avon Longitudinal Study of Parents and Children (ALSPAC) is a geographically based cohort that recruited pregnant women residing in Avon with an expected date of delivery between 1 April 1991 and 31 December 1992. A total of 14,541 pregnancies were initially enrolled, with 14,062 children born. This represented 80\u201390% of the eligible population\u2013see http:\/\/www.alspac.bris.ac.uk for further details [6]. Of these births, 13,988 were alive at 12\u00a0months. The population from ALSPAC used for this study consisted of 1,290 children: those who had DXA scans performed at aged 9.9\u00a0years, had measures of humeral dimensions performed and had data on pubertal stage available. Ethical approval was obtained from the ALSPAC Law & Ethics Committee, and Local Research Ethics Committees.\nMeasure of size at birth\nIn the immediate post-partum stage, whilst mother and child were still in hospital, trained ALSPAC staff measured crown-heel length with the Harpenden Neonatometer (Holtain Ltd., Crosswell, UK). Alternatively, this measure was collected from clinical records for babies who were not captured by the ALSPAC staff.\nMeasures of size at age 9.8 years\nChildren were seen in a research clinic at age 9.8\u00a0years (\u00b1\u20090.33\u00a0years). Parental consent and child\u2019s assent were obtained for all measurements made. Height was measured to the last complete millimeter (mm) using the Harpenden Stadiometer. Weight was measured to the nearest 50\u00a0grams (g) using the Tanita Body Fat Analyzer (model TBF 305, HealthCheck Systems, New York, USA). Total body less head (TBLH) bone area (cm2), TBLH bone mineral content (g), TBLH bone mineral density (g\/cm2), total body (TB) fat mass (kg) and TB lean mass (kg) were measured using a Lunar Prodigy dual-energy X-ray absorptiometer (Lunar Corp., Madison, WI, USA) on 7,444 children, using the default mode for all scans. The child was positioned carefully on the scanner and asked to lay their hands flat on the bed, palms down.\nMeasures of humeral dimensions\nThe present investigation was based on a subgroup of 393 children reporting fractures, for whom DXA scans were available at age 9.8\u00a0years, and an additional randomly selected group of 897 children, giving a total of 1,290 children. These children had originally been chosen for a study investigating the relationships between bone mass and fractures [5]. The measurer (EC) was blinded to the fracture status of the children. Customised settings were available on the Lunar Prodigy software and these were applied to the total body DXA image on screen. (No measures were performed during the DXA scan.) A region of interest (ROI) was drawn around the right humerus where possible (in case of movement artifact, the left humerus was used) after enlargement of the image to maximum magnification. The bone edge was detected visually with ease for the shaft and head of the humerus. At the distal end a straight line was drawn across the joint space from medial to lateral epicondyle, with the head of the ulna included within the humeral ROI. Where arm positioning was not ideal (such as palms not flat on the bed) the ROI was fitted as accurately as possible. The area (cm2) of the humeral ROI was recorded (Fig.\u00a01). Length of the humerus was obtained by use of an electronic ruler positioned between its upper and lower extremities. Average humeral width (cm) was calculated as area divided by length. The humeral aspect ratio (AR) was calculated as humerus width divided by length and then multiplied by 100, so the AR is the humerus width expressed as a percentage of humerus length.\nFig.\u00a01Total body DXA scan showing position of the region of interest (ROI) around the right humerus. a Total body scan image. b Image following maximal (i.e. fourfold) magnification, which was selected prior to placement of the ROI and measurement of length by electronic ruler\nThe precision of measurements of humeral geometry was calculated as the coefficient of variation (CV), based on ten scans with the measures repeated five times. The CV was 2.9% [95% confidence interval (CI): 2.1\u20133.7] for width, 1.5% (95% CI: 1.2\u20131.7) for length, and 3.2% (95% CI: 2.4\u20134.0) for humeral AR.\nOther measures\nThe mother\u2019s, partner\u2019s and grandparent\u2019s race and ethnic group and mothers\u2019 highest educational qualification were recorded at 32\u00a0weeks gestation as described elsewhere [7]. Gender was obtained from birth notifications. At the time of the DXA scan and measurement of the anthropometric variables, the child\u2019s age was calculated from the date of birth and date of attendance at the research clinic. Puberty was assessed by self-completion questionnaires using diagrams based on Tanner staging of pubic hair distribution for boys and girls, which we have previously found to show expected relationships with DXA measures in this cohort [8]. Prepuberty was defined as Tanner stage 1, and early puberty as Tanner stage 2.\nStatistical analyses\nResults from children reporting previous fractures and the randomly selected subgroup were pooled. Statistical analyses were performed with STATA 8.0. A two-tailed unpaired t-test was used to test the null hypothesis of no difference in the means for boys and girls. Linear regression was used to assess the associations between gender and humerus dimensions, which were adjusted for age on the day of the DXA scan and pubertal status. Additional analyses were performed following adjustment for TB fat mass and for fracture status.\nResults\nNo differences in gender, ethnicity, socio-economic status, body composition or humerus dimensions were found between the children with and without fractures (results not shown) so these results were pooled for all further analyses. Length at birth, age at DXA measurement, height, weight, TB fat and lean mass and TBLH bone area of the 1,290 children in whom humeral dimensions were measured are shown in Table\u00a01 according to gender and pubertal stage. There was no difference in age, height or weight between prepubertal boys and girls. However, boys had been longer at birth (by 0.3\u00a0cm on average), even after adjustment for gestational age and birth weight (P\u2009<\u20090.001). Prepubertal girls had a greater TB fat mass (P\u2009<\u20090.001), whereas prepubertal boys had a greater TB lean mass (P\u2009<\u20090.001). Puberty-related differences in size measures were also seen. For example, girls in early puberty (Tanner stage 2) were on average 4.4\u00a0cm taller and 5\u00a0kg heavier than prepubertal girls (Tanner stage 1). Boys\u2019 pubertal stage showed similar trends to those observed in girls, but differences in size measures were considerably smaller. \nTable\u00a01Mean age, height, weight, and DXA-derived total body fat mass and lean mass, and total body less head bone area for 648 boys and 642 girls with measurements of humeral size and dimensionsa\u00a0BoysGirls\u00a0Prepubertal, N\u2009=\u2009551Early pubertal,N\u2009=\u200997P value for difference between boys in pre- and early pubertyPrepubertal, N\u2009=\u2009548Early puberty, N\u2009=\u200994P value for difference between girls in pre- and early pubertyP value for difference between prepubertal boys and girlsP value for difference between boys and girls in early pubertyMeanSDMeanSDMeanSDMeanSDBirth length (cm)b50.91.550.91.50.99550.61.550.71.50.658<0.0010.316Age at DXA (years)9.80.39.90.30.6159.80.39.80.30.9460.6310.602Height (cm)139.46.1140.76.10.041138.76.0143.16.0<0.0010.0810.011Weight (kg)34.17.134.97.10.30334.27.339.27.3<0.0010.795<0.001TB fat mass (kg)7.34.97.74.90.4719.34.912.15.0<0.001<0.001<0.001TB lean mass (kg)25.33.025.63.00.31423.32.825.22.8<0.001<0.0010.332TBLH bone area (cm2)1,1411541,1611550.2611,1131571,217158<0.0010.0020.002aResults are shown separately for boys and girls, who are further subdivided according to results of Tanner stage self-completion questionnaire. P values shown are for the difference between prepubertal boys (Tanner stage 1) and boys in early puberty (Tanner stage 2); for the difference between prepubertal girls (Tanner stage 1) and girls in early puberty (Tanner stage 2); for the difference between prepubertal boys and girls; and for the difference between boys and girls in early puberty, all calculated by an unpaired Student\u2019s t-test. For TB fat mass, TB lean mass and TBLH bone area results are adjusted for age at DXA measurement, by linear regression.bBirth length adjusted for gestational age and birth weight by linear regression.\nWe then examined relationships between dimensions of the humerus, age, height and puberty. As expected, height was positively related to humerus, width and length based on analyses in boys and girls combined (P\u2009<\u20090.001). In spite of the relatively narrow age range of our study population, a positive association was also observed between age and width and length of the humerus (P\u2009<\u20090.001). In contrast, age and height were not related to humeral AR. Similar results were seen when boys and girls were analysed separately. Girls in early puberty had greater humeral width, length and area compared to prepubertal girls, but humeral AR in these two groups was similar (Table\u00a02). In contrast, no differences were observed in any measure of humeral geometry between pre- and early pubertal boys. \nTable\u00a02Humeral dimensions in prepubertal boys, boys in early puberty, prepubertal girls and girls in early pubertya\u00a0BoysGirls\u00a0Prepubertal, N\u2009=\u2009551Early pubertal, N\u2009=\u200997P value for difference between boys in pre- and early pubertyPrepubertal, N\u2009=\u2009548Early puberty, N\u2009=\u200994P value for difference between girls in pre- and early pubertyP value for difference between prepubertal boys and girlsP value for difference between boys and girls in early pubertyMeanSDMeanSDMeanSDMeanSDLength (cm)24.71.425.01.40.15924.91.425.91.5<0.0010.026<0.001Width (cm)1.920.21.930.20.6091.880.21.930.20.002<0.0010.985AR (%)7.780.77.760.70.6997.530.67.470.60.390<0.0010.006Area (cm2)47.76.048.46.10.29746.965.950.265.9<0.0010.0230.052aResults show mean and standard deviation for humeral length, width, aspect ratio (AR) and area. P values shown are for the difference between prepubertal boys (Tanner stage 1) and boys in early puberty (Tanner stage 2); for the difference between prepubertal girls (Tanner stage 1) and girls in early puberty (Tanner stage 2); for the difference between prepubertal boys and girls; and for the difference between boys and girls in early puberty, all calculated by an unpaired Student\u2019s t-test. All analyses are adjusted for age at DXA measurement by linear regression.\nWe then investigated the effects of gender on measures of humeral geometry according to pubertal stage, following adjustment for age of DXA scan. The humerus of prepubertal boys was slightly shorter (on average 0.2\u00a0cm), but of greater width (average of 0.04\u00a0cm) and area (on average 0.8\u00a0cm2), compared to prepubertal girls, as a result of which humeral AR was greater in prepubertal boys (an average of 3.2% greater, P\u2009<\u20090.001) (Table\u00a02 and Fig.\u00a02). Boys in early puberty still had a shorter humeral length, but a similar humeral width and a smaller area than girls in early puberty, as a result of which humeral AR remained greater in early pubertal boys (an average of 3.7% greater, see Fig.\u00a02). Similar gender differences were seen after adjustment for TB fat mass and fracture status (results not shown).\nFig.\u00a02Associations between humeral geometry, gender and puberty, as determined in 1,290 boys and girls. Figure shows mean\u2009\u00b1\u2009SD a humerus width (cm), b humerus length (cm) and c humerus aspect ratio (AR) according to gender and Tanner stage of puberty. P values are for the difference between boys and girls. All analyses are adjusted for age\nDiscussion\nHumeral width and length were positively related to age and height in boys and girls combined, and to pubertal status in girls, in this contemporary cohort of pre- and early pubertal children. These observations are similar to those previously reported for other DXA-derived measures of bone size in this cohort, such as TBLH and spinal bone area [7]. In contrast, age, height and pubertal status did not influence the ratio between humeral width and length, as reflected by humeral AR, presumably reflecting the action of mechanisms to ensure that skeletal shape remains constant as bones grow. In both pre- and early pubertal boys, humeral length was found to be shorter compared with girls, whereas humeral width was similar or greater, resulting in a greater humeral AR in boys. Taken together, these findings suggest that gender, but not puberty, affects the balance between periosteal and longitudinal growth. Hence, differences in overall skeletal shape between boys and girls appear to be established prior to puberty. However, from the present study we are unable to determine whether the AR changes during later pubertal stages, or during subsequent ageing.\nOur observation that Tanner stage was found to affect humeral geometry in girls but not boys presumably reflects the fact that boys and girls in Tanner stage 2 are not equivalent in terms of skeletal development. The finding that height and weight differences between Tanner stages 1 and 2 were considerably greater in girls compared to boys is consistent with this view. Therefore, analyses of differences in humeral geometry between Tanner stage 2 boys and girls may have limited validity, since these may not have fully accounted for gender differences in skeletal maturity that are likely to have been present. Nevertheless, since humeral AR was unaffected by Tanner stage in the age of children studied, these reservations are unlikely to affect the main conclusion from this study, namely that humeral AR is greater in boys compared to girls as assessed at age 9.9\u00a0years.\nThe gender differences in skeletal shape shown in Table\u00a02 (approximately 2\u20133%) were smaller than those observed in fat mass, lean mass and bone area as in Table\u00a01 (ranging from 7 to 27%). The larger gender differences in TBLH bone area (6.5%) compared with humeral area (2.7%) suggest that greater gender differences in bone area are present at other skeletal sites. Consistent with this conclusion, vertebral body size has been reported to be 11% [8] and 15% [9] larger in boys compared to girls. Our observation of an 8% gender difference in lean mass and 27% difference in fat mass, compared to the 1% gender difference in humeral length and 2% difference in width, perhaps reflects the strength of association between fat or lean mass and bone area [10].\nOur conclusion that gender differences in humeral shape are established prior to puberty is supported by a previous study in which greater humeral width was seen in prepubertal boys compared to girls, based on radiogrammetry [11]. In the present study, the humerus was selected as the most suitable site for providing an accurate measure of aspect ratio by analysis of total body DXA scans. Although other skeletal sites were not evaluated, we assume that equivalent gender differences in periosteal relative to longitudinal growth are established prior to puberty throughout the appendicular skeleton. Consistent with this suggestion, the metacarpals and proximal radius have been found to be wider in boys compared to girls at all stages of development, as assessed by analysis of radiographs [12, 13]. Furthermore, in a recent study of 128 boys and girls, boys had higher rates of periosteal expansion relative to girls, as measured prospectively over 20\u00a0months at the radial midshaft by pQCT, and this gender difference was similar in early, peri- and postpubertal children [3]. In a recent analysis of 18-year-old males and age-, height- and weight-matched females, long bone width was found to be greater at the hip and distal tibia as measured by DXA and pQCT, respectively, in boys compared to girls [14].\nAlthough our results suggest that gender differences in long bone width are due to more rapid periosteal apposition relative to longitudinal growth in boys prior to puberty, the precise timing of this gender effect is currently unclear. Review of the literature shows no evidence of gender differences in forearm bone width at birth [15], but studies of preschool and older children show conflicting results: Specker et al. [16] report no gender differences in radius width in children aged 1\u20136\u00a0years (based on 89 children), whereas Tanner et al. [17] found that the humerus is wider in boys compared to girls from age 3\u00a0years until the time of pubertal growth acceleration in girls (based on 505 children aged 3\u201318\u00a0years). It is also possible that the method used to measure bone size influences whether gender differences are found. For example, studies described above that used radiographs or pQCT identified gender differences [3, 11\u201313, 17], whereas those that used single photon absorptiometry did not [15, 16].\nPrepubertal gender differences in the relative rates of longitudinal and periosteal growth that we observed may be mediated by alterations in endocrine factors. For example, prepubertal girls have higher levels of insulin-like growth factor I, estradiol and testosterone concentrations compared to prepubertal boys [18], all of which are known to have effects on both longitudinal and periosteal bone growth. In terms of the potential influence of these differences on fracture risk, according to beam theory, columns with larger aspect ratios (i.e. ratio of width to length) have a reduced fracture risk than columns with smaller aspect ratios [19]. In addition, the ratio between periosteal diameter to long bone length provides an approximate estimate of critical buckling load, such that a lower aspect ratio results in a long bone which is more prone to failure by buckling [20]. Furthermore, in children, the majority of fractures occur at the distal forearm and can be divided into two main types: simple torus fractures and the \u2018greenstick\u2019 variety, both of which are associated with buckling or bulging on the side of the bone in compression [21].\nTherefore, theoretically, measurement of the ratio between long bone width and length from total body DXA scans as described here may provide an in vivo method for evaluating biomechanical strength of the skeleton. However, against this suggestion, boys have a higher fracture risk than girls in childhood [22], whereas girls have a smaller humeral AR. Furthermore, we found no relationship between humeral AR as measured in the present study and fracture risk [5]. One possible explanation for this lack of association is that our assumption that the humerus is cylindrical ignores gender differences in shape at the epiphysis or metaphysis which might contribute to fracture risk. On the other hand, humeral AR may predict fracture risk in certain adult populations, in view of evidence that bone width has previously been reported to be related to stress fractures in soldiers [23]. In light of our results, which suggest that humeral AR can be evaluated with relatively good precision, further studies are justified to determine whether this parameter represents a novel bone mineral density (BMD)-independent risk factor for upper limb fracture.\nThe measure of humeral length from which we derived humeral AR is likely to be relatively accurate, since the upper and lower ends of the humerus are generally clearly visible on total body DXA scans (see Fig.\u00a01). Alternative measurement techniques, such as pQCT, offer advantages over the approach described here, by measuring bone diameter directly, but do not provide a measure of bone length. Another limitation of the present study is that unlike girls, age 9.9\u00a0years appeared to be too young to evaluate possible effects of early puberty on skeletal development in boys. In future studies, we plan to repeat these analyses in older boys to confirm that as in girls, puberty increases humeral width and length whilst having no effect on humeral AR.\nIn conclusion, we have found that long bone shape, as reflected by humeral AR which we derived using a novel technique from total body DXA scans, is unaffected by age, height and puberty, as evaluated in a child cohort of relatively narrow age range and range of Tanner stages. This finding suggests that the ratio between longitudinal and periosteal growth is controlled to ensure it remains constant during rapid growth. However, humeral AR was related to gender, suggesting that the greater periosteal diameter of boys compared to girls, which is well recognised, is a consequence of gender differences in the balance between longitudinal and periosteal bone growth. Interestingly, these gender differences in humeral AR were present in prepubertal children, possibly resulting from prepubertal differences in sex hormone levels. Further studies are justified to determine whether humeral AR is an important determinant of biomechanical strength and fracture risk, particularly in adult populations.","keyphrases":["gender differences","epidemiology","growth and development","long bone dimensions","population studies"],"prmu":["P","U","R","R","R"]}
{"id":"Surg_Radiol_Anat-3-1-1820760","title":"Scapular deformity in obstetric brachial plexus palsy: a new finding\n","text":"While most obstetric brachial plexus palsy patients recover arm and hand function, the residual nerve weakness leads to muscle imbalances about the shoulder which may cause bony deformities. In this paper we describe abnormalities in the developing scapula and the glenohumeral joint. We introduce a classification for the deformity which we term Scapular Hypoplasia, Elevation and Rotation. Multiple anatomic parameters were measured in bilateral CT images and three-dimensional CT reconstruction of the shoulder girdle of 30 obstetric brachial plexus palsy patients (age range 10 months\u201310.6 years). The affected scapulae were found to be hypoplastic by an average of 14% while the ratio of the height to the width of the body of scapula (excluding acromion) were not significantly changed, the acromion was significantly elongated by an average of 19%. These parameters as well as subluxation of the humeral head (average 14%) and downward rotation in the scapular plane were found to correlate with the area of scapula visible over the clavicle. This finding provides a classification tool for diagnosis and objective evaluation of the bony deformity and its severity in obstetric brachial plexus palsy patients.\nIntroduction\nThe most commonly injured brachial plexus nerve root in obstetric injuries is the fifth cervical (C5) root. The supplied shoulder girdle musculature is therefore most commonly affected and indeed shoulder deformities comprise the most significant problems in this injury [2]. Within the context of shoulder deformity, the medial rotation contracture (MRC) deformity results in the greatest morbidity. The pathophysiology of deformity has been known for over 100 years: paralysis of the shoulder abductors and external rotators leads to relative dominance of already strong internal rotators of the shoulder, with eventual clinically observed changes of arm posture. The imbalance causes progressive deformity in the growing shoulder which may progress to a fixed medial rotation position of the humerus as well as posterior subluxation of the humeral head [3]. This constant position of the shoulder has a deleterious effect on glenohumeral development [9, 14, 17] and is accompanied by a characteristic flexion and MRC of the shoulder. Furthermore, scapular growth is impaired compared to the normal side [12]. The patients exhibit a persistent elbow-bent posture, pronation position of the forearm, and apparent shortening of the arm (Fig.\u00a01). In movement, there is loss of supination due to the abnormally situated arm in medial rotation, obvious elbow flare when the biceps is flexed, the \u201cbugler\u2019s position\u201d, and awkward external rotation. This secondary structural shoulder deformity develops early and may persist despite improvement in neurological status [14].\nFig.\u00a01At rest the child is noted to exhibit an internal rotation posture at the shoulder with elbow flexion and pronation of the forearm. There is apparent shortening of the arm\nThere have been previous radiologic demonstrations of scapular deformity as a sequela of obstetric brachial plexus palsy using plain radiographs or computed axial tomography were used, but attention was directed solely to the glenoid fossa and adjacent structures, without evaluating the body, spine and acromion process [8, 11, 12, 17].\nIn the current study, we describe dysplasia and malposition of the entire scapula in obstetric brachial plexus palsy patients. We introduce a classification of this deformity which we term collectively Scapular Hypoplasia, Elevation and Rotation (SHEAR).\nMethods\nPatients\nA total of 30 obstetric brachial plexus palsy patients with glenohumeral internal rotation were evaluated in the past 6\u00a0months. We reviewed the clinical data on these patients and diagnosed and classified the scapular elevation according to the results of clinical examination and analysis. There were 10 boys and 20 girls ranging in age from 10\u00a0months to 10.6\u00a0years. Three of the children had not undergone any surgery. Twenty-four of the children had undergone latissimus dorsi and teres major muscle transfer, subscapularis, pectoralis major and minor contracture releases and axillary nerve decompression and neurolysis for correction of abduction at our institute in the past. Twelve of the latter group and one that had not undergone the aforementioned procedure had undergone primary neurological repair surgery.\nMeasurements\nTransverse CT section and three-dimensional reconstructions of bilateral computerized tomograms (3D-CT) were used in the evaluation process. Trunk anterior and trunk posterior views of the whole shoulder girdle as well as superior trunk outlet and scapular posterior and medial views were examined. The contralateral scapulae were assessed for comparison.\nThe height (H) of the scapula was measured on the scapular medial view as the length of the medial border between the superior angle and the inferior angle. Width was measured along the spine of the scapula on the scapular posterior view. The total width (W) was measured from the lateral end of the acromion to the most medial part of the scapula. Width of the body of the scapula (w) was measured as the distance from the glenoid to the most medial part of the scapula (Fig.\u00a02a).\nFig.\u00a02Measurement of the different scapular variables. a The width is assessed in the posterior scapular view (left) along the axis of the scapular spine. The total width (W) is calculated as the length from the end of the acromion to the most medial aspect of the scapula. The scapular body width (w) is calculated as the distance from the mid glenoid to the most medial aspect of the scapula. The acromion length (acr) is also calculated on this view. The height (H) is measured along the medial border of the scapula in the medial scapular view (right). b Lines are drawn from the center of the glenoid cavity perpendicular to the vertebral axis line on the posterior trunk view. Superior displacement\u00a0=\u00a0A\/H. Downward\/upward rotation around an axis perpendicular to the scapular plane is defined as the difference between the angles \u03b1 defined by the vertebral axis and the extension of the lines connecting the center of the glenoid cavity and the medial end of the spine of the scapula. c In the anterior trunk view three lines are drawn: Line 1 is the line between the sternoclavicular joint and the center of the acromio-clavicular joint, line 2 is the line between the center of the acromio-clavicular joint and the inferior angle of the scapula, line 3 is the vertebral axis. The superior scapular angle \u03b3 is the angle defined between lines 1 and 2 and the inferior scapular angle \u03b4 is the angle between lines 2 and 3. d In the superior outlet view the anterior\/posterior rotation is calculated as the angle \u03d5 between the line connecting the acromio-clavicular joint to the superomedial angle and the line between the sterno-clavicular joint and the center of the acromio-clavicular joint (clavicular axis). e Schematic drawing showing the method of calculating glenoscapular angle (glenoid version \u03b8), posterior subluxation of the humeral head and spinoscapular angle (\u03b6). The scapular line that connects the medial aspect of the scapula and the mid glenoid is drawn. A second line is drawn connecting the posterior and anterior margins of the glenoid. 90\u00b0 are subtracted from the angle of the posterior medial quadrant defined by these lines to determine the glenoid version \u03b8. A line perpendicular to the scapular line is drawn and the percentage of posterior subluxation is defined as the ratio of the distance from the scapular line to the anterior portion of the head to the diameter of the humeral head (LM\/LN\u00a0\u00d7\u00a0100). The spinoscapular angle (\u03b6) is measured as the angle between the scapular line and the medial plane\nVertical displacement was measured on the trunk posterior view with reference to the contralateral side. Lines were drawn from the center of the glenoid cavity perpendicular to the vertebral axis line. Vertical displacement is the percentage of the difference between the levels of the two glenoids (A) divided by the height of the contralateral scapula (H). Positive sign denotes superior displacement and negative sign inferior displacement (Fig.\u00a02b).\nIn order to define the rotation of the affected scapula on an axis perpendicular to the scapular plane (downward\/upward) we calculated the angle (\u03b1) between the extension of the line connecting the mid glenoid to the base of the spine of the scapula and the vertebral axis line (Fig.\u00a02b). We also calculated two angles between three lines forming a triangle: Line 1 was drawn between the sterno-clavicular joint and the center of the acromio-clavicular joint, and line 2 between the center of the acromio-clavicular joint and the inferior angle of the scapula. Line 3 was the vertebral axis line. The superior scapular angle (\u03b3) is the angle defined between lines 1 and 2 and the inferior scapular angle (\u03b4) is the angle between lines 2 and 3 (Fig.\u00a02c). The rotation on the axis of the scapular spine (anterior\/posterior) was assessed on the superior outlet view by measuring the angle (\u03d5) of convergence of the longitudinal lines of the shaft of the clavicle and the line connecting the superomedial angle of the scapula to the acromio-clavicular joint (Fig.\u00a02d).\nThe total area of the scapula was calculated on the scapular posterior view. The area of the scapula visible on the anterior view above the clavicle with reference to the contralateral side was measured on the trunk anterior view. For distance and area measurements, graphic software (Universal Desktop Ruler, AVPSoft.com) was used.\nOn a transverse CT image at the mid-glenoid level a scapular line was constructed that connected the medial margin of the scapula to the middle of the glenoid fossa. The glenoscapular angle (glenoid version) was measured according to Friedman et al. [5] as the angle between the scapular line (connecting the scapular medial margin to the middle of the glenoid) and the line connecting the base of the anterior labrum and posterior labrum. By definition 90\u00b0 are subtracted from the posteromedial quadrant angle to define the glenoscapular angle (\u03b8) (Fig.\u00a02e). The same scapular line is used to determine the degree of humeral head subluxation. The greatest diameter of the head was measured (LN) as the distance of the scapular line to the anterior portion of the head (LM). The percentage of subluxation was calculated as the ratio of these distances multiplied by 100 (Fig.\u00a02e). Rotation about an approximately vertical axis (internal\/external) was assessed by measuring the spinoscapular angle (\u03b6) between the scapular line and the median plane.\nBoth the affected and contralateral sides were assessed and the values discussed take into account comparison between sides unless specifically indicated.\nStatistics\nDescriptive statistics were calculated for each variable, including mean, standard deviation and range. Comparisons of means for continuous variables were performed by the Pearson product correlation coefficients and the paired Student t test with Microsoft Excel 2003 software (Microsoft, Redmond, WA, USA). P values were two-tailed, and P values of <0.05 were considered to be significant.\nResults\nThe patients were noted to have unilateral scapular elevation as a result of downward rotation to various extents. The 3D-CT data for the patients as well as the significance value between affected and contralateral side and the correlation coefficient of each variable to the area of affected scapula visible above the clavicle are summarized in Table\u00a01. The area of scapula visible above the clavicle significantly correlated with downward rotational deformity (r\u00a0=\u00a0\u22120.65), affected to contralateral height ratio (r\u00a0=\u00a0\u22120.55), ratio of affected acromion to total width (r\u00a0=\u00a065), ratio of affected acromion to height (r\u00a0=\u00a00.85), affected to contralateral acromion length (r\u00a0=\u00a00.56), affected to contralateral area (r\u00a0=\u00a0\u22120.57), superior scapular angle (r\u00a0=\u00a0\u22120.84), inferior scapular angle (r\u00a0=\u00a00.83) and subluxation (r\u00a0=\u00a0\u22120.80).\nTable\u00a01Details of the computed tomography data on the patientsAffectedContralateralAffected to contralateralPraMeanRangeMeanRangeMeanRangeHypoplasiaAcromion as percentage of widthacr\/W\u00a0\u00d7\u00a010035.6\u00a0\u00b1\u00a05.6%24.4 to 48.9%32.4\u00a0\u00b1\u00a02.4%27.8 to 37.5%1.10\u00a0\u00b1\u00a00.170.70 to 1.58<0.0000.57Acromion as percentage of heightacr\/H\u00a0\u00d7\u00a010036.0\u00a0\u00b1\u00a06.6%22.4 to 55.6%30.5\u00a0\u00b1\u00a03.0%24.4 to 35.7%1.19\u00a0\u00b1\u00a00.240.79 to 1.73<0.0000.71Affected acromion\/control acromionacr\/acr1.04\u00a0\u00b1\u00a00.140.73 to 1.28NS0.56Percent scapular area visible above clavicle17.76\u00a0\u00b1\u00a014.35%0.61 to 53.98%Percent ratio affected to contralateral scapular area86.0 \u00b1\u00a06.4%73.1 to 103.3%\u22120.57Affected to contralateral height ratioH\/H0.89\u00a0\u00b1\u00a00.100.69 to 1.11<0.05\u22120.55Affected to contralateral width ratioW\/W0.95\u00a0\u00b1\u00a00.070.78 to 1.09NS\u22120.14Height to scapular body widthH\/w1.38\u00a0\u00b1\u00a00.200.95 to 1.811.40\u00a0\u00b1\u00a00.151.03 to 1.701.00\u00a0\u00b1\u00a00.130.80 to 1.30NS\u22120.30Affected to contralateral scapular body widthw\/w0.90\u00a0\u00b1\u00a00.070.76 to 1.00<0.05\u22120.35Height to total width ratioH\/W1.00\u00a0\u00b1\u00a00.130.68 to 1.261.07\u00a0\u00b1\u00a00.100.91 to 1.300.94\u00a0\u00b1\u00a00.130.69 to 1.17<0.05\u22120.35ElevationPercent vertical displacementA\/H\u22121.6\u00a0\u00b1\u00a09.33%\u221220.2 to 18.8%\u22120.03RotationDownward\/Upward displacement^\u03b159.7\u00b0\u00a0\u00b1\u00a013.5435\u00b0 to 83\u00b083.9\u00b0\u00a0\u00b1\u00a010.1267\u00b0 to 111\u00b0\u221224.2\u00a0\u00b1\u00a010.98\u221241 to \u22126<0.000\u22120.65Internal\/External displacement^\u03b6\u221235.2\u00b0\u00a0\u00b1\u00a08.5018\u00b0 to 55\u00b040.4\u00b0\u00a0\u00b1\u00a04.9330.5\u00b0 to 49\u00b0\u22126.5\u00b0\u00a0\u00b1\u00a07.92\u221220.5\u00b0 to 17\u00b0NS\u22120.22Anterior\/Posterior displacement^\u03d535.4\u00b0\u00a0\u00b1\u00a07.9320\u00b0 to 48\u00b043.2\u00b0\u00a0\u00b1\u00a08.0225\u00b0 to 54\u00b0\u22128.1\u00b0\u00a0\u00b1\u00a04.89\u221218\u00b0 to 4\u00b0<0.050.29Superior scapular angle^\u03b345.6\u00b0\u00a0\u00b1\u00a011.5020\u00b0 to 67\u00b058.3\u00b0\u00a0\u00b1\u00a07.3644\u00b0 to 81\u00b0\u221212.8\u00b0\u00a0\u00b1\u00a013.09\u221242\u00b0 to 11\u00b0<0.000\u22120.84Inferior scapular angle^\u03b431.1\u00b0\u00a0\u00b1\u00a016.51\u22122\u00b0 to 67\u00b09.7\u00b0\u00a0\u00b1\u00a07.70\u22123\u00b0 to 27\u00b021.4\u00b0\u00a0\u00b1\u00a017.72\u22129\u00b0 to 62\u00b0<0.0000.83Percent subluxationLM\/LN\u00a0\u00d7\u00a010025.7\u00a0\u00b1\u00a020.75%\u221227.7 to 53.0%49.0\u00a0\u00b1\u00a03.51%41.0 to 56.2\u221223.3\u00a0\u00b1\u00a021.21\u221276.0 to 4.6<0.000\u22120.80Glenoscapular angle^\u03b8\u221220.4\u00b0\u00a0\u00b1\u00a011.34\u221245\u00b0 to 0\u00b0\u22122.9\u00b0\u00a0\u00b1\u00a03.74\u221211\u00b0 to 5\u00b0\u221217.6\u00b0\u00a0\u00b1\u00a010.88\u221243\u00b0 to 1\u00b0<0.000\u22120.51The exact measurements (as abbreviated in the second column) are defined in Fig.\u00a02aCorrelation between ratio or difference of affected to contralateral side value to scapular area visible over clavicle\nAge did not significantly correlate with any of the parameters measured.\nVertical displacement was noted to be either superior (15 patients) or inferior (15 patients) and did not significantly correlate with any of the parameters measured.\nDownward rotational deformity was found to correlate with superior (r\u00a0=\u00a00.53) and inferior (r\u00a0=\u00a0\u22120.53) scapular angle.\nInternal\/external rotation did not significantly correlate with any of the parameters measured although only three patients had external rotation and two no rotation.\nAnterior\/posterior rotation did not correlate significantly with any of the parameters measured although only one patient had posterior rotation and one no rotation.\nArea of scapula was found to correlate with superior scapular angle (r\u00a0=\u00a00.60).\nAffected acromion to height ratio correlated with affected superior scapular angle (r\u00a0=\u00a0\u22120.81), inferior scapular angle (r\u00a0=\u00a00.83) and with subluxation (r\u00a0=\u00a0\u22120.80). Affected to contralateral acromion to height ratio correlated with superior scapular angle (r\u00a0=\u00a0\u22120.64), inferior scapular angle (r\u00a0=\u00a00.62) and with subluxation (r\u00a0=\u00a0\u22120.62).\nSuperior and inferior scapular angles correlated with subluxation (r\u00a0=\u00a00.63 and r\u00a0=\u00a0\u22120.63, respectively).\nSubluxation correlated with glenoscapular angle (r\u00a0=\u00a00.70).\nA grading scale was devised for assessing scapular deformity in brachial plexus palsy, based on the 3D-CT of the observed patients. The different stages are illustrated in Fig.\u00a03.\nFig.\u00a03The different SHEAR stages of scapular deformity as determined by three dimensional computer tomography. In SHEAR Grade 0 less than 2% of the scapula, less than 20% of the superior border and less than 6.5% of the medial border are visible above the clavicle. In SHEAR Grade 1 2\u20133.6% of the scapular area, 20\u201345% of the superior border and 6.5\u201316.5% of the medial border are visible over the clavicle. In SHEAR Grade 2 3.6\u201320% of the scapular area, 45\u201358% of the superior border and 16.5\u201328% of the medial border are visible above the clavicle. In SHEAR Grade 3 20\u201345% of the scapula, 58\u201368% of the superior border and 28\u201350% of the medial border are visible over the scapula. In SHEAR Grade 4 more than 42% of the area of the scapula, more than 68.5% of the superior border and more than 50% of the medial border are visible over the scapula\nDiscussion\nThe position of the affected scapula did not follow the symptoms and characteristics of Sprengel\u2019s deformity with congenital origin of scapular elevation. The hypoplasia and positioning of the scapula result from the brachial plexus injury and the apparent elevation is the result of downward rotation about an axis perpendicular to the scapular plane.\nThe lack of substantial forces applied to the scapula by the weakened scapula stabilizer muscles leads to a decreased rate of bone growth [8, 11]. The observed decreased height to total width is partly the result of enlargement of the acromion, which may be caused by traction through the deltoid [7]. The acromion was found to be always tapered in the presence of the mildest deformity and often beaked and its ratio to both the total scapular width and height were significantly larger than the contralateral side (both P\u00a0<\u00a00.000). The ratio of affected acromion to height correlated with the area of scapula visible over the clavicle (r\u00a0=\u00a00.85), with both affected superior scapular angle (r\u00a0=\u00a0\u22120.81) and inferior scapular angle (r\u00a0=\u00a00.83) and with subluxation (r\u00a0=\u00a0\u22120.80).\nIt seems that while the growth of height and width of the body of the scapula are impaired, the acromion follows a different pattern, of growth at the normal rate or at an aberrant rate if the deformity is more severe (correlation coefficient of affected to contralateral acromion and area of scapula visible over clavicle r\u00a0=\u00a00.56) and often impinges on the humeral head.\nThe vertical displacement which was either negative or positive did not significantly correlate with any of the parameters assessed implying that the apparent elevation of the scapula is the result of rotational displacement unlike congenital scapular elevation where the severity of the deformity correlates with the level of the shoulder joint [4].\nThe affected scapula is not only malpositioned, it is also hypoplastic. Previous radiological assessments have used oblique images of the scapula [8, 11] and no report has utilized 3D-CT and measured these characteristics. In fact, in posterior trunk views the hypoplasia looks more exaggerated than by measurement in the scapular posterior view because the view is affected by the internal rotation. The total area of the hypoplastic scapula averages 14% less than the contralateral scapula and correlates with the severity of the deformity (r\u00a0=\u00a0\u22120.57). While the rotation about a vertical axis was usually internal, in three instances it was external with no correlation to the severity of the deformity or to any other parameter assessed. Rotation about the axis of the scapular spine was in all but one instance anterior, forming a narrower scapuloclavicular space on the superior trunk outlet view.\nDuring the development of scapular dysplasia, both downward rotational deformity (angle of the spine of the scapula) and decreased height to width ratio contribute to change in superior scapular angle and inferior scapular angle and both indeed strongly correlated with area of scapula visible above clavicle (r\u00a0=\u00a0\u22120.75 and r\u00a0=\u00a00.83) and degree of subluxation (r\u00a0=\u00a00.63 and r\u00a0=\u00a0\u22120.63).\nThe clinical prognostic relevance of the various measurement methods is still under discussion [1, 13]. Global abduction has been correlated with glenoid version [17]. In the literature there seems to be no significant correlation between Mallet functional parameters other than global abduction and either radiographic parameters or age [13, 17] because active shoulder function is affected by the glenohumeral relationship, shoulder capsule, strength and physical properties of the muscles and the nervous system.\nIn many patients, different versions of muscle release of the contracted internal rotators with or without muscle transfer of latissimus dorsi and teres major to the teres minor is performed early in life so that the weakened deltoid and supraspinatus muscles can achieve active abduction. The success of this intervention is very impressive and the abduction is often significantly improved ([6, 10, 15] and our unpublished data). While glenohumeral incongruence is delayed after muscle contracture release ([15] and our unpublished data), internal rotation of the arm and winging scapula often develop. The lack of correlation between age and any of the parameters of the anatomical deformity in our study, unlike reports by others [17], may reflect the fact that most of our patients had undergone contracture releases of the latissimus dorsi, teres major, subscapularis, and pectoralis major and minor with tendon transfer of latissimus dorsi and teres major. While global abduction in our series was near normal, the forces exerted by the muscles on the two scapulas are not equivalent obviously upholding impaired growth to the affected scapula. The rate of progression of the deformity is individual to the child.\nThe rotation of the scapula causes positional changes in the acromio-clavicular joint. This causes impingement of the distal acromion and clavicle with the humeral head, resulting in the MRC positioning described above. The clinical implications of the SHEAR and resulting MRC are clear and have been described by Birch in detail [2]. The current standard of practice for treatment of MRC is muscle transfer if the shoulder is congruent [2, 16], corresponding to SHEAR 0\u20131 and humeral derotational osteotomy if the humeral head is incongruent corresponding to SHEAR 2\u20134 [16]. While derotational osteotomy may restore external rotation by placing the arm in a more functional position it addresses neither the scapular nor the glenohumeral deformities. Our findings that SHEAR score based on the percentage of scapula visible over the clavicle strongly correlates with rotation of the scapula, hypoplasia and subluxation, provides a convenient diagnostic tool to assess the presence of the MRC deformity. With the pathophysiology being thus established, anatomical correction of the deformity can be considered and planned, hopefully with improvement in functional parameters.\nConclusion\nScapular deformities common to the population of obstetric brachial plexus palsy patients due to muscular imbalances resulting from residual neurological deficit can be diagnosed and classified using the SHEAR classification and enable objective evaluation of the bony deformity and its severity as guide for treatment.","keyphrases":["scapula","obstetric","brachial plexus","acromion","paralysis","birth injuries"],"prmu":["P","P","P","P","P","M"]}
{"id":"Skeletal_Radiol-3-1-1934928","title":"The spine in Paget\u2019s disease\n","text":"Paget\u2019s disease (PD) is a chronic metabolically active bone disease, characterized by a disturbance in bone modelling and remodelling due to an increase in osteoblastic and osteoclastic activity. The vertebra is the second most commonly affected site. This article reviews the various spinal pathomechanisms and osseous dynamics involved in producing the varied imaging appearances and their clinical relevance. Advanced imaging of osseous, articular and bone marrow manifestations of PD in all the vertebral components are presented. Pagetic changes often result in clinical symptoms including back pain, spinal stenosis and neural dysfunction. Various pathological complications due to PD involvement result in these clinical symptoms. Recognition of the imaging manifestations of spinal PD and the potential complications that cause the clinical symptoms enables accurate assessment of patients prior to appropriate management.\nIntroduction\nPaget\u2019s disease (PD) or osteitis deformans is one of the commonest metabolically active bone diseases, second in prevalence only to osteoporosis, characterised by a disturbance in bone modelling and remodelling because of an increase in osteoblastic and osteoclastic activity. It is more common in people of Anglo Saxon origin and is less common in the Far East, India, Middle East and Africa. The overall prevalence of PD is 3\u20133.7% and increases with age [1\u20133]. By the age of 90\u00a0years, the prevalence increases to about 10% [2]. As the aetiology of PD continues to be the subject of debate, it is variously classified as an infection, metabolic disorder and a neoplastic process [1, 4\u201315]. However, strictly speaking, as the unaffected skeleton is metabolically normal, it does not fulfil the criteria for a true metabolic bone disorder.\nThe epidemiology of PD shows some significant changing trends in the clinical profilometry. First, recent studies have demonstrated that the incidence and prevalence of PD is gradually declining [16\u201318]. Second, there is a general trend towards reduction in the severity of the disease as measured by serum alkaline phosphatase levels [19, 20]. Third, there is a steady increase in the age at presentation by about 4\u00a0years per decade [18] and last, the proportion of patients with monostotic disease is increasing [18]. This in turn means that we are likely to see a higher incidence of monostotic vertebral involvement in the future.\nThe spine is the second most commonly affected site (53%) [6], after the pelvis (70%) [21\u201323]. The disease is polyostotic in 66% of cases and between 35% [24, 25] and 50% [7, 8] have spinal involvement. Using multimodal advanced imaging, this review article focuses on the pathological processes that underlie the varied spinal manifestations and complications of PD.\nPathomechanisms\nIn PD the loss of homeostatic control leads to increased osteoblastic and osteoclastic activity and constitutes the background for the main three phases. The initial lytic phase represents a mainly osteoclastic activity, the late osteoblastic phase is characterised by new bone formation, while the intervening mixed phase is seen when there is a combination of osteoblastic and osteoclastic activities [24, 26]. One more phase, \u201cinactive sclerotic phase\u201d, characterised by normal or decreased bone activity, has also been described [5], when the stimulation of new osteoblast and osteoclast formation ceases. Although the bone is metabolically inactive, it maintains a sclerotic coarsened architecture [27]. In bones with a low trabecular\/cortex ratio like the skull, femur and humerus, the early lytic phase is radiologically depicted by a clear leading edge at the interface with normal bone. The lytic phase is usually not detected in bones with a high trabecular\/cortex ratio like the vertebra, sacrum and pelvis [28]. Vertebral body involvement at radiological diagnosis is virtually always complete, and therefore the leading edge present in the other affected bones is not seen in the vertebra [29]. These phases can be evident in the same patient and at the same time in different bones including the vertebral column. Although progression of disease occurs within an affected bone, the sudden appearance of bone involvement at new skeletal sites years after the initial diagnosis is uncommon [30, 31].\nThe pathomechanisms and the dynamics involved in bone remodelling in PD of the appendicular and axial skeleton at the periosteal and endosteal surfaces have been previously described [3, 5, 32]. The enhanced abnormal osteoblastic activity results in periosteal and endosteal new bone formation (apposition). The abnormal osteoclastic activity on the endosteal surface results in bone resorption (absorption). The various combinations of these mechanisms give rise to four different patterns of bone remodelling at the periosteum\/endosteal interface leading to bone enlargement: periosteal and endosteal apposition; periosteal apposition and endosteal absorption; periosteal apposition with normal endosteal surface; and focal periosteal apposition \u2013\u201cpumice stone\u201d appearance (Figs.\u00a01, 2). The mechanisms in the spine commonly responsible for the changes on the periosteal and endosteal surfaces of the vertebral body and posterior neural arches are varied (Figs.\u00a01, 2, 3, 4). These various mechanisms are not exclusive of each other, but can occur in combination in the same vertebra at various borders. Usually, one of the pathomechanisms predominates in the involved vertebra. The most frequent mechanism of vertebral body expansion is periosteal apposition and endosteal absorption. The new bone formation predominates on the periosteal surface and it is responsible for the vertebral body enlargement, while the absorption on the endosteum results in an increased bone marrow space. Periosteal\/endosteal apposition and periosteal apposition with normal endosteal surface are two less common remodelling mechanisms seen in the vertebral body. In both, the apposition on the periosteal side results in vertebral body enlargement, but the bone marrow space is decreased or normal in size respectively. The least common mechanism of vertebral body expansion is focal periosteal apposition giving the \u201cpumice stone\u201d appearance (Figs.\u00a01, 2). Expansion of the vertebral bodies seen radiologically occurs in 63% of cases [25]. The commonest mechanisms in neural arch involvement are a combination involving periosteal and endosteal apposition or periosteal apposition and endosteal absorption (Figs.\u00a03, 4). In both of the mechanisms, the periosteal apposition causes a decrease in the size of the spinal canal resulting in spinal stenosis.\nFig.\u00a01Diagram depicting the osseous mechanisms involved in vertebral body enlargement in Paget\u2019s disease and its effect on the size of the marrow (dashed arrows) and cortex (solid arrows). A normal vertebra is depicted in the centre of the figure. a Periosteal apposition, normal endosteum resulting in thickened cortex, but with normal marrow size. b Periosteal apposition, endosteal resorption results in normal cortical thickness and an increased marrow size. c Periosteal apposition\/endosteal apposition results in a thickened cortex and reduced marrow size. d Focal periosteal apposition results in a focal \u201cpumice stone\u201d-like enlargementFig.\u00a02Axial CT sections in different patients showing the various mechanisms described in Fig.\u00a01 and their effect on marrow size (long white arrow) and cortical thickness (short white arrow). a Periosteal apposition, normal endosteum. b Periosteal apposition, endosteal resorption. c Periosteal and endosteal apposition. d Pumice stone type (dashed arrow) of focal periosteal apposition. Similar focal periosteal apposition of the spinous process is seenFig.\u00a03Diagram showing the periosteal and endosteal Pagetic osseous mechanisms involving the cortex of the spinal canal resulting in spinal canal narrowing. Normal cortical thickness (orange) of the spinal canal (white) is depicted at the top. a Expansion of bone due to periosteal apposition\/endosteal resorption results in a thin cortical outline (solid black arrow) of the narrowed spinal canal (dashed arrow). b Bony expansion due to periosteal apposition\/endosteal apposition results in a thickened cortical outline (solid black arrow) of the narrowed spinal canal (dashed arrow)Fig.\u00a04Axial CT images demonstrating the mechanisms in the posterior neural arch described in Fig.\u00a03 and their effect on cortical thickness (solid arrow) and marrow size (dashed arrow). a Periosteal apposition\/endosteal resorption. b Periosteal apposition\/endosteal apposition\nImaging manifestations\nPaget\u2019s disease of the spine can either involve a single level or more than one level. The lumbar spine and more commonly the L4 and L5 levels are the most frequently involved sites (58%) [33], more so than the thoracic (45%) and the cervical vertebrae (14%; Fig.\u00a05) [8, 23]. Involvement of the atlanto-axial region is very rare [8, 34, 35]. The vertebral body is almost always involved together with a variable portion of the neural arch. Isolated involvement of either the neural arch or the vertebral body is evident in only a small percentage of cases [25].\nFig.\u00a05Vertebral Paget\u2019s disease (PD) without expansion in two different patients. a PD of all the cervical vertebrae except C6. Note the absence of vertebral enlargement. There is sclerosis and loss of cortico-medullary differentiation of the vertebral bodies and the neural arches. b antero-posterior, c lateral radiographs and d axial CT through the L1 vertebra demonstrating sclerotic vertebra with no enlargement. The axial CT demonstrates trabecular and endosteal apposition, but no periosteal apposition accounting for the absence of enlargement. Diagnosis can be difficult and a biopsy (c) may be necessary in these cases\nOsseous changes (vertebral body)\nThe radiological appearance of vertebral body expansion is characterised on radiographs by an increase in the antero-posterior and lateral vertebral dimensions. However, the height of the vertebra is unchanged. The bony vertebral end-plates are subchondral condensations of trabecular bone and do not represent true bony cortex, i.e. there is no periosteum\/endosteum interface at the caudal\/cranial aspect of a normal vertebra. The sagittal and lateral enlargement is due to one of the previously described pathomechanisms in the corticated portions of the vertebral body. The pathomechanisms are best appreciated on CT (Fig.\u00a02), where the periosteal contour and the endosteal interface due to apposition and absorption are easily seen. Whatever the mechanism causing vertebral body remodelling, bone expansion is a common denominator in PD.\nUsually, the earliest phase seen radiologically in the vertebra is the mixed phase. The apparently \u201cearly\u201d radiographic appearance of vertebral body involvement in PD is thickening and hypertrophy of the trabecular bone [36, 37], parallel to the end plates (Fig.\u00a06), which can appear similar to a thickened cortex. CT reconstruction images can show this thickening and hypertrophy optimally. The combination of trabecular bone hypertrophy and thickening at the end-plates with apposition\/absorption on the periosteal\/endosteal surfaces at the anterior and posterior vertebral borders leads to the \u201cpicture frame\u201d sign [36]. Radiographs demonstrate increased density in the vertebral periphery and a relatively lucent centre in the vertebral body [36, 38, 39], best appreciated on lateral views and on sagittal CT reconstructions (Fig.\u00a06).\nFig.\u00a06a Lateral and b antero-posterior radiographs demonstrate expansion of the vertebra with characteristic sclerotic lines parallel to the end-plates due to trabecular hypertrophy, an \u201cearly\u201d sign of PD. c Lateral radiograph in a different patient demonstrates the \u201cpicture frame\u201d vertebra due to thickening of the cortex and trabecular hypertrophy at the end-plates\nProgression of the sclerotic phase in the spine leads to \u201civory vertebra\u201d, due to an increase in the density of the vertebral body, denser than the normal vertebral bodies. There is no change in the density and size of the adjacent intervertebral disc [40, 41]. Differential diagnoses of \u201civory vertebra\u201d include metastases, osteosarcoma, carcinoid and Hodgkin\u2019s lymphoma [29, 36]. The increased size of the vertebral body is a useful clue to the underlying diagnosis of PD. Biopsy may be necessary in some cases when there is no expansion (Fig.\u00a05).\nIn the exceptionally rare cases of vertebrae presenting in the lytic phase, there is marked osteopenia of the vertebra on radiographs, giving a \u201cghost vertebra\u201d appearance, because of the almost complete involvement of the vertebral body by the osteolytic process [42]. The lytic phase of PD involving the axis [43] and a rare pathological collapse of a purely lytic L5 vertebra [44] have been previously reported. It can be difficult to differentiate the lytic phase of PD from other causes of osteolysis and collapse. By demonstrating the cortical thickening, trabecular hypertrophy and vertebral expansion, CT confirms the lytic phase in the vertebral components due to the higher contrast resolution, the feasibility of \u201cbone window\u201d settings and multiplanar reconstructions.\nThe trabecular hypertrophy and cortical thickening caused by the osseous involvement by PD results in a variable degree of low signal on both T1- and T2-weighted images. In the severe cases of vertebral sclerosis (ivory vertebra appearance) the whole vertebra demonstrates a diffuse low signal on both T1- and T2-weighted sequences. More commonly however, the signal characteristics are heterogeneous on both T1- and T2-weighted sequences due to changes in the intervening marrow space. PD of the vertebra is easily missed or misinterpreted on MR images in the early stages. This is especially true when there is an absence of classic changes including vertebral enlargement and cortical thickening. This is mainly because PD is a disorder of bone and the vertebral marrow is only secondarily affected. MRI can show discrete areas of marrow signal alteration involving vertebral bodies with low non-specific signal on T1-weighted images and high signal on T2-weighted sequences. The marrow changes due to vertebral involvement by PD are described in detail later.\nOsseous changes (posterior elements)\nNeural arch involvement can be difficult to evaluate on radiographs alone. The best modality for neural arch assessment is CT with reconstructions.\nWhen periosteal and endosteal apposition occur, a markedly sclerotic cortex can be seen [5, 8]. However, when the basic mechanisms are periosteal apposition and endosteal absorption, CT demonstrates an enlarged marrow space of the neural arch delineated by a thin sclerotic line due to the periosteal apposition (Fig.\u00a04). Both processes result in a decrease in the spinal canal diameter.\nThe presumed epidural fat ossification as previously described in Paget\u2019s disease [45], results in the loss of the normal MR signal of epidural fat. This is probably a consequence of expanded Pagetic bone growth out of the neural arch components towards the spinal canal, reducing its size, rather than real ossification of the epidural fat. This can be misinterpreted as epidural fat ossification on radiographs and MR imaging and is best assessed on axial CT images (Fig.\u00a07). Sometimes, however, the fatty marrow changes in the expanded Pagetic posterior neural arch can be misinterpreted on MR imaging as epidural lipomatosis (Fig.\u00a08). CT again helps in the correct interpretation of this situation. This emphasises the need for a combination of imaging modalities to complement each other for accurate interpretation.\nFig.\u00a07a Sagittal T1-weighted MR image demonstrates PD in the L3 vertebral body and the posterior neural arch. The low T1 signal intensity mass (arrow) replacing epidural fat can be confused with epidural ossification. b However, an axial CT image confirms this to be due to expansion of the pagetic neural arch and not to ossification of the epidural fat. There is preservation of the intra-osseous fat as seen on both the MR and CT images, a useful discriminant from malignant infiltrationFig.\u00a08Paget\u2019s disease of T11 and T12 showing an increased amount of high MR signal (solid white arrow) in the posterior epidural space at these levels on a sagittal T2-weighted, b sagittal T1-weighted and c axial T1-weighted images. This can be mistaken for epidural lipomatosis. However, the d CT sagittal and e axial images demonstrate this to be due to the fat density (solid white arrow) within the expanded posterior neural arch involved in PD. The axial images (c,e) were obtained at the level of the tip of the solid arrows on sagittal images. There is also fusion of the vertebrae (dashed arrow) across the intervertebral disc. The combination of anterior and posterior vertebral involvement in this case resulted in severe spinal canal narrowing and cord compression. Note the high T1 signal indicating a high fatty marrow content within the pagetic T11 and T12 vertebrae\nBone scintigraphy using 99-Technetium substrates and SPECT can aid the diagnosis of PD, showing a typical distribution of the disease. Scintigraphy demonstrates increased uptake in all the affected vertebral components [46, 47] because of its sensitivity to osteoblastic activity and can be very useful in showing activity in the posterior neural arch. As a whole body imaging modality, it also provides information about the overall distribution of the disease. Moreover, the fourth sclerotic inactive phase described by Milgram is diagnosed essentially on a bone scan where radiographically affected bone does not demonstrate increased activity due to the metabolic inactivity of the lesion [27]. However, increased activity on bone scintigraphy is usually non-specific and requires comparison with radiographs\/CT. Various patterns of uptake are described on bone scintigraphy in PD [21, 46, 48, 49], all of which are non-specific.\nImaging techniques such as PET and PET-CT, can contribute to the differential diagnosis of PD from other disorders in the spine, especially when non-specific PD changes are incidentally discovered, and to assess disease activity after treatment.\nIn general 18F-fluoride positron emission tomography (PET) allows visualisation of the regional skeletal metabolic activity on the basis of the fluoride uptake on the bone surface when new bone formation and blood flow are greater [50]. The advantages of PET-CT on conventional nuclear bone scintigraphy include superior spatial resolution and more accurate quantification of bone activity. In PD, a general increase in global skeletal blood flow, measured by fluoride deposition, is seen associated with a regional increased uptake in the affected bone. Increased activity in the pagetic bone is usually recorded almost immediately after the injection and it remains high throughout the duration of the study. However, this finding is not specific to pagetic bony changes, giving rise to some false-positives. For this reason, the combination of non-specific activity on the PET image, the findings on the CT scan (PET-CT) and the correlation with other radiological examinations increases specificity [51].\nExtra-osseous changes\nPaget\u2019s disease can sometimes involve the soft tissue attached to the affected vertebra including the articular cartilage, the ligaments and the intervertebral disc [26, 45].\nThe articular facets are commonly involved in association with vertebral body and neural arch involvement. The affected facets are enlarged and sclerotic (Fig.\u00a09). The pagetic vertebral body involvement can interfere with the nutrition of the intervertebral disc leading to its degeneration due to sclerosis adjacent to the end-plates resulting in decreased diffusion [26].\nFig.\u00a09Facet involvement. a Axial CT section demonstrates incongruity at the facet joint (solid white arrow) due to an involved enlarged facet of one vertebra articulating with an uninvolved non-expanded adjacent vertebral facet. b Axial CT section in a different patient demonstrates advanced facet joint arthropathy (dashed white arrows) across two pagetic facets with complete loss of joint space and new bone formation contributing to spinal canal stenosis. This can progress to fusion across the facet joints\nOccasionally pagetic involvement of the spine can also produce extra-osseous extension resulting in ossification of the anterior\/posterior longitudinal ligaments and ligamenta flava. Lateral radiographs and CT sagittal reconstruction demonstrate the ossified anterior and posterior longitudinal ligaments. The ligamenta flava ossification are seen on the AP view as radio-opacities adjacent to the facet joints. Para-vertebral soft tissue swelling seen radiographically or by CT can be due to extra-medullary haematopoiesis. When PD of spine results in extra-medullary haematopoiesis [52], there is extra-osseous soft tissue with signal characteristics similar to the adjacent vertebral marrow. CT and MRI demonstrate the soft tissue mass adjacent to the vertebral body (see Pathological complications) in communication with the bone marrow within the vertebra [52].\nBone marrow\nPaget\u2019s disease is primarily a disorder of bone and not of the bone marrow. Secondary bone marrow changes do, however, occur. The marrow signal changes in established Paget\u2019s disease have been well described and vary with the stage of the disease [53]. Low signal on T1-weighted images and mild high signal on T2-weighted images (Fig.\u00a08) in the mixed hypervascular phase are seen. The sclerotic phase of PD results in low signal on both T1- and T2-weighted images in the vertebra due to increased trabecular thickness, sclerosis and marrow fibrosis (Fig.\u00a010). There is fatty transformation in the latter stages when there is high signal on both T1- and T2-weighted images (Fig.\u00a011). In the presence of osteolysis in pagetic vertebrae, a fat signal within the lesion is a useful clinical determinant between conservative management and biopsy in cases in which malignancy is suspected radiographically [54].\nFig.\u00a010a Initial scintigraphy for back pain demonstrates isolated increased uptake at a single vertebral level (T8). On initial inspection sagittal b T1-weighted and c T2-weighted MR images do not show any abnormality of the vertebral body. There is, though, some abnormal low signal from the posterior elements (black arrow). The diagnosis is still not clear. d However, a CT scan demonstrates the clear posterior vertebral (black arrow) sclerotic changes consistent with PD. Even on CT there are only minimal changes in the vertebral bodyFig.\u00a011On initial examination, a sagittal and b parasagittal T1-weighted, c sagittal and d parasagittal T2-weighted MR images of the lumbar spine do not demonstrate any obvious abnormality. e, f The radiographs, however, show classic pagetic changes of the L1 vertebra (dashed arrows) including vertebral expansion, sclerosis and cortical thickening. Review of the MRI shows some minor increased signal in the expanded L1 vertebral body on both T1 and T2 parasagittal images, suggestive of fatty marrow change (white arrows)\nWhenever vertebral bone marrow signal changes are seen on MRI, PD has to be considered in the differential diagnosis because of its non-specific and varied appearance in the bone marrow, and subtle bony alterations suggestive of PD have to be sought. Paget\u2019s disease can, however, also coexist with other disorders.\nWhen the bone marrow is not or only minimally involved (Fig.\u00a012) in PD, MRI will not reveal an appreciably altered signal on either T1- or T2-weighted images. The seemingly normal MR appearance of the vertebra does not exclude PD, but affirms only the normal appearance of the bone marrow, while the intervening trabecular bone is affected. For this reason, vigilance in recognising subtle signs like minor antero-posterior vertebral expansion and minimal cortical thickening is necessary as the signal characteristics within the centre of the vertebra may be entirely normal. MRI should be complemented by radiographs and CT in these instances (Figs.\u00a010, 11, 12). PD is fundamentally a bone disorder, where the bone marrow may or may not be involved, showing areas of sclerosis, fibrosis, increased vascularity, residual haematopoiesis and fatty marrow replacement, resulting in a wide spectrum of MR signal features. For this reason, all these described MR findings need the comparison of radiographs and CT images, because only these imaging modalities are actually capable of showing the specific and pathognomonic bony changes of PD. The aim of the integration of different techniques is to make a correct diagnosis and avoid unnecessary biopsy.\nFig.\u00a012a T1-weighted, b T2-weighted sagittal images of the lumbar spine demonstrate no marrow abnormality. There is only a subtle antero-posterior expansion of the L2 and L4. The diagnosis in these patients can be missed on initial MRI. c Lateral radiograph of the lumbar spine demonstrates the classic pagetic changes including vertebral expansion, trabecular hypertrophy and cortical thickening in L2 and L4. There is an incidental non-pagetic vertebral compression at L1. There is again preservation of the fat signal within the vertebrae involved in PD\nPathological complications\nPagetic bone is structurally weak. Despite this, the involved vertebral body and posterior neural arch still have to fulfil the biomechanical role they were designed for. The disease will alter the normal metabolic function in the bone while biomechanical forces in turn modify further the metabolic and structural changes that arise. Various clinical complications can occur secondary to vertebral PD resulting in back pain, spinal stenosis and neural dysfunction. Whilst each of these clinical symptoms can be caused by a variety of pagetic complications, it has to be stressed that PD and its complications can be entirely asymptomatic[21]. Moreover, the symptoms could be due to other coexisting spinal disorders.\nBack pain is the most common clinical symptom associated with PD of the spine (Table\u00a01). However, not all patients with PD of the spine are symptomatic [21]. Back pain in PD is due either to the disease itself or to the occurrence of different complications (Table\u00a01). Back pain due to the PD itself is caused by an increased blood flow and modelling\/remodelling process associated with the vertebral involvement including periosteal stretching and micro-fractures. In general, pagetic pain is a deep, dull ache or pain in the back that is unrelated to activity and not relieved by rest or non-steroidal anti-inflammatory medication. This pain is different from mechanical pain, which improves with rest and worsens with activity or a stressful posture of the back. It is also different from arthritic pain, characterized by aching and stiffness that is relieved by walking or by anti-inflammatory medication [24]. Between 11 and 54% [5, 55] of pagetic patients present with pain. In 24% of cases, back pain is due to PD itself [5, 8, 24]. In 50% of cases, it is due mainly to other complicating factors including fracture, spinal stenosis and facet arthropathy; less commonly it is due to spondylolysis with or without spondylolisthesis and discal involvement [26]. Another study found 88% of cases with back pain related to coexisting PD and osteoarthritis of the spine versus 12% of patients with pain attributed only to PD [56]. No particular difference in pain has been seen in multilevel involvement compared with single level involvement [24]. \nTable\u00a01Causes of back pain in Paget\u2019s diseasePeriosteal stretchingVascular engorgementMicrofracturesFacet arthritisIntervertebral disc diseaseOvert fractures of vertebrae, sacrumSpondylolysis\/-listhesisSarcoma\u2014very rare\nSpinal stenosis in PD of the spine has a prevalence of 33% of cases (Table\u00a02) [5]. It is graded as mild if it is just perceptible, moderate if the area of the spinal canal is reduced by less than 50% and severe if that area is reduced by more than 50% [24, 25]. Spinal stenosis can present clinically as back pain and\/or neurological dysfunction, as a consequence of cord (Figs.\u00a08, 13) or nerve root compression. In some cases, spinal stenosis is not associated with back pain. Patients with severe stenosis seen on imaging can present without clinical symptoms and patients with mild or moderate stenosis can present with back pain [24]. This is possibly explained by the adaptability of the thecal sac and its neural elements to severe spinal stenosis without significant loss of function [5, 24]. The proportion of symptomatic patients with spinal stenosis ranges from 26% [57] to 33% [5]. Seventy-five per cent of these cases present with pain, but without neurological dysfunction [25]. Expanded pagetic bone involving all vertebral components (vertebral body, neural arch and facet joints) is the commonest cause of spinal stenosis (Fig.\u00a013). It was reported by Wyllie in 1923 [58]. It is a consequence of a disturbance in the bone remodelling process, leading to a subsequent decrease in the spinal canal size [5, 24]. Less frequently, the stenosis can be due solely to expansion of the neural arch associated with the growth of pagetic bone involving the facets [8]. \nTable\u00a02Causes of neural dysfunction in Paget\u2019s diseasePosterior expansion of the vertebral bodyPosterior neural arch expansionFacet joint arthritis\/overgrowthLigament ossificationSpondylolisthesisFracture retropulsionExtra-osseous involvement\u2014PD, haematopoiesis, \u201cpseudosarcoma\u201d, malignancy\u201cArterial steal\u201d syndromeFig.\u00a013Sagittal T2-weighted MR image demonstrates cauda equina compression at the L1 level due to pagetic enlargement of the whole vertebra. Note the stenosis caused by expansion of both the vertebral body and posterior elements. Degenerative spondylolisthesis and stenosis at L4\/L5 is noted\nSpinal stenosis in Paget\u2019s disease has the unique radiographic feature of widening of the interpediculate distance on the antero-posterior view, with expansion of the vertebral body on the lateral view. CT is very useful for assessing the contribution of the facets and neural arch to the stenosis, not seen well on radiographs (Fig.\u00a014). CT also helps to assess the location of the expanded bone to determine if the stenosis is central or lateral and to assess the severity of the stenosis. In cases in which cord compression due to the spinal stenosis is suspected, the diagnostic tier has to be completed by performing an MRI scan (Figs.\u00a08, 13) [8, 24, 59]. Axial and sagittal images demonstrate the bone abnormalities, but they can also show myelomalacia in the spinal cord. The sagittal T2-weighted images show the pagetic bone compressing the cord with no evidence of normal CSF surrounding the cord. The axial images show the asymmetry or deformation in shape of the cord compressed by the expanded bone. Areas of myelomalacia demonstrate low cord signal on T1-weighted images and high signal on T2-weighted and STIR images, at the same level as the expanded pagetic bone.\nFig.\u00a014Post-myelographic lateral lumbar spine a radiograph and b axial CT scan demonstrate spinal block at the L1 vertebral level, which is pagetic. Note the severe degree of spinal stenosis despite the apparent increase in the interpedicular distance on the axial CT image\nNeural dysfunction can be due to spinal stenosis (33%) [24, 60], more frequently with thoracic spine involvement and less commonly with cervical spine involvement [61]. The less frequent neural involvement in lumbar spine involvement is due to the relatively capacious spinal canal in the lumbar region [5, 57, 62]. Although the pagetic process itself, together with compressive effects from the expanding bone, can give rise to symptoms and signs, other complications may develop and contribute to the symptoms complex of neural dysfunction (Table\u00a02). Patients presenting with neurogenic pain due to cord compression by expanded pagetic bone (Fig.\u00a020) can also respond well to medical treatment with calcitonin and bisphosphonates [63]. Surgical decompression is rarely necessary in cord compression as stenosis and neural dysfunction in PD may respond to medical treatment alone [5]. The \u201carterial steal phenomenon\u201d is another factor contributing to neural dysfunction. This is described as a deprivation of blood supply to the spinal cord due to the preferential blood flow of the pagetic vertebra (Fig.\u00a015) [64]. It is classified as a non-compressive spinal cord dysfunction and not directly related to pagetic bone growth and it can respond very well to treatment with calcitonin [64\u201366].\nFig.\u00a015Sagittal fat suppressed T1-weighted image after gadolinium-DTPA administration in the same patient as in Fig.\u00a08 demonstrates intense enhancement at both the T12\/L1 and the L3\/L4 levels (arrows). The enhancement in part reflects increased blood supply to the vertebra\nCompression fracture\nCompression fracture of the vertebral body is the commonest complication in PD of the spine and usually presents with sudden onset of back pain (Fig.\u00a016). It occurs more frequently in the lumbar spine [59], and rarely in the sacrum [59], the coccyx [67] and the odontoid peg.\nFig.\u00a016Lateral and anteroposterior radiograph of the lumbar spine demonstrates severe compression of a pagetic L3 vertebra with retropulsion into the spinal canal. Note the increased interpediculate distance as a hallmark of PD\nPara-vertebral swelling can be seen as an indirect sign of the vertebral fracture in the acute phase. The fracture line itself is best seen on CT with sagittal reconstructions. Vertebral fracture can also contribute to the pain related to spinal stenosis by two mechanisms: posterior vertebral body wall bulging and retropulsion (Fig.\u00a016) of a bone fragment into the spinal canal [8, 68], and cord compression. This can be recognised on radiographs in the lateral view and is associated with a decrease in the spinal canal size, but CT demonstrates this better.\nThe fractured vertebra in PD sometimes appears osteopenic on radiographs. This could be due to a fracture occurring in the lytic phase of PD. However, as described earlier, it is unusual to see the lytic phase of PD in the spine. The osteopenia is most likely due to fracture-induced osteolysis. MRI can also contribute to the diagnosis of a compression fracture, even if its findings are not specific. The fracture line is seen as a linear or curvilinear low signal on T1-weighted images and high signal on T2-weighted and STIR images. Oedema surrounding the fracture can be an indirect sign. It is seen as high signal on inversion recovery and T2-weighted images and low signal on T1-weighted images. When the fracture is not recent, the signal on the MRI scan will be low on both T1- and T2-weighted images because of the sclerosis of the bone. In general, diffusion weighted imaging can help to differentiate osteoporotic from malignant vertebral body collapse [69]. It is not yet known if benign fracture in PD can be mistaken for a malignant compression on diffusion weighted imaging given the associated underlying marrow changes prior to the occurrence of the fracture.\nFacet joint arthropathy\nFacet joint involvement can cause back pain, lateral spinal stenosis and neural dysfunction. Facet involvement can lead to facet joint arthropathy due to two mechanisms. One is the direct extension of PD into the articular cartilage similar to that seen in large synovial joints and the intervertebral disc [26], the other is the pagetic involvement of the facets leading to expansion of the facet and resultant incongruity with the apposing uninvolved facet (Fig.\u00a09). Facet joint arthropathy was noted in 17 out of 21 symptomatic patients with back pain in one study [25]. This gives rise to mechanical stress and early degenerative changes in the facet joint. This is one of the most important factors in back pain and also contributes to spinal stenosis. The spinal canal stenosis associated with facet joint arthropathy is typically lateral and can result in nerve root compression [5]. Patients with severe arthropathy often have symptoms of spinal stenosis, but they can be asymptomatic [24, 25]. Compared with radiography, CT gives a clearer depiction of facet joint changes (Fig.\u00a09), showing the loss of articular space, the destruction of cartilage associated with subchondral erosion and articular incongruity (Fig.\u00a09) [5, 26, 70]. Facet joint arthropathy may eventually lead to ankylosis of the articular space [25]. MR imaging can underestimate the bony changes associated with the facet involvement.\nSpondylolysis and spondylolisthesis\nBack pain may be also due to unilateral or bilateral spondylolysis if the pars interarticularis is involved in PD. PD, due to the associated modelling\/remodelling activity, results in an increased susceptibility to insufficiency fractures. A lateral radiograph may demonstrate the typical spondylolysis (Fig.\u00a017). However, due to the associated PD and sclerosis, this can be difficult to appreciate. CT is the best modality to detect this. Underestimation of this complication on radiographs and the relatively limited use of cross-sectional imaging in PD may be the reason for the under-reporting of this condition.\nFig.\u00a017Lateral radiographs in two different patients with PD showing two different mechanisms of spondylolisthesis: a spondylolytic (arrow) spondylolisthesis; b degenerative spondylolisthesis\nSpondylolisthesis in PD can be due to either spondylolysis or facet joint degeneration (Fig.\u00a017). It is important to differentiate between the two entities. The degenerative spondylolisthesis results in spinal stenosis and neural dysfunction. The lateral radiograph is quite useful for detecting spondylolisthesis and to establish its grade and severity. However, this can be underestimated if the vertebral alignment is not assessed carefully. An enlarged vertebra due to PD may slip anteriorly over a normal vertebra, but the posterior vertebral body alignment may still be preserved due to the posterior expansion. The assessment of the posterior neural arch alignment, however, will reveal the subtle slip in these cases. The standard lateral radiograph can be complemented by dynamic views in flexion and extension, which can reveal the presence of spondylolisthesis under stress. Spondylolisthesis, and particularly spondylolysis, can be confirmed on CT with sagittal reconstruction images.\nIntervertebral disc involvement\nPaget\u2019s disease can involve the intervertebral disc and cause intervertebral disc degeneration. The incidence of direct intradiscal transgression is about 10.7% [26]. In this study, 67% of patients with disc involvement presented with pain. Twenty-two percent were asymptomatic. Therefore, disc involvement can remain asymptomatic. Some studies describe the presence of para-vertebral soft tissue involvement without specific comment on the discal invasion by PD [45, 68]. PD transgressing the intervertebral disc occurs more often in the lumbar spine than in the thoracic and cervical spine. The mechanism is not very clear. Intervertebral disc degeneration can be caused mainly by the mechanical stress induced by asymmetry at the end plate attachment of annulus fibrosis between a pagetic and a normal vertebra, and also by direct invasion of the disc space by the pagetic process. Aggressive pagetic invasion at the disco-vertebral junction leads to pagetic tissue replacement of the cartilage end-plate and subsequently of the intervertebral disc. The direct resorption of the disc tissue with subsequent pagetic bone replacement in the disc is the most likely explanation [26]. PD can then invade the adjoining vertebra across the disc space. This can also occur by direct extension of PD along large pre-existing degenerative bridging osteophytes [26]. Radiological investigations in disc involvement show progressive disc space narrowing, loss of definition of the adjacent end-plates, sometimes indistinguishable from spondylodiscitis, and large degenerative osteophytes involved in PD (Figs.\u00a08, 18). Intervertebral disc involvement can eventually lead to pagetic vertebral ankylosis (PVA) with an incidence of 4.4% [26]. Ankylosis is commoner in men, affects the thoracic spine in over 50% of cases [71] and is usually asymptomatic. The fusion between the vertebral bodies is easily appreciated as being due to Pagetic changes. The mechanisms that promote intervertebral disc involvement in PD are also in play when extra-osseous involvement of the spinal ligaments and para-vertebral tissues is seen (Fig.\u00a019).\nFig.\u00a018Disc involvement. Serial radiographs 2\u00a0years apart in the same patient demonstrate progressive involvement of the L4\/L5 intervertebral disc in Paget\u2019s disease of the L5Fig.\u00a019Extra-osseous Paget\u2019s disease. a Sagittal CT reconstruction and b T1-weighted MR sagittal image demonstrate anterior longitudinal ligament ossification (white arrows). c, d Axial CT sections demonstrate ossification in the ligamentum flavum due to extra-osseous PD (black arrows) in a different patient. e Antero-posterior radiograph in another patient demonstrates paravertebral soft tissue swelling (dashed arrows) producing the \u201cpseudo-sarcoma\u201d appearance\nNeoplastic transformation\nNeoplastic transformation in pagetic bone is very rare in the spine (0.7%) [5, 8, 24, 72] and represents only 7% [73] of all sarcomatous degeneration in PD. It can present with back pain. Benign and malignant bone neoplasms can complicate Paget\u2019s disease. The majority of malignant transformations are osteosarcomas. They seem to be related to one or two genes on the chromosome 18q [5, 74, 75].\nRadiologically, this is seen on radiographs and CT images as a lytic lesion characterised by an aggressive pattern on a background of typical PD. It can be very difficult to diagnose this lesion on the radiographs because of the overlapping pagetic changes in the bone. However, as described earlier, it is unusual to see the lytic phase of PD in the absence of other complicating factors like a fracture. A para-vertebral soft tissue mass should always be regarded as possible neoplastic transformation associated with vertebral Paget\u2019s disease, but more frequently is due to benign causes (Table\u00a03). On MR axial and sagittal images the sarcomatous degeneration appears as a mass with irregular edges showing low signal on T1- and inhomogeneous high signal on T2-weighted images [8]. The preservation of the medullary fat signal on T1-weighted images excludes malignant degeneration and can be an extremely useful sign [54]. It usually shows inhomogeneous contrast enhancement after gadolinium administration because of its solid structure with internal vascularity. The other differential diagnosis to be considered is extra-medullary haematopoiesis. Less common is the \u201cpseudo-sarcoma\u201d appearance characterised by extra-cortical periosteal bone expansion or by a bulky juxta-cortical soft tissue mass similar in its characteristics on imaging to sarcomatous degeneration (Fig.\u00a019) [11, 76, 77]. Malignant transformation can contribute to spinal stenosis [5]. For this reason the radiological assessment is not only focussed on the presence of a malignant mass, but also on the presence of cord compression, well documented on sagittal T1- and T2-weighted MR images. The spine is a common site for metastatic disease and the secondary deposits may also involve bones previously involved in Paget\u2019s disease (Fig.\u00a020). A biopsy may be necessary to differentiate among these various entities. \nTable\u00a03Causes of paravertebral swelling in Paget\u2019s diseaseExtra-osseous extensionFracture haematomaExtra-medullary haematopoiesisPaget\u2019s sarcoma\/other tumoursPaget\u2019s pseudosarcomaFig.\u00a020Vertebral metastasis from colon carcinoma. Sagittal a T1- and b T2-weighted MR images demonstrate metastasis in L2 and L4 vertebral bodies seen as discrete lesions (white arrows) with low signal on T1- and high signal on T2-weighted images. A further epidural lesion (black arrow) is seen in the spinal canal posteriorly at L3. Note the pagetic changes with expansion of L2 and L3 vertebral bodies\nConclusion\nRecognition of the imaging manifestations of spinal PD and the potential clinical complications enables accurate assessment of patients prior to appropriate management. This knowledge should allow subtle PD to be identified on imaging when this is not suspected. This is especially relevant to MRI, as it has become the imaging modality of choice for investigating the spine. Patients presenting with back pain and spinal stenosis have to be assessed for pagetic complications before attributing the symptomatology to the disease itself.","keyphrases":["paget\u2019s disease","vertebra","spinal stenosis","magnetic resonance imaging","computed tomography"],"prmu":["P","P","P","M","M"]}
{"id":"Anal_Bioanal_Chem-3-1-1820759","title":"Certification of butyltins and phenyltins in marine sediment certified reference material by species-specific isotope-dilution mass spectrometric analysis using synthesized 118Sn-enriched organotin compounds\n","text":"A new marine sediment certified reference material, NMIJ CRM 7306-a, for butyltin and phenyltin analysis has been prepared and certified by the National Metrological Institute of Japan at the National Institute of Advanced Industrial Science and Technology (NMIJ\/AIST). Candidate sediment material was collected at a bay near industrial activity in Japan. After air-drying, sieving, and mixing the material was sterilized with \u03b3-ray irradiation. The material was re-mixed and packaged into 250 glass bottles (15 g each) and these were stored in a freezer at \u221230 \u00b0C. Certification was performed by use of three different types of species-specific isotope-dilution mass spectrometry (SSID\u2013MS)\u2014SSID\u2013GC\u2013ICP\u2013MS, SSID\u2013GC\u2013MS, and SSID\u2013LC\u2013ICP\u2013MS, with 118Sn-enriched organotin compounds synthesized from 118Sn-enriched metal used as a spike. The 118Sn-enriched mono-butyltin (MBT), dibutyltin (DBT), and tributyltin (TBT) were synthesized as a mixture whereas the 118Sn-enriched di-phenyltin (DPhT) and triphenyltin (TPhT) were synthesized individually. Four different extraction methods, mechanical shaking, ultrasonic, microwave-assisted, and pressurized liquid extraction, were adopted to avoid possible analytical bias caused by non-quantitative extraction and degradation or inter-conversion of analytes in sample preparations. Tropolone was used as chelating agent in all the extraction methods. Certified values are given for TBT 44\u00b13 \u03bcg kg\u22121 as Sn, DBT 51 \u00b1 2 \u03bcg kg\u22121 as Sn, MBT 67 \u00b1 3 \u03bcg kg\u22121 as Sn, TPhT 6.9 \u00b1 1.2 \u03bcg kg\u22121 as Sn, and DPhT 3.4 \u00b1 1.2 \u03bcg kg\u22121 as Sn. These levels are lower than in other sediment CRMs currently available for analysis of organotin compounds.\nIntroduction\nOrganotin compounds have wide ranging chemical and toxicological properties. They are widely applied as stabilizers of plastics, as fungicides and pesticides, and as marine antifoulants [1]. Use of tributyltin (TBT) and triphenyltin (TPhT) as marine antifouling agents has led to their almost global dispersal. Although use of TBT and TPhT has been controlled in Japan since 1989, the compounds are still prominent in the coastal sea waters of Japan. Thus, these organotins and their degradation products, di- and mono-organotins, in sediment are frequently monitored to evaluate organotin pollution in water environment. Quantification of organotin species in environmental samples is very difficult, because of their instability and the low concentrations present. Because long-term and worldwide monitoring is necessary for these compounds, comparability of the analytical results is required [2].\nThe National Metrology Institute of Japan (NMIJ) has already developed, in 2001, a marine sediment certified reference material (NMIJ CRM 7301-a) for analysis of mono, di, and tributyltins (MBT, DBT, and TBT) [3]. Certification was performed using two different types of species-specific isotope-dilution mass spectrometric (SSID\u2013MS) method\u2014gas chromatography coupled with inductively coupled plasma mass spectrometry (SSID\u2013GC\u2013ICP\u2013MS) [4] and SSID\u2013GC\u2013MS combined with a microwave-assisted extraction\u2014in which a mixture of 118Sn-enriched butyltins synthesized in our laboratory was used as a spike. In 2005 we developed a new marine sediment certified reference material, NMIJ CRM 7306-a, certified for di and triphenyltins (DPhT and TPhT) and the butyltins by using three different types of SSID\u2013MS method. In the SSID methods, 118Sn-enriched TPhT and DPhT newly synthesized from 118Sn-enriched metal, and a mixture of 118Sn-enriched butyltins previously synthesized from 118Sn-enriched metal [4] were used as spikes. The certification strategy for NMIJ CRM 7306-a was almost the same as that for NMIJ CRM7301-a [3] but with two improvements to ensure the reliability of the CRM. One was the sterilization of the material with \u03b3-ray irradiation. NMIJ CRM7301-a was not irradiation-sterilized because of possible degradation of the organotin compounds. Yang et al. reported that significant losses of butyltins in methanol occurs during \u03b3-ray irradiation whereas such degradation is minimal in a sediment matrix [5]. In our preliminary test, no substantial loss of organotins was observed on \u03b3-ray irradiation, and thus irradiation-sterilization was performed for CRM7306-a to ensure long term stability. The other improvement was that a total of six combinations of four extraction methods and three SSID\u2013MS methods were used for analysis of the organotin compounds to ensure the reliability of the certified values. The overview of the analysis for certification is shown in Fig.\u00a01. Although SSID\u2013MS methodology can correct most of the systematic errors that occur in the analysis, it can not compensate for non-quantitative extraction from the sample or for species re-conformation in the sample preparation process [6\u201311]. Indeed, quantitative extraction of MBT is not an easy task, because MBT is strongly adsorbed by sediment matrices, because of its polarity [12\u201314]. It has been reported that DBT is degraded during microwave-assisted extraction [6] and pressurized liquid extraction [7] when high microwave energy or high extraction temperatures are used, although species interconversion can be taken into account in SSID\u2013MS techniques that furnish data for more than one enriched isotope, as has been described by Encinar and co-workers [6\u20139]. In this certification, therefore, the four different types of extraction method, mechanical shaking, ultrasonic, microwave-assisted, and pressurized-liquid extraction were adopted to avoid possible analytical biases that caused by non-quantitative extraction and degradation or interconversion of analytes. SSID\u2013liquid chromatography (LC)\u2013ICP\u2013MS, was adopted as an SSID\u2013MS method in addition to SSID\u2013GC\u2013ICP\u2013MS and SSID\u2013GC\u2013MS, because organotin separation by LC does not need a derivatization step. Thus, use of SSID\u2013LC\u2013ICP\u2013MS can avoid potential degradation or interconversion of analytes in the derivatization step. This paper mainly describes SSID\u2013MS analysis for certification of NMIJ CRM 7306-a Marine Sediment for organotin compounds.\nFig.\u00a01Overview of analysis for certification\nExperimental\nPreparation of sediment material\nThe starting material for the CRM was collected in a bay near industrial activity in the Kyushu area, Japan. Approximately 300\u00a0kg surface sediment was sampled and the water was removed by filtration. The sediment material was air-dried for 2 weeks at room temperature (25\u201328\u00a0\u00b0C). After removal of visible external materials (rock, shell, etc.) the sediment material was ground in a high-purity alumina ball-mill. Sediment powder that passed through a 104-\u03bcm sieve was homogenized using a pan-type mixer. The sediment powder was then bottled in glass bottles (60\u00a0g each) and irradiation sterilized (60Co, 20\u00a0kGy). The irradiated powder was re-mixed and homogenized by use of a rocking mixer RM-10S (Aichi Electric, Japan) as a precaution against an inhomogeneous degradation of organotin compounds during sterilization. Finally, the powder was placed in amber glass bottles (15\u00a0g each) and stored at \u221230\u00a0\u00b0C.\nConversion to dry mass basis\nThe concentrations of the constituents of this CRM are given on a dry-mass basis. A dry mass correction factor for sample humidity was determined by drying the sample at 110\u00a0\u00b0C. After 5\u00a0h the sediment sample reached constant weight, so it was decided the drying time would be 6\u00a0h in this experiment. The dry mass correction factor at the time of certification was 0.959\u00b10.003 (average\u00b1standard deviation for ten different bottles).\nChemicals\nTributyltin (TBT) chloride, dibutyltin (DBT) chloride, and triphenyltin chloride (TPhT) were purchased from Wako Pure Chemicals (Osaka, Japan). Monobutyltin (MBT) chloride and diphenyltin chloride (DPhT) were purchased from Aldrich (Milwaukee, WI, USA). Tripropyltin (TPrT) chloride was purchased from Merck (Darmstadt, Germany). Standard solutions of each organotin compound except DPhT were separately prepared by dissolving each compound in methanol (pesticide analysis grade, Wako) to avoid any disproportionation reactions with other organotins. The standard solution of DPhT was prepared by dissolving DBTCl2 in 0.005\u00a0mol L\u22121 HCl in methanol, to prevent self-disproportionation. All standard solutions were stored at \u221220\u00a0\u00b0C and diluted working solutions were prepared daily before the analysis. Sodium tetraethylborate (NaBEt4) was purchased from Wako Pure Chemicals. A 5% m\/v solution of NaBEt4 was prepared in a glove box that was purged with N2 gas. Other chemicals used were of analytical reagent grade. Pure water prepared by use of a Milli-Q water-purification system (resistivity 18\u00a0M\u03a9 cm, Nihon Millipore Kogyo, Tokyo, Japan) was used throughout the experiments.\nSynthesis of the 118Sn-enriched organotin compounds\nThe mixture of 118Sn-enriched butyltin compounds used as the spike for the ID methods were synthesized from 118Sn-enriched tin metal (92% enriched) purchased from Trace Science International (Ontario, Canada). The synthetic procedure has been described previously [4]. 118Sn-enriched DPhT and TPhT were individually synthesized from 118Sn-enriched tin metal (98% enriched) purchased from Nippon Sanso (Tokyo, Japan). The synthetic procedures were almost the same as those described elsewhere [15]. A mixture of ca. 0.5\u00a0g 118Sn metal and ca. 2\u00a0g iodine was introduced into a 50-mL round-bottomed flask containing acetic acid (10\u00a0mL) and acetic anhydride (10\u00a0mL). A small crystal of potassium iodide was added as catalyst and the mixture was gently heated to reflux. After cooling in an ice bath, orange crystals of 118SnI4 (1.6\u00a0g) were collected. 118SnI4 was placed in a 50-mLround-bottomed flask containing 30\u00a0mL diethyl ether and 1\u00a0mol L\u22121 phenyl magnesium bromide in THF was added dropwise. After heating to reflux for 3\u00a0h the white-pink precipitate was collected by filtration after the Grignard reagent had been hydrolyzed with water. The solid was dissolved in dichloromethane and the solution was filtered to remove insoluble impurities. The dichloromethane was removed to leave 118Sn-tetraphenyltin (TePhT) as a white solid; this was washed with ethanol. 118SnI4 (0.25\u00a0g) and 118Sn-TePhT (0.5\u00a0g) were mixed in a glass tube and heated to 200\u00a0\u00b0C. The reaction products were dissolved in ethanol and the insoluble products were removed by filtration. By the addition of 20% potassium fluoride solution, 118Sn-TPhT fluoride was obtained as an insoluble salt. The fluoride was isolated by filtration, washed with a minimum amount of ethanol, and then treated with conc. hydrochloric acid and extracted with pentane. The extracts were dried with MgSO4 and the pentane was removed to yield 118Sn-enriched TPhT chloride as a white powder. 118Sn-enriched DPhT dichloride was prepared by treating 18Sn-enriched TePhT with HCl. The product was extracted with pentane. The extracts were dried with MgSO4 and the pentane was removed to yield 118Sn-enriched DPhT dichloride.\nExtraction procedure\nUltrasonic extraction\nThe ultrasonic extraction procedure for GC\u2013ICP\u2013MS and GC\u2013MS was as follows. The sediment sample (ca. 0.5\u00a0g) was placed in a PFA centrifuge tube and spiked with an appropriate amount of the spikes. Then 2\u00a0g NaCl, 12\u00a0mL toluene containing 0.1% tropolone, and 10\u00a0mL acetic acid\u2013methanol (1:1) were added to the tubes. The resulting mixtures were extracted in an ultrasonic bath for 30\u00a0min at 60\u00a0\u00b0C. After addition of 10\u00a0mL water the tubes were again shaken, for good phase separation, and then centrifuged at 3000\u00a0rpm for 5 min. Finally, the upper toluene layer was collected as the extract.\nFor LC\u2013ICP\u2013MS, the extraction solvent was replaced with 10\u00a0mL acetic acid\u2013methanol (1:1) containing 0.1% tropolone, and the same extraction procedure was performed.\nMechanical shaking extraction\nThe sediment sample (ca. 0.5\u00a0g) was placed in a PFA centrifuge tube and spiked with an appropriate amount of the spikes. Then 2\u00a0g NaCl, 12\u00a0mL toluene containing 0.1% tropolone, and 10\u00a0mL 0.5\u00a0mol L\u22121 HCl in methanol were added to the tubes, and the resulting mixtures were mechanically shaken for 60\u00a0min. After addition of 10\u00a0mL water the tubes were again shaken, for good phase separation, and then were centrifuged at 3000\u00a0rpm for 5\u00a0min. Finally, the upper toluene layer was collected as the extract.\nMicrowave-assisted extraction\nClosed-vessel microwave-assisted extraction (MAE) was performed. The microwave system used was Mars X (CEM, USA). The sediment sample (ca. 0.5\u00a0g) was placed in a PFA vessel and spiked with an appropriate amount of the spikes. Then 2\u00a0g NaCl, 12\u00a0mL toluene containing 0.1% tropolone, and 10\u00a0mL 1\u00a0mol L\u22121 acetic acid in methanol were added to the vessel. The microwave irradiation program was ramp to 120\u00a0\u00b0C in 10\u00a0min then hold for 10\u00a0min. The mixture was transferred to a PFA tube containing 10\u00a0mL water and the tube was shaken, for good phase separation, and then centrifuged at 3000\u00a0rpm for 5\u00a0min. Finally, the upper toluene layer was collected as the extract.\nPressurized liquid extraction\nPressurized liquid extraction (PLE) was performed with an ASE 200 instrument (Dionex, USA). The sediment sample (ca. 1\u00a0g) was placed in an 11-mL stainless extraction cell containing a glass filter and quartz sand and was spiked with an appropriate amount of the spikes. After filling the extraction vessel with quartz sand, the cell was placed in the PLE system. The extraction solvent used was 0.5\u00a0mol L\u22121 acetic acid in methanol containing 0.2% tropolone. The extraction conditions were: oven temp. 110\u00a0\u00b0C, pressure 10.3\u00a0MPa, static time and cycle was 5\u00a0min\u00d74 times. The extract obtained was transferred to a PFA tube containing 10\u00a0mL water and 12\u00a0mL toluene, and the tube was shaken, for good phase separation, and then centrifuged at 3000\u00a0rpm for 5\u00a0min. Finally, the upper toluene layer was collected as the extract.\nDerivatization procedure for GC\u2013ICP\u2013MS and GC\u2013MS\nThe extracted solutions were transferred to PFA centrifuge tubes and 25\u00a0mL ammonium-acetate buffer (0.5\u00a0mol L\u22121, pH 5) and 0.2\u00a0mL 5% NaBEt4 solution were added. The tubes were mechanically shaken for 20\u00a0min, for ethylation and extraction, and then centrifuged to achieve phase separation. The toluene layers were also transferred to the tubes and mixed with 2\u00a0g anhydrous sodium sulfate to remove the water.\nClean-up procedure\nClean-up on a Presep-C Florisil cartridge (Wako, Japan) was performed after the derivatization, except for LC\u2013ICP\u2013MS measurement. The toluene layer was evaporated to ca. 2\u00a0mL under a stream of N2 gas and loaded on to a cartridge previously conditioned with hexane. The eluent from the cartridge was collected in a 15-mL glass centrifuge tube. Hexane (6\u00a0mL) containing 5% (v\/v) diethyl ether was then also loaded on the cartidge and the eluent was collected in the glass tube. Finally, the collected eluent was evaporated to 0.2\u00a0mL under a stream of N2 gas and used as the measurement sample solution.\nFor the LC\u2013ICP\u2013MS measurement, a clean-up procedure using Presep-C18 cartridge (Varian, Australia) was performed. The extract was loaded on to a cartridge previously conditioned with acetone. The eluent from the cartridge was collected in a 15-mL glass centrifuge tube, evaporated to 0.2\u00a0mL under a stream of N2 gas, and used as the measurement sample solution.\nDetermination of organotin compounds by ID\u2013GC\u2013ICP\u2013MS\nThe GC\u2013ICP\u2013MS used was that the GC (Agilent 6890GC) was coupled with an ICP\u2013MS (HP4500, Yokogawa Analytical Systems, Tokyo, Japan) by means of a manufactured transfer-line (Yokogawa Analytical Systems). The GC column was HP-1\u00a0ms (30\u00a0m\u00d70.32\u00a0mm i.d., 0.32\u00a0\u03bcm film thickness). The measured masses were m\/z 118 and 120. The operating conditions and procedures for GC\u2013ICP\u2013MS were similar to those described elsewhere [4].\nThe concentrations of organotin compounds were calculated by inserting each value into the ID equation Eq. (1), based on a double-ID method [16, 17]: \nwhere Cx is the analyte concentration in the sample (mol g\u22121), mx the mass of sample (g) used for the sample-spike mixture, my the mass of spike solution (g) used for the sample-spike mixture,  the mass of spike solution (g) used for the standard-spike mixture, mz the mass of standard solution (g) used for standard-spike mixture; R is the 120Sn\/118Sn ratio in the sample\u2013spike mixed solution, R\u2032 is the 120Sn\/118Sn ratio in the standard\u2013spike mixed solution, Rx is the 120Sn\/118Sn ratio in the sample solution, Ry is the 120Sn\/118Sn ratio from the spike solution, Rz is the 120Sn\/118Sn ratio in the standard solution, w is the correction factor for dry mass; n is the number of replicate measurements, and k, Ky, and K\u2032 are the mass discrimination correction factors for each isotope ratio, which were calculated from the area ratio of 120Sn\/118Sn for TPrT in each chromatographic run [4]. P is the purity of each organotin chloride reagent used for preparing the standard solutions, D is the dilution factor for each organotin chloride in the gravimetric dilution of the standard solutions, Mw the molecular weight of each organotin chloride, B the procedure blank, and E the variation introduced by extraction to the analytical results.\nDetermination of organotins by ID\u2013GC\u2013MS\nThe GC\u2013MS used was Agilent model 6890GC\/5983MSD (Agilent Technologies, Wilmington, DE, USA) with an HP-5\u00a0ms column (30\u00a0m\u00d70.32\u00a0mm i.d., 0.25\u00a0\u03bcm film thickness). The measured masses were m\/z 231 and 233 for MBT, m\/z 261 and 263 for DBT and TBT, m\/z 301 and 303 for DPhT, and m\/z 349 and 351 for TPhT. The other operating conditions and procedures for GC\u2013ICP\u2013MS analysis were similar to those described previously [3].\nThe concentrations of organotins were calculated by inserting each value into Eq. (2), below, in which it was assumed that the relationship between mass ratio and peak ratio was linear. \nwhere Cx is the analyte concentration in the sample (mol g\u22121), mx the mass of sample (g) used for the sample-spike mixture, my the mass of spike solution (g) used for the sample\u2013spike mixture, RWL the ratio of the mass in the spike solution (g) to the mass in the standard solution (lower level), RWH the ratio of mass in the spike solution (g) to the mass in the standard solution (higher level), R the measured abundance ratio in the sample\u2013spike mixed solution, RL the measured abundance ratio in the standard\u2013spike mixed solution (lower level), RH the measured abundance ratio in the standard\u2013spike mixed solution (higher level), w the correction factor for dry mass, P the purity of each organotin chloride reagent used for the preparing the standard solutions, D the dilution factor for each organotin chloride in gravimetric dilution of the standard solutions, Mw the molecular weight of each organotin chloride, B the procedure blank, and E the variation introduced by the extraction to the analytical results. The mass bias correction was not performed for GC\u2013MS measurements. In this experiment we prepared several sample\u2013spike mixed solutions of different mass ratios and chose the solutions having values of RL and RH slightly lower and higher than the R value for calculation.\nDetermination of organotin compounds by ID\u2013LC\u2013ICP\u2013MS\nThe LC\u2013ICP\u2013MS system used consisted of a PU-712i HPLC pump (Inert model; GL Science, Tokyo, Japan), a Nanospace SI-2 auto-injection sampler (Siseido, Tokyo, Japan), and an Agilent 7500c ICP\u2013MS (Yokogawa Analytical Systems, Japan). The LC column used was a Mitysil RP-18 GP (150\u00a0mm\u00d72.0\u00a0mm i.d. 3\u00a0\u03bcm). All tubing used was 1\/16 in (0.13\u00a0mm i.d.) PEEK. The mobile phase was acetonitrile\u2013water\u2013acetic acid\u2013tetraethylammonium chloride\u2013tropolone 65:30:5:0.1:0.075 (%, v\/v). The mobile phase flow rate was 200\u00a0\u03bcL min\u22121 and the sample volume injected was 5\u00a0\u03bcL. The sample-introduction device for ICP\u2013MS was modified from the default settings as follows: a PFA \u03bcflow of 50\u00a0\u03bcL min\u22121 was used as a nebulizer, the double-path Scott type spray chamber was cooled to \u22125\u00a0\u00b0C, a 1.0\u00a0mm dimmer injector torch was used, and additional O2 (0.1\u00a0L min\u22121) gas was mixed with the argon axial gas flow via a T-adaptor. The LC column was directly connected via a 1\/16 PEEK adaptor. The concentrations of organotins were calculated by use of Eq. (1).\nHomogeneity study\nThe between-bottle homogeneity of the CRM was determined by analyzing sub-samples taken from ten bottles selected from the lot of 250 bottles. All the organotin compounds were determined by ID\u2013GC\u2013ICP\u2013MS after ultrasonic extraction. Analysis of variance (ANOVA) of the data was performed and mean squares within group (MSwithin) and among group (MSamong) were calculated. Then standard deviations between bottles (sbb) were calculated by use of Eq. (3): \nIf the measurement method was insufficient repeatable, the effect of analytical variation on the standard deviation between units ubb was calculated and used to estimate the inhomogeneity [18]. The ubb was calculated by use of Eq. (4): \nwhere denotes the degrees of freedom of MSwithin.\nResults and discussion\nHomogeneity study\nIn the homogeneity study, between-bottle inhomogeneity (sbb) was only observed for TPhT, so ubb was used as uncertainty-derived inhomogeneity for TPhT. A relatively large sbb value (7.1%) was obtained for DPhT, although this value was not very large compared with uncertainties from other sources, for example the uncertainty of analytical results and between-method variance, as is shown later. These results indicate that this material may be considered homogeneous for butyltin and phenyltin analysis.\nStability of organotins in this material\nWe have been monitoring the stability of the butyltin compounds in NMIJ CRM7301-a since 2001. All three butyltins were sufficiently stable during storage for five years at \u221230\u00a0\u00b0C even though CRM7301-a was not sterilized. The new CRM 7306-a contains almost the same butyltin concentrations as CRM7301-a, its sample composition was also similar, and it was sterilized with \u03b3-ray-irradiation. Thus, the butyltins in CRM 7306-a will also be stable for at least five years. Both the phenyltin and butyltin compounds in BCR CRM646, which was pasteurized at 100\u00a0\u00b0C, have been shown to be stable when the material is stored at below \u221220\u00a0\u00b0C [19]. Therefore, we decided to store CRM7306-a at \u221230\u00a0\u00b0C and to assess the stability of the material by measuring each organotin by ID\u2013GC\u2013ICP\u2013MS over a period of 1 year, and that for CRM7301-a.\nCharacterization of the synthesized 118Sn-enriched organotins\nCharacterization of the synthesized 118Sn-enriched organotins was performed by GC\u2013ICP\u2013MS after mixing of each, dilution with methanol, and ethylation with NaBEt4.\nFigure\u00a02 shows the GC\u2013ICP\u2013MS chromatograms obtained at m\/z 118 and 120 for the synthesized 118Sn-enriched organotins mixture. The 118Sn-enriched DPhT and TPhT could be synthesized individually. The molar ratio of the 118Sn-MBT, 118Sn-DBT, and 118Sn-TBT in the synthesized mixture measured by GC\u2013ICP\u2013MS was approximately 7:10:5.\nFig.\u00a02GC\u2013ICP\u2013MS chromatograms of 118Sn and 120Sn obtained for the mixture of 118Sn-enriched organotin compounds. The chromatogram of 118Sn was shifted 9\u00a0s and 7500\u00a0cps for clarity\nTen replicate measurements of the ethylated 118Sn-enriched organotin mixture by GC\u2013ICP\u2013MS were performed to obtain the 120Sn\/118Sn ratio for each compound. The mass bias was corrected with the in-run correction method with TPrT [4]. The 120Sn\/118Sn isotope ratios for 118Sn-butyltins were 0.055\u00b10.001 (mean\u00b1standard uncertainty, n=10), with no significant differences. The 120Sn\/118Sn isotope ratios for 118Sn-enriched DPhT and TPhT were 0.0020\u00b10.0002 and 0.00020\u00b10.0003, respectively.\nAssay for the standard solution of the organotins\nTo obtain a standard solution with a well-defined concentration, an assay was performed to determine the purity of the natural abundance organotin chloride reagents used to prepare the standard solutions. Details of the assay have been described elsewhere [4]. In brief, the amounts of organic and inorganic tin species impurities in the organotin chloride reagents were estimated by GC\u2013ICP\u2013MS. The inorganic impurities, except for Sn, were checked by ICP\u2013MS. The non-organotin organic impurities were also checked by GC\u2013FID. The water content of the organotin chloride reagents were evaluated with a Karl\u2013Fisher coulometric titrator (Model CA-05; Mitsubishi, Japan).\nThe main impurities in the organotin chloride reagents used are water and tin species. Small amounts of inorganic and organotin impurities were observed in both the ethylated MBT and ethylated DBT solutions, and significant amounts of several organotin impurities were observed in the ethylated TBT, ethylated TPhT, and ethylated DPhT solutions. Among the organotin impurities observed in each ethylated organotin solution, inorganic tin, MBT, DBT and tetrabutyltin (TeBT), MPhT, and DPhT were identified from their retention times in GC\u2013ICP\u2013MS measurement and isomers of TBT (iso-TBT) and dioctyldibutyltin (DOcDBT) were identified from the fragment-ion patterns obtained by GC\u2013MS. Two organotin impurities that could not be identified from their retention time were also observed in the ethylated-MBT solution, but the amounts of those were small (<0.03%). The purities of the organotin chloride reagent obtained were 99.4\u00b10.2% for MBT, 99.5\u00b10.2% for DBT, 96.8\u00b10.2% for TBT, 98.6\u00b10.2% for DPhT, and 98.8\u00b10.2% for TPhT.\nThe stability of the standard solution during certification is also important. Van et al. reported that DPhT in methanol was not stable because of its disproportionation reaction, although solutions of the butyltins and TPhT were found to be stable [20]. DPhT also reacted with MBT in mixed solutions in methanol. In contrast, Arnold et al. reported that DPhT in methanol containing 0.01\u00a0mol L\u22121 HCl was stable for six months [21]. Hence, the stability test for DPhT in methanol with and without 0.005\u00a0mol L\u22121 HCl was performed by GC\u2013ICP\u2013MS after storage at \u221220\u00a0\u00b0C for one month. Redistribution of DPhT to MPhT and TPhT was observed for the methanol solution, as has been described elsewhere [20]. In contrast, no degradation or redistribution of DPhT was observed for the methanol solution containing 0.005\u00a0mol L\u22121 HCl. Plazzogna et al. reported that the redistribution reaction between monomethyltrialkyltin (R3SnMe) and dimethyltin dichloride (Me2SnCl2) in methanol is occurred with dissociation of Me2SnCl2 [22]. They also reported that the redistribution reaction can be prevented by addition of chloride ion, as NaCl, which inhibits the dissociation of Me2SnCl2 in methanol. For DPhT in methanol the disproportionation reaction occurred with dissociation of DPhTCl2, and was prevented by addition of HCl, which inhibited the dissociation. Consequently, the standard solution of DPhT was prepared by dissolving DBTCl2 in methanol containing 0.005\u00a0mol L\u22121 HCl to prevent the self-disproportionation reaction.\nEvaluation of the degradation of DBT and TPhT during extraction\nBecause SSID\u2013MS methodology cannot compensate for non-quantitative extraction from the sample, aggressive conditions are required for solid\u2013liquid extraction of strongly adsorbed species, for example MBT. Degradation or interconversion of species can occur under the aggressive extraction conditions [6\u201311]. Indeed, it has been reported that degradation of DBT occurs during microwave assisted extraction [6] and pressurized liquid extraction [7] when high-energy microwaves or high extraction temperatures are used. Degradation of TPhT during sample pretreatment has also been reported [11, 12]. In this certification, therefore, degradation of DBT and TPhT during extraction was checked using an 117Sn-enriched DBT solution obtained from the LGC, UK [15] and the single solution of the synthesized 118Sn-enriched TPhT. Both 117Sn-enriched DBT and 118Sn-enriched TPhT solutions were used to spike the sediment sample and then the four extraction methods were performed. After derivatization and clean-up the extract was analyzed by GC\u2013ICP\u2013MS. Because the 118Sn-enriched TPhT solution does not contain any phenyltins, degradation of TPhT in each extraction was evaluated from the difference between the natural abundance isotope ratio and the measured ratios of 120Sn\/118Sn for DPhT. On the other hand, the 117Sn-enriched DBT solution contains small amounts (ca. 0.7%) of 117Sn-enriched MBT as an impurity. Therefore, the degradation of DBT in each extraction was evaluated from the difference between the natural abundance isotope ratios and the ratios of 118Sn\/117Sn for MBT, corrected for 117Sn-enriched MBT. In this experiment, the measured 120Sn\/118Sn ratios for DPhT obtained for all four extraction methods closely matched the natural abundance, and the results suggest that no significant degradation of TPhT to DPhT occurred during extraction. The degradation of DBT to MBT was also not observed in ultrasonic extraction and mechanical shaking extraction. Slight degradation of DBT to MBT was observed in microwave-assisted extraction MAE (0.8%) and in pressurized liquid extraction PLE (0.5%); these degradation levels were lower than those reported by Encinar [6, 7] (2\u20133% in both extractions under their optimized conditions). These differences could be because of different extraction conditions. The sample size used in our experiments was two to four times that used by Encinar, and the concentrations of acetic acid we used were lower. In addition, tropolone was used as a chelating reagent in all the four extraction methods. Formation of the tropolone-chelating complex might prevent degradation. The slight degradation of DBT observed would not significantly affect the analytical results obtained by use of MAE and PLE because the typical uncertainty of isotope-ratio measurement by GC\u2013ICP\u2013MS in this study was in the range 0.5 to 1%, and the relative standard deviations of the analytical results for DBT obtained by use of MAE and PLE were approximately 3%. As is shown later, the analytical results obtained by use of the four different extraction methods were in good agreement within the range of uncertainties. These results indicate that analytical biases caused by degradation or inter-conversion of analytes in the extraction would be negligible in this certification.\nAnalytical results obtained by each method\nDetermination of the organotin compounds in the candidate material by the six analytical methods was performed for the certification. Figures\u00a03, 4 to 5 show the GC\u2013ICP\u2013MS, GC\u2013MS, and LC\u2013ICP\u2013MS chromatograms obtained for the sample extract spiked with the 118Sn-enriched organotin compounds. It is apparent from Figs.\u00a03 and 4 that adequate sensitivity for all the organotins was obtained in GC\u2013ICP\u2013MS and GC\u2013MS measurements. Adequate sensitivity for the organotin compounds, except for DPhT, was also obtained in LC\u2013ICP\u2013MS; the sensitivity for DPhT was insufficient to obtain a reliable isotope-ratio measurement. The relative standard deviation for DPhT in LC\u2013ICP\u2013MS measurement was approximately 10% in triplicate measurement, and thus variation of the analytical results obtained was quite large. Thus, the analytical results for DPhT obtained by ID\u2013LC\u2013ICP\u2013MS were not adopted for the certification. The analytical results, with their uncertainties, obtained by both analytical techniques are summarized in Table\u00a01. The values were calculated as mass fractions (based on dry mass). The combined standard uncertainties for the analytical results obtained by each method were calculated by use of Eqs. (1) or (2). The uncertainties related to the standard solutions (uncertainty of P and D in Eqs. (1) and (2)) were not combined into uc because the same reagents were used for both analyses. They were combined later when the uncertainties in the certified values were calculated.\nFig.\u00a03GC\u2013ICP\u2013MS chromatograms of 118Sn and 120Sn obtained for the sample extract spiked with 118Sn-enriched butyltin and phenyltin compounds. The chromatogram of 118Sn was shifted 7\u00a0s and 2000\u00a0cps for clarityFig.\u00a04GC\u2013MS chromatograms of each measured mass obtained for the sample extract spiked with 118Sn-enriched butyltin and phenyltin compounds. The chromatogram for 118Sn was shifted 9\u00a0s and 500\u00a0cps for clarityFig.\u00a05LC\u2013ICP\u2013MS chromatograms of 118Sn and 120Sn obtained for the sample extract spiked with 118Sn-enriched butyltin and phenyltin compounds. The chromatogram of 118Sn was shifted 9\u00a0s and 2500\u00a0cps for clarityTable\u00a01Analytical results obtained by use of six combinations of extraction and measurement methods\u00a0\u00a0Result\u00b1combined standard ua (\u03bcg kg\u22121 as Sn)ExtractionbMeasurementTBTDBTMBTTPhTDPhTUSEGC\u2013ICP\u2013MS44.3\u00b11.151.2\u00b10.965.9\u00b11.17.8\u00b11.43.4\u00b10.3USEGC\u2013MS43.8\u00b11.750.7\u00b10.866.6\u00b11.75.8\u00b11.53.9\u00b10.4USELC\u2013ICP\u2013MS44.2\u00b11.750.7\u00b10.865.6\u00b11.77.4\u00b11.9\u2013cMAEGC\u2013ICP\u2013MS43.8\u00b11.552.1\u00b10.968.2\u00b11.27.0\u00b11.02.9\u00b10.13PFEGC\u2013ICP\u2013MS42.9\u00b11.250.1\u00b11.067.1\u00b10.86.3\u00b10.63.7\u00b10.2MWEGC\u2013ICP\u2013MS47.0\u00b11.352.5\u00b11.267.6\u00b10.88.7\u00b11.74.4\u00b10.8aCalculated by use of Eqs. (1) or (2)bUSE: ultrasonic extraction, MSE: mechanical shaking extraction, PFE: pressurized fluid extraction, MAE: microwave assisted extractioncTechnically invalid\nThe analytical results obtained by use of the six methods were in good agreement within the range of their uncertainties; this agreement may indicate there were no significant analytical biases between measurement and extraction techniques for all the analytes. Therefore, all the analytical results obtained were treated equally for calculation and evaluation of the certified values and their uncertainties.\nEstablishing certified values\nThe analytical results in Table\u00a01 were combined to provide the certified values listed in Table\u00a02. Certified values are the weighted means of these results from the six methods, where 1\/ui (ui is the uncertainty of each result) was used as a weight. Certified values are available for the concentrations of MBT, DBT, TBT, DPhT, and TPhT, as tin. \nTable\u00a02Certified values and their uncertainties for mass fractions of organotin compounds in NMIJ CRM 7306-a\u00a0TBTDBTMBTTPhTDPhTCertified value (mass fraction, \u03bcg kg\u22121 as Sn)4451676.93.4Relative standard uncertainty (%)\u00a0Calibration solution ucal0.3%0.3%0.3%0.5%1.0%\u00a0Analytical results uanal1.3%0.7%0.7%7.0%3.3%\u00a0Between method umethod1.6%1.0%0.2%\u2013\u2013\u00a0In-homogeneity sbb or ubb1.9%1.9%2.0%5.8%7.1%Combined uncertainty\u00a0Relative (%)2.8%2.3%2.1%9.1%7.9%\u00a0Absolute (\u03bcg kg\u22121 as Sn)1.31.21.40.60.3Expanded uncertainty U (k=2) (mass fraction, \u03bcg kg\u22121as Sn)3231.30.5\nUncertainty of the certified values\nThe uncertainties of the certified values included the combined effects of method imprecision, possible bias effects among methods, material inhomogeneity, and stability. Components of the uncertainty of each certified value are listed in Table\u00a02. Because the same reagents for calibration solutions were used for all the measurements, uncertainty of the calibration solutions (ucal) was combined with the uncertainty of the certified values separately from the uncertainty of the analytical results, as is described above. The property value was the weighted mean of the two results, and so the combined uncertainty of each analytical result (uanal) was given by Eq. (5): \nwhere xi are the results obtained by use of the six methods and wi are their weights. The between-method variance (umethod) was calculated from ANOVA on the data from the three techniques. The uncertainties derived from material inhomogeneity (ubb) were the estimates in the homogeneity study. As is described above, the butyltin and phenyltin compounds would be stable at lest five years at our storage temperature (\u221230\u00a0\u00b0C); we did not include the uncertainty from stability. The expanded uncertainty in each certified value is equal to U=kuc, where uc is the combined standard uncertainty, with coverage factor k=2, corresponding to 95% confidence intervals.\nComparison with other CRMs\nThe certified values for CRM7301-a and the CRM7306-a are summarized in Table\u00a03. In comparison with the CRM7301-a, the uncertainties of the certified values for DBT and MBT in CRM7306-a are significantly smaller, despite the similar concentrations of the compounds. The main contributors to the uncertainty in the certified values for DBT and MBT in CRM7301-a were ubb and uanal [3]. The homogeneity of CRM 7306-a was improved by remixing after X-ray sterilization, and the reliability of the analytical methods used for certification was also better than those used for CRM7301-a. Thus, these improvements may lead to the small uncertainties. \nTable\u00a03Certified values for butyltin and phenyltin in MNIJ7306-a and other CRMs certified by use of SSIDMS (unit:\u00a0\u00b5g\u00a0kg-1 as Sn)\u00a0TBTDBTMBTTPhTDPhTCRM7306-a44\u00b1351\u00b1267\u00b136.9\u00b11.23.4\u00b10.5CRM7301-a44\u00b1456\u00b1656\u00b16HIPA-178\u00b19SOPH-1125\u00b17174\u00b19PACS-2890\u00b11051047\u00b164\nMost recently, several sediment reference materials for butyltin analysis certified by use of SSID\u2013MS have been issued by the National Research Council Canada (NRCC, Canada). The certified values of the CRMs are also summarized in Table\u00a03. HIPA-1 and SOPH-1 were certified, and PACS-2 was recertified by NRCC. Their certified and re-certified values were based on results from the Comite Consultatif pour la Quantite de Matiere (CCQM) comparisons [23\u201325], in which we participated. These CRMs have certified values for DBT and TBT only, and their concentrations are higher than those in CRM7301-a and CRM7306-a.\nThe BCR 646 freshwater sediment for butyltins and phenyltins are also available from the European Commission Joint Research Centre (IRMM, Belgium), although SSID\u2013MS was not used for the certification. The certified concentrations for BCR CRM646 are higher than those for CRM7306-a.\nIn Japan, typical butyltin and phenyltin concentrations observed in environmental monitoring are not as high as they used to be. Reported TBT concentrations in sediment were below 80\u00a0\u03bcg kg\u20131, as Sn, in environmental monitoring by the Ministry of the Environment Japan in 1998 [26]. By using these CRMs properly, a wide concentration range can be covered in routine laboratories.","keyphrases":["certification","sediment","certified reference material","organotin compounds","species-specific isotope dilution"],"prmu":["P","P","P","P","R"]}
{"id":"Purinergic_Signal-3-1-2096771","title":"Chemotactic activity of extracellular nucleotideson human immune cells.\n","text":"Purinergic P2 receptors are a class of plasma membrane receptors that are express in many tissues and are ligated by extracellular nucleotides [such as adenosine triphosphate (ATP), adenosine diphosphate (ADP), uridine 5\u2013triphosphate (UTP) and uridine 5\u2013diphosphate (UDP)], which are released as a consequence of cell damage, cell stress, bacterial infection or other noxious stimuli. According to the molecular structure, P2 receptors are divided into two subfamilies: P2X and P2Y receptors. The P2X receptors are ligand-gated channels, whereas P2Y receptors are G-protein-coupled seven-membrane-spanning receptors. Several studies indicate that nucleotides play an important role in immune response modulation through their action on multiple cell types, including monocytes, mast cells, dendritic cells, neutrophils, and eosinophils. Recent work by our group and others identified extracellular nucleotides as chemotaxins for various human immune cells, including eosinophils, neutrophils and dendritic cells. In this review, we summarise recent findings in this field and put forward a hypothesis on the role of P2 receptors in the early recruitment of human immune cells to the site of inflammation.\nIntroduction\nCell migration plays a key role in a wide variety of biological processes, such as embryogenesis, development, angiogenesis, haematopoiesis, immune response and inflammation. In inflammation and host defence, the targeted trafficking of immune cells to tissues and\/or lymphoid organs is one of the essential steps. Migration of the different leukocytes is tightly controlled by chemokines. These chemotactic cytokines are secreted proteins with a molecular weight of 8\u20130 kDa, which direct cellular traffic along ingeniously regulated concentration gradient in the extracellular space. Based on amino acid alignments, chemokines are divided into four families. According to the position and the spacing of the first two conserved cysteines or the lack of them, these families are distinguished as either C, CC, CXC or CX3C. Until now, C and CX3C are composed of only one member each, lymphotactin and fractalkine, respectively, whereas CC and CXC each consist of numerous, well-characterised members [1, 2]. The chemotactic effects of these molecules are mediated due to their interactions with different specific serpentine receptors that span the plasma membrane seven times and belong to the G-protein-coupled receptor family.\nBesides chemokines, many constitutive molecules can regulate the function of leukocytes and thereby modulate immune responses, e.g. following tissue damage, intracellular localised substances can be released into the extracellular space. For this reason, an increase of the extracellular concentration of certain molecules can be a very simple sign of cell damage. However, for constitutive molecules to function as chemotaxins, it is essential that they are recognised by immune system migration cells. In the past few years, evidence has accumulated strongly suggesting that nucleotides fulfil these requirements. They are present at high concentrations (5\u20130 mM) in the cytoplasm of all cells, whereas in the extracellular compartment, their concentration is in the nanomolar range. Nucleotides can be released into the extracellular space via nonlytic mechanisms through regulated transport; e.g. adenosine triphosphate (ATP) has been reported to be secreted by different cell types in a broad variety of conditions, such as shear stress, endotoxin stimulation, or at sites of platelet aggregation [3, 4] Hence, in tissues, nucleotides are able to generate concentration-dependent gradients, which can serve as chemotactic signals for different immune cells, causing migration.\nP2 receptors\nP2 receptors are subdivided on the basis of pharmacological, functional and cloning data into two families: the P2YR and P2XR [5\u20138]. P2YR are seven-membrane-spanning, G-protein-coupled receptors, and eight different P2YR subtypes have been cloned so far (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y14) [5, 9, 10]. Activation of P2YR induces phospholipase C activation, inositol triphosphate generation, Ca2+ release from intracellular stores, and\/or stimulation\/inhibition of adenylate cyclase. Extensive pharmacological studies performed in P2Y transfected cells revealed that P2Y1, P2Y11, P2Y12 and P2Y13 selectively interact with ATP and\/or adenosine diphosphate (ADP), whereas uridine 5\u2013triphosphate (UTP) and uridine 5\u2013diphosphate (UDP) are inactive [11\u201315]. In contrast the P2Y2, P2Y4 and P2Y6 subtypes are responsive to uridine nucleotides [16\u201318]. Whereas ATP and UTP activate P2Y2 with similar efficiency, UTP and UDP are most the potent agonists at P2Y4 and P2Y6, respectively [16\u201318]. In addition, it has been shown that P2Y14 specifically responds to UDP glucose and related sugar nucleotides but not to ATP, ADP, UTP or UDP [5, 9, 19, 20]. P2YR have been shown to modulate multiple cell function of various human immune cells, including cytokine release from dendritic cells, reactive oxygen metabolite production from neutrophils and eosinophils or chemokine release from airway epithelial cells [20\u201323].\nP2XR are multimeric ligand-gated plasma-membrane ion channels activated by extracellular ATP and selective for monovalent and divalent cations [8, 24, 25]. At this time, seven different monomers have been cloned: the P2X1\u2013P2X7 subtypes. Activation of P2XR leads to increased plasma membrane permeability to ions (Na+, K+, and Ca2+) and induction of apoptosis in human immune cells [22, 26] . In contrast to P2YR, the only currently known physiological ligand for all P2XR subtypes is ATP.\nP2 receptors and migration of neutrophils\nThe first report that extracellular nucleotides can modulate human neutrophil function was a paper by Ward et al. showing that ATP and ADP can induce superoxide anion formation [27, 28]. Furthermore, it has been shown that human neutrophils or human promyelocytic HL60 cells respond to ATP, ATP\u03b3S and UTP with an increase in intracellular Ca2+ concentration via a pertussis toxin-sensitive G-protein receptor that coupled to the inositol phospholipid signaling system, suggesting involvement of P2Y subtypes [27, 29\u201333]. Activation of these intracellular signal-transduction systems is of great interest in the light of neutrophil recruitment to the site of inflammation, as increase in Ca2+ concentration is an important step in human neutrophil migration [34\u201336]. Furthermore, it has been shown that ATP also increases membrane expression of CD11b\/CD18 and adhesion to albumin-coated polystyrene latex beads [37].Up-regulation of these molecules enhances the adhesion of neutrophils to other cells, e.g. between neutrophils and pulmonary endothelial cells, and could be of relevance for neutrophil migration across the vessel wall. However, after earlier observations suggesting that nucleotides might be chemoattractant for human neutrophils, it was not until the end of the 1990s that ATP and UTP were shown, by Verghese et al. [103] to induce actin polymerisation and chemotaxis in human neutrophils via the activation of P2Y2 (formerly known as P2U) receptor.\nSurprisingly, the first reverse transcripaise polymerase chain reaction (RT-PCR) data revealed that human neutrophils express only P2Y4 and P2Y6 but not P2Y1 and P2Y2 receptors [38]. However, recent reports showed that human neutrophils also express messenger ribonucleic acid (mRNA) for the P2Y2 and P2Y11 receptor subtypes [39, 40]. Among the P2X receptors, so far, only the present of the P2X7 receptor has been shown by Northern blotting and immunocytochemistry [41]. Because increased extracellular nucleotide concentrations are associated with cell damage\/injury and human neutrophils are the initial cell type found at tissue injury sites, the conclusion could be drawn that extracellular nucleotides among other mediators are involved in the recruitment of neutrophils to the site of inflammation.\nP2 receptors and recruitment of human eosinophils\nBesides neutrophils, eosinophils also express P2 receptors. Several studies showed that human eosinophils express mRNA for the following P2Y and P2X subtypes: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y14, P2X1, P2X4 and P2X7 [23, 42\u201344]. Stimulation of P2 receptors expressed by eosinophils induces multiple cell responses, including production of reactive oxygen metabolites and secretion of eosinophil cationic protein (ECP) [23, 26].\nBurgers and colleagues [45] showed that ATP, secreted by thrombin-activated platelets, was able to raise eosinophil intracellular Ca2+ concentration and made the cells chemotact towards platelets. These seminal studies were later confirmed by the identification of eosinophil P2Y and P2X receptors [42, 44] and the observations that nucleotides also induce up-regulation of adhesion molecule CD11b and actin polymerisation to important features involved in blood eosinophil recruitment to tissue [23, 42, 44].\nIn addition to direct chemotactic influences, ATP might also have indirect effects on eosinophil recruitment. We recently showed that ATP and UDP induce secretion of interleukin (IL)-8 by eosinophils [26]. This chemokine is a potent attractor for eosinophils themselves (and neutrophils), i.e. they are able to recruit more cells to inflammation sties. Increased secretion of IL-8 has been described in eosinophils from patients with bronchial asthma or atopic dermatitis [46]. Moreover, IL-8 concentration in bronchoalveolar fluids from asthmatic patients is increased significantly in comparison with that of healthy subjects [46]; therefore, one can suggest the involvement of different nucleotides in the direct or IL-8-mediated recruitment of eosinophils and thus in the development and maintenance of allergic diseases.\nP2 receptors and mast cells\nMast cells are situated around blood vessels and nerves, especially at interfaces with the external environment, emphasising their role in immunity. They express several mRNAs that encode P2XR and P2YR subtypes [47, 48]. Human mast cells express P2X1 and P2X4, whereas the P2X7 receptor subtype is only expressed by human-cord-blood-derived mast cells when activated with anti-immunoglobulin (Ig)E [47]. Among the P2Y receptors, the presence of P2Y1, P2Y2, P2Y11, P2Y12 and P2Y13 subtypes were shown by RT-PCR [48]. Moreover, ATP and UTP enhance histamine release by human lung mast cells stimulated by cross-linkage of the fragment crystallisable (Fc)\u03b5RI [49]. This effect is attributed to the P2Y2 receptor. Data on chemotactic effects of nucleotides on mast cells are rare, but there is evidence that they might effect the migration of these cells. For example, McCloskey and colleagues showed that the nucleotides ADP, ATP and UTP are effective chemoattractants for rat-bone-marrow-cultured mast cells [50]. However, whether nucleotides can also induce migration of human mast cells remains to be elucidated\nP2 receptors and lymphocytes\nThe first evidence for a role of extracellular nucleotides in human lymphocyte responses has been present for some time [51\u201353], but a systemic analysis of the expression and function of P2 receptors in human lymphocytes was only started at the end of the 1980s [30, 54\u201357]. Human B lymphocytes express both P2X and P2Y receptors [58, 59]. The presence of different P2Y receptors is indicated by the ability of ATP and many other nucleotides to trigger Ca2+ release from intracellular stores [60, 61] and the finding of P2Y1, P2Y2, P2Y4 and P2Y6 receptor-specific mRNA in lymphocytes [38]. Different studies suggest that human B cells express at least P2X7 receptor [22], but the identification of other P2X receptor subtypes is limited by the absence of specific antibodies [62, 63]. Nevertheless, confocal microscopy studies using anti-P2X polyclonal antibodies suggest the presence of P2X1, P2X2, P2X4 and P2X7 subtypes on human B lymphocytes [59]. However, despite the presence of functional P2 receptors, data about chemotactic effects on B lymphocytes induced by nucleotides are still missing.\nFunctional and pharmacologic studies revealed that human peripheral T lymphocytes express P2X-like ATP-activated channels, most likely P2X1, P2X4 and P2X7, [22, 64, 65]. Functional activity of the P2X receptors on T cells has been shown by a large influx of Na+ and Ca++ from the extracellular medium caused by ATP and 3\u2013O-(4-benzoyl)benzoyl-ATP (Bz-ATP) [65].\nThe expression of P2Y receptors subtypes is still unclear, and whereas Baricordi and coworkers described a lack of functional P2Y receptors expression [65], recent studies using complete lymphocyte populations (T and B cells) could detected all the target genes for P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12 and P2Y13 [66]. However, a functional expression of different P2Y receptors on human T lymphocytes has not been proven.\nStudies showing that activation of the P2X7 receptor by ATP and Bz-ATP induced shedding of CD23 and L-selectin from B and T lymphocytes of a B-chronic lymphocytic leukaemia (B-CLL) patient and from normal subjects, a classic effect of chemoattractants [67\u201370], leads to the assumption that the P2X7 receptor might be involved in the transendothelial migration of lymphocytes [71, 72]. But elegant experiments from Chen and coworkers using P2X7 antagonist in in vitro migration assays indicated that ATP is neither a chemoattractant that stimulates transmigration of lymphocytes nor an agonist that mediates the global L-selectin loss during transendothelial migration [73]. In any case a direct effect of extracellular nucleotides on T and B lymphocyte migration has not yet been investigated and, therefore, the role of nucleotides in the recruitment of lymphocytes remains unclear.\nP2 receptor in monocyte\/macrophages\nAlthough the first report on a potential role of exogenous nucleotides on mouse macrophage function was a paper by Cohn and Parks from 1967 [74], a systemic investigation on human monocytes\/macrophages was only started in the 1990s [30, 38, 75\u201378]. Freshly isolated blood monocytes have been shown to express mRNA for the following P2Y and P2X subtypes: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 P2X1, P2X4 and P2X7 [38, 66, 79]. However, other investigations found a lack of functional P2X7 in these cells, whereas the receptor appears during maturation of monocytes to macrophages [22]. Besides P2X7 receptors, human macrophages or macrophage cell lines have been described to express P2Y2, P2Y4 and P2Y6 receptors [30, 80, 81].\nActivation of P2 receptors expressed by human monocytes\/macrophages induces multiple cell responses, including increase of intracellular calcium concentration, induction of apoptosis, generation of reactive oxygen intermediates, NO generation and secretion of IL-1\u03b2, tumour necrosis factor (TNF)-\u03b1 or IL-18 [22, 30, 77, 79, 82, 83]. In the light of migration, it is of great interest that extracellular ADP causes increased surface expression of MAC-1 (alpha M beta 2 integrin, CD11b\/CD18) on monocytes [84] and that nucleotides can induce the adherence of monocytes to surfaces [38], which could be of relevance for monocyte migration across the vessel wall. Accordingly, migration of the mouse macrophage line J774 towards ADP has been demonstrated [50]. Interestingly, Warny et al. demonstrated that UDP activates IL-8 gene expression and IL-8 release in human monocytic cells [85]. Because IL-8 is a central mediator in inflammation and an important chemotactic factor for various cells, including neutrophils, eosinophils and CD16+ natural killer (NK) cells [46, 86\u201389], UDP [through its action on dendritic cells (DC)] might be indirectly involved in the recruitment of these cells to the side of inflammation. However, at this time, no information on the chemotactic activity of nucleotides on human monocytes\/macrophages is available.\nP2 receptors in dendritic cells\nDCs are powerful antigen-presenting cells that circulate in the bloodstream or reside in peripheral tissues. They are characterised by a high antigen uptake capacity, recognition of constitutive or inducible endogenous \u2018danger signals\u2013provided by surrounding cells and a high responsiveness to chemotactic signals. The migratory ability of DCs is one of the main features in the initiation of immune responses. After acquiring antigens in the peripheral tissue, DCs migrate to the draining mediastinal lymph nodes to activate naive T cells [90\u201392]. Besides this \u2018classic\u2013action, some evidence also suggests that tissue-resident DCs are able to uptake tissue antigens and to migrate to the afferent lymph nodes, even in the absence of inflammatory conditions, thus contributing to tolerance maintenance [93, 94].\nIn the last few years, DCs came into the focus of researchers of the purinergic field, and the role of P2 receptors in DC migration came to the fore. RT-PCR analysis revealed that human DCs express a broad variety mRNAs for at least four subtypes of the P2X receptor family (P2X1, P2X4, P2X5, P2X7) and eight subtypes of the P2Y receptor family (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11 P2Y12 P2Y13, P2Y14) [95\u201399].\nFirst, studies by Liu et al. revealed that at least activation of the P2Y1, P2Y2 and P2Y4 mediates calcium release from intracellular storage. Furthermore, the observation that DCs redirect their dendrites towards a nearby patch pipette leaking ATP suggested that P2YR might mediate DC chemotactic response [96]. Indeed, ATP and UTP, probably via activation of the P2Y2, as well as ADP (via P2Y1?) turned out to be potent chemotactic stimuli for immature but not for mature DCs [100] In contrast, P2X receptor activation had only marginal chemotactic activity in both immature and mature DCs. Chemotaxis was paralleled by other intracellular signalling events, such as actin polymerisation and intracellular Ca2+ mobilisation. Recently, UDP could be added to the list of chemoattractant nucleotides, as it has been shown that UDP via binding to the P2Y6 receptor increased intracellular calcium, induced actin polymerisation and migration of immature, but again not mature, DCs [101]. The discrepancy between the responsiveness to extracellular nucleotides of immature and mature DCs could be explained by functional studies. They revealed a selective down-regulation of the Gi\/o protein-coupled chemotactic P2Y receptor responsiveness during maturation. Surprisingly, immature and mature DCs expressed similar amounts of mRNA for the purinergic receptor subtypes P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2X1, P2X4 and P2X7 [100]. DC maturation encompasses a coordinated down-regulation of inflammatory chemokine receptors (CCR1, CCR2, CCR5 and CXCR1) and induction of CCR7 and CXCR4 [2]. In addition, during maturation, functional down-regulation of chemotaxis-regulating P2Y receptors are uncoupled to chemotaxis-associated signal transduction pathways. As a result, DCs lose sensitivity to inflammatory chemokines as well as to the nucleotides ATP, ADP, UTP and UDP.\nIn addition, nucleotides can modulate the migration of DCs to chemokines, as immature and matured DCs stimulated with ATP gain the ability to migrate in response to CXC ligand (L)12 and CCL12 [102]. However, in contrast, Schnurr et al. reported that ATP through P2Y11 signalling could inhibit CCL21, induce migration of immature and mature monocyte-derived DCs and CD1a+ dermal DCs but not of CD1c+ peripheral blood DCs or IL-3R+ plasmacytoid DCs [98]. This controversy could be due to differences in the blood donors or preparation of monocyte-derived DCs in the different laboratories.\nBesides direct chemotactic effects on DCs, extracellular nucleotides are also involved indirectly via their action on DCs in the trafficking of other leukocytes through the release of chemokines. For example, ATP up-regulates the constitutive production of CCL22 [macrophage-derived chemokine (MDC)] and inhibits the lipopolysaccharide (LPS)-induced secretion of CXCL10 (IP-10) and CCL5 (RANTES), resulting in selectively impaired recruitment of type 1 but not type 2 T cells, suggesting a nucleotide-mediated communication between DCs and T cells an important event during antigen presentation in vivo [102]. In accordance, UDP can enhance the LPS-mediated release of chemotactic factor IL-8 via P2Y6 from mature DCs [101], so again, UDP might be indirectly involved in the recruitment of neutrophils, eosinophils and CD16+ NK cells to the side of inflammation.\nIn sum, activation of DCs by extracellular nucleotides leads to multiple cell responses, which results in a direct migration of DCs and also maybe indirect (DC-mediated) recruitment of other immune cells to the site of inflammation.\nConclusion\nOver the last few years, several studies have implied that extracellular nucleotides which are actively released or diffuse out of mechanically stressed, infected or injured cells might be involved in the early recruitment of immune cells to the site of inflammation\/cell damage. In these in vitro experiments, it has been shown that extracellular nucleotides (mainly by activating P2YR subtypes) are direct chemoattractants for human neutrophils, eosinophils and DCs and\/or they can modulate the chemokine production of eosinophils, monocytes and DCs, which might then influence the migration capacity of other immune cells. However, whether nucleotides play a role in the migration of immune cells in vivo still remains to be elucidated.","keyphrases":["nucleotides","immune cells","chemotaxins","purinergic receptors"],"prmu":["P","P","P","P"]}
{"id":"Histochem_Cell_Biol-4-1-2413111","title":"Tight junctions and the modulation of barrier function in disease\n","text":"Tight junctions create a paracellular barrier in epithelial and endothelial cells protecting them from the external environment. Two different classes of integral membrane proteins constitute the tight junction strands in epithelial cells and endothelial cells, occludin and members of the claudin protein family. In addition, cytoplasmic scaffolding molecules associated with these junctions regulate diverse physiological processes like proliferation, cell polarity and regulated diffusion. In many diseases, disruption of this regulated barrier occurs. This review will briefly describe the molecular composition of the tight junctions and then present evidence of the link between tight junction dysfunction and disease.\nIntroduction\nTight junctions (TJ) (zonulae occludentes) form a continuous, circumferential, beltlike structure at the boundary between the apical and the basolateral membrane domains in epithelial and endothelial cells. By constituting a regulated diffusion barrier for the paracellular pathway, TJs establish separate compartments in multicellular organisms and are also crucial for the exchange of substances between the internal and external cellular environment by the expression of tissue-specific transport proteins and channels. The transmembrane proteins constituting the TJs are attached to the cytoskeleton, thereby linking cell\u2013cell and cell\u2013substratum adhesion sites. In addition, cytoplasmic plaque proteins constitute scaffolds for TJ assembly or are involved in the regulation of processes like transcription, proliferation and differentiation into a tissue-specific regulated diffusion barrier in physiology and development. Many excellent reviews have summarised data regarding the molecular composition and function of TJs (Ebnet 2008; Gonzalez-Mariscal et al. 2003; Matter and Balda 2003a; Mitic and Anderson 1998; Schneeberger and Lynch 2004; Tsukita and Furuse 1999; Zahraoui et al. 2000), so that the scope of the current review is focused on disturbances of TJ function in human diseases.\nStructure of the tight junction\nAs the apicalmost part of the junctional complex (Farquhar and Palade 1963), the TJ forms a continuous, circumferential belt separating apical and basolateral plasma membrane domains, working as a barrier within the intercellular space and as a fence within the plasma membrane. In recent years, information on the molecular composition of TJs, in particular their transmembrane molecules, has accumulated, forming the basis of our current understanding of the structure and function of TJs in molecular terms (Fig.\u00a01). The morphology of TJs has been intensively analysed by freeze\u2013fracture electron microscopy (Staehelin 1973; Wolburg et al. 2003), where they appear as a set of continuous, anastomosing transmembraneous particle strands on the inner leaflet of the plasma membrane (P-face) with complementary vacant grooves on the outer leaflet (E-face) (Fig.\u00a02). The number and complexity of ramification of the network of TJ strands depends on the cell type, in sum yielding differences in permeability barrier function between different tissues (Staehelin 1973). However, since a direct linear relationship between the complexity of the TJ strand network and the measured transepithelial electrical resistance (TER) could not unambiguousy be established for all cell types analysed, it was predicted that the strands might contain pores that fluctuate between open and closed conformations, suggesting that the TJ strands appear to be remarkably dynamic (Claude 1978).\nFig.\u00a01Molecular composition of tight junctions. The transmembrane proteins occludin, the claudin(s) and junctional adhesion molecule-1 (JAM-1) constitute the barrier formed by TJs sealing the paracellular space. They appear to be interacting in a homophilic manner, and occludin seems to co-polymerase into claudin-based TJ strands. Claudins adhere with each other in a homotypic as well as a heterotypic manner. ZO-1, -2, and -3 bind the cytoplasmic tail of occludin and link the TJ to the actin cytoskeleton. Proteins of the ZO family can shuttle to the nucleus to influence transcriptional processes in cellular proliferation and differentiation. The ZO-proteins have also been shown to interact with claudins and provide molecular scaffolds for TJ assembly. Cingulin is a 140\u00a0kDa TJ plaque protein which assoicates with the actomyosin cytoskeleton. Its putative function is transduction of the mechanical force generated by the actomyosin cytoskeleton important for cellular differentiation. The Ras target AF-6 interacts with ZO-1 and serves as a. peripheral component of tight junctions in epithelial cells. Symplekin is a 126\u00a0kDa protein that occurs and probably functions in the nucleus as well as in the TJ plaques. Tyrosine phosphorylated Par3 regulates tight junction assembly and promotes cellular polarity by intracellular signalling. Localization of 7H6 TJ-associated antigen along the cell border of vascular endothelial cells has been shown to correlate with paracellular barrier function. Additional proteins have been localised to the TJs but a function has presently not been assigned. Moreover diverse signaling proteins are detected at the apical junctional complex but they are not uniquely confined to the TJFig.\u00a02Freeze\u2013fracture image of the rat intestinal epithelium. The freeze fracture electron micrograph showns the apical brush border, the intramembraneous particle strands of the TJs and the lateral cell surface. The replica shows a continous network of TJ strands. Adapted from (Achler et al. 1989) with permission from D. Drenckhahn, Universit\u00e4t W\u00fcrzburg. Bar 0.5\u00a0\u03bcm\nOccludin\nRecent identification of the TJ-specific integral membrane proteins forwarded our understanding of TJ molecular composition in mammals: occludin (ca. 60\u00a0kDa) was identified as the first integral membrane protein localised at TJs in chicken (Furuse et al. 1993) and then also in mammals (Ando-Akatsuka et al. 1996). The occludin transmembrane domain spans the membrane four times with a short cytoplasmic N-terminus and an especially long corboxy-terminal cytoplasmic domain (Fig.\u00a03a). Occludin localizes to the TJs, and its overexpression increases TER in mammalian epithelial cells (Balda et al. 1996). However, transfection of insect cells devoid of endogenous TJs with occludin cDNA surprisingly demonstrated that occludin is not by itself sufficient to form TJ strands but its expression merely produced focal homophilic adhesion sites (Furuse et al. 1996). Moreover, disruption of both occludin alleles in embryonic stem cells did not prevent the formation of an effective diffusion barrier and the polarisation of epithelial cells, as shown by freeze\u2013fracture analysis and TER assessment (Saitou et al. 1998). It was thus concluded that occludin is not required for the formation of TJ strands. However, occludin appears to interact, directly or indirectly, with claudins and is recruited into the long strands formed by coexpression of claudin-1 and claudin-2 (Furuse et al. 1998b, 1999). Balda et al. (1996) however reported that overexpression of both, wild type and COOH-terminally truncated occludin overexpression in cultured MDCK epithelial cells induces not only a modest increase in TER but also significant increase in the flux of the non-charged compound FITC-dextran 4\u00a0kDa. Using freeze\u2013fracture electron microscopy and immunocytochemistry, they were able to attribute this increase in paracellular permeability to an altered distribution and membrane topology between neighbouring cells.\nFig.\u00a03Transmembrane proteins of the tight junction. a Murine occludin is a 521 amino acid protein. The first extracellular domain may be involved in cell\u2013cell interaction containing a high tyrosine and glyine domain (triangles), acidic amino acids (orange-red), basic amino acids (green), neutral amino acids (cream). A second functional domain was assigned to the carboxy terminal 150 amino acids which appears to be responsible for the association of occludin with ZO-1. The occludin amino acid sequence is highly conserved between species (Ando-Akatsuka et al. 1996). b Claudins are four transmembrane domain proteins, containing two extracellular and one intracellular loop and a N- and C-terminal cytoplasmic domain. Claudins are a multigene family with so far 24 members identified in diverse species. In the first extracellular loop, claudins have a conserved common motif, GLWxxC(8\u201310aa)C (blue). Claudins further contain a PDZ-binding motif at the C terminus (orange) capable of bining to TJ plaque proteins like ZO-1. The claudins show a isotype-specific tissue expression pattern. c JAM-1 is an integral membrane protein expressed in endothelial and epithelial cells. Its extracellular domain can dimerize and bind homophilically. The intracellular domain (and in particular a PDZ-binding motif) enable JAM-1 to interact with structural and signaling proteins. JAM-1 is localized at the tight junctions of epithelial and endothelial cells and is involved in the regulation of junctional integrity and permeability. The polypeptide sequence of 299 amino acids has the typical feature observed in a type I integral membrane protein. A putative signal peptide may be cleaved between Leu23 and Val24 (pink), leaving 215 residues in the extracellular domain of the mature protein. A stretch of 17 hydrophobic residues (Ile 239 \u2192 Phe 255) presents a potential transmembrane region, and there are 45 residues in the cytoplasmic domain. The extracellular portion contains two domains with intrachain disulfide bonds typical of immunoglobulin-like loops of the V-type. Numerous PKC (blue) and Casein kinase II (yellow) phophorylation sites have been detected involved in platelet activation processes (Sobocka et al. 2000). Cys-43, -111, 146, -214 form dimer containing disulfide bridges and are labeled in black\nThe claudins\nSearching for the proteins forming the structural backbone of the TJ strands, the Tsukita group re-examined the junctional fraction prepared from chicken liver and discovered the first members of the claudin family: two 22-kDa proteins, claudin-1 and claudin-2 (Furuse et al. 1998a).\nThe claudins also have four transmembrane domains, but do not show any sequence similarity to occludin (Fig.\u00a03b). So far, 24 members of the claudin family have been identified in different species. Claudins appear to be expressed in a tissue-specific manner; some claudins are expressed only in specific cell types, e.g. claudin-5 is expressed primarily by vascular endothelial cells or claudin-11 appears to be expressed solely in oligodendrocytes and Sertoli cells (Morita et al. 1999). Moreover, most cell types express more than two claudin isoforms conferring different size and charge selectivity qualities by the amino acid sequence of their extracellular loops, and the combination of those isoforms results in cell- or tissue-type specific barrier function by \u201cheteropolymerisation\u201d into the resulting TJ strands (see below). As demonstrated by immunogold electron microscopy, there is accumulating evidence now that the claudins do constitute the TJ strands as observed by freeze\u2013fracture electron microscopy (Tsukita et al. 2001). Exogenous expression of claudins in fibroblasts devoid of endogenous TJs resulted in the formation of TJ strands.\nSummarizing existing data, there is clear evidence that the claudins constitute the backbone of TJ strands, while occludin, which itself cannot reconstitute such TJ strands, seems to play a permeability regulating role by incorporating itself into the claudin-based strands, a process, the mechanism of which however is not yet understood. The function(s) of occludin thus still remain to be elucidated.\nJunction-associated adhesion molecules (JAMs)\nA third class of integral membrane proteins comprises members of the immunoglobulin superfamily (Fig.\u00a03c), and can be subdivided into a group consisting of JAM proteins and a group consisting of CAR, CLMP, ESAM and JAM-4 (Ebnet 2008; Ebnet et al. 2004). To date, at least three JAM isoforms have been described, which are expressed differentially in epithelial and endothelial cells, but also in cells devoid of TJ strands like leukocytes (D\u2019Atri and Citi 2002). Except for JAM-1, little is known about the role of these proteins in TJ assembly and function. JAM-1, formerly F11 receptor (Sobocka et al. 2000) (Fig.\u00a03c), may be involved in immune cell transmigration or cell adhesion (Bazzoni et al. 2000) but has not been localized to the junction strands. A putative PDZ binding motif in the cytoplasmic domain of JAM-1 has been identified, which has been able to interact with PDZ domains of ZO-1, AF-6, PAR-3 and MUPP1 (D\u2019Atri and Citi 2002; Itoh et al. 2001). Moreover, JAM-1 has been shown to interact with cingulin. Thus, even though JAM-1 is not part of the TJ strands, it may well be involved in the propagation of signal cascades resulting from homophilic and heterophilic adhesion. A role for JAM-1 in the complex process of adhesion and transmigration of monocytes through endothelial cells has been demonstrated (Martin-Padura et al. 1998). As another function for the JAM proteins, a role in the assembly of TJs and regulation of paracellular permeability could be deciphered by ectopic transfection experiments (Cohen et al. 2001). Interaction of JAM-1 with occludin, so far not determined whether direct or indirect in nature, has been shown (Liu et al. 2000).\nTJ plaque proteins and the coordination of signaling at the TJ\nIn addition to their prime function as a regulated permeability barrier in the paracellular pathway and as fence in the plane of the membrane, TJs play a pivotal role in organizing such diverse processes as morphogenesis, cell polarity, cell proliferation, and differentiation, which require the coordination of signals impinging on and emanating from the plasma membrane. Mounting evidence suggests that the cytosolic TJ plaque is one of the sites in which such signaling is coordinated (Ebnet 2008; Gonzalez-Mariscal et al. 2003; Schneeberger and Lynch 2004). Since identification in 1986 of zonula occludens (ZO)-1 as the first TJ-associated protein (Stevenson et al. 1986), almost 30 additional proteins have been described associated with the cytoplasmic aspect of TJs (Schneeberger and Lynch 2004). They can be grouped into two major categories: The first are the peripherally associated scaffolding proteins like ZO-1 (ZO-2, ZO-3, AF6, and cingulin) that appear to organize the transmembrane proteins and couple them to other cytoplasmic proteins and to actin microfilaments. The second are numerous \u201csignaling\u201d proteins (ZONAB, RhoA, RalA, and Raf-1) proposed to be involved in junction assembly, barrier regulation, gene transcription, and perhaps other, presently undefined pathways.\nTJs as permeability barrier\nOne of the key functions of tight epithelia is the creation of a diffusion barrier by tight control of the transcellular and paracellular pathways. This is acomplished by asymmetrical distribution of transporters and channels for the transcellular route, and on the other side by regulation of diffusion through the paracellular space via integral TJ proteins (Turner 2006). Hereby, certain claudins participate in the formation of ion-selective pores: available data suggest that TJs on the one hand are permeable to small noncharged solutes up to a size of 0.43\u20130.45\u00a0nm in radius as shown by the use of the membrane-impermeant polyethylene glycol as a tracer, but investigations also showed that pores within the TJs are capable of discriminating between ion charge and size, which both are additionally dependent on concentration and pH (Schneeberger and Lynch 2004; van Bree et al. 1988).\nThe two extracellular loops of occludin consist solely of uncharched amino acid side chains (Fig.\u00a03a) represented mainly by an unusually high content of tyrosine and glycine (Furuse et al. 1993) so that a direct role for occludin in conferring charge selectivity is doubtful. However, occludin could increase electrical resistance by interaction with the extracellular domains of the differently charged residues of the different claudins (see below), since ablation of occludin is not without consequence despite the fact that the protein does not appear to be required for TJ strand formation. Occludin-null mice express a complex phenotype, which is, however, not manifested by structural or functional TJ abnormalities: in occludin \u2212\/\u2212 mice, TJs were not affected morphologically, and electrophysiological barrier function of intestinal epithelium was normal. However, histopathological abnormalities were detected in several tissues, i.e., chronic inflammation and hyperplasia of the gastric epithelium, calcification in the brain, testicular atrophy, the loss of cytoplasmic granules in striated duct cells of the salivary gland as well as the thinning of the compact bone, pointing to complex functions of the occludin protein (Saitou et al. 2000).\nRole of claudin proteins in formation of a TJ permeability barrier\nThe lack of charged amino acid residues within the extracellular loops of occludin segregates occludin from the candidates contributing directly to the formation of ion-selective pores. The different claudins whose extracellular loops span a wide range of pKion the contrary appear to be excellent candidates to mediate ion selectivity in TJ permeability (Fig.\u00a03b). Major insight into the distinct charge selectivity of the claudins came from the work of Furuse et al. (2001), who could relate the different levels of resistance between MDCK I cells (TER\u00a0>\u00a010,000\u00a0\u03a9cm2) and MDCK II cells (TER <200\u00a0\u03a9cm2) to a differential claudin isoform expression, while MDCK I cells express claudin-1 and -4 but not claudin-2, MDCK II cells additionally express claudin-2, which seems to mediate higher ion permeability by pore formation\/inducing strand breaks. Notably, transfection of the tight MDCK I with claudin-2 cDNA resulted in 20-fold decline in TER but not in the number of TJ strands.\nA wide range of further transfection and mutagenesis experiments introducing or ablating different claudins has been performed to date, and been partially helpful to elucidate the conductance conferring properties of the resulting pores (Matter and Balda 2003b). These informative observations must however be viewed with some caution because transfection and site-directed mutagenesis may lead to more than a change in net charge as for example an altered molecular conformation, or altogether the introduction of an unusual claudin protein could lead to disturbance of strand organisation within native strands. By the construction of chimera, Colegio et al. (2003) were moreover able to attribute ion selectivity of the claudins to distinct stretches within the primary amino acid sequence of the extracellular loops leading to alterations in TER and charge selectivity in transfection experiments. Taken together, available data strongly suggest a key role for the extracellular loops of the diverse claudins in the formation of ion selective pores within the TJ strands.\nRole of occludin in formation of a TJ permeability barrier\nThe role of occludin in TJs and a putative contribution to building up the TJ\u2019s ion selectivity (the ion pores) is unclear; it has been demonstrated that overexpression of occludin in MDCK cells reduces ion permeability but in the same time was shown to increase the transepithelial flux of the uncharged compound mannitol (Balda et al. 1996). Further observations then implicated that occludin may be a target for receptor-initiated signalling involving the Rho family of GTPases, and that by an additional not-yet-understood mechanism, occludin phosphorylation may regulate TJ permeability independently of cytoskeletal activity (Hirase et al. 2001).\nSome indirect evidence for a role of occludin in strengthening barrier properties arose from heterologous expression studies: when expressed in L-fibroblasts that lack endogenous cadherin-based adherens junctions and ZO-1, little adhesiveness was measured (Kubota et al. 1999). However, when overexpressed in fibroblasts, which do have well developed ZO-1-containing adherens-like junctions (NRK-fibroblasts), increased Ca2+-independent adhesion was observed, indicating that when interacting with ZO-1, occludin mediates adhesive functions (Van Itallie and Anderson 1997). Addition of neutralizing peptides of the peptide sequence of the second extracellular loop elicited competitive interference with cell\u2013cell-adhesion in a dose dependent manner, implicating that this loop directly participates in adhesive interaction (Wong and Gumbiner 1997).\nRelating epithelial malignancy to the loss of cell\u2013cell-adhesion, it was demonstrated that overexpression of oncogenic Raf 1 in rat salivary gland epithelial cells (Pa4\u2013Raf1) disrupts TJs and induces an oncogenic phenotype by downregulating the expression of occludin (Wang et al. 2005). Raf1-mediated transformation of Pa4\u2013Raf1 cells was subsequently shown to induce transcriptional downregulation of occludin and forced re-expression of occludin rescued the polarized phenotype of epithelial cells (Wang et al. 2007). Interestingly, forced expression of an occludin mutant lacking the second extracellular loop did not rescue the epithelial phenotype in vitro nor did it prevent tumor growth in vivo. These results demonstrate that the TJ protein occludin has a potent inhibitory effect on the Raf1-mediated tumorigenesis, and the second loop of occludin appears to be required for suppression of Raf1-induced tumor growth, potentially by mediating cell\u2013cell-adhesion as presented above contributing to contact inhibition of cell division.\nTJs and human disease\nModulation of barrier function by cytokines\nMany cytokines have been shown to influence epithelial and endothelial TJ function and the actin cytoskeleton both in vivo and in vitro. To name the most prominent, the interleukins IL-0, IL-1, IL-3, IL-4, as well as tumor necrosis factor alpha (TNF\u03b1) and interferon gamma (IFN\u03b3) could be demonstrated to influence TJ barrier function in epithelia and endothelia (Ahdieh et al. 2001; Oshima et al. 2001; Youakim and Ahdieh 1999). This review will emphasise the influence of a few selected cytokines on TJ structure\/function and its adjoining actin cytoskeleton in diseases like inflammatory bowel disease and inflammatory disease of the CNS (Table\u00a01).\nTable\u00a01Modulation of TJ barrier function in human diseaseClassificationTight junction protein affectedReferenceCancer\u00a0Breast cancer: invasive ductal cancerClaudin-1, ZO-1Itoh and Bissell (2003), detailed here\u00a0Prostate cancer: prostatic adenocarcinomasClaudins-1, -3, -4 and claudin-7Sheehan et al. (2007)\u00a0Thyroid neoplasma, follicular adenomaOccludin and claudin-1, -4 and -7Tzelepi et al. (2007)\u00a0Gastroesophageal reflux disease: Barrett\u2019s \u00a0esophagus (dysplasia)Claudin-18Jovov et al. (2007)\u00a0Lung cancer: Basaloid squamous carcinomaUltrastructural junctional dedifferentiationGilcrease and Guzman-Paz (1998)Inflammation\u00a0Inflammatory bowel disease: Morbus CrohnClaudins-2,-3, -5, and -8, ZO-1Zeissig et al. (2007), detailed here \u00a0Collagenous collitisOccludin, claudin-2 and -4Burgel et al. (2002)\u00a0Multiple sclerosisOccludin, claudin-5F\u00f6rster et al. (2007), detailed hereHereditary diseases\u00a0Heredit\u00e4ry deafnessClaudin-14Ben-Yosef et al. (2003), detailed here\u00a0Familial hypomagnesemiaClaudin-16Simon et al. (1999), detailed here\u00a0cystic fibrosisOccludin, claudin-1, -4, JAM, ZO-1Coyne et al. (2002)Vision loss\u00a0Diabetic eye disease: diabetic retinopathyOccludin, ZO-1Felinski and Antonetti (2005), detailed hereViral infection\u00a0Reoviral infection (hydrocephalus, encephalitis)JAM-1Forrest et al. (2003)Bacterial toxins\u00a0Clostridium perfringens enterotoxinClaudin-3 and -4McClane (2001), detailed here\nInflammatory bowel disease: Crohn\u2019s disease\nCrohn\u2019s disease ranks among the chronic inflammatory bowel diseases (IBD) with diarrhoea as a leading symptom. This is chiefly attributed to epithelial barrier dysfunction, leading to an increased loss of solutes in the form of \u201cleak-flux diarrhoea\u201d. Notably, the integrity of the intestinal barrier is not only impaired in patients with Crohn\u2019s disease but also many first-degree relatives of Crohn\u2019s disease patients present with increased intestinal permeability in the absence of clinical symptoms (Hollander 1993; Peeters et al. 1997; Yacyshyn and Meddings 1995). From this observation it was concluded that barrier dysfunction in Crohn\u2019s disease precedes the inflammatory process in contrast to many other inflammatory diseases, probably arising from genetic defects or driven by environmental factors like intestinal luminal antigens or bacterial toxins.\nThe epithelial barrier in the intestine is established by a single layer of epithelial cells. However, barrier dysfunction alone does not to seem be sufficient to cause Crohn\u2019s disease, since unaffected relatives of patients with Crohn\u2019s disease comparably present increased permeability, but do not show the abnormal mucosal inflammatory response reported of patients with Crohn\u2019s disease (Weber and Turner 2007). In healthy individuals, the intestinal mucosal barrier is organised through tight junction strands connecting the adjacent epithelial cells (Weber and Turner 2007). These TJs seal the paracellular space and an intact intestinal mucosa. TJ permeability is rate limiting for epithelial permeability and defects thereof are an established source for intestinal barrier defects as observed in Crohn\u2019s disease. The basic understanding of TJ function we developed is that they form an ion-selective and size-selective paracellular gate whose permeability varies throughout the gastrointestinal tract (see above). According to this, it is not surprising that the pattern of claudin isoforms expressed varies alongside the gastrointestinal tract (Nusrat et al. 2000). Recent studies now  show that the expression pattern of characteristic claudins is altered in Crohn\u2019s disease (Weber and Turner 2007) with a concomitant effect on the integrity of TJ strands: Zeissig et al. (2007) showed by freeze\u2013fracture electron microscopy that the overall number of TJ strands sealing the epithelial layer is reduced while concomitantly an increased number of strand breaks was noted in tissue from patiens with Crohn\u2019s disease (Zeissig et al. 2007). The expression of 12 claudin isoforms in the intestinal epithelium was investigated in healthy individuals and patients with Crohn\u2019s disease, leading to the insight that the expression of the claudins-3, -5, and -8 was decreased in active disease. On the contrary, expression of barrier-reducing claudin-2 was increased in histological samples from patients, which was particularly observed in the crypt epithelium. This altered pattern of claudin isoform expression was thus concluded to be partly responsible for the observed mophological and functional disruption of TJs. However, claudin isoform expression patterns were unaltered in patients with inactive Crohn\u2019s disease, rendering this observed change in expression patterns rather a consequence than a cause of active disease.\nIt is has been acknowledged for a long time that chronic inflammation in Crohn\u2019s disease is associated with the overproduction of proinflammatory cytokines. Searching for the nature of these pro-inflammatory mediators effecting the TJ disruption and dysfunction, investigators (Conyers et al. 1990; Heller et al. 2005; Prasad et al. 2005) had identified IL-13, IFN\u03b3 and TNF\u03b1 as factors in patient\u2019s serum lowering resistance and opening TJs. However, differential effects of the different cytokines crititical to IBD pathogenis have been reported from studies in cell culture models, indicating that the mechanisms by which IFN\u03b3 and TNF\u03b1 inrease paracelluar permeability differ from those used by IL-13 (Prasad et al. 2005). IFN\u03b3 and TNF\u03b1 exposure of model cell lines led to a reduction in claudin-2 expression, while IL-13 exposure induced increased claudin-2 expression. Those observed discrepancies, which might be based on the source of epithelial cell lines, the experimental design or the doses of cytokines applied are the focus of current investigation. Other lines of research are concentrated on gaining more information on the source of pro-inflammatory cytokines in IBD and the mode how those cytokines may work together to modify claudin expression pattern, either in vitro or in vivo (Weber and Turner 2007).\nAt the cell biological level, recent investigation demonstrated that IFN\u03b3 exposure promoted the endocytosis of occludin, JAM-1 and claudin-1 into early and recycling endosomes (Bruewer et al. 2005), an effect which appears to be mediated by RhoA\/Rho kinase, which further induced a myosin II-dependent formation of vacuoles in the apical cell moiety (Utech et al. 2005).\nAt the gene expression level, other research groups could demonstrate that TNF\u03b1 induced an increase in Caco-2 TJ permeability mediated by activation of the pro-inflammatory transcription factor NF\u03baB. NF\u03baB action leads to a downregulation of specifically ZO-1 gene expression and a redistribution of ZO-1 protein away from the intercellular junctions (Ma et al. 2004). A comparable effect could be described in the nonintestinal model epithelial cell line, MDCK, and in the blood brain barrier model cell line cEND, where also disruption of TJs following exposure of epithelial and endothelial cells to TNF\u03b1 could be demonstrated (F\u00f6rster et al. 2007; Poritz et al. 2004).\nBlood-brain barrier compromise in neuroinflammation\u2013multiple sclerosis\nHomeostasis of the central nervous system (CNS) microenvironment is essential for its normal function. It is maintained by the blood-brain barrier (BBB) which regulates the transport of molecules from blood into brain and backwards. The function of this highly specialised barrier is to (1) protect the brain from blood-borne substances and (2) to provide nutrient supply to the brain parenchyme by specialised transport systems (Rubin and Staddon 1999).\nThe main structures responsible for this barrier property are the TJ. TJ are strongly developed in endothelial cells of the BBB but only moderately formed between endothelial cells of the peripheral vasculature: leaky blood vessels in the body allow many molecules to cross through to tissue, but the tight construction of the vessels in the CNS guards against brain entry, leading to high electrical resistance in the range of 1,500\u20142,000\u00a0\u03a9cm2, depending on the species investigated. In BBB-forming brain capillary endothelial cells (BCECs) the TJ proteins occludin and claudin-5, claudin-3, claudin-1 and claudin-12 were demonstrated by immunofluorescence and western blot analysis (F\u00f6rster et al. 2007; Wolburg and Lippoldt 2002).\nUnder physiological conditions, brain microvascular endothelial cells form a barrier limiting the extravasation of cells of the immune system like monocytes, lymphocytes and other leukocytes. It has been shown in this context that leukocytes can transmigrate to the BBB without net changes in vascular permeability and cell\u2013cell contacts (Cramer 1992). However, increased leukocyte migration as in multiple sclerosis (MS) has been reported to lead to a re-organisation of the actin cytoskeleton and breakdown of the TJ proteins occludin and ZO-1 (Couraud 1998; F\u00f6rster et al. 2007). MS is characterised as a chronic and progressive CNS disease with a remitting-relapsing profile. In the course of the disease, both demyelination and microvascular inflammation have been described as central pathophysiological insults. From an animal model for MS, experimental allergic encephalomyelitis (EAE) (Korner and Sedgwick 1996), the inflammatory cytokines TNF\u03b1 and IL-1 have been delineated to be the key mediators inducing alterations in BBB permeability. Clinically matching data have been collected from MS patients, who show an elevation in TNF\u03b1 and\/or IL-1 in cerebrospinal fluid (CSF) (Weber and Rieckmann 1995). Cytokine-promoted opening of the BBB has been shown to occur due to degradation and decreased synthesis of TJ proteins (Chang and Werb 2001; Harkness et al. 2000; Silwedel and F\u00f6rster 2006; Yang et al. 2006), resulting in compromised junctional integrity. Particularly, the TJ proteins occludin and ZO-1 have been shown to be negatively affected by the presence of these pro-inflammatory cytokines. Searching for the mechanism of TJ protein degradation, consecutive studies showed, that in many neuroinflammatory conditions, including MS, the matrix metalloproteinases (MMP) play an important role in disrupting the BBB. Amongst these, it has been demonstrated that selectively, MMP-9 (gelatinase B) is increased in CSF levels in MS patients. Tissue inhibitors of metalloproteinases (TIMPs) form complexes with either activated MMPs or with their pro-forms after their secretion, thus regulating MMP activity under physiological conditions (Brew et al. 2000; Yong et al. 2001). Levels of TIMP-1 are however reduced in MS patients relative to control patients, suggesting an imbalance in MMP-9\/TIMP-1 ratios in MS (Avolio et al. 2005). MMP-9-mediated opening of the BBB then allows amplification of the inflammation, as demonstrated by radioisotopes (Kermode et al. 1990). Looking for therapeutical agents restoring the unbalaced MMP-9\/TIMP-1 ratios in MS, F\u00f6rster et al. (2007)  showed that glucocorticoid treatment reduces the levels of MMP-9 markedly in a cell culture model of the BBB, an effect which was shown to be based on glucocorticoid-mediated transcriptional activation of the TIMP-1 gene. This study further demonstrated that the administration of the glucocorticoids dexamethasone and hydrocortisone preserved the functional integrity of the TJs and adherens junctions under pro-inflammatory conditions, chiefly by maintaining the levels of the TJ components occludin, claudin-1, claudin-12, ZO-1 and VE-cadherin, while dexamethasone effects on claudin-5 were negligible (Blecharz et al. 2008; F\u00f6rster et al. 2007).\nAnother study demonstrated differential susceptibility of cerebral and cerebellar endothelial cell lines to BBB breakdown in response to inflammatory stimuli (Silwedel and F\u00f6rster 2006): increased fragility of the cerebellar BBB had been reported, as the typical caudal to rostral lesion development of MS and EAE with a preference of the cerebellum indicates (Cross et al. 1993; Juhler et al. 1984; Namer et al. 1993). Matching those studies, the authors could demonstrate a faster and more pronounced increase in permeability in the cerebellar BBB manifested by reduced TER and reduced TJ protein expression using the cerebellar BBB model cell line cerebEND (Silwedel and F\u00f6rster 2006). The stronger effects of TNF\u03b1 on endothelial cells of the cerebellum as compared the the cerebrum could be explained on the cellular level, where the TJ protein levels of claudin-5 and occludin were shown to be differently affected in cerebellar and cerebral microvascular endothelial cells: effects on occludin were more pronounced in cerebellar endothelial cells, while cerebral endothelial cells of the BBB showed a stronger decrease in claudin-5. Since it was shown that the main protein buiding up the barrier appears to be occludin, while claudin-5 protein, which is also well detectable in non-BBB endothelial cells, does not seem to influence barrier properties to the same extent (Foerster et al. 2002, 2005, 2006; Wolburg and Lippoldt 2002). One may speculate that the lower overall occludin contents of endothelial cells of the cerebellar BBB compared to the cerebral BBB can partly explain the greater sensitivity of the cerebellar BBB to inflammatory stimuli.\nHeredit\u00e4ry diseases\nHeredit\u00e4ry deafness: nonsyndromic recessive deafness DFNB29\nThe cochlea of the inner ear is divided into two compartments of different ionic composition. Hereby, the perilymph of the scala vestibuli and scala tympani is high in Na+ but low in K+ concentration, while the endolymph of the scala media resembles the intracellular ionic composition, being high in K+ but low in Na+ (Ferrary and Sterkers 1998). This ion gradient leads to the establishment of an 80\u2013100\u00a0mV endocochlear potential, necessary for the depolarisation of sensory hair cells, enhancing the sensitivity of mechano-activated channels at the top of stereocilia (Hudspeth 1989).\nBesides, hereditary defects in the gap junction proteins connexin-26 and connexin-43 (Sabag et al. 2005) an association between tight junctions and hearing loss was established. The importance of claudin-14 expression in the cochlea was demonstrated by the identification of two different CLDN14 mutations that cause nonsyndromic recessive deafness DFNB29 in two large consanguineous Pakistani families (Wilcox et al. 2001). By in situ hybridization and immunofluorescence labeling mouse claudin-14 expression in the sensory epithelium of the organ of Corti was observed. By the generation and characterisation of claudin-14 knockout mice as a model for autosomal-recessive deafness DFNB29 it was revealed that deafness occurred due to death of outer hair cells. Searching for the factors that promoted increased death of outer hair cells, morphology of the TJs of claudin-14 knockout mice was evaluated. It was demonstrated that TJ strands between supporting cells and between outer hair cells and supporting cells in the organ of Corti are still present and appear morphologically normal in freeze fracture replicas. The analysed TJ strands which are composed of multiple claudins were thus concluded to have rather a functional than  an ultrastructural defect (Ben-Yosef et al. 2003). Following the reasoning that claudins create charge-selective channels, Ben-Yosef characterised the electrophysiologic properties of claudin 14 by ectopically expressing it in MDCK cells, which revealed a strong discrimination of transfected monolayers against Na+ and K+ ions. Ben-Yosuf and coworkers thus concluded that the absence of claudin 14 from TJs in the organ of Corti might lead to elevated K+ concentration in the space of Nuel, which is normally filled with the K+-poor cortilymph and increased death of outer hair cells. Long-term exposure of the basolateral membranes of outer hair cells to high K+ concentrations had been shown to be toxic and eventually to lead to cell death before (Zenner 1986).\nBy the identification of two claudin-14 missense mutations that were unable to form TJs after ectopic expression of CLDN14 in L mouse fibroblastes, Wattenhofer et al. (2005) were then able to prove that the basis for the development of deafness is incorrect recruitment and TJ strand formation in cells expressing mutated CLDN14.\nFamilial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC)\nInherited and acquired defects in epithelial membrane transport are responsible for many human diseases. In the kidney, tubular claudins play a role in extracellular or paracellular permeability. In this context, it has been shown that mutations in claudin-16 (Paracellin-1) impair the function of TJs in the paracellular resorption of Mg2+ and Ca2+ but not of monovalent cations (Simon et al. 1999), probably founding the molecular basis for the development of familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC), an autosomal recessive renal tubular disorder. In FHHNC patients, the impaired tubular reabsorption of Mg2+ and Ca2+ in the thick ascending limb of the loop of Henle with an eventual progression can lead to end-stage renal disease (Lee et al. 2006). Functional tubular reabsorbtion mechanisms are vitally important, since a normal adult absorbs only 200\u00a0mg Ca2+ from the diet each day, but loses 8,000\u00a0mg to filtration across the glomerulus. To achieve normal calcium balance, the majority of the filtered load must be reabsorbed by the tubule, to approximately 90%, or 7,000\u00a0mg\/day and this occurs mainly via the paracellular pathway driven by a lumen positive potential across the tubular wall.\nThe structural backbone of the paracellular pathway must provide for the significant substrate selectivity and functional heterogeneity between nephron segments and thus also plays a pathophysiologic role in renal diseases. Clinically, the symptoms and gradual kidney damage caused by FHHNC cannot be alleviated by intravenous or oral supplementation with Mg2+, and kidney transplantation is often the only option at later stages of the disease.\nRecent studies (Simon et al. 1999) have reported that FHHNC is caused by mutations in a gene, which encodes the TJ protein, paracellin-1. Paracellin-1 (PLCN-1) belongs to the claudin family (claudin-16) that allows the paracellular transport of Mg2+ and Ca2+. As a member of the claudin family of TJ proteins claudin-16 exhibits four transmembrane spans with two extracellular loops and both termini located in the cytoplasm. To date, more than 20 distinct mutations have been identified, which affect either the trafficking of PLCN-1\/claudin-16, or its permeability properties (Kausalya et al. 2006).\nInterestingly, FHHNC patients do not exhibit the salt wasting or metabolic alkalosis that is found in Bartter\u2019s syndrome (Wong and Goodenough 1999). Based on these findings, it is tempting to hypothesize that PCLN-1 might form a Mg2+- and Ca2+ -conducting pore. It is therefore likely that PCLN-1 selectively mediates the barrier to divalent but not monovalent cations. Thus, not only can the paracellular barrier discriminate between charged and uncharged solutes, but it may also exhibit an even higher level of specificity distinguishing charge and ionic radius and size of the hydrate shell.\nAs to molecular investigations based on the use of diverse cell culture models for kidney epithelial cells, there is some discrepancy between results obtained upon overexpression of PLCN-1 in two different kidney epithelial cell lines used. In the canine MDCK cell line, another group found PCLN-1 to decrease Na+ permeability, to decrease TER and to enhance transepithelial Mg2+ transport (Ikari et al. 2006). Another recent study performed in the porcine proximal tubule LLC-PK1 cells suggests that paracellin-1 functions to modulate paracellular conductance and not transcellular transport. They hypothesise that PLCN-1 mediates mostly paracellular Na+ permeation to build up the positive lumen potential and that this lumen-positive potential would be the driving force for the reabsorption of Mg2+ (Hou et al. 2005).\nAs described for other molecules relevant to Mg2+ homeostasis such as ACDP2, (ancient conserved domain protein), a divalent metal transporter, or the Mg2+ transporter MagT1 (Wong and Goodenough 1999), the human PLCN-1 promoter appears to be responsive to external Mg2+ concentrations, since the renal epithelial cell specific PLCN-1 gene expression was shown to positively correlate with the amount of Mg2+ offered in the growth medium (Efrati et al. 2005).\nFurther, mutations in PLCN-1 can affect claudin-16 protein targeting at the TJs (Kausalya et al. 2006). The mutation T233R for example prevents interaction of claudin-16 with ZO-1 and results in lysosomal targeting of claudin-16 instead. Protein kinase A (PKA) mediates phosphorylation of claudin-16 at Ser217,  failure of which equally leads to dissociation of claudin-16 from ZO-1 and its translocation into the lysosomes (Ikari et al. 2006). In conclusion, although the mechanism of PLCN-1\/claudin-16-mediated Mg2+ and Ca2+ reabsorbtion is not yet fully understood, it is clear that these claudins play a crucial role in this process.\nRecently, Konrad et al. (2006) additionally unravelled a role for claudin-19 in the development of FHHNC. They had clinically characterized one Swiss and eight Spanish\/Hispanic families affected with severe hypomagnesemia due to renal wasting, nephrocalcinosis, and progressive renal failure but had to recognize that the individuals did not have any mutations in CLDN16. They showed that the renal phenotype of the affected patients was virtually undistinguishable from that of patients who presented with FHHNC with proven CLDN16 mutations. However, the affected individuals in these families also have severe visual impairment, characterized by macular colobomata, significant myopia, and horizontal nystagmus (Konrad et al. 2006). Konrad et al. were able to map a new locus for recessive renal magnesium loss on chromosome 1p34.2 and identified mutations in CLDN19, yet another member of the claudin family in patients affected with FHHNC and additionally, with severe ocular abnormalities. CLDN19, which encodes the tight-junction protein claudin-19 was demonstrated to be highy expressed in renal tubules and the retina. In an effort to understand the mechanisms underlying the roles of the two claudins in mediating paracellular ion reabsorption in the kidney, Goodenough and co workers (Hou et al. 2008) showed using pig kidney epithelial cells, that CLDN19 functioned as a Cl(\u2212) blocker, whereas CLDN16 functioned as a Na(+) channel. Mutant forms of CLDN19 were shown to be associated with FHHNC since they were unable to block Cl(\u2212) permeation. Coexpression experiments further elucidated that CLDN16 interacts with CLDN19 and that their association confers cation selectivity, suggesting a role for mutant forms of CLDN16 and CLDN19 in the development of FHHNC.\nVascular system\nDiabetic retinopathy\nThe barrier between the vascular lumen and neural layers in the retina and the brain parenchyme maintains a characteristic microenvironment and is essential for proper neuronal function. This blood-retinal barrier (BRB) consists of two anatomical entities: an inner BRB formed by junctions between endothelial cells of the retinal capillaries, and an outer BRB composed of TJs between retinal pigment epithelial cells. Clinical evidence from fluorescein angiography has demonstrated that the inner BRB appears to be the primary site of vascular leakage leading to macular edema.\nThe BRB is built up and maintained by TJ complexes that make up the blood vessels of the microvasculature (Mitic and Anderson 1998). The principal proteins found in the retinal endothelial TJs are occludin and claudin-5 (Dejana et al. 2001). Loss of the BRB integrity and vascular permeability leading to macular edema (Vitale et al. 1995) characterise diabetic retinopathy. Macular edema in turn is thought to be directly responsible for vision loss in diabetic retinopathy (Felinski and Antonetti 2005). Vascular endothelial growth factor (VEGF) is thought to be chiefly responsible for this increase in BRB permeability, albeit other permeabilizing factors like histamin or the cytokines IL-1 and TNF\u03b1 have also been implicated (Felinski and Antonetti 2005). Levels of VEGF, originally identified as an angiongenic factor serving as a mitogen for vascular endothelial cells have been recently correlated with increased vascular permeability (Felinski and Antonetti 2005), compatible with increased VEGF levels detected in the vitreous of diabetic patients for decades. There is enough evidence to show that diabetes and VEGF induce retinal vascular permeability by altering TJ complexes, evidenced for instance by the decrease in occludin content in the retinas of diabetic rats (Felinski and Antonetti 2005). In cultured primary bovine retinal endothelial cells, this observation was mirrored by the observation of decreased occludin contents after VEGF administration (Antonetti et al. 1998). Follow-up investigation underlined this and showed down-regulation of occludin in retinal endothelial cells in culture after VEGF stimulation. VEGF further activates PKC, which leads to increased occludin and ZO-1 phosphorylation (Harhaj et al. 2006). Phosphorylation of occludin and ZO-1 likely contribute to dysregulated endothelial paracellular permeability (Abbott et al. 2006). Regulation of PKC activity and TJ protein modifications may thus have therapeutic implications for treatment of diabetic retinopathy.\nRecent investigation has further revealed that alterations of the BRB may involve the active proteolytic breakdown of the endothelial cell TJs by MMPs as also evidenced in the case of MS (compare above) (Leppert et al. 2001). Specifically, elevated levels of MMP-9 have been seen when retinal endothelial cells were exposed to high glucose conditions as a cell culture model of diabetes, leading to proteloytic degradation of specifically the TJ protein occludin followed by disruption of the overall TJ complex (Giebel et al. 2005), rendering the regulation of MMP secretion and activity another therapeutical target in diabetic retinopathy.\nMultiple sclerosis\nSee above.\nTight junction alterations in cancer\nDecreased expression of claudin-1 correlates with malignant potential in breast cancer\nChanges in TJ function have been shown to be an early and key aspect in cancer metastasis (Ehler et al. 2001). Recent studies showed a role for claudin-1 in invasion and metastasis in mammary glandular differentiation (F\u00f6rster et al. 2002) and carcinogenesis (Morohashi et al. 2007).\nInterestingly, there is an absence or significant loss of claudin-1 expression in several established breast cancer cell lines. Claudin-1 expression in primary human mammary epithelial cells, in contrast to low or undetectable levels of expression in a number of breast tumors and breast cancer cell lines, points to claudin-1 as a possible tumor-suppressor gene. In sections from surgically resected breast specimens, a significant loss of claudin-1 protein in breast cancer cells could be shown by immunohistochemistry (Tokes et al. 2005). This finding suggests that claudin-1 may play a role in invasion and metastasis. The expression profile of claudin-1 in non-malignant versus tumor-derived cells has made this gene an interesting candidate for involvement in tumorigenesis, namely by acting as a suppressor of mammary epithelial proliferation.\nIn order to evaluate the CLDN-1 gene in sporadic and hereditary breast cancer, Kr\u00e4mer et al. (2000) characterized its genomic organization and screened the four coding exons for somatic mutations in 96 sporadic breast carcinomas and for germline mutations in 93 breast cancer patients with a strong family history of breast cancer. In addition, they compared the 5\u2032-upstream sequences of the human and murine CLDN1 genes to identify putative promoter sequences involved in down-regulation of the CLDN1 gene under malignant conditions and examined both the promoter and coding regions of the human gene in various breast cancer cell lines showing decreased claudin-1 expression. However, neither in sporadic or hereditary breast cancers, nor in breast cancer cell lines they found evidence for a role of aberrant claudin-1 in breast tumorigenesis and concluded that other regulatory or epigenetic factors could be involved in the down-regulation of the CLDN1 gene during breast cancer development.\nA correlation between TJ abnormalities and neoplasia can be supported by several observations: alterations in the number, appearance, and permeability of TJs have been demonstrated in various cancer types (Cochand-Priollet et al. 1998; Soler et al. 1999; Swift et al. 1983) and in pre-malignant mammary epithelial cells (F\u00f6rster et al. 2002). At the molecular level, the Drosophila lethal(1) discs-large-1 gene (dlg) product, which localises to TJ-related insect septate junctions has been shown to regulate epithelial cell proliferation. Genetic loss of dlg leads to a neoplastic overgrowth phenotype (Woods and Bryant 1991). The loss of expression of claudin-1 itself has been demonstrated in several mammary carcinoma cell lines (Swisshelm et al. 1999). The expression of the TJ plaque protein ZO-1 has been shown to be reduced in breast carcinomas or breast cancer cells (Hoover et al. 1998). These findings support the hypothesis that claudin-1 might be involved in the development of breast cancer and possibly in other epithelial tumors too.\nThe molecular pathways leading toward the loss of claudin-1 expression however, remain to be explained.\nBacterial toxins\nClostridium perfringens enterotoxin\nFood poisoning by Clostridium perfringens enterotoxin (CPE) leads to diarrhea symptoms classified as \u201cC. perfringens type A food poisoning and antibiotic-associated diarrhea\u201d. The enterotoxin protein has been elucidated to intrude the cells by interacting with epithelial TJ, including certain claudins. Toxin-induced cytolytic pore-formation is a prerequisite for this and involves residues in the N-terminal half of CPE protein, while residues near the C-terminus are required for binding to claudins. In detail, the single 35\u00a0kDa CPE polypeptide first causes cellular damage by altering plasma membrane permeability by the formation of an approximately 155\u00a0kDa CPE-containing pore complex necessary for the subsequent interaction with TJ proteins via its C-terminal receptor-binding region (McClane 2001). CPE was then perceived to affect TJ structure and function in a way that permeability properties of the epithelial layer are altered leading to diarrhea (McClane 2001).\nFurther investigation has determined the isoforms of claudins targeted by CPE. Sonoda et al. (1999) were able to show that the COOH-terminal half fragment of CPE (C-CPE) bound effectively to claudin-3 and claudin-4 in MDCK I cells expressing the claudins-1, -2, -3 and -4. They were further able to show that in the presence of C-CPE, reconstituted TJ strands gradually disintegrated and disappeared from their cell surface in C3L cells.\nAs a consequence, because diverse claudins are overexpressed on some human cancers, the toxin became interesting for targeting chemotherapy in the manner of a Trojan horse (Long et al. 2001; Michl et al. 2001).\nRecently, van Itallie et al. (2007) aimed to solve the structure of the CPE claudin-binding domain to advance its therapeutic applications. The structure was shown to be a nine-strand beta sandwich with previously unappreciated similarity to the receptor-binding domains of several other toxins of spore-forming bacteria, giving strong evidence for the presence of a common ancestor for several receptor-binding domains of bacterial toxins (Van Itallie et al. 2007). The very recent elucidation of the structure of a 14-kDa fragment containing residues 194 to the native COOH terminus at position 319 by X-ray diffraction to a resolution below 2\u00a0\u00c5 showed that the structure is a nine-strand \u03b2 sandwich with previously unappreciated similarity to the receptor-binding domains of several other toxins of spore-forming bacteria, i.e., the collagen-binding domain of ColG from Clostridium histolyticum and the large Cry family of toxins (including Cry4Ba) of Bacillus thuringiensis (Van Itallie et al. 2008). The authors speculate that the claudin-4 binding site is on a large surface loop between strands \u03b28 and \u03b29 or includes both of these strands. They further were able to clarify that the sequence crystallized binds to purified human claudin-4 with a 1:1 stoichiometry. The binding affinity was determined to be in the submicromolar range similar to that observed for binding of native toxin to cells (Van Itallie et al. 2008). These results could now provide a structural framework to advance therapeutic applications of the toxin.\nConclusion and perspectives\nThe discovery of the existence of occludin- and claudin-based TJs constituting a permeability barrier in epithelial and endothelial cells has paved the way for the elucidation of the molecular background of diverse human diseases the pathogenesis of which had been unclear before. It became clear that the presence of TJs is indispensable for tissue compartmentalisation and cellular homeostasis. Disturbances in TJ function are reflected in many diseases, but on the other hand, awareness of their involvement has facilitated therapy enormously. For instance, antiinflammatory or antimetastatic drugs could be developed based on their ability to repair broken barrier function. These strategies have become indispensable in the treatment of disease like MS, inflammatory bowel disease or diabetic retinopathy. Following the strategy of pathogenic agents, which use TJ proteins as a docking station to invade cells, even strategies for drug delivery techniques could be implemented. Future attempts will have to address the role of occludin and the exact contribution of the diverse claudins to the formation of ion selective pores, which are still open questions. A more thorough understanding will greatly facilitate diagnosis and the development of specific treatment regimens for diseases originating from impaired TJ function.","keyphrases":["tight junction","occludin","claudin","vascular","inflammation","cancer","hereditary","toxin"],"prmu":["P","P","P","P","P","P","P","P"]}
{"id":"Ann_Biomed_Eng-2-2-1705520","title":"Cellular and Matrix Mechanics of Bioartificial Tissues During Continuous Cyclic Stretch\n","text":"Bioartificial tissues are useful model systems for studying cell and extra-cellular matrix mechanics. These tissues provide a 3D environment for cells and allow tissue components to be easily modified and quantified. In this study, we fabricated bioartificial tissue rings from a 1 ml solution containing one million cardiac fibroblasts and 1 mg collagen. After 8 days, rings compacted to <1% of original volume and cell number increased 2.4 fold. We initiated continuous cyclic stretching of the rings after 2, 4, or 8 days of incubation, while monitoring the tissue forces. Peak tissue force during each cycle decreased rapidly after initiating stretch, followed by further slow decline. We added 2 \u03bcM Cytochalasin-D to some rings prior to initiation of stretch to determine the force contributed by the matrix. Cell force was estimated by subtracting matrix force from tissue force. After 12 h, matrix force-strain curves were highly nonlinear. Cell force-strain curves were linear during loading and showed hysteresis indicating viscoelastic behavior. Cell stiffness increased with stretching frequency from 0.001\u20130.25 Hz. Cell stiffness decreased with stretch amplitude (5\u201325%) at 0.1 Hz. The trends in cell stiffness do not fit simple viscoelastic models previously proposed, and suggest possible strain-amplitude related changes during cyclic stretch.\nINTRODUCTION\nMechanical stimuli are important for the health and maintenance of many biological functions.14 This is especially true in the cardiovascular system where the tissues and organs are undergoing constant cyclic loading. It is the cellular response to these mechanical forces that can either maintain healthy tissue or lead to a pathologic state.1\nMechanical stimuli have been used to create more functionally viable engineered tissues.2\u20135,9,15\u201317 It has been shown that constant amplitude cyclic stretch (a form of mechanical conditioning) of engineered tissue constructs can cause cells to reorient, increase cell proliferation, and produce more extracellular matrix (ECM).3 However, these studies generally have looked at how the tissue responds to mechanical conditioning over extended time.9,15 In addition, the studies generally quantify the mechanics using only the ultimate strength and modulus4,5,16 along with quantification of such biological factors as cell number, gene expression, and ECM components.2,3,17\nSince it is the cells that actively respond to mechanical loads, it is important to understand the effects of mechanical conditioning on the individual components of the tissue (cell and matrix) and how the mechanical properties change from the time that the conditioning is initiated. Our earlier work has shown that tissue forces drop with subsequent stretches during repeated cyclic stretching.19 However, whether this drop occurs in the cells and\/or matrix components is not known. Furthermore, it is not known how these drops are affected by different amplitudes and frequencies over many hours of continuous cyclic stretch.\nThe objective of this study is to measure the short-term (<24\u00a0h) mechanical response of collagen gels populated with cardiac fibroblasts to cyclic stretch at different amplitudes and frequencies. Although engineered tissue constructs may be composed of several cell and matrix components, we chose to use a simple system composed of a single cell type and a single defined ECM component to study the mechanics of tissue components during cyclic stretch. This system provides a 3D environment for the cells while allowing the components to be easily modified and quantified. The benefits of using collagen, the main structural component in cardiovascular tissue, are that its initial concentration can be easily modified and it is more stable than other ECM components such as fibrin.4,5,10 By using a single cell type we can avoid complicating effects due to mixed cell populations and clearly understand that cell type\u2019s contribution to the tissue mechanics. Cardiac fibroblasts are one of the two main cell types in cardiac tissue and are responsible for ECM maintenance and remodeling.7 These cells, along with the cardiac myocytes, undergo continuous cyclic stretch with each heartbeat. Thus, it is particularly useful to understand their cellular mechanics during cyclic stretch.\nBy measuring the cell forces under various cyclic stretch amplitudes and frequencies, we can better understand the relationship between the applied strains and the cells\u2019 mechanical response. Then we can model this behavior and be able to predict cellular responses to dynamic physiologic and pathologic loading conditions.\nMETHODS\nTissue Ring Fabrication\nChicken embryonic cardiac fibroblasts (CECF) were isolated from 10 day old chicken embryos (Charles River Laboratories, Wilmington, MA). Cells were maintained in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) supplemented with 3% fetal bovine serum (FBS), 50\u00a0units\/ml penicillin and 50\u00a0mg\/ml streptomycin (P\/S). Tissues were fabricated as described previously.20 Briefly, CECF from passage 2\u20138 were added to a collagen solution containing monomeric rat tail tendon collagen (Upstate Biotechnology Inc., Lake Placid, NY) neutralized with 0.1 N sodium hydroxide (NaOH) and 2\u00d7 concentrated DMEM. The final solution contained 1\u00a0mg\/ml collagen and one million cells\/ml. Then 1\u00a0ml of this solution was poured into cylindrical molds containing an inner mandrel (diameter=9.55\u00a0mm). After one hour of incubation, the solution gelled and the molds were filled with incubation medium containing DMEM supplemented with 10% FBS and P\/S. After five additional hours, the gels were removed from the molds and transferred to spacers (Fig. 1) placed in incubation medium. The spacers consisted of two fixed rods (2.07\u00a0mm diameter) separated by a distance to maintain the internal circumference of the rings at 30\u00a0mm. Medium was changed every other day.\nFIGURE 1.Cardiac fibroblast populated rings on spacers during static incubation (A) Day 0 [6\u00a0h] (B) Day 2 (C) Day 4 (D) Day 8. Mean ring width was 0.64\u00a0mm after 8 days incubation (see Fig. 4).FIGURE 2.Illustration of peak force versus time. The gray curve is sample data obtained for 10 stretch cycles. The black line plots the peak force for each cycle vs. time. This peak force line is used for many of the following figures.\nMechanical Tests\nAfter 2, 4, or 8 days of static incubation (Fig. 1) the width of the rings was measured and then the rings were removed from the spacers. The rings were then mounted on a mechanical tester described previously20 that allows independent programmable stretching of four rings while monitoring applied force. The test medium contained DMEM HEPES modification supplemented with 10% FBS and P\/S. All rings were placed on the tester at a baseline length that maintained the inner circumference of 30\u00a0mm. This was defined as 0% strain. In early tests, stretching began immediately after mounting the rings. This led to increases in peak force after initiation of stretch for low amplitudes at early incubation times. Upon further study, it was determined this was a result of the fresh media and FBS used in the test baths and not a result of a mechanical stretch induced response. In all further studies, the rings were mounted on the tester and held at baseline length for 3\u00a0h to allow them to reach equilibrium before testing began.\nEffect of Cyclic Stretch Amplitude\nFour rings were tested at each of the three days with each ring stretched either 5%, 10%, 20%, or 25% at 0.1\u00a0Hz using a triangular displacement waveform (constant strain rate during loading). The force was continuously monitored at 10\u00a0Hz over 14\u00a0h. For many of the figures, the peak force, defined as the maximum force during each cycle, is plotted vs. time (Fig. 2). We use the term stretch to refer to the amplitude of cyclic stretching, while we use the term strain to refer to the amount of deformation at a particular point in the loading curve.\nTo separate out the individual tissue component forces (cell and matrix), 2\u00a0\u03bcM Cytochalasin D (CytoD) was added either 3\u00a0h before or 12\u00a0h after initiation of stretch. CytoD disrupts actin filaments inside the cell and thus eliminates the majority of the cell-contributed force.21 The remaining force measured 120\u00a0min after addition of CytoD is termed the matrix force. Then the estimated cell force is calculated by taking the matrix force and subtracting that from the total tissue force without CytoD.\nFIGURE 3.Cell force calculation. Matrix force was measured by adding 2\u03bcM CytoD to rings prior to initiation of cyclic stretch. Cell force is calculated by taking the normalized matrix force (see Eq. 1) and subtracting it from the tissue force.\nTo estimate the cell forces during the initial 12\u00a0h of cyclic stretch, it is necessary to use two separate rings subject to identical stretching protocols. This is accomplished by subtracting the forces measured in a ring with CytoD added prior to cyclic stretching from the forces measured in a ring where CytoD was added after 12\u00a0h of cyclic stretch. We denote these forces FC0(t) and FC12(t) respectively and similarly, we denote the peak forces PC0(t) and PC12(t). However, the peak forces after 14\u00a0h of cyclic stretch, when both rings have been treated with CytoD, can still differ between rings subject to identical cyclic stretch protocols due to variations between rings. To account for this variation, we scale the forces in the ring to which CytoD was added before initiating stretch using the following formula:\nWe found that the shape of the loading\/unloading curve was similar whether CytoD was added before or after initiation of cyclic stretch. We then subtracted this scaled, or normalized, force curve from the force curve for the ring with CytoD added after 12\u00a0h to obtain an estimated cell force (Fig. 3):\nSingle Step Stretch and Hold Effect\nTo compare the effect of a single stretch to cyclic stretching, a step stretch (stretch to a given stretch magnitude in less than 0.1\u00a0s) of 5%, 10%, or 20% was applied and the rings were held at this stretched length for 8\u00a0h while the force was monitored. The tissue was then returned to its baseline length and the force was monitored for an additional 6\u00a0h. In another set of experiments, CytoD was added 3\u00a0h before or 12\u00a0h after either a 10% or 20% step stretch.\nEffect of Cyclic Stretch Frequency\nA similar procedure was followed to test frequency effects. Four rings were tested at each of the three days with each ring stretched at either 0.25\u00a0Hz, 0.1\u00a0Hz, 0.01\u00a0Hz, or 0.001\u00a0Hz at 10% stretch amplitude. The current tester\u2019s upper frequency limit is 0.25\u00a0Hz and thus unable to stretch at higher frequencies that are closer to physiologic values. CytoD was added either 3\u00a0h before or 12\u00a0h after initiation of stretch to separate out the cell and matrix components as a result of different frequencies of cyclic stretch.\nCell Number\nAfter the completion of testing, some rings were washed in phosphate buffered saline (PBS) and then placed in 1\u00a0ml lysis buffer (0.1% sodium dodecyl sulfate (SDS) in PBS). The samples were sonicated until the rings completely disintegrated to release the DNA from the cells. Finally, 30 \u03bcl of sample was placed in 3\u00a0ml of Hoechst working solution (30\u00a0nM Hoechst 33258 dye (Sigma, St. Louis, MO) in PBS). The fluorescence was then read by a spectrophotometer and cell number determined from a standard curve obtained from samples with known numbers of cells.\nImmunohistochemistry\nRings were removed from spacers after 2, 4, or 8 days static incubation and immediately placed in a solution of 4% paraformaldehyde for 30\u00a0min, washed with PBS and then permeabilized for 45\u00a0min in 0.2% Triton X-100 in PBS. This was followed by 1\u00a0h incubation in a blocking solution (2% normal goat serum and 0.02% sodium azide in PBS). After blocking, the rings were incubated overnight at 4\u00b0C in blocking solution containing TRITC phalloidin. Finally, the rings were washed again in PBS and mounted for viewing on a confocal microscope (Zeiss Confocor).\nRESULTS\nRing Width\nRing width was used as a metric for monitoring the extent of compaction during incubation. The initial width of the rings was approximately 10\u00a0mm. This width decreased until day 8 (Fig. 4(b)) when the average width was reduced to 0.64\u00a0mm. At this time the cross-sectional area of the ring appeared circular and the volume could be approximately calculated using a circular cross-sectional area and the circumference of the ring. A ring diameter of 0.64\u00a0mm corresponds to a 99% reduction in the initial tissue volume.\nFIGURE 4.Cell number and ring width during static incubation. (A) Cell number increases from the initial 1 million cells to 2.4\u00a0million cells after 8 days of static incubation. (B) Ring width decreased from an initial 10\u00a0mm to 0.64\u00a0mm after 8 days of static incubation, with a large amount of compaction occurring prior to day 2.\nCell Number\nRings started with a cell density of one million cells\/ml (one million cells per ring). This number increased steadily for 4 days to 2.3 million cells then increased only slightly to day 8 with a total cell number of 2.4 million (Fig. 4(a)). When combined with the 99% reduction in tissue volume, this translates into a final cell density of 234 million cells\/ml or a 234 fold increase in cell density.\nAmplitude Effect\nThe effect of increasing stretch amplitude on the peak tissue and matrix forces is shown in Fig. 5. At all amplitudes, the peak tissue forces immediately after initiating stretch (Fig. 5(a)) increased with static incubation time while the peak matrix forces were similar (Fig. 5(c)). After 12\u00a0h of cyclic stretch, both tissue (Fig. 5(b)) and matrix forces had decreased substantially, with a larger percentage decline in the matrix force (Fig. 5(d)). To examine the relationships between tissue, matrix, and cell forces more closely, figures in the remainder of this paper show data from rings statically incubated for 8 days prior to cyclic stretch unless otherwise noted. Similarities or differences to days 2 and 4 are discussed in the text. In addition, all curves are averages across repeated (n=2\u20137) experiments. Due to the large number of different conditions tested and the relatively low number of samples (exact numbers shown in Fig. 5(e), 10(e)) rigorous statistical analysis was not possible.\nTissue Force\nHigher stretch amplitudes resulted in higher initial peak forces (Figs. 5(a), 6(a), 6(b)). With each subsequent stretch, the peak force decreased with large drops during the first several stretches and sustained decreases during the first two hours, but only small further decreases in peak force over the next 10\u00a0h (Fig. 6(a)). In addition, this decrease in peak force was greater at higher stretch amplitudes both in terms of absolute force drop and percentage drop from first stretch. At days 2 and 4, after 12\u00a0h of stretch, higher amplitudes still have higher peak forces but the differences were smaller between the amplitudes than the first stretch differences (Fig. 5(a,b)).\nFIGURE 5.Peak tissue and matrix forces for different cyclic stretch amplitudes at 0.1 Hz. (A) Peak tissue force of the first cyclic stretch after 2, 4, or 8 days static incubation. Peak tissue force increases with cyclic stretch amplitude and incubation time. (B) Peak tissue force after 12 h of cyclic stretch after 2, 4, or 8 days static incubation. At day 8 the peak tissue force is similar regardless of cyclic stretch amplitude. (C) Peak matrix force of the first stretch after 2, 4, or 8 days static incubation. Peak matrix force increases with cyclic stretch amplitude. (D) Peak matrix force after 12 h of cyclic stretch after 2, 4, or 8 days static incubation. Higher amplitudes result in higher peak forces. (E) Number of rings averaged for data shown in Figures 5-8 at each amplitude and static incubation day. (F) Individual measurements used in panel B [Peak tissue force after 12 h, 8 days of incubation]. This shows that the trend within an experiment, i.e. rings fabricated on a single day, is consistent but the experiment to experiment variation in forces is large. Y-axis scales are different in each panel to emphasize the trends with amplitude and incubation time.FIGURE 6.Tissue force versus cyclic stretch amplitude after 8 days of static incubation. (A) Peak tissue force over 12\u00a0h of cyclic stretch. Peak tissue force dropped quickly after initiation of cyclic stretch followed by a continued slow decline. (B) First 5 tissue stretches after initiation of cyclic stretch. A large decline in tissue force and hysteresis occurred between the first and second stretch. (C) Last 5 tissue stretches after 12\u00a0h of cyclic stretch. The peak tissue force is similar at the four amplitudes.\nBy day 8, similar peak tissue forces were seen after 12\u00a0h of cyclic stretch regardless of stretch amplitude between 5% and 25% (Figs. 5(b), 6(a), 6(c)). The rings subject to lower stretch amplitudes (5% or 10%) were able maintain a positive baseline force (i.e. force at the baseline, or zero strain, length). At the highest stretch amplitude the rings had periods of zero force during both the loading and unloading of the ring, meaning that at low strains the rings were slack (Fig. 6(c)). After the first stretch (Fig. 6(b)), the loading curve remained nearly linear at 5% stretch amplitude, but became increasingly nonlinear at higher stretch amplitudes (Fig. 6(c)).\nFIGURE 7.Matrix force versus cyclic stretch amplitudes after 8 days of static incubation. (A) Peak matrix force over 12\u00a0h of cyclic stretch. Peak matrix force dropped quickly after initiation of cyclic stretch followed by a slow decline for the 12\u00a0h duration. (B) First 5 matrix stretches after initiation of cyclic stretch. A large decline in matrix force and hysteresis occurred between the first and second stretch. (C) Last 5 matrix stretches after 12\u00a0h of cyclic stretch. Matrix loading curve is highly nonlinear and peak force increased slightly with higher cyclic stretch amplitude.FIGURE 8.Cell force versus cyclic stretch amplitudes after 8\u00a0days of static incubation. (A) Peak cell force over 12\u00a0h of cyclic stretch. Peak cell force increased for 5% and 10% amplitudes while it decreased for 20% and 25% amplitudes. (B) First 5 cell stretches after initiation of cyclic stretch. (C) Last 5 cell stretches after 12\u00a0h of cyclic stretch. The peak cell force and cell stiffness decreased with increased amplitude. The loading curves are nearly linear for all amplitudes.FIGURE 9.Response to step stretch and hold after 8 days of static incubation. The tissue, matrix, and cell forces are shown after a step stretch of either 10% or 20%. The tissue is held at the stretched length for 12\u00a0h. The tissue and matrix force for a 20% step and hold is higher than that for a 10% step and hold. The cell force is identical for 10% and 20%.\nMatrix Force\nExcept for the first stretch, the matrix loading curves were nonlinear for all stretch amplitudes tested. Similar to tissue force, higher stretch amplitudes led to higher initial peak forces (Figs. 5(c), 7(b)). For all amplitudes, the peak force decreased with repeated cyclic stretching and this drop in peak force was larger at higher stretch amplitudes (Fig. 7(a)) but higher stretch amplitudes still maintained higher peak forces (Fig. 7(c)). For the first stretch, the ratio of peak forces at day 8 for 25% to 5% was 5:1, but this ratio decreased to 2:1 after 12\u00a0h of stretching, due to the peak matrix force dropping by a greater percentage at the higher amplitudes over time. The other distinct feature was the shape of the loading\/unloading curves.\nAfter several hours of stretching the shapes of the loading\/unloading curves were very similar for all stretch amplitudes, but the strain value at which the matrix force began increasing from zero during each loading cycle was shifted toward larger strain values. The amount of this shift was greater for higher stretch amplitudes (Fig. 7(c)). This meant that the matrix contributed to the tissue force during the loading only when the strain had reached a value from 3 to 7% less than its maximum value. This shape and shift is evident at days 2, 4, and 8. For all stretch amplitudes, the matrix is slack at low strains.\nCell Force\nCell force increased with increased incubation time. The increase in cell force after 12\u00a0h of stretch from 2 to 4 days of incubation was due to an increase in cell number (per cell force remained the same). However, the increase in cell force from days 4 to 8 could not be explained by the increase in cell number alone, indicating an increase in per cell force. The shape of the first force-strain curve for the cell differed from all subsequent curves. The initial baseline force was followed by a rise and then a leveling off with further increases in force seen only at the higher amplitudes (Fig. 8(b)). During the second stretch, the cell force loading curve was nearly linear. Unlike the tissue and matrix, the cell force loading curve retained a nearly linear shape throughout the 12\u00a0h of cyclic stretch even at 25% amplitude (Fig. 8(b,c)).\nAt day 2, the peak cell force initially drops for several cycles, but then gradually increases to the end of the test. The size of the initial force drop is dependent on the stretch amplitude. The trend is similar at day 4, except the cell force at 25% stretch remains fairly steady instead of increasing. At day 8, the peak cell force, after the initial drop, increases only for the 5% and 10% stretch, while it decreases for the 20% and 25% stretch (Fig. 8(a)).\nSingle Step Stretch & Hold Effect\nSimilar to cyclic stretching at different amplitudes, the larger magnitude step stretches led to higher initial forces (Fig. 9). The tissue force was higher than the matrix force immediately after the step at the same magnitude. However, both tissue and matrix force dropped quickly and to a greater extent for higher magnitudes. The matrix force was lower than the tissue force during the entire 12\u00a0h hold period at the same magnitude indicating that the cells contributed to the measured tissue force throughout the hold period (Fig. 9). The rate of decrease during the 12\u00a0h hold period is similar for both the tissue and matrix. This is evident in the fact that very quickly after the step stretch the cell force approaches a constant for the 12\u00a0h (Fig. 9). The cell force is approximately the same at both 10% and 20% step stretches during the 12\u00a0h held at the stretched length. For both step stretch magnitudes, the cell force at the end of the 12\u00a0h hold period is similar to the cell force at the baseline length (data not shown) immediately before the step stretch is applied.\nRings that did not have CytoD added before or during the step stretch were returned to their baseline lengths after being held at the stretched length for 8\u00a0h. The force dropped to zero but then began to recover with lower magnitude step stretches increasing their forces faster than higher magnitude step stretches. The recovered forces approached their baseline forces prior to stretch. After 6\u00a0h of monitoring force at baseline, rings stretched to 5% magnitude and held recovered more of their baseline force (84%) than rings stretched to 20% magnitude (42%).\nThe force decreases more rapidly in the step stretch and hold than the peak force drops in cyclic stretching at the same amplitude.\nFrequency Effect\nFigure 10 illustrates the trends in the peak tissue (Fig. 10(a, b)) and matrix force (Fig. 10(c, d)) either at the first stretch or after 12\u00a0h of cyclic stretch. It also compares the peak force trends over stretch frequencies and static incubation time. At all frequencies the peak tissue and matrix force increased with static incubation (Fig. 10).\nFIGURE 10.Peak tissue and matrix forces for different cyclic stretch frequencies at 10% amplitude. (A) Peak tissue force of the first cyclic stretch after 2, 4, or 8 days static incubation. Peak tissue force increased with cyclic stretch frequency and incubation time. (B) Peak tissue force after 12\u00a0h of cyclic stretch after 2, 4, or 8 days static incubation. At day 8 the peak tissue force increased slightly with increased frequency. (C) Peak matrix force of the first stretch after 2, 4, or 8 days static incubation. Peak matrix force increased slightly with increased cyclic stretch frequency. (D) Peak matrix force after 12\u00a0h of cyclic stretch after 2, 4, or 8 days static incubation. Peak matrix force similar regardless of cyclic stretch frequency. (E) Number of rings averaged for data shown in Figs.\u00a010\u201313 at each frequency and static incubation day. (F) Individual measurements used in panel B [Peak tissue force after 12\u00a0h and 8 days of static incubation]. This shows that the trend within an experiment (E#) is consistent but the experiment to experiment variation is large. Y-axis scales are different in each panel to emphasize the trends with frequency and incubation time.\nTissue Force\nInitially, higher frequencies led to higher peak tissue forces (Figs. 10(a), 11(a), 11(b)). After an initial drop, the peak force remained steady or increased at days 2 and 4. However, at day 8 after the initial drop the peak force continues to decline over the rest of the experiment (Fig. 11(a)). After 12\u00a0h, higher frequencies still resulted in higher peak forces (Figs. 10(b), 11(c)). Another difference is in the baseline forces, where lower frequencies had higher baseline forces than high frequencies. Thus, rings cyclically stretched at lower frequencies have lower measured tissue stiffness (slope of the loading curve).\nFIGURE 11.Tissue force versus cyclic stretch frequency at day 8. (A) Peak tissue force over 12\u00a0h of cyclic stretch. Peak tissue force dropped quickly after initiation of cyclic stretch followed by a continued slow decline. (B) First 5 tissue stretches after initiation of cyclic stretch. A large decline in tissue force occurred between the first and second stretch. (C) Last 5 tissue stretches after 12\u00a0h of cyclic stretch. The tissue stiffness increased with increasing cyclic frequency.\nMatrix Force\nInitially, higher frequencies led to higher peak matrix forces (Figs. 10(c), 12(a), 12(b)). These matrix forces quickly dropped over the first few hours and then continued a steady decline for the remainder of the cyclic stretch (Fig.\u200912(a)). At day 8, all the peak matrix forces quickly approached the same value about 30\u00a0min after the start of stretch (Fig. 12(a)). In addition, unlike the tissue and cell curves, there is little difference between the matrix curves at the different frequencies after 12\u00a0h except that lower frequencies contribute over a slightly larger range of strains but the peak force is still the same at all frequencies tested (Fig. 12(c)). As with the matrix force at different amplitudes, the loading curve was nonlinear and the matrix contributed only to the tissue force when the strain was within 4\u20136% of its maximum value.\nFIGURE 12.Matrix force versus cyclic stretch frequency at day 8. (A) Peak matrix force over 12\u00a0h of cyclic stretch. Peak matrix force dropped quickly after initiation of cyclic stretch followed by a slow decline for the 12\u00a0h duration. (B) First 5 matrix stretches after initiation of cyclic stretch. A large decline in matrix force occurred between the first and second stretch. (C) Last 5 matrix stretches after 12\u00a0h of cyclic stretch. Matrix loading curves were highly nonlinear and the peak forces was similar at all frequencies.\nCell Force\nSimilar to the first force-strain curves at different amplitudes, the cell force initially rose, then leveled off or even decreased at the highest frequency (Fig. 13(b)). At lower frequencies the slope of the loading curve was lower than that for higher frequencies (Fig. 13(c)). This is clear at day 8 when, for low frequency, the baseline cell force was higher and the peak cell force was lower than that of high frequencies (Fig. 13(c)). At the two lowest frequencies the cells were able to maintain a baseline force while at the two highest frequencies the baseline cell force was near zero. This leads to a cell stiffness that was higher at higher cyclic stretch frequencies. This was similar to the trend seen with the tissue stiffness.\nFIGURE 13.Cell force versus cyclic stretch frequencies at day 8. (A) Peak cell force over 12\u00a0h of cyclic stretch. (B) First 5\u00a0cell stretches after initiation of cyclic stretch. (C) Last 5\u00a0cell stretches after 12\u00a0h of cyclic stretch. Cell stiffness increased with increasing cyclic stretch frequency.\nImmunohistochemistry\nRings stained with a dye for actin cytoskeleton showed a distinct morphology that developed during the 8 days of incubation. Initially the cells were sparse with few cells on the outer layer of the rings (Fig. 14(a)). The inner core of the rings initially showed a higher cell concentration that had some alignment along the circumferential direction (Fig.\u00a014(b)). As the rings incubated, this outer layer that was approximately 25\u00a0\u03bcm thick increased in density but showed little preferential alignment (Fig. 14(c,e)). The inner core of the rings also had some increase in cell density but was noticeably less dense than the outer layer (Fig.\u00a014(d,f)). However, these cells were highly aligned in the circumferential direction (Fig. 14(d,f)).\nFIGURE 14.Confocal images at 2, 4, and 8 days of static incubation. F-actin fibers visualized using a TRITC phalloidin stain. (A) Outer surface (projection from 4\u201314\u00a0\u03bcm depth) after 2 days static incubation. (B) Inner core (projection from 150\u2013160\u00a0\u03bcm depth) after 2 days static incubation. (C) Outer surface (projection from 4\u201314\u00a0\u03bcm depth) after 4 days static incubation. (D) Inner core (projection from 61\u201371\u00a0\u03bcm depth) after 4 days static incubation. (E) Outer surface (projection from 10\u201315\u00a0\u03bcm depth) after 8 days static incubation. (F) Inner core (projection from 60\u201365\u00a0\u03bcm depth) after 8 days static incubation. Field of View: 230\u00a0\u03bcm \u00d7 230\u00a0\u03bcm. Circumferential direction is in the vertical direction.\nDISCUSSION\nWe have shown that a few hours after the initiation of stretch, higher cyclic stretch amplitude does not lead to higher cell or tissue forces. Thus, for the majority of the cyclic stretch duration, the peak stress experienced by the cells is independent of the stretch amplitude applied, at least in the range of 5%\u201325%. This is accomplished by the cells becoming less stiff at higher stretch amplitudes. The exact mechanism for this change in cell stiffness is unknown. We have found that the magnitude of the drop in tissue force after the addition of 2\u00a0\u03bcm CytoD is similar to the force drop after adding detergent (e.g. deoxycholate). Since the detergent treatment actually removes the cells,18 it is likely the actin cytoskeleton is primarily responsible for the change in cell stiffness. Whether this is a result of cytoskeletal remodeling and\/or reduction in active contraction\/relaxation of the myosin is not known.\nWhile the initial extracellular matrix consists entirely of collagen it is reasonable to assume that over 8 days of incubation time that additional cell-contributed matrix may be produced. We have incubated rings for 20 days prior to cyclic stretching and found that the matrix force-strain curves are similar to those after 8 days of incubation. Peak forces increase only a small amount at 5% or 10% stretch amplitude after 14\u00a0h of cyclic stretch at 0.1\u00a0Hz. These results suggest that the matrix mechanics do not change substantially even in the presence of cells that can produce additional matrix.\nIn addition, we treated some rings with 30\u00a0mM ribose after 10 days of incubation, and incubated them for an additional 10 days to non-enzymatically crosslink the collagen.9 This treatment increased the peak matrix force during cyclic stretch with a 3 to 4 fold increase after 14\u00a0h compared to the untreated rings incubated for 20 days. The shapes of the matrix force-strain curves changed somewhat, with a shorter period of slack during each force-strain curve. The increased peak force with glycation was similar to Isenberg and Tranquillo9 but comparing the shapes of the force-strain curves is difficult due to differences in experimental methods.\nWe also found that increased matrix stiffness due to crosslinking had little effect on cell force for 5% or 10% cyclic stretch amplitude at 0.1\u00a0Hz. Cell force-strain curves were almost identical for treated and untreated rings after 12\u00a0h of cyclic stretch. In 2-D cultures, cell traction depends upon the stiffness of the substrate.6 The cellular mechanism for this response is not clear, but Discher and co-workers suggest that both \u201coutside-in\u201d and \u201cinside-outside-in\u201d pathways may be involved. Whether 3-D culture conditions or cyclic stretching reduce the influence of matrix stiffness on cellular mechanics remains to be investigated.\nSince it is the cells that are responding to the mechanical conditioning it is important to realize that while the cells are undergoing a higher strain they are not undergoing a higher stress. So if the cells respond to stress, higher stretch amplitudes will not increase this response. Since both the cells and the tissues at different stretch amplitudes maintain similar stresses, it is reasonable to assume the cells are responding to maintain a certain range of mechanical stresses.\nSome limitations to estimating the cell-contributed force should be noted. Unlike the tissue and matrix force we are not able to measure the cell force directly but instead have to estimate it based on the direct measurement of the tissue and matrix. In addition, to estimate the cell force at more than a single time point, we must use the forces from different rings. Due to the ring to ring variations, it is necessary to normalize one ring to another to get quantitative values for the cell force. However, because this normalization occurs at 12\u00a0h, it is unknown how well this single point normalization works in predicting the cell force 12\u00a0h earlier during the initial stretches. Furthermore, the assumption that the cell force is simply the subtraction of the matrix force from the tissue force is only valid after a critical cell density is reached.11 At days 2 and 4 it is unlikely that this critical cell density is reached but it is surpassed based on cell density by day 8.\nUnlike the cells that respond to increased stretch amplitude by decreasing their stiffness, the matrix force-strain curves shift so that the matrix contributes only when the strain is within 3%\u20137% of the maximum value with the matrix contributing over a slightly larger range of strains at higher stretch amplitudes. This response of the matrix does not appear to depend upon the cells having an actin cytoskeleton, since we found a similar response when cytoD is added either before stretch is initiated or after 12\u00a0h of stretch. After 12\u00a0h of cyclic stretch, the peak matrix force is only slightly higher with increased stretch amplitude. With different frequencies of cyclic stretch, the peak matrix force is similar but the matrix contributes over a larger range of strains at lower frequencies. For a 10% stretch, the matrix contributes to the last 6% of the stretch at 0.001\u00a0Hz while contributing only to the last 4% at 0.25\u00a0Hz.\nThe higher baseline tissue and cell forces seen at lower frequencies (0.001\u00a0Hz and 0.01\u00a0Hz) are consistent with a simple viscoelastic model because the tissues and cells have more time to recover before being stretched again. The same is seen in the lower amplitudes at 0.1\u00a0Hz (5% and 10%) which are able to maintain a baseline force. These correspond to the lowest strain rates and may allow for recovery.\nFor a linear viscoelastic material undergoing continuous cyclic stretch, both higher stretch amplitudes and higher strain rates will lead to higher peak forces. For cyclic stretch at constant frequency, the highest stretch amplitude also corresponds to the highest strain rate. However, we observed that the peak cell force decreased as the amplitude (and the strain rate) increased. We also note that the decrease in peak cell force is a function of both stretch amplitude and strain rate because similar strain rates (5%\/s) at different amplitudes (e.g. 10% @ 0.25\u00a0Hz and 25% @ 0.1\u00a0Hz) show different tissue, matrix and cell responses In the case of the matrix at 25% @ 0.1\u00a0Hz, there is no positive matrix force until the strain increases beyond 10%; whereas there is matrix force below 10% strain during cyclic stretch at 10% @ 0.25\u00a0Hz.\nTo explain this complex behavior a model of cellular mechanics that includes strain-dependent and strain rate-dependent damage may be required. It is reasonable to assume large and\/or quick deformations may result in disruption of the actin cytoskeleton8 leading to damage, and thus a reduction in the cell force and stiffness. However, any damage model will be complicated by viscoelastic effects.\nOne possible mechanism for a strain dependent change in stiffness is an alteration in the amount of cell activation through a stretch-activated pathway. Two of the pathways that exist to activate myosin-light chain kinase and thus force generation in fibroblasts are a calcium dependent pathway and a Rho dependent pathway.13 To test the possible involvement of a calcium dependent pathway, we tested some rings in the presence of 50\u00a0\u03bcM or 500\u00a0\u03bcM of gadolinium chloride (GdCl3) to block stretch activated calcium channels.12 In the presence of 50\u00a0\u03bcM of GdCl3, day 2 rings showed a decrease in peak tissue force at both 5% and 10% cyclic stretch compared to untreated controls. This peak force decrease continued for the first 5\u20136\u00a0h of cyclic stretch then began to increase for the remainder of the 12\u00a0h of cyclic stretching but still did not return to the peak tissue force of untreated controls. However, for day 8\u00a0rings neither 50 nor 500\u00a0\u03bcM GdCl3 had an effect on the tissue force compared to untreated controls. Since GdCl3 reduces the tissue and cell force during the first several hours of cyclic stretch at day 2, it suggests that the calcium dependent pathway plays a role in maintenance of cell force at least during early tissue development. It is unclear what causes the cell force to increase after the initial decrease but it could be due to an upregulation of calcium independent pathways or incomplete inhibition of new or existing stretch activated channels. The lack of an effect of GdCl3 at day 8 suggests a reduced role of the calcium dependent pathway.\nIn addition, these results have important implications for the tissue engineering field, especially for those tissues that undergo continuous deformations such as in the cardiovascular system. By isolating the cell contribution to tissue force, we are able to provide a better understanding of the mechanical environment of the cell and aid in designing mechanical conditioning protocols that improve tissue properties. For example, the peak cell force is not constant during cyclic stretch and thus cell stimulation varies with time. Furthermore, if cell response is dependent on cell stress or force, increasing the amplitude of conditioning will not increase and may decrease the applied cell force.\nDue to the large changes in mechanical behavior after initiation of cyclic stretch, it is important to characterize engineered tissues\u2019 response to these loading conditions over longer time periods. Current mechanical characterization relies mostly on a few cycles of preconditioning followed by a test to failure.4,5,9,16 While failure tests are necessary, the response of the tissue and cells under physiologic loading is also vital. We have shown that mechanical properties may change dramatically over hours after initiation of stretch. These changes have implications on tissue function in vivo after implantation and these changes cannot be captured in simple failure tests.\nCONCLUSIONS\nWe have shown that bioartificial tissue constructs consisting of a single cell type and matrix component are a useful model system in which to explore tissue, matrix, and cell mechanics. The cell response to cyclic stretch is complex and depends on both the amplitude and frequency of cyclic stretch. The change in cell stiffness with amplitude behavior does not fit current cellular mechanics models. Future investigations may explore the microstructural basis for this behavior.","keyphrases":["cardiac fibroblasts","collagen","tissue engineering","cell mechanics"],"prmu":["P","P","P","P"]}
{"id":"Sleep_Breath-4-1-2194800","title":"Armodafinil improves wakefulness and long-term episodic memory in nCPAP-adherent patients with excessive sleepiness associated with obstructive sleep apnea\n","text":"Residual excessive sleepiness (ES) and impaired cognition can occur despite effective and regular nasal continuous positive airway pressure (nCPAP) therapy in some patients with obstructive sleep apnea (OSA). A pooled analysis of two 12-week, randomized, double-blind studies in nCPAP-adherent patients with ES associated with OSA evaluated the effect of armodafinil on wakefulness and cognition. Three hundred and ninety-one patients received armodafinil (150 or 250 mg) and 260 patients received placebo once daily for 12 weeks. Efficacy assessments included the Maintenance of Wakefulness Test (MWT), Cognitive Drug Research cognitive performance battery, Epworth Sleepiness Scale, and Brief Fatigue Inventory. Adverse events were monitored. Armodafinil increased mean MWT sleep latency from baseline to final visit by 2.0 min vs a decrease of 1.5 min with placebo (P < 0.0001). Compared with placebo, armodafinil significantly improved quality of episodic secondary memory (P < 0.05) and patients\u2019 ability to engage in activities of daily living (P < 0.0001) and reduced fatigue (P < 0.01). The most common adverse events were headache, nausea, and insomnia. Armodafinil did not adversely affect desired nighttime sleep, and nCPAP use remained high (approximately 7 h\/night). Adjunct treatment with armodafinil significantly improved wakefulness, long-term memory, and patients\u2019 ability to engage in activities of daily living in nCPAP-adherent individuals with ES associated with OSA. Armodafinil also reduced patient-reported fatigue and was well tolerated.\nIntroduction\nObstructive sleep apnea (OSA) syndrome includes excessive sleepiness (ES), fatigue, sleep fragmentation, impaired alertness, cognitive dysfunction, and mood disturbances [1, 2]. OSA is typically characterized by recurrent complete or partial airway collapse during sleep, resulting in frequent apnea and hypopneic events lasting at least 10\u00a0s [1]. Although the reported prevalence of OSA in the general population varies as a function of diagnostic criteria [2], OSA syndrome has been estimated to occur in 4 and 2% of middle-aged North American men and women, respectively [3]. OSA can lead to serious medical, social, and public safety consequences, and patients are at an increased risk for cardiovascular disease [4] and motor vehicle and occupational accidents [5, 6]. Quality of life and social functioning are also significantly impaired in this patient population [7, 8]. Fatigue, in particular, can contribute to reduced quality of life [1, 9]; thus, the assessment of patient-rated fatigue is an important outcome to evaluate and monitor in patients with OSA.\nNasal continuous positive airway pressure (nCPAP) therapy is the recommended standard of care for treating patients with moderate to severe OSA [10]. In a comprehensive review of 36 clinical studies, nCPAP therapy was shown to effectively reduce objective and subjective measures of sleepiness and improve quality of life in patients with moderate and severe OSA [11]. However, despite receiving adequate nCPAP therapy, half of patients with OSA still experience residual ES as measured by objective assessments as well as impairments in mood or cognition [12\u201318]. Cognitive impairment in patients with OSA is characterized by deficits in attention, learning and memory, and executive function (planning and problem solving) and may persist despite nCPAP therapy [12, 15\u201317]. A review of the literature suggests that while nCPAP therapy can substantially improve quality of life for patients with OSA, it has a smaller effect on improving cognitive performance [7].\nModafinil, a racemic mixture consisting of equal amounts of R- and S-enantiomers, has been shown to improve wakefulness in patients with OSA who experience residual ES despite regular nCPAP use [19, 20]. Pharmacokinetic studies have shown that the half-life of R-modafinil (10\u201314\u00a0h) is significantly longer than the half-life of S-modafinil (3\u20134\u00a0h) [21\u201323]. Armodafinil has higher plasma concentrations later in the day compared to modafinil on a \u201cmilligram-to-milligram\u201d basis and improves healthy subjects\u2019 wakefulness and ability to sustain attention for a longer period of time compared with modafinil [24]. Armodafinil has also recently been shown to improve wakefulness in patients with ES associated with narcolepsy in a 12-week double-blind, placebo-controlled study [25].\nRecent findings from two 12-week, double-blind, placebo-controlled studies of similar design indicate that armodafinil improved wakefulness and overall clinical condition in nCPAP-adherent patients with OSA and residual ES [26, 27]. In the present pooled analysis of the two OSA studies [26, 27], the effects on wakefulness, cognition, and fatigue and the safety and tolerability of armodafinil compared with placebo were evaluated in OSA patients adherent to nCPAP therapy.\nMaterials and methods\nStudy design and procedures\nData from two 12-week, multicenter, double-blind, placebo-controlled, parallel-group clinical studies were pooled for analysis. The pooling of these data was based on similarities in patient population, study design, and treatment duration in the two studies [26, 27]. One study was conducted in 36 centers across the USA, Australia, Russia, Germany, and France [26], and the other study was conducted in 37 centers across the USA and Canada [27]. The study protocols were approved by an Independent Ethics Committee or Institutional Review Board at each center. Both studies were conducted in accordance with the Good Clinical Practice: Consolidated Guideline [28] and national and local laws and regulations.\nPatients were randomized to receive armodafinil 150\u00a0mg (n\u2009=\u2009129) or placebo (n\u2009=\u2009130) daily in the first study [26] and armodafinil 150 (n\u2009=\u2009131) or 250\u00a0mg\/day (n\u2009=\u2009131), or placebo (n\u2009=\u2009130) in the second study [27]. Study drugs were initiated with a dose of 50\u00a0mg\/day on day\u00a01 and then increased in increments of 50\u00a0mg starting on day\u00a02 and every 2\u00a0days thereafter until the assigned dose was reached (i.e., 150\u00a0mg\/day on day\u00a04 for both studies or 250\u00a0mg\/day on day\u00a08 for the second study). The study drugs were taken once daily in the morning (before 08:00 and approximately 30\u00a0min before breakfast). Monitoring of patients\u2019 compliance with study drug administration was the responsibility of the investigator at each center and was assessed by completed study drug accountability records and reviews of patient diaries. nCPAP use at home was also monitored objectively by investigators at each visit to the study center, using the REMstar\u00ae Auto CPAP System (Respironics, Murrysville, PA). The REMstar Auto CPAP System was used in CPAP mode.\nPatient selection criteria\nMethods for selecting patients in both studies have been described previously [26, 27]. Men and women 18 to 65\u00a0years of age with a current diagnosis of OSA as defined by the International Classification of Sleep Disorders [1] were included in both studies. Patients were at least moderately ill (Clinical Global Impression of Severity of Illness rating greater than or equal to 4) [29] and had a complaint of ES. Patients were eligible if ES was determined to be pathological, based on an Epworth Sleepiness Scale (ESS) score greater than or equal to 10 [30]. All patients were receiving stable (greater than or equal to 4\u00a0weeks) and effective nCPAP therapy on a regular basis (greater than or equal to 4\u00a0h per night on greater than or equal to 70% of nights) during the 2-week evaluation period. The CPAP use was monitored using the REMstar Auto CPAP System. Effectiveness of therapy was determined by an apnea\u2013hypopnea index (AHI) less than or equal to ten events per hour on nighttime polysomnography (PSG).\nPatient exclusion criteria\nPatients were excluded if they had any of the following: a diagnosis of a sleep disorder other than OSA, symptoms of ES associated with a clinically significant uncontrolled medical or psychiatric condition as determined by the investigator, any disorder that might interfere with drug absorption, distribution, metabolism, or excretion, a history of drug or alcohol abuse, as determined by the Diagnostic and Statistical Manual of Mental Disorders IV criteria [31] or by a positive result from the urine drug screen given at screening and again at the final visit, caffeine consumption greater than 600\u00a0mg\/day, or a clinically significant sensitivity to central nervous system stimulants or modafinil. Women who were pregnant or breast feeding were also excluded. The use of any substance that could affect wakefulness or sleepiness (e.g., modafinil, sodium oxybate, melatonin, lithium, St. John\u2019s wort, methylphenidate, amphetamines, pemoline, antipsychotic agents, benzodiazepines, zolpidem, anticonvulsants, or barbiturates), use of other excluded agents (e.g., monoamine oxidase inhibitors, anticoagulants), use of clinically significant amounts of nonprescription drugs within 7\u00a0days of screening, and use of investigational drugs within 1\u00a0month of screening was prohibited. All patients provided written informed consent before participation in these studies and were compensated for their participation.\nAssessments\nEfficacy assessments\nEfficacy assessments were performed for both studies at baseline and weeks\u00a04, 8, and 12. The primary efficacy variables in the two individual studies were the change from baseline to final visit (week\u00a012 or last postbaseline measurement) in mean Maintenance of Wakefulness Test (MWT) [32, 33] sleep latency averaged across the first four tests and the proportion of patients with at least minimal improvement on the Clinical Global Impression of Change (CGI-C) [29]. The MWT was conducted as six separate 30-min sessions at 09:00, 11:00, 13:00, 15:00, 17:00, and 19:00. Sleep latency was defined as the time to onset of the first of three consecutive epochs of stage 1 sleep or the time to onset of any epoch of stages 2, 3, and 4 sleep or rapid eye movement sleep [34]. Sleep latencies were averaged across the first four tests (09:00, 11:00, 13:00, 15:00) and the last three tests (15:00, 17:00, 19:00) to distinguish between early- and late-day effects, respectively. The proportion of patients with at least a minimal improvement on the CGI-C was assessed to determine patients\u2019 overall clinical improvement in the individual studies; however, these data were not poolable for this analysis because of a substantial difference in the percentage of responders to placebo between the two studies.\nThe Cognitive Drug Research (CDR) battery of tests [35, 36] was administered by computer as six separate 25-min sessions conducted at 09:30, 11:30, 13:30, 15:30, 17:30, and 19:30. The CDR battery includes five memory tests (immediate word recall, delayed word recall, numeric working memory, word recognition, and picture recognition) and three attention tests (simple reaction time, choice reaction time, and digit vigilance). Two composite factors are derived from the memory tests in the CDR: quality of episodic secondary memory, a measure of long-term memory that measures the ability to recall verbal and visual information, and speed of memory, which assesses the time it takes to decide whether information is held in memory. Two composite factors are derived from the CDR for attention: power of attention, which measures the ability to focus attention and avoid distraction (concentration), and continuity of attention, which measures the ability to sustain attention (vigilance). Similar to the MWT, the CDR scores were averaged across the first four test sessions (09:30, 11:30, 13:30, 15:30) and the last three test sessions (15:30, 17:30, 19:30) to assess cognitive effects on early- and late-day measurements.\nThe patients\u2019 ability to engage in daily activities was assessed at each visit by the ESS [30]. ESS total scores range from 0 to 24, with higher scores indicating greater sleepiness. An ESS score greater than or equal to 10 indicates pathological sleepiness [30]. The severity and impact of patient-rated fatigue on daily functioning were assessed at each visit by the nine-item Brief Fatigue Inventory (BFI) [37] and were based on changes in global fatigue (average of all nine questions) and worst fatigue in the past 24\u00a0h (item 3). The rating scale for the BFI ranges from 0 (no fatigue) to 10 (as bad as you can imagine). A score of greater than or equal to 7 for either global fatigue or worst fatigue in the past 24\u00a0h is indicative of severe fatigue. Both the ESS and BFI were administered before the first MWT session (09:00) at each visit.\nSafety and tolerability assessments\nAdverse events were monitored and recorded by the study investigators at each center throughout both studies. Clinical laboratory tests (blood chemistry, hematology, urinalysis), vital signs (resting heart rate and systolic and diastolic blood pressure 3 and 13\u00a0h postadministration), and electrocardiograms were obtained at screening, baseline, and weeks\u00a04, 8, and 12. Physical examinations were performed at baseline and week\u00a012. Patients\u2019 use of nCPAP was monitored at least 2\u00a0weeks before baseline and throughout both studies using the Respironics REMstar Auto CPAP system. Effect on nighttime sleep was determined by PSG, which was performed the night immediately after the measurement of daytime MWT during the second screening visit and the final visit. The PSG was conducted for 8\u00a0h, starting within 30\u00a0min of the patient\u2019s usual bedtime but not earlier than 21:30.\nClinically significant elevations in resting systolic and diastolic blood pressure were defined a priori as greater than or equal to 140\u00a0mmHg with an increase of greater than or equal to 10% and greater than or equal to 90\u00a0mmHg with an increase of greater than or equal to 10%, respectively. Patients with worsening hypertension included those who had a history of hypertension at baseline and who started new antihypertensive medication and\/or increased the dose of previously used antihypertensive medication during the studies. Patients with newly diagnosed hypertension included those who had no history of hypertension at baseline and who started antihypertensive medication during the studies. Patients at risk for hypertension included those who had at least two clinically significant elevations in blood pressure readings between baseline and final visit.\nStatistical analysis\nDescriptive statistics were used to summarize continuous and categorical demographic variables. Efficacy assessments were analyzed at weeks\u00a04, 8, and 12 using observed cases and at final visit (week\u00a012 or last postbaseline measurement) using the last observation carried forward approach. Efficacy analyses included randomized patients who received at least one dose of study drug and had a baseline measurement with at least one postbaseline measurement on the MWT and CGI-C. Safety and tolerability analyses included all randomized patients who received at least one dose of study drug. All efficacy assessments were analyzed by analysis of variance (ANOVA) with treatment and study as factors. Tests of poolability for all continuous efficacy variables across the two studies were conducted using an ANOVA with treatment and study and treatment by study interaction as factors. Vital signs and data from nighttime PSG were analyzed by Wilcoxon rank-sum test. For nCPAP use, the change from baseline to on-treatment values was analyzed using one-way ANOVA. All statistical tests were two-tailed, and the 5% level of significance was used.\nResults\nPatient demographics and disease characteristics\nOf 658 patients randomized, 651 were included in the safety analysis, and 601 were evaluable for efficacy (Fig.\u00a01). Patient demographics and disease characteristics for all patients in the safety analyses are summarized in Table\u00a01. The mean body mass index was 36.6\u00a0kg\/m2. Patients reported high nightly nCPAP usage (mean [SD] hours of nightly use, armodafinil 6.9 [1.2] h; placebo 6.9 [1.0] h). The nCPAP therapy was effective, as shown by mean (SD) AHI values of 1.5 (3.3) and 1.2 (2.1) events\/h in the armodafinil and placebo groups, respectively. Overall, 41% of patients had a history of hypertension at baseline.\nFig.\u00a01Patient dispositionTable\u00a01Patient demographics and baseline characteristicsCharacteristicArmodafinil (n\u2009=\u2009391)Placebo (n\u2009=\u2009260)Age (years)Mean (SD)49.7 (9.0)50.3 (9.1)Sex, n (%)Men283 (72)183 (70)Women108 (28)77 (30)Race, n (%)White327 (84)224 (86)Black36 (9)21 (8)Asian6 (2)3 (1)Other22 (6)11 (4)Missing01 (<1)Weight (kg)Mean (SD)110.4 (24.6)111.2 (23.7)BMI (kg\/m2)Mean (SD)36.4 (8.0)36.9 (7.5)CGI-S, n (%)Moderately ill219 (56)138 (53)Markedly, severely, or extremely ill172 (44)122 (47)nCPAP (h)Mean (SD)6.9 (1.2)6.9 (1.0)AHIMean (SD)1.5 (3.3)1.2 (2.1)History of hypertensionn (%)159 (41)108 (42)AHI Apnea\u2013hypopnea index, BMI body mass index, CGI-S Clinical Global Impression of Severity of Illness, nCPAP nasal continuous positive airway pressure\nEffects on wakefulness\nMean (SD) sleep latency at baseline across the first four MWTs (09:00, 11:00, 13:00, 15:00) was 22.8 (8.4) and 23.2 (7.9) min for the armodafinil and placebo groups, respectively. At all study visits, armodafinil significantly improved mean sleep latency across the first four MWTs compared with placebo (Fig.\u00a02a). The mean change from baseline in sleep latency at final visit was 2.0\u00a0min for the armodafinil group compared with \u22121.5\u00a0min for the placebo group (P\u2009<\u20090.0001).\nFig.\u00a02Mean (SEM) change from baseline in sleep latency from the Maintenance of Wakefulness Test (MWT). a Sleep latencies averaged across the first four tests (09:00\u201315:00). b Sleep latencies averaged across the last three tests (15:00\u201319:00)\nArmodafinil also improved patients\u2019 ability to maintain late-day wakefulness (MWTs at 15:00, 17:00, 19:00) compared with placebo (Fig.\u00a02b). Mean (SD) sleep latency at baseline across the last three MWTs was 24.4 (7.8) and 24.6 (7.4) min for the armodafinil and placebo groups, respectively. The mean change from baseline in sleep latency at final visit was 1.1\u00a0min for the armodafinil group compared with \u22120.3\u00a0min for the placebo group (P\u2009<\u20090.05).\nEffects on memory and attention\nCompared with placebo, armodafinil significantly improved the quality of episodic secondary memory across the first four tests (09:30, 11:30, 13:30, 15:30) at all study visits (P\u2009<\u20090.05; Fig.\u00a03a). The mean (SD) change from baseline in quality of episodic secondary memory at final visit was 10.2 (31.7) U for the armodafinil group compared with \u22120.7 (46.3) U for the placebo group (P\u2009<\u20090.01). Differences between the treatment groups did not achieve statistical significance at the final visit for speed of memory, power of attention, or continuity of attention across the first four tests.\nFig.\u00a03Mean (SEM) change from baseline in quality of episodic secondary memory. a Scores averaged across the first four tests (09:30\u201315:30). b Scores averaged across last three tests (15:30\u201319:30)\nAcross the later three tests of the day (15:30, 17:30, 19:30), the difference in the quality of episodic secondary memory was statistically significant at week\u00a08 for armodafinil compared with placebo (P\u2009=\u20090.011; Fig.\u00a03b), with a trend toward significance at week\u00a012 (P\u2009=\u20090.081). The difference between treatment groups was not statistically significant at the final visit for the quality of episodic secondary memory. There were no significant changes from baseline to final visit between the armodafinil and placebo groups for speed of memory, power of attention, or continuity of attention across the last three tests.\nEffects on patients\u2019 ability to engage in activities\nThe mean (SD) ESS scores were high at baseline (15.4 [3.5] and 15.9 [3.5] for the armodafinil and placebo groups, respectively), despite the high nCPAP use as shown in Table\u00a01. Compared with placebo, treatment with armodafinil significantly improved patients\u2019 ability to engage in activities of daily living (ESS) at all visits (P\u2009<\u20090.0001; Fig.\u00a04). Nearly half of all patients (49%) in the armodafinil group responded to treatment (total ESS score less than 10 at final visit) compared with 26% in the placebo group.\nFig.\u00a04Mean (SEM) change from baseline in Epworth Sleepiness Scale total score\nEffects on fatigue\nThe mean (SD) scores for global fatigue from the BFI at baseline were 4.8 (1.9) and 4.9 (1.9) for the armodafinil and placebo groups, respectively. Armodafinil significantly improved global fatigue at all visits compared with placebo (P\u2009<\u20090.01; Fig.\u00a05a). Mean scores for worst fatigue in the past 24\u00a0h from the BFI at baseline were nearly identical for the armodafinil and placebo groups (7.2 [2.0] and 7.3 [2.0], respectively) and indicated severe fatigue (score greater than or equal to 7). Armodafinil significantly improved scores for worst fatigue in the past 24\u00a0h at weeks\u00a04 and 12 and at the final visit (P\u2009<\u20090.05 vs placebo; Fig.\u00a05b), with a trend toward significance at week\u00a08 (P\u2009=\u20090.056 vs placebo).\nFig.\u00a05Mean (SEM) change from baseline in fatigue. a Global fatigue. b Worst fatigue in the past 24\u00a0h. BFI Brief Fatigue Inventory\nSafety and tolerability\nArmodafinil was well tolerated, with a low incidence of adverse events, most of which were mild to moderate in nature. Headache was the most commonly reported adverse event, defined as occurring in greater than or equal to 5% in either group (Table\u00a02). Serious adverse events were considered by the investigator unlikely to be or not related to armodafinil (ulcerative colitis, n\u2009=\u20091; migraine, n\u2009=\u20091; worsening of Axis II disorder and mood disorder, n\u2009=\u20091; duodenal ulcer hemorrhage, n\u2009=\u20091). One serious adverse event was reported in the placebo group (gastroesophageal reflux disease). The most frequently occurring adverse events that led to discontinuation among patients receiving armodafinil were headache (n\u2009=\u20095) and nausea (n\u2009=\u20094).\nTable\u00a02Adverse events occurring in \u22655% of patientsAdverse event, n (%)Armodafinil (n\u2009=\u2009391)Placebo (n\u2009=\u2009260)Headache65 (17)20 (8)Nausea22 (6)10 (4)Insomnia22 (6)3 (1)Dizziness19 (5)4 (2)Anxiety20 (5)2 (<1)\nThere were no clinically significant changes from baseline to final visit in either the armodafinil group or the placebo group for laboratory values, electrocardiogram parameters, or physical examinations. There were also no meaningful changes (clinical or statistical) from baseline to final visit for systolic blood pressure (0.2 [14.2] mmHg for armodafinil vs \u22121.0 [14.6] mmHg for placebo), diastolic blood pressure (0.3 [9.3] mmHg for armodafinil vs \u22121.0 [10.0] mmHg for placebo), and heart rate (2.3 [9.6] bpm for armodafinil vs 1.4 [9.6] bpm for placebo).\nThe incidence of patients with newly diagnosed hypertension was less than 1% in the armodafinil group and less than 1% in the placebo group. The proportion of patients at risk for hypertension was similar for both treatment groups (18% of 391 patients, armodafinil group; 16% of 260 patients, placebo group). The incidence of patients with worsening hypertension was 3% of 391 patients in the armodafinil group and 2% of 260 patients in the placebo group.\nnCPAP use remained high (approximately 7\u00a0h\/night) throughout both of the studies. The duration of nCPAP use at final visit was reduced from baseline by a mean (SD) of \u22120.3 (0.7) h with armodafinil compared with \u22120.1 (0.6) h with placebo (a between-group difference of about 12\u00a0min; P\u2009<\u20090.0001). At final visit, AHI values remained low and were comparable between the armodafinil and placebo groups (P\u2009>\u20090.05 vs placebo). In addition, there were no significant changes from baseline in nocturnal PSG sleep variables between the armodafinil and placebo groups (P\u2009>\u20090.05; Table\u00a03).\nTable\u00a03Nocturnal polysomnography parametersaVariable (units), mean (SD)Armodafinil (n\u2009=\u2009391)Placebo (n\u2009=\u2009260)BaselineFinal VisitBaselineFinal VisitLatency to persistent sleep (min)22.3 (26.9)19.6 (20.5)21.3 (24.0)20.8 (21.4)Number of arousals, n20.0 (11.3)18.5 (10.2)18.7 (9.7)18.4 (10.4)Number of awakenings, n8.8 (4.7)9.2 (5.3)8.7 (5.1)9.6 (5.4)Sleep efficiency (%)82.4 (10.9)82.0 (12.0)82.0 (12.1)81.4 (11.2)Wake after sleep onset (min)66.6 (43.9)69.1 (48.5)68.7 (50.3)70.2 (46.5)Stage 1 (%)11.2 (6.4)10.5 (5.5)10.9 (6.1)10.6 (6.2)Stage 2 (%)59.3 (9.8)58.8 (11.2)58.8 (10.1)57.7 (11.2)Stage 3\/4 (%)10.6 (9.0)10.3 (9.0)10.8 (9.6)10.9 (10.2)REM (%)18.9 (6.9)19.8 (7.1)19.5 (7.2)20.7 (8.0)REM Rapid eye movementaNo significant differences between baseline and final visit were observed.\nDiscussion\nRecognizing and effectively managing ES in patients with OSA is essential for clinicians because of the profound impact that ES has on the patients, their families and coworkers, and the general public (e.g., increased risk of traffic accidents) [4\u20136]. Without appropriate treatment, ES adversely affects cognitive [15, 17], occupational [6], and social functioning [8]. Several studies evaluated in a comprehensive review [11] have shown that there is general improvement in wakefulness with nCPAP therapy, especially in patients with more severe OSA. Nonetheless, residual ES can persist even after effective nCPAP treatment [18], suggesting that there is need for adjunctive therapy to treat ES in these patients.\nThe present study re-evaluates the findings from two separate studies on the effects of armodafinil in patients with refractory ES associated with OSA and provides additional information regarding secondary analyses, for which the individual studies may not have had adequate statistical power. In the individual studies [26, 27], armodafinil was shown to significantly improve daytime mean sleep latency across the first four tests on the MWT and overall clinical condition as assessed by the CGI-C. In the pooled analysis, armodafinil significantly improved patients\u2019 ability to sustain wakefulness as objectively assessed by the MWT. Improvements in mean sleep latency were observed at the first visit at week\u00a04 and were maintained throughout the remainder of the study. The lack of a consistent effect of armodafinil on wakefulness across the last three MWTs (15:00, 17:00, 19:00) likely reflects the high mean sleep latency (approximately 24.5\u00a0min) at these times. Behavioral effects, causing poorer performance on the last test of the day, and high intertest variability in sleep latency may have also contributed to this finding [38].\nThe individual studies also included the clinician\u2019s subjective assessment of treatment effect on ES, the CGI-C. Data for the CGI-C, however, were not poolable for this analysis because of substantial differences in the proportion of patients considered responders to placebo between the two studies. In the individual studies, the proportion of patients considered responders to armodafinil and placebo was 71 and 53%, respectively, in one study [26] and 72 and 37%, respectively, in the other study [27].\nCognitive impairment is common in patients with OSA [17]. Although the etiology has not been definitively determined, decreased cortical activity because of impaired arousal or neuronal damage because of chronic intermittent hypoxia are possible causes [16, 39\u201341]. It is not yet clear what additional interventions may be effective in restoring cognitive function in patients with OSA. Findings from studies evaluating the effects of nCPAP therapy on memory and attention have yielded inconsistent results [15, 16, 40, 42, 43]. Unlike the findings from the individual armodafinil OSA studies [26, 27], the pooled analysis showed that adjunct armodafinil significantly improves long-term memory (i.e., quality of episodic secondary memory) at all study visits compared with placebo, indicating that treatment was associated with a greater ability to recall verbal and visual information. Interestingly, the long-term memory benefit occurred independent of a consistent benefit on attention and concentration. It is important to note that the CDR composite factors in both of the individual armodafinil OSA studies were secondary efficacy variables. Thus, although results from the pooled analysis provide a more precise estimate of the effects on long-term memory, further studies are needed to determine the potential role of armodafinil in improving cognitive function in nCPAP-adherent patients with OSA and associated ES.\nArmodafinil significantly improved patients\u2019 ability to engage in activities of daily living at all visits as measured by the ESS. The patient population studied in this pooled analysis had severe ES (mean ESS score greater than 15) despite effective and regular nCPAP therapy. At the final visit, 49 and 26% of patients receiving armodafinil and placebo, respectively, had ESS scores less than 10, indicating that nearly half of patients no longer had pathological sleepiness with adjunct armodafinil treatment.\nFatigue may be the presenting complaint of patients with ES [44] and is a common associated symptom [45, 46]. Armodafinil significantly reduced fatigue, a result similar to findings from the individual studies [26, 27]. Armodafinil reduced global fatigue from a baseline value of 4.8 to a final visit value of 3.6. Similarly, worst fatigue in the past 24\u00a0h was reduced from a baseline of 7.2 to 5.8 at final visit. These findings indicate that armodafinil significantly reduced the severity of global fatigue and worst fatigue in the past 24\u00a0h to the mild-to-moderate range of fatigue (BFI score less than 7 [37]).\nThis pooled analysis shows that armodafinil does not adversely affect nighttime sleep in patients with OSA even with its longer-lasting duration of action compared with modafinil on a milligram-to-milligram basis [24]. Armodafinil was well tolerated; in general, adverse events were rated as mild to moderate in nature, although some patients discontinued because of adverse events. There were some minor changes in vital signs; however, these effects were not considered to be clinically significant. The patient population selected for both studies was verified to be adherent to nCPAP therapy; thus, the primary pathology of OSA was being treated in an appropriate and effective manner throughout the course of the two studies. At the final visit, the duration of nCPAP use had decreased minimally from baseline compared with placebo. Average nCPAP use remained high (approximately 7\u00a0h\/night) and was effective (i.e., low AHI values). Adjunct armodafinil treatment did not affect arousals.\nFindings from the present pooled analysis are limited to patients with OSA who have residual ES despite regular and effective nCPAP therapy and should not be generalized to patients with OSA who are not receiving adequate nCPAP therapy or are not using it regularly. Additionally, the 12-week duration of treatment in these studies limits the applicability of observed results to a longer period of treatment. It should be recognized that armodafinil does not treat the underlying airway obstruction and should not be considered a replacement for nCPAP therapy in patients with OSA. Further research is needed to determine the role armodafinil may have in improving cognitive function and whether the significant reduction in fatigue observed in the pooled analysis will contribute toward improved quality of life in this patient population.\nIn conclusion, pooled data from two 12-week, double-blind, placebo-controlled studies showed that once-daily administration of armodafinil significantly improved wakefulness when used as adjunct therapy in nCPAP-adherent patients with residual ES associated with OSA. The effect on wakefulness with armodafinil was maintained throughout the day. Importantly, adjunctive treatment with armodafinil was associated with significant improvements in long-term memory and patients\u2019 ability to engage in daily activities. Armodafinil significantly reduced fatigue in the studied population and was well tolerated.","keyphrases":["armodafinil","wakefulness","sleepiness","fatigue","obstructive sleep apnea syndrome"],"prmu":["P","P","P","P","R"]}
{"id":"Childs_Nerv_Syst-3-1-1849423","title":"Paradigm shift in hydrocephalus research in legacy of Dandy\u2019s pioneering work: rationale for third ventriculostomy in communicating hydrocephalus\n","text":"Objective This study aims to question the generally accepted cerebrospinal fluid (CSF) bulk flow theory suggesting that the CSF is exclusively absorbed by the arachnoid villi and that the cause of hydrocephalus is a CSF absorption deficit. In addition, this study aims to briefly describe the new hydrodynamic concept of hydrocephalus and the rationale for endoscopic third ventriculostomy (ETV) in communicating hydrocephalus.\nIntroduction\nThe modern era of hydrocephalus research began with the experimental studies of Dandy and Blackfan [3] in 1914, which, until today constitutes an unsurpassed contribution to this field. By plugging the aqueduct, they produced hydrocephalus in dogs. The division of hydrocephalus into the obstructive and communicating type originates from their work. The bulk flow theory and the concept of cerebrospinal fluid (CSF) malabsorption as the cause of hydrocephalus are also based on their experiment [3]. However, Dandy strongly opposed the view that idiopathic communicating hydrocephalus is caused by an obstruction at the arachnoid villi. Such an obstruction cannot cause a higher pressure in the ventricles than in the subarachnoid space but would rather dilate the subarachnoid space. He also objected to the view that the CSF is absorbed by the arachnoid villi and instead proved that the CSF is absorbed by the capillaries [3, 4]. If this is true, the modern era of CSF and hydrocephalus research started and ended with Dandy, who left the pathophysiology of idiopathic communicating hydrocephalus unexplained for future exploration.\nBy going back to Dandy and following his lead of a capillary CSF absorption, a new understanding of hydrocephalus may be found. O\u2019Connel [19] and Bering [1] suggested that increased pulse pressure in the ventricles is the cause of communicating hydrocephalus. In 1978, Di Rocco and Pettorossi [5] verified that an intraventricular pulsating balloon causes communicating hydrocephalus in sheep. In 1993, Greitz re-valuated the physiology of the CSF circulation and hydrocephalus by using flow sensitive magnetic resonance imaging (MRI) and radionuclide cisternography [9]. He suggested that the capillaries absorb the CSF and that the distending force in the production of chronic hydrocephalus is an increased systolic pulse pressure in brain tissue [14].\nThe hydrodynamic concept of hydrocephalus\nClassification of hydrocephalus The classification is based on capillary absorption of the CSF [9\u201314]. Hydrocephalus is divided into two main groups, acute hydrocephalus and chronic hydrocephalus [14]. Acute hydrocephalus is caused by an intraventricular CSF obstruction in accordance with the conventional view. As opposed to that view, it is suggested that chronic hydrocephalus is caused by decreased intracranial compliance.\nCauses of decreased intracranial compliance Although the etiology of the decreased intracranial compliance such as CSF obstructions at the foramen magnum and adhesions in the subarachnoid space after intracranial bleedings, trauma, operations and infections is well known, the etiology remains unknown in many idiopathic and congenital cases of hydrocephalus. However, the final stage of chronic hydrocephalus, independent of its etiology, is uniform. The final stage is characterised by narrow capacitance vessels and reduced cerebral blood volume. This reduces cerebral blood flow, reduces intracranial compliance and enhances the intracerebral pulse pressure.\nIntracerebral pulse pressure theory of chronic hydrocephalus Chronic hydrocephalus consists of two subtypes, communicating hydrocephalus and chronic obstructive hydrocephalus [14]. The associated malabsorption of CSF is not involved as a causative factor in chronic hydrocephalus. The theory is based on one basic mechanism, i.e. that chronic hydrocephalus is caused by decreased intracranial compliance increasing the systolic pressure transmission into the brain parenchyma [9, 10, 12\u201314]. The increased systolic pressure in the brain is the cause of the distension of the brain and ventricles. The systolic expansion of the brain distends the brain towards the skull and simultaneously compresses the periventricular region of the brain. The rationale result is the predominant enlargement of the ventricles and a narrowing of the subarachnoid space.\nRationale for ETV in communicating hydrocephalus As described above, communicating hydrocephalus is caused by decreased compliance increasing the systolic pressure transmission into the brain. The systolic force compresses the brain including the intracranial capacitance vessels, i.e. the cerebral veins and capillaries. This results in a vicious cycle with narrow capacitance vessels and significantly reduced cerebral blood volume that causes further decrease in intracranial compliance and further increase in intra-cranial pulse pressure. It is obvious that an endoscopic third ventriculostomy (ETV) may interrupt the vicious cycle and reduce the systolic pressure in the brain simply by venting ventricular CSF through the stoma. The patent aqueduct in communicating hydrocephalus is too narrow to vent the ventricular CSF sufficiently. By reducing the pulse pressure, the ETV also reduces the compression of the capacitance vessels thereby restoring some degree of venous compliance, which in turn reduces the intracerebral pulse pressure further.\nETV versus shunting The proposed concept opens a new avenue in that ETV may be an effective treatment also in communicating hydrocephalus [8, 16\u201318]. It thus constitutes an interchangeable alternative to shunting. The primary aim of the treatment of chronic hydrocephalus is to restore intracranial compliance, which is achieved by both treatments [14]. Effective shunting is based on a slight over-drainage of CSF that ultimately causes a direct and forced dilation of the compressed veins [14]. Therefore, shunting is more effective in increasing intracranial compliance than ETV where the dilating effect on the veins works indirectly by reducing the systolic compressing force on the veins. Because ETV provides a more physiological treatment and has less late complications than shunting, it may be of benefit to increase the number of patients finally treated by ETV [2, 6\u20138, 15, 17, 18, 20]. In patients shunted under 1\u00a0year of age, ETV should be considered as an alternative treatment when they grow older. However, the final answer as to which is the optimal treatment must be based on randomized clinical studies.\nDiscussion\nThe aim of this paper is to briefly describe the hydrodynamic concept of hydrocephalus, which is based on Dandy\u2019s observation of capillary absorption of the CSF. Oi and Di Rocco [20] recently suggested that a minor CSF pathway through the brain parenchyma is the dominant CSF absorption site in the embryo, foetus and infant. The theory described here suggests that the minor pathway in the developing immature brain as well as the major pathway in adults are through the brain capillaries. It is beyond the scope of this brief communication to present the numerous physiological (and self-evident) evidences that the brain capillaries have the capacity to absorb fluids such as the CSF and its constituencies [3, 4, 9, 11, 12, 14]. This contrasts to the total lack of physiological evidence that the arachnoid villi can absorb the CSF. The CSF dynamics has been described in earlier published papers [9, 11, 12, 14].\nThe proposed concept indicates a paradigm shift in our view on hydrocephalus. It is suggested that chronic hydrocephalus is caused by decreased intracranial compliance rather than a CSF absorption deficit [14]. The associated CSF malabsorption is a secondary phenomenon to the decreased intracranial compliance. The only residue of the conventional view is acute hydrocephalus, which is caused by a CSF absorption deficit (of the periventricular capillaries). Normal-pressure hydrocephalus, aqueductal stenosis as well as communicating hydrocephalus in infants and adults are typical manifestations within the spectrum of chronic hydrocephalus. It should be emphasised that chronic hydrocephalus is an unstable hemodynamic disorder occurring at normal or slightly increased mean intracranial pressure with superimposed vascular pressure waves that can be fatal.\nThe theory may thus have identified decreased intra-cranial compliance as a new and potential universal cause of chronic hydrocephalus. Decreased compliance is common to all different types of chronic hydrocephalus whatever their etiology. If the theory is correct, there is hope for improved treatment of chronic hydrocephalus of unknown etiology. How come then that treatment failure rather is the rule than the exception of this devastating condition? The reason may be that shunting and ETV essentially represents symptomatic treatments of hydrocephalus. To improve therapy, we have to find treatments that are more effective in increasing compliance in cases with unknown etiology or are directed against the cause of the decreased compliance in cases with known etiology.","keyphrases":["hydrocephalus","cerebrospinal fluid","etv","pathophysiology","pulse pressure","compliance","normal-pressure hydrocephalus"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"J_Biomol_NMR-4-1-2268728","title":"BioMagResBank (BMRB) as a partner in the Worldwide Protein Data Bank (wwPDB): new policies affecting biomolecular NMR depositions\n","text":"We describe the role of the BioMagResBank (BMRB) within the Worldwide Protein Data Bank (wwPDB) and recent policies affecting the deposition of biomolecular NMR data. All PDB depositions of structures based on NMR data must now be accompanied by experimental restraints. A scheme has been devised that allows depositors to specify a representative structure and to define residues within that structure found experimentally to be largely unstructured. The BMRB now accepts coordinate sets representing three-dimensional structural models based on experimental NMR data of molecules of biological interest that fall outside the guidelines of the Protein Data Bank (i.e., the molecule is a peptide with 23 or fewer residues, a polynucleotide with 3 or fewer residues, a polysaccharide with 3 or fewer sugar residues, or a natural product), provided that the coordinates are accompanied by representation of the covalent structure of the molecule (atom connectivity), assigned NMR chemical shifts, and the structural restraints used in generating model. The BMRB now contains an archive of NMR data for metabolites and other small molecules found in biological systems.\nOrganization. The BioMagResBank (BMRB, http:\/\/www.bmrb.wisc.edu\/), the repository for experimental and derived data gathered from NMR spectroscopic studies of biological molecules (Ulrich et\u00a0al. 2008), has been a member of the Worldwide Protein Data Bank (wwPDB, http:\/\/www.wwpdb.org\/) since 2006 (Berman et\u00a0al. 2003). Other wwPDB partners include the Research Collaboratory for Structural Biology (RCSB PDB, http:\/\/www.pdb.org\/), the Protein Data Bank Japan (PDBj, http:\/\/www.pdbj.org\/), and the Macromolecular Structure Database at the European Bioinformatics Institute (MSD EBI, http:\/\/www.ebi.ac.uk\/msd\/). These four groups work closely together with the goal of maintaining a single Protein Data Bank archive of macromolecular structural data that is freely and publicly available to the global community.\nThe wwPDB Advisory Committee (wwPDBAC), which provides oversight and advice, meets yearly and currently is chaired by Stephen K. Burley. This committee consists of the leaders of the four sites plus two scientists nominated by each of the four partner sites. Additional ex officio members include scientists representing various stakeholder communities (e.g., International Union of Crystallography, International Council on Magnetic Resonance in Biological Systems, electron microscopy, structural genomics) and representatives of the international funding agencies that support the wwPDB member sites.\nOn matters pertaining to NMR spectroscopy, the wwPDB is advised by members of the wwPDB NMR Task Force (NMR TF), which is chaired by Robert Kaptein (a wwPDBAC member). Historically, members of this TF have been nominated by the wwPDB leadership. Additional interested persons are welcome to join and participate. The wwPDB NMR TF meets periodically (usually more than once each year) at international NMR conferences.\nData remediation. Together with other wwPDB partners, the BMRB participated in the recently completed remediation of the full PDB archive (Henrick et\u00a0al. 2008). Among the changes was adoption of uniform atom nomenclature in accordance with longstanding IUPAC recommendations (Markley et\u00a0al. 1998). BMRB\u2019s major contribution to the remediation was to parse and organize restraints for NMR structures into the NMR Restraints Grid (http:\/\/tang.bmrb.wisc.edu:8080\/WebModule\/MRGridServlet) (Doreleijers et\u00a0al. 2005).\nRequirement for structure factors and constraints. Over the past 2\u00a0years, the wwPDB organization, in consultation with other members of the crystallographic and NMR communities, reviewed policies concerning data deposition. These deliberations led to the decision, supported by the wwPDBAC and wwPDB NMR TF at their most recent meetings, that all PDB depositions must be accompanied by structure factors (for X-ray structures) and restraints (for NMR structures). These requirements, listed below, were formally announced on November 30, 2007, and are scheduled to go into effect on February 1, 2008.The wwPDB shall require deposition of structure factor amplitudes\/intensities (for crystal structure depositions) and\/or NMR restraints (for NMR structure depositions) in addition to atomic coordinates as a prerequisite for receiving a PDB ID.The wwPDB leadership shall inform the relevant journals of this new policy, and will suggest that Instructions to the Authors read as follows:\u201cFor papers describing structures of biological macromolecules, atomic coordinates and the associated experimental data (structure factor amplitudes\/intensities and\/or NMR restraints) must be deposited at a member site of the Worldwide Protein Data Bank (www.wwpdb.org): RCSB PDB (www.pdb.org), MSD-EBI (www.ebi.ac.uk\/msd), PDBj (www.pdbj.org), or BMRB (www.bmrb.wisc.edu). The PDB ID should be included in the manuscript. Authors must agree to release the atomic coordinates and deposited experimental data when the associated article is published. Questions relating to depositions should be sent to info@wwpdb.org.\u201d\nChemical shift depositions remain optional. These bodies also considered a proposal by members of the community that NMR structure depositions be accompanied by a BMRB deposition of assigned chemical shifts. Although it was decided that this requirement should not be made mandatory at present, the wwPDB NMR TF strongly recommend that these valuable data be deposited.\nSmall molecule NMR structures. At its 2006 meeting, the wwPDBAC discussed the question of small molecule NMR structures that do not comply with PDB guidelines. X-ray structures of such compounds are commonly deposited in the Cambridge Structural Database (CSD, http:\/\/www.ccdc.cam.ac.uk\/products\/csd\/), but no such archive is available for NMR structures. Subsequently, the wwPDBAC approached BMRB with a request that it consider supporting this effort. BMRB developed the following proposal, which was brought before its External Advisory Board in May 2007.\nBMRB will consider accepting coordinate sets representing three-dimensional structural models provided that the following criteria are met:The molecule falls outside the guidelines of the Protein Data Bank (i.e., the molecule is a peptide with 23 or fewer residues, a polynucleotide with 3 or fewer residues, a polysaccharide with 3 or fewer sugar residues, or a natural product)The molecule is of biological interestThe structural model(s) are based on experimental NMR dataThe coordinates are accompanied by:\nA representation of the covalent structure of the molecule (atom connectivity)Assigned NMR chemical shifts for the moleculeStructural restraints used in generating the structural model\nFor depositions meeting these criteria, BMRB encourages that authors submit their primary (time-domain) data, tables of NOEs, and other relevant information\nThis policy was announced at the most recent meetings of the wwPDBAC and wwPDB NMR TF.\nSmall molecule NMR structures meeting the above criteria can be deposited at the SMSDep website developed by BMRB and PDBj-BMRB (http:\/\/smsdep.bmrb.wisc.edu\/bmrb-adit\/). With small molecule structure depositions, the BMRB accession code serves to identify the structure as well as the supporting NMR data (questions about small molecule NMR structure depositions should be addressed to bmrbhelp@bmrb.wisc.edu).\nDepositor defined regions of local disorder in structures. At the request of the wwPDB NMR TF, BMRB created tags, enumerations, and formal PDBx dictionary (Westbrook et\u00a0al. 2005) entries for the following purposes: To separately specify the single structural model chosen by the authors to best represent the solution structure and to describe how it was derived.To describe uncertainty in the structural model (on a per-residue basis) and to denote residues found experimentally to be largely unstructured.To describe uncertainty in the structural model (on a per-atom basis) both qualitatively and quantitatively depending on the available data.\nA detailed draft was refined by RCSB PDB and circulated to the wwPDB NMR TF. These specifications make it possible for an author to clearly define those regions that are experimentally determined to be structured and those that are not. This information will be available in the mmCIF version of the PDB entry and can be used by software developers in displaying structures.\nMetabolomics data at BMRB. In collaboration with the Madison Metabolomics Consortium and the Human Metabolome Database (HMDB, http:\/\/www.hmdb.ca\/), BMRB has developed an archive of NMR data for metabolites and other small molecules found in biological systems (Ulrich et\u00a0al. 2008). A metabolomics deposition tool under construction at BMRB will enable others to submit data to add to this collection. Compounds are cross-referenced to PDB ligands, and the spectral information should be of interest to scientists studying macromolecule:ligand complexes. The site is mirrored by PDBj and CERM in Florence, Italy.","keyphrases":["structural restraints","nmr structure","metabolomics","archived nmr data","unstructured regions"],"prmu":["P","P","P","R","R"]}
{"id":"Ann_Surg_Oncol-3-1-2039827","title":"Preoperative Chemoradiotherapy with Capecitabine and Oxaliplatin in Locally Advanced Rectal Cancer. A Phase I\u2013II Multicenter Study of the Dutch Colorectal Cancer Group\n","text":"Background We studied the maximum tolerated dose (MTD) and efficacy of oxaliplatin added to capecitabine and radiotherapy (Capox-RT) as neoadjuvant therapy for rectal cancer.\nColorectal cancer is a major public health problem in the Western world and ranks as the third leading cause of death in both males and females. In 2000, more than 9000 new colorectal cancer patients were registered in the Netherlands, of whom 25% had rectal cancer.1 Surgical resection is the only curative treatment. However, following potentially curative resection, local recurrence rate varies between 5 and 40%.2\u20134 Total mesorectal excision (TME) is now the standard technique for primary resectable rectal cancer and has significantly improved local control.4\u20136 The cornerstone of the TME technique is the complete removal of the rectum and mesorectum to realize free circumferential resection margins. Adam showed in 1994 that the incidence of local recurrence 5 years after resection will rise from 10\u201378% in case of circumferential margin (CRM) involvement.7 So downstaging before the TME procedure may decrease the incidence of CRM involvement and local recurrence. In the Dutch TME trial, no tumor downstaging was detected in the week after 5 \u00d7 5 Gy.8 A recent Polish trial demonstrated that a radiotherapy schedule of 50.4 Gy combined with chemotherapy (5-FU\/Leucovorin) followed after 4\u20136 weeks by surgery resulted in a significant higher percentage of downstaging compared with short-term preoperative radiotherapy of 5 \u00d7 5 Gy followed by surgery within 7 days.9 So downstaging is dependent on both the total radiotherapy dose and the interval between the end of the radiotherapy and the surgery. In general, to achieve downstaging this interval needs to be at least 6 weeks and the dose needs to be at least 45 Gy.9 Although the obtained downstaging after neoadjuvant radiochemotherapy did result in a favorable prognosis,10 it was not clear until recently whether the addition of chemotherapy to preoperative radiotherapy could increase the amount of downstaging and thus improve local control. The evidence that the addition of chemotherapy to preoperative radiotherapy improves local control rates has recently been shown by two separate trials. The EORTC 22921 trial has a two by two factorial design and randomized between preoperative radiotherapy (45 Gy) versus preoperative chemoradiotherapy (45 Gy combined with 5-FU\/Leucovorin). The results demonstrated an increased local control rate for the chemoradiation arm: 91% versus 83%.11,12 A similar result was found in the French FFCD 9203 study, which randomized between preoperative radiotherapy (45 Gy) and preoperative chemoradiotherapy (45 Gy and 5-FU\/Leucovorin) and which showed local recurrence rates of 16.5% and 8%, respectively.13 Based on these studies, prolonged preoperative chemoradiation is considered the standard treatment for resectable locally advanced rectal cancers. The EORTC study and the French study employed 5FU bolus injection modulated with leucovorin. However 5FU administered by continuous infusion or orally (capecitabine\/ UFT) may be more effective and less toxic than 5FU administered by bolus injection.14\u201316 In parallel, the results of palliative chemotherapy in advanced colorectal cancer have been substantially improved by the combination of 5FU with oxaliplatin or irinotecan (reviewed by Punt17). Both in vivo and in vitro, oxaliplatin has been shown to have at least an additive interaction with radiotherapy in the management of digestive tract tumors.18\u201320 Incorporation of those drugs in combined treatment strategies could substantially improve the results obtained with bolus 5FU alone in downstaging and R0 resection rates in patients with locally advanced rectal cancer.\nWe investigated the combination of capecitabine and oxaliplatin given concomitantly with radiotherapy in patients with locally advanced rectal cancer. The optimal dose of oxaliplatin was first established in a phase I study, and in the following phase II study the efficacy of this regimen was tested.\nPATIENTS AND METHODS\nObjectives\nThe objective of the phase I study was to determine the maximum tolerated dose (MTD) of oxaliplatin in combination with a fixed dose of capecitabine and radiotherapy, and the objective of the phase II study was to determine the R0 resection rate and pathological complete response rate (pCR).\nEligibility Criteria\nEligibility criteria included histologically documented adenocarcinoma of the rectum within 15 cm from the anal sphincter, locally advanced stage T3 (distance to the endopelvic fascia < 2 mm) or T4 tumors based on computed tomography (CT) or magnetic resonance imaging (MRI) findings, performance status (Eastern Cooperative Oncology Group) 0\u20132, age >18 years old, adequate hematological, liver function, and other laboratory parameters (white blood cells 3.0 \u00d7 109\/L, platelets >100 \u00d7 109\/L, creatinin clearance > 50 mL\/min, bilirubin < 1.5 times the upper limit of the normal range (ULN), and written informed consent. Patients with reproductive potential should use adequate contraceptive measures. Patients were excluded in case of prior chemotherapy and\/or pelvic radiotherapy, acute bowel obstruction without colostomy, uncontrolled ischemic heart disease, peripheral neuropathy, or any uncontrolled serious systemic disease. The protocol was approved by the local ethics committees of all four participating hospitals. The study was performed within the framework of the Dutch Colorectal Cancer Group (DCCG).\nTREATMENT\nRadiotherapy\nAll patients received radiation delivered by an isocentric three- or four-field technique, using a linear accelerator of at least 10 MV. The patients were treated in either supine or prone position with a full bladder. The radiation field extended superiorly to the L5\/S1 junction and covered inferiorly the obturator foramina. The minimal inferior border extended 4\u20135 cm below the tumor. In case the tumor was located in the lower third of the rectum, the perineum was encompassed in the treatment field. The width of the AP-PA portals had to cover the lateral pelvic inlet with a margin of 1.5 cm. The entire sacrum was included with a dorsal margin of 1.5 cm. Anteriorly, the lateral fields had to encompass the tumor as determined by barium enema (optional) and pelvic CT scan. If there was clinical evidence of involvement of the bladder, the prostate, the cervix or the uterine body, not only the internal iliac nodes but also the external iliac nodes were included in the radiation field. Computerized dosimetry was routinely performed. Irradiation was delivered 5 days per week at a dose of 1.8 Gy\/day to a total dose of 45 Gy with a boost to the tumor in 3 fractions of 1.8 Gy to a total of 50.4 Gy.\nChemotherapy\nCapecitabine was administered orally twice daily at 1000 mg\/ m2 on days 1\u201314 and 25\u201338.\nOxaliplatin was given twice by intravenous infusion over 2 h, on the first day of radiotherapy and on day 29. Oxaliplatin doses were planned at 85, 100, and 135 mg\/m2. Each dose level was to be administered to at least three patients. If a dose-limiting toxicity (DLT) was observed in \u22651 patient, a total of 6 patients had to be treated at that dose level. The maximum-tolerated dose (MTD) was defined as the level at which \u22642 out of 6 patients experienced DLTs without compromizing radiotherapy and surgery and was the recommended dose in the phase II study.\nSurgery\nSurgical resection was performed 6\u20138 weeks after completion of the chemoradiation therapy. The resection was performed according to the principles of total mesorectal excision as described by Heald2 and Enker.21 These principles include sharp dissection under direct vision following the lipoma-like surface of the mesorectum. Proximal transection of the inferior mesenteric artery was in general performed distal to the collateral between the left colic artery and branches of the sigmoid artery. Anterior dissection in male patients was carried out in front of Denonvilliers\u2019 fascia. If tumor extended into the prostate a total exenteration was performed. In female patients, resection of the posterior vaginal wall was performed if necessary. A distal mesorectal margin of 2 cm was considered adequate if bowel continuity was to be restored.\nPathology\nPathology evaluation was standardized according to national guideliness. The CRM was assessed according to the method of Quirke et al.,7 and a margin of <1 mm from the primary tumor to the endopelvic fascia is considered positive.\nStatistics\nTwenty patients were to be included at the MTD to detect with a power of 80% a R0 resectability rate of >80% (with a 95% CI of 56\u201394%).\nMonitoring and Management During Treatment\nPhysical examination and evaluation of toxicity was performed weekly. Complete blood count, electrolytes, creatinine, and total protein were determined weekly. Standard antiemetic prophylaxis with 5HT3 antagonist prior to oxaliplatin infusion was used. Adverse events were classified according to the National Cancer Institute (NCI) Common Toxicity Criteria (CTC) Version 2.0. Global quality of life (QOL) assessment was evaluated weekly during the chemoradiotherapy using a visual analogue scale (VASQOL) in which in a single 10 cm line anchored on the left with \u201cworst\u201d and on the right with \u201cbest\u201d for QOL.22 Dose-limiting toxicities were defined as white blood cell (WBC) and platelets < 3.0 and < 100 \u00d7 109\/L, respectively, before start of the next treatment cycle with oxaliplatin after a maximum delay of one week; diarrhea grade > 2; oral mucositis grade >1; skin toxicity grade > 2 before start of next treatment cycle with capecitabine, despite a delay of dosing for 1 week; neurosensory toxicity grade > 2 despite a delay in dosing for 1 week; occurrence of any grade 4 toxicity; and any event that would compromise the administration of radiotherapy.\nRESULTS\nPatients Characteristics\nBetween June 2003 and December 2004, 22 patients with T3\/T4 rectal cancer from four different centers were enrolled in the study. Patient characteristics are shown in Table\u00a01. All patients were evaluable for toxicity during chemoradiation. One patient was lost to follow-up after chemoradiation (surgery was performed abroad). Therefore, 21 patients were evaluable for clinical outcome after surgery. Twenty patients (91%) received the planned dose of chemotherapy. All patients received the planned dose of radiotherapy. The median follow-up was 14 months.\nTABLE\u00a01.Patient characteristics (n = 22)CharacteristicsNo.Median age (years)58Range (years)45\u201370Men\/Women12\/10Performance status\u00a0\u00a003\u00a0\u00a0119Distance of tumor to anal verge (cm)\u00a0\u00a00\u2013511\u00a0\u00a05\u2013107\u00a0\u00a010\u2013153\u00a0\u00a0Unknown1T status\u00a0\u00a0T3a17\u00a0\u00a0T4b5a T3 beyond the fascia recti.b T4: into pelvic organs (1 ureter, 2 vagina, 2 prostate)\nToxicity\nAt the first oxaliplatin dose level of 85 mg\/m2, one patient experienced DLT (hospitalization for grade 3 nausea and grade 2 diarrhea). This dose level was then expanded to six patients, and DLT was observed in another patient (hospitalization for grade 3 diarrhea). Therefore, a dose of 85 mg\/m2 oxaliplatin was considered the MTD. In the additional 16 patients, two more patients experienced grade 3 diarrhea. Hand-foot syndrome, myelosuppression, and neurotoxicity were observed only in a minority of patients and were of mild severity. The grade 3\/4 toxicity data are shown in Table\u00a02.\nTABLE\u00a02.Toxicity in the study population (n = 22)StudyPhase IPhase IIDose oxaliplatin (number of patients)85 mg\/m2 (3)85 mg\/m2 (3)(22)Toxicity grade (NCI CTCV2.0)Toxicity grade (NCI CTCV2.0)Toxicity grade (NCI CTCV2.0)343434Leukopenia\u2013\u2013\u2013\u2013\u2013\u2013Neutropenia\u2013\u2013\u2013\u2013\u2013\u2013Thrombopenia\u2013\u2013\u2013\u2013\u2013\u2013Anemia\u2013\u2013\u2013\u2013\u2013\u2013Diarrhea\u2013\u20131\u20134\u2013Abdominal pain\u2013\u2013\u2013\u2013\u2013\u2013Nausea1\u2013\u2013\u2013\u2013\u2013Mucositis\u2013\u2013\u2013\u2013\u2013\u2013Neurotoxicity\u2013\u2013\u2013\u2013\u2013\u2013\nSurgery\nTME surgery was performed after a median of 47 days (range 4\u20138 weeks range in days) following the completion of chemoradiation. Mean hospitalization time was 14 days (range 9\u201395; 70% < 21 days). The main surgical complications were: major bleeding (1), rectal perforation (1), ureter lesion (1). Postsurgery treatment related complications were seen in 12 patients with mild wound infections (6), wound dehiscences (2), (sub)ileus (4), rhabdomyolysis (1), and in one patient a life-threatening multiorgan failure after a perforation in an abcess located near the anus praeter. Rhabdomyolysis is an unusual complication. In this patient it was probably caused by the operation procedure and not by this specific procedure as there were no other postoperative complications. Three patients (15%) needed reoperation for perforation (1) and pelvic abscesses (2). The 60-day mortality rate was nil.\nEfficacy\nTwenty-one patients with a T3\u2013T4 tumor based on CT or MRI underwent surgery with 10 abdominoperineal resections (APRs) and 10 low anterior resections (LARs); in one patient the tumor was not resectable. A R0 resection was achieved in 17 patients (81%, 95% CI 58\u201395%). A pCR was observed in 2 patients (10%, 95% CI 1\u201330%), and in one patient (5%) the surgical specimen only showed minimal microscopic disease. The postradiotherapeutic pathologic staging yielded in seven patients (33%; 95% CI 15\u201357%) a downstaging to pT0\u2013T2. After a median follow-up of 14 months, four patients have died due to metastatic disease and one patient has experienced a local recurrence.\nQuality of Life\nThe VASQOL score was measured weekly during chemoradiotherapy. The QOL decreased after 4 weeks treatment from 80% in week 4 to 60% in week 6 (p < .002, student-t test).\nDISCUSSION\nBased on the EORTC 22921 and FFCD 9203 phase III studies,11,13,23 preoperative chemoradiation with 5FU and 45 Gy or higher is now considered to be the standard of care for locally advanced rectal cancer. In these studies, 5FU was given as a daily bolus infusion in week 1 and 5. To improve on these results, we tested the feasibility and efficacy of the addition of oxaliplatin to capecitabine in this combined treatment strategy. Our recommended dose for the phase II study was oxaliplatin 85 mg\/m2 on day 1 and 29 plus capecitabine 1000 mg\/m2 twice daily during 14 days starting on day 1 and 25 in combination with radiotherapy at a total dose of 50.4 Gy. In our phase I\/II study, we found grade III diarrhea in 19%, a R0 resection in 81%, downstaging to T0\u20132 in 33%, and a pCR in 10% of patients. Table\u00a03 summarizes the studies with the addition of oxaliplatin to a fluoropyrimidine (IV or orally) and radiotherapy. These studies show a toxicity and efficacy profile comparable to the results from our study.\nTABLE\u00a03.Locally advanced rectal cancer, recent neoadjuvant studies with fluoropyrimidines and oxaliplatin in combination with radiotherapyStudyDesign (phase)StageNEBRT (Gy)CTR0 (%)pCR (%)Toxicity grade 3\u20134 (%)TotalDiarrheaMyelotoxicPreoperative radiotherapy vs. chemoradiotherapy (with 5 FU)EORTC 2292111,23IIIT3\/T4 101145\u2013\u2013538a17\u20131\u2013455FU pre\u20131454\u201334\u20139\u2013FFCD 920313IIIT3\/T476245\u20139043\u2013\u2013455FU pre911215\u2013\u2013Bujko9IIIT3\/T43165 \u00d7 5\u20138713\u2013\u201350.45FU pre961718\u2013\u2013Preoperative CRT with capecitabineKim30IIT3\/T4\/N+4550.4 Cap 3\u201331174\u2013Dunst31\/Glynne- Jones32IIT3\/T4\/N+9850.4 + 5.4Cap 1\u2013422410Chau33IIT3+, low T3, N27754Cap 1b99244 deathsYerushalmi16IIT3\/T4 (T2 10%)4650.4 5FU vs Cap 2\u201317443\u2013302Preoperative CRT with 5 FU based chemotherapy and oxaliplatinGambacorta34IIT33050Tomox\u20133013310Gerard26IIT2\/T3\/T44050.4FU + Ox 1\u2013151853Rodel35IIT3\u201343250.4Capox 479191680Carraro36IIT3\/T42250.4FU + Ox 2\u201314\u20132714Aschele25I\/IIT3\/T4\/N+2550.4FU + Ox 3892824164Glynne-Jones32I\/IIUnresectable9445Capox 278281310\u2013Machiels24IIT3\/T4\/N+4045Capox 18314\u201330\u2013Hospers (2006) This studyI\/IIT3\/T42250.4Capox8110\u201318\u2013Cap 1: 825 mg\/m22\u00d7 daily, 7 daysCapox 1: Cap 825 2\u00d7 daily, Monday\u2013Friday, oxali 50 weeklyTomox: raltitrexed 3, oxali 130, day 1, 19, 38Cap 2: 825 mg\/m2 2\u00d7 daily, Monday\u2013FridayCapox 2: Cap 650 2\u00d7 daily, 7 days, oxali 130 per 2 weeksFU + Ox 1: 5FU 350 daily, Oxali 130 weekly, week 1 and 5Cap 3: 825 mg\/m2 2\u00d7 daily,14 days (2\u00d7)Capox 3: Cap 825 2\u00d7 daily, 7 days, oxali 60 per 2 weeksFU + Ox 2: 5FU 375\/4 days, Oxali 25\/4 days, week 1 and 5Capox 4: Cap 825 2\u00d7 daily, 2 \u00d7 14 days, oxali 50, day 1, 8, 22, 29FU + Ox 3: 5FU 225 daily, Oxali 60 weeklya \u2265Grade 2 toxicity.b 12 weeks neoadjuvant CAPO.\nDiarrhea generally occurred during the last 2 weeks of chemoradiation, which was accompanied by a significant decrease in QOL. As shown in Table\u00a02, the addition of oxaliplatin increases the incidence of toxicities when compared with the use of 5FU alone. The observed postsurgical morbidity (mild wound infections excluded), the reoperations and the median hospitalization time in our study were 30%, 15%, and 14 days, respectively, and are comparable with other studies.24\u201326\nWe found a similar efficacy in pCR and R0 compared to other studies (Table\u00a03). However, comparing the efficacy of these regimes is hampered by a number of technicalities. These comprise differences in 1) staging techniques, 2) the determination of distance of the tumor to the endopelvic fascia, 3) pathology techniques, and 4) the difference in interval between neoadjuvant treatment and surgery. The staging techniques used to define the locally advanced stage of the primary tumor and lymph nodes are clinical examination, CT, MRI, endoultrasound or combinations, the use of ultrasmall particles of iron oxide (USPIOs).27 These different staging techniques may lead to variabilities in pretreatment staging and therefore compromise the quantitation of downstaging. Moreover, the tumor might be still visible on posttreatment imaging, but it may have become nonvital. The distance of the tumor to the endopelvic fascia will influence the local recurrence rate. The determination of a pCR and an R0 resection depends on the sophistication of the pathology techniques used, and the definition of a R0 resection (distance tumor to resection or to CRM) is not uniform. In the various clinical protocols, the time interval between chemoradiotherapy and surgery is not standardized, which may influence the degree of downstaging but also the ultimate results of local cure. Therefore, cross-study comparison should be interpreted with caution. The end points used in this study are known to have a different impact on clinical outcome: a pCR is associated with a low local recurrence rate, and prolonged DFS and is not significantly related to the cT and cN categories.28 R0 resection is known to be a highly relevant prognostic factor.29 Comparing our results on pCR and R0 to other oxaliplatin-containing studies, we found similar results; however, these were also observed using monotherapy with 5FU or capecitabine (Table\u00a02). Therefore, it is yet unknown whether the increased toxicity of adding oxaliplatin to a fluoropyrimidine in neoadjuvant chemoradiation schedules results in a clinical benefit for patients with rectal cancer.\nIn conclusion, this multicenter study demonstrated a neoadjuvant regimen with Capox-RT with an acceptable acute toxicity profile. Randomized phase III studies with in the standard arm 5FU will be necessary to show the true benefit of this approach. In such prospective studies, the standardization of the staging technique (i.e., the distance of the tumor to the endopelvic fascia), TME surgery, radiotherapy, and pathology should minimize the influence of these factors on the outcome.","keyphrases":["chemoradiation","radiotherapy","capecitabine","oxaliplatin","rectal cancer","phase i\u2013ii study"],"prmu":["P","P","P","P","P","R"]}
{"id":"Clin_Auton_Res-3-1-1858602","title":"Reduced brain perfusion and cognitive performance due to constitutional hypotension\n","text":"This review article includes a systematic evaluation of the empirical data concerning deficits in mental ability, brain perfusion, and cerebral functioning due to chronically low blood pressure. A number of studies have provided strong evidence for reduced cognitive performance in hypotension, particularly in the domains of attention and memory. EEG studies have demonstrated that the hypotension-related poorer mental ability is also reflected in diminished cortical activity. Contrary to convention, more recent research has suggested a deficient regulation of cerebral blood flow in persons with low blood pressure. In addition to reduced tonic brain perfusion, studies demonstrated insufficient adjustment of blood flow to cognitive requirements. Altogether, these findings suggest that more attention should be allocated to chronic hypotension in both research and clinical practice.\nIntroduction\nChronically low blood pressure is accompanied by a variety of complaints including fatigue, reduced drive, faintness, dizziness, headaches, palpitations, and increased pain sensitivity [1\u20134]. In addition, hypotensive individuals report cognitive impairment, above all deficits in attention and memory. Nevertheless, it is generally the case that in research, as well as in clinical practice, relatively little importance is ascribed to hypotension. One reason for this is that, despite mental symptoms, cerebral dysfunction generally is not taken into account [1]. This is a consequence of the current doctrine that low systemic blood pressure is compensated by autoregulatory processes which prevent reduced blood perfusion of the brain [5, 6].\nSome recent findings challenge this doctrine: reduced cognitive performance in hypotension has been demonstrated by neuropsychological testing, and EEG studies have revealed diminished cortical activity. Moreover, the assumption of unimpaired brain perfusion in hypotension no longer holds. In the present review the necessity of a reappraisal concerning hypotension is discussed in light of the relationship between blood pressure and cerebral functioning.\nConstitutional hypotension\nHypotension has been defined by the World Health Organization (WHO) [4] as a low blood pressure with a systolic reading below 110\u00a0mmHg in males and below 100\u00a0mmHg in females, regardless of diastolic blood pressure. The concept of constitutional hypotension refers to a chronic condition of inappropriately reduced blood pressure independent of the presence of further pathological factors. Both secondary hypotension (e.g. due to blood loss or medication) and orthostatic hypotension (caused by circulatory problems when assuming an upright position) are distinguished from the constitutional form [1, 6].\nConstitutional hypotension is relatively widespread in the general population. In a representative German sample of more than 7,000 subjects approximately 3% fulfilled the criteria of the WHO [c.f. 1]. In contrast to elevated blood pressure, which constitutes a significant risk factor for cardiovascular disease, hypotension is commonly not regarded as a severe condition. Nevertheless, its impact on personal well-being and quality of life has been shown in several epidemiological studies. For instance, Wessely et al. [7], as well as Pilgrim et al. [3] demonstrated impaired physical and mental health in the case of low blood pressure. Reduced health-related quality of life [8] and a heightened degree of depressiveness [9] were also reported by hypotensive individuals.\nChronic hypotension has been identified as a major risk factor in pregnancy [10]. Moreover, several studies focusing on the elderly population have reported associations between low blood pressure and the prevalence and incidence of Alzheimer\u2019s and vascular dementia [c.f. 11\u201314]. Whilst a few longitudinal studies examining this relationship have been published [15, 16], the causal role of low blood pressure in degenerative brain disorders has thus far not been proven [e.g. 13].1\nConcerning the etiology of hypotension, reduced liquid intake and low body weight may be considered [c.f. 1, 17]. A dysregulation of the autonomous nervous system may additionally be of significance. Various findings on reduced electrodermal activity [18] and increased heart rate variability [19] reflect diminished sympathetic tone and habitually heightened parasympathetic activity in persons with low blood pressure values. This is corroborated by findings on the effects of sympathomimetic drugs [20, 21]. In mixed samples of persons with constitutional and orthostatic hypotension, \u03b1 and \u03b2 adrenergic substances were shown to lead to blood pressure elevation accompanied by a reduction of subjective symptoms. A malfunction of the arterial baroreceptor system was postulated as a further etiological factor [22]. Responding to mechanical stretch of the vessel walls, the baroreceptors form part of a negative feedback loop (\u201cbaroreflex\u201d) compensating phasic blood pressure fluctuations [23]. Increased sensitivity of the baroreceptor system may result in stabilizing blood pressure at a lower level. In addition to findings in animals [24], this hypothesis is supported by a still unpublished study of our own group which yielded increased baroreflex sensitivity in the case of low blood pressure.\nCognitive deficits in constitutional hypotension\nTable\u00a01 presents the most relevant studies devoted to cognitive functioning in hypotension. In addition to the main results, the table includes information concerning the respective samples and assessment instruments.\nTable\u00a01Studies on cognitive performance in hypotension (SBP, systolic blood pressure; DBP, diastolic blood pressure)AuthorsMain focusSamplesAssessment instrumentsMain resultsRichter-Heinrich  et al. [18]Attentional performance in  hypotension and hypertension30 hypotensives (SBP\u00a0<\u00a0106\u00a0mmHg);  40 hypertensives (SBP\u00a0>\u00a0140\u00a0mmHg);  31 normotensive controls; age 16\u201340\u00a0yearsKonzentrations-Verlaufs-Test (course  of concentration test) [25]Reduced attentional performance in the  case of both lowered and elevated  blood pressureStegagno et al. [26]Attentional, memory and  arithmetic performance  in hypotension17 female hypotensives (SBP\u00a0<\u00a0100\u00a0mmHg);  19 normotensive controls; mean  age 23\u00a0yearsReaction times to acoustic stimuli;  digit span [27]; recall of word  lists [28]; serial subtractionsProlonged reaction times, poorer  performance on the verbal memory  and arithmetic tasks in hypotension,  no effect for digit spanCosta et al. [29]Attentional and memory  performance in hypotension26 female hypotensives (SBP\u00a0<\u00a0105,  DBP\u00a0<\u00a065\u00a0mmHg), 22 normotensive  controls; mean age 29\u00a0yearsAufmerksamkeits-Belastungs-Test  (attentional strain test) [30];  Zahlen-Verbindungs-Test (trail making  test) [31]; recall of word lists [28]Poorer attentional and memory  performance on each of the three  tests in hypotensionMorris et al. [32]Relationship between blood  pressure and cognitive  performance in elderly  personsRepresentative population sample covering  the total spectrum of blood pressure  (n\u00a0=\u00a05,816; age over 64\u00a0years)East Boston Memory Test [33]; Symbol  Digit Modalities Test [34]; Mini-Mental  State Examination [35]Weak U-shaped relationship between  blood pressure and cognitive  performanceWeisz et al. [22]Attentional performance in  hypotension25 female hypotensives (mean  SBP\u00a0=\u00a0102\u00a0mmHg); 25 normotensive  controls; age 19\u201344\u00a0yearsAttentional and Cognitive  Efficiency Battery [36]Reduced performance on a subtest  assessing cognitive flexibility  in hypotensionDuschek et al. [37]Attentional performance in  moderate hypotension26 borderline hypotensives (mean  SBP\u00a0=\u00a0112\u00a0mmHg); 29 normotensive  controls; mean age 26\u00a0yearsReaction times to acoustic stimuli;  Aufmerksamkeits-Belastungs-Test (attentional  strain test) [30]; Zahlen-Verbindungs-Test  (trail making test) [31]Prolonged reaction times and poorer  performance on the  \u201cAufmerksamkeits-Belastungs-Test\u201d in  moderate hypotension, no effects  for the \u201cZahlen-Verbindungs-Test\u201dDuschek et al. [38]Attentional and working  memory performance in  hypotension with motor  performance and  mood controlled40 hypotensives (SBP\u00a0<\u00a0105\u00a0mmHg in  women, SBP\u00a0<\u00a0110\u00a0mmHg in men);  40 normotensive controls; age  19\u201345\u00a0yearsTestbatterie zur Aufmerksamkeitspr\u00fcfung  (battery for the assessment of attention)  [39]; Motorische Leistungsserie (motor  performance series) [40]; Befindlichkeitsskala  (mood scale) [41]Poorer performance on six tests  assessing tonic and phasic alertness,  selective, divided and sustained  attention as well as working memory  in hypotensives with fine motor  performance and mood controlled\nA pioneering study investigating the relationship between blood pressure and cognitive abilities was conducted by Richter-Heinrich et al. [18]. They showed reduced performance of individuals with both lowered and elevated blood pressure on a test of concentration. A more comprehensive assessment was carried out by Stegagno et al. [26], in which poorer performances of hypotensive subjects on a verbal memory test and on an arithmetic task, as well as prolonged reaction times to acoustic stimuli were observed. Costa et al. [29] found reduced scores on two standard German paper\u2013pencil tests assessing selective attention and cognitive speed. Moreover, as in the Stegagno et al. [26] study, impaired verbal memory performance was documented.\nWeisz et al. [22] reported that female hypotensive subjects performed significantly worse than controls on a computer-based test measuring attentional flexibility [36]. Subjects with only a moderately decreased blood pressure were assessed by Duschek et al. [37]. In this sample, reduced attentional performance and prolonged reaction times were again found as compared to normotensive controls.\nDuschek et al. [38] investigated the relationship between low blood pressure and attentional abilities through the application of a multidimensional diagnostic approach. They presented their subjects with a battery of six computer-based tasks [39] focusing on tonic and phasic alertness, selective, divided and sustained attention, as well as working memory. Additionally, in order to control for possible confounders, a test battery examining fine motor abilities [40] and a mood questionnaire [41] were presented. Reduced performance of hypotensives was evident in each of the six cognitive tests. The significant differences between hypotensive and control subjects persisted even when the effects of motor performance and mood were controlled.\nAltogether, the existing data provide strong evidence for cognitive deficits related to constitutional hypotension, especially in the domains of attention and memory. The reduced performance seems to be a direct consequence of low blood pressure rather than an effect of impaired well-being related to this state. Up to now there have been no empirical data concerning specific effects of hypotension-related deficits on everyday life. Nonetheless, these findings raise concern regarding the impact of the deficits on attention-demanding activities (e.g. professional or academic), including public health-related functions (e.g. traffic safety) [42].\nIt would appear that both extremes of the blood pressure spectrum are accompanied by a decrease in cognitive ability. A number of studies concerned with elevated blood pressure showed reduced performance on various cognitive tests [43, 44]. This is in line with epidemiological studies reporting an inverted U-shaped relationship between blood pressure and cognitive performance in elderly persons [c.f. 11, 13, 32, 45, 46]. Therefore, it may further be hypothesized that there is a relatively small range of normal blood pressure in which the brain exerts its optimal function.\nNeuroelectrophysiological correlates of hypotension-related cognitive deficits\nCognitive deficits in low blood pressure states are also reflected in decreased cortical activity. In accordance with earlier results [22, 29], Duschek et al. [47] found the amplitude of the contingent negative variation (CNV) to be reduced in a hypotensive sample defined according to the WHO criteria [4] (c.f. Fig.\u00a01).\nFig.\u00a01The CNV occurs during the period between a warning signal (S1) and a second stimulus (S2) demanding a motor, verbal or cognitive response. As can be seen in the figure, its amplitude was found to be reduced in hypotensive subjects (N\u00a0=\u00a040) as compared to normotensive controls (N\u00a0=\u00a040); modified from [47]\nThe CNV is an evoked potential which is generally viewed as a neuroelectrophysiological correlative of attentional processing [48]. This is supported by pharmacological studies showing that the effects of various stimulant and sedative drugs on attention involve an increase or decrease, respectively, of the CNV amplitude [49, 50]. Moreover, attentional deficits due to brain lesions and psychiatric diseases are associated with a reduced CNV [51, 52]. In the Duschek et al. [47] study, the amplitude of the CNV proved to be negatively correlated with reaction time, which in turn was prolonged in the hypotensive group. This highlights that the CNV constitutes a brain electrical correlate of an aspect of cognitive functioning which is affected in hypotension.\nThe relationship between blood pressure and cortical activity is also reflected in a negative correlation between blood pressure and \u03b1 wave activity in the spontaneous EEG [47]. This suggests that low blood pressure is associated with a reduced tonic cortical arousal [53]. On the behavioral level, higher degrees of \u03b1 activity are known to be associated with generally diminished vigilance and preparedness to react [54].\nConsidering the physiological processes mediating the relationship between blood pressure and brain function, it must be taken into account that blood pressure can influence cortical activation processes via afferent projections [47, 55\u201357]. The brain continuously receives information about the state of the cardiovascular system by means of viscero-afferent fibers [23]. These afferent signals enter the brain via brainstem nuclei. From there, ascending pathways continue via hypothalamic and thalamic regions to cortical areas such as the anterior cingulate, the insula and the prefrontal lobe [58]. The latter areas, in particular the prefrontal cortex and the anterior cingulate are of crucial importance in the regulation of cortical arousal, as well as for attentional processes [59, 60]. Thus, these brain structures may represent functional interfaces between cardiovascular activity and attention [42].\nAlso neurochemical mediators should be regarded in the relationship between blood pressure and cortical activity. Noradrenaline is involved in the control of cortical arousal and attentional processes, as well as in the genesis of the CNV [59, 61]. Catecholamines are also of great influence in the regulation of blood pressure [62]. Bearing this in mind, one could speculate on the neurochemical level about a specific role of noradrenaline linking cardiovascular and cortical activation.\nCerebral blood perfusion in constitutional hypotension\nIt is generally assumed that in healthy individuals processes of autoregulation keep the cerebral blood flow constant within a wide range of arterial pressure. In order to ensure stable perfusion, cerebral resistance vessels constrict during increases and dilate during reductions in systemic blood pressure [5]. Autoregulation is considered to be a protective mechanism which prevents brain ischemia during blood pressure decrease, and guards against capillary damage and edema formation during periods of elevated blood pressure.\nUnder normal conditions the limits within which cerebral blood flow is assumed to be constant are approximately between a mean arterial pressure (MAP) of 60 and 150\u00a0mmHg [5, 63, 64]. If blood pressure is outside of this range, cerebral blood flow rises or falls with respective increases or decreases in blood pressure. Slight reductions of blood pressure below the lower limit of autoregulation can be compensated by an increase of the extraction coefficient of oxygen from the blood. Further reductions are accompanied by symptoms such as pallor and dizziness and ultimately lead to irreversible brain damage [5].\nMAP in individuals with constitutional hypotension usually does not decrease beyond the assumed lower MAP limit of autoregulation (60\u00a0mmHg). In light of this, low blood pressure in these subjects should be compensated, and cerebral blood flow should not be affected. This assumption was challenged by Duschek and Schandry [65]. They recorded blood flow velocities by means of transcranial Doppler sonography in both middle cerebral arteries (MCA) in hypotensive individuals, defined according to the WHO [4], and in normotensive control subjects. Contrary to the current doctrine, MCA blood flow at rest was found to be substantially bilaterally reduced in hypotensives (c.f. Fig.\u00a02).\nFig.\u00a02Mean flow velocities in the left and right middle cerebral arteries under resting conditions in hypotensive (N\u00a0=\u00a040) and control subjects (N\u00a0=\u00a040); bars represent standard errors [65]\nThe perfusion territory of the MCA includes subcortical areas, large fractions of the frontal and parietal lobes, as well as the temporal lobes [66]. Thus, the latter finding demonstrated that, despite autoregulation, blood pressure in subjects with constitutional hypotension is not sufficient in maintaining the perfusion of a large part of the brain at the level of normotensive individuals [see also 67].\nIn explaining this unexpected result, it must be noted that the determination of the lower limit of autoregulation was exclusively based on the experimental manipulation of blood pressure employing pharmacological methods, as well as head up-tilt [68\u201371]. However, such a transient reduction of blood pressure is most certainly not comparable to conditions of chronic hypotension. Additionally, in all of these studies global cerebral blood flow was assessed by means of the oxygen difference method according to Lennox and Gibbs [72], which may be less sensitive to perfusion changes than Doppler sonography.\nNevertheless, more recent findings suggest values for the lower MAP limit of autoregulation to be considerably higher than 60\u00a0mmHg [73\u201375]. In a critical review of the literature, Drummond [76] postulated an average lower limit of no less than 70\u00a0mmHg. Moreover, the limit seems to vary strongly across individuals. Waldemar et al. [77], for instance, reported an inter-individual range between 53 and 103\u00a0mmHg. In accordance with Duschek and Schandry\u2019s [65] data, the doctrine of stable cerebral perfusion down to the limit of a MAP of 60\u00a0mmHg can no longer be supported by the current state of research.\nA further important aspect of cerebral hemodynamics concerns the continuous adjustment of brain perfusion to current requirements. Due to the close coupling of neural activity and brain metabolism, cerebral activation processes are accompanied by changes in cerebral blood flow [78]. Neurovascular coupling is based on the contraction and dilation of small resistance vessels, resulting from the changing metabolic demands of neuron populations in the vicinity [64].\nWith regards to hypotension-related cognitive deficits, the extent of blood flow adjustment to mental activity was also tested in the Duschek and Schandry [65] study. Subjects were presented with a simple attentional task (motor reactions to visual stimuli cued by acoustic signals). During task execution blood flow velocities in the MCA of both hemispheres were recorded by means of functional transcranial Doppler sonography (for technical details see [66]).\nAs expected, mental activity was accompanied by a substantial increase of MCA perfusion. In control subjects, however, this increase was approximately 70% stronger than in hypotensives (c.f. Fig.\u00a03). It would seem that blood pressure in the latter group was not sufficient to enable adjustment of brain perfusion to cognitive demands, as seen in normotensives. Moreover, hypotensive participants showed prolonged reaction times, and a positive correlation was found between reaction speed and the extent of blood flow increase. This finding corroborates the significance of hemodynamic adjustment for optimal cognitive functioning.\nFig.\u00a03Changes of blood flow velocities in the left and right MCA during the execution of an attentional task in hypotensives (N\u00a0=\u00a040) and controls (N\u00a0=\u00a040). The subjects had to press a key in response to a visual imperative stimulus which was announced by a cuing tone. In both MCA the rise in flow velocity which occurred during the anticipation of the imperative stimulus was substantially less pronounced in hypotensives. A second flow velocity maximum visible after the motor reaction was also slightly reduced in hypotensives [65]\nFinal comments\nThere is strong evidence suggesting that chronically low blood pressure is accompanied by diminished cognitive performance, primarily involving attention and memory [26, 29, 38]. Recent research has provided an insight into the psychophysiological mechanisms of action underlying these deficits: EEG studies have demonstrated that the weaker cognitive performance is associated with reduced cortical activity [22, 29, 47]. Furthermore, deficient regulation of cerebral blood flow must be assumed in hypotension. In addition to diminished tonic brain perfusion, reduced adjustment of brain perfusion to cognitive demands was documented [65]. It stands to reason that, as a consequence of this situation, a diminished metabolic supply of the brain tissue accounts for the cognitive deficits.\nThe cognitive deficits can widely affect the every day life of hypotensive persons, and more attention should therefore be allocated to this topic within basic and clinical research, as well as in clinical practice [1, 17]. This finally leads to the issue of the treatment of hypotension. Empirical knowledge concerning the effectiveness of antihypotensive therapy continues to be scant. A variety of treatment strategies such as physical training, increase of liquid intake and pharmacological measures have been suggested [1]. In two clinical trials the administration of sympathomimetics was found to result in a reduction of subjective hypotensive symptoms [20, 21]. Recent data of our own group suggest that cognitive performance may also be enhanced by pharmacological blood pressure elevation [79]. Based on a placebo controlled design, the vasopressor agent midodrine was shown to raise cerebral blood perfusion as well as performance on a test assessing selective attention [30] in hypotensive subjects, defined according to the WHO [4]. The results of these pioneering studies are promising. Further research would appear worthwhile and should aim to establish precise guidelines which enable chronic hypotension to be efficiently and effectively dealt with in practice.","keyphrases":["cognitive","hypotension","blood pressure","cerebral blood flow","autonomic nervous system"],"prmu":["P","P","P","P","P"]}
{"id":"Apoptosis-4-1-2311383","title":"Protein kinase A-mediated CREB phosphorylation is an oxidant-induced survival pathway in alveolar type II cells\n","text":"Oxidant stress plays a role in the pathogenesis of pulmonary diseases, including fibrotic lung disease and cancer. We previously found that hydrogen peroxide (H2O2) initiates an increase in Ca2+\/cAMP-response element binding protein (CREB) phosphorylation in C10 alveolar type II cells that requires activation of extracellular regulated kinases 1\/2 (ERK1\/2). Here, we investigated the role of crosstalk between protein kinase A (PKA) and epidermal growth factor receptor (EGFR) in oxidant-induced signaling to ERK1\/2 and CREB in C10 cells. Application of H2O2 increased nuclear accumulation of PKA, and inhibition of PKA with H89 reduced oxidant-mediated phosphorylation of both CREB and ERK1\/2. Single cell measurements of cAMP and redox status, using a FRET-based biosensor and a redox-sensitive GFP, respectively, indicated that H2O2 increases production of cAMP that correlates with redox state. Inhibition of EGFR activity decreased both H2O2-induced CREB phosphorylation and translocation of PKA to the nucleus, suggesting that crosstalk between PKA and EGFR underlies the oxidant-induced CREB response. Furthermore, knockdown of CREB expression using siRNA led to a decrease in bcl-2 and an increase in oxidant-induced apoptosis. Together these data reveal a novel role for crosstalk between PKA, ERK1\/2 and CREB that mediates cell survival during oxidant stress.\nIntroduction\nLung epithelial cells, a target cell of inhaled oxidants, are subjected to a variety of environmental stresses, including oxidizing gases, particulates, and airborne microorganisms. Although the formation of oxidants is normally well regulated, excessive production may cause inflammation and cellular injury. Numerous studies suggest a role for oxidant stress in the pathogenesis of pulmonary diseases, including asthma, pulmonary fibrosis, and cancer [1].\nReactive oxygen species (ROS) are involved in multiple physiological processes through their capacity to regulate the expression of several growth factor receptors, protein kinases and phosphatases. In alveolar type II lung epithelial cells, hydrogen peroxide (H2O2) induces tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) [2] and leads to activation of protein kinase C (PKC) [3] and the mitogen activated protein kinases (MAPK) p38, c-Jun amino-terminal kinase (JNK), and extracellular signal-regulated kinase 1\/2 (ERK1\/2) [4, 5]. H2O2 also activates the expression of transcription factors, including Ca2+\/cyclic AMP-response element binding protein (CREB) [6] and members of the activator protein (AP-1) family, c-jun and c-fos [7], resulting in proliferation [4] and apoptosis [6, 8].\nCREB is a 43\u00a0kDa transcription factor belonging to the basic-leucine zipper (bZIP) family and is regulated by phosphorylation at serine residue 133 [9, 10]. Phosphorylation of CREB initiates recruitment of co-factors to the Ca2+\/cAMP-response element (CRE) that are necessary for transcriptional activation such as CREB-binding protein (CBP300) [11]. CREB activation is regulated by both Ca2+ and cAMP, which have been shown to regulate both ERK1\/2- and protein kinase A (PKA)-mediated CREB phosphorylation [6, 12, 13]. Many genes important for regulation of proliferation and apoptosis, including c-fos and bcl-2, respectively, contain CREs in their promoter [11].\nOur previous studies have shown that exposure of alveolar type II cells to bolus H2O2 or asbestos fibers results in CREB activation that is dependent on ERK1\/2 activation [6, 14]. Furthermore, we found that preventing CREB activation promoted cell survival and enhanced bcl-2 transcription, revealing a potential role for CREB in H2O2-mediated apoptosis [6]. Because of known crosstalk between PKA, ERK1\/2, and CREB in many cell types and the dose-dependent effects of H2O2, the goal of this study was to determine their relative importance in oxidant-induced signaling. The effects of transient bolus H2O2 as well as a peroxide generating system were tested to represent a range of oxidant concentrations that lead to cell proliferation and\/or apoptosis. The data support the hypothesis that PKA and EGFR are central regulators of oxidant\u2013induced ERK1\/2 and CREB activation and demonstrate a link between PKA, EGFR, and CREB in cell survival following exposure to H2O2 in alveolar type II cells.\nMaterials and methods\nCell culture and treatments\nC10 cells, a contact-inhibited, non-transformed murine alveolar type II epithelial cell line [15], were grown in CMRL 1066 medium supplemented with L-glutamine, penicillin\/streptomycin, and 10% fetal bovine serum (FBS) (GIBCO BRL, Rockville, MD). Cells were grown to 90% confluence, and then complete medium was replaced with CMRL 1066 medium supplemented with l-glutamine, penicillin\/streptomycin, and 0.5% FBS for 48\u00a0h before exposure to agents.\nH2O2 (Sigma, St. Louis, MO) was added to the medium at concentrations from 100\u00a0\u03bcM to 300\u00a0\u03bcM. Recombinant glucose oxidase (GO; 5\u00a0mU\/ml, 15\u00a0mU\/ml, or 30\u00a0mU\/ml) (Roche, Indianapolis, IN) was used to provide low level fluxes of H2O2 [16, 17]. Forskolin and epidermal growth factor (EGF) (Sigma, St. Louis, MO) (10\u00a0\u03bcM and 100\u00a0ng\/ml, respectively) were used as positive controls for induction of phospho-CREB and phospho-EGFR, respectively. Tumor necrosis factor \u03b1 (TNF\u03b1) (Calbiochem, LaJolla, CA) was used at 0.1\u00a0\u03bcg\/ml to induce apoptosis. Control cultures received medium without agents and were treated identically.\nThe ERK1\/2 inhibitor, U0126 (10\u00a0\u03bcM for 30\u00a0min prior to treatment), the EGFR tyrosine kinase inhibitor, Tyrphostin AG1478 (10\u00a0\u03bcM for 1\u00a0h prior to treatment), and the antioxidant enzyme, catalase (1000\u00a0U\/ml 1\u00a0h prior to treatment) were obtained from Calbiochem. The PKA inhibitor, H89 (10\u00a0\u03bcM for 1\u00a0h pre-treatment) was obtained from Biomol (Plymouth Meeting, PA). N-acetyl-l-cysteine (NAC; 10\u00a0mM 16\u00a0h prior to treatment) and DL-Buthionine-(S,R)-sulfoximine (BSO; 10\u00a0\u03bcM 16\u00a0h prior to treatment) were purchased from Sigma (St. Louis, MO).\nWestern blot analysis\nAfter C10 cells were exposed to agents as described above, the cells were washed twice with cold PBS and collected in 4X sample buffer (200\u00a0\u03bcM Tris, pH 6.8, 4% SDS, 4\u00a0mg\/ml bromophenol blue, 0.04% \u03b2-mercaptoethanol, 40% glycerol, 2\u00a0\u03bcM pyronin-Y). The amount of protein in each sample was determined using the RC\/DC protein assay (Bio-Rad). About 30\u00a0\u03bcg of protein was separated by a 10% SDS-PAGE and transferred to nitrocellulose. Western blots were performed as described previously [6] using antibodies specific to total and phosphorylated CREB (1:1000; rabbit polyclonal anti-CREB, Cell Signaling Technologies, Danvers, MA; 1:500; rabbit polyclonal anti-phospho-CREB, Cell Signaling Technologies), total and phosphorylated ERK1\/2 (1:1000; rabbit polyclonal anti-ERK1\/2, Cell Signaling Technologies; 1:500; rabbit polyclonal anti-phospho-ERK1\/2, Cell Signaling Technologies). Antibody binding was detected using horse radish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (1:5000; Jackson ImmunoResearch Laboratories, Inc., West grove, PA), followed by chemiluminescence (Kirkgaard and Perry Laboratories, Gaithersburg, MD). QuantityOne (Bio-Rad, Hercules, CA) was used to quantify band density, and intensity of phospho-CREB bands were normalized to the intensity of the corresponding total CREB bands.\nLive cell imaging of cAMP and redox potential\nC10 cells grown on glass coverslips in 6-well tissue culture dishes were transiently transfected with 8\u00a0\u03bcg of a plasmid encoding a unimolecular fluoresecent resonance energy transfer (FRET)-based cAMP biosensor, Epac1-camps (provided by Dr. Martin Lohse, University of W\u00fcrtzburg) [18, 19], or 1\u00a0\u03bcg of a plasmid encoding a redox-sensitive green fluorescent protein (GFP), roGFP2 (a gift from Dr. James Remington, University of Oregon) [20], using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), following the manufacturer\u2019s instructions. The transfection efficiency was approximately 50%.\nImaging experiments were conducted after 48\u201372\u00a0h of transfection, and the growth medium removed and replaced with Krebs-Ringer bicarbonate solution (KRB; 119\u00a0mM NaCl, 4.7\u00a0mM KCl, 2.5\u00a0mM CaCl2, 1\u00a0mM MgCl2, 1\u00a0mM KH2PO4, 25\u00a0mM NaHCO3 or 10\u00a0mM HEPES-NaOH (pH 7.40), and 2\u00a0mM glucose). Coverslips were placed into a heated microperfusion chamber mounted on the specimen stage of an inverted fluorescence microscope (Nikon TE-2000U) equipped with a CARV spinning disk confocal system (Atto Bioscience Inc., Rockville, MD). Cells were continuously superfused with Krebs-Ringer bicarbonate solution (2\u20135\u00a0ml\/min) at 37\u00b0C. Dithiothreitol (DTT) (Calbiochem, LaJolla, CA) was used to calibrate the redox minimum.\nFor imaging Epac1-camps FRET and roGFP2, cells were visualized with a Nikon Super Fluor 40\u00d7 objective. MetaFluor\/MetaMorph software was used for image acquisition and analysis (Universal Imaging). The Epac1-camps EYFP excitation wavelength was 440\u00a0nm. Dual emission ratio imaging at 485\u00a0nm and 535\u00a0nm for Epac1-camps and dual excitation ratio imaging at 400\u00a0nm and 490\u00a0nm for roGFP2 were accomplished using a computer-controlled high speed filter wheel (Lambda 10-2 optical filter changer with rotation every 60\u201380\u00a0ms, Sutter Instrument Co., Novato, CA). For roGFP2, a 505DRLP dichroic mirror and an emission filter, 535DF25, were used. Images (50\u2013250\u00a0ms exposure) were captured every 10\u00a0s with a 16-bit Cascade 650 digital camera (Roper Instruments, Trenton, NJ) and background-corrected by manual selection of background regions [19].\nThe ratio values were normalized to the average baseline values measured 1-min prior to application H2O2. Epac1-camps FRET decreases with increasing cAMP concentration, thus increased [cAMP]c is correlated with an increase in the relative emission ratio of ECFP\/EYFP (485\/535) [18]. Data were thus expressed as relative ratio 485\/535 for Epac1-camps and fold increase in relative ratio 400\/490 for roGFP2.\nImmunofluorescence in C10 cells\nC10 cells were grown on glass coverslips for all experiments. After experimental exposures, immunofluorescence to detect the catalytic subunit of PKA\u03b1 was performed as previously described [6, 21]. Briefly, cells were washed with phosphate buffered saline (PBS), fixed in 3.7% formaldehyde, permeablized with \u221220\u00b0C methanol and incubated with blocking solution containing 2% bovine serum albumin (BSA) in PBS. Cells were incubated with primary antibody (1:100; rabbit polyclonal PKA\u03b1 catalytic subunit antibody, Santa Cruz, Santa Cruz, CA) diluted in 2% BSA plus 0.1% Triton X-100 in PBS (BSA\/PBS-T) overnight at 4\u00b0C. Secondary antibody (1:400; AlexaFlour 568 goat-anti-rabbit IgG, Molecular Probes) diluted in BSA\/PBS-T was applied for 1\u00a0h at room temperature (RT), and followed by incubation with nuclear counterstain, YOYO-1 iodide (Molecular Probes, Carlsbad, CA; 1:10,000), 1\u00a0unit\/ml RNase, and 0.1% sodium azide in BSA\/PBS-T for 30\u00a0min at RT. Coverslips were mounted onto slides with AquaPolyMount (Polysciences, Inc. Warrington, PA). For each sample, confocal images were collected in fluorescence modes using a Bio-Rad MRC1024ES confocal scanning laser microscope (Bio-Rad, Hercules, CA). For quantification, a nuclear mask was generated in Corel Photopaint using the YOYO-1 image template, and pixel intensities for PKA\u03b1 were determined within the nuclear mask area as previously described [22].\nTransient transfections with small interfering RNA (siRNA)\nThe siCONTROL non-targeting siRNA #2 and SMARTpool mouse CREB siRNA (100\u00a0nM; Dharmacon, Lafayette, CO) were transfected into C10 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), following the manufacturer\u2019s instructions. After 4\u00a0h in transfection media, FBS was added to 10% and cells were incubated for 24\u00a0h. Cells were then transferred to reduced serum media (0.5% FBS) for 24\u00a0h prior to treatments.\nReal time quantitative PCR (RT qPCR)\nTotal RNA was extracted from C10 cells using the RNeasy\u2122 PLUS protocol for total RNA isolation from animal cells (Qiagen,Valencia, CA). cDNA was reverse transcribed from 500\u00a0ng total RNA using an Omniscript\u2122 Reverse Transcriptase RNase free DNase kit (Qiagen), with an oligo dT primer, according to the manufacturer\u2019s protocol. RT qPCR primers and probes for B-cell lymphoma-2 (bcl-2) and c-fos were obtained as Assays-on-Demand\u2122 kits from Applied Biosystems (Foster City, CA). PCR products were detected by TaqMan qPCR, as previously described [23]. Expression levels of target genes were determined using hypoxanthine-guanine phosphoribosyl transferase (hprt) as the internal standard. Samples were run in duplicate from 3 independent experiments and the comparative Ct (cycle threshold) method for relative quantity (RQ value) was used to calculate relative mRNA expression among samples.\nDetection and quantification of apoptosis\nApoptosis was detected by measuring single-stranded DNA using Apostain\u2122 as previously described [24]. Briefly, cell monolayers grown on glass coverslips were treated with 30\u00a0mU\/ml GO for 24\u00a0h, then fixed in methanol for 24\u00a0h at \u221220\u00b0C, boiled for 5\u00a0min in PBS containing 5\u00a0mM MgCl2, and then immersed in ice-cold PBS for 10\u00a0min. Cells were blocked with 40% FBS and then incubated with Apostain F7-26 (Alexis Biochemicals, San Diego, CA, 10\u00a0\u03bcg\/ml) followed by HRP-conjugated secondary antibody (goat anti-mouse IgM; Jackson Labortories, West Grove, PA, 1:400). To visualize secondary antibody binding, the peroxidase substrate DAB (Sigma) was used. Coverslips were mounted onto slides with AquaPolyMount (Polysciences, Inc. Warrington, PA) for subsequent examination using bright field light microscopy. To determine the numbers of apoptotic cells and total cell numbers per field, 5 random fields were evaluated per experimental condition at x200 total magnification.\nStatistical analysis\nStatistical analyses were performed using non-normalized data, and pair-wise comparisons between treatment groups were achieved using Student\u2019s t-test or Mann\u2013Whitney Rank Sum Test method for unequal variances. ANOVA (Holm\u2013Sidak method) was used for multiple comparisons. Differences were considered statistically significant at P\u00a0<\u00a00.05.\nResults\nCREB is phosphorylated after exposure to H2O2 and glucose oxidase\nWe have previously shown that H2O2 causes CREB phosphorylation in alveolar type II cells [6]. Here, we further show by Western blot that the CREB response to bolus addition of H2O2 (Fig.\u00a01a) and H2O2 generated by glucose oxidase (GO) (Fig.\u00a01b) is reduced when pretreated with the antioxidants NAC or catalase at effective concentrations documented previously in C10 cells [25].\nFig.\u00a01Bolus and glucose oxidase generated H2O2 mediate CREB phosphorylation in an oxidant-dependent manner. (a) C10 cells, pre-incubated with 1,000\u00a0U\/ml catalase (CAT) for 1\u00a0h, were treated with 10\u00a0\u03bcM forskolin or 100, 200, or 300\u00a0\u03bcM H2O2 for 10\u00a0min, and then analyzed by Western blot analysis for phospho-CREB (p-CREB). (b) C10 cells, pre-incubated with 10\u00a0mM NAC for 16\u00a0h or 1,000\u00a0U\/ml catalase (CAT) for 1\u00a0h, were treated with 10\u00a0\u03bcM forskolin or 5, 15, or 30\u00a0mU\/ml glucose oxidase (GO) for 4\u00a0h, and then analyzed by Western blot analysis for phospho-CREB (p-CREB). An antibody recognizing total CREB was used as a control for protein loading in A and B. Data are representative of 3 separate experiments\nH2O2-induced CREB phosphorylation is decreased after the reduction of PKA activity\nBecause PKA is an important regulator of CREB [9], we investigated its role in H2O2-induced CREB phosphorylation. Cells were examined after exposure to lower concentrations previously associated with expression of cyclin D (100\u00a0\u03bcM) and higher concentrations known to induce apoptosis (300\u00a0\u03bcM) [6, 26]. Exposure to H2O2 led to CREB phosphorylation in a concentration-dependent manner that was significantly decreased after reduction of PKA activity using the specific inhibitor H89 (Fig.\u00a02a, b). The pattern of CREB phosphorylation in response to H2O2 closely paralleled that seen in response to activation of PKA by forskolin. H89 also reduced H2O2-mediated ERK1\/2 activation, suggesting that PKA may regulate CREB activation directly or indirectly through ERK1\/2-mediated CREB activation.\nFig.\u00a02H2O2-mediated CREB activation is inhibited by the PKA inhibitor, H89. (a) C10 cells, pre-incubated with 10\u00a0\u03bcM H89 for 1\u00a0h, were treated with 10\u00a0\u03bcM forskolin or indicated concentrations of H2O2 for 10\u00a0min, and then analyzed by Western blot analysis for phospho-CREB (p-CREB), total CREB, phospho-ERK1\/2 (p-ERK1\/2) and total ERK1\/2. (b) Quantification of p-CREB band intensities from A corrected using the corresponding CREB band intensity and normalized to the untreated control. Data represent 4 separate experiments; *P\u00a0<\u00a00.05 when compared with corresponding condition without H89. (c) C10 cells, pre-incubated with 10\u00a0\u03bcM H89 for 1\u00a0h, were treated with 10\u00a0\u03bcM forskolin or indicated concentrations of glucose oxidase (GO) for 4\u00a0h, and then analyzed by Western blot for phospho-CREB (p-CREB), total CREB, phospho-ERK1\/2 (p-ERK1\/2), and total ERK1\/2. (d) Quantification of p-CREB band intensities from C corrected using the corresponding CREB band intensity and normalized to the untreated control. Data represent mean\u00a0\u00b1\u00a0SEM from 4 separate experiments; *P\u00a0<\u00a00.05 when compared with corresponding condition without H89\nSimilar to results with bolus addition of H2O2, H2O2 generated by GO stimulated CREB phosphorylation in a dose-dependent manner and the phosphorylation was significantly reduced by PKA inhibition at higher concentrations of GO (Fig.\u00a02c, d). However, unlike bolus addition, H2O2 generated by GO led to a dose-dependent increase in ERK1\/2 phosphorylation that was marginally sensitive to inhibition of PKA activity, suggesting that the duration of the stimulus may cause differences in signaling patterns.\nH2O2 stimulates cAMP production in C10 cells\nSingle cell imaging was utilized to determine whether H2O2 exerts a direct effect on upstream regulation of PKA through production of cAMP. Epac1-camps, a FRET-based cAMP biosensor, was expressed in C10 cells. Epac1-camps senses changes in cytoplasmic cAMP levels ([cAMP]c) through a cAMP-dependent conformational change resulting in reduced FRET between ECFP (485 emission) and EYFP (535 emission) [18, 19]. Exposure to H2O2 caused a monophasic increase in [cAMP]c (Fig.\u00a03a) that was comparable to the response elicited by direct activation of adenylyl cyclase with forskolin, but with a slower rise time (Fig.\u00a03c). H2O2 generated by GO stimulated a rise in [cAMP]c similar in magnitude to bolus addition of H2O2, but with a longer lag-time presumably due to the slower generation of oxidant (Fig.\u00a03b).\nFig.\u00a03H2O2 stimulates an increase in cAMP levels. C10 cells were transiently transfected with the cAMP FRET indicator Epac1-camps and then exposed to 10\u00a0\u03bcM 250\u00a0\u03bcM H2O2 (a), 15\u00a0mU\/ml glucose oxidase (GO) (b), or 10\u00a0\u03bcM forskolin (FSK) (c) for the times indicated. Data (means\u00a0\u00b1\u00a0SEM) are expressed as the relative ratio (485\/535) of the cAMP response from the live emission recording of a representative cell. Grey bars indicate time of addition and length of exposure. Results are representative of \u22659, 5, and 4 cells (A, B and C, respectively)\nSingle cell imaging was also used to measure redox status and correlate cAMP levels with redox status. The redox sensitive GFP variant, roGFP2, was expressed in C10 cells. Oxidation is detected by roGFP through a change in GFP excitation from 400\u00a0nm and 490\u00a0nm [20]. The time course of H2O2-mediated cAMP production correlated well with the level of intracellular oxidation measured by roGFP2 (Fig.\u00a04a). Upon removal of H2O2, the redox state returned to baseline approximately 36\u00a0min after the initial exposure (Fig.\u00a04b).\nFig.\u00a04Redox status correlates with cAMP level in cells responding to H2O2. C10 cells were transiently transfected with the redox indicator, roGFP2 and then exposed to 250\u00a0\u03bcM H2O2 for 10\u00a0min followed by 1\u00a0mM DTT (a) or 15\u00a0mU\/ml glucose oxidase (GO) for 65\u00a0min, followed by 200\u00a0\u03bcM H2O2, and then 10\u00a0mM DTT (b). Data (means\u00a0\u00b1\u00a0SEM) are expressed as the fold change in the excitation ratio at 400\/490\u00a0nm normalized to the average base-line ratio values measured 1\u00a0min before stimulation. Results are averages of \u226523 and 11 cells (A and B, respectively). Grey bars indicate time of addition and length of exposure, and WO indicates wash out with KREBs buffer\nEGFR tyrosine kinase activity is important for signaling from H2O2 to PKA and CREB\nIt has been shown that the protein tyrosine activity of EGFR is important for EGF-mediated stimulation of adenylyl cyclase [27]. Thus to determine the contributions of EGFR in the observed H2O2-induced CREB phosphorylation, cells were evaluated with or without pretreatment with the specific EGFR tyrosine kinase inhibitor, AG1478. Application of AG1478 significantly reduced CREB phosphorylation in response to bolus addition H2O2 and to H2O2 generated by higher concentrations of GO (Fig.\u00a05), suggesting that EGFR activity is involved in H2O2-induced CREB phosphorylation. As expected, reduction of EGFR activity also inhibited H2O2-induced ERK phosphorylation (Fig.\u00a05).\nFig.\u00a05Reduction of EGFR tyrosine kinase activity leads to the inhibition of both H2O2-induced CREB and ERK1\/2. C10 cells, pre-incubated with the EGFR tyrosine kinase inhibitor AG1478 (10\u00a0\u03bcM) for 1\u00a0h, were treated with 10\u00a0\u03bcM forskolin or 100\u00a0ng\/ml EGF for 5\u00a0min, the indicated concentrations of H2O2 for 10\u00a0min (a) or glucose oxidase (GO) for 4\u00a0h (c), and then analyzed by Western blot analysis for phospho-CREB (p-CREB), total CREB, phospho-ERK1\/2 (p-ERK1\/2), and total ERK1\/2. (b, d) Quantification of p-CREB band intensities from A and C respectively corrected using the corresponding CREB band intensity and normalized to the untreated control. Data represent mean\u00a0\u00b1\u00a0SEM of 3 separate experiments; *P\u00a0<\u00a00.05 when compared with corresponding condition without AG1478\nImmunofluorescence evaluation of activated PKA translocation to the nucleus was used to more directly test the effects of H2O2 on PKA activation and to further evaluate a role for EGFR. Exposure to H2O2 led to an increase in the PKA free catalytic subunit that was significant in the nucleus at 300\u00a0\u03bcM and similar to the response to forskolin. Inhibition of EGFR tyrosine kinase activity with AG1478 prevented the H2O2-induced increases at both 200\u00a0\u03bcM and 300\u00a0\u03bcM (Fig.\u00a06).\nFig.\u00a06Reduction of EGFR tyrosine kinase activity inhibits the H2O2-induced nuclear translocation of activated PKA. C10 cells, pre-incubated with 10\u00a0\u03bcM AG1478 for 1\u00a0h, were exposed to 10\u00a0\u03bcM forskolin and H2O2 for the indicated concentrations for 10\u00a0min. (a) The PKA free catalytic subunit (red) was detected by immunofluorescence and nuclei were stained with YOYO-1 (green). (b) Quantification of PKA immunofluorescence pixel intensity within nuclei; *P\u00a0<\u00a00.05 compared to control, #P\u00a0<\u00a00.05 compared to treatment, n\u00a0=\u00a03 experiments, 5 fields\/experiment\nDirect activation of CREB by PKA is a minor pathway in EGF-induced CREB phosphorylation\nBecause of the known crosstalk between EGFR, ERK1\/2, and PKA, we examined the role of PKA and ERK1\/2 in forskolin- and EGF-induced CREB activation. Treatment with both forskolin and EGF resulted in a significant increase in CREB phosphorylation when compared to control cells (Fig.\u00a07). Inhibition of PKA activity with H89 significantly decreased forskolin-induced CREB phosphorylation, but only blunted EGF-induced CREB phosphorylation. Inhibition of ERK1\/2 phosphorylation with the MEK inhibitor, U0126, also considerably decreased CREB phosphorylation. The inhibitory effects of H89 and U0126 were not additive towards either forskolin- or EGF-induced CREB phosphorylation, suggesting that direct activation of CREB through PKA may play a minor role in the activation of CREB. Furthermore, both forskolin and EGF induced ERK1\/2 phosphorylation, but only forskolin-mediated CREB phosphorylation was inhibited by H89, suggesting that PKA regulates CREB indirectly through the MAPK\/ERK1\/2 pathway.\nFig.\u00a07Crosstalk between PKA and ERK1\/2 signaling is important in regulating CREB phosphorylation. (a) C10 cells, pre-incubated with 10\u00a0\u03bcM H89, U0126, or H89 and U0126 for 1\u00a0h, were treated with 10\u00a0\u03bcM forskolin for 10\u00a0min or 100\u00a0ng\/ml EGF for 5\u00a0min, and then analyzed by Western blot analysis for phospho-CREB (p-CREB), total CREB, phospho-ERK1\/2 (p-ERK1\/2), and total ERK1\/2. (b) Quantification of phospho-CREB in A corrected using CREB intensities and phospho-ERK\/12 in A corrected using ERK1\/2 intensities; *P\u00a0<\u00a00.05 for DMSO treatment compared to AG1478 treatment; #P\u00a0<\u00a00.05 for DMSO control compared to treatment. Data represent mean\u00a0\u00b1\u00a0SEM of 3 separate experiments\nKnockdown of CREB using siRNA results in loss of oxidant-induced c-fos and bcl-2 transcription\nTo more directly assess the role of CREB in oxidant-mediated responses, an siRNA approach was used to reduce levels of CREB within C10 cells. Transfection with siCREB resulted in greater than 80% knockdown of both CREB mRNA and protein levels (Fig.\u00a08a, b). CRE-containing genes previously shown to be upregulated by H2O2 including bcl-2 and c-fos were found to be dose-dependently induced by GO. Induction was significantly reduced in cells transfected with siCREB (Fig.\u00a08c, d), suggesting that CREB is essential for the transcriptional response of these genes to H2O2.\nFig.\u00a08Knockdown of CREB using siRNA results in loss of oxidant-induced c-fos and bcl-2 transcription. (a) C10 cells were transfected with 100\u00a0nM scrambled siRNA (siControl) or siCREB followed by Western blot analysis to detect CREB. \u03b2-actin was used as a loading control. (b) Cells were transfected as in A, followed by treatment with 5 or 15\u00a0mU\/ml glucose oxidase (GO) for 6\u00a0h. Total RNA was extracted and cDNA was assessed by RT qPCR to quantify CREB, bcl-2 and c-fos mRNA levels. Shown are relative quantity (RQ) values normalized to the siControl sample. #P\u00a0<\u00a00.05 when compared to untreated siCon; *P\u00a0<\u00a00.05 when compared to treatment siCon\nKnockdown of CREB significantly increases the number of cells undergoing apoptosis following exposure to glucose oxidase\nRecently, we have shown that reduction of CREB activity, by introducing a phosphorylation-null CREB construct, led to a decrease in apoptosis [6]. To resolve whether CREB is essential for H2O2-induced apoptosis, C10 cells transfected with scrambled siRNA (siControl) or siCREB were treated with 30\u00a0mU\/ml GO for 8\u00a0h and apoptotic cells were identified by Apostain (Fig.\u00a09). H2O2 generated from GO induced a significant increase in the percentage of cells undergoing apoptosis compared to the control in both siControl and siCREB cells. Cells expressing siCREB had significantly higher levels of apoptosis than the siControl cells in both the untreated condition and after GO exposure, supporting a role for CREB in the regulation of apoptosis induced by H2O2. Interestingly, the apoptotic enhancing effect was not selective for H202-mediated apoptosis, as siCREB also enhanced the apoptotic effect of TNF\u03b1. Taken together, these results suggest that CREB plays an important role in the cellular response to oxidant stress including upregulation of survival genes and promoting cell survival.\nFig.\u00a09CREB is protective against oxidant-induced apoptosis. (a) C10 cells, transfected with siControl or siCREB, were exposed to 30\u00a0mU\/ml glucose oxidase (GO) or 0.1\u00a0\u03bcg\/ml tumor necrosis factor \u03b1 (TNF\u03b1) for 8\u00a0h followed by identification of apoptotic cells using Apostain. (b) Quantification of % Apostain positive cells was determined by scoring the average of 5 fields\/condition; *P\u00a0<\u00a00.05 when compared to untreated siCon; #P\u00a0<\u00a00.05 when compared to treatment siCon. Data represent mean\u00a0\u00b1\u00a0SEM of 4 separate experiments. Bar\u00a0=\u00a0100\u00a0\u03bcm\nDiscussion\nExposure to various environmental stresses, such as oxidative gases, metals, and particulates, initiates multiple physiological processes in alveolar type II cells. Oxidants have been implicated in the pathogenesis of lung cancer, pulmonary fibrosis, and asthma through their capacity to regulate an intricate network of protein kinases. Yet the relationship between oxidants and the pathogenesis of lung disease is unclear, primarily because of the lack of understanding of the mechanisms by which oxidants function in both normal physiological and disease states. In this study, we show for the first time that H2O2 exposure leads to an increase in cytoplasmic cAMP levels, followed by PKA-dependent CREB and ERK1\/2 phosphorylation in alveolar type II cells. Furthermore, we show that EGFR activity is necessary for H2O2-stimulated nuclear accumulation of activated PKA and for PKA-mediated CREB phosphorylation. Together these data suggest that PKA is a central hub for interplay between EGFR, ERK1\/2, and CREB signaling pathways activated by oxidant stress.\nSeveral studies have shown crosstalk between PKA and ERK1\/2 signaling pathways. This crosstalk appears to be cell type specific, since an increase in cAMP-dependent PKA activation stimulates ERK1\/2 in some cells, but suppresses it in others [28\u201330]. We have previously demonstrated that H2O2 exposure leads to ERK1\/2-dependent CREB phosphorylation in C10 alveolar type II cells [6]. Studies here have dissected the underlying mechanisms to reveal that H2O2-induces cAMP formation and PKA activation that are important for both ERK1\/2 and CREB phosphorylation.\nEGFR is overexpressed and activated in response to epithelial injury [31]. Its activation is believed to play an integral role in the cellular responses of lung epithelium to injury and oxidant stress, possibly by signal amplification through MAPK phosphorylation cascades. Oxidative modification of a reduced cysteine residue in the EGFR reversibly affects its activation [32, 33]. Moreover, Goldkorn and colleagues have shown that in alveolar type II cells, H2O2 induces EGFR phosphorylation on tyrosine residues [2]. These findings suggest that even though there is no specific receptor for oxidants, their signaling may be efficiently transduced through interactions with the EGFR.\nIn the present study, we show that EGFR activity is necessary for both activation of PKA nuclear translocation and subsequent CREB phosphorylation in response to H2O2. Mitogenic signals transmitted through the EGFR have been shown to involve PKA-dependent signaling cascades. Tyrosine phosphorylation of the EGFR requires an increase in cAMP levels and PKA function in many, but not all cell types [34, 35]. In addition, tyrosine kinase activity of EGFR is required for growth factor stimulation of adenylyl cyclase activity, which leads to the activation of PKA [27, 36]. PKA activity has also been shown to promote downstream Raf-1 function and subsequent ERK activation [37]. Crosstalk has also been seen from EGFR to adenylyl cyclase activity and PKA in airway epithelial cells via beta adrenergic receptors [38, 39]. Thus, communication between EGFR and PKA is potentially bi-directional.\nThe H2O2-mediated generation of cAMP and the CREB activation response to H2O2 increased with oxidant concentration but, with minor exception, were not dependent on whether oxidant was added by bolus or generated by glucose\/glucose oxidase. Although reduction of the CREB response by PKA or EGFR inhibition was only significant for the higher oxidant concentrations, a trend was observed with lower concentrations. While it is difficult to accurately predict the physiological significance of these oxidant levels, when put in context with previous findings, these observations suggest that CREB signaling through PKA and EGFR could participate in signaling that affects both oxidant-mediated proliferation and apoptosis.\nPreviously we demonstrated that expression of phosphorylation-incompetent CREB paradoxically increases bcl-2 transcription and reduces apoptosis following bolus H2O2 exposure [6]. Findings here show that siRNA-mediated knockdown of CREB results in a loss of bcl-2 mRNA and an increase in apoptosis in cells responding to an H2O2 generating system. The difference is likely due to the method of CREB inhibition, in that the dominant-negative CREB may have altered chromatin interactions with other transcription factors or caused quenching of the response. Putting these results in context with the findings of others, we propose that CREB activation through PKA signaling serves as a key survival pathway in cells responding to oxidant stress. PKA-and CREB-mediated gene expression may thus be important in regulating oxidant-mediated apoptosis resulting from lung pathologies, and future experiments will explore the importance of these signaling pathways in promoting changes in gene expression following lung epithelial cell injury or oxidant exposure.","keyphrases":["oxidative stress","transcription factor","pulmonary epithelium","lung fibrosis","fret biosensor"],"prmu":["P","P","R","R","R"]}
{"id":"Matern_Child_Health_J-2-2-1592145","title":"The Importance of Preconception Care for Women With Disabilities\n","text":"Overall estimates of the number of women with disabilities in the United States range from 16.8 to 28.6 million, or approximately one in every five women [1\u20133]. A large percentage of these women are in their reproductive years, and they often encounter greater obstacles to receiving health care than women without disabilities [4]. Some disabling conditions, such as spinal cord injury (SCI) and multiple sclerosis (MS), have low prevalence rates but are more common in the reproductive age group. Such conditions are often associated with a greater severity of disability and increased need for specialized services, including prenatal care [5]. Health care professionals need to be aware of the reproductive health care issues facing women with disabilities and take every opportunity to address them.\nSexuality and reproductive health issues have received inadequate attention for women with disabilities. Many women with disabilities desire children and are capable of conceiving, but face considerable pressure not to reproduce [6, 7]. Although a growing number of women with disabilities are becoming pregnant, anecdotal evidence suggests that many of these women encounter negative attitudes towards their pregnancies and report difficulty receiving comprehensive prenatal care [6].\nWomen with disabilities who become pregnant face similar issues and concerns as pregnant women without disabilities. Although many pregnant women experience problems with weight gain, fatigue, fluid retention, bladder dysfunction, and urinary tract infections, these problems may be more serious in women with disabilities [8]. All women are concerned about the health of their unborn child. Like other women, women with disabilities express concern about the possibility of giving birth to a child with disabilities and may seek genetic counseling. On the other hand, not all disabled women embrace the idea of genetic testing. To these women the idea of genetic counseling raises psychosocial issues that may be difficult for them to consider [6].\nSome disabling conditions pose unique problems during or after pregnancy. Complications associated with specific conditions, such as systemic lupus erythematosus (SLE), SCI, or MS, can affect pregnancy and should be evaluated prior to conception [8\u201312]. For example, an increased risk of fetal loss, growth retardation, premature placental aging with thrombosis, and preeclampsia are described in women with lupus [9]. Given the seriousness of these complications, ongoing monitoring and interventions to reduce them are critical. Pregnant women with SCI above the level of T6 also require special attention [7, 8, 10, 11]. Such women have an increased risk of autonomic dysreflexia during all stages of pregnancy and, if it is not recognized and treated, autonomic dysreflexia can be fatal [8, 11]. Although women with stable MS can safely carry a pregnancy to term, postpartum exacerbations of MS are quite common and occur in up to 30% of women within one month of delivery [11, 12]. Pregnancy management in women with disabilities should therefore include preconception counseling and evaluation to identify these and other potential problems that can occur throughout the course of the pregnancy.\nHealth care professionals need to know that physical barriers, such as the lack of accessible scales or examination tables, present enormous and recurring obstacles to obtaining adequate prenatal care. Also, communication barriers, particularly for those who are deaf or hard of hearing, affect a women's ability to receive appropriate medical care. In addition, there are some aspects of gynecologic health care such as informed consent, sedation, and contraceptive issues that are unique for women with developmental disabilities. A recent publication from the American College of Obstetricians and Gynecologists highlights ways to improve care for this group of women [7].\nPreconception care is associated with improved pregnancy outcomes. It is recommended that primary care providers assess all women of reproductive age for their preconception risk conditions and provide or refer individuals for interventions as appropriate [13]. When caring for a woman with a disability, a team approach involving the primary physician, obstetrician, anesthesiologist, neurologist, physiatrist, and other allied health professionals such as occupational and physical therapists, is recommended [6\u20138, 11]. This is particularly important, as many obstetricians are unfamiliar with disability-related complications. A multidisciplinary approach, which involves nursing and social work, can further improve pregnancy outcomes. This shared approach to pregnancy management can also increase the level of comfort for women with disabilities, by knowing that more than one health care provider is looking out for their well-being.\nPreconception counseling for women with disabilities should address the medical, psychological, and social impact of a pregnancy [10]. During preconception counseling, it is particularly important to evaluate the prenatal and postpartum social support systems available to women with disabilities. Body changes such as decreased mobility or bladder dysfunction increase the need for personal assistance with routine activities of daily living. Many women with disabilities also benefit from an occupational therapy evaluation to assess environmental barriers and explore adaptive equipment and techniques that facilitate breast feeding and infant care. Health care providers might want to recommend that women with disabilities explore creative parenting strategies such as those offered by Through the Looking Glass, a national resource center on parenting with a disability, located in Berkeley, California [6, 14].\nAn increasing number of women with disabilities are becoming pregnant. Despite this little is known about the reproductive experiences of these women. Population-based data describing the reproductive experiences of women with disabilities is virtually non-existent. Currently, disability is not regularly included in national or statewide surveys and surveillance tools related to pregnancy. Consequently, the true magnitude of health concerns, including reproductive issues, facing women with disabilities is largely unknown. To improve our understanding of these issues, disability should be included as a demographic variable in all national and state-level surveys addressing reproductive health issues.\nAll women of childbearing age, including women with disabilities, should have access to preconception care that addresses the medical, psychological and social impacts of pregnancy. Women with disabilities can and do have healthy pregnancies. Accurate knowledge of disabling conditions and a shared multidisciplinary approach to pregnancy management are critical components for good pregnancy outcomes among women with disabilities.","keyphrases":["disability","pregnancy","women's health"],"prmu":["P","P","R"]}
{"id":"Osteoporos_Int-2-2-1705543","title":"Effectiveness of bisphosphonates on nonvertebral and hip fractures in the first year of therapy: The risedronate and alendronate (REAL) cohort study\n","text":"Introduction Randomized clinical trials have shown that risedronate and alendronate reduce fractures among women with osteoporosis. The aim of this observational study was to observe, in clinical practice, the incidence of hip and nonvertebral fractures among women in the year following initiation of once-a-week dosing of either risedronate or alendronate.\nIntroduction\nOsteoporosis, a common skeletal disease in older populations, leads to more than a million fractures annually in the United States [1]. Nonvertebral fractures represent 75% of osteoporotic fractures seen in clinical practice [2]. The incidence of nonvertebral fractures, especially at the hip, increases rapidly with age [3]. In order to prevent these fractures, US clinical guidelines recommend that candidates for osteoporosis therapy be identified by screening the bone mineral density of all woman ages 65 and over (age 60 for high risk populations) [4].\nOral bisphosphonates are currently the most common therapy for osteoporosis [5]. While the three most utilized bisphosphonates (alendronate, risedronate, ibandronate) approved by the Food and Drug Administration have been shown to reduce vertebral fractures in randomized clinical trials, it is unknown if these three are equally effective in reducing nonvertebral fractures of real-world patients in clinical practice. A comparison of the results from the randomized clinical trials of each bisphosphonate, though limited by methodological differences between trials, suggests potential differences in degree of fracture reduction across bisphosphonates. In the primary analyses of the trials that followed patients for at least 3\u00a0years, risedronate significantly reduced the incidence of nonvertebral fractures by up to 39% [6, 7]; alendronate reduced the incidence of nonvertebral fractures by up to 21% [8\u201310]; ibandronate did not reduce nonvertebral fractures [11]. Post-hoc analyses of these trial data suggest that there are differences in the onset of fracture reduction. In those analyses, reduction of nonvertebral fractures began at 6\u00a0months for 5\u00a0mg daily dosing of risedronate [12] and at either 12\u00a0months (when fractures were recorded as adverse events) for 10\u00a0mg daily dosing of alendronate [13] or 24\u00a0months for 5\u00a0mg daily dosing of alendronate [14]. These possible differences in both the amount and the onset of fracture reduction between the bisphosphonates could arise from their differences in structure, potency, and binding properties [15].\nThe only direct comparison of bisphosphonates in a randomized clinical trial is based on surrogate endpoints (e.g., changes in bone mineral density and markers of bone turnover) [16]. However, the association between changes in these surrogates and subsequent fracture reduction is not consistent across studies [17, 18]. Unlike randomized clinical trials based on surrogate endpoints, observational studies of large populations provide the opportunity to use major disease endpoints (e.g., hip fracture) as the outcome of interest. The limitation of observational studies can be misleading results from bias arising from non-randomized treatment groups. This bias can be accounted for in part by statistically adjusting for known risk differences between groups. Furthermore, when different therapies are available to be prescribed for the same indication, there is at least some expectation of similarity in prognostic factors between treatment groups occurring naturally [19]. For example, observational studies have compared both the many antihypertensive drug therapies and many statins for reducing the disease endpoint of myocardial infarctions [20, 21].\nSince the once-a-week dosing regimens of both risedronate and alendronate have been available in the US since 2002, there is now an opportunity to observe their effect on reducing fractures in a large population of patients seen in clinical practice. Hence, we conducted an observational study across multiple US health plans to observe the incidence of hip and nonvertebral fractures among women ages 65 and over following initiation of therapy with once-a-week dosing of either risedronate or alendronate.\nMethods\nThe RisedronatE and ALendronate (REAL) cohort study was a retrospective observation of bisphosphonate patients within healthcare utilization records in the United States. The analysis plan was based upon an earlier report [22]. All authors had access to the data. For assurance of reproducibility [23], the analyses were independently replicated by the respective organizations of the authors. The reporting of this study is consistent with the STROBE guidelines [24].\nData source\nThe data source was commercially available datasets of healthcare utilization from the 1 health plan within Ingenix Lab\/Rx (Eden Prairie, MN; data through June 2004) and the 100 health plans of employers within MedStat Marketscan (Ann Arbor, MI; data through September 2004). These datasets contain a longitudinal history of patient-specific data including demographic information (sex, age, dates of dataset inclusion), clinical encounters (inpatient and outpatient services by associated procedures and diagnoses specified by the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM)), and outpatient pharmaceutical dispensations (retail and mail order specified by the national drug code (NDC)). To maximize sample size, the two datasets were combined for all analyses. At the time of data extraction for the current study, the combined datasets contained 12 million persons across 34 states in the US.\nStudy population\nWithin the data source, candidates for study inclusion were all women aged 65 and older with any use of once-a-week dosing of risedronate (35\u00a0mg; Actonel, P&G Pharmaceuticals) or once-a-week dosing of alendronate (35 or 70\u00a0mg; Fosamax, MSD) after July 2002, a date corresponding with contemporaneous commercial availability of once-a-week versions of both therapies (Fig.\u00a01). Patient exclusion criteria and rationale were: 1) less than 6\u00a0months (182\u00a0days) of health plan enrollment before their first bisphosphonate use after July 2002, a history period to measure baseline fracture risk; 2) less than 3\u00a0months (92\u00a0days) of health plan enrollment after their first bisphosphonate use, a minimum observation period with expectation of any fracture reduction (based on post-hoc analyses of clinical trials [12, 14]); 3) any bisphosphonate use (daily, weekly, or Paget's doses) during their 6\u00a0month history period, to include only patients who are new users of bisphosphonates; 4) diagnosis of malignant neoplasm, ICD-9-CM 140 - 208; or Paget's disease, ICD-9-CM 731.0 during either the 6\u00a0month history period or first 3\u00a0months of observation, to exclude patients with fracture risk not related to osteoporosis; and 5) discontinued therapy within the first 3\u00a0months, a minimum period of therapy adherence.\nFig.\u00a01Identification of the study population\nLength of observation\nObservation for a subject was censored at one of following end points, whichever occurred first: date of fracture, 12\u00a0months after date of first bisphosphonate prescription, end date of health plan enrollment, date of switch between bisphosphonate therapies or end date of therapy adherence. Adherence was measured as a function of the gaps between refills, which provides the best available measure within datasets of medical claims [25]. Once the gap between the completion of a 30-day supply and the start of a new prescription exceeded 15\u00a0days, the end date of therapy adherence was the prescription date before gap plus 45\u00a0days. For a 90-day supply, a gap of 45\u00a0days was allowed between completion and a new prescription, the end date of therapy adherence was the prescription date before gap plus 135\u00a0days. For the last prescription, the end date of therapy adherence was the date of last prescription plus 45\u00a0days for a 30-day supply and date of last prescription plus 135\u00a0days for a 90-day supply.\nFracture outcomes\nFrom the data source, two outcomes were identified: subjects with nonvertebral fractures collectively (hip, wrist, humerus, clavicle, pelvis, leg - sites previously specified [12]) and subjects with a hip fracture. Vertebral fractures were not included because the majority of them do not come to clinical attention and thus not systematically captured in the data source. In an attempt to identify incident fractures more likely related to osteoporosis, we used a series of exclusion criteria (Table\u00a01). A total of 109 hip and 507 nonvertebral fracture patients were available for analyses. \nTable\u00a01Identification of fractures outcomes in the study population (n\u2009=\u200933,830)\u00a0Hip fractureaNonvertebral fracturebSubjects with a medical claim for fracture during the observation period after initial bisphosphonate.135923Exclusion of medical claim if a fracture at the same site both before and after start of bisphosphonate therapy; in order to increase the likelihood of including only new fractures.\u221216\u2212368Exclusion of medical claim if a fracture at an unspecified sitec before the start of bisphosphonate therapy; in order to increase the likelihood of including only new fractures.\u22122\u22126Exclusion of medical claim if an open fractured; in order to decrease the likelihood of including traumatic fractures. \u22123\u221211Exclusion of medical claim if documented cause (E-codes) of injury is other than an accidental falle; in order to decrease the likelihood of including traumatic fractures.\u22125\u221231Subjects with a fracture outcome109507aInpatient ICD-9-CM codes (820.x, 733.14)bIn addition to inpatient hip fractures, inpatient and outpatient ICD-9-CM codes for fracture of the wrist (813.x, 733.12), humerus (812.x, 733.11), clavicle (810.x), pelvis (808.x), and leg (821.x, 823.x, 733.15, 733.16)cICD-9-CM code (733.10 or 733.19)dICD-9-CM code that is not 733.1x or where the 4th digit is not\u2009=\u2009.1, .3, .5, .9eICD-9-CM code with \u201cE\u201d classification (E880 - E888) for accidental falls\nStatistical analyses\nTo assess the comparability of baseline characteristics (see Table\u00a02 for specific definitions of demographic characteristics and health history) between the risedronate and alendronate cohorts, the chi-square test was used for dichotomous variables and the Wilcoxon rank sum test was used for continuous variables. \nTable\u00a02Comparison of baseline characteristics between cohorts in study\u00a0CohortsCharacteristicRisedronateAlendronatep-valueNumber of women subjects12,21521,615Duration of observation period Days (mean)226238< 0.001Age at study entryYears & months (mean)74 & 1074 & 7< 0.001 Ages 65 \u2013 74 (%)53.552.4 Ages 75 \u2013 84 (%)36.836.7 Ages 85 and over (%)9.711.0Medications \u2013 6\u00a0month historya Concomitant medications (mean)b4.03.6< 0.001 Gastrointestinal medication use (%)c26.220.1< 0.001 Estrogen use (%)d17.216.50.08 Other non-estrogen anti-osteoporotic use (%)e15.611.0< 0.001 Glucocorticosteroid use (%)f10.38.5< 0.001Medical encounters \u2013 6\u00a0month historya Office visits (mean)5.65.1< 0.001 Hospitalization (%)8.28.20.87 Osteoporosis diagnosis (%)g37.733.8< 0.001 Osteopenia diagnosis (%)h12.510.5< 0.001 Bone densitometry procedure (%)i47.441.5< 0.001 Gastrointestinal diagnosis (%)j15.412.3< 0.001 Rheumatoid arthritis diagnosis (%)k2.72.30.01aSix months before and including date of starting first bisphosphonate prescriptionbBased on number of therapeutic classes with a prescription [34]cBased on NDC codes for at least one prescription for either: H2 antagonists (ranitidine, cimetidine, famotidine, nizatidine); Proton pump inhibitors (omeprazole, esomeprazole, lansoprazole, pantoprazole); Cytoprotectives (misoprostol, sucralfate) [35]dBased on NDC codes for at least one prescription of estradiol, conjugated estrogen, esterified estrogen, or estropipateeBased on NDC codes for at least one prescription of calcitonin or raloxifenef Based on NDC codes for at least one prescription for triamcinolone, prednisone, prednisolone, methylprednisolone, dexamethasone, budesonide, betamethasone, cortisone, or hydrocortisonegICD-9 733.0xhICD-9 733.90 and no record of 733.0xiCPT 76070, 76075, 76076, 78350, 78351, ICD-9 88.98jMultiple ICD-9 codes [36]kICD-9 714.0\nFor the primary analysis, the main outcome measures were the 6 and 12\u00a0month incidence of nonvertebral fractures and hip fractures. Cox proportional hazard modeling (PROC PHREG, SAS Institute, Cary, NC) was used to compare the incidence of fractures between risedronate and alendronate cohorts, adjusting for potential differences in measurable risk factors for fractures. A parsimonious model for each outcome was developed to enhance precision of the parameter estimates and interpretation of results. The selection of variables to be included in the model was based on forward selection. These models were checked against models based on backward selection. The appropriateness of the proportional hazard assumption was assessed by graphical and numerical methods (ASSESS statement, SAS Institute).\nIn order to evaluate if the results of the primary analysis were dependent on methodology, sensitivity analysis were used to compare the incidence of fractures between risedronate and alendronate cohorts. These methods included: (1) an intent-to-treat analysis that observed all subjects for 12\u00a0months regardless of therapy adherence; (2) a proportional hazard model using the propensity score to adjust for differences in baseline fracture risk between cohorts; (3) use of different inclusion criteria for the study population; (4) use of different inclusion criteria for the study outcomes (see Fig.\u00a05 for specifics).\nResults\nThe risedronate cohort included 12,215 subjects on once-a-week dosing of 35\u00a0mg followed for a mean of 226\u00a0days on therapy. 37% of this cohort was censored before 12\u00a0months because of the end date of available data and 41% was censored for an end in therapy adherence. The alendronate cohort included 21,615 subjects on once-a-week dosing of 35\u00a0mg (8%) or 70\u00a0mg (92%) followed for a mean of 238\u00a0days on therapy. 33% of this cohort was censored before 12\u00a0months because of the end date of available data and 41% was censored for an end in therapy adherence. Upon start of bisphosphonate therapy, the two cohorts were different in several baseline characteristics (Table\u00a02). Statistically, the risedronate cohort was older, had more concomitant medications, had more use of glucocorticoids, and had more patients with rheumatoid arthritis than the alendronate cohort - characteristics that may increase fracture risk. Conversely, the risedronate cohort also had greater past use of calcitonin or raloxifene - a characteristic that may decrease fracture risk. Within the 12\u00a0months prior to the initiation of bisphosphonate therapy, similar percentages of the two cohorts had a diagnosis for a nonvertebral fracture and a clinical vertebral fracture, while a statistically larger percentage of the risedronate cohort had a diagnosis for a hip fracture than the alendronate cohort (Fig.\u00a02).\nFig.\u00a02Percent of patients with a clinical diagnosis of fracture before initiation of bisphosphonate therapy. 1ICD-9 codes 808.x, 810.x, 812.x, 813.x, 820.x, 821.x, 823.x, 733.10, -.12, -.14, -.19; 2ICD-9 codes 820.x, 733.14; 3ICD-9 codes 805.x, 806.x, 733.13; 4Subset (81%) of study population with available 12 month history. *Statistical difference (p<0.05) between cohorts\nDuring the 12\u00a0months of observation after the start of bisphosphonate therapy, 507 subjects had nonvertebral fractures. The site of nonvertebral fracture was wrist (30%), hip (21%), leg (17%), pelvis (15%), humerus (14%), and clavicle (3%). For the 109 women hospitalized with a hip fracture, the skeletal sites were intertrochanteric (46%), transcervical (28%), unspecified (20%), and trochanteric or subtrochanteric (6%).\nFor the primary analysis of nonvertebral fractures, the fracture incidence was similar between the risedronate and alendronate cohorts over the first 3\u00a0months of therapy (Fig.\u00a03). After 6\u00a0months of therapy, the risedronate cohort had a 19% lower (95% CI 0% \u2013 35%, p-value\u2009=\u20090.05) incidence of nonvertebral fracture than the alendronate cohort. After 12\u00a0months of therapy, the risedronate cohort had an 18% lower (95% CI 2% \u2013 32%, p-value\u2009=\u20090.03) incidence of nonvertebral fracture than the alendronate cohort (Table\u00a03).\nFig.\u00a03Cumulative incidence of nonvertebral fractures in patients treated with alendronate or risedronate for up to 1\u00a0yearTable\u00a03Cumulative incidence of fractures during therapyFracture typeCohort sizeNumber of women with a fracturePercent of women with a fractureaCrude rate ratiobAdjusted rate ratiob95% CIp-valueTime on therapy\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Cohort\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Nonvertebral6\u00a0Months\u00a0Alendronate21,6152531.31\u2013\u2013\u2013\u2013\u00a0Risedronate12,2151231.140.870.810.65\u20131.000.0512\u00a0Months\u00a0Alendronate21,6153432.30\u2013\u2013\u2013\u2013\u00a0Risedronate12,2151641.990.880.820.68\u20130.980.03Hip6\u00a0Months\u00a0Alendronate21,615540.29\u2013\u2013\u2013\u2013\u00a0Risedronate12,215190.170.630.540.32\u20130.910.0212\u00a0Months\u00a0Alendronate21,615800.58\u2013\u2013\u2013\u2013\u00a0Risedronate12,215290.370.680.570.37\u20130.870.01CI\u2009=\u2009confidence interval of adjusted rate ratioaProportion is based on Kaplan-Meier estimate of the survival function.bBased on Cox regression model.cBased on Cox regression model. Variables selected by forward stepwise selection where criteria for selection p\u2009<\u20090.1. Model for nonvertebral fractures included age, estrogen use, number of medications, rheumatoid arthritis diagnosis, and history of nonvertebral fractures. Model for hip fractures included age, estrogen use, number of medications, and history of hospitalization.\nFor the primary analysis of hip fractures, the fracture incidence was similar between the risedronate and alendronate cohorts over the first 3\u00a0months of therapy (Fig.\u00a04). After 6\u00a0months of therapy, the risedronate cohort had a 46% lower (95% CI 9% \u2013 68%, p-value\u2009=\u20090.02) incidence of hip fracture than the alendronate cohort. After 12\u00a0months of therapy, the risedronate cohort had a 43% lower (95% CI 13% \u2013 63%, p-value\u2009=\u20090.01) incidence of hip fracture than the alendronate cohort (Table\u00a03).\nFig.\u00a04Cumulative incidence of hip fractures in patients treated with alendronate or risedronate for up to 1\u00a0year\nThese differences between the risedronate and alendronate cohorts in the incidence of fractures were consistent across other methods of analysis (Fig.\u00a05). Depending on the method, the risedronate cohort had an estimated 6% to 23% lower incidence of nonvertebral fractures and an estimated 30% to 50% lower incidence of hip fractures than the alendronate cohort through 12\u00a0months of therapy. All of these other estimates were within the confidence intervals of the primary analyses.\nFig.\u00a05Sensitivity analysis: Rate ratio for fracture in the first year of therapy between patients on risedronate and patients on alendronate; results of the primary analysis and 4 other methods of analyses\nDiscussion\nIn this observational study across multiple US health plans, we observed that patients on once-a-week dosing of risedronate had a lower incidence of hip and nonvertebral fractures than patients on once-a-week dosing of alendronate. Differences in fracture incidence between these two cohorts of patients were observed at 6 and 12\u00a0months after initiating therapy.\nAs with all observational studies, systematic errors (e.g., selection bias, measurement misclassification) may be the basis for the observed results [26]. In this study, systematic errors may arise from differences in fracture risk between the 2 cohorts of patients at initiation of therapy. Between the two cohorts, there were statistical differences in measurable fracture risk characteristics (Table\u00a02), there are likely to be differences in known fracture risk characteristics not available within medical claims data (e.g., bone mineral density, family history, smoking history), and there are likely to be differences in unknown fracture risk characteristics (i.e., those that are controlled through randomized trials). The differences in measurable fracture risk characteristics, for which a greater percentage of the risedronate cohort has risk factors for fracture than the alendronate cohort suggesting bias towards higher fracture rates in the risedronate cohort, are inconsistent with observed results. The near unity in fracture incidence between the two cohorts during the first 3\u00a0months of therapy (Figs.\u00a03 and 4) - a period for which there is also unity in fracture incidence between bisphosphonate therapy and placebo in clinical trials [12, 14] - suggest that both cohorts had similar risk for fracture at initiation of therapy. However, differences in fracture risk at initiation of therapy between the two cohorts cannot be excluded.\nWithin healthcare utilization data, which are collected for purposes other than research, misclassification of fracture events and of therapy use are inevitable. As a check on the data, the rate of fracture events and therapy use of these health utilization data are in agreement with other data sources. In the current study, the annual fracture rates following initiation of therapy (\u22482.0% for nonvertebral fractures and \u22480.5% for hip fractures) are consistent with the annual rates in the treated population of clinical trials (between 2.0 and 2.3% for nonvertebral fractures and between 0.4% and 0.7% for hip fractures [6\u201310, 27]). In this study, risedronate patients constituted 25% [45,360\/(45,360\u2009+\u2009137,412)] of the bisphosphonate users in the utilization data compared to 24% [13.6 million \/ (13.6 million\u2009+\u200944.1 million)] of all bisphosphonate prescriptions in the US during the same period [28]. A good method for evaluating misclassification within healthcare utilization data is through a medical chart review. In a prior study, the proportion of fracture claims confirmed by chart review to be a fracture was highest for the hip relative to other fracture sites [29]. Since the effect of misclassification at these other fracture sites is likely to be no different between cohorts (i.e., misclassified exposure does not depend on cohort status), the study results for nonvertebral outcomes are likely more attenuated by misclassification than results for the hip [30].\nThe strength of observational studies can be the generalizability of results. In contrast, the generalizability of results from randomized trials to a real world setting can be limited by differences between the two in relation to expertise of health care provider, quality of medical care, course of therapy, and types of patients [31]. For example, it has been observed that the majority of patients considered candidates for osteoporosis therapy by their physician would not meet the eligibility criteria for inclusion in the randomized trials [32]. Since the population within the current observational study is drawn from multiple health plans in many US states and consists of subjects with a mixture of health characteristics (e.g., prior gastrointestinal comorbidities), the results are likely to be generalizable. Furthermore, the length of observation of therapy adherence (\u2248232\u00a0days) in the current study was consistent to the previously reported average duration of adherence to bisphosphonate therapy (245\u00a0days) [33].\nIn conclusion, within this observational study of clinical practice, a cohort of patients receiving risedronate had lower rates of hip and nonvertebral fractures during their first year of therapy than a cohort of patients receiving alendronate. These results do not appear to be explained by baseline differences in fracture risk between cohorts. In addition, the observed rates of fracture were consistent with the fracture rates in clinical trials. Thus it appears, patients receiving risedronate are better protected from hip and nonvertebral fractures during their first year of therapy than patients receiving alendronate.","keyphrases":["bisphosphonates","hip fractures","osteoporosis","nonvertebral fractures","epidemiology"],"prmu":["P","P","P","P","U"]}
{"id":"Apoptosis-4-1-2423419","title":"Identification and characterization of the human inhibitor of caspase-activated DNase gene promoter\n","text":"DNA fragmentation factor is a heterodimer complex of the nuclease CAD and its specific inhibitor ICAD, which can be activated during apoptosis to induce DNA fragmentation. Although ICAD expression levels have been quantified in a variety of human cancer cells, the mechanism of ICAD gene regulation remains unknown. In this study, we identified a 106-bp TATA-less region upstream of the transcription start site as a basal promoter of the human ICAD gene. An E-Box motif, which binds transcription factors of the basic helix-loop-helix\/leucine zipper family, is responsible for transcriptional activity, as demonstrated using mutated promoter-reporters. A chromatin immunoprecipitation assay further demonstrated that Myc binds to an endogenous ICAD promoter. The functional importance of Myc in the regulation of ICAD transcription was also demonstrated by knock-down of c-Myc and N-Myc gene expression, as well as their ectopic expression. Structural analysis of the human ICAD promoter and identification of factors which regulate its activity might further our understanding of the biological role of ICAD with respect to regulation of apoptosis and cancer development.\nIntroduction\nApoptosis or programmed cell death ensures the elimination of unwanted cells during normal development and homeostasis [1]. This process is progressively inactivated during malignant development and loss of the capacity for apoptosis is a hallmark of malignant cells. Chromatin condensation and internucleasomal DNA fragmentation are typical nuclear features and well-recognized events in apoptosis. DNA fragmentation factor (DFF) is a heterodimer protein composed of a 40-kDa caspase-activated DNase (CAD) otherwise known as DFF40, and its cognate 45-kDa inhibitor (inhibitor of CAD: ICAD or DFF45 [2\u20138]). Both human genes map to 1p36 [9, 10]. CAD is thought to be responsible for the majority of nuclear activity resulting in chromosomal DNA fragmentation. When apoptosis is activated, ICAD is cleaved by executor caspases, mainly caspase-3, into three fragments, after which it dissociates from CAD, resulting in CAD activation.\nThus, the DFF complex may play a role in malignant transformation, and up- or down-regulation of ICAD\/CAD expression might correlate with cancer aggression. Expression levels of ICAD have been examined in a variety of human cancers. For example, ICAD expression is down-regulated during the exponential growth phase of human colon carcinoma cells [11]. In some neuroblastomas, preferential ICAD expression is observed in low-stage, but not in their high grade [12]. Other research suggests that down-regulation of ICAD may contribute to tumor growth and lymph node metastasis in esophageal carcinoma [13], and that ICAD expression might serve as a marker of aggressive tumor behavior with an associated poor prognosis in ovarian cancer [14]. We have previously demonstrated that the hepatitis C virus core protein, which not only encodes the viral nucleocapsid but has a number of properties enabling persistent viral infection, induces gene expression of ICAD, thereby increasing steady-state levels of the ICAD protein [15]. To date, the mechanism of transcriptional regulation of the ICAD gene is not well understood. Analysis of the gene structure of mouse ICAD showed that a 118-bp flanking region of the ICAD gene is required for promoter activity [16]. In this study, we identified a functional promoter of the human ICAD gene and investigated the role of c-Myc and N-Myc in regulation of the ICAD promoter.\nMaterials and methods\nCell cultures\nHuman hepatoblastoma cells (Huh-7), human oral squamous carcinoma cells (HSC-2, HSC-3 and Ca22-9), mouse neuroblastoma cells (IMR-32 and GOTO) and mouse 3T3 cells were maintained in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) supplemented with 10% fetal bovine serum, 100\u00a0units\/ml of penicillin, and 100\u00a0mg\/ml of streptomycin and kept at 37\u00b0C in a 5% CO2 incubator.\nCloning of the 5\u2032-upstream region of the human ICAD gene\nHuman genomic DNA was isolated from Huh-7 cell lines using the QIAamp DNA Mini kit according to the manufacturer\u2019s instructions (QIAGEN). A 5\u2032-upstream region of the ICAD gene was amplified by PCR using primers based on the genomic DNA sequence of the human ICAD gene (GenBank accession No. NM_004401). Primers containing EcoRI sites were used as follows: forward (5\u2032-GAATTCAGGCTGGTCTCAAACTACTG-3\u2032) and reverse (5\u2032-GAATTCGATCTCGCCAGATTCTGGTA-3\u2032). The PCR reaction was carried out at 94\u00b0C for 1\u00a0min, 55\u00b0C for 1.5\u00a0min, and 72\u00b0C for 2\u00a0min, for total of 35 cycles. Each PCR product was purified and subcloned into a pGEM-T Easy vector (Promega), followed by sequencing using an ABI PRISM 310 automated DNA sequencer (Applied Biosystems).\n5\u2032 rapid amplification of cDNA ends (5\u2032-RACE) assay\nThe 5\u2032 ends of the ICAD transcript were cloned by 5\u2032-RACE using a Gene Racer RACE-Ready cDNA kit in accordance with the manufacturer\u2019s instructions (Invitrogen). Double-stranded cDNA molecules prepared from human liver poly(A)+ RNAs ligated by exposure to the adaptor were amplified in a primary PCR reaction using the adaptor primer 1 and the ICAD gene-specific antisense primer ICA1AS (5\u2032-GTGCTGTTCGCGGCTGTAGTT-3\u2032, nt \u2212147 to \u2212173), followed by a secondary PCR reaction using nested oligonucleotides, the adaptor primer 2, and the antisense ICAD specific primer ICA2AS (5\u2032-CACGGTGACTGGTGTCAGGGACTTATC-3\u2032, nt \u2212228 to \u2212254). PCR products were purified and cloned, followed by nucleotide sequencing as described above.\nPlasmid constructions\nThe 1-kb sequence containing the human ICAD promoter was excised by digestion of the above-mentioned pGEM-T vector with SacI and HindIII, and then cloned into the firefly-luciferase-expressing reporter plasmid pGL3-Basic (Promega), resulting in pLUC1005. A series of constructs with 5\u2032 end-deletions of the ICAD promoter were created by PCR amplification using the reverse primer (5\u2032-CAAGCTTGCCTCCACAAGGTGGGACCTG-3\u2032) and the following forward primers: 5\u2032-GGCTAGCCAGTACCCATTTCTGAAGAAG-3\u2032 (nt \u2212936 to +71), 5\u2032-GGCTAGCCCTCATTTGGGTCCATTTTCC-3\u2032 (nt \u2212622 to +71), 5\u2032-GGCTAGCCAGCTTTTTCAGACAGAATGG-3\u2032 (nt \u2212272 to +71), 5\u2032-GGCTAGCCAGCTTTTTCAGACAGAATGG-3\u2032 (nt \u2212205 to +71), 5\u2032-GGCTAGCCAGCTTTTTCAGACAGAATGG-3\u2032 (nt \u2212145 to +71), 5\u2032-GGCTAGCCAGCTTTTTCAGACAGAATGG-3\u2032 (nt \u2212106 to +71), 5\u2032-GGCTAGCCTATTTAGTTTGGTTAGTAAT-3\u2032 (nt \u221290 to +71), and 5\u2032-GGCTAGCCCAGATGGTAAATATACACAA-3\u2032 (nt \u221243 to +71). Each SacI\/HindIII fragment was inserted into the pGL3-basic vector to yield pLUC(\u2212936\/+71), pLUC(\u2212622\/+71), pLUC(\u2212272\/+71), pLUC(\u2212205\/+71), pLUC(\u2212145\/+71), pLUC(\u2212106\/+71), pLUC(\u221290\/+71) and pLUC(\u221243\/+71).\nTransfection and reporter assay\nHuh-7 cells were seeded at 5\u00a0\u00d7\u00a0104 cells\/well in 24-well plates and maintained at 37\u00b0C in a 5% CO2\/95% atmosphere. DNA transfection of cells with each ICAD-promoter-luciferase construct (1\u00a0\u03bcg) with an internal control vector pRL-TK (0.1\u00a0\u03bcg) (Promega) was performed with Trans IT LT-1 (Mirus) during 6-h of incubation. Cells were then rinsed with phosphate-buffered saline (PBS) 48\u00a0h after transfection, and luciferase activity was measured in the cell lysate using dual luciferase assay reagents (Promega) [17]. Firefly luciferase activity was standardized according to Renilla luciferase activity.\nChromatin immunoprecipitation\nChromatin immunoprecipitation assays were performed using the ChIP assay kit (Upstate). Briefly, cells in 100-mm dishes were grown to 70% confluency over 48\u00a0h. The chromatin from formaldehyde-fixed cells was sonicated and immunoprecipitated using mouse monoclonal anti-c-Myc or anti-N-Myc antibodies (Santa Cruz). The chromatin immunoprecipitate was analysed by PCR with the following primer pairs: F1 (5\u2032-CGAGCTCGGTATACATGCGTGTGCATCG-3\u2032) and R1 (5\u2032-CAAGCTTGCCTCCACAAGGTGGGACCTG-3\u2032) for amplifying the region from nt \u2212272 to \u221271 containing potential Myc-binding sites; and F2 (5\u2032-GAGATCAAAACTGCAGTGAG-3\u2032) and R2 (5\u2032-CACTGTTGGAGATTGTTCAG-3\u2032) for amplifying the region from nt \u2212789 to \u2212451 that does not contain Myc-binding sites.\nWestern blotting\nCells were washed with PBS and lysed in SDS sample buffer. Cell lysate samples were separated by 10% SDS-polyacrylamide gel electrophoresis and electrotransferred to a polyvinylidene difluoride membrane (Immobilion; Millipore). After blocking in nonfat milk solution (Blocking One; Nakaraitesk), the membranes were probed with monoclonal antibody against the ICAD protein (Santa Cruz), c-Myc (Sigma), or N-Myc (Santa Cruz), as the primary antibody for 1\u00a0h. After being washed, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse immunoglobulin as the secondary antibody, followed by visualization with SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology).\nsiRNA experiments\nsiRNAs for human c-Myc, N-Myc and the negative control were purchased from B-Bridge International. Each siRNA consisted of three different target sequences as follows: 5\u2032-GAGGAGACAUGGUGAACCA-3\u2032, 5\u2032-GAGAAUGUCAAGAGGCGAA-3\u2032, and 5\u2032-GAGAAUGUCAAGAGGCGAA-3\u2032 for c-Myc siRNA; 5\u2032-CGGAGAUGCUGCUUGAGAA-3\u2032, 5\u2032-CGGAGUUGGUAAAGAAUGA-3\u2032, and 5\u2032-CAGCAGUUGCUAAAGAAAA-3\u2032 for N-Myc siRNA; and 5\u2032-ATCCGCGCGATAGTACGTA-3\u2032, 5\u2032-TTACGCGTAGCGTAATACG-3\u2032, and 5\u2032-TATTCGCGCGTATAGCGGT-3\u2032 for the negative control. Huh-7 cells were transfected with 100\u00a0pmol\/ml siRNA using FuGENE6 (Roche) in Opti-MEM I (Invitrogen), after which the medium was replaced by standard DMEM after 6\u00a0h of transfection.\nRNA preparation and real-time quantitative RT-PCR\nTotal RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer\u2019s protocol. First-strand cDNAs were synthesized using the SuperScriptIII First-Strand Synthesis kit (Invitrogen) and then used as templates for real-time PCR. Quantitative PCR was performed using the ABI Prism 7700 sequence detection system using Taq-Man Gene Expression assays (Applied Biosystems). The standard curve was created using serially diluted total RNA obtained from Huh-7 cultures and \u03b2-actin was chosen as the internal standard to control for variability in amplification. Amplification was performed at 95\u00b0C for 10\u00a0min, followed by 40 cycles of amplification at 95\u00b0C for 15\u00a0s and 60\u00b0C for 60\u00a0s.\nResults and discussion\nLocalization of transcription start sites of the human ICAD gene\nPrecise localization of transcription start sites of the ICAD gene was examined in human hepatoma Huh-7 cells by 5\u2032-RACE analysis. A fragment of approximately 100\u00a0bp was obtained (Fig.\u00a01a) and cloned, after which sequencing analysis of several cDNA clones identified two of 5\u2032 termini (Fig.\u00a01b). These were located 71 and 68 nucleotides upstream from the translation start codon of the ICAD gene. From a database search of the DBTSS [18], we found that these are two of multiple sites for putative transcription initiation for the human ICAD gene. Although transcription may be initiated at both sites, the upper site was designated position +1 in this study.\nFig.\u00a01(a) Mapping of the transcription start sites by 5\u2032 RACE analysis. Following nested PCR reaction, a PCR product of 100-bp was detected by 1.5% agarose gel electrophoresis (lane 2). Lane 1: no template control; lane M: 100-bp DNA ladder marker. (b) The 5\u2032 flanking region of the human ICAD gene. The nucleotide sequence is numbered from the major transcription site, which is indicated by an arrow. Putative binding sites for transcription factors analyzed by the TRANSFAC database and search program were shown with underlines\nCharacterization of the ICAD gene promoter\nTo examine potential regulatory sequences involved in ICAD gene expression, a 1\u00a0kb 5\u2032-upstream region of the ICAD gene was sequenced and analyzed. The overall GC content of the 1-kb genomic DNA fragment was 46%, while GC content among the 200\u00a0bp proximal to the ATG start codon was approximately 62%. A search of transcription consensus motifs using the TRANSFAC database [19] demonstrated that none of the major eukaryotic promoter elements, TATA-, CCAAT-, or GC box, were located within 200\u00a0bp upstream of the transcription start site, although a TATA-like element was found more than 400\u00a0bp upstream from the start sites, indicating that the human ICAD gene has a TATA-less promoter. No CpG island, which is frequently observed in the TATA-less promoter, was found by screening with the CpG island searcher (http:\/\/cpgislands.usc.edu\/). Absence of a TATA-like sequence upstream of the transcription start site has also been reported for the mouse ICAD gene, suggesting that TATA-less promoters might be a common feature in transcriptional regulation of the ICAD gene.\nAs shown in Fig.\u00a01b, the 1-kb sequence contains potential binding sites for several transcription factors. The ability of the 5\u2032-upstream region of the ICAD gene to function as a promoter was assessed by its capacity to drive the expression of a luciferase reporter gene. A series of constructs in which genomic DNA fragments were fused to a promoterless firefly luciferase gene of the pGL3-basic vector were generated with the 3\u2032 end always terminating +71\u00a0bp from the transcription start site. The 5\u2032 ends began at bases \u2212936, \u2212662, \u2212272, \u2212205, \u2212145, \u2212106, \u221290, and \u221243 (Fig.\u00a02). Promoter activity was assessed by measuring luciferase activity in transiently transfected Huh-7 cells. Luciferase activity was normalized to Renilla luciferase activity. As shown in Fig.\u00a02 on the right, the region extending from \u2212936 to +71 had promoter activity since luciferase activity of the reporter gene was 45-fold that of the empty vector. A 5\u2032 deletion of the promoter sequence to \u2212662(pLuc(\u2212662\/+71)) enhanced promoter activity, and successive removal of nucleotides from \u2212272 to \u2212106 also enhanced promoter activity, suggesting the possibility of negative regulatory elements within the \u2212936\/\u2212622 and \u2212272\/\u2212106 regions. When the sequence from \u2212106 to \u221290 was removed (pLuc(\u221290\/+71)), promoter activity fell to parallel that of the pGL3 basic vector. This suggests the presence of a critical element in the region extending from \u2212106 to \u221290, thus, we have identified the region 106\u00a0bp upstream of the transcription start site as the basal promoter of the human ICAD gene.\nFig.\u00a02Human ICAD promoter drives reporter gene expression in transiently transfected Huh-7 cells. Deletion constructs of the upstream region of the ICAD gene linked to the firefly luciferase reporter gene (LUC) are illustrated in the left. Huh-7 cells were cotransfected with a firefly luciferase reporter and a Renilla luciferase internal control (pRL-TK). The relative luciferase activity (RLU) was obtained by normalizing the firefly luciferase activity to the Renilla luciferase activity, and is expressed as a percentage of RLU of pLuc(\u2212936\/+71). The average values are shown with standard deviation (SD) (n\u00a0=\u00a04\u00a0per construct)\nMyc and USF enhance ICAD promoter activity\nAs indicated in Fig.\u00a01b, a computer-based sequence analysis revealed that the 106\u00a0bp region contains potential binding sites for GATA-1, deltaE, Myc, and various upstream stimulatory factors (USF). Within this region, there is a canonical E-box sequence (5\u2032-CACGTG-3\u2032) located between \u2212103 and \u221298. E box motifs are known to bind to transcription factors of the basic helix-loop-helix\/leucine zipper (bHLH LZ) family, including Myc and USF [20\u201323]. We examined the potential role of E-box-binding proteins, Myc and USF, in potentiating ICAD gene transcription. Myc is bound to a partner protein Max via a bHLH LZ domain and the Myc-Max heterodimer activates transcription by binding to an E-box sequence [24, 25]. Max is present in greater amounts than Myc since the Myc transcript and protein have shorter half-lives compared to Max [26, 27]. Therefore, it is highly likely that Myc is the limiting, regulated component of the heterodimer. To examine the potential role of E box binding proteins, Huh-7 cells were co-transfected with luciferase reporter plasmids and expression vectors for c-Myc, N-Myc, USF1, and USF2 (Fig.\u00a03a). Luciferase activity of the \u2212145\/+71 promoter construct increased 2- to 5-fold, and 1.5- to 2-fold, when co-expressed with Myc or USF, respectively. The effect of c-Myc and N-Myc on activity of the \u221290\/+71 construct was less than for the \u2212145\/+71 construct. USF expression did not enhance activity of the \u221290\/+71 construct.\nFig.\u00a03(a) ICAD promoter activity after transient expression of c-Myc, N-Myc, USF1 and USF2. Huh-7 cells were cotransfected with either expression vector for c-Myc, N-Myc, USF1 or USF2 driven by the CMV promoter and pLuc(\u2212145\/+71), pLuc(\u221290\/+71) or pGL3 basic. Each firefly luciferase reporter plasmid (n\u00a0=\u00a04) was cotransfected in cells with pRL-TK for normalization of the reporter activity. RLU is expressed as a percentage of that of pLuc(\u2212145\/+71) in the absence of expressing plasmids for Myc and USF. (b) Effect of substitution mutation in the E-box element on the ICAD promoter activity. Cells were cotransfected with pLuc(\u2212106\/\u00b171) or an E-box-mutant, pLuc(\u2212106\/\u00b171)mt and either expression vector for c-Myc or N-Myc. pRL-TK was also used for normalization of the activity. RLU is expressed as a percentage of that of pLuc(\u2212106\/\u00b171) without over-expression of Myc\nTo further investigate involvement of the E box sequence in ICAD promoter activity, a reporter construct with an E box mutation (CCCGCG) was constructed and luciferase activity was examined in transfected cells (Fig.\u00a03b). The E box mutation resulted in marked down-regulation of reporter gene expression and the reporter activity expressed from the E-box mutant was little increased when co-expressed with Myc, suggesting a functionally important role of the \u2212106\/\u221290 region E box motif with regard to basal transcriptional activation of the ICAD promoter. These data indicates that E-box-binding proteins, especially Myc, actively participate in positive regulation of the human ICAD promoter.\nMyc binds to the ICAD promoter in vivo\nTo obtain direct evidence of an interaction between Myc and the ICAD promoter in vivo, we next examined binding of c-Myc and N-Myc to the 5\u2032-upstream region of the ICAD gene within the context of native chromatin in living cells by chromatin immunoprecipitation. An alignment of the sequence around putative Myc binding site of human ICAD promoter and the corresponding part of mouse ICAD sequence demonstrated conservation of Myc binding sequence in human and mouse (Fig.\u00a04a) [16]. Proteins were cross-linked to genomic DNA isolated not only from human (Huh-7) but mouse (IMR-32 and GOTO) cells, followed by immunoprecipitation with normal rabbit IgG or polyclonal antibodies to either c-Myc or N-Myc. IMR-32 and GOTO are neuroblastoma cells with N-Myc gene amplification [28]. The precipitated DNA was then subjected to PCR utilizing primers designed to amplify a 343-bp fragment (\u2212272\/+71) or a 338\u00a0bp fragment (\u2212789\/\u2212451) of the ICAD 5\u2032 flanking region. As shown in Fig.\u00a04b, the 343-bp DNA fragment was observed in the immunoprecipitate from Huh-7 cells following exposure to anti-c-Myc or anti-N-Myc, while amplification of the ICAD promoter fragment was not detected in the negative control immunoprecipitate. Under similar experimental conditions, we did not detect binding of c-Myc and N-Myc to the \u2212789\/\u2212451 region, which is not thought to contain a Myc-binding site. Similar results were obtained from IMR-32 and GOTO cells, which were immunoprecipitated with anti-N-Myc antibody. These results demonstrate that Myc forms a complex with the human ICAD promoter in cells, presumably through binding to the E-box sequence.\nFig.\u00a04(a) An alignment of the sequence around putative Myc binding site of human ICAD promoter and the corresponding part of mouse ICAD sequence. (b) Binding of Myc proteins in the endogenous ICAD promoter. Crosslinked chromatins isolated from Huh-7, IMR-32 and GOTO cells were immunoprecipitated with indicated antibodies (c-Myc, N-Myc) or an equivalent amount of mouse IgG. Recovered DNAs were purified and PCR-amplified with primers for nt \u2212272\/\u00b171 region or for nt \u2212789\/\u2212451 region. Input represents 1% of chromatin sample applied for immunoprecipitation\nMyc-dependent expression of the ICAD protein\nThe above data suggests that the ICAD promoter is regulated by endogenous Myc proteins. This assumption is supported by the loss-of-function studies shown in Fig.\u00a05a. We employed siRNA (small interference RNA) transfection to knock-down the expression of c-Myc or N-Myc in cells, after which we analyzed the effect of reduced Myc expression on steady state levels of the ICAD protein by Western blotting. Treatment with siRNA specific for either c-Myc or N-Myc markedly reduced ICAD expression without affecting the expression of \u03b2-actin. In contrast, control siRNA did not reduce ICAD levels. We further investigated the effect of over-expression of Myc on ICAD protein levels (Fig.\u00a05b). Transfection of Huh-7 and mouse 3T3 cells with a c-Myc or N-Myc expression vector enhanced ICAD protein levels with an effect that was less pronounced than in the loss-of-function studies, but reproducible nonetheless. These results suggest an important role of Myc proteins in positive regulation of ICAD expression.\nFig.\u00a05Myc-dependent expression of human ICAD. (a) Suppression of Myc expression decreases expression of ICAD protein. Huh-7 cells were transiently transfected with Myc siRNA (c-Myc, N-Myc) or the control siRNA (cont). Three days later, the cells were harvested and subjected to Western blotting. (b) Ectopic expression of Myc increases expression of ICAD protein. Cells (Huh-7 and mouse 3T3) were transfected with the expression vector for c-Myc or N-Myc, and after 3\u00a0days the cells were harvested for Western blotting. (c) Comparison of the mRNA expression of c-Myc and ICAD in Huh-7 cells and human oral squamous carcinoma cells (Ca22-9, HSC-2, and HSC-3). Levels of mRNA expression of ICAD and c-Myc were measured by quantitative real-time RT-PCR based on Taq Man chemistry. Results, relative to \u03b2-actin mRNA, are depicted as averages with SD (n\u00a0=\u00a03)\nFinally, we examined if there might be a correlation between ICAD and Myc expression levels in cancer cell lines. The mRNA expression of ICAD and c-Myc in human oral squamous cell carcinomas (Ca22-9, HSC-2, and HSC-3), in which ICAD expression has not been investigated to date, as well as in Huh-7 cells during their late exponential phase of growth, was analyzed by quantitative RT-PCR (Fig.\u00a05c). ICAD mRNA was readily detected in all cancer cells but the expression level was not consistent. A relatively high level of expression was observed in Ca22-9 and HSC-3 cells, while HSC-2 cells demonstrated the lowest. Interestingly, expression pattern of c-Myc mRNA in these cell lines showed a tendency similar to that of ICAD, suggesting an important role of Myc in ICAD expression in human cancers. A large scale analysis for investigating correlation between ICAD and Myc expression in a variety of cancer tissues obtained from patients is ongoing.\nSeveral studies have shown up- or down-regulation of ICAD in a variety of human cancers [11\u201314, 29, 30]. Although transcriptional deregulation is presumably involved in this aberrant expression of ICAD, little is known about the transcriptional regulation of ICAD in human cells. This work was done to characterize the human ICAD promoter and to examine transcription factors which may be involved in its regulation. We experimentally determined putative transcription start sites by employing the 5\u2032-RACE method, and identified a promoter region required for basal ICAD gene expression using promoter-reporter constructs with progressive deletions. A combined study of site-directed mutagenesis of a reporter construct and chromatin immunoprecipitation revealed the importance of an E box Myc-binding motif located at position \u2212103 to \u221298 from the transcription start sites, as well as an in vivo interaction between c-Myc and N-Myc and the proximal ICAD promoter. Furthermore, we demonstrated the functional importance of Myc proteins with regard to transcriptional regulation of the ICAD gene from studies examining ectopic expression of c-Myc and N-Myc, as well as with RNAi technology. We showed that expression levels of ICAD and Myc correlate in some tumors.\nc-Myc and N-Myc are transcription factors of the bHLH LZ family that bind to the E box sequence within promoters to control proliferation, cell differentiation, and apoptosis [25, 31, 32]. c-myc and N-myc genes are deregulated in a number of human cancers and influence proliferation and growth. A link between Myc and cancer is well established both in vivo and in vitro, and oncogenic activation of Myc has been observed to promote the development of a number of clinically significant cancers [25, 33]. However, the molecular and cellular mechanisms of Myc-mediated transformation are not fully understood. Although a variety of Myc target genes were identified and recruitment of Myc to the target promoters such as prothymosin and telomerase were shown [34], there has been no direct evidence on involvement of Myc in regulation of ICAD expression. It is known that c-Myc activation usually occurs during the later stages of carcinoma in humans. Conversely, in premalignant cells, c-Myc is a robust stimulator of apoptosis and programmed cell death [32]. One area of investigation into cancer cell death mechanisms centers on the mechanism by which c-Myc stimulates or suppresses apoptosis. For instance, Myc has been reported to potentiate apoptosis through both p53-dependent and -independent mechanisms [35, 36]. Myc controls the balance between pro- and anti-apoptotic factors at the level of the mitochondria, thereby regulating cytochrome C release and activation of downstream caspases [37, 38]. Involvement of Myc in up-regulation of ICAD expression as demonstrated in this study might present a novel mechanism of Myc-dependent inhibition of apoptosis. It is possible that elevated levels of ICAD in cells inhibit activation of endonuclear activity, thereby increasing the threshold for apoptosis signaling.\nKawane et\u00a0al. have reported on the structure and analyzed the promoter of a murine ICAD gene, by which they demonstrated that a 118-bp flanking region of the ICAD gene is required for its transcription [16]. The mouse sequence shares approximately 82% homology with a corresponding upstream region of the human ICAD gene. The mouse ICAD promoter has a number of potential binding sites for transcription factors, such as Ikaros, c-Rel, Myc, and Gfi-1 [16]. Conservation of the Myc-binding motif among human and murine promoters suggests a functional significance of Myc in transcriptional regulation of ICAD expression.\nIn conclusion, this is the first report to identify a functional promoter of the human ICAD gene, and to demonstrate that Myc proteins are able to positively regulate ICAD gene expression. Extensive apoptosis research to date has shown that tumor aggression depends on various defects in apoptosis signaling [39]. Further investigation into the molecular events linking Myc expression with ICAD gene regulation may provide insight into Myc\u2019s role in cell proliferation, transformation and apoptosis.","keyphrases":["promoter","icad","myc","gene expression","dff"],"prmu":["P","P","P","P","P"]}
{"id":"J_Mol_Biol-2-1-2279226","title":"Histone Modifications Influence the Action of Snf2 Family Remodelling Enzymes by Different Mechanisms\n","text":"Alteration of chromatin structure by chromatin modifying and remodelling activities is a key stage in the regulation of many nuclear processes. These activities are frequently interlinked, and many chromatin remodelling enzymes contain motifs that recognise modified histones. Here we adopt a peptide ligation strategy to generate specifically modified chromatin templates and used these to study the interaction of the Chd1, Isw2 and RSC remodelling complexes with differentially acetylated nucleosomes. Specific patterns of histone acetylation are found to alter the rate of chromatin remodelling in different ways. For example, histone H3 lysine 14 acetylation acts to increase recruitment of the RSC complex to nucleosomes. However, histone H4 tetra-acetylation alters the spectrum of remodelled products generated by increasing octamer transfer in trans. In contrast, histone H4 tetra-acetylation was also found to reduce the activity of the Chd1 and Isw2 remodelling enzymes by reducing catalytic turnover without affecting recruitment. These observations illustrate a range of different means by which modifications to histones can influence the action of remodelling enzymes.\nIntroduction\nPackaging DNA into nucleosomes and higher-order chromatin structures restricts access to the underlying genetic material. Thus, the manipulation of chromatin is a key step in many nuclear processes such as transcription, replication and repair. One of the means by which eukaryotes achieve this is through the use of ATP-dependent chromatin remodelling enzymes that non-covalently alter its structure. These enzymes are members of a diverse group of proteins named after the archetypal Saccharomyces cerevisiae Snf2 protein; the Snf2 family. Multiple members of this family of proteins are present in the sequenced genomes of eukaryotes of which the chromatin remodelling enzymes form distinct sub-groupings.1 Recent crystal structures of the catalytic domain of two Snf2 related proteins2,3 highlight structural similarities with the RecA domain found in a broad range of helicase related proteins. Snf2 proteins use the energy from ATP hydrolysis to alter DNA\u2013protein interactions. However, unlike bona fide helicases, the action of chromatin remodelling enzymes is not generally associated with separation of DNA strands. Instead they act to catalyse dynamic transitions in chromatin structure. This can result in a variety of different outcomes. In vitro, chromatin remodelling enzymes have been shown to cause alterations to DNA accessibility, effects on DNA and chromatin topology, partial or complete removal or exchange of histones from nucleosomes and translational changes in the position of the intact histone octamer along DNA.4\nHow these proteins interact with chromatin and the mechanism by which chromatin remodelling is achieved remains unclear. However, in many cases it is apparent that histone tail domains are important regulators of chromatin remodelling. Genetically, there are links between histone tails and remodelling. Deletion of the H2B tail bypasses the need for the SWI\/SNF complex in the regulation of several genes,5 and deletion of the H2A tail affects transcription of some SWI\/SNF dependent genes.6 Importantly, post-translational modifications of histone tails affect chromatin remodelling. The recruitment and activity of the SWI\/SNF complex in vivo is widely linked to that of the SAGA histone acetyltransferase complex.7\u20139 In addition, many chromatin remodelling complexes contain domains shown to bind modified amino acids, such as bromo- and chromodomains, PHD and WD40 domains.10 In these cases modifications have been proposed to act as epitopes that facilitate recruitment of proteins containing the appropriate recognition domains. For example, histone acetylation has been found to facilitate retention of SWI\/SNF on nucleosome arrays11 in a way that is dependent on bromodomains within the Snf2 protein.12\nThe majority of the best characterised modifications occur within the non-globular histone tail domains which protrude from the core of the nucleosome. These histone tails constitute up to 30% by mass of histones, but are not visible in the crystal structures of nucleosomes due to their high intrinsic flexibility and have been thought to be largely unstructured.13 Their importance is highlighted by the fact that deletion of histone tails has wide spread effects on chromatin structure and gene regulation.14\u201316 Histone modifications have been correlated with a variety of chromatin states. On a genome-wide basis, histone H3 K4 tri-methylation and H3 acetylation are strongly correlated with active transcriptional start sites, phosphorylated H2A.X foci mark sites of DNA damage, and H3 K9 tri-methylation promotes the formation of heterochromatin via interaction with HP1.10,17 This suggests that histone post-translational modifications are an important means by which cells spatially and temporally regulate specific loci independently of bulk chromatin.\nRigorously studying the effect of histone tail modifications in vitro has been hampered by the difficulty in isolating histones with defined and homogeneous modifications. To circumvent this we have adopted an approach to chemically synthesise modified histones by means of native chemical ligation. This technology has previously been applied to generating chromatin with uniform histone N-terminal tail modification patterns.18\u201320 Using this approach we show that histone modifications can affect nucleosome remodelling via distinct pathways. Tetra-acetylation of histone H3 results in a modest increase in the rate of intrinsic nucleosome repositioning. The ATP-dependent remodelling enzyme RSC shows a striking preference for H3 but not H4 tetra-acetylated chromatin, remodelling this 16-fold faster than the control. By measuring the kinetic parameters of the remodelling reaction with a real-time ATPase assay, we show that this is due to a lower Km value for H3 acetylated nucleosomes. In contrast, tetra-acetylation of histone H4 affects nucleosome remodelling by the Isw2 enzyme via altering the Kcat rather than the Km of the ATPase reaction. We also show that the yeast Chd1 enzyme is dependent on the H4 tail for efficient nucleosome remodelling in a manner similar to Isw2. Remarkably, histone H4 tetra-acetylation affects the rate of RSC catalysed nucleosome transfer in trans but not nucleosome sliding in cis. Thus, nucleosome modifications can affect enzyme catalysed remodelling reactions by altering the outcome of the reaction, through allosteric effects on ATPase activity and by acting as binding epitopes for recruitment.\nResults\nGeneration of modified chromatin\nModified histones were generated by ligating a bacterially expressed Xenopus laevis globular histone containing an N-terminal cysteine and a tail peptide with the modification of choice synthesised chemically as a C-terminal thioester. The ligation was based on the protocol described by Dawson & Kent21,22 and used thiophenol as the catalyst (Figure 1(a)). A key requirement of the globular histone is that it has an uncapped N-terminal cysteine available for ligation. One strategy that has been used previously to ensure that the initiator methionine is removed is the introduction of a protease cleavage site just before the cysteine to expose it post-translationally.20 Mass spectrometry (Supplementary Data, Figure 1) and the work of other laboratories19,23 show that removal of the initiator methionine in vivo by Escherichia coli methionine aminopeptidases is efficient when cysteine is the adjacent amino acid. This alleviates the requirement for more complex systems for generation of N-terminal cysteine residues.\nPolyacrylamide gel electrophoresis reveals that under the conditions used, ligation does not proceed to completion (Figure 1(b), lane 1). However, the full-length modified histone can be separated from the unreacted globular domain and tail peptide via multiple rounds of ion exchange chromatography to obtain material that was greater than 95% pure (Figure 1(b)). The overall efficiency of ligation and subsequent purification is approximately 20% (data not shown). When modified histones were combined with other core histones to form histone octamers and purified by size exclusion chromatography, they displayed identical elution profiles to unmodified octamers (data not shown). Modified histone octamers could be reconstituted onto DNA fragments to form nucleosomes. Interestingly, acetylation of H3 and to a lesser extent H4 reduced the migration of nucleosomes on native polyacrylamide gels (Figure 1(c)). This has been observed previously in the Bradbury laboratory using hyperacetylated histones purified from HeLa cells24 and suggests that linker DNA conformation may be affected. This altered electrophoretic migration is not due to the ligation procedure as nucleosomes containing methylated lysine residues did not show this behaviour (not shown).\nLysine acetylation can alter intrinsic nucleosome mobility\nAlthough nucleosomes are stable with respect to dissociation, they can undergo a range of dynamic fluctuations in their structure. For example, following thermal incubation nucleosomes are frequently observed to redistribute to thermodynamically favoured locations.25 This movement of nucleosomes from one translational position on DNA to another can be followed using native PAGE.26 Acetylated and control nucleosomes were reconstituted onto differentially labelled fluorescent DNA, mixed, incubated thermally and run on a native gel (Figure 2(a)). The different nucleosomes can then be visualised separately using selective excitation and emission filters. From this the proportion of nucleosomes at the initial and final position can be measured at each time point. The rate at which nucleosomes accumulate at their destination can then be plotted and a hyperbolic curve fitted to the data enabling the initial rate of redistribution to be estimated (Figure 2(b)\u2013(e)). This approach has proved more reliable at directly comparing the behaviour of different nucleosomes.27\nAlthough native thiol ligation results in a normal peptide bond without the introduction of unusual chemical moieties, it requires the presence of a cysteine residue at the point of ligation. For this reason, the unmodified control nucleosomes used as a reference are point mutants containing a cysteine at the equivalent position to the modified histones. The sites selected for ligation involved substitution of cysteine for amino acids with similar overall dimensions (e.g serine to cysteine). None the less it was important to show that introduction of these mutations did not affect the behaviour of nucleosomes. Supplementary Data, Figure 2 shows a comparison of the thermal mobility of wild-type and cystein point mutant nucleosomes showing that the cystein mutations themselves have no discernable effect. The effects of the cysteine point mutations were also compared to wild-type histone sequences in other assays described below and in all cases no significant effect was detected (Supplementary Data, Figures 2, 3, 4; and data not shown). As a histone bearing the relevant cysteine point mutation is most closely matched to the product of a peptide ligation reaction, we have generally presented data using the point mutant as a control rather than the wild-type histone sequence. However, we are not aware of any cases where the choice of control affects the conclusions drawn.\nH4 acetylated nucleosomes were observed to be repositioned at the same rate as control nucleosomes (Figure 2(b)). In contrast, nucleosomes containing acetylated H3 relocated twice as fast as the control (Figure 2(c) and (e)). To test to what extent this was due to the partial neutralisation of basic charge that occurs upon acetylation of lysine residues, a histone H3 construct with lysine to alanine mutations at the four acetylated positions (H3 K9, 14, 18, 23A) was compared to wild-type (Figure 2(d) and (e)). This nucleosome repositioned at the same rate as the control indicating that charge neutralisation is not the sole mechanism by which acetylation affects nucleosome mobility.\nThe RSC complex preferentially repositions tetra-acetylated H3 but not H4 nucleosomes\nRSC is an abundant and essential yeast chromatin remodelling complex that is closely related to SWI\/SNF.28 A notable feature of the RSC complex is that it contains almost half of the known bromodomains in the S. cerevisiae genome. As bromodomains within other proteins have been found to recognise specific patterns of histone acetylation,29,30 a distinct possibility is that histone acetylation will influence the action of the RSC complex. To test this, the ability of RSC to remodel either H3 or H4 acetylated chromatin relative to unmodified control chromatin was assayed. RSC showed a dramatic preference for H3 (K9, 14, 18, 23) tetra-acetylated chromatin compared to an unmodified control (Figure 3(a)). From the initial rates of remodelling derived from the plotted data, this was calculated to be 16(\u00b11.5)-fold faster than control (Figure 3(d)). When the rate of H4 (K5, 8, 12, 16) tetra-acetylated nucleosomes was measured, this was found to be indistinguishable from the rate of an unmodified control (Figure 3(b) and (d)).\nIt is also worth noting that although the rate at which the H3S28C control nucleosomes are shifted in Figure 3(a) is slower than the rate at which the H4V21C nucleosomes are shifted in Figure 3(b), this should not be interpreted as indicating that the S28C mutation reduces the rate at which nucleosomes are repositioned by RSC. The reason that S28C nucleosomes are moved slower is due to the preferential engagement of RSC with acetylated nucleosomes at the expense of non-acetylated nucleosomes in the reaction mixture, as they are essentially in competition with each other. Indeed, comparing the rate of acetylated nucleosomes to either wild-type or cysteine containing control nucleosomes shows the same effect (Supplementary Data, Figure 3) This illustrates the importance of only making direct comparisons between the two templates present in the same remodelling assay. In this case, our data show that H3 acetylation stimulates nucleosome slding whereas H4 acetylation does not. Thus, none of the seven bromodomains found within the RSC complex appear to interact with acetylated lysines within the H4 tail in a way that affects nucleosome sliding. This is consistent with the observation that the relative rate of remodelling of a H3 and H4 acetylated nucleosome is not faster than a nucleosome that is just acetylated on the H3 tail (Figure 3(c) and (d)).\nH3 tetra-acetylation influences remodelling by the RSC complex by affecting Km but not Kcat of ATP hydrolysis and acts largely via H3 K14 acetylation\nThe increased rate with which RSC repositions H3 acetylated nucleosome could be due to the modified lysine residues acting to recruit RSC or by allosterically affecting the remodelling reaction. To differentiate between these two options, the kinetic parameters of the ATP hydrolysis reaction were measured using a real-time fluorescent ATPase assay.31 The assay hinges on using a phosphate binding protein (PBP) labelled with a coumarin-based fluorescent dye, 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC), as a sensor for the amount of inorganic phosphate (Pi). On binding Pi, the labelled protein (MDCC-PBP) undergoes a shift in its emission wavelength coupled with a fivefold increase in fluorescence. When performed in a fluorimeter, this assay has the advantage of measuring ATP hydrolysis in real-time, from which kinetic parameters such as Km and Vmax are determined by non-linear fitting to the Michaelis\u2013Menton equation (Figure 4(a)\u2013(d)). We find that RSC has approximately threefold lower Km (tighter binding) for H3 acetylated nucleosome compared to the unmodified control, without affecting the Kcat of ATP hydrolysis (Figure 4(e)). This is consistent with preferential recruitment of RSC to H3 acetylated chromatin and the preferential binding of RSC to H3 acetylated nucleosomes (Supplementary Data, Figure 5). In contrast, RSC does not preferentially bind H4 acetylated nucleosomes, as the Km for these is the same as for the unmodified control (Figure 4(e)). Consistent with this, the Km value of the doubly H3, H4 acetylated nucleosome is indistinguishable from that of the H3 acetylated nucleosome (Figure 4(e)).\nPrevious work from the Cairns laboratory has suggested the tandem bromodomains of the RSC4p subunit of the RSC complex interact with acetylated H3 K14.32 To test whether the single acetylation of lysine 14 is responsible for the dramatic effect of H3 (K9,14,18,23) tetra-acetylation on RSC activity, the singly acetylated nucleosome was generated and put through the same ATPase assay described above. From Figure 4(e) it can be seen that as for H3 tetra-acetylated nucleosomes, the Kcat value remains unchanged. In contrast, the Km value was significantly lowered, almost to the levels of the tetra-acetylated construct, suggesting that acetylation at a single residue, K14, confers the majority of the effect observed upon tetra-acetylation of the H3 tail.\nThe H4 tail and its acetylation influence remodelling by Isw2 by affecting the Kcat of ATP hydrolysis\nSubfamilies of Snf2 family chromatin remodelling proteins contain different motifs capable of interacting with histones raising the possibility that they may be regulated in different ways by histone modifications. One of the defining characteristics of the Iswi subfamily of remodelling enzymes is that they require a particular epitope within the H4 tail for efficient remodelling activity.33\u201336 One of the lysine residues adjacent to this motif, H4 K16, can be acetylated, and there is evidence that this reduces the activity of ISWI containing remodelling complexes.19,37 When we compared the relative rate of mobilisation of H4 tetra-acetylated nucleosomes relative to an unmodified control by the yeast Isw2 enzyme, we found that the rate is approximately 1.5-fold slower (Figure 5(b) and (c)). This reduction, whilst significant, is not as severe as deletion of the H4 tail (Figure 5(a) and (c)). However, its biological importance is underlined in studies on flies, where defects in the genetic interaction between H4 K16 acetylation and ISWI result in large scale chromosomal abnormalities.37\nApplying the same kinetic analysis performed previously to RSC shows that deletion or acetylation of the H4 tail lowers the turnover rate of the hydrolysis reaction (Kcat) without affecting nucleosome binding (Km) (Figure 5(d)). We have confirmed this by performing gel shifts of either full length or gH4 nucleosomes with Isw2 and observe no difference in binding (Supplementary Data, Figure 6). This contrasts with the effect of histone acetylation on remodelling by the RSC complex and shows that histone modifications can affect chromatin-remodelling enzymes at different stages of the reaction cycle.\nThe yeast Chd1 enzyme requires the H4 tail for efficient chromatin remodelling\nChd1 belongs to a phylogenetic subfamily that is distinct from either of the previously studied enzymes1 and contains a chromodomain that has the potential to recognise histone modifications. To investigate the histone dependence of this enzyme, we first tested the effect of deleting individual histone tails. We found that Chd1 is unable to efficiently slide nucleosomes lacking the H4 amino terminal tail (approximately sixfold slower than control: Figure 6(a), compare lanes 1\u20136 with 7\u201312). This requirement for the H4 tail is similar to that seen with Iswi remodellers such as Isw2, although the magnitude of the effect is lower. Mutation of residues 16\u201319 to alanine caused a 4.4-fold reduction in Chd1 activity (Figure 6(b)). Although this effect is significant it does not fully match the effect of a H4 truncation raising the possibility that the amino acids recognised by Chd1 include residues in addition to those within the 16\u201319 region.\nAcetylation of residues within the H4 tail also affected Chd1's nucleosome remodelling activity. Indeed, when this was tested, there was a modest 1.4-fold reduction in the initial rate of remodelling of acetylated H4 versus unmodified nucleosomes (Figure 6(c) and (d)). This raised the question of whether, as in the case of Isw2, the H4 tail was an allosteric activator of ATP hydrolysis or whether it was required as a binding epitope to recruit Chd1 to nucleosomes. Comparing the ATP hydrolysis rates of Chd1 with full-length and gH4 nucleosomes revealed that binding (Km) was unaffected (see also Supplementary Data, Figure 6(b)) and it was again the catalytic turnover rate which decreased in the absence of the H4 tail (Figure 6(e)).\nH4 acetylation leads to loss of histones in trans\nStudies with the RSC and SWI\/SNF complexes in vitro have shown that in addition to being able to redistribute nucleosomes along DNA fragments, they can transfer histone octamers from one DNA fragment to another at lower efficiency. This entails the displacement of the histone octamer from one molecule of DNA and subsequent transfer onto a separate one. The SWI\/SNF complex has been implicated in the disassembly and subsequent reassembly of nucleosomes at the Pho5 promoter.38,39 This loss of nucleosomes at the promoter is correlated with spikes in histone acetylation levels, leading to the hypothesis that ATP-dependent remodellers may recognise this signal and promote transfer. To test whether there is a direct link between histone modification and octamer transfer, the efficiency of octamer transfer from different chromatin substrates by the RSC complex was measured.\nThe octamer transfer reaction involves incubating a \u201ccold\u201d donor nucleosome with a \u201chot\u201d 32P-labelled accepter DNA fragment in the presence of RSC and ATP. The reaction products were run on a native polyacrylamide gel and transfer is indicated by a shift in mobility of the \u201chot\u201d accepter DNA. If the accepter DNA has a histone octamer transferred onto it, it should now have the same mobility as a control nucleosome reconstituted onto the same fragment (Figure 7(a)).\nThis was quantified as the fraction of radiolabelled free DNA incorporated into nucleosomes, as measured by mobility on a native polyacrylamide gel (Figure 7(b) and (c)). The amount of octamer transfer catalysed increased over time and was dependent on both ATP (Figure 7(b), lanes 1\u20133) and RSC (data not shown). As expected, octamer transfer from H3 acetylated nucleosomes was greater compared to unmodified nucleosomes (Figure 7(b), compare lanes 1\u20133 and 4\u20136). This is consistent with RSC having a lower Km for H3 acetylated nucleosomes (Figure 4) and the preferential nucleosome sliding seen in Figure 3. Surprisingly, when we looked at H4 acetylated donor nucleosomes, these were transferred considerably more efficiently than unmodified nucleosomes. Indeed, the amount of transfer was almost as high as that of H3 acetylated nucleosomes (Figure 7(c)), despite RSC not showing any increased affinity towards H4 acetylated chromatin (Figure 4). A doubly H3 and H4 acetylated nucleosome was in turn transferred more efficiently than either of the single histone acetylations. This suggests that H4 acetylation does not act at the level of stimulating RSC directly but rather by predisposing the nucleosome to be remodelled in trans, perhaps by destabilising it. Consistent with this, we find that H4 acetylated nucleosomes are more prone to thermally induced histone H2A\/H2B dimer loss than H3 acetylated nucleosomes (Supplementary Data, Figure 7).\nDiscussion\nHistone acetylation alters the intrinsic dynamic properties of nucleosomes\nSince they were discovered in the 1960's histone modifications have been predicted to affect chromatin structure and gene regulation.40 Subsequent studies have revealed correlations between histone modifications and transcription. For example, the recent application of genome-wide microarrays to map histone modifications, reveals that modifications such as H3 acetylation are found at the promoters of active genes and that their levels correlate with that of transcription.41\u201346 There are two prevailing views as to how modifications such as acetylation affect transcription. The first is based on the recruitment of activators due to recognition via modification binding modules. The number of modifications and respective binding domains identified has resulted in the hypothesis that these may form a code, which is read to determine a specific response.47 Alternatively it has been suggested that lysine acetylation may have a structural effect on chromatin structure by neutralising the charge interactions between histone lysine residues and DNA, resulting in a more open conformation.48\u201350 Several lines of evidence suggest that this can destabilise chromatin fibres.19,51\u201353 Histone acetylation may also affect the structure of mononucleosomes. Several studies suggest that DNA within acetylated nucleosomes is more accessible.48,49,54 It has been shown that acetylated histone tails adopt a more \u03b1-helical conformation,55 and that acetylated nucleosomes have a reduced linking number,24 consistent with wrapping less DNA. However, in this view, the role of individual histone modifications has been less clear. Here we show that acetylation of the H3 tail increases the inherent thermal mobility of nucleosomes whereas acetylation of H4 does not (Figure 2(e)). This increase in mobility does not appear to be due to charge neutralisation alone, as mutation of the relevant lysine residues to alanine did not produce the same effect (Figure 2(d)). The mechanisms behind this are not immediately clear; however, it is interesting to note that acetylation of H3 but not H4 increased the distance between the arms of linker DNA within a mononucleosome, as measured by FRET.53 We have also recently observed that individual histone tails have non-redundant effects on nucleosome dynamics,27 suggesting that the relationship between histone modification and chromatin dynamics may be more complex than initially appreciated. It will be interesting to examine how these effects interact with each other to alter chromatin structure and function in vivo.\nHistone acetylation affects ATP-dependent chromatin remodelling through altering either the Km or Kcat of ATP hydrolysis\nAs their names suggest, ATP-dependent remodelling enzymes use the energy of ATP hydrolysis to alter chromatin structure. Based on the helicase domain of the catalytic subunit, chromatin remodelling complexes can be classified into a number of different subfamilies.1 Here we look at examples from three distinct subfamilies of chromatin remodellers: Chd1, Isw2 and RSC. The ATPase activity of these enzyme classes is stimulated by DNA and nucleosomes. Whereas it had been noted that histone tails and modifications affected their remodelling activity, it was not clear in many cases whether this was due to altered binding of modified nucleosomes or due to altered rates of ATP hydrolysis after binding. Using a real-time ATPase assay we show that depending on the particular enzyme involved either may be the case.\nAnalysis of the RSC complex confirmed that the improved remodelling of acetylated nucleosomes is due to higher substrate binding affinity and not due to increased ATP turnover (Figure 4(b)). This is consistent with previous observations of bromodomain containing remodelling enzymes, such as SWI\/SNF, which show improved binding to acetylated chromatin templates.12,56 Remarkably, given the large number of bromodomains, the RSC complex shows no improved binding of H4 tetra-acetylated nucleosomes but rather, specifically H3 acetylated nucleosomes. By using chromatin with defined modification patterns we show that mono-acetylation at lysine 14 results in an increase in binding almost to the level seen with H3 tetra-acetylation. Consistent with this the Rsc4p subunit of RSC interacts genetically with H3 K14 but not H3 K9.32 Studies from the Workman laboratory have shown that SWI\/SNF and RSC have increased affinity for nucleosomes that have been acetylated by the NuA4 HAT complex.12,56 We show here that RSC does not preferentially bind H4 K5,8,12,16 tetra-acetylated nucleosomes (Figure 4(b)); however, these observations can be reconciled by the fact that NuA4 shows HAT activity towards other histones besides H4, most notably H2A,57,58 and it may be this which results in the improved binding. For this reason, it would be interesting to test the affinity of RSC for nucleosomes containing acetylated H2A or H2B. Indeed, in high-resolution microarrays, H2A K7 acetylation but not H2B K16 acetylation is typically found in the same region of genes as H3 acetylation, and both are positively correlated with transcription.46\nIn the case of Isw2, we show that acetylation of H4 reduces nucleosome remodelling by lowering the Kcat of ATP hydrolysis and not by inhibiting nucleosome binding (Figure 5(c)). Thus, the unmodified H4 tail is required as an allosteric activator of ATP hydrolysis. This is consistent with the previous observation that acetylated H4 peptides have a reduced ability to stimulate ATP hydrolysis by dISWI in comparison to unmodified peptides.34 We also find that Chd1 activity is affected by modification and alteration to the H4 tail in a similar way to the Isw2 complex (Figure 6). It is notable that in the case of both Chd1 and Isw2, the effects of tail acetylation or truncation on ATPase activity are smaller than the effects on nucleosome sliding. It is possible that there is some amplification of the effect on ATPase activity due to multiple rounds of remodelling being required for repositioning. Alternatively, alterations to the H4 tail may reduce the efficiency with which these enzymes reposition nucleosomes in addition to altering their ability to hydrolyse ATP.\nPreviously, the requirement for the H4 tail for efficient nucleosome remodelling had only been observed for members of the Iswi subfamily of remodelling enzymes. However, there is evidence that there may be a significant degree of overlap in the function of Chd1 and Iswi subfamilies in S. cerevisiae. Isw1, Isw2 and Chd1 are all involved in transcription termination59 and their combined deletion is synthetic lethal with cellular stress.60 These complexes also reposition nucleosomes onto the same subset of thermodynamically favourable positions in vitro.61 Interestingly, like members of the Iswi subfamily, Chd1 is efficient in the generation of regularly spaced chromatin arrays,62 suggesting that a tightly coupled functional interaction with the H4 tail may be important for this activity. Studies with the similar Drosophila Mi-2 chromatin remodelling complex have shown that while its ATPase activity is stimulated by the assembly of histones into nucleosomes, this does not require histone tails.63 Whereas Mi-2 has similarity to Chd1, it is a member of a phylogenetically distinct group,1 suggesting that of the CHD-like remodellers, dependence on the H4 tail may be restricted to the Chd1 subfamily.\nH4 acetylation promotes octamer transfer in trans by the RSC complex\nA distinct activity of the SWI\/SNF and RSC complexes is their ability to remove nucleosomes and displace histone octamers in trans. The targeted removal of nucleosomes has been shown to be an important phenomenon in nuclear processes such as transcription64\u201366 and DNA repair67,68 and interestingly chromatin remodelling enzymes are involved in both.\nIn testing the role of histone modifications on octamer transfer in trans by RSC, we found that H3 tetra-acetylation resulted in a large increase in transfer (Figure 7(b) and (c)). This effect of H3 acetylation in promoting octamer transfer can be explained through improved recruitment due to the increased affinity for the modified nucleosome. Indeed, octamer transfer by the homologous SWI\/SNF complex has been shown to require its bromodomains to mediate octamer transfer of SAGA acetylated nucleosomes.69 An unexpected finding was that H4 tetra-acetylation also had a stimulatory effect (Figure 7(b) and (c)), in stark contrast to the results seen with nucleosome repositioning in cis (Figure 3(b) and (d)). However, the additional effect of H4 acetylation was confirmed by the fact that a doubly H3 and H4 acetylated nucleosome was transferred better still than a singly H3 acetylated nucleosome. Kinetic analysis of ATPase activity clearly shows that H4 tetra-acetylated nucleosomes are not bound any better than unmodified nucleosomes and they do not alter the rate of ATP hydrolysis by RSC (Figure 4(b)). Thus, the increase in octamer transfer seen with H4 acetylated nucleosomes is not due to the modification recruiting or stimulating RSC activity but rather by affecting the nucleosome in such a way as to make it easier to transfer. In agreement with this, we find that H4 acetylated nucleosomes show increased thermally induced H2A\/H2B dimer loss (Supplementary Data, Figure 7), indicating that this modification can affect chromatin dynamics independently of external factors. Interestingly, previous studies have shown that acetylation reduces the thermal stability of nucleosomes and H4 tetra-acetylation in particular reduces the thermal stability of nucleosomes almost to the level of bulk acetylation.70,71 The finding that histone acetylation improves the efficiency of histone octamer transfer by remodelling enzymes helps to explain discrepancies between previous studies of transfer efficiency. Previous studies that used native histones bearing modifications observed octamer transfer at a higher efficiency than was detected in another study using recombinant unmodified histones.72\nThe idea that acetylation of different histone tails may not be functionally equivalent is not a new one. Genome-wide microarray studies in yeast have shown that not all acetylation is positively correlated with transcription,42,46 and H4 acetylation does not substitute for H3 acetylation at Adr1-dependent genes.73 We propose that histone modifications may provide a means to facilitate histone eviction by ATP-dependent chromatin remodelling enzymes. Thus, acetylation, particularly of H4, may act in parallel with recruitment by activation domains to promote the removal of nucleosomes by remodelling enzymes such as RSC and SWI\/SNF.74 This may also have relevance to processes other than transcription such as DNA repair. Intriguingly, H4 acetylation is very rapidly targeted to these regions75,76 making it tempting to speculate that this modification also acts to promote nucleosome loss during DNA repair.\nExperimental Procedures\nPurification of remodelling enzymes\nTAP (tandem affinity purification) tagged yeast strains were either purchased from Euroscarf (Germany) for CHD1 and ISW2, or in the case of RSC the strain BCY211 was kindly supplied by Brad Cairns. Six litres of yeast were grown at 30\u00a0\u00b0C to an A600 of 2\u20132.5 in 3\u00d7yeast extract, peptone, adenine, d-glucose and frozen by dropwise addition into liquid nitrogen. Yeast cells were lysed by mechanical disruption using a blender (Waring) kept cold by addition of solid CO2. This was then thawed and purified using standard TAP protocols77 except using higher stringency wash buffers (20\u00a0mM Na-Hepes (pH 7.4), 350\u00a0mM NaCl, 10% (v\/v) glycerol, 0.1% (v\/v) Tween-20, 1\u00a0mM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride, 2.6\u00a0mM aprotinin, 2\u00a0\u03bcg\/ml leupeptin, 1\u00a0\u03bcM pepstatin). The purified eluate was concentrated using a Centricon YM-50 concentrator (Millipore) to 200\u2013300\u00a0\u03bcl, dialysed against wash buffer without protease inhibitors and stored as 10\u00a0\u03bcl aliquots at \u221280\u00a0\u00b0C. The purity of Isw2 and Chd1 is indicated by Stockdale et al.,61 the purity of RSC complex is illustrated in Supplementary Data, Figure 8.\nNative peptide ligation\nH3 \u03941-27 S28C (NCBI: CAD89679) and H4 \u03941-20 V21C (NCBI: CAD89677) were generated by sited directed mutagenesis. To ensure that the N-terminal cysteine within the globular histone was available for ligation, up to 10\u00a0mg of lyophilised histone was dissolved in 1\u00a0ml 6\u00a0M guanidine chloride(GnCl) (pH 7), 10\u00a0mM DTT, and incubated at 50\u00a0\u00b0C for 30\u00a0min. This was then dialysed extensively against three changes of 4l of 10\u00a0mM sodium acetate (pH 5.2), 1\u00a0mM EDTA, allowing at least 3\u00a0h per step. The fully reduced histone was then lyophilised and stored. Modified tail peptide thioesters were purchased from CSS-Albachem (Scotland) and aliquots of peptide in use stored at 10\u00a0mM in MilliQ water at \u221280\u00a0\u00b0C. The ligation conditions used were similar to those used by Dawson & Kent.21,22 The 1\u20132\u00a0mM globular histone was dissolved in 6\u00a0M GnCl, 0.2\u00a0M phosphate (pH 7.3) together with 0.4\u20130.5\u00a0mM thioester peptide and 2% (v\/v) thiophenol, typically to a final volume of 200\u2013300\u00a0\u03bcl. This was vortexed, and the reaction left at room temperature for 16\u201324\u00a0h. The reaction was stopped by adding DTT to 100\u00a0mM and dialysing the reactants against three changes of 500\u00a0ml SAUD0 buffer (7\u00a0M urea, 20\u00a0mM sodium acetate (pH 5.2), 5\u00a0mM 2-mercaptoethanol, 1\u00a0mM EDTA pH 8.0, 0\u00a0mM NaCl) using a 12\u201314\u00a0kDa MWCO dialysis membrane. The dialysed ligation mixture in SAUD0 buffer was spun to remove protein precipitate, and the supernatant loaded onto a 1\u00a0ml SOURCE 15S (Pharmacia) ion exchange column running at 1.5\u00a0ml\/min to separate unligated globular histone from the full length product. A stepwise elution using SAUD0 (buffer A) and SAUD2000 (buffer B) (7\u00a0M urea, 20\u00a0mM sodium acetate (pH 5.2), 5\u00a0mM 2-mercaptoethanol, 1\u00a0mM EDTA (pH 8.0), 2\u00a0M NaCl) was used to elute in as small a volume as possible. The H3 \u03941-27 S28C and H4 \u03941-20 V21C constructs elute at a conductivity of 14 milliSiemens (mS), equivalent to approximately 180\u00a0mM NaCl, whereas full length histones elute at less than 25mS, 500\u00a0mM NaCl. This is typically 9.2%, and 25% SAUD2000, but due to variations in buffer preparation, the concentration of the two buffers required to obtain this conductivity should be determined empirically. We found that histones can elute in multiple peaks even though they are identical by SDS\u2013PAGE and mass spectrometry, presumably representing differentially folded sub-species. The globular histone was eluted over 15\u00a0ml and the full-length histone over 10\u00a0ml, before the column was washed in SAUD2000 for 6\u00a0min in-between runs to remove tightly bound proteins and then re-equilibrated in buffer A. The relevant fractions are collected, diluted with SAUD0 to a final salt concentration below 100\u00a0mM NaCl and reloaded onto the column. We found that two to three rounds of purification were required to obtain greater than 95% pure ligated protein as determined by SDS\u2013PAGE (Gradipore, Australia). The final fractions are concentrated using a YM-10 centricon (Millipore) to approximately 200\u00a0\u03bcl and their concentration measured by absorbance at 276\u00a0nm.\nNucleosome reconstitution\nRecombinant Xenopus laevis histone proteins were expressed and purified as described.78 Where necessary site-directed mutagenesis was carried out using the Stratagene Quickchange kit. Nucleosomes were assembled by mixing equimolar amounts of histone octamer and DNA in high salt and performing stepwise dialysis into low salt as described.79 DNA was generated by preparative PCR using primers obtained from Eurogentec (Belgium) fluorescently labelled where appropriate. Nucleosomes were assembled on DNA fragments based on the MMTV nucleosome A positioning sequence80 or the synthetically selected 601 sequence.81 We have adopted a nomenclature in which the lengths of DNA extensions on either side of a nucleosome are indicated as numbers on either side of a letter that defines the core positioning sequence used. So 54A18 designates a nucleosome positioned on the MMTV nuc A positioning sequence with a 54\u00a0bp extension to the upstream side and a 18\u00a0bp extension to the downstream side. The oligos used to generate the 54A18 fragment were 5\u2032TATGTAAATGCTTATGTAAACCA and 5\u2032TACATCTAGAAAAAGGAGC; for the 54A54 fragment 5\u2032TATGTAAATGCTTATGTAAACCA and 5\u2032ATCACATGTGAAAGTTAAAAAA; for the 0W0 fragment 5\u2032CTGCAGAAGCTTGGTCCC and 5\u2032ACAGGATGTATATATCTG; for the 54W0 fragment 5\u2032TATGTCCATGCTCATGCC and 5\u2032ACAGGATGTATATATCTG; for the 36W36 fragment 5\u2032GGCGAATTCGAGCTCGGTAC and 5\u2032AGGTCGACTCTAGAGAATCC. The PCR fragments were purified by ion exchange chromatography on a 1.8\u00a0ml SOURCE 15Q (Pharmacia) column. Radiolabelled DNA was prepared using T4 polynucleotide kinase (New England Biolabs) and [\u03b3-32P]ATP (Molecular Bioproducts) according to the manufacturer's specifications.\nNucleosome remodelling\nThermal remodelling reactions were performed by incubating nucleosomes in a thin-walled 200\u00a0\u03bcl PCR tube (ABgene, UK) in 50\u00a0mM NaCl, 50\u00a0mM Tris (pH 7.5) at 47\u00a0\u00b0C in a PCR machine with a heated lid for the specified amount of time. At the end of the reaction, sucrose was added to 5% (w\/v) and the reactions placed on ice. The samples were separated on 0.2\u00d7 Tris\u2013Borate EDTA, 5% native polyacrylamide gels for 3.5\u00a0h at 300\u00a0V at 4\u00a0\u00b0C with pump recirculation. Gels were scanned in a Fuji Phosphoimager FLA-5100 and the bands quantified with Aida software (Fujifilm). For each time point the fraction of nucleosomes remodelled was calculated as the intensity of the destination position divided by the sum of the start and destination intensities. These data were then corrected to set the zero time point to zero percentage remodelled. The data points were plotted onto a graph and a hyperbolic curve of the form y\u00a0=\u00a0x\/(n+x) fitted non-linearly to the data using the Solver add-in for Excel over 1000 iterative cycles. The initial rate of remodelling was calculated by differentiation of the curve and solving for time equals zero. At this point the initial rates for unmodified and modified nucleosomes within the competition assay were divided by each other to describe the initial rate of remodelling relative to the unmodified control. The average and standard deviations were calculated from at least three independent repeats. ATP-dependent remodelling reactions were performed as for thermal repositioning except using a reaction buffer containing 50\u00a0mM NaCl, 50\u00a0mM Tris (pH 7.5), 3\u00a0mM MgCl2, 1\u00a0mM ATP. These were incubated at 30\u00a0\u00b0C with the amount of remodelling enzyme and for the time specified in the figure legends. The reactions were stopped using 500\u00a0ng of HindIII-digested bacteriophage lambda competitor DNA, adding sucrose to 5% (w\/v) and placed on ice. Nucleosome binding assays were performed in 50\u00a0mM Tris\u2013HCl (pH 7.5), 50\u00a0mM NaCl, 3\u00a0mM MgCl2 and 3% (w\/v) Ficoll-400 unless otherwise stated.\nATPase assay\nThe ATPase assay was performed as described.31 The reaction was measured in solution in real-time on a Cary Eclipse fluorimeter (Varian, Australia). The excitation and emission wavelengths were set to 430\u00a0nm and 465\u00a0nm, respectively, with a 5\u00a0nm slit width and polarisers used to compensate for anisotropy. A calibration curve using MDCC\u2013PBP at 5\u00a0nM and increasing amounts of inorganic phosphate was performed to determine the range across which the detection is linear. MDCC\u2013PBP was diluted using 10\u00a0mM Pipes (pH 7) to 50\u00a0nM, this was mixed with nucleosome and enzyme in a final buffer concentration of 50\u00a0mM NaCl, 50\u00a0mM Tris (pH 7.5), 1\u00a0mM MgCl2 to a final concentration of 5\u00a0nM. Enzyme and nucleosome concentrations are as stated in the Figure legends. Measurements were performed with 1\u00a0mM ATP which had previously been treated to remove inorganic phosphate contamination.31 However, identical results were obtained using 100\u00a0\u03bcM ATP which had not been treated with the phosphate mop. The measurement of the rate of hydrolysis was performed using the Cary Eclipse software and non-linear fitting of the Michaelis\u2013Menton equation to the data done within the Solver add-in for Excel over 1000 iterative cycles.\nOctamer transfer and dimer exchange\nOctamer transfer was performed under the same conditions as standard ATP-dependent remodelling. The 15\u00a0nM donor nucleosome (assembled on unlabelled 0W0 DNA) was incubated with 10\u00a0nM RSC and 1\u00a0nM radiolabelled acceptor 0W0 DNA for the specified amount of time in the presence of 1\u00a0mM ATP. The reactions were stopped by the addition of excess cold DNA and analysed by native PAGE. Thermal dimer exchange was performed as described.72 Briefly, this is performed as for nucleosome repositioning, except with a twofold molar excess of H3\/H4 tetrasomes assembled onto 147\u00a0bp \u2018601\u2019 DNA.81","keyphrases":["histone","snf2","chromatin","acetylation","nucleosome"],"prmu":["P","P","P","P","P"]}
{"id":"Pediatr_Surg_Int-_-Immediate_Access-2292794","title":"Adults with corrected oesophageal atresia: is oesophageal function associated with complaints and\/or quality of life?\n","text":"The aim of this study was to evaluate oesophageal function after correction of oesophageal atresia in adults, and to investigate the association between complaints, oesophageal function and quality of life (QoL). Twenty-five adults were included who participated in previous follow-up studies, during which complaints of dysphagia and gastro-oesophageal reflux (GOR), results of upper gastrointestinal endoscopy, oesophageal biopsies and QoL had been collected. Manometry was performed in 20 patients, 24 h pH-measurements were performed in 21 patients. pH-values (sample time 5 s) were calculated using criteria of Johnson and DeMeester. Associations were tested with ANOVA and \u03c72-tests. Ten patients (48%) reported complaints of dysphagia, seven (33%) of GOR. The amplitude of oesophageal contractions was low (<15 mmHg) in four patients (20%). pH-measurements showed pathological reflux in three patients (14%). Patients reporting dysphagia more often had disturbed motility (P = 0.011), and lower scores on the domains \u201cgeneral health perceptions\u201d (SF-36) (P = 0.026), \u201cstandardised physical component\u201d (SF-36) (P = 0.013), and \u201cphysical well-being\u201d (GIQLI) (0.047). No other associations were found. This study shows a high percentage of oesophageal motility disturbances and a moderate percentage of GOR after correction of oesophageal atresia. Patients reporting dysphagia, whom more often had disturbed motility, seemed to be affected by these symptoms in their QoL.\nIntroduction\nAt present, the survival rate of patients with oesophageal atresia (OA) is approximately 95% [1, 2]. With the decreased mortality, the interest in morbidity, especially the long-term results after correction of OA, has increased over the years.\nSeveral long-term follow-up studies have shown that long lasting gastro-oesophageal reflux (GOR) is a frequent problem after correction of OA, although intestinal metaplasia, as its theoretical consequence, is rare [3\u20136]. In these studies, GOR has been either diagnosed by upper gastrointestinal (GI) endoscopy with biopsies, by 24\u00a0h pH-measurements, or by both, showing varying degrees of GOR. Besides pH-measurements, oesophageal manometry has been performed in several studies, showing oesophageal motility disturbances in most patients [7\u201310]. The true impact of this finding on individual daily life is not clear.\nIn our centre, we have performed several long-term follow-up studies in a relatively large group of patients after correction of OA [5, 6]. Patients underwent upper GI endoscopy with biopsies and quality of life (QoL) measurements [11].\nThe first aim of this study was to evaluate the presence of GOR and oesophageal motility problems in a group of our adult patients treated for OA.\nThe second aim was to investigate if there was an association between complaints, oesophageal function, and QoL-measurements. This association has not been investigated before. Because of the influence of complaints of dysphagia and GOR on daily life, we hypothesised that patients with complaints have a poorer QoL than patients without complaints.\nPatients and methods\nTwenty-five patients over 18\u00a0years of age who participated in previous follow-up studies after correction of OA [5, 6, 11], and gave informed consent to participate, were included in this study. In these previous studies, data regarding the results of upper GI endoscopy, biopsies of the distal oesophagus, and QoL had been collected. From the QoL study, we used the results of the Medical Outcome Study 36-Item Short-Form Health Survey (SF-36) and of the gastro-intestinal quality of life index (GIQLI) [12, 13]. After approval of the study protocol by the Medical Ethical Committee, all patients received a written invitation to participate in the study.\nAll patients who gave their informed consent, underwent manometry and pH-measurements and were asked if they experienced difficulties swallowing solid foods (dysphagia) or experienced heartburn or retrosternal pain (GOR-related complaints). Oesophageal manometry was performed using the UPS-2020 measurement stationary system (MMS, Enschede, The Netherlands) with software version 7.\nThe pressure was measured with the Unisensor Microtip catheter type 8304-00-9980-D with three pressure transducers on a 5\u00a0cm distance from each other. The lower oesophageal sphincter basal or resting pressure (LOSP) and relaxation after swallowing, the motility in the oesophageal body after at least six wet swallows of 5\u00a0ml water, and the upper oesophageal sphincter pressure (UOSP) and relaxation were calculated. The amplitude of oesophageal body contractions was categorized as \u201clow\u201d (<15\u00a0mm Hg), \u201cmoderate\u201d (15\u201335\u00a0mm Hg), or \u201cnormal\u201d (>35\u00a0mm Hg). The encountered oesophageal motility disorders were classified according to the guidelines proposed by Spechler et al. [14]. Based on the basal LOSP, LOS relaxation, peristaltic wave progression, and distal wave amplitude, oesophageal motility disorders were classified into four categories: (1) \u201cinadequate LOS relaxation\u201d, (2) \u201cuncoordinated contraction\u201d, (3) \u201chypercontraction\u201d, and (4) \u201chypocontraction\u201d or \u201cineffective oesophageal motility\u201d.\npH-measurements were performed using the Comfortec dual channel pH probe (Sandhill Scientific), which was positioned with the pH measurement points 5 and 20\u00a0cm above the manometrically established upper border of the LOS. The position of the probe was checked by X-ray. Ambulatory pH measurement was performed during 24\u00a0h using the GORD pH-recorder (Sandhill Scientific) with a sample time of every 5\u00a0s. The pH values were calculated using the criteria of Johnson and DeMeester [15].\nThe data were entered into a database and analysis was performed using SPSS (Statistical Package of the Social Sciences) 10.0.1 for Windows. We tested for association between functional results and QoL by applying ANOVA and \u03c72-tests or Fisher\u2019s exact tests.\nDefinitions\nBefore testing for association, the results of functional tests were dichotomized. If upper GI endoscopy showed a normal oesophagus or grade I oesophagitis (according to the modified system of Savary-Miller) [16], this was scored as \u201cnormal\u201d. Grade II oesophagitis or worse was scored as \u201cabnormal\u201d. If the biopsies of the distal oesophagus showed normal oesophageal epithelium or mild reflux oesophagitis (according to Ismael-Beigi) [17], this was scored as \u201cnormal\u201d. Moderate oesophagitis or worse was scored as \u201cabnormal\u201d. As all patients were diagnosed as having \u201cineffective oesophageal motility\u201d, this variable could not be used. Instead, we used the amplitude of oesophageal body contractions as a measure of outcome of manometry, because decreased amplitude implies a defective peristaltic function of the oesophagus. The results were dichotomized as \u201cnormal\u201d or \u201cabnormal\u201d (moderate or low amplitude). The results of pH-measurements were also dichotomized as \u201cnormal\u201d or \u201cabnormal\u201d (minor or pathological reflux).\nResults\nPatient characteristics\nPatient characteristics are described in Table\u00a01. All patients had undergone a primary end-to-end anastomosis for Gross\u2019 type C OA. Two patients were using proton-pump inhibitors, they stopped taking their medication two days before the start of the study.\nTable\u00a01Patient characteristics of 25 patients participating in the studyMean (range) or n (%)Age (years)28.5 (18\u201342)Gender \u00a0Male16 (64%)\u00a0Female9 (36%)Concomitant congenital anomalies\u00a0None17 (68%)\u00a0Present8 (32%)\u00a0\u00a0Anorectal malformations3 (12%)\u00a0\u00a0Cardiac malformations4 (16%)\u00a0\u00a0Vertebral malformations1 (4%)\u00a0\u00a0Limb malformations2 (8%)\u00a0\u00a0Other malformations2 (8%)Anti-reflux procedure in past\u00a0No23 (92%)\u00a0Yes2 (8%)Anastomotic stenosis in past\u00a0No18 (72%)\u00a0Yes7 (28%)Current educational status\u00a0Primary school1 (4%)\u00a0Basic high school9 (36%)\u00a0Advanced high school7 (28%)\u00a0University5 (20%)\u00a0Unknown3 (12%)\nUnfortunately, it was impossible to perform manometry and pH-measurements in four patients. In one patient it was impossible to introduce the catheter through the nose due to resistance of the patient, who decided to withdraw from the study. In three out of four patients it was impossible to introduce the catheter due to oesophageal stricture. All of these three patients had been treated for anastomotic stricture in childhood, one patient had undergone anti-reflux surgery in childhood. Two of these three patients had complaints of dysphagia at the time of the study. The stricture of these three patients was treated with dilatation. Since dilatations may influence the results of manometry and pH-measurements, no measurements were performed in these patients. Due to a technical failure, the data of the manometry of one patient could not be retrieved.\nDysphagia was reported by 10\/21 patients (48%), GOR-related symptoms were reported by 7\/21 patients (33%).\nManometry\nThe data of 20 patients could be analyzed. The upper oesophageal sphincter (UOS) responded normally to swallowing in all patients. Mean UOS pressure was 30.8\u00a0\u00b1\u00a015.5\u00a0mm Hg.\nOesophageal contractions were observed in all patients. One or more propulsive contractions were observed in 14\/20 patients. All patients showed one or more non-transmitted contractions. Retrograde contractions were observed in 7\/20 patients. Mean minimum oesophageal body amplitude pressure was 20.7\u00a0\u00b1\u00a013.4\u00a0mm Hg. Mean maximum oesophageal body amplitude pressure was 32.0\u00a0\u00b1\u00a015.5\u00a0mm Hg. The amplitude of oesophageal body contractions was low (<15\u00a0mm Hg) in 4 patients (20%), moderate (15\u201335\u00a0mm Hg) in 10 (50%) and normal (>35\u00a0mm Hg) in 6 (30%).\nIn all patients, the LOS showed complete relaxation upon swallowing. Mean LOSP was 13.1\u00a0\u00b1\u00a07.2\u00a0mm Hg.\nAccording to the guidelines of Spechler et al., all patients were classified as having \u201cineffective oesophageal motility\u201d [16]. The LOS pressure was normal and LOS relaxation was complete in all patients. Wave progression varied from normal to absent progression, and the distal wave amplitude was low in \u2265\u00a030% of wet swallows (data not shown).\npH-measurements\nThe data of 21 patients could be analyzed. The results are described in Table\u00a02. pH-measurements showed a normal pattern in 17 patients (81%), minor reflux in 1 (5%), and pathological reflux in 3 (14%). In both of the patients who were taking proton-pump inhibitors, pH-measurements showed pathological reflux.\nTable\u00a02\u00a0Results of 24\u00a0h pH-measurements in 21 patientsProximal mean %\u00a0\u00b1\u00a0SDDistal mean %\u00a0\u00b1\u00a0SDTotal time pH\u00a0<\u00a040.2\u00a0\u00b1\u00a00.41.5\u00a0\u00b1\u00a02.2Upright time pH\u00a0<\u00a040.2\u00a0\u00b1\u00a00.62.2\u00a0\u00b1\u00a03.3Supine time pH\u00a0<\u00a040.0\u00a0\u00b1\u00a00.30.4\u00a0\u00b1\u00a01.0SD standard deviation\nAssociations\nThe association between symptoms and results of endoscopy, oesophageal biopsies, manometry, pH-measurements and QoL is shown in Table\u00a03. Patients reporting dysphagia, more often had disturbed motility (P\u00a0=\u00a00.011). These patients also had significantly lower scores on the domains \u201cgeneral health perceptions\u201d (P\u00a0=\u00a00.026), and \u201cstandardised physical component\u201d (P\u00a0=\u00a00.013) of the SF-36; and on the domain \u201cphysical well-being\u201d (P\u00a0=\u00a00.047) of the GIQLI.\nTable\u00a03Association between symptoms and results of endoscopy, oesophageal biopsies, manometry, pH-measurements, and quality of lifeComplaintsDysphagiaGORYesNoYesNoEndoscopy\u00a0Normal99612\u00a0Abnormal1212Oesophageal biopsies\u00a0Normal6547\u00a0Abnormal3526Manometry\u00a0Normal0*6*15\u00a0Abnormal10*4*68pH-measurements\u00a0Normal107512\u00a0Abnormal0422SF-36 scales\u00a0Physical functioning86.5 (19.2)94.1 (8.3)96.7 (5.2) 88.1 (16.4)\u00a0Physical role-functioning77.8 (38.4)100.0 (0.0)83.3 (40.8)92.9 (20.6)\u00a0Emotional role-functioning77.8 (44.1)96.7 (10.1)83.3 (40.8)90.5 (27.5)\u00a0Bodily pain78.2 (26.3)92.5 (13.9)85.5 (24.5)86.3 (20.5)\u00a0General health63.6 (23.6)**84.6 (15.1)**86.7 (14.8)70.2 (22.7)\u00a0Vitality50.0 (15.4)52.3 (12.9)58.3 (13.3)48.2 (13.2)\u00a0Social functioning88.9 (22.0)93.2 (18.8)97.9 (5.1)88.4 (23.2)\u00a0Mental health82.7 (15.781.5 (16.7)87.3 (12.5)79.7 (17.0)SF-36 component summary scales\u00a0PCS49.5 (7.4)***56.4 (3.4)***55.6 (4.9)52.3 (6.9)\u00a0MCS49.0 (10.9)49.5 (7.4)50.3 (7.9)48.8 (9.5)GIQLI scales\u00a0Physical well-being21.2 (3.1)****24.5 (3.7)****23.3 (3.8)22.9 (3.9)\u00a0GI symptoms64.1 (8.5)68.6 (7.8)68.0 (6.5)66.0 (9.0)\u00a0Social well-being12.0 (1.7)12.2 (0.4)12.0 (0.6)12.1 (1.3)\u00a0Emotional well-being15.1 (1.5)15.9 (0.5)15.5 (1.0)15.6 (1.2)Total score112.4 (13.3)121.3 (9.6)118.8 (10.3)116.6 (12.9)Data are shown as n, or mean (standard deviation)GOR gastro-oesophageal reflux, SF-36 Medical Outcome Study 36-Item Short Form Health Survey, PCS physical component summary, MCS mental component summary, GIQLI gastro-intestinal quality of life index*P\u00a0=\u00a00.011, **P\u00a0=\u00a00.026, ***P\u00a0=\u00a00.013, ****P\u00a0=\u00a00.047\nNo association was found between complaints of GOR and quality of life; nor between the results of endoscopy and the results of pH-measurement and\/or manometry; nor between the results of oesophageal biopsies and the results of pH-measurement and\/or manometry (data not shown). Problems with the initial surgical repair in childhood (i.e. anastomotic leak or stricture needing dilatation) did not influence the findings in the current study.\nDiscussion\nThe results of the present study confirm that oesophageal motility disturbances are frequently present after correction of OA. Low or moderate amplitude of oesophageal body contractions were found in 14\/20 patients (70%), all patients showed one or more non-transmitted contractions, and retrograde contractions were observed in 7\/20 patients (35%). All patients met the manometric features of \u201cineffective oesophageal motility\u201d, as described by Spechler et al. [14]. The manometric findings in the present study are comparable to those described in other studies [7\u201310].\nBased on pH-measurements, the prevalence of GOR in the current patient group is lower than expected. pH-measurements showed minor or pathological reflux in 4\/20 patients (20%). None of these patients had undergone anti-reflux surgery in the past. In other studies, the prevalence of GOR based on pH-measurements varies from 17 to 54% [7\u20139]. Unfortunately, the criteria used for diagnosing GOR and the age of the patient groups studied also vary between studies.\nThe question is, what the influence is of the disturbed oesophageal motility and GOR found in previous studies on the daily life of adults with corrected OA. This is the first study in which complaints and QoL after correction of OA have been combined with long-term studies of oesophageal function: endoscopy, oesophageal biopsies, manometry and pH-measurements. Patients reporting dysphagia more often had disturbed motility, and showed significantly lower scores on the domains \u201cgeneral health perceptions\u201d and \u201cstandardised physical component\u201d of the SF-36, and on the domain \u201cphysical well-being\u201d of the GIQLI. However, GOR-related complaints were not associated with disturbed oesophageal function, and did not influence QoL. It is important to consider that this group of patients has grown up with these symptoms, and may probably have gotten used to it. The fact that complaints of dysphagia affect the QoL and GOR-related complaints do not affect QoL may be explained by the influence of these complaints on daily activities such as eating.\nMotility problems after correction of OA were first reported by Haight [18] in 1957. The main cause of the abnormal oesophageal motility after correction of OA is not clear. Some studies propose a congenital nervous abnormality as the cause of motility disturbances. In the foetal rat model for OA, abnormalities were found in the course and branching pattern of the vagal nerves [19]. However, an acquired cause is also suggested, i.e. surgical damage to vagal fibres that innervate the oesophagus [20]. Abnormal oesophageal motility can cause symptoms of dysphagia. It can also worsen the effects of GOR, since malfunction of the peristaltic pump will result in a delayed clearance with a longer period of stasis of refluxed material in the oesophagus.\nIn conclusion, this study shows a high percentage of oesophageal motility disturbances and a moderate percentage of GOR after correction of OA. Only patients reporting dysphagia, who more often had disturbed motility, appeared to be affected by these symptoms in their QoL.","keyphrases":["oesophageal atresia","quality of life","manometry","ph-measurements","long-term follow-up"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_Arch_Psychiatry_Clin_Neurosci-3-1-1800370","title":"Deficit of social cognition in subjects with surgically treated frontal lobe lesions and in subjects affected by schizophrenia\n","text":"The ability of humans to predict and explain other people\u2019s behaviour by attributing independent mental states such as desires and beliefs to them, is considered to be due to our ability to construct a \u201cTheory of Mind\u201d. Recently, several neuroimaging studies have implicated the medial frontal lobes as playing a critical role in a dedicated \u201cmentalizing\u201d or \u201cTheory of Mind\u201d network in the human brain. In this study we compare the performance of patients with right and left medial prefrontal lobe lesions in theory of mind and in social cognition tasks, with the performance of people with schizophrenia.\nIntroduction\nSocial cognition refers to the ability to perceive, interpret and provide an adequate response to affective and other interpersonal cues [1]. Pioneering studies have found that schizophrenic subjects show social cognitive impairments, in particular in modifying their behaviour when interacting with other people and in recognizing social information cues [2]. People with schizophrenia also show a great deal of social naivety in interpersonal situations [3].\nThe ability to engage in competent social relations and to understand social information depends on the adequate functioning of a mental mechanism termed Theory of Mind (ToM), that allows people to understand and interpret their own and other people\u2019s mental states and hence to predict and explain their behaviour [4, 5]. Evidence from neuroimaging and neuropsychological studies has led researchers to conclude that ToM is subserved by dedicated brain systems, including the amygdala, the temporo-parietal junction, the orbital frontal cortex and, in particular, the medial frontal lobes [6, 7]. The results of these studies have been used to argue that the medial frontal lobes play a critical role in a dedicated mentalizing system [7, 8].\nNeuropsychological literature specifically relevant to the medial frontal cortex is scarce. Actually, only three studies have included patients with relatively focal medial frontal lobe damage and have sought to investigate correlations between specific frontal brain areas and performance in ToM tasks [9\u201311]. However, there are important limitations to these investigations. Stuss et al. [10] reported that patients with right frontal and bilateral frontal lobe damage were impaired in their ability to infer visual experience in others. The authors conclude that acquired brain damage to the medial frontal lobes does impact on ToM ability, but aetiology of brain damage for each patient is not reported, the text implies that most of the bifrontal group had suffered head traumas. It is often very difficult to assess the extent of brain damage reliably after head trauma as widespread damage can often occur through axonal shearing and other effects (e.g. [12]). The second neuropsychological study [9] reports that patients with left and right-sided lesions were equally impaired, while lesion size was unrelated to performance and no effect of lesion location was found when comparing patients with focal dorsolateral, medial or orbital frontal lesions.\nAlso, a recent group study of empathy in patients [11] included an assessment of performance on the Faux Pas Test, a probe of ToM ability. Patients with frontal lobe lesions were impaired in this task. Specifically, patients with ventromedial frontal lobe damage made significantly more errors than patients with posterior lesions or healthy controls in the Faux Pas Test.\nHowever, a detailed analysis of lesion sites associated with ToM deficits in these previous studies revealed a particularly important role for the right ventromedial prefrontal frontal lobe [11, 13]. Damage in these areas induces behavioural changes affecting personality (indifference), impaired social judgement, reduced affect and goal-directed behaviour, self-monitoring deficits [14\u201316].\nMore recent studies have provided evidence that is in disagreement with a single area hypothesis involved in ToM processing. In fact, the neural network involving the right and left temporo-parietal-junction (TPJ R and L); the posterior cingulated (PC), and medial prefrontal cortex (MPFC) seems to be crucial in processing the complex reasoning involved in mentalizing [17, 18]. Bird et al. [19] studied a patient affected by extensive damage of the medial frontal lobes reporting, interestingly, a dysexecutive syndrome with confabulation with preserved performance on some ToM tasks.\nSaxe and Wexler [20] in an fMRI study in normal subjects, described an equally selective profile of activation of the above-mentioned areas in a multicomponent pattern of activation fMRI methodology. Frontal lobe damage in patients has long been linked to impairments in social behaviour [21]; in fact, they have been described as presenting diminished social awareness and a lack of concern for social rules [15, 22].\nAn interesting approach to social competence deficits in schizophrenic people may be represented by the Machiavellian Intelligence Hypothesis (MIH, [23, 24]).\nAccording to this hypothesis, in the development of intelligence, social, rather than technical, efficiency represents the main selective pressure of human evolution [25]. Social efficiency is represented by the ability to understand the intentions and beliefs of others with the aim of deceiving and manipulating them to achieve relevant objectives, such as control of food sources or sexual partners [26]. Recent literature fosters that these abilities are localised in the ventromedial prefrontal cortex: selective damage to these areas causes a relevant impairment of interpersonal relationships and in regulating behaviour according to social rules [27\u201330].\nPatients with lesions to the orbitofrontal cortex also have disinhibited\/socially inappropriate behaviour. Grafman interpreted the patient\u2019s impairment in terms of an inability to access \u201csocial schema knowledge\u201d stored in the frontal lobes [31]. Social schema knowledge is thought to inhibit aberrant behaviour. Patients with orbitofrontal cortex lesions who cannot access social schema knowledge fail to inhibit aberrant behaviour, such as physical threats and aggression.\nThe prediction of a similar cognitive profile in terms of ToM abilities and social competence between frontal lesion subjects and schizophrenic people has been investigated and confirmed [6, 7]. An extensive and careful review recently published [32], reported a general agreement about the nature and extension of ToM dysfunctions in people affected by schizophrenia. These dysfunctions are symptoms related [33], disease specific and state independent.\nThe neural architecture of the social cognitive dysfunction of schizophrenia is of paradigmatic importance for the understanding of social cognitive dysfunction and, more importantly, for the understanding of the consequences at the behavioural level [34]. A previous seminal study provided evidence that structural orbitofrontal cortex abnormalities are related to social dysfunction in schizophrenic people [35]. Moreover this prefrontal area has been unequivocally involved in the social cognitive deficits associated with this disorder [36].\nHowever, empirically controlled investigations in which the cognitive profile of brain damaged patients was compared with schizophrenic subjects with an appropriate set of ToM and social intelligence tasks are lacking, thus leaving several crucial questions largely unresolved.\nIn the current study, we examined the performance of stabilized schizophrenic outpatients, inpatients with focal damage of left and right ventromedial prefrontal lobes and healthy controls, in ToM abilities, in social competence and tactical strategy (Machiavellian Intelligence), to clarify whether schizophrenic patients demonstrate impairment similar to ventromedial prefrontal lesion patients and whether their performance in these tasks can be differentiated from their performance in tasks sensitive to neuropsychological dysfunction, including \u201cexecutive\u201d functions. Our prediction is that an overlapping dysfunctional cognitive profile should emerge between brain damaged and schizophrenic subjects, when compared to healthy subjects. We also addressed several methodological issues raised by earlier studies using social cognition tasks created for adults and not for children.\nMethods\nParticipants\nThe subjects for this study included 18 adult neurosurgical patients with unilateral frontal lobe lesions who had undergone surgery at the Department of Neurosurgery of \u201cS. Salvatore\u201d Hospital, L\u2019Aquila, Italy. The patients were consecutively enrolled in the study from January 2003 to September 2005.\nOnly patients with exclusive frontal lesions were identified and brain damage was confirmed through neuroimaging, with pre- and postoperative CT scanning and MRIs.\nNine subjects with left side medial prefrontal cortex (LMPFC) lesions and nine subjects with right side medial prefrontal cortex (RMPFC) lesions were studied and underwent neuropsychological examinations (Fig.\u00a01A, B).\nFig.\u00a01(A) MRI T1 weight (axial view) with gadolinium showing low density abnormalities (meningioma excised, patients L.F.) orbitofrontal cortex on the left side. (B) CT scan image showing low density abnormalities (meningioma excised, patients L.G.) orbitofrontal cortex on right sides\nThirteen subjects had intra- or extra-axial tumours (72%); 3 had spontaneous haemorrhage (17%) and 2 (11%) had intracerebral haemorrhage from ruptured aneurysms.\nIn the RMPFC group seven patients had a tumour removed (four had a meningioma excised, 2 had a high grade glioma excised and 1 had an oligodendroglioma excised), one had a right anterior communicating artery (ACoA) aneurysm clipped, following rupture and one had spontaneous intracerebral haemorrhage with no evidence of arterial malformations. Of the LMPFC patients, six had a tumour removed (five had a meningioma excised and one had a high grade astrocytoma excised), two had spontaneous intracerebral haemorrhage with no evidence of arterial malformations and one had a left ACoA aneurysm clipped following rupture.\nThe location of the experimental group\u2019s lesions were defined anatomically as medial (Broadman area 9 and 46) and orbital (Broadman areas 10, 11, 12 and 25) and further classified according to the prefrontal sectors of functional significance into which the lesions encroached (Tables\u00a01, 2).\nTable\u00a01Socio-demographic details of subjects with ventromedial frontal lobe lesionsSubjectsSiteAgeEducationAetiologyM.DRight frontal lesion5613MeningiomaF.S.Right frontal lesion508MeningiomaL.GRight frontal lesion448MeningiomaP.G.Right frontal lesion635OligodendrogliomaT.RRight frontal lesion625Glioma grade IIIE.GRight frontal lesion698Spontaneous intracerebral haemorrhageS.MRight frontal lesion6413Glioma grade IVA.URight frontal lesion3518Intra-cerebral haemorrhage from aneurismal sac ruptureM.URight frontal lesion4418MeningiomaL.FLeft frontal lesion418MeningiomaL.PLeft frontal lesion305MeningiomaM.NLeft frontal lesion6813Spontaneous intra-cerebral haemorrhageM.M.Left frontal lesion5513Spontaneous intra-cerebral haemorrhageR.MLeft frontal lesion548MeningiomaD.SLeft frontal lesion628MeningiomaR.TLeft frontal lesion7313Astrocytoma grade IIIA.CLeft frontal lesion598MeningiomaR.GLeft frontal lesion388Intra-cerebral haemorrhage from aneurismal sac ruptureTable\u00a02Classification of the RMPFC and LMPFC experimental group according to the prefrontal sectors of functional significance into which the lesions encroachedPatientSexAge (years)AetiologyLesion locationFronto-orbitalMedialRF1F56Meningioma+RF2F50Meningioma+RF3M44Meningioma+RF4F63Oligodendroglioma+RF5M62Glioma grade III+RF6M69SIH+RF7F64Glioma grade IV+RF8F35IhAsc+RF9M44Meningioma+LF1M41Meningioma+LF2M30Meningioma+LF3F68SIH+LF4M55SIH+LF5M54Meningioma+LF6M62Meningioma+LF7M73Astrocytoma grade III+LF8F59Meningioma+LF9M38IhAsc+SIH\u00a0=\u00a0spontaneous intracerebral haemorrhage; IhAsc\u00a0=\u00a0intracerebral haemorrhage from aneurismal sac rupture\nSubjects were assessed with neuropsychological test batteries 20\u201340\u00a0days after surgery.\nA control group of schizophrenic subjects and a control group of psychiatrically and neurologically healthy subjects were studied as well.\nTwenty male patients, all native Italian speakers, whose symptoms satisfied the DSM-IV criteria for schizophrenia [37], participated in the study. They were all patients of the Department of Psychiatry of the University of L\u2019Aquila and diagnosis was re-confirmed on admission to (and 6\u00a0months after discharge from) the Day-Hospital (DH) using a non-structured interview conducted by two psychiatrists (M.C., R.R) referring to DSM-IV criteria.\nAll the subjects, whose assessment took place when clinically stable within a month of admission to the DH and establishment\/confirmation of diagnosis, were treated with maintenance antipsychotic drugs. The mean daily dose was 310.3 (SD 143.67) mg\/equivalents of Chlorpromazine [38]; this dose-equivalence with a typical antipsychotic is necessary in order to compare the different antipsychotic drugs as their strength may be different when administered to patients.\nTwenty neurologically and psychiatrically healthy control subjects (matched for age and education) were included. Exclusion criteria were: history of neurological disease including epilepsy, head trauma or mental retardation. All subjects provided informed consent to participate in the study.\nMaterials and procedure\nClinical assessment\nClinical assessment in the sample with MPFC lesions was performed by using a non-structured clinical interview and Brief Psychiatric Rating Scale (BPRS) version 4.0 translated into Italian by [39] and through the neuropsychiatric Inventory (NPI), a semistructured clinicians interview using the protocol described by [40].\nFor the schizophrenic sample, frequency and severity of current symptomatology was registered by using a non-structured clinical interview and Brief Psychiatric Rating Scale (BPRS) version 4.0 (Modified 24-item version, translated into Italian by [39] and Clinical Global Impression scale (CGI) [40].\nThe prevalent symptomatology was also investigated using the Scale for the assessment of Positive Symptoms (SAPS; [41]) and Scale for the assessment of Negative Symptoms (SANS; [42]).\nThe SAPS consists of 34 items and is divided into four subscales: hallucinations, delusions, bizarre behaviour and formal thought disorder. The SANS consists of 25 items and is designed to measure five domains: affective flattening or blunting, alogia, apathy, asociality and impaired attention.\nWe also evaluated social function with AD-Disability Assessment [40]. Socio-demographic and clinical data are reported in Table\u00a03.\nTable\u00a03Socio demographic, clinical details in the total sampleSchizophrenia (no. 20)Right medial prefrontal cortex lesion (no. 9)Left medial prefrontal cortex lesion (no. 9)Healthy controls (no. 20)Sex (M:F)7:135:42:715:6Age (years)42.7 (1.8)49.6 (10.3)46.4 (18.6)38.9 (2.8)Education (years)8.8 (0.8)9.2 (2.6)12.2 (3.97)13.4 (1.2)Day since surgery\u201391.4 (11.6)88.8 (13.7)\u2013IQ level88.8 (5.7)93.1 (2.4)98.7 (3.6)\u2013NPI total score6 (0)10 (0)9.8 (5.3)\u2013Duration of illness (years)12.30 (4.13)\u2013\u2013\u2013BPRS total score44 (11.1)52.5 (4.9)55.5 (17.5)\u2013C.G.I.4.5 (0.6)\u2013\u2013\u2013SANS50.42 (20.9)\u2013\u2013\u2013SAPS42.57 (19.1)\u2013\u2013\u2013Social function (AD)2.5 (1.1)\u2013\u2013\u2013The values are means; standard deviation are in parenthesis\nNeuropsychological assessment\nAll the schizophrenic subjects, with the exception of two positive schizophrenics, were also administered three neuropsychological tests for assessment of executive functions: the WCST, the Tower of London Test and a Phonemic Verbal Fluency Test (Table\u00a04).\nTable\u00a04Neuropsychological detailed of total sampleSchizophrenia (no. 20)Right medial prefrontal cortex lesion (no. 9)Left medial prefrontal cortex lesion (no. 9)Healthy controls (no. 20)Verbal fluency33.1 (10.4)21.2 (8.39)11.3 (4.19)38.7 (7.5)Tower of London29.8 (4.4)26.3 (6.5)19.2 (4.3)34.6 (1.9)WCST no. of category3.46 (2.41)2.66 (1.7)1.87 (2.1)5.7 (0.3)WCST % of perseverative errors27.66 (15.12)42.9 (22.6)50.7 (21.3)2.9 (2.6)Verbal memory19.1 (8.7)28.6 (8.01)8.3 (2.25)29.6 (3.01)The values are means; standard deviation are in parenthesis\nThe WCST (128 cards) was administered with standard instructions, as described by Spreen and Strauss [43], whereas scoring followed Heaton\u2019s [44] rules. Scoring and administration instructions for the Tower of London Test were those described by Krikorian et al. [45]. Scoring and administration instructions of the Phonemic Verbal Fluency Test were those described by Novelli et al. [46].\nVisuo-spatial intellectual level was assessed by means of Raven\u2019s Progressive Matrices [47].\nTheory of mind tasks\nFour stories were read to the participants individually to assess ToM competence. The stories were structured to assess the ability to understand first and second order false beliefs in adult subjects [9]. First order false beliefs require a subject to make an inference about the state of the world. To assess first order ToM two stories were used: The washing machine task [9] and The Cigarette Task [48].\nSecond order stories measure the capacity to understand other people\u2019s false beliefs. To assess second order ToM two stories were used; The Burglar [49] and The Wallpaper Story [9]. These stories were presented to the subjects in a series of cartoons in which the various actions of the characters are depicted in sequences.\nAll the subjects were asked a ToM question and three control questions:\nFalse belief test question. This was designed to elicit a response that demonstrated the ability to make inferences about another individual\u2019s mental state, namely, that a character in the story holds a false belief.Fact question. This was posed to determine whether subjects understood the actual sequence of relevant events that had occurred in the story which is in contrast with the sequence as understood by one of the characters in the story and that leads to his coming to a false conclusion.Memory question. This was used to assess whether memory for story details was approximately intact. The stories were the same for all the subjects examined. Each subject obtained a score ranging from 0 to 1 in the case of a correct answer to a False-belief question, to the fact question and to the memory question, where 0\u00a0=\u00a0incorrect answer; 1\u00a0=\u00a0correct answer. If the subject gave a correct answer to both the first order stories, s\/he had a global score for first order ToM equal to 1 (non-casual performance). For second order false belief stories we followed the same methods.\nSocial cognition tasks\nThe following tasks addressed two aspects of social cognition.\nThe ability to process the appropriateness of behaviour in different social contexts [21]; and the ability to use tactical strategy (Machiavellian Intelligence).\nSocial situations task\nThis task investigates the capacity to judge the appropriateness of behaviour that may induce anger in observers.\nProcedure. Nine short stories describing social situations incorporating behaviour were read by the patient. At various points in each story, the patient was asked to comment on how appropriate the behaviour was, giving a score from A to D. \u201cA\u201d scores meant that he judged the situation as normative. \u201cB\u201d to \u201cD\u201d scores meant that he judged the situation as a norm violation and indexed the extent of the violation (\u201cB\u201d scores being mild and \u201cD\u201d being serious). Blair and Cipollotti [21] report that previous piloting on a large, independent sample of healthy controls had resulted in the identification of a set of consistently identified normative situations and violations.\nTwo scores were obtained for this task: one referring to the number of normative situations and the other to the number of violations correctly identified. The third refers to the extent to which the patient judged the violations to be socially inappropriate.\nFor each situation, the participant obtained a score between 0 and 3, matching their response of \u201cA\u201d to \u201cD\u201d (i.e. \u201cA\u201d\u00a0=\u00a00, \u201cD\u201d\u00a0=\u00a03).\nMach IV Scale\nWe used the Mach IV Scale to assess the \u201cMachiavellian Intelligence\u201d of participants [50]. This is a self-report Likert scale, with scores ranging from 1 to 7 (where 1\u00a0=\u00a0I totally disagree; 4\u00a0=\u00a0no opinion; 7\u00a0=\u00a0I totally agree), composed of 20 items, each consisting of a statement.\nThe Mach IV scale is a method for assessing awareness and social functioning in a social context characterised by interpersonal deception (\u201cMachiavellianism\u201d).\nFrom the 20 statements of the Mach IV Scale we extracted two groups of items: (1) 5 items describing duplicity tactics, e.g.: \u201cIt is wise to flatter important people\u201d (\u201ctactics+\u201d) and (2) 4 items describing a disagreements with tactics duplicity, e.g.: \u201cWhen you ask someone to do something for you, it is best to give the real reason\u201d (\u201ctactics\u2212\u201d).\nThe items were labelled for coding as follows:\nPositive tactics (tactics+): subjects must agree with statements reporting the ability to manipulate other people\u2019s intentions and actions, according to Niccol\u00f2 Machiavelli\u2019s beliefs.\nNegative tactics (tactics\u2212): subjects must agree with statements reporting judgements of correct and honest behaviour; for this reason they cannot use \u201cintentional deception\u201d mechanisms involved in the Machiavellian Intelligence Hypothesis. These features depend on an accurate interpretation of even the most particular intention of respondents [32, 51].\nHuman nature components referring to \u201cpeople\u2019s knowledge\u201d, in particular the degree of cynicism concerning other people\u2019s intentions and decisions are strictly related to the ability to interpret other people\u2019s mental states.\nStatistical analysis\nOne-way ANOVA was used to compare demographic, clinical information and neuropsychological Assessment and social cognition tasks. The Kruskaal\u00a0\u00b1\u00a0Wallis test was used to analyse the level of significance of patients\u2019 scores on ToM tasks.\nResults\nNo differences among groups emerged for age [F(3,40)\u00a0=\u00a00.271, P\u00a0=\u00a00.751], years of education [F(3,38)\u00a0=\u00a00.387, P\u00a0=\u00a00.51] and estimated IQ [F(3,38)\u00a0=\u00a00.44, P\u00a0=\u00a00.64] (Table\u00a02).\nClinical assessment\nNeither were differences found between the four subgroups in age [F(3,38)\u00a0=\u00a00.271, P\u00a0=\u00a00.751, sex ratio, standard of education [F(3,38)\u00a0=\u00a00.387, P\u00a0=\u00a00.51], IQ [F(3,38)\u00a0=\u00a00.387, P\u00a0=\u00a00.51], in BPRS total scores [F(3,38)\u00a0=\u00a00.345, P\u00a0=\u00a00.72] and in NPI total score [F(3,38)\u00a0=\u00a00.748, P\u00a0=\u00a00.508].\nExecutive function\nThe three groups differed significantly in the planning Tower of London task [F(3,40)\u00a0=\u00a022.568, P\u00a0=\u00a00.000]; Verbal Fluency [F(3,40)\u00a0=\u00a040.023, P\u00a0=\u00a00.000], and No. of categories achieved in the WCST [F(3,40)\u00a0=\u00a09.578, P\u00a0=\u00a00.000] and perseverative errors [F(3,40)\u00a0=\u00a010.694, P\u00a0=\u00a00.000]. The performance data for the four groups on the tests of executive functioning are shown in Table\u00a03.\nThe LSD method was used for post hoc comparisons. This revealed that in Verbal Fluency both frontal groups (LMPFC and RMPFC) performed significantly worse than the schizophrenic group and control subjects. Bonferroni tests on the Tower of London task showed impaired performance for the LMPFC group only when compared to both the schizophrenic and healthy subjects control groups. The LMPFC group performed significantly worse than the RMPFC subjects.\nFirst-order false belief tasks\nFalse-belief test question: Groups differed significantly on the non-parametric Kruskall\u2013Wallis test: \u03c72(3)\u00a0=\u00a014.664, df\u00a0=\u00a03, P\u00a0<\u00a00.002. Post hoc (Bonferroni methods) comparisons showed that the RMPFC group\u2019s performance, differed significantly from that of the LMPFC (RMPFC vs. LMPFC mean differences\u00a0=\u00a0\u22120.665; P\u00a0<\u00a00.005) but not from schizophrenic subgroups, and that the overall percentage of correct scores for patient groups was significantly lower than those of the normal control group.\nFact questions: The percentage of correct scores revealed no significant overall differences between groups.\nMemory questions: The percentage of correct scores showed no significant group differences (Fig.\u00a02).\nFig.\u00a02Performance of study groups (schizophrenics, Right Medial Prefrontal Cortex\u2014RMPFC Lesion Subjects, Left Medial Prefrontal Cortex\u2014LMPFC Lesion Subjects and healthy controls) on First-order False Belief tasks\nSecond order theory of mind\nFalse-belief test question: Groups differed significantly: Kruskall\u2013Wallis \u03c72(3)\u00a0=\u00a011.72, df\u00a0=\u00a03, P\u00a0=\u00a00.008). Post hoc (Bonferroni methods) analyses revealed the most impaired performance for the RF and schizophrenia groups when compared to LMPFC (LMPFC vs. RMPFC mean differences\u00a0=\u00a0\u22120.598; P\u00a0<\u00a00.007; LMPFC vs. schizophrenics mean differences\u00a0=\u00a0\u22120.623; P\u00a0<\u00a00.000). However the overall percentage of correct scores for patient groups was significantly lower than those of the normal control group.\nFact questions: The percentage of correct scores revealed no significant overall difference between the groups.\nMemory questions: The percentage of correct scores showed no significant group differences (Fig.\u00a03).\nFig.\u00a03Performance of study groups (schizophrenics, Right Medial Prefrontal Cortex\u2014RMPFC Lesion Subjects, Left Medial Prefrontal Cortex\u2014LMPFC Lesion Subjects and healthy controls) on second-order False Belief tasks\nSocial situation task\nThe ANOVA comparison between RMPFC, LMPFC, schizophrenics and healthy controls showed statistically significant differences in the ability to identify normative situations [F(3,58)\u00a0=\u00a03.179, P\u00a0=\u00a06.073].\nPost hoc (Bonferroni methods) comparisons showed that the RMPFC group\u2019s performance, differed significantly from that of the LMPFC (RMPFC vs. LMPFC mean differences\u00a0=\u00a0\u22121.904; P\u00a0<\u00a00.001; LMPFC vs. schizophrenics mean differences\u00a0=\u00a01.623; P\u00a0<\u00a00.002) and healthy controls but not from schizophrenics. There were no significant differences between the four groups in the total score of the norm violations (Fig.\u00a04).\nFig.\u00a04Performances on social situation task in all groups (schizophrenics, Right Medial Prefrontal Cortex\u2014RMPFC Lesion Subjects and Left Medial Prefrontal Cortex\u2014LMPFC Lesion Subjects). The figure reports the scores in correct identification of normative situation and the mean scores of norm violations\nMach IV scale\nThe ANOVA comparison between RMPFC, LMPFC, schizophrenics and healthy controls showed statistically significant differences on the following Mach IV items (tactics+) item \u201cTrusting someone means getting into trouble\u201d [F(3,58)\u00a0=\u00a03.179, P\u00a0=\u00a00.035]; (tactics+) \u201cIt\u2019s hard to be successful without taking short-cuts\u201d [F(3,58)\u00a0=\u00a03.959, P\u00a0=\u00a00.000]; (tactics\u2212) \u201cThere is no need to deceive anyone\u201d [F(3,58)\u00a0=\u00a07.759, P\u00a0=\u00a00.021]; (tactics\u2212) \u201cIt is possible to be good in all situations\u201d [F(3,58)\u00a0=\u00a03.406, P\u00a0=\u00a00.027]. Post hoc multiple comparison (Bonferroni methods) showed that RMPFC score lower for items indicating agreement in strategic thinking (tactics+); than LMPFC, schizophrenics and controls showed higher scores for items indicating disagreement with strategic thinking (Tactics). Results are displayed in Fig.\u00a05.\nFig.\u00a05Performance on items of Mach IV Scale in the four groups for strategic thinking\nCorrelation analyses\nNo significant correlations were found between ToM first order and ToM second order questions and executive functions (verbal fluency, WCST number of categories and perseverative errors, Tower of London) in normal controls, and in subjects with left and right frontal lesions.\nNo significant correlations were found between the Mach IV scales (good tactical strategy and negative tactical strategy) and executive functions (verbal fluency, WCST no. of categories and perseverative errors, Tower of London) in normal controls, and in subjects with left and right MPFC.\nThere was a significant correlation between first order ToM scores and duration of illness (r\u00a0=\u00a0\u22120.375; P\u00a0<\u00a00.029); between first order ToM scores and SANS total scores (r\u00a0=\u00a0\u22120.562; P\u00a0<\u00a00.03); between first order and second order ToM scores and social functioning total scores (AD) (first order\u00a0=\u00a0\u22120.489; P\u00a0<\u00a00.036; second order r\u00a0=\u00a0\u22120.543; P\u00a0<\u00a00.029).\nThe significant correlation was also found in the schizophrenic sample between ToM performances and verbal fluency (r\u00a0=\u00a0\u22120.527; P\u00a0<\u00a00.000).\nNo significant correlations were found between the Mach IV scale and social cognition task and psychopathological and clinical variable (SANS, SAPS and CGI).\nDiscussion\nOne of the distinctive attributes of human social cognition is our propensity to build models of other people\u2019s minds: to make inferences about the mental states of others. Several neuroimaging studies have attempted to elucidate the neural substrates that support this distinctively human ability that is impaired in people with schizophrenia.\nThe main aim of the present paper is to establish whether patients affected by schizophrenia show an impairment in several social cognitive tasks as demonstrated in other researches [5, 52\u201354] and if this cognitive profile is comparable to patients with a unilateral brain lesion involving orbito-ventromedial areas of the frontal lobes.\nOur results are in line with other studies: in people with schizophrenia there was an impairment of social cognitive abilities and this deficit appears to be related to negative symptomatology [55] and to be a key determinant of functional outcome, including social outcome [56]. It has been suggested that theory of mind deficit make unable schizophrenic subjects to interact effectively with their social environment, but that a lack of certain aspects of social cognition will lead to social misperceptions.\nIn addition to these clinical and outcome goals, there is increasing interest in identifying the neural substrates that underlie social cognitive deficits in schizophrenia. For all of these reasons we compared the performances on social cognition tasks of schizophrenic subjects with the performances of MPFC subjects.\nAmong the several studies which investigated the effects of frontal lobe lesions (dorsolateral and ventromedial\/orbital) on performance in ToM tasks [9\u201311, 57\u201359], some of these including patients with bilateral frontal lobe damage, are limited because of a lack of detailed anatomical specification of lesion location [10, 11, 57]. Moreover, most of patients with bilateral lesions had suffered head trauma, an aetiology associated with rather diffuse brain damage that is particularly likely to impinge on orbitofrontal brain areas.\nThus, the present study strictly tests the hypothesis that the unilateral (right or left) medial frontal cortex is implicated in the neural network sub serving ToM [8] which is based on well established evidences suggesting the implication of the ventromedial frontal lobe areas in playing a critical role in a dedicated \u201cmentalizing\u201d or ToM network in human brains ([7, 28, 60] for a review).\nWe found out that subjects with RMPFC lesion are impaired in ToM tasks of \u201cfalse beliefs\u201d, showing thus a very similar cognitive dysfunctional profile to people affected by schizophrenia in all Theory of Mind tasks and in all social cognition tasks. A normal performance on control questions indicates an unimpaired comprehension of stories and suggest that the task was sensitive in detecting TOM impairments.\nIn addition, schizophrenics and subjects with RMPFC lesion also showed impairment in the social cognition tasks, in fact they both failed to discriminate in judging inappropriate behaviour likely to induce anger in observers. This was unlike patients with LMPFC who showed no impairment on any of these tasks.\nThis is clear evidence that the medial frontal cortex plays a critical role in a dedicated \u201cmentalizing\u201d brain network that underpins ToM ability [7, 8].\nOur findings are in agreement with previous \u201clesional\u201d studies, showing the association between right medial area damage and more severe ToM deficits [11, 13]. Siegal et al. [13] reported that ToM impairments seem to be associated with right hemisphere damage.\nIn the present study we report a dissociation in RMPFC damage patients who displayed a defective ToM performance in contrast to LMPFC patients. LMPCF subjects show lower performances than RMPCF subjects in other cognitive competences but have normal performance in ToM competences and our results confirm the results obtained by Siegal and Surian [61].\nIn addition, when a more sophisticated social ability is required in order to perform second order false belief tasks correctly, also LMPFC damaged subjects fail to perform at a normal level and show a statistically significant impairment, even though to a lesser degree, when compared to subjects with RMPFC and schizophrenic people. A possible explanation is that LMPFC is involved in more sophisticated mentalizing tasks and that an intact right hemisphere structure is nevertheless required [58]. Normal subjects and neurosurgical subjects with unilateral LMPFC lesions perform fairly well on tasks related to tactical strategy, showing correspondingly low scores on the ingenuity aspect of thinking.\nThe present study also provides further data on the neural prefrontal areas involved in social cognition tasks and in strategic thinking [21]. Social efficiency is represented by the ability to understand the intentions and beliefs of others with the aim of deceiving and manipulating them to achieve relevant objectives, such as control of food sources or sexual partners [26]. Recent literature fosters that these abilities are localised in the frontal cortex [27, 28]; the ability to recognise and manipulate hierarchical states to achieve some advantage would be localised in the amygdala and right hemisphere [62].\nSchizophrenic people and subjects with RMPFC lesion showed impaired performance on tactical strategy associated with relatively \u201chigh levels\u201d of social ingenuity and have an impaired ability to access \u201csocial schema knowledge\u201d which is stored in the frontal lobes [31]. Such patients cannot access social schema knowledge and fail to inhibit aberrant behaviour, such as physical threats and aggression [21, 63]. Interestingly a completely reversed pattern characterizes the performance of LMPFC lesion subjects and healthy controls, in fact we found out a complete disassociation of the neural prefrontal areas located in the medial part of the hemisphere in sub serving human ability to think strategically, indicating that the cortical organization related to tactical aspects of Machiavellian Intelligence is lateralized to the right hemisphere [64]. Our results are at slight variance with the pioneering study of Rowe et al. [9] who found a significantly impaired performance of both the RMPFC and LMPFC subjects in first and second order ToM tasks. In this study cortical lesions were non-exclusive medial hemisphere but includes subjects with dorsolateral prefrontal cortex (DLPC) damage.\nStudies of normal subjects have used a variety of imaging techniques, designs, and test materials, but especially PET and fMRI to define brain regions specifically activated during a ToM task [8, 65]. Such studies have consistently shown activation of the medial prefrontal gyrus (MPFG) and the Temporo Parietal Junction.\nFrith and Frith [7], reporting data on studies carried out in adults, have revealed an MPFC system of three components that are consistently activated during both implicit and explicit mentalizing tasks. This brain region is probably the basis of the decoupling mechanism that distinguishes mental state representations from physical state representations. We can speculate, according to the Edelman model [66, 67], that a hierarchical organization of mental operation, when disrupted at a specified level, impairs the integrity of final output via an interruption of the chain of events required to perform a task. This study is limited by the small number of patients with unilateral frontal lobe lesions due to the rarity of such lesion.\nDespite this limitation, this study provides further evidence that social competence is compromised in RMPFC subjects very closely to schizophrenics and these data seem to elucidate the possible neuroanatomic structure alteration present in schizophrenia. However, we are confident that there is a wide range of behavioural manifestations of frontal lobe dysfunction, and ToM impairments clearly cannot account for all of these, nor is it likely to be responsible for all reported difficulties in social cognition. In contrast, ToM tests are designed with the aim of isolating those aspects of social cognition associated with two-way reciprocal interactions that rely crucially on ToM ability and false belief tasks have facilitated the demonstration of a mentalizing impairment in subjects with lesions of the prefrontal cortex, which is independent of non-mental state inference.\nIn conclusion, our findings provide evidence that lesions to the right MPCF determine an incapacity to understand ToM \u201cfalse belief\u201d stories and to use tactical strategy and understanding of social schema. Other authors have reported the same results: subjects with right hemisphere damage, but not subjects with left hemisphere damage, had difficulties in performing simple theory of mind tasks [13, 68].\nThe difficulties shared by subjects with right hemisphere damage and young children on ToM tasks may have a similar origin [61]. These may both derive from a pragmatic deficit that prevents subjects from interpreting the implicit questions correctly, rather than from a conceptual deficit concerning the ability to represent mental states.\nThe response pattern of subjects with ventromedial prefrontal damage on ToM tasks adds new evidence to the growing literature on the effects of the right hemisphere on various pragmatic aspects of language production and comprehension [13].\nThe good performance of subjects with RMPCF lesions in executive function and verbal memory tasks suggests that their difficulties are due to a reduced sensitivity to the constraints that guide the interpretation and production of contextually appropriate utterances [61].\nIt may be concluded that the ToM disorder in RMPCF subjects does not stem from executive function or memory deficits and that deficits in ToM and executive functioning in subjects with frontal lobe lesions are not causally related, even though our findings are in disagreement with Channon and Crawford [69] who found a relationship between executive functioning and ToM ability in adults with damage to the frontal lobes [70].\nWe support two positions: first, that a specialized, discrete ToM module, or set of modules, is located in the frontal lobes, but is functionally independent and second that these deficits can co-occur, on the basis of the proximity of the respective underlying neural areas.","keyphrases":["social cognition","frontal lobe lesions","schizophrenia","theory of mind"],"prmu":["P","P","P","P"]}
{"id":"J_Gastrointest_Surg-4-1-2231408","title":"Helicobacter Genotyping and Detection in Peroperative Lavage Fluid in Patients with Perforated Peptic Ulcer\n","text":"Introduction and Objectives Certain Helicobacter pylori genotypes are associated with peptic ulcer disease; however, little is known about associations between the H. pylori genotype and perforated peptic ulcer (PPU). The primary aim of this study was to evaluate which genotypes are present in patients with PPU and which genotype is dominant in this population. The secondary aim was to study the possibility of determining the H. pylori status in a way other than by biopsy.\nIntroduction\nOver the past decades the incidence of perforated peptic ulcer (PPU) has declined in the western world. However, with an incidence varying between two and 10 per 100,000, it still is a problem in modern society.1 Moreover, mortality rates caused by gastric and duodenal ulcer perforation vary between 10 and 40% and zero and 10% respectively, and is higher among elderly patients.2,3 Several risk factors for PPU have been described such as smoking, alcohol abuse, and history of peptic ulcer disease (PUD).2 However, the main pathogenic factors are considered to be the use of non-steroidal anti-inflammatory drugs (NSAID) and the presence of H. pylori.2\nHelicobacter pylori are widespread bacteria, with a prevalence ranging from 25% in the industrialized world to more than 70% in developing countries.4,5 Most infected people remain asymptomatic; however, a small group of carriers will develop PUD.\nOf patients who have developed PPU, 70% will test positive for H. pylori,2 suggesting the pathogenesis of perforation is associated with the presence of H. pylori. In addition, it is shown that different genotypes of H. pylori are associated with different clinical manifestations like PUD and gastric cancer.6,7 Two well-known H. pylori genes that have been associated with PUD are the cytotoxin-associated gene (cagA) and the vacuolating cytotoxin gene (vacA).6,8\u201310\nVacA is present in all H. pylori strains and is associated with gastritis, PUD, and gastric carcinoma.10\u201312 It encodes for a vacuolating cytotoxin that causes epithelial cell injury and interferes with the immune system.13,14VacA contains at least two variable regions, the signal peptide (s)-region and the middle (m)-region. The s-region contains two allelic types, s1 and s2. The s1 strain has several subtypes, being s1a, s1b, and s1c.15 Two allelic types exist for the m-region, m1 and m2. The latter has two subtypes, m2a and m2b.16\nCagA is considered a marker for a genomic pathogenicity (cag) island that is associated with enhanced virulence.17\nIf PPU is associated with a specific H. pylori genotype it may be feasible to limit the patients undergoing antibiotic therapy to those who have this genotype. When this specific type is not present, another cause of PPU should be looked for and antibiotic therapy should not be started. This would mean cost reduction and, probably, a reduction in the speed of the development of antibiotic resistance.\nCurrently, gastric biopsy during endoscopy is a generally accepted method to diagnose H. pylori infection. However, patients with PPU will not undergo endoscopy but will generally be operated upon immediately. Taking a biopsy intraoperatively implicates a higher risk of bleeding and more difficult closure of the defect. Therefore, surgeons are reluctant to take a biopsy.\nThe primary aim of this study was to evaluate which genotypes are present in patients with PPU and if a genotype is dominant in this population. The secondary aim was to study the possibility of determining the H. pylori status in a manner other than by gastric tissue biopsy.\nMethods\nFrom 30 consecutive patients operated on for PPU serum samples, gastric tissue biopsies, lavage fluid, and fluid from the nasogastric tube were collected. These patients were treated in five different medical centers throughout the Netherlands. In each of these centers approval of the medical ethical committee was obtained. Immediately after collection, the materials were frozen at \u221220\u00b0C. One researcher performed the analysis and genotyping. For H. pylori genotyping, the presence of cytotoxin-associated gene (cagA) and the s- and m-region genotypes of the vacuolating cytotoxin gene (vacA) were determined.\nDNA was isolated according to Boom\u2019s method as described previously.18 A guanidine thiocyanate (GuSCN) solution was added to the collected material to induce lysis of the bacteria, releasing their DNA. After addition of the silica particles (Celite) the suspension was centrifuged. The silica particles, with the attached DNA, were washed with subsequently GuSCN-containing washing buffer, ethanol 70% and acetone. After drying, the DNA was eluted in an aqueous low salt buffer. The isolated DNA was amplified by means of polymerase chain reaction (PCR) and subsequently the presence of cagA and different types of vacA were analyzed by means of reverse hybridization on a strip (32). This assay consists of a nitrocellulose strip that contains dT-tailed oligonucleotide probes immobilized as parallel lines. For each strain, 10\u00a0\u03bcl of each PCR product (containing biotin at the 5\u2032 end of each primer) was denatured by the addition of an equal amount of 400\u00a0mM NaOH and 10\u00a0mM EDTA in a plastic trough. After 5\u00a0min, 1\u00a0ml of prewarmed hybridization solution (2\u00d7 SSC [1\u00d7 SSC is 0.15\u00a0M NaCl plus 0.015\u00a0M sodium citrate], 50\u00a0mM Tris\u2013HCl [pH 7.5], 0.1% SDS) was added, and a strip was submerged and incubated in a shaking water bath at 50\u00b0C for 1\u00a0h. The strips were washed with 2\u00a0ml of 2\u00d7 SSC-0.1% SDS for 30\u00a0min at 50\u00b0C. Subsequently, the strips were rinsed three times in phosphate buffer, and conjugate (streptavidin\u2013alkaline phosphatase) was added. After incubation at room temperature for 30\u00a0min, the strips were rinsed again and 4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate substrate was added. Hybrids are visible as purple probe lines. Interpretation of the hybridization patterns was performed visually. As a control, a \u03b2-globin PCR was performed. Patient related factors were obtained prospectively. Statistical analysis was performed with SPSS for Windows, version 11.0.\nResults\nA total of 30 patients were included of whom nine were women. The average age was 65\u00a0years, varying between 40 and 87. Ten patients (33.3%) were operated laparoscopically. The perforation was found prepyloric in 11 patients, at the site of the pylorus in eight patients and postpyloric in 11 patients.\nA total of five (16.7%) patients had a history of PUD. Ten patients (33.3%) used NSAID\u2019s, two patients (6.7%) used steroids, three patients (10.0%) used acid reducers, and one patient (3.3%) used a proton pump inhibitor (PPI) before admission to the hospital. The average hospital stay was 11.9\u00a0days, varying between 3 and 37\u00a0days.\nFluid from the nasogastric tube was obtained from 25 patients, lavage fluid from 26 patients, serum samples from 20 patients, and ulcer biopsies from 18 patients. The results of the genotyping are depicted in Table\u00a01.\nTable\u00a01Helicobacter pylori Status and GenotypeThe colors represent the \u03b2-globin and H. pylori status of the patient.\nThe \u03b2-globin determination was performed as a control. In nine samples of nasogastric tube fluid and in two samples of lavage fluid it was negative, rendering these results as unreliable. Therefore, these results were excluded from further analysis.\nTable\u00a02 represents the frequency of the individual genes and the allelic types found in the different samples by means of PCR and LiPA.\nTable\u00a02Frequencies of Individual Genes and Allelic TypesGenotypeFluid from Naso-Gastric TubeLavage FluidGastric Tissue BiopsyControl Non-UlcerNo.%No.%No.%%VacA s11090.91477.8777.846.9VacA s20015.5111.138.4VacA multiple19.1316.7111.114.7Total11100181009100100VacA m1654.5950.0555.629.4VacA m2436.4844.4333.355.9VacA incomplete genotype19.115.6111.10 (14.7 % incomplete)Total11100181009100100CagA positive981.81477.8555.647.1Total11100181009100100\u201cVacA multiple\u201d means that more than one allelic type or subtype has been found in one sample.In each different type of sample one incomplete genotype occurred, which is indicated as \u201cvacA incomplete\u201d. The \u201cControl non ulcer\u201d column represents the frequencies, found by van Doorn et al., in a population without PUD and is added to allow easy comparison.\nThese tables show that for vacA the allelic type s1 is predominantly present in all three types of samples. In the s1 positive strains, subtype s1a is predominant as depicted in Table\u00a03.\nTable\u00a03Distribution of the vacA s1 SubtypesVacA SubtypeFluid from Naso-Gastric TubeLavage FluidGastric Tissue BiopsyControl Non-UlcerNo.%No.%No.%%S1a880.01392.9571.481.3S1b220.017.1228.618.7S1c0000000Total10100141007100100The s1a subtype is predominant in all sample types.The \u201cControl non ulcer\u201d column represents the frequencies, found by van Doorn et al., in a population without PUD and is added to allow easy comparison.\nWith regard to the middle region of vacA the incidence of m1 allelic type is slightly higher; however, the difference is less outspoken compared to s1. The m2a was the only subtype that was found in the samples. In three samples, the genotyping was incomplete (Tables\u00a01 and 2), meaning that determination of the middle region was not possible. This was most likely caused by the small number of bacteria present in those samples.\nWith regard to the secondary aim of this study, analyzing possibilities to diagnose H. pylori presence in another fashion than through biopsy, the H. pylori status found in each type of sample was compared. A correlation was found between the H. pylori presence in biopsy and its presence in lavage fluid (Fisher\u2019s exact test, p\u2009=\u20090.015), indicating lavage fluid is a valid alternative for determination of H. pylori infection.\nThe sensitivity and specificity of the lavage fluid analysis was calculated, considering biopsy as a golden standard. Fourteen patients, of which the lavage fluid as well as the biopsy was analyzed, were included into this calculation (patients 2, 4, 5, 6, 8, 10, 15, 23\u201325, 27\u201330, Table\u00a01), which is shown in Table\u00a04. Of the remaining patients, either the biopsy or the lavage fluid was missing; therefore, these data cannot be used in the sensitivity\/specificity calculation.\nTable\u00a04Calculation of Sensitivity and Specificity of Lavage Fluid Analysis\u00a0BiopsyLavage fluid+\u2013Total+8210\u2013044Total8614Sens 8\/8\u2009=\u20091Spec 4\/6\u2009=\u20090.67\nThe sensitivity was 100%, which means that in case of the presence of H. pylori in the biopsy specimen, the lavage fluid analysis detected it in 100% of cases. The specificity of lavage fluid analysis was 66.7%, which means the chance for false-positives is over 30%. With regard to gender, age, BMI, history of PUD, location of perforation, complications after procedure, and use of steroids, PPI, or antihistaminic medication, no statistically significant correlation was found.\nDiscussion\nConcerning the role of H. pylori in the pathogenesis of PPU, some studies have been reported comparing the prevalence of H. pylori infection in patients with PPU to the prevalence in controls. They appear to be similar, suggesting that other factors like NSAID use play a role.19\u201321 However, the substantial genetic heterogeneity of H. pylori that has been revealed over the years leads to the hypothesis of a specific genotype causing PPU.5 Controls might test positive for H. pylori, but not develop PPU because it would not be this specific genotype that is isolated. This study of a selected population of patients, all with PPU, shows a limited diversity of H. pylori genotypes as represented by Table\u00a01.\nVacA s1 strains are predominantly present in the three sample types of which s1a is the predominant subtype. Concerning the vacA m-region, the m1 strains are found in a majority of cases; however, the difference is less convincing than for vacA s1. Except for the biopsy samples, the cagA positive strains were predominantly present is this population. In the biopsy samples, the frequency of cagA-positive strains seemed to be low; however, this number is distorted because in two of nine positive biopsies, a decent comparison with the other samples was not possible. In patient 5, genotyping of the lavage fluid and nasogastric tube fluid was incomplete, and for patient 15, the opposite was the case. This means that the actual incidence should be 71.4 % (5\/7).\nSummarising, these results shows that the vacA s1, cagA-positive strains were predominant in this population of patients with PPU. This finding is in accordance with literature reporting correlations between the presence of vacA s1, cagA-positive strains and PUD.6,10 Therefore, detection of the genotype vacA s1 does not specifically predict PPU; nevertheless, clinicians should be aware of this association.\nIn Tables\u00a02 and 3 the genetic distribution in a Dutch population without PUD, as found by van Doorn et al., are added for comparison. The frequencies found in this study for vacA s1, m1, and cagA-positive strains are clearly higher than in the non-PUD group, confirming the aforementioned hypothesis. However, with regard to the subtypes, Table\u00a03 shows an almost similar distribution of frequencies, suggesting that determination of the allelic subtype is of less importance.\nIn only 60% of patients biopsies could be analyzed. The reason for missing 40% is the restraint of the surgeon to take a biopsy when risk of bleeding and more difficult closure of the defect was estimated to be too high, which emphasizes the importance of finding an alternative. To do so, the H. pylori status of the patient as determined by biopsy was compared to the status as determined by analysis of nasogastric tube fluid, lavage fluid, and serum. A statistically significant correlation was found between the H. pylori status in biopsy and its status in lavage fluid (Fisher\u2019s exact test, p\u2009=\u20090.015). This finding suggests that determination of the H. pylori status can be done with lavage fluid as well, obviously without any risk of bleeding and closure related difficulties. The sensitivity is 100%, but the specificity is 66.7%. This could mean the chance for false-positives is over 30%, which is not optimal and could lead to therapy overshoot. However, considering the fact that with the lavage a larger area is sampled, rendering the chance of positive test results higher than in biopsy, it is more likely to find false negative biopsy results. This could lead to a therapy undershoot, which obviates the importance having an alternative for a biopsy.\nIn only two samples, both nasogastric tube fluids, a H. pylori genotype was isolated, while \u03b2-globin tested negative. In nine samples (seven nasogastric tube fluid, two lavage fluid) both \u03b2-globin and H. pylori tested negative. This means that either no humane cells were present in the samples, which is unlikely, or that an error in the PCR procedure had occurred. Because this was unclear these results were considered unreliable. Therefore, it still could be possible that nasogastric tube fluid is a good alternative for determining the H. pylori status as well.\nOverall, these results are positive, however they should be confirmed in a larger population.\nConclusion\nThis study shows that in a population of 30 patients with PPU, vacA s1, cagA positive strains are predominant. This finding is in accordance with literature reporting correlations between the presence of vacA s1, cagA-positive strains, and PUD. Therefore, detection of this genotype does not specifically predict PPU. Nevertheless, clinicians should be aware of this association.\nThis study shows as well that it is feasible to use intraoperative lavage fluid to determine the H. pylori status of the patient, implicating that biopsies, with a risk of bleeding and more difficult closure of the defect, are not necessary anymore. In addition, considering the fact that a larger area is sampled with lavaging, biopsies may result in more false negative results leading to insufficient therapy.","keyphrases":["genotype","peroperative lavage fluid","perforated peptic ulcer","peptic ulcer disease","h. pylori"],"prmu":["P","P","P","P","P"]}
{"id":"Int_J_Parasitol-1-5-1885961","title":"Gene silencing of the tick protective antigens, Bm86, Bm91 and subolesin, in the one-host tick Boophilus microplus by RNA interference\n","text":"The use of RNA interference (RNAi) to assess gene function has been demonstrated in several three-host tick species but adaptation of RNAi to the one-host tick, Boophilus microplus, has not been reported. We evaluated the application of RNAi in B. microplus and the effect of gene silencing on three tick-protective antigens: Bm86, Bm91 and subolesin. Gene-specific double-stranded (dsRNA) was injected into two tick stages, freshly molted unfed and engorged females, and specific gene silencing was confirmed by real time PCR. Gene silencing occurred in injected unfed females after they were allowed to feed. Injection of dsRNA into engorged females caused gene silencing in the subsequently oviposited eggs and larvae that hatched from these eggs, but not in adults that developed from these larvae. dsRNA injected into engorged females could be detected by quantitative real-time RT-PCR in eggs 14 days from the beginning of oviposition, demonstrating that unprocessed dsRNA was incorporated in the eggs. Eggs produced by engorged females injected with subolesin dsRNA were abnormal, suggesting that subolesin may play a role in embryonic development. The injection of dsRNA into engorged females to obtain gene-specific silencing in eggs and larvae is a novel method which can be used to study gene function in tick embryogenesis.\n1\nIntroduction\nThe cattle tick Boophilus microplus is an important pest of cattle in subtropical and tropical regions of the world (Estrada-Pena et al., 2006). Although all Boophilus species including B. microplus have been reclassified to the genus Rhipicephalus (Murrell and Barker, 2003), we maintain use of the previous genus assignment for the purpose of biological clarity. Besides causing direct production losses and leather damage, B. microplus transmits several cattle pathogens, including Babesia bovis, Babesia bigemina and Anaplasma marginale. Control of B. microplus depends primarily on the use of acaricides or genetically resistant animals. Both approaches have limitations, including development of acaricide resistance, environmental contamination, pesticide residues in food products, the expense of developing new pesticides and the difficulty of producing tick-resistant cattle while maintaining desirable production characteristics (Willadsen, 2004). Other tick control approaches which show promise are the use of biological control agents (reviewed by Samish et al., 2004) and anti-tick vaccines (reviewed by de la Fuente and Kocan, 2003; Willadsen, 2004).\nTwo commercial vaccines have been developed for control of tick infestations on cattle, TickGARD Plus\u00ae in Australia and Gavac\u00ae in Cuba. Both are based on the same recombinant antigen named Bm86, a glycoprotein of unknown function which is located predominantly on the surface of midgut digest cells (Gough and Kemp, 1993). This \u2018concealed\u2019 antigen is not naturally exposed to the host\u2019s immune system. Lysis of midgut digest cells occurs in ticks that feed on vaccinated cattle, resulting in leakage of blood meal into the tick hemocoel. The overall effect of the vaccine is on engorging female ticks and includes a decrease in the number and weight of replete ticks and oviposition. While Bm86-based vaccines were effective against several other tick species, including Boophilus annulatus (Fragoso et al., 1998; Pipano et al., 2003), Boophilus decoloratus, Hyalomma anatolicum anatolicum and Hyalomma dromedarii, they were not effective against Amblyomma variegatum, Amblyomma cajennense and Rhipicephalus appendiculatus (de Vos et al., 2001; Rodriquez and Jongejan, unpublished data).\nAnother \u2018concealed\u2019 antigen, Bm91, was shown to increase the efficacy of the Bm86 vaccine for B. microplus when co-administered (Willadsen et al., 1996). Bm91 is a low-abundance glycoprotein located in the salivary glands and midgut of B. microplus (Riding et al., 1994). The protein, a homologue of carboxydipeptidase, shares many biochemical and enzymatic properties with mammalian angiotensin converting enzyme, but its natural substrate has not been identified (Jarmey et al., 1995).\nMore recently, a protein first labeled 4D8 and now called subolesin was identified through Ixodes scapularis cDNA expression library immunisation as a potential tick-protective antigen. Immunisation trials using recombinant subolesin caused reductions of larval, nymphal and adult I. scapularis infestations (Almazan et al., 2003, 2005a,b). The protein was later found to be conserved among ixodid tick species. Characterisation of its function by RNA interference (RNAi) in I. scapularis, Amblyomma americanum, Rhipicephalus sanguineus, Dermacentor variabilis and Dermacentor marginatus suggested involvement of this protein in the modulation of blood ingestion and reproduction. Therefore, the generic name \u201csubolesin\u201d was introduced for the 4D8 proteins and \u201csubA\u201d for the subolesin-encoding gene (de la Fuente et al., 2006a). Gene silencing by RNAi of subA and Rs86, the homologue of Bm86, in R. sanguineus, revealed a synergistic effect in which the expression of both genes was silenced and resulted in decreased tick attachment, feeding and oviposition (de la Fuente et al., 2006c).\nRNAi is a conserved post-transcriptional gene-silencing mechanism present in ticks and a wide range of eukaryotes in which double-stranded RNA (dsRNA) triggers a sequence-specific degradation of cognate mRNAs. It has been an effective tool to study the function of tick proteins at the tick\u2013pathogen interface in a number of three-host tick species such as in I. scapularis which transmits Anaplasma phagocytophilum and Borrelia burgdorferi (Pal et al., 2004; Ramamoorthi et al., 2005; Sukumaran et al., 2006). RNAi was used to study the function of several tick salivary gland proteins involved in feeding of A. americanum (Aljamali et al., 2003; Karim et al., 2004), Haemaphysalis longicornis (Miyoshi et al., 2004) and I. scapularis (Narasimhan et al., 2004). The inducer of RNAi, dsRNA, is injected into nymphal or adult ticks which are then allowed to feed normally. Capillary feeding of dsRNA (Soares et al., 2005) or incubation of isolated tick tissues with dsRNA (Aljamali et al., 2003; Karim et al., 2005) are other methods used successfully to silence genes in ticks. These studies suggest that RNAi is systemic and effects gene silencing throughout the tick.\nIn one-host Boophilus ticks, with all life stages feeding and molting on the same host, alternative strategies are required to conduct gene silencing by RNAi as compared with three-host tick species which spend their non-parasitic life stages off-host. Herein, we examined two methods of dsRNA delivery and its effect on the one-host tick B. microplus: (i) injection of dsRNA into freshly molted females and (ii) injection of dsRNA into engorged females. The latter method caused gene-specific silencing in the oviposited eggs and larvae that hatched from these eggs. We believe this is the first report of the silencing of the expression of Bm86, Bm91 and subolesin in Boophilus ticks as quantified by real-time RT-PCR using two routes of dsRNA delivery.\n2\nMaterials and methods\n2.1\nExperimental animals\nThree Holstein\u2013Friesian calves, 5 months of age (#7793, #7794 and #7799), were used. All animals had no previous exposure to ticks. All tick feedings were approved by the Animal Experiments Committee (DEC) of the Faculty of Veterinary Medicine, Utrecht University (DEC No. 0111.0807).\n2.2\nTicks and tick feeding\nBoophilus microplus ticks originating from Mozambique were provided by ClinVet International (Pty), Bloemfontein, South Africa. The ticks were subsequently maintained on cattle at our tick rearing facility. Larvae were kept off-host at 20\u00a0\u00b0C with 95% relative humidity. Patches used for tick feeding with inner dimensions of 60\u00a0\u00d7\u00a085\u00a0mm and sewn to an open cotton bag were glued to the shaved back of calf #7793 and #7794 using Pattex\u00ae contact glue (Henkel Nederland, Nieuwegein, The Netherlands). A batch of larvae eclosed from 1500\u00a0mg of pooled eggs oviposited by 25 females (approximately 24,000 larvae), was divided on day 0 between two patches on calf #7794. Since males appear earlier from the nymphal stage than females, approximately 500 unfed males were collected on days 13 and 14 and 600 unfed females on days 14 and 15 and incubated at 27\u00a0\u00b0C with 95% relative humidity. Freshly molted females were subjected to injection of dsRNA on day 15 as described below. For gene silencing in engorged females and their progeny, 25 engorged females with an average weight of 261\u00a0mg (248\u2013272\u00a0mg) fed on calf #7794 were collected on day 21. Larvae which hatched from eggs laid by mock-injected, Bm86- and Bm91-dsRNA injected engorged females were fed in three patches on calf #7799.\n2.3\nRNA extraction and synthesis of tick cDNA for dsRNA preparations\nThe viscera of five partially fed B. microplus females were dissected in ice-cold PBS and immediately stored in 1\u00a0ml Tri reagent (Sigma\u2013Aldrich, Zwijndrecht, The Netherlands) at \u221280\u00a0\u00b0C. Total RNA was isolated and subsequently purified using the Nucleospin RNA II kit (Macherey-Nagel, D\u00fcren, Germany) in accordance with the reagent and kit manufacturer\u2019s directions. Total RNA concentration was determined spectrophotometrically and the material was stored at \u221280\u00a0\u00b0C before use. Complementary DNA was made with the Revertaid first strand cDNA synthesis kit (Fermentas, St. Leon-Rot, Germany) in accordance with the manufacturer\u2019s protocol using random hexamer primers. Control reactions were performed using the same procedures but without RT as a control for DNA contamination in the RNA preparations.\n2.4\nCloning and sequencing of the B. microplus subolesin gene\nCloning and sequencing of the subA gene from the Mozambiquan B. microplus strain was performed as described elsewhere (de la Fuente et al., 2006a). The sequence has been submitted to GenBank and can be retrieved under Accession No. DQ923495.\n2.5\ndsRNA synthesis\nOligonucleotide primers containing T7 promotor sequences at the 5\u2032-end for in vitro transcription and synthesis of dsRNA were used to PCR-amplify cDNA encoding B. microplus Bm86 (421\u00a0bp), Bm91 (417\u00a0bp) and subolesin (381\u00a0bp). All oligonucleotide primers used in this study were synthesised by Isogen Life Science, IJsselstein, The Netherlands and their sequences are shown in Table 1. PCR products were purified using the GfX PCR purification kit (Amersham) and used as templates to produce dsRNA using the T7 Ribomax Express RNAi system (Promega, Leiden, The Netherlands). dsRNA aliquots were stored at \u221280\u00a0\u00b0C until used.\n2.6\nInjection of ticks with dsRNA\nFreshly molted females were placed on double-sided sticky tape with the ventral sides upwards and injected into the anal aperture with 0.5\u00a0\u03bcl Bm86, Bm91 or subolesin dsRNA alone or a combination of Bm86 and subolesin dsRNA (6\u20139\u00a0\u00d7\u00a01011\u00a0molecules\/\u03bcl) using a 10\u00a0\u03bcl syringe with a 33\u00a0G needle (Hamilton, Bonaduz, Switzerland) mounted on a MM3301-M3 micromanipulator (World Precision Instruments (WPI), Berlin, Germany) and connected to an UMPII syringe pump (WPI). The tip of a 27\u00a0G needle was used to slightly pierce the anal aperture before the 33\u00a0G needle was inserted. The dsRNA was dissolved in injection buffer (10\u00a0mM Tris\u2013HCl, pH 7 and 1\u00a0mM EDTA). A control group was injected with injection buffer alone. The ticks were placed in an incubator at 27\u00a0\u00b0C with 95% relative humidity for 3\u201310\u00a0h following injection, before they were examined for mortality and placed in five separate patches, one for each group, on calf #7793. One hundred male ticks were placed in each patch simultaneously with the injected females. The ticks were checked twice daily and collected when they dropped from the host. Ticks still attached 14 days after the dsRNA-injection (day 29 after application of the larvae) were removed manually. All ticks were weighed separately within 1\u00a0h after collection and stored individually in 1.5\u00a0ml Eppendorf tubes with pierced lids at 27\u00a0\u00b0C and 95% relative humidity for oviposition. For the second experiment, engorged B. microplus females were injected with 5\u00a0\u03bcl of Bm86, Bm91 or subolesin dsRNA (1\u20132\u00a0\u00d7\u00a01012\u00a0molecules\/\u03bcl) or injection buffer alone in the right spiracular plate within 6\u00a0h after dropping off the host, using the same methods as described above, or left uninjected. They were stored individually in 2\u00a0ml Eppendorf tubes with pierced lids in an incubator at 27\u00a0\u00b0C and 95% relative humidity. Eggs were removed daily and each daily egg batch was stored separately under the same conditions.\n2.7\nAnalysis to confirm gene silencing by quantitative RT-PCR\nViscera was dissected from five females of each dsRNA-injected or mock-injected group after 6 days of feeding. Total RNA was isolated from these samples using Tri reagent and subsequently purified using the Nucleospin RNA II kit in accordance with the reagent and kit manufacturer\u2019s directions. Total RNA was isolated from 100\u00a0mg eggs (14 days after injection), 50\u00a0mg larvae (at 6 days and 5 weeks after hatching) laid by\/eclosed from the dsRNA- and mock-injected engorged females, 50\u00a0mg larvae at 10 weeks after hatching laid by the dsRNA- and mock-injected unfed females and from the dissected viscera of five females and five males which developed from 7-week-old larvae fed on animal #7799 using the same methods. cDNA from 1\u00a0\u03bcg of RNA (adults, eggs and 6-day-old larvae) and 0.3\u00a0\u03bcg of RNA (5-week-old larvae) was prepared using the Revertaid first strand cDNA synthesis kit (Fermentas) using random hexamer primers in accordance with the manufacturer\u2019s protocol. All samples were analyzed for transcription of target genes by quantitative real-time RT-PCR using primers Bm86h-F6 and Bm86h-R4, amplifying a 117\u00a0bp section of the Bm86 gene; Bm91-F2 and Bm91-R3, amplifying a 129\u00a0bp section of the Bm91 gene and Bm-subA-F2 and Bm-subA-R2, amplifying a 166\u00a0bp section of the subolesin gene. Tick \u03b2-actin was included as a control and used for normalisation. A 126\u00a0bp fragment was amplified using primers Actin-F2 and Actin-R. All primer combinations amplified a different part of the targeted genes than the sections which were used for dsRNA synthesis, circumventing re-amplification of any unprocessed dsRNA. Twenty-five microlitres of real-time PCRs were performed using the Quantitect SYBR green PCR kit in accordance with the manufacturer\u2019s protocol (Qiagen, Venlo, The Netherlands) on an iCycler real-time detection system (Bio-Rad Laboratories, Veenendaal, The Netherlands). Real-time PCR data were analyzed by iCycler IQ software version 1.0.\n2.8\nAnalysis to check for the presence of dsRNA in eggs\ncDNA from 1\u00a0\u03bcg of total RNA of eggs 14 days post-oviposition was screened by quantitative real-time RT-PCR for the presence of non-processed Bm86, Bm91 and subolesin dsRNA to see whether the injected dsRNA was incorporated into the eggs and could be re-amplified. Oligonucleotide primers located within the region used for dsRNA synthesis of the Bm86, Bm91 and subolesin genes were used for this purpose. The following primer combinations were used: Bm86-F7 and Bm86h-R3, amplifying a 121\u00a0bp section of the Bm86 gene, primers Bm91-F3 and Bm91-R1, amplifying a 128\u00a0bp region of the Bm91 gene, primers BmsubA-F2 and BmsubA-R1, amplifying a 121\u00a0bp section of the subolesin gene. Real-time RT-PCR conditions were identical to those used to confirm gene silencing.\n2.9\nStatistical analysis\nStatistical analysis of data from two quantitative RT-PCR experiments, the weights of ticks after feeding and oviposited egg masses, was performed using Microsoft Excel and consisted of an unpaired t-test with unequal variances. Tick mortality was compared between the dsRNA- and mock-injected ticks by \u03c72-test. P values of 0.05 or less were considered statistically significant.\n3\nResults\n3.1\nCloning and sequencing of the subolesin gene from B. microplus\nThe subolesin gene (subA) from the Mozambiquan B. microplus strain was cloned and sequenced. This gene was found to be 99\u2013100% identical to subA from B. microplus strains from Mexico and Brazil (de la Fuente et al., 2006a; our unpublished data).\n3.2\nRNAi in freshly molted B. microplus females\nFive groups consisting of 120 freshly molted B. microplus females were each injected with 0.5\u00a0\u03bcl of injection buffer in the following groups: (i) injection buffer alone, (ii) Bm86, (iii) Bm91, (iv) subolesin and (v) both Bm86 and subolesin-dsRNA. An average of 81 (76\u201382; 32.8% overall mortality) females were alive in each group 3\u201310\u00a0h following injection. These ticks were subsequently fed together on a calf with an excess of B. microplus males until the females became replete or for a maximum of 14 days. Tick weight after engorgement or manual removal, mortality rate, egg mass and hatching rate is presented in Table 2. A significant decrease in tick weight and oviposited egg mass, together with a higher mortality rate, was observed in the subolesin dsRNA injected groups compared with the control group (P\u00a0<\u00a00.01). Hatching rates were uniformly constant in the control, Bm86- and Bm91-dsRNA-injected groups (>90%), while in the ticks injected with subolesin dsRNA the hatching rate was lower (<20%). Gene silencing was confirmed by quantitative real-time RT-PCR (Fig. 1). The normalised transcript level of Bm86 was reduced with 86% in the Bm86-dsRNA-injected ticks and with 79% in the combined Bm86\/subolesin-dsRNA-injected ticks, compared with the normalised transcript level of the mock-injected group (Fig. 1A). For Bm91, the normalised transcript level was reduced with 90% in the Bm91 RNAi-silenced ticks compared with the control ticks. A significant decrease in the Bm91 transcript level of 58% was observed in the subolesin dsRNA-injected ticks (P\u00a0<\u00a00.01) as well, but this reduction was not observed in the combined Bm86\/subolesin-dsRNA-injected ticks (Fig. 1B). Normalised transcript levels of subolesin in the subolesin dsRNA- and combined Bm86\/subolesin dsRNA-injected ticks compared with the control group were reduced with 90% and 80%, respectively (Fig. 1C). No differences were observed in the normalised Bm86 and Bm91 transcript levels of 10-week-old larvae which hatched from the Bm86- and Bm91-dsRNA-injected ticks compared with the mock-injected ticks (results not shown).\n3.3\nRNAi in engorged B. microplus females\nFive groups of five engorged females each with an average weight of 261\u00a0mg (248\u2013272\u00a0mg) were injected with 5\u00a0\u03bcl of Bm86 dsRNA, Bm91 dsRNA, subolesin dsRNA, injection buffer alone or left uninjected within 6\u00a0h after dropping from the host. No reflux of the injected solution or hemolymph was observed from the puncture when the needle was gently withdrawn. Oviposition began in all groups within 3 days, except for one tick from the control-injected group and another tick from the Bm91 dsRNA-injected group which did not lay any eggs. The course of oviposition was not significantly influenced by the injection of dsRNA (Table 3), and dried or shriveled eggs were not observed, indicating that all eggs were successfully coated with a secretion from Gen\u00e9\u2019s organ. Interestingly, nearly all (>99.4%) eggs oviposited by engorged females injected with subolesin dsRNA showed an aberrant phenotype compared with those from the other groups. A typical example is shown in Fig. 2. Development of embryos in these eggs was not observed while many undifferentiated cells with some yolk cells were seen in Giemsa-stained egg crush smears. Most eggs did not hatch and eventually dried up and shriveled after 6\u20137 weeks of incubation at 27\u00a0\u00b0C\/95% relative humidity. The few eggs from this group which did develop and hatched normally (<0.6%) were all laid during the first day of oviposition.\nA 64% decrease of subolesin transcript levels was found in eggs from the subolesin dsRNA-injected engorged females (Fig. 3C). Interestingly, a 30-fold increase in the Bm86 transcript level and a 56% decrease in Bm91 transcript level were also found in these aberrant eggs (Figs. 3A and B). An 84% decrease in the number of Bm86 copies was observed in eggs laid by the engorged females injected with Bm86 dsRNA (Fig. 3A) and the transcript level of Bm91 was reduced by 97% in eggs from the Bm91 dsRNA-injected engorged females compared with the mock-injected control group (Fig. 3B), confirming gene-specific silencing in the eggs of dsRNA-injected engorged females.\nInjected dsRNA could be re-amplified from the eggs of dsRNA-injected engorged females when primers located within the dsRNA sections were used, instead of primers located downstream of the dsRNA region which were used to demonstrate gene silencing (Table 1). Results of quantitative real-time RT-PCR performed with these primers showed levels of Bm86, Bm91 and subolesin which were 10.5, 4.3 and 4.5 times higher, respectively, in the Bm86, Bm91 and subolesin injected groups than levels found in the mock-injected group (Fig. 4).\nGene silencing was observed in larvae 6 days after hatching; a decrease of 86% in Bm86 transcript level and of 91% in Bm91 transcript level in the Bm86- and Bm91 dsRNA-injected groups, respectively (Figs. 5A and B, grey bars). Subolesin transcript levels were not measured in the few larvae which hatched from the subolesin dsRNA-injected females due to their small number. Quantitative real-time RT-PCR analysis of RNA extracted from larvae 5 weeks after hatching (9 weeks after the initial injection of engorged females with dsRNA) revealed that genes remained silenced in both Bm86- and Bm91-silenced groups (Figs. 5A and B, black bars). This effect diminished over time, in particular for the Bm86-silenced group, in which transcript levels were now 67% lower compared with the control group. Bm91 transcript levels were still 90% lower in the Bm91-silenced group compared with the control group. When 7-week-old larvae from these three groups were fed in separate patches on a calf and total RNA from the viscera of adults which had developed from these larvae was analyzed by quantitative real-time RT-PCR, gene silencing was not observed (results not shown).\n4\nDiscussion\nThe production characteristics of the subolesin-silenced female B. microplus ticks corresponded with previous results from subolesin RNAi studies in other ixodid tick species, and typically resulted in decreased tick and egg mass weights and high mortality (de la Fuente et al., 2005, 2006a,c). The synergistic effect of combined silencing of the Bm86 and subolesin gene reported previously in R. sanguineus (de la Fuente et al., 2006c) could not be confirmed for B. microplus (Table 2).\nSignificant changes in the production characteristics of the Bm86- and Bm91-silenced females were not observed, suggesting that the deleterious effect on ticks feeding on Bm86- or Bm91-vaccinated cattle is not caused by a loss of function of the Bm86 or Bm91 protein alone. In fact, the protective effect of Bm86-based vaccines is through gut damage mediated by anti-Bm86 antibodies during tick feeding (Willadsen et al., 1989). These results suggest that the protection mechanisms of Bm86 and subolesin-based vaccines are different and may contribute to the increased efficacy of Bm86 and subolesin combined vaccines.\nQuantitative real-time RT-PCR performed on samples taken 6 days after dsRNA injection and feeding resulted in a decrease of 79\u201390% of the targeted gene transcript level compared with the mock-injected group. This result was comparable with previous semi-quantitative measurements of the gene silencing effect by dsRNA-injection in A. americanum. In this RNAi study, a decrease of \u223c90% to 50% in cystatin transcript level was observed from 24\u00a0h to 9 days of feeding (Karim et al., 2005). In the present study, although gene silencing was specific in the Bm86 and Bm91 dsRNA-injected groups, subolesin dsRNA injection resulted in both significantly decreased subolesin as well as Bm91 expression levels. This effect was not observed in the combined Bm86\/subolesin silenced group, which may be explained by a slight difference in midgut:salivary gland ratio in the dissected viscera between the groups. Since Bm91 is present in relatively high concentrations in salivary glands compared with midgut (Riding et al., 1994), a shift in the midgut:salivary gland ratio of the dissected viscera in favor of midgut tissue could result in the lower Bm91 expression levels we found in this specific sample. Alternatively, the effect of silencing subolesin expression may affect the expression of other genes. The pleiotropic effect on tick tissues in which subolesin expression has been silenced suggests that this gene may be involved in the regulation of multiple pathways in ticks (de la Fuente et al., 2006a).\nInjection of pilocarpine solution into the hemocoel via the spiracular plate of engorged ticks has been described for inducement of salivation in engorged B. microplus ticks (Bechara et al., 1988). These openings of the tracheae are sclerotised structures located posterior to the fourth pair of legs. Their rigid structure allows for puncturing and injection of small quantities of fluid by a fine needle without subsequent reflux of the injected solution, hemolymph or tissue. When we injected Bm86, Bm91 or subolesin dsRNA into the hemocoel of engorged females, the course of oviposition appeared to be unaffected. Only one Bm91 dsRNA-injected female and one mock-injected female tick died before ovipositing. Interestingly, embryogenesis was undisturbed in eggs oviposited by the Bm86 dsRNA-, Bm91 dsRNA- and mock-injected engorged females, but an aberrant development was seen in the majority of egg masses oviposited by the subolesin dsRNA-injected engorged females. This egg phenotype has not been described previously and suggests that subolesin plays a role in embryonic development. When total RNA isolated from these aberrant eggs was analyzed by real-time RT-PCR, significantly higher Bm86 levels and decreased Bm91 and subolesin levels were found. Again, these results suggest that subolesin may be a regulator of transcription in ticks.\nInjected dsRNA was detected in eggs from dsRNA-injected engorged females by real-time RT-PCR using primers located within the dsRNA section, indicating that unprocessed dsRNA is incorporated in the eggs. The suggested route of incorporation of exogenously produced yolk directly from the hemolymph into oocytes (Saito et al., 2005) may be followed for the incorporation of dsRNA into oocytes as well. Further experiments are needed to determine whether this dsRNA forms a reservoir of mobile silencing signals inducing gene-specific silencing in eggs, or whether small interfering RNAs are responsible for this effect. Other possible routes for the mobile silencing signal to enter the oocyte are through the pedicel in the ovary or, once the oocyte has ovulated, by contact with cells from the genital tract.\nSome eggs (<0.6%) found in the batches laid during the first day of oviposition by subolesin dsRNA-injected engorged females, hatched normally. It is likely that these eggs developed to a stage which was not accessible by the dsRNA prior to injection of the dsRNA. They may have been ovulated eggs which were not in direct contact with the hemolymph or ones in which the shell was hardened during the oocyte passage through the oviduct and thus became impermeable to dsRNA during this process (Diehl et al., 1982).\nAfter dsRNA was injected into the body cavity of B. microplus, the gene silencing effect spread throughout the organism and its progeny. This systemic RNAi has been associated with the sid-1 protein, a transmembrane protein which enables passive cellular uptake of dsRNA (Winston et al., 2002; Feinberg and Hunter, 2003). Unfortunately, our attempts to detect a B. microplus sid-1 homologue, as described previously in studies on presence of a sid-1 homologue in grasshopper species Schistocerca americana, were not successful (data not shown) (Dong and Friedrich, 2005). The only protein currently present in the B. microplus expressed sequence tag (EST) database (Guerrero et al., 2005) which is associated with the RNAi machinery is the nuclease Argonaute-2 (Ago-2), the central catalytic component of the RNA-induced silencing complex (RISC) in mammals and Drosophila (Liu et al., 2004; Miyoshi et al., 2005). Homologues of other RNAi-associated proteins such as Dicer, which is responsible for the cleavage of exogenous long dsRNA into short interfering RNA (siRNA), remain to be identified in B. microplus and other tick species as well.\nRNAi described herein provides an important tool to screen for tick-protective antigens in this one-host tick species, B. microplus (de la Fuente et al., 2005) and allowed for characterisation of the effect and function of tick protective antigens, as well as the role of genes involved in tick\u2013host\u2013pathogen interactions and the transmission of tick-borne pathogens (de la Fuente et al., 2006b). Initiation and completion of the B. microplus genome sequencing project would greatly enhance these kinds of studies (Guerrero et al., 2006), as well as the availability of the genomes from cattle and the pathogens transmitted by B. microplus, most notably: A. marginale (Brayton et al., 2005), B. bigemina and B. bovis, which are currently being sequenced. Although sequences of the dsRNAs used in this study do not contain any significant overlap with other known B. microplus genes, the possibility of off-target gene silencing effects cannot be excluded due to the limited amount of sequence data available. Availability of the complete B. microplus genome sequence data will facilitate screening for potential off-target effects. These can subsequently be minimised by avoiding the use of dsRNAs or siRNAs containing sequences which are present in multiple genes.\nThe effect of silencing tick genes suggested to be involved in embryogenesis such as vitellin degrading cysteine endopeptidase (Seixas et al., 2003), in the transovarial transmission of Babesia spp. or genes associated with acaricide resistance, which is measured in larvae by the Larval Packet Test (Li et al., 2003), could be studied using our method to silence genes in B. microplus embryos and larvae by injecting engorged females with dsRNA. These experimental approaches are more efficient and less labour intensive than the three other dsRNA delivery approaches into oocytes and embryos using microinjection (Wargelius et al., 1999), transgenic RNAi (Tavernarakis et al., 2000) and electroporation (Grabarek et al., 2002).","keyphrases":["bm86","bm91","subolesin","one-host tick","boophilus microplus","rnai"],"prmu":["P","P","P","P","P","P"]}
{"id":"Eur_Arch_Otorhinolaryngol-4-1-2217621","title":"Recurrent respiratory papillomatosis: an overview of current thinking and treatment\n","text":"Human papillomaviruses (HPV) infection in benign laryngeal papillomas is well established. The vast majority of recurrent respiratory papillomatosis lesions are due to HPV types 6 and 11. Human papillomaviruses are small non-enveloped viruses (>8 kb), that replicate within the nuclei of infected host cells. Infected host basal cell keratinocytes and papillomas arise from the disordered proliferation of these differentiating keratinocytes. Surgical debulking of papillomas is currently the treatment of choice; newer surgical approaches utilizing microdebriders are replacing laser ablation. Surgery aims to secure an adequate airway and improve and maintain an acceptable quality of voice. Adjuvant treatments currently used include cidofovir, indole-3-carbinol, ribavirin, mumps vaccine, and photodynamic therapy. The recent licensing of prophylactic HPV vaccines is a most interesting development. The low incidence of RRP does pose significant problems in recruitment of sufficient numbers to show statistical significance. Large multi-centre collaborative clinical trials are therefore required. Even so, sufficient clinical follow-up data would take several years.\nIntroduction\nSir Morrell Mackenzie (1837\u20131892) was the first to recognize papillomas as a lesion of the laryngo-pharyngeal system in children in the late 1800s. It is now apparent that these benign tumours may occur at other parts of the upper gastrointestinal and respiratory tracts, and in all age groups. It was not until the 1940s that Chevalier Jackson (1865\u20131958) first coined the term \u201cjuvenile laryngeal papillomatosis\u201d. The prevalence of laryngeal papillomatosis has been estimated at between four to seven cases per million person-years in the Western World [4, 5, 25, 43]. Furthermore, the incidence of recurrent respiratory papillomatosis (RRP) has been estimated at about 2 per 100,000 in adults and 4 per 100,000 in children [9]. The disease can be categorized into adult onset and juvenile onset forms. Age of first presentation of disease is usually in the teens (50%) for the juvenile onset form but can be as early as the first year of life. Initial presentation in the adult form tends to peak in the third and fourth decades.\nIt is now well established that human papillomaviruses (HPVs) are the aetiological agent of many benign and malignant tumours arising from epidermal tissues. They are a necessary cause of the second most common female cancer worldwide, cancer of the cervix [7, 45], and strongly associated with several other ano-genital cancers such anal, penile, vulval and vaginal carcinomas [17]. Furthermore, there is mounting evidence of at least some head and neck cancers associated with HPV infection [15, 17, 23]. These malignancies are associated with \u223c15 high risk (HR) types, in particular HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82. Benign tumours such as common warts, flat warts and genital warts are caused by low risk types such as HPV 1, 2, 3, 4, 6, 10, 11 and others. HPV 6 and 11 have been described as the dominant types found in RRP [10]. Despite the benign nature of these lesions, there is significant morbidity and occasional mortality due to multiple recurrences which necessitate hospital admission for surgical removal. Dissemination or extension of the growths into the lower airways indicates a poorer prognosis. The clinical behaviour is variable and lesions can regress, persist and in rare instances, progress to carcinoma if other environmental factors such as smoking or irradiation are involved.\nEpidemiology\nHuman papillomaviruses infection in benign laryngeal papillomas is well established. One Danish study showed that 95% of solitary laryngeal papillomas were positive for HPV DNA by in situ hybridization [26]. Another study from Hong Kong found that 59% of laryngeal papillomas showed the presence of HPV 6, 11, 16, 18 subtypes, with 6 and 11 the dominant types [10]. Malignant transformation of some lesions has been described in association with HPV 11 integration into the genome and mutation of p53 [32]. Infection with HPV 11 is more likely to be associated with the development of distal airways disease than HPV 6. Clinical manifestation of disease prior to three years of age is a further risk factor associated with distal spread of disease [1, 47].\nThe vast majority of RRP lesions are due to HPV types 6 and 11, and the reservoir for these types is the human ano-genital tract. Ano-genital HPV is the commonest sexually transmitted viral infection and the prevalence of clinically apparent genital warts is thought to be the \u201ctip of the iceberg\u201d of HPV infection. Koutsky et al. estimates that \u223c10\u201320% of the US population between 15 and 49\u00a0years of age have molecular evidence of infection and that another 60% have had prior infection [22]. In the UK, there were well over 81,000 incident genital wart infections reported from genitourinary medicine clinics in 2005 [16] and this has been increasing every year. Such high background prevalence suggests that there is a risk of mother to child transmission at the time of delivery, especially if there are noticeable genital warts. Data supporting this hypothesis showed that a history of maternal condylomata during pregnancy was associated with a 200-fold risk of RRP in the child [37]. An uncomplicated vaginal delivery in a mother with HPV infection has been estimated to carry a risk of transmission of 1:80\u20131:1,500 (median of 1:400) [34]. In one group of children with juvenile laryngeal papillomatosis, 54% had a maternal history of vulval condylomata at the time of delivery [13]. Another study found that in 77 mothers with condylomata at delivery, 9 children (11.6%) were later diagnosed with juvenile laryngeal papillomatosis [21].\nThe route of transmission is likely to be different in the juvenile onset and adult onset forms of RRP. Evidence for this has been suggested by a case control study, in which the risk factors for both forms were compared [20]. The authors found that adult onset patients were more likely to have had more sexual partners and oral sex than their controls. Patients with the juvenile form were more likely to have been born to teenage mothers and first-born children compared to their controls.\nAetiological and histopathological features\nPapillomatous lesions preferentially occur anatomically at the sites of \u201ctransformation zones\u201d, where squamous epithelia abut ciliated columnar epithelia but can infect anywhere in the respiratory tract [28]. The classical sites for recurrent disease in the upper aero-digestive tract would be the nasopharyngeal area of the soft palate, limen vestibuli, midzone of laryngeal area of the epiglottis, upper and lower margins of the ventricle, vocal fold undersurface, carina and bronchial spurs. It is interesting to note that papillomata have been observed at tracheotomy sites and tracts where the iatrogenic induction of change of epithelialization also occurs [19].\nHuman papillomaviruses are small nonenveloped viruses (\u223c8\u00a0kb), with a double stranded circular DNA genome encapsulated within an icosahedral capsid that replicate within the nuclei of infected host cells. The genome codes for 8\u201310 genes (median of eight). The late L1 and L2 genes code for the viral capsid proteins, the early proteins E1 and E2 are responsible for viral replication and transcription, and E4 appears to aid virus release from infected cells. The early genes E6 and E7 have transforming ability in in vitro assays for HR types but LR types have little to no ability for this feature [2, 33, 42].\nElectron microscopic analysis reveals the virion to be \u223c55\u00a0nm in diameter and the capsid to be comprised of 72 pentameric capsomers. The predominant protein in the capsid consists of the L1 protein, with a smaller proportion of L2 embedded deep within the protein shell. It is this L1 protein which provides the dominant antigenic epitopes recognized by neutralizing antibodies and forms the basis for the bivalent (GlaxoSmithKline) and quadrivalent (Merck, Hohenbrumn, Germany) vaccines currently available.\nThe virus is thought to bind to and gain entry to its host cell, the basal keratinocyte, by microtrauma or abrasions to the surface epithelium. The receptor has not been definitively identified but \u03b16-integrin and heparin sulphate may play important roles in viral entry [11, 18, 30].\nFollowing infection and uncoating, the virus is thought to maintain its genome as a low copy number episome in the basal cells. It has been suggested that expression of E1 and possibly E2, may be sufficient for basal maintenance of viral episomes [50]. Viral early proteins E6, E7, E1 and E2 are expressed at low level in early passage cell lines derived from naturally occurring low-grade cervical lesions and the viral genome is maintained at around 10\u2013200 copies per cell [8, 41].\nThe viral genome is amplified in differentiating keratinocytes via rolling-circle amplification that will synthesize sufficient viral genome for packaging [3, 12]. This requirement for differentiating epithelial cells is a key part of the virus life cycle but the normal restraint on cell cycle progression appears to be abolished by the E6 and E7 proteins and normal terminal differentiation is retarded [36]. These E6 and E7 effects on key apoptotic proteins such as Rb and p53 have been demonstrated in HPV 16 and other high-risk subtypes in in vitro assays [24, 27, 28]. HPV 6 and 11 E6 and E7 proteins do not readily bind to or degrade the p53 or Rb proteins [14, 31, 46]. This suggests that alternative mechanisms of altered cellular growth and proliferation may exist for the low-risk subtypes of HPV. Furthermore, there are little data on HPV 6 and 11 life cycles, replication, maintenance and viral production in respiratory cells. It is not known if these are similar to or different to disease in the ano-genital region.\nTreatment\nSurgical\nSurgical debulking is currently the treatment of choice; newer surgical approaches utilizing microdebriders are replacing laser ablation. Surgical excision aims to secure an adequate airway and improve and maintain an acceptable quality of voice [49].\nHPV is present in the normal macroscopically unaffected mucosa and it is currently not possible to distinguish infected cells with a normal appearance from uninfected epithelia. Repeated recurrences are frequent, however, repeated attempts to treat the papillomas may cause serious complications [40]. Current practice in the treatment of RRP was recently evaluated by a questionnaire in the UK [44]. Various lasers such as CO2, KTP, and pulsed dye were found to be the preferred method of surgical removal of RRP in children [49]. Spontaneous ventilation (65.3%) is the preferred method of anaesthesia.\nThe frequent recurrence of papillomas has resulted in the use of different adjuvant treatments alongside surgical removal of macroscopically obvious in the attempt to improve outcomes. In the future, advances in the understanding of the immune response to HPV may improve our treatment modalities and prevention strategies.\nAdjuvant treatment\nAdjuvant treatments currently used include cidofovir, indole-3-carbinol, ribavirin, mumps vaccine, and photodynamic therapy. As with surgical management, viral persistence occurs following treatment with these adjuvant modalities. Intralesional cidofovir may help control papilloma regrowth and reduce disease severity in many children with RRP [39]. In most cases, cidofovir would appear to be less efficacious in producing disease eradication. There appears to be little evidence to support prolonged treatment regimes (i.e. more than eight treatments) [35]. Subcutaneously injected cidofovir has been tested on cartilage in a rabbit model [39]. There was a positive dose-response relationship which existed for gross skin changes; however, there was no dose-response relationship for severity of change in the epithelium. Higher doses of cidofovir than commonly are used in the treatment of RRP may be safe, although the effects of repeat application and long-term complications are not yet known. In animals, cidofovir is carcinogenic (mammary adenocarcinoma in rats), embryotoxic and teratogenic [48]. Care must be taken in humans that the possibility of pregnancy is excluded when usage is considered. In view of the severe nephrotoxicity shown when intravenous cidofovir is administered to animals and humans, caution would be advised for repeated intralesional or subcutaneous applications for RRP treatment. The less common complications of bone marrow toxicity, iritis and uveitis may also arise and vigilance is required from clinicians.\nControlled trials failed to provide sufficient evidence to draw reliable conclusions about the effectiveness of antiviral agents as adjuvant therapy in the management of RRP. Further research is required before any specific antiviral adjuvant therapy can be recommended.\nFactors leading to virus activation in RRP have not been recognized, however, extra-oesophageal acid reflux disease (EERD) has been suggested as a possible factor, initially by Borkowski et al. [6], and then by a group from Harvard in 2005 [29]. There is clinical evidence suggesting a link between the presence of EERD and RRP. Inflammation induced by acid exposure may result in the expression of HPV in susceptible tissues. Therefore, treatment of EERD should be considered in all patients with difficult to control RRP with EERD.\nDespite currently available surgical and adjuvant management options, tracheotomy may become necessary in selected patients with extensive disease. Decannulation should be performed as early as possible to avoid further spread of viral infection and improve the quality of life. The primary cause of papilloma extension to the lower airways appears to be iatrogenic, i.e. the tracheotomies performed in children with laryngeal papillomatosis (92.5% of cases). This was reported in a case group of 448 children with RRP treated in St. Vladimir Moscow Children\u2019s Hospital between 1988 and 2003 [38].\nThe recent licensing of prophylactic HPV vaccines is a most interesting development. In particular, the quadrivalent vaccine from Merck & Sanofi-Pasteur (Gardasil\u00ae) which shows efficacy against HPV 6, 11, 16, 18 subtypes, may be anticipated to impact upon the incidence of RRP. The low incidence of RRP does pose significant problems in recruitment of sufficient numbers to show statistical significance. Large multi-centre collaborative clinical trials are therefore required. Even so, sufficient clinical follow-up data would take several years.","keyphrases":["recurrent respiratory papillomatosis","vaccination","hpv 6 and 11","human papilloma virus"],"prmu":["P","P","P","R"]}
{"id":"Eur_Radiol-4-1-2373858","title":"Contrast-enhanced magnetic resonance imaging of the breast: the value of pharmacokinetic parameters derived from fast dynamic imaging during initial enhancement in classifying lesions\n","text":"The value of pharmacokinetic parameters derived from fast dynamic imaging during initial enhancement in characterizing breast lesions on magnetic resonance imaging (MRI) was evaluated. Sixty-eight malignant and 34 benign lesions were included. In the scanning protocol, high temporal resolution imaging was combined with high spatial resolution imaging. The high temporal resolution images were recorded every 4.1 s during initial enhancement (fast dynamic analysis). The high spatial resolution images were recorded at a temporal resolution of 86 s (slow dynamic analysis). In the fast dynamic evaluation pharmacokinetic parameters (Ktrans, Ve and kep) were evaluated. In the slow dynamic analysis, each lesion was scored according to the BI-RADS classification. Two readers evaluated all data prospectively. ROC and multivariate analysis were performed. The slow dynamic analysis resulted in an AUC of 0.85 and 0.83, respectively. The fast dynamic analysis resulted in an AUC of 0.83 in both readers. The combination of both the slow and fast dynamic analyses resulted in a significant improvement of diagnostic performance with an AUC of 0.93 and 0.90 (P = 0.02). The increased diagnostic performance found when combining both methods demonstrates the additional value of our method in further improving the diagnostic performance of breast MRI.\nIntroduction\nBreast cancer is the most commonly diagnosed cancer in women and the most prevalent cancer worldwide [1]. In breast imaging, mammography is still the most commonly used imaging techniques both in screening for and staging of breast cancer. However, dynamic contrast-enhanced magnetic resonance imaging (MRI) is becoming an increasingly important imaging modality in the detection and staging of breast cancer. Because of its superior sensitivity for the detection of invasive breast cancer, MRI has become a very important modality in breast imaging [2\u20135].\nHowever, the classification of a lesion detected on MRI as benign or malignant still remains a challenge. Reported specificities in clinical studies range between 20% and 100% [6\u201315]. The main characteristics used for classification of detected lesions on MRI are the lesion morphology and the enhancement dynamics [4]. Dynamic evaluation is often based on late dynamic characteristics of enhancing lesions. In this approach, the decrease of signal intensity, often referred to as a type 3 curve or washout, is highly suggestive for breast cancer with the likelihood for malignancy of 87% [12]. This dynamic evaluation makes use of high-resolution T1-weighted MRI images with a relatively low time resolution of 42 s or more [3, 12, 16\u201320]. The high spatial resolution of these sequences is necessary for accurate morphologic evaluation. Irregular lesion contour, inhomogeneous internal enhancement and rim enhancement have been described as features indicating a malignancy [21].\nSchnall et al. [4] found focal mass margins and signal intensity to be a highly predictive imaging features.\nHowever, the combination of both dynamic and morphological parameters resulted in the highest diagnostic accuracy in multivariate analysis.\nThe evaluation of early enhancement using a high temporal resolution has also been a subject of study in breast MRI. Boetes et al. [6] found in a group of 87 lesions a sensitivity of 95%, a specificity of 86% and an overall accuracy of 93% based on early enhancement characteristics. In this study, a temporal resolution of 2.3 s was achieved using a turboFLASH sequence. The value of a high temporal resolution during initial enhancement was confirmed by Sardanelli et al. [22] who used a temporal resolution of 15 s during the first minute of enhancement. The overlap between malignant and benign enhancement curves was only 9% using the fast dynamic evaluation compared with 50% using a lower temporal resolution of 1\u00a0min.\nThe value of first pass high temporal resolution imaging for the differentiation of benign and malignant lesions was studied in a direct comparison of steady-state dynamic MRI (30 s temporal resolution) and first-pass imaging (2 s temporal resolution) of induced mammary tumors in female rats by Helbich et al. [23]. In their study, an estimate of first-pass perfusion using T2*-weighted imaging almost reached a significant difference between benign and malignant tumors. All other methods used, including T1-weighted first-pass imaging, failed to differentiate benign from malignant tumors. Gibbs et al. [24] also used a high temporal resolution (10.5\u201314.5 s) in the evaluation of small breast lesions and evaluated their data using a pharmacokinetic model. The incorporation of data from pharmacokinetic modeling in the evaluation of lesions improved diagnostic accuracy in their group.\nHigh temporal resolution sequences often cover a limited area of the breast [6, 24]. These imaging protocols are, therefore, less suitable in clinical MRI or screening. For this study, we adjusted the scanning protocol in order to obtain a high temporal resolution during initial enhancement while covering both breasts entirely. The aim of this study is to asses the value of pharmacokinetic parameters derived from fast dynamic contrast enhanced imaging during initial enhancement in differentiating between benign and malignant breast lesions on MRI.\nMaterials and methods\nPatient selection\nAll lesions detected on clinically performed breast MRI examinations in the period from January 2004 until June 2005 were initially included. All detected lesions were evaluated based on the following inclusion criteria: (1) histological confirmed diagnosis or (2) follow-up based on unchanged MRI morphology and enhancement characteristics during at least 24\u00a0months indicating a benign nature of the lesion [25]. Lesions that could not be classified as benign or malignant using these criteria were excluded. The protocol was approved by the institutional review board.\nImaging protocol\nAll patients were examined using a 1.5-Tesla MRI scanner (Sonata or Symphony, Siemens, Erlangen, Germany) in combination with a double breast coil. In premenopausal women, the MRI examination was performed in the second week of the menstrual cycle to minimize enhancement of normal glandular tissue [26]. Prior to the MRI examination, an intravenous catheter was inserted in the left or right arm. All patients were placed in the prone position with the breasts in the double breast coil and positioned at the isocenter of the magnet. After localizer images were obtained in three directions, low spatial resolution proton-density-weighted images were acquired in the transverse plane covering both breasts completely (TE 1.56, TR 800, FA 8, FOV 320, slices 24, TA 50\u00a0s, image resolution 3.9\u00a0mm\u2009\u00d7\u20091.3\u00a0mm\u2009\u00d7\u20094.0\u00a0mm). Subsequently, a coronally orientated high-resolution three-dimensional fast low-angle shot series (FLASH 3D) was acquired (TE 4, TR 7.5, FA 8, FOV 320, slices 120, TA 86 s, image resolution 1.3\u00a0mm\u2009\u00d7\u20091.3\u00a0mm\u2009\u00d7\u20091.3\u00a0mm). Thereafter, high temporal resolution T1-weighted images (turboFLASH) were recorded 22 times with identical spatial resolution and orientation as the proton-density-weighted images (TE 1.56, TR 66, FA 20, FOV 320, slices 24, TA 22\u2009\u00d7\u20094.1\u00a0s) during an intravenous bolus injection of a paramagnetic gadolinium chelate\u20140.2\u00a0mmol of gadoterate meglumine (Dotarem; Guerbet, The Netherlands) per kilogram of body weight\u2014which was administered with a power injector (Spectris; Medrad, Pittsburg, USA) at 2.5\u00a0ml\/s and followed by a 15-ml saline flush. Following these series, the FLASH 3D series was repeated four times. Total scan time for this protocol was 9\u00a0min 42 s, including the time needed to record localizer images.\nImage evaluation\nFor the evaluation, the MRI data were divided into two sets of dynamic data for each patient. The first dataset contained the high spatial resolution T1-weighted images (FLASH 3D) only. These were used for the evaluation of both lesion morphology as well as signal intensity versus time curves. This method will be further referred to as the \u2018slow dynamic\u2019 analysis. The second dataset contained the proton-density-weighted images, the high temporal resolution images as well as the precontrast high spatial resolution sequence. A high-resolution subtraction of the pre- and first postcontrast FLASH3D series prepared on the MRI scanner was also included in this dataset to aid in lesion detection. The proton-density-weighted sequence was necessary for an accurate estimation of the T1 value necessary for the quantitative analyses. The evaluation of this dataset will be further referred to as the \u2018fast dynamic\u2019 analysis. In this fast dynamic analysis, the last three postcontrast FLASH 3D series were not used.\nAll cases were evaluated prospectively by two experienced breast MRI radiologists (reader 1 and reader 2). Both readers had over 5\u00a0years of experience in dynamic breast MRI. The evaluation on the two workstations was performed independently in different sessions with at least a 2-month time interval between both sessions.\nFor the slow dynamic analysis, a dedicated breast MRI workstation was used (Dynacad, Invivo, Germany). This workstation creates subtraction images for all time points after contrast administration, of which the first is automatically displayed together with the precontrast T1 acquisition, both in a coronal orientation. Furthermore, axial reconstructions were displayed for both the subtracted and T1-weighted images with color overlays of wash-in\/wash-out enhancement characteristics projected over the T1-weighted images [27]. A maximum intensity projection and signal intensity versus time curves were also displayed. This display protocol resembles the protocol used in the clinical workflow of dynamic breast MRI in our hospital. A BI-RADS classification was assigned for each lesion based on their morphology and enhancement dynamics [28]. No clinical information, mammography or prior MRI data were provided to the readers during the evaluation of the cases.\nIn the fast dynamic analysis, a workstation, developed in-house for the evaluation of dynamic contrast enhanced MRI, was used [29, 30]. On this workstation, pharmacokinetic parameters derived from the high temporal resolution turboFLASH series were automatically calibrated, calculated and displayed using color overlays. Examples of the recorded high temporal resolution enhancement versus time curves are presented in Fig.\u00a01. In the preparation of this high temporal resolution data, each MRI signal enhancement\/time curve was first fitted to a general exponential signal enhancement model, as described previously [31]. This reduces a curve to model with the following five parameters: baseline (s0); start of signal enhancement (t0), which defines the onset of the exponential curve; time-to-peak (ttp), the exponential constant; peak enhancement (sp), the signal amplitude at which the exponential curve levels off; and late wash, defined as the slope of the late part of the exponential curve. The reduced signal enhancement\/time curve was converted to a reduced tracer concentration (mmol\/ml)\/time curve [31, 32], effectively converting sp to concentration tracer after initial rapid wash-in (often at a peak or plateau level) (Cgd,p). The reduced plasma concentration time curve was estimated using the reference tissue method [33]. Deconvolution of the plasma profile and estimation of pharmacokinetic parameters conformed to the theoretical derivations [34], but was implemented in the reduced signal space as: Ve\u2009=\u2009Cgd,ptissue\/Cgd,pplasma; kep\u2009=\u20091\/(ttptissue \u2013 ttpplasma). Ktrans\u2009=\u2009Ve \u00d7 Kep. Where Ve is an estimate of the extracellular volume [%], Ktrans, the volume transfer constant (1\/min), and kep, the rate constant (1\/min), between extracellular extravascular and plasma space. The subscript \u2018tissue\u2019 stands for a measurement in the tissue under investigation and the subscript \u2018plasma\u2019 for the reference tissue plasma estimate. The reference tissue was automatically determined by selecting a set of voxels in the whole image volume [relative enhancement, (sp-s0)\/s0] larger than 0.2 and smaller than 2.0). This was most often the pectoral muscle, sometimes the liver or spleen. The additionally recorded proton density images were used to correct for the coil profile. The data were presented on the workstation with high-resolution precontrast T1-weighted images in an axial, coronal and sagital reconstruction (FLASH 3D) as background. Color overlays were projected over the images representing Ktrans, kep and Ve parameter values that were based on the high temporal resolution images (turboFLASH). A subtraction image based on the pre- and first postcontrast FLASH 3D series was presented to aid in lesion detection. No criteria for differentiating between benign and malignant lesions were derived from the subtracted images. In this evaluation, the readers selected a region of interest (ROI) within the enhancing lesion. The ROIs were sphere-shaped and placed in an area within the lesion where the parameter values of Ktrans, Ve and kep were highest, based on the color-overlays. The outer limit of the lesion was used as a boundary of the ROI to rule out partial volume effects [35]. This method of ROI selection has previously been referred to as a hotspot method [36]. Each reader placed only one ROI per lesion. From this ROI, the workstation calculated the mean values for each of the pharmacokinetic parameters. Again, no clinical information, mammography or prior MRI data were provided to the readers during the evaluation of the cases. In case of multifocality, the tumor was analyzed as a single lesion.\nFig.\u00a01Relative enhancement versus time curves of a benign (a) and malignant (b) lesion. Note that the slope of enhancement and the level of enhancement is higher for the malignant lesion compared with the benign lesion. These fast dynamic acquisitions were analyzed as described in the Materials and methods section and resulted in the color overlays as presented in Figs. 4 and 5. The data used in this figure were respectively derived from a histopathology proven fibroadenoma and an invasive ductal carcinoma. The same lesions as presented in Figs.\u00a04 and 5\nStatistical analysis\nDifferences in pharmacokinetic parameter values between the malignant and benign group were evaluated using an independent sample t-test. The performance of both methods was compared using a receiver operator characteristic (ROC) analysis. From the slow dynamic analysis, the reader\u2019s final BI-RADS classification of the lesion was used in the ROC evaluation; from the fast dynamic analysis, the mean parameter values calculated from the ROI selected by each reader were used. Multivariate analysis was performed using logistic regression in order to evaluate the possible additional value of both methods to one another. Since the differentiation between benign and malignant lesions is more difficult in smaller lesions a subgroup of all lesions of 2\u00a0cm and smaller were also separately evaluated. The comparison of the various results, including the interobserver variability, was done by using the area under the ROC curve (AUC) as an estimate of diagnostic accuracy. A pairwise comparison was performed to evaluate differences in the AUC. P values <0.05 were considered to indicate statistical significance.\nResults\nA total of 870 consecutive clinical breast MRI examinations in 787 patients were performed. In these studies a total of 188 lesions were detected. Eighty-six lesions could not be included due to lack of histological diagnosis or insufficient follow-up. This resulted in a total of 102 lesions in 96 patients; 34 benign and 68 malignant lesions. The mean age was 51\u00a0years (range 28\u201374\u00a0years). Ninety-four lesions were included based on histological evaluation, eight lesions based on follow-up. Mean lesion size on MRI for the malignant group was 32\u00a0mm (range 9\u201390\u00a0mm) and this was 15\u00a0mm (range 5\u201350\u00a0mm) for the benign lesions. A total of 52 lesions were 2 cm or smaller; 25 malignant (mean lesion size 14\u00a0mm, range 6\u201320\u00a0mm) and 27 benign (mean lesion size 11\u00a0mm , range 5\u201320\u00a0mm).\nThe histological evaluation of the malignant lesions was in 14 cases based only on the core biopsy, in 14 cases based on an excision biopsy or breast saving surgery specimen and in 40 cases based on the mastectomy specimen. Histological proven benign lesions were in 19 cases based on a core biopsy and in seven cases based on an excision biopsy. Eight lesions were proven benign based on follow-up, mean follow-up was 37\u00a0months (range 24\u201352\u00a0months). The histological composition of the entire group is summarized in Table\u00a01. \nTable\u00a01Histological composition of the benign and malignant group of lesions (IDC\u2009invasive ductal carcinoma, DCIS\u2009ductal carcinoma in situ, ILC\u2009invasive lobular carcinoma)Benign (n\u2009=\u200934)Malignant (n\u2009=\u200968)Fibroadenoma11IDC47Fibrosis4DCIS14Adenosis3ILC7Inflammation2Ductal papilloma2Scar tissue1Hyperplasia1Hamartoma1Radial scar1Follow-up8\nImaging results\nIn the slow dynamic analysis reader 1 classified 25 lesions as a BI-RADS 2 (benign:malignant\u2009=\u200922:3), seven as BI-RADS 3 (4:3), 50 as BI-RADS 4 (6:44) and 20 an BI-RADS 5 (2:18). This was respectively 33 (24:9), 12 (4:8), 41 (5:36) and 16 (1:15) for reader 2. The ROC analysis for the slow dynamic analysis resulted in an AUC of 0.85 (95% CI\u2009=\u20090.773\u20130.918) and 0.83 (95% CI\u2009=\u20090.74\u20130.89) for reader 1 and 2, respectively.\nThe mean volume of the ROIs selected by the readers in the fast dynamic evaluation was 0.51\u00a0cm3 for reader 1 (range 0.15\u20131.94\u00a0cm3, SD 0.30) and 0.52\u00a0cm3 for reader 2 (range 0.15\u20131.94\u00a0cm3, SD 0.41). No significant difference was found for ROI size (P\u2009=\u20090.72). The pharmacokinetic parameters used in the fast dynamic analysis showed a significant difference between the benign and malignant group for both readers (Table\u00a02). The diagnostic performance of the fast dynamic analysis resulted in an AUC for Ktrans of 0.82 (95% CI\u2009=\u20090.735\u20130.905) and 0.82 (95% CI\u2009=\u20090.739\u20130.909) for reader 1 and 2. For Ve the AUC was 0.78 (95% CI\u2009=\u20090.682\u20130.873) and 0.77 (95% CI\u2009=\u20090.670\u20130.866) and for the kep parameter 0.72 (95% CI\u2009=\u20090.609\u20130.828) and 0.74 (95% CI\u2009=\u20090.629\u20130.841) for reader 1 and 2, respectively. Scatter plots of Ktrans and V displaying the parameter values of benign and malignant lesions found in the two readers are provided in Fig.\u00a02. The comparison of the diagnostic performance from the slow dynamic analysis with the single parameter fast dynamic analysis showed no significant differences for the Ktrans and V parameter. A significant difference was found for reader 1 between the slow dynamic analysis and the kep parameter (P\u2009=\u20090.02) , the slow dynamic analysis showing better results. This was not found for reader 2 (P\u2009=\u20090.08). \nTable\u00a02Mean pharmacokinetic parameter values categorized for malignant and benign lesions per reader. All parameter values proved significantly higher in the malignant group compared to the benign group (P\u2009<\u20090.01)\u00a0Benign (n\u2009=\u200934)95% CIMalignant (n\u2009=\u200968)95% CIReader 1Ktransa1.20.9\u20131.42.32.1\u20132.6Ve41.634.9\u201348.363.958.6\u201369.1kepa3.02.7\u20133.33.83.5\u20134.0Reader 2Ktransa1.31.0\u20131.52.52.2\u20132.8Ve44.637.2\u201352.067.162.0\u201372.3kepa3.02.6\u20133.33.93.7\u20134.2a1\/min.Fig.\u00a02Scatter plots from the extracellular volume (V) versus the transfer constant (Ktrans) for reader 1 (a) and reader 2 (b). Benign and malignant cases were clustered. Clusters were summarized with an iso-probability contour computed from the cluster mean and covariance at a squared normalized radius of 2. The continuous-line ellipsoid represents the benign subgroup, the dotted-line ellipsoid represents the malignant subgroup\nCombining the pharmacokinetic parameters (Ktrans, Kep and V) in a multivariate analyses resulted in an AUC of 0.83 (95% CI\u2009=\u20090.74\u20130.90) and 0.83 (95% CI\u2009=\u20090.74\u20130.90) for reader 1 and 2. No significant difference was found between the multivariate fast dynamic and the slow dynamic diagnostic performance (P\u2009=\u20090.49 and P\u2009=\u20090.85).\nThe multivariate analysis from all pharmacokinetic parameters combined with the slow dynamic analysis (combined analysis) resulted in an AUC of 0.93 (95% CI\u2009=\u20090.85\u20130.96) and 0.90 (95% CI\u2009=\u20090.83\u20130.95) for reader 1 and 2, respectively. The results from the combined analysis were significantly higher when compared with the fast dynamic analysis for both readers (P\u2009=\u20090.01 and P\u2009=\u20090.02). This was also found for the slow dynamic analysis (P\u2009=\u20090.02 for both readers). The ROC curves are presented in Fig.\u00a03.\nFig.\u00a03ROC curve for reader 1 (a) and reader 2 (b) displaying the fast dynamic, slow dynamic and combined analysis. No significant differences were found between the fast and slow dynamic analysis in both readers. A significant difference was found between the slow dynamic analysis and the combined analysis for both readers (P\u2009=\u20090.02 for both readers). The comparison between the fast dynamic analysis and the combined analysis also resulted in a significant difference for both readers (P\u2009=\u20090.01 and P\u2009=\u20090.02). No significant differences were found between the two readers\nIn the group of lesions of 2\u00a0cm and smaller, the slow dynamic analyses resulted in an AUC of 0.87 (95% CI\u2009=\u20090.75\u20130.95) for reader 1 and 0.79 (95% CI = 0.67-0.91) for reader 2. Overall, the fast analyses resulted in this group in an AUC of 0.83 (95% CI = 0.70\u20130.92) and 0.85 (95% CI = 0.72\u20130.93), respectively. No significant difference was found between the slow and fast dynamic analyses for both readers (P\u2009=\u20090.54 and P\u2009=\u20090.41). The combined analysis resulted in an AUC of 0.97 (95% CI = 0.88\u20130.99) and 0.94 (95% CI= 0.84\u20130.99), respectively. The results from the combined analysis were significantly higher when compared with the fast dynamic analysis for both readers (P\u2009<\u20090.01 and P\u2009=\u20090.04). This was also found when compared to the slow dynamic analysis (P\u2009=\u20090.03 and P\u2009<\u20090.01).\nNo significant differences were found between the two readers in any of the analyses. An example of a benign and a malignant lesion is presented in Figs.\u00a04 and 5.\nFig.\u00a04a Transverse reconstruction of the high-resolution subtraction sequence of the right breast. b Time versus relative enhancement curve of the slow dynamic series. Ktrans (c) and V (d) color overlay images of the right breast, including a scalar bar, to demonstrate the parameter values. The subtraction image shows a rounded, mostly sharp delineated lesion. The time versus signal intensity curve demonstrates a type 1 curve, indicative for a benign lesions. The readers classified this lesion as benign (BI-RADS 2) or probably benign needing follow up (BI-RADS 3) based on the slow dynamic analysis. The Ktrans and V parameter color overlays demonstrate relatively low values for both parameters (see Table 2 for comparison) indicative for a benign lesion. Histopathology proved this lesion to be a fibroadenomaFig.\u00a05a Transverse reconstruction of the high-resolution subtraction sequence of the right breast. b Time versus relative enhancement curve of the slow dynamic series. Ktrans (c) and V (d) color overlay images of the right breast, including a scalar bar, demonstrates the parameter values. The subtraction image shows a spiculated lesion retromammillar. The time versus signal intensity curve demonstrates a type 3 curve (wash-out) suggestive for a malignancy. Both readers classified this lesion as malignant (BI-RADS 4) based on the slow dynamic analysis. The Ktrans and V parameter color overlays demonstrate high values for both parameters (see Table 2 for comparison), indicative for a malignant lesion. Histopathology proved this lesion to be an invasive ductal carcinoma\nDiscussion\nIn this study, we demonstrated that the pharmacokinetic parameters derived from fast dynamic scanning during initial enhancement were a valuable additional tool for the differentiation between benign and malignant breast lesions on MRI. The pharmacokinetic parameters were significantly higher for the malignant group compared with the benign lesions (Table\u00a02). The diagnostic performance of the pharmacokinetic parameters was, compared with the results of the slow dynamic analysis, not significantly different. The combination of both methods, however, did improve the diagnostic performance significantly for both readers. These results were also found in the subgroup analysis of smaller breast lesions.\nThe slow dynamic analysis resembles the evaluation as routinely performed in the clinical workflow in our hospital. The performance of the workstation used in the slow dynamic evaluation has already been investigated and proven by Wiener et al. [27] in the evaluation of breast lesions prior to surgical treatment. Schnall et al. [4] evaluated the performance of both dynamic and morphological features in 854 women with 995 lesions. The results of their multivariate evaluation based on both morphological and relatively slow dynamic lesion characteristics resulted in a similar diagnostic accuracy (AUC values of 0.87 and 0.88) compared with the results obtained in the slow dynamic analyses of our study (0.85 and 0.83). Our results found in the slow dynamic analysis are, therefore, considered representative for the diagnostic performance of an experienced radiologist in this group of patients.\nIn the fast dynamic analysis, both the morphologic characteristics and slow dynamic characteristics were not included in the evaluation; instead, a quantitative analysis of pharmacokinetic parameters was used based on manual ROI placement within the lesion. In the literature, both a \u201chot-spot\u201d and \u201cwhole-tumor method for ROI placement are reported [36]. In this study, we used a hot-spot method. The importance of a consistent ROI placing strategy has been described by Liney et al. [36]. In our study, both readers were instructed with a simple ROI-placing strategy placing the ROI in an area with the highest parameter values guided by color overlays. Since no significant differences were found in the ROC analyses in any of the pharmacokinetic parameters used it is assumed that the performance of the fast dynamic analysis was not negatively affected by this manual ROI selection strategy. The optimal strategy of ROI selection within a breast lesion is a subject that still needs to be further investigated; this is beyond the scope of this study.\nGibbs et al. [24] found the use of quantitative pharmacokinetic parameters in the evaluation of sub 1-cm breast lesions to be beneficial. In their study of 43 women, a diagnostic accuracy of 0.92 was found combining the postcontrast images with the dynamic data in a logistic regression analysis. The exchange rate constant was found to be the best individual parameter with a diagnostic accuracy of 0.74. The Ktrans was also found to be the best individual parameter in our study with a diagnostic accuracy of 0.82. Furman-Haran et al. [18] concluded in their study of 141 lesions that the quantitative evaluation of perfusion parameters should be able to improve breast cancer diagnosis on MRI. In their study the Ktrans was also found to be the best discriminating parameter. Their analysis showed results of invasive ductal carcinoma versus fibroadenomas or fibrocystic changes. Unlike our study, the pharmacokinetic parameters used by Furman-Haran and coworkers were derived from high-resolution images with relatively a low temporal resolution of 2\u00a0min. Although our analysis used a more diverse histological distribution (Table\u00a01) compared with the results presented by Furman-Haran et al. [18], only a relatively small number of benign lesions could be included. The diverse histological distribution also resulted in the inclusion of benign lesions that do not necessarily cause a diagnostic dilemma in daily practice. This can be seen as a limitation of our study. In the subgroup analysis of smaller lesions, a more equal distribution between benign and malignant lesions was found. The analysis performed in this subgroup also proved the additional value of the fast dynamic analysis in classifying small breast lesions on MRI.\nThe three-time-point method used by Kelcz et al. [37] provides the reader with a composite image showing contrast uptake and wash-out characteristics related to the product of microvessel surface area and permeability, as well as to the extracellular volume fraction. In their study, the observers correctly diagnosed 27 of 31 malignant and 31 of 37 benign lesions (sensitivity 87%; specificity 84%) using the three-time-point method. The evaluation based on wash-in and wash-out curves in combination with morphology resulted in a sensitivity of 93% and a specificity of 82%. Our results not only demonstrate a similar performance of the pharmacokinetic analysis compared with the evaluation based on morphology and slow dynamics but also demonstrate the potential gain if both methods are combined. The results presented by Kelcz et al. [37] are, like other authors, again derived from high spatial resolution images with a relatively low temporal resolution of 2\u00a0min compared with our fast dynamic scanning protocol.\nWith a scanning protocol using only the fast dynamic evaluation and morphology the scantime could be reduced significantly when compared with a protocol including the evaluation of wash-out. This without loss of diagnostic performance when compared with the results of the slow dynamic analysis in our study and the results presented by other authors [4, 12, 18]. This reduction of scantime can in the future contribute to the cost-effectiveness of MRI screening. However, since the highest diagnostic performance was obtained by combining both the fast and slow dynamic analysis, further studies are needed before the scantime can be reduced.\nThe results presented in this study are our initial results using this scanning protocol. Therefore, no cut of values for the differentiation between benign and malignant lesions from the pharmacokinetic parameters were used in the evaluation or can be provided at this point. The results presented only show the potential of our method in differentiation between benign and malignant lesions in this group of patients. The value of our method needs to be further studied in a larger group, preferably using a more even distribution between benign and malignant cases and with lesions that can be classified on imaging as a BI-RADS 3 or higher.\nUnfortunately, the study design used did not allow a multivariate analysis combining the fast dynamic data with morphological characteristics. Also, the possible trade-off between the pharmacokinetic parameters based on initial enhancement and the wash-out based on late dynamic characteristics cannot be derived from these data. Both analyses will need to be performed in future projects in order to evaluate the full potential of the fast dynamic analysis as used in our study.In conclusion, pharmacokinetic parameters derived from fast dynamic imaging during initial enhancement have great potential in classifying enhancing lesions in the breast. In this study, the diagnostic performance for the fast dynamic analysis proved to be equal to the results of experienced radiologists using more common evaluation methods based on morphologic characteristics and slow dynamic enhancement characteristics. An increased diagnostic performance was found in combining both methods. This shows the additional value of this method in further improving the diagnostic accuracy of breast MRI.","keyphrases":["breast","pharmacokinetic","dynamic","mr"],"prmu":["P","P","P","U"]}
{"id":"Int_J_Legal_Med-3-1-1794629","title":"Variation of 52 new Y-STR loci in the Y Chromosome Consortium worldwide panel of 76 diverse individuals\n","text":"We have established 16 small multiplex reactions of two\u2013four loci to amplify 52 recently described single-copy simple Y-STRs and typed these loci in a worldwide panel of 74 diverse men and two women. Two Y-STRs were found to be commonly multicopy in this sample set and were excluded from the study. Of the remaining 50, four (DYS481, DYS570, DYS576 and DYS643) showed higher diversities than the commonly used loci and can potentially provide increased haplotype discrimination in both forensic and anthropological work. Ten loci showed occasional missing alleles, duplicated peaks or intermediate-sized alleles.\nIntroduction\nY-STRs have key roles in the fields of forensic genetics, anthropological genetics and genealogy because of their ability to discriminate between male lineages and provide information about the relationships between them [1, 2]. The Y chromosome haplotype reference database [3] provides a widely used compilation of haplotype information constructed from a \u201cminimal haplotype\u201d of nine loci or a \u201cminHt\u2009+\u2009SWGDAM core set\u201d of 11 loci (http:\/\/www.yhrd.org\/index.html). Some applications, however, require more Y-STRs. For example, a study of \u223c1,000 men from east Asia found that almost 3% (27\/1,003) shared the same 16-STR haplotype [4] and thus would not be distinguished by standard analyses. Most of the STRs on the Y chromosome have now been identified [5], and a set of 52 was highlighted that seemed particularly useful because their unit size was \u22653, they were single-copy, had a simple structure and showed variation in a set of eight diverse men. These additional loci proved to be useful in the east Asian study where 46 of them allowed a male lineage characteristic of the Qing Dynasty to be defined [4], but they clearly varied considerably in their diversity [4, 5] and may vary in other properties that affect their usefulness as well. In addition, it may often be impractical or impossible to type such a large number of markers. Further studies of these loci are therefore needed to identify the most useful subset. US population data for 16 of them have been presented [6], but data from other loci and populations are lacking. We have therefore established multiplex typing procedures for all of them and examined their variation in the Y Chromosome Consortium (YCC) worldwide panel of men [7].\nMaterials and methods\nThe YCC panel consists of 74 male and two female DNAs; the men may be broken down into 26 from Africa, 26 from Asia and the Americas and 22 from Europe or the Middle East. In addition, the haplogroup R individual previously typed with all of the new markers [5] was included in this study to facilitate consistent allele calling. DNA was amplified before use with the GenomiPhi whole genome amplification kit (Amersham Biosciences, Amersham, UK) according to the manufacturer\u2019s recommendations.\nA total of 52 polymorphic simple single-copy Y-STRs [5] were included in the present study. The published primers had been designed to operate under a common set of conditions and were therefore used in this study, except that a G was added to the 5\u2019 end of the unlabelled primer if it was not already present to facilitate non-templated addition of an A to the labelled product strand [8]. Loci were tested in silico for potential interactions between primers using the AutoDimer software [9], and suitable sets were assembled into small multiplexes for experimental assessment resulting in 16 multiplexes each consisting of 2\u20134 loci (Table\u00a0S1).\nPolymerase chain reactions (PCRs) were set up in 20\u00a0\u03bcl volumes containing 1\u00d7 PCR buffer (Invitrogen, Paisley, UK), 1.75\u00a0mM MgCl2, 200\u00a0\u03bcM deoxynucleotide triphosphates (dNTPs; Amersham Biosciences), 1.0 unit of Platinum Taq DNA polymerase (5\u00a0U\/\u03bcl, Invitrogen) with 10\u00a0pg\u20132\u00a0ng whole-genome-amplified DNA and primer pairs at the concentrations shown in Table\u00a0S1. Thermal cycling was carried out in an MJ Research (Genetic Research Instrumentation, Braintree, UK) DNA Engine Tetrad\u2122 2 starting with denaturation at 95\u00b0C for 15\u00a0min, followed by 20 cycles of touchdown PCR: 94\u00b0C for 30\u00a0s, 70\u00b0C for 45\u00a0s, 72\u00b0C for 1\u00a0min, with a 1\u00b0C decrease in annealing temperature every cycle and then 15 cycles of standard PCR (94\u00b0C for 30\u00a0s, 50\u00b0C for 45\u00a0s, 72\u00b0C for 1\u00a0min) and finishing with extension at 60\u00b0C for 45\u00a0min and storage at 4\u00b0C.\nProducts were analysed by mixing 1\u00a0\u03bcl of PCR product with 15\u00a0\u03bcl Hi\u2013Di formamide and 0.2\u00a0\u03bcl size marker (CXR 60\u2013400 bases, Promega UK, Southampton, UK) and running on 36\u00a0cm\u2009\u00d7\u200950\u00a0\u03bcm capillaries containing POP-4 polymer (Applied Biosystems) on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Warrington, UK). Electrophoresis was carried out at 3\u00a0kV for 3\u00a0s followed by 15\u00a0kV for 45\u00a0min with a run temperature of 60\u00b0C. Allele sizes were measured using GeneMapper v3.0 (Applied Biosystems). Most loci were sequenced because of the lack of previous sequence data, to confirm previous results or to investigate the structure of intermediate-sized sizes. Such alleles were amplified using unlabelled primers and sequenced by the Wellcome Trust Sanger Institute small-scale sequencing facility using standard methods.\nResults\nThe 52 Y-STRs were examined in the 76 YCC samples and haplogroup R control individual, but the analyses presented in this paper (Tables\u00a0S2, Tables\u00a0S3) are based only on the YCC data to facilitate comparisons with other YCC results [10]. As expected, no specific products were obtained from the two female YCC samples in the size range examined, and single peaks were seen in all males for 40 of the STRs. The other 12 loci showed more complex patterns (Table\u00a01). Products from four loci were missing in one (DYS525, DYS589, DYS636) or two (DYS556) individuals. These findings were reproducible and occurred in multiplex reactions that successfully amplified other loci, so that they may represent null alleles, but their structural basis remains to be determined, and they were treated conservatively as missing data in our analyses. \nTable\u00a01Loci showing multiple peaks, missing peaks or intermediate allelesLocusIntermediate alleleaCommentsDYF386S1Two peaks in many individuals, excluded from analysisDYF390S1Two peaks in many individuals, excluded from analysisDYS448Two peaks in two individualsDYS52210 (U2Ains)Intermediate-sized allele. This insertion converts the reference sequence, which can be written ATAG ATG (ATAG)10 into ATAG AT\u2009A\u2009G (ATAG)10, which therefore has 12 copies of the ATAG repeat, but differs from the regular allele 12DYS525No product in one individual, two peaks in one individualDYS53111 (D6Tins)Intermediate-sized alleleDYS549Two peaks in one individualDYS556No product in two individualsDYS567Two peaks in two individualsDYS576Two peaks in two individualsDYS589No product in one individualDYS636No product in one individualU upstream, D downstream, ins insertionaNomenclature based on the standard recommendations [1]\nTwo peaks were observed in many individuals for DYF390S1 and DYF386S1, and we interpreted these as duplicated loci that happened to have the same sized alleles in the small number of individuals examined before [5]; these two STRs were excluded from subsequent analyses. Five loci also showed two peaks of similar height in one (DYS525, DYS549) or two (DYS488, DYS567, DYS576) individuals, which may reflect rare duplications or somatic mutations in the YCC cell lines. In addition, two loci showed fragment sizes that did not fall into the expected size classes: DYS522 in one individual and DYS531 in 11 individuals corresponding precisely to haplogroup Q [7] and thus representing a variant characteristic of this haplogroup. The structural basis of these variants was determined by sequencing and found to arise from insertion events in the flanking sequences between the STRs and the primers (Table\u00a01). Null alleles, occasional duplications and intermediate alleles have been found in the standard Y-STRs [1], and so we concluded that 50 of the 52 new Y-STRs merited further consideration as loci for wider use.\nWe next examined the variation of these 50 STRs. The number of alleles ranged from two to 11, the diversity from 0.05 to 0.90 and the variance from 0.04 to 7.89 (Table\u00a02). All of these characteristics were correlated, probably because of their common dependence on the repeat count. To interpret the values obtained, we have compared them with published data on the standard single-copy loci in the YCC panel [10]. Of the new loci, four (DYS481, DYS570, DYS576 and DYS643) showed higher diversity than the most variable standard locus DYS390 (diversity\u2009=\u20090.79) and 15 showed higher diversity than DYS393 (diversity\u2009=\u20090.66; Table\u00a02). The discrimination of haplotypes that are not distinguished by the commonly used markers is a particularly useful property. As reported [10], eight pairs of YCC individuals carry haplotypes that are identical when the standard minimal set of Y-STRs is used. Two of these are from different populations (Mbuti Pygmy\/Bantu speaker; English\/German) and these were distinguished by seven and nine of the new loci, respectively. The other six pairs are from isolated populations, and these were distinguished by 2, 1, 1, 0, 0 and 0, respectively, of the new markers (Table\u00a0S4). Although a total of 15 loci contribute to this increased discrimination, all of the five distinguishable haplotypes could be separated using just two of the most variable loci, DYS570 and DYS576. \nTable\u00a02Variation of 50 new Y-STR loci in the YCC panelLocusMean repeat countNumber of allelesDiversityVarianceDYS48123.3110.907.87DYS57017.6110.863.89DYS57617.370.822.16DYS64311.190.822.33DYS48515.970.781.92DYF406S110.670.751.26DYS52211.260.741.03DYS58912.360.731.07DYS53311.260.721.03DYS54912.150.720.92DYS50512.160.711.00DYS50811.480.711.44DYS5259.970.710.95DYS53111.040.690.39DYS55611.760.671.03DYS57210.350.640.61DYS56511.560.640.71DYS5949.570.630.87DYS54011.440.620.52DYS48713.260.621.38DYS51110.950.620.73DYS5739.940.620.73DYS61712.450.621.06DYS49515.350.610.76DYS56710.460.610.72DYS49714.240.600.51DYS48813.460.580.91DYS49211.650.560.45DYS49012.970.562.43DYS56811.170.560.92DYS63611.350.560.49DYS53710.940.530.43DYS61811.840.510.37DYS63810.940.490.34DYS49112.150.460.40DYS5788.140.410.40DYS64011.230.390.39DYS47611.240.360.29DYS4949.040.350.28DYS6419.640.330.93DYS5549.050.310.36DYS57510.040.250.22DYS5838.030.220.12DYS5907.930.220.23DYS4807.930.220.16DYS56911.030.180.11DYS5309.120.170.09DYS5809.140.110.19DYS4728.020.080.04DYS5799.020.050.89Loci are ordered according to their diversity.\nDiscussion\nWe have investigated the properties of 52 new Y-STRs in a diverse worldwide set of males. We found that two of the Y-STRs were multicopy and thus not well suited to some applications and that the remaining 50 loci differed substantially in their properties. Our measurements of allele numbers, diversity and variance were overall consistent with the previous report [5]; correlation coefficients (R2 values) were 0.47, 0.58 and 0.67, respectively, but differed for some individual loci. The most variable Y-STR, in all respects, was DYS481, and this was not previously considered in detail because sequence data were not available before. Several other loci (e.g., DYS570, DYS576 and DYS643) may be particularly useful for increasing discrimination in forensic work, and the simple structure and mutational properties of this set make them the markers of choice for many population genetic studies. This is illustrated by considering the correlation between mean repeat count and variance in repeat number of the 50 simple loci: it was far higher (R2\u2009=\u20090.67) than the value reported for complex Y-STRs (R2\u2009=\u20090.34, [5]), suggesting that the simple STRs have simpler mutational mechanisms and may lead to more precise dates of lineages. The data in Table\u00a02 and Table\u00a0S3 now provide a basis for choosing the best simple loci and assembling them into a high-level multiplex reaction for more extensive population screening.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material. \nTable\u00a0S1\nMultiplex organization and primer concentrations (DOC 102 kb)\nTable\u00a0S2\nPCR product size range and allele range (DOC 92 kb)\nTable\u00a0S3\nHaplotypes of the YCC DNAs (XLS 58 kb)\n(Note: this table is provided as an Excel file and is the table mentioned in CE7. We transformed it into text because some journals insist on this, but the text version is difficult to interpret as CE5 highlights. The loci are ordered according to their positions in multiplexes 1\u201316 in \nTable\u00a0S1\nand \nTable\u00a0S2\n, and it seems most consistent to keep the same order for \nTable\u00a0S3\n). \nTable\u00a0S4\nSubdivision of minimal haplotypes by new Y-STRs (DOC 87 kb)","keyphrases":["y chromosome","dys481","dys570","dys576","dys643","intermediate allele","short tandem repeat (str)"],"prmu":["P","P","P","P","P","P","M"]}
{"id":"Purinergic_Signal-3-4-2072910","title":"Structural and functional evolution of the P2Y12-like receptor group\n","text":"Metabotropic pyrimidine and purine nucleotide receptors (P2Y receptors) belong to the superfamily of G protein-coupled receptors (GPCR). They are distinguishable from adenosine receptors (P1) as they bind adenine and\/or uracil nucleotide triphosphates or diphosphates depending on the subtype. Over the past decade, P2Y receptors have been cloned from a variety of tissues and species, and as many as eight functional subtypes have been characterized. Most recently, several members of the P2Y12-like receptor group, which includes the clopidogrel-sensitive ADP receptor P2Y12, have been deorphanized. The P2Y12-like receptor group comprises several structurally related GPCR which, however, display heterogeneous agonist specificity including nucleotides, their derivatives, and lipids. Besides the established function of P2Y12 in platelet activation, expression in macrophages, neuronal and glial cells as well as recent results from functional studies implicate that several members of this group may have specific functions in neurotransmission, inflammation, chemotaxis, and response to tissue injury. This review focuses specifically on the structure-function relation and shortly summarizes some aspects of the physiological relevance of P2Y12-like receptor members.\nIntroduction\nMost cellular effects of nucleotides are mediated through two main families of specific cell surface receptors, the ionotropic P2X and metabotropic P2Y nucleotide receptors. The metabotropic receptors belong to the superfamily of G protein-coupled receptors (GPCR). The GPCR superfamily comprises at least five structurally distinct families\/subfamilies (GRAFS classification) which share little sequence homology among each other [1]. The rhodopsin-like receptors (also called family A or 1) form the largest family in vertebrates. Eight different mammalian P2Y (P2Y1, 2, 4, 6, 11\u201314) receptor subtypes, all belonging to the rhodopsin-like receptors (family A), have been identified and proven to function as receptors for extracellular nucleotides so far [2]. Based on their preferential agonists, P2Y receptors have been subclassified into adenine nucleotide-activated (P2Y1, P2Y11, P2Y12, P2Y13), pyrimidine nucleotide-activated (P2Y4, P2Y6), ATP\/UTP-activated (P2Y2), and UDP-sugar-activated (P2Y14) receptors. Most structural and functional studies were performed with the classic P2Y receptors P2Y1 and P2Y2 [3]. However, in comparison to other GPCR, large-scale structure\/function data are rare, mainly due to difficulties in finding suitable P2Y-free heterologous expression systems [4]. Pharmacological characterization on both native and recombinant P2Y receptors is further hampered by the release of endogenous nucleotides and the hydrolysis or conversion of exogenous and endogenous nucleotides by ectoenzymes (for review see [5]). ATP and AMP are metabolized by cell surface enzymes, the ecto-NTPDase 1 (nucleoside triphosphate diphosphohydrolase 1, ectoapyrase, CD39, metabolizes ATP to AMP) and the 5\u2032-ectonucleotidase (CD73, metabolizes AMP to adenosine). In addition, a nucleoside diphosphokinase catalyzes the transfer of the gamma phosphate of nucleoside triphosphates to nucleoside diphosphates. For example, in the presence of ATP, nucleoside diphosphokinase catalyzes the conversion of UDP to UTP. These specific problems may have contributed to many false and controversial declarations of P2Y receptors being activated by nucleotides [6\u20139]. Recent success in expressing GPCR in yeast, which is discussed below, may circumvent these problems and may engage structure-function-relation studies.\nAlthough in-depth phylogenetic analysis of GPCR groups indicates clustering of P2Y receptors, when compared with other GPCR of family A [1, 10], the adenine nucleotide-activated P2Y1 and P2Y12, for example, display only minor structural relation at the protein level. Phylogenetic analysis even revealed that nucleotide specificity e.g. for ADP evolved independently in P2Y1 and P2Y12. So we suggest that grouping of P2Y receptors should be performed on phylogenetic aspects rather than on ligand specificity.\nBased on their amino acid sequences P2Y receptors can be subdivided into at least two groups (Fig.\u00a01). One comprises P2Y1, 2, 4, 6, 11, and the second group contains P2Y12\u201314. Additionally, several other structurally related GPCR, such as CysLT1R, GPR91, GPR99, and GPR34, cluster into both groups (Fig.\u00a01). These receptors are activated by structurally different ligands such as leukotrienes, organic acids, and phospholipids. Probably because of their structural relation several P2Y receptors display agonist promiscuity. It was shown that not only ADP but also leukotriene E4 (LTE4) activates P2Y12 at nanomolar concentrations [11]. Cysteinyl leukotriene receptors (CysLT1R and CysLT2R) have been described to be activated by UDP in addition to activation by cysteinyl leukotrienes [12, 13]. Further, there is evidence that CysLT1R antagonists, such as pranlukast and montelukast, inhibit activation of P2Y1 and P2Y6 by 2MeS-ADP (2-methylthio-ADP) and UDP, respectively [14]. Although P2Y12 and cysteinyl leukotriene receptors are clinically used targets for prevention of thrombocyte aggregation (clopidogrel) and treatment of asthma (montelukast), respectively, the structural basis and (patho)physiological relevance of dual\/multiple agonist specificity is obscure. Close structural relations between P2Y and other non-nucleotide receptors as well as the mentioned examples of ligand promiscuity indicate that nucleotides are probably only one of many natural agonists on the genuine P2Y receptors.\nFig.\u00a01Phylogenetic relation of human P2Y receptors. To evaluate the structural relation of P2Y receptors and related GPCR, amino acid sequences of human orthologs were aligned and phylogenetic relations were estimated using CLUSTAL W. The derived tree was essentially identical to former analysis [2, 10]. The P2Y12-like receptors cluster into a group (framed) that is distinct from other nucleotide receptors such as P2Y1 and P2Y2\nStructural evolution of P2Y12-like GPCR\nDiscovery and deorphanization of group members\nThe ADP-(P2Y12)-like receptor group is the most recently identified P2Y receptor group and includes P2Y12, P2Y13, and P2Y14 (see Fig.\u00a01, Table\u00a01). Based on high structural similarity, the lyso-phosphatidylserine receptor (GPR34) and several orphan GPCR (GPR34-like, GPR82, GPR87, GPR171) are considered members of the structural P2Y12-like receptor group (3,11). Most members of this group were deorphanized by the so-called reverse pharmacology approach. The orphan receptors predicted from sequence data are now often used to ascertain the ligands by testing them on tissue extracts and subsequent fractionation or on huge libraries of bioactive compounds, whereas, traditionally, the bioactive ligand was used to identify the receptor (classic approach).\nTable\u00a01Members of the P2Y12-like receptor familyReceptorG protein couplingNatural agonistTissue expressionP2Y12GiADP, CysLT-E4, phosphoribosyl pyrophosphatePlatelets, brain (glia) [19]P2Y13GiADP, diadenosine triphosphateSpleen and adult brain, lower expression in placenta, lung, liver, spinal cord, thymus, spleen, small intestine, uterus, stomach, testis, fetal brain, adrenal gland, lymph node, bone marrow, peripheral blood mononuclear cells, leukocytes [22, 45]P2Y14GiUDP-glucose, UDP-galactose, UDP-glucuronic acid, UDP-N-acetylglucosamineBrain (astrocytes, glia), placenta, adipose tissue, stomach, intestine, spleen, lung (epithelium), heart [15, 16, 71]GPR87Gin.kPlacenta, thymus, small intestine, colon, prostate, uterus, testis, peripheral blood leukocytes [28, 29]GPR171n.kn.kn.kGPR34Gilyso-phosphatidylserineBrain, glia cells, mast cells, spleen, heart, kidney, liver [27, 31, 34, 72]GPR82n.kn.kTestes, epididymis (unpublished own results)Based on phylogenetic analyses and structural similarities the seven GPCR clusters in a group which is distinct from other P2Y receptors (see Fig. 1). Although they share structural features it appears from current studies that not all members are receptors for nucleotides. The table lists the currently known receptor agonists, the receptor\u2019s G protein specificity and expression patternn.k not known\nAs the first member of the P2Y12-like receptor group P2Y14 (synonyms KIAA0001, GPR105, VTR 15\u201320) was identified [15, 16] and then deorphanized in the year 2000 [17]. UDP-glucose was found to activate P2Y14 by screening multiple receptors, each expressed in individual yeast strains, against a large library of over 700 known and putative natural GPCR agonists.\nIn 2001, P2Y12 (initially named SP1999) was discovered and identified as the platelet clopidogrel-sensitive ADP receptor by a number of groups [18\u201321]. The P2Y12 was deorphanized with both the classic and the reverse pharmacology approaches. Hollopeter et al. used subfractionated transcripts from platelets and isolated the cRNA (complementary RNA, encoding e.g. the P2Y12 receptor) which mediated ADP-induced increases in K+ current after injection into Xenopus oocytes [19]. In contrast, fractionated rat spinal cord extracts were assayed for Ca2+ mobilization in cells transiently transfected with P2Y12 and chimeric G\u03b1 subunit [21]. Almost in parallel, ADP was shown to be an agonist for P2Y13 (former GPR86), a very close structural neighbor of P2Y12 [22\u201324]. 2MeS-ADP is the most potent agonist at P2Y12 followed by ADP, adenosine 5\u2032-O-2-thiodiphosphate, and ATP. (N)-methanocarba-2-methylthio-ADP, a highly potent agonist at P2Y1, exhibited no and only minor agonist activity at P2Y12 and P2Y13, respectively [25].\nBased on structural relations GPR34 [26, 27], GPR82 [28], GPR87 [29], and GPR171 (synonym H963) [30] also belong to the P2Y12-like group (see Fig.\u00a01). Except for GPR34, which was recently identified as a receptor for lyso-phosphatidylserine [31] by a reverse pharmacology approach, all the other P2Y12-like receptors are still orphan GPCR.\nThe evolutionary origins\nBased on structural similarities P2Y12-like receptors cluster into a group distinct from other P2Y receptors (see Figs.\u00a01 and 2). This P2Y12-like receptor group can be subdivided into two subgroups. One subgroup encompasses P2Y12, P2Y13, P2Y14, GPR87, and GPR171, the other subgroup GPR34, GPR34-like, and GPR82 (Fig.\u00a02). This may suggest that both subgroups evolved from gene duplications starting from two related members. The grouping is further supported by shared genomic localization. Chromosomal clustering in the human genome is found for P2Y12, P2Y13, P2Y14, GPR87, and GPR171 at 3q24-3q25 as well as for GPR34 and GPR82 at Xp11.4. This clustering is evolutionary well preserved. In the zebrafish genome P2Y12, P2Y14\/GPR87, and GPR171 cluster at chromosome 15 and GPR34 type 2 and GPR82 are tandemly arranged at chromosome 9. Gene clustering is not only found for vertebrate P2Y12-like receptors but also for several other GPCR genes. Multiple copies of related GPCR genes, such as the human protease-activated receptors on 5q13, trace amine-associated receptors (TAAR, human 6q23.2), and CC-chemokine receptors (human 3p21.3), are the result of intrachromosomal gene duplications. The most impressive example is found in odorant receptors, where in humans, chromosome 11 contains nearly half of the odorant receptor repertoire, including a single cluster of more than 100 odorant receptors [32]. Gene amplification can be viewed as a dynamic and reversible regulatory mechanism that facilitates adaptation to variable environments. Clustered genes may confer selective benefits via their ability to be co-regulated and co-amplified. Indeed, even transcripts encoding a fusion protein of GPR34 and GPR82 have been observed (own unpublished results). However, the biological relevance of P2Y12-like receptor clustering in respect to transcriptional activity at their respective genomic loci, co-regulation, and transcript diversity need to be determined in the future.\nFig.\u00a02Evolutionary origin of P2Y12-like receptors. To subclassify and evaluate the phylogenetic relations between the P2Y12-like receptors, the amino acid sequences of orthologs from one species of each vertebrate class (if available) were aligned using CLUSTAL W (identity matrices) and a phylogenetic tree was constructed (1,000 iterations). Bootstrap values >600 were considered as significant to support a new branch\nSince P2Y12, P2Y14\/GPR87, GPR34, and GPR34-like are all found in sharks (Mustelus manazo, Carcharodon carcharias) and chimeras (Callorhinchus milii) one can assume that P2Y12-like receptors arose more than 450 million years (Myr) ago, before cartilaginous and bony fish split. Our own polymerase chain reaction (PCR) amplification attempts as well as the ongoing lamprey (Petromyzon marina) and sea urchin (Strongylocentrotus purpuratus) genome projects revealed no P2Y12-like sequences yet, suggesting their origin in the very early Gnathostomata or, less likely, a loss in agnate and all non-vertebrates.\nGene duplication can be a primary source of the genetic material from which genes with new functions evolve. One copy of a duplicated gene may become mutated and acquire unique functionality without risking the fitness of the organism ensured by the homolog. On the other hand, if not advantageous, continuous accumulation of mutations (neutral drift) will eliminate one of the genes, a process named pseudogenization. In fish genomes, there is only one respective ortholog with relation to P2Y12\/P2Y13 and to GPR87\/P2Y14. In the case of P2Y12\/P2Y13 the fish ortholog is more closely related to P2Y12 implicating that P2Y13 evolved later in tetrapod evolution probably by P2Y12 duplication. However, one cannot rule out that the gene was eliminated in the common ancestor of sharks and bony fishes. The evolutionary mechanism of P2Y14 and GPR87 evolvement remains unsolved since their structural relation to the fish orthologs is similar. The absence of P2Y14 in the African craw frog (Xenopus) genome but the presence of ortholog sequences in all other sequenced tetrapod genomes does not necessarily implicate that P2Y14 derived from GPR87 in early tetrapod evolution. Again, P2Y14 gene loss in amphibians must be considered.\nOrthologs of GPR34, GPR87\/P2Y14, and P2Y12 are found in all tetrapods and bony and cartilaginous fishes investigated so far. However, not all members of the P2Y12-like group appear to be present in all vertebrate classes. GPR82 has an ortholog in zebrafish but is not yet found in the genomes of pufferfishes and the lizard (Anolis carolinensis) genome. Although GPR171 is found in reptiles, birds, mammals, zebrafish, and chimera it appears to be absent in African craw frog, stickleback, and pufferfishes. This may implicate specific functions of some P2Y12-like receptors which are less important in some species or are compensated by other receptors and mechanisms.\nSignatures of gene inactivation (pseudogenization) are rarely found for members of the P2Y12-like group. Following gene duplication in the common ancestor of evolutionary basic fishes, like eels and carps [33], pseudogenization of one GPR34 subtype was identified in a salmon species (Keta salmon) (own unpublished results). The GPR34-like receptor is an evolutionary old P2Y12-like receptor being present in cartilaginous and bony fishes, amphibians, and birds but not in mammals (Fig.\u00a02). Sequences with residual relation to the GPR34-like receptor are found in the platypus genome but it is most likely a pseudogene.\nGenomic organization of P2Y12-like receptors\nComparison of transcript and genomic sequences provides information on intron\/exon structure of a GPCR gene. Since the gain or loss of spliceosomal introns are unique events in evolution, they can serve as markers for phylogenetic analysis. Further, such analyses may reveal splice variants and may be informative about the promoter structure and gene regulation. Introns are the basis of alternative splicing, exon skipping, and RNA editing events and, therefore, can contribute to receptor diversity at a supragenomic level.\nMost coding regions of human P2Y12-like receptors do not contain introns (Table\u00a02). One exception is GPR87 where the genomic sequence encoding the receptor\u2019s N terminus is interrupted by an intron in mammalian and avian orthologs. Another intron which is rarely removed (cryptic intron) has been identified in the N terminus-encoding sequence of GPR34 [34].\nTable\u00a02Genomic organization of P2Y12-like GPCR. n.a. not analyzed because of lack of mRNA informationMemberNumber of introns in the 5\u2032 non-coding regionNumber of introns in the coding regionApprox. size of genomic regionaP2Y12\u00a0Human1\u20132\u221247\u00a0kbp (2 different transcript starts)\u00a0Mouse4\u221246.5\u00a0kbp\u00a0Chickenn.a.\u2212bn.a. (>1\u00a0kbp)\u00a0African clawed frog\u22121 (N terminus)4\u00a0kbp\u00a0Zebrafishn.a.\u2212bn.a. (>1\u00a0kbp)\u00a0Stickleback11 (TMD2)2\u00a0kbp\u00a0Pufferfishn.a.1 (TMD2)>2\u00a0kbpP2Y13\u00a0Human\u22121 (rare transcript, N terminus, NM_176894) 3.2\u00a0kp (2 different transcript starts)\u00a0Mouse1\u22123\u00a0kbp\u00a0Chickenn.a.\u2212bn.a. (>1\u00a0kbp)\u00a0African clawed frog\u22121 (N terminus)14\u201319\u00a0kbpP2Y14\u00a0Human2\u221266\u00a0kbp (2 different transcript starts)\u00a0Mouse1\u20132\u221216\u00a0kbp\u00a0Chickenn.a.\u2212bn.a. (>1\u00a0kbp)GPR87\u00a0Human11 (N terminus)22.7\u00a0kbp\u00a0Mouse11 (N terminus)16\u00a0kbp\u00a0Chicken21 (N terminus)9.5\u00a0kbp\u00a0African clawed frogn.a.\u2212bn.a. (>1\u00a0kbp)P2Y14\/GPR87\u00a0Zebrafishn.a.\u2212bn.a. (>1\u00a0kbp)\u00a0Pufferfishn.a.\u2212bn.a. (>1\u00a0kbp)GPR171\u00a0Human2\u22125\u00a0kbp\u00a0Mouse1\u22124.5\u00a0kbp\u00a0Chicken1\u22122.3\u00a0kbp\u00a0Zebrafishn.a.\u2212bn.a. (>1\u00a0kbp)GPR82\u00a0Human2\u22124\u00a0kbp\u00a0Mouse2\u20133\u22126\u00a0kbp\u00a0Chickenn.a.\u2212bn.a. (>1\u00a0kbp)\u00a0African clawed frogn.a.\u2212bn.a. (>1\u00a0kbp)\u00a0Zebrafishn.a.\u2212bn.a. (>1\u00a0kbp)GPR34\u00a0Human3\u201341 cryptic (N terminus)8.2\u00a0kbp\u00a0Mouse3\u20134\u22129.1\u00a0kbp\u00a0Chicken1\u22122.5\u00a0kbp\u00a0African clawed frog2\u22125.2\u00a0kbp\u00a0Zebrafish\u00a0\u00a0Type 11\u22127.3\u00a0kbp\u00a0\u00a0Type 2\u2212\u22121.2\u00a0kbp\u00a0Pufferfishn.a.\u2212bn.a. (>1\u00a0kbp)Except for GPR87, human P2Y12-like receptor members contain no intron in the coding region. However, this intronless gene structure is not well preserved in all vertebrates. The table summarizes the number of introns within the 5\u2032 and coding region of selected vertebrate genes and estimates the size of the geneaBased on the 5\u2032 longest transcriptbComplete open reading frame within the genomic sequence but intron within the very N terminus cannot be ruled out because of lack of mRNA information\nThe genomic organization of the individual P2Y12-like receptor genes is not well conserved during vertebrate evolution (see Table\u00a02). For example, the P2Y12 gene gained an intron in bony fish evolution disrupting the open reading frame of the transmembrane domain 2 (TMD2)-encoding part. Similarly, one of the two GPR34 paralog genes acquired an intron in the more recent bony fish evolution [33]. The two long-standing alternative explanations for the origin of introns, the intron-early theory and intron-late theory, remain a matter of continuous debate not only for GPCR [35]. The intron-early theory suggests that introns are extremely ancient characteristics of genes and that early genes were created through the intron-mediated shuffling of exons. However, numerous gene and genome comparison studies provided evidence that at least some introns are more recently acquired (intron-late theory). The P2Y12-like receptor group is, therefore, a nice example where introns were acquired in the coding region in some species during more recent evolution.\nIn contrast to the coding region, the 5\u2032 region of most members of the P2Y12-like receptor group displays a distinct intron\/exon organization with sometimes multiple transcription starts. Also, the gene sizes strongly differ between the individual and even closely related receptors. Introns in the 3\u2032-untranslated region (UTR) of P2Y12-like receptors have not been found yet. Large and complex organized 5\u2032 non-coding regions of a gene may provide the basis for multiple promoter regions, cis-acting elements, and a variable 5\u2032 UTR of the mRNA. Alternative structures of the 5\u2032 UTR can contribute to expression regulation and alternative translation start points. For example, 5\u2032 UTRs often contain small open reading frames (ORF) which can be translated via leaky scanning at the ribosome. Such leaky scanning can reduce translation of the downstream main ORF as shown for several genes including GPCR [36, 37].\nIt is of interest to note that both genes, GPR34 and GPR82, are located in antisense orientation within a large intron of the CASK gene. This position is conserved during vertebrate evolution. The CASK gene encodes a calcium\/calmodulin-dependent serine protein kinase that is a member of the membrane-associated guanylate kinase (MAGUK) protein family. Since GPR34 and GPR82 transcripts are antisense orientated to CASK one can speculate that transcripts may regulate expression of CASK or vice versa. Such hypotheses will be addressed in receptor-deficient mouse models (see below) in the future.\nKey residues defining the individual member\nMany attempts have been made to identify structural signatures which are helpful in annotation and grouping of P2Y receptors. On the basis of available sequence data for validated P2Y receptors, key residues were extracted to define P2Y12-like and P2Y1-like receptor groups [11, 38, 39]. In recent studies, we have shown for several GPCR that a significant number of orthologs is required for identification of functional motifs and key residues [33, 40\u201342]. By mining public databases and by amplifying and sequencing P2Y12-like orthologs we have acquired large sets of sequences of P2Y12 (74 orthologs), P2Y13 (31 orthologs), P2Y14 (38 orthologs), GPR87 (51 orthologs), GPR171 (41 orthologs), GPR82 (34 orthologs), and GPR34 (133 orthologs) to determine structural conservation of the members and to identify amino acid sequence motifs and key residues that are unique for the P2Y12-like receptor group and the individual members (Fig.\u00a03). These data sets were obtained from vertebrate species ranging from evolutionary old bony and cartilaginous fishes to the more modern mammals representing 450 Myr of evolution. The overall identity between P2Y12-like members is rather low ranging from 19% amino acid identity (human GPR82 vs human GPR87) to 47% (human P2Y12 and human P2Y13). Between the respective fish and mammal receptor orthologs P2Y12 (~49%) shows the highest identity followed by GPR171 (~48%), GPR34 (~40%), and GPR82 (~35%). Since not all P2Y12-like GPCR have orthologs in all vertebrate classes, we analyzed the structural conservation within this receptor group by comparing Ka\/Ks values (ratio of the number of nonsynonymous substitutions per nonsynonymous site and the number of synonymous\u2014or silent\u2014substitutions per synonymous\u2014or silent\u2014site) of the P2Y12-like receptor ortholog set from species containing all group members (Table\u00a03). Although all P2Y12-like receptors display a purifying selection mode of evolution (Ka\/Ks <<1 indicates high conservation and elimination of deleterious mutations), there are significant differences between the members of this receptor group. P2Y12 and GPR87 were kept most conserved during evolution in birds and mammals. By contrast, GPR82 appears to be less constrained as already indicated by a relatively low conservation at the amino acid level (see above).\nFig.\u00a03Conserved residues in P2Y12-like receptors. To identify conserved group and member-specific positions the amino acid sequences of P2Y12 (74 orthologs), P2Y13 (31 orthologs), P2Y14 (38 orthologs), GPR87 (51 orthologs), GPR171 (41 orthologs), GPR82 (34 orthologs), and GPR34 (133 orthologs) were aligned using CLUSTAL W. Residues that are 100% conserved among the respective orthologs were boxed. Only a few positions are almost fully conserved among all members of the P2Y12-like group (position number refers to the relative numbering system by Ballesteros and Weinstein [73]): TMD1: Phe\/Tyr1.39, Phe1.57; TMD2: Leu2.43, Asn\/Asp2.50, Pro\/Ala2.58; TMD3: Tyr\/His3.33, Arg\/Gln3.50; TMD4: Trp4.50; TMD6: Cys\/Ser6.47; Pro6.50; TMD7: Asp7.49, Pro7.50, and the two Cys residues bridging extracellular loops 1 and 2. The approximate positions of the seven transmembrane domains (TMD) are given above the sequencesTable\u00a03Sequence conservation of P2Y12-like GPCRReceptorKa\/KsPi(mean \u00b1 SD)(mean \u00b1 SD)P2Y120.049\u2009\u00b1\u20090.0180.145\u2009\u00b1\u20090.022P2Y130.141\u2009\u00b1\u20090.0700.187\u2009\u00b1\u20090.023P2Y140.103\u2009\u00b1\u20090.0360.190\u2009\u00b1\u20090.024GPR870.046\u2009\u00b1\u20090.0270.134\u2009\u00b1\u20090.018GPR1710.078\u2009\u00b1\u20090.0120.158\u2009\u00b1\u20090.021GPR340.082\u2009\u00b1\u20090.0370.137\u2009\u00b1\u20090.026GPR820.202\u2009\u00b1\u20090.0490.167\u2009\u00b1\u20090.034To compare the sequence conservation of P2Y12-like receptors, ortholog sequences [relative positions 1.48 (in TMD1) to 7.68 (in C-term)] from 18 species were aligned and DNA polymorphism analyses were performed using DnaSP (version 4.1). The Ka\/Ks ratio is calculated from the number of nonsynonymous substitutions per nonsynonymous site (Ka) and the number of synonymous substitutions per synonymous site (Ks) for any pair of sequences. The nucleotide diversity (Pi) is the average number of nucleotide differences per site between two sequences. The orthologs of the following species were analyzed because their genome contained all members of the P2Y12-like group: Bos taurus, Equus caballus, Canis familiaris, Pteropus vampyrus, Ornithorhynchus anatinus, Monodelphis domestica, Callithrix jacchus, Pan troglodytes, Homo sapiens, Macaca mulatta, Microcebus murinus, Pongo pygmaeus, Tarsius syrichta, Cavia porcellus, Mus musculus, Rattus norvegicus, Tursiops truncates, Gallus gallus\nAs shown in Fig.\u00a03, members of the P2Y12-like group share only eight fully conserved residues (Phe1.57, Leu2.43, Trp4.50, Pro6.50, Asp7.49, Pro7.50, and both Cys residues bridging extracellular loops 1 and 2) when sequences of more than 400 receptors of this group are compared. Although the ADP receptors P2Y12 and P2Y13 share more than 50 fully conserved residues none of these conserved residues is exclusively found in P2Y12 and P2Y13 but rather present also in other P2Y12-like receptors (see Fig.\u00a03). Further, only a few residues are member specifically conserved (GPR171: Gln3.53 and Asn5.59; P2Y13: Met7.48; GPR82: Leu6.44 and Asp7.57; GPR34: Met7.52) and have not been found in other P2Y12-like receptors so far. These facts suggest that ligand and signaling specificity is determined by a combination of many, more or less conserved determinants. It has been proposed that His6.52\/Arg6.55 and Lys7.35\/Glu7.36\/Leu7.39 within TMD6 and the ECL3, respectively, may present such motifs required for nucleotide binding [38]. However, such residue combination is also present in some GPR87, GPR171, and GPR34 orthologs which are not activated by ADP. This does not rule out that these residues are involved in nucleotide binding of e.g. P2Y12 but it implicates additional positions which determine ligand specificity. In-depth structure-function analysis e.g. by mutagenesis studies are required to identify key positions and their structural properties.\nPhysiological relevance of P2Y12-like receptors\nFunctional characterization of P2Y12-like receptors in heterologous expression systems\nSignal transduction of the ADP receptor P2Y12 via pertussis toxin-sensitive Gi proteins and adenylyl cyclase inhibition is well established [22]. Similar Gi protein-coupling specificity was found for P2Y13 [22, 24], P2Y14 [43], and lyso-PS receptor GPR34 [31, 33]. Because adenylyl cyclase inhibition assays are usually less sensitive and robust several other experimental setups were established to measure function of P2Y12-like GPCR. It has been shown that G\u03b115 and G\u03b116 can be activated by a wide variety of GPCR [44]. The ability of G\u03b116 to bypass the selectivity of receptor\/G protein interaction was also useful to measure activation of P2Y13 [45]. It has been demonstrated that replacement of the four or five C-terminal amino acids of G\u03b1q with the corresponding G\u03b1i residues (referred to as G\u03946qi4 [46]) confers the ability to stimulate the PLC-\u03b2 pathway onto Gi-coupled receptors [47]. Successful heterologous expression and activation by applying chimeric G\u03b1qi4 has been demonstrated for P2Y12\u201314 and lyso-PS receptor GPR34 [25, 33, 48].\nWe have previously shown that P2Y12 as well as the lyso-PS receptor GPR34 display increased basal activity in functional assays when compared with other GPCR [33]. This high basal activity can be either discussed as the natural ground state of the receptor activity equilibrium or as the effect of receptor agonists that are present in the cell culture medium or that are released from cells. Analyzing mutations in the highly conserved DRY motif of P2Y12 we recently showed that basal activity is abolished in the DHY mutant but agonist-induced activation remains intact (Fig.\u00a04). We conclude that the basal activity of the ADP receptor is rather a genuine property of this receptor and not due to continuous stimulation by agonists. High basal activity appears to be a general feature of all members of the P2Y12-like receptor group. In our initial ortholog screen we identified two GPR87\/P2Y14 receptors, carp GPR87\/P2Y14 types 1a and 1b (AY241103, AY241102), which differ in only ten amino acid positions. Interestingly, carp GPR87\/P2Y14 type 1b displays higher basal activity and studies are ongoing to identify residues that promote constitutive activity.\nFig.\u00a04High basal activity is a genuine property of P2Y12. Basal activity of the wild-type P2Y12 (DRY motif) and a mutant P2Y12 (DHR motif) was determined in transiently transfected COS-7 cells. Gi coupling of P2Y12 was rerouted to IP production by co-transfection of a chimeric G protein G\u03946qi4 [46]\nBecause many standard mammalian expression systems endogenously express P2Y receptors clear-cut functional studies are difficult to perform. One exception is the 1321N1 human astrocytoma cell line which does not express P2Y receptors. However, transient expression of GPCR in 1321N1 cells is limited by low transfection efficiency and stable transfection of these cells is usually required. Heterologous expression of GPCR in yeast was initially established for large-scale purification of receptor proteins. These advances in the expression of heterologous GPCR in the yeast Saccharomyces cerevisiae have led to the development of sensitive and selective assays of ligand-induced GPCR activation (reviewed in [49]). To facilitate a more systematic genetic analysis of GPCR function, e.g., by saturating random mutagenesis, we took advantage of a yeast expression system in which parts of the mammalian GPCR signaling system (GPCR and chimeric G protein) are linked to a modified yeast pheromone pathway (Fig.\u00a05) [50]. The coupling of receptor activity to the genetically engineered yeast pathway allows for rapid and economical screening of substance libraries and randomly modified receptor libraries. Functional studies in yeast may have an advantage especially for P2Y receptors because one can (at least partially) circumvent specific problems in working with nucleotide receptors (see above). Only two NTPDases (GDA1 and YND1\/APY1) have been found within the entire yeast genome which are mainly expressed in the Golgi apparatus [51]. Although conversion of nucleotides by nucleotidases and ectoapyrases occur also in yeast and one cannot exclude conversion of extracellular nucleotides by yeast enzymes, there is no endogenous plasma membrane P2Y receptor in yeast which can mediate and, therefore, interfere with transmembrane signaling of heterologously expressed P2Y receptors in yeast. We and others have successfully used this system for expression and functional study of P2Y12 [52], P2Y14 [53], and lyso-PS receptor GPR34 (unpublished data). As shown in Fig.\u00a06a, P2Y12-expressing yeast cells grow only in the presence of the agonist 2-methylthioadenosine 5\u2032-diphosphate (2MeS-ADP).\nFig.\u00a05Functional expression and in vitro evolution of GPCR in yeast. Genetically modified yeast cells are transformed with a mammalian GPCR. Following agonist activation and constitutive receptor activity, the receptor couples to a chimeric G protein [backbone yeast G protein Gpa1 in which the C-terminal five amino acids were replaced by the respective mammalian G protein sequence (e.g., from the Gi protein)]. Activation of the chimeric G protein enables yeast cells to grow on histidine-free medium by utilizing parts of the yeast mating pathway [49]. Gpa yeast ortholog of the mammalian G protein alpha subunit, Ste4 yeast ortholog of the mammalian G protein beta subunit, Ste18 yeast ortholog of the mammalian G protein gamma subunit, Ste12 transcription factor that is activated by a MAP kinase signaling cascade, Far1 cell cycle regulator that directly inhibits the yeast cyclin-dependent kinase Cdc28-ClnFig.\u00a06Functional expression and random mutagenesis of P2Y12 in yeast. a The human P2Y12 receptor was transformed into modified yeast (see Fig.\u00a04). The agonist 2-methylthioadenosine 5\u2032-diphosphate (2MeS-ADP) induces a robust yeast cell growth. b The entire coding region of the human P2Y12 was subjected to random mutagenesis and transformed yeast cells were selected for growth in agonist-free U\u2212\/H\u2212medium. Colonies that grow under this condition contain a constitutively active P2Y12. Exemplarily, mutations of a triple mutant (L115Q\/F177S\/R224G) were individualized and tested separately for constitutive activity. The data indicate that L115Q mainly contributes to the constitutive activity of the triple mutant\nConstitutively active GPCR are useful tools in studying the action of inverse agonists and activation mechanisms in GPCR. A constitutively active P2Y12 was generated by replacing the endogenous C terminus with the corresponding part of the human P2Y1 receptor [54]. Pharmacological evaluation of several P2Y12 antagonists revealed AR-C78511 (an adenosine derivative) as a potent P2Y12 inverse agonist. Traditional mutagenesis approaches are limited to screen for activating mutations because the number of mutant proteins that can be investigated is usually relatively small, primarily because of technical reasons and the time and costs needed to generate and analyze large numbers of mutant receptors. To circumvent these limitations, yeast has emerged as a highly useful host for the in vivo reconstitution of mammalian GPCR. Therefore, we applied a random mutagenesis and screening approach in yeast to identify key residues in maintaining ground stage of the human P2Y12. PCR-based random mutagenesis was optimized to induce approximately four mutations in 1\u00a0kbp. Several dozen clones were selected and are now under in-depth investigation. For example, we identified a triple mutant P2Y12 (Leu115Gln\/Phe177Ser\/Arg224Gly), and individual characterization revealed Leu115Gln (TMD3) to be mainly responsible for constitutive activity in the yeast expression system (Fig.\u00a06b). These promising results in applying the yeast expression system await further efforts to identify functionally relevant determinants in P2Y12-like receptors.\nEstablishing in vivo function of P2Y12-like receptors using mouse models\nSelective receptor ligand and receptor-deficient animal models are suitable tools to evaluate the physiological relevance of distinct GPCR. Enormous efforts have been undertaken in the development of selective and clinically useful ADP and other nucleotide receptor ligands. But except for P2Y12, dissection of the physiological relevance of all other P2Y12-like GPCR is at the very early stage. Since the pharmacological properties of ligands have been reviewed in detail elsewhere [2] we only shortly summarize available data on receptor-deficient animal models.\nBased on the clinical success of irreversibly bound P2Y12 antagonists, such as clopidogrel, the pivotal role of ADP in arterial thrombogenesis is well established. Although the combined action of P2Y1 and P2Y12 is necessary for the full platelet aggregation response to ADP, mice deficient for P2Y12 already display reduced platelet adhesion\/activation, thrombus growth, and stability [18, 55]. Except for the altered platelet functionality P2Y12-deficient mice appear normal under standard laboratory conditions. In consent with this finding, we occasionally identified a frameshift (T insertion at base pair position 667) in the P2Y12 coding sequence of several individuals of the Asian house mouse (inbred laboratory strain of Mus musculus castaneus from of the Laboratoire G\u00e9nome Populations Interactions Adaptation at the Universite Montpellier, France) during ortholog screening which showed no obvious phenotype in standard laboratory captivity. The platelet function was, however, not tested. The identified P2Y12 inactivation in some Asian house mice may reflect either natural polymorphisms present in the wild population or a new polymorphism that has been introduced during captivity. To distinguish between these possibilities, we analyzed the position 667 in P2Y12 from 88 wild M. m. castaneus trapped in Taiwan [41]. All contained the intact P2Y12 allele indicating that the inactive P2Y12 allele is very rare or absent in this wild M. m. castaneus population and favors the hypothesis of its acquisition in captivity (unpublished observations).\nThe fact that receptor deficiency is per se compatible with viability and fertility appears to be true also for other P2Y12-like receptor-deficient species and mouse models. For example, mice individually lacking GPR82 and GPR34 are viable, fertile, and produced viable offspring (own unpublished observation). Further, there are several vertebrate classes and species naturally deficient in distinct P2Y12-like receptors. For example, P2Y13 is absent in fish genomes sequenced so far, GPR82 is not present in pufferfish genomes, and GPR171 is absent in pufferfish and Xenopus genomes.\nIt appears that many functions of P2Y12-like receptors are more distinct and their disclosure requires specific challenging conditions. Indeed, P2Y12-deficient mice revealed an unexpected phenotype when specifically challenged. CNS injury is accompanied by release of nucleotides, serving as signals for microglial activation or chemotaxis. Microglia cells express several purinoceptors, including P2Y12. Microglia in P2Y12-deficient mice showed significantly diminished directional branch extension toward sites of cortical damage in the living mouse. These results imply that P2Y12 is a primary site at which nucleotides act to induce microglial chemotaxis at early stages of the response to local CNS injury [56].\nVariants of P2Y12-like receptor genes within human populations\nActivating and inactivating mutations in GPCR have been made responsible for more than 30 different human diseases [57]. As expected from its pivotal role in platelet activation inactivating mutations in P2Y12 can cause a congenital bleeding disorder. Only a few missense (Met1Arg, Pro258Thr, Arg256Gln, Arg265Trp) and frameshifting (frameshift at amino acid position 240) mutations have been reported [19, 58\u201360]. Unexceptionally, the missense mutations found are at highly conserved positions and cause an impairment of receptor function. There are no other human diseases identified yet which are associated with dysfunction of P2Y12-like receptors.\nThe antithrombotic effect of clopidogrel is considerably variable and the P2Y12 gene was screened for possible sequence variants. Five nucleotide variations were found in the human P2Y12 gene, two of them silent substitutions in the coding region [61]. Several studies were initiated to investigate the impact of P2Y12 polymorphisms on atherosclerosis [62, 63] and clopidogrel efficiency in preventing neurological events [64] showing no or some association to one haplotype. In single nucleotide polymorphism (SNP) projects, such as HapMap [65], SeattleSNP etc., silent variations have been identified at amino acid position Val4, Asn6, Gly12, and Phe182 of P2Y12. For example the C\/T variation at amino acid position Phe182 is only found in the African population whereas the G\/T variation at position Gly12 is present only in European and Asian populations. One missense mutation (Glu330Gly; receptor C terminus) has been detected in SNP projects. This variant is absent in European and Asian populations but displays an allelic frequency of about 15% in the African population. The functional relevance of this missense mutation has not been studied yet but a Gly at position 330 is naturally found in many other P2Y12 orthologs including primate P2Y12 almost ruling out a specific input in receptor function.\nOne silent variant (C\/T at Ile59) and one substituting variant (T\/C at Met158Thr) were identified in the coding region of P2Y13. The Thr158 variant is less frequent in African populations (~4%) when compared to European (~21%) and Asian (~18%) populations. Met158 is not conserved and a Thr at this position is found in the dolphin (Tursiops truncates).\nOnly two silent substitutions (A\/G at Ala35 and T\/C at Phe240) have been identified in P2Y14 so far. The GPR171 gene contains two more frequent silent substitutions (T\/C at Tyr19, A\/C Thr58) and one missense variant (A\/G at Ile283Val, frequency about 6%) which is located within the DPXXY motif of TMD7. Ile283 is quite conserved among vertebrate GPR171 orthologs. The functional relevance of Val283 has not been studied yet but it occurs naturally in the zebrafish ortholog.\nIn the human GPR87 gene three silent polymorphisms (G\/A at Pro46, G\/A at Leu179, C\/T at Tyr355) and one nonsynonymous polymorphism (C\/T at Thr205Met) are present. The position 205 is not conserved and Met205 is found in several rodents. Only rare silent substitutions in the coding regions have been found in GPR82 (C\/T at Asp313) and in GPR34 (A\/G at Val296) [34].\nSNP stochastically occur in individual genomes and can amount to a reasonable frequency in populations by drift but also by selection. There are several approaches and methods which are suitable to distinguish between drift and selection. Population genetic models predict that selection can leave \u201cfootprints\u201d in closely linked genomic regions. Several methods were developed to detect signatures of selective sweeps in genomic sequences [66]. All methods require data on allele variation (mainly SNP) within populations. Large genome-wide analyses have scanned the human genome for signatures of positive selection on the basis of nonsynonymous and synonymous substitution ratios or single nucleotide polymorphism (SNP) data. Several loci which contain GPCR genes have been identified using such methods, but the P2Y12-like receptor-containing loci showed no strong signatures of recent positive selection in these studies [67\u201370].\nConclusion\nP2Y12-like receptors which are grouped mainly by phylogenetic relations have been an inherent part of the vertebrate GPCR repertoire since more than 450 Myr. Although sharing features in respect to structural determinants and signal transduction the activating ligands are heterogeneous such as nucleotides, nucleotide derivatives, leukotrienes, and phospholipids. We are at the very beginning of understanding the physiological importance of the individual members. The nature of the ligands, first functional data, and expression of several members in migrating cells point at functions in immunologic response and tissue damage response. Doubtless, upcoming receptor-deficient mouse models and selective receptor ligands will help to unveil the functions of P2Y12-like receptor members. Further, one should consider combined receptor-deficient mouse models within this group but also with other P2Y receptors to uncover phenotypes which are hidden by receptor redundancy, e.g., in the case of ADP receptors.","keyphrases":["evolution","nucleotide receptor","g protein-coupled receptor","orphan gpcr"],"prmu":["P","P","P","P"]}
{"id":"Breast_Cancer_Res_Treat-3-1-2001224","title":"Letrozole in the extended adjuvant setting: MA.17\n","text":"Relapse after completing adjuvant tamoxifen therapy is a persistent threat for women with hormone-responsive breast cancer. Third-generation aromatase inhibitors, such as letrozole, provide a new option for extended adjuvant hormonal therapy after 5 years of tamoxifen. MA.17 was conducted to determine whether letrozole improves outcome after discontinuation of tamoxifen. Postmenopausal women with hormone receptor-positive breast cancer (N = 5,187) were randomized to letrozole 2.5 mg or placebo once daily for 5 years. At a median follow-up of 30 months, letrozole significantly improved disease-free survival (DFS; P < 0.001), the primary end point, compared with placebo (hazard ratio [HR] for recurrence or contralateral breast cancer 0.58; 95% confidence interval [CI] 0.45, 0.76] P < 0.001). Furthermore, letrozole significantly improved distant DFS (HR = 0.60; 95% CI 0.43, 0.84; P = 0.002) and, in women with node-positive tumors, overall survival (HR = 0.61; 95% CI 0.38, 0.98; P = 0.04). Clinical benefits, including an overall survival advantage, were also seen in women who crossed over from placebo to letrozole after unblinding, indicating that tumors remain sensitive to hormone therapy despite a prolonged period since discontinuation of tamoxifen. The efficacy and safety of letrozole therapy beyond 5 years is being assessed in a re-randomization study, following the emergence of new data suggesting that clinical benefit correlates with the duration of letrozole. MA.17 showed that letrozole is extremely well-tolerated relative to placebo. Letrozole should be considered for all women completing tamoxifen; new results from the post-unblinding analysis suggest that letrozole treatment should also be considered for all disease-free women for periods up to 5 years following completion of adjuvant tamoxifen.\nIntroduction and rationale\nThere is a persistent risk of breast cancer recurrence following primary treatment [1\u20133]. Initially, patients with hormone receptor-positive (HR+) breast tumors have a lower risk of recurrence than those with HR\u2212 tumors, but with longer follow-up, the opposite may be the case [3, 4]. For example, Saphner showed that the significantly higher hazard of recurrence in HR\u2212 versus HR+ patients in the time period 0\u201312\u00a0years (P\u00a0<\u00a00.00001) could be explained by the higher risk of recurrence in years 0\u20135 for HR\u2212 patients (P\u00a0<\u00a00.0001). However, between years 3 and 4, the hazard of recurrence for HR\u2212 and HR+ patients crossed, and beyond 5\u00a0years was actually higher for HR+ patients (P\u00a0=\u00a00.00002) [4]. These data clearly indicate the need for continuous hormonal treatment for women with HR+ tumors.\nThe benefits of adjuvant hormonal treatment with tamoxifen were first demonstrated in the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-14 trial [5]. This large randomized, double-blind, placebo-controlled trial involving patients with node-negative, HR+ breast cancer demonstrated a significant prolongation of disease-free survival (DFS) among women treated with tamoxifen for 5\u00a0years, as compared with those receiving placebo. Updated results with longer follow-up demonstrated that the 5-year benefit in DFS with tamoxifen persisted through at least 10\u00a0years of follow-up, and a statistically significant survival benefit was also observed [6]. However, since the optimal duration of tamoxifen therapy was not known, patients who had completed 5\u00a0years of tamoxifen therapy and were disease-free were re-randomized to receive placebo or tamoxifen. Results published with a follow-up of 7\u00a0years after reassignment demonstrated a disadvantage in patients who continued tamoxifen compared with those who discontinued: DFS was 78 vs. 82%, respectively (P\u00a0=\u00a00.03), and overall survival (OS) was 91 vs. 94% (P\u00a0=\u00a00.07). Thus, extending tamoxifen treatment beyond 5\u00a0years was not deemed beneficial [7] nor recommended [8] when the MA.17 trial was initiated.\nWhile clearly in the best interest of patients, discontinuation of tamoxifen after 5\u00a0years creates a therapeutic dilemma because of the persistent risk of breast cancer recurrence. Relapse or appearance of new tumors after completion of tamoxifen therapy is relatively common in patients with HR+ tumors [1, 2, 4, 7]. The Oxford meta-analysis found that more than half of breast cancer recurrences and two thirds of breast cancer deaths occur after 5\u00a0years of adjuvant tamoxifen [3]. In the NSABP B-14 trial, the average annual rate of breast cancer recurrences was 8.9 per 1,000 patients who discontinued tamoxifen at 5\u00a0years [7]. Patients in whom tamoxifen is discontinued therefore require an alternative treatment option to provide continuing protection from recurrence.\nThe loss of efficacy seen with long-term tamoxifen therapy may result from the emergence of a hormone-independent tumor phenotype [9, 10] or the induction of hypersensitivity to estradiol [11, 12]. Tamoxifen is thought to be more susceptible than aromatase inhibitors (AIs) to this adaptive change because of its intrinsic agonist properties [12]. Furthermore, data from the NSABP B-14 trial suggest that residual tumor cells may become tamoxifen-resistant, and that continued use of tamoxifen might in fact stimulate their proliferation [6, 13].\nThe development of highly potent and selective third-generation AIs provided a new hormone therapy option for patients with HR+ breast cancer [14\u201316]. It is suggested in independent studies that Letrozole (Femara\u00ae) is the most potent of the AIs as determined by in vitro assays [17] and clinical studies of total body aromatization [18]. A recent study has demonstrated that the more complete inhibition of aromatase achieved by 2.5\u00a0mg of letrozole than by 1\u00a0mg of anastrozole results in a greater degree of suppression of estradiol [19]. The clinical efficacy of letrozole was initially demonstrated in patients with HR+ metastatic breast cancer. In this setting, first-line therapy with letrozole was shown to significantly improve outcome compared with tamoxifen therapy [20, 21]. While OS was not significantly improved (34 vs. 30\u00a0months, respectively), an exploratory analysis of the patients that did not cross over showed a longer survival benefit for letrozole-treated patients (35 vs. 20\u00a0months) [22]. Second-line therapy with letrozole has also demonstrated significant clinical benefits in patients with HR+ metastatic breast cancer in whom tamoxifen has failed [23], and in one randomized trial demonstrated a significantly higher response rate than anastrozole in this population [24].\nData from in vivo models using MCF-7 cells transfected with the aromatase gene have shown that letrozole is more effective than tamoxifen and devoid of the agonist action observed with the selective estrogen receptor modulator [25, 26]. Although the mechanisms of estrogen blockade are different for letrozole and tamoxifen, combining the two agents did not increase antitumor activity in the MCF-7 xenograft model [25]. The authors proposed that sequential administration of tamoxifen and letrozole would be a more effective strategy [25]. This hypothesis was recently confirmed in vivo [27]. Using the MCF-7 model, it was demonstrated that tumors progressing on tamoxifen responded to second-line letrozole therapy, but tumors that progressed on letrozole did not respond to second-line treatment with tamoxifen or fulvestrant [27].\nThe evident need to continue hormone therapy after discontinuation of tamoxifen in patients with HR+ primary breast cancer and the potential efficacy of AIs in tumors resistant to tamoxifen provided the rationale for testing adjuvant letrozole after 5\u00a0years of tamoxifen. This paper reviews the key results from the landmark MA.17 trial [28, 29] and discusses the advantages of letrozole treatment after the recommended 5\u00a0years of tamoxifen therapy have been completed. To date, letrozole is the only AI approved in the United States and Europe for extended adjuvant therapy.\nTrial design and patients\nMA.17 was a phase III, randomized, double-blind, placebo-controlled trial of letrozole as extended adjuvant therapy in postmenopausal women with primary breast cancer who had completed approximately 5\u00a0years of adjuvant tamoxifen therapy [28, 29]. The aim of the trial was to determine whether letrozole improves outcome after the discontinuation of adjuvant tamoxifen therapy. The MA.17 trial was led by the National Cancer Institute of Canada Clinical Trials Group and included the North American Breast Intergroup and the Breast International Group. Institutional review boards of participating institutions approved the study protocol, and all patients gave written informed consent.\nPatient population\nThe trial enrolled 5,187 postmenopausal women with early-stage breast cancer in whom 5\u00a0years of tamoxifen (range 4.5\u20136\u00a0years) therapy had been completed less than 3\u00a0months before enrollment. Eligible women had to have histologically confirmed, HR+ primary breast cancer. HR+ tumors were defined as estrogen receptor-positive (ER+) or progesterone receptor-positive (PgR+) as determined by a level of 10\u00a0fmol\/mg of protein or a positive immunohistochemical analysis. Women were defined as being postmenopausal if they were aged at least 50\u00a0years at the start of adjuvant tamoxifen therapy, were <50\u00a0years of age at the start of tamoxifen therapy but postmenopausal at the initiation of tamoxifen therapy, were <50\u00a0years at the start of tamoxifen therapy but had undergone bilateral oophorectomy, were premenopausal and <50\u00a0years at the start of tamoxifen therapy but became amenorrheic during chemotherapy or treatment with tamoxifen, or were any age but had postmenopausal levels of luteinising hormone or follicle-stimulating hormone prior to the study. All women had a good performance status and life expectancy of at least 5\u00a0years.\nRandomized trial design\nEligible women were randomly assigned to receive treatment with letrozole (2.5\u00a0mg) or placebo orally daily for 5\u00a0years (see Fig.\u00a01). Women were stratified according to the tumor HR status (positive or unknown), lymph-node status (negative, positive, or unknown), and receipt or nonreceipt of previous adjuvant chemotherapy. Exploratory sub-analyses were based on these stratification factors and two additional covariates (criteria for the definition of postmenopausal status at the start of tamoxifen treatment and duration of tamoxifen treatment).\nFig.\u00a01MA.17 randomized trial design\nEnd points and rules for interim analyses\nThe primary end point of the trial was DFS, defined as the time from randomization to the earliest recurrence of the primary disease (in the breast, chest wall, or nodal or distant metastatic sites) or the development of a new primary breast cancer in the contralateral breast. Secondary cancer and death without a recurrence or a diagnosis of contralateral breast cancer were not included as events. The trial was powered to detect a 2.5% improvement in 4-year DFS with letrozole (from 88 to 90.5%). Two interim analyses were scheduled, and stopping rules were specified a priori for interim monitoring [30].\nThe secondary efficacy end points of the trial were OS (defined as the time from randomization to death from any cause), annual incidence rate of contralateral breast cancer, long-term safety and tolerability, and overall and menopause-specific quality of life (QOL). In addition, distant DFS (DDFS), defined as the time from random assignment until the first observation of distant metastasis, was included as a secondary efficacy end point in the final analysis [29].\nQOL and long-term safety were assessed as secondary end points [31]. Adverse events were graded according to the Common Toxicity Criteria of the National Cancer Institute (version 2.0). QOL was assessed with the Medical Outcomes Study 36-Item Short Form General Health Survey (SF-36) and the Menopause-Specific Quality of Life questionnaire [32, 33]. The effects of letrozole on lipid profile and bone mineral density (BMD) were assessed annually in companion studies to MA.17 [34, 35].\nEfficacy of letrozole as extended adjuvant therapy\nA total of 5,187 patients were randomized to either letrozole (n\u00a0=\u00a02,593) or placebo (n\u00a0=\u00a02,594). Because of noncompliance, 10 patients in the letrozole and seven in the placebo arm were excluded from all analyses, leaving 5,170 patients (2,583 on letrozole and 2,587 on placebo) in the time to event analysis (50 patients deemed ineligible for several reasons and 33 with major protocol violations were included in the analysis). The final safety analysis excluded 21 patients who never received study medication, yielding a final safety population of 5,149 patients, 2,572 receiving letrozole and 2,577 receiving placebo [29]. Based on the 43% reduction in recurrence risk (P\u00a0=\u00a00.00008) with letrozole seen in the first interim analysis at 2.4\u00a0years\u2019 median follow-up [28], the data and safety monitoring committee recommended that the MA.17 trial be discontinued early, and the participants were informed of the results. The trial was unblinded in October 2003, and patients on placebo were given the opportunity to switch to letrozole. Updated efficacy results after a median follow-up of 30\u00a0months confirmed the significant clinical benefits of letrozole as extended adjuvant therapy [29]. The updated trial results and recent additional efficacy analyses of MA.17 are summarized below.\nLetrozole significantly improves outcome\nAt 30\u00a0months\u2019 follow-up, letrozole significantly improved DFS, the primary end point, compared with placebo (see Fig.\u00a02) [29]. The 4-DFS for patients receiving letrozole was 94.4%, compared with 89.8% for patients receiving placebo. The hazard ratio for recurrence or contralateral breast cancer was 0.58 (95% confidence interval [CI] 0.45, 0.76; P\u00a0\u2264\u00a00.76), representing a 42% reduction in risk for letrozole relative to placebo. The updated analysis also showed that letrozole produced a statistically significant improvement in DDFS (hazard ratio\u00a0=\u00a00.60; 95% CI 0.43, 0.84; P\u00a0=\u00a00.002), which may be regarded as a more meaningful end point than overall DFS; women with distant metastases inevitably die of breast cancer, and an improvement in DDFS may therefore translate into longer overall survival [36, 37]. Letrozole treatment non-significantly prolonged time to contralateral breast cancer incidence, resulting in a 37.5% relative risk reduction compared with placebo [29].\nFig.\u00a02Kaplan\u2013Meier curves for disease-free survival in the updated analysis of MA.17. N, number at risk; S, survival percent, with 95% confidence intervals (CIs) in parentheses. Reprinted from ref. [29] with permission\nThe prospectively planned subgroup analysis showed that letrozole significantly improved DFS in all patients, irrespective of nodal status. The reduction in risk of recurrence in node-positive tumors was 39% (hazard ratio\u00a0=\u00a00.61; 95% CI 0.45, 0.84), and 55% in those with node-negative tumors (hazard ratio\u00a0=\u00a00.45; 95% CI 0.27, 0.73). While OS was not significantly improved (hazard ratio\u00a0=\u00a00.82; 95% CI 0.57, 1.19; P\u00a0=\u00a00.3), letrozole significantly improved OS in patients with node-positive tumors (hazard ratio\u00a0=\u00a00.61; 95% CI 0.38, 0.98; P\u00a0=\u00a00.04) (see Fig.\u00a03), and this was the first survival advantage demonstrated by an AI in early breast cancer.\nFig.\u00a03Forest plots of the treatment effect (letrozole vs. placebo), in terms of overall survival, in subgroups defined by hormone receptor status, lymph node status, previous chemotherapy, menopausal criteria, and duration of tamoxifen treatment. For each subgroup, the hazard ratio for death from any cause is plotted as a solid square, and the area of the square is proportional to the variance of the estimated effect. The length of the horizontal line through the square indicates the 95% confidence interval (CI). The arrow at the end of the horizontal line indicates that the confidence interval is larger than the scale of the figure. Reprinted from ref. [29] with permission\nAdditional MA.17 analyses\nOptimal duration of extended adjuvant letrozole\nThe final MA.17 database, including all events up to the date of unblinding, was analyzed to examine the relationship between duration of treatment and outcomes [38, 39]. Data from this analysis have provided further evidence to support an extended duration of letrozole, as this cohort analysis found that the longer patients are exposed to extended adjuvant letrozole (at least out to 48\u00a0months), the greater the benefit [39].\nThe risk of disease recurrence increased over time in the placebo group, whereas in patients receiving letrozole, risk appeared to peak at around 2\u00a0years of treatment and decrease thereafter. In the overall patient population, hazard ratios for events in DFS and DDFS progressively decreased over time, favoring letrozole, with the trend being significant (P\u00a0<\u00a00.0001 and P\u00a0=\u00a00.0013, respectively). The trend for OS was not significant but was always <1. In the 2,360 patients with node-positive status, hazard ratios for DFS, DDFS, and OS all decreased over time, with tests for trend all showing significance (P\u00a0=\u00a00.0004, 0.0005, and 0.038, respectively). Considering the 2,568 patients with node-negative status, the hazard ratios for DFS decreased over time, with the test for trend being significant (P\u00a0=\u00a00.027), whereas the hazard ratios for DDFS and OS showed no significant change over time (see Table\u00a01).\nTable\u00a01Analysis of the hazard ratios for disease recurrence over time between the letrozole and placebo arms of MA.17Month after randomizationNo. at risk (letrozole\/placebo)Hazard rate (letrozole)Hazard rate (placebo)Hazard ratio (letrozole vs. placebo)a122,425\/2,4090.000930.001800.52 (0.40\u20130.64)241,555\/1,5300.001050.002360.45 (0.33\u20130.56)36768\/7230.000900.002610.35 (0.21\u20130.48)48244\/2310.000590.003060.19 (0.04, 0.34)Reprinted from ref. [38] with permission from ElsevieraHazard ratios <1 indicate values in favor of letrozole\nMA.17 ITT analysis\nThe intent-to-treat (ITT) analysis at 54\u00a0months\u2019 follow-up looked at all outcomes, including all events before and after the unblinding, based on the original randomization of letrozole versus placebo; it did not take into account whether or not patients in the placebo group switched to letrozole at the unblinding of the data. The results further showed that patients randomized initially to letrozole had fewer DFS events than those initially randomized to placebo [40]. Publication of the final analysis of this data are awaited, but these provisional results highlight the strong beneficial effect of extended adjuvant therapy with letrozole when started immediately after tamoxifen.\nImpact of HR status on clinical benefit\nA retrospective analysis was conducted to determine whether HR status had an effect on the outcome of letrozole in the extended adjuvant setting [41]. ER and PgR positivity were defined as \u226510\u00a0fmol\/mg protein or positive by immunocytochemical analysis. Preliminary results from 4,635 patients, based on local testing of HR status, showed that the reduction in risk of recurrence with letrozole compared with placebo was greatest in women with the most hormone-dependent tumors. The final results of this analysis await publication. These results should be interpreted cautiously, as this was an unplanned, retrospective analysis, and receptor levels were measured locally. Furthermore, from the outset, the analysis of outcomes in all other subgroups besides the ER+\/PgR+ (n\u00a0=\u00a03,809) patients was weakened by the low numbers of patients in these groups (e.g., ER+\/PgR\u2212, n\u00a0=\u00a0636) [42].\nMA.17 post-unblinding analysis\nThe trial was unblinded in 2003 because DFS values were met (stopping boundary nominal significance, P\u00a0=\u00a00.0008) and patients were given the opportunity to cross over. Post-unblinding analysis of MA.17 has provided additional efficacy data on patients who had crossed over from placebo to letrozole (n\u00a0=\u00a01,655), comparing them with patients who elected no treatment at the time of the unblinding (n\u00a0=\u00a0613) [43]. These patients had not received any hormonal therapy after discontinuing tamoxifen. Data were adjusted for baseline patient and disease variables including tumor size, nodal status, and prior adjuvant chemotherapy. A preliminary analysis suggests that letrozole has significant clinical benefits in patients in whom treatment with the AI is started after a prolonged period since the discontinuation of tamoxifen. The publication of these results is eagerly awaited as they may effect patient care worldwide.\nMA.17 re-randomization\nA re-randomization of all patients completing letrozole to receive a further 5\u00a0years of letrozole or placebo is under way to confirm this finding [40, 44]. An amendment to this protocol allows women completing 5\u00a0years of any AI to be re-randomized to a further 5\u00a0years of letrozole or not regardless of prior tamoxifen or its duration. The MA.17 re-randomization study should provide additional insights into the efficacy and safety of extending letrozole therapy beyond 5\u00a0years [45].\nSafety of letrozole in the extended adjuvant setting\nThe women included in this trial had been disease-free for approximately 5\u00a0years during treatment with tamoxifen and, therefore, the safety and tolerability of continued hormone treatment with letrozole was an important consideration when the MA.17 trial was designed. Furthermore, early unblinding of the trial has not prevented the collection of long-term safety data, and additional sub-studies are providing useful information on the safety profile of letrozole in the extended adjuvant setting. The safety of AIs is discussed in detail in the paper by Dr. Perez in this supplement.\nThe MA.17 trial showed that letrozole is extremely well tolerated relative to placebo. The most common adverse events reported were secondary to estrogen suppression and included hot flashes, myalgia, arthralgia, alopecia, and newly diagnosed osteoporosis. The increase in newly diagnosed osteoporosis (8.1% for letrozole vs. 6% for placebo; P\u00a0=\u00a00.003) [29] was predictable from the potent suppression of estrogens by third-generation AIs [17] and the association between estrogen levels and bone turnover [46\u201348]. Of note, no significant difference in clinical fracture rate was seen between letrozole and placebo groups (5.3 vs. 4.6%; P\u00a0=\u00a00.25) [29].\nMA.17B is a companion study designed to compare the effects of letrozole (n\u00a0=\u00a0122) and placebo (n\u00a0=\u00a0104) on BMD in the L2\u2013L4 (posteroanterior) region of the spine and hip [35]. At 24\u00a0months, patients receiving letrozole had a significant decrease in total hip BMD (\u22123.6 vs \u22120.71%; P\u00a0=\u00a00.044) and lumbar spine BMD (\u22125.35 vs. \u22120.70%; P\u00a0=\u00a00.008). Further follow-up is necessary to evaluate the long-term clinical implications of this modest increase in bone resorption and reduction in BMD in the spine and hip with letrozole compared with placebo. Prophylactic use of the bisphosphonate zoledronic acid is being studied as a means to prevent BMD loss [49, 50]. Results from two clinical trials have indicated that early use of zoledronic acid effectively prevents BMD loss in women receiving adjuvant letrozole [49, 51].\nEstrogen has a beneficial effect on lipid profiles, and it has been suggested that AIs may have a relatively unfavorable effect. However, MA.17L, a substudy of MA.17 (n\u00a0=\u00a0347), demonstrated that letrozole does not significantly alter serum cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, or lipoprotein (a) compared with placebo [34]. This is in agreement with the results of the main MA.17 trial, which showed no difference in hypercholesterolemia rates between placebo and letrozole [28]. Importantly, in the updated analysis of the MA.17 dataset, there were no significant differences between the letrozole and placebo arms in the incidence of hypercholesterolemia (16% in each arm; P\u00a0=\u00a00.79) or cardiovascular events (5.8 vs. 5.6%; P\u00a0=\u00a00.76) [29].\nThe QOL substudy was conducted in 3,612 patients treated in MA.17 (1,813 letrozole and 1,799 placebo) [31]. The analysis demonstrated that letrozole did not have an adverse impact on overall QOL, as determined by SF-36, which is an important and reassuring finding, as the extension of letrozole treatment for up to 10\u00a0years in this setting is now being tested [44].\nConclusions\nIn recent years, considerable progress has been made in developing more effective hormonal treatments for women with breast cancer and improving the efficacy demonstrated with tamoxifen [3]. The pioneering MA.17 trial has demonstrated the need for extended adjuvant therapy after 5\u00a0years of tamoxifen to reduce the risk of recurrence in postmenopausal women with HR+ breast cancer. On the basis of this trial, letrozole was approved as extended adjuvant therapy, and it is currently the only AI approved for this indication [22, 52].\nMA.17 demonstrated that extended adjuvant therapy with letrozole provides women further protection against relapse after the completion of tamoxifen. These findings support the concept that distant micrometastases that have survived 5\u00a0years of tamoxifen therapy remain highly estrogen-sensitive and responsive to extended adjuvant letrozole treatment. This is an important clinical benefit in view of the persistent risk of relapse beyond 10\u00a0years in patients with HR+ tumors [4]. In fact, the cohort analysis showed that in the placebo group, there was actually an increasing risk of disease recurrence over time after discontinuing prior tamoxifen [40]. The preliminary MA.17 ITT data suggest that the strongest beneficial effect is still obtained when starting letrozole within 3\u00a0months of completing tamoxifen [38], while the post-unblinding results suggest that women with hormone-dependent breast cancer who are prescribed letrozole following a prolonged delay after completing tamoxifen may experience a significant improvement in outcome [43]. Therefore, if a patient misses the chance to start letrozole within 3\u00a0months post tamoxifen, these results suggest that there is still a benefit to initiating letrozole therapy for up to 5\u00a0years following the discontinuation of tamoxifen. Importantly, women in all risk categories benefited in terms of reduced risk for recurrence of their cancer. Thus, in both node-positive as well as node-negative women, there was a strong improvement in DFS, and \u201clow risk\u201d status of the primary tumor should not preclude consideration of extended adjuvant therapy with letrozole. Extended therapy with adjuvant letrozole should therefore be considered for all women currently completing tamoxifen. In addition, in women who have completed tamoxifen within the last 5\u00a0years, introduction of letrozole for 5\u00a0years can be discussed because although we do not have level 1 evidence for benefit in this setting as yet, our post-unblinding analysis of MA17 strongly supports the potential for benefit in these women.\nAmong the clinical questions left unanswered are the optimal duration of letrozole and the long-term safety of extended adjuvant therapy in women leading a normal healthy lifestyle. Preliminary results of a cohort study analysis of MA.17 provide support for the use of extended adjuvant letrozole for at least up to 4\u00a0years [40, 53]. The MA-17 re-randomization study will assign patients completing 5\u00a0years of letrozole to a further 5\u00a0years of letrozole or placebo and should provide data on the efficacy and safety of extending letrozole therapy beyond 5\u00a0years [45].\nIn conclusion, HR+ breast cancer presents an unremitting threat that may require life-long hormone therapy. The optimal hormone treatment strategy is evolving based on the results of landmark clinical trials in the initial adjuvant [54, 55], sequential adjuvant [56], and extended adjuvant settings [28, 29]. The optimal agent, sequence of treatments, or combination of treatments will be able to provide the greatest improvement in OS with minimal acute and long-term toxicity. MA.17 has demonstrated that letrozole is highly effective and extremely well tolerated when given in the extended adjuvant setting. The results of the pivotal MA.17 trial have changed current clinical practice to extend letrozole protective therapy in thousands of breast cancer patients currently receiving 5\u00a0years of adjuvant tamoxifen. The benefits of long-term adjuvant letrozole treatment clearly outweigh any adverse events in postmenopausal women who have survived breast cancer after initial adjuvant therapy with tamoxifen. Long-term side effects and risks continue to be monitored and taken into account when any individual patient is being considered for extended adjuvant therapy.","keyphrases":["letrozole","ma.17","tamoxifen","breast cancer","aromatase inhibitors"],"prmu":["P","P","P","P","P"]}
{"id":"Purinergic_Signal-4-1-2245998","title":"P2 receptors in cardiovascular regulation and disease\n","text":"The role of ATP as an extracellular signalling molecule is now well established and evidence is accumulating that ATP and other nucleotides (ADP, UTP and UDP) play important roles in cardiovascular physiology and pathophysiology, acting via P2X (ion channel) and P2Y (G protein-coupled) receptors. In this article we consider the dual role of ATP in regulation of vascular tone, released as a cotransmitter from sympathetic nerves or released in the vascular lumen in response to changes in blood flow and hypoxia. Further, purinergic long-term trophic and inflammatory signalling is described in cell proliferation, differentiation, migration and death in angiogenesis, vascular remodelling, restenosis and atherosclerosis. The effects on haemostasis and cardiac regulation is reviewed. The involvement of ATP in vascular diseases such as thrombosis, hypertension and diabetes will also be discussed, as well as various heart conditions. The purinergic system may be of similar importance as the sympathetic and renin-angiotensin-aldosterone systems in cardiovascular regulation and pathophysiology. The extracellular nucleotides and their cardiovascular P2 receptors are now entering the phase of clinical development.\nIntroduction\nEver since the first proposition of cell surface receptors for nucleotides [1, 2], it has become increasingly clear that, in addition to functioning as an intracellular energy source, the purines and pyrimidines ATP, adenosine diphosphate (ADP), uridine triphosphate (UTP) and uridine diphosphate (UDP) can serve as important extracellular signalling molecules [3, 4] acting on 13 P2X homo- and heteromultimer ionotropic and 8 P2Y metabotropic receptor subtypes [5, 6] (Table\u00a01). To terminate signalling, ectonucleotidases are present in the circulation and on cell surfaces, rapidly degrading extracellular ATP into ADP, AMP and adenosine [7, 8]. Evidence is accumulating suggesting an important role for the purinergic system in cardiovascular regulation [9\u201315]. It stimulates vasoconstriction and vasodilatation, growth of vascular smooth muscle cells and endothelial cells, angiogenesis, is involved in vascular remodelling, stimulates platelet aggregation, regulates coagulation, inflammation and several aspects of cardiac function. It is involved in blood pressure regulation, development of myocardial infarction, heart failure and xenograft rejection. The physiological effects of the purinergic signalling system are dependent on the release of extracellular nucleotides, the degradation by ectonucleotides, the type of P2 receptors expressed on the cells, their desensitisation rates and their second messengers. P2 receptors have highly specific organ distributions and they can be rapidly up- or downregulated. The purinergic system may be of similar importance as the sympathetic and renin-angiotensin-aldosterone systems in cardiovascular regulation and pathophysiology. The involvement of purinergic signalling in a variety of different cardiovascular clinical conditions has been addressed to a limited extent previously [15\u201319] and the large number of new discoveries calls for a review to summarise the most important roles of the purinergic system in cardiovascular regulation and disease.\nTable\u00a01Receptor classification, intracellular signalling, ligands and selective agonists and antagonistsP2 subtypeI.c. signallingLigandSelective agonistSelective antagonistNon-selective antagonistP2Y1\u2191IP3ADP (ATP)MRS2365MRS 2179, MRS2500P2Y2\u2191IP3UTP = ATPMRS2498, UTP\u03b3S, INS3717Suramin > RB2P2Y4\u2191IP3UTP (=ATP in rodents)UTP\u03b3S, INS3717\u2013RB2 > SuraminP2Y6\u2191IP3UDPMRS2666, MRS2633, UDP\u03b2SMRS2578P2Y11\u2191IP3, \u2191cAMPATPAR-C67085MX, NF546NF157Suramin > RB2P2Y12\u2193cAMPADP\u2013Clopidogrel, prasugrel, AZD6140, INS50589, AR-C9931 (cangrelor)P2Y13\u2193cAMPADP\u2013MRS2211P2Y14IP3UDP-glucose, UDP-galactoseUDP-glucose, UDP-galactose\u2013P2X1Positive ion channelATP\u03b1,\u03b2-mATPNF023, NF449TNP-ATP, Ip5IP2X2Positive ion channelATP\u2013NF770Suramin, isoPPADS, RB2P2X3Positive ion channelATP\u03b1,\u03b2-mATPA317491, NF110SuraminP2X4Positive ion channelATPIvermectin potentiates\u2013TNP-ATPP2X5Positive ion channelATP\u2013\u2013Suramin, PPADSP2X6Positive ion channelATP\u2013\u2013\u2013P2X7Positive ion channelATP\u2013KN62, KN04, MRS2427Coomassie brilliant blue GEctonucleotidaseApyrase, human SolCD39ARC67156\nThe research field has grown rapidly since the term P2 receptor was coined [2]. In preparation of this review more than 2,700 references on P2 receptors and cardiovascular regulation were found in PubMed. Among the 15 receptor subtypes, one is the target for one of the most widely used medical drugs\u2014the platelet inhibitor clopidogrel (Plavix). As will be reviewed here, several of the other 15 receptors are promising drug targets to prevent cardiovascular disease.\nRegulation of vascular tone\nVasoconstriction produced by ATP released as a cotransmitter with noradrenaline (NA) from perivascular sympathetic nerves was recognised early [20, 21]. However, following the seminal discovery of endothelium-dependent vasodilatation by Furchgott in the early 1980s, it was shown that ATP acted on endothelial cells to release endothelial derived relaxing factor [later shown to be largely nitric oxide (NO)] resulting in vasodilatation [22] and dual purinergic neural and endothelial control of vascular tone established [23, 24].\nNeuronal regulation of vascular tone\nATP is, together with NA and neuropeptide Y (NPY), a cotransmitter in sympathetic neurons [11, 25, 26], and sensory-motor nerves during \u201caxon reflex\u201d activity release ATP to dilate or constrict vessels [27, 28] (Fig.\u00a01). The contribution of ATP to sympathetic contraction varies between vascular beds [25]. For example, it may mediate up to 50% of the neurogenic vasoconstriction via P2X1 receptors as seen in mesenteric arteries [29], about 70% in rabbit saphenous arteries and 100% in rabbit jejunal artery, while NA acts as a prejunctional modulator [30]. In the renal vasculature both P2X1 and P2Y2 receptors are important for neurogenic contraction. P2X1 receptors mediate contraction in afferent but are absent in the efferent renal arterioles [31, 32]. The result is that extracellular nucleotides selectively influence preglomerular resistance without having an effect on postglomerular tone. ATP is also released locally in the kidney in response to increased perfusion pressure, and in P2X1 receptor knockout mice auto-regulatory responses in kidney afferent arterioles are abolished, indicating an important role in renal glomerular pressure regulation [32, 33].\nFig.\u00a01P2 receptor-mediated regulation of the circulation. See text for details. Purines and pyrimidines are released on the luminal side from endothelial cells, platelets and red blood cells (RBC) in response to hypoxia, acidosis, adrenaline, shear stress and other stimuli. When the endothelial cell layer is intact, the response is vasodilatation by the endothelial release of nitric oxide (NO), endothelium-derived hyperpolarising factor (EDHF) and prostaglandins (Pgl). When the endothelium is damaged, platelets accumulate, release ATP and ADP and mediate vasoconstriction via P2 receptors on the vascular smooth muscle cell (VSMC). On the adventitial side sympathetic and sensory nerves mediate vasoconstriction. Extracellular nucleotides are rapidly degraded by NTPDase1 on endothelium reducing ATP to AMP, followed by conversion to adenosine by CD73 (not shown). In the subendothelium NTPDase2 is present, degrading ATP to ADP, maintaining the platelet activating and contractile effects. Ado adenosine, CGRP calcitonin gene-related peptide, SP substance P\nSympathetic nerves express inhibitory P2Y and P1 (A1) receptors indicating a P2 receptor-mediated negative feedback loop both directly by ATP and its degradation product adenosine [34, 35]. Ectonucleotidases are released from sympathetic nerves together with its substrate ATP, as a termination mechanism for the signalling [36]. Another balancing mechanism is the sympathicolytic effect seen in exercising skeletal muscle, which is important for increased blood flow in the working skeletal muscle during exercise, despite sympathetic NA release that normally would reduce blood flow. This sympathicolytic effect is mimicked by injection in the arterial lumen of ATP and UTP, inducing a vasodilatation that overrides sympathetic vasoconstrictor activity in human skeletal muscle, an effect not obtained by injection of adenosine [37], suggesting that ATP and UTP may mediate this important physiological mechanism.\nThe first evidence of the presence of P2 receptor subtypes suggested P2X receptors on vascular smooth muscle cells (VSMC) and P2Y receptors on endothelial cells [38]. Rat blood vessel immunohistochemistry and human mRNA quantification have shown the P2X1 receptor to be the highest expressed subtype in smooth muscle cells [39, 40]. It has been difficult to prove the importance of other contractile P2X receptors besides P2X1 due to the paucity of specific agonists and antagonists. However, contractile effects of UTP suggested that P2Y receptors were present on VSMCs and mediated contraction [41]. Using selective pyrimidines resistant to degradation, it has been possible to show that contractile effects are mediated by both UTP-sensitive P2Y2 and UDP-sensitive P2Y6 receptors [42]. Ectonucleotidases in the vessel wall rapidly degrade nucleotides and markedly reduce their contractile effects. Pyrimidine analogues more resistant to ectonucleotidases are powerful vasoconstrictors and up to 1,000-fold more potent than the endogenous ligands [42]. Similarly, a prominent more potent UDP contraction has been seen in NTPDase1 knockout mice, demonstrating the protective effect of ectonucleotidases against nucleotide contraction [43]. In human saphenous vein grafts, used during coronary bypass, a stable UDP analogue stimulates a strong contraction lasting for hours, which is not desensitised [44]. The reason why the P2Y6 receptor is not desensitised is the lack of serine residues in the C-terminal part of the P2Y6 receptor [45]. Neither \u03b11-adrenoceptors, nor P2Y6 receptors, are present in human coronary arteries, possibly to avoid deleterious vasospasm during ischaemia [44].\nVSMC express P2Y12 receptors that mediate contraction after stimulation with ADP [46]. At the mRNA level, the P2Y12 receptor is the highest expressed ADP receptor and the second highest expressed P2 receptor in human VSMC [46]. The contractions are not inhibited in patients medicated with clopidogrel. However, drugs with antagonistic effects on P2Y12 receptors that reach the peripheral circulation (AZD6140 and INS50589), affecting both platelets and VSMC, could be of double therapeutic benefit in their prevention of both thrombosis and vasospasm [46].\nIn conclusion, extracellular nucleotides mediate vasoconstriction when released from nerves on the adventitial side, or when released in the lumen when the endothelium is damaged (Fig.\u00a01). The most important contractile receptors on the VSMC are the ATP P2X1 receptor, the ATP\/UTP P2Y2 receptor, the UDP P2Y6 receptor and the ADP P2Y12 receptor (Fig.\u00a01 and Table\u00a02). To determine the relative physiological importance of these receptor subtypes in different human vascular beds is an important task for the future.\nTable\u00a02P2 receptor expression in the cardiovascular systemP2 subtypeEndotheliumVSMCHeartPlateletsRed blood cellsInflammatory cellsPerivascular sensory nervesPerivascular sympathetic nervesP2Y1High\u2013\u2013High(Turkey)MediumMedium (neg. feedback)P2Y2HighHighHigh\u2013\u2013HighLowPossibleP2Y4\u2013Possible\u2013\u2013\u2013P2Y6MediumMediumHigh\u2013\u2013P2Y11Possible\u2013High\u2013\u2013HighP2Y12\u2013Medium\u2013High\u2013LowP2Y13\u2013\u2013\u2013\u2013HighLowP2Y14\u2013\u2013\u2013\u2013\u2013P2X1\u2013HighMediumHigh\u2013MediumP2X2\u2013\u2013\u2013\u2013\u2013HighMedium (pos. feedback)P2X3\u2013\u2013\u2013\u2013\u2013HighP2X4High\u2013Low\u2013\u2013HighP2X5\u2013\u2013\u2013\u2013\u2013P2X6\u2013\u2013\u2013\u2013\u2013P2X7\u2013Possible\u2013\u2013LowHighUnless specified data refer to human tissue. High, medium or low expression represents an effort to summarise the results of a large number of mRNA, protein and pharmacological studies. Dash (\u2013) denotes lack of convincing evidence for expression. VSMC vascular smooth muscle cell\nEndothelial regulation\nShear stress and hypoxia are important stimuli of both ATP and UTP release from endothelial cells [47] (Fig.\u00a01). Extracellular nucleotides have several important effects mediated by activation of endothelial cells. Vasodilatation and decreased blood pressure by release of prostaglandins and NO has been demonstrated in several studies [5], but P2 receptors also mediate release of endothelium-derived hyperpolarising factor (EDHF), which relaxes VSMC by activation of potassium channels, with subsequent hyperpolarisation [48\u201350]. Both UTP and ATP reduce forearm vascular resistance in a prostaglandin and NO independent way [51], indicating an important role for EDHF in P2 receptor-mediated vasodilatation in man.\nThe P2Y1 receptor seems to be of major importance in most vascular beds [11]. However, several other P2 receptors are important for endothelial regulation. Pharmacology of vasodilatation and mRNA quantification in man indicates that P2Y2 and to a lesser degree P2Y6 also are important endothelial P2Y receptors [40, 52]. Knockout mice experiments recently confirmed this picture and demonstrated that the P2Y4 receptor does not mediate dilatation [53]. UTP and ATP are equipotent as vasodilators when infused in the human forearm circulation, indicating a role for both purinergic and pyrimidinergic receptors.\nThe P2X1 receptor is not expressed on the vascular endothelium and the evidence for other P2X receptors has been scarce, except for the P2X4 receptor which is the highest expressed P2 receptor in endothelium [40, 54]. Using antisense oligonucleotides the P2X4 receptor was shown to be important for shear stress-dependent Ca2+ influx via an ATP-dependent mechanism [55]. This indicates that ATP and P2 receptors may be of importance for shear stress-mediated effects, which is in agreement with the well-established release of ATP from endothelial cells during shear stress [56]. Vessel dilation induced by acute increases in blood flow is markedly suppressed in P2X4 receptor knockout mice [57]. Thus, endothelial P2X4 channels are crucial to flow-sensitive mechanisms that regulate blood pressure and vascular remodelling [57].\nExtracellular ATP in the circulation is rapidly degraded into ADP, AMP and adenosine by ectonucleotidases. Vascular NTPDase1 (CD39) is an endothelial cell membrane protein with both ecto-ATPase and ecto-ADPase activities [8, 58]. Ectonucleotidases are also released by shear stress from endothelial cells [59].\nReactive hyperaemia is the massive increase in blood flow that starts when a blood vessel is opened after a period of ischaemia. It is well known that ATP is released during ischaemia [7, 60]. Adenosine only mediates the late phase of the reactive hyperaemia as has been shown by infusion of adenosine deaminase or a selective A2A antagonist [61, 62]; ADP has been shown to mediate the mid-portion and peak of coronary reactive hyperaemia via endothelial P2Y1 receptors [63]. It has been proposed that different purines may mediate three phases of the reactive hyperaemia [63]. According to this hypothesis, ATP would mediate the first part of the hyperaemia via endothelial P2Y2 or P2X4 receptors. ATP would then be degraded to ADP, which mediates peak hyperaemia via endothelial P2Y1 receptors, followed by degradation of ADP to adenosine resulting in late phase hyperaemia mediated via A2A receptors on smooth muscle cells.\nRed blood cells as regulators of vascular tone\nThe matching of oxygen supply with demand requires a mechanism that increases blood flow in response to decreased tissue oxygen levels. Several reports suggest that the red blood cell (RBC) acts as a sensor for hypoxia and different mechanisms have been suggested by which the deoxygenated RBC stimulates vasodilatation [37, 64\u201366]. RBCs contain millimolar amounts of ATP and possess the membrane-bound glycolytic enzymes necessary for its production [67\u201369]. ATP is released in response to reductions in oxygen tension and pH [37, 64] (see Table\u00a03). It has been shown in vitro that vessels dilate in response to low O2 levels only when blood vessels are perfused with RBCs [70]. ATP is released in working human skeletal muscle circulation depending on the number of unoccupied haemoglobin O2 binding sites [64, 71]. Similar results have been shown in the coronary circulation of dogs [72]. The released ATP then binds to P2Y receptors on the endothelium and stimulates vasodilatation. Thus, the RBC functions as an O2 sensor, contributing to the regulation of blood flow and O2 delivery, by releasing ATP depending on the oxygenation state of haemoglobin.\nTable\u00a03Release of purines and pyrimidines in the cardiovascular systemSourceEndotheliumVSMCHeartPlateletsRed blood cellsInflammatory cellsVascular nervesStimuliShear stress, hypoxia, high glucoseHigh glucose, hypoxiaHypoxiaCollagen, thromboxane, other platelet activatorsHypoxia, adrenaline, deformationInfl. activatorsSympathetic nerves, sensory nerve collateralsMechanismVesicularUnknownUnknownVesicularUnknownUnknown or vesicularVesicularNucleotideATP, UTPATP, UTPATP, UTPADP > ATPATPATPATPTargetEndothelium VSMCVSMCCardiomyocytes, blood vessels, intrinsic cardiac neuronsPlatelets; endothelium or VSMCEndotheliumVSMC, endotheliumVSMCDegradationNTPDase1NTPDase2, NTPDase1NTPDase1NTPDase1NTPDase1NTPDase1NTPDase1 (co-released)Stimuli represents stimuli leading to release. Mechanism refers to mechanism of release at the cellular level. Target represents the cells that are regulated by the released purine or pyrimidine. Degradation refers to the degrading enzyme responsible for removing the purine or pyrimidine after the release. VSMC vascular smooth muscle cell\nADP activates a negative feedback pathway for ATP release from human RBCs via P2Y13 receptors [73]. Since blood consists of approximately 40% RBCs that contain a 1,000-fold higher ATP concentration than plasma (mmol\/l vs \u03bcmol\/l), even a minor release of ATP from the high intracellular concentrations could have major circulatory effects. A negative feedback system may therefore be of great physiological importance to mitigate ATP release. Another negative feedback system is the mechanism by which NO inhibits ATP release from erythrocytes, illustrating how NO may turn off ATP release [74]. NO is then scavenged by haemoglobin.\nFurthermore, the previous view of the RBC as a \u201cpassive bag that transports oxygen\u201d is challenged. It now turns out that it releases ATP in response to stimuli and, as with most important signalling systems, it has a negative feedback system to terminate its release. The described negative feedback pathway may be important to avoid high extracellular concentrations of ATP. At levels above 100\u00a0\u00b5mol\/l, ATP concentrations may exceed the catalytic capacity of ectonucleotidases and could, in fact, stimulate ATP release by increasing permeability of the RBC [75], probably via P2X7 receptors [76]. ATP may even release ATP from endothelial cells [77]. At high concentrations of ATP, a self-sustaining process may thus be instigated which may contribute to the irreversible stage of circulatory shock that can develop rapidly in severely ill patients. Similar mechanisms may be of importance in malaria because induction of the osmolyte permeability in Plasmodium-infected erythrocytes involves purinoceptor signalling [78].\nA mechanism of ATP release from RBCs, which has eluded researchers for many years, has recently been suggested by Locovei and co-workers as being mediated via the gap junction protein pannexin-1 [79]. Erythrocytes do not form gap junctions, instead pannexin-1 forms a mechanosensitive ATP-permeable channel that mediates osmotically induced ATP release.\nHypertension\nBlood pressure regulation by purinergic signalling is the net result of balancing contractile and dilatory effects as described above. ATP and UTP released on the luminal side, from endothelial cells and erythrocytes, stimulate vasodilatation, in contrast to release from nerves on the adventitial side, which results in vasoconstriction. ATP may also regulate blood pressure via renal mechanisms or brain stem regulation [31, 32].\nATP plays a significantly greater role as a sympathetic cotransmitter in spontaneously hypertensive rats (SHR) [80, 81], and there is increased responsiveness of the renal vasculature of isolated perfused rat kidneys to \u03b1,\u03b2-methylene ATP in SHR [82], while mesenteric vascular contractile reactivity to ATP via P2X1 and P2Y2 receptors is not altered in deoxycorticosterone acetate (DOCA)-salt hypertension [83]. In the aorta of SHR, endothelium-dependent relaxation to ATP is impaired because of the concomitant generation of an endothelium-derived contracting factor. Contractions to ATP were significantly potentiated in SHR aorta [84]. In hypertensive mature stroke-prone rats, NPY modulation of release of NA and ATP from sympathetic perivascular nerves in kidney vessels is impaired, which may account for the increased nerve-mediated responses [85]. Thus, animal models of hypertension reveal several alterations in purinergic signalling that may contribute to the development of hypertension.\nDiadenosine polyphosphates such as Ap4A, Ap5A and Ap6A are combinations of two adenosine molecules connected with four to six phosphate groups. They have been identified as vasocontractile agents [86], probably via actions on P2X1 and P2Y2 receptors. Ap5A and Ap6A are stored at higher levels in platelets from patients with hypertension and may contribute to their increased peripheral vascular resistance [87]. Up4A is a novel endothelium-derived vasoconstrictive factor more potent than endothelin in renal vasoconstriction [88]. It is released upon stimulation of the endothelium by acetylcholine, thrombin and mechanical stress and can be cleaved into either ATP or UTP to stimulate both P2X1 and P2Y2 receptors on VSMC resulting in increased blood pressure [88].\nAs mentioned above, the P2X4 receptor is the most abundantly expressed P2 receptor in the endothelium and mediates shear stress-stimulated vasodilatation. P2X4 receptor knockout mice have higher blood pressures and excrete smaller amounts of NO products in their urine than do wild-type mice [57]. The P2X4 receptor protein is upregulated in the placenta in preeclampsia [89]. Thus, shear stress-stimulated release of ATP acting on P2X4 receptors may be important for blood pressure regulation.\nThe importance of P2 receptors for hypertension will not be proven until tested in man. It would be of great interest to perform a clinical study on the effects of P2X1, P2Y2 or P2Y6 receptor antagonists in the treatment of hypertension.\nPulmonary hypertension\nErythrocyte release of ATP appears to regulate pulmonary resistance under some conditions [90], and patients with pulmonary hypertension have been found with impaired release of ATP from RBC [69]. Endothelium-dependent relaxation to ATP has been demonstrated in human pulmonary arteries [91]. On the other hand, ATP is a mitogen for pulmonary artery smooth muscle cells, which may be relevant for the pathophysiological basis of pulmonary hypertension [92]. Again, the balance between endothelial versus smooth muscle stimulation by ATP may regulate blood pressure in different directions and we are far from a full understanding of the role of the purinergic system in pulmonary hypertension.\nMigraine and vascular pain\nClassic migraine is associated with two distinct vascular changes: an initial vasoconstriction, followed by vasodilatation associated with pain. It has been proposed that purinergic signalling is involved in these changes. ATP released from perivascular sympathetic nerves may participate in producing the initial vasospasm mediated by P2 receptors in vascular smooth muscle. Cerebral arteries are strongly contracted by UTP and especially UDP via P2Y6 receptors. A UDP agonist might therefore have similar effects as the 5-HT1D agonists frequently used to treat migraine [93]. ATP released by endothelial cells acting on P2 receptors on endothelial cells mediating release of NO and producing vasodilatation may contribute to the phase of reactive hyperaemia following vasospasm [94]. It has been suggested that antagonists to P2X3 receptors, which are located on nociceptive sensory nerve endings in cerebral vessels, may be promising candidates for anti-migraine drug development [95]. A recent report demonstrates selective upregulation of nociceptive P2X3 receptors on trigeminal neurons by calcitonin gene-related peptide and suggests that this mechanism might contribute to pain sensitisation in migraine [96]. P2Y receptors on trigeminal sensory nerves have also been implicated in pain in migraine. It has been claimed that migraine attacks are characterised by a relative depletion of sympathetic NA stores in conjunction with an increase in release of ATP [97]. In a review in the Lancet in 1996, Burnstock proposed that vascular pain, as in angina, ischaemic muscle and pelvic pain in women, may be initiated by ATP released from microvascular endothelial cells during reactive hyperaemia, diffusing a short distance to activate P2X3 nociceptive receptors on sensory nerves in the adventitia [26].\nAtherosclerosis\nAtherosclerosis is the main cause of ischaemic stroke and cardiovascular disease and is now considered to be an inflammatory disease [98]. The formation of a plaque starts with the accumulation of cholesterol followed by invasion of macrophages taking up cholesterol and becoming foam cells. The plaque can be stabilised by smooth muscle cell formation of a fibrous cap that covers the lipid-rich region. However, stimulation of inflammation by oxidised low-density lipoprotein activates macrophages and dendritic cells into antigen-presenting cells, activating T lymphocytes resulting in release of cytokines and metalloproteinases degrading the fibrous cap. The end result is a vulnerable plaque and, when it ruptures, its highly thrombogenic contents activates platelets and causes the formation of a local thrombus occluding the artery or embolising, resulting in ischaemic stroke or myocardial infarction. Evidence both from basic research and from clinical studies indicates important involvement on several levels for purinergic signalling in the atherosclerotic process (Fig.\u00a02). Interestingly, fish oil components increase the release of ATP [99] and a high cholesterol diet decreases ATP release from arteries [100, 101].\nFig.\u00a02Functional roles of P2 receptors in the atherosclerotic inflammatory plaque and during restenosis. See text for details. Purines and pyrimidines acting on P2 receptors stimulate vascular inflammation both by actions on the endothelial cell (EC) and by effects on inflammatory cells. Furthermore, they stimulate vascular smooth muscle cell (VSCM) proliferation, the conversion to synthetic phenotype and production of matrix proteins. Mitogenic P2 receptors are upregulated by growth factors and cytokines. IL interleukin, MCP-1 monocyte chemoattractant protein-1, ICAM-1 intercellular adhesion molecule-1, TSP thrombospondin, IDO indoleamine 2,3-dioxygenase\nAtherosclerosis\u2014P2 receptor-mediated effects on inflammatory cells\nInflammatory cells express a large number of P2 receptors with multiple effects [102]. The final result is difficult to predict depending on the subtype of receptor expressed by a particular cell type and on the differentiation stage of the cell. The most important inflammatory cells for atherosclerosis are monocytes that differentiate into macrophages or dendritic cells in the plaque, and the T-helper and suppressor lymphocytes that coordinate the inflammatory reaction in the plaque [98].\nA large number of P2 receptors are expressed on T lymphocytes and macrophages [14, 103] and have been suggested to be important players in atherosclerosis [14]. Strong evidence for a functional role exists for P2X7, P2Y2 and P2Y11 receptors. The P2X7 receptor is mitogenic and anti-apoptotic for T lymphocytes [104, 105]. P2X7 is important for release of interleukin (IL)-1 [106], tumour necrosis factor [107] and L-selectin, an adhesion molecule important for lymphocyte binding to endothelium [108]. All of these effects are known to be important for atherosclerosis development. The severity of arthritis is reduced in P2X7 receptor knockout mice [109] and rheumatoid arthritis is coupled to increased incidence of myocardial infarction. It would be of great interest to examine the P2X7 receptor knockout mouse in an atherosclerosis model. ATP and UTP are chemotactic for dendritic cells probably via the P2Y2 receptor and may attract inflammatory cells to the vascular lesion [110]. The P2Y2 receptor enhances the oxidative burst in human macrophages [111]. P2Y1 receptor deletion in knockout mice reduces the atherosclerotic lesions and the plaque area occupied by macrophages in ApoE knockout mice [112]. Whether this is due to platelet inhibition, endothelial or an inflammatory mechanism requires further studies.\nATP inhibits CD4+ T cell activation via an increase in cyclic AMP (cAMP) probably via a P2Y11 receptor [113]. ATP acting on P2Y11 receptors regulates the maturation of human monocyte-derived dendritic cells and induces immunosuppression by inhibiting T-helper 1 cytokines and promoting T-helper 2 cytokines [114, 115].\nA polymorphism in the P2Y11 receptor has been shown to have clinical importance by increasing the risk of myocardial infarction [116]. The G-459-A polymorphism, carried by one fifth of the population, causes an Ala-87-Thr substitution in the P2Y11 ATP receptor and increases the risk of myocardial infarction by 21%. The odds ratio increased stepwise depending on the number of Thr-87 alleles, and in subgroups in which the genetic influence is known to be of increased importance, family history of acute myocardial infarction (AMI), early onset AMI or the combined group of early onset AMI with family history. The mechanism by which the polymorphism causes AMI seems to be coupled to increased inflammation because the Thr-87 variant of the P2Y11 receptor was coupled to elevated C-reactive protein (CRP) levels. CRP is a marker of inflammation and an independent prognostic risk factor for the development of AMI [98]. Thus, the P2Y11 receptor is important in the development of atherosclerosis via modulation of inflammation either via effects on T lymphocytes or macrophage cells.\nProinflammatory effects on the endothelium\nEven though P2 receptor-mediated activation of the endothelium stimulates release of NO, which inhibits inflammatory cells, it is now well established that important proinflammatory endothelial effects may also be triggered. A proinflammatory dysfunctional endothelium is crucial in the recruitment of monocytes to the atherosclerotic plaque and in the general extravasation and loss of peripheral resistance seen in sepsis. ATP stimulates neutrophil adherence to cultured endothelial cells [117]. ATP stimulates release of IL-6, IL-8, monocyte chemoattractant protein-1, growth-regulated oncogene \u03b1 and increased expression of intercellular adhesion molecule-1 in human microvascular endothelial cells [118]. UTP and ATP stimulate expression of proinflammatory vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells through activation of the P2Y2 receptor and increases the adherence of monocytic cells to human coronary endothelial cells, an effect that was inhibited by anti-VCAM-1 antibodies [119]. VCAM is important for the recruitment of monocytes and lymphocytes. The VCAM stimulation is caused by P2Y2 receptor-induced transactivation of the vascular endothelial growth factor receptor-2 [120]. These effects have been confirmed in an in vivo neointima model, in which perivascular infusion of UTP enhanced infiltration by macrophages [121].\nIn conclusion, ATP and UTP stimulate several inflammatory responses known to be important for atherosclerosis development (Fig.\u00a02).\nRestenosis and vascular smooth muscle cell proliferative disease\nVSMC proliferation contributes to plaque development, but it is probably beneficial by stabilising the plaque and forming a stronger fibrinous cap. However, there are several situations in which VSMC proliferation is important in vascular disease development: restenosis after balloon angioplasty, diabetic microvascular disease, chronic allograft rejection, pulmonary hypertension and possibly systemic hypertension. The latter is suggested since sympathetic nerves exert a trophic effect on vascular smooth muscle [122]. ATP is a cotransmitter from sympathetic nerves [25] and has been shown to be a more potent mitogen than the other cotransmitters NA and NPY [123].\nExtracellular ATP is a potent growth factor for VSMC by activation of G protein-coupled P2Y receptors [12, 123\u2013125]. UTP is also mitogenic, acting on P2Y2 receptors [123], as is UDP acting on P2Y6 receptors [124, 126, 127]. ATP is synergistic with polypeptide growth factors (e.g. platelet-derived growth factor, basic fibroblast growth factor) and insulin [123, 124]. The signal transduction is mediated via Gq proteins, phospholipase C and D, diacylglycerol, protein kinase C, extracellular signal-regulated kinase, phosphatidylinositol-3 kinase, MAPK\/ERK activity kinase, mitogen-activated protein kinases and Rho [12, 128, 129]. Several immediate early genes are activated and the cell is taken through different phases of the cell cycle [125, 130]. Sometimes, as with UDP acting on P2Y6 receptors, progression is stimulated to both the S and G2 phases, that is through the whole cell cycle [127].\nMitogenic effects have been demonstrated in rat, porcine, and bovine VSMC and cells from human coronary arteries, aorta, and subcutaneous arteries and veins [12, 13, 124, 131]. The trophic effects on VSMC and the abundant sources for extracellular ATP in the vessel wall make a pathophysiological role probable in the development of atherosclerosis, neointima formation after angioplasty, chronic allograft rejection, pulmonary hypertension, diabetic microvascular disease and possibly hypertension. In these processes interaction between the VSMC and the matrix is important. It is therefore interesting that the human P2Y2 receptor contains an integrin-binding domain (RGD) in its first extracellular loop and that interaction with integrins influences P2Y2 receptor-mediated activation of G proteins [132]. ATP stimulates release of matrix metalloproteinase-2 (MMP-2) from human aortic smooth muscle cells and MMP-2 has been implicated in aortic aneurysm pathogenesis [133]. MMPs open up the matrix for migrating VSMC. ATP and UTP are potent chemotactic agents stimulating VSMC migration via P2Y2 and P2Y6 receptors [134]. These mechanisms, together with growth-stimulating effects, explain why perivascular infusion of UTP in a neointima model enhanced neointimal development [121]. Overexpression of the ATP- and UTP-degrading enzyme NTPDase1 reduces neointima development in rat aorta [135]. Augmentation of NTPDase1 activity is an important adaptive response for cardiac allograft survival, with increased inflammation, platelet deposition and infarction rates in NTPDase-deficient mice [136]. The mechanism is dependent on increased expression of the atherosclerotic matrix protein osteopontin [134, 137].\nDiabetic microvascular disease\nSympathetic vascular dysfunction in early experimental juvenile diabetes was recognised many years ago. Two weeks after induction of streptozotocin diabetes in rats, there was prejunctional impairment of sympathetic neurotransmission and impaired ATP-mediated endothelial function in mesenteric arteries [138]. Altered relaxant responses to ATP in the corpus cavernosum of men and rats with diabetes have also been reported [139].\nAs mentioned above, red blood cells may stimulate vasodilatation via release of ATP, which may be important in maintaining perfusion in the microcirculation. Interestingly, in erythrocytes from humans with type 2 diabetes ATP release is impaired, which is consistent with the hypothesis that the defect in erythrocyte physiology could contribute to the vascular disease associated with this clinical condition [140].\nHigh extracellular glucose releases ATP and\/or UTP in endothelial cells and pancreatic \u03b2 cells [141, 142]. An increase in glucose from 5 to 15\u00a0mmol\/l results in a marked increase in the proatherogenic nuclear factor of activated T cells signalling pathway in VSMC [143]. The effect is mediated via glucose-induced release of ATP and UTP, which subsequently activate P2Y2 but also P2Y6 receptors (after degradation to UDP). Thus, nucleotide release is a potential metabolic sensor for the arterial smooth muscle response to high glucose. Diabetic patients experience microvascular disease characterised by increased wall-lumen ratio, mainly because of an increase in VSMC and have higher rates of restenosis after coronary angioplasty. High glucose-induced release of extracellular nucleotides, acting on P2Y receptors to stimulate VSMC growth via NFAT (nuclear factor of activated T cells) activation, may provide a link between diabetes and diabetic vascular disease [143].\nIn conclusion, ATP\/UTP\/UDP stimulate VSMC growth, migration, release of MMPs and osteopontin that may contribute to the development of restenosis, diabetic microvascular disease, chronic allograft rejection, pulmonary hypertension and possibly systemic hypertension (Fig.\u00a02).\nVascular remodelling and angiogenesis\nVascular remodelling\nP2 receptors may regulate VSMC phenotype and vice versa, and P2 receptor expression is markedly altered during phenotype changes [13]. The shift from a specialised contractile VSMC phenotype into a proliferating, matrix-producing, synthetic phenotype is a prerequisite for VSMC pathogenesis in vascular disease. Pacaud and co-workers found that the Ca2+-mobilising effects of 2-methylthioATP increased in VSMC during culture in serum, indicating upregulation of P2Y1 receptors in the transition from contractile to synthetic phenotype [144]. This was confirmed at the mRNA level where P2Y2 receptors were found to be upregulated, while P2X1 receptors were downregulated [145]. Mitogenic P2Y receptors are upregulated while ion channel receptors, with only contractile effects, are downregulated in the synthetic phenotype [145]. Growth factors, cytokines and interestingly also ATP are potent stimulators of P2Y2 receptor expression [146, 147]. Thus, factors of importance in the development of vascular disease increase mitogenic P2Y2 receptors; this is further supported by their upregulation in neointima after balloon angioplasty [148, 149]. ATP has a dual concentration-dependent effect on VSMC phenotype. Low ATP concentrations stimulate expression of genes specific for the contractile phenotype, while high ATP concentrations cause a phenotypic shift from the contractile to the synthetic phenotype, and this shift is dependent on a transient activation of protein kinase A, which inhibits activation of a serum response factor [150]. This previously unrecognised mechanism also appears to be important for the profound mitogenic effect of ATP. In intact human blood vessels examined in vitro, shear stress decreased contractile P2X1 receptor expression, but increased the expression of mitogenic P2Y2 and P2Y6 receptors in VSMC [151]. This mechanism could promote vascular growth and remodelling induced by shear stress as an adaptive response to increased flow. P2X4 receptor knockout mice are incapable of adaptive vascular remodelling, that is, a decrease in vessel size in response to a chronic decrease in blood flow [57].\nAngiogenesis\nVery little is known about the role of P2 receptors in angiogenesis, but there are some studies suggesting their involvement. Ischaemia stimulates release of ATP [7, 60] and it has been shown to be a growth factor for endothelial cells [13, 152]. ATP and UTP stimulate cytoskeletal rearrangements with consequent cell migration of human endothelial cells [153] and ATP stimulates vasa vasorum neovascularisation in pulmonary arteries [154]. Newly developed vascular endothelia express very high levels of NTPDase1, also seen under hypoxic conditions [155]. NTPDase1 knockout mice exhibit disordered cellular migration and angiogenesis [156].\nVaricose veins\nIt has been suggested that cell lysis, consequent to P2X7 receptor-induced pore formation, contributes to the disorganisation and decrease in contractile myocytes in the media of varicose veins [157]. Upregulation of P2Y1 and P2Y2 receptors and downregulation of P2X1 receptors on smooth muscle of varicose veins is associated with a shift from contractile to synthetic and\/or proliferative roles; this phenotypic change in smooth muscle leads to weakening of vein walls and may be a causal factor in the development of varicose veins [158], an interesting parallel to the receptor changes seen in response to increased shear stress (see above) [151].\nHeart\nATP is released in the heart as a cotransmitter together with catecholamines from sympathetic nerves, but it may also be released from other sources in the heart such as endothelium, platelets, RBC and ischaemic myocardium (Table\u00a03) [7, 60, 159]. P2 receptors are abundantly expressed in the foetal human heart [160] as well as in the adult human heart [60, 161].\nInotropy\nIn cardiomyocytes, ATP stimulates a pronounced positive inotropic effect and may also act in synergy with \u03b2-adrenergic agonists to augment myocyte contractility [60, 162\u2013165]. ATP stimulates an increase in cytosolic calcium and evidence for the involvement of inositol 1,4,5-trisphosphate (IP3)-coupled P2Y2 receptors and ion channel P2X receptors has been presented [162, 164\u2013167]. Ap4A, probably after degradation to ATP, increases force of contraction in human ventricular trabeculae [168].\nThe inotropic effects of ATP are dependent on both IP3 and cAMP [169]. The inotropic effects of catecholamines acting on \u03b2-receptors are mediated by an increase of cAMP and antagonists of these receptors are important drugs for the treatment of hypertension and reduce mortality in congestive heart failure. Similarly, ATP stimulates an increase in cAMP in cardiac myocytes and may act in synergy with the \u03b21-adrenergic agonist, isoproterenol, by differential activation of adenylyl cyclase isoforms [60, 170, 171]. However, the ATP receptor mediating this increase in cAMP has not been related to any particular P2 receptor subtype in cardiac cells [60]. It could be the P2Y11 receptor that is additionally coupled to Gs and activates adenylyl cyclase [60, 172\u2013174]. mRNA for the P2Y11 receptor has been detected in the human heart at high levels [161, 175].\nThe unstable agonist, UTP, has been shown to induce a positive inotropic effect in rat atria and in rat and guinea pig ventricular cardiomyocytes [164, 166, 176, 177]. Stable UTP and UDP analogues induce a pronounced inotropic effect on mouse cardiomyocytes [161]. The P2Y2 receptor is the most abundantly expressed receptor with very low levels of the P2Y4 receptor in the human heart [161], suggesting that the inotropic effects of UTP are mediated via P2Y2 receptors, while the UDP effects are mediated via the P2Y6 receptor [161]. These mechanisms are mediated via IP3-mediated signalling.\nIn conclusion, the inotropic effects in man are mediated via P2Y2 and P2Y6 receptors and a P2Y11-like receptor (Fig.\u00a03). P2X receptors are probably also involved, since several subtypes are expressed [60, 160], but it has not been possible to perform a clear receptor characterisation due to lack of selective antagonists.\nFig.\u00a03Functional roles of purines and pyrimidines acting on P2 receptors in the regulation of the heart. See text for details. a ATP, UTP and UDP exert inotropic effects on cardiomyocytes leading to increased cardiac output. b UTP and UDP stimulate hypertrophy of cardiomyocytes, while ATP can have apoptotic effects. c UTP protects against ischaemic injury and cardiomyocyte cell death. ATP-degrading enzymes preserve endothelial integrity and protect against allograft rejection. NTPDase nucleoside triphosphate diphosphohydrolase\nMyocardial infarction\nUsing microdialysis, ATP in the interstitial space has been estimated to be 40\u00a0nmol\/l, but the levels may increase markedly during electrical stimulation, ischaemia, challenge with cardiotonic agents, increase in blood flow, mechanical stretch and increased work load [60]. ATP is released from cardiomyocytes during reduced oxygen tension [60]. UTP and ATP are released from the heart during cardiac ischaemia [178] and patients with myocardial infarction have higher plasma levels of both ATP and UTP [161, 179]. There is a significant increase in ectonucleotidase activity (NTPDase1) in the hearts of patients with ischaemic heart disease [180] that could represent a compensatory mechanism against increased nucleotide levels during chronic ischaemia.\nIschaemia-reperfusion provokes barrier failure of the coronary microvasculature, leading to myocardial oedema, ATP may protect against reperfusion-induced coronary endothelial barrier damage and inhibition of ATP degradation enhances the stabilising effect of ATP on barrier function [181].\nThe common P2Y11 ATP receptor polymorphism, Ala-87-Thr, is associated with both increased CRP and increased risk of developing myocardial infarction [116]. Based on this association, we hypothesise that the P2Y11 receptor plays an important role in cardiovascular biology and in inflammatory disease (see above), causing myocardial infarction via a proinflammatory effect. However, increased inotropic effects may also contribute.\nAn interesting yin and yang situation for ATP and UTP has been revealed regarding hypertrophic effects. UTP but not ATP causes hypertrophic growth in neonatal cardiomyocytes [182] (Fig.\u00a03). In contrast, ATP inhibits hypertrophy and may even induce apoptosis and necrosis [163, 183]. The reason for the difference in effects could be due to activation of P2X receptors or cAMP stimulation by ATP [60, 161]. Both UTP and ATP transactivate epidermal growth factor receptors, but only ATP stimulates the hypertrophic marker genes atrial natriuretic peptide and myosin light chain 2 [184]. Similarly, UTP but not ATP protects cultured cardiomyocytes against hypoxic stress [185]. Since UTP is released during preconditioning [178], a role for UTP in the protective effects of preconditioning is plausible. Recently, Yitzhaki and co-workers were able to demonstrate prominent reductions in myocardial infarction size and improved rat heart function in vivo, by a single intravenous bolus dose of UTP before ischaemia [186] (Fig.\u00a03).\nCongestive heart failure\nThere are several reports of alterations or adaptations of purinergic signalling during congestive heart failure. The positive inotropic effect of ATP is impaired in heart failure, but reversed by the angiotensin-converting enzyme (ACE)inhibitor imidapril [187]. The contractile responses for the P2Y11 receptor agonist AR-C67085 are decreased in heart failure, suggesting a downregulation of this receptor function in cardiomyocytes in a similar manner as seen for \u03b21-receptors in congestive heart failure [169]. In human hearts, only the P2X6 receptor was altered in congestive heart failure [188]. In congestive heart failure P2X1 receptors are downregulated in VSMC in resistance arteries, which could represent a protective response against the increased sympathetic nerve activity and peripheral resistance seen in congestive heart failure [189].\nSeveral P2 receptors have been suggested as targets for pharmacological treatment of congestive heart failure. Overexpression of P2X4 receptors has a beneficial, life-prolonging effect in a heart failure model [190]. Both ATP and catecholamines are released from sympathetic nerves, acting through cAMP-stimulating receptors to mediate positive inotropic effects, stimulating the same intracellular mechanisms as adrenergic \u03b2-receptors. It is possible that agonists for the P2Y11 receptor could be used to improve cardiac output in patients with circulatory shock. However, an even more important drug candidate would be a P2Y11 receptor antagonist that may be beneficial in patients with congestive heart failure. The extracellular pyrimidines UTP and UDP may be inotropic factors in man, acting on P2Y2 and P2Y6 receptors stimulating the same intracellular pathways as angiotensin II. Synthetic agonists could thus be used as inotropic agents during circulatory shock and antagonists may have effects similar to angiotensin II receptor blockers, being beneficial in the treatment of hypertension and congestive heart failure.\nChronotropy and arrhythmia\nIt has been difficult to determine the chronotropic effects of ATP due to the dominating inhibitory effects of adenosine on the AV node. We know that ATP increases the contractile rate in neuron-myocyte co-cultures but the effect is markedly reduced in non-innervated myocyte cultures [191]. ATP could induce arrhythmia based on its increased automaticity and early after-depolarisations [60]. The P2X1 receptor is colocalised with connexin 43 in gap junctions that transmit the contractile stimulus between cardiomyocytes [192]. However, so far no firm evidence exists of an important role for P2 receptors for chronotropy or arrhythmia.\nPlatelets and coagulation\nPlatelets\nADP, released from erythrocytes or produced after ectonucleotidase breakdown of ATP released from erythrocytes and endothelial cells, was found to cause platelet aggregation as early as 1961, before the concept of P2 receptors was conceived [193]. Later, the effects were attributed to a single receptor, designated P2T, but it was not until 1998 that the three receptors involved were characterised [194\u2013196]. Two ADP receptors (P2Y12 and P2Y1) and one ion channel ATP receptor (P2X1) are expressed on platelets [197, 198] (Fig.\u00a04). The P2Y12 receptor is coupled to inhibition of cAMP. The molecular identifications of the P2Y12 receptor and generation of knockout mice revealed highly prolonged bleeding times, and their platelets aggregate poorly in response to ADP and display a reduced sensitivity to thrombin and collagen [199]. The P2Y1 receptor is responsible for ADP-induced shape change and weak, transient aggregation [200\u2013202], while the P2Y12 receptor is responsible for the completion and amplification of the response to ADP and to all platelet agonists, including thromboxane A2, thrombin and collagen. The platelets also express P2Y1 receptors that have been shown in knockout mice to be of similar importance as P2Y12 receptors [200, 203].\nFig.\u00a04P2 receptor-mediated regulation of coagulation and platelet aggregation. See text for details. ADP is an important mediator and positive feedback mechanism for platelet aggregation. Activated platelets stimulate coagulation, i.e. formation of a fibrin network. In contrast, ATP, ADP and UTP release tissue plasminogen activator (t-PA) from endothelial cells, resulting in degradation of the fibrin network. At the same time plasminogen activator inhibitor (PAI-1) is also released from endothelial cells by ATP stimulation, in turn inhibiting t-PA. Thus, P2 receptors regulate several balancing factors in haemostasis. Ado adenosine\nThe physiological importance of the P2X1 receptor is not fully elucidated. However, recent studies have demonstrated that P2X1 receptors can generate significant functional platelet responses, independently and in synergy with other receptor pathways. In addition, studies in transgenic animals indicate an important role for P2X1 receptors in platelet activation, particularly under conditions of shear stress and thus during arterial thrombosis [204]. Synergistic interaction between ATP and NA in stimulating platelet aggregation may have significant clinical implications and suggests a prothrombotic role for ATP in stress [205].\nNTPDase1 is an important inhibitor of platelet activation. Unexpectedly, NTPDase1-deficient mice had prolonged bleeding times with minimally perturbed coagulation parameters. Platelet interactions with injured mesenteric vasculature were considerably reduced in vivo and purified mutant platelets failed to aggregate to standard agonists in vitro [206]. This platelet hypofunction was reversible and associated with P2Y1 receptor desensitisation. In keeping with deficient vascular protective mechanisms, fibrin deposition was found at multiple organ sites in NTPDase1-deficient mice and in transplanted cardiac grafts. Endothelial ADPase activity is lost following ischaemia-reperfusion injury, xenograft rejection and inflammation resulting in increased levels of ATP and ADP [58, 207]. This may cause platelet aggregation but also other effects, such as mitogenic effects on VSMC. Infusion of systemic apyrase inhibits platelet aggregation and prolongs xenograft survival [208]. This has also been shown with adenoviral transfer of NTPDase1 [209]. NTPDase1 is also lost in vascular cardiac grafts subjected to oxidant stress. NTPDase2 only degrades ATP to ADP and has a prothrombotic effect. It is present in the subendothelium and can shift the balance in a prothrombotic direction after endothelial injury [210] (Fig.\u00a01).\nADP acting on P2Y12 receptors is not only important for platelet activation, it also stimulates vasoconstriction. Stable drugs with antagonistic effects on P2Y12 receptors, affecting both platelets and VSMC, could be of double therapeutic benefit in their prevention of both thrombosis and vasospasm [46].\nCoagulation\nThe prothrombotic effect of ADP on the platelets is counteracted by the effects of UTP and ATP on the endothelium that stimulates a substantial release of tissue-type plasminogen activator (tPA) with fibrinolytic effects [51, 211] (Fig.\u00a04). To make the picture even more complex, ATP also releases plasminogen activator inhibitor (PAI-1) which in turn inhibits tPA [212]. Thus, P2 receptors are involved at several stages of haemostasis, which is important for the development of atherosclerosis and thrombotic occlusions leading to myocardial infarction and stroke.\nClinical importance of P2 receptor-mediated regulation of platelets\nThe first ADP inhibitors, ticlopidine and clopidogrel, were developed before the cloning and identification of the platelet ADP receptors. Later, it was concluded that they were prodrugs, converted in the liver to a metabolite that binds irreversibly to the P2Y12 receptor resulting in non-competitive antagonism.\nTiclodipine has the disadvantage of a small risk of neutropaenia, so that clinically so far the most important contribution of drug development aimed at P2 receptors has been the beneficial effects of the platelet ADP receptor antagonist clopidogrel in atherosclerotic disease. In the CAPRIE trial, clopidogrel was even more effective compared to aspirin in preventing myocardial infarctions [213]. Clopidogrel, as an addition to aspirin, turned out to be necessary to prevent acute in-stent thrombosis in percutaneous coronary interventions. The CURE trial demonstrated that the addition of clopidogrel to aspirin reduced clinical events by 20% in acute coronary syndromes [213, 214]. This has also been shown for ST-elevation myocardial infarctions (COMMIT, CLARITY). However, long-term secondary prevention in high-risk groups with clopidogrel added to aspirin did not have significant benefits (CHARISMA). Clopidogrel combined with aspirin reduces cerebral emboli in patients undergoing carotid endarterectomy [215]. Clopidogrel (Plavix\u00ae) is now a blockbuster drug and one of the most sold drugs worldwide to the benefit of atherosclerotic patients. Nevertheless, clopidogrel is a rather weak antagonist at P2Y12 receptors with variable effects, often referred to as clopidogrel resistance. Newer P2Y12 receptor antagonists, such as prasugrel and AZD6140, have a stronger more consistent effect and are currently being studied in major clinical trials (TRITON and PLATO).\nClinical importance of purinergic signalling in cardiovascular disease: future directions\nThe P2 receptor family provides opportunities for drug development (see Table\u00a04). The large number of receptor subtypes and the increasing knowledge of their tissue distributions may make it possible to target a specific cardiovascular organ with limited side effects. So far, only P2Y12 receptor antagonists have been explored in patients, but with extraordinary success. Clopidogrel is one of the most prescribed drugs in the world for the treatment of thrombosis, stroke and myocardial infarctions in millions of patients. The following compounds, prasugrel and AZD6140, are even more potent P2Y12 receptor antagonists with clinical benefits currently being tested in phase III trials. Improved platelet inhibition can also be achieved with antagonists against the other two P2 receptors on platelets. Studies in P2Y1 and P2X1 receptor knockout mice and experimental thrombosis models using selective P2Y1 and P2X1 receptor antagonists have shown that, depending on the conditions, these receptors could also be potential targets for new antithrombotic drugs.\nTable\u00a04Cardiovascular disease targetsReceptor targetAgonist\/antagonist activity of therapeutic drugDiseaseVascular smooth muscle cellP2Y2AntagonistHypertension, restenosis, diabetic microvascular disease, subarachnoidal bleedingP2Y6AntagonistHypertension, restenosis, diabetic microvascular disease, subarachnoidal bleedingP2Y12AntagonistHypertension, vasospasmP2X1AntagonistHypertension, migraine, subarachnoidal bleedingP2Y6AgonistMigraineSolNTPDase1EctonucleotidaseRestenosis, xenograft rejectionEndothelial cellP2Y2AntagonistAtherosclerosis, inflammationP2X4AntagonistVascular remodelling (cancer?), preeclampsiaHeartP2Y2AntagonistCongestive heart failureP2Y6AntagonistCongestive heart failureP2Y11AntagonistCongestive heart failure, myocardial infarctionP2Y2AgonistShock, reduced cardiac output, cardiac protectionP2Y6AgonistShock, reduced cardiac outputP2Y11AgonistShock, reduced cardiac outputSolNTPDase1EctonucleotidaseHeart transplantation, myocardial infarctionInflammatory cellsP2Y2AntagonistAtherosclerosisP2Y11AntagonistAtherosclerosisP2X7AntagonistAtherosclerosisPlateletsP2Y1AntagonistMyocardial infarction, stroke, peripheral artery diseaseP2Y12AntagonistMyocardial infarction, stroke, peripheral artery diseaseP2X1AntagonistMyocardial infarction, stroke, peripheral artery diseaseSolNTPDase1EctonucleotidaseMyocardial infarction, stroke, peripheral artery diseaseRed blood cellP2Y13AntagonistImproved peripheral circulationP2X7AntagonistShockPerivascular nociceptive sensory nervesP2Y1AgonistMigraine, vascular painP2X3AntagonistMigraine, vascular painPerivascular sympathetic nervesP2Y1AgonistHypertensionP2X2AntagonistHypertensionThe table presents possible targets for the development of new pharmaceutical compounds for the treatment of cardiovascular diseases\nATP, UTP and UDP, acting on P2Y2 and P2Y6 receptors, are potent stimulators of inflammation, VSMC proliferation and migration. Antagonists to these receptors may be explored to protect against atherosclerosis, restenosis after balloon angioplasty, chronic transplant rejection and diabetic microvascular disease.\nIt is possible that hypertension and pulmonary hypertension could be treated with P2X1, P2Y2, P2Y6 or P2Y12 receptor antagonists. In the heart P2Y2, P2Y6 and P2Y11 receptor antagonists may have similar beneficial roles as angiotensin inhibition or \u03b2-blockers in the treatment of congestive heart failure. In the acute phase of a myocardial infarction, a UTP analogue could protect cardiomyocytes and limit infarct size. A polymorphism in P2Y11 receptors could be considered as a prognostic marker to identify patients at risk of myocardial infarction and drugs aimed at the receptor could be explored to prevent myocardial infarction.\nA promising strategy would be to use soluble NTPDase to prevent thrombosis, restenosis and xenograft transplantation. SolNTPDase1 protects against brain damage in a stroke model and a human genetically engineered solNTPDase has been developed that could be used in clinical trials. Possible clinical indications could be stroke, myocardial infarction and transplantation. P2X3 receptor antagonists could be used against migraine and vascular pain [see 19, 26].","keyphrases":["receptor","cardiovascular","atp","pathophysiology","p2x","p2y","purine","heart","pyrimidine"],"prmu":["P","P","P","P","P","P","P","P","P"]}
{"id":"Osteoporos_Int-3-1-1915640","title":"Vitamin K2 supplementation improves hip bone geometry and bone strength indices in postmenopausal women\n","text":"Summary Vitamin K mediates the synthesis of proteins regulating bone metabolism. We have tested whether high vitamin K2 intake promotes bone mineral density and bone strength. Results showed that K2 improved BMC and femoral neck width, but not DXA-BMD. Hence high vitamin K2 intake may contribute to preventing postmenopausal bone loss.\nIntroduction\nSince the discovery of osteocalcin, the vitamin K-dependent protein synthesized by bone tissue, attempts have been made to correlate bone vitamin K status with bone strength. The way in which these variables were measured varied in different studies. Obviously, the best way to assess bone strength is monitoring fracture incidence in a large study cohort over a long period of time. Unfortunately, this method is expensive and time consuming. Therefore, most authors have used bone mineral density as measured by DXA (DXA-BMD) as a surrogate marker for bone strength. A disadvantage of this technique is that it gives the amount of calcium per area, but does not take into account the three dimensions of the bone [1]. Findingreliable markers for bone vitamin K status turned out to be even more difficult. Dietary vitamin K may occur in different forms: K1 (also known as phylloquinone) and K2, which is a group name for a series of related compounds also known as menaquinones [2]. The various menaquinones differ structurally in the length of their isoprenyl side chain, which may contain four isoprenoid residues in menaquinones-4 (MK-4) up to nine residues in menaquinones-9 (MK-9). Still higher menaquinones have been described, but are increasingly rare in the diet. Dietary K1 originates mainly from green, leafy vegetables, whereas menaquinones are produced by bacteria and are mostly found in fermented foods such as cheese and the Japanese food natto (fermented soy beans) [3, 4]. Synthetic forms of vitamin K available in food supplements or pharmaceutical preparations are K1 and MK-4, recently also the natural MK-7 has become available. The recommended daily intake of vitamin K (all forms) is between 100 and 120\u00a0\u03bcg\/day, but this value is based on the hepatic vitamin K requirement for blood clotting factor synthesis. The requirements of other tissues have not yet been defined, but are probably higher.\nIn 1984, Hart and coworkers described a case-control study in which patients with femur neck fractures had lower circulating vitamin K1 concentrations than age- and sex-matched controls [5]. In a second study from the same authors it was found that subjects with low serum vitamin K1 concentrations had a lower DXA-BMD than those with normal or high serum vitamin K1 [6]. A potential bias in these studies is that low circulating vitamin K may be a marker for general poor nutritional status and that low DXA-BMD and increased fracture risk are associated with the combined deficiency of essential minerals, vitamins and proteins rather than with vitamin K alone.\nOthers have published population-based studies in which an inverse correlation was found between fracture risk and vitamin K1 intake [7, 8]. It was found that the association between vitamin K1 intake and fracture risk was much stronger than that between vitamin K1 intake and DXA-BMD [9]. A drawback of these studies is that no attempts were made to also monitor vitamin K2 intake. Although in the western society K2 forms only 10\u201320% of the total vitamin K intake [3, 4], it is absorbed from the food matrix much better than K1 and may cover 50% of the total vitamin K absorbed. Moreover, it has been demonstrated that vitamin K2 rather than K1 is preferentially taken up by extrahepatic tissues such as bone and arteries [10]. Hence also vitamin K1 intake seems to be a questionable marker for bone vitamin K status.\nPossibly the best marker available today is circulating undercarboxylated osteocalcin (ucOC). Either the absolute concentration, the ratio between ucOC and total osteocalcin, or the ratio between ucOC and carboxylated osteocalcin (cOC) are used in the literature. The group of Delmas [11\u201313] and Luukinen et al. [14] reported a number of independent studies suggesting a strongly increased fracture risk in populations with elevated serum ucOC; later studies by the French group [15] and others [9, 16] showed that ucOC was inversely associated with DXA-BMD. It is at least remarkable that in all approaches, associations between vitamin K status and fracture risk were demonstrated more readily than those between K status and DXA-BMD.\nIn a supplementation study with vitamin K1 alone no effect on DXA-BMD was observed during a 2-year treatment with 10\u00a0mg\/day [17]. In another study from the same group, however, a synergistic effect of 1\u00a0mg\/day of K1 with minerals and vitamin D [18] was found. In this study the rate of bone loss in postmenopausal women was reported to be 35% lower in the presence of minerals and vitamins K1 and D as compared to the group receiving minerals and vitamin D without vitamin K. A number of Japanese studies reported variable effects of vitamin K2 alone, but substantial beneficial effects if combined with vitamin D [19].\nIt has been reported by many different authors that in the healthy population serum osteocalcin is incompletely carboxylated, which is indicative for poor vitamin K status of bone [11, 14, 16, 20]. Based on our present knowledge of vitamin K-dependent proteins, it must be expected that non-carboxylated osteocalcin is not functional. Since the molecular function of osteocalcin has remained unclear thus far, we do not know whether it contributes to bone strength at all. From transgenic osteocalcin-deficient mice, we know that osteocalcin contributes to regulating the form and the dimensions of bone [21]. This may also be important for humans.\nThe importance of bone geometry for bone strength is rapidly gaining interest [22\u201326]. Although DXA-BMD is still the determinant most generally used in the clinical evaluation of hip fracture risk, it has been stipulated that its uncritical use may lead to size-related artifacts in the estimation of bone strength and in the identification of fracture risk [27]. As was mentioned by Heaney [28], the ultimate concern in studying bone status is bone strength. Holding other variables constant, strength will increase both as bone mass increases and as bone size increases. When estimating bone strength, two strategies to also compensate for the dimensions of bone have been proposed. First, it is encouraged that densitometric comparisons between groups are based on bone mineral content (BMC) rather than on DXA-BMD [28]. Second, bone dimensions are used as independent determinants for bone strength. Important geometric parameters are the hip axis length (HAL) and the femoral neck width (FNW). Patients with low DXA-BMD or after experiencing a hip fracture had an increased FNW, suggesting an attempt to compensate for the increased fracture risk at this critical site [26]. On the other hand, it seems obvious that at comparable DXA-BMD a larger FNW will positively contribute to bone strength. In this way it is understandable that also and increase of BMC may contribute to bone strength, although it should be reminded that it is not the mass per se but the distribution of mass that is crucial for bone strength. The importance of combining bone geometry with DXA-BMD for expressing bone strength and fracture risk was realized by Karlamangla et al., who developed mathematical equations for calculating indices for compression strength, bending strength, and impact strength of the femoral neck [29]. In these equations, DXA-BMD is combined with HAL, FNW, height and body weight. We have used the same equations to analyse the data from a placebo-controlled intervention trial in which we compared the effects of vitamin K2 on DXA-BMD and bone strength indices in postmenopausal women, who were recruited from the apparently healthy population and thus represent the large group of free living elderly subjects. Additional or synergistic effects of calcium and vitamin D were not included in the design of our study.\nSubjects and methods\nSubjects\nThe study was designed for apparently healthy, non-osteoporotic women. Participants were recruited by local newspapers. Inclusion criteria were female gender, age between 55 and 75\u00a0years at intake, Caucasian race, apparently healthy, at least 2\u00a0years postmenopausal and willingness to sign informed consent. Exclusion criteria were: a history of metabolic bone disease(s) or recent bone fractures (less than one year), low bone density (T-score < \u22122.5), ovariectomy, hysterectomy, oral anticoagulant treatment, hormone replacement therapy, treatment with bisphosphonates, calcitonin, prednisone, heparin, or vitamin K-containing vitamin concentrates and food supplements. Also subjects who had received an investigational new drug within the last 12\u00a0months were excluded from the trial. In total 325 women met the criteria and were randomized into our study. The participants were pre-stratified according to age: 105 in the age of 55\u201365\u00a0years, and 220 in the age of 65\u201375\u00a0years. The study protocol was approved by the University Hospital medical ethics committee.\nMasking\nParticipants were identified by a single randomization number according to the randomization schedule generated by the University Hospital Pharmacy using a computer-generated random permutation procedure in the software package SPSS. Participants were randomly assigned to treatment with either 45\u00a0mg\/day of MK-4 (menatetrenone, EISAI Co, Tokyo, Japan) or placebo, and compliance with treatment was decided from pill counts during home visits. The daily dose of MK-4 was given in three capsules of 15\u00a0mg each, which had to be taken at three time points spread over the day, preferably after the meal. The participant randomization codes were allocated sequentially in the order in which the participants were enrolled. After completion of all analyses the randomization code was disclosed to the investigator.\nMeasurements\nThe clinical end points chosen to evaluate the effect of the study treatment on bone were bone mineral density (DXA-BMD), bone mineral content (BMC) and bone strength indices of the femoral neck. DXA-BMD and BMC were measured at baseline and after 1, 2 and 3\u00a0years of treatment by dual energy X-ray absorptiometry (DXA) at the site of the left total hip and femoral neck, as well as the vertebrae (L2-L4) using a Hologic QDR 4500-A (Waltham MA, USA). The in vivo precision (CV) after repeated measurements in 10 healthy adults (30\u201340\u00a0years old) was 0.85% for the spine and 0.78% for the femur neck in short term (one day interval) and 1.03% and 0.98% in long term (one-year interval) precision measurements. The long-term precision of FNW assessment was 1.53%. At each time point, standing height and body weight were determined with standardized equipment and the body mass index (BMI) was calculated as the weight in kilograms divided by the square of the height in meters. The daily calcium intake was assessed using a validated food frequency questionnaire. Indices for compression strength (CSI), bending strength (BSI) and impact strength (ISI) at the site of the femoral neck were calculated from the mean femoral neck width (FNW) and hip axis length (HAL), together with height, weight and femoral neck DXA-BMD as described by Karlamangla et al. [29]. The mean FNW was obtained from the 1.5\u00a0cm wide femoral neck region of interest (area (cm2)\/1.5 (cm)). The HAL reflects the distance along the femoral neck axis from the lateral margin at the base of the greater trochanter to the inner pelvic brim. The equations used were: \nSample collection\nFasting blood was taken and 2-h fasting urine was collected at baseline and after 3, 6, 12 and 36\u00a0months of treatment. After blood was taken, it was allowed to clot at room temperature for half an hour. Serum was prepared after centrifugation at 3,000\u00a0rpm, during 15\u00a0minutes. It was subdivided in small aliquots and kept at \u221280\u00b0C until use. Urine was collected as the second morning void after an overnight fast.\nBiochemical measurements\nIn serum the following markers were tested, all based on enzyme immuno assays (EIA): total osteocalcin (t-OC, BioSource Europe SA); under-carboxylated osteocalcin (uc-OC) and carboxylated osteocalcin (c-OC) both obtained from Takara Shuzo Co Ltd., Shiga, Japan; bone-specific alkaline phosphatase (BAP, Alkphase B, Quidel\/Metra Biosystems San Diego, CA, USA); N-telopeptides of type I collagen (sNTX, Osteomark sNTx, Ostex International, Inc., Seattle, WA, USA) and 25-Hydroxy vitamin D (25-OH D, OCTEIA 25-hydroxy vitamin D, IDS, UK).\nIn the urine we measured free deoxypyridinoline by using an enzyme immunoassay (DPD, Quidel\/Metra Biosystems). Urinary calcium (Ca) and creatinine (creat) were assessed according to standard methods. Dpd and Ca were corrected for the urinary creat concentration.\nStatistics\nThe required numbers of participants in both age groups were assessed using separate power analyses (power of 90% and a significance level of 0.05). In the younger age group it was assumed that in the placebo group the mean bone loss is approximately 1.5% (4.5% in 3\u00a0years). The lowest detectable effect was set at an improvement of 30% (=3.15% bone loss after 3\u00a0years). Taking into account a drop-out frequency of 12% per year this group should consist of at least 76 subjects. In the older age group the mean bone loss was assumed to amount 1% per year (3% in 3\u00a0years). With a lowest detectable effect of 30% (=2.1% bone loss in 3\u00a0years) and a drop-out frequency of 19% per year, the minimal size of this group is 218 subjects.\nStatistical analysis was performed using the statistical package SPSS (SPSS Corp, Chicago, IL). The calculations are based on the intention-to-treat principle. Drop-outs were included in the analyses, with the last data available in all subsequent measuring points. All follow-up measurements were related to the baseline values and the proportional changes were calculated for each subject. Differences between and within the different groups were assessed with the unpaired and paired Student\u2019s t-test, respectively. All data are given as mean values \u00b1SE. The linear regression model was used to investigate the effect of treatment after adjustment for potential confounders. Change of FNW, HAL and bone strength indices after 3\u00a0years of treatment were used as dependent variables and age and BMI as independent variables.\nResults\nBaseline measurements and follow-up\nThree hundred fifty-one women were recruited for the study, 26 of whom were excluded before randomization for different reasons. After randomization there were 161 women receiving MK-4 and 164 women receiving placebo. The MK-4 group consisted of 54 women younger than 65\u00a0years of age and 107 women older than 65\u00a0years of age; in the placebo group these numbers were 51 and 113, respectively. The baseline characteristics of all participants in the placebo and MK-4 group are given in Table\u00a01. No significant differences were found between both groups. However, when comparing the two age groups there was an age-related increase in weight and BMI and an age-related decrease in height (Table\u00a02). DXA-BMD and BMC in the older age group were significantly lower at the site of the femoral neck, but not at the total hip and spine. From all serum markers for bone metabolism only BAP was higher in the older age group (borderline significant). Although there was an age-related decrease in serum vitamin D concentration, all participants had levels which were within the normal range at baseline (Tables\u00a01 and 2) and at all subsequent measurements (data not shown). Urinary DPD\/creat was markedly lower in the older age group compared to the younger group. \nTable\u00a01Baseline characteristics of all participants subdivided according to treatment\u00a0Placebo n\u2009=\u2009164MK-4 n\u2009=\u2009161P-valueAnthropomorphic variables:Age (years)66.0\u2009\u00b1\u20090.565.9\u2009\u00b1\u20090.40.9Years since menopause17.7\u2009\u00b1\u20090.717.2\u2009\u00b1\u20090.60.6Weight (kg)71.8\u2009\u00b1\u20091.070.3\u2009\u00b1\u20090.90.3Height (cm)162\u2009\u00b1\u20090.5161\u2009\u00b1\u20090.50.2BMI (kg\/m2)27.3\u2009\u00b1\u20090.327.1\u2009\u00b1\u20090.40.7Calcium intake (mg\/day)811\u2009\u00b1\u200926870\u2009\u00b1\u2009320.1Non-smoking (%)87871.0Bone density characteristics:DXA-BMD (g\/cm2) of: femoral neck0.688\u2009\u00b1\u20090.0070.706\u2009\u00b1\u20090.0080.1\u00a0Total hip0.842\u2009\u00b1\u20090.0090.861\u2009\u00b1\u20090.0080.1\u00a0Lumbar L2-L40.931\u2009\u00b1\u20090.0120.929\u2009\u00b1\u20090.0130.9BMC (g) of: femoral neck3.54\u2009\u00b1\u20090.043.65\u2009\u00b1\u20090.040.06\u00a0Total hip30.6\u2009\u00b1\u20090.4331.4\u2009\u00b1\u20090.390.2\u00a0Lumbar L2-L443.1\u2009\u00b1\u20090.7242.4\u2009\u00b1\u20090.760.6Hip geometry:Mean FNW (cm)3.43\u2009\u00b1\u20090.023.45\u2009\u00b1\u20090.0170.4HAL (cm)11.6\u2009\u00b1\u20090.0711.7\u2009\u00b1\u20090.060.2Bone strength indices:CSI (g\/kg.m)3.41\u2009\u00b1\u20090.0553.52\u2009\u00b1\u20090.0520.1BSI (g\/kg.m)1.00\u2009\u00b1\u20090.0171.04\u2009\u00b1\u20090.0170.2ISI (g\/kg.m)0.24\u2009\u00b1\u20090.0040.26\u2009\u00b1\u20090.0040.03Serum markers for bone metabolism:c-OC (ng\/mL)6.1\u2009\u00b1\u20090.26.2\u2009\u00b1\u20090.21.0uc-OC (ng\/mL)3.3\u2009\u00b1\u20090.23.3\u2009\u00b1\u20090.20.4t-OC (ng\/mL)13.2\u2009\u00b1\u20090.413.2\u2009\u00b1\u20090.50.7BAP (ng\/mL)23.1\u2009\u00b1\u20090.723.6\u2009\u00b1\u20090.80.2NTX (nM)10.8\u2009\u00b1\u20090.210.7\u2009\u00b1\u20090.30.525-OH D (nM)67.2\u2009\u00b1\u20092.372.3\u2009\u00b1\u20092.60.09Urine markers for bone metabolism:Creatinine (mmol\/L)3.5\u2009\u00b1\u20090.33.6\u2009\u00b1\u20090.21.0DPD \/ creat (\u03bcmol\/mol)7.4\u2009\u00b1\u20090.27.5\u2009\u00b1\u20090.20.7Calcium \/ creat (mol\/mol)0.29\u2009\u00b1\u20090.020.29\u2009\u00b1\u20090.010.7Only the BMC and the impact strength index of the femoral neck in the MK-4 group were slightly higher than in the placebo one. This difference is accounted for by expressing MK-4-induced changes as a percentage of the baseline values. All data are given \u00b1 SE.Table\u00a02Baseline characteristics of participants subdivided according to age\u00a0Age 55\u201365 n\u2009=\u2009105Age 65\u201375 n\u2009=\u2009220P-valueAnthropomorphic variables:Age (years)59.0\u2009\u00b1\u20090.369.3\u2009\u00b1\u20090.2Years since menopause9.7\u2009\u00b1\u20090.521.2\u2009\u00b1\u20090.4Weight (kg)67.4\u2009\u00b1\u20091.072.8\u2009\u00b1\u20090.80.0001Height (cm)163\u2009\u00b1\u20090.6161\u2009\u00b1\u20090.40.003BMI (kg\/m2)25.4\u2009\u00b1\u20090.428.1\u2009\u00b1\u20090.30.0001Calcium intake (mg\/day)816\u2009\u00b1\u200934858\u2009\u00b1\u2009270.4Non-smoking (%)88870.9Bone density characteristics:DXA-BMD (g\/cm2) of: femoral neck0.716\u2009\u00b1\u20090.0100.689\u2009\u00b1\u20090.0060.02Total hip0.871\u2009\u00b1\u20090.0120.842\u2009\u00b1\u20090.0070.03Lumbar L2-L40.940\u2009\u00b1\u20090.0150.925\u2009\u00b1\u20090.0110.4BMC (g) of: femoral neck3.69\u2009\u00b1\u20090.063.55\u2009\u00b1\u20090.030.02Total hip31.4\u2009\u00b1\u20090.630.7\u2009\u00b1\u20090.30.3Lumbar L2-L444.0\u2009\u00b1\u20090.942.1\u2009\u00b1\u20090.60.1Hip geometry:Mean FNW (cm):3.44\u2009\u00b1\u20090.0223.44\u2009\u00b1\u20090.0160.9HAL (cm):11.8\u2009\u00b1\u20090.0811.6\u2009\u00b1\u20090.060.03Bone strength indices:CSI (g\/kg m):3.75\u2009\u00b1\u20090.0683.31\u2009\u00b1\u20090.0390.0001BSI (g\/kg m):1.09\u2009\u00b1\u20090.0220.98\u2009\u00b1\u20090.0130.0001ISI (g\/kg m):0.27\u2009\u00b1\u20090.0050.24\u2009\u00b1\u20090.0030.0001Serum markers for bone metabolism:c-OC (ng\/mL)5.9\u2009\u00b1\u20090.26.3\u2009\u00b1\u20090.20.2uc-OC (ng\/mL)3.3\u2009\u00b1\u20090.23.2\u2009\u00b1\u20090.10.8t-OC (ng\/mL)13.9\u2009\u00b1\u20090.612.8\u2009\u00b1\u20090.40.1BAP (ng\/mL)21.8\u2009\u00b1\u20090.824.1\u2009\u00b1\u20090.70.05NTX (nM)11.0\u2009\u00b1\u20090.310.6\u2009\u00b1\u20090.20.225-OH D (nM)76.5\u2009\u00b1\u20092.766.3\u2009\u00b1\u20092.30.005Urine markers for bone metabolism:creatinine (mmol\/L)3.5\u2009\u00b1\u20090.33.6\u2009\u00b1\u20090.20.8DPD \/ creat (nmol\/mmol)8.2\u2009\u00b1\u20090.27.1\u2009\u00b1\u20090.20.0001Calcium \/ creat (mmol\/mmol)0.29\u2009\u00b1\u20090.020.29\u2009\u00b1\u20090.010.9The differences in all age-related variables were highly significant. All data are given\u00b1SE.\nAfter 3\u00a0years 257 women had completed the study: 133 women in the MK-4 group and 124 women in the placebo group. In total 68 women stopped during the study. This was 21% of the total. The largest group of participants (n\u2009=\u200930) stopped during the first 3\u00a0months of the trial. The most important reasons for discontinuation in this time period were various health problems (n\u2009=\u200918), low DXA-BMD (n\u2009=\u200917), lack of motivation (n\u2009=\u200910) and complaints about the capsules (n\u2009=\u20099). Subjects who stopped because of low DXA-BMD did so after consultation during the trial with their general practitioner; there were two complaints about the capsules in the MK-4 group and seven in the placebo group. Two participants died during the trial: one brain tumor and one cerebrovascular accident. Both casualties were in the placebo group. In total, 36 negative or positive side effects were mentioned by the participants. In the MK-4 group 16 women experienced negative effects and four experienced positive effects of the capsules; in the placebo group the numbers were 15 and one, respectively. The most frequent negative effects were gain of weight and gastro-intestinal complaints. However, there was no difference in total numbers of complaints between the MK-4 and the placebo group.\nEffect of MK-4 supplementation\nEffects on serum markers (if detectable) were maximal after 12\u00a0months of treatment, with no further increase of the difference from placebo at 36\u00a0months. In Fig.\u00a01 we have plotted the changes of bone markers in serum during the first year. As was to be expected, a statistically significant effect of vitamin K treatment was observed on the serum concentrations of cOC and ucOC. The markers for bone formation (tOC and BAP) were also significantly higher in the MK-4 group as compared to the placebo one. No effect was observed on markers for bone resorption, or on DXA-BMD at any of the sites measured (Fig.\u00a02a). Also urinary calcium excretion was not affected by MK-4 treatment (data not shown). It turned out that in the MK-4 group the BMC of the femoral neck decreased at a significantly lower rate than in the placebo one (Fig.\u00a02b), and that this effect was paralleled by a significant increase in the femoral neck width, suggesting bone accrual at this critical site (Fig.\u00a02c). The hip axis length (HAL) seemed to slightly increase with age in an MK-4 independent manner. From the DXA-BMD, the FNW and the HAL we have calculated the various indices for strength of the femur neck, and it resulted that MK-4 had a significant effect on all three indices (Fig.\u00a02d). Notably the bending strength remained almost constant after the three years of intervention.\nFig.1EffectofMK-4 on markers for bone metabolism during the first year of treatment. Bone formation markers are presented in graphs a\u2013d (cOC, ucOC, tOC and BAP, respectively) and bone resorption markers in graph E and F (NTX and DPD\/creat respectively). Closed symbols: MK-4; open symbols: placebo. Error bars represent SE. Significance of differences MK-4 compared to placebo: #: p\u2009<\u20090.05; ##: p\u2009<\u20090.005Fig.\u00a02Effects of MK-4 on bone after 3\u00a0years of treatment. All data are expressed as a percentage of the respective baseline values. Graph A shows the data of DXA-BMD of the femoral neck (DXA-BMDFN), total hip (DXA-BMDtotalhip) and lumbar spine (DXA-BMDLS); graph B shows the BMC of the femoral neck (BMCFN), total hip (BMCtotalhip) and lumbar spine (BMCLS); graph C shows the femoral neck width (FNW) and hip axis length (HAL) and graph D shows the indices of the compression strength (CSI), bending strength (BSI) and impact strength (ISI). Open bars: placebo; hatched bars: MK-4. Error bars represent SE. Significance of change from baseline: *: p\u2009<\u20090.05, **: p\u2009<\u20090.005; significance of difference MK-4 compared to placebo: #: p\u2009<\u20090.05, ##: p\u2009<\u20090.005\nUsing a linear regression model, we have tested the statistical significance of the MK-4 induced effects. As is shown in Table\u00a03, the differences in FNW, CSI, BSI and ISI between the MK-4 and placebo group were significant, both before and after adjustment for age and BMI. The effects on BMC, FNW and bone strength indices were observed for both age groups. As is shown in Fig.\u00a03 the loss of bone strength in the MK-4 supplemented group was almost negligible in the group of 65\u00a0years and older. \nTable\u00a03Effect of MK-4 treatment on geometrical variables\u00a0Unadjusted adjusted mean difference (SE)pMean difference(SE)pFNW (%)+1.30 (0.50)0.01+1.34 (0.50)0.009HAL (%)\u2212 0.25 (0.26)0.3+0.23 (0.22)0.9CSI (%)+2.26 (0.77)0.004+2.03 (0.76)0.008BSI (%)+3.80 (1.2)0.002+3.83 (1.2)0.001ISI (%)+1.98 (0.82)0.02+1.72 (0.79)0.03The increase\/decrease of each variable was calculated as a percentage from its baseline value. The differences between the change in MK-4 group and that in the placebo group are given. A positive value reflects a more pronounced effect in the MK-4, a negative value reflects a more pronounced effect in the placebo group. The values are given before and after adjustment for age and BMI, adjusted values are quoted at the mean values for age and BMI of the total group (age\u2009=\u200965.9\u00a0years; BMI\u2009=\u200927.2\u00a0kg\/m2).Fig.\u00a03Effect of MK-4 on bone strength indices in 2 different age groups (55\u201365 and 65\u201375\u00a0years of age). All data are expressed as the mean percent change relative to the respective baseline values after 3\u00a0years of treatment with either MK-4 or placebo. Graph A shows the compact strength index (CSI), graph B the bending strength index (BSI) and graph C the impact strength index (ISI). Open bars: placebo; hatched bars: MK-4. Error bars represent SE. Significance of change from baseline: *: p\u2009<\u20090.05, **: p\u2009<\u20090.005; significance of difference MK-4 compared to placebo: #: p\u2009<\u20090.05\nDiscussion\nIn this paper we demonstrate that the main effect of vitamin K2 on bone in the hip is an increase of the femoral neck BMC and width, resulting in maintenance of the calculated bone strength even at decreasing DXA-BMD after the menopause. This makes K2 to an interesting compound for combination therapy with other food supplements (calcium, vitamin D) or drugs (bisphosphonates) with known effects on also DXA-BMD. The potential synergism between K2 and other supplements\/drugs, however, requires further investigation.\nIn the literature there are many indications that vitamin Kcontributes to the maintenance of optimal bone strength. Besides various population-based studies and a number of clinical intervention trials investigating the effects of increased vitamin K intake, it has also been reported that subjects receiving vitamin K antagonists (oral anticoagulant therapy) have lower DXA-BMD and increased fracture risk [30\u201332]. Also experimental animals developed osteopenia upon treatment with the vitamin K antagonist warfarin [33]. On the other hand we must conclude, however, that in intervention trials the reported effects on DXA-BMD of vitamin K alone were small; most studies published thus far indicate that only in combination with calcium and vitamin D, vitamin K has a synergistic effect on DXA-BMD [18, 19, 34]. Osteocalcin is the most abundant vitamin K-dependent protein in bone, and the phenotype of transgenic osteocalcin-deficient mice suggests that it is primarily involved in the correct arrangement of hydroxyapatite crystals and in structuring the correct dimensions of bone [21]. Both factors may contribute to the total bone strength. Hence studies in which DXA-BMD is the only clinical endpoint do not give conclusive evidence on bone strength. If, for instance, the femoral neck DXA-BMD remains constant but increases in width the bone will become stronger and more resistant to fracture. Therefore, we have used mathematical procedures for calculating three indices for bone strength at the site of the femoral neck.\nDespite the very high dose of MK-4 used in the present trial, no effect was found on the DXA-BMD of the femoral neck, the total hip or the lumbar spine. On the other hand, significant positive effects were observed both on the femoral neck BMC and width. The relation between DXA-BMD, BMC and FNW can be seen from the formula: DXA-BMD=BMC\/area, where FNW is one of the dimensions of the surface area. In case FNW is increasing, the projected area of the DXA-BMD measurement will increase too. In case BMC remains constant, the DXA-BMD values will decrease. In case FNW increases and BMC increases adequately, DXA-BMD remains constant, with a concomitant increase of bone strength. The importance of the FNW for the calculated bone strength is obvious from the equations for all three bone strength indices. Our results showed that during the entire intervention period there was no or very little decrease in bone strength in the K2 treated group, whereas there was substantial loss of bone strength in the placebo one. The favourable effect was found both in the young postmenopausal group (55\u201365\u00a0years old) as well as in the older group (65\u201375\u00a0years old). Since together with vitamin D and calcium vitamin K was also reported to have a positive effect on DXA-BMD it might even be speculated that the combination of these supplements will result in an increase of bone strength. After we obtained these data, we re-analysed previous studies from our lab, and it turned out that vitamin K1 had only little or no effect on the FNW. It should be pointed out, however, that the doses used in these studies were 1\u00a0mg\/day and 10\u00a0mg\/day, which is considerably lower than the 45\u00a0mg\/day used in the present study.\nThe first question that may be raised is whether the very high dose of vitamin K used in our study is required to obtain the observed effect. The present recommendations for vitamin K intake vary between 90 and 120 micrograms per day, and in a dose of 45 milligrams per day the vitamin is used as a drug rather than as a food supplement. It should be realized, however, that MK-4 has a very short biological halflife time (1\u20132\u00a0h), which is the reason why it was given three times daily in a dose of 15\u00a0mg per helping. Even at this regimen its short halflife will result in fluctuating K2 serum levels, which may explain the requirement of a high dose. At this time no data are available on the efficacy of MK-4 at lower doses, for instance in the range between 100\u2013500 micrograms per day. A possible alternative to MK-4 might be using MK-7, the K2 vitamin most abundantly found in the Japanese food natto (fermented soy beans). MK-7 has probably a comparable effect as MK-4 [35], but it has a halflife in the circulation of 3\u00a0days, resulting in more constant plasma levels and accumulation in the blood and various tissues. Therefore, MK-7 is the most logical choice for use as a low-dose food supplement, because even at low intake relatively high blood and tissue levels can be obtained. Clinical trials in which the efficacy of MK-7 is tested are, however, lacking at this time. From our study it is not clear whether the high MK-4 regimen will increase vertebral bone strength in a similar way as observed for the DXA-derived femoral neck bone strength. To our knowledge no reliable formulas are available for calculating vertebral compression strength, the most important strength index for the vertebrae. However, the substantial and significant effect of MK-4 on the lumbar spine BMC warrants more elaborate studies in which the effect on vertebral fracture risk is investigated. A substantial risk reduction for vertebral fractures by long-term MK-4 treatment has also been reported by a number of Japanese studies [36, 37].\nAnother question that needs to be answered is why K2 has a much more pronounced effect than K1. One reason may be the different doses used: 45\u00a0mg\/day for MK-4 and 1\u201310\u00a0mg\/day for K1. Based on our present knowledge, all three doses are an excess; it seems not unlikely, therefore, that there is a qualitative difference between K1 and K2. The only difference between both vitamins is the aliphatic side chain, which does not influence the coenzyme function, but which does affect the distribution of vitamin K over the lipoproteins in the blood, and the transport to and absorption by the various tissues. It has been demonstrated in human volunteers that both K1 and K2 are transported to the liver via triglycerides, but that only K2 vitamins (both MK-4 and MK-7) are incorporated into the LDL fraction for transport to extrahepatic tissues [39]. It is not surprising, therefore, that in animal experiments it was found that K1 mainly accumulates in the liver, whereas MK-4 is far more abundant in other tissues including pancreas, bone and vessel wall [10]. In that perspective, K2 seems to be the most logical choice for supplementation because notably bone and arteries (and not the liver) were reported to have a sub-optimal vitamin K status in the majority of the healthy population.\nA limitation of this study is that it was conducted in non-osteoporotic women, in whom we tried to decrease bone loss and to maintain bone strength. Therefore, this study does not allow conclusions regarding beneficial effects of vitamin K2 on bone quality in women who have been diagnosed with osteoporosis already. A further limitation is that the study was exclusively conducted on women, so that no conclusions can be drawn for bone loss in men. A further limitation of our study was that the DXA-BMD of the spine was determined only in the postero-anterior (PA) position. This measurement includes not only the trabecular bone of the vertebral body, but also a substantial amount of cortical bone from posterior elements. During ageing, degenerative changes in the spine such as osteoarthritis or sclerosis of the endplates may occur. In those cases, PA projection may result in incorrect interpretation of the DXA-BMD and BMC values because the observed increase is due to degenerative changes and is not based on treatment effect. Lateral DXA-BMD measurements evaluate exclusively the vertebral body, allowing more sensitive detection of postmenopausal bone loss or DXA-BMD changes during therapy [38]. At the start of this study lateral DXA-BMD monitoring, however, was not yet available for clinical trials in our hospital.\nIn conclusion we have demonstrated that vitamin K2 is capable of improving both the BMC and bone geometry at critical sites, leading to maintenance of the DXA-derived femoral neck bone strength in postmenopausal women during a 3-year study period. Even in the very high doses used in our study, adverse side effects of MK-4 were minor and not different from the placebo group. Therefore, it seems desirable to evaluate the cost-benefits of supplementing low dose vitamin K2 to all postmenopausal women. The optimal dose, the preferred form (MK-4 or MK-7), and synergistic effects of complementary food supplements (calcium, vitamin D) or medication (bisphosphonates), however, require further research.","keyphrases":["vitamin k","menaquinone","fracture risk","bone mineral content","osteoporosis","femoral neck strength"],"prmu":["P","P","P","P","P","R"]}
{"id":"Eur_Arch_Psychiatry_Clin_Neurosci-2-2-1705499","title":"Epidemiologic and clinical updates on impulse control disorders: a critical review\n","text":"The article reviews the current knowledge about the impulse control disorders (ICDs) with specific emphasis on epidemiological and pharmacological advances. In addition to the traditional ICDs present in the DSM-IV\u2014pathological gambling, trichotillomania, kleptomania, pyromania and intermittent explosive disorder\u2014a brief description of the new proposed ICDs\u2014compulsive\u2013impulsive (C\u2013I) Internet usage disorder, C\u2013I sexual behaviors, C\u2013I skin picking and C\u2013I shopping\u2014is provided. Specifically, the article summarizes the phenomenology, epidemiology and comorbidity of the ICDs. Particular attention is paid to the relationship between ICDs and obsessive\u2013compulsive disorder (OCD). Finally, current pharmacological options for treating ICDs are presented and discussed.\nIntroduction\nSince the early 1990s, some researchers have suggested that the impulse control disorders (ICDs) might be conceptualized as a part of an obsessive\u2013compulsive spectrum based on their clinical characteristics, familial transmission, and response to both pharmacological and psychosocial treatment interventions [1\u20133]. Over a decade of study and scientific developments have led a DSM-V task force to consider two important changes: separating obsessive\u2013compulsive disorder (OCD) from the anxiety disorders and placing it in an autonomous category\u2014the obsessive\u2013compulsive spectrum disorders (OCSD); and creating several new autonomous disorders from those currently subsumed under ICDs not otherwise specified (ICD-NOS) [4], specifically including four new impulsive disorders, compulsive\u2013impulsive (C\u2013I) Internet usage disorder C\u2013I sexual behaviors, C\u2013I skin picking and C\u2013I shopping. They are called compulsive\u2013impulsive disorders due to the impulsive features (arousal) that initiate the behavior, and the compulsive drive that causes the behaviors to persist over time.\nThe relationship between OCD and the OC spectrum has been supported by studies over the past decade, although recent studies have also supported additional models. Recent neuroimaging (PET, fMRI etc.) and genetics studies have increased understanding of the biological and neuroanatomical characteristics of the ICDs and have supported both the OC spectrum model and suggested other models [5, 6]. The pharmacological options, moreover, have been expanded based on recent research; traditional treatment with the serotonin reuptake inhibitors (SRIs) supported the OC spectrum model, but recent research demonstrating the efficacy of different pharmacological interventions suggests that additional systems are involved and other models may be useful. For example, the efficacy of pharmacotherapies acting on different systems of neuromediators (opioid antagonists, mood stabilizers, dopamine reuptake inhibitors), support different theoretical models for the ICDs and make clear that it is valuable to look at the ICDs from different theoretical perspectives that suggest different mechanisms might be important and raise new research questions.\nICDs\u2019 phenomenology, epidemiology and relationship with OCD\nICDs are characterized by repetitive behaviors and impaired inhibition of these behaviors. Important defining criteria for these disorders include:The failure to resist an impulse to perform some act that is harmful to the individual or others;An increasing sense of arousal or tension prior to committing or engaging in the act;An experience of either pleasure, gratification, or release of tension at the time of committing the act.\nIn addition, there is usually a pattern of engaging in the abnormal behavior in spite of adverse consequences (e.g., criminal changes, impairment of normal functioning, etc.). To demonstrate that a relationship exists between ICDs and OCD, there should be evidence that OCD is overrepresented in patients with ICDs and\/or that ICDs are overrepresented in patients with OCD. Studies examining rates of OCD in patients with ICDs have reported inconsistent results, with some ICDs showing relatively high rates of comorbidity with OCD (trichotillomania, CI-shopping), and others demonstrating low rates (intermittent explosive disorder, pathological gambling, and C\u2013I sexual behaviors).\nPathological gambling (PG) is an impulse control disorder not otherwise specified (ICD-NOS) [4] that is characterized by recurrent and maladaptive patterns of gambling behavior that significantly disrupts the patient\u2019s functioning in the personal, familial, or vocational spheres. Recent studies suggest that the prevalence of PG is between 1% and 3% of the adult population [7, 8], and a meta-analysis [9] estimated that 86% of the population of the USA are recreational gamblers (Table\u00a01). The disorder usually starts during adolescence with a prevalence of approximately 4\u20137% in this population. However, over the last decade, there has been an unprecedented expansion of legalized gambling throughout North America, and, as a result, the prevalence of PG can be expected to increase. The disorder is currently more common in men than in women. Recent national studies on PG prevalence have also been conducted in New Zealand [10\u201312], Sweden [13, 14], Switzerland [15], Australia [16] and Great Britain [17], and despite the use of different methodologies and variable technical quality, problem gambling prevalence studies have shown a high degree of consistency in their general findings.\nTable\u00a01Prevalence estimates of impulse control disordersImpulse control disorderReferenceType of communityPrevalence reportedPathological GamblingGerstein et al. (1999)Adult population1\u20133%Welte et al. (2001)Adult populationTrichotillomaniaChristenson et al. (1991)College students1.5% males; 3.4 femalesPyromaniaKosky and Silburn (1984)Children and adolescents2.4\u20133.5%Kolko et al. (1988)Children and adolescentsJacobson (1995)Children and adolescentsIntermittent Explosive DisorderMonopolis and Lion (1983)Psychiatric surveys1\u20132%Coccaro et al. (2004)Adult populationLifetime 11.1%; 1 month 3.2%KleptomaniaGoldman (1991)Adult population0.6%C\u2013I Internet Usage Disorder\u2013\u2013\u2013C\u2013I ShoppingBlack et al. (2001)Adult population 2\u20138%C\u2013I Skin PickingDoran et al. (1985)Dermatologic patients2%Gupta et al. (1986)Dermatologic patientsC\u2013I Sexual BehaviorsShaffer and Zimmerman (1990)Adult population5\u20136%Coleman, 1991Adult population\nA crucial issue to consider is the high rate of comorbidity among pathological gamblers. Patients with PG, at least those seeking treatment, have been found to score significantly higher than control populations on measures of depression [18], and have high incidences of various psychiatric disorders, including bipolar, anxiety and substance use disorders [19]. This frequent comorbidity is not surprising given the psychopathological core features of PG: impulsivity, compulsive drive to gamble, addictive features such as withdrawal symptoms during gambling abstinence, and bipolar features such as urges, pleasure seeking and decreased judgment due to unrealistic appraisal of the individuals\u2019 own abilities. Several authors have noted the link between various core features of PG and neurobiological characteristics or treatment-response, and have conceptualized PG according to different models, thus placing it on different spectrums with the main psychiatric disorders of reference being OCD [1], addictive disorders [20], and affective disorders [21]. These models provide the theoretical rationale for the use of specific pharmacological treatments in PG. In addition, the models and related research findings may also suggest the presence of specific subgroups of patients with similar core features, comorbidity profiles and treatment-response within the population of pathological gamblers [22, 23]. The relationship between PG and OCD has allowed PG to be conceptualized as an OC spectrum disorder, within the impulsive cluster [1]. Patients with OC spectrum disorders, in fact, experience unpleasant feelings and physiological activation that result in an intense desire to perform a specific behavior in order to relieve the unpleasant feelings [24, 25]; this is the case in PG. In addition, a reduced capacity to resist gambling thoughts and urges leads to excessive gambling, in particular in the advanced phases of the disorder [26]. However, these patients differ from patients with OCD in important ways. Gambling behavior and thoughts are often experienced by these patients as ego-syntonic, while OCD obsessions and compulsions are generally ego-dystonic. In addition, the excessive doubt frequently experienced by OCD patients [24, 27, 28], as well as their harm avoidance, risk aversion and anticipatory anxiety [29], are not characteristic of pathological gamblers. OC spectrum disorders differ along the dimension of risk aversion vs. risk taking; the compulsive disorders are characterized by an overestimation of harm and by risk aversion while the impulsive disorders are characterized by an underestimation of risk and by risk seeking. Recently, the rate of comorbid OCD in individuals with PG was found to range from 1% to 20% [30] (Table\u00a02).\nTable\u00a02OCD rates in impulse control disordersImpulse control disorderReferenceRates of OCDPathological GamblingArgo and Black (2004)1\u201320%TrichotillomaniaChristenson and Mansueto (1999)3\u201327%Pyromania\u2013\u2013Intermittent Explosive DisorderMcElroy et al. (1998)22%KleptomaniaPresta et al. (2002)6.5\u201360%C\u2013I Internet Usage DisorderBlack et al. (1999)0% current; 10% lifetimeShapira et al. (2000)15% current; 20% lifetimeC\u2013I ShoppingChristenson et al. (1994)12.5\u201330%McElroy et al. (1998)C\u2013I Skin PickingSimeon et al. (1997)6\u201352%Arnold et al. (1998)Wilhelm et al. (1999)C\u2013I Sexual BehaviorsKafka and Prentky (1994)12\u201314%Black et al. (1997)\nPatients afflicted with trichotillomania (TTM) describe an overwhelming urge to pluck out specific hairs; when they do so, the anxiety is momentarily relieved but is quickly replaced by another compulsive urge to pluck and even greater anxiety [31]. The exact prevalence of TTM is unknown; however, estimates from university surveys suggest that 1.5% of males and 3.4% of females endorse clinically significant hair pulling, with .6% endorsing all diagnostic criteria of TTM [32] (Table\u00a01). The prevalence of non-clinical hair pulling behavior is even higher, up to 15.3%, in university surveys [33] (Table\u00a01). In describing the phenomenological similarities between OCD and TTM, Swedo [34] highlighted the egodystonic feeling and the resistance experienced by patients with TTM and OCD. In addition, patients with TTM recognize the behavior as senseless, undesirable and performed in response to increasing anxiety, with resultant tension relief. Furthermore, a higher than normal incidence of both OCD and TTM has been reported in first-degree relatives of patients with TTM [35], and comorbidity data also support a relationship between OCD and TTM [36, 37] (Table\u00a02). However, recent investigations [38, 39] have also included TTM in a spectrum of self-injurious behaviors (SIBs), including C\u2013I skin picking, and underscored the phenomenological link among these SIBs and the differences between TTM and OCD [39].\nIn pyromania there is impulsive, repetitive, deliberate fire setting without external reward (e.g., arson for money, revenge, as a political act). There are very few community sample studies of firesetting, which is understandable since it is illegal and thus likely to be kept secret. The majority of epidemiological studies have focused on pyromania in childhood and adolescence and have reported the prevalence to be between 2.4% [40] and 3.5% [41, 42] (Table\u00a01). In addition, several lines of evidence indicate that adolescent boys may be at higher risk for firesetting than adolescent girls [43, 44]. Among juveniles, firesetting is more prevalent in males than females, peaking between 12\u00a0years and 14\u00a0years [45]. Sixty percent of all fires in large U.S. cities are lit by individuals between 11\u00a0years and 18\u00a0years [46]. Besides young age, features such as temperament, parental psychopathology, social and environmental factors, and possible neurochemical predispositions [47] have been hypothesized to cause childhood pyromania. Some authors have noted a close link between firesetting and aggression [48] and between firesetting and antisocial behavior [49]. In addition, published data have shown high rates of conduct disorder among young arsonists [50]. Recent findings, moreover, revealed associations between firesetting and shyness, aggression and peer rejection [51]. No published studies of the relationship between pyromania and OCD in terms of comorbidity or family history are available.\nIntermittent explosive disorder (IED) is characterized by recurrent episodes of aggressive behavior that is out of proportion to psychosocial stressors and\/or provocation and that is not better accounted by another mental disorder, comorbid medical conditions, or the physiologic effects of a pharmacologic agent or other substance with psychotropic properties [4]. Despite its inclusion in DSM for more than two decades, there are few studies of the lifetime prevalence of IED in either psychiatric or community settings. Clinical surveys of psychiatric inpatients [52], and clinical treatment studies on IED [53] had found rates of IED in psychiatric settings ranging from 1% to 2%. Recently, however, Coccaro and colleagues reported much higher rates of IED, 11.1% lifetime prevalence and 3.2% 1-month prevalence, in a community sample of 253 individuals [54] (Table\u00a01). Based on these data, the authors estimated there are 1.4 million individuals with current IED in the US and 10 million with lifetime IED. As the authors suggested, prevalence rates so much higher than prior findings may reflect the changes in diagnostic criteria of IED from DSM-III [55] to DSM-IV [4] as well as the changes recently proposed in the development of research criteria for IED [56, 57]. A study by McElroy and colleagues reported rates of OCD in individuals with IED around 22% [58] (Table\u00a02); recent studies investigating the rates of IED in patients with OCD have given lower estimates [59\u201361].\nKleptomania is a disorder in which the individual impulsively steals even though there is need to do so (i.e., the individual has money to pay for the stolen items or does not need the stolen goods). Like other ICDs, kleptomania is characterized by an anxiety-driven urge to perform an act that is pleasurable in the moment but causes significant distress and dysfunction [62]. The prevalence of kleptomania in the U.S. is unknown but has been estimated at 6 per 1000 people. [63] (Table\u00a01). In addition, given the embarrassment surrounding kleptomania, it is often kept secret and thus goes undiagnosed [62]. Kleptomania is thought to account for 5% of shoplifting in the U.S. [64]. Based on total shoplifting costs of $10 billion in 2002 [65], this 5% translates into a $500 million annual loss to the economy attributable to kleptomania. This loss does not include the costs associated with stealing from friends and acquaintances or costs incurred by the legal system. Kleptomanic behavior carries serious legal consequences: approximately 2 million Americans are charged with shoplifting annually [66]. If kleptomania accounts for 5% of these, this translates into 100,000 arrests. Recent studies assessing the rate of OCD in patients with kleptomania have given widely differing estimates, ranging from 6.5% to 60% [67, 68] (Table\u00a02).\nC\u2013I Internet usage disorder, also referred as Internet addiction or problematic Internet use, has been proposed as an explanation for uncontrollable and damaging use of the Internet, and has only recently begun to appear in the psychiatric literature [69, 70]. People with problematic Internet use often report increasing amounts of time-spent web surfing, gambling, shopping or exploring pornographic sites. Others report spending time in chat rooms or corresponding by email. Frequently these people develop a preoccupation with the Internet, a need for escape to the Internet and increasing irritability when trying to cut back their Internet use. Ultimately, their attempt to cut back is unsuccessful. Functional impairments as a result of problematic Internet use include marital or family strife, job loss or decreased job productivity, legal difficulties or school failure [72]. Although diagnostic criteria for this disorder have been proposed, methods of assessing C\u2013I Internet usage disorder are limited. In addition, although increasing research is being conducted on the topic, several published articles contain information that has not been empirically researched [73]. For some individuals, their excessive Internet use may be entirely accounted for by another Axis I disorder such as PG or C\u2013I sexual behaviors; thus the Internet is functioning simply as another outlet for that disorder rather than being an additional disorder. Problematic Internet use has been reported in any age, social, educational, and economic range [74]. However, while previous studies tended to stereotype the classical Internet addicted patient as a young introverted man [75, 76], recent investigations have showed increasing rates of this disorder among women [74], as a result of the increased availability of the Internet. The prevalence of C\u2013I Internet usage disorder is not known. Most of the studies related to this condition have been conducted with small samples. People enrolled, moreover, frequently had comorbid psychiatric diagnoses. In a recent study [71], Shapira and colleagues found that all subjects with problematic Internet use also met DSM-IV criteria for ICD-NOS. Studies assessing comorbidity rates between OCD and C\u2013I Internet use reported estimates ranging from 10% to 20% for lifetime OCD and up to 15% for current OCD in Internet addicted patients [71, 77, 78] (Table\u00a02). Further investigations on the epidemiology of this disorder are needed to clarify the scale and demographic characteristics of C\u2013I Internet use.\nC\u2013I sexual behaviors (C\u2013ISBs) include repetitive sexual acts and compulsive sexual thoughts. The individual feels compelled or driven to perform the behavior, which may or may not cause subjective distress. Although generally not ego-dystonic, the behavior may interfere with several aspects of the patient\u2019s life, causing social or occupational impairment, or legal and financial consequences [79]. C\u2013ISBs involve a broad range of paraphilic or non-paraphilic symptoms [80]. Paraphilic C\u2013ISBs involve unconventional sexual behaviors in which there is a disturbance in the object of sexual gratification or in the expression of sexual gratification (e.g., exhibitionism, voyeurism). Non-paraphilic C\u2013ISBs, on the other hand, involve conventional sexual behaviors that have become excessive or uncontrolled [80]. The true prevalence of C\u2013ISBs remains unknown, given the hetereogeneity of these disorders as well as the secretiveness of the condition for the majority of the afflicted patients. Investigations conducted in the early 1990s reported prevalence estimates of C\u2013ISBs ranging from 5% to 6% of the US population [80, 81] (Table\u00a01). Male patients have been traditionally reported to be more afflicted than women by C\u2013ISBs [82, 83]. However, it is not clear how large this sex difference is and the extent to which the difference is due to men coming to the attention of professionals with greater frequency. Studies assessing the rates of OCD in patients suffering from C\u2013ISBs [79, 84] reported estimates around 12% and 14% (Table\u00a02).\nC\u2013I shopping, also referred as compulsive buying, is characterized by maladaptive preoccupations or impulses to buy or shop that are experienced as irresistible, intrusive and\/or senseless, accompanied by frequent episodes of buying items that are not needed and\/or that cost more than can be afforded. Frequently, these patients engage in these behaviors for longer periods of time than intended, and they experience distress and significant impairment in social and occupational performance. As specified for many other ICDs, the excessive buying or shopping behavior does not occur exclusively during periods of hypomania or mania [85, 86]. A recent study on C\u2013I shopping disorder estimated the prevalence of this disorder to be between 2% and 8% of the general adult population in the US [87]; 80% to 95% of those affected are female (Table\u00a01). Onset occurs in the late teens or early twenties, and the disorder is generally chronic. Previous studies investigating rates of OCD in patients with C\u2013I shopping reported rates of 12.5% to 30% [86, 88] (Table\u00a02); lower rates of compulsive buying have been found in patients with OCD (from 2.2% to 10.6%) [59\u201361], except for the study of Lejoyeux and colleagues (23.3%) [89].\nPatients with C\u2013I skin picking frequently present to dermatologists, and it has been estimated that about 2% of dermatology clinic patients may suffer from this condition [90, 91] (Table\u00a01). Prevalence in the general population or in psychiatric clinics is unknown. Skin picking is often not a transient behavior but may persist with a waxing and waning lifetime course. It should be considered pathological when it becomes habitual, chronic and extensive, leading to significant distress, dysfunction or disfigurement [38]. As reported by two recent studies, the majority of patients with C\u2013I skin picking are women and their condition is assumed to be chronic, with excoriations on both single or multiple sites [92, 93]; the face is the most common site of excoriation but picking can involve any area of the body. Both studies found the majority of patients experienced increasing tension before the act (79\u201381%), relief after the act (52\u201379%), or both (68\u201390%). Comorbid lifetime rates of skin picking in patients with trichotillomania were approximately 10% in both studies [92, 93], whereas comorbid lifetime OCD was present in rates ranging from 6% to 19%. Wilhelm and colleagues [94] reported rates of OCD around 52% in a sample of 31 patients with C\u2013I skin picking (Table\u00a02). As mentioned for trichotillomania, the inclusion of C\u2013I skin picking within a spectrum of self-injurious behaviors is receiving increasing support from clinical and neuroimaging studies [38].\nTreatment options for ICDs\nTreatment options for ICDs include both pharmacotherapy and psychotherapy. During the last decade, increasing research has been conducted on different pharmacological treatments across several ICDs; however, while the efficacy of various treatments has been investigated in double-blind studies for certain disorders (i.e., PG, IED, C\u2013I shopping), systematic research of clinical treatment is still lacking for other disorders (see Table\u00a03). In addition, a crucial issue to take into account when considering pharmacotherapy for patients with ICDs is the comorbidity with other psychiatric conditions such as affective and addictive disorders. The presence of bipolar or addictive comorbidity, in fact, will determine the most appropriate choice when different treatments have proven to be effective for a specific disorder.\nTable\u00a03Treatment options for impulse control disorders as reported in blinded and unblinded studiesImpulse Control DisorderDouble-blind studies (references)OutcomesOther treatment options as reported in open-label trialsPathological GamblingFluvoxamine vs. PC (Hollander et al. 2000;  Blanco et al. 2002)SSD for Fluvoxamine; No SSD  between Fluvoxamine and PC. Nefazodone, Bupropion, Citalopram,  Divalproex, TopiramateParoxetine vs. PC (Kim et al. 2002;  Potenza et al. 2003)SSD for Paroxetine; No SSD between  Paroxetine and PC. Lithium vs. PC (Hollander et al. 2005)SSD for Lithium;Naltrexone vs. PC (Kim et al. 2001)SSD for NaltrexoneTrichotillomaniaClomipramine vs. Desipramine  (Swedo et al. 1989)SSD for Clomipramine;Fluvoxamine, Citalopram, Venlafaxine,  Naltrexone, Lithium, CBTFluoxetine vs. PC (Christenson et al. 1991;  Streichenvein and Thornby 1995)No SSD between Fluoxetine and PCPyromania\u2013\u2013CBT and other psychotherapiesIntermittent  Explosive Disorder*Lithium vs. PC (Campbell et al. 1984 and  1995; Malone et al. 1998 and 2000) SSD for Lithium (in the Campbell\u2019  study of 1984, Lithium was  associated to Haloperidol)Clonidine*Divalproex vs. PC  (Hollander et al. 2003 and 2005) SSD for Divalproex*Fluoxetine vs. PC (Coccaro et al. 1997) SSD for Fluoxetine*Carbamazepine vs. PC (Foster et al. 1989) SSD for Carbamazepine*Phenytoin vs. PC (Barratt et al. 1997;  Stanford et al. 2001) SSD for Phenytoin*BBlockers vs. PC  (Greendyke et al. 1986a and 1986b) SSD for BBlockers*Risperidone vs. PC (Buitelaar et al. 2001;  Findling et al. 2001) SS for Risperidone*CBT vs. PC (Alpert et al. 1997)SSD for CBTKleptomania\u2013\u2013Fluoxetine, Paroxetine, Fluvoxamine,  Divalproex, Lithium, BenzodiazepinesC\u2013I Internet Usage  DisorderEscitalopram vs. PC  (Dell\u2019Osso et al. 2006**)SSD for EscitalopramPsychotherapyC\u2013I ShoppingFluvoxamine vs. PC (Black et al. 2000;  Ninan et al. 2000)No SSD between Fluvoxamine  and PC;Fluvoxamine, NaltrexoneCitalopram vs. PC (Koran et al. 2003)SSD for CitalopramC\u2013I Skin PickingFluoxetine vs. PC (Simeon et al. 1997;  Block et al. 2000)SSD for FluoxetineClomipramine, SertralineC\u2013I Sexual Behaviors\u2013\u2013Lithium, Tricyclics, Buspirone,  Fluoxetine, Nefazodone,  Sertraline, NaltrexoneSSD = statistically significant differences; CBT = cognitive behavioral therapy; PC = placebo* Studies on patients with impulsive aggression features, rather than with a proper DSM diagnosis of IED** Open-label study followed by double-blind discontinuation phase (Abstract)\nPG is a good example of the importance of comorbidity determining treatment. PG has demonstrated a good response to selective serotonin reuptake inhibitors (SSRIs), mood stabilizers and opioid antagonists in double-blind studies [22, 95\u201399] (Table\u00a03). Among all the antidepressants assessed so far, fluvoxamine [100], paroxetine [97, 98], citalopram [101], nefazodone [102], bupropion [103], (although only fluvoxamine and paroxetine in double-blind studies), the most convincing evidence is for the efficacy of the SSRIs. However, a major issue for this class of medication is the presence of bipolar spectrum comorbidity in some gamblers. This possibility needs to be carefully evaluated and excluded before treating pathological gamblers with antidepressants in order to avoid the possible reemergence of manic symptoms. The opioid antagonist naltrexone was effective in a double-blind trial, however, the risk of hepatotoxicity of this drug limits its use. Of note, the opioid antagonist nalmefene has shown to be efficacious in preliminary findings with better tolerability than naltrexone [104]. Patients with other addictive disorders (alcohol and other substances) and intense urges and craving might particularly benefit from opioid antagonists. Mood stabilizers and anticonvulsants (lithium and divalproex assessed in double-blind controlled trials) have shown good results in recent studies without any specific contraindications for their use across the different subtypes of gamblers. In addition, gamblers with consistent affective instability may particularly benefit from these treatments.\nPharmacological treatment of TTM is not well established and, although SSRIs seem to show the best efficacy and safety, double-blind controlled studies on their use have given mixed results (Table\u00a03). Clomipramine was found to be more effective than desipramine in a 10-week crossover study [105] conducted in the late 1980s. While subsequent uncontrolled studies found fluoxetine, fluvoxamine and citalopram to be efficacious in patients with hair pulling [106\u2013110], two controlled studies [111, 112] with fluoxetine could not replicate the positive findings reported with SSRIs in the open-label trials. Positive results have been also reported in uncontrolled studies with venlafaxine, lithium and naltrexone [113\u2013116] as well as in open-label augmentation studies with SSRIs and pimozide [117, 118]. However, treatment response is often disrupted by significant relapse during ongoing pharmacological treatment [117]. In a recent controlled study [119] comparing cognitive behavioral therapy (CBT) to clomipramine and placebo, CBT had a dramatic effect in reducing symptoms of TTM and was significantly more effective than clomipramine or placebo, underscoring the efficacy of behavioral as well as pharmacological treatment in hair pulling.\nTo our knowledge, no controlled pharmacological trial has been conducted in patients with pyromania. Non-pharmacological interventions for firesetters, including CBT [120], short-term counseling and day-treatment programs [121], have shown some efficacy. Undoubtedly, pyromania represents an ICD needing systematic pharmacotherapy research.\nTreatment options for IED include the use of mood stabilizers, phenytoin, SSRIs, \u03b2-blockers, \u03b12-agonists and antipsychotics (Table\u00a03). Actually the majority of trials with these compounds have been conducted on individuals with impulsive aggression rather than with a specific diagnosis of IED, and several authors still don\u2019t consider the current criteria for the diagnosis of IED to be adequate [122]. Nevertheless, the presence of impulsive aggression within the core features of IED allows us to put aside this nosographic debate. Among mood stabilizers, the most convincing evidence comes from controlled studies with lithium (especially in children and adolescents) [123\u2013127] and divalproex [128]. This last medication demonstrated significant efficacy in different populations of aggressive subjects [129, 130]. Carbamazepine has also shown some efficacy in a small double-blind study and in open-label trials [131, 132]. Phenytoin has showed positive results in two controlled double-blind studies [133, 134] at doses up to 300\u00a0mg\/d. With regard to SSRIs, a double-blind placebo controlled trial of fluoxetine [135] in patients with personality disorder showed reduced scores on measures of irritability and aggression in patients taking the active medication. B-blockers propranolol and pindolol have also shown positive results in controlled studies [136, 137], reducing aggressive behaviors in patients with brain damage, although their concomitant diagnosis of IED might be arguable as the aggressive behaviors may have a different etiology. The \u03b1-agonist clonidine was reported to decrease aggression in an open-label trial [138] with adolescents at dosages of 0.4\u00a0mg\/d, although the tolerability was a problem for some subjects. The atypical antipsychotic risperidone was also showed to be effective in treating aggression in controlled studies [139, 140]. Finally, controlled studies of behavioral interventions including CBT, group therapy, family therapy and social skill training have shown them to be valid treatments for aggressive patients [141, 142].\nThe pharmacological treatment of kleptomania includes SSRIs, mood stabilizers and opioid antagonists, although none of these medications have been tested in blinded, controlled trials so far (Table\u00a03). Among SSRIs, fluoxetine, alone or in combination with lithium or tricyclics, was shown to be effective in several case-reports [64, 143, 144], as were fluvoxamine and paroxetine [145\u2013148]. Mood stabilizer trials and reports in kleptomanic patients showed mixed results for lithium [64, 144, 145], valproic acid [64, 149] and carbamazepine [64]. The opioid antagonist naltrexone was reported to be effective in two different case reports [148, 150]. Finally the benzodiazepines clonazepam and alprazolam provided some evidence of efficacy in treating kleptomania [64, 147]. In conclusion, as discussed in a recent review [151], SSRIs seem to be the most promising treatment for kleptomania (19 of 30 cases of successful pharmacotherapy reported in the literature), either as monotherapy or in combination with other psychotropic drugs.\nGiven its recent recognition as a psychiatric problem, understandably no controlled pharmacological trials have been published on the treatment of C\u2013I Internet usage disorder so far. Recently, Sattar and Ramaswamy [152] reported the case of a 31-year-old man with severe Internet addiction successfully treated with escitalopram (10\u00a0mg\/d). Most treatment strategies for problematic Internet use have involved behavioral therapy techniques, which limit the amount of time on the Internet rather than requiring abstinence, as is done with many other addictions such as substance abuse. Self-help groups (both on and offline) are also being formed to address the problem. Our group has recently completed an open-label trial of escitalopram followed by a double-blind discontinuation phase in a population of C\u2013I Internet users with preliminary positive findings [153]. Given the increasing use of the Internet in the new generations, a growing prevalence and incidence of this disorder is arguable. Clinicians treating subjects with ICDs should always assess the presence of this disorder in these patients given the relationship between C\u2013I Internet use and some specific ICDs, such as pathological gambling and C\u2013I sexual behaviors [154, 155]. Finally, controlled studies are expected in order to investigate the treatment response of Internet addicted patients to pharmacotherapy and psychotherapy.\nAlthough C\u2013I sexual behaviors seem relatively common, controlled trials on pharmacological treatments for these disorders are still lacking, and the available literature on this topic consists essentially of open-label trials and case-report series (Table\u00a03). Positive findings have been reported with lithium and tricyclics [156\u2013158], SRIs [159\u2013162], buspirone [163, 164] and nefazodone [165]. As for other ICDs, the opioid antagonist naltrexone has recently shown to be efficacious in some case-reports [166]. Finally, different forms of psychotherapy have been shown to be effective for specific subtypes of C\u2013I sexual behaviors [167].\nThere is some evidence that C\u2013I shopping has been effectively treated with several different compounds (Table\u00a03). McElroy\u2019s group [86] reported on 20 patients that benefited from antidepressants, often in combination with mood stabilizers. Black [168] reported fluvoxamine to be effective in patients without comorbid major depression, suggesting that improvement was independent of the treatment of mood symptoms. Naltrexone was found to be effective in a case series [169]. Two double-blind placebo-controlled trials [170, 171] did not confirm the superiority of fluvoxamine over placebo. However, these studies had the patients in both conditions keep a log of their shopping; keeping logs is a therapeutic intervention in itself and may have led to the failure of the fluvoxamine and placebo groups to separate. An open-label trial of citalopram [172] and a subsequent open-label trial followed by double-blind discontinuation [173], neither of which using shopping logs, reported positive results. Studies comparing the efficacy of pharmacological treatment with psychotherapy have not been published yet.\nPatients suffering from C\u2013I skin picking often meet criteria for other psychiatric disorders (BDD and OCD), and frequently, due to medical complications of their psychopathology such as infection and scarring, they are referred to clinicians other than psychiatrists (i.e. dermatologists). The first controlled trial conducted by our group [97] found fluoxetine, at a mean dose of 55\u00a0mg\/d for 10\u00a0weeks, significantly superior to placebo in decreasing the behavior in 21 adults with chronic pathologic skin picking (Table\u00a03). More recently, a combined open-label and double-blind trial [174] confirmed the efficacy of fluoxetine in subjects with C\u2013I skin picking. Previously, a retrospective treatment review of BDD patients with skin picking indicated that SRIs were effective in about half of 33 patients, whereas other agents were not [175]. In a subsequent open-label study [176], sertraline (mean dose: 95\u00a0mg\/d) showed clinically significant improvement in 68% of 30 patients with skin picking after one month of treatment. Finally, uncontrolled psychodynamically oriented treatments and behavioral interventions have given mixed results described elsewhere [177].\nConclusions\nCurrent knowledge on ICDs in terms of epidemiology and pharmacological treatment varies notably across these disorders, with recent and continuing advances for some (i.e. pathological gambling and C\u2013I shopping), and anecdotal and obsolete data for others. Undoubtedly, given the high prevalence estimates of some ICDs (i.e. pathological gambling and C\u2013I sexual behaviors) as well as their comorbidity with other major psychiatric disorders, this group of disorders represents a global problem. Nevertheless, certain ICDs (i.e, pyromania, C\u2013I Internet usage disorder) still need systematic epidemiological and pharmacological research.\nStudying the relationships between specific ICDs and other major psychiatric conditions (i.e. OCD, bipolar disorders, addictive disorders) in terms of phenomenological issues and comorbidity patterns is not only of theoretical interest; indeed, it provides the rationale for the use of specific pharmacological treatments and behavioral interventions. From this perspective, more than one decade after its introduction, the conceptualization of ICDs as obsessive\u2013compulsive related disorders is still valid and has been confirmed by numerous studies; however, there is also evidence supporting the relationship between ICDs and addictive and affective disorders. Not only are the different models of conceptualizing the ICDs not mutually exclusive, but they can contribute to recognize specific subtypes within the disorders. As a result, different models of conceptualization of ICDs have led new developments in pharmacologic treatment of these disorders, with positive results obtained with mood stabilizers and opioid antagonists in addition to the SSRIs.","keyphrases":["impulse control disorders (icds)","kleptomania","pyromania","c\u2013i skin picking","obsessive\u2013compulsive disorder (ocd)","pathological gambling (pg)","c\u2013i internet usage disorder","trichotillomania (ttm)","intermittent explosive disorder (ied)","c\u2013i sexual behaviors (c\u2013isbs)","compulsive\u2013impulsive (c\u2013i) shopping"],"prmu":["P","P","P","P","P","P","P","P","P","P","R"]}
{"id":"Med_Oncol-4-1-2386844","title":"The effects inhibiting the proliferation of cancer cells by far-infrared radiation (FIR) are controlled by the basal expression level of heat shock protein (HSP) 70A\n","text":"We developed a tissue culture incubator that can continuously irradiate cells with far-infrared radiation (FIR) of wavelengths between 4 and 20 \u03bcm with a peak of 7\u201312 \u03bcm, and found that FIR caused different inhibiting effects to five human cancer cell lines, namely A431 (vulva), HSC3 (tongue), Sa3 (gingiva), A549 (lung), and MCF7 (breast). Then, in order to make clear the control system for the effect of FIR, the gene expression concerned to the inhibition effect by FIR were analyzed. In consequence, basal expression level of HSP70A mRNA was higher in A431 and MCF7 cells than in the FIR-sensitive HSC3, Sa3, and A549 cells. Also, the over expression of HSP70 inhibited FIR-induced growth arrest in HSC3 cells, and an HSP70 siRNA inhibited the proliferation of A431 cells by irradiation with FIR. These results indicate that the effect of a body temperature range of FIR suppressing the proliferation of some cancer cells is controlled by the basal expression level of heat shock protein (HSP) 70A. This finding suggested that FIR should be very effective medical treatment for some cancer cells which have a low level of HSP70. Still more, if the level of HSP70 in any cancer of a patient was measured, the effect of medical treatment by FIR can be foreseen for the cancer.\nIntroduction\nFar-infrared radiation (FIR), which causes heating, includes electromagnetic waves with wavelengths between 4 and 1,000\u00a0\u03bcm. Recently, there have been many studies of the effects of FIR on health and in the preservation of food. The available evidence indicates that whole-body irradiation by FIR has many biological effects. For example, hyperthermia (body temperature of 39\u201341\u00b0C) induced by whole-body FIR has been reported to substantially inhibit spontaneous mammary tumor growth in mice [1\u20134]. At normal temperature ranges (approximately 25.5\u00b0C), tumor growth in SHN mice can be inhibited by FIR [5, 6]. Furthermore, whole-body FIR irradiation is believed to improve human health and sleep by enhancing blood circulation in the skin [7, 8]. This is likely due to the ability of organic matter to absorb FIR at wavelengths between 7 and 12\u00a0\u03bcm.\nThe effects of FIR, and particularly whole-body FIR, remain unclear, because the experiments are easily affected by environmental changes in temperature and humidity and by the presence of bacteria. Therefore, we developed a chamber for raising animals that emits FIR upon heating and is capable of maintaining steady conditions. This system employs a sealed heater with a carbon\/silica\/aluminum oxide\/titanium oxide ceramic coating produced using a polycarbonate printing technique [9]. Using this system, we found that FIR inhibits tumor growth in the A431 tumor genesis model mouse by inhibiting the expression of matrix metalloprotease-1, 9, 10, and 13. Recent in\u00a0vitro studies by Teraoka et\u00a0al. found that FIR at wavelengths between 4 and 16\u00a0\u03bcm inhibits the growth of HeLa cells in\u00a0vitro at 37\u00b0C [10]. Despite these findings, the molecular mechanism by which FIR affects cellular gene expression remains unclear.\nThe lack of data on the effects of FIR on cells is due to the difficulty in stably irradiating cells with FIR under ideal culture conditions (i.e., 100% humidity, 37.0\u00a0\u00b1\u00a00.5\u00b0C, 5% CO2) and examining the effects of FIR at the cellular level. Therefore, using a polycarbonate printing technique, we developed a CO2 incubator with a sealed heater that has a carbon\/silica\/aluminum oxide\/titanium oxide ceramic coating and emits FIR upon heating [11]. This CO2 incubator can stably emit FIR at wavelengths between 4 and 20\u00a0\u03bcm (maximum at 7\u201312\u00a0\u03bcm) under conditions of 100% humidity, 37.0\u00a0\u00b1\u00a00.5\u00b0C, and 5% CO2. Our results using this incubator indicate that a body temperature range of FIR radiation suppresses the proliferation of HSC3, Sa3, and A549 cells. But, A431and MCF7 was not affected. These differences may be caused by any control system competing with FIR.\nTherefore, the gene expression concerning to the effect of inhibition by FIR are analyzed in the present study. Still more, the effect of over expressing and suppressing candidate FIR response genes HSP70 we examined.\nMaterials and methods\nFIR incubator\nAs previously reported [11], we fabricated an FIR radiant-panel incubator by coating a carbon\/silica\/aluminum oxide\/titanium oxide ceramic (radiation efficiency\u00a0>\u00a097%) using a polycarbonate printing technique (Bloodissue Co. Ltd. Tokushima, Japan). The incubator has a stably irradiate system with FIR at wavelengths between 4 and 20\u00a0\u03bcm (maximum at 7\u201312\u00a0\u03bcm) under conditions of 100% humidity, 37.0\u00a0\u00b1\u00a00.5\u00b0C, and 5% CO2 in air.\nCalculation of FIR absorbed per 10-cm tissue culture dish in the FIR incubator\nFourier Transform Infrared Spectroscopy (FTIR) analysis revealed that the ceramics coating inside the CO2 incubator emits FIR at 4\u00a0W m\u22121\u00a0str\u22121\u00a0\u03bcm\u22121 at wavelengths between 4.486 and 20.256\u00a0\u03bcm, with a maximal emission of 11.6\u00a0W m\u22121\u00a0str\u22121\u00a0\u03bcm\u22121 at 9\u00a0\u03bcm, which is >95% of the emission rate of an ideal black body. Since the ceramic coating was maintained at 40\u00b0C, the total generating energy, integrated over the entire range of wavelengths, was calculated to be 130.225\u00a0Wm\u22122\u00a0str\u22121. The total area of the FIR-emitting ceramic surface was 1.2385 (m2). Therefore, the total energy emitted into the incubator was 161.28366\u00a0W\/str. Assuming that FIR is emitted in all directions, the total emission was 2026.7502\u00a0J\/sec. Given that the volume of the CO2 incubator was 0.1257\u00a0m3 and the volume of culture medium was 6\u00a0ml, the amount of energy absorbed by each 10-cm culture dish was 0.09674\u00a0J\/sec. The surface area of each 10-cm culture dish was 78.5\u00a0cm2, so that the energy reaching the base of the dish was 0.001232\u00a0J\/sec\/cm2. Thus, over a 1-h period 4.4352\u00a0J\/h\u00a0cm2 was absorbed by each 10-cm culture dish.\nCell lines and cell culture\nA431 human epithelial vulva carcinoma cells and Sa3 human gingival squamous carcinoma cells were purchased from RIKEN Cell Bank (Tsukuba, Japan). A HSC3 human tongue squamous carcinoma cells, A549 human lung carcinoma cells, and MCF7 human breast carcinoma cells were purchased from Health Science Research Resources Bank (Sennan, Japan). A431, A549, and MCF7 cells were cultured in Dulbecco\u2019s modified Eagle\u2019s medium\/Ham\u2019s F-12 nutrient mixture (Sigma, St. Louis, MO, USA). HSC3 and Sa3 cells were cultured in Eagle\u2019s basal medium (Sigma). All culture medium was supplemented with 10% heat-inactivated fetal bovine serum, 100\u00a0\u03bcg\/ml penicillin G, 100\u00a0\u03bcg\/ml streptomycin sulfate, and 250\u00a0ng\/ml amphotericin B (Invitrogen, Carlsbad, CA, USA). Cells were maintained at 37\u00b0C in a humidified atmosphere of 5% CO2 in air. The medium were replaced every 2\u00a0days.\nMeasurement of cell number and growth\nCells (5\u00a0\u00d7\u00a0104) were plated in triplicate in 24-well plates (Nunc, Roskilde, Denmark). The attached cell populations were measured on day 8 using 0.2% Trypan blue and a hemocytometer. Incorporation of 5-bromo-2\u2032-deoxyuridine (BrdU) was used to determine the amount of DNA synthesis. DNA synthesis by proliferating cells was assessed using a BrdU labeling and detection kit III (Roche, Mannheim, Germany) according to the manufacturer\u2019s protocol. Briefly, cells (5\u00a0\u00d7\u00a0103 per well) were seeded in 96-well tissue culture plates (Nunc) and then placed in the FIR incubator for 4\u00a0days, and BrdU incorporation was measured during the logarithmic growth phase (i.e., before the cells were confluent) by treating the cells for 4\u00a0h at 37\u00b0C with 10\u00a0\u03bcM BrdU. BrdU incorporation was quantified by measuring the absorbance of the substrate reaction (405\u00a0nm) and the absorbance at the reference wavelength (590\u00a0nm) using an ImmunoMini NJ-2300 (System Instruments, Tokyo, Japan). Absorbance values directly correlated with the amount of DNA synthesis and therefore the number of proliferating cells.\nHistochemistry\nCells were grown on 22-mm2 glass coverslips in 6-well culture dishes (Nunc). After 4\u00a0days of FIR irradiation, the cells were observed with a CK40 phase contrast microscope (Olympus, Tokyo, Japan), fixed, and stained with hematoxylin and eosin. For immunofluorescent staining of heat shock protein (HSP) 70, cells were washed in Phosphate Buffered Saline (PBS), fixed for 20\u00a0min in 4% paraformaldehyde in PBS, washed three times for 5\u00a0min each in PBS, and blocked for 1\u00a0h at room temperature with 5% goat serum. Cells were incubated at 4\u00b0C overnight in 1:200 mouse monoclonal antibody to HSP70 (Stressgen, Victoria, Canada) in PBS containing 1\u00a0mg\/ml bovine serum albumin. After washing, the cells were incubated with 1:400 FITC (Fluorescein isothiocyanate)-labeled goat anti mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The localization of intracellular HSP70 protein was identified using a BX51 confocal microscope (Olympus) and a Cool SNAP CF digital camera (Roper Scientific, Trenton, NJ, USA) and calibrated using RS Image Express software (Roper Scientific).\nMicroarray studies and data analysis\nFour days after FIR irradiation, two control and two FIR-irradiated samples were prepared for microarray hybridization. Total RNA was extracted using a Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer\u2019s protocol. Agilent human 1A ver.2 microarray slides (Agilent Technologies, Palo Alto, CA, USA) were used for the hybridization. The quality of RNA samples was monitored using an Agilent 2100 bioanalyzer (200\u00a0ng each). To produce labeled cRNA (complementary RNA), high-quality RNA was amplified and labeled with Cy5-and Cy3-CTP (Amersham Biosciences, Buckinghamshire, UK) using a Low RNA Input Fluorescent Linear Amplification Kit (Agilent) according to the manufacturer\u2019s protocol. After the amplification and labeling, the dye incorporation ratio was determined using a Nanodrop spectrophotometer, and the ratios were within 10\u201320\u00a0pmol per \u03bcg cRNA, which is the range suggested by the manufacturer for hybridization. For hybridization, an Agilent 60-mer oligo microarray (Rev. 7, SSC Wash\/6-screw hybridization chamber) was used according to the manufacturer\u2019s protocol. Briefly, 750\u00a0ng Cy3-labeled control and 750\u00a0ng Cy5-labeled MPP+-treated sample were mixed and incubated for 17\u00a0h with an SSC-washed microarray slide from an Agilent In\u00a0situ Hybridization Kit. Sample pairs were dye-swapped and processed at the same time. The washed slides were immediately dried under a stream of ultrapure N2 in an ozone-free atmosphere. After drying, the slides were scanned using an Agilent Technologies Microarray Scanner with the PMT setting at 770 for Cy5 and 670 for Cy3, and the raw data were normalized and analyzed using GeneSpring 7.0 software (Silicon Genetics, Santa Clara, CA, USA). For normalization, per spot and per chip intensity-dependent (LOWESS) normalization was used to correct for the intensity-dependent ratio bias12. In addition, the following filters were applied to improve the quality of the data: eliminate saturated signal, eliminate non uniformity of background, eliminate non uniformity of feature, Feature Population Outlier, eliminate low-signal feature of background signal\u00a0+\u00a02.6\u00a0\u00d7\u00a0SD, and eliminate P-value\u00a0<\u00a00.01. Genes were further classified for process and function according to their GO term information (http:\/\/www.godatabase.org).\nStable transfection of HSP70A\nThe HSP70A expression vector pcDNA3-HSP70A containing the cDNA for full-length human HSP70A was a generous gift from Dr. Hector R. Wong (Children\u2019s Hospital Medical Center, Cincinnati, OH, USA). HSP70A cDNA was subcloned into the Xba I and Bam HI sites of pcDNA3.1(\u2212) (Invitrogen). Cells grown on 60-mm dishes were transfected with 8\u00a0\u03bcg of pcDNA3-HSP70A, or pcDNA3.1 (Invitrogen) using Lipofectamine 2000 (Invitrogen) according to the manufacturer\u2019s instructions. The transfected cells were selected with 400\u00a0\u03bcg\/ml G418 (Sigma), and clones formed were collected and maintained separately in medium supplemented with 400\u00a0\u03bcg\/ml G418.\nReverse transcription (RT)-polymerase chain reaction (PCR)\nTotal RNA was extracted using Trizol reagent (Invitrogen) following the manufacturer\u2019s instructions. The concentration and purity of the RNA were determined spectrophotometrically. One microgram of total RNA was reverse-transcribed into first-strand cDNA. Next, oligo(dT12\u201318) primer (Invitrogen), 10\u00a0mM dNTP mix (Invitrogen), 25\u00a0mM MgCl2 (Promega, Madison WI, USA), and 0.1\u00a0M dithiothreitol (Invitrogen) were added, and the mixture was incubated for 2\u00a0min at 42\u00b0C. Next, the RT reaction was performed by adding SuperScript II RT (Invitrogen) and incubating at 42\u00b0C for 50\u00a0min, followed by 70\u00b0C for 15\u00a0min. The reaction was terminated by rapid cooling on ice, after which RNA in the sample was degraded by treatment with RNase H (Invitrogen) at 37\u00b0C for 20\u00a0min. Polymerase chain reaction (PCR) was carried out using a 1-\u03bcl sample of the RT reaction and Ready Mix PCR Master Mix (AB gene, Epsom, Surrey, UK). The analyzed genes and the respective primer sequences were as follows: HSP70A, 5\u2032-TGTTCCGTTTCCAGCCCCCAA-3\u2032 (sense) and 5\u2032-GGGCTTGTCTCCGTCGTTGAT-3\u2032 (antisense); HSP70B, 5\u2032-CTCCAGCATCCGACAAGAAGC-3\u2032 (sense) and 5\u2032-ACGGTGTTGTGGGGGTTCAGG-3\u2032 (antisense); HSP70C, 5\u2032-TTGAGGAGGTGGATTAGGGGC-3\u2032 (sense) and 5\u2032-AGCCTTTGTAGTGTTTTCGCC-3\u2032 (antisense); glyceraldehyde-3-phosphate dehydrogenase (G3PDH), 5\u2032-ACCACAGTCCATGCCATCAC-3\u2032 (sense) and 5\u2032-TCCACCACCCTGTTGCTGTA-3\u2032 (antisense). PCR was carried out using cycles (30 for HSPs and 23 for G3PDH) of 94\u00b0C for 45\u00a0s, 58\u00b0C for 30\u00a0s (all HSPs) or 52\u00b0C (G3PDH), and 72\u00b0C for 90\u00a0s. Finally, primer extension was performed for 10\u00a0min at 72\u00b0C. A 10-\u03bcl sample of each PCR product was separated by electrophoresis on a 1.5% polyacrylamide gel in Tris borate\/EDTA buffer and stained with ethidium bromide.\nQuantitative real-time RT-PCR data analysis\nTo determine the level of HSP70A mRNA, quantitative real-time RT-PCR was carried out using a LightCycler and the Fast Start DNA Master SYBR Green I Kit (Roche). The reaction contained 50\u00a0ng of cDNA and 100\u00a0pmol of each primer in a final volume of 10\u00a0\u03bcl. The gene-specific primers were as follows: HSP70A, 5\u2032-TGTTCCGTTTCCAGCCCCCAA-3\u2032 (sense) and 5\u2032-GGGCTTGTCTCCGTCGTTGAT-3\u2032 (antisense); and \u03b1-actin, 5\u2032-ATAGCACAGCCTGGATAGCAACGTAC-3\u2032 (sense) and 5\u2032-CACCTTCTACAATGAGCTGCGTGTG-3\u2032 (antisense). The concentration of Mg2+ was 3\u00a0mM. In all cases, a first phase of denaturation was performed at 95\u00b0C for 10\u00a0min. Amplification was carried out for cycles of denaturation at 95\u00b0C for 10\u00a0s, hybridization for 10\u00a0s (58\u00b0C for HSP70A or 60\u00b0C for \u03b1-actin), and elongation at 72\u00b0C (20\u00a0s for HSP70A or 10\u00a0s for \u03b1-actin). Product specificity was evaluated by melting curve analysis. Fluorescence data were analyzed using LightCycler Software Ver. 3.5 (Roche). Crossing points were established using the second derivative method. The relative amount of target transcript in the sample was calculated by dividing the amount of target by the amount of internal standard (\u03b1-actin). Results were expressed as the target\/internal standard concentration ratio calculated from the calibration curve. Since the target and internal standard genes had different sequence and amplicon lengths, it was expected that they would show different PCR efficiencies. Therefore, the PCR efficiency (10\u22121\/m, where m is the slope from the calibration curve) was first established for each pair of primers. All reactions were performed in triplicate.\nHSP70 Enzyme-Linked Immuno Sorbent Assay (ELISA)\nHSP70 protein was quantified in cell lysates using a commercially available ELISA kit for human HSP70 (Stressgen). Cells (105 per well) were seeded in 6-well plates (Nunc). After 4\u00a0days, the cells were lysed, and all samples were assayed at optimal dilutions according to the manufacturer\u2019s instructions.\nProtein extraction and Western blotting\nCells (1\u00a0\u00d7\u00a0106) were grown in 60-mm tissue culture dishes (Nunc). After removing the cell culture medium from the culture dishes (Nunc) and washing the cells twice with cold PBS(\u2212), the cells were lysed in lysis buffer (20\u00a0mM Tris\u2013HCl, pH 7.5, 150\u00a0mM NaCl, 1\u00a0mM Na2EDTA, 1% Triton X-100, 2.5\u00a0mM sodium pyrophosphate, 1\u00a0mM \u03b2-glycerophosphate, 1\u00a0mM Na3VO4, and 1\u00a0\u03bcg\/mL leupeptin). Protein levels were measured by the Lowry method [13] using a DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Cell lysate containing 15\u00a0\u03bcg of protein for HSP70 was subjected to Sodium Dodesyl Sulfate (SDS)-polyacrylamide gel electrophoresis. Separated proteins were then transferred from the gel to a polyvinylidene difluoride membrane. After blocking with 5% skim milk in PBS-Tween, the membrane was incubated for 1\u00a0h at room temperature with primary antibody in PBS-T containing 5% skim milk, followed by three 10-min washes with PBS-T. Next, the membranes were incubated for 1\u00a0h at room temperature with horseradish peroxidase-labeled secondary antibody and washed three times for 10\u00a0min with PBS-T. Immunoreactive protein was detected using an ECL plus kit (Amersham Biosciences) and visualization by exposure to Hyperfilm (Amersham Biosciences). The primary antibodies used were rabbit anti-HSP70 (Stressgen), anti \u03b1-actin (Sigma), and the secondary antibody was horseradish peroxidase-conjugated anti-mouse (Zymed Laboratories, South San Francisco, CA, USA) or anti-rabbit IgG (Amersham Biosciences).\nSmall interfering RNA (siRNA)\nWe designed 21-nucleotide siRNAs targeting human HSP70A and HSP70C according to the manufacturer\u2019s instructions (Dharmacon, Lafayette, CO, USA) and corresponding to the sequence 5\u2032-AAGAACCAGGUGGCGCUGAAC-3\u2032. It was not possible to design a siRNA specific to HSP70A because its mRNA was highly homologous to the mRNA for HSP70C (see Table\u00a01). We used siCONTROL Non-Targeting siRNA (Dharmacon; 5\u2032-UAGCGACUAAACACAUCAAUU-3\u2032) as a negative control because it does not match the sequence of any known human or mouse genes. Cells (1\u00a0\u00d7\u00a0104\/well) were plated in 96-well culture dishes (Nunc) and cultured for 24\u00a0h at 37\u00b0C in a 5% CO2 atmosphere. When the cells reached 70\u201390% confluence, they were transfected with 100\u00a0nM siRNAs to HSP70A and HSP70C or control siRNAs complexed with Lipofectamine 2000 (Invitrogen) according to manufacturer\u2019s instructions. The growth medium was removed after 6\u00a0h, and the culture dish was transferred to the FIR incubator. After incubation for 48\u00a0h at 37\u00b0C in a 5% CO2 atmosphere, cell proliferation was assessed by BrdU incorporation.\nTable\u00a01Characterization of Hsp70 (A, B, and C)GeneLocationAmino acid homology to HSP70a (%)mRNA homology to Hsp70A (%)Alternative namesGenBankHsp70An6p21.3100100HSPA1A, Hsp72, Hsp70-1aM11717Hsp70Blq238484HSPA6, Hsp70BX51757Hsp70C6p21.39998HSPA1B, Hsp72, Hsp70-1bM5980aPercent amino acid homology to the HSP70 protein coded by Hsp70A\nStatistical analysis\nData are means\u00a0\u00b1\u00a0SE of replicate samples in single experiments or replicate experiments as described in the figure legends. Student\u2019s t-test was used for comparisons between two groups. Multiple group comparisons were performed by one-way ANOVA, followed by the Tukey\u2013Kramer multiple group comparisons test. All statistical analyses were performed using Statcel 2 software (OMS publishing, Saitama, Japan).\nResults\nFIR irradiation selectively inhibits the growth of specific cancer cell lines\nTo clarify the effect of FIR irradiation on the proliferation of cancer cell lines, we irradiated five cancer cell lines (A431, HSC3, Sa3, A549, and MCF7) with FIR and measured the number of live cells by Trypan blue dye exclusion. Although the proliferation of HSC3, Sa3, and A549 cells were significantly suppressed on day 8 of culture (45.75%, 74.63%, and 65.79%, respectively), FIR irradiation had little effect on the growth of A431, or MCF7 cells (Fig.\u00a01).\nFig.\u00a01Effect of FIR irradiation on cell growth of five cancer cell lines. Cells (1\u00a0\u00d7\u00a0105) were plated in 24-well dishes and cultured for 8\u00a0days. Cell numbers were counted every other day. Although proliferation of HSC3, Sa3, and A549 cells was suppressed on day 8 of culture, FIR irradiation had little effect on the proliferation of A431 and MCF7 cells. *P\u00a0<\u00a00.05 vs. unirradiated control cells\nMicroarray analysis and extraction of candidate gene for FIR control\nSeveral gene families showed high correlations between endogenous expression (signal in the microarray) and the growth rate, proteins involved in cell proliferation, cytoskeletal proteins, cell cycle components, and protein kinases. Especially, in the analysis of stress factor, some genes encoding the HSPs which were well known that they participate in the cellular resistance to stress were focused. Of the 35 HSP genes on the microarray, HSP70 showed the highest correlation with the growth rate (Fig.\u00a02a, b). We examined this further by real time RT-PCR, and we found that HSP70 is most highly expressed in A431 cells at all stages of the cell cycle. Lower levels of expression were found in HSC3 and Sa3 cells (Fig.\u00a02c). We did not find a statistically significant difference between the expression of this gene between control (unirradiated) and FIR-irradiated A431, HSC3, or Sa3 cells. An ELISA for HSP70 in untreated cells showed similar results, specifically, that the expression of HSP70A was higher in A431 cells than in the HSC3 or Sa3 cells (Fig.\u00a02d).\nFig.\u00a02Relationship between the expression of HSP70 and the suppression of proliferation by FIR irradiation. (a) Correlation between the signal from the microarray for the HSP family members in the control group and the correlation coefficient. (b) Correlation between the signal from the microarray for HSP70 and the growth rate in the control and the FIR-treated cells. (c) Expression of HSP70A mRNA in A431, HSC3, and Sa3 cells after 0.5, 1, 2, 4, 8, and 12\u00a0days of culture. The level of these mRNAs was determined by quantitative real-time RT-PCR. Bars represent the means and error bars the SD from three independent experiments. (d) Level of HSP70 protein on day 4 of culture. The concentration of HSP70 protein was measured by ELISA. Bars represent the mean, and error bars the SD (n\u00a0=\u00a04). *P\u00a0<\u00a00.01 vs. to A431 cells\nIncreased expression of HSP70A improves the survival of HSC3 cells after a limited exposure to FIR\nTo directly determine whether HSP70 can protect cells from FIR-induced cell death, we developed A431 and HSC3 cell lines stably expressing human HSP70A (A431-HSP70A and HSC3-HSP70A cells, respectively). Control cells were transfected with empty pcDNA3.1 (A431-Neo and HSC3-Neo; Fig.\u00a03a, b). In our initial experiments, we found that exposure of HSC3 and Sa3 cells but not A431 cells to limited FIR causes G2\/M arrest and induces partial hypertrophy to necrosis (data not shown). To determine whether increased expression of HSP70A confers protection against FIR, cell survival was examined in FIR-irradiated A431-HSP70, A431-Neo, A431-wt, and HSC3-HSP70, HSC3-Neo, and HSC3-wt cells. We found that over expression of HSP70A increased cell proliferation in A431 and HSC3 cells. Furthermore, the proliferation of FIR-irradiated and control (unirradiated) A431-HSP70A cells was similar (Fig.\u00a03c). The survival rate after 6\u00a0days of FIR irradiation was significantly higher in HSC3-HSP70A cells than in HSC3-Neo or HSC3-wt cells. In addition, the proliferation of FIR-treated HSC3-HSP70A cells was similar to that of control HSC3-HSP70A cells. BrdU incorporation was significantly higher in FIR-irradiated or control A431-HSP70A cells than in A431-Neo or A431-wt cells (Fig.\u00a03d). Although BrdU incorporation of FIR-irradiated HSC3-wt and HSC3-Neo cells was lower than in unirradiated HSC3-wt and HSC3-Neo cells, it was similar in FIR-irradiated and unirradiated HSC3-HSP70A cells. Surprisingly, BrdU incorporation by HSC3-HSP70A cells was significantly higher in both FIR-irradiated and unirradiated cells. These data demonstrate that over expression of HSP70A in HSC3 and A431 cells did not affect their proliferation, and also their morphology, even when they were irradiated with FIR (Fig.\u00a03e).\nFig.\u00a03Over expression of HSP70 prevents the suppression of cell growth and induction of cell hypertrophy by FIR. (a) Real-time RT-PCR of HSP70 expression. Cells overexpressing HSP70 were established. (b) Representative Western blots demonstrating increased expression of HSP70 in stably transfected A431 and HSC3 cells. Lane 1, wild-type cells; lane 2, cells transfected with empty pcDNA3.1(+); lane 3, cells transfected pcDNA3.1-HSP70A. (c) Cell proliferation of empty vector-transfected and HSP70-overexpressing cells. Cell proliferation was the same in FIR-irradiated and untreated A431 cells overexpressing HSP70. In contrast, in HSC3 cells, over expression of HSP70 prevented FIR inhibition of cell proliferation. (d) BrdU incorporation in wild-type, empty vector-transfected, and HSP70-overexpressing cells treated with FIR. (e) Hematoxylin and eosin staining of wild-type, empty vector-transfected, and HSP70A-overexpressing A431 and HSC3 cells after 5\u00a0days of transfection. Note that the morphology of HSP70A-transfected HSC3 cells was unaffected by FIR irradiation\nKnocking down HSP70A by using siRNA improves the deth of HSC3 cells after a limited exposure to FIR\nWe next examined the effect of knocking down HSP70A and HSP70C mRNA and HSP70 protein expression using siRNA. Transfection with HSP70A\/C siRNA effectively decreased HSP70A and HSP70C mRNA (Fig.\u00a04a) and protein levels (Fig.\u00a04b) in both A431 and HSC3 cells without affecting the level of HSP70B mRNA or protein. HSP70A\/C siRNA did not suppress BrdU incorporation in unirradiated A431 cells, but it suppressed BrdU incorporation in cells irradiated with FIR (Fig.\u00a04c). Similarly, the HSP70A\/C siRNA enhanced the suppression of BrdU incorporation by FIR irradiation. FIR irradiation also significantly suppressed BrdU incorporation in HSC3 cells transfected with the negative control siRNA (Fig.\u00a04c). These results indicate that a decrease in HSP70 protein mediates the ability of limited FIR to inhibit the proliferation of A431 and HSC3 cells.\nFig.\u00a04Inhibition of HSP70 expression and BrdU incorporation in A431 and HSC3 cells after 48\u00a0h of transfection with 100\u00a0nM HSP70 siRNA. (a) RT-PCR of HSP70A, HSP70B, and HSP70C in A431 and HSC3 cells after treatment with siRNAs. C, wild-type control cells; NC, cells transfected with negative control siRNA; siRNA, cells transfected with HSP70-siRNA. (b) Western blot analysis of HSP70 protein expression in A431 and HSC3 cells. The lanes are as shown in panel A. (c) Inhibition of HSP70 suppressed FIR-induced DNA synthesis in A431 cells. Cells were transfected with 100\u00a0nM HSP70 siRNA and grown for 48\u00a0h. Bars represent the mean and error bars the SD (n\u00a0=\u00a05). *P\u00a0<\u00a00.05). In HSC3 cells, the random siRNA slightly suppressed DNA synthesis, whereas the HSP70 siRNA strongly suppressed DNA synthesis\nDiscussion\nIn the present studies, we showed that FIR suppresses the proliferation of HSC3, Sa3, and A549 cell lines. Two other cell lines, A431 and MCF7, showed almost no growth arrest in response to FIR. The effect of FIR does not seem to be related to the cell type or source, because these three cell lines have different origins: HSC3 is from a tongue squamous cell carcinoma, Sa3 is from a gingival squamous cell carcinoma, and A549 is from a pulmonary adenocarcinoma. FIR was found to cause hypertrophy without apoptosis in all three sensitive cell lines, although there was a slight increase in necrosis in the Sa3 cells by histological observation. In addition, the expression of apoptosis-related genes was unchanged in the FIR-sensitive cells by microarray analysis. Overall, FIR seemed to cause changes in the cytoskeleton, suppress proliferation, and induce some necrosis without apoptosis. These results raise the question of why proliferation of A431 and MCF7 was not suppressed by FIR. Perhaps something in certain cancer cell lines is present for resistance to FIR. We then focused on genes encoding the HSPs, which are well known to participate in the cellular resistance to stress. We found that HSP70 showed the highest correlation with the growth rate of cancer cells affected by FIR in 35 HSP genes on the microarray system.\nThe expression of HSP70A mRNA was higher in FIR-insensitive A431 and MCF7 cell lines than in FIR-sensitive HSC3, Sa3, and A549 cell lines, although the gene expressions were not induced by FIR. This finding was confirmed by analysis of the expression of HSP70 mRNA and protein with real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. These results suggested that the effect on body temperature range by FIR, suppressing the proliferation of some cancer cells, may be controlled by the basal expression level of HSP70A. To test this hypothesis, the effects of overexpression and suppression of the HSP70 gene were studied. As a result, the overexpression of HSP70 inhibited FIR-induced growth arrest in HSC3 cells and an HSP70 siRNA inhibited the proliferation of A431 cells by FIR. These results confirmed that the effect on body temperature range by FIR, suppressing the proliferation of some cancer cells, is controlled by the basal expression level of HSP70A.\nHSP70 appears to be present in a variety of normal cell types and its expression may be induced by several stressors, such as hyperthermia, cardiac ischemia, infection, UV radiation, endotoxin, and nitric oxide to suppress or denature any foreign protein and restore an injured protein from lethal effects [12]. HSP70 seems to be particularly important for cancer cells. In human breast cancer, the expression of HSP70 correlates with increased cell proliferation, poor differentiation, lymph node metastases, and poor therapeutic outcome [13]. In\u00a0vivo animal studies and clinical trials have revealed that hyperthermia may serve as a powerful tool in the treatment of prostate cancer [14\u201319]; at the cellular level, hyperthermic stress induces HSPs. Moreover, chemotherapeutic agents such as cisplatin, adriamycin, and bleomycin, as well as \u03b3-radiation induce HSPs. HSP70 participates in cytoprotection and is associated with cellular resistance to lethal external effects [17\u201320]. However, in the present study, HSP70 was never induced by FIR. These results suggested that FIR has anti-tumor activity without inducing HSP70 as an anti-stress factor. This characteristic indicates that FIR may be suitable for medical treatment.\nNeutralization of the function of HSP70 or inhibition of its expression may inhibit tumor growth and\/or sensitize tumor cells to chemotherapeutic agents without affecting normal cells. Furthermore, inhibition of HSP70 expression reduces cell survival. Conversely, microinjection of an HSP70-neutralizing antibody increases the vulnerability of cells to damage by sub-lethal temperatures [21, 22]. This finding was consistent with our results for FIR and the suggestion of HSP70 as the acting mechanism. However, FIR may be regarded as a sub-lethal stress that avoids an anti-stress reaction in some cancer cells.\nThis is the first report suggesting that HSP70 may not rescue cells from the effects of FIR, although HSP70 is known to rescue cells from tumor necrosis, factor-induced caspase-independent programed cell death, heat shock, serum starvation, and oxidative stress [23]. This finding suggests that the effect of FIR may be a sub-lethal stress for cancer cells protected by HSP70 as an anti-stress protein. In other words, FIR may be a very effective medical treatment for some cancer cell lines that have a low level of HSP70. Furthermore, if the level of HSP70 in any cancer of a patient is measured, the effect of medical treatment by FIR on the cancer may be predicted.\nConclusion\nIt was found here for the first time the effect on body temperature range by FIR in several cancer cell lines in\u00a0vitro, which is controlled by endogenous HSP70 to protect cells from FIR-induced growth arrest. This finding suggests that FIR may be a very effective medical treatment for some cancer cell lines that have a low level of HSP70. Furthermore, if the level of HSP70 in any cancer of a patient is measured, the effect of medical treatment by FIR on the cancer may be predicted.","keyphrases":["far-infrared radiation (fir)","heat shock protein (hsp) 70","human cancer cell","over expression","sirna","co2 incubator"],"prmu":["P","P","P","P","P","P"]}
{"id":"Support_Care_Cancer-3-1-2071950","title":"Dying at home or in an institution: perspectives of Dutch physicians and bereaved relatives\n","text":"Introduction Previous studies have shown that most people prefer to die at their own home. We investigated whether physicians or bereaved relatives in retrospect differently appreciate the dying of patients in an institution or at home.\nIntroduction\nThe two most important events in life, that is, birth and death, relatively often occur at home in The Netherlands. Over the period 1995\u20132000, around one third of all Dutch births were home deliveries [1]. Further, in 2001, about 40% of all deaths have been found to occur at home, 40% in hospital, and about 20% in nursing homes [2]. Medical care in The Netherlands is strongly founded on home-based general practice. General practitioners or family physicians provide all basic medical care to outpatients and serve as a gate door to specialized care for patients with more complex health problems.\nIn 2003, around 45% of deaths due to cancer occurred at home in The Netherlands [3]. In the UK, the percentage of cancer deaths at home is lower and falling, from 27% in 1994 to 22% in 2003 [4]. In a study of the place of death of cancer patients in the Houston area, USA, 35% died at home [5]. End-of-life care is thus, especially in The Netherlands, rather often provided in the home situation by general practitioners, home-care nurses, and informal caregivers. Research has shown that dying at home is usually preferred over dying in an institution, in The Netherlands as well as in other countries [6]. Such a preference seems to be predominantly shaped by whether or not people have sufficient informal carer resources [7]. In contrast, people who are concerned about the medical management of their symptoms may appreciate the easy access to professional caregivers in an institutional setting. Concerns about burdening relatives have also been found to contribute to a wish to die in an institution [7]. The finding that people\u2019s wish to die at home becomes less predominant when death is nearing may be related to such concerns, as heavily burdening relatives with care duties is one of the most commonly recognized threats to a peaceful dying process [6]. A gradual shift in preference may also be due to the growing awareness of seriously ill people that dying sometimes involves problems and symptoms that are best treated by professional caregivers in an institutional setting. Finally, differential experiences of services also influence people\u2019s preferences [7].\nEnabling people to make genuine choices about their end-of-life care and to die at the place they prefer is often seen as a major challenge to current end-of-life care [8\u201310]. Home-care patients have been reported to have more control over the effects of their illness, medical care, and treatment received than patients receiving institutional care [11]. However, it is unclear if the relatively high home death rate in The Netherlands is really beneficial to the quality of death and dying. Little is known about the experiences of dying patients and their caregivers in different health care settings. We investigated to what extent dying in an institution or at home involves differences in care and its appreciation by physicians and bereaved relatives.\nMaterials and methods\nPatients\nThis study concerns a sub-sample of a cohort of 128 patients with advanced breast cancer, colorectal cancer, ovary cancer, lung cancer, or prostate cancer, who were followed for a study on end-of-life care needs and practices during the last stage of their lives [12]. Of these patients, 103 died within the time frame during which we were able to approach attending clinical specialists, general practitioners, and bereaved relatives for an after-death data collection. In 102 cases, physicians were willing to fill out a written questionnaire on the medical treatment and dying process of these patients: Questionnaires were filled out by clinical specialists in 30 cases, by general practitioners in 17 cases, and by both in 55 cases. Relatives who had been closely involved with the patient could be contacted in 91 cases; 63 gave their written consent to be personally interviewed at their own home. The reason for not participating was most often that relatives expected participation to be too burdensome. The patients for whom bereaved relatives participated in an interview had on average a longer disease duration (33 vs 21\u00a0months; p\u2009=\u20090.03) and were more often women (59 vs 28%; p\u2009=\u20090.004) than other patients.\nThe Medical Ethical Committee of the Erasmus MC, University Medical Center Rotterdam approved the research protocol.\nQuestionnaire for attending physicians\nAs soon as we were informed of the death of a patient, physicians received a self-developed written questionnaire addressing the end-of-life medical treatment and decision-making. The median time between the patient\u2019s death and filling out the questionnaire was 12.5\u00a0weeks (range, 1 to 59\u00a0weeks). The questions on end-of-life decision-making were based upon questionnaires that have proven to yield valid information in previous studies [2, 13]. In cases where both a clinical specialist and a general practitioner filled out a questionnaire, the information about the patients\u2019 anti-tumor treatment history, medical decision-making, and any \u2018negative\u2019 aspects of the dying process were considered to be additive.\nInterview with bereaved relatives\nInterviewers were trained to carry out interviews with vulnerable people during a 2-day course. The interviews were on average held 4.9\u00a0months after the patient\u2019s death (range, 0.8\u20139.5\u00a0months). The interview schedule included the following topics: personal characteristics of the bereaved relatives, such as age, sex, and relationship with the patient; symptoms of the patient, that is, loss of appetite, pain, fatigue, dyspnoea, nausea, mouth or mucous problems, incontinence, bedsores, confusion, anxiety, and depression; and whether or not the patient was treated for these symptoms. The patient symptom list was based upon the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire C30 [14, 15] and the Problems and Needs in Palliative Care questionnaire [16, 17] and completed with psychological symptoms. Further, contacts with health care professionals, admission to hospital or other care institutions, the actual and preferred place of death, and problems in end-of-life care during the last 3\u00a0days of life were assessed using scales from the Voices of Informal Carers-Evaluation of Services (VOICES) questionnaire [18]. Finally, we asked the bereaved relatives about the burden of care giving. We adapted several questions from the Caregiver Reaction Assessment to make them applicable for an after-death interview with bereaved relatives [19].\nStatistical analyses\nWe compared the characteristics of patients who died in an institution with the characteristics of patients who died at home. Student\u2019s t tests and \u03c72 tests were used to assess the statistical significance of differences between both groups. All analyses were done using the Statistical Package for the Social Sciences version 11.0.\nResults\nThe characteristics of all 103 deceased patients and of 63 patients for whom an after-death interview with a bereaved relative was available are presented in Table\u00a01. The mean age at death of all patients was 65\u00a0years; 48 patients (47%) were women. The most common types of cancer were lung cancer (48 patients) and breast cancer (19 patients). Of all 103 patients, 49 died within an institution: 35 patients died at a hospital department, 6 patients died after having been admitted to a department within a hospital, nursing home, or home for the elderly that was specialized in care for dying patients, 4 patients died within a general department of a nursing home or home for the elderly, and 4 patients died in a hospice. The remaining 54 patients died at home or in a home-like situation: 52 patients died in their own homes, 1 patient died in the home of a son, and 1 patient died during a holiday on a boat. The only significant difference between patients dying in an institution and patients dying at home concerned the percentage who had lived with a partner, which was 63% for patients who died in an institution and 83% for patients who died at home. All other characteristics of patients and relatives that are listed in Table\u00a01 were similar in both groups. The characteristics of the group of patients for whom a relative could be interviewed were also similar to the characteristics of the total group. The interviewed relative was the patient\u2019s spouse in 39 out of the 63 cases; in 19 cases, it was a son or daughter, and in 5 cases, another relative. The majority of relatives were women, and their mean age was 55\u00a0years.\nTable\u00a01Characteristics of patients and bereaved relativesCharacteristicsAll deceased patients (N\u2009=\u2009103)Deceased patients for whom a relative was interviewed (N\u2009=\u200963)Patients\u00a0Age at death, in years [mean (SD)]65 (11)64 (11)\u00a0Sex [n\/N (%)]\u00a0\u00a0Female48\/103 (47)37\/63 (59)\u00a0Living arrangement [n\/N (%)]\u00a0\u00a0With partner76\/103 (74)47\/63 (75)\u00a0Education [n\/N (%)]\u00a0\u00a0Lowa71\/102 (70)41\/63 (65)\u00a0Religion [n\/N (%)]\u00a0\u00a0Religious62\/103 (60)40\/63 (63)\u00a0Urbanization [n\/N (%)]\u00a0\u00a0Living in urban areab72\/103 (70)46\/63 (73)\u00a0Primary tumor site [n\/N (%)]\u00a0\u00a0Lung48\/103 (47)31\/63 (49)\u00a0\u00a0Breast19\/103 (18)14\/63 (22)\u00a0\u00a0Other36\/103 (35)18\/63 (29)\u00a0History of anti-tumor treatment [n\/N (%)]\u00a0\u00a0Surgery45\/102 (44)26\/62 (42)\u00a0\u00a0Chemotherapy\/hormone therapy84\/101 (83)51\/61 (84)\u00a0\u00a0Radiotherapy54\/101 (54)36\/61 (59)\u00a0Place of death\u00a0\u00a0Institution [n\/N (%)]49\/103 (48)29\/63 (46)\u00a0\u00a0\u00a0Hospital35\/4924\/29\u00a0\u00a0\u00a0End-of-life care unit6\/492\/29\u00a0\u00a0\u00a0Hospice4\/493\/29\u00a0\u00a0\u00a0Nursing home\/home for the elderly4\/49\u2013\u00a0\u00a0At home or in a home-like situation [n\/N (%)]54\/103 (52)34\/63 (54)\u00a0\u00a0\u00a0At patient\u2019s own home52\/5432\/34\u00a0\u00a0\u00a0Elsewhere2\/542\/34Bereaved relatives\u00a0Age at the time of dying of the patient, in years [mean (SD)]\u201355 (15)\u00a0Sex [n\/N (%)]\u00a0\u00a0Female\u201340\/63 (63)\u00a0Relative was patient\u2019s [n\/N (%)]:\u00a0\u00a0Spouse\u201339\/63 (62)\u00a0\u00a0Son or daughter\u201319\/63 (30)\u00a0\u00a0Other relationship\u20135\/63 (8)aLow education: lower vocational, lower secondary general education, or primary schoolbLiving in an urban area: patients who were treated as an outpatient in a hospital inside the Rotterdam area\nSome aspects of medical care are shown in Table\u00a02. In both groups, physicians reported that about two thirds of the patients had died peacefully. Patients who died in an institution had less often (37%) been ready to die than patients who died at home (71%). Agitation was a common problem during the dying process. Most patients in both settings had been unconscious before death. Physicians had discussed a number of end-of-life decisions each with about one third of their patients: This holds for decisions to forgo potentially life-prolonging treatment, intensive treatment of pain, and active euthanasia. Sedation was the only treatment option that was discussed slightly more often with patients who died in an institution. Life had actually been shortened due to the forgoing of potentially life-prolonging treatment or to the use of potentially life-shortening drugs in about one third of all cases in both settings. Such medical decisions were usually made with clear consent of the patient, and life was generally shortened by less than 1\u00a0week.\nTable\u00a02Dying in an institution or at home: the physician\u2019s perspective\u00a0Place of deathP value \u03c72 testIn institution (N\u2009=\u200949)At home (N\u2009=\u200954)N (%)Physician\u2019s evaluation of dying processa\u00a0Patient died peacefully28\/41 (68)29\/42 (69)0.94\u00a0Patient was able to say goodbye to relatives26\/40 (65)33\/44 (75)0.32\u00a0Patient was ready to die15\/41 (37)31\/44 (71)0.002\u00a0Patient died suddenly and unexpectedly13\/42 (31)14\/44 (32)0.93\u00a0Patient\u2019s dying was preceded by period of agitation25\/40 (63)29\/41 (71)0.43\u00a0Patient\u2019s dying was preceded by period of unconsciousness30\/39 (77)26\/42 (62)0.14Physician discussed with patient medical decisions that could shorten life\u00a0Forgoing treatment13\/49 (27)15\/50 (30)0.70\u00a0Intensified pain treatment16\/49 (33)16\/50 (32)0.95\u00a0Sedation15\/49 (31)8\/50 (16)0.09\u00a0Euthanasia18\/49 (37)19\/50 (38)0.90Life was possibly shortened due to\u00a0Forgoing treatment15\/47 (32)14\/52 (27)0.59\u00a0\u00a0With clear consent of the patient11\/1212\/13\u00a0\u00a0Life was shortened by more than 1\u00a0week4\/110\/9\u00a0Use of potentially life-shortening drugs16\/46 (35)13\/45 (29)0.55\u00a0\u00a0With clear consent of the patient12\/149\/10\u00a0\u00a0With the explicit goal of shortening life1\/164\/13\u00a0\u00a0Life was shortened by more than 1\u00a0week1\/132\/10Information as provided by clinical specialist (n\u2009=\u200985) and\/or general practitioner (n\u2009=\u200972). In case of conflicting answers concerning history of treatment, the information as provided by the clinical specialist prevailed. In all other cases, both sources were considered valid.aStatement was considered true if neither the clinical specialist nor the general practitioner considered it untrue.\nBereaved relatives reported in 25 out of 63 cases that the patient had preferred to die at home (Table\u00a03). Two patients had preferred to die in the hospital, 3 in a hospice, 2 elsewhere, and for 31 patients, the relative indicated that the patient had no clear preference. Most patients who had a preference died at the place they preferred, except for five patients who preferred home but died in the hospital. Patients who died in an institution had stayed there for on average 7\u00a0days. The number of transfers during 3\u00a0months before death was larger for patients who died in an institution (mean 1.9) than for patients who died at home (mean 1.2), and the number of medical specialties that were involved with the patient was also larger in institutions (mean 2.2) than at home (mean 1.8). Care involved on average six disciplines (medical and non-medical), both in institutions and at home.\nTable\u00a03Preferred and actual place of deathPreferred place of deathaActual place of deathTotal (N\u2009=\u200963)In institutionAt homeHospital (N\u2009=\u200924)Hospice\/end-of-life care unit (N\u2009=\u20095)At patient\u2019s own home (N\u2009=\u200932)Elsewhere (N\u2009=\u20092)Hospital22Hospice33At home52025Other place112No (clear) preference16112231aInformation as provided by bereaved relative\nDuring the last 3\u00a0days of life, a large proportion of patients in both settings suffered, according to the reports of bereaved relatives, from fatigue, loss of appetite, pain, dyspnoea, and mouth or mucus problems (Table\u00a04). Patients dying in an institution were somewhat more often incontinent and more often suffered from anxiety. Patients dying at home slightly more often had bedsores. There were no statistically significant differences between the settings in the degree to which symptoms were addressed with some form of medical treatment, except for loss of appetite, for which 6 of 22 patients who died in an institution received treatment, but only 2 of 30 patients who died at home (p\u2009=\u20090.04). The bereaved relatives\u2019 evaluation of end-of-life care was in general positive in both settings. Some relatives of patients who died in an institution felt they had not sufficiently been involved in the decision-making, whereas all relatives in the other group were satisfied about their level of involvement.\nTable\u00a04Dying in an institution or at home: perspective of bereaved relative\u00a0Place of deathP value t testP value \u03c72 testIn institution (N\u2009=\u200929)At home (N\u2009=\u200934)Number of days patient stayed at place of death [mean (SD)]7 (6)59 (35)0.000Number of transfers [mean (SD)]1.9 (1.7)1.2 (1.3)0.047Number of disciplines involved with patient [mean (SD)]6.1 (2.3)5.8 (1.9)0.64Number of clinical specialties involved with patient [mean (SD)]2.2 (1.2)1.8 (0.8)0.011Patient [n (%)]\u00a0Was fatigued22 (79)29 (88)0.33\u00a0Had loss of appetite22 (76)30 (88)0.20\u00a0Had pain20 (69)29 (85)0.12\u00a0Had dyspnoea18 (62)17 (52)0.40\u00a0Had mouth or mucous problems18 (62)16 (49)0.28\u00a0Was incontinent20 (69)13 (38)0.015\u00a0Had nausea9 (32)10 (30)0.88\u00a0Had bedsores5 (17)13 (38)0.07\u00a0Was confused14 (48)10 (29)0.12\u00a0Suffered from anxiety12 (41)5 (15)0.021\u00a0Was depressed3 (10)2 (6)0.54Evaluation of care during the last 3\u00a0days of life [n (%)]\u00a0Assistance with personal care was sufficient27 (93)32 (94)0.87\u00a0Nursing care was sufficienta26 (90)33 (97)0.23\u00a0Relative was involved in decision-making25 (89)33 (97)0.22\u00a0Relative was sufficiently involved in decision-making24 (89)33 (100)0.049\u00a0Patient might have disagreed with medical decision(s)4 (15)1 (3)0.10\u00a0Relative disagreed with medical decision(s)7 (24)7 (21)0.78\u00a0It had been clear that patient was dying18 (67)26 (79)0.29aIncluding patients who did not need professional nursing care\nFurther, of the bereaved relatives who had cared for a patient who died in an institution, 76% reported that it had significantly affected their own social life; this percentage was 94% for relatives who had cared for a patient who died at home (Table\u00a05). A substantial number of relatives had only received some or no support from other family members (38%), and the number of relatives who reported that their own health had been affected by caring for the patient was also quite large (38%). Caring for a dying relative rarely yielded financial problems. Nearly all relatives thought it very important that they had been involved in caring for the patient.\nTable\u00a05Dying in an institution or at home: experiences of bereaved relatives\u00a0Place of deathP value \u03c72 testIn institution (N\u2009=\u200929)At home (N\u2009=\u200934)n (%)How often did you see patient in the last months of life?0.78\u00a0Every day24 (83)29 (85)\u00a0Less than daily5 (17)5 (15)Did caring for patient affect your own social life?0.068\u00a0Yes22 (76)32 (94)\u00a0No7 (24)2 (6)Did caring for patient result in financial problems?0.17\u00a0Yes4 (14)1 (3)\u00a0No25 (86)33 (97)Did you receive support from your family in caring for patient?1.0\u00a0Much support16 (55)23 (68)\u00a0Some or no support13 (45)11 (32)Did caring for patient affect your own health?0.62\u00a0Yes12 (41)12 (35)\u00a0No17 (59)22 (65)Did caring for patient cost you a lot of energy?0.96\u00a0Often or all the time15 (52)15 (44)\u00a0Rarely or sometimes14 (48)19 (56)How important was being involved with care for patient for you?0.65\u00a0Very important26 (90)32 (94)\u00a0Important3 (10)2 (6)\nDiscussion\nMedical care for patients who are in the last stage of life has received much attention during the past decade in The Netherlands. The organization of health care services for terminally ill patients has greatly expanded during a 5-year period from 1998 through 2003, during which the government financially supported six university-based centers for the further development of end-of-life care. After this period, the government took the position that, whereas death and dying are common events, the provision of end-of-life care should be part of the professional skills of all physicians, including general practitioners providing end-of-life care to patients who are staying at home. For complex problems, general practitioners and other physicians can now, in many places, seek support from expert teams [20\u201322].\nOf the 103 patients in this study, who were, at inclusion, all treated as outpatients by a clinical specialist, about half died in their own home. Death in an institution mostly concerned a hospital, which was probably not, for all patients, foreseen as the place of dying. Patient characteristics did not clearly determine the place of dying in our study. Whether or not patients lived with a partner was the only variable that was significantly associated with the place of dying. Having access to informal care support is invariably found to be a strong determinant of being able to die at home [4]. Other factors that have been found to affect rates of dying at home are the health status and emotional capacity of the main carer, the availability and use of home-based end-of-life care services, the need for specialist symptom control, tumor type, distance to inpatient services, gender and age of the patient, the patient\u2019s socio-economic status, and strength and visibility of patient or carer preferences concerning the place and circumstances of dying [4, 23\u201326]. Obviously, the number of cases in our study was limited, and we did not assess all possibly relevant factors, which precludes firm conclusions on determinants of the place of dying. However, whereas access to the available public end-of-life care services, both institutional and home-based, is virtually unlimited in The Netherlands and financially covered by either private or public insurance, care needs of patients are probably a predominant factor.\nThe availability of in- and outpatient end-of-life care services is probably another important determinant of place of death. The number of patients who died in a specialized end-of-life care service in our study was limited. National statistics on the place of dying does not include hospices or end-of-life care units as a separate category. However, the percentage of cancer deaths inside such services as found in our study is probably comparable to the percentage in the total Dutch population. Recently, the number of beds in specialized end-of-life care services in The Netherlands has been rapidly increasing [27]. It is, therefore, likely that the proportion of cancer deaths in end-of-life care services will further increase in the coming years. However, the extent to which dying in an end-of-life care service will substitute dying in a hospital or dying at home is hard to predict. Probably, institutional end-of-life care services address very diverse needs of dying patients and their caregivers. Further, financial and other incentives that are aimed at setting the course for the supply of end-of-life care services may affect developments concerning the place of dying and end-of-life care as well [27].\nIt is remarkable that a substantial number of patients in our study did not have a clear preference concerning their place of dying. Obviously, we only have information about the preferences that patients had discussed with their interviewed relative. Patients may also have had wishes that relatives were not aware of. Nevertheless, the data in our study suggest that the place of dying is not a great concern for a substantial number of terminally ill cancer patients in The Netherlands.\nIn general, we found few differences in the evaluation by physicians and bereaved relatives of the dying phase of patients who died in an institution and patients who died at home. Patients who died in an institution were, according to their physicians, less often ready to die, which may be due to the fact that hospitalization is typically forgone in patients who are expected to die at short notice. A sudden and more rapid deterioration than expected may also have been among the reasons to admit patients to hospital shortly before dying in some cases. We did not find substantial differences between both settings in most end-of-life decision-making characteristics. Relatives in both settings quite often (21\u201324%) stated that they had not agreed with the decision-making. Relatives of patients who died in an institution somewhat less often than other relatives felt that they had been sufficiently involved in the decision-making. Dissatisfaction of relatives with end-of-life decision-making in an institutional setting has been described elsewhere too [28\u201330] and has been attributed to many factors, such as a lack of time of professional caregivers, lack of skills in communication, failure to recognize end-of-life decision-making as a subject that could be discussed, ethical barriers, and the lack of emotional support for relatives [28\u201332]. It is unclear if our finding that relatives of patients who died outside an institutional setting more often feel satisfied about their involvement in the decision-making process is due to better communication in the home situation. However, the general practitioner, who often has a longstanding relationship with patients and their families, typically plays a key role in end-of-life care at home and may be better able to adequately communicate with family than institutional caregivers [33]. Medical decision-making may also be less complex or controversial for patients who die at home.\nThe possibly rather complex decision-making in institutions is not associated with a higher prevalence of most symptoms. Fatigue, pain, and dyspnoea were among the most common symptoms in both settings, as has been found elsewhere too [34]. Incontinence was more common among patients dying in an institution, and this also holds for anxiety, which was reported as a problem for almost half of all patients dying in an institution. Incontinence and mental status have elsewhere also been found to be associated with a need of nursing home care [35]. In contrast, bedsores were more common at home.\nVirtually all relatives in both settings were satisfied about the personal and nursing care that had been provided to their dying relative. This may, to some extent, be indicative of the relative quality of end-of-life care in institutions and at home, but using satisfaction as an absolute indicator of the quality of palliative care services is quite problematic [36]. Further, most relatives were closely involved and appreciated their personal involvement very much, both for patients who died in an institution and patients who died at home. Nevertheless, caring for a dying relative often affected their own social life, especially when dying occurred at home, and took a lot of energy.\nOur study has several limitations. Firstly, the patients\u2019 perspectives on their own dying process is lacking. Bereaved family members\u2019 assessments are known to sometimes disagree with those of the patients\u2019, especially for subjective aspects such as psychological symptoms and pain [37]. This is also true for physicians\u2019 assessment of their patients\u2019 condition. Secondly, our group of patients is probably not representative for all patients dying from one of the five major types of cancer because patients with a very poor health status and patients who died shortly after the diagnosis of incurable cancer were not included in the cohort study upon which the data collection for this study was based. Moreover, data from bereaved relatives could only be obtained for 61% of all patients, which may have resulted in an overestimation of the degree of satisfaction of relatives. The fact that patients of whom no bereaved relative participated in the study had a shorter disease duration at inclusion than other patients suggests that a rapidly progressive disease process makes it more difficult for bereaved relatives to talk about the last phase of life of the patient.\nIn summary, we did not find major differences in the process and quality of dying between institutional settings and the home setting. Most patients with clear preferences concerning their place of dying were able to die at their preferred place. In about half of all cases, relatives were not aware of any preference of the patient concerning the place of dying, which is apparently not a major concern for many people. We conclude that the current situation in The Netherlands, in which the place of dying is mainly determined by the availability of informal caregivers at home and the care needs of the patient, involves no major threats to the process and quality of dying.","keyphrases":["place of death","cancer patients","end-of-life care","quality of dying"],"prmu":["P","P","P","P"]}
{"id":"Int_Urogynecol_J_Pelvic_Floor_Dysfunct-4-1-2335287","title":"Functional outcome after sacrospinous hysteropexy for uterine descensus\n","text":"The study aimed to evaluate urogenital symptoms, defecatory symptoms and quality of life before and after a sacrospinous hysteropexy for uterovaginal prolapse. Seventy-two women with symptomatic uterovaginal prolapse were treated with sacrospinous hysteropexy. Before and after surgery, urogenital and defecatory symptoms and quality of life were assessed with a validated questionnaire. Anatomical outcome was assessed by means of pelvic examination before and after surgery. The mean follow-up time was 12.7 months. Scores on all domains of urogenital symptoms and defecatory symptoms, except for the pain and fecal incontinence domain, improved significantly. Also, quality of life improved on all domains. No major complications were encountered.\nIntroduction\nIn the current debate on the optimal surgical treatment of a uterine descent, several vaginal and abdominal techniques have been described. In case of a vaginal vault prolapse, the sacrospinous ligament fixation has proven to be an effective treatment [1]. The sacrospinous ligament fixation can also be performed as primary treatment for a uterine descent, a technique that can be referred to as \u2018sacrospinous hysteropexy\u2019. This procedure has been described in women who wanted to preserve the uterus to retain fertility [2, 3]. Several studies have shown that the sacrospinous hysteropexy is anatomical efficient and safe and most women are highly satisfied about the procedure [4\u201310]. Outcome in these studies was mainly assessed in terms of anatomical results, and the majority of these studies did not evaluate urogenital symptoms and quality of life with validated questionnaires. So, although anatomical outcome of the sacrospinous hysteropexy appears to be good, we cannot conclude from current literature that this type of surgery is associated with a significant functional improvement of urogenital and defecatory symptoms. Measuring this functional outcome preoperatively and postoperatively was one of the recommendations for future research from a recent review on the subject [11]. The main objective of this study was to assess urogenital and defecatory symptoms and quality of life before and after sacrospinous hysteropexy. Secondary, we assessed anatomical outcome.\nMaterials and methods\nPatients\nIn the period between December 2001 and April 2005, 72 women underwent a sacrospinous hysteropexy at the University Medical Center Utrecht, The Netherlands.\nA woman was eligible for participation if she had a symptomatic pelvic organ prolapse and wanted surgical correction with preservation of the uterus. Exclusion criteria were abnormal uterus or ovaries on ultrasound examination, abnormal bleeding pattern and abnormal cervical cytology. All women answered a standardized questionnaire covering urogenital symptoms, defecatory symptoms and quality of life before and after surgery. Urodynamic assessment was performed in all women preoperatively. We did not try to diagnose occult stress incontinence. The study was approved by the local ethics committee.\nSurgery\nSurgery was performed by two experienced surgeons who at least had performed 20 sacrospinous hysteropexies before start of the study. The sacrospinous hysteropexy was performed unilaterally to the right ligament. A midline incision in the posterior vaginal wall is extended to the posterior part of the cervix. Through sharp and blunt dissection, the right sacrospinous ligament is made visible with the use of three Breisky retractors. Two non-absorbable Prolene\u00ae sutures (Ethicon, Sommerville, NJ, USA) are placed through the right sacrospinous ligament, approximately 2\u00a0cm median to the ischial spine, and subsequently placed through the posterior side of the cervix in the midline. An additional anterior and\/or posterior colporrhaphy (fascia plication), with absorbable Vicryl\u00ae 2-0 interrupted sutures (Ethicon, Sommerville, NJ, USA), was performed when indicated by the judgment of the individual gynecologist. In case of stress urinary incontinence, confirmed by urodynamic tests, a surgical procedure of the Tension-free Vaginal Tape (TVT, Ethicon, Sommerville, NJ, USA) was performed as described by Ulmsten [12]. All women received peri-operative thrombosis prophylaxis (anti-Xa) and a single dose of intravenous prophylactic antibiotic (amoxicillin\/clavunalic acid). Post-operatively, a 14-French Foley indwelling bladder catheter with a 5-ml balloon was placed in all women and removed after 1\u00a0day (in case of an additional anterior colporrhaphy after 3\u00a0days).\nMeasurements\nData collection took place in October\/September 2005, at least 3\u00a0months after surgery of the last participating woman. The following data were collected: age at the time of surgery; medical history; hospital stay and perioperative and postoperative complications. The anatomical outcome of all women was assessed with the Pelvic Organ Prolapse Quantification score (POP-Q) [13], which is described by the International Continence Society as a reliable and specific method to measure the pelvic organ support. Before surgery, POP-Q was performed by one of the two surgeons. After surgery, POP-Q was performed by one of three independent investigators. In the analysis, we dichotomized the POP-Q stage of prolapse into stage 1 or less and stage 2 or higher. Although we know that women with pelvic organ prolapse experience symptoms that do not necessarily correlate with the severity of prolapse, we have chosen this approach to try to separate potential clinical irrelevant from clinical relevant recurrences [14].\nUrogenital symptoms were measured before and after surgery with a standardized questionnaire, the Urogenital Distress Inventory (UDI), which has been validated for the Dutch population [15]. In this validation study on a large population-based sample, it was shown that the domain construction of the Dutch version was different from the original one. The following five domains were identified: urinary incontinence, overactive bladder, pain, obstructive micturition and prolapse. The scores of these domains vary between 0 and 100. A high score on a particular domain indicates more bothersome symptoms. The incidence of urinary incontinence before surgery was measured as follows: a woman was considered to have stress urinary incontinence if she replied positively to the question \u201cDo you experience urinary leakage related to physical activity, coughing or sneezing?\u201d. Urge urinary incontinence was scored if the question, \u201cdo you experience urinary leakage related to the feeling of urgency?\u201d was answered positively.\nAll patients completed a questionnaire, the Defecatory Distress Inventory (DDI) to assess the presence of defecation symptoms before and after surgery. This questionnaire was developed by our research group to assess the presence of defecation symptoms [16]. The DDI consists of 15 items about symptoms related to obstructive defecation, constipation, fecal incontinence and pain related to defecation. The questions were developed after studying the literature and international definitions, interviewing patients who presented with constipation or fecal incontinence, and by interviewing three experts in the field from the Department of Surgery and Department of Obstetrics and Gynecology from the University Medical Center Utrecht, The Netherlands. Eventually, a structured interview of the 15 selected items was held with 20 female patients. The DDI was used as, at present, there are no other Dutch validated questionnaires to measure quality of life related to defecation symptoms. The design on the questions is identical to those of the UDI with domain scores between 0 and 100. Again, a high score on a particular domain indicates more bothersome symptoms. The DDI was used in previous studies at our department [4, 9, 17].\nBefore and after surgery, disease-specific quality of life was measured with the Incontinence Impact Questionnaire (IIQ), validated for the Dutch population [15]. These questions cover the following five domains: physical functioning, mobility, emotional functioning, social functioning and embarrassment. The score ranged between 0 (best quality of life) and 100 (worst quality of life).\nEffect sizes were measured as a useful way to estimate whether an improvement on a particular domain was considered to have a small, moderate or large clinical relevance.\nStatistical analysis\nDescriptive statistics were used for the whole population. To compare scores on urogenital and defecatory symptoms before and after surgery a paired samples t test was used. The significance level was set at \u03b1 of 0.05. The effect size was calculated by Cohen\u2019s d test which is defined as the difference between two means divided by the pooled standard deviation for those means [18]. An effect size of 0.2 was considered to be small, 0.5 to be median and 0.8 or higher to be large [18]. Statistical analysis was performed in SPSS 12.0 for Windows.\nResults\nTable\u00a01 shows the characteristics of the 72 women. The fast majority had a combination of sacrospinous hysteropexy with an anterior colporrhaphy (87.5%). Five women (6.9%) had a sacrospinous hysteropexy of a stage 1 uterine descent. In these women, the surgeon decided to perform a sacrospinous hysteropexy during surgery because the uterine descent under anaesthesia was stage 2. The mean follow-up time was 12.7\u00a0months (median 11\u00a0months).\nTable\u00a01Patient characteristicsPatient characteristicsn\u2009=\u200972Age (years)a57.2 (11.9)Surgery for prolapse in medical history3 (4.2)Surgery for urinary incontinence in medical history4 (5.6)Urinary incontinence before surgeryb40 (55.6)\u00a0Urge incontinence9 (12.5)\u00a0Stress incontinence21 (29.2)\u00a0Combination stress and urge incontinence10 (13.9)Gynecological examination before surgeryDescensus uteri stage 2 or more67 (93.2)Cystoceles stage 2 or more57 (79.2)Rectoceles stage 2 or more19 (26.4)Enterocele1 (1.4)SurgerySacrospinous hysteropexy8 (11.1)Sacrospinous hysteropexy + anterior colporrhaphy54 (75)Sacrospinous hysteropexy + anterior and posterior colporrhaphy9 (12.5)Sacrospinous hysteropexy + posterior colporrhaphy1 (1.4)Additional TVT15 (20.8)Hospital stay (days)c3.5 (1\u20138)Follow-up in monthsc12.7 (3\u201333)Data are numbers (%)aMean (standard deviation)bAssessed with Urogenital Distress InventorycRange\nPerioperative and postoperative complications are shown in Table\u00a02. One woman needed repeated surgery for postoperative bleeding a couple of hours after the primary procedure. Total blood loss was estimated at 400\u00a0cc. There were no incidental bladder or rectal injuries. Of the 20 women (27.8%) who had urinary retention over 100\u00a0ml after removal of the indwelling bladder catheter, five women (6.9%) needed intermittent self-catheterisation more than 2\u00a0weeks after surgery. However, at 4\u00a0weeks, no women had significant urinary retention. This complication only occurred in women who underwent an anterior colporrhaphy. Seven women with cystitis received antibiotics and made an uneventful recovery. Five women (6.9%) developed right-sided buttock pain which persisted longer than 2\u00a0weeks. It resolved spontaneously within 6\u00a0weeks. No removal of the sacrospinal suture was required.\nTable\u00a02Complications related to surgeryPatient characteristicsn\u2009=\u200972Complications during surgery0 (0)Complications after surgery32 (44.4)\u00a0Second surgery because of bleeding1 (1.4)\u00a0Buttock pain13 (18.1)\u00a0\u00a0Buttock pain <2\u00a0weeks8 (11.1)\u00a0\u00a0Buttock pain >2\u00a0weeks5 (6.9)\u00a0Vaginal hematoma2 (2.8)\u00a0Urinary tract infection7 (9.7)\u00a0Retention bladder20 (27.8)\u00a0\u00a0Bladder catheterisation <2\u00a0weeks15 (20.8)\u00a0\u00a0Bladder catheterisation >2\u00a0weeks5 (6.9)\u00a0Vaginal adhesion3 (4.2)Data are numbers (%)\nDuring the follow-up period, a total of 16 women (22.2%) had a recurrent prolapse of one of the compartments. Five women (6.9%) had a recurrent prolapse of the uterus (four women stage 2, one woman stage 3). Ten women (13.9%) had a cystocele stage 2 or more (eight women stage 2, two women stage 3) and two women (2.8%) had a prolapse of the posterior compartment stage 2. All the women with a recurrent cystocele had had surgery of the anterior compartment combined with the hysteropexy, so there were no de novo cystoceles. The two women with a recurrent rectocele did not have surgery of the posterior compartment combined with the sacrospinous hysteropexy, so these can be considered as de novo rectoceles.\nTable\u00a03 shows the results of the UDI, DDI and IIQ domain scores before and after surgery. On all urogenital domains, there was significant improvement as well as on all quality of life domains. Symptoms on domain constipation and obstructive defecation also improved significantly. Large effect sizes were found on domain pain (effect size\u2009=\u20090.92) and genital prolapse (effect size\u2009=\u20092.0) of the UDI. The domain physical functioning and emotional health of the IIQ also showed a large effect size (0.82 and 0.79, respectively).\nTable\u00a03Urinary Distress Inventory (UDI), Defecatory Distress Inventory (DDI) and Incontinence Impact Questionnaire (IIQ)\u00a0Before surgerya (n\u2009=\u200972)After surgerya (n\u2009=\u200972)P valueEffect sizeUDIUrinary incontinence18.5 (24.2)10.5 (21.0)0.0120.35Overactive bladder29.5 (26.7)14.4 (18.6)0.0000.66Pain30.1 (26.7)9.5 (16.7)0.0000.92Obstructive micturition27.0 (28.3)10.8 (19.7)0.0000.66Genital prolapse56.6 (32.0)5.6 (17.4)0.0002.0DDIConstipation11.9 (18.8)6.3 (9.8)0.0210.37Obstructive defecation13.3 (19.7)8.3 (11.8)0.0160.22Pain4.5 (16.3)2.8 (8.3)0.4980.13Fecal incontinence8.6 (20.7)8.5 (14.4)0.9550.01IIQPhysical functioning26.0 (24.4)9.4 (15.1)0.0000.82Mobility25.0 (22.8)12.6 (18.4)0.0000.60Emotional health23.2 (23.3)8.0 (14.2)0.0000.79Social functioning14.8 (19.1)4.5 (11.8)0.0000.65Embarrassment11.2 (13.8)6.7 (11.1)0.0130.41Effect size: 0.2 = small effect, 0.5 = median effect, 0.8 = large effectaMean (standard deviation)\nIn addition to the table we made a sub-analysis for the 15 women who had additional surgery for urinary incontinence (TVT). These women improved significantly on the urinary incontinence domain after surgery (mean score: 26.7\u21926.7, p\u2009=\u20090.009). This improvement was not significant for the women without TVT surgery (mean score: 16.4\u219211.5, p\u2009=\u20090.162). On the other hand, the women with TVT surgery did not improve significantly on the overactive bladder domain (mean score: 24.3\u219216.23, p\u2009=\u20090.079) where the group without TVT did (mean score: 30.8\u219214.3, p\u2009=\u20090.000).\nDiscussion\nThe objective of this study was to assess quality of life and urogenital and defecatory symptoms before and after sacrospinous hysteropexy. The results show that a sacrospinous hysteropexy significantly reduced all urogenital and several defecatory symptoms and significantly improved quality of life. Effect sizes were large on domain genital prolapse and pain (UDI), and on domain physical functioning and emotional health (IIQ). It also anatomically cured the uterine descent in 93.1% of women.\nThe sacrospinous ligament fixation was first described by Sederl (1958) [19]. Later it became more popular by Richter and Albright [20] (Europe) and Randall and Nichols [21, 22] (USA). Several modifications of their techniques have been described since. The anatomical results of 2,256 women after a sacrospinous ligament fixation of the vaginal vault were recently reviewed [1]. Objective cure rates varied between 67 and 96.8%, and subjective cure rates varied between 70 and 98%. Our findings are in line with these results, although the review focused on the sacrospinous fixation of a vault prolapse. Subjective outcomes are underreported in most studies on the sacrospinous ligament fixation. We have shown prospectively that bladder and bowel function improves significantly after a sacrospinous hysteropexy.\nThere are a variety of reasons why women want to preserve their uterus. Among those reasons are: keeping their fertility, personal identity, but also the possibility that this kind of surgery might reduce operation time, estimated blood loss and postoperative recovery time [4, 23]. There are signs that removing the uterus may increase the risk of pelvic neuropathy, new onset urinary incontinence, bladder dysfunction and prolapse [24, 25, 26]. Several studies on the sacrospinous hysteropexy, as a technique in which the uterus is preserved, are available [3\u20136, 8\u201310, 27]. Among these studies, three were of prospective design [6, 8, 27], five were of retrospective design [3\u20135, 9, 10] and there was one case report [28]. One report described a different surgical technique and therefore cannot be compared with our study [2]. One study assessed risk factors for failure of sacrospinous hysteropexy [29] and another study only assessed sexual functioning after sacrospinous hysteropexy [30]. Anatomic success rates in these studies varied between 74 and 93.5%, which is comparable with our results. The main problems when comparing studies on the sacrospinous ligament fixation were recently debated by Morgan et al. [11]. They showed that there is a variety in definition of failure of sacrospinous ligament fixation due to differences in how anatomical outcomes are evaluated and which compartment of the vagina is considered. In our group, recurrent postoperative cystoceles, stage 2 or higher, were seen in 10 women (13.9%). This percentage is slightly lower than the 21.3% reported in the recent review [11]. However, our follow-up in the current study was relatively short and some recurrences may not have been detected in this timeframe.. In a previous retrospective study on the anatomical outcome of the sacrospinous hysteropexy by our group, we found a higher rate of recurrent cystoceles stage 2 or higher (30%) [9]. The follow-up period in this study was a mean of 23\u00a0months. The high rate of recurrent cystoceles may be related to the primary damage of neuromuscular support or may be the result of the retroverted axis of the vagina after sacrospinous hysteropexy. This last aspect, being regarded as an overcorrection, is held responsible for the high rate of cystoceles [27, 31]. However, in a study by Smilen et al. [32] the sacrospinous hysteropexy did not independently increase the risk of recurrent cystocele as compared to other surgical techniques.\nApart from true genital prolapse symptoms, urogenital symptoms and also bowel symptoms improved after the sacrospinous hysteropexy. Because the majority of women in our study had their sacrospinous hysteropexy combined with an anterior colporrhaphy, one may argue that it was this anterior repair that relieved symptoms, and not the sacrospinous hysteropexy. However, it was shown that pelvic organ prolapse and urogenital symptoms were only slightly correlated to the site and severity of the prolapse [14]. This lack of a clear correlation between the site of the pelvic organ prolapse and symptomatology makes it very difficult in combination surgery to contribute functional improvement to a certain intervention. All we can conclude from our results is that surgical procedures that involve a sacrospinous hysteropexy show good functional outcome.\nIt was shown that overactive bladder symptoms disappear after anterior repair in 60\u201382% of women [33]. We also found a marked improvement of overactive bladder symptoms after surgery in our group. However, this significant improvement was confined to the women who did not have a combined TVT procedure with their sacrospinous hysteropexy. Women who did have a TVT combined procedure experienced more bother on overactive bladder domain after surgery as compared to women without TVT surgery. This finding is consistent with literature on the TVT in which the development of overactive bladder symptoms after TVT surgery is reported to occur in up to 15% of women [34].\nWe have to keep this in mind when placing a TVT (prophylactic) in case of occult stress incontinence. After sacrospinous hysteropexy, postoperative complications occur, but none of them was life threatening. Most complications were self-limiting. The majority of postoperative complications were related to the bladder function. These complications did not occur in women who only had a sacrospinous hysteropexy. Therefore, it is likely that complications related to the bladder are the consequence of additional surgery and not the result of sacrospinous hysteropexy. The prevalence of buttock pain is estimated at 10 to 15% [35]. This pain can be explained by injury to surrounding nerves of the sacral plexus and branches of the pudendal nerve. In an anatomical study, the relationship of the pudendal nerve to the sacrospinous ligament was found to be variable (one branch of the pudendal nerve piercing through the ligament was found in 11%) [36]. Barksdale et al. also showed that nerve tissue is present and widely distributed within the sacrospinous ligament [37]. Therefore, although the placement of the suture two centimetres medial to the ischial spine protects against major nerve injuries, the complications of buttock pain cannot be prevented in all women. Fortunately, this buttock pain was shown to resolve spontaneously in most cases, as we also demonstrated in our series [9].\nThe strength of our study is that we measured urogenital and defecatory symptoms and quality of life in a large group of women who underwent a sacrospinous hysteropexy, with a validated questionnaire before and after surgery. There are some potential drawbacks that need to be discussed. First, there might be an indication bias. In our country, a vaginal hysterectomy is the standard surgical technique for correcting a uterine descent. Therefore, women that came to our hospital may have chosen specifically for this operation. They might have had high expectations of this procedure which could have influenced their outcome with respect to quality of life. Second, in some patients, follow-up time was limited to 3\u00a0months. Possibly, some recurrences had not yet developed at that time. Third, the study was performed in a single university hospital. The sacrospinous ligament fixation has become rapidly popular in our center and is performed by two surgeons. They are highly trained in performing the procedure. This might have influenced the outcome. Fourth, we did not compare the sacrospinous hysteropexy with another surgical technique to correct a uterine descent. Therefore, we cannot conclude that the sacrospinous hysteropexy is superior to other procedures. However, it is a safe and effective operation for women who wish to preserve their uterus at time of genital prolapse surgery.","keyphrases":["functional outcome","sacrospinous hysteropexy","quality of life","pelvic organ prolapse"],"prmu":["P","P","P","P"]}
{"id":"Exp_Brain_Res-3-1-1914235","title":"Error correction in bimanual coordination benefits from bilateral muscle activity: evidence from kinesthetic tracking\n","text":"Although previous studies indicated that the stability properties of interlimb coordination largely result from the integrated timing of efferent signals to both limbs, they also depend on afference-based interactions. In the present study, we examined contributions of afference-based error corrections to rhythmic bimanual coordination using a kinesthetic tracking task. Furthermore, since we found in previous research that subjects activated their muscles in the tracked (motor-driven) arm, we examined the functional significance of this activation to gain more insight into the processes underlying this phenomenon. To these aims, twelve subjects coordinated active movements of the right hand with motor-driven oscillatory movements of the left hand in two coordinative patterns: in-phase (relative phase 0\u00b0) and antiphase (relative phase 180\u00b0). They were either instructed to activate the muscles in the motor-driven arm as if moving along with the motor (active condition), or to keep these muscles as relaxed as possible (relaxed condition). We found that error corrections were more effective in in-phase than in antiphase coordination, resulting in more adequate adjustments of cycle durations to compensate for timing errors detected at the start of each cycle. In addition, error corrections were generally more pronounced in the active than in the relaxed condition. This activity-related difference was attributed to the associated bilateral neural control signals (as estimated using electromyography), which provided an additional reference (in terms of expected sensory consequences) for afference-based error corrections. An intimate relation was revealed between the (integrated) motor commands to both limbs and the processing of afferent feedback.\nIntroduction\nRecently, the relative importance of perceptual processes for motor control in general, and bimanual coordination in particular, has been intensely debated [e.g., see target article of Mechsner (2004) and associated commentaries]. Despite differences in the conceptual interpretation of empirical findings, there is ample evidence that perceptual factors affect bimanual coordination. For example, beneficial effects of augmented visual feedback on rhythmic bimanual coordination have been observed (Swinnen et al. 1997; Byblow et al. 1999; Mechsner et al. 2001), in combination with changes in concurrent neural activity (Debaere et al. 2003; Carson et al. 2005). In addition, it has been shown that perturbations of proprioception using tendon vibration result in disruption of the temporal coordination between the limbs (Verschueren et al. 1999; Steyvers et al. 2001). On the other hand, however, recent studies have indicated that important characteristics of rhythmic bimanual coordination are not primarily attributable to somatosensory feedback (Ridderikhoff et al. 2005b; Spencer et al. 2005), suggesting a key role for afference-independent (i.e., open-loop) control processes. Collectively, these findings motivated the present study on kinesthetic tracking, which (1) examined the closed-loop control processes underlying rhythmic bimanual coordination, and (2) addressed the potential interplay between open-loop and closed-loop control in the production of rhythmic bimanual movements.\nIn the present study, we focus on rhythmic bimanual movements performed at the same frequency. Such movements are characterized by the presence of only two stable coordination patterns that can be performed without training (Zanone and Kelso 1992), and that are defined in terms of the relative phase between the hands (\u03a6). In most cases, one pattern (in-phase coordination; \u03a6\u00a0=\u00a00\u00b0; simultaneous activation of homologous muscles) is more stable than the other pattern (antiphase coordination; \u03a6\u00a0=\u00a0180\u00b0; alternated activation of homologous muscles) (Kelso 1984; Swinnen 2002). These characteristics imply that the functional consequences of various control processes can be appreciated directly in terms of their contribution to the stability difference of in-phase and antiphase coordination. In general, these control processes can be classified according to three sources of interlimb interactions. One of these sources is afference-independent, and refers to open-loop processes involved in the integrated timing of the feedforward signals to both limbs. The other two sources are associated with closed-loop processes: a reflex-like interaction resulting in phase entrainment by contralateral afference, and intentional corrections of the timing based on the perceived error in the relative phase. This conceptual framework is motivated and described in detail elsewhere (Ridderikhoff et al. 2005b). In previous studies we found that the stability difference between in-phase and antiphase coordination depended mainly on the integrated timing of the feedforward control signals (Ridderikhoff et al. 2005b), and that phase entrainment by contralateral afference did not affect this difference (Ridderikhoff et al. 2005b, 2006). As regards the second afference-based source of interlimb interactions, timing corrections based on the perceived relative phase, a more complex picture emerged, which motivated the present study as discussed in the following paragraphs.\nKinesthetic tracking tasks have been used to assess the role of afference-based interactions between the limbs in rhythmic bimanual coordination, by examining the coordination of unilateral active rhythmic movements with motor-driven oscillations of the contralateral limb (Viviani et al. 1997; Stinear and Byblow 2001; Ridderikhoff et al. 2005b). In agreement with the stability characteristics of rhythmic bimanual coordination, two previous studies on kinesthetic tracking have demonstrated differences between in-phase and antiphase coordination. One study revealed a more variable relative phase and larger response times on a secondary task for antiphase coordination than for in-phase coordination (Stinear and Byblow 2001). The other study showed that the mean relative phase was more susceptible to an external stimulus (i.e., less stable) during antiphase coordination than during in-phase coordination (Ridderikhoff et al. 2005b). Thus, the study of kinesthetic tracking indicated that afference-based interlimb interactions may contribute to the stability difference between in-phase and antiphase coordination. In terms of the aforementioned (afference-based) sources of interlimb interaction these pattern-related stability differences should be attributed to corrections based on the perceived error in the relative phase, because phase entrainment by contralateral afference has been found to stabilize in-phase and antiphase coordination in equal measure (Ridderikhoff et al. 2005b; Ridderikhoff et al. 2006).\nIn our previous work we found that muscle activation patterns during kinesthetic tracking exhibited a striking similarity to the patterns observed in normal bimanual coordination, even though muscular activity was neither required nor of any consequence for the movement pattern of the motor-driven hand (Ridderikhoff et al. 2005b). In contrast, if motor-driven movements were used to induce phase entrainment by contralateral afference (i.e., when one limb was moved passively, but no coordination between the limbs was required), such activation patterns were not observed (Ridderikhoff et al. 2005b, 2006). Thus, the spontaneously adopted tendency of subjects to activate their muscles as if they were moving along with the motor-driven movement appeared directly related to the requirement of achieving a particular coordination pattern between the limbs. In principle, such spontaneous muscle activation in the driven limb may serve several purposes. It may be, for instance, that it reflects coactivation of gamma-motoneurons to enhance the sensitivity of muscle spindles (Gandevia et al. 1992). However, this possibility is less likely because several studies reported an increase of detection thresholds (Wise et al. 1998) and attenuation of muscle sense (Collins et al. 1998) during voluntary contractions (see Proske 2006 for a review). Alternatively, it may be that the activation of muscles in the motor-driven arm reflects the use of an internal control signal specifying the bimanual movement pattern. Considering that our previous work has indicated a crucial contribution of afference-independent interlimb interactions to the stability of the normal bimanual coordination (Ridderikhoff et al. 2005b), this internal control signal may well be generated reliably in an open-loop fashion. Hence, in the context of kinesthetic tracking this open-loop control signal may provide a suitable reference against which the error in the relative phasing (based on afferent signals) could be determined. Such involvement of motor commands in limb kinesthesis was recently demonstrated empirically (Gandevia et al. 2006), and has been advocated as a fundamental principle of motor control involving predictions of the movement\u2019s sensory consequences via efference copy (e.g., Wolpert and Ghahramani 2000; Scott 2004). Note that this second account of the EMG activity in the driven limb, which in fact served as a working hypothesis for the present study, implies an intimate relation between two of the aforementioned sources of interlimb interaction, viz. error correction based on the perceived relative phase, and the integrated timing of the feedforward signals (providing the reference pattern).\nThe aim of the present study was to examine the afference-based error corrections in detail using a kinesthetic tracking task, with a specific focus on the role of the muscle activity in the motor-driven arm, to elucidate the interplay of closed-loop and open-loop control processes in rhythmic bimanual coordination. We compared the performance during in-phase and antiphase coordination to further our understanding of the potential contribution of closed-loop control processes to the differential stability of these patterns. In addition, we examined the stability-related effects of bilateral muscle activation during kinesthetic tracking on the resulting coordinative stability. To address the latter issue we compared two conditions in which subjects were either instructed to keep the motor-driven limb relaxed, or to activate their muscles as if moving along with the imposed motor-driven movement. For the latter condition, the phase relations at the level of neural control signals (based on electromyographic data) and at the behavioral level (kinematics) were compared. The neural control signals represent the reference signal that may be used for the prediction of sensory consequences of the ongoing movement, whereas the kinematic phase relation reflects the actual quality of the performance. In both conditions, we performed an extensive analysis of the correlations between various kinematic variables to uncover the underlying structure of the timing corrections based on the (perceived) errors in the relative phase.\nMaterials and methods\nSubjects\nTwelve healthy subjects (6 male, 6 female; aged 19\u201331\u00a0years) volunteered to participate. All subjects were right-handed according to their scores on a common handedness questionnaire (Oldfield 1971). They had no previous experience with the task and reported no (history of) neurological disorders. The local ethics committee approved the experiment and all subjects gave their written informed consent before the experiment was conducted.\nApparatus\nSubjects sat in a height-adjustable chair with their elbows slightly flexed and their feet supported. Each forearm was placed in the apparatus in a neutral position (thumbs up and palms facing inward), and its position was restrained (by the support surface on the medial and ventral side, by two vertical foam-coated supports on the dorsal side, and by one horizontal foam-coated support on the lateral side) to prevent movements about the elbow. Both hands were fixated against the flat manipulanda using two Velcro straps, with all fingers extended. The apparatus only permitted flexion\u2013extension movements of the wrist in the horizontal plane. The right manipulandum was mounted on a potentiometer (Sakae, type FCP40A-5k, linearity 0.1%) to register wrist joint angles during active movement, while the left was connected to a servo-controlled motor that moved the hand passively. The potentiometer\u2019s output voltage was digitized by a 12-bit ADC (Labmaster DMA) and stored on a microcomputer at a sampling frequency of 1,000\u00a0Hz. The active movements were recorded with a precision of about 0.1\u00b0. The passive movements were generated using a DC brush motor (PARVEX, type RS440GR) that was controlled by a PC-mounted servo controller (ACS-Tech80, type SB214). The maximum torque of the motor was such that subjects were unable to alter the trajectory of the applied movements, and the maximum error in the trajectory of the passive movements was 0.26\u00b0. Subjects wore earmuffs with built-in stereo earphones (Bilsom 787, Flex II), which provided a moderate level of \u2018white\u2019 background noise to eliminate any auditory feedback from the motion of the motor. A white opaque screen was used to eliminate visual feedback of the hand movements.\nSurface electromyograms (EMG) were obtained from M. flexor carpi radialis (FCR), and M. extensor carpi radialis (ECR) of both arms. A bipolar arrangement of disposable electrodes (Medicotest, Ag\/AgCl-electrodes, square 5\u00a0\u00d7\u00a05\u00a0mm pick-up area) was attached with a center-to-center distance of 2\u00a0cm after cleansing and abrasion of the skin. The electrodes were positioned in the center of the muscle belly on the line from origin to insertion as determined by palpation. EMG signals were sampled at 1,000\u00a0Hz (TMS International, type Porti5-16\/ASD; 22 bits ADC) after band-pass filtering (0.5\u2013400\u00a0Hz), and stored on a microcomputer.\nProcedure\nSubjects were instructed to perform smooth oscillatory movements about the right wrist in such a way that (1) peak flexion and peak extension of both wrists were attained simultaneously (in-phase pattern), or (2) peak flexion of one hand coincided with peak extension of the other hand (antiphase pattern). To achieve this, the timing of the active right wrist movements had to be coordinated with the motor-driven movements of the left wrist. Only subjects that were able to perform both movement patterns in at least one of two selection trials at the start of the experiment were included (one candidate subject failed to meet this criterion). After the selection trials the EMG electrodes were applied, and subsequently all subjects performed maximum voluntary contractions (MVCs) by generating an isometric flexion or extension torque with each arm for approximately 3\u00a0s. For the purpose of normalization of the EMG, the maximum root mean square (RMS) value of two separate MVC measurements was used in the analysis.\nAdditional instruction was given to subjects with respect to the muscle activity in the left (driven) arm. Subjects were required either to keep the muscles of the left (driven) arm as relaxed as possible (relaxed condition) or to activate the muscles of the left arm as if they were moving along with the motor-driven manipulandum (active condition). The resulting 2 (Pattern)\u00a0\u00d7\u00a02 (Activity)\u00a0=\u00a04 conditions were performed in separate blocks of trials, the order of which was counterbalanced across subjects. Each block of trials started with at least four practice trials to familiarize the subjects with the task (if necessary, maximally four additional practice trials were allowed). Once the subject was able to perform the task properly, based on visual assessment by the experimenters, six experimental trials were performed that were used for the analysis.\nFor the motor-driven movements of the left wrist, sinusoidal trajectories with an average movement frequency of 1.4\u00a0Hz and mean amplitude of 35\u00b0 (i.e., a range of motion of 70\u00b0 about the neutral position of the wrist) were used. To create a challenging tracking task the period length and the amplitudes were varied within a trial. Because pilot experiments had shown that too much variability induced high levels of (mainly tonic) muscle activity in the relaxed condition, a moderate level of variability was selected, i.e., intermediate between natural variability and perfectly sinusoidal trajectories as used in previous studies (Ridderikhoff et al. 2005b; Stinear and Byblow 2001, respectively). Subjects started with a low level of variability in the selection trials (SDfrequency\u00a0=\u00a00.02\u00a0Hz; SDamplitude\u00a0=\u00a02\u00b0), which was increased in a step-wise fashion during the practice trials to the level of variability used in the experimental trials (SDfrequency\u00a0=\u00a00.03\u00a0Hz; SDamplitude\u00a0=\u00a05\u00b0). To assure a smooth start and finish of the trial, the amplitude of the motor-driven movements was gradually increased (from 0\u00b0) and decreased (to 0\u00b0) during the first and last 3\u00a0s of a trial, respectively. The duration of a trial was 30\u00a0s. To avoid transient effects and to exclude the cycles in which the amplitude of the passive movement was adjusted, the first 7 and the last 3\u00a0s of the trial were discarded, leaving 20\u00a0s per trial for analysis.\nData reduction (kinematics)\nFigure\u00a01 illustrates and defines the features of the time evolution of the joint angles on which the analyses of the kinematics were based. Because systematic differences in coordination were present depending on whether peak flexion or peak extension was chosen as reference, the relative phase between the hands (\u03a6) was calculated for each cycle as \nfor flexion and as \nfor extension, where ty,i and tx,i indicate the time of the ith peak flexion or peak extension of the right and left hand, respectively (for a similar method see, e.g., Carson et al. 1995). A positive relative phase meant that the right hand was lagging the left hand. Circular statistics (Mardia 1972) was used to calculate the mean and the circular standard deviation of the relative phase (SD\u03a6). The absolute error of the relative phase (AE\u03a6) was defined as the absolute difference between the mean relative phase and the required relative phase (0\u00b0 for in-phase; 180\u00b0 for antiphase).\nFig.\u00a01Main kinematic features of one full cycle of both hands, illustrated for the in-phase coordination pattern. Moments of peak flexion and extension at the start of the ith cycle in each hand are designated by tj,ik, where k indicates flexion (F) or extension (E), and j refers to the right hand (y) or left hand (x). The durations of the ith full cycle between two moments of peak excursion are designated by \u0394tj,ik. The duration of the ith half cycle is designated by \u03b4tj,il, where l indicates the orientation of the hand at the end of the half cycle: flexion (F) or extention (E). The (signed) error at the start of the ith cycle is designated by \u03b5ik. The definition of the (signed) errors depends on the coordination pattern and is defined in terms of the relative timing of corresponding peak excursions in both hands (flexion\u2013flexion and extension\u2013extension for in-phase; flexion\u2013extension and extension\u2013flexion for antiphase)\nIn a previous study (Ridderikhoff et al. 2005b), we showed that kinesthetic tracking performance can also be meaningfully evaluated in terms of correlations (RFC; FC\u00a0=\u00a0full cycle) between the signed timing error at peak flexion or peak extension (\u03b5i) and the duration of the following full cycle of the right (actively moving) hand (\u0394ty,i). The rationale behind this measure is that if an error in the relative phasing is detected at the start of the ith cycle, this error may be compensated for by adapting the duration of the next cycle, resulting in negative values of RFC. Motivated by the aforementioned differences between flexion and extension that were observed for the relative phase (see also \u201cResults\u201d), we also calculated the correlation between this error in the relative timing and the duration of the following half cycle of the right hand (RHC; HC\u00a0=\u00a0half cycle). RFC and RHC are intimately related in that the error correction made during a full cycle is the sum of the error corrections made during its two half cycles, provided that the latter two corrections are independent of each other. However, in general this proviso is not met, because part of the correction in the second half cycle may in fact compensate for errors that arose in the first half cycle, which is reflected by two additional temporal correlations. First, deviations in the duration of the first half cycle of the left (driven) hand (as a consequence of the variability of the imposed reference trajectory) may result in errors at the start of the second half cycle that lead to adaptation of the duration of the consecutive half-cycle of the right hand. This dependency is reflected in positive \u2018between-hands\u2019 correlations (Rxy) between the durations of a half-cycle of the left (driven) hand and the following half-cycle of the right hand. Second, if in each half-cycle the errors are overcompensated, a sequence of alternating longer and shorter half-cycles is obtained. Such a dependency is reflected by negative \u2018within-hand\u2019 correlations (Ryy) between the successive half-cycles of the right hand. Thus, positive values of Rxy and negative values of Ryy reflect dependencies between the error corrections in successive half cycles that reduce their contributions (RHC) to the full cycle error corrections RFC. These four temporal correlations (RFC, RHC, Rxy, and Ryy) provide information about the temporal structure of the performance in terms of the timing of peak flexion and peak extension of the wrist. As such, these measures are more closely related to the underlying control processes than the global performance measures based on the relative phase. Exact definitions of (the relations between) these measures can be found in the Appendix in terms of the underlying covariances.\nBecause systematic differences were found between the actual timing of peak flexion and that of peak extension, the effects of these two orientations on the error correction measures (RFC and RHC)\u2014as obtained for timing errors at peak flexion or peak extension of the right (actively moving) hand in relation to the duration of the following (full or half) cycle of this hand\u2014were examined as well. In addition, the effects of orientation on Ryy and Rxy were evaluated by comparing the correlations between the durations of the two successive half-cycles following either peak flexion or peak extension of the right (actively moving) hand. In this way, the implications of Ryy and Rxy for the effects of orientation that were observed for RFC could be discerned (see Appendix and Fig.\u00a04).\nData reduction (EMG)\nEMG records were bandpass filtered (10\u2013400\u00a0Hz) using a second-order bidirectional (zero-lag) Butterworth filter (Merletti et al. 1999). To visualize the average muscle activity within a cycle, eight bins were defined in relation to the continuous phase of the movement \u0398\u00a0=\u00a0arctan[(d\u03b8\/dt)\/(2\u03c0f\u03b8)], where \u03b8 and (d\u03b8\/dt) are joint angle and joint angular velocity, respectively, and f is the movement frequency. Thus, each bin represented an equal part of the phase of the hand oscillation. The first bin was centered around \u0398\u00a0=\u00a00\u00b0 (i.e., peak extension) and the fifth bin was centered around \u0398\u00a0=\u00a0180\u00b0 (i.e., peak flexion). For each bin the RMS of the EMG was calculated and normalized to that obtained for the MVC.\nIn addition to the comparison of conditions in terms of the average (normalized) amplitudes, the similarity of the rEMGs of homologous muscles was assessed using the weighted coherence. Specifically, the weighted coherence reflects the degree of similarity (or phase locking) of the activity bursts in the homologous muscles in terms of a weighted average of the coherence in the vicinity of the movement frequency (i.e., in the frequency band in which these bursts occur), yielding values between 0 (no phase locking) and 1 (perfect phase locking). Thus, the weighted coherence is an estimate of the strength of the interlimb coupling at the level of the neural control signals (Ridderikhoff et al. 2005b). In the present study it was used to evaluate differences in this regard between the relaxed and active conditions and between flexor and extensor muscles. The weighted coherence (Porges et al. 1980) of the full-wave rectified EMG (rEMG) of homologous muscles (CW) was calculated aswhere \u0394f defines a bandwidth around the movement frequency f (\u0394f\u00a0=\u00a00.1\u00a0Hz). Py is the power spectrum of the rEMG of the right arm, and Cxy is the coherence of the rEMGs of the homologous muscles in the left and right arm. The power spectra and the coherence were estimated with Welch\u2019s modified periodogram method (Oppenheim and Schafer 1975) using a Hanning window of three cycles.\nIn addition, the phase relations between rEMGs and joint angles \u03b8 were studied to compare the temporal relations observed at the level of the neural control signals to those observed at the behavioral level. This is of particular importance for the analysis of the contribution of the bilateral motor commands to the performance in the active condition. The phase shift between rEMG and \u03b8 was obtained from the cross-spectrum of rEMG and \u03b8 estimated at the movement frequency (using the same parameters as for the weighted coherence). Because flexion corresponded to negative values of \u03b8, the phase shift between the EMG of FCR and \u03b8 was adjusted by 180\u00b0 (cf. Ridderikhoff et al. 2004). Likewise, the relative phases between the rEMGs of homologous muscles were determined from the cross-spectrum of rEMGs of muscles in the left and right arm. The constant error of these relative phases was defined as the signed difference between the mean relative phase and the required relative phase (0\u00b0 for in-phase; 180\u00b0 for antiphase), with negative values indicating a relative phase lead of the activity in the right arm. The calculation of these measures required that the signals in question were phase and frequency locked, which implied that both measures could only be obtained for the active condition because only in this condition the rEMG of the muscles in the left (motor-driven) arm satisfied this requirement. For both measures (i.e., the phase shifts between rEMG and \u03b8, and the relative phases between rEMGs of homologous muscles) the values obtained for FCR and ECR were analyzed separately to examine differences in the relative timing of flexors and extensors in relation to effects of coordinative pattern (for both measures) and limb (for the phase shifts between rEMG and \u03b8). In addition, the values obtained for FCR and ECR were averaged to obtain a global measure for the phasing of the neural control signals to the limbs (Viviani et al. 1976). To visualize the main temporal relations in the movement system, the relative phase between the neural control signals, the phase shifts between the neural control signals and the movements of the left and right hand, and the average relative phase between the hands were examined in conjunction.\nIn the active condition, FCR of the left (driven) arm of two subjects showed substantial reactive activity comparable in magnitude to that observed in the relaxed condition (see Fig.\u00a05c; peak at bin 5, open symbols) in addition to the normal timing of muscle activation that was required in this condition (see Fig.\u00a05c; peak at bin 2, filled symbols). This reactive activity was to some extent present in most subjects (as indicated by the peaks in Fig.\u00a05, which was created without using the data of these two excluded subjects), but typically much less pronounced. For the two excluded subjects the large amplitude of the reactive activity resulted in a shift of the dominant frequency of the power spectrum of left FCR in this condition to twice the movement frequency (i.e., two bursts per cycle). The EMG data of these subjects were excluded from the analyses, because their inclusion resulted in a number of additional significant effects that could all be attributed to this reactive activity, but were not representative for the EMG data of the group as a whole. It should be noted, however, that all phenomena mentioned in the Results section were also observed for the excluded subjects.\nStatistical analysis\nStatistical analyses of the kinematics were performed using a 2 (Pattern: in-phase vs. antiphase coordination)\u00a0\u00d7\u00a02 (Activity: active vs. relaxed)\u00a0\u00d7\u00a02 (Orientation: flexion vs. extension) repeated measures analysis of variance (ANOVA). Also EMG-related measures were analyzed with repeated measures ANOVAs involving various, question-specific designs, which are described in the corresponding subsections of the Results. To facilitate the interpretation of the results obtained with ANOVA, paired-sample t-tests were used for post hoc analysis of the significant results (P\u00a0<\u00a00.05), and effect sizes (f) were calculated in terms of the partial \u03b72 (Cohen 1988). The correlations were transformed to normally distributed variables using the Fisher transform. The same transformation was applied to the weighted coherence (Rosenberg et al. 1989). Whereas the inferential analyses were based on the transformed values, the corresponding untransformed values (bounded on the interval [\u22121,\u00a01] for the correlations and on the interval [0,\u00a01] for the coherence) are presented for reasons of clarity.\nResults\nAdequate performance of a trial was determined using the following inclusion criteria: (1) the number of cycles performed by the right hand and the (driven) left hand should be the same (i.e., no phase wrapping); (2) AE\u03a6 should be smaller than 60\u00b0, and (3) the within-trial fluctuations of the relative phase should be within a range of 90\u00b0. In total 25 trials (8.7%) were excluded from the analysis: 9 trials in the active condition (3 trials for in-phase coordination, and 6 trials for antiphase coordination); 16 trials in the relaxed condition (7 trials for in-phase coordination, and 9 trials for antiphase coordination). In each condition all subjects were capable of performing the task in at least three trials in an adequate fashion.\nRelative phase\nStatistical analysis of AE\u03a6 (see Fig.\u00a02a) revealed a significant effect of Activity (F(1,\u00a011)\u00a0=\u00a05.38; P\u00a0<\u00a00.05; f\u00a0=\u00a00.70), indicating that AE\u03a6 was larger for the active condition (mean\u00a0\u00b1\u00a0between-subjects SD: 28.2\u00b0\u00a0\u00b1\u00a011.1\u00b0) than for the relaxed condition (20.6\u00b0\u00a0\u00b1\u00a012.5\u00b0). In addition, a significant Pattern\u00a0\u00d7\u00a0Orientation interaction was found (F(1,\u00a011)\u00a0=\u00a06.44; P\u00a0<\u00a00.05; f\u00a0=\u00a00.77). Post hoc analysis demonstrated two significant differences underlying this interaction, viz. between in-phase and antiphase coordination at peak extension (21.8\u00b0\u00a0\u00b1\u00a011.0\u00b0 vs. 26.8\u00b0\u00a0\u00b1\u00a011.1\u00b0, respectively) and between flexion and extension during antiphase coordination (24.0\u00b0\u00a0\u00b1\u00a010.0 vs. 26.8\u00b0\u00a0\u00b1\u00a011.1\u00b0, respectively). The same statistical results were obtained for the constant errors in relative phasing (not shown), because the errors were almost always in the same direction (i.e., negative), indicating that the right (actively moving) hand was leading in time.\nFig.\u00a02a Mean absolute error of the relative phase (AE\u03a6). b Standard deviation of the relative phase (SD\u03a6). Results obtained for the relative timing of peak flexion (black bars) and peak extension (gray bars) are shown for in-phase (IP) and antiphase (AP), both for the conditions in which the muscles of the left hand were active (AC) or relaxed (RE). Error bars indicate the standard error of the mean\nTo examine the stability of the coordinative patterns statistical analysis of SD\u03a6 (see Fig.\u00a02b) was conducted, which revealed significant effects of Activity (F(1,\u00a011)\u00a0=\u00a05.94; P\u00a0<\u00a00.05; f\u00a0=\u00a00.74) and Pattern (F(1,\u00a011)\u00a0=\u00a014.61; P\u00a0<\u00a00.005; f\u00a0=\u00a01.15). These effects resulted from, respectively, a larger SD\u03a6 in the relaxed condition than in the active condition (12.7\u00b0\u00a0\u00b1\u00a01.4\u00b0 vs. 11.7\u00b0\u00a0\u00b1\u00a01.4\u00b0) and a larger SD\u03a6 for antiphase coordination than for in-phase coordination (12.8\u00b0\u00a0\u00b1\u00a01.5\u00b0 vs. 11.6\u00b0\u00a0\u00b1\u00a00.87\u00b0).\nMovement amplitude\nTo examine whether the different activity levels of the muscles in the left (driven) arm had an effect on the amplitude of the movements about the right wrist, the range of motion (i.e., the difference in peak extension and peak flexion) was determined for each cycle, averaged per condition and subjected to a 2 (Pattern)\u00a0\u00d7\u00a02 (Activity) repeated measures ANOVA. The analysis solely revealed a significant effect of Activity (F(1,\u00a011)\u00a0=\u00a037.00; P\u00a0<\u00a00.001; f\u00a0=\u00a01.83), resulting from a larger movement amplitude in the active condition than in the relaxed condition (range of motion: 85.5\u00b0\u00a0\u00b1\u00a023.8\u00b0 vs. 58.6\u00b0\u00a0\u00b1\u00a014.4\u00b0, respectively).\nTemporal correlations between kinematic variables\nThe average correlations between the signed error and the duration of the following full cycle (RFC) or half cycle (RHC) are shown in Fig.\u00a03a, b, respectively. Statistical analysis of RFC revealed significant effects for Activity (F(1,\u00a011)\u00a0=\u00a07.05; P\u00a0<\u00a00.05; f\u00a0=\u00a00.80), Pattern (F(1,\u00a011)\u00a0=\u00a05.29; P\u00a0<\u00a00.05; f\u00a0=\u00a00.69) and Orientation (F(1,\u00a011)\u00a0=\u00a013.35; P\u00a0<\u00a00.01; f\u00a0=\u00a01.10). RFC was more pronounced (larger absolute values) for the active condition than for the relaxed condition (\u22120.76\u00a0\u00b1\u00a00.14 vs. \u22120.69\u00a0\u00b1\u00a00.14), for the in-phase pattern than for the antiphase pattern (\u22120.76\u00a0\u00b1\u00a00.14 vs. \u22120.69\u00a0\u00b1\u00a00.13), and for errors at peak flexion than for errors at peak extension (\u22120.75\u00a0\u00b1\u00a00.14 vs. \u22120.70\u00a0\u00b1\u00a00.14).\nFig.\u00a03Mean correlations between the signed timing error and the duration of the following full cycle (RFC, a) or half cycle (RHC, b) of the right (actively moving) hand. Results are shown for in-phase (IP) and antiphase (AP), both for the conditions in which the muscles of the left hand were active (AC) or relaxed (RE). RFC was calculated for full cycles following the error at two different orientations of the right (actively moving) hand: peak flexion (black bars) and peak extension (gray bars). RHC was calculated for the flexion half cycles following the error at peak extension (black bars) and the extension half cycles following the error at peak flexion (gray bars). Error bars indicate the standard error of the mean\nStatistical analysis of RHC revealed significant effects for Activity (F(1,\u00a011)\u00a0=\u00a013.33; P\u00a0<\u00a00.01; f\u00a0=\u00a01.10) and Pattern (F(1,\u00a011)\u00a0=\u00a013.02; P\u00a0<\u00a00.01; f\u00a0=\u00a01.09), indicating larger absolute values for the active condition than for the relaxed condition (\u22120.61\u00a0\u00b1\u00a00.17 vs. \u22120.50\u00a0\u00b1\u00a00.17), and for the in-phase pattern than for the antiphase pattern (\u22120.59\u00a0\u00b1\u00a00.17 vs. \u22120.52\u00a0\u00b1\u00a00.17). In addition, for RHC significant Activity\u00a0\u00d7\u00a0Pattern (F(1,\u00a011)\u00a0=\u00a06.14; P\u00a0<\u00a00.05; f\u00a0=\u00a00.75) and Pattern\u00a0\u00d7\u00a0Orientation (F(1,\u00a011)\u00a0=\u00a09.12; P\u00a0<\u00a00.05; f\u00a0=\u00a00.91) interactions were found. Post hoc analysis revealed that the first interaction effect resulted from a larger absolute value of RHC for in-phase coordination in the active condition than for the three other combinations of Activity and Pattern (see Fig.\u00a03b). The second interaction effect was due to a larger absolute value of RHC for the extension half-cycle in the in-phase pattern than for the three other combinations of Pattern and Orientation (see Fig.\u00a03b).\nThe main effects of Activity and Pattern were qualitatively the same for RHC and RFC, but the effect of Orientation was markedly different. An explanation of this difference was found in a formal analysis of the relation between the covariances1 underlying RHC and RFC, which demonstrated that (unlike differences related to Activity or Pattern) differences between flexion and extension in RHC are completely unrelated to those in RFC (see Appendix). Two additional factors are involved in the relation between RHC and RFC (see \u201cMaterials and methods\u201d and Appendix), which were captured by the within-hand and between-hands correlations of the durations of successive half cycles (Ryy and Rxy, respectively). The effect of Orientation in RFC can be attributed completely to (the covariances underlying) Ryy and Rxy (see Appendix). However, also for the other factors (Activity and Pattern) the relation between RHC and RFC may be affected by Ryy and Rxy, given the potential dependencies between error corrections in successive half-cycles. Thus, the temporal correlations were examined further in terms of the within-hand correlation Ryy and the between-hands correlation Rxy.\nAnalysis of the between-hands correlation Rxy (Fig.\u00a04b) revealed no significant effects. In contrast, significant effects of Activity (F(1,\u00a011)\u00a0=\u00a08.83; P\u00a0<\u00a00.05; f\u00a0=\u00a00.90) and Orientation (F(1,\u00a011)\u00a0=\u00a08.19; P\u00a0<\u00a00.05; f\u00a0=\u00a00.86) were found for the within-hand correlation Ryy. The effect of Activity revealed that the values of Ryy for the active condition were more negative than for the relaxed condition (\u22120.16\u00a0\u00b1\u00a00.17 vs. \u22120.02\u00a0\u00b1\u00a00.07). The effect of Orientation indicated that the correlations between the extension half cycle and the following flexion half cycle were more negative (\u22120.14\u00a0\u00b1\u00a00.10; gray bars in Fig.\u00a04a) than those between the flexion half cycle and the following extension half cycle (\u22120.02\u00a0\u00b1\u00a00.17; black bars in Fig.\u00a04a). The consequences of Ryy and Rxy for RFC are illustrated in Fig.\u00a04c, d, respectively, which show the contributions of the covariances underlying Ryy and Rxy to RFC, according to the relations derived in the Appendix. The temporal relations expressed by Ryy and Rxy both result in a reduction of RFC (i.e., they reduce the effects of the half cycle error corrections, see \u201cMaterials and methods\u201d); the effect of the former being larger than that of the latter (compare Fig.\u00a04c, d). Both correlations resulted in a larger reduction of RFC for errors at peak extension than for errors at peak flexion, which explains the aforementioned effect of Orientation obtained for RFC. In addition, the effect of Activity for Ryy revealed that the error corrections in successive half-cycles were more correlated for the active condition than for the relaxed condition. This implied that in particular in the active condition a part of the adjustments of the half-cycle durations (captured by RHC) compensated for overcorrection in the preceding half-cycle.\nFig.\u00a04Mean correlations of the durations of successive half-cycles obtained during in-phase (IP) and antiphase (AP) coordination, while the muscles of the left (driven) hand were active (AC) or relaxed (RE): a within-hand correlations (Ryy); b between-hands correlations (Rxy). c and d indicate the relative contributions of the covariances underlying the within-hand and between-hands factors (Cyy and Cxy, respectively) by expressing them as a fraction of the covariance underlying RFC (i.e., CFC): negative values indicate a reduction of the absolute value of RFC (see Appendix). To facilitate comparison the values of Ryy and Rxy are presented in the same order as the corresponding values of RFC in Fig.\u00a03. Error bars indicate the standard error of the mean. The legends of a and b also apply to c and d, respectively. Legends: FHC\u00a0=\u00a0flexion half cycle; EHC\u00a0=\u00a0extension half cycle; the arrows specify the temporal order of the half cycles. Whereas the second half cycle is always performed by the right hand, the first half cycle is performed either by the right hand (Ryy and Cyy, a and c) or by the left hand (Rxy and Cxy, b and d)\nEMG\nThe average amplitudes of the EMGs (normalized to MVC amplitude) during the movement cycle are presented in Fig.\u00a05, which exposes five noteworthy characteristics of the EMG data. First, in the right arm the EMG amplitudes were smaller in the relaxed condition than in the active condition (compare open and filled symbols, respectively, in Fig.\u00a05a, b), corresponding to the observed differences in range of motion between these conditions. Second, in line with the task requirements, the EMG amplitudes of the muscles in the left arm were in general much smaller in the relaxed condition than in the active condition (compare open and filled symbols, respectively, in Fig.\u00a05c, d). Third, for left FCR in the relaxed condition a distinctive (reactive) peak in the EMG amplitude was observed at bin 5, i.e., the moment at which the lengthening of the muscle started (open symbols in Fig.\u00a05c). Fourth, for left ECR in the relaxed condition the EMG amplitude showed a slight modulation during the movement cycle that mimicked that of normal activation patterns (compare open symbols in Fig.\u00a05d with the curves in Fig.\u00a05b). Fifth, both left FCR and ECR in the active condition (filled symbols in Fig.\u00a05c, d, respectively) were shifted leftward with respect to the (motor-driven) movement compared to the timing of right FCR and ECR when compared to the (self-generated) movement (Fig.\u00a05a, b, respectively). Note that this phase advance of the EMG in the left arm relative to the corresponding hand motion was possible because (in contrast to the right hand) the movements of the (motor-driven) left hand were completely independent of its muscle activity.\nFig.\u00a05Normalized EMG amplitudes (RMS values) of FCR (a right; c left) and ECR (b right; d left), averaged over subjects, at eight phases of the movement cycle. Each graph shows the muscle activity for the conditions in which the muscles of the left (driven) arm were either active (filled symbols) or relaxed (open symbols), for in-phase (triangles; solid lines) and antiphase coordination (circles; dashed lines) separately\nWeighted coherence\nThe weighted coherence of the rEMG of homologous muscles is shown in Fig.\u00a06. Statistical analysis using a 2 (Pattern)\u00a0\u00d7\u00a02 (Activity)\u00a0\u00d7\u00a02 (Muscle) repeated measures ANOVA revealed significant effects of Activity (F(1,\u00a09)\u00a0=\u00a0103.98; P\u00a0<\u00a00.001; f\u00a0=\u00a03.39) and Muscle (F(1,\u00a09)\u00a0=\u00a017.70; P\u00a0<\u00a00.01; f\u00a0=\u00a01.40), as well as a significant Activity\u00a0\u00d7\u00a0Pattern interaction (F(1,\u00a09)\u00a0=\u00a010.68; P\u00a0<\u00a00.05; f\u00a0=\u00a01.09). The effect of Activity revealed that the weighted coherence was larger in the active condition than in the relaxed condition (0.85\u00a0\u00b1\u00a00.25 vs. 0.54\u00a0\u00b1\u00a00.63). The effect of Muscle showed that the weighted coherence was larger for ECR than for FCR (0.77\u00a0\u00b1\u00a00.38 vs. 0.62\u00a0\u00b1\u00a00.54), indicating a greater similarity of the EMG activity for extensor than for flexor muscles. Post hoc analysis of the Activity\u00a0\u00d7\u00a0Pattern interaction revealed no significant differences between in-phase and antiphase coordination for either the active condition or the relaxed condition (or vice versa), although the weighted coherence was larger for in-phase than for antiphase in the active condition (0.86\u00a0\u00b1\u00a00.22 vs. 0.83\u00a0\u00b1\u00a00.32), but smaller for in-phase than for antiphase in the relaxed condition (0.51\u00a0\u00b1\u00a00.70 vs. 0.57\u00a0\u00b1\u00a00.60).\nFig.\u00a06Mean weighted coherence (CW) of the rectified EMGs of homologous muscles: FCR (black) and ECR (gray) during in-phase (IP) and antiphase (AP) coordination, both for the conditions in which the muscles of the left (motor-driven) hand were active (AC) or relaxed (RE). Error bars indicate the standard error of the mean\nTemporal relations in the active condition\nIn the active condition, the relative phase was determined at two levels: at the behavioral level (kinematics) and at the level of the neural control signals (based on rEMG). This distinction is important because for the right hand the phase shift between EMG activity and kinematics was constrained by the effector dynamics of the wrist (Ridderikhoff et al. 2004), whereas these constraints did not affect the phase shift for the left (motor-driven) hand. Hence, the two levels were not tightly coupled in this kinesthetic tracking task. To examine the relations between these levels, four different phase relations were studied in conjunction: the phase shifts between neural control signals and behavior for (1) the right hand and (2) the left hand, and the relative phase between the limbs at (3) the behavioral level and (4) the level of neural control signals. The results regarding the relative phase at the behavioral level were presented in the preceding. In the next two subsections the other phase relations (i.e., the phase shifts between rEMG and kinematics and the relative phase between rEMGs of homologous muscles) are presented. The results are combined in the final subsection to provide an overall picture of the temporal relations in the active condition across these different levels.\nPhase shifts between EMG and kinematics\nIn the active condition differences between the left and right hand were observed with respect to the timing of the muscle activity relative to the ongoing movement (see Fig.\u00a05). To analyze these differences, the phase shifts between rEMG and kinematics (see Table\u00a01; negative values indicate that the EMG is leading the kinematics) were examined using a 2 (Hand)\u00a0\u00d7\u00a02 (Pattern)\u00a0\u00d7\u00a02 (Muscle) repeated measures ANOVA. This revealed a significant effect of Hand (F(1,\u00a09)\u00a0=\u00a011.79; P\u00a0<\u00a00.01; f\u00a0=\u00a01.14), indicating larger phase shifts for the left hand than for the right hand (\u2212145.3\u00b0 vs. \u2212114.4\u00b0). In addition, significant Hand\u00a0\u00d7\u00a0Muscle (F(1,\u00a09)\u00a0=\u00a014.21; P\u00a0<\u00a00.01; f\u00a0=\u00a01.26), Hand\u00a0\u00d7\u00a0Pattern (F(1,\u00a09)\u00a0=\u00a06.24; P\u00a0<\u00a00.05; f\u00a0=\u00a00.83), and Hand\u00a0\u00d7\u00a0Pattern\u00a0\u00d7\u00a0Muscle (F(1,\u00a09)\u00a0=\u00a06.08; P\u00a0<\u00a00.05; f\u00a0=\u00a00.82) interactions were found. Post hoc analysis demonstrated that the Hand\u00a0\u00d7\u00a0Muscle interaction occurred because the phase shifts for ECR and FCR differed significantly for the right hand (\u2212101.5\u00b0\u00a0\u00b1\u00a021.2\u00b0 vs. \u2212127.4\u00b0\u00a0\u00b1\u00a014.9\u00b0), but not for the left (motor-driven) hand. The Hand\u00a0\u00d7\u00a0Pattern interaction post hoc analysis revealed that the phase shifts during in-phase and antiphase coordination differed significantly for the left (motor-driven) hand (\u2212149.9\u00b0\u00a0\u00b1\u00a023.7\u00b0 vs. \u2212140.6\u00b0\u00a0\u00b1\u00a027.2\u00b0), but not for the right hand. Post hoc analysis of the three-way interaction revealed that the phase shift for the left FCR during antiphase coordination deviated from the overall pattern: it was significantly different from the phase shift during in-phase coordination (in the absence of significant differences between in-phase and antiphase for any of the other muscles), and it was not significantly different from the phase shift for right FCR during antiphase coordination (whereas all other left-right comparisons yielded significant differences for both coordinative patterns).\nTable\u00a01Phase shifts between rectified EMG and joint angles in the active condition (mean\u00a0\u00b1\u00a0between-subjects SD) as determined from the cross-spectrum of these variables at the movement frequencyHandMuscleIn-phaseAntiphaseLeftFCR\u2212148.2\u00b0\u00a0\u00b1\u00a022.1\u00b0\u2212137.5\u00b0\u00a0\u00b1\u00a022.5\u00b0ECR\u2212151.7\u00b0\u00a0\u00b1\u00a027.8\u00b0\u2212143.7\u00b0\u00a0\u00b1\u00a033.5\u00b0RightFCR\u2212123.1\u00b0\u00a0\u00b1\u00a014.2\u00b0\u2212131.6\u00b0\u00a0\u00b1\u00a017.7\u00b0ECR\u2212103.5\u00b0\u00a0\u00b1\u00a017.7\u00b0\u221299.5\u00b0\u00a0\u00b1\u00a015.2\u00b0\nRelative phasing of EMG\nThe relative phase between the rEMG of homologous muscles in the active condition was calculated for FCR and ECR. The constant errors with respect to the reference values for in-phase (0\u00b0) and antiphase (180\u00b0) coordination (see Table\u00a02) were subjected to a 2 (Pattern)\u00a0\u00d7\u00a02 (Muscle) repeated measures ANOVA. The analysis revealed significant effects of Pattern (F(1,\u00a09)\u00a0=\u00a012.19; P\u00a0<\u00a00.01; f\u00a0=\u00a01.16), Muscle (F(1,\u00a09)\u00a0=\u00a016.20; P\u00a0<\u00a00.01; f\u00a0=\u00a01.32), and a significant Pattern\u00a0\u00d7\u00a0Muscle interaction (F(1,\u00a09)\u00a0=\u00a05.23; P\u00a0<\u00a00.05; f\u00a0=\u00a00.76). The effect of Pattern resulted in a significantly larger constant error for in-phase than for antiphase coordination (12.5\u00b0\u00a0\u00b1\u00a018.0\u00b0 vs. 0.3\u00b0\u00a0\u00b1\u00a024.0\u00b0; positive values indicate a relative phase lead of the activity in the left arm). The effect of Muscle indicated that the average constant errors for FCR and ECR were significantly different (\u22129.3\u00b0\u00a0\u00b1\u00a026.9\u00b0 vs. 22.2\u00b0\u00a0\u00b1\u00a020.6\u00b0). Post hoc analyses of the Pattern\u00a0\u00d7\u00a0Muscle interaction revealed a significant difference between the constant errors for in-phase and antiphase coordination for FCR but not for ECR (see values in Table\u00a02).\nTable\u00a02Mean constant errors in the relative phase for kinematics and EMG in the active condition (mean\u00a0\u00b1\u00a0between-subjects SD)LevelIn-phaseAntiphaseKinematics\u221222.7\u00b0\u00a0\u00b1\u00a010.4\u00b0\u221224.8\u00b0\u00a0\u00b1\u00a011.4\u00b0EMG\u00a0FCR0.1\u00b0\u00a0\u00b1\u00a026.2\u00b0\u221218.8\u00b0\u00a0\u00b1\u00a028.5\u00b0\u00a0ECR24.9\u00b0\u00a0\u00b1\u00a019.0\u00b019.4\u00b0\u00a0\u00b1\u00a024.7\u00b0\u00a0Mean12.5\u00b0\u00a0\u00b1\u00a018.0\u00b00.3\u00b0\u00a0\u00b1\u00a024.0\u00b0For the EMG the obtained values are presented for homologous FCR and ECR separately. The mean of the constant errors for FCR and ECR was adopted as the constant error of the neural control signal. Negative (positive) values indicate that the right limb is leading (lagging) the left limb\nRelative phasing of neural control signals and behavior\nTo gain insight into the neurobehavioral basis of error correction, we examined the phase relations between the neural control signals and the overt behavior in the active condition. For this purpose, the phasing of the neural control signals was estimated for each limb separately by taking the average of the values obtained for FCR and ECR (see Tables\u00a01, 2). The resulting phase relations are displayed schematically for in-phase and antiphase coordination in Fig.\u00a07, which illustrates the muscle-independent phase relations addressed in the previous two paragraphs, showing only the effects of Hand and Pattern. First, the phase shift between the neural control signal and the wrist movements was larger for the (motor-driven) left hand (dashed arrows) than for the right hand (solid arrows)\u2014significant effect of Hand. Second, the constant error in relative phase between the EMGs of homologous muscles was larger for in-phase than antiphase coordination (compare the corresponding diagrams)\u2014significant effect of Pattern. Third, it can be appreciated from the figure that the phase shift between EMG and kinematics (larger shifts in the left hand, i.e., longer dashed arrows, for in-phase than for antiphase coordination) depended on the combination of hand and pattern\u2014significant Hand\u00a0\u00d7\u00a0Pattern interaction. Finally, the diagrams indicate that, on average, the error in the relative phase was smaller for the neural control signal than for the kinematics (respective values are presented in Table\u00a02).\nFig.\u00a07Diagrams illustrating temporal relations in the active condition for in-phase (left panel) and antiphase (right panel) coordination. Circles represent signal sources: \u03b8 is the joint angle, and \u03be is the neural control signal (derived from the EMG, averaged over FCR and ECR). The length of the arrows indicates the phase shifts between the neural control signal and the kinematics (arrows point in the direction of time). The error of the relative phase \u03a6 is equal to the inclination angle of the line connecting the circles at either the kinematic (\u03b8) or the control level (\u03be). The target values of \u03a6 are indicated by the gray horizontal lines, and the dotted lines represent the relative error in \u03a6 in the relaxed condition (available for kinematics only)\nDiscussion\nThe goal of the present study was to gain more insight into the error correction processes as allegedly implicated in bimanual coordination (Ridderikhoff et al. 2005b). To this end, we examined the temporal adjustments of the actively moving (right) limb in a kinesthetic tracking task based on the perceived (error in the) relative phase between the limbs. In particular, subjects tracked their motor-driven left hand with the actively moving right hand to achieve in-phase or antiphase coordination, in conditions where the left hand musculature was activated (as if moving along with the reference movement) or kept as relaxed as possible. In addition, the relation between bimanual (between-limbs) and unimanual (within-limb) factors was examined. Unimanual and bimanual factors were dissociated by comparison of several (related) temporal correlations and by examining the phase shifts between neural control signals and kinematics both within and between limbs. In line with our research questions the analysis focused on two aspects of the measurements, which are discussed in detail in the next two subsections: (1) differences in error correction between in-phase and antiphase coordination, and (2) effects of muscle activity in the driven (left) limb on these error corrections and on the overall performance of the task. In the last two subsections, we will then address the observed differences between the left and right hand and between flexors and extensors in terms of the phase shifts between EMG and kinematics and the relative timing of flexion and extension. Before embarking on these discussions, it should be noted that the stability of the bimanual patterns was affected by the muscle activity in the motor-driven limb, as SD\u03a6 was larger in the relaxed condition than in the active condition. Furthermore, the variability of the relative phase (SD\u03a6) was smaller for in-phase than for antiphase coordination. This basic observation confirmed the result of an earlier study on kinesthetic tracking (Stinear and Byblow 2001) and indicated that also in normal bimanual coordination the stability difference of in-phase and antiphase coordination may be partly dependent on afference-based error corrections.\nError corrections\nWe performed an in-depth analysis of the corrections in the timing of the movements of the right (actively moving) limb based on the perceived errors in the relative phase. Since our analyses of the kinematics concerned the timing of discrete events (i.e., peak extension and peak flexion), the shortest possible time scale to study error corrections was related to compensatory adjustments of the duration of the half-cycle following the detected error. The related measure (RHC) indicated that the error corrections were more pronounced for in-phase than for antiphase coordination and that they were characterized by larger absolute values for the active condition than for the relaxed condition, in line with the observed effects for SD\u03a6. Additional analysis of the compensatory adjustments of the duration of the full cycle following the detected error (RFC) showed that both error correction effects persisted on a longer time scale. Therefore, we conclude that corrections in the movement timing of the right hand based on the perceived errors in the relative phase underlie the observed stability effects (SD\u03a6) of the examined coordination patterns (in-phase vs. antiphase) and muscle activity.\nIt is important to consider error corrections at different time scales, because the dependency of the corrections performed in successive half-cycles may vary over conditions (see \u201cMaterials and methods\u201d). The results revealed that in the present study these dependencies between successive half-cycles were reflected mainly by the within-hand correlation Ryy, indicating an important role for the unimanual coordinative processes underlying the rhythmic movements of the right hand. This role is of particular importance for the interpretation of the significant Pattern\u00a0\u00d7\u00a0Activity interaction observed for RHC, suggesting more pronounced error corrections during in-phase coordination in the active condition. Because the results obtained for Ryy demonstrated larger dependencies between successive half-cycles in the active condition than in the relaxed condition, the effects related to muscle activity were reduced at a longer time scale (RFC). As a consequence, the Pattern\u00a0\u00d7\u00a0Activity interaction was not significant for RFC and, thus, appeared to be of little functional relevance in terms of the coordinative stability. This illustrates how unimanual coordination (the flexion\u2013extension coupling in terms of Ryy) may impinge on the effects of interlimb interactions (the error corrections in terms of RHC) in bimanual coordination.\nTwo additional discrepancies between RHC and RFC were found in relation to effects of the orientation of the hand (flexion or extension). This was not surprising, because RHC and RFC are unrelated by definition with respect to these effects. As shown both analytically (for the underlying covariances, see Appendix) and empirically all differences between flexion and extension in RFC are attributable to the correlations between the durations of successive half-cycles within the right hand (Ryy) and between hands (Rxy). Specifically, the effect of orientation of the hand obtained for RFC was not related to error corrections based on the perceived relative phase, but rather reflected an asymmetry in the coupling of the unimanual flexion and extension half-cycles as captured by Ryy. In contrast, differences in the strength of the error corrections during the flexion and extension half-cycles are indicated by effects of orientation observed for RHC. Analysis of RHC revealed a significant Pattern\u00a0\u00d7\u00a0Orientation interaction, which indicated more pronounced error corrections in the half-cycle following peak flexion of the left (motor-driven) hand, in particular in the extension half-cycle during in-phase coordination (Fig\u00a03b). In line with this result, we observed the smallest errors in the mean relative phase at the end of the half-cycles with the most prominent error corrections (i.e., at peak extension during in-phase and at peak flexion during antiphase coordination; see Fig\u00a02a). A tentative explanation of these findings is that (relative) timing errors were detected most accurately at peak flexion of the driven hand and that, as a consequence, the corrections in the following half-cycle were most effective, leading to the highest accuracy in the relative phase at the end of these half-cycles.\nWith respect to the two adopted measures of error correction (RHC and RFC) these analyses demonstrated that both measures should be evaluated in conjunction to assess the net effect of error corrections in relation to the effects of movement pattern and muscle activity, whereas RHC offers additional insight into the differences between flexion and extension in this respect. The analyses of the temporal correlations revealed several important global characteristics of the afference-based error corrections underlying rhythmic bimanual coordination. Although the analyses demonstrated that error corrections occur during the next half-cycle, it is important to emphasize that the precise time course of the error corrections remains unknown. That is, these methods do not reveal how long it takes until the ongoing movements are adapted to compensate for perceived errors, except for showing that such changes occur within the next half-cycle. Although, in principle, it is possible that timing errors are detected throughout the cycle and not only at peak flexion and peak extension, there is ample evidence indicating that coordinated rhythmic movements are characterized by the presence of so-called anchor points (Beek 1989), that is, discrete points in the cycle that have particular significance for the control of timing. Furthermore, several studies have demonstrated that anchoring occurs when peak flexion and\/or peak extension are coinciding with an external stimulus (Byblow et al. 1994; Fink et al. 2000). These findings provide a rationale for the use of the moments of peak flexion and peak extension in the analysis of the temporal structure of the bimanual coordination pattern, and the instructions to subjects in terms of the timing of peak flexion and peak extension (see \"Materials and methods\") may have helped to establish such anchor points in the present study. However, further research regarding the existence of anchor points in the processing of kinesthetic feedback and the time course of the adjustments is required, for which the analyses in the present study may serve as departure point. For our current purposes, RHC and RFC provided important insights into the average amount of corrective activity that occurs at two relevant time scales in terms of the same kinematic measures that were used to assess the performance in terms of the relative phase between the hands.\nThe effects of muscle activity in the driven limb\nThe second objective of this study was to examine the neurobehavioral basis of error correction. To this aim, we manipulated the muscle activity in the motor-driven limb by instructing subjects either to activate their muscles in accordance with the ongoing motion or to keep them as relaxed as possible. The average EMG amplitudes (Fig.\u00a05) clearly showed that this manipulation of muscle activity was successful in the sense that subjects managed by and large to avoid activating the muscles in the motor-driven left hand in the relaxed condition. This was further underscored by the fact that the values of the weighted coherence of rEMGs of homologous muscles in the relaxed condition did not exceed those obtained in previous studies involving passive (i.e., motor-driven) hand movement (Ridderikhoff et al. 2005b; Ridderikhoff et al. 2006). In those studies subjects had been explicitly instructed to ignore the motor-driven movement (i.e., no coordination between the hands was required) in order to study phase entrainment by contralateral afference, implying that the resulting values of the weighted coherence can be regarded as a baseline for the effect of motor-driven movement per se. In contrast, in the active condition the changes in the EMG amplitude of the motor-driven hand resembled those observed for actively performed rhythmic wrist movements (as obtained for the right hand), and significantly larger coherence of the homologous muscle activity was observed.\nFor a proper evaluation of the adopted methodology it is relevant to note that the experiment actually involved a dual task paradigm, because in all conditions subjects were instructed to perform the required bimanual pattern and at the same time control the muscle activity in the driven arm. The additional cognitive load that is associated with performance of a secondary task may have augmented the intrinsic stability differences between in-phase and antiphase coordination (Temprado et al. 1999). However, the results did not indicate a differential effect of the two secondary tasks (active vs. relaxed condition) on the stability difference between in-phase and antiphase (i.e., no Activity\u00a0\u00d7\u00a0Pattern interactions were observed for SD\u03a6). From this we conclude that the additional cognitive load imposed by the secondary task was the same for the active and relaxed condition, and that the dual task paradigm did not confound the comparison between these conditions. In other words, we are confident that the observed differences between these conditions can be ascribed to the differences in muscle activity associated with these conditions, rather than differences in cognitive load. On the other hand, the dual task character of the present manipulations may explain why the present results regarding the SD\u03a6 of in-phase and antiphase coordination agreed with those reported in the study of Stinear and Byblow (1999), in which an additional amplitude matching of the movements was required, but not with our previous (single task) experiment on kinesthetic tracking (Ridderikhoff et al. 2005b).\nMuscle activity clearly had a beneficial effect on coordinative stability, as the variability of the relative phase was significantly smaller in the active condition than in the relaxed condition (Fig.\u00a02b). In addition, the adopted measures related to the error correction processes underlying kinesthetic tracking, showing larger compensatory adjustments of the timing of the right hand in the active condition (RHC and RFC; Fig.\u00a03), indicated that kinesthetic afference was processed more effectively in this condition. Consequently, it seems unlikely that the muscle activity during kinesthetic tracking that we observed in our previous study (Ridderikhoff et al. 2005b) was merely a byproduct due to neural cross-talk.2 However, in addition to these benefits of muscle activity, we also found that the absolute (and constant) errors in the mean relative phase were larger in the active condition than in the relaxed condition (Fig.\u00a02a), indicating that the coordinative pattern was performed less accurately in the former condition. Thus, muscle activation during kinesthetic tracking resulted in more pronounced and proficient error corrections in bimanual timing, but these corrections appeared to be performed with respect to less accurate reference values.\nIn explaining this reduced accuracy in the active condition it proved essential to dissociate the temporal relations between the limbs observed at the level of neural control signals and those observed at the behavioral level, because these levels may involve separate reference signals. In the relaxed condition only the relative phasing at the behavioral level (kinematics) is available as reference for the error corrections. In contrast, the presence of a bilateral neural control signal in the active condition implies that the relative phase between the neural control signals (or motor outflow) may also be used as reference (e.g., by means of anticipation of the sensory consequences of the ongoing movement based on efference copies). Comparison of the constant errors at the two levels (Table\u00a02) suggested an attraction toward the proper timing of the neural control signals, resulting in larger errors in the relative phase at the behavioral level (Fig.\u00a07). This observation also revealed a discrepancy between the relative phases at the two levels, which resulted from an inadequate timing of the neural control signals of the motor-driven hand as explained in the next subsection.\nThis interpretation of the results underscores that bimanual coordination benefited from muscle activity during kinesthetic tracking because the bilateral motor commands were used (e.g., via efference copy) as a reference signal for movement-elicited afference. The presence of this reference signal, which presumably allowed for prediction of the sensory consequences of the bimanual movement, resulted in more prominent error corrections, leading to a smaller variability of the relative phase in the active condition. Thus, the evidence suggested that an intimate sensorimotor integration, which has been proposed in the context of discrete goal-directed movements (e.g., Wolpert and Ghahramani 2000; Scott 2004), also underlies the coordination of rhythmic bimanual movements. In the context of the present study this interpretation can account for the observed effects of muscle activation on the stability as well as the accuracy of the relative phase.\nTiming of the EMG activity in the motor-driven arm\nUnlike the phase shifts between EMG and kinematics of the left (motor-driven) arm observed in the present study, the muscle activity observed during kinesthetic tracking in our previous study (Ridderikhoff et al. 2005b) was adequately timed with respect to the ongoing motor-driven movement. A possible explanation for this unexpected discrepancy between these two studies may be related to the instructions given to the subjects. In the active condition of the present study subjects were explicitly instructed to activate their muscles, whereas subjects did this spontaneously (i.e., without instruction) in our previous study. Given the instruction to activate the muscles as if moving along with the passive movement, subjects seemed to have aimed at generating contact forces that gave the impression that they were pushing or pulling the manipulandum in the correct direction. Comparison of the EMG amplitudes in the left (motor-driven) arm and the right (actively moving) arm (Fig.\u00a05) indicated that this was accomplished by an earlier activation of the muscles in the left arm (relative to the ongoing movement) and an increase of the EMG amplitude (i.e., an increase of the muscle torque). These changes substantially increased the forces acting between the hand and the manipulandum in the direction of the ongoing movement. Note also that the EMG activity was minimal at the moments of peak excursion, i.e., when the movement changed direction (see Fig.\u00a05). In other words, the results suggested that subjects in effect sought after the expected sensory consequences of moving something. This may be regarded as a misinterpretation of the instruction, because truly moving along requires the elimination of contact forces altogether.\nFlexor\u2013extensor differences during active rhythmic wrist movement\nIn addition to the effects of afference-based interlimb interactions (as reflected in the error corrections) the flexion and extension phases were also affected by the unimanual control processes underlying the rhythmic movement of the (right) wrist itself. In this regard, an asymmetry was found in the correlations between successive flexion and extension half-cycles (Ryy), indicating that the durations of the flexion half-cycles were adapted more to changes in the duration of the extension half-cycle than vice versa (Fig.\u00a04a). Studies on unilateral rhythmic movements have also reported an asymmetric relation between flexion and extension half-cycles in rhythmic finger movements (Balasubramaniam et al. 2004), and a more adequate temporal control of wrist flexion than wrist extension (Carson 1996; Carson and Riek 1998). An adaptive relation between the relative timing of FCR and ECR on the one hand and the resulting wrist movements on the other hand was also indicated by the analysis of the phase shifts between EMG and kinematics. Different phase shifts were obtained for FCR and ECR in the right hand but not in the left hand, in which timing of EMG and kinematics were unrelated. Such differences in the relative timing of FCR and ECR activity during rhythmic wrist movements have been reported before (Ridderikhoff et al. 2004), and may reflect adaptations to the different physiological properties of these muscles that would affect the movement trajectories unless compensated for. Furthermore, the EMG analysis revealed that the coherence between left and right ECR was larger than the coherence between left and right FCR, may be related to the finding that neural cross-talk effects were larger for extensor than for flexor muscles (Ridderikhoff et al. 2005a). These combined results point to a more independent control of the timing of FCR in the context of rhythmic wrist movement.\nConclusion\nThe present study revealed two characteristics of the error corrections based on the kinesthetically perceived relative phase that are relevant to the understanding of rhythmic bimanual coordination, and supplement the findings of our previous study on the sources of interlimb interaction involved in this type of task (Ridderikhoff et al. 2005b). First, the corrections were more effective during in-phase than during antiphase coordination, resulting in a smaller variability of the relative phase in the former condition. This effect may be attributable to differences in the kinesthetic perception of variability in in-phase and antiphase coordination (Wilson et al. 2003), and indicated that afference-based error corrections contribute to the well-known stability difference of these bimanual patterns (Kelso 1984; Swinnen 2002). Thus, although somatosensory feedback is not essential for the stability differences between in-phase and antiphase (Spencer et al. 2005), the present study showed that afference-based error corrections augment the stability difference resulting from open-loop processes underlying the integrated timing of the efferent signals to both limbs (Ridderikhoff et al. 2005b). Second, the corrections (and, as a consequence, pattern stability) were enhanced if the muscles in the motor-driven arm were activated as if the limb was moving along with the motor. On the other hand, the accuracy of the relative phasing between the limbs was decreased in this situation, indicating that the corrections were based on a different reference signal than in the relaxed condition. The most likely explanation for these findings was that the bilateral neural control signal provided a reference for the evaluation of the kinesthetic afference, in the sense that on the basis of this signal the sensory consequences (of the bimanual movement pattern) could be anticipated. A closed-loop control process that exploits this kind of prediction is apparently more effective in terms of the stabilization of the relative phase. This result suggests an intimate relation between the integrated timing of the feedforward signals (underlying the bilateral activation pattern if both hands are moving actively) and the use of kinesthetic feedback in rhythmic bimanual coordination","keyphrases":["bimanual coordination","electromyography","proprioception","rhythmic movement","kinesthesis"],"prmu":["P","P","P","P","P"]}
{"id":"Anal_Bioanal_Chem-4-1-2259237","title":"Discrimination of biofilm samples using pattern recognition techniques\n","text":"Biofilms are complex aggregates formed by microorganisms such as bacteria, fungi and algae, which grow at the interfaces between water and natural or artificial materials. They are actively involved in processes of sorption and desorption of metal ions in water and reflect the environmental conditions in the recent past. Therefore, biofilms can be used as bioindicators of water quality. The goal of this study was to determine whether the biofilms, developed in different aquatic systems, could be successfully discriminated using data on their elemental compositions. Biofilms were grown on natural or polycarbonate materials in flowing water, standing water and seawater bodies. Using an unsupervised technique such as principal component analysis (PCA) and several supervised methods like classification and regression trees (CART), discriminant partial least squares regression (DPLS) and uninformative variable elimination\u2013DPLS (UVE-DPLS), we could confirm the uniqueness of sea biofilms and make a distinction between flowing water and standing water biofilms. The CART, DPLS and UVE-DPLS discriminant models were validated with an independent test set selected either by the Kennard and Stone method or the duplex algorithm. The best model was obtained from CART with 100% correct classification rate for the test set designed by the Kennard and Stone algorithm. With CART, one variable describing the Mg content in the biofilm water phase was found to be important for the discrimination of flowing water and standing water biofilms.\nIntroduction\nWater quality assessment requires monitoring of carefully selected parameters. Usually water and sediment samples are collected in the hope that their chemical compositions will help to understand the nature of a given local or global environmental event. The chemical analysis of water indicates the water quality at the time of sampling, while analysis of sediments provides information about long-term environmental changes in aquatic systems. Biofilms reflect the environmental conditions in the recent past [1]. They are complex communities composed of microorganisms, which grow at almost any water\u2013substrate interface. Biofilms can accumulate metal ions and play an important role in the processes of sorption and desorption of chemical elements [2]; therefore, they can be very useful bioindicators of water quality.\nThe aim of this work is to investigate whether the biofilms grown in different aquatic systems can be discriminated on the basis of their chemical compositions. If the discrimination is possible, the next interesting question will be which measured parameters are responsible for it.\nFor the purpose of the study, biofilm samples originating from different water bodies such as flowing water, standing water or seawater were collected. The elemental concentrations of biofilms and the water phase extracted at the sampling locations were then analysed using inductively coupled plasma optical emission spectrometry (ICP-OES) and inductively coupled plasma mass spectrometry (ICP-MS), respectively. To obtain useful information on the data collected, several pattern recognition techniques are going to be applied. These are principal component analysis (PCA), classification and regression trees (CART), discriminant partial least squares regression (DPLS) and uninformative variable elimination\u2013DPLS (UVE-DPLS). PCA is an unsupervised pattern recognition method that aims to compress and to visualise the data structure, which allows for an easy interpretation of relationships between samples and the measured parameters. The supervised pattern recognition methods like CART, DPLS and UVE-DPLS aim to develop classification or decision rule(s) using a set of samples with known group origin. Then the classification rule(s) determines the belongingness of unknown samples to the available groups [3]. The application of such approaches in our study will provide a better understanding of the accumulation behaviour of different biofilms.\nExperimental\nDescription of the sampling procedure\nThe biofilm samples investigated can be divided into two groups: a group of systematically sampled biofilms and a group of uniquely sampled biofilms. The samples of the group of systematically sampled biofilms can be further split into two subgroups, namely, biofilms grown on polycarbonate plates and biofilms developed on natural substrates. A detailed description of the samples collected is presented in Table\u00a01.\nTable\u00a01Description of the biofilm and water samples collectedGroup of biofilmsSubgroup of biofilmsCharacter of the water phaseNumber of samplesSystematically sampled biofilmsBiofilms grown on polycarbonate platesf\u2014the Saale river11s\u2014the Teich pond22Biofilms grown on natural substratesf\u2014the Leutra river13Uniquely sampled biofilmsBiofilms grown on natural substratesf\u2014Celle (a, b), Lauscha (a, b, c), Oberp\u00f6llnitz, Falken, London, Munich, New York, Geithain, Steinach, Juquitiba12s\u2014Chemnitz, New Hampshire, Bossow, Metebach, Erfurt, Rippachtal6m\u2014Travem\u00fcnde (a, b), Punta Skala, Nin, Majorca, Damp, Steinbeck7a, b and c denote different sampling locations.f a body of flowing water, s a body of standing water, m a body of seawater\nThe biofilm samples collected in the Saale river, in a pond and in the Leutra river were systematically sampled, i.e. the biofilm and water samples were gathered within a definite period of time. The Saale river was chosen as a body of flowing water. The river flows through highly populated regions, which are heavily industrialised and are subjects of typical geogenic and anthropogenic pollution [4]. The Saale water and biofilm samples were collected within a 2-year period (from September 2003 till October 2005). The sampling point was situated in the village of Kunitz located downstream of Jena (Thuringia, Germany). This place was preferred, because it reflects a characteristic pollution of the nearby town. The Saale river is there about 1.5\u00a0m deep and Kunitz is remote from civilisation. These conditions facilitated the sampling campaign.\nA small pond of 0.75-m depth was selected as a typical example of a standing water body. It was located in the city of Jena. The sampling campaign duration was the same as for the sampling campaign in the Saale river. At these two locations, the biofilms were artificially grown on polycarbonate plates (10\u00a0cm \u00d7 10\u00a0cm) exposed vertically to the water (in the Saale river, in a streaming direction). The plates were fixed into polypropylene boxes, approximately 10\u00a0cm under the water surface and 1\u00a0m away from the riverbank. After a definite time of exposure, the biofilm samples were immediately transferred into plastic boxes filled with the river or pond water and transported to the laboratory. The plates were then washed with bidistilled water and the biofilm samples were scraped off the whole surface of the polycarbonate plates using a Teflon spatula. The river and pond water samples were collected every 2\u00a0weeks.\nThe Leutra river, located in the city of Jena, was chosen as the second example of a flowing water body. The stony bed of the river, its small depth and good accessibility facilitated the sampling campaign. The biofilm samples from the Leutra river were scraped off the riverbed stones using a plastic spatula. They were placed into polyethylene bottles and transported to the laboratory. The sampling campaign at the Leutra river was held in autumn 2005 and in spring 2006. Additionally, water samples were collected.\nThe sampling procedure, for water and uniquely sampled biofilms, was the same as that carried out for the Leutra river. The locations of the sampling sites were selected according to the availability of a suitable sampling device. The samples collected were also placed into polyethylene bottles and transported to the laboratory.\nAnalytical procedure\nThe biofilm samples collected were air-dried at 105\u00a0\u00b0C. Then, the samples containing 10\u201350\u00a0mg biofilm powder were dissolved in 3\u00a0ml of 70\u201372% perchloric acid and were heated for 3\u00a0h at 50\u00a0\u00b0C. The remaining dry matter of each sample was further dissolved in water so that the resulting solution was up to 5\u00a0ml. A Fisons Instruments (Beverly, MA, USA) Maxim 112 inductively coupled plasma optical emission spectrometer was used to analyse Al, Ca, Fe, K, Mg, Mn, Na and Sr, while Cd, Co, Cr, Cu, Ni, P, Se and Zn were determined with a PerkinElmer (Wellesley, MA, USA) Elan 6000 inductively coupled plasma mass spectrometer. An external aqueous calibration was adopted for the analysis by ICP-OES, while a standard addition procedure was used for the element analysis by ICP-MS. All contents correspond to the sample dry weight. The trueness of the measurements was tested by analysing a certified reference algae material. The element contents were certified for an aqua regia digestion. Additionally, the element contents were determined after microwave digestion with nitric acid. No differences between these two digestion methods were obtained. All the measurements were done in triplicate and the relative standard deviation of the technique was 10\u201315% for all the biofilms, indicating good repeatability of the measurements.\nTheory\nClassification and regression trees\nThe CART method was proposed by Breiman et al. [5], for data modelling and classification. Depending on the type of the response variable, y (categorical or continuous), either classification or regression trees are built. In the present study, we will focus on constructing classification trees only. The goal of CART is to form a set of mutually exclusive regions in the data space, containing as homogeneous groups of objects as possible. This is achieved by finding optimal splits of some suitable explanatory variables at a given threshold value, such that a defined impurity function is minimised. The impurity function measures the homogeneity of each node obtained from the split. It takes the lowest value for pure nodes [5]. The nodes are split while a specified number of objects are not present in the child nodes or the nodes are not pure. A node which cannot be split any further is called a terminal node. One of the most popular impurity functions is entropy [5] and it is the function used in our study.\nOwing to a binary data splitting, the results of CART can easily be visualised as a binary tree, which consists of a number of nodes symbolising subgroups of data objects.\nIn order to ensure good prediction properties of the constructed tree, the number of the tree nodes should be optimal. Selection of the optimal number of nodes relies upon a deletion of some nodes from the tree, which is done by means of the so-called cost-complexity pruning [5].\nDiscriminant partial least squares\nThe DPLS approach aims to relate a set of n explanatory variables (predictors), X (m \u00d7 n), to a dependent variable, y. The dependent variable, y, is either a discrete variable, representing the belongingness of m objects to two defined groups denoted by \u22121 and 1, or a binary variable.\nThe popularity of the DPLS method in chemometrics is due to its attractive properties. DPLS can successfully deal with multicollinearity in the data by constructing a few (f) latent factors, T (m \u00d7 f), which maximise the covariance between X and y [6].\nIn order to obtain DPLS models with good prediction abilities, an optimal number of factors should be chosen. The optimal number of factors is usually found with the help of a cross-validation procedure [6]. The model with the smallest root mean square error of cross-validation (RMSCV) is to be selected. The goodness of model fit is indicated by a root mean square (RMS) error, whereas the success of the prediction is expressed by a root mean square error of the test set. Moreover, the performance of DPLS depends on the set of samples used for its construction. The model set has to cover all possible sources of data variance. Furthermore, DPLS is sensitive to the number of objects used to build the model. Its performance is optimal when the model set contains two groups with the same number of objects [7]. The predictive ability of the model built also depends on the quality of the variables measured.\nUninformative variable elimination\u2013discriminant partial least squares\nUsually, the samples collected are characterised by a large number of variables in order to ensure a detailed description of the event studied. However, some of the experimental variables may be irrelevant for the particular discriminant problem. Such uninformative variables, which have a high variance, but small covariance with y, lead to a DPLS model with unsatisfactory predictive ability. Therefore, finding an optimal set of variables by discarding the uninformative variables from the data can substantially improve the DPLS model by a decrease of the prediction error for test samples or\/and a decrease of model complexity. The variable selection method used in our study is UVE-DPLS [8]. With UVE-DPLS, variables with unstable regression coefficients are removed. In order to estimate the stability of the regression coefficients, a matrix, N (m \u00d7 p), containing at least p=300 random variables, is augmented with the matrix of experimental variables, X (m \u00d7 n), which results in a matrix, Z, of dimension m \u00d7 n+p. To keep the influence of the variables added negligible, their elements are generated from the normal distribution and are multiplied by a small constant with magnitude 1\u2009\u00d7\u200910\u201310. The matrix of regression coefficients is constructed by the use of a leave-one-out cross-validation procedure, i.e. m PLS models are built and each one by using m-1 objects. The stability of a variable is determined by the ratio of the mean of m regression coefficients and their standard deviation. The variables with absolute stabilities of regression coefficients below a given cutoff value are uninformative and are deleted from the data. The cutoff value is defined as the largest absolute value of all stability values for the random variables added.\nThe goodness of a discrimination model is characterised by the percentage of correct classification or the so-called correct classification rate. It is commonly agreed that the higher the correct classification rate, the better the model. Additionally, one should consider sensitivity and selectivity of the model. For a two-class problem for instance, sensitivity is defined as the percentage of correctly classified samples of class A, while selectivity is the percentage of correctly classified samples of class B.\nResults and discussion\nAll the data collected were organised in a matrix, X, of dimension 71 \u00d7 34. All 71 biofilm samples (Table\u00a01) were characterised by the contents of 17 chemical elements analysed in the biofilms and in the water phases extracted at the sampling locations. Three groups of samples were distinguished depending on the type of water body, namely flowing water, standing water and seawater.\nFirstly, PCA was used for an overall exploration of the data structure. PCA is an unsupervised approach, and is frequently employed for data compression and visualisation [9]. With PCA, the original data matrix, X (m \u00d7 n), is decomposed into two matrices: a scores matrix, T (m \u00d7 n), the columns of which contain principal components (PCs) and a loadings matrix, P (n \u00d7 n). PCs are found as linear combinations of explanatory variables by maximising the variance of projected data. The loadings matrix, P, describes the contributions of each variable to the constructed PCs.\nPrior to the PCA analysis, the explanatory variables were autoscaled, because they had been measured in different units. Autoscaling is performed by subtracting the column mean from each data element and dividing it by the corresponding standard deviation. It gives variables the same importance in the PCA analysis. The results of PCA for autoscaled data are presented in Fig.\u00a01.\nFig.\u00a01Principal component analysis of the data set containing the uniquely sampled biofilms and the systematically sampled biofilms: a scree plot of the cumulative percentage of data variance explained by the consecutive principal components (PC), b projection of biofilms on the plane defined by PC 1 and PC 2, c projection of biofilms on the plane defined by PC 1 and PC 3, d projection of variables on the plane defined by PC 1 and PC 2 and e projection of variables on the plane defined by PC 1 and PC 3\nThe first three PCs explain about 50% of the total data variance (Fig.\u00a01a). Figure\u00a01a indicates that the compression is not very effective, because the data variance is distributed over all PCs. However, some general trends in the data structure can be revealed.\nAll the seawater samples are differentiated from the standing water and flowing water samples in the PC 1\u2013PC 2 score plot (Fig.\u00a01b). The sea samples can be divided into two subgroups. The first subgroup contains the samples from Steinbeck (Germany), Travem\u00fcnde (Germany) and Damp (Germany), located in the Baltic Sea, while the second subgroup includes the samples from Punta Skala (Croatia), Nin (Croatia) and Majorca (Spain), situated in the Mediterranean Sea. The standing water and flowing water biofilm samples overlap. The flowing water biofilm sample from Munich can also be distinguished from all the other samples along PC 2. This distinction is even more evident along PC 3 (Fig.\u00a01c). Looking at the loading plots, shown in Fig.\u00a01d and e, one finds the reasons for the objects\u2019 distributions observed in the score plots. PC 1 represents the Sr, Cu, Mg, Se, K and Na contents in the water phase (W-Sr, W-Cu, W-Mg, W-Se, W-K, W-Na). This factor can be related to the salt content of the water phase and is conditionally called the \u2018salt\u2019 factor. The second PC, PC 2, is mainly associated with Fe and Mg (Fig.\u00a01d). These elements are basic components participating in the biofilm formation. PC 3 reflects the Mn, Zn, Cd, Pb, Fe, and Co contents in the water phase. The presence of Cd and Pb is usually a result of environmental contamination and that is why PC 3 is associated with the anthropogenic influence. From the information obtained from the score and loading plots, it follows that the water phase of the biofilm grown in seawater is indeed richer in dissolved salts than the water phase of the standing water and flowing water bodies. The levels of Fe and Mg in sea biofilms are also higher in comparison with those for the other biofilms. The salt content, pH and temperature of water vary at different sampling locations and they influence the biofilm formation. It was reported in [10] that Mg strongly influences attachment and biofilm structure. The surface colonisation and biofilm depth increase with the increasing Mg concentration. The biofilms collected in Punta Skala, Nin and Majorca contain higher levels of dissolved salts in the water phase and higher Fe and Mg contents in comparison with the biofilms collected in Travem\u00fcnde, Damp and Steinbeck. The sample originating from Munich shows a high anthropogenic influence, i.e. it has higher Mn, Zn, Cd, Pb, Fe and Co contents in the water phase and lower Fe and Mg contents in the biofilm in comparison with the other samples.\nIn order to see whether the biofilms developed in standing water could be distinguished from the biofilms grown in flowing water, supervised approaches such as CART, DPLS and UVE-DPLS were applied. Furthermore, it was important to determine if the models constructed could predict the origin of new biofilm samples and how well. Another question to be answered was what variables are responsible for an eventual discrimination of groups. Only seven biofilms were grown in seawater; therefore, they were excluded from the forthcoming analysis.\nTo construct a reliable discriminant model and to test its predictive ability, the data were divided into two subsets (model and test) with the Kennard and Stone [11, 12] and duplex [13] algorithms enabling a uniform subset selection. In the Kennard and Stone method, objects in the model set are selected sequentially, starting with the object closest to the data mean. The next object included in the subset is the one situated furthest away from the first one. The third object selected is the most distant one from the objects selected in the model set. The selection of objects continues while a predefined number of objects are not assigned to the model set. The remaining objects form the test set. As a similarity measure, the Euclidian distance was used. With the duplex algorithm, the two most distant objects in the data are found and included in the model set. The next two most distant objects are assigned to the test set. The remaining objects are consecutively added to the subsets, switching over to the most distant unassigned object with respect to the model set and to the most distant unassigned object with respect to the test set. The Kennard and Stone algorithm ensures that the objects in the model set cover all possible sources of data variance, while the duplex method guarantees the representativeness of both subsets. Selection of model and test sets should be done for each group separately. When a preprocessing procedure is required, the selection of objects is applied to preprocessed data. In our study, the model and test sets were selected using autoscaled data in order to remove the scale differences among variables while evaluating the Euclidean distances among objects. It should be mentioned that the performance of CART is not influenced by autoscaling. In our study, the model set of dimension 42 \u00d7 34 contains 21 flowing water and 21 standing water biofilm samples, whereas the test set of dimension 22 \u00d7 34 includes 15 biofilms of flowing water and seven biofilms of standing water.\nResults of CART, DPLS and UVE-DPLS for model and test sets designed with the Kennard and Stone algorithm\nTo trace the importance of variables responsible for the discrimination of both groups, a classification tree was built. After tenfold cross-validation, an optimal tree, containing two terminal nodes, was selected. The cross-validation error is 14%, indicating a relatively good predictive ability of the constructed tree shown in Fig.\u00a02.\nFig.\u00a02Classification tree constructed for 42 biofilm model samples with target variable describing the type of the water (flowing, f, or standing, s), in which the biofilms were grown\nSince there is only one split in the tree, the discriminant problem is rather simple and the most discriminative variable describes the Mg content in the water phase (W-Mg). As mentioned before, Mg plays an important role during the biofilm formation [10]. All the model set samples, belonging to the group of standing water (17 samples), have Mg content in water phase below 37\u00a0mg g\u22121. The remaining samples (21 flowing water biofilms and four standing water biofilms) are placed in the left terminal node, which results in a misclassification error of 9.5% for the complete tree. Although four model standing water biofilms are recognised as flowing water biofilms, the constructed classification tree provides a correct classification of 100% for the test samples (Table\u00a02). Therefore, the model yields fairly high sensitivity (percentage of correct classification of the test flowing water biofilms) and selectivity (percentage of correct classification of the test standing water biofilms).\nTable\u00a02Correct classification rate (CCR), sensitivity and selectivity of the modelsSelection of model and test setsKennard and StoneDuplexTechniqueCARTaDPLSUVE-DPLSbCARTcDPLSUVE-DPLSdFlowing water vs. standing water samplesCCR (%)100.081.890.986.486.486.4Sensitivity (%)100.073.386.7100.0100.0100.0Selectivity (%)100.0100.0100.057.157.157.1CART classification and regression trees, DPLS discriminant partial least squares regression, UVE uninformative variable eliminationaSelected variable: W-MgbSelected variables: W-Mg, W-Ca and W-SrcSelected variable: W-MgdSelected variables: Fe, Mg, Al, W-Cr, W-Cu, W-Mg, W-Ca, W-Sr and W-K\nAdditionally, good discrimination results can be obtained when the primary split is made on the variable describing the Ca content in the water phase (W-Ca). This variable is a competitive variable selected after removing W-Mg. The split on W-Ca leads to a total misclassification error of 14.3%. The presence of Ca has been shown to have an influence on mechanical properties of biofilms [14].\nIn the next step of the investigation, DPLS was considered, in order to check if a discrimination model using linear combinations of explanatory variables can perform better than CART. The DPLS model has complexity 1. The RMSCV is 0.95 and RMS error is 0.64. The DPLS model constructed allows for 81.8% correct classification of the test set samples. The analysis of the misclassified test samples indicates that four out of 15 (26.7%) flowing water biofilms collected in the Leutra river are incorrectly predicted as standing water biofilms; therefore, the model has a lower sensitivity (73.3%) than the CART model. All the test samples belonging to the group of standing water biofilms are well predicted, which again indicates the high selectivity (100%) of the model constructed (Table\u00a02). An improved DPLS model was obtained by use of the UVE-DPLS approach, after discarding the uninformative variables. The one-factor UVE-DPLS model constructed with three informative variables (W-Mg, W-Ca, W-Sr), offers a total correct classification of 90.9% for the test set. It yields a selectivity of 100% and a better sensitivity (86.7%) in comparison with the DPLS model, because only two out of 15 (13.3%) biofilms grown in the flowing water of the Leutra river are now assigned to the group of standing water biofilms (Table\u00a02).\nThe best discrimination results are obtained from CART, even though this model shows a misclassification error of 9.5% for the complete tree. Since the splits are done in a univariate way, the correlation between variables is not taken into account. Therefore, CART provides unsatisfactory results when a linear combination of variables is responsible for discriminating the samples. This, however, cannot be verified unless multivariate approaches such as DPLS and UVE-DPLS are used. Although CART and UVE-DPLS have different objective functions, common variables are selected as essential for the discrimination. The primary variable, W-Mg, and two competitive variables, W-Ca and W-Sr, in CART are also selected by UVE-DPLS.\nResults of CART, DPLS and UVE-DPLS for model and test sets designed with the duplex algorithm\nResults of CART, DPLS and UVE-DPLS were obtained using data designed with the duplex algorithm, which ensures the representativeness of the model and test sets.\nThe classification tree built has two terminal nodes and the primary split is again made on the variable representing the Mg content (W-Mg) in the water phase. The cross-validation error is 7.1%. Two out of 42 model set samples are wrongly classified, which leads to a misclassification error of 4.8% for the complete tree. Compared with the previous results, the constructed tree shows a better performance for the model set samples, but worse prediction rates (Table\u00a02); therefore, the model has again a fairly high sensitivity (100%), but quite low selectivity (57.1%).\nThe DPLS model constructed for the data designed by the duplex algorithm shows slightly better prediction ability (86.4%) than the model built for the data designed by the Kennard and Stone algorithm (81.6%). It presents a better sensitivity (100%), but a reduced selectivity, with only 57.1% of standing water samples being well recognised. A discriminant model characterised by relatively high sensitivity and selectivity parameters is to be preferred over a model with a high sensitivity and a low selectivity. Therefore, the UVE-DPLS model for data designed by the Kennard and Stone algorithm is to be favoured (Table\u00a02). All the methods allow a correct prediction for 86.4% of samples. The samples collected at Chemnitz and White Dak Pond, Metebach, are improperly classified by all methods. In fact, this is not a striking observation though when the data contain some samples that are different in comparison with the majority of samples. These samples are always assigned to the model set using the Kennard and Stone method and then the test samples are correctly predicted. Using the duplex method, we assigned some atypical samples to the test set, which results in a construction of models with too pessimistic predictive abilities.\nResults of CART, DPLS and UVE-DPLS for biofilm samples grown on natural substrates\nAnother important issue to be discussed is whether the biofilm samples grown on natural substrates (see the group of uniquely sampled biofilms in Table\u00a01) can be used to derive similar conclusions as those drawn using the whole data. If this is possible, the sampling procedure will be carried out in a simpler way, which will be less time-consuming and relatively low in price.\nFor an initial inspection of the data structure, PCA was considered. PCA was applied to autoscaled data (25\u2009\u00d7\u200934) containing only uniquely sampled biofilms and the results are presented in Fig.\u00a03Fig.\u00a03Principal component analysis of the data set containing the uniquely sampled biofilms: a scree plot of the cumulative percentage of data variance explained by the consecutive PCs, b projection of biofilms on the plane defined by PC 1 and PC 2, c projection of biofilms on the plane defined by PC 1 and PC 3, d projection of variables on the plane defined by PC 1 and PC 2 and e projection of variables on the plane defined by PC 1 and PC 3\nThe first three PCs account for 59.1% of the total data variance (Fig.\u00a03a). Similar to PCA of the whole data, the compression is not very effective. The biofilms grown in seawater can again be distinguished along PC 1 (Fig.\u00a03b). Moreover, two subgroups of sea biofilms are distinguished along PC 1 (Fig.\u00a03c). The content of the subgroups is the same as before. The biofilm sample collected in Munich is again found far away from all the other samples. Another extreme biofilm sample, collected in the Aller river (Celle, Germany), appears along PC 3. Regarding the variable loadings (Fig.\u00a03d, e), PC 1 is again associated with the salt content of the biofilm water phases, while PC 2 probably is now linked to the contamination of the biofilm water phases, because the variables W-Zn, W-Cd, W-Ni, W-Fe, W-Co, W-Mn and W-Pb possess high loading values. PC 3 consists of Zn, Cd and Pb, which are usually associated with an anthropogenic influence and this factor is therefore associated with contaminants accumulated by the biofilm. Summarising the results of PCA, one can additionally point out that the biofilm samples collected in Majorca, Punta Skala and Nin are richer in Zn, Cd and Pb in comparison with the remaining sea biofilm samples. Moreover, the highest Zn, Cd and Pb contents are characteristic for the biofilm sample from Celle.\nIn order to construct the CART, DPLS and UVE-DPLS models, only data of natural biofilms grown in flowing water and in standing water were considered. Since the number of samples in each group is small (Table\u00a01), the models were used for an exploratory purpose only. Because of this, the predictive abilities of the models were not tested using an independent test set.\nThe complete classification tree with three terminal nodes is shown in Fig.\u00a04. The primary split is made on the variable describing the Pb content in the water phase (W-Pb). W-Pb is the most discriminant variable. The next split on variable Al corrects the improper assignment of one sample and it is of a lower importance. Owing to the small number of samples, the required tenfold cross-validation procedure could not be applied and, therefore, the cross-validation error was not reported. All the biofilms grown in standing water are well classified, but two biofilm samples grown in flowing water are wrongly classified, which results in a total classification rate of 88.9%. The incorrectly classified samples originate from Steinach (Germany) and Geithain (Germany).\nFig.\u00a04Classification tree constructed for 18 biofilm samples with target variable describing the type of the water (flowing, f, or standing, s), in which the biofilms were grown\nThe DPLS model constructed has complexity 1. RMSCV is 1.78 and RMS error accounts for 0.59. Two samples are incorrectly classified. One of them belongs to the biofilms of standing water and originates from Chemnitz (Germany), while the other one is the biofilm collected in the flowing water body (S\u00e3o Louren\u00e7o) located in Juquitiba (Brazil). The DPLS model built yields a total classification rate of 88.9%. It should be emphasised that DPLS can lead to a too optimistic result when the number of variables outnumbers the number of samples [7]. A remedy for this problem is to reduce the number of variables by the use of a feature selection technique, e.g. UVE-DPLS. The UVE-DPLS model has RMSCV of 0.95. One variable, namely W-Pb, is selected. However, all biofilms grown in flowing water are correctly classified with the model constructed, but all biofilms grown in standing water are improperly classified.\nConclusions\nDiscrimination between sea biofilms and the remaining standing water and flowing water biofilms is straightforward by investigating the score plots obtained from PCA. The loading plots emphasise the expected higher salt content of the water phases extracted from the sea biofilms as well as their higher levels of Fe and Mg in comparison with the other biofilms. A further discrimination between flowing water and standing water biofilms is possible by means of supervised methods like CART, DPLS and UVE-DPLS. The best discriminant model is obtained from CART. One variable describing the Mg content in the water phase (W-Mg) is enough to build a model with 9.5% misclassification error. All test samples selected by the Kennard and Stone algorithm are correctly classified using the constructed CART model. The DPLS and UVE-DPLS methods do not outperform CART for the data set studied and, therefore, it can be pointed out that a linear combination of explanatory variables does not lead to a better prediction for new samples. Moreover, CART appears as a very simple and efficient discriminant technique leading to a straightforward data interpretation in terms of explanatory variables. Hence, CART can be considered as a pilot discriminant approach. When the CART model is not satisfactory, one can apply discriminant methods, such as DPLS and UVE-DPLS, or if necessary to use a nonlinear multivariate classifier like, e.g., support vector machines.\nAll discriminant models, CART, DPLS and UVE-DPLS, lead to 86.4% correct classification for the test set designed by the duplex algorithm. However, CART uses only one variable (W-Mg), UVE-DPLS selects nine variables and DPLS uses all explanatory variables to build the model.\nDiscrimination of flowing water and standing water biofilms that are uniquely sampled, using CART, DPLS and UVE-DPLS models, is done only for a better understanding of the data collected. For a definite conclusion whether these two groups of samples can be discriminated, more samples are required to properly validate the discriminant models.","keyphrases":["biofilms","classification and regression trees","chemometrics","environmental pollution","uninformative variable elimination\u2013discriminant partial least squares regression"],"prmu":["P","P","P","R","R"]}
{"id":"J_Autism_Dev_Disord-4-1-2268724","title":"Face and Emotion Recognition in MCDD Versus PDD-NOS\n","text":"Previous studies indicate that Multiple Complex Developmental Disorder (MCDD) children differ from PDD-NOS and autistic children on a symptom level and on psychophysiological functioning. Children with MCDD (n = 21) and PDD-NOS (n = 62) were compared on two facets of social-cognitive functioning: identification of neutral faces and facial expressions. Few significant group differences emerged. Children with PDD-NOS demonstrated a more attention-demanding strategy of face processing, and processed neutral faces more similarly to complex patterns whereas children with MCDD showed an advantage for face recognition compared to complex patterns. Results further suggested that any disadvantage in face recognition was related more to the autistic features of the PDD-NOS group rather than characteristics specific to MCDD. No significant group differences emerged for identifying facial expressions.\nIntroduction\nSocial cognition is a crucial component of healthy adjustment, and deficits have been reported in a range of psychiatric disorders in children and adults (for review, see Blair 2003; Phillips et\u00a0al. 2003a; Walker 1981). A core deficit in children with autism spectrum disorders, or Pervasive Developmental Disorder (PDD) (as defined in the DSM-IV; (APA 1994) relates to social cognition, important aspects of which are, for example, the processing of faces and facial expressions. Encompassed by the overarching category of PDD is the diagnosis of Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS). Children with PDD-NOS form a heterogeneous group characterized by autistic-like symptoms of varying severity such as marked impairments in social interaction, communication and\/or rigid and stereotyped behavior patterns, but fail to meet full criteria for autistic disorder (APA 1994; Walker et\u00a0al. 2004). Within this heterogeneous group of PDD-NOS children, Cohen and colleagues highlighted the existence of a number of children with disturbances in various areas of functioning such as the regulation of state and arousal (i.e. anxiety and fears), social relations (i.e. detached, aggressive, clingy), and thought disorders (i.e. magical thinking, unusual thoughts, and difficulties in separating fantasy from reality) (Cohen et\u00a0al. 1986). These children have been described in the past as schizotypal, having borderline disorder, childhood schizophrenia, childhood onset PDD or atypical PDD (for review see Ad-Dab\u2019bagh and Greenfield 2001). The description of this group was refined through a specific set of diagnostic criteria, and the term \u2018Multiplex Developmental Disorders\u2019 (MDD) was proposed (Cohen et\u00a0al. 1986). Subsequently, the criteria were altered and the term was modified to \u2018Multiple Complex Developmental Disorder\u2019 (MCDD) (Towbin et\u00a0al. 1993). Although Cohen and colleagues positioned MDD within the broader classification of Pervasive Developmental Disorder (PDD), they also recognized the overlap with several other DSM-III (APA 1980) disorders (i.e. Avoidant Disorder, Overanxious Disorder, Schizotypal Disorder) (Cohen et\u00a0al. 1986). To date, there is little research on these children, however further study is warranted since MCDD children may be at risk for a poor outcome in adulthood including Axis II disorder (Lofgren et\u00a0al. 1991), and schizophrenia spectrum disorders (van Engeland and van der Gaag 1994). This study aims to directly compare children with MCDD to those with PDD-NOS on two measures of social cognition: face recognition and identification of facial expressions. Both of these skills are important to examine given the difficulties in social functioning reported in children with MCDD. Such studies are valuable since any emerging differences in social-cognitive functioning between these two groups of children would further support the validity of MCDD as a separate diagnostic construct. Further validation of the concept of MCDD would emphasize the importance of investigating etiology and efficacy of treatments for MCDD separately from PDD-NOS.\nOn a symptom level, children with MCDD can be distinguished from other developmental disorders. A study examining the medical charts of children with MCDD, children with autism, and children with externalizing and internalizing disorders, reported that MCDD children, compared to children with autism, were more aggressive, more anxious, showed more psychotic thinking, and suspiciousness. Autistic children were more disturbed in their social interaction and communication and displayed more stereotyped and rigid behavior than MCDD children (van der Gaag et\u00a0al. 1995). There is also evidence that children with MCDD or autism have elevated levels of formal thought disorder compared to children with Attention Deficit Hyperactivity Disorder (ADHD) and anxiety disorders (van der Gaag et\u00a0al. 2005). A recent study directly comparing the symptom profiles of children with MCDD and PDD-NOS, reported that the greatest group differences (i.e. largest effect sizes) were found for psychotic thought problems (de Bruin et\u00a0al. 2007). MCDD children, in comparison to PDD-NOS children, experienced more paranoia, incoherent thoughts and delusions. Furthermore, children with MCDD also had a higher frequency of anxiety disorders and disruptive behavior disorders according to the Diagnostic Interview Schedule for Children (Version IV) (DISC-IV) (i.e. Oppositional Defiant Disorder, and Conduct Disorder) compared to children with PDD-NOS. De Bruin and colleagues (2007) applied research criteria to assess MCDD and PDD-NOS independently of one another, and found that a greater percentage of children with PDD-NOS met criteria for a diagnosis of autism or autism spectrum disorder (ASD) and scored higher on measures of reciprocal social interaction and communication deficits as assessed using the Autism Diagnostic Observation Schedule-Generic (ADOS-G) (Lord et\u00a0al. 1999). Findings suggest that children with MCDD, compared directly to those with PDD-NOS, can be differentiated on various dimensions, one of which (i.e. psychotic thought problems) might resemble difficulties experienced by adults with schizophrenia.\nChildren with MCDD also demonstrate psychophysiological differences compared to children with autism, ADHD, dyslexia, and normally developing children as assessed by Event-Related Potentials (ERP) obtained during performance on a visual oddball task (Kemner et\u00a0al. 1999). There is evidence for a blunted cortisol response to psychosocial stress compared to normally developing children (Jansen et\u00a0al. 2000a) and children with autism (Jansen et\u00a0al. 2003). Although both MCDD and autistic children are characterized by abnormal reactions to their social environment, group differences in cortisol response to psychosocial stress suggests that the disorders may have different etiological backgrounds or perhaps may be different neurobiological conditions. A blunted response to psychosocial stress among children with MCDD could also be partly due to higher levels of comorbid conduct disorders. Similar blunted cortisol responses to psychosocial stress have also been reported in adults with schizophrenia (Jansen et\u00a0al. 1998, 2000b, 2003). This suggests that children with MCDD may possess a biological vulnerability to this disorder that may not be evident among children with PDD\u2019s (at least autism).\nFindings to date on the symptom and biological\/psychophysiological profiles suggest that children with MCDD form a group who may not be well placed under the umbrella of the PDD\u2019s, since there is evidence that they may be at risk for developing schizophrenic spectrum disorders later in life. Despite the differences seen among children with MCDD compared to other clinical groups on a symptom, psychophysiological, and neurobiological level, no studies to our knowledge have assessed social-cognitive functioning in these children. The only study to investigate a related area, examined formal thought disorder in MCDD children compared with other clinical groups (van der Gaag et\u00a0al. 2005). Although children with MCDD may demonstrate similar core social deficits as seen in children with PDD-NOS, parallels between the symptoms and biological reactivity in children with MCDD, and adults with schizophrenia spectrum disorders, suggest that a different pattern of performance on social cognitive tasks might emerge compared to children with PDD-NOS. We aimed to examine whether children with MCDD could be differentiated from children with PDD-NOS on two important aspects of social cognition: face recognition and the identification of facial expressions.\nFace Recognition\nSerra and colleagues provided empirical evidence that a more time-consuming, controlled, attention-demanding strategy may characterize face processing in children with PDD-NOS by demonstrating that children with PDD-NOS were significantly slower in face recognition than age-matched normally developing children while the recognition of abstract visuo-spatial patterns did not discriminate between the groups (Serra et\u00a0al. 2003). Klin and colleagues studied face recognition in children with autism, PDD-NOS, and non-PDD disorders (Klin et\u00a0al. 1999). Findings revealed a specific deficit in face recognition in children with autism (which could not be attributed to general cognitive ability), but no specific face recognition deficit in children with PDD-NOS. However, these authors did not investigate processing times.\nTo our knowledge, no studies have examined face processing in children with MCDD, nor have any directly compared children with MCDD to those with PDD-NOS. We can only speculate on the results for MCDD children. However, if children with MCDD are distinct from those with PDD-NOS, we might expect them to show a different pattern of face recognition compared to children with PDD-NOS only (Serra et\u00a0al. 2003), which might be characterized by less controlled time-consuming processing.\nIdentification of Facial Expression\nFacial expression recognition has not been previously examined in children specifically diagnosed with MCDD. Considering that children with MCDD have been previously described as schizotypal or having a diagnosis of childhood schizophrenia (see Ad-Dab\u2019bagh and Greenfield 2001), a study that most closely approximates the investigation of facial expression recognition in MCDD is one examining emotion expression recognition in children with schizophrenia, aggression, anxiety\/depression, and normally developing children (Walker 1981). Walker (1981) reported that children with schizophrenia were less accurate in recognizing emotional expressions compared to the other groups.\nDeficits in identifying facial expressions have been reported in children with PDD (mainly autism) (Castelli 2005; Celani et\u00a0al. 1999; Hobson et\u00a0al. 1988). Although a review by Blair (2003) on facial expression recognition in neuro-cognitive disorders noted that once participants with autism were matched to controls on mental age, impairments in emotion expression recognition disappeared. Few studies have probed emotion processing in children with PDD-NOS. One study comparing normally intelligent PDD-NOS children with school children on emotion recognition (i.e. face, posture, and gesture recognition), found no differences between the two groups (Serra et\u00a0al. 1998).\nBased on the above findings, and given the fears and anxieties inherent to the classification of MCDD, one might predict specific differences, particularly in the identification of threatening expressions (i.e. fearful and angry expressions) compared to children with PDD-NOS. This is based on previous research indicating that adults and children with elevated levels of anxiety demonstrate biases in processing anger\/threatening expressions (Hadwin et\u00a0al. 2003; Mogg et\u00a0al. 2004).\nAims and Predictions of the Study\nIf robust social cognitive differences would emerge, in light of the symptom profile and biological\/psychophysiological differences between the groups, there would be further evidence for recognizing a subcategory of MCDD within the DSM-V as already suggested by others (van der Gaag et\u00a0al. 1995). Considering MCDD as qualitatively distinct from PDD-NOS has implications for treatment. Viewing MCDD as a subgroup of PDD-NOS may focus treatment on the improvement of social skills, whereas if the emphasis in MCDD was on the thought disorder and its relation to psychotic development as well as on anxiety symptoms, a more medication-focused approach might be preferred. However, if social cognitive skills were quite the same, such a finding could also suggest that the diagnostic boundaries of the DSM-categories are weak: people with schizoid\/psychotic symptoms might have a neurobiological condition different from that of people with only PDD-symptoms though sharing important PDD core features. Such a finding would stress the necessity for profiling patients along various dimensions including their psychophysiological responsiveness and (social) cognitive capacities.\nWe aimed to address whether children with MCDD significantly differ from children with PDD-NOS on two facets of social-cognitive skills: face recognition and the identification of facial expressions. Children with MCDD and PDD-NOS were carefully selected using explicitly outlined research criteria. Based on previous research:We predicted that children with MCDD would differ from those with PDD-NOS on recognizing neutral faces in comparison to complex patterns. More specifically, if indeed children with MCDD are not well-placed under the PDD\u2019s, we would expect them to be faster and more accurate on face recognition compared to children with PDD-NOS, but perform similarly on especially complex pattern recognition.Children with MCDD would show differences in the identification of facial expressions, particularly a bias toward processing fear and anger expressions, as compared to children with PDD-NOS. Any significant differences found should not be explained by differences in mental age.\nMethods\nParticipants\nThe study sample was selected from 503 children, aged 6\u201312\u00a0years old, who were consecutively referred to the outpatient department of child and adolescent psychiatry, between July 2002 and September 2004. Referrals were comprised of a large variety of child psychiatric disorders (externalizing disorders, internalizing disorders, PDDs). Research criteria for MCDD and PDD-NOS were rated incompletely for 12 (2.4%) children, who were excluded from further analyses. Complete MCDD and PDD-NOS criteria were rated for 491 children.\nTwenty-nine (5.9%) children met research criteria for a diagnosis of MCDD. Eleven children (44%) meeting MCDD research criteria also met research criteria for PDD-NOS. These children were placed in the MCDD group. The parents of four of these children refused to participate in the study. Seventy-nine children met research criteria for a diagnosis of PDD-NOS without meeting research criteria for MCDD (PDD-NOS group). These children did not meet DSM-IV criteria for autism or Asperger syndrome. Children with an IQ score of less than 70 were not administered the neuropsychological battery; two children in the MCDD group, and 13 children in the PDD-NOS group were not administered the neuropsychological tasks on this basis. A further two children meeting MCDD criteria had missing data for the face recognition (FR) task and identification of facial expressions task (IFE). Thus, the MCDD group included a total of 21 children. In addition to the children without neuropsychological data due to a low IQ, five children in the PDD-NOS group were missing data for the FR task, and four were missing data for the IFE task, resulting in a total of 61 PDD-NOS children with FR task data, and 62 children with IFE task data.\nEthics\nParticipation was voluntary, and informed consent was signed by all parents\/caretakers prior to participation in the study. Children who were 12\u00a0years old also signed the consent forms themselves. The Medical Ethics Committee of the Erasmus Medical Center approved the study.\nMCDD and PDD-NOS Research Criteria\nExplicit research criteria for MCDD and PDD-NOS were used (see Table\u00a01) (Buitelaar et\u00a0al. 1999a). Nine different child and adolescent psychiatrists were involved in rating these research criteria. Ratings were based on semi-structured interviews with the parents\/caretakers and individual psychiatric observation of the child. Assessment information spanned from early development to current level of social, communicative, and adaptive functioning. School, relevant medical, and psychological assessment information were obtained as well. Immediately after all diagnostic procedures had been completed, MCDD and PDD-NOS research criteria were ticked as present or absent, and subsequently an algorithm, of which the rater was unaware, was used to determine whether the thresholds for research diagnoses of MCDD and PDD-NOS were met. For a detailed review of the development of the MCDD and PDD-NOS research criteria see Buitelaar and van der Gaag (1998).\nTable\u00a01Research criteria used to identify children with MCDD and PDD-NOS (from Buitelaar and van der Gaag 1998)MCDD1PDD-NOS2(1) Impaired regulation of affective states and anxietiesA2. (1) Qualitative impairment in social interaction(a) Unusual or peculiar fears and phobias, or frequent idiosyncratic or bizarre anxiety reactions(a) Marked impairment in the use of multiple nonverbal behaviors, such as eye-to-eye gaze, facial expression, body postures, and gestures to regulate social interaction(b) Recurrent panic episodes, or flooding with anxiety(b) Failure to develop peer relationships appropriate to developmental level(c) Episodes of behavioral disorganization punctuated by markedly immature, primitive, or violent behaviors(c) A lack of spontaneous seeking to share enjoyment, interests, or achievements with other people (e.g. by a lack of showing, bringing, or pointing out objects of interest)(d) Lack of social and emotional reciprocity(2) Impaired social behavior(2) Qualitative impairments in communication(a) Social disinterest, detachment, avoidance, or withdrawal(a) In individuals with adequate speech, marked impairment in the ability to initiate or sustain a conversation with others(b) Markedly disturbed and\/or ambivalent attachments(b) Stereotyped and repetitive use of language or idiosyncratic language(3) The presence of thought disorder(3) Restricted repetitive and stereotyped patterns of behavior, interests, and activities(a) Irrationality, magical thinking, sudden intrusions on normal thought process, bizarre ideas, neologism, repetition of nonsense words(a) Stereotyped and repetitive motor mannerisms (e.g. hand or finger flapping or twisting, or complex whole-body movements)(b) Perplexity and easy confusability. overvalued ideas, including fantasies of omni-potence, paranoid preoccupations, overengagement with fantasy figures, referential ideationB. Does not meet criteria for autistic disorder or for other specific pervasive developmental disorder1A total of 5 or more items from 1, 2, and 3, with at least one item from (1), one item from (2), and one item from (3)2A total of 3 or more items from (1), (2), and (3), with at least one item from (1)\nAn interrater reliability study was conducted for 30 randomly selected children (27%). Two clinicians independently rated all MCDD and PDD-NOS research criteria. Agreement between the raters on the presence or absence of a PDD-NOS diagnosis was good (\u03ba\u00a0=\u00a0.62). Agreement for MCDD diagnosis could not be calculated, as MCDD did not occur once in this subsample.\nMaterials\nProcedure\nChildren were assessed on two occasions, separated by a week. Testing was conducted in a quiet room in the outpatient department of the hospital. On the first occasion, the full Weschsler Intelligence Scale for Children (WISC-R) was administered, taking on average, 2\u00a0h per child. The social-cognitive tasks were administered on the second visit the following week as part of a larger neuropsychological battery taking approximately one and a half hours. Children were always tested in the morning to minimize the effects of fatigue and to maximize concentration.\nIntelligence: Weschsler Intelligence Scale for Children (WISC-R)\nThe full WISC-R (revised for use in the Netherlands) was administered to the children. Based on the full-scale IQ score, each child\u2019s mental age was computed using the following formula: Mental age\u00a0=\u00a0(age * full scale IQ score)\/100. Mental age (MA) was included as a covariate since it has been shown to be an important mediator for group differences in social cognition (see Buitelaar et\u00a0al. 1999b; Happe 1995).\nBaseline Speed (BS)\nA simple reaction time task was employed to obtain a baseline measure for the speed (BS) of responding with the response key to ensure children understood how to respond using the response key. Children were required to press a key with the index finger of their dominant hand when a square was presented. Thirty-two trials were administered. This task was a subtest of the computerized Amsterdam Neuropsychological Tasks (ANT version 2.1; de Sonneville 1999). The total baseline speed and the standard deviation (SD) of the BS were calculated. The SD provides a measure of the variability of performance. A higher SD could indicate less attention to the task.\nFace Recognition\nThe Face Recognition (FR) subtest of the ANT 2.1 battery (de Sonneville 1999) was selected to measure the speed and accuracy of recognizing neutral faces. This task has previously been administered in studies assessing face recognition in children with PDD-NOS (Serra et\u00a0al. 2003) and in normally developing children (de Sonneville et\u00a0al. 2002). In the FR task, a target (neutral) face was presented for 2.5\u00a0s. Following the presentation of the target face, a set of four photographs of individuals was presented and children were required to indicate (using a two-key response panel) whether or not the target individual appeared in the set of four (see Fig.\u00a01a). The sex and age category of the target (i.e. boys, girls, men or women) matched those of the subsequently shown set of four faces to be judged. In half of the trials (i.e. 20), the target individual did appear in the set of four and participants were required to press a \u2018yes\u2019 key (\u2018target\u2019 condition), and in 20 trials the target individual did not appear in the subsequent set of four, requiring participants to press the \u2018no\u2019 key (\u2018non-target\u2019 condition). Reaction time (RT) data and accuracy (assessed by calculating the proportion of correct trials out of the maximum score) were calculated for target and non-target conditions.\nFig.\u00a01(a) Face Recognition (FR) task. A neutral face (target stimulus) is presented for 2,500\u00a0ms, followed by a 500\u00a0ms delay. A display set of four neutral faces is then presented. The child must indicate (using a two-button response key) whether the target face is present in the display set (target condition) or not (nontarget condition). Reaction time and accuracy (proportion correct, with a maximum value of 1) data were calculated for target and non-target conditions. (b) Pattern Recognition (PR) task. The children must indicate whether a target pattern is present in one of four patterns presented in a display set. Presentation time parameters and outcome variables are the same as in the FR task. Two PR tasks are presented: (1) patterns involving a similar level of complexity to the FR task (i.e. Complex PR), (2) patterns of a dissimilar (i.e. Easy PR) level of difficulty compared to the FR task\nPattern Recognition\nA subtest of the ANT 2.1 battery assessing pattern recognition (PR) was administered. The task consisted of two conditions, one containing visuo-spatial patterns that are dissimilar and hence easily distinguishable (easy condition), and the other containing complex patterns that are quite similar and therefore hardly distinguishable (complex condition) (see Fig.\u00a01b). The same manner of responding was used as in the FR task, and the same four outcome variables were generated: RT and accuracy for target and non-target conditions. As with the FR task, there were 20 trials for the target condition and 20 trials for the non-target condition, each for the easy condition and the complex condition. Easy and complex patterns as target and non-target trials were presented in a random manner.\nIdentification of Facial Expressions\nThe \u201cIdentification of Facial Expressions\u201d(IFE) subtest of the ANT 2.1 was employed to probe emotion processing (see Fig.\u00a02). Children were required to respond as to whether a face displayed a particular target emotion (by pressing a \u201cyes\u201d button) or not (by pressing the \u201cno\u201d button). Four conditions were administered, each corresponding to a target emotion (i.e. happy, sad, anger, and fear). For each condition, children were instructed to focus on a particular emotion, and to respond whether the face demonstrated that particular emotion or not (i.e. for the happy condition, children were to respond \u201cyes\u201d if the face was happy or to press the \u201cno\u201d button if the face displayed a different emotion). Each emotion condition consisted of 40 trials, 20 of which were the target emotion (requiring a \u201cyes\u201d response) and 20 of which were a random selection of other emotions (requiring a \u201cno\u201d response). The images were digitized photographs of four adult identities (two men and two women). Four outcome variables were calculated for each emotion category: (1) RT for target condition (when the target emotion is presented) (2) RT for non-target condition (when the target emotion is not presented) (3) Accuracy in the target condition (4) Accuracy in the non-target condition. Accuracy (calculated for target and non-target conditions separately) was assessed by calculating the proportion of correct trials out of the maximum score.\nFig.\u00a02Examples of different expressions in the Identification of Facial Expressions (IFE) task. Children are presented with four different tasks (each corresponding to one of four target emotions: happy, sad, anger, and fear). For each task, children are required to focus on a particular emotion, and to judge whether the face displays a specific target emotion. The target consists of an adult face expressing one of four emotions. When the face matches the emotion a \u2018yes\u2019 response is required, when the face does not match the emotion, a \u2018no\u2019 response is required. A total of 40 trials per emotion condition were presented, with half of those trials requiring a \u2018yes\u2019 response (target), and half requiring a \u2018no\u2019 response (nontarget). RT and accuracy (i.e. proportion correct) for target and non-target conditions were calculated\nStatistical Analysis\nA series of multivariate General Linear Models (GLM) was conducted to compare children with MCDD to those with PDD-NOS-only on the various outcome variables of BS, PR, FR, and IFE tasks. For each task (except for the BS task which yielded reaction times only), separate repeated-measures analyses of covariance (ANCOVAs) were performed on the measures for accuracy and speed of processing. Assumptions underlying the use of parametric statistics were examined for each outcome variable. Where assumptions of normality were violated, transformations were applied. Only variables representing accuracy (for target and non-target conditions) were transformed. For these, we applied the arc sin transformation, since this is appropriate for proportional data (Howell 1997). Following these transformations, data were appropriate for the use of parametric statistics. For the repeated-measures ANCOVA\u2019s, Wilk\u2019s lambda and corresponding F-statistics and significance are presented where Mauchly\u2019s test of sphericity was not significant. Where this test was significant, the corrected Greenhouse-Geisser degrees of freedom and significance levels are presented. Two-tailed tests were used. Effect sizes (small: \u22650.02 and \u2264.06 ; medium: >.06 and \u22640.13; large: \u22650.14) were estimated using partial Eta squared (\u03b7p2) (Stevens 1992).\nIf any significant group differences were found, we repeated the analyses while covarying for the effects of mental age (MA) to exclude the possibility that the differences could be explained by variations in general intelligence.\nResults\nGroup Characteristics\nChildren in the MCDD group did not significantly differ from the PDD-NOS group on age (F(1, 78)\u00a0=\u00a02.29, p\u00a0=\u00a00.14), sex distribution (Fisher\u2019s Exact\u00a0=\u00a01.00), VIQ (F(1, 82)\u00a0=\u00a00.22, p\u00a0=\u00a00.64), PIQ (F(1, 82)\u00a0=\u00a00.59, p\u00a0=\u00a00.44), FIQ (F(1, 82)\u00a0=\u00a00.04, p\u00a0=\u00a00.84) or MA (F(1, 78)\u00a0=\u00a01.07, p\u00a0=\u00a00.30). Means (SD) are presented in Table\u00a02. There were no significant group differences in baseline speed (BS) (F(1, 79)\u00a0=\u00a00.21, p\u00a0=\u00a00.65) or SD of BS (F(1, 79)\u00a0=\u00a00.59, p\u00a0=\u00a00.44). This indicates that children in the PDD-NOS group were not significantly different from those in the MCDD group in terms of their basic ability to use the response key or basic attention to the task.\nTable\u00a02Frequencies, Means (SD) for the MCDD and PDD-NOS groups for age, sex, and IQPDD-NOS, N\u00a0=\u00a062MCDD (all with MCDD), N\u00a0=\u00a021 \u2018Pure\u2019 MCDD subsample, N\u00a0=\u00a013 Mean (SD)Chronological age (years)9.22 (1.82) 9.89 (1.49) 9.87 (1.47) Male\/Female (n)54\/818\/310\/3Mental age (years)8.83 (2.10) 9.35 (1.60) 9.43 (1.77)IQ: WISC-RVerbal IQ95.54 (14.26) 97.33 (17.75) 98.77 (21.31)Performance IQ97.57 (16.64) 94.52 (12.55) 94.46 (10.15)Total IQ95.97 (14.39) 95.24 (14.17) 96.00 (14.52)\nFace Recognition (FR) Compared with Pattern Recognition (PR)\nOur first hypothesis was that children with PDD-NOS would be less accurate and slower in processing neutral faces compared to children with MCDD. Such a group difference was not expected for the recognition of abstract visuo-spatial patterns. Means and standard deviations (SD) for accuracy and speed of performance on both the face recognition (FR) and pattern recognition (PR) task are presented in parts 1 and 2 of Table\u00a03, and generally indicate a slower speed of processing in the PDD-NOS group (for both face recognition and pattern recognition) while differences in accuracy appear less pronounced.\nTable\u00a03Means (SD) for face recognition, pattern recognition, and identification of facial expressions tasksTask variablesPDD-NOSMCDD-allPure MCDDRT (ms): Mean (SD) Proportion accurate: Mean (SD)Face recognition (FR)N\u00a0=\u00a062N\u00a0=\u00a021N\u00a0=\u00a013Targets2155.29 (625.54)1862.71 (469.08)1853.12 (468.23)0.76 (0.17)0.83 (0.17)0.86 (0.11)Non-targets2751.15 (730.77)2599.61 (697.01)2701.30 (742.33)0.84 (0.12)0.84 (0.14)0.82 (0.15)Pattern recognition (PR)N\u00a0=\u00a061N\u00a0=\u00a021N\u00a0=\u00a013Easy PR: Targets1826.3 (519.12)1689.90 (509.77)1785.92 (544.13)0.96 (0.07)0.97 (0.05)0.98 (0.02)Easy PR: Non-targets1449.47 (470.34)1332.83 (356.20)1364.01 (392.80)0.85 (0.23)0.89 (0.17)0.87 (0.20)Complex PR: Targets2627.03 (723.78)2536.89 (763.48)2656.76 (886.98)0.83 (0.23)0.80 (0.26)0.78 (0.29)Complex PR: Non-targets3318.54 (984.58)3084.97 (845.74)3129.71 (1016.1)0.76 (0.20)0.84 (0.13)0.83 (0.14)Identification of Facial Expressions (IFE)N\u00a0=\u00a062N\u00a0=\u00a021N\u00a0=\u00a013Happy: Targets1001.03 (358.12)977.91 (347.91)1103.95 (380.76)0.92 (0.09)0.95 (0.04)0.95 (0.05)Happy: Non-targets1322.07 (482.98)1145.38 (352.43)1231.97 (394.17)0.95 (0.06)0.94 (0.06)0.95 (0.06)Sad: Targets1345.73 (419.14)1217.74 (414.07)1311.15 (467.99)0.69 (0.25)0.77 (0.21)0.81 (0.15)Sad: Non-targets1691.06 (657.64)1482.22 (412.78)1569.99 (382.69)0.78 (0.19)0.86 (0.13)0.86 (0.14)Anger: Targets1221.14 (513.73)1115.36 (275.29)1172.91 (296.49)0.73 (0.20)0.75 (0.21)0.79 (0.19)Anger: Non-targets1538.94 (614.53)1417.10 (361.06)1535.36 (347.95)0.89 (0.15)0.89 (0.13)0.87 (0.15)Fear: Targets1351.81 (573.98)1301.85 (491.01)1327.11 (586.66)0.79 (0.18)0.78 (0.24)0.80 (0.26)Fear: Non-targets1476.66 (519.16)1321.81 (300.87)1377.08 (355.14)0.82 (0.20)0.87 (0.14)0.87 (0.15)\nTo test our hypothesis, two types of repeated measures analyses were conducted, each on our measures of accuracy and speed of processing separately. In the first type we compared the easy condition of the PR task with the performance of the FR task, in the second type we compared the complex condition of the PR with the FR task. Each analysis included two within-subjects variables: (1) \u201ctask\u201d (FR versus PR) and (2) \u201cresponse type\u201d (target versus non-target). The between-subjects variable was group. Significant group by task interactions would indicate that the groups differ in their manner of processing neutral faces as compared to abstract patterns.\nConcerning performance on the easy condition of pattern recognition as compared to face recognition, there were no significant interactions involving group (i.e. group*task*response type or group*task) nor a significant main effects of group for either accuracy or speed of processing. Irrespective of group, a significant interaction between task and response type for RT (Wilk\u2019s lambda\u00a0=\u00a00.26, F(1, 80)\u00a0=\u00a0231.10, p\u00a0<\u00a00.001; \u03b7p2\u00a0=\u00a00.74), indicated that children took substantially longer to perform the FR task compared to easy PR, and this effect was particularly strong for the non-target condition.\nComparison of the complex condition of the PR task with face recognition revealed a significant interaction between group, task (PR versus FR), and response type (target versus non-target) (Wilk\u2019s lambda\u00a0=\u00a00.94, F(1, 80)\u00a0=\u00a05.07, p\u00a0=\u00a00.03; \u03b7p2\u00a0=\u00a00.06). Children with PDD-NOS were less accurate in recognizing the presence of a target face in the response set compared to children with MCDD, whereas this difference was not evident when children had to recognize a target pattern amongst hardly distinguishable other patterns (see Fig.\u00a03). This interaction bordered on significance after controlling for MA (p\u00a0=\u00a00.05; \u03b7p2\u00a0=\u00a00.05). Such results did not emerge for speed of processing: there were no significant interactions; group*task*target (p\u00a0=\u00a00.91; \u03b7p2\u00a0<\u00a00.01) or group*task (p\u00a0=\u00a00.73; \u03b7p2\u00a0<\u00a00.01), nor was there a significant main effect of group (p\u00a0=\u00a00.23; \u03b7p2\u00a0=\u00a00.02). Irrespective of group, children processed faces faster than complex patterns (Wilk\u2019s lambda\u00a0=\u00a00.64, F(1, 80)\u00a0=\u00a044.85, p\u00a0<\u00a00.001; \u03b7p2\u00a0=\u00a00.36).\nFig.\u00a03Accuracy of PDD-NOS versus MCDD children for face recognition (FR) and complex pattern recognition (PR). This figure is based on raw (untransformed) error rates, without covarying for MA. Error bars represent standard error of the mean\nOur first hypothesis was therefore partially supported: children with PDD-NOS were less accurate than children with MCDD in recognizing that a neutral face was present in the response set, but this effect was not seen when they were asked to note whether an abstract pattern was present amongst similar ones (i.e. complex condition). There was, however, no group difference in task dependent RTs.\nIdentification of Facial Expressions (IFE) Task\nOur second hypothesis was that children with MCDD would demonstrate differences in the identification of facial expressions compared to those with PDD-NOS, particularly in the identification of fear and anger expressions. Means and standard deviations (SD) for both measures of speed and accuracy are presented in the third part of Table\u00a03 showing a generally slower speed of processing in the PDD-NOS group while differences in accuracy appear less pronounced. To examine group differences in children\u2019s identification of facial expressions, two repeated-measures analyses were conducted (one for accuracy and the other for speed of processing). The within-subjects factors included (1) emotion-category (four levels: happy, sad, anger, fear) and (2) response type (target versus non-target). The between-subjects factor was group.\nContrary to our expectations, children with MCDD did not significantly differ from those with PDD-NOS on emotion-processing; there were no significant main effects of group for accuracy (p\u00a0=\u00a00.22) or for RT (p\u00a0=\u00a00.18), nor any significant interactions between group*emotion-category for either accuracy (p\u00a0=\u00a00.16) or RT (p\u00a0=\u00a00.98) data. We therefore found no support for our second hypothesis that children with MCDD would process facial expressions of emotion (particularly anger and fear faces) differently compared to children with PDD-NOS.\nReanalyses Including \u2018Pure\u2019 MCDD Versus PDD-NOS Children\nBecause approximately 40% of the children with MCDD also fulfilled research criteria for PDD-NOS, it is plausible that MCDD status might have been confounded with PDD-NOS. To ensure that the results were not simply due to the overlap between children with MCDD, and PDD-NOS, we repeated the above analyses, however this time comparing a sub-sample of the MCDD children who fulfilled research criteria for MCDD but not PDD-NOS (\u2018pure MCDD\u2019; n\u00a0=\u00a013), to children with PDD-NOS (n\u00a0=\u00a061). For emotion-processing (i.e. Identification of Facial Expression task), similar results were obtained as with the larger MCDD group; no significant results involving group (\u2018pure MCDD\u2019 versus PDD-NOS) emerged. Yet, re-analysis of the face versus pattern recognition performances revealed a group difference not previously found. This difference relates to the comparison between complex pattern recognition and face recognition; the significant interaction between task*response type*group previously found for only the accuracy data now emerged for RT data as well (Wilk\u2019s lambda\u00a0=\u00a00.94, F(1,72)\u00a0=\u00a04.77, p\u00a0=\u00a00.03; \u03b7p2\u00a0=\u00a00.06). Children with PDD-NOS compared to the \u2018pure\u2019 MCDD group were slower in recognizing faces in the target condition, whereas no such group difference was found for complex pattern recognition. The interaction remained significant after adjusting for the effect of MA (p\u00a0=\u00a00.04; \u03b7p2\u00a0=\u00a00.06). All other results were similar when reanalyzed using this more \u2018pure\u2019 MCDD group.\nDiscussion\nPrevious research suggests that children with MCDD may form a group of children that is, based on symptom, biological, and psychophysiological profiles, well-distinguishable from those with autism, externalizing, and internalizing disorders. This group, though possibly at risk for poor prognosis in adulthood, including schizophrenia spectrum disorders, is currently subsumed under the larger, heterogeneous category of PDD-NOS. Yet, there is debate as to whether this group should be considered a separate diagnostic category within DSM-V (van der Gaag et\u00a0al. 1995). To our knowledge, only one other study has directly compared children with PDD-NOS to those with MCDD, examining symptom differences between the two groups of children (de Bruin et\u00a0al. 2007). The present study contributes to literature on this under-studied and somewhat controversial diagnosis, by examining whether children with MCDD can be differentiated from children with PDD-NOS on two domains of social-cognitive functioning; face recognition and the identification of facial expressions. The ability to recognize individuals, and to process the emotional cues of others quickly and accurately is a crucial component of social functioning and development (for reviews, see Blair 2003; Herba and Phillips 2004).\nFace Recognition\nOur first hypothesis postulated that children with PDD-NOS would demonstrate poorer performance in face recognition than children with MCDD, but that the two groups would perform similarly on detecting complex patterns. To test this hypothesis we adopted an approach used by Serra and colleagues (2003) which provided evidence that children with PDD-NOS may use a more attention-demanding strategy of face processing compared to typically developing children who were suggested to process faces more automatically. In that study, children with PDD-NOS showed an only small discrepancy in speed of processing complex patterns and faces, whereas typically developing children showed a greater advantage for face recognition compared to pattern recognition. We therefore predicted that children with MCDD, if indeed distinct from those with PDD-NOS, would demonstrate a more specific advantage for face recognition compared to complex pattern recognition, whereas children with PDD-NOS would show a smaller difference between processing faces and complex patterns. Our results are somewhat consistent with this hypothesis. When comparing easy pattern recognition with the recognition of unfamiliar faces, no group differences emerged; overall, children were faster and more accurate to recognize easy patterns compared to faces. However, when comparing the recognition of complex patterns with the recognition of faces, children with PDD-NOS appeared to process faces more similarly to complex patterns especially in the target condition whereas children with MCDD demonstrated greater accuracy for processing faces compared to complex patterns. This effect remained significant after adjusting for MA. Moreover, when investigating the \u2018pure MCDD\u2019 group (children who met criteria for MCDD, but not PDD-NOS), the advantage for face recognition could be shown to also exist for their speed of processing. These results suggest that MCDD-specific characteristics on their own (see Table\u00a01: impaired regulation of affective states, impaired social behavior, and thought disorder) might not be associated with a more attention-demanding strategy of face recognition. In contrast, children with PDD-NOS, consistent with their symptoms of impaired non-verbal behavior, lack of social and emotional reciprocity, and lack of spontaneous affiliation with other people, appeared to process facial information in a way quite similar to how they process complex patterns. De Bruin and colleagues (2007), using data from the same participants as in the present study, reported that children in the MCDD group (36.0%) fulfilled criteria for ADOS-G classifications of autism or autism spectrum to a lesser extent compared to children in the PDD-NOS group (62.2%). Hence, the more time-consuming and therefore attention-demanding strategy of face processing described by Serra and colleagues and seen in our group of children with PDD-NOS could be due to the more \u2018autistic\u2019 characteristics of our PDD-NOS group. Face recognition appeared to be less attention-demanding in our MCDD group that did not meet the diagnostic criteria for a PDD-NOS. Our results therefore suggest that MCDD symptoms are associated with disadvantages in especially the speed of recognizing unfamiliar faces only to the extent to which they are accompanied by social problems severe enough to meet the diagnostic criteria for PDD-NOS.\nIdentification of Facial Expressions\nGiven the extensive fears and anxieties inherent to the diagnosis of MCDD compared to autism or PDD-NOS, we expected significant group differences to be evident in emotion-processing. We predicted that these group differences would be particularly marked for identifying facial expressions of fear and anger expressions, since previous research has indicated that aberrant patterns in processing certain emotions are specific to the symptoms of the particular disorder (Phillips et\u00a0al. 2003a), and that adults and children with high levels of anxiety demonstrate biases toward processing threatening facial expressions (Hadwin et\u00a0al. 2003; Mogg et\u00a0al. 2004). Yet, we found no support that children with MCDD differed significantly from those with PDD-NOS in identifying fear or anger expressions, nor for any of the other emotion-categories we investigated. Taking the model of emotion-processing by Phillips and colleagues proposing the following three main components (Phillips et\u00a0al. 2003b): (1) identification of the emotional significance of a stimulus; (2) production of an affective state; and (3) regulation of an affective state, we must note that only the first component was tested in the current paper. Evidently, the notable differences between children with MCDD and those with PDD-NOS found on a symptom level (de Bruin et\u00a0al. 2007) do not translate to a more basic level of emotion recognition. Differences between the two groups in the production and regulation of affective states are nevertheless quite likely, since especially children with MCDD appear to have substantial difficulties regulating their affective state. Future work should incorporate tasks that allow for the examination of each of the three of the above-mentioned components of emotion-processing. In order to provoke changes in affective state such a task should take into account real-life situations or experiences that may trigger strong emotional reactions in these children. Such a task might help to extract further information on how the symptoms associated with MCDD may or may not discriminate emotion-processing ability from those with PDD-NOS.\nStrengths and Limitations\nTo our knowledge this is the first study to directly compare children with MCDD to those with PDD-NOS (selected from a range of child psychiatric disorders) on social-cognitive functioning using well-validated research criteria for MCDD and PDD-NOS. A strength of this study was the selection of children with MCDD and PDD-NOS on the basis of explicit research criteria. Earlier studies on the neuro-cognitive profiles of children resembling MCDD included ill-defined groups representative of broad categories of disorder. Other studies examining symptom profiles and thought disorder using clearly defined groups of MCDD children selected these children from a sample of children with PDD, implying that MCDD can only occur within the broader category of PDD (van der Gaag et\u00a0al. 1995, 2005). We screened children from a larger sample of outpatients, and applied the research criteria for both MCDD and PDD-NOS independently from one another. Therefore, a diagnosis of MCDD could occur if the child did not meet criteria for PDD, consistent with earlier work suggesting that only approximately half of the children with MCDD also met criteria for PDD-NOS (Towbin et\u00a0al. 1993). Furthermore, PDD-NOS has in many studies been assessed as a \u2018default\u2019 diagnosis of the DSM when children did not quite meet the diagnosis for any of the other PDD subtypes rather than being explicitly defined on its own (see Walker et\u00a0al. 2004). Explicit research criteria and not a DSM default option were applied in this study to identify children with PDD-NOS.\nHowever, we were also faced with a number of limitations. Since MCDD is not currently a DSM-IV diagnosis, all MCDD children had been assigned other clinical diagnoses. These diagnoses, based on DSM-IV and the DISC, included PDD-NOS, anxiety disorders (including separation anxiety and obsessive compulsive disorder), disruptive behavior disorders, in addition to ratings of psychotic thought problems (rated by the Child and Adolescent Functional Assessment Scale (CAFAS) and the CBCL thought problems subscale) (see also de Bruin et\u00a0al. 2007). Approximately 40% of the MCDD children also met research criteria for PDD-NOS. Thus, although the PDD-NOS children in this study did not meet criteria for MCDD, some of the children in the MCDD group also met the criteria for PDD-NOS. Our results of an advantage for the speed of recognizing neutral faces compared to complex patterns among children in the \u2018pure MCDD\u2019 group suggest that the more attention-demanding strategy of face recognition used by children with PDD-NOS may be dependent to a larger extent on their \u2018autistic-like qualities\u2019 rather than specific characteristics of MCDD. It is also noteworthy that these results emerged despite the small number of participants in this \u2018pure MCDD\u2019 group. Future studies could aim for a purer comparison by using groups of PDD-NOS (without MCDD) and MCDD (without PDD-NOS). A further limitation was the relatively small sample size for the MCDD group. This was mainly due to the low prevalence rate of MCDD. However, another study assessing formal thought disorder in children with MCDD included a similar sample size (van der Gaag et\u00a0al. 2005) and found higher rates of formal thought disorder in children with MCDD compared to clinical or healthy control groups. Furthermore, de Bruin et\u00a0al. (2007) directly compared children with MCDD to PDD-NOS, and reported differences in symptom profiles using similar-sized groups while standard deviations in the dependent measures relative to their means (i.e. the variance coefficients) were greater in that study than those of, for example, the reaction times in our study. This suggests that the hypothesized group differences could have been smaller in order to be detected as significant. We are therefore not inclined to interpret the few significant differences in our study as being due to a power problem. We cannot exclude the possibility that children with MCDD and PDD-NOS use different cognitive strategies to achieve the same end level of performance. The use of neuro-imaging technologies and ERP studies would help to rule out this possibility, and may yield greater insight into the mechanisms underlying social cognition in these children.\nConclusion\nDespite the existence of thought disorder in children with MCDD, the symptom level differences between PDD-NOS and MCDD children, and the biological\/psychophysiological differences between MCDD children and other comparison groups, we found little evidence that children with MCDD are clearly distinguishable from those with PDD-NOS on the identification of facial expressions. Surprisingly, the high rates of anxieties and fears clinically characteristic of children with MCDD did not translate to any significant effects on our emotion-processing task. Further work is needed to probe whether more subtle emotion-processing differences exist. Such studies should focus on examining children\u2019s processing of emotional stimuli within a context more relevant to \u2018real-life\u2019 as well as detailed evaluation of children\u2019s emotion regulation ability.\nThe only significant difference to emerge was that children with MCDD not meeting the criteria for a PDD diagnosis demonstrated fewer errors and a faster processing of unfamiliar neutral faces compared to children with PDD-NOS who processed faces more similarly to how they processed complex patterns. This suggests a disadvantage in face processing being related to the autistic characteristics of the PDD-NOS. Based on these findings, it is recommended that the impact of autistic features (amount and severity) are carefully considered when evaluating a child with MCDD symptoms since such features may yield relevant information about the child\u2019s social cognitive abilities. Ideally, future work should include prospective designs which follow up children with MCDD who do and do not demonstrate autistic characteristics.","keyphrases":["emotion recognition","mcdd","pdd-nos","face recognition","pervasive developmental disorders","facial expression recognition"],"prmu":["P","P","P","P","P","P"]}
{"id":"Appl_Microbiol_Biotechnol-4-1-2266783","title":"Degradation of 4-fluorophenol by Arthrobacter sp. strain IF1\n","text":"A Gram-positive bacterial strain capable of aerobic biodegradation of 4-fluorophenol (4-FP) as the sole source of carbon and energy was isolated by selective enrichment from soil samples collected near an industrial site. The organism, designated strain IF1, was identified as a member of the genus Arthrobacter on the basis of 16S ribosomal RNA gene sequence analysis. Arthrobacter strain IF1 was able to mineralize 4-FP up to concentrations of 5 mM in batch culture. Stoichiometric release of fluoride ions was observed, suggesting that there is no formation of halogenated dead-end products during 4-FP metabolism. The degradative pathway of 4-FP was investigated using enzyme assays and identification of intermediates by gas chromatography (GC), GC\u2013mass spectrometry (MS), high-performance liquid chromatography, and liquid chromatography\u2013MS. Cell-free extracts of 4-FP-grown cells contained no activity for catechol 1,2-dioxygenase or catechol 2,3-dioxygenase, which indicates that the pathway does not proceed through a catechol intermediate. Cells grown on 4-FP oxidized 4-FP, hydroquinone, and hydroxyquinol but not 4-fluorocatechol. During 4-FP metabolism, hydroquinone accumulated as a product. Hydroquinone could be converted to hydroxyquinol, which was further transformed into maleylacetic acid and \u03b2-ketoadipic acid. These results indicate that the biodegradation of 4-FP starts with a 4-FP monooxygenase reaction that yields benzoquinone, which is reduced to hydroquinone and further metabolized via the \u03b2-ketoadipic acid pathway.\nIntroduction\nDuring the past decades, widespread application of fluoroaromatic compounds as agrochemicals and pharmaceuticals has lead to an increased occurrence of environmental contaminants containing fluorine (Key et al. 1997). Fluorinated compounds are rare in nature (Harper and O\u2019Hagan 1999). The stability of the carbon\u2013fluorine bond (116\u00a0kcal\/mol in CH3F, compared to 81\u00a0kcal\/mol for the carbon-chlorine bond in CH3Cl) makes most fluorine-containing compounds much more resistant to biodegradation than their unsubstituted analogs (Key et al. 1997). Furthermore, the van der Waals radius of fluorine is small (1.47\u00a0\u00c5, in between that of a hydrogen and an oxygen). Yet, the electronegativity of fluorine causes strong polarization of C\u2013F bonds, and fluorine substituents may be involved in biological interactions (Howard et al. 1996).\nRegardless of the recalcitrance of most fluoroaromatics to biodegradation, several bacterial cultures have been described that aerobically degrade fluorobenzoic acids (Oltmanns et al. 1989; Engesser et al. 1980; Harper and Blakley 1971; Schlomann et al. 1990). Research on bacterial fluorophenol degradation has been limited to studies with whole cells, cell extracts, or purified enzymes from Rhodococcus species that were obtained by enrichment with other aromatic compounds as a growth substrate (Boersma et al. 1998, 2001; Bondar et al. 1998, 1999; Finkelstein et al. 2000). The fluorobenzene-degrading organism Rhizobiales F11 could grow on 4-fluorophenol (4-FP), but information on the pathway of 4-FP metabolism is lacking.\nCometabolic degradation of difluorophenols and trifluorophenols by several Rhodococcus species is initiated by a phenol hydroxylase that catalyzes ortho-hydroxylation, resulting in the formation of the respective fluorocatechol, which is then cleaved by an intradiol dioxygenase to produce fluoromuconate (Bondar et al. 1998). Conversion of 4-FP by whole cells of the phenol-degrading organism Rhodococcus opacus 1cp resulted in the formation of 4-fluorocatechol, 1,2,3-trihydroxy-5-fluorobenzene, and fluoromuconates (Finkelstein et al. 2000). Yeasts and fungi that are able to cometabolically transform fluorinated phenols have also been described. Whole cells of Exophiala jeanselmei transformed 4-FP into 4-fluorocatechol and 3-fluoromuconate (Boersma et al. 1998). Penicillium frequentans metabolized monofluorophenols in the presence of glucose or phenol. The metabolism of meta-or para-fluorophenols yielded the corresponding catechol and 4-carboxymethylenebut-2-en-4-olide (Hofrichter and Schreibner 1993; Hofrichter et al. 1994). None of these organisms could utilize fluorophenols as a growth substrate.\nTo our knowledge, studies on the metabolism of fluorophenols by a bacterial culture that is capable of using such as compound as a sole source of carbon and energy have not been reported. In the present paper, we describe the isolation and characterization of a bacterial strain growing on 4-FP as the sole source of carbon and energy. Based on the identification of several intermediates, a metabolic route for the degradation of 4-FP by this strain is proposed.\nMaterials and methods\nMedia and growth conditions NB medium contained 8\u00a0g of Nutrient Broth (Difco) per liter. Mineral salts medium (MM) contained per liter 5.3\u00a0g Na2HPO4 12H2O, 1.4\u00a0g KH2PO4, 0.2\u00a0g MgSO4 7H2O, 0.5\u00a0g (NH4)2SO4, 5\u00a0ml trace metals solution (Janssen et al. 1985), and 10\u00a0mg of yeast extract (Difco Laboratories). When required, the solid medium was obtained by adding 15\u00a0g\/l of Difco agar. Strain IF1 was grown at 30\u00b0C on a rotary shaker (180\u00a0rpm). Cultures were grown in 100-ml flasks filled to 25% of their volume and were closed with Teflon-lined screw caps. E. coli cells were grown in Luria\u2013Bertani medium (LB) at 37\u00b0C on a rotary shaker.\nEnrichment and isolation of 4-FP-degrading cultures A variety of soil samples, collected from different sites in The Netherlands that are contaminated with halogenated aliphatic compounds (such as monochlorobenzene, hexachlorobenzene, and trichloropropane), were used as the initial inoculum for the 4-FP enrichments. The soil samples were used to inoculate flasks containing 30\u00a0ml of sterile minimal salts medium and 1\u00a0mM of 4-FP, supplied in the liquid phase as the sole carbon and energy source. The cultures were incubated at room temperature on a rotary shaker (150\u00a0rpm), and 40% of the suspension was transferred to a new flask containing fresh medium every 15\u00a0days. During this time, growth (optical density at 600\u00a0nm) and liberation of fluoride were monitored. Samples of the enrichment culture were periodically spread onto minimal salts agar plates containing 1\u00a0mM 4-FP and onto NB plates as soon as growth on 4-FP was established. Pure cultures were obtained by repetitive streaking onto solid MM containing 4-FP and tested separately for growth on 1\u00a0mM 4-FP liquid medium. Growth and fluoride release were again monitored to verify 4-FP degradation. Strains capable of 4-FP degradation as a sole source of carbon and energy were used for further experiments. Strain IF1 was deposited at Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands, under accession number NCCB 100218.\nSequencing of the 16S rRNA gene For cloning of the 16S ribosomal ribonucleic acid (rRNA) gene, a single colony of strain IF1 was directly used for polymerase chain reaction (PCR) amplification. The primers 63f (5\u2032-CAGGCCTAACACATGCAAGTC-3\u2032) and 1387r (5\u2032-GGGCGGWGTGTACAAGGC-3\u2032; Marchesi et al. 1998) were used for PCR amplification. The PCR reaction mixture (50\u00a0\u03bcl) contained Taq PCR buffer, 2.5\u00a0mM MgCl2, 20\u00a0pmol of each appropriate primer, 200\u00a0mM of each deoxyribonucleotide triphosphate, 1\u00a0U Taq DNA polymerase, and biomass of strain IF1. The PCR conditions were 94\u00b0C for 10\u00a0min followed by 1\u00a0min at 95\u00b0C, 1\u00a0min at 55\u00b0C, 1.5\u00a0min at 72\u00b0C, and 5\u00a0min at 72\u00b0C. The resulting fragments were cloned into the pCR4-TOPO vector (Invitrogen, Carlsbad, CA) and transformed into E. coli TOP10 cells. The transformed cells were plated on LB plates containing 0.5\u00a0mg\/ml of ampicillin, and the positive colonies were used for plasmid isolation and sequencing.\nPhylogenetic analysis Alignments of the 16S rRNA gene were made using sequences downloaded from the Ribosomal database project II (RDP II; Cole et al. 2005), after searching for nearest neighbors using the sequence match tool. Further searches were conducted using BLAST and FASTA of the European Molecular Biology Laboratory (EMBL) database for 16S rRNA gene sequences that are closely related to the 16S rRNA gene of strain IF1 but not available from the RDP II. Alignments were subsequently made using the profile alignment option in ClustalX (Thompson et al. 1997), refined using BioEdit ver 7.0.5.2 (Hall 1999), and subsequently parsed through Gblock (Castresana 2000) to remove ambiguously aligned sections and increase the robustness of the data. Phylogenetic trees were determined using the neighbor-joining (NJ) method. Evolutionary distances for the global tree were calculated using the Kimura-2-parameter model with a transition\/transversion ratio of 2 (Fig.\u00a01). Further trees were constructed in Phylip version 3.6a3 (J. Felsenstein), using maximum parsimony and maximum likelihood methods. All NJ trees were tested statistically by means of bootstrap analysis.\nFig.\u00a01Phylogenetic tree of the 16S rRNA gene sequence of strain IF1. The scale bar represents 0.1 fixed mutation per site. Bootstrap values were derived from 1,000 analyses. The DNA sequences were aligned using ClustalX, and the tree was constructed by the neighbor-joining program from a similarity matrix of pairwise comparisons made by using the Kimura-2-parameter algorithm. Tree a shows the global position of the strain IF1 while tree b shows the Bayesian tree and the more precise relationship of strain IF1 to other members of the genus Arthrobacter. The values in parenthesis in this tree are the posterior probabilities for the nodes that show where IF1 resides\nPreparation of cell extracts Cells were grown in MM with 4\u00a0mM 4-FP, harvested by centrifugation at 5,000\u2009\u00d7\u2009g for 15\u00a0min, washed with ice-cold TD buffer (0.1\u00a0M Tris\u2013HCl, pH\u00a07.5, and 0.1\u00a0M 1,4-dithiothreitol), and stored at \u221220\u00b0C until further use. The frozen cells were thawed, resuspended in TD buffer, and incubated with lysozyme (50\u00a0mg\/ml) for 1\u00a0h at 30\u00b0C. A French press was used to disrupt the cells, and the crude extracts were centrifuged at 10,000\u2009\u00d7\u2009g for 15\u00a0min to separate the soluble from the particulate fraction. The supernatant was used for further experiments. Protein concentrations were determined with the Biorad protein assay kit.\nEnzyme assays Catechol 1,2-dioxygenase and catechol 2,3-dioxygenase activities were measured spectrophotometrically at 260 and 375\u00a0nm, respectively. The reaction mixtures contained 0.1\u00a0mM catechol, TD buffer, and cell-free extract (0.1\u00a0mg of protein) in a final volume of 1\u00a0ml. 4-Fluorocatechol 1,2-dioxygenase was measured as catechol 1,2-dioxygenase but with 4-fluorocatechol instead of catechol as the substrate.\nThe 4-FP monooxygenase activity was measured spectrophotometrically by following the consumption of NADH at 340\u00a0nm in a reaction mixture containing cell-free extract (0.1\u00a0mg protein), 0.1\u00a0mM 4-FP, 0.1\u00a0mM NADH, and buffer in a total volume of 1\u00a0ml. The observed rates were corrected for substrate-independent NADH oxidation.\nHydroxymuconic semialdehyde dehydrogenase was measured at 375\u00a0nm. Reaction mixtures contained (in a final volume of 1\u00a0ml) about 0.05\u00a0mM freshly prepared hydroxymuconic semialdehyde, TD buffer, 0.1\u00a0mM NAD, and cell-free extract (0.1\u00a0mg of protein). Hydroxymuconic semialdehyde was obtained by incubation of catechol with the cell-free extract of Pseudomonas putida mt-2 as described previously (Mars et al. 1998).\nHydroquinone dioxygenase was assayed spectrophotometrically by monitoring the change in absorbance between 230 and 330\u00a0nm in a reaction mixture of 1\u00a0ml final volume containing 0.1\u00a0mM hydroquinone, TD buffer, and cell-free extract (0.1\u00a0mg of protein). Hydroquinone hydroxylase and hydroxyquinol dioxygenase were assayed by using a fiber optic oxygen sensor. Reaction mixtures contained 1\u00a0mM of substrate, MM, and cell suspension (0.5\u00a0mg\/ml protein) in a final volume of 1.25\u00a0ml.\nOxygen uptake measurements Strain IF1 was grown with glucose or 4-FP as the sole carbon source and harvested by centrifugation at 6,000\u2009\u00d7\u2009g for 15\u00a0min at 4\u00b0C. Cells were resuspended in MM, and O2 consumption was measured with a fiber optic oxygen sensor (MOPS-1, ProSense BV, Hannover, Germany). All reactions were performed in a stirred vessel at room temperature. The reaction mixtures contained 1\u00a0mM of substrate (4-FP, hydroquinone, 4-fluorocatechol, hydroxyquinol, or catechol), MM, and cell suspension (0.5\u00a0mg\/ml protein) in a final volume of 1.25\u00a0ml.\nAnalytical methods For capillary gas chromatography (GC), 4\u00a0ml samples were extracted with 1\u00a0ml of diethyl ether. A model 6890 gas chromatograph (Hewlett-Packard) equipped with a flame ionization detector and a HP-5 column (Agilent 19091J-413, 30\u00a0m\u2009\u00d7\u20090.25\u00a0mm\u2009\u00d7\u20090.25\u00a0\u03bcm) were used for the analysis. GC-mass spectrometry (MS) analysis was carried out with a model 5973 mass selective detector (Hewlett-Packard) coupled to a HP 6890 series injector and a HP1 column (Agilent 19091Z-433; 30\u00a0m\u2009\u00d7\u20090.25\u00a0mm\u2009\u00d7\u20090.25\u00a0\u03bcm). High-performance liquid chromatography (HPLC) was carried out using a Chrompack C18 column (10\u00a0cm\u2009\u00d7\u20095\u00a0mm) connected to a Jasco UV-1575 detector, which monitored absorbance at 214 and 254\u00a0nm, and operated with Jasco PU-980 pumps and a Jasco AS-1555 sampler. The mobile phase was water\/acetonitrile (70:30), 5\u00a0mM potassium phosphate (pH\u2009=\u20093), and 340\u00a0mg\/l sodium dodecyl sulfate, and the flow rate was 1\u00a0ml\/min.\nLiquid chromatography (LC)-MS was carried out with a ZMD Micromass spectrometer, equipped with a XTerra MS, Symmetry Shield C8 column (4.6\u2009\u00d7\u2009150\u00a0mm), a Waters 996 photodiode array detector, and a Waters 2690 separations module. Samples of 20\u00a0\u03bcl were analyzed, and compounds were isocratically eluted at a flow rate of 1\u00a0ml\/min with a solution of water\/acetonitrile (80:20) and 10\u00a0mM formic acid.\nConcentrations of free fluoride in the culture supernatants were measured with a fluoride electrode (model 96-09, Thermo Russell, Scotland). Fresh sodium fluoride standards were prepared for calibration curves.\nChemicals 4-FP (>98%) was obtained from Sigma-Aldrich (Steinheim, Germany). All chemicals were of the highest purity grade available (Sigma-Aldrich; Acros Organics, Geel, Belgium). The compound 4-fluorocatechol was kindly provided by Dr. Erik de Vries. Purity of 4-FP, 4-fluorocatechol, and hydroquinone was checked by HPLC.\nNucleotide sequence accession numbers The 16S rRNA sequence of strain IF1 was deposited at GenBank with the accession no. DQ425093.\nResults\nIsolation of a 4-FP-degrading bacterium To obtain a bacterial culture that is able to use 4-FP as carbon and energy source for growth, enrichments were performed and followed over time. Two months of selective enrichment by repeated transfer of samples from a culture that displayed growth on 4-FP to fresh 4-FP containing media resulted in a microbial consortium that was capable of growth on 4-FP as a sole source of carbon and energy. Samples of the consortium were repeatedly plated onto NB agar plates and MM agar plates containing 4-FP. This procedure resulted in the isolation of three pure strains named IF1, IF2, and IF3. The strains were restreaked on MM supplemented with 4-FP and inoculated in liquid cultures with 1\u00a0mM 4-FP. Fluoride liberation was observed for all three strains but not in control incubations to which no bacterial inoculum was added. All the three strains showed growth, as monitored at 600\u00a0nm, and thus were able to use 4-FP as a sole carbon and energy source. After 5\u00a0days of incubation in liquid culture supplied with 1\u00a0mM 4-FP, strain IF1 reached 100% fluoride release and an optical density (OD) of 0.119 at 600\u00a0nm. Strain IF2 released 76% of the fluorine and reached an OD600 of 0.09, while strain IF3 reached an OD of 0.076 and released 54% of the theoretical amount of fluoride ions. When a mixed culture of the three strains in liquid media was used, an OD600 of 0.217 and 100% fluorine release were measured after 4\u00a0days. Apparently, the mixed culture contained, even after prolonged adaptation, organisms with varying efficiencies of 4-FP utilization. Because strain IF1 showed the highest degradation rates combined with stoichiometric release of fluoride, this organism was chosen for further studies.\nMicrobiological characterization Strain IF1 is a Gram-positive motile bacterium with a rhodococcus lifecycle. The optimal temperature for growth is 30\u00b0C. The 16S rRNA gene sequence was determined. Initial searches against the RDP II and EMBL rRNA databases resulted in very close associations with members of the genus Arthrobacter, the closest of which were the quinaldine-degrading strain Arthrobacter sp. KA1-1 (Overhage et al. 2005) and the 4-nitroguaiacol-degrading actinobacterium Arthrobacter nitroguajacolicus sp. nov (Kotouckova et al. 2004). The 16S rRNA gene sequences of these organisms were greater than 99% identical to IF1\u2019s. The phylogenetic analysis places the isolates in the phylum Actinobacteria and the genus Arthrobacter (Fig.\u00a01). The subtree that were obtained shows the detailed relationship of isolate IF1 to other members of the genus Arthrobacter. The topologies of all the trees that were obtained during statistical analysis were in agreement and clearly placed this isolate in the genus Arthrobacter (data not shown). Furthermore, the change in form during the growth cycle between rod and coccus is typical of the lifecycle of the Arthrobacter genus.\nCatabolic activities of Arthrobacter sp. strain IF1 To study the degradation potential of strain IF1, a range of organic compounds were tested as growth substrates. Cells were inoculated into MM, and different organic substrates were added at a concentration of 1\u00a0mM. After 72-h incubation growth, substrate disappearance and halide release were measured.\nGrowth and substrate removal were found when catechol, hydroquinone, hydroxyquinol, benzoate, phenol, 4-fluorocinnamic acid, and 4-nitrophenol were used as substrates. The organism did not grow on 2-fluorophenol, 3-fluorophenol, 4-chlorophenol, 4-bromophenol, 4-iodophenol, fluoroacetate, trifluoroacetate, fluoroacetamide, trifluoroethanol, or on 2-bromoethanol. The fact that strain IF1 is capable of growth on catechol, hydroquinone, and hydroxyquinol but not on 4-fluorocatechol indicates that 4-fluorocatechol is not the most likely intermediate in the 4-FP pathway, although toxic effects could also play a role.\nTo test the range of 4-FP concentrations tolerated by strain IF1, experiments were conducted in sealed flasks with 4-FP at concentrations of 1 to 7\u00a0mM as a sole carbon and energy source. Control assays without 4-FP showed no growth or release of fluoride, and sterile controls showed no abiotic loss of 4-FP. Between 1 and 4\u00a0mM 4-FP, the substrate was completely consumed, stoichiometric release of fluoride was observed, and biomass increased linearly with the amount of 4-FP added (Fig.\u00a02a). This indicates that the degradation of 4-FP by strain IF1 does not give large amounts of fluorinated dead-end products. Concentrations of 4-FP above 4\u00a0mM caused a toxic effect on the growth of IF1 (Fig.\u00a02b). The use of 5\u00a0mM 4-FP promoted growth, but a longer lag time was observed. At 7\u00a0mM 4-FP, no growth occurred even after 15\u00a0days of incubation.\nFig.\u00a02Fluoride release and biomass formation by strain IF1 with different concentrations of 4-FP. a Stoichiometric fluoride release. Symbols: squares, optical density at 600\u00a0nm; circles, fluoride concentration. b Growth in the presence of different levels of 4-FP. The initial concentrations used were: triangles, 1\u00a0mM 4-FP; circles, 4\u00a0mM 4-FP; diamonds, 5\u00a0mM 4-FP; squares, 7\u00a0mM 4-FP\nGrowth on 4-FP and formation of metabolites A batch culture of strain IF1 supplied with 1\u00a0mM 4-FP as the only source of carbon and energy was monitored in time. Growth was accompanied by an increase in biomass, decrease in 4-FP, and formation of fluoride (Fig.\u00a03). After 120\u00a0h, there was complete conversion of 1\u00a0mM 4-FP, and 1\u00a0mM fluoride was formed, indicating that there was no transient accumulation of fluorinated intermediates over the whole growth period. When cells grown on 4-FP were incubated in the presence of the iron chelator 2,2\u2032-dipyridyl, no 4-FP degradation occurred, and no fluoride was released in the medium. This indicates that initial or further enzymes involved in 4-FP metabolism require ferrous ions for activity.\nFig.\u00a03Biodegradation of 4-FP and fluoride liberation in a batch culture of strain IF1. The carbon source used is 1\u00a0mM 4-FP. Symbols: triangles, 4-FP concentration; diamonds, optical density; squares, fluoride concentration\nTo isolate intermediates of the degradation of 4-FP, samples from a batch culture containing 4\u00a0mM 4-FP were taken at appropriate intervals and analyzed by GC, HPLC, and LC-MS. Metabolites that were detected were numbered in order of time of appearance in the culture. GC analysis indicated that at least four metabolites were formed and degraded over time (Table\u00a01). Metabolite I, the earliest product that was observed, had the same retention time as an authentic hydroquinone standard and metabolite II had the same retention time as a standard of hydroxyquinol. GC-MS analysis showed that the mass spectrum of metabolite I was indeed similar to that of the hydroquinone standard, with a molecular ion peak at 110 and at 55\u00a0m\/z. Metabolite II gave a molecular ion peak at 126 and 63\u00a0m\/z, which is typical for hydroxyquinol, and the spectrum coincided with that of a standard. Metabolites V and VI were detected by GC but did not ionize in GC-MS under the conditions tested and were not identified.\nTable\u00a01Retention times in GC and HPLC analyses of metabolic intermediates formed by cells of strain IF1 growing on 4-FP and of some authentic standardsCompoundGC retention time (min)HPLC retention time (min)am\/z of observed fragment ions detected by GC-MSMetabolitesI20.71.3110, 81, 54II24.31.1126, 80, 52III\u2013c0.75\u2013IV\u20132.6\u2013V16.5\u2013\u2013VI19.4\u2013\u2013VII\u20133.1b159, 80dAuthentic standards4-FP15.73112, 83, 574-Fluorocatechol14.42128, 82, 51Hydroquinone20.71.3110, 81, 54Hydroxyquinol24.31.1126, 80, 52Catechol19.1\u2013\u2013aElution followed at 254\u00a0nmbAnalyzed after separation on a Symmetry Shield C8 column (4.6\u00a0mm\u2009\u00d7\u2009150\u00a0mm) as described in \u201cMaterials and methods\u201d for LC-MS analysisc\u2013 Not determineddLC-MS analysis\nWhen the supernatant of IF1 cells growing on 4\u00a0mM 4-FP were subjected to HPLC analysis, four peaks were detected (Table\u00a01). Metabolite I had the same retention time as the hydroquinone standard, and metabolite II coeluted with an authentic standard of hydroxyquinol. Hydroquinone was detected in culture supernatants by HPLC between 7.5 and 51\u00a0h (Fig.\u00a04). LC-MS analysis of the extracted samples of the supernatant revealed the presence of a compound (VII) of which the negative ion spectrum shows a molecular ion peak at 159\u00a0m\/z and a peak at 80\u00a0m\/z, which are expected for the negative ionization of 3-oxoadipate.\nFig.\u00a04Accumulation of hydroquinone during growth of strain IF1 on 4\u00a0mM 4-FP. The optical density (diamonds), 4-FP concentration (squares), and hydroquinone concentration (x marks) are indicated. Hydroquinone was identified by GC-MS and quantified by GC-FID\nThe above results suggest that 4-FP is initially converted to hydroquinone. The most likely enzyme involved in such a conversion is a 4-FP monooxygenase.\nEnzyme activities To test inducibility of enzymes involved in 4-FP degradation, cells of Arthrobacter sp. strain IF1 were grown on glucose or 4-FP, washed, and tested for oxygen consumption in the presence of different substrates. Washed suspensions of cells grown on 4-FP rapidly oxidized 4-FP, hydroquinone, and 1,2,4-benzenetriol (hydroxyquinol) without a lag. Catechol and 4-fluorocatechol did not stimulate oxygen uptake (Table\u00a02). This result indicates that 4-fluorocatechol and catechol are not likely intermediates and that hydroquinone and hydroxyquinol are. Glucose-grown cells did not oxidize any of the aromatic substrates tested with the exception of hydroxyquinol. This observation implies that hydroxyquinol may be converted by a constitutive oxygenase, whereas most other enzymes seem inducible. Cells grown with hydroxyquinol showed complete conversion of benzenetriol in the presence or absence of 2,2\u2032-dipyridyl, indicating that the putative hydroxyquinol oxygenase does not require ferrous ions to be active and that oxidation beyond this compound is not inhibited by the chelator. The formation of hydroquinone, which can be readily oxidized by 4-FP-grown cells, is in agreement with the involvement in 4-FP degradation of a 4-FP monooxygenase that is induced by 4-FP.\nTable\u00a02Substrates oxidized by 4-FP and glucose grown cells of Arthrobacter sp. strain IF1aAssay substrateO2 uptake (\u03bcmol\/min\/mg of protein) by whole cells after growth onFluorophenolGlucose4-Fluorophenol0.356<0.001Hydroquinone0.313<0.001Hydroxyquinol0.1950.1564-Fluorocatechol<0.001<0.001Catechol<0.001<0.001a Oxygen consumption was measured with an oxygen sensor as described in Materials and Methods. All substrate were used at a concentration of 1\u00a0mM\nTo study in more detail the degradation pathway of 4-FP by strain IF1, cell-free extracts of IF1 grown on 4-FP were investigated for the presence of several enzyme activities (Table\u00a03). When catechol 1,2- and 2,3-dioxygenases were tested for, no activity was found. Furthermore, no activity was detectable for 4-fluorocatechol dioxygenase. Consequently, the pathway does not proceed through catechol or a substituted catechol. When cell extracts were assayed for 4-FP monooxygenase, activity could be found in the presence of NADH but not in the presence of NADPH as the reducing cosubstrate. This observation indicates that the cells contain an NADH-dependent 4-FP monooxygenase activity. HPLC assays of incubations of cell extracts with 4-FP showed that substrate conversion was complete. The formation of the putative 4-FP monooxygenase was induced by 4-FP because no activity was found in extracts of cells grown in the presence of glucose.\nTable\u00a03Specific activities of enzymes in crude extracts of strain IF1 pregrown in 4-FP or glucoseAssay substrateEnzyme testedSpecific activity (U\/mg of protein) after growth on4-FluorophenolGlucoseCatechol1,2-Dioxygenase<0.01<0.012,3-Dioxygenase<0.01<0.014-FluorocatecholDioxygenase<0.01<0.014-FluorophenolMonooxygenase0.12<0.01HydroquinoneDioxygenase<0.01<0.01Monooxygenase0.313<0.01Hydroxymuconic semialdehydeDehydrogenase<0.01<0.01HydroxyquinolDioxygenase0.1950.156\nWhen hydroquinone was used as an assay substrate, its typical spectrophotometric peak at 288\u00a0nm was not replaced by a peak absorbing at 290 to 320\u00a0nm, which would have pointed to formation of hydroxymuconic semialdehyde. Thus, no hydroquinone dioxygenase was induced, or its product was rapidly further converted. The latter was ruled out by the observation that added hydroxymuconic semialdehyde was not converted by cell extracts of strain IF1, indicating that no hydroxymuconic semialdehyde dehydrogenase was induced during growth in the presence of 4-FP. These results make it unlikely that hydroxymuconic semialdehyde is an intermediate in the 4-FP pathway by strain IF1.\nWhen cell extracts were assayed for hydroxyquinol degradation, complete conversion of the substrate was observed with HPLC. When the reaction was monitored spectrophotometrically, the peak at 287\u00a0nm, typical of hydroxyquinol, was substituted by a peak at 245\u00a0nm, typical of maleylacetate. This indicates the involvement of a hydroxyquinol oxygenase that converts hydroxyquinol into maleylacetate, which is further converted into 3-oxoadipate.\nDiscussion\nThis study reports the isolation and characterization of Arthrobacter strain IF1, an organism capable of growth with 4-FP as a sole source of carbon and energy. The biodegradation of 4-FP by strain IF1 was analyzed by GC, GC-MS, HPLC, LC-MS, oxygen uptake experiments, and measurements of enzymatic activities. A metabolic pathway is proposed on the basis of the results of this study (steps 1 and 2, ortho-cleavage) and by analogy with other systems (Fig.\u00a05). We suggest that the degradation of 4-FP in strain IF1 starts with the conversion by a monooxygenase to benzoquinone, which is immediately reduced to hydroquinone. Hydroquinone then undergoes a further hydroxylation to form hydroxyquinol. This benzenetriol is the ring fission substrate, which is transformed by ortho-cleavage and yields maleylacetate, which is further converted to 3-oxoadipate.\nFig.\u00a05Proposed pathway for 4-fluorophenol degradation by Arthrobacter sp. strain IF1. Hydroquinone and hydroquinole were identified as intermediates by GC-MS\nUp to now, two main metabolic routes for the aerobic degradation of halogenated phenols have been described in the literature. In bacteria that degrade mono- and dichlorophenols, a degradation pathway is usually observed in which the substituted phenol is hydroxylated to the corresponding catechol, which is followed by ortho-cleavage of the aromatic ring (Haggblom 1992; Hollender et al. 1997; Wieser et al. 1994). On the other hand, biodegradation pathways in which the substituted phenol is converted via hydroquinone to maleylacetate have been found, mainly in organisms that grow on polyhalophenols or 4-nitrophenol (Kiyohara et al. 1992; Xun et al. 1992; Kadiyala and Spain 1998; Nordin et al. 2005; Perry and Zylstra 2007). Most studies have focused on the organisms and pathways that involve degradation via catechols.\nA clear indication for the first step in the 4-FP catabolic pathway of strain IF1 was obtained by mass spectroscopic analysis of culture supernatants, which indicated the formation of hydroquinone as an early nonfluorinated intermediate. This result, in combination with the enzyme assays and oxygen uptake experiments, suggests the involvement of an inducible 4-FP monooxygenase that is NADH dependent, although the first expected product of this reaction, i.e., benzoquinone, was not detected. Monooxygenation of an aromatic substrate carrying an electron-withdrawing halogen group or a nitro-substituent can result in simultaneous hydroxylation and dehalogenation or nitrite removal. This has, for example, been described for the conversion of tetrafluoro-p-hydroxybenzoate by para-hydroxybenzoate hydroxylase (Husain et al. 1980) and the oxidation of 2,4-6-trifluorophenol by a monooxygenase from Ralstonia eutropha JMP134 (Xun and Webster 2004). The quinone that is produced is chemically reduced by NADH to a hydroxyquinol, leading to a net stoichiometry of two NADH oxidized per halide or nitrite that is released (Fig.\u00a05). As in our case, no transient accumulation of a benzoquinone is usually observed, indicating that benzoquinone reduction is rapid. Other examples of halophenol degradation with formation of hydroquinone derivatives are the degradation of trichlorophenol to 2,6-dichlorohydroquinone by a strain of Pseudomonas pickettii (Kiyohara et al. 1992), the degradation of pentachlorophenol by a Sphingobium sp. through hydroxylation by a flavoprotein monooxygenase (Xun and Orser 1991), and the degradation of 4-chlorophenol by an Arthrobacter sp. (Bae et al. 1996).\nOur results further indicate that hydroquinone is not the ring fission substrate because no enzymatic activities for hydroquinone dioxygenase or hydroxymuconic semialdehyde dehydrogenase were found. Instead, upon conversion of hydroquinone by strain IF1, we observed the formation of a transient metabolite that was identified as hydroxyquinol, indicating that a second hydroxylation takes place before ring fission. The production of hydroxyquinol could be due to the action of a separate hydroquinone monooxygenase, or it could be caused by a second hydroxylation step by the 4-FP monooxygenase. Conversion of hydroquinone to hydroxyquinol before ring fission was also suggested for strains Arthrobacter that degrade 4-nitrophenol (Perry and Zylstra 2007) and 4-chlorophenol (Nordin et al. 2005). Ring fission of hydroxyquinol would produce maleylacetate, which we did not detect as an intermediate in 4-FP degradation, but a transient metabolite was found with mass properties identical to those of 3-oxoadipate, which is expected to be the next intermediate in hydroxyquinol degradation.\nMost cometabolic transformations of fluorophenols described in the literature involve the initial action of a phenol hydroxylase that results in the formation of fluorocatechols, which are subsequently transformed into fluoromuconates through a catechol 1,2-dioxygenase. The conversion usually proceeds with lactonization and elimination of fluoride (Boersma et al. 2001; Bondar et al. 1999; Finkelstein et al. 2000; Boersma et al. 1998; Bondar et al. 1998). Our results suggest that Arthrobacter strain IF1, which grows on 4-FP, possesses a new pathway for the degradation of 4-FP that involves immediate defluorination by a monooxygenase. Strain IF1 avoids the accumulation of possible toxic metabolites such as 3- or 4-fluorocatechol by this initial dehalogenation.","keyphrases":["4-fluorophenol","biodegradation","fluoride","hydroquinone","monooxygenase"],"prmu":["P","P","P","P","P"]}
{"id":"Neurosurg_Rev-3-1-2077911","title":"Foramen magnum meningiomas: detailed surgical approaches and technical aspects at Lariboisi\u00e8re Hospital and review of the literature\n","text":"Foramen magnum meningiomas are challenging tumors, requiring special considerations because of the vicinity of the medulla oblongata, the lower cranial nerves, and the vertebral artery. After detailing the relevant anatomy of the foramen magnum area, we will explain our classification system based on the compartment of development, the dural insertion, and the relation to the vertebral artery. The compartment of development is most of the time intradural and less frequently extradural or both intraextradural. Intradurally, foramen magnum meningiomas are classified posterior, lateral, and anterior if their insertion is, respectively, posterior to the dentate ligament, anterior to the dentate ligament, and anterior to the dentate ligament with extension over the midline. This classification system helps to define the best surgical approach and the lateral extent of drilling needed and anticipate the relation with the lower cranial nerves. In our department, three basic surgical approaches were used: the posterior midline, the postero-lateral, and the antero-lateral approaches. We will explain in detail our surgical technique. Finally, a review of the literature is provided to allow comparison with the treatment options advocated by other skull base surgeons.\nIntroduction\nMeningiomas are common neoplasms representing 14.3 to 19% of all intracranial tumors [63]. Among all the meningiomas, only 1.8 to 3.2% arises at the foramen magnum (FM) level [3]. Nevertheless, meningiomas are the most commonly observed FM tumors, representing 70% of all benign tumors [13, 15\u201319, 55, 64]. Most of the time, these are strictly intradural. Ten percent have an extradural extension: Most are intra- and extradural, and a few may be entirely extradural [9, 13, 15\u201319, 24, 25, 56, 60, 61].\nThe lesion is often large when discovered because of their slow-growing rate, their indolent development, the difficulty of the diagnosis leading to a long interval since the first symptom, and the wide subarachnoid space at this level [7].\nThe prerequisite for treating FM meningiomas (FMMs) is the perfect knowledge of the surgical anatomy. We will then first detail a comprehensive review of the relevant anatomy of the FM area with special emphasis on the vertebral artery (VA) V3 and V4 segments. Second, we will describe our classification system. This classification system helps for determining the best surgical approach and for anticipating the position of lower cranial nerves. The technical aspects of our surgical approaches will then be detailed extensively. Finally, we provide a summary of the relevant literature, detailing surgical results and surgical approaches advocated by other skull base surgeons and discussing the extent of bone resection and the need for fusion.\nSurgical anatomy\nLimits of the FM\nA meningioma is considered to be located into the FM region if its insertion zone is mainly situated into the FM area. According to the FM limits that we have previously used, the FM area is defined by these landmarks [13, 18, 19] (Fig.\u00a01):\nAnterior border: lower third of the clivus and upper edge of the body of C2Lateral borders: jugular tubercles and upper aspect of C2 laminasPosterior border: anterior edge of the squamous occipital bone and C2 spinous processFig.\u00a01Illustration of the foramen magnum anatomy through a postero-lateral approach. The skin incision (dotted line) extends on the midline just upper to the occipital protuberance and curves laterally toward the pathological side. The right vertebral artery has been elevated from the C1 posterior arch. The C1 posterior arch has been resected on the pathological side, and a suboccipital craniectomy has been performed. The dura matter has been opened. 1 CN IX\u2013X\u2013XI, 2 PICA, 3 CN XII, 4 vertebral artery V4 segment, 5 C1, 6 dentate ligament, 7 vertebral artery V3 segment\nThe VA V3 and V4 segments\nThe VA V3 and V4 segments are important vascular structures whose anatomy must be perfectly known when approaching FMMs.\nIn fact, after piercing the lateral aspect of the occipito-atlantal dura mater, the VA V3 segment becomes the V4 segment and then joins the contralateral one to form the basilar artery [8].\nThe VA V3 segment anatomy and techniques of approach, exposure, and transposition have been extensively described [8]. In summary, the VA V3 segment, also called the \u201csuboccipital segment,\u201d extends from the C2 transverse process to the FM dura mater. Its course is divided in three portions: a vertical portion, between the transverse processes of C2 and C1, a horizontal portion in the groove of the posterior arch of the atlas, and an oblique portion on leaving this groove up to the dura mater [8].\nAlong its course, this VA V3 segment can be at the origin of small collateral branches: in the middle of the posterolateral aspect of the C1\u2013C2 portion, corresponding to the C2 radicular artery, a small muscular branch coursing posteriorly at the upper exit of the C1 transverse foramen, the posterior meningeal artery, and the posterior spinal artery, just before its dural penetration. This last branch enters the dural foramen where the C1 nerve root exits the spinal canal [50] and can also be bound with the VA by fibrous dural bands. The posterior spinal artery may also arise from the initial intradural part of the VA V4 segment or from the posteroinferior cerebellar artery (PICA) [11, 33, 43, 50]. Then, this branch runs medially behind the most rostral attachment of the dentate ligament [50]. On reaching the lower medulla, it divides into ascending and descending branches, providing, respectively, the vascular supply to a part of the medulla oblongata and the spinal cord [50]. The VA V3 segment is surrounded by a periosteal sheath that invaginates into the dura when piercing the FM lateral dura, thus creating a double furrow for 3\u20134\u00a0mm. In fact, the periosteal sheath is in continuity with the outer layer of the dura. At this level, the VA is attached to the periosteal sheath, and the adventitia is adherent to the double furrow forming a sort of distal fibrous ring [8]. In such a way, despite being the most mobile segment, the VA V3 segment is fixed at its extremities.\nAnatomical relations of the VA are modified by head movements of rotation, as well as during surgical positioning. In neutral position, the vertical and horizontal portions of the V3 segment are perpendicular. On the contrary, after head rotation, as required during an anterolateral approach, both segments are stretched and run parallel, only separated by the posterior arch of the atlas, because the C1 transverse process is pushed anteriorly by this movement, away from the C2 transverse process [8].\nAbnormalities of the VA V3 segment are important to know and to identify preoperatively:\nIn 40% of the subjects, the VAs size is different: One side is dominant, and the other is hypoplastic or atretic [8].The VA V3 segment can end at the PICA or at the occipital artery [12]. In 20% of the cases, the PICA originates extracranially, arising from the VA above C1, between C1 and C2, or even in the V2 segment [29, 36].The VA V3 segment can have an intradural course that corresponds to a VA duplication: One atretic portion follows the normal course, but the main portion pierces the dura between C1 and C2 [23, 26, 30, 40, 53].A proatlantal artery consists of a persistent congenital anastomosis between the carotid artery and the VA; it is often associated with an atretic proximal VA and an extracranial origin of the PICA [12, 36, 48].Finally, the groove of the arch of the atlas can be turned into a tunnel if the occipitoatlantal membrane is calcified or ossified, raising some difficulties to expose the horizontal portion at this level [23, 47].\nThe VA V4 segment ascends from the dura up to the anterior aspect of the pontomedullary sulcus where it joins the controlateral one to form the basilar artery. Initially, the VA V4 segment faces posteriorly and medially the occipital condyle, the hypoglossal canal, and the jugular tubercle. Later, the VA V4 segment lies on the clivus and runs in front of the hypoglossal and the lower cranial nerves rootlets.\nAt the FM level, the VA V4 segment is at the origin of several branches: the PICA, the anterior spinal artery, the anterior and posterior meningeal arteries.\nThe PICA is the main VA branch and originates at, above, or below the FM level [56]. The anterior spinal artery starts near the vertebrobasilar junction. Most of the time, the anterior spinal arteries of both sides joins together above the FM level near the lower end of the olives, before descending through the FM and running on the anterior midline [56]. In some cases, one artery is dominant, or no fusion exists between both arteries, only one of these arteries forming the anterior spinal artery.\nThe vascular supply of the FM dura originates from the anterior and posterior VA meningeal branches and the meningeal branches of the ascending pharyngeal and occipital arteries [11, 42, 50]. Infrequently, meningeal branches are coming from the PICA, the posterior spinal artery, and the VA V4 segment. The anterior meningeal branch arises from the VA at the level of the third interspace. The posterior meningeal artery originates from the VA posterosuperior aspect when turning around the lateral mass of the atlas, above the posterior arch of the atlas, just before penetrating the dura, or just at the beginning of the V4 segment [8, 11, 50]. The ascending pharyngeal artery, a branch of the external carotid artery, gives meningeal branches passing into the hypoglossal canal and the jugular foramen. The meningeal branch of the occipital artery is inconstant and runs through the mastoid emissary foramen [50].\nLower cranial nerves\nThe glossopharyngeal (CN IX), vagus (CN X), and spinal accessory (CN XI) nerves arise from rootlets of the postolivary sulcus and join the jugular foramen, passing ventral to the choroid plexus protruding from the foramen of Luschka and dorsal to the VA [49].\nThe accessory nerve is composed of rootlets originating from the spinal cord and the medulla. Spinal rootlets join together to form the main trunk, which ascends through the FM running behind the dentate ligament and unites with the upper medullary rootlets. Anastomoses with the dorsal roots of the upper cervical nerves are frequent, that with the C1 nerve root being the most common and largest one [49]. The upper medullary rootlets penetrate directly the jugular foramen [49].\nThe hypoglossal nerve (CN XII) arises from rootlets of the preolivary sulcus. This nerve runs anterolateral through the subarachnoid space and pass behind the VA to reach the hypoglossal canal. Rarely, the VA separates the CN XII rootlets [49].\nThe dentate ligament\nThe dentate ligament is a white fibrous sheet that extends from the pia mater, medially, to the dura mater, laterally. It forms arches leaving passage to the VA for the first one and the second cervical nerve root for the second one.\nOur classification system of foramen magnum meningiomas\nMeningiomas are considered to be located in the FM area if their base of insertion is mainly located within the FM limits. This definition excludes tumors invading secondarily the FM region (Figure 2).\nFig.\u00a02Classification of foramen magnum meningiomas. Foramen magnum meningiomas are classified according to their compartment of development, their dural insertion, and their relation to the vertebral artery. The relation to the vertebral artery permits to anticipate the displacement of the lower cranial nerves. Tumors growing below the vertebral artery push the lower cranial nerves at the superior aspect of the lesion. On the other hand, tumors developed above or on both sides of the vertebral artery displace the lower cranial nerves in all directions and their position can then not be anticipated. Three basic surgical approaches are used. The extent of bone removal is delimited by the dotted lines\nThe definitive objective of a classification system is to define preoperatively the surgical strategy based on preoperative imaging characteristics of the lesion. The surgical strategy in cases of FMMs is the surgical approach but also the anticipation of modified vital structure position.\nIn our classification system, FMMs can be classified according to their compartment of development, their dural insertion, and to their relation to the VA [18].\nAccording to the compartment of development, FMMs can be subdivided in:\nIntraduralExtraduralIntra- and extradural\nIntradural meningiomas are the most commonly observed. Extradural meningiomas like at any other locations are very invasive, into the bone, the nerves and vessels sheaths, and soft tissues. The VA sheath and even its adventitia can also be infiltrated. This raises some difficulties and explains the higher incidence of incomplete removal as compared to intradural meningiomas [31, 32, 39, 56, 60, 62].\nAccording to the insertion on the dura, FMMs can be defined in the antero-posterior plane as:\nAnterior, if insertion is on both sides of the anterior midlineLateral, if insertion is between the midline and the dentate ligamentPosterior, if insertion is posterior to the dentate ligament\nAnterior meningiomas push the spinal cord posteriorly. Therefore, the surgical opening between the neuraxis and the FM lateral wall is narrow, and the drilling must extend to the medial part of the FM lateral wall to improve the access. In almost every case, no drilling of the lateral mass of the atlas and occipital condyle is necessary. Exceptionally, anterior meningiomas of small size without anterior compartment enlargement need more bone resection but never includes more than one fifth of these elements. On the other hand, lateral meningiomas displace the neuraxis posterolaterally and widely open the surgical access; therefore, the drilling has never to be extended into the lateral mass of the atlas or the occipital condyle.\nFinally, surgical strategies vary according to the relation to the VA, FMM having the possibility to develop:\nAbove the VABelow the VAOn both sides of the VA\nMeningiomas are more often located below the VA. In this case, the lower cranial nerves are always pushed cranially and posteriorly. There is no need to look for them. They will come into view on reaching the superior tumoral part. On the other hand, if the lesion grows above the VA, the position of the lower cranial nerves cannot be anticipated; the nerves may be displaced separately in any direction. After partial debulking of the tumor, one has to look for them so as to identify and protect them during the tumor resection. In case of tumoral development on both sides of the VA, a similar problem may exist with the position of the lower cranial nerves. Moreover, the dura around the VA penetration may be infiltrated by the tumor. As previously mentioned, the dura is normally adherent to the adventitia, and complete resection of the tumor at this level is hazardous. In this case, which is rarely observed, it may be safer to leave a cuff of infiltrated dura around the VA and to coagulate this zone.\nSurgical aspects\nPreoperative and perioperative considerations\nStandard preoperative workup includes magnetic resonance imaging (MRI), computed tomography (CT) scan, and sometimes angiography.\nOn MRI, gadolinium-enhanced sequences help to precisely delimit the dural attachment zone, the tumor, and its relation to neural and vascular structures. On T2-weighted images, the presence of an arachnoid plane between the tumor and the neuraxis is sometimes visible.\nBone windows CT scan is helpful in case of extradural extension to investigate bone erosion and to schedule preoperatively the need for fusion.\nConventional angiography is generally useless. There are only two indications for preoperative angiography:\nIf a highly vascularized tumor is suspected and embolization is contemplatedTo perform a balloon occlusion test in case of VA encasement (extradural or recurrent meningioma and meningioma inserted around the VA). In our experience, it has never been necessary to occlude the VA.\nIntraoperative neurophysiological monitorings have been used by several surgeons [3, 7] and includes somatosensory-evoked potentials, brainstem auditory-evoked potentials, and electromyographic monitoring of lower cranial nerves, by recordings through an endotracheal tube (CN X) and with a needle in the sterno-mastoid (SM) muscle (CN XI) and the tongue (CN XII).\nSurgical approaches and techniques at Lariboisi\u00e8re Hospital\nThe midline posterior approach\nThe midline posterior approach is indicated for posterior meningiomas, whatever their intra- and extradural extension, if they remain posterior to the plane of the dentate ligament and medial to the VA [15\u201317]. In such cases, the neuraxis is pushed anteriorly.\nThe patient may be in the sitting, ventral, or lateral position. To decrease venous bleedings, the sitting position is preferred at Lariboisi\u00e8re Hospital as far as there is no contraindication such as a patent foramen ovale; air embolism is prevented by hypervolemia and G-suit.\nThe skin is incised on the midline from the occipital protuberance down to the upper cervical region. The midline avascular plane is opened between the posterior muscles, up to the occipital protuberance and down to the spinous process of C2. Bone opening is performed using a drill and Kerrison rongeurs and is always limited to the lower part of the occipital bone and the posterior arch of the atlas. The dura is then incised in a T- or Y-shaped fashion and retracted with stitches.\nPostero-lateral approach\nThe postero-lateral approach is preferred for any intradural process located laterally and\/or anteriorly to the neuraxis and for extradural lesions developed on the posterior part of the lateral FM wall [15\u201317]. For tumors extending far beyond the anterior midline, the postero-lateral approach has the advantage to allow a bilateral approach in the same stage (Fig.\u00a03, 4, and 5).\nFig.\u00a03a\u2013c Preoperative MRI. A large lateral foramen magnum meningioma displaces the neuraxis. d, e Postoperative CT scan. The meningioma has been completely resected. The spinal cord has regained a normal shape. f Reconstructed 3D CT scan after contrast administration. The resection of the posterior arch of the atlas is visible on the right side. The lateral mass of the atlas (star) was left intact. The vertebral artery (arrow) has been elevated from the C1 posterior arch (compare with the left side) during the dissectionFig.\u00a04a, b Preoperative MRI. A large anterior foramen magnum meningioma severely compresses the neuraxis, which is reduced to a crescent (star). c, d Postoperative MR images confirm the complete resection of the tumorFig.\u00a05Surgical steps during a postero-lateral approach. a The left vertebral artery V3 segment (black arrow) has been elevated from the lateral part of the C1 posterior arch (white arrowhead). The medial portion has been resected up to the midline (black arrowhead). The dural entrance of the vertebral artery, where the V3 segment becomes the V4 one, is delineated by the dotted line. b The dura matter has been incised. The inferior contraincision extends inferiorly to the site of entrance of the vertebral artery into the dura matter. The C1 posterior rootlets are identified (black arrowhead). The inferior portion of the meningioma (white star) severely compresses the spinal cord (black star). c The vertebral artery V4 segment (white arrow), the spinal accessory nerve (black arrowheads), and the XIIth cranial nerve (white arrowhead) have been controlled. Of note, the dura matter (black star) is stretched over the left C1 lateral mass by a stitch to enlarge the lateral access and after the occipital craniotomy, the fall of the left cerebellum is prevented by a blade (white star). d After the complete removal of the meningioma, both vertebral arteries are visible. On the left side, we observe the section of a feeding vessel (white arrow). The right PICAs is visible (black arrow) as well as the Xth (black star) and the XIIth (black arrowhead) cranial nerves\nThe postero-lateral approach is a lateral extension of the midline posterior approach. The patient must be carefully positioned in the same position as during a posterior midline approach. The head must be placed in neutral position. Any flexion must be avoided because it decreases the space in front of the neuraxis and therefore may worsen the compression and the neurological condition. The vertical midline skin incision is identical, but the incision is curved laterally on the tumoral side just below the occipital protuberance toward the mastoid process. The posterior muscles are divided along the occipital crest and retracted laterally to expose the occipital bone, the posterior arch of the atlas, and the C2 lamina, if required. The exposure may be extended on a limited way on the contralateral side.\nVA exposure At this step, the VA running above C1 needs to be exposed to safely resect the C1 posterior arch up to the C1 lateral mass. The exposure of the horizontal segment of the VA V3 segment progresses from the midline of the posterior arch of the atlas laterally toward the atlas groove. The medial border of the groove is clearly marked by a step with a decrease in the height of the posterior arch. The safest way to expose the VA is to dissect strictly in the subperiosteal plane. The periosteum of the C1 posterior arch needs to be elevated from medial to lateral and inferiorly to superiorly. Working in this manner permits to expose the posterior aspect of the posterior arch of the atlas, to bring into view the step at the medial end of the groove and then to elevate the VA from the atlas groove. In fact, the VA V3 segment is surrounded by a venous plexus; both artery and veins are enclosed in a periosteal sheath. Both provide protection against VA damage. If tearing occurs, venous bleedings can be controlled by direct bipolar coagulation on the VA periosteal sheath. Dissection at the superior aspect of the VA is more difficult because there is no good landmark and the periosteal sheath is in continuity with the ligaments covering the occipital condyle. Cutting of the ligaments must proceed a few millimeters above the VA on the occipital condyle by following the superior aspect of the VA sheath. The occipitoatlantal membrane covering the groove of the posterior arch of the atlas can be calcified or ossified, turning the groove into a tunnel, raising some difficulties to expose the horizontal portion at this level [23, 47]. After being fully exposed, the VA can be gently displaced superiorly to show the lateral mass of the atlas.\nBone opening The posterior arch of the atlas is resected with rongeurs from the midline toward the transverse process. To obtain a decompression of the neuraxis before the tumoral resection, the posterior arch of the atlas must also be resected beyond the midline toward the controlateral side. By this way, hyperpressure induced even by gentle manipulation during resection are not transmitted to the neuraxis. The lower part of the occipital bone is drilled or resected with rongeurs laterally toward the sigmoid sinus and also beyond the midline.\nThe lateral extension of the bone opening is established according to the position of the meningioma. In case of lateral meningiomas, the spinal cord is displaced toward the contralateral side, and then any resection of the FM lateral wall is not necessary. In case of anterior meningiomas, the spinal cord is pushed posteriorly, and the surgical corridor has to be enlarged laterally by drilling up to the medial side of the C1 lateral mass and\/or the occipital condyle. Drilling further the lateral mass of the atlas or the occipital condyle is exceptionally necessary. In the exceptional case where the opening must be enlarged, no more than 20% of the medial part of the FM lateral wall has to be drilled. In such a way, stabilization is never required.\nThe cranio-caudal bone resection is scheduled according to the position of the tumor regarding to the VA. The drilling has to be extended toward the lateral mass of the atlas if the tumor grows below the VA, to the jugular tubercle and the occipital condyle if it grows above, and toward both parts of the FM lateral wall, if it encircles the VA on both sides.\nDura opening The most adequate dural incision is a curvilinear incision starting at the inferolateral corner then running vertically at a paramedian level (5 to 10\u00a0mm from the midline) and curving toward the superolateral corner. This type of incision permits to take the maximum benefit of the bone opening without contraincision and is generally easy to close. Keeping the dura over the neuraxis provides protection during the surgical dissection. Moreover, arachnoid connections between the dura and the medulla are left undissected to prevent an anterior fall of the neuraxis during tumor resection.\nIntradural step Lateral meningiomas are directly brought into view and their dural attachment directly accessible. Contrarily, anterior meningiomas are partially hidden by the neuraxis. The first step of the intradural stage, before beginning tumor resection, is to identify several important structures and to open the surgical access to the lesion by completely releasing the neuraxis. Important structures are the VA, the accessory nerve, and the dentate ligament. The VA is identified by following the course of the V3 segment where it pierces the dura matter. The first two digits of the dentate ligament have to be divided. To further improve the surgical access, the first cervical nerve root can be divided distal to its connections with the accessory nerve.\nAt this step, the surgical technique must be individualized according to the tumor location: above, below, or on both sides of the VA.\nAs previously mentioned, in cases of tumor developed below the VA, the lower cranial nerves are always pushed cranially and posteriorly by the tumor. These nerves will then be found at the superior pole of the lesion at the end of the surgery. The resection must start at the caudal aspect of the meningioma with the goal to release the dural attachment and to suppress the vascular supply first; then, the tumor is debulked in a dry surgical field with a sucker, an ultrasound aspirator, or a laser, according to the tumor consistency. When liberating the dural insertion, it is important to keep undetached a small part of the base, at the side of the neuraxis, to avoid free movement of the lesion, which can be responsible for inadvertent damage to the neuraxis during the remnant resection. When hollowing the tumor, a small layer is also kept with its capsule against the neuraxis. This part will be resected at the last surgical step and under better conditions when the meningioma is completely devascularized and the surgical field widely open.\nIf the tumor is developed above the VA, two special points must be taken into consideration: the displacement of the lower cranial nerves and the dissection of the VA branches. Indeed, in such location, the displacement of the lower cranial nerves cannot be anticipated. To prevent damage, the rootlets must be under control on the side of the jugular foramen and then followed along their courses more or less adherent to the meningioma. With the lesion being progressively debulked, the nerve rootlets can be more easily mobilized, often inferiorly, to allow a more confident tumor resection at some distance from fragile nervous structures. The tumor dissection from the VA branches, especially the PICA, is another difficulty encountered only with tumors developed above the VA. Precise knowledge of the patient anatomy based on preoperative investigation is mandatory.\nIf the meningioma encases the VA, the technique is identical as described above. Special consideration is nevertheless required if the meningioma has its insertion on the dura surrounding the VA penetration. The dural resection is better achieved by progressing from the extradural side toward the intradural aspect, along the VA because the VA invaginates into the dura with its periosteal sheath. This furrow can be resected as much as the VA adventitia is not invaded.\nDural closure A watertight dural closure is required to prevent a postoperative cerebrospinal fluid (CSF) leakage. The closure is generally easy with the curvilinear incision. If necessary, a dural patch using the suboccipital aponeurosis achieves a perfect closure. In any case, the muscular and aponeurosis layers must be tightly closed.\nAntero-lateral approach\nThe antero-lateral approach is rarely used in our department as we consider that it is a good choice only in some meningiomas with extradural extension through the bony structures [15\u201317].\nThe antero-lateral approach to the craniocervical junction has been extensively described in the article focusing on the surgical exposure of the VA V3 segment and will not be detailed again for this reason [8]. Some points required nevertheless special consideration. The head has to be slightly extended with a rotation of 60\u00b0 toward the opposite side. Because of this rotation, VA segments above and below the C1 posterior arch run parallel, separated by this bone. Opening the C1 transverse foramen is mandatory for VA transposition.\nBone opening When completely liberated, the VA can be pulled out and transposed, bringing into view the FM lateral wall as the occipital condyle, the anterior and posterior arches of the atlas, the lateral mass of the atlas, and the C1\u2013C2 joint become visible.\nThe exposure must now be adapted to each case, depending on the location and extension of the meningioma. The odontoid process can be reached by passing over the C1\u2013C2 joint. The mastoid process can be resected and small bridging bone removed to open completely the jugular foramen from the end of the sigmoid sinus, to the jugular tubercle just underneath the junction with the jugular bulb, up to the beginning of the internal jugular vein.\nDura opening The dural opening is most of the time out of concern because the antero-lateral approach is mainly indicated for extradural meningiomas. When lesions are developed in both extradural and intradural compartment, the dura is already opened by the lesion and must only be enlarged.\nDural closure Dural closure can be a considerable problem in cases where the dural defect is anteriorly located at the level of the clivus, the anterior arch of the atlas, and the odontoid process.\nPostoperative complications\nThe antero-lateral approach could induce a transient dysfunction of the accessory nerve by the nerve manipulation, responsible for pain along the trapezius muscle and weakness of the trapezius muscle and\/or the SM muscle. During this approach, manipulation of the sympathetic chain could also be at the origin of a transient Horner\u2019s syndrome.\nVA damage has never been observed. In case of tumoral encasement, a balloon occlusion test must be realized.\nPreservation of the lower CNs can be hazardous. If CN IX and X are damaged, postoperative swallowing problems must be anticipated.\nReview of the literature\nPublished series\nYasargil has reviewed series published from 1924 to 1976 and counted 114 cases of FMMs. Since then, more than 400 cases of FMMs have been reported in the literature [2\u20137, 10, 18\u201322, 27, 35, 37, 38, 41, 44\u201346, 52, 54, 55, 57, 58]. George et al. [16, 19] reported on a series in the French literature of 106 craniocervical meningiomas from 21 hospitals. The largest single-center series have been published by Meier et al. [38] in 1984, Samii et al. [55] in 1996, and George et al. [18] in 1997. Table\u00a01 summarizes published series over the last 20\u00a0years in the English literature.\nTable\u00a01Review of the literature of published series of FMMs over the last 20\u00b0years in the English literatureAuthorYearNb ptFMM location (%)Recurrence (%)Va encasement (%)ApproachVA transpResection JTPartial mastNb CR (%)Extent CRInstability (%)Outcome (%)Resection (%)Transient morbidity (%)Permanent morbidity (%)Mortality (%)FURecurr (%)AnteriorLateralPosteriorImprovedUnchangedWorsenedTotalSubtotalGilsbach19875100\u2013FLY1\/3100.00.0200.0Guidetti19881782.40100.01211.0Sen, Sekhar19905802080\u2013ELYYY1001\/3\u20131\/2020206060.040.06020.0Crockard199131003333TONo00331000.066.0100.010066.020.633.0Kratimenos1993810012.50\u2013FLY1\/3087.512.5025.0\u2013\u2013Babu19949100ELYYY1001\/3\u20131\/2088.811.278.05611.19.40.0Akalan1994812.5087.50PM008812100.000.0Bertalanffy199619100\u2013FL SO TCY1001\/30100.00.000.0\u2013\u2013Samii199638955540PM, LSO17.501\/3063.030.037.056.021.05.0George1997404552.502.5038100Partial0902.507.5087.510.00.007.557.60.0Pirotte19986100\u2013Y1001\/2\u20131\/30100.00.01717.0Sharma1999105050PM, FL00Yes100.015.0Salas199924100\u2013TC\/ELTJYY1001\/3066.033.0\u20130.014.8\u2013Arnautovic20001810011.10\u2013TCY1001\/2\u20131\/30891175.012.55511.116.640.05.5Roberti200121EL TC76.024.021.509.5Goel20011710059SO11.801\/3\u20131\/4010082.018.060.0Nanda20026100\u2013FL00100100.00.000.043.00.0Marin Sanabria2002772.5028.50TO, SO, TC291\/3\u20131\/2080.0020100.00.072.5514.0Parlato20037\u2013Y<1\/2086.0%14.00.024.00.0Boulton2003106010300070201090.010.040.0100.0Pamir20042291940FL951\/3095.54.5274.500.040.00.0Margalit200518100Lat50Partial (9\/18)0.0Bassiouni200625325711443FL0096.04.040.084.073.20.0CR Condyle resection, EL extreme-lateral, FMM foramen magnum meningioma, FL far lateral\/postero-lateral, FU follow-up, JT jugular tubercle, mast mastoidectomy, Lat lateral, Nb number, pt patient, Recurr recurrence, SO suboccipital, TC transcond dylar, TO transoral, transp transposition, VA vertebral artery, Y yes\nHeterogenicity of published series\nA heterogenous group of patients constituted the published studies. Several studies included not only FMMs but also other FM pathologies such as neurofibromas, schwannomas, as well as other various lesions leading result analysis precarious [2, 4, 10, 35, 38, 41, 45, 57, 58]. Table\u00a01 summarizes also the high variability of tumor location, rate of VA encasement, rate of tumor recurrence, and surgical approaches. The proportion of anterior FMMs is comprised between 12.5 [2] and 100% [3, 27, 41]. The subdivision between anterior and lateral FMM is not always detailed in studies including 90 to 100% of antero-lateral FMMs [4, 6, 20, 21, 35, 44, 46, 54, 55, 57]. Posterior FMMs were always included in studies with anterior or antero-lateral lesions with a variable proportion from 2.5, 5, 9, 11, 28.5, 30, up to 50% [5, 7, 18, 37, 44, 55, 58]. The rate of VA encasement can be higher than 59% and the rate of tumor recurrence up to 80% [21, 57] (Table\u00a01).\nSurgical approaches, extent of bone resection, and need for fusion\nFMMs are undoubtedly challenging tumors, requiring special considerations because of the vicinity of the brainstem, medulla oblongata, lower cranial nerves, and the VA. Several approaches have been advocated. The definite goals are to achieve the largest tumor removal and the lowest morbidity rate as possible. Minimizing the morbidity is obtained by choosing the appropriate exposure. The approach must allow adequate controls of important neurovascular structures, without exposing to unnecessary risks.\nThere is no discussion about the best surgical approach of posterior FMMs. The posterior approach is the best option, is well known by neurosurgeons, and is associated with a low morbidity rate.\nThe debate about the best surgical approach is more open for lateral and mainly anterior FMMs.\nThe transoral approach has been reported sporadically [10, 39]. Despite providing access to the anterior part of the craniocervical junction, this approach has several drawbacks in case of intradural lesions: increased risk of CSF fistula and meningitis after crossing of the contaminated oral cavity, poor access to laterally extending tumors resulting in a low rate of complete resection, and increased risk of posteroperative instability and velopalatine insufficiency [10, 39].\nThe two main surgical approaches reported in the literature are the far-lateral approach [28], also called postero-lateral approach or lateral suboccipital approach, and the extreme-lateral approach, also named antero-lateral approach. As detailed by Rhoton [51], the far-lateral approach is a lateral suboccipital approach directed behind the sternocleidomastoid muscle and the VA and just medial to the occipital and atlantal condyles and the atlanto-occipital joint. The extreme lateral approach is a direct lateral approach deep to the anterior part of the sternocleidomastoid muscle and behind the internal jugular vein along the front of the VA. In fact, both approaches permit drilling of the occipital condyle but provide a different exposure because of the differences in the approach direction.\nThe extreme-lateral transcondylar approach for FMMs was first reported by Sen and Sekhar [57] in 1990. Further publications of the same experienced skull base surgeons followed [4, 52, 54]. Salas et al. [54] reported in 1999 several variations of the extreme-lateral approach based on a series of 69 patients, including 24 meningiomas. The lesions were removed most of the time through a partial transcondylar approach and more rarely through transfacetal, retrocondylar, and extreme transjugular approaches. During the partial transcondylar approach, the posterior one third of occipital condyle and superior facet of C1 were drilled away. These authors also performed a partial mastoidectomy [4, 54, 57]. Arnautovic et al. [3] published their experience in 2000 on a series of 18 ventral FMMs. They used also the extreme-lateral transcondylar approach. The condyle drilling ranged approximately from one third to one half of the condyle, without causing craniocervical instability. The extreme-lateral trancondylar approach requires the VA transposition to reach and drill the occipital condyle [3, 4, 27, 45, 54, 57].\nAs others [5, 6, 20, 21, 27, 35, 41, 44, 55, 58], we advocate rather the postero-lateral approach, also called far-lateral approach, even for anterior intradural FMMs. During this approach, the VA is controlled in the horizontal portion of the V3 segment, above the C1 posterior arch. VA transposition is rarely performed and has only been reported in one series [46]. The extent of FM lateral wall drilling is variable, in fact directly proportional to the tumor extension to the contralateral side. In the literature, the occipital condyle resection varies between 0 and 66% [27, 41, 44, 46, 55]. In a recent publication, Bassiouni [5] classified judiciously surgical approaches in two groups: transcondylar or retrocondylar if the occipital condyle is not drilled. In our review of the literature (Table\u00a01), we found nine series in which the retrocondylar approaches was used to reach anterior or anterolateral meningiomas [2, 5, 7, 21, 35, 37, 41, 55, 58]. In four of these series, a retrocondylar approach was not used in all cases but in 89.2, 82.5, 71.5, and 50% [21, 35, 37, 55]. The five other series are homogeneous and only included patients treated through a retrocondylar approach [2, 5, 7, 41, 58]. Of these, three studies reported complete resection in 100% of the cases [2, 41, 58]. In the two others, complete resection was noted in 90 and 96% of the cases, and the remaining lesions were resected subtotally [5, 7]. Surgical results were good, and surgical morbidity and mortality rates low [2, 5, 7, 41].\nExtradural lesions can be treated either by posterolateral or anterolateral approach (Fig.\u00a02). The extent of drilling during this procedure can be larger but is only dictated by the tumoral invasion and must be limited to the destroyed or invaded bone. In such a way, the question of instability is only a preoperative concern. In our experience of tumors located at the craniocervical junction, we have not observed any instability if less than half of the C0\u2013C1 and C1\u2013C2 joints were resected [15]. In our experience, VA transposition was merely performed in selected cases of FMMs with extradural extension and never for intradural lesions.\nSurgical results\nMorbidity\u2013mortality\u2013prognosis factors\nIn the Yasargyl\u2019s review of the literature of series published before 1976, the overall mortality rate was approximately 13% but could be as high as 45% in some series [34].\nOver the last 20\u00a0years, the overall mortality is 6.2%. The mortality rate is comprised between 0 and 25% (Table\u00a01). Mortality rates higher than 10% were mainly observed in small series [4, 27, 37, 46, 57, 58].\nIn the Yasargil\u2019s review, a good outcome was noted in 69% and a fair and poor outcome, respectively, in 8 and 10%. In series larger than ten patients published over the last 20\u00a0 years (Table\u00a01), neurological improvement, stability, and worsening were noted, respectively, in 70\u2013100, 2.5\u201320, and 7.5\u201310% of the cases. The permanent morbidity rate is comprised between 0 and 60%. The permanent morbidity rate is lower through a far-lateral approach (0\u201317%), either transcondylar or retrocondylar, than through an extreme-lateral transcondylar approach (21%\u201356%, considering series without recurrent tumor). Lower cranial nerves dysfunctions are the most frequently encountered preoperative deficits. These deficits have the propensity to recover even completely postoperatively [3\u20135], except in cases of en plaque meningiomas or recurrent tumor [55].\nSeveral factors lead the surgical procedure still more difficult, then influencing negatively the morbidity rate: anterior tumor location [18, 55], tumor size (smaller lesions are more difficult to resect because the surgical corridor is small), tumor invasiveness, extradural extension [18], VA encasement [22], absence of arachnoidal sheath [5, 14, 65], and adherences in recurrent lesions [4, 5, 55, 57].\nRate of tumoral resection\nBased on a multicentric study from 21 hopitals, George et al. [19] reported 77, 16, and 7%, respectively, of complete, subtotal, and partial removals. Over the last 20\u00a0years, most of the studies reported complete or subtotal removal of the tumor [2, 4\u20137, 10, 18\u201322, 27, 35, 37, 41, 44\u201346, 52, 54, 57, 58]. Factors limiting the resection completeness are adherences of the lesion to vital structures, VA encasement and invasiveness of the lesion. Adherences are observed during repeated surgery and explain the lower rate of complete tumor resection (60\u201375% of Simpson grade 1) in surgical series in which a high rate of recurrent tumors are included [3, 55, 57]. Eventually, in recurrent tumors, some authors advocate leaving a small tumor remnant to preserve a low morbidity rate [5, 18]. On the other hand, Arnautovic et al. [3] favored radical removal of recurrent tumors with the goal of providing a relatively long and stable postoperative course, even at the price of frequent but transient morbidity induced by lower cranial nerves dysfunction. Arnautovic et al. [3] have also demonstrated that the rate of complete removal is higher at first surgery than when treating recurrence, advising then to be more aggressive at the first surgical presentation. VA encasement was noted in 38 to 59% in some series [5, 18, 21, 44, 55]. This factor was recognized as an independent factor of incomplete removal [55]. The location of the meningioma, either intraextradural or extradural, reflects the tumoral invasiveness. These tumors are less favorable to be completely resected than pure intradural lesions [19, 39]. In the French cooperative study [19], the rate of complete removal of intradural, extradural, and intraextradural meningiomas was, respectively, 83, 50, and 45%.\nOur experience\nOur experience has been published in 1997 after the treatment of 40 FMMs operated in the period 1980\u20131993 [18]. Twenty-four lesions were intradural, two were extradural, and four were intraextradural. Eighteen were considered anterior, 21 lateral, and one was posterior. The tumor was above the VA in four cases, below in 20 cases, and on both sides in 16 cases. The posterolateral approach was used in 31 cases, the anterolateral one in five cases, and the posterior midline in four cases. The rates of complete resection for intradural and extradural lesions were, respectively, 94 and 50%. Postoperatively, the clinical condition improves in 90% of patients, remains stable in 2.5%, and worsened in 7.5%. Our present experience over the last 25\u00a0years is now based on 97 FMMs (unpublished data). Complete removal was achieved in 86% and subtotal removal in 11%. Subtotal removals were due to extradural extension or to recurrent cases. The rate of complete removal increased up to 94% when selecting only intradural lesions treated at first presentation. We mainly use the far-lateral retrocondylar approach for intradural FMMs. In some cases, the drilling of the FM lateral wall has to be performed for intradural anterior meningiomas but remains limited at worst to the medial 20% of the FM lateral wall. Whatever, this extent of drilling has to be tailored according to the tumor characteristics. We consider nevertheless that more bone resection is never necessary because of the anatomical anterior position of the lateral mass of the atlas and the occipital condyle. Our attitude has been reinforced by cadaveric study, which has demonstrated that increasing the bone drilling is not associated with a significant widening of the surgical corridor [41]. In fact, resecting one third and one half of the occipital condyle increases the visibility respectively by 15.9 and 19.9\u00b0. Two anatomic reports have demonstrated that condyle resection allows a wider angle of exposure to gain the anterior foramen area [1, 59]. However, these studies did not consider the fact that in surgical approaches to anterior lesions, space-occupying lesions can enlarge the surgical corridor. We consider that the extreme-lateral approach could be associated with a higher morbidity rate than the far-lateral approach. The exposure allowed by the far-lateral retrocondylar or partial transcondylar approach is adequate for resecting even anterior intradural FMMs. The supposed benefit in term of exposure provided by the extreme transcondylar approach is counterbalanced by the risks associated with the CN XI dissection, the VA transposition, and the condyle drilling.\nConclusions\nFMMs are challenging tumors in the vicinity of the brainstem, the VA, and lower cranial nerves. Several surgical approaches are possible, each one with specific indications. The drilling of the FM lateral wall required during the approaches is always limited and by itself should not be at the origin of any instability. Postoperative complications can be dramatic and must be anticipated.","keyphrases":["foramen magnum","meningioma","surgical approach","tumor","vertebral artery"],"prmu":["P","P","P","P","P"]}
{"id":"J_Autism_Dev_Disord-4-1-2268726","title":"Brief Report: The Use of WAIS-III in Adults with HFA and Asperger Syndrome\n","text":"The WAIS III was administered to 16 adults with high functioning autism (HFA) and 27 adults with Asperger syndrome. Differences between Verbal Intelligence (VIQ) and Performance Intelligence (PIQ) were not found. Processing Speed problems in people with HFA appeared. At the subtest level, the Asperger syndrome group performed weak on Digit Span. Comprehension and Block Design were relative strengths. In the HFA group, performance on Digit-Symbol Coding and Symbol Search was relatively poor. Strengths were found on Information and Matrix Reasoning. The results suggest that the VIQ-PIQ difference cannot distinguish between HFA and Asperger syndrome. WAIS III Factor Scale and Subtest patterning provides a more valid indicator.\nIntroduction\nOver the past few years, interest in HFA and Asperger syndrome in adults with normal intelligence has increased markedly. However, not much is known about the cognitive profiles of these groups. Only a few studies exist about adults who function relatively well in society and have been diagnosed late in life (Howlin 2004; Vermeulen 2002). The present study aims to assess the cognitive profiles of this relatively well-functioning subgroup by means of the Wechsler Intelligence Scale III (WAIS III, Wechsler 1997).\nIn WAIS III, the intelligence pattern is described at three levels: The first level contains Performance Intelligence and Verbal Intelligence. The second level consists of the four factor scales: Verbal Comprehension, Perceptual Organization, Freedom from Distractibility and Processing Speed. The third level contains the subtests. The following paragraphs summarizes research on the intelligence profiles of adults with the autistic disorder or Asperger syndrome on the basis of these three levels.\nThe Performance IQ (PIQ)\u2013\u2013Verbal IQ (VIQ) dichotomy has been incorrectly used for years to underpin the diagnosis of autistic disorder or Asperger syndrome. It is questionable whether the two constructs should even be applied in general, because research did not support the construct validity of the VIQ-PIQ dichotomy (Taub 2001).\nFor adults with HFA, studies on WAIS-R have yielded contradictory results (Minshew et\u00a0al. 1992; Siegel et\u00a0al. 1996; Vermeulen 2002), which may reflect the validity problems of the VIQ-PIQ dichotomy (Arnau and Thompson 2000; Taub 2001).\nThe factor scale level is of great importance in assessing cognitive abilities because factor analytic studies indicate that the factor scales give the best estimates of the four factors underlying intelligence (Arnau and Thompson 2000; Ryan and Paolo 2001).\nNo studies have been performed on WAIS-III profiles for adults with HFA or Asperger syndrome as far as we know. Therefore no information is available on the results of factor scales in these groups. This leads to the conclusion that the most important factors of the intelligence pattern for adults with HFA or Asperger syndrome are still unknown.\nOn subtest level, some studies on WAIS or WAIS-R found low Comprehension versus high Block Design scores (Goldstein et\u00a0al. 2001; Rumsey and Hamburger 1988). A relatively high variability between the subtests scores in adults with HFA has also been reported (Siegel et\u00a0al. 1996).\nIn summary, research shows that among adults with HFA or Asperger syndrome, results of VIQ-PIQ differences vary and may be influenced by the validity problems of the VIQ-PIQ dichotomy. The factor scale scores and the subtest patterns provide a better representation of the intelligence pattern.\nAims of the Present Study\nThe present study aims to acquire insight into the WAIS III profiles of normal intelligent adults with HFA and Asperger syndrome. Profiles in the total group and differences between the two diagnostic groups will be examined.\nMethods\nProcedure\nAll participants were recruited from the GGZ (Mental Health Center) Eindhoven and Oost-Brabant. The participants met the criteria for Asperger syndrome or HFA. Participants with relevant neurodevelopmental conditions and genetic conditions were excluded, as were institutionalized patients and patients with a Full Scale IQ below 80.\nSubjects \nThe mean Full Scale IQ of the participants was 110.16, individual scores varied between 83 and 145 (see Table\u00a01).\nTable\u00a01Characteristics of participantsIQ and ageMSDRange\u00a0\u00a0\u00a0\u00a0Full scale IQ110.1616.0583\u2013145\u00a0\u00a0\u00a0\u00a0Mean age41.9310.6720\u201360Diagnosisn%\u00a0\u00a0\u00a0\u00a0Autistic disorder1637.2 \u00a0\u00a0\u00a0\u00a0Asperger syndrome2762.8 Gender\u00a0\u00a0\u00a0\u00a0Male3990.7 \u00a0\u00a0\u00a0\u00a0Female49.3 Education\u00a0\u00a0\u00a0\u00a0Lower\/middle education1841.9 \u00a0\u00a0\u00a0\u00a0Higher education2558.1 Employment status\u00a0\u00a0\u00a0\u00a0Employed or retired3069.8 \u00a0\u00a0\u00a0\u00a0Studying12.3\u00a0\u00a0\u00a0\u00a0Unemployed1227.9 Current living circumstances\u00a0\u00a0\u00a0\u00a0Lives with partner2353.5 \u00a0\u00a0\u00a0\u00a0Lives independently 1227.8 \u00a0\u00a0\u00a0\u00a0Sheltered living24.7 \u00a0\u00a0\u00a0\u00a0Lives with parents 614.0 \nAll individuals ranged in age from 18 to 60\u00a0years. The mean age was 41.93. Of all participants, 25 finished higher education and 30 individuals had work. 23 participants lived together with a partner. The relatively large number of participants who had a relationship, worked and were well educated emphasizes the relatively high level of functioning in this group.\nAssessment of Disorder \nHetero-anamnestic information was gathered using the Dutch version of the Autistic Disorder Diagnostic Interview, revised version (ADI-R, Lord et\u00a0al. 1994), administered by psychologists who were officially trained in the administration and scoring of the instrument. To gather anamnestic information, a semi-structured interview was used to assess presence of the DSM-IV criteria of HFA and Asperger syndrome (APA 1994). Because of the controversial nature of the DSM-IV criteria (Ghaziuddin et\u00a0al. 1992; Mayes et\u00a0al. 2001), additional questions were used to differentiate between HFA and Asperger syndrome, based on the diagnostic criteria of Gillberg and Gillberg (1989) and ICD-10 (WHO 1993).\nAssessment of Intelligence \nThe intelligence profile was assessed using the Dutch translation of the WAIS III (Wechsler 1997). The WAIS-III has excellent psychometric properties (Sattler and Ryan 1999) and has been validated for the Dutch population (Wechsler 1997).\nResults\nAnalyses were done at the three WAIS-III levels: VIQ versus PIQ, the four factor scales and all subtests. Preliminary analysis included checks for normality, linearity, influential data points and assumptions of repeated measures. No serious deviations were found. T-tests showed that both diagnosis groups were comparable in education and work status, as well as in gender distribution.\nDifferences Between WAIS III VIQ and PIQ\nDifferences between VIQ and PIQ for all participants and both diagnostic groups were analyzed by means of paired t-tests. No statistically significant effects were found for any of the investigated groups (see Table\u00a02).\nTable\u00a02VIQ and PIQ differences in the total group and in diagnostic groupsVIQPIQMean difference nMSDMSDTotal group110.3013.83108.4218.211.8843Asperger111.41 13.57112.5217.281.1127Autistic disorder108.4414.49101.50 18.136.9416\nDifferences Between Factor Scale Scores\nFactor Scale profiles were studied within the total group and between the two diagnostic subgroups by means of repeated measures analysis of variance. Mauchly\u2019s test indicated that the assumption of sphericity was not met. Therefore the degrees of freedom were corrected using the Huynh\u2013Feldt correction (\u03b5\u00a0=\u00a0.89). Post-hoc comparisons using the Sidak adjustment for multiple comparisons showed that the main effect of the WAIS III Factor Scale was statistically significant (F(2.7,109.7)\u00a0=\u00a07.0, p\u00a0<\u00a00.001). An interaction effect of differences in Factor Scale mean by diagnostic group was also found (F(2.7, 109.7)\u00a0=\u00a02.7, p\u00a0=\u00a00.05). To find out which differences in WAIS III Factor Scale means added to the significant main effect, post hoc pairwise comparisons were done. This showed that the main effect in the total group can be attributed to Processing Speed being significantly lower than Verbal Comprehension (p\u00a0<\u00a0.01) and Perceptual Organization (p\u00a0<\u00a0.005).\nPost hoc pairwise comparisons were done for the two diagnostic groups to analyse the \u2018within group\u2019 effect. In the Asperger group, no significant differences in Factor Scale mean scores were found. The HFA group however, showed a significant lower Processing Speed compared to Verbal Comprehension (p\u00a0<\u00a0.01), Perceptual Organization (p\u00a0<\u00a0.01) and Freedom from Distractibility (p\u00a0<\u00a0.05) (see Table\u00a03).\nTable\u00a03Factor scale scores for the total group and the diagnostic groupsFactor scaleMSDNVerbal comprehensionAutistic disorder107.5*12.116Asperger syndrome110.811.927Total109.6*12.043Perceptual organizationAutistic disorder105.0*18.716Asperger syndrome111.813.027Total109.3*15.543Freedom from distractibilityAutistic disorder105.1*18.216Asperger syndrome107.215.427Total106.416.343Processing speedAutistic disorder91.8*17.416Asperger syndrome106.519.427Total101.0*19.843*p\u00a0< .05.\nDifferences Between WAIS III Subtest Scores\nThe Subtest profiles were explored within the total group and between the two diagnostic subgroups by means of a repeated measures analysis of variance. The assumption of sphericity was not met. Therefore the degrees of freedom were corrected using the Huynh\u2013Feldt correction (\u03b5\u00a0=\u00a0.82). Post-hoc comparisons were performed using the Sidak adjustment for multiple comparisons. The results (see Table\u00a04) showed a significant main effect of the type of Subtest (F(10.7,438.7)\u00a0=\u00a04.8, p\u00a0<\u00a00.001).\nTable\u00a04Mean standardized subtest scores for the total groupSubtest scores MSDnVocabulary11.63*2.56443Similarities11.422.49043Arithmetic11.77*3.04643Digit span10.723.26843Information12.42*2.77943Comprehension12.53*2.77243Letter-number sequencing10.982.95643Picture completion10.883.25343Digit-Symbol Coding9.81*3.43843Block design12.02*3.56243Matrix reasoning11.98*2.45443Picture arrangement11.533.73143Symbol search10.37*3.97043Object assembly11.163.08643*p\u00a0< .05.\nAn interaction effect of Subtest by diagnosis was also found (F(10.7, 438.7)\u00a0=\u00a02.1, p\u00a0<\u00a00.05), indicating that the patterning of the WAIS III subtest mean scores for the two diagnostic groups differs. Table\u00a05 and 6 show the mean Subtest scores and standard deviations for the HFA group and the Asperger syndrome group.\nTable\u00a05Mean standardized subtest scores for the autistic disorder groupSubtest Scores MSDNVocabulary11.312.49616Similarities10.941.76916Arithmetic11.443.70516Digit span11.313.40016Information12.13*3.28416Comprehension11.752.17616Letter-number sequencing10.253.15216Picture completion10.814.07016Digit-Symbol Coding8.38*3.03016Block design10.563.44416Matrix reasoning11.44*2.82816Picture arrangement10.193.67416Symbol search8.44*3.48316Object assembly9.883.32416*p\u00a0< .05.Table\u00a06Mean standardized subtest scores for the Asperger syndrome groupSubtest scoresMSDnVocabulary11.812.63227Similarities11.702.82627Arithmetic11.962.63827Digit Span10.37*3.20027Information12.592.48527Comprehension13.00*3.01327Letter-number sequencing11.412.80527Picture completion10.932.74527Digit-Symbol Coding10.673.43127Block design12.89*3.40127Matrix reasoning12.302.19827Picture arrangement12.333.59527Symbol search11.523.84727Object assembly11.932.71627*p\u00a0< .05.\nPost hoc pair wise comparisons showed that the main effect in the total group can be attributed to the fact that Digit-Symbol Coding was significantly lower than Vocabulary (p\u00a0<\u00a0.05), Arithmetic (p\u00a0<\u00a0.05), Information (p\u00a0<\u00a0.005), Comprehension (p\u00a0<\u00a0.005), Block Design (p\u00a0<\u00a0.05) and Matrix Reasoning (p\u00a0<\u00a0.005). Furthermore, Symbol Search was lower than Information (p\u00a0<\u00a0.05) and Comprehension (p\u00a0<\u00a0.05).\nPost hoc pair-wise comparisons were also performed for the two diagnostic groups to analyze the \u2018within group\u2019 effect. The two groups showed significant differences in Subtest scores. In the Asperger syndrome group, Digit Span was lower than Comprehension (p\u00a0=\u00a0.005) and Block Design (p\u00a0<\u00a0.05).\nIn the HFA group performance was significantly higher in Information compared to Digit-symbol Coding (p\u00a0<\u00a0.05) and Symbol Search (p\u00a0<\u00a0.05). Furthermore, Digit-Symbol Coding was lower than Matrix Reasoning (p\u00a0<\u00a0.05).\nDiscussion\nWAIS VIQ Versus PIQ\nNo significant differences were found between VIQ and PIQ in the total group nor in the two diagnostic subgroups. The results are in line with factor analytic studies showing that the VIQ-PIQ dichotomy is not valid for general populations (Arnau and Thompson 2000; Taub 2001).\nWAIS III Factor Scale Level\nThe Asperger syndrome group was characterized by a flat Factor Scale profile in the Asperger syndrome group, while the HFA group performed significant low in Processing Speed. A low Processing Speed indicates problems in speed of processing visual information (Wechsler 1997). Adults with HFA apparently need more time than other people to process and integrate visual information and to act on this information.\nThe Processing Speed performance of the HFA group might be influenced by problems with top-down processing and ignoring irrelevant details, which are characteristic of people with HFA (Happ\u00e9 2005; Shah and Frith 1993). In order to maintain an overview of what they are doing, they work slowly.\nWAIS III Subtest Level\nAnalyses showed different Subtest patterns in the HFA and the Asperger syndrome groups. The HFA group performed significantly low in Digit-Symbol Coding and Symbol Search. These two subtests together form the Processing Speed Factor. The low scores for these subtests represent the problems in speed of processing visual information as described in the preceding paragraph.\nThe HFA group showed significantly high performance in Information and Matrix Reasoning. High scores for Information are in line with the fact that people with autism usually acquire much factual knowledge (Happ\u00e9 1999).\nMatrix Reasoning taps nonverbal perceptual reasoning. Matrix Reasoning is the only Perceptual Organization subtest without a time limit and is possibly not influenced by low Processing Speed performance scores. The good performance of the HFA group can probably be attributed to their visual-spatial strengths (Lincoln et\u00a0al. 1995; Tsatsanis 2005) and to the absence of a time limit for this subtest.\nIn the Asperger group, scores for Digit Span were relatively low. Digit Span taps working memory capabilities (Wechsler 1997), which can been defined as \u2018the ability to hold in mind past states of the environment and past actions while currently performing an action\u2019 (Russell 1997). People with autism or Asperger syndrome tend to store information in details instead of using strategies, which often leads to problems in retaining information (Happ\u00e9 2005; Minshew et\u00a0al. 1992; Tsatsanis 2005). Low Digit Span scores in the Asperger group may reflect problems in applying strategies to retain information.\nThe Asperger syndrome group performed significantly well on Comprehension. High scores on Comprehension in this group seem to contradict former research results (Klin et\u00a0al. 2005; Mayes and Calhoun 2003; Siegel et\u00a0al. 1996). However, people with Asperger syndrome often try to function in society by analyzing social situations at a cognitive level, which has been described as using an \u2018explicit theory of mind\u2019 (Frith and Happ\u00e9 1999). A extremely well developed explicit theory of mind may have caused the Asperger syndrome group to have such high scores on Comprehension.\nThe Asperger Syndrome group also performed significantly well on Block Design. Strengths in Block Design have often been reported in studies of people with HFA or Asperger syndrome (Happ\u00e9 2005; Shah and Frith 1993). This has been attributed to strengths in processing unconnected stimuli outside a meaningful context, which go together with the central coherence problems seen in people with autistic impairment (Shah and Frith 1993).\nConclusions\nThe present study found people with Asperger syndrome to differ significantly from people with HFA in WAIS III Factor Scale profiles and WAIS III Subtest patterning. In people with HFA Processing Speed problems were found. Further, the HFA and Asperger syndrome group showed different subtest patterns. The present study supports the idea that HFA and Asperger syndrome can be differentiated empirically at the level of intellectual functioning. This lends support to the hypothesis that HFA and the Asperger syndrome are two separate disorders.","keyphrases":["wais-iii","asperger syndrome","high functioning autism","intelligence","processing speed"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_Arch_Otorhinolaryngol-3-1-2099165","title":"Severe delayed posttonsillectomy haemorrhage due to a pseudoaneurysm of the lingual artery\n","text":"A 3.5-year-old child is presented with severe, recurrent haemorrhages after a tonsillectomy. The haemorrhages were caused by a pseudoaneurysm of the lingual artery, which was visualised more than 2 weeks postoperatively by angiography. The pseudoaneurysm was successfully embolised by coils. Coiling is an adequate therapeutic option for severe posttonsillectomy haemorrhage due to arterial damage.\nIntroduction\nPostoperative haemorrhage is the most common serious complication of adenotonsillectomy and its incidence is around 3% [1]. Intraoperative bleeding (<24\u00a0h) may be related to the surgical technique or a bleeding diathesis. Delayed postoperative haemorrhage (>24\u00a0h) predominantly occurs on the fifth to seventh-day postoperatively when the slough separates from the granulating fossa. The most critical posttonsillectomy haemorrhages are due to arterial dissections and aneurysms. In this report we present a boy with a severe, delayed posttonsillectomy haemorrhage due to a pseudoaneurysm of the lingual artery.\nCase report\nAn otherwise healthy 3.5-year-old boy was brought in with severe oral bleeding. Nine days prior to the presentation he had undergone an adenotonsillectomy for chronic adenotonsillitis in another hospital. The tonsils had been removed with cold dissection and sutures had been placed in both tonsil areas. The procedure and the immediate postoperative period went uncomplicated. On the fifth postoperative day he had been admitted for a one-night observation because of haemorrhage from the mouth. During the admission no active bleeding was observed and his haemoglobin level of 4.9\u00a0mmol\/l (normal values: 6.5\u20138.4\u00a0mmol\/l) was treated conservatively.\nThe second postoperative bleeding started 9\u00a0days postoperatively, late at night when the patient was lying in bed. There were no provoking events. The bleeding lasted around 10\u00a0min but had already stopped at arrival in our emergency room. We saw a pale and restless boy. Intra-oral examination in general anaesthesia showed lacerated pharyngeal muscles in the left tonsillar fossa and a loose suture was removed. The right tonsillar fossa revealed diffuse bleeding from the pharyngeal muscles without pulsating masses. The nasopharynx and tongue base showed no abnormalities. The haemoglobin was increased from 2.9 to 8.2\u00a0mmol\/l by transfusion with two units of packed red blood cells. The coagulation values were within the normal range. The postoperative period was uncomplicated, so he was discharged after 5\u00a0days, 15\u00a0days after the primary intervention, with dietary advices.\nOne day later the boy arrived by ambulance with heavy bleeding from the mouth. He was pale, shivering and drowsy. Again, the severe bleeding started without a provoking moment and persisted for about 15\u00a0min. Immediately, after arrival at our emergency department, he was re-explored intra-orally under general anaesthetics. We saw a bluish, pulsating mass of 5 by 12\u00a0mm low in the right tonsillar fossa without active bleeding. The faucial pillars on the right side were approximated without tearing the mucosa or induration of the aneurysm. However, during extubation a massive bleeding occurred. The boy was re-intubated, and with digital oral pressure during 15\u00a0min haemostasis was achieved.\nThe patient was stabilised and an arteriography was performed. A pseudoaneurysm of the right lingual artery was visualized with selective catheterisation using a vertebral catheter (Fig.\u00a01). The pseudoaneurysm was coiled distally and proximally using a microcatheter (Excelsior 10, Boston scientific, Natick, MA, USA) and microcoils (GDC 10, Boston Scientific, Natick, MA, USA). The close relation of the proximal part of the pseudoaneurysm to the entrance of the facial artery made coiling of the facial artery inevitable. The pseudoaneurysm was coiled successfully and no bleeding focus was seen on the control arteriography (Figs.\u00a02, 3). During the procedures blood transfusion was needed to restore the low haemoglobin level.\nFig.\u00a01Pseudoaneurysm of the right lingual artery seen from right-anterior side (3D-surface rendering)Fig.\u00a02Arteriography of pseudoaneursym of the right lingual artery seen from right-anterior sideFig.\u00a03After coiling of the pseudoaneurysm, including the facial artery; compare to Fig.\u00a02\nThe boy was extubated without any bleeding and 4\u00a0days after the embolisation he was discharged in a good clinical condition. At a follow-up examination, he did not report any intra-oral or neurological abnormalities.\nDiscussion\nTrue aneurysms develop through congenital or acquired weakness of all three layers of the vascular wall. False or pseudoaneurysms may arise due to localised arterial wall laceration caused by blunt or penetrating trauma. The intima or adventitia layer of the vessel wall is dissected, which creates a periarterial haematoma. Pseudoaneurysms after tonsillectomy may be triggered by blunt or direct trauma during dissection or due to placing ligation sutures.\nHaemorrhages from posttonsillectomy pseudoaneurysms of the lingual, facial and internal carotid artery have been observed within 5\u00a0h postoperatively to the eighth postoperative day in a few cases [2, 3]. In those reports no intra-oral pulsating masses were observed. The pseudoaneurysms were shown with angiography and treated with embolisation. Mitchell et al. reported a case of fulminant bleeding during the tonsillectomy procedure from an aberrant lingual artery with a pseudoaneurysm, which was treated with embolisation [4]. A pulsating neck mass can also be the first symptom of a pseudoaneurysm, which has been seen in two cases without any bleeding [5, 6].\nIt is striking that the above-mentioned cases of posttonsillectomy pseudoaneurysms concerned children under the age of 10\u00a0years. No cases of posttonsillectomy haemorrhage due to pseudoaneurysms in adults have been reported. This suggestive higher incidence of pseudoaneurysms in children might result from the smaller anatomy and thinner pharyngeal muscles, and a subsequent higher risk of damaging the large vessels.\nSince the angiography of our patient did not show any aberrant courses of arteries, trauma to the artery during dissection or placing the suture in the tonsillar fossa could have caused the pseudoaneurysm in our patient. In the two first oral inspections no masses were observed, only after the third bleeding (16th day postoperatively) a pulsating mass was seen. This suggests a gradual development of the aneurysm. False aneurysms are known to expand when the periarterial clot dissolves and more blood flows into the periarterial space [5]. The size of the lesion to the vessel wall might determine the amount of blood and whether the pseudoaneurysm is large enough to be observed. Maybe the vessel lesion in our patient was small, which made the pseudoaneurysm grow slowly. Although no real provoking events seem to have initiated the bleedings, lying down in bed might have raised the blood pressure in the carotid artery, which made the aneurysm leak. A new clot made the severe short lasting bleeding stop.\nSevere posttonsillectomy haemorrhage is treated with external ligation of the internal or external carotid artery or endovascular coiling of the injured artery. Although a randomized controlled trial has not been performed, embolisation seems to have advantages over external ligation [7]. Firstly, the diagnostic evaluation can be combined with direct therapeutic intervention. Furthermore, coiling is more selective. This is especially convenient in posttonsillectomy haemorrhages, where several branches of the external or internal carotid artery could be damaged. The third advantage is that embolisation is less mutilating and has less risk of damaging the vagal and accessory nerves. The potential complications of embolisation are perforation of the vessel with leakage of coils, ischemia of mucosa or nerves, or vasospasm. External ligation seems to be the only quick option in massive bleeding and unstable patients.\nConclusion\nPseudoaneurysms are life-threatening and should be considered in severe posttonsillectomy haemorrhage. Pseudoaneurysms do not always present as intra-oral or cervical pulsating masses, and can develop gradually. If the technique and technical crew are available, interventional arteriography is strongly recommended in severe posttonsillectomy haemorrhage. It is diagnostic and therapeutic at the same time and more selective than surgical ligation.","keyphrases":["posttonsillectomy haemorrhage","pseudoaneurysm","lingual artery","embolisation"],"prmu":["P","P","P","P"]}
{"id":"J_Med_Internet_Res-5-4-1550579","title":"How do Consumers Search for and Appraise Information on Medicines on the Internet? A Qualitative Study Using Focus Groups\n","text":"Background Many consumers use the Internet to find information about their medicines. It is widely acknowledged that health information on the Internet is of variable quality and therefore the search and appraisal skills of consumers are important for selecting and assessing this information. The way consumers choose and evaluate information on medicines on the Internet is important because it has been shown that written information on medicines can influence consumer attitudes to and use of medicines.\nIntroduction\nConsumers frequently use the Internet as an information source and it has been reported that 80% of adult Internet-users have accessed it for general health information [1]. More specifically, 36% of Internet-using consumers have used the Internet as a source of information on medicines [1].\nIt is broadly acknowledged that health information on the Internet is of variable quality as evidenced by the large number of studies that have explored the quality of consumer health information on the Internet [2]. This is to be expected because the Internet is a free medium. It has also been widely postulated that consumers searching for health information are in danger of being harmed by poor-quality information even though there is little evidence of this [3]. A consumer's risk for encountering poor-quality health information is purportedly related to the proportion of poor-quality information on the Internet and the consumer's ability to filter out this information [2]. As the quality of information on the Internet cannot be controlled, the more-imperative issue is the ability of consumers to search through information and assess its quality so they are able to avoid untrustworthy information [4]. An Australian study suggested that consumers found it difficult to describe how they distinguished good-quality information on medicines from poor-quality information on medicines on the Internet [5]. However, this study was limited by a small (N = 9), select sample and did not explore in-depth the way consumers searched for and selected information on medicines.\nThere is little information concerning consumer Internet-search behavior for health information. One study reported that participants mainly select Web sites that looked and read professionally and preferred understandable Web sites from official sources that used scientific references [6]. When participants were observed while searching for health information on the Internet, it was found that they mainly used search engines and were described as having \"suboptimal\" search skills [6]. This study reported that participants did not find blatantly-incorrect health information in their searches [6]. This indicates that they had used selection criteria to decide on the Web sites, though the criteria were not fully described in this paper.\nConsumer use of information on medicines is an important issue because written information on medicines has been shown to influence consumer attitudes towards their medicines, and affect their medicine-taking behavior [7]. Furthermore, medicines, unlike general health issues, have overtly-commercial imperatives, which may influence the information available. Since the Internet has become a common source of information on medicines, it is important to identify the way consumers are using it. Therefore the aim of this study was to explore consumer use of Internet-based information on medicines. In particular, the objectives were to:\nexamine consumer attitudes to the availability and quality of Internet-based information on medicines;\nexplore consumer reasons for using this information;\nexplore consumer experiences in searching for and appraising information on medicines;\ninvestigate the self-reported impact and application of this information.\nThis paper will present results from the broader study on consumer experiences in searching for and appraising Internet-based information on medicines.\nMethods\nSelection of Method\nFocus groups were selected to address the study aims because they are useful for time-efficient, in-depth exploration of issues surrounding topics where there is little information [8- 10]. Since there is little known about how consumers use Internet-based information on medicines, focus groups were an ideal method for exploring this issue. The results of focus groups are not intended to be statistically generalizable, but are used to reveal the range of consumer opinions and attitudes.\nResearch Instrument\nAn interview guide consisting of general themes constructed from the literature was prepared (Table 1). This paper focuses on results ensuing from the exploration of themes 4, 5, and 6. The interview guide was composed of open-ended questions that addressed various issues pertaining to consumer use of Internet-based information on medicines; the questioning route was designed to stimulate discussion [11- 13].\nTable 1\nThemes for focus group interview guide*\nGeneral opinions about the Internet as a source of information on medicines.\nExperiences in using the Internet to seek information on medicines.\nReasons for seeking information on medicines.\nThe methods and process of searching for information on medicines.\nOpinions and critique of the information found.\nExperiences in the evaluation of the quality of Internet-based information on medicines.\nFeelings after reading the information.\nActions taken as a result of reading the information.\nPerceived benefits and drawbacks of the Internet as a source of information on medicines\n* This paper focuses on results ensuing from the exploration of themes 4, 5, and 6.\nThe interview guide and questioning route was pretested with a convenience sample of consumers (N = 13) to test for interpretation, appropriateness, and comprehensiveness, and to establish face and content validity. No significant changes were made to the interview guide or questioning route as a consequence of this pretest.\nParticipant Recruitment\nAfter approval was granted by the Human Research Ethics Committee of The University of Sydney, participants were enlisted for the focus groups by a recruitment agency. Participants were recruited from the agency's database of consumers across metropolitan Sydney, Australia via telephone using a screening questionnaire.\nConsumers were deemed to be eligible for this study if they had sought Internet-based information on medicines in the preceding 12 months. This bounded reference period was applied to allow for a suitable recall of past events [14] while also allowing enough time for consumers to have used the Internet for this purpose. Inclusion criteria required that participants were 18 years of age or over, did not require a translator to take part in focus group discussions, did not have training as a health professional, and did not have specialist Internet training. Participants were financially reimbursed for their time and travel expenses.\nStudy Design\nSix focus groups were conducted in a number of locations around metropolitan Sydney in March to May 2003.\nTo approximate a representative cross section of consumers, participants were recruited with the intention of including subjects from both genders and across different age groups. Focus groups were age stratified to achieve a level of homogeneity within each group. The use of stratification may increase congruency between participants, thereby allowing a more comfortable discussion [11,15]. Eight persons were recruited for each focus group to ensure that groups were large enough to motivate a discussion, yet small enough allow for all opinions to be heard [11]. The number of groups needed was not determined beforehand because data was collected until saturation occurred (the point where no new themes emerged) [10]. In this study, saturation occurred by the sixth focus group.\nThe focus groups were facilitated by a skilled moderator while 2 assistant moderators observed and took notes. The group discussions were 1 to 1.5 hours in duration and were digitally sound recorded after permission was obtained from all participants. The recordings were transcribed verbatim. Participants also completed a demographics questionnaire that collected data on Internet usage.\nData Analysis\nThe verbatim transcripts were entered into NVivo qualitative software [16] and thematically content analyzed using a grounded theory approach. The grounded theory approach is an inductive approach to analyzing qualitative data, where ideas and emerging themes are systematically coded to generate theory [17].\nResults\nThis paper presents participants' responses to themes 4, 5, and 6 (Table 1). Responses to other themes are currently unpublished.\nDemographics\nForty-six consumers participated in this study. The age of the participants ranged from 18 to 67 years, with a median of 41 years (interquartile range, 21 years) and a mean of 41.7 years (standard deviation, 12.7 years). Fifty-seven percent of the participants were female. The majority of the participants were employed full-time (58.7%) and about a fifth were either retired or full-time homemakers. Almost half the sample (47.8%) had occupations that could be classified as managers, professionals, or associate professionals [18]. A high proportion of the sample (65.2%) had completed further educational qualifications beyond high school, and 23.9% of the sample had a bachelors or postgraduate degree.\nData on participant usage of the Internet is presented in Table 2. The majority of participants had a few years experience in using the Internet and over half had accessed it from both their home and workplace. In addition to using the Internet for information on medicines, most participants also used it for general health information and for services such as e-mail.\nData on participant usage of the Internet for information on medicines is presented in Table 3. In addition to using the Internet, many participants also reported using other media such as magazines for information on medicines. This variety of information sources has also been seen in another Australian study on consumer use of Internet-based general health information [19]. Even though most participants (82.6%) were seeking information for themselves, many reported also searching for other family members. This was also reflected in the aforementioned Australian study that showed that 63% of Internet-using consumers sought health information mainly for themselves [19].\nTable 3\nParticipant usage of the Internet for information on medicines (N = 46 participants)\nCharacteristic\nUsage\nFrequency, Number of Participants\nRelative Frequency, (% of Participants)\nMedia sources of information on medicines (more than one category could be selected)\nInternet Magazines Television Books Radio\n46 31 23 22 9\n100.0 67.4 50.0 47.8 19.6\nPerson that Internet medicine information was used for (more than one category could be selected)\nSelf Spouse\/partner Child Parent Another relative Friend\n38 24 19 17 13 6\n82.6 52.2 41.3 37.0 28.3 13.0\nHealth categories for which information on medicines had been sought for (more than one category could be selected)\nAllergies Arthritis\/joint pain Asthma Cancer Skin disorders Hormones Other miscellaneous Child health Diabetes High cholesterol Immunization Pain and injury High blood pressure Mental health Digestion\/stomach disorders Infections Migraine Osteoporosis Alzheimer's disease Dementia\n20 14 14 13 12 11 11 10 10 9 9 9 8 8 6 6 6 6 5 5\n43.5 30.4 30.4 28.3 26.1 23.9 23.9 21.7 21.7 19.6 19.6 19.6 17.4 17.4 13.0 13.0 13.0 13.0 10.9 10.9\nTable 2\nParticipant usage of the Internet (N = 46 participants)\nCharacteristic\nUsage\nFrequency, Number of Participants\nRelative Frequency, % of Participants\nLength of experience in the use of the Internet\nMore than 5 years 4 to less than 5 years 3 to less than 4 years 2 to less than 3 years 1 to less than 2 years Less than 1 year\n17 13 7 4 4 1\n37.0 28.3 15.2 8.7 8.7 2.2\nLocation of Internet access\nHome and work Home only Work only\n25 16 5 \n54.3 34.8 10.9\nActivities that the Internet is used for (more than one category could be selected)\nInformation on medicines E-mail Health information Travel information\/booking Banking\/financial services News, weather, sport Job or study related research Real estate Shopping\u2014product research Games and hobbies Chat or instant messaging Shopping\u2014purchasing Purchasing medicines \n46 45 43 40 36 34 34 33 33 26 22 19 7\n100.0 97.8 93.5 87.0 78.3 73.9 73.9 71.7 71.7 56.5 47.8 41.3 15.2\nSearching for Internet-Based Information on Medicines\nSearch Engines\nAll participants had used a search engine to find information on medicines. Most participants had a single favorite search engine that they would always use, but a few reported using more than one search engine to find the information they required.\nThe choice of search engines was determined by many different factors ranging from the default search engine on their browser to active selection based on self-developed criteria. Numerous participants were influenced by the search engine that was used by coworkers, for example:\nGroup 4, Participant 8\nI saw it on this guy's computer and . . . I thought 'Oh, I'm going to use this'. That's how I started it at work.\nSome participants also reported that their browser automatically defaulted to a certain search engine and a few participants were unable to identify the search engine they used, for example:\nGroup 3, Participant 6\nCouldn't tell you [the search engine] really. I just log on and use whatever comes on.\nMany participants used search engines recommended by family and friends.\nThere were certain determinants that led some participants to actively choose a specific search engine. These included perceptions of the credibility of the search engine, ease of use, relation with services such as e-mail, and a lack of advertising. These determinants did not necessarily include perceived quality of the information on medicines obtained through their use.\nA few participants reported using AltaVista [20] because they thought it had an educational advantage, for example:\nGroup 1, Participant 1\nIt's got an educational edge, that's my experience. When I was at university doing my second degree, that was one that was sort of promoted as credible I suppose.\nSome participants preferred to use Ask Jeeves [21] because they could enter the searches in a question or statement format rather than using search terms.\nMany participants reported using Yahoo! [22] because it appeared as a default homepage, was used as a personal e-mail account, or was advertised through other media. Yahoo! and Google [23] were also said to be useful for Australian-only searches.\nGoogle was undoubtedly the search engine the majority of participants used most and preferred. This was especially true of the younger participants. The common perception was that Google appeared to be straightforward and did not focus on advertising, for example:\nGroup 4, Participant 6\nIt's just got less [rubbish]. It seems to be direct to what you want. I think that other [search engines] always have these categories and they always have suggestions for buying things and stuff like that but Google's pretty much straight to the point. It's simple.\nParticipants also commented that this search engine was useful for suggesting spelling corrections when errors were made, as medicine names were sometimes difficult to spell. A few participants reported preferring Google as their search engine of choice specifically for health-related searches but were unable to explain reasons for their preference.\nOther search engines used by participants were metasearch engine Dogpile [24], Australian metasearch engine Search66 [25], Australian-based search engine Web Wombat [26], and ninemsn [27], the Australian-based access to search engine MSN Search [28]. Many participants who used metasearch engines were unaware of the difference between these and normal search engines.\nGenerally, although a variety of search engines were used by participants when seeking information on medicines, the majority of participants used the same few dominant search engines. Participants generally preferred search engines with less advertising, and would continue to use the same search engine if they were successful in their searches. Most participants used the same search engine that they used for nonhealth information, and were usually influenced by what was used by friends, family, and colleagues.\nSearch Processes\nParticipants displayed a large variation in the process of searching for information on medicines.\nMost participants found information by typing the name of the medicine (drug name or brand name) into the search engine. A few participants felt this was the only way of finding information on a medicine, for example:\nInterviewer\nHow do you put in your searches?\nGroup 6, Participant 2\nMedicines are really specific to just the name.\nOther participants reported looking for broader information, for example:\nGroup 1, Participant 2\nI often use a more general [search]. I might use something like 'women's health' or something. And I like to see a whole range of things . . . rather than targeting specifically . . . and then I choose within that.\nThe information found through this type of search was said to be less specific to one medicine and had more general or comparative information.\nSome participants used more-advanced search techniques such as quotation marks, phrases, and extra words to narrow down their searches. They displayed an understanding of how these techniques helped to focus their searches, for example:\nGroup 4, Participant 6\nIf you type it in with quotation marks, it'll search for those words together whereas if you type them separately, it'll just search for them anywhere.\nParticipants reporting advanced skills were generally observed to be those who were younger or those who had greater experience of the Internet through work or study.\nHowever, it was clear that search skills varied significantly. The following interchange illustrates the mixed levels of understanding as to how search engines work:\nGroup 5, Participant 4\n[You need to] ask a specific question . . . 'What are the side effects?' rather than typing in 'penicillin'.\nParticipant 6\nYeah, you really have to do a whole sentence. A whole statement.\nParticipant 3\nI would type in 'penicillin side effects'.\nParticipant 4\n'Then it could hit on 'penicillin' or it could hit on 'side effects'.\nThe uninformed way in which some participants agreed upon what they considered to be optimal search skills was obvious in the group discussions. The majority of participants in this study who reported searching using less-than-optimal techniques\u2014such as typing in whole questions\u2014tended to be nonworkers, for example, full-time homemakers or retirees.\nThe search skills of participants varied widely and these differences may affect the resulting information that participants encounter. Searching via a search engine however, was not the only way of finding information on the Internet on medicines.\nOther Methods of Finding Internet-Based Information on Medicines\nSome participants mentioned ways of finding information on medicines in addition to using search engines.\nA few participants said that they guessed the Web sites of medicines by typing the name of the medicine in the address bar in the format of www.[brand name or drug name].com.\nSeveral participants found information on medicines from Web sites recommended by family and friends, and from seeing advertisements in seniors' and health publications. Some reported bookmarking favorite Web sites for future reference and a few subscribed to mailing lists at health-related Web sites.\nOne participant described searching for information on medicines using online journals. Although aware that the information was not aimed at consumers, this participant still chose to use this means to search for pertinent information on medicines:\nGroup 2, Participant 2\nI actually searched via . . . the professional journals . . . And I guess that was a little bit harder to do it that way because . . . reading through the journals was quite difficult. I tend to just go to the abstracts.\nParticipants reported using a variety of search skills to obtain information on medicines. However, the important issue was how they selected and appraised the information.\nAppraising Internet-Based Information on Medicines\nSelecting Internet-based Information on Medicines\nParticipants described different ways of choosing which Web site to visit when selecting from the numerous results obtained from using a search engine. Some worked down the list of results from the first one while others looked for keywords in the Web site descriptions or for the Web site's recency. Often participants made a judgment based on the URL (Web page address) of the result, for example:\nGroup 4, Participant 1\nI actually like looking at the actual web address, just seeing how professional it is. Like if it's some silly thing, I won't bother going into it.\nMany participants also reported looking for indicators in the Web site address to determine whether it belonged to a government, a university, an official organization, or a pharmaceutical company.\nEven though most participants said they would not go beyond the first page of the search results, one expressed the opinion that the best information was in the middle of the results and not on the first few pages. This participant had the erroneous opinion that the first pages of results are older and that results appeared mainly in the order in which the information had been created.\nMany participants reported looking for the country of origin of the information and preferred information generated from their country of residence, for example:\nGroup 4, Participant 2\nIf I'm searching for a medication . . . and it brought up some things and I noticed it was in Australia, I click on that.\nThese participants felt that Australian information would be more applicable to them and professed an awareness of health-setting issues such as differences in the brand names and availability of medicines in different countries. However, others had more confidence in United States-based information because they believed that this was where most new research was undertaken.\nIt was clear that most participants did not pay conscious attention to how they selected Internet-based information on medicines, with one referring to the process as \"a vibe\" that you obtain through experience. Another described this as a feeling that \"things have a look of credibility.\" Similarly, many participants had trouble in articulating their selection process, for example:\nGroup 5, Participant 3\nI find that sometimes I get to a site and I think 'Gee, this is a good site, but I don't know how I got there.' You know what I mean? You fluke it.\nDespite the inability of many participants to express how they selected information on medicines, many were able to express what they would not select. Participants reported quickly rejecting sites that were slow to load, sites that contained too many graphics, and sites that had pop-up advertisements.\nThe process of selecting information on medicines varied among the participants. It appeared that all participants had their own criteria for selecting and rejecting information which may or may not appear logical to others. Credibility of the source, however, appeared to be a common determinant in the criteria of all participants.\nCredibility of the Source of Internet-Based Information on Medicines\nParticipants expressed conflicting opinions about the credibility of the source of Internet-based information on medicines. Many participants regarded information produced by pharmaceutical companies to be the \"official\" information on a medicine and therefore trusted this the most, while many others were suspicious of a possible information bias, for example:\nGroup 1, Participant 7\nIf you're looking at [a pharmaceutical company website], they've got factories throughout the world, they're a pretty good company so . . . you know that they've done so much research it's credible information.\nGroup 1, Participant 6\nIf it's a pharmaceutical company, they're gonna put a good stance on their drug.\nMany other participants preferred information that originated from what they considered to be impartial and reputable sources such as government, professional, or disease-focused organizations, or university Web sites. A few participants also reported looking for credentials such as the author's qualifications when assessing the credibility of the information provider.\nA small number of participants preferred information written by other consumers who had personal experiences in taking the medicine. However, most participants expressed that they would be less likely to trust information on medicines generated by other consumers, for example:\nGroup 1, Participant 6\nThere are chat rooms . . . if you've ever been prescribed such and such a medication; you'll get people from all around the world . . .\nParticipant 2\nDo you not find that a bit dangerous because everything is rather specific to each person's body?\nParticipant 6\nOh yeah, but it would be comparable to having a chat with some of your friends.\nSome participants felt that the authorship of Internet-based information on medicines should be regulated and feared the reliability of the information because there was \"no watchdog\" for the information published on the Internet while others regarded it as analogous to the way they would trust information given in common conversation and therefore felt comfortable using information in this context.\nThe credibility of the source of information on medicines was a strong determinant in the selection process. However, in addition to the source participants evaluated information using criteria described in the next section.\nEvaluating Internet-Based Information on Medicines\nParticipants evaluated information on medicines using criteria such as the motive for the information, the language used, and the applicability to their needs.\nAlmost all participants were skeptical to some degree of Internet-based information on medicines. Many participants professed a universal need for consumers to inherently distrust this information, and to interpret it accordingly. One participant stated that it is important to also consider why the information is on the Internet:\nGroup 1, Participant 6\nWhat are the motives? Are they conflicting, credible? Whoever has posted it, are they trying to make a profit?\nOther participants described the obviously difficult-to-believe nature of some of this information and looked for signs of conspiratorial or misleading language when deciding whether to trust the information, for example:\nGroup 2, Participant 8\nIf it says 'hazard free' and 'completely no side effects', for example, I'm more likely to disbelieve than believe that\nIn addition to this awareness of unreliable information on medicines, many participants also expressed an understanding that the information they find may not necessarily be applicable to them and that the information should not be used at face value, for example:\nGroup 2, Participant 7\nThe thing with medicines is there's no sort of right or wrong . . . Everyone's different, everyone's going to have a different reaction.\nGroup 2, Participant 6\nWhen you ask the doctor, they tell you 'well, [the side effects] happen but it's not like that', I think what happens is that the information is not tailored for myself. It's general information.\nPertinent to this appraisal was the information-filtering process described by participants:\nGroup 2, Participant 4\nIt's always better to try and take as much information and try and sift out what's useless\nGroup 1, Participant 6\nWhen they're talking about people using this medicine, 'ninety-eight percent will die within five years' . . . you have to take that and filter it through a whole bunch of other variables . . . and whether [the information] is not terribly well informed or completely informed.\nOne common way in which some participants were able to filter information on medicines was to use other Web sites for comparison and cross-checking, for example:\nGroup 4, Participant 1\nI always go to two or three sites.\nAlthough participants reported methods of evaluating information, many expressed a difficulty in their evaluation, for example:\nGroup 2, Participant 4\nHow do you [figure] out what's useful?\nGroup 3, Participant 7\nHow do you know what's reliable and what's not?\nUltimately, despite an awareness of the shortcomings and difficulties in evaluating the quality of information on medicines, all participants saw the Internet as an important resource for this information, for example:\nGroup 1, Participant 1\nI think as patients you expect immediate information and the Internet, whether it's credible or not, it's the fact that people can get it.\nDiscussion\nThe issue of consumer use of Internet-based information on medicines is important because it has been shown that written information on medicines can be interpreted by consumers in ways that may lead to anxiety or apprehension [7,29- 32], and a refusal of prescribed medicines [33]. Conversely, it has been shown that written medicine information increases consumer knowledge about their medicines [29,34- 36] and that well-informed consumers with an increased understanding of the purpose of their medicines may have improved compliance and satisfaction with their therapy [29,31,37- 40].\nHowever, studies on consumer use of written information on medicines have evaluated standardized information on medicines such as that produced by pharmaceutical companies, government or professional bodies, or health care practitioners [7]. In contrast, this study explored Internet-based information, which is neither standardized nor subject to universal quality control. Furthermore, medicines in particular are subject to commercial considerations that may have an impact on the motives for and quality of information. Therefore, the impact of Internet-based information on consumer use of medicines may differ from that reported from consumer use of standardized written information on medicines.\nThe reported search skills of these participants were comparable to those of participants observed while searching for general health information [6] in that they mainly searched using simple strategies in a search engine and chose results primarily from the first page of search results. Although this similarity is not surprising, it does illustrate the overlap between appraising general health information and specifically medicines-related information. Indeed, it was not always possible for consumers in this study to speak on issues surrounding searching for and appraising information on medicines without speaking about other health-related issues.\nParticipants in this study searched for information on medicines using a range of search techniques from simple 1-word searches and advanced techniques to suboptimal techniques. However, although some participants had little understanding of how search engines worked and possessed suboptimal search skills, a few participants described proficient search skills. Contrary to findings where consumers were observed to use information not applicable to their health setting [6], participants generally reported a strong awareness of the limitations of non-Australian information due to health-setting limitations pertinent to medicines use.\nParticipants were conscious that there was an abundance of poor-quality information on medicines on the Internet. They were also predominantly aware that information on the use of medicines and on the incidence of side effects is often based on individual factors that should not be seen as applicable to everyone. Therefore, while consumer evaluation skills have been referred to as \"meager\" [41], the assumption that consumers believe everything they read does not take into account those participants who are savvy about issues such as bias, commercialism, and the lack of regulation of Internet-based information on medicines.\nHowever, the fact that many participants searched for information on a medicine by typing the brand name into a search engine would indicate that it was highly likely that they encountered the Web site of a pharmaceutical company on the first page of results [42], which raises the matter of consumer ability to interpret information on medicines that may not be comparative and unbiased in nature and not aimed at an Australian audience. Even though results from this study would indicate that many participants were aware of these limitations, others still viewed a pharmaceutical company Web site as the official, and therefore exclusive, information on a medicine; this indicates that some consumers may be unaware of or uninterested in information on medicines produced by alternate sources. Nevertheless, it has been suggested that consumers are more likely to search for alternate sources, rather than relying on product brands, as they become more experienced using the Internet [43].\nIt is clear that there was a variety of skills among participants. Many had not been conscious of some of the issues surrounding the process of searching for and appraising information on medicines and did not undertake this process in the most-constructive way. Furthermore, there have been few studies in the literature that have sought to educate consumers on strategies for effective use of the Internet for health information [44- 47].\nLimitations in This Study\nThere are several important limitations in this study.\nFirst, as this information is self-reported, consumers may not actually search for and appraise information in the same way as they describe. Such a discrepancy was demonstrated when participants in an observational study were reported to be less likely to look for the sources of the information than was apparent from claims in focus groups [6]. However, participants in that observational study were not searching for information that they would personally use; this may have meant that they were less concerned about the quality of the information.\nSecond, the bounded period of 12 months in the inclusion criteria may be too long for consumers to correctly remember details of how they searched for and chose information. It might have been beneficial to actually perform a search as an activity to stimulate the participants' memories.\nThird, participants in group situations may feel compelled to provide socially-desirable answers that are not necessarily accurate. In this study, we sought to minimize this by informing participants that their results would be confidential and that they were welcome to speak about anything they felt even if they disagreed with someone else. However, this does not negate the problem. Although the use of individual interviews may help to minimize this discrepancy, this method is more time-consuming and cannot use group interaction for the generation of ideas.\nLast, certain actions are intuitive and therefore difficult to articulate. Most participants were not able to adequately describe their search and appraisal processes, which suggests that this process may largely be a form of tacit or implied knowledge.\nTherefore, future research needs to take into account actual observed (rather than reported) search and appraisal skills of consumers who are seeking information on medicines for their own use.\nConclusion and Future Research\nThe results of this study show that consumers may benefit from greater awareness and education on the significance of good search and appraisal skills for information on medicines so that this process is deliberate and conducted with thought rather than being random and tacit. Furthermore, there is evidence that consumers may support education that shows them how to search for information on medicines on the Internet [48]. However, health promotion and education needs to take into account the variety of consumer skills in both searching for and critically evaluating information. Pharmacists are in an ideal position to provide consumer training as they frequently counsel consumers on medicines [49] and have consumers present them with information from the Internet [19]. However, to successfully deliver this program, pharmacists need to be trained in these skills . Furthermore, the impact of pharmacist education on consumers' searches for Internet-based information on medicines and appraisal of that information needs to be evaluated. Therefore, future research by this team will be on the development of a health-promotion program for pharmacists to train consumers to search for and appraise Internet-based information on medicines.","keyphrases":["consumers","information","medicines","internet","focus groups","drugs","qualitative research"],"prmu":["P","P","P","P","P","P","R"]}
{"id":"Immunogenetics-4-1-2206249","title":"Variation analysis and gene annotation of eight MHC haplotypes: The MHC Haplotype Project\n","text":"The human major histocompatibility complex (MHC) is contained within about 4 Mb on the short arm of chromosome 6 and is recognised as the most variable region in the human genome. The primary aim of the MHC Haplotype Project was to provide a comprehensively annotated reference sequence of a single, human leukocyte antigen-homozygous MHC haplotype and to use it as a basis against which variations could be assessed from seven other similarly homozygous cell lines, representative of the most common MHC haplotypes in the European population. Comparison of the haplotype sequences, including four haplotypes not previously analysed, resulted in the identification of >44,000 variations, both substitutions and indels (insertions and deletions), which have been submitted to the dbSNP database. The gene annotation uncovered haplotype-specific differences and confirmed the presence of more than 300 loci, including over 160 protein-coding genes. Combined analysis of the variation and annotation datasets revealed 122 gene loci with coding substitutions of which 97 were non-synonymous. The haplotype (A3-B7-DR15; PGF cell line) designated as the new MHC reference sequence, has been incorporated into the human genome assembly (NCBI35 and subsequent builds), and constitutes the largest single-haplotype sequence of the human genome to date. The extensive variation and annotation data derived from the analysis of seven further haplotypes have been made publicly available and provide a framework and resource for future association studies of all MHC-associated diseases and transplant medicine.\nIntroduction\nThe MHC has long been believed to be the most important region in the human genome with respect to infection, inflammation, autoimmunity and transplant medicine (Lechler and Warrens 2000). This was recently confirmed by the largest genome-wide association study carried out to date for seven common diseases, including two autoimmune diseases (type 1 diabetes and rheumatoid arthritis) and one inflammatory disease (Crohn\u2019s disease). The highest associations were found between the MHC and these two autoimmune diseases (The Wellcome Trust Case Control Consortium 2007). The complex aetiology of MHC-associated disease coupled with high density, polymorphism, linkage disequilibrium (LD) and frequent non-Mendelian inheritance of gene loci have made it challenging to identify variations that cause or contribute to disease phenotypes. Additional limiting factors have been our incomplete knowledge of the allelic variation of genes and regions flanking the nine classical human leukocyte antigen (HLA) loci and the lack of a single haplotype reference sequence, the original reference sequence being a composite of multiple MHC haplotypes (Mungall et al. 2003; The MHC Sequencing Consortium 1999).\nRecognizing that the future identification of variants conferring susceptibility to common disease is critically dependent on fully informative polymorphism and haplotype maps, the MHC Haplotype Consortium formed in 2000 with the aim to generate these critical data and to make them publicly available as a general resource for MHC-linked disease studies. Similar efforts, but with different experimental approaches, were also carried out in Japan (Shiina et al. 2006) and the USA (Smith et al. 2006). To develop the resource, eight HLA-homozygous MHC haplotypes were selected on the basis of conferring either protection against or susceptibility to two autoimmune diseases, type 1 diabetes and multiple sclerosis, and that represented common haplotypes in European populations. In the subsequent years, incremental data, materials and tools comprising this resource have been released (Allcock et al. 2002; Horton et al. 2004; Stewart et al. 2004; Traherne et al. 2006) and have contributed towards the construction of a high-resolution LD map and a first generation of HLA tag single nucleotide polymorphisms (SNPs; de Bakker et al. 2006; Miretti et al. 2005) and the identification of a second MHC susceptibility locus for multiple sclerosis (The International Multiple Sclerosis Genetics Consortium; Yeo et al. 2007). In this paper, we report the final account of this international effort, including, analysis of the last four of the eight haplotypes, up-to-date variation statistics, gene annotation, population-specific aspects and a detailed description of the databases and tools for viewing and accessing the data in the context of existing genome annotation.\nMaterials and methods\nVariation analysis\nThe method previously reported for comparison of MHC haplotype sequences (Stewart et al. 2004; Traherne et al. 2006) was extended to cover all eight haplotypes. Briefly, the most suitable method proved to be a clone by clone comparison using the discrepancy-list option of the cross_match program (Green, unpublished; http:\/\/www.phrap.org\/), an implementation of the Smith\u2013Waterman sequence alignment algorithm (Smith and Waterman 1981), using the alignment of a haplotype clone sequence with the appropriate overlapping reference sequence from a PGF clone or clones. All variations were submitted to dbSNP using the submitter handle SI_MHC_SNP and user identifiers of the form [PGF BAC clone sequence version]_[position in PGF BAC clone sequence]_[variation change]. Thus, AL662890.3_6645_TC indicates a substitution in which the base T at base position 6645 in AL662890.3 (PGF BAC 308K3) was substituted by C in the other haplotype. In the case of indels, the \u2018variation change\u2019 consists of \u2018i\u2019 or \u2018d\u2019 (for insertion or deletion), followed by a numerical value for the length of the indel, in turn followed by the inserted or deleted sequence if this were of 12 or fewer bases. For longer indels, an X value is given, which refers to a look-up table (http:\/\/www.sanger.ac.uk\/HGP\/Chr6\/MHC\/Xfile). Thus, AL662890.3_7470_d8TACACACA indicates a deletion in AL662890.3 after base 7470 of the eight bases \u2018TACACACA\u2019. Further, AL662890.3_10559_i5ATATT indicates an insertion in AL662890.3 starting after base 10559 of the five bases \u2018ATATT\u2019. AL662890.3_7475_d14X1 indicates a 14-base deletion after base 7475 in AL662890.3 of a sequence coded as X1 which is \u2018ATACACACACACAC\u2019.\nMajor indel sequences, appearing as breaks in the cross_match discrepancy lists between two clones from difference haplotypes, were extracted and subjected to analysis by RepeatMasker to detect the presence of retrotransposible elements.\nGene annotation\nThe finished genomic sequence for each of the eight haplotypes was analysed using a modified Ensembl pipeline (Searle et al. 2004). CpG islands were predicted on unmasked sequence. Interspersed and tandem repeats were masked out by RepeatMasker (Smit, AFA, Hubley, R & Green, P. RepeatMasker Open-3.0. 1996\u20132004, http:\/\/www.repeatmasker.org) and Tandem Repeats Finder (TRF; Benson 1999), respectively. The sequence was then BLAST searched (BLAST, basic local alignment search tool; Altschul et al. 1990) using a vertebrate set of complementary DNAs (cDNAs) and expressed sequence tags (ESTs) from the European Molecular Biology Laboratory (EMBL) nucleotide database (Kulikova et al. 2007), followed by the re-alignment of significant hits. Non-redundant proteins were aligned similarly. Protein domain matches were provided through alignment of Pfam to the genomic sequence using Genewise (Birney et al. 2004), thereby providing protein domain data to the annotator. Ab initio gene predictions were performed by Genscan (Burge and Karlin 1997) and Fgenesh (Salamov and Solovyev 2000), and potential transcriptional start sites were predicted by Eponine (Down and Hubbard 2002). Analysis results were displayed, and annotation was performed through an in-house annotation software system.\nGenes were manually annotated according to the human and vertebrate analysis and annotation (HAVANA) guidelines (http:\/\/www.sanger.ac.uk\/HGP\/havana\/) using evidence based on comparison with external databases as of August 2005. All gene structures are supported by transcriptional evidence, either from cDNA, EST, or protein. In general, annotations are supported by best-in-genome evidence. Haplotype-specific evidence is assigned where possible. As with previous MHC annotation (Stewart et al. 2004; Traherne et al. 2006), some olfactory receptors have been built upon protein homology alone because of their restricted expression.\nLocus and variant types were annotated according to established standards (Harrow et al. 2006), with the modification that, within the MHC region, the artefact locus has been used to tag historically annotated structures that are no longer deemed valid.\nHLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQA1, and HLA-DQB1 allele types were assessed by comparison against the IMGT\/HLA database (http:\/\/www.ebi.ac.uk\/imgt\/hla\/; Marsh et al. 2005).\nAnnotation status of haplotypes\nThe PGF, COX, and QBL haplotypes have already been annotated in detail (Stewart et al. 2004; Traherne et al. 2006). It was decided, however, to re-annotate and update this annotation to maintain consistency between all eight haplotypes with the current supporting evidence and pipeline analyses. The SSTO haplotype was manually annotated de novo. The new annotation from the PGF haplotype was projected through a DNA\u2013DNA alignment to each of the remaining haplotypes (APD, DBB, MANN and MCF) where possible. This projection was checked thoroughly and non-alignable regions were manually adjusted (including the C4 and HLA-DRB1 hypervariable regions). Polyadenylation sites and signals were not annotated for haplotypes APD, DBB, MANN and MCF because of time constraints. In the main, however, these features may be assumed to correspond to the same positions as in the first four haplotypes.\nCombination of variation and annotation data\nBy employing a series of Perl scripts, the array of haplotype variation was combined with the annotation of gene loci, repeat elements and microsatellites, extracted from the Vertebrate Genome Annotation (VEGA) database in general feature format (GFF; http:\/\/www.sanger.ac.uk\/Software\/formats\/GFF\/), to determine the variation status of all loci.\nDistribution of sequenced HLA haplotypes in Europeans\nTo assess the distribution of sequenced haplotypes at the population level, 180 founder haplotypes were reconstructed using genotypic data from Centre d\u2019Etude Polymorphisme Humain (CEPH) trios (de Bakker et al. 2006). A ~214\u00a0kb segment spanning the HLA\u2013DRB1\u2013DQB1 genes was selected for the analyses. This segment, represented by 54 SNPs, is delimited by rs2187823 and rs2856691, with NCBI build 36 chromosome 6 coordinates 32547486 and 32761413, respectively. Phased haplotypes with known HLA\u2013DRB1\u2013DQB1 alleles were then used to construct a neighbor-joining tree (Kumar et al. 2001) and a phylogenetic network (Bandelt et al. 1999).\nResources\nAll sequences presented in this paper have been submitted to the EMBL\/GenBank\/DNA Data Bank of Japan (DDBJ) database and allocated accession numbers. For clarity, all bacterial artificial chromosome (BAC) clones are referred to using their accession numbers. The annotation of each haplotype has been entered in the VEGA database and is accessible through its browser (http:\/\/www.VEGA.sanger.ac.uk). All variations from the study were submitted to dbSNP (http:\/\/www.ncbi.nlm.nih.gov\/SNP) using the submitter handle SI_MHC_SNP.\nBAC clones from the CHORI-501 (PGF) and CHORI-502 (COX) libraries can be requested from BACPAC resources (http:\/\/www.bacpac.chori.org\/). Clones from the other libraries can be requested from john.elliott@ualberta.ca.\nThe web site for the MHC Haplotype Project provides links to various data resources (http:\/\/www.sanger.ac.uk\/HGP\/Chr6\/MHC\/).\nDAS sources for all substitutions and indels are available from http:\/\/www.das.ensembl.org\/das as follows:\nens_35_COX_SNP ens_35_COX_DIPens_35_QBL_SNP ens_35_QBL_DIPens_35_SSTO_SNP ens_35_SSTO_DIPens_35_APD_SNP ens_35_APD_DIPens_35_DBB_SNP ens_35_DBB_DIPens_35_MANN_SNP ens_35_MANN_DIPens_35_MCF_SNP ens_35_MCF_DIP\nThese can be accessed via the VEGA browser.\nResults and discussion\nVariation analysis\nOne of the main aims of the MHC Haplotype Project was to generate a comprehensive variation map of this most variable region of the human genome. To achieve this, eight haplotypes were sequenced and subjected to variation analysis. Table\u00a01 details the lengths of the sequence contigs, the number of sequence gaps and the allelic types of major HLA loci for each haplotype. Of the eight haplotypes sequenced, three have already been described: PGF and Cox (Stewart et al. 2004) both of which formed single contigs of approximately 4.7 Mb, and QBL (Traherne et al. 2006), of approximately 4.2 Mb but with five gaps. The remaining haplotypes sequenced all contained gaps, their coverage ranging from 2.33 Mb (DBB with 28 gaps) to 4.19 Mb (MANN with 10 gaps).\nTable\u00a01Haplotype sequence contig length, number of gaps and HLA allele typesHaplotypeLength (bp)GapsHLA-AHLA-BHLA-CHLA-DQA1HLA-DQB1HLA-DRB1PGF47548290A*03010101B*070201Cw*07020103DQA1*010201DQB1*0602DRB1*150101COX47318780A*01010101B*080101Cw*070101DQA1*050101DQB1*020101DRB1*030101QBL42492725A*260101B*180101Cw*050101DQA1*050101DQB1*020101DRB1*030101APD416096516A*01010101\u2013\u2013\u2013\u2013\u2212DBB233010128A*02010101\u2013Cw*06020101DQA1*0201DQB1*030302DRB1*070101MANN419101410A*290201B*440301Cw*160101DQA1*0201DQB1*0202DRB1*070101MCF408741315[A*020101]B*15010101Cw*030401DQA1*0303DQB1*030101\u2013SSTO370424922A*320101B*44020101Cw*050101DQA1*030101DQB1*030501DRB1*040301Sequence length (bp) and number of gaps in each haplotype sequence, together with the HLA gene types obtained by BLAST against the IMGT\/HLA database. Dashes or data in square brackets indicate the absence or the partial presence, respectively, of a gene owing to a sequence gap.\nFor the variation analysis, each of the above haplotypes was compared with the PGF reference sequence, resulting in the identification of 44,544 variations (37,451 substitutions and 7,093 indels, Table\u00a02), which have all been submitted to dbSNP. The success of this exercise is illustrated by the fact that examination of this public database (NCBI dbSNP build 127, March 2007) showed that there were only a further 19,598 variations, submitted by other laboratories, in this region which were not identified by this project. In accordance with the annotation that we also generated for each haplotype (see below), the variations shown in Table\u00a02 were further classified as untranslated region (UTR), exonic, intronic, intergenic and eight more sub-categories (Table\u00a03). Coding substitutions, which are of particular interest with respect to altered functionality, were further classified as synonymous, non-synonymous conservative, or non-synonymous non-conservative and grouped depending on whether they affected HLA or other genes (Table\u00a04). The actual variations and affected amino acids can be viewed using the VEGA browser as illustrated in Fig.\u00a01 and described in the corresponding section later on. In addition, we have analysed all haplotype sequences for inversions, which represent another important variation category that has been linked to genomic disorders (Shaw and Lupski 2004). Using Ssaha2 (Ning et al. 2001), we found no evidence of any inversion polymorphism within the generated sequences but could not exclude large-scale (e.g. involving entire MHC) inversions with breakpoints outside the MHC regions sequenced here.\nFig.\u00a01Annotation and variation data in VEGA. VEGA \u2018overview\u2019 (a), \u2018detailed view\u2019 (b) and \u2018basepair view\u2019 (c) example of the variation in the OR2J1 locus in which a STOP codon is present in all haplotypes except MCFTable\u00a02Distribution of substitutions and indels amongst haplotypesHaplotypeSubstitutionsIndelsALLCOX15,9672,39318,360QBL15,2822,36017,642SSTO14,9822,30017,282APD4,2306834,913DBB14,2551,97516,230MANN12,1021,65413,756MCF10,7901,54512,335Overall37,4517,09344,544Number of variations found by comparing the PGF haplotype sequence with each of the other haplotype sequences in turn.Table\u00a03Distribution of substitutions and indels within different sequence regions amongst haplotypesSequence regionBase pairsCOXQBLSSTOAPDDBBMANNMCFSIDSIDSIDSIDSIDSIDSIDCoding247,5053538503193802740351640193482UTR155,960382344385933135389326393033530931Intronic1,283,4723,1415713,1355902,6585056021472,8975092,1853932,126404Total intragenic1,686,9373,8766134,0766683,3695427141563,5745542,8894372,783437Pseudogenic57,223235152262122719101819110109611310Pseudogenic intron63,1085075422027215181582025822981317913Transcript exon78,0921903020733119227181361788167015Transcript intron332,7051,2431971,1862161,05315585291,2451921,08116126853REPEATS:LINEs608,4292,1102212,0152402,388255755932,0972172,0841931,530164SINEs428,5671,3814281,3164011,3113853461341,229318928241936271Other repeats487,8632,6052072,5182292,514207925562,7481992,1981772,170169Total in repeats1,524,8596,0968565,8498706,2138472,0262836,0747345,2106114,636604Microsatellite15,18518616895852221981429607661719068All above3,297,59012,3331,93311,8591,92011,4181,8013,16953311,5381,6059,5361,3158,1391,200Other intergenic996,7203,6344603,4234403,5644991,0611502,7173702,5663392,651345Total4,754,82915,9672,39315,2822,36014,9822,3004,23068314,2551,97512,1021,65410,7901,545Variations shown in Table\u00a02 ascribed to sequence regions identified during annotation. These included exonic, UTR and intronic regions of coding; pseudogenic and transcript loci; repeat elements, microsatellites and other intergenic regionsS Substitution, ID indelTable\u00a04Codon variation caused by substitutions in HLA and other gene lociCodons variation by virtue of substitutionsCOXQBLSSTOAPDDBBMANNMCFHLAOtherTotalHLAOtherTotalHLAOtherTotalHLAOtherTotalHLAOtherTotalHLAOtherTotalHLAOtherTotalSynonymous498113071106177725712912425666913559791388052132Non-synonymousTotal Conservative12576201184121305164722361927461207619614491235147562036842110102721749239131111829674010777601378235117Non-conservative573491824913172331058917533689673198652186Total174157331255227482236129365205171186145331203170373227108335Coding substitutions analysed for their effects on protein sequences and listed in by haplotype for HLA genes (HLA-A HLA-B HLA-C HLA-DRB1 HLA-DRA HLA-DQA1 HLA-DQB1 HLA-DPA1 HLA-DPB1) and for all other genes according to the changes they induced in codons as either synonymous, non-synonymous conservative, or non-synonymous non-conservative changes.\nGene annotation\nThere have been several previous annotations of the gene content of the MHC (Horton et al. 2004; Mungall et al. 2003; Stewart et al. 2004; The MHC Sequencing Consortium 1999; Traherne et al. 2006). The maximum region annotated in this study extends from the telomeric ZNF452 gene in the MHC extended class I region (COX haplotype) to the centromeric ZBTB9 gene just telomeric of the MHC extended class II region (PGF and SSTO haplotypes). The PGF haplotype (Stewart et al. 2004) remains the longest complete MHC haplotype, encompassing 320 annotated loci with 1,267 variants. The number of variants ascribed to each locus-type is listed in Table\u00a05. A comparison of the statistics for loci in each haplotype is shown in Table\u00a06.\nTable\u00a05Splice-variant statistics for PGF annotationTypeNo.Total splice variants1,267Coding523Unprocessed_pseudogene50Processed_pseudogene41Expressed_pseudogene7Transcript271Putative71Retained_intron263Nonsense_mediated_decay30Artefact11Total loci320Splice variants annotated in the PGF haplotype.Table\u00a06Gene annotation statistics for eight MHC haplotypesLocus typePGFCOXQBLSSTOAPDDBBMANNMCFCoding16515915013182146129150Transcript2828262619262722Putative181815156161214Pseudogenes total9895939859929575Unprocessed5048485336525342Processed4142403919343728Expressed75564655Artefact111110110000Total loci320311294281166281264261Total variants1,2671,1911,1551,0585681,1389601,115Annotation statistics for loci in each haplotype. For definitions of locus types see \u201cMaterials and methods\u201d.\nVEGA database and browser\nThe VEGA database provides access to gene annotation of the eight MHC haplotype sequences, a valuable public resource and a means of integrating annotation and variation data. The VEGA database also provides the facility to download nucleotide or peptide sequences for genes of interest, by selecting \u2018export cDNA\u2019 or \u2018export peptide\u2019 from the menu obtained by clicking on gene cartoons in the VEGA \u2018detailed view\u2019 or \u2018basepair view\u2019 window. From these, any desired alignments can be made. Variation data may be viewed in the browser linked to a distributed annotation system (DAS) source of any given variation (see \u201cMaterials and methods\u201d). This is illustrated An example of the use of this browser to view a C to T substitution is illustrated for the OR2J1 locus (Fig.\u00a01). An overview of the genomic environment is given in Fig.\u00a01a, showing the gene within a cluster of olfactory gene loci on chromosome 6. The detailed view (Fig.\u00a01b) shows OR2J1 with associated variations in all haplotypes. The basepair view (Fig.\u00a01c) illustrates the presence of the C\/T substitution in all haplotypes except MCF, and its positioning above the translated sequence, at the first position of a CAG codon, indicating the presence of a stop codon instead of glutamine.\nAnnotation changes\nIn addition to loci annotated in the previous studies, newly recognised with official Hugo Gene Nomenclature Committee (HGNC) symbols have also been annotated. These have included the mitochondrial coiled\u2013coil domain protein 1 gene MCCD1 (Semple et al. 2003) and the related unprocessed pseudogenes MCCD1P1 and MCCD1P2, as well as the zinc-finger and BTB domain-containing protein gene ZBTB9, annotated at the very centromeric boundary of the sequenced region.\nThe C6orf21 gene (De Vet et al. 2003; XXbac\u2013BPG32J3.17-001) of the MHC class III region was annotated as a separate locus from the adjacent centromeric locus LY6G6D (splice variants XXbac\u2013BPG32J3.4-001 and XXbac\u2013BPG32J3.4-002). There was, however, a further coding splice variant of LY6G6D (XXbac\u2013BPG32J3.4-004), which spanned not only the other LY6G6D splice variants but also C6orf21, suggesting that this is a possible so-called chimeric transcript (Parra et al. 2006).\nHLA-DRB1 hypervariable region\nOf the five newly annotated MHC haplotypes, APD alone exhibited the HLA\u2013DRBDR52 antigenic specificity found on DRB1*3, DRB1*05 (DRB1*11 and DRB1*012) and DR6 (DRB1*13 and DRB1*14) haplotypes and encoded by HLA\u2013DRB3, whereas the remainder (SSTO, DBB, MANN and MCF) exhibited the DR53 specificity, encoded by HLA\u2013DRB4, here annotated for the first time in genomic sequence. The HLA\u2013DRB53 sequences included three known loci (HLA\u2013DRB4, HLA\u2013DRB7 and HLA\u2013DRB8), as well as three novel pseudogenes (DASS\u2013218M11.1, DASS\u201323B5.1 and DASS\u201323B5.2). DASS\u201323B5.1 corresponds to a pseudogene derived from the gene for the protein kinase, interferon-inducible double-stranded RNA dependent activator (Chida et al. 2001) for which the symbol PRKRAP1 has now been recognised. A further processed pseudogene, FAM8A5P (Jamain et al. 2001), was also annotated in the DR53 specificity.\nHLA-V and HLA-P\nOur analysis showed that the two unprocessed class I pseudogenes HLA-V and HLA-P ( previously HLA-75 and HLA-90, Geraghty et al. 1992) should in fact be merged together; individually they merely represented the 5\u2032 and 3\u2032 portions of a single unprocessed pseudogene, separated by repeat elements. According to our annotation guidelines (see \u201cMaterials and methods\u201d), the newly merged locus was assigned the symbol from the 3\u2032 component, in this case, HLA-P. Best-in-genome nucleotide evidence was found to support five transcript variants at the 5\u2032 end, which, together with evidence for continued locus-transcription, led us to designate the locus as a transcribed pseudogene. Because transcription appears to still occur at this locus, it was, therefore, designated as a transcribed pseudogene. A further six expressed pseudogenes were identified in the MHC region (HLA\u2013DPB2, HLA-J, CYP21A1P, HLA\u2013DRB6, HLA\u2013L and PPP1R2P1).\nRCCX hypervariable region\nThis module within the MHC class III region, named for its gene content (RP-C4A\/B-CYP21-TNXB), may be duplicated or triplicated (Chung et al. 2002), and the pseudogenes CYP21A1P, TNXA and STK19P contain the complement component gene, C4, in either or both of the two versions, C4A and C4B (Awdeh and Alper 1980). This gene may also be present in either long (C4AL, C4BL) or short (C4AS, C4BS) forms depending on the presence or absence of an inserted HERVC4 element in intron 9. Contrary to our previous annotation (Stewart et al. 2004) see also legend to (Fig.\u00a02), the PGF haplotype now appears to possess an arrangement in which C4AL precedes C4BL, whereas COX has a single module with C4BS and QBL has a single module with C4AS (Traherne et al. 2006). For the new haplotype sequences reported in this paper, SSTO was bimodular with two copies of C4BL, whereas DBB was bimodular with C4AL followed by C4BS. Although a sequence gap was present in MCF, this haplotype appeared to be bimodular in that, although the telomeric copy of the C4 gene could not be identified, there was evidence for the pseudogenes CYP21A1P, TNXA and STK19P in a telomeric module. The second centromeric module in MCF contained C4AL. The RCCX region in the APD and MANN haplotypes was incomplete because of sequence gaps.\nC6orf205\nVariability in the C6orf205 gene has been reported to consist of extension of the minisatellite in exon 2 from 27 copies in PGF and COX to 31 copies in QBL (Traherne et al. 2006). In the newly annotated haplotypes, we found the minisatellite to extend to 29 in MANN. The APD, DBB and MCF possessed 27 copies. There was a sequence gap in this region in the SSTO haplotype.\nMICA\nThe known allelic polymorphism of MICA reported for the DRB1*03 QBL cell line sequence, in which a four-base insertion (GCGT) extended the open reading frame in coding exon 5 haplotype (Traherne et al. 2006), was also present in the DRB1*07 MANN haplotype. The insertion was absent from PGF, COX and SSTO. No sequence was available in APD, DBB and MCF for this gene.\nPPP1R2P1\nThe intronless pseudogene PPP1R2P1 reported to have a full-length open reading frame in the PGF, COX and QBL haplotypes (Stewart et al. 2004; Traherne et al. 2006) was found to have a similar open reading frame in the DBB and MANN haplotypes but to have the frameshift mutation seen in the original chromosome reference sequence (Mungall et al. 2003) in the SSTO, APD and MCF haplotypes.\nPSORS1C1\nThe QBL haplotype remains the only one in which there was a single nucleotide deletion in a polyC tract of exon 5 (Traherne et al. 2006). DBB, MANN and MCF resembled PGF and COX. No sequence was available for this gene in SSTO or APD.\nPOU5F1\nThe PGF haplotype has been reported to have a disrupted start codon for alternative splice variant of POU5F1 (Traherne et al. 2006). This disruption was not present in COX or QBL nor was it present in the further haplotypes reported in this paper, namely SSTO, DBB, MANN and MCF. APD had no sequence in this region.\nOR2J1\nThis olfactory receptor OR2J1 has been reported to have both functional and non-functional alleles (Ehlers et al. 2000), the latter the result of a premature stop codon at amino acid position 194 introduced by a substitution in the coding sequence. In our annotation, we found the PGF and MCF haplotypes to contain the full-coding sequence, whereas the COX, QBL SSTO, APD, DBB and MANN haplotypes to contain the truncated sequence as an unprocessed pseudogene (see above and Fig.\u00a01).\nOther annotation differences\nOther loci included in the current but not the previous PGF annotation were HCG4P11, HCG4P8, HCG4P7, HCG4P5, HCG4P3 and the loci without symbols listed in Table\u00a07. Previously annotated loci not annotated in this study or considered artefacts because they did not reach our current standards of annotation included HLA-X, C6orf215, HCG2P7, HCG8, HCP5P2, HCP5P3, HCP5P6, HCP5P12, HCP5P13, HCP5P14, HCP5P15, HCG8 and HCG26.\nTable\u00a07Other newly annotated lociLocusLocus typeXXbac-BCX196D17.5TranscriptXXbac-BPG116M5.14PutativeXXbac-BPG116M5.15PutativeXXbac-BPG116M5.16PutativeXXbac-BPG118E17.9PutativeXXbac-BPG126D10.10Processed pseudogeneXXbac-BPG126D10.11Processed pseudogeneXXbac-BPG13B8.10TranscriptXXbac-BPG13B8.9Unprocessed pseudogeneXXbac-BPG154L12.4PutativeXXbac-BPG181B23.4TranscriptXXbac-BPG181M17.4PutativeXXbac-BPG246D15.8TranscriptXXbac-BPG248L24.10Unprocessed pseudogeneXXbac-BPG248L24.9Processed pseudogeneXXbac-BPG249D20.9PutativeXXbac-BPG250I8.13TranscriptXXbac-BPG254F23.5PutativeXXbac-BPG254F23.6PutativeXXbac-BPG254F23.7TranscriptXXbac-BPG254F23.7PutativeXXbac-BPG27H4.7TranscriptXXbac-BPG27H4.8TranscriptXXbac-BPG294E21.7Processed pseudogeneXXbac-BPG296P20.14PutativeXXbac-BPG296P20.15PutativeXXbac-BPG299F13.14PutativeXXbac-BPG308J9.3TranscriptXXbac-BPG308K3.5PutativeXXbac-BPG308K3.6TranscriptXXbac-BPG309N1.15Unprocessed pseudogeneXXbac-BPG32J3.18PutativeXXbac-BPG8G10.2Unprocessed pseudogeneDAQB-12N14.5TranscriptDAQB-331I12.5PutativeDAQB-335A13.8TranscriptNewly annotated loci without HGNC symbols.\nNon-canonical splice sites\nEight variants within six loci were shown to exhibit haplotypic variation at their splice sites (canonical to non-canonical motif; Table\u00a08). These variations may affect the gene expression at the post-transcriptional level. Hoarau et al. (2004, 2005) have already described the differential splicing within the HLA\u2013DQA1 locus, and this can clearly be seen by comparing the new HLA\u2013DQA1 annotation through the VEGA genome browser.\nTable\u00a08Haplotype variation at splice sitesGeneVariantAffected exonsDonor*Acceptor*dbSNP cluster IDBest evidencePGFQBLCOXSSTODBBAPDMANNMCFTRIM3123\/4ggttggrs28400887cDNANCNCNCCNDNCNCCTRIM3152\/3ggttggrs28400887ESTNCNCNCCNDNCNCCC4B73\/4ggtcgg\u2013ESTNCNDNCCNCNDNDNDC4A73\/4ggtcgg\u2013ESTNCNCNDCNCNDNDNCHLA-DQA144\/5ggtcggrs707947cDNACCCNCNCNDNCNCHLA-DQA154\/5ggttaa\/caars3667cDNANCNCNCCCNDCCHLA-DRB122\/3gatcagrs9271083ESTNCCCCCNDCNDGene loci and variants that are affected by disruptive variations at splice sites. C Canonical splice site (donor\u2009=\u2009ngt; acceptor\u2009=\u2009nag), NC non-canonical, and ND no data (gene absent or gap). Donor and acceptor variable nucleotides in bold with equivalent dbSNP cluster ID number given in column to right. The C4A and C4B genes are, for these purposes, effective duplicates of each other. The two TRIM31 variants share the same splice site (but differ elsewhere in structure). The two HLA\u2013DQA variants share the same donor but have alternative acceptors. Note the mutually exclusivity of these variants amongst the haplotypes (Hoarau et al. 2004; Hoarau et al. 2005).\nCombination of variation and annotation data\nThe data for sequence contig length, gaps, variation rate within haplotypes and PGF coding gene annotation have been combined in the map in Fig.\u00a02. This illustrates the concentration of variation around the HLA gene loci, specifically in 3\u00a0areas: around HLA-F, HLA-G and HLA-A; around HLA-C and HLA-B; and around HLA-DRB1, HLA-DQA1, HAL-DQB1, HLA-DQA2 and HLA-DQB2. The variation status of genes of the PGF haplotype is shown in Table\u00a09.\nFig.\u00a02Variation and annotation map of eight MHC haplotypes. The map represents the complete reference sequence (orange bar split into three 1.6\u00a0Mb sections) labelled PGF and marked with a scale (Mb) and approximate megabase positions on the NCBI36 build of chromosome 6 (grey milestones). Below the reference sequence are arrows representing gene positions and orientations colour-coded for variation status (invariable, black; with synonymous variation only, green; with non-synonymous, conservative variation, red; with non-synonymous, non-conservative variation, purple; see Table 8) and their symbols on a band denoting MHC class (extended class I, green; class I, yellow; class III, pale orange; class II, light blue; extended class II, pink; outside MHC, pale grey). Above the reference sequence, coloured bands represent the sequences of the other seven haplotypes (COX, orange; QBL, mauve; APD, yellow; DBB, green; MANN, light blue; SSTO, dark blue; MCF, purple) with sequence gaps in dark grey; the RCCX hyper-variable region shown with green (C4A block) and\/or red (C4B block) or black (block absent), and the HLA\u2013DRB hyper-variable region in shades of blue-green. Above each haplotype bar, a bar-graph represents total variation between the haplotype and the reference sequence (total variations\/10\u00a0kb) in dark red. Re-examination of the sequence AL645922 from the PGF haplotype, which contains the RCCX region, has shown that the original assembly was erroneous. Correction of these errors leads us now to the conclusion that the C4A gene precedes the C4B gene in this clone sequence. This new gene order is reflected in Fig.\u00a02Table\u00a09Variation status of the main coding variant of each gene in the PGF haplotype annotationInvariableSynonymous variation onlyNon-synonymous variationConservative variationNon-conservative variationABCF1BAT1aAGERBAT2AGPAT1BAT5BRD2aBAT3AIF1C2BTNL2BAT4APOMCREBL1C6orf21C4AATP6V1G2DAXXC6orf27C4BB3GALT4DDR1aCFBC6orf10C6orf134GNL1DOM3ZC6orf100C6orf136aGPSM3DPCR1C6orf15C6orf26GTF2H4EGFL8C6orf205C6orf48HLA-DOAaEHMT2C6orf25CLIC1HSPA1BFKBPLC6orf47CSNK2BLY6G6CGABBR1CCHCR1CUTAMSH5HLA-DMACDSNCYP21A2PBX2HLA-DOBCOL11A2DDAH2POU5F1HLA-DQB2DHX16FLOT1PPP1R11HLA-DRAHLA-AHLA-DPA1PRR3HSPA1AHLA-BHLA-DRB5RING1LY6G6DHLA-CHSD17B8RNF5MCCD1HLA-DMBKIFC1aRXRBMOGaHLA-DPB1LSM2SYNGAP1OR11A1HLA-DPB2LST1TRIM10OR2H2HLA-DQA1LTBTRIM26OR2J1HLA-DQA2LY6G5CTRIM27OR2J2HLA-DQB1LY6G6ETRIM39aOR2J3HLA-DRB1MAS1LVPS52PHF1HLA-EMRPS18BZBTB12PSMB9HLA-FNCR3ZBTB9RPP21HLA-GNEU1ZNRD1SFTPGHSPA1LNRMSKIV2LIER3OR2B3SLC44A4KIAA1949OR2H1TAP2LTAOR2W1TRIM15LY6G5BPFDN6WDR46MDC1PPP1R10ZBTB22MICAPRRT1ZNF311MICBPSMB8bNFKBIL1RDBPNOTCH4RGL2OR10C1RPS18OR12D2SLC39A7OR12D3STK19OR5U1TNFOR5V1TUBBPPT2ZFP57PSORS1C1PSORS1C2RNF39TAP1TAPBPTCF19TNXBTRIM31TRIM40UBDVARSVARSLGene coding sequences may be invariable (no recorded variation), have synonymous variation only (variation at the nucleotide but not the peptide level) or have non-synonymous variation (variation at both the nucleotide and peptide level), which in turn, may be conservative or non-conservative variation according to the criteria of positive or negative values in the BLOSUM62 matrix. The main coding variant is that numbered 001 in the VEGA database except for LY6G6E and HLA-DPB2 where the main variant is not coding. C4A and C4B were excluded from calculation of variation because the order of these genes in the PGF sequence precluded alignment with other haplotype sequences. Nevertheless, alignment of the coding sequences for each gene separately showed that there were non-synonymous, non-conservative variations. HLA-DRB5 is present in this study only in the PGF haplotype and, therefore, here appears invariableaCoding genes where the main variant does not harbour non-conservative, non-synonymous variation but other variants do (BAT1 BRD2 DDR1 C6orf136 HLA-DOA MOG KIFC1 and TRIM39).bSimilarly, coding genes where the main variant does not harbour conservative non-synonymous variation but other variants do (PSMB8).\nAs well as the variations reported above, major indels revealed as breaks in cross_match discrepancy lists and analysed by RepeatMasker are given in Table\u00a010. Many of these have been previously reported (Dangel et al. 1994; Dunn et al. 2003; Dunn et al. 2002; Gaudieri et al. 1999; Horton et al. 1998; Kulski and Dunn 2005; Stewart et al. 2004). These indels were most frequently but not exclusively associated with AluY elements.\nTable\u00a010Major indels in the form of retrotransposible elementsChr6 pos\u2019nFlanking lociPresence in haplotypeDetailsPGFCOXQBLSSTOAPDDBBMANNMCF29002370TRIM27:C6orf100CCCC??CCComplex region (A)29440424OR5V1:OR12D3\u2713\u2713?\u2713??XXAluYa529784097C6orf40:HCP5P15\u2713X\u2713\u2713?XXXAluYa5\/8 175..30429788451Within HCP5P15XX\u2713X?\u2713\u2713XAluYa5\/8 176..31029794763HCP5P15:HLA-F\u2713XX\u2713?XXXSVA_E plus simple rpt.s29922942HLA-G:MICF\u2713X\u2713\u2713\u2713\u2713\u2713\u2713L1ME3B 5940..616529954495MICF:HLA-H\u2713XXXXXXXHERVK9 inserted in MER930008633HLA-K:HLA-21\u2713XX\u2713XX\u2713?SVA E\/F plus simple rpt.30106475HCG8:ETF1P1X\u2713XX\u2713\u2713XXAluYb830547387SUCLA2P:RANP1XXX\u2713?XX?AluJb 1..283 and parts of MLT1D\/L1PBa31079582C6orf205:HCG22XX\u2713XXX?XAluYb8 37..29731117638C6orf205:HCG22\u2713XX\u2713\u2713X\u2713\u2713AluY (whole & part) and MER63 1017..106231301931HCG27:HLA-C\u2713\u2713X\u2713?\u2713\u2713\u2713HERV3 part (6489...7339)31320352HCG27:HLA-C\u2713XXX?XXXSVA_F 349..850 plus GC rich rpt.31358220RPL3P2:WASF5PXX\u2713X?XXXAluY 35..30631400900WASF5P:HLA-B\u2713\u2713\u2713\u2713?XXXAluSp plus L1PREC2 part (3205...4617)31405648WASF5P:HLA-B\u2713X\u2713\u2713?XxxHERVIP10F (part) and AluSg (only cf CX DB)31418854WASF5P:HLA-B\u2713\u2713\u2713\u2713?\u2713\u2713XL1PA5 part (5503..5876)31530995MICA:HCP5\u2713X?\u2713??X?SVA B\/F plus simple rpt.s32421915within C6orf10\u2713XX\u2713XX\u2713XAluYb832486228BTNL2:HLA-DRA\u2713\u2713\u2713\u2713\u2713XXXL1P1\/L1HS parts32655545HLA-DRB1 intron 5\u2713xxX?\u2713\u2713?AluYa5 within more or less partial LTR1232660731HLA-DRB1 intron 1X\/X\u2713\/XX\/X\u2713\/\u2713?\u2713\/\u2713\u2713\/\u2713?Tigger4\/AluSx32661119HLA-DRB1 intron 1CCCC?CC?Complex region (B)32663167HLA-DRB1 intron 1X\/\u2713\u2713\/\u2713\u2713\/\u2713\u2713\/X?\u2713\/X\u2713\/X?AluSq\/AluY32669534HLA-DRB1:HLA-DQA1CCCC?CC?Complex region (C)32679461HLA-DRB1:HLA-DQA1\u2713XXX?XX?AluY32693271HLA-DRB1:HLA-DQA1\u2713\u2713\u2713\u2713?X\u2713?L1PA4 (parts)32697545HLA-DRB1:HLA-DQA1XXXX?\u2713\u2713?L1HS 7..603232701428HLA-DRB1:HLA-DQA1\u2713X\u2713\u2713?xXxL1PA2 part and from CX: MER2B and AluY32728179HLA-DQA1: HLA-DQB1CCCC?CCCComplex region (D)32739664within HLA-DQB1XX\u2713X?X\u2713XAluY32743646HLA-DQB1: MTCO3P1XXXX?\u2713XXLTR1332746780HLA-DQB1: MTCO3P1XXXX?\u2713X\u2713L1PA4 (parts)32751442HLA-DQB1: MTCO3P1XXXX?X\u2713XLTR5_Hs32753489HLA-DQB1: MTCO3P1\u2713\u2713\u2713\u2713?X\u2713XL1PA10 268..4888 around L1PA4 (part)32756020HLA-DQB1: MTCO3P1XXXX?X\u2713XLTR5_Hs32764047HLA-DQB1: MTCO3P1\u2713\u2713\u2713\u2713?X\u2713XAluSx32765930HLA-DQB1: MTCO3P1XXXX?X\u2713XAluYa532785062MTCO3P1:HLA-DQB3\u2713\u2713\u2713\u2713?XXXTigger4 (Zombi)\/L1HS (parts) and T-rich32795150MTCO3P1:HLA-DQB3XXXXX\u2713X\u2713AluY32796573MTCO3P1:HLA-DQB3XXXXX\u2713X\u2713AluY32815974HLA-DQB3: HLA-DQA2X\u2713XX\u2713XXXAluYa532857369HLA-DQB2:HLA-DOB\u2713X\u2713\u2713XX\u2713XAluYg632881426HLA-DQB2:HLA-DOBXX?\u2713\u2713XX?AluYa532887265HLA-DQB2:HLA-DOB\u2713X?XXX\u2713\u2713LTR42 and parts of L1MC5 and AluSc 3..10533201559within HLA-DPB2\u2713XXX\u2713?X?AluYb833234360HCG24:COL11A2\u2713\u2713\u2713??\u2713\u2713XAluY (1..293) AluJb (26..306)Where there was a break in the cross_match discrepancy list match between two clones, the inserted sequence was extracted and subjected to analysis by RepeatMasker to assess the number of major indels that were a result of retrotransposible elements. Chromosome 6 position (NCBI35\/36) of the inserted sequence was that of the midpoint where the sequence was an insertion in PGF or the position before the deletion in PGF. Flanking loci were retrieved from the annotation. Insertion in a haplotypes is indicated by \u2018\u2713\u2019, deletion by \u2018X\u2019, complex regions by \u2018C\u2019. Where there is a sequence gap in a haplotype corresponding to the indel, this is shown by \u2018?\u2019. Four complex deletion\/insertion events are listed: A, B, C and D; for details, see text.\nFour of these major indels were complex and designated as complex regions A, B, C and D in Table\u00a010. They include three known regions from the comparison of the PGF and COX haplotypes (Stewart et al. 2004). Complex region A (involving MIR, MER41B, MER115, AluSx, Flam_C, AluSg, AluY, AluSx, L2 and MER38 elements) maps between TRIM27 and C6orf100 and was found to be deleted in COX but present in PGF, QBL, SSTO, MANN and MCF. Complex region B (involving L2 and AluY elements) maps to intron 1 of HLA\u2013DRB1 and was also found by comparing PGF with COX, QBL, DBB, MANN and SSTO. Complex region C (SVA and low-complexity repeat elements) maps between HLA\u2013DRB1 and HLA\u2013DQA1 and was noted in COX as a deletion of the SVA and low-complexity repeats. Whereas, DBB, MANN and SSTO displayed the same deletion, as well as a telomeric deletion of AluSx\/MIRb, QBL had both deletions plus that of an intervening 2.5\u00a0kb sequence containing Alu, L3 and MLT1A1 elements. Complex region D maps between HLA\u2212DQA1 and HLA\u2212DQB1 and is more complicated than previously reported. At the telomeric end, PGF lacks an L1PA4 fragment of >300\u00a0bp that is present in COX, QBL, SSTO and MCF and is also absent in DBB and MANN where it is interrupted by about 1.3\u00a0kb of SVA sequence. Centromeric to this PGF contains an AluSx, an AluY and an AluYd2, flanked by long interspersed nuclear element repeats, all deleted in the other haplotypes. Further towards the centromere there is an L1MA7 fragment, into which in PGF alone there are insertions of an AluSx followed by an AluY; a subsequent AluSg present in all haplotypes contains an insertion of 795\u00a0bp of SVA sequence in just COX and QBL. Finally, at the centromeric end of this region, PGF uniquely contains intact MER11C and LTR5 elements.\nRepresentation of haplotypes within European populations\nThe eight haplotypes analysed in this study were selected on the basis of their association with type 1 diabetes and multiple sclerosis and their high population frequencies. To determine how representative these haplotypes are with respect to SNP haplotypic diversity in a population, we determined their distribution in the haplotypic tree space in the European population.\nFor this analysis, we selected a segment of ~214\u00a0kb, spanning the HLA\u2013DRB1 and HLA\u2013DQB1 genes in a population of European ancestry with known HLA allelic data (de Bakker et al. 2006). Phylogenetic analysis of 180 founder haplotypes derived from genotypic data (54 substitutions) shows that the eight haplotypes selected as part of the MHC Haplotype Project share identical HLA alleles over most of the tree space (Fig.\u00a03a), representing almost the entire variation observed in the population assayed with the exception of two branches (DRB1*1103\u2013DQB1*0301 and DRB1*0101\u2013DQB1*0501).\nFig.\u00a03Clusters of haplotypes in the European haplotypic diversity. Phylogenetic relationship of 180 founder SNP haplotypes from CEPH trios spanning a 214-kb segment of the MHC class II region, including the HLA-DRB1 and HLA-DQB1 genes (54 substitutions from rs2187823 to rs2856691). a Sequenced haplotypes are widely distributed in this NJ tree and represent the vast majority of the variation in the population sampled. Four-digit alleles are indicated for the corresponding DRB1 and DQB1 genes in each haplotype ID label to highlight the HLA haplotypic distribution based on the underlying nucleotide variation. The NJ tree was constructed using pairwise genetic distances considering the Kimura 2-parameters model without correction for rate variation among sites as implemented in the MEGA2 software (Kumar et al. 2001). b Each haplotype sequenced is associated to a single haplotype cluster. This phylogenetic network (Bandelt et al. 1999) also shows that clusters (shaded area) are constituted by one central haplotype and its derivatives. Circles represent individual haplotypes, and the size of the circle is proportional to the haplotype frequency. The length of the lines connecting nodes is relative to the distance between them, e.g. distances within shaded areas (clusters) never exceed three mutation steps. Cluster of haplotypes sharing HLA alleles with sequenced cell lines are named accordingly: COX and QBL: DRB1*0301 DQB1*0201\u2013PGF: DRB1*1501 DQB1*0602\u2013APD: DRB1*1301 DQB1*0603\u2013MCF: DRB1*0401 DQB1*0301\u2013DBB: DRB1*0701 DQB1*0303\u2013SSTO: DRB1*0403 DQB1*0302\u2013MANN: DRB1*0701 DQB1*0202. HLA haplotypes DRB1*1103\u2013DQB1*0301 and DRB1*0101\u2013DQB1*0501 indicate the two major haplotype clusters not represented in the MHC haplotype project data\nHaplotype diversity in this sub-population is restricted to relatively few haplotype clusters (Fig.\u00a03b). Each cluster consists of a founder haplotype, depicted by the most frequent and centrally located haplotype within the cluster. Recently derived haplotypes show lower frequencies and are connected to the central haplotype by relatively few mutation steps (in this case, up to three). This phylogenetic network clearly shows that all the sequenced haplotypes occupy central positions in their respective haplotypic groups. Inferences about phylogenetic relationships between haplotype clusters are, however, only approximate as a consequence of recombination events.\nIt should also be noted that SNP haploptypes derived from CEPH pedigrees of European ancestry by no means represent an exhaustive sampling of European diversity. Nevertheless, the sampling has been shown to represent the European population in the UK reasonably well (Ke et al. 2005). In conclusion, our analysis demonstrates that the HLA haplotypes selected for the MHC Haplotype Project are ancestral haplotypes, representative of MHC diversity in the European population.\nConclusion and outlook\nThe MHC Haplotype Project has succeeded in providing a new public resource for immune-linked disease and population genetic studies. First reports from studies using the resource indicate that it adds significant power to the identification and fine-mapping of disease-associated variations (Yeo et al. 2007). The data have also contributed to the recent identification of a first set of HLA tag SNPs, which hold great promise for future applications in clinical settings, e.g. to complement or replace classical HLA-typing in transplant medicine (de Bakker et al. 2006). While costs and other limitations of the current (capillary) sequencing technology have restricted our study to only few (eight) MHC haplotypes, the number of new variations found, combined with the fact that no variation plateau has yet been reached, indicates that there are many more variations to be discovered. The recent introduction of several new and massively parallel sequencing platforms (for review, see Bentley 2006) has created the opportunity to do just that by re-sequencing haplotypes and, eventually, entire genomes at the population level and as integral part of case control studies. Because of its wide-ranging medical importance, the MHC can be expected to be among the first regions of the human genome to be sequenced in this way. Such sequencing will provide the critical, and until now missing, data to identify causal variations and their underlying mechanisms on an unprecedented scale.","keyphrases":["haplotype","major histocompatibility complex","polymorphism","population genetics","retroelement","genetic predisposition to disease"],"prmu":["P","P","P","P","U","M"]}
{"id":"Acta_Neuropathol_(Berl)-2-2-1705477","title":"The muscle protein dysferlin accumulates in the Alzheimer brain\n","text":"Dysferlin is a transmembrane protein that is highly expressed in muscle. Dysferlin mutations cause limb-girdle dystrophy type 2B, Miyoshi myopathy and distal anterior compartment myopathy. Dysferlin has also been described in neural tissue. We studied dysferlin distribution in the brains of patients with Alzheimer disease (AD) and controls. Twelve brains, staged using the Clinical Dementia Rating were examined: 9 AD cases (mean age: 85.9 years and mean disease duration: 8.9 years), and 3 age-matched controls (mean age: 87.5 years). Dysferlin is a cytoplasmic protein in the pyramidal neurons of normal and AD brains. In addition, there were dysferlin-positive dystrophic neurites within A\u03b2 plaques in the AD brain, distinct from tau-positive neurites. Western blots of total brain protein (RIPA) and sequential extraction buffers (high salt, high salt\/Triton X-100, SDS and formic acid) of increasing protein extraction strength were performed to examine solubility state. In RIPA fractions, dysferlin was seen as 230\u2013272 kDa bands in normal and AD brains. In serial extractions, there was a shift of dysferlin from soluble phase in high salt\/Triton X-100 to the more insoluble SDS fraction in AD. Dysferlin is a new protein described in the AD brain that accumulates in association with neuritic plaques. In muscle, dysferlin plays a role in the repair of muscle membrane damage. The accumulation of dysferlin in the AD brain may be related to the inability of neurons to repair damage due to A\u03b2 deposits accumulating in the AD brain.\nIntroduction\nAggregation of neuronal proteins is a common mechanistic theme in neurodegenerative diseases [13]. Whether due to point mutations or post-translational modification, normally soluble proteins are altered and form insoluble fibrillar aggregates [18]. Alzheimer\u2019s disease (AD), the most common form of dementia, is characterized pathologically by abundant diffuse and neuritic plaques throughout most cortical regions. Amyloid \u03b2-protein (A\u03b2) is a 39\u201343 amino acid peptide cleaved from a larger precursor protein (amyloid precursor protein or APP) that deposits extracellularly as senile plaques either as loose, nonfibrillar diffuse plaques, or as a more compacted, fibrillar form, with dystrophic neurites coursing through the plaque [25]. Other aggregated proteins found in the AD brain include the microtubule-associated protein tau [11] as neurofibrillary tangles and the presynaptic protein \u03b1-synuclein [13] in the form of Lewy bodies.\nThe alteration, deposition and aggregation of amyloid proteins may be an ill-fated response to the disease process or themselves lead to a cascade of cellular responses ultimately resulting in neuronal dysfunction and death.\nNonbrain protein aggregates have been described in muscle disease. For example, amyloid-like deposits have been described in inclusion body myositis [2]. Other protein aggregates found in muscle disease include desmin-related myopathies, actinopathies and myosinopathies [30]. Aggregates containing these proteins have not been described in brain disease. Another muscle protein associated with myopathy is dysferlin. Dysferlin is a plasma membrane and cytoplasmic vesicle-associated protein implicated in three adult-onset muscle diseases (limb-girdle dystrophy, type 2B, Miyoshi myopathy and distal anterior compartment myopathy)[4, 9, 22, 24]. Although described in peripheral nerve [30] and demonstrated by Western blot in human and mouse brain [21], there is little known about the function of dysferlin in the brain or its role in neurodegenerative disease. We describe the pattern of dysferlin expression in the normal and AD brain and examine dysferlin accumulation in association with amyloid deposition in the brains of AD patients.\nMaterials and methods\nCase materials\nCase materials were derived from research participants in a longitudinal study of healthy aging and dementia who were studied postmortem. The diagnostic criteria are consistent with probable AD reported by the National Institute of Neurological and Communicative Disorders and Stroke and Alzheimer\u2019s Disease and Related Disorders Association [26] and confirmed by autopsy to meet high probability of AD according to National Institute of Aging-Reagan criteria [28]. The clinical dementia rating (CDR) was used to determine the presence or absence of dementia and stage its severity [27]. The global CDR is derived by a synthesis of individual ratings in six cognitive and functional categories where CDR\u00a0=\u00a00 indicates no dementia and CDR\u00a0=\u00a00.5, 1, 2, or 3 corresponds to very mild, mild, moderate, or severe dementia [27]. The CDR is closely correlated with the presence of dementia pathology at autopsy [14]. The Washington University Human Studies Committee approved all procedures.\nTwelve brains were examined. Three cases were rated as nondemented (CDR 0) with a mean age of 87.5\u00a0years. Nine AD brains were examined; three each of mild (CDR 1), moderate (CDR 2) and severe (CDR 3). The mean age of the demented cases was 85.9\u00a0years with mean disease duration of 8.9\u00a0years (Table\u00a01). Control cases were all Braak stage I or II [6]. Sampling during postmortem exam was done from 29 different cortical, subcortical and brainstem regions. Paraffin-embedded sample for this study were taken from midfrontal, cingulate and entorhinal regions; three sections were stained for each antibody with appropriate controls [15]. Biochemistry was performed on midfrontal gyrus adjacent to the section taken for immunohistochemistry for each case.\nTable\u00a01Sample characteristics and findings at postmortem examinationCaseAge (years)SexCDRDisease duration (years)Brain wt (g) Braak scoresCERAD criteriaNIA\u2013Reagan criteriaNeurofibrillaryAmyloid192F0\u20131,120IIA\u2013\u2013285F0\u20131,100IA\u2013\u2013392F0\u20131,210IA\u2013\u2013485F181,200IVCDefiniteHigh599M121,230VBDefiniteHigh695M1141,360VCDefiniteHigh781M216790VICDefiniteHigh891M2111,580VBDefiniteHigh990M271,170IVBDefiniteHigh1086M3101,310VICDefiniteHigh1179F38770VICDefiniteHigh1288M3111,130VICDefiniteHigh\nImmunohistochemistry\nImmunohistochemistry was done as previously described [14, 15]. Briefly, serial 6\u00a0\u03bcm sections of paraformaldehyde-fixed, paraffin-embedded tissue were prepared and incubated overnight at 4\u00b0C with antibodies against dysferlin (Novacastra Laboratories), tau (17026, gift from J. Trojanowski), and amyloid \u03b2-protein (6E10, Signet). Sections were developed by avidin\u2013biotin complex method (Vector Laboratories) for immunohistochemistry with DAB (brown), Vector Red (pink) and Vector SG (blue) as chromogens. Alexa-Red and Green (Jackson Laboratories) antibodies were used for immunofluorescence. Sections were examined with a Nikon E800 Research microscope, and images were digitized and viewed with AnalySIS analytic software (Soft Imaging Systems, GmbH).\nWestern blots\nWestern blotting and biochemical fractionation were performed as described previously [16]. Briefly, two sections of gray matter (0.3\u00a0g) from the midfrontal cortex of AD and normal age-matched control brains were dissected from underlying white matter. After dissection, gray matter was divided into two samples (Fig.\u00a01). One sample was homogenized in RIPA (150\u00a0mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40, 50\u00a0mM Tris, pH 8, 20\u00a0mM, NaF, 2\u00a0mM EGTA, 0.5% levamisole, 1\u00a0mM NaVO4) to estimate total dysferlin. The other sample was homogenized and subjected to sequential extraction buffers of increasing protein extraction strength. For each buffer, pellets were washed twice and supernatants pooled. Samples were first homogenized in 2\u00a0ml\/gram of tissue of high-salt (HS) buffer (50\u00a0mmol\/l Tris, pH 7.4, 750\u00a0mmol\/l NaCl, 10\u00a0mmol\/l NaF, 5\u00a0mmol\/l ethylenediaminetetraacetic acid with protease inhibitors) and centrifuged at 100,000g for 30\u00a0min. The pellets were re-extracted and the supernatants were pooled. The pellets were sequentially extracted twice with 2\u00a0ml\/gram of HS buffer\/1% Triton X-100 (HS\/T) and once with 1\u00a0ml\/gram sodium dodecyl sulfate (SDS) sample buffer (1% SDS, 10% sucrose, 10\u00a0mmol\/l Tris, pH 6.8, 1\u00a0mmol\/l ethylenediaminetetraacetic acid, 40\u00a0mmol\/l dithiothreitol). The pellets were extracted with 0.67\u00a0ml\/g 70% formic acid (FA) and disrupted with two sequential 2-s sonication bursts. FA was evaporated in a vacuum centrifuge (Eppendorf). SDS sample buffer (0.67\u00a0ml\/g) was added to the dried pellets, followed by vigorous vortex, and the pH was adjusted with NaOH. SDS sample buffer was added to the HS and HS\/Triton fraction and all of the samples were boiled for 5\u00a0min. The FA samples were centrifuged at 13,000g for 5\u00a0min to remove insoluble debris. Five microliters of each fraction was loaded in separate lanes for SDS-polyacrylamide gel electrophoresis followed by Western blot analysis with antibodies again dysferlin. The immune complexes were visualized with the use of the ECL Plus kit (Amersham) according to the manufacturer\u2019s protocol. The autoradiographs obtained were scanned and the band intensity quantified utilizing TotalLab software (Nonlinear Dynamics, Newcastle on Tyne, UK). Statistical analyses were performed by analysis of variance using SPSS statistical software.\nFig.\u00a01Schematic representation of sequential protein extraction buffers. Samples are divided into equal portions. The first sample is homogenized in RIPA buffer and blotted to estimate total dysferlin levels. The other sample is homogenized in sequential extraction buffers of increasing protein extraction strength. The first extraction is in high-salt (HS) buffer. The supernatants are saved and the pellet is extracted with HS buffer\/1% Triton X-100 (HS\/T) buffer. After saving the supernatants, the resultant pellet is homogenized in sodium dodecyl sulfate (SDS) sample buffer. Resulting pellets are finally extracted with 70% formic acid (FA). Supernatants are blotted to estimate differing levels of dysferlin solubility with the HS fraction representing the most soluble phase and the FA fraction representing the most insoluble phase\nResults\nLocalization of dysferlin in normal (CDR 0) brain\nMicroscopic evaluation of the dysferlin staining in the normal (CDR 0) brain localized expression to the cytoplasm of large pyramidal neurons of layers III, V and VI in the neocortex (Fig.\u00a02a), layer II neurons of the entorhinal cortex, CA 1\u20133 neurons in the hippocampus and large projection neurons in the amygdala (not shown). Dysferlin was not localized to astrocytes, endothelial cells or microglia (arrowheads in Fig.\u00a02b). Little to no dysferlin neuritic staining was seen in control cases (Braak stage I or II).\nFig.\u00a02Localization of dysferlin in brains of nondemented subjects. a, b Demonstrates the localization of dysferlin immunoreactivity in the brain. There is a neuronal expression of dysferlin localized in cytoplasm of large pyramidal (layers III, V, VI) neurons in the frontal cortex at low power (a). Little to no neuritic staining was detected. Dysferlin expression is confined to neurons (arrow, DAB brown in b) and is not seen in glial cells stained with GFAP (small arrowheads, Vector Red pink in b). Magnification of (a) is 100\u00d7 and the scale bar in (a)\u00a0=\u00a050\u00a0\u03bcm. Magnification of (b) is 400\u00d7 and the scale bar in (b)\u00a0=\u00a020\u00a0\u03bcm\nDysferlin in the AD brain\nDysferlin neuritic accumulation is found throughout the AD brain and in double-label studies it is localized in A\u03b2 neuritic plaques in the neocortex (Fig.\u00a03a). Double-labeled studies demonstrate co-localization of dysferlin (brown in Fig.\u00a03b) and A\u03b2 (pink in Fig.\u00a03b) in the hippocampus.\nFig.\u00a03Localization of dysferlin with amyloid pathology in the AD brain. a, b Demonstrates the location of the dysferlin accumulation in the AD brain with double-labeled studies. a Diffuse dysferlin accumulation (DAB, brown) and A\u03b2 (DAB-nickel, black) in the frontal cortex. Using VectorRed (pink, Vector Laboratories) as the chromogen for A\u03b2 and DAB (brown, Vector Laboratories) as the chromogen for dysferlin, the pathologic deposition is localized as neuritic pathology in A\u03b2 neuritic plaques in the neocortex hippocampus (b). Magnification of (a) is 100\u00d7 and the scale bar in (a)\u00a0=\u00a020\u00a0\u03bcm. Magnification of (b) is 200\u00d7 and the scale bar in (b)\u00a0=\u00a020\u00a0\u03bcm\nDysferlin neuritic pathology occurs in conjunction with tau neuritic pathology\nIn Fig.\u00a04, we demonstrate that neurites in plaques may comprise tau, dysferlin or both. Triple-labeled studies of midfrontal (Fig.\u00a04a), cingulate (Fig.\u00a04b) and entorhinal (Fig.\u00a04c) cortex illustrate the relationship of tau (pink, arrowheads) and dysferlin (brown, arrows) within an A\u03b2 plaque (blue). Double-labeled immunofluorescent studies (Fig.\u00a04d\u2013f) demonstrate that tau (green in 4d) and dysferlin (red in Fig.\u00a04e) have both distinct and overlapping (yellow in Fig.\u00a04f) epitopes in neuritic plaques. Dysferlin does not appear to co-localize with the more abundant tau dystrophic neurites.\nFig.\u00a04Localization of dysferlin with tau pathology in the AD brain. a\u2013f Demonstrates the location of the dysferlin and tau accumulation in the AD brain with triple-labeled immunohistochemistry studies (a\u2013c) and double-labeled immunofluorescence studies (d\u2013f). a Midfrontal, b cingulate and c entorhinal demonstrate tau (Vector Red, pink, arrowheads), dysferlin (DAB, brown, arrows) and A\u03b2 (Vector SG, blue). Note the distinction between tau-positive and dysferlin-positive structures. Double-labeled immunofluorescence studies demonstrated neurites within amyloid plaques. Neurites may comprise tau (green, d), dysferlin (red, e) or overlapping epitopes (yellow in merged image of f). Magnification of (a\u2013f) is 600\u00d7 and the scale bar in (d)\u00a0=\u00a020\u00a0\u03bcm\nBiochemistry\nWestern blot analysis following RIPA extraction demonstrated the presence of dysferlin as 230\u2013272\u00a0kDa bands in all brains examined (Fig.\u00a05a); however, there was a decrease in the total dysferlin content in CDR 3 (severe AD) brains compared to CDR 0 (nondemented). There is a progressive decrease in total dysferlin with progressive disease (r\u00a0=\u00a00.998, P\u00a0=\u00a00.001). Western blot analysis following serial extraction of proteins demonstrated a change in the solubility state of dysferlin across the spectrum of dementia (Fig.\u00a05b). No dysferlin could be demonstrated in the HS fraction. In contrast to the control CDR 0 brains, however, there was a significant reduction of dysferlin in the HS\/T fraction in the demented cases (CDR 1 and 2) and was undetectable at the CDR 3 level of severity (F\u00a0=\u00a02,991, P\u00a0<\u00a00.001). In the SDS-soluble fraction from the demented cases, there was an accumulation of 230\u2013272\u00a0kDa bands with dementia progression, that is a more insoluble form of dysferlin was found in severe CDR 3 brains compared to milder stages of dementia (CDR 1) or in nondemented individuals (F\u00a0=\u00a03,183, P\u00a0<\u00a00.001). Dysferlin did not aggregate or accumulate in the FA-soluble fractions of the control or AD brains.\nFig.\u00a05Western Blot analysis of dysferlin in nondemented and AD brains. a Nondemented (CDR 0) and AD brains (mild\u00a0=\u00a0CDR 1, moderate\u00a0=\u00a0CDR 2, and severe\u00a0=\u00a0CDR 3) were extracted in RIPA as described in the Materials and methods. Five microliters of each extraction were loaded into separate lanes of a 12% SDS-polyacrylamide gel electrophoresis and after transfer to nitrocellulose membranes were probed with antibodies against dysferlin. The blot demonstrates the presence of dysferlin in both normal and AD brains with declining levels of protein across disease progression. Band intensities were quantified and compared in a bar graph. There is a linear decrease in total dysferlin associated with disease progression (r\u00a0=\u00a00.998, P\u00a0=\u00a00.001). b The same brains were sequentially extracted with HS, HS\/T, SDS and FA as described in the Materials and methods section as loaded onto 12% SDS-polyacrylamide gel electrophoresis as described earlier. Dysferlin was not seen in either the HS or FA fractions. Dysferlin was demonstrated prominently in HS\/T fraction in the CDR 0 brains, with a significant reduction (F\u00a0=\u00a02,991, P\u00a0<\u00a00.001) of dysferlin in the HS\/T fraction in the demented cases (CDR1 and 2) and was nearly undetectable in severe AD (CDR 3) In contrast, the SDS fraction representing insoluble phases of dysferlin demonstrated significant increases (F\u00a0=\u00a03138, P\u00a0<\u00a00.001) in the 230\u2013272\u00a0kDa bands as dementia progressed from mild (CDR 1) to severe (CDR 3)\nDiscussion\nDysferlin is a new protein described in the AD brain associated with neuritic plaques. Dysferlin is widely expressed in the brain and is found in intracytoplasmic compartments in pyramidal neurons in normal brains. Dysferlin begins to accumulate in dystrophic neurites in the AD brain at mild stages of dementia (CDR 1). Dysferlin is found at all stages of disease with greater accumulation in more advanced disease (CDR 3).\nSignificantly, our Western blot analysis demonstrated dysferlin in RIPA-extracted fraction in all cases; however, in the serial extracted fractions an alteration in the solubility state of dysferlin was found in demented cases (CDR\u00a0>\u00a01) compared with nondemented cases. This coincides with the finding of neuritic pathology by immunohistochemistry. The HS\/T fraction represents the transmembrane cytoskeletal fraction of normal dysferlin and decreases with advanced stages of dementia. This may represent the loss of membrane integrity and alteration in cytoskeleton structure. In contrast, the SDS-soluble fraction represents more insoluble forms of the protein suggesting aggregation of dysferlin may be related to disease progression. This alteration of solubility and accumulation is seen in other neurodegenerative diseases (e.g. Parkinson\u2019s disease, multiple system atrophy, dementia with Lewy bodies) [15, 31].\nDysferlin is a member of ferlin family, homologous to fer1 protein in C. elegans [5]. It is highly expressed in muscle but little is known about the role of the protein in the brain. Several members of the ferlin family have been mapped. Dysferlin is found on chromosome 2p13 [1] and deficiency is associated with three clinical phenotypes: Limb-Girdle muscular dystrophy, type 2B, Miyoshi myopathy and distal anterior compartment myopathy [20, 22, 24]. Another member of the ferlin family, Otoferlin is mapped to chromosome 2p23 and is associated with autosomal recessive nonsyndromic deafness [31]. Two other members of the ferlin family have been described: Myoferlin (10q24) [7] and FerlL4 (20q11) [12]. Myoferlin is a type II transmembrane protein upregulated in Duchenne muscular dystrophy and may have a role in muscle regeneration and repair [10]. The function of Fer1L4 is unknown.\nDysferlin is thought to play a role in muscle membrane maintenance and repair [3, 4, 9, 12, 19]. In C. elegans, the Fer1 protein plays a role in vesicle trafficking and membrane fusion and can bind to phospholipids in a calcium-dependent manner [22]. In dysferlin null-mice, muscle cells are defective in repairing membrane disruptions leading to degeneration [4, 21, 23]. If dysferlin has a similar membrane repair function in neurons, then it is possible that the deposition of dysferlin in AD may be related to the inability of neurons to repair damage due to accumulating A\u03b2 pathology throughout the progression of AD. The findings reported suggest that there may be common mechanisms of membrane repair in degenerative diseases of muscle (e.g. dystrophinopathies and sarcoglycanopathies) and brain (e.g. AD).\nThe aggregation of normally soluble proteins in fibrillar lesions is the neuropathologic hallmark of many neurodegenerative diseases. Whether the aggregates or their precursors are the actual toxic species is still under debate [8]; however, it is likely that the conversion of proteins from a soluble to insoluble state impairs the long-term viability of neurons. In addition, because dysferlin appears to accumulate in conjunction with A\u03b2 deposition and disease progression it may be able to serve as a marker of disease progression. Continued efforts aimed at understanding abnormalities in the misfolding of protein leading to neurodegenerative disease such as A\u03b2 and tau, or proteins associated with aggregation such as dysferlin will provide insights into disease mechanisms underlying neurological disorders characterized by abundant filamentous lesions.","keyphrases":["dysferlin","alzheimer\u2019s disease","amyloid \u03b2-protein","protein aggregation","tau protein","neurodegeneration"],"prmu":["P","P","P","P","R","U"]}
{"id":"Eur_J_Pediatr-4-1-2190787","title":"Concordance between school outcomes and developmental follow-up results of very preterm and\/or low birth weight children at the age of 5 years\n","text":"Introduction Long-term follow-up studies have revealed a high frequency of developmental disturbances in preterm survivors of neonatal intensive care who were formerly considered to be non-disabled. These developmental disturbances interfere with the acquisition of everyday skills and, in particular, with normal school functioning.\nIntroduction\nThe survival rate of very preterm (VPT\u2009<\u200932 completed weeks of gestation) and very low birth weight (VLBW\u2009<1500\u00a0g) children has steadily improved during the last decades, but the prevalence of major disabilities or handicaps in these children has remained stable. Follow-up studies have shown that VPT and VLBW infants are at risk with regard to neurological dysfunctioning such as cerebral palsy and mental retardation [6, 17, 31, 33]. Even more children of these groups experience difficulties in intellectual, speech and language skills as well as clumsiness and attention and behaviour problems, all of which can affect school functioning [4, 12, 13, 16, 19, 24, 26].\nSignificant\/severe neurosensory dysfunctioning is usually detected early, with the result that children with major handicaps often attend a school for special education from school entry. Minor developmental impairments and disabilities, however, frequently go undetected until school age (4\u00a0years in The Netherlands). These children start their school career in mainstream education. Once at school, however, certain learning problems may become apparent during the first 2\u00a0years (the kindergarten years), but more often these appear only when the children start formal academic training [15, 22, 34]. Several reasons may explain this phenomenon of 'growing into educational deficits' [2, 9, 18, 20, 27].\nMild developmental disturbances usually pose no problem for the child until greater demands have to be met at school or when more detailed evaluations are made. In addition, the cumulative effect of the failure to acquire basic skills and the resulting declining motivation may play an important role. In The Netherlands, schools are focused strongly on the early detection of children who need special assistance. After 1\u00a0year of education, at the age of 5, teachers usually have a good impression of both the cognitive and language development of their children and of their motor skills and behaviour. Standardized tests are used to assess children who are not doing well at school, and the data from these tests are used to underpin the need for special help for these children. Additionally, many neonatal intensive care units (NICUs) have follow-up programmes for premature children in which standardized assessments of different domains enable an overall evaluation of the child's development.\nWithin the framework of follow-up research we assessed a group of VPT and VLBW children at the age of 5\u00a0years in different developmental domains. Perinatal, social and economic data as well as information on school functioning were available. This provided the opportunity to compare school outcomes and developmental follow-up outcomes, assuming that developmental skills mediate the effects of biological and social risk factors on school performance [35]. We studied the degree of agreement between follow-up assessments and school outcomes as well as the most important characteristics of the children (perinatal, developmental and socio-economic) in this context. Our aim was to obtain a understanding of the (potential) developmental disturbances in order to facilitate an adequate and timely signalling of children who need special help to meet their developmental capacities.\nMethods\nStudy population\nThe study population consisted of 768 infants of less than 32\u00a0weeks of gestation and\/or weighing less than 1500 g who were born between October 1992 and December 1994 and treated in three Dutch neonatal intensive care units: the Radboud University Nijmegen Medical Centre, the Academic Medical Centre, Amsterdam, and the M\u00e1xima Medical Centre, Veldhoven. Mortality before the age of 5\u00a0years was 131 (17%). Forty-six children (6%) were excluded because they had participated in another study. Thirty-three children (4%) with severe or moderate cerebral palsy, blindness, mental retardation, chromosomal abnormalities, inborn errors of metabolism, personality developmental disorders and\/or attendance at special schools or institutions were excluded. Sixty children (8%) of non-Dutch parents were excluded from the analyses since school problems could be caused by language and cultural problems. As a result, 498 children (65%) qualified for the study.\nSchool performance and socio-economic situation\nA questionnaire was mailed to the parents of the participating children 1\u00a0month before follow-up assessments were to be carried out; these were returned when the family attended the outpatient clinic for the assessment. The paediatrician initiated the follow-up assessment by checking the questionnaire and exploring the answers with the family in more depth. Within the framework of the present study we analysed questions addressing school performance and educational level of the parents (low, middle, high). School outcome was defined in two categories: (1) normal (mainstream education without problems, which means no remedial teaching or other forms of extra help) or (2) problematic (mainstream education with remedial teaching or special mainstream education; i.e. schools for children with mild learning, behavioural or cognitive problems). The parents were asked to fill in the school items together with the schoolteacher. The educational level of the parents was used in this study as a measure of the social and economic situation (SES) because it is the best indicator in predicting school outcomes in The Netherlands [5].\nPerinatal data\nPerinatal data were prospectively collected during admission in the NICU, stored in the NICU databases and retrieved for data analysis. The following individual characteristics and perinatal variables were considered: gender, multiple birth, gestational age, birth weight, small for gestational age (SGA: birth weight below the 10th centile), low 5-min Apgar score (<7), intracranial haemorrhage grade 3 and 4 (ICH) [32], periventricular leucomalacia (PVL), bronchopulmonary dysplasia (BPD; oxygen dependency at 36\u00a0weeks postconceptional age or at 28\u00a0days of life), ante- or postnatal use of steroids and length of stay in NICU.\nAssessment at the age of 5\u00a0years\nClinical assessments were carried out on the health and neurological functioning of the children (not reported in this article) and four developmental domains: cognition, language, motor functioning and behaviour. A paediatrician, a child psychologist and a paediatric physical therapist assessed the children. Appointments were scheduled at random.\nCognitive development was assessed with the Revised Amsterdam Children\u2019s Intelligence Test (RAKIT) for children aged 4\u201311\u00a0years (short version). This version has a correlation of 0.93 with the full-scale test. The concurrent validity with the Wechsler Intelligence Scale for Children-Revised (WISC-R) is 0.86 for total IQ (Intelligent Quotient) [3].\nThis test included logical reasoning, word knowledge, visual-motor integration and word fluency. It also included visual synthesis for children younger than 5.2\u00a0years and visual analysis and memory for children aged 5.2\u00a0years or older. The norm score (IQ score) of the test is 100 with a standard deviation (SD) of 15. All scores higher or equal to 85 are classified as normal, while scores below 1 or 2 SD\u2019s are classified as mild or severe cognitive problems, respectively.\nThe Dutch Language Screening Test assessed language and speech. This test consists of 39 items covering the use of vocabulary, comprehension, memory and production of language, use of plurals, prepositions and pronunciations. The total score varies from 0 to 52, with a score \u226418 considered to be normal, that of 19\u201325 considered to indicate mild problems and that of >25 to indicate severe problems [7]. The Movement Assessment Battery for Children (Movement ABC) was used to assess motor skills. A total motor impairment score (range: 0\u201340) was computed by summing the scores on all motor tasks. A total score \u226410.5 (15th centile) was considered to be normal, from 11.0 through to 17.0 (5th centile) considered to indicate children at risk and >17.0 to indicate abnormal motor development [11, 23].\nTo assess behavioural outcomes, we used the Child Behaviour Checklist for children aged 4\u201318\u00a0years (CBCL). This is a standardized parental questionnaire used to describe the skills and behavioural problems of children. It comprises 113 descriptions of behavioural problems, with each description scored as: 0 (not true), 1 (somewhat or sometimes true) or 2 (very true or often true). A total problem score is obtained by summing all items. The severity of the behaviour problems can be the borderline and clinical cut-off point, corresponding with standardized norm scores of 60 and 63, respectively [1, 30]. A normal score is <60, a mild problematic score is \u226560 and \u226463, and a severe problem score is >63.\nStatistical analysis\nIn order to compare the results of the follow-up assessments and school outcomes, we cross-tabulated categorical data originating from the individual developmental test results of the children with school outcomes (normal or problematic). Normal follow-up outcomes were defined as four normal developmental scores or only one mild developmental problem score. Problematic follow-up outcomes were defined as two mild developmental problem scores, one severe developmental problem score or two or more severe developmental problem scores.\nCross-tabulation was also used to describe the relation between the follow-up assessment outcomes and the school outcomes and the nominal variables (chi-square for testing). A one-way analysis of variance was used to assess within each of the follow-up outcomes the differences between the two types of school outcomes on the interval variables (F test for testing). The general linear model (GLM) was used for multivariate testing of the differences between the two school outcome groups within each of the two follow-up outcome groups.\nResults\nOf the 498 children included in this follow-up study, 143 (29%) were not assessed for various reasons (Table\u00a01). Consequently, all outcome data, including developmental data, were collected on 355 children at the age of 5\u00a0years. No significant differences were found in perinatal data between assessed, partly assessed and unassessed children, with the exception of multiple births in that they were overrepresented in the assessed group [14]. \nTable\u00a01Eligible and assessed children\u00a0nPercentagenPercentageCohort 1992\u20131994768100\u00a0\u00a0Died13117\u00a0\u00a0Excluded because of participation in an other study466\u00a0\u00a0Excluded because of severe handicap and\/or in Special Schools334\u00a0\u00a0Children of non-Dutch parents608Eligible49865498100\u00a0\u00a0Address unknown255\u00a0\u00a0Moved outside the country51\u00a0\u00a0Treated in another hospital61\u00a0\u00a0Impossibility to make a convenient appointment296\u00a0\u00a0Refusal by the parents388\u00a0\u00a0Assessment not fully performed408Assessed35571\nMutual correlations between child characteristics and perinatal variables were present but rather low. The percentage of children with all follow-up assessment results being normal or with only one mild developmental problem score was 64% (n=228); 36% (n=127) of the children had at least two mild problems or one or more severe problems according to the four developmental tests. No school problems were reported for 222 children (63%), while 133 children (37%) were reported to school problems as reflected in the need for special help (remedial teaching or other forms of extra help or specialized mainstream education). One hundred and seventy-five children (49% of the study group) had normal school outcomes and normal follow-up outcomes and 80 children (23% of the study group) had problematic follow-up outcomes and problematic school outcomes. These results show that for 72% (n=255) of the children there was a positive relation between school outcomes and the results of the follow-up assessments. Fifty-three children (15% of the study group) had school problems, while their developmental test scores were normal, and 47 children (13% of the study group) did not receive any extra help at school, although they had test scores that fell in the problematic range. However, for 100 children (28%) there was no concordance between the school outcomes and follow-up assessment results (Table\u00a02). A significant difference (\u03c72=66.44, p=0.00) was found between the two school outcome groups, with more developmental problems in the school problem group. Children who received special help at school although they had obtained normal follow-up assessment results, differed significantly from the children with normal follow-up results without special assistance. This subgroup comprised more boys and SGA children who had lower Movement ABC scores (motor skills) and, in particular, lower language screening test scores. \nTable\u00a02Follow-up outcomes versus school outcomesSchool outcomes:Normal: n=222 (100%)Problematic: n=133 (100%)Total: n=355 (100%)Follow-up outcomes:\u00a0\u00a0Normal: n=228 (64%)\u00a0\u00a0\u00a0Four normal developmental scores124 (56%)26 (20%)\u00a0\u00a0\u00a0One mild developmental problem score51 (23%)27 (20%)\u00a0\u00a0Subtotal175 \u219249%53 \u219215%\u00a0\u00a0Problematic: n=127 (36%)\u00a0\u00a0\u00a0Two mild developmental problem scores8 (4%)10 (7%)\u00a0\u00a0\u00a0One severe developmental problem score36 (16%)54 (41%)\u00a0\u00a0\u00a0Two or more severe developmental problem scores3 (1%)16 (12%)\u00a0\u00a0Subtotal47 \u219213%80 \u219223%\nFor children who did not receive any extra help at school, even though problematic follow-up assessments were found, the results differed significantly from those who did receive extra help. This subgroup was less preterm, had higher Apgar scores, had had BPD less often and was treated less frequently with steroids during the neonatal period. Their scores on the Movement ABC, the language screening test and, in particular on the RAKIT (intelligence test) were better (Tables\u00a03 and 4). Within each of the follow-up assessment groups (normal or problematic assessment results) the MANOVA showed significant differences between the two school outcome groups (F=3.47, p<0.01, df=6.22 and F=3.29, p<0.01, df=6.12, respectively). \nTable\u00a03Means, standard deviations (SD) and one-way analyses of variance for normal and problematic school outcomes\u00a0Follow-up outcomesSchool outcomesNormalProblematicFMeanSDMeanSDGestational ageNormal30.081.8530.501.542.33Problematic30.692.0129.902.303.82*Birth weightNormal1286.24319.201314.28307.09.32Problematic1225.09306.431241.22364.36.06NICU stayNormal27.2022.3325.0716.53.42Problematic26.9120.8236.9437.172.89RAKIT IQNormal104.8211.77102.3910.641.83Problematic95.1712.1986.7412.9213.19***Movement ABCNormal6.114.317.814.236.44*Problematic15.307.7118.389.103.80*Language scoreNormal8.294.6810.785.7410.48***Problematic11.557.8615.488.486.73*CBCL Total problem scoreNormal47.228.8348.068.25.38Problematic55.3210.7055.0512.06.02*p<0.05, ***p<0.001Table\u00a04Comparison of child's characteristics, perinatal data and parents\u2019 education with normal and problematic school outcomes\u00a0Follow-up outcomesSchool outcomes\u03c72NormalProblematicPercentagePercentageMaleNormal42574.10*Problematic6463.01Multiple birthNormal35441.43Problematic3236.20SGANormal23374.29*Problematic4253.25Apgar scoreNormal1572.27Problematic47318.73*ICHNormal502.55Problematic46.21PVLNormal22.04Problematic261.09BPDNormal46.24Problematic0168.40**Neonatal steroidsNormal10.31Problematic0115.62*Parents education-highNormal4236.73Problematic39262.15Parents education-middleNormal4751.11Problematic3848.90Parents education-lowNormal1113.18Problematic2326.10*p<0.05, **p<0.01\nDiscussion\nThe developmental skills of children are largely expressed in school functioning [25]. Therefore, it would be expected that there is a great concordance between the assessments of the NICU follow-up teams and school teams with respect to the identification of children who need special assistance at school. The results of this study support this expectation with respect to children with good developmental outcome assessments and those with obvious problematic developmental outcomes. While the latter group mainly consisted of children with very low gestational ages and\/or serious perinatal problems, some were being faced with the direct adverse consequences of the use of neonatal steroids [36]. It is likely that the problems of these children and the genuine concern of parents and teachers has offered them the extra attention and assistance they required.\nChildren with relatively normal developmental outcomes who, notwithstanding this, received extra help at their schools, still had language and\/or motor problems that apparently interfered with school functioning, thereby causing schoolteachers to provide them with extra attention and help. The overrepresentation of boys in this group, as in both groups with problematic developmental outcomes, is a frequent finding [8] and because their behavior is often more demonstrative there is a stronger need to deal with. More children who were SGA were also found in this group. A number of these were still small at the age of 5\u00a0years, which may have led to overprotection or the conviction that the child needs more time or help to be ready for reading, writing or arithmetic [10, 21]. Further research should reveal if the problems of this latter group of children are attributable to not being ready for learning (pre) academic skills or to still being too playful, or whether it concerns serious learning and developmental problems.\nThe limited number of children with developmental problems who were not signalled by the school did not have special characteristics with the exception of having slightly better developmental outcomes and\/or having not been born extremely premature, having not been subjected to neonatal steroids and\/or having had a somewhat less complicated neonatal period. It is possible that their developmental problems did not interfere with school functioning or they were able to compensate for them, but it is also possible that their parents and\/or teachers did not signal their problems adequately. Parents are sometimes very relieved that the first difficult and worrisome years are over and unconsciously cut themselves off from new problems or both parents and teachers lower their expectations and demands because of overprotection. We did not find that less well-educated parents had more difficulties in mobilizing extra help for their children than relatively well-educated parents. Children of the former displayed developmental problems more often, but they received extra help and assistance in the same ratio as children of relatively more highly educated parents. Based on this line of reasoning a possible overrepresentation of children of less well-educated parents in the unassessed group would not change the degree of concordance between the follow-up results and school outcomes.\nOne may question whether our developmental tests were sensitive enough to register minor neurological signs. In the overall study the paediatrician did a neurological examination (the modified Touwen examination) [28]. This examination classified 120 (34%) of the children participating in this study with neurological problems (disabling cerebral palsy, non-disabling cerebral palsy, minimal neurological dysfunction and gross motor retardation). Ninety-five (93%) of these children were also classified as problematic by one or more of the other instruments. While these neurological data provide a more complete picture, they do not improve the degree of concordance between school outcome and follow-up outcome (70%).\nOf the preterm children tested at age 5\u00a0years, one-half were found to have problems in the developmental assessments or in school functioning. This outcome confirms the findings of others that follow-up research in more than one developmental domain in combination with school outcomes shows a more problematic outcome picture [29].\nSchools are doing quite well in identifying children with and without developmental problems, but the need for longitudinal multidisciplinary follow-up programmes in different developmental domains remains. Information that promotes the understanding of the development of these children during their school career provides schools and parents with more tools for early detection and will facilitate the design and evaluation of intervention programmes.","keyphrases":["school outcome","low birth weight children","premature children","school performance","developmental outcome","very preterm children"],"prmu":["P","P","P","P","P","R"]}
{"id":"Clin_Auton_Res-3-1-1797060","title":"The association of heart rate recovery immediately after exercise with coronary artery calcium: the coronary artery risk development in young adults study\n","text":"We tested whether slower heart rate recovery (HRR) following graded exercise treadmill testing (GXT) was associated with the presence of coronary artery calcium (CAC). Participants (n = 2,648) ages 18\u201330 years at baseline examination underwent GXT, followed by CAC screening 15 years later. Slow HRR was not associated with higher odds of testing positive (yes\/no) for CAC at year 15 (OR = 0.99, p = 0.91 per standard deviation change in HRR). Slow HRR in young adulthood is not associated with the presence of CAC at middle age.\nIntroduction\nThe prognostic value of slow heart rate recovery (HRR) after exercise in predicting cardiovascular disease (CVD) events has been established [5, 11, 12, 13, 15]. Initial increases in heart rate with exercise are due to parasympathetic withdrawal while sympathetic activation is responsible for heart rates greater than a 100\u00a0beats\/minute. In the first two minutes following cessation of exercise, the rapid decrease in heart rate is principally determined by parasympathetic reactivation [1, 8]. Although slow HRR is associated with less autonomic nervous system responsiveness, the underlying mechanisms linking slow HRR to increased cardiovascular morbidity are not well understood. It is possible that slow HRR is associated with a higher susceptibility for atherosclerosis. Prior studies of patients referred for cardiac angiography for suspected ischemic heart disease (IHD) suggest an association between slow HRR and higher atherosclerotic burden [15]. Further, slow HRR has been observed to be associated with several risk factors for atherosclerosis [2, 3, 10]. However, the relationship between HRR and atherosclerosis in the general population has not been studied using a prospective study design. In a cohort of healthy young adults, we investigated whether slow HRR following a graded exercise treadmill test (GXT) was associated with the presence of coronary artery calcium (CAC), a marker of subclinical atherosclerosis, when assessed 15\u00a0years later.\nMethods and statistical analysis\nThe Coronary Artery Risk Development in Young Adults study (CARDIA) is a longitudinal study designed to investigate the origins of cardiovascular disease in young adulthood [6]. Beginning in 1985, 5,115 African-American and Caucasian individuals [(African-American (52%) and women (54%)] ages 18\u201330 were recruited at sites in Birmingham, Alabama; Chicago, Illinois; Minneapolis, Minnesota; and Oakland, California. All participants gave informed consents prior to enrollment.\nAt baseline, a symptom-limited maximal GXT was administered using the Balke protocol [14]. The test included nine 2-minute stages of increasing difficulty with participants encouraged to exercise to exhaustion, followed by a recovery period at a speed of 3.2\u00a0km\/hour at 0% grade. HRR was defined as the difference between the maximum HR and HR at 2-minutes into recovery [10]. Participants were ineligible for exercise testing if they were on cardioactive medications, had a resting systolic or diastolic pressure >160 or >100\u00a0mmHg, or were febrile at time of examination. The rate of energy expenditure for the completion GXT was estimated and reported in metabolic equivalents (METs), as previously described [14]. Information on physical activity was collected by interview using a standardized questionnaire.\nAt the year 15 follow-up examination, returning participants (n\u00a0=\u00a03,043) underwent coronary artery computed tomography (CT) scanning for the measurement of CAC. Mean HRR did not differ between those who did and did not return to the year 15 examination (42.8 versus 42.5\u00a0bpm, respectively; p\u00a0=\u00a00.55). Details of the scanning procedures have been described elsewhere [4]. Briefly, using standardized protocols, two scans were obtained for each participant (1\u20132\u00a0minutes apart) using electron beam CT scanners at the Chicago and Oakland sites and multidetector-row CT scanners at the Birmingham and Minneapolis sites. Calcium scores were calculated across each coronary artery and then summed across all the arteries. The final CAC score of positive scans was calculated as the mean of the two CAC scores obtained from each of the scans.\nParticipants were sequentially excluded from this analysis for the following reasons: use of medications that affect heart rate (HR) (n\u00a0=\u00a027), unavailable GXT or CAC data (n\u00a0=\u00a0661), missing data on blood pressure, lipids, glucose, or smoking (n\u00a0=\u00a0353), pregnancy (n\u00a0=\u00a027) or those absent at the year 15 exam (n\u00a0=\u00a01,399). Following exclusions, 2,648 participants remained.\nBaseline characteristics were compared across sex-specific HRR tertiles. For continuous variables, test for linear trend was performed with HRR as a continuous variable using linear regression models. The Cochran-Armitage test was used to check for linear trend in binomial proportions across the HRR categories. Next, logistic regression was used to estimate the odds of the presence of CAC (defined as a CAC score >0) in relation to year 0 HRR (independent variable). HRR was modeled both as a continuous variable and in tertiles (fastest HRR tertile as the reference). Statistical significance was determined at P\u00a0<\u00a00.05. All analyses were conducted using SAS version 9.1 (SAS Institute Inc, Cary, NC).\nResults\nDemographic characteristics of the study sample, by 2-minute HRR tertiles, are presented in Table\u00a01. Mean 2-minute HRR (standard deviation) for men and women were 44.3 (11.4) and 41.7 (11.5)\u00a0bpm, respectively. Participants with slower HRR had less favorable GXT performance characteristics, a higher resting heart rate (both at year 0 and year 15) and reported less physically activity (both at year 0 and year 15).\nTable\u00a01Baseline characteristics according to tertiles of 2-minute heart rate recovery (N\u00a0=\u00a02,648)aBaselineBaseline sex-specific HRR tertiles1 (slow)23 (fast)P trendNumber of women479463521NANumber of men392391402NAHRR (median [min, max])Women31.0 (1.0, 36.0)41.0 (37.0, 45.0)51.0 (46.0, 116.0)NAMen34.0 (\u22126.0, 39.0)44.0 (40.0, 48.0)54.0 (49.0, 97.0)NAAge (years)25.4 (3.5)25.1 (3.6)25.0 (3.6)0.003African-American, N (%)360 (41.3)383 (44.9)435 (47.1)0.014Body Mass Index\u00a0kg\/m224.9 (5.2)24.1 (4.2)23.8 (4.0)<0.001Resting heart rate (bpm) at year 071.5 (11.4)69.3 (10.2)66.1 (9.7)<0.001Resting heart rate (bpm) at year 1568.3 (10.8)68.2 (12.1)66.5 (11.3)<0.001Maximum heart rate (bpm)180.1 (15.8)180.0 (14.6)181.0 (13.9)0.001Estimated METs at peak exercise11.8 (3.0)12.1 (2.7)12.5 (2.7)<0.001Total physical activity score (exercise units) at year 0375.8 (273.0)428.6 (317.5)459.7 (307.3)<0.001Total physical activity score (exercise units) at year 15325.4 (278.1)346.9 (288.0)382.4 (296.6)<0.001aValues are expressed as mean (standard deviation) unless otherwise indicated. HRR\u00a0=\u00a0heart rate recovery. NA\u00a0=\u00a0not applicable. METs\u00a0=\u00a0metabolic equivalents\nThe prevalence of CAC in the study sample was 9.0% (n\u00a0=\u00a0239). Mean HRR at year 0 did not differ between those who had positive CAC scores versus those who had a CAC score of 0 at year 15 (42.8\u00a0bpm, for both). The unadjusted odds ratio (OR) for having a CAC score >0 for those in the slowest HRR tertile compared to those in the fastest HRR tertile was not significantly greater than 1.00 (Table\u00a02). Similar findings were observed when HRR was studied as a continuous variable.\nTable\u00a02Unadjusted odds ratio (95% confidence interval) for the presence of coronary artery calcium (score >0) at year 15 by baseline 2-minute heart rate recoveryTotal sampleCaucasian menCaucasian womenAfrican-American menAfrican-American womenOdds Ratio (95% CI) for threshold models based on HRR tertilesTertile 1 (slow)1.13 (0.81\u20131.57)0.90 (0.54\u20131.50)1.12 (0.53\u20132.33)1.11 (0.52\u20132.36)1.44 (0.60\u20133.49)Tertile 21.20 (0.87\u20131.66)0.90 (0.54\u20131.50)0.90 (0.41\u20132.01)1.55 (0.78\u20133.09)1.37 (0.57\u20133.32)Tertile 3 (fast)1.00 (Ref)1.00 (Ref)1.00 (Ref)1.00 (Ref)1.00 (Ref)Odds Ratio (95% CI) for HRR as continuous variablePer SD change in HRRa0.99 (0.87\u20131.13)1.04 (0.84\u20131.28)0.86 (0.65\u20131.15)0.95 (0.70\u20131.30)0.88 (0.60\u20131.27)HRR\u00a0=\u00a0heart rate recovery. CAC\u00a0=\u00a0coronary artery calcium. CI\u00a0=\u00a0confidence interval. SD\u00a0=\u00a0standard deviation aSD of HRR was 11.5\u00a0bpm\nIn secondary analysis, similar findings were observed when the presence of higher CAC burden (defined as a score >100 [n\u00a0=\u00a034]) was studied, as well as when quartiles or quintiles of 2-minute HRR were analyzed. Lastly defining HRR at 1-minute into recovery also did not result in any association between HRR and CAC.\nConclusion\nSlower HRR in young adulthood is not associated with the presence of CAC when assessed 15\u00a0years later in middle age (average age 40\u00a0years). Moreover, mean HRR at baseline did not differ between those with and without measurable CAC at year 15. While previous studies have examined the relationship of HRR with CAD events this study is first to investigate the relationship of HRR with a measure of subclinical atherosclerosis.\nSlow HRR has been associated with higher incident all-cause mortality, sudden cardiac death (SCD), and CVD events; however, the underlying mechanisms that link these relationships together are not known [5]. Morshedi-Meibodi et al. [12] using data from the Framingham Heart Study (FHS) observed slow HRR to be associated with coronary heart disease events (defined as acute coronary syndromes or SCD), suggesting a possible relationship between slow HRR and ischemic processes. Similarly, slow HRR has been observed to be associated with several risk factors for atherosclerosis [2, 3, 10]. In contrast to findings from the FHS, Jouven et al. [9] observed slow HRR to be related only to SCD and not death from myocardial infarction. Due to over lapping and discrepancy in clinical end points used in prior HRR studies, it cannot be determined whether slow HRR is associated with atherosclerosis or an increased susceptibility to lethal cardiac arrhythmias.\nThe ability of coronary CT to assess the global burden of atherosclerosis in young adults is uncertain, and CAC is only a subset of atherosclerosis, representing calcified plaques which tend to be more stable than their lipid-rich counterparts [7]. Despite these limitations, our study suggests that slow HRR in young adulthood is not related to subclinical atherosclerosis at middle age, which supports the hypothesis that slow HRR is associated with mechanisms related to cardiac arrhythmia rather than atherosclerosis.","keyphrases":["heart rate","heart rate recovery","coronary artery calcium","autonomic nervous system","atherosclerosis","sudden cardiac death"],"prmu":["P","P","P","P","P","P"]}
{"id":"Biodegradation-3-1-2151777","title":"Substrate specificity of a long-chain alkylamine-degrading Pseudomonas sp isolated from activated sludge\n","text":"A bacterium strain BERT, which utilizes primary long-chain alkylamines as nitrogen, carbon and energy source, was isolated from activated sludge. This rod-shaped motile, Gram-negative strain was identified as a Pseudomonas sp. The substrate spectrum of this Pseudomonas strain BERT includes primary alkylamines with alkyl chains ranging from C3 to C18, and dodecyl-1,3-diaminopropane. Amines with alkyl chains ranging from 8 to 14 carbons were the preferred substrates. Growth on dodecanal, dodecanoic acid and acetic acid and simultaneous adaptation studies indicated that this bacterium initiates degradation through a Calkyl\u2013N cleavage. The cleavage of alkylamines to the respective alkanals in Pseudomonas strain BERT is mediated by a PMS-dependent alkylamine dehydrogenase. This alkylamine dehydrogenase produces stoichiometric amounts of ammonium from octylamine. The PMS-dependent alkylamine was found to oxidize a broad range of long-chain alkylamines. PMS-dependent long-chain aldehyde dehydrogenase activity was also detected in cell-free extract of Pseudomonas strain BERT grown on octylamine. The proposed pathway for the oxidation of alkylamine in strain BERT proceeds from alkylamine to alkanal, and then to the fatty acid.\nIntroduction\nPrimary fatty amines contain a nitrogen atom attached to one long alkyl chain. Commercial primary alkylamines are usually mixtures of homologs because the sources of the hydrophobic groups are fatty acids derived from palm oil, coconut oil or tallow. The alkyl chains therefore vary in both chain length and degree of unsaturation. Alkylamines are primarily introduced into the environment through emissions during industrial production. The alkylamines produced in high volumes are toxic to aquatic organisms (Newsome et\u00a0al. 1991; Schultz et\u00a0al. 1991; Finlay and Callow 1997). Microbial degradation of primary fatty amines is therefore important for removal in biological wastewater treatment systems and to maintain low environmental concentrations.\nYoshimura et\u00a0al. (1980) studied the biodegradability of various fatty amines in the MITI test. The MITI test assesses the ready biodegradability by determining the biological oxygen demand (OECD 1992). Ratios of the biological oxygen demand and theoretical oxygen demand >0.6 were achieved in MITI tests, demonstrating the susceptibility of alkylamines (C8 to C18) to biodegradation (Yoshimura et\u00a0al. 1980). Dodecylamine was also demonstrated to be biodegradable in another ready biodegradability test, i.e. Closed Bottle test (OECD 1992). The biodegradation of dodecylamine started immediately and reached \u223c80% within a week (van Ginkel et\u00a0al. 1995). The results obtained in ready biodegradability tests are far more satisfactory when supported by pure culture studies. Until now two pure cultures of bacteria have been shown to degrade primary fatty amines (Yoshimura et\u00a0al. 1980; Selig et\u00a0al. 1999). Possible intermediates of alkylamine degradation were not identified in these studies. Therefore, much remains to be learned about the substrate specificity of long-chain alkylamine degrading micro-organisms and the biodegradation pathway.\nThe aim of this study was to investigate how alkylamines are degraded by micro-organisms using a pure culture capable of utilizing alkylamines as sole carbon and energy source. Substrate specificities of the pure culture and cell-free extracts catalysing the initial degradation steps are reported.\nMaterials and methods\nChemicals\nSamples of octylamine, decylamine, dodecylamine, tetradecylamine, hexadecylamine, octadecylamine cocoamine and tallowamine (Armeen\u00ae) were provided by Akzo Nobel Surfactants, Stenungsund, Sweden. All other chemicals were of reagent-grade quality and obtained from Sigma Aldrich, Zwijndrecht, The Netherlands or Akcross Organics, Geel, Belgium. The biochemicals were purchased from Boehringer, Mannheim, Germany.\nActivated sludge\nActivated sludge used as inoculum was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This activated sludge plant treats predominantly domestic wastewater.\nMedia\nThe mineral salts medium used for isolation and growth experiments contained the following in 1\u00a0l of deionized water; 1.55\u00a0g K2HPO4, 0.85\u00a0g NaH2PO4, 0.5\u00a0g NH4Cl, 0.1\u00a0g MgSO4\u00a0\u00b7\u00a07H2O and 0.1\u00a0ml trace solution described by Vishniac and Santer (1957). The medium was sterilized by autoclaving for 20\u00a0min, together with any added growth substrate (1\u00a0g\u00a0l\u22121). The volatile primary amines, i.e. octylamine and decylamine were added after autoclaving the mineral salt medium. When these volatile primary amines were used as growth substrate, silica gel was added to give a final concentration of 32\u00a0g\u00a0l\u22121. Silica gel was added to reduce the concentration of alkylamines in the water phase.\nEnrichment, isolation and growth\nAerobic long-chain alkylamine degrading micro-organisms were isolated from an enrichment culture developed from activated sludge. The enrichment culture was obtained in batch culture flasks containing 200\u00a0ml mineral salts medium and 1.0\u00a0g\u00a0l\u22121 dodecylamine inoculated with 5\u00a0ml of activated sludge. After five subcultures a bacterium in this culture was streaked to purity on agar plates containing mineral salts medium, 1.0\u00a0g\u00a0l\u22121 dodecylamine and 15\u00a0g\u00a0l\u22121 agar. The cultures and agar plates were incubated at 30\u00b0C. The growth rate of strain BERT was recorded by measuring the increase in turbidity in culture flasks with a Hach Ratio XR turbidimeter (Hach, Loveland, CO, USA). The ability of the strain to grow on other substrates was tested using similar media in which dodecylamine was substituted with possible intermediates or other fatty amine derivatives.\nCulture conditions and preparation of washed cell suspensions and cell-free extracts\nCells were grown on octylamine as sole nitrogen, carbon and energy source in a continuous culture. This continuous culture was run at 30\u00b0C in a 2-l fermentor (Applikon, Schiedam, The Netherlands) with a working volume of 1.0\u00a0l. The impeller speed was 500\u00a0rpm. The mineral salt medium was pumped continuously into the fermentor at a rate of 1.0\u00a0l\u00a0day\u22121 by means of a peristaltic pump. Octylamine was introduced separately to the fermentor with a syringe pump and teflon tube giving a final concentration of 1.0\u00a0g\u00a0l\u22121. This set-up was required because of transport of the alkylamine through silicone tubes. Cells were also cultivated in the fermentor with acetate, octanal and octanoate under the same conditions but with a mineral salts medium containing the respective organic compounds. The pH of the reactor was maintained at 7.\nExperiments on the nitrogen balance were also conducted in the fermentor fed with a mineral salts medium without ammonium chloride. The nitrogen recovery was estimated from the amount of nitrogen leaving the fermentor as biomass, ammonium and total nitrogen. The total nitrogen represents the residual substrate and\/or water-soluble nitrogen-containing compound formed during the biodegradation process.\nCells were harvested from the continuous cultures by centrifugation at 25,000g for 10\u00a0min and washed three times with 15\u00a0mM phosphate buffer, pH 7.0. Washed cells (10\u00a0ml) were disrupted by a French pressure cell at 35,000\u00a0psi (Thermo IEC, Needham Heights, MA, USA) After one pass the insoluble matter was precipitated at 50,000g for 20\u00a0min.\nOxygen consumption\nThe endogenous and substrate-dependent oxygen uptake rates by washed cell suspensions were determined in a polarographic oxygen monitor (Yellow Springs Instruments, Yellow Springs, OH, USA). The polarographic oxygen monitor consisted of a thermostated vessel with a magnetic stirrer, and an oxygen electrode to measure oxygen depletion. Cells were resuspended in phosphate buffer at 30\u00b0C in a final volume of 5\u00a0ml. After 5\u00a0min necessary to determine the endogenous respiration, the substrate-dependent respiration was measured by injection of 0.1\u00a0ml of a 1.0\u00a0g\u00a0l\u22121 substrate solution into the vessel. Suspensions of alkylamines with more than ten carbons and octanal were heated prior to injection.\nEnzyme assays\nAll enzyme assays were performed at 30\u00b0C. Spectrophotometric enzyme assays were carried out in a Shimadzu UV 160A spectrophotometer (Shimadzu, Kyoto, Japan) in 1\u00a0cm light-path cuvettes. The oxygen uptake was measured with a polarographic oxygen monitor (Yellow Springs Instruments).\nAlkylamine dehydrogenase (phenazinium methyl sulfate (PMS)-dependent) was assayed by recording the oxygen uptake in a polarographic oxygen monitor. The reaction mixture (volume 5\u00a0ml) contained 0.8\u00a0mM PMS and 0.7\u00a0g\u00a0l\u22121 protein in a phosphate buffer (15\u00a0mM; pH 7). The reaction was started by injecting alkylamine in the reaction vessel at a concentration of 4.0\u00a0mM. Alkylamine dehydrogenase (PMS-dependent) activity was also determined by colorimetric detection of ammonium in a reaction vessel containing 8.0\u00a0mM octylamine, 0.8\u00a0mM PMS and 0.5\u00a0g\u00a0l\u22121 protein in a phosphate buffer (15\u00a0mM; pH 7). The enzyme assays were carried out at least twice and the difference in specific activity was less than 20%.\nAlkylamine dehydrogenase (NAD(P)-dependent) activity was spectrophotometrically assayed by measuring the increase of NAD(P)H at 340\u00a0nm. The reaction mixture consisted of 1.3\u00a0mM NAD(P), a phosphate buffer (15\u00a0mM; pH 7) and cell-free extract (1.4\u00a0g\u00a0l\u22121\u00a0protein) in a total volume of 3\u00a0ml. The alkylamine dehydrogenase (NAD(P)-dependent) activity was measured under anaerobic conditions to minimize NADH oxidase activity. For that purpose the contents of the cuvettes were stoppered with Suba Seal\u00ae septa (Sigma Aldrich, Zwijndrecht, The Netherlands) and flushed with nitrogen gas.\nAldehyde dehydrogenase activity was also measured by spectrophotometric and a respirometric assays. Aldehyde dehydrogenase (PMS-dependent) activity was assayed respirometrically. The reaction mixture (volume 5.0\u00a0ml) contained a phosphate buffer (15\u00a0mM; pH 7), 0.8\u00a0mM PMS, cell-free extract (0.7\u00a0g\u00a0l\u22121\u00a0protein). The reaction was started by injecting octanal in the reaction vessel giving 4.0\u00a0mM.\nAldehyde dehydrogenase (NAD(P)-dependent) activity was determined by measuring the increase in absorbance at 340\u00a0nm. Reaction mixtures contained phosphate buffer (15\u00a0mM; pH 7), 0.15\u00a0\u03bcM aldehyde, 1.0\u00a0mM NAD and 0.1\u00a0mM KCN and enzyme solution in a volume of 3\u00a0ml.\nAnalyses\nAmmonium was determined colorimetrically by forming indophenol blue with hypochlorite and salicylate in the presence of sodium nitroferricyanide as catalyst (Verdouw et\u00a0al. 1978).\nProtein was quantified by bicinchoninic acid method. Cells were first lysed by incubating at 95\u00b0C with 1.0\u00a0M NaOH. The protein concentration was estimated by using the Bio-Rad Protein assay kit with bovine serum albumin as standard protein. Dissolved organic carbon and total nitrogen were quantified with a Shimadzu TOC apparatus (Shimadzu).\nOctylamine was analysed by ion chromatography with a Dionex DX-120 (Dionex, Sunnyvale, CA, USA). The Dionex DX-102 was equipped with an IonPac CS14 (4\u00a0mm) analytical column, a 25\u00a0\u03bcl sample loop, a CSRS-I in the external water mode and a CDM-3 flow-through conductivity cell with a DS4 detection stabilizer. The DX-120 was operated at a column temperature of 20\u00b0C and a detector temperature of 35\u00b0C. The eluent was deionized water with 5.0\u00a0mM methylsulphonic acid and 2.5% acetonitrile. The eluent flow rate was 1.0\u00a0ml\u00a0min\u22121.\nThe dry weight of the micro-organisms was determined by washing and concentrating a known volume from the continuous culture through centrifugation (25,000\u00a0g for 30\u00a0min). The concentrated biomass on a preweighed watch glass was dried for 1.5\u00a0h at 104\u00b0C.\nResults\nIsolation and characterization\nA culture capable of growth on dodecylamine was readily enriched from activated sludge through repeated transfers of cells to fresh mineral medium containing dodecylamine. Dodecylamine plates streaked with dilutions of this enrichment culture enabled the isolation of bacteria. The predominant isolate BERT was a Gram-negative, motile, non-spore-forming rod, 2.0\u20133.5\u00a0\u03bcm long and 0.6\u20130.8\u00a0\u03bcm wide. Oxidase, catalase, urease and alcohol dehydrogenase were present. The strain was able to utilize the following substrates: glucose, phenylacetate, citrate, malate, mannose mannitole and gluconate. It did not grow on maltose, trehalose, m-inositol, citraconate, erytritol, sorbitol, d-xylose, d-tartrate and l-arabinose. The strain was not capable of reducing nitrate. The identity of the primary alkylamine degrading bacterium was determined by the profile of cellular fatty acids and partial 16S rRNA gene sequencing. The profile of the cellular fatty acids is typical for the RNA group I of the genus Pseudomonas. The partial sequences have shown a similarity of 99.3% to Pseudomonas putida, P. plecoglosscida and P. alcaligenes. These characteristics only allow to place strain BERT within the RNA group I of the genus Pseudomonas.\nGrowth\nA number of fatty amine derivatives were tested for their ability to support growth of Pseudomonas strain BERT. The strain was found to be able to grow on a wide range of long-chain primary amines, i.e. octylamine (in the presence of silica gel), decylamine (in the presence of silica gel), dodecylamine, tetradecylamine, octadecylamine, cocoamine oleylamine and tallowamine as sole source of carbon. Coco-1,3-diaminopropane and dodecyl-1,3-diaminopropane\u2014both in the presence of silica gel\u2014were also used as carbon and energy source by Pseudomonas strain BERT whereas dodecyldimethylamine, didodecylamine, didodecymethylamine, dodecyltrimethylammonium chloride did not support growth. Mineral salts media amended with possible intermediates of dodecylamine degradation such as dodecanal, dodecanoic acid and acetate supported growth of Pseudomonas strain BERT.\nDepletion of dodecylamine to non-detectable concentrations from the nitrogen-free mineral salts medium in batch cultures and the concurrent growth of the isolate clearly demonstrate that this isolate also used alkylamines as its nitrogen source (data not shown).\nOctylamine was used as growth substrate in a number of experiments because this amine is soluble in water at a concentration of 1.0\u00a0g\u00a0l\u22121. Use of octylamine allowed, for instance, accurate measurement of the increase in turbidity due to growth. From the growth curve, a doubling time of 4\u00a0h was estimated (data not shown). Growth yield obtained with Pseudomonas strain BERT was 0.4\u00a0g dry weight of cells per g of octylamine utilized. The fate of the nitrogen of octylamine in terms of ammonium, biomass-nitrogen, nitrogen-containing soluble microbial products and residual octylamine was assessed in a continuous culture. Total nitrogen, ammonium and biomass-nitrogen were measured in triplicate. Approximately 25\u00a0\u00b1\u00a03% of the nitrogen was recovered as biomass-nitrogen. Octylamine-nitrogen was converted for 60\u00a0\u00b1\u00a04% into ammonium. Less than 1% of the nitrogen in the effluent was present as octylamine. Approximately 10\u00a0\u00b1\u00a02% of the nitrogen was recovered as water-soluble nitrogen-containing products.\nRespiration experiments\nThe alkylamine-degrading isolate was grown on a number of different substrates as sole source of carbon after which respiration rates of washed cell suspensions were examined with a variety of substrates in order to identify possible intermediates of the degradation pathway (Table\u00a01). Washed cell suspensions of Pseudomonas strain BERT grown with octylamine were capable of oxidizing octylamine, octanoate octanal and acetate. Whole cells of Pseudomonas strain BERT also showed octylamine-dependent oxygen uptake when grown with octanal, and octanoate. However, strain BERT grown with acetate did not respire octylamine. Finally, Pseudomonas strain BERT grown on octanal, octanoate and actate displayed significant activities with the alkanal and alkanoate tested (Table\u00a01).\nTable\u00a01Oxidation of various potential intermediates of alkylamine degradation by washed cell suspensions of strain BERT grown on octylamine, octanal, octanoate and acetateSubstrateGrowth substrateOctylamineOctanalOctanoateAcetatenmol\u00a0min\u22121\u00a0mg\u22121\u00a0proteinOctylamine8525440Octanal6243660Octanoate84539041Acetate39354150Rates of oxygen uptake are expressed as nmoles\u00a0O2\u00a0min\u22121\u00a0mg\u22121 protein after correction for endogenous respiration. The endogenous respiration of the strain grown on octylamine, octanal, octanoate and acetate were 8, 4, 8 and 6\u00a0nmol\u00a0O2\u00a0min\u22121\u00a0mg\u00a0protein\u22121, respectively\nThe ability of octylamine-grown strain BERT to oxidize alkylamines was also evaluated (Table\u00a02). Octylamine-grown cells were capable of oxidizing all alkylamines with alkyl chains ranging from 3 to 18 carbon atoms. The highest activity was observed when hexylamine, octylamine and decylamine were added to the reaction vessel. The oxidation rate of nonylamine was comparable to the activity found with octylamine. Propylamine was respired at a low rate. Low activities were also detected with alkylamines with more than 14 carbon atoms. All other fatty amine derivatives except for dodecyl-1,3-diaminopropane were not oxidized by strain BERT grown on octylamine (Table\u00a02).\nTable\u00a02Oxidation of various alkylamines by washed cell suspensions and alkylamine dehydrogenase (PMS-dependent)SubstrateWashed cell suspensionCell-free extractnmol\u00a0min\u22121\u00a0mg\u22121\u00a0proteinButylamine145Hexylamine8830Octylamine8533Decylamine7634Dodecylamine6731Tetradecylamine5735Hexadecylamine3118Octadecylamine125Nonylamine8535Propylamine73Methylamine00Didecylamine00Decyldimethyamine00Decyltrimethylammonium 00Dodecyl-1,3-diaminopropane165Rates of oxygen uptake are expressed as nmoles\u00a0O2\u00a0min\u22121\u00a0mg\u22121\u00a0protein. The rates for the washed cell suspensions are corrected for the endogenous respiration of octylamine-grown cells, i.e. 8\u00a0nmol\u00a0O2\u00a0min\u22121\u00a0mg\u00a0protein\u22121\nEnzymatic activities\nActivities of enzymes that may be involved in alkylamine metabolism were tested in cell-free extracts prepared from cells grown on octylamine. Extracts of octylamine\u2013grown cells released ammonium in stoichiometric amounts from octylamine in the presence of PMS (Fig.\u00a01). In another experiment the presence of PMS induced the consumption of 0.08\u00a0mM oxygen upon the addition of 0.17\u00a0mM octylamine. The activity of the PMS-dependent alkylamine dehydrogenase with dodecylamine was 31\u00a0nmol\u00a0min\u22121\u00a0mg\u22121\u00a0protein. NAD and NADP could not replace the artificial electron acceptor. The substrate specificity of cell-free extracts catalysing the conversion of alkylamines was studied by measuring the oxygen consumption in the presence of PMS. The enzyme was active towards alkylamines with alkyl chains with 8\u201318 carbons. Propylamine, nonylamine and dodecyl-1,3-diaminopropane also acted as substrate for the enzyme. Among the primary alkylamines examined, the most preferred were alkylamines with alkyl chains length ranging from 8 to 12 carbons. Methylamine secondary, tertiary and quaternary fatty amine derivatives were no adequate substrates (Table\u00a02). Demonstration of alkanal dehydrogenase activity in cell extracts was achieved by incubating cell-free extracts with octanal and PMS and measuring the oxygen consumption. An activity of 19\u00a0nmol\u00a0O2\u00a0min\u22121\u00a0mg\u22121 protein was measured. Alkanal dehydrogenase activity was not detected with other electron acceptors, i.e. NAD and NADP, despite repeated attempts.\nFig.\u00a01Formation of ammonium from 0.78\u00a0mM octylamine by cell-free extract of octylamine-grown cells in the presence (open square) and absence (filled square) of PMS. The protein concentration in the assay was 0.5\u00a0g\u00a0l\u22121\nDiscussion\nThe fate of long-chain alkylamines in the environment is largely dependent on the ability of micro-organisms to metabolize these compounds. Although degradation of alkylamines in OECD ready biodegradability tests has been observed (Yoshimura et\u00a0al. 1980; OECD 1992; van Ginkel et\u00a0al. 1995), little is known about the intermediates in the biodegradation process and the enzymes involved in the biodegradation. In this paper, we describe the properties of the newly isolated Pseudomonas strain BERT, which is capable of degrading long-chain alkylamines. Yoshimura et\u00a0al. (1980) also isolated a Pseudomonas strain. Both Pseudomonas strains degraded primary alkylamines with varying alkyl chain lengths. Pseudomonas species are noted for their metabolic diversity and are often isolated from enrichments designed to identify bacteria that degrade anthropogenic organic compounds. Another species known to utilize a long-chain alkylamine as sole source of carbon and energy is from the genus Rhodococcus isolated from top soil (Selig et\u00a0al. 1999).\nA nitrogen mass balance obtained in a continuous culture already, strongly indicated that octylamine a water soluble long-chain alkylamine is completely (ultimately) biodegradable because almost all of the octylamine-nitrogen was converted into biomass-nitrogen and ammonium. Other evidence of complete degradation of alkylamines has been obtained through the proposed biodegradation pathway. The biodegradation of alkylamines was assumed to proceed either via a Calkyl\u2013N cleavage or oxidation at the far-end of the alkyl chain. Based on the induction pattern, and PMS-dependent dehydrogenase activity with alkylamines, the biodegradation of alkylamines is suspected to involve a cleavage of the Calkyl\u2013N bond, which yield the respective alkanals and ammonium. The alkanals are oxidized by a PMS-dependent alkanal dehydrogenase to the respective fatty acids. The induction of long-chain alkanal dehydrogenase activity by growth of the bacterium on octylamine demonstrates that alkanals are significant intermediates. A PMS-dependent long-chain alkanal dehydrogenase was also detected in Acinetobacter calcoaceticus HO1-N grown on hexadecane and hexadecanol (Fox et\u00a0al. 1992). At present it is unknown if both the alkanal and the alkylamine are dehydrogenated by the same enzyme in Pseudomonas strain BERT. Fatty acids produced by the alkanal dehydrogenase are channelled into the \u03b2-oxidation cycle (Ratledge 1994). The degradative pathway shown in Fig. 2 is inducible because octylamine is not oxidized by acetate-grown cells (Table\u00a01). The degradation pathway is quite similar to those described for alkyltrimethylammonium salts, alkyldimethylamines and alkylbis(2-hydroxyethyl)amines (van Ginkel 1996, 2003). This biodegradation pathway provides evidence of total mineralization of alkylamines as indicated by the nitrogen mass balance and the ready biodegradability test results (Yoshimura et\u00a0al. 1980; van Ginkel et\u00a0al. 1995).\nFig.\u00a02Proposed pathway for the degradation of alkylamines in Pseudomonas strain BERT; (1) alkylamine dehydrogenase, (2) aldehyde dehydrogenase. The fatty acids formed are channelled into the \u03b2-oxidation cycle\nPMS, an artificial electron acceptor is generally used to assay enzymes known as quinoprotein enzymes. Physiological electron acceptors are cytochrome c, protein-bound haem and ubiquinone. Methylamine dehydrogenases (de Beer et al. 1980; Duine et\u00a0al. 1990), short-chain alkylamine dehydrogenases (Shinagawa et\u00a0al. 1988) and a long-chain alkanal dehydrogenase (Fox et\u00a0al. 1992) have been identified as quinoproteins. Methylamine dehydrogenases catalyse the oxidative deamination of methylamine to methanal and ammonium (McIntire et\u00a0al. 1990). The PMS dependent removal of ammonium from alkylamines in strain BERT may be catalysed by an enzyme similar to methylamine dehydrogenases although no activity towards methylamine was detected (Table\u00a02). Zhu et\u00a0al. (2000) demonstrated that the conversion of one amino acid of methylamine dehydrogenase altered the substrate preference of methylamine dehydrogenase to a large extent. The preferred substrates of the mutated enzyme are alkylamines with at least seven carbons. Long-chain alkylamines are probably also oxidized by an alkylamine dehydrogenase isolated from a butylamine-grown Pseudomonas sp. although the highest activity was found with butylamine (Shinagawa et\u00a0al. 1988). A quinoprotein was also found in a P. putida catalysing the deamination of propylamine and butylamine both naturally occurring alkylamines (Adachi et\u00a0al. 1998). Only a long-chain alkylamine dehydrogenase is required to convert anthropogenic alkylamines into compounds, which can enter an existing pathway. This enzyme may have evolved from methylamine dehydrogenases or short-chain alkylamine dehydrogenases involved in the degradation of naturally occurring amines.\nA major feature of Pseudomonas strain BERT is its broad substrate specificity with respect to primary alkylamines. Activities of washed cell suspensions and alkylamine dehydrogenase activities in cell-free extracts demonstrate that alkylamines with alkyl chain lengths ranging from 6 to 10 carbons are preferred. Odd alkyl chain amines were also respired by Pseudomonas strain BERT. Water-insoluble long-chain alkylamines are oxidized at a lower rate by washed cell suspensions and alkylamine dehydrogenase of strain BERT. This probably reflects the low bioavailability of the long-chain alkylamines. Limited bioavailability of long-chain alkylamines (C12 to C18) can also be concluded from the non-toxicity of the alkylamines to strain BERT at a concentration of 1.0\u00a0g\u00a0l\u22121. In batch cultures growth of Pseudomonas strain BERT on octylamine and decylamine was only possible in the presence of silica gel. Silica gel reduces the toxicity of fatty amine derivatives through adsorption onto the silica gel (van Ginkel et\u00a0al. 1992; Selig et\u00a0al. 1999).\nThe ease with which Pseudomonas strain BERT was isolated and the high growth rate of strain BERT are consistent with studies showing ready biodegradability of many primary amines (Yoshimura et\u00a0al. 1980; van Ginkel et\u00a0al. 1995). Based on the biodegradation pathway and the broad substrate specificity, significant differences in the biodegradability of alkylamines with varying chain lengths are not expected. However, degradation of primary alkylamines does differ in ready biodegradability tests (Yoshimura et\u00a0al. 1980). This can be explained by biocidal effects and limited bioavailability. Reading across of the ready biodegradability test results enabled by this study reveals that all long-chain alkylamines\u2014both saturated and unsaturated\u2014are readily biodegradable. Future research should focus on identifying the intermediates and purifying the enzymes involved in the degradation of alkylamines.","keyphrases":["substrate specificity","long-chain alkylamines","pseudomonas sp","biodegradation pathway"],"prmu":["P","P","P","P"]}
{"id":"J_Struct_Funct_Genomics-3-1-1769342","title":"Coverage of whole proteome by structural genomics observed through protein homology modeling database\n","text":"We have been developing FAMSBASE, a protein homology-modeling database of whole ORFs predicted from genome sequences. The latest update of FAMSBASE (http:\/\/daisy.nagahama-i-bio.ac.jp\/Famsbase\/), which is based on the protein three-dimensional (3D) structures released by November 2003, contains modeled 3D structures for 368,724 open reading frames (ORFs) derived from genomes of 276 species, namely 17 archaebacterial, 130 eubacterial, 18 eukaryotic and 111 phage genomes. Those 276 genomes are predicted to have 734,193 ORFs in total and the current FAMSBASE contains protein 3D structure of approximately 50% of the ORF products. However, cases that a modeled 3D structure covers the whole part of an ORF product are rare. When portion of an ORF with 3D structure is compared in three kingdoms of life, in archaebacteria and eubacteria, approximately 60% of the ORFs have modeled 3D structures covering almost the entire amino acid sequences, however, the percentage falls to about 30% in eukaryotes. When annual differences in the number of ORFs with modeled 3D structure are calculated, the fraction of modeled 3D structures of soluble protein for archaebacteria is increased by 5%, and that for eubacteria by 7% in the last 3 years. Assuming that this rate would be maintained and that determination of 3D structures for predicted disordered regions is unattainable, whole soluble protein model structures of prokaryotes without the putative disordered regions will be in hand within 15 years. For eukaryotic proteins, they will be in hand within 25 years. The 3D structures we will have at those times are not the 3D structure of the entire proteins encoded in single ORFs, but the 3D structures of separate structural domains. Measuring or predicting spatial arrangements of structural domains in an ORF will then be a coming issue of structural genomics.\nIntroduction\nGenome sequencing projects provided a huge number of amino acid sequences without functional information (Stein 2001). To discover biological functions of those proteins, both computational predictions and biochemical experiments are necessary (Tsoka and Ouzounis 2000). Most of the proteins perform functions after forming specific 3D structures, and therefore protein 3D structure is one of the most valuable sources of information to predict protein function (Domingues et\u00a0al. 2000; Xie and Bourne 2005). Protein function prediction based on 3D structures, especially protein surface structures, with evolutionary and\/or physicochemical characteristics have been extensively studied (Lichtarge and Sowa 2002; Campbell et\u00a0al. 2003; Kinoshita and Nakamura 2003; Laskowski et\u00a0al. 2003; Ota et\u00a0al. 2003; Pieper et\u00a0al. 2006). However, determining protein structures of all the function-unknown proteins for applying these types of study is not practical.\nProteins are classified into a large number of \u2018families\u2019 based on the amino acid sequence similarity (Dayhoff 1972), and proteins with similar amino acid sequences are known to have similar 3D structures (Chothia and Lesk 1986), all because the proteins in a family are evolutionary related (Doolittle 1995). Once we have 3D structure of at least one of the proteins in a family, then 3D structures of other proteins in the same family can be computationally deduced by \u2018homology modeling\u2019 (Burley 2000; Baker and Sali 2001). Based on this logic, structural genomics (SG) projects, which are to determine protein 3D structures of representatives for each family have been proposed and launched (Vitkup et\u00a0al. 2001; Brenner 2000; Burley and Bonnano 2002). In homology modeling, corresponding residues between an amino acid sequence of structure unknown protein (target) and that of 3D structure known protein (template) in the same family are determined by sequence alignment and every residue in a template protein is replaced by that in a target protein (Marti-Renoma et\u00a0al. 2000).\nSG projects have been providing new protein structures (Todd et\u00a0al. 2005; Xie and Bourne 2005; Chandonia and Brenner 2006). Protein Data Bank (PDB) (Berman et\u00a0al. 2000) now contains more than 390 3D structures for function unknown or hypothetical proteins (Stark et\u00a0al. 2004). Protein function predictions based on 3D structures determined by SG projects are also in progress (Goldsmith-Fischman and Honig 2003; Liu et\u00a0al. 2005; Petrey and Honig 2005). There are some projects that focus on a specific species and try to determine the 3D structures of whole proteins encoded in the genome of the species (Kim 2000; Yokoyama et\u00a0al. 2000; Kim et\u00a0al. 2003). Those projects provide a considerable number of 3D structures in a single protein family. This results in providing multiple templates for a single protein family and it can improve quality of homology modeling (Contreras-Moreira et\u00a0al. 2003).\nWe have developed FAMSBASE; a database for homology modeling 3D structures of whole proteins predicted on whole genome sequences, since 2001 (Yamaguchi et\u00a0al. 2003; http:\/\/daisy.nagahama-i-bio.ac.jp\/Famsbase\/). FAMSBASE contains results of homology modeling by FAMS, a full automatic modeling software (Ogata and Umeyama 2000). Sequence alignments between whole ORFs and proteins in PDB are based on GTOP (Kawabata et\u00a0al. 2002).\nWe report here the update of the database including differences in the amount of structural data from the previous version, estimation of the time that whole ORFs predicted out of genome sequences are covered by homology modeling 3D structures and upcoming issues for utilizing those modeled structures.\nMethods\nData update of FAMSBASE\nCorrespondence between ORFs derived from whole genome sequences and protein amino acid sequences whose 3D structures are known is provided by GTOP database (Kawabata et\u00a0al. 2002). The update in May 2005 of FAMSBASE is based on February 2004 version of GTOP. Protein 3D structures in PDB by November 2003 are used for homology modeling templates. FAMS (Ogata and Umeyama 2000) is applied by Umeyama et\u00a0al. to pair-wise alignments between a predicted ORF sequence and an amino acid sequence with known 3D structure, and a 3D structure is modeled. All the results are stored in FAMSBASE.\nAssessing annual difference of data in FASBASE\nBased on the amount of data in FAMSBASE in 2001 and the amount of increase in the following years, a due year for whole proteome 3D structure models is estimated. Estimation is done residue-wise, not ORF-wise, since modeled structures in FAMSBASE are often limited to structural domains. In this report, structural domains refer to SCOP domains (Andreeva et\u00a0al. 2004). All ORFs predicted out of genome sequences are divided into soluble and membrane proteins. The division is carried out by SOSUI (Hirokawa et\u00a0al. 1998), and a protein with one or more transmembrane regions is classified into a membrane protein. The number of residues of whole soluble proteins encoded in the genome sequence (G) of species i is denoted as S, and the number of residues of whole membrane proteins is denoted as M. The number of residues included in modeled 3D structures of soluble and membrane proteins are denoted as S3 and M3, respectively. For a certain genome Gi, the coverage of modeled 3D structures in whole soluble proteins is then S3\/S\nand the coverage for whole membrane proteins is M3\/M. The coverage is summarized in different kingdoms of life as in the following equations;\nBoth figures are calculated based on the data at the different times of FAMSBASE update, gradients in figures are then calculated, and the figures are extrapolated up to the year that coverage reaches to 100.\nIt is getting to be known that not all ORFs assume stable 3D structures. Some parts of ORFs are considered to be natively disordered (Oldfield et\u00a0al. 2005; Dyson and Wright 2005). Hence it is unlikely that coverage by homology modeling reaches to 100. We, therefore, estimate disordered regions in whole ORFs by DisEMBL (Linding et\u00a0al. 2003) and omit these disordered regions from the calculation.\nNon-overlap multiple model structures in single ORFs\nModeled 3D structures in FAMSBASE are often limited to structural domains. To find an ORF of which most of the entire 3D structure is modeled in pieces of structural domains, an ORF covered by non-overlapping three or more modeled 3D structures in eukaryotic genome is surveyed based on the following criteria; (1) 70% or more residues in the ORF are included in one of the modeled 3D structures, (2) the ORF contains three or more non-overlapping modeled structures, and (3) the sequence identity between a template protein and a target domain is no less than 25%. At the time of FAMSBASE building, five model structures are at most built for each ORF (Yamaguchi et\u00a0al. 2003). Therefore, the expected number of modeled structures in the above criteria is between three and five.\nPrediction of domain interfaces\nThe 3D structure in pieces for a single ORF needs to be assembled to model the entire 3D structure. For this procedure, a prediction of domain interfaces of each 3D structure is needed. A hydrophobicity index based on protein 3D structures is built for domain interface prediction. Hydrophobicity of amino acid residue is measured by buriedness of a residue inside the protein 3D structures. A representative 4,529 chains in PDB among which sequence identities are less than 30% were selected and solvent accessibility of each residue is calculated on a monomer state. For each amino acid residue type i (i\u00a0=\u00a01,...,20), the number of residue with accessibility no less than b (=0.0\u00a0\u2212\u00a01.0) is counted (Sb,i). Database derived hydrophobicity index (Ib,i) is obtained by;\nb is set to 0.15 to maximize the difference of Ib,i among different residues. The index has good correlation with Kyte and Doolittle hydrophobicity index (Kyte and Doolittle 1982). The index I0.15,i is assigned to every residue on the surface (accessibility no less than 0.15) of a modeled 3D structure. The hydrophobicity of each residue on a surface of a protein is then obtained by averaging the assigned values of residues within 7.0\u00a0\u00c5 from the residue in concern. A hydrophobic patch on the surface of the modeled structure is found as a cluster of surface residues with the hydrophobicity no less than 0.0.\nResults and discussion\nCoverage of whole protein space by homology modeling\nThe latest update of FAMSBASE at May 2005 uses protein 3D structures deposited to PDB by the end of Nov. 2003 and ORFs predicted from genome sequences deposited by February 2004 (http:\/\/daisy.nagahama-i-bio.ac.jp\/ Famsbase\/). The latest FAMSBASE contains 1,396,272 modeled 3D structures of 368,724 ORFs derived from 17 archaebacterial, 130 eubacterial, 18 eukaryotic and 111 phage genomes; in total 276 genomes. Five models at maximum are built for each ORF in FAMSBASE. Those five models are the structure for the same or different regions in the ORF. When multiple models are built for the same region of ORF, we can evaluate the reliability of the model. When the model based on different templates have the similar 3D structures, then the 3D structure would be reliable. When the structures are different, the modeled structure would be less reliable. We further test the quality of modeled 3D structure by ProsaII (Sippl 1993) and find that about 72% of the modeled 3D structures are energetically ranked as number one and comparable to experimentally determined 3D structures. Some of the structures that fail the test are structures of a part of a large protein, mostly structural domains of large proteins. It is difficult to assess the quality of this type of domain structures, because interfaces of the domain for other parts of the protein are exposed in the modeled structures. Tendency of amino acid residue appearance in the interface is supposed to be different from that at the surface as we discuss down below.\nIn the genome of 276 species, 734,193 ORFs are predicted. Therefore, in FAMSBASE, 3D structure of 50% (368,724\/734,193) of ORFs have been built and stored (Table\u00a01). These are about 47% of ORFs in archaebacterial genomes, about 52% in eubacterial genomes and about 49% of eukaryotic genomes.Table\u00a01Number of ORFs and those with modeled 3D structures in 276 genomesSpeciesORFModel%ArchaeaArchaeoglobus fulgidus DSM43042,4071,23351.2Aeropyrum pernix K12,69478929.3Halobacterium sp. NRC-12,6051,19545.9Methanosarcina acetivorans C2A4,5442,12446.7Methanocaldococcus jannaschii DSM26611,77087549.4Methanopyrus kandleri AV191,68778446.5Methanosarcina mazei Goe13,3711,63448.5Methanothermobacter thermautotrophicus1,86999853.4Nanoarchaeum equitans Kin4-M53626449.3Pyrococcus abyssi Orsay1,78494252.8Pyrobaculum aerophilum IM22,6051,04740.2Pyrococcus furiosus DSM 36382,0651,03550.1Pyrococcus horikoshii OT32,06187942.6Sulfolobus solfataricus P22,9941,36545.6Sulfolobus tokodaii 72,8261,22843.5Thermoplasma acidophilum DSM17281,47884457.1Thermoplasma volcaniumGSS11,52683955.0sum38,82218,07546.6EubacteriaAquifex aeolicus VF51,55392959.8Nostoc sp. PCC 71206,1322,76545.1Agrobacterium tumefaciens C585,3013,01756.9A. tumefaciens C58 (Dupont)5,4023,02856.1Bacillus anthracis str. Ames5,3112,46346.4Buchnera aphidicola Sg55241074.3B. aphidicola50738575.9Bordetella bronchiseptica RB504,9942,93458.8Borrelia burgdorferi1,63953532.6Bacillus cereus ATCC 145795,2552,53448.2Candidatus Blochmannia floridanus58344776.7Bacillus halodurans C-1254,0662,12752.3Bradyrhizobium japonicum8,3174,44953.5Bifidobacterium longum NCC27051,73198556.9Brucella melitensis 16M3,1981,80156.3Bordetella parapertussis4,1852,52560.3B. pertussis Tohama I3,4472,17963.2Bacillus subtilis 1684,1062,15352.4Brucella suis 13303,2641,67751.4Bacteroides thetaiotaomicron VPI-54824,8162,46251.1Buchnera sp. APS57443676.0Clostridium acetobutylicum ATCC8243,8482,05353.4Coxiella burnetii RSA 4932,04592545.2Chlamydophila caviae GPIC1,00550550.2Caulobacter crescentus3,7372,08455.8Corynebacterium diphtheriae NCTC131292,2721,16551.3Corynebacterium efficiens YS-3142,9981,51350.5Corynebacterium glutamicum ATCC 130323,0991,55450.1Campylobacter jejuni1,63489354.7Chlamydia muridarum Nigg91148353.0Clostridium perfringens 132,7231,47054.0Chlamydophila pneumoniae AR391,11649544.4Chlamydophila pneumoniae CWL0291,05249647.1Chlamydophila pneumoniae J1381,06950146.9Chlamydophila pneumoniae TW-1831,11350145.0Chlorobium tepidum TLS2,2521,16651.8Clostridium tetani E882,4321,30653.7Chlamydia trachomatis D\/UW-3\/CX89448554.3Chromobacterium violaceum ATCC 124724,3852,34353.4Deinococcus radiodurans R13,1021,57950.9Escherichia coli K-12 MG16554,2842,39856.0E. coli O157:H75,4472,60747.9E. coli O157:H7 EDL9335,4492,62948.2E. coli CFT0735,3792,55847.6Enterococcus faecalis V5833,2651,56848.0Fusobacterium nucleatum ATCC 255862,0671,01148.9Geobacter sulfurreducens PCA3,4451,90255.2Gloeobacter violaceus PCC 74214,4302,20849.8Haemophilus ducreyi 35000HP1,71786550.4Helicobacter hepaticus ATCC 514491,87590248.1Haemophilus influenzae Rd1,7091,03860.7Helicobacter pylori 266951,56674147.3Helicobacter pylori J991,49174750.1Listeria innocua Clip112623,0431,64153.9Leptospira interrogans serovar4,7251,71936.4Lactococcus lactis IL14032,2661,25455.3Listeria monocytogenes EGD-e2,8461,65358.1Lactobacillus plantarum WCFS13,0091,64754.7Mycobacterium bovis subsp.3,9202,01851.5Mycoplasma gallisepticum R72637151.1Mycoplasma genitalium G3748030563.5Mycobacterium leprae TN1,60591857.2Mesorhizobium loti MAFF3030997,2813,82952.6Mycoplasma penetrans1,03747245.5Mycoplasma pneumoniae M12968833348.4Mycoplasma pulmonis UAB CTIP78239850.9Mycobacterium tuberculosis H37Rv3,9182,03652.0Mycobacterium tuberculosis CDC15514,1871,99047.5Nitrosomonas europaea ATCC 197182,4611,36655.5Neisseria meningitidis MC582,0251,01650.2Neisseria meningitidis Z24912,0651,02549.6Oceanobacillus iheyensis HTE8313,4961,89254.1Phytoplasma asteris, OY strain75442356.1Pseudomonas aeruginosa PAO15,5663,20657.6Porphyromonas gingivalis W831,90994449.4Photorhabdus luminescens laumondii4,6832,28648.8Prochlorococcus marinus MED41,71293354.5Prochlorococcus marinus MIT93132,2651,12249.5Prochlorococcus marinus marinus1,88293949.9Pasteurella multocida PM702,0141,23761.4Pseudomonas putida KT24405,3502,96855.5Pseudomonas syringae pv. tomato str.5,6082,93852.4Pirellula sp. 17,3252,58835.3Rickettsia conorii Malish 71,37457241.6Rhodopseudomonas palustris4,8142,73956.9Rickettsia prowazekii Madrid E83449859.7Ralstonia solanacearum GMI10005,1162,69852.7Streptococcus agalactiae2,1241,15954.6Streptococcus agalactiae NEM3162,0941,17456.1Staphylococcus aureus Mu502,7481,45152.8Staphylococcus aureus N3152,6241,44755.1Staphylococcus aureus MW22,6591,41053.0Streptomyces avermitilis7,6714,00152.2Streptomyces coelicolor A3(2)8,1544,19551.4Staphylococcus epidermidis ATCC 122282,4851,30352.4Shigella flexneri 2a 3014,4522,30651.8Shigella flexneri 2a str. 2457T4,0682,15953.1Sinorhizobium meliloti 10216,2053,49956.4Streptococcus mutans UA1591,9601,13658.0Shewanella oneidensis MR-14,7782,29147.9Streptococcus pneumoniae R62,0941,10152.6Streptococcus pneumoniae TIGR42,0431,13555.6Streptococcus pyogenes SF3701,69695656.4Streptococcus pyogenes MGAS82321,84599654.0Streptococcus pyogenes MGAS3151,86598652.9Streptococcus pyogenes SSI-11,86197652.4Salmonella typhi CT184,7672,34749.2Salmonella typhimurium LT24,5542,45754.0Salmonella enterica subsp. enterica4,3232,26352.3Synechocystis sp. PCC 68033,1671,67953.0Synechococcus sp. WH 81022,5171,24349.4Thermosynechococcus elongatus BP-12,4751,30352.6Thermotoga maritima MSB81,8461,05156.9Treponema pallidum subsp.1,03151750.1Thermoanaerobacter tengcongensis MB4T2,5881,40354.2Tropheryma whipplei TW08\/2778349463.1Tropheryma whipplei str. Twist80849961.8Ureaplasma urealyticum61130349.6Vibrio cholerae N169613,8281,97151.5Vibrio parahaemolyticus RIMD 22106334,8322,46150.9Vibrio vulnificus CMCP64,5372,46154.2Vibrio vulnificus YJ0165,0282,49949.7Wigglesworthia brevipalpis61144172.2Wolinella succinogenes DSMZ 17402,0441,20859.1Xanthomonas axonopodis pv. citri 3064,4272,37453.6Xanthomonas campestris pv. campestris4,1812,28754.7Xylella fastidiosa 9a5c2,8321,15840.9Xylella fastidiosa Temecula12,0361,06652.4Yersinia pestis CO924,0832,11651.8Yersinia pestis KIM4,2812,12349.6sum396,126206,31152.1EukaryotesArabidopsis thaliana28,72314,39450.1Caenorhabditis briggsae14,7137,06348.0Caenorhabditis elegans22,2208,84139.8Ciona intestinalis15,8657,99450.4Drosophila melanogaster18,3029,54152.1Danio rerio26,58716,44361.8Encephalitozoon cuniculi1,99688744.4Guillardia theta Nucleomorph63230748.6Homo sapiens (ENSEMBLE)28,06315,46755.1Leishmania major Friedlin1736235.8Mus musculus24,92814,38257.7Neurospora crassa10,0883,80037.7Oryza sativa16,7244,51727.0Plasmodium falciparum 3D75,2681,90536.2Rattus norvegicus28,68216,74058.4Saccharomyces cerevisiae5,8692,91349.6Schizosaccharomyces pombe5,2612,80753.4Takifugu rubripes rubripes37,45215,20240.6sum291,546143,26549.1Phages\/Viruses18646817.444AHJD2114.844RR2.8t2525120.2933W80911.3A11872912.5A5111100.0Aeh13315115.4APSE-154611.1B11119.1B10317423.5Bcep7816158.2BF238112.5bIL1706423.1bIL2856258.1bIL28661711.5bIL30956610.7bIL31029413.8bIL31122627.3bIL31227311.1BK5-T6369.5Bxb1861214.0C23925.1Cp-12827.1\u03d5CTX4748.5D29791519.0D3941111.7Rb1549612.2\u03d5g1e49612.2GA-13538.6Gh-1421228.6H-19B22418.2HF2114119.6HK02257814.0HK62058610.3HK97611016.4HP14137.3HP23638.3K1394449.1KVP403815715.02,38957712.3L-413C40410.0L5851214.1\u03bb661827.3A261813.1Mu53611.3N15601321.7Mycoplasma virus P11100.0Enterobacteria phage P11100.0P242511.9P2236925.0P2758915.5P33549612.2P412216.7P60801316.3PA0134514.7PaP369811.6\u03d5KZ306258.2\u03d5Ch19899.2\u03d5YeO3-12591322.0\u03d510551815.7\u03d5C3155814.5\u03d53626501020.0\u03d5E125711216.9\u03d5ETA66812.1\u03d5NIH1.155610.9\u03d5PV8365913.8\u03d5SLT621219.4\u03d5adh63812.7\u03d5BT155916.4\u03d5A1122501020.0P682229.1\u03d5KMV481122.9PM22214.5PRD122418.2\u03a8M23113.2\u03a8M10037410.8PY54671014.9PZA27414.8R1t50612.0RB692565621.9RB492724918.0Rd47612.8RM3781461711.6PVL62812.9Sfi112514.0V53713.2SIO134617.6Sk15411.9SP620630.0SP \u03b2c21853317.8SPP110676.6MM153611.3ST64B56814.3ST64T65913.8720146817.4DT147714.9O12055747.0Sfi1945613.3Sfi2150918.0Stx2165116.7T3441022.7T42785820.9T7581017.2TM48955.6TP901-156712.5Tuc200956712.5Ul365858.6VHML57814.0VpV2626746.0VT2-Sa821113.4W\u03d54449.1Sum7,699107313.9Total734,193368,72450.2\nWhen a modeled 3D structure is counted based on the number of amino acid residues, not on the number of ORFs, a different aspect emerges. Figure\u00a01 shows the percentage of amino acid residues per ORF included in the modeled structures. ORFs without a modeled structure are omitted. Of archaebacterial and eubacterial genomes, in 60% of ORFs, more than 80% of the residues are included in modeled 3D structures, however, of eukaryotic genomes, only in 30% of ORFs, more than 80% of the residues are included (red and blue sections in Fig.\u00a01). The proportion of residues in modeled 3D structure can be measured by the number of residues in a typical structural domain as shown in SCOP (Andreeva et\u00a0al. 2004). The average size of protein domain is around 100\u2013150 residues (Copley et\u00a0al. 2002). In ORFs with modeled structures, a continuous region of residues with one domain or more remains as structure unknown in only about 18% of ORFs of archaebacterial and eubacterial genomes, whereas in about 60% of ORFs of eukaryotic genomes, the regions with one domain or more remain as structure unknown.Fig.\u00a01Percentage of amino acid residues included in modeled 3D structures in each ORF is classified by 10% bins and shown in pie charts. ORFs without a modeled structure are not included. A number of ORFs with modeled structures and an average length of the ORFs are shown at the center of each pie chart. Sections bordered by thick black lines indicate that the unmodeled region in the ORF is no less than the size of a domain (about 150 residues)\nAnnual difference of model structures\nIn FAMSBASE of 2001, 38% of amino acid residues in all ORFs of archaebacterial and 40% of eubacterial genomes were included in modeled 3D structures (Yamaguchi et\u00a0al. 2003). In the current update of FAMSBASE based on data by around 2004, 42% of amino acid residues in all ORFs in archaebacterial and 46% of eubacterial genomes are included in modeled structures. In eukaryotic genomes, 24% of amino acid residues in 2003, and 26% in 2004 are included in modeled 3D structures. Those figures can be used to estimate the time when modeled 3D structures of whole proteins predicted from genomes are obtained. The estimation for the time obtaining the whole soluble and membrane proteins are treated separately, because the speed of structure determination for soluble and membrane proteins seems to differ. The assumption for the estimation is that the speed for structure determination would stay the same and no new protein family would appear.\nFor eubacterial genomes, 72.6% of residues in whole ORFs are predicted by SOSUI (Hirokawa et\u00a0al. 1998) to encode soluble proteins and 27.4% to encode membrane proteins. This ratio is not so different from the previous prediction by Krogh et\u00a0al. (2001). Of about 40% of whole eubacterial ORF that were with modeled 3D structures in 2001, approximately 90% were soluble proteins and 10% were membrane proteins. Therefore, about 50% (=0.40\u00a0\u00d7\u00a00.90\/0.726) of the whole soluble proteins were modeled. Of the whole membrane proteins in eubacterial genome, about 15% (=0.40\u00a0\u00d7\u00a00.10\/0.274) were modeled. In 2004, those figures are grown to 57% and 19%, respectively. In eubacterial whole ORFs, about 19.9% of amino acid residues are predicted to be included in disordered region by DisEMBL (Linding et\u00a0al. 2003). Some of these regions are included in the modeled structures. These regions are either incorrectly predicted regions or incorrectly modeled regions. Assuming that the disordered regions without modeled 3D structures are correctly predicted, 10.8% of amino acid residues in soluble proteins were disordered and we would never obtain 3D structures of those regions. Then, by extrapolating the coverage of soluble proteins up to 89.2% (100\u201310.8) with the current growth rate, we can estimate that, by the year 2017, whole soluble proteins encoded in eubacterial genomes can be modeled (Fig.\u00a02). Whole soluble proteins of archaebacterial genome can be modeled by 2021 and those of eukaryotic genomes, by 2031.Fig.\u00a02Annual differences of modeled structures classified by kingdoms of life. The percentage is the number of amino acid residues included in modeled structures over the whole number of residues in predicted sequences for soluble and membrane proteins in each kingdom. (S) stands for soluble proteins and (M) stands for membrane proteins. Some of the residues are predicted to be in a disordered region. The percentage of residues in disordered regions is shown at the top\nOrengo et\u00a0al. (1999) showed percentage of ORFs with protein 3D structures as between 30 and 46% in 1999. The genome sequences known by 1999 were mostly derived from prokaryotic species and the known protein 3D structures were mostly soluble proteins. Therefore, the figures they presented in 1999 should correspond to the figures of archaebacterial and eubacterial soluble proteins. When we extrapolate the figures of archaebacterial and eubacterial soluble proteins to the past in Fig.\u00a02, the figures are around 40% in 1999, indicating that their figures approximately lie on the extrapolated lines.\nThe current estimation indicates that we will obtain 3D structures of whole soluble proteins of eubacteria in 11\u00a0years and archaebacteria in 15\u00a0years. This estimation does not take into account the acceleration of structure determination speed by automation (McPherson 2004; DeLucas et\u00a0al. 2005), which makes the due days closer to the present. For membrane proteins, speed of structure determination has been drastically accelerated by recent technical innovations (Kyogoku et\u00a0al. 2003; Lundstrom 2004; Walian et\u00a0al. 2004; Dobrovetsky et\u00a0al. 2005), and therefore we will not linearly extrapolate the present status to estimate the due day for membrane proteins.\nFrequency of template structure in use\nWhen the template 3D structures used in FAMSBASE are classified by SCOP superfamily, which is a group of proteins that have low sequence identities but whose structural and functional features suggest that a common evolutionary origin is probable (Lo Conte et\u00a0al. 2002), and frequencies of superfamilies in use are counted, \u2018P-loop containing nucleoside triphosphate hydrolases\u2019 superfamily is found to be the most frequent one; 7,532 times (about 12%) in whole archaebacterial model structures, 77,806 (about 10%) in eubacterial structures and 35,468 times (about 6%) in eukaryotic structures. The templates that follow in frequency in archaebacterial and eubacterial protein structures are \u2018NAD(P)-binding Rossmann fold domains\u2019, \u20184Fe\u20134S ferredxin\u2019, and \u2018PLP-dependent transferases\u2019 superfamilies. In eukaryotic protein structures, \u2018protein-kinase\u2019, \u2018immunoglobulin\u2019 and \u2018C2H2 and C2HC zinc fingers\u2019 superfamilies, which appear specifically in eukaryotic genomes, follow the top.\nDifferences in distribution of frequency of templates in different kingdoms of life are evident, when frequencies in use of template are plotted in descending order (Fig.\u00a03). In any kingdoms of life, the frequencies of the most and the second most used templates exceed those of the remaining templates. The frequencies of templates in use drops first in archaebacterial protein structures and then in eubacterial protein structures. The descending curve of eukaryotic template frequency is less steep compared with the others, indicating that one template can produce a large number of domain 3D structures in eukaryotic ORFs. In other words, a significant number of proteins encoded in eukaryotic genomes are originated by domain duplication, as Koonin et\u00a0al. (2000) demonstrated. Superfamilies with the 3D structures and with many copies in eukaryotic genomes, but seldom in prokaryotic genomes are \u2018protein kinase-like\u2019, \u2018immunoglobin\u2019, \u2018RNA-binding domain\u2019, \u2018C2H2 and C2HC zinc fingers\u2019, \u2018WD40-repeat\u2019, \u2018glucocorticoid receptor-like\u2019, \u2018homeodomain-like\u2019, \u2018PH domain-like\u2019, \u2018RING-box\u2019, \u2018L domain\u2019, \u2018ankyrin repeat\u2019, \u2018ARM repeat\u2019, \u2018cytochrome P-450\u2019 and \u2018EF-hand\u2019 superfamilies. These superfamilies are transcription factors, protein\u2013protein interaction mediators and response factor for toxic substances, mostly known to be unique to eukaryotes.Fig.\u00a03Frequency of template usage in descending order. Horizontal axis is a template and the vertical axis is a frequency of templates in use. Red line is a template usage in archaebacteria, blue line is eubacteria and green line is eukaryotes\nThe \u2018P-loop containing nucleoside triphosphate hydrolases\u2019 superfamily outnumbering other superfamily in template frequency corresponds to the previous finding that the enzyme is highly frequently used in every kingdom of life (Leipe et\u00a0al. 2003). When biological functions of these ORFs with the 3D structure of \u2018P-loop containing nucleoside triphosphate hydrolases\u2019 superfamily are classified, about half of the proteins are ABC transporters in archaebacterial and eubacterial proteomes, but numbers of G-proteins and motor proteins in eukaryotic proteomes are noticeable (Fig.\u00a04).Fig.\u00a04Protein family distribution of \u2018P-loop containing nucleoside triphosphate hydrolases\u2019 superfamily in each kingdom. In the three pie charts, the section with the same color is a category of the same family except for the white section\nIn the last two years, new protein structures were determined and contributed to an increase in the number of templates for homology modeling. A part of those template structures are listed in Table\u00a02. A part of those top 15 templates contributed a lot for the growth of modeled 3D structure database. In Table\u00a02, 3D structure derived from SG projects is rare. The ratio of SG products in Table\u00a02 is the same as that in PDB (Editorial Board, Nature Structural & Molecular Biology 2004). As the SG projects in US and Europe have proceeded to phase 2 (Service 2005), SG products are expected to contribute to increase in the number of templates in the near future. The qualities of protein 3D structures, namely, size, resolution, R-factors and so forth, derived from SG projects were compared with those in PDB and no obvious compromise in quality of SG products were found (Todd et\u00a0al. 2005). The quality of homology modeling based on products of SG projects in the future, therefore, will be expected to be no less than the current quality.Table\u00a02Top 15 modeling templates in the newly determined 3D structures between 2002 and 2003PDBIDChainNumber of uses as a templateSGaProtein name1q12A7,031NMaltose\/maltodextrin transport ATP-binding protein MalK1l2tA6,529NHypothetical ABC transporter ATP-binding protein Mj07961oxxK3,948NABC transporter ATP-binding protein GlcV1pf4A3,202NTransport ATP-Binding Protein MsbA1nr0A2,640YActin interacting protein 1 Aip11ixcA2,495NLysR-type regulatory protein CbnR1ld8A2,410NFarnesyltransferase \u03b1 subunit1ji0A2,331YABC transporter1oywA2,251NATP-dependent DNA helicase; RecQ helicase1kt1A2,198NFk506-binding protein FKBP511mt0A1,961NHaemolysin secretion ATP-binding protein; ATP-binding domain1mdbA1,745N2,3-dihydroxybenzoate-AMP ligase DhbE1nnmA1,730NAcetyl-CoA synthetase1gxrA1,715NTransducin-like enhancer protein 1 Esg11uohA1,706N26S proteasome non-ATPase regulatory subunit 10a\u00a0PDB entry seemingly derived from the SG projects judged by description in PDB file is tagged Y, and the remaining entry is tagged N\nWhole structure and function of proteins from homology modeling of domain structures\nProtein function prediction, especially studies on enzyme specificity, based on homology modeling structures is intensively carried out in the field of drug design and related fields (Goldsmith-Fischman and Honig 2003; Kopp and Schwede 2004). Those studies are mostly based on homology modeling of domain structures. As mentioned above, most of the eukaryotic protein structures in FAMSBASE are 3D structures of structural domains, not the entire coding regions (Fig.\u00a01). Protein functional sites are often located at a cleft of domains (Laskowski et\u00a0al. 1996), and therefore understanding relative location of domains will be a critical issue. Xie and Bourne (2005) and O\u2019Toole et\u00a0al. (2003) also pointed out this problem and mentioned, \u201ceven if all the domains of a multiple-domain query sequence have determined structures, the individual structures will not enable accurate modeling of how they associate together in the structure of the entire proteins (O\u2019Toole et\u00a0al. 2003).\u201d\nFigure\u00a05 shows all eukaryotic ORFs whose 3D structures are mostly modeled in pieces. There are three types of enzymes and four types of cell surface receptors. A protein structure of ENSP00000264705 which is an ORF found in human genome can be modeled based on Escherichia coli carbamoylphosphate synthetase (CPS) and Pyrococcus abyssi asparatate transcarbamoylase (ATC). E. coli CPS is composed of a large subunit and a small subunit. CPS and ATC are the first and the second enzymes, respectively, in pyrimidine biosynthesis pathway. In mammalian genomes, those proteins are coded by a single gene and active in a hexamer form (Serre et\u00a0al. 2004). Interactions between the large subunit domain and the small subunit domain of human CPS are conjectured to be the same as those between the large and the small subunits of E. coli CPS. N-terminal residues of the large subunit and the C-terminal residues of the small subunit are spatially located close in E. coli CPS, which permits the two chains to be chemically connected without disrupting subunit interfaces. To be active, human CPS should form a hexamer supramolecule and the interfaces for the supramolecule formation should be predicted from the modeled 3D structures. At the moment, the interfaces are unknown.Fig.\u00a05Eukaryotic ORFs with multiple model structures covering more than 70% of entire protein. In each of the bar representation of proteins, a black box is a region with 3D structure. A name and PDB ID of a template structure and amino acid sequence identity between template and target domains are given below the black box. A yellow box is a putative signal peptide and green box is a putative transmembrane region. Template and modeled structures of ENSMUSP00000019416 were shown on the right side of the figure. Each domain is colored by hydrophobicity. A hydrophilic residue is in green and a hydrophobic residue is in red. A buried residue is in deep blue\nENSMUSP00000019416 is an ORF found in mouse genome and encodes a putative cell surface receptor. The protein is predicted to consist of six consecutive Ig-fold domains. There is a putative transmembrane helix at the C-terminal region of the protein. Two consecutive Ig-fold domains are modeled without overlap, and no pieces of information for relative orientation of three modeled structures have been found. Information of interaction sites of those domains is required to build the entire structure of the protein and to predict a target molecule of this receptor. Computational analyses of domain interfaces and of protein\u2013protein interfaces have been targets for extensive study for a long time, and some general characteristics have been found. One of them is the hydrophobicity of the interfaces (Wodak and Janin 2002). Hydrophobic clusters on the surface of modeled structures of ENSMUSP00000019416 are shown in right side of Fig.\u00a05. One of the template structures, Nkp46 ectodomain, has hydrophilic surface (green) around the C-terminal residues of the domain, however the modeled structure has a hydrophobic surface (orange) at the corresponding area. The other template structure, LIR-1 D1D2, has a hydrophilic surface around the N-terminal residues of the domain, however the modeled structure has a hydrophobic surface at the corresponding area. The surfaces uniquely turned into hydrophobic in modeled structures are close to the residues that are chemically bonded in the target protein, and therefore both of the areas likely form interfaces of the two domains. The modeled structure based on LIR-1 D1D2 domain has another hydrophobic surface around the C-terminal residues, which may interact with CD158j-like domain located at the C-terminal side of the domain.\nAccuracy of homology modeling\nThere are at least three major issues that affect accuracy in homology modeling; the best template selection, accuracy of an amino acid sequence alignment between template and target protein sequences and the accuracy of structure building procedure itself (Contreras-Moreira et\u00a0al. 2005). Accuracy of the alignment is high, when sequence identity of template and target proteins is higher than 30%, and alignment of proteins with identity less than 30% is known to be less reliable, thereby accuracy of homology modeling deteriorates (Kopp and Schwede 2004). FAMS has been shown to construct relatively accurate model structures, even with low sequence identity between template and target sequences in CAFASP2, the homology modeling competition (Iwadate et\u00a0al. 2001; Yamaguchi et\u00a0al. 2003). A distribution of sequence identity between amino acid sequences of template and target proteins in FAMSBASE is shown in Fig.\u00a06. Half of the model structures in FAMSBASE rely on alignments of sequence identity less than 20%. Figure\u00a06 suggests that the current 3D structure database does not contain good enough structures for high quality homology modeling. SG projects will eventually provide better template structures, and improvement in target selection, alignment and modeling methods are also in pursuit to overcome the difficulties in homology modeling (John and Sali 2003; Wallace et\u00a0al. 2005).Fig.\u00a06Distribution of sequence identity between template and target amino acid sequences in FAMSBASE\nConclusion\nConstruction of database of whole genome homology modeling clarified that protein 3D structures of about 50% of the protein coding regions in whole genome can now be modeled. Maintaining the current speed of 3D structure determination, it will take, at most, 11\u00a0years to have enough templates to cover whole soluble proteins of eubacterial genomes, and 25\u00a0years to cover those of eukaryotic genomes. The current advancement in technologies of protein structure determination is expected to make these due times closer to the present. What we obtain at those times are not the 3D structures of entire proteins, but domain structures in pieces. A homology modeled domain structure is now in use of predicting domain functions, but predicting spatial arrangement of domains in a protein will be an important issue for function prediction.","keyphrases":["structural genomics","genome","homology modeling","p-loop","domain duplication","domain interactions"],"prmu":["P","P","P","P","P","R"]}
{"id":"Cancer_Causes_Control-2-2-1705485","title":"Hypothesis: hair cover can protect against invasive melanoma on the head and neck (Australia)\n","text":"The anatomic distribution of cutaneous melanoma reflects people\u2019s levels and patterns of sun exposure. While examining trends of incident invasive melanomas by site in recent decades in Australia we noted significant increases in incidence on the ears but not the face or any other site in women younger than 40 years, by 6% (95% confidence interval [CI] 2\u201310%) per year, and 40\u201359 years by 7% (95% CI 4\u201310%) per year. Men of the same age showed no corresponding changes in ear melanoma. However incidence rates of ear melanoma in general were fourfold higher in males than females in Australia. Further, using data from the National Cancer Institute\u2019s Surveillance, Epidemiology and End Results (SEER) Program, rates of invasive melanoma on the ear were found to be sevenfold higher in males than females in the US population in the same period. Higher rates of scalp and neck melanomas were also seen in men and women in both populations. We therefore speculated that the isolated rises of ear melanoma in younger women in Australia, and the higher overall rates of ear, scalp and neck melanoma in men compared with women, reflect differences in hair coverage. We tested the specific hypothesis that hair cover reduces sun exposure of the ears using experimental manikins and found that hair cover of the ear reduced solar ultraviolet-B exposure by 81% [SE \u00b18] compared with uncovered ears. We conclude that hair cover can protect against invasive melanoma on the ear and may similarly protect on the scalp and neck. When discretionary, hair may be an important additional factor to be considered for melanoma prevention.\nIntroduction\nSun exposure is the major environmental cause of melanoma [1] and so the anatomic distribution of melanoma in Caucasian populations largely reflects relative levels of sun exposure of different body sites [2]. Similarly in a given population, changes in site distribution of melanoma have long been known to offer clues about change in people\u2019s sun-related behavior [3]. For example Magnus [4] analyzed melanoma incidence rates by age cohort and tumor site in Norway 1955\u20131977 and found that trunk and lower limb melanomas had increased much more than face and neck melanomas among younger generations, consistent with changes in clothing and sun-tanning habits in the first half of the twentieth century. Here we describe an isolated rise in the incidence of melanoma of the ear in women under 60\u00a0years of age in Australia. This distinct increase of invasive melanoma on a discrete site indicated some diminution in covering of the ear in young women in preceding decades. While a change in hat wear was one possible explanation, we speculated that a change in hair cover may also explain the observed rise, and that less hair coverage of the head among men in general may explain their higher relative incidence of melanoma on the ears and scalp compared with women [2]. When we searched for published data regarding the sun protection factor of hair we found none. Hence we set out to test the specific hypothesis that hair cover protects the ear from sun exposure using experimental manikins.\nRecent trends in melanoma in Australia\nBased on data for 109,062 invasive melanomas assembled by the Australian Institute of Health and Welfare from cancer registries in all Australian states and territories, trends in incidence of cutaneous melanoma in Australia 1982\u20131999 by sex and site were examined. Sites of invasive melanoma were pre-classified by cancer registries according to a standard protocol. For each site annual notifications were provided with age in 5-year bands. Population estimates by age and sex were obtained for each year from the Australian Bureau of Statistics. Annual sex-specific incidence rates of melanoma age-standardized to the WHO World Population were computed for each site and relative rates of increase were calculated for three age bands, less than 40\u00a0years, 40\u201359\u00a0years and 60\u00a0years and over, by regressing the logarithms of these rates on years.\nThe average relative change in melanoma incidence rates over all sites each year was estimated as 3.2% (99% CI 3.0\u20133.4%) in males and 1.5% (99% CI 1.3\u20131.8%) in females but increases varied by sex and age. Highest increases were seen in men 60\u00a0years and over (4.4% per year, 95% CI 4.2\u20134.6%), followed by women 60\u00a0and over and men 40\u201359\u00a0years (2.9% per year), and then by women aged 40\u201359\u00a0years (1.5% per year) (Table\u00a01). In contrast persons under 40\u00a0years showed little to no relative increase in invasive melanoma in the last two decades: young men showed a 1.4% annual increase of borderline significance and women under 40\u00a0years showed virtually no change in melanoma incidence (Table\u00a01).\nTable\u00a01Age-standardized incidence of melanoma on head and neck sites and all sites in Australia 1982\u20131986 and 1995\u20131999\u00a0by age and sexSiteAge (years)No. of melanomasIncidence rates per 100,000% Annual relative increase (95% CI)1982\u201319861995\u20131999MalesEar<401790.150.222.0 (\u22120.9\u20135.0)40\u201359 4260.951.31 1.9 (0.0\u2013 3.8)\u226560 1,1153.296.464.2 (3.0\u2013 5.4)Face<40 3920.340.502.6 (\u22120.7\u20134.5)40\u201359 1,1252.483.85 3.2 (2.1\u20134.4)\u226560 3,54611.0620.303.7 (3.0\u20134.4)All sites<40 10,7318.9111.981.4 (1.0\u20131.8)40\u201359 19,89843.3864.652.8 (2.5\u20133.1)\u226560 27,95785.34166.534.4 (4.2\u20134.6)FemalesEar<40 1060.080.176.1 (2.4\u20139.9)40\u201359 1300.230.537.0 (3.6\u201310.4)\u226560 2260.580.921.9 (\u22120.8\u20134.6)Face<404510.400.540.9 (\u22120.9\u20130.7)40\u2013598402.482.340.0 (\u22121.3\u20131.4) \u2265603,5089.7315.282.4 (1.8\u20133.1)All sites<4013,49413.4915.290.3 (0.1\u20130.7)40\u20135917,32844.2454.691.5 (1.2\u20131.8)\u22656019,65455.7487.052.9 (2.6\u20133.2)\nRecent site-specific trends in melanoma on the head and neck in Australia\nSite-specific trends in melanoma for males and females within each age group reflected the above trends for all sites combined, such that men 60\u00a0and over generally had the highest relative increases on each anatomic site and women under 40, the least (data not shown for trunk and limbs). The striking exception was a significant 6.1% increase (95% CI 2.4\u20139.9%) in incidence rates of melanoma on the ears in women under 40\u00a0years based on 106\u00a0invasive tumors diagnosed in the study period. A similar rise of 7% (95% CI 3.6\u201310.4%) was seen for ear melanomas in women 40\u201359\u00a0years based on 130\u00a0invasive tumors (Table\u00a01). The incidence rates of melanoma on the face in young women and those under 40\u201359\u00a0years showed little change (0.9% and 0.0% respectively), and similarly on the scalp and neck (data not shown).\nIn contrast, young men showed no significant relative increases of melanoma on the ear or any other sites on the head and neck in the same period. Men aged 40\u201359\u00a0years also showed no significant relative increases of melanoma on the ear but modest relative increases in melanoma on the face of around 3% per year (Table\u00a01) and also on the scalp and neck.\nSex-specific differences in incidence rates of melanoma on the head and neck\nBetween 1982 and 1999 in Australia the overall age-standardized incidence rate of ear melanoma in males was 0.91 (99% CI 0.85\u20130.97) per 100,000, 4\u00a0times higher than the corresponding rate of 0.24 (99% CI 0.21\u20130.26) per 100,000\u00a0in females. The male excess was seen in all age groups though the difference in rates of ear melanoma between young men and young women was small by the end of the study period, compared with the sevenfold sex-specific difference seen in older men and women over 60. The incidence rate of scalp and neck melanoma (sites pre-combined) in males was 2\u00a0times higher than in females (2.18, 99% CI 2.09\u20132.27 and 1.08, 99% CI 1.02\u20131.14\u00a0per 100,000\u00a0respectively) though again the differences were narrowed in the youngest age group. Incidence rates of melanoma on the face were slightly higher overall in males than females (2.62, 99% CI 2.52\u20132.72 and 2.06, 99% CI 1.98\u20132.14\u00a0per 100,000\u00a0respectively) but were no different in men and women under 40\u00a0years (Table 1).\nDifferences in incidence rates of melanoma on the ear, scalp, face and neck were seen even more clearly in an earlier unrelated study of the Queensland data for 1987 [2] where site-specific incidence rates of melanoma had been calculated and further adjusted for relative body surface area. Incidence of invasive melanoma on the ear in Queensland males was 209 per unit area of skin per 100,000\u00a0per year compared with 50 per unit area of skin per 100,000 per year in females, again a fourfold difference. On the scalp there was a 20-fold greater incidence in males than females and on the neck a fourfold difference. Again rates on the face in males and females showed very little difference in Queensland in 1987 [2].\nTo assess whether comparable patterns were seen in other populations, age-standardized incidence rates of melanoma per 100,000 for 1982\u20131999 were calculated for sub-sites of the head and neck for the regions covered by the National Cancer Institute\u2019s Surveillance, Epidemiology and End Results (SEER) Program of population-based cancer registries [5]. In males compared with females there was a sevenfold higher incidence rate of ear melanoma and a threefold higher rate of scalp and neck melanoma compared with a less than twofold higher incidence rate of melanoma on the face.\nHypothesis\nThe systematically higher incidence of invasive melanoma on the ear, scalp and neck compared with the face in males and females is consistent with the general hypothesis that hair cover can offer long-term protection of the skin from sun exposure. We tested the specific hypothesis that hair cover of the ear protects it from solar ultraviolet (UV) radiation by conducting an experiment using manikin head-forms in which the difference in solar UV radiation to ears with and without hair cover was measured. We used four manikin head-forms: three wearing human hairpieces (blonde, brown and black) covering the ears and one uncovered (Fig.\u00a01). These were set facing north on a concrete floor and exposed to natural sunlight in Brisbane (27\u00b0S) from 11 am to 1 pm Australian Eastern Standard Time daily for five consecutive days in May 2005. Cloud cover was zero and surface reflectivity less than 7%. Sun exposure was measured by UV-sensitive polysulfone dosimeters placed on the ears and nose of each manikin.\nFig.\u00a01Measurement of solar ultraviolet radiation on ears in relation to hair cover using head-forms and polysulfone dosimeters\nEars covered by hair received on average 81% [SE \u00b18] less solar UV radiation than bare ears when exposed to the sun. As a control site on the face, the corresponding solar UV exposures to the noses of the manikins were monitored (Fig. 1) and showed a slight variation during the experiment in the reverse direction, namely a 5% [SE \u00b13.4] increase in UV radiation to the wigged manikins compared with the bare manikin.\nDiscussion\nThese experimental data support our hypothesis that hair cover offers substantial sun protection of the skin of the ear and by extrapolation, the skin of the scalp and back of the neck as well if hair is worn long. Taken together with the recently observed change in incidence of invasive melanoma on the ear in women in Australia and the consistent pattern in Australia and the United States of lower incidence of melanoma in females on the scalp, ear and neck\u00a0\u2013\u00a0sites that are more frequently covered or screened by hair than in males, the evidence suggests that hair cover can indeed protect against invasive melanoma on these sites.\nOther explanations for the observed site- and sex-specific patterns can be considered. Change in diagnostic trends was unlikely to explain the observed rise in ear melanomas in younger women since in situ melanomas were not included in the analyses. One possibility is that the distinct increase of ear melanomas in Australian women under 60 was due to the loss of some other form of physical protection. For example the rise in popularity of caps in the 1980s resulted in a decrease in cover of the ear but not the face in young women who wore brimmed hats in preceding decades [6]. If this were the sole explanation, some analogous rise in the rate of ear melanomas in men might have been expected as an aftermath as well, but has been observed neither in Australia nor the US. Another cause of increasing sun exposure of the ears in young women may have been the upsurge in women\u2019s participation in outdoor sport that also occurred in the 1970s and 1980s [7] if caps rather than hats were worn. However even if change from hats to caps did explain the recent rise in incidence of ear melanomas in Australian women, difference in hat wear between the sexes does not explain the systematically higher rates of invasive melanoma in men than in women across the various sub-sites of the head and neck with less discrepant rates on the face. (Indeed facial hair cover in men with beards may mitigate the effect of overall higher outdoor exposure of men than women for facial melanomas). We also speculated that younger women\u2019s hair styles may have changed over time and become shorter leading to greater exposure of the ears and neck, but there is little evidence available to address this question.\nFinally in view of the known sex-specific differences in melanoma incidence on sites such as the scalp, it is surprising that the sun-protection factor of human hair has never been measured before. This absence of quantitative data contrasts with the extensive information available about the sun-protection provided by clothing and sunscreens [8, 9]. If our general hypothesis is confirmed, hair cover of the ears in particular would add another potential means of melanoma prevention, especially in high-risk populations, among individuals in whom amount of hair cover is discretionary.","keyphrases":["hair","protection","melanoma","ear","solar ultraviolet radiation"],"prmu":["P","P","P","P","P"]}
{"id":"Plant_Mol_Biol-4-1-2238787","title":"Enhancement of stress tolerance in transgenic tobacco plants constitutively expressing AtIpk2\u03b2, an inositol polyphosphate 6-\/3-kinase from Arabidopsis thaliana\n","text":"Inositol phosphates (IPs) and their turnover products have been implicated to play important roles in stress signaling in eukaryotic cells. In higher plants genes encoding inositol polyphosphate kinases have been identified previously, but their physiological functions have not been fully resolved. Here we expressed Arabidopsis inositol polyphosphate 6-\/3-kinase (AtIpk2\u03b2) in two heterologous systems, i.e. the yeast Saccharomycescerevisiae and in tobacco (Nicotiana tabacum), and tested the effect on abiotic stress tolerance. Expression of AtIpk2\u03b2 rescued the salt-, osmotic- and temperature-sensitive growth defects of a yeast mutant strain (arg82\u0394) that lacks inositol polyphosphate multikinase activity encoded by the ARG82\/IPK2 gene. Transgenic tobacco plants constitutively expressing AtIpk2\u03b2 under the control of the Cauliflower Mosaic Virus 35S promoter were generated and found to exhibit improved tolerance to diverse abiotic stresses when compared to wild type plants. Expression patterns of various stress responsive genes were enhanced, and the activities of anti-oxidative enzymes were elevated in transgenic plants, suggesting a possible involvement of AtIpk2\u03b2 in plant stress responses.\nIntroduction\nDuring their life plants are often challenged with various types of abiotic stresses. Proper physiological and biochemical responses to such stresses are controlled by an array of stress-dependent signal transduction pathways (Xiong et\u00a0al. 2002). Several signaling molecules and second messengers including calcium ions (Ca2+; Knight et\u00a0al. 1996; Knight et\u00a0al. 1997) and inositol 1,4,5-trisphosphate (IP3; Takahashi et\u00a0al. 2001) have been demonstrated to be important elements of signaling pathways that link the perception of stress to various types of downstream responses. As one of the central molecules in cell signaling, IP3 is subject to phosphorylation and dephosphorylation by specific inositol phosphate kinases and phosphatases, respectively, which helps to control intracellular IP3 concentration (Pattni and Banting 2004).\nIn higher plants, over-expression of an inositol polyphosphate 5-phosphatase (Ins5Pase), possibly leading to intracellular degradation of IP3, modulated stress and ABA signal transduction (Perera et\u00a0al. 2002; Burnette et\u00a0al. 2003). Likewise, mutation of an inositol polyphosphate 5-phosphatase gene (CVP2) results in increased sensitivity to ABA and vein patterning defects in cotyledons (Carland and Nelson 2004). IP3 can also be hydrolyzed by FRY1-like inositol polyphosphate 1-phosphatases (Ins1Pases; Xiong et\u00a0al. 2002). The fry1 mutant of Arabidopsis accumulated around ten-fold more IP3 than the corresponding wild type, exhibited hypersensitivity towards ABA, and was compromised in tolerance to freezing, drought and salt stresses (Xiong et\u00a0al. 2001). These data demonstrate the importance of inositol polyphosphate phosphatases in IP3 metabolism and IP3-mediated stress signal transduction. However, evidence for a role of inositol polyphosphate kinases in relation to stress tolerance is currently lacking in plants.\nInositol 1,4,5-trisphosphate 3-kinase (IP3K) and more generally inositol polyphosphate kinases play an essential role in cellular signal transduction and maintenance of Ca2+ homeostasis by phosphorylating inositol 1,4,5-trisphosphate (IP3) to inositol 1,3,4,5-tetrakisphosphate (IP4; Communi et\u00a0al. 1995). Both IP3 and IP4 are second messengers responsible for Ca2+ mobilization from intracellular stores (Berridge 1997). IP4 itself is also able to regulate cytosolic Ca2+ concentration by promoting Ca2+ sequestration (Hill et\u00a0al. 1988). In Arabidopsis thaliana, IP3K is encoded by two distinct genes, AtIpk2\u03b1 and AtIpk2\u03b2, which are ubiquitously expressed in mature tissues (Stevenson-Paulik et\u00a0al. 2002; Xia et\u00a0al. 2003). Both of the Arabidopsis IP3Ks use IP3 as a substrate and display dual-specificity inositol polyphosphate 6-\/3-kinase activities that successively phosphorylate IP3 to generate inositol 1,3,4,5,6-pentakisphosphate (IP5) predominantly via an inositol 1,4,5,6-tetrakisphosphate (IP4) intermediate (Stevenson-Paulik et\u00a0al. 2002; Xia et\u00a0al. 2003). Recently it was reported that AtIpk2\u03b1 plays a role in pollen germination and root growth (Xu et\u00a0al. 2005), while AtIpk2\u03b2 functions in axillary shoot branching through the auxin signaling pathway (Zhang et\u00a0al. 2007) and phytate synthesis (Stevenson-Paulik et\u00a0al. 2005).\nAlthough AtIpk2\u03b2 has well been characterized at the biochemical level and has been shown to participate in plant development, it remained unclear whether it is also involved in stress signaling. In this study we demonstrate that AtIpk2\u03b2 restores the salt-, osmotic- and temperature-sensitive growth defects of a yeast mutant strain (arg82\u0394) deficient for the ARG82\/IPK2 gene that encodes inositol polyphosphate multikinase. We also show that constitutive expression of AtIpk2\u03b2 in transgenic tobacco enhances its tolerance towards various abiotic stresses. Thus, inositol polyphosphate kinase encoded by AtIpk2\u03b2 very probably plays an important role in signaling pathways controlling the cellular response to abiotic stresses.\nMaterials and methods\nExpression of AtIpk2\u03b2 in yeast mutant arg82\u0394\nThe S. cerevisiaearg82\/Ipk2 deletion strain (derived from BY4741; Mat a; his3\u03941; leu2\u03940; met15\u03940; ura3\u03940; YDR173c::kanMX4) was obtained from EUROSCARF (http:\/\/www.web.uni-frankfurt.de\/fb15\/mikro\/euroscarf\/). In this strain, the ARG82\/IPK2 allele is disrupted through insertion of the kanamycin resistance gene, kanMX4. The AtIpk2\u03b2 open reading frame was cloned into yeast expression plasmid pYX212. The resulting plasmid, pYX212-AtIpk2\u03b2, was used to transform the yeast mutant. The wild type strain BY4741 was used as control. Ten-fold serial dilutions (starting at OD600\u00a0=\u00a01.0) of each strain were plated on YPD medium (1% yeast extract, 2% peptone, 2% glucose) supplemented with either NaCl (0.4 or 0.8\u00a0M) or mannitol (0.4 or 0.8\u00a0M), and incubated at 30\u00b0C for 48\u00a0h. For growth at 37\u00b0C, yeast cells were grown on media containing no extra NaCl or mannitol. Each experiment was repeated at least three times. For growth in liquid YPD medium, cells were cultured at 30\u00b0C for three days to saturation (OD600\u00a0=\u00a012.0). Five \u03bcl of the cell culture were used to inoculate 3\u00a0ml of synthetic defined minimal medium supplemented with different concentrations of NaCl or mannitol. Cell density was measured at A600.\nQuantitative real-time PCR analysis of AtIpk2\u03b2 expression in Arabidopsis\nTen-day-old Arabidopsis seedlings grown on MS medium were used for different treatments as described previously (Shi and Zhu 2002). RNA was extracted with TRIZOL Reagent (Invitrogen) and reverse transcription of RNA was carried out using M-MLV RTase Synthesis Kit (TaKaRa). Quantitative real-time PCR was performed using the Rotor-Gene 3000 Series real time DNA amplification system under the following conditions: 95\u00b0C for 10\u00a0s; 40 cycles of 95\u00b0C for 5\u00a0s, 60\u00b0C for 20\u00a0s. AtIpk2\u03b2-specific primers were used (Table\u00a01). Melting curve analysis was included to verify specificity of the DNA amplification. Expression of the reference gene Actin, which served as quantifying control, was monitored using gene-specific forward and reverse primers (Table\u00a01). Data analysis was performed with Rotor-Gene software 6.0.\nTable\u00a01Gene-specific primers used in this studyPrimer namePrimer sequenceFor yeast expression vectorpYX-F5\u2032-CTCCCATGGTAATGCTCAAAGTCCCTG-3\u2032pYX-R5\u2032-GATTGTCGACCTAGCGCCCGTTCTC-3\u2032For real-time RT-PCRAtIPK2\u03b2-F 5\u2032-CCACGGTTTCGTTGGGGTTC-3\u2032AtIPK2\u03b2-R 5\u2032-GTTACAACCGCCATAAACCTCTG-3\u2032AtActin2-F 5\u2032-CATCCTCCGTCTTGACCTTGC-3\u2032AtActin2-R 5\u2032-CAAACGAGGGCTGGAACAAG-3\u2032For RT-PCRNtActin-F 5\u2032-TTACGCCCTTCCTCATGCAATT-3\u2032NtActin-R 5\u2032-GGCGCCACCACCTTGATCTTC-3\u2032AY562132-F5\u2032-GTAGCATTGTTGGTGGTGGTGTG-3\u2032AY562132-R5\u2032- ACCGTGGAGCAGTCAATGGAAG-3\u2032AY554169-F5\u2032- ACAGTCGTGAGCATTCCCAAC-3\u2032AY554169-R5\u2032-CCAAACCTTCTGTGCTACCTC-3\u2032AY554170-F5\u2032-CCGGAGTGAAGGGGATGG-3\u2032AY554170-R5\u2032-CAAGCAATGTGAATGGTATGTGAG-3\u2032NtERD10B-F5\u2032-CAATTTAGTGCAGGCCAGGC-3\u2032NtERD10B-R5\u2032-GGTCCATGGTGGCCAGGAAG-3\u2032NtERD10C-F5\u2032-GGGTAGCGCAAACGTGGAG-3\u2032NtERD10C-R5\u2032-CTTTTCCCTCAGCCTCGTGC-3\u2032\nVector construction and plant transformation\nTo create a Cauliflower Mosaic Virus (CaMV) 35S plant expression construct, the 903\u00a0bp long AtIpk2\u03b2 cDNA containing the complete open reading frame was inserted via XbaI\u2013SalI sites into the binary vector pBinAR-HPT. The resulting construct was introduced into Agrobacterium tumefaciens (strain LBA4404). Tobacco (Nicotiana tabacum cv. SR-1) leaf disks were infected with the transformed Agrobacteria. After two days of co-cultivation, the explants were transferred to regeneration medium containing 1-mg\/l BAP, 0.1-mg\/l NAA, 50-mg\/l hygromycin and 200-mg\/l ampicillin. Regenerated shoots were separated from the calli and transferred onto rooting medium containing MS salts, 0.1-mg\/l NAA and 50-mg\/l hygromycin. Rooted shoots were transplanted into soil. Seeds were harvested and homozygous plants were screened on a 12-h light\/12-h dark photoperiod.\nSalt stress tolerance tests\nWild type and homozygous T2 transgenic seeds were surface sterilized with a solution of 10% commercial bleach (0.525% sodium hypochlorite) for 10\u00a0min, and washed three times with sterile water. For germination assays, seeds were plated on MS medium supplemented with different concentrations of NaCl (0, 100 and 200\u00a0mM). For root growth measurements, 10-day-old seedlings cultured on solid MS medium were transferred to MS medium supplemented with different concentrations of NaCl. Plates were oriented vertically with seedlings kept upside down. Three replicates were performed for each experiment. Root length was recorded after seven days of treatment. To monitor stress effects on the growth of wild type and AtIpk2\u03b2 expressing plants, 15-day-old seedlings were transferred to MS medium supplemented with 0, 100, 200 or 300\u00a0mM NaCl, and cultured at 25\u00a0\u00b1\u00a02\u00b0C with cool white fluorescent light (\u223c125\u00a0\u03bcmol\u00a0m\u22122\u00a0s\u22121) under short day condition (12-h light\/12-h dark). For salt stress treatments and ion measurements, wild type and transgenic seedlings were transferred to pots containing soil. Plants were watered bi-weekly with 1\/8 concentrated MS salt solution supplemented with or without 200-mM NaCl and grown at 25\u00a0\u00b1\u00a02\u00b0C with cool white fluorescent light (\u223c125\u00a0\u03bcmol\u00a0m\u22122\u00a0s\u22121), 70% humidity, under long-day conditions (16-h light\/8-h dark) for 6\u00a0weeks. Mature plants treated in the same way were also used to determine cellular Na+ concentrations. The third leaf from the top of each plant was collected and dried at 90\u00b0C for 24\u00a0h, and the dry weight of each sample was measured. The dried leaf material was immersed in 0.1-M HAc, followed by water bath incubation at 90\u00b0C for 2.5\u00a0h, and analyzed for Na+ content using atomic absorption spectrophotometry. For hydroponic salt tolerance experiments, wild type plants and two independent transgenic lines (T2 generation) were grown hydroponically in Hoagland solution. Plants were grown at 25\u00a0\u00b1\u00a02\u00b0C with cool white fluorescent light (\u223c125\u00a0\u03bcmol\u00a0m\u22122\u00a0s\u22121), 70% humidity, under long-day conditions (16-h light\/8-h dark). The nutrient solution was replaced weekly.\nOxidative stress experiments\nMalondialdehyde (MDA) concentration was determined as a biomarker for oxidative stress. To measure MDA concentration, leaf samples from plants grown at 200-mM NaCl were ground in 5\u00a0ml of 0.1% trichlor acetic acid (TCA) and mixed with 5\u00a0ml of 0.5% thiobarbituric acid. The samples were then boiled for 10\u00a0min, cooled down to room temperature and centrifuged at 1,200\u00a0\u00d7\u00a0g. The cleared supernatant was analyzed by monitoring the difference in absorbance at A532 and A600. For oxidative damage experiments, leaf disks of 1.5-cm diameter were excised from healthy and fully expanded leaves of two-month-old tobacco plants. The disks were floated in a 10-ml solution with different concentrations of H2O2 or water (control) for 72\u00a0h. Chlorophyll a and b concentrations were measured as described previously (Lichtenthaler 1987). The treatment was carried out with continuous white light at 25\u00a0\u00b1\u00a02\u00b0C. The experiment was repeated at least three times.\nEnzyme assays and protein determination\nEnzyme assays were carried out spectrophotometrically using a nucleic acid and protein analyzer (Du 640, Beckman Coulter). One-month-old seedlings were treated with H2O or 1% H2O2 for 12\u00a0h, and ground in extraction buffer (1\u00a0mM ascorbate in 50-mM potassium phosphate buffer, pH 7.8) at a ratio of 0.1\u00a0g leaf material\/1\u00a0ml ice-cold extraction buffer. The slurry was transferred to a centrifuge tube and centrifuged at 20,000\u00a0\u00d7\u00a0g at 4\u00b0C for 20\u00a0min. The supernatant was used for enzyme activity assays and total protein determination as described previously (Badawi 2004; Bradford 1976). Ascorbate peroxidase (APX) activity was determined by measuring the rate of ascorbate oxidation at A290 in a 100-\u03bcl-reaction mixture (1-mM EDTA in 50-mM potassium phosphate buffer, pH 7.8, 0.2-mM ascorbate, 20-mM H2O2 and 5-\u03bcl soluble protein extract). APX activity is expressed as micromoles of ascorbate oxidized per minute per milligram of protein. Catalase (CAT) activity was measured by monitoring the decrease in absorbance at A240 caused by the disappearance of H2O2 in a 100-\u03bcl-reaction mixture (1-mM EDTA in 50-mM potassium phosphate buffer, pH 7.8, 20-mM H2O2 and 5-\u03bcl protein extract). The activity of superoxide dismutase (SOD) was measured at A560, in a 3-ml reaction mixture (13.05-mM dl-methionine, 0.1-\u03bcM EDTA, 0.075-mM Nitro Blue tetrazolium chloride and 2-\u03bcM riboflavin in 50-mM phosphate buffered saline, pH 7.8, and 100-\u03bcl protein extract).\nWhole plant drought tolerance test\nWild type and transgenic plant seeds were surface sterilized as described above. Seedlings germinated on MS medium were transplanted to soil. After two months, healthy plants of the same size and age were pooled into two groups. For the first group (12 individual plants, each grown in a 6.5-inch pot), plants were irrigated with 60\u00a0g\/l PEG-6000 to simulate drought stress. After 21\u00a0days, the plants were photographed. Simultaneously, plants of the second group were subjected to drought stress by omitting watering. After three weeks, the plants were re-hydrated and observed for recovery. Photos were taken 21\u00a0days after initiation of the stress treatment. The relative humidity was maintained at \u223c60%. The experiments were repeated twice with three replicates in each experiment.\nFreezing tolerance test\nThree-week-old seedlings of wild type and transgenic plants grown on MS medium were cultured at 4\u00b0C for one day under long day condition (16-h light\/8-h dark). After cold acclimation, the plants were left at \u221220\u00b0C for 1 or 2\u00a0h, transferred immediately to 4\u00b0C for another 12\u00a0h (overnight). Subsequently, plants were kept in the greenhouse at 25\u00b0C and observed further. Photographs were taken seven days after initiation of the recovery growth in the greenhouse.\nDetermination of proline concentration\nFour-week-old seedlings grown on MS medium were transferred onto filter paper in a Petri dish saturated with 100- or 200-mM NaCl, incubated at 25\u00a0\u00b1\u00a02\u00b0C for 24\u00a0h under continuous white light. After the treatment, proline content was determined as described previously (Bates and Waldren 1973).\nWestern blot analysis\nLeaves from either Arabidopsis or tobacco plants were ground in liquid N2 and suspended in 1.5\u00a0ml of 30\u00a0mM potassium phosphate buffer (pH 7.5) containing 40-mM NaCl and 3-mM dithiothreitol (Powell 1986). Homogenates were centrifuged for 5\u00a0min at 12,000\u00a0\u00d7\u00a0g. Supernatants were transferred to new tubes, and protein concentrations were determined as described previously (Bradford 1976). A 10-\u03bcg sample of total protein was mixed with loading buffer, boiled for 3\u00a0min, and size-fractionated through electrophoresis in a 12.5% SDS polyacrylamide gel. Separated proteins were transferred to a PVDF membrane (Millipore Corporation) according to the manufacturer\u2019s instructions. A polyclonal antibody against AtIpk2\u03b2 protein purified from recombinant E. coli was raised in rabbits. Antiserum was purified by IgG affinity purification. The anti-AtIpk2\u03b2 antiserum was used at a 1:5,000 dilution in TBST. Peroxidase-labeled affinity purified secondary antibody isolated from a pool of serum from goats immunized with purified rabbit IgG was used at a 1:1,000 dilution in TBST (KPL Corporation).\nStress gene expression analysis using reverse transcription PCR\nThree-week-old tobacco seedlings grown on MS agar plates were treated with 300-mM NaCl for 5\u00a0h. Total RNA was isolated with TRIZOL Reagent (Invitrogen) and used for reverse transcription (RT) to generate first-strand cDNA. RT was performed on 2-\u03bcg total RNA per reaction using ReverTra Ace (TOYOBO). A portion of the resulting cDNA was then subjected to PCR amplification using gene specific primers (Table\u00a01). Expression level of the Nicotiana tabacum ACTIN gene, which served as reference, was monitored with NtActin forward and reverse primers (Table\u00a01).\nStatistical analysis\nFor statistical analyses, the Student\u2019s t-test was used to generate every P-value. The alpha level was 0.05. The tests were one-tailed. The data were normalized and all samples were normally distributed with homogeneity of variance.\nResults\nYeast complementation with AtIpk2\u03b2\nIn S. cerevisiae, the ARG82\/IPK2 gene encodes an inositol polyphosphate kinase (Odom et\u00a0al. 2000). The S. cerevisiaearg82\/Ipk2 (arg82\u0394) mutant strain that lacks Ipk2 activity has slowed growth at 30\u00b0C and is unable to grow at 37\u00b0C (Odom et\u00a0al. 2000). We and others have reported previously that AtIpk2\u03b2 rescues its growth defects at 37\u00b0C (Stevenson-Paulik et\u00a0al. 2002; Xia et\u00a0al. 2003). This result led us to postulate that AtIpk2\u03b2 may also be able to rescue its growth defects when other abiotic stresses were imposed. We observed that under adverse growth conditions such as high salt concentration or upon osmotic stress treatment, growth of the arg82\u0394 mutant was severely impaired when compared to that of the wild type (Fig.\u00a01a). To test whether AtIpk2\u03b2 restores the growth defect of the mutant strain under these conditions, we transformed it with the ArabidopsisAtIpk2\u03b2 cDNA. Expression of AtIpk2\u03b2 almost completely restored growth of the arg82\u0394 mutant under both kinds of stresses (Fig.\u00a01a). It also restored growth at 37\u00b0C (Fig.\u00a01a), as reported earlier (Stevenson-Paulik et\u00a0al. 2002; Xia et\u00a0al. 2003). We also performed experiments using liquid media and observed again, that AtIpk2\u03b2 restores growth under conditions of abiotic stress (Fig.\u00a01b).\nFig.\u00a01Expression of AtIpk2\u03b2 in S. cerevisiaearg82\u0394. (a) AtIpk2\u03b2 rescues growth defects of the yeast arg82\u0394 mutant under high salt or mannitol stress, and at 37\u00b0C. Ten-fold serial dilutions of the strain were plated and incubated at 30\u00b0C for 48\u00a0h on YPD medium supplemented with either 0.4- or 0.8-M NaCl, or with 0.4- or 0.8-M mannitol. Plates incubated at 37\u00b0C contained YPD medium without extra NaCl or mannitol. WT, wild type; arg82\u0394, arg82\u0394 mutant; arg82\u0394\u00a0+\u00a0AtIpk2\u00df, arg82\u0394 mutant harbouring plasmid pYX212-AtIpk2\u03b2. (b) Liquid yeast cultures. Cells were grown for three days to saturation in YPD medium (OD600\u00a0=\u00a012.0). Five \u03bcl of cell culture were then used to inoculate 3\u00a0ml of defined synthetic minimal medium supplemented with different concentrations of NaCl or mannitol. Cell density was determined at various time points as absorbance at 600\u00a0nm. Cells were grown at 30\u00b0C. WT, wild type; arg82\u0394, arg82\u0394 mutant; arg82\u0394\u00a0+\u00a0AtIpk2\u00df, arg82\u0394 mutant harbouring pYX212-AtIpk2\u03b2; arg82\u0394\u00a0+\u00a0vector, arg82\u0394 mutant harbouring pYX212\nAtIpk2\u03b2 is abiotic stress regulated in Arabidopsis\nWe were interested to know whether different kinds of stresses such as salinity, cold and drought, or treatment with ABA would affect AtIpk2\u03b2 transcript levels in Arabidopsis plants. To this end we performed quantitative real-time PCR (qRT-PCR). In ten-day-old seedlings grown on MS medium, AtIpk2\u03b2 transcript level increased slightly upon exposure to cold or drought stress, but decreased in response to salt or ABA treatment (Fig. S1A in Supplementary Material). These results showed that AtIpk2\u03b2 is a stress responsive gene potentially involved in different abiotic stress- and ABA-triggered signaling pathways in plants. We further tested AtIpk2\u03b2 protein level by Western blot analysis. Fourteen-day-old Arabidopsis seedlings grown on MS medium were subjected to different stress treatments for 24\u00a0h. In seedlings treated with 500-mM mannitol, AtIpk2\u03b2 protein level increased slightly, however no significant changes in protein level were detected in seedlings subjected to cold or NaCl stress (Fig. S1B in Supplementary Material).\nConstitutive expression of AtIpk2\u03b2 in transgenic tobacco\nTo test whether constitutive expression of AtIpk2\u03b2 affects abiotic stress tolerance in plants, we introduced its open reading frame (Fig.\u00a02a) into the genome of tobacco (Nicotiana tabacum cv. SR-1) by Agrobacterium tumefaciens-mediated transformation. Expression of AtIpk2\u03b2 in the transgenic plant was controlled by the Cauliflower Mosaic Virus (CaMV) 35S promoter. Forty-six independent transgenic plants (T0 generation) were obtained; six lines were grown to produce seeds. Transgenic plants homozygous for the 35S:AtIpk2\u03b2 transgene were obtained and three lines (S13, S14 and S15) were chosen for further experiments. Constitutive expression of AtIpk2\u03b2 did not affect overall plant morphology although the transgenic plants grew slightly faster than wild type plants under normal growth condition. Western blot analyses were performed to test for the presence of AtIpk2\u03b2 protein. A 33-kDa band corresponding to AtIpk2\u03b2 was identified in Arabidopsis plants; similarly, 33-kDa AtIpk2\u03b2 protein was detected in the transgenic but not the wild type tobacco lines (Fig.\u00a02b), indicating that it was successfully expressed in the genetically modified plants.\nFig.\u00a02Construct used for tobacco transformation and Western blot analysis of AtIpk2\u03b2 in wild type and transgenic tobacco plants. (a) Schematic map of the binary construct used for tobacco transformation. Expression of AtIpk2\u03b2 is driven by the Cauliflower Mosaic Virus 35S promoter. Ocs, ocs terminator; RB and LB, right and left border of T-DNA, respectively. (b) Western blot analysis. Lane 1, wild type Arabidopsis; lane 2, wild type tobacco; lanes 3\u20137, transgenic AtIpk2\u03b2 expressing tobacco lines S11, S13, S14, S15, and S18. Coomassie Bright Blue stained gel is shown below the Western blot to demonstrate equal loading of protein\nExpression of AtIpk2\u03b2 increases salt tolerance of transgenic tobacco plants\nWe first studied the salt tolerance of AtIpk2\u03b2 expressing plants during germination and early stages of seedling development. Seeds of wild type and the three homozygous transgenic lines S13, S14 and S15 were germinated on MS medium containing different concentrations of NaCl. Germination rates of wild type and transgenic seeds were similar when cultured on normal MS medium (Fig.\u00a03a). However, germination of wild type seeds was significantly impaired on MS medium supplemented with 100- or 200-mM NaCl, whereas germination of transgenic seeds was less strongly affected. After eight days on high salt MS medium (100-mM NaCl), only \u223c28% of the wild type seeds, but more than 70% of the seeds obtained from AtIpk2\u03b2 expressing plants germinated (Fig.\u00a03b). When sown on MS medium supplemented with 200-mM NaCl, only \u223c7% of the wild type seeds, but \u223c30% of the transgenic seeds germinated after 18\u00a0days (Fig.\u00a03c). At low salt concentration (50-mM NaCl), wild type and transgenic seeds displayed similar germination rates (data now shown). At 150-mM NaCl, both wild type and transgenic seeds germinated. However, development of wild type seedlings was impaired; plants produced smaller leaves and less root biomass than did transgenic seedlings (Fig.\u00a03d). To further test the effect of salt on growth, 15-day-old wild type and transgenic seedlings were transferred to MS medium supplemented with NaCl at different concentrations. After two additional months, strong growth retardation was observed in wild type seedlings. Inhibition of growth was less evident in transgenic lines (Fig.\u00a03e). A root-bending assay has previously been used to test the effect of elevated salt concentration on root growth (An et\u00a0al. 2007; Zhu et\u00a0al. 1998). At 50-mM NaCl on MS medium, root growth of both wild type and transgenic lines was accelerated, but it was inhibited at increasing NaCl concentrations. At 150-mM NaCl, inhibition of root growth was significantly more pronounced in wild type than in transgenic plants (Fig.\u00a03f, g).\nFig.\u00a03Germination rate and early seedling development of transgenic plants. (a\u2013c) Percentage of germinating seeds of wild type (WT) and transgenic T2 plants (S13, S15) grown on MS medium supplemented with 0-, 100- or 200-mM NaCl. Results are presented as means and standard errors from three independent experiments (\u2265100 seeds of each line were sown for each experiment). (d) Wild type (WT) and transgenic seeds (S13, S15) geminated on MS medium supplemented with 50 or 150-mM NaCl. Photo was taken three weeks after seeds were sown. (e) Wild type (WT) and transgenic lines (S13, S14, S15) grown on MS medium supplemented with different concentrations of NaCl for 2\u00a0months. (f) Root bending assay of wild type (WT) and transgenic plants (S13, S15) on MS medium supplemented with 0 or 150-mM NaCl. Representative pictures are shown. (g) Primary root lengths of wild type (WT) and transgenic plants (S13, S15) in the presence of different salt concentrations. Root growth under control condition (0-mM NaCl) was set to 100%. The results are shown as means and standard errors from three independent experiments (\u226515 seeds of each line were sown for each experiment). *** indicates significant differences in comparison to the wild type at P\u00a0<\u00a00.001 (Student\u2019s t-test)\nWe also performed salt tolerance experiments with wild type and two transgenic lines grown in the greenhouse in soil. The plants were watered bi-weekly with 1\/8 concentrated MS salt solution supplemented with or without 200-mM NaCl. After six weeks, distinct differences were observed between wild type and transgenic plants exposed to 200-mM NaCl (Fig.\u00a04a). Although both sets of plants flowered under conditions of salt stress, wild type plants developed much shorter stems (Fig.\u00a04b), produced fewer pods (Fig.\u00a04c), and produced seeds that were not viable. We determined endogenous Na+ levels in leaves and roots of wild type and transgenic plants grown in the absence or presence of 200-mM NaCl. Both wild type and transgenic plants exposed to salt contained elevated levels of Na+ ions in leaves and roots (Fig.\u00a04d), however, ion concentrations were not significantly different between wild type and transgenic plants (the same was true for plants grown under normal conditions). We also grew tobacco plants in hydroponic culture. In the absence of salt stress, no overt morphological difference was observed between wild type and transgenic plants. However, growth of wild type tobacco was severely inhibited in the presence of 300-mM NaCl (Fig.\u00a04e), leading to a depression of shoot height by about one third, and of plant fresh weight by about 50% in comparison to AtIpk2\u03b2 expressing tobacco plants after a two-month stress treatment (Fig.\u00a04f). All these results indicated that AtIpk2\u03b2 alleviated the negative effects imposed by salt stress on plant growth.\nFig.\u00a04Salt tolerance test to compare wild type and AtIpk2\u03b2 expressing tobacco plants. For each experiment, twelve plants of each line were used. (a) Photograph of representative wild type (WT) and transgenic plants (S13, S15) watered with 200-mM NaCl for six weeks. (b), (c) Shoot height and pod numbers of wild type (WT) and transgenic (S13, S15 ) plants treated with 200-mM NaCl. * and ** indicate significant differences in comparison to the wild type at P\u00a0<\u00a00.05 and P\u00a0<\u00a00.01, respectively (Student\u2019s t-test). (d) Na+ content in leaves and roots of wild type (WT) and transgenic plants (S13, S15) grown in soil supplied with 0 or 200-mM NaCl solution. (e) Photograph of a representative wild type (WT) and transgenic line S15 hydroponically grown in nutrient solution supplemented with 300-mM NaCl for two months. (f) Shoot height and (g) fresh weight of wild type (WT) and transgenic lines S13 and S15 grown for two months under control (0-mM NaCl) or high salt (300-mM NaCl) conditions. Results are presented as means and standard errors from three independent experiments. ** indicates significant differences in comparison to the wild type at P\u00a0<\u00a00.01 (Student\u2019s t-test)\nTransgenic plants showed increased tolerance to osmotic, drought, freezing temperature and oxidative stress\nIn order to determine whether expression of AtIpk2\u03b2 affects osmotic stress tolerance in plants, we watered wild type and transgenic tobacco plants (lines S13 and S15) with PEG-6000 (60\u00a0g\/l) as described previously (Eltayeb et\u00a0al. 2006). Under such experimental conditions, wild type plants were less vigorous, flowered earlier and produced smaller leaves than AtIpk2\u03b2 expressing plants (Fig.\u00a05a). We also tested the effect of water shortage on plant growth. After a 21-day drought period, wild type plants completely wilted, while transgenic plants were less severely affected (Fig.\u00a05b). At the end of the drought tolerance test, the plants were re-watered and grown further to allow seed setting. Transgenic plants produced larger shoots than wild type plants (Fig.\u00a05c).\nFig.\u00a05Effect of drought and osmotic stress on plant performance. For each experiment, twelve plants of wild type (WT) and each transgenic line (S13, S15) were used. (a) Wild type and transgenic plants grown in soil were watered with 60\u00a0g\/l PEG-6000. Photograph was taken after 21\u00a0days of treatment. (b) Water was withheld for 21\u00a0days to impose drought stress. (c) Drought stressed plants were re-watered and grown for seed setting. (d) Proline content of wild type and transgenic plants after treatment with 100 or 200\u00a0mM NaCl for 24\u00a0h. Results are presented as means and standard errors from three independent experiments. ** indicates significant differences in comparison to the wild type at P\u00a0<\u00a00.01 (Student\u2019s t-test)\nProline accumulation in response to osmotic or salinity stress has been well documented in prokaryotic and eukaryotic organisms (Schobert 1997). Here, we compared the proline content of wild type and transgenic plants treated with 100- or 200-mM NaCl. Although proline accumulated in a dose-dependent manner in both wild type and transgenic plants, proline concentration was slightly higher in transgenic plants in the presence of 200-mM NaCl (Fig.\u00a05d).\nThe results presented in Fig.\u00a04 show that AtIpk2\u03b2 expression increased the salt tolerance of transgenic tobacco plants. Since salt stress exerts osmotic stress to plant cells in addition to its ionic toxicity, we further determined if AtIpk2\u03b2 expression confers elevated tolerance to oxidative stress on transgenic plants. Leaf disks from wild type and transgenic tobacco plants were exposed to increasing concentrations of H2O2 for 72\u00a0h. As shown in Fig.\u00a06, transgenic plants exhibited a strong tolerance to oxidative stress. Leaf disks from wild type plants were almost completely bleached at the end of the stress treatment, whereas those from transgenic plants showed significant resistance to H2O2-caused damage (Fig.\u00a06a).\nFig.\u00a06Oxidative stress and short term freezing tolerance of wild type (WT) and transgenic tobacco plants (S13, S15). (a) Leaf disks were incubated in different concentration of H2O2 (1% and 2%, respectively) under continuous white light for 72\u00a0h. H2O2 induced leaf yellowing is delayed in the transgenic plants. (b) Three-week-old seedlings were stored at \u221220\u00b0C for 2\u00a0h, and then transferred to normal temperature for further growth. Photograph was taken seven days after the freezing treatment. (c) Quantification of the seedling survival rate (determined seven days after the freezing treatment). Results are presented as means and standard errors from three independent experiments. Fore each experiment, 40 seedlings of each line were used. *** indicates significant difference in comparison to the wild type at P\u00a0<\u00a00.001 (Student\u2019s t-test). (d) Chlorophyll content was determined in leaf disks of wild type and T2 transgenic plants kept in H2O2 for 72\u00a0h. Disks floated in water served as control. Results are preans and standard errors from three independent experiments. ** and *** indicate significant differences in comparison to the wild type at P\u00a0<\u00a00.01 and P\u00a0<\u00a00.001, respectively (Student\u2019s t-test)\nTo further examine the effect of constitutive AtIpk2\u03b2 expression on plant performance, the tolerance towards freezing temperature was analyzed. To this end, three-week-old seedlings were kept at \u221220\u00b0C for 1 or 2\u00a0h, respectively, and subsequently allowed to recover for seven days in the greenhouse. Most transgenic plants resumed normal growth after the 2\u00a0h treatment at \u221220\u00b0C (Fig.\u00a06B), but only very few wild type plants did so. When treated for 1\u00a0h at \u221220\u00b0C, the damage was less severe, but again transgenic plants grew better than wild type plants after transfer back to the greenhouse (data not shown). Transgenic seedlings showed a superior survival rate after short-term \u221220\u00b0C treatment (Fig.\u00a06c). In the presence of oxidative stress, chlorophyll loss was significantly delayed in AtIpk2\u03b2 expressing plants compared to that of the wild type (Fig.\u00a06d).\nLipid hydroperoxidation is an effective indicator of cellular oxidative damage (Yoshimura et\u00a0al. 2004). Changes in the rates of lipid hydroperoxide production induced by oxidative stress were measured by determining MDA content in leaf disks. At high salinity (200-mM NaCl), MDA concentration markedly increased in wild type plants, whereas only a marginal increase was observed in the two transgenic lines (Fig.\u00a07a). Although superoxide dismutase (SOD) activity was largely unaffected in wild type and transgenic plants treated with 1% H2O2 (Fig.\u00a07b), a slight elevation of APX activity was observed in transgenic plants (Fig.\u00a07c). Also, catalase (CAT) activity increased in transgenic plants (Fig.\u00a07d). These results indicate that constitutive expression of AtIpk2\u03b2 enhanced tolerance to oxidative stress-induced membrane hydroperoxidation in transgenic tobacco plants.\nFig.\u00a07MDA level, anti-oxidative enzyme activity, and expression level of stress regulated genes in transgenic plants. (a) MDA levels in wild type and transgenic lines S13, S15 after treatment with 200-mM NaCl. (b\u2013d) One-month-old seedlings were incubated for 12\u00a0h in 1% H2O2 or water (control) under continuous white light. SOD, APX and CAT activities were determined. Results are presented as means and standard errors from three independent experiments. * and ** indicate significant differences in comparison to the wild type at P\u00a0<\u00a00.05 and P\u00a0<\u00a00.01, respectively (Student\u2019s t-test). (e) Two-week-old seedlings of wild type and transgenic lines (S13, S15) were used for RNA extraction. For the salt stress experiments, seedlings were treated with 300-mM NaCl for 5\u00a0h before RNA isolation. The transcriptional levels of five stress genes were determined by RT-PCR analyses. The stress genes used for the tests are as follows: lipid transfer protein (AY562132); fructose-bisphosphate aldolase (AY554169); raffinose synthase family protein\/seed imbibition protein (AY554170); group 2 LEA proteins NtERD 10B (AB049336) and NtERD 10C (AB049337)\nIncreased stress responsive gene expression in transgenic tobacco plants\nMany genes responding to abiotic stress were cloned in recent years and used as molecular markers to monitor the activity of stress-signaling pathways in plants (Kasuga et\u00a0al. 2004). The stress experiments described above suggested that AtIpk2\u03b2 plays an important role in regulating stress tolerance in plants. To address whether or not AtIpk2\u03b2 affects the expression of stress responsive genes, we investigated the transcriptional levels of five marker genes in wild type and transgenic tobacco plants by RT-PCR. The marker genes chosen are identical or homologous to those encoding lipid transfer protein, fructose-bisphosphate aldolase, raffinose synthase, chloroplast ATPase, and group 2 LEA proteins of tobacco. As shown in Fig.\u00a07, transcript levels of these genes were higher in transgenic plants than in the wild type under both, normal and salt stress conditions (Fig.\u00a07).\nDiscussion\nA number of genes encoding Ins(1,4,5)P3 3-kinase or Ins(1,4,5)P3 dual specificity 6-\/3-kinases were cloned and biochemically characterized in the past years. Examples include genes from yeast (Odom et\u00a0al. 2000), animals (Bertsch et\u00a0al. 1999), human (Dewaste et\u00a0al. 2000), and higher plants (Stevenson-Paulik et\u00a0al. 2002; Xia et\u00a0al. 2003; Xu et\u00a0al. 2005). It has recently been reported that in yeast (S. cerevisiae) inositol polyphosphate kinase (Kcs1p) is required for resistance to salt stress, cell wall integrity, and vacuolar morphogenesis (Dubois et\u00a0al. 2002), and that overexpression of D-IP3K1, a Drosophila IP3-kinase gene, confers resistance of flies to H2O2-, but not to paraquat-induced oxidative stress (Monnier et\u00a0al. 2002). Further genetic studies indicated that the protective effect conferred by elevated D-IP3K1 expression is mainly due to a lowered IP3 level and thus reduced Ca2+ ion release from internal stores, rather than an increased IP4 level.\nBased on these observations, we were interested to know whether constitutive expression of AtIpk2\u03b2 confers improved resistance to abiotic stresses on higher plants. Since the Arabidopsis AtIpk2\u03b2 protein shares high amino acid sequence identity with the yeast IPK2 enzyme, which functions as a transcriptional regulator (also called Arg82 or ArgRIII) and displays dual-specificity InsP3-InsP4 kinase activity (Odom et\u00a0al. 2000), we first produced transgenic Arabidopsis (ecotype Columbia-0) plants overexpressing AtIpk2\u03b2. Surprisingly, these plants did not exhibit a significant improvement to salt or osmotic stress in our preliminary studies (data not shown). This may be due to the possible overlapping\/redundant functions of the two Arabidopsis inositol polyphosphate kinases (AtIpk2\u03b1 and AtIpk2\u03b2) in\u00a0vivo, as suggested earlier by the York group (Stevenson-Paulik et\u00a0al. 2002). To examine this possibility, we further compared the growth of Arabidopsis wild type as well as transgenic AtIpk2\u03b2 over-expresser (35S::AtIpk2\u03b2) and knock-out mutant (atipk2\u03b2, SALK_104995, ABRC) plants. All three types of plants had similar growth phenotypes when subjected to salt or osmotic stress (data not shown). Here we introduced AtIpk2\u03b2 into tobacco plants and found that transgenic lines developed superior tolerance to various types of abiotic stresses.\nPreviously, AtNHX1, a vacuolar Na+\/H+ antiporter gene from Arabidopsis, was over-expressed in tomato and canola to improve the salt tolerance of transgenic plants (Zhang and Blumwald 2001). However, a comparative analysis of Na+ and K+ accumulation in leaves and roots of transgenic AtNHX1 over-expressers and wild type plants grown under high-salt conditions was not performed because the high (200\u00a0mM) NaCl concentration used in these experiments was lethal to the wild type plants. In the study reported here, the relatively high salt tolerance of wild type tobacco plants made it possible to perform such an analysis. In the presence of 200-mM NaCl, despite a similar increase of Na+ concentration in wild type and transgenic plants, slightly more Na+ accumulated in leaves after long-term salt stress (Fig.\u00a04d). This phenomenon is different from what has been seen in transgenic wheat overexpressing AtNHX1 (Xue et\u00a0al. 2004). Salinity causes two major damages to plant cells, water deficit resulting from the relatively high solute concentrations in the soil, and ion toxicity due to altered K+\/Na+ ratios as well as excessive Na+ and Cl\u2212 contents (Apse and Blumwald 2002). The Na+\/H+ exchange activity of AtNHX1 is regulated in a Ca2+- and pH-dependent manner as reported earlier (Yamaguchi et\u00a0al. 2005). The increased accumulation of salt in both leaves and roots suggests that unlike AtNHX1, which mainly works through sodium compartmentation, AtIpk2\u03b2 may function through both sodium extrusion and compartmentation controlled by Ca2+-dependent signal transduction, although the precise mechanism of this still remains to be elucidated.\nProline contributes to osmotic adjustment (LeRudulier et\u00a0al. 1989) and the protection of macromolecules during dehydration (Yancey et\u00a0al. 1982), and as a hydroxyl radical scavenger (Hong et\u00a0al. 2000). After treatment with 200-mM NaCl, but not 100-mM NaCl, proline accumulated to slightly higher levels in transgenic than in wild type tobacco plants (Fig.\u00a05d). An increase in proline content was also observed in salt tolerant Arabidopsis with suppressed levels of proline degradation (Nanjo et\u00a0al. 1999), transgenic salt tolerant tobacco plants with enhanced levels of proline biosynthesis (Kishor et\u00a0al. 1995), and transgenic salt tolerant tomato plants overexpressing AtNHX1 (Zhang and Blumwald 2001). So, it is possible that the elevated concentration of proline in transgenic plants helps to protect anti-oxidative enzyme thus alleviating the negative effects imposed by salt on transgenic AtIpk2\u03b2 plants.\nSalt-stress-induced generation of reactive oxygen species (ROS) has been thought as one of the major causes of adverse effects of high salinity. ROS are products of chloroplast and mitochondrial metabolism produced during stress; they cause membrane damage resulting in electrolyte leakage (Apse and Blumwald 2002). At 200-mM NaCl, a relatively strong increase of MDA production was observed in wild type plants, but only a slight increase was seen in the two transgenic lines (Fig.\u00a07a). Furthermore, catalase (CAT) activity increased significantly in transgenic plants after treatment with 1% H2O2 (Fig.\u00a07d). These results led us to hypothesize that constitutive expression of AtIpk2\u03b2 improves cell membrane integrity. Evidence supporting this hypothesis was obtained from stress tolerance experiments with transgenic Arabidopsis overexpressing aldehyde dehydrogenase, or transgenic tobacco overexpressing Chlamydomonas glutathione peroxidase (Yoshimura et\u00a0al. 2004).\nThe induction of stress-related genes has been taken as a hallmark of stress adaptation in plants (Thomashow 1999). Earlier studies have shown that lipid transfer proteins are induced by osmotic stress (Smart et\u00a0al. 2000). Genes encoding plastidic aldolase are induced by salt (Yamada et\u00a0al. 2000). Transgenic plants overexpressing such enzymes exhibit improved tolerance to the respective stresses. Here, we observed that constitutive expression of AtIpk2\u03b2 under the control of the CaMV 35S promoter triggered an increased expression of various stress responsive genes in transgenic tobacco. Already in the absence of salinity stress, transcript levels of genes encoding raffinose synthase family protein\/seed imbibition protein (AY554170) and group 2 LEA protein NtERD 10C (AB049337) were found to be slightly elevated in the transgenic plants. At high salinity, the expression levels of genes encoding lipid transfer protein (AY562132) and raffinose synthase family protein\/seed imbibition protein (AY554170) increased in the transgenic lines. These results suggest that AtIpk2\u03b2 acts in one or more signal transduction pathway(s) thereby affecting the activity of stress-related genes and stress tolerance.\nMany stresses evoke an elevation of the cytosolic calcium ion concentration, including salinity (Knight et\u00a0al. 1997), drought (Knight et\u00a0al. 1997), cold (Knight et\u00a0al. 1996), and oxidative stress (Sanders et\u00a0al. 2002). Previously, it has been shown that salinity and osmotic stress induce a rapid and transient increase of IP3 levels (DeWald et\u00a0al. 2001;Takahashi et\u00a0al. 2001), and the reported accumulation of IP3 occurs during a time frame similar to that observed for stress induced Ca2+ mobilization. Therefore, the possibility exists that enhanced abiotic stress tolerance of transgenic AtIpk2\u03b2 plants results from changes of Ca2+ dependent signaling. Whether this is indeed the case has to be analyzed in future studies\nPlant growth and productivity are severely affected by abiotic stress. Augmented resistance towards various environmental stresses has been a desirable goal for genetic improvement of plants. Stress tolerance can for example be improved by altering the expression of stress-responsive genes in transgenic plants. Representatives include genes encoding chitinase and glucanase (Hong and Hwuang 2006), betaine aldehyde dehydrogenase (Yang et\u00a0al. 2005), tonoplast and plasma membrane Na+\/H+ antiporters (Zhang and Blumwald 2001; Apse and Blumwald 2002), protein kinases (Xiong and Yang 2003), transcription factors (Waller et\u00a0al. 2006), peroxidases (Yoshimura et\u00a0al. 2004), DNA helicase (Sanan-Mishra et\u00a0al. 2005), and DNA binding protein (polypeptide; Kalifa et\u00a0al. 2004). Although the precise mode of action of AtIpk2\u03b2 in plant responses to stress remains elusive, the results in this work provide direct evidence that alteration of AtIpk2\u03b2 expression can significantly modify plant tolerance to various abiotic stresses, indicating a great potential of this gene for engineering stress tolerant crops.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nQuantitative real-time RT-PCR and Western blot analyses of AtIpk2b in wild type Arabidopsis seedlings in response to different stresses. Fourteen-day old seedlings grown on MS agar plates were treated with 300-mM NaCl or 500-mM mannitol, or kept at 4\u00b0C for 24\u00a0h. (A) Quantitative real-time RT-PCR analysis of AtIpk2b transcript level. The experiments were repeated three times independently and the average calculated. Error bars represent standard error of the mean. (B) Western blot analysis of AtIpk2b expression level. Coomassie Bright Blue stained gel is shown below the Western blot to demonstrate equal protein loading. (TIF 457\u00a0kb)","keyphrases":["stress tolerance","transgenic tobacco","atipk2\u03b2","arabidopsis thaliana","inositol phosphate","ip3"],"prmu":["P","P","P","P","P","P"]}
{"id":"Biochim_Biophys_Acta-2-1-2258317","title":"Neuronal calcium sensor proteins are unable to modulate NFAT activation in mammalian cells\n","text":"Calcium activated gene transcription through Nuclear Factor of Activated T-cells, (NFAT) proteins, is emerging as a ubiquitous mechanism for the control of important physiological processes. Of the five mammalian NFAT isoforms, transcriptional activities of NFATs 1-4 are stimulated by a calcium driven association between the ubiquitous phosphatase calcineurin and the calcium-sensing protein calmodulin. Published in vitro evidence has suggested that other members of the calmodulin super-family, in particular the neuronal calcium sensor (NCS) proteins, can similarly modulate calcineurin activity. In this study we have assessed the ability of NCS proteins to interact directly with calcineurin in vitro and report a specific if weak association between various NCS proteins and the phosphatase. In an extension to these analyses we have also examined the effects of over-expression of NCS-1 or NCS-1 mutants on calcineurin signalling in HeLa cells in experiments examining the dephosphorylation of an NFAT-GFP reporter construct as a readout of calcineurin activity. Results from these experiments indicate that NCS-1 was not able to detectably modulate calcineurin\/NFAT signalling in a live mammalian cell system, findings that are consistent with the idea that calmodulin and not NCS-1 or other NCS family proteins is the physiologically relevant modulator of calcineurin activity.\n1\nIntroduction\nCalcineurin (CN) [1] is a serine\/threonine specific protein phosphatase present in all eukaryotes that is responsible for the modulation of numerous cell signalling pathways. Calcineurin interacts with the ubiquitous EF-hand containing calcium (Ca2+) sensor calmodulin (CaM) [2] and this obligate association drives calcium dependent phosphatase activity, one of the primary cellular targets of which are the Nuclear Factor of Activated T-cell (NFAT) family of transcription factors. This family of gene regulatory proteins is composed of five members (NFAT1-5) [3,4], that were first identified as fundamental components of the recombinatorial immune system of vertebrates [5], but which have now been implicated in a far broader range of diverse and important physiological processes. Expression of NFAT proteins is not restricted to cells of the immune system, demonstrated by their presence and effect on key functions in a selection of other cell and tissue types including brain and muscle. In a neuronal setting, NFATs have been implicated in axonal remodelling, synaptic plasticity and memory function [6-9]. Transcriptional events mediated by activated NFATs have been linked to aspects of cardiovascular development [10,11] and they have also been suggested to drive proliferative and apoptotic pathways in epithelial cells, fibroblasts, pre-adipocytes, pancreatic \u03b2-cells and osteoblasts [12-16]. The discovery that NFAT proteins can influence cell cycle regulation has led to the proposal that they may also be involved in certain aspects of tumorigenesis [17].\nNFAT proteins in unstimulated cells are heavily phosphorylated on multiple serine residues by of a number of constitutively active kinases [18]. This modification acts to mask a nuclear localisation signal and under such circumstances NFATs remain predominantly cytosolic and transcriptionally inactive. Stimulation of cells with agonists coupled to the phospholipase-C\/IP3 generating pathway, depletion of endoplasmic reticulum Ca2+ stores and subsequent sustained elevation of intracellular Ca2+ concentration ([Ca2+]i) due to opening of plasma membrane Ca2+ release activated Ca2+ channels elicits the rapid dephosphorylation of NFAT proteins, their nuclear import and upregulation of target gene expression [3]. Under such circumstances, the Ca2+ signal is integrated upstream of NFAT by CN in conjunction with CaM and it is this protein complex that represents the critical regulatory mechanism for NFAT dephosphorylation and hence activation of NFAT dependent gene expression. The relationship between CN\/CaM and NFATs is so deeply intertwined that nuclear translocation of NFAT proteins has been employed as a robust assay of CN activity in intact mammalian cells [19-21] and has even found application in large scale screens for proteins involved in calcium induced calcium release [22].\nCaM is the primordial member of a superfamily of small EF-hand containing Ca2+ binding proteins that function to transduce spatial and temporal cellular Ca2+ signals. Other members of the CaM family include the neuronal calcium sensor (NCS) subgroup of neuronal\/neuroendocrine specific proteins [23,24]. Although only distantly related (21% sequence identity between CaM and NCS-1) published in vitro studies have identified potential overlap in target protein interactions between CaM and NCS-1 including an apparent positive interaction of both proteins with CN [25]. Since CaM exhibits high and ubiquitous expression these observations led to the speculation that NCS proteins may simply represent redundant or alternative modulators of classical CaM targets. Evidence from our laboratory and others has gone some way to disproving this idea with the identification of NCS specific binding proteins that, at present, have no documented interaction with CaM [26]. Further evidence for distinct functionality of the NCS protein family is apparent from their higher Ca2+ affinity compared to CaM, their differential cellular\/tissue distributions and their non-redundancy revealed in genetic studies [23,27]. Together these data indicate that NCS proteins possess the requisite biochemical properties to allow them respond to unique Ca2+ signals and to bind to distinct effector proteins. The possibility exists however that overlap between CaM and NCS target specificities may be of physiological significance and in the case of CN, identification of a functionally relevant interaction with NCS proteins would be of great interest in view of recent findings implicating CN activity in aspects of neuronal function.\nIn order to resolve some of the outstanding questions concerning potentially redundant functions of NCS proteins we have examined whether or not they are able to elicit CN activation in the physiologically relevant setting of intact mammalian cells using NFAT activation as a coupled reporter. We present in vitro biochemical data indicating that various recombinantly expressed NCS family members are able to directly bind CN although to lower levels compared with CaM. Consistent with these data we have also determined that expression of NCS-1, mutants of this protein that have previously been demonstrated to disrupt NCS-1 function in vivo, along with other NCS family members have no effect on NFAT dephosphorylation in live HeLa cells in response to elevation of [Ca2+]i. These data are supported by studies indicating that the nuclear translocation of NFAT on elevation of [Ca2+]i to cell nuclei is unaffected in cells expressing NCS-1 or NCS-1 mutants. We therefore suggest that NCS proteins, although exhibiting a detectable in vitro affinity for CN, may not be likely to act as physiologically relevant modulators of CN activity or NFAT mediated gene transcription in intact cells.\n2\nMaterials and methods\n2.1\nRecombinant proteins\nAll recombinant GST fusion proteins used in this study were expressed and purified as previously described [26]. Purified recombinant calcineurin was obtained from Sigma (Poole, UK) or Biomol (Exeter, UK). Unless otherwise stated all chemicals were of analytical grade and obtained from Sigma.\n2.2\nSmall Scale GST protein binding assays\nIn all binding assays, recombinant GST-fusions proteins (1\u00a0\u03bcM) were immobilised by incubation with 40\u00a0\u03bcl of glutathione cellulose (GST cellulose) resin (50% bead slurry, Bioline, London, UK) that had been pre-washed in binding buffer (KCl 50\u00a0mM, HEPES 20\u00a0mM (pH 7.4), EGTA 5\u00a0mM, NTA 5\u00a0mM, CaCl2 4.6\u00a0mM (giving a free [Ca2+] of 1\u00a0\u03bcM)) (+Ca2+ conditions) or binding buffer with no added CaCl2 (- Ca2+ conditions) by incubation with constant agitation for 30\u00a0min\/4\u00a0\u00b0C. Recombinant calcineurin (1\u00a0\u03bcM) or bovine brain cytosol (\u223c\u00a01\u00a0mg, dialysed against the appropriate\u00a0\u00b1\u00a0Ca2+ binding buffer) were then added to immobilised GST proteins in a 100\u00a0\u03bcl total binding reaction volume and samples incubated with constant agitation for 2\u00a0hrs at 4\u00a0\u00b0C. GST cellulose beads were pelleted by centrifugation (5,000\u00a0rpm\/1\u00a0min\/4\u00a0\u00b0C) and washed with 1\u00a0ml of the appropriate binding buffer. This wash step was repeated three times and final bead pellets extracted into 50\u00a0\u03bcl SDS dissociation buffer (125\u00a0mM HEPES pH (6.8), 10% (v\/v) sucrose, 10% (v\/v) glycerol, 4% (w\/v) SDS, 1% \u03b2-mercaptoethanol, 2\u00a0mM EDTA) and boiling for 5\u00a0min. Samples were resolved on SDS-PAGE (12.5% gel) and transferred to nitrocellulose filters for western blotting by transverse electrophoresis. Bound calcineurin was detected using a monoclonal antibody (1:1000, Sigma) followed by incubation with anti-mouse-HRP (1:400, Sigma) and visualisation with ECL reagents. All western blots were quantitated using ImageJ (National Institutes of Health) densitometry software.\n2.3\nLarge scale GST protein binding assays\nThis binding assay is essentially as described in [26]. Briefly, each recombinant GST-fusion protein (5\u201310\u00a0mg), including free GST as control, was immobilised onto 3\u00a0ml glutathione-Sepharose 4B resin that had been pre-washed extensively with binding buffer by incubation for 2\u00a0h at 4\u00a0\u00b0C with constant agitation. Clarified bovine brain cytosolic extract was applied to the GST affinity columns and binding allowed to proceed for 16\u00a0h at 4\u00a0\u00b0C with constant agitation. Each column was washed with a minimum 50\u00a0volumes of binding buffer and specific Ca2+-dependent binding proteins eluted using binding buffer containing no added Ca2+(calcium-free or CF buffer). An additional high-salt elution step with binding buffer supplemented with 1\u00a0M NaCl and no added Ca2+ (High Salt or HS buffer) was used to isolate potential Ca2+-independent binding interactions. Eluted protein fractions were concentrated by methanol precipitation and pellets extracted into SDS dissociation buffer by boiling for 5\u00a0min. All western blots were quantitated using ImageJ (National Institutes of Health) densitometry software.\n2.4\nSulfo-SBED cross-linking experiments\nThe manufacturers protocol provided by Pierce (Rockford, IL, USA) was followed with minor modifications. All steps, except those indicated, were carried out in the dark. Bait recombinant protein of interest (\u223c5\u00a0mg) was dialysed against binding buffer overnight at 4\u00a0\u00b0C. Immediately before use, the contents of one tube of No-Weigh Sulfo-SBED (Pierce) was dissolved in 22\u00a0\u03bcl of DMSO, this was then added to 1\u00a0ml of dialysed protein. Sulfo-SBED\/protein mixes were incubated at room temperature for 30\u00a0minutes, centrifuged briefly to remove any precipitated, hydrolysed, Sulfo-SBED, and dialysed overnight against binding buffer. Bovine brain cytosol (500\u00a0\u03bcl, \u223c4\u00a0mg protein) dialysed against binding buffer was added to 500\u03bcl of the Sulfo-SBED\/protein mix. This mixture was incubated on a rotator at room temperature for 1\u00a0hour. After this step, samples were exposed to long-wave UV illumination (365\u00a0nm) at a distance of \u223c5\u00a0cm from source for 15\u00a0minutes. During this step an appropriate volume of Neutravidin beads (Pierce) were washed extensively in binding buffer, 100\u00a0\u03bcl added to the bait\/cytosol mixture and samples incubated on a rotator for 1\u00a0hour at room temperature. Bait\/cytosol mixes were then centrifuged at 13,000\u00a0rpm for 1\u00a0minute, supernatants removed and beads washed with 1\u00a0ml of binding buffer. This step was repeated 6 times. Disulfide bond reduction was then achieved by incubating with 1\u00a0ml of 50\u00a0mM DTT for 1\u00a0hour at room temperature. Beads were subsequently centrifuged and washed extensively with binding buffer. After the final wash, 100\u00a0\u03bcl of SDS dissociation buffer was added to the beads and samples boiled for 10\u00a0minutes. Samples were separated on SDS PAGE (12.5% gel) and western blotted with a monoclonal anti-calcineurin antibody (1:1000, Sigma) followed by detection with anti-mouse-HRP (1:400, Sigma) and ECL reagents.\n2.5\nHeLa cell cultures and transfections\nHeLa cells were grown in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM, Invitrogen, Paisley, UK) containing 5% foetal bovine serum (Invitrogen), 1% non-essential amino acids (Invitrogen), and 1% penicillin\/streptomycin (Invitrogen) at 37\u00a0\u00b0C in an atmosphere of 5% CO2 and maintained at \u223c\u00a01,000,000 cells per 75\u00a0cm2 flask. 24\u00a0hrs prior to transfection, cells were seeded onto glass cover slips on a 24-well plate at \u223c\u00a050,000 cells per well. Transfection mixtures comprised 3\u00a0\u03bcl Fugene\u2122 (Roche, UK) per 1\u00a0\u03bcg plasmid DNA in a 100\u00a0\u03bcl total volume of DMEM and were incubated at room temperature for 30\u00a0minutes prior to being added drop-wise to cells. Cells were assayed 24-48\u00a0hrs post-transfection. The NFAT-GFP reporter construct was a kind gift from Professor Anjana Rao (Harvard Medical School, Boston, MA, USA).\n2.6\nIonomycin induced NFAT translocation\nTransfected HeLa cells were washed twice in Krebs buffer (NaCl 145\u00a0mM; HEPES 20\u00a0mM (pH 7.4); D-(+)-Glucose 10\u00a0mM; KCl 5\u00a0mM; CaCl2 3\u00a0mM; MgCl2 1.3\u00a0mM; NaH2PO4 1.2\u00a0mM). Cells were subsequently incubated in 500\u00a0\u03bcl Krebs containing 3\u00a0\u03bcM ionomycin or 100\u00a0\u03bcM histamine for required times before either solubilisation in SDS dissociation buffer, to investigate the phosphorylation state of NFAT via Western blotting, or fixing in 4% formaldehyde to observe the resulting sub-cellular localisation of NFAT post-treatment via confocal microscopy. For western blotting all samples were resolved on SDS-PAGE (12.5% gel) and proteins transferred to nitrocellulose filters by transverse electrophoresis. Filters were probed with monoclonal anti-GFP (1:1000, JL-8 clone, Invitrogen) followed by incubation with anti-mouse-HRP (1:400, Sigma) and protein visualised with ECL reagents.\n2.7\nConfocal microscopy\nFor confocal laser scanning microscopy, transfected cells were examined with either a Leica TCS-SP-MP microscope or a Leica TCS-SP2-AOBS microscope (Leica Microsystems, Heidelberg, Germany) using a 22\u00a0\u03bcm pinhole and a 63\u00d7 water immersion objective with a 1.2 numerical aperture. EGFP was imaged using excitation at 488\u00a0nm and light collection at 500-550\u00a0nm.\n3\nResults\n3.1\nDirect binding of calcineurin to NCS proteins\nA previous in vitro study demonstrated that NCS-1 is capable of stimulating the phosphatase activity of CN but did not examine whether the two proteins directly associate. To confirm a direct interaction we first performed a series of in vitro binding assays utilising various recombinantly expressed and purified NCS proteins to act as baits and where either purified recombinant CN or bovine brain cytosol were employed as a source of prey protein. Initially, purified CN (1\u00a0\u03bcM) was incubated in the presence of various GST-tagged NCS proteins (also at 1\u00a0\u03bcM) in binding buffer containing 1\u00a0\u03bcM free Ca2+ (Fig. 1a, upper panel). CaM was included in this assay as a positive control for CN binding (Fig. 1a, upper panel, Calmodulin Sepharose). CN was observed to bind directly to GST-NCS-1, GST-Hippocalcin, GST-Neurocalcin-\u03b4 and GST-CaBP1 but not GST-KChIP1, despite equal loading of the proteins (Fig. 1a, lower panel), indicating some degree of interaction specificity. It was also apparent from this analysis that, under identical experimental conditions, seven-fold less CN was bound by GST-tagged NCS proteins than by CaM. We next examined if GST-tagged NCS proteins could efficiently interact with CN present as a component of a complex protein mixture. GST control protein, GST-NCS-1 and GST-CaBP1 (Calcium Binding Protein-1, a calcium sensor more closely related to CaM than the NCS family proteins [28]) were incubated with bovine brain cytosolic protein extract in a large scale pull-down experiment [26] in the presence of 1\u00a0\u03bcM free Ca2+ and Ca2+ dependent binding partners eluted by application of Ca2+ free buffer containing EGTA and NTA (Fig. 1b, Calcium Free (CF) lanes). An additional elution step using 1\u00a0M NaCl supplemented buffer was also applied to the samples to elute potential Ca2+ independent binding proteins (Fig. 1b, High Salt (HS) lanes). Western blotting for the presence of CN in eluates confirmed a strictly Ca2+ dependent interaction with both GST-NCS-1 and GST-CaBP1. No binding was detectable to GST control protein. The signal for CN in the bovine brain cytosolic extract was compared in the same experiment (Fig. 1b, Bovine brain cytosol) and it was clear that CN was not enriched over the brain extract in the Ca2+ free eluates.\nTo provide an independent method to examine the interaction between NCS proteins and CN, we exploited a recently developed bi-functional cross-linking reagent [29] which permits the transfer of a biotin tag from a bait protein of interest to any specifically interacting prey proteins captured following a binding reaction. Samples from a GST-Neurocalcin-\u03b4 binding assay prepared via this method were probed with an anti-CN antibody and the presence of the phosphatase confirmed only in samples treated with the cross-linker (Fig. 1c, (+) Sulfo) and no CN was detectable in samples devoid of the reagent (Fig. 1c, (-) Sulfo). Despite use of cross-linking to maintain any low affinity interactions, CN was not enriched in the eluate over brain cytosol input into each condition (Fig. 1c (+) Sulfo vs. Bovine brain cytosol).\nThe data presented in Fig. 1a suggests that the affinity of CN for CaM may be substantially greater than for the NCS proteins tested. In order to independently verify these observations we further examined the binding of CN from bovine brain cytosol to GST control protein and GST-NCS-1 in direct comparison to CaM (Fig. 1d). We observed Ca2+ dependent binding of CN to CaM (Fig. 1d, Calmodulin Sepharose (+)) that was significantly enriched in the eluate compared to bovine brain cytosolic extract, in contrast, at this exposure of the western blot, binding to GST-NCS-1 was barely detectable. On longer exposure to film, NCS-1 bound CN was detectable although it should be noted that results obtained from this small scale binding assay are not directly comparable to data presented in Fig. 1b from a large scale binding assay which employed a greater amount of bovine brain cytosolic prey protein. These data are consistent with a high affinity Ca2+ dependent interaction between CaM and CN in contrast to a comparatively weak interaction between NCS-1 and CN.\n3.2\nOverexpression of NCS proteins has no effect on the dephosphorylation of an NFAT-GFP reporter construct in response to cytosolic Ca2+ elevation in HeLa cells\nAlthough our in vitro studies pointed to CaM as most likely the predominant interacting\/activating partner for CN, we speculated that the situation in the complex physiological setting of mammalian cells might not reflect the results obtained from such a comparatively simple analysis. We therefore attempted to resolve this issue through an examination of a possible interaction between CN and NCS proteins in intact HeLa cells. For these experiments we obtained a GFP tagged NFAT reporter construct [30], encoding the phosphorylated regulatory domain of the phosphatase, that undergoes a detectable dephosphorylation and nuclear translocation in response to sustained increases in [Ca2+]i. Our rationale was that should there be a functionally relevant NCS protein\/CN interaction in cells then there might be discernable alterations in dephosphorylation kinetics\/nuclear translocation of the NFAT reporter in cells [30,31] over-expressing NCS proteins as observed for other NCS-1 specific functions [32-35].\nIn initial studies, we saw no effect on NFAT dephosphorylation or nuclear translocation of over-expression of wild-type NCS-1, hippocalcin or neurocalcin-\u03b4. Since NCS-1 had been identified in a previous report [25] as an in vitro activator of CN enzymatic activity we decided, however, to focus on this NCS family member for this part of the study. A second reason for examining NCS-1 in further detail also related to the fact that we had at our disposal several mutant NCS-1 constructs one of which has been previously characterised as a dominant inhibitor of NCS-1 function [32]. Such mutants, we reasoned, might assist in unmasking potential functional interactions between NCS-1, CN and NFAT. The first of these mutants, NCS-1E120Q, has been employed in a number of studies due to its ability to interact normally with NCS-1 effectors however, as a consequence of impaired Ca2+ binding and conformational change, it can then exert a dominant inhibitory phenotype [32-36]. The second NCS-1 mutant we employed in this study, NCS-1G2A, is myristoylation deficient and, unlike wild type NCS-1 which is constitutively membrane associated through this lipid modification, is entirely cytosolic [37,38]. This protein was included in these studies due to the fact that the majority of cellular CN is similarly cytosolic and hence we hypothesised that the G2A mutation may exaggerate any effects of a potential NCS-1\/CN interaction.\nIn an initial analysis, HeLa cells were co-transfected with NFAT-GFP along with control empty vector, NCS-1 or NCS-1 mutant constructs (Fig. 2a). Cells were treated for 7\u00a0minutes with ionomycin\/Ca2+ to elevate [Ca2+]i, lysed and NFAT-GFP detected by western blotting. A band corresponding to the fully phosphorylated form of NFAT was apparent in 0 time point samples from all transfections (Fig. 2a, P, upper arrow). Dephosphorylation of NFAT to two more rapidly migrating species after sustained [Ca2+]i for 7\u00a0minutes was observable in all transfected samples and there was no detectable difference in the dephosphorylation pattern obtained between control transfected cells and NCS-1 or NCS-1 mutant transfected conditions (Fig. 2a, DeP, lower arrows). These data are fully consistent with results obtained from parallel confocal imaging analyses of NFAT-GFP nuclear translocation in HeLa cells transfected and treated in an identical manner but which were then fixed for microscopy (Fig. 2b). For all transfection conditions at time 0 of Ca2+\/ionomycin treatment NFAT-GFP was excluded from cell nuclei and was diffusely cytosolic (Fig. 2b, control, left hand panels), compatible with the presence of the phosphorylated form of the protein (Fig. 2a). After 7\u00a0minutes of Ca2+\/ionomycin treatment NFAT-GFP had redistributed to cell nuclei for all transfection conditions (Fig. 2b, ionomycin, right hand panels) again consistent with the rapid dephosphorylation of the protein (Fig. 2a).\nWe additionally verified that all NCS-1 expression constructs used in these studies over-expressed in HeLa cells to similar extents when co-expressed with NFAT-GFP (Fig. 2c). Western blotting with an anti-NCS-1 antiserum [39] of total cell lystates from HeLa cells transfected identically to those used in functional assays (Figs. 2a and b) highlighted essentially equivalent levels of over-expression of NCS-1, NCS-1G2A and NCS-1E120Q proteins. There was no detectable NCS-1 protein expression in control, pcDNA transfected, cells (Fig. 2c, pcDNA).\n3.3\nOverexpression of NCS-1, NCS-1E120Q and NCS-1G2A has no effect on the dephosphorylation of an NFAT-GFP reporter construct in response to cytosolic Ca2+ elevation in HeLa cells over an acute time course\nOur analyses suggested that, over a 7\u00a0minute time course, none of the NCS-1 constructs under examination were capable of affecting the dephosphorylation of NFAT-GFP in response to [Ca2+]i elevation and, by inference, nor were they able to influence CN activity in intact mammalian cells. These whole cell data were consistent with our in vitro binding studies however we decided to complete our investigation with two further analyses, firstly examining whether or not NCS-1 might exert a far more subtle effect on the CN\/NFAT pathway over an acute time course of [Ca2+]i elevation (Fig. 3a). In these experiments, HeLa cells were again transfected with NFAT-GFP along with control or NCS-1 expression constructs however dephosphorylation was monitored over a short 5\u00a0minute time course. For all constructs tested NFAT dephosphorylation was clearly apparent by the 5\u00a0minute time point (a 25\u00a0minute time point was also included to demonstrate complete dephosphorylation of NFAT-GFP) as determined by gel shift of the NFAT-GFP. Analysis of these earlier time points indicated there was no discernable difference in the pattern of dephosphorylation between any of the NCS-1 constructs tested and control transfected cells.\nIn these experiments the calcium induced elevation in cytosolic free Ca2+ was artificially generated by high concentrations of ionomycin and external Ca2+. Since NCS-1 has a high affinity for Ca2+ (300\u00a0nM) one possibility is that it could activate CN under conditions of limited Ca2+ elevation thus increasing the overall cellular sensitivity to a Ca2+ signal. We completed, therefore, our analyses with an examination as to whether over-expression of NCS-1 or its mutants could influence NFAT dephosphorylation in response to a physiological stimulus. Histamine is a well characterised HeLa cell agonist that generates oscillatory intracellular Ca2+ signals [40] and activation of protein kinase C with phorbol esters has been shown to potentiate NFAT driven gene expression elicited by such Ca2+ oscillations [41-43]. We stimulated HeLa cells transfected identically to those in Figs. 2 and 3 with 100\u00a0\u03bcM histamine in both the presence and absence of 50\u00a0nM PMA over a three hour time course and assessed the phosphorylation state of NFAT-GFP by western blotting. No dephosphorylation of NFAT-GFP was detectable in these experiments in control vector expressing cells indicating that the stimulus was below the threshold for activation of CN\/NFAT-GFP dephosphorylation. Significantly, cells expressing NCS-1, NCS-1E120Q or NCS-1G2A also failed to exhibit dephosphorylation of NFAT-GFP indicating that over-expression of NCS-1 did not enhance the sensitivity of the CN\/NFAT signalling pathway to Ca2+ signals under these conditions (data not shown).\nFor NCS-1E120Q to be able to exert a dominant negative effect it would have to bind to CN. In order to validate our use of the NCS-1E120Q mutant as a dominant negative construct in this study we therefore generated a recombinantly purified GST-tagged fusion construct encoding this protein which was used to confirm its ability to bind to purified CN (Fig. 3b). GST-NCS-1E120Q retained almost wild-type CN binding activity in this assay although with less strict Ca2+-dependency validating its use in functional assays of NFAT dephosphorylation and nuclear translocation. For GST-NCS-1 we observed a 13.7-fold increase in binding of CN in the presence of Ca2+ compared to conditions where Ca2+ was absent. In contrast, there was only a 2.2-fold increase in CN binding to GST-NCS-1E120Q in the presence of Ca2+.\n4\nDiscussion\nDiverse Ca2+ signals are transduced and decoded within cells by a variety of Ca2+-sensing proteins. Of these Ca2+ responsive proteins the most intensively studied and best understood is calmodulin (CaM) [2]. CaM is the prototypical member of a super-family of evolutionarily diverse EF-hand containing Ca2+ sensors to which the NCS sub-family belongs [23]. Whilst the NCS proteins are only distantly related to CaM, comparative studies have identified significant in vitro overlap in their effector specificities [25]. It was proposed that NCS proteins might represent a set of redundant or alternative modulators of bone fide CaM target interactions [25] but the ubiquitous and high level expression of CaM would make this doubtful.\nCalcineurin (CN) is a cellular serine\/threonine phosphatase critical to the activation of NFAT transcription factors and hence in the control of a variety of fundamental physiological processes [3]. CaM has been identified as the essential co-activator for CN function, however CN has also been identified as a potential target for NCS-1 [25] and as such represents a candidate dual CaM\/NCS effector.\nA series of possibilities exist as a direct consequence of these observations. Firstly, NCS proteins could indeed simply represent redundant modulators for CaM targets and as such might be expected to elicit responses and to exhibit biochemical properties closely approximating those observed with CaM. Secondly, NCS proteins could feasibly interact with CaM targets in biochemically distinct ways to modulate their activities in specific, CaM independent, manners leading to the overall generation of unique physiological endpoints. The latter has been suggested for dual regulation of voltage-gated Ca2+-channels by both CaBP1 and CaM [44]. With specific regard to possible CN\/NFAT activation by NCS-1, this possibility is intriguing in light of data identifying NFAT dependent gene expression as important to aspects of neuronal cell function. Thirdly, the possibility also exists that interactions observed between NCS proteins and CaM targets are physiologically irrelevant in cells. In this study we have attempted to resolve these issues through an investigation of firstly the biochemical characteristics of NCS protein interactions with CN, and secondly by examining the potential consequences of NCS protein function on activation of the classical CaM-CN-NFAT pathway in mammalian cells.\nIn a series of in vitro biochemical assays we were able to show that there exists a selective and Ca2+ dependent interaction between various NCS proteins and both purified recombinant CN, and CN derived from bovine brain cytosolic protein extract. We noted during these analyses however that significantly more CN bound to immobilized CaM. These data are consistent with the findings of Schaad et al. indicating that NCS-1 is able to stimulate the phosphatase activity of CN in vitro but to a lesser extent than observed for CaM activation. In experiments utilising bovine brain cytosol as a source of prey protein the difference in binding of the NCS proteins for CN in comparison to CaM was more greatly magnified, and whilst considerable quantities of CN could be affinity purified by a calmodulin sepharose column, the amount of CN associated with NCS family members under identical experimental conditions was considerably lower. Previous work using a biotinylated protein overlay assay also found much less binding of CN to NCS-1 compared to CaM [45]. These data led us to conclude that the CaM\/CN interaction was likely to be of a higher affinity than for interactions between CN and NCS proteins. Indeed, published biochemical parameters suggest that the in vitro Kd for CaM\/CN interaction may be \u2264\u00a00.1\u00a0nM [46] with that reported for NCS-1\/CN being 350\u00a0nM [25]. The in vitro affinity of CaM for CN is therefore three orders of magnitude greater than that determined for NCS-1 which would agree with the binding data presented in this study. Importantly, CaM is expressed at 5-200 fold higher concentrations in cells than NCS-1 [25] and so CN would be predicted to be bound to CaM rather than NCS-1 at elevated [Ca2+]i levels.\nTo assess whether NCS proteins were capable of modulating CN activity in a cellular environment we employed the dephosphorylation and nuclear translocation of a characterised NFAT-GFP reporter construct as a probe of CN phosphatase activity. Co-expression of NCS-1 did not alter the basal phosphorylation state of NFAT-GFP compared with control transfected cells nor did it exert a detectable effect on NFAT-GFP dephosphorylation\/nuclear translocation after sustained elevation of [Ca2+]i during single endpoint and acute time course analyses. Co-expression of the NCS proteins Hippocalcin, Neurocalcin-\u03b4 [26], and the more closely related CaBP1 (which shares 56% similarity with CaM [47]) were similarly without effect in analyses of NFAT-GFP dephosphorylation (data not shown). From these analyses we cannot formally exclude the possibility that NCS family proteins may activate a pool of cellular CN that does not signal via NFAT dephosphorylation. This seems unlikely however taking into account our biochemical data and the fact that in numerous other studies assessing diverse triggers of CN activity, concomitant activation of the NFAT pathway is always detected.\nIn an extension to these studies we also examined the effects of two NCS-1 mutant proteins, NCS-1E120Q and NCS-1G2A, on NFAT-GFP dephosphorylation in HeLa cells. We were able to demonstrate that the E120Q mutant of NCS-1 [32] retained approximately wild type binding properties with respect to interaction with purified CN. However, this protein, which has been routinely observed to exert a dominant negative phenotype in various other studies of NCS-1 function [32-35], was unable to generate detectable alterations in NFAT-GFP dephosphorylation. In an effort to try and understand if NCS-1 was unable to mediate an in vivo modulation of CN activity due to its restricted membrane localisation we also examined NFAT-GFP phosphorylation in the presence of co-expressed NCS-1G2A, a protein that fails to become myristoylated and as a result remains cytosolic. Since the bulk of cellular CN is also cytosolic we reasoned that expression of the G2A mutant might bias any interaction with CN to generate an observable alteration in dephosphorylation of NFAT-GFP. Our analysis of this mutant indicated that it elicited no significant change in the kinetics of NFAT-GFP dephosphorylation compared to control transfected cells. In related experiments we tested the ability of NCS-1 and NCS-1 dominant inhibitory mutants to influence NFAT dephosphorylation in response to physiological stimuli. We observed no detectable changes in NFAT dephosphorylation in the presence of over-expressed NCS-1 proteins suggesting that in addition to sustained elevation of cytosolic free Ca2+, NCS-1 proteins are similarly unable to stimulate NFAT dephosphorylation in response to agonist derived Ca2+ signals that were below the threshold for significant activation of CN and NFAT dephosphorylation.\nCollectively our findings would argue that, although NCS proteins are capable of interacting with CN in vitro, they may be unable to act as functionally relevant activators of the enzyme in a cellular context. The possibility remains that there may be some level of interplay between NCS proteins and CaM within protein complexes, exemplified by observations that both CaM and CaBP1 interact with IP3-receptors [40,48,49], that both NCS-1 and CaM are able to interact with Ca2+ channels [50] and G-protein coupled receptor kinases [34,51,52] and that CaM, NCS-1 and CaBP1 have all been shown to modulate the activity of the transient receptor potential channel, TRPC5 [35]. It is impossible to formally rule out direct modulation of CN by NCS proteins under all physiological conditions based on the data presented in this study however we have provided data to challenge the idea that CN is a cellular target of the NCS protein family and instead that CaM appears the more likely regulator of this key signalling phosphatase.","keyphrases":["calmodulin","calcineurin activity","nfat signaling","ncs family proteins","ca2+, calcium","nfat, nuclear factor of activated t-cells","ncs, neuronal calcium sensor","cabp1, calcium binding protein-1","cam, calmodulin","cn, calcineurin","gst, glutathione-s-transferase","kchip1, potassium channel interacting protein-1","gfp, green fluorescent protein","sds, sodium dodecyl sulphate","page, polyacrylamide gel electrophoresis","hrp, horse radish peroxidase","hepes, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid","edta, ethylenediaminetetraacetic acid","egta, ethylene glycol-bis(2-aminoethylether)-n,n,n\u2032n\u2032-tetra-acetic acid","nta, n,n-bis(carboxymethyl)glycine","pma, phorbol 12-myristate 13-acetate"],"prmu":["P","P","P","P","R","R","R","M","R","R","M","M","M","M","M","M","M","M","M","M","M"]}
{"id":"Anal_Bioanal_Chem-3-1-1805043","title":"Analysis of pharmaceuticals in wastewater and removal using a membrane bioreactor\n","text":"Much attention has recently been devoted to the life and behaviour of pharmaceuticals in the water cycle. In this study the behaviour of several pharmaceutical products in different therapeutic categories (analgesics and anti-inflammatory drugs, lipid regulators, antibiotics, etc.) was monitored during treatment of wastewater in a laboratory-scale membrane bioreactor (MBR). The results were compared with removal in a conventional activated-sludge (CAS) process in a wastewater-treatment facility. The performance of an MBR was monitored for approximately two months to investigate the long-term operational stability of the system and possible effects of solids retention time on the efficiency of removal of target compounds. Pharmaceuticals were, in general, removed to a greater extent by the MBR integrated system than during the CAS process. For most of the compounds investigated the performance of MBR treatment was better (removal rates >80%) and effluent concentrations of, e.g., diclofenac, ketoprofen, ranitidine, gemfibrozil, bezafibrate, pravastatin, and ofloxacin were steadier than for the conventional system. Occasionally removal efficiency was very similar, and high, for both treatments (e.g. for ibuprofen, naproxen, acetaminophen, paroxetine, and hydrochlorothiazide). The antiepileptic drug carbamazepine was the most persistent pharmaceutical and it passed through both the MBR and CAS systems untransformed. Because there was no washout of biomass from the reactor, high-quality effluent in terms of chemical oxygen demand (COD), ammonium content (N-NH4), total suspended solids (TSS), and total organic carbon (TOC) was obtained.\nIntroduction\nMost pharmaceutical substances are, by nature, biologically active and hydrophilic, in order that the human body can take them up easily, and persistent, to avoid degradation before they have a curing effect. Depending on the pharmacology of a medical substance it will be excreted as a mixture of metabolites, as unchanged substance, or conjugated with an inactivating compound attached to the molecule [1]. When they enter a wastewater-treatment plant, xenobiotics are not usually completely mineralized. They are either partially retained in the sludge, or metabolized to a more hydrophilic but still persistent form and, therefore, pass through the wastewater-treatment plant (WWTP) and end up in the receiving waters. Their removal in WWTPs is variable and depends on the properties of the substance and process conditions (e.g. sludge retention time (SRT), hydraulic retention time (HRT), temperature) [2, 3]. Levels of many pharmaceutically active compounds (PhACs) are barely reduced and they are, therefore, detected in WWTP effluents. The presence of PhACs in surface, drinking, and wastewaters is well documented in the literature [1, 4\u201312]. Although present at low concentrations in the environment, drugs can have adverse effects on aquatic organisms. These effects are chronic rather than acutely toxic, and depend on exposure (bioavailability), susceptibility to the compound in question, and the degradability of the compound [13].\nTo ensure compliance with future discharge requirements, upgrading of existing wastewater-treatment facilities and implementation of new technologies is envisaged as the next step in improvement of wastewater treatment. In the last ten to fifteen years the use of membranes in wastewater reclamation has attracted much interest. Membrane technology has become a technically and economically feasible alternative for water and wastewater treatment, especially because of high SRTs achieved within compact reactor volumes. In the MBR the concentration of microorganisms can be increased to up to 20\u00a0mg L\u22121. Because of this high biomass concentration the rate of degradation is higher and specialists are grown for problematic compounds. Another advantage of membrane treatment is separation of suspended solids by membranes, so they are not limited by the settling characteristics of the sludge. The performance, in terms of effluent quality, is believed to be better, but there is a striking lack of knowledge about the behaviour of trace-pollutants. Literature data on this subject is still very limited and contradictory [2, 11, 14\u201316]. Ultrafiltration membranes do not enable greater retention of the drugs investigated in this study as a result of filtration effects\u2014the molecular sizes of the compounds selected are at least a factor of 100 smaller than the pore size of the membranes. Additional removal of hydrophobic compounds by membranes can, nevertheless, be expected, because they are adsorbed by particles deposited as a layer on the membrane surface; this effect for hydrophilic compounds is not yet very well defined, however [11, 17].\nThe objectives of this work were detection of target pharmaceuticals in wastewater influents and effluents, observation of their elimination in the CAS process, and comparison with results obtained for a laboratory-scale MBR provided with a plate-and-frame submerged membrane module. The pharmaceutical products investigated were analgesics and anti-inflammatory drugs (ibuprofen, ketoprofen, naproxen, diclofenac, indomethacin, acetaminophen, mefenamic acid, and propyphenazone), lipid regulators and cholesterol-lowering statin drugs (clofibric acid, gemfibrozil, bezafibrate, pravastatin, and mevastatin), antibiotics (erythromycin, azithromycin, sulfamethoxazole, trimethoprim, and ofloxacin), psychiatric drugs (fluoxetine and paroxetine), an antiepileptic drug (carbamazepine), \u03b2-blockers (atenolol, sotalol, metoprolol, and propranolol), anti-histaminics (famotidine and loratidine), anti-ulcer agents (lansoprazole and ranitidine), an anti-diabetic (glibenclamide), and a diuretic (hydrochlorothiazide). These compounds had different physicochemical properties (i.e. neutral, ionic, hydrophilic, hydrophobic). Their chemical structures and CAS numbers are listed in the Appendix. If their behaviour during wastewater treatment could be more reliably related to process design and operating conditions, process performance could possibly be improved by alteration of these conditions in accordance with the types of molecule that are difficult to eliminate.\nExperimental\nMaterials and standards\nChemical standards of carbamazepine, lansoprazole, loratidine, famotidine, trimethoprim, ofloxacin, atenolol, metoprolol, azithromycin dihydrate, erythromycin hydrate, fluoxetine hydrochloride, ranitidine hydrochloride, sulfamethoxazole, propranolol hydrochloride, indomethacin, acetaminophen, mefenamic acid, clofibric acid, bezafibrate, mevastatin, and sotalol hydrochloride were purchased from Sigma\u2013Aldrich (Steinheim, Germany), propyphenazone, pravastatin, and paroxetine hydrochloride from LGC Promochem (London, UK), ketoprofen, diclofenac, gemfibrozil, ibuprofen, and naproxen from Jescuder (Rub\u00ed, Spain), glibenclamide from SIFA Chemicals (Liestal, Switzerland), and hydrochlorothiazide from Pliva (Zagreb, Croatia). All pharmaceutical standards were of high-purity grade (>90%).\nIsotopically labelled compounds used as internal standards were 13C-Phenacetin, from Sigma\u2013Aldrich, mecoprop-d3, from Dr Ehrenstorfer (Augsburg, Germany), and ibuprofen-d3, atenolol-d7, and carbamazepine-d10 from CDN Isotopes (Quebec, Canada).\nAll solvents (methanol, acetonitrile, and water) were HPLC-grade and were purchased from Merck (Darmstadt, Germany), as also was hydrochloric acid (HCl, 37%), ammonium acetate (NH4Ac), and acetic acid (HAc). Nitrogen for drying, purity 99.995%, was from Air Liquide (Spain).\nStock solutions of individual standards (1\u00a0g L\u22121) and internal standards were prepared in methanol and stored at \u221220\u00a0\u00b0C. Stock solutions of ofloxacin, pravastatin, and sulfamethoxazole were renewed monthly because of their limited stability. A standard mixture in which the compounds were at a concentration of approx. 20\u00a0mg L\u22121 was prepared from the stock solutions. Further dilutions of this mixture were prepared in 25:75 (v\/v) methanol\u2013water and were used as working standard solutions. A mixture of internal standards prepared by dilution of individual stock solutions in methanol was used for internal standard calibration.\nMembrane bioreactor (MBR)\nA submerged MBR of approximately 21\u00a0L active volume equipped with two flat sheet membranes (A4 size, area 0.106\u00a0m2, pore size 0.4\u00a0\u03bcm), purchased from Kubota (Osaka, Japan), was installed in a municipal WWTP in Rub\u00ed (Barcelona, Spain). Although the nominal porosity of the membranes was 0.4\u00a0\u03bcm (microfiltration) a fouling layer of proteins and microorganisms formed on the surface of the membranes reduced the effective porosity to 0.01\u00a0\u03bcm, which brought the type of filtration into the ultrafiltration range [17].\nThe MBR was operated in parallel with the CAS process (aeration tank and secondary settling tank). The biocenosis of the MBR was grown from inoculated sludge from the municipal WWTP (aeration basin) and cultivated over a period of approximately 1\u00a0month to reach steady-state conditions. The hydraulic retention time was set to 14\u00a0h by regulating the effluent flow and the SRT was infinite, because no sludge was discharged from the reactor.\nThe laboratory-scale MBR was operated dynamically in intermittent permeation mode\u2014cycles of permeation for 8\u00a0min interrupted with 2\u00a0min of halt. Influent and permeate flows were controlled by use of flow meters and computer-controlled pumps. Continuous aeration was provided by means of a sparger pipe situated at the bottom of the reaction vessel; the oxygen concentration was kept between 1 and 2\u00a0mg L\u22121. The temperature inside the reactor was 20\u2009\u00b1\u20092\u00a0\u00b0C throughout sampling.\nWastewater-treatment plant (WWTP)\nRub\u00ed WWTP was designed for 125,550 inhabitant equivalents. During the sampling programme the WWTP was operating with an average daily flow of 22,000\u00a0m3 day\u22121. A mixture of municipal, hospital, and industrial wastewater is treated. Treatment consists of pretreatment, preliminary treatment, primary sedimentation, and secondary (biological) treatment. Pretreated wastewater goes through a physical process of settling in a primary clarifier. Secondary treatment occurs in pre-denitrification (anaerobic) and nitrification (aerobic) tanks, and two secondary clarifiers. Secondary sludge is recirculated to a primary clarifier which improves the settling characteristics of the primary sludge and increases sludge age. A mixture of primary and secondary (activated) sludge is processed (thickening, dewatering) and anaerobically digested, and biogas produced is used to heat a digester. The hydraulic retention time of CAS treatment in WWTP Rub\u00ed, calculated for the average daily flow, is approximately 12\u00a0h. During the sampling programme the plant was operating with an SRT of approximately 3\u00a0days. WWTP effluent is discharged into the river Riera de Rub\u00ed, which flows into the Mediterranean sea.\nSampling and sample preparation\nSampling was conducted during May and June, 2005. Twenty-eight samples were analyzed. All wastewater samples were taken as time-proportional grab-samples, bearing in mind the HRT of the MBR and CAS processes. The sampling points were: \nprimary sedimentation tank effluent, as the inflow to the conventional treatment plant and membrane bioreactor,CAS effluent, andMBR effluent.\nWastewater samples were collected, in amber glass bottles pre-rinsed with ultra-pure water, as 24-h composite samples; the volume depended on the type of sample (100\u00a0mL for influent wastewater and 200\u00a0mL for effluent). Immediately on arrival at the laboratory they were filtered through 1-\u03bcm glass fibre filters and then through 0.45\u00a0\u03bcm Nylon membrane filters from Whatman (UK). The target compounds were extracted in one step, by a method described elsewhere [18], using a Baker vacuum system (J.T. Baker, The Netherlands) and Waters (Milford, MA, USA) Oasis HLB cartridges (60\u00a0mg, 3\u00a0mL) previously conditioned at neutral pH with 5\u00a0mL methanol then 5\u00a0mL deionised water (HPLC grade). Elution was performed twice with 4\u00a0mL methanol at a flow of 1\u00a0mL min\u22121. The extracts were then evaporated under a nitrogen stream and reconstituted with 1\u00a0mL 25:75 (v\/v) methanol\u2013water.\nChemical analysis\nLC analysis was performed with a Waters (Milford, MA, USA) 2690 HPLC system coupled to a Micromass Quattro (Manchester, UK) triple quadrupole mass spectrometer equipped with a Z-spray electrospray interface. Chromatographic separation was achieved on a Purospher Star RP-18 endcapped column (125\u00a0mm\u2009\u00d7\u20092.0\u00a0mm, particle size 5\u00a0\u03bcm) and a C18 guard column, both from Merck.\nA specific multi-residue analytical method was set up for measurement of the concentrations of the pharmaceutical compounds in wastewaters [18]. Analysis was performed in multiple-reaction-monitoring (MRM) mode, in both positive and negative electrospray-ionization mode. This method was refined for analysis of hydrochlorothiazide and glibenclamide. MRM transitions selected for each compound are summarized in Table\u00a01. In accordance with the performance characteristics defined in EU Commission Decision 2002\/657\/EC for confirmation and identification of pharmaceuticals when using LC\u2013tandem MS as the instrumental technique, a minimum of three identification points are required. When using LC\u2013MS\u2013MS (QqQ) analysis two MRM transitions are sufficient to confirm the identity of a compound. The MRM ratio, calculated as the relationship between the abundances of both transitions and the LC retention time are also criteria used to confirm the presence of an analyte in the samples. In this study, therefore, transitions between a precursor ion and the two most abundant fragment ions were chosen for each analyte when working in MRM mode, resulting in four identification points, enough to conform with the aforementioned EU directive. When poor fragmentation was observed for the compounds, only one transition could be monitored. Confirmation of the identities of these was achieved by matching their LC retention times with those of standards. Shifts in retention times were less than 3%, so the confirmation was regarded as sufficiently reliable. For internal standards only one transition was selected, because they were isotopically labelled compounds unlikely to be found in environmental samples. \nTable\u00a01MRM transitions of the compounds analyzedCompoundMRM 1MRM 2MRM 3Ibuprofen205\u2192161Ketoprofen253\u2192209253\u2192197Naproxen229\u2192170229\u2192185Diclofenac294\u2192250294\u2192214Indomethacin356\u2192297356\u2192255Acetaminophen152\u2192110152\u219293Mefenamic acid240\u2192196240\u2192180Propyphenazone231\u2192201231\u2192189Clofibric acid213\u2192127213\u219285Gemfibrozil249\u2192121Bezafibrate360\u2192274360\u2192154Pravastatin447\u2192327Mevastatin391\u2192185391\u2192159Carbamazepine237\u2192194237\u2192192Fluoxetine310\u219244310\u2192148Paroxetine330\u2192192330\u2192123Lansoprazole370\u2192252370\u2192205Famotidine338\u2192189338\u2192259Ranitidine315\u2192176315\u2192130Loratidine383\u2192337383\u2192267383\u2192259Erythromycin734.5\u2192158734.5\u2192576.4Azithromycin749.5\u2192591.4749.5\u2192158Sulfamethoxazole254\u219292254\u2192156Trimethoprim291\u2192230291\u2192261Ofloxacin362\u2192316Atenolol267\u2192190267\u2192145Sotalol273\u2192255273\u2192213Metoprolol268\u2192133268\u2192159Propranolol260\u2192166260\u2192183Hydrochlorothiazide296\u2192269296\u2192205Glibenclamide494\u2192369\nTo compensate for matrix effects from sample matrices internal standard calibration and adequate dilution of sample extracts were used, on the basis of the previously published method [18].\nRecoveries of the method were determined by spiking. Influent samples and CAS and MBR effluents were spiked in triplicate with a standard mixture of selected compounds to a final concentration of 1\u00a0\u03bcg L\u22121. Spiked samples and a blank sample were analysed by the above mentioned method. Recoveries of the target pharmaceuticals are listed in Table\u00a02, with method detection limits (MDL) for influent and effluent samples. MDLs and method quantification limits (MQL) were calculated on the basis of signal-to-noise ratios (S\/N) of 3 and 10, respectively. \nTable\u00a02Mean recoveries of the selected compounds and method detection limits (MDL) in ng L\u22121CompoundRecovery (%)MDL (ng L\u22121)InfluentMBR effluentCAS effluentInfluentMBR and CAS effluentIbuprofen131 (18.1)a68.8 (11.0)90.4 (11.0)98.020.0Ketoprofen62.8 (2.94)71.3 (3.11)59.1 (0.897)19074.0Naproxen49.2 (20.0)59.4 (1.28)53.4 (2.31)79.020.0Diclofenac83.3 (1.17)94.9 (10.0)95.0 (12.6)16040.0Indomethacin113 (2.95)120 (5.63)110 (3.78)15031.0Acetaminophen123 (17.0)108 (10.5)56.0 (7.61)20.95.35Mefenamic acid93.3 (1.95)92.1 (1.02)91.5 (5.29)5.701.85Propyphenazone60.0 (8.00)71.0 (4.00)71.0 (4.00)4.801.45Clofibric acid86.0 (10.8)104 (6.87)74.5 (1.40)16.33.75Gemfibrozil91.0 (8.47)87.5 (1.36)108 (17.2)8.702.20Bezafibrate106 (3.43)94.4 (9.30)89.4 (4.62)18.54.35Pravastatin85.6 (2.56)78.0 (12.2)96.0 (19.5)12030.9Mevastatin103 (8.61)134 (15.6)123 (9.86)9.301.30Carbamazepine84.0 (7.84)89.5 (5.20)88.0 (9.24)2.200.600Fluoxetine46.7 (2.34)93.7 (17.6)59.0 (1.60)19.81.70Paroxetine62.2 (2.15)109 (5.73)71.4 (1.49)3.500.650Lansoprazole70.0 (10.0)87.0 (5.00)86.0 (4.00)10.94.20Famotidine58.2 (7.76)55.4 (6.30)66.6 (5.39)3.100.40Ranitidine41.5 (9.85)75.8 (14.8)125 (11.7)1.400.300Loratidine72.6 (1.81)78.0 (6.97)64.5 (4.98)8.002.75Erythromycin67.7 (3.15)50.0 (13.0)66.6 (12.0)12.42.00Azithromycin30.0 (7.00)73.0 (2.00)63.0 (3.00)1.000.300Sulfamethoxazole33.7 (2.76)95.5 (9.24)78.3 (1.08)16.13.10Trimethoprim58.8 (3.29)128 (6.58)60.8 (3.87)1.300.350Ofloxacin142 (19.0)135 (5.45)138 (4.47)29.37.85Atenolol83.5 (33.8)60.8 (10.8)131 (15.5)1.700.750Sotalol47.1 (2.91)31.9 (3.05)52.0 (3.63)4.800.700Metoprolol36.7 (1.44)120 (2.64)76.7 (1.43)6.301.60Propranolol60.2 (0.506)90.8 (4.02)70.5 (5.27)2.600.300Hydrochlorothiazide39.8 (7.43)58.9 (1.62)73.4 (22.9)4.500.900Glibenclamide100 (11.7)107 (10.3)98.5 (11.7)19.22.30aThe relative standard deviation (%) of the recoveries is given in parentheses (n\u2009=\u20093)\nResults and discussion\nIt is well documented that WWTPs are major contributors of pharmaceuticals in the environment. WWTP Rub\u00ed mainly receives domestic, hospital, and industrial wastewater and effluent concentrations of several monitored compounds exceed \u03bcg L\u22121 levels. Ranges of output loads for WWTP Rubi for each pharmaceutical and mean values (g day\u22121) are reported in Table\u00a03. The quantities of pharmaceuticals discharged into the environment are calculated by multiplying the detected effluent concentrations by an average daily flow rate of 22,000\u00a0m3 day\u22121. The total amount of pharmaceutical compounds discharged by WWTP Rubi into the environment exceeded 300\u00a0g day\u22121 (an average value). The most abundant compounds, with average individual loads of 21\u201356\u00a0g day\u22121, were the anti-inflammatory drugs ibuprofen, naproxen, and diclofenac, the lipid regulators gemfibrozil and bezafibrate, the diuretic hydrochlorothiazide, and the \u03b2-blocker atenolol. \nTable\u00a03Average daily output loads of the investigated pharmaceuticals for Rub\u00ed WWTPPharmaceuticalEffluent load (g day\u22121)MeanRangeAnalgesics and anti-inflammatory drugsNaproxen37.010.8\u201376.9Ketoprofen17.111.4\u201336.3Ibuprofen56.37.39\u2013137.9Diclofenac27.317.3\u201343.8Indomethacin1.93nd\u20132.73Acetaminophen4.551.06\u20139.2Mefenamic acid0.440.27\u20130.85Propyphenazone0.680.43\u20130.96Anti-ulcer agentRanitidine2.770.55-5.30Psychiatric drugParoxetine0.08nda\u20130.16Antiepileptic drugCarbamazepine5.211.44-6.71AntibioticsOfloxacin6.932.40\u201311.2Sulfamethoxazole3.061.42\u20135.81Erythromycin2.290.95\u20134.51\u03b2-blockersAtenolol21.07.70\u201333.2Metoprolol3.321.14\u20135.43DiureticHydrochlorothiazide33.721.2\u201346.0Hypoglycaemic agentGlibenclamide0.74nd\u20130.98Lipid regulator and cholesterol lowering statin drugsGemfibrozil54.330.1\u201373.9Bezafibrate21.610.9\u201350.8Clofibric acid1.750.40\u20133.43PravastatinndndaNot detectable (below the LOQ)\nTo assess the efficiency of elimination by the MBR, substance-specific analysis must be performed and the bulk properties DOC and COD of wastewater influents and effluents must also be determined. The performance of the MBR system is summarized in Table\u00a04. The data are presented for the sampling period. Removal efficiencies of 98.7% for TSS and 90.4% for total COD were achieved during the membrane process. Average effluent ammonia concentration was 1.01\u00a0\u03bcg L\u22121 in the MBR effluent, compared with 48.41\u00a0\u03bcg L\u22121 in the CAS effluent. It is known that membrane processes are quite efficient at removing COD and TOC from wastewater [19, 20]. Improved COD removal is attributed to the combination of complete retention of particulate material by the membrane, including suspended COD and high molecular weight organisms, and to avoidance of biomass washout problems common in activated sludge systems. Consequently, stable conditions are provided for growth of specialized microorganisms which are the able to remove poorly biodegradable components. \nTable\u00a04Summary of the performance of the MBR systemPropertyInfluentMBR effluentCAS effluentTSS (mg L\u22121)119.2 (17.37)a1.600 (1.770)26.72 (15.69)CODtotal (mg L\u22121)508.2 (124.3)48.58 (22.47)111.6 (53.35)TOC (mg L\u22121)67.67 (24.29)10.89 (3.470)27.33 (13.75)N-NH4 (mg L\u22121)49.13 (15.79)1.010 (0.4200)48.41 (12.87)pH7.52 (0.300)7.08 (0.270)7.63 (0.160)aValues are averages from n\u2009=\u200916 samples, with standard deviations in parentheses\nOf 31 pharmaceutical products included in the analytical method, 22 were detected in the wastewater entering WWTP Rub\u00ed. Box plots of measured concentrations of each pharmaceutical are showed in Figs.\u00a01, 2 and 3. Ten measured values are given for influent and MBR effluent concentrations and eight for CAS effluent (data are missing for two sampling programmes). For each variable the box has lines at the lower quartile (25%), median (50%), and upper quartile (75%) values. The whiskers are the lines extending from each end of the box to show the extent of the data up to 1.5 times the interquartile range (IQR). Outliers are marked with + symbols.\nFig.\u00a01Removal, during MBR and CAS treatment, of the analgesics and anti-inflammatory drugs naproxen (a), ketoprofen (b), ibuprofen (c), mefenamic acid (d), diclofenac (e), indomethacin (f), acetaminophen (g), and propyphenazone (h)Fig.\u00a02Removal during MBR and CAS treatment of the antibiotics ofloxacin (a), sulfamethoxazole (b), and erythromycin (c), the \u03b2-blockers atenolol (d) and metoprolol (e), the anti-ulcer agent ranitidine (f), the antiepileptic drug carbamazepine (g), and the psychiatric drug paroxetine (h)Fig.\u00a03Removal during MBR and CAS treatment of the lipid regulator and cholesterol-lowering statin drugs gemfibrozil (a), bezafibrate (b), clofibric acid (c), and pravastatin (d), the diuretic hydrochlorothiazide (e), and the hypoglycaemic agent glibenclamide (f)\nThe highest influent concentrations (\u03bcg L\u22121) were measured for the analgesics and anti-inflammatory drugs naproxen, ibuprofen, ketoprofen, diclofenac, and acetaminophen, the antihyperlipoproteinaemic drugs gemfibrozil and bezafibrate, the \u03b2-blocker atenolol, and the diuretic hydrochlorothiazide. For other compounds input concentrations were usually in the range 10\u2013100\u00a0ng L\u22121. Because the low concentrations measured were sometimes close to the limits of quantification, any hypothesis about the efficiency of their elimination is questionable. Mean removal was, nevertheless, calculated for each of the pharmaceutical compounds; the results are presented in Table\u00a05. The most important removal pathways of organic compounds during wastewater treatment are: \nbiotransformation\/biodegradation,adsorption by the sludge (excess sludge removal), andstripping by aeration (volatilization).Table\u00a05Mean removal of selected pharmaceuticals by the MBR and CAS processesCompoundElimination (%) in:MBRaCASbAnalgesics and anti-inflammatory drugsNaproxen99.3 (1.52)85.1 (11.4)Ketoprofen91.9 (6.55)51.5 (22.9)Ibuprofen99.8 (0.386)82.5 (15.8)Diclofenac87.4 (14.1)50.1 (20.1)Indomethacin46.6 (23.2)23.4 (22.3)Acetaminophen99.6 (0.299)98.4 (1.72)Mefenamic acid74.8 (20.1)29.4 (32.3)Propyphenazone64.6 (13.3)42.7 (19.0)Anti-ulcer agentsRanitidine95.0 (3.74)42.2 (47.0)Psychiatric drugsParoxetine89.7 (6.69)90.6 (4.74)Antiepileptic drugsCarbamazepineNo eliminationcNo eliminationAntibioticsOfloxacin94.0 (6.51)23.8 (23.5)Sulfamethoxazole60.5 (33.9)55.6 (35.4)Erythromycin67.3 (16.1)23.8 (29.2)B-blockersAtenolol65.5 (36.2)No eliminationMetoprolol58.7 (72.8)No eliminationDiureticsHydrochlorothiazide66.3 (7.79)76.3 (6.85)Hypoglycaemic agentsGlibenclamide47.3 (20.1)44.5 (19.1)Lipid regulator and cholesterol lowering statin drugsGemfibrozil89.6 (23.3)38.8 (16.9)Bezafibrate95.8 (8.66)48.4 (33.8)Clofibric acid71.8 (30.9)27.7 (46.9)Pravastatin90.8 (13.2)61.8 (23.6)a,bValues are averages, with relative standard deviations (%) in parentheses, for n\u2009=\u200910a or n\u2009=\u20098b samplescCompounds were classified as \u201cno elimination\u201d if elimination was less than 10%\nBecause of the low values of the Henry coefficients (KH) of the compounds investigated [21], the fraction removed by volatilization can be neglected [16]. The two processes abiotic (adsorption) and biotic degradation (transformation by microorganisms) could not be distinguished, because no method was developed for analysis of the target compounds in sludge. The term \u201cremoval\u201d is therefore used here for conversion of a micropollutant to compounds other than the parent compound.\nElimination efficiency of the laboratory-scale MBR and the full-scale CAS process was comparable for naproxen, ibuprofen, acetaminophen, hydrochlorothiazide, and paroxetine. All were removed to a large extent by both systems (removal was greater than 80% except for hydrochlorothiazide, for which it was between 56 and 85%). Hydrochlorothiazide and paroxetine were eliminated slightly better by conventional treatment. Similar results for the behaviour of these drugs during conventional treatment have been reported by several authors [2, 3, 9, 11].\nFor ketoprofen, diclofenac, bezafibrate, and gemfibrozil removal by the MBR system was very high and uniform (>90%), with the exception of two sampling programme. It is assumed this variation could have been a result of reduced microbial activity or altered sorption and flocculation conditions. No plausible explanation can be given for the drastically reduced efficiency of removal of clofibric and mefenamic acid by MBR in two sampling programmes; otherwise these were eliminated with efficiencies between 65 and 90%. High and steady removal (>80%) in the MBR was also observed for ranitidine and ofloxacin. In conventional treatment all these pharmaceuticals were eliminated with a wide range of efficiencies, always lower than those obtained by the MBR. Better removal of readily biodegradable micropollutants by the MBR could be because of the smaller flock size of the sludge, which enhances mass transfer by diffusion and therefore increases elimination. Taking into consideration the composition of sludge originating from a membrane bioreactor (specialized microorganisms, large amount of active biomass in suspended solids) improved removal is to be expected; this was confirmed by our experiments.\nA possible explanation of substantially greater attenuation of diclofenac by the MBR (average removal efficiency 87% compared with 50% in CAS) could be the greater age of the MBR sludge. Improved removal is observed with increasing solids retention time [14]. Another explanation could be greater adsorption potential of the MBR sludge, because the organic matter content is greater than for CAS sludge. According to results from the EU project Poseidon [22], adsorption processes affect elimination of diclofenac. Literature data on this matter is still very contradictory. Clara et al. reported poor removal of diclofenac in laboratory-scale WWTPs whereas in full-scale plants removal varied from less than 20% to between 60 and 80% for some of the facilities investigated [2]. Heberer et al. [7] reported low removal efficiencies in a WWTP whereas Ternes et al. documented significant (69%) elimination of diclofenac [8].\nRemoval of carbamazepine was, in contrast, very poor (<20%), and effluent concentrations for both MBR and CAS were frequently greater than influent levels. Poor elimination of this neutral drug has been reported by many authors [9, 11, 23, 24]. Glucuronide conjugates of carbamazepine can, presumably, be cleaved in sewage, thus increasing environmental concentrations [8].\nRates of removal of the antibiotic sulfamethoxazole were very variable in both treatments investigated. According to Drillia et al. its microbial degradation will depend on the presence of readily biodegradable organic matter in wastewater; this varies during both MBR and CAS treatment [25]. Also, a substantial amount of sulfamethoxazole enters WWTPs as its human metabolite N4-acetylsulfamethoxazole, which can possibly be converted back to the original compound during treatment [26].\nEfficiency of removal of atenolol, metoprolol, pravastatin, erythromycin, and indomethacin varied in both MBR and CAS treatment. This could not be explained. Fluctuation of elimination efficiency was also observed for propyphenazone (44.8\u201382.9% for MBR and 6.82\u201362.6% for CAS) and glibenclamide (14.8\u201373.7% for MBR and 11.9\u201379.7% for CAS).\nEffluent concentrations greater than those recorded for the influent could be explained by the presence of input conjugate compounds that are transformed into the original compounds during treatment. Because these conjugates were not included in the analysis, no firm conclusion can be made about their biotransformation, especially because sampling inaccuracy can also lead to errors.\nConclusion\nSeveral pharmaceutical products (e.g. ibuprofen, naproxen, acetaminophen, ketoprofen, diclofenac, bezafibrate, gemfibrozil, ranitidine, ofloxacin, hydrochlorothiazide, and paroxetine) with high rates of attenuation can be expected to be completely removed from wastewater by adsorption or degradation, or a combination of both, during membrane treatment. For most of the compounds investigated MBR effluent concentrations were significantly lower than in the effluent from conventional treatment. Elimination of hydrochlorothiazide and paroxetine was slightly better in CAS treatment. Some substances (e.g. carbamazepine) were not removed by either MBR or CAS treatment. No relationship was found between the structures of target compounds and their removal during wastewater treatment, however. The range of variation of the efficiency of removal by the MBR system was small for most of the compounds; in conventional treatment greater fluctuations were observed and removal efficiency was found to be much more sensitive to changes in operating conditions (temperature, flow rate, etc).\nAlthough membrane technology seems a promising means of removal of pharmaceutical compounds, the MBR process investigated would not completely halt discharge of micropollutants. Membrane treatment processes should be optimized by modification of the membranes (variation of the materials and reduction of molecular mass cut-off limits) and\/or by modification of the treatment process (inoculation of special microorganisms). The efficiencies of diverse microbial populations in elimination of selected pharmaceuticals, and optimization of design and operating conditions of a laboratory-scale MBR will be the main objectives of our future investigations. That would provide guidelines for scale-up of a biological pilot plant and its evaluation by integration into an industrial process water-recycling system. Because of the current lack of information on the behaviour of pharmaceuticals in surface and wastewaters, however, further studies are required on the occurrence, fate, and effects of these substances in the environment.","keyphrases":["pharmaceuticals","membrane bioreactor","removal efficiency","wastewater treatment","conventional activated sludge treatment"],"prmu":["P","P","P","P","R"]}
{"id":"J_Biol_Inorg_Chem-4-1-2359831","title":"DNA cleavage and binding selectivity of a heterodinuclear Pt\u2013Cu(3-Clip-Phen) complex\n","text":"The synthesis and nuclease activity of a new bifunctional heterodinuclear platinum\u2013copper complex are reported. The design of this ditopic coordination compound is based on the specific mode of action of each component, namely, cisplatin and Cu(3-Clip-Phen), where 3-Clip-Phen is 1-(1,10-phenanthrolin-3-yloxy)-3-(1,10-phenanthrolin-8-yloxy)propan-2-amine. Cisplatin is not only able to direct the Cu(3-Clip-Phen) part to the GG or AG site, but also acts as a kinetically inert DNA anchor. The nuclease activity of this complex has been investigated on supercoiled DNA. The dinuclear compound is not only more active than Cu(3-Clip-Phen), but is also capable of inducing direct double-strand breaks. The sequence selectivity of the mononuclear platinum complex has been investigated by primer extension experiments, which reveal that its interaction with DNA occurs at the same sites as for cisplatin. The Taq polymerase recognizes the resulting DNA damage as different from that for unmodified cisplatin. The sequence-selective cleavage has been investigated by high-resolution gel electrophoresis on a 36-bp DNA fragment. Sequence-selective cleavages are observed in the close proximity of the platinum sites for the strand exhibiting the preferential platinum binding sites. The platinum moiety also coordinates to the other DNA strand, most likely leading only to mono guanine or adenine adducts.\nIntroduction\nDNA is a target for numerous antitumor drugs [1, 2]. Reversible or irreversible modifications of the nucleic acids can lead to disruption of the transcription and\/or replication, initiating ultimately the death of cancer cells. Cisplatin [3] and bleomycin are among these efficient anticancer drugs [4]. The mechanism of action is different for both drug substances; cisplatin primarily induces distortions upon binding to DNA, whereas bleomycin is able to generate DNA strand scissions.\nSince the discovery of bleomycin [5], numerous metal complexes have been synthesized that are able to produce DNA cleavage. Typical examples are iron(II)\u2013edta complexes, manganese(III)\u2013porphyrin and Cu, Co, Ru and Rh complexes with phenanthroline [6\u20139]. For instance, Cu(phen)2+ in the presence of dihydrogen peroxide efficiently cleaves double-stranded DNA through the oxidative attack on deoxyribose units from the minor groove [10, 11]. The consequent DNA-cleavage products include 5\u2032-monophosphate and 3\u2032-monophosphate ester termini, free bases, 5-methylene furanone, and a small amount of 3\u2032-phosphoglycolate [12\u201314]. Dihydrogen peroxide can be generated by Cu(phen)22+ in close proximity to the DNA strands, in the presence of a reductant and molecular oxygen [15]. The nuclease activity of Cu(phen)2 has been enhanced with the synthesis of 1-(1,10-phenanthrolin-3-yloxy)-3-(1,10-phenanthrolin-8-yloxy)propan-2-amine (3-Clip-Phen, 4) based on the covalent linkage of two phenanthroline units through their 3-position, leading to an increase of 60 times compared with the nuclease activity of Cu(phen)2 itself [15]. However, similarly to Cu(phen)2, the copper complexes of 3-Clip-Phen have no sequence selectivity, and cleave the DNA in a single-stranded fashion. The amine group of 3-Clip-Phen has been functionalized with different groups, such as a distamycin analog or various DNA intercalators [16, 17]. Thus, the resulting complexes show enhanced cleaving activities, and the complexes with the distamycin analog exhibit excellent targeting properties toward A\u00b7T boxes [18].\nRecently, it has been shown that Cu(3-Clip-Phen) (3) attached to a cisplatin motif is able to perform direct double-strand cuts, thanks to the DNA-anchoring platinum moiety [19]. Platinum complexes have received considerable attention since the discovery of the antiproliferate activity of cisplatin in 1969 [20]. It is generally accepted that the distortion of DNA generated upon binding of cisplatin is largely responsible for its antitumor properties [3]. Subsequent drug activation via intracellular aquation reactions results in a variety of stable bifunctional DNA\u2013platinum(II) adducts. 1,2-Intrastrand cross-links between two adjacent guanine bases d(GG) or between an adenine and a guanine residue d(AG) are primarily formed. The platinum center of complexes with a cis motif preferentially coordinates to the N7 position of both adenine and guanine in the major groove of DNA [21\u201324].\nIn the present paper, the preparation of a bifunctional complex [CuPt[N1-(3-(1,10-phenanthrolin-3-yloxy)-1-(1,10-phenanthrolin-8-yloxy)propan-2-yl)ethane-1,2-diamine]Cl4, 2] containing both a cis-bis(amine)Pt(II) moiety and a nuclease active 3-Clip-Phen group is reported (Fig.\u00a01). The bridge connecting the platinum and copper moieties is very short and therefore rigid in contrast to that in the previous report, where the complexes had flexible linkers in order to have a major\u2013minor groove interaction [19]. The platinum and the 3-Clip-Phen adjacent parts are now separated by a short linker. Consequently, in this case either the platinum species will not interact with its preferential site (the major groove) or the 3-Clip-Phen moiety will not interact with its preferential site (the minor groove). The platinum component plays two roles: (1) it acts as a DNA anchor, thus allowing the Cu(3-Clip-Phen) moiety to perform cleavages in the close proximity of the Pt\u2013DNA adducts, and (2) it induces a sequence-selective binding of the heterodinuclear complex. Accordingly, the achievement of double-strand DNA breaks is potentially increased, because the single-strand cuts are in the close proximity of the platinum adduct and sequence-selective cleavage may be expected.\nFig.\u00a01Complexes 1\u20133\nIn the present study, the binding property of the platinum moiety and the cleavage selectivity and activity of this novel heterodinuclear complex 2 were investigated by agarose gel electrophoresis and high-resolution analysis with a 36-bp DNA fragment (Fig.\u00a02). The results obtained are compared with those achieved with cisplatin and the Cu(3-Clip-Phen) complex.\nFig.\u00a02Nucleobase sequences of oligonucleotide I (ODN I), oligonucleotide II (ODN II) and the primer used for the binding experiments. The preferential binding sites of cisplatin are indicated by arrowheads\nMaterials and methods\nPreparation of Pt[N1-(3-(1,10-phenanthrolin-3-yloxy)-1-(1,10-phenanthrolin-8-yloxy)propan-2-yl)ethane-1,2-diamine]Cl2 and 2\nAll reagents and solvents were commercially available and used without further purification. Cu(3-Clip-Phen) (3) was prepared as previously described [15]. The 1H NMR chemical shifts of standard solvent molecules have been omitted in all redrawn spectra for clarity.\n2-(2-(3-(1,10-Phenanthrolin-3-yloxy)-1-(1,10-phenanthrolin-8-yloxy)propan-2-ylamino)ethyl)isoindoline-1,3-dione\n3-Clip-Phen (4) (100\u00a0mg, 0.22\u00a0mmol) was dissolved in 5\u00a0mL of dimethylformamide (DMF). One equivalent of N-(2-bromoethyl)phthalimide (5) (56.7\u00a0mg, 0.22\u00a0mmol) and 1\u00a0equiv of N-ethyldiisopropylamine (38\u00a0\u03bcL, 0.22\u00a0mmol) were added to 4. The reaction mixture was heated to 100\u00a0\u00b0C for 2\u00a0days. After the mixture had cooled to room temperature, 10\u00a0mL of dichloromethane (DCM) was added, and the organic phase was washed three times with 10\u00a0mL of distilled water. After being dried over Na2SO4 and evaporation under reduced pressure, the resulting crude product was purified by column chromatography (SiO2, DCM, MeOH, NH4OH, 95:5:0.5), to yield 2-(2-(3-(1,10-phenanthrolin-3-yloxy)-1-(1,10-phenanthrolin-8-yloxy)propan-2-ylamino)ethyl)isoindoline-1,3-dione (6) as an off-white powder (yield\u00a034%). 1H NMR (DCCl3, 300\u00a0MHz) \u03b4 9.05 (dd, 2H, J\u00a0=\u00a04.32, 1.67\u00a0Hz), 8.81 (d, 2H, J\u00a0=\u00a02.83\u00a0Hz), 8.10 (dd, 2H, J\u00a0=\u00a08.07, 1.62\u00a0Hz), 7.72 (dd, 2H, J\u00a0=\u00a05.43, 3.05\u00a0Hz), 7.67 (d, 2H, J\u00a0=\u00a08.88\u00a0Hz), 7.62 (d, 2H J\u00a0=\u00a08.86\u00a0Hz), 7.54 (dd, 2H, J\u00a0=\u00a06.09, 4.74\u00a0Hz), 7.48 (m, 4H), 4.27 (d, 4H, J\u00a0=\u00a05.18\u00a0Hz), 3.84 (t, 2H, J\u00a0=\u00a06.06\u00a0Hz), 3.54 (m, 1H), 3.13 (t, 2H, J\u00a0=\u00a06.12\u00a0Hz) ppm. 13C NMR (DCCl3, 75\u00a0MHz) \u03b4 168.4, 153.7, 150.1, 146.0, 142.4, 140.3, 135.73, 133.8, 132.0, 129.3, 127.1, 125.9, 123.0, 122.0, 114.9, 67.3, 55.6, 45.4, 37.8\u00a0ppm. Low-resolution mass spectrometry (MS) (electrospray ionization, ESI, more than 0) m\/z 621.00 [(M\u00a0+\u00a0H)+; calcd for C37H29N6O4+: 621.66], 642.94 [(M\u00a0+\u00a0Na)+; calcd for C37H28N6O4Na+: 643.65] Anal. calcd for C37H28N6O4\u00b71.6 H2O: C, 68.42; H, 4.84; N, 12.94. Found: C, 68.34; H, 4.86; N, 13.21.\nN1-(3-(1,10-Phenanthrolin-3-yloxy)-1-(1,10-phenanthrolin-8-yloxy)propan-2-yl)ethane-1,2-diamine\nTwo equivalents of hydrazine (15.6\u00a0\u03bcL, 0.32\u00a0mmol) were added to a solution of 6 (100\u00a0mg, 0.16\u00a0mmol) in 5\u00a0mL of pure ethanol. The mixture was refluxed overnight. The ethanol was evaporated under reduced pressure, and the crude product was purified by column chromatography (SiO2, DCM, MeOH, NH4OH, 90:10:1) to give N1-(3-(1,10-phenanthrolin-3-yloxy)-1-(1,10-phenanthrolin-8-yloxy)propan-2-yl)ethane-1,2-diamine (7) as a light-brown powder (yield 73%). 1H NMR (MeOD-d3, 300\u00a0MHz) \u03b4 9.00 (dd, 2H, J\u00a0=\u00a04.41, 1.63\u00a0Hz), 8.84 (d, 2H, J\u00a0=\u00a02.84\u00a0Hz), 8.34 (dd, 2H, J\u00a0=\u00a08.10, 1.67\u00a0Hz), 7.90 (d, 2H, J\u00a0=\u00a02.86\u00a0Hz), 7.83 (m, 4H), 7.65 (dd, 2H, J\u00a0=\u00a08.10, 3.26\u00a0Hz), 4.51 (d, 4H, J\u00a0=\u00a04.47\u00a0Hz), 3.58 (m, 1H), 3.07 (t, 2H, J\u00a0=\u00a05.64\u00a0Hz), 2.97 (t, 2H, J\u00a0=\u00a05.23) ppm. 13C NMR (MeOD-d3, 75\u00a0MHz) \u03b4 155.6, 150.6, 146.3, 143.0, 140.5, 137.6, 131.0, 128.7, 128.2, 127.4, 123.5, 116.7, 69.1, 57.5, 41.9\u00a0ppm. Low-resolution MS (ESI\u00a0>\u00a00) m\/z 490.98 [(M\u00a0+\u00a0H)+; calcd for C29H27N6O2+: 491.56] Anal. calcd for C29H26N6O2\u00b71.9 CH2Cl2: C, 56.93; H, 4.61; N, 12.89. Found: C, 56.79; H, 5.13; N, 13.23.\nPt[N1-(3-(1,10-phenanthrolin-3-yloxy)-1-(1,10-phenanthrolin-8-yloxy)propan-2-yl)ethane-1,2-diamine]Cl2\nTo a solution of 7 (70.4\u00a0mg, 0.14\u00a0mmol) in 6.5\u00a0mL of DMF was added a solution of 0.8\u00a0equiv of K2PtCl4 (47.69\u00a0mg, 0.11\u00a0mmol) in 3.25\u00a0mL of de-ionized H2O. The reaction mixture was stirred for 6\u00a0h at room temperature. The off-white precipitate was filtered off and washed with 30\u00a0mL of deionized water, 30\u00a0mL of MeOH and 20\u00a0mL of diethyl ether to give Pt[N1-(3-(1,10-phenanthrolin-3-yloxy)-1-(1,10-phenanthrolin-8-yloxy)propan-2-yl)ethane-1,2-diamine]Cl2 (1) as an off-white powder (yield 42%). 1H NMR (DMSO-d6, where DMSO is dimethyl sulfoxide, 300\u00a0MHz) \u03b4 9.04 (d, 2H, J\u00a0=\u00a02.77\u00a0Hz), 8.88 (d, 2H, J\u00a0=\u00a02.45\u00a0Hz), 8.45 (d, 2H, J\u00a0=\u00a08.00\u00a0Hz), 8.05 (br, 2H), 7.95 (m, 4H), 7.70 (dd, 2H, J\u00a0=\u00a07.99, 4.35\u00a0Hz), 4.49 (br, 4H), 4.07 (br, 5H) ppm. 195Pt NMR (DMSO-d6) \u03b4 \u22122,317 (complex), \u22123,292 (complex with one DMSO coordinated) ppm. IR (neat): \u03bd\u00a0=\u00a03,049 (br); 1,591 (s); 1,506 (s); 1,424 (s); 1,236 (s); 1,198 (s); 1,102 (s); 1,018 (s); 876 (s), 834 (s), 728 (s), 332 (s), 325 (s)\u00a0cm\u22121. Anal. calcd for C29H26Cl2N6O2Pt\u00b7DMF: C, 46.33; H, 4.01; N, 11.82 Found: C, 47.54; H, 4.43; N; 11.70; the deviation in calculated percentage C is ascribed to traces of solvent other than DMF.\nCuPt[N1-(3-(1,10-phenanthrolin-3-yloxy)-1-(1,10-phenanthrolin-8-yloxy)propan-2-yl)ethane-1,2-diamine]Cl4\nCuCl2 (17.0\u00a0mg, 0.1\u00a0mmol) was added as a solid to a suspension of 1 (75.7\u00a0mg, 0.1\u00a0mmol) in DMF (25\u00a0mL). The reaction was stirred overnight at 50\u00a0\u00b0C. DMF was partially evaporated under reduced pressure, and the crude compound was precipitated in 100\u00a0mL of diethyl ether. The solid material was filtered off and washed with 3\u00a0\u00d7\u00a020\u00a0mL of diethyl ether, and dried overnight at 50\u00a0\u00b0C under reduced pressure to give 2 as a green powder (yield 93%). X-band electron paramagnetic resonance (solid state): g\u00a0=\u00a02.1191. UV\u2013vis (H2O) \u03bbmax 282 (40,100), 321 (12,500), 333 (10,100), 347 (3,280) \u00a0nm. High-resolution MS (m\/z): [2M\u20132Cl] calcd for (C58H52Cl6N12O4Pt2Cu2)2+ 853.00852, 853.5096, 854.0097, 854.5087, 855.0088, 855.5072, 856.0073, 856.5083, 857.0100, 857.5067; found 853.0117, 853.5117, 854.0114, 854.5110, 855.0107, 855.5105, 856.0103, 856.5101, 857.0098, 857.5097.\nCytotoxicity\nThe cytotoxicity of complexes 1 and 2 has been determined for breast (MCF7), two glioblastoma (Hs683 and U373), two colorectal (HCT-15 and LoVo) and lung (A549) cancer cell lines (see supplementary material for experimental details and results).\nSolutions of complexes for experiments with DNA\nOne millimolar DMSO solutions of the complexes investigated were prepared, and were subsequently diluted with Milli-Q water.\nNuclease activity on supercoiled DNA\nOne millimolar DMSO solutions of the complexes investigated were diluted to, respectively, 200, 400 and 1,000\u00a0nM with Milli-Q water. Five microliters of the complex solution was added to 10\u00a0\u03bcL of supercoiled \u03a6X174 DNA (Invitrogen, 7\u00a0nM, 40\u00a0\u03bcM base pairs) in 6\u00a0mM NaCl, 20\u00a0mM sodium phosphate buffer (pH 7.2), and incubated for 20\u00a0h at 37\u00a0\u00b0C. To initiate the cleavage, 5\u00a0\u03bcL of a 20\u00a0mM mercaptopropionic acid solution in water was added, and the resulting reaction mixture was incubated at 37\u00a0\u00b0C for 1\u00a0h. The reaction was quenched at 4\u00a0\u00b0C, followed by the addition of 4\u00a0\u03bcL of loading buffer (glycerol with bromophenol blue) prior to the loading on a 0.8% agarose gel containing 1\u00a0\u03bcg\u00a0mL\u22121 ethidium bromide. The gels were run at a constant voltage of 70\u00a0V for 90\u00a0min in 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris) borate edta (TBE) buffer containing 1\u00a0\u03bcg\u00a0mL\u22121 ethidium bromide. The gels were visualized under a UV transilluminator, and the bands were quantified using a Bio-Rad Gel Doc 1000 apparatus interfaced with a computer.\nTime-course experiments of DNA cleavage\nFifty microliters of the complex solution was added to 100\u00a0\u03bcL of supercoiled \u03a6X174 DNA (Invitrogen, 7\u00a0nM, 40\u00a0\u03bcM base pairs) in 6\u00a0mM NaCl, 20\u00a0mM sodium phosphate buffer (pH 7.2), and the resulting reaction mixture was incubated for 20\u00a0h at 37\u00a0\u00b0C. To initiate the cleavage, 50\u00a0\u03bcL of 20\u00a0mM mercaptopropionic acid was added, and a sample was taken out every 10\u00a0min. Four microliters of loading buffer (glycerol with bromophenol blue) was added, and the sample was directly frozen in liquid nitrogen. When all samples had been collected, they were loaded on a 0.8% agarose gel containing 1\u00a0\u03bcg\u00a0mL\u22121 ethidium bromide.\nAnalyses with 5\u2032-32P-end-labeled DNA\nOligonucleotides (ODNs) I and II and the primer (Fig.\u00a02) were purchased from Eurogentec, and purified on a 15% polyacrylamide (acrylamide to bisacrylamide ratio 19:1) gel. The denaturating gel contained 7\u00a0M urea. The migrations was performed in the presence of TBE buffer at 2,500\u00a0V for 3\u00a0h. The concentrations of single-stranded ODNs were determined by UV titration at 260\u00a0nm [25]. The ODNs were end-labeled with 32P using standard procedures with T4 polynucleotide kinase (New England BioLabs) and [\u03b3-32P]ATP for the 5\u2032-end, before being purified on a MicroSpin G25 column (Pharmacia) [26].\nComparison of the platinum\u2013ODN adducts formed with the different complexes\n5\u2032-end-labeled ODN I (2\u00a0\u03bcM) was annealed to 1\u00a0equiv of its complementary strand ODN II in 1,100\u00a0\u03bcL of Tris\u2013HCl (20\u00a0mM, pH 7.2) by heating to 90\u00a0\u00b0C for 5\u00a0min, followed by slow cooling to room temperature. Then, 60\u00a0\u03bcL of this solution was incubated with 60\u00a0\u03bcL of the complex solution (6 or 20\u00a0\u03bcM) for 20\u00a0h at 37\u00a0\u00b0C, followed by precipitation with 100\u00a0\u03bcL of sodium acetate buffer (3\u00a0M, pH 5.2) and 1,300\u00a0\u03bcL of cold ethanol. Pellets were rinsed with ethanol and lyophilized. Samples were dissolved in formamide containing bromophenol blue and xylene cyanol and heated for 3\u00a0min at 90\u00a0\u00b0C before being loaded on the gel. Platinum\u2013DNA adducts were analyzed by denaturing 20% polyacrylamide gel electrophoresis, then by phosphorimagery.\nComparison of the sequence-selective binding by primer extension experiments with Taq polymerase\nODN I (2\u00a0\u03bcM) was annealed to ODN II (2\u00a0\u03bcM) in 1,100\u00a0\u03bcL of Tris\u2013HCl (20\u00a0mM, pH 7.2) by heating to 90\u00a0\u00b0C for 5\u00a0min, followed by slow cooling to room temperature. Sixty microliters of this solution was then incubated with 60\u00a0\u03bcL of the complex solution (6 or 20\u00a0\u03bcM) for 20\u00a0h at 37\u00a0\u00b0C, followed by precipitation with 100\u00a0\u03bcL of sodium acetate buffer (3\u00a0M, pH 5.2) and 1,300\u00a0\u03bcL of cold ethanol. Pellets were rinsed with ethanol and lyophilized. For primer extension, an aliquot of the redissolved samples (0.25\u00a0\u03bcM) was annealed with 5\u2032-end-labeled primer (0.25\u00a0\u03bcM) and 1 equiv of ODN I (0.25\u00a0\u03bcM) in the enzyme buffer (the buffer contains a small amount of reductant, but not enough to induce DNA cleavage by complex 2) before the addition of 250\u00a0\u03bcM dGTP, dCTP, dATP and dTTP and 2.5\u00a0U of Taq polymerase (final concentrations are given, the total volume was 10\u00a0\u03bcL). One equivalent of unmodified ODN I was added in order to displace the ODN II from the duplex and replace it with the labeled primer. The samples were reacted at 37\u00a0\u00b0C for 30\u00a0min, and 1\u00a0\u03bcL of edta (0.2\u00a0M) was subsequently added. Samples were dissolved in formamide containing bromophenol blue and xylene cyanol and heated for 3\u00a0min at 90\u00a0\u00b0C, before being loaded on the gel. Five microliters of sample was then analyzed by denaturing 20% polyacrylamide gel electrophoresis and phosphorimagery. The Maxam and Gilbert [27] sequencing scale, including a final scale of T4 polynucleotide kinase digestion to remove 3\u2032-end phosphates, was used to analyze the DNA fragments. The sequencing experiments show, in addition to a strong, easily assigned G signal, also a weak A signal.\nComparison of the cleavage patterns of ODN I\u2013ODN II induced by the copper complexes\nThe 5\u2032-end-labeled 36mer target (2\u00a0\u03bcM) was annealed to 1 equiv of its complementary strand in 1,100\u00a0\u03bcL of Tris\u2013HCl (20\u00a0mM, pH 7.2) by heating to 90\u00a0\u00b0C for 5\u00a0min, followed by slow cooling to room temperature. To 60\u00a0\u03bcL of this solution was added 60\u00a0\u03bcL of complex 1, 2 or 3 solutions (20\u00a0\u03bcM). Some of the samples involving complex 2 were incubated for 19\u00a0h, followed by the addition of 1\u00a0equiv of CuCl2 per complex and subsequent incubation (1\u00a0h). The other samples were incubated for 20\u00a0h at 37\u00a0\u00b0C. Next, all samples were precipitated with 100\u00a0\u03bcL of sodium acetate buffer (3\u00a0M, pH 5.2) and 1,300\u00a0\u03bcL of cold ethanol. Pellets were rinsed with ethanol and lyophilized, then dissolved to 1.33\u00a0\u03bcM in Tris\u2013HCl buffer (13.3\u00a0mM, pH 7.4). For the cleavage experiments, to 15\u00a0\u03bcL of this solution was added 5\u00a0\u03bcL of a 0.8\u00a0mM ascorbate solution (5\u00a0\u03bcL of water was added to the controls). The samples were incubated at 37\u00a0\u00b0C for 1\u00a0h, followed by precipitation in 20\u00a0\u03bcL of sodium acetate buffer (3\u00a0M, pH 5.2) containing 1\u00a0\u03bcg of salmon testes DNA and 180\u00a0\u03bcL of cold ethanol. Pellets were rinsed with ethanol and lyophilized. In order to study the DNA cleavage mechanism, additional treatments were performed on some samples: (1) heating at 90\u00a0\u00b0C in 50\u00a0\u03bcL of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)\u2013NaOH buffer (0.1\u00a0M, pH 8.0) for 30\u00a0min, followed by ethanol precipitation; (2) heating at 90\u00a0\u00b0C in 50\u00a0\u03bcL of piperidine (0.2\u00a0M in water) for 30\u00a0min, followed by lyophilization. Samples were dissolved in formamide containing bromophenol blue and xylene cyanol and heated for 3\u00a0min at 90\u00a0\u00b0C, before being loaded on the gel. Samples were analyzed by denaturing 20% polyacrylamide gel electrophoresis, then phosphorimagery. The Maxam and Gilbert [27] sequencing scale was used to analyze DNA fragments. The sequencing experiments show, in addition to a strong, easily assigned G signal, also a weak A signal.\nResults and discussion\nDesign and synthesis of a heterodinuclear platinum\u2013copper complex\nA ditopic ligand 7 was designed to favor the simultaneous coordination of a platinum and a copper entity; hence, the resulting bifunctional complex would combine the ability to form a kinetically inert coordination bond with DNA, thanks to its cis-Pt moiety, with the cleavage properties of Cu(3-Clip-Phen). Two coordination compounds, namely, the platinum complex 1 and the heterodinuclear platinum\u2013copper complex 2 (Fig.\u00a01) were prepared with the ligand 7. The binding ability of the platinum unit and the cleavage selectivity of the Cu(3-Clip-Phen) moiety were investigated. The results were compared with those for the known Cu(3-Clip-Phen) complex (3). The short separation between the Pt and the Cu centers in complex 2 most likely affects the preference for platinum to bind in the major groove, and in the minor groove for copper. Accordingly, the coordination of the platinum moiety in the major groove will force the Cu(3-Clip-Phen) component to bind in the major groove, and vice versa.\nThe general synthetic pathway to prepare complexes 1 and 2 is depicted in Scheme\u00a01. The selective and complete platination of the ethylenediamine unit using 1\u00a0equiv of K2PtCl4 was monitored by 195Pt\u00a0NMR and 1H NMR. The in situ reaction of the resulting platinum derivative 1 with 1\u00a0equiv of CuCl2 produces the heterobimetallic complex 2.\nScheme\u00a01Preparation of complexes 1 and 2. Reagents and conditions as follows: i dimethylformamide (DMF), diisopropylethylamine, 100\u00a0\u00b0C, 2\u00a0days; ii ethanol, H2N\u2013NH2, reflux, overnight; iii K2PtCl4, MeOH\/water, room temperature, 6\u00a0h; iv CuCl2, DMF, 50\u00a0\u00b0C, overnight\nCleavage of supercoiled DNA\nThe relaxation of supercoiled circular \u03a6X174 DNA (form I) into its relaxed (form II) and linear (form III) conformations was monitored to compare the aerobic cleavage abilities of complexes 2 and 3, in the presence of a reducing agent (Fig.\u00a03). First, the complexes were incubated for 20\u00a0h, to allow the formation of platinum\u2013DNA adducts. The nuclease activity was subsequently initiated by the addition of 5\u00a0mM mercaptopropionic acid. Complex 2 exhibits a markedly higher nuclease activity than Cu(3-Clip-Phen) (3) (compare lanes 5 and 9 in Fig.\u00a03). Indeed, at complex concentrations of 100\u00a0nM, most supercoiled DNA has reacted to form circular and linear DNA in the case of 2, while Cu(3-Clip-Phen) only generates a small amount of form II (Fig.\u00a03, lanes 4 and 8). Moreover, almost all the DNA has reacted to form smaller DNA fragments (migrating as a smear) at a complex concentration of 250\u00a0nM for 2 (Fig.\u00a03, lane 5), whereas no smear is observed for Cu(3-Clip-Phen) (Fig.\u00a03, lane 9). Interestingly, at a concentration of 100\u00a0nM for complex 2, form III is detected before the total disappearance of form I (Fig.\u00a03, lane 4) [19]. This result indicates that the heterodinuclear platinum\u2013copper complex is able to perform direct double-strand cuts since form I is still detected in the reaction. Such double-strand breaks are highly cytotoxic, since the cells have difficulties to repair such damage [28, 29]. Cu(3-Clip-Phen) is only able to perform repetitive single-strand cuts [15]. Interestingly, the cleavage activity of complex 2 is also more efficient compared with that of the platinum\u2013copper complexes previously reported by our group [19].\nFig.\u00a03Comparative experiments of the oxidative cleavage of \u03a6X174 plasmid DNA performed by 2 and 3. Lane 1 control DNA. Lane 2 250\u00a0nM solution of 2. Lane 3 50\u00a0nM 2, in the presence of 5\u00a0mM mercaptopropionic acid (MPA). Lane 4 100\u00a0nM 2, in the presence of 5\u00a0mM MPA. Lane 5 250\u00a0nM 2, in the presence of 5\u00a0mM MPA. Lane 6 250\u00a0nM 3. Lane 7 50\u00a0nM 3, in the presence of 5\u00a0mM MPA. Lane 8 100\u00a0nM 3, in the presence of 5\u00a0mM MPA. Lane 9 250\u00a0nM 3, in the presence of 5\u00a0mM MPA\nTime-course studies of DNA cleavage by complexes 2 and 3 were carried out to further investigate the direct double-strand cleavage event (Fig.\u00a04). Complex 3 at a concentration of 250\u00a0nM generates a maximum of 80% of circular DNA (form II) after a reaction time of about 1\u00a0h [19]. Around 20% of linear DNA (form III) is produced via the action of 3 after 70\u00a0min. Remarkably, the formation of form III is only observed after a reaction time of 30\u00a0min when already 60% of form II has been produced. For a 150\u00a0nM solution of complex 2, linear DNA (form III) is generated from the initial stages of the cleavage reaction, with supercoiled DNA (form I) still being present (Fig.\u00a04). Also, only a maximum of 60% of form II is generated. These features reflect the ability of 2 to perform direct double-strand breaks, most likely as a result of the binding of the platinum moiety to DNA, allowing the copper part to achieve more than one oxidative cleavage in the close proximity of the platinum coordination site.\nFig.\u00a04Time-course experiments of DNA cleavage (20\u00a0\u03bcM in base pairs) over a period of 70\u00a0min. Complex 2 (150\u00a0nM) was incubated for 24\u00a0h, followed by activation with 5\u00a0mM MPA and air\nAnalysis of the platinum adducts on ODN\u00a0I of the ODN\u00a0I\u2013ODN\u00a0II duplex target\nHigh-resolution analyses with a 36-bp DNA duplex (ODN\u00a0I\u2013ODN II) were performed to investigate the coordination of the platinum component of the bifunctional complexes to DNA, (Fig.\u00a05a). The sequence of this duplex was chosen to include GG and AG sites (which are the two major binding sites of cis-Pt(II) complexes) on one strand (ODN I). The results obtained after incubation of the complexes with the duplex labeled on the 5\u2032-end of ODN I for 24\u00a0h were analyzed by polyacrylamide gel electrophoresis under denaturing conditions and are shown in Fig.\u00a05a. Denaturing conditions allowed the analysis of the Pt\u2013DNA adducts on ODN I of the duplex.\nFig.\u00a05a Polyacrylamide gel electrophoresis (PAGE) analysis of the Pt\u2013ODN I adducts of the ODN\u00a0I\u2013ODN\u00a0II duplex target (1\u00a0\u03bcM). ODN I was 5\u2032-end-labeled with 32P-phosphate. The complexes were incubated with the DNA for 24\u00a0h before analyses. Lane 1 ODN I. Lane 2 3\u00a0\u03bcM cisplatin. Lane 3 10\u00a0\u03bcM cisplatin. Lane 4 3\u00a0\u03bcM 1. Lane 5 10\u00a0\u03bcM 1. Lane 6 3\u00a0\u03bcM 2. Lane 7 10\u00a0\u03bcM 2. b Phosphorimager data of a DNA sequencing gel comparing the sequence specificity of cisplatin, 1 and 2. All the samples were extended using Taq polymerase, starting from the 5\u2032-end-labeled primer. Lane 1 blank experiment; Lane 2 3\u00a0\u03bcM cisplatin; Lane 3 10\u00a0\u03bcM 1. Lane 4 10\u00a0\u03bcM 2. It is noteworthy that the GTA and GGAC sites give the sequence of the opposite strand that induced the stopping of the primer extension (Figs.\u00a0S1, S2). c Nucleobase sequence of ODN I, and indication of the damage sites induced by cisplatin, 1 and 2. Large arrows and small arrows represent, respectively, major and minor stop sites\nMolecules able to irreversibly bind to DNA will retard the rate of migration of the modified ODN, compared with that of the free ODN; therefore, ODN\u2013Pt adducts appear as retarded bands on gels. The incubation with 3\u00a0equiv of cisplatin clearly reveals an impeded mobility of the ensuing cisplatin\u2013ODN I adduct (Fig.\u00a05a, lane 2). Only traces of free ODN I were detected; 89% of the ODN I had been modified. The incubation of the DNA duplex with 10\u00a0equiv of cisplatin results in a total conversion of ODN I (Fig.\u00a05a, lane 3). Complex 1 shows the formation of Pt\u2013ODN I adducts (Fig.\u00a05a, lanes 4 and 5). Two distinct bands are clearly observed when 10\u00a0equiv of complex 1 is incubated with the DNA target, indicating the formation of Pt\u2013ODN I adducts. The quantification of free ODN I reveals that as much as 84% of this DNA fragment has reacted. The use of complex 2 leads to comparable results, with the conversion of 88% of the initial ODN I (Fig.\u00a05a, lane 7). However, the reaction between ODN I and complex 2 produces a smear (i.e., a range of products) on the gel. Our previously reported compounds showed reduced formation of Pt\u2013ODN adducts under similar experimental conditions compared with complexes 1 and 2 [19].\nSequence selective binding of complexes 1 and 2 compared with cisplatin\nPrimer extension experiments were performed to investigate the sequence-selective binding of the platinum units to the ODN I fragment of the ODN I\u2013ODN II DNA duplex (Figs.\u00a05b, c, S1, S2). The platinum complexes that did not react with DNA were removed prior to the start of the primer extension experiments. Nevertheless, it is possible to have more than one platinum moiety coordinating the ODN strand, since the concentrations of cisplatin, complex 1 and complex\u00a02 were, respectively 3 and 10\u00a0times higher than the concentration of the ODN I\u2013ODN II DNA duplex. Taq polymerase has proven to be a valuable tool for the determination of the sequence selectivity of various platinum complexes [23, 30\u201339]. Cisplatin inhibits the enzymatic polymerization at the anticipated GG and AG sites (Fig.\u00a05b, c), but the majority of cisplatin is detected at the GG site. It should be noted that once the ODN I strand contains two Pt adducts on both the GG and AG site, the enzyme stops only at the GG site. Therefore, it is possible that the amount of modified AG sites is underestimated. Nevertheless, not all of the ODN I has been modified by the complexes (Fig.\u00a05a). It is therefore reasonable to say that the majority of the ODN I contains only one Pt adduct and that the AG site is indeed the minor site of interaction. The stop sites observed for complex 1 are also located for a major part at the GG base pairs and for a minor part at the AG base pairs. Complex 2 shows only stops at the GG binding site. These results indicate that the platinum moiety interacts with its preferential binding site. The difference between cisplatin and complexes 1 and 2 is the precise point at which the peak intensity occurs at the damaged site. The stops of the Taq polymerase are mainly located at the A base before the GG site for cisplatin, complex 1 and complex\u00a02. However, the enzyme stops induced at the GG site are more equally distributed among the GAC site in the cases of complexes 1 and 2 (Fig.\u00a05b, c; the size of the arrows is an indication of the damage intensity). The binding of complexes 1 and 2 apparently produces bulky adducts, thus allowing a partial stop at the second nucleotide before the classic position, at the GG adduct, since an increase of the reaction time with the enzyme (from 30 to 120\u00a0min, Fig.\u00a0S3; complex 1) induces a bypass of the stop at the C base (associated with a decrease of the peak intensity when compared with that for G). The difference between cisplatin and complex 1 is more pronounced at the AG site. Although it is the minor binding site, the damage induced by cisplatin almost exclusively takes place at the G base, at the 5\u2032-end of ODN I. The Taq polymerase stops for complex 1 are mainly observed at the A base.\nCleavage of the ODN I strand of the ODN I\u2013ODN II duplex\nThe cleavage of the ODN I\u2013ODN II duplex with complexes 1\u20133 was investigated by polyacrylamide gel electrophoresis, the target being labeled on the 5\u2032-end of ODN I (Fig.\u00a06). To the best of our knowledge, studies of such bifunctional complexes have been reported only twice [40, 41].\nFig.\u00a06a PAGE analysis of cleavage of ODN I of the ODN I\u2013ODN II duplex (1\u00a0\u03bcM) by compounds 1\u20133 (10\u00a0\u03bcM when unspecified). The cleavage reactions were initiated with ascorbate (200\u00a0\u03bcM) in aerobic conditions or by heating for 30\u00a0min at 90\u00a0\u00b0C in aqueous 0.2\u00a0M piperidine. The Maxam\u2013Gilbert sequencing reactions A\u00a0+\u00a0G (lane 1) and G (lane 2) were performed to determine the cleavage sites. On the top of the gel are indicated the conditions used during the experiments (details are given in \u201cMaterials and methods\u201d). Lanes 3\u20136 are for controls without complexes. Lanes 17\u201322 are for experiments that were performed in the presence of 1\u00a0\u03bcM 3. b High-contrast picture of lanes 8, 10, 11, 13, 15 and 16 in a allowing easier observation of the the cleavage pattern. \u0394pH 8 was a heating step of 30\u00a0min at 90\u00a0\u00b0C in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)\u2013NaOH buffer (0.1\u00a0M, pH 8.0). Unlinked complexes 1 and 2 were removed in a precipitation step with ethanol before the induction of cleavage\nComplexes 1 and 2 were preincubated for 20\u00a0h to allow the coordination of the platinum moiety to the DNA target. For complex 1, the preincubation was subsequently followed by the coordination of 1\u00a0equiv of copper, since the copper-free complex does not show any nuclease activity. The noncoordinated complexes were removed by precipitation before the cleavage was initiated by the addition of ascorbic acid (0.2\u00a0mM) under aerobic conditions. Additional treatments (HEPES pH 8.0 and piperidine) were carried out.\nThe noncovalent interaction of Cu(3-Clip-Phen) (3) with DNA permits its abstraction from the ODNs by a simple precipitation step. A tenfold excess of 3 (compared with the ODNs) does not generate cleavage products after the precipitation step (Fig.\u00a06, lane 24), while 1\u00a0equiv of 3 without precipitation exhibits significant cleavage (Fig.\u00a06, lane 20). The platinum-containing complexes 1 and 2 form kinetically inert bonds with DNA, and therefore cannot be removed from the ODN during the precipitation step. In contrast, the unreacted complexes are eliminated.\nAs expected, the cleavage pattern of 3 is nonspecific, [18] as about 20% of the target is oxidized at all nucleotides (Fig.\u00a06, lane 20). The cleavage achieved by complex 2 in the presence of ascorbic acid results in the full and partial disappearance of, respectively, the Pt\u2013ODN I adducts and the ODN I band (Fig.\u00a06, lane 10). The total cleavage amounts to nearly 80%, and its pattern is different from the features resulting from the action of 3, but no clear sequence selectivity is observed. Accordingly, a smear of products is noted in contrast to 3, suggesting that the mechanism of the cleavage is different. This observation may be explained by either of the following possibilities: (1) some cuts are due to platinum adducts of complex 2 positioned on the other strand of the duplex (ODN II); (2) the extensive cleavage observed most likely indicates that the ODN I fragment experiences more than one cut; therefore, complex 2 should have been released after repetitive cuts from ODN I and should be able to cut the free ODN.\nTo verify this hypothesis, the experimental conditions were adjusted to introduce fewer platinum adducts onto the target (20% against the 80% of modification of ODN I shown in Fig.\u00a06), and less ascorbate (100\u00a0\u03bcM) was used to limit the recleavage events. However, the resulting cleavage pattern appears to be similar to that observed with the former conditions (Fig.\u00a08).\nOne equivalent of CuCl2 was added to complex 1, after the formation of Pt\u2013DNA adducts, to analyze the cleavage selectivity of the resulting compound. A comparatively less efficient cleavage was achieved using complex 1 with extra added CuCl2, when compared with complex 2 (compare lanes 10 and 15 in Fig.\u00a06), possibly as a result of the partial coordination of the copper ions to 1. Similarly to 2, the cleavage pattern obtained with 1 is found to be significantly different from that of 3 (Fig.\u00a06, lane 20). Interestingly, the free ODN I is not affected during the cleavage process, as only the ODN fragments containing platinum adducts are cleaved. Furthermore, a sequence-selective cleavage (associated with the formation of fragments of ODN I including probably the 3\u2032-phosphate end, since they comigrated as Maxam and Gilbert sequencing fragments) is observed in the close vicinity of the GG and AG sites (indicated as black bars in Fig.\u00a06, left). The intensity of the bands is much stronger at the GG site, reflecting the results of the primer extension experiments previously obtained. The four base pairs neighboring the GG site in the 5\u2032-direction are also affected by the cleavage of complex 1. In the 3\u2032-direction, no clear bands are observed, but a smear is observed, which can be explained as follows: the cleavage products remain coordinated to the complex and have therefore a totally different mobility, compared with the Maxam\u2013Gilbert sequencing fragments. The apparent smear observed in the 3\u2032-direction supports this assumption, since such behavior is observed for Pt\u2013ODN I adducts including the copper complex (Fig.\u00a05a, lane 6), and can therefore be expected for cleavage fragments that coordinate to the complex.\nA heating step in HEPES buffer (90\u00a0\u00b0C; pH 8.0) is often used to cleave the metastable products resulting from the oxidation of deoxyribose. This treatment of the cleavage products obtained with complexes 1\u20133 did not show a strong increase of the DNA cuts (Fig.\u00a06, respective comparisons between lanes 15, 16 and 10 and 11, 20 and 22). Therefore, products of strand cleavage are essentially observed during this analysis. However, the smears observed in lanes 10 and 15 are partially converted into bands, indicating that some cleavage products, arising from the oxidation of deoxyribose, are sensitive to alkaline conditions.\nMore drastic alkaline heating (as with piperidine) allows one to detect the oxidations of the nucleobases that do not induce direct DNA cleavage [6]. Surprisingly when this treatment is applied to complexes 1 (with addition of 1\u00a0equiv of copper) and 2, clear cleavages are observed, although the systems had not been incubated with ascorbate (Fig.\u00a06, lanes 13 and 8, respectively). This phenomenon is dependent of the presence of copper ion, since it is not observed for Pt\u2013ODN I adducts with complex 1 without the addition of CuCl2 (results not shown). Therefore, these alkaline conditions are sufficient to induce a redox activity of the copper complex part of the hybrid molecule [this phenomenon has not been observed with Cu(3-Clip-Phen), which can be removed during a precipitation step preceding the heating in the presence of piperidine]. Interestingly, only the Pt\u2013ODN I adducts are cleaved; none of the free ODN I is degraded and selective cleavages are observed around the position of adducts detected during primer extension experiments. Unfortunately, this activity of the copper complexes covalently linked to the target does not allow us to analyze whether or not the dual Pt\u2013Cu complexes perform the oxidation of nucleobases.\nCleavage of the ODN II strand of the ODN I\u2013ODN II duplex\nThe same experiments were performed with the ODNI\u2013ODNII duplex labeled on the 5\u2032-end of ODN II (Fig.\u00a07). Similar amounts of Pt\u2013ODN II adducts are formed with complexes 1 (Fig.\u00a07, lane 8) and 2 (Fig.\u00a07, lane 3). Most likely, only parts of the platinum components of complexes 1 and 2 are bound on ODN II at the preferential AG site, and the cleavages do not seem to be restricted to one selective site. Other adducts are probably also present. The occurrence of platinum\u2013guanine monoadducts on ODN II (which is particularly rich in guanine bases) may explain the results observed. Further investigations are required to confirm these proposals.\nFig.\u00a07PAGE analysis of cleavage of ODN II of the ODN I\u2013ODN II duplex (1\u00a0\u03bcM) by compounds 1\u20133. The cleavage reactions were initiated with ascorbate (200\u00a0\u03bcM) in aerobic conditions or by heating for 30\u00a0min at 90\u00a0\u00b0C in aqueous 0.2\u00a0M piperidine. The Maxam\u2013Gilbert sequencing reactions A\u00a0+\u00a0G (lane 1) and G (lane 2) were performed to determine the cleavage sites. On top of the gel are indicated the conditions used during the experiments (details are given in \u201cMaterials and methods\u201d). \u0394pH 8 was a heating step of 30\u00a0min at 90\u00a0\u00b0C in HEPES\u2013NaOH buffer (0.1\u00a0M, pH 8.0). Unlinked complexes 1 and 2 were removed in a precipitation step with ethanol before the induction of cleavage\nBoth complex 1 with added copper and complex\u00a02 show extensive cleavage activities in the presence of ascorbic acid in air (Fig.\u00a07, lanes 11 and 6, respectively). In the experiments with complex 2, the Pt\u2013ODN II adducts have fully reacted, while the free ODN II is only partly altered. The cleavage fragments are poorly resolved on the gel (a smear is essentially observed), and treatment with HEPES or piperidine fails to improve their analysis. These cleavage fragments are composed of modified DNA. It can be reasonably proposed that the fragments include Pt\u2013ODN II adducts and that these adducts produce a smear during the migration in polyacrylamide gel electrophoresis. All these results indicate that the platinum moieties of complexes 1 and 2 bind, as expected, to the preferential platinum site on ODN II, but also and mainly to single A and G nucleobases.\nComparison between the cleavage of duplex and single-stranded DNA\nA comparison between the cleavage patterns resulting from the action of complex 2 on single-stranded ODN I and on the duplex ODN I\u2013ODN II was made to appraise the influence of the formation of Pt\u2013ODN II adducts on the resulting cleavage (Fig.\u00a08). Pt\u2013ODN I adducts are formed when the single strand is used, and these adducts are essentially positioned at the GG site of ODN I (the results of the primer extension are not shown). For this study, only 20% of the platinum adducts on ODN I of the duplex and 40% of the platinum adducts on the single strand were created in order to have a maximum of one adduct per DNA target, and thus to simplify the analysis. The cleavage was induced by the addition of a small quantity of ascorbate (100\u00a0\u03bcM) to disfavor recleavage events.\nFig.\u00a08Phosphorimager scanning of the PAGE-cleavage patterns of ODN I by complex 2. The cleavage was performed on either the ODN I\u2013ODN II duplex (5\u2032-end-labeled on ODN I) or on single-stranded ODN I. Cleavage was induced by the addition of ascorbate (100\u00a0\u03bcM) after the removal of the unlinked complex during a precipitation step with ethanol. The clear band area on the 3\u2032-side of the GG site of the duplex is probably due to a covalent adduct of complex 2 on ODN II (the complementary strand of the duplex). 3\u2032-Phosphoglycolate cleavage fragments are labeled with an asterisk. The other clear bands were attributed to fragments of ODN I including 3\u2032-phosphate ends\nThe cleavage of the ODN I fragment of the duplex results in a cleavage pattern exhibiting various moderate peaks in the 3\u2032-region of the GG base pair of ODN I. The cleavage of single-stranded ODN I by complex 2 gives rise to a smear in the same region. The smears observed characterize cleaved ODN I products that are still coordinated to complex 2. Accordingly, the weak peaks in this region of the DNA sequence are most likely due to the cleavage of complex 2\u2013ODN II adducts on both strands. Since the conditions used favor a maximum of one Pt\u2013ODN adduct per duplex, these cleavages on nonmodified ODN I probably originate from complex 2\u2013ODN II adducts.\nThe results are summarized in Fig.\u00a09. Importantly, the cleavage of ODN I by Pt\u2013ODN II adducts appears to be partially responsible of the visible nonselective cleavage of the duplex by complex 2.\nFig.\u00a09Summary of cleavage events due to platinum\u2013copper dual complex 2 on double-stranded DNA. The labeled strand of the DNA duplex that can be observed from denaturing PAGE experiment is labeled with an asterisk\nInterestingly, the weak peaks observed on the 5\u2032-side of the cleavage fragments (and shown with an asterisk in Fig.\u00a08) probably correspond to DNA fragments with 3\u2032-phosphoglycolate extremities. Indeed, these species are observed at the position of the cleavage products of the duplex produced by the action of complex 3, and which have been previously characterized [15]. Such fragments were earlier identified as the result of the oxidation of the C4\u2032 position of 2-deoxyribose. Further investigations are required to confirm these proposals.\nConcluding remarks\nThe novel heterodinuclear platinum\u2013copper complex 2 exhibits improved nuclease activity, compared with its parent compound Cu(3-Clip-Phen) (3), and is able to perform double-strand breaks. The platinum moiety acts as an anchor to DNA, forcing the Cu(3-Clip-Phen) group to generate cuts in close proximity to the Pt\u2013DNA adducts, hence even allowing direct double-strand breaks. Mechanistic investigations on a 36-bp DNA fragment (ODN I\u2013ODN II) revealed that platinum adducts are indeed formed with both complexes 1 and 2. The platinum moiety of complexes 1 and 2 binds to GG (primarily) and AG sites, like cisplatin. Nevertheless, the Taq polymerase enzyme stops at different base pairs for cisplatin and complexes 1 and 2. This feature suggests that complexes 1 and 2 induce different distortions (compared with cisplatin) upon their binding to DNA, most likely owing to the bulkiness of the 3-Clip-Phen moiety. Complex 1 with added copper (1\u00a0equiv) shows a sequence-selective cleavage, in the close proximity of the platinum adducts. A sequence-selective cleavage is also observed for complex 2, but to a lesser extent compared with complex 1. The opposite strand also contains Pt\u2013DNA adducts, but no clear sequence selectivity is observed, most likely owing to the lack of preferential platinum binding sites. These atypical platinum adducts lead to the partial damage of the other DNA strand, as is clearly evidenced by comparison of the cleavage products on a single-stranded or duplex DNA.\nInterestingly, it has been found that piperidine is also able to activate the Cu(3-Clip-Phen) component of the bifunctional platinum\u2013copper complexes, a feature so far not reported. Thus, a good sequence-selective cleavage is observed upon treatment of complex 1 with piperidine. Reasonable sequence selectivity is achieved with complex 2. The investigation of the mechanism of cleavage by complex 2 is currently in progress.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nESM1 (PDF 89.7 kb)","keyphrases":["dna","heterodinuclear","platinum","ditopic","copper"],"prmu":["P","P","P","P","P"]}
{"id":"Int_J_Colorectal_Dis-4-1-2386752","title":"Impact of transanal endoscopic microsurgery on functional outcome and quality of life\n","text":"Introduction Transanal endoscopic microsurgery (TEM) is a minimal invasive technique for local excision of rectal tumours. The procedure is performed via a rectoscope with a diametre of 4 cm. The aim of this prospective study was to assess both functional outcome and quality of life after TEM.\nIntroduction\nLocal excision of benign rectal tumours is safer compared to radical surgery and considered treatment of choice [1\u20133]. Several techniques have been developed for local excision, with the transanal technique according to Parks as the most frequently used [1, 4]. Other techniques used are the dorsal transcoccygeal approach (Kraske) and the dorsal transsphincteric approach (York\u2013Mason) [5\u20139]. Each procedure has its own (dis)advantages, and none of the procedures mentioned is able to achieve local excision of tumours throughout the entire rectum. Transanal endoscopic microsurgery (TEM) demonstrated to be a safe procedure capable of overcoming this shortcoming. In early publications, even distal sigmoid tumours could be locally excised with excellent results. Moreover, recurrence rates are minimal compared to other local techniques. As a result, the indication for local excision of rectal tumours has expanded dramatically [10\u201313].\nFew studies have addressed functional outcome following TEM, and with the operation rectoscope with a length of 12 or 20\u00a0cm and a diametre of 40\u00a0mm, scepticism towards post-operative faecal continence remains. In manometric studies after TEM, there seems to be a detrimental, temporary impact on internal sphincter functioning, although without clinical significance [14\u201316].\nCataldo et al. [17] recently performed a prospective study on faecal continence and incontinence-specific quality of life after TEM using standardised surveys. They stated TEM does not result in significant alterations. These results are promising, especially with a relative short duration of follow-up of 6\u00a0weeks in this study. As known from other types of rectal surgery, incidence of faecal incontinence diminishes with time [18]. This could imply results after TEM may even improve with longer follow-up.\nQuality of life is increasingly recognised as the ultimate endpoint when assessing clinical outcomes after different surgical interventions because it measures the patient\u2019s perspective. The precise impact of the TEM procedure on quality of life has not been well studied. This prospective study was set out to provide a comprehensive insight into the impact of TEM on functional outcome and quality of life.\nPatients and methods\nBetween January 2004 and January 2006, a consecutive series of 50 patients were referred for a TEM procedure. All patients were evaluated pre-operatively according to a standard protocol including rigid rectoscopy, tumour biopsy and endorectal ultrasound. If TEM was considered feasible, patients were eligible for this study. Informed consent had to be given before inclusion. Local medical ethical committees approved this study. A full-thickness excision was always performed. The portion of the tumour located within the sphincter musculature was excised in partial thickness. Before and at least 6\u00a0months after the TEM procedure, patients were asked to fill out a questionnaire to assess anorectal functioning and quality of life. All data were collected by an independent research coordinator not previously involved in the patients\u2019 care. We recorded the demographics, operative details, post-operative length of stay, post-operative complications and functional outcome for each participant. We evaluated functional outcome by means of a detailed questionnaire based on the Faecal Incontinence Severity Index (FISI) [19]. This system, developed by Rockwood, uses two basic components: the type of incontinence and its frequency. FISI scores range from 0 (total continence) to 61 (complete incontinence to solid stool on a daily basis). We used the validated weighting scores that are based on patients\u2019 input.\nQuality of life was evaluated using both the EuroQol EQ-5D and the Faecal Incontinence Quality of Life (FIQL) score. The EuroQol EQ-5D consists of a so-called index score representing the societal value of the health state and has a scale ranging from 0 (no quality of life) to 100 (optimal quality of life). The EuroQol EQ-5D also uses a visual analogue scale, the EQ-VAS, representing the patient perspective. This scale ranges from 0 (no quality of life) to 100 (optimal quality of life). The EuroQoL EQ-5D scores were compared with a sex- and age-matched, community-based sample of healthy persons without co-morbidity [20]. The FIQL score as described by Rockwood et al. [21] measures specific quality of life issues expected to affect patients with faecal incontinence. This instrument is composed of 29 questions within four domains: lifestyle issues, coping\u2013behaviour, depression and self-perception and embarrassment. The scores in the FIQL range from a minimum score of 1 to a maximum of 4, for all of the scales (1\u2009=\u2009quality of life alteration present most of the time, 4\u2009=\u2009none of the time). Data are presented as medians and standard deviations. Changes within groups were evaluated using the nonparametric one-sample Wilcoxon\u2019s signed-rank test. Comparison of these changes between groups was conducted using the Mann\u2013Witney U test. The Spearman\u2019s correlation coefficient was used for correlation between the different findings. A P-value of \u22640.05 was considered statistically significant.\nResults\nFifty patients were eligible and informed consent was obtained. Three patients were excluded. In two patients, TEM could not be performed because of bulky tumour or technical problems. An additional patient underwent low-anterior resection for locally recurrent disease within 6\u00a0months of the TEM. The remaining 47 patients completed both questionnaires and were included for analysis. All of these patients were alive without evidence of recurrent disease. The group consisted of 22 males and 25 females. Median age was 67\u00a0years (range 40\u201384). Pre-operative diagnosis was villous adenoma in all patients. Median distance from the distal tumour margin to the dentate line was 7\u00a0cm (range 0\u201315\u00a0cm) and median tumour size was 20\u00a0cm2 (range 4\u201353\u00a0cm2). The median rate of captured circumference of the rectal wall was 40% (range 5\u201380; Table\u00a01). Median operative time, defined as beginning when the rectoscope was inserted and ending when it was removed, was 55\u00a0min (range 10\u2013140). Complications developed in 4 of 47 (8.5%) patients. Two patients had urinary retention, one patient a urinary tract infection and one suffered from a low hemoglobin rate requiring blood transfusion. No reoperations were necessary and mortality rate was zero. Median length of stay was 4\u00a0days (range 3\u20139\u00a0days; Table\u00a02). Definite histopathological examination of the resected specimens revealed an adenoma in 44 patients and an invasive carcinoma in three patients (pT1 in two patients and pT2 in one patient). These three patients were reluctant to undergo major surgery and were observed with rectoscopy and endorectal ultrasound every 3\u00a0months without signs of recurrence at 6\u00a0months after TEM. In three adenomas, excisional margin was considered microscopic irradical, resulting in 94% of tumours being radically excised.\nTable\u00a01Patient and tumour characteristicsCharacteristicsNumberMale\/female (N\u2009=\u200947)22\/25Median age in years (range)67 (40\u201384)Median distance from dentate line in centimetres (range)7 (0\u201315)Median tumour size in square centimetres (range)20 (4\u201353)Median capture of circumference of rectal wall in per cent (range)40 (5\u201380)Table\u00a02Procedure-related characteristicsCharacteristicsNumberMedian duration of operation in minutes (range)55 (10\u2013140)Complications4\/47 (8.5%)Urinary retention2Urinary tract infection1Blood transfusion1ReoperationsNoneLength of hospital stay in days (range)4 (3\u20139)\nSix months after surgery, mean FISI scores were found to be decreased (pre-operative, 10; post-operative, 7 (p\u2009\n<\u20090.01)), depicting an improvement in faecal continence (Fig.\u00a01). Overall, when pre-operative and post-operative FISI scores were compared, 24 patients improved, 16 patients were unchanged and seven deteriorated. Operation time or tumour size did not influence the change in FISI score. There was a significant correlation between the decrease in FISI score and tumour height (p\u2009=\u20090.02). Reduction of FISI was significantly greater in patients with a tumour location within 7\u00a0cm from the dentate line (p\u2009=\u20090.01; Table\u00a03). Mean scores and ranges of the EuroQol EQ-5D are presented in Table\u00a04. Mean general quality of life score from the patients\u2019 perspective (EQ-VAS) was found to be significantly higher 6\u00a0months after TEM (p\u2009<\u20090.02). The observed changes in EQ-VAS showed no correlation with the post-operative alterations in FISI scores or tumour characteristics. Mean pre-operative EQ-VAS score in our group was lower compared to the mean EQ-VAS score of the sex- and age-matched general population (p\u2009=\u20090.02). Post-operative EQ-VAS score was comparable with the general population. Mean index score (social perspective) remained the same (p\u2009=\u20090.09). Both pre- and post-operative EQ-5D index scores were similar to those of the sex\u2013age-matched general population. Comparing the change from baseline in FIQL scores, a statistically significant improvement was observed in two of the four domains (embarrassment; p\u2009=\u20090.03 and lifestyle; p\u2009=\u20090.05). The domains of lifestyle, coping and behaviour and embarrassment were correlated with the FISI (all p\u2009<\u20090.05; Table\u00a05). Overall, EQ-5D and FIQL scores were not affected by age and gender of the patients. Surgical aspects and tumour characteristics did not influence the outcome.\nFig.\u00a01Mean Faecal Incontinence Severity Index (FISI) scores (\u00b1SEM) before and after Transanal Endoscopic Microsurgery (TEM)Table\u00a03Mean FISI scores; numbers in parentheses are standard deviationsFISI scorePre-operativePost-operativeStatistical significanceOverall10 (2)6 (2)p\u2009<\u20090.01Duration of operation <55\u00a0min (N\u2009=\u200924)9 (4)7 (3)p\u2009=\u20090.24Duration of operation >55\u00a0min (N\u2009=\u200923)12 (3)4 (2)p\u2009=\u20090.17Tumours <7\u00a0cm from dentate line (N\u2009=\u200921)16 (5)5 (2)p\u2009=\u20090.01Tumours >7\u00a0cm from dentate line (N\u2009=\u200926)6 (2)7 (3)p\u2009=\u20090.43Median tumour size <20\u00a0cm2 (N\u2009=\u200927)12 (4)6 (3)p\u2009=\u20090.12Median tumour size >20\u00a0cm2 (N\u2009=\u200920)8 (3)6 (3)p\u2009=\u20090.32Lower values indicate better anorectal functioningTable\u00a04Mean EuroQoL EQ-5D scores; numbers in parentheses are standard deviations\u00a0Control groupPre-operativePost-operativeStatistical significanceEQ-VAS82 (7)77 (14)82 (11)p\u2009=\u20090.02Index score86 (6)84 (11)89 (9)p\u2009=\u20090.09EQ-VAS represents the patients\u2019 perspective on quality of life; index score represents the societal value on quality of life. Higher scores indicate higher quality of life. Both scores are compared with a healthy sex- and age-matched control group.Table\u00a05Mean FIQL scores; numbers in parentheses are standard deviationsFIQLSPre-operativePost-operativeStatistical significanceLifestyle3.7 (0.3)3.9 (0.3)p\u2009=\u20090.05Coping3.6 (0.5)3.8 (0.4)p\u2009=\u20090.10Depression3.7 (0.3)3.9 (0.4)p\u2009=\u20090.08Embarrassment3.1 (0.3)3.7 (0.4)p\u2009=\u20090.03Higher scores indicate higher quality of life.\nDiscussion\nIn rectal adenomas, TEM has emerged as the procedure of choice because of its safety and low local recurrence rates. Especially compared to radical surgery, TEM has proven its safety [22, 23]. However, possible adverse effects of TEM have to be addressed. The use of a rectoscope with a 4-cm diametre, introduced transanally, has lead to substantial scepticism regarding impact on anorectal functioning. In earlier studies, we already showed TEM to be superior to total mesorectal excision regarding post-operative defecation disorders, although this did not result in improved quality of life [24]. In the present study, TEM resulted in improved faecal continence as measured by the FISI. This apparent paradox may be attributed to pre-operative tumour symptoms such as mucinous or bloody discharge, prolapse, tenesmi and\/or urge, giving rise to incontinence-like symptoms. Post-operative improvement of continence was most significant in tumours within 7\u00a0cm from the dentate line but disappeared in our study in tumours above 7\u00a0cm from the dentate line. Kreis et al. [25] performed manometric studies after TEM and found a significant reduction in anal resting pressure 1\u00a0year post-operative and a temporary reduction in anal squeezing pressure, resulting in a temporary rise in urge\u2013incontinence. Kennedy et al. [26] found a significant reduction in anal resting pressure 6\u00a0weeks after TEM. This reduction was significantly correlated with duration of the procedure, but mean continence score was not changed after TEM. Both of the above studies however did not use validated questionnaires on faecal continence, and therefore comparison with our study is difficult. Cataldo et al. [17] reported on the impact of TEM on functional outcome and incontinence-specific quality of life using the same questionnaires. No significant alteration was found in faecal continence after TEM. The discrepancy between both studies may be explained by the relative short interval between the TEM procedure and post-operative questioning of 6\u00a0weeks in the Cataldo series. Also, in his study, indications for TEM were heterogeneous which may have influenced results. The positive effect of TEM on faecal continence in our series may be explained by the differences in pre-operative FISI score between both studies (10 versus 2.4), depicting more continence problems among the patients in our series. Another explanation may be the differences in tumour distance from the dentate line (present series median 7\u00a0cm, Cataldo series 11\u00a0cm). Also, in our series, tumours were larger (median 20\u00a0cm2 versus 8.75\u00a0cm2). Because tumours were larger in our series, more extensive resections were performed, often in tumours located within the sphincter apparatus. These latter resections were already shown to influence rectoinhibitory reflex, reflex sphincter contraction, rectal sensitivity and compliance [16]. Further analysis within our series upon this issue showed only tumour distance from the dentate line of less than 7\u00a0cm to be a significant contributing factor. These results however are based upon low number of patients and therefore solid conclusions cannot be drawn. Although in our study TEM resulted in a significant improvement in continence, the post-operative FISI was still worse compared to the Cataldo series (7 versus 2.4). Regarding quality of life, Cataldo found TEM was of no significant influence. In our series, mean general quality of life score from the patients\u2019 perspective, EQ-VAS, was significantly higher after TEM. This improvement could not be explained by improved FISI scores but probably by lower pre-operative EQ-VAS scores as compared to healthy controls. Another explanation may be the rejoice phenomena, that is, patients are relieved the tumour has been excised, and in most cases an adenoma was found [27]. However, because of the low number of invasive carcinomas in our series this is purely theoretical. The societal value of general quality of life, EQ-5D, remained unchanged. Measuring quality of life using the FIQL questionnaires resulted in a significant improvement in two of the four FIQL domains (embarrassment and lifestyle). Moreover, the domains of lifestyle, coping and behaviour and embarrassment were all significantly correlated with the FISI.\nIn conclusion, how are these results to be interpreted? This study supports the hypothesis that rectal tumours give rise to incontinence-like symptoms, especially in low-lying rectal tumours. After the tumour is excised using the TEM technique, faecal continence improves. TEM itself does not improve continence but also does not deteriorate faecal continence. Mean quality of life from the patients\u2019 perspective following TEM is improved.\nBased on, as we know, the only two studies addressing anorectal functioning and quality of life after TEM in one study, it can be concluded that TEM does not impair faecal continence. Also, quality of life is not negatively influenced by the TEM procedure itself, and therefore TEM is the procedure of choice in all rectal adenomas.","keyphrases":["functional outcome","quality of life","tem","local excision","rectal neoplasms"],"prmu":["P","P","P","P","M"]}
{"id":"J_Headache_Pain-4-1-2245994","title":"Treatment of migraine attacks based on the interaction with the trigemino-cerebrovascular system\n","text":"Primary headaches such as migraine are among the most prevalent neurological disorders, affecting up to one-fifth of the adult population. The scientific work in the last decade has unraveled much of the pathophysiological background of migraine, which is now considered to be a neurovascular disorder. It has been discovered that the trigemino-cerebrovascular system plays a key role in migraine headache pathophysiology by releasing the potent vasodilator calcitonin gene-related peptide (CGRP). This neuropeptide is released in parallel with the pain and its concentration correlates well with the intensity of the headache. The development of drugs of the triptan class has provided relief for the acute attacks but at the cost of, mainly cardiovascular, side effects. Thus, the intention to improve treatment led to the development of small CGRP receptor antagonists such as olcegepant (BIBN4096BS) and MK-0974 that alleviate the acute migraine attack without acute side events. The purpose of this review is to give a short overview of the pathological background of migraine headache and to illustrate the mechanisms behind the actions of triptans and the promising CGRP receptor blockers.\nThe trigemino-cerebrovascular system\nPain-sensitive structures such as the intracranial vessels and the meninges, especially the dura mater, are supplied with sensory nerve fibers. In fact they are innervated by the ophthalmic ramus of the first branch of the trigeminal nerve that arises from pseudounipolar neurons located in the trigeminal ganglion. Those neurons project onto second order sensory neurons in the trigmenial nucleus caudalis (TNC) in the brain stem and its related extensions down to the C2-level called the trigeminocervical complex [1]. From this region a signal is transmitted to the ventroposterior thalamus leading to activation in cortical areas, including frontal cortex, insulae and cingulate cortex. This results in the experience of pain (Fig.\u00a01). In addition, a migraine active region has been pointed out in the brainstem by using positron emission tomography (PET) [2\u20134]. Thus, an involvement of the trigemino-cerebrovascular system in the transmission of nociceptive information to the central nervous system is a viable position.\nFig.\u00a01In migraine attacks, sensory nerves, which innervate the cerebral blood vessels, are activated to antidromically release calcitonin gene-related peptide. They originate from the first branch of the trigeminal nerve that arises from pseudounipolar neurons located in the trigeminal ganglion (TG). Those neurons project onto second order sensory neurons in the trigeminal nucleus caudalis (TNC) from where a signal is transmitted to the cortex\nThe trigemino-cerebrovascular reflex\nThere is a dense supply of CGRP-containing nerve fibers originating in the first division of the trigeminal ganglion that innervate intracranial vessels. Experimental studies have shown that induction of vasoconstriction provoked rapid normalization of the vascular tone due to\u2013\u2013in part\u2013\u2013reflex activation of the trigeminal nerve. Vasoconstriction triggers an antidromic release of sensory trigeminal neuropeptides, mainly CGRP, being a strong intracranial dilatator [5, 6]. After denervation there is no alteration in the contractile response of the vessel but the time to attain initial basal tone is markedly prolonged. This is of importance as headache may be initiated by a spreading wave of depression of cortical neurons resulting in contraction of some parts of the cerebral circulation. This phenomenon has now been shown in man [7] and could activate the trigeminal vascular system to release CGRP to reestablish the vascular tone. Thus, the trigemino-cerebrovascular reflex is probably part of a counter-balancing system, which is able to mediate dilatation of brain vessels and sustain cerebral blood flow [8]. This may assure the maintenance of local brain blood flow within normal limits as the cerebral circulation requires high and constant flow. Activation of the neurons in the trigeminal ganglion leads on the other hand to an activation of neurons in the trigeminocervical complex that project to the central nervous system (CNS) and mediate the central aspects of pain [1, 9].\nCalcitonin gene-related peptide\nCGRP is a neuropeptide consisting of 37 amino acids and is predominantly expressed in the nervous system. It originates from the calcitonin gene encoding calcitonin and CGRP depending upon alternative splicing.\nHistochemical studies of cerebral perivascular nerve fibers and human trigeminal ganglia revealed that almost half of the cell bodies contain CGRP [10, 11]. Although many other signal molecules are located in the trigeminal ganglion, CGRP is the most abundant in man [12] and can be seen as a marker for trigeminal activity. Furthermore it is the only transmitter reliably released in acute migraine attacks at present. There was a marked increase in CGRP levels during migraine attacks, but no changes could be observed for other neuropeptides such as vasoactive intestinal peptide (a marker for parasympathetic activity), neuropeptide Y (sympathetic activity) and substance P (sensory activity) when their levels were analyzed in the cranial venous outflow during migraine attacks [8, 13].\nIn migraine attacks elicited within clinical studies by administration of nitroglycerin a good correlation has been described between increased levels of CGRP and the intensity of the headache [14, 15]. But it is noteworthy to mention that nitroglycerin elicits head pain, but not always genuine migraine attacks. There may be a need for the subject to be in a \u201cprone phase\u201d, where the trigemino-cerebrovascular system might be hyper-reactive as it was demonstrated in cluster headache [16].\nTriptans\nThe increased cranial venous levels of CGRP observed in conjunction with migraine attacks thus indicate activation of the trigeminal system. Administration of sumatriptan\u2013\u2013belonging to the highly effective anti-migraine drugs called triptans or 5-HT1B\/1D agonists-results in alleviation of the headache and normalization of the cranial venous CGRP levels [8]. They are thought to act mainly as powerful vasoconstrictors of cerebral vessels via 5-HT1B receptors, which have been shown to be expressed in the medial smooth muscle cell layer of the human middle meningeal [17] and cerebral arteries [18].\nIn addition, triptans may bind to presynaptic 5-HT1B\/1D receptors on trigeminal afferents to inhibit nerve activity and hence reduce CGRP release. 5-HT1B\/1D receptors were found on trigeminal sensory fibers [19], trigeminal ganglion cells [20] and in the trigemino-cerebrovascular system in man [21]. Furthermore, the 5-HT1B\/1D receptors were co-localized with CGRP in the human trigeminal ganglion [20].\nAutoradiographic studies using labeled zolmitriptan confirmed the presence of 5-HT1B\/1D receptors in certain brain stem nuclei that are involved in cranial nociceptive processing [22] and are likely to be activated during migraine.\nThus, it can be presumed that a potential relaxation of intracranial blood vessels during migraine attacks can be normalized by triptans, which activate the cerebrovascular 5-HT1B receptors and thereby cause vasoconstriction. By stimulating 5-HT1B\/1D receptors, triptans inhibit the release of CGRP from the perivascular nerve terminals and the trigeminal ganglion cells. Activation of 5-HT1B\/1D receptors at the level of the CNS (trigeminal nucleus caudalis) may interrupt central aspects of the headache process.\nRecently the relationship between pharmacokinetic and pharmacodynamic aspects of triptans in migraine was investigated. It has been observed that subcutaneous administration of sumatriptan is more effective than oral application. The difference in efficacy is most likely due to the rapid initial rise in plasma level after subcutaneous administration [23]. Furthermore migraine attacks may influence gastric stasis leading to delayed absorption of drugs [24, 25]. Thus, to increase efficacy of treatment, attention should be paid to the development of migraine drugs, which can be quickly absorbed.\nTriptans cause sometimes cardiovascular adverse effects as they also act as constrictors in coronary arteries. But triptans are considered as more powerful vasoconstrictors in human isolated cerebral arteries than in coronary arteries. They are at least threefold more potent [26]. The reason for the higher potency and efficiency of triptans in intracranial arteries could be due to at least three differences. First there is a higher number of 5-HT1B receptors in those vessels compared to peripheral arteries [18, 27]. Secondly there are few, if any, 5-HT2A receptors in intracranial vessels whereas the 5-HT2A receptor population dominates in coronary arteries [27]. Thirdly, there might be potential differences in coupling of the 5-HT1B receptors between cerebral and coronary arteries. In consideration of the fact that triptans are weak constrictors in coronary arteries [28, 29] and have little impact on coronary blood flow, the clinical consequences in healthy subjects are minor. However, small contractions may anyhow have proportionally greater impact in diseased artery with luminal narrowing. Triptans are hence contra-indicated in patients with known cardiovascular disease.\nSensitization in migraine\nTreatment with triptans achieves good results in patients suffering from sensitization. Sensitization is defined as an increased afferent activity in response to an unchanged stimulus [30] and both peripheral and central sensitization may play a role in migraine attacks. Peripheral sensitization of meningeal perivascular nociceptors arising from trigeminal neurons can clinically explain phenomena like intracranial hypersensitivity (worsening pain during physical activity) and the throbbing element in migraine pain. This may be due to normally innocuous meningeal nociceptors becoming hyper-responsive to rhythmic fluctuation in intracranial pressure that is produced by normal arterial pulsations [31, 32].\nCentral sensitization of nociceptive second order neurons in the trigeminocervical complex can be the reason for extracranial tenderness and cutaneous allodynia. The latter is an abnormal sensory state in which otherwise innocuous stimuli are sensed as painful [31].\nTwo-thirds of patients develop allodynia during or even after a migraine attack [33]. Based on clinical studies new guidelines for triptan treatment in migraine has been established. They include that allodynia-free patients can receive good results from triptan treatment at any time during the migraine attack. Furthermore, patients suffering from allodynia can expect excellent results from triptans before allodynia gets established. Therefore they should take triptans as early as possible during the attack. After the onset of allodynia, migraineurs can still benefit from triptans, but pain relief may be incomplete [34\u201336].\nHowever, a slightly different view has been expressed in a recent study [37]. After subcutaneous administration of sumatriptan, there was no difference in alleviation of migraine pain irrespective if the drug was given early or late. Therefore, it was suggested that the sensitization is of minor importance and that the relatively poor effect of sumatriptan late in migraine attacks is primarily due to gastric stasis [37].\nCGRP receptors\nLocal vasodilatation and release of CGRP from the vascular nerve fiber endings\u2013\u2013caused by electrical field stimulation or capsaicin treatment [38, 39]\u2013\u2013are attenuated by administration of triptans, but also by application of CGRP receptor blockers [40]. The proof that CGRP plays a key role in migraine was provided by demonstrating that the potent CGRP receptor antagonist olcegepant (BIBN4096) could abort acute migraine attacks to a comparable degree as sumatriptan [41, 42]. In addition, CGRP receptors are located at several sites, which play an important role during migraine, including the cerebrovasculature [43\u201345], the trigeminocervical complex within the brainstem [46] and the trigeminal ganglion [47].\nFunctional CGRP receptors consist of three components, namely a seven-transmembrane domain G-protein coupled receptor called calcitonin receptor-like receptor (CLR), a receptor activity-modifying protein (RAMP) and a receptor component protein (RCP). CLR shares 55% sequence identity with the calcitonin receptor. Co-expression of CLR with RAMP1 composes the CGRP receptor, while co-expression of CLR with either RAMP2 or RAMP3 represents the adrenomedullin (AM) receptor or possibly a combined receptor (CGRP and AM). Apart from contributing to the receptor specificity, RAMP1 is required for glycosylation and transport of CLR to the plasma membrane [48\u201350]. In addition, RAMP1 seems to be functionally rate limiting for CGRP receptor activity in the trigeminovascular system. It was proposed that elevated RAMP1 expression could sensitize the trigeminal ganglion of individuals to CGRP actions such as CGRP release and neurogenic inflammation [47].\nThe third component of the CGRP receptor, the accessory protein RCP, is required for proper biological function as it is involved in coupling the receptor to downstream signaling pathways like the protein kinase A (PKA) pathway [51]. Activation of CGRP receptors in cultured trigeminal ganglion cells increased the cAMP levels, which in turn led to elevated CGRP promoter activity and increased CGRP mRNA levels. These results point to an autoactivation of CGRP expression and indicate that elevated release of CGRP in the trigeminal system may create a self-sustaining feedback loop [47].\nCGRP receptor antagonists olcegepant and MK-0974\nThe excellent correlation between CGRP release and migraine headache has long pointed towards the potential usefulness of a specific CGRP receptor antagonist in the treatment of primary headaches [9, 52]. Thus, a series of small molecule CGRP receptor blockers were developed. Among those is olcegepant, which demonstrates extremely high affinity for human CGRP receptors, even higher affinity than the endogenous ligand [53]. Therefore it was selected for further pharmacological investigations. Olcegepant was shown to be a pure antagonist for the human CGRP receptor and to be selective against a broad panel of enzymes and receptors such as calcitonin, amylin or adrenomedullin receptors [54].\nIn isolated human temporal, middle meningeal and cerebral arteries olcegepant effectively inhibits CGRP induced dilatation [44, 55]. That may be due to the block of the CGRP receptor as mRNA for CLR and RAMP1\u20133 has been found in human middle meningeal arteries, cerebral arteries as well as in microvessels [56] and CGRP receptor components could be detected in the human cerebral vasculature [43]. In addition, olcegepant dose-dependently inhibited vasodilatation induced by electrical stimulation of the trigeminal ganglion in primates [54].\nOne major advantage of a CGRP receptor blocker is the lack of vasoconstrictor ability. But blockade of the receptor of a strong vasodilatation involves a theoretical risk of causing both peripheral and cerebral vasoconstriction, especially as high affinity binding sites for CGRP have been reported in the heart, liver, spleen, intestine, urogenital system, skeletal muscles, and the skin [57\u201359]. But olcegepant in doses up to 10\u00a0mg did not cause any clinical relevant changes of vital signs like blood pressure and pulse rate, in the ECG or in forearm blood flow in healthy volunteers [60]. Detailed studies of peripheral arteries such as mesenteric and coronary arteries [44, 61] revealed that those arteries show weaker responses to CGRP and lower degree of antagonism by olcegepant. This is possibly related to a lower number of CGRP receptors. In addition, a study with seven healthy volunteers disclosed that olcegepant had no influence on cerebral blood flow or on the blood flow velocity in the middle cerebral artery [62]. It was concluded that CGRP receptor blockade has no effect on the cerebral circulation in man.\nHowever, it is still not clear where olcegepant acts as a CGRP antagonist. It has been reported to inhibit (1) dilatation of dural arteries initiated by CGRP [63], (2) CGRP-induced hypotension [40], and (3) increase in facial blood flow after trigeminal ganglion stimulation [54]. In contrast, olcegepant did not significantly inhibit changes in tone of cerebral arterioles or of local cortical cerebral blood flow [40]. Those results indicate that olcegepant poorly penetrates the blood-brain barrier, but is very effective in preventing vasodilatation of vessels without this feature [64]. Thus, this CGRP receptor blocker was suggested to exert its antagonistic effects mainly extracerebral, which correlates with results from a study of healthy volunteers [65]. In contrast, it was recently shown that CGRP-induced relaxation of the rat middle cerebral artery can only be inhibited by abluminally administered olcegepant as olcegepant was prevented from reaching the CGRP receptors by the arterial endothelium [66]. These results suggest that the anti-migraine effect of this CGRP receptor blocker is on the abluminal side of the blood-brain barrier. This is supported by the fact that olcegepant only has an antimigraine effect in high doses.\nAnyhow, olcegepant was effective in a dose of 2.5\u00a0mg in the treatment of acute migraine attacks [41]. The overall treatment effect revealed a responder rate (a moderate to severe headache going to mild or no headache) of 60% at 2\u00a0h after the start of the infusion compared with 27% in the placebo group (Fig.\u00a02). Thus, intravenous olcegepant was found to be as efficacious as oral sumatriptan in the relief of acute attacks of migraine [42].\nFig.\u00a02The anti-migraine effect of the CGRP receptor antagonists MK-0974 and olcegepant and the triptans rizatriptan and sumatriptan compared to placebo at 2-h pain relief. The overall treatment effect of MK-0974 (p\u00a0=\u00a00.015) and rizatriptan (p\u00a0=\u00a00.010) showed significance versus placebo (left part) [69]. A dose of 2.5\u00a0mg of olcegepant also showed significant superiority over placebo (p\u00a0=\u00a00.001) [41]. Data of sumatriptan from another study [42] were added to demonstrate the similar efficacy of olcegepant compared to triptans\nSignificant superiority over placebo was seen regarding the pain-free rate at 2\u00a0h, the sustained pain relief at 24\u00a0h and the improvement of nausea, photophobia and phonophobia (Fig.\u00a03). The response was still rising at 4\u00a0h pointing at a long duration of action. At 24\u00a0h the pain-free rate was better than with triptans, suggesting a lower grade of rebound.\nFig.\u00a03Comparison of the sustained pain free-rate at 24\u00a0h of the CGRP receptor antagonists MK-0974 and olcegepant, the triptans rizatriptan and sumatriptan, and placebo. MK-0974 displayed superior efficacy versus placebo (p\u00a0<\u00a00.001) [69]. In order to compare the efficacy of olcegepant to a triptan, data of sumatriptan from another study [42] were added\nThe rate of recurrence was 19% as compared to a placebo rate of 46%. This is clearly lower than the recurrence rate of triptans which usually is around 28% [42].\nSafety and tolerability studies also showed that olcegepant is well tolerated. Adverse events were, in general, minor and dose-dependent. Transient mild paresthesia was the single most frequent adverse event [60].\nOne major disadvantage of olcegepant remains in its structure. It is a hydrophilic molecule, has poor penetration across the blood-brain barrier and is not suitable as an oral drug. Considering the crucial advantages of an oral formulation of a drug, a small-molecule, non-peptide CGRP antagonist called MK-0974 was recently developed for the acute treatment of migraine attacks [67]. MK-0974 was, among others, tested in a noninvasive pharmacodynamic model in rhesus monkeys, in which endogenous CGRP release and a vasodilatatory response are achieved by topical application of capsaicin to the forearm of the rhesus monkey [68]. Infusion of different CGRP receptor antagonists inhibited the increase in local dermal blood flow and showed effectiveness with MK-0974 being the most potent among them (>3.5-fold) [67]. In addition, MK-0974 displayed a low clearance (7.0\u00a0mL\u00a0min\u22121\u00a0kg\u22121) and a good i.v. half-life (2.8\u00a0h). The compound was finally chosen as a clinical candidate, when it showed >10,000-fold selectivity in assays including more than 160 receptors, transporters, and enzymes [67].\nRecently, MK-0974 has been tested in a randomized, double-blind, placebo-and active-controlled, outpatient study with a two-stage, dose-ranging design to test safety and effectiveness [69]. Three hundred and thirty patients suffering from migraine with and without aura were treated with MK-0974 (in doses of 25\u2013600\u00a0mg), rizatriptan (10\u00a0mg) or placebo. As doses below 300\u00a0mg were insufficient, they were discontinued. Thus, the overall treatment effect was defined as the average of the 300, 400, and 600\u00a0mg doses and showed significance vs. placebo on the primary endpoint (p\u00a0=\u00a00.015), which was defined as pain relief at 2\u00a0h after treatment (Fig.\u00a02).\nMK-0974 displayed also superior efficacy vs. placebo for secondary endpoints such as pain freedom at 2\u00a0h (overall treatment effect p\u00a0<\u00a00.001), sustained pain relief at 24\u00a0h (overall treatment effect p\u00a0<\u00a00.001) or sustained pain freedom at 24\u00a0h (overall treatment effect p\u00a0<\u00a00.001) (Fig.\u00a03). In addition, MK-0974 provided relief of migraine-associated symptoms such as photophobia, phonophobia and nausea.\nRizatriptan (10\u00a0mg) was used as an active control and demonstrated effectiveness vs. placebo (p\u00a0=\u00a00.010) on the primary endpoint defined as pain relief at 2\u00a0h. By comparison, 300 and 600\u00a0mg doses of MK-0974 appeared to work as well as rizatriptan or showed even numerical superiority regarding the sustained pain freedom at 24\u00a0h and the sustained pain relief at 24\u00a0h.\nConcerning tolerability and safety, MK-0974 was generally well-tolerated. The reported side effects were mainly nausea, dizziness and somnolence, but the incidence of adverse experiences seemed to be comparable to the placebo group. In addition, there was no increase in adverse events with increasing dose.\nThus, MK-0974 showed only mild side effects, but significant relief of migraine pain 2\u00a0h after treatment compared to placebo and the relief was sustained through 24\u00a0h [69].\nConclusion\nThe novel CGRP receptor antagonists appear to be effective in the treatment of moderate and severe migraine attacks. They do not only provide pain relief at 2\u00a0h, but also show efficacy regarding the secondary endpoints such as pain freedom, improvement of associated symptoms and functional disability [41, 69]. In addition, they show a prolonged effect of action compared to the triptans. That may suggest not only less rebound headache but perhaps also a prophylactic possibility. Furthermore, only minor adverse effects were reported and no cardiovascular side effects could be seen so far. This is probably due to the absence of vasoconstrictor properties and it also suggests that vasoconstriction is not necessary to abort acute migraine attacks. This lack of direct vasoconstrictor activity may represent an important advantage over the triptans. Further development of drugs against the CGRP receptor will offer a new promising way of treatment.","keyphrases":["trigemino-cerebrovascular system","cgrp","triptan","olcegepant","mk-0974"],"prmu":["P","P","P","P","P"]}
{"id":"Plant_Cell_Rep-4-1-2413081","title":"Agrobacterium tumefaciens-mediated transformation of poinsettia, Euphorbia pulcherrima, with virus-derived hairpin RNA constructs confers resistance to Poinsettia mosaic virus\n","text":"Agrobacterium-mediated transformation for poinsettia (Euphorbia pulcherrima Willd. Ex Klotzsch) is reported here for the first time. Internode stem explants of poinsettia cv. Millenium were transformed by Agrobacterium tumefaciens, strain LBA 4404, harbouring virus-derived hairpin (hp) RNA gene constructs to induce RNA silencing-mediated resistance to Poinsettia mosaic virus (PnMV). Prior to transformation, an efficient somatic embryogenesis system was developed for poinsettia cv. Millenium in which about 75% of the explants produced somatic embryos. In 5 experiments utilizing 868 explants, 18 independent transgenic lines were generated. An average transformation frequency of 2.1% (range 1.2\u20133.5%) was revealed. Stable integration of transgenes into the poinsettia nuclear genome was confirmed by PCR and Southern blot analysis. Both single- and multiple-copy transgene integration into the poinsettia genome were found among transformants. Transgenic poinsettia plants showing resistance to mechanical inoculation of PnMV were detected by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Northern blot analysis of low molecular weight RNA revealed that transgene-derived small interfering (si) RNA molecules were detected among the poinsettia transformants prior to inoculation. The Agrobacterium-mediated transformation methodology developed in the current study should facilitate improvement of this ornamental plant with enhanced disease resistance, quality improvement and desirable colour alteration. Because poinsettia is a non-food, non-feed plant and is not propagated through sexual reproduction, this is likely to be more acceptable even in areas where genetically modified crops are currently not cultivated.\nIntroduction\nPoinsettia, Euphorbia pulcherrima Willd. Ex Klotzsch, is a contemporary symbol of Christmas in most parts of the world. Since it was introduced to the United States in 1825 from Mexico, poinsettia has become the primary potted flower produced and sold in North America, Europe, Asia and Australia (Ecke et al. 2004; Williams 2005). Today, Europe and North America represent the largest volume of production and sales, but demand is growing quickly in the Australian region as poinsettia becomes more popular each year (Williams 2005). Global production of poinsettia has exceeded hundreds of millions and is still expanding, indicating its economic and market potential for the floral industry.\nGenetic engineering is an important tool for breeding ornamental plants with addition of desirable traits such as novel colour, better quality and resistance to pathogens and insects (Mol et al. 1995; Deroles et al. 2002; Hammond 2006; Hammond et al. 2006). This technology has been successfully utilized in the production of a number of important ornamental crops e.g. blue roses (Yoshikazu 2004), novel carnations (http:\/\/www.florigene.com), transgenic gladiolus (Kamo et al. 1997) and improvement of chrysanthemums (Teixeira da Silva 2004). To date, transgenic ornamentals of over 30 genera have been produced by different transformation approaches (Hammond 2006; Hammond et al. 2006). However, there are only a few reports describing genetic transformation of poinsettia: one was the US patent 7119262 (Smith et al. 1997) using the biolistic transformation approach, while the other two were electrophoresis-based transformation attempts (Vik et al. 2001; Clarke et al. 2006). Biolistic transformation requires the use of a gene gun device (Sanford et al. 1987) and tends to generate transformants with a high transgene copy number, complex transgene loci and unpredictable silencing of the transgene (Herrera-Estrella et al. 2004). Electrophoresis of DNA into meristems on a living plant was described as a simple method to generate transformants by avoiding tedious tissue culture work and was utilized in producing transgenic orchid (Griesbach 1994). However, no stable transgenic poinsettia was ever produced using electrophoresis, regardless of the strong transient expressions that were detected in both studies (Vik et al. 2001; Clarke et al. 2006). Thus, Agrobacterium-mediated transformation for poinsettia was developed in the present study.\nPoinsettia mosaic virus (PnMV) is a single-stranded, positive-sense RNA virus (Bradel et al. 2000) that belongs to the family Tymoviridae (Dreher et al. 2005). Infection of poinsettia plants with PnMV results in mosaic symptoms during parts of the growing season (Fulton and Fulton 1980), which in turn decreases the commercial value of this ornamental plant. Thus, growers are interested in the potential benefits of growing PnMV-free poinsettias. PnMV-free poinsettia plants can be obtained by heat treatment or in vitro culture of apical meristems, which are time-consuming and cost-ineffective methods. An additional problem is that PnMV-free poinsettia tends to be rapidly reinfected, although no vector is known (Blystad and Fl\u00f8istad 2002; Siepen et al. 2005). There is therefore a need for a new and effective alternative approach, like Agrobacterium-mediated transformation, which can overcome these difficulties.\nRNA silencing is a mechanism by which transcription or translation of a gene is suppressed. It is known to occur in plants, fungi and animals (Fire et al. 1998; Waterhouse et al. 1998; Baulcombe 2005). It is triggered by double stranded RNA (dsRNA) molecules (Meister and Tuschl 2004), which are subsequently recognized and cleaved by the host-encoded endoribonuclease dicer (Bernstein et al. 2001) into small interfering RNA (siRNA) molecules of 21\u201326 nucleotides in length (Hamilton and Baulcombe 1999). These siRNAs, in conjunction with the RNA-induced silencing complex (RISC), target RNA molecules with homologous sequences for sequence-specific degradation (Hammond et al. 2000; Bernstein et al. 2001; Tabara et al. 2002). In plants, RNA silencing can be achieved by genetic transformation with gene constructs that express highly transcribed sense, anti-sense or self complementary hairpin RNA (hpRNA) containing sequences homologous to the target gene (Smith et al. 2000; Wesley et al. 2001; Helliwell and Waterhouse 2003). RNA silencing has been efficiently used to generate resistance against plant viruses in many plant species including ornamentals (Metzlaff et al. 1997; Tenllado et al. 2004; Bucher et al. 2006; Hammond et al. 2006).\nIn this study, we report the development of an A. tumefaciens-mediated transformation method for poinsettia, which has never previously been described for this plant species. This method could facilitate the improvement of poinsettia by introducing new traits into existing commercial poinsettia cultivars in order to meet market demands. Using this approach, in combination with RNA silencing technology, transgenic PnMV resistant poinsettia plants carrying PnMV-derived hpRNA constructs were produced for the first time.\nMaterials and methods\nPlant materials\nEuphorbia pulcherrima, poinsettia cv. Millenium, plants were kindly supplied by the J. Kristiansen nursery, Grimstad, Norway, in the year 2000. The original stock plants were subjected to heat therapy to eliminate PnMV (Fl\u00f8istad and Blystad, unpublished). PnMV-free cv. Millenium cuttings were grown in the greenhouse under a photoperiod of 16\u00a0h light and 8\u00a0h dark with a temperature of 22\u00b0C. Internode stem explants from 8 to 10-week-old cv. Millenium plants were used for somatic embryogenesis and A. tumefaciens-mediated transformation.\nSomatic embryogenesis of poinsettia cv. Millenium\nInternode stem explants 5\u201315\u00a0mm long from cv. Millenium plants were excised and used in the establishment of somatic embryogenesis for poinsettia prior to transformation. In Experiments 1 and 2, the explants were surface sterilized for 10\u00a0min in 3% NaOCl and then rinsed three times with sterilized deionized and autoclaved H2O for 5, 10 and 15\u00a0min according to Preil (1994). Because of a large number of infections encountered in Experiments 1 and 2, the stem explants were sterilized in Experiment 3 using 70% ethanol for 1\u00a0min followed by 5\u00a0min with 1% NaOCl and 3 rinsings with sterile deionized and autoclaved water for 3, 10 and 20\u00a0min, a modification of the protocol of Preil (1994). The modified protocol was repeated twice before being utilized in Experiment 3 and also in subsequent transformation experiments. After sterilization, stem segments of ca. 1\u20131.5\u00a0mm thickness were placed on callus induction (CI) medium [MS medium (Murashige and Skoog 1962) supplemented with 0.2\u00a0mg\u00a0l\u22121 BAP and 0.2\u00a0mg\u00a0l\u22121 CPA and 30\u00a0g\u00a0l\u22121 sucrose, Table\u00a01] for 8\u201310\u00a0days. Embryogenic calli were then transferred to somatic embryo induction (SEI) medium (MS medium contains 0.3\u00a0mg\u00a0l\u22121 NAA and 0.15\u00a0mg\u00a0l\u22121 2iP and 30\u00a0g\u00a0l\u22121 sucrose, Table\u00a01) for somatic embryogenesis. The somatic embryos emerged after an average of 12\u00a0weeks were then transferred to somatic embryo maturation (SEM) medium (MS basal medium containing 0.05 mg\u00a0l\u22121 BAP and 30\u00a0g\u00a0l\u22121 sucrose, Table\u00a01). Shoots and plantlets derived from somatic embryos were subsequently cultured on root induction (RI) medium consisting of 1\/2 strength MS, 2\u00a0mg\u00a0l\u22121 IAA and 20\u00a0g\u00a0l\u22121 sucrose or on hormone free 1\/2 strength MS (HFMS) medium supplemented with 20\u00a0g\u00a0l\u22121 sucrose for root induction (Table\u00a01). Plants with well-developed roots were transferred to soil and grown in the greenhouse at 22\u00b0C. The above culture conditions and media compositions for somatic embryogenesis of cv. Millenium were developed according to Preil (1994) with a number of modifications described above. Detailed information regarding media compositions is summarized in Table\u00a01.\nTable\u00a01Media names and compositions modified according to Preil (1994)MediumMSSucrose (%)CPA (mg\u00a0l\u22121)BAP (mg\u00a0l\u22121)NAA (mg\u00a0l\u22121)2iP (mg\u00a0l\u22121)IAA (mg\u00a0l\u22121)CIFull strength30.20.2\u2013\u2013\u2013SEIFull strength3\u2013\u20130.30.15\u2013SEMFull strength3\u20130.05\u2013\u2013\u2013RI1\/2 strength2\u2013\u2013\u2013\u20132HFMS1\/2 strength2\u2013\u2013\u2013\u2013\u2013CI Callus induction medium, SEI somatic embryo induction medium, SEM somatic embryo maturation medium, RI root induction medium, HFMS hormone free MS medium for root induction\nLight conditions were 23\u00a0\u03bcE\u00a0m\u22122\u00a0s\u22121 for callus and somatic embryos and 30\u00a0\u03bcE\u00a0m\u22122\u00a0s\u22121 for plantlets in RI and HFMS media with a 16\u00a0h photoperiod. Temperature was constant at 24\u00b0C. A light microscope connected to a digital camera was used to follow the development of the somatic embryos.\nAgrobacterium strain and hairpin (hp) RNA constructs\nThe disarmed A. tumefaciens strain LBA4404 (Hoekema et al. 1983; Invitrogen, California, USA) was utilized throughout the study. Three hpRNA constructs, named as pCP, pR2 and pR3, were generated. Construct pCP targeted the viral coat protein (CP), whereas constructs pR2 and pR3 targeted two distinct regions within the viral RNA-dependant RNA-polymerase (RdRp) (Fig.\u00a01a). Briefly, constructs pCP, pR2 and pR3 were generated by amplifying the corresponding fragments from the viral genome and introducing the appropriate restriction sites. The following restriction sites were introduced: XhoI\u00b7KpnI (for sense orientation) and ClaI\u00b7XbaI (for antisense orientation) for R2 and R3 fragments, and XhoI\u00b7EcoRI (sense) and ClaI\u00b7XbaI (antisense) for CP fragment. The primers used are presented in Table\u00a02. Each amplified fragment was 500\u00a0bp long. Subsequently, the fragments were inserted into pHANNIBAL vector (kindly provided by CSIRO Plant Industry, Canberra, Australia) in sense and antisense orientations, interrupted by an intron pyruvate orthophosphate dikinase (pdk) gene (Fig.\u00a01b) as described by Helliwell and Waterhouse (2003). The three expression cassettes were cloned into pART27 (Gleave 1992, kindly provided by CSIRO Plant Industry, Canberra, Australia), a binary plasmid vector, at Not1 site under the control of CaMV 35S promoter and with ocs terminator (Helliwell and Waterhouse 2003), generating hpRNA constructs pCP, pR2 and pR3 (Fig.\u00a01b). For selection, the neomycin phosphotransferase II (nptII) gene conferring kanamycin resistance was used under the control of nopaline synthase promoter (Nos-P) and with Nos terminator (NosT, Fig.\u00a01b) in the binary plasmid expression vector pART27. Sequence analysis was utilized to verify the junctions and orientations in the expression vectors pCP, pR2 and pR3. All three plasmid vectors were introduced into A. tumefaciens strain LBA 4404 by electroporation according to the manufacturer\u2019s instructions (Invitrogen, California, USA).\nFig.\u00a01Schematic representation of PnMV genome (a) and hairpin (hp) RNA constructs used in the current study (b). The locations of the primers used to generate the constructs as well as for screening purposes are presented over the PnMV genome. Sequences of the primers are presented in Table\u00a02. The location of the probe used to detect siRNA is presented below the PnMV genome (see \u201cMaterials and methods\u201d). CP coat protein region, R2 and R3 RNA-dependent RNA-polymerase (RdRp) regions of the PnMV genomeTable\u00a02Primers used in this studyPrimer nameRestriction sitesSequenceaLocationR2F-XhKXhoI, KpnIctcgagggtaccTTTAGCAAAACGCAGCACAAAATCA4,371\u20134,395R2R-CXbClaI, XbaIcatcgattctagaTCTCCAGACACCATGATTGGGTG4,848\u20134,870R3F-XhKXhoI, KpnIctcgagcggtaccTTCGCTTTAAAACAGAAAGCACCA4,939\u20134,962R3R-CXbClaI, XbaIcatcgatgtctagaGCCTCGTAGCTTGGTTGGGTT5,418\u20135,438CPF-XhEXhoI, EcoRIctcgaggaattcAACCACGTCGACTCCACTCCAT5,478\u20135,499CPR-CXbClaI, XbaIatcgattctagaAGCTTGCCGCTCACCAGCAC5,958\u20135,977aPrimer sequences are presented in the 5\u2032\u20133\u2032 orientation. Sequences complementary to the viral genome are presented in uppercase letters whereas non-complementary restriction sites included in each primer are presented in lowercase letters. The primers used for screening purposes are identical to the ones presented but do not include the non-complementary restriction sites\nTransformation of poinsettia stem explants\nA. tumefaciens strain LBA4404, harbouring the plasmid pCP, pR2 or pR3, was grown overnight in 15\u00a0ml liquid LB medium supplemented with 50\u00a0mg\u00a0l\u22121 kanamycin (Sigma-Aldrich, St Louis, USA) at 28\u00b0C with shaking at 200\u00a0rpm until an OD600 of 0.6\u20130.8 was reached. The bacterium suspension was collected at 2,700\u00a0rpm for 10\u00a0min, washed twice with MS basal medium supplemented with 2% sucrose (MS-2), and resuspended in 10\u00a0ml MS-2.\nThe internode stem explants taken from 8 to 10-week-old poinsettia plants derived from cuttings were disinfected as described above, excised into stem segments with 1\u20131.5\u00a0mm thickness and inoculated with Agrobacterium suspension for 5\u00a0min with gentle shaking. After the infection, stem segments were blotted briefly with sterile filter paper and placed on CI medium at 24\u00b0C in the dark for 72\u00a0h without selection. After co-cultivation, the explants were blotted gently on sterile filter paper and transferred to the CI medium with selection and light conditions as described above.\nSelection and regeneration of transgenic plants\nAfter 8\u201310\u00a0days on CI medium supplemented with 500\u00a0mg\u00a0l\u22121 claforan (Aventis Pharma Ltd, Norway) and 10\u00a0mg\u00a0l\u22121 kanamycin, the explants were transferred to SEI medium supplemented with 400\u00a0mg\u00a0l\u22121 claforan and 25\u00a0mg\u00a0l\u22121 kanamycin for somatic embryogenesis. The protocol for somatic embryogenesis is detailed above. The somatic embryos obtained after about 12\u00a0weeks were then transferred to the SEM medium supplemented with 400\u00a0mg\u00a0l\u22121 claforan and 10\u00a0mg\u00a0l\u22121 kanamycin. Shoots and plantlets derived from somatic embryos were subsequently cultured on RI medium or on hormone free 1\/2 strength MS (HFMS) medium (Table\u00a01) for root induction and were subsequently transferred to soil and grown in the greenhouse at 22\u00b0C.\nScreening of transgenic poinsettia plants \nScreening of transgenic poinsettia plants was carried out by PCR. The primer pairs used to amplify the CP, R2 and R3 fragments were the same as those for vector constructions, but did not include any additional restriction site (Table\u00a02). HotStarTaq PCR kit purchased from Qiagen (Valencia, California) was used in PCR screening of transformants. Twenty micro litres of reaction mixture containing 2\u00d7 HotStarTaq Mastermix, 0.4\u00a0\u03bcM of each primer, 0.1\u00a0\u03bcg template DNA and H2O were subjected to PCR amplification under the following conditions: 15\u00a0min at 95\u00b0C (1 cycle), 30\u00a0s at 95\u00b0C, 30\u00a0s at 55\u00b0C, 1\u00a0min at 72\u00b0C (35 cycles) and a final extension 10\u00a0min at 72\u00b0C (1 cycle) according to the manufacturer\u2019s instructions. All the PCR amplification was performed using Applied Biosystems 96 Thermal Cycler (Applied Biosystems). PCR products were analysed by electrophoresis on 0.8% (W\/V) agarose gels.\nSouthern blot analysis\nTo confirm the stable integration of transgenes into the poinsettia genome and to evaluate transgene copy number, Southern blot analysis was performed, basically as described by Sambrook et al. (1989). Total genomic DNA was isolated from young leaves of control plant and the putative transformants using a modified CTAB protocol as described by Rogers and Bendich (1988). Ten micrograms of genomic DNA was digested with the restriction enzyme HindIII for 4\u00a0h and separated on a 1% (W\/V) TBE agarose gel overnight at 37\u00a0V followed by transfer onto Gene Screen Transfer membrane (NEN\u2122 Life Science Products Inc., Boston, MA, USA). The HindIII site is not present in the pCP, pR2 and pR3 vectors. The HindIII site from the pHANNIBAL vector was removed during the vector construction. Membranes were hybridized overnight with 32P-labelled probe of 1.5\u00a0kb in size targeting the CP, R2 and R3 regions (Fig.\u00a01a). The probe was generated by amplifying a 1.5\u00a0kb fragment that included the R2, R3 and CP regions used to generate the hairpin RNA constructs pR2, pR3 and pCP. This allowed us to use one probe instead of three probes for southern blot analyses of pR2, pR3 and pCP transformants.\nNorthern blot hybridization of low molecular weight RNA\nTotal RNA was extracted using the Trizol reagent (Invitrogen, USA) as recommended by the manufacturer. Low molecular weight (LMW) RNA was separated from high molecular weight RNA by precipitation with 1\u00a0volume of 4\u00a0M LiCl4 at 4\u00b0C overnight. After centrifugation, the LMW RNA-containing supernatant was transferred to a new tube and precipitated with 1\u00a0volume of isopropanol. LMW RNA concentrations were measured using a GeneQuant II spectrophotometer (Amersham Biosciences, UK) and RNA quality was verified by agarose gel electrophoresis. LMW RNA (approximately 30\u00a0\u03bcg) was mixed with 1\u00a0volume of Tris-borate\u2013EDTA\u2013urea sample buffer (Biorad, USA) and heated at 100\u00b0C for 5\u00a0min. Subsequently, LMW RNA was separated on a 15% polyacrylamid tris-borate\u2013EDTA\u2013urea gel and transferred to a Hybond-N membrane (Amersham Biosciences, UK) overnight. The membranes were hybridized with a digoxigenin (DIG)-labelled (Roche Diagnostics, Germany) RNA probe (Fig.\u00a01a), previously cleaved by alkaline hydrolysis to generated fragments of approximately 50\u00a0bp as described by Hamilton and Baulcombe (1999). After hybridization the membranes were washed at room temperature, 2\u00a0\u00d7\u00a015 min in 5\u00d7 SSC\u00a0+\u00a00.5% SDS and 2\u00a0\u00d7\u00a015\u00a0min 1\u00d7 SSC\u00a0+\u00a00.5% SDS and then exposed to films.\nVirus inoculation and detection\nSap from poinsettia and N. benthamiana plants systemically infected with PnMV was used as an inoculum. Approximately 0.5\u00a0g of infected leaf tissue was ground with 4\u00a0ml of inoculation buffer [0.03\u00a0M sodium phosphate buffer (pH 8)] and mechanically inoculated to leaves of three to four-leaves stage plants lightly dusted with carborundum. Plants were grown in the greenhouse at 22\u201324\u00b0C.\nDetection of PnMV infection by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was carried out at 6 and 10\u00a0weeks post-inoculation basically as described by Clark and Adams (1977). The uppermost fully expanded leaves of each plant were collected in polyvinyl bags (Bioreba, USA). Extraction buffer [100\u00a0mM phosphate buffer (pH 7.4), 20\u00a0mM Tris, 137\u00a0mM NaCl, 3\u00a0mM KCl, 2% PVP 24\u00a0kD, 0.05% Tween 20, and 0.02% NaN3] was added to the bags (1\/10 w\/v) and the samples were macerated. A measure of 100\u00a0\u03bcl of the homogenates were transferred to wells of microtitre plates previously coated with 100\u00a0\u03bcl of PnMV IgG (Agdia, USA) diluted 1\/1,000 in coating buffer [50\u00a0mM carbonate\u2013bicarbonate buffer (pH 9.6) and 0.02% NaN3], followed by incubation at +4\u00b0C overnight. Microtitre plates were washed with washing buffer [10\u00a0mM phosphate buffer (pH 7.4), 140\u00a0mM NaCl, 3\u00a0mM KCl, and 0.05% Tween 20] and 100\u00a0\u03bcl of alkaline phosphatase conjugated PnMV IgG (Agdia, USA) diluted 1\/1,000 in extraction buffer was added to each well. Microtitre plates were incubated at +4\u00b0C overnight. Subsequently, they were washed with washing buffer and 100\u00a0\u03bcl of substrate buffer [1\u00a0M diethanolamine (pH 9.8) and 0.02% NaN3] containing p-nitrophenyl phosphate (0.5\u00a0mg\u00a0ml\u22121) was added. Absorbance values were recorded at 405\u00a0nm with an Expert plus microtitre plate reader using the Kim software (ASYS HITECH, Austria). Values were considered positive when the A450 value was twofold higher than that of the negative control.\nResults\nEstablishment of somatic embryogenesis for poinsettia cv. Millenium\nAn efficient protocol for somatic embryogenesis of poinsettia cv. Millenium was developed. Stem explants of cv. Millenium responded well to the induction media and a large number of somatic embryos were effectively induced (Table\u00a03; Fig.\u00a02a\u2013d). In Experiments 1 and 2, somatic embryos could only be induced from 21 to 37% of the explants due to infection, whereas more than 75% of the explants produced somatic embryos in Experiment 3 after the modification of the disinfection protocol. The average numbers of somatic embryos on each embryogenic callus varied from 5.3\u00a0\u00b1\u00a02.4 and 5.8\u00a0\u00b1\u00a03.1 for Experiments 1 and 2, respectively, to 7.2\u00a0\u00b1\u00a04.1 for Experiment 3 (Table\u00a03). The highest induction of somatic embryos was found in Experiment 3 with 18 somatic embryos on a single callus. When globular stage somatic embryos (Fig.\u00a02a, arrowed) reached the mature stage (Fig.\u00a02b), they became loosely attached to the calli. Shoots derived from somatic embryos were subsequently cultured on RI medium or HFMS medium (Fig.\u00a02c) for root induction and were transferred into the greenhouse when the roots were well established (Fig.\u00a02d). The three experiments conducted and the efficiency of somatic embryogenesis are summarized in Table\u00a03.\nTable\u00a03Experiments and efficiency of somatic embryogenesisExperimentNo. of explants% of embryogenic explantsRange of SE\/embryogenic explantMean SE\/embryogenic explantaNo. of regenerated plantletsb1200212\u2013105.3\u00a0\u00b1\u00a02.4702200372\u2013145.8\u00a0\u00b1\u00a03.11353350753\u2013187.2\u00a0\u00b1\u00a04.1768SE somatic embryosaValues are presented with their corresponding standard deviationbRegenerated plantlets after 14\u00a0weeksFig.\u00a02Somatic embryogenesis in poinsettia cv. Millenium: a embryogenic structure and globular stage somatic embryos (arrows) that appeared on the callus (bar 1\u00a0mm), b cotyledonary stage of somatic embryos (bar 1\u00a0mm), c plantlets deriving from somatic embryos on RIM medium; and d regenerated plants established in the greenhouse\nThe RI and HFMS media used for root induction did not show any clear differences in the percentage (85 and 87% for RI and HFMS, respectively) of plants that developed roots 3\u00a0weeks after being transferred to the rooting media.\nDevelopment of the A. tumefaciens-mediated transformation method\nPrior to the production of transgenic poinsettia plants, a number of optimizations were carried out to reveal the most suitable conditions for inoculation and co-cultivation, concentration of antibiotic for eliminating A. tumefaciens after co-cultivation, and kanamycin selection without inhibiting somatic embryogenesis and regeneration of plants. Results showed that the optimal inoculation time was 5\u00a0min with gentle shaking, while the most suitable co-cultivation time was 72\u00a0h (Table\u00a04). Using this combination, 26 out of the 80 (33%) explants tested PCR positive after inoculation, co-cultivation and 2\u00a0weeks on claforan (Table\u00a04). Those explants were followed and showed normal embryogenesis and regeneration as compared to the control (data not shown). Therefore, these conditions were utilized throughout all the transformation experiments. When inoculation time exceeded 20\u00a0min followed by a co-cultivation period of 6\u00a0days, only 4% of the explants survived due to the overgrowth of Agrobacterium on the surfaces of the explants (Table\u00a04).\nTable\u00a04Number of PCR positive explants that survived 2\u00a0weeks after Agrobacterium inoculation and co-cultivation treatments. A total of 80 explants were used in each treatmentCo-cultivation time (days)Inoculation time 5\u00a0min10\u00a0min20\u00a0min29 (11%)13 (16%)21 (26%)326 (33%)21 (26%)20 (25%)610 (13%)8 (10%)3 (4%)\nFor elimination of Agrobacterium growth after co-cultivation, the optimized concentration of claforan was 500\u00a0mg\u00a0l\u22121 for the CI medium, whereas 400\u00a0mg\u00a0l\u22121 claforan was an adequate concentration for the SEI and SEM media. For selection of transformed cells, 25\u00a0mg\u00a0l\u22121 kanamycin was found optimal for the SEI medium, while 10\u00a0mg\u00a0l\u22121 kanamycin was optimal for the CI and SEM media.\nProduction of transgenic poinsettia plants, molecular analyses and resistance assays\nTo produce transgenic poinsettia plants, 5 transformation experiments including 868 stem segment explants were carried out using the established inoculation, co-cultivation and selection conditions described above. Of those explants, 228 (26.3%) somatic embryo-derived plants were obtained. The results are summarised in Table\u00a05.\nTable\u00a05Summary of five Agrobacterium-mediated transformation experiments on poinsettia cv. Millenium with pCP, pR2 and pR3 constructsExperimentaNo. of explantsNo. of regenerated plantsNo. of transformantsbTransformation efficiency (%) c1 (CP)1855431.62 (R3)1326932.33 (R3)1726663.54 (R2)2542131.25 (R2)1251832.4aCP, R3, and R2 represent transformation experiments with constructs pCP, pR2 and pR3, respectivelybTransformants verified by PCR and Southern blot analysiscNumber of transformants\/total number of explants transformed\nPCR analysis was conducted for screening the putative transformants. All 228 regenerated plants were analysed by PCR and 18 transformants were revealed. Of these, three possessed pCP constructs, while six and nine contained pR2 and pR3 constructs, respectively (Fig.\u00a03a\u2013c). Transformation frequency varied from 1.2 to 3.5% with an average transformation frequency of 2.1% (Table\u00a05).\nFig.\u00a03PCR analysis. PCR positive transformants detected with primer pairs for (a) CP, (b) R2 and (c) R3 fragments respectively. Lane A1 1\u00a0kb marker; lane A2 non-transformed plant; lanesA3\u20135 are independent CP-transgenic lines 11-1, 11-2 and 3-1; laneA6 plasmid control. Lane B1 1\u00a0kb marker; lane B2 non-transformed plant; lanes B3-8 are R2-transgenic lines 72-1, 72-2, 75-2, 79-1, 84-2, and 84-4; lane B9 plasmid control. Lane C1 1\u00a0kb marker; lane C2 non-transformed plant, lanes C3-11 are R3 transgenic lines 18-1, 30A, 38-1, 40A, 40B, 41-2, 41-5, 56-2 and 62-1; lane C12, plasmid control. The 500\u00a0bp band of the 100\u00a0bp marker is depicted with an arrow\nSouthern blot analysis was performed on selected PCR positive plants with satisfactory characteristics for studying transgene integration and estimating the transgene copy number. Results of Southern blot analysis confirmed the stable integration of transgene and both single- and multiple-copy transgene integration into the poinsettia genome were detected among the transformants (Fig.\u00a04). Of the eight transformants analysed, six showed single copy integration of transgene (lanes 2\u20136 and 9 in Fig.\u00a04) with fragment size greater than the length of intact T-DNA (ca 7.5\u00a0kb). However, two pR3 transformants (lanes 7\u20138 in Fig.\u00a04) showed multiple-copy transgene integration with both large (intact T-DNA) and small fragments (partial, non intact T-DNA).\nFig.\u00a04Southern blot analysis of selected PCR positive transformants carrying pCP, pR2 and pR3 constructs. The HindIII-digested total genomic DNA was probed with a 1.5\u00a0kb probe homologous to the region of CP, R2 and R3 fragments (Fig.\u00a01) allowing us to analyse all the three different types of transformants (CP, R2 and R3 transformants) at the same time. Lane1 plasmid control; lanes2-4 CP transformants 11-1, 11-2 and 3-1; lanes5-6 R2 transformants 72-2, 79-1; lanes 7-9 R3 transformants 38-1, 40B, 18-1; lane10 negative control\nFurthermore, Northern blot analysis of low molecular weight RNA was carried out on the transgenic plants harbouring constructs pCP, pR2 and pR3, prior to inoculation. Transgene-derived siRNA molecules of 21\u201326 nucleotides length were detected in transgenic lines pCP 11-1, pCP 3-1, pR2 72-1, pR2 72-2 and pR3 18-1 (Fig.\u00a05). This data indicated that the transgene had been transcribed and converted into siRNAs (Fig.\u00a05). Due to limitations in the amount of LMW RNA from transgenic pR3 lines 40B, 41-2, 62-1 and 41-5, these lines were not included in the experiment.\nFig.\u00a05Northern blot analysis of low molecular weight (LMW) RNA to detect small interfering RNAs (siRNA) in non-inoculated transgenic plants and non-inoculated control. aLane 1 control; lanes 2\u20133 CP transgenic lines 11-1, 3-1; lanes 4\u20135 R2 transgenic lines 72-1,72-2; lane 6 R3 transformant, 18-1. Ribosomal RNA is presented in (b) to indicate presence of LMW RNA in the gel. Lane 1 control; lanes 2 and 3 PCP transformed plants; lanes 4 and 5 PR2 transformed plants; lane 6 PR3 transformed plant\nTransgenic plants and non-transformed controls were mechanically inoculated with PnMV at a three to four leaves stage. The upper non-inoculated leaves were tested by DAS-ELISA at 6 and 10\u00a0weeks post-inoculation. Results showed that the control plants were systemically infected with PnMV, whereas the tested transgenic lines carrying pCP and pR2 constructs remained virus free (Table\u00a06). However, of the five pR3-containing transgenic lines studied, two lines remained virus free, while the remaining three lines were systemically infected (Table\u00a06).\nTable\u00a06Immunological detection of PnMV on non-transformed and transformed poinsettia plants inoculated with PnMVConstructTransgenic plantaELISAb6\u00a0wpi10\u00a0wpiR272-10.10.172-20.10.2R318-10.10.240B0.82.441-20.52.662-10.10.241-50.72.8CP11-10.10.23-10.10.1Control +10.82.820.82.7Control \u221210.10.220.20.230.20.240.10.1wpi Weeks post inoculationaOne plant per transgenic line was challenged in this assaybELISA absorbance values (405\u00a0nm) after 60\u00a0min of incubation at room temperature are presented. Samples with twice the value of the highest negative control sample were deemed positive\nDiscussion\nSignificance of genetic engineering in control of virus diseases in ornamentals\nThe application of genetic engineering in the floriculture industry has clearly become instrumental and rewarding in meeting the demand for novel desirable traits. A number of genetic engineering approaches have been developed for ornamental plants (Griesbach 1994; Kamo et al. 1995; Deroles et al. 1997; Zuker et al. 1998; Kamo et al. 2000; Kishimoto et al. 2002; Kim et al. 2004; Teixeira da Silva 2004; Hammond 2006 and the current study) to facilitate the improvement of ornamental crops with better quality and enhanced resistance against diseases and pests. Effective resistance against viruses conferred by transgenes has shown significant potential because no chemical can be applied for control of virus diseases. Regardless of the importance, only a few reports have been published demonstrating the virus resistance obtained in ornamentals through genetic engineering (Kamo et al. 1997; review by Hammond et al. 2006). Furthermore, many of the ornamentals were transformed only with marker genes like gus or selectable antibiotic or herbicide resistant markers (Deroles et al. 2002). Using the Agrobacterium-mediated transformation protocol developed in the current study, we have obtained PnMV resistant transgenic poinsettia lines expressing viral sequences targeting the CP and RdRp regions of the PnMV genome. To our knowledge, no PnMV resistance has previously been obtained using classical or molecular breeding approaches, demonstrating the potential of the transgenic lines produced in the present study for the poinsettia industry.\nSomatic embryogenesis and Agrobacterium-mediated transformation in poinsettia cv. Millenium\nOne of the reasons that transformation of ornamental plants has lagged somewhat behind that of major crops is the lack of efficient regeneration systems, as these are key components leading to the successful development of transformation protocols. Moreover, both transformation and regeneration can be cultivar- or even genotype-dependent. Therefore, we developed an efficient somatic embryogenesis system for poinsettia cv. Millenium prior to transformation, although a number of somatic embryogenesis systems were reported previously for old cultivars such as Angelika, Franzi and Diamond (Preil and Beck 1991; Preil 1994; Osternack et al. 1999). A recent study on somatic embryogenesis of cv. Freedom Red and Freedom White demonstrated the influence of cultivar and even plant genotype on somatic embryogenesis in poinsettia (Castellanos et al. 2006), indicating the necessity of developing an effective regeneration system for the individual cultivar prior to the development of transformation systems.\nPoinsettia, like other Euphorbia species, produces latex, a milky solution exuded from cut surfaces. When explants were excised from greenhouse grown poinsettia plants, the latex was released from the cutting points, making surface sterilization difficult. In Experiments 1 and 2, a very low percentage of explants produced somatic embryos due to a severe infection found in the callus induction culture (Table\u00a03). This is likely to have been the result of a combination of latex and insufficient sterilization based on the sterilization procedure described for cv. Angelika, Franzi and Diamond (Preil 1994), which might not be optimal for cv. Millenium (Table\u00a03). A modified protocol was thus used for surface sterilization in Experiment 3 as well as in the five transformation experiments, and the efficiency of somatic embryogenesis was greatly improved (see Table\u00a03), demonstrating the significance of optimization of sterilization and regeneration protocols for the individual cultivar.\nIn addition to the availability of an efficient regeneration system, successful A. tumefaciens-mediated transformation depends on several factors, e.g. inoculation and co-cultivation times with A. tumefaciens, antibiotic selection and regeneration. The optimized infection conditions and co-cultivation times in our study (Table\u00a04) are consistent with the range of published reports on ornamental plants, where 5\u201330\u00a0min infection time and 1\u20136\u00a0days co-cultivation have been reported (Aida et al. 1999; Kishimoto et al. 2002; Cui and Ezura 2003; Kim et al. 2004; Teixeira da Silva 2004). However, in our study, 6\u00a0days co-cultivation led to A. tumefaciens overgrowth and explant death, thus differing from the results for Begonia, in which 6\u00a0days co-cultivation is required (Kishimoto et al. 2002). These data indicate that co-cultivation times vary among different plant species.\nAntibiotic selection is another key factor influencing the establishment of a transformation protocol. To eliminate Agrobacterium after co-cultivation, several antibiotics such as cefotaxim (claforan), carbenecillin, and timentin have been used in previous studies. The influence of the type of antibiotic and\/or concentration on each transformation system varies greatly (Ishida et al. 1996; Nauerbey et al. 1997; Sunikumar and Rathore 2001). In the present study, optimised concentrations of claforan in the CI, SEI and SEM media effectively eliminated Agrobacterium. For selecting transformed cells, the nptII gene was used. Although selection media containing 50 and 100\u00a0mg\u00a0l\u22121 kanamycin have been documented in the production of transgenic plants (Kishimoto et al. 2002; Kim et al. 2004; Wu et al. 2005), those concentrations severely inhibited the development of embryogenic calli and somatic embryos in poinsettia. In our study the optimal concentrations of kanamycin were 10 and 25\u00a0mg\u00a0l\u22121. In the case of Agrobacterium-mediated transformation of Platanus acerifolia, kanamycin at 20\u00a0mg\u00a0l\u22121 was found effective for the inhibition of regeneration in non-transformed shoots (Li et al. 2007), suggesting the variability of antibiotic selection in each transformation system and the necessity of optimization of antibiotic selection for each plant species.\nAlthough transgenic PnMV resistant poinsettias were produced using the Agrobacterium-mediated transformation protocol developed in the current study, the transformation frequency was fairly low (Table\u00a05), suggesting that further improvement is required. This will be carried out in the near future.\nSouthern blot analysis confirmed the integration of transgenes into the poinsettia genome and detected both single- and multiple-copy integration of the transgenes among the transformants (Fig.\u00a04). The majority (six out of eight) of the transformants showed single copy transgene integration with fragment sizes greater than that of the intact T-DNA (ca 7.5\u00a0kb), indicating that the intact T-DNA was integrated into the poinsettia genome. However, the bands with sizes around 4\u20136\u00a0kb, smaller than that of intact T-DNA, were also found in the transgenic lines with multiple insertions (lanes 7 and 8 in Fig.\u00a04).\nAccording to Afolabi et al. (2004) and Zhu et al. (2006), insertion of non-intact T-DNAs were found in more than 70% of the transgenic rice lines causing 14\u201321% of the loci to contain only part of the T-DNA, due to T-DNA truncation. Moreover, T-DNA rearrangement is well documented in many plant species including both dicots and monocots (Deroles and Gardner 1988; Puchta et al. 1992; Azhakanandam et al. 2000; Yin and Wang 2000; Rai et al. 2007). Almost 50% of the 27 transgenic rice lines studied showed rearrangement of T-DNA inserts according to Rai et al. (2007). Consequently, such T-DNA truncations and\/or rearrangements could also possibly occur in our study causing partial T-DNA insertion. Since Agrobacetrium-mediated transformation of poinsettia has never been described before, there is no available information regarding T-DNA transfer and integration into the poinsettia genome.\nThe hp RNA constructs and PnMV resistance in transgenic poinsettia cv. Millenium \nA number of effective approaches have been utilized to control viral diseases (Goldbach et al. 2003). Among these, coat protein-mediated and RNA silencing-based resistance have been shown to be efficient transgenic approaches to engineer virus resistance in plants (Powell-Abel et al. 1986; Barker et al. 1998; Waterhouse et al. 1998; Smith et al. 2000; Dom\u00ednguez et al. 2002; Lu et al. 2003). Furthermore, according to previous studies, hpRNA-induced RNA silencing is more efficient than sense- or antisense-mediated silencing (Chuang and Meyerowitz 2000).\nIn the current study, the establishment of Agrobacterium-mediated transformation enabled us to transform poinsettia plants with intron-containing hpRNA constructs homologous to the PnMV genome. PnMV resistant transgenic poinsettia lines showing no systemic infection were identified after mechanical inoculation as determined using the DAS-ELISA assay (Table\u00a06). The presence of transgene-derived siRNA molecules in non-inoculated transgenic poinsettia plants carrying pCP, pR2 and pR3 constructs, and the absence of these in the non-transformed control plants, indicates that the transgenes were expressed and subsequently cleaved. This data indicates that RNA silencing underlies the PnMV resistance observed in our study (Fig.\u00a05). As mentioned previously, LMW Northern analysis included only one pR3 line (i.e.18-1) due to the limitations in the amount of RNA available for the remaining lines. Therefore, the underlying reason for lack of resistance in some of the pR3-containing transformants (Table\u00a06) needs to be clarified by further studies.\nPoinsettia is a non-food, non-feed and vegetatively propagated ornamental plant. Pollen mediated transgene outflow is restricted, a significant advantage over sexually propagated plants. The combination of genetic engineering and RNA silencing technologies for the generation of virus-resistant transgenic ornamentals is therefore appealing, especially in an industry in which quality is so important. Transgenic vegetatively propagated ornamentals like PnMV resistant transgenic poinsettia are likely to be more acceptable even in areas where genetically modified crops are currently not cultivated.\nIn summary, we have developed an A. tumefaciens-mediated transformation method for poinsettia. Using this approach, transgenic PnMV resistant transgenic lines expressing PnMV-derived hpRNA constructs were produced. Phytoplasma, the poinsettia branch-inducing factor for good branching (\u201cfree-branching\u201d) and for a compact growth habit (Lee et al. 1997), is currently being reintroduced into the transgenic poinsettias. Candidate transgenic lines with PnMV resistance and desired ornamental values will be selected for further study with the future goal of possible commercialization. The methodology developed here could facilitate the future engineering of poinsettia to meet customers\u2019 expectations for new colours and other desirable traits.","keyphrases":["agrobacterium tumefaciens","transformation","euphorbia pulcherrima","poinsettia mosaic virus","somatic embryogenesis"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_Arch_Otorhinolaryngol-3-1-1914238","title":"Quality of life and functional status in patients with cancer of the oral cavity and oropharynx: pretreatment values of a prospective study\n","text":"We assessed the pretreatment health-related quality of life (HRQOL) and functional status of patients with advanced oral and oropharyngeal cancer. Eighty patients were investigated. HRQOL was assessed by EORTC QLQ-C30\/QLQ-H&N35 questionnaires. Functional status assessment comprised speech and oral function tests. The results revealed a wide range of HRQOL and functional deficits before treatment. HRQOL appeared to be related to some extent to tumor site (patients with oral tumors reported more pain compared to patients with oropharyngeal tumors) and tumor classification (patients with T3\u2013T4 tumors reported more trouble opening the mouth and felt more ill compared to patients with T2 tumors). Comorbidity appeared to have a major impact. Patients with comorbidity had significantly worse scores on several scales\/items on both the EORTC questionnaires. Functional deficits were related to tumor site, classification and comorbidity. Patients with oral cavity tumors (versus oropharyngeal tumors), patients with T3\u2013T4 tumors (versus T2 tumors), and patients with comorbidity (versus without comorbidity) scored significantly worse on several speech and oral function tests. Impaired speech and oral function appeared to be clearly related to global quality of life (QLQ-C30) and self-reported speech (QLQ-H&N35). Many patients with advanced oral and oropharyngeal cancer have compromised HRQOL and functional status before the start of treatment. In addition to tumor site and tumor classification, comorbidity appears to have a major impact on HRQOL and functional status. Knowledge of pretreatment HRQOL and functional status levels is useful for better understanding the impact of treatment on these outcomes over time.\nIntroduction\nThe most important outcome for cancer patients is overall survival. However, the disease and its treatment often have a major impact on health-related quality of life (HRQOL) and functional status in patients with head and neck cancer [1]. Therefore, HRQOL and functional status are important aspects to consider in treatment evaluation. To interpret outcome following treatment, it is necessary to assess HRQOL and functional status following diagnosis, but before the start of treatment [2].\nStudies on pretreatment HRQOL [2\u20135] and functional status [6, 7] often include heterogeneous groups of head and neck cancer patients. However, there are substantial differences between patient groups that are related to tumor site and stage [7]. Patients with advanced oral or pharyngeal cancer, for example, often have the poorest HRQOL and functional status [5, 8]. Pretreatment HRQOL among patients with oral or oropharyngeal cancer is often compromised, although it tends to be better compared to HRQOL following treatment [9\u201311]. There is also evidence that functional status, including oral function, speech and swallowing abilities is significantly deteriorated before treatment [12\u201315]. Comorbidity is another important factor that varies substantially among subpopulations of patients, and can have a significant influence on the choice of initial treatment, the care that patients receive, and on treatment outcomes [16]. Therefore, comorbidity should be taken into consideration evaluating the HRQOL of patients, both at time of diagnosis, and over the course of treatment [17\u201319].\nThe primary objective of the present study is to assess pretreatment HRQOL and functional status in relation to tumor site, tumor classification and comorbidity, in a well-defined group of patients with advanced oral and oropharyngeal cancer.\nPatients and methods\nPatients\nBetween January 1998 and December 2001, 92 consecutive patients diagnosed with stage II-IV oral or oropharyngeal squamous cell carcinomas were asked to participate in the study. The planned treatment was composite resection with microvascular soft tissue transfer (i.e., radial forearm free flap) for the reconstruction of surgical defects, and radiotherapy on indication. Exclusion criteria were age greater than 75\u00a0years, serious cognitive impairment and lack of basic fluency in the Dutch language. Twelve patients declined to participate, resulting in a final sample of 80 patients (response rate\u00a0=\u00a087%). All patients were treated at the Department of Otolaryngology\/Head and Neck Surgery of the VU University Medical Center, Amsterdam, The Netherlands.\nData collection and study measures\nAll data were collected prior to the start of treatment, including sociodemographics (age and gender), disease stage, comorbidity, HRQOL, and functional status. Comorbidity was assessed dichotomously (yes or no) by review of medical records and on the basis of self-report, and was noted if one or more relevant medical ailments accompanied the primary medical illness. The comorbid conditions were cardiovascular, respiratory, gastro-intestinal, renal, endocrine, neurological, and immunological disorders, previous malignancy and considerable weight loss or alcohol abuse. For example, cardiovascular problems such as a myocardial infarct or hypertension, respiratory problems such as restrictive lung disease or COPD, or endocrine disorders such as diabetes mellitus with insulin usage were defined as relevant comorbid conditions.\nHRQOL was assessed by means of the European Organization for Research and Treatment of Cancer (EORTC) QLQ-C30 questionnaire (version 2.0) [20] and the EORTC head and neck cancer module QLQ-H&N35 [21]. The QLQ-C30 is composed of multi-item scales and single items assessing five areas of functioning (physical, role, emotional, cognitive, and social), fatigue, pain, emesis, dyspnea, insomnia, appetite loss, constipation, and diarrhea. Overall quality of life and the perceived financial impact of the disease and treatment are also assessed. The head and neck cancer-specific QLQ-H&N35 module comprises seven symptom scales: pain, swallowing, senses, speech, social eating, social contact, and sexuality. There are 11 additional, single items covering problems with teeth, opening the mouth wide, dry mouth, sticky saliva, cough, feeling ill, weight loss, weight gain, use of nutritional supplements, feeding tubes, and painkillers. The scores of both the QLQ-C30 and of the QLQ-H&N35 are linearly transformed to a scale of 0\u2013100, with a higher score indicating a higher (i.e., more positive) level of functioning or global HRQOL, or a higher (i.e., more negative) level of symptoms or problems.\nThe QLQ-C30 data of the patients were compared with published normative data from the general population of Norway [22]. Comparisons were made with the normative subsample that corresponded closely to the age and gender distribution of the patient sample (i.e., age range of 50\u201359\u00a0years and 48% female). The QLQ-H&N35 results were compared with reference values derived from a randomly selected sample of the Swedish general population [8].\nSpeech and oral functional status\nSpeech analyses were performed according to a standardized speech assessment protocol. Speech recordings of a read aloud text were performed in a sound-treated room and digitized using Cool Edit PRO 1.2 (Adobe Systems Incorporated, San Jose, CA, USA) with 22\u00a0kHz sample frequency and 16\u00a0bit resolution. Recording level was adjusted for each speaker to optimize signal-to-noise ratio. All recordings were made with a mouth-to-microphone distance of 30\u00a0cm. A computer program was developed to perform blinded randomized speech evaluation and to score overall intelligibility, and quality of articulation and nasal resonance. Overall intelligibility was assessed on a 10-point scale ranging from poor to excellent by two trained speech therapists. Scores below six were defined as insufficient intelligibility (according to the Dutch educational system). To obtain more insight into the cause of decreased intelligibility, evaluation of the quality of articulation and nasal resonance was performed by the same two speech therapists on a 4-point scale ranging from deviant (score 1\u20133) to normal (score 4). Speech rate was measured by calculating words per minute on a read aloud standardized text.\nOral function was evaluated by a trained investigator (blinded for the clinical data regarding tumor site and stage) according to a protocol described by Teichgraeber et al. [23]. All assessments were based on 5-point scales ranging from poor to excellent (transformed scores ranging from 0\u2013100). The oral function evaluation included three tests; (1) tongue mobility (mean score of tongue straight out, elevation of tongue tip, elevation of the base of the tongue, tongue deviation left and right, symmetry left and right, and tongue withdrawal), (2) lip mobility (mean score of general movement, spreading and rounding, symmetry left\/right, lip closure without speech, and lip closure during speaking), and (3) diadochokinesis (mean score of repetitive motion ability concerning tongue movement left\/right, up\/down, and in\/out, and repeating the syllables \/ta\/ \/cha\/, and \/ka\/). Tongue and lip strength were quantified by a calibrated digital voltameter. To measure tongue strength the patient was asked to push the tongue against a metallic disc with the lips positioned around a cylinder, and to resist the force. To measure lip strength, the patient was asked to keep his lips around a button with a string attached to it on which the examiner pulled. Scores ranged from 0 (no strength) to 0.5\u00a0mV (normal strength).\nStatistical analyses\nChi-square tests were used to assess associations between the independent variables tumor site, classification and comorbid condition. Student\u2019s t-tests were performed to test for differences between the patient sample and the normative samples. These tests were based on the mean scores of the study and normative samples and the standard deviations of the study sample only, because no standard deviations of normative samples were available. Student\u2019s t-tests (HRQOL) and Mann\u2013Whitney tests (functional status) were performed to determine the impact of tumor site (oral versus oropharyngeal), tumor classification (T2 versus T3\u2013T4) and comorbidity (yes or no). Spearman correlation coefficients (r) were calculated to investigate the association between self-reported HRQOL (i.e., the QLQ-C30 global quality of life scale and the QLQ-H&N35 speech scale) and speech and oral functional status. Statistical significance was defined as a P-value less than or equal to 0.05.\nResults\nSample description\nPatient characteristics are shown in Table\u00a01. The patients\u2019 age ranged from 23 to 74\u00a0years (mean 58\u00a0years). Forty-one percent of the sample was female. One patient was operated on for a recurrent tumor after prior transoral excision (1\u00a0year earlier) in which the rTNM stage was used, and one patient had undergone previous radiotherapy (3\u00a0years earlier) for a neck node of an unknown primary. Four patients had a synchronous second primary tumor, and in these cases the stage of the largest tumor was used. An equal percentage (50%) of patients had a tumor originating on the left or right side, and in the majority of patients (73%) the tumor did not extend over the median line. All 80 patients completed the EORTC questionnaires, speech rate tests (word count) and oral function tests (mobility and strength). In one patient speech recording could not be done due to logistical problems, and in three patients speech recordings were inadequate for interpretation due to technical problems. Speech quality analyses could thus be performed on 76 patients. Presence of comorbid conditions and tumor classification were significantly associated. Significantly more patients with comorbidity were diagnosed with a larger tumor (\u03c72\u00a0=\u00a010.37, P\u00a0<\u00a00.01). No other significant associations were observed between tumor site, tumor classification, age, gender and comorbidity.\nTable\u00a01Characteristics of 80 patients included in this studyAge\u00a0(years)\u00a0Range23\u201374\u00a0\u00a0Mean58n (%)Gender\u00a0Male47 (59)\u00a0Female33 (41)General condition\u00a0Comorbidity48 (60)\u00a0No comorbidity32 (40)Tumour site\u00a0Oral cavity38 (47)\u00a0Oropharynx42 (53)T\u00a02 35 (44)\u00a03 42 (52)\u00a04 3 (4)N\u00a0024 (30)\u00a0116 (19)\u00a02a2 (3)\u00a02b30 (38)\u00a02c6 (7)\u00a03 2 (3)\nHealth-related quality of life\nCompared to the general population, the patients with oral or oropharyngeal cancer scored significantly worse on 5 (out of 15) scales or single items regarding the QLQ-C30: role functioning (P\u00a0=\u00a00.000), emotional functioning (P\u00a0=\u00a00.000), pain (P\u00a0=\u00a00.026), insomnia (P\u00a0=\u00a00.016) and appetite loss (P\u00a0=\u00a00.003). Conversely, patients scored significantly better regarding social functioning (P\u00a0=\u00a00.009), fatigue (P\u00a0=\u00a00.030), emesis (P\u00a0=\u00a00.001), and diarrhea (P\u00a0=\u00a00.001). Regarding the QLQ-H&N35, the patients scored significantly worse on 7 (out of 10) scales or single items compared to the general population: pain (P\u00a0=\u00a00.000), swallowing (P\u00a0=\u00a00.000), senses (P\u00a0=\u00a00.050), social eating (P\u00a0=\u00a00.000), teeth (P\u00a0=\u00a00.009), opening the mouth wide (P\u00a0=\u00a00.000) and sticky saliva (P\u00a0=\u00a00.005). Conversely, the patients scored significantly better on two items: coughing (P\u00a0=\u00a00.002) and feeling ill (P\u00a0=\u00a00.025).\nRegarding tumor site, the QLQ-C30 results were comparable between oral cavity and oropharynx cancer patients. Only the pain score was significantly worse (P\u00a0=\u00a00.027) for patients with oral cavity tumors as compared to patients with oropharynx tumors. The QLQ-H&N35 also revealed that patients with oral cavity tumors had significantly more pain (P\u00a0=\u00a00.002); furthermore, problems with their teeth appeared to be different (P\u00a0=\u00a00.001).\nNo statistically significant differences were observed for any of the QLQ-C30 scores as a function of tumor classification (T2 versus T3\u2013T4). Regarding the QLQ-H&N35, patients with T3\u2013T4 tumors scored significantly worse on opening the mouth (P\u00a0=\u00a00.009) and reported feeling more ill (P\u00a0=\u00a00.035) compared to patients with T2 tumors.\nRegarding comorbidity (Table\u00a02), patients with one or more comorbid conditions scored significantly worse on QLQ-C30 physical functioning (P\u00a0=\u00a00.002), global quality of life (P\u00a0=\u00a00.016), fatigue (P\u00a0=\u00a00.014), pain (P\u00a0=\u00a00.024), constipation (P\u00a0=\u00a00.042) and diarrhea (P\u00a0=\u00a00.017) compared to patients without comorbidity. Additionally, based on the QLQ-H&N35 data, patients with comorbidity reported significantly more pain (P\u00a0=\u00a00.007), trouble with social eating (P\u00a0=\u00a00.036), teeth problems (P\u00a0=\u00a00.034), problems with opening the mouth (P\u00a0=\u00a00.013) and feeling ill (P\u00a0=\u00a00.003). The use of pain medication was significantly higher among patients with than those without comorbidity (75 versus 50%; P\u00a0=\u00a00.025).\nTable\u00a02EORTC QLQ-C30 and QLQ-H&N35 scores for comorbidityComorbidityNoYesMean (SD) n\u00a0=\u00a032Mean (SD) n\u00a0=\u00a048PEORTC QLQ-C30\u00a0Physical functioning95.0 (12.4)81.7 (21.4)0.002\u00a0Role functioning85.4 (18.3)79.2 (24.9)0.228\u00a0Cognitive functioning89.6 (18.3)87.2 (19.2)0.574\u00a0Emotional functioning69.5 (23.4)72.2 (24.6)0.626\u00a0Social functioning93.2 (13.3)88.5 (21.5)0.275\u00a0Global quality of life82.6 (19.6)71.5 (19.7)0.016\u00a0Fatigue13.9 (20.0)26.9 (24.1)0.014\u00a0Emesis1.6 (4.9)1.4 (4.7)0.874\u00a0Pain22.4 (25.3)37.8 (31.8)0.024\u00a0Dyspnea6.3 (19.7)10.4 (18.4)0.338\u00a0Insomnia36.5 (36.3)31.3 (39.1)0.550\u00a0Appetite loss7.3 (20.3)18.7 (31.4)0.072\u00a0Constipation5.2 (20.9)18.7 (32.9)0.042\u00a0Diarrhea1.0 (5.9)8.3 (16.1)0.017\u00a0Financial impact2.1 (8.2)7.6 (20.9)0.157EORTC QLQ-H&N35\u00a0Pain27.1 (17.6)41.5 (25.5)0.007\u00a0Swallowing14.6 (22.6)24.1 (22.6)0.068\u00a0Senses 6.3 (14.5)8.7 (21.5)0.577\u00a0Speech 9.4 (14.1)13.0 (17.5)0.336\u00a0Social eating14.6 (25.3)27.4 (27.1)0.036\u00a0Social contact3.1 (7.0)3.7 (6.1)0.674\u00a0Sexuality16.1 (24.9)16.3 (27.6)0.981\u00a0Teeth13.5 (26.6)30.6 (38.8)0.034\u00a0Opening mouth7.3 (20.3)25.7 (37.2)0.013\u00a0Dry mouth15.6 (26.8)22.2 (28.6)0.303\u00a0Sticky saliva12.5 (29.0)21.5 (31.1)0.196\u00a0Coughing6.3 (13.2)14.6 (21.6)0.055\u00a0Feeling ill0 (0)9.7 (18.1)0.003% Yes% YesPain medication50.075.00.022\nSpeech and oral functional status\nAbnormal scores were observed in 17% of the patients regarding overall intelligibility, in 25% regarding nasality and in 37% regarding articulation. Functional results in relation to tumor site, classification and comorbidity are shown in Table\u00a03. Regarding tumor site, patients with oral cavity tumors scored significantly worse on intelligibility (P\u00a0=\u00a00.015), articulation (P\u00a0=\u00a00.039), nasality (P\u00a0=\u00a00.040), tongue and lip mobility (P\u00a0=\u00a00.000, 0.009), diadochokinesis (P\u00a0=\u00a00.004), and tongue strength (P\u00a0=\u00a00.001) compared to patients with oropharyngeal tumors. Regarding tumor stage, patients with T3\u2013T4 tumors scored significantly worse on intelligibility (P\u00a0=\u00a00.011), articulation (P\u00a0=\u00a00.003), tongue mobility (P\u00a0=\u00a00.001), diadochokinesis (P\u00a0=\u00a00.012), and tongue and lip strength (P\u00a0=\u00a00.021, 0.018) than patients with T2 tumors. Patients with comorbidity scored significantly worse on articulation (P\u00a0=\u00a00.043), diadochokinesis (P\u00a0=\u00a00.026), and tongue strength (P\u00a0=\u00a00.045) as compared to patients without comorbidity.\nTable\u00a03Functional status tests for tumour site, tumour stage and comorbidityTumour siteTumour stageComorbidityOral cavityOropharynxT2T3\u2013T4NoYesMean (SD) n\u00a0=\u00a037Mean (SD) n\u00a0=\u00a039PMean (SD) n\u00a0=\u00a032Mean (SD) n\u00a0=\u00a044PMean (SD) n\u00a0=\u00a029Mean (SD) n\u00a0=\u00a047PSpeech\u00a0Intelligibility\u00a0\u00a0(0\u201310)6.0 (1.3)6.7 (1.0)0.0156.8 (1.0)6.0 (1.2)0.0116.6 (1.0)6.2 (1.3)0.132\u00a0Articulation\u00a0\u00a0(0\u20134)3.4 (0.8)3.7 (0.6)0.0393.8 (0.4)3.3 (0.8)0.0033.8 (0.4)3.4 (0.8)0.043\u00a0Nasality\u00a0\u00a0(0\u20134)3.6 (0.6)3.8 (0.4)0.0403.8 (0.4)3.7 (0.6)0.9113.7 (0.4)3.7 (0.5)0.771n\u00a0=\u00a038n\u00a0=\u00a042n\u00a0=\u00a035n\u00a0=\u00a045n\u00a0=\u00a032n\u00a0=\u00a048\u00a0Rate\u00a0\u00a0Words per minute183 (31.6)182 (38.5)0.988189 (33.8)178 (35.8)0.201189 (31.4)179 (38.1)0.233n\u00a0=\u00a038n\u00a0=\u00a042n\u00a0=\u00a035n\u00a0=\u00a045n\u00a0=\u00a032n\u00a0=\u00a048Oral functions\u00a0Mobility\u00a0\u00a0Tongue (0\u2013100)82.3 (20.0)96.4 (5.4)0.00096.0 (5.8)84.8 (19.3)0.00193.9 (9.9)89.9 (14.4)0.209\u00a0\u00a0Lip (0\u2013100)87.4 (5.7)99.7 (0.9)0.00999.4 (2.0)98.0 (5.1)0.14099.3 (2.2)98.5 (3.9)0.239\u00a0\u00a0Diadoch (0\u2013100)87.4 (20.7)97.2 (6.1)0.00497.5 (5.0)88.7 (19.6)0.01297.4 (5.1)91.5 (15.4)0.026\u00a0Strength\u00a0\u00a0Tongue (0\u20130.5)0.2 (0.2)0.4 (0.2)0.0010.4 (0.2)0.3 (0.2)0.0210.4 (0.2)0.2 (0.2)0.045\u00a0\u00a0Lip (0\u20130.5)0.3 (0.1)0.4 (0.2)0.3280.4 (0.2)0.3 (0.1)0.0180.4 (0.2)0.3 (0.1)0.370\nCorrelations between self-report and observer rated data\nSignificant correlations (P\u00a0<\u00a00.01) were observed between self-reported global quality of life scale (QLQ-C30) and observer ratings of intelligibility (r\u00a0=\u00a00.41) and articulation (r\u00a0=\u00a00.36) (Table\u00a04). Additionally, statistically significant (P\u00a0<\u00a00.05) but relatively low correlations were found between self-reported speech (QLQ-H&N35) and observer ratings of intelligibility (r\u00a0=\u00a0\u22120.28), articulation (r\u00a0=\u00a0\u22120.24), nasality (r\u00a0=\u00a0\u22120.27) and diadochokinesis (r\u00a0=\u00a0\u22120.28).\nTable\u00a04Correlations between functional status tests and QLQ-C30 global quality of life scale\/QLQ-H&N35 speech problemsScalesQLQ-C30 global quality of lifeQLQ-H&N35 speech problemsCorrelation coefficient (r) n\u00a0=\u00a076PCorrelation coefficient (r) n\u00a0=\u00a076PSpeech\u00a0Intelligibility0.4110.000\u22120.2850.013\u00a0Articulation0.3550.002\u22120.2420.035\u00a0Nasality0.1520.190\u22120.2700.019n\u00a0=\u00a080n\u00a0=\u00a080\u00a0Rate0.1290.267\u22120.1420.221n\u00a0=\u00a080n\u00a0=\u00a080Oral functions\u00a0Mobility\u00a0\u00a0Tongue0.0780.490\u22120.1880.095\u00a0\u00a0Lip0.1570.164\u22120.1760.119\u00a0\u00a0Diadochokinesis0.1090.337\u22120.2800.012\u00a0Strength\u00a0\u00a0Tongue0.1880.095\u22120.1750.121\u00a0\u00a0Lip0.0560.624\u22120.0490.665\nDiscussion\nPretreatment HRQOL and functional status were investigated in a well-defined sample of patients with advanced oral and oropharyngeal cancer. Deteriorated HRQOL and functional status before treatment have been reported in earlier studies, albeit for less well defined patient groups [9\u201315]. The results of the present study indicate a wide range of HRQOL and functional deficits in patients with advanced oral and oropharyngeal cancer before treatment. Impaired speech and oral function, as assessed objectively, was associated significantly with self-reported global HRQOL and speech problems, which is in accordance with the conclusions of Rogers et al. and Karnell et al. [14, 24].\nCompared to the general population, patients scored significantly worse on 5 of 15 scales or items of the EORTC QLQ-C30 but, conversely, they scored significantly better on four others. Regarding the QLQ-H&N35, patients scored significantly worse on most scales or items but significantly better on coughing and feeling ill. The significantly better scores for patients compared to the reference groups on some scales or items may be explained by the fact that people from the general population may have (other) chronic conditions as well. Alonso et al. [25] reported that, 55% of the general population has one chronic health condition, and 30% has more than one chronic condition. They also found that comorbidity can have a substantial impact on HRQOL, and stated that the presence of comorbidity limits the ability to attribute HRQOL deficits to one specific disease (e.g., to head and neck cancer).\nIn head and neck cancer patients, comorbidity has proven to be an important factor associated with complications and mortality rates [26\u201329]. Studies on the impact of comorbidity on functional status in head and neck cancer patients are lacking. Studies that have examined the impact of comorbid status on HRQOL are scarce, have yielded conflicting results, and have examined the post-treatment period only. Pourel et al. [17] found no significant association between comorbidity and HRQOL in 113 long-term survivors 2\u00a0years after treatment for oropharyngeal carcinomas. Similarly, Taylor et\u00a0al. [30] found no impact of comorbidity on work-related disability among 384 patients after treatment for head and neck cancer. However, Terrel et\u00a0al. [19] in a study on HRQOL of 570 head and neck cancer patients after treatment, reported a clear effect of comorbidity in patients with two or more comorbid conditions. In our study on patients with oral or oropharyngeal carcinomas before treatment, comorbidity was present in 60% of the patients, and proved to have a major impact on HRQOL and functional status. Patients with comorbidity had significantly worse scores on several general and head and neck specific quality of life aspects and on speech and oral function tests compared to patients without comorbidity. Unfortunately, the cause of comorbidity was not systematically noted in the present study; future studies may provide more insight into the relation between the cause of comorbidity and HRQOL.\nThe impact of tumor site and classification on HRQOL appeared to be limited, with patients with oral cavity tumors reporting more pain, and patients with T3\u2013T4 tumors reporting more trouble opening the mouth and feeling more ill. With regard to tumor site, comparisons with earlier studies are difficult because most studies included patients with oral cavity tumors only or reported on heterogeneous samples not stratified by (sub)sites [4, 9\u201311]. Regarding tumor classification, other studies have reported worse HRQOL for patients with higher tumor classifications and stages before treatment [2, 14].\nTumor site and classification were found to have a clear impact on functional status. Patients with oral cavity tumors (versus oropharyngeal tumors) and patients with T3\u2013T4 tumors (versus T2 tumors) had worse speech and oral function scores, which is in accordance with the results of earlier studies [13, 14].\nIn conclusion, we observed compromised HRQOL and functional deficits among patients with advanced oral and oropharyngeal cancer before the start of treatment. In addition to the impact of tumor site and classification, comorbidity proved to have a major impact on HRQOL and functional status. Prospective studies are needed to obtain insight into the relation between pretreatment HRQOL and functional status and outcome after treatment, and the relationship between changes in HRQOL and functioning over time and tumor site, tumor classification, and comorbid conditions.","keyphrases":["quality of life","functional status","speech","head and neck cancer","microvascular reconstruction"],"prmu":["P","P","P","P","R"]}
{"id":"Crit_Care-6-5-130146","title":"Antithrombin III in patients admitted to intensive care units: a multicenter observational study\n","text":"Introduction The administration of antithrombin III (ATIII) is useful in patients with congenital deficiency, but evidence for the other therapeutic indications of this drug is still uncertain. In Italy, the use of ATIII is very common in intensive care units (ICUs). For this reason we undertook an observational study to determine the pattern of use of ATIII in ICUs and to assess the outcome of patients given this treatment.\nIntroduction\nAntithrombin III (ATIII) is a recognized treatment for patients with congenital ATIII deficiency [1,2,3,4,5] (see also the approval of this indication by the Food and Drug Administration); in contrast, the evidence supporting its use for other clinical indications is uncertain [6,7,8,9,10].\nIn Italian hospitals this drug is widely used in patients admitted to intensive care units (ICUs), who are generally given ATIII for the treatment of sepsis or disseminated intravascular coagulation (DIC). The approval of ATIII by the Italian Ministry of Health was granted nearly 10 years ago (before the profound reform of the Drug Regulatory Agency made by the Italian Ministry of Health in 1993) and has remained unchanged since then. This approval of ATIII was rather generic and included 'congenital deficiency of ATIII and all clinical conditions that can cause an acquired deficiency of ATIII'.\nThree small randomized studies [7,8,9] and one large international trial [10] assessed the effectiveness of ATIII in sepsis, but none of these trials found a significant benefit in terms of reduced morbidity or mortality. As regards congenital deficiency, the effectiveness of ATIII is fairly well documented [1,2,3,4,5], but these patients are rare. The other clinical indications (such as acute thrombosis or thromboembolism, prevention of DIC in hepatic coma, and treatment of bleeding episodes in cirrhosis) are supported by a small series of very preliminary studies (see, for example, the Drugdex databank, CD-ROM Drugdex, volume 110; Micromedex, Englewood, Colorado, USA).\nTo achieve a better definition of the current use of ATIII in Italian hospitals and to generate naturalistic data (based on routine practice) about the outcome of this treatment, we undertook a multicenter observational study.\nMethods\nDesign of the study and aims\nThe study was based on a multicenter observational design. From 20 May to 20 July 2001 all consecutive patients admitted to ICUs in 20 Italian hospitals and treated with ATIII were enrolled in the study. The study had the following aims: (1) surveying the use of ATIII in patients admitted to ICUs; (2) determining the outcome of patients treated with ATIII; and (3) comparing the results obtained from our observational study with those previously found in the randomized controlled trials (RCTs).\nA meta-analysis was also conducted to summarize the information deriving from four RCTs [7,8,9,10] that studied the effectiveness of ATIII in sepsis.\nData collection\nThe following information was recorded from each patient enrolled in the study: (1) demographic characteristics (age, sex, weight); (2) congenital deficiency (y\/n); (3) baseline ATIII level; (4) ward of first admission in the hospital; (5) clinical indication for using ATIII (sepsis or DIC or any other clinical condition); (6) daily dose and duration of treatment with ATIII; (7) outcome of hospitalization (alive or dead); and (8) concurrent administration of antibiotics and\/or heparin.\nAnalysis\nThe information collected from each patient was analyzed by standard descriptive statistics. In the subgroup of patients with sepsis, the in-hospital mortality rate observed in our study was compared with that previously reported by the four RCTs. All rates were presented together with their 95% confidence interval (CI), which was calculated by using Equations 1.26 and 1.27 of Fleiss [11].\nResults\nThe overall number of patients who were admitted to ICUs during the study period was 1648. Of these patients, 216 (13%) were enrolled in our study. The characteristics of these 216 patients are presented in Table 1.\nThe clinical indication for using ATIII was sepsis (n = 56), DIC (n = 50), or other (n = 101). Table 1 also reports separate information for the subgroup of 56 patients treated for sepsis.\nThe duration of ATIII therapy did not differ at levels of statistical significance between patients treated for different clinical indications (P = 0.57 according to an analysis of variance). The daily dose of ATIII showed a difference between sepsis and other indications (Table 1).\nTable 2 reports the outcome of hospitalization according to clinical indication. With regard to the use of ATIII in patients with sepsis, Figure 1 shows the percentage mortality rate (with 95% CI) observed in our study, together with the rates found in four previous studies [7,8,9,10].\nSubgroup analyses within the patient cohort of our study did not identify any relationship between mortality and patient characteristics. The administration of heparin, which Warren et al. [10] found to have some implications for outcome, did not influence mortality in our patient series: mortality was 19.6% in the 107 patients who received heparin, compared with 30.5% in the 95 patients who did not receive this drug (P = 0.10) by Fisher's exact test; mortality was 28.6% in the 28 patients with sepsis who received heparin, compared with 42.3% in the 26 patients with sepsis who did not receive this drug (P = 0.39).\nDiscussion\nThe main scientific value of our observational and prospective study lies in its naturalistic design; the population of patients that we studied was in fact drawn from the everyday practice of more than 20 hospitals and was intentionally free from specific exclusion criteria.\nIn interpreting our outcome data, one disadvantage is that the group treated with ATIII was not compared with any reference group observed prospectively within our research; neither did we include any retrospective control group not treated with the drug. However, historical retrospective controls would have raised profound problems of matching the retrospective data with the prospective ones. A prospective enrollment of controls not treated with ATIII was not feasible because the therapeutic policy of the ICUs involved in our study was to administer ATIII to virtually all patients with a diagnosis of sepsis or DIC.\nRegardless of our statistical indexes, a 'first-look' comparison between the data on sepsis produced by the previous RCTs (including four treatment groups and four control groups) and those observed in our naturalistic study indicates a complete overlap of the various survival rates and of their respective 95% CIs. This qualitative impression (Figure 1) is in agreement with the meta-analysis shown in Figure 2 (see Appendix2 for details of its methodology).\nThis meta-analysis gave the following results: summary odds ratio 0.98; 95% CI 0.83\u20131.15, P = 0.80; \u03c72 for heterogeneity 1.86; 3 degrees of freedom; P = 0.60. In this meta-analysis, the large-scale trial by Warren et al. [10] outweighed the other three small RCTs in that Warren's trial included 93% of the overall cohort of the four RCTs. In the light of the above data, there seems to be no clinical benefit in administering ATIII to critical patients with sepsis; in this context, one crucial point is that the most recent large-scale trial gave very clear results and was negative. The other clinical indications reported in our patients' series were more difficult to interpret because of the nearly complete lack of previous controlled studies exploring these therapeutic issues.\nThere has been a lively debate in the literature on the relative merits of observational studies and RCTs in providing useful evidence of clinical effectiveness [12,13,14]. Although the great majority of researchers stick to the concept that RCTs are the gold standard, common sense suggests that having information both from RCTs and from observational studies is better than having information from RCTs only. In this framework, our study advances knowledge about the use of ATIII in critical patients.\nIn conclusion, our findings based on an observational prospective study and on an updated meta-analysis of the previous RCTs do not support the use of this drug in ICU patients with sepsis.\nKey messages\nAntithrombin III (ATIII) is a recognized treatment for patients with congenital ATIII deficiency; in contrast, the evidence supporting its use for other clinical indications is uncertain.\nIn Italian hospitals this drug is widely used in patients admitted to intensive care units (ICUs), who are generally given ATIII for the treatment of sepsis or disseminated intravascular coagulation.\nThree small randomized studies and one large international trial have assessed the effectiveness of ATIII in sepsis, but none of these trials has found a significant benefit in terms of reduced morbidity or mortality.\nOur findings, based on an observational prospective study and on an updated meta-analysis of the previous randomized controlled trials, do not support the use of this drug in ICU patients without congenital deficiency.\nCompeting interests\nIn 2001 our research group received a grant from Eli-Lilly (Italy) to conduct an original study on factors influencing length of stay in critical patients with sepsis. In Italy, anti-thrombin III is marketed by Aventis-Behring and by Baxter.\nAppendix 1: Gruppo di Studio sull'antitrombina III (The Antithrombin Study Group)\nThe Antithrombin Study Group includes the study coordinators (A Messori, F Vacca, M Vaiani, S Trippoli, Laboratorio di Farmacoeconomia, c\/o Azienda Ospedaliera Careggi, Firenze) and a total of 51 participants. The names and addresses of the participants involved in the project were the following (all located in Italy): R Banfi, M Cecchi, E Cini, D Dupuis, T Falai, R Fornaini, A Ipponi, ML Migliaccio, F Pelagotti, L Rabatti, I Ruffino, R Silvano, E Tendi (Firenze, four hospitals); P Becagli, M Monciatti (Empoli); B Bozzone, R Casullo, F Cattel, S Pardossi, R Passera, S Stecca, U Tagliaferro (Torino, two hospitals); P Di Bartolomeo, T Faggiano, M Lattarulo (Bari); N Caboni, A Cannas (Cagliari); A Plescia, M Sorci (Rimini); L Bonistalli, M Puliti (Prato); B Ciammitti, M Costantini, F Mammini (Terni); L De Cicco, G Mazzaferro (Napoli); P Marrone, R Tetamo (Palermo); P Beneduce, MG Celeste, P Fiorani, S Galeassi, G Guaglianone, A Pecere, L Ragni (Roma, two hospitals); SM Germinario (Andria); O Basadonna, L Todesco (Camposampiero, Padova); R Calle-gari, M Pegoraro (Asolo); E Lamura (Ancona).\nAppendix 2: Methodology of the meta-analysis\nA MedLine search (PubMed, ) was performed to cover the period from January 1980 to November 2001. The search was limited to the studies published in English and was based on four index terms combined with the following Boolean syntax: \"antithrombin III\" AND (sepsis OR septic shock OR \"disseminated intravascular coagulation\"). This search was supplemented by examining the Drugdex databank (CD-ROM Drugdex, volume 110; Micromedex, Englewood, Colorado, USA).\nEligible studies were included if they met the following criteria: patients were admitted to an ICU; randomized design; diagnosis of sepsis, septic shock or DIC; assessment of survival. The odds ratio was used as the main index to assess the treatment effect within each trial and to generate the overall results of the meta-analysis. The calculation of the summary odds ratios was based on a random-effect model [15,16]. Heterogeneity was assessed as described previously [17].\nAbbreviations\nATIII = antithrombin III; CI = confidence interval; DIC = disseminated intravascular coagulation; ICU, intensive care unit; RCT = randomized controlled trial.","keyphrases":["antithrombin iii","sepsis","disseminated intravascular coagulation","septic shock"],"prmu":["P","P","P","P"]}
{"id":"Int_J_Colorectal_Dis-3-1-2134973","title":"Colonic irrigation for defecation disorders after dynamic graciloplasty\n","text":"Background and aims Dynamic graciloplasty (DGP) improves anal continence and quality of life for most patients. However, in some patients, DGP fails and fecal incontinence is unsolved or only partially improved. Constipation is also a significant problem after DGP, occurring in 13\u201390%. Colonic irrigation can be considered as an additional or salvage treatment for defecation disorders after unsuccessful or partially successful DGP. In this study, the effectiveness of colonic irrigation for the treatment of persistent fecal incontinence and\/or constipation after DGP is investigated.\nIntroduction\nDynamic graciloplasty (DGP) is a proven effective treatment for fecal incontinence. Success rates vary from 42\u201392% [1\u20135]. Comparison of results is difficult because the outcome of this method seems to be influenced by the etiology of fecal incontinence, pre-existing stoma, length of follow-up, method of stimulation, and the surgeon\u2019s experience [1, 6]. DGP significantly improves quality of life and anal continence for most patients [2]. Quality of life and patient satisfaction scores correlate significantly with continence scores [7, 8]. However, in some patients, fecal incontinence is unsolved or only partially improved after DGP.\nMorbidity rates are high after DGP: complications associated with the technique can often be prevented or treated, but other complications like reduced sensitivity of the rectum by destruction of sensory nerves can be very hard to treat and often result in DGP failure [4, 8, 9]. Morbidity rates are lower and success rates higher in the hands of surgeons experienced in the technique [3, 10].\nConstipation is a significant problem after graciloplasty, occurring in 13\u201390% of the patients after DGP [6\u20138, 11, 12]. Constipation due to technical failure, like a gracilis wrap that is too tight, can be treated by revisional surgery. Other causes of constipation can first be treated with dietary measures and medication. In case of pelvic floor dysfunction, biofeedback training can be started [9]. When this first line treatment is unsuccessful, retrograde colonic irrigation can be considered. Colonic irrigation can also be used as an additional therapy or salvage therapy for persistent fecal incontinence after failed or partially failed surgery [13]. Not much is known about the success rate of retrograde colonic irrigation, as there is only a limited number of published studies [13, 14].\nAntegrade irrigation through a colostomy or appendico-cecostomy is another technique for colonic irrigation, with a reasonable success rate of 64\u201385% [15\u201317].\nIn this study, the effectiveness of colonic irrigation for the treatment of persistent fecal incontinence and\/or constipation after DGP was investigated.\nMaterials and methods\nPatients with DGP and postoperative defecation disorders were selected for colonic irrigation between January 1999 and June 2003. The patients were offered colonic irrigation as additional or salvage therapy, as a colostomy was the final option for these patients. The inclusion criteria were invalidating fecal incontinence and\/or constipation after DGP and a signed informed consent; exclusion criteria were patients not willing to perform the irrigation because of either embarrassment or wish for colostomy or patients physically and\/or mentally not capable of performing irrigation.\nAll patients visited the outpatient clinic of the University Hospital Maastricht. Relevant physical and medical history were collected. The patients could be divided in six groups according to the etiology of fecal incontinence before DGP: congenital, trauma (rupture\/anal surgery), pudendopathy (PNTML\u2009>\u20092.6\u00a0ms), spinal cord lesion, cancer (abdominal perineal resection in rectumcarcinoma), and prolapse. The patients were asked to fill out a questionnaire. This questionnaire contained questions concerning the method of irrigation, the time needed for irrigation, the amount of water used, the frequency of irrigation, added substances to the irrigation water, and side effects of colonic irrigation. The endpoint was defined that irrigation had to be successful and satisfying for the patients. Successful irrigation was defined as reaching continence for feces (pseudo-continence) and\/or complete resolution of straining, feeling of incomplete evacuation, bloating abdomen, and abdominal pain during irrigation.\nThe patient was considered satisfied when he or she indicated that the colonic irrigation rendered a major improvement of the quality of life. The improvement of quality of life was measured by a visual analog scale\/specific questions within the questionnaire. The irrigation was performed in most patients as retrograde irrigation; only four patients performed antegrade irrigation through an appendico-cecostomy or a colostomy. Patients with fecal incontinence can gain continence as a result of colonic irrigation. This is called pseudo-continence because these patients are only continent, as there is no fecal filling of the rectum and distal colon. The definition of resolved constipation was no straining and no feeling of incomplete evacuation after defecation. Defecation occurred during irrigation in most of these patients.\nThe Biotrol\u00ae Irrimatic pump (Braun\u00ae; Fig.\u00a01) or the irrigation bag (Braun\u00ae; Fig.\u00a02) were used for colonic irrigation. The Irrimatic pump is an irrigation pump using a flexible tube with a cone-shaped end. The tube is introduced either in the anal canal in case of retrograde irrigation or placed in an appendico-cecostomy or colostomy in case of antegrade irrigation. The pump can hold a maximum of 2\u00a0l. The patients were instructed to start the irrigation daily with 500\u00a0ml of water. Thereafter, the frequency and the amount of water were adjusted until a satisfactory result was achieved. Water used for irrigation was at body temperature; too cold water can cause abdominal cramps or collapse and too hot water can cause burns. Soap or laxatives could be added if necessary. The pump works on a storage battery, and the speed of water ejection can be regulated. The working mechanism of the irrigation bag is similar, except for the water ejection, which is induced by gravity.\nFig.\u00a01Biotrol\u00ae Irrimatic pump (Braun\u00ae)Fig.\u00a02Irrigation bag (Braun\u00ae)\nData analysis was performed by using SPSS 14.0 (SPSS release 14.0, SPSS, Chicago, USA), with the Mann\u2013Whitney U test. P\u2009\u2264\u20090.05 was considered statistically significant. Data are given as the mean values with the standard deviation.\nResults\nThe questionnaire was sent to 74 patients. Fourteen patients did not wish to participate in the study for various reasons, and 14 patients did not return the questionnaire. Forty-six patients (62%) could be included for analysis of which 37 were female (80%), and these patients had an overall mean age of 59.3\u2009\u00b1\u200912.4\u00a0years. Complications of the DGP occurred in 23 (50%) patients of which 10 patients had two or more complications. Twenty-two patients suffered from constipation, 35 patients were still incontinent for feces, and seven patients had pain in the IPG pocket, leg, or anus.\nThe medical history besides the DGP is mentioned in Table\u00a01. On average, the patients started the irrigation 21.39\u2009\u00b1\u200938.77\u00a0months after the DGP. Eight patients already used irrigation before DGP.\nTable\u00a01Medical historyMedical historyNumber of patients (percentage of total)Anal rupture during delivery13 (28%)Anal repair14 (30%)Prolapse operation19 (41%)Hysterectomy17 (37%)Abdomino-perineal resection for rectum carcinoma8 (17%)Sigmoid resection for sigmoiditis2 (9%)Cholecystectomy8 (17%)Anus atresia1 (2%)\nTwenty-four (52%) patients used irrigation as an additional therapy for fecal incontinence in partially failing DGP. Eleven (24%) patients used irrigation for constipation and 11 (24%) patients for both. Most patients (91%) used the irrigation pump. Three patients added soap and one patient an enema to the water to achieve a better result. These were patients with constipation or a combination of fecal incontinence and constipation.\nIrrigation was usually performed in the morning (70%) and sometimes in the evening (16%). The frequency of irrigation was 0.90\u2009\u00b1\u20090.40 times per day. The amount of water used for irrigation was 2.27\u2009\u00b1\u20091.75\u00a0l with duration of 39\u2009\u00b1\u200923\u00a0min. Seventy-four percent of the patients with fecal incontinence irrigated in the morning, 17% in the evening, and 9% twice a day. For the patients with constipation, these numbers are, respectively, 45, 18, and 36%. Ninety percent of the patients with a combination of defecation disorders used the colonic irrigation in the morning.\nThere was no significant difference in the frequency of irrigation between patients with fecal incontinence and patients with constipation (0.92 vs 0.98\/day; P\u2009=\u20090.108). The amount of water used for irrigation was higher in patients with fecal incontinence compared to patients with constipation, but this difference was not significant (2.31 vs 1.91\u00a0l; P\u2009=\u20090.484). There was no significant difference in the time needed for irrigation between patients with fecal incontinence and those with constipation (40.8 vs 32.9\u00a0min; P\u2009=\u20090.154).\nOverall, 81% of the patients were satisfied with the irrigation, 80% of the patients with fecal incontinence, 90% of the patients with constipation, and 72% of the patients with a combined defecation disorder. Thirty-seven percent of the patients with fecal incontinence reached (pseudo-) continence, and in 30% of the patients, the constipation completely resolved (Fig.\u00a03). In 29% of the patients with a combined defecation disorder, the constipation resolved and they were (pseudo-) continent. Overall, satisfaction was related with symptomatic improvement. Only one patient with good symptomatic improvement was not satisfied because he felt that the irrigation was time-consuming and not practical.\nFig.\u00a03Success of rectal irrigation for defecation disorders\nFour patients, two with fecal incontinence, one with constipation, and one with both, performed antegrade irrigation through a (3) colostomy or an (1) appendico-cecostomy with good results, and all indicated that the irrigation improved their quality of life.\nThe patients could be divided in six groups according to the underlying cause of fecal incontinence before DGP (Table\u00a02). When the different etiologies before DGP were compared with the result during irrigation, the patients with a rupture or anal surgery had the worst results of irrigation. The patients with a spinal cord lesion seemed to have the best results.\nTable\u00a02Success of rectal irrigation in different etiologies for fecal incontinence before DGPEtiologyNumber of casesSuccess (%) during irrigationCongenital11 (100%)Trauma143 (21%)Pudendopathy155 (33%)Spinal cord lesion53 (60%)Abdomino-perineal resection for rectum carcinoma83 (37%)Prolapse20 (0%)Total4515 (33%)\nSide effects of the irrigation were reported in 61% of the patients: leakage of water after irrigation (43%), abdominal cramps (17%), and distended abdomen (17%). Twenty-four (53%) patients changed the irrigation method during their course to improve the results. Seven (16%) patients stopped the rectal irrigation: in five patients, the result was unsatisfactory; two other patients did not need the irrigation anymore because the complaints resolved completely. Finally, two patients received a permanent stoma.\nDiscussion\nColonic irrigation can be used effectively to treat defecation disorders when other conservative treatments fail or in addition to unsuccessful or partially successful surgical treatment [18].\nColonic irrigation is usually forgotten as an alternative conservative treatment for defecation disorders, although many health care takers are familiar with colonic irrigation, particularly, for its use as perioperative colonic cleansing [19, 20].\nRetrograde colonic irrigation is performed through the anorectum or via a colostomy. Only a few publications addressed to retrograde colonic irrigation are found in literature. Briel et al. [13] found a success rate of 38% of retrograde colonic irrigation for fecal incontinence and a significant improvement in quality of life. Recently, another study reported a success rate of 41% in patients with fecal incontinence and 65% in patients with constipation [18]. The success rate is based on patient satisfaction. Although patient satisfaction is the primary goal of treatment, it is a subjective measure. In future research, this should be combined with objective measures such as validated quality of life questionnaires in a prospective study design.\nAntegrade colonic irrigation is especially known for the treatment of evacuation disorders in small children and can be performed through an appendico-cecostomy (MACE) or a cecostomy button [16, 21\u201323]. Alternative enteral access is a sigmoid tube or transverse colonic conduit for patients with a left colonic evacuation disorder [24\u201326]. The most common problems of MACE are stoma stenosis and leakage [16, 17]. The results in our hospital of antegrade irrigation by an appendico-cecostomy were described earlier [23]. In this study, four patients performed antegrade irrigation via a colostomy or a MACE with good results. O\u2019Bichere et al. performed an experimental study investigating the effect of retrograde vs antegrade colonic irrigation in pigs. That study demonstrates that colonic emptying is more efficient with antegrade irrigation compared to retrograde irrigation [27]. Although a reasonable success rate can be achieved with antegrade irrigation (64\u201385%) [15\u201317], retrograde colonic irrigation is preferred above antegrade irrigation as initial treatment because of its non-invasive nature and benign complications.\nThe amount of water used for irrigation was higher in patients with fecal incontinence compared to patients with constipation. This can be explained by the difficulty of retaining water in the rectum in case of fecal incontinence. Theoretically, the amount of water retained in the rectum is higher in patients with constipation. The grade of colonic emptying by irrigation in constipated patients by means of scintigraphy was investigated by Christensen et al. [14]. The effect of retrograde colonic washout was significantly better in idiopathic fecal incontinence compared to idiopathic constipation, and its effect correlated with the extent to which the irrigation fluid had entered the colorectum.\nSurgical treatment of fecal incontinence gives an overall success of 50\u201384%, depending on the etiology of fecal incontinence and type of surgical procedure [28\u201332]. Success percentages in DGP vary from 42\u201392% [1\u20135]. The success rate of 80% of colonic irrigation is comparable to the success rate of surgical treatment for fecal incontinence. Sixty-one percent of the patients that received the questionnaire completed it. Even when we assume that the patients who did not complete the questionnaire were dissatisfied with the treatment (bad case scenario), the success rate in the present study would still be 50%. It is important to realize that this is a selected group of patients with persistent incontinence and\/or constipation after DGP. Regarding the fecal incontinence, this group of patients represents the 8\u201358% (assuming 42\u201392% success after DGP) with insufficient results of DGP [1\u20135].\nRegarding the different underlying causes for fecal incontinence before DGP, the patients with a trauma to the anal sphincter have the lowest success rate and the patients with a spinal cord lesion have the highest success rate. It is not possible to draw conclusions about the statistical significance of these results because of the limited group size.\nPrevious studies already reported an increased incidence of constipation after DGP [3, 6, 8, 11, 12].\nIn this study, we included 22 patients that were constipated after DGP, of which 11 patients had constipation in combination with persistent fecal incontinence. It is important to realize that many patients are incontinent for feces after periods of severe evacuation problems. These evacuation problems are present again after treatment of the fecal incontinence. Its etiology is not well understood, but is likely to be multifactorial [33, 34]. This is known after all surgical procedures for fecal incontinence [35]. Constipation can be due to technical problems such as a tight graciloplasty, which can be solved by revision of the DGP. It is also possible that nerve tissue around the anus sustains damage during the operation, causing pudendopathy which cannot be treated by revisional surgery. Previous surgery in the pelvis, like an abdomino-perineal resection (eight patients in this study), is known to have a very high rate of obstructed defecation, probably due to reduced rectal sensitivity and pelvic floor dysfunction [36]. Constipation related to physical impairment, like a disturbed sensation and\/or motility because of a congenital cause or degeneration, is difficult to treat [9]. These patients are usually served well with colonic irrigation.\nSurgical treatment of constipation is troublesome and only indicated in a selected group of patients with intractable constipation. The bowel frequency usually increases, but abdominal pain and bloating persists in most patients [37]. In this study, 90% of the patients were satisfied with the irrigation, and in 30%, the constipation resolved with colonic irrigation.\nThe final step of treatment after the colonic irrigation in the patients of this study would be a colostomy or ileostomy. In the study of Norton et al., the majority of the patients with fecal incontinence managed by a colostomy reported to be positive about the stoma. However, a few could not adapt to the stoma and disliked it intensely [38]. Harris et al. investigated patients with fecal incontinence or constipation managed with a stoma. Many of these patients reported that lifestyle restrictions were imposed by the stoma, and almost half of the patients felt stigmatized. Up to a third of these patients had significant depression, especially the younger women [39]. Cultural issues also have an important impact on the acceptance of a stoma [40]. Keeping these disadvantages of a definitive stoma in mind, colonic irrigation offers an attractive alternative, which is worthwhile trying before proceeding to a definitive stoma.\nConclusion\nColonic irrigation is an effective alternative for the treatment of persistent fecal incontinence after DGP and\/or recurrent or onset constipation additional to unsuccessful or (partially) successful DGP. Colonic irrigation is an undervalued and often forgotten treatment option, which deserves its rightful place among the other treatment modalities.","keyphrases":["colonic irrigation","dynamic graciloplasty","fecal incontinence","constipation"],"prmu":["P","P","P","P"]}
{"id":"Calcif_Tissue_Int-3-1-1914224","title":"The -1997 G\/T and Sp1 Polymorphisms in the Collagen Type I alpha1 (COLIA1) Gene in Relation to Changes in Femoral Neck Bone Mineral Density and the Risk of Fracture in the Elderly: The Rotterdam Study\n","text":"The COLIA1 Sp1 polymorphism has been associated with bone mineral density (BMD) and fracture. A promoter polymorphism, -1997 G\/T, also has been associated with BMD. In this study, we examined whether these polymorphisms alone and in the form of haplotypes influence bone parameters and fracture risk in a large population-based cohort of elderly Caucasians. We determined the COLIA1 -1997 G\/T (promoter) and Sp1 G\/T (intron) polymorphisms in 6,280 individuals and inferred haplotypes. Femoral neck BMD and BMD change were compared across COLIA1 genotypes at baseline and follow-up (mean 6.5 years). We also investigated the relationship between the COLIA1 polymorphisms and incident nonvertebral fractures, which were recorded during a mean follow-up period of 7.4 years. Vertebral fractures were assessed by radiographs on 3,456 genotyped individuals. Femoral neck BMD measured at baseline was 3.8% lower in women carrying two copies of the T-Sp1 allele (P for trend = 0.03). No genotype dependent differences in BMD loss were observed. In women homozygous for the T allele of the Sp1 polymorphism, the risk of fragility fracture increased 2.3 times (95% confidence interval 1.4\u20133.9, P = 0.001). No such association was observed with the promoter polymorphism. In men, no association with either the Sp1 or the -1997 G\/T promoter polymorphism was seen with BMD or fracture. High linkage disequilibrium (LD; D\u2032 = 0.99, r2 = 0.03) exists between the two studied polymorphisms. We observed three haplotypes in our population: haplotype 1 (Gpromoter\u2013Gintron) frequency (f) = 69%, haplotype 2 (Gpromoter\u2013Tintron) f = 17.6%, and haplotype 3 (Tpromoter\u2013Gintron) f = 13.4%. Haplotype 2 was associated with a 2.1-fold increased risk of fragility fracture in women (95% confidence interval 1.2\u20133.7, P = 0.001). We confirm that the COLIA1 Sp1 polymorphism influences BMD and the risk of fracture in postmenopausal Caucasian women. In contrast, we found no independent effect of the -1997 G\/T promoter polymorphism on BMD or fracture.\nIntroduction\nOsteoporosis is a multifactorial disease with both genetic and environmental determinants. It is characterized by a reduction in bone mineral density (BMD) and microarchitectural deterioration of bone tissue, which leads to an increased risk of fracture in later life [1, 2]. Being a predictor of bone fragility and susceptibility to fracture, BMD is used for the diagnosis of osteoporosis [3, 4]. The risk of fracture is dependent not only on BMD but also on geometry, architecture, material properties, and mass distribution [5].\nThe skeletal determinants of osteoporotic fracture risk such as BMD, bone loss, and bone geometry are all subject to strong genetic influences [2, 6, 7]. It has been estimated from twin studies that 60-80% of the variance in BMD is attributable to genetic factors [8, 9]. Several genes are thought to be involved in the pathogenesis of osteoporosis. Collagen type I alpha 1 (COLIA1) is one of the most prominent candidate genes, which has been consistently associated with osteoporosis in different populations [10\u201312]. COLIA1 encodes the alpha 1 chain of collagen type I, which is the most abundant structural protein in the bone matrix; rare mutations in this gene cause osteogenesis imperfecta, a mendelian disorder presenting with moderate to severe bone fragility [13, 14].\nPreviously, Grant et al. [15] identified a relatively common guanine to thymidine (G\u2192T) polymorphism in the first intron of COLIA1. This polymorphism affects one of the binding sites of the transcription factor Sp1 and results in increased expression of collagen type I alpha 1 in bone matrix in T carriers [11] .We and others have shown that the T allele is associated with osteoporosis, lower BMD [10, 15], and increased fracture risk [10, 12, 16, 17]. Moreover, in a very large prospective meta-analysis of individual data, we observed that the Sp1 polymorphism in the COLIA1 gene is associated with reduced BMD and incident vertebral fractures independent of BMD [18]. In addition to the Sp1 polymorphism, Garcia-Giralt et al. [19] described two polymorphisms within the COLIA1 promoter region: -1997 G\/T and -1663 indelT. The study showed in a small cohort of postmenopausal Spanish women that the T allele of the -1997 G\/T polymorphism was significantly associated with a 7.5% decreased lumbar spine BMD and a 12% decreased femoral neck BMD. Furthermore, they analyzed compound genotypes including three polymorphic sites. However, this small study of the promoter polymorphisms in women investigated the relation in form of compound genotypes, was not extended to haplotypes of promoter and Sp1 polymorphisms, and most importantly, did not analyze fractures, the clinically most relevant end point of osteoporosis.\nTherefore, we investigated the influence of both COLIA1 polymorphisms independently and in the form of haplotypes in relation to baseline femoral neck BMD, change in BMD with follow-up, and risk of vertebral and nonvertebral fractures in a large population-based cohort of elderly Caucasian men and women.\nMaterials and Methods\nStudy Population\nThis study was embedded in the Rotterdam Study, a population\u2013based cohort study in which all residents of the Rotterdam suburb Ommoord aged 55 years and older were invited to take part. The design of the study has been described elsewhere [20]. Written informed consent was obtained from all participants, and the Medical Ethics Committee of the Erasmus Medical Center approved the study. Baseline data collection was conducted in January 1990 and June 1993, while two follow-up assessments were performed between July 1993 and January 1996 and from July 1996 until December 1999. A total of 7,983 subjects participated in the study (response rate 78%), and for the present study, we examined 6,280 individuals who were genotyped.\nStudy Design\nThis study was performed in three steps. In the first step, we performed a cross-sectional analysis where we examined the relation between the genotype and baseline BMD (n = 5,737). In the second step, we performed a longitudinal analysis to study change in BMD between baseline and the second follow-up (mean 6.5 \u00b1 standard deviation [SD] 0.6 years, n = 2,670). In the third step, we looked at the relation between COLIA1 polymorphisms and the risk of incident fracture. We studied incidence of nonvertebral fracture (mean follow-up 7.4 \u00b1 3.3 years, n = 6,280) and vertebral fractures assessed by radiographs both at baseline (1990\u20131993) and at follow-up visit\u2019 between 1997 and 1999, thoracolumbar radiographs of the spine were available for 3,469 individuals in a mean follow-up of 6.4 years.\nMeasurements\nBMD (g\/cm2) of the hip and L2-L4 of the lumbar spine were measured by dual-energy X-ray absorptiometry (DXA) using a Lunar DPX densitometry (DPX-L) (Lunar Radiation, Madison, WI) and reanalyzed with DPX-IQ software, under standard protocols. Methods, quality assurance, accuracy, and precision issues of the DXA measurements have been described previously [21]. The relative change of BMD from baseline was estimated as the difference in BMD between assessment periods divided by the BMD at baseline.\nHeight (cm) and weight (kg) were measured with a stadiometer at the initial examination, in standing position wearing indoor clothes without shoes. Body mass index (BMI) was computed as weight divided by height (kg\/cm2).\nFracture Follow-up\nInformation on incident nonvertebral fractures was collected from baseline (1990\u20131993) until January 1, 2002 (mean follow-up \u00b1 SD 7.4 \u00b1 3.3 years, n = 6,280).\nNonvertebral fracture events were retrieved from computerized records of the general practitioners (GPs) in the research area. Research physicians regularly followed participant information in GP records outside the research area and made an independent review, encoding all reported events. Subsequently, a medical expert in the field reviewed all coded events for final classification. We excluded fractures that were considered nonosteoporotic, i.e., caused by cancer and high trauma, including fractures of the hand, foot, skull, and face. Subsequently, we analyzed separately \u201cfragility\u201d fractures, which were defined as any fracture of the hip, pelvis, or proximal humerus that had occurred with minimal trauma at older age (mean >75 years).\nVertebral Fracture Assessment\nBoth at baseline and at first follow-up, between 1997 and 1999, thoracolumbar radiographs of the spine were obtained. The follow-up radiographs were available for 3,456 individuals, who survived an average of 6.4 (SD = 0.4) years after the baseline visit and who were still able to come to our research center. All follow-up radiographs were scored for the presence of vertebral fracture by the McCloskey\/Kanis method, as described previously [22].\nGenotyping\nGenomic DNA was extracted from samples of peripheral venous blood according to standard procedures. Genomic DNA (1\u20132 ng) was dispensed into 384-well plates using a Sciclone ALH3000 pipetting robot (Caliper, Mountain View, CA). Genotypes were determined using the Taqman allelic discrimination assay. The Assay-by-Design service (http:\/\/www.appliedbio-systems.com) was used to set up a Taqman allelic discrimination assay for the COL1PR-1997 polymorphism (primers: Fw, GCCTCCGGAGGGTGTCA, Rv, AAGGAGAGCAATTCTTACAGGTGTCT; probes: FAM-CCTGAGGGATGGAA-MGB, VIC-CCTGAAGGATGGAAG-MGB. The polymerase chain reaction (PCR) mixture included 1\u20132 ng of genomic DNA in a 2 \u03bcL volume and the following reagents: FAM and VIC probes (200 nM), primers (0.9 \u03bcM), and 2\u00d7 Taqman PCR master mix (ABgene, Epsom, UK). Reagents were dispensed in a 384-well plate using the Deerac Equator NS808 (Deerac Fluidics, Dublin, Ireland). PCR cycling was performed in 384-well PCR plates in an ABI 9700 system (Applied Biosystems, Foster City, CA) and consisted of initial denaturation for 15 minutes at 95\u00b0C, 40 cycles with denaturation of 15 seconds at 95\u00b0C, and annealing and extension for 60 seconds at 60\u00b0C. Results were analyzed by the ABI Taqman 7900HT using the sequence detection system 2.22 software (Applied Biosystems). To confirm the accuracy of genotyping results, 332 (5%) randomly selected samples were regenotyped with the same method. No inconsistencies were observed. For Sp1 (intron1) an assay was set up using Primer Express Software version 2.0 (Applied Biosystems, Foster city, CA). Forward and reverse primer sequences were 5\u2032-GTTGTCTAGGTGCTGGAGGTT-3\u2032 and 5\u2032-GGCGAGGGAGGAGAGAAGG-3\u2032. The PCR mixture included 5 ng of genomic DNA in a 4\u00a0\u03bcL volume and the following reagents: FAM-CCCGCCCACATTCCCTGG-MGB probes (250 nM), TET-CCCGCCCCCATTCCCTGG-MGB probe (500 nM), primers (300 nM), 2\u00d7 Taqman PCR master mix (Applied Biosystems). PCR cycling was performed in 384-well PCR plates in the ABI 9700 PCR system and consisted of initial denaturation for 15 minutes at 95\u00b0C, 40 cycles with denaturation of 15 seconds at 95\u00b0C, and annealing and extension for 60 seconds at 60\u00b0C. Results were analyzed by the ABI Taqman 7900HT using the sequence detection system 2.1 software (Applied Biosystems). To confirm the accuracy of the genotyping results, 332 randomly selected samples were genotyped for a second time with the same method. All polymorphisms had an error rate lower than 1%.\nStatistical Analysis\nHardy-Weinberg equilibrium of the COLIA1 polymorphism genotypes was tested using the GENEPOP package [23]. Linkage disequilibrium (LD) between each pair of alleles at both polymorphic loci was calculated as D\u2032 and r2 [24].\nWe stratified all analyses by gender, considering peak bone mass, changes in BMD, and fractures, following age- and sex-specific patterns. Baseline BMD and BMD rate of change were compared across COLIA1 polymorphisms using univariate analysis of variance (ANOVA). Corrections were made for age and BMI. Trend analysis assuming an underlying additive genetic model was done for the presence of zero, one, or two copies of the associated allele, incorporating the genotype variable as a continuous term in a general linear regression model. For the analysis of nonvertebral fracture follow-up data, we computed the incidence rates of fracture among genotypes and used Cox\u2019s proportional hazards model, adjusting for age and BMI to estimate risk of fracture. For vertebral fractures, odd ratios with 95% confidence intervals (95% CIs) were calculated using logistic regression models since no data on the exact time of occurrence could be determined.\nWe used HaploStats (available at http:\/\/www.cran.r-project.org\/) to estimate the frequency of inferred haplotypes and investigate the association of haplotypes with BMD and the risk of fractures. We restricted the analysis to haplotypes with an inferred frequency of more than 0.02. The first haplotype, which was most frequent, was used as reference.\nSignificant P values were 0.05 or lower. Finally, model assumptions were verified and model residuals checked for goodness-of-fit. SPSS 11.0 (SPSS, Chicago, IL) was used for the analyses.\nResults\nAllele and genotype frequencies of the -1997 G\/T and Sp1 polymorphisms were in Hardy-Weinberg equilibrium (P = 0.61 and P = 0.10, respectively). General characteristics of the study population at baseline and follow-up are shown in Table\u00a01.\nTable\u00a01General characteristics of the study population at baseline and second follow-upWomenMenBaselineSecond follow-upBaselineSecond follow-upNumbern = 3,374n = 1,724n = 2,452n = 1,287Age (years)68.3 \u00b1 8.272.7 \u00b1 6.867.6 \u00b1 7.772.2 \u00b1 6.5Height (cm)161.1 \u00b1 6.8160.6 \u00b1 6.4174.6 \u00b1 6.8174.0 \u00b1 6.7Weight (kg)69.3 \u00b1 11.470.3 \u00b1 12.278.2 \u00b1 10.879.5 \u00b1 11.3BMI (kg\/m2)26.7 \u00b1 4.127.2 \u00b1 4.425.6 \u00b1 3.026.3 \u00b1 3.2FN-BMD (g\/cm2)a0.83 \u00b1 0.140.80 \u00b1 0.130.92 \u00b1 0.140.90 \u00b1 0.14Lumbar spine BMD (g\/cm2)1.03 \u00b1 0.18-1.16 \u00b1 0.20-FN-BMD changen = 1,527n = 1,143FN-BMD change (relative % of baseline year)b\u22120.84 \u00b1 1.09-Values are means \u00b1 SD. Anthropometric measurement based on 5,826 individuals at baseline and 3,011individuals at follow-upaBMD measurements based on 5,737 individual at baseline and 2,670 individual at second follow-upbFemoral neck (FN) BMD change was measured between second follow-up and baseline. Second follow-up measurements were performed on average 6.5 (SD = 0.6) years after baseline\nBaseline BMD by COLIA1 Genotypes\nIn both genders, age, height, weight, and BMI did not differ significantly between genotypes for the -1997 G\/T and Sp1 polymorphisms (data not shown). Table\u00a02 shows the BMD values according to COLIA1 genotypes in men and women. Femoral neck BMD was 3.8% lower (mean difference = 0.03 g\/cm2, P = 0.09) in women who were homozygous carriers of the Sp1 T allele compared to noncarriers, with evidence for an allele dose effect (P for trend = 0.03) (Table\u00a02). No association was found between the -1997 promoter polymorphism and lumbar spine BMD or femoral neck BMD in men or women (Table\u00a02). We did not observe any significant association between the Sp1 and -1997 promoter polymorphisms with changes in BMD during follow-up in men or women (Table\u00a02).\nTable\u00a02BMD measurements by COLIA1 genotypes at baseline and follow-upPromoter -1997 G\/TIntron 1 Sp1 G\/TGGGTTTP*GGGTTTP*Menn = 1,663n = 494n = 37n = 1,484n = 655n = 55Femoral neck (g\/cm2)0.92 \u00b1 0.130.92 \u00b1 0.140.90 \u00b1 0.141.000.92 \u00b1 0.140.92 \u00b1 0.140.90 \u00b1 0.120.45Lumbar spine (g\/cm2)1.16 \u00b1 0.191.17 \u00b1 0.201.15 \u00b1 0.220.961.17 \u00b1 0.191.16 \u00b1 0.201.16 \u00b1 0.200.91Numbern = 668n = 233n = 16n = 606n = 293n = 18FN-BMD change (relative % of baseline year)\u22120.46 \u00b1 0.91\u22120.36 \u00b1 0.93\u22120.38 \u00b1 0.600.62\u22120.39 \u00b1 0.93\u22120.56 \u00b1 0.87\u22120.30 \u00b1 0.820.01Womenn = 2,157n = 710n = 53n = 1,971n = 858n = 91Femoral neck (g\/cm2)0.83 \u00b1 0.140.84 \u00b1 0.130.85 \u00b1 0.130. 220.83 \u00b1 0.130.82 \u00b1 0.140.80 \u00b1 0.140.09**Lumbar spine (g\/cm2)1.03 \u00b1 0.181.04 \u00b1 0.181.01 \u00b1 0.180.691.04 \u00b1 0.181.04 \u00b1 0.191.01 \u00b1 0.200.52Numbern = 820n = 298n = 24n = 773n = 340n = 29FN-BMD change (relative % of baseline year)\u22120.84 \u00b1 1.11\u22120.83 \u00b1 1.01\u22120.78 \u00b1 1.030.85\u22120.85 \u00b1 1.08\u22120.80 \u00b1 1.04\u22120.74 \u00b1 1.490.55Values are expressed as mean \u00b1 SD. Adjustments for age and BMI. Femoral neck (FN) BMD change was measured between second follow-up and baseline*P for ANOVA. **For trend linear regression: P = 0.03\nRisk of Fracture by COLIA1 Genotypes\nThe relation between risk of fracture and COLIA1 Sp1 polymorphism is shown in Table\u00a03. Women with two copies of the T allele of the Sp1 polymorphism had a 2.3 times higher risk of fragility fracture (95% CI 1.4-3.9, P = 0.001). Adjustment for femoral neck BMD did not essentially modify the association. A similar association was observed in men who were homozygous carriers of the Sp1 T allele, which was borderline significant (risk ratio = 2.3, 95% CI 0.9\u20135.8, P = 0.07). For the -1997 promoter polymorphism no association was found with any type of fracture in either men or women (Table\u00a03).\nTable\u00a03Risk of fractures by COLIA1 genotypesCOLIA1 genotypesTypes of fractureEvent (%)Risk ratio (95% CI)GGGTTTGG (reference)GT vs. referenceTT vs. referenceMenPromoter -1997 G\/TNonvertebral147\/1952 (7.5)49\/592 (8.3)1\/46 (2.2)1.0 (reference)3.18 (0.45\u201322.74)3.44 (0.47\u201329.94)Fragility72\/1952 (3.7)14\/592 (2.4)1\/46 (1.8)1.0 (reference)1.46 (0.20\u201310.55)0.94 (0.12.7.14)Vertebral100\/1056 (9.5)35\/340 (10.3)3\/28 (10.7)1.0 (reference)1.07 (0.71\u20131.61)1.23 (0.36\u20134.18)Intron 1 Sp1 G\/TNonvertebral91\/1254 (7.3)39\/574 (6.8)4\/44 (9.1)1.0 (reference)0.97 (0.71\u20131.33)1.40 (0.65\u20133.00)Fragility36\/1254 (2.9)14\/574 (2.4)2\/44 (4.5)1.0 (reference)0.94 (0.58\u20131.51)2.34 (0.94\u20135.85)Vertebral68\/719 (9.5)31\/350 (8.9)2\/22 (9.1)1.0 (reference)0.83 (0.55\u20131.24)1.59 (0.60\u20134.22)WomenPromoter -1997 G\/TNonvertebral528\/2721 (19.4)182\/879 (20.7)8\/63 (12.7)1.0 (reference)1.53 (0.76\u20133.09)1.66 (0.82\u20133.36)Fragility216\/2721 (7.9)73\/879 (8.3)4\/63 (6.3)1.0 (reference)1.13 (0.42\u20133.05)1.19 (0.44\u20133.26)Vertebral159\/1320 (12.0)52\/445 (11.7)5\/35 (14.3)1.0 (reference)0.98 (0.70\u20131.37)1.31 (0.49\u20133.45)Intron 1 Sp1 G\/TNonvertebral279\/1626 (17.2)121\/710 (17.0)17\/76 (22.4)1.0 (reference)1.05 (0.90\u20131.23)1.34 (0.92\u20131.23)Fragility98\/1626 (6.0)42\/710 (5.9)10\/76 (13.2)1.0 (reference)1.01 (0.78\u20131.31)2.33 (1.39\u20133.87) Vertebral98\/891 (11.0)51\/401 (12.7)3\/34 (8.8)1.0 (reference)1.21 (0.88\u20131.65)1.37 (0.56\u20133.35)Adjustments for age and BMI\nHaplotype Analysis\nHigh LD exists between the -1997 G\/T and Sp1 polymorphisms, as assessed by D\u2032 measure (D\u2032 = 0.99, r2 = 0.03). We observed in our population three -1997 G\/T, Sp1 G\/T haplotype alleles (Fig.\u00a01): haplotype 1 (Gpromoter\u2013GIntron), 69.0%; haplotype 2 (Gpromoter\u2013TIntron), 17.6%; and haplotype 3 (Tpromoter\u2013GIntron), 13.4%. Haplotype 4 (Tpromoter\u2013TIntron) was not present (Fig.\u00a01). The strong LD between the -1997 G\/T and Sp1 G\/T polymorphism and the virtual nonexistence of haplotype 4 suggests there is absence of ancestral recombination in the region. We observed a borderline significant association (P = 0.06) between haplotype 2 (Gpromoter\u2013TIntron) and femoral neck BMD in women. Women who carried haplotype 2 (Gpromoter\u2013TIntron) had lower (\u22120.01 mg\/cm2) femoral neck BMD compared with those who carried haplotype 1 (Gpromoter\u2013GIntron).\nFig.\u00a01Schematic representation of the COLIA1 gene with the structural-1997 G\/T polymorphism in the promoter region and G\/T Sp1 polymorphism at binding site, with observed haplotype frequencies in the Rotterdam Study\nThe relation between risk of fracture and COLIA1 haplotypes is shown in Table\u00a04. Women with haplotype 2 (Gpromoter\u2013TIntron) had a 2.1 times higher relative risk of fragility fracture (P = 0.03); in men the increase in risk was 2.0 (P = 0.31). These results were essentially unchanged after adjustment for femoral neck BMD. For haplotype 3 (Tpromoter\u2013GIntron) we found no association with any type of fracture in either men or women.\nTable\u00a04Risk of fracture by COLIA1 haplotypesTypes of fractureMen, OR (95% CI)Women, OR (95% CI)Haplotype 1Haplotype 2Haplotype 3Haplotype 1Haplotype 2Haplotype 3Nonvertebral1 (reference)1.29 (0.55\u20133.02)0.00 (0.00-\u221e)1 (reference)1.31 (0.83\u20132.07)0.61 (0.29\u20131.29)Fragility1 (reference)2.01 (0.71\u20135.67)0.00 (0.00-\u221e)1 (reference)2.12 (1.23\u20133.66)0.80 (0.29\u20132.23)Vertebral1 (reference)1.64 (0.62\u20134.31)1.19 (0.35\u20134.00)1 (reference)1.23 (0.51\u20132.95)1.31 (0.50\u20133.42)Adjustments for age and BMI. Haplotype 1, (Gpromoter\u2013GIntron); haplotype 2, (Gpromoter\u2013TIntron); haplotype 3, (Tpromoter\u2013GIntron)\nDiscussion\nIn this large population-based study, we found that the Sp1 polymorphism influences the risk of fragility fracture in elderly women, with a similar yet not significant effect in men. Similarly, women homozygous for the T allele had 3.8% lower BMD at baseline. The -1997 G\/T polymorphism showed no independent effect on fracture risk or BMD levels in both genders. The haplotype analysis showed an association with BMD and fracture in women, which appeared to be driven by the effect of the Sp1 polymorphism.\nA study in Spain [19] showed that the -1997 G\/T polymorphism located in the promoter region of the COLIA1 gene was associated with BMD in postmenopausal women of Spanish origin. In addition, analysis of compound genotypes of the three studied polymorphisms (-1997 G\/T, Sp1, and -1663 indelT) suggested that the lowest value for BMD corresponded to GG homozygous at -1997 and heterozygous at the other two loci. Furthermore, in another report, the same group observed a possible functional mechanism for the -1997 G\/T polymorphism [25]. Our population-based study suggests there is no independent effect of the -1997 polymorphism on BMD and the risk of fractures.\nRecently, Stewart et al. [26] examined the three polymorphisms of the COLIA1 gene in forms of haplotypes in postmenopausal women. In contrast with our study, they observed an association between reduced BMD values and the promoter \u20131997G\/T polymorphism. Since the promoter -1997G\/T polymorphism is in strong LD with the Sp1 polymorphism, the observed association of haplotype 2 is driven by the SP1 polymorphism.\nWe showed that the association between fragility fractures and Sp1 polymorphism is significant only in women. We also found that the association between fragility fracture and the Sp1 polymorphism was independent of femoral neck BMD. A possible explanation for an increased risk of fracture is the different number of fractures between men and women. There are a higher number of fractures in women compared to men (13.2% in women and 4.5% in men). This suggests that other underlying biological mechanisms beyond BMD levels, such as the role of microarchitecture and composition of mineral crystals in bone tissue, might explain the increased fracture risk [11, 27, 28]. Biomechanical testing of bone samples from heterozygous individuals with the GT genotype showed reduced bone strength compared to the homozygous GG genotype and a slight reduction in mineralization of bone [11]. Presence of the T allele in the COLIA1 Sp1 binding site leads to an abnormal relative level of COLIA1\/COLIA2, which may reduce bone quality and quantity [11]. Accordingly, we assume that a weaker network of abnormal collagen cross-linking may generate a three-dimensional unstable condition that may be responsible for its relatively greater risk of fragility fracture in elderly women homozygous for the Sp1 T allele. Similarly, it is likely that the Sp1 polymorphism drives these associations since evidence of functionality of this polymorphism has been reported previously.\nIn the Genetic Markers for Osteoporosis (GENOMOS) Study, which is the largest study examining the Sp1 polymorphism (n = 20,786) in relation to osteoporosis, an association between the Sp1 polymorphism and a 1.3 times incident risk of vertebral fractures was also observed. An effect of the -1997 promoter polymorphism due to power limitations cannot be fully excluded and should be subject to study in a larger population like that of GENOMOS.\nOur present study has some limitations. Survival bias may play a role if individuals who were lost to follow-up were associated to genotype. Considering this selection bias, a possible relationship of the COLIA1 polymorphisms with changes in BMD cannot be fully excluded.\nIn conclusion, we observed an increased risk of fragility fractures in women carriers of the COLIA1 Sp1 T allele. In contrast, the -1997 G\/T polymorphism by itself appears to have no influence on fracture or BMD in postmenopausal women, though the role of power limitations cannot be excluded.","keyphrases":["colia1","bone","bone mineral density","fracture","osteoporosis"],"prmu":["P","P","P","P","P"]}
{"id":"BMC_Struct_Biol-4-_-331416","title":"Consensus structural models for the amino terminal domain of the retrovirus restriction gene Fv1 and the Murine Leukaemia Virus capsid proteins\n","text":"Background The mouse Fv1 (friend virus) susceptibility gene inhibits the development of the murine leukaemia virus (MLV) by interacting with its capsid (CA) protein. As no structures are available for these proteins we have constructed molecular models based on distant sequence similarity to other retroviral capsid proteins.\nBackground\nThe Fv1 gene is one of a series of mouse genes that control the susceptibility of mice to murine leukaemia virus (MLV) [1-3]. The gene acts in the cell to restrict virus replication [4] through a mechanism that is still uncertain. Genetic studies suggest that the target for the Fv1 gene product is the capsid protein (CA) of MLV [5,6] and it is thought to interact with CA after entry of the virus into the cell but before integration and formation of the provirus.\nWhen cloned and sequenced [7], the Fv1 gene was found to have sequence similarity to endogenous retroviruses of the HERV-L and MuERV-L families [7,8]. Based on its position within the Gag gene of these endogenous elements, it appears that Fv1 encodes a capsid-like protein. This structural assignment of the Fv1 gene is consistent with its function as it can be postulated that the gene product might act as a dominant negative mutation and interfere with the MLV capsid function [9]. Sequence alignments have been made between Fv1 and other retroviral capsid proteins [8] but besides one region of clear similarity, called the Major Homology Region (MHR), there is otherwise little that is conserved across the full family of retroviral (and related lenti-virus) CA sequences.\nThere are now several known structures for retroviral capsid proteins in the Protein Databank (PDB). While some of these are only fragmentary, a selection can be extracted that gives a reasonable phylogenetic spread across the retroviruses, with examples from three out of six genera of orthoretroviruses. In all the known structures, the CA protein has an all-\u03b1 type structure consisting of two domains: a larger N-terminal and smaller C-terminal domain with a short extended linker-region between them. As this linker enters the C-terminal domain it incorporates the MHR. There is considerable variation in the orientation of the domains and in the conformation of the loop-regions between \u03b1-helices, particularly in the N-terminal domain.\nIn this work, we have exploited these multiple structures to construct consensus molecular models using threading methods both for the Fv1 gene product (FV1) and its target protein, the MVL CA. As threading takes known and predicted structure into account, it should provide better alignments for the regions that lie outside the MHR. However, as these methods are still far from perfect, we have constructed a model based on each known structure and the degree to which these agree has been used to assess the confidence of different parts of the model. As the threading method we have used has some 'free' parameters (such as the gap-penalty) we have introduced a novel modelling strategy in which the parameters are varied to give maximum agreement among the resulting models.\nResults and Discussion\nThe sequence alignments compiled on the proteins of known structure using the \u03a8-BLAST\/QUEST search strategy (Methods Sectn.) were used in the modelling of both the MLV CA and FV1 sequences. These varied from 4 to 7 sequences (Table 1). Not unexpectedly, the alignments are to some extent similar, in particular each contains the sequence of the HIV-1 CA structure [1e6jP]. (QUEST is biased to retain sequences of known structure). Greatest overlap exists between the sequence sets of the two human viruses, with the HTLV-I sequences being a subset of the HIV-1 sequences (Table 0(d) and Table 0(c)). While it would be possible to extend and realign these sub-families based on structure comparisons, they were left unaltered so as to be equivalent to the MLV and FV1 alignments described below. This allows control modelling tests to be directly comparable to those performed for sequences of unknown structure.\nTable 1\nTemplate sequences selected for alignment. The \u03a8-BLAST\/QUEST search strategy (Methods Sectn.) when started with the probe sequence of the PDB structure indicated by \"SEED\" selected the sequences indicated in each subtable: (a) EIAV [1eia], (b) RSV [1d1dA], (c) HIV-1 [1e6jP] and (d) HTLV-I [1qrjA]. The sequences are identified by their gene identification (gi) number (first column) and their local source databank identifier. The sequence fragment (automatically extracted by QUEST) is given as a range of residue numbers.\n(a) EIAV\n(b) RSV\nSEED\n1eia\n12084543\npdb-1E6J\n0\u2013210\n6358699\ngb-AAF07324\n131\u2013342\n8072301\ngb-AAF71968\n0\u2013155\n6815746\ngb-AAF28696\n0\u2013173\n6649692\ngb-AAF21520\n5\u2013224\n12084543\npdb-1E6J\n3\u2013210\n120850\nsp-P18041\n97\u2013362\n27803398\ngb-AAO21890\n120\u2013281\n294961\ngb-AAA74706\n116\u2013381\n5106563\ngb-AAD39752\n81\u2013346\nSEED\n1d1dA\n(c) HIV-1\n(d) HTLV-I\n6358699\ngb-AAF07324\n129\u2013340\nSEED\n1qrjA\n22037894\ngb-AAM90230\n148\u2013359\n12084543\npdb-1E6J\n0\u2013210\nSEED\n1e6jP\n22037894\ngb-AAM90230\n144\u2013370\n532325\ngb-AAA99545\n50\u2013224\n9886907\ngb-AAG01643\n0\u2013222\n9886907\ngb-AAG01643\n0\u2013211\nMLV Modelling\nThe databank search using the MLV sequence as a probe provided a useful collection of six sequences (Table 1(a)) which, with the MLV probe sequence itself, were passed through the PsiPred secondary structure prediction protocol (Methods Sectn.). When the predicted secondary structures were viewed on the aligned sequences, several distinct \u03b1-helices were apparent, consistent with the protein having an all-\u03b1 type structure similar to the other capsid proteins of known structure (Figure 1).\nThe MLV alignment with predicted secondary structure was matched in the MST program with the four capsid proteins of known structure along with their associated aligned sequences. This was done for each parameter combination specified in Methods Sectn. and the quality of the agreement among the four resulting models quantified using both the Fu and Fw measures defined in Methods Sectn. There was reasonable overall agreement between the two measures of the parameter combinations (Figure 2) and with otherwise no basis on which to prefer one measure over the other, the combined measure (Fv) was used to select the parameters on which the final models were calculated. These were S = 7, G = 30.\nFigure 1\nMLV sequence alignment with the sequence of 1qrjA and related sequences. The alignment is displayed twice in different colouring schemes. In the top two panels, (a) the amino half is coloured firstly by predicted secondary structure (red = \u03b1, green = \u03b2) and then below using a different colour for each amino acid type (Taylor, 1997; black = gap). The known helices (N1\u2013N5) marked as red lines (minor helices are orange) between the two blocks. In the lower two panels (b) the carboxy half of the alignment is shown in a similar way. The sequence identifiers are given below the alignment in the same order as they are aligned. The mid-line divides the 1qrjA sub-family from the MLV sub-family.\nFigure 2\nMLV model agreement. The degree of similarity among the four MLV CA models is plotted for different parameter combinations of MST. Combinations resulting in better agreement have colours towards the red end of the spectrum. (a) Using the unweighted RMSd measure (Fu) and (b) Using the weighted RMSd measure (Fw). (See Methods Sectn. for details.) (c) Combined score (Fu\u00b7Fu). The MST parameters varied were X-axis: S = 0\u21929 (in steps ofone) and Y-axis: G = 10\u219290 (in steps of 10). (See Methods Sectn. for details.) The best combined score is at: S = 7, G = 30.\nN-terminal domain\nThe multiple structure alignment of the four models showed good agreement. In the N-terminal domain there were two extensive regions over which all models were aligned in register, covering helices N2-N3 and N5 (including the preceding short helix in the loop region). Two alternate registers were observed for helices N1 and N4, with relative shifts of 3 and 4 residues, respectively. The models based on 1qrjA (HTLV-I) and 1d1dA (RSV) were in complete agreement and the summed Fw score indicated that 1d1dA provided the best consensus model.\nGiven that the alignment of the capsid protein sequences is ambiguous, the superposition of the models on the structures from which they were derived provides a better way to assess whether there is any significant sequence similarity that could be used as a basis on which any one model might be preferred over the others. The PSId values were: 5.6, 18.5, 10.5, 13.0 for 1d1dA, 1qrjA, 1eia and 1e6jP, respectively. (No differences were observed whether using the standard version or the sequence-biased version of SAP).\nAlthough the 1d1dA model was the best consensus representative, it has only 5.6% sequence similarity with MLV and the higher sequence similarity of the 1qrjA based model with its template (18.5%) was considered sufficient to justify its adoption as the preferred model over the 1d1dA model. Structurally, both were very similar (1.4 \u00c5 wRMSd) with the only significant structural difference being a slight reorientation of the helical region in the loop preceeding the final helix. This structure for the 1qrjA based model is shown along with its comparison with the 1d1dA model in Figure 3.\nFigure 3\nConsensus model for the MLV N-domain. (a) The model for the MLV CA N-domain based on 1qrjA shown as a \u03b1-carbon trace with predicted \u03b1-helices coloured red (some fragments of predicted \u03b2-structure are coloured green). The molecule is in approximately the same orientation as in Figure 8(a) and residues identified by Stevens et al. (2003) are marked as small spheres. (b) The models based on 1qrjA and 1d1dA are shown superposed and coloured from blue (amino) to red (carboxy). Feint dashed lines connect identical residues. The wRMSd = 1.4 (uRMSd = 5.8) over 123 residues.\nIt can be seen from Figure 3(a) that there is good location of the predicted and model helices with deviations occurring only at the ends of some helices and into the loop regions at the 'top' of the molecule. The ends of helices N4 and N5 and the loops at this end of the molecule also encompass the mutations identified as affecting the sensitivity of the virus to Fv1 [29].\nC-terminal domain\nWith its relatively unambiguous MHR, all the models of the C-terminal domain were in complete agreement over the first half of the domain. The more C-terminal half, however, was less consistent due to a combination of its generally less conserved nature combined with uncertainty in the location of the terminus in some of the sequences.\nAs the C-terminal domain has been shown to be less important in determining the property of virus susceptibility, further effort was not expended to try and refine the alignment at the carboxy terminus of the molecule, especially in the more difficult alignment of the FV1 sequence described below.\nFV1 Modelling\nThe databank search using the FV1 sequence as a probe provided a less useful collection of only two distinct sequences (Table 1(b)). Although other sequences were found these were rejected by QUEST as being too similar to those retained.\nThe FV1 alignment with predicted secondary structure was matched to the four capsid proteins of known structure as described above for each MST parameter combination and agreement among the four models monitored using the F scores (Methods Sectn.). These were 50.3 and 131.874 for Fu and Fw while the corresponding score obtained for the MLV models were 78.7 and 188.0, respectively. The greater variation among the FV1 models was undoubtedly due to uncertainty in the placement of the large 'unstructured' region. Nevertheless, there was reasonable overall agreement between the two measures and both found maximal model agreement when S = 9, G = 20 (Figure 4).\nFigure 4\nFV1 model agreement. The degree of similarity among the four FV1 CA models is plotted for different parameter combinations of MST. (a) Using the unweighted RMSd measure (Fu) and (b) Using the weighted RMSd measure (Fw). (See Methods Sectn. for details.) (c) Combined score (Fu\u00b7Fu). The MST parameters varied were X-axis: S = 0\u21929 (in steps of one) and Y-axis: G = 10\u219290 (in steps of 10). (See Methods Sectn. for details.) The best combined score is at: S = 9, G = 20.\nWhen the predicted secondary structures were viewed on the aligned sequences, although several distinct \u03b1-helices were apparent, these had a less direct correspondence with those expected for a capsid protein (Figure 5). In particular, there is little predicted \u03b1-structure for helix N4 and the alignment around N5 is ambiguous. If the secondary structure prediction were to be believed, this might indicate the introduction of a large insertion, or given that there were less sequences on which to base the predictions, it is possible that the predictions are not as accurate as those obtained for the MLV sequences.\nFigure 5\nFV1 sequence alignment with the sequence of 1e6jP and related sequences. The alignment is displayed twice in different colouring schemes. In the top two panels, the amino half is coloured by predicted secondary structure (red = \u03b1, green = \u03b2) and amino acid type (Taylor, 1997). See the legend to Figure 2 for further details. with the known helices (N1\u2013N5) marked as red lines between (minor helices are orange). The carboxy half of the alignment is displayed in a similar way in the lower panels. The sequence identifiers are given below the alignment in the same order as they are aligned. The mid-line divides the 1e6jP sub-family from the FV1 sub-family.\nN-terminal domain\nThe multiple structure alignment of the four models showed unexpectedly good agreement. As in the MLV models, two regions were aligned in register over all the models. These included most of helix N4 and the following loop region then, after only one shift, the full register was regained for helix N5. The sum-of-F scores for each model indicated that 1eia provided the best consensus, followed by 1e6jP (to which it was most similar). The PSId values were: 9.3, 7.8, 6.6, 15.9 for 1d1dA, 1qrjA, 1eia and 1e6jP, respectively. As the 1e6jP model aligns better on its target by almost 10 PSId, this was considered sufficient to justify its adoption as the preferred model over the 1eia based model. Both models are similar in the core region (1.3 \u00c5 wRMSd) but with the significant differences in the extensive loop regions at the 'top' of the molecules. This structure for the 1e6jA based model is shown along with its comparison with the 1eia model in Figure 6.\nFigure 6\nConsensus model for the FV1 N-terminal domain. (a) The model for the FV1 N-domain based on 1e6jP shown as a \u03b1-carbon trace with predicted \u03b1-helices coloured red (some fragments of predicted \u03b2-structure are coloured green). The molecule is in approximately the same orientation as in Figure 8(a). (b) The models based on 1e6jP and 1eia are shown superposed and coloured from blue (amino) to red (carboxy). Feint dashed lines connect identical residues. The wRMSd = 1.3 (uRMSd = 5.4) over 124 residues. Note: the modelling program rescales the secondary structure prediction values so that there is the same proportion of predicted structure as measured secondary structure on the model.\nIt can be seen from Figure 5 and Figure 6(a) that there is reasonable location of the predicted and model helices except for helix N4 which is almost completely unpredicted. Otherwise deviations occurred at the ends of helices and in the loop regions at the 'top' of the molecule.\nTable 2\nTarget sequences selected for alignment. The \u03a8-BLAST\/QUEST search strategy (Methods Sectn.) when started with the two target sequences indicated by \"SEED\", selected the sequences indicated in each subtable: (a) MVL and (b) FV1. The sequences are identified as in Table 1. (* the C-terminal domain of this sequence appears to be replaced by an oncogene.)\n(a) MLV\nSEED\nAAD55051\n215\u2013432\ngi-120892\nsp-P03330\n207\u2013423\ngi-2393894\ngb-AAC58239\n206\u2013434\ngi-419481\npir-A46312\n199\u2013423\ngi-323873\ngb-AAA43041\n203\u2013418*\ngi-7548235\ngb-AAA4306\n206\u2013422\ngi-5726238\ngb-AAD48375\n156\u2013352\n(b) FV1\ngi-7521942\nT29096\ngi-23485357\ngb-EAA20381.1\ngi-3913713\nsp-P70213\nFV1 MOUSE\nControl Models\nTo help assess the accuracy of the models constructed above, the protocols described in the Methods sections were applied to each of the sequences of known structure. For each protein, this results in four models based on the other structures (and its own structure). Each of these models, was then compared to the known structure using the modified SAP program and the RMSd\/PSId measures reported in Table 3.\nTable 3\nControl model similarity. The similarity of the capsid protein domains are tabulated as: uRMSd\/PSId, both as calculated by the SAP program. The proteins are identified as in Table 4. The column \"params\" gives the MST parameter values S, G at which the four models in each row had maximum agreement as measured by the combined agreement score Fv (Methods Sect n.).\nstr \\ seq\nD1D\nQRJ\nE6J\nEIA\nparams\nD1D\n0.60\/78.9\n4.14\/90.0\n2.05\/56.7\n2.41\/38.3\n9,10\nQRJ\n5.58\/100.\n0.32\/100.\n3.58\/88.4\n5.51\/100.\n3,10\nE6J\n1.88\/95.2\n1.70\/84.7\n0.31\/100.\n1.39\/100.\n7,10\nEIA\n2.00\/93.3\n2.00\/100.\n1.28\/100.\n0.35\/100.\n8,20\nWith few exceptions, the wRMSd\/PSId values indicated generally good models and by visual inspection, all had the correct fold. The models based on their own structures were almost identical to their templates with the exception of 1d1dA. When the uWRMSd values of the models against the known structure are plotted against number of residues ranked by local score (Figure 7) it can be seen that for 1d1dA the error rises sharply after 85 positions. Visual inspection revealed that this was due to a misaligned segment in the large loop region. The majority of models exhibited a gradual increase in wRMSd with increased error mounting as the loop regions were introduced. Two models that ran markedly below this trend were those based on 1eia and 1e6jP (22% identity), the two most similar proteins. A few models were also markedly worse. Two of these were derived from the 1qrj alignment based models which had a distinctly lower weight on the structure component (S weight) in the MST alignment.\nFigure 7\nControl model uRMSd values. Each model was compared with its known structure and the local residue matches calculated by SAP were ranked. The uRMSd was calculated for increasingly larger sets of ranked residues and plotted against set size. This means that the best fitted residues in each comparison are found to the left of the plot with increasing divergence towards the right. Four models are plotted for each of four CA proteins of known structure: 1d1dA = red, 1qrjA = green, 1eia = purple, 1e6jP = blue. The best models are those of sequences built on their own structure. Three of these remain low throughout their length while one has poor loops and rises towards the right. Above these are two models for the most similar sequences of 1e6jP and 1eia built on each others structure (22% identity). Most models lie in the mid range with a few accumulating early errors due to shifts in the core regions.\nConclusions\nBased on relative degrees of sequence similarity among the control models (Table 3(b)) and the MLV and FV1 sequences (MLV:1qrj = 18.5%, FV1:1e6jP = 15.9%), it would be expected that the models constructed for the MLV sequence would fall in the mid-range of the spread in quality observed in Figure 7 \u2013 typically, a good core model (3 \u00c5 RMS over 80 residues) with increasing divergence in the more variable loop regions. This is where the majority of the control models lie which were all constructed from sequence similarities that are generally lower than either of the above relationships used to model the MLV or FV1 sequences.\nWhile a similar confidence might be hoped for the FV1 model, given its overall lower sequence similarity to the proteins of known structure and the less consistent nature of its secondary structure prediction, it is more likely that it will be of lesser quality \u2013 corresponding more to the poorer models constructed among the control proteins (Figure 7). Typically, this would include shifts in the core helices (giving the characteristic immediate rise in the traces in Figure 7) with further shifts in the loop regions. Despite this, as with all of the control models, it is likely that the core fold of the protein should remain unaltered.\nThis study has shown that reasonable models can be constructed for both the FV1 and its target MLV protein based on other retroviral capsid proteins. Although this result was suggested by the existence of the MHR in both sequences, the fluid nature of retroviral genomes does not necessarily constrain the preceding domain to remain constant in structure or even remain at all. Despite only weak sequence similarities in this region, the addition of multiple sequences with predicted secondary structure has allowed plausible models to be constructed.\nThese models can now be used in the interpretation of experimental studies on the mode of action of retroviral susceptibility. As will be reported in more detail elsewhere [29], a series of amino acids in CA affecting the CA \u2013 FV1 interaction have been identified in the loops at the 'top' of the N-terminal domain (Figure 3). Based on the model, they suggest a potential FV1 binding domain in the MLV CA. Experiments are currently under way to test this prediction by crystallographic studies.\nFor many years, the Fv1 gene has been the only known intracellular non-immune natural defense against a retroviral infection. Recently, two additional genes, Ref1 and Lv1, with antiviral activity have been described in human cells [30,31]. Phenotypically, they resemble Fv1 [32] but the genes themselves remain to be characterised. Understanding the mechanism of Fv1 action will provide insights into how natural defences to retroviral infection might be deployed against HIV.\nMethods and Data\nData\nSequence Data\nAll sequences were extracted from (and searches were made over) the non-redundant protein sequence databank (NRDB) at the National Centre for Biotechnology Information (NCBI) as it was found on 28th of January 2003.\nThe sequence of the MLV used was the gag protein AAD55051 (GI:5881091) [10] and a region was extracted from residues 215\u2013432, corresponding to the CA protein.\nThe sequence of the Fv1 gene [7] was taken from FV1_MOUSE (GI:3913713). The region corresponding to the CA was identified as residues 100\/120\u2013340\/360 where the inner numbers represent the probable core of the protein. This range corresponds to the region of highest similarity to the MuERV-L sequence [8]. The leading 100 residues of the polyprotein may correspond with a relic matrix protein. Perhaps because of this, there is no obvious protease cleavage motif [11] to give any indication of the true terminus. However, in other situations this has not always been an accurate guide [12].\nStructural Data\nThe structures of capsid proteins were extracted from the PDB [13] with the aid of the FSSP structure comparison database [14]. Of the six structures in the FSSP alignment, only four extended over the full length of the two structural domains. There were as follows, with their PDB code (and chain delimiter, if any) shown in brackets: Rous Sarcoma Virus (RSV) [1d1dA] [15], Human T-cell Leukemia Virus (HTLV-I) [1qrjA] [16,17], Equine Infectious Anemia Virus (EIAV) p26 [1eia] [18] and Human Immunodeficiency Virus (HIV-I) p24 [1e6jP] [19].\nThe common core of the N-terminal domains of these proteins (in the numbering of the PDB structure) was defined as: 1d1dA 15\u2013148, 1qrjA 16\u2013129, 1eia 16\u2013145 and 1e6jP 16\u2013146. These fragments will be distinguished below as: 1d1dAn, 1qrjAn, 1eia-n and 1e6jPn and each terminates 8 or 9 residues before the conserved glutamine of the MHR. The N-terminal domain can be described as having five \u03b1-helices (N1...N5) with a long 'disordered', partly helical, loop between helices N4 and N5. For ease of reference below, this region will be called the 'top' of the molecule and its representation in the Figures will preserve this orientation.\nThe C-terminal domains were defined as: 1d1dA 152\u2013224, 1qrjA 132\u2013204, 1eia 149\u2013220, 1e6jP 149\u2013220 and were distinguished by the suffix \"c\". The common core of this domain consists of an extended strand leading into the MHR region followed by four helices designated C1...C4.\nDespite their different sizes, both the N and the C domains have the same fold, perhaps suggesting an ancient gene duplication. This is most obvious in the HIV structure [1e6jP] where the domains can be superposed with 4.6 (2.0) unweighted (weighted) RMSd over 68 residues.\nThe similarity of sequence and structure over these domains was calculated using the SAP structure comparison program [20] for each pair of like domains (Table 4). The N-terminal domains have clearly related structures (3\u20136 \u00c5 RMSd) but have no significant sequence similarity (over 20%) except for the EIAV and HIV-1 structures. The smaller C-terminal domain (which contains the MHR) has greater overall sequence and structural similarity compared to the N-terminal domain (mostly over 20% with 2\u20133 \u00c5 RMSd) and over 40% for the EIAV\/HIV-1 pair.\nTable 4\nCapsid protein similarity. The similarity of the capsid protein domains are tabulated as: (a) RMSd (unweighted) and (b) PSId, both as calculated by the SAP program. Upper-right triangle: over the common core of the C-terminal domain and Lower-left triangle: over the common core of the N-terminal domain. The proteins are identified as: D1D = RSV [1d1dA], QRJ = HTLV-I [1qrjA], E6J = HIV-I [1e6jP], EIA = EIAV [1eia ].\n(a) structure\n(b) sequence\nN \\ C\nD1D\nQRJ\nE6J\nEIA\nN \\ C\nD1D\nQRJ\nE6J\nEIA\nD1D\n2.88\n2.64\n2.46\nD1D\n21.9\n22.2\n14.7\nQRJ\n3.83\n2.11\n2.39\nQRJ\n12.5\n30.6\n26.4\nE6J\n6.21\n4.76\n1.40\nE6J\n8.6\n10.8\n41.7\nEIA\n5.75\n4.31\n3.22\nEIA\n11.6\n12.5\n22.0\nSequence Databank Searches\nInitially, each probe sequence was compared against the sequence databank using the \u03a8-BLAST program [21] with a significance level set at 0.001 and 5 cycles of iteration. When the probe is a retroviral sequence, the number of hits found by \u03a8-BLAST can be large (typically over 1000). These were reduced to manageable numbers by the use of the search program QUEST which is similar to \u03a8-BLAST but incorporates a multiple sequence alignment stage in its iterations to exclude redundant sequences as well as excluding poorly related or incomplete sequences [22]. The alignments produced by QUEST typically contain between 6\u201312 sequences (including the probe sequences), none of which have more than 60% sequence identity (PSId) with each other.\nThe sequences retained by QUEST are selected on the basis of associated biological information, with those including useful annotation and structural data being given preference over those with no annotation or keywords such as \"hypothetical\". The filters are part of the MULTAL sequence alignment program [23] which are fully described in Ref. [24].\nSecondary Structure Prediction\nThe multiple alignments resulting from the \u03a8-BLAST\/QUEST search protocol were passed to the program PsiPred , Version 2.3) [25]. This program normally performs its own databank searches using \u03a8-BLAST to build-up an alignment. Given the problems described above that arise when searching with retroviral sequences, the PsiPred program was used locally to search only a database consisting of the sequences that had already been selected by QUEST.\nEach sequence in the alignment was taken in turn and used as a probe against this small local database. As the \u03a8-BLAST parameters used by PsiPred were more restrictive than those used in the full search (only 3 cycles) and there are fewer sequences in the databank, each sequence may only find those to which it is more closely related. This introduces some variation into the predictions which provides a useful indication of the confidence of each predicted secondary structure element (SSE).\nMultiple Sequence Threading\nThe alignment gathered on the probe sequence was then aligned with a protein structure using the multiple sequence threading MST program [26]. This program uses multiple sequence and structural information to automatically construct an \u03b1-carbon molecular model for the probe sequence with some limited remodelling in regions of insertion and deletion.\nTemplate Sequence Alignments\nThe MST program can incorporate multiple aligned sequences along with both the probe sequence and the template structure. The latter were gathered in an identical manner to the probe sequence using the \u03a8-BLAST\/QUEST search protocol described above. Each search against the NRDB was started with the sequence of the protein of known structure and the resulting multiple alignments examined 'by-eye' in the light of the known secondary structures. If any large insert had been made in a secondary structure element (SSE) then it was assessed whether the gap could be shifted outside the SSE without significant loss of residue matches. Similarly, if a large insert (more than 6 residues) was made by any sequence other than the probe sequence (of known structure) then the insert was reduced to six residues by removing the positions with most gaps.\nParameter Choice\nThe MST program has parameters that allow different weights to be attached to the matching contribution of the sequences, their secondary structures, residue exposure and the residue packing in the resulting model. There is also a gap-penalty. The best values for these weights depends on the number and degree of relatedness among both the probe and the template sequences [26]. Rather than vary all these parameters individually, the weights on the structural components (secondary structure, exposure and packing) were 'ganged' together into a single parameter reflecting the contribution of structural terms relative to the sequence matching component. This gave two parameters: S (for structure) and G (the gap-penalty). Previously, the structural parameters had all been scaled into the same range so a value of S = 3 corresponds to a value of 3 for each individual weight. Although the gap-penalty is correlated with S, it cannot be linked in the same way without the risk of missing good alignments.\nIn the current application, there was more than one available template structure and advantage was taken of this by constructing models based on all available templates and choosing the MST parameters such that the agreement among the models was greatest. The parameters were varied over the ranges: S = 0\u21929 (in steps of one) and G = 10\u219290 (in steps of 10).\nMeasuring Model Agreement\nWhatever the parameters for MST, all the models constructed from the same probe have an identical sequence. These might therefore be compared using the \u03b1-carbon RMSd based on a one-to-one (100 PSId) sequence equivalence. However, using this simple measure, a 'trivial' shift in space in which, say, an \u03b1-helix shifts by one turn relative to another \u03b1-helix might result in a large RMSd between what are, topologically, similar models. It is better to allow a local relative shift in sequence of four residues to restore the spatial equivalence at the expense of residue identity.\nTo implement this trade-off between RMSd and PSId, the models constructed for each parameter combination were compared against each other using the program SAP [20]. This program calculates both a weighted (Rw) and unweghted (Ru) RMSd for the two structures being compared and reports the percentage sequence identity of the alignment. The weighted RMSd down-weights regions of weak similarity which are mainly loop regions that can have large relative displacements. Despite its origins [27], in its current implementation the SAP program  does not include a sequence matching component and this was restored (for sequence identity only) by doubling the local residue pair score for identical residue types and otherwise halving all other residue match scores in the initial score matrix.\nA score reflecting match quality (f) was calculated as: f = M\/(1+R), where M is the PSId measured over the positions aligned by SAP and R is one of the RMSd measures. Identical structures would score 100. For a set of N models, a sum was calculated over the (N2 - N)\/2 pair combinations giving an overall measure of agreement (F) among the set. For a set of four models that align perfectly (100 PSId) with 2 \u00c5 RMSd, the overall score obtained would be 200. This score was calculated for both the wRMSd and uRMSd values (giving Fw and Fu, respectively) and a combined score (Fv) as the product of Fw and Fu.\nWhile this procedure provides a general method for choosing parameter values, in the current application to a multi-domain protein it was not meaningful to calculate the RMSd over the full atomic model (because of relative domain movements). Instead, the agreement was calculated over the more distantly related N-terminal domain.\nSelecting a consensus model\nAlthough any model in the set could be taken as a representative, it is best to try and select one that, by some criteria, can be considered to be the most representative. To do this, we compared each pair of models usuing the structure comparison protocol described in the previous section. This provides a pairwise alignment based on structure, and even though each model has an identical sequence, the structural alignment may not match-up identical residues. The pairwise alignment were then combined into a multiple structure alignment [28] and as the models all have an identical sequence, their relative shifts can be seen easily. Rather than use a pure structure or sequence based measure of similarity between the proteins, the score F was devised in the previous section (Methods Sectn.) that combines both a sequence and a structural component. This was used to find the model with the greatest sum-of-scores to the others.\nAn alternative selection test was also considered of selecting the model that had greatest sequence similarity when superposed with the template structure from which it was derived. As most of the sequence similarities considered below lie in the 'twilight-zone', the latter option was only used when one model was clearly better than the others. For this, we choose the criterion that it had to be 10 PSId points clear of its 'rivals'.\nAbbreviations\nFv1\/FV1, gene\/gene-product of Friend Virus susceptibility locus-1;\nMLV, Murine Leukaemia Virus;\nCA, CApsid protein;\nHERV-L, Human Endogenous RetroVirus (L family);\nMuERV-L, Murine Endogenous RetroVirus (L family);\nMHR, Major Homology Region;\nNCBI, National Centre for Biotechnology Information;\nNRDB, Non-Redundant DataBank;\nMST, Multiple Sequence Threading (program);\nSSE, Secondary Structure Element;\nPSId, Percent Sequence Identity;\nPDB, Protein DataBank;\nRMSd, Root-Mean Square deviation;\nwRMSd, weighted Root-Mean Square deviation;\nuRMSd, unweighted Root-Mean Square deviation;\nFigure 8\nCapsid protein domains. The structures of the common core of the capsid proteins are shown using the RSV protein [1d1dA] as a representative. (a) The N-terminal domain, coloured blue\u2192red from amino\u2192carboxy termini with the five major helices represented by thickened lines. (b) The C-terminal domain, represented as in part (a) but with the MHR region marked in white.","keyphrases":["fv1","mlv","virus capsid protein models threading"],"prmu":["P","P","R"]}
{"id":"Cardiovasc_Intervent_Radiol-3-1-2062488","title":"Embolization with the Amplatzer Vascular Plug in TIPS Patients\n","text":"Vessel embolization can be a valuable adjunct procedure in transjugular intrahepatic portosystemic shunt (TIPS). During the creation of a TIPS, embolization of portal vein collaterals supplying esophageal varices may lower the risk of secondary rebleeding. And after creation of a TIPS, closure of the TIPS itself may be indicated if the resulting hepatic encephalopathy severely impairs mental functioning. The Amplatzer Vascular Plug (AVP; AGA Medical, Golden Valley, MN) is well suited for embolization of large-diameter vessels and has been employed in a variety of vascular lesions including congenital arteriovenous shunts. Here we describe the use of the AVP in the context of TIPS to embolize portal vein collaterals (n = 8) or to occlude the TIPS (n = 2).\nPortal vein collaterals supplying esophageal varices may give high risk for secondary rebleeding. Those collaterals might require embolization to decrease this risk, despite transjugular intrahepatic portosystemic shunt (TIPS) placement. Embolization of the collaterals may be particularly difficult when these vessels are large. Moreover, after creation of a TIPS, closure of the TIPS itself may be indicated when hepatic encephalopathy severely impairs mental functioning. The Amplatzer Vascular Plug (AVP; AGA Medical, Golden Valley, MN) is a device well suited to embolize large vessels and has been employed in a variety of vascular lesions such as pulmonary arteriovenous malformations and congenital shunts [1, 2].\nThis report describes the use of the AVP in the context of TIPS to embolize portal vein collaterals (n\u00a0=\u00a08) or to occlude the TIPS (n\u00a0=\u00a02).\nDescription of Procedures\nEmbolization of Portal Vein Collaterals\nEight patients were referred for TIPS, seven because of multiple recurrent bleeding episodes from esophageal varices (n\u00a0=\u00a05), gastric fundus varices (n\u00a0=\u00a01), or both (n\u00a0=\u00a01) that could not be managed by endoscopic therapy. One other patient (n\u00a0=\u00a01) was in hypovolemic shock because of recurrent massive bleeding from rectal hemorrhoids. Three TIPS procedures were elective and five were done as emergency procedures for uncontrolled bleeding after initial hemodynamic stabilization had been achieved using blood transfusions, endoscopic treatment, intravenous octreotide infusion, and\/or placement of a Sengstaken double balloon tube, alone or in combination. TIPS procedures were carried out under general anesthesia.\nAs an adjunct procedure, we obliterated the portal vein collaterals supplying the bleeding varices using AVP embolization (Figs.\u00a01 and 2). This was done either before or after creation of the TIPS based on operator preference and clinical circumstances. Embolization early in the procedure with early control of the potential bleeding site was considered relevant in emergency procedures in hemodynamically unstable patients. Also, at least theoretically, AVP embolization may be done more easily prior to TIPS whenever the collateral target vein is still widened because of portal hypertension and easily opacified because of preferential flow. After the creation of a TIPS both these phenomena may have been altered.\nFig.\u00a01Portal venous angiogram in a 53-year-old patient referred for TIPS creation because of recurrent variceal bleeding not controlled by medication plus multiple treatments with endoscopic band ligation and sclerotherapy. The varices are supplied by a single coronary veinFig.\u00a02The same patient as in Fig. 1 immediately following TIPS creation and AVP embolization of the coronary vein (a; thin arrow). The unsubtracted image (b) shows the radiopaque sclerosing agent from prior endoscopic sclerotherapy (fat arrow) in the esophageal varices\nWhenever AVP embolization preceded TIPS placement, the guiding catheter for AVP delivery was introduced transhepatically into the right portal vein. At our institution the portal vein is routinely catheterized prior to TIPS with a 3- or 4-Fr catheter to visualize the portal vein as a fluoroscopic target for transjugular transhepatic puncture. For transhepatic AVP placement this catheter was exchanged for a 6- to 8-Fr hepatic introducer sheath. After AVP embolization and initial creation of the hepatic-to-portal vein shunt tract for TIPS, the hepatic introducer sheath was withdrawn in multiple small successive steps over the course of the remainder of the procedure, so that a solid sealing clot could form in the hepatic parenchymal tract. No additional embolization of the tract was performed. Whenever vein embolization was performed after the creation of a TIPS, AVP placement was done via the transjugular route.\nIn all patients, the large-diameter coronary\/gastric vein supplying extensive esophageal\/gastric varices was embolized using a single AVP plug 10, 12, or 14 mm in diameter. For placement, a 6- to 8-Fr guiding catheter was first negotiated into the proximal part of the coronary vein. Then the AVP device was advanced through the guiding catheter and initially deployed by retracting the guiding catheter but without detaching the device. A control injection was made, and if needed, the AVP was retrieved into the guiding catheter and repositioned. When in a satisfactory position, the AVP was detached by anticlockwise rotation of the AVP guide wire to unscrew it from the AVP device proper. Within minutes of deployment, the collateral flow through the coronary vein had completely stopped. TIPS creation was done by placement of e-PTFE-covered stent-grafts (Viatorr; Gore, Flagstaff, AZ) 8\u201310 mm in diameter and 6\u00a0\u00b1\u00a02 cm long, resulting in a postprocedural portosystemic pressure difference of 5\u201310 mm Hg. All patients were hemodynamically stable immediately after the procedure.\nDuring follow-up there were no recorded episodes of rebleeding with a follow-up of 1 year maximum. The patient who was treated for bleeding rectal hemorrhoids died after 3 days because of combined liver and respiratory failure. Another patient who received emergency TIPS while in hypovolemic shock due to massive unstoppable bleeding from esophageal varices died 46 days after the procedure because of liver and kidney failure complicated by ileus.\nTIPS Occlusion\nA 47-year-old male was referred for closure of a TIPS that had been created 3 years earlier for alcoholic Child A liver cirrhosis with recurrent episodes of esophagus variceal bleeding. He now presented with problems of amnesia, difficulties concentrating, and a general, overwhelming feeling of tiredness. Spectral EEG findings were consistent with grade 2 hepatic encephalopathy. The symptoms had not improved using conservative treatment. At angiography, a patent TIPS was seen, with a portosystemic pressure difference of 10 mm Hg. Closure of the TIPS with a 14-mm AVP device (Fig.\u00a03) resulted in a dramatic improvement in cognitive functioning and normalization of the spectral EEG. Two years after closure of the TIPS, the patient is still free of variceal bleeding. Recently, however, he developed esophageal varices grades I\u2013II, which are currently treated with band ligation. This will be repeated every 2 weeks until complete obliteration.\nFig.\u00a03AVP device for TIPS occlusion in a patient with life-incapacitating hepatic encephalopathy. The 14-mm-diameter AVP is positioned through a guiding catheter in the central portion of the 10-mm-diameter e-PTFE stent-graft (a). By retraction of the guide wire, the unconstrained AVP sets in the intended place (b). At this point, the AVP can still be retracted into the guiding catheter if in an unsatisfactory position. The AVP is fully deployed by unscrewing the guide wire from the AVP device proper (c)\nAnother patient, a 65-year-old male, had been referred for TIPS placement for refractory ascites following liver transplantation, which required weekly paracentesis. In the days following placement of a 10-mm-diameter e-PTFE-covered stent-graft TIPS, liver failure developed, necessitating its closure. Closure of the TIPS was technically successfully achieved using a 16-mm-diameter AVP but did not substantially improve the clinical status. Because of liver failure and refractory ascites, the patient underwent a second liver transplantation 2 days after TIPS occlusion.\nDiscussion\nIn this report we have described the use of AVP embolization in the context of TIPS. For closure of collateral veins supplying bleeding varices, we consider the AVP a useful device in a defined subset of patients. The AVP\u2019s relative advantages are its efficiency to occlude large-diameter vessels in a single-step procedure and its reliability of placement. Unlike standard embolization coils, the AVP can be positioned precisely, its position can be checked with contrast injections before actual detachment, and it can be repositioned if required. Migration of the AVP during or after deployment is unlikely. Disadvantages of the AVP include the higher cost of the device itself and of the additionally required guiding catheter. Furthermore, the 6- to 8-Fr guiding catheter markedly increases the bulk of the introducer system relative to the 4- to 5-Fr catheter alternatively used for coil embolization. In our practice we are inclined to use the AVP whenever there is a single, large-diameter collateral vein which would otherwise require the insertions of multiple embolization coils and\/or whenever accurate placement of the embolic device close to the portal vein may be critical.\nEmbolization of portal vein collaterals to esophageal varices may be indicated even after successful portal venous decompression by creation of a TIPS. Especially in the life-threatening emergency situation of acute esophageal bleeding in hemodynamically unstable patients, we feel more confident providing additional control of the bleeding site by hemodynamically separating the weak-walled varices from the high-flow and potentially high-pressure portal venous bed. TIPS has proven efficient in the secondary prevention of variceal bleeding but this protection is not absolute. The rebleeding rate of TIPS alone (without embolization) has been reported as between 9% and 40.6%, with a median of 18.4% [3]. These values apply to bare-stented TIPS and initial experience suggests that the rebleeding rate is lower with e-PTFE-covered stented TIPS [4]. Whether or not performing embolization of the portal vein collaterals as an adjunct procedure provides additional protection against rebleeding is unknown. Under the assumption that it does, we routinely combine TIPS with embolization of all venous collaterals supplying the esophageal varices when creating a TIPS in the context of variceal bleeding.\nAnother potential use for the AVP device is for closure of the TIPS itself, in circumstances where hepatic encephalopathy and\/or liver failure because of the shunt may prove to be life-incapacitating. Here, the effectiveness of the AVP in occluding large-diameter high-flow vessels, its resistance to migration, and its ease of placement are particularly helpful.\nA potential concern when occluding a TIPS is the acute increase in portal venous pressure, which can lead to recurrence of variceal bleeding and ascites and, also, to hemodynamic changes with increased cardiac loading [5].\nAs mentioned, we routinely place a transhepatic catheter into the portal vein as a target for puncturing the portal vein during TIPS creation. In our experience, this increases the control over the exact entrance point into the portal vein and also reduces the number of puncture attempts while creating the TIPS tract. We have found that the reduced procedure time for the TIPS procedure proper more than makes up for the extra time it takes to insert this initial transhepatic catheter into the portal vein. We have encountered no complications because of this approach in more than 100 TIPS procedures.\nTo our knowledge, this is the second reported use of the AVP in the portal venous system. In a recent case report, Kessler and Trerotola described a patient with gastric varices and TIPS in whom they used the AVP device for transhepatic obliteration of the portal vein collateral supplying the varices [6], a patient similar to one of our patients with flow-mediated gastric varices. We would like to emphasize that the AVP may be considered also for embolization of the more common collateral veins to esophageal varices and, if need be, the TIPS stent itself.","keyphrases":["embolization","amplatzer vascular plug","transjugular intrahepatic portosystemic shunt","varices","interventional radiology"],"prmu":["P","P","P","P","U"]}
{"id":"Cult_Med_Psychiatry-2-2-1705533","title":"Making muslim babies: Ivf and gamete donation in sunni versus shi\u2019a islam\n","text":"Medical anthropological research on science, biotechnology, and religion has focused on the \u201clocal moral worlds\u201d of men and women as they make difficult decisions regarding their health and the beginnings and endings of human life. This paper focuses on the local moral worlds of infertile Muslims as they attempt to make, in the religiously correct fashion, Muslim babies at in vitro fertilization (IVF) clinics in Egypt and Lebanon. As early as 1980, authoritative fatwas issued from Egypt\u2019s famed Al-Azhar University suggested that IVF and similar technologies are permissible as long as they do not involve any form of third-party donation (of sperm, eggs, embryos, or uteruses). Since the late 1990s, however, divergences in opinion over third-party gamete donation have occurred between Sunni and Shi\u2019ite Muslims, with Iran\u2019s leading ayatollah permitting gamete donation under certain conditions. This Iranian fatwa has had profound implications for the country of Lebanon, where a Shi\u2019ite majority also seeks IVF services. Based on three periods of ethnographic research in Egyptian and Lebanese IVF clinics, this paper explores official and unofficial religious discourses surrounding the practice of IVF and third-party donation in the Muslim world, as well as the gender implications of gamete donation for Muslim marriages.\nIntroduction: Medical anthropology and science and technology studies\nIn recent years, medical anthropologists have been turning their ethnographic attention to issues of science and technology, including how science is produced, where it is produced, by whom, and when. Anthropologists have also highlighted how scientific and technological advancements spread around the world, gradually being incorporated into the lives of ordinary people in many global sites. In order to document the production and reproduction of new scientific and technological discoveries, medical anthropologists have begun to take their ethnographic research projects into previously unexplored terrains, including hospitals, scientific research laboratories, clinical consultation rooms, and other \u201cbehind the scenes\u201d places where the culture of science and technology is perhaps best revealed. Furthermore, most of the medical anthropologists working in this field have documented the ways in which the scientific and the divine seem to have intermingled, not only among the patients who turn to religious theodicies to make sense of their suffering, but also among the scientists and clinicians who deliver medical services and attempt to offer patients hope. These recent medical anthropological efforts to document the intersections of science, technology, and religion are clearly reflected in the theme of the 2004 annual meeting of the American Anthropological Association: \u201cMagic, Science, and Religion.\u201d Furthermore, this special issue of Culture, Medicine and Psychiatry reflects the ongoing importance of religion to science, medicine, and biotechnology, including in the Muslim world.\nMedical anthropology has much to offer to discussions of science, medicine, and religion, as demonstrated in a number of award-winning books on these subjects. To take but a few recent examples, in Testing Women, Testing the Fetus: The Social Impact of Amniocentesis in America, medical anthropologist Rayna Rapp (1999) reveals the efforts of more than ten years\u2019 worth of extensive ethnographic research conducted at New York City hospitals, genetic counseling and testing centers, and genetics laboratories themselves. Her book focuses on the difficult decision-making of pregnant women from multiple ethnic, religious, and economic backgrounds, who are asked by clinicians and advised by genetic counselors to undergo amniocentesis in order to detect genetic anomalies in their fetuses. Although the scientific field of genetics is burgeoning with excitement, Rapp\u2019s intent in Testing Women, Testing the Fetus is to show how women who are expected to use new genetic tests during pregnancy are put in the difficult position of being \u201cmoral pioneers:\u201d namely, they are forced to make often heart-wrenching moral decisions about what constitutes an acceptable human life.\nWhereas Rapp\u2019s book focuses on decisions about bringing life into the world, a book by medical anthropologist Margaret Lock asks us to consider decisions about ending lives considered \u201cbrain dead.\u201d Twice Dead: Organ Transplants and the Reinvention of Death, Lock\u2019s (2002) ethnography, examines the history of organ transplantation in North America over the past 20\u00a0years. Scientific advancements in transplant technology have made it possible for North American surgeons to perform thousands of transplants each year. In the majority of these cases, individuals diagnosed as \u201cbrain dead\u201d are the source of the organs, without which transplants could not take place. Lock asks her readers to consider what is meant by \u201cbrain death,\u201d and how it might be viewed differently in another culture, namely, Japan. Despite the availability of the necessary technology and expertise, Japanese society has not accepted the notion of brain death as a sufficient criterion of death, following a well-informed but heated public debate on the topic. Although brain death was legally recognized in Japan in 1997, it is authorized only in limited circumstances, meaning that organ transplantation in Japan is not widespread. Drawing on extensive interviews conducted over ten years with Intensive Care Unit (ICU) physicians, transplant surgeons, organ recipients, donor families, Japanese political activists opposed to the recognition of brain death, and members of the general public in both Japan and North America, Lock shows us that death itself is not a self-evident, biological event. Rather, it is surrounded by morally troubling cultural, medical, legal, and political dimensions.\nLock\u2019s book clearly demonstrates the degree to which medical anthropology is concerned with comparative cultural perspectives, particularly in an era of rapid globalization. Indeed, medical anthropology as a discipline can be said to focus on the \u201clocal in the global.\u201d In other words, local considerations, be they cultural, social, economic, or political, shape and sometimes curtail the way that Western-generated scientific technologies are both offered to and received by non-Western subjects. New forms of science and technology are not transferred into cultural voids when they reach places like Japan. Thus, the assumption on the part of global producer nations that new biotechnologies\u2014as value-free, inherently beneficial medical technologies\u2014are \u201cimmune\u201d to culture and can thus be appropriately transferred and implemented anywhere and everywhere is subject to challenge once local formulations, perceptions, and actual consumption of these technologies are taken into consideration (Inhorn 2003a).\nIndeed, the global spread of biotechnologies provides a particularly salient but little discussed example of what anthropologist Arjun Appadurai (1996) has termed a \u201ctechnoscape,\u201d or the \u201cglobal configuration, also ever fluid, of technology, and the fact that technology, both high and low, both mechanical and informational, now moves at high speeds across various kinds of previously impervious boundaries.\u201d Appadurai reminds us that this movement of technologies around the globe is both a deeply historical and an inherently localizing process. In other words, globalization is not enacted in a uniform manner around the world, nor is it simply culturally homogenizing\u2014necessarily \u201cWesternizing\u201d or even \u201cAmericanizing\u201d in its effects. The global is always imbued with local meaning, such that local actors, living their everyday lives at particular historical moments in particular places, mold the very form that global processes take (Freeman 2000).\nThis acknowledgment of the importance of locality in the global dispersion of modern biotechnologies has been a theme of much recent work in medical anthropology. Similarly, much of this anthropological concern with locality has to do with local moral systems, or what medical anthropologist\/psychiatrist Arthur Kleinman (1995) has called \u201clocal moral worlds\u201d of pain and suffering. According to Kleinman (45), local moral worlds are \u201cmoral accounts, [which] are the commitments of social participants in a local world about what is at stake in everyday experience.\u201d Through an \u201cethnography of experience,\u201d Kleinman urges medical anthropologists to pay close attention to the moral issues that may accompany the arrival of new biotechnologies around the globe. In his recent powerful and path-breaking book, What Really Matters: Living a Moral Life amidst Uncertainty and Danger, Kleinman (2006) asks us to carefully consider what it really means to live a \u201cmoral life,\u201d particularly in the midst of life-or-death decision-making.\nThis focus on the \u201clocal moral\u201d is found in another award-winning book on the topic of IVF. Titled Reproducing Jews: A Cultural Account of Assisted Conception in Israel, this book by medical anthropologist Susan Martha Kahn (2000; see also her article in this special issue) takes us into the often arcane world of Jewish Halakhic law, where male rabbis legislate on the appropriate uses of IVF for their followers. Kahn carefully describes how these rabbinical debates and decisions affect the actual practice of Israeli IVF. For example, third-party donation of gametes, including sperm donation, is allowed, since Jewishness is seen to be conferred through the mother\u2019s side, particularly through the act of gestating and birthing the baby. However, most conservative rabbis prefer that non-Jewish donor sperm be used, to prevent adultery between a Jewish man and a Jewish woman and to prevent future genetic incest among the offspring of anonymous donors in this small, intermarrying country. Furthermore, debates have revolved around whether surrogacy should be allowed for infertile couples, using single or married surrogates. Generally speaking, single Jewish women are preferred as surrogates, both to avoid the implications of adultery for married surrogate women and to confer Jewishness through a Jewish woman\u2019s gestation of the fetus. Finally, because the Jewish state is pronatalist\u2014with the state subsidizing up to six cycles of IVF or up to the birth of two IVF children for any given Jewish patient\u2014rabbis have generally been permissive when it comes to single career women, as well as lesbian Jewish mothers, conceiving children through assisted conceptive means.\nKahn\u2019s fascinating and frankly funny book details the sometimes dizzying rabbinical arguments regarding morally appropriate and inappropriate reproduction. In so doing, the book bespeaks the importance of local religious moralities in the contemporary world of Israeli assisted conception. There, doctors in many clinics serving orthodox Jewish patient populations attempt to practice IVF according to the moral dictates set forth by religiously conservative rabbis. The IVF laboratories in these clinics are full of orthodox women called maschigots, who literally peer over the shoulders of laboratory technicians to make sure that the correct sperm and correct eggs are being united\u2014so as not to produce a mamzer, or an illegitimate child. In her book, Kahn is explicit in stating that the American consumer model of free-market reproductive medicine has yet to take hold in Israel, with its concern over religious guidelines. Nonetheless, Israel\u2019s relative permissiveness over the use of donor gametes, surrogacy, and single and lesbian motherhood stands in stark contrast to the Muslim Middle East, including both neighboring Egypt and Lebanon, where I have conducted my own ethnographic research on IVF.\nIvf in the muslim middle east\nWith this background in mind, it is my goal in this article to take readers into the heart of the Muslim Middle East, thereby showing how the practice of IVF in Israel, the country located between Egypt and Lebanon, differs significantly from that in both of its Muslim neighbors. During more than two years of medical anthropological fieldwork in Egypt (1988\u201389, 1996) and Lebanon (2003), I conducted in-depth, ethnographic interviews with infertile Muslim IVF patients, both husbands and wives, now totaling nearly 500 patient couples. Furthermore, since the mid-1990s, I have focused my research on the rapid expansion of IVF technologies into this region of the Muslim world. Indeed, it is fair to assert that since the birth in 1978 of Louise Brown, the world\u2019s first test-tube baby, IVF has spread around the globe, reaching countries far from the technology-producing nations of the West. Perhaps nowhere is this globalization process more evident than in the 22 nations of the Muslim Middle East, where a private IVF industry is flourishing. As of 2003, Egypt alone (population 70 million) boasted 50 IVF centers, and the tiny country of Lebanon (population 4 million) boasted more than 15 IVF centers, one of the highest per capita concentrations in the world. These global metrics are impressive compared to the IVF technology-producing nation of Israel, where 24 IVF centers cater to a population of 6 million (Kahn 2000).\nIn both Egypt and Lebanon, as in other Muslim countries, nonbinding but authoritative Islamic religious proclamations called fatwas have profoundly affected the practice of IVF in ways that are not commonly seen in the West. Indeed, in the Muslim world, infertile couples are usually extremely concerned about making their test-tube babies in the religiously correct fashion. To that end, they seek out the \u201cofficial\u201d Islamic opinion on the practice of IVF in the form of a fatwa. In recent years, many such fatwas on a wide variety of reproductive health issues have been issued in Egypt and other Muslim countries (Lane and Rubinstein 1991; Zuhur 1992). But as we shall see in this paper, major divergences have occurred between Sunni and Shi\u2019ite religious authorities regarding the permissibility of third-party gamete donation, with new fatwas emerging from the Shi\u2019ite world in recent years. It is the differences between Sunni and Shi\u2019a Islam with regard to third-party gamete donation that constitute the focus of this article.\nSunni Islam and IVF\nIVF was first practiced in the Sunni Muslim world, with clinics opening in the mid-1980s in Egypt, Saudi Arabia, and Jordan, all Sunni-majority countries. The Grand Shaikh of Egypt\u2019s famed religious university, Al Azhar, issued the first widely authoritative fatwa on medically assisted reproduction on March 23, 1980. This fatwa\u2014issued only two years after the birth of the first IVF baby in England but a full six years before the opening of Egypt\u2019s first IVF center\u2014has proved to be truly enduring in all its main points (Inhorn 2006a). In fact, the basic tenets of the original Al-Azhar fatwa on IVF have been upheld by other fatwas issued since 1980 and have achieved wide acceptance throughout the Sunni Muslim world. Sunni Islam, it must be emphasized, is the dominant form of Islam found in the Middle Eastern region and throughout the Muslim world. Between 80 and 90 percent of the world\u2019s Muslims are Sunni, and more than 90 percent of Egypt\u2019s citizens are Sunni Muslims, the rest being predominantly Coptic Christian.\nThe degree to which these official Sunni Islamic fatwas on IVF have affected the actual practices of the Middle Eastern medical profession is also quite striking. For physicians, the dominant Sunni religious opinion on IVF has been made known to the Middle Eastern medical community through the writings of Gamal I. Serour, one of three founding members of the first Egyptian IVF center and the director of Al-Azhar\u2019s International Islamic Center for Population Studies and Research. In article after article (Serour 1992, 1994, 1996; Serour and Omran 1992; Serour, El Ghar, and Mansour 1990, 1991; Serour, Aboulghar, and Mansour 1995), Serour has spelled out the main points of the Sunni Islamic position on medically assisted conception, as follows: Artificial insemination with the husband\u2019ssemen is allowed, and the resulting child is the legal offspring of the couple.In vitro fertilization of an egg from the wife with the sperm of her husband followed by the transfer of the fertilized embryo(s) back to the uterus of the wife is allowed, provided that the procedure is indicated for a medical reason and is carried out by an expert physician.No third party should intrude into the marital functions of sex and procreation, because marriage is a contract between the wife and husband during the span of their marriage. This means that a third party donor is not allowed, whether he or she is providing sperm, eggs, embryos, or a uterus. The use of a third party is tantamount to zina, or adultery.Adoption of a donor child from an illegitimate form of medically assisted conception is not allowed. The child who results from a forbidden method belongs to the mother who delivered him\/her. He or she is considered to be a laqit, or an illegitimate child.If the marriage contract has come to an end because of divorce or death of the husband, medically assisted conception cannot be performed on the ex-wife even if the sperm comes from the former husband.An excess number of fertilized embryos can be preserved by cryopreservation. The frozen embryos are the property of the couple alone and may be transferred to the same wife in a successive cycle, but only during the duration of the marriage contract.Multifetal pregnancy reduction (or so-called selective abortion) is only allowed if the prospect of carrying a high-order pregnancy (i.e., twins, triplets, or more) to viability is very small. It is also allowed if the health or life of the mother is in jeopardy.All forms of surrogacy are forbidden.Establishment of sperm banks is strictly forbidden, for such a practice threatens the existence of the family and the \u201crace\u201d and should be prevented.The physician is the only qualified person to practice medically assisted conception in all its permitted varieties. If he performs any of the forbidden techniques, he is guilty, his earnings are forbidden, and he must be stopped from his morally illicit practice.\nBut to what degree are these fatwa declarations\u2014particularly the explicit prohibition on any form of third-party donation of reproductive materials\u2014actually followed by physicians in the Muslim world? A global survey of sperm donation among assisted reproductive technology centers in 62 countries provides some indication of the degree of convergence between official discourse and actual practice (Meirow and Schenker 1997). In all of the Muslim countries surveyed in the mid-1990s\u2014including the Middle Eastern countries of Egypt, Iran, Kuwait, Jordan, Lebanon, Morocco, Qatar, and Turkey, as well as a number of non-Middle Eastern Muslim countries, including Indonesia, Malaysia, and Pakistan\u2014sperm donation in IVF and all other forms of gamete donation were strictly prohibited. As the authors of this global survey, Meirow and Schenker (1997), state, \u201cIn many Islamic countries, where the laws of Islam are the laws of the state, donation of sperm was not practiced. AID [Artificial Insemination, Donor] is considered adultery and leads to confusion regarding the lines of genealogy, whose purity is of prime importance in Islam\u201d (134).\nIn summary, in the Sunni-majority countries of the Middle East and the rest of the Sunni Muslim world, prohibitions against gamete donation have been enacted either in law or in professional medical codes of ethics. As a result, gamete donation is not legally practiced in the Sunni Muslim world, with clinics turning away (or referring to other European countries) couples who require these services. In Sunni Muslim countries such as Egypt, the thought of using donor gametes is reprehensible to most infertile Muslim (and even Christian) couples, who agree completely with the bans on third-party donation dictated by the religion and upheld through medical codes of ethics (Inhorn 2003b). As shown in the next section, however, the situation is changing for Shi\u2019ite Muslims, whose leading cleric has taken a bold step in a new direction.\nShi\u2019a Islam and IVF\nFor Shi\u2019ite Muslims, attitudes toward gamete donation have changed considerably since the late 1990s. Shi\u2019a is the minority branch of Islam found in Iran, parts of Iraq, Lebanon, Bahrain, Syria, and Saudi Arabia, as well as Afghanistan, Pakistan, and India. It has been much in the news lately because of the United States\u2013led war in Iraq, the conflict between Lebanon and Israel, and the current tensions between the United States and Iran. Until recently, most Shi\u2019ite religious authorities have supported the majority Sunni view: namely, they have agreed with Sunni clerics who say that third-party donation should be strictly prohibited.\nIn the late 1990s, however, the Supreme Jurisprudent of the Shi\u2019a branch of Islam, Ayatollah Ali Hussein Khamanei, the handpicked successor to Iran\u2019s Ayatollah Khomeini, issued a fatwa effectively permitting donor technologies to be used. This fatwa has proved to be very significant for those Shi\u2019a who follow the lead of Ayatollah Khamanei in Iran. This would include Lebanon\u2019s Hizbullah leaders, who consider Ayatollah Khamanei to be their marja\u2019 taqlid, or spiritual reference (literally, source of emulation).\nWith regard to egg donation, Ayatollah Khamanei stated in his initial fatwa that egg donation \u201cis not in and of itself legally forbidden.\u201d But he stated that both the egg donor and the infertile mother must abide by the religious codes regarding parenting. Thus, the child of the egg donor has the right to inherit from her, as the infertile woman who received the eggs is considered to be like an adoptive mother.\nWith regard to sperm donation, Ayatollah Khamanei said in his original fatwa that the baby born of sperm donation will follow the name of the infertile father rather than the sperm donor. However, as with egg donation, the donor child can only inherit from his biological father, the sperm donor, since the infertile father is considered to be like an adoptive father.\nThe situation for Shi\u2019ite Muslims is actually much more complicated than this, however, given two Shi\u2019ite religious practices called ijtihad and mutca. Unlike Sunni Muslim scholars who are scripturally based in their thinking, Shi\u2019ite religious authorities give precedence to a form of individual religious reasoning known as ijtihad. Through the use of caql, or intellectual reasoning, various Shi\u2019ite ulama have come to their own conclusions regarding the rightness or wrongness of gamete donation. Some Shi\u2019ite clerics continue to prohibit gamete donation for their followers, while others have allowed it under certain conditions. As many scholars of Shi\u2019a have noted (Cole 2002; Tober 2004), the practice of ijtihad has allowed a certain flexibility and pragmatism toward new technological developments, including IVF and a number of other new medical technologies (e.g., contraception, organ transplants, transgender surgery). Furthermore, ijtihad has ultimately led to great heterogeneity of opinion and practice within the Shi\u2019ite community.\nAdditionally, Shi\u2019a Islam allows a form of temporary marriage called mutca (also called sigheh in Iran), which is not recognized by Sunni religious authorities (Zuhur 1992). In Shi\u2019a Islam, mutca is a union between an unmarried Muslim woman and a married or unmarried Muslim man, which is contracted for a fixed time period in return for a set amount of money. It is practiced in Iran (Haeri 1989), as well as in other parts of the Shi\u2019ite world. In the past, middle-aged and older women who were divorced or widowed often engaged in mutca marriages for financial support. In Iran, following the loss of men during the devastating, eight-year Iran\u2013Iraq war, former Iranian President Rafsanjani recommended mutca as a means of protecting the large numbers of single or widowed women who had no other source of income. For Shi\u2019ite men, mutca marriages could be contracted while traveling, or as a way of achieving marital variety and sexual pleasure (Haeri 1989). Since the arrival of donor technologies, however, mutca has also been invoked to make egg donation legal within the parameters of marriage.\nWithin this context of ijtihad and mutca, Shi\u2019a religious authorities who now accept the idea of donation, but are strict in their interpretation of how donation should be practiced, argue that when a couple needs a donor, they should go to a Shi\u2019ite religious court, where a decision can be made on a case-by-case basis;there should be a determination about which religious \u201creference\u201d (i.e., source of spiritual emulation) the infertile couple follows;the decision should be made in the presence of witnesses, the IVF doctor, and with the agreement of both parties (the infertile couple and the donor);the husband should do a mutca marriage with the egg donor for the period of time in which the whole procedure (egg retrieval to embryo transfer) is taking place, because polygyny is legal in Islam and avoids the implications of zina, or adultery;but because a married Shi\u2019ite Muslim woman cannot marry another man other than her husband (since polyandry is illegal in Islam), she cannot do a mutca marriage with a sperm donor. Technically, the child born of a sperm donor would be a laqit, or out-of-wedlock child, without a family name and without a father. Thus, in theory, only widowed or otherwise single women should be able to accept donor sperm, in order to avoid the implications of zina. However, in the Muslim countries, single motherhood of a donor child is unlikely to be socially acceptable (Inhorn 1996; Zuhur 1992).\nBe that as it may, divergent gamete donation practices are beginning to emerge in the Shi\u2019ite Muslim world, as religious authorities come to their own conclusions about third-party donation. Among Shi\u2019ite religious scholars, the major disagreements, or religious \u201csticking points,\u201d revolve around the following set of issues: whether sperm donation should be allowed at all;whether the child should follow the name of the infertile father or the sperm donor;whether the child should inherit from the infertile father or the sperm donor;whether donor children and their \u201csocial\u201d parents are related at all, and, if not, whether they could potentially marry each other, which has implications for proper comportment in domestic life (e.g., bathing, veiling, etc.);1whether donation is permissible at all if the donors are anonymous;whether a financial transaction should be allowed between gamete donors and recipients;whether the husband of an infertile woman needs to do a temporary mutca marriage with the egg donor, then divorce her after the embryo transfer (48 to 72 hours later), in order to avoid zina. For his part, Khamanei clearly stipulates that mutca marriage is not required, for he believes that zina requires the physical act of intercourse (Clarke 2006); andwhether the wife of an infertile husband can temporarily divorce her infertile husband, remarrying him after accepting sperm from a donor (Tremayne, personal communication, 5\/5\/06).\nThese disagreements of opinion have played out in interesting ways. As shown in anthropologist Morgan Clarke\u2019s (2006) recent research on the Shi\u2019ite religious discourses surrounding gamete donation, many Shi\u2019ite ulama do not agree with Khamanei\u2019s permissive fatwa on donor technologies, because they do not regard him as a brilliant legal thinker. For example, Ayatollah Muhammad Husayn Fadlallah, Lebanon\u2019s most prominent Shi\u2019ite religious figure, does not agree with Ayatollah Khamanei\u2019s permission of sperm donation, although he agrees with the permission of egg donation. Neither of them requires the use of mutca marriages to solve the zina issue (Clarke 2006).\nAyatollah Fadlallah\u2019s positions opposing sperm donation but supporting egg donation square with the dominant religious discourse in Iran. There, the religious rulings regarding gamete donation have evolved quickly, with sperm donation now effectively banned (Soraya Tremayne, personal communication, 23 July 2004, 31 July 2004).2 Namely, a law on gamete donation passed in 2003 in the Iranian parliament (majlis) and approved by the Guardian Council (i.e., a religious \u201cwatchdog\u201d body that endorses every bill before it becomes law) has restricted gamete donation to married persons. Even though the law is brief (less than one page), it states clearly and succinctly who can and cannot donate and receive gametes. Egg donation is allowed, as long as the husband marries the egg donor temporarily\u2014thereby ensuring that all three parties are married. Sperm donation, on the other hand, is legally forbidden, because a sperm donor cannot temporarily marry an already married woman whose husband is infertile. Quite interestingly, however, embryo donation\u2014which involves both sperm and egg from another couple\u2014is allowed in order to overcome both male and female infertility. Because an embryo comes from a married couple and is given to another married couple, it is considered hallal, or religiously permissible.\nThe social and biological implications of embryo donation are quite interesting. For Iranian couples unable to produce a child because of male infertility, embryo donation allows them to bypass the problem of the husband\u2019s weak (or absent) sperm. However, embryo donation does not allow a presumably fertile wife of an infertile husband to contribute her own ova, in effect severing her biological ties to the donor child. Furthermore, and most strikingly, embryos donated from another married couple involve both egg and sperm donation. Even though direct sperm donation is bypassed via the injection of another man\u2019s sperm into the wife\u2019s womb\/ova, embryo donation still disrupts male paternity and involves the acceptance by an already married woman of another man\u2019s (and woman\u2019s) gametes. Moreover, a woman\u2019s acceptance of another woman\u2019s egg is effectively like gestational surrogacy, which is strictly prohibited in Sunni Islam. Indeed, in recent months, cases of surrogate motherhood have occurred in Iran, despite the lack of firm legislation regarding this practice.\nWhether these problematic complications of embryo donation have been carefully thought through by the religious and legal authorities in Iran is unclear. Based on her path-breaking research carried out at Iranian IVF clinics, anthropologist Soraya Tremayne (2005, 2006) notes, \u201cMy first and possibly superficial interpretation at this stage can only be that the moral, ethical and legal aspects of the whole matter have not yet been thought through and the consequences dawned on either the doctors and legislators, or on people themselves. It seems to me that ethics will follow, rather than lead the issue. These operations have been in place only for the past ten years ... and the cases are too new to cause problems as yet\u201d (personal communication, 23 July 2004).\nIt is interesting to note that many Shi\u2019ite religious leaders both inside and outside of Iran do not agree with the relative Iranian \u201cpermissiveness\u201d vis-\u00e0-vis donor technologies; instead, they abide by the dominant Sunni Muslim ban on all forms of third-party donation. For example, I recently attended a two-day conference in Tehran, Iran, on \u201cGamete and Embryo Donation,\u201d sponsored by the Avesina Research Institute in association with the Law and Political Science Faculty of the University of Tehran.3 The conference provided a fascinating example of \u201cijtihad in action,\u201d with some ulama, dressed in their stately robes and turbans (black for the sayyids, or descendants of the Prophet Muhammad), arguing against the moral permissibility of embryo and gamete donation. The disagreements generated in public between \u201cpro\u201d and \u201ccon\u201d ulama were also debated in the more private recesses of the conference. For example, a Shi\u2019ite shari\u2019a judge from Bahrain, who was staying at our guest residence, took great pains to describe to me his opposition to all forms of gamete donation. To prove this point, he provided me with a copy of his book on Islamic personal status law, which had been translated into English and which supported his anti\u2013gamete donation position based on evidence from the traditional Islamic scriptures. According to him, Iranian clergy, who speak Farsi rather than Arabic, are not as familiar with the original Islamic scriptures (in Arabic) that demonstrate the immorality of third-party donation. Thus, in his view, some Iranian clergy are \u201cinnovating\u201d in ways that are religiously unacceptable, and that are at odds with the rest of the Muslim world.\nA case in point: ayatollahs Ali al-Sistani and Muhammad Sa\u2019id al-Tabataba\u2019i al-Hakim, both Shi\u2019ite religious authorities in Iraq, advise caution against third-party donation practices, viewing them as largely unacceptable (Clarke 2006). Indeed, Ayatollah al-Sistani\u2019s son, Muhammad Rida Al-Sistani, has devoted an entire volume of richly documented legal analysis to this debate, providing \u201can invaluable resource for other scholars\u201d (26). According to Clarke, \u201cSistani\u2019s work, while perhaps posing more questions than clear answers, opens up for other scholars a fascinating window into this area of Shi\u2019ite jurisprudential debate, at a time when the Western media are just waking up to the vibrant engagement Shi\u2019ite scholars have had with other such new technologies\u201d (26).\nIndeed, the degree to which some Shi\u2019ite clergy are \u201cpushing the envelope\u201d in the realm of reproductive science and technology is quite remarkable. At the recent gamete donation conference in Iran, some Iranian clergy and physicians present advocated for future laws permitting all forms of donation as well as surrogacy. Once passed into law, gamete donation of all kinds will be difficult to stop. Meanwhile, in the absence of formal legislation, some IVF physicians in Tehran\u2014as well as in Shi\u2019ite-dominant Lebanon, which is closely following the Iranian lead\u2014are using the legal vacuum and the original \u201cpermissive\u201d fatwa of Ayatollah Khamanei to practice all forms of gamete donation among their desperate infertile patients. As noted by Clarke (2006) for Lebanon, \u201cDoctors keep Khamanei\u2019s fatwa collection on the shelves of their surgeries to demonstrate the permissibility of such procedures to skeptical Muslim patients; and many such patients have profited from it to undertake donor sperm and egg procedures, even surrogacy arrangements, with a clear conscience\u201d (26).\nMuslim patient opposition to donation\nAlthough donor law and practice are headed in interesting new directions in both Iran and Lebanon, it must be reiterated that the vast majority of Muslims, both Shi\u2019ite and Sunni, do not accept the idea of third-party gamete donation. Why are they opposed to donation?\nIn the hundreds of interviews that I have conducted since 1996 in Egypt (with Sunni Muslims) and Lebanon (with both Sunni and Shi\u2019ite Muslims), the majority of infertile couples were clear that donation is haram, or forbidden by the religion (Inhorn 2006b). Patient concerns revolve around three sets of related issues: (1) the moral implications of third-party donation for marriage, (2) the potential for incest, and (3) the moral implications of donation for kinship and family life.\nWith regard to marriage, Islam is a religion that can be said to privilege\u2014even mandate\u2014heterosexual marital relations. As is made clear in the original Al-Azhar fatwa, reproduction outside of marriage is considered zina, or adultery, which is strictly forbidden in Islam.\nAlthough third-party donation does not involve the sexual \u201cbody contact\u201d of adulterous relations, or presumably the desire to engage in an extramarital affair, it is nonetheless considered by most Islamic religious scholars to be a form of adultery, by virtue of introducing a third party into the sacred dyad of husband and wife. It is the very fact that another man\u2019s sperm or another woman\u2019s eggs enter a place where they do not belong that makes donation of any kind inherently wrong and threatening to the marital bond.\nThe other aspect of third-party donation that troubles marriage is the potential for incest among the offspring of unknown donors. Moral concerns have been raised about the potential for a single anonymous donor\u2019s offspring to meet and marry each other, thereby undertaking an incestuous union of half-siblings.\nThe final moral concern voiced by Muslim IVF patients is that third-party donation confuses issues of kinship, descent, and inheritance. As with marriage, Islam is a religion that can be said to privilege\u2014even mandate\u2014biological inheritance. Preserving the \u201corigins\u201d of each child\u2014meaning its relationship to a known biological mother and father\u2014is considered not only an ideal in Islam, but a moral imperative. The problem with third-party donation, therefore, is that it destroys a child\u2019s lineage, which is immoral in addition to being psychologically devastating.\nMuslim IVF patients use the term \u201cmixture of relations\u201d to describe this untoward outcome. Such a mixture of relations, or the literal confusion of lines of descent introduced by third-party donation, is described as being very \u201cdangerous,\u201d \u201cforbidden,\u201d \u201cagainst nature,\u201d \u201cagainst God\u201d\u2014in a word, haram, or morally unacceptable. It is argued that donation, by allowing a \u201cstranger to enter the family,\u201d confuses lines of descent in patrilineal Islamic societies. For men in particular, ensuring paternity and the \u201cpurity\u201d of lineage through \u201cknown fathers\u201d is of paramount concern (Inhorn 2006b). As one Sunni Muslim man, a high school biology teacher, summarized the problem:\nThe most important thing is that we are Muslims. If there is faith in carrying out this operation using sperm from the husband and ova from the wife, then this is okay. We cannot accept what happens in the West. We heard some women \u201chire the womb\u201d of another woman, or take sperm. According to our religion, this is called ikhtilat in-nasab, \u201cmixing relations.\u201d We consider it some kind of zina, prostitution. Because there are many hadiths from the Prophet Muhammad that confirm this. If you put your sperm in another woman besides your wife, you go to hell. This is adultery. There is a hadith on adultery. \u201cIf you put your sperm in another woman other than your wife, you are going to commit a sin.\u201d People asked the Prophet, \u201cHow?\u201d He said, \u201cIf you put it in your wife, you are going to be rewarded from Allah.\u201d They said, \u201cYes.\u201d He told them, \u201cBut this is also the case if you put it in the wrong womb. You are going to have punishment.\u201d\nIn addition to the consequences of mixed bloodlines and adultery, bringing such donor children into the world is considered unfair to the children themselves, who would never be treated with the love and concern parents feel for their \u201creal\u201d children. Such a child could only be viewed as a bastard\u2014an ibn haram, literally \u201cson of sin.\u201d Thus, a child of third-party donation starts life off as an \u201cillegal\u201d child. The child is deemed illegitimate and stigmatized even in the eyes of his or her own parents, who will therefore lack the appropriate parental sentiments (Inhorn 2006b). As one Sunni Muslim IVF patient stated:\nMy baby must be mine, and from my husband. This is logical. A mother will never feel this is her child if it is from another [man\u2019s] donated sperm or ova. It\u2019s only natural. Everything must occur naturally. If the child is from the father and mother, they will feel this is actually our baby. If not, we\u2019ll not be a family. The feeling of the baby, and our feelings. You will feel like you\u2019re acting, making a movie, living a life that\u2019s not true. This is our feeling. Of course, people take babies [through adoption], but this is not so common here in Egypt. We don\u2019t even want to think about this point! We are making our trial [of IVF] and hoping God will help us.\nIndeed, the firm conviction that parenthood of a donor child is an impossibility is clearly linked to the legal and cultural prohibitions against adoption throughout the Muslim world. The Islamic scriptures, including the Qur\u2019an, encourage the kind fostering of orphans but do not allow legal adoption as it is known in the West, whereby a child takes its adoptive parents\u2019 surname and is treated as one\u2019s own child (Inhorn 1996; Sonbol 1995; Zuhur 1992). In the Muslim Middle East, few IVF patients, either Sunni or Shi\u2019ite, will contemplate adoption, stating with conviction that it is \u201cagainst the religion\u201d and that the adopted child \u201cwon\u2019t be my own son\u201d (Inhorn 2006b). Even though legal adoption is practiced in Iran\u2014with the child receiving a birth certificate in the adoptive couple\u2019s name after a six-month period of adjustment\u2014the social and cultural resistances to adoption remain strong in that country, making this a \u201clast resort\u201d for infertile Iranian couples (Janet Heindl, personal communication, 8 July 2004; Tober 2004).\nNonetheless, Iran\u2019s acceptance of adoption has clearly paved the way for gamete donation, with Ayatollah Khamanei\u2019s initial fatwa deeming the infertile couple to be like adoptive parents. Indeed, Iran\u2019s clearly adventurous path regarding both adoption and donation is leading to social transformations in other parts of the Shi\u2019ite Middle East, in ways described in the following section.\nMarriage and gender relations\nIn considering infertility and IVF in the Muslim world, it is important to ask: What happens to infertile Middle Eastern Muslim couples who are not allowed to adopt and who do not accept the use of donor gametes? In the absence of adoption and gamete donation, infertile Muslim couples have no choice but to turn to IVF and other assisted reproductive technologies to solve their infertility problems using their own gametes.\nIn the Middle Eastern Muslim world, marriage is highly valued, and nearly all adults marry if possible in most Middle Eastern countries (Population Reference Bureau 2004; Zuhur 1992). Middle Eastern societies are also pronatalist\u2014they highly value children for numerous reasons and expect all marriages to produce them (Inhorn 1996). Thus, the notion of a married couple living happily without children is unthinkable. Children are desired from the beginning of marriage in most cases, and are usually loved and cherished once they are born.\nAs a result, childless couples are often under tremendous social pressure to conceive. In the Muslim world, infertile women often live in fear that their marriages will \u201ccollapse,\u201d for Islamic personal status laws consider a wife\u2019s barrenness to be a major ground for divorce. Although Islam also allows women to divorce if male infertility can be proven, a woman\u2019s initiation of divorce continues to be so stigmatizing that women rarely choose this option unless their marriages are truly unbearable (Inhorn 1996). Instead, they often \u201ccover\u201d for their infertile husbands, accepting the social responsibility for the infertility and diffusing the embarrassment of their husbands\u2019 reproductive emasculation (Inhorn 2003b, 2004).\nThe emergence of the revolutionary new IVF technology called intracytoplasmic sperm injection (ICSI), however, has ironically increased the potential for divorce in the Muslim Middle East. Namely, with ICSI, infertile men with very poor sperm profiles\u2014even azoospermia, or lack of sperm in the ejaculate\u2014are now able to produce \u201cbiological\u201d children of their own. As long as a single viable spermatozoon can be retrieved from a man\u2019s body, including through painful testicular aspirations and biopsies, this spermatozoon can be injected directly into the ovum under a high-powered microscope. What ICSI requires, then, is high-quality ova, despite low-quality sperm. However, the wives of many of these men, who have \u201cstood by\u201d their infertile husbands for years, even decades in some cases, may have grown too old to produce viable ova for the ICSI procedure. In the absence of adoption or of any kind of egg donation, infertile Muslim couples with a reproductively \u201celderly\u201d wife face four difficult options: (1) to remain together permanently without children; (2) to legally foster an orphan, which is rarely viewed as an acceptable option; (3) to remain together in a polygynous marriage, which is rarely viewed as an acceptable option by women themselves; or (4) to divorce so that the husband can have children with a younger wife.\nIn my research in Egypt and Lebanon, the first option has proven to be the most common\u2014namely, infertile husbands and their 40-something wives often love each other deeply, and remain together in long-term marriages without producing any children. Thus, divorce is not the immediate consequence of infertility that it is stereotypically portrayed to be. Because of the Sunni Islamic restrictions on the use of donor eggs, however, as well as lack of acceptance of this option among some segments of the Shi\u2019ite population, at least some Muslim men are choosing to divorce or take a second wife, believing that their own reproductive destinies lie with younger, more fertile women.\nThat being said, in the Shi\u2019ite Muslim world, including in Iran and Lebanon, at least some Shi\u2019ite couples are beginning to receive both donor gametes and donor embryos,4 as well as donating their gametes to other infertile couples. For infertile Shi\u2019ite couples who accept the idea of third-party donation\u2014as well as for Ayatollah Khamanei, who originally introduced the idea of donation to the Muslim world\u2014the introduction of donor technologies has been described as a \u201cmarriage savior,\u201d helping to avoid the \u201cmarital and psychological disputes\u201d that may arise if the couple\u2019s case is otherwise untreatable. Such disputes are clearly dramatized in the popular Iranian film Laila, which documents the painful separation of an otherwise happily married but infertile couple, and which was released in the mid-1990s before the Khamanei fatwa permitted such marriages to be saved through the use of donor technologies.\nIn Iran today, donor egg and donor embryo programs have been set up at most IVF clinics. Donor eggs come from three sources: other IVF patients, relatives,5 and unmarried women who agree to participate as egg donors in one-day mutca marriages for a fee. Such marriages only require a witness and are not officially registered; thus, they take place in confidence in the back rooms of IVF clinics. Indeed, donors who wish to remain anonymous enter these mutca marriages only by written agreement, without ever meeting the recipients of their eggs or their temporary husbands. They receive their money following egg harvesting (usually about U.S. $550), provide no personal information about themselves to the recipient couple, receive no information about the recipient couple, and \u201cgo about their business\u201d (Soraya Tremayne, personal communication, 31 July 2004). In short, egg donation\u2014as well as embryo donation from other couples\u2014is largely a financial transaction in Iran, with very little regulation or control over who donates or how donation is enacted.\nThe same is not true for the receiving of embryos. According to Tremayne, the recent law in Iran specifies clearly that couples desiring an embryo as a result of infertility must apply in writing to a court in order to receive permission for embryo transfer. The law specifies that the couple must be morally sound and suitable as parents and must be Iranian citizens, much like the law governing adoption in Iran (Janet Heindl, personal communication, 8 July 2004). Still, the donor embryo law is so new that most IVF clinics in Iran do not yet own a copy of the legislation and are not necessarily abiding by the legal requirements for donor embryo transfer at their clinics. If the husband is infertile, the couple simply receives another couple\u2019s embryos, with most donor couples choosing to remain anonymous. As Tremayne (31 July 2004) states,\nI did not get the impression that people desperate to have a child thought very far about the issues of kinship and family relations. As far as I could see, the donation is considered more a financial transaction than a donation\/gift, and once you have paid the couple to buy their embryo, or paid the temporary wife for her egg, they have no further claims on you and this is the end of the story. The forms filled by the donors leave it to them to decide whether they want to give their name or not.\nIn Lebanon where I have conducted my own research, the situation is very similar, despite the lack of a national law governing any aspect of IVF or third-party donation.6 At Lebanese IVF clinics providing donor technologies, some of the donors are other IVF patients (mostly Shi\u2019ite Muslims who accept the idea of donation), some are friends or relatives (including egg-donor sisters), and some are anonymous donors, who provide their ova for a fee. In at least one clinic catering to a largely conservative Shi\u2019ite clientele, some of these donors are young non-Muslim, American women who travel to Lebanon for extra payment to donate their eggs anonymously to infertile Lebanese couples. Ironically, those most likely to receive these \u201cAmerican eggs\u201d are conservative Shi\u2019ite couples, who accept the idea of donation because they follow the teachings of Ayatollah Khamanei in Iran. Thus, in Lebanon, those most likely to follow the spiritual guidance of Ayatollah Khamanei\u2014and, hence, to receive American donor eggs\u2014are generally members of or sympathizers with Lebanon\u2019s Hizbullah political party, which is officially described by the U.S. administration as a terrorist organization!\nFurthermore, quite interestingly, in multisectarian Lebanon, the recipients of these donor eggs are not necessarily only Shi\u2019ite Muslim couples. Some Sunni Muslim patients from Lebanon and from other Middle Eastern Muslim countries such as Egypt and Syria are quietly slipping across transnational borders to \u201csave their marriages\u201d through the use of donor gametes, thereby secretly \u201cgoing against\u201d the dictates of Sunni Muslim orthodoxy. Such border crossing has also been noted by Clarke (2006) for Lebanon; he writes, \u201cIndeed patients come from other countries to benefit from this relatively relaxed regime\u201d (26). The same is true in Iran, where, according to IVF clinic staff, scores of Persian Gulf Arabs from countries such as Saudi Arabia and Kuwait are traveling to Tehran in pursuit of donor gametes.\nConclusion\nIn conclusion, it is fair to state that global reproductive \u201ctechnoscapes\u201d (Appadurai 1996) are becoming increasingly expansive as we enter this new millennium. As this article has tried to show, the Muslim world\u2014generally positioned on the receiving end of global reproductive technology transfers\u2014has nonetheless embraced assisted reproductive technologies with considerable enthusiasm while, at the same time, reconfiguring them in accordance with the local religious moralities so important in this region.\nAlthough generally portrayed as monolithic, Islam itself takes several different forms, as evident in this essay. In the Sunni Muslim world, which includes most Middle Eastern countries, the use of IVF and related assisted reproductive technologies has clearly led to an entrenchment of deeply held religious beliefs about the importance of biologically based kinship, family life, and parenthood. Yet the globalization of these technologies to the Shi\u2019ite Muslim world has fundamentally altered understandings of the ways in which families can be made and the ways in which marriages can be saved through the uses of assisted reproductive technologies.\nFor Shi\u2019ite Muslims, in particular, the frankly adventurous attitude on the part of some Shi\u2019ite religious leaders toward third-party donation has led to a potential transformation in gender relations among infertile Muslim couples. For example, in Lebanon, the recent Shi\u2019ite fatwas allowing egg donation have been a great boon to marital relations. There, both fertile and infertile men with \u201creproductively elderly\u201d wives are lining up at IVF clinics to accept the eggs of donor women. Furthermore, in multisectarian Lebanon, the recipients of donor gametes are not necessarily only Shi\u2019ite Muslim couples, but include some Sunni Muslim and Christian couples as well.\nIn short, the arrival of donor technologies in the Muslim Middle East has led to a brave new world of reproductive possibility never imagined when these technologies were first introduced there nearly 20\u00a0years ago. These technologies have engendered (1) significant medical transnationalism and reproductive tourism; (2) mixing of gametes across ethnic, racial, and religious lines; and (3) the birth of thousands of ICSI and, now, donor babies to devout infertile Muslim couples. Infertile couples have begun to reconsider traditional notions of biological kinship, even if \u201csocial parenthood\u201d of a donor child is still not widely embraced (Inhorn 2006b). And because donor technologies are now widely available in both Iran and Lebanon, the power of the Sunni Muslim ban on third-party donation is being weakened across the region, with some infertile Sunni Muslim couples reconsidering their own antidonation moral stances. As a result, Shi\u2019ite gametes are finding their ways into Sunni bodies, an interesting variation on the \u201cmaking of Muslim babies.\u201d\nIn my view, these multiple transformations are powerful indicators of the profound social effects that reproductive technologies may engender in the new world order. As the assisted reproductive technologies become further entrenched in the Muslim world, and additional forms of global reproductive technology become available, it is important to examine the new local moral worlds that are likely to arise in response to this variant of globalization. The pace of change evident in the production of assisted reproductive technologies themselves\u2014as highlighted on the recent Nova special called \u201c18 Ways to Make a Baby\u201d\u2014as well as the rapid spread of these technologies into far reaches of the non-Western world is, indeed, striking. Thus, as one science and technology studies scholar, David Hess (1994), rightly observes, \u201cAnthropology brings to these discussions a reminder that the cultural construction of science is a global phenomenon, and that the ongoing dialogue of technoculture often takes its most interesting turns in areas of the world outside the developed West\u201d (16).\nNotes\nTo overcome the difficulties of raising a biologically unrelated donor child, some Shi\u2019ite physicians in Lebanon are arguing for a variety of novel solutions, including inheritance through gifts and bequests; institutions of rida\u2019 (milk kinship), whereby the mother of a donor child becomes related to it through breastfeeding; and the notion of legal guardianship of foster children (Clarke 2006).\nI am deeply grateful to Soraya Tremayne, who, fresh from fieldwork in Iran, has engaged in a lively e-mail discussion with me and has provided invaluable information on the practices of IVF and gamete donation in Iran since 2004. I have tried to represent her findings as accurately as possible in this paper.\nIn Iran, I want to thank Mohammad Jalal Abassi-Shavazi, Mohamad Mehdi Akhondi, and Pegah Ebadi for so generously inviting and hosting me at the Avesina Research Center and University of Tehran conference on embryo and gamete donation.\nIn Lebanon, anonymous sperm donation\u2014using frozen sperm from overseas sperm banks or fresh sperm samples from mostly medical and graduate students\u2014is \u201cquietly\u201d practiced at IVF clinics. One of my azoospermic Lebanese male informants produced a donor child in this way, and several others, both Muslim and Christian, had also made the decision to use donor sperm.\nIn Iran, women commonly bring their sisters as potential egg donors. But this is not allowed, as Islam is explicitly against the marriage of one man to two living sisters. Apparently, men also bring their brothers as potential sperm donors in Iran. According to Tremayne, she observed one case where the husband did so without his wife\u2019s knowledge; the wife believed that she was receiving her husband\u2019s sperm instead of that of her brother-in-law.\nSome leading members of the Lebanese medical community are pushing for a law that bans all forms of third-party donation in the country. However, this law has yet to be debated in the Lebanese parliament and is unlikely to pass, according to some sources.","keyphrases":["gamete donation","islam","medical anthropology","in vitro fertilization","middle east"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_Arch_Otorhinolaryngol-4-1-2279156","title":"Surgery did not improve the subjective neuropsychological symptoms of patients with incidentally detected mild primary hyperparathyroidism\n","text":"Primary hyperparathyroidism (PHPT) is known to cause diverse subjective symptoms, in addition to those related to osteitis fibrosa cystica and kidney stones. The treatment of the disease ameliorates the subjective symptoms and improves the patients\u2019 quality of life. In this prospective study, patients undergoing surgery for incidentally detected, mild, asymptomatic PHPT were assessed to determine whether subjective neuropsychological symptoms are improved even in patients with \u201casymptomatic\u201d PHPT. From October 1995 to March 2004, 25 patients who had one or more neuropsychological symptoms preoperatively and were followed up 1 year after parathyroidectomy were enrolled. The subjective symptoms were identified using questionnaires distributed to patients; eight questions were used to determine the presence or absence of psychoneurological symptoms. Compared to their preoperative status, patients responded that their general health perceptions 1 year after surgery were improved (13 cases, 52%), unchanged (11 cases, 44%), or aggravated (1 case, 4%). There were no statistically significant differences in the patients\u2019 responses before and after surgery with respect to individual neuropsychological symptoms, such as \u201ctiring easily, \u201cforgetfulness,\u201d \u201cdecreased concentration,\u201d \u201cdepression,\u201d \u201cirritability,\u201d \u201cuneasiness,\u201d and \u201csleeplessness.\u201d Therefore, subjective neuropsychological symptoms did not improve in otherwise asymptomatic PHPT patients following parathyroidectomy. However, patients\u2019 questionnaire responses may not reflect their actual status as accurately as laboratory examination results. Overall, 52% of patients were subjectively satisfied with surgery; this may result from patients\u2019 expectations of treatment.\nIntroduction\nSince calcium concentration is now included in the serum biochemical analyses done by multichannel autoanalyzers, it is possible to detect patients with hypercalcemia. Consequently, an increased incidence of mild cases of primary hyperparathyroidism (PHPT) has been noted. More than 80% of PHPT patients are essentially \u201casymptomatic\u201d [1, 2]. Currently, resection of the pathologic parathyroid gland continues to be the sole therapeutic modality available for curing PHPT. However, the surgical procedure is not necessarily easy for surgeons with limited experience, particularly in certain patients with mild PHPT. Under these circumstances, several investigators [1, 3, 4] have reported, based on surveys, on the natural history of patients with asymptomatic PHPT who had been conservatively followed up. Some patients reported no signs or symptoms during observation periods of various durations, although some patients developed complications specific to PHPT that required surgery. PHPT has been known to produce diverse subjective symptoms, in addition to those related to osteitis fibrosa cystica and kidney stones. The treatment of the disease ameliorates these symptoms, which improves patients\u2019 quality of life (QOL).\nFrom the clinical standpoint, based on patient self-reporting after surgery, most surgeons have had the impression that parathyroidectomy improves psychological and mental deficits. This prospective study of patients undergoing surgery for incidentally detected, mild, asymptomatic PHPT determined whether subjective neuropsychological symptoms improved postoperatively in patients with asymptomatic PHPT.\nPatients and methods\nFrom October 1995 to March 2004, 62 patients with PHPT were treated at the Cancer Institute Hospital. All patients underwent surgery. In 60 patients, the pathological parathyroid glands were successfully removed; the pathology was adenoma in 58 patients, hyperplasia in none, and cancer in 2. Of the 58 patients whose parathyroid adenoma was removed, 7 had kidney stones and 14 had osteitis fibrosa cystica. Thirty-seven patients without any signs and\/or symptoms of classic PHPT were diagnosed as \u201casymptomatic\u201d, because all were incidentally found to have hypercalcemia on laboratory testing, and their subsequent serum examinations showed inappropriately elevated intact parathyroid hormone concentrations without concomitant clinical, biochemical, or radiological evidence of osteitis fibrosa cystica or renal stone colic. Of these 37 patients, 34 reported one or more neuropsychological symptoms preoperatively; 3 patients reported no neuropsychological symptoms preoperatively, and these 3 patients were excluded from the postoperative survey. Twelve months after successful parathyroidectomy, 25 patients responded to our questionnaire (Fig.\u00a01) distributed at the outpatient clinic or by mail. They included 2 males and 23 females, ranging in age from 48 to 76 (mean 64) years. The other 9 patients were requested to respond by mail. Of these 9 patients, 3 gave no response, and 6 responded to less than half of the questions; thus, these 9 patients were excluded from the study.\nFig.\u00a01Survey of primary hyperparathyroid patients (pts) treated surgically during an 8.5-year period (1995\u20132004)\nPreoperative bone mineral density was measured in the lumbar spine (L2\u2013L4) using a dual-energy X-ray absorptiometer. The serum calcium level was 10.1\u201312.4 (mean 11.0) mg\/dl, the urinary calcium excretion rate was 51\u2013497 (mean 246) mg\/24\u00a0h, the creatinine clearance rate was 40\u2013183 (mean 90.8) ml\/min, and the bone mineral density was 0.2 to \u22124.8 (mean \u22122.5) SD.\nThe subjective symptoms were assessed by distributing questionnaires that contained eight questions dealing with the presence or absence of neuropsychological symptoms (Table\u00a01). The eight questions were formulated based on a textbook [5] and dealt with \u201ctiring easily, \u201cforgetfulness,\u201d \u201cdecreased concentration,\u201d \u201cdepression,\u201d \u201cirritability,\u201d \u201cuneasiness,\u201d \u201csleeplessness,\u201d and \u201cgeneral health\u201d. \u201cUneasiness\u201d was defined as feeling worried or unhappy about a particular situation, especially because one thinks something bad or unpleasant might happen. The questionnaires were distributed before and 1\u00a0year after surgery. To ensure that the patients understood the questions, the meaning of each question was thoroughly explained prior to surgery. To ascertain patients\u2019 perceptions of their general health, they were asked whether they felt better 1\u00a0year after surgery than before surgery. Patients were asked to select one of the following responses to each question: the symptom is totally absent or occurs rarely (score 0); the symptom is slight or occurs only occasionally (score 1); or the symptom has recently been aggravated or occurs frequently (score 2).\nTable\u00a01Questionnaire contents1. Do you tire easily?2. Are you forgetful?3. Do you lack the ability to concentrate?4. Do you feel depressed?5. Do you feel irritable?6. Do you feel uneasiness?7. Do you experience sleeplessness?8. How do you perceive your general health compared with that before surgery?aaThis question was asked only once, 1\u00a0year postoperatively\nStatistical analysis\nA two-sided Wilcoxon\u2019s signed-rank test was used to detect statistically significant differences among the responses; the significance level was set at P\u00a0<\u00a00.05.\nResults\nGeneral health perceptions 1\u00a0year after parathyroidectomy\nWhen compared to their preoperative responses, the patients\u2019 general health perceptions 1\u00a0year after surgery were improved in 13 cases (52%), unchanged in 11 cases (44%), and aggravated in 1 case (4%); these changes were statistically significant (P\u00a0=\u00a00.0013).\nComparison of the individual subjective neuropsychological symptoms\u2019 grade \nTable\u00a02 shows the preoperative and 1-year postparathyroidectomy comparison of individual subjective neuropsychological symptoms. Preoperatively, 17 patients (68%) reported \u201ctiring easily,\u201d and 1\u00a0year postoperatively the symptom had resolved in 6 and was still reported by 11, although all 11 perceived the symptom as mild. However, of the 8 patients who did not report tiring easily preoperatively, 5 reported a mild degree of tiring easily 1\u00a0year postoperatively. These changes in the number of patients who reported tiring easily were not statistically significant.\nTable\u00a02Comparison of pre- and postoperative scoresNeuropsychological symptomsPreoperative1\u00a0year postoperativeP valueNo. of patientsScore012Easily tiredScore083500.2411257025140No. of patients259160ForgetfulnessScore061500.811558224040No. of patients256172Decreased concentrationScore074300.311466224040No. of patients2510132DepressionScore01210110.171844025221No. of patients251672IrritabilityScore01312100.4511155121001No. of patients251762UneasinessScore010820111347222011No. of patients2512103SleeplessnessScore01173111936025014No. of patients2510105\nChanges in the number of patients reporting other neuropsychological symptoms, such as \u201cforgetfulness,\u201d \u201cdecreased concentration,\u201d \u201cdepression,\u201d \u201cirritability,\u201d \u201cuneasiness,\u201d and \u201csleeplessness,\u201d before and after surgery were also not statistically significant (Table\u00a02).\nDiscussion\nIn normal subjects, the serum calcium concentration is always kept within a narrow normal range and plays an important role in the physiological control of neuropsychological functions. In PHPT patients, even in those with the mild form, the abnormally elevated serum calcium concentration returns to the normal level postparathyroidectomy. It has been assumed that some subjective improvement in neuropsychological function must occur following surgery.\nIn the present prospective study of patients with incidentally detected, mild PHPT, 52% of all patients who had successful parathyroidectomy reported improved general health 1\u00a0year after surgery. This result was statistically significant.\nPasieka and Parsons [6] reported that general health improved following surgery in 60% of PHPT patients. Talpos et al. [7] studied 53 patients with asymptomatic hyperparathyroidism who were randomized into a parathyroidectomy group or an observation alone group; they were evaluated every 6\u00a0months for 2\u00a0years using the SF-36 health survey. The 28 patients who had parathyroidectomy had improved social and emotional function scores compared to the observation alone group. Edwards et al. [8], who studied 100 patients with PHPT, found that parathyroidectomy for hyperparathyroidism was associated with significant lasting improvement in subjective symptoms; they stated that the potential for long-term improvement of these QOL symptoms was a valid indication for parathyroidectomy. However, contrary to reports of long-term postoperative improvement in subjective symptoms, Okamoto et al., using the GHQ-28 in 26 patients with mild PHPT, found no improvement in symptoms except for severe depression 24\u00a0months after surgery, though they found improvement in the total GHQ score, somatic symptoms, anxiety, and severe depression 3\u00a0months following parathyroid surgery [9]. Okamoto et al. hypothesized that the transient, short-term improvement in symptoms after surgery may have been a consequence of patients\u2019 expectations of treatment.\nThe results of the present study showed that approximately half of the patients reported improvement in general health perceptions, and there was no statistically significant improvement in any of the individual neuropsychological symptoms. The subjective satisfaction with surgery reported by 52% of our patients may also reflect their expectations of treatment.\nBollerslev et al., in a randomized study using the SF-36 involving 191 patients with asymptomatic PHPT who were assigned to surgery or medical observation, found that \u201cno benefit of operative treatment, compared with medical observation, was found\u201d [16]. In the present study, the numbers of patients required for comparison of differences before and after surgery, using a significance level of 0.05 and a power of 0.80, were calculated as follows: \u201ctiring easily\u201d, n\u00a0=\u00a039; \u201cforgetful\u201d, n\u00a0=\u00a0382; \u201cdecreased concentration\u201d, n\u00a0=\u00a062; \u201cdepression\u201d, n\u00a0=\u00a055; \u201cirritability\u201d, n\u00a0=\u00a0165; \u201cuneasiness\u201d, n\u00a0=\u00a01492; and \u201csleeplessness\u201d, n\u00a0=\u00a01878. Therefore, between 39 and 1,878 patients would be required to have adequate statistical power to determine \u201cwhether surgery improves psychoneurological symptoms\u201d. The sample size of the present study was 25 patients, which was too low to determine \u201cwhether surgery improves psychoneurological symptoms\u201d. However, the present findings were similar to those of Bollerslev et al.\nThere is a recognized need to determine the surgical indications for PHPT. In 1990, the guidelines of the US National Institutes of Health (NIH) were published. The results from a questionnaire given to the members of the Society of Surgical Endocrinology of America showed that several practices were not precisely in accordance with the 1990 NIH guidelines [11]. Sywak et al. conducted a comparative study involving one group that met the criteria of the 1990 NIH guidelines and one group that did not. They reported that nonspecific symptoms, such as fatigue, depression, irritability, mood swings, and forgetfulness, improved in both groups [12]. The guidelines were revised in 2002, and a 2002 workshop panel promulgated six criteria for PHPT surgery [13]. Since there is uncertainty concerning the specificity of subjective symptoms, the NIH conference excluded them from the criteria for parathyroidectomy. Although the present study did not find that subjective neuropsychological symptoms were improved in patients with asymptomatic PHPT following parathyroidectomy, patients\u2019 responses to questionnaires may not reflect their actual status as accurately as laboratory test results, such as the serum calcium level, the urinary calcium excretion rate, the creatinine clearance rate, and bone mineral density.\nNevertheless, obtaining information on changes in subjective symptoms is clinically important. The difficulty in quantitatively evaluating nonspecific symptoms has been previously identified, and some studies have used the Visual Analogue Scale [6, 14] or the SF-36 [7, 15, 16]. We devised an original questionnaire based on a textbook [5]. This questionnaire has not been validated as a research tool and was not tested on the normal population. However, the five questions related to \u201ctiring easily, \u201cforgetfulness,\u201d \u201cdepression,\u201d \u201cirritability,\u201d and \u201cgeneral health\u201d are identical to those used by Pasieka et al. [6, 14], and the other three questions dealing with \u201cdecreased concentration,\u201d \u201cuneasiness,\u201d and \u201csleeplessness\u201d are all easily understandable neuropsychological symptoms. To ensure that all patients understood the questions, the meaning of each question was thoroughly explained to the patient prior to surgery; however, 1\u00a0year postoperatively, the questionnaires were simply sent by mail to the patients with no further explanations. To allow nonspecific symptoms to be more readily evaluated, simple, quantitative methods are required.\nBollerslev et al. [10] reported that asymptomatic patients with mild PHPT have more psychological symptoms than normal controls and decreased QOL. In the present study, of the 37 patients who underwent parathyroid surgery for \u201casymptomatic\u201d PHPT, 34 had one or more neuropsychological symptoms preoperatively, though it was not confirmed that these symptoms were due to the PHPT itself. The results of the survey done 1\u00a0year after successful parathyroidectomy did not show any definite improvement in any of the individual neuropsychological symptoms. The main reason for this might be the mild nature of the patients\u2019 PHPT. Two reasons may explain why six patients gave incomplete answers and three patients did not respond to the second questionnaire: the questionnaires were mailed to patients who had stopped visiting the out-patient clinic, and patients may have found the questionnaire bothersome. If the patients had been truly symptomatic, they would have visited our hospital.","keyphrases":["subjective symptoms","parathyroidectomy","asymptomatic primary hyperparathyroidism"],"prmu":["P","P","R"]}
{"id":"Int_J_Legal_Med-4-1-2226061","title":"Stable RNA markers for identification of blood and saliva stains revealed from whole genome expression analysis of time-wise degraded samples\n","text":"Human body fluids such as blood and saliva represent the most common source of biological material found at a crime scene. Reliable tissue identification in forensic science can reveal significant insights into crime scene reconstruction and can thus contribute toward solving crimes. Limitations of existing presumptive tests for body fluid identification in forensics, which are usually based on chemoluminescence or protein analysis, are expected to be overcome by RNA-based methods, provided that stable RNA markers with tissue-specific expression patterns are available. To generate sets of stable RNA markers for reliable identification of blood and saliva stains we (1) performed whole-genome gene expression analyses on a series of time-wise degraded blood and saliva stain samples using the Affymetrix U133 plus2 GeneChip, (2) consulted expression databases to obtain additional information on tissue specificity, and (3) confirmed expression patterns of the most promising candidate genes by quantitative real-time polymerase chain reaction including additional forensically relevant tissues such as semen and vaginal secretion. Overall, we identified nine stable mRNA markers for blood and five stable mRNA markers for saliva detection showing tissue-specific expression signals in stains aged up to 180 days of age, expectedly older. Although, all of the markers were able to differentiate blood\/saliva from semen samples, none of them could differentiate vaginal secretion because of the complex nature of vaginal secretion and the biological similarity of buccal and vaginal mucosa. We propose the use of these 14 stable mRNA markers for identification of blood and saliva stains in future forensic practice.\nIntroduction\nHuman body fluids such as blood and saliva are the most common sources of biological trace material found at a crime scene. Reliable tissue identification in forensic casework is important as it provides crucial insights into crime scene reconstruction and can thus contribute towards solving crimes. Blood stains are routinely tested in forensic practise using various methods including the tetrabase (4,4-bis(dimethylamino)diphenylmethane) test [1], the Kastle\u2013Meyer phenolphthalein test, the tetramethylbenzidine test [2], the orthotolidine test [3], or the luminol (3-aminophthalhydrazide) chemoluminescence test [4], with the latter especially appropriate for detecting blood stains after cleaning attempts [2, 5]. All these presumptive\u2014thus indicative but not identifying\u2014tests take advantage of the peroxidase-like activity of the heme unit of the hemoglobin molecule in human blood. Therefore, false-positive results can be caused by the presence of strong oxidants, such as chlorine-containing detergents or by true peroxidases (e.g., from plants) [6].\nSaliva stains are usually detected in forensic practise via an enzymatic amylase test using Phadebas [7] or with a recently developed enzyme-linked immunosorbent assay-based method [8]. However, because of amylase degradation, the time window for the successful performance of such tests can be limited [9]. Furthermore, no amylase assay can distinguish between salivary amylase and amylases from other tissues (pancreatic, urinary, etc.); therefore, the tests for saliva identification are only presumptive (similar to existing blood identification tests).\nOn the other hand, methods for identification and quantification of mRNA are already well established, although mostly outside the forensic field. These methods make massive multiplex gene expression profiling possible\u2014among many other applications\u2014for the discovery of tissue-specific mRNA markers. The major concern of using mRNA markers for forensic applications is their assumed high susceptibility to degradation. However, recent studies using a few selected genes demonstrated that it is possible to isolate total RNA of sufficient quality and quantity from biological stains that are several months or even years old [10\u201312]. It has also been suggested, although with limited evidence so far, that different types of mRNA seem to follow different rates of degradation [13]. It is assumed that the degradation process of mRNA is influenced by many external and internal factors, including structural peculiarities like the presence of AU-rich elements (ARE motifs), protein binding properties, and cellular localization [14, 15]. However, detailed knowledge on the molecular reasons for differences in RNA degradation between different types of RNAs as well as between mRNAs of different genes is currently lacking and further investigations are sorely needed.\nAlthough a small number of mRNA markers has been tested for tissue identification in forensic science [16\u201319], no systematic study has yet been performed. In addition, the identification of candidate markers in previous studies was based on a mixed literature and database search, apparently without strict criteria of selection, considering only a limited number of genes and tissues, and not taking into account RNA degradation levels. Furthermore, expressed sequence tags databases, which were used previously, like the Cancer Genome Anatomy Project [18], are expected to provide heavily biased information on candidate genes because of the nonrandom character of representation of clone libraries.\nTo find stable mRNA markers for body fluid identification in forensic practice, we performed a systematic and comprehensive whole-genome gene expression analysis on time-wise degraded blood and saliva stains using the Affymetrix U133 plus2 GeneChip. This expression array contains >54,000 mRNA probe sets, which encompass most, if not all, known and predicted human genes. Tissue-specific expression patterns of the most promising candidate genes from the array analyses were further confirmed using the GNF SymAtlas expression database [20], which covers about 100 human tissues, and finally verified by quantitative real-time polymerase chain reaction (PCR) in blood and saliva as well as in other body fluids relevant for forensic casework, i.e., semen and vaginal secretion.\nMaterials and methods\nSample collection\nAliquots of 5\u00a0ml of whole blood and saliva were collected from each of five healthy volunteers (four men and one woman) of western European genetic origin under informed consent before their inclusion in the study. Native blood was collected without anticoagulation treatment to avoid disturbing effects of anticoagulation reagents on gene expression. In each sample, 75 cotton swabs were immersed. Special care was taken to shorten the time between collection and swab absorption to avoid blood coagulation. After complete absorption of the fluids, swabs were left until dry on a bench top at room temperature. When dry, the swabs were stored in dust-free nonhumid conditions (but subjected to normal daylight) for different time intervals. Swabs were visually inspected and sorted out to ensure similar liquid content between individual swabs. After 0, 1, 3, 7, 14, 21, 57, and 180\u00a0days, swabs were stored at \u221280\u00b0C until RNA isolation. For the time interval 0\u00a0days, samples were frozen immediately after drying. Semen and vaginal secretion samples were collected from one male and one female individual absorbed with cotton swabs and dried overnight before RNA isolation.\nRNA isolation\nRNA was isolated using the Qiagen RNeasy kit (Qiagen Benelux B.V.) according to the manufacturer\u2019s instructions with minor modifications. These included cutting up the cotton swab into 1\u2009\u00d7\u20091-mm pieces and soaking them in RLT buffer for 1\u00a0h at 4\u00b0C before the extraction. Trial experiments to lengthen this incubation time up to 24\u00a0h did not reveal any improvement in respect to RNA quantity and quality (data not shown).\nMicroarray hybridization and gene expression data analysis\nBefore hybridization to Affymetrix U133 plus2.0 GeneChip arrays (Affymetrix, Santa Clara, CA), RNA isolated from blood and saliva stains was amplified using the Ambion MEGAscript T7 two-cycle amplification kit (Applied Biosystems, The Netherlands). Amplification, labeling, hybridization, washing, and scanning were performed by the microarray core facility of the Erasmus MC Center for Biomics according to Affymetrix specifications. Background subtraction and probe signal summarization were calculated according to the robust multiarray analysis algorithm [21] using the R Bioconductor software [22]; the resulting log2 signal values were back-transformed to linear scale. Presence\/absence calls for individual probe sets were calculated with the mas5calls function of the Bioconductor mas package. Because the constant global mean assumption does not hold true for arrays hybridized to differentially degraded RNA samples, the normalization of the signal intensities between samples was performed using the nonhuman control genes present on Affymetrix arrays (spiked-in probes). Normalization factors for each array were inferred from the average signal intensities of bioB, bioC, bioD, and Cre control probe sets. Analysis of differential gene expression was performed using the significance analysis of microarrays (SAM) algorithm [23] implemented in the TM4 software [24]. In the saliva dataset, we selected only genes with signal intensities above 50 (which is below the usually applied background threshold in expression array experiments) that had a signal intensity below 50 in the blood dataset. The selection of blood-targeted genes was done in a similar manner but with different criteria, the lower intensity limit in blood was set to 1,000 to reasonably restrict the number of candidates.\nReal-time PCR\nFirst strand cDNAs were synthesized with SuperScript\u00ae III RTS First-Strand cDNA Synthesis Kit (Invitrogen BV, The Netherlands) using total RNA as a template. The primers were designed with Primer3 software [25] so that forward and reverse primers were complementary to different exons of the respective genes and most closely located to the 3\u2032-end of the corresponding RefSeq cDNA (Electronic Supplementary Material Table S1). Real-time PCR reactions with the SuperScript\u00ae III Platinum\u00ae SYBR\u00ae Green One-Step qPCR Kit (Invitrogen BV) were performed on an ABI 7300 PCR machine (Applied Biosystems, The Netherlands) using the following parameters: initial denaturation at 94\u00b0C for 10\u00a0min, followed by 45 cycles of denaturation at 94\u00b0C for 15\u00a0s, and a final annealing\/elongation at 60\u00b0C for 30\u00a0s. Melting profiling and agarose gel electrophoresis were used to confirm the specificity of the primers and the absence of DNA contamination. Quantification of the amplified cDNA yield in comparative blood and saliva PCRs was done by the standard curve method. PCR experiments with semen and vaginal secretion were quantified using delta Ct (dCt) method. In both cases, GAPDH gene was used as an endogenous control to normalize the amplification signal between the samples from different tissues and individuals. Time points were compared to each other without normalization: Assuming the temporal degradation of all RNA molecules, no internal control gene could be used, and the only proper way to normalize RT-PCR signals was to use the same amount of template in each reaction. We found that this requirement holds true for our experiments because the GAPDH expression variability between different samples from the same tissue was relatively low (CV <25%, data not shown), which is probably because of approximately the same amount of blood or saliva absorbed with cotton swabs during material collection.\nResults and discussion\nMicroarray expression data\nAs expected, hybridization signals demonstrated high variability between individuals; however, the most striking differences were observed between the different tissues. Signal intensities in blood samples were on average about five times higher than in saliva (174.2\u2009\u00b1\u20091.9 in blood samples vs 26.9\u2009\u00b1\u20090.7 in saliva; Wilcoxon test rank sum p\u2009<\u20090.001). In addition, at the time-point zero, the number of the probe sets called as present according to the Affymetrix algorithm was, on average, more than three times higher in blood than in saliva (30.2%\u2009\u00b1\u20090.9 vs 9.3%\u2009\u00b1\u20090.6; t test p\u2009<\u20090.001). The SAM test with stringent parameters (false discovery rate was set to 0%) showed that, both in blood and saliva experiments, no genes demonstrated significant expression differences in a time range of 0\u201357\u00a0days of stain storage. Only few genes (37 and 10 significantly differential genes for saliva and blood, respectively) appeared to be differentially expressed at 180\u00a0days in comparison to other time points. This suggests that in dried blood and saliva, mRNA molecules remain relatively stable for a long period. Recent studies of Heinrich et al. [26] also revealed poor correlation between RNA degradation and postmortem time intervals.\nSelection of tissue-specific markers\nThe initial selection of tissue-specific genes was performed using the normalized signal intensities of microarray hybridizations averaged across the five biological replicates at the zero experimental time point. About 500 apparent saliva-specific and 1,000 apparent blood-specific candidate genes were selected. Further refinement of tissue-specific gene sets was achieved by probing the selected candidates against the GNF SymAtlas tissue database [20] after excluding all cell lines from the database retaining only tissues and organs for the analysis. Genes were selected only if they were highly and exclusively expressed in the target tissue(s) based on the GNF SymAtlas database. For blood, target tissue in the database was defined as whole blood; while for saliva, the target tissues were salivary gland, tongue, trachea, and tonsils. The selection criteria were as follows: high expression (signal intensity >1,000) in target tissue and low expression (signal intensity <200) in nontarget tissues. Using these criteria and combining data from expression array experiments as well as GNF SymAtlas database verification, we identified six saliva-targeted genes and 15 blood-targeted genes that were highly expressed only in target tissues (or respective organs) but not, or nearly not, in the nontarget tissues (Electronic Supplementary Material Figure\u00a0S1a, S1b, S1c).\nRT-PCR confirmation of tissue-specific markers\nTo confirm the microarray results, real-time PCR experiments were designed for the 21 best candidate markers selected from array hybridizations and database searches and performed using RNA extracted from aged blood and saliva stains, also providing a method suitable for forensic applications. In agreement with the array results, all 21 markers analyzed showed good amplification in the target tissue but no or only marginally detectable amplification in the nontarget tissue (Fig.\u00a01a\u2013c). Among the candidate markers, only the PPL gene that was targeted for saliva demonstrated significant expression overlap with blood and therefore was excluded from further experiments. Our results demonstrate that, irrespective of the stain storage time, sufficient RT-PCR amplification was observed in all samples, even in the samples from the longest storage time tested (180\u00a0days), indicating marker stability over long periods of sample storage time. The only exception was the CCR2 gene for which no amplification was detected in the blood stains stored for 180\u00a0days and was therefore excluded from further analyses. A plausible explanation for this peculiarity could be the location of the PCR-amplified region, which is more than 1\u00a0kb distant from the 3\u2032 end of the mRNA because of the very long untranslated region of CCR2. Apparently, the degree of degradation of the CCR2 mRNA after 180\u00a0days of sample storage was too high to allow its efficient reverse transcription using the oligo(dT) method that targets the 3\u2032end of the molecule. This observation highlights the necessity to design PCR primers for the most 3\u2032-proximal part of the mRNA molecule for successful amplification of cDNA fragments in degraded samples.\nFig.\u00a01a, b RT-PCR results for blood-targeted genes in blood and saliva stains. c RT-PCR results for saliva-targeted genes in saliva and blood stains. Genes were selected based on expression microarray results and GNF SymAtlas database. Expression values for each time point were averaged across three male and three female RNA samples; no gender-specific expression differences were detected (t test p\u2009<\u20090.05). B indicates blood; S indicates saliva; samples were processed after complete drying of blood and saliva at 0, 21, 57, and 180\u00a0days, respectively\nExpression of the candidate markers in other body fluids\nFor additional confirmation of the tissue-specificity, we tested by RT-PCR the expression patterns of our candidate RNA markers in other body fluids that might be observed in a forensic case, i.e., vaginal secretion and semen. According to the GNF SymAtlas database, all our markers targeted for blood and saliva are not expressed in testis nor in uterus tissues. In agreement, our dedicated RT-PCR experiments revealed that two of the saliva-targeted mRNA markers (SPRR3 and SPRR1A) show no detectable expression in semen (after 50 RT-PCR cycles), and the remaining three (KRT4, KRT6A, and KRT13) show vast overexpression in saliva compared to semen (ddCt\u2009>\u200915, Fig.\u00a02a), keeping with the assumption of high saliva specificity of the five proposed mRNA markers. SPRR1A and SPRR3 genes both encode cornified envelope precursor proteins and are predominantly expressed in oral and esophageal epithelia, where they are strictly linked to keratinocyte terminal differentiation [27]. Keratins 4, 6A, and 13 are known as one of the major structural proteins of oral mucosa [28, 29].\nFig.\u00a02a RT-PCR for saliva and blood-targeted genes in semen stains. b RT-PCR for saliva and blood targeted in vaginal secretion stains. Delta Ct (dCt) values were calculated as follows: dCt\u2009=\u2009Ct (candidate gene)\u2009\u2212\u2009Ct (endogenous control, GAPDH gene). Low dCt values correspond to high expression level of the specific mRNA. Gray bars correspond to the samples from target tissues for selected genes (either blood or saliva); black bars correspond to samples from nontarget tissues (either vaginal secretion or semen). Dotted bars represent the cases were amplification was not detected after 50 cycles, in this case, the expression values were arbitrary set to Ct value of 25 (plot maximum)\nFor the 14 blood-targeted genes, we observed no detectable amplification in semen for nine genes (CASP1, AMICA1, C1QR1, ALOX5AP, AQP9, C5R1, NCF2, MNDA, ARHGAP26), keeping with the assumption of high blood specificity of the respective mRNA markers. These genes encode the proteins with important functions in different types of blood cells. They are known to be highly or even specifically expressed in peripheral leukocytes (AQP9, NCF2, CASP1, C5R1, C1QR1, ALOX5AP [30\u201335]) and myelocytes or hematopoietic cells (MNDA, ARHGAP26, AMICA1 [36\u201338]). However, five genes demonstrated only slightly differential or even comparable expression in blood and semen (CD36, CCR1, PF4, BIN2, and ALOX5), not expected given the information provided by the GNF database, and were therefore excluded from the final list of blood-specific markers. Thus, our microarray-based genome-wide approach to find tissue-specific mRNA markers identified the genes that are functionally relevant for the target tissues.\nFurthermore, and not surprisingly, RT-PCR of all saliva- and blood-targeted markers in samples from vaginal secretion revealed gene expression at a level comparable to that in blood and saliva samples (Fig.\u00a02b). The natural occurrence of blood cells in vaginal secretion most likely explains the expression of our blood-targeted markers in vaginal secretion, whereas the high biochemical and histological similarity of oral and vaginal epithelia [40] makes the similarity of gene expression patterns between both tissues plausible. It should be pointed out that mRNA markers previously claimed to be useful for the identification of vaginal secretion such as HBD-1 [18] and MUC4 [18, 19] are known to be abundant also in oral epithelial cells and the salivary transcriptome [41\u201343]. Furthermore, Nussbaumer et al. [19] ruled out the potential to differentiate saliva and vaginal secretion using solely MUC4. Our results, together with previous findings, suggest that establishing mRNA markers expressed exclusively in vaginal secretion could be a challenging if not impossible task.\nComparison with previously suggested mRNA markers\nInterestingly, tissue-specific genes, as identified here, do not overlap with the ones previously suggested for blood and saliva stain identification [18, 19]. This could be explained by the experimental setup and the systematic (but not ad hoc) approach of this study, namely, the degraded biological material analysed and the Affymetrix microarray platform applied. In contrast to previous studies, we restricted our marker ascertainment to those genes, which retained structural mRNA integrity during the stain dry-out process as well as the subsequent long-term storage of 180\u00a0days. This allows future application of detection of these markers in forensic stains of unknown age, at least up to an age of 6\u00a0months, but expectedly longer. Furthermore, our saliva-specific candidate genes were derived from mouth and pharynx epithelial cells, unlike the previously suggested STATH and HTN3 genes that are expressed in the salivary gland [18]. Secreted mRNAs that are abundant in fresh saliva are more prone to fast degradation by extracellular RNAses [39]; they are therefore not expected to be present in dried stains, explaining why they were not detected by the relatively low-sensitive microarray hybridization method used in this study. The SPTB and PBGD genes, previously proposed as blood-specific markers [18], do not demonstrate any overexpression relative to other tissues in whole blood according to the GNF SymAtlas database (data not shown).\nConclusions\nIn summary, whole-genome expression analysis in time-wise degraded samples from blood and saliva stains in combination with RT-PCR verification of various forensically relevant body fluids has resulted in the identification of stable tissue-specific mRNA markers from five genes for saliva (SPRR3, SPRR1A, KRT4, KRT6A, and KRT13) and nine genes for whole blood (CASP1, AMICA1, C1QR1, ALOX5AP, AQP9, C5R1, NCF2, MNDA, and ARHGAP26). For the first time, mRNA markers were ascertained considering almost the entire human transcriptome and based on experimental data of genome-wide gene expression as well as considering the degradation stability of mRNAs. We could demonstrate that the candidate genes identified here provide informative mRNA markers for blood and saliva identification for stains up to 180\u00a0days of age. We would like to propose their application in forensic case work (with the potential practical limitation of coamplification in vaginal secret) for stains of at least 6\u00a0months of age. However, we expect that the proposed mRNA markers will successfully identify older blood and saliva stains (respective experiments are currently in progress). Finally, we would like to remark that tissue identification in forensics should be performed in a reciprocal way; so that a tissue is identified because of the presence of markers specific for the relevant tissue together with the absence of markers specific for all other tissues in question. Clearly, more research should be dedicated towards finding the most suitable markers for tissue identification in forensics.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nTable\u00a0S1\nRT-PCR primers for the genes amplified (DOC 79\u00a0kb).\nFigure\u00a0S1a\nGNF Symatlas expression profiles of blood-specific genes (GIF 15\u00a0kb)\nHigh resolution image file (TIF 82\u00a0kb)\nFigure\u00a0S1b\nGNF Symatlas expression profiles of blood-specific genes (GIF 14\u00a0kb)\nHigh resolution image file (TIF 77\u00a0kb)\nFigure\u00a0S1c\nGNF Symatlas expression profiles of saliva-specific genes (GIF 12\u00a0kb)\nHigh resolution image file (TIF 74\u00a0kb)","keyphrases":["rna markers","blood","saliva","body fluid identification","gene expression","biological traces"],"prmu":["P","P","P","P","P","P"]}
{"id":"Anal_Bioanal_Chem-3-1-1802725","title":"Non-destructive analysis of museum objects by fibre-optic Raman spectroscopy\n","text":"Raman spectroscopy is a versatile technique that has frequently been applied for the investigation of art objects. By using mobile Raman instrumentation it is possible to investigate the artworks without the need for sampling. This work evaluates the use of a dedicated mobile spectrometer for the investigation of a range of museum objects in museums in Scotland, including antique Egyptian sarcophagi, a panel painting, painted surfaces on paper and textile, and the painted lid and soundboard of an early keyboard instrument. The investigations of these artefacts illustrate some analytical challenges that arise when analysing museum objects, including fluorescing varnish layers, ambient sunlight, large dimensions of artefacts and the need to handle fragile objects with care. Analysis of the musical instrument (the Mar virginals) was undertaken in the exhibition gallery, while on display, which meant that interaction with the public and health and safety issues had to be taken into account.\nIntroduction\nAdvances in analytical technology have led to increasing interest in the scientific examination of precious artefacts and antiquities. These examinations may be for a variety of reasons, most commonly fundamental interest in the materials and techniques used by the artist, or understanding their conservation, condition or degradation processes. Analytical data (e.g. from spectroscopic examinations) can, by material identification, assist in dating or authenticating art objects or works of art. The availability of these techniques is therefore very important and it enables us to solve specific questions concerning the conservation, restoration and history of artworks [1\u20133].\nIn conservation science, analytical techniques which are non-destructive or micro-destructive are particularly important. Raman spectroscopy is such a technique, allowing material identification from particles down to 1\u00a0\u03bcm. This method has already been used for the investigation of different artefacts, including panel paintings [4, 5], glass [6], wall paintings [7, 8], manuscripts [9\u201311] and historical biomaterials [12, 13]. Although this technique used to be expensive and highly specialised, it is becoming increasingly more accessible to large museums owing to instrumental improvements and lower costs [14\u201317]. However, regional museums often still do not have access to this kind of analytical facility. Use of mobile equipment is one solution to this problem, since several institutions may benefit from a single instrument. In addition, mobile fibre-optic-based Raman instrumentation may facilitate the analysis of artefacts that are difficult or impossible to bring to the laboratory, such as wall paintings on the vault of a chapel [8].\nIn this work we evaluate the use of a mobile fibre-optic-based Raman instrument to perform investigations in a museum context. Different types of artefacts were investigated in a variety of locations, testing and demonstrating the flexibility in instrumental set-up. In particular the experimental conditions during the examination of a virginals on exhibition were challenging. Instrument stability after travelling overseas (between Belgium and Edinburgh, UK) was also thoroughly evaluated.\nExperimental\nObjects were investigated in the conservation and research laboratories, stores and an exhibition gallery of the National Museums of Scotland (NMS), Edinburgh, as well as in the Burrell Collection (Glasgow) (31 October\u201313 November 2005). For these surveys mobile Raman equipment was used, which has been described extensively elsewhere [19]. The core of the equipment consists of a SpectraPro-150i 150-mm spectrometer and a thermoelectrically cooled charge-coupled device detector (Roper Scientific\/Princeton Instruments). The system is also equipped with a 785-nm diode laser for excitation, which has a maximal output power of 300\u00a0mW at the source. In order to avoid damaging the artefacts during these investigations, and depending on the object and the colour of the area under investigation, the output power was limited to a maximum of 10\u00a0mW (measured at the surface). The accumulation time ranged from 10\u00a0s to 1\u00a0h for weak Raman scatterers. For these investigations the 600\u00a0lines\/mm grating was selected. Due to non-ideal focussing conditions, the expected minimal laser spot size of ca. 25-\u03bcm diameter (when working with a \u00d76 infinity-corrected objective lens) could not be reached. In practice we worked with a larger spot size of ca. 50\u00a0\u03bcm. For each area at least 10 spectra were recorded and evaluated in order to tackle paint inhomogeneity at this scale. The experimental configuration was adapted to suit the artefact under study. For example, the probe head could either be mounted horizontally or vertically on the articulating arm and extension tubes could be used on the probe head to facilitate access to the artworks [8, 18].\nResults and discussion\nWhen testing the versatility of a mobile spectrometer in a museum context, the technique needs to be applied to a broad range of artefacts and questions. Throughout this text we will provide several examples, illustrating different facets of this approach. Table\u00a01 gives an overview of the investigated artefacts, together with the analytical challenges that these investigations represent. \nTable\u00a01Overview of the different artefacts that were investigated in this work and the analytical challenges they representInvestigated artefactAnalytical challengeEgyptian sarcophagus (NMS) (A.1907.569A)Pigment identification through a thick varnish layerEgyptian sarcophagus (Glasgow) (1895.167.a)Pigment analysis with strong interference from sunlight. Transport to a different museum, analyse, and return in 1\u00a0dayDean panel (H.KL 72)Analysis of a varnished paintingKalighat painting (A.1912.122)Analysis of work on paperIndian textile banner (K.2005.234)Analysis of a textile artefact of large dimensions with a thin and friable painted decorationMar virginals (H.LT 122)Investigation in the exhibition gallery open to the public\nThe examination of Egyptian sarcophagi, both in the National Museums of Scotland (Edinburgh) and in the Burrell Collection (Glasgow Museums & Art Gallery) illustrate the comparative ease of transportation of the instrument. The capability of the technique to analyse pigments on artefacts covered by a thick varnish layer as well as to undertake the investigation with interference from sunlight are shown. During the examination of a Renaissance painted panel (one of the panels from Dean house, Edinburgh), strong inteference from a fluorescing varnish layer was also encountered. The possibilities of the technique for the non-invasive investigation of fragile artefacts on paper are illustrated by the yellow pigment identification on Indian Kalighat paintings and on a painted textile banner. During the latter survey the articulating arm was used to allow analysis of a large artefact displayed horizontally. Finally, working in the galleries represented an interesting experience as it involved undertaking analytical research whilst taking into account ambient conditions and visitors\u2019 curiosity. This approach is illustrated by the study of the Mar virginals.\nPigment analysis of antique Egyptian sarcophagi\nAn antique Egyptian sarcophagus [A.1907.569 A], dating from the twenty first\/second Dynasty (1069\u2013715B.C.), was investigated at the NMS by using mobile Raman spectroscopy (Fig.\u00a01a). Samples from this artefact had been examined before by Fourier transform (FT) Raman spectroscopy [19]. The aim of the present study was firstly to investigate whether non-destructive mobile Raman spectroscopy confirmed the previous findings. Secondly we wished to investigate the thick yellowed varnish layer, to see how it influenced the investigation, to establish when it was most likely to have been applied and what its visual effect was on the underlying colours.\nFig.\u00a01a Experimental set-up for the investigation of an Egyptian sarcophagus at the NMS. b Stack plot of the baseline-corrected Raman spectrum obtained from the yellow areas of this sarcophagus and the reference spectrum of orpiment (As2S3). (Experimental conditions for the spectrum of the artefact: 60 accumulations of 1\u00a0s, \u00d76 objective, 785\u00a0nm, ca. 5\u00a0mW at the surface)\nAs expected, the yellow varnish generated serious interference of fluorescence radiation which overwhelmed the weaker Raman signal. Unfortunately it was not possible to identify its composition and therefore determine if it was original or whether it was the result of a previous conservation treatment. Nevertheless different pigments on the artefact could be identified. Figure\u00a01b shows that the baseline-corrected Raman spectrum of the omnipresent yellow pigment on this sarcophagus is orpiment (As2S3). Other pigments that could be identified were hematite (Fe2O3), calcite (CaCO3) and carbon black (C). This is in agreement with the findings by Eremin et al. [19].\nAlthough positive results were obtained, it was impossible to record a satisfactory Raman spectrum of the green and blue painted areas of the sarcophagus. It is very likely that Egyptian blue was used in this artefact; however, when stimulated with a 785-nm laser this pigment does not yield a Raman spectrum but gives rise to serious fluorescence background. Eremin et al. [19] indeed identified Egyptian blue with FT Raman spectroscopy (although the sample did not come from exactly the same area), so this illustrates one of the disadvantages of using mobile equipment. In contrast to many laboratory instruments, mobile instrumentation is usually equipped with a single laser, which makes it impossible to switch to another laser wavelength to overcome fluorescence interference.\nA second Egyptian sarcophagus [1895.167.a] was investigated at the Burrell Collection in Glasgow, illustrating one of the advantages of using mobile equipment: several collections can share one instrument. The sarcophagus (Fig.\u00a02a), dating from the ancient Egyptian New Kingdom (1570\u20131070B.C.), was in the conservation lab, with high levels of natural light. The conservator wished to know the identity of the yellow pigment on the cover of the sarcophagus, since orpiment (As2S3) may degrade when exposed to high levels of light. Figure\u00a02b shows that the yellow pigment on the sculptured head and hands of the sarcophagus lid indeed was indeed orpiment (As2S3), whereas the yellow pigment on the other areas was limonite (FeOOH\u00b7 nH2O).\nFig.\u00a02a Experimental set-up for the investigation of an Egyptian sarcophagus in the conservation lab of the Burrell Collection, Glasgow. Ambient sunlight interfered with the investigations. b Stack plot of two Raman spectra obtained from the sculptured head and hands of this sarcophagus. (Experimental conditions: \u00d76 objective, 785\u00a0nm, ca. 5\u00a0mW at the surface, 120 accumulations of 1\u00a0s)\nPigment investigation of a painted panel from Dean House, Edinburgh\nMobile Raman spectroscopy was applied to identify the pigments in one of the Renaissance painted panels from Dean House in Edinburgh. The \u201cDeans Panels\u201d are thought to date from around 1627 [20]. The panel representing the sense of hearing (KL 72) is exhibited on a wall, at a height of about 3\u00a0m, which would have required the construction of scaffolding in the gallery. Therefore it was considered more practical to remove the panel temporarily from the exhibition and bring it to the conservation lab for analysis. The painted surface was found to be covered with a varnish layer, which contributed to the fluorescence background, although it still was possible to identify the pigments in the investigated areas of the artefact.\nVermilion (HgS) was observed in the red areas, whereas orange areas merely contained red lead (Pb3O4). Brownish red areas were painted with a hematite (Fe2O3)-containing paint. On several occasions, mixtures of two of these pigments were encountered. Black areas contained carbon black (C), whereas the white regions were painted by using lead white (2PbCO3\u00b7Pb(OH)2). Pink zones consisted of a mixture of the latter pigment with vermilion; vermilion could also be identified in the purple regions (the blue component of which could not be identified). Finally, the yellow areas examined were painted by using lead\u2013tin yellow, type I (Pb2SnO4). All these pigments are consistent with the early modern origin of the artwork.\nIdentification of the yellow pigment from an Indian Kalighat painting\nThe yellow pigment from different areas from an Indian Kalighat painting was examined by using mobile Raman spectroscopy. These are nineteenth century Indian watercolour and opaque media paintings on industrial paper. Since paper artefacts are fragile, special care has to be taken not to damage them during handling and positioning of the spectrometer. Gloves were worn and positioning of the probe head was performed with great care. On all the yellow painted areas of the Kalighat paintings that were examined, the same yellow pigment was encountered. Figure\u00a03 shows the Raman spectrum obtained, along with the reference spectrum of chrome yellow (PbCrO4). It is clear that the yellow pigment in the Kalighat paintings is chrome yellow.\nFig.\u00a03Stack plot of the Raman spectra obtained from a yellow area from an Indian Kalighat painting and the reference spectrum of chrome yellow (PbCrO4). (Experimental conditions for the spectrum of the artefact: 8 accumulations of 150\u00a0s, \u00d76 objective, 785\u00a0nm, ca. 3\u00a0mW at the surface)\nAnalysis of a painted banner\nThe National Museums of Scotland has a large (1,080\u00d72,030\u00a0mm) textile banner of uncertain provenance in its collection. Stylistic interpretations of the polychrome design suggest an Indian origin. The banner depicts, on a green background, colourful imagery of the different stages on the way to heaven (Fig.\u00a04b, detail). Because of its large dimensions and the absence of appropriate non-destructive analytical methods, no previous investigation had been made and little was known of the materials that have been applied. The mobile Raman spectrometer was therefore used to identify the pigments.\nFig.\u00a04a Experimental set-up for the investigation of a large textile banner. b Lower part of the investigated textile banner. c Raman spectrum of an orange area of the textile banner, together with the reference spectra of massicot (PbO) and red lead (Pb3O4). (Experimental conditions for the spectrum of the artefact: 1 accumulation of 150\u00a0s, \u00d76 objective, 785\u00a0nm, ca. 10\u00a0mW at the surface). d Raman spectrum of a red area of the textile banner and reference spectrum of vermilion. (Experimental conditions: 1 accumulation of 150\u00a0s, \u00d76 objective, 785\u00a0nm, ca. 5\u00a0mW at the surface)\nIn order to be able to examine a range of positions on the surface of the banner, the articulating arm was used for macro-positioning of the probe head, while the artefact was placed horizontally on a table (Fig.\u00a04a). All the paint surfaces were thin, and in several areas the paint was friable or had already been lost. Red areas gave rise to the Raman spectrum of vermilion (HgS, Fig.\u00a04d), whereas the orange areas yielded a more complex spectrum, which consists of a combination of massicot (PbO) and red lead (Pb3O4) (Fig.\u00a04c). Red lead is an orange pigment, whereas massicot has a more yellowish shade. It is not clear whether massicot was intentionally added to the paint or whether the latter pigment arose as a side-product of the synthesis of red lead. Other pigments that were positively identified in the banner were calcite (CaCO3), carbon black (C), lead white (2PbCO3\u00b72 H2O), azurite (2CuCO3\u00b7Cu(OH)2) and anatase (TiO2). Under the ambient conditions of the conservation laboratory, which did not have full light exclusion, and using the 785-nm laser, it was impossible to obtain a spectrum of sufficient quality to identify the green areas on the artefact. One solution to overcome this problem would be to use a Raman instrument with a different laser, with a smaller absorption cross section for the green pigments.\nInvestigation of an early keyboard instrument, the Mar virginals\nThe Mar virginals is an early keyboard instrument, which, although probably made in the Low Countries, has a long association with Lady Mary Stewart, countess of Mar and could have been made in Scotland. It probably dates from between A.D. 1560 and 1660, and its lid and soundboard contain three separate painted panels, the central one depicting Orpheus playing to the animals. We wished to compare the paintings on the lid to those on the soundboard, since similar or different pigment compositions may support or disprove the idea that the lid is contemporary to the virginals.\nThis artefact had to be analysed while on display in the museum gallery, which enabled us to evaluate the feasibility of undertaking the analytical work in a public space. It was necessary to ensure that there were no health and safety risks, especially from direct or scattered light from the laser beam, as well as keeping the electronics and power supply cables and units away from enquiring fingers. The visiting public were clearly intrigued with what was going on and to help explain this, a poster informed the visitors about the experiments.\nWorking in the gallery also meant that the ambient conditions were far from ideal. During this study, two issues severely hampered the Raman investigations: background light and spatial limitations. It was not possible to reduce the intensity of background light to avoid interference which tended to overwhelm the spectrum, nor on this occasion to undertake the work overnight. And since the Mar virginals was not taken out of its display case, little space was available to align the probe head with the painted surface, the cramped conditions meaning that great care had to be taken not to touch the artefact. To maximise flexibility the probe head was mounted on an articulating arm and extension tubes [8] were used to approach the painted surface (Fig.\u00a05).\nFig.\u00a05Experimental set-up for the investigation of the Mar virginals\nNevertheless, despite these non-ideal conditions, the pigments could be identified. White lead (2PbCO3\u00b7Pb(OH)2) is omnipresent: almost all the recorded Raman spectra contain the intense Raman band at 1,056\u00a0cm\u22121, whereas the less intense bands of white lead at 260, 203 and 154\u00a0cm\u22121 are only observed in a few spectra. Apparently, this pigment was applied to modify the hue of the coloured areas, making them appear brighter. From the intense Raman bands at 254, 283 and 343\u00a0cm\u22121, it is clear that the red areas were painted with the red pigment vermilion (HgS), whereas the bright yellow areas were painted by using orpiment (As2S3). Carbon black (C) is the black pigment which was used in the painted decoration.\nIn general, the blue areas of the painting did not yield Raman spectra of sufficient quality to allow identification. The reasons for this are multiple. In general, blue and green pigments absorb the red (785\u00a0nm) laser light which hinders the recording of a spectrum. In addition there was a large amount of background radiation, both from the gallery lights and from fluorescence, which was probably caused by the varnish layer. It is very likely that the blue pigment azurite (2CuCO3\u00b7Cu(OH)2) is used in the artwork, since this pigment was very commonly used in Renaissance period: however, unfortunately azurite happens to be a weak Raman scatterer when using a 785-nm laser, which means that small amounts of background radiation easily overwhelm the Raman spectrum.\nAlthough only a few areas on the soundboard could be examined, the pigments on the lid were in good agreement with those on the soundboard. Despite the fact that this research project should be considered as a pilot study, this tends to support the thesis that the lid and the soundboard originate from the same instrument, or at least were painted at a similar period. However other approaches, such as stylistic studies, or taking samples to matching the spore-element compositions of the paint from the lid and the soundboard [9], are needed to be more definite. Since the investigation of this rare musical instrument was performed in one day, time was confined and only a limited number of areas could be examined. In fact, except for the absence of lead\u2013tin yellow, none of the pigments were rare for an artwork of this period.\nConclusions\nThis work reports on the application of mobile Raman spectroscopy to the non-invasive investigation of a range of object of arts in a museum context. The different cases presented here illustrate different obstacles and analytical challenges that are often encountered during direct Raman investigations of museum objects. The investigation of two antique Egyptian sarcophagi shows the possibilities of the technique to deal with artefacts covered by a thick varnish layer as well as the investigation with the interference of ambient sunlight in the conservation laboratory. The investigation of the Dean House panel illustrates the possibilities of pigment investigation although a fluorescing varnish layer is present; the suitability of the method to analyse fragile artefacts was demonstrated by the investigation of Indian Kalighat paintings on paper; the textile banner was both fragile and large. Finally, the Mar virginals has been examined for the first time directly in the exhibition gallery with normal lighting conditions and with visitors present. All these examples illustrate that non-destructive, mobile Raman spectroscopy is able to cover a broad field of research questions on artistic and historical objects, in a range of different experimental conditions, encountered in a museum context.","keyphrases":["raman spectroscopy","conservation science","mobile raman equipment","art analysis","non-destructive investigation"],"prmu":["P","P","P","R","R"]}
{"id":"Ann_Biomed_Eng-2-2-1705526","title":"The Effects of Time Varying Curvature on Species Transport in Coronary Arteries\n","text":"Alterations in mass transport patterns of low-density lipoproteins (LDL) and oxygen are known to cause atherosclerosis in larger arteries. We hypothesise that the species transport processes in coronary arteries may be affected by their physiological motion, a factor which has not been considered widely in mass transfer studies. Hence, we numerically simulated the mass transport of LDL and oxygen in an idealized moving coronary artery model under both steady and pulsatile flow conditions. A physiological inlet velocity and a sinusoidal curvature waveform were specified as velocity and wall motion boundary conditions. The results predicted elevation of LDL flux, impaired oxygen flux and low wall shear stress (WSS) along the inner wall of curvature, a predilection site for atherosclerosis. The wall motion induced changes in the velocity and WSS patterns were only secondary to the pulsatile flow effects. The temporal variations in flow and WSS due to the flow pulsation and wall motion did not affect temporal changes in the species wall flux. However, the wall motion did alter the time-averaged oxygen and LDL flux in the order of 26% and 12% respectively. Taken together, these results suggest that the wall motion may play an important role in coronary arterial transport processes and emphasise the need for further investigation.\nIntroduction\nThe formation of an atherosclerotic plaque is dependent upon a range of factors including local variations in haemodynamics, the transport of chemical species and cells from the blood to the arterial wall and their accumulation within the arterial wall.3,21,38The local susceptibility of coronary arteries to atherosclerosis has been putatively linked to the physiological motion arising out of their anatomical attachment to the epicardial surface of the beating heart.7,8Coronary arteries exhibit highly complex wall motion patterns such as bending, stretching, twisting, vessel torsion and vessel displacement due to the cardiac and respiratory motion of the heart.7,22 Despite their seemingly important role, to date, only a handful of studies have looked at the effects of wall motion on atherogenesis.\nEarly studies investigated the axial and lateral movement of a straight tube model of the coronary arteries and showed that the wall shear stress (WSS) patterns were significantly affected by the axial movement and not so by the lateral movement.4,23Other studies examined the effects of time dependent curvature on the flow field and demonstrated that the flow patterns were affected by the wall motion due to instantaneous changes in curvature and that the WSS patterns were strongly dependent on the frequency of curvature variation.18,24,32,33Another study considered the cyclic flexion of coronary arteries and reported that the plaque advancement was proportional to the flexion angle.37\nMore recently, studies have concluded that the dynamic curvature effects could play an important role in the flow and WSS patterns in curved and in bifurcating arteries.28,40,41Nevertheless, these studies reported that the temporal WSS variations produced by the wall motion was only secondary to that caused by the pulsatility of blood flow.28,41So far studies have only paid attention to the haemodynamic effects of wall motion. However, it has now come to light that the transport of atherogenic macromolecules such as LDL and solutes such as oxygen from the blood to the arterial wall play an important role in atherogenesis.38 Interestingly, with the exception of a single study, no other past study has investigated the effects of species transport to the moving walls of coronary arteries.17 Even in that study only the effects of diameter variation (<6% of mean diameter) on oxygen transport was considered.\nHence, the aim of our work was to determine the effects of wall motion on mass transport patterns in coronary arteries with physiological flow. For our investigation, we chose a geometric model of the left anterior descending coronary artery (LAD) because of its high predilection to atherosclerosis among the major coronary arteries.12 Computer aided design (CAD) models of the LAD were employed in this study as opposed to realistic geometries to simplify the complexity of the problem definition, solution and analysis of results so as to gain a fundamental understanding of the haemodynamic effects of wall motion in coronary arteries. The species of interest in this study were low-density lipoprotein (LDL) and oxygen.\nMethods\nComputational Model\nA 3-D computational model of the LAD (Fig.\u00a01) was constructed based on the average LAD dimensions (diameter (d)\u00a0=\u00a00.36\u00a0cm and mean radius of curvature (Rmean)\u00a0=\u00a04\u00a0cm),9,15,16using the CFD pre-processor Gambit (v2.0.4 \u2013 Fluent Inc., Lebanon, NH). The total length (l) of the LAD considered in this study was 4.68\u00a0cm which was equivalent to 13 tube diameters. In order to make the simulation as simple as possible, the model was assumed to be rigid with a circular cross section of constant radius and a constant uniplanar curvature over its entire length at any instant in time. An unstructured grid composed of hexahedral mesh elements with a thin boundary layer was generated using the Cooper meshing scheme2 with strict adherence to mesh quality metrics.\nFigure\u00a01.A schematic model of the left anterior descending coronary artery (LAD).\nBlood Flow Boundary Conditions\nThe flow boundary conditions controlling the model included no slip at the tube wall, velocity inlet at the inlet, symmetry condition at the plane of symmetry and a traction free outflow condition. The LAD inlet velocity profile was assumed to be parabolic. A pulsatile physiological LAD inlet velocity waveform (Fig.\u00a02) derived from a previous publication by Marcus et al.22 was applied at the inlet boundary through a user defined function. The time-averaged mean velocity (\u03bd), mean flow rate (qo) Reynolds number (Re) and Dean number (\u03ba) corresponding to this flow waveform were 9.715\u00a0cm\/s, 0.99\u00a0ml\/s, 105 and 22.3. The Dean number is defined as below\nFigure\u00a02.The LAD inlet flow and curvature waveform.\nThe Womersley parameter (\u03b1) of the flow was 2.78 and the period of a cardiac cycle (T) was 0.8\u00a0s. Blood was considered to be an incompressible, homogeneous, Newtonian fluid with a viscosity (\u03bc) of 0.0035\u00a0Pa\u00a0s and a density (\u03c1) of 1050\u00a0kg\/m3. Past arterial flow dynamic studies have shown that these assumptions are reasonable enough to use in haemodynamic studies.16,29\nWall Motion Boundary Conditions\nThe wall motion boundary condition used in this study was based on the model presented by Santamarina et al.32 The main components of coronary artery motion are solid body motion and deformation. The deformation parameter (\u03b5) was calculated as below  where R is the radius of curvature of the LAD. The present study used a deformation parameter of 0.6 which falls within the physiological range of LAD deformations.15 A sinusoidal function as given below was used to specify the time dependent variation of curvature of the LAD (Fig.\u00a02)  where \u03c9 is the angular frequency of deformation. A user-defined function was written to apply this curvature waveform as a grid motion boundary condition. The tube inlet was fixed constant in the simulations at all times. This allowed the centre of curvature to vary sinusoidally in a cardiac cycle.\nMass Transfer Boundary Conditions\nThe mass transport of species in the arterial lumen is mathematically described using the three dimensional convection-diffusion equation  where C is the concentration of species, u is the velocity vector and D is the diffusivity of species in the blood.\nIn this study, the transport of LDL and oxygen from the blood to the endothelium (fluid phase), and across the endothelium (transendothelial) were modelled. The fluid phase transport of both LDL and oxygen takes place via convection and diffusion. However, the transendothelial transport of LDL and oxygen are governed by different mechanisms due to their relative size and the endothelial function. The semi permeable endothelial membrane, acts as a barrier for the entry of macromolecules such as LDL into the arterial wall, but allows blood plasma and small solutes such as oxygen to pass through it.10 This is reflected in the permeability of LDL (K\u00a0=\u00a02\u00a0\u00d7\u00a010\u22128cm\/s) which is five orders of magnitude smaller than that of oxygen (K\u00a0=\u00a02\u00a0\u00d7\u00a010\u221203cm\/s).36 This indicates the need for modelling LDL and oxygen transport differently through appropriate mass transport boundary conditions.\nLDL transport was modelled as a concentration polarisation effect, a phenomenon which has been demonstrated to occur in large and medium sized arteries.5,11,39Concentration polarisation is the accumulation and elevation of endothelial surface concentration of LDL due to plasma filtration through the semi permeable endothelium. LDL mass transport boundary conditions applied in this study were uniform inlet concentration, Co (=1.2\u00a0mg\/ml of blood),36 a zero gradient in concentration at the outlet and mass conservation of LDL at the luminal surface of the endothelium39 described as  where Vw is the plasma filtration velocity normal to the wall (=4\u00a0\u00d7\u00a010\u22126cm\/s),36Cw is the endothelial surface concentration of LDL, n is the unit vector normal to the vessel wall, D is the physiological LDL diffusivity (=5\u00a0\u00d7\u00a010\u22128cm2\/s)39 and K is the overall mass transfer coefficient of LDL which was considered equivalent to the physiological endothelial permeability of LDL. Equation (5) was rearranged as below to implement a wall reaction boundary condition in the CFD solver. \nIn this study, oxygen transport was modelled by applying a passive transport law for oxygen flux at the wall.31 It is known from previous estimates that the diffusion velocity of oxygen is orders of magnitude greater than the filtration velocity.1 Hence, the flux of oxygen to the wall is essentially the diffusive flux. To satisfy flux continuity at the wall the diffusive flux of oxygen to the wall should be equal to the wall flux of oxygen which can be modelled with a constant permeability wall boundary condition.31 Oxygen mass transport boundary conditions employed in this study were uniform inlet oxygen mass fraction (=0.005),20 a zero gradient in outlet concentration and a constant permeability wall boundary condition31 which is given as  where the oxygen diffusivity was 1\u00a0\u00d7\u00a010\u22125cm2\/s1.\nNumerical Method\nThe commercial CFD solver Fluent (v6.1.22 \u2013 Fluent Inc., Lebanon, NH) was used to simulate species transport in the moving LAD model. Fluent uses a finite volume technique to solve the three-dimensional (3-D) unsteady equations of momentum, mass and species conservation. QUICK scheme was employed to solve the flow and species transport equations. The time discretization and pressure\u2013velocity coupling were achieved through second order time implicit, and PISO schemes respectively. The dynamic mesh model feature of the Fluent was used to implement the deformation of the model according to Eq. (3). The dynamic mesh model uses an arbitrary Lagrangian\u2013Eulerian approach to move the mesh nodes. At each time step, the volume mesh is updated automatically by the solver based on the new co-ordinates of the boundary zones that were calculated by the user-defined function. At the beginning of each simulation, the radius of curvature of the model was at its maximum (=6.4\u00a0cm). At t\u00a0=\u00a00.2 s and 0.6 s the radius of curvature of the model was equivalent to its mean value.\nModelling Approach\nTo separate the effects of blood flow pulsatility and wall motion on mass transport patterns in coronary arteries so as to determine their relative importance, we undertook the following four different numerical simulations:Case 1: Steady flow inlet (qo\u00a0=\u00a00.99\u00a0ml\/s) in the static geometry with the mean radius of curvature (R\u00a0=\u00a04\u00a0cm).Case 2: Pulsatile flow inlet in the static geometry with the mean radius of curvature (R\u00a0=\u00a04\u00a0cm), to isolate the haemodynamics effects of flow pulsatility.Case 3: Steady flow inlet (qo\u00a0=\u00a00.99\u00a0ml\/s) with dynamic geometry, to isolate the haemodynamics effects of wall motion.Case 4: Pulsatile flow inlet with dynamic geometry, to study the combined effects of pulsatility and wall motion.\nModel Validation\nThe computational method was validated by simulating Santamarina et al.\u2019s32 coronary artery motion study in a curved model of exactly similar dimensions under identical flow and wall motion conditions. The flow and wall shear stress (WSS) patterns agreed well with that of Santamarina et al\u2019s study and the maximum difference in the wall shear rate was less than 2%. The mesh and time step independence of the solutions were investigated by successively refining the grid and the time step size by a factor of two. Computational solutions obtained with three different mesh densities (80,310, 156,861 and 310,464 nodes) and time step sizes (0.025\u00a0s, 0.0125\u00a0s and 0.00625\u00a0s) were then compared against each other (Fig.\u00a03). Mesh and time step independence were considered to have been achieved when the maximum difference between the time-averaged normalised outer and inner wall flux of LDL (Cw\/Co) between the successive simulations was less than 2% at any axial location. The outer and inner walls were defined as the wall regions farthest and shortest from the centre of curvature to represent epicardial and myocardial surface of the curved coronary arteries. This study was only carried out for LDL transport in the dynamic model with pulsatile flow because this was the computationally most challenging problem (Schmidt number, \u03bc\/\u03c1D\u00a0=\u00a06.7\u00a0\u00d7\u00a0105 & Peclet number, dv\/D\u00a0=\u00a07\u00a0\u00d7\u00a0107) encountered in this study. Based on these criteria, a mesh containing 156,861 nodes with a time step size of 0.0125\u00a0s was chosen as the optimum case for simulation.\nFigure\u00a03.Comparison of the time-averaged normalised LDL wall flux predicted with three different meshes and time step sizes to demonstrate mesh (top panel) and time step (bottom panel) independence of the computational solutions.\nResults\nNumerical simulations of oxygen and LDL transport in a 3-D curved tube model of the LAD were carried out under physiological flow and mass transport conditions. The computational solutions concerned the prediction of flow, WSS and mass transport patterns. The results will be presented only for the first 10 diameters length as this length is more relevant to realistic coronary flow situation as discussed elsewhere.32\nVelocity Patterns\nThe temporal velocity magnitude (coloured contour plots) and secondary flow patterns (vector plots) in the static and dynamic models of the LAD are shown in Fig.\u00a04. The fundamental flow patterns in a curved tube flow situation with a characteristic velocity skewing towards the outer wall of bend and the formation of Dean vortices were clearly seen in all the models (Fig.\u00a04). Although both the pulsatility (Fig.\u00a04b) and wall motion (Fig.\u00a04c) produced time dependent velocity skewing, the flow pulsation had a greater effect on velocity skewing than did wall motion. The secondary flow was also affected by both the pulsatility and wall motion (Fig.\u00a04e and f). However, the wall motion induced time dependent curvature (Fig.\u00a04f) seemed to have a major influence on the secondary flow patterns. Adding the dynamic motion to the pulsatile flow reduced skewing over the entire cycle (Fig.\u00a04d). Moreover, this combination led to a reduction in secondary flow when the time dependent vessel curvature was small and vice versa during the peak curvature phases (Fig.\u00a04g). In general, both the velocity skewing and secondary flow increased with increase in curvature and the flow rate (Fig.\u00a04).\nFigure\u00a04.Temporal axial and secondary flow patterns in a plane located at 9\u00a0diameters from the inlet in the (a) steady flow static model (R\u00a0=\u00a04\u00a0cm, qo\u00a0=\u00a00.99\u00a0ml\/s), (b and e) pulsatile flow static model (R\u00a0=\u00a04\u00a0cm), (c and f) steady flow dynamic model (qo\u00a0=\u00a00.99\u00a0ml\/s), and (d and g) pulsatile flow dynamic model. The outer and inner walls are located at the top right corner and top left corner of individual figures respectively.\nWSS Patterns\nThe effects of pulsatility and wall motion on the instantaneous WSS distribution in a cardiac cycle at two axial locations (5D & 9D) are plotted in Fig.\u00a05. The effect of motion in steady flow at the outer wall was to increase the WSS when the curvature was maximum. Along the inner wall the opposite effects were seen with a drop in the WSS at the maximum curvature. The wall motion also produced asymmetric WSS distribution over time in the steady flow dynamic model despite the imposed symmetric wall motion curve.\nFigure\u00a05.Temporal axial inner and outer WSS distribution at 5\u00a0diameters (top panel) and 9\u00a0diameters (bottom panel) from the LAD inlet.\nThe pulsatility of blood flow caused time varying WSS distribution along the outer and inner walls. The WSS increased with the pulsatile flow rate and overall this increase was greater along the outer wall. Hence peak WSS was noted along the outer wall at maximum flow rate. Together, wall motion and flow pulsatility produced instantaneous variations in the outer and inner WSS which were somewhat similar to the WSS distribution in the pulsatile flow static model. Although the gross distribution of WSS primarily followed the flow rate curve, the dynamic curvature effects were evident in the central section where the location and magnitude of peak WSS were different from that of the pulsatile flow static model.\nThe time-averaged axial WSS distribution at the outer and inner walls in all the models is shown in Fig.\u00a06. In general, the spatial dependence of time averaged WSS was evident in all the models. In the steady flow static model the outer WSS increased to a peak and thereafter was found to decrease very gradually. In contrast, the inner WSS decreased to a minimum and was thereafter found to increase slowly. In comparison, the flow pulsatility caused a big increase in the outer WSS in the pulsatile flow static model. However, the wall motion only produced a little change in the outer WSS in the steady flow dynamic model.\nFigure\u00a06.Time-averaged axial WSS distribution along the inner and outer walls of the LAD.\nThe combined effects of wall motion and pulsatility caused an increase in the outer WSS up to its peak value and a subsequent decrease in the WSS in comparison with that of the pulsatile flow static model, and an overall increase in the outer WSS when compared to the steady flow models. Along the inner wall the flow pulsatility and the wall motion produced contrasting effects whereby the former decreased the WSS and the later increased the WSS. Due to this opposing effect of wall motion and flow pulsatility their combination had only caused smaller variations in the WSS distribution.\nOxygen Transport\nThe time dependent non-dimensional oxygen wall flux (KCw\/KCo) distribution at 5D and 9D is presented in Fig.\u00a07. The oxygen wall flux did not vary appreciably over time in a cardiac cycle. At any instance the outer wall oxygen flux was higher than that at the inner wall. These patterns were similar in all the models studied. When compared to the oxygen wall flux in the steady flow static model, pulsatility caused a reduction and wall motion an increase in the oxygen wall flux along the inner wall. Along the outer wall the effects of pulsatility and wall motion were opposite to this. In total, the net effect of pulsatility and wall motion was an increase in oxygen flux at the inner wall and decrease at the outer wall when compared to the steady flow static model. This effect was more prominent in the distal region (Fig.\u00a07c and d).\nFigure\u00a07.Temporal normalised oxygen flux to the inner and outer walls at 5\u00a0diameters (top panel) and 9\u00a0diameters (bottom panel) from the LAD inlet.\nThe time-averaged non-dimensional oxygen wall flux to the outer and inner walls of all the four models are shown in Fig.\u00a08. The overall effects of pulsatility and wall motion were similar to that predicted with the time dependent oxygen wall flux distribution. The effect of flow pulsation was to decrease the oxygen flux to the inner wall. In contrast, the wall motion contributed to an increase in oxygen flux in the distal region of the inner wall. Hence the combined effects of wall motion and flow pulsatility were to decrease oxygen flux in the proximal and medial regions of the inner wall and a very gradual increase distally.\nFigure\u00a08.Time-averaged normalised oxygen wall flux distribution along the inner and outer walls of the LAD.\nAlong the outer wall after an initial sharp drop the flow pulsation caused a gradual increase and then a gradual decrease in the oxygen flux. The wall motion produced a net reduction in the oxygen flux to the outer wall from the inlet. When both the wall motion and flow pulsatility were included the oxygen flux decreased sharply closer to the inlet followed by a gradual increase in the medial region and a gradual decrease thereafter. Nevertheless, in all the models the time-averaged outer wall oxygen flux was more than that to the inner wall.\nLDL Transport\nThe time dependent non-dimensional LDL wall flux (KCw\/KCo) distribution at 5D and 9D is plotted in Fig.\u00a09. Like oxygen wall flux the LDL wall flux did not exhibit notable variations over time. The flow pulsatility caused an increase in LDL flux to the inner wall and a decrease in LDL flux to the outer wall when compared to the LDL flux distribution in the steady flow static model. In contrast, the wall motion induced LDL wall flux was lower along the inner wall. However, along the outer wall it produced a differential distribution whereby the LDL wall flux was lower than that in the steady flow static model at 5D and vice versa at 9D (Fig.\u00a09c and d). At 9D the outer wall LDL flux was greater than that along the inner wall. Also the outer wall flux in the steady flow dynamic model was higher than that in the pulsatile flow static model. Hence the net effect of wall motion and pulsatility produced LDL wall flux distribution in the pulsatile flow dynamic model such that it was between the range of LDL wall fluxes caused by the pulsatility and wall motion separately.\nFigure\u00a09.Temporal normalised LDL flux to the inner and outer walls at 5\u00a0diameters (top panel) and 9\u00a0diameters (bottom panel) from the LAD inlet.\nThe time-averaged non-dimensional LDL wall flux to the outer and inner walls of all the four models are shown in Fig.\u00a010. In all the models the time-averaged LDL wall flux distribution was seen to vary appreciably with respect to the axial distance from the inlet. The flow pulsatility caused an increase and decrease in the flux along the inner and outer walls respectively when compared to the steady flow static model. In contrary, the wall motion caused a reduction in the inner wall LDL flux when compared to that of the steady flow static model. Whereas the outer wall LDL flux was lower and higher than that in the steady flow static model in the medial and distal regions respectively. The combined effects of pulsatility and wall motion led to a substantial increase in the outer wall LDL flux in the distal regions. In the proximal and medial regions the outer wall LDL flux was lower than that to the inner wall. However, the inner wall LDL flux increased to a maximum and was then seen to drop off to a value lower than that along the outer wall in the distal part.\nFigure\u00a010.Time-averaged normalised LDL flux distribution along the inner and outer walls of the LAD.\nDiscussion\nTo determine the effects of wall motion, a factor which has received less attention in the previous mass transport studies in coronary arteries, mass transport patterns were computed in an idealised moving model of the LAD. In this study, LDL and oxygen transport were simulated because of their important role in atherogenesis. The flow patterns reported in the current study (Fig.\u00a04) qualitatively agree with the findings of past experimental and computational studies in that they all demonstrate a general trend of velocity skewing towards the outer wall of curvature and the appearance of in-plane secondary flow.16,27,29,32The centripetal acceleration of the fluid due to the arterial curvature was primarily responsible for these flow patterns (Fig.\u00a04a). This basic flow feature was further altered by the flow pulsatility (Fig.\u00a04b and e) and the dynamic curvature (Fig.\u00a04c and f). Their relative role on secondary flow can be characterised using the non-dimensional Dean number (\u03ba).\nFrom the definition of the Dean number (Eq. 1), it is clear that higher the flow rate or smaller the radius of curvature the stronger the Dean vortices will be. This was evident in the figures as the magnitude of skewing and the strength of Dean vortices increased with increasing flow rate and curvature and were maximum at maximum flow rate in the pulsatile flow static model and at maximum curvature in the steady flow dynamic model. Therefore, the combination of flow pulsatility and wall motion were responsible for the appearance of time dependent flow patterns in the pulsatile flow dynamic model (Fig.\u00a04d and g). The flow patterns seen in this study are qualitatively similar to that of Santamarina et al\u2019s work which considered the effects of dynamic curvature.32\nThe flow patterns at t\u00a0=\u00a00.2\u00a0s and 0.6\u00a0s in the steady flow dynamic model where the time dependent curvature was equal to the mean curvature appeared to be different from that in the steady flow static model (Fig.\u00a04c and f). This suggests that a quasi-static model cannot predict the flow patterns accurately and that the dynamic wall motion could be as important as the instantaneous geometry. Similar findings were reported by Ethier et al. who compared the flow patterns in frozen geometries and in a moving model of the right coronary artery segment.41\nThe WSS patterns calculated in the pulsatile flow static models are similar to those reported by other studies on coronary artery flow.16,27,29Since the WSS is directly proportional to the gradient of velocity the WSS patterns followed the trends of velocity distribution. The velocity skewing towards the outer wall subjected the outer and inner walls to higher and lower WSS respectively except up to a distance of 1D where the entrance effects were dominant (Fig.\u00a06). The instantaneous and mean WSS distribution in the pulsatile flow dynamic model closely followed that of the pulsatile flow static model except in the central section where the curvature was high. These results suggest that the effects of wall motion on temporal WSS patterns are only secondary to the pulsatile flow effects and agree well with the findings of other dynamic model studies.28,41\nConcentration polarisation of LDL has been demonstrated to occur in large arteries by several past experimental and numerical studies.5,11,39The build-up of LDL concentration at the apical surface of the endothelium due to concentration polarisation could increase the influx of LDL into the arterial wall.5,35 This, in turn, could initiate a sequence of biological and biophysical events that are associated with atherogenesis.5,25 Therefore, LDL transport was modelled as a concentration polarisation effect and the results were presented as normalised LDL wall flux (KCw\/KCo).\nThe present study also considered oxygen transport because hypoxia or low oxygen tension in the arterial wall has long been considered to be a factor responsible for atherogenesis.1,17It induces endothelial cell apoptosis, causes breakdown of endothelial barrier and forms interendothelial gaps which may cause lipid infiltration into the arterial wall. Recent evidences suggest that hypoxia may also increase vascular permeability to macromolecules by up regulating vascular endothelial growth factor (VEGF) release by vascular cells.38 Hence oxygen transport was modelled with a passive transport law for oxygen flux at the wall.31 To describe oxygen transport patterns normalised oxygen wall flux (KCw\/KCo) was calculated.\nIn general, the mass transport patterns were similar in all the models. The establishment of low velocity, low WSS regions along the inner wall due to reasons discussed earlier led to an overall elevation of LDL flux and a reduction in oxygen flux to the inner wall. In contrast, high axial flow and high WSS along the outer wall due to axial velocity skewing caused a reduction in LDL flux and an increase in oxygen flux to the outer wall. The higher the WSS the higher the convection and the convective transport of oxygen. Hence the oxygen flux to the outer wall was more than that to the inner wall. The opposite effects were noted for LDL transport because the degree of concentration polarisation is reduced where high flow and high WSS prevail.39 As a result, the LDL accumulation was generally lower along the outer wall and higher along the inner wall. The diffusive transport of LDL and oxygen however, had minor influence on their transport. This was especially true because of their constant low diffusion coefficients. These results qualitatively agree with mass transport studies in curved arterial transport models.17,17,39\nDespite the large time dependent variations in the blood flow and WSS patterns, the flow pulsation only had a minimal effect on oxygen (Fig.\u00a07) and LDL (Fig.\u00a09) transport to the arterial wall. Consequently, the time-averaged oxygen and LDL wall fluxes (Figs.\u00a08 and 10) in the static pulsatile flow model were only slightly different from their instantaneous values. This less significant pulsatile effects on time-averaged mass transport patterns have also been reported by other studies.11,19,34\nSchneiderman et al.34 simulated oxygen transfer in straight arterial segments and concluded that the pulsatile flow has negligible effect on oxygen transport to the wall. Ma et al.19 studied the effect of pulsatility on mass transfer in a sudden expansion region and reported that the time-averaged pulsatile mass transfer patterns were similar to the steady flow mass transfer patterns. Fatouraee et al.11 simulated LDL concentration polarisation in a straight tube model of a carotid artery under pulsatile flow conditions. The variation of LDL wall concentration in their study was less than 0.014%.\nTime dependent variations in mass transport patterns were also not present in the dynamic models (Figs.\u00a07 and 9). As a result, the magnitude of time-averaged oxygen and LDL wall fluxes were only slightly different from their corresponding time dependent flux values (Figs.\u00a08 and 10). Although the wall motion did not significantly affect the temporal mass transfer patterns in the dynamic models, an overall change in the mass transfer patterns was evident (Figs.\u00a07 and 9). This change was also reflected in the time-averaged mass transfer patterns in the dynamic models (Figs.\u00a08 and 10). The percentage change in time-averaged oxygen wall flux and LDL wall flux along the inner and outer walls between the static and dynamic models under both pulsatile and steady flow conditions was in the order of 26% and 12% respectively.\nThe fact that the pulsatility did not alter mass transfer patterns and that there were variations in mass transport patterns in the dynamic models suggest that the wall motion may indeed be important in determining the time-averaged mass transport patterns in the dynamic models. These variations were not significant in the proximal regions of the LAD model. However, considerable changes in the mass transport patterns were present in the medial and distal regions of the LAD (Figs.\u00a08 and 10). This regional difference occurred due to the specification of fixed inlet boundary condition. Although this is an effect of the fixed inlet boundary condition these results once again indicate that strong motion of the coronary arteries could have a significant impact on the luminal mass transport patterns.\nThe species transport however, does not entirely depend on the WSS distribution. Karino et al drew a scatter plot to establish a relationship between the WSS and LDL surface concentration.39 The scatter in their data suggests that the species transport is also influenced by global flow patterns, path line of fluid flow, spatial location and residence time of LDL at a site. This was also found in the present study which noticed different values of LDL and oxygen wall flux for the same WSS value along the outer and inner walls (Figs.\u00a06, 8 and 10).\nThe inner walls of a curved artery where low WSS prevails is known to be a predilection site for atherosclerosis.14,21 LDL and oxygen transport studies have reported impaired oxygen flux and enhanced LDL accumulation in the low shear regions.5,17,17,39Similar to these findings the present study also predicted co-localisation of low WSS regions, high LDL flux regions and hypoxic regions along the inner wall of curvature (Figs.\u00a06 and 10) in both the static and dynamic models. Hence our results suggest that even when the wall motion is considered the susceptibility of inner wall regions of the curved arteries to atherosclerosis is maintained.\nBack et al.1 investigated the relationship between oxygen transport and lipoprotein accumulation in various arterial configurations. They noticed a correspondence between oxygen deprivation (hypoxia) and enhanced lipoprotein accumulation in decelerated flow regions and separation locations and concluded that the oxygen transport behaves in an opposite way to lipoprotein accumulation. This contrasting behaviour of oxygen and LDL transport was also noticed in our present study. It would be interesting to study the indirect effects of hypoxia on LDL transport through pathways involving enhancement of endothelial permeability for macromolecules.38\nThe present study assumed a constant endothelial permeability of oxygen and LDL which, in reality, is spatially variable and is dependent on the concentration of species at the arterial wall surface and the WSS.26,36Pulsatile variations in the WSS has been shown to alter the morphology of endothelial cells, increase macromolecular permeability to LDL, promote transendothelial diffusion and intimal entrapment of atherogenic particles and enhance oxygen wall flux.13,21Therefore, it is possible that the temporal variations in WSS caused both by the pulsatility of blood flow and wall motion may have an effect on LDL and oxygen transport if shear dependent variations in physiological parameters including the endothelial permeability are considered in mass transport studies. So far their effects on species transport have only been considered in static models30,36and it remains to be investigated how these factors would influence mass transport in moving arteries. This will be the subject of our future study.\nThe limitations of this study centre on the assumptions that were made in the construction of computational models which include idealized geometry, wall motion, and biophysical factors governing LDL and oxygen transport. An idealised, uniformly curved model without complex geometric features such as tortuosity, twisting and non-uniform cross sections was used. Inclusion of these geometric features would be expected to create local regions of flow acceleration, deceleration, separation, recirculation and secondary motion1,27 which may exacerbate or reduce the effects of wall motion on haemodynamics and species transport. While we accept that the neglect of complex geometry may introduce some simplifications in the results it should be recognised that the geometric simplification enabled us to isolate the effects of geometry and to concentrate on the role of wall motion on species transport.\nThe results predicted in this study are specific to the flow and wall motion used in our model and therefore would be expected to change if a different flow or wall motion were imposed. For instance, if the flow and wall motion were more out of phase it may be expected that this would reduce the instantaneous Dean number and therefore one might expect the impact of the dynamic wall motion to be decreased from what was predicted. Although the wall motion effects were secondary to the pulsatile flow effects this does not mean that the wall motion is unimportant. Given the considerable variability in coronary artery motion, geometry and flow patterns in vivo there may be instances where wall motion could play a more significant role and therefore its effects should be considered on a case by case basis.\nOur study assumed that the non-Newtonian effects caused by the presence of red blood cells are negligible and therefore used constant diffusivities for LDL and oxygen. However, the effective diffusivity of a species changes due to the shear induced microscopic motion of red blood cells.6 In regions of high WSS, the effective diffusivities of LDL and oxygen would increase. This would in turn enhance oxygen wall flux but reduce LDL wall flux due to LDL concentration polarisation. Therefore, the magnitude of LDL and oxygen wall fluxes predicted in our study would be expected to change but the general patterns of flux will remain the same.\nConclusion\nIn this study, we have incorporated the effects of idealised motion into a simulation of oxygen and LDL transport in the LAD. The LDL and oxygen transport were modelled differently due to their differences in in-vivo transport mechanisms. We carried out simulations under both the steady and pulsatile flow conditions in the static and dynamic models to quantify the relative importance of pulsatility and wall motion on mass transport patterns. This study showed that the effects of wall motion on mass transport in the coronary arteries could be modelled in the commercial CFD code Fluent.\nOur results predicted elevation of LDL flux, impaired oxygen flux and low wall shear stress (WSS) along the inner wall of curvature, a region known for its predilection to atherosclerosis. The temporal variations in velocity and WSS patterns in the dynamic model did not influence time dependent mass transport and were only secondary to the pulsatile flow effects. However, wall motion may influence mass transport if shear dependent variations in species permeability and other factors are considered in the calculations. Nevertheless, the wall motion did alter the time-averaged mass transfer in the medial and distal regions in the order of 26% and 12% for oxygen and LDL transport when compared to the corresponding static models. Taken together, these results suggest that wall motion may play an important role in coronary arterial transport processes. However, future studies on more physiologic models are warranted to gain a fundamental understanding of the role of wall motion on mass transport and atherosclerosis.","keyphrases":["mass transport","wall motion","wall shear stress","oxygen transport","cfd","coronary artery motion","ldl transport","coronary atherosclerosis"],"prmu":["P","P","P","P","P","P","P","R"]}
{"id":"Bioinformation-1-2-1891631","title":"A database for medicinal and aromatic plants of JK (Jammu and Kashmir) in India\n","text":"High throughput screening of small molecules for a given drug target is achieved using plant materials of medicinal value. Therefore, it is important to document the availability and location of such medicinal plants in the form of a database. Here, we describe a web database containing information (botanical name, common name, local name, botany, chemistry, folklore medicinal use and medicinal uses) about the medicinal and aromatic plants available in JK (Jammu and Kashmir). The database is available for free in public domain.\nBackground\nMedicinal plants documented in the literature from thick vegetations of the world are routinely used for high throughput \nscreening in small molecular drug discovery. A large number of such plants are known to be recognized in a discrete manner. \nTherefore, it is important to store information related to medicinal and aromatic plants of JK in a database. This will help in \nthe use and exploitation of the plant materials for drug discovery. Some of the plants available in this region are also known \nto have aromatic value. Here, we describe the development and use of a database containing information on medicinal and aromatic \nplants from JK.\nMethodology\nDataset\nFolklore medicinal usage and other associated data were manually collected from the inhabitants (tribal\/gujjars\/local \nclinicians) of JK through direct person to person survey for each plant species. The current dataset contains information for \nabout 133 plant species.\nDatabase Interface\nThe database interface is designed for searching the dataset using a PHP (a programming language that allows web developers \nto create dynamic content that interacts with databases) server enabled script for keywords such as botanical name, local name, \nfolklore use and medicinal use. The database is accessible for free through the internet. \nDatabase Design\nThe database is developed in MySQL on a Linux Platform and updated regularly.\nSearch result\nThe search output for a given query (for example, diabetes) is given in Figure 1.\nUtility\nThe database finds utility in the selection of medicinal plant species for high throughput screening in drug discovery.","keyphrases":["medicinal","aromatic","plants","jammu and kashmir","india","botanical name","common name","folklore"],"prmu":["P","P","P","P","P","P","P","P"]}
{"id":"Intensive_Care_Med-4-1-2244699","title":"The influence of corticosteroids on the release of novel biomarkers in human endotoxemia\n","text":"Objective Sepsis intervention studies need better patient stratification methods, and one way to realize this is the introduction of stable biomarkers. A set of recently developed novel biomarkers, based upon precursor-fragments of short-lived hormones, was previously shown to be increased during sepsis. However, it is not known whether these biomarkers are influenced by sepsis intervention strategies. Therefore we investigated the markers in a model of human endotoxemia intervened by increasing doses of prednisolone.\nIntroduction\nDuring the past decade numerous sepsis intervention strategies have been introduced; only few of them, however, have shown beneficial effects. Major problems in sepsis intervention trials were mixed results and unclear risk stratification methods. The lack of clear stratification methods was especially disturbing, since the effectiveness of intervention in these studies correlated closely with disease severity and risk of mortality\u00a0[1]. Within this context, there is a\u00a0need for biomarkers to predict disease severity and mortality risk.\nDuring sepsis a\u00a0wide range of inflammatory proteins is released and in theory all could be used as a\u00a0biomarker. The vast majority of these proteins is biologically active though and as a\u00a0consequence rapidly cleared from the circulation. A\u00a0promising new approach is the measurement of stable prohormone fragments of bioactive peptide-hormones\u00a0[2\u20134]. In theory, these fragments often have no known biological function during sepsis and are therefore less rapidly cleared and less sensitive to small homeostatic changes\u00a0[5\u20137].\nThe most prominent prohormone sepsis marker is procalcitonin (PCT), which represents the precursor hormone of calcitonin. Its diagnostic and prognostic properties were superior to C-reactive protein (CRP) in multiple studies\u00a0[8]. In addition, PCT responded to sepsis intervention strategies and represented a\u00a0promising marker for monitoring of treatment effect\u00a0[9,\u202f10]. In recent years, additional assays have been developed for measurement of stable prohormone fragments, such as mid-regional pro-adrenomedullin (MR-proADM)\u00a0[5], derived from the precursor of adrenomedullin (ADM); mid-regional pro-atrial natriuretic peptide (MR-proANP), derived from the precursor of atrial natriuretic peptide (ANP)\u00a0[6]; and C-terminal proAVP (CT-proAVP), derived from the precursor of arginine\u2013vasopressin (AVP; also known as the antidiuretic hormone)\u00a0[7]. Levels of these biomarkers were higher in samples from patients with sepsis\u00a0[2\u20134]. Yet it is not known whether these levels are influenced by a\u00a0potential therapeutic sepsis intervention. In order to investigate the response of the novel biomarkers to a\u00a0sepsis intervention strategy we applied the model of human endotoxemia and pre-treated subjects by increasing doses of corticosteroids, which is one of the few relatively successful, and among the most cost-effective, septic shock intervention strategies to date\u00a0[11].\nMethods\nSubjects and study design\nThe study was designed according to the requirements of the Declaration of Helsinki and approved by the institutional scientific and ethics committees, and written informed consent was obtained from all subjects. The study was performed simultaneously with an investigation of the effects of prednisolone on activation of the cytokine network, coagulation and hemodynamic variables in the same cohort\u00a0[12]. Thirty-two healthy male volunteers [mean (\u00b1\u202fSE) age 23.9\u202f\u00b1\u202f0.7 years] were admitted to a\u00a0hospital clinical research unit. Screening tests were all normal. Prednisolone (prednisolone sodium phosphate oral solution 5\u202fmg\/ml; prepared by institutional pharmacy) was administered orally at a\u00a0dose of 0, 3, 10 or 30\u202fmg (n\u202f=\u202f8 per group) at 2\u202fh prior to LPS injection. All participants were challenged at t\u202f=\u202f0\u202fh with LPS (Escherichia coli lipopolysaccharide, lot G; US Pharmacopeia, Rockville, MD) as a\u00a0bolus intravenous injection at a\u00a0dose of 4\u202fng\/kg, which is a\u00a0standardized dose\u00a0[13].\nAssays\nBlood was collected at intervals from 2\u202fh before LPS injection to 24\u202fh thereafter. Blood was immediately centrifuged (4\u202f\u00b0C, 10\u202fmin, 3000\u202frpm) and plasma was stored at \u201320\u202f\u00b0C until assayed. Uptake of orally administered prednisolone was measured by HPLC-MS\/MS technology (CombinatoRx, Inc., Boston, MA, USA). MR-proADM, MR-proANP, CT-proAVP and PCT were measured by chemiluminescence label-coated tube-based sandwich immunoassays (Brahms Diagnostica, Berlin, Germany) as previously described\u00a0[5\u20137,\u00a014].\nStatistical analysis\nDifferences between treatment groups were analyzed using a\u00a0non-parametric mixed model approach (repeated-measures ANOVA). The effects reported are treatment effects adjusted for time effects. p values <\u202f0.05 were considered statistically significant. Values presented are given as mean\u202f\u00b1\u202fSE.\nResults\nPharmacokinetic data\nAs shown in Fig.\u202f1, uptake of orally administered prednisolone was demonstrated by a\u00a0dose-dependent increase in plasma levels during endotoxemia. The levels peaked from 1\u202fh before LPS administration to 1\u202fh after LPS administration (peak levels 302\u202f\u00b1\u202f34\u202fng\/ml, 190\u202f\u00b1\u202f19\u202fng\/ml and 71\u202f\u00b1\u202f5\u202fng\/ml in the groups receiving prednisolone 30, 10 and 3\u202fmg respectively; t\u202f=\u202f\u20131\u202fh).\nFig.\u00a01Prednisolone. Mean (\u00b1\u202fSE) values of prednisolone concentrations after LPS administration (4\u202fng\/kg IV, t\u202f=\u202f0\u202fh) to healthy male volunteers, preceded by oral administration of prednisolone 3\u202fmg (circles), 10\u202fmg (triangles), or 30\u202fmg (squares) at t\u202f=\u202f\u20132.5\u202fh\nNovel prohormone biomarkers\nMR-proADM levels increased after LPS administration from 0.31\u202f\u00b1\u202f0.02\u202fnmol\/l at baseline to a\u00a0peak value of 1.05\u202f\u00b1\u202f0.11\u202fnmol\/l in the control group at t\u202f=\u202f4\u202fh (Fig.\u202f2). The rise was inhibited after prednisolone 30\u202fmg and 10\u202fmg pre-treatment (levels at t\u202f=\u202f4\u202fh: 0.65\u202f\u00b1\u202f0.06\u202fnmol\/l and 0.65\u202f\u00b1\u202f0.1\u202fnmol\/l respectively; p\u202f<\u202f0.05), but not after the low dose of prednisolone 3\u202fmg. MR-proANP levels rose from 42.4\u202f\u00b1\u202f2.2\u202fpmol\/l at baseline to 118.4\u202f\u00b1\u202f17.2\u202fpmol\/l in the control group at t\u202f=\u202f4\u202fh. These levels were reduced after prednisolone 30\u202fmg and 10\u202fmg (peak levels: 91.5\u202f\u00b1\u202f9.9\u202fpmol\/l and 64.8\u202f\u00b1\u202f5.6\u202fpmol\/l at t\u202f=\u202f2\u202fh and t\u202f=\u202f4\u202fh, respectively; p\u202f<\u202f0.05). CT-proAVP levels showed a\u00a0modest increase from 12.5\u202f\u00b1\u202f4.0\u202fpmol\/l to 18.0\u202f\u00b1\u202f4.8\u202fpmol\/l in the control group at t\u202f=\u202f4\u202fh. The rise was inhibited after prednisolone 30\u202fmg (peak level: 6.1\u202f\u00b1\u202f1.0\u202fpmol\/l; p\u202f<\u202f0.05) and not significantly changed by 10\u202fmg (peak level: 10.6\u202f\u00b1\u202f3.4\u202fpmol\/l; p\u202f<\u202f0.1). In contrast, prednisolone 3\u202fmg pre-treatment was associated with enhanced CT-proAVP release (peak level 34.7\u202f\u00b1\u202f11.5\u202fpmol\/l; p\u202f<\u202f0.05). PCT levels increased from a\u00a0baseline level of 0.04\u202f\u00b1\u202f0.02\u202fng\/ml to a\u00a0peak value of 16.7\u202f\u00b1\u202f2.6\u202fng\/ml in the control group. Peak PCT levels all occurred at t\u202f=\u202f24\u202fh and were not significantly affected by prednisolone pre-treatment (11.9\u202f\u00b1\u202f3.0\u202fng\/ml, 13.1\u202f\u00b1\u202f2.5\u202fng\/ml and 17.6\u202f\u00b1\u202f3.5\u202fng\/ml in the 30, 10 and 3\u202fmg group, respectively).\nFig.\u00a02Novel prohormone biomarkers. Mean (\u00b1\u202fSE) values of MR-proADM, MR-proANP, CT-proAVP and PCT after LPS administration (4\u202fng\/kg IV, t\u202f=\u202f0\u202fh) to healthy male volunteers, preceded by oral administration of prednisolone 3\u202fmg (open circles), 10\u202fmg (triangles), 30\u202fmg (squares) or, in the control group, 0\u202fmg (filled circles) at t\u202f=\u202f\u20132.5\u202fh. *\u202fp\u202f<\u202f0.05 vs. control group. NS, non significant\nDiscussion\nThe primary objective of this study was to determine the effect of increasing doses of prednisolone on the release of novel biomarkers during human endotoxemia. We show here that prednisolone pharmacologic concentrations peaked at the time of LPS infusion and that the release of MR-proADM, MR-proANP and CT-proAVP was subsequently inhibited in a\u00a0dose-dependent way. The levels of PCT were not significantly inhibited within the study period of 24\u202fh. However, previously inhibition of PCT was shown by anti-inflammatory agents within a\u00a0period of 168\u202fh\u00a0[9]. The levels of CT-proAVP were, remarkably, increased after prednisolone 3\u202fmg, yet the reason for this remains unclear because the pattern was unique to this biomarker.\nThe increased levels of MR-proANP, MR-proADM and CT-proAVP reflect activation of their mature counterparts. Difficulties in measurement of the mature hormones made it difficult to determine their roles during sepsis\u00a0[5\u20137]. The currently investigated biomarkers are produced after proteolytic processing of the same prohormone precursors as the mature hormones; thus, they reflect production levels of mature hormones in stoichiometric concentrations\u00a0[7,\u202f15]. In theory, the biomarker assays may measure levels of both cut prohormone fragments and full-length prohormones. However, the meaning of this relationship remains unclear in plasma because no well-defined methods are currently available for measurement of full-length prohormone plasma levels.\nThe biomarker levels in this study were dose-dependently influenced by corticosteroid intervention, which is important for treatment effect monitoring. The development of new biomarkers is needed for improvement of sepsis stratification methods, which influence trial outcomes. Several sepsis intervention strategies have shown promise\u00a0[16]; in this study corticosteroids were selected because they aim primarily at inhibition of the immune response, and for this intervention in particular, effects are dose-dependent and improvement of patient stratification methods is urgently needed\u00a0[11,\u202f17]. Prednisolone is among the most frequently applied corticosteroids\u00a0[18]; the oral route is convenient and potentially safer than intravenous administration\u00a0[19].\nThe doses of prednisolone used in our model were chosen according to common dose regimens for prednisolone\u00a0[18]. These doses were relatively low (equivalent to 12, 40 and 120\u202fmg hydrocortisone) in comparison to doses used for sepsis intervention (hydrocortisone 200\u2013300\u202fmg per day), yet sufficient to reduce the release of the investigated biomarkers during endotoxemia. Of note, it has been demonstrated that the effects of orally administered doses of prednisolone do not differ from those of comparable doses of intravenously administered hydrocortisone\u00a0[20].\nThe human endotoxemia model differs from sepsis in many aspects: it uses a\u00a0single-dose drug intervention and bolus intravenous challenge of LPS, it is self-limiting, and it lacks an infectious source\u00a0[13]. The study subjects are healthy young people not using any co-medication. The intervention must be administered before LPS injection in order to reach clinically relevant drug concentrations during endotoxemia because LPS is extremely quickly cleared from the circulation. Due to these differences, data cannot be directly extrapolated to the sepsis field and must always be confirmed in patient studies. Nonetheless, the model is the best available in healthy humans and has proven useful to obtain a\u00a0proof of principle of the actions of drugs and\/or to study mechanisms that contribute to the activation of inflammatory pathways\u00a0[13]. With these limitations in mind, we demonstrate here that the levels of the novel biomarkers MR-proADM, MR-proANP, CT-proAVP and PCT are all increased after LPS injection, and that the levels of MR-proADM, MR-proANP and CT-proAVP can be inhibited by prednisolone. These results add to their potential as biomarkers in sepsis.","keyphrases":["corticosteroids","sepsis","biological markers","endotoxin"],"prmu":["P","P","R","U"]}
{"id":"J_Mol_Model-3-1-2039871","title":"Optimization of parameters for semiempirical methods V: Modification of NDDO approximations and application to 70 elements\n","text":"Several modifications that have been made to the NDDO core-core interaction term and to the method of parameter optimization are described. These changes have resulted in a more complete parameter optimization, called PM6, which has, in turn, allowed 70 elements to be parameterized. The average unsigned error (AUE) between calculated and reference heats of formation for 4,492 species was 8.0 kcal mol\u22121. For the subset of 1,373 compounds involving only the elements H, C, N, O, F, P, S, Cl, and Br, the PM6 AUE was 4.4 kcal mol\u22121. The equivalent AUE for other methods were: RM1: 5.0, B3LYP 6\u201331G*: 5.2, PM5: 5.7, PM3: 6.3, HF 6\u201331G*: 7.4, and AM1: 10.0 kcal mol\u22121. Several long-standing faults in AM1 and PM3 have been corrected and significant improvements have been made in the prediction of geometries.\nIntroduction\nOver the past 30 years, NDDO-type [1, 2] semiempirical methods have evolved steadily. The earliest of these methods was MNDO [3, 4], which itself was a major advance over even earlier non-NDDO methods such as MINDO\/3 [5]. The main advantage of MNDO over earlier methods was that the values of the parameters were optimized to reproduce molecular rather than atomic properties. When it first appeared, MNDO was immediately popular because of its increased accuracy, but, with the passage of time, various limitations were found, among the most important of which was the almost total absence of a hydrogen bond. As hydrogen bonding is essential to life, this particular fault essentially precluded MNDO being used in modeling biochemistry.\nIn 1985 an attempt, AM1 [6], was made to improve MNDO by adding a stabilizing Gaussian function to the core-core interaction to represent the hydrogen bond. Despite the fact that this was an over-simplification of a very complicated phenomenon, the overall effect was similar, and for the first time NDDO methods gave a good, albeit limited, model of hydrogen bonding.\nIn the course of the next several years, improvements were made to the method of parameter optimization. The result of this was the PM3 method [7\u201310], which culminated in the parameterization of all the elements in the main group in 2004 [11]. At the same time, various changes to the original set of approximations used in MNDO were proposed, the most important of which were the addition of d-orbitals to main-group elements [12, 13] and the introduction of diatomic parameters. Work started on the transition metals, and parameters for some of these have been reported [14, 15]. More recently, parameter sets tailored to reproduce specific phenomena such as the binding energy of nucleic acid base pairs [16], iron complex catalyzed hydrogen abstraction [17], phosphatase-catalyzed reaction barriers [18], and the redox properties of iron containing proteins [19] have been developed.\nBecause of the way advances in NDDO developments occurred, in terms of the modifications of the approximations and the extensions to specific elements or groups of elements, there has been an inevitable lack of consistency. The aim of the current work was three-fold: to investigate the incorporation of some of the reported modifications to the core-core approximations into the NDDO methodology; to carry out a systematic global parameter optimization of all the main group elements, with emphasis on compounds of interest in biochemistry; and to extend the methodology by performing a restricted optimization of parameters for the transition metals. This resulted in the development of a new method, consisting of the final set of approximations used and the optimized parameters. This method will be referred to as parametric method number 6, or PM6. The name PM6 was chosen to avoid any confusion with two other unpublished methods, PM4 and PM5.\nTheory\nDespite the apparent complexity of semiempirical methods, there are only three possible sources of error: reference data may be inaccurate or inadequate, the set of approximations may include unrealistic assumptions or be too inflexible, and the parameter optimization process may be incomplete. In order for a method to be accurate, all three potential sources of error must be carefully examined, and, where faults are found, appropriate corrective action taken.\nReference data\nIn contrast to earlier methods, in which reference data was assembled by painstakingly searching the original literature, the current work relies heavily on the large compendia of data that have been developed in recent years. The most important of these are the WebBook [20], for thermochemistry, and the Cambridge Structural Database [21] (CSD), for molecular geometries.\nDuring the early stages of the current work, consistency checks were performed to ensure that erroneous data were not used. These checks revealed many cases in which the calculated heats of formation were inconsistent with the reference heats of formation reported in the NIST database. On further checking, many of these reference data were also found [22, 23] to be inconsistent with other data in the WebBook. In those cases where there was strong evidence of error in the reference data, the offending data were deleted, and the webbook updated [24].\nFor molecular geometries, gas phase reference data are preferred, but in many instances such data were unavailable, and recourse was made to condensed-phase data. Provided that care was taken to exclude those species whose geometries were likely to be significantly distorted by crystal forces, or which carried a large formal charge, condensed-phase data of the type found in the CSD were regarded as being suitable as reference data.\nBecause earlier methods used only a limited number of reference data, most of the cases where the method gave bad results were not discovered until after the method was published. In an attempt to minimize the occurrence of such unpleasant surprises, the set of reference data used was made as large as practical. To this end, where there was a dearth or even a complete absence of experimental reference data, recourse was made to high level calculations. Thus, for the Group VIII elements, there are relatively few stable compounds, and the main phenomena of interest involve rare gas atoms colliding with other atoms or molecules, so reference data representing the mechanics of rare gas atoms colliding with other atoms was generated from the results of ab-initio calculations. Additionally, there is an almost complete lack of thermochemical data for many types of complexes involving transition metals, so augmenting what little data there was with the results of ab-initio calculations was essential.\nUse of Ab-Initio results\nAb-initio calculations provide a convenient source of reference data; for this work, extensive use has been made of results of Hartree Fock and B3LYP density functional [25, 26] methods (DFT), both with the 6\u201331G(d) basis set for elements in the periodic table up to argon. For systems involving heavier elements, the B88\u2013PW91 functional [27, 28] was used with the DZVP basis set. Within the spectrum of ab-initio methods these methods are not particularly accurate; many methods with larger basis sets and with post-Hartree-Fock corrections are more accurate. However, the methods used in this work were chosen because they were regarded as robust, practical methods, allowing many systems to be modeled in a reasonable amount of time, a condition that could not be achieved with the more sophisticated ab-initio methods.\nProcedure used in deriving \u0394Hf\nReference heats of formation, \u0394Hf, for compounds and ions of elements for which there was a paucity of data were derived from DFT total energies in two stages. In the first stage, a basic set of \u223c1,400 well-behaved compounds, for which reliable reference values of experimental \u0394Hf were available, was assembled. Only compounds containing one or more of the elements H, C, N, O, F, P, S, Cl, Br, and I were used. For this set, a root-mean-square fit was made to the reference \u0394Hf using the calculated total energies, Etot and the atom counts. Thus, the error function, S, in Eq.\u00a0(1) was minimized.\nIn this expression, the Ci are constants for each atom of type i, and the ni are the number of atoms of that type.\nIn the second stage, the contribution to the total energy of compounds containing element X arising from the elements in the first stage was removed using the coefficients from Equation (1). A second RMS fit was then performed. In this, the function minimized, S, was the RMS difference between the reference \u0394Hf of compound X and the values predicted from the DFT energy, Eq.\u00a0(2).\nIn this expression, the only unknown is the multiplier coefficient Cx. After solving for Cx, the \u0394Hf of any compound of X could then be predicted as soon as its DFT total energy was evaluated.\nTraining set reference data\nThe training set of reference data used was considerably larger than that used in parameterizing PM3 [7, 8], where approximately 800 discrete species were used. In optimizing the parameters for PM6, somewhat over 9,000 separate species were used, of which about 7,500 were well-behaved stable molecules. The remainder consisted of reference data that were tailored to help define the values of individual parameters or sets of parameters.\nUse of rules in parameter optimization\nMost reference data can be expressed as simple facts. Indeed, all the earlier NDDO methods were parameterized using precisely four types of reference data: \u0394Hf, molecular geometries, dipole moments, and ionization potentials. During the development of PM6, however, the use of other types of reference data was found to be necessary. Because of their behavior, these new data are best described as \u201crules.\u201d In this context, a rule can therefore be regarded as a reference datum that is a function of one or more other data. To illustrate the use of a rule, consider the binding energy of a hydrogen bond in the water dimer. By default, the weighting factor for \u0394Hf for normal compounds is 1.0\u00a0kcal mol\u22121. With this weighting factor, average unsigned errors in the predicted \u0394Hf of the order of 3\u20135\u00a0kcal mol\u22121 would be acceptable, particularly as the spectrum of values of \u0394Hf spans several hundreds of kilocalories per mole. However, the binding energy of a hydrogen bond in a water dimer is only 5\u00a0kcal mol\u22121. To have an average unsigned error (AUE) of 4\u00a0kcal mol\u22121 in the prediction of hydrogen bond energies would render such a method almost useless for modeling such phenomena.\nOne way to increase the importance of the hydrogen bond in water would be to increase the weight for the \u0394Hf of the water molecule, \u221257.8\u00a0kcal mol\u22121, and the water dimer system, ca. \u2212120.6\u00a0kcal mol\u22121. While this would have the intended effect of increasing the weight of the hydrogen bond energy, it would also have the undesired effect of increasing the weight of the \u0394Hf of water.\nAn alternative would be to express the \u0394Hf of the water dimer in terms of the \u0394Hf of two individual water molecules. The difference between the two \u0394Hf, that of water dimer and that of two isolated water molecules, would be the energy of the hydrogen bond. If the weight assigned to this quantity were then increased, it would increase the weight for the hydrogen bond energy without also increasing the weight for the \u0394Hf of water. Such a reference datum is referred to here as a rule. That is, rules relate the \u0394Hf of a moiety to that of one or more other moieties. Thus, in the above example, the simple reference datum H, representing the \u0394Hf of an isolated water molecule, could be expressed as:\nUsing a rule-based reference datum to represent the strength of the hydrogen bond, and giving a weight of 10 to the hydrogen bond energy, the \u0394Hf of the water dimer would then be defined as\nIn this expression, HH2O was the calculated \u0394Hf, in kcal mol\u22121, of an isolated water molecule. This rule could be interpreted as \u201cThe calculated strength of the hydrogen bond formed when two water molecules form the dimer should be 5\u00a0kcal mol\u22121, and the importance should be 100 times that of ordinary heats of formation.\u201d\nRules are very useful in defining the parameter hypersurface. Examples of such tailoring are as follows:\nCorrecting qualitatively incorrect predictions\nDuring the parameterization of transition metals, some systems were predicted to have qualitatively the wrong structure. For example, [CuIICl4]2\u2212 was initially predicted to have a tetrahedral structure, instead of the D2d geometry observed. To induce the parameters to change so as to make the D2d geometry more stable than the Td geometry, a rule was added to the set of reference data for copper compounds. This rule was constructed using the results of B3LYP calculations on [CuIICl4]2\u2212. First, the total energies of the optimized B3LYP structure and that of the structure resulting from the semiempirical calculation were evaluated. The difference between these energies was then used in constructing the rule. In this case, the rule was that \u201cThe \u0394Hf of the geometry predicted by the faulty semiempirical method should be n.n kcal mol\u22121 more than that of the B3LYP geometry.\u201d When such a rule was included in the parameter optimization, with an appropriate large weight, any tendency of the parameters to predict the incorrect geometry resulted in a large contribution to the error function. That is, with the new rule in place, there was a strong disincentive to prediction of the incorrect structure. Usually one rule was sufficient to correct most qualitative errors, but for a few complicated structures more than one rule was needed. The commonest need for multiple rules occurred when, initially, one rule was used to correct a faulty prediction and, after re-optimizing the parameters, the geometry optimized to a new structure that was distinctly different from either the correct structure or the incorrect structure covered by the rule. When that happened, the procedure just described was repeated, and a new rule added to the set of reference data to address the new incorrect structure. In extreme cases, several such rules might be needed, each one defining a geometry that was incorrect and should therefore be avoided.\nRare gas atoms at sub-equilibrium distances\nFor some elements, specifically those of Group VIII, there is an understandable shortage of useful experimental reference data. In addition, most simulations involving these elements are likely to involve a rare-gas atom dynamically interacting with another atom or with a molecule at distances significantly less than the equilibrium distance. This makes determining the potential energy surface at sub-equilibrium distances important. As with hydrogen bond energies, the energies involved in this domain are likely to be in the order of a few kcal mol\u22121. The shape of the potential energy surface (PES) can readily be mapped using DFT methods. By selecting two or three representative points on this PES, reference data rules can be constructed that describe the mechanical properties of the interactions. As with hydrogen bonding, a large weight can be assigned to these rules.\nUse of rules to restrain parameter values\nIn general, uncharged atoms that are separated by a distance sufficiently large so that all overlaps between orbitals on the two atoms are vanishingly small will not interact significantly, and what interaction energy exists would arise from VDW terms: of their nature, these are mildly stabilizing. Although statements of this type are obviously true, when they are expressed as rules and added to the training set of reference data they can help define the parameter values. For a pair of atoms, A and B, a simple diatomic system would be constructed in which the interatomic separation was the minimum distance at which any overlaps of the atomic orbitals would still be insignificant. The electronic state of such a system would then be the sum of the states of the two isolated atoms. Thus, if both A and B were silicon, then, since the ground state of an isolated silicon atom is a triplet, the combined state would be a quintet. Because the two atoms do not interact significantly, a rule could then be constructed that said \u201cThe energy of the diatomic system is equal to the addition of energies of the two individual systems.\u201d By giving this rule a large weight, any tendency of the method to generate a spurious attraction or repulsion between the atoms would be prevented.\nAtomic energy levels\nIn keeping with the philosophy that a large amount of reference data should be used in the parameter optimization, spin-free atomic energy levels were used for most elements. The exceptions were carbon, nitrogen, and oxygen, where there were enough conventional reference data that the addition of atomic energy levels would not significantly improve the definition of the parameter surface.\nNDDO approximations do not allow for spin-orbit coupling. Therefore, spin-free levels were needed. For a few elements, there were insufficient spin states to allow the spin-free energy levels to be calculated. For all the remaining elements, spin-free energy levels were calculated.\nIn Moore\u2019s compendia [29\u201331] of atomic energy levels, observed emission spectra were used in determining the energy levels of the various states of neutral and ionized atoms. Most of these energy levels were characterized by three quantum numbers: the spin and orbital angular momenta, and the \u201cJ\u201d or spin-orbit quantum number. The starting point for determining the spin-free atomic energy levels for a given element consisted of identifying each complete manifold of atomic energy levels for that element, that is, each set of levels split by spin-orbit coupling. If all members of the set were present, i.e., all energy levels from L+S to |L\u2212S|, then the weighted barycenter of energy could be calculated. The spin-free energy level, E, was derived from the spin-split levels E(S,L,J) using Eq.\u00a0(3).\nIn those cases where the ground state of an atom was itself a member of a spin-split manifold, the barycenter of the ground state manifold was calculated and used in re-defining the spin-free ground state. For all elements except tungsten, this change in definition was benign. There is a 7S3 level present in tungsten that is located only 8.4\u00a0kcal mol\u22121 above the ground state. This puts it inside the 5DJ, manifold, which has a barycenter at 12.7\u00a0kcal mol\u22121. The effect of this was that, on going from a spin-split to a spin-free ground state, the ground state changed from 6d25d4 or 5D to 6d15d5 or 7S, and the 5D state now became an excited state with an energy of 4.4\u00a0kcal mol\u22121. To allow for this, a corresponding change was made to the ground state configuration in the PM6 definition of tungsten.\nWhere there were relatively few other reference data, the singly-ionized, and, in rare cases, the doubly-ionized, spin-free states were also evaluated and used as reference data.\nEach energy level contributed one reference datum to the training set. Most atoms have a large number of atomic energy levels, so in order to minimize the probability that a level might be incorrectly assigned, each level was labeled with three quantum numbers: the total spin momentum, the total angular momentum, and the principal quantum number for these two quantum numbers. These were compared with the corresponding values calculated from the state functions. Since each set of three quantum numbers is unique, the potential for miss-assignment was minimized. In rare cases, particularly during the early stages of parameter optimization, two states with the same total spin and angular quantum numbers would be interchanged, with the result that the calculated principal quantum number would also be interchanged. All such cases always involved the ground state, and were quickly identified and corrected.\nApproximations\nMost of the approximations used in PM6 are identical to those in AM1 and PM3. The differences are:\nCore-core interactions\nIn the original MNDO set of approximations, two changes were made to the simple point-charge expression for the core-core repulsion term. Beyond about five \u00c5ngstroms, there should be no significant interaction of two neutral atoms. However, in MNDO, the two-electron, two-center  integrals and the electron-core interactions do not converge to the exact point charge expression; instead, they are always slightly smaller. To prevent there being a small net repulsion between two uncharged atoms, the core-core expression is modified by the exact 1\/RAB term being replaced by the term used in the  integrals. An additional term is needed to represent the increased core-core repulsion at small distances due to the unpolarizable core. These two changes can be expressed as the MNDO core-core repulsion term as shown in Eq.\u00a0(4).\nThis approximation works well for most main-group elements, but when molybdenum was being parameterized, Voityuk [14] found that the errors in heats of formation and geometries were unacceptably large, and good results were achieved only when a diatomic term was added to the core-core approximation, as shown in Eq.\u00a0(5).\nWhen PM3 parameters for elements of Groups IA were being optimized, the MNDO approximation to the core-core expression was found to be unsuitable. In these elements there is only one valence electron so the core charge is the same as that of hydrogen. A consequence of this was that the apparent size of these elements was also approximately that of a hydrogen atom, in marked contrast with observation. For these elements, diatomic core-core parameters were also found to be essential.\nFurther examination showed that when diatomic parameters were used, there was always an increase in accuracy; therefore, in the current work, Eq.\u00a0(4) was replaced systematically by Eq.\u00a0(5).\nAs the interatomic separation increased, Voityuk\u2019s equation converged to the exact point-charge interaction, as expected. However, for rare gas interactions, an increase in accuracy was found when the rate of convergence was increased by the addition of a small perturbation. Subsequently, the perturbed function was found to be generally beneficial. Because of this, the general form of the core-core interaction used in PM6 is that given in Eq.\u00a0(6).\nAt normal chemical bonding distances, Eqs.\u00a0(5) and (6) have essentially similar behavior, but at distances of greater than about 3\u00a0\u00c5 the effect of the perturbation is to make the PM6 function significantly smaller than the Voityuk approximation.\nd-orbitals on main-group elements\nThiel and Voityuk have shown [13] that a large increase in accuracy results when d-orbitals are added to main-group elements that have the potential to be hypervalent. During preliminary stages of this work, d-orbitals were excluded from main-group elements, and the parameters were optimized. This work was then repeated but with d-orbitals on various main-group elements. The results were in accordance with Thiel\u2019s observation: the accuracy of the method increased significantly. Because of this, d-orbitals were added to several main-group elements: the value of the increased accuracy far outweighs the extra computational cost.\nThe effect of the addition of d-orbitals was fundamentally different between main-group elements and transition metals. For main-group elements, the effect of d-orbitals is merely a perturbation: to a large degree the chemistry of these elements is determined by the s and p atomic orbitals. This is not the case with transition metals, where the d-orbitals are of paramount importance and the s and p orbitals are of only very minor significance. In recognition of the importance of the s and p shells in main-group chemistry, specific parameters are used for the five one-center two-electron integrals. Conversely, for the transition metals, the values of these integrals are derived directly from the internal orbital exponents.\nUnpolarizable core\nAs noted earlier, the NDDO core-core interaction is a function of the number of valence electrons. For elements on the left of the periodic table these numbers are small and can cause the elements to appear to be too small. This was part of the rationale behind the adoption of Voityuk\u2019s diatomic core-core parameters. However, even the Voityuk approximation failed during parameter optimization when, in rare cases, a pair of atoms would approach each other very closely. Examination of these catastrophes indicated that the cause was the complete neglect of the unpolarizable core of the atoms involved. To allow for its presence, the core-core interaction for all element pairs was modified by the addition of a simple function, fAB, based on the first term of the Lennard-Jones potential [32]. A candidate function was constructed, Eq.\u00a0(7), using the fact that, to a first approximation, the size of an atom increases as the third power of its atomic number.\nThe value of c was set to 10\u22128, this being the best compromise between the requirements that the function should have a vanishingly small value at normal chemical distances. That is, under normal conditions the value of the function should be negligible, and at small interatomic separations the function should be highly repulsive, i.e., that it should represent the unpolarizable core.\nIndividual core-core corrections\nFor a small number of diatomic interactions, the general expression for the core-core interaction was modified in order to correct a specific fault. Because it is desirable to keep the methodology as simple as possible, modifications of the approximations were made only after determining that the existing approximations were inadequate. The diatomic specific modifications were:\nO\u2013H and N\u2013H\nIn the original MNDO formalism, the general core-core interaction, Eq.\u00a0(4), was replaced in the cases of O\u2013H and N\u2013H pairs with Eq.\u00a0(8).\nAn unintended effect of this change was that at distances where hydrogen-bonding interactions are important, the diatomic contribution to the \u0394Hf is greater than if the general approximation, Eq.\u00a0(4), had been used. This contributed to a reduced hydrogen-bonding interaction in MNDO, and was a contributor to the need for modified core-core interactions in AM1 and PM3.\nIn PM6, the MNDO core-core approximation is replaced by Voityuk\u2019s diatomic expression, but even with that modification, the resulting hydrogen bond interaction energy was too small. In an attempt to increase it, the Voityuk approximation was replaced by Eq.\u00a0(9).\nAt normal O\u2013H and N\u2013H separations, approximately 1\u00a0\u00c5, Eqs.\u00a0(5) and (9) have similar values, but at hydrogen bonding distances, \u223c2\u00a0\u00c5, the contribution arising from the exponential term is significantly reduced, resulting in a corresponding increased hydrogen bond interaction energy.\nC\u2013C\nAfter optimizing all parameters, it was found that compounds containing yne groups, -C\u2261C-, were predicted to be too stable by about 10\u00a0kcal mol\u22121 per yne group. This error was unique to compounds with extremely short C\u2013C distances, and in light of the increased emphasis on accurately reproducing the properties of organic compounds, the C\u2013C core-core term was perturbed by the addition of a repulsive term. This term was optimized to correct the error in the yne groups and to have a negligible effect on all other C\u2013C interactions. The optimized form of the C\u2013C core-core interaction is given in Eq.\u00a0(10).\nSi\u2013O\nDuring testing of PM6, neutral silicate layers of the type found in talc, H2Mg3Si4O12, were found to be slightly repulsive instead of being slightly bound. An attempt was made to correct for this error by adding a weak perturbation to the Si\u2013O interaction, illustrated by Eq.\u00a0(11).\nNitrogen sp2 pyramidalization\nAlthough PM6 predicted the degree of pyramidalization of primary amines correctly, it overestimated the pyramidalization of secondary and tertiary amines. The degree of pyramidalization of these amines was decreased by adding a function to make the calculated \u0394Hf more negative as the nitrogen became more planar, as shown in Eq.\u00a0(12).\nIn this equation, the angle \u03d5 is a measure of the non-planarity of the nitrogen environment, and is given by 2\u03c0 minus the sum of the three contained angles about the nitrogen atom. For planar sp2 secondary and tertiary amines, this correction amounted to 0.5\u00a0kcal mol\u22121 per nitrogen atom.\nMore elements\nThe NDDO basis sets of many of the elements parameterized in PM6 have not previously been described. For all elements except hydrogen, which has only an s orbital, the basis set consists of an s orbital, three p orbitals, and, for most elements, a set of five d orbitals. Slater atomic orbitals are used exclusively; these are of form:\nWhere \u03be is the orbital exponent, n is the principal quantum number (PQN), and the Ylm(\u03b8, \u03d5) are the normalized real spherical harmonics. The PQN are those of the valence shell, i.e., the set of atomic orbitals most important in forming chemical bonds. For PM6, the PQN used are shown in Table\u00a01. For most main-group elements, the s and p PQN are the same, and, when d orbitals are present, all three PQN are the same: that is, the PQN are (ns, np, nd). For transition metals, the d PQN is one less than that of the s and p shells, i.e., (ns, np, (n\u20131)d). An exception to this generalization occurs in the elements of Group VIII. Here, the valence shell is completely filled, so in all chemical interactions that could occur between an atom of a Group VIII element and any other atom, electron density could only migrate from the Group VIII element to the other atom. That is, when a rare gas element forms any type of chemical bond it would necessarily become slightly positive. This is an unrealistic result. In order to allow rare gas atoms to have the potential of being slightly negative, the set of valence orbitals was changed from (ns, np) to (np, (n+1)s), for the elements Ne, Ar, Kr, and Xe. Helium is the only exception to this change, because it does not have a \u201c1p\u201d valence shell. For helium, the valence shell used was (1s, 2p), this being considered the best compromise.\nTable\u00a01Principal quantum numbers for atomic orbitals\u00a0spd\u00a0spdH1Kr54He12Rb55Li22Sr55Be22Y554B22Zr554C22Nb554N22Mo554O22Tc554F22Ru554Ne32Rh554Na33Pd554Mg33Ag554Al333Cd55Si333In55P333Sn55S333Sb555Cl333Te555Ar43I555K44Xe65Ca44Cs66Sc443Ba66Ti443La665V443Lu665Cr443Hf665Mn443Ta665Fe443W665Co443Re665Ni443Os665Cu443Ir665Zn44Pt665Ga44Au665Ge44Hg66As444Tl66Se444Pb66Br444Bi66\nParameter optimization\nBackground\nThe objective of parameter optimization is to modify the values of the parameters so as to minimize the error function, S, Eq.\u00a0(13), representing the square of the differences between the values of reference data, Qref(i), and the values calculated using the semiempirical method, Qcalc(i), with appropriate weighting factors, gi.\nThis process is initiated by rendering the reference data in the training set dimensionless. The default conversion factors are given in Table\u00a02, with weighting factors for reference data represented by rules being much larger, typically in the order of 5\u201320\u00a0kcal mol\u22121.\nTable\u00a02Default weighting factors for reference dataReference dataWeight\u0394Hf1.0\u00a0mol.kcal\u22121Bond length0.7\u00a0\u00c5\u22121Angle0.7 degrees\u22121Dipole20 Debye\u22121I.P.10\u00a0eV\u22121ElementsMultiplierCore1.0Organic0.9Main group0.8Transition metals0.7\nThe elements were divided into four sets: core elements, (H, C, N, and O), other elements important in organic chemistry (F, Na, P, S, Cl, K, Br, I), the rest of the main group, and the transitions metals. Elements were assigned to the different sets based on their presumed degree of importance in biochemistry, and this importance was converted into a weighting factor to be used in the parameterization optimization procedure. Reference data representing species consisting only of core elements were given their default weight. When other elements were present, the weight was set to the default weight times the smallest multiplier shown in Table\u00a02. Thus the default weight for a reference datum involving tetramethyllead, Pb(CH3)4, would be multiplied by 0.8 reflecting the fact that this species contains an element in the main group set.\nFor a given set of parameters, P, optimization proceeds by calculating the values of all the Qcalc(i), their first derivatives with respect to each parameter, P(j), and the second derivatives with respect to every pair of parameters. Evaluating these quantities is time-consuming, and considerable effort was expended in minimizing the need for explicit evaluation of these functions. The most efficient strategy developed [7] involved assuming that, in the region of parameter space near to the current values of the parameters, the values of the first derivatives of the Qcalc(i) with respect to P were, at least to a first approximation, constant. By making this assumption the values of the parameters could then be updated using perturbation theory. Because the assumption is only valid in the region of the starting point in parameter space, periodically the focus was moved to the new point in parameter space and a complete explicit re-evaluations of all the functions performed. The parameter optimization process terminated when the scalar of the first derivatives dropped below a preset limit. This process was fully automated, and for given sets of reference data and parameters, parameter optimization could be performed rapidly, easily, and reliably.\nSequence of optimization of parameters\nNotwithstanding the reliability of the parameter optimization procedure, a simple global optimization of all the parameters for all 70 elements involving about over 9,000 discrete species was found to be impractical because of the large number of derivatives involved. Such an optimization would involve over 2,000 parameters and over 10,000 reference data. The set of second derivatives alone would consist of 2\u00d71010 terms. With more powerful computers, evaluating such large sets of derivatives might be practical some day, but even then, one faulty reference datum or one faulty initial parameter value would ruin an optimization run. The strategy of parameter optimization was approached with great caution, and the procedure finally adopted was as follows:\nBecause the elements H, C, N, and O are of paramount importance in biochemistry, and because large amounts of reference data are available, the starting point for parameter optimization involved the simultaneous optimization of parameters for these four elements. For the purposes of discussion, this set of four elements will be called the \u201ccore elements\u201d.\nOnce stable parameters had been obtained, parameters for other elements important in organic chemistry were optimized in two stages. First, the parameters for the core elements were held constant, and parameters for the elements F, P, S, Cl, Br, and I were optimized one at a time. Then all parameters for all ten elements were simultaneously optimized. This set (the organic elements) was then used as the starting point for parameterizing the rest of the main group.\nThe same sequence was followed for the rest of the main-group elements. That is, parameters for each element were optimized while freezing the parameters for the organic elements. Then, once all the elements had been processed, all parameters for all of the 39 main-group elements, plus zinc, cadmium, and mercury, were optimized simultaneously.\nWhen parameters for the transition metals were being optimized, all parameters for the main group elements were held constant. There were several reasons for this. Most importantly, the reference data for the transition metals, particularly the thermochemical data, was of lower quality, so one consideration was to prevent the transition metals from having a deleterious effect on the main-group elements. Another important consideration was that most compounds involving transition metals also involved only elements of the organic set. Since parameters for these elements had been optimized using a training set consisting of all the main-group elements, the values of the optimized parameters would likely be relatively insensitive to the influence of the small number of additional reference data involving transition metals.\nIn general, all parameters for a given element were optimized simultaneously; this was both efficient and convenient. In some optimizations, specifically those involving a new element, only sub-sets of parameters were used. Three main sub-sets were used:\nParameters that determine atomic electronic properties\nFor most elements, atomic energy levels are determined by six parameters: the one-electron one-center integrals Uss, Upp, Udd, and the internal orbital exponents \u03b6sn, \u03b6pn and \u03b6dn. If the heat of ionization and sufficient atomic energy level data were available, these quantities could be uniquely defined; there would be no need for the use of molecular reference data. These parameters were the first to be optimized whenever an optimization was started for an element that had not previously been parameterized\nParameters that determine molecular electronic properties\nTwo of the more important electronic molecular properties are the dipole moment, which indicates the degree of polarization within a molecule, and the ionization potential. These properties are determined primarily by 12 parameters: the six parameters that determine atomic electronic properties and six additional parameters: \u03b2s, \u03b2p, and \u03b2d and the Slater orbital exponents \u03b6s, \u03b6p, and \u03b6d. In the second stage of parameter optimization, the first six parameters were held constant at the values defined using atomic data and the second set optimized. During this operation, all geometries were fixed at their reference values.\nParameters that determine geometries\nAs soon as an initial optimized set of electronic parameters was available, the diatomic and other core-core parameters could be optimized. The most efficient process was to optimize these parameters initially without allowing the electronic parameters or the molecular geometries to optimize. If geometries were allowed to optimize, optimization of the core-core parameters would be slowed considerably, because of the tight dependency of the optimized geometries on the values of the core-core parameters, and vice versa.\nAs soon as all parameters had been optimized using fixed geometries, the geometries were allowed to relax and the parameters that determine geometry re-optimized. After that there would be three sets of incompletely optimized parameters: the six atomic electronic parameters, the six molecular electronic parameters and the core-core parameters. The only remaining operation was the simultaneous optimization of all the parameters. If the training set of reference data was insufficient to unambiguously define the values of all the parameters, then, at that stage, the potential existed for the parameters to become ill-defined. An example of this would be where there were too few atomic energy levels to allow all six parameters in the first set to be defined. To allow for this, a penalty function was added to each parameter. If the values of a parameter exceeded pre-defined limits, the error function S was incremented by a constant times the square of the excess. No penalty was applied if the value of a parameter was between the pre-defined limits; that is, no bias was applied to the numerical value of a parameter. During the early stages of simultaneous optimization of all the parameters for a given element the penalty function was used frequently. In the later stages the penalty function was invoked rarely, and then only when there was a distinct shortage of reference data.\nResults\nParameters for PM6\nPM6 atomic and diatomic parameters for the 70 elements are presented in Tables\u00a03 and 4, respectively. Not all elements have all parameters: where monatomic parameters are missing, the associated approximations were not used. For diatomic parameters, where an atom-pair is missing, no representatives of that type of bond were used.\nTable\u00a03PM6 parameters for 70 elementsTable\u00a04Diatomic core\u2212core parameters\nAccuracy\nComparison with other semiempirical methods\nUsing the program MOPAC2007 [33], an extensive comparison was made between the results obtained using PM6 and those from PM5, PM3, and AM1. This comparison was started by generating tables of reference data (that is, \u0394Hf, geometries, ionization potentials (I.P.s), and dipole moments) and differences between the calculated and reference values, using each of the four methods presented here. Because of their size they are provided in the supplementary material. To simplify navigating within the tables, all species are listed in the order of their empirical formula.\nAverage unsigned errors (AUE) for \u0394Hf for each element parameterized at the PM6 level are shown in Table\u00a05, together with AUE for PM5, PM3, and AM1. The number of data used in each average varies depending on the elements available in each method. AM1 boron [34] uses a different core-core interaction expression from the other elements and was not used. AUE for bond-lengths are shown in Table\u00a06. In those cases where a calculated bond-length was very large, indicating that the bond had broken, the bond-length was not used in the analysis. If such data had been used, the resulting statistics would have been misleading. AUE for angles are shown in Table\u00a07. Errors in angles for many elements that form very ionic, i.e., labile, bonds are of less importance than errors involving elements that form strong covalent bonds. The angles subtended by such bonds are often determined largely by the electronic structure of the atom. Information on the accuracy of prediction of molecular electronic structure can also be inferred from the AUE of dipole moments, Table\u00a08, and ionization potentials, Table\u00a09.\nTable\u00a05Average unsigned errors in calculated heats of formation (kcal mol\u22121)ElementPM6No.PM5No.PM3No.AM1No.Hydrogen7.29303913.89234017.09234021.122270Helium0.0010.0010.0010.001Lithium7.988315.318318.028318.8482Beryllium5.923429.063429.583418.5134Boron6.4412210.8112011.84120\u2013\u2013Carbon7.31282813.03215515.06215519.422123Nitrogen8.22106716.4576120.9676124.23744Oxygen8.42175816.59124320.13124427.681229Fluorine8.4949722.3135021.2535037.40334Neon0.0010.0010.0010.001Sodium5.72408.57399.473910.7738Magnesium9.846612.076617.946618.7166Aluminum7.617517.497519.157518.9975Silicon6.51989.289612.809617.0095Phosphorus8.2011016.019817.369820.0695Sulfur8.8142715.4033018.4433026.38323Chlorine8.2867016.6939018.7139023.06383Argon0.0010.0010.0010.001Potassium6.534312.33429.364228.3841Calcium11.874328.684343.444363.2043Scandium10.3352\u2013\u2013\u2013\u2013\u2013\u2013Titanium10.2085\u2013\u2013\u2013\u2013\u2013\u2013Vanadium14.2959\u2013\u2013\u2013\u2013\u2013\u2013Chromium14.0960\u2013\u2013\u2013\u2013\u2013\u2013Manganese12.7744\u2013\u2013\u2013\u2013\u2013\u2013Iron18.3176\u2013\u2013\u2013\u2013\u2013\u2013Cobalt15.5142\u2013\u2013\u2013\u2013\u2013\u2013Nickel15.1051\u2013\u2013\u2013\u2013\u2013\u2013Copper13.0047\u2013\u2013\u2013\u2013\u2013\u2013Zinc5.565417.845432.935437.0654Gallium7.514729.124737.584746.8747Germanium9.836712.206715.866719.1267Arsenic6.944915.224916.684917.3449Selenium4.402539.582539.712532.0025Bromine7.3733017.2019925.0419928.22199Krypton0.0010.0010.0010.001Rubidium10.912416.572421.472429.3323Strontium7.723852.4638103.163857.2138Yttrium13.2851\u2013\u2013\u2013\u2013\u2013\u2013Zirconium11.1846\u2013\u2013\u2013\u2013\u2013\u2013Niobium8.5751\u2013\u2013\u2013\u2013\u2013\u2013Molybdenum13.4170\u2013\u2013\u2013\u201335.7769Technetium15.1450\u2013\u2013\u2013\u2013\u2013\u2013Ruthenium13.8756\u2013\u2013\u2013\u2013\u2013\u2013Rhodium20.9232\u2013\u2013\u2013\u2013\u2013\u2013Palladium11.6547\u2013\u2013\u2013\u2013\u2013\u2013Silver4.6714\u2013\u2013\u2013\u2013\u2013\u2013Cadmium3.493834.663861.9238\u2013\u2013Indium7.335431.535429.835432.1654Tin7.147716.837717.107720.2177Antimony5.415830.985834.615835.0058Tellurium8.204535.664546.804522.9145Iodine7.2327923.7717625.9017636.55175Xenon0.0010.0010.0010.001Cesium6.894037.014035.224055.3339Barium12.123798.2037154.6537161.0937Lanthanum10.3737\u2013\u2013\u2013\u2013\u2013\u2013Lutetium7.6824\u2013\u2013\u2013\u2013\u2013\u2013Hafnium8.5237\u2013\u2013\u2013\u2013\u2013\u2013Tantalum14.3736\u2013\u2013\u2013\u2013\u2013\u2013Tungsten7.3828\u2013\u2013\u2013\u2013\u2013\u2013Rhenium10.4057\u2013\u2013\u2013\u2013\u2013\u2013Osmium6.4619\u2013\u2013\u2013\u2013\u2013\u2013Iridium10.2125\u2013\u2013\u2013\u2013\u2013\u2013Platinum11.6177\u2013\u2013\u2013\u2013\u2013\u2013Gold12.8232\u2013\u2013\u2013\u2013\u2013\u2013Mercury5.945116.395117.675119.7551Thallium10.424432.634473.964573.1845Lead7.924418.084414.184416.7144Bismuth7.745399.885328.9553119.2353Table\u00a06Average unsigned errors in bond lengths (\u00c5)ElementPM6No.PM5No.PM3No.AM1No.Hydrogen0.0442380.0562190.0322170.035181Helium0.25160.45960.18240.6555Lithium0.1751110.1911100.1671100.171105Beryllium0.076420.131420.067420.08542Boron0.0271160.0431160.066122\u2013\u2013Carbon0.05711910.0666930.0516340.063628Nitrogen0.0906630.1453090.1242590.163253Oxygen0.09511630.1226250.1035770.117571Fluorine0.0633960.0962460.0692510.101228Neon0.35350.18220.06210.0301Sodium0.229330.200330.208300.14029Magnesium0.0891060.0671060.1671050.073106Aluminium0.045770.120720.098700.13870Silicon0.039970.056940.074950.07790Phosphorus0.0391410.078920.073920.08387Sulfur0.0943590.1072160.0912070.134200Chlorine0.0696720.0982830.0952840.130285Argon0.25840.3031\u2013\u2013\u2013\u2013Potassium0.139460.135470.148470.28146Calcium0.133670.177690.151670.10260Scandium0.05390\u2013\u2013\u2013\u2013\u2013\u2013Titanium0.078140\u2013\u2013\u2013\u2013\u2013\u2013Vanadium0.090168\u2013\u2013\u2013\u2013\u2013\u2013Chromium0.08089\u2013\u2013\u2013\u2013\u2013\u2013Manganese0.083107\u2013\u2013\u2013\u2013\u2013\u2013Iron0.102117\u2013\u2013\u2013\u2013\u2013\u2013Cobalt0.107100\u2013\u2013\u2013\u2013\u2013\u2013Nickel0.065133\u2013\u2013\u2013\u2013\u2013\u2013Copper0.174130\u2013\u2013\u2013\u2013\u2013\u2013Zinc0.076770.084770.098770.14276Gallium0.048800.105810.192810.13581Germanium0.0381310.0451310.0561330.068133Arsenic0.073720.069700.080720.09972Selenium0.056560.094550.071540.06154Bromine0.1043580.1061840.1461820.136184Krypton0.05960.41730.62330.6023Rubidium0.413360.498370.176340.23036Strontium0.087560.199550.128320.24247Yttrium0.13269\u2013\u2013\u2013\u2013\u2013\u2013Zirconium0.06365\u2013\u2013\u2013\u2013\u2013\u2013Niobium0.06088\u2013\u2013\u2013\u2013\u2013\u2013Molybdenum0.10489\u2013\u2013\u2013\u20130.09584Technetium0.07884\u2013\u2013\u2013\u2013\u2013\u2013Ruthenium0.073113\u2013\u2013\u2013\u2013\u2013\u2013Rhodium0.16268\u2013\u2013\u2013\u2013\u2013\u2013Palladium0.080120\u2013\u2013\u2013\u2013\u2013\u2013Silver0.15141\u2013\u2013\u2013\u2013\u2013\u2013Cadmium0.159540.179550.12150\u2013\u2013Indium0.039770.085770.155770.10277Tin0.073960.065960.078960.08794Antimony0.060920.169910.083910.13592Tellurium0.070800.162790.123770.12279Iodine0.1442860.1371470.1461450.175141Xenon0.62080.58440.47220.7936Cesium0.258400.335430.372250.35843Barium0.202510.228470.207480.26151Lanthanum0.25347\u2013\u2013\u2013\u2013\u2013\u2013Lutetium0.05060\u2013\u2013\u2013\u2013\u2013\u2013Hafnium0.07142\u2013\u2013\u2013\u2013\u2013\u2013Tantalum0.07459\u2013\u2013\u2013\u2013\u2013\u2013Tungsten0.14157\u2013\u2013\u2013\u2013\u2013\u2013Rhenium0.068108\u2013\u2013\u2013\u2013\u2013\u2013Osmium0.07250\u2013\u2013\u2013\u2013\u2013\u2013Iridium0.16971\u2013\u2013\u2013\u2013\u2013\u2013Platinum0.057140\u2013\u2013\u2013\u2013\u2013\u2013Gold0.15884\u2013\u2013\u2013\u2013\u2013\u2013Mercury0.143640.110640.135630.13964Thallium0.202590.248550.208450.26843Lead0.140530.167530.121530.12551Bismuth0.142810.616750.225820.68275Table\u00a07Average unsigned errors in bond angles (Degrees)ElementPM6No. in setPM5No. in setPM3No. in setAM1No. in setLithium7.79286.82283.53289.4828Beryllium6.61146.44146.94145.9814Boron3.27314.41314.6131\u2013\u2013Carbon2.501342.791342.751312.25131Nitrogen7.32378.01376.75357.9431Oxygen12.145911.125810.17539.5742Fluorine8.32316.18326.34324.672Sodium21.0042.8743.4345.324Magnesium8.44247.282414.23247.1024Aluminum4.05205.26207.21194.3319Silicon5.25353.37352.81342.8834Phosphorus3.24354.40356.01355.0735Sulfur5.23465.64455.42415.0541Chlorine3.65519.47510.31514.805Potassium17.901110.271112.931112.7511Calcium14.991611.351616.811618.0615Scandium7.9832\u2013\u2013\u2013\u2013\u2013\u2013Titanium7.8639\u2013\u2013\u2013\u2013\u2013\u2013Vanadium7.4644\u2013\u2013\u2013\u2013\u2013\u2013Chromium3.7719\u2013\u2013\u2013\u2013\u2013\u2013Manganese6.0226\u2013\u2013\u2013\u2013\u2013\u2013Iron11.2130\u2013\u2013\u2013\u2013\u2013\u2013Cobalt10.6829\u2013\u2013\u2013\u2013\u2013\u2013Nickel10.4448\u2013\u2013\u2013\u2013\u2013\u2013Copper10.7744\u2013\u2013\u2013\u2013\u2013\u2013Zinc10.922714.41278.162713.3427Gallium4.431810.861814.431813.8418Germanium4.58525.37528.95525.7152Arsenic6.29366.52366.48365.0336Selenium7.272416.162412.37235.4623Bromine12.64420.03419.2133.273Rubidium9.691110.201121.03116.6811Strontium18.162532.912532.922531.0025Yttrium12.2934\u2013\u2013\u2013\u2013\u2013\u2013Zirconium10.3612\u2013\u2013\u2013\u2013\u2013\u2013Niobium6.5423\u2013\u2013\u2013\u2013\u2013\u2013Molybdenum8.1527\u2013\u2013\u2013\u20138.7327Technetium4.9622\u2013\u2013\u2013\u2013\u2013\u2013Ruthenium6.9334\u2013\u2013\u2013\u2013\u2013\u2013Rhodium10.6622\u2013\u2013\u2013\u2013\u2013\u2013Palladium9.1946\u2013\u2013\u2013\u2013\u2013\u2013Silver23.369\u2013\u2013\u2013\u2013\u2013\u2013Cadmium15.231013.521020.0910\u2013\u2013Indium4.47177.21175.30174.9417Tin3.06344.09343.743411.8134Antimony6.494112.24416.84417.4041Tellurium4.85257.00255.33257.8725Iodine8.33112.55120.6614.531Cesium15.50128.521219.381211.7512Barium28.651028.431037.041036.1710Lanthanum9.2514\u2013\u2013\u2013\u2013\u2013\u2013Lutetium7.0826\u2013\u2013\u2013\u2013\u2013\u2013Hafnium5.6410\u2013\u2013\u2013\u2013\u2013\u2013Tantalum9.8815\u2013\u2013\u2013\u2013\u2013\u2013Tungsten10.909\u2013\u2013\u2013\u2013\u2013\u2013Rhenium7.3932\u2013\u2013\u2013\u2013\u2013\u2013Osmium12.6710\u2013\u2013\u2013\u2013\u2013\u2013Iridium7.8618\u2013\u2013\u2013\u2013\u2013\u2013Platinum5.9272\u2013\u2013\u2013\u2013\u2013\u2013Gold13.5916\u2013\u2013\u2013\u2013\u2013\u2013Mercury20.201520.991518.471521.4915Thallium5.731010.281019.951025.3810Lead4.33205.24204.61203.5719Bismuth8.012521.74258.282533.9925Table\u00a08Average unsigned errors in dipole moments (D)ElementPM6No.PM5No.PM3No.AM1No.Hydrogen0.622660.802650.642220.50204Lithium0.78160.95160.79160.5216Beryllium1.6311.4910.2710.531Boron0.66170.66170.7317\u2013\u2013Carbon0.512190.622180.411760.42165Nitrogen0.61480.66480.46400.5539Oxygen0.991981.271961.05740.7475Fluorine0.801241.111210.59630.6959Sodium1.3460.8061.9761.266Aluminium0.3311.5011.7610.531Silicon0.21111.09110.72110.2911Phosphorus0.83140.79140.37100.8710Sulfur0.62281.01280.74210.7021Chlorine0.991031.271000.77470.8443Potassium0.4440.3441.3040.584Calcium0.7341.1241.2340.334Scandium1.119\u2013\u2013\u2013\u2013\u2013\u2013Titanium1.028\u2013\u2013\u2013\u2013\u2013\u2013Vanadium0.828\u2013\u2013\u2013\u2013\u2013\u2013Chromium1.989\u2013\u2013\u2013\u2013\u2013\u2013Manganese1.0611\u2013\u2013\u2013\u2013\u2013\u2013Iron1.6114\u2013\u2013\u2013\u2013\u2013\u2013Cobalt1.046\u2013\u2013\u2013\u2013\u2013\u2013Nickel1.4015\u2013\u2013\u2013\u2013\u2013\u2013Copper1.1110\u2013\u2013\u2013\u2013\u2013\u2013Zinc0.2140.1840.1640.164Gallium0.2011.8111.3510.641Germanium0.63230.63230.55230.5923Arsenic0.3760.9960.3560.376Selenium0.66100.94100.61100.8010Bromine0.90881.34871.01370.5039Rubidium1.8462.4361.6560.446Strontium1.6461.3162.5561.516Yttrium1.708\u2013\u2013\u2013\u2013\u2013\u2013Zirconium0.948\u2013\u2013\u2013\u2013\u2013\u2013Niobium0.9110\u2013\u2013\u2013\u2013\u2013\u2013Molybdenum1.098\u2013\u2013\u2013\u20131.488Technetium1.7413\u2013\u2013\u2013\u2013\u2013\u2013Ruthenium1.1312\u2013\u2013\u2013\u2013\u2013\u2013Rhodium1.096\u2013\u2013\u2013\u2013\u2013\u2013Palladium0.978\u2013\u2013\u2013\u2013\u2013\u2013Silver1.989\u2013\u2013\u2013\u2013\u2013\u2013Cadmium0.4222.2220.672\u2013\u2013Indium0.4730.7830.7531.363Tin0.28130.41130.88130.8113Antimony0.5550.7750.4850.615Tellurium0.4720.7520.3121.352Iodine1.03771.54771.48281.2230Cesium1.2593.4791.8990.879Barium1.77111.29111.93111.1111Lanthanum1.238\u2013\u2013\u2013\u2013\u2013\u2013Hafnium0.636\u2013\u2013\u2013\u2013\u2013\u2013Tantalum0.975\u2013\u2013\u2013\u2013\u2013\u2013Tungsten0.9214\u2013\u2013\u2013\u2013\u2013\u2013Rhenium0.7613\u2013\u2013\u2013\u2013\u2013\u2013Osmium0.638\u2013\u2013\u2013\u2013\u2013\u2013Iridium0.968\u2013\u2013\u2013\u2013\u2013\u2013Platinum1.078\u2013\u2013\u2013\u2013\u2013\u2013Gold0.7814\u2013\u2013\u2013\u2013\u2013\u2013Mercury0.6390.7790.6390.679Thallium0.8931.3530.4532.433Lead0.7360.7660.4160.826Bismuth0.4283.2181.1483.408Table\u00a09Average unsigned errors in ionization potential (eV)ElementPM6No.PM5No.PM3No.AM1No.Hydrogen0.432260.402260.602260.52217Lithium0.89120.88121.29120.5912Beryllium0.5270.2970.9370.457Boron0.31110.34111.0111\u2013\u2013Carbon0.412300.392300.542300.54227Nitrogen0.55430.45430.53430.4842Oxygen0.62720.56720.63720.6969Fluorine0.64670.65670.74670.8565Sodium0.3450.3451.4350.514Magnesium0.9741.0541.1041.414Aluminum0.6230.2930.4030.693Silicon0.43110.81110.70110.6811Phosphorus0.49130.47130.64130.5613Sulfur0.52460.51460.48460.6246Chlorine0.48620.58620.57600.6157Potassium0.2340.5040.5440.343Calcium0.7411.2410.5210.411Scandium3.731\u2013\u2013\u2013\u2013\u2013\u2013Titanium0.091\u2013\u2013\u2013\u2013\u2013\u2013Zinc0.3250.3550.9950.495Gallium0.5230.7331.2831.163Germanium0.70130.49130.93131.0513Arsenic0.6950.3150.6250.795Selenium0.38100.29100.47101.2210Bromine0.28330.39331.20330.4932Rubidium0.1830.3930.9330.223Strontium0.6310.3810.1410.261Cadmium0.3350.4650.395\u2013\u2013Indium0.6320.8622.0620.832Tin0.70140.48141.22140.4414Antimony0.4450.9051.1650.545Tellurium0.4330.2030.2530.703Iodine0.47290.46290.48290.8929Cesium0.5840.7141.3741.114Barium0.0810.9710.0810.751Mercury0.51120.43120.74120.4912Thallium0.3030.4630.8030.533Lead0.56130.47130.93130.6513Bismuth0.9851.2850.7251.665\nComparison of the accuracy of PM6 with the other NDDO methods PM5, PM3, and AM1, was made more complicated by the fact that different sets of elements were available in each method. To allow a simple comparison, therefore, average unsigned errors (AUE) for the four common properties for various subsets are presented in Tables\u00a010, 11, 12, 13 and 14. To ensure a valid comparison the same number of data were used in each method, except for AM1 in \u201cwhole of main group\u201d, where data for cadmium and boron were not used.\nTable\u00a010Average unsigned errors in \u0394Hf for various sets of elements (kcal mol\u20131)Set of elementsNo.PM6RM1PM5PM3AM1H, C, N, O11574.644.895.605.659.41H, C, N, O, F, P, S, Cl, Br, I 17745.056.576.758.0512.57Whole of main group31886.1615.2717.7622.3470 elements44928.01Table\u00a011Average unsigned errors in bond lengths for various sets of elements (\u00c5)Set of elementsNo.PM6RM1PM5PM3AM1H, C, N, O4130.0250.0220.0330.0210.031H, C, N, O, F, P, S, Cl, Br, I7120.0310.0360.0440.0370.046Whole of main group26360.0850.1210.1040.13170 elements51540.091Table\u00a012Average unsigned errors in angles for various sets of elements (Degrees)Set of elementsNo.PM6RM1PM5PM3AM1H, C, N, O1003.13.13.32.52.7H, C, N, O, F, P, S, Cl, Br, I2443.24.04.33.83.4Whole of main group9008.08.68.58.870 elements16817.9Table\u00a013Average unsigned errors in dipole moments for various sets of elements (D)Set of elementsNo.PM6RM1PM5PM3AM1H, C, N, O550.380.220.310.260.26H, C, N, O, F, P, S, Cl, Br, I1310.370.330.500.360.38Whole of main group3130.600.860.720.6570 elements5690.85Table\u00a014Average unsigned errors in I.P.s for various sets of elements (eV)Set of elementsNo.PM6RM1PM5PM3AM1H, C, N, O990.450.400.410.510.45H, C, N, O, F, P, S, Cl, Br, I2290.470.410.440.510.56Whole of main group3830.500.490.680.6370 elements3850.50\nComparison with AM1*\nWinget, et al. [15], developed AM1* parameters for P, S, and Cl, in which Voityuk\u2019s diatomic parameters were used for all atom-pairs involving P, S, and Cl with H, C, N, O, F, P, S, Cl and Mo. In the AM1* method, all parameters for elements other than the ones being optimized are held constant at the AM1 values. As such, AM1* could be regarded as a hybrid method: parameters for a few individual elements are re-optimized, in this case with some changes in the set of approximations, while holding the parameters for the other methods constant at their AM1 values. Tables comparing individual P, S, and Cl species calculated with AM1* and PM6 are given in the supplementary material. A summary of the statistical analysis is given in Table\u00a015. Winget et al. also reported AM1* parameters for titanium and zirconium [15]. These parameters were not used in the comparison given here because the set of approximations used was incompatible with the set used in PM6.\nTable\u00a015Average unsigned errors in phosphorus, sulfur, and chlorine\u00a0\u0394Hf (kcal mol\u22121)Bond length (\u00c5)Dipole (D)I.P. (eV)Angles (Degrees)PM6AM1*No.PM6AM1*No.PM6AM1*No.PM6AM1*No.PM6AM1*No.Phosphorus8.319.1900.0220.051560.570.49100.510.81122.53.319Sulfur6.510.61990.0290.060710.360.64140.520.50453.14.134Chlorine6.118.21560.0250.106690.550.60100.520.62253.414.64\nComparison with RM1\nIn 2006, ten elements, H, C, N, O, F, P, S, Cl, Br, and I, that had been parameterized at the AM1 level were re-parameterized [35]; the result was a new method, RM1. No changes were made to the set of approximations used, so that, for example, P, S, Cl, Br, and I used only the s-p basis set. That is, RM1 was functionally identical to AM1. A statistical analysis showed that RM1 was more accurate than any of the other NDDO methods, and therefore was the method of choice for modeling organic compounds. An indication of the effect of the current changes to the set of approximations can be obtained by comparing the AUE for PM6 and RM1 in Tables\u00a010, 11, 12, 13 and 14.\nVoityuk reported the parameterization of molybdenum [14] at the AM1* level. These parameters were added to the standard AM1 parameters and were used in the analysis.\nComparison with high-level methods\nA comparison of PM6, HF 6\u201331G(d) and B3LYP 6\u201331G(d) errors in predicted \u0394Hf for 1373 compounds is given in the supplementary material. Only compounds containing the elements H, C, N, O, F, P, S, Cl, and Br were considered, these being the more important elements in biochemistry. Ab-initio \u0394Hf were obtained from the calculated total energies by the addition of a simple atomic correction and conversion from atomic units to kcal mol\u22121. No allowance was made for thermal population effects, zero point energies, etc., the assumption being made that such effects could be absorbed into the atomic corrections.\nA statistical analysis of errors in thermochemical predictions for the three methods is given in Table\u00a016. A check was also done to verify that the error distribution was approximately Gaussian. The resulting histogram, shown in Fig.\u00a01, shows that the distribution is indeed Gaussian.\nTable\u00a016Statistical analysis of errors in predicted \u0394Hf for various methods (kcal mol\u22121)StatisticPM6B3LYP*HF*Median3.263.755.10AUE4.445.197.37RMS6.237.4210.68No. of molecules in set: 1373* Basis set: 6\u201331G*Fig.\u00a01Histogram of errors in calculated \u0394Hf\nHydrogen bonding\nOne of the commonest forms of hydrogen bonding involves a hydrogen atom attached to an oxygen atom and forming a weak bond to a distant oxygen atom. The simplest, well-characterized case is that of the water dimer. In an exhaustive analysis of this system, Tschumper, et. al. [36], characterized this system using CCSD(T) and a large basis set. They identified and characterized ten stationary points on the 12-dimensional potential energy surface of the dimer and determined that the lowest energy conformer of the water dimer was 5.00\u00a0kcal mol\u22121 more stable than two isolated water molecules. A comparison of the relative heats of formation of these points calculated using NDDO methods is shown in Table\u00a017. The AUE for the various methods are as follows: PM6: 1.35\u00a0kcal mol\u22121, PM5: 3.35, PM3: 2.16, and AM1: 1.67.\nTable\u00a017Relative energies of conformers of water dimerStructureRef.Relative \u0394Hf (kcal mol\u22121)PM6PM5PM3AM1(Non-planar open Cs)*\u22125.00\u22123.96\u22120.24\u22122.79\u22122.811 (Non\u2212planar open Cs)0.000.000.000.000.002 (Open Ci)+0.520.830.500.910.643 (Planar Open Cs)0.570.660.250.930.464 (Cyclic Ci)0.700.290.112.10\u22120.945 (Cyclic C2)0.950.770.392.63\u22120.516 (Cyclic C2h)0.990.590.212.71\u22120.677 (Triply Hydrogen Bonded1.810.93\u22121.851.16\u22120.958 (Non-planar Bifurcated3.572.67\u22120.831.711.269 (Non-planar Bifurcated1.790.73\u22121.951.15\u22120.8710 (Planar Bifurcated C2v2.711.42\u22121.771.28\u22120.05*: Relative to two isolated water molecules+: Structures 2 \u2013 10 are relative to Structure 1\nThe energies of various different types of hydrogen bonds were estimated from the energy released when the two small molecules involved associate to form a hydrogen-bonded system. Table\u00a018 lists the values predicted using B3LYP and the NDDO methods.\nTable\u00a018Comparison of B3LYP and PM6 hydrogen bond energies (kcal mol\u22121)Hydrogen-bonded systemRefPM6PM5PM3AM1Ammonia - ammonia\u22122.94\u22122.34\u22120.77\u22120.67\u22121.41Water - methanol\u22124.90\u22125.12\u22122.59\u22120.20\u22124.52Water - acetone\u22125.51\u22125.25\u22122.43\u22122.22\u22124.09Water, dimer, linear (O\u2013H\u2013O = 180\u00b0)\u22125.00\u22123.69\u22121.57\u22123.49\u22123.16Water, dimer\u22125.00\u22124.88\u22122.43\u22121.95\u22125.01Benzene dimer, T-shaped\u22122.34\u22120.83\u22120.22\u22120.56\u22120.07Water - acetate anion\u221219.22\u221218.72\u221212.28\u221215.77\u221215.91Water - formaldehyde\u22125.17\u22124.22\u22122.17\u22122.73\u22123.40Water - ammonia\u22126.36\u22124.32\u22122.75\u22121.53\u22122.90Water - formamide\u22128.88\u22127.60\u22124.14\u22124.33\u22127.54Formic acid, dimer\u221213.90\u221210.03\u22124.75\u22128.65\u22126.44Water - methylammonium cation\u221218.76\u221214.90\u22128.94\u221210.48\u221214.36Formamide - formamide\u221213.55\u221210.83\u22124.46\u22126.08\u22128.14Acetic acid, dimer\u221214.89\u221210.33\u22124.50\u22128.70\u22126.44\nNitrogen pyramidalization\nA well-documented fault in PM3 nitrogen was its exaggerated degree of pyramidalization when in the sp2 configuration. This is dramatically evident in N-methylacetamide, where the H-N-C\u2013C torsion angle should be 180\u00a0\u00b0, but is predicted by PM3 to be 136\u00a0\u00b0. That is, the nitrogen, instead of being in a planar environment, is predicted to be highly pyramidal. The results of a survey of 19 molecules that contain sp2 nitrogen are presented in Table\u00a019.\nTable\u00a019Average errors in pyramidalization of nitrogen (Torsion angle about nitrogen, in degrees)StatisticPM6PM3AM1RM1Average signed error\u22121.7\u221213.60.29.7Average unsigned error5.015.03.519.1\nTransition metals\nOptimizing parameters for transition metals was not as straightforward as for the main group elements. As with the main group compounds, there is a wealth of structural reference data on transition metal complexes. However, unlike main group compounds, there is a distinct shortage of reliable thermochemical data. To alleviate this shortage, the thermochemical data that was available was augmented by the results of DFT calculations. It was recognized, however, that these derived reference data were likely to be of a lower accuracy than the experimental data. Many transition metal complexes are also highly labile; a consequence of this was that some moieties that are known to exist in the solid phase were predicted to be unstable in the gas phase, at least at the PM6 level of calculation. In most cases, such moieties had a high formal charge, therefore, without any countercharge, their instability in isolation is understandable. When an intrinsically unstable ion was identified, it was removed from further consideration.\nMost transition metal compounds also have extensive UV-visible properties, arising from d-d transitions and from charge-transfer excitations, the presence of these absorption bands being indicative of the existence of low-lying electronic excited states. The self-consistent field (SCF) equations frequently did not converge unless special techniques were used. One of these, using the direct inversion of the iterative sub-space [37], or DIIS, would frequently yield an SCF when other methods failed. However, as a result of the way it works, the DIIS converged the wavefunction to the nearest stationary point, not necessarily to the lowest energy point. Because of the potential existence of multiple low-lying excited states, special care had to be taken when the DIIS technique was used. Conversely, the tendency to converge to the nearest stationary point was an advantage when electronic states of transition metal atoms were being optimized. In several instances, the lowest energy wavefunction corresponded to a hybrid of s, p and d atomic orbitals that did not transform as any irreducible representation of the group of the sphere. In those cases, the wavefunction could be induced to converge to the correct spherical harmonic solution by using the DIIS procedure.\nSets of transition metals\nFor the purpose of discussion, the set of 30 transition metals can be partitioned into eight of the groups of the Periodic Table, with each group containing one or more triads of elements. A detailed discussion of each element is impractical because of the wide range of compounds in transition metal chemistry. The following section, therefore, will be limited to systems where PM6 does not work well, and to systems illustrative of the structural chemistry of specific elements.\nGroup IIIA: Scandium, Yttrium, Lanthanum, and Lutetium\nPossibly because of its scarcity, only a few experimental thermochemical reference data for scandium compounds were available for use in the parameterization. What reference data existed were augmented by the results of DFT calculations and with a large number of atomic energy levels for the neutral and ionized atom. Only the chemistry of ScIII was studied. Most bond lengths involving scandium were reproduced with good accuracy (for example tri(\u03b75\u2212cyclopentadienyl)-scandium, Fig.\u00a02), the exception being the coordination complex [Sc(H2O)9]3+ which PM6 predicts to decompose to [Sc(H2O)7]3+ plus two water molecules.\nFig.\u00a02Tri(\u03b75\u2212cyclopentadienyl)-scandium Reference value in parenthesis\nAs with scandium, very few thermochemical reference data were found for yttrium or lanthanum. To compensate for this, extensive use was made of the CSD. The chemistry of lutetium is similar to that of lanthanum, with the principal difference being that whereas LaIII has an empty 4f shell, in LuIII that shell is completely filled. Since the 4f shell is, at least chemically, virtually inert, lutetium could be regarded as a conventional transition metal, and was therefore included in this work.\nGroup IVA: Titanium, Zirconium, and Hafnium\nIn contrast to all the elements of Group IIIA, titanium is plentiful, and an abundance of reference data on TiIII and the more common TiIV is available. These data include many tetrahedral and octahedral inorganic complexes as well as organotitanium compounds. Most bond lengths are reproduced with good accuracy, the exceptions being the Ti-H bond in TiH4, where the predicted value, 1.36\u00a0\u00c5, is 0.37\u00a0\u00c5 shorter than the reference, and coordination complexes which involve oxygen forming a purely dative bond to titanium. In this latter case, the Ti-O bond is typically too long by 0.1 to 0.3\u00a0\u00c5.\nThe behavior of zirconium and hafnium is similar to that of titanium.\nGroup VA: Vanadium, Niobium, and Tantalum\nMost of the structural chemistry of vanadium in its five common oxidation states, 0, II, III, IV, and V, are reproduced with good accuracy. The common VO5 structure which occurs in bis(Acetylacetonato)-oxo-vanadium(iv), where vanadium forms a double bond to one oxygen atom and single bonds to the other four, is reproduced accurately, the V=O distance being 1.58\u00a0\u00c5 (reference, 1.56), the V-O distance 2.03\u00a0\u00c5 (1.97), and the O-V=O angle: 104.5\u00a0\u00b0 (105.9).\nNot all systems were reproduced with such accuracy. When there are several ligands around a vanadium atom, the effects of steric crowding are over-emphasized, and PM6 incorrectly predicts that one of the metal-oxygen bonds would break. An example is bis(bis(\u03bc2-trifluoroacetato-O,O\u2032)-(\u03b75-cyclopentadienyl)-vanadium), where each vanadium atom extends bonds to four oxygen atoms and one cyclopentadienyl. In this system, PM6 predicts that one of the V-O bonds would break.\nIn the heavier elements there is an increased tendency to form highly symmetric polynuclear complexes. An example is the tantalum dication, [Ta6Cl12]2+. This is predicted to have an octahedral structure in modest agreement with the DFT result (Fig.\u00a03).\nFig.\u00a03Calculated structure of the complex ion [Ta6Cl12]2+ Reference value in parenthesis\nTransition metal complexes usually have one or more unpaired electrons; such systems can only be modeled using an open shell method such as unrestricted Hartree Fock (UHF) or restricted Hartree Fock followed by a configuration interaction (RHF-CI) correction. The UHF method is faster and more reliable, and is the method of choice when only simple properties such as heats of formation or geometries are of interest. For [M6X12]2+, M = Nb or Ta, X = Cl or Br, UHF predicts an almost octahedral complex, a very slight distortion lowering the symmetry to D4h. This distortion is also reflected in the asymmetric charge distribution. When RHF-CI is used, the geometry converges on the exact Oh structure.\nGroup VIA: Chromium, Molybdenum, and Tungsten\nMost Cr\u2013O and Cr\u2013N bonds are reproduced well, as illustrated by [CrIII(EDTA)]- in Fig.\u00a04. The organometallic bond in chromium hexacarbonyl is 1.90\u00a0\u00c5, which is in good agreement with the crystal structure, 1.92\u00a0\u00c5, found in FOHCOU01[21].\nFig.\u00a04Chromium Ethylenediaminetetraacetate anion, [Cr(III)(EDTA)]\u2212\nThe octacyano-molybdate(IV) moiety, [MoIV(CN)8]4\u2212, is a stable eight-coordinate organometallic molybdenum complex ion whose geometry in the crystal is that of a slightly distorted square antiprism. Rather unexpectedly, this structure was reproduced by PM6, the expectation being that in the absence of crystal field forces the structure would have optimized to a geometry which has a higher symmetry, i.e., converged to the exact D4d geometry. The predicted Mo-C distance was 2.22 versus 2.16\u00a0\u00c5, again in unexpectedly good agreement for an ion with such a large formal charge.\nMolybdenum forms the cluster anion [Mo6(\u03b73-Cl8)Cl6]2\u2212 in which the six molybdenum atoms form a regular octahedron. PM6 successfully reproduces this structure, and predicts the following distances: Mo-Mo: 2.30 (2.63), Mo-\u03b73Cl: 2.75 (2.56), and Mo-Cl: 2.50 (2.43\u00a0\u00c5).\nThe trioxide of molybdenum can form polyoxometalates, a typical example of which is the \u03b1-keggin heteropolyoxyanion [SiO4@MoVI12O36]4\u2212. In this structure, shown in Fig.\u00a05, each Mo forms a double bond with one oxygen, single bonds to four other oxygen atoms, and what can only be described as a third of a bond to a sixth oxygen that is part of the SiO4 unit. Despite the apparently high symmetry, Td, this system has only a center of inversion. This low symmetry is reproduced by PM6.\nFig.\u00a05\u03b1-Keggin structure of tetraconta-oxo-silicon-dodeca-molybdenum, [SiO4@Mo12O36]4\u2212 Crossed-eyes stereo; Mo=O: 1.77\u00a0\u00c5 (1.69), Mo-O: 2.00 (1.85), Si\u2013O: 1.52 (1.64) (Ref. in parentheses)\nPM6 predicts the structures of all three hexacarbonyls with good accuracy, but gives qualitatively the wrong structures for the dinuclear decacarbonyls. This failure to qualitatively predict the structure of the polynuclear carbonyls occurred frequently during the survey of the transition metals.\nGroup VIIA: Manganese, Technetium, and Rhenium\nLike many other transition metals, manganese can form sepulchrates, closo polyhedral complexes of general structure 3, 6, 10, 13, 16, 19-hexaaza-bicyclo(6.6.6)icosane. In contrast to the more common open hexadentate chelates of manganese, e.g. [MnII(EDTA)]2\u2212, the metal atom in a sepulchrate is extremely tightly bound, and cannot be removed without destroying the organic framework. A simple sepulchrate is shown in Fig.\u00a06. PM6 predicts the Mn-N distance with good accuracy but gets the twist angle incorrect. A DFT calculation reproduced the twist angle found in the crystal, which suggests that the error in the twist angle cannot be attributed to the neglect of crystal packing forces.\nFig.\u00a06[Sepulchrate-manganese(III) ]3+ (3,6,10,13,16,19-Hexaaza-bicyclo(6.6.6)icosane)-manganese(III) \u00a7: CSD entry: HAFBUL\nAlthough there is a large amount of structural information on technetium compounds, there is a distinct shortage of thermochemical data. To make up for this, almost all the reference heats of formation of representative technetium compounds were derived from DFT calculations. Only one heat of formation was used in this derivation, that of the isolated technetium atom, therefore the reference values used almost certainly include a systematic error that may amount to many kilocalories per mole. Consequently, the reference heats of formation and the errors in PM6 predicted heats of formation of technetium compounds should be taken cum granus salis. However, this should not be construed as implying that they are meaningless: because reactions are balanced, when heats of reaction are evaluated, any systematic errors in the heats of formation are cancelled out.\nOne of the more important technetium species is the pertechnetate ion, [TcO4]\u2212, used in nuclear medicine. In this ion, PM6 predicts the Tc-O distance to be 1.73\u00a0\u00c5, in good agreement with the DFT value of 1.76\u00a0\u00c5.\nGroup VIIIA: Iron, Cobalt, Nickel, Ruthenium, Rhodium, Palladium, Osmium, Iridium, and Platinum\nThe geometries of most compounds of this large group were reproduced with modest to good accuracy, including the iron-porphorin complex, Fig.\u00a07, of the type found in heme. The main exception is iron pentacarbonyl, Fe(CO)5, which in its equilibrium geometry is known unambiguously to be of point-group D3h, and which PM6 predicts to be equally unambiguously C4v. When this error was discovered, attempts were made to correct the fault by adding a rule to the training set for iron. This rule stated that \u201cThe C4v geometry was 28.7\u00a0kcal mol\u22121 higher in energy than the D3h geometry,\u201d 28.7\u00a0kcal mol\u22121 being the difference between the energies of the two structures calculated using DFT. However, even when a very large weighting factor, 20.0, was used, the C4v structure remained more stable than the D3h, albeit the error in the relative energies was decreased. During this optimization errors in all other iron compounds increased significantly. Rather than accept a general deterioration in the predicted properties of iron compounds, the rule was removed from the training set.\nFig.\u00a07trans-7,8-Dihydro-2,3,7,8,12,13,17,18-octaethylporphyrinato-iron (II) Reference value (CSD entry BUYKUB) in parenthesis\nThe well-known red complex nickel dimethylglyoxime is normally encountered in the quantitative analysis of inorganic nickel in solution. At the center of the molecule is the planar structure NiN4 structure, which is frequently found in nickel compounds in biochemical systems. PM6 predicts this with good accuracy (Fig.\u00a08).\nFig.\u00a08Nickel Dimethylglyoxime Reference value (CSD entry NIMGLO10) in parenthesis\nOne of the first polyhapto organometallic complexes discovered was Zeise\u2019s salt. In the anion, [PtCl3(\u03b72-C2H4)]\u2212, platinum forms a synergic bond with an ethylene molecule. The calculated and X-ray structures of this complex are shown in Fig.\u00a09.\nFig.\u00a09Zeise\u2019s Salt, trichloro-(\u03b72-ethene)-platinate Reference value (CSD entry XIVSAK) in parenthesis\nGroup IB: Copper, Silver, and Gold\nCopper phthalocyanine is an extremely stable blue dyestuff. As with nickel dimethylglyoxime, the planar CuN4 moiety at the center of the porphyrin ring is typical of many copper species of importance in biochemistry. PM6 reproduces it with very good accuracy (Fig.\u00a010).\nFig.\u00a010Copper phthalocyanine Reference value (CSD entry CUPOCY16) in parenthesis\nDimethyl gold cyanide tetramer provides a good example of a square-planar AuIII complex. In this system, each gold atom forms covalent single bonds of length 1.99\u00a0\u00c5(2.01) to the carbons of the methyl groups, a weaker, longer bond of length 2.12\u00a0\u00c5(2.23) to the carbon of the cyanide group, and a still longer bond, 2.27\u00a0\u00c5(2.23) to the nitrogen atom.\nGold also forms small planar clusters. PM6 predicts that neutral clusters of up to about nine gold atoms should be planar, an example being the D6h Au7 cluster, in which the Au-Au distance is predicted to be 2.71\u00a0\u00c5(2.01). Clusters of up to 12 gold atoms are also predicted to be stable, provided the cluster has a single negative charge.\nGroup IIB Zinc, Cadmium, and Mercury\nThese elements have completely filled d shells; therefore the valence shell can be limited to the s and p orbitals. As such, they behave like main-group elements.\nDiscussion\nMethodological changes\nDuring the development of PM6, only very minor changes were made to the set of approximations. The main change was in the construction of the training set used for parameter optimization. One of the most important changes was the use of rules in the training set to define chemical information that was not a function of any single molecule. In earlier methods the training set had included only standard reference data. Of their nature, such data could not allow for chemical facts that were independent of any one moiety. For example, the strength of a hydrogen bond is of great importance in biochemistry, but it could not be expressed in terms of a single species. By use of rules, the value of some chemical quantity could be related to that of another. In the case of hydrogen bonding, the heat of formation of the water dimer was made a function of the heat of formation of two separated water molecules.\nRules were particularly useful when elements of the three transition metal series were being optimized. Many complexes of these elements are highly labile, and, in the early stages of parameter optimization, there was a strong tendency for the optimized geometry of such complexes to be qualitatively incorrect. Faults of this kind could not be corrected by simply increasing the weight assigned to the correct geometry, so rules were developed to indicate that the faulty geometries were indeed incorrect. Specific points on the potential energy surface were selected, and from single-point high level calculations, the relative energy of these points above the minimum was evaluated. The points selected were precisely those qualitatively incorrect geometries resulting from the use of the then-current parameters. The fact that the incorrect geometry was predicted by high level methods to be of higher energy than the correct geometry was then added to the set of rules. A good example of such a rule was the rule concerning Fe(CO)5 mentioned above, in which the only datum that was defined referred to the relative energies of the compound in two different symmetries. No reference was made to the bond lengths, or bond angles. With such a rule in place, the parameters could be re-optimized to minimize the error arising from the rule, with the effect that the energy of the incorrect symmetry increased relative to that of the correct symmetry. In the majority of cases, one rule of this type was sufficient; less frequently, two rules were used, and, in rare cases, even more rules were necessary.\nAnother change was the use of very large reference data training sets. In earlier parameterizations, the training set used was deliberately made as small as possible. Only when the resulting method was used in a survey of species not used in the training set could the predictive power of the method be determined. The training set used in the development of PM6 was designed to be considerably larger than the survey set. The rationale for this was that, by including in the training set reference data for unconventional species, e.g., non-equilibrium and hypothetical species, a greater region of the error-function surface could be defined. This would in turn, result in a better definition of the values of the parameters. That this is useful can be evidenced by the recent work in parameterizing chlorine at the AM1* level, where the compound 1,1\u2032,2-trichloro-1,2,2\u2032-trifluoroethane, C2Cl3F3 has a reported \u0394Hf of \u2013173.7\u00a0kcal mol\u22121, but the value predicted using AM1* was \u2013273.9\u00a0kcal mol\u22121. That is, the AM1* value was in error by over 100\u00a0kcal mol\u22121. If this compound had been included in the training set, it is highly likely that the error would have been significantly reduced.\nAlthough over 10,000 reference data were used in the PM6 training set, there are several indications that even this large number is still inadequate for the definition of the values of the parameters, and that an even larger training set would be highly desirable. In light of this, work has begun on identifying species to be added to the training set. During the testing of PM6, several faults were found in the method. Some of these were quickly traced to specific core-core parameters. One of the hydrogen atoms in the complex [ScIII (H2O)7]3+ was predicted to readily move toward the central atom with the result that a Sc-H bond was formed. Such faults could easily be corrected by the addition to the training set of appropriate reference data from high-level calculations. This was done in several instances, and the specific error was corrected, but this action then also required all the testing to be re-started. Because this was a time-consuming process, when faults were found near the end of the testing phase, the decision was taken that the fault should be noted, as in the Sc-H error mentioned here, and to take no further action at that time.\nA different type of error, found only near the end of testing, was the unrealistically large p electron population of some transition metals. The values of the parameters that determine the p population are defined using two very different groups of reference data: atomic energy levels and conventional properties of polyatomics. If atomic energy levels were excluded from the parameter optimization, then the p population would become very small; but if atomic energy levels were excluded, then the resulting method would not be suitable for reproducing such levels. The decision to use all available atomic energy levels in the training set was a value judgement. In the next training set, it is likely that the result of this decision-making process will be different.\nDetecting faults in semiempirical methods is difficult, and rather than wait until all errors of this type were found and fixed, a process that could potentially take several more years, the decision was made to freeze the parameters at their current value. Obviously, PM6 still has many errors; some have already been described. Work has already started in an attempt to correct them.\nElimination of computational artifacts\nEarlier NDDO methods, particularly PM3 and AM1, produced artifacts in potential energy surfaces as a result of unrealistic terms in the core-core approximation, specifically in the set of Gaussian functions used. In PM6, only one Gaussian-type correction to the core-core potential is allowed, and, consequently, the potential for these artifacts has been reduced. On the other hand, because PM6 uses diatomic parameters, the likelihood of readily-characterized errors involving specific pairs of atoms, e.g. Sc and H, as mentioned earlier, is increased. Errors of this type can be easily eliminated by a re-parameterization of the faulty diatomic.\nThere are over 450 sets of diatomic interactions parameterized in PM6, covering most of the common types of chemical bonds. But the number of potential bonds is much larger: given 70 elements, there are almost 2500 diatomic sets. If a molecule contains two elements for which the diatomic interaction parameters are missing, then, provided the elements are well separated, say by more than 4 \u00c5ngstroms, the absence of the parameters will not be important. If the two elements were near to each other, then the diatomic core-core parameters would be needed. This would involve generating a small training set of reference data that included a few examples of the type of interaction involved, and optimizing the two terms in the diatomic interaction.\nThis ability to add diatomic parameter sets to PM6 without modifying the underlying parameterization has the advantage that more and more types of interaction can be added without changing the essential nature of the method.\nAccuracy\nPM6, being the most recent member of the NDDO family of approximate semiempirical methods, is understandably the most accurate. The development of each new method has been guided by the knowledge of the documented faults found in the earlier methods. This is reflected in the steady decrease in AUE of simple organic compounds, from 12.0\u00a0kcal mol\u22121 for AM1 to 4.9\u00a0kcal mol\u22121 for PM6.\nSeveral low-energy phenomena are predicted more accurately by PM6, with the most important of these being the prediction of the energies and geometries involved in hydrogen bonding. One consequence of this increased accuracy is that the lowest energy conformer of acetylacetone is now correctly predicted to be the ene-ol structure, and not the twisted di-one configuration.\nDespite the improvement in hydrogen bonding, a significant error was found in the balance of energies involved in forming zwitterions of hydroxyl and amine groups. This is best illustrated by the dimer of 2-aminophenol, where PM6 predicts that the zwitterion should be 3.6\u00a0kcal mol\u22121 more stable than the neutral form, but higher level calculations indicate that the neutral form should be 17.7\u00a0kcal mol\u22121 more stable than the zwitterion. In the solid state, CSD entries AMPHOM01 \u2013 AMPHOM10 [21], 2-aminophenol exists as the neutral species.\nIn general, however, average unsigned errors in \u0394Hf have steadily decreased as semiempirical methods have evolved. Earlier NDDO methods such as PM3 and AM1 had AUE significantly larger than the 6\u201331G* Hartree Fock method. With the advent of PM5 and RM1 errors were intermediate between HF and B3LYP. In the current work, AUE in \u0394Hf are lower than those of both B3LYP and HF 6\u201331G*. This increase in accuracy of prediction of \u0394Hf relative to higher level methods should not be construed as disparaging those methods: semiempirical methods in general, and PM6 in particular, were parameterized to reproduce \u0394Hf. The performance of these methods when applied to non-equilibrium systems, in particular transition states, is likely to be very inferior to that of B3LYP or HF 6\u201331G*.\nAs a result of the current work, there is a clear strategy for further improving the accuracy of semiempirical methods. All three potential sources of error need to be addressed. Regarding reference data, considerably more data are needed than were used here. This would likely come from increased use of high-level theoretical methods: methods significantly more accurate than those used here would obviously be needed in any future work. Parameter optimization can be performed with confidence and reliability, particularly when well-behaved systems are used. In all cases examined where problems were encountered in parameter optimization, problems also occurred in the normal SCF calculation in MOPAC2007. This implies that as faults in the SCF procedure are corrected, faults in parameter optimization would also be removed.\nPermanent errors\nNotwithstanding the optimism just expressed, not all errors can be eliminated by better data and better optimizations. Despite strenuous efforts, some calculated quantities persistently failed to agree with the reference values. Many potential causes for these failures were investigated. In each case the weight for the offending quantity was increased considerably and the parameter optimization re-run. When that was done, the specific error decreased, but errors elsewhere increased disproportionately. Since the final gradient of the error function was acceptably small, it followed that the parameter optimization was not in error. The reference data were checked to ensure that they were in fact trustworthy. Because two of the three possible origins of error had been eliminated, the inescapable conclusion was that there is a fault in the set of approximations. The most serious of these faults was the qualitatively incorrect prediction of the geometry of the exceedingly simple system, iron pentacarbonyl.\nConclusions\nThe NDDO method has been modified by the adoption of Voityuk\u2019s core-core diatomic interaction parameters. This has resulted in a significant reduction in error for compounds of main-group elements, and, together with Thiel\u2019s d-orbital approximation, allows extension of the NDDO method to the whole of the transition metal block.\nThe accuracy of PM6 in predicting heats of formation for compounds of interest in biochemistry is somewhat better than Hartree Fock or B3LYP DFT methods, using the 6-31G(d) basis set. For a representative set of compounds, PM6 gave an average unsigned error of 4.4\u00a0kcal mol\u22121; for the same set HF and B3LYP had AUE of 7.4 and 5.2\u00a0kcal mol\u22121, respectively.\nThe potential exists for further large increases in accuracy. This would likely result from the increased use of accurate reference data derived from high-level methods, and from the development of better tools for detecting errors at an early stage of method development.\nElectronic supplementary material\nTables of errors in predicted heats of formation, geometries, dipole moments, and ionization potentials obtained using PM6, AM1, PM3, PM5, RM1, AM1*, HF 6-31G*, and B3LYP 6-31G* for individual species are provided, together with references for all reference data used. These data were used in generating the statistics presented in the discussion on accuracy.\nESM\nDOC 23.7\u00a0MB","keyphrases":["nddo","pm6","parameterization","transition metals"],"prmu":["P","P","P","P"]}
{"id":"Neuropsychologia-2-1-2394569","title":"Cortical circuits for silent speechreading in deaf and hearing people\n","text":"This fMRI study explored the functional neural organisation of seen speech in congenitally deaf native signers and hearing non-signers. Both groups showed extensive activation in perisylvian regions for speechreading words compared to viewing the model at rest. In contrast to earlier findings, activation in left middle and posterior portions of superior temporal cortex, including regions within the lateral sulcus and the superior and middle temporal gyri, was greater for deaf than hearing participants. This activation pattern survived covarying for speechreading skill, which was better in deaf than hearing participants. Furthermore, correlational analysis showed that regions of activation related to speechreading skill varied with the hearing status of the observers. Deaf participants showed a positive correlation between speechreading skill and activation in the middle\/posterior superior temporal cortex. In hearing participants, however, more posterior and inferior temporal activation (including fusiform and lingual gyri) was positively correlated with speechreading skill. Together, these findings indicate that activation in the left superior temporal regions for silent speechreading can be modulated by both hearing status and speechreading skill.\n1\nIntroduction\nAuditory speech processing reliably engages perisylvian regions, particularly in the left hemisphere (e.g., Scott & Johnsrude, 2003). In hearing people, perisylvian regions are also recruited for silent speechreading. In particular, silent speechreading elicits activation in superior temporal regions, including middle and posterior portions of the superior temporal gyrus, its dorsal and ventral surfaces (i.e., lateral sulcus and superior temporal sulcus or STS, respectively) and the middle temporal gyrus (Bernstein et al., 2002; Calvert et al., 1997; Calvert et al., 1999; Calvert, Campbell, & Brammer, 2000; Ludman et al., 2000; MacSweeney et al., 2000; Paulesu et al., 2003; Pekkola et al., 2005; Ruytjens, Albers, van Dijk, Wit, & Willemsen, 2006), and inferior frontal regions (Buccino et al., 2004; Campbell et al., 2001; Nishitani & Hari, 2002; Paulesu et al., 2003; Watkins, Strafella, & Paus, 2003). Generally, seen speech appears to engage similar circuits to those activated when speech is heard. This includes portions of the superior temporal cortex reliably involved in processing auditory information. Activation in this region also appears to be modulated by speechreading skill. Hall, Fussell, and Summerfield (2005) did not find marked activation in the superior temporal gyrus at the group level when hearing adults observed silently spoken sentences, as compared to viewing facial gurning. However, their participants varied greatly in their ability to speechread, and a positive correlation was found between activation in the left posterior superior temporal gyrus and speechreading skill.\nDeaf people can outperform hearing people in comprehending seen speech (Bernstein, Demorest, & Tucker, 2000; Mohammed, Campbell, MacSweeney, Barry, & Coleman, 2006). Nevertheless, earlier reports suggested that superior temporal activation for speechreading was less reliably observed in deaf than in hearing people (MacSweeney et al., 2001; MacSweeney et al., 2002). However, the group size for these studies was small (n\u00a0=\u00a06), and so there may not have been sufficient statistical power to detect activation in this region. Furthermore, while the speechreading task in MacSweeney et al.'s (2002) study was easy (identify spoken numbers between 1 and 9), it was compared with a relatively high-level task\u2014counting numbers of meaningless mouth actions. In contrast, a separate study by Sadato et al. (2005), reported activation in superior temporal regions in both hearing and deaf participants viewing speech-like actions. Here, the stimulus was a cartoon avatar opening and closing its mouth to form different vowel-like patterns, which participants may have interpreted as phonological gestures.\nThe present study is the first to examine patterns of activation in deaf people who are proficient speechreaders while they searched for a speechread target embedded in lists of unrelated words. We anticipated that both hearing status and speechreading ability, measured outside the scanner, may determine the extent of activation in perisylvian regions. This was explored in two complementary ways. First, the group comparison between deaf and hearing activation patterns was assessed with speechreading skill entered into the analysis as a covariate. Speechreading skill was assessed using the Test of Adult Speechreading (TAS, Mohammed et al., 2006). By \u2018partialling out\u2019 individual differences in speechreading ability, we hoped to establish whether activation in brain regions could be modulated as a function of hearing status, irrespective of speechreading skill. Second, we used correlational analysis to establish, for each group in turn, which regions were sensitive to variations in speechreading skill.\nTo summarise, this study examines cortical correlates for the perception of lists of speechread words under lexical target detection conditions. We aimed to identify regions that may be activated during observation of silently spoken lexical items that are not drawn from a closed set, and when the contrast (baseline) condition was a speaker at rest. The questions posed were: (1) To what extent do prelingually deaf people who are proficient signers and speechreaders show activation in superior temporal regions, including auditory cortical processing regions? (2) Are the patterns of activation different in deaf and hearing people? (3) In which regions is speechreading ability positively correlated with activation?\n2\nMethod\n2.1\nParticipants\nThirteen (six female; mean age: 27.4; age range: 18\u201349) deaf adults were tested. All were congenitally (severely or profoundly) deaf (81\u00a0dB mean loss or greater in the better ear over four octaves, spanning 500\u20134000\u00a0Hz). Across the group, the mean hearing loss in the better ear was 103\u00a0dB. They were all native signers, having acquired British Sign Language (BSL) from their deaf signing parents. Thirteen (six female; mean age: 29.4; age range: 18\u201343) hearing, monolingual speakers of English were also tested. All participants were right-handed with no known neurological or behavioural abnormalities. Non-verbal IQ was measured using the Block Design subtest of the WAIS-R. Speechreading was measured using the Test of Adult Speechreading (TAS). The TAS comprises three subtests of silent speechreading in English: word identification, sentence identification, and short story identification (Mohammed et al., 2006). Independent-samples t-tests showed that deaf and hearing participants did not differ on non-verbal IQ (p\u00a0>\u00a00.1). However, deaf participants scored significantly higher than hearing non-signers on the TAS (t (24)\u00a0=\u00a04.779, p\u00a0<\u00a00.001), confirming earlier findings (Mohammed et al., 2006) with an independent sample of participants. Participant characteristics are summarised in Table 1. Standard scores (TAS z-scores) were derived from the populations reported in Mohammed et al.'s (2006) study, together with those for the present study. These scores were calculated separately for deaf and for hearing groups. Standard scores were used in order to correct for the differences in statistical distribution of scores within the deaf and the hearing groups, and were used in the correlational analyses (see Table 1).\nAll participants gave written informed consent to participate in the study according to the Declaration of Helsinki (BMJ 1991; 302: 1194) and the study was approved by the Institute of Psychiatry\/South London and Maudsley NHS Trust Research Ethics Committee.\n2.2\nStimuli\nStimuli were full-colour motion video of silently mouthed English words. Stimuli were modelled by a deaf native signer of BSL, who spoke English fluently (i.e., a BSL-English bilingual). The model was viewed full-face and torso. The words to be speechread were piloted on adult hearing volunteers who were not scanned. The final stimuli comprised only those words that were speechreadable by the hearing pilots. Stimuli consisted of both content words (nouns) and descriptive terms (both adjectival and adverbial).\n2.3\nfMRI experimental design and task\nThe speechreading task was one of four conditions presented to participants. The other three conditions comprised signed language (BSL) material (not reported here). The speech stimuli were presented in blocks, alternating with blocks of the other three experimental conditions (30-s blocks for each condition), and with a 15-s baseline condition. The total run duration for all four conditions and baseline was 15\u00a0min. Both deaf and hearing participants were given the same target-detection task and instructions. During the speechreading condition, participants were instructed to watch the speech patterns produced by the model and to try to understand them. They were required to make a push-button response whenever the model was seen to be saying \u2018yes\u2019. This relatively passive task was chosen in preference to a \u2018deeper\u2019 processing task (such as semantic classification) for several reasons. First, it allowed for relatively automatic processing of non-target items to occur (as confirmed in post-scan tests). Second, it ensured similar difficulty of the task across stimulus conditions. As hearing non-signers would not be able to perform a semantic task on the sign stimuli, using a sparse target detection task enabled all participants to perform the same task during all experimental conditions. Over the course of the experiment, participants viewed 96 stimulus items, 24 in each of the four experimental conditions. Items were not repeated within the same block and were pseudorandomised to ensure that repeats were not clustered at the end of the experiment. Each participant saw five blocks of the speechreading condition.\nThe baseline condition comprised video of the model at rest. The model's face and torso were shown, as in the experimental conditions. During the baseline condition, participants were directed to press a button when a grey fixation cross, digitally superimposed on the face region of the resting model, turned red. To maintain vigilance, targets in both the experimental and baseline conditions occurred randomly at a rate of one per block. Prior to the scan, participants practiced the tasks and were shown examples of the \u2018yes\u2019 targets outside the scanner using video of a model and words that were similar but not identical to those used in the experiment. Following the experiment, a sample of the hearing participants (8 of 13) and all of the deaf participants were asked to identify the items they had seen.\nStimuli in the experimental conditions appeared at a rate of 15 items per block. The rate of articulation across all experimental conditions, including the speechreading blocks, was approximately one item every 2\u00a0s. All stimuli were projected onto a screen located at the base of the scanner table via a Sanyo XU40 LCD projector and then projected to a mirror angled above the participant's head.\n2.4\nImaging parameters\nGradient echoplanar MRI data were acquired with a 1.5-T General Electric Signa Excite (Milwaukee, WI, USA) with TwinSpeed gradients and fitted with an 8-channel quadrature head coil. Three hundred -weighted images depicting BOLD contrast were acquired at each of the 40 near-axial 3\u00a0mm thick planes parallel to the intercommissural (AC-PC) line (0.3\u00a0mm interslice gap; TR\u00a0=\u00a03\u00a0s, TE\u00a0=\u00a040\u00a0ms, flip angle\u00a0=\u00a090\u00b0). The field of view for the fMRI runs was 240\u00a0mm, and the matrix size was 64\u00a0\u00d7\u00a064, with a resultant in-plane voxel size of 3.75\u00a0mm. High-resolution EPI scans were acquired to facilitate registration of individual fMRI datasets to Talairach space (Talairach & Tournoux, 1988). These comprised 40 near-axial 3\u00a0mm slices (0.3\u00a0mm gap), which were acquired parallel to the AC-PC line. The field of view for these scans was matched to that of the fMRI scans, but the matrix size was increased to 128\u00a0\u00d7\u00a0128, resulting in an in-plane voxel size of 1.875\u00a0mm. Other scan parameters (TR\u00a0=\u00a03\u00a0s, TE\u00a0=\u00a040\u00a0ms, flip angle\u00a0=\u00a090\u00b0) were, where possible, matched to those of the main EPI run, resulting in similar image contrast.\n2.5\nData analysis\nThe fMRI data were first corrected for motion artefact, then smoothed using a Gaussian filter (FWHM 7.2\u00a0mm) to improve the signal to noise ratio over each voxel and its immediate neighbours prior to data analysis. In addition, low frequency trends were removed by a wavelet-based procedure in which the time series at each voxel was first transformed into the wavelet domain and the wavelet coefficients of the three levels corresponding to the lowest temporal frequencies of the data were set to zero. The wavelet transform was then inverted to give the detrended time series. The least-squares fit was computed between the observed time series at each voxel and the convolutions of two gamma variate functions (peak responses at 4 and 8\u00a0s) with the experimental design (Friston, Josephs, Rees, & Turner, 1998). The best fit between the weighted sum of these convolutions and the time series at each voxel was computed using the constrained BOLD effect model suggested by Friman, Borga, Lundberg, and Knutsson (2003) in order to constrain the range of fits to those that reflect the physiological features of the BOLD response1.\nFollowing computation of the model fit, a goodness of fit statistic was derived by calculating the ratio between the sum of squares due to the model fit and the residual sum of squares (SSQ ratio) at each voxel. Permutation testing, as well as its freedom from many of the distributional assumptions of parametric tests, also offers the possibility of testing a number of statistics that are not easily testable parametrically. The SSQ ratio is such a statistic and is a simplified substitute for the F statistic suggested by Edgington (1995) that avoids the necessity of calculating the residual degrees of freedom of the time series following model fitting.\nThe data were permuted by the wavelet-based method described by Bullmore et al. (2001) with the exception that, prior to permutation, any wavelet coefficients exceeding the calculated threshold (as described by Donoho and Johnstone (1994)) were removed. These were replaced by the threshold value. This step reduces the likelihood of refitting large, experimentally unrelated components of the signal following permutation.\nSignificant values of the SSQ were identified by comparing this statistic with the null distribution, determined by repeating the fitting procedure 20 times at each voxel and combining data over all intracerebral voxels. This procedure preserves the noise characteristics of the time series during the permutation process, and the global assessment of the null distribution performed in this way provides good control of Type I error rates (Bullmore et al., 2001). The voxel-wise SSQ ratios were calculated for each subject from the observed data and, following time series permutation, were transformed into standard space (Talairach & Tournoux, 1988) as described previously (Brammer et al., 1997; Bullmore et al., 1996). The Talairach transformation stage was performed in two parts. First, each participant's fMRI data was realigned with their own high resolution -weighted images using a rigid body transformation. Second, an affine transformation to the Talairach template was computed. The cost function for both transformations was the maximization of the correlation between the images. Voxel size in Talairach space was 3\u00a0mm\u00a0\u00d7\u00a03\u00a0mm\u00a0\u00d7\u00a03\u00a0mm.\n2.6\nGroup analysis\nIdentification of active 3-D clusters was performed by first thresholding the median voxel-level SSQ ratio maps at the false positive probability of 0.05. The activated voxels were assembled into 3-D connected clusters and the sum of the SSQ ratios (statistical cluster mass) was determined for each cluster. This procedure was repeated for the median SSQ ratio maps obtained from the wavelet-permuted data to compute the null distribution of statistical cluster masses under the null hypothesis. The cluster-wise false positive threshold was then set using this distribution to give an expected false positive rate of <1 cluster per brain (Bullmore et al., 1999).\n2.7\nANOVA\nDifferences between the groups were calculated by fitting the data at each voxel in which all participants had non-zero data using the following linear model, Y\u00a0=\u00a0a\u00a0+\u00a0bX\u00a0+\u00a0e, where Y is the vector of BOLD effect sizes for each individual, X is the contrast matrix for the particular inter-group contrast required, a is the mean effect across all individuals in the groups, b is the computed group difference and e is a vector of residual errors. The model is fitted by minimising the sum of absolute deviations rather than the sums of squares to reduce outlier effects. The null distribution of b is computed by permuting data between groups (assuming the null hypothesis of no effect of group) and refitting the above model. This permutation method thus gives an exact test (for this set of data) of the probability of the value of b in the unpermuted data under the null hypothesis. The permutation process permits estimation of the distribution of b under the null hypothesis of no mean difference. Identification of significantly activated clusters was performed by using the cluster-wise false positive threshold that yielded an expected false positive rate of <1 cluster per brain (Bullmore et al., 1999).\n2.8\nANCOVA\nAnalysis of covariance was used to address behavioural differences between the deaf and hearing participants in relation to the patterns of activation for the speechreading condition (see Table 1). Differences in responses (R) were inferred at each voxel using the linear model, R\u00a0=\u00a0a0\u00a0+\u00a0a1H\u00a0+\u00a0a2X\u00a0+\u00a0e, where H codes the contrast(s) of interest between groups, X is a covariate and e is the residual error. Maps of the standardized coefficient (size of group difference) (a1), were tested for significance against the null distribution of a1 (no effect of group membership) generated by repeatedly refitting the above model at each voxel following randomization of group membership (H).\n2.9\nCorrelational analysis\nIn order to examine the relationship between brain activation and speechreading skill, correlational analysis was performed between the BOLD effect data for each individual and the Test of Adult Speechreading (TAS) z-score. These were calculated separately for each group. Pearson product\u2013moment correlation coefficients were calculated between the observed behavioural and BOLD effect data. The null distribution of correlation coefficients was then computed by permuting the BOLD data 100 times per voxel and then combining the data over all voxels. Median voxel-level maps were computed at the false probability of 0.05 and cluster-level maps, where r was significant, were computed such that the expected false positive rate was <1 cluster per brain.\n3\nResults\n3.1\nBehavioural data\nAll participants completed the behavioural (target detection) task in the scanner reasonably accurately. Deaf participants identified the speechreading targets more accurately than hearing participants (mean accuracy (max\u00a0=\u00a05), deaf\u00a0=\u00a04.69, hearing\u00a0=\u00a03.85, t(24)\u00a0=\u00a02.99, p\u00a0=\u00a00.007). Speechreading target identification was slower in deaf than hearing participants (mean RT, deaf\u00a0=\u00a01192.63\u00a0ms, hearing\u00a0=\u00a0920.08\u00a0ms, t(24)\u00a0=\u00a04.15, p\u00a0<\u00a00.001). Following scanning, participants were presented with the experimental stimuli. The deaf participants identified more words than the hearing participants (mean percent correct identification, deaf\u00a0=\u00a069%, hearing\u00a0=\u00a046%), t(19)\u00a0=\u00a04.11, p\u00a0=\u00a00.001). The behavioural data suggest that deaf participants\u2019 greater accuracy in identification of non-target items (as indicated by the post-scan test) may have interfered with their processing of the target (as indicated by the relatively slow reaction times to targets in the scanner).\n3.2\nfMRI data\n3.2.1\nSpeechreading vs. baseline\nIn both deaf and hearing groups, extensive activation was observed in fronto-temporal cortices, bilaterally (Table 2, Fig. 1). In deaf participants, activation in the left superior temporal cortex was focused at the border between the posterior superior temporal gyrus and the transverse temporal gyrus (BA 42\/41) and extended to the middle (BA 21) and inferior (BAs 37, 19) temporal gyri and the supramarginal gyrus (BA 40). This cluster of activation also extended to inferior (BAs 44, 45) and middle (BAs 6, 9) frontal gyri and precentral gyrus (BA 4). In the right hemisphere, a cluster of activation focused in the superior\/middle temporal gyri (BA 22\/21) extended to BAs 42 and 41 and posterior inferior temporal gyrus (BAs 37, 19). Activation in the right frontal cortex was focused in the precentral gyrus (BA 6) and extended to the inferior (BAs 44, 45) and middle (BAs 46, 9) frontal gyri. Additional activation was observed at the border of the medial frontal gyrus and the anterior cingulate gyrus (BA 6\/32).\nIn hearing participants, we observed activation focused in the left middle temporo-occipital junction (BA 37) and in the right superior\/middle temporal gyrus (BA 22\/21). These clusters of activation extended to include the superior and transverse temporal gyri (BAs 22, 42, 41), the postcentral gyri (BA 43) and the middle and inferior temporal (BAs 21, 37, 19, 20) and cerebellar gyri. In the left hemisphere, this cluster also extended to the supramarginal gyrus (BA 40). In both hemispheres, clusters in the inferior parietal cortex were focused in the supramarginal gyrus (BA 40). These clusters extended to angular (BA 39) and middle occipital (BA 19) gyri. The cluster in the right hemisphere extended medially to the border of the dorsal posterior cingulate gyrus (BA 31). Activation in frontal cortices was focused in the precentral gyrus (BA 4\/6) of the left hemisphere and in the inferior frontal gyrus (BA 44) of the right hemisphere. In both hemispheres, frontal activation included the inferior (BAs 44, 45 47) middle (BA 46) and superior (BA 9) frontal gyri and the precentral gyrus (BAs 4, 6). In the right hemisphere, the frontal cluster extended anteriorly to the border of the frontal pole (BA 10). Additional activation was observed in the right medial frontal gyrus (BA 6), extending to medial BA 8 and anterior cingulate gyrus (BAs 24 and 32).2\n3.2.2\nDeaf vs. hearing\nDeaf native signers displayed significantly greater activation in left and right superior temporal cortices than hearing non-signers. In the left hemisphere, the cluster of activation (116 voxels) was focused at the border between the posterior superior temporal gyrus (i.e., planum temporale) and the transverse temporal (i.e., Heschl's) gyrus (BA 42\/41; x\u00a0=\u00a0\u221254, y\u00a0=\u00a0\u221222, z\u00a0=\u00a010). In the right hemisphere, the cluster (61 voxels) was focused at the border between the superior and middle temporal gyri (BA 22\/21; x\u00a0=\u00a051 y\u00a0=\u00a0\u22127 z\u00a0=\u00a0\u22123). Hearing non-signers showed greater activation than deaf signers in the right prefrontal cortex (128 voxels, focused in BA 44; x\u00a0=\u00a040, y\u00a0=\u00a011, z\u00a0=\u00a026).\nWhen speechreading performance, as indicated by individual TAS z-score, was entered as a covariate into this analysis, deaf participants displayed greater activation than hearing participants in the left temporal cortex. The cluster of activation (120 voxels) was focused at the border between the posterior superior temporal gyrus (i.e., planum temporale) and the transverse temporal (i.e., Heschl's) gyrus (BA 42\/41; x\u00a0=\u00a0\u221254, y\u00a0=\u00a0\u221222, z\u00a0=\u00a010). The focus of this cluster was verified using probabilistic maps provided by Penhune, Zatorre, MacDonald, and Evans (1996) (25\u201350% probability of Heschl's gyrus) and Westbury, Zatorre, and Evans (1999) (26\u201345% probability of planum temporale). Based on these probability maps, 15 voxels within this cluster, displayed \u226550% probability of being located in Heschl's gyrus, and five voxels showed \u226546% probability of being in planum temporale. This cluster also extended into the posterior lateral portion of the superior temporal gyrus (BA 22) and the middle and posterior portions of the superior temporal sulcus and middle temporal gyrus (BA 21; see Fig. 2). No brain regions were significantly more active in hearing than deaf participants when speechreading was a covariate in the analysis.\n3.2.3\nCortical activation for speechreading: correlations with speechreading skill\nSpeechreading skill, as measured by performance on the Test of Adult Speechreading (TAS), varied considerably across participants (Table 1). Several brain regions were significantly positively associated with TAS z-scores in both deaf and hearing groups.\n3.2.4\nDeaf group\nIn the deaf group, ten clusters of activation (\u22655 voxels) were positively associated with speechreading skill (see Table 3). In the temporal lobe, clusters in the superior temporal cortex were focused in the lateral portion of the transverse temporal gyrus (BA 41) in the right hemisphere, and in the superior temporal gyrus (BA 42) in the left hemisphere. However, although the Talairach and Tournoux (1988) atlas suggests that this cluster incorporates the transverse temporal gyrus, the probability map of this region provided by Penhune et al. (1996) suggests otherwise. In fact, only one voxel (in the left hemisphere cluster) displayed a \u226550% probability of being located in this region (Penhune et al., 1996). Both clusters extended to include the posterior superior temporal gyrus (BAs 42, 22). Additional areas showing significant correlation included the middle portion of the right middle temporal gyrus (BA 21). In the frontal cortex, correlations were observed in the middle frontal gyri of both hemispheres (BA 6). In the right hemisphere, correlations were also observed in the dorsolateral prefrontal cortex (BA 46), precentral gyrus (BA 6\/4) and in the anterior insula. Additional correlations were observed in the anterior cingulate gyrus (BA 32\/24) and the cerebellum.\n3.2.5\nHearing group\nIn the hearing group, clusters of activation that were positively correlated with TAS z-scores included the fusiform (BA 37) and lingual (BA 18) gyri of the right hemisphere and the right postcentral gyrus (BA 4). Additional positive correlations were observed in the posterior cingulate gyrus (BA 23).\n4\nDiscussion\nDeaf participants were better speechreaders than hearing participants, both in terms of their TAS performance (Table 1) and, when tested post-scan at identifying the words presented in the scanner. The finding that deaf people can be better speechreaders than hearing individuals is not new (Bernstein et al., 2000; Mohammed et al., 2006). Deaf people, including deaf people who use a signed language, rely on speechreading, whether hearing-aid supported or un-aided, to communicate in the wider hearing community. In contrast, in hearing people, where the auditory channel dominates for speech identification, reliance on silent seen speech is generally unfamiliar and unpractised. In the present study most participants, whether deaf or hearing, could speechread much of the spoken material, and it can be assumed, therefore, that some of what they were shown in the scanner was lexically processed\u2014albeit more in deaf than in hearing participants. Interpretation of the imaging data must bear these considerations in mind. Covariance and correlational analyses allow the behavioural and neuroimaging results to be aligned.\nThe group-level analyses, conducted separately for the deaf and hearing groups, contrasted silent speechreading with a low-level target detection task. As such, these analyses cannot allow unambiguous interpretation of the specificity of such activation in relation to speechreading alone, but they do suggest a general pattern against which the group differences can be explored. In hearing people, the pattern of activation replicates that which has been observed in many previous studies, showing extensive activation across the temporal cortex. While some of this activation must relate to visual movement detection and to the perception of biological motion, especially in posterior and inferior regions (see, for example, Zeki et al., 1991), it is likely that much of the activation in superior temporal regions relates to speechreading, since several studies contrasting speechreading with a higher-level baseline, such as observing non-speech-like mouth movements, report enhanced activation in this region (e.g., Calvert et al., 1997; Paulesu et al., 2003). The present study found that, in both hearing and deaf participants, activation associated with speechreading words included the dorsal surface of the superior temporal cortex including the junction of the superior temporal gyrus and the lateral portion of the transverse temporal (Heschl's) gyrus (BA 42\/41). Spatial smoothing intrinsic to transforming data into standard brain space may limit the spatial resolution in this study. Thus the finding that activation for silent speechreading included the lateral portion of Heschl's gyrus must be interpreted with caution. Nevertheless, this finding is consistent with previous neuroimaging research that delineated this region on individual brains (Pekkola et al., 2005). In addition, left inferior frontal regions were activated when observing speech silently. This has also been observed where the contrasts were with higher-level conditions such as watching non-vocal mouth actions (Buccino et al., 2004; Campbell et al., 2001; Paulesu et al., 2003; Watkins et al., 2003) and may reflect the operation of mirror neuron systems in the observation of speech actions.\nThe finding of superior temporal activation for speechreading in deaf people extends earlier studies exploring the neural organisation of processing a variety of oral gestures in hearing people. This pattern of superior temporal activation found in the present study is consistent with the findings recently reported by Sadato et al. (2005), who presented deaf participants with simple segmental utterances including vowel-like lip shapes. At first sight, the present results do not fit with those we have previously reported using a closed stimulus set, covert articulation and a gurning control condition conducted with a small group of deaf people (MacSweeney et al., 2001; MacSweeney et al., 2002). However, we did report activation within right superior temporal regions, when analysis combining the data from two experiments allowed for an increase in power (MacSweeney et al., 2002). A further study involving a larger group of deaf participants, and manipulating task, baseline condition and stimuli, will help establish whether our previous studies simply lacked power or whether task and stimulus factors systematically affect the extent to which superior temporal regions are recruited during silent speechreading in those born profoundly deaf.\n4.1\nDeaf vs. hearing\nWhen hearing non-signers were compared with deaf signers, and speechreading skill (which differed between the groups) was entered as a covariate (Fig. 2) greater activation was observed for the deaf than hearing group in left middle-posterior superior temporal regions. This cluster of activation was focused at the border between the posterior and transverse temporal gyri (BA 42\/41) and extended to the middle and posterior portions of the superior temporal gyrus and sulcus, and middle temporal gyrus. No regions showed greater activation in hearing than deaf participants. In hearing people, the role of the posterior superior temporal sulcus (p-STS) has been proposed as a key \u2018binding site\u2019, responsible for cross- and supra-modal processing of co-incident auditory and visual streams in audiovisual speech processing (Calvert et al., 1999; Calvert et al., 2000). However, in deaf people, p-STS cannot play this role, since the association between seen and heard speech in deaf people is variable and relatively unsystematic. In the present study, not only was activation in this region observed in the absence of audition; it was greater in deaf than hearing people. One possibility is that activation by seen speech in p-STS is sensitive to the dominant speech modality within this multimodal region. That is, activation by silent speech in this region may be greater in deaf people because the region has developed to be sensitive to visual speech, while for hearing people it has developed to be sensitive to auditory speech characteristics, with visual speech as a secondary function. Structural imaging of the connections between p-STS and visual and auditory cortices in deaf and hearing individuals could be employed to test this hypothesis.\nA non-mutually exclusive possibility is that greater activation in superior temporal regions for deaf than hearing individuals reflects a more general plasticity of these regions in deaf people. Several studies suggest that brain regions considered specialised for audition can be recruited for processing stimuli from other modalities in deaf people (e.g., Fine, Finney, Boynton, & Dobkins, 2005; Finney, Fine, & Dobkins, 2001; Sadato et al., 2005). While the extent and specificity of primary auditory cortex recruitment by visual events remains unclear (Bavelier, Dye, & Hauser, 2006), some studies (e.g., MacSweeney et al., 2004) suggest that perception of signed language, and even of non-linguistic biological movement, can recruit regions within superior temporal cortex to a greater extent in deaf native signers than in hearing people exposed to a signed language from birth (hearing native signers).\n4.2\nCorrelations of activation with individual differences in speechreading skill\nTAS speechreading scores and post-scan speechreading of the items seen in the scanner were positively correlated (deaf: r\u00a0=\u00a00.476, p(1-tailed)\u00a0=\u00a00.05; hearing: r\u00a0=\u00a00.673, p(1-tailed)\u00a0=\u00a00.034); thus we can infer that the higher the TAS score, the more likely it is that participants would have processed the speechread material lexically. However, TAS scores were not normally distributed across the two groups. For this reason, standard scores (TAS-z) derived for each group formed the basis for exploring the relationship between speechreading skill and cortical activation. Within each group, different patterns of association were observed. In deaf participants, the correlational analyses showed that activation in the posterior portion of the superior temporal gyri (as well as middle temporal and middle frontal gyri) was positively associated with speechreading.\nIn the hearing participants, who were less able and more varied speechreaders than the deaf participants, speechreading skill was positively associated with activation in the right lingual and posterior cingulate gyri, which is consistent with findings from Hall et al. (2005). Additional activations displaying a positive correlation with speechreading skill included the right postcentral and inferior temporal (fusiform) gyri, perhaps suggesting relatively greater involvement of articulatory skill and face processing in hearing individuals\u2019 speechreading, respectively.\nTaken together, these data show that hearing status is an important determinant of activation in left superior temporal regions when words are speechread. In particular, silent speechreading elicits greater activation in the left middle and posterior portions of the superior temporal cortex, including the superior and middle temporal gyri and the lateral portion of the transverse temporal gyrus in deaf than hearing people, even when speechreading skill is held constant. However, speechreading skill can moderate this activation, showing a positive relationship in deaf but not hearing participants. The relatively small group sizes used in the correlational analysis (n\u00a0=\u00a013 in each group), however, require that this interpretation should be provisional. Hall et al. (2005) did not find reliable activation in superior temporal gyrus for silent speechreading in contrast to viewing facial gurning in a group of 33 hearing participants, who also varied widely in speechreading skill. However, they did report a reliable positive correlation between speechreading skill and activation in this region. The inference from that study together with the present one must be that, when speechread material is linguistically processed, superior temporal regions within the left hemisphere are likely to be recruited. Additionally, the present study shows that it was deaf rather than hearing people who showed this relationship most clearly, and where individual differences in speechreading skill made an additional impact, despite the range of speechreading skill being larger in the hearing than the deaf group.\nWe have shown that, when auditory regions are not activated by acoustic stimulation, they can nevertheless be activated by silent speech in the form of speechreading. This finding may have some practical as well as theoretical significance. Current practice in relation to speech training for prelingually deaf children preparing for cochlear implantation emphasises acoustic processing. In auditory-verbal training, the speaking model is required to hide her or his lips with the aim of training the child's acoustic skills (e.g., Chan, Chan, Kwok, & Yu, 2000; Rhoades & Chisholm, 2000). Thus, a neurological hypothesis is being advanced which suggests that the deaf child should not watch spoken (or signed) language since this may adversely affect the sensitivity of auditory brain regions to acoustic activation following cochlear implantation. Such advice may not be warranted if speechreading activates auditory regions in both deaf and hearing individuals.\nSpeechreading gives access to spoken language structure by eye. It therefore has the potential to impact positively on the development of auditory speech processing following cochlear implantation. While there are few consistent correlates of improved post-implant speech processing in prelingually deaf cochlear implantees, efficiency in speechreading is implicated. For example, pre-implant silent speechreading skills are positively associated with general speech and language outcomes (Bergeson, Pisoni, & Davis, 2005). The possibility that superior temporal regions in deaf individuals, once tuned to visible speech, may then more readily adapt to perceiving speech multimodally should be seriously considered when recommendations concerning pediatric cochlear implantation procedures are being developed.","keyphrases":["speechreading","deafness","fmri","brain","sign language","language"],"prmu":["P","P","P","P","P","P"]}
{"id":"Matern_Child_Health_J-2-2-1592158","title":"Preconception Care and Treatment with Assisted Reproductive Technologies\n","text":"Couples with fertility problems seeking treatment with assisted reproductive technologies (ART) such as in vitro fertilization should receive preconception counseling on all factors that are provided when counseling patients without fertility problems. Additional counseling should address success rates and possible risks from ART therapies. Success rates from ART are improving, with the highest live birth rates averaging about 40% per cycle among women less than 35 years old. A woman\u2019s age lowers the chance of achieving a live birth, as do smoking, obesity, and infertility diagnoses such as hydrosalpinx, uterine leiomyoma, or male factor infertility. Singletons conceived with ART may have lower birth weights. Animal studies suggest that genetic imprinting disorders may be induced by certain embryo culture conditions. The major risk from ovarian stimulation is multiple gestation. About one-third of live-birth deliveries from ART have more than one infant, and twins represent 85% of these multiple-birth children. There are more complications in multiple gestation pregnancies, infants are more likely to be born preterm and with other health problems, and families caring for multiples experience more stress. Transferring fewer embryos per cycle reduces the multiple birth rate from ART, but the patient may have to pay for additional cycles of ART because of a lower likelihood of pregnancy.\nCurrent knowledge\nFertility problems affect about 10% of couples in developed countries [1\u20133]. These couples should receive preconception counseling on all factors that are discussed during preconception counseling for patients without fertility problems. Additional counseling should address success rates and possible risks from the specific fertility treatments being considered because of the couple\u2019s diagnoses and preferences. To provide an example of an approach to preconception counseling for the infertile couple, treatment with assisted reproductive technologies (ART) such as in vitro fertilization (IVF) is reviewed here.\nAbout 1% of births in the United States are the result of ART [4]. ART care represents a unique opportunity for preconception counseling because the moment of conception is easily discernable. Counseling should address expected success rates from ART based on the woman and the man\u2019s risk profile. Older maternal age is the strongest risk factor for not achieving a successful outcome from ART. Live birth rates average about 40% per cycle among women less than 35 years old, and gradually decrease to about 6% per cycle among women over age 42 who use their own oocytes [4]. Older women who accept donor oocytes increase their chance of a live birth to approximately the rate seen in the age group of the donor woman [5]. Cumulative live birth rates account for the fact that many couples return for further treatment if the first cycle of ART is unsuccessful. For couples who complete two to three ART cycles, cumulative live birth rates are about 50\u201360% for women 35 years old or younger, declining to about 30% by age 40 [6]. As men\u2019s age increases, the time required for a couple to conceive lengthens, even after controlling for the woman\u2019s age and other risk factors for reduced fertility [7].\nThe chance of having a live birth from ART therapy is influenced by the health habits and the infertility diagnoses of the couple. Current tobacco smoking by women decreases ovarian function and is manifested by increased basal levels of follicle stimulating hormone. Such women produce fewer oocytes during ART and have lower pregnancy rates [8, 9]. Current smoking by the male partner also decreases pregnancy rates through direct effects on sperm and by exposing the woman partner to side-stream smoke. Increasing body mass index (BMI) is associated with decreasing ovarian response to follicle stimulating hormone, resulting in fewer oocytes collected in a given IVF cycle, as well as increasing rates of early pregnancy loss [10]. Although no strong evidence associates underweight with adverse ART outcomes, maternal underweight (BMI < 18.5\u00a0kg\/m2) is risk factor for low birth weight in the general population.\nAs the number of specific infertility diagnoses increases for a couple, pregnancy and live birth rates from ART decrease. Moreover, certain infertility diagnoses are somewhat more difficult to overcome than others during treatment. Live birth rates after ART tend to be slightly higher if the only diagnosis is endometriosis, and slightly lower if the only diagnosis is uterine leiomyoma or hydrosalpinx [4, 6, 11]. Leiomyoma may decrease uterine receptivity, mainly when an intramural or submucosal fibroid distorts the endometrial cavity. The mechanism by which hydrosalpinx worsens the prognosis in ART is not understood, but tubal fluid in hydrosalpinx has embryotoxic properties and salpingectomy improves live birth rates [12]. Male factor infertility is present in about 40% of couples presenting for ART, and may be a barrier to achieving pregnancy unless intracytoplasmic sperm injection (ICSI) is used [4, 6, 11].\nThe most common adverse outcome from infertility treatment is multiple gestation. ART produced 11,544 multiple birth deliveries in the United States during 2002 [4]. Of all the ART infants born that year, 45.7% were twins and 7.6% were triplets or higher order multiples. Although health outcomes are good to excellent in many twin pregnancies, mothers of twins experience more pregnancy complications, lasting infant neurologic impairment may occur from very preterm birth, and the neonatal death rate of twins is 5\u201310 times greater than of singletons. For triplets, 90% of births are preterm and the relative risk of death in the first month of life is increased 20-fold [13].\nTo place the risk of multiple gestation from ART in perspective, estimates are available on the proportion of multiple births from unassisted conception and from treatment with ovulation medications without ART. These estimates have been made by subtracting the rate of multiple births during the era before ovulation-inducing drugs (unassisted conception) and the rate of multiple births resulting from ART from the total rate of multiple births, adjusted for trends in maternal age [4, 14]. Such an analysis shows that about 65% of twins and 15\u201318% of triplets are from natural conception each year. About 20% of twins and 35\u201340% of triplets are probably from ovulation induction without ART, and about 15% of twins and 40\u201345% of triplet births result from ART [14].\nIn general, women with the best prognosis for achieving a live birth from ART are younger than 35 years, use freshly fertilized embryos from their own eggs, and have good quality embryos\u2014a marker of the latter is having more embryos available than are transferred. For this group, recent surveillance statistics showed that transferring one, two, three or four embryos resulted in live birth rates of 47.4, 51.8, 49.6 and 45.5%, respectively; the singleton rates among these births were 100.0, 60.4, 52.8 and 40.7%, respectively [4]. By age 38\u201340 years the prognosis is poorer among all women who use freshly fertilized embryos from their own eggs. Transferring one, two, three or four embryos in this group resulted in live birth rates of 10.4, 23.3, 28.9 and 29.9% respectively; the singleton rates among these births were 97.4, 81.2, 79.2 and 70.4%, respectively. Thus, for women with the best prognosis, transferring more embryos provides little improvement in live birth rates with an increasing risk of multiples. For women with a poorer prognosis, additional embryos may improve live birth rates up to a point, and the risk for multiples may increase less dramatically [4].\nDuring ART, ovarian hyperstimulation syndrome is associated with production of a large number of follicles. Usually the only symptoms are modest abdominal pain. More severely affected women, however, experience increased capillary permeability that leads to ascites and in rare cases, death. Symptoms requiring hospitalization occur in 0\u20133% of IVF cycles [15]. Ovarian cancer risk from medications used to stimulate folliculogenesis has been studied because of concerns that this treatment may trigger growth of ovarian cortical cells through the process of healing at the sites of ovulation. Although studies show that women treated with ART have about a 50% increase in ovarian cancer risk compared to the general population, when compared to control women with infertility who did not receive ART, ovarian cancer risk is not elevated [16]. Thus it appears that infertility itself may be the risk factor for ovarian cancer, rather than ART.\nSingletons conceived with ART may be at greater risk of being born prematurely, or to be small for gestational age [17]. Available studies have been limited by lack of data on key confounders. When matching is used to control for the most important confounders, the risk difference between spontaneously conceived singletons and singletons conceived with ART remains, but appears to be relatively small. The rates of fetal growth restriction differ by about 3.7%, with an overall rate of growth restriction of 8.6% among spontaneous singletons compared to a rate of 12.3% among singletons conceived with ART. The rates of very preterm birth differ by about 1.3%, with an overall rate of very preterm birth of 0.7% for spontaneous singletons compared to 2.0% for singletons conceived with ART [17].\nIn some studies, an increased risk of birth defects has been noted among IVF children compared to the general population, but in other studies the difference tends to disappear when risk factors for both infertility and birth defects such as older parental age and multiple gestation are controlled [18]. Infertility is sometimes caused by sex chromosome aberrations, such as Y chromosome deletions that lead to reduced sperm counts in men [18, 19]. Sperm of men with reduced fertility has more aneuploidy [20]. Whether treatment of male factor infertility by ICSI will cause fertility problems in male offspring cannot be known until long-term follow-up is available.\nAdverse embryo culture conditions can cause imprinting disorders in mice during a procedure similar to ART. This lends biologic plausibility to imprinting disorders as a possible adverse effect of IVF [18, 19, 21]. Imprinting is the mechanism by which congenital disorders such as Beckwith-Wiedemann syndrome, Angelman syndrome and Prader-Willi syndrome occur. These syndromes have been reported in children conceived with ART, although the rarity of the syndromes and problems generalizing from animal experiments to humans makes a causal relationship difficult to establish. In an Australian study that used data from a national genetics testing laboratory, 37 cases of Beckwith-Wiedemann syndrome occurred in 1.3 million live births, a rate of one in every 35,580 births [22]. Four of the cases were associated with IVF compared to a matched control group in which one mother had IVF (P < 0.05). In a cohort study from the Danish national registries for IVF and for all births, however, no increase in imprinting diseases was linked to ART [23].\nRecommendations for action\nReproductive endocrinologists use a combination of clinical factors to advise couples of the likelihood of success from infertility treatment, and they use clinical tests such basal follicle stimulating hormone levels to further characterize prognosis. Professional practice guidelines are available that recommend transferring fewer embryos among the couples with greatest risk for multiple gestation [24].\nProfessional guidelines note the elements to be considered when educating patients during the informed consent process for ART [25]. Reproductive endocrinologists routinely counsel patients about the risks from ART, including multiple gestation, ovarian hyperstimulation syndrome, physical injury during procedures, and success rates [26]. It may be prudent to counsel patients about emerging scientific issues as new research is published, such as concerns about embryo culture and ICSI. This would be similar to the approach used for other potential concerns. That is, even though a causal relationship between ART treatment methods and development of ovarian cancer has not been substantiated, the majority of reproductive endocrinologists inform patients of the issue [26].\nEffectiveness of interventions\nLittle research exists on comprehensive preconception counseling for infertility patients. Studies that have examined counseling about the most common adverse outcome, multiple births, have found that many infertility patients prefer to have twins and sometimes triplets rather than a single infant [27\u201329]. A randomized trial of a new educational intervention was ineffective at reducing this preference, although other research has found that raising awareness of the risks of multiple births reduces patient preference for multiples [27, 28].\nIntegration with other preconception services\nRoutine preconception counseling for women and men should include information on the many well-documented risk factors for experiencing infertility later in life. When patients who have trouble conceiving present for ART, reinforcing these concepts and combining preconception care at the key decision-making points should help maximize health outcomes for parents and their future children.","keyphrases":["assisted reproductive technologies","in vitro fertilization","preconception counseling","infertility","multiple","pregnancy","pregnancy complications"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Biogerontology-4-1-2367389","title":"Caloric restriction counteracts age-dependent changes in prolyl-4-hydroxylase domain (PHD) 3 expression\n","text":"Caloric restriction remains the most reproducible measure known to extend life span or diminish age-associated changes. Previously, we have described an elevated expression of the prolyl-4-hydroxylase domain (PHD) 3 with increasing age in mouse and human heart. PHDs modulate the cellular response towards hypoxia by regulating the stability of the \u03b1-subunit of the transcriptional activator hypoxia inducible factor (HIF). In the present study we demonstrate that elevated PHD3, but not PHD1 or PHD2, expression is not restricted to the heart but does also occur in rat skeletal muscle and liver. Elevated expression of PHD3 is counteracted by a decrease in caloric intake (40% caloric restriction applied for 6 months) in all three tissues. Age-associated changes in PHD3 expression inversely correlated with the expression of the HIF-target gene macrophage migration inhibitory factor (MIF), which has been previously described to be involved in cellular HIF-mediated anti-ageing effects. These data give insight into the molecular consequences of caloric restriction, which influences hypoxia-mediated gene expression via PHD3.\nIntroduction\nOne of the hallmarks of ageing is the decline of physiological functions. This is accompanied with progressive organ failure and an increased incidence of age-related diseases. Caloric restriction remains the most reproducible measure known to extend the maximum lifespan and to retard a variety of age-associated changes in several species (Speakman and Hambly 2007). Recent evidence suggests that caloric restriction, besides preventing accumulation of energy-related metabolites like reactive oxygen species or advanced glycation end products, triggers a regulatory response (Koubova and Guarente 2003). As a result of the limited energy intake, a modulated expression of genes involved in various regulatory integrative functions has been discovered. In a recent study we described age-related changes in the expression of the prolyl-4-hydroxylase domain (PHD) 3 and as a consequence in the expression of the hypoxia-inducible factor (HIF)-1\u03b1 transcription factor (Katschinski 2006; Rohrbach et al. 2005).\nPHD3 and HIF belong to the cellular oxygen sensing system and regulate therefore the adaptation of cells towards a decreased oxygen supply via hypoxia-inducible gene expression. This involves genes associated with angiogenesis, pH control, glucose metabolism, oxygen transport etc. (Wenger et\u00a0al. 2005). In this regard, it should be noted that some ageing-associated diseases such as ischemic diseases, atherosclerosis and cancer are associated with an altered oxygen supply. Moreover, the tolerance to ischemia and hypoxia is reduced in elderly (Abete et\u00a0al. 1999; Bosch-Marce et\u00a0al. 2007; Mariani et\u00a0al. 2000; Paolucci et\u00a0al. 2003).\nHIF is a heterodimer comprising the oxygen labile \u03b1-subunit and the oxygen-independently expressed \u03b2-subunit, which is also called ARNT. The expression of HIF-1\u03b1 is regulated by three described PHDs, i.e. PHD1, PHD2 and PHD3, at the protein level (Ivan et\u00a0al. 2001; Jaakkola et\u00a0al. 2001). PHD-dependent hydroxylation is directly depending on the oxygen availability (Bruick and McKnight 2001; Epstein et\u00a0al. 2001). Hydroxylation of HIF-1\u03b1 allows binding of the von Hippel Lindau tumor suppressor protein, which triggers rapid ubiquitination and degradation (Maxwell et\u00a0al. 1999). An age-dependent decline of HIF-1\u03b1 protein levels and HIF-target gene expression has been described in different organs like rat cerebral cortex, mouse heart, carotid body and smooth muscle cells isolated from rabbit aorta (Di Giulio et\u00a0al. 2005; Rivard et\u00a0al. 2000; Rohrbach et\u00a0al. 2005). The age-related expression pattern of PHDs in other organs besides heart is not known.\nCaloric restriction has been consistently described to challenge biological ageing (Rohrbach et\u00a0al. 2006b). Regarding the HIF system, it is interesting to note that caloric restriction counteracts age-related changes in the angiogenic response and can protect cardiomyocytes from hypoxic death in rodent models of ischemic diseases (Ahmet et\u00a0al. 2005; Facchetti et\u00a0al. 2007). To gain more insight into the impact of nutrient availability on the adaptive transcriptional response towards hypoxia, we quantified the expression of the HIF-1\u03b1 regulating PHD1\u20133 in heart, liver and skeletal muscle in young and old rats undergoing a well-defined protocol of caloric restriction.\nMaterials and methods\nAnimals and diet protocol, 40% caloric restriction for 6 months\nMale young (4\u00a0months) and senescent (22\u00a0months) Sprague-Dawley rats were obtained from Charles River (Germany), caged individually with a light\/dark cycle of 12\u00a0hrs and with tap water ad libitum. Food (AltrominR 1244; 2550\u00a0cal\/g) was offered ad libitum. Prior to the application of the specific diet protocols, daily food intake of the normal standard diet was monitored for 14 days and averaged for each rat individually. Thereafter, rats at the age of 6 months (young rats) or at the age of 24 months (old animals) were randomly assigned to one of the following diets for the next six months: rats on \u201ccontrol diet\u201d received their individual prediet average of AltrominR 1244 (2,550\u00a0cal\/g), but not more, in order to avoid any degree of diet-induced obesity. Rats subjected to caloric restriction received also their prediet average, but of a calorically reduced, fibre-rich diet (AltrominR 1344\/1500; 1,550\u00a0cal\/g). Thus, in young rats on control diet (n\u00a0=\u00a06), the daily energy intake during six months was 64.8\u00a0\u00b1\u00a02.2\u00a0kcal and in young rats on -40% caloric restriction (n\u00a0=\u00a06) the daily intake was 37.8\u00a0\u00b1\u00a01.9\u00a0kcal. In old rats the daily energy intake amounted to 57.9\u00a0\u00b1\u00a01.8\u00a0kcal with control diet (n\u00a0=\u00a03) and to 36.6\u00a0\u00b1\u00a00.1\u00a0kcal with 40% caloric restriction (n\u00a0=\u00a03). All animals were fasted for 12\u00a0hours before killing.\nRNA extraction, RT-reaction and quantification by PCR\nThe RNA was isolated from left ventricle, liver and skeletal muscle (M. gastrocnemicus) tissue as described before (Rohrbach et\u00a0al. 2007). Integrity and quality of the RNA was confirmed by agarose gel electrophoresis and the concentration determined by measuring UV-absorption.\nReal time PCR\nReverse transcription (RT) of RNA samples was carried out for 30\u00a0min at 42\u00b0C. Real-time PCR and data analysis were performed using the Mx3000P Multiplex Quantitative PCR System (Stratagene). DNA amplification was performed as follows: initial denaturation at 95\u00b0C for 10\u00a0min, 40 cycles of amplification (denaturation at 95\u00b0C for 30\u00a0s, annealing at 60\u00b0C for 60\u00a0s, and extension at 72\u00b0C for 60\u00a0s), followed by a denaturation at 95\u00b0C for 60\u00a0s and a melting curve over the range from 55\u00b0C up to 95\u00b0C. Fluorescence data were collected at the end of the annealing stage of amplification. We performed Real-time PCR of PHD1, PHD2, PHD3, MIF-1 and 18S rRNA (18S rRNA Control kit, Yakima Yellow\u00ae-Eclipse\u00ae Dark Quencher, Eurogentec) in samples derived from rat left ventricles, skeletal muscle and liver. The following primers were used: PHD1 sense CGTGAGGCATGTTGACAATC, PHD1 antisense AACACCTTTCTGTCCCGATG; PHD2 sense TACAGGATAAACGGCCGAAC, PHD2 antisense GGCTTGAGTTCAACCCTCAC; PHD3 sense GGCCGCTGTATCACCTGTAT, PHD3 antisense TTCTGCCCTTTCTTCAGCAT; MIF sense CAGAACCGCAACTACAGCAA, MIF antisense GAACAGCGGTGCAGGTAAGT. Each assay was performed in duplicate and validation of PCR-runs was assessed by evaluation of the melting curve. All data of mRNA are given as relative units of 18S rRNA concentrations.\nWestern blot\nLiver tissue was rapidly homogenized in a buffer containing 50\u00a0mmol\/l Tris\u00b7HCl, 1% SDS, 1\u00a0mmol\/l sodium-orthovanadate, 5\u00a0mmol\/l EGTA, 1\u00a0mmol\/l PMSF, 1\u00a0\u03bcg\/ml aprotinin, and 1\u00a0\u03bcg\/ml leupeptin. Proteins were quantified using the bicinchoninic acid protein assay (Pierce). Protein (50\u00a0\u03bcg) in 2\u00d7 Laemmli SDS sample buffer were boiled for 5\u00a0min and after centrifugation loaded onto a SDS-PAGE gel. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 100\u00a0V for 90\u00a0min. The filters were blocked with 0.01% Tween, 2% nonfat milk, and then incubated with antibodies directed against MIF (Abcam) and GAPDH (Abcam).\nStatistical analysis\nFor statistical comparison of age groups or the influence of caloric restriction, a non-paired t-test was used. A P-value\u00a0<\u00a00.05 was considered to represent a significant difference.\nResults\nPrevention of age-dependent increase of PHD3 in heart, liver and skeletal muscle\nAge-dependent changes of PHD1-3 expression were investigated in three different organs obtained from young (12\u00a0months old) and old (30\u00a0months old) rats. PHD1 and PHD2 mRNA expression did not differ significantly between the two age groups neither in heart, liver nor in skeletal muscle (Figs.\u00a01 and 2). However, in all three different organs investigated, there was a significant increase in PHD3 mRNA expression in the tissues obtained from the old rats compared to the young animals (Fig.\u00a03).\nFig.\u00a01Effect of age and caloric restriction on PHD1 mRNA expression. Tissue samples (heart, liver and skeletal muscle (M. gastrocnemicus)) were obtained from young (6 months old) and old (24 months old) rats with or without a 40% caloric restriction applied for 6 months. Subsequently, RNA was extracted and PHD1 mRNA quantitated by real time PCRFig.\u00a02Effect of age and caloric restriction on PHD2 mRNA expression. Tissue samples (heart, liver and skeletal muscle (M. gastrocnemicus)) were obtained from young (6 months old) and old (24 months old) rats with or without a 40% caloric restriction applied for 6 months. Subsequently, RNA was extracted and PHD2 mRNA quantitated by real time PCRFig.\u00a03Effect of age and caloric restriction on PHD3 mRNA expression. Tissue samples (heart, liver and skeletal muscle (M. gastrocnemicus)) were obtained from young (6 months old) and old (24 months old) rats with or without a 40% caloric restriction applied for 6 months. Subsequently, RNA was extracted and PHD3 mRNA quantitated by real time PCR\nYoung and old rats were subsequently challenged by caloric restriction. Daily caloric intake was reduced to 40% in young and old animals for 6 months. Control animals received their prediet average caloric intake. Using this regimen, we previously have observed that thioredoxin reductase 2 is significantly reduced in ageing skeletal and cardiac muscle and renormalized after caloric restriction (Rohrbach et\u00a0al. 2006a). Whereas no significant effect of caloric restriction was observed on the expression of PHD1 or PHD2 (Figs.\u00a01 and 2), the reduced food intake significantly counteracted the age-dependent increase in PHD3 expression in heart, liver and skeletal muscle (Fig.\u00a03). PHD3 levels in old rats after caloric restriction was comparable to the PHD3 expression levels in young animals.\nCaloric restriction affects the expression of the macrophage migration inhibitory factor\nNumerous HIF-target genes, including erythropoietin, vascular endothelial growth factor, carbonic anhydrase IX etc., have been identified (Wenger et\u00a0al. 2005). Recently, the expression of the macrophage migration inhibitory factor (MIF) has been demonstrated to be inducible by hypoxia. In addition, functional HIF binding sites in the promoter have been identified. In a recent study evidence was provided that HIF-1 plays a critical role in delaying the onset of senescence in rodent cells via transcriptional activation of MIF and thereby inhibition of the p53-mediated pathway (Welford et\u00a0al. 2006). To gain insight into the functional consequences of age and caloric restriction-mediated changes on the HIF-induced signal transduction pathways, we analyzed the mRNA expression of MIF as a function of age and caloric restriction. In line with the suggestion that HIF-dependent gene expression is hampered with increasing age, MIF RNA and protein expression was significantly lower in the heart, liver and skeletal muscle obtained from the old rats compared to the expression found in the young rats (Figs.\u00a04, 5). Restriction of food intake reversed the age-dependent decline in MIF expression (Fig.\u00a04). MIF levels after caloric restriction were comparable to the expression levels in young rats in all three tissues investigated.\nFig.\u00a04Effect of age and caloric restriction on MIF mRNA expression. Tissue samples (heart, liver and skeletal muscle (M. gastrocnemicus)) were obtained from young (6 months old) and old (24 months old) rats with or without a 40% caloric restriction applied for 6 months. Subsequently, RNA was extracted and MIF mRNA quantitated by real time PCRFig.\u00a05Effect of age and caloric restriction on MIF protein levels. Liver tissue samples were obtained from young (6 months old) and old (24 months old) rats with or without a 40% caloric restriction applied for 6 months. Subsequently protein was extracted and MIF and GAPDH protein levels were investigated by immunoblot analysis\nDiscussion\nCaloric restriction has been described to extend life in a variety of different mammalian and non-mammalian species (Masoro 2000). In addition, restriction of food intake delays the progression of several age-associated diseases. Identification of age-dependently expressed genes is one of the most direct approaches in ageing research. In a previous study, we identified PHD3 as one age-dependently expressed gene in human and mouse heart (Rohrbach et\u00a0al. 2005). The data presented here demonstrate that PHD3 expression is additionally increased in rat heart, liver and skeletal muscle with increasing age. These findings indicate that age-dependent changes in PHD3 expression are tissue- and species-independent. Furthermore our data presented here demonstrate that PHD3 expression is decreased by caloric restriction in old rats, whereas no change in the expression of PHD1 or PHD2 was observed. Previous findings indicate that each PHD-isoform displays its own tissue and cell-line-specific expression pattern (Appelhoff et\u00a0al. 2004). In addition, the susceptibility of the PHD isoform expression by change in the oxygen availability differs greatly. Whereas PHD3 is highly inducible by hypoxia, there is just a slight hypoxia-mediated induction described in case of PHD2. In case of PHD1 no hypoxia-inducible expression has been observed (Appelhoff et\u00a0al. 2004; Marxsen et\u00a0al. 2004). Including the age-dependent and caloric restriction-induced changes in PHD3 expression, PHD3 seems to be the most flexible PHD isoform regarding stimuli-induced change in expression.\nIn an unbiased screen using white adipose tissue it was found that energy restriction lowers the expression of genes linked to angiogenesis indicating that hypoxia-inducible gene expression is affected by caloric restriction (Higami et\u00a0al. 2006). Similarly, the age-dependent and caloric restriction-induced expression of PHD3 described here seems to have consequences. Quantification of the HIF-target gene MIF demonstrates a decrease of MIF expression in heart, liver and skeletal muscle in the old animals, which was reversed by caloric restriction. In line with our observation, in previously published microarray experiments increased levels of MIF RNA were found in liver lysates from young adult mice treated with caloric restriction or with a methionine-deficient diet as well as in young mice of the long-lived Snell dwarf stock (Dozmorov et\u00a0al. 2002; Miller et\u00a0al. 2005; Miller et\u00a0al. 2002). Since MIF affects cellular senescence (Welford et\u00a0al. 2006), changes in PHD3 expression with increasing age seem to be one important feature of the molecular senescence phenotype. This conclusion is in line with a recent report demonstrating increased HIF-1\u03b1 hydroxylation in aged versus young primary murine fibroblasts (Chang et\u00a0al. 2007). Together with a previously published study demonstrating that under hypoxic conditions senescent cells failed to induce HIF-1\u03b1 compared to presenescent cells our data indicate that the age-dependent changes in the oxygen sensing system may indeed have impact for the ageing process (Coppe et\u00a0al. 2006).\nUp to now a reduced protein expression of HIF-1\u03b1 with increasing age has been demonstrated in smooth muscle cells isolated from rabbit aorta, rat cerebral cortex, mouse lung, liver, kidney, heart and in the carotid body (Di Giulio et\u00a0al. 2005; Frenkel-Denkberg et\u00a0al. 1999; Rivard et\u00a0al. 2000; Rohrbach et\u00a0al. 2005). A decreased HIF-1\u03b1 expression was also demonstrated in tissue lysates from ischemic limbs comparing 20 months old mice with 2 months old mice (Bosch-Marce et\u00a0al. 2007). Hwang et\u00a0al described a diminished HIF-1\u03b1 stabilization in old rats in response to hypoxia, although in nomoxic breathing animals there was an age-related increase in basal levels of HIF binding to DNA. Finally, mouse and human fibroblasts isolated from different age groups express less HIF-1\u03b1 as a matter age (Chang et\u00a0al. 2007). However, it also should be noted that Kang et\u00a0al demonstrated an increased HIF activity and HIF-1\u03b1 expression in old rats and conflicting to the presented results a decrease in HIF activity as a result of caloric restriction in liver lysates (Kang et\u00a0al. 2005). Comparing the chow protocols, these rats were fed a soybean protein diet, which may have resulted in the described increase in redox metabolites and HIF-1\u03b1 protein expression. The degree and management of caloric restriction is not described (Hwang et\u00a0al. 2007). Taken collectively, several papers indicate a diminished HIF-response with increasing age with some exceptions, which may be tissue or diet-specific.\nWhereas ageing thus is mostly associated with a decreased hypoxia-inducible gene expression, in endometrial cancer cells mutation of PHD2 has been associated with increased expression of HIF-1\u03b1 and immortality (Kato et\u00a0al. 2006). Downregulation of HIF-1\u03b1 by overexpression of functional PHD2 altered the cancer cell phenotype and led to the occurrence of senescence markers (Kato et\u00a0al. 2006). The impact of the HIF-system for cell fate decisions additionally was described by Welford et\u00a0al demonstrating in a well defined nontransformed\/nonimmortalized knock out cell model that HIF-1 delays the onset of senescence via transcriptional activation of MIF and inhibition of p53 dependent pathways (Welford et\u00a0al. 2006). Alltogether these data demonstrate the necessity of tight regulation of the HIF system to prevent unrestricted cell proliferation or early cellular senescence.","keyphrases":["ageing","prolyl-4-hydroxylase domain","hypoxia","hif-1\u03b1"],"prmu":["P","P","P","P"]}
{"id":"Neurosci_Lett-1-5-1885960","title":"p53 is upregulated in Alzheimer's disease and induces tau phosphorylation in HEK293a cells\n","text":"p53 and tau are both associated with neurodegenerative disorders. Here, we show by Western blotting that p53 is upregulated approximately 2-fold in the superior temporal gyrus of Alzheimer's patients compared to healthy elderly control subjects. Moreover, p53 was found to induce phosphorylation of human 2N4R tau at the tau-1\/AT8 epitope in HEK293a cells. Confocal microscopy revealed that tau and p53 were spatially separated intracellularly. Tau was found in the cytoskeletal compartment, whilst p53 was located in the nucleus, indicating that the effects of p53 on tau phosphorylation are indirect. Collectively, these findings have ramifications for neuronal death associated with Alzheimer's disease and other tauopathies.\nTau is the major microtubule-associated protein (MAP) in neurons and functions in the formation and maintenance of axons by influencing microtubule organization. In adult human brain, there are six isoforms of tau generated by alternative mRNA-splicing. Tau has zero, one or two amino-terminal inserts and either three or four repeats of a microtubule-binding domain situated towards the carboxy-terminus [7]. Tau splicing and phosphorylation are developmentally regulated. Only the shortest tau isoform is expressed in foetal brain [8] and foetal tau is more extensively phosphorylated than tau from adult brain [10,15]. Phosphorylated tau is less efficient at promoting microtubule assembly [12,14] and elevated levels of phosphorylated tau correlate with increased microtubule dynamics associated with plasticity during development [1]. Increased tau phosphorylation is also a characteristic feature of Alzheimer's disease (AD) and tauopathies such as frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) [7]. In these disorders, normally soluble tau is present as paired-helical filaments (PHFs), which in turn aggregate to form neurofibrillary tangles (NFTs).\np53 is a tumor suppressor protein, which induces cell cycle arrest or apoptosis. Normally, p53 is maintained at low levels by murine double minute-2 (MDM2) or the human homologue (HDM2), which inhibit the transcriptional activity of p53 and promote degradation of p53 via the proteasome [2]. Activation of p53 involves stabilization of the protein by post-translational modifications, which disrupts the interaction between p53 and MDM2. Several studies have reported an increase in p53 immunoreactivity in sporadic AD [11,13] especially in subpopulations of cortical neurons undergoing neurofibrillary degeneration [5]. Furthermore, p53\u2212\/\u2212 mice display a reduction in tau phosphorylation [6]. These findings prompted us to investigate the effects of p53 on tau phosphorylation in vitro.\nThe following antibodies and plasmids were used in this study: mouse anti-\u03b2 actin, which was from Sigma (UK). Rabbit anti-total tau from Dakocytomation (UK). Tau-1 monoclonal antibody, which was a gift from Professor L. Binder (Cognitive Neurology and Alzheimer's Disease Center, Northwestern University, USA). PHF-1 monoclonal antibody, which was a kind gift from Dr. P. Davies (Albert Einstein College of Medicine, NY, USA). AT270 and AT8 monoclonal antibodies, goat anti-mouse IgG and goat anti-rabbit IgG were from Santa Cruz Biotechnology (USA). Anti-p53 (clone DO7) was from Novocastra Laboratories (UK). Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG were from Invitrogen (UK). p53, which was a generous gift from Dr B. Vogelstein (John Hopkins, USA). 2N4R tau was a gift from Professor J. Woodgett (Ontario Cancer Institute, Toronto, Canada) BAX-Luc and p21waf-Luc were from Dr. T. Soussi (Universite P.M. Curie, Paris).\nHuman embryonic kidney 293a cells (HEK293a) (Quantum Biotechnologies, Canada) were cultured in low glucose Dulbecco's modified essential medium (Invitrogen, UK) containing 10% fetal bovine serum (Autogen Bioclear, UK), 2\u00a0mM l-glutamine, 100\u00a0IU penicillin, and 100\u00a0mg\/ml streptomycin.\nPost mortem brain tissue from the superior temporal gyrus was provided by the MRC London Brain Bank for Research on Neurodegenerative Diseases. Use of human tissue was approved by the South London and Maudsley Research Ethics Committee and was conducted in accordance with the Declaration of Helsinki. Control subjects had no history or symptoms of neurological disorder. All AD cases were neuropathologically confirmed, using conventional histopathological techniques and diagnosis was performed using the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) criteria. Total protein was extracted (50\u00a0\u03bcg) from 17 non-demented elderly controls and 53 AD cases and p53 levels were determined by Western blotting using a specific anti-p53 antibody (1:1000). p53 expression was normalised to \u03b2-tubulin by standard densitometric procedures. Values for control and AD were compared using an unpaired two-tailed t-test, *p\u00a0<\u00a00.05. For tau phosphorylation experiments HEK293a cells plated in a 6 well plate were transfected with OptiMEM (100\u00a0\u03bcl) containing FuGene 6 (5\u00a0\u03bcl) and cDNA constructs (500\u00a0ng of each) encoding human 2N4R tau independently or in combination with p53. Cells lysates were harvested 24\u00a0h after transfection. Western blotting was performed using the following primary antibodies; rabbit anti-total tau (1:10,000), mouse anti-phospho-Ser199\/Ser202\/Thr205 (tau-1, 1:1000), mouse-anti-phospho-Ser202\/Thr205 (AT8, 1:500), mouse anti-phospho-Ser396\/Ser404 (PHF-1) (1:1000) or mouse anti-phospho-Thr181 (AT270, 1:1000) according to standard protocols with HRP-conjugated secondary antibodies and enhanced chemiluminescence reagents (Amersham Pharmacia, UK). To ensure equal loading membranes were reprobed with mouse anti-\u03b2 actin. All experiments were performed in triplicate.\nFor sub-cellular localisation experiments HEK293a cells were transfected with human 2N4R tau independently or in combination with p53 as described above. The following day the cells were fixed in ice-cold methanol and stained according to standard protocols. Cells were incubated with rabbit anti-total tau (1:500) or mouse anti-p53 (1:250) before being incubated with the appropriate fluorescent secondary antibody (1:200). Nuclei were counter-stained with Hoescht 33342. Immunofluorescence was captured using a Zeiss LSM510 meta-confocal microscope. All experiments were performed in triplicate; therefore, figures shown are representative of a single experiment.\nThe transcriptional effects of p53 were examined by reporter gene assay. Four wells of HEK293a cells plated in a 48 well plate were transfected by adding 25\u00a0\u03bcl of a master-transfection mix to the culture medium. The master-mix contained 100\u00a0\u03bcl of OptiMEM (Invitrogen, UK), 4\u00a0\u03bcl FuGene 6 (Roche, UK), 400\u00a0ng of firefly either BAX-Luc or p21waf-Luc (luciferase-based reporter DNA), 50\u00a0ng phTK-Renilla luciferase (Promega, UK) to control for transfection efficiency and 800\u00a0ng of p53. Appropriate amounts of empty vector DNA were included where necessary to maintain constant DNA concentrations. Twenty-four hours post-transfection the firefly and Renilla luciferase activities were sequentially measured using Dual-Glo reagents (Promega, UK) in a Wallac Trilux 1450 Luminometer (Perkin-Elmer, UK). Firefly values were divided by the Renilla value from the same well to control for non-specific effects. Data for each set of four replica transfections was averaged, the control in each set normalized to 1 and data presented as fold increases over control. Each assay was repeated three times.\np53 levels were quantified by Western blotting in the superior temporal gyrus from a large cohort of patients comprising of 53 AD cases and 17 control subjects. In accordance with previous reports that had examined smaller patient numbers [5,11,13] we found that p53 immunoreactivity was indeed significantly elevated in tissue from AD patients compared to healthy elderly controls (Fig. 1A). Densitometry revealed an approximate 2-fold increase in p53 expression in AD (Fig. 1B). Next we explored the effects of p53 on tau phosphorylation. The 2N4R isoform of human tau was exogenously expressed in HEK293a cells (which do not contain endogenous tau) alone or in combination with p53. Co-expression of tau with p53 resulted in an increase in tau phosphorylation as demonstrated by a decrease in electrophoretic mobility using an anti-total tau antibody, which detects all tau isoforms independently of their phosphorylation state (Fig. 2A, panel a). Tau can be phosphorylated at a number of serine (Ser) and threonine (Thr) residues; therefore, we subsequently used a number of phosphorylation-specific anti-tau antibodies to examine the effects of p53 on tau further. There was an almost complete reduction of tau-1 immunoreactivity in HEK293a cells co-transfected with p53 and tau in comparison to cells expressing tau alone (Fig. 2A, panel b). Tau-1 recognises a number of amino acids including Ser199, Ser202 and Thr205 [4] when de-phosphorylated, therefore a decrease in immunoreactivity at this site reflects an increase in tau phosphorylation. Consistent with this, p53 induced an increase in tau phosphorylation as evidenced using the AT8 antibody, which recognizes tau phosphorylated at Ser202\/Thr205 (Fig. 2A, panel c). In contrast, no changes in tau phosphorylation in the presence of p53 were observed using PHF-1 (Fig. 2A, panel d) or AT270 (Fig. 2A, panel e) monoclonal antibodies. Immunoblotting for \u03b2-actin illustrated that equal amounts of protein had been loaded across lanes (Fig. 2A, panel f).\nConfocal microscopy demonstrated that p53 and tau are compartmentally separated (Fig. 2B). p53 (red) exhibits a diffuse nuclear localisation, whilst tau (green) is present in the cytoskeletal compartment when expressed both independently and in combination. This suggests that the effects of p53 on tau phosphorylation are indirect and most likely attributable to the transcription of a p53 target gene. We verified the transcriptional properties of p53 in HEK293a cells using BAX-Luc and p21waf-Luc (Fig. 2C), which are luciferase reporter constructs derived from known p53 target genes.\nIn summary, our findings demonstrate that p53 is upregulated in the superior temporal gyrus in AD and that p53 induces tau phosphorylation at the tau-1\/AT8 epitope in HEK293a cells. We infer that the effects of p53 on tau phosphorylation are indirect as evidenced by the compartmental segregation of the two proteins. Therefore, pathological and sustained expression of p53 in adult brain might promote excessive and prolonged tau phosphorylation, which in turn might precipitate the formation of NFTs and neuronal death. Interestingly, we [9] have previously shown that TAp73 induces tau phosphorylation in HEK293a cells at the tau-1 and at the PHF-1 epitopes, which suggests that a similar mechanism of action might be shared by other p53 family members. Pertinent to our observations, it has recently been demonstrated that the expression of p53 is in part mediated by the transcriptionally active intracellular domain (ICD) of the \u03b2-amyloid precursor protein (APP), the generation of which is dependent upon the \u03b3-secretase activities of presenilin-1 [3]. It is feasible then that in sporadic and familial AD, which both exhibit increased A\u03b2 production, the concomitant increase in APP-ICD generation could lead to an increase in p53 expression and increased tau phosphorylation. Such a scheme forges a link between the two neuropathological hallmarks of AD, senile plaques and NFTs. In addition, it has also recently been demonstrated that A\u03b2 itself, in particular the 42 amino acid form, binds the p53 promoter and enhances transcription [13]. Thus, p53 seems to play a pivotal role in AD, implying that modulation of cell death pathways might be of therapeutic benefit in AD and indeed in other age related neurological disorders.","keyphrases":["p53","alzheimer's disease","tau","microtubules"],"prmu":["P","P","P","P"]}
{"id":"Crit_Care-8-3-468887","title":"Clinical review: Influence of vasoactive and other therapies on intestinal and hepatic circulations in patients with septic shock\n","text":"The organs of the hepatosplanchnic system are considered to play a key role in the development of multiorgan failure during septic shock. Impaired oxygenation of the intestinal mucosa can lead to disruption of the intestinal barrier, which may promote a vicious cycle of inflammatory response, increased oxygen demand and inadequate oxygen supply. Standard septic shock therapy includes supportive treatment such as fluid resuscitation, administration of vasopressors (adrenergic and nonadrenergic drugs), and respiratory and renal support. These therapies may have beneficial or detrimental effects not only on systemic haemodynamics but also on splanchnic haemodynamics, at both the macrocirculatory and microcirculatory levels. This clinical review focuses on the splanchnic haemodynamic and metabolic effects of standard therapies used in patients with septic shock, as well as on the recently described nonconventional therapies such as vasopressin, prostacyclin and N-acetyl cysteine.\nIntroduction\nResearch interest has focused on the intestinal and hepatic circulations in various models of shock, and particularly in septic shock. The splanchnic area is reported to be the 'motor' of multiple organ failure [1] and the 'canary' of the body [2]. In fact, because of its peculiar vascular anatomy, the hepatosplanchnic area is jeopardized during septic shock, which may potentially lead to a vicious circle of inflammatory responses, culminating in multiple organ failure syndrome.\nThe present clinical review briefly discusses the splanchnic vascular anatomy and focuses on the different therapeutic approaches that have been proposed to promote perfusion of the gastrointestinal tract during resuscitation of patients with septic shock. When possible and reasonable, we propose therapeutic recommendations.\nReferences were obtained from Medline database (from the earliest records to 2003). We used the following keywords: gastric mucosal pH or pHi, splanchnic, haemodynamics, microcirculation, sepsis, septic shock, vasoactive drugs, dobutamine, dopamine, norepinephrine, epinephrine, dopexamine vasopressin, terlipressin, prostacyclin, N-acetyl cysteine, dialysis and haemofiltration. We also reviewed the reference lists of all available review articles and primary studies to identify references not found in computerized searches. We placed emphasis on prospective, randomized, controlled clinical trials.\nAnatomy of hepatosplanchnic vascular bed\nThe splanchnic vasculature includes both serial and parallel vascular beds (Fig. 1). The gut is perfused by the coeliac trunk and mesenteric arteries, and is drained via the portal system. The liver has a unique and special blood supply that includes both arterial (the common hepatic artery) and venous (the portal vein) inflow. The portal vein supplies 75\u201380% of the liver blood flow and the hepatic artery supplies 20\u201325%. Physiologically, there is an interdependent response with a compensatory blood flow between the portal vein and the hepatic artery called the hepatic arterial buffer response [3]. The hepatosplanchnic blood flow accounts for 25\u201330% of the cardiac output [4], and the regional oxygen extraction is slightly higher than the whole body oxygen extraction. During sepsis or septic shock, splanchnic oxygen extraction is increased compared with nonseptic patients (44% versus 30%), which leads to an increase in the hepatic venous\/mixed venous haemoglobin oxygen saturation gradient [4]. In clinical practice it is generally not possible to determine portal venous flow in isolation, and measurements are taken from the hepatosplanchnic region as a whole. The flow is estimated at bedside by the method of primed, constant infusion of indocyanine green (ICG) with hepatic venous catheterization [5].\nThe intestinal villus is supplied by a single, unbranched arterial vessel that arborizes at the villus tip into a network of surface capillaries drained by a central villus vein. This anatomical arrangement allows countercurrent exchange and shunting of diffusible molecules such as oxygen, and hypoxia may occur at the tip of the villus even during moderate decreases in macrocirculatory flow [6]. In addition, intestinal villi perfusion is highly heterogeneous, as suggested by the wide range of intestinal surface oxygen saturation [7].\nIn patients with sepsis, splanchnic blood flow usually increases in proportion to cardiac output [8] and is associated with decreased hepatic vein oxygen haemoglobin saturation. Two different interpretations are possible: first, the increase in splanchnic blood flow is insufficient to meet the increased oxygen consumption; and second, hepatic arterial blood flow is reduced as a consequence of the hepatic arterial buffer response. The latter hypothesis is supported by the observations of De Backer and coworkers [9], who demonstrated that there is usually no net lactate production from the hepatosplanchnic area. In addition, the observation that splanchnic blood flow is increased does not rule out an impairment in microvascular blood flow [10-12] or the presence of cytopathic hypoxia [13].\nIn normal conditions the partial carbon dioxide tension (PCO2) gap, which is defined as the difference between mucosal PCO2 measured with a tonometer and arterial PCO2, is low. In case of inadequate mucosal blood flow, whether tissue hypoxia is present or not, the PCO2 gap increases. Levy and coworkers [14] recently reported that a PCO2 gap greater than 20 mmHg was associated with poor outcome in patients with septic shock. Unfortunately, there is no apparent correlation between PCO2 gap and global or regional haemodynamic measurements in septic patients [15] because the PCO2 gap mirrors both variations in microvascular flow [10] and in carbon dioxide metabolism [16]. For these reasons variations in PCO2 gap must be interpreted with caution.\nTherapeutic strategies\nFluid challenge\nThe mainstay of supportive treatment in patients with severe sepsis or septic shock is maintenance of adequate fluid balance, titration of appropriate oxygen delivery, and adequate perfusion pressure [17]. Hypovolaemia is a common clinical occurrence in intensive care medicine and results from several mechanisms such as fluid loss, haemorrhage, vasoplegia and capillary leak syndrome. This explains why fluid replacement therapy is a key component in the treatment of severe sepsis and septic shock. Although there is no consensus regarding the ideal type of fluid replacement, colloids are efficient in this indication [18].\nThere are few clinical studies focusing on the effects of colloids on splanchnic haemodynamics. In a randomized study conducted in patients with sepsis, Boldt and coworkers [19] assessed the effects on tonometric gastric mucosal acidosis of hydroxyethyl starch and albumin targeted to maintain pulmonary artery occlusion pressure between 12 and 18 mmHg. In hydroxyethyl starch treated patients cardiac index, oxygen delivery and consumption increased, and gastric intramucosal pH (pHi) remained stable whereas it decreased in albumin treated patients. In three other studies [20-22] conducted in patients with sepsis and septic shock, fluid challenges performed with hydroxyethyl starch neither altered the PCO2 gap nor influenced splanchnic haemodynamics. Moreover, a randomized comparison of hydroxyethyl starch and gelatin in haemodynamically stable septic patients revealed a beneficial effect of gelatin on the PCO2 gap [20]. These studies suggested no better effect of one colloid over the others on splanchnic haemodynamics, and the use of colloids must be weighed against their side effects [23].\nRed blood cell transfusions are commonly used in intensive care units (ICUs) to enhance systemic oxygen delivery. However, proof of improved utilization of oxygen by peripheral tissues, especially in the splanchnic area, is lacking. Silverman and Tuma [24] reported the absence of improved gastric pHi with red blood cell transfusions in 21 septic patients. Moreover, there is an inverse association between the change in gastric pHi and the age of the transfused blood [25]. Finally, a recent report in 15 septic patients showed that red blood cell transfusion failed to improve oxygen utilization measured either using Fick's equation or by indirect calorimetry, and gastric pHi remained unaltered [26].\nAdrenergic drugs\nThe choice of vasoactive drugs in sepsis and septic shock is controversial. There is no evidence that any one vasoactive drug is more effective or safer than any other. Larger trials are needed to elucidate existing clinically significant differences in morbidity and mortality. A multicentre trial, which is currently ongoing, is comparing the effects of epinephrine with a combination of a fixed dose of dobutamine in addition to norepinephrine.\nDopamine alone or versus norepinephrine (Table 1)\nThe infusion of low-dose dopamine (defined as a dose lower than 5 \u03bcg\/kg per min administered to normotensive patients) may not improve gut mucosal perfusion. In fact, Nevi\u00e8re and coworkers [27] showed that low-dose dopamine decreased gut mucosal blood flow in septic patients. Furthermore, other investigators [27-30] reported that either pHi or PCO2 gap were unchanged in patients with sepsis treated with low-dose dopamine. The effects on liver blood flow may also be variable; Maynard and coworkers [30] observed that dopamine did not affect ICG clearance and monoethylglycinexylidide (MEGX) formation from lidocaine. Interestingly, the effects of dopamine on splanchnic blood flow may differ according to basal splanchnic perfusion. Low-dose dopamine increased splanchnic blood flow that was low at baseline (seven patients) but not when splanchnic perfusion was preserved (four patients) [28]. The very small number of patients in each group limited these observations. Recently, Jakob and coworkers [31] reported that dopamine administration titrated to achieve a 25% increase in cardiac output induced a significant increase in splanchnic blood flow from 0.9 to 1.1 l\/min per m2, which was associated with a significant reduction in splanchnic oxygen consumption.\nThe results are even more controversial when dopamine is used at higher doses to restore blood pressure. Ruokonen and coworkers [32] observed that dopamine increased splanchnic blood flow and metabolism in some but not all patients with septic shock. In some patients, the same group of investigators [33] also observed an increase in hepatic vein oxygen saturation, suggesting an improvement in the balance between oxygen supply and demand during dopamine administration. However, in a pilot study, Marik and Mohedin [34] reported that dopamine administered at doses up to 25 \u03bcg\/kg per min even decreased pHi. Given the very small number of patients included in these studies, no definite conclusions can be drawn regarding the effects of dopamine on splanchnic blood flow in septic patients.\nComparison of the effects of norepinephrine and dopamine is difficult because norepinephrine is often combined with dobutamine, and study results are conflicting. Ruokonen and coworkers [32] reported unpredictable effects on splanchnic blood flow in patients with septic shock with norepinephrine, whereas dopamine induced a consistent increase in splanchnic blood flow. By contrast, in the randomized study reported by Marik and Mohedin [34], conducted in 20 septic patients with hyperdynamic septic shock, dopamine was reported to induce a decrease in pHi when compared with norepinephrine. More recently, De Backer and coworkers [35] reported the effects of dopamine, norepinephrine and epinephrine on the splanchnic circulation in moderate and in severe septic shock, and the main results are as follows. In moderate septic shock cardiac index was similar in dopamine-treated and norepinephrine-treated patients, and higher in epinephrine-treated patients, whereas splanchnic blood flow was the same with the three drugs. The gradient between mixed venous and hepatic venous oxygen saturation gradient was the lowest with dopamine, while PCO2 gaps were identical. In patients with moresevere septic shock cardiac index was greater and splanchnic blood flow lower with epinephrine than with dopamine and epinephrine; mixed venous and hepatic venous oxygen saturation gradient was greater with epinephrine, whereas PCO2 gap remained unaltered by any of the treatments.\nGiven the available data (summarized in Table 1), no definite conclusions can be drawn regarding differences between dopamine and norepinephrine on splanchnic blood flow and metabolism in patients with septic shock.\nDobutamine alone or combined with norepinephrine versus epinephrine (Tables 2 and 3)\nIn patients with sepsis, a retrospective study conducted by Silverman and coworkers [24] identified a beneficial effect of dobutamine infusion on pHi [24]. Two years later Gutierrez and coworkers [36] reported an increase in pHi with dobutamine infusion in patients with sepsis syndrome who initially had low pHi. This beneficial effect, confirmed in other studies [37-39], was not related to an increase in splanchnic blood flow induced by dobutamine [39,40]. Creteur and colleagues [41] reported that dobutamine decreased the PCO2 gap in septic patients with a high gradient between the mixed venous and hepatic vein oxygen saturation (>20%), whereas PCO2 gap was not affected in patients when this gradient was less than 20%. This suggests that patients with the most severe alterations in hepatosplanchnic blood flow are also prone to decreased mucosal perfusion.\nDobutamine usually, but not without exception, increases splanchnic perfusion [40-42]. The effects on splanchnic metabolism are more variable [39] and may depend on the adequacy of splanchnic perfusion at baseline. In patients with septic shock, De Backer and coworkers [43] reported that splanchnic oxygen consumption increased during dobutamine administration only in patients with an increased gradient between hepatic venous and mixed venous oxygen saturation.\nCombinations of dobutamine and other catecholamines have often been studied, in particular in association with norepinephrine for its effects on \u03b2-receptors, with the aim of modulating hepatosplanchnic haemodynamics. Indeed, in patients with sepsis, changing from norepinephrine (\u03b1-agonist and \u03b2-agonist) to phenylephrine (pure \u03b1-agonist), titrated to produce similar global haemodynamic measurements, led to a decrease in splanchnic blood flow, splanchnic oxygen delivery and gastric pHi. These changes were associated with decreased rates of liver lactate uptake and glucose production [44].\nWhether dobutamine has a specific effect on the splanchnic circulation is still debated. In a cross-over study conducted in eight patients with septic shock, Meier-Hellmann and coworkers [45] showed that epinephrine caused lower splanchnic flow and oxygen uptake, lower gastric pHi, and higher hepatic vein lactate concentration than did the combination of dobutamine and norepinephrine. Duranteau and coworkers [11] compared the effects of epinephrine, norepinephrine and the combination of norepinephrine and dobutamine in patients with septic shock on gastric mucosal flow, as assessed using a laser Doppler technique. Epinephrine and dobutamine\u2013norepinephrine led to a significant increase in gastric mucosal flow as compared with norepinephrine alone, but these findings were not corroborated by those reported by Seguin and coworkers [46]. Moreover, in patients with septic shock resistant to dopamine, the combination of norepinephrine and dobutamine, in comparison with epinephrine alone, restored gastric pHi more quickly and limited the increase in arterial lactate concentration. However, there was no difference in gastric mucosal PCO2 gradients between groups at 24 hours of treatment [38].\nThe preferential effect of dobutamine on splanchnic blood flow was not confirmed by Reinelt and coworkers [42], who studied the effects of dobutamine on fractional splanchnic flow and hepatic glucose production in septic patients resuscitated adequately with fluid and norepinephrine. Their results showed a parallel increase in splanchnic blood flow and cardiac index, unaltered splanchnic oxygen consumption and decreased rate of endogenous production of hepatic glucose. These findings suggest that splanchnic blood flow is increased in well resuscitated septic patients, and that a dobutamine test is able to reveal a oxygen delivery\/consumption dependency [41,43] but it cannot exclude intraorgan blood flow redistribution at the microcirculatory level. The inadequacy of blood flow distribution is mirrored by the absence of correlation between splanchnic blood flow and the PCO2 gap.\nReported data on the effects of dobutamine and norepinephrine on splanchnic haemodynamics are summarized in Tables 2 and 3, respectively.\nRecommendations regarding use of adrenergic drugs\nWe suggest that both dopamine and norepinephrine can be given to septic shock patients as first-line catecholamine drugs but that their use must be weighed against the undesired neuroendocrine side effects of dopamine [45]. Epinephrine should be reserved for use as rescue therapy. If norepinephrine is chosen as the first agent, then the addition of dobutamine may be considered.\nDopexamine\nDopexamine hydrochloride is a dopamine analogue with vasodilating effects that may be useful in improving splanchnic microcirculation in septic shock. Twenty-five ventilated patients with systemic inflammatory response syndrome were randomly assigned to receive either a 2-hour infusion of dopexamine (1 mg\/kg per min) or of dopamine (2.5 \u03bcg\/kg per min) after baseline measurements of gastric pHi, MEGX formation from lidocaine and ICG disappearance rate. Dopexamine had no effects on systemic measurements but it significantly increased pHi and ICG plasma disappearance, suggesting a selective increase in splanchnic blood flow and improved hepatic function, as indicated by increased MEGX concentration [30]. A previous study from the same group showed that dopexamine at higher doses (4\u20136 \u03bcg\/kg per min) raised gastric pHi together with a nonsignificant increase in ICG clearance [47]. Temmesfeld-Wollbr\u00fcck and coworkers [7] employed reflectance spectrophotometry for direct assessment of the microvascular haemoglobin saturation and haemoglobin concentration in the gastric mucosa in patients with septic shock. Compared with healthy control individuals, patients with septic shock exhibited a reduced microvascular haemoglobin saturation with a wide distribution and with tailing of the histogram to severely hypoxic values in spite of high whole body oxygen delivery. This microvascular disturbance was associated with reduced microvascular haemoglobin concentration and a lower gastric pHi. Short-term infusion of 2 \u03bcg\/kg per min dopexamine in 10 patients with septic shock increased both microvascular haemoglobin saturation and concentration, whereas whole body oxygen uptake and gastric pHi remained unaltered.\nOther investigators did not confirm these beneficial effects. Hannemann and coworkers [48] reported the effect of incremental doses (0.5\u20134 \u03bcg\/kg per min) dopexamine on splanchnic circulation in 12 patients with severe sepsis haemodynamically controlled with fluid challenge and dobutamine. Splanchnic blood flow increased proportionally to cardiac output but dopexamine lowered gastric pHi in a dose-dependent manner in all patients [49]. Finally, in 12 septic shock patients haemodynamically controlled with norepinephrine, dopexamine titrated to increase cardiac output by 25% [50] increased median splanchnic blood flow whereas the fractional splanchnic blood flow was significantly reduced, and none of global or regional oxygen exchange or PCO2 was altered. In addition, those investigators found no influence of dopexamine on metabolic parameters either [51]. Given these discrepancies, it is reasonable to recommend further investigations into dopexamine before it may be routinely used in septic shock.\nOther vasoactive drugs\nVasopressin and terlipressin\nPhysiologically, vasopressin (a nonapeptide that is released from the neurohypophysis) plays a minor role in blood pressure regulation. Clinical data revealed that the initially very high plasma concentrations of vasopressin decrease during prolonged sepsis [52].\nIn the past few years clinical studies showed that blood pressure can be rapidly restored in septic shock using vasopressin, but this is mainly at the expense of cardiac output [53]. Nevertheless, in 2000 the American Heart Association and International Liaison Committee on Resuscitation recommended (grade IIB) continuous vasopressin infusion in refractory septic shock [54]. However, the effects of vasopressin on regional (i.e. splanchnic) blood flow are discussed controversially.\nIn 1997, Landry and coworkers [52] reported on the continuous infusion of vasopressin (1.8\u20133.0 IU\/hours) in five patients with septic shock. In all patients, blood pressure was rapidly restored and urine output increased in three. Patel and coworkers [55] randomly assigned 24 patients with septic shock to a double-blind 4-hour infusion of norepinephrine or vasopressin, and open-label vasopressors were titrated to maintain blood pressure. Although norepinephrine dosage could be significantly lowered in the vasopressin group, blood pressure and cardiac index were maintained in both groups. Urine output did not change in the norepinephrine group but increased substantially in the vasopressin group. Similarly, creatinine clearance did not change in the norepinephrine group but increased by 75% in the vasopressin group. Finally, gastric mucosal PCO2 gradient did not change significantly in either group.\nRecent results from Klinzing and coworkers [56], however, indicate that vasopressin may lead to a different blood flow distribution pattern in the splanchnic area as compared with norepinephrine. In 12 patients with septic shock, vasopressin was administered at a dose of 0.06\u20131.8 IU\/min to replace norepinephrine completely. As a result, cardiac index and systemic oxygen uptake decreased significantly. Total splanchnic blood flow tended to decrease, while splanchnic blood flow expressed as percentage of cardiac output as well as the PCO2 gap were doubled [56]. By contrast, the increase in gastric PCO2 gap suggests that blood flow may have been redistributed away from the mucosa, and therefore it does not appear beneficial to directly replace norepinephrine with vasopressin in septic shock. Clinical data also suggest that low-dose vasopressin (0.04 IU\/min) to compensate for endogenous deficiency could be a beneficial strategy [57-60], as was recently demonstrated by D\u00fcnser and coworkers [61], who randomly assigned 48 patients with catecholamine-resistant vasodilatory shock to receive a combined infusion of vasopressin and norepinephrine or norepinephrine alone. Vasopressin-treated patients had significantly lower heart rate, norepinephrine requirement and incidence of new onset tachyarrhythmias. Mean arterial pressure, cardiac index and stroke volume were significantly greater, and the PCO2 gap was significantly lower in patients treated with this combination. However, these patients also presented with a significant increase in plasma bilirubin concentration, suggesting an impaired liver blood flow and\/or a depressed hepatic function mediated by vasopressin.\nMore recently, terlipressin (glycinpressin), a long-acting vasopressin analogue, was proposed as a treatment for septic shock. O'Brien and coworkers [62] reported their clinical experience with terlipressin (1\u20132 mg) as rescue treatment in eight patients with refractory septic shock. Those investigators reported a rapid and 24 hour lasting stabilization in blood pressure, with a significant reduction in norepinephrine but a significant decrease in cardiac index. In that study, seven patients required renal replacement therapy and four patients died during their stay in the ICU. However, optimism regarding these findings must be tempered somewhat [63], in particular because detrimental effects on splanchnic blood flow have been described. Auzinger and coworkers [64] studied seven patients with catecholamine-refractory septic shock and subsequent infusion of terlipressin using gastric tonometry. During the 24-hour intervention period, terlipressin was administered as an intermittent bolus (1\u20133 mg). Although no changes occurred in lactate levels, the PCO2 gap progressively increased over 72 hours.\nBoth vasopressin and terlipressin are potent vasoconstrictors and both are able to restore blood pressure in vasodilatory or septic shock. However, the effects on splanchnic blood flow are not yet fully elucidated. Clearly, adequacy of volume resuscitation is a major prerequisite for maintenance of microcirculatory blood flow. The currently available data suggest that both substances administered to compensate for endogenous vasopressin deficiency may be beneficial. Although the armamentarium for treatment of septic shock is enriched by such substances, it remains unclear whether administration during septic shock decreases morbidity or improves survival, and further research is warranted.\nEnoximone\nModulation of the cytokine response by catecholamines might be a mechanism by which decreased morbidity and mortality are achieved with supranormal oxygen delivery in high-risk surgical patients [65]. Phosphodiesterase III inhibitors have positive inotropic, vasodilating and anti-inflamatory properties, and they may avoid the development of tolerance to catecholamines as a result of \u03b2-receptor desensitization.\nIn a prospective, double-blind study [66], 44 patients with septic shock and conventional resuscitation were randomly assigned to receive dobutamine or enoximone to maximize left ventricular stroke work index. At 12 and 48 hours after baseline measurements, liver blood flow was assessed with hepatic venous catheterization, liver function was derived from appearance in plasma of MEGX, and release of tumour necrosis factor-\u03b1 was determined to assess the severity of ischaemia\/reperfusion injuries. There was a similar increase in cardiac index, systemic oxygen delivery and consumption, and liver blood flow in the two groups. Fractional splanchnic blood flow decreased slightly but significantly in dobutamine-treated patients, whereas it remained unchanged in enoximone-treated patients. In the latter group liver oxygen consumption and MEGX kinetics were significantly higher at 12 hours but not at 48 hours. The release of hepatic tumour necrosis factor-\u03b1 after 12 hours of dobutamine treatment was twice as high (P < 0.05) as during enoximone treatment, suggesting a faster anti-inflammatory effect of enoximone. These interesting findings on hepatosplanchnic effects of phosphodiesterase III inhibitors were not confirmed by other studies, and further investigations are needed if these agents are to be recommended for routine clinical use.\nProstacyclin\nProstacyclin or its stable analogue iloprost are vasodilator substances with platelet aggregation inhibiting and cytoprotective properties. Administration of prostacyclin by the intravenous route was shown to increase oxygen delivery and consumption in septic patients [67] and to improve gastric pHi [68], as did aerosolized prostacyclin in patients with septic shock and pulmonary hypertension treated with epinephrine or norepinephrine [69]. Finally, Lehmann and coworkers [70] reported restored plasma ICG clearance without harmful effect on systemic haemodynamics in patients with septic shock treated with iloprost.\nMore recently Kiefer and colleagues [71] reported the hepatosplanchnic effects of iloprost in 11 patients with septic shock requiring norepinephrine. Iloprost was incrementally infused to increase cardiac index by 15%, which significantly increased splanchnic blood flow in parallel, without a major fall in mean arterial pressure. Iloprost induced a decrease in endogenous glucose production rate without change in the hepatic clearance of the glucose precursors alanine, pyruvate and lactate. Similarly, the PCO2 gap was not altered. The authors avoided mean arterial pressure drop by careful exclusion of hypovolaemia before inclusion, but still the increment in iloprost doses was limited by the decrease in arterial partial oxygen tension, which raises many questions in patients with acute respiratory distress syndrome. These interesting findings on hepatosplanchnic effects of such vasodilators need further investigation before these agents may be recommended for routine clinical use [72].\nNitroglycerin\nOpening the microcirculation using a vasodilator is an alternative approach for treatment of the jeopardized microcirculation in patients with sepsis or septic shock. Data reported by Sprock and coworkers [73] suggest that the use of intravenous nitroglycerin results in improved sublingual microvascular flow, as assessed by orthogonal polarization spectral imaging. However, one cannot assume that the sublingual microcirculation necessarily behaves like the whole splanchnic microcirculation does.\nN-acetyl cysteine\nN-acetyl cysteine (NAC) administration was associated with a decrease in gastric pHi in septic patients [74,75] and prevented the decrease in pHi in septic patients under hyperoxic stress [76]. In a randomized, double-blind study conducted in septic shock patients, NAC given within the first 24 hours after admission to the ICU was shown to improve cardiac index and splanchnic blood flow and MEGX concentration, and to decrease gastric mucosal PCO2 gap, whereas it did not influence fractional splanchnic blood flow [75]. Nevertheless, these positive effects of NAC on the splanchnic circulation must be balanced against several negative studies. Indeed, NAC was reported to depress cardiac performance in septic patients [77], and it even worsened mortality rate when it was given more than 24 hours after hospital admission [78]. Is NAC a 'double edged sword'? This question should be answered before its use in daily practice can be recommended.\nExtracorporeal renal support\nPublications related to this topic are scarce. In 11 critically ill patients mechanically ventilated and treated with inotropic support, intermittent dialysis increased the PCO2 gap [79]. In contrast, in two recent studies conducted in patients with acute renal failure [80] and septic shock [81], the PCO2 gap remained unaltered whereas cardiac index and stroke volume, as well as splanchnic blood flow, transiently decreased [80]. Although improved cardiovascular stability during continuous veno-venous haemofiltration in comparison with intermittent dialysis has been demonstrated in retrospective studies [82], the superiority of continuous haemofiltration over hemodialysis on splanchnic circulation has not been proven [81].\nConclusion\nIn this review we summarize different, and potentially opposing, approaches to management of splanchnic circulation in patients with septic shock. However, in these studies the measurements were focused on the effect of the drug on splanchnic blood flow or a surrogate such as the PCO2 gap, but none of these studies reported convincing results with respect to mortality and\/or morbidity.\nCompeting interests\nNone declared.\nAbbreviations\nICG = indocyanine green; ICU = intensive care unit; MEGX = monoethylglycinexylidide; NAC = N-acetyl cysteine; PCO2 = partial carbon dioxide tension; pHi = intramucosal pH.","keyphrases":["septic shock","supportive treatment","adrenergic drugs","splanchnic circulation","nonconventional treatments"],"prmu":["P","P","P","P","R"]}
{"id":"Ann_Hematol-3-1-2040174","title":"Rituximab therapy for chonic and refractory immune thrombocytopenic purpura: a long-term follow-up analysis\n","text":"The aim of this study was to evaluate the long-term response to rituximab in patients with chronic and refractory immune thrombocytopenic purpura (ITP). Adults with ITP fail to respond to conventional therapies in almost 30% of cases, developing a refractory disease. Rituximab has been successfully used in these patients. We used rituximab at 375 mg\/m2, IV, weekly for a total of four doses in 18 adult patients. Complete remission (CR) was considered if the platelet count was >100 \u00d7 109\/l, partial remission (PR) if platelets were >50 \u00d7 109\/l, minimal response (MR) if the platelet count was >30 \u00d7 109\/l and <50 \u00d7 109\/l, and no response if platelet count remained unchanged. Response was classified as sustained (SR) when it was stable for a minimum of 6 months. Median age was 43.5 years (range, 17 to 70). Median platelet count at baseline was 12.5 \u00d7 109\/l (range, 3.0 to 26.3). CR was achieved in five patients (28%), PR in five (28%), MR in four (22%), and two patients were classified as therapeutic failures (11%). Two additional patients were lost to follow-up. The median time between rituximab therapy and response was 14 weeks (range, 4 to 32). SR was achieved in 12 patients (67%). There were no severe adverse events during rituximab therapy. During follow-up (median, 26 months; range, 12 to 59), no other immunosuppressive drugs were used. In conclusion, rituximab therapy is effective and safe in adult patients with chronic and refractory ITP. Overall response rate achieved is high, long term, and with no risk of adverse events.\nIntroduction\nIn immune thrombocytopenic purpura (ITP), platelets are coated with an IgG autoantibody that prompts its premature destruction and, as a result, different grades of peripheral thrombocytopenia and clinical bleeding become evident [5, 23]. Steroids are the first-line treatment for acute ITP. In case of failure to steroids, splenectomy induces a 70 to 80% response rate [10]. However, almost 30% of adults with ITP fail to respond to conventional therapies [steroids, IV immunoglobulin (IVIg), splenectomy, or immunosuppressive drugs] and, eventually, they develop a chronic refractory disease [5, 12, 23]. Refractory patients are defined as those who failed standard-dose steroids and splenectomy, requiring further treatment due to unsafe platelet counts (<30\u2009\u00d7\u2009109\/l) or clinical bleeding. These patients are unlikely to be cured, although spontaneous remissions sometimes occur [17]. If a patient becomes refractory, some alternatives are available such as danazol, dapsone, azathioprine, high-dose steroids, or chemotherapy combinations like CHOP regimen. However, these options are not quite useful because if response is achieved it is but for a brief period and sustained responses are scarce [23]. Hematopoietic stem cell transplantation has been occasionally used in some cases, but this option represents a high-risk procedure [14]. At the end, these patients may respond poorly to subsequent therapy, may have significant morbidity from the disease and its treatment (bleeding and infections secondary to immunosuppressive therapy), and the mortality rate at 10\u00a0years may rise up to 20% [5, 11]. On the other hand, these patients have higher hemorrhagic risk imposed by trauma or surgery. Therefore, new treatment strategies for these patients are needed.\nRituximab is an anti-CD20 chimeric murine-human monoclonal antibody. CD20 is a B-lymphocyte membrane protein absent on other normal cells. This monoclonal IgG-kappa antibody is used in non-Hodgkin lymphomas because it induces apoptosis or direct lysis of B cells [19]. In ITP, B cells are responsible of autoantibody production and the subsequent platelet opsonization that allows its destruction. Therefore, if B cell clones are eliminated, thrombocytopenia could be reverted.\nThere are some reports informing that rituximab is useful in refractory ITP showing a high overall response rate, with complete remission (CR) and partial remission (PR) rates ranging from 48 to 54% [4, 8, 9, 18, 20\u201322]. We report herein our long-term experience with rituximab therapy in chronic and refractory ITP.\nMaterials and methods\nIn a 5-year period, we prospectively administered rituximab to all patients with chronic and refractory ITP who were unresponsive to prednisone, azathioprine, vincristine, danazol, and high-dose dexametasone, among other therapies, including those unsuccessfully splenectomized. Patients must have a platelet count of <30\u2009\u00d7\u2009109\/l to be considered for this therapy.\nRituximab was given in doses of 375\u00a0mg\/m2, diluted in 500\u00a0ml 5% dextrose\u2013water solution in a 4-h continuous IV infusion. This regimen was indicated weekly for four doses after prophylactic application of antipyretic and antihistamine premedication administered 30\u00a0min before rituximab infusion (paracetamol, 500\u00a0mg, PO; diphenhydramine, 30\u00a0mg, PO; and hydrocortisone, 100\u00a0mg, IV). Outpatient follow-up was scheduled monthly for 6\u00a0months. Long-term follow-up was performed every 2\u00a0months.\nResponse was classified as complete (CR) if the platelet count was >100\u2009\u00d7\u2009109\/l, partial (PR) if platelet count >50\u2009\u00d7\u2009109\/l, minimal response (MR) if platelet count was <50\u2009\u00d7\u2009109\/l and >30\u2009\u00d7\u2009109\/l, and no response (NR) if there was no change from the baseline platelet count. Response was sustained (SR) when it was maintained for a minimum of 6\u00a0months.\nNumerical variables were expressed using the median and range number. The probability to achieve more than 50\u2009\u00d7\u2009109\/l and 100\u2009\u00d7\u2009109\/l platelets and the median time from response to end of follow-up was calculated using the Kaplan and Meier method.\nBefore starting treatment, all patients signed an informed consent authorization. The procedures done in this protocol were in accordance with the Helsinki Declaration of 1975. The protocol was approved by the Ethics Committee of the participating hospitals.\nResults\nEighteen patients were candidates for this therapy, and all of them were suitable for evaluation because they received the planned four doses of rituximab. Patient characteristics and response to rituximab are shown in Table\u00a01. Median age for the whole group was 43.5\u00a0years (range, 17 to 70). Median platelet count at baseline was 12.5\u2009\u00d7\u2009109\/l (range, 3.0 to 26.3). Median follow-up was 26\u00a0months (range, 12 to 59). Mean number of treatment regimens received before rituximab was 5.5 (range, 3 to 8). Fifteen patients (83%) had failed to splenectomy.\nTable\u00a01Patient characteristics and outcomesPatient\/age\/genderEvolutiona (months)Previous treatmentsPC at baseline (\u00d7109\/l)PC at 12\u00a0months (\u00d7109\/l)Time to response (weeks)Response typeSR1\/42\/F37P, S, Az, D, IFN3.014624CRYes2\/43\/F84P, S, Az, D, IFN, V5.012324CRYes3\/30\/F48P, S, Az, D, IFN, V12.534. 516MRYes4b\/27\/F38P, S, Az, D, IFN24.47816PRbYesb5\/70\/M60P, Az, D, V7.8NRNRNRNo6\/43\/F96P, S, Az, D, IFN, V10.137.78MRYes7\/53\/F96P, S, Az, D, IFN, V12.55412PRYes8\/36\/F264P, S, Az, D, IFN, V, OE, IVIg5.030.64MRYes9\/17\/F38P, S, Az, D, IFN, V, OE6.51294CRYes10\/57\/F26P, Az, D8.3648PRYes11\/36\/F25P, S, Az, D, IFN, V7.04920MRNo12\/63\/F120P, Az, D, V26.3NRNRNRNo13\/53\/F348P, De, D, S, Az22.01128CRYes14\/22\/F266P, S, D, IFN, IVIg12.66712PRNo15\/56\/F137P, D, S, Az18.42234CRYes16\/35\/F38P, D, Az, S23.211.40NRNo17\/52\/F41P, D, S16.551.94PRYes18\/24\/F60O, D, S24.0NRNRNRNoPC Platelet count, P prednisone, S splenectomy, Az azathioprine, D danazol, IFN interferon alpha 2b, V vincristine, De dexamethasone, OE opsonized erythrocytesaTime from ITP diagnosis to rituximab therapybA second course of rituximab was given.\nCR was achieved in five patients (28%), PR was obtained in another five (28%), MR was seen in four (22%) patients, and two patients were considered treatment failures (11%). Two patients were lost to follow-up (11%). Ten patients (55%) achieved >50\u2009\u00d7\u2009109\/l platelets. Median time from the first rituximab dose to achievement of any response was 14\u00a0weeks (range, 4 to 32). Twelve patients (67%) showed SR beyond the sixth month of follow-up. As expected from the results depicted in Table\u00a01, we were unable to find any correlation between the ITP duration and the type of response to rituximab. Figure\u00a01 shows the median platelet counts during follow-up in three different groups of patients classified according to their pattern of response (CR, PR, and MR). The median time to achieve >50\u2009\u00d7\u2009109\/l platelets was 5\u00a0months (95%CI\u2009=\u20090.5 to 11.6\u00a0months), and the median time to achieve >100\u2009\u00d7\u2009109\/l platelets was not reached (Fig.\u00a02). One patient relapsed 22\u00a0months after the first course of rituximab. She received steroids for another 14\u00a0months; she did not obtain response, and a second course of rituximab was given using the regimen described previously. She achieved a new PR 6\u00a0months after stopping therapy. After 22\u00a0months of follow-up, PR still remains in this patient.\nFig.\u00a01Platelet count achieved after first dose of rituximab (time 0 first dose of rituximab). Diamonds, CR; ovals, PR; triangles, MRFig.\u00a02Time required to obtain platelet counts >50\u2009\u00d7\u2009109\/l (a) or >100\u2009\u00d7\u2009109\/l (b) after first dose of rituximab in adult patients with chronic and refractory ITP\nMedian time of response duration was 54\u00a0months (95%CI\u2009=\u200915\u201393\u00a0months) for patients with CR, 18\u00a0months (95%CI\u2009=\u20098\u201328\u00a0months) for patients with partial response, and 12\u00a0months (95%CI\u2009=\u20097\u201317\u00a0months) for those individuals with minimal response. Difference was statistically significant between patients in CR vs those achieving PR or MR (p\u2009<\u20090.05). No difference was observed between PR and MR (Fig.\u00a03).\nFig.\u00a03Median duration of response for patients who achieved complete remission (CR; 54\u00a0months, 95%CI\u2009=\u200915\u201393\u00a0months), partial remission (PR; 18\u00a0months, 95%CI\u2009=\u20098\u201328\u00a0months), or minor response (MR; 12\u00a0months, 95%CI\u2009=\u20097\u201317\u00a0months)\nSide effects related to the first dose of rituximab such as fever, chills, and respiratory symptoms were common (8 out of 18 patients, 43%). There were no severe adverse events during drug administration. Neither hemorrhagic events nor infections were recorded. Neutropenia, an occasional long-term side effect of rituximab therapy, was never recorded in our group of patients (Fig.\u00a04). In fact, the lower neutrophil count recorded during the study was 1.3\u2009\u00d7\u2009109\/l in patient 7 after 6\u00a0weeks of treatment with rituximab. No patient needed additional immunosuppressive therapy other than rituximab. During the whole follow-up period, no other illnesses have been recorded in this group of patients.\nFig.\u00a04Mean (squares), maximum (diamonds), and minimum (triangles) neutrophil counts through the follow-up of patients receiving rituximab for chronic and refractory ITP\nDiscussion\nBecause almost 30% of individuals with ITP do not respond to the first- and second-line therapies, they live with low platelet counts, a situation that carries a high risk of hemorrhage and, eventually, a short life expectancy. It has been largely demonstrated that ITP with persistent low platelet counts carries a grave prognosis [6, 13]. Efforts to increase the platelet count to at least 30\u2009\u00d7\u2009109\/l include IVIg, anti-D immunoglobulin, cyclophosphamide, polychemotherapy such as the CHOP regimen, thrombopoietin, interleukin-11, dapsone, alpha interferon, plasma exchange, and bone marrow transplantation [23]. Using these therapies, the response rate is low and the patient is exposed to unnecessary risks [10]. When the first ITP patient successfully treated with rituximab was informed, it drew the attention of physicians because this regimen offered high response rate and low toxicity. In subsequent reports, researchers have found similar results.\nThe aim of this study was to evaluate the response rate achieved in 18 patients with chronic and refractory ITP after rituximab treatment as well as the evolution of these patients to learn about the possible long-term side effects associated with the use of this drug, information that is almost inexistent in the literature. We observed 12 favorable responses (67%) and at least a stable clinical evolution in two additional patients (11%), for a global response rate of 78%. Based on the characteristics of the evolution of our group of patients, we may emphasize some points of interest: (1) Response rate obtained was quite satisfactory considering the history of chronicity and refractoriness of the patients; (2) the monoclonal antibody was well tolerated and caused only mild allergic reactions that could be easily managed with antihistaminics and paracetamol; (3) most of the patients achieve SR; and (4) increase of platelet count was not always immediate (median time to response\u2009=\u200914\u00a0weeks), a fact that slightly disagrees with previous reports that found shorter time to response (3 to 8\u00a0weeks; Table\u00a02) [4, 8, 20]. In fact, in a recent systematic review about the efficacy of rituximab in adult patients with ITP, it was found that median time to response was 5.5\u00a0weeks (ranges 2 to 18\u00a0weeks) [2]. We do not have an explanation for this discrepancy. After a careful analysis, we did not find significant differences between the results obtained in the systematic review and our results in terms of the variables that may affect the time to response to rituximab, namely, age, sex, previous use of corticosteroids, number of treatments before rituximab, rituximab dose and schedule, period between diagnosis and rituximab administration, duration of ITP before rituximab, and pretreatment platelet counts. Although splenectomy may not be a significant predictor of response to rituximab as previously suggested [2, 4, 8], it should be noted that 83% of the patients included in our report had had splenectomy as compared with 50.5% of the patients in the systematic review. Therefore, history of splenectomy seems to be the only factor related to a different time to response to rituximab in our series.\nTable\u00a02Patterns of response in patients with chronic and refractory ITP (from [1, 6, 18])ReferenceNumber of patientsOR (%)CR (%)PR (%)Median time to response (weeks)Mayo clinic1250428?USA\u2013Italy575331228Denmark353318152\u20138Peru22684027?Mexico1856282814OR Overall response, CR complete response, PR partial response\nOf course, we have no data about all possible variables influencing the pattern of response to rituximab. For example, we do not show information about B-cell counts and platelet autoantibodies before and after rituximab therapy, two variables that may influence the response as observed in other trials. The expected therapeutic effect of rituximab is a reduction in specific platelet-associated autoantibodies and the consequent increase in platelet counts. Although rituximab has not been always associated with a reduced load of platelet autoantibodies and most of the publications reporting this effect are small series of cases [1], the high overall response rate obtained in our study allows us to believe that rituximab effectively decreased the B-cell counts as well as the levels of platelet autoantibodies.\nThree patterns of response to rituximab have been proposed: early (before the fourth dose of rituximab), intermediate (7 to 11\u00a0weeks after rituximab), and delayed (>13\u00a0weeks after rituximab) [8]. In our study, the probability to achieve a platelet count >50\u2009\u00d7\u2009109\/l occurred at a median of 5\u00a0months, so we speculate that the complete inhibition of antibody formation and restoration of platelet counts with rituximab may occur after at least 5\u00a0months (95%CI\u2009=\u20090.5 to 11.6\u00a0months) from the first dose of the antibody (Fig.\u00a02). However, some patients may achieve a quite delayed response (as long as 1\u00a0year after therapy), a situation in which it is important to wait a reasonable time period before another treatment is planned.\nA previous report informed the results obtained in patients treated with the same regimen as we used [4]. After a median follow-up of 47\u00a0weeks, the authors observed a lower overall response rate of 44% (CR\u2009=\u200918%, PR\u2009=\u200915%, MR\u2009=\u200910%); most of the responses were sustained. They observed two response patterns: (1) an early-response group in which responses appeared within the first 2\u00a0weeks after the first dose of rituximab; (2) a late-response group characterized by an increase in platelets several weeks after rituximab. Finally, after a median follow-up of 72.5\u00a0weeks, the response rate was 54% with a majority of SR [8]. In a prospective trial performed in pediatric patients with chronic ITP, treatment with rituximab produced an increase of >50\u2009\u00d7\u2009109\/l platelets in 11 of 36 children (31%) [3]. Median time to response was 1\u00a0week (range, 1 to 7\u00a0weeks); however, a 6% incidence of serum sickness was observed. More recently, one of the largest experiences in ITP patients treated with rituximab was published [15]. An increase of >50\u2009\u00d7\u2009109\/l platelets was observed in 55% of the patients (CR\u2009=\u200946%, SR\u2009=\u200935%). The only predictive factor for SR was to achieve CR. Patients who were treated more intensively (more than three different treatments) and those with a longer ITP duration (>10\u00a0years from diagnosis) had the worse response. In this study, non-splenectomized patients had a higher early response rate than those splenectomized. Although some of these results are similar to those reported in our study, we believe that contrasting results seen in our study are partially explained by differences in the inclusion and response criteria used, in the evolution time of the disease, and in the number and type of treatments given before rituximab.\nAs early relapses may be seen in ITP patients treated with rituximab and because re-treatment with this monoclonal antibody offers good results [16], this drug has been used as a long-term maintenance treatment. Based on long-lasting remissions achieved using one or two doses of rituximab [7] and the results of late responders, we feel that a single dose of 375\u00a0mg\/m2 every 6\u00a0months could be an alternative for this purpose.\nOn the other hand, because of the mechanism of action of rituximab and its interference with the immune system, we searched for possible long-term complications associated with its use, namely, neoplasias, chronic infections, or autoimmune diseases. Immune status is a major concern in patients treated with rituximab being the induction of low CD20+ counts and hypogammaglobulinemia (specifically low levels of immunoglobulin M), two previously reported secondary effects. Although we do not have data about these two immunological variables, the lack of severe infectious diseases during the long-term follow-up of our patients allows us to speculate that no severe immune abnormalities were developed in our series. As we previously stated, during the follow-up period, no associated illnesses or pathological phenomena were observed. To our knowledge, this seems to be the first report about the lack of long-term complications associated with rituximab.\nIn conclusion, rituximab is a long-term, safe, and effective alternative treatment of chronic and refractory ITP patients. Prospective randomized clinical trials are needed to elucidate the efficacy of the drug in comparison to splenectomy in early stages of the disease","keyphrases":["rituximab","refractory itp","anti-cd20","chronic itp"],"prmu":["P","P","P","P"]}
{"id":"Exp_Brain_Res-3-1-1914230","title":"Joint-action coordination in transferring objects\n","text":"Here we report a study of joint-action coordination in transferring objects. Fourteen dyads were asked to repeatedly reposition a cylinder in a shared workspace without using dialogue. Variations in task constraints concerned the size of the two target regions in which the cylinder had to be (re)positioned and the size and weight of the transferred cylinder. Movements of the wrist, index finger and thumb of both actors were recorded by means of a 3D motion-tracking system. Data analyses focused on the interpersonal transfer of lifting-height and movement-speed variations. Whereas the analyses of variance did not reveal any interpersonal transfer effects targeted data comparisons demonstrated that the actor who fetched the cylinder from where the other actor had put it was systematically less surprised by cylinder-weight changes than the actor who was first confronted with such changes. In addition, a moderate, accuracy-constraint independent adaptation to each other\u2019s movement speed was found. The current findings suggest that motor resonance plays only a moderate role in collaborative motor control and confirm the independency between sensorimotor and cognitive processing of action-related information.\nIntroduction\nGiven the strong inclination that people have towards behavioral mimicry when they are jointly involved in walking or speaking (Chartrand and Bargh 1999; Lakin et al. 2003), can we also demonstrate co-actors adopting each other\u2019s movement patterns in the simple task of transferring objects? If an object relevant for my own actions is placed in front of me at a high speed, shall I then automatically pick it up and transfer it also with a high speed or will I stick to my own, preferred speed? Furthermore, will an observer be sensitive to the height of the movement trajectory with which an object is placed within reach as a potential cue of that object\u2019s weight?\nDetailed questions surrounding the potential transfer of movement kinematics in a shared action sequence as specified above have not yet been systematically investigated (cf. Sebanz et al. 2006). The little work that has been done in this area was limited to unintentional synchronization of arm movements in pendulum swinging (Schmidt and O\u2019Brien 1997), incidental entrainment of leg movements when dyads solve a puzzle through dialogue (Shockley et al. 2003), and the kinematic characteristics of cooperative versus competitive grasping movements (Georgiou et al. 2007).\nA recent model of joint action by Oztop et al. (2005) that makes relevant claims regarding joint-action coordination in sequential task performance presumes that when a person observes someone else\u2019s actions she automatically will activate her own action system not only to understand the behavior of the actor being observed (Rizzolatti and Craighero 2004) but also to infer that actor\u2019s intentions (cf. Wolpert et al. 2003; Iacoboni et\u00a0al. 2005). The example Oztop et\u00a0al. (2005) mentioned in this context concerns the implied meaning that is conveyed by the specific way in which people may handle a tool. For example, whereas holding a hammer with a usual grip around its handle is very likely to signal the intent of hammering, holding the hammer with a power grip around its face communicates the intention to handing over the tool to someone else rather than start hammering with it oneself. Of course, which semantics are primed by the way in which a tool is handled will depend on the shared knowledge of how the tool is used for the general purpose it was designed for (see also Cuijpers et\u00a0al. 2006).\nIn the present study we were neither interested in what a particular act might convey to an observer in terms of action semantics (Oztop et\u00a0al. 2005) nor how cooperative or competitive contexts are differentially reflected in the prehension kinematics of dyads (Georgiou et\u00a0al. 2007). Instead, we focused on the extent to which an observer in her subsequent movements is influenced by specific kinematic features of a co-actor\u2019s movements bringing the object within reach. Since the participants in the present study were asked to perform complementary acts rather than identical ones this research question is therefore only indirectly related to topics like behavioral mimicry and imitation (Koski et\u00a0al. 2003).\nOur reasoning about the possible transfer of kinematic movement parameters from one actor to another was that if during action observation people internally simulate the movements that they observe (Gallese and Goldman 1998; Rizzolatti and Craighero 2004), then the kinematic features of these simulated movements might as well be re-used for a related, self-generated movement that, in time, closely follows the observed one even when this future movement is complementary in nature. Economizing on parameter remapping costs formed the basis of this expectation (cf. Rosenbaum et al. 1986).\nAs regards inference making we were interested in the extent to which people derive the weight of an object when seeing someone else picking up and transporting the object. A similar matter has been investigated in an fMRI study by Grezes et al. (2004) but their experiment was primarily designed to find the brain correlates of action observation in the context of deception. To this end these researchers used pictures of an accomplice lifting boxes as if they were heavy or when they were actually heavy. In the present study, however, deceit was not the issue of investigation. Moreover, the object-weight dependent variations in movement kinematics were more subtle than in the Grezes et\u00a0al. (2004) study and related studies on box lifting by Hamilton et\u00a0al. (2007) and Kingma et\u00a0al. (2005) as we will explain next.\nTo gain further insight into the extent to which task-dependent kinematic variations affect the subsequent complementary actions of a co-actor, we exploited two widely investigated motor-control paradigms, viz. the speed-accuracy trade-off (Fitts 1954; Fitts and Peterson 1964) and size\u2013weight illusion (Charpentier 1891). Figure\u00a01 depicts the transportation task that we used for this purpose. First, one of the two actors\u2014the \u2018putting-actor\u2019\u2014was asked to pick up a vertically positioned cylinder from a nearby location on the table and to put that cylinder into a circular-shaped target area in the middle of the table, i.e., within reach for the other actor. Subsequently, the other actor\u2014the \u2018fetching-actor\u2019\u2014was asked to fetch the cylinder from where the putting-actor had left it, and to reposition it, also in a circular-shaped, nearby target area. The fetching-actor was instructed not to pick up the cylinder before the putting-actor had released it.\nFig.\u00a01Top view of experimental setup depicting the experimental task. The dashed squares represent the starting areas for the (right) hand movements of both actors. At the filled circle a cylinder was positioned which had to be picked up by the putting-actor, repositioned in the central circular target area where the fetching-actor was to pick it up and place it in the target circle in the vicinity of her own starting area\nThree task variables were manipulated, viz. the size of the target areas in which the actors were to successively reposition the cylinder and the size and weight of the to-be-transported cylinder. The size of the target areas was expected to modulate the actors\u2019 movement-speed and the size and weight properties of the cylinders were thought to affect the height with which the actors would lift the cylinders during transport. Before elaborating on our rationale it is important to mention that task conditions were kept constant within trial blocks of three repetitions to examine how quickly the dyads adapted to the between trial-block changes in the experimental conditions. All factors other than the repetition factor were randomized across the trial series and dyads, and at the start of each trial block the participants were unaware of the experimental conditions of that particular block (see \u201cMethod\u201d).\nTwo types of potential transfer of movement kinematics were foreseen in our experimental task. First, it was expected that an incorrectly anticipated weight of a to-be-picked up cylinder would be reflected in the height with which an actor would lift that cylinder (cf. Grezes et al. 2004). A lighter than expected cylinder was assumed to be lifted higher than a heavier one and vice versa, i.e., a heavier than expected cylinder was assumed to be lifted less high than a lighter one. Furthermore, if the fetching-actor would infer the weight of the cylinder that was transported by the putting-actor by observing her movements, then the fetching-actor was expected to have a more precise initial estimate of the weight of the to-be-transported cylinder than the putting-actor. The degree to which any surprise effect due to a wrongly anticipated object weight would transfer from the putting-actor to the fetching-actor thus formed a test as to whether or not the fetching-actor had actually inferred the cylinder weight. In repetitions two and three of each trial block both actors could rely, of course, on their sensorimotor memory (see Nowak and Hermsdorfer 2003) as regards the object weight since in these trials they already had handled that particular cylinder in the previous (two) trial(s) of the trial block.\nTo investigate the predicted object-weight dependent interpersonal kinematic transfer effects, we factorially combined two cylinder weights (light vs. heavy) with two cylinder sizes (small vs. large) yielding four cylinders. The size\u2013weight illusion that results from this particular combination of object features reflects the fact that people perceive a smaller of two equally-massed objects as heavier. For one of the suggested explanations of this illusion, we refer to Flanagan and Beltzner (2000). The size\u2013weight illusion is so prominent that it also yields its typical effects when wielding a small and a large object of equal weight in the left and right hand simultaneously suggesting the distorted perception is due to sensory invariants rather than inferences (Amazeen and Turvey 1996). Irrespective of any size\u2013weight illusion effects, we expected the cylinder-weight variations to result in larger between trial adaptation effects on the height with which the participants would lift the cylinders than the cylinder-size variations (Gordon et\u00a0al. 1991a, b). We thus examined the extent to which the weight of an object carried from one position to another would be inferred by an observer. If the observer, i.e., the fetching-actor, would not infer the weight of the transferred cylinder, then the surprise-effect expressed in the lifting height of the fetching-actor should be equally large as that in the putting-actor. Conversely, should the fetching-actor correctly infer the weight of the object by observing the putting-actor lifting and repositioning the cylinder, then the fetching-actor should be able to anticipate the cylinder\u2019s weight better and not show such large surprise effects as the putting-actor would.\nA second manipulation targeted the extent to which the two actors would adopt each other\u2019s movement speed in the face of variations of movement-accuracy constraints. If merely by observing someone else\u2019s movements, one would be inclined to adopt a similar speed in a subsequent complementary movement, then confronting two actors with different target area sizes was expected to modulate their speed differently than when the actors would be confronted with these target areas individually. The rationale here was based on Fitts\u2019 law, which states that one\u2019s movement speed systematically varies with the size of the target area (Fitts 1954; Fitts and Peterson 1964; Mottet et\u00a0al. 2001). The smaller the target area to reach for, the lower the movement speed due to the higher accuracy demands that are associated with small target areas. In the present study we manipulated the target area sizes of the first and the second actor separately, where the size was either small (diameter of 9\u00a0cm) or large (diameter of 18\u00a0cm). If both actors had identical (either small or large) target area sizes, their movement speeds were most likely to become similar under the assumption that there would be a strong tendency to adopt each others\u2019 speed. If this tendency of movement speed transfer was strong enough, then violations of Fitts\u2019 law in the fetching-actor could be expected under conditions in which the two actors were confronted with dissimilar target area sizes.\nIn sum, in the present study consisting of a sequential motor task involving two actors we tested whether lifting height\u2014a parameter that potentially could reveal an object\u2019s weight\u2014would be picked up by an observing co-actor and determine her subsequent handling of that object and whether she would be inclined to adapt her speed to the observed speed, or, alternatively, whether the interpersonal transfer of kinematic parameters would be incidental in nature, i.e., unrelated to the task constraints with which the individuals of the dyad were confronted.\nMethod\nParticipants\nFourteen right-handed adult dyads participated in the experiment. Their ages ranged from 18 to 30\u00a0years; mean age was 22\u00a0\u00b1\u00a02\u00a0years. The 28 participants, 22 females and 6 males, formed 8 female dyads and 6 dyads of mixed gender. They received either course credits or payment (6\u00a0euros per hour) for their participation. Each participant signed an informed consent form. The study was approved by the local ethics committee and performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.\nMaterials\nA Microsoft Windows-2000 controlled PC was used to control the stimulus events and data collection. Two synchronized 3D infrared motion-tracking systems (Optotrak\u2122, Northern Digital, Waterloo, Canada) were used to record the displacements of the wrist, thumb and index finger of the right hand of the two actors. Three infrared-light emitting diodes (IREDs) were used per participant. The IREDs were fixated on the wrist joint and the distal phalanxes of the index finger and thumb of the right arm of the participants. The IRED-position data were sampled at a rate of 100\u00a0Hz with accuracy better than 0.2\u00a0mm in X, Y and Z directions. Each participant wore a pair of earphones through which a repetitive sound was presented at approximately 60\u00a0dB throughout the experiment. This prevented the participants from speaking to each other and hearing any task-related noise, e.g., due to the cylinders making contact with the tabletop at completion of the transport phases. Each participant also wore a facial mask to avoid communication by means of facial cues. The participants were seated comfortably at opposite sides of a table on top of which a 1-cm thick marble plate was placed to prevent any object-weight information being conveyed from one actor to the other by means of vibrations via the table surface.\nFour cylinders of 25\u00a0cm height were used, two with a diameter of 2.5\u00a0cm and two with a diameter of 6.5\u00a0cm. Two cylinders had a lightweight of 230\u00a0g and two cylinders had a weight of 835\u00a0g. To avoid any potential confounding between the effects of cylinder and target-area size all cylinders were given a 4-mm thick, circular base of 8\u00a0cm in diameter. This ensured that the thin and thick cylinders had the same size support base and could be repositioned in a comparable stable way which was a factor that also could contribute to the accuracy constraints since the participants were asked to place the cylinders on small and large circular target areas that were clearly marked on the table surface.\nProcedure and task\nThe experiment consisted of three sections. In the first section one of the two participants performed 48 single-actor control trials consisting of picking up and repositioning in random order, but blocked (three repetitions per block), the four cylinders without the presence of the other participant. In the second section, the other participant performed 48 single-actor control trials, also alone. In the third section the two participants of the dyad performed 96 joint-action experimental trials. The 48 control trials per participant resulted from three blocked replications (n\u00a0=\u00a03) of each of the factorial combinations of the factors size of starting area (small vs. large), size of target area (small vs. large), cylinder weight (light vs. heavy) and cylinder size (small vs. large). The number of joint-action experimental trials was twice the number of single-actor control trials. In the joint-action experimental trials each participant was given in half the trials the role of putting the cylinder in the centre of the table, and in the other half of the trials the role of fetching the cylinder from the centre of the table (see Fig.\u00a01). The role was randomly assigned to the trial blocks of three repetitions.\nIn their control trials the participants of dyads 1\u20139 (arbitrary numbered) performed the fetch-part of the experimental task, i.e., at the start of their control trials they were confronted with a cylinder that was placed in the middle of the table and their task was to pick it up and place it in the area which was closest to their own starting position. Dyads 10\u201314 performed in their control trials the put-part of the experimental task, i.e., at the start of each trial they found the cylinder on the area closest to themselves and they were instructed to put the cylinder in the target area at the centre of the table. The data analyses showed that there were no systematic differences between the two groups of dyads as far as the targeted research questions concerned so the comparisons with the control trial data will not be addressed further in the remainder of this paper.\nThe experiment was conducted by two experimenters who sat behind the participants. Each experimenter had a clear view on a separate computer screen which displayed information that specified the experimental conditions of every trial. In-between trials the participants were asked to close their eyes. After both participants had closed their eyes, the experimenters put the appropriate target pads on the table as well as the appropriate cylinder in the starting area of the putting-actor. Then, the two experimenters simultaneously tapped the participants on their lower arm, which indicated that they could open their eyes to start performing the trial. At the same time one of the experimenters started the movement recording.\nThe standard event sequence in a joint-action experimental trial was as follows. At the start of each trial one of the two actors, i.e., the putting-actor, found in a location close to the starting position of the right hand a cylinder that had to be picked up and moved to the target area in the centre of the tabletop (see Fig.\u00a01). Only after the putting-actor had released the cylinder was the fetching-actor allowed to pick up the cylinder and to reposition it in a target area adjacent to her. The action sequence was to be completed within 8\u00a0s during which the displacements of the wrist and finger IREDs on both actors were sampled at a rate of 100\u00a0Hz. After every 48 trials, or whenever they asked, the participants were offered a 10-min break.\nData analysis\nThe raw IRED position data were smoothed with a second-order, low-pass Butterworth filter with a cut-off frequency of 8\u00a0Hz. Subsequently, the position data were segmented on the basis of the tangential velocity\u2013time functions of the IREDs on the wrists of the two actors (see top-left graph in Fig.\u00a02).\nFig.\u00a02Kinematics derived from position data of infrared-light emitting diode (IRED) on tip of the thumb, tip of the index finger and wrist as obtained in a prototypical trial. Green (light grey) functions are from the putting-actor, red (dark grey) functions are from the fetching-actor. The top-left graph shows the tangential wrist-velocity time functions, the top-right graph a 3D rendering of finger-thumb displacements, the bottom-left graph shows the aperture-time functions, and the bottom-right graph shows the height (in cm above the table surface) of the midpoint between the thumb and index-finger IREDs\nThe movement sequence of each actor was segmented into three intervals, the first interval corresponding to grasping the cylinder starting with the right hand in the home-position on the edge of the table and ending with finger and thumb making contact with the object, the second interval corresponding to the transportation of the cylinder to the target area (see put and fetch legends in Fig.\u00a02), and the third interval corresponding to the participant\u2019s hand returning to the home-position. The intervals were detected by a semi-automatic search procedure for relevant local minima in the tangential velocity profiles of the wrist movements. The time indexes thus found could be adjusted with an interactive computer program using a cross-hair whenever the experimenter considered the segmentation inaccurate.\nFollowing segmentation, the data analyses focused on the displacements of the IREDs located on the tip of the thumb and index finger of each of the two actors. The top-right graph of Fig.\u00a02 shows a 3D-graph of the typical finger\u2013thumb displacements of the putting-actor (in green or light grey) and the fetching-actor (in red or dark grey). The three cylinders were added to this graph post hoc to indicate the phasing of the displayed movement patterns. The bottom-left graph of Fig.\u00a02 shows the aperture-time functions of both actors. This figure was used to inspect the grasping behavior of the participants. Systematic aperture overshoots just prior to object contact and upon object release were visually checked by the experimenter to verify whether the task of each actor was performed as requested.\nThe bottom-right graph of Fig.\u00a02 shows the height in the Z-dimension (perpendicular to the table surface) of the midpoint between the 3D positions of the IREDs on the thumb and index finger of each actor. This parameter reflected the surprise effects due to unexpected between-trial cylinder-weight changes on the lifting height, which was defined as the difference in the vertical position of the midpoint between the tips of the digits at the time of grasping the cylinder and the maximum height realized during transportation. In the second interval of each of the three-segment movement patterns, i.e., the green function for the putting-actor and the red function for the fetching-actor, this lifting height reflected the height with which the participants lifted the cylinder while transporting it to their target area.\nThe maximum lifting height (in cm) and the mean tangential wrist speed (in cm\/s) realized by each actor during cylinder transport were used for further analyses. Statistical evaluation of the data consisted of repeated measures ANOVAs of which the designs are described in the relevant paragraphs in the results section or targeted t tests to evaluate specific hypotheses in more detail.\nResults\nEffects of cylinder properties\nFigure\u00a03 shows the means and standard errors of the lifting heights realized by the putter and fetcher as a function of cylinder mass, cylinder size and the repetition factor. Table\u00a01 summarizes the results of the repeated measures ANOVA that was conducted to evaluate these effects with a factorial design consisting of the within-dyad factors 2 roles (put vs. fetch)\u00a0\u00d7\u00a02 object masses (light vs. heavy)\u00a0\u00d7\u00a02 object sizes (small vs. large diameter)\u00a0\u00d7\u00a03 repetitions. As expected, the light cylinders were lifted higher than the heavy ones [F(1,13)\u00a0=\u00a086.5, P\u00a0<\u00a00.001]. This effect interacted with object size [F(1,13)\u00a0=\u00a014.4, P\u00a0<\u00a00.01] in line with the size\u2013weight illusion. For the light cylinders it was the smaller one that was lifted higher than the larger one. For the two heavy cylinders, it was the larger one that was lifted higher than the smaller one. The interaction between object mass and object size on lifting height\u2014reflecting the size\u2013weight illusion effect\u2014was most pronounced in repetition 1 as confirmed by the first-order interaction between object mass and repetition [F(2,26)\u00a0=\u00a0123.8, P\u00a0<\u00a00.001] but remained present in repetitions 2 and 3 as reflected by the second-order interaction between object mass, object size and repetition [F(2,26)\u00a0=\u00a03.78, P\u00a0<\u00a00.05], confirming the robustness of the size\u2013weight illusion.\nFig.\u00a03Means and standard errors of the lifting height (in cm) as a function of role (putting vs. fetching), object diameter (small vs. large) and object weight (light vs. heavy) for repetitions 1, 2 and 3 separatelyTable\u00a01Results of repeated measures Anova on the lifting height according to a 2 role (putting vs. fetching)\u00a0\u00d7\u00a02 object mass (230 vs. 835\u00a0g)\u00a0\u00d7\u00a02 object sizes (2.5 vs. 6.5\u00a0cm in diameter)\u00a0\u00d7\u00a03 repetitions factorial designFactordfFPRole1,13<1NSObject mass1,1386.5<0.001Object size1,137.1<0.05Repetition2,2658.0<0.001Role\u00a0\u00d7\u00a0object mass1,13<1NSRole\u00a0\u00d7\u00a0object size1,131.3NSObject mass\u00a0\u00d7\u00a0object size1,1314.4<0.01Role\u00a0\u00d7\u00a0object mass\u00a0\u00d7\u00a0object size1,13<1NSRole\u00a0\u00d7\u00a0repetition2,26<1NSObject mass\u00a0\u00d7\u00a0repetition2,26123.8<0.001Role\u00a0\u00d7\u00a0object mass\u00a0\u00d7\u00a0repetition2,26<1NSObject size\u00a0\u00d7\u00a0repetition2,263.67<0.05Role\u00a0\u00d7\u00a0object size\u00a0\u00d7\u00a0repetition2,26<1NSObject mass\u00a0\u00d7\u00a0object size\u00a0\u00d7\u00a0repetition2,263.78<0.05Role\u00a0\u00d7\u00a0object mass\u00a0\u00d7\u00a0object size\u00a0\u00d7\u00a0repetition2,26<1NS\nNo main effect or interaction of role (put vs. fetch) was found (all Fs\u00a0<\u00a01), which was not unexpected since cylinder-weight increases and decreases were expected to have counteracting effects on the lifting heights by the putter and fetcher if any interpersonal transfer would take place at all. Targeted paired t tests confirmed that there were systematic interpersonal transfer effects of lifting height between putter and fetcher as will be described next.\nA detailed time-series analysis of the lifting heights which the dyads realized indicated that the fetching-actor benefited from movement observation. The results of this analysis are shown in Fig.\u00a04. In those cases in which from one trial block to the next the cylinder\u2019s weight changed from heavy to light and the putting-actor showed a \u2018surprise\u2019 effect as reflected by an increase of her lifting height between these two trials of, on average, 4.69\u00a0cm, the fetching-actor showed a similar surprise effect but to a smaller extent. This reduced surprise effect in the fetching-actor of, on average, 3.67\u00a0cm, is shown in the left-hand graph of Fig.\u00a04. The incidence of this effect was relatively high. It happened in 88% of those trials (i.e., N\u00a0=\u00a099) in which a heavy-to-light cylinder-weight change occurred. A two-tailed t test comparing the lifting heights on trial i\u00a0\u2212\u00a01 and trial i (being the third trial of each trial block and the first of the subsequent block, respectively) for the putting-actor revealed that her surprise effect was statistically significant [t(98)\u00a0=\u00a015.98, P\u00a0<\u00a00.01]. For the fetching-actor, the smaller surprise-effect was also statistically significant [t(98)\u00a0=\u00a013.54, P\u00a0<\u00a00.01]. A repeated measures ANOVA confirmed that the relevant interaction between role (putter vs. fetcher) and trial (i\u00a0\u2212\u00a01\u00a0=\u00a0heavy and i\u00a0=\u00a0light) was statistically significant [F(1,13)\u00a0=\u00a07.24, P\u00a0<\u00a00.05].\nFig.\u00a04Means and standard errors of the lifting height (in cm) of the putting-actor and the fetching-actor. Left-hand graph: whenever the putting-actor showed a lifting-height increase from trial i\u00a0\u2212\u00a01 to trial i due to an unexpected mass reduction between these trials, the fetching-actor also showed this surprise effect but less strong. Trials i\u00a0\u2212\u00a01 were the third trials of the trial blocks; trials i were the first trials of the subsequent trial blocks. Right-hand graph: similarly, whenever the putting-actor showed a lifting-height decrease from trial i\u00a0\u2212\u00a01 to trial i due to an unexpected mass increase between these trials, the fetching-actor also showed this surprise effect, but again, not as strong as the putting-actor did\nWe found a similar effect when focusing on those trial pairs in which the cylinder-weight changed from light to heavy and the putter showed a \u2018surprise\u2019 effect as reflected by a decrease of her lifting height between those trials. The results of this analysis are shown in the right-hand graph of Fig.\u00a04. Whereas the putting-actor proved to lift the cylinder, on average, 2.21\u00a0cm less high when the cylinder turned heavier from trial i\u00a0\u2212\u00a01 to trial i, the fetching-actor did so as well but, on average, for only 0.97\u00a0cm. The incidence of this phenomenon was at chance level, i.e., in 54% (N\u00a0=\u00a061) of the cases in which such a cylinder-weight change occurred in the experiment. A two-tailed t test comparing the lifting heights on trial i\u00a0\u2212\u00a01 and trial i for the putting-actor and fetching-actor separately again revealed that the effects were statistically significant for both actors [t(60)\u00a0=\u00a012.08, P\u00a0<\u00a00.01, t(60)\u00a0=\u00a03.69, P\u00a0<\u00a00.01, respectively]. A repeated measures ANOVA confirmed that the relevant interaction between role (putter vs. fetcher) and trial (i\u00a0\u2212\u00a01\u00a0=\u00a0light and i\u00a0=\u00a0heavy) was statistically significant [F(1,13)\u00a0=\u00a031.08, P\u00a0<\u00a00.001].\nThe time-series analyses on light-to-heavy cylinder weight changes and vice versa were corroborated by the results of dyad-based linear regression analyses in which the lifting height of the putting-actor in the first trial of the trial blocks was used as predictor for that of the fetching-actor. In contrast to the repeated measures ANOVA, the regression analysis took into account lifting-height co-variations in both directions, i.e., increases as well as decreases. An example of these data is depicted in Fig.\u00a05. The slopes of the resulting regression equations turned out to be smaller than 1.0 for all 14\u00a0dyads showing that the putting-actor was more surprised by a cylinder-weight change than the fetching-actor. For nine of the dyads the positive slope proved statistically significant (P\u00a0<\u00a00.05). On average, the slopes amounted to 0.43 (R2\u00a0=\u00a00.25), indicating that the fetching-actors were less surprised by any cylinder-weight change than the putting-actors.\nFig.\u00a05Example of the linear regression analysis between the lifting-height data generated by the putter and fetcher of 1 of the 14\u00a0dyads participating in the study. The data concern the first trial within trial blocks of three repetitions of the experimental conditions. The regression analyses revealed the robustness of the observed reduction of the size\u2013weight illusion due to movement observation in the dyads studied. The dashed line with slope\u00a0=\u00a01 represents the situation in which the lifting height of the fetcher would equal that of the putter\nEffects of target-area size variations\nFigure\u00a06 shows the average effects on the mean tangential wrist velocity of the putting and fetching-actors as a function of the manipulations of the size of the target regions where the cylinders had to be placed. The second part of the sequential action under study, i.e., fetching the cylinder from the middle of the table, proved for no apparent reason overall to be performed somewhat slower than the first part, i.e., putting the cylinder in the middle of the table. The performance speeds of the participants were independently tuned to the sizes of the target areas with which the actors were confronted. In line with Fitts\u2019 law, small target areas elicited low speeds and large ones yielded high speeds. This relationship applied to both actors whether or not they were confronted with similar or dissimilar accuracy constraints. Table\u00a02 summarizes the repeated measures ANOVA to evaluate these effects with a 2 roles (put vs. fetch)\u00a0\u00d7\u00a02 sizes of putting target region (\u2018putting-size\u2019; small vs. large)\u00a0\u00d7\u00a02 sizes of fetching target region (\u2018fetching-size\u2019; small vs. large) factorial design. The absence of the first-order interaction between putting-size and fetching-size (F\u00a0<\u00a01) and the second-order interaction between the factors role, putting-size and fetching-size confirm the robustness of these findings.\nFig.\u00a06Means and standard errors of the mean wrist speed (in cm\/s) as a function of role (putting vs. fetching) and target area size (S\u00a0=\u00a0Small, L\u00a0=\u00a0Large; p\u00a0=\u00a0put, f\u00a0=\u00a0fetch). The labels with subscript p reflect the size of the target area of the putting-actor and labels with subscript f reflect the size of the target area of the fetching-actorTable\u00a02Results of repeated measures ANOVA on the mean wrist speed (in cm\/s) according to a 2 role (putting vs. fetching)\u00a0\u00d7\u00a02 putting-size (small vs. large target area)\u00a0\u00d7\u00a02 fetching-size (small vs. large target area) factorial designFactordfFPRole1,1318.60<0.01Putting-size1,1375.07<0.001Fetching-size1,1333.90<0.001Role\u00a0\u00d7\u00a0putting-size1,1320.21<0.001Role\u00a0\u00d7\u00a0fetching-size1,1322.03<0.001Putting-size\u00a0\u00d7\u00a0fetching-size1,13<1NSRole\u00a0\u00d7\u00a0putting-size\u00a0\u00d7\u00a0fetching-size1,13<1NS\nA trial-by-trial inspection of the speed changes of both actors revealed, however, a moderate degree of performance-speed adaptation by the fetching-actor to that of the putting-actor. Figure\u00a07 shows the results of this analysis. If from one trial to the next the size of the target areas remained constant for both actors but the putting-actor increased her performance speed for some reason, the fetching-actor followed this incidental speed increase of the putting-actor only partially as shown in the left-hand graph of Fig.\u00a07. The incidence of this co-variation in performance speed between the two actors was below chance level, i.e., it happened in 34% (N\u00a0=\u00a0150) of the cases in which the phenomenon could theoretically occur. Nevertheless, the changes in speed were, in both actors, statistically significant [t(149\u00a0=\u00a07.63, P\u00a0<\u00a00.02 and t(149)\u00a0=\u00a05.95, P\u00a0<\u00a00.01, two-tailed, for the putting-actor and fetching-actor, respectively).\nFig.\u00a07Means and standard errors of the wrist speed (in cm\/s) of the putting-actor and the fetching-actor. Left-hand graph: whenever the putting-actor showed a task-unrelated speed increase from trial i\u00a0\u2212\u00a01 to trial i, the fetching-actor also showed such a speed increase. The trials involved concerned the second and third trials of the trial blocks in which no task conditions changed. Right-hand graph: similarly, whenever the putting-actor showed a task-unrelated speed decrease from trial i\u00a0\u2212\u00a01 to trial i, the fetching-actor followed this speed decrease\nThe reverse co-variation of performance speeds happened slightly more often. The right-hand graph of Fig.\u00a07 shows that whenever the putting-actor reduced her movement speed from trial i\u00a0-\u00a01 to trial i during which the size of the target regions remained constant, the fetching-actor again partially followed this speed decrease. This co-variation in speed occurred in 42% (N\u00a0=\u00a0186) of the cases in which this effect could occur. For the putting-actor and fetching-actor the speed decreases that were isolated here were again both statistically significant [t(185)\u00a0=\u00a05.95, P\u00a0<\u00a00.01 and t(185)\u00a0=\u00a03.32, P\u00a0<\u00a00.01, two-tailed, respectively].\nLinear regression analyses in which the mean wrist speeds of the putting-actor as observed in the second and third trials of the trial blocks were used as predictor for that of the fetching-actor confirmed the just described results. The slopes of the regression equations proved positive for 12 of the 14\u00a0dyads indicating that the fetching-actor followed the speed changes of the putting-actor in direction but only partially in size. For 10 of the 14\u00a0dyads the positive correlations proved statistically significant (P\u00a0<\u00a0.05). R2s in these analyses ranged between 0 and 0.33 (mean R2 was 0.11) indicating the target-size independent speed relationship between the two actors was weak.\nCombined effects of size\u2013weight illusion and speed-accuracy trade-off\nAn inspection of the combined effects of the three task constraints that were manipulated revealed that even though on average the heavier cylinders were transported at a systematically lower speed than the lighter cylinders (mean\u00a0=\u00a043.78\u00a0cm\/s; SD\u00a0=\u00a08.37\u00a0cm\/s for the light cylinders and M\u00a0=\u00a038.14\u00a0cm\/s; SD\u00a0=\u00a07.06\u00a0cm\/s for the heavy cylinders), the speed variations due to the variations in the size and weight of the cylinders did not differentially affect the movement-speed variations as a result of the target-area size variations. Lifting-height variations due to the spatial accuracy-constraint manipulations were marginal (<2\u00a0mm) and also did not systematically influence the lifting-height variations due to the size-illusion effect.\nDiscussion\nThe present study examined the extent to which at the level of movement kinematics a transfer of performance parameters takes place between co-actors involved in transferring objects. The results of the main analyses that we conducted to assess the effect sizes of the experimental manipulations were largely negative. Object-mass variations induced lifting-height variations of equal size in both actors. Target-region size based speed variations also did not show interpersonal transfer. These results suggest that in sequential joint actions the tuning to each others movement parameters is absent and thus demonstrate the limits of automatic movement simulation by observation as suggested by mirror-neuron theories (Gallese and Goldman 1998; Gallese et al. 2004; Rizzolatti and Craighero 2004). However, it remains to be seen whether the mirror-neuron system represents observed actions with a precision that allows for direct imitation. This does not appear to be the case in macaque monkeys. One could claim that any hypothetical system in general could infer the goals\/intentions of observed movements without explicitly representing, say, the speed of the movement.\nMore detailed analyses of incidental interpersonal transfer effects indicated that the situation was more complex than that. When focusing on the effects of between-trial cylinder-weight changes, the actor who was confronted with such changes first showed a systematically larger surprise-effect than the actor who was asked to transport the cylinder after the first actor had done so. A key finding here was that through the realized trajectory height during object transportation, the object mass was picked up by movement observation and integrated into the observer\u2019s movement plan. Our prediction was therefore confirmed, i.e., the movements of an actor are likely to affect a subsequent, complementary movement generated by an observer.\nRegarding the size of the effects it proved that the object property mass was, via lifting height, a more prominent factor determining task performance of the co-actors than movement speed. The linear regression analyses of the between-trial lifting-height changes due to cylinder-mass changes yielded larger proportions of explained variance when correlating the behavior of the fetching-actor with that of the putting-actor than the comparable analyses involving the incidental speed changes. With respect to goals and means of task performance, the dominance of object-mass over target-width makes sense given that the object is more likely to function as a goal in the task of handing over objects than the movements needed to perform such a task (Cuijpers et\u00a0al. 2006). In a similar vein, the different sizes of the effects of object mass and movement speed could be taken to provide support for the importance of goal-inference making in collaborative joint action over and above a quasi-automatic, direct-matching based process of movement imitation or \u2018motor resonance\u2019 (Flanagan and Johansson 2003; Iacoboni et\u00a0al. 1999; Koski et\u00a0al. 2003) (Fig.\u00a08).\nFig.\u00a08Example of the linear regression analysis between the wrist-speed data generated by the putter and fetcher of 1 of the 14\u00a0dyads participating in the study. The data concern the second and third trials within trial blocks of three repetitions of the experimental conditions. The regression analyzes revealed the robustness of the observed, task-constraint independent covariation of movement speed between the two actors. The dashed line with slope\u00a0=\u00a01 represents the situation in which the wrist speed of the fetcher would equal that of the putter\nOur interpretation of the current results does rely, of course, on the validity of both the weight-change effects and the co-variation of the actors\u2019 movement speeds. It could be argued that the fetcher\u2019s movement speed covaried with that of the putter due to a non-specific, visually primed increase in attention rather than the direct consequence of perceiving the putter\u2019s object-transportation movements. However, attentional mechanisms are an unlikely explanation for our observations since the only visual cue that could have increased the attention in the fetcher was the putter\u2019s movement speed. Whether the object-weight related adaptation in the fetcher to the observed movement of the putter was due to the inference of the weight of the object or to the observed kinematics per se, also remains a matter of discussion. If it was the former, then motor resonance (i.e., mirror-neuron system) may not be responsible for a direct transfer of the kinematics parameters.\nThe effects of the object and the accuracy constraints differ in one fundamental aspect. The expected information transfer for the object is valid for the programming of the movement, while any hypothesized transfer of speed, by simple copying the observed movement to reduce dimensionality violates Fitts\u2019 law. This implies that our hypothesis about the transfer of speed was either not valid or that the effect is intrinsically short lived because the duration of the effect is overruled by intrapersonal constraints. In other words, while the information about weight change is very relevant for movement programming, the examined speed changes were not.\nApparently, movements that are being observed in a joint-action task can be used for multiple purposes. First, the observation might lead to internal simulation in order to understand the behavior that is being observed (Rizzolatti and Craighero 2004). Second, the internally simulated movements might provide clues as to the intentions associated with the observed behavior (Oztop et al. 2005; Wolpert et al. 2003; Iacoboni et\u00a0al. 2005). Third, the observed movements might form the basis for prediction as to what an actor might do in the near future (Csibra 2005). Fourth, observed movements might provide clues about the task constraints at hand. In the present experiment, lifting height was such a parameter that indicated to the observer the weight of the cylinder that was transported. Fifth, observed movements may provide a global scaling parameter of ones own future actions, specifically if these actions are complementary to those performed by the actor that is being observed. Further research into the factors that determine the relative importance of these various roles which action observation may play in joint action is clearly needed.\nOur study also suggests that kinematic parameters picked up during movement observation may even affect a subsequent movement that is performed in a workspace that is totally differently oriented than the workspace of the actor being observed. This finding extends the findings of the recent study by Van Schie et al. (2004) who showed that action observation activates cortical motor areas as if the observer had performed the task herself, i.e., when an observer who is facing an actor sees that actor move with her right hand the right cortical motor areas of the observer become activated indicating that the observer would have executed the task with her left hand. These findings suggest that movement simulation is egocentric and viewpoint-dependent.\nOur demonstration of interpersonal action coordination in complementary joint action was restricted to the successive transportation of a cylinder by two actors. The movements preceding and succeeding the actual transportation of the cylinder could also have been scrutinized for signs of action coordination. However, the amplitudes of these movements by the two actors were hardly comparable. Moreover, the dyads involved in this study showed, not surprisingly, also the tendency to wanting to start task performance simultaneously. The preference of people to adopt either in-phase or out-of-phase timing patterns is not only prevalent in individual, inter-limb coordination tasks but also when people perform tasks together (Schmidt and O\u2019Brien 1997; Shockley et al. 2003). These tendencies clearly co-exist alongside the casual transfer of kinematic movement parameters in dyadic action sequences.","keyphrases":["coordination","grasping","joint action","size\u2013weight illusion","fitts\u2019 law"],"prmu":["P","P","P","P","P"]}
{"id":"J_Gastrointest_Surg-3-1-1852373","title":"Impact of Solitary Involved Lymph Node on Outcome in Localized Cancer of the Esophagus and Esophagogastric Junction\n","text":"Node-positive esophageal cancer is associated with a dismal prognosis. The impact of a solitary involved node, however, is unclear, and this study examined the implications of a solitary node compared with greater nodal involvement and node-negative disease. The clinical and pathologic details of 604 patients were entered prospectively into a database from1993 and 2005. Four pathologic groups were analyzed: node-negative, one lymph node positive, two or three lymph nodes positive, and greater than three lymph nodes positive. Three hundred and fifteen patients (52%) were node-positive and 289 were node-negative. The median survival was 26 months in the node-negative group. Patients (n = 84) who had one node positive had a median survival of 16 months (p = 0.03 vs node-negative). Eighty-four patients who had two or three nodes positive had a median survival of 11 months compared with a median survival of 8 months in the 146 patients who had greater than three nodes positive (p = 0.01). The survival of patients with one node positive [number of nodes (N) = 1] was also significantly greater than the survival of patients with 2\u20133 nodes positive (N = 2\u20133) (p = 0.049) and greater than three nodes positive (p < 0001). The presence of a solitary involved lymph node has a negative impact on survival compared with node-negative disease, but it is associated with significantly improved overall survival compared with all other nodal groups.\nIntroduction\nCarcinoma of the esophagus carries a dismal prognosis, and for patients presenting with localized resectable disease, multivariate analysis has established that the presence or absence of involved lymph nodes confers the greatest prognostic significance.1 In surgical management, the extent and type of lymphadenectomy undertaken varies from no formal lymphadenectomy to two and three field dissection.2\u20135 The presence and extent of lymph node involvement is important as selective approaches may be considered depending on the nodal stage at presentation. In early tumors, for instance, the sentinel node concept initially developed in melanoma and breast cancer was explored to help identify patients who may not require lymph node dissection.6\u20138 The advent of minimally invasive esophagectomy may also highlight the need to subselect patients for lymphadenectomy.9\nIn the observations of the senior author (JVR), patients with solitary involved lymph nodes may achieve good outcomes, and this hypothesis was evaluated in this analysis of a large prospective database. We report herein that the cohort with a solitary node involved had cancer outcomes closer to node-negative disease than other node-positive subgroups, and suggest that this represents a distinct prognostic subgroup.\nPatients and Methods\nThe study population consisted of all patients with tumors of the esophagus and esophagogastric junction who underwent surgical resection, either alone or preceded by neoadjuvant chemoradiation, between 1993 and 2005. Patients receiving multimodal therapy received cisplatin, 5-fluorouracil, and external beam radiotherapy (40\u201344\u00a0Gy, 2\u20132.67\u00a0Gy\/fraction) as previously described.10 Data concerning the clinical and pathologic parameters for all patients was obtained from a detailed prospective database maintained by a full-time data manager. Pathologic parameters analyzed included the location of the tumor, tumor morphology, i.e., adenocarcinoma or squamous cell carcinoma, histological differentiation (grade), TNM staging, number and site of involved lymph nodes, and R classification after surgical resection. Staging of tumors was performed according to the American Joint Committee on Cancer TNM system.11\nA subtotal esophagectomy was performed with a sutured anastomosis either in the right thorax (two-stage) or neck (three-stage). All cases underwent a formal abdominal lymphadenectomy and mediastinal lymph node dissection up to and including the subcarinal nodes. Thoracic nodes were submitted separately to abdominal nodes.\nStatistical Analysis\nData are presented as frequencies, means, and percentages. ANOVA was used for comparison of the four demographic groups. Survival probability was estimated using the Kaplan\u2013Meier method. Survival was calculated from the date of clinical diagnosis to date of death or date last seen. In the multivariate analysis, independent prognostic factors for survival were determined by using a Cox regression hazard model. Two analyses were performed, one for all patients and the other exclusive to node-positive patients. All statistical analyses were performed using Stata software (version 9.1 for Windows, Statcorp, TX). A p value <0.05 was considered statistically significant.12\nResults\nPatients\/histology\nSix hundred and four patients underwent surgery for localized malignancy of the esophagus or esophagogastric junction. The mean age was 62\u2009\u00b1\u200910.4 (median\u2009=\u200964, range 56 to 70). Four hundred and twelve (68%) patients were men. The mean number of lymph nodes examined per specimen was 12\u2009\u00b1\u20096 (median\u2009=\u200910, range\u2009=\u20096 to 55). Two hundred and eighty-nine patients (48%) had node-negative disease [number of nodes (N)\u2009=\u20090], 84 (14%) had one node positive (N\u2009=\u20091), 84 had two or three nodes positive, and 147 (24%) had greater than three nodes positive (N\u2009>\u20093). In patients with one involved node, in all cases the node was adjacent to the tumor, mediastinal for esophageal tumors, and periesophageal or along the left gastric artery for junctional tumors (Tables\u00a01 and 2). \nTable\u00a01Demographics of Nodal SubgroupsHistologic DataN\u2009=\u20090 (n\u2009=\u2009289)N\u2009=\u20091 (n\u2009=\u200984)N\u2009=\u20092\u20133 (n\u2009=\u200984)N\u2009>\u20093 (n\u2009=\u2009147)Tumor site (%)Lower esophagus138 (47)39 (46)37 (44)57 (39)EG junction80 (28)35 (42)33 (39)75 (51)Middle esophagus55 (19)10 (12)12 (14)11 (7)Upper esophagus16 (6)02 (3)4 (3)Morphology (%)Adenocarcinoma140 (48)51 (61)57 (68)113 (77)Squamous cell carcinoma140 (48)29 (35)25 (30)32 (22)Others9 (4)4 (5)2 (1)2 (1)Treatment (%)Multimodal therapy129 (44)28 (33)24 (29)21 (14)Surgery alone161 (56)56 (76)60 (71)125 (86)Residual tumor (%)R0: no residual tumor250 (86)71 (85)64 (76)108 (73)R1: residual tumor found39 (13)13 (15)19 (23)39 (27)Rx: unknown1 (1)\u20131 (1)\u2013Pathological stage (%)Stage 053 (18)\u2013\u2013\u2013Stage I59 (20)1 (1)\u2013\u2013Stage II170 (59)21 (25)25 (30)16 (11)Stage III5 (2)58 (29)53 (63)110 (76)Stage IV1 (1)4 (5)6 (7)20 (13)pT stage (%)Tx3 (1)02 (3)1 (0.5)Tis12 (4)000T040 (14)1 (1)2 (3)2 (1)T156 (19)5 (6)4 (5)3 (2)T235 (12)16 (19)18 (21)12 (8)T3138 (48)60 (71)54 (64)120 (82)T46 (2)2 (3)4 (5)8 (5)EG = esophagogastricTable\u00a02Histology of Nodal SubgroupsHistologic DataN\u2009=\u20090N\u2009=\u20091N\u2009=\u20092\u20133N\u2009>\u20093AdenoSCCAdenoSCCAdenoSCCAdenoSCCn\u2009=\u2009140N\u2009=\u2009140n\u2009=\u200951n\u2009=\u200929n\u2009=\u200957n\u2009=\u200925n\u2009=\u2009113n\u2009=\u200932No%No%No%No%No%No%No%No%Tumor siteLower Esophagus64(46)66(47)19(52)15(52)23(40)13(52)39(35)17(53)EG Junction73(52)6(4)31(10)3(10)33(58)0074(65)1(3)Middle Esophagus3(2)52(37)1(28)8(28)1(2)10(40)0010(31)Upper Esophagus0016(11)0(10)3(10)002(8)004(13)TreatmentMultimodal80(57)46(34)23(45)5(17)20(35)4(16)19(13)3(10)Surgery alone60(43)93(66)28(55)24(83)37(65)21(84)94(87)28(90)Path stageStage 029(21)18(13)000000000000Stage 142(30)15(10)1(2)0000000000Stage 266(47)102(73)15(29)4(14)19(33)5(20)15(13)1(3)Stage 32(1)4(3)32(63)24(83)35(61)17(68)82(73)27(84)Stage 4001(1)3(6)1(3)3(6)3(12)16914)3(10)Unknown1(1)0000000000001(3)pT stageTx2(1)1(1)0000002(8)001(3)Tis9(6)0000002(4)000000T019(14)17(12)1(2)003(5)002(2)00T139(29)15(11)4(8)0014(24)003(3)00T216(11)19(14)12(23)3(10)36(63)4(16)11(10)1(3)T353(38)84(60)33(65)26(90)2(4)17(68)91(80)28(88)T42(1)4(2)1(2)00002(8)6(5)2(6)Adeno = adenocarcinoma, SCC = small cell carcinoma, EG = esophagogastric\nTwo hundred and two patients (33%) had multimodal therapy and 402 patients (67%) had surgery alone. Of the multimodal cohort, 129 (64%) were ypN0 on histopathologic assessment, 28 (14%) had one node positive, 24 (12%) had two to three positive nodes, and 21 (10%) had greater than three positive nodes. The attainment of an R0 resection was significantly greater in patients with none or one node involved compared with both other groups (p\u2009<\u20090.05). The majority of patients in all groups had pT3 tumors, 48% in the pN0 group compared with 71, 64, and 82% in the N\u2009=\u20091, N\u2009=\u20092\u20133, and N\u2009>\u20093 groups, respectively (p\u2009<\u20090.05). One hundred and forty (62%) of the squamous cell carcinoma cohort were node-negative (N\u2009=\u20090) compared with 140 (39%) of cases with adenocarcinoma (39%) (p\u2009<\u20090.05).\nSurvival\nThe median survival for all patients was 20\u00a0months at a median follow-up of 19\u00a0months (3\u2013167). Patients who were node-negative (N\u2009=\u20090) had a median survival of 26\u00a0months (Table\u00a03), compared with 16\u00a0months when one node was positive (p\u2009=\u20090.03). Patients who had two to three nodes positive had a median survival of 11\u00a0months, and 8\u00a0months in patients who had greater than three nodes positive (p\u2009=\u20090.01; N\u2009=\u20092\u20133 vs N\u2009>\u20093). The survival of patients with one node positive (N\u2009=\u20091) was significantly greater than the survival of patients with 2\u20133 nodes positive (p\u2009=\u20090.04) and the cohort with greater than three involved nodes (p\u2009<\u20090.0001). \nTable\u00a03Univariate and Multivariate Analysis: All PatientsVariablesNo. of PatientsMedian Survival (moths)p Valuea (Univariate)HR95% CIap valueb (Multivariate)HR95% CITreatmentSurgery only401130.0771\u2013\u2013\u2013Multimodal203190.840.69\u20131.02Tumor siteUpper esophagus25160.3711\u2013\u2013\u2013Middle esophagus87140.9460.980.58\u20131.66Lower esophagus268140.6581.160.69\u20131.81EG junction224140.6241.130.69\u20131.84Depth of invasionT05755<0.00110.6521T168260.5371.160.73\u20131.830.4720.710.21\u20132.3T281260.4191.200.77\u20131.850.5731.110.31\u20133.94T337311<0.0012.281.60\u20133.260.8711.400.79\u20132.41T4197<0.0014.342.46\u20137.680.6492.591.42\u20134.08No. of nodes028926<0.0011<0.00110.63\u20131.87184160.0381.361.02\u20131.820.7741.080.83\u20132.432\u201338411<0.0011.911.45\u20132.520.2021.421.07\u20133.18>31478<0.0012.612.08\u20133.290.0271.84HistologySquamous361140.9161Adenocarcinoma224130.5961.050.87\u20131.28\u2013\u2013\u2013Other19260.4830.800.44\u20131.48Stage05355<0.00110.1181I63550.7470.920.56\u20131.510.5760.680.18\u20132.59II230200.0371.491.02\u20132.170.5081.550.42\u20134.69III22510<0.0012.711.86\u20133.950.5271.680.34\u20135.58IV316<0.0016.163.72\u201310.20.1823.141.14\u20137.76Residual tumorR049217<0.00110.0521R111081.701.37\u20132.121.250.99\u20131.58a\u03c72bCox regressionHR = hazard ratio, CI = 95% confidence intervals, EG = esophagogastric\nThe 1-, 3-, and 5-year survival of the pN0 group was 78, 51, and 44%, respectively (Fig.\u00a01). Where one node was involved, survival was 67, 41, and 35%, respectively. Where two to three nodes were involved, the 1-, 3-, and 5-year survival was 57, 25, and 13%, respectively, and where greater than three nodes were involved, this was 40, 14, and 8%, respectively.\nFigure\u00a01Overall survival by number of nodes positive.\nUnivariate analysis (Table\u00a03) revealed nodal status, pT stage, pathologic stage, and R status as predictors of survival. Multivariate analysis revealed nodal status alone to significantly (p\u2009<\u20090.0001) impact on survival. By this analysis the hazards ratio increased from 1.08 for one involved node to 1.42 for two to three involved nodes, and 1.84 for greater than three nodes.\nExcluding node-negative patients, univariate analysis (Table\u00a04) revealed pT stage, pathologic stage, R status, and number of nodes as predictive of survival. By multivariate analysis (Table\u00a05), pathologic stage (p\u2009=\u20090.010) and number of nodes were significant determinants of survival. Compared with the cohort with one involved node, the hazard ratio for two to three nodes was 1.56 (p\u2009=\u20090.049) and 2.06 (p\u2009=\u20090.007) for greater than three nodes. \nTable\u00a04Univariate Analysis: Node-positive AloneVariablesNo. of PatientsMedian Survival (moths)p valuea (Univariate)HR95% CITreatmentSurgery only241110.23410.63\u20131.11Multimodal74110.84Tumor siteUpper esophagus9180.6501Middle esophagus32100.5561.310.54\u20133.18Lower esophagus130100.1831.750.77\u20133.98OG junction144120.3501.480.65\u20133.36Depth of invasionT05110.0011T11280.9171.060.33\u20133.41T246240.1761.120.43\u20131.78T3235110.7571.430.74\u20132.14T41450.1572.230.74\u20136.78HistologySquamous86110.6381Adenocarcinoma221110.6381.070.81\u20131.40Other830.8481.070.49\u20132.35Stage1\u2013II6319<0.0011III\u2013IV251102.011.43\u20132.83Residual tumorR0259120.0351R16191.331.02\u20131.73No. of nodes18417<0.00112\u2013384130.0211.671.06\u20132.29>31479<0.0012.531.50\u20133.62a\u03c72HR = hazard ratio, CI = 95% confidence intervalsTable\u00a05Mutivariate Analysis: Node-positive OnlyVariablesp valuea (Multivariate)HR95% CIDepth of invasionT01T10.5440.820.31\u20131.75T20.6791.230.74\u20131.81T30.3131.490.99\u20132.21T40.2021.831.39\u20133.24StageI\u2013II0.0101III\u2013IV1.590.82\u20133.06No. of nodes112\u201330.0491.561.21\u20132.35>30.0072.061.51\u20132.82Residual tumorR00.2831R11.220.80\u20131.79aCox regressionHR = hazard ratio, CI = 95% confidence intervals\nDiscussion\nCancers of the esophagus and esophagogastric junction are aggressive tumors, which are typically diagnosed at an advanced stage of disease progression.13 This large retrospective review of a tertiary center\u2019s experiences over 12\u00a0years highlights the importance of lymph node involvement in the prognosis of these tumors. The study shows that the presence of a solitary node, although a significantly negative factor compared with pN0 disease, is associated with significantly improved median and 1-, 3-, and 5-year survival compared with cohorts of patients with greater nodal involvement. The 5-year survival, for instance, was 35% compared with 13 and 8%, respectively, for cohorts with two to three positive nodes and greater than three positive nodes.\nThere is no uniform consensus on the number of lymph nodes that must be sampled. In a study by Ito et al.,3 the median number of lymph nodes examined per specimen was 6 (range 0 to 35) and only 20% of patients had at least 15 lymph nodes examined. In this study, the median number of lymph nodes examined per specimen was 12 (range 6 to 55), and 24% of the patients had at least 15 lymph nodes examined. These results appear consistent with practice in the United States where an analysis of the National Cancer Database indicated that only 18% of patients undergoing surgery for gastric cancer have more than 15 lymph nodes analyzed.14 In this Unit, lymph node clearance involves a D2 dissection of abdominal nodes, and wide mediastinal clearance to the carina and paratracheal node dissection if they appear involved. No cervical dissection is performed, consistent with recommendations from another group.15 It is acknowledged that variation in lymph node yield may mask stage migration, particularly in a retrospective analysis, but the standardization of lymphadenectomy is likely to minimize the impact of this potential bias.\nThe association between extent of nodal involvement and outcome is well described.16\u201318 No study to our knowledge has previously focused on the impact of one positive node on outcome in esophageal cancer. The observation, however, of the unique prognostic significance of a solitary involved node was recently reported.19 In a study of 187 patients with esophageal adenocarcinoma treated with neoadjuvant chemoradiotherapy, Gu et al.19 at the MD Anderson observed from their analysis that patients with a solitary involved node had better overall and relapse-free survival compared with other nodal groups. Moreover, the 5-year survival outcomes and 2-year relapse-free survival was not significantly different from the node-negative cohort. Although in our series survival figures were better for node-negative patients than patients with a solitary involved node, the overall pattern of outcome data in our series is consistent with the report from the Anderson group, with prognosis in this cohort closer to node-negative than other node-positive subgroups.\nThe clinical implication of this finding is not clear at this time, but it should, at minimum, encourage a more optimistic view of patients who have a solitary lymph node identified after adequate lymphadenectomy, as approximately 35% of patients with this pathologic stage may be cured. In the future, it is possible that advances in endoscopic US staging, fluorodeoxyglucose PET, and sentinel node assessment may improve pre- and intraoperative assessment of nodal involvement, defining node-negative, solitary involved node and micrometastatic-involved subgroups, and selective lymphadenectomy and minimally invasive approaches may be evaluated in these situations. This demands prospective evaluation, but it may be noteworthy that all involved nodes in the solitary involved node cohort were close to the primary site and may possibly have been identified as sentinel nodes.\nIn conclusion, this study shows that in a large cohort of patients, lymph node status and the number of lymph nodes positive at the time of surgical resection is directly linked to survival. Extensive nodal involvement is confirmed as carrying a dismal prognosis, but greater optimism is justified where a solitary involved lymph gland defines the pN stage after an adequate lymphadenectomy.","keyphrases":["lymph node","survival","lymphadenectomy","esophagectomy"],"prmu":["P","P","P","P"]}
{"id":"Diabetologia-4-1-2292423","title":"Increasing overall physical activity and aerobic fitness is associated with improvements in metabolic risk: cohort analysis of the ProActive trial\n","text":"Aims\/hypothesis Our aim was to examine the association between change in physical activity energy expenditure (PAEE), total body movement (counts per day) and aerobic fitness (maximum oxygen consumption []) over 1 year and metabolic risk among individuals with a family history of diabetes.\nIntroduction\nThe metabolic syndrome has been defined as a cluster of closely related cardiovascular risk factors including visceral obesity, insulin resistance, hyperglycaemia, hypertension, hypertriacylglycerolaemia and low HDL-cholesterol [1]. The combination of these risk factors has been shown to predict type 2 diabetes, cardiovascular disease and all-cause mortality [2], with physical inactivity identified as a major underlying risk factor. Given the rising prevalence of obesity and type 2 diabetes, the metabolic syndrome represents a potentially large public health burden [3, 4]. Previous research has shown that low levels of physical activity are associated with the metabolic syndrome [5, 6], and that objectively measured physical activity energy expenditure (PAEE) predicts progression towards the metabolic syndrome independently of obesity [7], with some evidence of interaction by cardiorespiratory fitness [8, 9]. Additionally, lifestyle interventions targeting physical activity as a modifiable risk factor have shown positive effects upon various metabolic components [10]. However, physical activity remains poorly measured in most epidemiological studies, with many relying on self-reporting [11]. Furthermore, it is unclear whether metabolic risk is most effectively reduced by increases in overall energy expenditure (EE), fitness or total body movement. A better understanding of these relationships would inform the development and targeting of individual and population-based interventions to reduce risk of diabetes and related metabolic disorders.\nThe ProActive (UK) trial is an explanatory trial of a theory-based intervention to promote physical activity among individuals reporting low levels of activity and who are at increased risk of the consequences of a sedentary lifestyle due to their family history of type 2 diabetes. The main trial results are presented elsewhere [12]. We recently observed a cross-sectional association between total body movement and clustered metabolic risk in this cohort [13] and are now extending these observations to examine the association between change in objectively measured PAEE (measured by individually calibrated heart rate [HR] monitoring), physical activity (total body movement measured by accelerometry) and aerobic fitness and clustered metabolic risk in the ProActive trial cohort over a period of 1\u00a0year.\nMethods\nThe ProActive trial Full details of the study have been reported elsewhere [14]. In brief, ProActive aimed to evaluate the efficacy of a theoretical, evidence- and family-based intervention programme to increase physical activity among individuals defined as high-risk through having a parental history of type 2 diabetes. Potential participants were identified via diabetes registers and medical records of family history in 20 general practices in the East Anglia region of the UK. Three hundred and sixty-five individuals aged 30\u201350\u00a0years and reporting low levels of activity were randomly assigned to one of three interventions: brief written advice (control group), or a behavioural-change programme at two levels of intensity delivered either by telephone (distance) or face-to-face in the family home. The theory-based intervention programme [15] was delivered by trained facilitators and aimed to support increases in physical activity through the introduction and facilitation of a range of self-regulatory skills. Main trial results indicated no significant difference in the 1\u00a0year change in objectively measured daytime physical activity expressed as a ratio to resting EE between the three trial arms [12]. Consequently, the three trial arms were pooled and a cohort analysis conducted. Complete data on PAEE, aerobic fitness, anthropometry and biochemistry were available in 365 participants at baseline and 321 participants at follow-up, in addition to socio-demographic information. Total body movement measured by accelerometry was also assessed in a subsample of participants (n\u2009=\u2009192) at baseline and follow-up and the data were included in these analyses. The measure of socioeconomic status (SES) was based on age at finishing full-time education (above or below 16\u00a0years). Ethics approval was obtained from the Eastern England Multi-centre Research Committee (MREC) and all participants gave written informed consent.\nAnthropometric and metabolic tests Participants attended the study centre after an overnight fast and a sample of venous blood was taken. Fasting plasma glucose, serum insulin levels and lipid profiles (cholesterol, triacylglycerol, HDL- and LDL-cholesterol) were measured using the hexokinase method and standard enzymatic methods, as described previously [14]. Weight was measured on standard calibrated scales and height was measured using a rigid stadiometer. BMI was calculated as weight (kg) divided by height (m) squared. Waist circumference (cm) was measured over light indoor clothing as the mid-point between the lower costal margin and the level of the anterior superior iliac crests. Body fat percentage was measured by bio-electrical impedance (Bodystat, Isle of Man, UK), and systolic and diastolic blood pressures were measured using an automated Accutorr sphygmomanometer (Accutorr, Cambridge, UK).\nAssessment of aerobic fitness, PAEE and total body movement Aerobic fitness (maximum oxygen consumption []) was predicted as oxygen uptake at maximal HR (220 minus age [years]) by extrapolation of the regression line established during the individual calibration for the relationship between oxygen consumption and HR during a submaximal graded treadmill exercise test. O2 uptake and CO2 production was measured continuously by indirect calorimetry throughout the test (Vista XT metabolic system; Vacumed, Ventura, CA, USA).Resting EE (REE) was calculated by the Weir formula [16] using  and  measurements obtained in the fasting state, after approximately 10\u00a0min of supine rest using the same indirect calorimetry system as previously described. PAEE was measured using the flex HR method [8]. Flex HR was calculated as the mean of the highest resting HR and the lowest HR while exercising. This point was used in the analysis of free-living minute-by-minute HR data to discriminate between rest and exercise. Below the flex HR point, EE was assumed to be equivalent to REE. EE above the flex point was predicted from the individual HR\u2013EE regression line. Participants wore HR monitors (Polar Electro, Kemple, Finland) continuously during the waking hours over the following 4\u00a0days. PAEE was calculated by subtracting REE from the estimated average daily EE, and thereafter averaged over the 4\u00a0day period. PAEE is body size dependent, e.g. it requires more energy to move a heavier body. Similarly, aerobic fitness is highly correlated with body size or mass, e.g. larger individuals have a higher absolute aerobic capacity. Therefore, body size is a confounding factor that needs to be normalised for when analysing associations between EE variables and metabolic outcomes [17]. As such, both baseline and follow-up measures of aerobic fitness and PAEE were expressed relative to fat-free mass (FFM; kJ kg\u22121 FFM min\u22121) to adjust for between-individual differences in body size [17, 18].Free-living total body movement was assessed with an MTI Actigraph (formerly known as the CSA activity monitor) model WAM7164 (Manufacturing Technology, Fort Walton Beach, FL, USA). The accelerometer was worn over the same 4\u00a0day period as the HR monitor. Participants who did not manage to record at least 500\u00a0min\/day of activity for at least 3\u00a0days were excluded from analyses. A customised program was used for data reduction and further analyses (MAHUffe; http:\/\/www.mrc-epid.cam.ac.uk). The outcome variable derived from accelerometry presented in this study is total body movement (counts per day), adjusted for monitored time, which is an indicator of the overall level of physical activity.\nCalculation of the metabolic syndrome z score A summary score of clustered metabolic risk based on WHO criteria [1] was calculated by summing standardised values for waist circumference, triacylglycerol, fasting insulin and glucose, systolic blood pressure and the inverse of HDL-cholesterol [7\u20139]. Variables were standardised by subtracting the sample mean from the individual mean and dividing by the SD. Baseline and follow-up z scores were computed with the same transformation, e.g. using the mean and SD of baseline values. This continuously distributed metabolic risk variable (zMS) was also calculated without the adiposity component (i.e. waist circumference, zMS-ob). This clustered metabolic risk score increases statistical power as variables were not dichotomised, and includes a continuous measure of glycaemia (unlike some other measures of vascular risk [19]). Additionally, the International Diabetes Federation criteria were used to define metabolic syndrome as a dichotomous variable [1]. This score predicts hard clinical endpoints [20].\nStatistical analyses Descriptive summary statistics were calculated separately for men and women using means and SDs at baseline and follow-up. t tests were used to examine whether there were any differences in baseline characteristics between those with and without follow-up data, as well as those with missing accelerometer data. Fasting insulin and triacylglycerol values were log transformed (ln) due to their non-normal distribution. In order to characterise change in physical activity and fitness in the ProActive cohort, we described the association between exposure variables using correlation coefficients. We then examined the proportion of metabolic syndrome and the mean clustered metabolic risk score among participants who increased and decreased their PAEE, fitness and total body movement over 1\u00a0year. We used linear regression to model change in PAEE and fitness (both per unit FFM), and total body movement ([total counts per day]\/1,000, adjusted for monitored time), against individual subcomponents of the clustered metabolic risk score at follow-up (waist circumference, blood pressure, fasting triacylglycerol, insulin and glucose, and the inverse of HDL-cholesterol). These models were adjusted for age, sex, waist circumference (except when waist circumference was modelled as the outcome), smoking status, SES (assessed by self-reporting) and baseline phenotype, and were presented as standardised \u03b2-coefficients. We then tested whether change in PAEE, fitness and total body movement was associated with the clustered metabolic risk score at follow-up. The first model (obesity dependent) included all metabolic subcomponents (zMS) and was adjusted for age, sex, smoking status, SES and baseline zMS, while the second model excluded waist circumference from the zMS (obesity independent zMS-ob) and was adjusted for age, sex, smoking status, SES, baseline zMS-ob and waist circumference. Finally, we examined whether there was evidence of interaction between change in PAEE, total body movement and aerobic fitness and clustered metabolic risk, as well as interaction by age and sex. All data were analysed in continuous form, although some data were dichotomised for illustrative purposes. All analyses were completed using Stata Version 8.0. (STATA, College Station, TX, USA).\nResults\nTable\u00a01 shows the anthropometric and metabolic characteristics of participants with complete data for baseline and follow-up (n\u2009=\u2009321) stratified by sex. More women met the inclusion criteria and agreed to take part in the study than men. Participants with missing data at follow-up were slightly shorter than those with complete data (difference\u2009=\u20093.1\u00a0cm, p\u2009<\u20090.05) but not significantly different for all other baseline variables. There were also no significant differences in baseline characteristics between participants with and without accelerometer data (data not shown). In general, men were significantly taller and heavier than women but had a lower percentage body fat at baseline. In terms of the six metabolic syndrome subcomponents, waist circumference, systolic blood pressure, triacylglycerol, fasting glucose and insulin were all significantly higher in men, while HDL-cholesterol was significantly higher in women. Statistically significant differences were also observed for PAEE (kJ kg\u22121 FFM min\u22121) and  (ml kg\u22121 FFM min\u22121), with men achieving higher levels of physical activity and fitness even after adjustment for FFM. There were no significant differences in total body movement between men and women at baseline.\nTable\u00a01Anthropometric and metabolic characteristics of ProActive participants with complete baseline and follow-up data (n\u2009=\u2009321), stratified by sex\u00a0MenWomenBaseline (n\u2009=\u2009129)Follow-up (n\u2009=\u2009129)Baseline (n\u2009=\u2009192)Follow-up (n\u2009=\u2009192)Age (years)40.2 (5.8)N\/A40.8 (6.1)N\/AHeight (cm)177.8 (6.8)177.8 (7.0)163.4 (6.1)a163.2 (5.9)cWeight (kg)89.3 (15.5)89.5 (15.6)73.7 (14.6)a74.0 (15.2)BMI (kg\/m2)28.2 (4.3)28.3 (4.4)27.6 (5.2)27.8 (5.4)dBody fat (%)25.6 (5.3)25.8 (5.2)34.6 (6.9)a34.8 (7.1)Waist circumference (cm)100.3 (11.1)100.9 (11.5)87.9 (11.8)a89.2 (12.5)cSystolic blood pressure (mmHg)127.1 (11.3)124.5 (11.3)d120.6 (13.8)a116.8 (13.3)cDiastolic blood pressure (mmHg)81.4 (8.8)79.6 (9.7)d76.2 (9.3)a73.9 (9.9)cTriacylglycerol (mmol\/l)1.8 (1.6)1.7 (1.3)1.2 (0.6)a1.1 (0.7)Total cholesterol (mmol\/l)5.2 (1.0)5.3 (1.0)5.1 (0.9)5.2 (1.0)HDL-cholesterol (mmol\/l)1.2 (0.3)1.2 (0.3)1.6 (0.4)a1.6 (0.4)Fasting plasma insulin (mmol\/l)70.2 (71.6)72.2 (52.3)55.0 (35.6)b56.3 (35.4)Fasting plasma glucose (mmol\/l)5.1 (0.9)5.2 (1.0)4.7 (0.5)a4.8 (0.5)dPAEE (kJ kg\u22121 FFM min\u22121)0.13 (0.07)0.14 (0.08)d0.10 (0.08)b0.11 (0.07) (ml kg\u22121 FFM min\u22121)60.7 (10.54)61.2 (11.42)57.10 (10.72)b58.06 (10.38)Daily physical activity (total count)e265,000 (79,000)275,000 (106,000)269,000 (109,00)280,000 (110,000)Data are means (SD)ap\u2009<\u20090.001, bp\u2009<\u20090.05 for women vs men at baseline; cp\u2009<\u20090.001, dp\u2009<\u20090.05 for baseline vs follow-up (separately in men and women)eThese values are based on data from n\u2009=\u2009192 ProActive participants for whom accelerometer data were available at baseline and follow-up\nFor men, there were no significant differences in mean height, weight, BMI, body fat, waist circumference, triacylglycerol, HDL-cholesterol, fasting insulin and glucose from baseline to follow-up. The only metabolic subcomponent that changed significantly over time was systolic blood pressure, which reduced from 127.1 to 124.5\u00a0mmHg. In terms of the activity variables, men significantly increased their physical activity (PAEE) from 0.125 to 0.139\u00a0kJ kg\u22121 FFM min\u22121, but increases in fitness and total body movement did not achieve statistical significance. For women, there were no significant differences in mean weight, body fat, triacylglycerol, total cholesterol, HDL-cholesterol and fasting insulin from baseline to follow-up. Mean height decreased very slightly, while BMI increased from 27.6 to 27.8\u00a0kg\/m2 and waist circumference increased from 87.9 to 89.2\u00a0cm. Values for both diastolic and systolic pressure decreased significantly, while fasting plasma glucose increased slightly from 4.7 to 4.8\u00a0mmol\/l. Increases over the year in PAEE, total body movement and fitness did not reach statistical significance. There was no significant change in the use of cardiovascular therapies during follow-up (data not shown).\nThere was no evidence for a correlation between change in fitness and change in PAEE (r\u2009=\u20090.1) or total body movement (r\u2009=\u20090.0). There was limited evidence for a positive correlation between change in PAEE and total body movement (r\u2009=\u20090.3). The overall prevalence of metabolic syndrome was 20% at both baseline and follow-up, although there was some movement between groups. The proportion of metabolic syndrome at follow-up was higher among participants who failed to increase their PAEE, fitness and total body movement over 1\u00a0year (Fig.\u00a01a). Similarly, clustered metabolic risk scores were lower among individuals who managed to increase their PAEE, fitness and total body movement during follow-up (Fig.\u00a01b). None of the differences between these groups reached statistical significance but were in the expected direction of effect.\nFig.\u00a01a Proportion of ProActive participants with metabolic syndrome at 1\u00a0year follow-up by those who increased and decreased their PAEE, fitness and total body movement. b Mean metabolic summary score for ProActive participants at 1\u00a0year follow-up by those who increased and decreased their PAEE, fitness and total body movement (bars represent 95% CIs)\nTable\u00a02 shows the crude and adjusted associations between change in PAEE (kJ kg\u22121 FFM min\u22121),  (ml kg\u22121 FFM min\u22121) and total body movement ([counts per day]\/1,000) with standardised subcomponents of the clustered metabolic risk score and the whole score (zMS) at follow-up. As all outcomes are expressed in the same unit (SD), it is possible to directly compare the magnitude of associations between the different components of activity with each of the outcomes, assuming they are measured with the same degree of measurement error. In adjusted analyses, fasting glucose, insulin and HDL-cholesterol at follow-up were significantly associated with a change in fitness over time; on average, individuals who increased their fitness had reduced levels of glucose and insulin, and increased levels of HDL-cholesterol at follow-up. Similarly, fasting glucose and insulin were associated with change in total body movement over time. All other subcomponents of the clustered metabolic risk score failed to show an association with change in either physical activity, fitness or total body movement. In adjusted analyses, change in fitness, but not change in PAEE, was associated with clustered metabolic risk at follow-up with (zMS; p\u2009=\u20090.034) and without the obesity component (zMS-ob; p\u2009=\u20090.04). In addition, change in total body movement from baseline to follow-up was associated with clustered metabolic risk in both the overall (zMS; p\u2009=\u20090.004) and obesity-independent score (zMS-ob; p\u2009=\u20090.01).\nTable\u00a02Crude and adjusted associations (standardised \u03b2-coefficients) between change in PAEE,  and total body movement with standardised subcomponents of the metabolic summary score and the whole score (zMS) at follow-up in the ProActive cohort over 1\u00a0yearOutcome (SD)Standardised \u03b2-coefficient (95% CI)Change in PAEE (n\u2009=\u2009318)Change in  (n\u2009=\u2009318)Change in total body movement (n\u2009=\u2009191)CrudeAdjustedaCrudeAdjustedaCrudeAdjustedaWaist circumference\u22120.061 (\u22120.172, 0.051)\u22120.025 (\u22120.065, 0.016)\u22120.074 (\u22120.185, 0.037)\u22120.017 (\u22120.058, 0.024)\u22120.117 (\u22120.261, 0.027)\u22120.019 (\u22120.071, 0.032)Systolic blood pressure0.062 (\u22120.047, 0.171)0.074 (\u22120.004, 0.152)\u22120.017 (\u22120.126, 0.093)\u22120.035 (\u22120.113, 0.044)\u22120.017 (\u22120.158, 0.125)0.012 (\u22120.094, 0.118)Logged triacylglycerol\u22120.008 (\u22120.127, 0.110)\u22120.024 (\u22120.100, 0.051)\u22120.059 (\u22120.178, 0.060)\u22120.009 (\u22120.084, 0.067)\u22120.026 (\u22120.180, 0.128)\u22120.054 (\u22120.149, 0.041)Logged fasting insulin\u22120.064 (\u22120.175, 0.046)\u22120.024 (\u22120.093, 0.045)\u22120.094 (\u22120.203, 0.016)\u22120.069 (\u22120.137, \u22120.002)b\u22120.097 (\u22120.239, 0.045)\u22120.101 (\u22120.186, \u22120.017)bFasting plasma glucose\u22120.005 (\u22120.133, 0.123)\u22120.024 (\u22120.114, 0.067)\u22120.139 (\u22120.266, \u22120.012)b\u22120.090 (\u22120.180, 0.000)b\u22120.163 (\u22120.328, 0.002)\u22120.147 (\u22120.259, \u22120.035)bHDL-cholesterol (inverse)\u22120.053 (\u22120.165, 0.059)\u22120.056 (\u22120.125, 0.014)\u22120.115 (\u22120.227, \u22120.004)b\u22120.097 (\u22120.166, \u22120.029)b\u22120.027 (\u22120.167, 0.113)\u22120.026 (\u22120.121, 0.069)zMS\u22120.031 (\u22120.112, 0.050)\u22120.017 (\u22120.054, 0.020)\u22120.081 (\u22120.160, \u22120.001)b\u22120.056 (\u22120.093, \u22120.020)b\u22120.074 (\u22120.176, 0.028)\u22120.066 (\u22120.111, \u22120.021)bzMS-ob\u22120.023 (\u22120.103, 0.058)\u22120.012 (\u22120.053, 0.029)\u22120.083 (\u22120.163, \u22120.004)b\u22120.062 (\u22120.102, \u22120.022)b\u22120.065 (\u22120.166, 0.037)\u22120.068 (\u22120.119, \u22120.016)baAll coefficients adjusted for age, sex, smoking status, SES, waist circumference and baseline phenotype, except for waist circumference, which was adjusted for age, sex, smoking status, SES and baseline phenotypebp\u2009<\u20090.05\nThere was no evidence of interaction between change in EE and aerobic fitness (p\u2009=\u20090.28), EE and total body movement (p\u2009=\u20090.13) and aerobic fitness and total body movement (p\u2009=\u20090.13) with clustered metabolic risk at follow-up. While there was no evidence of a significant interaction between age or sex and change in the activity variables, the \u03b2-coefficients for change in PAEE appeared stronger in men than in women, and also in older rather than younger individuals.\nDiscussion\nOur results suggest that small increases in physical activity, measured using accelerometry and aerobic fitness, were associated with improvement in clustered metabolic risk over a 1\u00a0year period in middle-aged individuals at high risk of developing type 2 diabetes. Our results were independent of age, sex, smoking status, socioeconomic group and baseline phenotype. This confirms our earlier cross-sectional result [13] in a prospective analysis, which provides stronger inferential evidence for the association between total body movement and metabolic risk. Further, this result is supported by our finding that the proportion of metabolic syndrome at follow-up was higher, albeit non-significantly, among groups who failed to increase their PAEE, fitness and total body movement over 1\u00a0year.\nAlthough PAEE was not significantly associated with clustered metabolic risk in our analyses, we were able to demonstrate an association between overall body movement (i.e. physical activity) assessed by accelerometry and clustered metabolic risk. This is a novel finding given that previous prospective associations between objectively measured physical activity and metabolic risk were observed in a population of healthy middle-aged Europids [7], rather than the overweight, sedentary younger adults with a high risk of developing type 2 diabetes described in this cohort. We also observed that the benefits of increased fitness and total body movement acted primarily through changes in serum glucose, insulin and HDL-cholesterol, which may suggest that blood pressure is less sensitive to changes in activity and fitness. In a comparable study, Ekelund et al. [21] observed that an increase in activity was associated with fasting insulin, triacylglycerol, 2\u00a0h glucose and clustered metabolic risk but not with systolic or diastolic blood pressure. Contrary to our findings, previous literature has shown an association between PAEE and clustered metabolic risk, both cross-sectionally [8] and prospectively over a period of 5.6\u00a0years, independently of fitness [7]. This apparent discrepancy might be explained in a number of ways. First, while the trial was powered to detect a difference in physical activity-related EE between trial arms equivalent to 2 metabolic energy equivalent (MET) h\/day or approximately 30\u00a0min of brisk walking, the sample size was relatively small for a cohort analysis and may not have been large enough to detect smaller but still biologically important differences. Nonetheless, all the PAEE \u03b2-coefficients were in the same direction of effect as those for the accelerometry data but they did not reach statistical significance.\nSecond, the precision in the methods we used to assess PAEE and body movement may affect the observed associations. PAEE from individually calibrated HR monitoring is an integrated measure of EE above rest calculated from free-living HR data. In sedentary populations, much of the daytime is spent in the region around the flex HR, which is used to discriminate between rest and physical activity. The association between HR and EE is less precise in this region, which may influence the accuracy of predicted PAEE on an individual level. Furthermore, the flex HR method may be sensitive to the fitness level of the people under investigation [22]. In contrast, accelerometry measures the vertical accelerations of the body, i.e. physical work, and is likely to be less sensitive to the characteristics of the population in terms of a sedentary lifestyle and fitness level. If most of the activities performed are locomotor activities, such as walking, measurement of body movement by accelerometry may be superior to HR monitoring when examining associations with disease risk. Ideally, the two different measurement techniques should be combined into one single piece instrument [23].\nFinally, by standardising the clustered metabolic risk score, we assumed each component made an equal contribution towards defining metabolic risk. It is unlikely that each component is equally strongly associated with metabolic risk and some variables will be more important that others in different populations. It would therefore be ideal to weight each component of the score, but data are currently unavailable.\nOur results suggest that small, feasible changes in physical activity (total body movement) and fitness in an at-risk population may prevent progression towards the metabolic syndrome. This supports previous literature, which has demonstrated an inverse relationship between fitness, physical activity and risk of developing metabolic syndrome in a number of settings [5, 24\u201326]. At a population level, our findings have important implications. This group of middle-aged, slightly overweight individuals, identified through primary care registers via their first degree family history of diabetes, represents an accessible population who might benefit from increased physical activity and fitness levels. Primary care or public health practitioners might target this group for preventive action. Findings can also be extrapolated to inform the characteristics of public health initiatives. Increasing total body movement can be achieved through small changes in lifestyle activity, such as taking the stairs and parking the car further away from work, which may be more palatable to sedentary and at-risk populations than targeting changes in moderate to vigorous intensity activities. Regular physical activity participation also has multiple positive effects upon other diseases in addition to metabolic syndrome, such as stroke, coronary heart disease and cancer [27, 28]. However, currently there is limited evidence regarding the effectiveness of lifestyle interventions for individuals, at-risk groups or populations in the treatment and prevention of metabolic syndrome and this is an area of research that warrants further investigation. It is important to clarify whether metabolic risk is most effectively reduced by increases in overall EE, fitness or total body movement to inform the development and nature of preventive interventions.\nThis study had several methodological strengths. The ProActive trial allowed objective measurement of several components of physical activity and fitness over a period of 12\u00a0months in a well-defined group of individuals accessed through primary care who were at risk of diabetes. There was a high follow-up rate (88%) and standardised measures were used throughout. We used objective measures of physical activity, which reduces the error and bias commonly associated with self-report measures and our objective measures of EE, physical activity (PAEE, total body movement) and fitness have been extensively validated in the laboratory and during free-living conditions [18].\nIn conclusion, small increases in physical activity assessed by accelerometry and in aerobic fitness were associated with improved metabolic risk over 1\u00a0year. Further research is needed to clarify the relationship between physical activity, EE, fitness and metabolic risk to inform advice and intervention development. We may need even more precise methods of measurement to capture the true dose effect of such relationships.","keyphrases":["physical activity","fitness","metabolic risk","metabolic syndrome","cardio-respiratory fitness","syndrome x"],"prmu":["P","P","P","P","M","M"]}
{"id":"Oecologia-4-1-2270366","title":"Scaling of offspring number and mass to plant and animal size: model and meta-analysis\n","text":"The scaling of reproductive parameters to body size is important for understanding ecological and evolutionary patterns. Here, we derived allometric relationships for the number and mass of seeds, eggs and neonates from an existing model on population production. In a separate meta-analysis, we collected 79 empirical regressions on offspring mass and number covering different taxa and various habitats. The literature review served as a validation of the model, whereas, vice versa, consistency of isolated regressions with each other and related ecological quantities was checked with the model. The total offspring mass delivered in a reproductive event scaled to adult size with slopes in the range of about 3\/4 to 1. Exponents for individual seed, egg and neonate mass varied around 1\/2 for most heterotherms and between 3\/4 and 1 for most homeotherms. The scaling of the progeny number released in a sowing, clutch or litter was opposite to that of their size. The linear regressions fitted into a triangular envelope where maximum offspring mass is limited by the size of the adult. Minimum seed and egg size scaled with weight exponents of approximately 0 up to 1\/4. These patterns can be explained by the influence of parents on the fate of their offspring, covering the continuum of r-strategists (pelagic\u2013aquatic, arial, most invertebrates, heterotherms) and K-strategists (littoral\u2013terrestrial, some invertebrates, homeotherms).\nIntroduction\nScaling of processes and patterns to body size has fascinated biologists for centuries (e.g., Peters 1983; Damuth 2007). Plant and animal characteristics have been correlated to organisms\u2019 body mass (m) by simple scaling relationships of the form a\u00b7mb or, if log transformed, log(a)\u00a0+\u00a0b\u00a0log(m). Properties include various reproductive parameters, such as the mass and number of the offspring, important for understanding life history strategies of species. The scaling of reproduction reflects several important trade-offs, e.g., investment of energy in somatic versus gonadic growth, in continuous or intermittent release and in many small or a few large offspring.\nStudies have derived empirical regressions for offspring mass and number within one taxon (see review by Peters 1983). In addition, several partly contradictive physical, biochemical and ecological constraints related to parental energy investment, have been hypothesized to be responsible for the observed patterns (May 1978; Gordon 1989; Reiss 1989; Charnov and Downhower 1995; Hendry et al. 2001; Charnov 2001, 2002; Aarssen 2005). Although the trade-offs in reproductive investment reflect universal dilemmas that every species has to face, most work so far has covered either several progeny characteristics within a single species group or a single offspring parameter within at most two taxa (Moles et al. 2005a; Grubb et al. 2005).\nIn the present paper, we will focus on the relationship of offspring mass and number to adult weight for miscellaneous groups of species. The aim is to obtain a set of straightforward equations that are coherent with allometric theory and underpinned by empirical regressions available in literature. To this end, we will first derive body-size functions for the mass and number of offspring delivered in a reproductive event which comply with allometric macroecology (e.g., West et al. 1997; Hendriks 1999). Next, we will compare the acquired relationships to independent empirical regressions collected in a meta-analysis. Separating model development and testing allows for a comparison of theoretical and empirical evidence, reducing weaknesses of both. In this approach, model predictions of, e.g., offspring mass, are underpinned by independent measurements. Likewise, isolated empirical regressions for this reproductive parameter are checked for consistency with body-size dependence of related ecological factors, such as population production or survival.\nIn our analysis, we will seek a balance between model transparency and accuracy, needed for underpinning assumed mechanisms and identifying exceptions. The equations are not intended to cover detailed or speculative differences, observed in empirical regressions for sufficient taxa. In our interpretation, we will follow the classical distinction between r-strategists and K-strategists (MacArthur and Wilson 1967; Pianka 1970) as well as more recent applications thereof (Winemiller and Rose 1992; McCann and Shuter 1997; Cox and Moore 2005). However, it should be emphasized that the traditional classification of many small offspring versus few large offspring as used in the present paper is no more than a convenient way to structure the discussion.\nMaterials and methods\nRegression collection and treatment\nAllometric regressions derived from data before 1983 were collected from an extensive review (Peters 1983). Papers published afterwards were obtained by screening books and by an electronic literature search in Web of Science in 2006. All body-size correlations acquired for the meta-analysis were checked in the original papers. Collection and treatment of data by the authors was assumed to be adequate. Where multiple types of regressions were reported, we took the least-squares equations, to increase compatibility with the other studies. If regression characteristics were not given or suspected to be wrong, we derived them from the original data or digitized graphs.\nTo allow for comparison, all values were converted to fresh biomass, using dry\u2013wet weight fractions. A database containing dry matter fractions of 110 species, collected by the present authors for various purposes, yielded averages (with 95% confidence intervals) of 20% (18\u201322%) for herbs, 22% (19\u201325%) for invertebrates, 25% (18\u201332%) for cold-blooded vertebrates and 30% (21\u201339%) for warm-blooded vertebrates. In addition, the literature reports means of 53% for wood, 15\u201321% for fruit, 87% for seeds, 23 for insects, 19% for marine invertebrates, 25% for fish and 29% for birds and mammals (Peters 1983; Suzuki 1999). Although the samples may be somewhat biased towards particular taxa, average levels do not deviate substantially. In the present analysis, we therefore used dry\u2013wet weight fractions of 20% for fruits, forbs, herbs and invertebrates, of 25% for cold-blooded vertebrates, of 30% for warm-blooded vertebrates, of 40% for shrubs and trees (wood and leaves together) and of 80% for seeds. Differences between species around these values have, on average, a negligible influence on the intercept of the regressions. Even more apparent, conversion does not affect the slope of the allometric relationships. Seeds mass was multiplied by 80\/20%\u00a0=\u00a04 to arrive at the fresh weight of sprouts with a water content that is comparable to that of eggs and neonates. Since total plant weight is not used as the independent variable for vegetation regressions, we took the closest alternatives, being the above-ground or stem mass. In one study on insects, animal size was expressed on a length basis instead of a weight basis (Berrigan 1991). For these regressions, length (m) was converted to weight (kg) as mass\u00a0=\u00a01\/152\u03c0\u00a0\u00d7\u00a0103\u00a0length3, assuming that the total size equals 1.5 times the elythra length and twice the abdomen length.\nRate constants for reproduction of plants were reported as annual standing reproductive mass delivered by all plants, including both reproducing and non-reproducing individuals. We assumed that the seed and fruit mass are renewed each year. For animals, total clutch mass reflected the total reproductive mass released in an event or present in ovaries, sometimes calculated in the original studies as a product of the total number of eggs (neonates) in a clutch (litter), the number of eggs per clutch (neonates per litter) and the egg (neonate) weight.\nTo allow for comparison between plants, invertebrates, cold-blooded vertebrates and warm-blooded vertebrates, we calculated the arithmetic average of all slopes (b) reported for each taxonomic group. To correct for differences in the amount of information included in the regressions, we also computed an average weighted by the number of data. For instance, the weighted average of a regression with b\u00a0=\u00a00.5 and n\u00a0=\u00a010 and another with b\u00a0=\u00a00.3 and n\u00a0=\u00a020 was calculated to be 0.5\u00a0\u00d7\u00a010\/30\u00a0+\u00a00.3\u00a0\u00d7\u00a020\/30\u00a0=\u00a00.37. As we are interested in both the absence and presence of links to organism mass, weak and insignificant correlations were included in the analysis as well. Where used, however, they will be explicitly mentioned in the text.\nModel development\nThe relationship of offspring mass and size to adult weight can also be derived theoretically from previous modeling efforts (Hendriks 2007). The starting point in this model is production because coefficients and slopes of allometric regressions for this parameter differ only slightly for various groups of species. The rate constant for population turnover (production kp), measured as the production\/biomass ratio (P\/B) (kg\u00a0kg\u22121\u00a0day\u22121) is related to adult mass (m) as: \nThe exponent (\u03ba) was close to 1\/4 for a wide range of cold-blooded species but tended to be somewhat higher for homeotherms (Peters 1983; Hendriks 1999, 2007). The coefficient (\u03b3p) represents the intercept at the standard temperature of 20\u00b0C and has a universal value of 7.5\u00a0\u00d7\u00a010\u22124\u00a0kg\u03ba\u00a0day\u22121 for all species (Hendriks 2007). Following biochemical reaction kinetics, the intercept increases by a factor of qT as a function of temperature (Gillooly et al. 2001). Production kp by warm-blooded species with a body temperature of 37\u00b0 is qT\u00a0=\u00a03.5 times higher than that of equally sized cold-blooded species at 20\u00b0C (Hendriks 2007).\nProduction kp is allocated to somatic and gonadic growth. We will consider the rate constant for reproduction (reproduction kpr) to be a fraction (prp) of total production kp according to: \nIn Spermatophyta, 7% of the total dry weight production is diverted to reproduction via seeds (Niklas and Enquist 2002). After correction to a dry\u2013wet weight fraction of 20% as described in the methods section, the reproductive fraction (prp) equals 28% (Table\u00a01). In animals, prp varies between 0 for juveniles and 1 for adults without somatic growth. Filling in the slope and the intercepts, yields the reproduction rate constant as a function of m (Fig.\u00a01).\nTable\u00a01Main factors used in the equationsSymbolDescriptionUnitValuea, source\u03b3pScaling coefficients for productionkg\u03ba\u00a0day\u221217.5\u00a0\u00d7\u00a010\u22124 (Hendriks 2007)kp, kprRate constants for production and reproductionday\u22121Equation 1\u03baScaling exponent\u2013p,c,w1\/4, w1\/3 (Hendriks 2007)mAdult masskgVariablemrTotal reproductive mass in a batchkgEquation 5meSeed, egg or neonate masskgmin(me)\u00a0=\u00a010\u221210, i10\u221210, p,acv10\u22127, tcv10\u22123\u2026 mr\/3prlFraction inter-reproductive period of average life time\u20130.2prpFraction of production directed to reproduction\u2013p0.28 (Niklas and Enquist 2002), i,v1R0Fecundity, number of offspring released in a reproductive eventno. individual\u22121Equation 6qTTemperature quotient\u2013p,c1, w3.5 (Hendriks 2007)\u03c4rInter-reproductive perioddayEquation 4aSuperscripts: p plants, c cold-blooded, w warm-blooded, i invertebrates, v vertebrates, a aquatic, t terrestrialFig.\u00a01Reproduction rate constants (kpr) (kg\u00a0kg\u22121\u00a0day\u22121) decrease with adult mass (m) (kg) to the power of about 1\/4\u20131\/3. Model estimations (thick lines with letter) as well as empirical regressions (thin lines) for vascular plants (A, green dotted), invertebrates (B, yellow dashed-dotted), cold-blooded vertebrates (C, blue dashed) and warm-blooded vertebrates (D, red solid), as listed in Table\u00a02. d\u22121 Day\u22121\nUsually seeds, eggs and neonates are not delivered continuously but released in discrete batches, i.e., sowings, clutches and litters. The total offspring mass (mr) accumulated in a period between two successive reproductive events (\u03c4r) equals: \nThe inter-reproductive interval (\u03c4r) might be related to the organisms\u2019 metabolism and to environmental conditions. Initially, we did not find empirical regressions that link \u03c4r to organism size. However, many other biological time parameters, including gestation and weaning time of homeotherms, scale to m with an exponent of \u03ba (Peters 1983; Hendriks 1999). For an average mammal, the age at birth, weaning off and average death is at about 2, 3, 50% of the total period from conception to death, respectively (Peters 1983). \u03c4r, covering at least gestation and weaning (2\u00a0+\u00a03%), thus equals at least (2\u00a0+\u00a03%)\/50%\u00a0=\u00a010% of the average lifetime \u03c4l, i.e., \u03c4r\u00a0=\u00a0prl\u00b7\u03c4l\u00a0>\u00a010%\u00b7\u03c4l. To allow for additional time to recover and mate we set the inter-reproductive period at a fraction (prl)\u00a0=\u00a020% of the average lifetime \u03c4l. This lemma is indirectly demonstrated by one long-term study on metabolism, reproductivity and average lifetime (\u03c4l) of earthworms (Mulder et al. 2007). In fact, although no evidence of a correlation between \u03c4r and the offspring was found, \u03c4l and m were strongly correlated. Following a more general theory, \u03c4l can indeed be defined here as the mean residence period in the population and can thus be replaced by 1\/kp (Odum 1983). In this way, \u03c4r can now be assumed to equal: \nFilling in the parameter values for mammals yields \u03c4l\u00a0=\u00a00.3\/(3.5\u00a0\u00d7\u00a07.5\u00a0\u00d7\u00a010\u22124\u00a0m\u22121\/4)\u00a0=\u00a0114\u00a0m1\/4. This is a factor of 2 larger than the value of 55\u00a0m0.23 (r2\u00a0=\u00a00.37, n\u00a0=\u00a041) derived from a compilation of data on South American mammals that was published after our estimation (Pereira and Daily 2007). Obviously, this relationships needs to be confirmed for other species groups.\nSubstituting Eq. 4 into Eq. 3 yields the reproductive mass (mr) accumulated between batches as a linear function of adult size (m) as (Fig.\u00a02): Fig.\u00a02Clutch or litter (reproductive) mass (mr) (kg) increases with m (kg) to the power of about 3\/4 to 1. Legend as in Fig.\u00a01. Regressions listed in Table\u00a03\nThe total reproductive mass derived from Eq. 5 can be divided into many small or a few large young. At one end of the interval, species called \u201cr\u201d-strategists here, maximize the number of young per female (R0) up to a limit set by the absolute minimum size [min(me)] of a seed, egg or neonate. The existence of such a threshold has been attributed to various restrictions but, to our knowledge, supporting quantitative evidence is still lacking. Since each individual starts off as a single cell, the average size of parental cells may serve as a first indication for the minimum egg or seed mass (me). The weight of a cell is reported to be in the range of 10\u221215\u201310\u221211\u00a0kg for plants and animals (Raven 1998; West et al. 2001; Mulder et al. 2005). Lower taxa with multi-cellular organization, such as mycorrhizal conidia and fruit bodies of basidiomycetes, actually release offspring as single cells (Mulder et al. 2003). Higher species reproduce by multi-cellular sexual spores, seeds, eggs and neonates, with sizes at or above that range. With this variability in mind, min(me) was tentatively set at the minimum observed for the taxon covered (Table\u00a01). At the other side of the range, \u201cK\u201d-type organisms invest all reproductive mass into a few young. In this strategy, seed, egg and neonate mass are maximized by dividing the total reproductive mass over a few offspring. Low values are noted for, e.g., birds with an average of 4.8 eggs per clutch and for mammals with means of 2.7\u20132.8 neonates per litter (Blueweiss et al. 1978; Ernest 2003). Apparently, the risk of premature death is typically spread over, on average, at least three young, so that the maximum mass of individual offspring [max(me)] is calculated as max(me)\u00a0=\u00a0mr\/3\u00a0=\u00a0prp\u00b7prl\u00b7m\/3. With seed, egg and neonate mass ranging between min(me) and max(me), we can now derive R0 as: \nThe number of seeds, eggs and neonates (R0) is thus expected to scale to m with exponents in the range of 0\u20131, for K-strategists and r-strategists, respectively. The corresponding intercepts are prp\u00b7prl\/min(me) and 3, respectively. Intermediate values indicate that both types of species are present in the sampled taxon. If strategies are uniformly distributed among species, one expects a mean slope of 1\/2, with the geometric average of prp\u00b7prl\/min(me) and 3 as a coefficient. Vice versa, the mass of a seed, egg or neonate (i.e. me) varies between min(me)\u00a0\u221d\u00a0m1 and max(me) \u00a0\u221d\u00a0m0, also with an average at m1\/2 (Figs.\u00a03, 4).\nFig.\u00a03Egg or neonate mass (me) (kg) increases with m (kg) to a power varying around 1\/2 for cold-blooded animals and in the range of 3\/4\u20131 for warm-blooded animals. Legend as in Fig.\u00a01; in addition, model estimations for organisms in general (E, dashed-dotted, me\u00a0=\u00a00.0000046\u00a0m1\/2, me\u00a0=\u00a00.2\u00a0m). Regressions listed in Table\u00a03Fig.\u00a04Seed, egg or neonate number in ovaries, clutch or litter (R0) (young\/individual) increases with m (kg) to a power varying around 1\/2 for cold-blooded animals and close to 0 for warm-blooded animals. Legend as in Fig.\u00a01; in addition, model estimations for organisms in general (E, dashed-dotted, R0\u00a0=\u00a045,000\u00a0m1\/2, R0\u00a0=\u00a01). Regressions as listed in Table\u00a03\nEquations 4\u20136 apply to cases where \u03c4r is related to the metabolism of the organisms. Alternatively, \u03c4r may depend on cyclic processes in the environment. Since optimal conditions for offspring usually occur in specific seasons, synchronization of reproduction to annual cycles of temperature, light or food availability by e.g., hibernation or fetal growth retardation, is most likely. With \u03c4r\u00a0=\u00a0365\u00a0days, Eq. 3 becomes: indicating that the mass released in a yearly sowing, clutch or litter (mr) scales to m with an exponent of 1\u2212\u03ba. Organisms with an intrinsic \u03c4r close to 1\u00a0year can exploit such synchronization most readily. Smaller species may somewhat postpone their reproduction, increasing the gonadic mass released. Larger species may do the opposite, decreasing the slope of the regression towards 3\/4. Fitting Eq. 1 into 4 and writing m explicitly gives: \nUsing the typical values for the parameters (Table\u00a01), cold-blooded and warm-blooded organisms with a \u03c4r of 365\u00a0days are expected to have an m of about 4 and 500\u00a0kg, respectively. Organisms that are much smaller mature sufficiently fast to allow multiple generations within a year or growing season. Species which are substantially larger need more than 1\u00a0year to produce one egg or neonate.\nResults\nReproduction rate constant\nWe will now compare the derived equations to the empirical regressions obtained in the meta-analysis. The few correlations available for gonadic growth show that reproduction kpr indeed decrease significantly with increasing adult mass (P\u00a0<\u00a00.0001\u2026<0.002, Table\u00a02). Average slopes for plants and animals are in the range of \u22120.16 to \u22120.39 (Table\u00a04). Slopes for warm-blooded species tend to be steeper than those for cold-blooded species. The empirical regressions for seed plants were derived from one study, with annual reproductive mass expressed as a function of foliage and stem mass because total weight correlations were not provided (Fig.\u00a01; Niklas and Enquist 2003). The invertebrate regressions apply to short-term studies on different types of copepods. The outlier noted in field experiments with sac spawners was not confirmed by other correlations, including those for high food levels (Hirst and Bunker 2003, p 1,995). Low intercepts for fish and mammals apply to long-term observations, including intervals without gonadic growth (Charnov 2001; Charnov et al. 2001). High values were noted for reproductive growth in gestation periods (Payne and Wheeler 1968; Blueweiss et al. 1978). Reproduction within primates is slower, as earlier noted for production (Table\u00a02, no.\u00a034; Western and Ssemakula 1982; Hendriks 2007).\nTable\u00a02kpr (kg\u00a0female\u00a0kg\u22121\u00a0day\u22121) as a function of m (kg) according to empirical regressions (y\u00a0=\u00a0amb) collected in the meta-analysis. For abbreviations, see Table 1NoTaxonabnr2PSource1Spermatophyta1.8\u00a0\u00d7\u00a010\u22124\u22120.162790.75<0.0001Niklas and Enquist (2003)2Spermatophyta3.0\u00a0\u00d7\u00a010\u22124\u22120.334180.75<0.0001Niklas and Enquist (2003)11Copepoda9.7\u00a0\u00d7\u00a010\u22124\u22120.2530810.06<0.001Hirst and Bunker (2003)12Copepoda1.3\u00a0\u00d7\u00a01000.174520.05<0.001Hirst and Bunker (2003)13Copepoda2.7\u00a0\u00d7\u00a010\u22123\u22120.26350.320.002Ki\u00f8rboe and Sabatini (1995) 14Copepoda1.2\u00a0\u00d7\u00a010\u22123\u22120.26100.720.002Ki\u00f8rboe and Sabatini (1995)21Osteichthyes3.0\u00a0\u00d7\u00a010\u22124\u22120.191390.74<0.0001Charnov et al. (2001)31Mammalia4.9\u00a0\u00d7\u00a010\u22124\u22120.331920.89<0.0001Charnov (2001)32Mammalia1.9\u00a0\u00d7\u00a010\u22123\u22120.43920.65<0.0001Blueweiss et al. (1978)33Mammalia3.6\u00a0\u00d7\u00a010\u22123\u22120.40300.90<0.0001Payne and Wheeler (1968)34Primates8.4\u00a0\u00d7\u00a010\u22124\u22120.44150.93<0.0001Payne and Wheeler (1968)\nThe differences between regressions for the reproduction kpr (kg\u00a0kg\u22121\u00a0day\u22121) follow the trends expected from the model, based on average turnover kp (Eq. 2). That is to say, slopes \u2212\u03ba for both reproduction and production tend to be close to \u22121\/4 for heterotherms and at or below \u22121\/3 for homeotherms (Table\u00a04; White and Seymour 2004; Hendriks 2007). Intercepts for seed growth in plant populations, including all life stages, follow Eq. 2, independently confirming the value of 28% for the prp. The reproduction rate regressions for animals are expressed on a female basis. With the exception of deviations noted for some regressions on copepods and primates, levels for true gonadic growth in heterotherms are within a factor of 3 of the model, based on a \u03b3p of 7.5\u00a0\u00d7\u00a010\u22124\u00a0kg\u00a0kg\u22121\u00a0day\u22121 previously derived for production (Fig.\u00a01). In general, the variability of the intercepts for reproduction reflects differences in methods, conditions and species and is similar to the variability observed for, e.g., individual growth or consumption (Hendriks 2007). Keeping this variability in mind, we might approximate reproduction by 0.28\u00a0kp in plants and 1\u00a0kp in adult animals as predicted by Eq. 2 of the model.\nTotal offspring mass in a reproductive batch\nWith average production kp as a predictor of reproduction, we can now compare measurements and estimations for the reproductive masses and numbers (Fig.\u00a02). Regressions indicate that total seed, egg and neonate weight increase with parent size (P\u00a0<\u00a00.0001\u2026<0.05 in Table\u00a03, Fig.\u00a02). Most 95% confidence intervals for the average of the slopes encompass 3\/4 or 1, but the whole animal size range can only be covered by an exponent of 1 (Table\u00a04). The outcome for plants is dominated by a regression with a slope of 0.67 observed for fruit versus stem mass (1 in Table\u00a03). Expressed on leaf mass, annual standing reproductive weight in plants scaled to 0.84 (Niklas and Enquist 2003). Comparing weighted averages, the mean slope for invertebrates 0.95 is significantly higher than the confidence interval calculated for plants and cold-blooded vertebrates (Table\u00a04). The confidence intervals noted for exponents of birds and mammals cover 3\/4 but not 1. All intercepts for cold-blooded and warm-blooded species are close to each other with the exception of one low value noted for fish (Fig.\u00a02, Table\u00a03; Stolz 2005). This outlier, however, reflects year-round observations while other regressions apply to peak gonad mass. Similar intercept differences were noted for the reproduction kpr.\nTable\u00a03Reproductive mass [sowing, clutch, litter (mr); seed, egg, neonate (me)] (kg) as well as the number of offspring (seed, egg, neonate; R0) as a function of m (kg), according to empirical regressions (y\u00a0=\u00a0amb) collected in the meta-analysis. Values in italics approximated by us because not\/wrongly reported in the original study. Offspring mass or number measured in event (clutch, litter) or organ (ovary, fruits). n.a. Not available, n.s. not significant; for other abbreviations, see Tables 1 and \u00a02No.Taxone, omr (kg)me (kg)R0 (no.\/female)Sourceabnr2Pabnr2pabnr2P1Spermatophytaao1.1\u00a0\u00d7\u00a010\u221210.674180.75<0.0001Niklas and Enquist (2003)2Angiospermaeao1.1\u00a0\u00d7\u00a010\u221211.02150.87<0.00015.8\u00a0\u00d7\u00a010\u221260.23150.080.302.1\u00a0\u00d7\u00a01051.01150.490.004Aarssen and Jordan (2001)3Spermatophytao5.7\u00a0\u00d7\u00a010\u221221.08130.94<0.00011.0\u00a0\u00d7\u00a010\u221250.60130.260.076.3\u00a0\u00d7\u00a01020.43130.200.13Greene and Johnson (1994)4Angiospermaeo5.5\u00a0\u00d7\u00a010\u221220.95570.68<0.00011.4\u00a0\u00d7\u00a01030.562200.38<0.0001Shipley and Dion (1992)5Spermatophytao8.0\u00a0\u00d7\u00a010\u221250.472240.43<0.001Moles et al. (2005b)6Spermatophytao4.6\u00a0\u00d7\u00a010\u221260.602260.25<0.0001Grubb et al. (2005)11Rotifera\u20133.6\u00a0\u00d7\u00a010\u221250.60430.550.001Walz et al. (1995)12Copepoda\u20134.0\u00a0\u00d7\u00a010\u221230.93210.87<0.001Ki\u00f8rboe and Sabatini (1995)13Copepoda\u20131.2\u00a0\u00d7\u00a010\u221250.62410.75<0.001Ki\u00f8rboe and Sabatini (1995)14Crustaceae2.5\u00a0\u00d7\u00a010\u221221.16230.99<0.00015.2\u00a0\u00d7\u00a010\u221280.24220.82<0.00012.5\u00a0\u00d7\u00a01040.59230.59<0.0001Blueweiss et al. (1978)15Dipteraao6.7\u00a0\u00d7\u00a010\u221211.00480.75<0.052.1\u00a0\u00d7\u00a010\u221260.31480.19<0.052.1\u00a0\u00d7\u00a01050.62480.19<0.05Berrigan (1991)16Hymenopteraao1.1\u00a0\u00d7\u00a010\u221210.96310.80<0.051.4\u00a0\u00d7\u00a010\u221230.89310.75<0.057.9\u00a0\u00d7\u00a01010.07310.02n.s.Berrigan (1991)17Coleopteraao6.7\u00a0\u00d7\u00a010\u221220.79560.82<0.051.5\u00a0\u00d7\u00a010\u221230.69560.72<0.054.6\u00a0\u00d7\u00a01010.10560.05n.s.Berrigan (1991)18Araneomorphaee1.3\u00a0\u00d7\u00a01001.09130.900.00012.7\u00a0\u00d7\u00a010\u221250.43330.620.00014.7\u00a0\u00d7\u00a01040.66390.700.0001Marshall and Gittleman (1994)19Aphidaee1.2\u00a0\u00d7\u00a01030.161370.35<0.0001Llewellyn and Brown (1953)20Cephalopoda\u20132.2\u00a0\u00d7\u00a010\u221250.26180.080.25Wood and O\u2019dor (2000)21Osteichthyeso1.3\u00a0\u00d7\u00a010\u221221.04200.85<0.001Stolz (2005)22Osteichthyes-Reptiliaoe1.1\u00a0\u00d7\u00a010\u221210.92850.86<0.00011.2\u00a0\u00d7\u00a010\u221250.43630.820.00071.2\u00a0\u00d7\u00a01040.551210.56<0.0001Blueweiss et al. (1978)23Osteichthyeso1.5\u00a0\u00d7\u00a010\u221211.0890.91<0.0001McCann and Shuter (1997)24Osteichthyeso4.4\u00a0\u00d7\u00a010\u221220.86200.91<0.0001McCann and Shuter (1997)25Osteichthyese4.7\u00a0\u00d7\u00a01040.791080.62<0.0001Winemiller and Rose (1992)26Osteichthyese1.4\u00a0\u00d7\u00a01050.781080.35<0.0001Winemiller and Rose (1992)27Salamandraee2.2\u00a0\u00d7\u00a010\u221220.64740.89<0.012.0\u00a0\u00d7\u00a010\u221250.25250.190.027.1\u00a0\u00d7\u00a01020.31250.250.01Kaplan and Salthe (1979)28Salamandrae\u20131.8\u00a0\u00d7\u00a010\u221240.37250.58<0.0001Kaplan and Salthe (1979)29Salamandrae\u20137.2\u00a0\u00d7\u00a010\u221240.49210.550.0001Kaplan and Salthe (1979)30Reptiliae1.5\u00a0\u00d7\u00a010\u221210.88350.96n.a.7.5\u00a0\u00d7\u00a010\u221230.42350.70<0.00012.1\u00a0\u00d7\u00a01010.48540.78<0.0001Blueweiss et al. (1978)31Avese2.9\u00a0\u00d7\u00a010\u221210.641140.69<0.0016.9\u00a0\u00d7\u00a010\u221220.67760.88<0.0014.1\u00a0\u00d7\u00a0100\u22120.081140.10<0.001Cabana et al. (1982)32Avese2.1\u00a0\u00d7\u00a010\u221210.742200.85<0.00015.3\u00a0\u00d7\u00a010\u221220.771600.83<0.00014.9\u00a0\u00d7\u00a0100\u224801000n.a.Blueweiss et al. (1978)33Avese1.9\u00a0\u00d7\u00a010\u221210.723500.79n.a.5.4\u00a0\u00d7\u00a010\u221220.772300.93n.a.Western and Ssemakula (1982)34Mammaliae1.1\u00a0\u00d7\u00a010\u221210.84760.93<0.0014.0\u00a0\u00d7\u00a010\u221220.941140.94<0.001Cabana et al. (1982)35Mammaliae1.6\u00a0\u00d7\u00a010\u221210.821100.97<0.00015.6\u00a0\u00d7\u00a010\u221220.922000.94<0.00012.7\u00a0\u00d7\u00a0100\u224801000n.a.Blueweiss et al. (1978)36Mammaliae1.1\u00a0\u00d7\u00a010\u221210.79920.95n.a.5.1\u00a0\u00d7\u00a010\u221220.94910.94<0.00012.4\u00a0\u00d7\u00a0100\u22120.14910.74<0.0001Western and Ssemakula (1982), Sacher and Staffeldt (1974)37Mammaliae2.7\u00a0\u00d7\u00a010\u221220.71950.88<0.013.9\u00a0\u00d7\u00a01000.001000n.s.Millar (1977)38Mammaliae3.4\u00a0\u00d7\u00a0100\u22120.16290.54<0.001Millar and Zammuto (1983)39Mammaliae2.2\u00a0\u00d7\u00a01020.9540.98<0.00012.8\u00a0\u00d7\u00a0100\u22120.12630.32<0.0001Purvis and Harvey (1995)40Primatese8.3\u00a0\u00d7\u00a010\u221220.851000.93<0.0001Ross (1998)Table\u00a04Allometric scaling of reproductive parameters as listed in Tables\u00a02 and 3 plotted in Figs.\u00a01\u20134. Measured arithmetic mean and data-weighted mean (in italics) for the regression exponent b with 95% confidence interval and model values. For abbreviations, see Tables 1 and \u00a02ParameterReproduction rateBatch massOffspring massOffspring numberSymbolpap\u00b7kpmrmeR0mr\/meUnitd\u22121kgkgNumber of individualsEmpirical averagePlants\u22120.33, \u22120.160.93 (0.65\u20131.22)0.48 (0.20\u20130.75)0.66 (\u22120.10 to 1.43)0.450.78 (0.65\u20130.91)0.53 (0.45\u20130.60)0.58 (0.38\u20130.78)0.25Invertebrates\u22120.15 (\u22120.49 to 0.19)1.00 (0.83\u20131.17)0.55 (0.35\u20130.75)0.32 (\u22120.04 to 0.68)0.45\u22120.20 (\u22120.33 to \u22120.07)0.95 (0.87\u20131.03)0.56 (0.51\u20130.62)0.27 (0.13\u20130.42)0.39Cold-blooded vertebrates\u22120.190.90 (0.74\u20131.06)0.39 (0.28\u20130.50)0.58 (0.26\u20130.91)0.510.85 (0.78\u20130.91)0.40 (0.27\u20130.53)0.67 (0.54\u20130.81)0.45Birds0.70 (0.56\u20130.84)0.74 (0.59\u20130.88)\u22120.04 (\u22120.55 to 0.47)\u22120.04Mammals\u22120.39 (\u22120.53 to \u22120.25)0.82 (0.75\u20130.88)0.88 (0.77\u20131.00)\u22120.08 (\u22120.28 to 0.11)\u22120.06Model valueCold-blooded\u22121\/411\/21\/2Warm-blooded\u22121\/4 to \u22121\/3(3\/4)\u20131(3\/4)\u201310\nFor the overall range, empirical slopes for heterotherms approach the value of 1 expected from Eq. 5 of the model. In addition, the intercepts for plants and animals are at the level expected from the coefficients prl\u00b7prp of Eq. 5. Exponents collected for homeotherms and some large-sized heterotherms tend towards 3\/4, suggesting synchronization to annual cycles as explained by Eq. 7. The steep lines observed for invertebrates in comparison to the flatter slopes of cold-blooded vertebrates confirm the prediction that heterotherms of ca. 4\u00a0kg are likely to experience some kind of synchronization. In addition, exponents for mammals, including large species weighting up to 500\u00a0kg, are close to 3\/4. Exponents for birds are smaller and obviously their weight range does not include this value.\nSeed, egg and neonate mass\nRegressions show that seed and egg masses are significantly related to adult size for most heterothermic species groups (P\u00a0<\u00a00.05, Table\u00a03). Exceptions are noted for some correlations that cover a small size range or a few data (P\u00a0\u2265\u00a00.07;Table\u00a03, nos. 2 and 3 and 20). The slope varies within a small interval around the mid-point value of 1\/2 (Fig.\u00a03). Averages for invertebrates are higher than those for plants and cold-blooded vertebrates, mainly because regressions on egg-carrying copepods and wasps have slopes closer to 1 (Table\u00a03, 12 and 16; Berrigan 1991; Ki\u00f8rboe and Sabatini 1995). The egg and seed masses (me) for plants, invertebrates and (semi-)aquatic vertebrates of the same size are within 1 order of magnitude of each other (Fig.\u00a03). Eggs of aquatic crustaceans tend to be smaller than those of terrestrial spiders and insects with equal body size (Table\u00a03, nos. 12\u201314\u00a0<\u00a014\u201318). Likewise, intercepts for cold-blooded vertebrates increase in the sequence of fish, water-bound salamanders, land-dwelling salamanders and reptiles (Table\u00a03, 22\u00a0<\u00a027\u00a0<\u00a028\u00a0\u2264\u00a029\u00a0<\u00a030; Blueweiss et al. 1978; Kaplan and Salthe 1979). Warm-blooded animals distribute their reproductive effort over a small and size-invariant R0, leading to exponents for egg and neonate mass (me) between 3\/4 and 1 (Fig.\u00a03).\nThe partitioning of the reproductive mass over seeds, eggs and neonate thus follows a combination of both strategies distinguished in the model. The empirical regressions collected are within the minimum and maximum boundaries set by Eq. 6 of the model (Fig.\u00a03). The slopes for plants, most invertebrates and all cold-blooded vertebrates vary around \u00bd indicating some intermediate value between min(me)\u2013m0 and max(me)\u2013m1. Egg-carrying copepods, wasps, birds and mammals largely consists of K-strategists, with egg and neonate mass (me) exponents near those noted for clutch and litter mass (mr). Almost all regressions are above the intermediate intercept calculated by the model for an overall minimum offspring size min(me) of 10\u221210\u00a0kg (Table\u00a01). Using typical minimum values min(me) of 10\u22127\u00a0kg for plants, fish and amphibians and of 10\u22123\u00a0kg for reptiles rather than the overall 10\u221210\u00a0kg gives a better fit of the model.\nSeed, egg and neonate number\nThe number of seeds and eggs released by heterotherms scales to size with exponents largely between 1\/2 and 1 (Table\u00a04, Fig.\u00a04). The difference between intercepts reflects the fraction of the lifetime covered, being near 100% for herbs and a few percent of less for trees, reflecting lifetime and batch fecundity (R0), respectively (Table\u00a03, 2 vs. 3\u20134; Shipley and Dion 1992; Greene and Johnson 1994; Aarssen and Jordan 2001). The amount of eggs delivered by crustaceans, flies, spiders and aphids is significantly correlated to adult size but those for wasps and beetles are size-independent (Table\u00a03, nos. 14\u201315, 18\u201319 vs. 16\u201317; Llewellyn and Brown 1953; Blueweiss et al. 1978; Berrigan 1991; Marshall and Gittleman 1994). The low intercept for vertebrate heterotherms applies to reptiles (Table\u00a03, 30; Blueweiss et al. 1978). The clutch and litter size of warm-blooded species does not increase with adult size. In fact, four out of seven regressions have slightly negative slopes.\nAs expected, average slopes of regressions for R0 are in the same range as those calculated for the ratio of total and individual offspring mass (mr\/me) (Table\u00a04). The largest deviation between arithmetic means is noted for plants and equals 0.66\u22120.45\u00a0=\u00a00.21. However, this difference is due to one study in which sowing weight and number but not seed weight scale significantly to m (b\u00a0=\u00a01.01\u20261.02, P\u00a0<\u00a00.00001\u20260.004, vs. b\u00a0=\u00a00.23, P\u00a0=\u00a00.30). With parameters set at typical values, the model follows these trends (Table\u00a01).\nDiscussion\nData variability\nThe meta-analysis yielded regressions for various species groups. However, important phylogenetically lower taxa, such as bacteria, algae, fungi, mosses, ferns, Protozoa and annelids have not been included in allometric relationship on reproductive parameters. Budding or binary fission in unicellular organisms yields new cells that become independent at about 30\u201350% of the parent cell volume, respectively (Woldringh et al. 1993). Unicellular taxa thus maximize the mass rather than the number of their offspring. Fungi, mosses and ferns release spores that are at the lower end of the palynological range noted for seed plants (e.g., Nilsson 1983; Haig and Westoby 1991; Moore et al. 1991; Mulder and Janssen 1999). Their strategy is thus comparable to that of some seed plants, such as orchids. Additional study of the size-related reproduction of these cryptogams is required.\nPlant offspring is characterized by seed and fruit mass with or without ancillary tissues, while adult mass refers to leaves and\/or stems. Animal studies either cover the whole annual cycle or reproductive periods only. In addition, deviations from average values of other factors, such those for length\u2013weight conversion or life-stage may increase variability.\nSlopes on the trade-off between reproduction and production\nDespite uncertainties, clear patterns on the average trends emerge (Fig.\u00a05). Reproduction kpr (kg\u00a0kg\u22121\u00a0day\u22121) decrease with size, as expected from the model. According to Eq. 2, reproduction is considered to be a fraction (prp) of the production kp, representing the P\/B of a population. Reproduction kpr by homeotherms has steeper slopes and larger intercepts in comparison with heterotherms, as earlier noted for production kp (Fig.\u00a02, Table\u00a02). It indicates that the trade-off between somatic and gonadic growth is rather invariant to size and metabolism. The fraction of the production directed to reproduction (prp) was found to be 28% for plants after conversion to seedling wet weight, indeed independent of the species weight (Niklas and Enquist 2002). The similarity between slopes for reproduction and overall population production indicates that such a relationship also exists for animals. However, the derivation of such a fraction is yet impeded by large variability among intercepts, by a lack of information on many heterothermic species and by the focus on adult animals instead of whole populations.\nFig.\u00a05Theoretical (lines) and empirical (dotted area) trends of seed, egg or neonate mass (mr, me) (kg) and number (R0) versus m (kg) for several species groups\nSlopes on the trade-off between offspring number and mass\nThe meta-analysis in the present paper shows that the mass released in a single reproductive event (mr) (kg) is a constant fraction of the parents\u2019 weight for a remarkable variety of plant and animal species (Table\u00a03, Fig.\u00a02). Over the whole range of species studied, the slope is close to 1 but exponents for specific groups, such as homeotherms and possibly plants, are smaller. Ignoring differences in slopes and intercepts, about 20% of the adult mass is released in a sowing, clutch or litter (Eq. 5). This supports, though not necessarily implies, the existence of a size-independent prp.\nIn addition to the trade-off between somatic and gonadic growth, organisms may divert their reproductive mass over many small or a few large offspring (Fig.\u00a05). In other words, the R0 is inverse proportional to seed, egg or neonate mass (me). As elaborated in the Model development section, one may distinguish between species with r-tactics that maximize the number of offspring with increasing adult mass (R0~m1, me~m0) and K-strategists that amplify seed, egg or neonate weight (me~m1, R0~m0). Slopes of regressions on R0 and me of the offspring generally vary around the intermediate value of 1\/2, expected if species with an r- and K-strategy are equally distributed (Table\u00a03). However, regressions on egg-carrying copepods, wasps, birds and mammals have exponents close to 1 for egg and neonate size (me) and near 0 for R0 (Table\u00a03). These species groups thus largely consist of K-strategists.\nGraphically, seed, egg and neonate mass are confined to a triangular envelope that contains small species with small offspring and large species with small or large offspring (Figs.\u00a03\u20135). Such a pattern has been previously recognized in original data on plants and fish. The triangle was found to be right-angled for fish, with a minimum egg size [min(me)] of 10\u22127\u00a0kg independent of the m (e.g., Duarte and Alearaz 1989). In plants, the envelope was reported to be obtuse-angled, where the 5%-tile and the 95%-tile of seed size scaled to total plant mass with exponents in the range of 0.21\u20130.32 and 0.38\u20130.68, respectively (Grubb et al. 2005; Moles et al. 2005b). The slopes for the lower boundary encompass the value of 1\/4, known for scaling of biological time variables (Peters 1983; Hendriks 1999). Although the period to adulthood has been suggested to limit egg and seed size, such a relationship may be more complex than a 1\/4 power scaling to size (see, e.g., Kiflawi 2006). In addition, some proof for the underlying mechanisms, including an explanation for its absence and presence in different species, as well as taxon-specific slope values, is yet lacking.\nSimilar considerations apply to the upper boundary. The egg and neonate mass (me) in warm-blooded species is proportional to the weight of their parents. Each of the slopes is steeper than observed for clutch or litter mass (mr). However, confidence intervals for the averages indicate that 3\/4 may be the appropriate exponent in addition to 1, at least within small size ranges such as noted for birds. Slopes of 3\/4 can be understood from inter-reproductive periods that synchronize to annual cycles rather than to adult mass (Eq. 7). Yet, the difference between cold-blooded (\u22481\/2) and warm-blooded [(3\/4)\u20131] species indicate that metabolism-related mechanisms may be important too. Just as size of warm-blooded adults appears to be constrained by heat exchange, neonate mass may be also be determined by thermodynamic principles. However, the exploration and underpinning of other values for the slopes is beyond the scope of the present paper. For now, we assume that the lower and upper end of the range scale to between 0 and 1. Most species groups contain both an r- and K-strategists yielding an average of slope of about 1\/2, whereas some animal taxa were shown to scale to 1. Future refinement by thermodynamic principles may lead to a smaller range between, e.g., 1\/4 and 3\/4, still yielding average slopes around 1\/2.\nIntercepts on the trade-off between aquatic and terrestrial habitats\nHaving described the possible values of the slopes, we will now take a look at the intercepts. The variability of the exponents excludes detection of subtle differences between coefficients. Yet, there is an evident increase in egg size from aquatic to terrestrial habitats, both among invertebrates and vertebrates. The difference has historically been attributed to oxygen limitations as its diffusion in air is 10,000 times faster than in water (Hendry et al. 2001). However, experiments within the same species do not confirm this explanation (Einum et al. 2002). We therefore suggest that an alternative, more universal, mechanism may be more important. A parent is more willing to invest in the size of a seed or egg when it can influence its fate. Organisms have little control of transport and development in large homogeneous compartments such as water and air. Consequently, clutches of aquatic animals consist of many small eggs (Fig.\u00a04; Blueweiss et al. 1978; Kaplan and Salthe 1979; Berrigan 1991; Winemiller and Rose 1992; McCann and Shuter 1997; Wood and O\u2019dor 2000). Even within fish, pelagic species reproduce by smaller and more eggs than demersal counterparts of equal size (Duarte and Alearaz 1989). The intercepts for the amphibians increase in the sequence of pond breeders that attach eggs to plants followed by stream and land breeders with nests (Table\u00a03, 27\u00a0<\u00a028\u00a0<\u00a029; Kaplan and Salthe 1979). Within the heterogeneous terrestrial environment, land animals can direct their offspring to sites that provide shelter or food to avoid wasting reproductive mass in unfavorable patches (Blueweiss et al. 1978; Berrigan 1991; Marshall and Gittleman 1994). The small seed size of terrestrial plants in comparison to egg masses in reptiles can be understood from the extra dispersal function of propagules in the former case.\nObviously, other factors that covary with the water\u2013land gradient may be responsible for the observed patterns. For instance, the dry\u2013wet weight fraction of adults increases from aquatic to terrestrial habitats and a similar trend might exist in their offspring too. However, this explanation, and the related interpretation, is unlikely to cover the orders of magnitude of the noted differences.\nIntegration of theoretical and empirical evidence\nIntegrating these trends, we can now derive common patterns within the triangular envelope of offspring and adult mass (Fig.\u00a05). In general, r-strategists are found amongst small adults (Fig.\u00a05, left-hand side) with many small offspring in aquatic environments (Fig.\u00a05, lower end). Large adults (Fig.\u00a05, right-hand side) with a few large young live in terrestrial habitats (Fig.\u00a05, upper end). Obviously, these patterns cannot cover all reproductive and non-reproductive characteristics associated with r\u2212K gradients. For instance, ruderal plants typically reproduce as r-strategists, but more K-type species, especially in close canopies, may also regenerate via wind dispersal of small seeds (Grime et al. 1997; Grime 2001; Aarssen 2005). In addition, seed mass is known to be negatively correlated with the leaf size or the life span of 640 plant species (D\u00edaz et al. 2004). Such results for plants strongly imply the existence of correlated traits in other taxa and demand further investigation.","keyphrases":["reproduction","body size","macroecology","fecundity","allometric scaling"],"prmu":["P","P","P","P","R"]}
{"id":"Bioinformation-2-2-2174422","title":"TeCK Database: A comprehensive collection of telomeric and centromeric sequences with their associated proteins\n","text":"Telomeres and centromere are two essential features of all eukaryotic chromosomes. They provide function that is necessary for the stability of chromosomes. We developed a comprehensive database named TeCK, which covers a gamut of sequence and other related information about telomeric patterns, telomere repeat sequences, centromere sequences and centromeric patterns present in chromosomes. It also contains information about telomerase ribo-nucleoprotein complexes, centromere binding protein and centromere DNA-binding protein complexes. The database also includes a collection of all kinetochore-associated proteins including inner, outer and central kinetochore proteins. The database can be searched using a user-friendly web interface.\nBackground\nTelomeres [1] are a series of repeated DNA sequences located at the end of chromosomes, with strand asymmetry in GC \ncontent, resulting in one G-rich strand and one C-rich strand. They serve to assure that a chromosome is replicated properly during cell division. During this process some of the \ntelomeres are lost. Eventually little or no telomere remains, and the cell dies. [2] Telomerase is an enzyme that adds specific \nDNA sequence repeats, (\u201cTTAGGG\u201d in all vertebrates) to the 3\u2019 end of DNA strands, in the telomere regions at the ends of chromosomes, thus preventing it from getting shorter. \nCentromeres [3] are DNA sequences contained in the heterochromatin, responsible for the segregation of each chromosome into \ndaughter cells during cell division. The kinetochore [4] assembled on the centromere links the chromosome to the mitotic \nspindle during mitosis. As a cell tends to become cancerous [5], it divides more often, and its telomeres become very short, \ncausing death of cells, which can be prevented by activating the enzyme telomerase. By understanding these regions of chromosomes, cancer and aging can be treated effectively and \nthus is a potential target of drug design approach. Hence, a database for analyzing and interpreting  chromosomal information is essential. So far there had been no attempts to \nannotate and curate these parts of chromosomes.\nMethodology\nTECK is developed using MySQL [6] a relational database management system that serves as the backend for storing data. \nIIS (Internet Information Server) is used as the web server and PHP5 (Hypertext preprocessor) [7] a widely used scripting \nlanguage driven by Zend engine is used as the web interface.\nThe nucleotide information is retrieved from NCBI, [8] a national resource repository for molecular biological information. \nAbout 160 entries are obtained for both telomere and centromere. The information about the associated proteins is retrieved from SWISSPROT, [9] \na protein database. About 154 entries are obtained for telomerase, 162 entries are obtained for centromere binding proteins and 170 entries are obtained for kinetochore. Hyperlinks are \nprovided to the corresponding databases for each entry with their IDs displayed. \nEach entry is given an unique ID called TeCK ID consisting of three parts, the first part indicating the class and the second depicting the organism and the last denoting the position of \nthat particular entry in the table. About 16 tables had been created all of which are linked using their unique TeCK ID. Each protein entry is described using twenty parameters such as protein name, \ngene name, source, lineage, function, other interacting proteins etc. The nucleotide information is described using parameters such as chromosome number, keywords, lineage, the articles along with \nthe author details and references. BLAST [10] is a search tool for comparing biological sequences. A stand alone BLAST Program obtained from NCBI \nhas been installed for searching similar sequences.\nAccessibility\nTeCK database can be accessed via internet and the screen shot of the homepage is displayed [Figure 1A]. There are three ways by which the user can query the database. The first one is the keyword \nsearch in home page that can be performed for five categories (Telomere, Centromere and its associated proteins along with Kinetochore proteins) by giving any text data related to organism name, protein name, \nfunction etc. to retrieve the required data. The second one is the advanced search option for specific requirements [Figure 1B], where the user can fill the form by specifying the input parameters such as \nSwissProt, Interpro, Pfam, BLOCKS, PDB, ModBase IDs or TeCk ID. Alternatively, the users can also browse the database [Figure 1C] by organisms and category. On selection of the required query, a result page \ncontaining the number of hits and the list of all entries ID with a short description is displayed. Detailed information can be obtained by clicking each entry ID [Figure 1D]. The sequences are displayed in \nFASTA format and the retrieval can be done using a separate \u2018retrieve in FASTA format\u2019 link. BLASTn can be done against TeCK database & NCBI and BLASTp against TeCK database, SwissProt & PDB.\nUtility\nAs TeCK database includes name, function, sequence, subunits, and other literature information. The data for TeCK database were obtained from various curated databases and hence TeCK database helps end users \nto study, compare, analyze and interpret them and finds utility in control of cancer and ageing.\nCaveats\nIt should be noted that the consistency depends on the source of the original data.","keyphrases":["telomere","centromere","chromosome","kinetochore proteins"],"prmu":["P","P","P","P"]}
{"id":"Oecologia-3-1-1915602","title":"Impact of foliar herbivory on the development of a root-feeding insect and its parasitoid\n","text":"The majority of studies exploring interactions between above- and below-ground biota have been focused on the effects of root-associated organisms on foliar herbivorous insects. This study examined the effects of foliar herbivory by Pieris brassicae L. (Lepidoptera: Pieridae) on the performance of the root herbivore Delia radicum L. (Diptera: Anthomyiidae) and its parasitoid Trybliographa rapae (Westwood) (Hymenoptera: Figitidae), mediated through a shared host plant Brassica nigra L. (Brassicaceae). In the presence of foliar herbivory, the survival of D. radicum and T. rapae decreased significantly by more than 50%. In addition, newly emerged adults of both root herbivores and parasitoids were significantly smaller on plants that had been exposed to foliar herbivory than on control plants. To determine what factor(s) may have accounted for the observed results, we examined the effects of foliar herbivory on root quantity and quality. No significant differences in root biomass were found between plants with and without shoot herbivore damage. Moreover, concentrations of nitrogen in root tissues were also unaffected by shoot damage by P. brassicae larvae. However, higher levels of indole glucosinolates were measured in roots of plants exposed to foliar herbivory, suggesting that the development of the root herbivore and its parasitoid may be, at least partly, negatively affected by increased levels of these allelochemicals in root tissues. Our results show that foliar herbivores can affect the development not only of root-feeding insects but also their natural enemies. We argue that such indirect interactions between above- and below-ground biota may play an important role in the structuring and functioning of communities.\nIntroduction\nOver the past 20\u00a0years, studies exploring community-level processes have been based primarily on bi- and tri-trophic interactions in above-ground systems, usually involving associations among plants, insect herbivores, and their natural enemies such as parasitoids or predators (Price et\u00a0al. 1980; Turlings et\u00a0al. 1990; Vet and Dicke 1992; Dicke 1999). While providing valuable data on a range of selective forces shaping community structure and function, these studies have generally ignored biological processes that occur below-ground. Recently, however, it has become apparent that above-ground trophic interactions can be strongly influenced by interactions between the host plant and soil-dwelling organisms (Gange and Brown 1989; Masters and Brown 1992, 1997; Masters et\u00a0al. 1993; Masters 1995; Gange 2001; Van der Putten et\u00a0al. 2001; Bardgett and Wardle 2003; Bezemer et\u00a0al. 2003). For instance, recent studies have shown that soil-dwelling organisms\u2014such as root-feeding insects, arbuscular mycorrhiza fungi, and nematodes\u2014can also influence the growth, development, and behavior of higher trophic levels below-ground, including parasitoids (Masters et\u00a0al. 2001; Gange et\u00a0al. 2003; Wurst and Jones 2003; Bezemer et\u00a0al. 2005; Guerrieri et al. 2005) and even hyperparasitoids in the fourth trophic level (Soler et\u00a0al. 2005).\nIt is also known that above-ground herbivory can affect the development of below-ground insect herbivores (reviewed by Blossey and Hunt-Joshi 2003), when the shared host plant has previously been exposed to intense shoot damage. For example, Tindall and Stout (2001) reported that severe damage by insect foliar herbivores was correlated with a significant reduction in the body size of an insect herbivore feeding on root tissues of the same host plant, compared with conspecifics developing on roots of plants without foliar herbivores. Other studies have shown similar effects (Masters et\u00a0al. 1993; Hunt-Joshi and Blossey 2005). However, the effects of foliar herbivory on the development of natural enemies in the soil, such as predators and parasitoids, remain largely unexplored.\nThus far, the effects of above-ground herbivory on the performance of below-ground consumers have been explained mainly by quantitative changes in the amount of root biomass, when an extreme amount of leaf damage results in partial or complete death of the root system. However, minor levels of shoot damage, while not leading to a significant reduction in root biomass, may still affect the quality of the root tissues influencing the development of root-associated organisms (reviewed in Bezemer and Van Dam 2005). Furthermore, interactions between above- and below-ground herbivores may also be mediated by qualitative changes in the shared host plant. For example, insect herbivory frequently leads to a systemic defense response within the host plant, resulting in enhanced concentrations of secondary plant compounds in plant organs that have not been damaged by herbivory (Karban and Baldwin 1997). Secondary plant compounds commonly have a negative impact on herbivore performance (Karban and Baldwin 1997). Several studies have shown that root herbivory can result in enhanced concentrations of secondary plant compounds in the foliage, although the reverse, e.g., the effects of foliar herbivory on changes in concentrations of root secondary plant compounds, has rarely been reported (reviewed in Bezemer and Van Dam 2005). Moreover, herbivory can induce a stress response within the host plant, which can lead to a reallocation of plant compounds such as carbohydrates and soluble nitrogen between root and shoot tissues, and the concentration of these compounds is known to directly affect insect growth parameters (Masters et\u00a0al. 1993; Masters and Brown 1997). It is important to stress that the mechanisms described above are not necessarily mutually exclusive.\nIn this study, we examined the effects of above-ground herbivory on the development and survival of a below-ground insect herbivore and its endoparasitoid. Our study is based on interactions involving a naturally occurring system in Western Europe. The black mustard, Brassica nigra L. (Brassicaceae), is a widely distributed annual crucifer that is common along rivers and on disturbed sites in The Netherlands (Schaminee et\u00a0al. 1998). It is attacked by several adapted specialist herbivores, including the large cabbage white butterfly, Pieris brassicae L. (Lepidoptera: Pieridae) whose larvae feed on the shoots and flowers of this and related species (Harvey et\u00a0al. 2003). Mustard plants are also attacked by specialized root-feeding herbivores, including the cabbage root fly, Delia radicum L. (Diptera: Anthomyiidae) (Coaker and Finch 1971). Trybliographa rapae (Westwood) (Hymenoptera: Figitidae) is one of the main parasitoids of D. radicum in Europe (Lahmar 1982). The main aim of this investigation was to determine whether the growth, development, and survival of D. radicum and its endoparasitoid, T. rapae, are affected by foliar herbivory by P. brassicae. We also examined whether above-ground herbivory leads to qualitative and\/or quantitative differences in root tissues. We compared levels of root biomass in plants with and without shoot damage, as well as levels of nitrogen in root tissues. Finally, we compared levels of allelochemicals (glucosinolates) in root tissues of plants under the two treatments. We hypothesized that above-ground herbivory by P. brassicae would result in reduced root-plant quality which would negatively affect the performance of D. radicum. Because T. rapae is a koinobiont parasitoid, D. radicum larvae continue feeding and growing during much of the parasitoid\u2013host interaction (reviewed in Harvey 2005). As a consequence of this close association between host and parasitoid, we hypothesized that the effects of P. brassicae on D. radicum would be carried over to T. rapae. Since parasitoids are highly susceptible to very small changes in the quality of the hosts, the effect on the parasitoid should be stronger than that on the host.\nMaterials and methods\nStudied species\nBrassica nigra, like other members of the Brassicaceae, possesses potent inducible direct defenses via the production of glucosinolates and their breakdown products; thus, the plant provides excellent potential for the study of multitrophic interactions (Van Dam et\u00a0al. 2003). Pieris brassicae is a specialist chewing foliar herbivore that feeds on several species of wild and cultivated crucifers. The female butterfly typically lays broods of 7\u2013140 eggs on the underside of a leaf. After hatching, the larvae feed gregariously until the third instar, when they disperse on the food plant. Delia radicum is a specialist chewing root herbivore that feeds on several species of crucifers. Females oviposit near the root-stem interface. The newly hatched maggots feed by boring galleries in the upper roots of the plant. Trybliographa rapae is a fairly specialized solitary koinobiont endoparasitoid that parasitizes L1\u2013L3 instar larvae of D. radicum, as well as several closely related species. Adult parasitoid females forage via antennation, ovipositor probing, and vibrotaxis in order to find their hosts (Vet and Van Alphen 1985). The wasps readily parasitize larvae that are actively feeding in the root galleries but refuse hosts extracted from the root (Jones 1986). Females use their sharp, but flexible, ovipositor to explore infested roots to find hosts and evaluate their quality (Neveu et\u00a0al. 2000).\nSeeds of B. nigra were collected from a single population in the Northwest of Wageningen, The Netherlands. Seeds were surface sterilized and germinated on a bed of glass pearls (pearls of 1\u00a0mm diameter). One week after germination, seedlings were transplanted into 1.2-l pots, filled with soil collected from a restoration area that was abandoned in 1996, at De Mossel in Ede, The Netherlands. The sandy loam soil was sieved (2\u00a0cm) and mixed with 10% white sand (to improve the drainage of the soil), and sterilized using gamma radiation (25 KGray) to eliminate all soil organisms. The plants were grown in a greenhouse, at 70% relative humidity, at 22\u00a0\u00b1\u00a01\u00b0C (day) and 16\u00a0\u00b1\u00a01\u00b0C (night) on a 16-h:8-h day:night cycle. Natural daylight was provided by metal halide lamps (225\u00a0\u03bcmol\u00a0s\u22121\u00a0m\u22122 PAR; 1 lamp per 1.5\u00a0m2). Plants were watered daily and supplemented with nutrients (Hoagland solution) once when the plants were 3\u00a0weeks old. Pieris brassicae was obtained from the insect culture maintained at the Laboratory of Entomology of Wageningen University, The Netherlands. D. radicum and T.rapae were obtained from insect cultures maintained at the Laboratory of Ecobiology of Insect Parasitoids, of Rennes University, France.\nExperimental design and insect measurements\nTo test the effect of above-ground herbivores on the performance of soil insects, we reared D. radicum and its parasitoid T. rapae on roots of plants previously exposed (treatment) and not exposed (control) to P. brassicae larvae. Twenty-five days after transplanting, three newly hatched L1 larvae of P. brassicae were carefully placed with a small brush on the youngest fully developed leaf of each plant, and enclosed in a cylindrical clip cage (5.5\u00a0cm diameter) in order to ensure that the damage was localized. After 3\u00a0days, the larvae were moved to another new leaf on the plant. The larvae were allowed to feed for a further 3\u00a0days, after which each larva and clip cage was removed. Thus, each foliar-damaged plant was exposed to feeding by three L1 P. brassicae larvae for a total of 6\u00a0days. A second set of plants was kept undamaged and served as control. To standardize the possible effects of the clip cages on plant shoots, they were also placed on control plants, following the same methods as for the foliar-damaged plants. A total of 72 plants were used in the experiment (36 plants with and 36 without foliar herbivore damage).\nOne day after removing the P. brassicae larvae, four L1 D. radicum larvae were introduced to each plant by carefully placing the larvae with a brush onto the soil surface immediately adjacent to the stem. Using a magnifying glass, each plant was carefully checked 30\u00a0min after inoculation, to ensure that all the root fly larvae had successfully entered into the soil. For each plant, two of the larvae were healthy and two had been parasitized by T. rapae within approximately the prior 12\u00a0h. In a previous study, we reported that there was no significant difference in final root biomass in B. nigra plants with initial densities of five unparasitized D. radicum larvae per plant when compared with undamaged plants (Soler at al. 2005). Furthermore, parasitized larvae consume less plant material and attain smaller final masses than healthy hosts (Grandgirard 2003); thus, we assumed that plant root biomass would not be a limiting factor for root herbivore\/parasitoid development. It is also important to stress that larvae of D. radicum are not cannibalistic (A.M. Cortesero, unpublished observations), making it possible for unparasitized and parasitized larvae to share a host plant.\nTo obtain parasitized D. radicum larvae, approximately 300 first instar larvae were offered to 60 mated T. rapae females over the course of 4\u00a0h. To facilitate and ensure high rates of parasitism, a slice of B. napus (turnip) root of 1.5\u20132\u00a0cm in height (15\u00a0cm diameter) that had previously been infested with the larvae of D. radicum was offered to the parasitoid females. The larvae were collected from the root after 4\u00a0h, and 144 larvae were then transferred to the experimental B. nigra plants. Because direct observations of parasitism are almost impossible in T. rapae, 100 additional larvae from the infested turnip slice were removed and placed on a new turnip, where parasitoid emergence was monitored. From these 100 larvae, 92 T. rapae adults emerged, and none produced D. radicum adults.\nFollowing the below-ground inoculation of unparasitized and parasitized D. radicum larvae, experimental plants were placed individually into large meshed cylindrical cages (height 1\u00a0m, diameter 35\u00a0cm) to ensure that newly emerged adult flies and wasps did not escape and could be immediately captured. Plants from the two treatments were randomly distributed in the greenhouse and were moved around the room every 2\u20133\u00a0days to avoid any potential effects of positioning on insect development. The cages were checked twice daily (once in the morning and once in the afternoon) to record the emergence of adult flies and parasitoids. Newly emerged adults of D. radicum and T. rapae were collected with an aspirator. At emergence, the following data were recorded: (1) development time between inoculation and adult eclosion in days and (2) adult body size, based on the measurement of hind tibia length in both the herbivore and parasitoid. Hind tibia length has been commonly used as a measure of adult size in many insects, especially parasitoids (Godfray 1994; Harvey et\u00a0al. 1994; Bezemer and Mills 2003). The hind tibia lengths of D. radicum and T. rapae were measured using a calibrated slide under a stereomicroscope. Adult survival (calculated as the proportion of insects that emerged relative to the number of larvae introduced in each pot) was also recorded.\nPlant responses\nBecause the measurement of glucosinolate and nitrogen concentrations requires the destructive sampling of plants, root samples were taken from an additional set of plants (nine plants per treatment). The plants in the additional set were treated in exactly the same way as the foliar-damaged and control plants described above. Root material was collected 1\u00a0h before the other set of experimental plants was inoculated with parasitized and unparasitized D. radicum larvae to evaluate the quality of the control and foliar-damaged plants that larvae encountered after inoculation. After carefully washing the roots of each plant, the fine roots were separated from the main root. Roots less than 1\u00a0mm thick were considered fine (with a hair-like appearance) and those thicker than 1\u00a0mm were considered to be main roots. The roots were immediately frozen at \u221280\u00b0C, then freeze-dried and ground. Glucosinolate contents were determined using high performance liquid chromatography (HPLC) as described by Van Dam et\u00a0al. (2004). Nitrogen content was determined using a Carlo Erba\/Fisons, type EA1108, CHN analyzer. The total root biomass of the experimental plants was determined at the end of the experiment. The roots of each plant were harvested, oven-dried at 70\u00b0C and, subsequently, weighed on a microbalance.\nStatistical analysis\nThe effects of foliar herbivory (with or without) and larval status (parasitized or unparasitized) on survival of D. radicum and T. rapae were tested using a generalized linear mixed model (with binomial distribution and logit link function, including plant as random term). The effects of foliar herbivory on developmental time and hind tibia length of D. radicum and T. rapae were tested using a generalized linear model (with normal distribution and identity link function). The effects of foliar herbivory and root type (primary or secondary roots) on plant quality (nitrogen and glucosinolate levels) were tested using a linear mixed model, including plant as random term to consider that the fine and main roots were sampled from the same plants. To test the effect of foliar herbivory on plant quantity (root biomass), we used analysis of variance. Normality, independence, and homogeneity of variance were checked by inspection of the residuals after model fitting. Analyses were carried out in Statistica and Genstat.\nResults\nFoliar herbivory significantly affected the survival of the root herbivore D. radicum and its parasitoid T. rapae (Wald\u00a0=\u00a012.17, P\u00a0<\u00a00.001). Relative to control plants, the survival of D. radicum and T. rapae decreased by more than 50%, when the host plants were previously exposed to foliar herbivory (Fig.\u00a01), and the effect was qualitatively similar for root herbivore and parasitoid responses indicated by a non-significant interaction term (Wald\u00a0=\u00a00.49, P\u00a0=\u00a00.48). There was a significant effect of foliar herbivory on D. radicum and T. rapae adult size, measured as hind tibia length (Wald\u00a0=\u00a05.88, P\u00a0=\u00a00.02). Both herbivore and parasitoid adult sizes were reduced in the presence of foliar herbivores on the shared host plant (Fig.\u00a02). Again, the negative effect of foliar herbivory did not differ between host and parasitoid (interaction term Wald\u00a0=\u00a00.02, P\u00a0=\u00a00.86). Development times of D. radicum and T. rapae were not significantly affected by foliar herbivory (Wald\u00a0=\u00a03.34, P\u00a0=\u00a00.07).\nFig.\u00a01Mean (\u00b1SE) survival of D. radicum (herbivore) and T. rapae (parasitoid) on B. nigra plants that had been exposed to foliar herbivory (by P. brassicae) (shaded bars), and on control plants (open bars). Within herbivore and parasitoid, significant differences (P\u00a0<\u00a00.05) between treatments are indicated by different lettersFig.\u00a02Mean (\u00b1SE) hind tibia length of D. radicum (herbivore) and T. rapae (parasitoid) on B. nigra plants that had been exposed to foliar herbivory (by P. brassicae) (shaded bars), and on control plants (open bars). Within herbivore and parasitoid, significant differences (P\u00a0<\u00a00.05) between treatments are indicated by different letters\nPlants under both treatments demonstrated similar root biomass (F1,30\u00a0=\u00a00.008, P\u00a0=\u00a00.92; Fig.\u00a03a). Root-nitrogen levels also did not differ significantly between control plants and plants exposed to foliar herbivory (Table\u00a01). Independent of the treatment, nitrogen levels were significantly lower in secondary than in primary roots (Table\u00a01). Foliar herbivory significantly affected the levels of indole glucosinolates (neoglucobrassicin and glucobrassicin) in the roots (Table\u00a01). In the presence of foliar herbivory, the levels of neoglucobrassicin (Fig.\u00a04a) and glucobrassicin (Fig.\u00a04b) significantly increased in both primary and secondary roots. Independent of the treatment, levels of neoglucobrassicin and glucobrassicin were significantly higher in secondary than in primary roots. Concentrations of gluconasturtin (Fig.\u00a04c) and sinigrin (Fig.\u00a04d) did not differ significantly between the roots of control plants and plants that had been exposed to foliar herbivory (Table\u00a01). Independent of the treatment, gluconasturtin levels were significantly higher in primary than in secondary roots. Sinigrin levels did not differ significantly between primary and secondary roots.\nFig.\u00a03Mean (\u00b1SE) root biomass (a) and percentage of nitrogen in primary and secondary roots (b) of Brassica nigra plants that had been exposed to foliar herbivory (by P. brassicae) (shaded bars) and in those of control plants (open bars )Table\u00a01Approximate F-test (F and P values) for the fixed effects and variance component estimates from REML (restricted maximum likelihood); analysis of the effect of foliar herbivory (by P. brassicae) and root-type on nitrogen and glucosinolate levels of B. nigra rootsFactorsNitrogenNeoglucobrassicinGlucobrassicinGluconasturtinSinigrinFPFPFPFPFPFoliar herbivory (1)1.60.215.0<0.00115.3<0.0010.50.41.70.2Root-type (2)28.4<0.00128.5<0.00189.5<0.0014.570.030.0010.9Interaction (1.2)0.40.52.90.080.20.50.70.31.40.2Fig.\u00a04Mean (\u00b1SE) levels of glucosinolates (\u03bcmol\u00a0g\u22121 dry mass) (a neoglucobrassicin, b glucobrassicin, c gluconasturtin, d sinigrin) in primary and secondary roots of Brassica nigra plants exposed to foliar herbivory (by P. brassicae) (shaded bars) and in those of control plants (open bars). Within each root type, significant differences (P\u00a0<\u00a00.05) between treatments are indicated by different letters\nDiscussion\nThis study provides evidence that shoot-feeding insects, via systemic changes in the root quality of the shared host plant, can influence the development of root-feeding insects sharing the host plant. Moreover, the effects can be carried over at least one trophic level higher, affecting the performance of a parasitoid. We found that foliar herbivory was significantly correlated with a reduction in successful development of Deliaradicum and Trybliographarapae. Survival of the herbivore and its parasitoid was two to three times higher on plants that were undamaged above-ground than on plants that were previously exposed to foliar herbivory. In addition, when compared with those on control plants, newly emerged adult D. radicum and T. rapae were significantly smaller when the insects developed on plants that had been initially damaged by foliar herbivores. Several authors have argued that body size is the main target of selection in parasitoids and other insects, because it is correlated with life-history and demographic characters such as searching and mating efficiency, longevity, and fecundity (Godfray 1994; Harvey et\u00a0al. 1994; Bezemer and Mills 2003; Harvey 2005). Consequently, our results reveal not only that D. radicum and T. rapae suffer from increased mortality on plants previously exposed to foliar herbivory compared with control plants, but that fitness of the surviving adults is also compromised.\nIn a recent study based on the same multitrophic system, we reported that root herbivory by D. radicum reduced the performance of the foliar feeder P. brassicae (Soler et\u00a0al. 2005). However, several others found no significant relationship between root herbivory and the performance of above-ground herbivores (Moran and Whitham 1990; Salt et\u00a0al. 1996; Hunt-Joshi and Blossey 2005), and yet others found a positive relationship (Masters and Brown 1992). These results clearly show that the effects of below-ground herbivores on their above-ground counterparts are likely to be association-specific, although this contrasts with the rather consistent negative effect thus far observed in most reciprocal studies of above-ground herbivory on below-ground insects (this study; Moran and Whitham 1990; Masters and Brown 1992; Salt et\u00a0al. 1996; Tindall and Stout 2001; Hunt-Joshi and Blossey 2005). More work is clearly required to elucidate possible patterns in interactions between above- and below-ground systems, and to determine whether there are any clear patterns emerging from them.\nWe also found that the amount of root biomass did not differ significantly between the foliar-damaged and control plants, suggesting that the negative effects of above-ground herbivory by P. brassicae on the development of D. radicum and T. rapae were based on qualitative, rather than quantitative, changes in the root system. Because only three young caterpillars were allowed to feed from the foliage for a limited period of time, the physical amount of shoot tissue removed was negligible (R. Soler, personal observation). Changes in root biomass as a result of source\u2013sink relationships within the plant are likely to occur when above-ground herbivore damage is much more extensive (Hunt-Joshi and Blossey 2005). Root nitrogen levels were also not significantly affected by foliar herbivory, and thus it is highly unlikely that changes in this important (and often limiting) primary plant compound were responsible for the observed effects.\nHowever, levels of indole glucosinolates were significantly increased by about 30% in plants that had been exposed to foliar herbivores. Glucosinolates and their breakdown products, including indoles, have been shown to be involved in reducing plant quality for both generalist and specialist insect herbivores and nematodes (Li et\u00a0al. 2000; Van Dam et\u00a0al. 2005). Although indoles constituted only a small fraction of total glucosinolates in the plant (with sinigrin being by far the most prevalent), studies with other secondary compounds have shown that only trace amounts in the diet are required to exert strong effects on the development of herbivores and their parasitoids (Barbosa et\u00a0al. 1986). Consequently, the negative relationship between above-ground herbivory and the development of D. radicum may be, at least partially, correlated with increased levels of indole glucosinolates in the roots of the shared host plant that were induced by feeding damage from P. brassicae larvae above ground.\nIt has been well established that allelochemicals in the herbivore\u2019s diet can exhibit negative effects on the morphology, development, size, and survival of their predators and parasitoids (reviewed in Harvey 2005; Ode 2006). T. rapae is a koinobiont parasitoid of D. radicum and, therefore, after being parasitized, Delia larvae continue feeding and growing until the final larval instar has been reached. At this time, the developing parasitoid larvae begin destructive feeding and exponential growth (Harvey et\u00a0al. 1994). At the end of the host\u2019s larval development, allelochemicals that may be present in the hemolymph and other tissues, such as fat body, are rapidly consumed by the koinobiont parasitoid (Harvey 2005). Due to the tight linkage between host and koinobiont parasitoid development, in our study, increased mortality and reduced adult size observed in the T. rapae adults on plants that had been exposed to foliar herbivory could probably be attributed to a corresponding increased mortality and reduced size of its host D. radicum. Parasitoids, in particular, are highly susceptible to very small changes in the quality of the host\u2019s internal biochemical environment (Harvey 2005). Other studies have shown that parasitoids can be more affected than their hosts by the quality of the host plant (Harvey et\u00a0al. 2003; Soler et\u00a0al. 2005). This appears not to be the case in our studied trophic chain, since we found comparable effects of foliar herbivory on the root herbivore and its parasitoid.\nIn summary, this study has reported that herbivore damage of plant shoots can have significant negative effects on the development of below-ground herbivores and their natural enemies. Combined with the results of a previous study based on the same system, we have demonstrated that these effects are largely reciprocal (Soler et\u00a0al. 2005). Our research further highlights the importance of integrating the below-ground domain with above-ground systems in order to better understand the myriad of factors that shape the evolution, assembly, and functioning of communities and ecosystems (Wardle et\u00a0al. 2005). Studies over larger spatial scales have already shown that above-ground herbivores can influence the community structure of the below-ground system and vice-versa (De Deyn and Van der Putten 2005; Wardle et\u00a0al. 2004). Further studies, combining interactions between above-ground and below-ground communities in a multitrophic framework, and which seek to explore both processes and mechanisms, are required if we are to develop a better understanding of the many complex factors that influence community structure and function.","keyphrases":["delia radicum","trybliographa rapae","glucosinolates","above-\/below-ground interactions","plant\u2013insect interactions"],"prmu":["P","P","P","M","M"]}
{"id":"Exp_Brain_Res-3-1-2137946","title":"Children with cerebral palsy exhibit greater and more regular postural sway than typically developing children\n","text":"Following recent advances in the analysis of centre-of-pressure (COP) recordings, we examined the structure of COP trajectories in ten children (nine in the analyses) with cerebral palsy (CP) and nine typically developing (TD) children while standing quietly with eyes open (EO) and eyes closed (EC) and with concurrent visual COP feedback (FB). In particular, we quantified COP trajectories in terms of both the amount and regularity of sway. We hypothesised that: (1) compared to TD children, CP children exhibit a greater amount of sway and more regular sway and (2) concurrent visual feedback (creating an external functional context for postural control, inducing a more external focus of attention) decreases both the amount of sway and sway regularity in TD and CP children alike, while closing the eyes has opposite effects. The data were largely in agreement with both hypotheses. Compared to TD children, the amount of sway tended to be larger in CP children, while sway was more regular. Furthermore, the presence of concurrent visual feedback resulted in less regular sway compared to the EO and EC conditions. This effect was less pronounced in the CP group where posturograms were most regular in the EO condition rather than in the EC condition, as in the control group. Nonetheless, we concluded that CP children might benefit from therapies involving postural tasks with an external functional context for postural control.\nIntroduction\nPoor postural control has been suggested to underlie the delays and deviations in motor skill acquisition and development observed in children with cerebral palsy (CP) (e.g., Berger et al. 1984; Liao et al. 1997; Liao and Hwang 2003). In particular, children with CP encounter problems during static upright standing in altered sensory environments (Cherng et al. 1999; Rose et al. 2002) and when rapid weight shifts during standing are required, either in gait initiation (Stackhouse et al. 2007) or in reaction to external perturbations (Nashner et al. 1983; Woollacott and Burtner 1996).\nPostural control is often assessed by means of posturography, that is, the quantitative analysis of centre-of-pressure (COP) trajectories as measured with a force platform. Several descriptive statistics of the COP time series, which are often derived by averaging out the assumed noisy or random character of postural sway, have been shown to change with various motor, sensory and cognitive processes involved in the control of standing (e.g., Horak 2006; Woollacott and Shumway-Cook 2002). In the past two decades, however, new concepts and methods for studying postural control have been introduced based on the assumption that the act of maintaining an erect posture may be viewed as a stochastic process (Collins and De Luca 1993; Newell et al. 1997; Riley et al. 1999; Frank et al. 2001). Based on these insights, new measures have been developed for examining the time-varying or dynamical characteristics of COP trajectories. These dynamical measures may be more informative with regard to changes in postural control with task constraints, aging or pathology than the more global summary statistics that have been typically employed for this purpose, or at least provide important additional information (see Baratto et al. 2002; Raymakers et al. 2005; Roerdink et al. 2006).\nRecent research has suggested that the regularity of COP trajectories is a particularly revealing and theoretically important dynamical measure. As a case in point, Roerdink et al. (2006) found that COP trajectories were more regular (as indexed by reduced sample entropy) in stroke patients than in healthy elderly while COP trajectories became progressively less regular with recovery. These findings may be viewed in line with the notion of pathological regularity versus healthy complexity (e.g., Goldberger 1996, 1997). Moreover, when performing a secondary cognitive task COP trajectories became less regular (Roerdink et al. 2006). The latter finding was replicated in young healthy adults, albeit only in the more attention-demanding eyes closed condition (Donker et al. 2007). Based on these results, a direct relation was proposed between the regularity of COP time series and the amount of attention invested in postural control (or, inversely, between COP regularity and the degree of automatism of postural control; see Donker et al. 2007; Roerdink et al. 2006). In particular, the introduction of a secondary cognitive task withdraws attention from the regulation of posture (i.e., towards the performance of the attention-demanding secondary task), resulting in less regular COP trajectories. This interpretation is in line with the claim and corresponding findings of Wulf and colleagues that adopting an external focus of attention is beneficial for the performance and learning of motor skills, whereas an internal focus of attention (e.g., directing attention to one\u2019s own body and bodily movements) can be detrimental as it may disrupt the \u2018automatisms\u2019 with which well-learned skills are normally performed (for a review see Wulf and Prinz 2001; see also Wulf et al. 2001; McNevin and Wulf 2002; McNevin et al. 2003).\nWhen applied to CP children this insight may imply that their postural control may be improved by means of instructions or task conditions that direct their attention away from the regulation of posture itself to its consequences in the world. Providing CP children with concurrent visual feedback about postural sway may help create a more external focus of attention (i.e., the external consequences of postural sway become readily apparent via visual cursor movements). Visual feedback of postural sway provides a functional context for postural control (e.g., minimise cursor movements), which is likely to enhance the performance of the primary task (cf., Lin et al. 1998; Van der Weel et al 1991; Volman et al. 2002). For example, Volman et al. (2002) showed that the quality of reaching movements of the affected arm in children with spastic hemiparesis could be enhanced when reaching to press a light switch to turn on a light (task with functional context) compared to reaching to a marker (non-functional task). We therefore expected that postural control could benefit from postural visual feedback as it constitutes a task with a functional external context for postural control, inducing a more external focus of attention. Although postural visual feedback is sometimes used by physical therapists to train postural control and steadiness (i.e., the ability to maintain a given posture with minimal sway, Nichols 1997), evidence for this practice is limited (e.g., Rougier 2003; Ledebt et al. 2005). The recent conceptual and methodological developments in the analysis of COP trajectories alluded to in the preceding provide an alternative approach for studying the consequences of concurrent visual feedback for postural control and thus of its potential for possible therapeutic applications.\nMotivated by this combination of theoretical and practical considerations, the aim of the present study was (1) to compare postural sway fluctuations of children with CP with that of typically developing (TD) children and (2) to investigate the effect of visual information on postural sway in CP children and in TD children. For this purpose, the availability of visual information was manipulated relative to standing with eyes open (EO condition) by means of visual deprivation (standing with eyes closed, EC condition) and external provision of COP feedback (FB condition). Two kinds of measures were used to analyse the COP trajectories recorded during those conditions, the one scale-dependent and the other scale-independent. The conventional, scale-dependent measures pertained to the \u2018amount of sway\u2019, whereas the more recent scale-independent measures pertained to the \u2018regularity of sway\u2019 (see Methods section for details). We hypothesised that (1) compared to TD children, CP children exhibit a greater amount of sway and more regular sway and (2) concurrent visual feedback (providing a functional external context for postural control, creating a more external focus of attention) decreases both the amount of sway and sway regularity in TD and CP children alike, while closing the eyes (increasing task difficulty through deprivation of visual information, promoting a less automatic mode of postural control) has opposite effects.\nMethods\nParticipants\nTen children with congenital hemiplegia or cerebral palsy (CP children; six boys, four girls; mean age\u00a0=\u00a07\u00a0years, range\u00a0=\u00a05\u201311\u00a0years), reduced to nine in the analyses (see below) and nine typically developing children (TD children; five boys, four girls; mean age\u00a0=\u00a08\u00a0years, range\u00a0=\u00a05\u201311\u00a0years) without known motor impairments or movement-related disorders volunteered to participate in the experiment. All children and their parents gave their informed consent prior to participation. The experiment was carried out in compliance with the Helsinki Declaration and was endorsed by the VU University Medical Centre Amsterdam.\nProcedure and apparatus\nChildren stood upright on a 1\u00a0m\u00a0\u00d7\u00a01\u00a0m custom-made strain gauge force plate1 with their arms hanging relaxed alongside the body. Their feet were placed parallel to the anterior\u2013posterior axis of the force plate and the distance between the feet corresponded to the width of the pelvis. Children wore their own shoes, including ankle-foot ortheses and\/or insoles to correct for leg-length discrepancies.\nChildren participated in three conditions that were presented in random order: standing upright with eyes open (EO condition), standing upright with eyes closed (EC condition) and standing upright with visual COP feedback (FB condition). As explained, the FB condition was introduced to provide an external functional context for postural control, creating a more external focus of attention. To this end, a 2.5\u00a0m\u00a0\u00d7\u00a02.5\u00a0m vertical screen was placed in front of the child at a distance of 1.3\u00a0m. On this screen the force plate was depicted as a 40\u00a0cm\u00a0\u00d7\u00a040\u00a0cm square onto which the COP was projected as a red dot. The children were asked to keep this dot within the target area, which consisted of a 4\u00a0cm\u00a0\u00d7\u00a04\u00a0cm square. Deprivation of visual postural sway information was effectuated by blindfolding the children in the EC condition. Trial duration was 60\u00a0s. Participants performed each condition once, with the proviso that, as part of a separate study, five CP children performed each condition twice (the first trial was used for further analyses). Because in the latter set of recordings trial duration was 30\u00a0s, the first 3,000 samples of all trials were used in the analyses to avoid differences in the reliability of the dependent measures. One CP child did not want to be blindfolded and was therefore excluded from further analyses, which rendered the number of CP children equal to the number of control subjects.\nData analysis\nPrior to all analyses, the mean was subtracted from mediolateral and anterioposterior COP trajectories, which transformed the original time series into x and y time series, respectively. Subsequently, x and y time series were bi-directionally filtered (second-order low-pass Butterworth filter, cut-off frequency 12.5\u00a0Hz) and the resultant distance time series r was constructed using \n(see Prieto et al. 1996), with i\u00a0=\u00a01, 2, 3,..., N and N indicating the total number of analysed samples in the COP time series (i.e., 3,000 samples).\nThe \u2018amount of sway\u2019 was quantified by means of two conventional, scale-dependent measures (see Prieto et al. 1996). First, the average COP distance to the origin of the mean-centred posturogram was determined by taking the mean of the r time series (i.e., mean amplitude rmean in mm). Second, sway path length (SP in mm) was determined by taking the sum of the distances between consecutive points in the conventional posturogram, using .\nTo examine the structure of COP trajectories in more detail, independent of its size or scale, x and y were normalised to unit variance by dividing those time series by their respective standard deviations \u03c3x and \u03c3y, resulting in a normalised posturogram. Subsequently, two scale-independent COP measures were quantified. First, the sway path of the normalised posturogram (SPn) was determined in a similar manner as described above for the conventional posturogram. Because the posturograms were normalised to its size, differences in SPn could only be the result of changes in the structure of the posturogram, with a larger SPn indicating a larger amount of \u2018twisting and turning\u2019 or \u2018curviness\u2019 in the COP trajectory (cf., Donker et al. 2007). Second, COP regularity was quantified by calculating the sample entropy (cf., Richman and Moorman 2000; Lake et al. 2002; see also Roerdink et al. 2006), which is a slightly improved version of the approximate entropy (cf., Pincus 1991). The sample entropy (SEn) is the negative natural logarithm of an estimate of the conditional probability that subseries (epochs) of length m (in our case, m\u00a0=\u00a03) that match pointwise within a specific tolerance also match at the next point (for a more formal and detailed explanation see Richman and Moorman (2000; Lake et al. 2002). In other words, smaller SEn values imply more regular COP time series, that is, a greater likelihood that sets of matching epochs in a time series will be followed by another match within a certain tolerance. On the other hand, highly irregular COP time series are characterised by the fact that sets of matching epochs tend to be followed by data samples of different values, resulting in larger SEn estimates. In the present study, SEn was quantified from the r time series, which was first normalised to unit variance. To optimise the choice of the tolerance for a given m (see also Roerdink et al. 2006) we applied the approach of Lake et al. (2002) and selected the median value of the optimal tolerance over all trials (in our case, 0.05). The code used for calculating sample entropy was obtained from PhysioNet2 (Goldberger et al. 2000).\nTo rule out the possibility that COP trajectories exhibited regular or deterministic patterns by chance, we compared the SEn estimates obtained for the COP trajectories to those obtained for surrogate data (cf., Theiler et al. 1992). We used time-randomisation to preserve the probability distribution of the data (e.g., mean, variance) while destroying the temporal correlations in the data, resulting in very large values for SEn. In contrast, phase-randomisation randomises the phases of the data in the Fourier domain but preserves the probability distribution of the data and its spectral power distribution (cf., Kantz and Schreiber 2004). Consequently, estimates of SEn for phase-randomised COP data should be somewhat increased, although less pronounced as for the time-randomised COP data.\nStatistics\nA repeated-measures analysis of variance (ANOVA) was conducted on all dependent variables with group as between-subject factor (two levels: CP and TD children) and condition as within-subject factor (three levels: EO, EC and FB conditions). The effect of randomisation was evaluated using a one-way repeated measures ANOVA with the factor randomisation (three levels: original, phase-randomised and time-randomised time series), including each individual time series as a sample. Effect sizes for main and interaction effects are reported as partial eta squared (\u03b5p2). Besides significant effects (P\u00a0<\u00a00.05), also tendencies towards significance (P\u00a0<\u00a00.10) are reported in view of the limited sample size. Paired-samples t-tests were used for post-hoc analyses of significant condition or randomisation effects. The analyses were performed using SPSS (SPSS, Inc., Chicago IL, USA).\nResults\nThe results (F, P and \u03b5p2 values) of the group by condition ANOVA for the dependent COP variables are presented in Table\u00a01. In Fig.\u00a01, the group by condition effects are summarised for all dependent measures. CP children\u2019s sway amplitude was greater than that of TD children (i.e., rmean\u00a0=\u00a012.8\u00a0mm and 5.7\u00a0mm, respectively, although this difference was strictly speaking not significant, P\u00a0=\u00a00.062). Neither a significant main effect of condition nor a significant group by condition interaction effect was observed for rmean. Furthermore, no significant main or interaction effects were observed for the sway path in the conventional posturogram (SP; mean 1,367\u00a0mm, standard error 108\u00a0mm), whereas the sway path in the normalised posturogram (SPn) differed significantly between groups and conditions in the absence of a significant interaction (see Table\u00a01). Specifically, SPn was significantly larger for the TD children (328) than for the CP children (212). Moreover, post-hoc analysis revealed that concurrent visual feedback significantly increased SPn (310) compared to both standing with eyes open (SPn\u00a0=\u00a0259: t(17)\u00a0=\u00a02.41, P\u00a0<\u00a00.05) and standing with eyes closed (SPn\u00a0=\u00a0243: t(17)\u00a0=\u00a02.49, P\u00a0<\u00a00.05).\nTable\u00a01Main and interaction effects of group (between-subject factor, two levels: CP and TD children) and condition (within-subject factor, three levels: EO, EC and FB) on mean sway amplitude (rmean), sway path of the conventional posturogram (SP), sway path of the normalised posturogram (SPn) and sample entropy (SEn)GroupConditionInteractionF(1, 16)aP-value\u03b5p2F(2, 32)aP-value\u03b5p2F(2, 32)aP-value\u03b5p2rmean4.010.0620.201.35ns0.081.11ns0.06SP1.86ns0.100.13ns0.010.13ns0.01SPn11.11<0.0050.415.12<0.020.242.04ns0.11SEn13.49<0.0050.465.53<0.010.263.230.0560.17ns not significantaIn case the assumption of sphericity was violated, the number of degrees of freedom was adjusted using the Huynh\u2013Feldt method (corrected degrees of freedom are not listed here)Fig.\u00a01Effects of group (CP and TD children, represented by black and grey bars, respectively) and condition (EO, EC and FB) on the \u2018amount of sway\u2019 (upper panels, mean sway amplitude rmean and sway path of the conventional posturogram SP) and on the \u2018regularity of sway\u2019 (lower panels, sway path of the normalised posturogram SPn and sample entropy SEn; note that lower SEn values imply more regular posturograms). Error bars represent the standard error\nAs can be seen in Table\u00a01, the main effects of group and condition were both significant for sample entropy, while the group by condition interaction tended towards significance (P\u00a0=\u00a00.056). SEn was significantly larger for the TD children (1.01) than for the CP children (0.71). Moreover, post-hoc analysis revealed that the FB condition significantly increased SEn (0.98) compared to both standing with eyes open (SEn\u00a0=\u00a00.86: t(17)\u00a0=\u00a02.37, P\u00a0<\u00a00.05) and standing with eyes closed (SEn\u00a0=\u00a00.83: t(17)\u00a0=\u00a02.42, P\u00a0<\u00a00.05). The observed tendency of the group by condition interaction towards significance (see Fig.\u00a01, lower right panel) may be due to the fact that for CP children SEn increased gradually over EO, EC and FB conditions (0.71, 0.73 and 0.76, respectively), whereas in the TD children SEn first reduced in the EC condition (0.93) and then increased for the FB condition (1.21) as compared to standing with eyes open (1.01).\nThe surrogate analysis revealed that SEn differed significantly with randomisation (F(2, 106)\u00a0=\u00a02915.90, P\u00a0<\u00a00.001, \u03b5p2\u00a0=\u00a00.98). Post-hoc t-tests indicated that SEn differed significantly over all comparisons (all t(53)\u00a0>\u00a07.2, all P\u00a0<\u00a00.001), indicating that the regular or deterministic patterns observed in the original data did not occur by chance. SEn was on average (standard error) 0.89 (0.04), 1.06 (0.04) and 3.44 (0.02) for original, phase-randomised and time-randomised COP data, respectively.\nDiscussion\nIn the present study we compared posturograms of children with CP with those of TD children under three conditions. Across conditions, we expected the posturograms of CP children to reflect a greater amount of sway and more regular sway than those of TD children (Hypothesis 1). Furthermore, in line with the recently postulated relation between the amount of attention invested in posture and COP regularity, we expected that concurrent visual COP feedback, providing a functional external context for postural control leading to a more external focus of attention, would result in a decrease in both the amount of sway and sway regularity, whereas the EC condition, in which visual information is absent, would have opposite results (Hypothesis 2). The data were largely in agreement with both hypotheses, as will be discussed next. Furthermore, the finding that SEn estimates were significantly increased after time- and phase-randomisation of the original data ruled out the possibility that the regular or deterministic properties observed in the original data occurred by chance.\nCP children exhibit greater and more regular sway than TD children\nPosturogram characteristics of children with CP differed markedly from those of TD children (see Fig.\u00a01). As expected (Hypothesis 1), the CP children exhibited a larger amount of sway than the TD children. In particular, a near significant (P\u00a0=\u00a00.062, accompanied by a large effect size) increment in mean sway amplitude (rmean) was observed in the CP children compared to that in the TD children (see Table\u00a01 and Fig.\u00a01). Although, strictly speaking, not significant due to the large interindividual variations, this effect suggests that \u2018postural steadiness\u2019 is reduced in CP children, consistent with previous results on balance control in CP children (e.g., Cherng et al. 1999; Ferdjallah et al. 2002; Liao and Hwang 2003; Rose et al. 2002). An interesting and unexpected finding was that the total COP excursion (i.e., SP) did not differ significantly between CP and TD children. In combination with the apparent increase in sway amplitude, this finding hints at the presence of marked differences in the dynamical structure of posturograms between CP and TD children. To expose those differences, we focused on the sway regularity using two scale-independent measures. Scale-independence was achieved by normalising the data to unit variance. As expected, sway path length of the normalised posturogram was significantly reduced in CP children, indicating less \u2018twisting and turning\u2019 in the posturogram (see also Donker et al. 2007). Relatedly, resultant COP trajectories of the CP children were more regular than those of the TD children, as indexed by significantly lower sample entropy values. Recently, increased regularity in COP trajectories has been reported for a variety of pathological conditions, including athletes with a sports-related cerebral concussion (Cavanaugh et al. 2005 using approximate entropy), patients with Parkinson\u2019s disease (Schmit et al. 2006 using recurrence quantification analysis) and stroke patients (Roerdink et al. 2006 using sample entropy). These findings are in line with the more general notion of \u2018dynamical diseases\u2019 (Glass and Mackey 1988; Belair et al. 1995) and the \u2018pathological regularity versus healthy complexity\u2019 hypothesis (Goldberger 1996, 1997; Goldberger et al. 2002; Kyriazis 2003; Lipsitz 2002), according to which less \u2018complex\u2019 or more \u2018regular\u2019 physiological time series reflect less effective physiological control. In a similar vein, the observed increased COP regularity in CP children may be viewed as the dynamical signature of poor postural control.\nEffect of visual information\nWe further expected that the \u2018amount of sway\u2019 would be reduced in the presence of concurrent visual COP feedback, providing a supposedly beneficial functional external context for postural control (cf., Van der Weel et al. 1991; Volman et al. 2002; Wulf and Prinz 2001), as opposed to deprivation of visual information in standing with eyes closed. However, no significant main effects of condition were found for the scale-dependent COP measures (i.e., mean amplitude and sway path, see Table\u00a01). This unexpected result is in line with some recent studies showing that the use of visual COP feedback to facilitate quiet standing has no effect on conventional COP parameters in TD children (Lebiedowska and Syczewska 2000) and healthy elderly and stroke patients (Dault et al. 2003) alike. These findings suggest that participants may require some practice to benefit from visual COP feedback. This may explain why, in spite of the apparent lack of immediate effects, Ledebt et al. (2005) found that balance training with visual feedback improved CP children\u2019s quiet standing ability as well as their functional limits of stability as assessed by a weight-shifting task.\nIn contrast to the results obtained for the \u2018amount of sway\u2019 under the various conditions, the scale-independent measures pertaining to the \u2018regularity of sway\u2019 did show significant differences between the FB condition and the EO and EC conditions. This indicates that the dynamical structure of the posturogram must have been different in the FB condition compared to the EO and EC conditions, which was precisely what we anticipated when formulating our expectation that sway would be less regular in the FB condition (i.e., longer sway path of the normalised posturogram and higher sample entropy) and more regular in the EC condition (Hypothesis 2). Sway path of the normalised posturogram was indeed significantly longer with visual feedback than for standing with eyes open  or closed, indicating more twisting and turning (see also Donker et al. 2007). In parallel, sample entropy increased with concurrent visual feedback compared to standing with eyes open and eyes closed. Thus, in spite of the fact that the \u2018amount of sway\u2019 did not change significantly over conditions, the observed changes in \u2018regularity of sway\u2019 were as expected: sway was more regular in the EC condition and less regular in the FB condition.\nOur expectations concerning the \u2018regularity of sway\u2019 with conditions were derived from the proposed direct relation between the amount of attention invested in posture and the regularity of COP trajectories (cf., Donker et al. 2007; Roerdink et al. 2006). This interpretation is in line with the insight of Wulf and colleagues that adopting an external focus of attention, i.e., a focus on the consequences of one\u2019s movement in the external world, is beneficial for the performance and learning of motor skills (McNevin et al. 2003; McNevin and Wulf 2002; Wulf and Prinz 2001; Wulf et al. 2001). Specifically, we expected that providing CP children with concurrent visual feedback about postural sway would help shift the focus of attention to the external consequences of postural sway (i.e., cursor movements) by constituting a functional external context for postural control (e.g., minimise cursor movements). Providing a functional context for the task at hand generally enhances its performance (e.g., Lin et al. 1998; Van der Weel et al. 1991; Volman et al. 2002). In line with these notions, we expected that standing with concurrent visual feedback, a functional task inducing a more external focus of attention, would lead to less regular sway compared to the EO condition, whereas standing with eyes closed would lead to more regular sway in the EC condition compared to the EO condition because task difficulty increased due to the absence of visual information. This predicted pattern of results was only found for the TD children and not for the CP children (see lower right panel of Fig.\u00a01). As it turned out, COP regularity in the CP children gradually decreased from the EO condition (most regular), via the EC condition, to the FB condition (least regular). As a result of this difference in group behaviour, a strong tendency towards a significant group by condition interaction on sample entropy was found. From this latter observation one could tentatively infer that CP children\u2019s posture actually improved when standing with eyes closed compared to standing with eyes open. In support of this somewhat counter-intuitive inference, Rose et al. (2002) showed that the percentage of CP children with abnormal sway characteristics did not differ between EO and EC conditions, while the number of sway path outliers even reduced when standing with eyes closed in that particular CP group. Following a similar line of interpretation, Newell et al. (1997) suggested that very young children may create self-induced perturbations when using visual information in controlling posture, a phenomenon which may be particularly prominent in CP children given that the development of proper neural control mechanisms is strongly delayed compared to TD children (viz., Forssberg 1999). From the opposite SEn results between CP and TD children with eyes open and eyes closed it is fair to conclude that postural control in CP children is organised differently than in TD children, especially with regard to the use of normal sway-induced visual information.\nIt has been suggested that creating an external focus of attention would allow for a more automatic control of movements (e.g., Donker et al. 2007; McNevin and Wulf 2002; Roerdink et al. 2006), which is often found to be more efficient (e.g., Blanchard et al. 2005; McNevin et al. 2003). Although no significant main effect of condition on mean amplitude was found, the finding that during quiet standing with visual COP feedback sample entropy increased (i.e., less regularity in the posturogram) in both TD and CP children supports this idea. Moreover, it corroborates the suggestion that visual COP feedback may be advantageous when seeking to improve postural control, although it must be emphasised again that it requires training (see Ledebt et al. 2005).\nConclusion\nThe findings of the present study indicate that postural control in CP children is deteriorated compared to TD children, most likely due to a slowed development of neural control mechanisms in CP children. Moreover, visual deprivation (EC condition) influenced sway characteristics differently in CP and TD children, testifying to the altered nature of postural control in CP children. In line with the proposed relation between the amount of attention invested in posture and COP regularity, we found in CP and TD children alike that, compared to the EO and EC condition, COP trajectories were less regular in the presence of visual COP feedback, corresponding to a functional context mediated external shift in the focus of attention. Future studies should examine the merits of adopting an external focus of attention to enhance postural control in CP children in training or therapy, for example by providing visual COP feedback leading to a posture specific functional task or by introducing an attention-demanding secondary cognitive task.","keyphrases":["cerebral palsy","regularity","postural control","attention","centre-of-pressure trajectories"],"prmu":["P","P","P","P","R"]}
{"id":"Dig_Dis_Sci-3-1-1914242","title":"Gastrointestinal Endoscopic Terminology Coding (GET-C): A WHO-Approved Extension of the ICD-10\n","text":"Technological developments have greatly promoted interest in the use of computer systems for recording findings and images at endoscopy and creating databases. The aim of this study was to develop a comprehensive WHO-approved code system for gastrointestinal endoscopic terminology. The International Classification of Diseases, 10th edition (ICD-10), and the ICD-10 clinical modification (ICD-10-CM) were expanded to allow description of every possible gastrointestinal endoscopic term under conditions defined by the WHO. Classifications of specific gastrointestinal disorders and endoscopic locations were added. A new chapter was developed for frequently used terminology that could not be classified in the existing ICD-10, such as descriptions of therapeutic procedures. The new extended code system was named Gastrointestinal Endoscopic Terminology Coding (GET-C). The GET-C is a complete ICD-10-related code system that can be used within every endoscopic database program for all specific endoscopic terms. The GET-C is available for free at http:\/\/www.trans-it.org\/.\nIntroduction\nTechnological developments and the introduction of hospital information systems have greatly promoted interest in the use of computer systems for recording findings and images at endoscopy. Several database systems have been developed for this purpose, most working only as a report generator. These systems are, however, hampered by the lack of a specific code system for endoscopic diagnoses and terminology. As a result of this shortcoming, the composition of reports and storage of data differ considerably between systems. Systems use different database structures and variable terminology and are thus not compatible or comparable.\nThe International Classification of Diseases (ICD) is designed to promote international comparability in the collection, processing, classification, and presentation of mortality statistics. This ICD is developed, published, and maintained by the World Health Organization (WHO). The system is, in its current setting, not applicable for endoscopy, as it does not include descriptive terminology. However, endoscopic findings are classified according to their appearance. Adequate description of this appearance in uniform terminology is essential for the interpretation of endoscopy reports and allows important conclusions to be made with respect to therapy and prognosis. Thus it is important to uniformly store and code endoscopic findings. This is, however, hampered by the lack of a specific, uniformly applicable code system for endoscopic terminology and findings. In addition, only about 80% of report systems are able to use a code in their database [1].\nDifferent coding systems for medical data are being used. The ICD, 9th edition (ICD-9), and the clinical modification of the ICD-9 (ICD-9-CM) have already been used and proven to be of value in a specific and defined gastrointestinal endoscopic setting [2]. The ICD has been revised periodically to incorporate changes in the medical field. The tenth edition (ICD-10) differs from the ninth edition in several ways, although the overall content is similar. First, the ICD-10 has alphanumeric categories rather than numeric categories. Second, some chapters have been rearranged, some titles changed, and conditions have been regrouped. Third, the ICD-10 has almost twice as many categories as the ICD-9. The ICD-10 has been available since 1992 and was translated into Dutch in 1997 [3]. Other medical specialities, such as oncology and dentistry, have produced an extended code system for their field of interest based on the ICD-10 [4].\nThe aim of this project is to develop a comprehensive code system for gastrointestinal endoscopic terminology, based on the widely accepted ICD-10 code system, that can be used in any gastrointestinal endoscopic information system.\nMethods\nAs a basis for the new coding system the latest edition of the ICD, the ICD-10, was chosen. The reason for choosing this code system was that a predecessor has already been in use for some gastrointestinal endoscopic databases [2]. In addition, the current version of the ICD is now accepted in a majority of countries throughout the world [5].\nExtensions of the ICD-10 are made according to the recommendations of the WHO, without changing the structure of the ICD-10. The first four characters are never changed, but one or more characters are added behind these first four descriptors. In this way it is always possible to delete the extension to come back to the original ICD-10. We used the Dutch translation of the ICD-10. Also, the draft version of the ICD-10-CM of the National Center for Health Statistics (NCHS) was studied, which is available on the Internet [6]. Conflicts with this ICD-10 CM system were avoided, to rule out coding conflicts.\nA working group, the TRANS.IT working group, was founded, comprised of representatives from two academic hospitals (Utrecht, Amsterdam) and four general hospitals. This working group used the Endobase III system (Olympus). In this system, the endoscopist can choose among three methods of writing reports. Besides the Minimal Standard Terminology (MST) [7], text-blocks and standard reports are also available. A standard report is a complete report based on a diagnosis or a combination of diagnoses. With text-blocks, different portions of the text are combined to compose a complete report. These two variants enable more rapid generation of reports and are well accepted and widely used in the Netherlands [8].\nIn the TRANS.IT project group, which was founded as a peer reference group, standard reports, text-blocks, and linkage to the new code system were discussed.\nA list of every endoscopic finding, intervention, or complication described using one of the three methods of report writing available for TRANS.IT users was generated. All items on this list were linked to a specific code.\nResults\nWe first generated a list of the different fields necessary for evaluation of endoscopic data (Table\u00a01). The program automatically generated the data during the composition of endoscopic reports. All these fields are necessary to generate a comprehensive anonymous database with endoscopic information for extensive research. In addition, demographic data such as gender and age of patients and specialty of referring doctor are recorded.Table 1Different fields that need a specific code in endoscopic information systemsReason for endoscopyMedication useSedation and medication during endoscopyPreparationProcedure for investigationEndoscopic diagnosis\/findingsTherapeutic and diagnostic interventionsHistology resultsTherapy startedAdvice to referring doctorComplications\nWe included all available standard reports, text-blocks, and diagnoses of Minimal Standard Terminology (MST) accepted by the TRANS.IT group. The endoscopic findings, interventions, and complications were extracted and linked to a specific code. In this way, a total of 316 standard reports and 1571 text-blocks were coded, to assure coding of every item within this project.\nIn total, 2593 different items were extracted and received a specific code. Of these items, 630 (24%) could be coded with the existing ICD-10. Thus, 1963 new codes were required and added to the ICD-10 system, in order to be able to specifically code every endoscopic term.\nThe ICD-10 used was originally designed for mortality and discharge statistics. The problem with that design is that the structure used in the ICD-10 is not very detailed. This is true especially for gastrointestinal endoscopic terms, which do not always include a mortality risk but, most of the time, descriptive items essential for prognosis and therapy. For example, specific descriptions of peptic ulcers are relevant for determination of outcome risks, therapy, and prognosis. We therefore introduced the Forrest classification for description of gastric and duodenal ulcers [9]. We thus, for instance, changed the diagnosis K25.2 (acute gastric ulcer with both hemorrhage and perforation) to K25.21 (acute gastric ulcer with spurting bleeding and perforation; Forrest Ia) in the Gastrointestinal Endoscopic Terminology Coding (GET-C; Table\u00a02).Table 2Comparison of ICD-10 and GET-C codes for gastric ulcerICD-10DescriptionGET-CDescriptionK25.0Gastric ulcer, acute with hemorrhageK25.0Gastric ulcer, acute with hemorrhageK25.01Gastric ulcer, acute with spurting bleeding (Forrest Ia)K25.02Gastric ulcer, acute with nonspurting active bleeding (Forrest Ib)K25.1Gastric ulcer, acute with perforationK25.1Gastric ulcer, acute with perforationK25.2Gastric ulcer, acute with both hemorrhage and perforationK25.2Gastric ulcer, acute with both hemorrhage and perforationK25.21Gastric ulcer, acute with spurting bleeding and perforation (Forrest Ia)K25.22Gastric ulcer, acute with nonspurting active bleeding and perforation (Forrest Ib)K25.3Gastric ulcer, acute without hemorrhage or perforationK25.3Gastric ulcer, acute without hemorrhage or perforationK25.31Gastric ulcer, acute with visible vessel (Forrest IIa)K25.32Gastric ulcer, acute nonbleeding with overlying clot (Forrest IIb)K25.33Gastric ulcer, acute with hematin-covered basis (Forrest IIc)K25.34Gastric ulcer, acute with clean ulcer ground (Forrest III)K25.5Gastric ulcer, chronic or unspecified with perforationK25.5Gastric ulcer, chronic or unspecified with perforationK25.6Gastric ulcer, chronic or unspecified with both hemorrhage and perforationK25.6Gastric ulcer, chronic or unspecified with both hemorrhage and perforationK25.61Gastric ulcer, chronic or unspecified with spurting bleeding and perforation (Forrest Ia)K25.62Gastric ulcer, chronic or unspecified with non- spurting active bleeding and perforation (Forrest Ib)K25.7Gastric ulcer, chronic without hemorrhage or perforationK25.7Gastric ulcer, chronic without hemorrhage or perforationK25.71Gastric ulcer, chronic with visible vessel (Forrest IIa)K25.72Gastric ulcer, chronic non-bleeding with overlying clot (Forrest IIb)K25.73Gastric ulcer, chronic with hematin-covered basis (Forrest IIc)K25.74Gastric ulcer, chronic with clean ulcer ground (Forrest III)K25.9Gastric ulcer, unspecified, without hemorrhage or perforationK25.9Gastric ulcer, unspecified, without hemorrhage or perforationK25.91Gastric ulcer, unspecified, with visible vessel (Forrest IIa)K25.92Gastric ulcer, unspecified, nonbleeding with overlying clot (Forrest IIb)K25.93Gastric ulcer, unspecified, with hematin-covered basis (Forrest IIc)K25.94Gastric ulcer, unspecified, with clean ulcer ground (Forrest III)\nBesides such an adaptation of the ICD-10 system, endoscopy is still developing and new terms and endoscopic techniques are continuously being created. For example, new disease classifications are being introduced from time to time, such as the semirecent Los Angeles classification for reflux esophagitis [9, 10].\nFor several fields, there was no ICD-10 code available, and the item thus could not be categorized under an existing ICD-10 code. For example, the procedure for investigation and interventions could not be covered within the ICD-10 or even the ICD-10-CM system. Because these items are essential for good analysis of endoscopic data, a new chapter with the same structure as the ICD-10 was developed. The items in this new chapter start with the letters OG. We chose to categorize therapeutic interventions in this new chapter also because new therapies are developed frequently and can now be adapted.\nAdaptations were made in different chapters of the ICD-10. In Chapter 1, Certain Infectious and Parasitic Diseases (A00\u2013B99), the exact locations in the gastrointestinal tract of some specific infections were added. In Chapter 2, Neoplasms (C00\u2013D48), besides the exact locations in the gastrointestinal tract, the endoscopic characteristics of polyps were described and coded in more detail. It is important to register whether a polyp is pedunculated, sessile, or flat or has a villous endoscopic aspect. Also, the number of polyps found in a specific part of the intestine must be coded, because of important diagnostic and therapeutic options for patients and the follow-up of these patients.\nIn Chapter 9, Diseases of the Circulatory System (I00\u2013I99), hemorrhoids and varices were described according to a classification. Different gradings of protrusion were used for hemorrhoids. Paquet\u2019s endoscopic classification for esophageal varices was coded [11].\nMost changes were, however, made in Chapter 11, Diseases of the Digestive System (K00\u2013K93). The description of esophagitis was divided into different origins and severity. Gastric and duodenal ulcers were coded according to the Forrest classification and their specific location in the stomach or duodenum. Gastritis and duodenitis were coded according to their specific location and the endoscopically suspected cause. Inflammatory bowel diseases were coded according to their endoscopic severity and location. Some complications related to gastrointestinal procedures were coded in more detail.\nIn Chapter 18 symptoms, signs, and abnormal clinical and laboratory findings that are an indication for endoscopic investigations were coded in more detail. In Chapter 21 postoperative situations important for gastrointestinal endoscopic investigations such as gastric and colon operations were extensively coded.\nIn the new, thirteenth chapter of the GET-C, about 446 new codes were generated. These codes start with the letters OG. The first part of this chapter includes coding of various indications for endoscopy that could not be categorized in the ICD-10. The second part consists of specific codes for detailed locations in the gastrointestinal tract. The third part includes procedures, which are divided into diagnostic and therapeutic procedures. Diagnostic procedures include, for example, sampling of histology and culture specimens. Examples of therapeutic procedures are different kinds of polypectomies, endoscopic mucosal resections, placement of endoprotheses, and dilatation of stenoses. Another aspect of these therapeutic interventions was used for the management of gastrointestinal bleeding such as injection therapy with or without coagulation, band ligations, and clipping. Preparation and procedure for the endoscopic examination are also coded in this chapter.\nThe extensions of the GET-C were checked by the Dutch translation board of the ICD-10 to preclude any conflicts between the two code systems.\nDiscussion\nThe increased use of computer systems in health care and the need for communication between these systems necessitate the availability of generally accepted code systems. Good coding provides epidemiological information for research purposes. Coding is of importance for hospitals and professionals because most of the financial systems used in health care are based on different codes. In addition, good coding makes statistical analysis of different diagnoses and collection of rare diagnoses easier. With an internationally accepted code, it is possible to compare and share information in one field of interest. Most currently used coding systems are regional or national. Only some coding systems, such as the ICD-10, are translated into different languages and used in different countries.\nFor endoscopic report writing and endoscopic databases, different systems have been developed. These systems are now being used at many hospitals. We used the Endobase III system from Olympus Europe in our project to compose reports and record different codes. The system runs as a network version with different workstations as well as a stand-alone unit.\nThe Endobase III system enables the generation of reports via three pathways; in addition to standard reports and text-blocks, the latest translated version of the MST is also used to compose an endoscopic report. Thus it is essential that all differently generated data are coded in the same way to enable analysis of the data anonymously. In this system, all standard reports and text-blocks are linked to one or more specific GET-C codes. The choice of a standard report or text-block leads directly to the recording of the correct GET-C code in the Endobase III database. Due to the structure of the MST, the automatic link for it is more complex to realize. We chose to link the diagnoses with the GET-C code, which is separately selected within the MST by the endoscopist at the end of the report. Automatic linking is essential to ensure correct and complete selection of codes and to make the system workable for endoscopists.\nIn the TRANS.IT project, an anonymous central database of endoscopic investigations was built. In this central database, only coded data instead of free text can be collected, for privacy reasons. After 3 years of use, a database of about 120,000 investigations using the same coding system has been built by this working group. The GET-C system allows the encoding of any data collected during endoscopic investigations and recorded in any endoscopic database. By extending the ICD-10 with respect to the structure developed by the WHO, it is always possible to extract the original ICD-10 code from the GET-C. Thus, it is possible to link the endoscopic database with other health-care systems throughout the world.\nBecause endoscopy is an evolving medical specialty, new techniques are continuously becoming available. These require new codes, which will be discussed within the TRANS.IT project.\nThe Dutch Association of Gastroenterologists has accepted the GET-C. The GET-C will be available for use in other systems and was discussed at the WHO Family of International Classifications Network meeting in October 2004 [12]. The GET-C is available for free at http:\/\/www.trans-it.org. We hope that this coding system will help to make endoscopic databases and endoscopic report writing programs more meaningful.","keyphrases":["terminology","coding","database","international classification of diseases","icd-10 clinical modification","gastrointestinal endoscopy","10th edition (icd-10)"],"prmu":["P","P","P","P","P","R","R"]}
{"id":"Int_J_Med_Sci-4-2-1796947","title":"Genetic polymorphisms in the nucleotide excision repair pathway and lung cancer risk: A meta-analysis\n","text":"Various DNA alterations can be caused by exposure to environmental and endogenous carcinogens. Most of these alterations, if not repaired, can result in genetic instability, mutagenesis and cell death. DNA repair mechanisms are important for maintaining DNA integrity and preventing carcinogenesis. Recent lung cancer studies have focused on identifying the effects of single nucleotide polymorphisms (SNPs) in candidate genes, among which DNA repair genes are increasingly being studied. Genetic variations in DNA repair genes are thought to modulate DNA repair capacity and are suggested to be related to lung cancer risk. We identified a sufficient number of epidemiologic studies on lung cancer to conduct a meta-analysis for genetic polymorphisms in nucleotide excision repair pathway genes, focusing on xeroderma pigmentosum group A (XPA), excision repair cross complementing group 1 (ERCC1), ERCC2\/XPD, ERCC4\/XPF and ERCC5\/XPG. We found an increased risk of lung cancer among subjects carrying the ERCC2 751Gln\/Gln genotype (odds ratio (OR) = 1.30, 95% confidence interval (CI) = 1.14 - 1.49). We found a protective effect of the XPA 23G\/G genotype (OR = 0.75, 95% CI = 0.59 - 0.95). Considering the data available, it can be conjectured that if there is any risk association between a single SNP and lung cancer, the risk fluctuation will probably be minimal. Advances in the identification of new polymorphisms and in high-throughput genotyping techniques will facilitate the analysis of multiple genes in multiple DNA repair pathways. Therefore, it is likely that the defining feature of future epidemiologic studies will be the simultaneous analysis of large samples.\n1. Introduction\nSporadic cancer is a multifactorial disease that results from complex interactions between many genetic and environmental factors 1. This means that there will not be a single gene or single environmental factor that has large effects on cancer susceptibility. Environmental factors (e.g. tobacco smoke, dietary factors, infectious agents and radiation) add to the carcinogenic load to which humans are exposed, but exact numbers for added risk are generally less well established.\nCancer is the result of a series of DNA alternations in a single cell or clone of that cell, which leads to a loss of normal function, aberrant or uncontrolled cell growth and often metastasis. Several of the genes that are frequently lost or mutated have been identified, including genes that function to induce cell proliferation under specific circumstances (e.g. the ras and myc proto-oncogenes) and those which are programmed to halt proliferation in damaged cells (e.g. the TP53 and RB1 tumor suppressor genes). Other mutations in genes involved in DNA repair are also necessary. About 150 human DNA repair genes have been identified to date 2, but the real number is probably higher, since less than 50% of known and putative genes have an identified function. The association between defects in DNA repair and cancer was established by Cleaver in 1968 3, who showed that xeroderma pigmentosum (XP) is caused by deficient nucleotide excision repair (NER). For more than a quarter of a century after that it was thought that only rare syndromes, such as XP, Cockayne syndrome (CS) and ataxia telangiectasia, were associated with DNA repair defects 4. Novel, common polymorphisms in DNA repair genes are continuously being identified, and these polymorphisms may play a pivotal role in sporadic carcinogenesis. A growing body of literature, including observations of inter-individual differences in measures of DNA damage, suggests that these polymorphisms may alter the functional properties of DNA repair enzymes.\nAt least four pathways of DNA repair operate on specific types of damaged DNA. Base excision repair (BER) operates on small lesions, while the NER pathway repairs bulk lesions. Mismatch repair corrects replication errors. Double-strand DNA break repair (DSBR) actually consists of two pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ). The NHEJ repair pathway involves direct ligation of the two double strand break ends, while HR is a process by which double-strand DNA breaks are repaired through the alignment of homologous sequences of DNA. The following sections review the literature on DNA repair genes in more detail, specifically those involved in the NER pathway.\nNER is a versatile DNA repair system that removes a wide range of DNA lesions including UV-induced lesions. There are two subpathways in NER. One is transcription-coupled DNA repair (TCR), which preferentially removes DNA damage that blocks ongoing transcription in the transcribed DNA strand of active genes. The other is global genome repair (GGR), which removes lesions throughout the genome, including those from the nontranscribed strand in the active gene 5. Three rare, autosomal recessive inherited human disorders are associated with impaired NER activity: XP, CS and trichothiodystrophy (TTD) 6. XP has been studied most extensively. XP patients develop skin tumors at an extremely high frequency (1000 fold increased incidence as compared to normal individuals) because of their inability to repair UV-induced DNA lesions. These clinical findings are associated with cellular defects, including hypersensitivity to killing and the mutagenic effects of UV and the inability of XP cells to repair UV-induced DNA damage 7. Approximately 80% of XP patients who have been classified have a defect in the NER pathway. These patients are said to have \"classical\" XP, in contrast to the remaining 20% of patients who are designated as XP variants (XPV) and most likely have a defect in post-replication repair. In XPV patients, DNA replication stops or is interrupted at sites of UV-damage. Furthermore, de novo DNA synthesis opposite cyclobutane pyrimidine dimer lesions is prone to errors, leading to the fixation of multiple DNA mutations and ultimately to cancer. Seven different DNA NER genes, which correct seven distinct genetic XP complementation groups (XPA, XPB (ERCC3), XPC, XPD (ERCC2), XPE, XPF (ERCC4) and XPG (ERCC5, this gene causes CS)) and XPV have been identified 6. XPA, ERCC3\/XPB, ERCC2\/XPD, ERCC4\/XPF and ERCC5\/XPG have a defect in TCR and GGR, while XPC and XPE have a defect in GGR only. ERCC6 and ERCC8 are also known as CS type B (CSB) and CSA, respectively. Approximately 20% of patients have been assigned to the CSA complementation group Essentially CS shows some overlap with certain forms of XP. In contrast to XP and TTD, however, the NER defect in CS is limited to the TCR pathway. As with XP, TTD involves mutations in XP genes, usually XPD, which encodes a component of the transcription factor TFIIH 8. However, it has been suggested that the functions of XPD associated with TTD are distinct from those of XPD associated with XP. Approximately half of the patients with TTD display photosensitivity, correlated with the NER defect.\nThe aim of this article is to review and evaluate associations between genes in the NER pathway and lung cancer risk, focusing on genes encoding five key enzymes in this pathway: XPA, ERCC1, ERCC2\/XPD, ERCC4\/XPF and ERCC5\/XPG.\n2. Materials and methods\n2-1. Identification and eligibility of relevant studies\nWe conducted MEDLINE, Current Contents and Web of Science searches using \"XPA\", \"ERCC1\", \"ERCC2\/XPD\", \"ERCC4\/XPF\", \"ERCC5\/XPG\", \"lung cancer\" and \"polymorphism\" as keywords to search for papers published (from January 1, 1966 through May 31, 2006). Additional articles were identified through the references cited in the first series of articles selected. Articles included in the meta-analysis were in any language, with human subjects, published in the primary literature and had no obvious overlap of subjects with other studies. We excluded studies with the same data or overlapping data by the same authors. Case-control studies were eligible if they had determined the distribution of the relevant genotypes in lung cancer cases and in concurrent controls using a molecular method for genotyping. Using the MEDLINE database, we identified 5 genetic epidemiological studies 9-13 that provided information on lung cancer occurrence associated with the XPA G23A polymorphism (one of the identified 6 candidate studies was excluded due to overlapping data 11). We identified 5 studies of the ERCC1 T19007C polymorphism (all of 5 candidate studies were independent 13-17). We gathered 18 articles on the ERCC2 312\/751 polymorphisms found through literature searches and checked their references for additional relevant studies. Of the relevant 18 studies, 2 studies appeared to be on populations already reported 14, 18, 19, leaving 15 independent studies (11 studies for the Asp312Asn polymorphism 11, 13, 14, 17-24 and 14 studies for the Lys751Gln polymorphism 11, 13, 14, 17-19, 21-28. Less than 5 studies each have been reported on the ERCC1 C8092A, ERCC4\/XPF Arg415Gln, ERCC4\/XPF Ser835Ser, ERCC5\/XPG His46His, ERCC5\/XPG Asp1104His SNPs.\n2-2. Data extraction and assessment of study quality\nFor each study, characteristics such as authors, year of publication, ethnic group of the study population, source of control population, number of genotyped cases and controls, crude odds ratio (OR) and the method for quality control of genotyping were noted. For studies including subjects of different ethnic groups, data were extracted separately for each ethnic group whenever possible.\nMethods for defining study quality in genetic studies are more clearly delineated than those for observational studies. We assessed the homogeneity of the study population (Caucasian or Asian).\n2-3. Meta-analysis\nData were combined using both a fixed effects (the inverse variance-weighted method) and a random effects (DerSimonian and Laird method) models 29. The Cochrane Q statistics test is used for the assessment of heterogeneity. The fixed effects model is used when the effects are assumed to be homogenous, while the random effects model is used when they are heterogenous. In the absence of between-study heterogeneity, the two methods provide identical results. The presence of heterogeneity can result from differences in the selection of controls, age distribution, prevalence of lifestyle factors, histologic type of lung cancer, stage of lung cancer and so on. The random effects model incorporates an estimate of the between-study variance and tends to provide wider CIs when the results of the constituent studies differ among themselves. As the random effects model is more appropriate when heterogeneity is present 29, the summary OR and prevalence were essentially based on the random effects model. The meta-analyses were performed on crude ORs, since the adjusted ORs were not comparable because of the inclusion of different covariates in the multivariate regression models. Using individuals with the homozygous common genotype as the reference group, we calculated ORs for individuals with the heterozygous genotype and homozygous rare genotype separately whenever possible (information available in at least two studies). In some cases, we combined the heterozygous genotype with the homozygous rare genotype due to a low prevalence of the rare allele in several polymorphisms. The Q statistic was considered significant for P<0.10 30, 31. Publication bias is always a concern in meta-analysis. The presence of publication bias indicates that nonsignificant or negative findings remain unpublished. To test for publication bias, both Begg's 32 and Egger's 33 tests are commonly used to assess whether smaller studies reported greater associations than larger studies. Publication bias is considered significant for P<0.10. Publication bias may be always a possible limitation of combining data from various sources as in a meta-analysis. The idea of adjusting the results of meta-analyses for publication bias and imputing \"fictional\" studies into a meta-analysis is controversial at the moment 34. Sutton et al. concluded that publication or related biases did not affect the conclusions in most meta-analyses because missing studies changed the conclusions in less than 10% of meta-analyses 34. All of the calculations were performed using STATA Version 8.2 (Stata Corporation, College Station, TX) software.\n3. Results\n3-1. DNA repair capacity and lung cancer risk\nCigarette smoke contains several thousand chemicals that are known to chemically modify DNA 35 and lead to the formation of mutations 36. Most of these compounds are procarcinogens that must be activated by Phase I enzymes, such as cytochrome P450s. All activated carcinogens can bind to DNA and form DNA adducts that are capable of inducing mutations and initiating carcinogenesis. The capacity to repair DNA damage induced by activated carcinogens appears to be one of the host factors that may influence lung cancer risk. A critical cellular response that counteracts the carcinogenic effects of DNA damage is DNA repair. As stated earlier, there are several known pathways of DNA repair, all of which act to remove DNA lesions and prevent mutations, thereby restoring genetic integrity.\nSeveral studies have investigated whether reduced DNA repair capacity (DRC) is associated with an increased risk of cancer 37. The reduced DRC of benzo(a)pyrene-7,8-diol-9,10-epoxide (an active form of benzo(a)pyrene)-DNA adducts is associated with an increased risk of lung cancer (2.1-fold, 95% confidence interval (CI) = 1.5 - 3.0) 38. The reduced DRC has been shown to be associated with a 5.7-fold (95% CI = 2.1 - 15.7) increased risk of developing lung cancer 39. Likewise, the reduced DRC of bleomycin-induced damage was found to be associated with an increased risk of lung cancer 40. These studies suggested that a low DRC of various DNA repair mechanisms predisposes individuals to lung cancer, and this realization prompted us to search for defined DNA repair activities that may be risk factors for lung cancer. Polymorphisms in DNA repair genes may be associated with differences in the DRC of DNA damage and may influence an individual's risk of lung cancer, because the variant genotype in those polymorphisms might destroy or alter repair function.\n3-2. XPA G23A polymorphism and lung cancer risk\nThe heterotrimeric replication protein A (RPA) is required for NER and may play an important role in the damage recognition process. The XPA protein is required for NER and is involved in the DNA damage recognition process. Both RPA and XPA preferentially bind damaged DNA, and because RPA and XPA directly interact in the absence of DNA, the RPA-XPA complex has been implicated as a key component in the earliest stage of damage recognition 41. There is also evidence that the XPC-hHR23B protein complex may initiate recognition of DNA damage for the global genomic repair pathway of NER 42. Recent evidence also implicates the damaged DNA binding protein heterodimer in damage recognition, because the complex binds damaged DNA with high affinity 43 and can dramatically increase the repair rate of certain DNA adducts, including cyclobutane pyrimidine dimers, in conjunction with XPA and RPA 44.\nThe XPA maps on chromosome 9, at 9q22.3. In the XPA gene, a polymorphic site was identified that was in the 5' untranslated region (UTR) of the gene and which consisted of a G-to-A (or A-to G) substitution in the fourth nucleotide before the ATG start codon (dbSNP rs 1800975) 45. SNP alleles with higher frequencies are more likely to be ancestral than less frequently occurring alleles although there may be some exceptions. As the 23G allele was more prevalent than the 23A allele (Table 1), we regarded the 23G allele as ancestral (wild-type or major) allele for descriptive purposes (the XPA 23 polymorphism caused by the G-to-A substitution is the XPA G23A polymorphism). The polymorphism, termed the XPA G23A polymorphism (at position 23 in the transcript, four nucleotides upstream of the start codon), is in the Kozak sequence near the start codon and thus may affect the XPA protein levels in cells 46. A functional association between the XPA G23A polymorphism and DRC has been reported 10. It has been shown that healthy subjects with at least one 23G allele have significantly higher DRC. When the combined A\/A and A\/G genotype was used as the reference, the G\/G genotype was associated with a significantly decreased risk of lung cancer (adjusted OR = 0.56, 95% CI = 0.35 - 0.90) in Koreans 9. A significant protective effect of the combined G\/A and G\/G genotypes on lung cancer risk was reported in Americans (adjusted OR = 0.69, 95% CI = 0.53 - 0.90) and Mexican-Americans (adjusted OR = 0.32, 95% CI = 0.12 - 0.83) 10. Likewise, a protective and nonsignificant effect was seen among Germans 11 and Danes 12. As compared with the combined G\/A and A\/A genotypes, the G\/G genotype was, however, associated with a significantly increased risk of lung cancer (adjusted OR = 1.59, 95% CI =1.12 - 2.27) in a Norwegian population 13. Summary frequencies of the 23A allele among all and Caucasian populations, based on the random effects model, were 0.368 (95% CI = 0.308 - 0.429) and 0.352 (95% CI = 0.277 - 0.428), respectively (Table 1). Summary ORs for the G\/A genotype and G\/G genotype among 5 studies in 7 populations were 0.73 (95% CI = 0.61 - 0.89) and 0.75 (95% CI = 0.59 - 0.95), respectively (Table 1). Evidence for heterogeneity was absent in both analyses. Among Caucasian studies, the summary ORs for the G\/A genotype and the A\/A genotype were 0.72 (95% CI = 0.58 - 0.89) and 0.82 (95% CI = 0.61 - 1.11), respectively. The Cochrane Q test for heterogeneity did not show a statistical significance. The Egger's test was statistically significant for publication bias in a subgroup analysis of Caucasians (P = 0.073, G\/A genotype vs. G\/G genotype).\nTwo studies investigated associations between cigarette smoking and the G23A polymorphism in relation to lung cancer. When stratifying by smoking status, there was a significant protective effect for current smokers who possessed the G\/G genotype (adjusted OR = 0.23, 95% CI = 0.07- 0.71) but not for former or never smokers 9. Ever smokers (current and former) with at least one copy of the 23G allele showed a significantly reduced risk of lung cancer (adjusted OR = 0.68, 95% CI = 0.51 - 0.91) among Caucasians 10. The presence of the 23A polymorphism, however, was associated with a statistically significant reduced risk in subjects who smoked >29 pack-years (OR = 0.53, 95% CI = 0.17 - 0.97) 13. Interactions between cigarette smoking and the polymorphism were not determined in the studies 9, 10, 13. No associations were seen between the G23A polymorphism and any histologic types of lung cancer 11, while the G\/G genotype was associated with a significantly decreased risk for small cell lung cancer (OR = 0.23, 95% CI = 0.07 - 0.71) 9.\nThe XPA G23A polymorphism may, thus, be a promising SNP for lung cancer. It is thought that cigarette smoking modifies the association between DNA repair polymorphisms, as well as metabolic polymorphisms, and lung cancer risk. Since interactions between the G23A polymorphism and smoking have not been fully elucidated, further studies are needed to better understand the associations between the XPA G23A polymorphism and lung cancer risk.\n3-3. ERCC1 polymorphisms and lung cancer risk\nThe ERCC1 coding region is 1.1 kb long and comprises 10 exons. This gene is located on 19q13.2 - q13.3. Shen et al. 47 have identified polymorphisms of three of the exons of the ERCC1 gene, all of which resulted in silent mutations. No amino acid substitutions were observed among the ERCC1 polymorphisms 48. The functional effects of the silent polymorphisms in ERCC1 have not been fully elucidated; however, some of the variant alleles of the polymorphisms in DNA repair genes may be associated with the reduced DRC. The studies have focused on polymorphisms of the 3\u2032 UTR (C8092A, dbSNP no. rs3212986) and codon 118 (Asn118Asn, T19007C, dbSNP no. rs11615) in ERCC1.\nFor the T19007 C (Asn118Asn) polymorphism, although the T\/T genotype generates the less commonly associated triplet codon sequence encoding the amino acid and has been termed the \"variant\" by convention, the T\/T genotype indeed has been reported to occur at higher frequencies. Hence, the T\/T genotype is used as reference in this paper. The C\/C genotype of the C8092A polymorphism is used as reference on the same score.\nThe C\/C genotype of the T19007C polymorphism was associated with a significantly decreased risk of lung cancer (adjusted OR = 0.32, 95% CI = 0.19 - 0.55) in a Norwegian population 13. A lack of association between the T19007C polymorphism and lung cancer risk was observed in a Danish population 14, a large American population 15, a Chinese population 16 and a nonsmoking European population 17. As shown in Table 2, summary frequencies of the 19007T allele among all and Caucasian populations, based on the random effects model, were 0.499 (95% CI = 0.387 - 0.611) and 0.575 (95% CI = 0.529 - 0.622), respectively. The summary ORs for the T\/C genotype and the C\/C genotype were 0.82 (95% CI = 0.62 - 1.08) and 0.72 (95% CI = 0.46 - 1.11), respectively. Even if the analysis was restricted to Caucasian studies, the ORs did not materially change. The Cochrane Q test for heterogeneity showed a statistical significance in any analysis. In comparison of the T\/C genotype with the T\/T genotype, the Begg's test was statistically significant in an overall analysis (P = 0.086) and a subgroup analysis of Caucasians (P = 0.089).\nTwo studies examined an interaction between the T19007C polymorphism and cigarette smoking. When stratified by smoking status, the interaction between smoking and the polymorphism was not statistically significant 15, 16. Only one study provides information on the T19007C polymorphism and lung cancer risk in histologic types. There was no difference in risk estimates according to the histological type of lung cancer 16.\nAs for the C8092A polymorphism, no association was found between the polymorphism and lung cancer risk in Norwegians 13 and Americans 15. The C8092A and T19007C polymorphisms have been reported to be in linkage disequilibrium 15.\nAlthough harboring at least one 19007C allele may be associated with a deceased risk of lung cancer, the protective effect of the 19007C allele needs to be confirmed in other independent studies. Furthermore, additional studies are needed to detect the function of the ERCC1 polymorphisms.\n3-4. ERCC2\/XPD polymorphisms and lung cancer risk\nThe ERCC2\/XPD protein plays a role in the NER pathway, which recognizes and repairs a wide range of structurally unrelated lesions such as bulky adducts and thymidine dimers. ERCC2\/XPD works as an ATP-dependent (5'\u21923') helicase joined to the basal TFIIH complex used to separate the double helix. The ERCC2\/XPD protein is necessary for normal transcription initiation and NER. ERCC2\/XPD maps on chromosome 19, at 19q13.3 and covers 21.14 kb. Mutations in the ERCC2 gene can diminish the activity of TFIIH complexes, giving rise to repair defects, transcription defects and abnormal responses to apoptosis 49.\nA number of polymorphisms in the ERCC2\/XPD gene have been reported. Whereas polymorphisms in the codons 199, 201 and 575 are rare, those in codons 156, 312, 711 and 751 are common. Two ERCC2\/XPD polymorphisms, Asp312Asn (db SNP no. rs1799793) and Lys751Gln (db SNP no. rs13181), have mainly been investigated in relation to phenotypic endpoints relevant to lung carcinogenesis. With regard to the Asp312Asn polymorphism, most of the reported data indicate a higher level of DNA adducts in subjects with the Asn allele. The interpretation of this finding is a lower DRC for the Asn allele than the Asp allele. This is also true for the ERCC2\/XPD Lys751Gln polymorphism. The Gln allele is associated with a higher DNA adduct level or lower DRC.\nThe Asp\/Asp genotype of the ERCC2\/XPD Asp312Asn polymorphism was found to have an increased risk of lung cancer when the combined Asp\/Asn and Asn\/Asn genotypes served as reference (OR = 1.86, 95% CI =1.02 - 3.40) in Polish men 20. A large American lung-cancer study also reported an elevated risk (adjusted OR = 1.5, 95% CI = 1.1 - 2.0; Asn\/Asn genotype vs. Asp\/Asp genotype) 18. Likewise, Chinese subjects homozygous for the Asn\/Asn genotype had an increased risk of lung cancer (adjusted OR = 10.33, 95% CI = 1.29 - 82.50) compared with subjects homozygous for the Asp\/Asp genotype 19. No association with this polymorphism was seen in an admixed population 21, a small Swedish population 22 and among Finnish smoking men 23. Two meta-analyses have been published in 2004 50 and 2005 51, respectively. Both of them are based on the same published data from 6 individual case-control studies 18-23. The first meta-analysis showed that individuals with the Asn\/Asn genotype had a 27% (95% CI = 1.04 - 1.56) increased risk of lung cancer compared with individuals with the Asp\/Asp genotype. The results supported the hypothesis that individuals with the Asn\/Asn genotype are at higher risk of developing lung cancer 50. The second meta-analysis was somewhat different from the first one, because unadjusted ORs were summarized in the first one. The summary OR associated with the Asn\/Asn genotype was 1.18 (95% CI = 0.84 - 1.67). No significant association between the ERCC2\/XPD Asp312Asn polymorphism and lung cancer was found in the second meta-analysis 51. Regardless, these meta-analyses indicate that the excess lung cancer risk from the Asn\/Asn genotype may be less than 30%.\nFive studies have been reported since the publication of these two meta-analyses. They revealed that the Asp312Asn polymorphism was not associated with lung cancer risk in Germans 11, Norwegians 13, Danes 14, Europeans 17 and Chinese 24.\nAs shown in Table 3, the summary frequency of the 312Asp allele among Caucasians (0.645, 95% CI = 0.572 - 0.719) was significantly lower than that among Asians (0.936, 95% CI = 0.925 - 0.946). Summary ORs associated with the ERCC2\/XPD Asp312Asn polymorphism are also shown in Table 3. No significant association between lung cancer and the heterozygous Asp\/Asn genotype was found for all of the studies combined or by ethnicity. The Cochrane Q test for heterogeneity did not show a statistical significance in all analyses. Although no evidence of publication bias was found in overall analyses, both Begg's (P= 0.035) and Egger's (P = 0.003) tests showed a statistical significance in a subgroup analysis of Caucasians (Asn\/Asn genotype vs. Asp\/Asp genotype).\nWhen stratifing by smoking dose, the risk of lung cancer was significantly higher in light-smokers with the Asp\/Asp genotype than in those with the Asn\/Asn genotype 20. Similar findings were not seen for never-smoker or heavy-smokers 20. A significant interaction between smoking (smoking status, pack-years and duration) and the polymorphism was observed in one study 18 but not in two other studies 16,19. Stratification analysis revealed that the increased risk was mainly confined to squamous cell carcinoma of the lung, with the ORs being 20.50 (95% CI = 2.25 - 179.05) for the 312Asn\/Asn genotype 19.\nTable 4 shows the association between the ERCC2 Lys751Gln polymorphism and lung cancer risk. The Gln\/Gln genotype was associated with an increased risk for lung cancer compared with the 751Lys\/Lys genotype (adjusted OR = 2.71, 95% CI = 1.01 - 7.24) in Chinese 19. Stratification analysis revealed that the increased risk was mainly confined to lung squamous cell carcinoma, with the OR being 4.24 (95% CI = 1.34 - 13.38) for the Gln\/Gln genotype 19, however. Although David-Beades et al. reported that the Gln\/Gln genotype was associated with a significantly increased risk of lung cancer in Caucasians (USA), a multivariate-adjusted OR was no longer significant 25. No association with the Lys751Gln polymorphism was seen in two Caucasian populations 18, 22, an admixed population 21, a Finnish population 23, African-Americans 25, a Chinese population 26 and a Korean population 27. The meta-analysis by Hu et al. (2004) showed that the Gln\/Gln genotype had a 21% (95% CI = 1.02 - 1.43) increased risk of lung cancer compared with individuals with the Lys\/Lys genotype 51. The meta-analysis by Benhamou and Sarasin (2005) reported that the summary OR for the Gln\/Gln genotype was 1.18 (95% CI = 0.95 - 1.47) 51. Both of the meta-analyses were based on the same published data from 8 individual case-control studies 18, 19, 21-23, 25-27. No significant association between the Lys751Gln polymorphism and lung cancer was found in the two meta-analyses 51. These meta-analyses indicate that the excess lung cancer risk from the Gln\/Gln genotype may be about 20%. Six studies 11, 13, 14, 17, 24, 28 have been reported after the two meta-analysis. Danish subjects with the Gln\/Gln genotype were at a 2.01-fold (95% CI = 1.20 - 3.35) higher risk of lung cancer risk than those with the Lys\/Lys genotype 14. Similarly, the Gln\/Gln genotype was associated with significantly increased risk of lung cancer (adjusted OR = 1.60, 95% CI = 1.10 - 2.30) in Norwegians 13. German individuals with the Gln\/Gln genotype were at a borderline increased risk (adjusted OR = 1.59, 95% CI = 0.95 - 2.67) 11. However, individuals with the Gln allele had a 61% (95% CI = 14 - 83) reduction of lung cancer risk in a Chinese population 24. No association with the Lys751Gln polymorphism was seen in a European cohort 17 and in non-Hispanic Caucasians (USA) 28.\nThe summary frequency of the 751Lys allele among Caucasians (0.634, 95% CI = 0.614 - 0.655) was significantly lower than that among Asians (0.843, 95% CI = 0.763 - 0.924). A statistically significant ethnic difference was observed between Caucasians and Asians. Summary ORs for the Gln\/Gln genotype and Lys\/Gln genotype were 1.06 (95% CI = 0.97 - 1.16) and 1.30 (95% CI = 1.14 - 1.49), respectively. Evidence of publication bias was absent in all of the analyses. The effect of the Gln\/Gln genotype on lung cancer risk was stronger in Caucasians (OR = 2.25, 95% CI = 0.97 - 5.23) than in Asians (OR = 1.02, 95% CI = 0.20 - 5.27). This may only be due to a difference in sample sizes. Reasons for this difference in risk among different ethnic populations are as yet unknown but, if real, may be related to other genetic or environmental factors. The Cochrane Q test for heterogeneity showed a statistical significance among Asian studies (P = 0.040, Gln\/Gln genotype vs. Lys\/Lys genotype).\nThere was no interaction between smoking (smoking status, pack-years and duration) and the polymorphism 14, 19, 26, 27. Although the Lys\/Lys genotype was associated with a statistically significant increased risk (OR = 2.0, 95% CI = 1.15 - 3.41) among subjects who smoked>29 pack-years, an interaction between cigarette smoking and the polymorphism was not determined 13. When stratified by histological type, no statistically significant association between the polymorphism and lung cancer risk was found 26, 27.\nSeveral studies have investigated the possible association of ERCC2\/XPD Asp312Asn and Lys751Gln polymorphisms with lung cancer with inconsistent results. The Lys751Gln polymorphism has been more studied than the Asp312Asn polymorphism, because the frequency of the 751Gln allele is more prevalent than the 312Asn allele. The Asp312Asn polymorphism is in linkage disequilibrium with the Lys751Gln polymorphism 19, 20, 21, however. The inconsistent associations in previous studies of the ERCC2\/XPD polymorphisms could be due to differences in study populations, the small sample sizes of earlier studies and possible environmental interactions.\n3-5. ERCC4\/XPF polymorphisms and lung cancer risk\nERCC4\/XPF is an essential protein in the NER pathway, which is responsible for removing UV-C photoproducts and bulky adducts from DNA. Among the NER enzymes, ERCC4\/XPF and ERCC1 are also uniquely involved in removing DNA interstrand cross-linking damage. The ERCC4\/XPF-ERCC1 complex, which makes incisions at the 5\u2032 end of DNA loops, may contribute to the repair of large trinucleotide repeat containing loops that are generated due to replication slippage and that are too long to be repaired by the postreplicative DNA mismatch repair system 52. Polymorphisms in enzymes involved in large loop repair could be responsible for the observed variation in the stability of similar-sized trinucleotide repeat disease alleles among different individuals. The ERCC4\/XPF gene is evolutionarily conserved. Extensive homology exists between human ERCC4\/XPF, Drosophila Mei-9, Saccharomyces cerevisiae RAD1, and S. pombe Rad16 53, all of which have similar functions in NER.\nThe ERCC4\/XPF gene contains 11 exons, spans 28.2 kb and is located on chromosome 16p13.2 - p13.13. Several polymorphisms exist in the coding region of ERCC4\/XPF, a few of which have been associated with cancer risks. Genetic instability of simple repeated sequences might also be influenced by the ERCC4\/XPF polymorphisms. The ERCC4\/XPF G1244A polymorphism is a G-to-A change in exon 8 (Arg415Gln, dbSNP no. rs1800067) that results in a change from arginine to glutamine. The ERCC4\/XPF polymorphism in exon 8 has been reported to be associated with an increased risk for developing breast cancer 54. The T2505C polymorphism is a T-to-C change in exon 11 (Ser835Ser, dbSNP no. rs1799801) that results in no amino acid change (serine is conserved) 55. Functionally significant SNPs in the ERCC4\/XPF gene may also contribute to individual differences in the fine details of DNA repair. A lack of association was found between the G1244A (Arg415Gln) polymorphism and lung cancer risk (adjusted OR = 1.11, 95% CI = 0.59 - 2.07; Arg\/Gln genotype vs. Arg\/Arg genotype) in Koreans 9. The C\/C genotype of the T2505C polymorphism was nonsignificantly associated with an increased risk of lung cancer (adjusted OR = 1.71, 95% CI = 0.52 - 5.58) in Chinese 24.\n3-6. ERCC5\/XPG polymorphisms and lung cancer risk\nERCC5\/XPG is responsible for a 1186 amino acid structure-specific endonuclease activity that is essential for the two incision steps in NER. The ERCC5\/XPG nuclease has been suggested to act on the single-stranded region created as a result of the combined action of the XPB helicase and the ERCC2\/XPD helicase at the DNA damage site. In human cells, ERCC5\/XPG catalyses an incision approximately 5 nucleotides 3' to the site of damage but is also involved non-enzymatically in the subsequent 5' incision. It is further involved in the stabilization of a pre-incision complex on the damaged DNA.\nThe ERCC5\/XPG gene contains 17 exons, spans 32 kb and is located on chromosome 13q32.3 -q33.1. Several polymorphisms in the coding sequence of the EECC5\/XPG gene have been identified. The association between lung cancer and two common polymorphisms, T335C (His46His, dbSNP no. rs1047768) and G3507C (Asp1104His, dbSNP no. rs17655), have been investigated. The functional effects of these two SNPs are still unknown. However, it is likely that the SNPs in the coding DNA sequences may result in a subtle structural alteration of the ERCC5\/XPG activity and modulation of lung cancer susceptibility.\nThe Asp\/Asp genotype of the Asp1104His polymorphism was associated with a significantly decreased risk of lung cancer (adjusted OR = 0.60, 95% CI = 0.38 - 0.95) in a Korean population 56. Similarly, the Asp\/Asp genotype was inversely associated with lung cancer (adjusted OR = 0.65, 95% CI = 0.39 - 1.1) in an admixed population (composed mostly composed of whites) 57. However, the Asp\/Asp genotype was not associated with lung cancer risk in a Chinese population 24. As for T335C polymorphism, the C\/C genotype was associated with a significantly increased risk of lung cancer (adjusted OR = 1.79, 95% CI = 1.19 - 2.63) in Norwegians 13 but not in Chinese 24.\n4. Discussion\nEpidemiological studies of common polymorphisms in DNA repair genes, if large and unbiased, can provide insight into the in vivo relationships between DNA repair genes and lung cancer risk. Such studies may identify empirical associations which indicate that a polymorphism in a gene of interest has an impact on lung cancer, independent of metabolic regulatory mechanisms and other genetic and environmental variability. Findings from epidemiological studies can complement in vitro analyses of the various polymorphisms, genes, and pathways. In addition, epidemiological studies of common polymorphisms can lead to an increased understanding of the public health dimension of DNA-repair variation.\nWe conducted a systematic literature review to evaluate the associations between sequence variants in DNA repair genes and lung cancer risk. We found an increased risk of lung cancer among subjects carrying the ERCC2\/XPD 751Gln\/Gln genotype (OR = 1.30, 95% CI = 1.14 - 1.49). The Gln allele of the ERCC2\/XPD Lys751Gln polymorphism is associated with a higher DNA adduct level or lower DNA repair efficiency, except in research published by Duell et al. (2000) who found no correlation between the ERCC2\/XPD Lys751Gln polymorphism and the level of polyphenol-DNA adducts in human blood samples 58. Matullo et al. (2003) demonstrated a higher level of DNA adducts, measured by 32P-postlabeling, in lymphocytes from nonsmokers with the ERCC2\/XPD 751Gln\/Gln genotype 59. Similarly, Palli et al. (2001) reported a higher level of DNA adducts in workers with at least one Gln allele who were exposed to traffic pollution in comparison with workers with the two common alleles 60. An increased number of aromatic DNA adducts was found by Hou et al. (2002) in peripheral blood lymphocytes from subjects with the ERCC2\/XPD 312Asn and ERCC2\/XPD 751Gln alleles 22. The combined Asn\/Asn and Gln\/Gln genotypes showed a higher level of DNA lesions than did other genotypes.\nIn contrast, we found a protective effect of the XPG G23A G\/G genotype (OR = 0.75, 95% CI = 0.59 - 0.95) on lung cancer risk. The G23A polymorphism itself may alter the transcription and\/or translation of the gene. Because this polymorphism is located in the vicinity of the translation initiation codon, it may alter translation efficiency. The nearby proximal nucleotides to the AUG initiation codon are important for the initiation of translation because the 40S ribosomal subunit binds initially at the 5'-end of the mRNA 61. The consensus sequence around the start codon is GCCRCCAUGG, which is known as the Kozak consensus sequence 62. The R at position -3 and the G just downstream of the start codon are especially important, and the lack of these bases leads to read-through of the start codon 63. However, there has been no precise explanation of the mechanism by which the recognition of the start codon is aided by a purine at position -3 62, which is the core nucleotide of the Kozak consensus. The polymorphism XPA G23A is a G\/A transversion occurring 4 nucleotides upstream of the start codon of XPA and possibly improving the Kozak sequence 9. The sequences (CCAGAGAUGG) around the predicted initiator methionine codon of the XPA gene agree with the Kozak's consensus sequence at positions -3 and +4 64. Although both the A and polymorphic variant G nucleotides at the -4 position of the XPA gene do not correspond to the original consensus Kozak sequence containing the nucleotide C at position -4, it is possible that a nucleotide substitution of A to G at position -4 preceding the AUG codon may affect ribosomal binding and thus alter the efficiency of XPA protein synthesis. To investigate whether the transition from G to A changes the translation efficiency, an in vitro transcription\/translation analysis and a primer extension assay of the initiation complex will be necessary in the future. Furthermore, a functional association between the G23A polymorphism and DRC was reported 10, which showed significantly higher repair efficiency in healthy subjects with at least one G allele. An alternative explanation could be that the protective XPA 23G allele is in linkage disequilibrium with an allele from an adjacent gene which is the true susceptibility gene.\nSeveral DNA repair pathways are involved in the maintenance of genetic stability. The most versatile and important one is the NER pathway, which detects and removes bulky DNA adducts, including those induced by cigarette smoking 65. However, there are several conflicting reports on the association between this polymorphism and lung cancer risk among various populations. Although the reasons for the inconsistencies in the studies are not clear, possible explanations are: 1) low frequency of the \"at-risk\" genotype, which would reduce the statistical power of the studies and 2) small size of the studies. Ethnic differences in the roles of the polymorphism may be caused by gene-gene interactions, different linkages to the polymorphisms determining lung cancer risk and different lifestyles.\nThe most important problems facing lung cancer research are identifying \"at-risk\" individuals and implementing clinical surveillance, prevention practices, and follow-up care. Repair pathways play an important role in lung cancer risk, and genetic variations may contribute to decreased DRC and lung cancer susceptibility. Although the increased\/decreased risk associated with individual DNA repair SNPs may be small compared to that conferred by high-penetrance cancer genes, their public health implication may be large because of their high frequency in the general population. It is thus essential that epidemiological investigations of DNA repair polymorphisms are adequately designed. Unfortunately a fairly good number of studies are limited by their sample size and subsequently suffer from too low power to detect effects that may truly exist. Also, given the borderline significance of some associations and multiple comparisons that have been carried out, there is a possibility that one or more findings are false-positives 66. Large and combined analyses may be preferred to minimize the likelihood of both false-positive and false-negative results. In addition, controls should be chosen in such a way that, if they were cases, they would be included in the case group; when controls are matched to cases, it is essential to account for matching in the analysis. When appropriate, confounding factors should be controlled for, with particular consideration of race and ethnicity. An additional major concern is the grouping of genotypes for calculation of ORs. Without functional data to dictate genotype groupings, it seems prudent to present two ORs per polymorphism (one for heterozygotes vs. common-allele homozygotes and one for rare-allele homozygotes vs. common-allele homozygotes) so that dominant, codominant, or recessive patterns may be elucidated.\nContinued advances in SNP maps and in high-throughput genotyping methods will facilitate the analysis of multiple polymorphisms within genes and the analysis of multiple genes within pathways. The effects of polymorphisms are best represented by their haplotypes. Data from multiple polymorphisms within a gene can be combined to create haplotypes, the set of multiple alleles on a single chromosome. None of the studies reviewed here reported haplotype associations, although several studies analyzed multiple polymorphisms within a gene, sometimes with inconsistent results. The analysis of haplotypes can increase the power to detect disease associations because of higher heterozygosity and tighter linkage disequilibrium with disease-causing mutations. In addition, haplotype analysis offers the advantage of not assuming that any of the genotyped polymorphisms is functional; rather, it allows for the possibility of an ungenotyped functional variant to be in linkage disequilibrium with the genotyped polymorphisms 67. An analysis of data from multiple genes within the same DNA-repair pathway (particularly those known to form complexes) can provide more comprehensive insight into the studied associations. Such an analysis may shed light on the complexities of the many pathways involved in DNA repair and lung cancer development, providing hypotheses for future functional studies. Because of concerns over inflated type I error rates in pathway-wide or genome-wide association studies, methods of statistical analysis seeking to obviate this problem are under development 68. The ability to include haplotype information and data from multiple genes, and to model their interactions, will provide more powerful and more comprehensive assessments of the DNA repair pathways.\nThis review, which is limited by the bias against publication of null findings, highlights the complexities inherent in epidemiological research and, particularly, in molecular epidemiological research. There is evidence that some polymorphisms in DNA repair genes play a role in carcinogenesis, most notably the ERCC2\/XPD Lys751Gln and XPA G23A polymorphisms. The variant allele of each of the three polymorphisms was associated with about a 30% decrease or increase in lung cancer risk. Although the summary risk for developing lung cancer in individuals of each genotype may not be large, lung cancer is such a common malignancy that even a small increase in risk can translate to a large number of excess lung cancer cases. Therefore, polymorphisms, even those not strongly associated with lung cancer, should be considered as potentially important public health issues. In addition, it is important to keep in mind that a susceptibility factor in one population may not be a factor in another. There are differences in the prevalence of DNA repair polymorphisms across populations. In a population where the prevalence of an \"at-risk\" genotype in a given polymorphism is very low, the \"at-risk\" allele or \"at-risk\" genotype may be too infrequent to assess its associated risk. At a population level, the attributable risk must be small simply because it is an infrequent allele. Finally, the major burden of lung cancer in the population probably results from the complex interaction between many genetic and environmental factors over time. Most environmental carcinogens first require metabolic activation by Phase I enzymes to their ultimate forms which then bind to DNA, forming aromatic-DNA adducts that are thought to be an early step in tumorigenesis. On the other hand, these activated forms are detoxified by Phase II enzymes. Thus, genetically determined susceptibility to lung cancer may depend on the metabolic balance among Phase I enzymes, Phase II enzymes and DNA repair enzymes 69. Further investigations of the combined effects of polymorphisms between DNA repair genes and drug-metabolizing genes may also help to clarify the influence of genetic variation in the carcinogenic process. Consortia and international collaborative studies, which may be a way to maximize study efficacy and overcome the limitations of individual studies, are needed to help further illuminate the complex landscape of lung cancer risk and genetic variations.","keyphrases":["genetic polymorphism","nucleotide excision repair","lung cancer","meta-analysis"],"prmu":["P","P","P","P"]}
{"id":"J_Chem_Ecol-4-1-2239252","title":"Barbarea vulgaris Glucosinolate Phenotypes Differentially Affect Performance and Preference of Two Different Species of Lepidopteran Herbivores\n","text":"The composition of secondary metabolites and the nutritional value of a plant both determine herbivore preference and performance. The genetically determined glucosinolate pattern of Barbarea vulgaris can be dominated by either glucobarbarin (BAR-type) or by gluconasturtiin (NAS-type). Because of the structural differences, these glucosinolates may have different effects on herbivores. We compared the two Barbarea chemotypes with regards to the preference and performance of two lepidopteran herbivores, using Mamestra brassicae as a generalist and Pieris rapae as a specialist. The generalist and specialist herbivores did not prefer either chemotype for oviposition. However, larvae of the generalist M. brassicae preferred to feed and performed best on NAS-type plants. On NAS-type plants, 100% of the M. brassicae larvae survived while growing exponentially, whereas on BAR-type plants, M. brassicae larvae showed little growth and a mortality of 37.5%. In contrast to M. brassicae, the larval preference and performance of the specialist P. rapae was unaffected by plant chemotype. Total levels of glucosinolates, water soluble sugars, and amino acids of B. vulgaris could not explain the poor preference and performance of M. brassicae on BAR-type plants. Our results suggest that difference in glucosinolate chemical structure is responsible for the differential effects of the B. vulgaris chemotypes on the generalist herbivore.\nIntroduction\nGlucosinolates and their breakdown products are involved in plant defense against a wide variety of potential plant enemies. They can also serve as feeding and oviposition stimulants for specialist herbivores (Chew 1988; Louda and Mole 1991; Wittstock et al. 2003). The composition of glucosinolates is genetically variable within plant species, and influences the feeding choices of insect herbivores (Kroymann et al. 2001; Lambrix et al. 2001). A heritable glucosinolate polymorphism in Barbarea vulgaris results in two discrete chemotypes (Van Leur et al. 2006). In this study, we investigate the effect of these two chemotypes on the preference and performance of two lepidopterans.\nThe most common and genetically dominant chemotype of B. vulgaris forms mainly (S)-2-hydroxy-2-phenylethylglucosinolate (glucobarbarin, BAR-type). Although depending on the presence of cofactors like ESP, pH, or metal ions (Burow et al. 2006), the most likely initial hydrolysis product of glucobarbarin is an isothiocyanate. Due to the 2-hydroxylation of the glucosinolate side chain, it spontaneously cyclizes to 5-phenyloxazolidine-2-thione (Kjaer and Gmelin 1957). This glucosinolate breakdown product is known to reduce infection by the soil fungus Plasmodiophora brassicae (Ludwig-M\u00fcller et al. 1999). Hardly anything is known about other ecological activities of oxazolidine-2-thiones (Wittstock et al. 2003).\nThe less abundant and genetically recessive chemotype of B. vulgaris contains mainly 2-phenylethyl glucosinolate (gluconasturtiin, NAS-type). 2-Phenylethyl glucosinolate is also present in Arabidopsis thaliana and other Brassicaceae (Reichelt et al. 2002), predominantly in root tissues (Sang et al. 1984). Its most likely breakdown product is 2-phenylethyl isothiocyanate. Isothiocyanates are the predominant breakdown products of glucosinolates and are generally toxic to various herbivores (Wittstock et al. 2003). 2-Phenylethyl isothiocyanate negatively affects a broad range of phytophages, e.g., nematodes (Potter et al. 1999, 2000; Serra et al. 2002; Lazzeri et al. 2004), snails (Kerfoot et al. 1998), flies, aphids, mites (Lichtenstein et al. 1962), fungi (Sarwar and Kirkegaard 1998), and several generalist and specialist Lepidoptera (Wadleigh and Yu 1988; Borek et al. 1998). Despite counter-adaptations of specialists to reduce or circumvent negative effects of glucosinolates (Ratzka et al. 2002; Wittstock et al. 2003), isothiocyanates can still reduce survival and growth, and increase development time of specialists (Agrawal and Kurashige 2003). In contrast to the oxazolidine-2-thiones formed in BAR-type plants, which can increase the incidence of goiter in mammals, the 2-phenylethyl isothiocyanate formed in NAS-type plants has chemopreventive effects against tumorigenesis in mammalian organisms (Canistro et al. 2004).\nBased on the specific biological effects of the expected breakdown products of glucobarbarin and gluconasturtiin, we hypothesized that NAS-type and BAR-type B. vulgaris plants have differential effects on insect herbivores. Generally, it is expected that chemical plant defenses are more effective against generalist herbivores than against specialists (Cornell and Hawkins 2003). Therefore, we compared the preference and performance on the two chemotypes of a generalist (Mamestra brassicae) and a specialist (Pieris rapae) herbivore, which are both well-studied, important crucifer pests (Theunissen et al. 1985; Finch and Kienegger 1997).\nM. brassicae is a generalist feeding on plants in 70 species and 22 families, of which Brassicaceae are among the most preferred (Rojas et al. 2000). Even though M. brassicae can detect glucosinolates by receptor cells on the sensilla (Wieczorek 1976), and its oviposition is stimulated by damaged cabbage plants (Rojas 1999), no physiological adaptations of this species to glucosinolates have been yet described. The larvae of P. rapae, on the other hand, can detoxify glucosinolates by shifting hydrolysis products from isothiocyanates to less toxic nitriles by using the myrosinase directing nitrile-specifier protein (NSP). This enables P. rapae to consume foliage that is otherwise well defended (Wittstock et al. 2003, 2004). Female adults of P. rapae can detect intact glucosinolates in leaves of Brassicaceae with specialized receptor cells, and are stimulated to lay eggs on glucosinolate containing plants.\nWe assessed herbivore preference on the level of adult oviposition and larval feeding. Female oviposition preference initially determines the host of the larvae (Akhtar and Isman 2003). As the isothiocyanates from NAS-type plants are expected to be more toxic than the oxazolidine-2-thiones produced by BAR-type plants, we expected M. brassicae to be repelled by NAS-type plants and to prefer BAR-type plants for oviposition. Oviposition of P. rapae is affected by glucosinolates at the leaf surface (Renwick et al. 1992; Van Loon et al. 1992) and known to be stimulated by glucobarbarin as well as by gluconasturtiin in a dose-dependent way (Chew 1988; Huang and Renwick 1994; Huang et al. 1994a, b).\nAs larvae can eventually leave their initial host (van Dam et al. 2000), we also assessed larval preference. If the B. vulgaris chemotypes differ in toxicity or palatability, we expect to see the largest effect on larval preference and larval performance for the unadapted generalist M. brassicae and no or minor effects on the glucosinolate specialist P. rapae.\nAlthough we observed no differences in morphology, growth, or germination between the chemotypes (van Leur and van Dam, unpublished results), pleiotropic effects or close linkages could possibly cause other genes and metabolites to be consistently different between the chemotypes. As the nutritional value of plants is important for herbivore performance and preference (Simpson and Simpson 1990; Berenbaum 1995), additionally, we analyzed the sugar content and amino acid level of the tissue on which the herbivores were feeding.\nMaterials and Methods\nPlant Material B. vulgaris seeds were collected from 10 individual BAR and their nearest neighbor NAS-type maternal plants, which were freely cross pollinated in a natural population of B. vulgaris. The population was located in Elderveld, The Netherlands (51.95\u00b0N; 5.87\u00b0E) and consisted of 22% NAS-type plants (Van Leur et al. 2006). We selected offspring of maternal NAS-type plants \u201cEL44\u201d (68% BAR-type offspring) and \u201cEL13\u201d (62% BAR-type offspring). Plants were grown in a glasshouse, at 21\u00b0C (day) and 16\u00b0C (night), with 60% relative humidity and natural daylight supplemented with sodium lamps to maintain the minimum PAR at 225\u00a0\u03bcmol m\u22122 s\u22121 with a photoperiod of 16:8 (L:D). One week after germination on glass beads, seedlings were transplanted to a mixture of peat soil (Potgrond 4, Lentse Potgrond BV., Lent, The Netherlands) and 20% sand. After 2\u00a0weeks, the seedlings were transplanted to 1.1-l pots, watered, and fertilized regularly with half strength Hoagland\u2019s nutrient solution with a doubled KH2PO4 content.\nChemical Analysis For quantification of glucosinolates, soluble sugars, and amino acids, one global extraction was used. In a 2-ml Eppendorf tube, 50.0\u00a0mg of lyophilized finely ground plant material were dissolved in 1.0\u00a0ml 70% MeOH in water (v\/v), vortexed, and immediately boiled for 5\u00a0min to kill the remaining myrosinase activity. Tubes were placed in an ultrasonic bath for 15\u00a0min and centrifuged (10\u00a0min 10,000\u00a0rpm). The extraction was repeated for the pellet omitting the boiling step. For each sample, both supernatants were combined in a new 2-ml Eppendorf tube and supplemented individually with 70% MeOH to attain the average mass (N\u2009=\u20093) of a 2-ml Eppendorf tube that contained 2.0\u00a0ml 70% MeOH. This \u201cstock\u201d extract was stored at \u221220\u00b0C until further analysis.Half (1.0\u00a0ml) of the stock extract was used for glucosinolate analysis and applied to a DEAE-Sephadex A 25 column (EC 1990), desulfated with arylsulfatase (Sigma, St. Louis, MO, USA) and separated on a reversed phase C-18 column on HPLC with a CH3CN\u2013H2O gradient as described in van Dam et al. (2004). Glucosinolate analysis was performed with a PDA detector (200\u2013350 nm) with 229\u00a0nm as the integration wavelength. Desulfoglucosinolate peaks were identified by comparison of HPLC retention times and UV spectra with authentic standards isolated from B. vulgaris as previously described (Agerbirk et al. 2001), as well as standards kindly provided by M. Reichelt, MPI Chemical Ecology, and a certified rape seed standard (Community Bureau of Reference, Brussels, code BCR-367R). The same response factor was used for glucobarbarin as for gluconasturtiin (Buchner 1987). To calculate glucosinolate concentrations in the plant tissue, the obtained values were multiplied by two and divided by dry mass.To analyze soluble sugar content, a 10-\u03bcl aliquot of the stock extract was diluted with 990\u00a0\u03bcl MilliQ water. Soluble sugars were analyzed by injecting 5\u00a0\u03bcl of the diluted extract on Dionex HPLC system, equipped with a Carbopac PA1 column (2\u2009\u00d7\u2009250\u00a0mm) and a Carbopac PA1 guard column (2\u2009\u00d7\u200950\u00a0mm, Dionex, Sunnyvale, CA, USA). An isocratic gradient mixture of 10% 1\u00a0M NaOH and 90% MilliQ water was used to separate sugars at a flow rate of 0.25\u00a0ml\/min. Column temperature was kept at 20\u00b0C. A \u201c10-ppm\u201d reference solution that contained 54.9\u00a0\u03bcM sorbitol and manitol, 29.21\u00a0\u03bcM trehalose, sucrose, and melbiose, and 55.51\u00a0\u03bcM glucose and fructose, was diluted to obtain 7.5, 5, and 2.5\u00a0ppm calibration standards to obtain a reference curve. After every 10 samples, an additional standard was injected to check for deviations of retention times and the calibration curve. To calculate the molar concentration of sugars in the plant tissue, the concentration values were multiplied by 200 and divided by dry mass.Amino acids were analyzed on a Dionex HPLC system by integrated pulsed amperometric detection. An aliquot of 20\u00a0\u03bcl of the stock extract was diluted with 980\u00a0\u03bcl MilliQ. Of this diluted extract, 25\u00a0\u03bcl were injected, and amino acids were separated with a ternary gradient (see DIONEX application update 152, Method 1, standard AAA gradient; condition 60\/2 in Hanko and Rohrer 2004) on a 2\u2009\u00d7\u2009250\u00a0mm AminoPac\u00a9 PA10 column with a 2\u2009\u00d7\u200950\u00a0mm AminoPac\u00a9 PA10 Guard column (Dionex, Sunnyvale, CA, USA). Eluents, flow rates, waveform, and working electrode conditions were all as specified under Method 1 in Dionex application update 152 and in Hanko and Rohrer (2004). The Sigma AA-S-18 amino acid standard (Sigma, St Louis, MO, USA) that contained 17 amino acids was supplemented with asparagine, glutamine, and tryptophane (2.5\u00a0\u03bcmol\/ml each) to obtain a reference sample that contained the 20 most common amino acids. This reference solution was diluted to obtain calibration standard ranging from 1\u20138\u00a0\u03bcM for each amino acid, except for cysteine, which had a range of 0.5\u20134\u00a0\u03bcM. After every 10 samples, an additional standard was injected to check for deviations of retention times and the calibration curve. To calculate the molar concentration of the amino acids in the plant tissue, the concentration values were multiplied by 200 and divided by dry mass.To determine the chemotype of each plant, glucosinolates were extracted from the first full grown leaf and analyzed on HPLC as described above. When the peak area of glucobarbarin divided by the peak area of gluconasturtiin was >10, the plant was considered a BAR-type. When this ratio was <0.1, it was considered a NAS-type.\nOviposition Insects were obtained from the Laboratory of Entomology of Wageningen University the Netherlands. Stock colonies of P. rapae and M. brassicae were maintained on Brassica oleracea var. gemnifera L., cultivar Cyrus, in a climatized room at 20\u201322\u00b0C, 50\u201370% relative humidity, and a photoperiod of 16:8 (L:D). We used ca. 4-month-old half-sib plants of family EL44 that were clipped 1\u00a0month before use to ensure abundant fresh leaf material. At the start of the experiment, plants were moved from the glasshouse to a climatized room at 21\u00b0C (day) 16\u00b0C (night), 60% relative humidity, illuminated to 200 PAR at plant height (Philips Master TLD 50W\/840\u00a0HF and 60\u00a0W lights) and a photoperiod of 16:8 (L:D). In the same room, 1- to 3-day-old adult insects were held in a mesh cage (40\u2009\u00d7\u200945\u2009\u00d7\u200965\u00a0cm3) provided with sugar solution. Males and females were held together to mate for at least 24\u00a0h. Oviposition preference was assessed by introducing individual pairs into one of the 11 oviposition mesh cages (40\u2009\u00d7\u200945\u2009\u00d7\u200965\u00a0cm3) each containing one BAR-type and one NAS-type plant and a source of sugar. Oviposition was checked every day at 10.30 and 15.30\u00a0h. M. brassicae oviposition was recorded 24\u00a0h after the first egg up to 7\u00a0days after introduction (N\u2009=\u200968). P. rapae oviposition was recorded for at least 5\u00a0h after the first egg and up to a maximum of 24\u00a0h in total (N\u2009=\u200935). Eggs deposited on the cage, pot, or on the label were not included in the analyses. To obtain sufficient replicates, four randomly chosen plants were used twice for M. brassicae, but oviposition preference was never tested on the same combination of plants.\nLarval Preference For P. rapae as well as for M. brassicae, we used plants from EL44 and EL13 half sibs (one plant per chemotype per half-sib family and per herbivore). Two 0.9\u00a0cm diameter discs of each chemotype were cut from fully expanded leaves and placed in a circle (in an alternated design) in a 12\u00a0cm diameter Petri dish. We tested neonate larvae and 5-day-old larvae, which, until use, were feeding on Brassica oleracea var. gemnifera L., cultivar Cyrus leaves. The larvae were released individually at the center of the Petri dish with equal distance to all leaf discs (N\u2009=\u200920 per species per age). After 4\u00a0h, the amount of leaf material consumed was recorded visually and categorized as follows: 0 = no damage, 1 = only consumption of the lower leaf layers, 2 = less then 10% was consumed, 3 = between 10% and 50% of the leaf disc was consumed, and 4 = more than 50% was consumed. We were not able to assign consumption categories in the experiment with neonates because they caused so little damage, but 5-day-old larvae showed distinct feeding patterns.\nLarval Performance Larval performance was tested by forcing neonate larvae to stay on selected plants and measuring larval biomass and survival every other day. We selected forty 6-week-old plants (20 EL44 half sibs and 20 EL13 half sibs; 10 of each chemotype per half-sib family). To obtain a total of 80 plants, every plant was multiplied by cutting it into two halves and growing each plant in fresh pots. After 3\u00a0weeks, the plants were used to test performance of M. brassicae. Larvae were kept on the plant using 25\u00a0mm diameter meshed clip cages. After 4\u00a0days, these clip cages were replaced by 55\u00a0mm diameter meshed clip cages. After 8\u00a0days, M. brassicae larvae had consumed such large amounts of leaf tissue that the experiment had to be stopped. The remaining larvae were removed and weighed. All plants were clipped and after 3\u00a0weeks of regrowth used to asses performance of Pieris in the same way as we did for M. brassicae. However, P. rapae larvae were kept on the plants till day\u00a018 when most larvae had pupated. Until day\u00a04, each larva was feeding on a single leaf that we harvested to measure the consumed leaf area and sugar, glucosinolate, and amino acid content. These samples were considered to provide an estimate for the chemotype differences over the first 8\u00a0days of feeding.The long-term performance of M. brassicae larvae was studied in a second experiment, in which three neonate larvae were placed on one plant (15 BAR and 15 NAS-type plants of EL 13). The larvae could freely move on the plant, but could not move to other plants due to a plastic cylinder and a water barrier around each plant. After 8 and 13\u00a0days, we determined larval mass and the number of dead or lost larvae. After 13\u00a0days on BAR-type plants, all but five larvae had died or were lost, so that we stopped the experiment. As dead larvae had often dried out, we excluded their biomass from all calculations.\nStatistical Analysis In the oviposition preference experiments, total egg load may differ among individuals. Therefore, the number of eggs on each chemotype per individual female was treated as a paired sample. Per paired sample, we considered the chemotype with the highest egg load as the preferred type. Overall preference was tested with a Sign test. The number of eggs per plant and number of clusters per plant were not normally distributed, and were analyzed with the Wilcoxon matched-pair signed-ranks test. As there were no paired observations for cluster size, these data were analyzed with the Mann\u2013Whitney U test.To analyze larval preference, we compared the average food consumption category between the two chemotypes by using the Wilcoxon matched-pair signed-ranks test.In the larval performance experiments, the larval masses were not normally distributed and therefore analyzed for differences between the chemotypes with separate Mann\u2013Whitney U tests for each day followed by Bonferroni correction for multiple comparisons (for M. brassicae \u03b1\u2009=\u20090.05\/4\u2009=\u20090.0125; for P. rapae \u03b1\u2009=\u20090.05\/8\u2009=\u20090.0062). Data on the leaf consumption of each herbivore on the two chemotypes were also tested with the Mann\u2013Whitney U test. The relationships between larval biomass and leaf consumption and between larval biomass and total glucosinolate concentration were tested by using Pearson product\u2013moment correlations. The data were log-transformed to get a normal distribution. Before analysis of variance (ANOVA) total glucosinolate, sugar, and amino acid content data were log-transformed to meet the assumptions of ANOVA. Statistical analyses were performed with STATISTICA (data analysis software system), version 7.1. (StatSoft (2005); http:\/\/www.statsoft.com).\nResults\nOviposition Preference Generalist and specialist adult herbivores did not prefer one chemotype over the other. Of the 68 M. brassicae females tested, 33.8% oviposited on the plants, 47.1% preferred to oviposit on the cage, whereas 19.1% did not oviposit at all within 1\u00a0week. The chemotypes did not receive significantly different numbers of eggs (Table\u00a01). M. brassicae, which is a gregarious species, deposited a total of 19 clusters on BAR-type and 19 on NAS-type plants. The number of clusters per plant and the average cluster size, ranging from 6 to 465 eggs per cluster, were not significantly different between chemotypes (Table\u00a01).\nTable\u00a01Oviposition of Mamestra brassicae and Pieris rapae on BAR-type and NAS-type Barbarea vulgaris\u00a0 Mamestra brassicaePieris rapaeBARNASPZBARNASPZPreferencea14110.8380.20412190.4720.720Eggsb220\u2009\u00b1\u200940.9193\u2009\u00b1\u200951.70.3990.84326.9\u2009\u00b1\u200918.226.6\u2009\u00b1\u200914.50.9220.100Clusters\/plantc1.36\u2009\u00b1\u20090.21.73\u2009\u00b1\u20090.30.9510.061Cluster sized162\u2009\u00b1\u200923.4112\u2009\u00b1\u200932.90.8000.254aTimes when that chemotype received most eggs in a pairwise comparisonbAverage number of eggs per plant \u00b1 SEcAverage number of clusters per plant \u00b1 SEdAverage number of eggs per cluster \u00b1 SEOf the 35 P. rapae pairs tested, 32 females oviposited on plants, two females did not oviposit at all, and one female died. With an average of 53.5\u2009\u00b1\u20095.1 eggs per female, 61% deposited most eggs on NAS-type plants, and one deposited an equal amount of eggs on both chemotypes. The average numbers of eggs deposited on each chemotype did not significantly differ between chemotypes (Table\u00a01). In total, BAR-type plants received 861 eggs and NAS-type plants 850 (Table\u00a01).\nLarval Preference Of the 20 5-day-old M. Brassicae larvae tested, only one preferred to feed on BAR-type leaf discs, whereas 19 preferred the NAS-type (Sign test: Z\u2009=\u20093.67, P\u2009<\u20090.001). This preference for NAS-type leaf discs was confirmed when corrected for the consumed quantity (feeding categories). M. brassicae larvae consumed more from NAS-type leaf discs than from BAR-type (Fig. 1; Wilcoxon matched-pairs test on feeding categories: M. brassicae N\u2009=\u200920, Z\u2009=\u20093.743, P\u2009<\u20090.001). In contrast, the 20 5-day-old P. rapae larvae did not show a significant preference. Five larvae preferred NAS-type, 11 BAR-type, and four consumed from BAR-type as well as NAS-type leaf discs (Sign test: Z\u2009=\u20091.25, P\u2009=\u20090.211). Although P. rapae larvae consumed more from BAR-type leaves, the difference in feeding categories was not significant (Fig.\u00a01; Wilcoxon matched-pairs test on feeding categories: P. rapae N\u2009=\u200920, Z\u2009=\u20091.629, P\u2009=\u20090.103). Consequently, we found no significant preference of P. rapae larvae for either chemotype.\nFig.\u00a01Consumption of BAR-type (grey bars) and NAS-type (white bars) leaf discs by 5-day-old larvae of Mamestra brasscicae and Pieris rapae (feeding category averaged per Petri dish \u00b1 SE)\nLarval Performance and Chemotypes Over the first 8\u00a0days, the larval biomass accumulation of the generalist M. brassicae was affected by chemotype (Mann\u2013Whitney U test: P\u2009<\u20090.001 for each day of recording). When forced to stay on NAS-type plants, M. brassicae larvae grew exponentially (Fig.\u00a02a). On BAR-type plants, however, M. brassicae larvae hardly increased in biomass and were moribund. This difference in performance was positively correlated to the difference in leaf consumption after 4\u00a0days of feeding (correlation analysis: r\u2009=\u20090.942, r2\u2009=\u20090.887, P\u2009<\u20090.01). Consequently, M. brassicae larvae caused more damage to NAS-type leaves than to BAR-type (Fig.\u00a03a; Mann\u2013Whitney U test: U\u2009=\u200912, Z\u2009=\u2009\u20137.583, P\u2009<\u20090.001). Larval survival after 8\u00a0days on the plants was in line with these results. On NAS-type plants, all larvae were still alive, whereas on BAR-type plants 37.5% of the M. brassicae larvae had died. The results of the second experiment, in which the larvae could move freely on the plants, confirmed the results of the clip cage experiment. After 13\u00a0days, 89% \u00b110 of the larvae had stayed and survived on NAS-type plants and weighed 42.13\u2009\u00b1\u20092.69\u00a0mg, whereas only 20% \u00b110 of the larvae had stayed and survived on BAR-type plants and weighed only 4.11\u2009\u00b1\u20090.02\u00a0mg.\nFig.\u00a02Biomass accumulation of aMamestra brassicae and bPieris rapae larvae during 8\u00a0days of feeding BAR-type (grey bars) and NAS-type (white bars) Barbarea vulgaris plants \u00b1 SEFig.\u00a03Leaf characteristics after 4\u00a0days of larval feeding of Mamestra brassicae (left graphs) or Pieris rapae (right graphs) on Barbarea vulgaris \u00b1 SEM: a and b consumed leaf area (cm2), c and d total glucosinolate content, e and f total sugar content, g and h total amino acid content. Results are depicted per chemotype (grey bars BAR-type plants; white bars NAS-type plants) and per half-sib family (no hatching EL13, hatching EL44)In contrast to M. brassicae larvae, P. rapae larvae grew and survived equally well on both chemotypes. P. rapae larval biomass accumulation over time showed no significant difference between chemotypes over the first 18\u00a0days (Fig.\u00a02b; Mann\u2013Whitney U test: P\u2009>\u20090.05 for each day of recording) and was positively correlated to leaf consumption after 4\u00a0days (correlation: r\u2009=\u20090.768, r2\u2009=\u20090.589, P\u2009<\u20090.01). Leaf damage due to P. rapae feeding was not different between the chemotypes (Fig.\u00a03b; Mann\u2013Whitney U test: U\u2009=\u2009673, Z\u2009=\u2009\u22121.217, P\u2009=\u20090.223). After 8\u00a0days, larval mortality was 7.5% on NAS-type and 12.5% on BAR-type plants. On day\u00a014, the first P. rapae larvae started pupating. After 16\u00a0days on BAR-type plants, 45% had pupated, and on NAS-type plants this was 47.5%. At the end of the experiment, after 18\u00a0days, larval weights were still not significantly different on the chemotypes (on BAR-type: 161.9\u2009\u00b1\u20097.37\u00a0mg, on NAS-type: 175.24\u2009\u00b1\u20095.63\u00a0mg; Mann\u2013Whitney U test: U\u2009=\u2009238, Z\u2009=\u2009\u22121.131, P\u2009=\u20090.265).\nLarval Performance and Total Glucosinolate Content The leaves on which larvae were feeding differed in glucosinolate composition (glucobarbarin vs. gluconasturtiin) and in total glucosinolate content (Fig.\u00a03c,d). In both chemotypes, we detected glucobarbarin, gluconasturtiin, glucosibarin, glucobrassicin, 4-methoxyglucobrassicin, and neoglucobrassicin. In both families, BAR-type leaves had on average 1.5 times higher total glucosinolate levels than NAS-type leaves (Fig.\u00a03c; ANOVA type effect: F1,143\u2009=\u200922.831, P\u2009<\u20090.001). Because M. brassicae fed more on NAS plants, overall a negative\u2014but weak\u2014correlation of total glucosinolate content with the consumed leaf area was observed (correlation: r(X,Y)\u2009=\u2009\u22120.26, r2\u2009=\u20090.065, P\u2009<\u20090.05). To analyze in more detail whether total glucosinolate level determines larval consumption and performance, we also examined the correlation within each chemotype. Within chemotypes, the range of glucosinolate concentrations was substantial (factor 6\u20137), but there was no negative correlation between glucosinolate level and larval biomass (Fig.\u00a04; Correlation: within BAR, r(X,Y)\u2009=\u20090.15, r2\u2009=\u20090.024, P\u2009=\u20090.932; within NAS, r(X,Y)\u2009=\u20090.28, r2\u2009=\u20090.078, P\u2009=\u20090.090). Moreover, larvae on BAR-type leaves with similar low levels of glucosinolates as NAS-types consumed and performed considerably worse (Fig.\u00a04; glucosinolate levels <35\u00a0\u03bcmol g\u22121 dry mass). There was also no difference in total glucosinolate level from BAR-type leaves on which the larvae survived or died (with living larvae: 31.32\u2009\u00b1\u20092.95\u00a0\u03bcmol g\u22121, with dead larvae: 32.81\u2009\u00b1\u20096.49\u00a0\u03bcmol g\u22121; ANOVA: F1,29\u2009=\u20090.059, P\u2009>\u20090.05; Fig.\u00a04). As the performance of P. rapae larvae was similar among chemotypes and families, there was no correlation of larval biomass with total glucosinolate level.\nFig.\u00a04Correlation of total glucosinolate content and the larval biomass after 4\u00a0days feeding of Mamestra brassicae experiment on NAS-type (white dots), BAR-type (black dots), and dead larvae of on BAR-type (black crosses) Barbarea vulgaris plants with their correlations per chemotype (black line) and their 95% confidence intervals (dotted lines)\nLarval Performance and Nutritional Value Total sugar content of both chemotypes was composed of five consistently detected sugars, which were glucose, sucrose, sorbitol, fructose, and trehalose (from high to low average concentration). Total amino acid content of both chemotypes was composed of 11 consistently detected amino acids, which were threonine, isoleucine, arginine, serine, glutamate, aspartate, glutamine, asparagine, phenylalanine, tyrosine, and histidine (from high to low average concentration). Because the individual sugars and amino acids showed effects similar to the total levels (results not shown), we only present data on total levels.In contrast to the total glucosinolate level, sugar and amino acid content did not differ between the chemotypes (Fig.\u00a03e\u2013h). Although in the M. brassicae experiment the total sugar content was overall higher in BAR-type plants, there was a significant interaction between chemotype and family (Fig.\u00a03e; ANOVA type effect: F1, 75\u2009=\u200912.014, P\u2009<\u20090.001; type \u00d7 family F1, 75\u2009=\u200910.252, P\u2009<\u20090.05). Similar interactions between chemotype and family were shown for amino acid content of the plants in the M. brassicae experiment and for sugars and amino acid content of plants in the P. rapae experiment. In all chemotype \u00d7 family interactions, the BAR-type of the EL13 half-sib family had higher levels of primary metabolites than the NAS-type of that family (Fig.\u00a03e\u2013h). In the EL44 half-sib family, the sugar and amino acid level differences of the chemotypes were negligible in plants during the M. brassicae experiment or even opposite to those of the EL13 during the P. rapae experiment. As sugar and amino acid content did not vary consistently with chemotype, these cannot explain the differences in larval performance and preference between the chemotypes.\nDiscussion\nNAS and BAR chemotypes of B. vulgaris differentially affected preference and performance of the generalist herbivore M. brassicae but not the specialist P. rapae. Larvae of M. brassicae grew exponentially and had 100% survival on NAS-type leaves, but hardly grew and had a high mortality when feeding on BAR-type plants. As the dose-dependent effect of total glucosinolate content on herbivores is well-known (Li et al. 2000; Agrawal and Kurashige 2003; Mewis et al. 2006), the poor performance of M. brassicae could have been caused by the on average 1.5 times higher total glucosinolate content of BAR-type plants compared to NAS-type plants (Van Leur et al. 2006). However, we did not observe any dose-dependent effect within the chemotypes. Moreover, M. brassicae larvae on BAR-type plants with total glucosinolate levels similar to NAS-type plants still performed significantly worse. Hence, we exclude total glucosinolate level as explanatory factor for the differences found between chemotypes on larval biomass and leaf consumption.\nIn addition to total glucosinolate content, sugar and amino acid contents differed between chemotypes, but the pattern was inconsistent among half-sib families. The statistical interaction between chemotype and family found for sugar and amino acid levels was not found for herbivore performance and preference (Fig.\u00a03). Therefore, total sugar content and total amino acid content can not explain the observed effects on M. brassicae.\nIn a different B. vulgaris polymorphism, described by Agerbirk et al. (2003b), there were P- and G-type plants that differed in resistance to Plutella xylostella. Other than our BAR\/NAS polymorphism, the P-type and G-types also differed in trichome density, did not co-occur in natural populations, were hard to cross, and neither of the Danish types had a high content of gluconasturtiin in the leaves (Agerbirk, personal communication).The resistance of the G-type to P. xylostella was due to a difference in saponin content (Agerbirk et al. 2003a). Based on these findings, we compared saponin levels between BAR and NAS-type plants by using LC-TOF-MS (see supplemental data). This analysis revealed that the levels of the saponin described by Agerbirk et al. (2003a) and a saponin described by Shinoda et al. (2002) did not differ between the NAS and BAR chemotypes (supplemental data). Therefore, we exclude these saponins as an explanatory factor for the poor performance of M. brassicae on BAR-type plants.\nThe observed effects are likely caused by difference in glucosinolate structures and their break-down products. BAR-type plants were more toxic and deterrent to M. brassicae than the NAS-type plants. This suggests that M. brassicae can deal effectively with gluconasturtiin and isothiocyanates, but not with glucobarbarin and resulting oxazolidine-2-thiones. The latter compounds have received hardly any attention in chemical\u2013ecological literature. Generalist herbivores possess broad-spectrum detoxification enzymes such as P450 enzymes and mixed-function oxidases (MFO) that enable them to deal with a wide range of allelochemicals, including glucosinolates (Schoonhoven et al. 1998; Li et al. 2000). Which detoxification mechanism is present in M. brassicae and why it can handle the presumably more toxic isothiocyanates, but not the oxazolidine thiones is unknown. The non-different performance of P. rapae on the chemotypes may indicate that NSP enzymes are equally effective in redirecting the hydrolysis pathways of both chemotypes towards the generally less toxic nitriles (Wittstock et al. 2004). Identification of the bioactive compounds could be acquired by using bioassay-guided fractionation.\nNeither herbivore species significantly preferred one of the chemotypes as a host plant for oviposition. Even though isothiocyanates are known to elicit anemotaxis in herbivores at extremely low concentrations (Finch and Skinner 1982), the concentrations of volatile cues emitted by our undamaged plants may have been below the detection limit (Finch et al. 1978). Upon damage or induction by herbivores, volatile levels may rise and affect oviposition preference (Rothschild and Schoonhoven 1977; Bruinsma et al. 2007). Next to chemical suitability, the surface on which to oviposit can be an important factor (Renwick and Chew 1994). In our experiment, however, leaf surface structure was unlikely to affect oviposition preference between chemotypes because the chemotypes did not have apparent differences in leaf surface (e.g., trichomes) in contrast to the completely different B. vulgaris polymorphism reported by Agerbirk et al. (2003b).\nAs M. brassicae larvae performed poorly on BAR-type plants, the lack of oviposition preference of adults seems to be non-adaptive. The discrepancy between larval performance and oviposition preference on B. vulgaris has also been shown for the Diamondback moth (Plutella xylostella) (Serizawa et al. 2001; Badenes-Perez et al. 2006). It may occur when insects or plants are new to an area and there has not been enough time for evolutionary adaptation (Agosta 2006). Whether this is the case for M. brassicae and the B. vulgaris chemotypes is unknown.\nCo-evolutionary theory suggests that the variation of plant defense compounds is maintained by sequential adaptations of specialist herbivores and plants (Agrawal et al. 1999; Cornell and Hawkins 2003). The good performance of P. rapae on both chemotypes suggests that neither chemotype is effectively defended against this specialist. On the contrary, BAR-type plants are defended against the generalist M. brassicae. Although we have no experimental evidence indicating which chemotype is the newest form, the effective defense of BAR-type plants against M. brassicae suggests an evolutionary adaptive step in which BAR-type evolved from NAS-type. This matches with the biosynthetic origin of these glucosinolates. Gluconasturtiin is presumably the precursor that is hydroxylated to produce glucobarbarin. Moreover, gluconasturtiin occurs in five times more genera than glucobarbarin (Fahey et al. 2001). This does not preclude, however, that the NAS-types found locally in the Netherlands are due to a loss-of-function mutation from the BAR-type. In natural populations of B. vulgaris, there are only 0\u201322% NAS-type plants (Van Leur et al. 2006). This results in a potentially limited genetic basis for NAS-type plants compared to BAR-type plants, which could contribute to linkage disequilibrium between the BAR\/NAS locus and other loci (for instance closely linked loci). However, the natural population from which we selected seed batches for these experiments had a phenotype frequency close to a Hardy\u2013Weinberg equilibrium (22% potential NAS-type fathers observed instead of 25% expected). We estimate the chance of linkage disequilibrium effects in our experiments to be relatively small. Additionally, there are no a priori indications of closely linked loci that affect herbivore performance.\nFor the plant, the benefit of increasing its defense against M. brassicae will be larger than increasing its defense against P. rapae. Being gregarious and larger, M. brassicae larvae are more harmful to the chosen individual (many larvae feeding from one plant) and will more strongly reduce plant fitness than the solitary P. rapae (only one or few larvae feeding on a plant). Therefore, irrespective of the degree of specialization of the herbivores, it will be more important for B. vulgaris to be defended against M. brassicae than against P. rapae.\nBased on our results, we would expect BAR-type plants in the field to suffer less from herbivory than NAS-type plants. Besides being the chemotype that is most severely damaged by M. brassicae larvae, the NAS chemotype is also the recessive genotype. Therefore, if herbivorous insects are the only selective force, we expect that natural selection will drive natural populations towards 100% BAR-types. European populations of B. vulgaris indeed are mainly dominated by BAR-type plants, but a minority number of populations has still up to 22% NAS-type plants. This indicates that in these populations there may be other factors that play a role in maintaining this chemical polymorphism. Other factors that may differ between the two types and that determine plant fitness, e.g., belowground herbivory, higher trophic level interactions, and inter- and intraspecific competition, need to be included in future studies.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nESM\n(DOC 52.5 kb)","keyphrases":["barbarea vulgaris","glucosinolates","performance","mamestra brassicae","pieris rapae","oviposition","polymorphism","co-evolution"],"prmu":["P","P","P","P","P","P","P","U"]}
{"id":"Histochem_Cell_Biol-4-1-2323033","title":"Cell fusions in mammals\n","text":"Cell fusions are important to fertilization, placentation, development of skeletal muscle and bone, calcium homeostasis and the immune defense system. Additionally, cell fusions participate in tissue repair and may be important to cancer development and progression. A large number of factors appear to regulate cell fusions, including receptors and ligands, membrane domain organizing proteins, proteases, signaling molecules and fusogenic proteins forming alpha-helical bundles that bring membranes close together. The syncytin family of proteins represent true fusogens and the founding member, syncytin-1, has been documented to be involved in fusions between placental trophoblasts, between cancer cells and between cancer cells and host cells. We review the literature with emphasis on the syncytin family and propose that syncytins may represent universal fusogens in primates and rodents, which work together with a number of other proteins to regulate the cell fusion machinery.\nCell fusions in the developing mammal\nThe fusion between the sperm and the egg marks the beginning of a new individual and ensuing development of either man or beast depends upon a number of additional cell fusion events (reviewed by Ogle et al. 2005). Thus, mononuclear cytotrophoblasts fuse to form the syncytiotrophoblast layer of the placenta in man and certain other mammals. Syncytiotrophoblasts control the exchange of gases, nutrients and waste products between the fetus and the mother, protect the fetus against the maternal immune system and are also responsible for the production of hormones, which like chorionic gonadotropin, regulate the continuation of pregnancy. In addition, in the developing individual, myoblasts fuse to form multinucleated skeletal muscle fibers, while cells of monocytic origin fuse to form osteoclasts, which participate in bone sculpturing and remodeling as well as in the regulation of serum calcium concentrations.\nCell fusions contribute to tissue repair\nIt is well-known that skeletal muscle is regenerated through fusion of muscle fibers with satellite cells (Bischoff 1994) and that macrophages may fuse to form multinucleated giant cells with enhanced phagocytic capabilities in response to injury and antigenic challenges (Vignery 2005). Recent data indicate that additional cell fusions may contribute to tissue repair in the adult (Vassilopoulos et al. 2003; Wang et al. 2003). Bone-marrow-derived (BMD) cells have been shown to fuse with hepatic cells, nerve cells and gastrointestinal cells and the theory has been put forward that such fusions may serve to repair damaged or corrupted cells (Alvarez-Dolado et al. 2003; Rizvi et al. 2006; Vassilopoulos et al.2003; Wang et al. 2003). However, it is still debated whether such fusions are important to tissue repair and whether they engage BMD stem cells or more differentiated fusogenic cells like monocytes\/macrophages (reviewed by Vignery 2005). Intriguingly, the accumulation of macrophages in injured organs may reflect not only the need to remove debris but also to repair compromised cells through heterotypic fusions (Vignery 2005). Moreover, in recent transplant experiments, irradiated mice showed evidence of fusions between crypt stem cells and donor BMD cells (Rizvi et al. 2006). In thus transplanted mice, representatives of all cell lineages derived from intestinal crypt stem cells (absorptive columnar, enteroendocrine, goblet and Paneth cells) showed expression of chromosomal markers characterizing donor BMD cells. This phenomenon appeared to depend upon tissue injury since expression of donor cell markers never was detected in non-irradiated mice (Rizvi et al. 2006). An alternative to fusions has been suggested by Holmgren et al. (1999) and de la Taille et al. (1999), who have demonstrated that cells phagocytosing apoptotic cells may acquire functional DNA. Moreover, a mechanism of cell\u2013cell invasion (entosis) was recently described and could potentially transfer DNA between cells (Overholtzer et al. 2007). Thus, viral as well as normal sequences from apoptotic cells have been found to be active also in phagocytosing cells (Holmgren et al. 1999). This observation challenges our understanding of apoptosis as an efficient way of clearing corrupted or alien DNA.\nCell fusions during viral infections\nInfections with enveloped viruses such as HIV-1 may also lead to cell fusions. The viral genome encodes envelope (Env) proteins, which bind to cell surface receptors and assist the virus in entering the cell. The infected cell commences to synthesize Env proteins, which, upon insertion in the plasmalemma, engages receptor proteins in neighboring cells and initiate fusions. Interestingly, modifications of the cytoplasmic tail of several Env proteins appear to modify fusions. Thus, proteolytic cleavage of the cytoplasmic tail regulates fusions and also cellular signaling events, such as tyrosine kinase activity, may be involved in regulation of virally induced cell fusions (Kubo et al. 2003).\nReceptor proteins may bind Env proteins from different classes of viruses. Thus, a cell infected by a specific virus becomes resistant to infections with other viruses that bind to the same receptor. This phenomenon, which probably reflects receptor saturation, has led to the grouping of retroviruses into different subtypes, which bind to the same receptor. Other types of viruses encode fusogenic proteins (like influenza hemagglutinin), which do not bind to surface receptors, but which liberate a fusion peptide in the acidic milieu of endosomes and thus initiate viral\u2013host cell fusion following endocytic uptake of viral particles. Both these proteins and the Env proteins are referred to as class I viral fusion proteins and contain hydrophobic fusion peptides buried within their sequences. Following receptor engagement or endosomal acidification the fusion peptides are liberated by proteolytic cleavage. The cleaved peptides undergo conformational changes, resulting in hairpin-like, alpha-helical bundles, which bring the viral and cell membranes into close apposition and thereby facilitates fusion (reviewed by Chen and Olson 2005).\nCancer\u2013host cell fusions\nIn 1911 Aichel suggested that cancer cells might spontaneously fuse with host cells to produce hybrids with supernumerary chromosomes that could evolve into cells of increased malignancy. Subsequent studies have confirmed this prediction by demonstrating that cells with mixed phenotypes spontaneously appear in co-cultures of normal and malignant cells (Barski and Cornefert 1962; Busund et al. 2002; Mortensen et al. 2004; Wakeling et al. 1994) (Figs.\u00a01, 2, 3). Additionally, transplanted tumor cells of animal (Busund et al. 2003; Chakraborty et al. 2000; Fortuna et al. 1990; Kerbel et al. 1983; Larizza et al. 1984a; Wiener et al. 1974a, b) or human (Goldenberg et al. 1974; Goldenberg and Pavia 1982; Mortensen et al. 2004) origin may fuse with and acquire phenotypic characteristics of normal host cells (Fig.\u00a04). In some of the in vivo studies, data suggested that the putative fusion partner was of macrophage or endothelial origin but, in most cases, definite identification was not achieved. In a few cases, it has also been demonstrated that the fused cells expressed genetic markers of both parental cell types (Goldenberg et al. 1974; Mortensen et al. 2004; Wiener et al. 1974a, b) (Figs.\u00a02, 3). Mortensen et al. (2004) demonstrated that, initially after fusion, bi- or multinucleated cells formed and that such cells had the parental genomes seggregated in diffferent nuclei (heterokaryons) (Fig.\u00a02a\u2013e). Subsequently, mitotic figures appeared showing an admixture of the parental chromosomes (Fig.\u00a02f\u2013j). Eventually, cells with the parental genomes mixed in a single nucleus (synkaryons) were detected (Fig.\u00a02k\u2013o). In agreement with the notion that synkaryons appear after mitotic divison of heterokaryons, synkaryons are usually detected in pairs (Fig.\u00a03). A few recent studies present compelling evidence that cells with genetic characteristics of hybrid cells also may appear in human tumors (Andersen et al. 2007; Chakraborty et al. 2004; Yilmaz et al. 2005; Streubel et al. 2004). Thus, in two cases of renal carcinomas, arising in bone marrow transplant recipients, some tumor cells showed genetic markers characterizing the healthy donor (Chakraborty et al. 2004; Yilmaz et al. 2005). Moreover, in patients with multiple myelomas, osteoclasts have been shown to contain an admixture of nuclei, of which some possess tumor-specific chromosomal translocations while others are devoid of translocations (Andersen et al. 2007). Finally, lymphoma-specific genetic abnormalities were described in endothelial cells in B cell lymphomas (Streubel et al. 2004). Although these data do not formally prove that tumor\u2013host cell hybrids did form in these patients, the combined results from studies in vitro and in vivo do present compelling evidence that cell fusions do occur in tumors (reviewed by Pawelek 2005).\nFig.\u00a01Cancer\u2013endothelial cell fusion. Co-culture of human breast cancer (MCF-7) cells and human umbilical vein endothelial cells (HUVEC). Cells were stained for the cancer cell marker cytokeratin (red, c), the endothelial cell marker vimentin (green, b) and DNA (bisbenzimide, blue, d). Note in the merged image (a) one fused, multinucleated cell reacting for both cytokeratin and vimentin (orange-yellow)Fig.\u00a02Chromosomal markers and cell fusions. Co-cultures of human breast cancer cells and bovine endothelial cells. The cultures were subjected to fluorescent in situ hybridization (FISH) with probes recognizing all bovine chromosomes (green; b, g, l) and all human chromosomes (red; c, h, m) and counter-stained for DNA with DAPI (blue; d, i, n) and observed in differential interference contrast (DIC; e, j, o). Merged red-green images are shown in a, f, k. Note that initially, binuclear cells (heterokaryons) form, having one bovine and one human nucleus (a\u2013e). Occasionally, tri- or multinuclear cells with different admixtures of bovine and human nuclei are also detected. At longer times after mixing, mitotic figures, containing an admixture of bovine and human chromosomes appear (f\u2013j) and, eventually, cells with a single nucleus, containing an admixture of the two genomes in mixture (synkaryons) are detected (k\u2013o)Fig.\u00a03Chromosomal markers and cell fusions. Mixed culture of human breast cancer cells and bovine endothelial cells submitted to double FISH as in Fig.\u00a02 (a double FISH; b combined DAPI and DIC). Low power micrograph showing a pair of synkaryons with the bovine and human genomes admixed in single nuclei (arrows). In addition, nuclei hybridizing only for the bovine (green) or only for the human (red) genome occurFig.\u00a04Cancer\u2013host cell fusion in vivo. a, b Section from lung of a nude mice injected with human breast cancer (MDA-MB-231) cells in the tail vein (Mortensen et al. 2004). The section underwent FISH for the mouse genome (red) and the human genome (green) (a) and DNA was counterstained with DAPI (b; blue). Note one nucleus in which the human and mouse genome co-localize (arrow). C: Similar section, stained with an antibody detecting human (but not mouse) p53 (red; p53 is mutated and overexpressed by the breast cancer cells), an antibody to beta-catenin (to mark cell membranes) and for DNA with bisbenzimide (blue). Note a micrometastasis of human breast cancer cells having violet (red\u00a0+\u00a0blue) fluorescent nuclei. d Section stained for human p53 (red) and the endothelial marker von Willebrand factor (green) and DNA (blue). Note a human cancer cell with a violet (red\u00a0+\u00a0blue) nucleus showing membrane-staining for von Willebrand factor. Since von Willebrand factor is not normally expressed by the breast cancer cells, this image is suggestive of a fusion between a human breast cancer cell and a mouse endothelial cell. Similar results were obtained using double FISH for the human and mouse genome and imunofluorescent staining for von Willebrand factor (described by Mortensen et al. 2004)\nThe essential question regarding cancer\u2013host cell fusions is, of course, if they are relevant to the patient. In fact, there are two opposing views. The first is based on early experiments on fusions induced to occur between cancer cells and normal cells in culture. With few exceptions, such experiments revealed that malignancy was suppressed (Harris et al. 1969; Harris 1988; Stanbridge 1976; Wiener et al. 1974a, b). These studies were, in fact, seminal to the discovery of tumor suppressor genes (reviewed by Anderson and Stanbridge 1993). Since tumor suppressor genes, like p53 and Rb, frequently are inactivated in cancer cells, fusions would present cancer cells with unperturbed tumor suppressors from the normal fusion partner and consequently initiate cell cycle arrest or apoptosis. Although, this certainly applied for the cell types studied in the contributions cited above, it may not be a general rule. Thus, production of monoclonal antibodies depends upon the fact that it is possible to fuse antibody-producing spleen cells with myeloma cells to obtain hybridomas that retain the unlimited proliferative ability of the tumor cell partner and the antibody production of the normal cell (Kohler and Milstein 1975). In fact, several studies documented that some fusions may lead to cells of increased malignancy (Barski and Cornefert 1962; Busund et al. 2003; Chakraborty et al. 2001; De Baetselier et al. 1981; Kerbel et al. 1983; Larizza et al. 1984b; Pawelek 2000; Rachkovsky et al. 1998). Possibly, the genetic make-up of tumors may dictate the outcome of individual cancer\u2013host cell fusions. Moreover, fusions lead to aneuploidy and chromosomal instability, which characterizes most cancers and may, by itself, stimulate carcinogenesis (Duelli et al. 2007).\nMechanisms behind cell\u2013cell fusions\nFusions between normal cells\nCell fusion events must be extremely well controlled. Due to their major importance to fertilization, placentation, muscle development, bone structure, calcium homeostasis and the immune defense system, much effort has gone into elucidating mechanisms underlying cell\u2013cell fusions. Additionally, the potential role of cell fusions as a repair mechanism and the role of cell fusions in cancer development and progression have further stimulated research in this field. In spite of this, much less is known about cell\u2013cell fusion mechanisms than is known about how intracellular membranes fuse through v- and t-SNAREs. Interestingly, engineered flipping of the v- and t-SNARE machinery has been shown to promote cell\u2013cell fusions (Weber et al. 1998). However, it is evident that this mechanism is not a physiological mediator of cell\u2013cell fusions. Interestingly, v- and t-SNAREs act similar to class I viral fusion proteins in that they form bundles of alpha-helices, which result in membrane apposition and fusion (Blumenthal et al. 2003; Jahn et al. 2003). Of the many proteins, which to date have been shown (or proposed) to be involved in cell fusions in mammals, only the syncytin family appears to use a similar alpha-helical mechanism. That they do is not surprising in view of the fact that syncytins represent conserved endogenous retroviral Env sequences. The founding family member, syncytin-1, was discovered as a protein capable of mediating fusions between cytotrophoblasts into syncytiotrophoblasts (Blond et al. 2000; Mi et al. 2000). This capability may have contributed to a high degree of evolutionary conservation of the syncytin-1 sequence. Syncytin-1 represents the Env protein of the human endogenous retroviral (HERV) W sequence, which entered the primate genome 25\u201340 million years ago. In contrast, most other retroviral sequences inserted in our genome have been subject to inactivating changes and probably represent garbage sequences. Molecular studies have shown that syncytin-1 (Env W) shares a structure similar to class I viral fusion proteins, especially in the region of the N- and C-terminal heptad repeats (NHR and CHR), and shares a common fusion mechanism with these proteins (Chang et al. 2004; Gong et al. 2005). A synthetic peptide derived from the CHR is also capable of inhibiting syncytin-1-mediated fusions by perturbing this mechanism (Chang et al. 2004). Syncytin-1 binds to the D-type retroviral receptor ASCT-2 (Blond et al. 2000) and may use another neutral amino acid transporter (ASCT-1) as an auxiliary receptor (Lavillette et al. 2000). Syncytin antibodies, syncytin-1 downregulation through antisense oligonucleotides and the syncytin-1 CHR peptide have been shown to inhibit fusions between trophoblast-derived cells (Blond et al. 2000; Chang et al. 2004; Mi et al. 2000). Agents increasing cellular levels of cAMP or cAMP analogues have been shown to promote cytotrophoblast fusions in vitro (Keryer et al. 1998) and such agents are also known to elevate protein and mRNA levels of syncytin-1 in isolated cytotrophoblasts (Frendo et al. 2003). Also estradiol may regulate syncytin-1 expression (Carino et al. 2003) and the placenta-specific transcription factor GCMa interacts with two upstream sites in the HERV-W 5\u2032-long terminal repeat and stimulates syncytin-1 transcription (Yu et al. 2002). Additionally, experimentally induced truncations in the cytoplasmic tail of syncytin-1 increases its fusogenicity (Drewlo et al. 2006), similar to what has been observed for some virally derived Env proteins (reviewed by Kubo et al. 2003). However, if modifications in the cytoplasmic tail of syncytin-1 are of physiological importance for regulating fusogenicity has yet to be demonstrated.\nA second syncytin-family member, syncytin-2, was subsequently found also to be expressed in the placenta (Blaise et al. 2003). Syncytin-2 also represents a highly conserved endogenous retroviral envelope gene and is derived from the HERV FRD sequence (Blaise et al. 2003). Immunocytochemical studies have localized syncytin-1 primarily to syncytiotrophoblasts as well as to cytotrophoblasts (Fig.\u00a05). The exact localization is somewhat controversial (reviewed by Potgens et al. 2004), which may reflect differences in antibodies, fixation procedures and controls. A major point of concern is also the degree of cross-reactivity to the related sequences in syncytin-2, which recently was localized to a subpopulation of cytotrophoblasts (Malassin\u00e9 et al. 2008). The syncytin-1 receptor protein ASCT-2 was recently localized to cytotrophoblasts (Hayward et al. 2007). More studies on the exact distribution of syncytin expression in the placenta using syncytin-1 and -2 specific antibodies seem warranted in order to exactly localize where fusions are likely to occur.\nFig.\u00a05Human term placenta immunocytochemically stained with a syncytin-1 peptide antiserum. Staining occurs in the syncytiotrophoblasts, which predominate at this stage. Nuclei are lightly counterstained with haematoxylin\nTrophoblast cell fusions are, however, not unique to the primate placenta. Thus, also in the mouse placenta, trophoblasts fuse to form syncytiotrophoblasts but mice do, of course, not express HERV sequences. Amazingly, an in-silico search of the mouse genome unraveled the existence of two murine endogenous retroviral (MERV) Env genes, labeled syncytin-A and -B (Dupresssoir et al. 2005). These genes were also fusogenic and orthologous genes were present in additional species of muridae (rats, gerbils, voles and hamsters) and appear to have entered the rodent lineage some 20 million years ago (Dupresssoir et al. 2005). They were expressed in the placenta and at least syncytin-A has been shown to be involved in the formation of syncytiotrophoblasts in mice (Gong et al. 2007). This represents an amazing example of parallel acquisition of retroviral genes of importance to reproduction in primates and rodents and poses the question whether also other species may have acquired similar viral genes of importance to cell fusions. The ability of syncytins to induce cell fusions may not be their only physiologic role. Thus, recent studies have shown that while syncytin-2 and syncytin-B also possess immunosuppressive activity, syncytin-1 and syncytin-A do not (Mangeney et al. 2007). Syncytin-1 expression was originally detected in the placenta and testis (Blond et al. 2000; Mi et al. 2000) but subsequent studies have revealed the presence of syncytin-1 also in the brain (Antony et al. 2004) and in breast, colon and endometrial cancers (Bjerregaard et al. 2006; Strick et al. 2007, Larsen, Talts, Andersen, Bjerregaard and Larsson: work in progress). Syncytin-1 may also regulate production of inflammatory mediators (Antony et al. 2004).\nA number of additional molecules have been shown to be important to cell fusions. In fact, in all systems studied so far, from mating yeast to man, a bewildering array of mechanisms have been identified (see Chen and Olson 2005; Chen et al. 2007; Oren-Suissa and Podbilewicz 2007 for recent reviews). Molecules potentially involved in mammalian cell fusions include ADAM (a disintegrin and metalloproteinase domain) 12 (meltrin alpha), which has been associated with myoblast and osteoclast cell fusions (Abe et al. 1999; Galliano et al. 2000; Gilpin et al. 1998; Yagami-Hiromasa et al. 1995). In addition, ADAMs 1 and 2 (fertilins) may be involved in sperm\u2013oocyte fusions, but do not seem indispensable for this function and, in man, the fertilin alpha gene is dysfunctional (Chen and Olson 2005; Cho et al. 1997, 1998, Evans et al. 1998; Jury et al. 1997, 1998). In contrast, the tetraspanin protein CD9 is needed for sperm\u2013oocyte fusions (Le Naour et al. 2000). CD9 is also expressed by BeWo trophoblast tumor (choriocarcinoma) cells and has been linked both to BeWo invasiveness and to invasion during mouse embryo implantation (Hirano et al. 1999; Liu et al. 2006). CD9 has, together with another tetraspanin family member, CD81, also been linked to myoblast fusion and myotube maintenance (Tachibana and Hemler 1999). Moreover, antibodies to either CD9 or CD81 have been shown to block fusions induced by Mason\u2014Pfizer monkey virus\u2014a D-type retrovirus (Duelli et al. 2005). Tetraspanins are known to organize other proteins into membrane microdomains and may link to the actin cytoskeleton via EWI (Glu-Trp-Ile) and ERM (ezrin\u2013radixin\u2013moesin) proteins (Hemler 2003; Sala-Vald\u00e9s et al. 2006). Possibly, their role as organizers of other proteins into microdomains may play a role in their involvement with cell fusions (Zivyat et al. 2006). They do not express characteristics of fusogenic proteins like the syncytins, SNAREs and class I viral envelope proteins, but are coexpressed with syncytin-1, at least in BeWo cells. A macrophage fusion receptor (MFR, SIRPalpha), resembling CD4\u2013the cell surface receptor for HIV\u2014has been identified in macrophages (Saginario et al. 1995, 1998; van den Berg et al. 2005). MFR, which belongs to the immunoglobulin superfamily, binds another member of this family, CD47. CD47 is also structurally related to proteins expressed by Vaccinia and Variola viruses (Chen et al. 2007). Whereas expression of MFR is restricted to myeloid cells and neurons, CD47 is ubiquitously expressed. MFR is transiently induced in macrophages at the onset of fusion while CD47 expression is constant. It has been hypothesized that CD47 initially binds to a long form of MFR to secure recognition and then switches to bind a shorter form to bring the plasma membranes closer (5\u201310\u00a0nm) for fusion (Vignery 2005). Additionally, or alternatively, CD47 may promote calcium entry by forming a membrane pore (reviewed in Chen et al. 2007). Also the hyaluronan receptor, CD44, is induced transiently when macrophages start to fuse. CD44 and MFR are subsequently cleaved by proteases during fusion. As mentioned above, CD9 and CD81 may also contribute to macrophage fusion. Additionally, DC-STAMP seems to be required for macrophage fusions. Mice lacking DC-STAMP are osteopetrotic and lack multinucleated osteoclasts and giant cells (Yagi et al. 2005). DC-STAMP is a seven-transmembrane receptor, somewhat similar to the HIV co-receptor CXCR4, but a ligand has yet to be identified.\nThese and many more molecules, including integrins, vacuolar ATPase and receptors and their ligands, as well as different signaling intermediates have been associated with cell fusions. With the exception of the syncytins, most of the mammalian molecules so far studied do not fulfill strict criteria for fusogens and several are dispensable for fusions (reviewed by Chen and Olson 2005; Chen et al. 2007; Oren-Suissa and Podbilewicz 2007). However, this may reflect molecular redundancy and does not definitely exclude that these molecules may participate in fusions. It is noteworthy that, in both man and mouse, two different syncytins are expressed and are both fusogenic. It is possible that, in the mammalian system, fusion requires a flotilla of molecules organized into membrane microdomains by proteins like CD9, encompassing receptors\/ligands, signaling entities, proteases and fusogens of retroviral origin that are capable of forming alpha-helical bundles, which bring membranes closer. Syncytins and related retroviral envelope sequences may, in this connection, function both as fusogens and receptor ligands. However, it seems unlikely that as irreversible an event as a cell\u2013cell fusion should depend upon a single receptor\u2013ligand interaction. Thus, both facilitatory and inhibitory factors are expected to be part of the flotilla.\nCancer cell fusions\nStudies by Mortensen et al. (2004) documented that human breast cancer cells fused with endothelial cells in culture. Stimulated by studies showing that syncytin-1 was involved in cytotrophoblast cell fusions (Blond et al. 2000; Mi et al. 2000), we examined whether a similar mechanism could account for cancer\u2013endothelial cell fusions. Expression of syncytin-1 was documented in the breast cancer cell lines examined (MCF-7 and MDA-MB-231 cells; Bjerregaard et al. 2006, SK-BR-3 cells: Talts, Bjerregaard and Larsson: unpublished data). Moreover, we found that both tumor cells and endothelial cells expressed the syncytin-1 receptor ASCT-2. Use of phosphorthioate-protected syncytin-1 antisense oligonucleotides downmodulated syncytin-1 expression as measured by either quantitative RT-PCR or Western blotting and inhibited breast cancer\u2013endothelial cell fusions, whereas a scrambled oligonucleotide control was without effect. Additionally, the syncytin-1 CHR peptide, referred to above, also inhibited the fusions whereas a control peptide was without effect (Bjerregaard et al. 2006). However, neither the antisense nor the CHR peptide experiments effected a total inhibition of cancer-endothelial cell fusions. There may be several reasons to this. Thus, the antisense oligonucleotide did not totally downmodulate syncytin-1 levels and could therefore not be expected to decrease cell fusions to zero and the lack of total inhibition by the CHR peptide could potentially be ascribed to proteolytic degradation. However, we also detected that the breast cancer cells produced syncytin-2 and cannot exclude that also this molecule contributed to the cancer\u2013host cell fusions. Further experiments using shRNA-directed downmodulation of both syncytin-1 and -2 as well as of additional putative fusogenic retroviral sequence are now underway to test this. Nevertheless, these data provide strong evidence that syncytin-1 is involved in mediating cancer\u2013endothelial cell fusions in vitro.\nWe next examined two series of human breast cancer patients for tumoral expression of syncytin-1 using a polyclonal antiserum raised to a synthetic nonapeptide derived from the syncytin-1 sequence. In addition, tumors were also screened for expression of ASCT-2 using a peptide antiserum. Preabsorption of the antisera with the corresponding peptides, but not with irrelevant peptides, abolished staining (Larsson et al. 2007b) (Fig.\u00a06). The results showed that 38% of all breast cancer samples showed detectable staining for syncytin and that endothelial cells expressed ASCT-2 (Fig.\u00a06). Moreover, significant expression of ASCT-2 was detected also in many tumor cells (Fig.\u00a06). The degree of syncytin immunostaining was visually graded using coded specimens and statistical analysis showed that it correlated positively with disease-free survival of the patients (Larsson et al. 2007b). Multivariate analysis included age dichotomized at 40\u00a0years, tumor size dichotomized at 20\u00a0mm, grade and adjuvant therapy and identified syncytin expression as an independent prognostic indicator of increased disease-free survival Also when used as a continuous variable, syncytin expression emerged as a significant prognostic indicator for disease-free survival in the Cox model (P\u00a0=\u00a00.02) (Larsson et al. 2007b).\nFig.\u00a06Human breast cancers immunocytochemically stained for syncytin-1 (a) or with antigen-preabsorbed antiserum (b) and for ASCT-2 (c, e) or with antigen-preabsorbed antiserum (d). Nuclei are lightly counterstained with haematoxylin. Note presence of syncytin immunoreactivity in tumor cells and of ASCT-2 immunoreactivity in endothelial cells (arrows) and in tumor cells\nThe involvement of syncytin-1 in tumor cell fusion events was subsequently confirmed by Strick et al. (2007), working with endometrial carcinomas. In their study, downmodulation of synctytin-1 expression also inhibited fusions between endometrial tumor cells. In agreement with findings on placental cells (vide supra), both cAMP elevating agents and estrogens upregulated syncytin-1 expression. However, only cAMP elevating agents stimulated cell fusions (Strick et al. 2007). This apparent discrepancy may possibly be ascribed to the fact that estrogen treatment upregulated TGFbeta, which interfered with syncytin-induced fusions. Thus, estrogen did, in fact, stimulate cell fusions if TGFbeta was immunoneutralized. Conversely, additions of TGFbeta 1 or 3 reduced fusions induced by cAMP-elevating agents. This effect was observed both in endometrial carcinoma cells and in trophoblast-derived cells. These results show that both cAMP and estrogens positively may regulate syncytin-1 expression in tumor cells and that the TGFbeta family may negatively regulate the fusogenic effects of syncytin-1 in both trophoblasts and cancer cells (Strick et al. 2007).\nThe role of syncytin-1 and cell fusions in cancer needs further study. Thus, our data show that syncytin-1 is not the only fusogenic protein expressed by breast cancer cells and the results presented by Strick et al. (2007) show that additional regulators, such as TGFbeta isoforms may be important modulators of cell fusions. So far, our breast cancer data indicate that syncytin-1 expression constitutes a positive prognostic factor. This is not the same as to say that cell fusions may be universally beneficial to cancers. First, syncytins may have additional effects within the tumor environment (Larsson et al. 2007a, b). Secondly, it seems likely that several factors within the cancer and its stroma (inactivated tumor suppressor genes, activated oncogenes, expression of fusogens and of CD9 and CD81 as well as TGFbeta) may act together to bring about a tumor profile that may be as diverse as the one demonstrated by the cell fusion experiments referred to above. Interestingly, however, expression of CD9 has been analyzed in a number of tumors and seems, like syncytin expression, to predict a good prognosis (Funakoshi et al. 2003; Hashida et al. 2002; Higashiyama et al. 1997; Houle et al. 2002; Miyake et al. 1995; Uchida et al. 1999). We propose that syncytins may be fusogens of importance to both trophoblast and cancer cell fusions and that they possibly also may mediate additional cell fusion events, acting in concert with other molecules with both enhancing and inhibitory regulatory effects.","keyphrases":["cell fusion","cancer","syncytin","placenta","env-w"],"prmu":["P","P","P","P","U"]}
{"id":"Planta-4-1-2249615","title":"Tie-dyed1 and Sucrose export defective1 act independently to promote carbohydrate export from maize leaves\n","text":"tie-dyed1 (tdy1) and sucrose export defective1 (sxd1) are recessive maize (Zea mays) mutants with nonclonal chlorotic leaf sectors that hyperaccumulate starch and soluble sugars. In addition, both mutants display similar growth-related defects such as reduced plant height and inflorescence development due to the retention of carbohydrates in leaves. As tdy1 and sxd1 are the only variegated leaf mutants known to accumulate carbohydrates in any plant, we investigated whether Tdy1 and Sxd1 function in the same pathway. Using aniline blue staining for callose and transmission electron microscopy to inspect plasmodesmatal ultrastructure, we determined that tdy1 does not have any physical blockage or alteration along the symplastic transport pathway as found in sxd1 mutants. To test whether the two genes function in the same genetic pathway, we constructed F2 families segregating both mutations. Double mutant plants showed an additive interaction for growth related phenotypes and soluble sugar accumulation, and expressed the leaf variegation pattern of both single mutants indicating that Tdy1 and Sxd1 act in separate genetic pathways. Although sxd1 mutants lack tocopherols, we determined that tdy1 mutants have wild-type tocopherol levels, indicating that Tdy1 does not function in the same biochemical pathway as Sxd1. From these and other data we conclude that Tdy1 and Sxd1 function independently to promote carbon export from leaves. Our genetic and cytological studies implicate Tdy1 functioning in veins, and a model discussing possible functions of TDY1 is presented.\nIntroduction\nSugars synthesized in photosynthetic leaf cells must be transported into the veins for distribution to nonphotosynthetic tissues. Maize (Zea mays) is a C4 plant which displays Kranz anatomy in its leaves (Esau 1977). Veins are surrounded by bundle sheath cells which in turn are surrounded by mesophyll cells. Mesophyll and bundle sheath cells cooperatively perform the reactions of photosynthesis and carbon assimilation. Sucrose is synthesized in the cytoplasm of mesophyll cells (Lunn and Furbank 1999), diffuses through plasmodesmata into bundle sheath cells, and then into vascular parenchyma cells (Russin et al. 1996). Maize is an apoplastic phloem loading species (Evert et al. 1978), whereby sucrose is exported from the vascular parenchyma cells to the apoplast by an unknown mechanism (see Lalonde et al. 2004 for discussion). Sucrose is then imported into phloem companion cells and\/or sieve elements by sucrose transporters located in the plasma membrane (Aoki et al. 1999; Lalonde et al. 2004; Sauer 2007; Scofield et al. 2007). The energy to transport sucrose into the phloem is derived from H+-ATPases that generate the proton motive force across the plasma membrane (Bush 1993; Gaxiola et al. 2007). In addition, the abundance of the sucrose transporter that controls phloem loading is transcriptionally regulated by a sucrose-sensing signal transduction pathway (Chiou and Bush 1998; Vaughn et al. 2002; Ransom-Hodgkins et al. 2003). If carbon transport into the vein is blocked, carbohydrates accumulate in the photosynthetic cells, leading to down-regulation of photosynthetic gene expression and reduced chlorophyll levels (chlorosis) (Sheen 1990; Goldschmidt and Huber 1992; Riesmeier et al. 1994; Krapp and Stitt 1995; Burkle et al. 1998; Gottwald et al. 2000; Jeannette et al. 2000). Even though the transport pathway for assimilated carbon has been studied in maize leaves for almost 40\u00a0years (Hofstra and Nelson 1969), the mechanisms that regulate sucrose export from leaves remain poorly understood.\nTo date, two recessive maize mutants have been described with defects in carbon export from leaves. tie-dyed1 (tdy1) and sucrose export defective1 (sxd1) mutants develop nonclonal chlorotic leaf sectors that hyperaccumulate starch and soluble sugars (Russin et al. 1996; Provencher et al. 2001; Braun et al. 2006). Other mutants with variegated leaves have been characterized, but none have carbohydrate accumulation within the chlorotic regions (Rhoades and Carvalho 1944; Fisher and Eschrich 1985; Madore 1990; Yu et al. 2007). tdy1 and sxd1 both display growth-related defects such as reduced plant height and inflorescence development due to the retention of carbohydrates in leaf tissues. However, their phenotypes also show significant differences. sxd1 mutant leaves progressively exhibit chlorosis which initiates at the leaf tip and basipetally spreads toward the base. Interestingly, in sxd1 mutants, only the leaf minor veins are affected in phloem loading (Russin et al. 1996). The tdy1 mutant phenotype is distinct from sxd1 mutants in that large chlorotic regions develop throughout leaf blade tissue. Further, tdy1 chlorotic regions form only during a limited time as the leaf emerges from the whorl and, once formed, do not expand (Braun et al. 2006). Lastly, in tdy1 mutants, chlorotic tissues are often bounded by lateral veins, implicating this vein class in limiting the expansion of a tdy1 chlorotic sector (Baker and Braun 2007).\nSxd1 encodes tocopherol cyclase, an enzyme functioning in tocopherol (vitamin E) biosynthesis, and sxd1 mutants lack tocopherols (Sattler et al. 2003). Absence of tocopherols results in callose deposition over the plasmodesmata at the bundle sheath-vascular parenchyma cell interface of leaf minor veins (Russin et al. 1996; Botha et al. 2000; Provencher et al. 2001). This occludes these passageways and prevents sucrose from moving into the vein, resulting in the build-up of carbohydrates in photosynthetic cells. The function of tocopherols in preventing callose deposition in the phloem is evolutionarily conserved. RNAi-mediated suppression of tocopherol cyclase activity in potato results in callose deposition in vascular-associated cells and carbohydrate accumulation in the photosynthetic cells (Hofius et al. 2004). In addition, in Arabidopsis tocopherol cyclase mutants and other vitamin E deficient mutants, callose is deposited in phloem parenchyma transfer cells and the photosynthetic cells accumulate carbohydrates if the plants are subjected to nonfreezing low temperatures (Maeda et al. 2006). It is not known how lack of tocopherols causes callose deposition.\nBecause both mutants show defects in carbon export from leaves, we examined whether Tdy1 and Sxd1 function in the same pathway to limit leaf carbohydrate accumulation. Using aniline blue fluorescence microscopy and transmission electron microscopy (TEM) we determined that carbohydrate accumulation in tdy1 mutant leaves does not result from a similar impediment to phloem loading as in sxd1 mutants. To genetically test whether the two genes function in the same pathway, we analyzed F2 families segregating both mutations. We observed that the double mutant plants showed an additive interaction for growth-related phenotypes and soluble sugar accumulation indicating that the two genes function in separate genetic pathways. To determine if Tdy1 functions in the same biochemical pathway as Sxd1, we measured tocopherol levels in mutant plants. We found that tdy1 mutants contain wild-type levels of tocopherols, indicating that Tdy1 does not function in the same biochemical process as Sxd1. Thus, from the combination of cellular, genetic and biochemical investigations we conclude that Tdy1 and Sxd1 function independently to promote carbon export from leaves. Additionally, genetic and cytological studies suggest that Tdy1 functions within the veins, and a model of possible TDY1 functions is discussed.\nMaterials and methods\nPlant materials\nPlants were grown in the summer in the Rock Springs Agronomy Farm, Pennsylvania State University, and in the winter in Juana Diaz, Puerto Rico. The tdy1-Reference (tdy1-R) (Braun et al. 2006) and sxd1-1 (Russin et al. 1996) mutations were backcrossed to the B73 inbred line five times prior to crossing together. F2 families were generated by crossing tdy1-R and sxd1-1 mutant plants and self-pollinating the F1 individuals.\nMorphological analyses\nPlant height was measured from the soil surface to the tip of the central spike of the tassel. Tassel length was measured from the node of the flag leaf to the tip of the central spike. Anthesis was recorded as the day of first pollen shed. Plants were scored as having produced ears if either (1) ears were clearly visible, (2) silks were visible emerging from the leaf sheath, or (3) ears developed such that they were identifiable by peeling back the ear leaf sheath. For wild-type and tdy1-R single mutants n\u00a0=\u00a018 for all measurements. For sxd1-1 single mutants, 12 of the 16 plants produced a tassel that shed pollen, and 13 produced an ear. For tdy1-R; sxd1-1 double mutants, 7 of the 15 plants shed pollen and 8 made an ear. Barren tassels that did not shed pollen were not included in the tassel length or anthesis measurements.\nAniline blue staining\nLeaf strips 1\u00a0cm in width were cut from mutant and wild-type leaves. The abaxial epidermis and the mesophyll cells directly beneath it were gently scraped away with a single-edge razor to expose the inner leaf tissues. The leaf strip was placed on a slide, briefly stained with a 0.05% aniline blue and immediately viewed using a 360\u2013370-nm excitation filter and a 420-nm long pass emission filter on a Nikon Eclipse 80i fluorescent microscope. Epifluorescent illumination was provided by a 100-W mercury lamp, and images were recorded using a DXM1200F Nikon digital camera.\nTEM analyses\nFor tissue fixation, leaf samples were diced into 3\u00a0\u00d7\u00a01\u00a0mm pieces, placed in 4% glutaraldehyde, 1% paraformaldehyde, 0.3% Tween 20, 50\u00a0mM sodium cacodylate, pH 7.4, and vacuum infiltrated on ice for 4\u20136\u00a0h. Samples were post-fixed in 1\u20132% osmium tetroxide at 4\u00b0C overnight, subsequently dehydrated through a graded acetone series and embedded in Spurr\u2019s epoxy resin (Spurr 1969). Thin sections (90\u2013100\u00a0nm) were cut on a Leica Ultracut E ultramicrotome with a glass knife and lifted onto 200-mesh copper grids. The grids were stained in 2% uranyl acetate, followed by Reynold\u2019s lead citrate and observed with a JEOL JEM 1200 EXII at an accelerating voltage of 80\u00a0kV.\nChlorophyll quantification\nRelative levels of total chlorophylls were quantified from leaves using a SPAD 502 Data Logger Chlorophyll Meter (Spectrum Technologies). For each tissue, 30 samples were measured, and the experiment was performed three times. Representative data from one replicate are shown.\nSugars and starch quantification\nLeaf tissues were collected at the end of the photoperiod, weighed and frozen at \u221280\u00b0C. Carbohydrate extraction was performed according to Dinges et al. (2001). Sugars and starch were quantified using commercial assay kits according to the manufacturer\u2019s instructions (R-Biopharm). Six samples from each tissue type were measured in triplicate.\nFor visualizing starch in leaves, samples were collected at the end the photoperiod, decolored by boiling in 95% ethanol and stained with iodine-potassium iodide (IKI) (Ruzin 1999).\nTocopherol quantification\nLeaf discs of 11\u00a0mm were harvested with a cork borer from leaf tissues, weighed and immediately frozen on dry ice. Total lipids were extracted from collected tissues and tocopherols measured according to (Sattler et al. 2003).\nResults\ntdy1-R and sxd1-1 single mutants both display variegated chlorotic leaves\ntdy1-R and sxd1-1 mutants show striking parallels in their leaf phenotypes. Both develop chlorotic sectors that hyperaccumulate starch and soluble sugars in leaf blades (Fig.\u00a01). However, there are also distinctions. Whereas wild-type leaves have a uniform, dark green color, sxd1-1 mutant leaves develop a chlorotic phenotype that shows a continuum of expression from strongest at the tip to mildest toward the base (Fig.\u00a01a, b). In addition, at the tip and leaf margins, the chlorotic tissues progressively accumulate anthocyanin and excess starch (Fig.\u00a01b, e). In sxd1-1 leaves, no clear, delineated boundaries occur between green and chlorotic regions. Conversely, tdy1-R mutants display a variegated pattern of chlorotic and normal appearing green regions throughout leaf blade tissue (Fig.\u00a01c). Additionally, the boundaries between tdy1-R chlorotic and normal appearing green tissue are sharp and distinct, and they tend to occur at lateral veins (Baker and Braun 2007). As in sxd1-1 mutants, chlorotic regions in tdy1-R mutants hyperaccumulate starch relative to green tissues (Fig.\u00a01f). tdy1 and sxd1 are the only known leaf variegation mutants that hyperaccumulate carbohydrates in any plant; therefore, we investigated whether Tdy1 and Sxd1 function in a common pathway influencing carbon partitioning in leaves.\nFig.\u00a01tdy1-R and sxd1-1 leaves display chlorotic sectors that hyperaccumulate carbohydrates. a Wild-type leaf showing uniform dark green color. b sxd1-1 mutant leaf displaying a chlorotic gradient, strongest at the tip to mildest toward the leaf base. Anthocyanins accumulate in the chlorotic tissues at the leaf tip and margins. c tdy1-R mutant leaf containing variegated chlorotic and green sectors throughout the leaf blade. d\u2013f Cleared, IKI stained leaf tissues showing that mutant chlorotic regions hyperaccumulate starch. d Wild type. e sxd1-1. f tdy1-R. Scale bars represent 4\u00a0cm\nCarbohydrate accumulation in tdy1-R mutants occurs by a different mechanism than in sxd1-1 plants\nTo address whether the two genes function in the same pathway, we investigated if the mechanism for carbohydrate accumulation was similar in the two mutants. Carbohydrate accumulation in sxd1-1 mutants results from a blockage in the symplastic pathway of sucrose movement (Russin et al. 1996; Botha et al. 2000; Provencher et al. 2001). Specifically, at the bundle sheath\u2013vascular parenchyma cell interface of leaf minor veins, callose is deposited over the plasmodesmata in bundle sheath cells. This blockage prevents sucrose from being loaded into the phloem and results in carbohydrate accumulation in the photosynthetic cells. To determine if the tdy1-R leaf phenotype is similarly caused by callose deposits plugging the plasmodesmata, we performed aniline blue staining and fluorescence microscopy. Aniline blue binds callose and fluoresces a blue\u2013white color under UV light. In wild-type plants, only the sieve plates in the end walls of phloem sieve elements stain positively for callose (Fig.\u00a02b). As previously reported, sxd1-1 mutants display many punctate callose deposits at the vascular parenchyma-bundle sheath cell interface of minor veins (Fig.\u00a02c) (Botha et al. 2000). In tdy1-R chlorotic regions, no ectopic callose deposits were detected at this interface (Fig.\u00a02d). Further, no callose deposits were observed in any cells of minor or lateral veins in tdy1-R chlorotic tissues, nor in any cells of tdy1-R green regions (data not shown). These data suggest that tdy1-R mutants do not accumulate carbohydrates due to callose blocking symplastic sucrose movement.\nFig.\u00a02Aniline blue staining of ectopic callose deposits in leaf minor veins. a Cross section of a wild-type minor vein shown by UV autofluorescence indicating the cell types and orientation of view (arrow) for b\u2013e. Abaxial cells were removed below the black dotted line to view the bundle sheath\u2013vascular parenchyma cell interface. X xylem, P phloem, VP vascular parenchyma, BS bundle sheath, M mesophyll. b\u2013eAniline blue fluorescence images of paradermal sections along minor veins. b Wild-type cells lack punctate callose deposits indicating no blockages along the symplastic pathway. The bright fluorescence observed in the phloem corresponds to sieve plates (arrowhead). c sxd1-1 minor veins contain many callose deposits at the bundle sheath\u2013vascular parenchyma cell interface (arrow). d tdy1-R chlorotic tissue lacks callose deposition over plasmodesmata in any cells. e tdy1-R; sxd1-1 double mutants contain callose deposits (arrow) at the bundle sheath\u2013vascular parenchyma cell interface comparable to the level seen in sxd1-1 single mutants. Scale bar in a represents 50\u00a0\u03bcm, and in b\u2013e 10\u00a0\u03bcm\nAlthough we did not observe ectopic callose deposition in any cells in tdy1-R mutant leaves using aniline blue staining, it is possible that symplastic sucrose transport is precluded due to a different defect in plasmodesmatal structure. To investigate this possibility, we performed TEM to inspect the plasmodesmata ultrastructure along the symplastic pathway. In minor veins from wild-type leaves, the plasmodesmata between bundle sheath and vascular parenchyma cells were unobstructed, spanned the cell wall and connected the cytoplasms between the two cells (Fig.\u00a03a). Consistent with previous reports, minor veins from leaf tips expressing the sxd1-1 mutant phenotype showed occlusions over the plasmodesmata on the bundle sheath cell side of the bundle sheath\u2013vascular parenchyma cell interface (Fig.\u00a03b) (Russin et al. 1996; Botha et al. 2000; Provencher et al. 2001). In tdy1-R mutant leaves, we found no occlusions over the plasmodesmata or alterations in their structure between the bundle sheath and vascular parenchyma cells in minor veins (Fig.\u00a03c). In examining plasmodesmata along the symplastic pathway at all cellular interfaces in tdy1-R leaves, we did not observe any structural perturbations or occlusions (Fig.\u00a03e\u2013j). These data indicate that carbohydrate retention in tdy1-R mutant leaves does not appear to result from physical blockage or plasmodesmatal structural changes. Together, the aniline blue staining and TEM studies indicate that the cellular basis for the tdy1-R and sxd1-1 phenotypes is different.\nFig.\u00a03TEM images of cellular interfaces along the symplastic pathway of minor veins. a-d Bundle sheath\u2013vascular parenchyma cells. Arrows indicate the location of the plasma membrane in the bundle sheath cell. a Wild-type plasmodesmata span the cell wall, lack occlusions, and connect with the plasma membrane. b sxd1-1 chlorotic leaf tips contain occlusions over the plasmodesmata on the bundle sheath cell side of the cell wall. c tdy1-R chlorotic sectors contain normal appearing plasmodesmata that lack occlusions. Note the plasmodesmata span the cell wall and connect with the plasma membrane. d tdy1-R; sxd1-1double mutant chlorotic tissues contain deposits over the plasmodesmata in the bundle sheath cell similar to sxd1-1 single mutants. Additional cellular interfaces along the symplastic pathway of wild type (e, g, i) and tdy1-R chlorotic tissues (f, h, j) are shown. In e\u2013j the plasmodesmata spanned the cell wall connecting the cytoplasms of adjacent cells and lacked occlusions. e, f BS\u2013BS cells, g, h BS\u2013M cells, i, j M\u2013M cells. BS bundle sheath, M mesophyll. Scale bars represent 200\u00a0nm\nDouble mutant plants show more severe growth defects than either single mutant\nBoth tdy1-R and sxd1-1 single mutant plants have growth-related defects due to the retention of carbohydrates in leaves. To determine whether the two genes function in the same genetic pathway promoting carbon export from leaves, we created F2 families segregating both mutations. Both mutations are recessive and the F2 segregation ratio fits the expected 9:3:3:1 prediction, indicating that neither mutant shows epistasis (Table\u00a01). sxd1-1 is a molecular null allele lacking transcripts (Provencher et al. 2001), and based on dosage analysis, tdy1-R is a genetic null allele (Braun et al. 2006). If Tdy1 and Sxd1 act in a linear genetic pathway, we would expect that the phenotype of the double mutant would be similar to one or the other mutant. If the two genes function in separate pathways, then we predict that the double mutant would be more severe than either single mutant.\nTable\u00a01Genetic segregation in F2 familiesFamily #TotalWild typetdy1-Rsxd1-1tdy1-R; sxd1-1\u03c72DB 55039348221672.0P\u00a0>\u00a00.05DB 55129552231552.1P\u00a0>\u00a00.05DB 55239355181550.6P\u00a0>\u00a00.05Total2811556346172.9P\u00a0>\u00a00.05\u03c72 analyses of tdy1-R and sxd1-1 segregation in three F2 families conform to a 9:3:3:1 expectation. Observed numbers of individuals are shown\nTo examine the interactions between tdy1-R and sxd1-1, we quantified the growth parameters of plant height, tassel size, time to anthesis and ear production (Fig.\u00a04, Table\u00a02). At the whole plant level, the double mutants express a more severe phenotype and show a stronger reduction in growth characteristics than either single mutant (Fig.\u00a04a). Compared to wild-type, tdy1-R single mutants have an 11% decrease, sxd1-1 mutants have a 24% reduction, and the tdy1-R; sxd1-1 double mutant plants show the greatest reduction in plant height of 35.5%. Similar to plant height, tassel size is reduced 13% in tdy1-R plants, 29% in sxd1-1 mutants, and 50% in tdy1-R; sxd1-1 double mutants (Fig.\u00a04c, Table\u00a02). In addition, approximately half of the tdy1-R; sxd1-1 double mutant plants produced very reduced tassel branches lacking spikelets (Fig.\u00a04d). This phenotype was observed in 25% of the sxd1-1 single mutants. Correlated with the tassel height defects, both mutations retarded time to flowering. Both tdy1-R and sxd1-1 single mutants first shed pollen eight days later than wild-type siblings (Table\u00a02). Double mutant plants were delayed even further by approximately another 5\u00a0days. Similarly, wild-type and tdy1-R mutant plants invariably made ears, and sxd1-1 mutants produced ears 81% of the time. In contrast, only 53% of the double mutant plants produced an ear (Table\u00a02). All of the growth defects observed in the single and double mutants are consistent with a restriction in assimilates transported to the growing portions of the plants. The double mutant plants showed the most severe phenotypes suggesting they have a greater reduction in carbohydrates exported out of leaves than in either single mutant. In support of this, the leaves of tdy1-R; sxd1-1 double mutant plants are more strongly chlorotic than either single mutant, with a corresponding reduction in the amount of green tissue (compare Figs.\u00a04b with 1b, c). In double mutant leaves, the tip to base gradient of chlorosis and anthocyanin pigmentation characteristic of the sxd1-1 phenotype is superimposed on the tdy1-R chlorotic and green sectored pattern. Double mutant leaves still contain some green tissues located at the leaf base as in sxd1-1 single mutants; however, the boundaries between the chlorotic and green regions are sharp and distinct as in tdy1-R mutants (Fig.\u00a04b). Thus, the double mutant leaves express the phenotypes of both single mutants, and double mutant plants display stronger growth retardation than either single mutant.\nFig.\u00a04Phenotypic comparisons among plants from a F2 family segregating tdy1-R and sxd1-1 mutants. In a and c the order from left to right corresponds to wild type, tdy1-R, sxd1-1, and tdy1-R; sxd1-1 double mutant. a Double mutants show the greatest reduction in plant height compared with either single mutants or wild-type siblings. b Double mutant leaves are severely chlorotic with lighter green tissues restricted to the leaf base. Distinct borders between the chlorotic and green tissues characteristic of tdy1-R are visible at the leaf base. The strong tip to base chlorosis gradient of sxd1-1 expression is evident. c Tassel height is most strongly reduced in tdy1-R; sxd1-1 double mutants compared with sxd1-1 and tdy1-R single mutants or wild-type siblings. d Approximately half of the tdy1-R; sxd1-1 double mutant plants produced barren tassels lacking spikelets. Scale bars represent 4\u00a0cmTable\u00a02Morphometric analyses in F2 familiesPhenotypePlant heightwt%Tassel lengthwt% AnthesisEars (%)Wild type220.4a\u00a0\u00b1\u00a00.3910055.2a\u00a0\u00b1\u00a00.1410071.9a\u00a0\u00b1\u00a00.06 100tdy1-R196.8b\u00a0\u00b1\u00a00.9289.247.9b\u00a0\u00b1\u00a00.28 86.880.3b\u00a0\u00b1\u00a00.13 100sxd1-1168.9c\u00a0\u00b1\u00a01.2476.639.3c\u00a0\u00b1\u00a00.1871.280.3b\u00a0\u00b1\u00a00.0981tdy1-R; sxd1-1142.2d\u00a0\u00b1\u00a00.7964.526.8d\u00a0\u00b1\u00a00.34 48.684.9c\u00a0\u00b1\u00a00.1353Values are the means\u00a0\u00b1\u00a0SE. Plant height and tassel length are measured in cm. Anthesis is measured as the number of days after planting until first pollen shed. Percent ears indicates the frequency each phenotypic class produced an ear. Different letters in superscript within a column denote statistical significance determined using Student\u2019s t test\nSince double mutants have more extensive chlorosis in their leaves, we ascertained whether the callose deposits at the vascular parenchyma-bundle sheath cell interface observed in sxd1-1 single mutants were more severe in the double mutants. Using aniline blue fluorescence, the double mutants showed no enhancement of the callose deposition phenotype and appeared similar to sxd1-1 single mutants (Fig.\u00a02c, e). Similarly using TEM, we found blockages over the plasmodesmata on the bundle sheath cell side of the bundle sheath\u2013vascular parenchyma cell interface of minor veins in the double mutants comparable to what we observed in sxd1-1 single mutant leaves (Fig.\u00a03b, d). In neither case was there any increase in the severity of the phenotype. Hence, tdy1-R and sxd1-1 do not display a synergistic interaction as the double mutant leaves express both single mutant phenotypes. Rather, these data suggest that the phenotypes observed in the double mutants are additive, and that the genes act in independent pathways.\nLeaf tips of double mutant plants have the greatest reduction in chlorophyll levels\ntdy1-R and sxd1-1 single mutants both show variegated chlorotic regions in their leaves (Fig.\u00a01b, c). We quantified total chlorophyll levels to determine whether the double mutants were more strongly affected than either single mutant (Table\u00a03). We observed a decreasing series in chlorophyll content with the strongest reduction in the tips of double mutant leaves\u00a0<\u00a0the chlorotic regions of either tdy1-R or sxd1-1 single mutant leaves\u00a0<\u00a0the green base of sxd1-1 or double mutant leaves\u00a0<\u00a0green regions of tdy1-R mutants or wild-type leaves. The leaf bases of sxd1-1 mutants and the double mutants displayed a mild chlorosis phenotype with approximately 70% as much chlorophyll as wild-type leaves indicating that these tissues are affected by the sxd1-1 mutation. As the tips of double mutant leaves showed a more severe chlorosis phenotype than either single mutant, it suggests that Tdy1 and Sxd1 function in separate pathways.\nTable\u00a03Chlorophyll quantification in F2 plantsPhenotypeChlorophyllWild type55.7a\u00a0\u00b1\u00a00.55tdy1-R green54.8a\u00a0\u00b1\u00a00.57sxd1-1 green41.0b\u00a0\u00b1\u00a00.75tdy1-R; sxd1-1 green42.2b\u00a0\u00b1\u00a00.67tdy1-R chlorotic20.9c\u00a0\u00b1\u00a00.62sxd1-1 chlorotic21.5c\u00a0\u00b1\u00a00.66tdy1-R; sxd1-1 chlorotic11.4d\u00a0\u00b1\u00a00.64Chlorophyll levels were measured in different phenotypic (green or chlorotic) tissues from different genotypes. Values represent the means\u00a0\u00b1\u00a0SE in relative units (n\u00a0=\u00a030). Statistical significance was determined using Student\u2019s t test and indicated by different letters in superscript\nSugar accumulation in double mutant leaves is greater than in either single mutant\nBoth single mutants accumulate excess starch in the chlorotic regions of their leaves (Fig.\u00a01e, f). To investigate whether Tdy1 and Sxd1 act in the same or separate genetic pathways limiting carbohydrate accumulation in leaves, we determined the starch and soluble sugars content in different phenotypic regions of F2 plants (Fig.\u00a05; Table\u00a0S1 in electronic supplementary material). For both the green regions and the chlorotic regions, we found that the double mutants contained similar amounts of starch as the single mutants. In contrast, we determined that the levels of soluble sugars differed with respect to genotype and phenotype. As previously reported, we found green regions of tdy1-R leaves had sucrose, glucose and fructose levels indistinguishable from wild type (Braun et al. 2006). However, green leaf bases of the double mutants and sxd1-1 single mutants contained approximately fourfold higher levels of glucose and fructose as wild type or tdy1-R green tissues. Among the chlorotic leaf tissues, the double mutant leaf tips had the highest concentrations of sugars, with approximately 33% greater amounts of glucose, and a significant increase in sucrose content compared with either single mutant. Double mutant leaf tips also contained \u223c30% more fructose than sxd1-1 leaf tips and approximately two-fold greater levels than in tdy1-R chlorotic regions. Overall, sugar concentrations in green tissues of double mutant leaves are more similar to sxd1-1 leaf bases than to tdy1-R green regions, whereas in chlorotic tissues, the double mutants contain more soluble sugars than either single mutant. Because the double mutants show greater individual sugar accumulation than either single mutant, these data support the hypothesis that Tdy1 and Sxd1 function in independent pathways promoting carbon export from leaves.\nFig.\u00a05Carbohydrate quantification in leaves of wild-type, tdy1-R, sxd1-1 and double mutant plants. Units for all panels are mg carbohydrate\/g fresh weight. Values are the means of 18 samples\u00a0\u00b1\u00a0SE. Abbreviations are wt, wild-type, dm, tdy1-R; sxd1-1 double mutant. Different letters indicate statistically significant differences between samples determined using Student\u2019s t test\nTdy1 does not function in the same biochemical pathway as Sxd1\nSxd1 encodes tocopherol cyclase, the penultimate enzyme in the vitamin E biosynthesis pathway, and sxd1 mutants lack tocopherols (Sattler et al. 2003). To determine whether Tdy1 functions in the same biochemical pathway as Sxd1, we quantified tocopherol levels in tdy1-R plants (Table\u00a04). We found no statistically significant changes in tocopherol quantities in tdy1-R chlorotic or green regions in comparison to wild-type tissue. sxd1 mutants had undetectable levels of tocopherols as previously reported (Sattler et al. 2003). Hence, tdy1-R does not have a lesion in tocopherol synthesis or accumulation, and Tdy1 does not function in the same biochemical pathway as Sxd1.\nTable\u00a04Tocopherol quantificationPhenotypeTocopherolWild type118.6a\u00a0\u00b1\u00a03.59tdy1-R green101.0a\u00a0\u00b1\u00a03.17tdy1-R chlorotic112.3a\u00a0\u00b1\u00a06.17sxd1-1ndValues are means of six samples\u00a0\u00b1\u00a0SE measured in \u03bcg\/g fresh weight. nd indicates none detected. Values indicated by letter \u201ca\u201d in superscript were not statistically different as determined using Student\u2019s t test\nDiscussion\nTdy1 and Sxd1 independently promote carbon export in maize leaves\ntdy1 and sxd1 are the only known mutations in plants that confer a variegated leaf phenotype with sectors that hyperaccumulate carbohydrates. Using a combination of biochemical, cytological and genetic analyses we investigated whether Tdy1 and Sxd1 act in a common pathway. Whereas sxd1 mutants lack tocopherol, we determined that tdy1-R mutants accumulate wild-type levels of tocopherols. Therefore, Tdy1 does not function in the same biochemical process as Sxd1. sxd1 mutants accumulate carbohydrates in leaf tissues due to callose being deposited over the interface of bundle sheath-vascular parenchyma cells in leaf minor veins which blocks sucrose symplastic movement. Using aniline blue fluorescence microscopy we did not identify any ectopic callose deposition in tdy1-R mutant leaf tissues. Moreover, using TEM we did not observe any structural alterations to the plasmodesmata, indicating that the symplastic loading pathway is not physically impeded. This suggests that tdy1-R mutants do not accumulate carbohydrates due to an occlusion, but rather due to a different cellular mechanism. This was confirmed by double mutant analyses which showed that the two mutations had an additive interaction. We found that the double mutants expressed both single mutant leaf phenotypes, that the reductions in plant height, tassel size and chlorophyll levels were additive, and that the accumulation of sugars in chlorotic leaf tissues of the double mutants was greater than in either single mutant. In addition, half of the double mutants produced ears whereas the tdy1-R mutant frequency was indistinguishable from wild type and 81% of the sxd1-1 single mutants made ears. The increased frequencies in the failure to make ears and production of barren tassels observed in the double mutants are most likely explained by an additive retention of carbohydrates in leaf tissues and a concomitant failure to export sucrose to the developing inflorescences. Collectively, these data lead us to conclude that Tdy1 and Sxd1 function independently, and that tdy1 defines a distinct genetic pathway affecting carbon partitioning in maize leaves.\nChlorotic leaf tissues do not retain sink identity\nWe previously suggested that one possibility to explain the excess carbon accumulation in tdy1-R chlorotic leaf sectors was a block in the developmental transition from sink to source identity, such that the chlorotic sectors continued to import photoassimilates from neighboring green tissues (Braun et al. 2006). If the chlorotic tissues were to remain sinks, the large amounts of green source tissues could potentially provide the carbohydrates accumulating in the chlorotic regions. However, based on our genetic analyses, our data do not support this hypothesis. In the tdy1-R; sxd1-1 double mutants, the amount of green leaf tissue is greatly reduced and is restricted to a narrow region at the very base of the leaves. Additionally, this green tissue displays mild chlorosis and an increase in carbohydrate accumulation indicating that it is perturbed in carbon export. As this tissue is proximal to and up to a meter distant from the chlorotic tip of adult leaves, it is highly unlikely to be providing the carbohydrates that accumulate in the cells near the tip. Furthermore, half of the double mutant plants produced a tassel and an ear. These are sink tissues dependent on the photosynthetic source leaves for their carbohydrates. Because the great majority of the area of double mutant leaves is severely chlorotic, yet still must be capable of exporting photoassimilates to produce the developing reproductive tissues, our data argues against the chlorotic tissues remaining sinks and does not support the hypothesis that Tdy1 functions to regulate the sink to source transition. Rather, the new data lead us to favor the hypothesis that the cells in the tdy1 chlorotic region are source tissues that are partly blocked in carbon export capacity.\nA model for TDY1 function\nIn wild-type maize leaves, sucrose is synthesized in mesophyll cells, diffuses through plasmodesmata into bundle sheath cells and then into the vascular parenchyma cells (Fig.\u00a06a). Sucrose is exported from the vascular parenchyma cell to the apoplast, and then loaded into the phloem by sucrose transporters in the plasma membrane of the companion cell and\/or sieve element (Lalonde et al. 2004; Sauer 2007). sxd1 mutant leaf tips are blocked in this pathway at the bundle sheath\u2013vascular parenchyma cell interface (Fig.\u00a06b) (Russin et al. 1996; Botha et al. 2000; Provencher et al. 2001).\nFig.\u00a06Model for TDY1 functions in phloem loading of sucrose. Diagrams represent the pathway of sucrose movement from photosynthetic cells into the phloem. M mesophyll, BS bundle sheath, VP vascular parenchyma, CC companion cell, SE sieve element. Green and yellow ovals in M and BS cells represent chloroplasts. White circles within chloroplasts represent starch grains. Magenta box on VP plasma membrane represents sucrose efflux transporter, and blue boxes on CC and SE plasma membranes depict sucrose transporters. Arrows indicate the direction of sucrose movement. a Wild-type tissue with normal, unimpeded sucrose movement into the phloem. b sxd1-1 mutant chlorotic tissue has callose deposits (red box) over the plasmodesmata at the BS\u2013VP cell interface blocking sucrose movement and resulting in carbohydrate accumulation in M and BS cells. c tdy1-R chlorotic tissue has normal appearing plasmodesmata and lacks callose deposits. We hypothesize that either the sucrose efflux transport step is inhibited (red X) or the CC\u2013SE sucrose transporters (orange X) fail to load sucrose into the phloem. This would result in the carbon hyperaccumulation observed in the photosynthetic cells\ntdy1 chlorotic sectors accumulate excess carbohydrates indicating that they have a defect in phloem loading. In agreement with this, we previously localized the site of TDY1 function to the innermost tissue layer of leaves comprised of the veins, bundle sheath cells, and interveinal mesophyll cells (Baker and Braun 2007). Due to the limits of the experiment it was not possible to further delineate where within this tissue layer TDY1 acts. However, the cytological investigations presented resolve this ambiguity. As we observed no blockages or alterations to plasmodesmatal structure along the sucrose symplastic pathway in tdy1-R leaves, the carbon accumulation points to a defect at a later step in phloem loading. Nonetheless, even though the plasmodesmata appear unobstructed, we cannot be certain that they are functional. However, we propose that a molecular blockage of plasmodesmata precluding sucrose movement is an unlikely scenario for the following reason. In sxd1 mutants, specific plasmodesmata are occluded and unable to transit sucrose, causing the vascular parenchyma cells to appear plasmolysed due to their low osmotic concentration (Russin et al. 1996). Accordingly, we carefully inspected for, but never observed, plasmolysis of any vascular parenchyma, mesophyll or bundle sheath cells in tdy1 mutants in our microscopic analyses. Therefore, these data suggest that sucrose is able to pass freely between cells along the symplastic pathway, and hence that TDY1 likely functions in the veins (Fig.\u00a06c).\nFrom the variegated phenotype of tdy1 mutants, we hypothesize that TDY1 acts as a regulator of sucrose transport rather than as a transporter itself, as loss of transporter function leads to uniform carbohydrate accumulation and chlorosis phenotypes (Riesmeier et al. 1994; Burkle et al. 1998; Gottwald et al. 2000; see Braun et al. 2006; Baker and Braun 2007 for discussion of mechanisms for tdy1 variegation). Several possibilities are envisioned for Tdy1 function. The first possibility is that Tdy1 may regulate sucrose efflux out of vascular parenchyma cells. Failure to export sucrose to the apoplast would create a bottleneck restriction leading to the build-up of sucrose in the vascular parenchyma cells and, in turn, to carbohydrate accumulation in the bundle sheath and mesophyll cells. The second possibility is that Tdy1 may function to regulate the activity of the H+-ATPases or sucrose transporters within the companion cells and sieve elements. Lack of function of either the H+-ATPases or sucrose transporters results in failure to load sucrose into the phloem and thereby to excess accumulation in the apoplast. Sucrose accumulation in the apoplast would shift the equilibrium for sucrose export from vascular parenchyma cells leading to increased concentration within these cells, and ultimately to carbon accumulation in the photosynthetic cells. In support of this, antisense expression or mutation of a phloem H+-ATPase or sucrose transporters causes an accumulation of carbohydrates in photosynthetic cells and chlorosis (Riesmeier et al. 1994; Burkle et al. 1998; Gottwald et al. 2000; Zhao et al. 2000). A third possibility is that TDY1 may function in the sucrose signal transduction pathway that regulates the abundance of the sucrose transporter controlling phloem loading (Chiou and Bush 1998; Vaughn et al. 2002; Ransom-Hodgkins et al. 2003).\nOur analyses of the tdy1-R mutant phenotype and genetic interactions between tdy1-R and sxd1-1 have shown that Tdy1 acts independently of Sxd1. Thus, the tdy1 mutation identifies a separate genetic pathway limiting carbon accumulation in maize leaves. Future work characterizing the molecular function of Tdy1 will determine the mechanism by which Tdy1 influences carbon partitioning. Understanding the control of carbon export from leaves will lead to novel strategies to manipulate carbon allocation and biomass deposition which may have applications in the production of biofuels.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\n(DOC 31 kb)","keyphrases":["tie-dyed1","sucrose export defective1","maize","carbohydrate accumulation","carbon partitioning"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_J_Nutr-2-2-1705523","title":"Effect of sesamin on serum cholesterol and triglycerides levels in LDL receptor-deficient mice\n","text":"Background Sesamin, a major lignan from sesame seeds has been associated with cholesterol reduction in previous reports, but recent studies suggested differences in the response to sesamin intake depending on the model studied as well as the nature of the sesamin preparation used.\nIntroduction\nThe research on natural substances affecting cholesterol metabolism for prevention of hypercholesterolemic atherosclerosis has particular therapeutic importance. In particular, the identification of dietary components that can be added to foods to lower or regulate cholesterol levels has gained special interest.\nSesame seeds in general and its major lignan sesamin in particular, have been associated with various biochemical actions, mainly related to lipid metabolism, including a hypocholesterolemic effect in both human and animal-based trials (see [1, 2] for review). Although the general hypolipidemic effect of dietary sesamin seems to be clear in studies with rats, recent studies pointed out discrepancies in the response to sesamin between animal species [3] raising doubts about a possible extrapolation of these results to humans. Additionally, the heterogeneous nature of the sesamin preparations used in the different studies seems to be another point to consider, since different biological actions and mechanisms have been proposed for the two epimers of dietary sesamin [4]. In this study, we aimed to further investigate the hypocholesterolemic action of sesamin minimizing the above described variables and using only pure sesamin epimer and selecting low density lipoprotein (LDL) receptor-deficient mice as experimental animals. This model aims to reproduce homozygous familial hypercholesterolemia and it is characterized by decreased removal of cholesterol from the circulation due the lack of functional LDL receptors [5] that leads to increased serum cholesterol levels.\nMaterials and methods\nAnimal model\nFemale ldlr\u2212\/\u2212 mice (strain number, B6.129S7-Ldlrtm1Her) quality SPF were obtained from the Jackson Laboratory (Bar Harbor, Maine, US), and maintained at the Central Animal Laboratory of Raisio Benecol Ltd. (Raisio, Finland) in accordance with European guidelines (European Treaty Series No. 123, EU No 609\/86, Official Journal of the European Communities No. L 358) approved by the Animal Care and Use Committee of the University of Turku (Finland) approval number 1263\/02. Animals were 17\u201318\u00a0weeks at the beginning of the study\/15\u201320\u00a0g and after 4\u00a0weeks of acclimatization, mice were housed (Polycarbonate Macrolon III, Scanbur AS, Denmark) in groups of 1\u20132\/cage and randomly given free access to I\u2013IV diets for 4\u00a0weeks. Ambient temperature was 21\u00a0\u00b1\u00a03\u00b0C, humidity ranged 55\u00a0\u00b1\u00a015% and illumination consisted in 12-h dark\/light cycle. Free access to community tap water was allowed, except during the experiments. Blood samples were collected for the determination of the concentrations of serum cholesterol and triglycerides at the beginning and at the end of the 4-week dietary intervention. Mice were sedated with CO2\/O2-mixture and the blood samples were collected from the orbital sinus (at the beginning) or through a cardiac puncture (at the end). At the beginning of the study the blood was collected into 500\u00a0\u00b5l gel tubes and allowed to clot at least for 1\u00a0h. Samples were then centrifuged for 15\u00a0min at 4000\u00a0rpm. Serum was separated and stored at \u221220\u00b0C until analyses. At the end of the intervention, blood samples were collected in heparin tubes and centrifuged within 1\u00a0h. The plasma samples were frozen at \u221220\u00b0C and stored until analyses. About 50\u00a0\u00b5l was used for cholesterol and triglyceride analyses and the rest was stored at \u221220\u00b0C for further analyses. During the intervention, the animals were weighed twice a week and observed daily (morning and afternoon) for general well being.\nTest compounds and diets\nPlant-derived stanol ester was produced by Raisio Benecol Ltd. (Raisio, Finland). Sesamin epimer (CAS No. 607\u201380-7, >95% purity, Fig.\u00a01A) was synthesized by Industrial Research Ltd. (Wellingtown, New Zealand). During acclimatization\/isolation period the diet was RM1 (E) SQC, (Special Diet Service, Witham Essex, UK). Experimental atherogenic diets containing 0.25% of cholesterol were prepared at Raisio Benecol Ltd. based on Clinton-Cybulsky diet premix (D12106pxc, Research Diets Inc., New Brunswick, NJ) in which the test compounds were administered, as follows: control (I) diet (n\u00a0=\u00a08), stanol ester (II) diet (n\u00a0=\u00a08, 7\u00a0mg\/kg), sesamin (III) diet (n\u00a0=\u00a08, 1\u00a0mg\/kg) and combined (IV) diet (n\u00a0=\u00a07, 7\u00a0mg\/kg stanol ester and 1\u00a0mg\/kg sesamin). Diets were stored at +2 to +8\u00b0C and allowed to warm to room temperature before delivery to the animals (twice a week) in 300-ml glass cups attached on the cage floor.\nFig.\u00a01Chemical structure of sesamin epimers (A) sesamin and (B) episesamin (or asarinin)\nRationale for dose selection\nFat and cholesterol levels in the atherogenic diet were based on the literature [6]. The use of stanol ester as control test model has been proven several times to induce cholesterol reduction (Raisio Benecol Ltd., unpublished results) and the concentration in this study was selected to exert sufficient but not the maximal response, enabling the possible combination effect with sesamin. Sesamin concentration, that was adopted from previous reports [7\u20139] allowing comparison of the results was also considered as a rationale level to be used in clinical trials.\nAnalysis\nSerum cholesterol and triglycerides were determined by Ecoline CHOD-PAP and Ecoline 25 GPO-PAP assay kits (1.14856.0001, Merck KGaA, Darmstadt, Germany), respectively. Control serum was Qualitrol HS N (Merck KGaA, Darmstadt, Germany). Serum metabolic profile of lignans was carried out by HPLC with coulometric electrode array detection [10] and the absence of plant lignans other than sesamin epimer in the diets was confirmed by isotope-dilution GC-MS [11].\nStatistics\nGraph Pad Prism software version 3.02 (GraphPad Software Inc., CA) was used for statistical data treatment. The differences in changes from the basal concentrations during the intervention between the groups were analyzed by ANOVA. Pair-wise comparisons between the groups were performed by Tukey\u2019s Multiple Comparison Test. Two-way ANOVA was used to analyze differences in the body weights between the groups. A P-value less than 0.05 was considered as statistically significant. All analyses were performed as two-sided tests.\nResults\nBody weight gain\nThe body weights of the animals increased in all groups during the 4-week intervention period (Fig.\u00a02). The increment tendency in group I was less than those in the other groups but analysis did not reveal a significant group\u00a0\u00d7\u00a0week interaction (P\u00a0=\u00a01.00). However, ANOVA showed a significant group effect (P\u00a0=\u00a00.0002), which indicates that the body weights differed between the groups during the intervention.\nFig.\u00a02Body weights of the animals in the different treatment groups during the study (n\u00a0=\u00a07\u20138)\nEffects of the atherogenic diet and test compounds on serum cholesterol and serum triglyceride concentrations\nThe atherogenic diet (group I) induced an almost 3-fold increase in the s-cholesterol levels but no effect was seen in the triglyceride levels. Stanol ester alone (group II) or together with sesamin (group IV) significantly attenuated the elevation of the cholesterol levels. Sesamin alone (group III) either normalize the elevation of cholesterol levels nor did it enhance the effect of stanol ester (group II versus group IV). Diet-induced changes in the triglyceride levels did not differ between groups (P\u00a0=\u00a00.89). Results are presented in Fig.\u00a03.\nFig.\u00a03S-cholesterol and -triglyceride concentrations before (Basal) and after (Intervention) the 4-week intervention with experimental diets and the difference between basal and Intervention levels (Difference). **P\u00a0<\u00a00.001 in comparison with Group 1 (Tukey\u2019s Multiple Comparison Test)\nEffects of the atherogenic diet and test compounds on plasma lignan profile after intervention\nPresence of sesamin epimer in the diets resulted in the appearance of enterolignans in plasma, although concentrations varied greatly between animals (Table\u00a01). Significantly increased levels of enterodiol (P\u00a0=\u00a00.00), but not enterolactone were found.\nTable\u00a01Enterolignan (enterolactone and enterodiol) values after 4-week intervention period nmol\/l (Mean\u00a0\u00b1\u00a0SEM, n\u00a0=\u00a08)GroupnENLaENDENL\u00a0+\u00a0ENDI, Control84.61\u00a0\u00b1\u00a00.406.11\u00a0\u00b1\u00a02.109.18\u00a0\u00b1\u00a02.14II, Stanol ester83.01\u00a0\u00b1\u00a00.3510.0\u00a0\u00b1\u00a04.6913.0\u00a0\u00b1\u00a04.85III, Sesamin88.01\u00a0\u00b1\u00a00.98569\u00a0\u00b1\u00a054.2**577\u00a0\u00b1\u00a055.0**IV, Stanol ester\u00a0+\u00a0sesamin76.54\u00a0\u00b1\u00a01.99388\u00a0\u00b1\u00a090.7**394\u00a0\u00b1\u00a091.8****P\u00a0<\u00a00.001 in comparison with Group 1 (Tukey\u2019s Multiple Comparison Test)aENL, Enterolactone; END, enterodiol\nDiscussion\nAs one of the major components of sesame seeds, the lignan sesamin has received a great deal of interest regarding its potential as a hypocholesterolemic agent, especially after the positive results reported by Hirata et al. [7] in humans. Thereafter, different approaches have been used to confirm this observation in which different sources of dietary sesamin have been used. Studies using sesame seed-based diets provided results that are difficult to discuss since the hypocholesterolemic effect could not be primarily attributed to sesamin. As for the studies using pure sesamin supplementation, the preparations used so far contained a mixture of sesamin epimers (sesamin and episesamin in a 1:1 ratio) that they are known now to possess a different behavior both in their metabolic fate [12] and biological action [4] supporting the need to differentiate between different sesamin epimers in future studies.\nA clear hypocholesterolemic effect elicited by sesamin (alone or in combination with vitamin E) was reported in studies conducted in rats [9, 13\u201315] or in cultured rat cells [16] and similarly, other biological effects proposed for sesamin [17\u201326], have also been studied using rats as experimental animal. Very few studies though, have used other animals than rats to test the effects of sesamin [27, 28]. In the recent study of Kushiro et al. [3] where sesamin was tested in different experimental animals, results for rats were confirmed, but no effect was observed in hamsters or mice. Only one study has investigated the hypocholesterolemic effect of sesamin in humans [7] finding a significant effect of dietary sesamin in the reduction of total cholesterol and LDL-cholesterol. In this study however, dietary sesamin was administered together with vitamin E and therefore the observed effect might be due to the reported synergism between this two dietary components [14] and since no separated sesamin-group was included in the study, the hypothetical action of sesamin could not be confirmed.\nIn order to minimize the commented variables, in the present experiment only pure sesamin epimer was used and additionally, transgenic mice were selected for they offer the most convenient model to investigate the two proposed mechanisms for the hypocholesterolemic effect of sesamin i.e. reduction of endogenous cholesterol synthesis and reduction of dietary absorption. Due to the lack of functional LDL-receptors, the animals used in this trial have a spontaneously elevated serum cholesterol concentration, and in addition, they are prone to diet-induced modulation of the serum cholesterol concentration [5, 6]. Hepatic HMG-CoA reductase is the rate-limiting enzyme in the cholesterol biosynthetic pathway and its inhibitors are very effective in lowering plasma cholesterol. Sesamin has been reported to inhibit HMG-CoA reductase in rats [13] and through this mechanism it has been proposed to reduce plasma cholesterol. However, in this study no reduction of serum cholesterol or triglyceride levels was observed in the group supplemented with sesamin alone (group III) and therefore it is concluded that sesamin does not affect HMG-CoA reductase in this animal model. Similarly, cholesterol supplementation (0.25%, w\/w) for 4\u00a0weeks increased plasma total cholesterol levels approx. 3-fold in the absence of a functional LDL receptor. However, the introduction of stanol ester in the atherogenic diet significantly reduced the total plasma cholesterol level, independently of the presence of sesamin. It is known that plant stanols inhibit the absorption of dietary cholesterol and the reabsorption (enterohepatic circulation) of endogenous cholesterol from the gastrointestinal tract [29]. It seems though that sesamin does not affect dietary cholesterol absorption in this animal model.\nThe metabolism of sesamin seems to play an essential role in its further biological action. It has been recently suggested that in humans, sesamin is absorbed through the portal vein reaching the liver where extensive metabolism takes places generating demethylenated (catechol) derivatives secreted as glucuronidates via the bile [26]. The same metabolites were found after in vitro fermentation of pure sesamin standard with human fecal inoculum and furthermore extensive in vivo conversion to enterolactone was reported after sesame seed supplementation in humans [11]. We hypothesized then that the absorption of sesamin could certainly take place via the portal vein and that transformation to catechol derivatives could be possible, as well as further absorption to the general circulation where they may exert the biological actions proposed for sesamin. Nevertheless, in this study as in our previous trial [11], extensive conversion of sesamin to enterolignans was observed suggesting that non-absorbed sesamin and possibly biliary catecholic sesamin derivatives undergo metabolism by gut microflora leading to the production and further absorption of enterolignans. Postprandial levels of sesamin in plasma differed considerably between rats [12] and humans [11], suggesting a different metabolic pathway depending on species.\nSo far, our data suggest that a limited absorption or a too rapid enterohepatic circulation of sesamin could be the explanation for the lack of antihypercholesterolemic effect in mice\/hamsters and possibly in humans but this hypothesis needs to be confirmed. Furthermore, the characteristics of our animal model did not allow to study a third mechanism for cholesterol reduction that sesamin could use; the increment of LDL-receptor activity. This hypothetical mechanism has been suggested for other dietary components [30], and it is a likely explanation of the negative results reported in this paper.\nIn conclusion, possible confounding factors have been minimized with the use of a specific animal model and the selection a pure sesamin epimer. It can be therefore stated that sesamin does not seem to affect cholesterol biosynthesis or absorption in mice. The so far contradictory results hinder the extrapolation to humans, and only a clinical trial with separate epimeric forms and the elucidation of the complete metabolic pathway of sesamin in humans will contribute to clarify the possible utilization of sesamin as a hyocholesterolemic agent.","keyphrases":["sesamin","cholesterol","triglycerides","lignans","stanol ester","enterolignans"],"prmu":["P","P","P","P","P","P"]}
{"id":"Oecologia-4-1-2373415","title":"Swan foraging shapes spatial distribution of two submerged plants, favouring the preferred prey species\n","text":"Compared to terrestrial environments, grazing intensity on belowground plant parts may be particularly strong in aquatic environments, which may have great effects on plant-community structure. We observed that the submerged macrophyte, Potamogeton pectinatus, which mainly reproduces with tubers, often grows at intermediate water depth and that P. perfoliatus, which mainly reproduces with rhizomes and turions, grows in either shallow or deep water. One mechanism behind this distributional pattern may be that swans prefer to feed on P. pectinatus tubers at intermediate water depths. We hypothesised that when swans feed on tubers in the sediment, P. perfoliatus rhizomes and turions may be damaged by the uprooting, whereas the small round tubers of P. pectinatus that escaped herbivory may be more tolerant to this bioturbation. In spring 2000, we transplanted P. perfoliatus rhizomes into a P. pectinatus stand and followed growth in plots protected and unprotected, respectively, from bird foraging. Although swan foraging reduced tuber biomass in unprotected plots, leading to lower P. pectinatus density in spring 2001, this species grew well both in protected and unprotected plots later that summer. In contrast, swan grazing had a dramatic negative effect on P. perfoliatus that persisted throughout the summer of 2001, with close to no plants in the unprotected plots and high densities in the protected plots. Our results demonstrate that herbivorous waterbirds may play a crucial role in the distribution and prevalence of specific plant species. Furthermore, since their grazing benefitted their preferred food source, the interaction between swans and P. pectinatus may be classified as ecologically mutualistic.\nIntroduction\nHerbivores may have considerable impact on the distribution and population dynamics of plants (e.g. Maron and Crone 2006) as well as vegetation community structure (e.g. Augustine and McNaughton 1998), but non-grassland examples of such impacts are few. Similarities between aquatic and terrestrial systems and their dependence on top\u2013down regulation are increasingly being appreciated by ecologists (Cyr and Pace 1993; Chase 2000). However, in these broad-scale inter-system comparisons, the predation of zooplankton on algae still plays a front-stage role in the representation of aquatic plant\u2013animal interactions. The importance of vertebrate grazing on submerged macrophytes community structure remains disputed. Among the studies reported to date on interactions between aquatic herbivores and plants, few have considered the possibility of herbivory mediating competition between plants (but see Lauridsen et al. 1993; Van Donk and Otte 1996; Santamar\u00eda 2002; LaMontagne et al. 2003), and none have experimentally shown that this can lead to a specific herbivore-induced vegetation distribution and composition. Considering that the impact of herbivory on community structure in terrestrial systems increases with grazing intensity (Augustine and McNaughton 1998), the study of some aquatic plant\u2013animal interactions with sometimes extremely high grazing pressures on both above- and belowground parts may prove rewarding.\nPotamogeton pectinatus and P. perfoliatus are two species of aquatic macrophytes typical of eutrophic lakes, often co-occurring, with a more or less cosmopolitan distribution. They hibernate mainly vegetatively with rhizomes and turions (P. perfoliatus) or specialised tubers (P. pectinatus) in the sediment (Fig.\u00a01). Many species of waterbirds rely on the leaves and seeds of these two species of water plants (Cramp and Simmons 1986). Among waterbird foragers, swans form a special group of consumers that are also capable of uprooting and consuming the belowground parts of P. pectinatus and P. perfoliatus. In particular, Bewick\u2019s (Cygnus columbianus bewickii) and whooper swans (C. cygnus) are highly dependent on the nutrient-rich tubers of P. pectinatus (Nolet and Drent 1998; Nolet et al. 2001, 2002; Beekman et al. 2002).\nFig.\u00a01Vegetative reproductive organs of Potamogeton perfoliatus and P. pectinatus. The tubers of P. pectinatus may considerably vary in size between approximately 2 and 16\u00a0mm. The depicted tuber is 15\u00a0mm long\nThe spatial distributions and segregation of P. pectinatus and P. perfoliatus have been studied extensively. Typically, P. pectinatus is found in shallower waters than P. perfoliatus. Lehmann et al. (1997) argued that the higher tolerance of P. pectinatus to wave exposure in shallow water and the higher tolerance of P. perfoliatus to light attenuation in deep water could be a major causal factor of this distribution of the two species. Scheffer et al. (1992) was able to explain about 50% of the variation in the distribution of both species by considering water depth, transparency, spring water temperature and exposure to waves for P. pectinatus and water depth and transparency for P. perfoliatus. During extensive studies on the distribution of aquatic vegetations and swan foraging in two of our major research sites where P. pectinatus and P. perfoliatus co-occur (Pechora Delta, in Arctic Russia, and Lake Ringsj\u00f6n, in southern Sweden), we noticed that P. perfoliatus occurred not only in deep water but also in very shallow water seldom visited by tuber-feeding swans. We thus hypothesised that swan foraging, which is limited to areas of intermediate depth (Beekman et al. 1991; Nolet et al. 2001; Sandsten 2002), at least partly determines the distribution of P. perfoliatus.\nIn order to investigate how swan feeding on tubers of P. pectinatus affects the growth of P. perfoliatus, we monitored vegetation and swan foraging and performed a transplant experiment in Eastern Lake Ringsj\u00f6n, Sweden. Potamogeton perfoliatus was planted in a P. pectinatus stand after which the development of both species was followed in the presence and absence of swan grazing.\nMethods\nEastern Lake Ringsj\u00f6n (55\u00b052\u2032N, 13\u00b032\u2032E) is a eutrophic, 20.5-km2 large lake with a maximum and mean depth of 16.4 and 6.1\u00a0m, respectively. The experiment was set up in Fulltofta Bay (0.7\u00a0km2, <1\u00a0m deep; Fig.\u00a02), which is a wind exposed, shallow and sandy part of Eastern Lake Ringsj\u00f6n, with an aquatic vegetation that nearly exclusively consists of P. pectinatus and P. perfoliatus. Every year from September to November, Fulltofta Bay hosts large numbers of migrating waterbirds (up to 1600 individuals, Sandsten 2002). Many of these migrating waterbird species, notably swans (whooper swans C. cygnus, Bewick\u2019s swans C. columbianus bewickii, and mute swans C. olor), feed on tubers of P. pectinatus and rhizomes and turions of P. perfoliatus by digging in the sandy sediment (e.g. Cramp and Simmons 1986; Nolet and Drent 1998; Sandsten 2002). Coot (Fulica atra) and various species of ducks (mallard Anas platyrhynchos, pintail Anas acuta, wigeon Anas penelope, goldeneye Bucephala clangula, tufted duck Aythya fuligula, pochard Aythya ferina) may also forage on tubers, but nearly always in association with swans since they are unable to dig up the tubers from the sediment by themselves (e.g. Cramp and Simmons 1986; Sandsten 2002). Geese (mainly Anser anser) roost frequently in the area, but they very seldom feed on tubers (Cramp and Simmons 1986; personal observations, H. Sandsten).\nFig.\u00a02Map of Lake Ringsj\u00f6n with an enlargement of Fulltofta Bay and an overview of the bird-monitored and plant-monitored areas as well as the plant-experimental site. Enlargement of the plant-experimental site shows the six replicates of three treatments, with gray squares representing plots where no P. perfoliatus was planted and no protection from waterbirds was provided until 18 December 2000 at which time all treatments were covered with metal nets on the bottom (treatment p). Black squares represent plots with planted P. perfoliatus that were continuously protected by nets (treatment \n). White squares represent plots with planted P. perfoliatus that were unprotected from 8 August to 18 December 2000 (treatment P)\nTo quantify our impression that P. perfoliatus occurs only in very shallow and relatively deep parts of Fulltofta Bay, we mapped the occurrence of P. pectinatus and P. perfoliatus in relation to water depth in Fulltofta Bay on 10 August 2000 (Fig.\u00a02). Vegetation and water depths were examined on 173 points regularly spaced (at 20\u201330\u00a0m distance from point to point) in an area (0.12\u00a0km2) running from the shore to a depth of 1.2\u00a0m. The geographical position of each sampling point was measured with a GPS (Garmin Personal Navigator 12 XL). On each of the points we investigated plant occurrence three times by throwing a 50\u00a0\u00d7\u00a050-cm metal frame into the water and diving down to look for plants. For P. pectinatus, a cover index was used, where 0 was no ramets m\u22122, 1 was 1\u201320\u00a0ramets\u00a0m\u22122, 2 was 21\u201380\u00a0ramets\u00a0m\u22122 and 3 was >80\u00a0ramets\u00a0m\u22122. We counted all shoots emerging from the sediment surface as ramets. Potamogeton perfoliatus shoots were less frequent than P. pectinatus shoots and seldom found in the frames\u2014possibly because of less favourable growing conditions but at least in part because of its morphology and growth strategy. We therefore used data on absence\/presence in the proximity (within a radius of 10\u00a0m) of the sampling point. The data were entered into a geographic information system, GIS (Surfer 6.04) and interpolated over the entire monitored surface using intermediate distance interpolation.\nSwans were counted over an area of 0.54\u00a0km2 from an observation point (Fig.\u00a02) south of Fulltofta Bay on 5, 17 and 30 August, 10 and 23 September, 5, 18 and 28 October and 9 November 1999.\nSwans foraging on tubers tread water to create a water current that flushes the sediment and uncovers tubers and rhizomes, resulting in the appearance of pits and piles of sediment on the bottom at the sites where foraging activity has taken place. Wave action will ultimately level the sediment again (Sandsten 2002). Depth heterogeneity was used as an indirect measure of waterbird foraging on tubers in the sediment. In order to investigate the timing of swan foraging in relation to water depth we measured depth heterogeneity along five fixed transects running parallel to the shore within the plant-monitored area. The transects were aimed to be at equidistant intervals parallel to the shore, but in fact they were located at a distance of 110, 180, 260, 340 and 400\u00a0m from the shore (mean depths were 37, 45, 60, 75 and 90\u00a0cm on 5 August 1999). Depth heterogeneity was obtained by calculating the SD from 5\u00a0\u00d7\u00a010 measurements of water depth along each transect. Within every second metre along the 11-m-long transect ten water depths were measured at a distance of 10\u00a0cm, resulting in five SD from each transect. These five SD were entered as replicates from each transect in the statistical analysis and were also the basis for the graphical presentation of \u201cmean standard deviation\u201d. Measurements were conducted prior to and during the main swan grazing period on 5, 17 and 30 August, 10 and 23 September, 5 and 18 October and 1 and 12 November 1999. Depth heterogeneity in relation to time of measurement and mean water depth was statistically analysed using repeated measures ANOVA (Statistica ver. 5.1; StatSoft, Tulsa, OK). Normal distribution of residuals was checked with a probability plot, and heteroscedasticity of variances was checked for with Levene\u2019s test. If the variances were not homogenous or the plots of residuals were not linear, the data were transformed to meet the assumptions of ANOVA.\nThe transplant experiment consisted of three separate treatments with six replicate plots (1.4\u00a0\u00d7\u00a01.4\u00a0m), all situated in an established (at least for 5\u00a0years) stand of P. pectinatus in Fulltofta Bay (Fig.\u00a02). The experiment ran from 31 May 2000 until 15 August 2001. One treatment,  consisted of continuously protected plots where rhizomes with ramets of P. perfoliatus had been planted among the P. pectinatus plants. The second treatment, P, consisted of plots where ramets of P. perfoliatus had also been planted among the P. pectinatus plants, but where swans were allowed to graze during the autumn migratory period (8 August until 18 December 2000). The last treatment, p, consisted of P. pectinatus plots without P. perfoliatus where swans were allowed to graze permanently. This last treatment was designed as a control treatment to check for any effects of the planting of P. perfoliatus on P. pectinatus. Ramets of P.\u00a0perfoliatus were collected in the eastern part of the nearby Lake Krankesj\u00f6n (55\u00b042\u2032N, 13\u00b028\u2032E) by scuba diving on 30 May 2000 just after onset of sprouting. The collected ramets with roots and intact meristems and varying in size from 30 to 70\u00a0cm were stored in plastic bags at 5\u00b0C overnight. On the following day, 22 ramets (density 11\u00a0m\u22122) with roots and intact meristems were planted in each of the  and P plots. The randomly selected ramets were distributed evenly over the plots and planted at a depth of 10\u00a0cm. The plots were subsequently protected with a 1.6-m high plastic net (mesh size 25\u00a0\u00d7\u00a032\u00a0mm). The p plots were not protected since summer grazing on above- and belowground biomass of P. pectinatus is considered negligible (Santamar\u00eda 2002).\nIn all 18 plots, the above- and belowground biomass of the vegetation was surveyed by scuba diving and core sampling prior to the migratory and main grazing period of the swans, on 8 August 2000. After the main swan-grazing period (verified by counts on 18 July, 8 and 18 August, 8 September, 4 and 24 October and 2 November 2000, yielding 22, 18, 34, 10, 10, 10 and 10 swans km\u22122, respectively), the belowground biomass was again surveyed by core sampling on 18 December 2000. Finally, the effect of swan grazing  on the aboveground plant biomass during the autumn of 2000 was again assessed by scuba diving on 6 June and 15 August 2001 the following growth season. Aboveground biomass was evaluated by counting all ramets emerging from the sediment in two perpendicular transects (5\u00a0cm wide, 2.0\u00a0m long) running diagonally across each plot. Belowground biomass was sampled using four sediment core samples (diameter\u00a011 cm, length 30\u00a0cm) in each plot. Roots, rhizomes and tubers of P. pectinatus and roots and rhizomes of P. perfoliatus were dried at 105\u00b0C for 24\u00a0h before dry weight (DW, g) measurements were taken. In December, we wanted to retain most of the P. pectinatus tubers for other purposes. Consequently, we first measured the fresh weight (FW, g) of each collected tuber and then assessed the DW on a subsample of randomly selected tubers. The regression of these DW estimates on FW was used to estimate total tuber DW of the whole sample (DW\u00a0=\u00a00.2953 FW, P\u00a0<\u00a00.0001, R2\u00a0=\u00a00.958, n\u00a0=\u00a033).\nLake Ringsj\u00f6n is usually covered with ice during the winter, which poses a potential threat to the fences around the plots. Therefore, on 18 December 2000, when the majority of birds had left the bay, the fences of treatment  were removed. Subsequently all plots, of treatments P and p, were covered with metal nets (mesh width 10\u00a0\u00d7\u00a010\u00a0cm) to protect them from belowground winter grazing by swans during occasional periods when the lake was not ice-covered. These bottom-covering nets remained in place throughout the experiment, and counting of ramets in 2001 was carried out without removing the nets.\nThe experimental data were analysed with analysis of variance, ANOVA, in Statistica ver. 5.1. Heteroscedasticity and residuals were examined, and transformations were carried out using the same methods as for the depth heterogeneity data. Tukey\u2019s honestly significant difference (HSD) was used to test all pairwise comparisons among means when the overall treatment effect in the ANOVA was (near) significance.\nResults\nVegetation and water depth mapping\nThe shallowest (<20\u00a0cm) and deepest (>95\u00a0cm) parts of Fulltofta Bay were void of submerged macrophytes (Fig.\u00a03). Potamogeton pectinatus grew at depths varying from 20\u00a0to 95\u00a0cm, with the highest ramet densities at intermediate water depths. In contrast, P. perfoliatus was found at relatively few points and mainly in deep water (>70\u00a0cm). Some P. perfoliatus was also found in shallow water (20\u201330\u00a0cm) but except for one finding at a depth of 46\u00a0cm, no P. perfoliatus was found at intermediate water depths (30\u201370\u00a0cm).Fig.\u00a03Maps on vegetation and water depth in Fulltofta Bay, Lake Ringsj\u00f6n. The vertical line shows the same point in the three maps. The upper map shows the distribution of P. perfoliatus on deep and shallow water. Grey represents areas where the probability of encountering P. perfoliatus on the sampling points was larger than 50%. The middle map shows the distribution and abundance of P. pectinatus. The lower map is a three-dimensional presentation of water depths in the monitored area\nSwans\nFigure\u00a04 (upper panel) shows swan densities in Fulltofta Bay during the autumn of 1999; swan grazing followed a similar temporal pattern in 2000 (Sandsten 2002). Depth heterogeneity largely coincided with swan densities and increased with time, indicating that swan grazing on P. pectinatus tubers increased from August to early November (Fig.\u00a04, lower panel). Depth heterogeneity was significantly affected by time and the interaction between water depth and time [F8,160\u00a0=\u00a014.38 P\u00a0<\u00a00.001, F32,160\u00a0=\u00a03.65 P\u00a0<\u00a00.001, F4,20\u00a0=\u00a01.80 P\u00a0=\u00a00.168; respectively; data was ln (Y\u00a0+\u00a00.1) transformed]. The interaction between time and water depth indicates that over time the swans\u2019 main foraging depth changed. As shown in Fig.\u00a04, birds started foraging on macrophytes at shallow depths (37 and 45\u00a0cm) in August, shifting to deeper areas (60, 75 and 90\u00a0cm) in October and November. During the last days of October, stormy weather events evened out the sediment, resulting in little depth heterogeneity being recorded on 1 November.Fig.\u00a04The upper panel shows swan densities in Fulltofta Bay (area 0.54\u00a0km2) over the period August\u2013November 1999. Swans include the whooper swan (Cygnus cygnus), Bewick\u2019s swan (C. columbianus bewickii) and mute swan (C. olor) The lower panel shows water depth heterogeneity measured as 5 SD of water depth at five locations with different water depths. The columns show mean\u00a0+\u00a0standard error of these SD. Depth heterogeneity is used as an indirect measure of swan foraging on tubers in the sediment\nTransplant experiment\nDuring the summer following planting, P. perfoliatus established well in the  and P plots, as indicated by the August 2000 sampling (Fig.\u00a05) prior to the peak swan-grazing period (compare Fig.\u00a04). A comparison of the  and P treatments revealed that neither belowground biomass nor ramet density of P. perfoliatus differed significantly at the start of the experiment (Table\u00a01; Fig.\u00a05). As expected, no P. perfoliatus was found in the p plots where no P. perfoliatus had been planted.Fig.\u00a05Above- and belowground biomass (means and standard errors) of P. pectinatus and P. perfoliatus in the transplant experiment during various times of the year in 2000 and 2001.  six plots where P. perfoliatus was planted in May 2000 and where above- and belowground biomass was protected against waterbird herbivory until December 2000, P six plots where P. perfoliatus was planted in May 2000 and above- and belowground biomass was protected against waterbird herbivory until August 2000, p six \u201cunplanted\u201d plots without protection against herbivores until December 2000. All 18 plots were protected from waterbirds after December 2000 with metal nets covering the bottomTable\u00a01ANOVA table for above- and belowground data of Potamogeton perfoliatus and P. pectinatus sampled in the three treatments on 8 August 2000 before the peak of waterbird herbivorySource of variationPotamogeton sp.FdfPRamets (number m\u22122)P. pectinatusa0.621,10NSP. perfoliatus a1.61,10NSP. pectinatus b1.02,15NSBelow-ground DW (g m\u22122)P. pectinatusa0.861,10NSP. perfoliatusa2.21,10NSP. pectinatusb3.32,15NS (0.065)*Tubers (number m\u22122)P. pectinatusa3.01,10NSP. pectinatusb1.72,15NS*Tukey\u2019s HSD multiple comparison between treatments resulted in a near significant effect (P\u00a0=\u00a00.058) for the comparison between  and p; NS, not significantaPlots without planted P. perfoliatus excludedbAll plots included\nThe August sampling also showed a healthy stand of P. pectinatus in all plots before the peak swan-grazing period. Although there seemed to be a tendency for P. pectinatus to perform worse in the presence of P. perfoliatus, no significant differences in belowground DW, ramet density or P. pectinatus tuber density were detected between the treatments (Table\u00a01; Fig.\u00a05).\nIn December, when the majority of swans had left the lake, belowground DW was again estimated (Fig.\u00a05). Potamogeton perfoliatus was found in three of the  and none of the other plots. Although this result suggested a negative impact of swan grazing on P. perfoliatus, it did not allow for any meaningful statistical analysis. In December, P. pectinatus belowground biomass and tuber density were higher in the  plots than in the P plots [F1,10\u00a0=\u00a07.7, P\u00a0<\u00a00.05 after ln(Y\u00a0+\u00a01) transformation, and F1,10\u00a0=\u00a012, P\u00a0<\u00a00.001 after square root (Y\u00a0+\u00a01) transformation, respectively], revealing the effect of tuber consumption by swans.\nRamet densities in the plots were counted in June 2001. There were significant treatment effects both on P. pectinatus and P. perfoliatus, and both species were present at higher densities in the  plots than in the P plots (Fig.\u00a05; Table\u00a02). In August 2001 there was still a difference in P. perfoliatus ramet density but not in P. pectinatus (Fig.\u00a05; Table\u00a02).Table\u00a02ANOVA table on P. pectinatus and P. perfoliatus ramet density in P and  plots on 6 June and 15 August 2001Source of variationPotamogeton sp.FdfPRamets (n m\u22122) in JuneP.\u00a0pectinatus431,10***Ramets (n m\u22122) in JuneP.\u00a0perfoliatus5651,10***Ramets (n m\u22122) in AugustP.\u00a0pectinatus1.11,10NSRamets (n m\u22122) in AugustP.\u00a0perfoliatus451,10******P\u00a0<\u00a00.001; NS, not significantAll data is ln(Y\u00a0+\u00a01) transformed\nDiscussion\nOur vegetation mapping study supports our earlier impression that the distribution of P. perfoliatus is limited to those areas where the pressure of swan grazing is low. It also shows that P. pectinatus grows at intermediate water depths, while P. perfoliatus is found mainly in deep water and occasionally at shallow depths. This is consistent with reports in the literature that P. perfoliatus mainly grows deeper than P. pectinatus (Scheffer et al. 1992; Lehmann et al. 1997), although we found no other report on P. perfoliatus growing in very shallow water.\nDepth heterogeneity substantiated our impressions of the spatial variation in foraging activity of the swans in Fulltofta Bay. Swans showed a tendency to forage at intermediate depths that are largely void of P. perfoliatus. They started to forage in relatively shallow water and moved to deeper parts, where foraging is more expensive (Nolet et al. 2001), later in the season. A similar phenomenon was observed in Bewick\u2019s Swans foraging on P. pectinatus in the Russian White Sea (Nolet and Drent 1998). This shift into deeper water over time may not only have been a consequence of depletion\u2014it may also have been related to a decrease in the water table by 0.2\u00a0m from August to early November (Swedish Meteorological and Hydrological Institute, unpublished data). When the spatial variation in foraging activity was considered over the whole season, there was a tendency for intermediate water levels to be used more frequently than the shallow and deep parts of the bay. This result is in accordance with those from an experiment on tuber depletion at different water depths in Fulltofta Bay (Sandsten 2002) and with previous observations on P. pectinatus foraging swans elsewhere (Beekman et al. 1991; Nolet et al. 2001). The observed tendency of swans to preferentially forage at intermediate depths becomes even more striking when we study the depth heterogeneity in Fulltofta Bay during the month of October 1999, when swan numbers were peaking.\nThe results of the transplant experiment showed that subterranean swan grazing had a dramatic negative effect on P. perfoliatus and, consequently, on the spatial depth-related distribution of P. pectinatus and P. perfoliatus in Fulltofta Bay and possibly elsewhere. When faced with increased subterranean herbivory, P. pectinatus seems to increase the depth at which its tubers are buried\u2014likely as a defence mechanism to reduce tuber loss (Klaassen and Nolet 2007; Santamar\u00eda and Rodr\u00edguez-Giron\u00e9s 2002). However, during the August\u2013December 2000 period, P. pectinatus winter biomass was considerably reduced as a result of swan foraging. Both belowground tuber biomass and tuber numbers were negatively affected. At the same time, swan foraging also had a marked negative effect on the occurrence of P. perfoliatus in the following year. Potamogeton perfoliatus had virtually disappeared from the unprotected sites where waterbirds had free access: in December, directly after herbivory, no belowground remnants of P. perfoliatus were detected; in June and August of the following year, the aboveground presence of P. perfoliatus was marginal in the unprotected plots, which contrasted sharply with the situation in the plots that had been continuously protected from subterranean herbivory by swans. Although not as dramatic as in P. perfoliatus, tuber herbivory by swans also had an effect on the aboveground biomass of P. pectinatus in the spring, but differences in aboveground biomass between protected and unprotected plots were no longer detectable by August 2001. The small number of tubers remaining in P was probably sufficient to produce a large number of tubers during the following autumn. This result is consistent with observations by LaMontagne et al. (2003) and the model of Jonz\u00e9n et al. (2002), which suggest that tuber herbivory in winter leads to a lower density of ramets the following spring, but that compensation (or even overcompensation; compare Nolet 2004) of tuber production may occur the following autumn.\nIn Sweden, the investments of P. perfoliatus and P. pectinatus in vegetative reproduction are large. It is highly likely that the difference in their wintering strategy determines their susceptibility to subterranean foraging by swans and, consequently, their prevalence in sites regularly frequented by digging swans. The small, round and relatively robust tubers of P. pectinatus are a highly preferred food source by foraging swans (e.g. Cramp and Simmons 1986; Sandsten 2002; Nolet and Drent 1998). However, if missed and not eaten, they are very likely to survive the digging and uprooting activity of the swans. In contrast, the rhizomes and turions of P. perfoliatus are long, interconnected and relatively fragile. Compared to the solitary tubers of P. pectinatus, they are easily detected and, if not eaten, fragmented by the digging and uprooting of swans.\nThe experiment reported here was mainly designed to study competition between P. pectinatus and P. perfoliatus under autumnal swan grazing. However, during the early experimental phase, which was without grazing until August 2000, no effect of P. perfoliatus on P. pectinatus growth was observed. Nevertheless, it is rather conceivable that both species compete for nutrients and light and that the removal of P. perfoliatus by swans will ultimately be of benefit to P. pectinatus. Therefore, the swans ultimately increase the profitability of the vegetation to themselves by promoting the nutrient-rich tuber-producing P. pectinatus. Although subterranean feeding in P. pectinatus stands may also favour the establishment of new macrophyte species (LaMontagne et al. 2003), the current findings suggest that tuber-feeding herbivores may actually stimulate the development of P. pectinatus by mediating competition with other macrophyte species, notably P. perfoliatus. This outcome implies a potentially mutualistic relationship, at least in an ecological sense (sensu J\u00e4remo et al. 1999), between a predator (swan) and its prey (P. pectinatus). Following J\u00e4remo et al.'s (1999) classification of mutualistic plant\u2013herbivore interactions, the current relationship may represent a \u201cplant\u2013herbivore antagonism\u201d, where P. pectinatus has adapted itself better than competing plants to subterranean herbivory (by producing specialised clonal propagules and hiding; Klaassen and Nolet 2007) thus obtaining a relative fitness advantage over less well-adapted species such as P. perfoliatus. However, since overcompensation to subterranean grazing may occur (Santamar\u00eda and Rodr\u00edguez-Giron\u00e9s 2002; Nolet 2004), the relationship between P. pectinatus and swans may also be considered as an \u201cevolution of overcompensation relationship\u201d, in which P. pectinatus has an absolute fitness advantage to grazing, whereas its competitors have not.\nOur data support the view that waterbirds may play a crucial role in the prevalence of certain plant species with potential consequences for the functioning of the system as a whole (e.g. Engelhardt and Ritchie 2001). Reviewing the (terrestrially-skewed) literature, Augustine and McNaughton (1998) concluded that the impact on vegetation communities tends to increase with grazing intensity. In aquatic systems, belowground grazing may be facilitated by the generally softer and easily penetrable sediments, notably if grazers make use of water jets to uncover the vegetative plant parts. Grazing intensity may thus reach very high levels in these systems, resulting in very rapid alterations in vegetation composition that are uncommon in terrestrial systems.","keyphrases":["potamogeton","cygnus","subterranean herbivory","mutualism","predator\u2013prey interactions"],"prmu":["P","P","P","U","M"]}
{"id":"Eur_Spine_J-2-2-1602193","title":"Osteoma in the upper cervical spine with spinal cord compression\n","text":"Osteoma is a common benign tumor. It occurs dominantly at the skull bone. Outside skull osteoma is rare, and primary intra-canal osteoma is extremely rare. To the author\u2019s knowledge, only 14 cases of osteomas of the spine had been reported, in which only seven cases were in English literature. The authors reported two rare cases of intra-canal osteoma of the upper cervical spine with cord compression. Included are pertinent history, physical examination, rontgenographic evaluation before and after operation, surgical interventions, pathological study, and outcome. The available literature is also reviewed. On systemic examination and rontgenographic study, these two cases were found to have bone tumor in the upper cervical canal. Surgical interventions were performed, one with an en bloc excision, the other with a subtotal excision. The pathological study demonstrated a diagnosis of osteoma. After a follow-up with 20 and 15 months, the clinical symptoms of both cases significantly improved.\nIntroduction\nOsteomas are rare, slow growing lesions composed of compact bone that occur almost exclusively in the skull [2, 7], most often in the inner and outer table of the calvarium or in the bones of the face or mandible [1, 12, 13]. The prevalence of this disease amounting those who have had sinus radiographs has been estimated to be 0.42% [7]. Although these lesions may cause sinusitis or exophthalmos when they arise from the wall of the nasal sinuses or the orbit, cranial osteomas often cause no symptoms and are discovered incidentally.\nPrimary intra-canal osteomas are extremely rare. To the authors\u2019 knowledge, at the time of writing, only 14 cases of osteomas of the spine had been reported, in which only seven cases were in the English literature [4\u20136, 8, 9, 11].\nThe purpose of this article is to report on two patients who presented with cervical intra-canal osteoma.\nCase report\nCase One\nA 56-year-old man had presented with paresis and weakness of his extremities after a hyperextension injury of the cervical spine in August 2002. The man had been asymptomatic prior to the accident.\nPhysical examination demonstrated limitation in neck rotation, and motor weakness of the extremities with strength 3\/5 in upper extremities and 4\/5 in lower extremities. He could eat with chopsticks to a limited degree. He could walk. However, he needed cane or aid on stairs. Hypoesthesia was present at the upper extremities, bilaterally. The biceps tendon reflex, patellar tendon reflex, and Achilles tendon reflex were hyperactive, and the Hoffman and Babinski reflex were positive bilaterally. Sustained ankle clonus was present, bilaterally. Neurological examination was consistent with upper neuron cell injury. The Japanese orthopedic association (JOA) score for cervical myelopathy was 11.\nPreoperative imaging study demonstrated a cortical like solid lesion. The results were shown in Fig.\u00a01a\u2013e.\nFig.\u00a01a Cervical A\u2013P view X-ray showed densification at left side at C2\u20133 level. b Cervical lateral view X-ray showed high density at C2\u20133 level, the facet joints between C2\\C3 were obscure. c CT scan through the intervertebral foramen between C2 and C3 showed the tumor occupied more than 60% canal and the vertebral shape changed, caused by the chronic pressure. d Three dimensions CT reconstruction showed the three-dimension relationship between the tumor and the cervical spinal canal. e Middle sagittal plane T1WI MRI showed the spinal cord was pressed by the tumor. The signal of the tumor was low. f The same plane as c after operation, showed the whole decompression of the spinal canal, part of the tumor, near the vertebral artery was left. g The same plane as e after operation, showed the whole decompression of the cord and spinal canal. h, i The pathological study showed that the lesion was composed of uniformly dense, compact, cortical-like mature lamellar bone. H: \u00d7100, I: \u00d7400\nWe performed a leminectomy of C2 and C3. In the operation, we found that the tumor was adhesive with the left side vertebral artery. To avoid the injury of the artery, part of the tumor, near the vertebral artery, was left. However the decompression of the spinal cord was enough (Fig.\u00a01f\u2013g). The pathological study showed that the lesion was composed of uniformly dense, compact, cortical-like, mature lamellar bone (Fig.\u00a01h\u2013i), and a diagnosis of osteoma was made. Two weeks after the operation, this patient\u2019s clinical symptoms improved significantly. Then, with hard cervical brace protection, the patient was permitted to ambulate. He noted gradual improvement of strength in his upper extremity. Three months later, the brace was removed. Twenty\u00a0months later, the muscle strength was 5\/5 in all extremities. He could eat with chopsticks, but awkwardly. He could walk with no disability. No hypoesthesia was present. The patient\u2019s JOA score increased from 11 to 16 (Table\u00a01).\nTable\u00a01JOA score of the two cases before and after therapyBefore therapyFollow-upCase 1Case 2Case 1Case 2Motor\u00a0Upper extremity2133\u00a0Lower extremity2243Feeling\u00a0Upper extremity1122\u00a0Trunk2222\u00a0Lower extremity2222Sphincter2233Total11101615\nCase two\nA 49-year-old man had experienced paresis and weakness of his extremities after a weight-hit injury of the head in November 2002. The man had been asymptomatic before the accident. Physical examination showed moderate motor weakness and hypoesthesia of the upper extremity, with strength 3\/5 in upper extremities and 4\/5 in lower extremities. He could eat with spoon but could not with chopsticks. He could walk. However, he needed a cane or aid on stairs. The feel of trunk and lower extremity is fair. The biceps, patellar, and Achilles tendon reflexes were hyperactive, and the Hoffman and Babinski reflexes were positive bilaterally. Ankle clonus was noted continuously on both sides.\nPreoperative imaging study demonstrated lamina fracture of C3, traumatic instability of C3\u20135, and a cortical like solid lesion at the left side of C2 lamina. The results were shown in Fig.\u00a02a\u2013c.\nFig.\u00a02a Cervical lateral view X-ray showed high density at C2\u20133 level, the facet joints between C2\\C3 were obscure, traumatic instability of C3\u20135. b CT scan through C2 showed the tumor at the left side of the lamina, occupied about 20% canal, and the left side lamina of the C2 became thicker. c Sagittal plane T2WI MRI showed the spinal cord was pressed by the tumor. The signal of the tumor was low. d Three-dimension CT reconstructive image showed complete decompression\nWe performed a laminectomy of C3\u2013C4 and part of C2, as well as internal fixation of C3\u20135, using pedicle screw system. The tumor was removed completely and the spinal cord was decompressed. The pathological study showed that the lesion was also composed of uniformly dense, compact, cortical-like, mature lamellar bone without nidus (not shown). Lateral view X-ray after operation showed the reconstruction of the stability by the pedicle screw system and lamina decompression of C3\u20134 and part of C2. Three dimension CT reconstructive image showed complete decompression of the spinal cord (Fig.\u00a02d).\nA month after the operation, this patient\u2019s clinical symptoms improved significantly. Then, with hard cervical brace protection, the patient was permitted to ambulate, and he noted gradual improvement of strength in his upper extremity also. Three months later, the brace was removed. After 15\u00a0months, the muscle strength was 4\/5 in upper extremity and 5\/5 in lower extremity. He could eat with chopsticks, but awkwardly. He could walk without cane or aid, but slowly. No hypoesthesia was present. The JOA score increased from 10 to 15 (Table\u00a01).\nDiscussion\nCongenital disease, rheumatoid arthritis, fracture and dislocation, and other systemic diseases can cause the compression of the upper cervical spinal cord [14]. However, osteoma is one of the rare reasons. Osteoma is defined by WHO as a benign lesion consisting of well differentiated mature bone tissue with a predominantly laminar structure, and showing very slow growth. Osteomas are bosselated, round to oval sessile tumors that project from the subperiosteal or endosteal surfaces of the cortex [3]. They are composed of a composite of woven and lamellar bone that is frequently deposited in a cortical pattern with haversian-like systems. Some variants contain a component of trabecular bone in which the intertrabecular spaces are filled with hematopoietic marrow [10]. Osteoma usually occurs in the skull. Only occasionally have these lesions been reported in extracranial locations (Table\u00a02). In 1980, Pecker et al. reported the first osteoma in cervical inter-vertebral foramen. They used a Lateral interscalenic approach to excise the lesion [8]. In 1986, Lantsman reported six cases of osteoma in spine, but the article was in Russian, no more details could be reviewed [4]. In 1993 [5] and 1996 [6] Laus reported the same case in Italian and English. To excise the lesion, they performed an anterior prevascular extra oral approach. In 1996 [9], Peyser reported 11 cases of extracranial osteoma, in which five were in spine, one in the cervical spine, two in the lumbar spine and two in the scrum spine. In 1998 [11], Rengachary reported that another osteoma affected the cervical spine.\nTable\u00a02Cases reported in the literatureReferencesArticle languageCase numberSite of the lesionRecurredRengachary [11]English1CervicalNoPeyser [9]English5Cervical (1), lumbar (2), sacrum (2)One pain recurredLaus [6]English1CervicalNoLaus [5]Italy1CervicalNoLantsman [4]Russian6SpineNoPecker [8]French1CervicalNoTotal14aaLaus M. reported the same case in English and Italy\nThe diagnosis of intra-canal osteoma is still confusing. In Peyser\u2019s series [9], none of the cases had correct diagnosis before the operation. In our cases, the rontgenographic study before operation could give some clues, but the final diagnosis still depended on the pathological findings. Histologically, the reactive bone induced by infection and trauma may be similar with a true osteoma [10]. However the differential diagnosis can be made by the tumor\u2019s site and pertinent history. Traumatic reactive bones are most frequently observed in relation to the femur, beneath the quadriceps, in relation to the adductor magnus (the so-called rider\u2019s bone) or in relation to the medial collateral ligament of the knee joint. The condition is also not infrequent at the elbow, the exostosis forming in the intermuscular planes of the brachialis following dislocation. Injury leads to the formation of a subperiosteal hemorrhage, the periosteum being detached by muscular traction. If the periosteum remains intact, the hematoma may become absorbed; but if the periosteal reapposition and the absorption of the blood clot are prevented, traumatic bone overgrowth may occur. However, this condition could not occur at the endosteum. In the case of infective reactive bones, an infection history of relevant site should be noted.\nThe clinical features of osteoma in the spine may be confusing. Cranial osteomas often cause no symptoms and are discovered incidentally, although these lesions may cause sinusitis or exophthalmus when they arise from the wall of the nasal sinuses or the orbit. It would be expected that the symptoms of spinal osteoma would depend on the location of the lesion and its relationship with the peripheral tissue. As Rengachary [11] and Laus [5, 6] reported, the symptom of the spinal osteoma was caused from spinal cord or nerve root pressure. However in Peyser\u2019s series all but one of the patients was symptomatic and had dull, aching pain at the time of the initial presentation. In our series, the two cases had no symptom before the injuries. It may be because osteomas are benign and very slow-growing lesions. And the spinal cord and never roots may adapt to the chronic pressure. Since the lesions cause spinal canal stenosis, they can cause the spinal cord to be more easily damaged by minor injury.\nThe growth potential of osteoma remains unclear, as we know the cranial osteomas grow very slowly. Peyser\u2019s excellent work showed us the growth potential feature of the spinal osteoma [9]. In their series, one patient who had a juxtacortical osteoma of the cervical spine, showed no major change on serial CT scans performed twelve years apart. However, another patient\u2019s sacral osteoma showed significant growth on serial radiographs performed 18\u00a0years apart. The method of surgical management of an osteoma depends on the growth feature of the tumor. Cranial osteoma does not need surgical excision if there are no symptoms. The aim of surgical management of spinal osteoma is decompression and stabilization if instability exists. Rengachary and Laus performed total excision of the lesion, with no tumor recurrences. Peyser performed three total and two subtotal excisions, in the case series of osteomas, none recurred. However in one of the patients, partial resection of an osteoma from the body of the fifth lumbar vertebra resulted in no relief of pain and necessitated an additional procedure to remove the remainder of the lesion. In our series, it was relatively easy to perform an en bloc excision in the second case. In the first case, in view of a patient\u2019s age and risk of vertebral artery injuries, a subtotal excision was performed, also with satisfied short time outcome. However, the follow-up was not long enough to observe the recurrence.\nConclusion\nThe incidence of intra-canal osteomas is rare. It can cause upper cervical myeolopathy. The rontgenographic study before operation can give some clues, but the final diagnosis still depends on the pathological findings. Surgical treatment, either en bloc or subtotal excision of the lesion, may be effective.","keyphrases":["osteoma","cervical spine","intra-canal","myeolopathy","operative treatment"],"prmu":["P","P","P","P","R"]}
{"id":"J_Abnorm_Child_Psychol-4-1-1915634","title":"Homotypic Versus Heterotypic Continuity of Anxiety Symptoms in Young Adolescents: Evidence for Distinctions Between DSM-IV Subtypes\n","text":"Objective: to investigate homotypic and heterotypic longitudinal patterns of symptoms of separation anxiety disorder (SAD), generalized anxiety disorder (GAD), social phobia (SoPh), panic disorder (PD), and obsessive compulsive disorder (OCD) in young adolescents from the Dutch general population.\nIntroduction\nAnxiety disorders are among the most prevalent psychiatric disorders in children and adolescents (Costello et\u00a0al., 1996; Essau et\u00a0al., 2000; Verhulst et\u00a0al., 1997), are persistent, and are associated with impaired functioning (Canino et\u00a0al., 2004; Ferdinand & Verhulst, 1995; Ferdinand et\u00a0al., 1995; McGee & Stanton, 1990; Pine et\u00a0al., 1998; Verhulst et\u00a0al., 1997). The high degree of comorbidity amongst anxiety disorders in children and adolescents seems to point in the direction of one taxonomic construct, instead of a number of separate disorders. High comorbidity rates have been reported by many authors (Essau et\u00a0al., 2000; Masi et\u00a0al., 1999; Newman et\u00a0al., 1996; Verduin & Kendall, 2003). Evidence for a higher order factor that explains the presence of different types of anxiety has been found in children (Nauta et\u00a0al., 2004) and adults (Hettema et\u00a0al., 2005; Krueger, 1999; Vollebergh et\u00a0al., 2001). Negative affectivity (NA) (Chorpita, 2002; Clark, 2005; Clark & Watson, 1991; Lonigan et\u00a0al., 1999, 2003) may be one of the higher order factors that may explain the finding of heterotypic continuity. NA represents displeasurable engagement with the environment and a sense of high subjective distress (Lonigan et\u00a0al., 2003), and is often considered as a temperament trait that is associated not only with anxiety, but with depression as well (Clark, 2005).\nUsing data collected at the first assessment wave of a study that was also used to conduct the research that is being described in the present manuscript, support was found for the presence of one single anxiety dimension, instead of a number of separate anxiety concepts (Ferdinand et\u00a0al., 2006). The sample of this previous study consisted of 10- to 12-year-olds from the Dutch general population, who completed a self-report questionnaire for anxiety symptoms. Based on item scores on this questionnaire, latent class analysis did not detect classes of individuals with, for instance, high scores on items tapping separation anxiety, and simultaneously low scores on items tapping panic or social anxiety. Instead, high scores on one anxiety dimension implicated high scores on the other anxiety dimensions as well. However, other studies found evidence for separate anxiety dimensions. By performing factor-analyses, several authors found that DSM-IV anxiety disorders, such as generalized anxiety disorder, separation anxiety disorder, social phobia, and panic disorder represent different problem dimensions in children and adolescents (Chorpita & Daleiden, 2000; Muris et\u00a0al., 2002; Spence, 1997).\nLongitudinal studies can provide valuable information regarding taxonomic constructs. For instance, it was found that symptoms of social phobia in adolescents predicted similar symptoms in adulthood (Pine et\u00a0al., 1998). The prediction of a disorder by the same disorder is called homotypic continuity. However, social phobia symptoms also predicted simple phobia in adulthood. The prediction of a disorder by another disorder is called heterotypic continuity (Costello et\u00a0al., 2003). Several mechanisms may explain heterotypic continuity. Heterotypic continuity may occur by chance. In other words, disease A may disappear, and disease B may occur subsequently, as a coincidence. However, in that case, continuity would not be reflected in statistical significance. More likely reasons for heterotypic continuity would be that disease A would be the cause of disease B, or that disease A and B share a common vulnerability factor.\nThe aim of the present study was to investigate homotypic and heterotypic longitudinal patterns of symptoms of separation anxiety disorder, generalized anxiety disorder, social phobia, panic disorder, and obsessive compulsive disorder in young adolescents from the Dutch general population. For this purpose, individuals from a community sample, who were assessed for the first time when they were aged 10 to 12 years, were followed up across a period of two years. At both assessments, anxiety symptoms were assessed with a self-report questionnaire. Given mixed results of previous studies, we did not formulate specific hypotheses regarding the level of homotypic or heterotypic continuity of different types of anxiety.\nMethods\nSample and procedure\nThe TRacking Adolescents\u2019 Individual Lives Survey (TRAILS) is a prospective cohort study of Dutch early adolescents aged 10\u201312 years, who are followed biennially. The present study used data from the first (2001\u20132002) and second (2003\u20132004) assessment wave. The TRAILS target sample consisted of young adolescents from five municipalities in the North of the Netherlands, including both urban and rural areas. More details about the sample selection have been published elsewhere (de Winter et\u00a0al., 2005).\nOf all subjects who were approached at wave 1 (N\u00a0=\u00a03,145), 6.7% were excluded. The exclusion criteria were (1) adolescent incapable to participate because of mental retardation or a serious physical illness or handicap and (2) Dutch-speaking parent or parent surrogate not available, and not feasible to administer a part of the measurements in parent\u2019s own language. Of the remaining 2,935 young adolescents, 24% did not want to cooperate, and 76.0% cooperated with the study at wave 1 (N\u00a0=\u00a02,230, mean age 11.09 years, SD .55, with 50.8% girls). Most frequent reasons for non-response were \u2018not interested\u2019 (33.8%), participation in other research or unfavorable experiences with research (15.4%), too much of a burden on the child (12.2%), lack of time (10.3%), concerns about privacy and confidentiality (8.0%), and the child\u2019s refusal to participate because friend(s) did not participate (4.0%). In 34 cases (1.2%) we failed to contact anyone of the household (de Winter et\u00a0al., 2005). Responders and non-responders did not differ with respect to the proportion of single parent families, or the prevalence of teacher-rated problem behavior. Furthermore, no differences between responders and non-responders were found regarding associations between socio-demographic variables and mental health outcomes (de Winter et\u00a0al., 2005). To assess anxiety symptoms, the Revised Child Anxiety and Depression Scale (RCADS) (Chorpita & Daleiden, 2000) was used at wave 1, and also at wave 2. For 20 cases, RCADS data were not obtained at wave 1 because respondents were not present during the measurements that were conducted in the classrooms, and could not be reached afterwards. Hence, RCADS data of 2,210 pre-adolescents were available at wave 1.\nAt the second assessment wave, following similar procedures as at wave 1, RCADS information was obtained from 2,067 individuals. This was 95.5% of those for whom wave 1 RCADS information had been collected (51.4% girls). To examine possible selective attrition, a stepwise logistic regression analysis was performed with \u2018wave 2 RCADS information available\u2019 as a dependent variable, and wave 1 age, sex, and the wave 1 RCADS Total Anxiety score (that was constituted by summing scores on the five anxiety dimensions that were assessed with the RCADS in the present study, see below) as possible predictors. The RCADS Total Anxiety score and sex did not predict attrition. However, younger age predicted attrition significantly (odds ratio\u00a0=\u00a0.17, Wald\u00a0=\u00a093.1, p\u00a0<\u00a0.001; Model chi-square\u00a0=\u00a0109,551, df\u00a0=\u00a01, p\u00a0<\u00a0.001). Cox and Snell R-square of the regression model was .048, which indicated that the effect of age was small. Further, most importantly, the level of anxiety at the initial assessment did not influence cooperation at wave 2.Table 1RCADS itemsSADSoPhFears being alone at homeWorried when does poorly at thingsScared to sleep aloneWorried when somebody angryScared to sleep away from homeWorried will do badly at schoolFears being away from parentsWorried about mistakesWorried in bed at nightWorried what others thinkTrouble going to schoolScared to take a testAfraid of being in crowded placesWorried might look foolishAfraid to talk in front of classAfraid to look foolish in front of peopleGADPDWorried something awful will happen to familySuddenly trouble breathing without reasonWorried bad things will happen to selfWhen has a problem, feels shakyWorried something bad will happen to selfSuddenly trembling, shaking without reasonThinks about deathSuddenly dizzy, faint without reasonWorried about thingsWhen has a problem, stomach feels funnyWorried about what will happenWhen has a problem, heart beats really fastSuddenly feeling scared without reasonSuddenly heart beats too fast without reasonWorried suddenly get scared without reasonOCDCan\u2019t get silly\/bad thoughts out of headKeeps checkingHas to think thoughts to stop bad eventsHas to do things over and over againHas to do things just right to stop bad eventsBothered by bad\/silly thoughts or images\nMeasures\nThe Revised Child Anxiety and Depression Scale (RCADS) (Chorpita & Daleiden, 2000) is a revision of the Spence Children\u2019s Anxiety Scale (SCAS) (Spence, 1997). It is a self-report questionnaire with 47 items, that are scored on a 4-point scale (0\u00a0=\u00a0never, 1\u00a0=\u00a0sometimes, 2\u00a0=\u00a0often, 3\u00a0=\u00a0always). The questionnaire covers six scales, corresponding with DSM-IV dimensions of anxiety disorders and depressive disorder. The following five scales were used for the present study: separation anxiety disorder (SAD), generalized anxiety disorder (GAD), social phobia (SoPh), panic disorder (PD), and obsessive compulsive disorder (OCD) (see Table 1). The scale major depressive disorder (MDD) was not used. The internal consistencies of the scales that were used were (respectively at wave 1\/wave 2) .66\/.59 for SAD, .80\/.72 for GAD, .78\/.88 for SoPh, .75\/.72 for PD, and .68\/.66 for OCD. The factor structure\u2014for all six scales together\u2014that was originally based on data from 1,641 children and adolescents from a community sample from Hawaii (Chorpita & Daleiden, 2000), was confirmed by confirmatory factor analysis in the TRAILS sample at wave 1 (fit indices of NNFI\u00a0=\u00a0.96, RMSEA\u00a0=\u00a0.05, and SRMR\u00a0=\u00a0.05, indicating an adequate fit to the sample data) (Ferdinand et\u00a0al., 2006). The association of RCADS dimensions of anxiety with corresponding DSM-IV anxiety disorders was supported by previous research (Nauta et\u00a0al., 2004).\nStatistical analyses\nFirst, to obtain information regarding comorbidity between different types of anxiety problems in the study sample, correlations among wave 1 RCADS scale scores were computed for each sex. Then, Pearson correlations were computed between wave 1 and wave 2 RCADS scale scores, separately for each sex group. Correlations provide insight in the associations between measures. However, by just computing correlations, it can not be judged if continuity is homotypic or heterotypic. For instance, the magnitude of a correlation between wave 1 SAD and wave 2 SoPh scores depends on the correlation between wave 1 SAD and wave 1 SoPh scores. The higher correlations between wave 1 SAD and wave 1 SoPh are, the higher the correlation between wave 1 SAD and wave 2 SoPh will be. In other words, if assessment of continuity would solely be based on correlations, comorbidity at wave 1 would artificially inflate estimations of the extent of heterotypic continuity between wave 1 and wave 2.\nTo correct for the effects of wave 1 comorbidity rates, regression analyses were conducted. First, it was assessed which part of continuity in anxiety problems was typically homotypic. For this purpose, for scores on each of the five RCADS scales at wave 2, a set of regression analyses was conducted, with wave 2 RCADS SAD, GAD, SoPh, PD, and OCD scores as dependent variables. These analyses were conducted to investigate how much of the variance in a specific RCADS scale score at wave 2 was not accounted for by an overall elevation in different types of anxiety at wave 1, but instead, specifically by its own counterpart at wave 1. We will now describe the regression analyses that were conducted for wave 2 SAD. Those for GAD, SoPh, PD, and OCD were similar. In the first block of the analyses, wave 1 scores on GAD, SoPh, PD, and OCD were entered simultaneously as predictors. Then, in a second block, wave 1 scores on the SAD scales were added, to see how much of the variance in wave 2 scores was predicted specifically by wave 1 SAD scores, and not by scores on the other RCADS scales at wave\u00a01. This variance reflects specific homotypic continuity. In the third block, sex was added. In the fourth block, an interaction between sex and SAD was added. If this interaction was significant, analyses were conducted for girls and boys separately. For each next block, the variance that was accounted for by the variable in this block was computed (R2).\nSecond, it was assessed which part of continuity in anxiety problems was specifically heterotypic. For this purpose, for scores on each of the five RCADS scales at wave 2, a set of regression analyses was conducted, with wave 2 RCADS SAD, GAD, SoPh, PD, and OCD scores as dependent variables. These analyses were conducted to investigate how much of the variance in a specific RCADS scale score at wave 2 was not accounted for by its own counterpart at wave 1, but instead, by the other wave 1 anxiety scale scores. We will now describe the regression analyses that were conducted for wave 2 SAD. Those for GAD, SoPh, PD, and OCD were similar. In the first block of the analyses, wave 1 SAD scores were entered as predictor. Then, in the second block, scores on wave 1 GAD, SoPh, PD, and OCD scales were added, to see how much of the variance in wave 2 scores was specifically predicted by other RCADS scales at wave 1. This variance reflects specific heterotypic continuity. In the third block, sex was added.\nTo judge the magnitude of effects, Cohen\u2019s rules for effects sizes can be used (Cohen, 1988). According to Cohen, R2 between 1.0% and 5.9% is small, between 5.9% to 13.8% medium, and above 13.8% large.\nResults\nMeans and standard deviations at wave 1 were calculated for SAD (mean=.375, SD=.356), GAD (mean=.666, SD=.454), SoPh (mean=.779, SD=.427), PD (mean=.428, SD=.363), and OCD (mean=.597, SD=.445). Means and standard deviations were also calculated for wave 2 SAD (mean=.236, SD=.291), GAD (mean=.485, SD=.427), SoPh (mean=.684, SD=.465), PD (mean=.301, SD=.321), and OCD (mean=.339, SD=.348). Means reflect mean item scores for each RCADS scale.\nCorrelations among wave 1 RCADS scale scores for each sex separately are presented in Table 2. It is shown that all correlations were in a close range, almost similar across sexes, and generally above .50 which can be regarded as high (Cohen, 1988). For both sexes the highest correlations were found between PD and OCD (r=.61 in boys and girls), and the lowest between SAD and OCD (boys r=.47; girls r=.51).Table 2Correlations among wave 1 RCADS scale scoresWave 1 RCADS scaleWave 1 RCADS scaleGAD b\/gSoPh b\/gPD b\/gOCD b\/gSAD.52\/.58.52\/.52.51\/.52.47\/.51GAD\u2014.59\/.54.55\/.54.58\/.58SoPh\u2014.56\/.55.54\/.53PD\u2014.61\/.61OCD\u2014Note. Correlations are presented for boys (b), girls (g) separately.Table 3Correlations between wave 1 and wave 2 RCADS scale scoresWave 1 RCADS scaleWave 2 RCADS scaleSAD b\/gGAD b\/gSoPh b\/gPD b\/gOCD b\/gSAD.30\/.38.23\/.32.35\/.29.28\/.27.21\/.25GAD.27\/.27.34\/.38.29\/.28.22\/.25.23\/.27SoPh.29\/.29.35\/.29.42\/.41.29\/.28.25\/.25PD.22\/.29.25\/.31.24\/.27.32\/.42.25\/.29OCD.23\/.30.25\/.32.28\/.27.27\/.32.31\/.37Note. Correlations are presented for boys (b), girls (g) separately.\nCorrelations between wave 1 and wave 2 RCADS scale scores can be found in Table 3. For instance, in boys, the correlation between wave 1 and wave 2 SAD scores was .30, whereas the correlations between wave 1 GAD, SoPh, and PD scores and wave 2 SAD scores were .27, .29, and .22 respectively. Hence, heterotypic correlations were almost as high as the homotypic correlation. In girls, a similar result was found for wave 2 SAD. Heterotypic correlations ranged between .27 and .29, whereas the homotypic correlation was .38. Similar relatively small discrepancies between homotypic and heterotypic correlations were found for wave 2 GAD, SoPh, and PD scores.Table 4Specific homotypic continuity (prediction by target scale). Prediction of wave 2 RCADS scale scores by wave 1 scale scores and sexWave 2 RCADS scaleSADGADSoPhPDOCDWave 1 predictorsR2\/F change\/p1R2\/F change\/p1R2\/F change\/p1R2\/F change\/p1R2\/F change\/p1Non-target scales (block 1).119\/69.68\/.000.150\/91.19\/.000.132\/78.35\/.000.126\/74.19\/.000.106\/61.22\/.000Target scale (block 2).050\/123.57\/.000.023\/58.06\/.000.069\/178.54\/.000.037\/90.39\/.000.025\/59.20\/.000Sex (block 3).023\/58.92\/.000.036\/93.32\/.000.037\/100.65\/.000.032\/81.21\/.000.009\/21.34\/.000Target-scale*sex (block 4).001\/2.06\/ns.002\/5.12\/.024.000\/.81\/ns.009\/23.81\/.000.002\/5.38\/.020Models for girls\/boys separately in case of interaction target-scale*sex\u2003Girls\u2003\u2003Non-target scales (block 1)\u2014.145\/44.70\/.000\u2014.128\/38.75\/.000.109\/32.82\/.000\u2003\u2003Target scale (block 2)\u2014.024\/31.06\/.000\u2014.060\/77.54\/.000.038\/47.13\/.000\u2003Boys\u2003\u2003Non-target scales (block 1)\u2014.136\/39.32\/.000\u2014.107\/29.72\/.000.089\/24.41\/.000\u2003\u2003Target scale (block 2)\u2014.018\/20.85\/.000\u2014.020\/22.82\/.000.021\/23.05\/.000Note. Target-scale---Wave 1 RCADS scale identical to the wave 2 RCADS outcome scale in the regression model (prediction indicating specific homotypic continuity). Non-target scales---all other wave 1 RCADS scales, not identical to the wave 2 RCADS outcome scale. R2---explained variance.\nThe results of the regression analyses are presented in Tables 4 and 5. Analyses that were aimed at assessing specific homotypic continuity (Table 4) indicated that variances reflecting homotypic continuity were 2.3% for SAD, 3.6% for GAD, 3.7% for SoPh, 3.2% for PD, and .9% for OCD. Analyses aimed as specifically investigating heterotypic continuity revealed variances of 2.0% for SAD, 4.2% for GAD, 1.3% for SoPh, 2.3% for PD, and 2.7% for OCD (Table 5).\nIn analyses aimed at homotypic continuity, some anxiety*sex interactions were significant. A marked sex difference was found for PD. Table 4 shows that homotypic continuity was much stronger for girls (homotypic R2=6.0%) than for boys (homotypic R2=2.0%).\nDiscussion\nThe present study assessed homotypic and heterotypic continuity of symptoms of separation anxiety disorder, generalized anxiety disorder, social phobia, panic disorder, and obsessive compulsive disorder in individuals from a community sample, who were assessed for the first time when they were aged 10 to 12 years, and for the second time two years later. Variances reflecting homotypic continuity were roughly equal to those reflecting heterotypic continuity for SAD (2.3% versus 2.0%) and GAD (3.6% versus 4.2%). Variances reflecting homotypic continuity were larger than for heterotypic continuity for SoPh (3.7% versus 1.3%) and PD (3.2% versus 2.3%), and smaller for homotypic than for heterotypic continuity for OCD (.9% versus 2.7%) (Tables 4 and 5). Applying cross-sectional research designs, previous studies found high comorbidity rates among different types of anxiety problems (Essau et\u00a0al., 2000; Masi et\u00a0al., 1999; Newman et\u00a0al., 1996; Verduin & Kendall, 2003). The present study extended the knowledge about the taxonomy of anxiety problems in young adolescents with longitudinal data. In accordance with previous cross-sectional work, SAD, GAD, SoPh, PD, and OCD symptoms appeared to be intertwined in a longitudinal fashion as well. However, considerable homotypic continuity was found as well.\nSeparation anxiety\nFor SAD, homotypic and heterotypic continuity were roughly equal. Previous studies indicated considerable comorbidity between SAD and the other anxiety problems, especially with SoPh (Compton et\u00a0al., 2000). In the present study comorbidity rates at wave 1 were high as well, not only with SoPh, but also with other types of anxiety. This suggests that SAD and the other anxiety problems may represent two sides of the same coin. However, homotypic continuity was as strong as heterotypic continuity, which supports the usefulness of SAD as a separate diagnostic construct. The distinction between SAD and other types of anxiety was further supported by another study, in which we conducted latent class analysis to assess the boundaries between SAD and SoPh in referred 8- to 11-year-olds (Ferdinand et\u00a0al., 2006). Four different classes of individuals were detected; those with (1) low SAD and SoPh item scores on a self-report questionnaire, (2) high SAD and high SoPh item scores, (3) high SAD and low SoPh scores, and (4) low SAD and high SoPh scores. This also supported the idea that, despite high comorbidity rates, SAD may at least partially represent a separate phenomenon, that, for instance, may be subject to specific etiological influences that differ from influences that affect the course of other anxiety problems.Table 5Specific heterotypic continuity (prediction by non-target scales). Prediction of wave 2 RCADS scale scores by wave 1 scale scores and sexWave 2 RCADS scaleSADGADSoPhPDOCDWave 1 predictorsR2\/F change\/p1R2\/F change\/p1R2\/F change\/p1R2\/F change\/p1R2\/F change\/p1Target scales (block 1).391\/373.39\/.000.375\/337.53\/.000.436\/483.57\/.000.380\/347.98\/.000.337\/263.45\/.000Non-target scale (block 2).020\/83.77\/.000.042\/86.59\/.000.013\/103.79\/.000.023\/80.00\/.000.027\/82.20\/.000Sex (block 3).027\/81.60\/.000.041\/90.95\/.000.040\/107.45\/.000.028\/82.80\/.000.012\/55.90\/.000Note. Target-scale---Wave 1 RCADS scale identical to the wave 2 RCADS outcome scale in the regression model. Non-target scales---all other wave 1 RCADS scales, not identical to the wave 2 RCADS outcome scale (prediction indicating specific heterotypic continuity). R2---explained variance.\nGeneralized anxiety\nFor GAD, homotypic continuity was almost as strong as heterotypic continuity. This is\u2014at least partly\u2014consistent with a previous study (Pine et\u00a0al., 2001) in adolescents from the general population indicating that separation anxiety disorder and social phobia in adolescents did not predict future generalized anxiety disorder. This suggested rather strong homotypic continuity of GAD. However, continuity of GAD was more heterotypic than in the Pine et\u00a0al. study, because in our study, heterotypic continuity was also considerable. This contrast may be due to methodological issues such as differences in sample characteristics (Pine et\u00a0al. investigated older adolescents), assessment procedures (Pine et\u00a0al. applied standardized interviews instead of self-report questionnaires), or statistical approach (Pine et\u00a0al. used categorical diagnostic samples whereas the present study used dimensional scale scores). Further, Pine et\u00a0al. did not use a block design for their regression analyses, but included all predictors in a forward stepwise logistic regression analysis. So, in essence, they did not test if one predictor predicted future GAD, over and above the effect of other predictors.\nSocial phobia\nCompared to heterotypic continuity, homotypic continuity of SoPh symptoms was relatively strong. Pine et\u00a0al. (2001) found that SoPh, but also GAD, in adolescence predicted future SoPh, independently of other types of anxiety, whereas SAD did not. Remarkably, in their study, GAD was a better predictor of future SoPh than SoPh itself. The aforementioned methodological differences between the Pine et\u2009al. study versus the present study may explain differences between findings.\nPanic\nHomotypic and heterotypic continuity of PD did not differ much. Analyses for boys and girls separately (Table 4) indicated that homotypic continuity was higher in girls than in boys. Previous studies already indicated that panic disorder tends to have a chronic course, in children as well as in adults (Biederman et\u00a0al., 1997; Bruce et\u00a0al., 2005). The prevalence of full blown panic disorder, and even of panic attacks, in adolescents is very low (Essau et\u00a0al., 1999). The present study nevertheless suggests that in girls, the disorder may already begin with a chronic homotypic course at a very young age which is in accordance with the higher prevalence in females versus males (Goodwin et\u00a0al., 2005), and with studies that retrospectively investigated age at onset, and that often point to childhood or adolescence as a starting point (Sheehan et\u00a0al., 1981).\nObsessive compulsive disorder\nComorbidity rates of OCD with other anxiety disorders are generally high (Heyman et\u00a0al., 2001; Tukel et\u00a0al., 2002), which was supported by the correlations among the wave 1 RCADS scale scores. The present study showed that heterotypic continuity of OCD symptoms was higher than homotypic continuity. To our knwoledge, previous studies that assessed homotypic continuity of OCD symptoms versus heterotypic continuity with other types of anxiety in young adolescents are not available, so we cannot compare our findings with previous work. Homotypic continuity was somewhat stronger in girls than in boys. This seems to suggest that, longitudinally, OCD symptoms in boys correlate differently with comorbid conditions than OCD symptoms in girls. Again, we were not able to find previous work on this topic. Future studies are needed to investigate if the differences between boys and girls we found can also be found in other samples, countries, and cultures.\nPractical implications\nIn many studies, anxiety disorders are treated as one group of disorders (Barrett et\u00a0al., 2001; Lipman et\u00a0al., 2001; MacMillan et\u00a0al., 2001; Roza et\u00a0al., 2003; Shortt et\u00a0al., 2001), and, even, some widely used assessment instruments do not contain scales that tap different anxiety dimensions (Achenbach, 1991a, b). Previous studies found considerable associations between different types of anxiety symptoms, which suggested the presence of one higher order factor (Nauta et\u00a0al., 2004). Several studies with adults also found evidence for a higher order factor that explained the presence of different types of anxiety (Hettema et\u00a0al., 2005; Krueger, 1999; Vollebergh et\u00a0al., 2001). Given the magnitude of heterotypic continuity in the present study, a higher order factor is likely to be present. However, the present study also showed that considerable homotypic continuity is present as well, occurring separately from a general propensity for high anxiety levels. This indicates that each type of anxiety problem may, at least partly, represent a distinct taxonomic construct. Homotypic continuity was found specifically for SAD, GAD, and SoPh, and for PD in girls. This may indicate that SAD, GAD, SoPh, and PD represent diagnostic constructs that are at least partially distinct. Hence, in clinical practice, instruments are needed that measure different anxiety dimensions separately. Intruments that just assess on single anxiety dimension may not be sufficient. Further, the distinctions between different anxiety constructs indicate that, despite the evidence that similar treatment methods are generally efficacious for different types of anxiety problems, each type of anxiety might require a slightly different treatment approach, and development of specific treatment modules.\nLimitations\nThe sample consisted of young adolescents only. For older adolescents, different homotypic and heterotypic continuities could apply. Furthermore, questionnaires were used instead of clinical interviews; information about the presence or absence of DSM-IV (American Psychiatric Association, 1994) clinical diagnoses was not obtained. Even though RCADS symptom dimensions have proved to reflect corresponding DSM-IV anxiety disorders (Nauta et\u00a0al., 2004), still, it may be the case that different results would have been obtained if DSM-IV diagnoses, that take account of the level of functional impairment, would have been used instead of RCADS scale scores.\nSince different informants may provide different information, this study would have been more valuable if information regarding symptoms of different types of anxiety would also have been gathered from parents or teachers. Unfortunately, such information was not available.\nConclusion\nIn the present study\u2019s sample of young adolescents from the Dutch general population, evidence for homotypic continuity was found, especially for symptoms of separation, social, and generalized anxiety, and for symptoms of panic disorder in girls.","keyphrases":["anxiety","adolescence","taxonomy"],"prmu":["P","P","P"]}
{"id":"Mol_Immunol-1-5-1994210","title":"Modulation of Aire regulates the expression of tissue-restricted antigens\n","text":"Intrathymic expression of tissue-restricted antigens (TRAs) has been viewed as the key element in the induction of central tolerance and recently, a central role for the autoimmune regulator (Aire) has been suggested in this process. The aim of this study was to establish whether down or up-regulation of Aire leads to alterations in TRA expression and whether this is limited to thymic epithelial cells. This study also characterized whether TRAs follow Aire expression during normal development, and whether thymic microenvironment plays a role in the expression of Aire and TRAs. We did several in vivo and in vitro experiments to manipulate Aire expression and measured expression of four TRAs (Trefoil factor-3, Insulin-2, Major urinary protein-1 and Salivary protein-1) by real-time RT-PCR. Aire had an allele dose-dependent effect on TRA expression in the thymuses of mice from two strains, C57BL\/6J and Balb\/c, but had no effect on TRA expression in the lymph nodes. In the thymus, Aire and TRAs were both localized in the medulla and were co-expressed during normal development and involution. In the primary stromal cells as well as thymic epithelial cell line, the adenoviral over-expression of Aire resulted in an increase in TRA expression. By manipulating in vitro organ-cultures we showed that thymic microenvironment plays a dominant role in Aire expression whereas TRAs follow the same pattern. The data underline a direct role for Aire in TRA expression and suggest that modulation of Aire has a potential to control central tolerance and autoimmunity.\n1\nIntroduction\nThymus has an essential role in establishing immune tolerance. Previous studies have demonstrated that tissue-restricted antigens (TRAs) are expressed in thymus and that this expression is needed for the deletion of self-reactive T cells (Kyewski and Klein, 2006). A central feature in this process is the promiscuous expression of TRAs by epithelial cells in thymic medullary region, where the TRAs are presented and encountered by the thymocytes, leading to the induction of tolerance either by clonal deletion or functional inactivation (Derbinski et al., 2001). In this context, medullary thymic epithelial cells (mTEC) highly express MHC molecules with costimulatory signals and act as professional antigen presenting cells (APCs) in thymus. A detailed study of the gene expression pattern in mTEC revealed that many of the TRAs and, in particular, almost all putative autoantigen targets of experimental animal models and human diseases are expressed by mTEC (Derbinski et al., 2001; Gotter and Kyewski, 2004). Altogether, the pool of promiscuously expressed genes in thymus appears to be highly diverse including tissue and sex-specific genes and genes specifically involved in development (Kyewski and Klein, 2006).\nAn important molecule in regulation of TRA expression in mTEC is autoimmune regulator (Aire) (Nagamine et al., 1997). The Aire protein has several features such as SAND and PHD finger domains that are characteristic to proteins involved in transcriptional control and has been reported to bind directly to DNA (Kumar et al., 2001) and to a common transcriptional regulator and histone acetyltransferase, CREB binding protein (CBP) (Pitkanen et al., 2000). In the thymus and cell lines, the Aire protein is subcellularly located to the nuclear bodies (Bjorses et al., 1999; Heino et al., 1999), which have been associated with several functions, including modulation of chromatin structure, transcriptional control, DNA repair and antiviral response (Everett and Chelbi-Alix, 2007). Initial studies have shown the Aire protein to be predominantly expressed in mTECs and suggest it has a role in regulation of immune tolerance (Blechschmidt et al., 1999; Heino et al., 1999). In humans, mutations in AIRE cause autoimmune-polyendocrinopathy-candidiasis ectodermal dystrophy (APECED), a syndrome characterized by the presence of autoantibodies to multiple self antigens and lymphocytic infiltration of endocrine glands, leading to autoimmune endocrine disorders (Perheentupa, 2006; Peterson and Peltonen, 2005). In agreement with the human disease, the Aire deficient mice have autoantibodies and tissue infiltration, although the full development of autoimmune disease appears to depend on the genetic background of the mouse (Anderson et al., 2002; Kuroda et al., 2005; Ramsey et al., 2002). The Aire deficiency affects negative selection since there is a complete failure to delete the organ-specific thymocytes in this mouse model (Liston et al., 2003). More importantly, the microarray analysis of mTEC population shows a decreased or abolished expression of multiple tissue specific genes in the Aire deficient mouse suggesting thus that Aire plays a role in modulating TRAs in the mTEC (Anderson et al., 2002; Derbinski et al., 2005; Jiang et al., 2005).\nThis study aims to further clarify whether Aire can directly regulate the TRA expression by analyzing the expression of four antigens in several experimental settings where Aire's expression has been modulated. The study aims to establish whether there is a dose-dependent correlation between the number of Aire allele copies and TRA expression level in thymic epithelial cells, and whether TRAs are co-expressed with Aire during thymic development and involution. We also studied whether the over-expression of Aire as a sole factor is sufficient to induce TRA expression and whether thymic microenvironment plays a role in the expression of Aire and TRAs.\n2\nMaterial and methods\n2.1\nMice and cell cultures\nAire deficient mice (C57BL\/6J and Balb\/c background) were generated at The Walter and Eliza Hall Institute (Melbourne, Australia). The inserted targeting construct containing LacZ gene replaced mouse Aire exon 8. For genotyping, the genomic DNA was extracted using JetQuick Tissue DNA Spin Kit (Genomed), and wild-type (WT) and knockout (KO) alleles were amplified using primers: 1042 5\u2032-cagaagaacgaggat-3\u2032, 1045 5\u2032-cagactgccttggga-3\u2032 or 1043 5\u2032-ctgtcttctgtgaaggcttctagg-3\u2032. As shown in Fig. 1A, primers pair 1042\/1043 and 1043\/1045 detect WT and KO alleles, respectively. Thymuses from 4- to 6-week-old WT, Aire HET (heterozygote) and Aire KO mice were used. Embryonic (E13.5, E15.5 and E17.5), newborn, neonatal D11 and adult (6 weeks, 6 months and 12 months) mouse tissues were used in developmental dynamics analysis. Mice were maintained at the mouse facility of the Institute of Molecular and Cell Biology, Tartu University. TEC 1C6 cell line (Mizuochi et al., 1992) was kindly provided by G. Holl\u00e4nder (University of Basel, Switzerland). Human embryonic kidney (HEK293) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100\u00a0U\/ml penicillin, 100\u00a0\u03bcg\/ml streptomycin and 0.25\u00a0\u03bcg\/ml amphotericin B (Gibco BRL).\n2.2\nEGFP and Aire adenovirus construction and infection\nThe pAdTrack-CMV (Stratagene) vector expressing enhanced green fluorescence protein (EGFP) gene was used as pAd-GFP plasmid. The mouse Aire gene was amplified from pcAire vector (Heino et al., 2000) using the primers: mAire-5-SalI 5\u2032-tttgtcgac agatggcaggtggggatggaatg-3\u2032 and mAire-3-NotI_stop 5\u2032-tttgcggccgctcaggaagagaagggtggtgtc-3\u2032 and cloned into SalI and NotI sites of pAdTrack-CMV resulting in AdAire-GFP. HEK293 cells (Invitrogen), which constitutively express AdEasy deleted E1 genes in-trans, were used for expression analysis of adenoviral vectors and for virus growth. To make recombinant adenoviruses, pAd-GFP and pAdAire-GFP plasmids were recombined with pAdEasy-1. The amplification and harvest process was repeated to generate higher titer viral stocks. Subsequently, the lyzates from several amplification steps were purified by CsCl gradient centrifugation (He et al., 1998). Virus bands was collected and mixed with 2\u00d7 preservation buffer (10\u00a0mM Tris pH 8.0, 100\u00a0mM NaCl, 0.1% BSA, 50% glycerol). We quantified the Ad-GFP and AdAire-GFP virus particles by absorbance measurement at 260\u00a0nm to be equivalent to 1012 particles with virus titers >2\u00a0\u00d7\u00a0108\u00a0pfu\/ml. The adenoviruses were next verified for their expression by infection of HEK293 cells for 48\u00a0h and analyzed by immunoblotting with rabbit polyclonal anti-mouseAire and mouse monoclonal anti-G3PDH (Ambion). The signal was detected by Supersignal\u00ae West Pico Chemoluminescent Substrate (Pierce Biotechnology) according to the manufacturer's instructions. For adenoviral infection of thymic primary culture and TEC 1C6, the cells were infected with Ad-GFP and AdAire-GFP at approximately 70% confluence. Cells were incubated in 500\u00a0\u03bcl serum-free OptiMEM (100\u00a0U\/ml penicillin, 100\u00a0\u03bcg\/ml streptomycin and 0.25\u00a0\u03bcg\/ml amphotericin B) and infected with equal amounts of virus for 1\u00a0h. The infection rate was further assessed by quantitative real-time PCR analyzing intake of viral genomic DNA.\n2.3\nThymic stromal cell isolation\nSmall cuts were made into the capsules of thymi dissected from eight to twelve 4-week-old mice and thymocytes were released by repetitive pipeting. The remaining thymic fragments were incubated in 0.5\u00a0mg\/ml dispase\/collagenase (Roche) and 1.5\u00a0\u03bcg\/ml DNase I (AppliChem) in PBS at 37\u00a0\u00b0C for 20\u00a0min, with gentle agitation using a Pasteur pipette every 5\u00a0min. Released cells were collected to separate fractions and fresh enzyme solution was added four times. The cells were resuspended in 5\u00a0mM EDTA in 10\u00a0ml of RPMI-1460. Isolated cells were used either for stromal cell isolation or for growing thymic primary culture. For thymic primary culture, the isolated cells were cultured for 4 days in DMEM supplemented with 10% FCS. For stromal cell isolation cells were counted in each fraction. To obtain 2\u00a0\u00d7\u00a0108 cells required for isolation, the final fraction was used first and then collected backwards through the collected fractions. The fractions were pooled and passed through a 100\u00a0\u03bcm mesh to remove clumps.\n2.4\nCell sorting\nFor CD45 depletion CD45 MicroBeads (Miltenyi Biotec) were used according to manufacturers instructions. For cortical epithelial cell (cTEC) isolation, the CD45\u2212 cells were stained with H213-HB Ab (anti-CDR1) followed by goat anti-rat IgG Microbeads (Miltenyi Biotech) and AutoMACS separation (isolation mode: Possel-S). The positive fraction (CDR1+) contained magnetically bound cTEC-s. For mTEC isolation CDR1\u2212 cells were stained with G8.8 (anti-EpCAM, generated from a G8.8 hybridoma cell line) followed by goat anti-rat IgG Microbeads (Miltenyi Biotec) and separation as previously described. The purity of mTEC and cTEC was >80% as assessed by staining with anti-CD45 FITC (30F11, Miltenyi Biotech) and anti-I-Ab PE (AF6-120.1, BD Biosciences) using FACSCalibur flow cytometer (BD Biosciences).\n2.5\nImmunofluorescence and microscopy\nCryostat sections (5\u03bcm) of fresh-frozen 10-day-old mouse thymus were thaw-mounted onto SuperFrost Plus microscope slides (Menzel\u2013Gl\u00e4zer) and fixed in cold acetone (\u221220\u00a0\u00b0C) for 5\u00a0min. Sections were permeabilized in PBS\/0.5% Triton X-100\/1% normal goat serum (DAKO) for 15\u00a0min. Slides were blocked with 1% normal goat sera for 20\u00a0min at room temperature and incubated with rat monoclonal G8.8 (1:100) or rabbit polyclonal anti-mAIRE (1:2000) and then incubated with Alexa Fluor 594-conjugated goat anti-rat IgG (H\u00a0+\u00a0L) or Alexa Fluor 488-conjugated goat anti-rabbit (Fab)2 (both from Molecular Probes, Eugene), followed by washing six times in PBS. The slides were incubated with 15\u00a0\u03bcg\/ml DAPI (Roche) and mounted with fluorescent mounting medium (DAKO). The images were acquired by fluorescence microscopy (Eclipse TE2000-4; Nikon, Melville, NY).\n2.6\nThymic reaggregate organ culture\nReaggregated thymic organ-cultures were done as described previously (Jenkinson et al., 1992). Briefly, thymic stromal cells from E17.5 C57BL\/6 mice were prepared by disaggregating fetal thymic lobes, which had been previously cultured for 7 days in 1.45\u00a0mM deoxyguanosine (Sigma, St. Louis, MO) using 1\u00d7 trypsin (Life Technologies, Grand Island, NY). Reaggregates were formed by mixing together stromal cells and thymocytes at a 3:1 cell ratio and cultured for 3 days at 37\u00a0\u00b0C.\n2.7\nReal-time PCR\nRNA was isolated using TRIzol (Invitrogen, Life Technologies) and reverse-transcribed to cDNA using the SuperScript\u2122 III Reverse Transcriptase (Invitrogen, Life Technologies). Real-time PCR was performed with the ABI Prism 7900HT Fast Real-Time PCR System instrument (Applied Biosystems) using qPCR SYBR Green Core Kit (Eurogentec) according to the manufacturer's instructions except that 2\u00a0mM MgCl2 concentration was used. The amplification program included an initial denaturation step at 95\u00a0\u00b0C for 10\u00a0min, followed by denaturation at 95\u00a0\u00b0C for 15\u00a0s, and annealing and extension at 60\u00a0\u00b0C for 1\u00a0min, for 45 cycles. SYBR Green fluorescence was measured after each extension step, and the specificity of amplification was evaluated by melting curve analysis. Primers used to amplify specific gene products from murine cDNA were K2-8 sense, 5\u2032-aggagctcattccgtagctg-3\u2032; K2-8 antisense, 5\u2032-tctgggatgcagaacatgag-3\u2032; Aire 11\/12, 5\u2032-ccccgccggggaccaatctc-3\u2032; Aire 12\/13, 5\u2032-agtcgtcccctaccttggcaagc-3\u2032; Tff3 sense, 5\u2032-tacgttggcctgtctccaag-3\u2032; Tff3 antisense, 5\u2032-cagggcacatttgggatact-3\u2032; Ins2 sense, 5\u2032-gacccacaagtggcacaac-3\u2032; Ins2 antisense, 5\u2032-tctacaatgccacgcttctg-3\u2019, Mup1 sense, 5\u2032-tctgtgacgtatgatggattcaa-3\u2019; Mup1 antisense, 5\u2032-tctggttctcggccatagag-3\u2032; Spt1 sense, 5\u2032-aacttctggaactgctgattctg-3\u2032; Spt1 antisense, 5\u2032-gaggcctcattagcagtgttg-3\u2032. The relative gene expression levels were calculated using the comparative Ct (\u0394\u0394Ct) method (according to Applied Biosystems), where the relative expression is calculated as , and where Ct represents the threshold cycle. Every sample was run in three parallel reactions.\n3\nResults\n3.1\nDecrease in Aire expression down-regulates the TRA expression in a dose-dependent manner\nWe initially set out to confirm by real-time PCR the previously published array results (Derbinski et al., 2005) demonstrating decreased TRA mRNA levels in the Aire deficient mouse. In order to study whether the self antigen expression is dependent on Aire we chose four TRAs; Tff3, Ins2, Mup1 and Spt1, which were downregulated in the Aire deficient mouse according to publicly available microarray data (Derbinski et al., 2005). Throughout the study, we normalized our data to the expression level of keratin 8 (K8) mRNA, which in thymus is specifically expressed in epithelial cell fraction and is not influenced by Aire gene expression (Anderson et al., 2002; Derbinski et al., 2005). The real-time PCR analysis showed almost complete absence of TRA mRNA signal in C57BL\/6 Aire KO thymus samples, which was uniformly seen with all four antigens studied (Fig. 1B). Furthermore, Aire allele dose-dependency was observed, as heterozygous mouse thymus consistently showed lower expression levels compared to the WT thymus levels. The expression level of all four TRAs in heterozygous mice thymus was approximately 10\u201320% of the expression level in WT mice. In order to determine whether Aire's effect on TRA expression depends on the genetic background, we also measured expression levels of the four TRAs in Aire KO and Aire HET mice backcrossed to Balb\/c WT mice (Fig. 1C). Again, we observed a clear allele dose-dependency for all TRAs studied and almost no expression of the TRAs in the Aire KO mouse.\nWe next determined whether Aire has a similar effect on TRA expression in the lymph nodes and quantified the expression of TRAs in the lymph nodes from C57BL\/6 mice. Although we could clearly detect Aire mRNA in the lymph nodes at level that was even higher than the one of the whole thymus (Fig. 2A), most of the analyzed TRAs were undetectable or close to the detection limit. The higher expression of Aire in lymph nodes was relative to the epithelial cell marker K8, limiting the detection of Aire mRNA signal to the epithelial cell fraction. However, the mRNA signal for the Ins2 was clearly present in lymph node samples but, interestingly, did not depend on the presence of Aire (Fig. 2B).\nIn order to establish whether Aire co-localizes in the thymus with TRAs, we purified the thymic mTEC based on the cell-surface marker EpCAM (Fig. 3A) and analyzed the expression of the TRA genes. As seen in Fig. 3B, the expression of the Tff3, Ins2, Mup1 and Spt1 antigens was limited to the mTEC population, i.e. the cell population of Aire expression. The cTEC population showed a very low expression for all four TRAs both in the WT as well as Aire KO mouse. Collectively these data show that Aire dose-dependently regulates TRA expression in thymus but not in the lymph nodes, and confirms by real-time PCR the previously published microarray data, suggesting that both Aire and TRAs are predominantly expressed in thymus medullary epithelium.\n3.2\nTRAs follow the expression of Aire during normal development and involution\nThus far, the expression of Aire and TRAs has been studied in fetal or neonatal mice using WT versus Aire KO mice. If the expression of self antigens is directly dependent on Aire, this should be evident throughout the development of thymic tissue. However, the thymic cell content and volume changes significantly during development. To limit our analysis to the epithelial cell subsets only, we normalized our data again to the K8 gene. Thymuses from different embryonic, neonatal, young or adult developmental stages were analyzed for the Aire and TRA expression (Fig. 4). Very low Aire expression was detectable already at day E13.5 but showed a significant increase at E15.5. Thus, the start of Aire expression coincides with the influx of the first wave of the hematopoietic cells to the thymus. We observed the highest expression level at postnatal D11 and a gradual decrease thereafter until the very last time-point studied. Aire expression was, however, clearly present even in 12-month-old mice. The expression of TRAs closely followed the pattern of Aire reaching their peak at D11 followed by an obvious decrease, suggesting a role for Aire in their regulation.\n3.3\nOver-expression of Aire results in an increase in TRA expression\nAlthough the lack of Aire has been shown to have a negative effect on TRA expression, the ability of Aire as a single factor to up-regulate TRA expression has not been demonstrated. We used an adenoviral expression system (AdAire-GFP versus Ad-GFP) to determine whether the specific over-expression of Aire has any effect on TRAs. Infection with the AdAire-GFP resulted in an increased production of Aire protein (Fig. 5A). The AdAire-GFP infection also resulted in an increase of expression of all four TRAs studied, which was detected in primary thymic stromal cells (Fig. 5B) as well as in thymic medullary epithelial cell line TEC 1C6 (Fig. 5C). The results demonstrate that Aire can indeed act as a single inducer of TRAs in thymic epithelial cells even in the absence of signals from other cell-types normally present in thymus.\n3.4\nThymic microenvironment is needed for the expression of Aire and TRAs\nAlthough the precise mechanism is not fully understood, it has been demonstrated that the complex 3D structure of thymus is essential for mTECs to function properly (Anderson et al., 2006). To study whether the microenvironment plays a role in Aire and TRA expression, we used ex vivo culture of thymocyte-depleted dissaggregated and reaggregated thymic stromal cells. As shown in Fig. 6, the disaggregation of thymus to 2D culture resulted in a dramatic decrease in expression of Aire as well as most of the TRAs. This effect was, at least partly, reversed by reaggregation suggesting a critical role for the thymic microenvironment in Aire as well as TRA expression.\n4\nDiscussion\nIn this paper, we report Aire dependent expression of four TRAs in mouse thymus. TRA expression was previously reported to be substantially decreased in Aire deficient mTEC subpopulations by microarray analysis (Anderson et al., 2002; Derbinski et al., 2005), which prompted us to follow the expression of four TRAs as marker antigens. All four genes have highly selective tissue specific expression. For example, Tff3 is restricted to mucin producing epithelial cells, with high expression in stomach and intestine (Hoffmann and Jagla, 2002; Karam et al., 2004), Spt1 is expressed in salivary and lacrimal glands (Dickinson et al., 1989) and Mup1 is expressed in liver but also in salivary, lacrimal and mammary glands (Shaw et al., 1983). Of the two insulin genes present in the mouse genome and encoded from separate loci, we selected Ins2 due to its high expression in the thymus as well as pancreatic beta cells, and previously reported Aire dependent expression pattern (Chentoufi and Polychronakos, 2002; Derbinski et al., 2005). Furthermore, it has been reported that Ins2 deficient mouse with low insulin expression in thymus has T cell reactivity to proinsulin (Chentoufi and Polychronakos, 2002).\nThe expression signal of all four TRAs tested was readily detectable in whole thymus and sorted mTEC samples by real-time PCR analysis. Interestingly, the TRA expression in heterozygous mouse thymus, both on C57Bl\/6 and Balb\/c background, was repeatedly only 10\u201320% and not 50% of the expression level seen in WT mouse, which would be expected when one of the Aire genomic alleles remains intact. This expression at lower levels than expected in the heterozygous mouse suggests Aire haploinsufficiency in regulation of target TRA genes. In haploinsufficiency of transcriptional regulators, only one intact gene copy is not sufficient for the functional activity of the regulated target gene product. The phenomenon has been explained by a stochastic expression model where diploid cells have a higher probability than haploid cells in maintaining the abundance of an expressed gene product above a low threshold level (Cook et al., 1998; Kaern et al., 2005). Further support to Aire haploinsufficiency comes from the report by Liston et al (Liston et al., 2004) demonstrating that the loss of one copy of the Aire allele in TCR-insHEL double transgenic mice caused severe functional defects in negative selection of autoreactive T cells and resulted in pancreatic cell insulitis, with diabetes incidence comparable to the Aire KO mouse. It should be noted, however, that the activation of target genes by a transcriptional factor in vivo can be dependent on the specific gene and the physiological context, such as in the case of cardiac transcription factor Nkx2\u20135 (Jay et al., 2005).\nA recent study reported that lymph node stroma can also express Aire as well as a range of TRAs and contributes accordingly to tolerance induction (Lee et al., 2007). Here we report that, although Aire as well as Ins2 expression is clearly present in the lymph nodes of WT mice, the lack of Aire does not lead to a decrease in Ins2 expression in the Aire KO mice. The data suggests that, unlike in the thymus, other Aire-independent factors are likely to control Ins2 expression in the lymph nodes. Regarding the rest of the TRAs studied, we found very low, if any, expression by real-time PCR, which did not allow us to quantify the changes.\nIn order to determine whether the thymic expression of Aire and TRAs follow the same pattern during development, we monitored the expression throughout mouse development from E13.5 to 12 months. We detected an increase in Aire expression at day E15.5, which is in concordance with earlier reported results (Sousa Cardoso et al., 2006). The expression was at its highest at D11 and decreased thereafter, but was still present even in 12-month-old mouse tissue. The expression of TRAs followed a similar pattern to Aire expression, indicating a correlation between the amount of Aire and TRAs. The data demonstrate that the dynamics of Aire and TRA expression closely follow the dynamics of thymic function in general, being most active during the postnatal period and followed by a gradual decline in activity (Gray et al., 2006).\nAire dependent TRA expression is further illustrated by adenoviral experiments enforcing Aire expression in thymic epithelial cells. We show that over-expression of Aire as a single factor is sufficient to induce the expression of all four TRAs studied, providing evidence that modulation of Aire can directly lead to alterations in TRA levels and may thus also affect the maintenance of central tolerance. The finding that Aire, in addition to primary thymic stromal cells, can also modulate TRA levels in the thymic medullary epithelial cell line, suggests that there is no need for other cell types for the Aire-induced up-regulation of TRAs to occur.\nThe disruption of normal thymic architecture is known to affect the expression pattern and functionality of thymus, and it has been suggested that interactions between epithelial cells and thymocytes control the development of the thymic microenvironment and T cell development (Van Ewijk et al., 1994). Although the maturation of thymic epithelial stroma during the fetal period apparently occurs independently of thymocyte-derived signals (Jenkinson et al., 2005) and is mainly regulated by thymic mesenchyme (Jenkinson et al., 2003), thymocytes deliver signaling molecules, which are needed to maintain the normal adult thymic microenvironment. For example, lymphotoxin that signals through the lymphotoxin receptor and directs the alternative NfkappaB pathway, is needed for development of the thymic medullary compartment. Consequently, lymphotoxin receptor deficient mouse thymus had subnormal levels of Aire and TRAs (Boehm et al., 2003; Chin et al., 2003). Thymic medullary atrophy and lower expression of Aire and TRAs have been reported in mouse models deficient in several genes involved in the NFkappaB pathway, such as TRAF6, NIK, RelB or p52 suggesting an important role of this pathway in development of thymic medulla (Akiyama et al., 2005; Burkly et al., 1995; Kinoshita et al., 2006; Zhang et al., 2006). A recent study suggests that Aire deficiency may also cause changes in the organization and composition of the medullary epithelial compartment (Gillard et al., 2007). Thus, it is presently unknown whether the reduced levels of TRA expression seen in Aire KO mice are predominantly the result of changes in transcriptional activity or changes in thymic epithelial cell development. In this study, we show a sustained Aire and TRA expression in 2-deoxyguonosine treated FTOC, which rapidly disappeared after the disruption of the three-dimensional thymic meshwork into two-dimensional culture. Aire as well as TRA expression was regained in RTOC, however, the presence of thymocytes did not further augment this effect. These results show that Aire and TRA expression is dependent on the three-dimensional structure of epithelial microenvironment. This expression, however, seems to be independent of the presence of thymocytes being in line with previous data demonstrating the Aire expression signal in RAG deficient and CD3etg26 transgenic mice, in which T-cell development is blocked (Jenkinson et al., 2005; Zuklys et al., 2000).\nIn conclusion, we show that Aire has a dose-dependent effect on TRA expression in thymus but not in the lymph nodes. Both, Aire as well as TRAs localize in the thymic medulla and are co-expressed during normal development and involution. We also show that Aire can directly induce TRA expression in medullary epithelial cells although the thymic microenvironment plays a crucial role for the maximal expression to occur. Our data suggest a clear correlation between the expression of Aire and TRAs and indicate that approaches to stimulate Aire expression in thymic epithelium could be considered to modulate tolerance induction to peripheral antigens.","keyphrases":["tissue-restricted antigens","autoimmune regulator","autoimmunity","thymus"],"prmu":["P","P","P","P"]}
{"id":"Eur_J_Nutr-2-2-1705483","title":"Diet supplementation for 5 weeks with polyphenol-rich cereals improves several functions and the redox state of mouse leucocytes\n","text":"Background Cereals naturally contain a great variety of polyphenols, which exert a wide range of physiological effects both in vitro and in vivo. Many of their protective effects, including an improvement of the function and redox state of immune cells in unhealthy or aged subjects come from their properties as powerful antioxidant compounds. However, whether cereal-based dietary supplementation positively affects the immune function and cellular redox state of healthy subjects remains unclear.\nIntroduction\nNowadays, consumption of natural products is a matter of major importance as regards health. Indeed, consumption of fruit and vegetables was already associated with a reduced risk of chronic diseases and age-related functional decline several years ago [1]. Nevertheless, less attention has been focused on the importance of phytochemicals from cereals in maintaining health. It is well known that the nutritional status is a central factor contributing to immune system competence [2] and that an adequate function of the immune system is related to health promotion and disease prevention [3]. Nutrients that have traditionally been shown to be required for correct immune system function include different types of vitamins, zinc, copper, and selenium. However, there is an increasing amount of data showing the implication of the so-called \u201cnon-nutrients\u201d, i.e., phytochemicals, in the immune system responses [4]. Further, the phytochemicals found in natural products have been involved in protecting against a wide range of oxidative stress-related chronic diseases [5]. The overproduction of oxidants and the overwhelming of the antioxidant defence lead to oxidative stress, that can cause oxidative damage to large biomolecules, such as proteins, DNA, and lipids, resulting in an increased risk of disease [5, 6]. For this reason, to forestall an excessive production of reactive oxygen species (ROS), sufficient amounts of antioxidants need to be consumed. However, the generation of ROS is a key part of normal immune system function [7]. These molecules play a key role in the defence against infectious agents and seem to be involved in regulating many biological processes, serving as second messengers [8]. Thus, it is important to maintain a balance between ROS generation and antioxidant defences within immune cells to ensure a correct functioning of the immune system and help to prevent the onset of chronic diseases.\nCereals naturally contain a wide variety of polyphenols such as the hydroxycinnamic acids, ferulic, vanillic, and p-coumaric acids [9], which show a strong antioxidant power and may help to protect from oxidative stress and, therefore, can decrease the risk of contracting many diseases. Flavonoids are present in small quantities, even though their numerous biological effects and their implications for inflammation and chronic diseases have been widely described [10, 11]. The mechanisms of action of polyphenols go beyond the modulation of oxidative stress-related pathways [6]. Cereal phytochemicals exert their health benefits through multifactorial physiological mechanisms, including enhancement of the immune system and hormones, facilitation of substance transit through the digestive tract, metabolism, modulation and antioxidant, antibacterial and antiviral activities [12].\nIt is known that unhealthy or aged subjects are more susceptible to infection, showing an impairment of function and redox state of immune cells and that such impairment can be counteracted by antioxidant administration [13, 14]. However, whether dietary antioxidant supplementation affects the functional and redox state of immune cells of healthy subjects remains unclear. Not all subjects are likely to benefit in the same way from supplementation, i.e., there are certain groups within the apparently healthy, general population that benefit from adequate intakes of certain compounds [15].\nSince there is an increasing interest in consuming products naturally rich in phytochemicals able to provide health benefits and prevent a wide variety of diseases, in this context we have investigated in the present work the effects of diet supplementation for 5\u00a0weeks with different types of cereal fractions naturally rich in polyphenols on certain parameters of immune function and redox state of peritoneal leukocytes from adult healthy mice.\nMaterial and methods\nAnimals\nOne hundred-fifty female ICR (CD-1) mice (Mus musculus) (Harlan Ib\u00e9rica, Barcelona, Spain), which were 24\u00a0weeks old on arrival to our laboratory, were used. Mice were checked periodically by animal care personnel and were specific pathogen free, as tested by Harlan according to FELASA recommendations, and did not show any sign of malignancy or other pathological processes. The animals were randomly housed in polyurethane boxes, at a constant temperature (22\u00a0\u00b1\u00a02\u00b0C) in sterile conditions inside an aseptic air negative-pressure environmental cabinet (Flufrance, Cachan, France), on a 12\/12\u00a0h reversed light\/dark cycle. All animals were fed water and standard Sander Mus (A04 diet from Panlab L.S., Barcelona, Spain) pellets ad libitum. The diet was in accordance with the recommendations of the American Institute of Nutrition for laboratory animals. Mice were treated according to the guidelines of the European Community Council Directives 86\/6091 EEC. Although we have previously observed that the oestrous cycle phase of mice has no effect on this experimental assay, all females used in the present work were at the beginning of dioestrous.\nExperimental groups\nThe animals were marked for their individual follow-up. Five experimental groups, one control (n\u00a0=\u00a030) and four supplemented (30 mice per group), were used. The supplementation started at 28\u00a0weeks of age. Then, the supplemented groups were fed for a period of 5\u00a0weeks, and at the age of 33\u00a0\u00b1\u00a02\u00a0weeks samples were obtained to evaluate certain parameters of function and redox state of peritoneal leukocytes. The experiments were carried out in January at a constant laboratory temperature of 25\u00a0\u00b1\u00a02\u00b0C. Food intake was measured weekly at the same time (9:00\u201311:00\u00a0h). Each animal was fed weekly 50\u00a0g of diet (control or supplemented) and, after 1\u00a0week, the uneaten remains were weighed. Body weights were determined once every week. No significant differences on food consumption and body weight among diet groups during the study were observed.\nCereal products and preparation of extracts\nAll cereal products (wheat germ, buckwheat flour, fine rice bran and wheat middlings, named B, C, D and E; respectively) used in this study were provided by Danone Vitapole (France). The samples were stored and kept at \u221220\u00b0C prior to analysis. Moisture content of the cereal samples was evaluated by drying at 130\u00b0C for 90\u00a0min. The analyses were done in duplicate. The different extracts were analyzed by Danone Vitapole (France). Briefly, the cereal products were sequentially extracted with water at 50\u00b0C followed by 80% methanol and ethyl acetate. Extraction with each solvent was carried out twice for 30\u00a0min under N2, in the dark and constant agitation (200\u00a0rpm). The ethyl acetate extracts were evaporated under vacuum and re-dissolved in 80% methanol. The final insoluble pellet was dried. The soluble fractions and the insoluble pellet were stored at 4\u00b0C (in the dark and under N2) prior to analysis of polyphenols and determination of antioxidant activity.\nAnalysis of total polyphenols and antioxidant activity\nThe amount of total phenolic compounds in all extracts was determined by Danone Vitapole (France), according to the Folin\u2013Ciocalteu method [16]. The total phenolic concentration is calculated from a standard curve using a range of concentrations of gallic acid. The results are expressed as milligrams of gallic acid equivalents per gram of material. The antioxidant in vitro activity of the extracts obtained from selected cereal products was evaluated using an Oxygen Radical Absorbance Capacity (ORAC) procedure (Brunswick Laboratories, Wareham, MA, USA). Values are expressed as \u03bcmol of Trolox equivalents (TE) per g of dry matter. This assay was conducted in triplicate. There was also a positive correlation between ORAC and total polyphenol content (r2\u00a0=\u00a00.843).\nPreparation of diets\nThe control group only received a standard maintenance diet (AO4 diet from Panlab L.S. Barcelona, Spain; see Table\u00a01) and the remaining groups received 80% of control diet plus 20% of cereal fractions naturally rich in polyphenols (B, C, D and E; see Table\u00a01). Control and supplemented diets were prepared by mixing the standard maintenance diet or one of the cereal fractions with water, forming a biscuit-shape preparation. Both types of preparations were daily turned upside down and maintained in a dark room for 3\u00a0days to obtain a dry preparation. Then, the humidity of the different diets was measured and each of them was stored at 4\u00b0C for less than 3\u00a0weeks, avoiding exposure to light in order to prevent their oxidation.\nTable\u00a01Nutritional composition of standard (control) and experimental (B, C, D and E) dietsDiet ingredientsa (mg\/100g)ControlBCDEHFLFbHFLFbLFbLFbHFLFbPolyphenols\u00a0\u00a0Gallic acid\u2014\u2014\u2014\u20140.49\u2014\u2014\u2014\u2014\u00a0\u00a0Catechin\u2014\u2014\u201410.80.192.043.34\u2014\u2014\u00a0\u00a0p-HB acid\u20140.270.99\u20140.78\u2014\u20141.04\u2014\u00a0\u00a0Vanillic acid\u20140.5413.191.82\u20141.93\u20140.860.46\u00a0\u00a0p-coumaric acid\u2014\u2014\u20140.210.161.2818.910.060.68\u00a0\u00a0Sinapic acid\u2014\u2014\u2014\u20140.454.73\u2014\u2014\u2014\u00a0\u00a0Ferulic acid\u201425.9755.600.420.0911.7569.1923.8363.06\u00a0\u00a0Quercetin\u2014\u20140.17a\u20142.42a\u2014\u2014\u2014\u2014\u00a0\u00a0Rutin\u2014\u20143.94a\u201411.85a\u2014\u2014\u2014\u2014\u00a0\u00a0Oryzanol\u2014\u2014\u2014\u2014\u20144.03a\u2014\u2014Vitamins\u00a0\u00a0Vitamin A0.45\u2014\u2014\u2014\u2014\u00a0\u00a0Vitamin D30.0038\u2014\u2014\u2014\u2014\u00a0\u00a0Vitamin E213.650.21.335.55\u00a0\u00a0Thiamin0.11.870.411.811.48\u00a0\u00a0Riboflavin0.4\u2014\u2014\u2014\u2014\u00a0\u00a0Niacin2\u2014\u2014\u2014\u2014\u00a0\u00a0Pantothenic acid1.5\u2014\u2014\u2014\u2014\u00a0\u00a0Pyridoxin0.40.870.20.180.68\u00a0\u00a0Biotin0.01\u2014\u2014\u2014\u2014\u00a0\u00a0Folacin0.12914569103\u00a0\u00a0Cobalaminec2\u2014\u2014\u2014\u2014\u00a0\u00a0Choline50\u2014\u2014\u2014\u2014Fatty acids\u00a0\u00a0Palmitic acid460\u2014\u2014\u2014\u2014\u00a0\u00a0Palmitoleic acid39\u2014\u2014\u2014\u2014\u00a0\u00a0Stearic acid130\u2014\u2014\u2014\u2014\u00a0\u00a0Oleic acid650\u2014\u2014\u2014\u2014\u00a0\u00a0Linoleic acid1,390\u2014\u2014\u2014\u2014\u00a0\u00a0Linolenic acid130\u2014\u2014\u2014\u2014Minerals\u00a0\u00a0Calcium88025107862\u00a0\u00a0Phosphorum590\u2014\u2014\u2014\u2014\u00a0\u00a0Chloride310\u2014\u2014\u2014\u2014\u00a0\u00a0Sodium250\u2014\u2014\u2014\u2014\u00a0\u00a0Potasium600\u2014\u2014\u2014\u2014\u00a0\u00a0Magnesium140259174910203Proteinsd15.433.611.521.316.85Carbohidratesd101.521.167.47.428Fibred3.97.73.140.429.3Each control (non-supplemented) animal received a 100% of A04 maintenance diet (Panlab, Barcelona, Spain). Each supplemented animal received an 80% of A04 maintenance diet plus 20% of cereal fractions (B, C, D or E) naturally rich in polyphenols. B\u00a0=\u00a0wheat germ; C\u00a0=\u00a0buckwheat flour; D\u00a0=\u00a0fine rice bran; E\u00a0=\u00a0wheat middlings. ORAC (\u03bcTrolox Equivalents\/g): B\u00a0=\u00a0106; C\u00a0=\u00a081; D\u00a0=\u00a0121; E\u00a0=\u00a049. Total polyphenols content (eq mg gallic acid\/g): B\u00a0=\u00a011.15; C\u00a0=\u00a08.48; D\u00a0=\u00a015.24; E\u00a0=\u00a09.35. HF (mg\/100g)\u00a0=\u00a0hydrosoluble fraction; LF (mg\/100mg)\u00a0=\u00a0lyposoluble fraction; p-HB\u00a0=\u00a0p-hydroxybenzoic. amg\/100\u00a0g. bmg\/100\u00a0mg. c\u03bcg\/100\u00a0g. dg\/100\u00a0g\nCollection of peritoneal leukocytes\nPeritoneal suspensions were obtained between 8:00 and 10.00\u00a0h, without sacrificing the animals, following the method previously described [17]. Peritoneal leukocytes (macrophages and lymphocytes) were collected, identified, quantified by their morphology using optical microscopy (40\u00d7) and their concentration was adjusted depending on the function assayed [14, 18]. The cellular viability, determined in each experiment using the trypan-blue exclusion test, was in all cases higher than 95%.\nLeukocyte functions\nThe chemotaxis assays were performed according to a technique previously described [14]. The results were expressed as chemotaxis index (C.I.) by counting the number of macrophages and lymphocytes at the lower face of the filters of the Boyden\u2019 chambers [14]. The intracellular superoxide anion (O2\u00b7\u2212) levels were evaluated as described elsewhere [17]. The results were expressed as nmol of O2\u00b7\u2212 per 106 cells. The lymphoproliferation assay and the determination of interleukin-2 (IL-2) levels were carried out following the methods previously described [14]. The proliferation results were expressed as percentage of stimulation by the mitogens Concanavalin A (Con A) or lipopolysaccharide (LPS) with respect to spontaneous proliferation that was considered to be 100. In the case of IL-2 the results were expressed as pg\/ml.\nLeukocyte redox state\nTotal glutathione and its oxidized form (GSSG) were spectrophotometrically evaluated according to [17]. The results are expressed as nmol\/106cells. To calculate the GSSG\/GSH ratio, the GSH (reduced form) was obtained by substracting the GSSG values from the total glutathione values. The secretion of mouse tumor necrosis factor alpha (TNF\u03b1) was measured in culture supernatants stimulated with LPS, using an ELISA kit (HyCult Biotechnology, Uden, The Netherlands). The results were expressed as pg\/ml, being the minimum detectable value 8\u00a0pg\/ml. Catalase activity (CAT) was carried out according to [19], slightly modified. Samples of peritoneal suspension (adjusted to 106 cells\/ml) were resuspended in 50\u00a0mM phosphate buffer, sonicated and centrifuged at 3200\u00a0g (4\u00b0C) for 20\u00a0min. The reaction was initiated by the addition of H2O2 and spectrophotometrically measured at 240\u00a0nm (25\u00b0C) for 50\u00a0s. The results were expressed as U\/106 cells. Malondialdehyde (MDA) levels were evaluated according to a technique previously described [20], slightly modified, and quantified by high performance liquid chromatography (HPLC). Aliquots of peritoneal suspension (adjusted to 106 cells\/ml) resuspended in 50\u00a0mM KH2PO4 (Sigma) pH 6.8 were sonicated and added to a mixture reaction that contained 0.44\u00a0M ortophosphoric acid (H3PO4, Fluka, Steinheim, Germany), 2-thiobarbituric acid (0.6%), (TBA, Sigma) and 3\u00a0mM butylated hydroxytoluene (BHT, Sigma). It was heated at 95\u00b0C for 30\u00a0min and after cooling on ice, the samples were extracted adding n-butanol (Panreac), centrifugated at 13,000\u00a0rpm (4\u00b0C) for 5\u00a0min and 50\u00a0\u03bcl of supernatants (organic phase) were injected in the HPLC column (Novapack C18, 15\u00a0cm\u00a0\u00d7\u00a03.9\u00a0mm, Waters). As mobile phase 50\u00a0mM KH2PO4 pH 6.8\/methanol (90\/10 v\/v) was used. The flow rate of the mobile phase was adjusted to 0.4\u00a0ml\/min. MDA was monitored at \u03bb\u00a0=\u00a0532\u00a0nm in a WATERS 486 ultraviolet detector. Standard curves were prepared fresh daily using MDA bis (dimethyl acetal, Sigma). The results were expressed as nmol\/106cells.\nStatistical analysis\nStatistical analysis was performed using the software SPSS 11.5 (Chicago, IL, USA). The data are expressed as the mean\u00a0\u00b1\u00a0standard deviation (S.D.) of the eight values corresponding to the number of experiments. Each value is the mean of the data from assays performed in duplicate. The normality of the samples was confirmed by the Kolmogorov\u2013Smirnov test and the homogeneity of variances by the Levene test. The data were analysed by the one-way analysis of variance (ANOVA) followed by the Tukey test. The Kruskall Wallis test for non-parametric data was used. The minimum level considered statistically significant was P\u00a0<\u00a00.05.\nResults\nImmune system response\nFigure\u00a01 shows the chemotaxis capacity, lymphoproliferative response to the T-cell mitogen Concanavalin A (ConA) or the B-cell mitogen LPS and release of IL-2 of peritoneal lymphocytes from adult female mice supplemented for 5\u00a0weeks with cereal fractions naturally rich in polyphenols. Mice supplemented with cereals showed a strongly significant increase of lymphocyte chemotaxis compared with that of control mice (P\u00a0<\u00a00.001 with B, D and E and P\u00a0=\u00a00.031 with C) (Fig.\u00a01a). As regards lymphoproliferative response to Con A (Fig.\u00a01b), mice supplemented with any of the cereals B (P\u00a0=\u00a00.027), C (P\u00a0=\u00a00.002), D (P\u00a0<\u00a00.001) or E (P\u00a0<\u00a00.001) showed a significantly higher capacity compared with that of control mice. The supplementation with the cereals C (P\u00a0<\u00a00.001), D (P\u00a0=\u00a00.024) or E (P\u00a0<\u00a00.001), but not with B, was able to increase lymphoproliferation in response to LPS with respect to that of non-supplemented mice (Fig.\u00a01c). The secretion of IL-2 by peritoneal leukocytes from mice supplemented with cereal B (P\u00a0<\u00a00.001), C (P\u00a0=\u00a00.004) or E (P\u00a0=\u00a00.006) was higher than that of control mice (Fig.\u00a01d). The chemotaxis capacity of macrophages increased significantly in cereal E-supplemented mice in comparison with that of control mice (434\u00a0\u00b1\u00a039 versus 290\u00a0\u00b1\u00a042, P\u00a0<\u00a00.001). The intracellular superoxide anion (O2\u00b7\u2212) levels in non-stimulated peritoneal leukocytes significantly increased after supplementation for 5\u00a0weeks with cereal B and C with respect to non-supplemented mice (Controls: 24\u00a0\u00b1\u00a05; B: 42\u00a0\u00b1\u00a07, P\u00a0<\u00a00.001; C: 41\u00a0\u00b1\u00a07, P\u00a0<\u00a00.001). The release of intracellular O2\u00b7\u2212 from peritoneal leukocytes stimulated with latex beads was significantly higher in mice supplemented with cereals B, C and E (Controls: 52\u00a0\u00b1\u00a05; B: 63\u00a0\u00b1\u00a04, P\u00a0=\u00a00.045; C: 67\u00a0\u00b1\u00a08, P\u00a0=\u00a00.002 E: 64\u00a0\u00b1\u00a07, P\u00a0=\u00a00.021).\nFig.\u00a01(a) Chemotaxis capacity of peritoneal lymphocytes, (b) lymphoproliferation in ConA (1\u00a0\u03bcg\/ml)-stimulated 48\u00a0h-culture supernatants, (c) lymphoproliferation in LPS (1\u00a0\u03bcg\/ml)-stimulated 48\u00a0h-culture supernatants and (d) release of interleukin-2 (IL-2) in supernatants of 48\u00a0h-cultures of peritoneal leukocytes stimulated with Con A (1\u00a0\u03bcg\/ml) from adult female ICR (CD-1) mice supplemented for 5\u00a0weeks with cereal fractions (B, C, D or E) naturally rich in polyphenols. Bars show the mean\u00a0\u00b1\u00a0SD of eight values, corresponding to the same number of experiments, being each value the mean of duplicate assays. *P\u00a0<\u00a00.05 **P\u00a0<\u00a00.01 and ***P\u00a0<\u00a00.001 versus the control group. Lymphoproliferation in response to both mitogens, Con A and LPS, was expressed as % of stimulation with respect to spontaneous proliferation, the latter considered as 100. Spontaneous lymphoproliferation (in counts per min): Control\u00a0=\u00a01245\u00a0\u00b1\u00a0253; B\u00a0=\u00a01348\u00a0\u00b1\u00a0239; C\u00a0=\u00a01071\u00a0\u00b1\u00a0171; D\u00a0=\u00a01186\u00a0\u00b1\u00a0166; E\u00a0=\u00a01052\u00a0\u00b1\u00a0169\nCellular redox state\nThe content of oxidized glutathione (GSSG) and the GSSG\/GSH ratio, the levels of TNF\u03b1, CAT activity and MDA levels in peritoneal leukocytes are shown in Fig.\u00a02. All cereal fractions studied decreased significantly the GSSG levels (P\u00a0<\u00a00.001) compared to those of control mice (Fig.\u00a02a). The GSSG\/GSH ratio (Fig.\u00a02b) was significantly decreased after supplementation with any of the cereals B (P\u00a0<\u00a00.001), D (P\u00a0=\u00a00.002) or E (P\u00a0<\u00a00.001) compared with that of non-supplemented mice. Supplementation for 5\u00a0weeks with any of the cereals did not cause changes in the GSH levels of peritoneal leukocytes with respect to the values of controls (2.29\u00a0\u00b1\u00a00.23). The secretion of TNF\u03b1 decreased in leukocytes from mice supplemented with cereal C (P\u00a0=\u00a00.005), D (P\u00a0=\u00a00.007) and E (P\u00a0=\u00a00.004) in comparison with that of non-supplemented mice (Fig.\u00a02c). The CAT activity of peritoneal leukocytes from mice supplemented with cereal C (P\u00a0<\u00a00.001), D (P\u00a0=\u00a00.003) and E (P\u00a0=\u00a00.040) was higher than the activity of this antioxidant enzyme in control mice (Fig.\u00a02d). Finally, the lipid oxidative damage in peritoneal leukocytes, evaluated as MDA levels (Fig.\u00a02e), was lower (P\u00a0<\u00a00.001) in mice supplemented for 5\u00a0weeks with cereals than in the control mice.\nFig.\u00a02(a) Oxidized glutathione (GSSG) content, (b) GSSG\/GSH ratio, (c) tumor necrosis factor (TNF\u03b1) levels in LPS-stimulated (1\u00a0\u03bcg\/ml) culture supernatants, (d) catalase (CAT) activity and (e) malondialdehyde (MDA) levels in peritoneal leukocytes from adult female ICR (CD-1) mice supplemented for 5\u00a0weeks with cereal fractions (B, C, D or E) naturally rich in polyphenols. Bars show the mean\u00a0\u00b1\u00a0SD of eight values, corresponding to the same number of experiments, being each value the mean of duplicate assays. *P\u00a0<\u00a00.05 **P\u00a0<\u00a00.01 and ***P\u00a0<\u00a00.001 versus the control group\nDiscussion\nIt is widely accepted that antioxidant compounds may help to protect against oxidative stress, with resulting health promotion and disease prevention [21]. Moreover, an adequate intake of these compounds can enhance certain aspects of immune function, which is related to health [4]. Thus, it is becoming clear that diet supplementation with appropriate amounts of antioxidants enhances the immune function and improves the redox state of unhealthy subjects [15]. However, whether antioxidant supplementation has these effects in healthy subjects, still remains unknown. In the present study the cereal fractions used were able to improve several aspects of immune function. Thus, important events of innate immune response, i.e., migration capacity (chemotaxis) and microbicidal activity (intracellular superoxide anion levels), appear to be effectively affected by the supplementation with cereals naturally rich in polyphenols. Akbay et al. [22] showed that the three flavonoid glycosides from Urtica dioica L., quercetin-3-O-rutinoside, kaempherol-3-O-rutinoside and isorhamnetin-3-O-glucoside increased, in vitro, the chemotaxis and the intracellular killing activity of neutrophils, using methods similar to those of the present work. In our study, the fraction E is only able to enhance macrophage chemotaxis, whereas all the cereal fractions were able to show a strong stimulatory effect on this function in lymphocytes. This fact suggests that the polyphenols present in cereal fractions could exert a cell-dependent effect on migration capacity. Since a higher NF-\u03baB expression is linked to an increased production of migratory inhibitory factor (MIF), thus being responsible for a decreased migration capacity [13], and several polyphenols are able to inhibit NF-\u03baB activation [23], it seems plausible that some of the polyphenols present in the cereals used in our study could enhance the chemotaxis capacity through their action on NF-\u03baB activation. All cereal fractions used in the present work, with the exception of D, have been shown to increase the microbicidal activity of peritoneal leukocytes. Since p-hydroxybenzoic acid is not present in fraction D, this compound could be responsible for that effect. However, to date, there is no evidence of a microbicidal activity increase following p-hydroxybenzoic acid administration.\nLymphoproliferation is a crucial event of the acquired immune response. Several studies have demonstrated a strong stimulatory effect on this capacity and on the cytokine well-known as enhancer of proliferative response, namely the IL-2, after antioxidant supplementation. Thus, Bub et al. [4] showed an increase in both functions in peripheral blood mononuclear cells (PBMC) from healthy subjects after diet supplementation with fruit juices rich in polyphenolic compounds. In ethanol-fed mice, Cheshier et al. [24] demonstrated an increased IL-2 production by mitogen-stimulated splenocytes after in vitro administration of Pycnogenol\u00ae, a commercial combination of phenolic compounds from pine bark. To date, no studies on the effect of the isolated polyphenolic compounds of the cereals used in the present research on these functions have been carried out.\nGlutathione (GSH) plays important roles in antioxidant defence, nutrient metabolism and regulation of many cellular events, and its deficiency contributes to the oxidative stress-related pathogenesis of several chronic diseases [25]. Both the oxidized glutathione (GSSG) content and the GSSG\/GSH ratio, the latter being considered a sensitive marker of cellular oxidative stress [26], were significantly decreased after supplementation with all the cereal fractions, although fraction C only showed a tendency to diminish the GSSG\/GSH ratio. However, the GSH content of peritoneal leukocytes was not changed after cereal-based supplementation, suggesting that the lower GSSG\/GSH ratio observed is primarily due to diminutions of the GSSG content. Rosenblat et al. [27] demonstrated that isoflavonoid glabridin (25\u00a0\u03bcg\/kg\/day) supplementation was able to reduce the GSSG content in murine peritoneal macrophages. However, the same authors also observed increased GSH levels in these immune cells.\nTumor necrosis factor is one of the most common proinflammatory cytokines produced predominantly by macrophages that has been reported to increase in many oxidative stress-related chronic diseases [28]. Our study showed that each of the cereal fractions, except B, was able to decrease the secretion of TNF\u03b1 by peritoneal leukocytes. In agreement with Wang and Mazza [29], catechin at a concentration of 31\u00a0\u03bcM inhibited the TNF\u03b1 production of LPS\/IFN\u03b3-activated RAW 264.7 macrophages. However, our results suggest that p-coumaric acid could also have an important anti-inflammatory role because this common phenolic acid is present in each cereal fraction, except the B. Moreover, in rats with endotoxemia, two different doses (75, 150\u00a0mg\/kg, i.p.) of a water extract of propolis, a natural product rich in p-coumaric acid, were able to inhibit TNF\u03b1 production in plasma [30]. Nevertheless, other authors did not observe any effect on TNF\u03b1 production in whole blood cultures in the presence of phenolic compounds like p-coumaric acid at the concentrations of 10\u22127\u201310\u22124\u00a0M [31]. The most probable mechanism triggered by phenolic compounds for blocking LPS-induced production of TNF\u03b1 by macrophages could be the inhibition of NF-kB activation [23].\nCatalase plays a fundamental role in protecting cells against oxidative damage [32]. Decreased CAT activity may compromise the overall enzymatic defence system. In the present study, each of the cereal fractions increased CAT activity in peritoneal leukocytes, although fraction B only showed a tendency to increase this enzymatic activity. In other study, the renal CAT activity was significantly increased in rats fed black rice, one of the most important cereals rich in phenolic acids [33]. Khan et al. [34] showed that following oral feeding (0.2% w\/v) for 30\u00a0days of polyphenols from green tea, a popular beverage rich in catechin, CAT activity significantly increased in the small bowel, liver and lungs of mice. In fact, fractions C and D (the fractions rich in catechin) showed the greatest effect. The present data suggest that p-coumaric acid, which is present in almost all the cereal fractions (C, D and E) might also have a relatively important role in modulating the CAT activity. Indeed, an oral administration of p-coumaric acid (100\u00a0mg\/kg) for five days ameliorated the CAT activity in the heart from rats treated with doxorubicin, an anticancer antibiotic [35]. This potential protective action of p-coumaric acid is likely to be due to its free radical scavenging power.\nROS generation is able to promote oxidative damage to fatty acids present in biological membranes via an autocatalytic process known as lipid peroxidation. Malondialdehyde is one of the end-products of this process [36], which is related to the onset of chronic diseases [37]. In the present work, all the cereal fractions strongly decreased the MDA levels in peritoneal leukocytes. It has been observed that highly reactive and destructive hydroxyl radicals are effectively scavenged by oral administration of p-coumaric acid (317\u00a0mg\/day) for 30\u00a0days in rats [38], which might decrease the potential damage to biological membranes by lowering MDA production. Several authors have shown that rutin, quercetin or catechin, which are some of the flavonoids present in the cereals used in the present work, are able to decrease lipid oxidative damage [39, 40]. The same happens with ferulic acid, the most common phenolic acid in cereal cell walls, which inhibit peroxidation [41]. Red wine polyphenols, together with ascorbic acid, inhibit lipid peroxidation in human muscle tissue [42]. The same authors demonstrated that, in the presence of catechin, ascorbic acid at pH 3.0 works in a sinergystic way preventing lipid oxidative damage and \u03b2-carotene cooxidation. This fact might explain the central role that consuming natural products rich in polyphenolic antioxidants, like cereals, has in promoting health and preventing the onset of chronic diseases.\nThe present data demonstrate the importance of polyphenol-rich cereal consumption because not only has shown to be very effective for modulating several parameters of immune function, but also is able to improve to a large extent the redox state of immune cells from mice. More importantly, we have demonstrated that cereal-based antioxidant supplementation can exert beneficial effects in healthy mice and thus we suggest that normal healthy populations may benefit from this supplementation. Indeed, a recent study has provided evidence for potential protective effects of moderate consumption of polyphenol-rich red wine in healthy subjects [43]. It has been reported that subjects susceptible to infections or suffering from chronic inflammatory processes showing an impairment of function and redox state of immune cells likely to benefit from antioxidant administration [13, 14]. However, it is difficult to know whether there is changes or not in the healthy mice from the present study may be a reliable predictor for the nutrient-immune requirements in disease-associated, unhealthy mice. To confirm this possibility we should know the optimal combination and quantities of nutrients, the potential interactions that might occur between these nutrients and the most appropiate time at which that immunonutrition should be provided. In words of Berger [15], \u201cthe efficiency of supplementation is a question of timing. Antioxidant nutrients cannot cure an installed disease, but they may prevent its promotion\u201d.\nIt is important to point out that the cereal fractions used by us contain other compounds, both nutrients and non-nutrients, such as vitamins, minerals or fibre and, therefore, we cannot rule out that they can result in health benefits. However, the present cereal fractions show a good correlation coefficient between total antioxidant capacity and content of phenolic compounds, thus suggesting that polyphenols are largely responsible for the antioxidant activity of the different cereal fractions, which ultimately may help to protect from oxidative stress and, consequently, can decrease the risk of many potential diseases. Nevertheless, it is important to take into account some issues. First, the doses used play a crucial role, since it is known that excessive amounts of antioxidant compounds can impair the immune function [44] or even becomes potentially prooxidants [45]. Yet, we have not used \u201cmegadoses\u201d, but nutritional doses of polyphenols. Second, the present recommendation is not a single polyphenol-based supplementation to modulate the antioxidant status or the immune system, but it is the combination of different substances as the best way to provide protective action [46, 47]. Therefore, the cereals used by us contain a complex natural mixture of polyphenols of different size, polarity and solubility. Third, we can speculate that one of the reasons for the protective effects of cereals could be that the metabolism of polyphenols carried out by the gut microflora [48] is able to provide immune protection beyond the gut. Fourth, it should not be ruled out the possibility that synergistic and\/or additive effects occur. Several studies have shown that polyphenols can work synergistically with other antioxidant compounds [42, 49]. Thus, it is of great physiological importance that many polyphenols of different solubility will interact in the body cells and compartments, with antioxidants recharging adjacent antioxidants in an integrated manner [50]. Finally, we consider the present nutritional intervention in animals as preventive, but not therapeutic, since it is able to maintain the normal functional and redox status of the body cells from a general population of mice. Therefore, we do not deliver a cereal-based antioxidant supplement to unhealthy subjects, i.e., in conditions caused by excessive free radicals generation. In conclusion, we strongly believe that polyphenols naturally present in cereals may have important implications for health preservation, by acting, al least partly, as modulators of immune function and redox state. Although we emphasize the importance of further studies on this subject, we suggest the use of this type of supplementation for the general population since \u201cprevention is a much more effective approach that treatment of disease\u201d.","keyphrases":["polyphenols","health promotion","peritoneal leukocytes","leukocyte functions","leukocyte redox state"],"prmu":["P","P","P","P","P"]}
{"id":"Psychopharmacologia-3-1-2080349","title":"Medial prefrontal serotonin in the rat is involved in goal-directed behaviour when affect guides decision making\n","text":"Rationale Across species, serotonin (5-HT) depletion in the prefrontal cortex (PFC) has been shown to cause impaired performance on tests of cognitive flexibility and the processing of affective information (e.g. information with an \u2018emotional\u2019 content). While recent work has explored the specific role of the orbital PFC herein, the role of the medial PFC remains unclear.\nIntroduction\nCertain aspects of cognitive flexibility (i.e. the ability to adapt behaviour, see for instance, Fuster 1980; Kolb 1984; De Bruin et al. 1994; Dalley et al. 2004) depend critically on central serotonin (5-HT). Recent experiments in humans with tryptophan depletion, a method resulting in a transient decrease in 5-HT synthesis, show impairments in the ability to learn changed stimulus\u2013reward associations (Rogers et al. 1999) and in the performance on tests of cognitive flexibility (Rubinsztein et al. 2001). Lesion studies in non-human primates corroborate these findings, showing that it is the 5-HT innervation of the prefrontal cortex (PFC) that is critical for adapting behaviour when stimulus\u2013reward contingencies are reversed in a two-choice discrimination (reversal learning; Clarke et al. 2004, 2005). Similar observations have been made after central 5-HT depletion in rats, indicating that 5-HT depletion leads to impaired flexibility (Harrison et al. 1999), increased perseverative responding (Beninger and Phillips 1979; Morgan et al. 1993) and impaired impulse inhibition (Winstanley et al. 2004, 2006).\nIn addition to effects on cognitive flexibility, tryptophan depletion has also been reported to lead to impairments in affective processing (i.e. the ability to evaluate and integrate emotional content, see for instance, Hariri et al. 2006) and reward discrimination (Rubinsztein et al. 2001; Rogers et al. 2003; see also Roberts and Wallis 2000). Such changes in affective processing (see also Remijnse et al. 2005) have, however, not yet been reported in studies involving selective prefrontal 5-HT lesions in either primates or in rats. Elucidation of the neural substrates underlying this special role of 5-HT in affective processing is one aim of the current study.\nA second issue that is central to studies of the PFC in cognitive flexibility is that of specific contributions of the different cortical sub-areas. Evidence for a functional dissociation within the PFC regarding cognitive flexibility comes from studies across species, and it is important to note that the functional differentiation (specificity) of the anatomical subdivision of the rat brain PFC is, to a large extent, comparable to that of the primate PFC (Uylings et al. 2003). In terms of homology between the primate and rodent PFC, we believe, given the data reviewed in Uylings et al. (2003), that the rat medial PFC (mPFC) shows a functional overlap with both lateral and medial frontal regions of the primate brain.\nThe orbital PFC has been shown to support reversal learning in both primates (Iversen and Mishkin 1970; Butters et al. 1973; Dias et al. 1996; Clarke et al. 2004; Kringelbach et al. 2003; Hornak et al. 2004) and rats (Schoenbaum et al. 2002; Chudasama and Robbins 2003; McAlonan and Brown 2003) and is implicated in the coding of reward-related information in non-human primates (Hikosaka and Watanabe 2000; Tremblay and Schultz 2000; Schultz 2006), rats (Schoenbaum et al. 2003; Bohn et al. 2003) and humans (O\u2019Doherty et al. 2001; Kringelbach 2005).\nThe mPFC, on the other hand, supports rule switching and attentional set shifting in rats (De Bruin et al. 1994; Joel et al. 1997; Ragozzino et al. 1999a,b; Birrell and Brown 2000). This functional dissociation between reversal learning and attentional set shifting is also observed in primates, in which the latter is dependent on the lateral PFC (Dias et al. 1996; see also Robbins 2005). However, this particular distinction between PFC functions might be rather specific to visual- and odour-based learning in rats (Chudasama and Robbins 2003; Schoenbaum et al. 2002) and primates (Clarke et al. 2005, 2007), as several groups have reported an involvement of the mPFC in reversal learning using spatial discriminations (Kolb et al. 1974; Li and Shao 1998; Salazar et al. 2004; De Bruin et al. 2000). Furthermore, the mPFC has been shown to be involved in the initial processing of reward-related information, as recording of single neuron activity indicated this area to be involved in the coding of reward information in relation to spatial cues (Hok et al. 2005). From this, we conclude that the mPFC may have a specific role in spatial reversal learning. On a more general level, these findings have been paralleled and extended by human studies. Knutson et al. (2005), for example, reported on mPFC-mediated reward coding, and evidence for a mPFC involvement in implementation of that (reward) knowledge in existing stimulus\u2013reward contingencies comes from Ridderinkhof et al. (2004).\nGiven the fact that 5-HT is involved in reversal learning supported by the orbital PFC (see above) and that 5-HT lesions lead to functional deficits such as perserverative responding and loss of impulse inhibition (Beninger and Phillips 1979; Soubri\u00e9 1986; Harrison et al. 1999; Dalley et al. 2002; Winstanley et al. 2004, 2006), which are also observed after lesioning of the mPFC (Morgan et al. 1993; Passetti et al. 2002; Salazar et al. 2004), we feel that investigation of the involvement of 5-HT in the mPFC in spatial reversal learning is warranted. This forms the second main aim of these experiments.\nGiven the primate literature on the involvement of 5-HT in affective processing and of the mPFC in the implementation of reward information, we hypothesise that the medial prefrontal 5-HT is selectively involved in cognitive flexibility when affect guides decision making (i.e. when \u2018emotional\u2019 content drives decision making). Moreover, based on the \u2018reversal\u2019 literature, we hypothesise that 5-HT in the mPFC is especially important for successful behavioural adaptation when the stimulus modality is spatial but not odour-based.\nTo test our hypotheses, we conducted an experiment in which we selectively destroyed 5-HT terminals in the mPFC of male Wistar rats by means of local infusion of the toxin 5,7-dihydroxytryptamine (5,7-DHT). Both sham controls and lesioned animals were then assessed on a series of behavioural tests in which functions thought to underlie cognitive flexibility and affective processing were addressed (De Bruin et al. 2000; Schoenbaum et al. 2006).\nTo explore the hypothesis that mPFC 5-HT is involved in affective processing, we tested the impact of altered reward value on reversal learning. For half of the sham and control animals, we switched the reward presented after a correct response from non-preferred reward pellets to preferred pellets at the time of the first reversal. Our expectation was that the introduction of this affective shift, on top of the reversal, would cause lesioned animals to show altered task performance because of an inability to accurately process the altered reward information.\nFor the hypothesis that stimulus modality determines specific involvement of the mPFC, we tested reversal learning across different stimulus modalities.\nIn line with our studies on mPFC lidocaine inactivation (De Bruin et al. 2000), we subjected the animals to a test of spatial discrimination and reversal learning. In addition, we included a second, operant, odour-based go\/no-go reversal task. Based on our hypothesis regarding selective involvement of the mPFC in spatial learning, we expected impaired reversal learning in the spatial but not in the odour-guided task in lesioned animals.\nTo gain more insight in the mechanisms that might underlie impaired cognitive flexibility, we tested the effect of the lesion on extinction learning and impulse inhibition, and we assessed the animals\u2019 behaviour during extinction of the \u2018spatial\u2019 task and responding on \u2018go\u2019 and \u2018no-go\u2019 trials in the \u2018odour\u2019 task.\nMaterials and methods\nAll experiments were approved of by the Animal Experimentation Committee of the Royal Netherlands Academy of Arts and Sciences and were carried out in agreement with Dutch Laws (Wet op de Dierproeven 1996) and European regulations (Guideline 86\/609\/EEC).\nSubjects\nSubjects were 32 male outbred Wistar rats (Harlan\/CPB, Horst, The Netherlands), weighing 175\u2013200\u00a0g upon arrival. The animals were socially housed in groups of four in standard type IV Macrolon cages, where they were kept under a reversed day\/night cycle (dimmed red light from 7\u00a0a.m. until 7\u00a0p.m., white light from 7\u00a0p.m. until 7\u00a0a.m.) for the duration of the experiment. After surgery, the animals were food restricted (16\u00a0g\/animal per day) to maintain their body weight at 90% of free-feeding weight. Water was available ad libitum throughout the experiment.\nSurgery\nTwo weeks after arrival, the animals were randomly assigned to either the experimental lesion groups or the sham groups and subjected to surgery. The aim was to selectively destroy the serotonergic (5-HT) innervation of the mPFC. To prevent loss of dopaminergic (DA) and noradrenergic (NA) terminals in the target area because of the treatment, all animals were intraperitoneally injected with 20\u00a0mg\/kg Desipramine HCl (Sigma, Zwijndrecht, The Netherlands) dissolved in MilliQ water (10\u00a0mg\/ml) 30\u00a0min before surgery. The weight of the animals at the time of surgery was 247\u2013289\u00a0g. Anaesthesia was induced with 5% isoflurane in oxygen and maintained for the duration of the surgery with 2% isoflurane. For the surgical procedure, the animals were mounted in a stereotactic frame with the toothbar set at \u22122.5\u00a0mm. Bilateral stainless steel cannulas (outside diameter 0.3\u00a0mm), connected to a microinfusion pump (801 syringe pump, Univentor, High Precision Engineering, Zejtun, Malta) with flexible PEEK tubing (0.51\u00a0mm outside diameter, 0.013\u00a0mm inside diameter; Aurora-Borealis, Schoonebeek, The Netherlands), were then placed in the mPFC at an angle of 12\u00b0, anterioposterior +30, lateral \u00b116; ventral \u221240 (Paxinos and Watson 1998).\nAfter cannula placement, 0.5\u00a0\u03bcl of either 5,7-DHT (5,7-DHT creatinine sulphate salt, Sigma; 32\u00a0\u03bcg\/\u03bcl, dissolved in 0.1% ascorbic acid) or vehicle was infused into the target area over a period of 1\u00a0min. After infusion, the cannulas were kept in place for an additional 2\u00a0min to allow for diffusion. The wound was closed with surgical stitches after the cannulas were removed from the brain. Post-operative pain reduction was achieved with Finadyne (flunixin meglumide 50\u00a0mg\/ml, Schering-Plough, Segr\u00e9, France), 0.01\u00a0ml\/100\u00a0g body weight subcutaneously, approximately 2\u00a0h after surgery. After surgery, the animals were returned to their home cages.\nApparatus\nTwo behavioural tasks were used. For the first (two-lever discrimination) task, locally constructed operant chambers were equipped with a set of two retractable levers positioned to the left and right of a food dispenser (distance lever\u2013feeder, 10\u00a0cm). A house light was placed on the opposite wall, two lights were positioned above the levers, signalling their presentation, and one inside the food dispenser, signalling the delivery of a 45\u00a0mg food pellet (Noyes Formula AI or P, Research Diets, New Brunswick, NJ). Nose pokes were detected by an infrared sensor located inside the food well. Operation of the Skinner boxes was controlled by a personal computer running the Med-pc\u2122 software (Med Associates, Sanddown Scientific, Middlesex, UK).\nFor the second (odour-based go\/no-go) task, commercially available operant chambers (Med Associates, Sanddown Scientific) were equipped with a retractable lever and an odour-sampling unit positioned on opposing walls (spaced 34\u00a0cm apart), allowing the presentation of distinct (non-aversive natural) odours. A food dispenser, from the same company, was positioned next to the odour-sampling port. Trial lights indicated odour, lever and reward presentation.\nPellet rewards\nTo manipulate the affective value of the rewards, we used two different types of pellet rewards (Noyes \u2122 AI and P; for additional information see www.researchdiets.com) that were shown to be differentially preferred by the animals (see \u201cPellet preference\u201d). Before the start of the experiment, all animals were familiarised with both types of pellets in their home cage. During the reversal phases of both tasks, the preferred pellet reward was only available to half of the animals (for groups, see below).\nPellet preference\nTo establish whether male Wistar rats showed preference for a specific food pellet, we pre-tested three types of rewards (Noyes \u2122 formula AI, P, and FP; see Fig.\u00a01) in a separate group of eight animals. The testing apparatus was a standard-size T-maze with trap doors in which both arms were baited. Five daily habituation sessions were given during which the animals were allowed to freely explore the entire T-maze and familiarise themselves to the position of the two different rewards that were tested that day. Subsequently, the animals were given the choice between the two arms, each baited with one of the three types of reward (15 trials). In these trials, the trap door was lowered after an arm entry was made to prevent the rat from entering the second arm. All three possible combinations of pellets were given over 3 consecutive days to all rats. To avoid \u2018sequence\u2019 effects, the rats were pseudo-randomly assigned to a particular pellet order. The animals were housed and food restricted as the experimental animals.\nTable\u00a01Results of HPLC analysis of sham and 5-HT-lesioned animals in seven brain areas\u00a0Treatment5-HTDANAmPFC\u201415,7-DHT63.2\u2009\u00b1\u20098.6 (9.1%)**35.5\u2009\u00b1\u20097.1 (66.9%)417.6\u2009\u00b1\u200941.1 (66.6%)**Sham691.5\u2009\u00b1\u2009119.553.1\u2009\u00b1\u20098.0627.0\u2009\u00b1\u200956.8mPFC\u201425,7-DHT68.8\u2009\u00b1\u200914.9 (28.0%)**60.9\u2009\u00b1\u200911.1 (93.0%)446.3\u2009\u00b1\u200941.8 (104.8%)Sham245.3\u2009\u00b1\u200946.565.5\u2009\u00b1\u20095.0425.8\u2009\u00b1\u200925.4mPFC\u201435,7-DHT42.5\u2009\u00b1\u20096.9 (18.0%)*71.1\u2009\u00b1\u200919.1 (70.3%)272.6\u2009\u00b1\u200923.0 (74.1%)Sham236.0\u2009\u00b1\u200970.1101.1\u2009\u00b1\u200933.7367.7\u2009\u00b1\u200948.0olPFC\u201415,7-DHT319.1\u2009\u00b1\u2009125.2 (107.2%)11.3\u2009\u00b1\u20091.6 (87.5%)239.4\u2009\u00b1\u200915.7 (93.4%)Sham297.6\u2009\u00b1\u200928.012.9\u2009\u00b1\u20091.8256.4\u2009\u00b1\u200913.0olPFC\u201425,7-DHT175.6\u2009\u00b1\u200940.5 (80.4%)23.7\u2009\u00b1\u20093.4 (74.2%)293.6\u2009\u00b1\u200942.9 (126.6%)Sham218.3\u2009\u00b1\u200932.332.0\u2009\u00b1\u20094.5231.8\u2009\u00b1\u200914.0olPFC\u201435,7-DHT278.2\u2009\u00b1\u200996.5 (107.7%)77.1\u2009\u00b1\u200914.0 (94.7%)235.5\u2009\u00b1\u200919.7 (93.5%)Sham258.2\u2009\u00b1\u200925.681.4\u2009\u00b1\u20099.8251.9\u2009\u00b1\u200920.4M-25,7-DHT138.7\u2009\u00b1\u200919.2 (72.1%)*53.4\u2009\u00b1\u20096.9 (74.6%)199.4\u2009\u00b1\u200914.7 (112.3%)Sham192.3\u2009\u00b1\u200915.171.5\u2009\u00b1\u200910.2177.5\u2009\u00b1\u20099.5Tissue concentrations of serotonin (5-HT), dopamine (DA) and noradrenaline (NA) in three adjacent (anterior\u2013posrterior) medial prefrontal (mPFC 1\u20133) and orbital prefrontal (olPFC 1\u20133) areas, and control area (M-2) in sham (n\u2009=\u200916) and 5,7-DHT- (n\u2009=\u200914) lesioned animals. Group averages (\u00b1SEM) are presented in ng\/g*Significant group differences p\u2009<\u20090.01**Significant group differences p\u2009<\u20090.05\nExperimental procedure\nApproximately 9\u00a0days after surgery, a period that a pilot experiment had indicated to be necessary to achieve maximum 5-HT depletion (data not shown), the behavioural experiments started. On experimental days, which typically lasted from 9\u00a0a.m. to 5\u00a0p.m., the animals were transferred to the experimental room, where they remained in their home cages until testing. In both the animal facility and the experimental room, a radio played to mask background noises.\nBefore the behavioural testing, both lesion and sham groups were further divided to create a total of four groups. Two groups, one lesion and one sham, received non-preferred pellets throughout the experiment, and two groups started receiving non-preferred pellets but only received preferred pellets from the start of the first reversal.\nThe two behavioural tasks were applied consecutively in the same order to all animals. At the start of the second task, all animals received non-preferred pellets again.\nBehavioural task 1. Two-lever discrimination task. (weeks\u00a01\u20134)\nThis task was used to assess the effects of mPFC 5-HT depletion on the acquisition of an operant response, discrimination between two spatial stimuli and reversal and extinction of the acquired responses (De Bruin et al. 2000).\nFirst, the animals were trained, in a maximum of nine (64 trials) shaping sessions, to press a lever for a food reward. During this shaping phase, a single lever was presented at each trial (randomly the left or right lever). In the course of the sessions, the animals were required to increase the number of lever responses per trial from a fixed ratio (FR) of one to three responses to obtain a reward. All of the trials in the two-lever spatial discrimination and reversal task (including shaping) were discrete choice trials; that is, the lever(s) were retracted upon responding or, in case of an omission, at the end of the trial duration (60\u00a0s). The inter-trial interval was set at 25\u00a0s.\nThereafter, a second lever was introduced, and the animal had to learn to press either the left or right lever to obtain a food reward (two-lever discrimination). Pressing the incorrect lever or omitting a response (i.e. not responding within 60\u00a0s) led to trial termination and an inter-trial interval timeout. After reaching criterion, i.e. obtaining 90% of the food pellets in a session, the animals were subjected to serial reversals. For two of the four groups, this coincided with the switch from non-preferred to preferred pellets. During this reversal phase, the rewarded and non-rewarded levers were reversed daily for 4 consecutive days. The final phase was an extinction phase in which both levers were presented but none were rewarded.\nOn both the two-lever discrimination and the serial reversal tests, the animals were tested twice daily: a morning and an afternoon session. The extinction phase consisted of a single daily session for 4 consecutive days. All sessions comprised eight blocks (with eight trials each), which were divided for analysis into two sets of four blocks. Session duration ranged from 45\u00a0min (two-lever discrimination) to 2\u00a0h (extinction).\nBehavioural task 2. Odour go\/no-go inhibition task (weeks\u00a05\u20139)\nThis task was used to assess the acquisition of a two-odour discrimination, the inhibition of lever pressing and the reversal of the acquired discrimination.\nIn this task, the animals were first trained to sample an odour in an odour port and subsequently press a lever once for a food reward (FR1). This was achieved in four (30 trial) sessions over 2\u00a0days. Once they acquired these two operant behaviours, they were trained in six (30 trial) daily sessions to discriminate between two odours, one indicating a \u2018go\u2019 trial in which the animal was required to press the lever to obtain a food reward and another odour that signaled a \u2018no-go\u2019 trial in which the animal had to withhold pressing the lever to obtain a food reward. Failure to press the lever on a \u2018go\u2019 trial or inhibit a lever response on a \u2018no-go\u2019 trial, as well as a failure to sample the odour or respond within 60\u00a0s (omission), triggered a 30-s inter-trial timeout during which the house light was turned off and no pellet was rewarded. The final phase was a single reversal in which the stimulus\u2013response contingencies were switched and the animals were required to reverse their previously acquired go\/no-go responses. For two of the four groups, this coincided with the switch from non-preferred to preferred pellets. In this final phase, the animals were tested in 24 (45 trial) daily sessions over 12\u00a0days. The number of trials per session during this phase was increased to 45 to facilitate task acquisition.\nBehavioural measures\nPellet preference test The mean cumulative number of arm entries\/pellet choices per rat were grouped and compared over all, 15 trials, sessions.\nGeneral activity To assess the possible effects of the lesion on general activity, the groups were compared regarding average number of head entries into the food dispenser and lever presses per block. These measures were taken during testing on the two-lever discrimination task. To exclude the influence of performance on these activity measures, data from the last acquisition session (both sets) were used. In this session, the performance of both groups was stable and statistically identical.\nTests of cognitive flexibility The data obtained from both behavioural tasks were taken as measures of performance in the following fashion: the amount of obtained rewards as a fraction of the amount of obtainable rewards was taken as an index of overall performance on all test phases except the extinction phase of the two-lever discrimination. For this phase, the total number of lever responses was taken as the index, and, as this measure is prone to interference from pre-existing differences between groups in the total number of lever presses, the amount of lever presses per block of eight trials was expressed as a percentage of the mean number of lever presses per group in the same block on the previous day.\nResponse accuracy as an index of the amount of response errors was scored as the number of correct lever presses relative to the total amount of lever presses. This measure, together with the number of lever omissions, was taken to explain sub-optimal performance.\nPerformance on the two-odour go\/no-go task was further divided into success and failure of responding on the \u2018go\u2019 trials and the \u2018no-go\u2019 trials as a measure of inhibition.\nThe effects of reward type (preferred vs non-preferred pellets) on reversal learning were measured by comparing performance scores of the reversal phase of both groups.\nThe success of task acquisition of any test phase was established by calculating the aforementioned performance. In case of the extinction phase, the success on the test was measured by the total number of lever presses.\nLesion evaluation\nNeurochemistry After all experimental procedures were completed, the animals were numbed with a mixture of O2\/CO2, after which they were decapitated. Dissection of the brain and removal of the regions of interest immediately followed decapitation. Both the medial (prelimbic, infralimbic and anterior cingulate cortex) and orbitolateral (ventral orbital, ventrolateral orbital, lateral orbital, agranular insular) PFC were completely removed in three adjacent (anterior\u2013posterior from frontal pole to genu of the corpus callosum) slices as well as a single control area (motor cortex, M-2; Paxinos and Watson 1998; Uylings et al. 2003). After dissection, all samples were stored in \u221280\u00b0C until analysis. All medial and orbitolateral PFC areas were analysed separately to gain more insight into the spread of the lesion. Tissue from both hemispheres were, however, analysed together.\nTissue samples were weighed and homogenised in ice-cold 0.1\u00a0mol\/l perchloric acid and centrifuged for 15\u00a0min at 14,000\u00a0rounds\/min. After centrifuging, the unfiltered supernatants were transferred to an autosampler and 20-\u03bcl aliquots were injected onto a column for high-performance liquid chromatography (HPLC) analysis (Waters 600E pump and Waters 717plus autosampler, Waters Chromatography b.v., The Netherlands; Decade VT-03 electrochemical detector, Antec Leyden, The Netherlands) and Shimadzu Class-vp\u2122 software (version 5.03, Shimadzu Duisburg, Germany).\nThe mobile phase consisted of 0.06\u00a0mol\/l sodium acetate, 9\u00a0mmol\/l citric acid, 0.37\u00a0mmol\/l heptanesulphonic acid and 12.5% methanol. The flow rate was kept constant at 0.65\u00a0ml\/min. Separation of 5-HT, DA and NA from other components was achieved with a Supelcosil column (LC-18-DB 25\u00a0cm\u2009\u00d7\u20094.6\u00a0mm\u2009\u00d7\u20095\u00a0\u03bcm), with a 2-cm guard column of the same material (Supelco Superguard \u2122, Supelco, USA) kept at a constant temperature of 28\u00b0C. Quantification was achieved by means of electrochemical detection (oxidation potential set at +0.65\u00a0V). The transmitter\/metabolite content was calculated against a calibration curve of external standards.\nThe lowest detectable concentration in the supernatant of our main transmitter of interest, 5-HT, was 800\u00a0pg\/ml, at a signal to noise ratio of 2:1.\nData analysis\nPellet preference The data of the pellet-preference experiment were analysed using a Friedman rank-order test with the cumulative number of choices per pellet as rank sums. Post-hoc testing consisted of two-group Mann\u2013Whitney U comparisons. p values (two-tailed) were set at p\u2009<\u20090.01.\nBehavioural tasks 1 and 2 Behavioural data taken during these experiments were analysed using SPSS for Windows (version 11.0; SPSS, Gorinchem, The Netherlands). Shaping data from both behavioural tasks were analysed as the number of trials needed to reach a performance level of greater than 90%. Acquisition and reversal data obtained from both behavioural tasks were arcsine transformed to reduce the interference from ceiling effects (Microsoft Excel, Seattle, WA). The data of the extinction phase of the two-lever discrimination, where no ceiling effect was present, were not transformed. Based on previous results, in which effects of local mPFC inactivation were often observed only in one half of a learning session, we divided the data of each session of the two-lever discrimination task in two sets of four blocks, each consisting of eight trials (De Bruin et al. 2000). In contrast, data from the go\/no-go task execution were analysed between sessions and not between sets or blocks because the observed learning in this task was slow and group differences spread out over multiple days. Because of technical problems, some data points were lost.\nFor the data of both tasks, a repeated-measures analysis of variance (ANOVA) was used with treatment (control\/lesion) and pellet (AI\/P) as \u2018between-subjects\u2019 factors and block as the \u2018within-subjects\u2019 factor. When a \u2018between-subjects\u2019 effect was found, a one-way ANOVA and Student\u2013Newman\u2013Keuls (SNK) test was used to test for group differences. For the shaping and acquisition phases (which preceded the introduction of the different pellets), the behavioural data of both control groups and lesion groups were combined. This was done to increase power and because both control and lesion groups received the same pellet. In case a treatment effect was found, an independent-samples t test was used as the post-hoc test. Activity measures over the last acquisition session were analysed in a similar fashion.\nNeurochemical data Transmitter tissue content was analysed against a calibration curve of external standards. Group comparisons were made using one-way ANOVA, and post-hoc testing was done with an independent-samples t test.\nResults of the 5,7-DHT mPFC lesion experiment\nPellet preference Figure 1 shows the mean cumulative scores or arm entries for the three pellet types tested. Comparison of rank sums using a Friedman rank-order test revealed significant group differences (F(9,2)\u2009=\u200912.667, p\u2009=\u20090.002). Repeated post-hoc Mann\u2013Whitney U tests revealed a food preference for pellet type \u2018P\u2019 over pellet types \u2018FP\u2019 and \u2018AI,\u2019 p\u2009<\u20090.001.\nFig.\u00a01Pellet preference test. Rats were tested over 3 consecutive days in a T-maze for pellet preference. Mean cumulative number of arm entries\/pellet choices is displayed. Asterisk, different from \u2018AI\u2019 and \u2018FP,\u2019 p\u2009<\u20090.001\nNeurochemical examination Postmortem tissue analysis of brain tissue revealed that 5-HT levels were reduced in lesioned animals compared to control rats in all mPFC areas. Table\u00a01 summarises the results. Spread to neighbouring areas was limited to a small but significant reduction of 5-HT in M-2. Pre-surgery desipramine administration prevented significant loss of DA terminals in all inspected areas, although reductions in both medial and orbital areas were observed. A modest but significant reduction in NA was found in the most frontal mPFC area (med-1). Two animals were excluded from the experiment and further data analysis for showing insufficient loss of 5-HT in the mPFC (i.e. less than 30%) or loss of transmitter in neighbouring areas (i.e. more than 50% of both DA and NA).\nBody weight and general activity Post-surgery body weight did not differ between the lesion group and animals that underwent sham surgery (data not shown). The behavioural measures, head entries into the food dispenser and average number of lever presses, taken to assess overall activity of the treatment groups, did not differ, respectively (F(1,28)\u2009=\u20090.719, p\u2009=\u20090.404 and F(1,28)\u2009=\u20090.001, p\u2009=\u20090.977). Table\u00a02 shows the averages for both parameters.\nTable\u00a02General activity measures\u00a0ShamLesionHead entries into food dispenser15.48\u2009\u00b1\u20091.4317.38\u2009\u00b1\u20091.76Lever presses26.71\u2009\u00b1\u20090.7126.73\u2009\u00b1\u20090.39The average number (\u00b1SEM) of head entries into the food dispenser and the average number of lever presses for sham control and lesioned animals. No statistical differences of the lesion on these parameters were observed.\nBehavioural analysis\nEffects of mPFC 5-HT depletion on the two-lever discrimination task\nShaping During the initial shaping phase, the animals learned to press a lever for a food reward. No differences between any of the groups were observed on number of trails needed to reach criterion (i.e. >90% of pellets obtained; F(3,28)\u2009=\u20091.597, p\u2009=\u20090.215). Shaping took maximally nine sessions spread out over 5\u00a0days. Performance, expressed as a percentage of obtained rewards, exceeded 90% before initiation of the next test phase.\nTwo-lever discrimination Shaping on the operant task was followed by a two-lever discrimination. A main effect of block, showing that learning took place, was found over all blocks of the morning session (F(7,168)\u2009=\u200917.019, p\u2009=\u20090.000) after which performance levelled off. Initially, the overall performance of the sham controls fell behind that of lesioned animals (interaction effect), which made fewer errors in the second set of the morning session (F(1,24)\u2009=\u20095.599, p\u2009=\u20090.026; see Fig.\u00a02). An independent-samples t test over this set showed blocks 2 and 3 to differ between lesion and control groups, respectively (t\u2009=\u2009\u22122.509; p\u2009=\u20090.025 and t\u2009=\u2009\u22122.534; p\u2009=\u20090.024). No difference in the number of omissions was observed (F(1,24)\u2009=\u20091.045, p\u2009=\u20090.316). Group differences had disappeared by the second session.\nFig.\u00a02Acquisition of the two-lever discrimination. Depicted is the percentage of correct responses for both lesion and control groups on the first acquisition day. White and grey bars indicate sets of four blocks (32 trials). Asterisk indicates group differences, p\u2009<\u20090.05\nOverall performance over the second set of the final acquisition session showed that all groups (four) had obtained at least 90% of the rewards and did not differ significantly in performance (F(1,23)\u2009=\u20090.980, p\u2009=\u20090.334; see Fig.\u00a03a).\nFig.\u00a03Performance on the final acquisition phase and initial reversal. a Performance on the final acquisition sessions. All groups obtained at least 90% of the rewards over the last set of the afternoon session, and no group differences were found. b Performance over the first reversal sessions. Control animals that switched to preferred pellets fell behind the other groups. White and grey bars indicated sets of four blocks (32 trials). Asterisk indicates group differences, p\u2009<\u20090.05\nSpatial reversal After reaching criterion on the two-lever discrimination, the animals were subjected to four serial reversals. As each group (lesion and control) was now divided into a non-preferred- and preferred-pellet group, the number of groups was four from now on. A main effect of block (F(7,133)\u2009=\u200951.715, p\u2009=\u20090.000; not treatment or pellet, p\u2009>\u20090.3) was observed over the morning session of the first reversal (F(1,19)\u2009=\u200951.715, p\u2009=\u20090.000). During the afternoon session, a block effect over both sets taken together indicated that learning continued (F(7,140)\u2009=\u200922.669, p\u2009=\u20090.000). Main effects on performance for both treatment (F(1,23)\u2009=\u20095.245, p\u2009=\u20090.032) and pellet (F(1,23)\u2009=\u20096.158, p\u2009=\u20090.021) as well as a treatment\/pellet interaction (F(1,23)\u2009=\u20095.701, p\u2009=\u20090.026) were observed over the first set. A one-way ANOVA with SNK post-hoc test revealed that sham animals receiving preferred pellets obtained fewer pellets than any other group in blocks 1 (F(3,27)\u2009=\u20094.163, p\u2009=\u20090.017), 2 (F(3,27)\u2009=\u20095.176, p\u2009=\u20090.007) and 4 (F(3,27)\u2009=\u20095.520, p\u2009=\u20090.012; see Fig.\u00a03b). No differences for treatment, pellet or interaction were found over the second set of the afternoon session (p\u2009>\u20090.3). A covariate analysis revealed that the effects found during the reversal could not be attributed to a pre-existing performance difference between groups.\nTo examine if the group differences could be attributed to either an increase in errors or an increase in omissions, further analyses were performed. The data indicated that the control animals that were switched to preferred pellets exhibited a decrease in accurate lever pressing, i.e. increased errors (F(1,24)\u2009=\u20094.638, p\u2009=\u20090.042; see Fig.\u00a04), as opposed to increased omissions (F(1,24)\u2009=\u20091.606, p\u2009=\u20090.213), in this group in the first set of the afternoon session. After the initial reversal, all groups performed in similar fashion in the three subsequent reversals.\nFig.\u00a04Omissions and response accuracy in the first reversal of the two-lever spatial discrimination task. The graph shows the percentage omissions (top) and percentage correct responses (bottom) during the initial reversal. Decreased lever press accuracy (increased errors) of control animals that receive preferred pellets is seen during the first set of the afternoon session. White and grey bars indicated sets of four blocks (32 trials). Post-hoc testing revealed the second block of trials in this set to be statistically different from all other groups. No differences were observed for the percentage of omissions\nExtinction After four consecutive reversals, the animals were subjected to an extinction phase during which lever pressing was no longer rewarded. When comparing the total amount of lever presses corrected for baseline lever pressing, all groups showed a steady decrease over both sets of each of the four sessions (main effect of block: F(7,175) between 29.237 and 10.048; p\u2009=\u20090.000; see Fig.\u00a05). There were, however, no main effects of treatment or pellet or any interaction effect between the groups (p\u2009>\u20090.2). Over the course of 4\u00a0days, lever pressing was never totally extinguished. Analysis of the uncorrected extinction data yielded identical results.\nFig.\u00a05Lever responding during extinction of the two-lever spatial discrimination task. The cumulative number of lever presses per group during the extinction phase of the two-lever discrimination task, when lever pressing is no longer rewarded is shown. Although a decrease is seen over each individual session, lever pressing is never totally abolished. White and grey bars indicated sets of four blocks (32 trials). No significant group differences were found\nEffects of mPFC 5-HT depletion on the odour-based go\/no-go task\nShaping During the initial shaping phase, the animals learned to sample an odour and press a lever for a reward. Both groups acquired the responses (i.e. >90% of pellets obtained) in maximally eight sessions, spread out over 6\u00a0days, without showing any treatment effect (F(3,28)\u2009=\u20090.667, p\u2009=\u20090.590).\nTwo-odour discrimination After shaping, the animals were trained, over six sessions, to respond to one odour with a lever press, while inhibiting a lever press after sampling a second, different, odour. Results show an overall increase in performance over these sessions, i.e. a main effect of block (F(5,140)\u2009=\u200916.074, p\u2009=\u20090.00) for all groups (data not shown). No effect of treatment was found (F(1,28)\u2009=\u20091.375, p\u2009=\u20090.251). Response preference for either the \u2018go\u2019 or \u2018no-go\u2019 response were not observed.\nOdour reversal After the acquisition of the odour discrimination, the stimulus\u2013response contingencies were reversed, and the animals had to switch their response to the odours. In this treatment, the two groups were again divided into sub-groups for preferred and non-preferred pellets. In contrast to the quickly acquired spatial reversal, the odour reversal took 12 sessions to acquire (reaching \u2018pre-reversal\u2019 performance). Figure 6 shows the performance of all groups.\nFig.\u00a06Performance on the final acquisition phase and reversal of the two-odour go\/no-go discrimination task. a Performance over the four final acquisition sessions. All groups obtained at least 90% of the rewards over the final four acquisition sessions, and no group differences were found. b Performance over the reversal sessions. Control animals that were rewarded non-preferred pellets fell behind the other groups. Asterisk indicates group differences, p\u2009<\u20090.05\nOver 6 consecutive test days (12 sessions), main effects of both session (F(1,10)\u2009=\u200940.163, p\u2009=\u20090.000) and treatment (F(1,10)\u2009=\u20099.025, p\u2009=\u20090.013) as well as a treatment\/pellet interaction were observed (F(1,10)\u2009=\u20095.764, p\u2009=\u20090.037). Further analysis (one-way ANOVA and SNK post-hoc test) revealed that in sessions 7 (F(3,13)\u2009=\u20096.851, p\u2009=\u20090.009), 8 (F(3,13)\u2009=\u20093.898, p\u2009=\u20090.044) and 9 (F(3,13)\u2009=\u20095.563, p\u2009=\u20090.017), control animals receiving non-preferred pellets obtained fewer rewards than all other groups.\nFurther analysis of the data showed that unlike the initial reversal task, none of the groups differed in response accuracy (F(1,10)\u2009=\u20090.158, p\u2009=\u20090.699). However, a treatment\/pellet interaction (F(1,10)\u2009=\u20095.898, p\u2009=\u20090.036) was observed for the average number of omissions (see Fig.\u00a07). Post-hoc testing showed this measure in sessions 8 and 10 to differ significantly between sham animals on non-preferred rewards and all other groups, respectively (F(3,13)\u2009=\u20094.647, p\u2009=\u20090.028 and F(3,13)\u2009=\u20094.194, p\u2009=\u20090.037).\nFig.\u00a07Omissions and response accuracy during reversal learning of the two-odour go\/no-go discrimination task. Mean percentage of omissions (top) and percentage correct responses (bottom) per group during the \u2018odour-reversal\u2019 are shown. An increase in the number of omissions is seen for control animals that received non-preferred pellets on sessions 8 and 10. No differences were observed for the percentage of correct responses\nDiscussion\nWith the current experiment, we found support for our hypothesis that medial prefrontal 5-HT is selectively involved in cognitive flexibility when affect guides decision making. Local depletion of mPFC 5-HT prevented the reversal learning impairments that were induced by a change in the reward value during operant behaviour. This effect was observed both in spatial two-lever discrimination reversal learning and during the reversal of the odour-based go\/no-go task. In the former, control animals that were switched to preferred pellets showed increased erroneous lever pressing compared to all other groups, whereas in the latter, non-switched control animals showed increased omissions. Apparently, the lesion rendered the animals less sensitive to changed reward value. Similar effects, however, were not observed during extinction learning, when rewards were no longer available.\nTaken together, these data suggest that depletion of mPFC 5-HT impairs goal-directed behaviour by rendering rats less capable of responding to a change in reward value (incentive learning; Balleine and Dickinson 1998; Balleine 2005; Niv et al. 2006), without disturbing their ability to detect the presence or absence of a reinforcer and to utilise this information to adapt behaviour accordingly.\nThe data moreover indicate that this effect occurs independent of the stimulus modality of the behavioural tasks, thereby opposing our hypothesis that reversal deficits induced by mPFC 5-HT lesioning would be limited to spatial tasks, as opposed to odour-guided ones.\nIn the current experiment, we lesioned 5-HT terminals in the mPFC by means of a local microinjection of the toxin 5,7-DHT. HPLC analysis showed that this procedure was successful as a marked loss of serotonergic innervation (70\u201390%), compared to sham controls, which was observed in the target area. This reduction was observed without spread to neighbouring areas whilst sparing both DA and NA innervation.\nTwo behavioural paradigms were employed to study cognitive flexibility and affective processing. First, we used a task previously developed in our lab with which we showed mPFC involvement in spatial reversal learning (De Bruin et al. 2000). A second, symmetrically reinforced go\/no-go odour reversal task was newly designed for the current experiments. These tasks were chosen to give insight into reversal learning across different stimulus modalities and to allow the study of different aspects of reversal learning, such as inhibitory processes involving both response extinction (spatial discrimination task) and impulse inhibition (odour-based, go\/no-go task). Data showed that whilst the stimulus discriminations for both tasks were relatively quickly acquired, reversal learning took significantly longer for the odour stimuli. Whereas the \u2018spatial\u2019 lever press reversal was acquired within a day, the \u2018odour\u2019 go\/no-go reversal took up to 12\u00a0days. These differences are in line with existing literature on the acquisition of the spatial lever-press task (De Bruin et al. 2000; Van der Meulen et al. 2003) and a comparable odour-based go\/no-go task (Schoenbaum et al. 2006).\nOur hypothesis that medial prefrontal 5-HT depletion would have a stronger impact on reversal learning in the \u2018spatial\u2019 task than in the \u2018odour\u2019 task could not be proven.\nWhen comparing the performance of lesioned animals to that of control animals, we were unable to find any indication for impaired reversal learning on either task. Not only did 5-HT lesioned animals make fewer errors during the initial acquisition, these animals showed to be fully capable of acquiring both types of reversals, demonstrating intact cognitive flexibility across stimulus modalities when the reward identity remains constant. These findings suggest that in rats, the acquisition of a spatial reversal task, which depends on the mPFC, does not depend on 5-HT innervation of that area. This contrasts the finding that in primates, the orbital PFC involvement in visual reversal learning does depend on 5-HT.\nTo test our hypothesis that mPFC 5-HT is involved in affective processing, we used a pellet switch to induce a change in affective value of the reinforcer during both reversal tasks. The rationale for this was that the impact of the serotonergic lesion in the mPFC on cognitive flexibility would be greater in a situation where the affective value of the reward can be used to guide decision making. Whereas in primate literature, the term \u2018affective shifts\u2019 has been used for a switch in the value associated with the stimulus (Dias et al. 1996), we wanted to introduce a switch in affective value of the reinforcer. Similar to the experiments of Tremblay and Schultz (2000), we changed the relative reward value from non-preferred, although still salient, to preferred (see also Watanabe 1996), at the time of the reversal.\nTo determine food preference, food-deprived rats were pre-tested on three types of food reward in a T-maze, where all rats chose Noyes \u2122 pellet P over AI. These were subsequently implemented in the task as \u2018preferred\u2019 and \u2018non-preferred\u2019 rewards, respectively, to examine the effect of this affective shift in both groups during reversal learning. To exclude a \u2018novelty\u2019 effect, the animals were acquainted with both types of pellets before testing. As we observed pellet switch-induced performance effects in control rats during both tasks, we conclude that the pellet rewards were sufficiently different. The possibility that the lesion induced an indifference to either reward does not seem likely. If lesioned animals were in fact indifferent to reward outcome, they would have performed as non-switched control animals throughout the experiment, but this was not the case (see below).\nDuring initial \u2018spatial\u2019 reversal learning, lesioned animals behaved as non-switched controls, showing a quick reversal, without performance deficits on reversal learning per se. We did, however, observe that switching intact animals to preferred rewards resulted in reduced performance on the reversal task compared to lesioned animals. These results suggest that in intact animals behavioural adaptation is at least partly guided by reward value and, in this particular case, further complicates acquisition of the reversal.\nA second instance of such a pellet-switch-induced performance deficit was observed during the \u2018odour\u2019 reversal. Whereas the initial reversal impairment in switched control animals was due to increased errors, during the \u2018odour\u2019 reversal, non-switched control animals fell behind in performance because of increased omissions.\nAs the pellet switch did not affect performance of lesioned animals at any time, we pose that an inability to use affective information (i.e. pellet value) in lesioned animals underlies the observed group differences. These data suggest that initial behavioural adaptation guided by altered reward outcome is dependent on medial prefrontal 5-HT.\nIn the following section, we will first discuss why, in our opinion, a number of possible mechanisms cannot explain the observed effects induced by the pellet switch.\nWe will then propose how our observations fit and extend current literature.\nAmong the possible causes for these effects, altered associative learning is not a likely one. Although lesioned animals did make fewer errors during the acquisition of the spatial discrimination task, an effect that has also been observed by others (e.g. Ward et al. 1999), and suggests possible improved stimulus\u2013outcome learning, performance of neither lesioned group (switch or non-switched) exceeded performance of the control animals during the subsequent testing. Moreover, the performance impairments observed in the control group occurred only during the early stages of reversal learning, leaving the acquisition of subsequent (spatial) reversals or stimulus\u2013outcome learning unaffected (see also Harrison et al. 1999).\nDespite reports on involvement of both the mPFC (Salazar et al. 2004, Winstanley et al. 2006) and 5-HT (Beninger and Phillips 1979; Soubri\u00e9 1986) in impulsive responding and extinction processes, our tests indicate that reduction in mPFC 5-HT does not lead to decreased extinction learning or increased impulsivity. No effects of the lesion were observed during extinction of lever pressing or increased lever pressing during the go\/no-go inhibition task. Although these results do not seem to fit the current literature on 5-HT in impulsivity, recent findings by Chamberlain et al. (2006) in human subjects show that impulse inhibition might be mediated via the NA system, rather than the classically implicated 5-HT. Therefore, the possibility that reversal learning improved in lesioned animals through altered inhibition or extinction processes is unlikely. Furthermore, subsequent analysis showed no difference between groups on general motor activity, as assessed by the average number of lever presses and nose pokes into the food dispenser.\nThe possibility that a motivational effect of 5-HT depletion underlies the observed deficits does not seem likely either. Although the control animals showed an increase in the number of omissions towards the end of the go\/no-go task, an effect suggesting decreased motivation towards the end of the experiment for the non-preferred reward, none of the groups (lesion or sham) showed any sign of increased or decreased lever pressing during the preceding test phases, suggesting that motivational processes were intact in lesioned animals. These findings are supported by other reports where motivational changes after general 5-HT depletion in humans (Evers et al. 2005), primates (Clarke et al. 2004) and rodents (Harrison et al. 1999) were not observed either.\nEarlier work by others (e.g. Roberts et al. 1994) suggests that (global) 5-HT depletion may induce increased reinforcing efficacy for both psychomotor stimulants and food rewards (but also see Tran-Nguyen et al. 2001). The evidence for such an effect is, however, inconclusive, and the observed changes can also be ascribed to increased impulsivity and insensitivity to punishment (e.g. Harrison et al. 1997). The current data, moreover, does not support these findings, as lesioned animals showed \u2018normal\u2019 extinction learning, whereas decreased extinction learning would be expected in case of increased reinforcing efficacy.\nFrom these findings, we conclude that the behavioural effects are not due to a generalized decrement of overall functioning but are rather specific to an inability to effectively incorporate reward value. These results suggest that mPFC 5-HT depletion induce a specific deficit in goal-directed behaviour that is limited to the ability to use current reward value to guide behaviour (Balleine and Dickinson 1998; Niv et al. 2006).\nHuman literature provides a framework in which we think the current findings can be placed. Miller (2000) has described the PFC as a control centre over overt behaviour that regulates \u2018active maintenance of patterns of activity that represent goals and the means to achieve them.\u2019 Later work by Miller and Cohen (2001) and Ridderinkhof et al. (2004) extends this model by incorporating different cortical sub-areas and describes how these areas connect to combine (reward-related) information and lead to goal-directed behaviour. In this model, the orbital PFC can be seen as the area where reward-related information is encoded (as discussed in \u201cIntroduction\u201d) and the mPFC functions as an area that regulates or signals the orbital PFC to implement performance adjustments (based on reward information).\nA unified model for the rodent PFC, similar to that of Miller for the human PFC, has not been proposed, but the available experimental data obtained in rodent areas that show functional overlap with the human mPFC (Uylings et al. 2003) parallel and extend these findings. Balleine and Dickinson (1998), for example, showed that lesions of the prelimbic area (part of the mPFC) render the animals insensitive to variations in the contingency between a response and a specific reward. Moreover, the same authors (De Wit et al. 2006) showed that inactivation of the mPFC leads to impaired resolving of response conflict through loss of inhibitory function, without any apparent impairment in discriminative function for foods or loss of reinforcing function of food pellets and sugar. Further indications for a parallel between the human and rodent mPFC comes from Cardinal et al. (2001), who showed that lesions of the mPFC yielded what Dalley et al. (2004) later described as a flattening of the shift from large-delayed rewards to small-immediate rewards. Although the authors ascribed this to a \u2018timing impairment,\u2019 these findings fit the described, human, model of mPFC-mediated performance monitoring and adjustment of behaviour. Finally, Hok et al. (2005) reported on neurons recorded in the mPFC that encode the motivational salience of places. These results add to the experimental evidence presented earlier of orbital PFC-mediated reward encoding by showing that reward-related information is also processed in the mPFC. Together, the presented rodent data support a role for the mPFC in the monitoring, processing and implementation of reward-related information, as suggested for the human mPFC.\nExperiments concerning the underlying neurochemical mechanisms involved in the processing of reward-related stimuli have mainly focused on DA. Direct evidence for DA involvement comes from measurements of DA ventral tegmental area neurons in non-human primates that show reactivity to reward (Schultz 1998), probability of reward (Fiorillo et al. 2003) and adaptive neural firing in response to reward-predicting stimuli (Tobler et al. 2005). These findings are further supported by a recent study that shows midbrain activation in humans during reward anticipation (Wittmann et al. 2005).\nThe presented data, however, show that cognitive flexibility, when affect guides decision making, might be dependent on intact serotonergic projections. Although we cannot rule out possible compensatory mechanisms, our analysis shows that DA innervation in the lesioned animals remained intact. Support for our findings can be found in human studies where global depletion of 5-HT through tryptophan depletion caused altered processing of reward cues, lesading to unfavourable choice behaviour in a gambling task (Rogers et al. 2003).\nThe current experiments support previous notions on the mPFC as a performance monitoring structure, responsible for the implementation of adjustments in cognitive control. Moreover, they extend the current model by implicating 5-HT, in addition to DA, in cognitive control.\nWith regards to the initial research questions whether cognitive flexibility is dependent on mPFC 5-HT, the present data suggest that in both the spatial and the odour tasks, the lesioned animals are fully capable of acquiring a reversal, show appropriate extinction learning in the absence of a reinforcer and exert normal inhibitory control. The current observations, however, also suggest that when the value of a reinforcer changes but not its presence or absence, mPFC 5-HT is necessary to adapt the behaviour accordingly.","keyphrases":["cognitive flexibility","reversal learning","affective processing","5,7-dht","medial prefrontal cortex"],"prmu":["P","P","P","P","R"]}
{"id":"Immunome_Res-1-_-1289288","title":"AntiJen: a quantitative immunology database integrating functional, thermodynamic, kinetic, biophysical, and cellular data\n","text":"AntiJen is a database system focused on the integration of kinetic, thermodynamic, functional, and cellular data within the context of immunology and vaccinology. Compared to its progenitor JenPep, the interface has been completely rewritten and redesigned and now offers a wider variety of search methods, including a nucleotide and a peptide BLAST search. In terms of data archived, AntiJen has a richer and more complete breadth, depth, and scope, and this has seen the database increase to over 31,000 entries. AntiJen provides the most complete and up-to-date dataset of its kind. While AntiJen v2.0 retains a focus on both T cell and B cell epitopes, its greatest novelty is the archiving of continuous quantitative data on a variety of immunological molecular interactions. This includes thermodynamic and kinetic measures of peptide binding to TAP and the Major Histocompatibility Complex (MHC), peptide-MHC complexes binding to T cell receptors, antibodies binding to protein antigens and general immunological protein-protein interactions. The database also contains quantitative specificity data from position-specific peptide libraries and biophysical data, in the form of diffusion co-efficients and cell surface copy numbers, on MHCs and other immunological molecules. The uses of AntiJen include the design of vaccines and diagnostics, such as tetramers, and other laboratory reagents, as well as helping parameterize the bioinformatic or mathematical in silico modeling of the immune system. The database is accessible from the URL: .\nIntroduction\nThere is a vast, and ever increasing, volume of important information that has accumulated from decades of experimental analysis within immunology. This will only become compounded as high-throughput techniques begin to impinge upon the immunological biosciences. The only efficient way for this information to be properly utilized requires the development of databases that store it and systems that use it. Although the type of data archived may alter from case to case, nonetheless the creation, use, and manipulation of databases containing biologically important information is the most crucial feature of current bioinformatics, both as it supports the genomic and post-genomic revolutions and as a discipline in its own right. There is nothing new in developing databases focusing on immunology: many spotlighting the in-depth sequence analysis of individual immunomacromolecules have existed for some time [1]. Functional or epitope-orientated databases are a more recent development. Examples include the now defunct MHCPEP database [2], FIMM [3], SYFPEITHI [4], the HIV sequence database [5], the HLA ligand database [6], the EPIMHC database [7], and the MHCBN database [8].\nAn epitope is any molecular structure that can be recognised by the immune, or other biological, system. Epitopes, or the antigen from which they are derived, can be composed of protein, carbohydrate, lipid, nucleotide, or a combination thereof. It is through recognition of foreign, or non-self, epitopes that the immune system can identify and, hopefully, destroy pathogens. Hitherto, peptide epitopes have been the best studied, and have, traditionally, have been categorized as either T cell or B cell epitopes. T cell epitopes are peptides presented to the cellular arm of the immune system via the MHC-peptide-TCR complex. B cell epitopes represent surface regions of an antigen that are bound by soluble or membrane-bound antibodies. If this region of a protein antigen is comprised of residues distally separated within the primary structure, and brought into local proximity by protein folding, then it is termed a discontinuous or conformational B cell epitope. Linear or continuous B cell epitope residues are sequential in both primary structure and thus as a region on the proteins' surface. Such epitopes are predominantly identified by antigen-specific antibody cross-reactivity with peptides.\nThere is a need to create a databank for the wider disciplines of immuno-vaccinologists, which can act as a central repository and resource. Our aim is to complement other databanks [2-8] and thus we have developed AntiJen, a computational information resource for immunology and vaccinology that integrates quantitative kinetic, thermodynamic and biophysical data, with functional and cellular information. AntiJen v2.0, a development of our earlier database system JenPep [9,10], contains functional data on T cell and B cell epitopes. Moreover, the B cell archive is now sub-divided into linear and conformational epitopes. These epitopes form the basis of the humoral immune response and, unlike T cell epitopes, methods of prediction are often inaccurate [11]. A more in-depth B cell epitope archive should aid the development of prediction strategies. Antigen recognition by the Major Histocompatibility Complex (MHC) is vital to T cell activation hence, the inclusion of thermodynamic data on the binding of peptides to MHC molecules and T Cell Receptor (TCR) binding to peptide-MHC (pMHC) complexes. This data is complemented by kinetic data based on the same molecular interactions. Data on antigen processing and presentation is also included in AntiJen. Binding data derived from peptide interactions with the Transporter Associated with Antigen Processing (TAP transporter) are included in the archive. Additionally, quantitative specificity data from position-specific peptide libraries is included. AntiJen also incorporates thermodynamic data on protein-protein interactions, within an immunological context, such as co-receptor and superantigen binding, plus interactions with the MHC. All of these interactions are, potentially, key factors for the successful computational design of vaccines.\nAntiJen also contains biophysical data, including diffusion coefficients and cell surface copy numbers, on a variety of immunological molecules. Such data provides insight into the number of target receptors, which is an important, if under explored, component of binding between cells. Indeed, the number of molecules expressed on the membrane can alter depending on disease. The final addition to the databank focuses upon antigen binding to antibodies. One key innovation is a greatly increased compendium of experimental conditions, which, in conjunction with a greatly enhanced search capacity, consolidates our databases as a unique, value-added data source, fostering developments within both in silico immunology and the wider community of immunovaccinology. The database is available from the URL: .\nDatabase development\nRelative to the database system used for JenPep [9,10], the interface to AntiJen is entirely new, having been completely rewritten. AntiJen has been designed and implemented using postgreSQL, a system comprising a relational database and database server, and has thus established increased database robustness, creating an improved infrastructure for foreseeable issues of data storage and data growth. Data within AntiJen is structured into twenty-four normalised tables. Each is category specific and holds either statistical or experimental data. Additional tables accommodate the keyword data \u2013 which powers our protein-orientated antigen search and allows integration of the BLAST search \u2013 and there is also a structural data table to accommodate links to external structural databases. The user interface consists of a series of HTML forms. The search requests from these forms target PERL scripts integrated with SQL which in turn query the database.\nDatabase content\nCompared to its progenitor JenPep [9,10], the data archived in AntiJen v2.0 has grown considerably in depth (additional data types such as experimental conditions), breadth (addition of new data to existing databases), and scope (addition of extra sub-databases containing novel kinds of information). Additions to AntiJen have been derived from exhaustive searching of the primary literature, to give a dataset of > 31,000 entries. AntiJen v2.0 now consists of 11 sub-databases; details of the different databases are given in Table 1. The relative sizes of the databases and the growth from JenPep are summarised in Table 2.\nTable 1\nAntiJen sub-databases and content.\nDATABASE\nCONTENT\nT Cell Epitopes\nContains T Cell epitope peptides (known binders).\nB Cell Epitopes\nContains B Cell linear and conformational epitope peptides.\nMHC-Peptide\nBinding data relating to antigenic peptides and MHC interactions.\nTCR\nBinding data relating to antigenic peptides \u2013 TCR \u2013 MHC interactions.\nTAP\nBinding data relating to antigenic peptides and TAP interactions.\nKinetics\nKinetic binding data for MHC peptide interactions.\nIPPI\nBinding data for a collection of immunological protein interactions.\nDiffusion Coefficient\nCollection of Diffusion and Friction coefficients for surface peptides.\nCopy Number\nNumber\/Abundance of cell surface molecules.\nPeptide Libraries\nRelative binding data for antigenic peptide amino acid substitutions.\nAntibody-Peptide\nA variety of antibodies known to bind proteins.\nTable 2\nSize of AntiJen relative to JenPep. The number of peptides for each category in the AntiJen database is given, distinguishing between class I and class II categories, where appropriate. Growth versus JenPep 1 and 2, the progenitors of AntiJen, is included. For certain data categories, most obviously TAP binding data, re-evaluation of the quality of data within JenPep has seen it decrease rather than increase, however the expansion of the data is clearly seen.\nDATABASE\nJenPep v1.0\nJenPep v2.0\nAntiJen v1.0\nAntiJen v2.0\nClass 1\nClass 2\nTotal\nClass 1\nClass 2\nTotal\nClass 1\nClass 2\nTOTAL\nClass 1\nClass 2\nTOTAL\nT cell epitotes\n1266\n795\n2061\n2060\n1158\n3218\n2247\n1578\n3825\n2402\n1585\n4158\nMHC peptide binding\n3196\n2652\n5848\n6411\n5925\n12336\n6853\n7772\n14625\n7304\n8114\n15454\nTAP peptide binding\n432\n441\n408\n1106\nB cell epitotes\n816\n1295\n3541\nTCR \u2013 peptide-MHC\n49\n375\n124\n594\n527\n253\n782\nMHC peptide kinetics\n704\n243\n947\n897\n294\n1150\nIPPI\n805\n2675\nCopy Number\n161\n243\n414\nDiffusion coefficients\n759\nPeptide Libraries\n897\nAntibody\n395\nAntiJen contains both generic and dataset-specific data. For each entry, we record the peptide sequence (eg. YTSDYFISY) of the epitope using the standard one-letter code, its length (9 in this case), and, by linking to the sequence database Swiss-Prot  or NCBI , the antigen to which the peptide sequence most closely matches (in the case of YTSDYFISY: C-ests-1 (p54), SWISS-PROT code P41156). The description of the antigen is, wherever possible, obtained directly from the literature. AntiJen is also linked to PUBMED. This allows us to record the original citation associated with the data. For example, for YTSDYFISY, we cite: Journal of Immunol 1994 volume 152 pages 3913\u20133924, PUBMED ID 8144960. For the T cell epitope, MHC ligand, and TCR-pMHC complex categories, we also record, for each peptide, the MHC restriction in terms of the host species, class (class I vs. class II), and, where the data is available, the serotype and allele. For YTSDYFISY, these data would be human, class I, HLA-A1, and A*0101.\nEntries within AntiJen are, in turn, linked to external databases, which enables further in-depth cross referencing. As we have said, protein sequence identifiers, which may be the source of an antigenic peptide or immunological co-receptor, link directly to details in the Swiss-Prot database [12] or the NCBI protein database. Journal references can be viewed via a link to the PUBMED database , and thus to full literature references, where available. AntiJen also links to structural data, currently derived from the MPID database [13] and the Protein Data Bank [14]. The database aims to provide access to background data where available.\nAllele identifiers serve as a link to the IMGT\/HLA database  at the European Bioinformatics Institute [15]. Inherent variability in the way MHC alleles are named within the primary literature  prevents us from unambiguously standardizing nomenclature within AntiJen. HLA nomenclature follows that of the HLA Informatics Group . An allele is named using a defined pattern. For example, for HLA-A*0101: the HLA-A refers to the HLA locus; the first 01 to the serologically recognized A1 antigen and the final 01 to the individual HLA allele protein sequence. AntiJen stores the antigen classification (i.e. HLA-A1) and, when available, the specific allele. We have often encountered problems with the nonstandard allele reporting. A 4-digit HLA name necessarily implies the two digit serological antigen, a two digit classification clearly does not imply a specific allele.\nDuring database compilation, a sequence search allows us to identify the protein from which an epitope sequence originates. However, because epitopes are generally short, their sequences may be present in several potential antigens: in orthologues, paralogues, or in totally unrelated sequences. As epitopes are processed from whole proteins via a complex proteolytic pathway, one can use the sequence context to infer preferred proteasomal or endoplasmic protease cleavage patterns, but not if its context is defined incorrectly. Moreover, assuming that AntiJen is used subsequently to assign the antigenic status of proteins, wrongly identifying particular proteins as antigens can lead to the percolation of annotation errors [16,17].\nAntiJen is, where possible, a quantitative database archiving continuous measures of binding. This is a fundamental feature of several sub databases, such as the MHC ligand and pMHC-TCR databases. The binding of an immunological macromolecule to a peptide or other biomacromolecule is quantified as are other receptor-ligand interactions:\nR+L\u2194RL\nHere R is the receptor (an MHC or TCR), L the ligand (peptide or pMHC), and RL, the receptor-ligand complex (pMHC or pMHC-TCR complex). The rate of the forward reaction is proportional to [L] [R]. The rate of the reverse reaction is proportional to [RL] as no other species are involved in dissociation. At equilibrium, the forward and reverse rates are equal, and so using kon and koff as the respective constants:\nkon[R][L] = koff[RL]\nRearranging:\nHere KD is the equilibrium dissociation constant, which represents the concentration of ligand that occupies 50% of the equilibrium receptor population, and KA is the equivalent association constant.\nExperimentally, the measurement of equilibrium dissociation constants is often addressed using radio-ligand binding assays. Saturation radio-ligand binding assays measure equilibrium binding, at a range of peptide concentrations, to establish affinity (Ka) and receptor number (Bmax). Competitive binding experiments determine binding at a single labelled ligand concentration in the presence of a range of concentrations of unlabelled ligand. AntiJen records a hierarchy of different binding measures in its different sub-databases. Equilibrium constants are the most dependable and sit atop this hierarchy. Next come IC50 values, which can be obtained from a competitive radio-ligand or fluorescence assay. These are the Fmost commonly reported binding measures.\nValues obtained from radio-ligand or fluorescence methods may be significantly different. IC50 values for a peptide may vary between experiments depending on the intrinsic affinity and concentration of the standard radiolabelled reference peptide, as well as the intrinsic affinity of the test peptide. IC50 values vary with the equilibrium dissociation constant, at least within a single experiment. In practice, the variation in IC50 is often small enough that values can be compared between experiments. For the peptide discussed above, YTSDYFISY, the radiolabelled IC50 value recorded in AntiJen is 5.3 nM. BL50 values are also obtained from a peptide binding assay and are commonly reported. They are the half maximal binding levels calculated from mean fluorescence intensities of peptides binding to MHCs bound on the surface of RMA-S or T2 cells. Cells, pre-incubated with peptides, are labelled with a fluorescent monoclonal antibody. An overview of the thermodynamic and kinetic binding data within AntiJen is given in Table 3.\nTable 3\nAntiJen Thermodynamic and Kinetic Data. An overview of the 6 AntiJen databases that provide binding data. It must also be noted that several of the databases contain additional data not present in any of the other databanks.\nMHC-Peptide\nKinetics\nIPPI\nTAP\npMHC-TCR\nAntibody\nTOTAL\nIC50\n8562\n0\n247\n1000\n0\n4\n9813\nKon\n0\n188\n563\n0\n157\n87\n995\nKoff\n0\n146\n610\n0\n150\n101\n1007\nKD\n359\n156\n1143\n16\n227\n70\n1971\nKa\n65\n0\n37\n0\n28\n132\n262\nt1\/2\n0\n207\n72\n0\n148\n0\n427\nAntiJen also now contains experimental conditions, such as temperature and pH. A summary of this data is given in Table 4. The accuracy of data depends greatly upon the experimental method used. The grouping of data with respect to specific experimental techniques allows a more thorough assessment of training sets. Figure 1 shows the distribution of data for each type of analysis with respect to each database. The MHC Kinetics and TAP databases highlight the problems outlined above. The kinetics database contains data determined from over 14 methods while the TAP database is derived from 4 methods, with radiolabelled assays accounting for 80% of the data.\nTable 4\nExperimental conditions and associated information archived in AntiJen. Number of recorded experimental conditions stored within the AntiJen database. For each condition (temperature, pH, etc.) we show here the number of entries within a particular sub-database. [Standard] is the concentration of labelled standard peptide in an assay. Likewise, [competitor] is the concentration of competitor peptide within a competition assay. [peptide] is the concentration of peptide in a kinetic experiment. The Method category refers to a standard procedure used to perform a particular assay. Differences in the number of recorded data, relative to figures in Table 1, arise primarily from the omission of key details from particular papers. Archiving of experimental conditions is on-going.\nDATABASE\nTOTAL\npH\nTemperature\n[standard]\nStand. peptide seq.\n[competitor]\nMethod\n[peptide]\nMHC Binding\n15454\n6679\n9831\n10893\n12796\n5007\n1251\nMHC Kinetics\n1150\n677\n1101\n1149\n606\nTAP Binding\n1106\n22\n243\n1092\n1101\n86\n981\nTCR-pMHC\n782\n426\n632\n668\nIPPI\na\n2675\n726\n1371\n2600\nCopy Number\n414\n183\n278\n414\nPeptide Libraries\n897\n897\n897\nDiffusion Coefficient\n759\n321\n668\n736\nAntibody\n395\n119\n115\n372\nFigure 1\nThe distribution of experimental methods applied within each database. The number of different experimental methods and the abundance of data relating to the method is shown within the figures. The 'OTHERS' category refers to methods for which there is a relatively small number of entries.\nThe compilation process has highlighted the considerable inconsistency within the immunological literature regarding the recording of such fundamental data. AntiJen contains, however, a direct, verbatim transcription of data from the primary literature. As such, we do not attempt, as a matter of policy, the comprehensive and retrospective correction of potential errors. To undertake such correction would only compound any errors, introducing the kind of percolating inconsistencies so much a feature of other database systems [16,17]. Further inaccuracies may stem from our logistic inability to verify data independently, therefore we must trust those values reported in the literature.\nSubsidiary Databases in AntiJen\nThe AntiJen database contains a number of sub-databases. Each of these contains data on different aspects of the biological function and\/or biophysical properties of different classes of immunomacromolecule. We describe the nature and content of each sub database below.\nB Cell Epitopes\nEpitopes are the principal chemical moieties recognized by the immune system. Although the importance of non-peptide epitopes, such as carbohydrates and lipids, is now increasingly well understood, peptidic B cell and T cell epitopes remain the principal tools by which the intricacy of the immune response can be explored. B cell epitopes are regions of the surface of a protein, or other biomacromolecule, recognized by soluble or membrane-bound Antibody molecules. In developing AntiJen, we have discarded the contents of our previous B cell archive and constructed one de novo. It contains an entirely new data set with a substantially different data structure. There are two forms of epitopes: linear and discontinuous. A linear B cell epitope is composed of a single stretch of sequential residues. A discontinuous B cell epitope is composed of sequentially separate residues brought into close proximity in a conformationally-dependent arrangement. The data we archive is primarily focused upon linear epitopes. This is due to the far greater amount of experimental data available for linear epitopes, which reflects both the relatively facile experiments needed to identify them and an implicit belief in their utility as potential vaccine epitopes. By contrast, discontinuous epitopes are thought to be more prevalent within folded proteins, but are far more challenging to determine experimentally. The archive catalogues the sequence of binding peptides, and also gives the length and source: TTGDVIASS, a 9 amino acid peptide from Escherichia coli non-fimbrial adhesion. Residues identified as important in binding to the antibody are recorded. This may correspond to a whole peptide or a subsequence, such as TTGDVI in the above example. The peptides are also categorized in terms of their relative observed immunodominance. Antibodies host organism and isotype are recorded. The current B cell epitope archive contains 3,541 epitopes.\nT Cell Epitopes\nT cell epitopes are short peptides bound by major histocompatibility complexes (MHC) and subsequently recognized by T cells. Epitopes recognized by both CD4+ and CD8+ T cells are included in the database. Such epitopes can be identified in many different ways. However, this diversity of measurement imposes a certain need for consistency, necessitating the requirement for recording a range of different experimental methods. The archive has expanded to include 4,158 entries. The entries contain the epitopes, ranging in length from 4 to 38 amino acids, peptide information, detailing the source, with links to Swiss-Prot and the corresponding MHC restriction data such as Serotype, Allele and Class. Additionally, the peptides are categorized in to groups such as Allergens, Bacterial, Cancer, Human, Viral and Self peptides.\nMHC \u2013 Peptide binding\nAntiJen continues to archive quantitative data on the thermodynamics of peptide interactions [18,19], and it has expanded in number and content, with additions such as experimental conditions, plus specific Standard and Competitor peptide concentrations used in the assays. The current archive contains 15,454 entries. The sequence of the binding peptide, along with the source, plus relevant MHC restriction data is recorded. The restriction alleles currently include those from Human, Mouse, Rat, Rhesus Monkey, Cotton-top Tamarin, and Chimpanzee. AntiJen contains IC50 values, binding affinity measurements from competitive binding assays, for which the standard and competitor peptides and concentrations are recorded, plus BL50 values, calculated from peptide stabilizing assays. Where possible, antibodies and the concentrations used to calculate BL50 values are archived. Additionally, but on a somewhat smaller scale, equilibrium association (KA) and dissociation (KD) constants are recorded for peptide-MHC interaction. Melting temperatures (Tm) and signal wavelength are also recorded; this is the temperature and wavelength at which 50% of the MHC protein is denatured as measured by circular dichroism. AntiJen also records so-called Weak\/Non-binders. This indicates that the peptide has been tested in an MHC restriction assay and has been found to exhibit a binding affinity, i.e. an IC50 value > 10,000 nM for a radio-ligand assay, so low that it can be categorized as inactive.\npMHC-T Cell Receptor interaction\nThe TCR sub-database contains 782 entries, which records thermodynamic and kinetic binding data for the interaction of peptide-MHC (pMHC) complexes with TCRs. Different MHCs exhibit a distinct selectivity for certain peptide sequences. T cell receptors, in their turn, also exhibit different affinities for peptide-MHC complexes. The entries contain epitope, peptide source and MHC restriction data, as described above, plus TCR structure information, located at the MMBD database . Furthermore, any mutations are noted and a designated name for the TCR is archived. In addition, the peptides are recorded as either agonists or antagonists. The binding data is given as equilibrium constants (KD), EC50 values, rate of association (Kon), rate of dissociation (Koff), association constant (KA) and the half-life (t1\/2) of the TCR-peptide interaction.\nTAP Binding\nThis dataset contains binding data for the interactions between peptides and the TAP transporter, one of the principle steps in antigen presentation. As with the peptide-MHC database, the data is established from competitor binding experiments, based on labelled assays. Therefore, standard and competitor peptide sequences and their concentrations are recorded. The binding data is given as IC50 and KD values. The database currently contains 1,106 entries, with peptides from Human, Rat and Mouse sources. Based on IC50 values > 10,000 nM, the peptides are categorized as weak\/non-binders. The entries have increase in number from the level found in JenPep, although several entries were removed in an effort to increase the accuracy and consistency of the archive (Table 2).\nPeptide-MHC Kinetics\nAntiJen's kinetics sub-database, which contains 1,150 entries, mostly relates to Class I MHC data. It records measurements for forward and reverse rate constants for complexation events. This complements the thermodynamic measurements on peptide-MHC binding described above. The data currently focuses upon both the half-lives of binding interactions, as well as association and dissociation rate constant values (Kon and Koff) for the recorded epitopes. Additionally, concentrations of the peptide, MHC and TAP are archived. The half-life for radioisotope labelled \u03b22-microglobulin dissociation from an MHC class I complex, as measured at 37\u00b0C, is also archived. This is a kinetic measurement rather than a thermodynamic one, although it is often assumed that the greater the half-life the stronger the peptide-MHC complex. The half-life (t1\/2) equals:\nHere the t1\/2 corresponds to the dissociation of the MHC-\u03b22 microglobulin complex rather than the kinetics of the protein-ligand interaction, but is still peptide dependent, as well as kinetic in nature.\nImmunological Protein-Protein Interactions\nThe immune system is built around protein interactions therefore we developed another new sub-database which deals with Immunological Protein-Protein Interactions (IPPI). This archive contains 2,675 entries based on a variety of binding data, such as Kon and Koff rates, for a range of macromolecules implicated in physiological or pathological interactions, as well as KD, KA and IC50 values. The molecules include receptors such as CD4 or CD8 molecules, superantigens and other microbial virulence factors, cytokine receptors and cell adhesion molecules. The entries detail both protein partners involved in the binding interaction, with links provided to the NCBI-Entrez database. Additional data for MHC receptors is archived, whereby the reactive epitope is recorded and the co-receptors are categorized into viral, bacterial and self peptides. MHC data outlined in the previous databases is given, where appropriate including any mutations to the MHC.\nAntibody \u2013 Protein Binding\nAntiJen also contains a further sub-database, which comprises thermodynamic data relating to antibody-antigen binding. The dataset contains 395 entries for antigen proteins and antibodies, mostly derived from viral and mammalian sources. Reported values were obtained using radiolabelled assays and BIAcore analysis. This archive should aid in the selection of antibodies and peptides for in vitro studies. The entries list the antibodies and the binding\/kinetic data, consisting of KD and KA values and to a lesser extent Kon, Koff and IC50 values.\nPeptide Libraries\nThis archive further complements our MHC binding databases by indicating the relative contribution of residues within peptide libraries to MHC binding. 897 entries contain quantitative specificity data derived from position specific peptide libraries [20]. This catalogues the relative effect on affinity, in the form of IC50, log relative SD50 and log SI values, of all substitutions, at all peptides positions, against a random sequence backdrop. All of the libraries relating to a known peptide binder are designated a name within AntiJen, this usually consists of the author and year of publication. The archive contains the core peptide, along with the mutation position and the substituted amino acid. The corresponding MHC data is given as mentioned above.\nDiffusion Co-efficients\nTo further increase the range of data archived, AntiJen also contains 759 records of cellular biophysical data, in the form of diffusion co-efficients, recorded as cm2s-1, for a diversity of cell surface molecules, including MHCs (Mouse and Human), viral peptides and other receptors [21]. The molecules are either chemically or fluorescently labelled and then measured using one of two methods: Single Particle Tracking (SPT) or Fluorescence Photobleaching Recovery (FPR). SPT monitors the lateral motion of a labelled molecule while FPR measures the rate of subsequent infiltration from a photobleached section of the membrane. Friction co-efficient data is also given, which measures of the velocity and force applied to an antibody-coated bead. Records contain the cell or cell type where diffusion is occurring, the name of the diffusing protein along with the form of labelling applied. Furthermore, specific experimental data is given such as antibody bead size. In this case the diffusion of the beads is monitored. Increasingly, data relating to photobleaching is included, such as beam power, bleaching duration, pre- and post-bleach time, etc..\nCopy Numbers\nThe final sub-database contains 414 measures of cell surface populations of different molecules, called cell surface copy numbers hereafter. This database focuses on an array of molecules, including Class I MHCs (22) and TCRs [23]. The entries are given as number and type of MHC molecules, number of MHC-peptide complexes or abundance of peptides associated with each MHC serotype, generally defined by mass spectroscopy. The entries list the cell type, the antibody bound to the MHC and, if appropriate, the binding epitope. This only applies to the number of MHC-peptide complexes and the abundance of peptides associated with each MHC.\nSearching the database\nSearch mechanisms within AntiJen are significantly improved and allow either a detailed or a broad search from a simple user interface. From our experience with JenPep, we recognize that accessibility to the data in a user friendly manner is a vital requirement, and have improved our current search mechanisms and developed new search interfaces. Two different search mechanisms are available. One is based on BLAST [24] and the other is a bespoke system, allowing several alternative searches. Within a typical search, the user-entered search criteria are carried from an HTML form to a category specific PERL\/SQL script, which performs the database queries.\nThe BLAST search allows querying of a peptide or nucleotide sequence against the proteins contained in AntiJen; all entries containing data within AntiJen which are relevant to a protein sequence are linked via the BLAST output. A local database of protein sequences is searched with BLASTP or BLASTX [24] using the BLOSUM64 matrix. All BLAST control variables are fixed. An HTML Front-End (Figure 2), where a sequence can be entered or uploaded, connects to a web server-based PERL\/CGI scripts, which interacts with BLAST. An annotated version of the default BLAST output is produced and links to AntiJen entries via SWISS-PROT [12] accession codes, which act as a query within a Keyword search. This allows AntiJen entries to be viewed directly from the BLAST output.\nFigure 2\nOverview of the different search methods within AntiJen. The example search is focused upon an MHC ligand. The MHC ligand data can be searched directly (A) from a link on the AntiJen homepage, a broad search and specific search is available. A search for the epitope AMALLRLPLV, has one hit (D), this leads to the entry (G). All of the other sub-databases can be searched in this manner from the homepage. The two other searches are more generalised. A Keyword search (B) carries out a broad search on the whole database, for the criteria \u2013 Bacteria. This search gives 139 hits (E) and all of the sub-database entries can be selected from this output. The final search method is a BLAST search (C). The peptide (or nucleotide) sequence is queried against a local protein database. The output (F) provides links to the sub-databases relative to the protein.\nAt present, peptide string, keyword, and protein name index searches are available within the bespoke system, which allows querying of individual peptide sequences or at the level of whole protein antigens. An overview of the AntiJen search systems is given in Figure 2 and 3. Epitopes, MHC, TCR, TAP peptide binding and kinetics databases can all be searched using sequence strings. The search protocol first returns an epitope list and a count of epitope matches. Subsequently, experimental criteria can be accessed for each selected epitope. Peptides can be searched using an amino acid orientated query: a sparse peptide string, similar in form to a peptide binding motif [3] or a PROSITE pattern [25], is used to identify all matching sequences. See Figure 4. Alternately, a list of protein antigens within AntiJen can be searched using keywords; the thermodynamic binding data such as MHC-peptide, TCR-pMHC and TAP, plus the B and T cell archives, related to the search criteria can then be selected from the matches based upon the SWISS-PROT accession codes [12], displaying all corresponding entries in the database.The other search method, allows the IPPI, diffusion co-efficients, peptide libraries, antibody-protein and copy number sub-databases to be searched using an index method, within a user-friendly HTML drop-down menu. Each of these methods can also be moderated using subsidiary search filters, data size ranges, and result presentation alternatives, such as peptide length or IC50 values. Minimum and maximum values can be used to restrict results, as can selection of MHC restriction alleles.\nFigure 3\nSearchable database types within AntiJen. The database contains 3 types of searchable sub database: a set of Antigens searchable by keyword, various databases of functional and thermodynamic data searchable by peptide sequence, and a database of immunological protein-protein interactions searchable through an index. Peptide sequence searches can be explicit or \"motif\" based. Searches can also be focussed by setting the value ranges for properties, such as IC50 etc, recorded in the databases. Currently there is a link between all the thermodynamic and kinetic databases and between them and the database of antigens. Only antigens with data in one of the other sub-databases are included in the antigen database. Links to external databases are also indicated. The BLAST search provides an overall search of the databases except the Protein-Protein Interactions archive.\nFigure 4\nSparse peptide sequence search. Examples of the two search types available in AntiJen: (Search 1) a substring query and (Searches 2 and 3) PROSITE-like sparse queries allowing sets of variable (indicated by asterisks) and alternative (encased in square brackets) amino acids in the search. In our example, each query is an extension of the previous search. The initial search type returns a single hit, which is a weak binder. By introducing variable amino acid positions within the query string, the second query permits access to a larger data set with 39 hits being returned. The third search utilises both variable and alternative amino acid request functionality and returns 1996 hits. The number of entries returned can be reduced by specifying the epitope length, limiting IC50 values and restricting by one MHC allele. This search is constrained to peptides of amino acid length 9, which returns 280 hits. Constraining further by MHC allele HLA-A*0201, reduces this to 120. An additional constraint using the IC50 data filter, and requesting values below 500 only (the epitope range), reduces this again to 60.\nDiscussion\nThe name given to our new database, AntiJen, reflects a shift from a peptide orientated database structure, which was inherent within our earlier JenPep database, to one which can properly balance its focus on both protein antigens and isolated peptides. As such, it represents an important, integrated, immunological data resource. AntiJen now provides broad insight into both T cell and B cell mediated antigen recognition. In addition, through the auspices of the IPPI sub-database, the database also throws light on co-stimulation by co-receptors and gives important insights into the innate immune response. Our approach to protein-protein interactions, focusing on measured affinities, complements other methods, such as the Yeast-2-Hybrid system, which, while giving greater volumes of data, has problems of accuracy [26]. This, while not devoid of experimental artifact, gives a usefully different perspective on cataloguing protein-protein interactions. The further addition of weak binding notation to the MHC-peptide and TAP provides a greater overview of the nature of antigenic epitopes. This is further improved by the addition of the peptide libraries database, whereby key peptide residues can be highlighted. New databases have expanded the breadth of AntiJen to include biophysical data such as diffusion co-efficients and cellular data such as abundance of molecules. The antibody-antigenic protein sub-database will also provide a key resource for in vivo and in vitro studies, aiding in the selection of antibodies and peptide\/protein targets.\nAntiJen distinguishes itself from the other specific binding databases [2-8] in several ways. Firstly, more data is recorded; our MHC-peptide database contains over 2,000 more entries than MHCPEP [2] and 10,000 more entries than EPIMHC [7]. Additionally, we have not restricted our archive to only high binders or to a specific category, as seen in EPIMHC and the HIV sequence databases [5]. Furthermore, AntiJen is currently a curated database, which is constantly expanding.\nMost obviously, AntiJen is useful in the design of epitope and subunit vaccines. Additionally, AntiJen is helpful in the design of clinical diagnostics and other laboratory reagents, such as the selection of peptides for tetramer design. AntiJen is also useful in the parameterization of mathematical models in theoretical immunology [27]. The redevelopment of the database has focused not only on content, but also on infrastructure. The current system, based on epitope string, keyword and index searches, along with an overall BLAST search, plus the redesigned HTML interface, leads to much greater accessibility and usability. Finally, the database acts as a repository of quantitative, continuous data, for the development of data-driven in silico predictive models, such as prediction of epitopes and MHC binding [18,19,28,29] through QSAR modeling.\nFuture work\nFuture tasks in the development of AntiJen, fall into two principle categories: eliminating deficiencies, errors, and inconsistencies within the database and simultaneously reinforcing it by expanding its depth, breadth, and scope. We also need to monitor updates within external databases, so that any alterations are mirrored within the archive. Like all other such repositories, AntiJen is prone to both systematic and random errors within the data accumulation process. User feedback and our interactions with immunologists will hopefully address persisting errors. Deficiencies in our database include our current inability to encode chemically or post-translationally modified peptides, non-natural MHC mutants and non-amino acid peptidomimetic MHC ligands. Additionally, it would also be interesting to complement our existing data on TAP binding with information on antigen presentation pathways, such as proteasomal and cathepsin cleavage patterns. Moreover, the compilation of B cell or antibody epitope data is an area ripe for robust development. Linear and conformational B cell epitopes are very much larger in number than our current compilation, leaving us scope to greatly increase recorded epitopes.\nConclusion\nThe development of a database is always a work in progress. Not simply because the easily accessible literature is typically always increasing, but also because of the desire to capture as much of the existing, but hidden, literature, as possible. In the post-genomic era, the database has formed the bedrock and language of bioinformatics; increasingly databases are coming to underpin our modern understanding of biology as a whole. Traditionally, databases have arisen as a response to need, answering the individual and idiosyncratic questions posed by biologists. However, the history of bioinformatics databases has shown the extraordinarily diverse ways in which archived data can be used.\nIn creating AntiJen, we were motivated partly by our desire, and the desire of collaborators, to use the data within it to build predictive in silico models [16,17,28,29], and partly by a more altruistic desire to generate a useful, integrated database system with a quantitative focus. AntiJen has many potential uses throughout the immunological discipline, from immunoinformaticans to experimental immunologists and vaccinologists. By increasing the degree to which data is machine readable and web accessible, we open up new, and previously unthought-of, avenues for the bioinformatic exploration of immunological data.\nAntiJen is a primary data resource, amongst the most complete of its kind, yet, like SWISSPROT [12] or GenBank [30] decades ago, it is still relatively small and offers much scope for improved annotation. We see the database as a foundation from which to consolidate, through time, thus achieving a comprehensive resource of immunological data.","keyphrases":["vaccines","b cell epitopes","mhc","antibodies","t cell epitopes","tcr","mhc-peptide binding"],"prmu":["P","P","P","P","P","P","R"]}
{"id":"Pflugers_Arch-4-1-2270919","title":"Endocytic pathways: combined scanning ion conductance and surface confocal microscopy study\n","text":"We introduce a novel high resolution scanning surface confocal microscopy technique that enables imaging of endocytic pits in apical membranes of live cells for the first time. The improved topographical resolution of the microscope together with simultaneous fluorescence confocal detection produces pairs of images of cell surfaces sufficient to identify single endocytic pits. Whilst the precise position and size of the pit is detected by the ion conductance microscope, the molecular nature of the pit, e.g. clathrin coated or caveolae, is determined by the corresponding green fluorescent protein fluorescence. Also, for the first time, we showed that flotillin 1 and 2 can be found co-localising with ~200-nm indentations in the cell membrane that supports involvement of this protein in endocytosis.\nIntroduction\nThe role of biological cell plasma membrane endocytic pits, e.g. clathrin-coated pits [19] and caveolae [15], has been extensively studied over the last decades and still remains a hot topic. They not only provide cells with a means of transport across the membrane but also work as signalosomes aggregating receptors and ion channels [4, 17]. More recently, another membrane-microdomain-associated protein flotillin (1 and 2), traditionally thought to be associated with various receptors such as insulin receptor [1], has been shown to be responsible for clathrin- and caveolins-independent endocytosis [6], providing an alternative route of trafficking through the cell membrane. Whilst it is well known that clathrin and caveolin form basket-shaped pits in cell membranes [17, 25], the surface structures created by flotillin remain unknown.\nCurrent methods for endocytic pit function and dynamics studies include confocal microscopy, total internal reflection (TIR) microscopy [6, 20, 26], scanning and transmission electron microscopy [25] and atomic force microscopy [5]. Although fluorescence confocal microscopy is the one of the most commonly used microscopy techniques in cell biology, it provides limited information about single endocytic pit due to its lack of resolution. TIR has better vertical resolution and signal-to-noise ratio and has been used for single endocytic event following [2]. However, TIR is limited to observation of events that occur on the basal cell membrane only since it relies on imaging to a depth of about 100\u00a0nm from the glass surface and is not applicable to studying apical cell surfaces. In addition, using fluorescence-based microscopy techniques, it is impossible to determine the precise location of the endocytic pit relative to the cell membrane since the membrane position is hard to define.\nOn the other hand, electron microscopy techniques have more than sufficient resolution and provide much more detailed information about the surface topography than optical images since the cell membrane can be imaged. However, these require fixation and specialised treatments of the cells, which potentially can alter the structure of interest and are thus incompatible with live studies.\nScanning probe microscopy techniques may address a number of these issues by providing topographical data of surfaces. Atomic force microscopy has been used to study cell surfaces but, due to the direct interaction of its probe with the sample, imaging soft and mobile cell surfaces is difficult, limiting most studies to fixed cells or isolated membrane leaflets which are more rigid [5]. Scanning ion conductance microscopy (SICM), which uses a glass micropipette as an imaging probe [9], has no such disadvantage and allows non-contact visualisation of the topography of living cells [13, 14]. This has recently been combined with simultaneous confocal fluorescence imaging, resulting in a new technique\u2014scanning surface confocal microscopy (SSCM) [7]. Also, since its inception, the resolution of the technique has been significantly improved, making it possible to visualise structures as small as protein complexes on surfaces of live cells [23]. This novel technique allows us to identify fluorescence located on the surface of the cell and localise it with topographically observed nanometre-scale structures (unpublished results).\nMaterials and methods\nSSCM\nSSCM is based on a combination of SCM and SICM. SICM is a scanning probe microscopy technique[14, 23] in which the ion current flowing into a nanopipette is used to control the vertical (z axis) position of the cell relative to the pipette tip. As shown diagrammatically in Fig.\u00a01a (not to scale), in SSCM, the cell is moved up and down in the z direction while scanning in the x and y directions, so its surface is always the same distance from the nanopipette. A laser is passed up a high numerical aperture objective so that it is focused just at the tip of the nanopipette, and a pinhole is positioned at the image plane so that the confocal volume is just below the pipette, as described [22]. Thus, a fluorescence image of the cell surface is obtained in a single scan, as well as a simultaneously captured image of the cell topography.\nFig.\u00a01Topographical imaging of endocytic pits in living cells by SICM. a Schematic diagram of the scanning ion conductance microscope. b SICM topographical image of live Cos-7 cell. c High resolution topographical SICM image of live Cos-7 cell membrane revealing numerous pits. d High resolution topographical SICM image of a fixed Cos-7 cell membrane revealing numerous pits. e Zoomed image showing a single pit (top). Topographical profile of a pit in a live cell (bottom). f Zoomed image showing two pits (top). Topographical profile of pits in fixed cell (bottom)\nThe SCIM scanning head was developed in collaboration with Ionscope Limited, UK and mounted on a Nikon TE2000-U Inverted Microscope (Nikon Co., Japan). The sample holder was attached to a 100-\u03bcm HERA XY Nanopositioning System (Physik Instrumente (PI) GmbH & Co., Germany) used for lateral scanning. Vertical measurement and modulation was provided by 12-\u03bcm LISA XY Nanopositioning System (Physik Instrumente (PI) GmbH & Co., Germany). Both piezo stages were mounted on 25-mm translation stage DC motors (Physik Instrumente (PI) GmbH & Co., Germany) to provide coarse lateral and vertical approach. The setups were controlled via a computer with a SBC6711 DSP board equipped with A4D4 ADC\/DAC modules (Innovative Integration, USA) using SICM software v. 1.2.00 (Ionscope Limited). The time to acquire a 512\u2009\u00d7\u2009512 pixel image was approximately 10\u00a0min.\nTwo types of nanopipettes were used for the experiments. For low resolution images, nanopipettes with internal diameters ~150\u00a0nm were pulled from borosilicate glass capillaries. High resolution imaging was made using quartz nanopipettes with internal diameters ~70\u00a0nm. The nanopipettes were made from 1.00-mm outer diameter by 0.5-mm inner diameter capillaries with inner filament (Sutter Instrument, USA) using a laser-based Brown\u2013Flaming puller (model P-2000, Sutter Instrument, San Rafael, CA, USA).\nThe nanopipettes, backfilled with phosphate-buffered saline (PBS) and lowered in PBS, produced a resistance of approximately 300\u00a0M\u03a9 for quartz and 100\u00a0M\u03a9 for borosilicate pipettes. The maximum ion current measured using an Axopatch 200B (Axon Instruments, USA) was ~0.7\u00a0nA for quartz and ~1.5\u00a0nA for borosilicate pipettes. The set-point for imaging was 1% of the maximum of modulated ion current.\nThe excitation light source was provided by a GPNT-02 laser diode (532-nm wavelength, IQ1A 635-nm laser; Power Technology Inc., USA). The optical recording system consisted of a Nikon TE2000-U Inverted Microscope equipped with a \u00d7100 1.3-N.A. oil-immersion objective. The excitation light was fed through an epi-fluorescent filter block and emitted light was collected by a photomultiplier with a pinhole (model D-104-814; Photon Technology International, UK).\nImage processing and data analysis\nMatching VLP topographical structure to its corresponding fluorescent signal was done as follows: fluorescent confocal images were threshold to subtract the background, and the positions of individual fluorescent spots were marked by arrowheads and the multiple spots (where individual signals could not be resolved) circled. All positional markers were then grouped into one template and placed over the simultaneously recorded topographical image. As the result of this procedure, those topographical features having corresponding fluorescent signals were marked.\nImage contrast enhancement included slope correction and high-pass filtration performed similarly as previously described [10, 18]. The slope was calculated and subtracted from images by least squares algorithm [21] resulting in image flattening. A high-pass filter is a filter that passes high frequencies efficiently but reduces frequencies lower than the certain specified (cutoff) frequency. When applied to a 3D image, high-pass filtering results in finer, i.e. high frequency, details to stand out because larger features are eliminated.\nCell culture and plasmids\nMonkey Cos-7 cells were routinely maintained at 37\u00b0C in 5% CO2, using DMEM (GIBCO\/BRL) containing 5% (vol.\/vol.) FCS. The plasmid DNA used in the experiments were in pCi (Promega) containing either caveolin-green fluorescent protein (GFP), clathrin-GFP, flotillin-1-GFP or flotillin-2-GFP.\nTransfection procedure\nCos-7 cells (1\u2009\u00d7\u2009106 cells per flask) were plated into a T25 flask and incubated overnight at 37\u00b0C in DMEM containing 5% FCS. Cells were washed prior to transfection with PBS, and complexes of Lipofectamine (Invitrogen) and plasmid DNA at a ratio of 1\u00a0\u03bcl to 1\u00a0\u03bcg were added in Optimem (Invitrogen) without FCS to the cells. For flotillin transfections, a mixture of flotillin-1- and flotillin-2-GFP plasmids were complexed together and added to the cells. After 2\u00a0h, the media was replaced with DMEM containing 5% FCS. After 24\u00a0h, the cells were trypsinised and plated onto coverslips (5\u2009\u00d7\u2009104 per well) and incubated at 37\u00b0C overnight. Cells where then either used for live imaging or fixed for 20\u00a0min with 3% formaldehyde containing 5% sucrose.\nResults\nImaging of the cell surface using SICM\nIn order to access the apical surface of Cos-7 cells, we used a basic arrangement of the SICM. A schematic diagram of the microscope is presented in Fig.\u00a01a (not to scale), while a detailed description of the instrument can be found in \u201cMaterials and methods\u201d section. SICM [9, 13, 14] is a type of scanning probe microscopy that uses a glass nanopipette as a sensitive probe to acquire a topographical image of the surface. Distance feedback-control is based on the ion current that flows between electrodes in the nanopipette and the bath, and this current is reduced as the pipette approaches the surface. This allows non-contact imaging of live cells in a physiological buffer.\nUsing the SICM technique, we are able to either examine the morphology of the whole cell or achieve high resolution images of the cell surface. Scanning the complete cell surface gives insight into the number of macrostructures present on the cell surface, for example microvilli, and can be used to probe cell surface dynamics [8]. The morphology of the cell also has an impact on the resolution of SICM images, since large structures and steep slopes make small features difficult to resolve. A typical SICM image of live untransfected (control) Cos-7 cell in physiological conditions is presented in Fig.\u00a01b. Cos-7 cells have very few macrostructures visible on the surface, and although the highest part of the cell membrane above the nucleus (light area, Fig.\u00a01b) generates a steep slope, there are also flat peripheral areas. High resolution scans of these peripheral areas generate images containing a number of indentations of the cell surface (Fig.\u00a01c). Observed indentations range from 50- to 160-nm diameters (see an example in Fig.\u00a01e) and could present endocytic pits formed by clathrin and\/or caveolin. The mobility of these indentations was relatively high compared to the present scan rate of our SICM as shown later in the paper; therefore, we used fixed cells to identify particular endocytic pits and imaged them at a higher resolution. A high resolution image of a fixed Cos-7 cell presented in Fig.\u00a01d reveals similar indentations as seen in Fig.\u00a01c but at higher clarity and contrast. A digitally zoomed image is presented in Fig.\u00a01f and shows two pits (top) with the corresponding topographical profiles (bottom).\nFig.\u00a02Topographical and fluorescent imaging of clathrin coated pits in fixed clathrin-GFP transfected Cos-7 cells by scanning surface confocal microscope. a Schematic diagram of SSCM. b Topographical image of cell. c Fluorescent image of the clathrin-GFP transfected cell shown in b. d 3D representation of overlaid topographical and fluorescent images shown in b and c, respectively. e High resolution topographical image of cell surface revealing numerous clathrin-coated pits. f Same topographical image as in e but inverted and presented in a red palette. g Overlaid inverted topographical and fluorescent images shown in f and g, respectively. The image reveals that the pit topography matches the clathrin-GFP fluorescence. h The distribution of clathrin-coated pit width calculated from the SICM topographical images\nScanning surface confocal microscopy\nIn order to identify what pit indentations detected topographically by SICM corresponded to what endocytic pits, clathrin or caveolin, we transfected Cos-7 cells with corresponding GFP construct and then used scanning surface confocal microscopy to study the transfected cells.\nA schematic diagram of the microscope is presented in Fig.\u00a02a (not to scale). Basically, SSCM is a combination of SICM and scanning confocal microscope [7]. SSCM also uses a glass nanopipette to acquire a true topographical image of the surface and a combination of a laser, pinhole and photomultiplier to gather fluorescent information from the cell surface, removing any out-of-focus fluorescence. Because the laser beam is always focused on the very tip of the nanopipette, topographical and fluorescent data are spatially aligned, thus enabling correlation between topographical features and fluorescent signals to be found.\nImaging clathrin-coated pits in membranes of fixed cells using SSCM\nCells transfected with clathrin-GFP were scanned, at low and high resolution, to examine the distribution of clathrin-coated pits on the cell surface. A typical topographical image of clathrin-FGP transfected fixed Cos-7 cell, together with its fluorescent image, is presented in Fig. 2b and c, respectively. An overlay of topographical and fluorescent images is shown in Fig.\u00a02d. Peripheral areas of the cell contain punctated GFP fluorescence and had a flat topography. A high resolution topographical scan of these cell surfaces at a 7\u2009\u00d7\u20097-\u03bcm scan (Fig.\u00a02e) reveals numerous indentations ranging from 58 to 159\u00a0nm with an average depth of 102\u00a0nm (see distribution in Fig.\u00a02h).\nIn order to identify what proportion of the imaged pits were real clathrin-coated pits, we overlaid the topographical and fluorescent data. To make pit indentations that appear as dark spots in topographical images, visible when overlaid with the fluorescence image, the topographical image was inverted and red palette was applied (Fig.\u00a02f). An overlaid, inverted topography red palette and clathrin-GFP fluorescence image is presented in Fig.\u00a02g. As can be seen, there is a \u2018cloud\u2019 of green fluorescent signal around almost every single red spot that corresponds to indentations in the topography, giving a high degree of co-localisation between these structures and fluorescent puncta. Therefore, we can conclude that the majority of observed indentations are clathrin-coated pits. Analysis of the images revealed that 102 out of 115 (88.7%) detected endocytic pits co-localised with clathrin-GFP fluorescent spots. Presumably, this is because we are also detecting other types of pits that are not clathrin-coated. Reverse analysis showed that 11 out of 113 (9.7%) detected clathrin-GFP fluorescence spots did not match pit indentations. This could reflect the fact that clathrin-coated pits become highly mobile and move away from the surface inside the cell as soon as they bud off the cell membrane [16]. At higher magnification, we could also see clustering of the clathrin-coated pits in round patches (data not shown) that is similar to observations made by Frankel et al. [5].\nImaging caveolae in membranes of fixed cells using SSCM\nUsing SSCM, the surface of caveolin-GFP transiently transfected cells was then examined. Low resolution topographical, fluorescent and overlaid images are presented in Fig. 3a,b and c, respectively. Higher resolution studies (Fig.\u00a03d and e) revealed indentations in the cell membrane. These pits were slightly smaller than the clathrin-coated pits, ranging between 52 and 90\u00a0nm and giving a mean depth of 69\u00a0nm (see distribution in Fig.\u00a03f).\nFig.\u00a03Topographical and fluorescent imaging of caveolin pits in fixed caveolin-GFP transfected Cos-7 cells by SSCM. a Topographical image of cell. b. Fluorescent image of caveolin-GFP transfected cell shown in a. c 3D representation of overlaid topographical and fluorescent images shown in a and b, respectively. d High resolution topographical image of cell surface. e High resolution fluorescent image of caveolin-GFP transfected cell shown in d. f Caveolin pit width distribution histogram calculated from SICM topographical images. g Digital zoom of the topographical image shown in d (dotted square) revealing numerous caveolin pits. h Same zoomed topographical image as in g but inverted and presented in a red palette. i Overlaid inverted topographical image shown in h and digitally zoomed fluorescent image from the area shown in e (dotted square). The image reveals that pits match the caveolin-GFP fluorescence\nOn closer examination using digitally zoomed images [topographical (Fig.\u00a03g), topographical inverted and in red palette (similar to one described for clathrin) (Fig.\u00a03h) and an overlay of the topography and fluorescence image (Fig.\u00a03i)], it could be seen that the indentations in the cell surface co-localise with fluorescent signal. However, compared to clathrin-coated pits, identification of caveolae is more problematic. In all experiments conducted, the fluorescent images have a punctate appearance. However, in only two out of 26 experiments did fluorescence and structure co-localise. In those experiments, 139 pits were observed on the cell surface and only 13 of them (9.35%) were identified as matching caveolin-GFP fluorescent signal. This is in good agreement with the previous measurement that 90% of the pits identified in the topographic image on the cell surface are clathrin-coated and indicates that the other 10% are caveolae.\nOur observation that we detect many fluorescence spots from caveolin-GFP that do not correspond to pits in the SICM image suggests that many caveolae are below the cell surface or present on the surface but not resolvable. By transmission electron microscopy, they have been identified as 50\u2013100\u00a0nm in size [15]; however, this is often the size of the whole structure, with the neck being much smaller [25]. It has even been suggested that there is a proteinaceous cap covering the neck of the caveolae [25]. If this is the case, and the cap could influence the flow of ions, then we would not be able to image caveolae structures on the cell surface unless the cap is open.\nImaging flotillin in membranes of fixed cells using SSCM\nTo our knowledge, flotillin 1 and 2 has not been yet associated with cell membrane structure that could be detected topographically by any microscopy technique. However, it has been shown that this protein is present in lipid rafts [1] and is responsible for clathrin- and caveolin-independent endocytosis [6]. Here, we examined Cos-7 cells transfected with flotillin 1 and 2 by SSCM for the presence of topographically detectable membrane features that could potentially co-localise with flotillin-GFP fluorescent signal.\nLow resolution topographical and confocal images together with overlaid image of flotillin-GFP-transfected Cos-7 cells are presented in Fig.\u00a04a,b and c, respectively. Higher-resolution topographical images of the cell surface (Fig.\u00a04d and g) revealed 200\u2009\u00b1\u200920-nm indentations. Figure 4e presents inverted red palette topographical image of Fig.\u00a04d. Inverted red palette topography and fluorescence overly reveals that some indentations co-localise with the flotillin-GFP fluorescent signal (Fig.\u00a04f, arrows). Remarkably, we have found that larger and higher-intensity fluorescent spots correlated with smooth protrusions from the cell surface (Fig.\u00a04g,h and i, hollow arrows). The image presented in Fig.\u00a04h is a high-pass filtered topographical image of the same data as in Fig.\u00a04g.\nFig.\u00a04Topographical and fluorescent imaging of fixed flotillin-GFP transfected Cos-7 cells by SSCM. a Topographical image of cell. b. Fluorescent image of flotillin-GFP transfected cell shown in a. c 3D representation of overlaid topographical and fluorescent images shown in a and b, respectively. d High resolution topographical image of the cell surface revealing numerous indentations. e Same topographical image as in d but inverted and presented in red palette. f Overlaid inverted topographical image shown in e and high resolution fluorescent image of flotillin-GFP acquired from the same area. The image reveals that some indentations on the cell surface match flotillin-GFP fluorescence (white arrows). g High resolution topographical image of cell surface revealing numerous indentations (solid arrows) as well as two protrusions (hollow arrows). h Same topographical image as in g but high-pass filtered. i High resolution fluorescent image of caveolin-GFP transfected cell shown in g. The arrows point to indentations that match flotillin-GFP fluorescence. Hollow arrows point to protrusions that match flotillin-GFP fluorescence\nImaging endocytic pits in membranes of living cells using SSCM\nIn order to test whether SSCM can identify particular endocytic pits in membranes of live cells, we performed a series of experiments with live clathrin-GFP transfected Cos-7 cells. Figure 5a and b presents normal and inverted red palette topographical images of a live cell. When overlaid with fluorescence, the inverted red palette topography shows that almost all topographically detected pits co-localise with clathrin-GFP fluorescence. There are fluorescence spots that are not round, but elongated in shape that do not match pit indentations on the surface. These spots of fluorescence probably reflect fast-moving clathrin vesicles right under the cell membrane.\nFig.\u00a05Live topographical and fluorescent imaging of clathrin coated pits in clathrin-GFP transfected Cos-7 cells by SSCM. a High resolution topographical image of live cell membrane revealing numerous clathrin-coated pits. b. Same topographical image as in a but inverted and presented in red palette. c Overlaid inverted topographical image shown in a and fluorescent image of the same area. The image reveals that, on live cells, we can detect that the pits\u2019 topography match clathrin-GFP fluorescence. d Sequence of topographical images of live cell membrane revealing dynamics of the clathrin-coated pits. The images are separated by 10\u00a0min\nFigure 5d shows a sequence of three topographical images acquired from the same area of a cell with 10-min intervals. As can be seen, the indentations that correspond to endocytic pits are highly mobile and appear on or disappear from the surface of the cell membrane. It is beyond the current time resolution of our SSCM to follow the dynamics of these pits. However, this is the first time that endocytic pits are resolved topographically on the surface of live cell.\nDiscussion\nBy combining high resolution ion conductance imaging of the cell surface topography with fluorescence confocal imaging, we can identify the molecular nature of endocytic pits on the surface of living cells and measure the topography of the pits. For the first time, we showed that flotillin 1 and 2 is involved in the formation of ~200-nm-size indentations in the cell membrane. This observation is important evidence in support of the involvement of this protein in clathrin- and caveolin-independent endocytosis.\nWe have found on Cos-7 cells that about 89% of the detected pits are clathrin-coated and 9% are caveolae, leaving a small percentage to be presented by flotillin pits. In each particular case, cell preparation transfection could introduce some deviation of clathrin\/caveolin\/flotillin proportion comparing to untransfected control. The fact that clathrin-coated pit formation is dependent on multiple factors [11, 24] provides indirect evidence that transfection may not influence the amount of pit formation. In contrast, it has been shown that expressing the caveolin in cells that do not contain this protein is enough to form caveolae [3]. However, there are other studies indicating that, although in caveolin transfected cells the total amount of produced caveolin is increased, the concentration of caveolin in the cell membrane remains unchanged [12]. The sizes of the pits we have measured are in good agreement with those obtained by electron microscopy.\nWe have also shown that it is possible to apply our method to live cells. The clathrin-coated pits show fast dynamics on the time scale of our current imaging but suggest that improvements in the speed of imaging should allow us to follow the dynamics of endocytic pits on living cells with the intriguing possibility of directly imaging the endocytic process in real time.","keyphrases":["fluorescence","caveola","endocytosis","membrane topology","membrane transport"],"prmu":["P","P","P","M","R"]}
{"id":"Sleep_Breath-3-1-1794626","title":"Review of oral appliances for treatment of sleep-disordered breathing\n","text":"Between 1982 and 2006, there were 89 distinct publications dealing with oral appliance therapy involving a total of 3,027 patients, which reported results of sleep studies performed with and without the appliance. These studies, which constitute a very heterogeneous group in terms of methodology and patient population, are reviewed and the results summarized. This review focused on the following outcomes: sleep apnea (i.e. reduction in the apnea\/hypopnea index or respiratory disturbance index), ability of oral appliances to reduce snoring, effect of oral appliances on daytime function, comparison of oral appliances with other treatments (continuous positive airway pressure and surgery), side effects, dental changes (overbite and overjet), and long-term compliance. We found that the success rate, defined as the ability of the oral appliances to reduce apnea\/hypopnea index to less than 10, is 54%. The response rate, defined as at least 50% reduction in the initial apnea\/hypopnea index (although it still remained above 10), is 21%. When only the results of randomized, crossover, placebo-controlled studies are considered, the success and response rates are 50% and 14%, respectively. Snoring was reduced by 45%. In the studies comparing oral appliances to continuous positive airway pressure (CPAP) or to uvulopalatopharyngoplasty (UPPP), an appliance reduced initial AHI by 42%, CPAP reduced it by 75%, and UPPP by 30%. The majority of patients prefer using oral appliance than CPAP. Use of oral appliances improves daytime function somewhat; the Epworth sleepiness score (ESS) dropped from 11.2 to 7.8 in 854 patients. A summary of the follow-up compliance data shows that at 30 months, 56\u201368% of patients continue to use oral appliance. Side effects are relatively minor but frequent. The most common ones are excessive salivation and teeth discomfort. Efficacy and side effects depend on the type of appliance, degree of protrusion, vertical opening, and other settings. We conclude that oral appliances, although not as effective as CPAP in reducing sleep apnea, snoring, and improving daytime function, have a definite role in the treatment of snoring and sleep apnea.\nIntroduction\nTreatment of sleep-disordered breathing (i.e. snoring, upper airway resistance syndrome, sleep apnea syndrome) can be divided into four general categories. These include: (1) lifestyle modification, i.e. weight loss, cessation of evening alcohol ingestion, sleep position training, (2) upper airway surgery, (3) oral appliances, and (4) CPAP. Although the latter category provides the most reliable therapeutic modality and is the most widely used method to treat sleep-disordered breathing today\u2014it is also the most cumbersome one. Many patients, particularly young non-apneic snorers, find it unappealing, difficult to tolerate, and unacceptable. The only other non-invasive alternative, which can produce favorable results within a short time, is oral appliances.\nAlthough there are several reviews of oral appliances, which have appeared since the start of the new millennium [1\u20136], including a recent review and practice parameters for treatment of snoring and sleep apnea [124, 125], this treatment modality is still underutilized. Even the dentists who are primary providers of this treatment, lack education in this area. For example, Bian [7] surveyed 500 general dentists in the state of Indiana and found that 40% \u201cknew little or nothing about oral appliances for treatment of obstructive sleep apnea\u201d. Unarguably, the knowledge about oral appliances among dentists and physicians varies geographically, being higher in large urban centers, which provide more educational opportunities locally, but the results of the survey certainly indicate a need for more education in this area.\nThis review will summarize our current state of knowledge of the efficacy of oral appliances for the treatment of snoring and obstructive sleep apnea. We shall not limit this review to a simple summary of the effect of oral appliances on nocturnal respiration, but will also examine other aspects of this therapy, such as the reduction in symptoms, vascular consequences, side effects, and compliance. However, the main objective of this review will remain to be the examination of the current data dealing with the efficacy of oral appliance therapy for the treatment of snoring and sleep apnea.\nHistorical aspects\nGeorge Cattlin [8] was probably the first person who seriously thought that the route of breathing may influence sleep quality and daytime function. He attributed good health of the native North American Indians, compared to their immigrant European counterparts, to the fact that they are taught, from the early age, to breathe through the nose rather than the mouth. He pointed out that breathing through the nose promotes more restful and better quality sleep, which translates into better daytime function and better general health. After the publication of his book, there appeared many patents describing devices designed to promote nasal breathing. Some of the early patented appliances are shown in Fig.\u00a01.\nFig.\u00a01Examples of early oral appliances\nHowever, modern published clinical work begins in 1903, when Pierre Robin first described a device, called the \u201cmonoblock\u201d, for the treatment of glossoptosis [9]. More than 30\u00a0years later, he used an oral appliance to reposition the mandible [10]. For the next 50\u00a0years, little work was done in this field. It took almost another 50\u00a0years to start using oral appliances for the treatment of snoring and sleep apnea when Cartwright and Samelson [11] described the tongue retaining device in 1982. This work stimulated further investigations, resulting in many subsequent studies, many of which will be summarized in this review.\nTypes of appliances\nAlthough the type and number of specific appliances may be bewildering and is still growing (Table\u00a01), all may be divided into three general groups: soft palate lifters (SPL), tongue retaining devices (TRD), and mandibular advancement appliances (MAA). The first category is virtually no longer in use today. The second category is used very seldom, mainly if there are dental reasons precluding the construction of MAA. The last category (MAA) is by far the most common type of dental appliance in use today. It protrudes the mandible forward, thus preventing or minimizing upper airway collapse during sleep. These devices can be either fixed (i.e. the protrusion distance cannot be changed), or variable (i.e. protrusion can be increased or decreased). The final protrusion distance represents a delicate balance between side effects and efficacy. For this reason, the construction and fitting of the appliance should be done by a dentist with an expertise in this area who is familiar with different appliances, is capable of selecting the appropriate one based on the dental examination and has access to a sleep laboratory where the objective efficacy of the appliance can be verified. \nTable\u00a01Examples of oral appliancesOral appliancesThe EquilizerJasper jumperEsmarkThe SilencerPM PositionerTPEKlearwayTongue locking applianceSnoreExNAPAAdjustable soft palate lifterHAPTAPZ-training applianceTessiTOPSSnore-no-moreSnore GuardSNOARElastometricSilent NightHerbstSUADTheraSnore\nMechanism of action of oral appliances\nMuch was written on how and why oral appliances may improve snoring and sleep apnea. The results show that upper airway obstruction during sleep may occur at any site between the nasopharynx and the larynx. The most common sites of obstruction are behind the base of the tongue (retroglossal) and behind the soft palate (retropalatal). This partial or complete occlusion of the upper airway during sleep is a consequence of abnormal anatomy and physiology (i.e. the airway is narrow and \u201cfloppy\u201d). There is still a lively debate as to the relative contributions of abnormal anatomy vs abnormal physiology in the pathogenesis of upper airway obstruction during sleep [12, 13]. This debate is partially fuelled by the fact that airway narrowing or even a complete occlusion is a normal physiological event during sleep. In patients with sleep apnea, this normal response is exaggerated. The question then becomes whether this exaggerated response is due to abnormal neuromuscular control superimposed on the otherwise anatomically normal airway, or is the airway anatomically narrower than normal without any abnormalities in the neuromuscular control. It is now an accepted fact that a combination of abnormal anatomy and physiology is necessary to produce pathological repetitive narrowing (or complete occlusion) of upper airway during sleep\u2014i.e. sleep apnea.\nGiven that sleep apnea and snoring are a consequence of abnormal anatomy and physiology of the upper airway, is there evidence that oral appliances can correct these abnormalities, at least in some patients? There are several studies, not only during wakefulness, but also during sleep, indicating that advancing the mandible forward can enlarge the airway and reduce pharyngeal collapsibility [14\u201319, 120, 123] in normal subjects and patients with sleep apnea. These investigations confirmed the effect of oral appliances on upper airway properties. Some authors suggested that measurements of airway pressures during sleep may even predict the beneficial response to oral appliances. For example, recently, Battagel et al. [19] performed sleep nasendoscopy in 27 patients with sleep apnea. The mandible was gently advanced by 4\u20135\u00a0mm to simulate the effect of the mandibular appliance. The authors suggested that this procedure may help to determine whether a particular patient is a candidate for oral appliance therapy. Similarly, Ng et al. [123] measured upper airway pressures during natural sleep in 12 patients with obstructive sleep apnea to identify the site of airway collapse. The authors found that oropharyngeal, rather than velopharyngeal collapse, was predictive of the beneficial response to oral appliance. However, another study of 25 patients with sleep apnea where esophageal pressure was measured during sleep, found no significant differences in nadir esophageal pressure or cephalometric parameters between the patients who responded to oral appliance therapy and those who did not [120].\nIt is safe to conclude at this time that anatomical changes in the oropharynx, produced by mandibular advancement, result in the alterations of the intricate relationships between different muscle groups controlling the upper airway caliber. In some patients with sleep apnea these alterations may prevent the obstruction, in others\u2014worsen the obstruction, and yet in others, particularly in those with low level obstruction, the part of the airway where the obstruction occurs may be unaffected. There is currently no reliable way to predict the outcome of treatment with oral appliance in individual patients and therefore to select appropriate candidates for this treatment. Clinical features also do not seem to offer much help in trying to predict who will respond to oral appliances, or just the opposite, to identify patients who are not likely to respond to this therapy. When two expert maxillofacial surgeons examined (in a blind fashion) 100 patients with sleep apnea to determine if there were any contraindications to mandibular advancement devices, they found that primary contraindications were present in 34% of patients [20]. This relatively high rate of contraindications and disagreements between individual experts only point out that a team approach is necessary to select the proper treatment for patients with sleep apnea.\nResults of clinical trials\nThe onset of the new millennium carried forward the momentum started by Cartwright and Samelson [11] resulting in the increased use of oral appliances for the treatment of snoring and sleep apnea. However, the emphasis on the type of appliances has changed. Tongue retaining devices are currently seldom used, being almost completely replaced by the mandibular advancement appliances. The latter are sometimes also called mandibular repositioners, protruders, devices, splints, prosthesis, etc.\u2014but the common feature of all these appliances is their ability to adjust the degree of mandibular advancement to achieve resolution of snoring and sleep apnea.\nIn what follows, we shall summarize the results of clinical trials employing oral appliances for the treatment of sleep apnea and snoring. In addition, we shall review and summarize the information regarding changes in daytime function as a result of using the appliance, clinical and dental side effects, and compliance with treatment.\nOral appliances for the treatment of sleep apnea\nTable\u00a02 summarizes the results of individual studies using oral appliances starting with the 1982 polysomnographic study of TRD by Cartwright and Samelson [11] until the present. The only criterion for the inclusion of a particular study into this table, and thus the only common feature of all studies listed, was the availability of at least partial results of nocturnal monitoring of respiration with and without oral appliance. Otherwise, the studies are highly variable in their design, methodology, data analysis, outcome definition and assessment and presentation of results. This makes the interpretation of individual results, and particularly any attempt to summarize all of them, very challenging. Before describing the methods of analysis and presenting the summary of the data, it is very important to keep in mind the following points. \nTable\u00a02Clinical studies with oral appliances: 1982\u20132005ReferenceNTypeAHIbaseAHIapplSuccess rate (%)Response rate (%)Comments[11]14TRD56271414Case series; AI[22]16TRD54331932Case series; AI[23]16TRD\u2013\u2013\u201356Case series; all patients had SMR or UPPP; AI[24]1TRD795100\u2013Case report; AI[25]7MAA37125743Case series; Esmarch prosthesis; AI[26]44MAA5023\u201359Case series; Esmarch prosthesis; AI[27]5TRD4894060Case series; NAPA; AI[28]12MAA54362517Case series; modified functional appliance[29]1MAA359100\u2013Case report; mandibular repositioning appliance[30]12TRD37175817Case series[31]1MAA572100\u2013Case report; mandibular repositioning appliance[32]15TRD2711730Case series[33]14MAA3294357Case series; fixed splint; AI[34]20MAA47203540Case series; fixed dental orthosis[35]12MAA5019\u2013\u2013Case series; fixed prosthesis; AI[36]2MAA3075050Two case reports; intra-oral fixed prosthesis; RDI[37]16MAA3796925Case series; NAPA; RDI[38]24MAA48127313Prospective case series; Herbst-like; RDI[39]19MAA3513\u2013\u2013Case series of CPAP failures; Herbst[40]20MAA57262040Case series; Esmarch; AI[41]12MAA4530\u2013\u2013Case series; Herbst vs MR (muscle relaxation) appliance; result for Herbst; ODI4[41]12MAA4541\u2013\u2013Case series; Herbst vs MR (muscle relaxation) appliance; result for MR; ODI4[42]51MAA3218\u2013\u2013Case series; mandibular advancement splint fixed 75% of maximum protrusion[43]4MAA200110\u201375Case series; fixed intra-oral prosthesis; apneas\/night[44]30MAA6531\u2013\u2013Case series; Esmarch; AI[45]21MAA34201924Crossover, comparing AMP device with CPAP[46]19MAA20106811Randomized, prospective crossover comparing the Snore-Guard with CPAP[47]23MAA37185230Case series; mandibular repositioning device;most patients were CPAP failures; RDI[48]1MAA343100\u2013Case report; elastometric sleep appliance[49]14TRD383000Case series: SnorEx appliance; RDI[50]20MAA25145521Randomized, crossover comparing the AMP device with CPAP[51]8MAA44126313Case series; mandibular advancing positioner[52]1MAA534100\u2013Case report; fixed dental appliance[53]14MAA44\u2013\u2013Fixed mandibular splint; maximum protrusion; median AHI[53]9MAA71\u2013\u2013Fixed mandibular splint; 70% of maximum protrusion; median AHI[54]44MAA2596416Case series, prospective; mandibular advancement device[55]18MAA42156111Case series of UPPP failures; Herbst[56]25MAA3397212Case series;mandibular positioning device; RDI[57]15MAA19320\u2013\u2013Case series of snorers; mandibular advancement device; snores\/h of sleep[58]14MAA3657121Case series; Serenox[59]41MAA1812783Prospective, randomized, parallel groups comparing dental appliance with UPPP[60]75MAA44125128Case series; TAP appliance[61]11MAA4510\u2013\u2013Case series; dental appliance[62]15MAA288\u2013\u2013Case series; Klearway appliance[63]28MAA53213236Case series; elastic mandibular advancement device[64]37MAA26115015Case series; three fixed appliances with 2, 4 and 6\u00a0mm protrusion; ODI4[65]8MAA72361350Case series comparing MAA (Snore-Guard) with TRD and with soft palate lifter (SPL)[65]5TRD5044\u2013\u2013Ibid[65]2SPL4757\u2013\u2013Ibid[66]10MAA41126030Case series; Herbst[67]38MAA33125518Case series; Klearway[68]24MAA23967\u2013Randomized, crossover comparing Herbst vs monoblock; results given for Herbst[69]39MAA17859\u2013Case series; SnorBan[70]256MAA43185414Case series; mandibular advancement device[71]22MAA40125923Case series; modified functional appliance[72]24MAA30143825Randomized, placebo-appliance-controlled, crossover; mandibular advancement splint[73]22MAA286\u2013\u2013Case series; either Herbst or monoblock[74]72MAA43225322Case series; adjustable mandibular splints[75]25MAA3.41.8\u2013\u2013Randomized, placebo-appliance-controlled, crossover series of snorers; snoring measured on a 0\u20134 scale[76]33MAA25958\u2013Prospective case series; mandibular advancement device[77]23MAA2185222Randomized, crossover, comparing 4\u00a0mm with 14\u00a0mm inter-incisal opening; results for 4\u00a0mm opening[78]7MAA67204343Case series; Herbst-like[79]32MAA187639Randomized, parallel groups comparing UPPP and MAA[80]26MAA1888812Case series; Karwetzky activator[81]34MAA227\u2013\u2013Case series; Karwetzky activator; median AHIs[82]73MAA27123627Randomized, crossover, placebo-appliance-controlled[83]6MAA136830Case series; titration study; results for maximum protrusion; Klearway[84]20MAA181430\u2013Randomized, crossover, comparing CPAP with ISAD appliance[85]48MAA311547\u2013Randomized, crossover, comparing CPAP with mandibular repositioning splint[86]20MAA382333\u2013Randomized, crossover, placebo-appliance-controlled; mandibular advancement splint[87]34MAA294\u2013\u2013Case series comparing patients on CPAP who switched to MAA[88]24MAA22870\u2013Prospective randomized crossover, comparing MAS with CPAP; soft one-piece mandibular advancement splint[89]40MAA50165228Prospective, randomized, parallel groups comparing 75% and 50% of mandibular protrusion; results for 75% group[90]26MAA196734Prospective, randomized, parallel groups comparing 75% and 50% of mandibular protrusion; results for 75% group[91]25MAA38154424Case series; The Silencer appliance[92]80MAA2114\u2013\u2013Randomized, crossover, controlled, comparing mandibular advancement splint with CPAP and with placebo tablet[93]44MAA46126418Case series; titration protocol; Herbst-like[94]19MAA34173711Case series; titration protocol; Klearway[95]277MAA21854\u2013Case series; mandibular advancement devices[96]11MAA32\u2013\u2013Prospective case series of non-apneic snorers; Herbst; ODI4[97]20MAA84\u2013\u2013Case series; fixed mandibular advancement device[98]34MAA203940Case series of consecutive patients; TAP appliance[99]17MAA2515\u2013\u2013Case series of patients with CHF; mandibular advancement device[19]19MAA3287911Case series; Herbst[100]16MAA4624\u2013\u2013Prospective, randomized, crossover comparing Twin Block and Herbst; median AHI [101]251MAA2916\u2013\u2013Mail survey of 544 patients; RDI; mainly Klearway, few mandibular repositioners, fewer TRDs[114]21MAA3425538Case series; Klearway appliance[115]92MAA18\u2013\u2013\u2013Case series; questionnaires; bed partners\u2019 replies recorded[120]25MAA35.98.260\u2013Median AHI; case series; 6\u00a0weeks use; split polysomnography[121]4MAA49.511.77525Case series[122]73MAA24.412.255\u2013Prospective, randomized, placebo-appliance-controlled 4\u00a0weeks study[123]12MAA229.258\u2013Case series[126]161MAA1865922Case series; OSA defined as AHI\u2009>\u20095\nFirst, Table\u00a02 contains the studies from the two extreme ends of the spectrum of scientific rigor. At the highest end of the spectrum there are prospective, randomized, crossover, controlled (either against placebo-appliance or another treatment modality) studies. At the lowest end of the spectrum are individual case reports. Some studies are prospective case series, but most studies are retrospective analysis of series of cases.\nSecond, the investigations listed in Table\u00a02 form a very inhomogeneous group with respect to several variables: time of follow-up study, type of polysomnography, which respiratory variables measured, presentation of results, type of oral appliance used, missing data, etc. The time interval between the diagnostic and \u201cwith appliance\u201d polysomnography varied from a few hours to a few months. In some studies, split polysomnography (i.e. diagnostic part followed by \u201cwith appliance\u201d part) was carried out, while in others, the two sleep studies were separated by a couple of days to several months. In some investigations, formal in-hospital polysomnography was performed, while in others, only at-home monitoring of oxygen saturation was carried out. In some of the earliest investigations, only the apnea index (AI) was measured. Later investigations reported the oxygen desaturation index (number of times per hour of sleep that oxygen saturation falls by more the 4% from the baseline\u2014ODI4). Recent investigations all reported either the apnea\u2013hypopnea index (AHI) or the respiratory disturbance index (RDI). Most investigations presented only the mean values, rather than the individual data, and some presented only the median values. Oral appliances used by the investigators included tongue retaining devices, soft palate lifters, and fixed and variable mandibular advancement appliances. In the vast majority of investigations, the mandibular advancement appliance was used, less than ten investigations employed the TRD, and only one investigation used the soft palate lifter. In many investigations, not all patients who had a diagnostic sleep study also had a follow-up \u201cwith appliance\u201d study.\nClearly, the above factors have a significant effect on the analysis of the pooled data presented in Table\u00a02.\nThe first step in analyzing the results of individual investigations is to decide on which outcome variables to analyze. Since we are interested in sleep apnea, the following four variables are an obvious choice\u2014baseline index of respiration (we shall denote this as AHIbase), \u201cwith appliance\u201d index of respiration (denoted as AHIappl), success rate defined as the reduction of AHIbase to a value less than the defining value for sleep apnea, and response rate defined as the reduction of AHIbase by greater than 50% while still remaining higher than the defining value for sleep apnea.\nTable\u00a02 lists 89 distinct investigations, involving a total of 3,027 patients. Inspection of the individual results indicates the widest possible variability. Respiration is analyzed in terms of AI, ODI4, AHI, or RDI. The definition of sleep apnea was based either on AI\u2009<\u20095, AHI\u2009<\u200910, RDI\u2009<\u200910 or ODI4\u2009<\u200910. Success and response rates were defined differently in many studies; however, whenever possible we extracted the information to calculate the success and response rates according to the standard definition given above. In other words, no matter what definition of sleep apnea was employed in a particular study, i.e. whether it was defined as AI\u2009<\u20095, AHI\u2009<\u200910, or ODI\u2009<\u200910, etc.\u2014the success rate we calculated was based on the percent of patients in whom AHIappl was less than 10, and the response rate was calculated as the percent of patients in whom 10\u2009<\u2009AHIappl\u2009<\u20090.5\u2009\u00d7\u2009AHIbase.\nClearly, it is not possible to carry out a meta-analysis of these studies because of the differences in study design, data collection, statistical analysis and presentation of the data. Even the simple descriptive statistics based on the pooled data must be interpreted with caution due to the methodological differences listed above. To obtain the general information about the efficacy of oral appliances, we analyzed the results of individual investigations in several different ways, as follows.\nFirst, we rejected all case reports [24, 29, 31, 48, 52] and all studies, which did not report the mean AI, the mean AHI, or the mean RDI [43, 53, 57, 64, 75, 81, 96, 100, 120]. These studies reported either the total number of apneas per night, snoring only, median AHI, or ODI4, etc. This procedure left a total of 75 studies involving 2,832 patients. We used this \u201cpooled\u201d data to calculate the \u201cpooled\u201d means for each outcome variable\u2014AHIbase, AHIappl, success rate and response rate. If an individual study did not report this outcome variable\u2014it was not used in the calculation of pooled means. For example, to calculate the AHIbase, we only had to reject 1 study [23] where this variable was not reported, thus leaving 74 studies with 2,816 patients. To calculate the AHIappl, we had to reject 2 studies [23, 115], thus leaving 73 studies with a total of 2,724 patients. Similarly, looking at Table\u00a02, we can easily see which studies had to be rejected to calculate the success and the response rates. The results are shown in Table\u00a03. We note that oral appliance reduced the AHIbase from 31 to 14. The success and response rates were 54% and 21%, respectively. \nTable\u00a03Summary of the outcome variables for studies listed in Table\u00a02VariableResultNo. of patientsNo. of studiesAHIbase312,81674AHIappl142,72473Response rate21%1,57751Success rate54%2,08759\nA second way to analyze the data presented in Table\u00a02 is to select only those investigations where all four outcome variables (AHIbase, AHIappl, success rate, response rate) were reported. This left 49 studies involving 1,517 patients. It is interesting to note that the results shown in Table\u00a04, are almost identical to what was found in a larger dataset. \nTable\u00a04Summary of results for complete studiesResults for complete studiesNo. of studies49No. of patients1,517AHIbase35AHIappl14Response rate20%Success rate54%\nA third way to analyze the data is to select only the randomized, crossover, placebo-controlled studies. There are only five such studies\u2014all done after the year 2000 and all using the mandibular advancement appliance. Inactive appliance was used as placebo in four studies [72, 82, 86, 122], and a pill was used as placebo in one study [92]. In the latter study, only the mean AHIs were reported, but not the success and response rates. The results are shown in Table\u00a05. We note that: (1) only patients with mild to moderate sleep apnea were studied, (2) in two out of five studies, the mean AHI was reduced by less than 50%, (3) the success and response rates differ depending on the cut-off value of the AHI (either five or ten), (4) for AHI\u2009=\u200910 cut-off, the success rate is very similar to that of the uncontrolled trials, but the response rate is lower. The overall conclusion from these placebo-controlled crossover trials is that oral appliances significantly improved sleep apnea by reducing AHIbase from 25 to 14 with the combined success and response rates of 64%. \nTable\u00a05Summary of results if randomized, crossover, placebo-controlled studiesReferenceNAHIbaseAHIapplSuccess rate (%)Response rate (%)Comments[72]2430143825For AHI\u2009=\u20095 cut-off5417For AHI\u2009=\u200910 cutoff[82]7327123627For AHI\u2009=\u20095 cutoff[86]2038233010Identical results for AHI\u2009=\u20095 and AHI\u2009=\u200910 cutoffs[92]802114\u2013\u2013Tablet used as placebo; CPAP arm was also present[122]73241236\u2013For AHI\u2009=\u20095 cutoff55\u2013For AHI\u2009=\u200910 cutoffSummary27025143524For AHI\u2009=\u20095 cutoff5014For AHI\u2009=\u200910 cutoff\nOne of the issues facing the effective use of oral appliances is our ability to determine the appropriate degree of protrusion necessary to resolve apnea and snoring. Until recently, the only way to do so was to advance the mandible forward as much as is tolerated by the patient, to carry out polysomnography and hope that sleep apnea is eliminated. Unlike CPAP, which may be controlled remotely without waking the patient, the design of most appliances requires removing it from patient\u2019s mouth to change the degree of protrusion. This wakes up the patient repeatedly, resulting in a high probability that the titration study will be unsuccessful, thus wasting valuable resources. However, several \u201ctitration\u201d protocols were described recently to eliminate this problem. Fleury et al. [93] studied a protocol where this titration was done at home, over a period of several weeks, by advancing the mandible in 1\u00a0mm steps and recording symptoms and ODI4. The effective protrusion was defined as that which results in either resolution in symptoms or reduction in ODI4 to below ten. Using this protocol, they were able to determine the protrusion, which abolished sleep apnea in 64% of patients. Tsai et al. [94] described a remotely controlled mandibular advancement device, which could be titrated during the night in much the same way that the CPAP is titrated. The mandible was advanced remotely during the night in 1\u00a0mm increments until respiratory events were eliminated. The success was confirmed subsequently by carrying out all night polysomnography with the oral appliance set to the effective protrusion determined during the titration study. The positive predictive value of this titration protocol was 90%. On the other hand, Kuna et al. [114] found that the titration protocol was not predictive of the response during chronic use. In this investigation, 9 out of 21 patients with OSA achieved reduction in AHI to less than 10 during titration. However, none of them demonstrated the same beneficial response during longer use of the appliance at home with the effective protrusion determined during the titration night. These results indicate that oral appliance titration to predict the amount of mandibular advancement required to reduce AHI to less than ten is still imprecise and must be used with caution when determining the appliance settings for home use. Nevertheless, the application of titration protocols is a new and important development in this field, which may improve the success of oral appliance therapy by identifying patients who are likely to respond to this treatment.\nPredicting who will respond to the oral appliance therapy is not yet possible, although there are several studies where the differences in various parameters (mainly weight and measures of airway size and collapsibility) were studied in responders and non-responders [39, 116, 117]. The best correlates were always weight and oropharyngeal airway size. However, although these studies provide useful information, particularly with respect to the factors that determine airway occlusion, they do not as yet provide us with a method to predict who will respond to this therapy.\nOral appliances vs CPAP\nSince CPAP remains as the \u201cgold standard\u201d treatment of sleep apnea, the objective success rate of any other treatment must be judged against it. There are seven randomized, crossover studies, which compared mandibular advancement appliances against CPAP. The results are summarized in Table\u00a06. We note that the findings of all such studies are remarkably consistent\u2014CPAP results in better improvement in AHI than oral appliances. In all studies except one, CPAP normalized the respiration, bringing AHI to less than ten. In one study where AHI with CPAP remained above ten, the highest pressure used was 10\u00a0cm H2O, which is probably too low. However, patients subjectively prefer oral appliances over CPAP. In five out of seven studies, the patients expressed preference for an oral appliance, in one study neither treatment was preferred and in another study CPAP was preferred. \nTable\u00a06Randomized, crossover, CPAP vs oral appliance studiesReferenceNAHIbaseAHIapplAHI CPAPComments[46]192010468% of patients were satisfied with OA vs 62% with CPAP (p\u2009<\u20090.05)[45]21342011OA preferred[50]202514465% preferred OA, 30% preferred CPAP[84]2018144\u201cPatients identified oral appliance as being easier to use\u201d[85]4831158\u201cNeither treatment was significantly preferred by patients\u201d[88]242283\u201c...17 out of the 21 subjects who completed both arms of the study preferred the MAS\u201d[92]8021145\u201cAlthough subjects reported that CPAP was the most difficult treatment to use, they felt that it was the most effective and overall preferred it to the MAS, which was in turn preferred to placebo\u201dSummary23224146Oral appliance preferred overall\nThere are 2 additional investigations [87, 102], which are not listed in Table\u00a02 because of missing data regarding follow-up sleep study. Nevertheless, these investigations provide interesting information regarding the comparison of oral appliances and CPAP. Smith and Stradling [87] attempted to determine whether oral appliances can substitute CPAP at least for 1\u00a0month. The authors found that patients achieved similar reduction in ODI4 with CPAP (from 29 to 1) as with oral appliance (from 29 to 4). However, the patients did not like their oral appliance and were refusing to use it. Out of 50 patients on CPAP who were switched to oral appliance, only 11 were still using it by end of 1\u00a0month. Most patients discontinued its use because of discomfort, side effects, or treatment failure. This study therefore favors CPAP. On the other hand, McGown et al. [102] carried out a questionnaire survey of 126 patients treated with oral appliances. There were 41 patients who had tried both CPAP and oral appliance; 71% preferred oral appliance, 19% preferred CPAP, and 10% were unsure. This study favors oral appliance.\nWe conclude therefore that CPAP is more effective than oral appliances in reducing AHI, but despite this, most patients prefer oral appliances, undoubtedly because they find them to be less cumbersome than CPAP.\nOral appliances vs other treatments\nSince 1988 there were several studies [21, 53, 59, 64, 65, 68, 77, 79, 89, 90, 100], which compared either different types of appliances, different degrees of protrusion or different inter-incisal distance. The results are listed in Table\u00a07. Columns labeled \u201cbase\u201d and \u201cappl\u201d show the AHIbase and AHIappl, respectively. The column labeled \u201ccomp\u201d gives the AHI measured when other, i.e. \u201ccomparison\u201d treatment was used. The type of oral appliance employed and the precise variables that were compared are described in the last column. Since most studies employed parallel group design, baseline AHI before comparison treatment is also shown in this column. \nTable\u00a07Studies comparing oral appliances to treatments other than CPAPReferenceNAHICommentsBaseApplComp[53]2343.50.8Parallel groups; OA=MAA with max protrusion, comp=MAA with 70% of maximum protrusion, but double inter-incisal opening; baseline AHI\u2009=\u20097 for comp group[59]4118610Parallel groups; OA=MAA, comp=UPPP; prospective, randomized, baseline AHI\u2009=\u200920 for UPPP group; results at 12\u00a0months[64]37261711Single group; OA=MAA with 2\u00a0mm protrusion, comp\u2009=\u20096\u00a0mm protrusion; ODI4 recorded[65]5503044Single group; OA=MAA, comp=TRD; only 5\/8 patients agreed to try TRD[65]2473557Single group; comp=SPL; only 2\/8 patients agreed to try SPL[68]242398Crossover, randomized; OA=MAA (Herbst), comp=monoblock[21]105510Parallel groups: OA=TRD, comp=somnoplasty; baseline RDI same for both groups[77]2321810Crossover, randomized; OA=MAA with 4\u00a0mm inter-incisal opening, comp=MAA with 14\u00a0mm opening[79]7218714Parallel groups, randomized; OA=MAA, comp=UPPP; baseline AHI for UPPP group\u2009=\u200920; results at 4\u00a0years[89]84471716Parallel groups, randomized; OA=MAA with 50% protrusion, comp=MAA with 75% protrusion; baseline AHI for 75% group\u2009=\u200950; results at 6\u00a0months[90]551666Parallel groups, randomized; OA=MAA with 50% protrusion, comp=MAA with 75% protrusion; baseline AHI for 75% group\u2009=\u200919; results at 12\u00a0months[100]16462534Crossover, prospective, randomized; OA=Herbst, comp=Twin Block; median AHIs reportedSummary392261112\nExamination of the individual investigations reveals that when oral compliances are compared to each other, either two different appliances or the same appliance with different degrees of protrusion or opening\u2014it is cleat that the efficacy (objective and subjective) is very much dependent on the type of appliance and the degree of advancement. This further emphasizes the point that oral appliance therapy should be carried out by a dentist with expertise in this field who is familiar with different types of appliances and can select the most appropriate one for the particular patient. There is no \u201cbest\u201d appliance. The best one is that which is comfortable to the patient and achieves the desired efficacy.\nThere are several studies, mainly case series, comparing oral appliances with surgical treatments. Comparisons with UPPP [59, 79] demonstrated the superiority of oral appliances. At 1\u00a0year follow-up, sleep apnea was resolved (AHI\u2009<\u200910) in 78% of the oral appliances group and 51% of the UPPP group. With longer follow-up, this success rate deteriorates. At 4\u00a0years follow-up, 63% of the oral appliance group and 33% of the UPPP group continue to have AHI\u2009<\u200910. One parallel group study of oral appliance (TRD) vs radiofrequency ablation (somnoplasty) found that both treatments significantly reduced sleep time spent with loud snoring. However, there was no difference between oral appliance and somnoplasty [21]. A recent small case series of 4 patients (out of 43 treated with oral appliances) who elected maxillomandibular advancement surgery [121] showed that initial AHI\u2009=\u200950 dropped to 12 with oral appliance and to 2 after surgery.\nOral appliances for the treatment of snoring\nAlthough everyone can recognize snoring, it proved to be a very elusive entity to measure objectively. One can define the sound properties (i.e. frequency spectrum and intensity), relationship to breathing (i.e. waxing and waning sound, generally during inspiration), and measure this sound during sleep. However, subjective recognition of sound, which satisfies some pre-defined \u201csnoring\u201d criteria as de facto snoring depends very much on the listener. This is contrary to the case of apneas or even hypopneas, whose definition is independent of any subjective perception. These difficulties with the definition of snoring are the reasons why objective measurement of sound is seldom a routine part of polysomnography. However, snoring is the cardinal symptom of sleep apnea. In fact, it is frequently the only reason why these patients come to the sleep clinic in the first place. Consequently, when polysomnography does not reveal sleep apnea in these patients, the physician still has to deal with their snoring. Unfortunately, this is often ignored by physicians.\nThe most frequent scenario is that a patient is referred to a sleep specialist because of snoring, polysomnography is carried out, no sleep apnea is found, the patient is reassured, advised to loose weight, stop smoking and drinking alcohol, embark on an exercise program, and discharged from the clinic. Sometimes this advice, dispensed in the form of preprinted sheets, is given also to non-obese non-smokers. Clearly, the patient leaves unhappy, the referring physician is dissatisfied with the help received from the specialist and nothing was accomplished to justify the expense incurred in the process of investigations. For apneic snorers, the problem is simpler because treatment with CPAP will abolish snoring.\nNon-apneic snorers without daytime symptoms do not tolerate CPAP well. Many of them will agree to try it, but the majority will stop using it after a short time (generally a few weeks to a few months). Oral appliances therefore constitute an attractive alternative for the treatment of snoring. In fact, they were originally invented precisely for that reason.\nMany, but not all investigations of oral appliances comment on their efficacy in reducing snoring. Recognizing that the objective measurement of sound during polysomnography may not correspond to the perception of this sound as being snoring\u2014subjective assessment is generally employed. This assessment varies from simply asking an informal question \u201cis your snoring improved?\u201d, to employing a more formal method, which is usually a visual scale (analogue or digital) to rate snoring. Unfortunately, in almost all studies, the answers to these subjective questions are given by the snorers who of course are unaware of their snoring, rather than by the bed partner. Although snoring is recorded as the patient\u2019s chief complaint, it is really not the patient\u2019s complaint at all\u2014it is the complaint of the bed partner. The implicit assumption in most investigations is that the patient\u2019s responses reflect those of the bed partner. Nevertheless, the efficacy of snoring treatment must be assessed by the same bed partner, not by the snorer. This poses great logistical problems in carrying out appropriate investigations. However, some support for this assumption of equivalence between the responses of snorers and listeners is provided by a recent questionnaire data of Bates and McDonald [115] who found that 70% of snorers and 70% of the bed partners reported improvement in snoring after using a mandibular repositioning splint for 3\u00a0months.\nTable\u00a08 lists 47 investigations, which utilized oral appliances. The only common feature among these investigations, and the reason why other investigations were not included\u2014is that all of the listed ones contained a specific comment regarding snoring. The types of snoring assessment carried out in these investigations ranged from an informal question about snoring to rating the snoring using a visual scale and objective sound measurement together with subjective assessment. Very few investigations included objective measurement of snoring. Investigations with subjective assessment of snoring using questionnaires form a very inhomogeneous group because they used different types of questionnaires with different rating methods. Given these differences in methodology, it is not possible to rigorously summarize the results. However, certain generalizations can be made. \nTable\u00a08SnoringReferenceNTypeSnoring measureCommentsBaseAppl[27]5TRD\u201cSnoring decreased or completely disappeared\u201d[28]12MAA\u201c8\/12 reported substantial reduction of sonorous sleeping[31]1MAA\u201cAfter appliance insertion...immediate...reduction in snoring\u201d[34]68MAA8.51.5Snoring severity assessed subjectively (max score\u2009=\u200910); snoring eliminated in 42%[35]12MAASnoring reduced, although never eliminated, in 79%[38]24MAA7.6Snoring on a scale from 0 to 10; improvement also on a scale from 0 to 10\u2014result\u2009=\u20094.3[42]51MAA9.48.2No. of snores\/min; snoring eliminated in 8\/51, improved in 43\/51[46]25MAASnoring less than \u201cmoderate\u201d in 19\/25 pts[47]23MAA\u201c...20\/23 patients (87%) reported subjective improvement...in snoring\u201d[49]23TRD\u201cVisual analog scores of snoring...were also reduced significantly\u201d in 6\/23 (23%) subjects[53]23MAA\u201c...loud snore duration was reduced from a median of 27.1\u00a0min to 11.4\u00a0min\u201d[54]44MAA\u201cSnoring was satisfactorily reduced in\u201d 37\/44 patients (84%)[57]15MAA19320Median snores\/h given; snoring loudness and time spent snoring also improved[102]132MAA\u201cSnoring was reported...to be satisfactorily controlled in 107 (81%)...[58]14MAA6\/14\u2014no snoring; 8\/14\u2014mild snoring[59]41MAA0.70.5No. of snores\/h of sleep at baseline and 12\u00a0months follow-up (NS)[60]75MAA\u201cDramatic reduction in the attributes of snoring was achieved\u201d[66]112MAA76\/112 (68%) snoring either eliminated or acceptable[68]24MAA5033No of snores\/h sleep; results for Herbst appliance; 19\/20 disturbed by snoring at baseline, vs 9\/20 with appliance[21]10TRD113Percent of time spent in loud snoring[69]39MAA\u201cTime with snoring dropped significantly from 16.3% to 6.6%\u201d[71]22MAASnoring eliminated in 13\/22, significantly reduced in 5\/22; success rate\u2009=\u200918\/22 (82%)[104]53MAAQuestionnaire survey; 27\/53 were still using the device at 1\u00a0year, and 22 were satisfied (42%)[105]21MAAQuestionnaire survey; 22 patients fitted with appliance; 43% thought it reduced snoring, 48%\u2014no benefit[102]126MAAQuestionnaire survey; \u201c80 out of 94 patients reported improvement in snoring\u201d[73]22MAA5924No. of snores\/h of sleep; subjective improvement as well[75]25MAA3.41.8Randomized controlled vs placebo-appliance; non-apneic snorers; frequency of snoring (nights\/week), p\u2009<\u20090.05; 15\/25 greatly improved with MAA vs 2\/25 with placebo[76]33MAA\u201c19\/33 had short-term satisfactory treatment results with the device\u201d[72]28MAA402242Randomized, controlled vs placebo-appliance; snores\/h of sleep (p\u2009<\u20090.005); mean snoring intensity significantly reduced; \u201cthe majority of patients reported substantial improvement in snoring (70%)[82]73MAA366207Randomized, controlled vs placebo-appliance; snores\/h of sleep (p\u2009<\u20090.0001); mean and maximum snoring intensity significantly reduced; significant subjective reduction in snoring[86]16MAA3.12.6Randomized, placebo-controlled crossover; frequency of snoring (nights\/week) (p\u2009=\u20090.07); no significant difference in loudness of snoring[79]32MAA0.70.5Duration of snoring\/h of sleep (p\u2009<\u20090.01)[80]26MAA\u201cThe patients and their bed partners thought that...snoring...improved...\u201d[84]20MAA5536Snoring epochs\/h (p\u2009<\u20090.01); randomized crossover study vs CPAP[89]40MAA0.860.57Duration of snoring\/h of sleep (p\u2009<\u20090.001); comparison of two protrusions; results for 75% protrusion group[90]26MAAComparison of two protrusions; results for 75% protrusion group; \u201cproblems with apneas and snoring...decreased by...79%...\u201d[91]25MAA\u201c...snoring...patients have benefited from oral appliance therapy and their spouses will testify to the same\u201d[93]44MAASubjective assessment; \u201con average, a mean reduction of 90% of the intensity of snoring was reported by the patients\u201d[95]619MAA\u201cIt is estimated that 50% of the 619 snorers and sleep apnea patients had treatment success or subjective beneficial effects...\u201d[96]11MAA24075Noise level measured; \u201c10 out of 11 subjects had a significant reduction in snore noise sound level...\u201d[97]20MAA9.06.8VAS 0\u201310 scale (p\u2009<\u20090.05); result at 6\u00a0months follow-up; subjectively 14\/20 were satisfied[106]110MAAQuestionnaire survey; 37 out of 77 patients who returned questionnaire thought snoring was satisfactorily controlled[99]17MAA5316p\u2009=\u20090.02; snoring time[19]25MAA15\/25 snoring markedly improved[100]16MAA14464Snores\/h; however, VAS 0\u201310 scale\u2013no difference[101]251MAA75% of 191 users of appliance reported control of snoring; 43% of non-users of appliance also thought snoring was controlled[115]53MAASnoring was reported to be improved by 70% of the responding bed partners\nFirst, the majority of the investigations concluded that oral appliances are beneficial in reducing snoring in the majority of patients. Second, all of the randomized, placebo-appliance-controlled studies except one [86] found significant reduction in snoring, independently of whether it was assessed objectively or subjectively. Johnston et al. [86] did not find a significant difference in either the loudness (measured using the VAS 0 to 5 scale) or the frequency (nights\/week) of snoring. However, an earlier study from the same group [75], employing similar methodology but different patient population (non-apneic snorers), did demonstrate significant reduction in snoring. This further illustrates the difficulties with subjective assessment of snoring in different patient populations. Even in the same patient population, there is a discrepancy between objective measurement and subjective perception, as found by Lawton et al. [100].\nWe shall present the summary of the individual investigations listed in Table\u00a08 as follows. First, we selected only those investigations where a numerical value describing snoring with and without appliance was given. These results are shown in Table\u00a09. There are 18 studies involving 529 patients. All, except one [21], employed the mandibular adjustment appliance. All of these investigations give a \u201cnumber\u201d to quantify snoring. We note the diversity of measurement of snoring in each study\u2014the \u201cnumber\u201d in Table\u00a09 represents either a VAS score, number of snorers\/h (or min) of sleep, amount of time spent with loud snoring per hour of sleep or per night, number of nights per week spent with disturbing snoring or noise level, etc. However, we can calculate the percent change between the baseline night and the \u201cwith appliance\u201d night, displayed in the last column of Table\u00a09. We note that despite the diversity of snoring measurements, in all investigations the percent change is negative\u2014which indicates that the investigators always found reduction in snoring with oral appliances. The mean reduction in snoring using the pooled data was 45%. \nTable\u00a09Studies with measurement of snoringReferenceNSnoring measureExplanation of snoring measurementPercent changeBaseAppl[34]688.51.5Visual analogue scale 0\u201310\u221282[42]519.48.2Number of snores\/min\u221213[57]1519320Number of snores\/h\u221290[59]410.70.5Number of snores\/h\u221229[68]245033Number of snores\/h\u221234[21]10113Percent of sleep time spent in loud snoring\u221273[73]225924Number of snores\/h\u221259[75]253.41.8Nights\/per week with disturbing snoring\u221247[72]28402242Number of snores\/h\u221240[82]73366207Number of snores\/h\u221243[86]163.12.6Nights\/per week with disturbing snoring\u221216[79]320.70.5Minutes of snoring\/h of sleep\u221229[84]205536Snoring epochs\/h of sleep\u221235[89]400.860.57Minutes of snoring\/h of sleep\u221234[96]1124075Noise level\u221269[97]209.06.8Visual analogue scale 0\u201310\u221224[99]175316Total snoring time\u221266[100]1614464Number of snores\/h of sleep\u221256Summary529\u221245\nEffect of oral appliances on daytime function\nIn assessing the effect of oral appliances on sleep apnea syndrome, it is not sufficient to focus only on the apnea\/hypopnea index or snoring. We must also demonstrate the effect of this treatment on daytime function, which is almost always compromised in patients with sleep apnea and sometimes in non-apneic snorers also. There are several tools used to assess daytime function, but there is little consistency in using these tools in investigations involving oral appliances.\nTable\u00a010 summarizes the results of investigations where some assessment of daytime function was performed, no matter how primitive. The methods of assessment differ in each investigation. Some relied on answers to questions regarding daytime sleepiness and tiredness, others utilized visual scales, yet other investigations measured response time to various tasks, etc. After 1995, the majority of investigations employed the Epworth sleepiness score (ESS). The results generally show improvement in daytime symptoms with oral appliances. \nTable\u00a010Functional assessmentReferenceNTest or questionResultCommentsBaseAppl[11]14Daytime function\u2013\u201314\/14 reported improvement[25]7Sleepiness\u2013\u2013Improved[26]44Vigilance0.50.4Reaction time (p\u2009<\u20090.05)[27]5Daytime sleepiness\u2013\u2013\u201cDaytime somnolence was eliminated or diminished markedly\u201d[28]12Daytime somnolence\u2013\u2013\u201c9\/12 patients reported increased alertness and\/or reduction in daytime sleepiness\u201d[34]63Prevalence of daytime sleepiness\u2013\u2013\u201c51% of these patients reported no more sleepiness with orthosis use\u201d[35]12Daytime sleepiness\u2013\u2013\u201cDaytime sleepiness was improved...in all but two patients\u201d[33]14Symptom score (including sleepiness)5.51.1Significant(p\u2009<\u20090.001) improvement in daytime symptoms[38]24Sleepiness and improvement using 0\u201310 Likert scale6.4\u2013Improvement on 0\u201310 Likert scale\u2009=\u20094.5 at 36\u00a0months[42]51Patients tired; patients sleepy4430[44]30No. of mistakes in vigilance test7.63.7p\u2009<\u20090.05[45]21EDS daytime symptoms2.41.61\u20135 scale; p\u2009<\u20090.0001) for all symptoms[46]25Prevalence of EDS84%40%p\u2009<\u20090.005; significant reduction in prevalence of other symptoms[47]23Patients with EDS2320[49]14EDS using VAS\u2013\u2013Reduction in scores (p\u2009<\u20090.05) in 6 of 14 patients who were compliant with treatment[50]20ESS10.34.7p\u2009<\u20090.05; EDS improved in 13\/20 patients[53]14ESS124.5Median score, p\u2009<\u20090.005[53]9ESS74Median score, p\u2009<\u20090.005[54]44Patients with daytime sleepiness4434[58]14Patients with moderate and severe daytime somnolence100[59]41Daytime sleepiness on 1\u20135 scale\u2013\u2013Prospective, randomized, UPPP group and OA group; \u201cin comparison with their baseline values...a significant (p\u2009<\u20090.001) reduction in subjective daytime sleepiness\u201d[60]75ESS117p\u2009<\u20090.0005[107]90Quality of life (vitality+contentment+sleep)12994Significant improvement compared to baseline; two parallel groups\u2014OA vs UPPP; no difference in vitality and sleep[66]112No. of patients \u201crefreshed by sleep\u201d\u201366\/114\u201cMost of the regular users had an improvement in their quality of sleep and day time somnolence...\u201d[68]24ESS13.18.6p\u2009<\u20090.001; identical result for two different MAAs[71]22No. of patients whose sleepiness disappeared17\/22\u201c17 (85%) of 22 patients reported subjective improvement in excessive daytime sleepiness\u201d[72]24ESS10.13.9p\u2009<\u20090.01[73]22ESS127.5p\u2009<\u20090.05 at 12 to 30\u00a0months follow-up[75]24ESS7.56.5p\u2009<\u20090.01; randomized placebo-controlled crossover trial of non-apneic snorers[76]19No. of patients reporting reduction in EDS13\/19[77]23ESS1812p\u2009<\u20090.0001; identical result for two different MAAs[81]26Questionnaire: EDS1.61Scale from \u22123 (maximum deterioration) to +3 (maximum improvement)[87]34ESS137.7After 28\u00a0days, only 11 patients continued to wear MAA; initial ESS based on 34 patients, final\u2014on 11[88]24ESS13.49.0p\u2009<\u20090.001; randomized crossover vs CPAP[85]48ESS1412NS; randomized crossover vs CPAP; extensive tests of daytime function; \u201cthese results do not support these MRS devices as first-line treatment for sleepy patients with SAHS\u201d[82]73ESS119p\u2009<\u20090.0001); \u201cthe proportion of patients with normal subjective sleepiness was significantly higher with the MAS than with the control device (82 vs 62%, p\u2009<\u20090.01), but this was not so for objective sleepiness (48% vs 34%, p\u2009=\u20090.08)\u201d[86]18ESS12.611.6NS; randomized placebo-controlled crossover trial of apneic snorers[89]42ESS11.57.5p\u2009<\u20090.001; prospective randomized comparing 50% and 75% protrusion; result for 75%; initial value\u201442 patients, final value\u201440[90]55Questionnaire: EDSRandomized comparison of two protrusions; \u201c82% of patients in 50% group (n\u2009=\u200929) and 84% in 75% group (n\u2009=\u200926) reported a decrease in daytime sleepiness\u201d[96]29ESS9.46.9p\u2009<\u20090.001[92]80ESS10.29.2p\u2009<\u20090.001; randomized vs placebo pill and CPAP[93]40ESS12.05.1p\u2009<\u20090.001[97]20ESS8.85.4p\u2009<\u20090.05[98]42ESS106p\u2009<\u20090.02; median values[19]27ESS96p\u2009<\u20090.001; median values[100]16ESS108Median values[101]161ESS117In users of OA; in 90 non-users\u2014ESS fell from 11.1 to 8.1[115]67Concentration, energy levels, sleep quality, ESS9.7\u2013ESS given; 29\u201359% of responders reported improvement[122]73ESS and full battery of neuropsychological measures5.04.2Total score of all self-report measures given; prospective, randomized, placebo-appliance-controlled 4\u00a0weeks study\nBecause of the diversity of methods assessing daytime function, it is difficult to pool and summarize the individual data. One way of doing this is to select only those investigations, which employed identical methods of assessment and summarize the mean results, as was done in the previous tables. The most common single way of assessing daytime function in most investigations was the ESS. These investigations are listed in Table\u00a011. There are 23 of them involving a total of 962 patients. However, not all investigations could be used in calculating pooled data. We rejected four investigations [19, 53, 98, 100], involving 108 patients where only the median and not the mean values of the ESS were reported. Consequently, we are left with 19 investigations involving 854 patients. As a group, these patients were only mildly sleepy with the mean ESS of 11.2. In all investigations, the ESS dropped with the use of oral appliances. For the entire group, there was a significant reduction in the ESS from 11.2 to 7.8. \nTable\u00a011Functional assessment using ESSReferenceNESSCommentsBaseAppl[50]2010.34.7p\u2009<\u20090.05; EDS improved in 13\/20 patients[53]14124.5Median score, p\u2009<\u20090.005[53]974Median score, p\u2009<\u20090.005[60]75117p\u2009<\u20090.0005[68]2413.18.6p\u2009<\u20090.001; identical result for two different MAAs; [72]2410.13.9p\u2009<\u20090.01[73]22127.5p\u2009<\u20090.05 at 12 to 30\u00a0months follow-up [75]247.56.5p\u2009<\u20090.01; randomized placebo-controlled crossover trial of non-apneic snorers[77]231812p\u2009<\u20090.0001; identical result for two different MAAs[87]34137.7After 28\u00a0days, only 11 patients continued to wear MAA; initial ESS based on 34 patients, final ESS\u2014on 11[88]2413.49.0p\u2009<\u20090.001; randomized crossover vs CPAP[85]481412NS; randomized crossover vs CPAP; extensive tests of daytime function; \u201cthese results do not support these MRS devices as first-line treatment for sleepy patients with SAHS\u201d[82]73119p\u2009<\u20090.0001); \u201cthe proportion of patients with normal subjective sleepiness was significantly higher with the MAS than with the control device (82% vs 62%, p\u2009<\u20090.01), but this was not so for objective sleepiness (48% vs 34%, p\u2009=\u20090.08)[86]1812.611.6NS; randomized placebo-controlled crossover trial of apneic snorers[89]4211.57.5p\u2009<\u20090.001; prospective randomized comparing 50% and 75% protrusion; result for 75%; initial n\u2009=\u200942 patients, final n\u2009=\u200940 patients[96]299.46.9p\u2009<\u20090.001)[92]8010.29.2p\u2009<\u20090.001; randomized vs placebo pill and CPAP[93]4012.05.1p\u2009<\u20090.001[97]208.85.4p\u2009<\u20090.05[98]42106p\u2009<\u20090.02; median values[19]2796p\u2009<\u20090.001; median values[100]16108Median values[101]161117Result for users of OA; in 90 non-users\u2014ESS fell from 11.1 to 8.1[122]739.17.1Prospective, randomized, placebo-appliance-controlled 4\u00a0weeks studySummary85411.27.8References 19, 53, 98\u2013100 were excluded (no mean values were given)\nIn two investigations [85, 86], both randomized, crossover and controlled (one vs placebo and another one vs CPAP) reduction in ESS was not significant. Engleman et al. [85] carried out a very extensive study of daytime function comparing the effect of oral appliance to CPAP. Functional assessment included maintenance of wakefulness test, measures of daytime sleepiness and symptoms, measures of well-being (using the SF-36 questionnaire, HADS anxiety and depression score), and cognitive performance. The results favored CPAP in 7 out of 21 variables (including the ESS, AHI, effectiveness and symptoms), and showed no difference between CPAP and oral appliance in other variables (including the maintenance of wakefulness tests, cognitive performance and treatment preference). Johnston et al. [75, 86] compared oral appliance to placebo, but did not carry extensive investigations of daytime function\u2014only the ESS and a 5-point scale describing how refreshed the patients felt in the morning. The authors found significant improvement compared to placebo, but only in non-apneic snorers [75], not in patients with sleep apnea [86].\nIn all other randomized, crossover, controlled studies there was a statistically significant improvement in the ESS, but not in other subjective measures of daytime performance. In fact, none of the studies demonstrated a significant improvement in all of the subjective outcomes studied. This is not surprising because almost all studies comparing placebo treatment with active treatment, no matter what it is, always demonstrate a significant placebo effect.\nProbably the most complete assessment of neuropsychological function was carried out by Naismith et al. [122] in a prospective, randomized, placebo-appliance-controlled study of 73 patients treated for 4\u00a0weeks. The authors demonstrated significant improvement in the measures of self-reported sleepiness, fatigue and energy levels, but no improvement in the measured speed\/vigilance (except for the improved reaction time), attention\/working\/verbal memory or visuospatial\/executive functioning. Walker-Engstrom et al. [107] compared the quality of life in two parallel groups of patients with sleep apnea 1\u00a0year after treatment with either oral appliance or UPPP. The ESS was not measured, but there was other extensive assessment of three quality of life dimensions (vitality, contentment and sleep). Both groups improved compared to the baseline. There was no difference in vitality and sleep dimensions between the two groups, but the UPPP group was more content than the oral appliance group.\nThe effect of oral appliances on daytime function was not studied as fully and extensively as for CPAP. For example, there are no studies comparing driving simulator performance in patients treated with oral appliance, no studies comparing multiple sleep latency or maintenance of wakefulness. Recognizing the limited nature of the data\u2014the conclusion from all of the investigations taken as a group must be that oral appliances improve daytime function, although they are not necessarily superior or consistently preferred than other treatments such as CPAP and UPPP.\nEffect of oral appliances on vascular disease\nNumerous investigations examined the relationship between sleep apnea and vascular events, such as coronary artery disease, hypertension, and cerebro-vascular disease. Fewer, but still many investigations were carried out to examine the effect of the treatment of sleep apnea with CPAP on changes in these conditions.\nQuite the opposite situation is seen regarding the effect of oral appliances on vascular disease. There are only three studies examining the effect of the treatment of sleep apnea with oral appliance on blood pressure. Both studies employed the randomized, controlled, crossover design. The first study was carried out by Gotsopoulos et al. [113] specifically for the purpose of examining the effect of 4\u00a0weeks treatment with a mandibular advancement splint on 24-h blood pressure in 67 patients with sleep apnea (mean AHI\u2009=\u200927). There was approximately 3.5\u00a0mmHg drop in the systolic and diastolic blood pressure with treatment, but only during wakefulness. There was no change in blood pressure during sleep. The second study by Barnes et al. [92] compared the effect of 3\u00a0months treatment with oral appliance to CPAP and placebo (a tablet). In 110 patients with sleep apnea (mean AHI\u2009=\u200921), the 24\u00a0h blood pressure was measured. Treatment with oral appliance (but not with CPAP or placebo tablet) resulted in the significant reduction in nighttime diastolic blood pressure by 2.2\u00a0mmHg. There were no changes in diastolic blood pressure during wakefulness and no changes in systolic blood pressure either during wakefulness or sleep. The third study was carried out by Yoshida [126] who measured blood pressure in 161 patients with sleep apnea before and after 60\u00a0days of treatment with oral appliance. There was a statistically significant drop in blood pressure from 132.0\/82.1 to 127.5\/79.2\u00a0mmHg. Regression analysis demonstrated weak, but significant correlation between the mean arterial and baseline blood pressures and the reduction in AHI. This area of investigation is still in its infancy, and undoubtedly, more results will be forthcoming in the future.\nThere are no rigorous studies of the effect of oral appliances on other vascular diseases. Eskafi et al. [99] carried out a single night, unattended, home sleep study in 17 patients with sleep apnea (mean AHI\u2009=\u200925) and congestive heart failure with periodic breathing before and after intervention with a mandibular advancement device. The authors found improvement in sleep apnea (mean AHI reduced from 25 to 15), but no improvement in periodic breathing or left ventricular ejection fraction after 6\u00a0months of treatment.\nThere is not enough evidence at the present time to draw any conclusions regarding the effect of oral appliance therapy on vascular disease. This remains a very interesting area of investigation. Given the differences in intra-thoracic pressure as a consequence of CPAP vs oral appliance, it is possible that results obtained with oral appliances therapy will be different from those obtained with positive pressure therapy.\nSide effects\nAlmost every study describing oral appliances comments on the side effects voiced by patients. The type of side effects and their frequency depend on the questions asked, the rating scale, the number of patients in the study, etc. Some studies specifically focused on the side effects and compliance with treatment [101, 102, 106, 110], others simply asked a few questions about the side effects. Table\u00a012 summarizes some of the common side effects; there is also a reference to the study, which reported the highest frequency of this particular side effect. \nTable\u00a012Patient reported side effects of oral appliancesSide effectPercent of patientsReference reporting maximum percentDifficulty in chewing11\u201319[101]Excessive salivation9\u201360[106]Dry mouth14\u201386[73]Tooth discomfort11\u201359[73]Tongue discomfort6\u20138[101]Jaw discomfort8\u201341[73]Gum discomfort1\u20132[101]Headache2\u201327[89]Occlusive changes41[76]TMJ pain37[102]Masseter muscle pain45[66]No side effects at all100[39]; 14 subjects\nExcessive salivation, mouth, and teeth discomfort are the most common side effects reported. However, patients seldom consider these side effects troublesome. Provided that this complaint is addressed by the dentist and the appliance is adjusted, they continue to use it. In many cases the side effects are transient and disappear with continued use. When patients stop wearing the oral appliances it is mainly because of ineffectiveness, rather than because of side effects, although in some studies [95, 101] up to 40\u201350% of patients discontinued the use of the appliances because of the side effects. One study [39], involving only 14 patients all using the Herbst appliance, reported that none of the patients had any side effects! The conclusion, based on the results of most studies, is that when oral appliances are properly constructed by the dentist with expertise in this area, they are relatively comfortable in the majority of patients.\nThere are also dental effects of oral appliances. It is still not entirely clear if long-term use of oral appliances will lead to permanent adverse dental changes, particularly when used in children. There are several studies addressing various dental\u2013skeletal changes of oral appliances using various imaging techniques. One of the most common effects, commented upon in many studies, is the degree of vertical and horizontal overlap of the teeth (overjet and overbite, respectively). These results are summarized in Table\u00a013. There are 11 distinct studies, involving 694 patients with mean follow-up time of 43\u00a0months. Two studies [78, 118] were rejected because only the median results were given, and one study [127] was rejected because no mean values for overjet and overbite were shown, only changes in these parameters over a period of 3\u00a0years. A summary of the remaining data involving 389 patients with mean follow-up of 39\u00a0months shows that the overbite is reduced from 3.8 to 2.4\u00a0mm and the overjet is reduced from 4.0 to 2.7\u00a0mm. It is clear that dental\u2013skeletal effects of oral appliances are certainly present, but the long-term results and their clinical significance are unknown at this time. The recent studies of Marklund [118] and de Almeida et al. [119] described patients who were using mandibular advancement appliances for more than 5\u00a0years. Their results suggest that orthodontic changes (1) are variable (favorable in some and unfavorable in others), (2) are clinically relevant, and (3) might be predictable from the initial dental characteristics of the patients and the type of device. \nTable\u00a013Dental effects: overjet and overbiteReferenceNF\/U (months)OverjetOverbiteCommentsBaseF\/UBaseF\/U[39]19134.0\u22123.02.96.3Herbst appliance[108]32244.54.13.63.5[71]2265.971.083.97\u22128.01After correction for magnification error[109]87304.253.194.093.07Effects evident already at 6\u00a0months[73]22143.33.14.03.6Median results at follow-up[110]47283.93.63.22.8Significantly larger changes compared to reference group[110]28314.53.93.83.3Ibid[81]34304.43.13.62.5[111]30483.53.14.33.8NS; compared to UPPP[112]20303.842.634.432.61Effects evident at 6\u00a0months[90]2912\u2013\u20132.52.4NS; for 50% protrusion; same for 75% protrusion[118]187603.53.03.02.80Median values; overbite change NS; orthodontic side effect increase with treatment time and more frequent use[119]31892.120.452.70.4670 patients followed-up for 7.4\u00a0years; measurements made from models; \u201cunfavorable change\u201d group[119]10892.752.94.453.87As above; \u201cno change\u201d group[119]29893.952.724.472.52As above; \u201cfavorable change\u201d group[127]6736\u2013\u2013\u2013\u2013Only changes, but not baseline values in overjet and overbite are given (\u22120.8 and \u22120.6, respectively over 3\u00a0years); small but significant reductions observed mainly during the first yearSummary389394.02.6References [73, 118] were excluded418373.72.4\nCompliance\nCompliance with oral appliances depends strictly on the balance between the perception of benefit and side effects. Most patients treated with oral appliances have relatively mild sleep apnea and relatively few daytime symptoms; the main reason for treatment was snoring. Consequently, the perception of benefit is generally that of the bed partner, whereas the side effects are experienced by the wearer of the appliance. This is why the assessment of compliance is a complex issue. In some cases, although the appliance is quite comfortable, the patient may stop wearing it if the bed partner is no longer present or no longer complains of snoring.\nTable\u00a014 shows the results of studies, which provide compliance data. All of them except one [49] employed mandibular advancement devices. There is a very wide variability between individual investigations\u2014from as little as 4% to as high as 76% at the end of 1\u00a0year. \nTable\u00a014Compliance with oral appliancesReferenceNF\/U (months)Compliance (%)[34]71771\u201375[35]24124\u20135[38]243650\u201375[39]192468\u201393[47]294155\u201370[49]23621[103]1913152\u201376[107]451282[66]173945\u201370[70]2563190[71]226100[76]336258[102]1662242\u201356[79]454862[81]8618\u20132430\u201353[89]741272\u201376[106]1102240\u201357[95]6301275\u201376[101]5446830\u201364[115]92368[118]4506056Summary3,1073356\u201368\nThe largest study is that of de Almeida et al. [101]. It is based on a mail survey of 544 patients, of whom 251 returned the questionnaire on the average of almost 6\u00a0years after the construction of the appliance. The majority of patients were fitted with MAAs, although some had TRDs. At the time of follow-up, 161 patients continued to use the appliance. Assuming \u201cthe worse case scenario\u201d (i.e. all those who did not return the questionnaire were no longer using the appliance) the compliance rate is 161\/544\u2009=\u200930%, while in the \u201cbest case scenario\u201d the compliance rate is 161\/251\u2009=\u200964%. Among those who used the appliance, 82% of bed partners were satisfied with this treatment; even among the non-users of appliances, 46% of bed partners were satisfied. The main reasons for discontinuing the use of the appliance were discomfort (44%) and perception of little or no benefit (34%).\nPooled data summarizing all 21 reviewed studies involving 3,107 patients, showed that at the end of 33\u00a0months, 56\u201368% of them continued to wear the appliance.\nConclusions\nOral appliances used to date constitute a relatively heterogeneous group of devices for the treatment of sleep apnea and non-apneic snoring. It is this heterogeneity, which partly accounts for the variability in their benefit and side effects. Another reason for variability is the diverse methodology employed in different studies. The evidence available at present indicates that oral appliances successfully \u201ccure\u201d mild-to-moderate sleep apnea in 40\u201350% of patients, and significantly improve it in additional 10\u201320%. They reduce, but do not eliminate snoring. Side effects are common, but are relatively minor. Provided that the appliances are constructed by qualified dentists, 50\u201370% of patients continue to use them for several years. Their effectiveness is inferior to CPAP. It is similar to surgical procedures, but these are invasive, (although not particularly dangerous) and irreversible. The effect of oral appliances on the vascular consequences of sleep apnea is not known.\nThe place of oral appliances in the spectrum of treatment options for apneic and non-apneic snorers was extensively discussed in various reviews and guidelines, including the most recent report by the American Academy of Sleep Medicine [124, 125]. The current review does not alter those conclusions. It simply illustrates the marked variability of individual responses to oral appliance therapy, and therefore the necessity to approach each patient on an individual basis. Patients with sleep apnea should be informed about all treatment options. In some cases, the decision is simple; after informing patients about all available options, a strong and clear recommendation can be given by the health care practitioner. In other cases, the decision regarding treatment is arrived at only after individual consideration of all the factors\u2014urgency of clinical situation, reimbursement plan available to patient, risk factors and the patient\u2019s ability or motivation to modify them, patient\u2019s preferences, and a possibility of having a trial of treatment with oral appliance and CPAP. There are patients with severe sleep apnea successfully treated with oral appliances, just as there are non-apneic snorers with or without upper airway resistance syndrome, successfully treated with CPAP. The decision regarding treatment in each individual patient is best made by medical practitioners with experience in sleep medicine who are aware of all options, and who are preferably a part of a specialized sleep disorders center.\nAn important issue, not addressed in this review, is the underuse of oral appliances currently. This is due in part to the lack of qualified dentists working in this area and in part to reimbursement policies. At present, the majority of government-sponsored and private health care providers will cover (fully or partially) the cost of CPAP, whereas very few, if any, health care plans will cover the cost of oral appliances. Considering that this treatment approach is the only non-invasive alternative to CPAP, it is important to continue to lobby health care providers to enable this treatment for qualified patients.","keyphrases":["oral appliance","sleep apnea","continuous positive airway pressure"],"prmu":["P","P","P"]}
{"id":"Dig_Dis_Sci-3-1-1914298","title":"Triggering for Submaximal Exercise Level in Gastric Exercise Tonometry: Serial Lactate, Heart Rate, or Respiratory Quotient?\n","text":"Gastric exercise tonometry is a functional diagnostic test in chronic gastrointestinal ischemia. As maximal exercise can cause false-positive tests, exercise buildup should be controlled to remain submaximal. We evaluated three parameters for monitoring and adjusting exercise levels (heart rate [HR], respiratory quotient [RQ], and serial lactate measurements) in 178 tests in both healthy volunteers and patients suspected of gastrointestinal ischemia. Exercise levels above submaximal occurred in 20% of HR-, 2% of RQ-, and 5% of lactate-monitored tests (P<0.05 for HR vs. RQ and lactate). Low levels were seen in 5% of HR-, 10% of RQ-, and 41% of lactate-monitored tests (P<0.01 for lactate vs. HR and RQ). High levels resulted in 43% false-positive tonometry results compared to 19% of all tests (P<0.001); low levels did not result in more false negatives (5% vs. 6%). Although RQ monitoring yielded the greatest proportion of optimal exercise tests, serial lactate monitoring is our method of choice, combining optimal diagnostic accuracy, low cost, and simplicity.\nIntroduction\nFor diagnosis in patients with suspected chronic gastrointestinal ischemia, we have previously shown that gastric exercise tonometry can be used as a functional test providing information about the adequacy of the gastrointestinal mucosal perfusion [1, 2]. In these studies it was demonstrated that during 10\u00a0min of submaximal exercise, gastric ischemia occurred only in patients with splanchnic artery stenosis. An extreme exercise level may cause false-positive tests, as shown in a study in healthy volunteers, where lactate levels exceeding 8\u00a0mM resulted in gastric ischemia in 50% [3]. Furthermore, it may be conceivable that exercise of very low intensity can lead to false-negative results in exercise tonometry used for diagnosing chronic gastrointestinal ischemia. In order to prevent false-positive and false-negative tonometry tests, the exercise intensity should be monitored throughout the test, and if necessary, adjustments of the workload should be made in order to obtain an optimal exercise test.\nExercise intensity can be monitored by various parameters including arterial plasma lactate concentration, decrease in serum arterial base excess (BE) or bicarbonate concentration (which are both directly related to lactate level), heart rate (HR), and respiratory parameters [respiratory gas exchange ratio (RQ) = carbon dioxide output (VCO2)\/oxygen uptake (VO2)] [4].\nIn this study we evaluated and compared three consecutive time periods in each of which a different parameter was used for monitoring the exercise intensity and adjusting the workload in order to obtain a submaximal exercise test. Initially HR monitoring was used; in the second period exercise intensity monitoring and adjustment were guided by monitoring of RQ. In the final period exercise intensity was monitored by serial rapid lactate measurements.\nSubjects and methods\nIn 10 volunteers (5 females, 5 males; mean age, 25 years; range, 23\u201328 years) and 157 patients (59 males, 98 females; median age, 55 years; range, 13\u201382 years), 178 tonometry exercise tests were performed. The volunteers were tested as part of a more extensive study investigating the effect of two different exercise levels on gastric tonometry [3]. All patients were suspected of having symptomatic chronic gastrointestinal ischemia. Their clinical presentation was that of unexplained abdominal pain, weight loss, diarrhea, or gastric ulcers. Gastric tonometry exercise testing was performed as a diagnostic function test in addition to duplex sonography and selective angiography of the splanchnic vessels. Some of the patients in this study have been previously described in a publication by our group investigating the diagnostic potential of gastric exercise tonometry [1].\nThe procedure of gastric tonometry exercise testing was described in detail in a previous study [3]. In short, a standard nasogastric tonometry catheter was inserted and connected to an automated air tonometry device that was set up to measure intragastric partial carbon dioxide pressure (PCO2) every 10\u00a0min. All subjects were studied after a fasting episode of 4\u00a0hr. Ranitidine, 100\u00a0mg, was given intravenously 90\u00a0min prior to exercise testing. A radial artery catheter was introduced in the nondominant arm to allow sequential arterial blood sampling.\nExercise was performed on an electromagnetically braked bicycle ergometer (Lode, Groningen, Netherlands).\nMonitoring exercise intensity and adjusting the workload\nThe maximal workload (Wmax) was estimated using standard criteria, i.e., age, sex, and weight [5]. The exercise episode was started at 10% of Wmax, and in the first 4\u20136\u00a0min the workload was increased every minute by 10% of Wmax. The workload was intended to remain constant thereafter at a submaximal exercise level. Three different approaches for monitoring the exercise intensity and\u2014if necessary\u2014guiding additional adjustments of the workload were evaluated in consecutive periods.\nFirst period: HR-triggered tests\nExercise intensity was monitored by measuring HR, aiming for a HR in the last 4\u20135\u00a0min of 80% of maximal predicted HR (HRmaxpred). HRmaxpred was calculated by the following formula: HRmaxpred = 210 \u2212 0.65, age [6].\nSecond period: Respiratory gas exchange ratio (RQ)-triggered tests\nThe workload adjustments were guided by measurement of the RQ, aiming for an RQ of 1.0 in the last 4\u20135\u00a0min of the exercise episode. This RQ was chosen as it indicates the anaerobic threshold\nThird period: Lactate-triggered tests\nIn 40 of the RQ-triggered tests serial rapid lactate measurements were done during exercise at t\u00a0=\u00a00, 4, 6, 8, and 10\u00a0min. These measurements were not used for guiding the exercise intensity, but to develop a lactate-based exercise triggering protocol. From the results of rapid serial lactate measurements in the RQ-triggered period, an algorithm was developed for triggering the exercise intensity (see Table 1). We aimed for a lactate concentration of 4\u00a0mM in the last 4\u00a0min of the test. In parallel with RQ triggering at an RQ value of 1.0, this lactate level resembles the anaerobic threshold, at which redistribution of blood flow patterns is initiated.Table 1Algorithm for lactate-guided exercise intensity adjustmentsStart at 10% of predicted WmaxIncrease workload by 10% of Wmax every minute (at t = 1, 2, 3, 4, 5, and 6\u00a0min)Lactate measurements at t = 4, 6, 8, and 10\u00a0minIf lactate >3\u00a0mM, decrease workload by 10% of WmaxIf lactate not >1\u00a0mM, increase 2 \u00d7 10% of Wmax\nIn the first two periods (HR and RQ triggering) the tests were performed at the pulmonary function department of our hospital. During these exercise tests a 12-lead electrocardiogram was recorded (Case 12; Marquette Electronics Inc., Milwaukee, WI, USA). HR was recorded every minute. Breath-by-breath oxygen uptake (VO2) and RQ were measured by a respiratory gas analyzer system (Oxycon-\u03b1; Jaeger, Bunnik, the Netherlands) and recorded every 30\u00a0sec.\nIn the third period (rapid serial lactate triggering) the test was performed at the gastroenterology function department; HR was monitored, but respiratory parameters were not monitored.\nArterial blood samples for determination of base excess (BE), bicarbonate (blood gas analyzer; Radiometer ABL520, Copenhagen, Denmark), and lactate (enzymatic assay; Cobas Fara; Roche Diagnostics, Branchburg, NJ, USA) were drawn, in parallel with the 10-min tonometry interval, before and immediately at the end of the 10-min exercise episode. Rapid serial lactate measurements were performed using a small portable lactate analyzer (Accutrend; Roche Diagnostics, Almere, the Netherlands), specifically developed for use during exercise testing. Using this device, measurement results were available within 60\u00a0sec after blood sampling.\nThe tests evaluated in the present study were all aimed at a submaximal level: lactate level (or BE decrease) at or just above 4\u00a0mM (the anaerobic threshold) and not exceeding 8\u00a0mM. After comparison with arterial lactate measurements (see Results), BE decrease during exercise was used as the parameter assessing the exercise intensity (Fig. 1). The optimal exercise level was a BE decrease of between 3 and 7\u00a0mM. Less than 3\u00a0mM and more than 7\u00a0mM were regarded as low and high exercise levels, respectively.Fig. 1Relationship between arterial lactate concentration after exercise and BE decrease at the end of the exercise period\nFor comparing the results of HR and RQ measurements with BE decrease as the indicator of the exercise intensity reached, the following parameters were used: maximum RQ reached (RQmax), total time RQ>1 (RQt>1), maximum heart rate reached (HRmax) as a percentage of HRmaxpred, and total time of HR >80% of HRmaxpred (HRt>80).\nStatistics\nAll values are given as mean\u00a0\u00b1\u00a0SD unless otherwise stated. P values <0.05 were considered statistically significant. Correlations between lactate and BE and lactate and bicarbonate decrease, and between both HR and RQ parameters and exercise intensity as measured by BE decrease, were calculated using Spearman\u2019s rank test. For comparison of HR and RQ parameters with exercise level (as divided into low, optimal, or high), one-way ANOVA with Bonferroni post hoc analysis was used. Group comparisons of the tonometry results of high and low exercise levels versus all tests and the diagnostic accuracy of tonometry in the different monitoring episodes were performed using chi-square testing.\nResults\nHR triggering\nIn 39 tests (10 in healthy volunteers and 29 in patients) HR triggering was used. In 22 (56%) of these HR-triggered tests the mean HR in the last 4\u00a0min equaled the target HR of 80%\u00a0\u00b1\u00a05% of HRmaxpred. In nine tests (23%) the target HR was not reached; in five of these the subjects were using \u03b2-blocking medication. In eight tests (20%) the HR was >85% of HRmaxpred.\nThe resulting exercise levels as measured by BE decrease are listed in Table 2. In three tests the exercise intensity was too low. In two the target HR was not reached; one of the subjects used \u03b2-blockers. Two tonometry results were true negative; the other was false positive.Table 2Resulting exercise intensities for the three triggering regimesExercise level(BE decrease, <3\u00a0mM)(BE decrease, 3\u20137\u00a0mM)(BE decrease, >7\u00a0mM)Triggering methodLowTargetHighHR (39 tests)3 (8%)27 (69%)9 (23%)*RQ (84 tests)8 (10%)74 (88%)2 (2%)Lactate (55 tests)23 (41%)**29 (53%)*3 (5%)Note. Values are given as numbers (percentage of total for triggering method). *P < 0.05 and **P < 0.001 comparing the three triggering regimes.\nOf the eight tests with an erroneously high exercise level, in only one test was the HR too high. The other six tests in which the HR was too high resulted in the submaximal exercise levels aimed for. When analyzing the 110 tests in which both HR and RQ were recorded, only a weak correlation was observed both between HRmax and exercise intensity and between HRt>80 and exercise intensity as measured by BE decrease (r=0.23 and r=0.20, respectively; P<0.05). There was no significant difference for the HR-derived parameters comparing low, target, and high exercise intensity. Figure 2 displays the comparison of HRmax for the three exercise intensity groups.Fig. 2HRmax (95% confidence interval) at low, target, and high exercise levels HRmax is expressed as percentage HRmaxpred\nRQ triggering\nIn 84 tests RQ triggering was used. In 67 (80%) of these tests the target RQ of 1.0 was reached while the mean RQ in the last 4\u00a0min was 1.0\u00a0\u00b1\u00a05%. In 11 tests (13%) the target RQ was not reached and in 6 tests (7%) the mean RQ in the last 4\u00a0min exceeded the target RQ by more than 5%.\nOf the eight tests with a low exercise level, the target RQ was not reached in four. In both tests with a too high exercise intensity, the mean RQ in the last 4\u00a0min was >1.05.\nWhen analyzing all 110 tests in which RQ was monitored, positive correlations were found for comparing RQmax and RQt>1 with BE decrease (r\u00a0=\u00a00.34 and r\u00a0=\u00a00.38, respectively; P\u00a0<\u00a00.0005). When divided into low, target, and high exercise intensity groups, both RQmax and RQt>1 showed significant differences among the three groups (P\u00a0<\u00a00.0005; one-way ANOVA with Bonferroni post hoc analysis). Figure 3 displays the comparison of RQmax for the three exercise intensity groups.\nLactate triggering\nIn 55 tests lactate triggering was used. In 26 of these tests the target lactate level of 4.0\u00a0mM in the last 2\u20134\u00a0min was not reached. This resulted in a BE decrease below 3\u00a0mM in 19 tests. In two tests the rapid lactate measurements exceeded 8\u00a0mM at t\u00a0=\u00a06 or 8\u00a0min. In one, the resulting exercise level was too high (BE decrease>7\u00a0mM). Of the 27 tests in which the target lactate levels were reached (at least 4\u00a0mM at t\u00a0=\u00a06 or 8\u00a0min, and remaining below 8\u00a0mM), the resulting exercise levels were too low in 7 and too high in 2.\nArterial lactate vs BE decrease and bicarbonate decrease\nIn 78 tests both arterial lactate levels and blood gas parameters were obtained. A high degree of correlation of both BE decrease and bicarbonate decrease with lactate level after exercise was found: r\u00a0=\u00a00.93 and r\u00a0=\u00a00.89, respectively (P\u00a0<\u00a00.0005). Figure 1 displays the relation between arterial lactate and BE decrease after exercise. Mean paired difference between lactate and BE decrease was 0.7 (SD, 0.7)\u00a0mM, and that between lactate and bicarbonate decrease was 1.4 (SD, 0.9)\u00a0mM.\nComparison accuracy of exercise tonometry at low, target, and high exercise levels\nAs shown in Table 1, lactate triggering resulted in significantly more low exercise levels compared to the other two regimes (P\u00a0<\u00a00.001).Fig. 3RQmax (95% confidence interval) at low, target, and high exercise levels\nFalse-negative tonometry results were found in 10 of all 178 tests (6%). Low exercise levels did not result in an increase in false-negative tonometry tests: in only 2 of 10 (5%) false-negative tests was the exercise level too low; 1 test was RQ-triggered, the other lactate-triggered.\nTonometry results were false positive in 34 of all 178 tests (19%). Of the 14 tests with a too high exercise level, in 6 (43%) the tonometry tests were false positive (P\u00a0<\u00a00.001 comparing high exercise level vs. all tests). Three of these tests were HR-triggered (but HR did not exceed the target HR), two RQ-triggered, and one lactate-triggered.\nThe sensitivity and specificity of tonometry exercise testing for gastrointestinal ischemia did not differ among the three groups (82% and 73%, respectively).\nDiscussion\nFor optimal diagnostic accuracy, the exercise level in gastric exercise tonometry can be monitored and adjusted by RQ measuring or, alternatively, by serial arterial lactate measurement. Although the latter resulted in more tests with lower than desired exercise levels, this had no influence on diagnostic accuracy.\nThe greatest problem relating to exercise level in gastric exercise tonometry is the above-submaximal, or maximal, test, as this is associated with 43% false-positive tests in this study. In maximal exercise, splanchnic blood flow is severely reduced and can lead to reduction below the level required to maintain aerobic metabolism. Although data on the splanchnic vascular responses to maximal exercise have not been established, ischemia has been established frequently in these circumstances [3, 7]. In a study on splanchnic blood flow in trained volunteers, mean superior mesenteric artery blood flow fell by 38\u201349% after a 10-min exercise test at 70% of maximal workload [8]. It has been shown that the flow reduction in the celiac artery exceeds that of the superior mesenteric artery but still does not exceed about 50% [9]. Therefore the normal splanchnic flow after submaximal exercise remains well above the normal lower level of normal splanchnic blood flow, which is estimated as approximately 30% of the normal baseline value [10]. This explains why submaximal exercise does not result in ischemia in subjects with normal splanchnic vessels. Gastric ischemia during submaximal exercise can be seen in patients with splanchnic stenoses or in patients with reduced cardiac output [1, 11]. Thus, for practical clinical purposes 10\u00a0min of submaximal exercise seems adequate to distinguish between normal and pathological gastrointestinal vascular responses.\nSome exercise should be performed to allow for blood flow redistribution and reduction of the splanchnic blood flow and, thus, provocation of ischemia. However, the minimally required level is currently unknown. Even with very low-intensity exercise in healthy humans a significant shift in blood flow from the abdominal viscera to the exercising muscles was observed [12]. Thus submaximal exercise as currently advocated might not be necessary, and lower lactate levels may suffice for diagnostic testing.\nTriggering on RQ measurements resulted in the highest proportion of tests within the target range but has disadvantages. It requires a pulmonary function laboratory with specific devices for measurement of exhaled carbon dioxide and inhaled oxygen, making the test expensive and more complicated. The advantage of RQ monitoring in minimizing the proportion of below-target exercise level did not result in a lower number of false-negative tests in this study.\nThe major advantages of the serial lactate measurement are its simplicity and low cost. This rapid test can be performed by paramedic personnel and enables determination of lactate within 60\u00a0sec. This enables adequate monitoring and triggering of exercise levels. A gastric tonometry exercise test, a routine procedure at our hospital in patients suspected of chronic gastrointestinal ischemia, takes 15\u00a0min of a doctor\u2019s time and 2\u00a0hr for a registered nurse. Moreover, the currently available air tonometry device allows for accurate and reproducible measurements without the hassle and problems associated with classically used saline tonometry [13].\nIn conclusion, by using RQ and serial lactate measurements, adequate exercise levels for gastric exercise tonometry can be achieved. Rapid lactate measurements and the presented algorithm for gastric exercise tonometry are feasible for daily clinical practice.","keyphrases":["exercise level","gastric exercise tonometry","tonometry","chronic gastrointestinal ischemia","exercise test","diagnosis"],"prmu":["P","P","P","P","P","P"]}
{"id":"Dig_Dis_Sci-3-1-1914244","title":"Melanoma of the Anus Disguised as Hemorrhoids: Surgical Management Illustrated by a Case Report\n","text":"Introduction\nAnal mucosal melanoma is a rare tumor that constitutes only 0.4\u20131.6% of all melanoma manifestations [1]. Prognosis is very poor, with a median survival of less than 2 years, despite curative surgery [2, 3]. Females are more likely to be affected than males and most patients present in the sixth or seventh decade of their lives [1]. Diagnosis is not always straightforward, and is often accidental after surgical treatment for presumed benign disease such as hemorrhoidectomy or lateral internal sphincterotomy, provided that tissue is submitted to the department of pathology [4]. Unfortunately, the tumor is often widely metastasized at the time of initial diagnosis [5, 6]. Here we present a patient who was referred to the surgeon for treatment of hemorrhoids but was diagnosed with anal melanoma.\nCase report\nA 62-year-old black man with a history of prostatism was referred to our hospital by his general practitioner with the diagnosis of bleeding hemorrhoids. For a 10-week period he noticed a swelling protruding from the anus with daily bleeding from the anus. Defecation was problematical because of pain, but the stool had a normal aspect. Inspection of the anus showed a painful swelling with a diameter of 1.5\u00a0cm with a dark necrotic aspect surrounding the anus below the dentate line. Rectal palpation was impossible because of pain. At the outpatient department the swelling was excised under local anesthesia of the entire anus (anal block). Because of the atypical aspect of the swelling, the material was submitted for pathologic examination, which revealed a melanoma of the anus. Further staging of the tumor was not possible based on this material because of fragmentation of the tissue.\nA sigmoidoscopy was performed but showed no further lesions. In addition, an abdominal and thoracic computed tomographic (CT) scan with contrast showed no signs of tumor around the anorectal region and no signs of intraabdominal, lymphatic or thoracic metastases. After the patient was diagnosed with having melanoma of the anus, examination under general anesthesia was performed. Inspection of the anal mucosa showed two additional lesions at 2 and 3 o\u2019clock with the sacrum at 6 o\u2019clock (Fig. 1). A wide local excision (WLE) including these lesions was performed with a margin of 2\u00a0cm, en bloc with 25% of the circumference of the rectal wall (Fig. 1). The total excised tissue was 6\u00d77\u00a0cm. Pathologic examination revealed two small foci of the melanoma with a depth of maximal 3.5\u00a0mm. All margins were free of tumor. The patient recovered well from the operation. After 1-year follow-up by means of outpatient department visits and proctoscopy every 3 months, and thoracic and abdominal CT-scan twice a year, there were no signs of recurrent tumor.Fig. 1Melanoma of the anus: wide local excision. A=melanoma\nDiscussion\nPatients with anorectal melanoma usually present with symptoms of rectal bleeding and an anorectal mass and are often misdiagnosed as having hemorrhoids, as was illustrated by the present case report [4]. Therefore, on any atypical anorectal lesion, biopsy should be performed in order to prevent delayed diagnosis.\nFor the operative management of anorectal melanoma, two options are available: a wide local excision (WLE) or a more extensive abdominal perineal resection (APR). The choice between these surgical procedures is controversial. The main arguments in favor of APR are its ability to control lymphatic spread and to create bigger excision margins, resulting in an assumed lower local recurrence rate. But, in contrast to WLE, APR is associated with mortality, considerable postoperative morbidity (4% hemorrhage, 11\u201316% wound infection and 14\u201324% wound dehiscence) and the need for a permanent colostomy [7\u201311].\nDespite attempted curative surgery, the median survival for anorectal melanoma is only 20 months and most patients die within 5 years regardless of the type of intervention used [12]. Therefore, quality-of-life issues must be given consideration when making treatment decisions.\nDroesch et\u00a0al performed a systematic review of the literature, including 14 studies with a total of 301 patients [12]. Wide local excision was performed in 129 patients, and 172\u00a0patients underwent APR. Local recurrence developed in 47% of patients after WLE and in 23% of patients after APR. Although these data suggest a trend towards better locoregional control after APR, the difference was not statistically significant. In addition, there was no difference in overall survival between WLE and APR (median survival 21\u00a0months for WLE and 17 months for APR).\nBullard et\u00a0al found an opposite trend with a rate of 50% of local recurrence after APR, compared to 18% after WLE in 15 patients [5]. Postoperative radiotherapy may improve locoregional control after wide local excision. In the retrospective study of Ballo et\u00a0al, actuarial 5-year local control was 74% and nodal control was 87% in 23 patients who received postoperative radiotherapy after wide local excision [13]. Definitive assessment of the efficacy of adjuvant radiation therapy requires further prospective studies.\nIn general, it is assumed that the stage of the disease, especially the tumor thickness, is the main prognostic factor [13\u201315]. Therefore, Weylandt et\u00a0al suggested that the decision between WLE and APR should be governed by the tumor thickness [16]. In patients with a tumor thickness below 1\u00a0mm, a local sphincter-saving excision with 1\u00a0cm safety margin would be appropriate; and in cases of a tumor between 1 and 4\u00a0mm, a wide local excision with a margin of 2\u00a0cm seems to be adequate. Patients with a tumor thickness above 4\u00a0mm or invasion of the internal sphincter muscle should be treated with APR. In the case report mentioned in this article, the tumor depth was 3.5\u00a0mm and excision margins were free of tumor, thus no additional APR was performed.\nIn order to evaluate the deepness of the lesion preoperatively, endorectal ultrasonography can be performed [6]. Accuracy in evaluating tumor depth of rectal cancer ranges from 81 to 94%, and accuracy in detecting lymph node metastasis ranges from 58 to 80% [17]. However, its accuracy in evaluation of anorectal melanoma remains unproved and is still evaluated [5].\nAt the time of diagnosis, up to one-third (16\u201333%) of the patients have disseminated disease [3]. For these patients, palliative treatment with chemotherapy might be a treatment option. Kim et\u00a0al reported a series of 18 patients with metastatic anorectal melanoma treated with cisplatin, vinblastine, dadabazine, interferon alpha-2b and interleukin-2 [18]. Major response was seen in 44% of the patients, and complete response occurred in 11%, with a median overall survival of 12.9 months. Yeh et\u00a0al used a different treatment regimen of temozolomide, cisplatin and liposomal doxorubicin in a patient with stage IV anal mucosal melanoma [19]. This patient showed a remarkable response to chemotherapy, with minimal residual disease and excellent quality of life at 12 months after the start of treatment. However, this therapy still has to be further evaluated and currently no standard systemic therapeutic regimen exists for metastatic anorectal melanoma.\nIn conclusion, anorectal melanoma represents both a diagnostic and therapeutic challenge to physicians given its non-specific presentation and rarity. It is associated with poor prognosis, regardless of the type of intervention used. Therefore, the overall treatment goal should be to optimize the quality of life. Since wide local excision is a more limited intervention associated with at least comparable survival compared to APR and no need for permanent colostomy, wide local excision is recommended as primary therapy if negative surgical margins can be achieved. APR should be reserved for patients in whom the tumor is thicker than 4\u00a0mm and\/or involves the anal sphincter.","keyphrases":["diagnosis","treatment","wide local excision","melanoma anus","abdominoperineal resection"],"prmu":["P","P","P","R","M"]}
{"id":"J_Urban_Health-2-2-1705510","title":"Exploring Barriers to \u2018Respondent Driven Sampling\u2019 in Sex Worker and Drug-Injecting Sex Worker Populations in Eastern Europe\n","text":"Respondent driven sampling (RDS) has been used in several counties to sample injecting drug users, sex workers (SWs) and men who have sex with men and as a means of collecting behavioural and biological health data. We report on the use of RDS in three separate studies conducted among SWs between 2004 and 2005 in the Russian Federation, Serbia, and Montenegro. Findings suggest that there are limitations associated with the use of RDS in SW populations in these regions. Findings highlight three main factors that merit further investigation as a means of assessing the feasibility and appropriateness of RDS in this high risk population: the network characteristics of SWs; the appropriate level of participant incentives; and lack of service contact. The highly controlled and hidden nature of SW organizations and weak SW social networks in the region can combine to undermine assumptions underpinning the feasibility of RDS approaches and potentially severely limit recruitment. We discuss the implications of these findings for recruitment and the use of monetary and non-monetary incentives in future RDS studies of SW populations in Eastern Europe.\nIntroduction\nIn many countries in Eastern Europe large scale social and economic transition over the past 15\u00a0years has been associated with widespread poverty and increasing levels of unemployment and population migration, particularly among women.1,2 Such transition has also been associated with shifts in patterns of sexual behaviour and sexual relationships, including increased commercial sex work, the growth of informal sex work economies and associated human trafficking.3 Additionally, a number of countries in the former Soviet Union have experienced a rapid or explosive spread of HIV associated with injecting drug use.4\u20137 These epidemics are potentially exacerbated by close links between sex work and injecting drug use.2,8\u201311 This has caused concerns that high levels of sexual mixing between injecting drug users (IDUs) and their sexual partners in settings of high HIV and sexual transmitted infection (STI) prevalence may transform HIV infection from concentrated epidemics among risk populations to generalised epidemics.9,10 It is therefore essential to assess the diffusion of HIV and related risk behaviour in hidden vulnerable populations, such as IDUs and sex workers (SWs), as accurately as possible.\nThere has been increased and recent interest in an approach to sampling hidden populations known as respondent driven sampling (RDS).12,13 One of the claimed advantages of RDS over other methods of sampling hidden populations, such as time\u2013location sampling, is that it requires little in-depth formative research among study populations.13,14 RDS begins with a set number of non-randomly selected seeds (members of the target population). Seeds recruit their peers (other target population members who make up their social network) who in turn recruit their peers into the study. This occurs through successive waves of recruits which, it is argued, become increasingly more representative of the underlying population as the recruitment progresses. Since RDS claims to generate a representative sample from non-randomly selected seeds, many researchers have assumed that sampling can proceed without detailed prior knowledge about the local target population.13,14\nRDS has proved feasible and successful in recruiting hidden populations of IDUs in a variety of settings, resulting in the rapid acquisition of long and varied recruitment chains.11,14\u201319 RDS has also been used in several countries to collect both behavioural and biological data from SWs (e.g., Vietnam, India) and men who have sex with men (e.g., Bangladesh, Cambodia, Uganda, United States).20\u201324 However, little is known about the feasibility of RDS as a method of recruiting hidden populations of SWs in settings where the organisation of sex work and access to SWs is highly controlled and where there is little or no contact between the target population and local services. Drawing upon three case studies in Eastern Europe targeting SWs and IDUs involved in SW (IDU\/SWs), we explore the feasibility of RDS, identifying some barriers to successful recruitment. These findings have implications for the direction of future research exploring the feasibility of RDS methods.\nMethods\nUsing RDS, data on HIV prevalence and associated risk factors were gathered from SWs in Serbia and Montenegro and from IDUs involved in sex work in Russia (see Table\u00a01). SWs in Serbia and Montenegro were given three uniquely numbered coupons with which to recruit other SWs. In Russia, although no RDS study directly targeting SWs was carried out, RDS was used to sample SWs\/IDUs in an RDS study among IDUs. Incentives were provided to participants completing the survey (primary incentive) and for recruiting other eligible participants (secondary incentive). Participants in Belgrade were also offered a rapid HIV test and pre- and post-test voluntary and confidential counselling (VCT). Those with preliminary-positive tests for HIV were referred to a local clinic for confirmatory testing. The studies were undertaken by local fieldwork teams coordinated as part of larger internationally funded research initiatives.\nTable\u00a01.Three RDS studies among sex workers and injection drug using sex workersCountryCity  (population size)Formative researchRecruitment criteria  for SWsNumber of  seedsSample reached  (target)Year, study  durationSerbiaBelgrade  (about 2 million)Literature search;  questions on network  characteristics and RDS  study feasibility posed  during individual  interviews with SWs in  2005 (n=10)Exchanged sex for  drugs, money or  other goods in  last 30\u00a0days  (irrespective of gender  or sexual orientation)Six initially,  six added later209 (400)2005,  2\u00a0monthsMontenegroPodgorica  (about 300,000)Literature search;  questions on network  characteristics and RDS  study feasibility posed  during in-depth interviews  with SWs in 2005 (n=4)Exchanged sex for drugs,  money or other goods  in last 30\u00a0days  (irrespective of gender or  sexual orientation)One to start with,  one added  later2 (150)2005,  2\u00a0monthsRussian  FederationTogliatti  (740,636)Literature search [relied on  previous qualitative study  of IDUs conducted in 2001  (n=57) and epidemiological  survey of IDUs including SWs  (n=426)]Exchanged sex for drugs,  money or other goods  and injected drugs in  last 4\u00a0weeksOne, 35 SWs recruited  by a female non-SW  IDU seed36 (not set)2004,  1.5\u00a0months\nAll studies were approved by appropriate local ethical committees and international human subject review boards. Participants were required to agree to an informed consent before participating. Participants were not linked to the study with any personal identifiers, and all participant information was kept confidential and anonymous.\nPrior to data collection, formative research, comprising a combination of key informant interviews and a literature review, was undertaken to assess the local feasibility of the RDS study identifying the optimal study set up (Table\u00a01, column 3). The formative research elicited data on size estimates of the sex work population; description of how sex work is organised (sectors of sex work, freedom of movement, average earnings and prices charged for services in each sector); and the legal situation surrounding sex work. In Serbia and Montenegro, researchers also asked SWs about the size of their social networks in order to fine-tune questions on which RDS analysis would be based. A social network was defined as the number of SWs, 18\u00a0years and older, each SW knows and has seen in the past week, month, three months, six months, or year.\nResults\nAs shown in Table\u00a01, a total of 209 SWs were recruited in Belgrade (Serbia), two in Podgorica (Montenegro) and 36 in Togliatti (Russian Federation).\nCase 1: Serbia\nRDS was conducted in the autumn of 2005 in Belgrade, Serbia\u2019s capital, by a local fieldwork team with a limited access to SW networks. Estimates of the size of the SW population in the municipality, based on police records and anecdotal estimates, vary widely from 900 to 3,000 SWs.2,24,25\nSex work is illegal. Findings from in-depth interviews of SWs suggest that there are approximately ten areas located near the main transit routes in and out of Belgrade where street SWs operate in small groups. Qualitative data suggests that the majority of SWs operate independently, advertising their services in newspapers or through referrals via existing client networks, potentially making them harder to reach through RDS.\nFormative interviews also found that there was little mixing of SWs across different sex work sectors (street, agency, and SWs working independently), among street SWs themselves (transvestite, Roma and Serbian\/Montenegrin heterosexual SWs) and among SWs working independently. However, in addition to a lack of strong connection among SWs and their independent working situations, the study team undertaking formative qualitative research associated SWs\u2019 reluctance to participate in the study to perceptions of inadequate incentive (10 euros for 1\u00a0hour of their time) and a general mistrust associated with coming into contact with \u2018official\u2019 agencies. However, the same interviews suggested that in principle SWs would be interested in participating in a survey involving the collection of both behavioural and biological data through HIV testing. Interviewees indicated that they knew approximately 20 other SWs, which again suggested that RDS would be feasible.\nThe RDS study had a target of 400 SWs, both male and female. The sample needed to be large enough for a statistically reliable analysis of risk factors and HIV prevalence, should RDS analysis show that the sample is reasonably representative. The interview site was on the premises of a non-governmental organisation (NGO) located in central Belgrade easily accessible by public transport. It was a part of a large apartment block with a separate entrance. The site was open everyday from 3 to 9 p.m. and could accommodate three to four participants an hour because of the computer administered questionnaire. After two months, the study ceased, having reached only 209 SWs. In total, 12 seeds were used during the study. Of the initial six seeds only three produced any subsequent waves. The rate of recruiting increased noticeably after an RDS study of IDUs being implemented at the same site had ceased. Due to the difficulties experienced by the research team in locating new seeds through other means, four participants with dense social networks were drawn from the seventh wave of two of the most productive recruitment chains and used as new seeds. This resulted in two more productive chains that ultimately reached seven waves.\nThe average SW network size reported during the study was 12. In response to the question, \u201cwhere do you most often solicit your clients?,\u201d the majority of SWs replied that they solicited on the street (43%), followed by referral from an agency, pimps, friends or other SWs (28.9%) and by telephone (25.2%). Importantly, the take-up of VCT provided as part of the RDS study was high (71.8%), and SWs indicated that they participated because of the HIV testing, rather than the monetary incentive.\nCase 2: Montenegro\nThere are no estimates of the number of SWs in Podgorica, the Republic\u2019s capital. Sex work is ostensibly illegal. Through formative rapid assessment no street-based SWs were identified and the majority of SWs were found to work independently from their homes. Key informant interviews (IDUs, ex-IDUs, taxi drivers, public health officials, NGO representatives and journalists) identified the only semi-public sex work sites as being approximately six illegal brothels, registered as lap dancing venues, in which most of the SWs also lived. At least one such brothel was found not to allow SWs to leave the premises. Indications were that local police also \u2018moonlighted\u2019 as security guards in these brothels. Other links between brothels and public services included taxi drivers, beauty salons and hotels. The intersection of the police and sex work networks, the small size of the city and the high number of implicated actors suggested that SWs could be easily monitored and controlled.\nPrevious attempts by the local NGOs to conduct research among SWs in Podgorica had failed. Key informants, interviewed during formative rapid assessment, reported knowing at least one SW or their pimps but in almost all cases introductions were denied or never materialized. Those conducting the formative rapid assessment received strong advice from local experts and key informants\u2014including NGOs, police, and taxi drivers\u2014against approaching brothel owners because of the highly controlled nature of SW venues in the city. After two weeks of trying to make a contact, four SWs were accessed via their drug user networks, the only entry points into SW networks available to researchers. These SWs were interviewed at their place of work (brothel and pimp\u2019s flat). Each of the four interviewed SWs independently estimated that there were approximately 200 SWs in the city, many of them also drug injectors, most of whom worked independently, advertising their services via newspapers and television and via client recommendation but who were not open about their SW status. All four SWs interviewed indicated that they would participate in a RDS study and would invite others to do so, and each estimated they had personal regular contact with between five and ten SWs.\nThe four SWs participating in the formative rapid assessment were invited to participate in the RDS study as seeds. Only one of the four participated but failed to recruit anyone else. She reported having had contact with ten SWs in 4\u00a0weeks, but no additional seeds were secured. Despite recruiting an additional SW (who required complete anonymity fearing being identified as a SW by others to his family and who had not seen other SWs in the past month), the RDS study failed to identify a sufficient pool of seeds willing to participate.\nCase 3: Russia\nThe RDS study of IDUs and IDU\/SWs was conducted in May 2004 in Togliatti City. Togliatti City is the second city of Samara Region situated approximately 1,000\u00a0km south of Moscow. Togliatti was selected as a study site because evidence suggests a recent increase in the number of IDUs and SWs and an explosive spread of HIV associated with injecting drug use.6,11\nApproximately 2,000 SWs are estimated to operate in Togliatti, half of whom work from the street.6 Despite ambiguous Russian legislation towards sex work, it is not tolerated by the wider community or the police. The city has been noted for its intense street policing of drug users and SWs, often involving fines levied under administrative codes, such as possession of drugs, causing a public nuisance, or lack of official residency permit, rather than criminal codes.26,27 Street-based SWs in Togliatti operate openly along the main roads and highways. Two popular locations are Moskovsky Prospect where approximately 70 women are working at any one time, and Pobeda Street where about 100 women work.\nIn a study conducted in Togliatti City in 2001, 50% of female IDUs (n=155) reported ever exchanging sex for goods or money, of whom 86% (n=66) were currently SWs.6 Although no stand-alone RDS studies of SWs have been conducted in Russia, attempts have been made to boost participation of IDU\/SWs in RDS studies of IDUs motivated by the evidence of high proportion of SWs who also inject drugs in other cities.28\u201330\nA cross-sectional survey of IDUs and female IDU\/SWs was undertaken in Togliatti in 2004 as a follow up to the 2001 baseline survey. A total of 476 IDUs were sampled during a 5-week period in May 2004 using RDS. Among these, 55 (11%) reported ever exchanging sex for money, drugs or goods, and of these, 36 in the past month, which was lower than expected. At the start of the field work, seven seeds were selected, of which one seed was female who had exchanged sex in the past month. SWs were explicitly asked to recruit other SWs, and non-SWs were encouraged to recruit either SWs or non-SWs (Table\u00a01, column 4). This first SW seed produced no recruits and was lost to follow up. Another female non-SW started a chain that recruited a total of 22 female IDUs who had ever exchanged sex, of whom 13 had exchanged sex in the past month. This female non-SW seed produced a total of 135 recruits by the end of the study equivalent to 28% of the total sample, of which 15% (20) were SWs and who reported working on the street. Among the other seeds, only a handful of current SWs were recruited (n=4). Towards the end of the project more coupons were distributed to female SWs in an attempt to increase the SW sample (n=5), none of which produced any recruits. In order to increase the sample of SWs in the survey, 21 SWs were subsequently identified and interviewed without coupons.\nData on the number of female SWs within an individual\u2019s IDU network were unfortunately not provided. The mean IDU network size among SWs was 19.5 (SD 14.2) and among non-SWs 27.1 (SD 33.0). Only one SW recruited through RDS reported working from an apartment; all others were street-based SWs. We expected that RDS would enable us to recruit more hidden populations of SWs such as those working from apartments or hotels. The fact that SW seeds failed to produce any waves of SWs or were lost to follow up, indicates that SW networks may not be as well connected.\nField notes show that initial attempts to access street SWs at Moskovsky Prospect and Pobeda Street had failed primary because of intensified police action in the area. A local officer of the Federal Security Service believed he had been recently infected with HIV by a SW from the area, and, in revenge, he and his colleagues enforced a repressive policy of fines and arrests among SWs. Recruiting SWs towards the end of the survey was more successful when policing practices returned to normal. Meanwhile, the sample target was reached for non-SW IDUs.\nDiscussion\nDespite undertaking formative research in the planning of these RDS studies, we found that RDS methods were unable to attain sufficient samples of SWs in Serbia, Montenegro, and Russia. A number of inter-related factors emerged as important to the success of RDS: low motivation of SWs to participate due to their perception of inadequate incentive; low-density SW networks combined with high level of independence in the organisation of sex work; a tightly controlled small number of sex work venues; lack of contact between SWs and local services in the context of high levels of mistrust of \u2018official\u2019 agencies; and difficulties emerging in the organisation of the RDS studies of SWs at the same time as a parallel RDS study of IDUs.\nWhereas in other settings (e.g., Vietnam, north-eastern India, Brazil) SWs have reacted favourably to nominal monetary incentives in RDS studies, including incentives which fall below the rate charged for sex,1617 it is possible that the incentives offered were perceived as being too low. In order to earn primary and secondary incentives, which together in all three studies amounted to about a half-an-hour SW\u2019s rate, they had to leave their work and travel to and from the fixed interview site at least twice plus spend at least an hour involved in the study. It is possible that SWs in Eastern Europe may be less interested in the incentives offered since they have comparatively higher earnings.\nIndications are that free HIV\/STI health assessments and referral for treatment should be explored in addition to the incentive. The high acceptance of VCT in Belgrade, for example, is a good example of how provision of services can be more effective than monetary incentives in recruiting survey participants. If incentives are purely monetary, they should be adequate to entice SWs to participate; yet this is often not feasible given the risk of high incentives also encouraging the attempted participation of non-SWs.\nUnlike other RDS studies conducted in Vietnam,16 SWs in Belgrade felt uncomfortable being interviewed at a location where a concurrent study of IDUs was being conducted. Based on anecdotal information and the fact that SWs in Belgrade started participating in the study once the IDU study had finished, we believe that SWs were dissuaded from going to a location that was used by IDUs. This problem can be assessed during formative research using focus groups with SWs to assess whether they would feel comfortable sitting in a waiting room with a group of male and female IDUs.\nPerhaps the main reason for the poor success of RDS recruitment among SWs in these three settings is the characteristics of SW networks. RDS methodology requires that the target population form sufficiently large and dense networks. However, the information on the network sizes of a few SWs gathered through our formative research are not easy to interpret since there is no known cut-off point for determining an appropriate network size or density for RDS. Based on the results of our studies, SWs in our three study areas often work independently, thus forming smaller, more isolated, networks. Those working on the street and in brothels are also under tight control by managers, pimps and police thus restricting their movement and weakening network density. On the other hand, prevalent societal norms\u2014a form of social control\u2014prevent SWs who work independently from revealing their SW status and participating. The low recruitment rate encountered in our studies might be overcome by allowing studies to run for a much longer time\u2014perhaps as long as six months or a year\u2014allowing SWs to gain trust in the investigators.\nIn the studies described above, interview sites were rented and staff employed for a fixed amount of time. A preferable setting is one with on-going services for SWs that can be used as an interview site for a long period of time allowing recruits to come at a slow rate. However, time was unlikely a major factor contributing to difficulties in RDS recruitment in the cities where our studies were conducted.\nThe dearth of well-established projects targeting SWs in Eastern Europe means that greater effort should be invested prior to RDS studies to identify a greater number of seeds and engender SWs\u2019 trust. In some cases existence of a well-established service with established contacts with local SW communities might be a decisive factor. For example, this was the case in a RDS study among SWs in Tbilisy, Georgia (January 2006, written communication, Johnston LG). In addition to promoting trust, such service organisations can provide additional networking opportunities for loosely connected SWs.\nConclusion\nGiven the cost of conducting second-generation sero-surveillance among highly vulnerable populations, formative research is important for establishing the appropriate type of sampling and reward system. We found the making available free and confidential HIV testing services was an effective incentive, and perhaps more so than monetary incentives alone. Although formative research would require some additional costs, it can increase efficiency in a more extensive and successful study.\nDuring formative research, we suggest that policy makers, service providers and target populations be consulted to determine the visibility of sex work, as low visibility can be associated with tight control of the sex work industry and weak networking among SWs, indicating difficulties using RDS. If researchers find it difficult to recruit SWs for formative study, the RDS study is likely to fail. Depending on the timescale, funding and interest among existing service providers, additional services may be provided well ahead of a RDS study in order to access SW respondents.\nFurther investigation is needed into the ethnography of SW networks in this region, especially on network composition and service access. In the case of IDUs who exchange sex for money, goods, or drugs, it is important to understand their primary network linkage\u2014injecting drug use or sex work\u2014and how they represent their \u201cother identity\u201d within each network. Incentives need to be better identified in advance of RDS studies, specifically as they relate to level of income, opportunity costs, and trust in the investigators and seeds. Without this cultural and legal sensitivity, surveillance for HIV among this vulnerable population will not provide adequate information on epidemic spread or program effectiveness.","keyphrases":["respondent driven sampling","sex workers","russia","serbia","montenegro","hidden populations"],"prmu":["P","P","P","P","P","P"]}
{"id":"Ann_Hematol-4-1-2174523","title":"Association between long travel and venous thromboembolic disease: a systematic review and meta-analysis of case-control studies\n","text":"The term \u201ceconomy-class syndrome\u201d defines an infrequent episode of venous thromboembolism (VTED) related to a long travel, namely by plane. However, this relation has not clearly been demonstrated by investigators. We carried out a systematic review and a meta-analysis of cases-control studies that had studied this topic. We realised a systematic review of the literature and selected all the case-control studies published. Two authors carried out a methodological evaluation according to the Scottish Intercollegiate Guidelines Network items (concordance was analysed by weighted kappa index), and a systematic analysis of the potential biases of each study was assessed. We carried out the meta-analysis with the data extracted from the studies. We recovered eight cases-control studies. The relation between the antecedent of a long travel and subsequent VTED varied from OR = 1.1 to OR = 4.0 and was found to be significant in four studies. The studies were highly heterogeneous in methodology and so the results obtained about the relation between the long travel and the VTED and the score at SIGN50. Two meta-analysis were carried out: only with travels by plane in which the relation was not significant (OR = 1.21; CI 95%, 0.95\u20131.55) and with all types of transport, with a slightly significant relation (OR = 1.46; CI95%, 1.24\u20131.72). We may deduce from this systematic review that there does exist a weak association between episodes of VTED and a long travel, but not by plane specifically. The heterogeneity and the methodological quality of the studies published preclude of more robust conclusions.\nIntroduction\nVenous thromboembolic disease (VTED) has been associated with many risk factors, one of which is the antecedent of a long travel, most often by plane. This association was previously suggested by Homans [1] and was believed so self-evident that it gave rise to the term \u201ceconomy-class syndrome\u201d [2]. Subsequently, the description of clinical cases related to other means of transport led to the coining of another more appropriate generic term, \u201ctraveller\u2019s thrombosis\u201d [3].\nThe scenario for this syndrome has varied from the alarm caused by the apparently high frequency of fatal pulmonary embolism (PE) occurring after a prolonged flight, as reported in the media, to the scepticism of a weak, although likely association, reported in various scientific studies. The published reports have ranged from descriptions of individual cases to cohort studies and even clinical trials. However, the low incidence of episodes of VTED after a long travel makes the design of a case-control study particularly useful for evaluating the suggested association [4].\nOur objective was to carry out a systematic review and meta-analysis of case-control studies to analyse the association between long travels and the development of VTED.\nMaterials and methods\nWe carried out a bibliographic search using a combination of keywords and MeSH headings in the Medline, Embase and Cochrane Library bibliographic databases. We selected only case-control studies, in any language, with no limit on the date of publication, and performed a cross search of the references cited in these studies. The corresponding author named in each study was requested to inform us of any relevant data that had not been described in the original manuscript.\nTwo authors (JTS and AJP) carried out a descriptive analysis of the studies identified in the search and evaluated their quality according to the checklist proposed by the Scottish Intercollegiate Guidelines Network [5]. Concordance in the evaluation of the items on this list was analysed using the respective weighted kappa indexes.\nWe also analysed the limitations and potential biases that might reduce the validity of the studies examined. The sources of bias analysed were grouped into three categories: exposure misclassification (recall bias), selection bias (including detection bias, effect misclassification, survival bias, self-selection bias and Berkson\u2019s bias) and confounding factors. Memory bias was defined as the fact that when subjects know they have suffered VTED, it makes them more likely to remember prior exposure to the antecedent of a long travel. Effect misclassification was minimised by considering only those studies in which VTED was diagnosed by objective complementary examination. Detection bias was suspected when the odds ratio (OR) in the days immediately after the travel was less than that corresponding to subsequent days. We only selected data published for VTED incidence at 30\u00a0days after the travel because was the time lapse most frequently analysed in the studies. Survival bias was considered to be that occurring when the study included prevalent cases, which could give rise to confounding of the variables related to the origin and those concerning the prognosis of the disease. Self-selection bias was considered to be present if the controls participated on their own initiative in the study and were not selected in a consecutive or random way; thus, they might be related to the results being sought. Berkson\u2019s bias would be derived from the use of hospital patients as controls.\nThe confounding factors were judged to be influential if no account were taken of the possibility that the exposure factor and the disease might be related through a third variable which was related to each of the other two.\nFrom the results included in the published studies and the data supplied by the different authors, we were able to perform a meta-analysis of the studies that had estimated the risk ratio of deep-vein thrombosis (DVT) and of DVT and\/or PE either after a travel (by any means of transport) or only after a travel by plane. The OR was used as a measure of association, with a confidence interval of 95%. The hypothesis of homogeneity was evaluated using X [2] and Der Simonian\u2013Laird tests, and fixed and random effects methods were used, respectively, for the cases of homogeneity and heterogeneity between the studies. We assessed possible publication bias using Begg and Egger tests and carried out a sensitivity analysis by evaluating the influence of the absence of each individual study on the global measure of association of all the other studies.\nThe computer softwares used for this study were SPSS 11.5 (SPSS, Chicago, IL, USA) and EpiDat 3.1 [6].\nResults\nNine case-control studies were recovered; of these, we analysed the data from seven [7\u201314], as the other one [15] corresponded to a preliminary analysis for one of the seven. One of these [7] was made up of three sub-studies, but for reasons of consistency, we only included the data for two of these in the meta-analysis, as in the third one, the authors assessed patients outpatients and inpatients with PE. Table\u00a01 shows the main characteristics of all these studies. The relation between the antecedent of a long travel and subsequent VTED varied from OR\u2009=\u20091.1 to OR\u2009=\u20094.0 and was found to be significant in four studies [8, 9, 11, 14]. In them, the OR ranged from 2.1 (1.1\u20134.0) to 4.0 (2.0\u20137.9).\nTable\u00a01Principal characteristics of the case-control studies evaluating the association between a long travel and venous thromboembolic diseaseStudy, yearFerrari, 1999Samama, 2000Dimberg, 2001Arya, 2002Hosoi, 2002Ten Wolde, 2003Martinelli, 2003Cannegieter, 2006Period of study1992\u201319951990\u201319911995\u201319982000\u201320012000\u201320011997\u201320001999\u201320011999\u20132000Origin of casesPatients admitted with VTEDDVT, symptomatic outpatientsEmployees of the World Bank with confirmed DVTDVT, symptomatic outpatientsDVT, symptomatic outpatientsDVT outpatients; PE outpatients and hospital admissionsPatients with VTED during previous 12 months and examined for possible thrombophiliaVTED, outpatients and admissionsOrigin of controlsPatients admitted in Cardiology Department, age-matchedViral syndrome, matched by age and genderEmployees of the World Bank without DVT, matched by month and year of diagnosisHospital attention for compatible symptoms; DVT excludedHospital attention for compatible symptoms; DVT excludedHospital attention for compatible symptoms; DVT or PE excludedFriends or partners, volunteers, of hospital patientsPartners of casesNo. of travel\/cases (%)39\/160 (24.4%)62\/494 (12.6%)3\/17 (17.6%)20\/185 (10.8%)15\/101 (14.9%)8\/130 (6.2%)31\/210 (14.8%)233\/1906 (12.2%)No. of travel\/controls (%)12\/160 (7.5%)31\/494 (6.3%)163\/489 (33.3%)31\/383 (8.1%)13\/106 (12.6%)38\/959 (4.1%)16\/210 (7.6%)182\/1906 (9.5%)OR (95% CI)4.0 (2.0\u20137.9)2.1 (1.4\u20133.4)0.4 (0.1\u20131.5)1.4 (0.8\u20132.5)1.25 (0.56\u20132.7)1.6 (0.7\u20133.5)2.1 (1.1\u20134.0)2.1 (1.5\u20133.0)Type of transportVariousVariousOnly planeVariousVariousVariousOnly planeVariousDuration of travelAt least 4 h (not stratified)Not stated (\u201clong travel\u201d)Not statedStratified into 3 and 8 hours>3 hoursStratified from 3\u20135h to >16 hStratified into 8 hAt least 4 h, stratified into 4 hLapse between travel and VTED (weeks)43442448Principal limitationsBerkson\u2019s bias, Controls age\u2013matchedNot specific to assess risk of travel, Confounding, Controls matched by age and genderOnly international travels were evaluated (not duration of travel)Selection of controlsConfounding, Not differentiated by duration of travelBerkson\u2019s biasSurvival bias, Matched by age, gender and academic level, Self\u2013selection bias, Recall biasMemory bias, NO objective clinical information about controls\nWith respect to means of transport, two studies [9, 12] only assessed travel by plane, while two [7] MEGA examined separately each means of transport. Another study [13] distinguished travel by plane from all other means of transport, while another two [10, 11] considered various means of transport jointly, but did not distinguish those corresponding to cases from controls, and in one study [8], any means of transport was eligible for consideration without further specification.\nThe duration of the travel was addressed in different ways: two studies [8, 12] made no reference to this question; two studies [11, 15] only considered travels with a duration exceeding 4 h, and another, those exceeding 3 h [13]. The other three studies [7, 9, 10] considered different durations. The lapse of time between the travel and the diagnosis of VTED was 2\u00a0weeks in one study [13] and 4\u00a0weeks for the others, except that of Samama et al. [8] which did not specify the period exactly and MEGA study [14] that included 8\u00a0weeks before. In the study by Dimberg et al. [12], two periods were analysed (15 and 30\u00a0days), but in our own analysis, only the data corresponding to 30\u00a0days were taken into consideration, this period being equivalent to the 4\u00a0weeks used in the other studies.\nFour studies [7, 9, 11, 14] analysed cases of both DVT and of PE, while the others only considered patients with clinical presentation of VTED in the form of DVT. The selection of cases was generally that of patients with symptoms of DVT and\/or PE. One study [12] used a register of days off work, kept by medical insurance companies for the staff of the company in which the study was carried out. The rate of prior history of a long travel, among the cases, ranged from 6.2% [7] to 24.4% [11].\nDiverse methods were used for selection of controls. Three studies [7, 10, 13], selected outpatients who, after attending hospital with symptoms compatible with DVT and\/or PE, were found not to have these diseases; in another study [11], the controls were hospital patients, matched by age, who had been admitted for other reasons during the same period of time as the cases. Two studies chose outpatient controls, matched by age and sex with the cases, one selecting patients with symptoms of respiratory viriasis [8] and the other using relatives and friends of patients who had suffered VTED [9]. Another study [14] chose the partners of the cases as controls. Finally, Dimberg et al. [12] chose ten control cases, matched by month and year of diagnosis of DVT, from among the staff of the same company that employed the cases. The study by Ferrari et al. [11] even used different exclusion criteria for cases and for controls such that anticoagulant treatment was an exclusion criterion for the former but not for the latter. The rate of prior occurrence of a long travel, among the controls, ranged from 4.1% [7] to 33.3% [12].\nWith respect to the possible biases present in each of the studies, we highlight the following:\nRecall or classification bias: the study by Martinelli et al. [9] could suffer from bias in this sense, as one of the exclusion criteria was the absence of a prior occurrence of VTED during the previous 24\u00a0months. In general, this bias is consubstantial with the studies of cases and controls such that the cases tend to relate the disease with the antecedent of interest.Detection bias: only one study [12] analysed two periods of time in which DVT had occurred after a long travel, namely at 15 and 30\u00a0days. The OR for the former period was greater than that for the latter, and thus, this form of bias was not present here.Misclassification bias: in all the studies, only cases of VTED that had been objectively diagnosed and for which the appropriate complementary examinations were taken into consideration, and thus, their correct classification was ensured. In three [7, 10, 13], the controls were derived from persons who, having sought medical treatment for symptoms compatible with DVT and\/or PE, were found not to suffer either of these after the same objective examination used to confirm the cases. In others, the controls presented no symptoms of DVT and\/or PE and so were also assumed to be true controls.Survival bias: this bias type could have occurred in the study by Martinelli et al. [9], as the cases chosen were those patients who had suffered an episode of venous thromboembolism during the previous 2\u00a0years and for whom a study of thrombophilia was performed; thus, there was, in fact, a selection of all the potential incidental cases of DVT during this period.Self-selection bias: in the study by Martinelli et al. [9], the controls were recruited from the relatives and friends of the cases, a circumstance that could have negatively influenced the relation being studied, as the sample was obtained from a population that was socially related to the cases, and thus, exposure factors could have been shared. In MEGA study [14], the controls were partners of the cases, and the evaluated relation might have been diminished.Berkson\u2019s bias: this bias might have been present in the study by Ferrari et al. [11], as the controls chosen were patients admitted to hospital with chest pain, which could have meant they had a lower probability of prior travel; this pathology was, therefore, a potential limiting factor. Other studies [7, 10, 13] could also have been affected by this form of bias in that the controls they chose were patients who had attended hospital with symptoms compatible with those of DVT and\/or PE; objective examination subsequently determined that neither pathology was present. The study by ten Wolde et al. [7] included cases of VTED present in patients who had been admitted to hospital for other reasons, which reduced the number of days before the episode of VTED in which the patient could have travelled.\nThe methodological evaluation of the studies is shown in Table\u00a02. Overall, the evaluation was appropriate in five studies [7, 10, 12\u201314], while results were less satisfactory in three [8, 9, 11] mainly because of the inadequate choice of controls and because the study design did not enable the authors to achieve a good estimate of the risk being studied. There was a high degree of agreement between the reviewers concerning the quality criteria of the cases and controls (weighted kappa index\u2009=\u20090.8).\nTable\u00a02Methodological evaluation of the studies included in the systematic review in accordance with SIGN 50 criteriaCharacteristicFerrari 1999Samama 2000Dimberg 2001Arya 2002Hosoi 2002Ten Wolde 2003Martinelli 2003Cannegieter 2006Internal validityClear, appropriate questionsGAGGGGAGSelection of subjectsCases and controls from comparable populationsAAA-GA-GGAPGIdentical exclusion criteria for cases and controlsP-AAAGGGAGParticipation rate by cases and controls95-NS80NSNS74-79NS91-NS83-77Comparison between participants and non-participantsNSNSNSNSNSNSNSNSCases are defined and clearly differentiated from controlsA-GGPAGG-APG-AIt is clearly stated that the controls are non-casesAGPPG-AG-APPEvaluationKnowledge of exposure did not influence designation of casesAAAPG-APAAThe exposure is measured in a standard, valid wayAPA-PG-AG-AG-APAConfoundingIdentification of main confounding factorsP-APAP-AAA-PP-AAStatistical analysisIdentification of confidence intervalsYesYesYesYesYesYesYesYesOverall assessmentControl of bias and confounding factors+\/++++\/+++\/++\/++++++++\/+++Confidence that the overall effect is due to the exposure being investigated++++++++++\/++++++\/+++The results are applicable to the target group of patients being studied+++++\/++++++++\/+++++++++++A single evaluation is shown when the two reviewers agree; otherwise, both evaluations are given.G Good, A adequate, P poor, NS not stated\nResults of the meta-analysis The studies were varied as regards the episode of VTED included (DVT and\/or PE), the controls chosen, the means of transport evaluated and the estimated duration of the travels. These variations influenced the performance of the meta-analysis. Thus, we decided to carry out two meta-analyses, on the basis of the similarity between study designs, i.e., on the one hand, studies of patients with DVT (with or without PE), considering any type of transport, and on the other hand, those studies examining only cases in which the subjects travelled by plane (Table\u00a03).\nTable\u00a03Results obtained in the studies, by type of transport\u00a0All types of transportOnly planeOther typesCasesControlsCasesControlsCasesControlsDVTSamama62\/494 (12.6)31\/494 (6.3)NSNSNSNSHosoi15\/101 (14.9)13\/106 (12.6)9\/101 (8.9)12\/106 (11.3)6\/101 (5.9)1\/106 (0.9)Aryaa20\/185 (10.8)31\/383 (8.1)16\/185 (8.6)29\/383 (7.6)4\/185 (2.2)2\/383 (0.5)Ten Woldeb8\/130 (6.2)38\/959 (4.1)NSNSNSNSDimbergcNANA17\/30 (56.7)489\/891 (54.9)NANADVT and\/or PEFerrari39\/160 (24.4)12\/160 (7.5)NSNSNSNSMartinelliNANA31\/210 (14.8)16\/210 (7.6)NANACannegieterd233\/1906 (12.2)182\/1906 (9.5)86\/1906 (4.5)72\/1906 (3.8)147\/1906 (7.7)110\/1906 (5.8)NA Not applicable, NS not statedResults are expressed as number of patients travelling \/ total (%).aArya: Travels of more than 3\u00a0hbTen Wolde. Restricted to patients capable of travellingcDimberg: More specific case definition of the two analyseddCannegieter: Venous thromboembolism episodes within the 8\u00a0weeks after travel\nMeta-analysis of studies including patients with DVT or DVT+PE, all types of transport Six studies [7, 8, 10, 11, 13, 14] were examined. The Der Simonian\u2013Laird test of heterogeneity gave a result of p\u2009=\u20090.011, with an estimated 75% total variance due to variance between the studies. The global estimator with a model of random effects produced an OR of 1.46 (95%CI, 1.24\u20131.72). No publication bias was recorded (the Begg and Egger tests results were not significant). The sensitivity analysis revealed that no study influenced significantly on the final OR. Figure\u00a01a shows the forest plot graph corresponding to this meta-analysis.\nFig.\u00a01Forest plot for studies that evaluated the antecedent of any type of transport and only travels by plane. a Travels by any type of transport. b Travels by plane\nMeta-analysis of studies including patients with DVT or DVT+PE, only travels by plane This meta-analysis included five studies [9, 10, 12\u201314]. The results of the Der Simonian\u2013Laird heterogeneity test were not significant, which suggests there was little heterogeneity between the variances of the studies. The OR estimator produced a non-statistically significant value of 1.21 (95%CI, 0.95\u20131.55). Again, no publication bias was detected, and the sensitivity analysis showed that none study was influential on the OR. Figure\u00a01b shows the forest plot graph corresponding to this meta-analysis.\nDiscussion\nThe results from our analysis reveal a slight association between long travels and the development of DVT or PE, although this association disappears when only travels by plane are considered. The published case-control studies were characterised by considerable heterogeneity, especially in the selection of controls, and there were some errors in the study design that could have led to bias in estimating the effect of the association being studied. A meta-analysis of case-controls studies has recently been published. In that study, we did not find any relationship between a long-distance travel and venous thromboembolic disease when all the means of transportation were valued or only by plane, but not all published studies were included [14].\nThe pathogenic basis of the \u201ceconomy class syndrome\u201d lies in a series of predisposing environmental factors within aircraft (low humidity, relative hypoxia and low barometric pressure) that would facilitate the relative dehydration of passengers; these factors would be accompanied by mechanical ones, such as immobility, the narrowness of the seats and the prolonged maintenance of a seated position, with flexion and compression of the poplitea region [15\u201317]. The description of episodes of DVT related to other means of transport produces a loss of plausibility of the environmental factors associated solely with travels by plane and puts the prime focus on purely mechanical factors and on the characteristics of the patients themselves. Moreover, a recently published study [18] found no variation at all between different coagulation factors after exposure to the environmental conditions resembling in a prolonged travel by plane. Nevertheless, in one recent systematic review, Philbrick et al. [19] emphasised the presence of prothrombotic risk factors and a flight duration longer than 6\u00a0h as predisposing to suffer a VTE episode.\nThe patient-related variables refer to a greater frequency of VTED risk factors. Thus, various studies have reported that a high percentage of the patients involved were obese, higher or smaller, aged more than 50\u00a0years, had antecedents of VTED, were taking oral contraceptives or presented a state of hypercoagulability or thrombophilia [2, 11, 15, 18, 19]. This result implies that there is a relation between transport-related factors and those concerning to the patients themselves, and that this is relevant to the development of an episode of VTED.\nStudies that have sought to analyse this association have obtained divergent results mainly because of the different study designs; this would explain the high variability in the OR obtained, which ranged from 1.1 to 4.0. Moreover, these studies were heterogeneous in various methodological respects, and this could have influenced the final results; some [8, 11] matched the cases and controls by sex and age, which limits the influence of these variables in determining the association under study; in the study by Martinelli et al. [9], the cases were a selection of all the possible incident cases within a given period; in another [11], the controls selected were not derived from the same population as that of the cases; the means of transport and the duration of the travel, as well as the type of VTED episode evaluated were different in all the studies; and, in general, no multivariate or stratified analysis was carried out to attribute the increased risk of developing an episode of VTED to the long travel.\nAn interesting aspect from the methodological point of view is the possible selection bias that might have entered these studies: those patients (the cases) who travelled presented fewer VTED risk factors than did the controls, i.e. they were basically more healthy. Not only could this have led to the relation between the development of an episode of VTED and the antecedent of having made a long travel being underestimated, but it might even appear that the prior travel had a protective effect, as was the case in the study by Dimberg et al. [12]. One way of minimising this type of bias could have been to include only those patients who would physically have been capable of making a long travel; this was only actually done in the case of the study by ten Wolde et al. [7]. Another possibility could be to adjust for this variable (which might be considered that of comorbidity) by means of multivariate analysis.\nGiven the methodological heterogeneity of the published studies of cases and controls, it was not possible to carry out a detailed meta-analysis of different aspects of the relation under study, and so its scope was limited to those studies that assessed the association between DVT or between DVT and PE and prolonged travels by plane or by any other form of transport. In any case, the association that was found in the latter case was only weak, and there was a large degree of variation in the methodological quality applied in the various studies.\nA noteworthy question is the antecedent of a prolonged travel made by the controls, with variations in all the studies of 6.3\u20138.1%, except in the case of the study by Dimberg et al. [12]; in the latter case, the scenario chosen for analysis led to the choice of a population in which a high proportion (33.3%) made international travels, an aspect that could have weakened any potential association. Similarly, the rate of incidence of a lengthy travel among the cases ranged from 6.7 to 14.8%, although in two studies, this figure was surprisingly high, 17.6% in that by Dimberg et al. [12] and 24.4% in the study by Ferrari et al. [11].\nThe methodological quality of the studies examined in this systematic review means that we must be cautious concerning the results reported. Although there does seem to be a likely relation between a long travel and the development of an episode of VTED, such an association must be of such a magnitude that a small bias or modification to the study could increase or decrease the strength of the association recorded.\nIn conclusion, we may deduce from this systematic review that there does exist a real, but weak, association between episodes of VTED and the antecedent of a lengthy travel, and this relation with the travels by plane is only nearly significant. The heterogeneity and the methodological quality of the studies published on the question limit the robustness of the conclusions obtained.","keyphrases":["long travel","venous thromboembolism","economy-class syndrome"],"prmu":["P","P","P"]}
{"id":"J_Assoc_Res_Otolaryngol-3-1-1915593","title":"Frequency-Dependent Properties of a Fluid Jet Stimulus: Calibration, Modeling, and Application to Cochlear Hair Cell Bundles\n","text":"The investigation of small physiological mechano-sensory systems, such as hair cells or their accessory structures in the inner ear or lateral line organ, requires mechanical stimulus equipment that allows spatial manipulation with micrometer precision and stimulation with amplitudes down to the nanometer scale. Here, we describe the calibration of a microfluid jet produced by a device that was designed to excite individual cochlear hair cell bundles or cupulae of the fish superficial lateral line system. The calibration involves a precise definition of the linearity and time- and frequency-dependent characteristics of the fluid jet as produced by a pressurized fluid-filled container combined with a glass pipette having a microscopically sized tip acting as an orifice. A procedure is described that can be applied during experiments to obtain a fluid jet\u2019s frequency response, which may vary with each individual glass pipette. At small orifice diameters (<15 \u03bcm), the fluid velocity of the jet is proportional to the displacement of the piezoelectric actuator pressurizing the container\u2019s volume and is suitable to stimulate the hair bundles of sensory hair cells. With increasing diameter, the fluid jet velocity becomes proportional to the actuator\u2019s velocity. The experimentally observed characteristics can be described adequately by a dynamical model of damped fluid masses coupled by elastic components.\nINTRODUCTION\nSensory hair cells are the primary mechano-detectors in several mechano-sensory organs in vertebrates such as the hearing- and vestibular organ and the lateral line system in fish and amphibians. The hair bundle is the mechano-sensitive organelle of a hair cell and consists of a well-organized arrangement of villi on the apical side of the cell, called stereocilia, in some types of hair cells together with a true cilium, the kinocilium. In most mechano-receptor organs, the hair cells are covered by an overlying tectorial structure bathed in fluid often mechanically coupling many hair bundles (Pumphrey 1950; van Netten 1997). These tectorial structures, when driven by fluid flow, convey the mechanical signals present in the fluid flow to the hair bundles. In the mammalian cochlea, for instance, sound signals are converted to vibrations of the cochlear endo- and perilymph, which drive the cochlear partition, causing a motion of the stereocilia. Deflections of a bundle of stereocilia, which pivot at their base, induce changes in the open probability of mechanically gated ion channels located in the tips of the stereocilia (e.g., Hudspeth 2000; Fettiplace and Ricci 2006). The combined mechanics and hydrodynamics of the hair bundles and tectorial structure determine an organ\u2019s sensitivity to a specific aspect of the excitatory fluid motion (e.g., van Netten 2006).\nTo quantitatively study the mechanics of tectorial structures and individual hair bundles and the mechano-electrical transduction process of hair cells, a well-defined mechanical stimulus is required. Due to the small dimensions of most tectorial structures, manipulation at a microscopic spatial resolution is desirable, especially when stimulating an individual hair bundle. A commonly used mechanical stimulus for individual hair bundles consists of an electromagnetic or piezoelectric actuator driving an elongated microscopic fiber or glass probe pushing against the hair bundle (e.g., Strelioff and Flock 1984; Crawford and Fettiplace 1985; Howard and Ashmore 1986; Howard and Hudspeth 1988; Russell et al. 1992; Kennedy et al. 2003, 2005). The probe\u2019s tip may adhere to the cell membrane of the kinocilium or tallest stereociliar row allowing for force application to the bundle in both directions. A stiff probe enables direct displacement of the bundle with virtually the same amplitude as the driving element, overruling the intrinsic bundle mechanics, and can be used to study transducer channel kinetics (e.g. Holton and Hudspeth 1986; Assad and Corey 1992; Vollrath and Eatock 2003; Kennedy et al. 2003). Alternatively, a more compliant probe with a stiffness comparable to that of the hair bundle can be used. Although under such conditions the application of displacement may become less direct, the stiffness of the probe can be designed as to not dominate the intrinsic mechanics of the hair bundle. Consequently, it allows for estimating the bundle\u2019s intrinsic mechanical characteristics like its compliance or resistance (Crawford and Fettiplace 1985; Howard and Ashmore 1986; Howard and Hudspeth 1988; Jaramillo and Hudspeth 1993; Benser et al. 1996; Kennedy et al. 2005). Viscous forces do, however, attenuate the efficiency of a compliant probe at higher frequency stimuli, limiting their use up to a certain cut-off frequency (Crawford and Fettiplace 1985; Howard and Hudspeth 1987). As several types of hair cells and most mechano-detecting structures in mechano-receptor organs are fluid-driven, a fluid coupling between stimulator and mechano-receptor is an attractive natural alternative to direct-contact stimulation with a probe. Well-defined fluid displacements can be obtained using a stimulus sphere attached to a piezoelectric element. Submerged in water, a sphere vibrating with a constant displacement amplitude may generate a near-field water flow that has a constant displacement amplitude as a function of frequency up to the order of a kilohertz (van Netten 1991, 2006). It does, however, stimulate very inefficiently at larger distances, as the stimulus strength in the near field measured as water displacement amplitude (A) is proportional to the third power of the ratio of stimulus sphere radius (a) to the distance (r) to its centre (i.e. ; e.g. van Netten 2006). To compensate for this reduced efficiency at larger distances, a stimulus sphere with larger radius (a) may be used. Large spheres, however, are less practical in many situations in which small experimental chambers and objective lenses do not provide sufficient space. To obtain a more targeted but sufficiently strong mechanical stimulus, a fluid jet emerging from a glass pipette with a small opening at its tip may provide a practical alternative solution.\nThe use of a fluid jet to mechanically excite individual hair bundles may have several advantages over stimulating with glass probes, noticeably yielding a better defined resting position of the bundle, a more even application of force, and in the case of lymph-driven hair cells, a more natural physiological stimulus. Also the asymmetry in force that may occur in pushing and pulling the cell membrane of the stereocilia, inevitably related to the direct contact of a glass probe, can be avoided. It is feasible that glass probes allow wider stimulating bandwidths although (lateral) resonances have been reported that may limit the high frequency response to the order of 10\u00a0kHz (e.g., Beurg et al. 2006; see also Discussion section). Fluid jet stimuli have been considered as effective force stimuli, whereas (stiff) probe excitation present displacement stimuli to the bundle (Vollrath and Eatock 2003). Such differences in stimulation may also lead to differences in speed of engagement of the transduction apparatus.\nIn one of the first reports on the use of a fluid jet in mechano-reception research, it was applied to drive the cupula of a fish lateral line neuromast (Jielof et al. 1952). The 4-mm pipette opening diameter used in that study was relatively wide compared to later, more miniaturized models, which were designed to displace individual hair bundles (Flock and Orman 1983). A first step to calibrating the frequency dependence of a fluid jet was set by Saunders and Szymko (1989). They used glass micro-beads with a density close to that of water that were captured in the hydrodynamic flow field and monitored their motion by stroboscopic-illuminating microscopy. Later developments of fluid jet-producing devices were utilized to displace single inner or outer hair cell bundles or lateral line cupulae and were in some cases pressure-calibrated (Kros et al 1992; Denk and Webb 1992; G\u00e9l\u00e9oc et al. 1997; Nicolson et al. 1998; Vollrath and Eatock 2003).\nIn the present work, a calibration procedure of a newly designed piezoelectrically driven fluid jet device is presented. A compliant glass fiber with a resin sphere attached to its tip is used as a sense probe to measure the dynamic fluid displacement output of the fluid jet device. The motion of the sense probe, which is reflecting the dynamics of the fluid jet, is monitored using a displacement measurement method implemented in a transmitted-light microscope. The procedure, which can be applied in an experimental setting, enables the determination of the frequency dependence of the fluid jet. This is essential to correct experimental data obtained using the same fluid jet as a stimulus. To understand the physical factors of the fluid jet-producing device governing the frequency response of the produced fluid jet, a theoretical model of the device has been developed. The results of the model were compared to the measured data and show that the fluid resistance of the orifice at the pipette\u2019s tip is a key parameter, influencing the amplitude and phase characteristics of the emerging fluid jet. As an example of its usefulness, the fluid jet system was applied to induce transducer currents in mouse cochlear hair cells, showing the importance to correct for the phase delay of the fluid jet, especially at high frequencies. This example also shows that fluid jet dynamics may lead to resonance, which in practice limits the use of the present design to a bandwidth of approximately 5.5\u00a0kHz.\nMATERIALS AND METHODS\nFluid jet-producing device\nThe device used for the generation of a fluid jet is shown in Figure\u00a01A and its individual parts in Figure\u00a01B. The main parts are a Perspex fluid container (C) and a brass rear end (R). Three screws (S) pull both parts together, sandwiching a piezoelectrically driven brass disc (P, \u00d8\u2009=\u200918\u00a0mm, thickness, 0.35\u00a0mm, resonance frequency \u2248 8.3\u00a0kHz, Conrad Electronic SE, Hirschau, Germany) in a configuration that is clamping the disc at its rim. The Perspex container is cone-shaped and is closed by the piezoelectric disc at the rear end. A rubber o-ring (O) between the Perspex and the piezoelectric disc provides a fluid-tight seal. When driven by a voltage signal, the piezoelectric disc produces forces that move the fluid in or out of the container.\nFIG.\u00a01.The fluid jet-producing device. A Picture of the assembled fluid jet-producing device mounted on an x,y,z-micromanipulator. B Disassembled device showing its separate parts: R Rear brass element, P piezoelectric disc, O o-ring, S screws, C Perspex container, W rubber cone washer, Sp Perspex spacer, Sc Perspex screw cap, Pi glass pipette. The calibration bar indicates 1\u00a0cm.\nAt the front end of the Perspex container, a glass pipette (Pi, outer \u00d8\u2009=\u20091.5\u00a0mm, inner \u00d8\u2009=\u20091.17\u00a0mm) is inserted. The screw cap (Sc) applies pressure on two cone washers (W) separated by a Perspex spacer (Sp) to produce a fluid tight seal and fixes the glass pipette in a mechanically stable way. In most applications, the pipette was narrowed at its tip using an electrode puller. Tip diameters were varied between 6 and 62\u00a0\u03bcm and tapering lengths were kept at approximately 3\u20134\u00a0mm. Also, in some experiments, untreated glass pipettes without tapered tips were used so that their effective opening equaled the inner diameter of the glass pipette (\u00d8\u2009=\u20091.17\u00a0mm). The total length of the glass pipette was about 5\u00a0cm of which about 1.5\u00a0cm was clamped inside the Perspex container. To facilitate horizontal application of the produced fluid jet in the experimental chamber, the glass was bent 1\u00a0cm from the tip over an angle of about 20\u00b0 so that its tip could be aligned within the horizontal plane (Fig.\u00a01A). For inducing transducer currents in hair cells (see Applications), straight glass pipettes were used.\nTo obtain an air-free filling of the fluid container in the Perspex body part, it was filled with a low viscosity silicone fluid (200 Fluid 5\u00a0CS, Dow Corning, Midland, MI, USA), which has low surface tension properties. The silicone fluid was degassed under vacuum conditions for several hours before use to prevent dissolved gas from creating bubbles inside the fluid chamber. The glass pipette was filled with de-mineralized water, or in the case of physiological experiments, with extracellular fluid (bath solution). Inserting the filled glass pipette into the Perspex body part generated a pressure increase inside the fluid container, replacing a volume of the water in the back side of the pipette by silicone fluid. During this temporary overpressure, the fluid jet device was transferred to its position in the set-up, and the pipette tip was submerged in the bath solution preventing air from entering the tip.\nWhen prepared, as described above, the instrument also benefits from the high resistivity (\u22481015\u00a0Ohm cm) of the silicone fluid creating an electrical resistance between the brass disc and the bath solution, thereby, preventing electrical cross-talk of the piezoelectric control signal to the possible electrophysiological recordings obtained from a preparation. Additional electrical shielding was obtained by connecting the brass disc, which is facing the preparation, to the electrical ground. The rear end of the fluid jet device, which together with the grounded brass disc enclosed the piezoelectric material, was also grounded, thereby, shielding off the applied voltages.\nA shaft connected to the rear end of the fluid jet device was used to mount the device on an x,y,z-micromanipulator (Fig.\u00a01A). The tip of the jet pipette was lowered in the bath solution and positioned under visual or microscope guidance. All calibration measurements were done approximately 5\u00a0mm above the bottom and below the surface.\nSignal generation\nSine wave stimuli at exponentially distributed frequencies ranging from 1 to 1,000\u00a0Hz were generated at 32 points per period using the full amplitude range of a 16-bit D\/A converter (Ariel, DSP 16, Highland Park, NJ, USA). These signals were subsequently attenuated to the desired amplitude and filtered (8 pole Bessel, 3988, Krohn-Hite Corporation, Brockton, MA, USA) at eight times the stimulus frequency. Frequencies used to probe resonances exceeding 1\u00a0kHz were derived from a signal generator (PM 5129; Philips, Eindhoven, The Netherlands). Signals for exciting hair cells were generated using a 16-bit D\/A converter with dedicated software (Power 1401 and Signal 2.0 software package, Cambridge Electronic Design Ltd, Cambridge, UK).\nSense probes\nSense probes used for the detection of fluid displacement consisted of compliant glass fibers (Borosilicate glass capillaries GC150T-10, Harvard apparatus Ltd., UK) produced with a micropipette puller (P-97 Sutter instrument Co., Novato, CA, USA) with a small sphere that was produced by dipping the glass fiber tip into a resin (Bison, Silicone universal transparent, Goes, The Netherlands). After curing the resin, sphere diameters ranged from 10 to 60\u00a0\u03bcm. The fibers were about 1\u00a0cm in length with a gradually decreasing diameter from about 100\u00a0\u03bcm to approximately 2\u00a0\u03bcm at their tips. The fibers were glued to a stiff glass capillary that was subsequently mounted on an x,y,z-micromanipulator. The mechanical frequency responses of the sense probes to fluid displacement were obtained by stimulating the sense probe with the fluid flow produced by a glass stimulus sphere (\u00d8 1.1\u00a0mm) attached to a piezoelectric element. The frequency response of this stimulus sphere was separately determined using a laser interferometer (see Motion detection).\nWhen stimulating the same sense probe with a fluid jet, the center of the fluid jet was aimed at the center of the resin sphere. The distance between the jet-producing pipette tip and the resin sphere of the sense probe was at least one diameter of the sphere. Based on the frequency responses of the sense probes, their overall stiffness coupling to the stiff glass tube held in the x,y,z-micromanipulator was estimated to be of the order of 10\u22124\u00a0N\/m.\nMotion detection\nThe motion of the stimulus sphere and the sense probe were measured with a heterodyne laser interferometer coupled to a fixed-stage transmitted light microscope mounted on a vibration isolated table. The objective lens (\u00d740\u2009WI, NA 0.8, Zeiss, Oberkochen, Germany) of the microscope (modified Axiotron, Zeiss, Oberkochen, Germany) focuses two coherent laser beams (HeNe 127, Newport Spectra Physics GmbH, Darmstadt, Germany) aligned in parallel so that their beam waists intersect in the plane of focus creating a measuring volume with microscopic dimensions (\u00d8\u2009\u2248\u20094\u00a0\u03bcm).\nA frequency difference of 400\u00a0kHz between the two laser beams produced by two Bragg cells (1201E, Isomet Corporation, Springfield, VA, USA) driven at 40.0 and 40.4\u00a0MHz, creates a moving fringe pattern in the measuring volume. Light scattered by a stationary object, therefore, fluctuates in intensity at a carrier frequency of 400\u00a0kHz (fhet). Additional motion of the object induces a phase modulation of these intensity fluctuations, which is displacement-dependent.\nThe back-scattered laser light from an irregularity on the surface of the object was detected by a photomultiplier (model H6780 02, Hamamatsu Photonics, Japan) coupled to an I\u2009\/\u2009V converter. The output, containing the carrier frequency, fhet, phase-modulated in proportion to the object\u2019s displacement, was band-pass filtered with a centre frequency at fhet and electronically demodulated using a modified frequency demodulator (OFV 3000, Polytec GmbH, Waldbronn, Germany, equivalent r.m.s velocity noise was 0.1\u00a0\u03bcm\/s). Time-varying phase modulations were thus treated as frequency modulations (i.e., Doppler shifts), which was appropriate, as the phase changes per time of the carrier were small compared to the carrier frequency itself. Under these conditions, the output signal of the demodulator is a calibrated linear measure of the velocity of the object within the range of 10\u22121 to 103\u00a0\u03bcm\/s. For low frequency stimuli (<10\u00a0Hz), a digital phase demodulator (OVD-20, Polytec GmbH, Waldbronn, Germany) was used, which has an output proportional to the displacement of the object with a 32-nm resolution.\nThe demodulator signals were low-pass filtered (3988, 8 pole Bessel, Krohn-Hite Corporation, Brockton, MA, USA) at eight times the frequency of stimulation and were amplified before being digitized using a 16-bit A\/D converter (DSP 16, Ariel, Highland Park, NJ, USA) with a sample frequency at 32 times the frequency of stimulation. Displacement responses consisting of usually ten consecutive stretches, each 16 periods in length, were averaged on-line by the data acquisition board. The first 4\u00a0s of a response to a stimulus were not recorded to prevent effects of onset transients.\nThe averaged response waveform was stored on hard disc. A fast-Fourier transform (FFT) was used to extract the amplitude and phase at the frequency of stimulation from this averaged waveform.\nIn some experiments, a Position Sensitive Detector (PSD; PSM2-2 On-Trak, Photonics Inc, Lake Forest, CA, USA) was used to measure displacements of uncalibrated sense probes at frequencies beyond 1\u00a0kHz, using a A\/D converter with dedicated software (Power 1401 and Signal 2.0 software package, Cambridge Electronic Design Ltd, Cambridge, UK).\nHair cell electrophysiology\nExperiments were performed on apical-coil outer hair cells obtained from acutely isolated organs of Corti taken from neonatal C57Bl\/6 mice (Harlan Netherlands B.V., Horst). Animal procedures conformed to Dutch governmental rules and the guidelines of the University of Groningen Institutional Animal Committee (RuG-DEC). Transducer currents in response to fluid jet stimulation were measured under whole-cell voltage clamp with an Axopatch 200B patch clamp amplifier (Axon Instruments Inc, Molecular Devices, Foster City, CA, USA) at \u221284\u00a0mV holding potential. Further details on the procedures and solutions used can be found elsewhere (Kros et al. 1992; van Netten et al. 2003).\nModel of fluid jet dynamics\nSchematics of a mechanical model illustrating the principal physical elements of the fluid jet-producing device are shown in Figure\u00a02. The device is shown to work with only minor nonlinear distortion (e.g., Fig.\u00a03) so that the system may be adequately analyzed in the frequency domain. In this section, all dynamic variables will therefore be considered to vary harmonically with angular frequency, \u03c9. A piezoelectrically driven disc, with mass Mpiezo and stiffness Spiezo is used to pressurize the fluids in the jet-producing device. A force, F0, on the brass diaphragm of the piezoelectric disc is produced in proportion to the applied voltage, V0. This causes a displacement of the disc, Xc, so that the fluid in the Perspex container is pressurized. Compressibility of the fluid is neglected (see also Discussion section).\nFIG.\u00a02.Mechanical model representation of the fluid jet-producing device. F\n0 is the piezoelectrically induced force, proportional to the applied voltage, V\n0. The piezoelectric element consists of a brass disc (dark gray) and a smaller ceramic plate. The spring, S\npiezo, and a resistive element, R\nc, together with the mass of the piezo, M\npiezo, and the mass of the container fluid, M\nc (cone-shaped), affect the fluid displacement amplitude, X\nc, in the Perspex container. The fluid displacement, X\nc, is amplified through a lever arm, with ratio L, into L\u00b7X\nc, which drives the fluid in the glass pipette. The same lever ratio applies to the forces F\n1 and F\n2. The displacement response of the fluid mass in the pipette, X\np, is determined by the pipette\u2019s spring, S\np, the pipette\u2019s tip resistive element, R\np, and the mass of the pipette fluid, M\np. X\np is taken as a proportional measure of the displacement of the jet fluid.FIG.\u00a03.Sense probe displacements induced by the fluid jet. A Sense probe displacement in response to a 106-Hz vibrational fluid jet stimulus as a function of time. An average of ten traces is shown. B Fast Fourier transform of the average waveform shown in A with a fundamental component at 106\u00a0Hz and a second harmonic component at 212\u00a0Hz, which is more than 40\u00a0dB less in amplitude than the fundamental component. Total harmonic distortion (THD) was calculated using , where a\n1 is the response amplitude at the fundamental frequency and a\nn is the amplitude of the nth harmonic component, as determined from the FFT calculated from the averaged waveform of measured sense probe responses.\nThe fluid in the Perspex container has a mass, Mc. Fluid mass appears in the dynamic equations (e.g., Eq.\u00a01a) as a product of mass times its acceleration. The fluid mass in the cone-shaped Perspex container can be considered as the sum of a series of cross-sectional fluid slices with variable radius, so that the fluid mass of each slice is proportional to the square of its radius. Assuming that the equation of continuity holds in combination with incompressibility means that the fluid flux along the series of slices is conserved. The velocity of each fluid slice and also its acceleration must therefore be inversely proportional to the square of its radius, so that the product of mass and acceleration of each slice is independent of radius. This means that the overall product of mass and acceleration of the fluid in the cone-shaped Perspex container can be dynamically modeled with a cylinder with length L1 (7\u00a0mm), equal to the cone\u2019s actual length and a diameter D1 (13.4\u00a0mm) equal to the cone\u2019s base. Therefore, the mass of the fluid in the Perspex container is modeled as  and its effective acceleration as . Here \u03c1 (1,000\u00a0kg\/m3) denotes the fluid density. The fluid mass in the container Mc (0.987\u2009\u00d7\u200910\u22123\u00a0kg) sums with that of the piezoelectric disc Mpiezo (0.741\u2009\u00d7\u200910\u22123\u00a0kg), resulting in Mtot\u2009=\u2009Mc\u2009+\u2009Mpiezo (1.728\u2009\u00d7\u200910\u22123\u00a0kg). A resistive element, Rc, in series with mass Mc is included, which is related to friction of the fluid flow in the Perspex container and the motion of the piezoelectric disc. The mass in the cylindrical glass pipette is described by  (53.8\u2009\u00d7\u200910\u22126\u00a0kg), where L2 (50\u00a0mm) and D2 (1.17\u00a0mm) are the length and inner diameter of the glass pipette, while this mass has a displacement Xp. The overall effect of the conservation of flux in the conical Perspex container is the transformation of the effective displacement of fluid in the Perspex container into that of the glass pipette. The transformation factor equals the ratio of the (maximum) cross-sectional areas of the two fluid compartments  (\u2248131). The force ratio of this transformation is governed by the same ratio, . The transformed fluid displacement, , is not completely transferred into displacement of the fluid mass in the pipette, Xp, as the elastic cone washers around the glass pipette absorb it partly. This is represented by the compliant element, Sp. A resistive element, Rp, describes the viscous friction in the pipette, which is dominated by the tip resistance. The resulting fluid displacement, Xp, is taken as a proportional measure of the fluid displacement of the jet emerging from the pipette tip. Again, assuming the equation of continuity, this proportion is related to the square of the ratio of the pipette\u2019s tip diameter to the glass pipette\u2019s cross-sectional diameter (D2). However, the absolute displacement of the jet is strongly dependent on the distance to the pipette tip (See also Absolute fluid jet velocity in the Discussion section).\nThe governing equations relating the piezoelectric force, F0, to the fluid displacements Xc and Xp, utilizing the usual notation for time derivatives, are then:\nand\nChanging to the frequency domain, V0, F0, F1, F2, Xc, and Xp are all assumed to vary harmonically with time so that they are proportional to the factor exp(i\u03c9t). This factor will be omitted in the further analysis so that only (complex) amplitudes can be written for these variables. Displacement of the fluid emerging from the jet pipette, Xp, as a function of angular frequency, \u03c9\u2009=\u20092\u03c0f, and piezoelectrically induced force, F0, is then described by:\nwith a relative transformation factor, T(\u03c9), between the displacement in the container and the pipette given by:\nInspection of the denominator of Eq.\u00a02a shows that two minima may occur as a function of \u03c9\u2009=\u20092\u03c0f, corresponding with a low (fl) and high (fh) resonance frequency. The high resonance frequency, fh, can be found by minimizing the term between square brackets, assuming that T(\u03c9) varies only slowly around fh. This frequency is therefore related to Mtot, the summed mass of piezoelectric disc and mass of the fluid in the Perspex container, and the parallel combination of Spiezo, and the up-transformed stiffness of the pipette, L2Sp. Together, they amount to the stiffness , in accordance with Mtot\u2019s position in between these two parallel elastic elements (Fig.\u00a02). The related high-resonance frequency is consequently given by:\nAt relatively low frequencies the frequency dependent terms of the factor between square brackets (Eq.\u00a02a) are relatively small so that this factor reduces to Spar. Then Xp can be approximated in the low frequency range by:\nwhere Sser is the series spring combination, of Sp and Spiezo\u2009\/\u2009L2 given by:\nIt then follows from Eq.\u00a04a that the low-frequency resonance, fl, is given by:\nEquation\u00a04c shows that the low frequency oscillation is due to the mass of the fluid in the pipette, Mp, that is elastically supported by the effective stiffness, Sser, which is formed by the series combination of Sp and the down-transformed piezo-stiffness, Spiezo\u2009\/\u2009L2, in line with their series configuration (Fig.\u00a02). The parameters Spiezo, Rc, Sp, and Rp were determined from fitting the full model (Eqs.\u00a02a and 2b) to the measured data. The fluid masses were determined from their actual physical sizes (D1\u2009=\u200913.4\u00a0mm; D2\u2009=\u20091.17\u00a0mm) and lengths (L1\u2009=\u20097\u00a0mm; L2\u2009=\u200950\u00a0mm).\nRESULTS\nLinearity of the fluid jet\nFigure\u00a03A shows a typical sinusoidal displacement of the sense probe driven by a fluid jet produced with a pipette tip diameter of 62\u00a0\u03bcm. The average sense probe displacement of 10 consecutive traces is given each containing 16 periods at a frequency of 106\u00a0Hz. The waveform clearly shows the 16 periods of the fundamental frequency with negligible harmonic distortion. The extent of the distortion was determined by the Fast Fourier Transform (FFT) calculated of the waveform shown in Figure\u00a03A. Figure\u00a03B shows the resultant frequency content of this waveform up to 600\u00a0Hz. The fundamental frequency is clearly visible at 106\u00a0Hz and rises about three orders of magnitude above the noise floor. The second harmonic component is visible, but has an amplitude more than 40\u00a0dB below the component at the fundamental frequency. Higher harmonics are even smaller. The calculated total harmonic distortion in this case was \u221232.7\u00a0dB (see legend Fig.\u00a03).\nFluid jet calibration\nTo determine the frequency-dependent motion of a fluid jet produced with a pipette, we used the following procedure. The vibrational displacement of the sense probe induced by the fluid jet was measured as a function of stimulus frequency. The frequency-dependent amplitude and phase characteristics obtained this way are, however, contaminated with frequency-dependent characteristics of the sense probe and other possible frequency-selective components of the equipment. To isolate the fluid jet frequency response, three additional frequency responses were measured in this procedure, which are displayed in Figure\u00a04.\nFIG.\u00a04.Schematic representation of the fluid jet correction procedure. A Each column (1\u20134) represents a type of measurement needed to complete the correction. The frequency responses measured with the sense probe contain frequency-dependent properties of ED (equipment, demodulator), S (stimulus sphere), SP (sense probe), or FJ (fluid jet) as a function of frequency. Results represented in a column are used to correct the measured response in the next column, indicated by the long arrows and produce results pointed at with a short arrow. B Displacement amplitude and phase for each measurement (1\u20134) described in (A). Each solid line is the result of a correction (except for column 1) and is used in the next column to correct the measured frequency response (symbols), both for the amplitude and the phase response.\nThe first step (Fig.\u00a04A1) is to obtain the frequency-dependent properties of the demodulator (D) including the additional signal measuring and conditioning equipment (E) like (anti-alias) filters and amplifiers. For this step, a voltage-controlled oscillator (VCO) was used to generate a frequency-modulated 400\u00a0kHz carrier signal, which was sent to the demodulator. These signals of the VCO at various known modulation frequencies simulate a calibrated output of the photomultiplier similar to operating the laser interferometer. The resulting response, ED(f), characterizing demodulator (D) and equipment (E) is presented in Figure\u00a04B1, as a function of frequency, f. It shows a nearly constant amplitude as a function of frequency and a clear phase change starting at about 300\u00a0Hz as a result of the used anti-alias filter.\nThe second step characterizes the frequency response of the stimulus sphere, S(f). The laser interferometer is used to directly measure its displacement amplitude and phase as a function of input frequency with fixed voltage amplitude. This response, consisting of ED(f)\u2009\u00d7\u2009S(f) (Fig.\u00a04B2; data points) can now be divided by ED(f), obtained in step 1, to isolate the pure stimulus sphere response, S(f) (Fig.\u00a04B2; solid line). The resonance at approximately 900\u00a0Hz, originating from the mass of the stimulus sphere and its elastic attachment, can clearly be discerned. The fluid displacement produced by a vibrating stimulus sphere can be taken proportional to the displacement of the sphere S(f) (e.g., van Netten 2006).\nIn the third step, the same stimulus sphere vibrating in water is used to hydrodynamically displace the sense probe, which has frequency characteristics SP(f) that subsequently will be used to probe the fluid jet displacement. The sense probe displacement resulting from sphere stimulation, ED(f)\u2009\u00d7\u2009S(f)\u2009\u00d7\u2009SP(f), as measured by the laser interferometer is thus contaminated with equipment, i.e., demodulator and stimulus sphere characteristics. This response is divided by the previously (step 2) isolated stimulus sphere response, S(f), resulting in the sense probe frequency response ED(f)\u2009\u00d7\u2009SP(f) (Fig.\u00a04B3; solid line), which is intentionally still contaminated with the equipment response characteristics, ED(f).\nIn the fourth step, this result, ED(f)\u2009\u00d7\u2009SP(f), is used to divide the fluid jet-driven sense probe response measured by the interferometer ED(f)\u2009\u00d7\u2009FJ(f)\u2009\u00d7\u2009SP(f), thus eventually isolating the amplitude and phase of the pure fluid jet displacement response, FJ(f), as a function of the frequency (Fig.\u00a04B4; solid line).\nEffects of tip diameter\nMeasurements\nThe dimensions of the object that has to be stimulated largely determine the diameter of the tip of the glass pipette to be used for producing the fluid jet. Mechanically stimulating an individual hair bundle usually requires pipette tip (i.e., orifice) diameters smaller than 10\u201315\u00a0\u03bcm, whereas some tectorial structures like a lateral line neuromast may require tip openings of up to 1\u00a0mm. A narrow tip increases the outflow resistance and thus decreases the fluid flow of the jet as evidenced by the larger voltage amplitudes that have to be applied across the piezoelectric disc to obtain equal displacement amplitudes of the sense probe.\nAnother important consequence of the tip diameter is its influence on the fluid flow frequency characteristics of the fluid jet. To illustrate this calibrated fluid jet characteristics obtained as described in the previous section are shown for different tip diameters in Figure\u00a05. This figure also shows the amplitude and phase response of the resulting fluid displacement when using a pipette without a tip restriction (Fig.\u00a05A). In this case, the glass pipette was cut at the same length as the pipettes used in Figure\u00a05B and C, but was not tapered at its tip. Its outflow diameter therefore equals the inner diameter of the glass pipette (1.17\u00a0mm). The characteristics of the fluid jet generated using such an unrestricted tip in response to signals applied to the piezoelectric disc within a range of frequencies and with a fixed amplitude, have a constant displacement amplitude up to a certain frequency (about 100\u00a0Hz) and the jet thus effectively behaves as a displacement stimulus in this frequency range (Fig.\u00a05A, data points). At higher frequencies, the amplitude starts increasing and approaches a resonance at about 150\u00a0Hz. Beyond this resonant frequency, the displacement amplitude decreases at a rate of about \u221240\u00a0dB\/dec, which is reminiscent of a second-order system driven beyond resonance. Around the resonant frequency, the phase rotates over an angle of 180\u00b0, which is consistent with these second-order characteristics.\nFIG.\u00a05.Effect of tip diameter on fluid jet response. Data points are the measured amplitude and phase responses as a function of frequency using a pipette without a tip restriction (A), a 56-\u03bcm tip (B), or a 7-\u03bcm tip (C). The solid lines are fits to the data with the model description (Eqs.\u00a02a and 2b) of the jet-producing device (Fig.\u00a02). Fixed parameters based on physical sizes and properties: L\n1\u2009=\u20097\u00a0mm; L\n2\u2009=\u200950\u00a0mm; D\n1\u2009=\u200913.4\u00a0mm; D\n2\u2009=\u20091.17\u00a0mm; \u03c1\u2009=\u20091,000\u00a0kg\/m3. Fixed parameters: S\npiezo\u2009=\u20092.0\u2009\u00d7\u2009106\u00a0N\/m; R\nc\u2009=\u20097\u00a0Ns\/m. Varied parameters: A\nS\np\u2009=\u200985\u00a0N\/m, R\np\u2009=\u20090.0165\u00a0Ns\/m; F\n0\u2009=\u20097.0\u00b710\u22123\u00a0N, (B) S\np\u2009=\u20099\u00a0N\/m, R\np\u2009=\u20090.09\u00a0Ns\/m; F\n0\u2009=\u200962\u00b710-3\u00a0N, (C) S\np\u2009=\u200911\u00a0N\/m, R\np\u2009=\u20092\u00a0Ns\/m; F\n0\u2009=\u200952\u00b710-3\u00a0N.\nA different jet response is produced with a pipette with an intermediate tip restriction (56\u00a0\u03bcm; Fig.\u00a05B, data points). At low frequencies, the jet\u2019s fluid displacement is proportional to the voltage applied to the piezoelectric disc, as evidenced by the constant amplitude and the almost zero phase. With this more restricted pipette tip, the displacement amplitude, however, starts declining beyond 15\u00a0Hz, changing towards a slope of about \u221220\u00a0dB\/dec with an according \u221290\u00b0 phase rotation in the mid-frequency range. At about 400\u00a0Hz, the slope of the amplitude starts to decrease further and also the phase lag changes to values more negative than \u221290\u00b0. With this more restricted tip diameter (56\u00a0\u03bcm), no clear resonance is evident as was the case with the larger diameter (1.17\u00a0mm).\nIn the case of an extremely restricted tip with a diameter of 7\u00a0\u03bcm, the fluid jet device produces a displacement amplitude response, which declines with about \u221220\u00a0dB\/dec over the measured frequency range (1\u20131,000\u00a0Hz; Fig.\u00a05C data points), so that effectively, the velocity amplitude of the fluid jet is constant within this frequency range. Consistent with these characteristics is the almost constant phase lag of \u221290\u00b0, except at the higher frequencies where it starts to decline further. These frequency characteristics reflect an overdamped system. In the case of a small pipette tip diameter, therefore, the resulting dynamics lead to a fluid jet of which the velocity is proportional and in phase with the voltage applied to the piezoelectric disc within the measured frequency range.\nModel calculations\nTo get a better understanding of the physical factors influencing the output characteristic of the jet-producing device, the measured data were described by a mechanical model of the masses of the fluids related to the two main compartments of the device, i.e., the Perspex container part and the pipette (see Materials and Methods). The mass of the piezoelectric disc (Mpiezo) is combined with the fluid mass contained in the Perspex fluid container (Mc), because they can be considered to move in unison, having a total mass . The fluid mass in the glass pipette is indicated with Mp. These two effective masses, Mtot and Mp, are mechanically coupled by a lever and a spring Sp (Fig.\u00a02). The displacement amplitude of the mass in the pipette, Xp, is considered to be proportional to the displacement of the fluid jet emerging from the pipette tip and is calculated as a function of frequency of the force produced by the piezoelectric disc. The force is assumed to be proportional to the electrical signal applied to the piezoelectric disc. The results of this model were subsequently fitted to the measured data points (Figs.\u00a05A-C, solid lines).\nThe model results support the measurements in showing that the value of the resistance controlled by the tip of the pipette, Rp, strongly influences the fluid jet response characteristics. When fitting the model to the data obtained for different tip diameters, the value of the resistance, Rp, increases more than two orders of magnitude (Fig.\u00a05A solid lines, Rp,small\u2009=\u20090.0165\u00a0Ns\/m; Figure\u00a05C solid lines, Rp,large\u2009=\u20092\u00a0Ns\/m), when the pipette without tip restriction (\u00d8large\u2009=\u20091.17\u00a0mm) is compared with the most restricted pipette (\u00d8small\u2009=\u20097\u00a0\u03bcm). Fitting the flow produced by the pipette tip with intermediate opening (\u00d8\u2009=\u200956\u00a0\u03bcm) results in an intermediate resistance value (Fig.\u00a05B, solid lines, Rp\u2009=\u20090.09\u00a0Ns\/m). Besides these expected changes in Rp, the value of Sp needed to be modified (9 to 85\u00a0N\/m) for obtaining proper model fits to the data. The cone washers (Fig.\u00a01; denoted with W) are the most compliant structures in the assembly to which part of the elastic stiffness Sp can most likely be attributed. The variation in Sp found between individual fluid jets could be due to the variability with which the cone washers were positioned during the insertion of the pipette and subsequently tightened. In addition, we cannot exclude the variability in the amount of gas or air bubbles present in the silicone fluid causing these variations in Sp between individual fluid jets. Except for Sp and Rp, all other parameters of the model were kept fixed in fitting the results with different pipette tip dimensions (Fig.\u00a05).\nResonance frequencies\nUnless overdamped by resistive elements, a combination of a spring and a mass leads to a resonance phenomenon and is accompanied by a \u2212180\u00b0 phase rotation and an amplitude response slope change of \u221240\u00a0dB\/dec around the resonance frequency. The present model, including two sets of masses and elastic elements, therefore predicts two possible resonant frequencies, fl, and fh (see Materials and Methods). The high resonance frequency, fh, is beyond the calibrated frequency range considered (1\u00a0kHz). This resonance is related to the summed mass (Mtot) of the fluid in the Perspex container (Mc) and that of the piezoelectric disc (Mpiezo) combined with the parallel stiffness (Spiezo) of the piezoelectric disc and the up-transformed stiffness of the cone washers (L2Sp), as effectively acting on the fluid mass in the Perspex container (Fig.\u00a02; Eq.\u00a03). The existence of a second resonance at higher frequencies was confirmed by applying both sinusoidal signals and voltage steps to the piezoelectric disc of the fluid jet device. The displacement of a (uncalibrated) sense probe in front of a small pipette tip (\u224810\u00a0\u03bcm) of the fluid jet device driven by a voltage step clearly shows a resonance at about 5.65\u00a0kHz (Fig.\u00a06). The same value was found using a series of sinusoidal voltages applied to the piezoelectric disc. The high-resonance frequency also coincides with the resonance frequency of the back side of the piezoelectric disc measured directly using laser interferometry, while the disc was being clamped and operated in the same fluid jet device (not shown).\nFIG.\u00a06.High frequency fluid jet resonance. Response of the fluid jet to an unfiltered step driver voltage applied to the piezoelectric disc, showing a high resonance frequency, f\nh\u2009=\u20095.65\u00a0kHz. (solid line, calibration as indicated). Parameters used in the model (dashed line): S\npiezo\u2009=\u20092.12\u2009\u00d7\u2009106\u00a0N\/m; R\nc\u2009=\u20094\u00a0Ns\/m; S\np\u2009=\u20096.4\u00a0N\/m, R\np\u2009=\u20090.402\u00a0Ns\/m; F\n0\u2009=\u20092.5\u2009\u00d7\u200910\u22123\u00a0N.\nIn the case of the unrestricted pipette with small damping, the low resonant frequency, fl, was observed (Fig.\u00a05A), together with the accompanying \u2212180\u00b0 phase rotation within the measured frequency range. This low-frequency resonance, fl, (see Eq.\u00a04c) can be attributed to the mass of fluid in the pipette (Mp) and the combined series stiffness, Sser, that is associated with the compliant cone washers or air bubbles (Fig.\u00a01, C) and the down-transformed piezo stiffness Spiezo\/L2, which effectively also acts on Mp. The resonance frequency resulting from substituting the fitted parameters of Figure\u00a05A in Eq.\u00a04c yields fl\u2009=\u2009152\u00a0Hz, similar to the measured value (\u2248150\u00a0Hz).\nApplications\nMechanical excitation of sensory hair cells using the fluid jet\nTo measure transducer currents at acoustic frequencies, whole-cell voltage-clamp recordings of individual outer hair cells were performed in acutely prepared organs of Corti of mice (e.g., G\u00e9l\u00e9oc et al. 1997; van Netten et al. 2003). The hair bundles were stimulated by the present fluid jet device using narrow tips (\u00d8\u2009<\u200915\u00a0\u03bcm), so that fluid jet velocities were proportional to the voltage applied to piezoelectric disc (e.g., Fig.\u00a05C). The pipette\u2019s tip was aimed at the hair bundle, the mechano-receptive structure of a hair cell, at an angle of about 20\u00b0 with the apical plate of the hair cell, and at a distance of approximately 15\u00a0\u03bcm from the hair bundle.\nFigure\u00a07 shows examples of inward transducer currents measured in an apical mouse outer hair cell. The hair bundle was excited with 16 periods of a pure sinusoidal fluid stimulus at 100\u00a0Hz and at 1,000\u00a0Hz, as produced by the micro-fluid jet device. The driver voltage and resulting jet velocity are symmetric, whereas the measured transducer currents (Fig.\u00a07B; 100\u00a0Hz; Figure\u00a07C; 1,000\u00a0Hz) are asymmetric. This is expected as the transducer channels only gate ionic currents when they are deflected in the direction of the tallest hair bundles (e.g., Hudspeth 2000; Fettiplace and Ricci 2006).\nFIG.\u00a07.Application of the fluid jet to excite sensory hair cells. A Schematic representation for recording transducer currents in cochlear outer hair cells. Indicated are the fluid jet\u2019s pipette tip and the hair cell under whole-cell voltage-clamp configuration with a patch pipette at the lateral side. The sinusoidal driver voltage (DV) applied to the piezoelectric disc is indicated. Positive voltages correspond with the fluid moving out of the chamber towards the hair bundle so that it bends in the excitatory direction towards the tallest stereocilia. B Transducer currents in response to a fluid stimulus at 100\u00a0Hz. (C) Transducer currents in response to a fluid stimulus at 1,000\u00a0Hz from the same cell, and using the same fluid-jet pipette as in B. D Responses of transducer currents (dashed, 100\u00a0Hz; solid, 1,000\u00a0Hz) shown on an extended time axis in relation to two stimulus periods of the driver voltage (DV, thin black line upper trace). The two transducer currents have a comparable magnitude but show a different delay in activation with respect to the maxima of the driver voltage (vertical thin lines), which is caused by the different phase delays of the fluid jet at 100 and 1,000\u00a0Hz. The measured delays are 0.0194 and 0.171 periods, for respectively, the 100\u00a0Hz and the 1,000\u00a0Hz stimulus, and correspond to a phase delay of 7\u00b0 and 62\u00b0 respectively. E Step driver voltage (DV) applied to the piezoelectric disc of the device and the evoked transducer current showing a 5.65-kHz oscillation produced by resonance of the fluid in the Perspex container.\nIn Figure\u00a07D, two periods of the traces from Figure\u00a07B and C are shown on an extended time scale. For both frequencies, there are delays in peak transducer current with respect to the maximum stimulus (vertical lines). The measured delays are 194\u2009\u00b1\u2009155\u00a0\u03bcs and 171\u2009\u00b1\u200915\u00a0\u03bcs (mean\u2009\u00b1\u2009SD, n\u2009=\u200916), for, respectively, the 100\u00a0Hz and the 1,000\u00a0Hz stimulus. This corresponds to a phase delay of 7\u2009\u00b1\u20096\u00b0 at 100\u00a0Hz and to 62\u2009\u00b1\u20095\u00b0 at 1,000\u00a0Hz, which can be attributed to the frequency-dependent phase delay of the fluid jet (Fig.\u00a05C; see also Discussion).\nHair cells also reliably transduce the (high-frequency) resonance of the fluid jet device in response to an unfiltered step applied to the piezoelectric disc as can be seen in Figure\u00a07E. The hair cell response shows an initial step transducer current followed by an oscillation of 5.65\u00a0kHz. This frequency is identical to the resonance observed while applying an unfiltered fluid jet stimulus to the sense probe (Fig.\u00a06) and thus illustrates the need for proper filtering of the voltage step to avoid fluid jet resonance.\nDISCUSSION\nReliability of the calibration method\nThe flow of microscopic fluid jets produced by several types of devices has been used in hair cell research for several decades (Flock and Orman 1983; Saunders and Szymko 1989; Kros et al. 1992; R\u00fcsch et al. 1994; G\u00e9l\u00e9oc et al. 1997; Vollrath and Eatock 2003; van Netten et al. 2003). In almost all cases, the mechanical output characteristics are assumed to be constant in terms of fluid displacement or velocity but generally lack detailed data on these actual output characteristics.\nIn the present study, a method was developed to dynamically calibrate a microscopic fluid jet using a flexible sense probe that was independently calibrated by means of an additional stimulus device (stimulus sphere), providing its detailed frequency selectivity within a relevant frequency range. This information is necessary to properly analyze the displacements of objects induced by a fluid jet. Because changes in the tip diameter of the jet-producing pipette alter the frequency response, a calibration for each individual pipette is necessary especially when applied at higher frequencies (e.g., Fig.\u00a07D). The developed calibration procedure using a sense probe has been shown to be practical within the frequency range tested (1\u20131,000\u00a0Hz). This calibration procedure intrinsically produces results for the flow of the jet in a free and unobstructed fluid field. This is related to the principle used, which is based on equivalent forces acting on a sense probe when stimulated with either a fluid jet or a sphere with known (free) flow field.\nThe frequency-dependent characteristics of a particular sense probe can be determined well in advance of the actual physiological experiment, and the sense probes can be stored for later use. During an experiment, a sense probe can be positioned in close proximity of the preparation to enable calibration of the fluid jet directly after the stimulation of the preparation without having to move the pipette tip out of the water. Essential for this procedure is that the fluid jet\u2019s frequency response does not change over time. A potential danger is that the pipette tip gets (partially) blocked by dirt floating in the bath solution. Such an additional restriction of the tip leads to changed jet response characteristics, precluding a proper correction of the measured data. However, in practice, such a blockage in the glass pipette\u2019s tip can be monitored by microscopic visualization.\nDisplacement or velocity output depends on tip size\nFrom the experiments on fluid jets produced by various glass pipettes, it appears that the pipette\u2019s tip size is the dominant parameter determining the fluid jet output characteristics both in terms of the overall output and frequency selectivity (Fig.\u00a05). One of the most relevant features of this stimulus device is whether it is the fluid displacement or velocity of the jet that follows the voltage applied to the device. Our results show that with a large tip diameter (unrestricted pipette) the displacement of the fluid jet is proportional to the displacement of the piezoelectric disc in a low frequency range (<100\u00a0Hz). This displacement mode is accompanied with resonance behavior (Fig.\u00a05A). A restriction of the pipette\u2019s tip diameter critically damps the system, preventing it from resonating. Under such conditions (e.g., \u00d8 56\u00a0\u03bcm; Fig.\u00a05B), it produces a constant fluid displacement amplitude as a function of frequency of the input signal up to about 10\u201320\u00a0Hz. Beyond this frequency, the displacement amplitude falls off with \u221240\u00a0dB\/dec and is accompanied by a phase rotation that may reach \u2212180\u00b0 at high frequencies. An even higher restriction of the tip diameter (\u00d8 7\u00a0\u03bcm; Fig.\u00a05C) results in a fluid displacement that declines with about \u221220\u00a0dB\/dec from low (1\u00a0Hz) to high (1,000\u00a0Hz) frequencies with a fixed phase delay of about 90\u00b0. This means that the velocity of the fluid jet follows the voltage applied to the piezoelectric disc. This is the most effective mode for producing a hydrodynamic stimulus to excite micro-sized objects such as sensory hair bundles.\nComparison of measured fluid jet characteristics with model\nA physical representation of the fluid jet-producing device as two damped fluid masses coupled via a lever and loaded with springs describes the behavior of the fluid jet emerging from the pipette\u2019s tip quite well (Fig.\u00a05 solid lines). It follows from fitting the model response to the measured data that the dynamics of the fluid in the pipette, rather than that in the larger Perspex container, dominantly determines the overall frequency response characteristics below 1\u00a0kHz. In this frequency range the output can be approximated with Eq. 4a, which shows the importance of Rp and Mp in relation to the frequency selectivity. It also shows that the overall output at frequencies around and below the low resonance frequency () is inversely proportional to the stiffness of the piezoelectric disc .\nThe high resonance frequency of the fluid jet was found at 5.65\u00a0kHz (Fig.\u00a06). The value of this resonance frequency was confirmed by laser interferometric measurements on the back side of the piezoelectric disc under the same fluid load conditions. When the fluid was removed from the Perspex body and no pipette was inserted, the piezoelectric disc resonated at about 8.3\u00a0kHz. As in this configuration only the disc is moving, we could estimate its effective dynamic mass value in combination with the value of Spiezo to be Mpiezo\u2009=\u20090.741\u2009\u00d7\u200910\u22123\u00a0kg. This appears to be 65% of the physical mass of the disc (1.14\u2009\u00d7\u200910\u22123\u00a0kg), which seems to be a reasonable fraction if the circular disc is moving in a mode with a circular node imposed by its peripheral clamping support.\nThe compressibility of the fluid in the Perspex container and that in the pipette has been neglected. This can be justified as the obtained maximum stiffness values of the elastic elements associated with these masses, Spiezo (\u223c2\u2009\u00d7\u2009106\u00a0N\/m) and Sp (\u223c85\u00a0N\/m), are significantly smaller than the effective stiffness values associated with their compressibility, 4.4\u2009\u00d7\u2009107 and 4.7\u2009\u00d7\u2009104\u00a0N\/m, respectively. These effective stiffness values, Seff, were evaluated by considering the compressibility along the long axis of a fluid-filled cylinder having length, L, and cross sectional area, A, and can be shown to equal Seff\u2009=\u2009KA\u2009\/\u2009L, with K (=\u20092.2\u2009\u00d7\u2009109\u00a0Pa) the bulk modulus of water (Lide 2005).\nHighly restricted pipettes with tip diameters of the order of 15\u00a0\u03bcm or less strongly overdamp the system and produce a velocity output which is proportional to the force produced by the piezoelectric disc and therefore to the voltage signal applied (Fig.\u00a05C). The model therefore predicts the transition in fluid jet characteristics from a displacement stimulus in the situation of a large tip diameter (small tip resistance) to a velocity stimulus due to tip restriction. Figure\u00a08 compares these model results in more detail for increasing tip resistance, Rp. At a relative low pipette resistance (Rp\u2009=\u20090.02\u00a0Ns\/m), a constant displacement amplitude of the jet is produced at frequencies below about 80\u00a0Hz showing resonance around 150\u00a0Hz. Increasing the resistance to Rp\u2009=\u20090.07\u00a0Ns\/m, critically damps the system making it a useful displacement stimulus device up to about 100\u00a0Hz. Gradually increasing the resistance Rp (2 to 20\u00a0Ns\/m) changes the fluid jet apparatus into a velocity-producing device in the full frequency range considered (1\u20131,000\u00a0Hz) at the expense of the jet\u2019s flow amplitude. This velocity output corresponds with a slope of \u221220\u00a0dB\/dec in the displacement amplitude response.\nFIG.\u00a08.The effect of increasing pipette resistance. Series of modelled frequency responses showing fluid displacement output as a function of the frequency of the voltage signal put across the piezoelectric disc. Parameter values: L\n1\u2009=\u20097\u00a0mm; L\n2\u2009=\u200950\u00a0mm; D\n1\u2009=\u200913.4\u00a0mm; D\n2\u2009= \u20091.17\u00a0mm; \u03c1 \u2009=\u20091,000\u00a0kg\/m3; S\npiezo\u2009=\u20092.0\u2009\u00d7\u2009106\u00a0N\/m, R\nc\u2009=\u20097\u00a0Ns\/m, S\np\u2009=\u200955\u00a0N\/m, values of pipette resistance (R\np in Ns\/m) are given next to each curve.\nAbsolute fluid jet velocity\nThe calibration method described was focused on the relative frequency selectivity of the fluid jet emerging from the tip of the glass pipette. The absolute fluid velocity that can be obtained not only depends on the tip diameter, but it also varies strongly with the measurement position with respect to the pipette\u2019s tip. This means that it is not useful to afterwards calibrate the absolute fluid velocity on a sense probe as done for determining the relative frequency sensitivity. Nevertheless, an estimate of the order of magnitude follows directly from curves like Figure\u00a05C or alternatively by visualizing the trajectories of small (\u00d8\u2009=\u20091.0\u00a0\u03bcm) latex microbeads in the flow field (e.g., Sauders and Szymko 1989). Our observations, for instance, show that a sensitivity of about 1\u00a0mm\/s per volt applied to the piezoelectric disc is obtained at a distance of about 30\u00a0\u03bcm in front of the pipette tip (\u00d8\u2009=\u200915\u00a0\u03bcm). From such calibrations, we estimate that the fluid flow velocity, required to produce an open probability of the transducer channels of 0.5, is about 2\u00a0mm\/s. Linearly extrapolating this to the maximum voltages that may be applied to the piezo-electric element (100\u00a0V) without damaging it, leads to a maximal velocity in the order of 200\u00a0mm\/s.\nApplications\nAs shown in Figure\u00a07, a fluid jet produced by the apparatus using a restricted tip diameter (<15\u00a0\u03bcm) can be adequately used to effectively evoke robust transducer currents in individual sensory hair cells. It is known from previous studies (van Netten et al. 2003; Ricci et al. 2005) that the speed of the transducer apparatus in mammalian outer hair cells is well exceeding the range of frequencies (1 to 1,000\u00a0Hz) considered here. This implies that the transducer apparatus is capable of following signals up into the kiloHertz range without delay. The observed delays in transducer currents (Fig.\u00a07D) can therefore be attributed to the frequency selectivity of the fluid jet. Figure\u00a05C illustrates that the difference in phase delay at 1,000\u00a0Hz (\u2212130\u00b0) compared to that at 100\u00a0Hz (\u221280\u00b0) amounts to about 50\u00b0, when using restricted pipettes. The observed time delays of the hair cell responses (Fig.\u00a07D) in stimulus periods (0.0194 at 100\u00a0Hz and 0.171 at 1,000\u00a0Hz) or in terms of phase delays (7\u00b0 at 100\u00a0Hz and 62\u00b0 at 1,000\u00a0Hz, a difference of 55\u00b0) are therefore in line with the measured phase difference of the fluid jet at these frequencies. As is also apparent from Figure\u00a05C, the amplitude response of the fluid jet is quite comparable in terms of velocity of the jet at 100 and 1,000\u00a0Hz, in line with the equal magnitude of the measured transducer currents at these frequencies. These results support our notion that a proper calibration of the fluid jet characteristics is necessary for a correct interpretation of the (phase) measurements.\nA relevant issue related to the application to hair cell stimulation is the proper mechanical stimulus to excite a sensory hair bundle. Based on the micromechanics and hydrodynamics of an individual hair bundle, it has theoretically been argued that a hair bundle\u2019s displacement response is flat as a function of the frequency of a fluid velocity stimulus up to a cut-off frequency determined by its stiffness and its size (G\u00e9l\u00e9oc et al. 1997; van Netten 1997). The pipettes used to displace individual bundles have opening diameters that are matched to the dimensions of the bundle resulting in diameters of about 15\u00a0\u03bcm or below. When applying a (filtered) step voltage, the accompanying high resistance of the pipette tip results in a jet velocity proportional to the step and therefore produces an adequate stimulus to statically displace individual hair cell bundles inducing step displacements of the bundle. If unfiltered, the velocity output may, however, be contaminated with a ringing (5.65\u00a0kHz) caused by resonance of the fluid in the Perspex fluid container (Fig.\u00a07E). Adequate low-pass filtering of the applied voltage signal can prevent these oscillations, but will inevitably put an upper limit to the displacement rise time of a hair bundle that can be achieved (see also next section).\nA second consequence of tip restriction is the decreased output response. Much larger voltages have to be applied to the piezoelectric disc to produce similar fluid jet displacements. This consequence of tip restriction was previously recognized by Saunders and Szymko (1989) who showed a square relation between fluid displacement amplitude and tip diameter. Remarkably, they did not report differences in the frequency response of their fluid jet due to tip diameter. Their calibrating method did not give precise information about the phase of the response and lacks relevant information on tip size. Their reported displacement frequency responses have a slope of \u221210\u00a0dB\/dec, which makes their apparatus neither a true displacement nor a true velocity-producing device.\nWhen a preparation is only stimulated at a single frequency, the detailed frequency response of the fluid jet is less relevant as long as the desired displacement and related phase of the object can be established. Harmonic distortion of the produced fluid jet might, however, cause a problem in the investigation of a system\u2019s nonlinearities. Although the harmonics produced by the described device are relatively small (e.g., Fig.\u00a03; THD\u2009<\u2009\u221230\u00a0dB) and comparable to the fluid jet distortion reported by Saunders and Szymko (1989), they may nevertheless preclude the direct measurement of transducer-related hair bundle nonlinearities from harmonic analyses as predicted for specific types of hair cells (e.g. van Netten and Kros, 2000).\nBandwidth limitations of the fluid jet-producing device\nThe calibration method described here is restricted to about 1,000\u00a0Hz because of the frequency range of the stimulus sphere used to calibrate the sense probes, which resonates at about 900\u00a0Hz and produces only a limited fluid displacement beyond resonance (Fig.\u00a04B2). However, it may be possible to design a method to mechanically calibrate the sense probes into the supra-kHz range so that the jet\u2019s output can also be calibrated in an extended frequency bandwidth.\nIn the velocity-producing mode, resonance of the fluid mass, Mc, in the Perspex container may limit the use of the device beyond the high resonance frequency (5.65\u00a0kHz), where the fluid jet velocity output declines with \u221220\u00a0dB\/ dec. It may seem therefore that a fundamental restriction of the application of the fluid jet produced by the apparatus described lies in the restricted bandwidth imposed by the piezoelectric disc used. The piezoelectric disc\u2019s intrinsic resonance behavior under dry unloaded conditions, i.e., while being clamped at its rim similarly as in the operational fluid jet device but without the fluid load imposed, is about 8.3\u00a0kHz. However, using the model, it can be shown that this is not a fundamental limit for its dynamic use, as the high-frequency resonance is also determined by the stiffness Sp, which contributes via up-transforming via the lever with a factor L2 to the stiffness of the moving fluid in the Perspex container determining the high-resonance frequency (Eq.\u00a03).\nEnhanced (high) frequency characteristics, i.e., a higher resonance or cut-off frequency (fh) in the velocity mode using small pipette tip diameters, may thus be expected from designs with a smaller fluid mass (Mc) in the Perspex container, improved lever action (higher L), and overall stiffer elastic elements (Spiezo, Sp), although the latter will decrease the overall output of such devices. In practice, this means that at the expense of a smaller output faster fluid jet performance may be obtained by using smaller but stiffer (ultrasonic) piezo elements.\nConclusions\nThis work emphasizes the importance of the calibration of a jet-producing stimulus device and demonstrates the importance of the outflow restriction for the jet response characteristics. Not only the jet output is decreased with a higher tip resistance, but more importantly, its output characteristic changes from a displacement to a velocity fluid stimulus in relation to the input voltage of the device. A simple mechanical model that adequately describes the fluid jet\u2019s characteristics can help understanding the physical parameters underlying these characteristics and can thus facilitate optimizing the design of this type of hydrodynamic stimulus device for particular applications.","keyphrases":["hair cell","lateral line","micromechanics","micro fluid jet","hydrodynamic excitation","calibrated fluid sensing"],"prmu":["P","P","P","M","R","R"]}
{"id":"Diabetologia-4-1-2235909","title":"A randomised, 52-week, treat-to-target trial comparing insulin detemir with insulin glargine when administered as add-on to glucose-lowering drugs in insulin-naive people with type 2 diabetes\n","text":"Aims\/hypothesis This 52-week multinational, randomised, open-label, parallel-group, non-inferiority trial compared clinical outcomes following supplementation of oral glucose-lowering drugs with basal insulin analogues detemir and glargine in type 2 diabetic patients.\nIntroduction\nPeople with type 2 diabetes inadequately controlled by oral glucose-lowering drugs can achieve clinically relevant improvements in HbA1c with the addition of insulin therapy [1\u20133]. However, patients and healthcare providers are often reluctant to initiate insulin due to concerns over injections, fear of hypoglycaemia and additional weight gain, and also because insulin treatment is perceived as complex and an added burden to diabetes management [4, 5]. Moreover, once insulin is initiated, recommended targets for glycaemic control (HbA1c\u2009<\u20096.5\u20137.0%) are often not met [5\u20137].\nIn recent years, the basal insulin analogues glargine and detemir have been introduced. These were developed to improve upon the limitations of NPH insulin (NPH) and other conventional basal insulins, which have an inadequate duration of action, a marked peak glucose-lowering effect and variability in response from one injection to another [8]. These analogues might help to overcome some of the barriers to insulin initiation and optimisation, including concerns over hypoglycaemia and weight gain.\nSeveral recent studies have assessed basal insulin as an add-on therapy to oral glucose-lowering drugs, comparing either insulin glargine or insulin detemir with NPH, and using titration algorithms based on glucose monitoring [1, 9\u201313]. These studies have demonstrated that simple regimens involving a once or twice daily injection of a basal insulin analogue can achieve clinically important improvements in glycaemic control similar to those achievable with NPH, but with less risk of hypoglycaemia. Insulin detemir has consistently shown less body weight gain than NPH when used in this way, as well as when used in basal plus mealtime insulin therapy [14, 15], whereas a weight advantage has been reported in only a few of the comparative trials of glargine vs NPH, as for example in the recent LANMET study [11].\nInsulin glargine is licensed only for once daily use as a basal insulin for people with diabetes. Insulin detemir, in contrast, is available for once or twice daily use. Glucose clamp comparisons between these insulins have given contradictory information on whether their duration of effect is comparable [16, 17]. The only direct comparison in patients with type 2 diabetes suggests very similar pharmacodynamic profiles at clinically relevant doses [16], but methodological issues remain controversial. The objective of the current study was to compare treatment with insulin detemir and insulin glargine in line with their licensed indications as add-on therapy to oral glucose-lowering agents in insulin-naive patients with type 2 diabetes.\nMethods\nStudy protocol This 52-week, parallel-group trial was conducted in 2003 and 2004 at 80 sites in Europe and the USA and included 582 insulin-naive people with type 2 diabetes, who were randomised (1:1) and treated with insulin detemir (Levemir; Novo Nordisk, Bagsv\u00e6rd, Denmark) or insulin glargine (Lantus; sanofi-aventis, Paris, France) as add-on therapy to oral glucose-lowering drugs. The trial was conducted in accordance with the Declaration of Helsinki and principles for Good Clinical Practice and was approved by ethics committees\/review boards in all countries. All participants gave written informed consent.Concealed randomisation was carried out by an automatic telephone response system and was stratified according to oral glucose-lowering drug mono- or combination-therapy at entry. An open-label design was required to allow twice daily administration of insulin detemir if needed, according to the dosing algorithm targets for pre-dinner plasma glucose (PG) concentrations (Table\u00a01). Glargine was only administered once daily at bedtime as per study protocol and in accordance with its licence [18]. To reduce potential bias, HbA1c results were only disclosed to investigators at randomisation and at trial end.\nTable\u00a01Algorithm used for insulin dose titrationAlgorithmAdjustment of insulin dose (U)If positive response to previous dose adjustmentIf no response to previous dose adjustmentaEvening insulin dose adjustment\u00a0Average pre-breakfast self-monitored PG\u00a0\u00a0>10.0\u00a0mmol\/l+12+12\u00a0\u00a09.1\u201310.0\u00a0mmol\/l+8+10\u00a0\u00a08.1\u20139.0\u00a0mmol\/l+6+8\u00a0\u00a07.1\u20138.0\u00a0mmol\/l+4+6\u00a0\u00a06.1\u20137.0\u00a0mmol\/l+2+2\u00a0If one self-monitored PG measurement\u00a0\u00a03.1\u20134.0\u00a0mmol\/l\u22122\u20132\u00a0\u00a0<3.1\u00a0mmol\/l\u22124\u20134Morning insulin dose adjustmentb\u00a0Average pre-dinner self-monitored PG\u00a0\u00a0>10.0\u00a0mmol\/l+8+8\u00a0\u00a09.1\u201310.0\u00a0mmol\/l+6+8\u00a0\u00a08.1\u20139.0\u00a0mmol\/l+4+6\u00a0\u00a07.1\u20138.0\u00a0mmol\/l+2+4\u00a0\u00a06.1\u20137.0\u00a0mmol\/l+2+2If one self-monitored PG measurement\u00a0\u00a03.1\u20134.0\u00a0mmol\/l\u20132\u20132\u00a0\u00a0<3.1\u00a0mmol\/l\u20134\u20134a Non-responses: the average self-monitored PG level is increased and\/or within the same range as at the last contactbSome insulin detemir-treated participants only+, insulin dose titrated up; \u2013, insulin dose titrated down\nParticipants Insulin-naive men and women with type 2 diabetes and the following characteristics were recruited: \u226518\u00a0years old, \u226512\u00a0months disease duration, BMI\u2009\u2264\u200940.0\u00a0kg\/m2 and HbA1c 7.5\u201310.0%. For inclusion, they had to be receiving one or two oral agents (metformin, insulin secretagogues, \u03b1-glucosidase inhibitors) \u22654\u00a0months on at least one-half the maximum recommended dose, according to local guidelines. Exclusion criteria included treatment with thiazolidinediones (due to labelling restrictions in Europe), use of more than two oral agents within 6\u00a0months, hypoglycaemic unawareness or other medical conditions likely to interfere with trial conduct. Withdrawal criteria included pregnancy, HbA1c\u2009>\u200911.0% after the first 12\u00a0weeks of treatment and initiation of medication interfering with glucose metabolism.\nStudy medications Oral glucose-lowering therapy, diet and physical activity were recommended to remain stable during the study; no meal-time insulin was allowed. Basal insulin was initiated once daily in the evening at a dose of 12\u00a0U and titrated according to a structured treatment algorithm. In line with its licence, people allocated to insulin detemir were allowed to receive an additional morning insulin dose if pre-dinner PG was >7.0\u00a0mmol\/l, but only if pre-breakfast PG was <7.0\u00a0mmol\/l or nocturnal hypoglycaemia (major episode or PG\u2009\u2264\u20094.0\u00a0mmol\/l) precluded achievement of the fasting plasma glucose (FPG) target. Insulin detemir was administered with a pen-injector (FlexPen, Novo Nordisk) 1\u00a0h before to 1\u00a0h after dinner, and if needed within 30\u00a0min of breakfast. Glargine was given once daily at bedtime, via a pen-injector in the EU (OptiPen Pro 1, sanofi-aventis) and with syringes in the USA. The evening dose of insulin detemir was administered somewhat earlier than that of insulin glargine, to allow for the possibility that recipients might be switched to a twice daily schedule, in which case a more even distribution of the dose across 24\u00a0h would be achieved.\nTitration of basal insulin Participants attended 16 scheduled visits and nine telephone contacts over 1\u00a0year. Evening insulin doses were titrated throughout the trial to a FPG target \u22646.0\u00a0mmol\/l in the absence of hypoglycaemia. An identical pre-dinner glucose target was applied for people administering a morning dose of insulin detemir (Table\u00a01). Participants measured capillary PG using glucose meters (Medisense Xtra; Abbott, Wiesbaden, Germany). Dose adjustments were to be based on the average of three self-measurements before breakfast (and before dinner if on twice daily insulin detemir). During the first 12\u00a0weeks, participants had weekly investigator contact. A titration committee monitored the algorithm for insulin dose optimisation and reviewed prescribed insulin doses periodically. This committee was not informed of the treatment insulin being used, but this would have been evident for participants on twice daily insulin detemir.\nOutcome measures The primary endpoint was baseline-adjusted HbA1c at end of treatment. Secondary variables included clinic FPG, within-participant variation in PG, ten-point self-measured PG profiles, proportion of participants achieving HbA1c\u2009\u2264\u20097.0% with and without hypoglycaemia, change in body weight, incidence of hypoglycaemia, adverse events and standard safety parameters.\nAnalyses and assessments HbA1c was analysed by HPLC (Bio Rad, Munich, Germany [EU analyses] and Bio Rad, Hercules, CA, USA [US analyses], DCCT-harmonised), with a reference range 4.3\u20136.1%. Clinic FPG was measured centrally by a hexokinase method (Gluco-quant; Roche, Mannheim, Germany). Body weight was measured using calibrated scales. Participants recorded ten-point PG profiles during the last week of treatment. Hypoglycaemia was classified as \u2018major\u2019 if assistance from another person was required, \u2018minor\u2019 if confirmed by PG\u2009<\u20093.1\u00a0mmol\/l or \u2018symptoms only\u2019 if PG\u2009\u2265\u20093.1\u00a0mmol\/l or no measurement was made.\nStatistical analyses The sample size was based on non-inferiority of insulin detemir relative to insulin glargine for HbA1c after 52\u00a0weeks. To achieve a power of 95% with an expected SD for change in HbA1c of 1.1% and allowing a dropout rate of 15%, it was originally planned to randomise 466 patients. However, this was extended to 566 patients to allow for potential regional differences between the EU and US. The possibility of a treatment \u00d7 region interaction was tested, but eliminated from the model as no significant interaction was found. Non-inferiority was accepted if the upper limit of the two-sided 95% CI for the difference in HbA1c (detemir\u2013glargine) was less than 0.4%-units, a value decided in discussion with the Food and Drug Administration (FDA) for the entire insulin detemir phase III study programme.Unless otherwise specified, all analyses were based on the intention-to-treat (ITT) population, this being all randomised patients exposed to insulin detemir or insulin glargine. A significance level of 5% was used for all analyses. Participants were regarded as being on once- or twice-daily insulin detemir according to the regimen used at trial completion. Statistical analyses were not made for these subsets of patients, as this was non-randomised and protocol-determined; results are given for observational comparison only.Analyses of HbA1c, clinic FPG and change in weight after 52\u00a0weeks were by ANOVA with treatment, region and oral glucose-lowering therapy as fixed effects and baseline (randomisation) value as covariate. The last observation carried forward (LOCF) principle was specified for HbA1c and clinic FPG in non-completers treated \u226512\u00a0weeks (time of the first on-treatment measurements). Change in weight was estimated based on all participants completing the trial. Additional weight analyses were prepared for the ITT population, applying the same LOCF principle or using imputed data, by treatment-specific linear regression, from week\u00a012 onwards to account for continuing weight change and limit the influence of differences in withdrawal rates and times between treatments. Fisher\u2019s exact test was used to compare the numbers of patients on insulin detemir and insulin glargine with an HbA1c\u2009\u2264\u20097.0% without symptomatic hypoglycaemia confirmed by PG <4.0\u00a0mmol\/l or any single value <3.1\u00a0mmol\/l during the last 3\u00a0months of treatment.The ten-point self-monitored PG profiles were analysed for parallelism using repeated measures ANOVA depending on treatment, region, oral glucose-lowering therapy, time and treatment \u00d7 time interaction as fixed effects. Within-participant variation in self-measured fasting and pre-dinner PG was determined from four measurements for each during the last week of treatment. Hypoglycaemic episodes during the 52\u00a0week treatment period were analysed as recurrent events in a Cox regression analysis using a gamma frailty model (Splus 2000; Insightful, Seattle, WA, USA). Nocturnal episodes (2300 to 0600\u00a0hours) were analysed separately. Adverse events were summarised using descriptive statistics. All other analyses were performed using SAS version 8.2 (SAS Institute, Cary, NC, USA).\nResults\nParticipants Of 892 patients screened, 582 were randomised, while 289 did not fulfil the selection criteria and 21 withdrew consent. In each treatment arm, 291 patients were randomised and treated, of whom 231 (79%) and 252 (87%) completed the trial on insulin detemir and insulin glargine, respectively (Fig.\u00a01). The higher withdrawal rate observed with insulin detemir was partly due to adverse events, ten of which were considered possibly or probably related to trial drug (Electronic supplementary material [ESM] Table 1). These included two cases of possible cutaneous allergy, five of injection site reactions and one each of injection site inflammation, hypoglycaemia and weight increase. Adverse event withdrawals with possible or probable relation to glargine included one case each of pruritus, myalgia, hyperglycaemia and hypoglycaemia. Details of all adverse events leading to withdrawal are provided in ESM Table 2. Other diabetes-related reasons for withdrawal with insulin detemir included HbA1c 12.0% (n\u2009=\u20091) and multiple hypoglycaemic episodes (n\u2009=\u20091). Other reasons for withdrawal included the closure of a trial site and various circumstantial events unrelated to the trial medications.\nFig.\u00a01Patient disposition during the trial and consequent analysis setsBaseline characteristics (Table\u00a02) were comparable between the two treatment groups. Participants completing the trial on once or twice daily insulin detemir had a baseline HbA1c of 8.60 and 8.66%, respectively.\nTable\u00a02Clinical characteristics of type 2 diabetic participantsCharacteristicsDetemirGlargineRandomised\/exposed\/ITT, n (%)291 (100)291 (100)Men\/women, n166\/125171\/120Ethnicity (n)Black2212White250263Asian-Pacific islander77Other129Oral glucose-lowering drugs, n (%)Monotherapy73 (25)70 (24)\u00a0Metformin32 (11)33 (11)\u00a0Insulin secretagogues41 (14)37 (13)Combination therapy218 (75)221 (76)\u00a0Metformin + secretagogue212215\u00a0Metformin + alpha glucosidase inhibitor31\u00a0Secretagogue + alpha glucosidase inhibitor34\u00a0Secretagogue + secretagogue (SU + glinide)\u20131Age (years)58.4 (10.2)59.4 (9.6)Weight (kg)a87.4 (16.6)87.4 (17.4)BMI (kg\/m2)30.6 (4.8)30.5 (4.6)Duration of diabetes (years)9.1 (6.1)9.1 (6.4)HbA1c (%)a8.64 (0.78)8.62 (0.77)C-peptide (nmol\/l)0.87 (0.56)0.85 (0.55)Values are n (%) or mean (SD)aBefore randomisationSU, sulfonylurea\nInsulin dosing Of participants treated with insulin detemir, 104 (45%) completed the trial on one daily injection, while 129 (55%) administered an additional morning dose. The majority of patients administering insulin detemir twice daily (n\u2009=\u2009103) were transferred to this regimen within 12\u00a0weeks of treatment. At the time of transfer, about two-thirds of the patients adding a morning injection fulfilled the formal dose transfer guidelines, the other one-third were changed through investigator discretion. Participants using insulin detemir twice daily administered about 40% of their daily insulin dose before breakfast. After 52\u00a0weeks the mean daily dose of insulin detemir (n\u2009=\u2009227) was 0.78\u00a0U\/kg (0.52\u00a0U\/kg on once daily [n\u2009=\u2009102] and 1.00\u00a0U\/kg on twice daily [n\u2009=\u2009125]) and the mean daily dose of glargine (n\u2009=\u2009248) was 0.44\u00a0IU\/kg.\nGlycaemic control HbA1c decreased by 1.5% with both insulins and was comparable after 52\u00a0weeks at 7.2% (n\u2009=\u2009268) and 7.1% (n\u2009=\u2009275) for detemir and glargine respectively (Table\u00a03), with an estimated difference of 0.05 (\u22120.11, 0.21)%, thereby meeting the criteria for non-inferiority for insulin detemir vs glargine. HbA1c was similar in participants completing the study on once or twice daily insulin detemir (7.1 and 7.1%). Clinic FPG was 10.8\u00a0mmol\/l at baseline in both arms and was comparable at end of treatment with insulin detemir and insulin glargine (7.1 and 7.0\u00a0mmol\/l) (Table\u00a03). The profile of change in HbA1c and FPG was very similar (Fig.\u00a02a). Mean self-monitored FPG was comparable between treatments, being 6.1\u00a0mmol\/l with insulin detemir and 6.0\u00a0mmol\/l with insulin glargine; self-monitored PG pre-dinner (6.8 and 7.0\u00a0mmol\/l) was also similar.\nFig.\u00a02Change in HbA1c with time (a). Black circles, insulin detemir; white circles, insulin glargine. b Mean ten-point self-monitored PG profiles during the last week of treatment. Triangles, insulin detemir (once daily); squares, insulin detemir (twice daily); black circles, insulin detemir (all patients); white circles, insulin glargineTable\u00a03Glycaemic control in type 2 diabetic patients treated with insulin detemir or insulin glargine\u00a0DetemirGlargineDifference (95% CI) (detemir\/glargine)p valueCompleters on once dailyCompleters on twice dailyAllAllParametersnMeannMeannMeannMeanHbA1c % (SE)a1047.12 (0.11)1277.06 (0.10)2687.16 (0.08)2757.12 (0.08)0.05 (\u20130.11, 0.21)\u2013Clinic FPG (mmol\/l)a1047.27 (0.31)1276.73 (0.25)2687.14 (0.21)2726.98 (0.21)0.16 (\u20130.26, 0.58)\u2013HbA1c \u22647.0%, n (%)10352 (51)12769 (54)248129 (52)259135 (52)\u20131.00HbA1c \u22647.0% without hypoglycaemia, n (%)10331 (30)12748 (38)24882 (33)25990 (35)\u20130.71Within-participant variation (mmol\/l)Pre-breakfast (mmol\/l)b1030.93 (15.0)1251.15 (19.6)2381.06 (17.5)2571.03 (17.3)\u20130.45\u2003Pre-dinner (mmol\/l)b1031.27 (19.8)1251.84 (26.4)2381.60 (23.6)2581.55 (22.0)\u20130.41aMean (SE); n=number of patients for whom data are availablebWithin-participant SD (CV%)Within-participant variation in self-monitored PG pre-breakfast and pre-dinner did not differ significantly between insulin detemir and insulin glargine (Table\u00a03). The overall shape of the ten-point self-monitored PG profiles during the last week of treatment was parallel for insulin detemir (n\u2009=\u2009218) and insulin glargine (n\u2009=\u2009246) (NS) and appeared identical, regardless of treatment regimen (Fig.\u00a02b).Of participants in both arms, 52% achieved HbA1c\u2009\u2264\u20097.0%, while 33% treated with insulin detemir and 35% treated with insulin glargine did so in the absence of hypoglycaemia. Fasting and pre-dinner PG targets of \u22646.0\u00a0mmol\/l were achieved by 25 and 20% of participants treated with insulin detemir and insulin glargine (NS), respectively; the pre-breakfast target alone was achieved by 46 and 58% (p\u2009<\u20090.01) and the pre-dinner target by 38 and 30%, respectively (p\u2009<\u20090.05).\nHypoglycaemia The risk of hypoglycaemia of any type was comparable between treatments (Table\u00a04). The overall rate of hypoglycaemia was low at 5.8 vs 6.2 episodes per patient-year with insulin detemir versus insulin glargine (relative risk: 0.94 [96% CI 0.71, 1.25]), while the rate of nocturnal hypoglycaemia was only 1.3 episodes per patient-year with both insulins. Adjustment for HbA1c did not affect the outcome of the analyses. Major hypoglycaemic episodes were rare with both insulins, especially at night, and could not be statistically analysed.\nTable\u00a04Hypoglycaemic episodes in type 2 diabetic patients treated with insulin detemir or insulin glargineEvents \u00a0Insulin detemir (n\u2009=\u2009291)Insulin glargine (n\u2009=\u2009291)Relative risk (detemir\/glargine) (95% CI)Participants, n (%)Episodes (n)Rate (per patient-year)Participants, n (%)Episodes (n)Rate (per patient-year)All182 (63)15215.8191 (66)16706.20.94 (0.71\u20131.25)\u00a0Nocturnal95 (33)3521.393 (32)3501.31.05 (0.69\u20131.58)Major5 (2)90.08 (3)80.0\u2013\u00a0Nocturnal3 (1)50.04 (1)40.0\u2013Minor135 (46)7372.9151 (52)7862.91.05 (0.75\u20131.46)\u00a0Nocturnal73 (25)2120.871 (24)1920.71.17 (0.75\u20131.83)Symptoms only137 (47)7603.0133 (46)8663.20.88 (0.61\u20131.25)\u00a0Nocturnal48 (17)1280.549 (17)1510.60.88 (0.50\u20131.54)No statistical analyses were performed on the small numbers of major events.\nBody weight Weight gain in participants completing 52\u00a0weeks of treatment was lower with insulin detemir (n\u2009=\u2009230) than with insulin glargine (n\u2009=\u2009252) (3.0 [SE\u2009=\u20090.4] vs 3.9 [SE\u2009=\u20090.4] kg, p\u2009=\u20090.01). Change in weight analysed by LOCF (detemir n\u2009=\u2009268, glargine n\u2009=\u2009275) confirmed this lower weight gain (2.7 vs 3.5\u00a0kg, p\u2009=\u20090.03), as did analysis with imputation for non-completers (2.8 vs 3.5\u00a0kg, p\u2009=\u20090.04). Patients completing the study on once daily insulin detemir (n\u2009=\u2009104) had a weight gain of 2.3 (SE\u2009=\u20090.5) kg, whereas those treated twice daily (n\u2009=\u2009126) gained 3.7 (SE\u2009=\u20090.4) kg, similar to glargine (Fig.\u00a03).\nFig.\u00a03Mean weight change from baseline at week 52 in patients completing treatment on insulin detemir once or twice daily (and overall) and insulin glargine. ***p\u2009<\u20090.001; \u2020p\u2009<\u20090.012\nAdverse events and other safety measures Serious adverse events were less frequent with insulin detemir (42 patients with 47 events) than with glargine (53 patients with 73 events). However, only five serious events with insulin detemir (two hypoglycaemia, one each of hypothyroidism, injection-site reaction and a motor vehicle accident) and four with glargine (three hypoglycaemia, one hyperglycaemia) were considered by local investigators to be probably or possibly related to trial products. A participant treated with insulin detemir was found dead in bed (no autopsy report available), but had a history of myocardial infarction. A patient using glargine was hospitalised after the study for pulmonary fibrosis and died of cryptogenic fibrosing alveolitis. Adverse events recorded as serious tended to be of a wide-ranging disparate nature, with no clear pattern of between-treatment differences. Full details of all serious adverse events and events recorded as possibly or probably related to the study insulins are given as in the ESM Tables\u00a01, 2, 3, and 4).The only differences in adverse events, judged as possibly or probably related to trial drugs, between treatments were: injection-site disorders (13 patients [4.5%] on insulin detemir compared with four [1.4%] on glargine), allergic reactions (three patients on detemir vs one on glargine) and skin disorders including pruritus and rash (six patients on insulin detemir vs one on glargine). There were no differences in standard safety parameters between treatments.\nDiscussion\nThe results of this first head-to-head comparison of insulin detemir and insulin glargine as add-on for treatment with oral glucose-lowering agents in people with type 2 diabetes using forced insulin titration suggest that clinically significant and similar improvements in glycaemic control can be achieved with both analogues, together with a similarly low risk of hypoglycaemia. Insulin doses were higher with insulin detemir overall, partly as a result of the doses used by those taking it twice daily. The withdrawal rate appeared to be higher with insulin detemir (21 vs 13%), which was partly accounted for by adverse events (8 vs 4%), this excess being primarily due to injection site reactions. Insulin detemir was associated with a modest relative reduction in weight gain, consistent with observations in previous comparisons of this analogue with NPH in type 2 diabetes [12, 13, 19, 20]. In the present study, the between-treatment difference in weight was primarily accounted for by those patients completing the study on once-daily insulin detemir. It is, however, possible that differences in eating pattern were contributory factors in both the insulin detemir dosing schedule and the different levels of weight gain observed between the two insulin detemir dosing schedules. The mechanism(s) responsible for relatively lower weight gain observed here and previously with insulin detemir remain the object of speculation [14, 15, 21, 22].\nThe present study was designed to compare the two basal insulin analogues as they had been used in previous studies and according to label. Thus, insulin glargine was given once daily at bedtime regardless of glycaemic profile, while for insulin detemir an option was provided for adding a second dose, primarily based on pre-dinner blood glucose level following a structured insulin titration protocol. The decision to use twice-daily insulin detemir could not be attributed to hypoglycaemia, as patients who were switched to a twice-daily regimen had on average 3.7 hypoglycaemic episodes per patient-year prior to transfer, compared with 4.7 episodes per patient-year in patients completing treatment on once-daily insulin detemir. The frequency of nocturnal hypoglycaemia was also lower in relevant patients before transfer (0.7 vs 0.9 episodes per patient-year).\nInterestingly, the initiation of insulin with once-daily insulin detemir has recently been tested by Philis-Tsimikas et al. in type 2 diabetes patients on oral glucose-lowering agents, resulting in HbA1c reductions of 1.5% (morning injection) and 1.6% (evening injection) from baseline values of 9.1 and 8.9%, respectively [13]. These improvements in glycaemic control approach the magnitude of HbA1c reduction shown in the current study and in previous studies of once-daily insulin glargine [9\u201311], although the insulin detemir doses were higher at trial-end and the absolute HbA1c values achieved at trial-end were relatively high.\nThe design of the current study does not allow definitive conclusions for comparison of once and twice daily dosing with insulin detemir, although these post hoc observations do suggest that once daily administration can be an appropriate starting regimen for people using insulin detemir as add-on to oral glucose-lowering drug therapy. However, with dose optimisation, a significant proportion of patients may eventually need a twice-daily regimen, guided by structured glucose monitoring. Further studies are required to better define the differences between these two basal insulin analogues on the basis of a similar insulin administration regimen, once daily only, for both insulins. Alternatively, if the option of adding a second insulin dose is available for both insulins, then a properly designed study protocol should allow addition of this second insulin injection only when a normal or much lower FPG target is achieved. Indeed, the use of lower FPG targets (e.g. \u22645.5\u00a0mmol\/l, as has been used previously) should also enable a higher proportion of patients to reach guideline HbA1c targets.\nIn conclusion, the use of insulin detemir or insulin glargine as add-on to oral glucose-lowering therapy resulted in comparable HbA1c improvements and a similarly low risk of hypoglycaemia. Further comparisons of detemir and glargine are required to fully understand how each may relatively benefit defined groups of patients starting on insulin.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nESM 1\n(PDF 12.4\u00a0kb)\nESM Table 1 \nTreatment emergent adverse events probably or possibly related to trial product by system organ class, ITT cohort (PDF 20.2\u00a0kb)\nESM Table 2\nAdverse events leading to withdrawal (PDF 17.2\u00a0kb)\nESM Table 3\nTreatment emergent serious adverse events by system organ class, ITT cohort (PDF 23.1\u00a0kb)\nESM Table 4\nTreatment emergent serious adverse events preferred term, occurring in more than one subject, ITT (PDF 14.6\u00a0)","keyphrases":["insulin detemir","insulin glargine","type 2 diabetes","hypoglycaemia","body weight","oral glucose-lowering agents","fasting plasma glucose","glucose variability","glucose control","insulin supplementation"],"prmu":["P","P","P","P","P","P","P","R","R","R"]}
{"id":"Matern_Child_Health_J-2-2-1592143","title":"Social Marketing: Planning Before Conceiving Preconception Care\n","text":"Social marketing approaches can help to shape the formation of and to create demand for preconception care services. This article describes four components of social marketing, often referred to as the 4 P\u2019s, that should be carefully researched and set in place before a national effort to launch and sustain preconception care services is pursued. First, the product or package of services must be defined and adapted using the latest in scientific and health care standards and must be based on consumer needs and desires. Second, the pricing of the services in financial or opportunity costs must be acceptable to the consumer, insurers, and health care service providers. Third, the promotion of benefits must be carefully crafted to reach and appeal to both consumers and providers. Fourth, the placement and availability of services in the marketplace must be researched and planned. With the application of market research practices that incorporate health behavior theories in their exploration of each component, consumer demand for preconception care can be generated, and providers can take preconception care to the market with confidence.\nIntroduction\nPreconception care (PCC) is a critical component of maternal and child health that does not currently have a strong foothold in our health care delivery system [1\u20133]. A more detailed discussion of how preconception care supports preconception health (PCH) and ultimately healthy pregnancies and babies is found in the introduction of this volume. In order to market a health product or service, it is important to understand what people want or need before it is developed, and then to provide them with the opportunity to meet those needs in a way that is compelling to them and also improves a mutually beneficial outcome for patients and their providers: a healthy baby. Of course, unlike commercial products, the outcome of preconception care services (a healthy pregnancy and a healthy baby), cannot be guaranteed; there are many factors that influence pregnancy and birth outcomes that are outside a mother or healthcare provider\u2019s realm of control. Despite the risk of adverse outcomes, there is much a woman, her partner, and her health care provider can do together preconceptionally to optimize pregnancy and birth outcomes. Social marketing is defined as \u201cthe application of commercial marketing technologies to the analysis, planning, execution, and evaluation of programs designed to influence voluntary behavior of target audiences in order to improve their personal welfare and that of society\u201d (4). The social marketing process involves identifying an effective \u201cmarketing mix\u201d of product, price, promotion, and place with the goal of offering an exchange that has clear and compelling benefits, minimal barriers, and an advantage over the competition. In plain language, that means setting up a situation so that future parents, health care professionals, and health insurers can all benefit and support a common goal of healthy women, healthy pregnancies, and healthy babies. To accomplish this, resources first need to be dedicated to conducting audience and market research using a systematic approach at all levels. Often, health behavior theories can be used to shape the development of new products or services as well as to evaluate their effectiveness in achieving intended outcomes. Table 1 lists some of the theories and frameworks that can be applied to addressing the 4 P\u2019s of preconception care services (5).\nTable 1Theories and frameworks applicable to guiding research on the 4 P\u2019s of social marketing preconception care servicesMarketing componentTheory or frameworkFocus of the theory or frameworkRelevant constructs or conceptsProductSocial marketingThe application of marketing techniques to the design, implementation, and evaluation of programs designed to facilitate voluntary exchanges of mutual benefit and improved personal well-being and societal welfareFormative research; Market segmentation; Competitive analysisDiffusion of InnovationThe spread of new ideas, products, or services within a society or from one society to another; focus on innovationRelative advantage; Compatibility; Complexity; Trialability; ObservabilityPriceSocial cognitive theoryThe interaction between personal factors, environmental factors, and human behaviorReciprocal determinism; Behavioral capability; Expectations; Self-efficacy; Observational learning; ReinforcementsHealth belief modelThe perceptions of threat posed by a health problem, the benefits of avoiding the threat, and factors influencing the decision to actPerceived benefits; Perceived barriers; Cues to action; Self-efficacyPromotionDiffusion of innovationThe spread of new ideas, products, or services within a society or from one society to another; focus on diffusionCommunication channels; Social systems\/networksPrecaution adoption process modelThe journey from lack of awareness to action and maintenance in dealing with hazards; acknowledge inherent riskUnawareness; Unengaged; Decision-making; ActingPlaceCommunication theoryThe different types of communication that affect health behaviorMedia Exposure; Media effects; Agenda setting; FramingNote. For easy-to-understand information about the theories, frameworks, constructs, and concepts listed in this table, go to http:\/\/www.nci.nih.gov\/theory\/pdf for a downloadable copy of NCI\u2019s \u201cTheory at a Glance\u2014A guide for health promotion practice\u201d\nViewing health care services as a business, and using language such as \u201cmarketing\u201d and \u201csales,\u201d may be uncomfortable to some who prefer to view the process of having a baby as a natural, emotional and deeply personal part of the human experience. However, the notion of providing preconception care as a service could very well benefit from a well-conceived and constructed social marketing research plan and execution that acknowledges and embraces the emotions of the patient and her partner. Thus, thinking about developing a social marketing plan to support preconceptional health care could help to ensure that babies are born with the best start in life.\nOne example of how the business market has taken advantage of this type of opportunity is the development of a bank that preserves umbilical cord blood (UCB). The original idea in 1988 was a not-for-profit resource bank of donated cord blood for those who might need it for transplantation purposes (6). However, since that time, enterprising companies have created a market and generated the \u201cneed\u201d for cord blood storage for a couple\u2019s own child\u2014even though the chances they will personally need it are roughly 1 in 10,000. Companies who forecast a profit in this service use direct marketing strategies to expectant couples through diaper companies and magazine subscriptions. Attempts to patent the cryopreservation processes and limit competition have raised legal, ethical and clinical questions\u2014but the market to sell the idea of cord blood collection and preservation is lucrative and the demand is increasing (7). Advocates of preconception care can learn important lessons from the UBC preservation business marketing executives who market, sell and provide services to a similar audience.\nProduct\nPreconception care (PCC) is the product or concept we are trying to \u201csell\u201d as a precursor for assuring PCH: The need for PCC is well-supported by research in the health field (1, 8, 9). The product has been described in a number of publications as including a \u201cbundle\u201d of services most often including screening of the following elements: reproductive history; medications and potential teratogens; environmental exposures; age, family medical and genetic history; infections and immunizations; social habits and risk behaviors; diet and exercise; and any chronic medical conditions (10, 11). Preconception care is supported by a number of practitioner specialties (1, 12, 13) and was related to a Healthy People 2000 goal (14). Whereas some aspects of PCC have more evidence linking them to improved outcomes (e.g., folic acid for NTDs and glycemic control for diabetes) and others have less evidence (e.g., genetic counseling and smoking cessation), the need for a more integrated approach to health prior to conception enjoys a broad range of support (15\u201318). Nevertheless, there has been little support of PCC in a systematic manner in the US or other countries as a standard of care (2, 19\u201321). A social marketing approach can provide an important support for that uptake by beginning with a critical first step\u2013defining the attributes of the \u201cproduct\u201d we are selling from a potential consumer\u2019s point of view\u2014which involves formative research with consumers. Key questions for the research may include: what do women and their partners want, when are PCC services optimally delivered, and what needs would have to be met in order for women or couples to value PCC services? This approach dictates that it is important to understand the perspective of the patient (and her partner) as more than just as consumers of health care services, but also by understanding their other perceived needs and desires in order to provide a satisfactory exchange. Couples may not view the decision to start a family as a medical decision, so this may be one reason the health care delivery system is more likely to be engaged some time after conception. In fact, couples may assess their financial readiness to start a family before their physical, emotional, or relationship readiness for this life-altering decision. The research that is an integral part of the social marketing process can help us to understand the decision-making dynamic (or lack thereof) that couples may engage in prior to conception. While only 50% of pregnancies are planned in the U.S., understanding how couples think about pregnancy may be a good place to start. Frishman suggests other \u201cwindows of opportunity\u201d that could be explored among women including when women inquire about birth control, obtain negative pregnancy tests, or have recently delivered a baby (22). Wallerstedt et\u00a0al. suggest that preconceptional health promotion and interconceptional counseling may be even more beneficial for parents who have had previous perinatal or infant loss (23). Social marketing research can help provide an understanding of the motivations and barriers for and against preconception care. A social ecological perspective (24) encourages program planners to look at multiple levels of influence: 1) intrapersonal or individual level; 2) interpersonal or social level; 2) institutional or organizational level; 4) community level; 5) and public policy or societal level. A major corporation preparing to launch a new product for women who might become pregnant (e.g., a new home pregnancy test) would never begin without spending substantial time and resources on understanding the target market and identifying the product attributes that will appeal to that market, while also knowing what the competitive environment is before they make investments in product development that might not have a profitable return. Product attributes can have a major impact on both the speed and extent of a product\u2019s uptake by a target population. Rogers\u2019 diffusion of innovation theory (25) identified a number of product attributes including relative advantage (is the product better than what it will replace?), compatibility (does the product fit with the intended audience?), complexity (is the product easy to use?), trialability (can the product be \u201ctried out\u201d before making a decision to purchase it?), and observability (are the results of the product visible and measurable?). Program planners can position their products effectively in the marketplace if they maximize their product\u2019s appeal by addressing these important product attributes.\nPrice\nThe next aspect of social marketing research to increase the supply of and demand for preconception care is price\u2014defining the costs to patients, providers and insurers, measured in time and\/or money, in order to have quality and timely preconception care. Once the product attributes have been defined and developed to address PCC services in a mutually satisfactory way for women, health care professionals and insurers, then appropriate pricing is vital to allow for uptake. Lowering the intangible (non-financial) costs to patients can be facilitated by using some common standards of social marketing: the most effective health messages often incorporate emotional connections and values to make behavior change more appealing. Also, the best social marketing approaches make it easy for people to make changes that the service providers want them to\u2014in this case in order to be healthy, and have a healthy pregnancy and baby. In addition to appeal and ease, some marketers would say that a new product would have to have relative advantage over products currently in the marketplace. \u201cNew and improved\u201d labeling does matter when a product is introduced. A product\u2019s relative advantage can often be framed as more appealing and\/or easier, but at its core it must meet a customer need or desire. Appealing and easy actions to improve health are perceived as less costly by the patient or customer. Social cognitive theory (26) which addresses the interaction between personal, environmental, and behavioral factors is commonly used to understand many facets of a person\u2019s expectations of outcomes from performing a behavior, environmental contexts (helps and hindrances) related to the behavior, experiences in performing the behavior (positive or negative), and efficacy (ability\/confidence) in performing the behavior. These are just a few of the ways in which the pricing can build on the positive product attributes developed in the first step. It also helps that the outcome\u2014a healthy pregnancy and baby\u2014is a highly valued and very tangible benefit for those who want it.\nThe pricing for a health care organization is more complicated. Cost-effectiveness studies and business case studies need to document how the organization\u2019s PCC service costs compare with treatment of preventable health conditions. Balancing financial overhead and time pressures in these times of managed care may be a real challenge\u2014and particularly so for reaching women who may have unplanned pregnancies. The greatest challenge may be weighing the intangible costs and benefits against the financial costs: How much preconceptional folic acid counseling is worth a child being born without a lifelong disability like spina bifida? How many smoking cessation sessions balance out the costs of a premature\/low birth weight baby who might spend his first months of life in the NICU? How many fewer patients might a health care professional have time to see and bill for when pregnancy counseling they do for people with genetic conditions takes up more time? And what additional patient education messages, addressing chronic or acute health issues, are not being delivered during a regular well visit because the health care professional\u2019s time is being used discussing preconception health issues? The health care provider medical office may well not be the best and\/or most efficient way to market preconception care services to consumers in many cases. The practicing physician may also not be the first or best person to deliver the preconception care message. The goal of a good pricing strategy is to find an effective and innovative way to reach the target market while reducing the costs to both the health care delivery system and the consumer.\nPromotion\nAfter the product has been defined as a generally acceptable package of PCC services, and the price to individual and organization is determined to be mutually beneficial, how can a social marketing approach diffuse it into common practice and social norms? This is where the promotion comes into play among two key audiences: 1) people who would like to be providers (or sellers) of the product and 2) people who would be consumers (or buyers) of the product. Often, promotion is thought to focus solely on creating demand among consumers for a particular product, and the promotion process is what most people typically think of as marketing or advertising. However, whereas most businesses prefer to have products \u201con the shelf\u201d to meet the needs of consumers proactively, consumers are more influential than ever before in demanding that a product appears on their vendor\u2019s shelves. PCC could very well become a product that consumers themselves create demand for\u2014especially since the health care industry already sees the value of preconception care services but has not built a product offering that delivers what they believe to be important. Once the product and pricing attributes are developed, then promoting the product among potential buyers and sellers could be done using a number of communication theories that provide guidance on what types of communication messages and media vehicles (e.g. communication channels) can achieve in terms of exposure and effects. In fact, trade shows and product expositions are a major venue for new products to be introduced to businesses who sell products. Health care provider\/insurer conferences, continuing medical education efforts, and medical school training could be venues for promoting PCC services among potential providers (27). Because the product attributes have already been defined as a benefit, and pricing has been addressed in the previous step, promotion should be a matter of convincing both buyers and sellers that they are acting in their own best interests. After promotion has helped to generate consumer demand for the product, PCC is an opportunity for current health care professionals, insurers and those in the business of health to expand their services\u2014as in a classic new product offering. An important way for organizations to justify offering these services is to demonstrate an immediate benefit (perhaps in sales of other products and services) in addition to longer term benefits (perhaps fewer complicated pregnancies, lower malpractice premiums, fewer patients who need life-long care). And of course, the actual benefits would be determined through a process of market research with stakeholders\u2014such as patients, health professionals and insurers (Table 2). Addressing current barriers and how to overcome them is a major contribution of the promotion activities. Using innovative ways to promote preconception care services not only can be modified from general market promotions but also by taking advantage of existing opportunities to deliver tailored \u201cgateways\/pathways\u201d to PCC services. These could vary from fertility treatment centers to other family planning arenas. Whether a woman is seeking to become pregnant or trying to avoid becoming pregnant (but preserving her options for the future), she is a consumer who may be fertile ground, so to speak, for preconception care services. A challenging part of promoting PCC services will be figuring out how to move the thinking of a \u201cnow-oriented\u201d consumer towards more \u201cfuture-oriented\u201d events; especially because future outcomes could happen earlier than planned\/anticipated. This promotion could take advantage of additional opportunities such as involving male partners and other influential people in women\u2019s lives\u2014and also taking a lifelong approach to health (13, 28). Another challenge to promoting PCC services will be addressing the ethical aspects of promoting a product that cannot guarantee outcomes: Not every woman who chooses and uses preconception care services will be able to have a healthy pregnancy or a healthy baby. In fact, it might even be the case that unhealthy women (e.g. those with chronic diseases or other medical conditions) are the first to want and use preconception care services because they know that their poor health status makes pregnancy a hazardous situation for themselves and their baby. The importance of recognizing hazards and taking precautionary actions to protect health, which are addressed in the precaution adoption process model (29), may be relevant in promoting PCC services in a manner that acknowledges pregnancy as having inherent risk for both the mother and the unborn baby.\nTable 2Sample social marketing questions for selected stakeholders of preconceptional care servicesConsumersHealth care providersInsurersProduct \u2013 What action must be taken by each audience (e.g., product, behavior, or policy)What does PCC services need to look and feel like for consumers to want\/use\/demand them?What does PCC services need to look and feel like for busy health care providers to want to offer them?What does PCC services need to look and feel like for health insurers to want to cover this service?Pricing \u2013Why would they want to do it?What is the value of PCC services and what would they be willing to pay for them?What is the value for offering PCC services to patients and what would it cost to offer them?What is the return on investment for including PCC services as a covered service?Promotion \u2013How will each audience be told about the what, why, where, and how?How will PCC services be publicized and made available to consumers in a way that they are attractive, affordable, and accessible?How will PCC services be presented to health care providers so that they are willing and able to offer them?How will PCC services be presented to insurers so that they are willing and able to reimburse for them?Place \u2013Where can they do the action needed?What settings\/locations are most acceptable, appropriate, and accessible for providing PCC services?What settings\/locations are most acceptable, appropriate, and accessible for offering PCC services?What forums or conferences can instigate productive discussions about including PCC services as a covered expense?\nPlace\nIt is useful to ask how PCC services could be placed, or distributed, to women across their reproductive lifespan in a manner that addresses social marketing\u2019s call toward mutually satisfactory exchanges, and how the placement of PCC services could affect their delivery. What components of PCC might change across the life span (e.g. early health promotion\/contraception\/STD infection\/vaccinations\/others)? The high level of unintended pregnancies in the US\u201440\u201360% for adults, 95% for teens (30)\u2014is also a concern, because any approach that requires a self-selection into the audience will miss about half of the pregnancies. Some authorities have suggested a lifelong approach to preconceptional health\u2014throughout the lifespan of a women\u2019s potential fertility (say, 10\u201350 years of age) and an approach that may move women from one set of services and questions into another (2, 31). Social marketing can help by determining in the early phases what women and health care professionals want, and thus ensuring that supply can be ready when the demand is articulated. Health care professionals need to be involved in the design of those PCC services that they would deliver, and must do so in the context of what they would want to deliver\u2014and those they would benefit from. Women report that one of their most influential sources of health information is their own health care professional, who knows them, their medical history and their concerns (32). Health care professionals and their medical staff may be the front line \u201csales people\u201d for preconceptional care; if doctors never offer their patients the opportunity to \u201cbuy\u201d this product, it will go nowhere. Medical professionals who are trained on the benefits of the product (e.g., by inclusion in medical school training and on medical board exams) and rewarded for using it (by reducing malpractice premiums for example) will find it more acceptable and worthwhile to integrate the practice into the time pressures of the busy medical office. Once a physician selection is made, patients visit for an acute problem, management of a chronic condition, and for routine checkups. Placement market research will help to define how PCC services may fit into each of these areas. This integrated vision of PCC could become part of the core training curricula for various medical professionals\u2014more than an option during continuing education. This strategic preparation for placement also ensures that when the increased demand from the consumer begins to be evident at the health care provider office, the medical professionals in that office will know how to respond to that demand, and have the knowledge and systems in place to do so. Existing \u201cbundles\u201d of services that can be prepackaged to respond to this demand have in some cases been developed (33, 34). If the research in the first phase shows that the busy medical office is not the best location for providing PCC services, then alternate placement sites must be identified.\nConclusion\nThe best marketing and sales happen when the product has the qualities that a consumer wants and that a seller wants to provide. PCC must meet the needs and desires of patients, health professionals and insurers in order to be successfully adopted in the United States. For women and their families, that may mean believing that a healthy, wanted baby is in their future. For doctors, that may mean finding a way to give healthy women the preventive services they want and need while still dealing with the urgent care of their sick patients. For insurers it may mean a balance in service provision to meet both short and long term financial goals. But market research is needed to actually define exactly what the benefits are to each of those groups. And it is likely to be that the satisfactory exchange is more than just a purely medical application of clinical services. Consumers across the US are willing to spend $3.00 to drink what is essentially a 50 cent cup of coffee, but they are experiencing something more than the product\u2019s primary attributes (e.g., flavor, caffeine) but also the positive associations with the product (e.g., funky, cool). In the same way, consumers can tell us how they would like to experience preconception health care services (35). All of us need to understand that if we market and sell it as the best experience possible, PCC could contribute to healthier babies, fewer medical treatment costs, and a healthy next generation that thinks about pregnancy in a whole different way.","keyphrases":["social marketing","preconception care","behavior theories","preconception health","health communication"],"prmu":["P","P","P","P","R"]}
{"id":"Clin_Oral_Investig-3-1-1797073","title":"Deposition of fluoride on enamel surfaces released from varnishes is limited to vicinity of fluoridation site\n","text":"The aim of the in-situ study was to determine fluoride uptake in non-fluoridated, demineralized enamel after application of fluoride varnishes on enamel samples located at various distances from the non-fluoridated samples. All enamel samples used were demineralized with acidic hydroxyethylcellulose before the experiment. Intra-oral appliances were worn by ten volunteers in three series: (1, Mirafluorid, 0.15% F; 2, Duraphat, 2.3% F and 3, unfluoridated controls) of 6 days each. Each two enamel samples were prepared from 30 bovine incisors. One sample was used for the determination of baseline fluoride content (BFC); the other was treated according to the respective series and fixed in the intra-oral appliance for 6 days. Additionally, from 120 incisors, each four enamel samples were prepared (one for BFC). Three samples (a\u2013c) were placed into each appliance at different sites: (a) directly neighboured to the fluoridated specimen (=next), (b) at 1-cm distance (=1 cm) and (c) in the opposite buccal aspect of the appliance (=opposite). At these sites, new unfluoridated samples were placed at days 1, 3 and 5, which were left in place for 1 day. The volunteers brushed their teeth and the samples with fluoridated toothpaste twice per day. Both the KOH-soluble and structurally bound fluoride were determined in all samples to determine fluoride uptake and were statistically analyzed. One day, after fluoridation with Duraphat, KOH-soluble fluoride uptake in specimen a (=next) was significantly higher compared to the corresponding samples of both the control and Mirafluorid series, which in turn were not significantly different from each other. At all other sites and time points, fluoride uptake in the enamel samples were not different from controls for both fluoride varnishes. Within the first day after application, intra-oral-fluoride release from the tested fluoride varnish Duraphat leads to KOH-soluble fluoride uptake only in enamel samples located in close vicinity to the fluoridation site.\nIntroduction\nIt is well established that fluoride products play an important role in the prevention and remineralization of carious lesions. Topical fluoride applications in the form of varnishes and varnishes may lead to appreciable acquisition of fluoride on and in both enamel and dentin samples, which were directly treated with the agent [4, 6, 14, 21, 25]. Especially, the KOH-soluble fluoride deposit on the surfaces of dental hard tissue might be elevated depending on the fluoride concentration and composition of the fluoride varnish applied. However, the KOH-soluble fluoride deposit is continuously dissolved in the oral cavity due to the influence of saliva. This leads to a drastic reduction of the deposit within a period of about 1\u00a0week after application of the fluoride agent [2, 5, 14].\nAfter the use of fluoride-containing products, also, salivary fluoride levels are increased for a certain period of time depending on the concentration and application form used. This effect is important because fluoride levels above 0.04\u00a0ppm in the surrounding solution of the dental hard tissue have been shown to be related to lower risk of caries progression in clinical studies [16, 17]. The fluoride distribution in the oral cavity showed site-specific variations, which was exemplarily demonstrated by Weatherell at al. [24] after dissolving a fluoride tablet in the oral vestibulum. With oral fluoride-containing mouthrinses, salivary fluoride levels remain elevated for up to 12\u00a0h after application [8, 13, 23, 26]. The use of fluoride mouthwashes twice daily leads to a sustained elevated fluoride level even between the daily applications [12]. Fluoride dentifrices can cause increase in salivary fluoride content for a period of about 60\u201390\u00a0min [3, 11, 26]. Also, after application of fluoride varnishes onto tooth surfaces, salivary fluoride levels remain increased for up to 32\u00a0h [13, 18, 22].\nThe above-mentioned studies had made clear that fluoride-treated tooth surfaces and salivary fluoride levels are increased after administering local fluoride regimes. Besides these effects, it may be speculated that fluoride applications also lead to fluoride uptake at tooth surfaces that were not directly treated or covered with fluoridation agents such as varnishes. This fact could especially be important for demineralized surfaces, which were inaccessible to direct fluoride application such as inter-proximal areas. Due to toxicological and practical reasons, application of fluoride varnishes is mostly limited to some sites of the dentition. Therefore, the aim of the present study was to determine fluoride transfer from fluoride-varnished enamel surfaces to demineralized enamel surfaces located at various distances from the fluoridation site.\nMaterials and methods\nEnamel samples\nEnamel cylinders (4\u00a0mm in diameter) were prepared from the buccal surface of bovine incisors. The cylinders were ground flat and polished, thereby removing about 200-\u03bcm depth of the enamel as controlled with a micrometer. Afterwards, all specimens were sterilized with gamma irradiation (25\u00a0kGy). For rehydration, the sterilized samples were stored in synthetic saliva [1] for a minimum of 2\u00a0weeks. Then, all samples were demineralized with acidic hydroxyethylcellulose (pH\u00a04.8, 3\u00a0days).\nAccording to this procedure, each two enamel samples were prepared from 30 bovine incisors and each four samples were gained from additional 120 incisors. One sample from each tooth was used for the determination of baseline fluoride content of the respective tooth. The remaining samples of the 30 bovine incisors were later treated according to series 1\u20133. The remaining samples of the 120 bovine incisors were used for the determination of intra-oral fluoride transfer and acquisition.\nSet-up of the study\nThe study was designed and conducted according to the guidelines of Good Clinical Practice. Ethical approval of the study was granted by the Ethics Committee of the University of G\u00f6ttingen (2\/9\/01). Ten panellists (aged 21 to 29\u00a0years) participated in the study. They were all residents of G\u00f6ttingen (fluoride content in domestic water: <0.2\u00a0ppm F). The subjects had been instructed to avoid fluoride uptake. The participants had a stimulated salivary fluoride rate within the normal range of 1.0\u20135.0\u00a0ml\/min. Further inclusion criteria were that they had at least 20 teeth, no faulty dental restorations, no glass ionomer fillings and no current dental caries activity.\nIntra-oral, lower jaw appliances with two buccal aspects were worn by ten volunteers in three series of 6\u00a0days each. In each series, one enamel sample was placed for 6\u00a0days in one buccal aspect of the appliance after treatment according to the respective series (=central, fluoridated sample). With each five of the participants, the fluoridated samples were placed in the right buccal aspect; with the remaining subjects, the fluoridated samples were fixed in the left aspect of the appliance. A washout phase of 1\u00a0week elapsed between the three series.\nIn series 1 and 2, 0.1\u00a0g of the fluoride varnish Mirafluorid\u00ae (1) or Duraphat\u00ae (2) was either applied on the central enamel sample (Table\u00a01). Care was taken to avoid contamination of the remaining enamel samples of the intra-oral appliances with the varnish applied. After application of the varnish, the intra-oral appliances were immediately inserted into the oral cavities. In series 3, the respective samples were not fluoridated with varnish and served as controls. \nTable\u00a01Ingredients and composition of the fluoride varnishes Mirafluorid\u00ae and Duraphat\u00ae according to manufacturers\u00a0Mirafluorid\u00aeDuraphat\u00aeFluoride content0.12%2.26%Fluoride formSodium fluorideSodium fluorideCetylaminehydrofluorideBis(hydroxyethyl)-aminpropyl-N-hydroxyethyl-octadecyl-amindihydrofluoridepH4.6NeutralSolventSolvent-freealcohol (33.14%)BaseWater-soluble polymerNatural resins (colophonium, mastix, shellac)Other ingredients\u2013Wax, saccharine, flavourManufacturerHager & Werken, Duisburg, Germany Colgate-Palmolive, Hamburg, Germany\nEach three unfluoridated samples were placed into each appliance at different sites (a\u2013c): (a) directly neighboured to the central, fluoridated specimen (=next), (b) at 1-cm distance (=1\u00a0cm) and (c) in the opposite buccal aspect of the appliance (=opposite). These three samples were replaced after 1, 3 and 5\u00a0days by new unfluoridated samples, which were removed after 1\u00a0day intra-oral exposition. Thus, each one sample was worn at the respective site for the following periods: days 0\u20131, days 1\u20132, days 3\u20134 and days 5\u20136. The central samples, which were fluoridated on day 0, were not removed from the appliances before completion of the experiment.\nThe volunteers brushed their teeth and the samples with a pea-size amount of fluoridated toothpaste (0.125% fluoride as amine fluoride) twice per day (elmex\u00ae, GABA, L\u00f6rrach, Germany) during the experiment to simulate the common practice of using a fluoridated dentifrice and to follow the recommendations of the Ethical Committee, which did not allow detaining a fluoride dentifrice to the participants. They began to use the dentifrice 3\u00a0days before the start of the trial. After toothbrushing, the enamel samples in the appliances were carefully cleaned with ten brushing strokes using the toothbrush (impregnated with the remaining dentifrice slurry) that had been used for brushing of the teeth before. A minimum of 8\u00a0h elapsed between application of the respective varnish onto the central specimen and first brushing of the samples. During the experiment, the appliances were worn night and day except during meals, during which, they were stored in 0.9% saline.\nFluoride analysis\nImmediately after removal from the intra-oral appliances, the enamel samples were cleaned with a soft toothbrush under running tap water and analyzed for KOH-soluble and structurally bound fluoride. The fluoride was assayed by a specific fluoride electrode (Orion Research, Cambridge, USA). The structurally bound fluoride was determined in two successive layers of 30\u00a0\u03bcm each. The layers were removed by means of a special grinding technique described in detail previously [15]. The amount of KOH-soluble fluoride was determined by the method of Caslavska et al. [9]. The assays were prepared as described in those studies, resulting in a 0.9-ml sample solution for KOH-soluble fluoride and 3-ml solution for structurally bound fluoride determination, respectively. Also, fluoride content of the fluoridated samples was assessed after the experimental period.\nStatistics\nAccording to the manufacturer, sensitivity of the fluoride electrode is given as 1\u00a0\u03bcmol\/l in a solution. Bioanalytical guidelines recommend disregarding the measurements that are close to the sensitivity of an assessment method [20]. An international cooperation study [7] determining the lowest level of sensitivity for measurements with a fluoride electrode showed that measurements below 4\u00a0\u03bcmol\/l (equivalent to 0.544\u00a0\u03bcg\/cm2 KOH-soluble fluoride and 604.8\u00a0\u03bcg\/cm3 structurally bound fluoride) were unstable, not reproducible and showed high standard deviations and coefficients of variation (>20%). Therefore, these readings were regarded as to be \u201cbelow detection limit\u201d. Thus, fluoride uptake was not calculated by subtracting the baseline fluoride content of the respective tooth, which was below detection limit, from the measured value recorded for the specimen.\nAll groups were compared regarding their number of readings above detection limit using chi-square tests. For those groups, in which more than 50% of the readings were above the detection limit, discriminations were conducted using the rank-sum test according to Wilcoxon and Mann\u2013Whitney. The level of significance was set at P\u2009<\u20090.05.\nResults\nIn Table\u00a02, the percentages of fluoride measurements in the respective groups, which were above the detection limits, are given. It is obvious that most of the groups had more than 50% of their readings below the detection limit. Chi-square tests proved that the sample groups with more than 50% readings above detection limit were statistically significantly different from the remaining groups, which in turn did not differ significantly from each other. \nTable\u00a02Percentages of measurements above detection limitsa for KOH-soluble and structurally bound fluoride in two successive layers in enamel specimens worn in intra-oral appliances at different sites and for different periods (days 0\u20136, days 0\u20131, days 1\u20132, days 3\u20134 or days 5\u20136) after the application of either Duraphat, Mirafluorid or no fluoride varnish (control) on the central specimen on day 0\u00a0\u00a0KOH-soluble F\u22121st Layer (0\u201330\u00a0\u03bcm)2nd Layer (31\u201360\u00a0\u03bcm)PeriodSiteMirafluorid (%)Duraphat (%)Control (%)Mirafluorid (%)Duraphat (%)Control (%)Mirafluorid (%)Duraphat (%)Control (%)0\u20136Central90100010100101010000\u20131Next10600030003000\u201311\u00a0cm10300050001000\u20131Opposite00001000001\u20132Next20100101000001\u201321\u00a0cm20000000001\u20132Opposite10000000003\u20134Next100001000003\u201341\u00a0cm10000000003\u20134Opposite10000000005\u20136Next0000000005\u201361\u00a0cm10000000005\u20136Opposite000000000aDetection limits (KOH-soluble F\u2009=\u20090.544\u00a0\u03bcg\/cm2, structurally bound fluoride\u2009=\u2009604.8\u00a0\u03bcg\/cm3)\nIn Table\u00a03, KOH-soluble fluoride and structurally bound fluoride data of the experimental groups are given, for which more than 50% of the readings were above detection limit. As mentioned above, chi-square tests had proven that these values were statistically significantly different from baseline content and from the remaining groups. For the remaining groups, fluoride contents are not given because statistical data such as mean value or median are not reasonable to calculate for values, which are not reliably measurable by definition. Acquisition of KOH-soluble fluoride was detectable in the central, fluoridated samples of both groups, Mirafluorid and Duraphat, with the latter once accumulating statistically significantly more KOH-soluble fluoride (P\u2009=\u20090.00015). The central, fluoridated Duraphat samples also showed significant uptake of structurally bound fluoride in both enamel layers with significantly higher uptake in the first layer compared to the second one (P\u2009=\u20090.02836). One day after fluoridation, the samples located next to the Duraphat-varnished central specimens also accumulated measurable amounts of KOH-soluble fluoride. The KOH-soluble precipitate on these samples was statistically significantly less pronounced compared to the central specimens (P\u2009=\u20090.00015). The samples located next to the Duraphat-treated showed measurable KOH-soluble fluoride acquisition at days 0\u20131. They, therefore, differed significantly from both the control and Mirafluorid series, which did not show measurable fluoride acquisition at this time point and were, in turn, not significantly different from each other. At all other sites and time points, fluoride uptake in the enamel samples was not different from controls for both fluoride varnishes. \nTable\u00a03Mean \u00b1 standard deviation (median; minimum\u2013maximum) of KOH-soluble fluoride and structurally bound fluoride (in 1st and 2nd layer) given for the experimental groups with more than 50% of the respective measurements above detection limit\u00a0\u00a0KOH-soluble F\u2212 (\u03bcg\/cm2)1st Layer (\u03bcg\/cm3)2nd Layer (\u03bcg\/cm3)PeriodSiteMirafluoridDuraphatDuraphatDuraphat0\u20136Central0.67\u2009\u00b1\u20090.19a (0.62; 0.54\u20131.1)8.38\u2009\u00b1\u20093.18a,c (8.82; 3.65\u201314.5)2237\u2009\u00b1\u2009576b (2,282; 1,233\u20133,315)1636\u2009\u00b1\u2009487b (1,503; 1,110\u20132,646)0\u20131Next\u2013*0.83\u2009\u00b1\u20090.56c (0.76; 0.16\u20132.12)\u2013*\u2013**Less than 50% readings above detection limitSignificantly different values in the same line (aKOH-soluble F\u2212, bstructurally bound F\u2212) or ccolumn are marked.\nDiscussion\nAs stated in \u201cIntroduction\u201d, several investigations had demonstrated that intra-orally applied fluoride regimes may cause fluoride release into saliva. In a previous study, Dijkman et al. [10] had demonstrated that no measurable fluoride enrichment could be detected in untreated sound enamel controls located in a distance of 3\u00a0mm from fluoride varnish-treated samples after a period of 1\u00a0week. The present study made clear that within the first day after application the highly concentrated fluoride varnish Duraphat KOH-soluble fluoride deposition in close vicinity to the fluoridation occurs. However, in consistence with the investigation by Dijkman et al. [10], no measurable fluoride transfer could be revealed after the first day. Although, when interpreting the results of the present study, it should be noted that a more sensitive method for fluoride determination might have shown slightly different results.\nIn the present study, no fluoride acquisition could be detected in samples located at a distance of 1\u00a0cm from the fluoridated site or fixed in the opposite vestibulum of the participants. This means that fluoride transfer via saliva was strictly limited to the close neighbourhood of the fluoridation site. This finding corresponds to a previous study, which demonstrated that significant elevation of salivary fluoride concentrations is more or less restricted to the fluoridation site [24].\nWith exception for the samples directly treated with Duraphat, no measurable acquisition of structurally bound fluoride was observed in any other sample group. In a previous study, application of Mirafluorid did not also result in uptake of structurally bound fluoride [2]. However, also the samples which were located next to the specimens treated with Duraphat for 1\u00a0day and which showed a KOH-soluble fluoride deposit did not show increase of structurally bound fluoride. This finding could be explained by the fact that formation of structurally bound fluoride is a time-dependent process during which some of the KOH-soluble deposit is dissolved, allowing fluoride to diffuse into the underlying enamel and to be structurally incorporated into the enamel crystallites [19]. However, in these samples, structurally bound fluoride was assessed already 1\u00a0day after insertion into the appliance. It might be speculated that with longer exposition in the oral cavity, structurally bound fluoride might have been formed by dissolution from the KOH-soluble fluoride deposit.\nIn the present study, demineralized enamel specimens were used for assessing fluoride acquisition. Recent studies had proven that fluoridation measures lead to significantly higher uptake of fluoride in demineralized samples as compared to sound enamel [1]. It could, therefore, be assumed that fluoride acquisition of sound enamel surfaces due to fluoride transfer in the oral cavity might be actually more limited as compared to the present results.\nUnder clinical conditions, a varnish is often applied onto more sites, which might elevate salivary fluoride to a presumably higher extent as in the present study, where the varnishes were applied to a single site only.\nDue to the results of the present study, it could be concluded that within the first day after application, intra-oral fluoride release from the tested fluoride varnish Duraphat leads to KOH-soluble fluoride uptake only in demineralized enamel samples, which are located in close vicinity to the fluoridation site. Owing to the negligible transfer of fluoride from the varnish to more remote sites, this finding means, for the clinical situation, that all sites in the oral cavity requiring fluoridation have to be directly treated with the varnish.","keyphrases":["fluoride","enamel","koh-soluble","saliva","transfer"],"prmu":["P","P","P","P","P"]}
{"id":"Acta_Neuropathol_(Berl)-3-1-2039857","title":"MLC1 is associated with the Dystrophin-Glycoprotein Complex at astrocytic endfeet\n","text":"Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a progressive cerebral white matter disease with onset in childhood, caused by mutations in the MLC1 gene. MLC1 is a protein with unknown function that is mainly expressed in the brain in astrocytic endfeet at the blood\u2013brain and cerebrospinal fluid\u2013brain barriers. It shares its localization at astrocytic endfeet with the dystrophin-associated glycoprotein complex (DGC). The objective of the present study was to investigate the possible association of MLC1 with the DGC. To test this hypothesis, (co)-localization of DGC-proteins and MLC1 was analyzed by immunohistochemical stainings in gliotic brain tissue from a patient with multiple sclerosis, in glioblastoma tissue and in brain tissue from an MLC patient. In control tissue, a direct protein interaction was tested by immunoprecipitation. Results revealed that MLC1 is co-localized with DGC-proteins in gliotic brain tissue. We demonstrated that both MLC1 and aquaporin-4, a member of the DGC, were redistributed in glioblastoma cells. In MLC brain tissue, we showed absence of MLC1 and altered expression of several DGC-proteins. We demonstrated a direct protein interaction between MLC1 and Kir4.1. From these results we conclude that MLC1 is associated with the DGC at astrocytic endfeet.\nIntroduction\nMegalencephalic leukoencephalopathy with subcortical cysts (MLC, MIM 604004) is a childhood leukoencephalopathy with autosomal recessive inheritance [21]. Patients develop macrocephaly during the first year of life, followed by slow motor deterioration with cerebellar ataxia and spasticity [21]. Magnetic resonance imaging (MRI) shows diffusely abnormal, mildly swollen cerebral white matter and subcortical cysts in anterior temporal and frontoparietal areas [21]. Histopathologic and electron microscopic examination of brain tissue reveals countless intramyelinic vacuoles [22].\nFrom early on, the striking similarity in MRI features of MLC and congenital muscular dystrophy (CMD) with merosin deficiency (CMD type 1A, MDC1A) was noted [21]. Patients with MDC1A also have diffusely abnormal, mildly swollen cerebral white matter and sometimes anterior temporal cysts (Fig.\u00a01) [23]. Microscopic examination of the brain of a patient, who was later genetically confirmed to have MDC1A (B. Echenne, personal communication) also revealed myelin vacuolation [8]. MDC1A is caused by mutations in the LAMA2 gene, encoding the laminin-\u03b12 chain of merosin [9]. In contrast to MDC1A, MLC is not associated with muscle disease [21]. We hypothesized that MLC could be the \u201cbrain-only\u201d variant of MDC1A. We could, however, not confirm linkage of MLC to the LAMA2 locus and instead demonstrated that mutations in another gene, MLC1, cause MLC [10]. MLC1 encodes a plasma-membrane protein of unknown function. Recently, the MLC1-protein was found to be located in astrocytic endfeet at the blood\u2013brain and cerebrospinal fluid\u2013brain barriers [3, 19].\nFig.\u00a01MRI of the brain of a patient with MLC (a\u2013c) and MDC1A (d\u2013f). The axial T2-weighted images (a, b, d, e) reveal extensive signal abnormalities and mild swelling of the cerebral white matter in both MLC (a, b) and MDC1A (d, e). The corpus callosum and internal capsule are relatively preserved in both cases (a, d). The sagittal T1-weighted images reveal a large cyst in the anterior temporal white matter and smaller cysts in the frontal subcortical white matter in the MLC patient (c). There are small cysts in the same regions in the MDC1A patient (f). Both MLC and MDC1A patients had a genetically confirmed diagnosis\nOther proteins localized at astrocytic endfeet are those of a multi-subunit complex called dystrophin-associated glycoprotein complex (DGC) [12, 20]. The DGC is expressed in various tissues and has been characterized best in skeletal muscle where it connects the cytoskeleton of muscle fibers to the extracellular matrix [14]. Mutations in different DGC components lead to various muscular dystrophies. In a subset of muscular dystrophies, the congenital muscular dystrophies (CMDs), the muscular phenotype is often combined with brain involvement, including white matter abnormalities, most notably in MDC1A [23].\nThe brain DGC is involved in the adhesion of perivascular glia to the extracellular matrix [20]. At the astrocytic endfeet, the DGC consists of four main components [6, 16, 29]: (1) \u03b1- and \u03b2-dystroglycan, which form the backbone of the DGC by forming a transmembrane link between the actin cytoskeleton and the extracellular matrix. \u03b1-Dystroglycan is a heavily glycosylated peripheral membrane protein that binds to extracellular ligands such as merosin and agrin. \u03b2-Dystroglycan is a transmembrane protein that anchors the extracellular \u03b1-dystroglycan to the plasma membrane and interacts intracellularly with dystrophin (and\/or utrophin, which can replace dystrophin) and syntrophin. (2) Dystrophin and\/or utrophin, are linker molecules that can form a bridge between actin and \u03b2-dystroglycan. (3) \u03b1-Dystrobrevin, which is linked to dystrophin and\/or utrophin binds to syntrophin. (4) Syntrophin, which binds to the water channel aquaporin-4 and the potassium channel Kir4.1 is responsible for the polarized expression of these proteins in astrocytic endfeet.\nThe striking similarities in brain MRI features and pathology between MLC and MDC1A, together with the specific localization of both MLC1 and merosin at astrocytic endfeet, brought us to the hypothesis that there may be an association between MLC1 and DGC in the brain. In the present paper we report on our studies to test this hypothesis. We investigated the co-localization of MLC1 and DGC-proteins in gliotic white matter, because MLC1 staining is more intense in gliotic brain tissue than in normal tissue [3]. Previous studies in humans and mice have shown that if members of the DGC are mutated or missing, the complex may be disrupted, which affects the localization of other protein members [1, 13, 16, 27]. We therefore also tested our hypothesis by studying the localization of MLC1 and several DGC-proteins in glioblastoma and MLC brain tissue. Besides, we assessed a direct association between MLC1 and DGC-members by co-immunoprecipitation experiments.\nMaterials and methods\nImmunohistochemical staining of human brain tissue\nHuman control brain tissue specimens, both frozen and paraffin-embedded, consisting of neocortex and white matter, were obtained at autopsy from patients without neurological disease and neuropathologic abnormalities. Additionally, frozen brain tissue was obtained from a multiple sclerosis patient to study gliotic white matter in which MLC1 has a normal localization but MLC1 expression is increased [3]. During life, all patients or their next of kin had given consent for autopsy and the use of brain tissue for research purposes.\nHuman glioblastoma multiforme tissue (frozen) and brain biopsy tissue from MLC patients (both frozen and paraffin-embedded) were obtained for diagnostic purposes and used for research purposes with informed consent of patients and families. Light-microscopy of glioblastoma tissue was used to select areas that display characteristic glioblastoma features, including necrosis, giant cells and endothelial proliferation, for further studies. Brain biopsies were obtained in the following MLC patients: EL18 (decreased MLC1 expression [2], frozen tissue), EL649 (homozygous for the c.733G\u00a0>\u00a0C (p.Ala245Pro) mutation; paraffin-embedded tissue), and EL746 [compound-heterozygous for the c.268-1G\u00a0>\u00a0A and c.597\u00a0+\u00a01G\u00a0>\u00a0A mutations, leading to splice-defects and truncation of the protein (p.Cys90-Val107del and p.Val200-Ala238del); paraffin-embedded tissue].\nImmunohistochemistry was performed as described previously [7] with antibodies against the following proteins: MLC1 (polyclonal; 1:200 [3]); merosin (monoclonal; 1:500 [11]); aquaporin-4 (polyclonal; 1:200, Chemicon, Huissen, The Netherlands); Kir4.1 (polyclonal; 1:200, USBiological, Swampscott, Massachusetts), \u03b2-dystroglycan (monoclonal; 1:100, Santa Cruz, Heidelberg, Germany), \u03b1-dystroglycan (monoclonal; 1:100, UpstateCellSignaling solutions), agrin (monoclonal; 1:2500 [26]); glial fibrillary acidic protein (GFAP, polyclonal; 1:500, DAKO, Heverlee, Belgium) and NeuN (monoclonal; 1:1600, MAB377, Chemicon). In all slides, except the slides used for double stainings, the nuclei are counterstained with Haematoxylin. Double immunofluorescence staining was performed as described [3] with agrin, \u03b2-dystroglycan (monoclonal; 1:100) or syntrophin (monoclonal; 1:100, Affinity BioReagents, Raamdonksveer, The Netherlands) versus MLC1. In negative controls the primary antibodies were omitted. Double-stainings were performed as described previously [25]. In frozen tissue double-stainings were done with agrin (red) antibodies versus AB4 (blue, polyclonal; 1:200, Lab Vision\u2013NeoMarkers, Fremont, CA, USA) antibodies and in paraffin tissue with agrin or \u03b1-dystroglycan antibodies versus NeuN or GFAP. For the agrin (brown)\/NeuN (blue) double-stainings, agrin was visualized with biotinylated secondary antibodies (DAKO) and NeuN with poly-HRP (Immunologic, Duiven, The Netherlands). For the agrin (red)\/GFAP (blue) double-stainings, agrin was detected with poly-HRP (Immunologic) and GFAP with biotinylated secondary antibodies (DAKO). In negative controls the primary antibodies were omitted.\nImmuno-precipitation and western blotting\nPreparation of brain tissue and immunoprecipitation experiments were performed as previously described [5] without antibodies as a negative control, with 1\u201315\u00a0\u03bcg mMLC1-C (home made), with the nonspecific CHOP (Santa Cruz, Heidelberg, Germany) as an extra negative control, and with Kir4.1 (USBiological, Swampscott, Massachusetts) antibodies. Western blots were incubated with the Kir4.1 antibodies overnight at 4\u00b0C and complexes were visualized using ECL Western Blot detection reagents (Amersham, Buckinghamshire, United Kingdom).\nResults\nCo-localization of MLC1 and DGC-members\nTo test whether MLC1 co-localizes with DGC-proteins at astrocytic endfeet, we performed double-immunofluorescent staining experiments in gliotic brain tissue with the transmembrane protein MLC1 (Fig.\u00a02a, d, g; green) versus the extracellular matrix protein agrin (Fig.\u00a02b; red), the intracellular protein syntrophin (Fig.\u00a02e; red), and the transmembrane protein \u03b2-dystroglycan (Fig.\u00a02h; red). There was partial overlap (yellow) between all three DGC-proteins (red) and MLC1 (green). The co-localization (Fig.\u00a02i; yellow) of MLC1 (Fig.\u00a02g; green) and \u03b2-dystroglycan (Fig.\u00a02h; red) was most striking. If MLC1, agrin, \u03b2-dystroglycan or syntrophin was omitted, fluorescent labeling was only visible for the appropriate isotype, thereby excluding cross-reactivity (not shown). On omission of the primary antibody no staining was observed (not shown).\nFig.\u00a02Co-localization of MLC1 and DGC-proteins. a\u2013i Double immuno-fluorescence labelling of MLC1 (a, d, g, green), agrin (b, red), syntrophin (e, red), and \u03b2-dystroglycan (h, red) in gliotic human brain tissue. There is partial overlap (c, f, i, yellow) between MLC1 and these DGC-proteins. The overlap (i, yellow) between the transmembrane proteins MLC1 (green) and \u03b2-dystroglycan (red) is most striking. In all panels the ocular magnification and 10\u00a0\u03bcm scale bars are indicated in the lower right hand corner\nMLC1 and aquaporin-4 are redistributed in glioblastoma cells\nStaining normal human brain tissue with anti-MLC1 and anti-aquaporin-4 antibodies confirmed their specific expression around blood vessels, consistent with the previously described perivascular localization (Fig.\u00a03a, c) [3, 15]. In glioblastoma tissue, both MLC1 and aquaporin-4 had altered expression patterns. The proteins were no longer confined to astrocytic endfeet, but were redistributed over the entire glioblastoma cell (Fig.\u00a03b, d). Anti-agrin staining showed expression of the protein in the extracellular matrix around blood vessels of all sizes in normal brain tissue (Fig.\u00a03e), whereas it was exclusively expressed around larger blood vessels in glioblastoma tissue (Fig.\u00a03f). Double staining of agrin (red) with AB4 (blue), an endothelial marker, in glioblastoma tissue confirmed agrin and AB4 staining around larger blood vessels (Fig.\u00a03g) and absence of agrin around small vessels (Fig.\u00a03g, boxed area). This is in line with previously published results [27]. On omission of the primary antibody no staining was observed (not shown).\nFig.\u00a03Localization of MLC1 in glioblastoma tissue. Immunohistochemical staining of MLC1, aquaporin-4 (AQP4) and agrin in control brain (a, c, e) and tumor tissue (b, d, f, g). a MLC1 has a perivascular localization in normal tissue. b In glioblastoma tissue, the restriction of MLC1 to perivascular areas is lost and the protein has an intracellular location in glioblastoma cells. cAQP4 is expressed in perivascular astroglial endfeet in control brain. dAQP4 is localized in glioblastoma cells. e, f Agrin is expressed in the extracellular matrix around blood vessels of all sizes in normal tissue (e) and only around large vessels in glioblastoma tissue (f). g Double-staining of agrin (red)\/AB4 (blue) in glioblastoma tissue shows agrin and AB4 (endothelial marker) staining around larger blood vessels. The boxed area in panel g shows that agrin staining is absent around small vessels. In all panels the ocular magnification and 10\u00a0\u03bcm scale bars are indicated in the lower right corner\nAbsence of MLC1 and altered expression of DGC-proteins in MLC brain tissue\nAnti-MLC1 staining revealed perivascular expression of the protein in control brain tissue (Fig.\u00a04a) [3] and absence of staining in MLC tissue (Fig.\u00a04b). The distribution of merosin, \u03b2-dystroglycan and aquaporin-4 was similar in control (Fig.\u00a04c, e, g) and MLC tissue (Fig.\u00a04d, f, h). In control tissue, Kir4.1 showed a perivascular localization in configuration suggesting presence in distal atroglial processes (Fig.\u00a04i), while in MLC patient tissue Kir4.1 additionally showed a diffuse cytoplasmic staining of astrocytes (Fig.\u00a04j). In control tissue, agrin (Fig.\u00a04k) and \u03b1\u2013dystroglycan (Fig.\u00a04m) were seen in the perivascular extracellular matrix, whereas these proteins were observed both in the perivascular extracellular matrix and within cells in MLC tissue (Fig.\u00a04l, n).\nFig.\u00a04Localization of proteins associated with the DGC in MLC patients. In control tissue the perivascular staining of MLC1 is visible (a), but no staining is observed in MLC brain tissue (b). Normal perivascular basal lamina staining with merosin is seen in control (c) and MLC (d) brain tissue. \u03b2-dystroglycan has a normal perivascular distribution in both control (e) and MLC (f) brain tissue. Aquaporin-4 (AQP4) has a normal perivascular distribution in both control (g) and MLC (h) brain tissue. Kir4.1 is present in distal astrocytic processes around blood vessels in control tissue (i). In MLC tissue (j) Kir4.1 is expressed both in distal astrocytic processes around blood vessels and in cell bodies of astrocytes (boxed area). Agrin is expressed in the extracellular matrix around blood vessels in control tissue (k). In MLC tissue (l) agrin is expressed in both the extracellular matrix around blood vessels (boxed area) and in cells, as indicated by arrows. Immunostaining for \u03b1\u2013dystroglycan shows a normal perivascular distribution in control tissue (m) and expression of \u03b1\u2013dystroglycan around blood vessels (boxed area) and in cells in MLC tissue (arrows in n). o Double-staining of NeuN (blue)\/agrin (brown) in MLC tissue shows cytoplasmic agrin staining in NeuN positive cells (arrows). p Double-staining of Agrin (red)\/GFAP (blue) in MLC brain tissue. The picture on the left shows an astrocyte that is only GFAP positive (arrow), while the picture on the right shows an astrocyte that is both GFAP and agrin positive (arrow, brown staining in cell body). In all panels the ocular magnification, 10\u00a0\u03bcm scale bars and patient numbers are indicated in the lower right corner\nThe cells expressing these latter two DGC-proteins had the morphological characteristics of neurons. Double stainings of agrin and \u03b1-dystroglycan with NeuN, a neuronal marker, in MLC brain tissue confirmed that both agrin (brown) (Fig.\u00a04o) and \u03b1-dystroglycan (not shown) co-localized with NeuN (blue). Although, most redistribution appeared to be in neurons, double stainings of agrin (red) and GFAP (blue) showed that also some astrocytes displayed positive cell body staining for agrin (Fig.\u00a04p). On omission of the primary antibody no staining was observed (not shown).\nDirect protein interaction between MLC1 and Kir4.1\nTo verify interaction of DGC-proteins with MLC1, immunoprecipitation experiments were performed using human control brain lysates. Figure\u00a05 shows that after immunoprecipitation with both anti-MLC1 and anti-Kir4.1 antibodies and probing the precipitate for the presence of Kir4.1, a band at 200\u00a0kDA was detected, the size expected for Kir4.1. This band was not present in a pull-down without antibodies or with an unrelated antibody (anti-CHOP). Of concern in this experiment was the use of cross-linking reagents, since this might cause nonspecific interactions. When cross-linking reagents were left out, we failed to detect interactions, which is in agreement with previous studies on interaction between DGC-members [5].\nFig.\u00a05Co-immunoprecipitation (IP) of human brain lysates. Antibodies used for IP are indicated above the figure. The dash indicates pull-down without antibodies; anti-CHOP was used as a nonspecific antibody. The resulting Western blot was probed with antibodies against Kir4.1. The arrowhead shows the position of Kir4.1 at 200\u00a0kDa and the asterisk indicates a nonspecific band that is pulled down with all the antibodies used\nDiscussion\nThe DGC is important in linking cytoskeletal proteins to the extracellular basal lamina. It is present in several tissues including muscle, heart, nerve and brain. The composition of this complex shows some differences for different tissues. Structural defects in a number of DGC-members cause muscular dystrophies. Best known is Duchenne muscular dystrophy, related to mutations in dystrophin. Another muscular dystrophy is the CMD MDC1A, in which the MRI features are strikingly similar to those of MLC. MDC1A is caused by mutations in the laminin-\u03b12 chain of merosin [9]. A specific group of CMD variants is known as \u201cdystroglycanopathies\u201d and includes Walker\u2013Warburg syndrome, Fukuyama type of CMD, muscle\u2013eye\u2013brain disease, CMD type 1C and type 1D. They are characterized by hypoglycosylation of \u03b1-dystroglycan [14]. MDC1A and most of the dystroglycanopathies lead to both muscle disease and brain involvement, including cerebral white matter abnormalities.\nWe hypothesized that MLC1 could be associated with the DGC in the brain. To strengthen our hypothesis, we showed co-localization between MLC1 and DGC-proteins around blood vessels by immunofluorescent stainings. A common feature of the DGC is that mutations affecting one of its components often lead to destabilization of the complex with reduced or altered expression of other DGC-members. For instance, in muscle and brain tissue of patients with a dystroglycanopathy, immunostaining is reduced for \u03b1-dystroglycan as well as merosin and agrin [13]. In patients with Duchenne muscular dystrophy and Fukuyama type of CMD, decreased expression of multiple DGC-proteins has been found in muscle and brain [4, 17, 18]. In brain tissue of syntrophin, knock out mice altered expression of aquaporin-4 has been reported [16]. In glioblastoma tissue, abnormal expression of aquaporin-4 and syntrophin over the entire surface of glioblastoma cells and absence of \u03b1-dystroglycan from glial structures have been reported [27, 28]. Agrin was only present around larger vessels but absent around small vessels in glioblastoma tissue [27, 28]. We decided to use these DGC destabilization phenomena to test our hypothesis. Because of the co-localization of MLC1 and DGC-members at perivascular regions and the disassembly of the DGC in glioblastoma tissue we were interested in whether MLC1 is redistributed in this type of brain tumor. We, therefore, tested the expression pattern of MLC1 by immunohistochemical staining in glioblastoma tissue. We confirmed previous findings for agrin and aquaporin-4 [27, 28] and demonstrated that MLC1, like aquaporin-4, is redistributed in glioblastoma cells. We were also interested whether MLC1 mutations would lead to destabilization of the complex with reduced or altered expression of DGC-members in MLC brain. We demonstrated the absence of MLC1 and altered expression of Kir4.1, agrin and \u03b1-dystroglycan in brain tissue of MLC patients, whereas merosin, \u03b2-dystroglycan and aquaporin-4 retained their normal perivascular localization. Agrin and \u03b1-dystroglycan were redistributed in both neurons and astrocytes, although most positive staining cells appeared to be neurons. All the above results provide circumstantial evidence for an association between MLC1 and the DGC. To show a direct protein\u2013protein interaction we performed co-immunoprecipitation experiments that revealed an association between MLC1 and Kir4.1. All together, the above findings provide strong evidence for an association between MLC1 and the DGC.\nSo far almost all defects in DGC-proteins have been associated with a muscular dystrophy, which is absent in MLC. This difference can be ascribed to a difference in DGC composition for muscle as compared to brain. Whereas merosin is expressed both in muscle and brain, MLC1 is not expressed in muscle (http:\/\/biomed.ngic.re.kr\/cgi-bin\/cards\/carddisp?MLC1&search\u00a0=\u00a0KIAA0027) Both in MLC and MDC1A, the water content of the affected white matter is abnormally high due to intramyelinic vacuole formation [8, 22, 24]. The DGC is crucial for anchoring of water and potassium channels at the perivascular endfeet [15]. Destabilization of the DGC may lead to changes in glial polarity, disturbances of the blood\u2013brain barrier and alterations in ion and water homeostasis of the brain [27, 28], and result in an increased myelin water content. Our findings may, therefore, have implications for the pathophysiology of white matter abnormalities observed in MLC, MDC1A and the dystroglycanopathies.","keyphrases":["mlc1","astrocytes","endfeet","dystrophin-associated glycoprotein complex","leukodystrophy"],"prmu":["P","P","P","P","U"]}
{"id":"Pediatr_Nephrol-3-1-1805046","title":"Clinical predictors of neurocognitive deficits in children with chronic kidney disease\n","text":"The purpose of the study was to explore associations between neurocognitive function and chronic kidney disease (CKD)-related clinical characteristics. Twenty-nine children, ages 7 to 19 years, with an estimated creatinine clearance (eCrCl) of 4\u201389 ml\/min per 1.73 m2 body surface area were enrolled. Intellectual function (IQ), memory, and attention were measured and expressed as age-based standard scores. Clinical data were obtained by physical examination, laboratory testing, parental questionnaires and medical chart review. Pearson correlations and standard Student\u2019s t-tests were used to identify significant (P < 0.05) relationships between targeted clinical variables and neurocognitive scores. Increased CKD severity correlated with lower IQ (P = 0.001) and memory function (P = 0.02). Memory function was lower in children with longer duration of disease (P = 0.03). Similarly, IQ scores were lowest when kidney disease had started at a younger age (P = 0.03) and with a greater percent of life with CKD (P = 0.04). Our findings provide preliminary evidence that increased disease severity, longer duration of disease, and younger age of onset of kidney disease potentially place children with CKD at increased risk of neurocognitive deficits. Additional investigation is required to better quantify these risk factors, particularly regarding how much variability is accounted for by these specific risk factors.\nIntroduction\nNeurocognitive difficulties have long been observed in the chronic kidney disease (CKD) population [1]. While identification and treatment of specific comorbidities of CKD (e.g., anemia) have yielded improvement in the overall functioning of this population, reports of neurocognitive deficits in children with CKD continue to appear. These neurocognitive deficits undoubtedly will have significant lifelong implications for the CKD population as they move into adulthood. A recent review by Groothoff [2] examined the long-term outcomes of children diagnosed with CKD and identified persistent difficulties with a limited knowledge base, memory, and concentration into adulthood, as well as lower educational attainment. A better understanding of potential risk factors for cognitive impairment and decline in the pediatric CKD population could allow for earlier intervention and, possibly, less morbidity, particularly for certain cognitive deficiencies.\nStudies aimed at investigating neurocognitive impairment in children with CKD have identified a wide range of delays in motor and cognitive development. Measures of general intelligence, memory and attention have been used most frequently to look at cognitive function in the pediatric CKD population. Most studies have demonstrated lower IQ scores among children with end stage renal disease (ESRD) than in unaffected siblings [3] or the general population [4, 5] and also when pre- and post-transplantation performances are compared [6]. Memory deficits also have been identified in children with mild CKD as well as ESRD [7]. Fennell et al. [7] also reported in this same study that there was no significant decline in measures of attention among these children; however, improved attention performance has been demonstrated in children with ESRD after transplantation [6]. Observations of hyperactivity at school also have been noted in 50% of a study population assessing cognitive outcomes following dialysis during infancy [4]. The measures of cognitive performance in each of these studies varied widely but, when viewed collectively, suggest that children with CKD are vulnerable to cognitive deficiencies in IQ, memory and attention.\nOne thing to note, however, is that most studies to date have focused primarily on children with ESRD, leaving unanswered whether children with mild to moderate CKD also suffer this same vulnerability. Cognitive deficiencies in children with CKD could arise through a gradual process, proportional to the level of kidney dysfunction, or may develop once a filtration threshold has been passed. It seems logical that the severity of CKD might be proportional to the degree of cognitive impairment, but the data are not yet available to support this. The stage of neurologic development at the time of disease onset or the cumulative time children spend with CKD may also impact on the degree or type of cognitive impairment experienced. To our knowledge, no published study has yet focused specifically on isolating risk factors to identify which children with CKD are most at risk for cognitive decline. The primary goal of this study was to explore associations between clinical aspects describing CKD and selected neurocognitive test scores in a sample of children and adolescents with CKD. In addition, this study should provide the foundation for identifying specific clinical risk factors for subsequent neurodevelopmental dysfunction.\nMethods\nParticipants\nStudy participants were recruited from a university-based pediatric nephrology practice. The participants included children between 7 and 19\u00a0years of age, with an estimated creatinine clearance (eCrCl) by Schwartz formula [8] of less than 90\u00a0ml\/min per 1.73\u00a0m2 body surface area for at least 3\u00a0months duration. Renal transplant recipients and children with profound developmental delays were excluded. Following parental consent and patient assent, 29 children meeting these criteria underwent neurocognitive testing at a single point in time. A standardized physical examination and laboratory sample collection were also performed within 2\u00a0weeks of cognitive testing. A questionnaire providing demographic and historic information was completed by a parent or legal guardian. The study was reviewed and approved by the University of North Carolina Institutional Review Board.\nIncluded among the study sample were 17 children with obstructive uropathy or structural congenital renal anomalies, seven with glomerulonephritis, one with calcineurin inhibitor toxicity, one who had sustained ischemic injury, one with renal insufficiency of undetermined etiology, one child with Alport\u2019s syndrome, and one with cystinosis. Participants were evenly divided with respect to gender and disease severity. Approximately half the study sample had mild to moderate CKD with eCrCl ranging from 31\u201389\u00a0ml\/min per 1.73\u00a0m2 body surface area. Kidney disease onset was documented from birth in 59%. The majority (59%) were hypertensive, and, among these children, 70% were on antihypertensive therapy, and 65% had measured blood pressure above the 95th percentile at the time of examination. The mean hemoglobin level was 12.7\u00a0mg\/dl, with only three (10%) below 11\u00a0mg\/dl, with values of 10.8\u00a0mg\/dl, 10.7\u00a0mg\/dl and 10.1\u00a0mg\/dl. The general characteristics of our study sample are described in Table\u00a01. All the participants attended school in a regular classroom setting, and two reported having an individualized education plan (IEP) at the time of participation. \nTable\u00a01Demographic characteristics and medical variables of the study sample (PD peritoneal dialysis, HD hemodialysis)Study sample characteristicsNumber (n\u2009=\u200929)aRangeAge (years)12.5 (3.2)7\u201319Male (%)52Caucasian (%)52eCrCl (ml\/min per 1.73\u00a0m2 body surface area)32 (29)4\u201389ESRD (13 PD, 1 HD) (%)48Age at disease onset (years)4.4 (5.9)0\u201316Duration of CKD (years)6.7 (4.7)0.2\u201315Percent of life with CKD69 (39)1\u2013100Hypertensive (%)59Hemoglobin (mg\/dl)12.7 (1.5)10\u201317aMean (standard deviation) or percent\nNeurocognitive instruments\nAll participants completed a battery of intellectual, attention, and memory tasks as part of a larger neuropsychological evaluation. To gain an estimate of overall intellectual functioning, we administered the Wechsler Abbreviated Scale of Intelligence [9]. The Gordon Diagnostic System [10], a computerized continuous performance test, was also administered to gain an estimate of attention functioning. Within the memory domain, the Wide Range Assessment of Memory and Learning [11] was employed to assess short-term memory across verbal and visual domains. Age-based standard scores (mean\u2009=\u2009100, standard deviation\u2009=\u200915) were generated for variables from each of these tasks. In all instances, higher scores represented higher function for each cognitive test result. All tasks were administered by trained examiners who were supervised by a child neuropsychologist.\nThe Wechsler Abbreviated Scales of Intelligence (WASI) is built on the well-known Wechsler measures of global intellectual functioning [9]. It was designed for ages 6\u00a0years through adulthood, and employs a fluid-crystal model of intelligence. Four subtests comprise the WASI and include vocabulary, block design, similarities, and matrix reasoning. All four subtests were administered to gain a brief full scale IQ score.\nThe Gordon Diagnostic System (GDS) is a computerized continuous performance test that measures various aspects of visual attention [10]. For this study, the vigilance task was administered, which requires the individual to respond to specific visual stimuli (e.g., the number 1 followed by the number 9) from a series of numbers presented at a rate of approximately one per second. This task yields standard scores for the number correct, correct variability, commissions, and commission variability. For this study, the total correct standard score was employed.\nThe Wide Range Assessment of Memory and Learning (WRAML) is a comprehensive memory battery of tests [11]. It includes nine different subtests that measure short-term verbal memory (story memory, sentence memory, number\/letter), short-term visual memory (picture memory, design memory, finger windows), and new learning capabilities (sound symbol, visual learning, verbal learning). A general memory index is also computed from the nine subtests. Normative data are available for ages 6 through 18\u00a0years. The WRAML was administered and scored according to standardized procedures, with the general memory index being employed for this study.\nClinical measures\nDisease severity, age of onset, and duration of renal disease, coded as time in years and as percent of life with CKD, as well as the presence of anemia or hypertension were explored as potential risk factors of interest, given their association with detrimental cognitive outcomes in the CKD literature. Clinical data were collected through physical examinations and laboratory tests performed within 2 weeks of cognitive testing. Age of disease onset, as well as duration and percent of life with CKD, were determined through record reviews and were based on the date of primary disease diagnosis.\nThe Schwartz formula [8], using creatinine and height measurements, was used to estimate the eCrCl for non-dialysis patients. Creatinine clearance and urea kinetics were calculated for peritoneal dialysis patients from a 24\u00a0h fluid collection. All but three of the peritoneal dialysis patients exceeded Kidney Disease Outcomes Quality Initiative (K\/DOQI) guidelines for maintaining a Kt\/V of at least 2.0, and those not achieving this level ranged from 1.8 to 1.9 [12]. For our continuous analysis, we represented the eCrCl from the 24\u00a0h fluid collection from dialysis patients (incorporating residual renal function for the six dialysis patients who still produced urine) on a continuum with the eCrCl from the Schwartz formula that had been calculated for CKD patients. The one hemodialysis participant had a Kt\/V of 1.34 and received dialysis four times a week. For analysis, we imputed this participant\u2019s eCrCl, designating the mean creatinine clearance value from anuric peritoneal dialysis participants. For the dichotomous analysis, \u201cmild to moderate\u201d disease was categorized as an eCrCl of 30 to 89\u00a0ml\/min per 1.73\u00a0m2 body surface area, representing those patients with stage 2 or 3 CKD as defined by K\/DOQI guidelines [12]. Those with an eCrCl of less than 30\u00a0ml\/min per 1.73\u00a0m2 or those on dialysis, consistent with stage 4 or 5 CKD, were categorized as \u201csevere CKD\/ESRD\u201d.\nAnemia was defined as a hemoglobin level of less than 11\u00a0mg\/dl, reflecting the normative ranges of hemoglobin for children aged 9\u00a0years and above and the K\/DOQI guidelines that specify a target hemoglobin level of 11\u201312\u00a0mg\/dl in CKD [13]. Hypertension was defined as either the regular use of antihypertensive medications or the presence of a blood pressure greater than the 95th percentile for age, height, and gender [14].\nData analysis\nInitial summary statistical analysis was used to describe the total study sample. This included both descriptive analysis and graphical examination of all relevant variables. To address the primary research question for this study, we used bivariate analysis comparing neurocognitive scores and the clinical characteristics of the study sample, using Pearson\u2019s correlations for all continuous variables. Trend analysis based on linear regression was also used when continuous variables were compared graphically. For those variables that, by definition, were dichotomous (e.g., hypertension), Student\u2019s t-tests were used to compare the mean neurocognitive scores between categories. For those clinical variables for which an argument could be made either to express continuously or to dichotomize on the basis of a normal range (e.g., hemoglobin), both correlations and t-tests were used.\nResults\nAn initial inspection of the neurocognitive test scores showed that the sample of children and adolescents with CKD was mildly variable, with most of the participants falling within the low average to average range on most tasks. For the full study sample, the mean IQ score was 91 (range 61 to 117, SD\u200916), and the mean memory score was 88 (range 65 to 128, SD\u200916), both falling within the low average to average range when compared with normative expectations. The mean attention score of 96 fell well within the average range but did show much more variability (range 37 to 120, SD\u200923).\nDisease severity\nDisease severity, represented by the eCrCl, yielded the strongest relationship with both IQ (r\u2009=\u20090.57, P\u2009=\u20090.001) and memory (r\u2009=\u20090.45, P\u2009=\u20090.02), demonstrating that higher eCrCl correlated with higher scores in these cognitive domains. Figures\u00a01 and 2 illustrate the linear trend in the relationship between the continuous representation of disease severity and both IQ and memory scores. The correlation between attention and eCrCl was not significant. Additionally, lower IQ measures among severe CKD\/ESRD participants in our study were evident when compared dichotomously with those with mild to moderate CKD (P\u2009=\u20090.0008), and the same relationships held for memory scores (P\u2009=\u20090.009) and attention scores (P\u2009=\u20090.012), despite the fact that attention was not linearly associated with eCrCl. Figure\u00a03 illustrates the mean cognitive scores for categories of disease severity. While a downward trend with increasing severity is observed across all cognitive domains, statistical analysis confined to only the 15 non-dialysis patients yielded similar but less robust correlations among all areas of cognition, with IQ being the strongest (r\u2009=\u20090.46, P\u2009=\u20090.08).\nFig.\u00a01Relationship between IQ and estimated renal functionFig.\u00a02Relationship between memory and estimated renal functionFig.\u00a03Mean cognitive scores by level of renal disease\nAge at disease onset and CKD duration\nIncreased CKD duration in years was associated with lower memory scores (r\u2009=\u2009\u22120.40, P\u2009=\u20090.03). Age at disease onset did not correlate significantly with IQ, memory, or attention scores in our original analysis. However, it was noted that many participants in our study sample had presented with already severe disease, making the date of disease diagnosis less reflective of the actual age at which reduced renal function began. In a secondary exploratory analysis, we repeated our analysis using only those subjects (n\u2009=\u200910) for whom we had a clearly documented date on which the eCrCl dropped below 90\u00a0ml\/min per 1.73\u00a0m2. Among these patients were six with obstructive uropathy or structural congenital renal anomalies, three with glomerulonephritis, and one with cystinosis. In this secondary analysis we observed that the direction of prior relationships was preserved, with a stronger relationship between IQ and both age at disease onset (r\u2009=\u20090.69, P\u2009=\u20090.03) and percent of life with CKD (r\u2009=\u2009\u22120.66, P\u2009=\u20090.04). This may indicate that younger age at onset of CKD and longer percent of life with CKD correlate with lower IQ scores.\nHypertension and anemia\nTable\u00a02 summarizes the results of our analysis of hypertension and anemia. The dichotomous separation of these two variables did not produce significant group differences. These findings suggest that the presence of anemia or hypertension at the time of testing did not correlate with differences in neurocognitive test results between study participants with or without these clinical features. In a secondary analysis we also explored designation of hypertension, defined initially as either the use of antihypertensive medications or the presence of hypertension at the time of examination, on the basis of the presence of each criterion separately, and we found no differences. Additional examination of anemia revealed that only three patients had a hemoglobin level below the anemia definition of < 11\u00a0mg\/dl, with values of 10.8\u00a0mg\/dl, 10.7\u00a0mg\/dl and 10.1\u00a0mg\/dl. Consequently, we were unable to assess anemic patients across a wide range of hemoglobin (Hb) values. Within our sample of hemoglobin ranging between 10.1\u00a0mg\/dl and 17.0\u00a0mg\/dl, no association between our cognitive domains and hemoglobin was observed. \nTable\u00a02Relationships between hypertension, anemia and cognitive measuresNumber (n\u2009=\u200929)IQaGeneral memoryaAttentionaTotal cohort91 (16)88 (16)96 (23)Non-hypertensive90 (18)87 (16)89 (24)Hypertensive92 (15)89 (17)100 (21)Non-anemic91 (16)89 (16)98 (22)Anemic (<11\u00a0mg\/dl)92 (17)81 (23)76 (20)aFor each represented test among the normal population, the mean is 100 with a standard deviation of 15. No significant differences on Student\u2019s t-tests for hypertension or anemia.\nThe use of a contemporary blood pressure or hemoglobin value to represent the hypertension or anemia status of our participants evaluated the potential effect of these factors on their cognitive performance on the day of testing. Day-to-day changes in blood pressure could cause changes in cognitive function. Another approach is to use time-averaged hemoglobin and blood pressure values as markers of long-term strain on cognitive skills. Therefore, we also did a retrospective chart review of all blood pressure and hemoglobin values recorded in the 6 months prior to the study visit and generated a mean value for each of these two variables. On the basis of these time-averaged values, we repeated our analysis. In the blood pressure range represented, hypertension remained unrelated to any of the cognitive variables. Hemoglobin levels, however, demonstrated a subtle correlation with memory performance in the continuous analysis (r\u2009=\u20090.41, P\u2009=\u20090.03). Dichotomous analysis of anemia yielded a significant difference in cognitive scores only when defined as a time-averaged hemoglobin level less than 10.5\u00a0mg\/dl (mean memory score of 67 among anemic patients versus 91 among non-anemic participants, P\u2009=\u20090.01). However, this must be interpreted with caution, since only three participants fulfilled this criterion for anemia.\nWe should also acknowledge that there is a high degree of correlation among the results of the individual cognitive domains tested. Among the children included in our study, 12 (41%) had no cognitive scores greater than one standard deviation below the normative mean of 100, eight (28%) deviated in this fashion in a single cognitive domain, three (10%) did so in two areas, and six (21%) did so in all three areas. The level of overlap is, in part, likely due to the nature of cognitive testing, whereby testing in one area is, to some degree, dependent upon function in another area. The fact that there is not complete overlap, however, indicates that cognitive testing can distinguish among these deficits with some level of specificity.\nDiscussion\nNeurocognitive deficits among children with ESRD have been demonstrated in the literature, with some suggestion that key clinical variables can be associated with the level and, perhaps, the pattern of these deficits. The primary purpose of this study was to examine the relationship between targeted clinical variables, based on the available literature, and selected neurocognitive functions as defined by IQ, memory, and attention. Further, the current study examines these relationships across the full spectrum of CKD, including the mild to moderate stages of CKD. While it is recognized that some of the etiologies leading to CKD in our patients, namely cystinosis, calcineurin inhibitor toxicity, and ischemic injury, could potentially be independently associated with some level of cognitive dysfunction, in all three cases the children did not demonstrate evidence of other neurologic signs that would indicate central nervous system damage from their underlying disorders. Additionally, these children are representative of a typical pediatric CKD population, and any contribution from the presence of their comorbid conditions would not compromise the validity of our findings.\nIn the current study, several key findings were evident. First, disease severity significantly correlated with IQ and memory, while duration of disease significantly correlated with memory in the expected direction. Age at onset and percent of life with CKD did not initially correlate with any of the neurocognitive indices. When the age at onset and percent of life with the disease were examined in a secondary analysis restricted to a group of participants with more precisely defined onset of true decreased renal function, there was a clear trend that suggested that the younger the age at onset and the greater the percent of life with CKD, the lower the IQ score. The finding that younger age at renal insufficiency onset is significantly related to lower IQ complements previous observations in two prior studies of children with ESRD [3, 15]. Lawry et al. [15] found that among 24 children, including both dialysis and transplant patients, younger age at ESRD onset correlated with lower IQ scores. Brouhard et al. [3] also found that younger age at renal diagnosis was associated with lower achievement scores, although not specifically with lower IQ scores.\nAlso interesting to note is the wide range of IQ and memory scores among the ESRD subset of our study sample, as can be observed in Figs.\u00a01 and 2. This might reflect the presence of other clinical variables, such as duration on dialysis, dialysis adequacy, or presence of residual renal function, which could also influence cognitive performance. A similar wide range of results among children with ESRD has been observed in other study samples, such as that of Brouhard et al. [3], in which children with renal disease had scores that included some in the average range but still had mean IQ and achievement scores significantly lower than their healthy sibling controls. These findings support the hypothesis that disease severity plays a strong role in predicting neurocognitive vulnerability, yet also suggest that there might be additional contributing variables characterizing end stage disease that warrant further investigation. In an effort to explore this issue further, the sub-analysis confined to the relationship between disease severity and cognitive outcomes in the non-dialysis participants demonstrated a more subtle downward trend of scores across all cognitive domains that did not reach statistical significance. This was likely, in part, due to the reduction in power resulting from our limiting the analysis to only 15 participants. It also might suggest that therapy with dialysis itself contributes to cognitive dysfunction.\nThe literature has been more ambiguous with regard to attention, and our findings did not demonstrate a significant deficit in attention. Our sample did, however, demonstrate significantly more variation in the attention measure among children with CKD than one would anticipate in the general population, as exhibited by the large standard deviation in our study sample. This suggests the possibility that there may be a subset of children with CKD who are more vulnerable to attention-related problems. More extensive exploration of the various components of attention may be required to explore this observation further.\nHypertension, as a single measure or time-averaged, was not associated with the neurocognitive outcomes evaluated. Hypertension in the general pediatric population has been correlated with decreased performance in tasks requiring memory, attention, and concentration [16]. Using school-aged children from a population-based US survey who were diagnosed with hypertension on the basis of systolic or diastolic blood pressure above the 90th percentile for age, height and gender, Lande et al. [16] showed that hypertension was significantly associated with lower neurocognitive scores representative of short-term memory, attention and concentration problems. In our study sample of children with CKD, the majority of our participants were on antihypertensive therapy, so any existing hypertension in our study sample was at least partially treated, thus significantly restricting the range of variance available for correlation analysis. Furthermore, some antihypertensive therapies, for instance clonidine, have the potential to cause sedating effects in children but can actually help concentration in other children. The effects of medications on just a few children could appear amplified in our small study sample, and, thus, a true relationship could be obscured.\nThe hemoglobin level at the time of testing did not predict cognitive function. Time-averaged hemoglobin concentrations less than 10.5\u00a0mg\/dl were associated with lower memory performance. It has also been well established that anemia is associated with decreased cognitive function in adult patients with renal disease [17, 18]. Stivelman\u2019s review on the topic highlights several studies investigating the use of erythropoietin in uremic adult patients. Increasing hematocrit was associated with improved neurocognitive measures [17]. For example, Marsh et al. [18] used erythropoietin in 24 adult hemodialysis patients to improve their average hematocrit from 23% to 36%, resulting in significantly improved neuropsychological test scores reflecting memory, learning and attention. In our study sample of children with CKD, only three children had a low hemoglobin level, and these were between 10.1\u00a0mg\/dl and 10.8\u00a0mg\/dl. The mean scores for memory and attention among those three children were 8 points and 22 points lower, respectively, than those for non-anemic children, suggesting that there could be a relationship that would require a larger sample size to identify. In the normal or near-normal range, detrimental effects of lower hemoglobin levels are likely more subtle and, thus, less likely to be captured in analysis of a small sample. Our time-averaged hemoglobin analysis supported this as well.\nThis is the first study to date that focuses specifically on identifying potential risk factors for neurocognitive decline in children and adolescents with CKD. Its value is further enhanced by the inclusion of children with mild to moderate CKD, thus extending findings to a wider range of pediatric CKD patients. Increased disease severity, duration of CKD, and younger age at CKD onset were identified as potential risk factors for the targeted neuropsychological functions of IQ and memory. Our results further suggest a significant linear relationship with disease severity, with IQ scores continuously declining as disease severity worsens. Not only does this finding argue against a threshold effect, it implicates disease severity as an important risk factor for neuropsychological dysfunction. These findings also suggest the possible utility of these variables in a cumulative risk model to predict neurocognitive dysfunction in CKD and lay the foundation for exploration of such a model. Finally, these findings emphasize the importance of conducting further research in neurocognitive development among children with CKD. This includes research both to more clearly predict which children with CKD are most vulnerable to neuropsychological dysfunction and to identify specific correlates of this dysfunction. While there have been great strides in improving quality and length of life among children with CKD, a better understanding of those processes that interfere with normal neurodevelopment is a critical element of providing optimal care to these children.","keyphrases":["pediatrics","developmental delay","chronic renal insufficiency","cognitive deficits"],"prmu":["P","P","R","R"]}
{"id":"Breast_Cancer_Res_Treat-3-1-2001225","title":"A decade of letrozole: FACE\n","text":"Third-generation nonsteroidal aromatase inhibitors (AIs), letrozole and anastrozole, are superior to tamoxifen as initial therapy for early breast cancer but have not been directly compared in a head-to-head adjuvant trial. Cumulative evidence suggests that AIs are not equivalent in terms of potency of estrogen suppression and that there may be differences in clinical efficacy. Thus, with no data from head-to-head comparisons of the AIs as adjuvant therapy yet available, the question of whether there are efficacy differences between the AIs remains. To help answer this question, the Femara versus Anastrozole Clinical Evaluation (FACE) is a phase IIIb open-label, randomized, multicenter trial designed to test whether letrozole or anastrozole has superior efficacy as adjuvant treatment of postmenopausal women with hormone receptor (HR)- and lymph node-positive breast cancer. Eligible patients (target accrual, N = 4,000) are randomized to receive either letrozole 2.5 mg or anastrozole 1 mg daily for up to 5 years. The primary objective is to compare disease-free survival at 5 years. Secondary end points include safety, overall survival, time to distant metastases, and time to contralateral breast cancer. The FACE trial will determine whether or not letrozole offers a greater clinical benefit to postmenopausal women with HR+ early breast cancer at increased risk of early recurrence compared with anastrozole.\nIntroduction\nThe aromatase inhibitors (AIs) have proven to be a powerful drug class for use in hormone-sensitive breast cancer and have shown superiority over the selective estrogen-receptor modulator (SERM) tamoxifen in preclinical models of hormone-dependent breast cancer [1] and in randomized controlled trials in patients with advanced breast cancer [2] and early breast cancer [3, 4]. Initial adjuvant therapy with either letrozole (Femara\u00ae) or anastrozole (Arimidex\u00ae) was shown to be significantly more effective than tamoxifen in both the Breast International Group (BIG) 1-98 and Anastrozole and Tamoxifen Alone or in Combination (ATAC) randomized controlled trials in postmenopausal women with localized breast cancer [3, 4].\nIn the BIG 1-98 primary core analysis, patients with hormone receptor-positive (HR+) tumors randomized to receive letrozole initially were compared with those assigned to receive tamoxifen initially (N\u00a0=\u00a08,010). After a median follow-up of 25.8\u00a0months, 351 events had occurred in the letrozole group (n\u00a0=\u00a04,003) and 428 events in the tamoxifen group (n\u00a0=\u00a04,007), with 5\u2013year disease-free survival (DFS) estimates of 84.0% and 81.4%, respectively. Letrozole significantly reduced the risk of breast cancer recurrence (hazard ratio\u00a0=\u00a00.81; 95% confidence interval [CI] 0.70, 0.93; P\u00a0=\u00a00.003), especially the risk of distant recurrence (hazard ratio\u00a0=\u00a00.73; 95% CI 0.60, 0.88; P\u00a0=\u00a00.001) [3]. An analysis limited to patients randomized to either letrozole-only or tamoxifen-only arms (N\u00a0=\u00a04,922) was recently published and allows for more direct comparisons with results from other trials of continuous therapy with a single endocrine agent [5]. Results from the letrozole-only or tamoxifen-only arms were consistent with those published for the primary core analysis and showed that letrozole significantly reduced the risk of DFS events (hazard ratio\u00a0=\u00a00.82; 95% CI 0.71, 0.95; P\u00a0=\u00a00.007) [5, 6] and the risk of distant metastases. After a median follow-up of 51\u00a0months, 352 DFS events (14.3%) were observed in the letrozole-only group (n\u00a0=\u00a02,463), compared with 418 DFS events (16.9%) in the tamoxifen-only group (n\u00a0=\u00a02,459) [5, 6].\nThe ATAC trial, which compared anastrozole with tamoxifen for 5\u00a0years, did not have receptor positivity as a study requirement. After a median follow-up of 68\u00a0months (n\u00a0=\u00a06,186), anastrozole significantly prolonged DFS (575 events with anastrozole vs. 651 events with tamoxifen; hazard ratio\u00a0=\u00a00.87, 95% CI 0.78, 0.97; P\u00a0=\u00a00.01) and time-to-recurrence (402 vs. 498 events; hazard ratio\u00a0=\u00a00.79, 95% CI 0.70, 0.90; P\u00a0=\u00a00.0005), and significantly reduced the risk of developing distant metastases (324 vs. 375 events; hazard ratio\u00a0=\u00a00.86, CI 0.74, 0.99; P\u00a0=\u00a00.04) and contralateral breast cancers (35 vs. 59 events; 42% reduction, 95% CI 12, 62; P\u00a0=\u00a00.01) in the intent-to-treat (ITT) population [4]. However, neither the time to distant recurrence nor distant DFS (DDFS) were significantly improved with anastrozole in the HR+ population [7].\nWhile both letrozole and anastrozole have been evaluated extensively in early breast cancer, no head-to-head trial of these two AIs has been conducted in this setting. This report will focus on the design of the Femara versus Anastrozole Clinical Evaluation (FACE) trial, and describe how it will prospectively address potential efficacy and safety differences between the two AIs.\nRationale for head-to-head trial\nAn American Society of Clinical Oncology technology assessment concluded that AIs should be included in the adjuvant treatment of postmenopausal women with HR+ breast cancer [8] but did not recommend one AI over another. The National Comprehensive Cancer Network has also recommended initial adjuvant therapy with an AI (specifically letrozole or anastrozole) as an alternative to tamoxifen [9]. Some evidence suggests that AIs may not be equivalent, even though they belong to the same pharmacologic class of agents; differences have been reported in terms of potency, suppression of aromatization, antitumor effects, and pharmacogenomics. However, whether or not one AI is superior in treating early breast cancer is not known.\nRelative potency\nBhatnagar and colleagues compared the aromatase-inhibiting potency of letrozole and anastrozole in a variety of aromatase-containing cellular endocrine and tumor models [10, 11]. While letrozole and anastrozole were approximately equipotent in a cell-free aromatase system (human placental microsomes), letrozole was found to be more potent than anastrozole in inhibiting intracellular aromatase in intact rodent cells (50% inhibitory concentration [IC50] 20 vs. 600\u00a0nM, respectively), normal human adipose fibroblasts (0.8 vs. 14\u00a0nM), and human cancer cell lines (MCF-7Ca 0.07 vs. 0.82\u00a0nM and JEG-3 0.07 vs. 0.99\u00a0nM). Miller and colleagues reported that letrozole and anastrozole were more potent than aminoglutethimide in\u00a0vitro against tumor samples obtained from postmenopausal women with breast cancer, with letrozole demonstrating the lowest IC50 (2\u00a0nM, 8\u00a0nM, and 20\u00a0\u03bcM, respectively) [12]. Letrozole was compared with anastrozole in\u00a0vivo in athymic mice inoculated with MCF7 cells [13]. Tumor volumes increased to 145.9% in controls and decreased to 22.4% with letrozole 10\u00a0\u03bcg, and to 95.6% or 78.2% with anastrozole 10 or 60\u00a0\u03bcg, respectively. These results are consistent with a higher in\u00a0vitro potency of letrozole in cell-based assays [13].\nSuppression of aromatization\nThe effects of letrozole and anastrozole on suppression of total-body aromatization and plasma estrogen concentrations have been compared in patients with metastatic breast cancer [14, 15]. Levels of aromatase were detectable in 11 of 12 patients during treatment with anastrozole (mean percentage inhibition in the whole group, 97.3%) but in none of the 12 patients during treatment with letrozole (>\u00a099.1% suppression in all patients; Wilcoxon, P\u00a0=\u00a00.0022, comparing the two drug regimens). Suppression of estrone and estrone sulfate was found to be significantly greater during treatment with letrozole compared with anastrozole (P\u00a0=\u00a00.019 and P\u00a0=\u00a00.0037, respectively). Another study conducted in 54 postmenopausal women with invasive breast cancer showed that more complete inhibition of aromatase was achieved by 2.5\u00a0mg of letrozole than 1\u00a0mg of anastrozole, resulting in significantly greater suppression of estradiol (P\u00a0<\u00a00.0001) [15]. Thus, letrozole reduces estradiol levels to a greater degree than anastrozole, but it is not known whether this difference is clinically relevant.\nBreast cancer proliferation\nBiological changes in breast tumors occurring within 14\u00a0days of starting treatment may predict the efficacy of different endocrine agents in the adjuvant setting and could prove to be useful surrogate markers to compare drug efficacy [16]. A study of neoadjuvant endocrine therapy compared the effects of letrozole and anastrozole on the expression of HRs and markers of tumor proliferation in postmenopausal women with estrogen receptor (ER)-positive breast cancer [16]. Neoadjuvant letrozole and anastrozole decreased overall ER expression (Allred score) after 14\u00a0days, but more cases showed a reduction in progesterone receptor (PgR) expression following letrozole treatment (75\/106) than with anastrozole treatment (65\/102). Furthermore, only letrozole significantly reduced proliferation at lower Allred ER expression levels (scores 2\u20135). This is a potentially important finding, because it has been suggested that a greater suppression in proliferation may lead to improved DFS [17].\nClinical activity\nLetrozole and anastrozole have not been directly compared in the adjuvant setting, but data from a randomized, head-to-head trial in patients with advanced breast cancer are available [18]. Postmenopausal women with advanced breast cancer (N\u00a0=\u00a0713) that had progressed either during antiestrogen therapy or within 12\u00a0months of completing that therapy were randomized to receive letrozole (2.5\u00a0mg per day) or anastrozole (1\u00a0mg per day). Letrozole was significantly superior to anastrozole in terms of overall response rate (19.1% vs. 12.3%, P\u00a0=\u00a00.013), but there were no significant differences in median time to progression, the primary end point of the trial. Both agents were well-tolerated, and there were no significant differences in safety.\nAnastrozole and letrozole in the adjuvant setting have demonstrated superiority to tamoxifen in significantly reducing the risk of recurrence. In the ATAC trial, at 68\u00a0months\u2019 median follow-up, anastrozole significantly reduced the risk of distant metastases in the ITT population by 14% (P\u00a0=\u00a00.04) but not significantly in the HR+ patient subgroup (hazard ratio\u00a0=\u00a00.84; P\u00a0=\u00a00.06) [4]. A recent study of the recurrence rates after 2.5 and 5\u00a0years from the ATAC study showed that there were fewer recurrence events with anastrozole at these time points due to reductions in contralateral, primary, loco-regional, and distant recurrences [19]. In addition, at 25.8\u00a0months\u2019 median follow-up in the BIG 1-98 trial, letrozole significantly reduced the risk of distant metastases by 27% (P\u00a0=\u00a00.001) in the HR+\u00a0population, and another analysis of the early risk of relapse in 5,980 patients, with a median follow-up of 25\u00a0months, showed that letrozole reduced distant recurrences early on [3, 20]. The recently reported analysis of letrozole-only and tamoxifen-only arms in the BIG 1-98 trial showed that the time to distant metastases advantage for letrozole was consistent with these findings from the primary core analysis [5]. These data are potentially important, because the development of distant metastases directly translates into decreased survival. The ATAC trial showed a 3% relative improvement in overall survival (OS) (P\u00a0=\u00a00.7) with anastrozole at 68\u00a0months of follow-up, while a 9% relative improvement in OS (P\u00a0=\u00a00.35) was seen with letrozole at 51\u00a0months of follow-up [3, 5].\nSubset analyses of randomized trials comparing letrozole or anastrozole with either tamoxifen or placebo demonstrated differences between these AIs and suggested that specific patient populations may derive differing degrees of benefit from a particular AI. In the trial, retrospective subgroup analyses with a median follow-up of 33\u00a0months revealed no significant benefit of anastrozole over tamoxifen in patients with node-positive tumors and with prior chemotherapy [21], and these findings were confirmed in the 4-year update of the ATAC trial. Thus, the hazard ratio for risk of recurrence in patients with four or more positive nodes was 0.96 (95% CI 0.72, 1.25), and in patients with prior chemotherapy, it was 0.98 (95% CI 0.76, 1.28), indicating no differences between treatments [22, 23]. No analyses in similar subgroups were presented in the 68-month update [4].\nProspectively planned subgroup analysis revealed a benefit of letrozole over tamoxifen in patients who had received chemotherapy and in those with node-positive tumors [3, 5]. In the former subset, letrozole reduced the risk of recurrence after 5\u00a0years (hazard ratio 0.70; 95% CI 0.54, 0.92; P\u00a0=\u00a00.01). In the node-positive subset, letrozole reduced the risk of an event ending a period of DFS by 29% (hazard ratio 0.71, 95% CI 0.59, 0.85; P\u00a0<\u00a00.001). The advantage for letrozole in these patient subsets was confirmed in the recent analysis of the letrozole-only and tamoxifen-only arms of BIG 1-98 [5]. Interestingly, with longer follow-up of 51\u00a0months in this monotherapy analysis, there was an emerging benefit in the node-negative group, as letrozole reduced the relative risk of recurrence by 12% in this patient population. The MA.17 trial, evaluating the efficacy of extended adjuvant letrozole therapy, although not positive for OS in the overall population, demonstrated that OS was statistically significantly improved with letrozole among lymph node-positive breast cancer patients compared with placebo (hazard ratio 0.61; 95% CI 0.38, 0.98; P\u00a0=\u00a00.04) [24].\nThe question of whether one third-generation AI is superior for the adjuvant treatment of postmenopausal women with HR+\u00a0breast cancer remains, as does the question of whether there are any specific patient populations who derive particular benefit from a specific AI. Patients with early breast cancer can be assigned to risk groups on the basis of clinical and pathological characteristics. In the St. Gallen Guidelines [25], node-positive patients are considered to be in the intermediate- or high-risk group depending on the number of positive nodes and human epidermal growth factor receptor-2 (HER2) expression (see Table\u00a01). The guidelines state that endocrine therapy with an AI is a recommended option for patients with node-positive tumors who are in the intermediate- or high-risk groups. As high-risk patients are at greater risk of relapse, a drug specifically effective in this patient population would provide the oncology community with valuable information that may alter the outcomes of these patients.\nTable\u00a01Risk categories for early breast cancer according to the St. Gallen Guidelines. Reprinted from [25], with permission from the European Society for Medical OncologyLow risk Steroid hormone receptors expression, node-negative, and all of the following features:pT\u00a0\u2264\u00a02\u00a0cmGrade 1No peritumoral vascular invasionHER2\/neu gene neither overexpressed nor amplifiedAge \u226535\u00a0yearsIntermediate riskNode-negative and at least one of the following features:pT\u00a0>\u00a02\u00a0cmGrade 2\u20133Peritumoral vascular invasionHER2\/neu gene either overexpressed or amplifiedAge < 35\u00a0yearsNode positive (1\u20133 involved nodes) and HER2\/neu gene neither overexpressed nor amplifiedHigh riskNode positive (1\u20133 involved nodes) and HER2\/neu gene either overexpressed or amplifiedNode-positive (4 or more involved nodes)HER2 human epithelial growth factor receptor 2pT pathological tumor size (i.e. size of the invasive component)\nIs one AI superior in early breast cancer?\nMicroarray analysis was used to study the effects of various hormone therapies on ER+\u00a0MCF-7 cells, stably transfected with the aromatase gene (MCF-7aro cells) [26]. The study found that hormonal stimulation of gene expression can be counteracted by treatment with AIs (letrozole and anastrozole) and an anti-estrogen (tamoxifen), but that each agent had its own unique effects on gene expression (see Fig.\u00a01), suggesting possible differences between letrozole and anastrozole [26]. Although differences between letrozole and anastrozole have been demonstrated in preclinical models, it is widely recognized that preclinical findings do not always translate into clinical results, and that comparisons in one treatment setting or subpopulation cannot be extrapolated to another. A prospective trial is therefore needed to address the question of whether one AI is superior to another.\nFig.\u00a01Changes in inhibitor-responsive genes after treatment with letrozole, anastrozole, or tamoxifen. The Venn diagrams show the numbers of genes responsive to individual inhibitors in hormone-regulated genes. Reprinted from [26], with permission from the American Association for Cancer Research\nFACE was designed to test whether there is a preferable AI for the adjuvant treatment of postmenopausal women with HR+\u00a0and lymph node-positive cancer [27]. Node-positive patients were selected, because this population has a higher risk of relapse, and recurrence events occur earlier than in node-negative patients [20, 28, 29]. Thus, conducting the FACE trial in patients with lymph node-positive early breast cancer will provide an answer more quickly than conducting a trial in a broader population that includes patients with node-negative tumors.\nFACE trial design\nFACE is a phase IIIb open-label, randomized, multicenter trial [30]. The primary objective of the trial is to compare DFS at 5\u00a0years for letrozole and anastrozole. Secondary objectives are to assess safety, OS, time to distant metastases, and time to contralateral breast cancer [27].\nPatients\nThe trial is recruiting 4,000 patients from up to 250 international sites. Eligible patients are postmenopausal women with HR+ and lymph node-positive tumors who have recently undergone surgery for primary breast cancer (pathologic or clinical stage IIA, IIB, or IIIA). All patients must provide written informed consent.\nHR+\u00a0tumors are defined as tumors with any detectable ER or PgR expression by institutional standards. Patients who are PgR+ and ER\u2212 are eligible for the trial. Pathologic assessment of axillary lymph nodes is determined by sentinel node biopsy and\/or axillary lymph node dissection. Patients are stratified according to the number of involved lymph nodes and HER2 tumor status. Adjuvant trastuzumab is permitted in patients with HER2+ tumors. Other inclusion criteria include World Health Organization performance status of 0 or 1, lipid panel (fasting total cholesterol and triglycerides)\u00a0\u2264\u00a0grade 1 (National Cancer Institute Common Terminology Criteria for Adverse Events v3.0), and adequate hematologic, hepatic, and renal function.\nPatients with T4 tumors, metastatic disease, contralateral breast cancer including ductal carcinoma in\u00a0situ, or evidence of disease progression are excluded. Other exclusion criteria include prior neoadjuvant endocrine therapy; hormone replacement therapy (except intravaginal estradiol preparations) not stopped at least 4\u00a0weeks before randomization; adjuvant anti-estrogen therapy for\u00a0>\u00a01 month immediately following surgery, radiotherapy, and\/or chemotherapy; breast cancer chemoprevention with anti-estrogens if\u00a0<\u00a018\u00a0months between stopping and diagnosis of breast cancer; and therapy with any hormonal agent, such as raloxifene, for management of osteoporosis.\nRandomized trial design and treatments\nEligible patients are randomized to receive either letrozole 2.5\u00a0mg or anastrozole 1\u00a0mg daily for up to 5\u00a0years (see Fig.\u00a02). The date of randomization must be no more than 12\u00a0weeks from completion of surgery or 4\u00a0weeks after completion of adjuvant chemotherapy. Treatment assignments are balanced based on the number of lymph nodes (1\u20133, 4\u20139, 10+) and HER2 status (positive, negative, or unknown). Treatment will commence within 30\u00a0days of randomization and following the completion of standard chemotherapy (if given) and concurrently with radiotherapy (if given). Patients receive treatment with the allocated AI for up to 5\u00a0years or until disease recurrence\/relapse. Recurrence and survival will be assessed every 12\u00a0months.\nFig.\u00a02FACE randomized trial design\nEfficacy end points\nThe primary end point is DFS, defined as the time from the date of randomization to the date of the first documentation of re-occurrence of invasive breast cancer in local, regional, or distant sites; new invasive breast cancer in the contralateral breast; or death from any cause.\nSecondary efficacy end points include OS, defined as the time from the date of randomization to date of death from any cause; breast cancer-free survival, defined as the time from date of randomization to the date of death due to breast cancer; time to development of distant metastases, defined as the time from date of randomization to the date of the first development of any recurrent or metastatic disease in sites other than the local mastectomy scar, the ipsilateral breast in case of breast conservation, or the contralateral breast; and time to development of contralateral breast cancer, defined as the time from date of randomization to the date of the first development of any disease in the contralateral breast.\nAlthough the FACE trial, co-chaired by Drs. Ian Smith and Joyce O\u2019Shaughnessy, is an open-label trial, analysis of the data in a blinded fashion will make the data from this trial comparable with that obtained in a single-blinded trial. Both patients and their physicians will know which drug is being taken, but the analysis of the data will be conducted blinded to study treatment. The sponsor of the trial will not have access to the database, and all efficacy analyses will be conducted by an independent academic organization (the Instituto Nazionale Tumori, Milan, Italy), which will receive the data in a blinded manner. The data will be reviewed by an Independent Data Monitoring Committee, chaired by Professor Martine Piccart. The Independent Data Monitoring Committee will then make recommendations to a Trial Steering Committee chaired by Dr. Kathy Pritchard. The Independent Data Monitoring Committee will decide when the data will be released. The final analysis will be performed after the expected total number of DFS events have occurred. This is anticipated to be 7\u00a0years after the start of the study, following an accrual period of about 2\u00a0years and a minimum of 5\u00a0years of further follow-up. There are two planned interim analyses, scheduled to occur after one third and subsequently after two thirds of the maximum number of events have been observed. The interim analyses will be conducted after 320 and 639 events, respectively, have been recorded. In addition, analyses of secondary end points will be conducted at the interim time points.\nFACE is powered to detect a 3.5% absolute difference between the two treatment arms in DFS at 5\u00a0years. The 3.5% difference corresponds to a hazard ratio of 0.83 in favor of letrozole, corresponding to 5-year DFS values of 80.0% and 76.5% for letrozole and anastrozole, respectively.\nSafety end points\nGeneral patient safety and drug tolerability will be evaluated. Adverse events are recorded at every visit and graded for severity using the National Cancer Institute Common Terminology Criteria for Adverse Events v3.0. A checklist of adverse events is used to solicit adverse event information from patients.\nSafety analyses specifically include cardiovascular events and bone fracture events. All patients are evaluated clinically for osteoporosis and fracture risks. Bone mineral density testing is recommended at least every 2\u00a0years for all patients during study therapy by dual X-ray absorptiometry, peripheral dual X-ray absorptiometry, or ultrasound densitometry. Osteoporosis may be managed as clinically indicated using calcium supplements, vitamin D, or bisphosphonates. Measurements of fasting serum lipids are obtained at 6 and 12\u00a0months and then annually thereafter for the duration of the study treatment. Other laboratory assessments include hematology and blood chemistry.\nOther head-to-head studies\nOther trials that are directly comparing AIs are also under way. A randomized phase III trial [31] is comparing neoadjuvant therapy with exemestane, letrozole, or anastrozole in postmenopausal women undergoing surgery for stage II or stage III breast cancer. Another ongoing randomized trial, MA.27 [32], has been designed to compare the event-free survival of postmenopausal women with HR+ primary breast cancer when treated with exemestane or anastrozole. Results from these trials and ongoing pharmacogenomic studies [26] will also help individualize AI therapy for early breast cancer.\nThe study of inherited genetic polymorphisms that affect drug response and toxicity promises to help physicians individualize hormone treatment. For example, \u201cslow metabolizers\u201d of tamoxifen may have a worse outcome in the adjuvant setting with tamoxifen treatment than \u201cfast metabolizers,\u201d suggesting that these patients might be better treated with an AI [33\u201335]. Polymorphisms in tamoxifen metabolizing cytochrome P (CYP) 2D6 gene affect the plasma concentration of tamoxifen active metabolites: women with the CYP2D6 *4\/*4 or wt\/*4 genotype could have lower benefit of tamoxifen treatment and tend to have a higher risk of disease relapse [35, 36]. Genetic polymorphisms in the aromatase gene, CYP19, have recently been characterized [37]. Eighty-eight polymorphisms were identified, resulting in 44 haplotypes. Functional genomic studies revealed that polymorphisms may lead to changes in aromatase activity and altered affinity for AIs. These findings indicate that genetic variation in CYP19 might contribute to variation in the pathophysiology of estrogen-dependent disease. Clinical trials have been initiated to study the impact of genetic differences on response to AI therapy and may eventually lead to patient-specific selection of therapy based on optimizing efficacy and toxicity.\nConclusions\nLetrozole and anastrozole have both demonstrated superior efficacy compared with tamoxifen as initial therapy for early breast cancer [3, 4]. Preclinical and clinical evidence suggests that AIs do not have identical pharmacodynamic profiles, but it is not known whether one agent may be more effective as adjuvant therapy for early breast cancer. Differences in potency in preclinical studies, and the reduction in distant metastases in the BIG 1-98 study, suggest the potential for clinical efficacy differences between AIs. Based on the results of these trials, international guidelines now recommend adjuvant hormone therapy with an AI [9, 25] in patients with an increased risk of early recurrence. The FACE trial is addressing an important medical question in the oncology community: whether or not letrozole offers greater clinical benefit to postmenopausal women with HR+\u00a0early breast cancer at increased risk of early recurrence compared with anastrozole. Results from the FACE trial may refine the treatment strategies for treating breast cancer in postmenopausal women.","keyphrases":["letrozole","aromatase inhibitors","anastrozole","tamoxifen","breast cancer","femara"],"prmu":["P","P","P","P","P","P"]}
{"id":"Photosynth_Res-3-1-1769344","title":"Self-assembled monolayer of light-harvesting core complexes of photosynthetic bacteria on an amino-terminated ITO electrode\n","text":"Light-harvesting antenna core (LH1-RC) complexes isolated from Rhodospirillum rubrum and Rhodopseudomonas palustris were successfully self-assembled on an ITO electrode modified with 3-aminopropyltriethoxysilane. Near infra-red (NIR) absorption, fluorescence, and IR spectra of these LH1-RC complexes indicated that these LH1-RC complexes on the electrode were stable on the electrode. An efficient energy transfer and photocurrent responses of these LH1-RC complexes on the electrode were observed upon illumination of the LH1 complex at 880 nm.\nIntroduction\nWhen light energy is absorbed in vivo by purple bacterial light-harvesting (LH) complexes it is rapidly transferred to the reaction centers (RC) where the light energy is efficiently used to drive chemical reactions (Ke 2001). In most types of purple bacteria there are two types of antenna complexes: peripheral LH2 complexes and the LH1 complexes (Ke 2001). The structure of the LH2 complex of Rhodopseudomonas acidophila strain 10050 has been resolved to a resolution of 2.0\u00a0\u00c5 (McDermott et\u00a0al. 1995). This LH2 complex consists of a ring of nine heterodimeric subunits. However, such high-resolution structure has not been determined for the LH1 complex yet. There are, however, low-resolution projection structures produced by transmission electron microscopy (TEM) (Karrasch et\u00a0al. 1995) of two-dimensional (2D) crystals of the LH1 complex and a 4.8\u00a0\u00c5 X-ray crystal structure of the LH1-RC core complex (Roszak et\u00a0al. 2003). TEM analysis of the LH1 complexes revealed two types of complex, monomeric complexes from Rhodospirillum rubrum (R. rubrum) (Karrasch et\u00a0al. 1995) and dimeric complexes from Rhodobacter sphaeroides (R. sphaeroides) (Jungas et\u00a0al. 1999). The recent crystal structure of the LH1-RC \u2018core\u2019 complex from Rhodopseudomonas palustris (Rps. palustris) reveals that the LH1 complex surrounds the contours of the RC so that the \u2018core\u2019 complex has an overall oval rather than a circular shape (Roszak et\u00a0al. 2003). This structure showed the RC surrounded by the LH1 complex which consisted of 15 pairs of transmembrane helical \u03b1- and \u03b2-polypeptides and their coordinated BChls. The complete closure of the RC by the LH1 is prevented by a single transmembrane helix called W. Atomic force microscopy has also been used to observe antenna complexes in both natural and reconstituted membranes (Scheuring et\u00a0al. 2001, 2003, 2004; Fotiadis et\u00a0al. 2004; Bahatyrova et\u00a0al. 2004a, b; Stamouli et\u00a0al. 2003). Scheuring et\u00a0al. observed the LH1 complex as a minor component together with the major LH2 complexes from Rubrivivax geratinosus (Scheuring et\u00a0al. 2001) in reconstituted membranes and intact LH1-RC core complexes in native photosynthetic membranes (chromatophore) from Rhodopseudomonas viridis (Scheuring et\u00a0al. 2003) and Rhodospirillum photometricum (Scheuring et\u00a0al. 2004). Fotiadis et\u00a0al. observed the LH1-RC core complex from R. rubrum in membranes formed from Escherichia coli lipids (Fotiadis et\u00a0al. 2004). Bahatyrova et\u00a0al. showed that the LH1 complexes of a mutant lacking the RC from R. sphaeroides which form circular, elliptical, and even polygonal ring shapes as well as arcs and open rings (Bahatyrova et\u00a0al. 2004b) and that the LH1 complexes are positioned to function as an energy collection hub from the LH2 complexes in native membranes (Bahatyrova et\u00a0al. 2004a).\nOur understanding of charge separation and energy transfer in these LH2 and LH1-RC core complexes has enabled the first steps to be taken towards generating artificial systems that convert light energy into usable electrical current. Previous attempts to produce an artificial, energy-converting electrode system used either the LH1 complexes (Ogawa et\u00a0al. 2002) or RC (Blankenship et\u00a0al. 1995) immobilized on the electrodes. Until now, there have only been a few attempts to immobilize intact \u2018core\u2019 complexes, consisting of both the LH1 and the RC components together, onto an electrode (Ogawa et\u00a0al. 2004; Das et\u00a0al. 2004).\nWe have recently developed a procedure to create a self-assembled monolayer (SAM) of reconstituted LH1 complexes on a transparent indium tin oxide (ITO) electrode modified with 3-aminopropyltriethoxysilane (APS) using electrostatic interactions or hydrogen bonding between the electrode surface and the anionic LH1 polypeptides at pH 8.0 (Ogawa et\u00a0al. 2002). The near infra-red (NIR) absorption spectrum showed that the LH1 complex was stable when immobilized onto the electrode. Our current work extends this approach to the native LH1-RC core complexes. LH1-RC \u2018core\u2019 complexes isolated from R. rubrum and Rps. palustris were successfully assembled on an ITO electrode modified with APS (APS-ITO). Efficient energy transfer and photocurrent responses could be observed upon illumination at 880\u00a0nm.\nMaterials and methods\nGrowth of R. rubrum and Rps. palustris bacterium\nThe photosynthetic bacteria, R. rubrum strain S1 and Rps. palustris strain 2.1.6 were grown anaerobically in the light in modified Hutner\u2019s media as previously described (Roszak et\u00a0al. 2003; Visschers et\u00a0al. 1991).\nPurification of the LH1 complex of R. rubrum\nChromatophores of R. rubrum were prepared as previously described (Visschers et\u00a0al. 1991). Carotenoid was extracted from chromatophores using benzene. Usually approximately 30\u00a0mM OG (1\u00a0mM Tris, pH 7.5) was added to dissolve the chromatophores until the near IR absorption band shifted from 873 to 820\u00a0nm. Then the resultant aqueous solution was applied to a Sephadex G-100 column (1.5\u00a0cm\u00a0i.d.\u00a0\u00d7\u00a075\u00a0cm) to separate the RC and, LH1 complexes with carotenoids and subunit LH\/BChl a complexes without carotenoid (B820 complexes). The RC was eluted immediately after the void volume and then the LH1 complexes with carotenoid. Finally the B820 complexes were collected. Absorbance (\u03bbmax) of the LH1 complex in the absence of carotenoid (15\u00a0mM OG at 25\u00b0C): 372\u00a0nm (0.86), 586 (0.23), 870 (1.14). Absorbance (\u03bbmax) of the LH complex in the presence of carotenoid (20\u00a0mM OG at 25\u00a0\u00b0C): 372\u00a0nm (0.86), 475 (0.19), 512 (0.22), 546 (0.19), 586 (0.235), 875 (0.9).\nIsolation and purification of the core complex of R. rubrum and Rps. palustris\nThe LH1-RC core complexes isolated from R. rubrum and Rps. palustris were purified essentially as described previously (Roszak et\u00a0al. 2003). These LH1-RC core complexes from R. rubrum and Rps. palustris were initially solubilized by the addition of LDAO to 0.4% or 1% v\/v in 20\u00a0mM Tris\u2013HCl pH 8.0, respectively. The LH1-RC core complex of Rps. palustris was then separated from the LH2 complexes by sucrose density centrifugation, and was further purified by ion exchange chromatography by DE52 cellulose column. The OD880 of the isolated LH1-RC core complexes was adjusted to 0.3.\nPreparation of the core complex assembled on APS-ITO electrodes\nThe basic methods for this have been reported previously (Ogawa et\u00a0al. 2002). Transparent indium tin oxide electrodes were cleaned by immersion in piranha solution (H2O2:H2SO4\u00a0=\u00a03:7). We obtained APS-ITO electrodes by reacting 3-aminopropyltriethoxysilane with the surface of ITO electrodes in dry benzene at 80\u201390\u00b0C for 4\u00a0h. The OD880 of the isolated core was adjusted to 0.3. APS-ITO electrodes were immersed in the LH complex and the core complex solutions in Tris\u2013HCl pH 8.0 for 6\u00a0h at 4\u00b0C, and then rinsed with Tris\u2013HCl pH 8.0. The core complexes were immobilized, as a self-assembled monolayer on an APS-ITO electrode.\nNear IR, FT-IR, and fluorescence spectra\nNear IR spectra were recorded with Hitachi U-2000 and U-3500. Fluorescence spectra were measured with a Nippon Roper fluorometer by using a halogen tungsten light bulb (TS-428 DC), a single monochromator (SP-150M) for selection of the excitation wavelength, a monochromator (SP-306) and a CCD detector (Spec 10-100 BR\/LN) to detect the emitted fluorescence. The slits were set at 0.50\u00a0mm for the LH1 complexes in OG. Slits were set at 1.00\u00a0mm for the LH1 complex on the electrode. The samples were measured at 25\u00b0C. FT-IR spectra were recorded with Perkin-Elmer Spectrum 2000.\nPhotocurrent measurements\nPhotocurrents were measured at \u22120.2\u00a0V (versus Ag\/AgCl) in a home made cell that contained three electrodes; an APS-ITO electrode incorporating the core complex as a working electrode, an Ag\/AgCl (saturated KCl) as a reference electrode, and a platinum flake as a counter electrode. The working electrode was illuminated with a halogen lamp unit, AT-100HG, through a monochromator, SPG-120S (Shimadzu). The solution consisted of 0.1\u00a0M phosphate buffer (pH 7.0), containing 0.1\u00a0M NaClO4 and 5\u00a0mM methyl viologen.\nResults and discussion\nFigure\u00a01 shows the NIR absorption spectra of the isolated R. rubrum (a) and Rps. palustris (b) core complexes in 20\u00a0mM Tris\u2013HCl buffer pH 8.0 OG micelle (dotted line) and assembled onto an APS-ITO electrode (solid line), respectively. These spectra show that these core complexes have the absorption maximum at 880\u00a0nm with two smaller peaks at 800 and 760\u00a0nm. The former peak is attributable to the overlap of bacteriochlorophyll a (BChla) in the LH1 complex (880\u00a0nm) and the reaction center BChla dimer \u2018special pair\u2019 (870\u00a0nm) and the latter two peaks to the BChla called \u2018accessory\u2019 (800\u00a0nm) and bacteriopheophytin (760\u00a0nm) in the RC, respectively (Blankenship et\u00a0al. 1995). The NIR absorption spectra of these core complex on the electrode indicate that these complexes were not denatured when assembled onto an APS-ITO. In the previous study it was apparent that when the RC of R. rubrum was assembled, by itself, on the electrode it was relatively labile (Matsumoto et\u00a0al. 1999). In present study, the complete core complex proved to be quite stable, when assembled onto the electrode. The enhanced stability of the RC surrounded by the LH1 complex probably results from supportive interactions between the two complexes.\nFig.\u00a01NIR absorption spectra of the isolated R. rubrum (a) and Rps. palustris (b) core complexes in 20\u00a0mM Tris\u2013HCl buffer pH 8.0 OG micelle (dotted line) and assembled onto an APS-ITO electrode (solid line)\nTable\u00a01 shows the NIR absorption and fluorescence bands of the LH1 complex of R. rubrum and the LH1-RC core complexes of R. rubrum and Rps. palustris in OG micelles and on an APS-ITO electrode. The fluorescence bands of these core complexes on the electrode are identical to those in the OG micelles, again indicating that these complexes are stable when assembled onto the APS-ITO electrode. Interestingly, when illuminating at 880\u00a0nm the fluorescence emission of BChla molecules in the LH complex of R. rubrum on the APS-ITO was strongly quenched, due to the presence of the RC of R. rubrum. This indicates that an efficient energy transfer from BChla in the LH1 complex to the RC in the core complex is still occurring on the electrode (data not shown) (Ogawa et\u00a0al. 2004). FT-IR spectra of the LH complex of R. rubrum and the LH1-RC core complexes of R. rubrum and Rps. palustris assembled on the APS-ITO show the absorptions at 1650 and 1550\u00a0cm\u22121. These bands can be assigned to the amide I (C=O stretching vibration) and amide II (N\u2013H deformation vibration) bands of \u03b1 helical conformation, respectively (Miura et\u00a0al. 1998). These results indicate that the LH polypeptides are in the same \u03b1 helical configurations on the ITO electrode as in OG micelles (Parkes-Loach et\u00a0al. 1988; Kashiwada et\u00a0al. 2000).\nTable\u00a01NIR absorption and fluorescence bands of the LH1 complex of R. rubrum and the LH1-RC core complexes of R. rubrum and Rps. palustrisIn OG micelleOn APS-ITOAbsorbance (nm)Fluorescence (nm)Absorbance (nm)Fluorescence (nm)R. rubumLH1870890870890R. rubumLH1-RC880900880900Rps. palustrisLH1-RC878900878900\nIn summary, all of these results indicate that the LH1 and LH1-RC core complexes were not denatured by binding to the APS-ITO surface. Similar results were obtained using Langmuir\u2013Blodgett (LB) films to lay down LH1 complex membranes as a layer on a glass substrate (Iida et\u00a0al. 2000). The LH1 complex of R. rubrum and the LH1-RC core complexes of R. rubrum and Rps. palustris on the APS-ITO electrode were stable enough to handle at 4\u00b0C in the dark condition least 24\u00a0h.\nFigure\u00a02 shows the time course of the photocurrent generated from the LH1-RC core complex, LH1 complex or the RC of R. rubrum assembled onto an APS-ITO when the electrode was illuminated with a pulse of light at 880\u00a0nm. It is clear in Fig.\u00a02 that an enhanced photocurrent was observed for the LH1-RC core complex. In contrast no photocurrent was observed for either LH1 complex or the RC. Under our experimental conditions a cathodic photocurrent was observed, implying that one-way electron transfer from pigments in the LH1-RC core complex to methyl viologen was occurring as shown in Fig.\u00a03 (Nagata et\u00a0al. 2003; Imahori et\u00a0al. 2000).\nFig.\u00a02Time course of the photocurrent of the LH1-RC core complex, LH1 complex or the RC complex of R. rubrum on an APS-ITO electrode when the electrode is illuminated with pulsed light (880\u00a0nm) firing continuously for 30\u00a0sFig.\u00a03Schematic drawing of LH1-RC core complexes on an APS-ITO electrode generated cathodic photocurrent which shows the electron flow from the complex to methyl viologen (left) according to the cathodic photocurrent as shown in Fig. 2. Energy diagram for cathodic photocurrent generation by the LH1-RC core complex (right)\nFigure\u00a04 shows excitation spectrum of the photocurrent density (dots) and the NIR absorption spectra (solid line) from R. rubrum (a) and Rps. palustris (b) LH1-RC core complexes assembled onto an APS-ITO, respectively. These photocurrent responses showed a maximum at the wavelength corresponding to the absorption bands of the complex. Interestingly, an enhanced photocurrent was observed especially upon illumination at 880\u00a0nm for both R. rubrum (a) and Rps. palustris (b) LH1-RC core complexes. The quantum yield of the photocurrent was 0.05% for both the R. rubrum and Rps. palustris complexes (Imahori et\u00a0al. 2000). When the LH1 complex of R. rubrum alone, was immobilized on the electrode, the observed photocurrent was mainly generated by light absorbed at 770\u00a0nm, i.e. from monomeric BChla (Nagata et\u00a0al. 2003). Furthermore, when the RC complex of R. rubrum only was immobilized on the electrode, an efficient photocurrent was not observed upon illumination at 880\u00a0nm as shown in Fig.\u00a02. Thus, the enhanced photocurrent observed at 880\u00a0nm in the assembled LH1-RC core complex can be ascribed to energy transfer from the LH1 to the RC and then electron transfer from the electrode to the RC as shown in Fig.\u00a03 (Ogawa et\u00a0al. 2004). This data indicates that the LH1-RC core complex was well organized on the ITO and the photocurrents were driven by light that was initially absorbed by the LH components.\nFig.\u00a04Photocurrent density (dots) and NIR absorption spectrum (solid line) of LH1-RC core complexes form: (a) R. rubrum and (b) Rps. palustris assembled on an APS-ITO electrode\nIn conclusion, the SAM method is clearly successful in allowing assembly of functional LH1-RC core complexes on the electrode. This has been confirmed by NIR absorption spectroscopy, demonstrating that the photocurrent response, which is derived from electron transfer between the RC and the electrode, is enhanced by illumination at 880\u00a0nm.","keyphrases":["electrode","energy transfer","photocurrent","light-harvesting complex","light harvesting-reaction center complex","photosynthesis"],"prmu":["P","P","P","R","M","U"]}
{"id":"Int_Arch_Occup_Environ_Health-4-1-2413126","title":"Perceived work-related stress and early atherosclerotic changes in healthy employees\n","text":"Objective This study was conducted to investigate the relationship between perceived work-related stress and preclinical atherosclerosis.\nIntroduction\nStress can no longer be recognized as a problem of an individual only. Due to its versatile presence in every human\u2019s life, often resulting in health complications, stress has become a social problem and the role of work-related stress in this puzzle should not be underestimated. Coronary heart disease (CHD) is on the list of stress-related health problems.\nAtherosclerosis, the basis of CHD, develops in a long-lasting, multifactorial and complex process. In the etiopathogenesis of atherosclerosis, apart from traditional factors like lipids, carbohydrate metabolic disorders, hypertension and lifestyle (smoking, alcohol use and limited physical exercise), immunological disorders, as a consequence of reciprocal interactions in the psycho-neuro-immunological system, have been recently brought up (George and Shoenfeld 1997; Bednarska-Makaruk and Pasierski 2000; Kiecolt-Glaser and Glaser 1991; Amengual et al. 2001; Patryka et al. 2001; J\u0119dryka-G\u00f3ral et al. 2002; J\u0119dryka-G\u00f3ral 2003; Dinan 2005). Early atherosclerosis can even occur in young healthy people (Pasierski 1999).\nFor a long time now, occupational medicine has been interested in the relationship between work-related stress and CHD. Studies undertaken in this field have been based on western as well as eastern european populations (Jonsson et al. 1995; Hammer et al. 1998; Bosma et al. 1998, Kristenson et al. 1998; Kivimaki et al. 2002; Smith et al. 2005; Malinauskiene et al. 2005). Although there is a large body of literature on clinically overt CHD and its relation to work-related stress, there have been very few studies on preclinical CHD in those circumstances (Lynch et al. 1997; Nordstrom et al. 2001; Rosvall et al. 2002; Hintsanen et al. 2005).\nThe current study aimed to fill this gap and to assess the relationship between work-related stress and preclinical atherosclerotic changes in healthy employees. We considered carotid artery intima-media thickening and\/or plaque presence as a surrogate of coronary atherosclerosis. To detect these changes \u201cgold-standard\u201d methodology, i.e., B-mode carotid ultrasound examination, was applied. Because of the complex nature of the pathogenesis of CHD, other contributing factors have not been disregarded.\nMethods\nOrganization of the study\nFirst, all participants took part in a psychological study in which the level of stress and coping were evaluated with relevant questionnaires (see below). Then, the participants consecutively presented themselves for a clinical visit in which a medical examination took place and a blood sample was taken. Tests for biochemical parameters (see below) were performed on the same day as the blood sample was taken, according to routine laboratory procedures. Sera for immunological parameters (see below) were collected and stored according to international laboratory standards, then tested in runs (up to 40 sera per day) to minimize day-to-day variations.\nParticipants\nOne hundred healthy managers and 50 office workers (F:M\u00a0=\u00a050:50) aged 35\u201365 were studied. All of them were investigated for the presence of individual (age, obesity, hypertension, diabetes, smoking, alcohol use and low physical activity) and family (obesity, hypertension, diabetes, heart attack and stroke) risk factors for atherosclerosis. The studied individuals\u2019 detailed characteristics are presented in Table\u00a01.\nTable\u00a01Characteristics of studied groupsManagers (n\u00a0=\u00a0100)Office workers (n\u00a0=\u00a050)Total (n\u00a0=\u00a0150)Number50 (50%)25 (50%)75 (50%)\u00a0Females\u00a0Males50 (50%)25 (50%)75 (50%)Age47.2\u00a0\u00b1\u00a06.850.16\u00a0\u00b1\u00a06.4648.68\u00a0\u00b1\u00a06.63\u00a0Mean (years) \u00a0Minimum343534.5\u00a0Maximum656464.5Individual risk factors26 (26%)23 (46%)49 (32%)\u00a0\u00a0Hypertension\u00a0\u00a0Obesity19 (19%)17 (34%)36 (24%)\u00a0Diabetes1 (1%)1 (2%)2 (1.3%)Alcohol use\u00a0Every day3 (3%)03 (2%)\u00a0\u00a02\u20133\u00d7a week23 (23%)2 (4%)25 (16.7%)\u00a0\u00a0Less69 (69%)47 (94%)116 (77.3%)\u00a0\u00a0Never5 (5%)1 (2%)6 (4%)\u00a0Smoking cigarettes49 (49%)30 (60%)79 (52.7%)\u00a0Hormone replacement therapy8 (8%)4 (8%)12 (8%)Physical activity\u00a0\u00a02\u20133\u00d7a week26 (26%)12 (24%)38 (25%)\u00a0\u00a01\u00d7in 7\u201310\u00a0days32 (32%)14 (28%)46 (30.7%)\u00a0\u00a0Less42 (42%)24 (48%)66 (44%)Family risk factors41 (41%)31 (62%)72 (48%)\u00a0Hypertension\u00a0Obesity45 (45%)23 (46%)68 (45.3%)\u00a0Diabetes31 (31%)12 (24%)43 (28.7%)\u00a0Heart attack36 (36%)21 (42%)57 (38%)\u00a0Stroke30 (30%)17 (34%)47 (31.3%)\nInstruments\nWork-related stress was operationalized as perceived pressure caused by inappropriate working conditions. It was measured by the \u201cSources of pressure in your job\u201d scale that is a part of the short version of the Occupational Stress Indicator (OSI-2) by Cooper et al. Widerszal-Bazyl\u2019s (2001) Polish adaptation of OSI-2 was used in the study. The scale consists of 40 items on potential sources of pressure at work; respondents are required to rate each source on six-point response scales, in which 1\u00a0=\u00a0it is very definitely not a source (of pressure) to 6\u00a0=\u00a0it is very definitely a source. Those 40 items were divided into eight subscales concerning the following stress factors: workload (e.g. \u201cHaving to work very long hours\u201d, \u201cTaking work home\u201d), relationships (e.g. \u201cInadequate guidance and backup from superiors\u201d, \u201cFeeling isolated\u201d), home\u2013work balance (e.g. \u201cPursuing a career at the expense of home life\u201d), recognition (e.g. \u201cUnderpromotion\u2014working at a level below my level of ability\u201d), organizational climate (e.g. \u201cCharacteristics of the organization\u2019s structure and design\u201d, managerial role (e.g. \u201cManaging or supervising the work of other people\u201d), personal responsibility (e.g. \u201cHaving to take risks\u201d), hassle at work (e.g. \u201cAttending meetings\u201d). The scale made it possible to assess the global level of work-related stress (the sum of scores for all items) as well as to assess the level of a given kind of work stress, e.g. workload (the sum of scores for items related to workload). The reliability of the Polish version of the total scale was very high: Cronbach\u2019s \u03b1\u00a0=\u00a00.95. It was also satisfactory for most subscales: workload \u03b1\u00a0=\u00a00.95, relationships \u03b1\u00a0=\u00a00.89, home\u2013work balance \u03b1\u00a0=\u00a00.81, recognition \u03b1\u00a0=\u00a00.75, organizational climate \u03b1\u00a0=\u00a00.63, managerial role \u03b1\u00a0=\u00a00.57, personal responsibility \u03b1\u00a0=\u00a00.78, hassle at work \u03b1\u00a0=\u00a00.54. (Widerszal-Bazyl 2001).\nCoping was measured with the \u201cHow you cope with the stress you experience\u201d scale that is also part of OSI-2, too. It consists of 10 items on potential coping strategies; respondents are required to rate the extent to which they actually use them as ways of coping with stress. Answers are given on six-point scales, from 1\u00a0=\u00a0I never use it to 6\u00a0=\u00a0I use it very extensively. Some coping strategies included into the scale dealt with coping through control (e.g. \u201cEffective time management\u201d), and some with coping through social support (e.g. \u201cTalk to understanding friends\u201d). The reliability of the Polish version of the total coping scale was Cronbach\u2019s \u03b1\u00a0=\u00a00.70, and reliability of its subscales: 0.70 (for coping through control) and 0.50 (for coping through social support). Therefore, the total index of coping was used in the analysis, as well as the index of coping through control.\nPrior to the study, appropriate approval from the Local Ethics Committee was obtained. Participants were provided with detailed written information on the study\u2019s objectives and the methods that were going to be used; subsequently they signed their informed consent.\nSerum levels of biochemical (total cholesterol, LDL, HDL, TG, glucose) and serological risk factors of atherosclerosis (anticardiolipin, anti-\u03b22 GPI, anti-oxLDL, anti-HSP and anti-hsCRP antibodies) were evaluated.\nAnticardiolipin antibodies Patients\u2019 sera were tested with ELISA for the presence of anticardiolipin antibodies, according to the modified Harris method (Luft et al. 1990). The results were presented as OD units. Reference ranges of aCL were established by studying sera from 100 healthy donors. The cut-off value was established as OD\u00a0=\u00a0x\u00a0+\u00a04SD estimated for sera of healthy blood donors; it was 0.109 for the IgG class and 0.156 for the IgM class.\nAnti-\u03b22 glycoprotein\u00a0I (\u03b22 GPI) antibody levels were measured with a commercial DIASTAM\u2014FBGP 200 from Axis-Shield (Cat. No. FBG200) test. The results were expressed as OD units. Reference ranges of anti-\u03b22 glycoprotein I antibodies were established by studying sera from 152 healthy Caucasian donors (Axis-Shield Diagnostics Ltd. examination). The cut-off value was established as OD\u00a0=\u00a00.195.\nAnti-HSP antibody levels (IgG class) were estimated with non-commercial methodology, after necessary modification, according to Tsoulfa et al. (1989), with the use of McLean reagents. The cut-off value was established as x + 4SD estimated for sera of healthy blood donors; it was OD\u00a0=\u00a00.423. The estimation of IgM and IgA class antibodies was performed in the same manner.\nAnti-oxLDL IgG antibody levels were measured with ELISA with the use of a commercial test from OLAB (Cat. No. BI-20032). The cut-off value was established as 200 arbitrary units.\nhsCRP concentration was measured with ELISA with the use of commercial tests from EUCARDIO Laboratory Inc. (Cat. No. ZZ C4011E). The cut-off value was established as 4000\u00a0ng\/ml.\nIntima-media thickness (IMT) and atherosclerotic plaque in carotid arteries were searched using a computer analysis of B-mode ultrasound images. Both carotid arteries were measured with a 7.5-MHz linear-array transducer, part of Hewlett Packard Sonos. Plaque was defined as intima-medial thickening larger than 50% in comparison to the surround area (Pignoli et al. 1986)\nStatistical analysis of data was performed with SPSS v. 11.5. Means comparisons were carried out with a t test for independent groups. In bivariate correlation analysis Pearson\u2019s r coefficients were calculated. The level of significance was set at \u03b1 <\u00a00.05.\nResults\nAssessment of work-related stress\nThe level of work-related stress measured with OSI-2\u2019s Sources of pressure scale was as follows: managers\u2014mean\u00a0=\u00a0154 (\u03c3\u00a0=\u00a025), for office workers\u2014mean\u00a0=\u00a0145 (\u03c3\u00a0=\u00a028), for both groups\u2014mean\u00a0=\u00a0151(\u03c3\u00a0=\u00a025). According to Polish sten norms, the sten scale (\u201csten\u201d stands for standard ten) consists of ten units. Each unit equals 0.5 of a standard deviation. It is, assumed that stens five and six means average results, stens seven to ten high results, and stens one to four low results (Canfield 1951). Developed for Polish managers (Widerszal-Bazyl 2001), the managers\u2019 mean falls on sten six, whereas the office workers\u2019 mean falls on sten five (Table\u00a02). As a rule, stens five and six are interpreted as average results.\nTable\u00a02Results of psychological examinationStudied groupsWork-related stressCopingMeanStensMeanStensManagers154664056Office workers145553855Total151654056\nAssessment of coping\nThe level of coping measured with OSI-2\u2019s Coping scale was as follows: managers\u2014mean\u00a0=\u00a040, office workers\u2014mean\u00a0=\u00a038, both groups\u2014mean\u00a0=\u00a040. According to Polish sten norms the managers\u2019 mean falls on sten five for men and on sten six for women. The office workers\u2019 mean falls on sten five (for both genders). Thus, it can be said that the coping results were in the average range.\nLaboratory findings\nThe abnormality most frequently disclosed in biochemical findings were elevated levels of total cholesterol, LDL and triglycerides (in 52, 39 and 22% of the studied individuals, respectively). Decreased levels of HDL and increased levels of glucose were less frequent and were found in 12% of cases each.\nOf the immunological parameters studied, oxLDL antibodies were most frequently found (in 26% of cases), followed by an elevated level of hsCRP (in 11% of cases). In a few cases antibodies to \u03b22 GPI and HSP-65 (IgG class) were found (in 1% and 3% of studied cases, respectively). None of the studied individuals was positive for aCl. For six sera which were borderline or positive for HSP-65 IgG class, IgM and IgA class antibodies were searched. Only in three cases were the results weakly positive (two in IgM and one in IgA class).\nUltrasound examination of carotid arteries\nChanges in the ultrasound examination of carotid arteries confirming the presence of early atherosclerosis are shown in Table\u00a03. Plaque was found in 43 (29%) cases: in 26 managers (26%) and in 17 office workers (34%); groups did not differ statistically. The distribution of the mean values of the IMT measure was slightly skewed to the right. The mean values of IMT in managers and office workers were 0.0620\u00a0\u00b1\u00a00.014 and 0.0610\u00a0\u00b1\u00a00.012\u00a0mm, respectively; groups did not differ statistically.\nTable\u00a03Results of ultrasound examination of carotid arteries in studied groupsManagers (n\u00a0=\u00a0100)Office workers (n\u00a0=\u00a050)Total(n\u00a0=\u00a0150) Number of individuals without changes74 (74%)33 (66%)107 (71%) Number of individuals with plaque26 (26%)17 (34%)43 (29%) Intima-media measurement values 0.0620\u00a0\u00b1\u00a00.0140.0610\u00a0\u00b1\u00a00.0120.0618\u00a0\u00b1\u00a00.013\nCorrelation between IMT and the presence of plaque in carotid arteries with atherosclerosis risk factors\nPositive correlation was found between IMT and age (r\u00a0=\u00a00.42; P\u00a0<\u00a00.001), diastolic blood pressure (r\u00a0=\u00a00.20; P\u00a0<\u00a00.05), LDL (r\u00a0=\u00a00.28; P\u00a0=\u00a00.001), anti-HSP antibodies (r\u00a0=\u00a00.24; P\u00a0<\u00a00.05), whereas negative correlation was revealed with aCl\u2014IgG (r\u00a0=\u00a0\u20130.20; P\u00a0<\u00a00.05) and global work-related stress level (r\u00a0=\u00a0\u22120.26; P\u00a0<\u00a00.01). Among the stressors studied, statistical significance was shown for interpersonal relations (r\u00a0=\u00a0\u22120.23; P\u00a0<\u00a00.00), work\u2212home balance (r\u00a0=\u00a0\u22120.19; P\u00a0<\u00a00.05), managerial role (r\u00a0=\u00a0\u22120.24; P\u00a0<\u00a00.01) and organization climate (r\u00a0=\u00a0\u22120.22; P\u00a0<\u00a00.01). The presence of plaque correlated positively with age (r\u00a0=\u00a00.42; P\u00a0<\u00a00.001), smoking (r\u00a0=\u00a00.18; P\u00a0<\u00a00.05), LDL (r\u00a0=\u00a00.30; P\u00a0<\u00a00.01) and negatively with global work-related stress level (r\u00a0=\u00a0\u22120.28; P\u00a0<\u00a00.01). Statistical significance was shown for the following stressors: work load (r\u00a0=\u00a0\u22120.24; P\u00a0<\u00a00.01), interpersonal relations (r\u00a0=\u00a0\u22120.18; P\u00a0<\u00a00.05), work\u2212home balance (r\u00a0=\u00a0\u22120.33; P\u00a0<\u00a00.01) and responsibility (r\u00a0=\u00a0\u22120.25; P\u00a0<\u00a00.01) (see Table\u00a04).\nTable\u00a04Correlations between IMT and the presence of plaque in carotid arteries and atherosclerosis risk factors (Pearson\u2019s r)Risk factorIMTPlaqueAge 0.42**0.43**BMI 0.120.04Systolic blood preasure 0.130.06Diastolic blond preasure 0.20*0.10Cigarette smoking 0.130.18*Physical activity\u22120.020.07LDL0.28**0.30**HDL \u22120.16\u22120.05Triglycerides0.04 0.05Glucose 0.080.02hsCRP 0.050.02Anti-HSP 0.160.19aCl\u2013IgG \u22120.20*\u22120.10aCl\u2013IgM \u22120.14\u22120.15Anti-oxLDL \u22120.10\u22120.17Workload \u22120.15 \u22120.24**Relationships\u22120.23** \u22120.18*Home\u2013work balance \u22120.19*\u22120.33**Managerial role \u22120.24*\u22120.14Personal responsibility \u22120.14\u22120.25**Hassle at work \u22120.11\u22120.14Recognition \u22120.25**\u22120.15Organizational climate \u22120.22**\u22120.15Total index of coping \u22120.12\u22120.01Coping through control \u22120.050.02Global level of stress\u22120.26**\u22120.28*** Correlation is significant at 0.01 (2-tailed)** Correlation is significant at 0.05 (2-tailed)\nCorrelation between LDL\/smoking and global stress level\nPearson\u2019s analysis showed a negative relation between LDL and global stress level (r\u00a0=\u00a0\u22120.17; P\u00a0<\u00a00.05), and between smoking and global stress level (r\u00a0=\u00a0\u22120.17; P\u00a0<\u00a00.05).\nWork-related stress, coping, lifestyle and IMT\nPearson\u2019s analysis was performed in two triangles: (a) work-related stress\u2013coping\u2013IMT, and (b) work-related stress\u2013healthy lifestyle\u2013IMT. No statistically significant relations were shown either between work-related stress and coping, or between coping and IMT (P\u00a0>\u00a00.05), or between work-related stress and healthy lifestyle (no smoking, no excessive use of alcohol, high physical activity), or between healthy lifestyle and IMT (P\u00a0>\u00a00.05).\nDiscussion\nA multidisciplinary approach to assessing risk factors for atherosclerosis was considered by us in an earlier study (J\u0119dryka\u2013G\u00f3ral et al. 2006). On the basis of a deep multilevel analysis we were able to show that in healthy individuals, as in CHD patients, individual and biochemical risk factors were likely to play a leading role in the development of early atherosclerosis. Psychosocial stress should also be considered; however, inflammatory-immunological factors have not been found to be an independent predictor.\nAs inflammatory-immunological factors, antibodies to oxLDL and HSP-65, hsCRP as well as anticardiolipin and anti-\u03b22 GPI antibodies were considered. In the extensive literature of the subject, these parameters are recognized as directly involved in the pathogenesis of atherosclerosis (George and Shoenfeld 1997; Kiecolt-Glaser, Glaser 1991; Amengual et al. 2001; Patryka et al. 2001; J\u0119dryka-G\u00f3ral et al. 2002; J\u0119dryka-G\u00f3ral 2003).\nThe present study was undertaken to extend the investigation of psychosocial stress and atherosclerosis. We looked in depth for correlation between preclinical atherosclerosis and work-related stress; correlations with other risk factors of CHD were done in parallel.\nAs expected, we found correlation between IMT and age, diastolic blood pressure, LDL and anti-HSP antibodies, and correlation between plaque and age, smoking and LDL.\nSurprisingly, our results revealed that early atherosclerotic changes negatively correlated with the level of global job strain and some of its components (interpersonal relations, work\u2013home balance, managerial role, organization climate\u2014for IMT; work load, interpersonal relations, work\u2013home balance, responsibility\u2014for plaque).\nAmbiguous results have been previously reported in other, although very few, studies where work, psychosocial factors and carotid atherosclerosis were studied with ultrasound. Rosvall et al.\u2019s (2002) hypothesis that work-related stress characterized by high psychological demands and low decision latitude was associated with increased carotid atherosclerosis could not be confirmed either for women or for men. On the other hand, Lynch et al.\u2019s (1997) prospective study showed that men who experienced work-related stress (demanding work and low economic rewards) had significantly greater progression of carotid atherosclerosis than more advantaged men. Hintsanen et al. (2005) provided data that job strain (a joint effect of job demands and job control) was associated with increased IMT in men but not in women. Similarly, Nordstrom et al. (2001) showed that in men with greater work-related stress (workplace demands and intrusion of work concerns into home life) the risk of focal lesions or intima-media thickness in coronary artery increased, whereas in women stress was not related to the prevalence of early atherosclerosis. Hlatky et al. (1995) disclosed that job strain (high psychological demands and low decision latitude) did not correlate with the presence of coronary disease in angiography, either for men or for women.\nBy searching for a relationship between work-related stress and atherosclerosis, we wanted to prove that the correlation found between early atherosclerotic changes and LDL and smoking is, at least partly, caused by a higher stress reaction in the studied individuals. It was assumed that individuals with a level of high stress undertook unhealthy behaviour: smoking or excessive food intake. However, for both LDL and smoking the correlation with stress was negative. One should keep in mind that only 39% of the studied individuals had elevated levels of LDL and 47% reported smoking. Therefore, these results should be interpreted causally.\nWe explored in depth the negative correlation we found between IMT and the presence of plaque, and the level of work-related stress. Being aware that OSI-2 measured perceived stress, another hypothesis was formulated; persons with a high level of work-related stress (conscious stress) undertake preventive activities at the level of coping or healthy lifestyle.\nPerceived work-related stress as a risk factor for a clinically overt CHD has been investigated in multiple studies. Some prospective studies showed an association of work-related stress with an incidence of CHD (Bosma et al. 1998; Kivimaki et al. 2002; Kivimaki et al. 2005); whereas others did not (De Bacquer et al. 2005; Rosvall et al. 2002; Eaker et al. 2004). The largest INTERHEART study covering 11\u00a0119 cases and 13\u00a0648 controls from 52 different countries all over the world confirmed this association with regard to work, home, financial and major life stress (Rosengren et al. 2004). There are studies which, indicated that perceived work-related stress had profound impact on internal organs, sensual organs, the locomotor system and skin health problems (Cheng et al. 2001; Lindgren et al. 2002).\nIn the literature of the subject, coping is closely linked to stress. The most common meaning of coping is, an effort to solve problems and to seek reduction of tolerance to stress. The CATS theory (cognitive activation theory of stress) offered a new approach to coping (Ursin, Eriksen 2004; Eriksen et al. 2005). This theory assumed two cognitive reformulations of the learning theory (stimulus\u2013stimulus learning\u00a0=\u00a0classical conditioning and response learning\u00a0=\u00a0instrumental conditioning). Both reformulations are essential to understanding the relationship between learning, activation and relations between stress and health. The stress response (an alarm in the homeostatic system) results in behaviors that aim to cope with a situation. The level of alarm depends on what outcome of a stimulus is expected and the specific responses available for coping. Response outcome expectancy might be positive (coping), negative (hopelessness) or none (helplessness). Hence, coping defined as acquired positive outcome expectancy has some predictive value for stress and health.\nAccording to Weidner and Cain (2003) research on how people cope with stress situations disclosed avoidant mechanisms (denial, distraction, excessive alcohol consumption) in men, whereas more cardioprotective strategies (depression, asking for help)\u2014in females.\nStress management goes far beyond coping strategies. Toobert et al.\u2019s (2002) results disclosed success of a complex intervention program to reduce CHD risk, which included improvement of diet, stress management, social support, smoking and physical activity.\nIn our study on preclinical atherosclerosis and work-related stress, we were not able to confirm the hypothesis about the above-mentioned prophylactic activities. An analysis of correlation showed no significant relations between work-related stress and coping, between coping and IMT, between work-related stress and healthy lifestyle, or between healthy lifestyle and IMT.\nA relatively novel conception was presented by de Lange et al. (2005), who claimed that the relationship between stressful work and psychological well-being may be reciprocal. Their results showed that mental health might influence employees\u2019 perceived work characteristics. It is likely that the employees in our study had been recruited from that part of working society who felt fit to undertake ambitious tasks of managers and office workers and who developed job adaptive mechanisms over time.\nThe most probable interpretation of the negative correlation between perceived work- related stress and preclinical atherosclerosis is that in the case of individuals with a low level of perceived work-related stress, somatization of stress took place, i.e., stress is not perceived at the conscious level but it leads to somatic effects (e.g., IMT).\nWe are aware of some limitations of our study. Firstly, the results regarding work-related stress were based on self-reported data, which per se could always be a matter of some bias. Secondly, the number of employees studied was limited to 150. Voluntary participation of employees and fully unselected method of recruitment can guarantee the objectivity of the results. Thirdly, it was a cross-sectional study. A prospective type of research on risk assessment is preferable nowadays. However, our study did not aim to assess risk of CHD but to find correlation between work-related stress and early atherosclerosis, so we believe the measure taken for this purpose was appropriate.\nWe are more than convinced that to achieve full understanding of negative correlation between work-related stress and early atherosclerosis requires further interdisciplinary studies and we would be happy to continue exploration into this intriguing field.","keyphrases":["work-related stress","early atherosclerosis","carotid arteries"],"prmu":["P","P","P"]}
{"id":"Purinergic_Signal-4-2-2377318","title":"Loss of apical monocilia on collecting duct principal cells impairs ATP secretion across the apical cell surface and ATP-dependent and flow-induced calcium signals\n","text":"Renal epithelial cells release ATP constitutively under basal conditions and release higher quantities of purine nucleotide in response to stimuli. ATP filtered at the glomerulus, secreted by epithelial cells along the nephron, and released serosally by macula densa cells for feedback signaling to afferent arterioles within the glomerulus has important physiological signaling roles within kidneys. In autosomal recessive polycystic kidney disease (ARPKD) mice and humans, collecting duct epithelial cells lack an apical central cilium or express dysfunctional proteins within that monocilium. Collecting duct principal cells derived from an Oak Ridge polycystic kidney (orpkTg737) mouse model of ARPKD lack a well-formed apical central cilium, thought to be a sensory organelle. We compared these cells grown as polarized cell monolayers on permeable supports to the same cells where the apical monocilium was genetically rescued with the wild-type Tg737 gene that encodes Polaris, a protein essential to cilia formation. Constitutive ATP release under basal conditions was low and not different in mutant versus rescued monolayers. However, genetically rescued principal cell monolayers released ATP three- to fivefold more robustly in response to ionomycin. Principal cell monolayers with fully formed apical monocilia responded three- to fivefold greater to hypotonicity than mutant monolayers lacking monocilia. In support of the idea that monocilia are sensory organelles, intentionally harsh pipetting of medium directly onto the center of the monolayer induced ATP release in genetically rescued monolayers that possessed apical monocilia. Mechanical stimulation was much less effective, however, on mutant orpk collecting duct principal cell monolayers that lacked apical central monocilia. Our data also show that an increase in cytosolic free Ca2+ primes the ATP pool that is released in response to mechanical stimuli. It also appears that hypotonic cell swelling and mechanical pipetting stimuli trigger release of a common ATP pool. Cilium-competent monolayers responded to flow with an increase in cell Ca2+ derived from both extracellular and intracellular stores. This flow-induced Ca2+ signal was less robust in cilium-deficient monolayers. Flow-induced Ca2+ signals in both preparations were attenuated by extracellular gadolinium and by extracellular apyrase, an ATPase\/ADPase. Taken together, these data suggest that apical monocilia are sensory organelles and that their presence in the apical membrane facilitates the formation of a mature ATP secretion apparatus responsive to chemical, osmotic, and mechanical stimuli. The cilium and autocrine ATP signaling appear to work in concert to control cell Ca2+. Loss of a cilium-dedicated autocrine purinergic signaling system may be a critical underlying etiology for ARPKD and may lead to disinhibition and\/or upregulation of multiple sodium (Na+) absorptive mechanisms and a resultant severe hypertensive phenotype in ARPKD and, possibly, other diseases.\nIntroduction\nMonociliated ductal epithelial cells are receiving much attention due to their remodeling in polycystic kidney diseases, role in other cystic diseases of the kidney and other tissues, and in sensory physiology [1\u201312]. Cilia and flagella from lower organisms have important roles in sensory physiology in response to flow, touch, chemical and osmotic stimuli [1\u20135, 15, 16]. MDCK cells and other cell and tissue models of the renal collecting duct have been essential in characterizing cilium-derived cell calcium (Ca2+) signals [17\u201330]. It appears likely that this cilium-affected Ca2+ signal is derived from Ca2+ entry from extracellular stores and Ca2+ release from intracellular stores; the latter, perhaps, an ER cisternae near the basal body immediately beneath the monocilium [17\u201330]. Previously thought to be a vestigial organelle [1\u20135] or morphological marker for the cortical collecting duct (CCD) principal cell (PC) [31], the apical central monocilium is likely a sensor for ductal epithelia [5].\nRecently, our laboratory has shown that epithelial sodium channel (ENaC)-mediated sodium (Na+) absorption is upregulated in mutant cilium-deficient orpk CCD PC monolayers versus genetically rescued cilium-competent controls [32]. ENaC activity is present under open-circuit voltage and short-circuit current measurements in rescued cell monolayers with a well-formed cilium, but the electrical signals were three- to sixfold less than mutant monolayers [32]. One of our working hypotheses is that an inhibitory signal is lost (when the monocilium is not well formed) that is responsible for tonic attenuation of ENaC function [32]. Indeed, flow-induced Ca2+ signals have been shown recently by Praetorius and Leipziger not to be due to the cilium of MDCK cells per se but to autocrine ATP signaling that is stimulated by pressure pulses and responsible for Ca2+ spark and wave signal formation [33]. Immature MDCK cells without discernible cilia and mature MDCK cells with cilia responded similarly [33]. Alternatively, Satlin and coworkers have compelling data that monocilia do confer flow-based Ca2+ signals in isolated perfused CCDs from control mice versus mutant Tg737orpk mice [20]. In multiple different preparations from tissue to renal epithelia to heterologous cells, flow-induced Ca2+ signals have been observed [17\u201330]. However, the concept that an autacoid might mediate these cilium-specific effects has not been addressed.\nHerein, we show that the fully formed monocilium does confer a more robust Ca2+ signal in rescued cell monolayers versus mutant cell monolayers that are deficient in well-formed cilia. This finding agrees with the majority of the literature examining this phenotype [17\u201330]. However and more importantly, we show that stimulated or regulated ATP release is impaired when the monocilium is malformed. Ionomycin-, hypotonicity-, and mechanically induced ATP release are more robust in cilium-competent monolayers versus cilium-deficient monolayers. Varying the order of stimuli also revealed that cell Ca2+ influences the mechanically induced secreted ATP pool and that hypotonic cell swelling and other mechanical stimuli trigger the release of the same ATP pool. Finally, the flow-induced Ca2+ signal in this cell model requires autocrine ATP release and signaling, as it was blocked by the ATPase\/ADPase scavenger, apyrase. These data may reconcile the different conclusions within the Praetorius and Leipziger study [33] and the study of Satlin and coworkers [20]. To our knowledge, this is the first report linking the sensory apical central and nonmotile cilium to ATP secretion.\nMaterials and methods\nCell culture The collecting duct principal cells derived from an Oak Ridge polycystic kidney (orpk) mouse model of autosomal recessive polycystic kidney disease (ARPKD) and the genetically rescued cells with the wild-type orpkTg737 gene were a generous gift from Dr. Bradley Yoder (University of Alabama at Birmingham). The \u201cmutant 1\u201d cell clone (94D pcDNA 3.1 cells), the \u201crescued 1\u201d cell clone (94D BAP737-1 cells), and the \u201crescued 2\u201d cell clone (94D 737-2 cells) were handled identically and were grown under G418 selection on 6.5-mm diameter (Corning Costar) or 12-mm diameter filter supports (Millicell CM) and bathed in Dulbecco\u2019s modified Eagle\u2019s medium nutrient mixture F-12 HAM with L-glutamine and 15\u00a0mM hydroxyethylpiperazine ethanesulfonic acid (HEPES) (Sigma) [32]. This medium was supplemented with 5% fetal bovine serum (FBS) and contained (per 1,000\u00a0ml) dexamethasone (100\u00a0\u03bcl\/l of 2\u00a0mg\/ml stock), interferon-\u03b3 (25\u00a0\u03bcl of 800\u00a0U\/\u03bcl stock), T3 (10\u00a0\u03bcl\/l of 13\u00a0mg\/ml stock), G418 (500\u00a0\u03bcl of 400\u00a0\u03bcg\/ul stock), penicillin-streptomycin (10\u00a0ml of 100X stock), and ITS (10\u00a0ml of 0.5\u00a0mg\/ml insulin transferrin selenium concentrated stocks). The cells continued to be bathed on both sides of the filter until a monolayer tight to fluid formed. Measurement of the resistance of the filters was used as an indicator of the formation of a mature monolayer. Once the monolayers were formed for at least 12 h, the cells were then acceptable for the experimental assay.\nIn addition to these original clones developed in the Yoder laboratory, we also generated two new clonal lines from the original 94D mixed mutant CCD cell cultures, namely \u201cmutant 2\u201d and \u201cmutant 3.\u201d The only difference between these two subsequent clones and the original \u201cmutant 1\u201d clone is the fact that the \u201cmutant 1\u201d clone is stably transfected with an \u201cempty\u201d pcDNA 3.1 vector that confers G418 resistance. \u201cMutant 2,\u201d \u201cmutant 3,\u201d and the mixed mutant cultures are grown in a medium without G418. We also studied a \u201crescued B2\u201d clone that was stably transfected and rescued with the wild-type Tg737 gene but we derived from a different mutant CCD originally, 94E. The generation of these additional clones was described in a previous publication [32].\nBioluminescence detection of ATP released from epithelial monolayers In every assay performed, there was an initial basal ATP measurement followed by subsequent ATP measurements in response to different stimuli added in different sequences. ATP release assays were performed mainly on well-polarized cell monolayers grown in clear polyester 6.5-mm diameter filter supports. Mutant versus rescued cell monolayers were studied side by side in each ATP release protocol. The use of luciferase-luciferin to indirectly measure the ATP concentration has been published previously in detail [34]. Every drug prior to experimentation was tested to ensure that they did not interfere with the luciferase enzyme activity [34\u201337]. There was also no change in cell viability with any of these maneuvers as has also been reported previously [34\u201337]. Each experiment began with a basal ATP measurement or the addition of Opti-MEM I medium (GIBCO-BRL) with 1\u00a0mg\/ml luciferase-luciferin reagent (Sigma) being added to the apical or basolateral side of the filter\u2019s monolayer. Basolateral ATP release was negligible; therefore, all data reported are apical or luminal ATP secretion. Basal levels of ATP release were measured for 6 min in 15-s, nonintegrated photon collection periods in a TD-20\/20 Luminometer (Turner Designs) [34\u201337]. In different orders of addition, we used the following stimuli: (1) ionomycin (2\u00a0\u03bcM) (to increase the intracellular calcium concentration), (2) distilled water with 1\u00a0mg\/ml luciferase-luciferin (to dilute the osmotic strength of the Opti-MEM I medium), and (3) intentionally harsh pipetting in the center of the apical surface of the cell monolayer (to induce a mechanical stimulus on the apical membrane). Normally, addition of drug or distilled water (or the same volume of medium as a volume addition control) is performed or dispensed very slowly along the wall of the plastic support that holds the permeable filter so as not to disrupt the tight, confluent monolayer. Therefore, by quickly pipetting the medium up and down onto the monolayers, a mechanical stimulation is induced. Luminescence was measured for 6\u00a0min after each stimulus. All experiments ended with the addition of hexokinase to eliminate any ATP left in the medium. All assays were performed at room temperature.\nFura-2\/AM imaging of cytosolic free calcium in a cell monolayer-based perfusion system Fura-2\/AM imaging was performed as described previously [38\u201340]. However, to remain faithful to the study of polarized cell monolayers, we designed a cell monolayer-based perfusion chamber system where 12-mm diameter Snapwell Transwell filter supports are inserted into a homemade perfusion chamber designed to accommodate this special filter support. Apical and basolateral perfusion are then performed separately through independent injection ports and separate ejection ports are subjected to vacuum. Response to changes in apical flow from 1\u00a0ml\/min (a \u201cslow\u201d flow) to 5\u00a0ml\/min (\u201chigh\u201d flow) were performed to induce a flow-induced Ca2+ transient akin to that observed by many other laboratories. We assessed mutant and rescued cell monolayers in parallel during all protocols. We also assessed the flow-induced Ca2+ transient signal in the absence of apical extracellular Ca2+ and in the presence of gadolinium chloride and apyrase. It was not possible to calibrate the Fura-2 fluorescence signal to real free cytosolic Ca2+ values because of ionomycin contamination of the flow chamber. As such, fluorescence ratio values are shown. We give estimates of what the free Ca2+ may be based on previous calibrations of the same cells grown as nonpolarized cells.\nMaterials All reagents and drugs were purchased from Sigma. Larger 12-mm diameter filter supports were obtained from Millipore. Smaller 6.5-mm diameter filter supports were obtained from Corning Costar. It should be noted that we are using less luciferase:luciferin detection reagent than in past studies [34\u201337].\nResults\nBasal ATP release or secretion is not different between mutant and rescued CCD PC monolayers Basolateral ATP release was near background and was negligible in both mutant and rescued cell monolayers. This result does not mean that ATP is not released across the basolateral cell surface. ATP may, in fact, be released but \u201ctrapped\u201d between the cell monolayer and the filter support and\/or the detection reagent may not gain sufficient access to this \u201ctrapped\u201d space. Therefore, only apical-directed ATP release was studied exclusively in the detection experiments below. Regardless of the filter support used to form cell monolayers and the amount of detection reagent included in the real-time experiments, basal or unstimulated or constitutive ATP release was low and not different between the panels of mutant and rescued cell monolayers studied. There was agreement between all mutant clones and all genetically rescued clones in all protocols below. As such, the data are pooled for mutant cell monolayers and rescued cell monolayers. Basal ATP release data are summarized in the bar graph in Fig.\u00a01 as bioluminescence in arbitrary light units (ALU). In total, the amounts of ATP released were below 50\u00a0nM. A standard curve was run with each series of ATP release experiments. Thus, basal ATP secretion is not different across the apical membrane of mutant and rescued cell monolayers and the amount of ATP released is barely sufficient to engage the most sensitive P2Y or P2X cell surface receptors.\nFig.\u00a01Basal ATP release across the apical cell surface is not different between mutant cilium-deficient and genetically rescued cilium-competent orpk kidney cell monolayers. In three separate preparations of mutant versus rescued cell monolayers on different filter supports and exposed to different concentrations of luciferase:luciferin detection reagent, ATP release was not different between the cilium-deficient and cilium-competent cell monolayers (n\u2009=\u200912 for left set of bars; n\u2009=\u20096 for center and right datasets). In this study, we had a breakthrough with regard to what filter support to use for our ATP release experiments studying cell monolayers on filter supports. We found that the 6.5-mm diameter filter support (the same as used in open-circuit and Ussing chamber experiments studying ENaC upregulation [32]) was ideal for these real-time ATP release assays into the apical medium because the apical cell surface was as close as it could be and fully exposed to the luminescence detector in the base of the chamber. The left set of bars is data derived from this filter support and procedure. All other data in subsequent figures derive from this preparation. A diagram shows how the filter support was handled and placed into the luminometer chamber. After washing of serum-containing medium away from the monolayer, a small volume of Opti-MEM 1 medium (15\u00a0\u03bcl) is added in a drop to the lid of a 35-mm dish to hydrate the preparation and to promote adherence to the lid of the dish; 200\u00a0\u03bcl of Opti-MEM 1 medium containing 1\u00a0mg\/ml lyophilized luciferase:luciferin reagent are placed within the cup in contact with the apical surface of the cell monolayer. Detection is then begun for bioluminescence due to secreted ATP from the cell monolayer into the apical space in continuous 15-s collection periods delayed only by the time to push the \u201cstart\u201d button on the luminometer and to record the luminescence to an Excel spreadsheet. The standard curve with which approximations were made as to the secreted ATP concentrations was performed with each dataset, was compiled, and is as follows: 10\u221210 M ATP, 0.052\u2009\u00b1\u20090.005 ALU...10\u22129 M ATP, 0.476\u2009\u00b1\u20090.042 ALU...10\u22128 M ATP, 4.95\u2009\u00b1\u20090.51 ALU...10\u22127 M ATP, 51.2\u2009\u00b1\u20094.8 ALU...10\u22126 M ATP, 455\u2009\u00b1\u200923 ALU...10\u22125 M ATP, 3956\u2009\u00b1\u2009311 ALU...10\u22124 M ATP and 10\u22123 M ATP, >9999 ALU (exceeded limit of detection of luminometer). It is important to note that these are estimates of secreted ATP because we do not add inhibitors of ecto-ATPases and it is likely that our ATP release signal is diminished significantly by ATP degradation before detection. The left set of bars show basal ATP secretion from the 6.5-mm diameter clear polyester filter support without feet that is immediately above the light sensor and bathed in 15\u00a0\u03bcl of serosal medium. The middle set of bars show ATP secretion from a 12-mm polyester clear filter support with feet and bathed in 200\u00a0\u03bcl of serosal medium. The third set of bars at right show ATP secretion from a polycarbonate filter support with feet and bathed in 200\u00a0\u03bcl of serosal medium\nIonomycin-stimulated ATP release is more robust in rescued versus mutant cell monolayers We first assessed the effect of an increase in intracellular free calcium (Ca2+) on ATP release. This first study was done in part because of the emerging role of the primary central monocilium in mediating Ca2+ transients, sparks, and waves in cell monolayers derived from extracellular and intracellular stores [17\u201330]. This cilium-derived Ca2+ signal is triggered by touch or flow across this organelle and is mediated, at least in part, by the polycystin proteins, PC-1 and PC-2 [17\u201330]. Polycystin-2 is a part of the TRPP subfamily of TRP genes and is a distant relative of the transient receptor potential or TRPC gene family of Ca2+ entry channels [17\u201330]. Ionomycin (2\u00a0\u03bcM) applied to the apical surface of well-polarized cell monolayers triggered a slow, monophasic rise in ATP release over 3\u20135 min (see typical time courses below in Figs.\u00a06 and 8). This response was a similar phenotype to that observed in previous studies by our laboratory in human vascular endothelial cell monolayers [37]. In mutant monolayers deficient in monocilia, an increase in cell Ca2+ increased extracellular ATP from below 50\u00a0nM to 0.4\u00a0\u03bcM. Figure\u00a02 shows data as bioluminescence in arbitrary light units (ALU) with corresponding estimated ATP for each step of the protocol based upon parallel standard curves with known amounts of ATP. In sharp contrast, rescued monolayers responded to ionomycin stimulation with a rise in secreted ATP from approximately 50\u00a0nM to 2\u00a0\u03bcM, three- to fivefold higher sustained amounts on average than in mutant monolayers. The data in Fig.\u00a02 also group the data from the panels of mutant and rescued clones which were in agreement. Taken together, these data show that cell Ca2+-stimulated ATP release is more robust when a well-formed central monocilium is present. In fact, the \u201creleasable\u201d pool of ATP that is sensitive to Ca2+ appears intact in cilium-competent monolayers and deficient in cilium-deficient monolayers.\nFig.\u00a02Ionomycin-induced ATP release across the apical cell surface is markedly attenuated in mutant cilium-deficient versus genetically rescued cilium-competent orpk kidney cell monolayers. Six to nine cell monolayers were assessed at left for the summary data derived from the 6.5-mm diameter filter support preparation. The fold difference in ionomycin (2\u00a0\u03bcM, added in a 2-\u03bcl bolus along the side of the plastic wall into the apical medium)-induced ATP release in rescued cell monolayers over mutant monolayers is graphed for the dataset at left along with three additional datasets. Although the filter supports, amount of detection reagent, and magnitude of luminescence measured differed between preparation to preparation (n\u2009=\u20096 each), the fold difference in the ionomycin effect was constant. The asterisk reflects P\u2009<\u20090.05 by paired Student\u2019s t-test; the cross reflects P\u2009<\u20090.05 significance by analysis of variance (ANOVA) and Tukey\u2019s ad hoc test. The statistical analysis and results are given similarly in all other figures\nHypotonicity-induced ATP release is more robust in rescued versus mutant cell monolayers Hypotonic challenge and resultant cell swelling and regulatory volume decrease (RVD), a shared mechanism of cell volume regulation by all cells [41\u201344], is a robust stimulus for ATP release [41\u201346]. It is a shared sentiment by most that ATP release precedes and triggers, at least in part, RVD mechanisms such as opening of parallel K+ and Cl\u2212 channels to mediate rapid KCl efflux and to lower cell volume back to basal levels [41\u201346]. As such, we used hypotonic challenge to assess ATP release in mutant cilium-deficient cell monolayers versus rescued cilium-competent cell monolayers. Figure\u00a03 summarizes these data. These stimuli followed the ionomycin treatment. We show other typical real-time courses below when hypotonic challenge was performed in the absence of ionomycin (see Fig.\u00a07). In all monolayers, hypotonic challenge of the apical surface (50% dilution of the medium osmolality with distilled water containing 1\u00a0mg\/ml detection reagent) triggered an increase in ATP release that had transient and sustained components. In mutant monolayers, hypotonic challenge increased ATP detected in the medium from approximately 0.4\u00a0to 3\u00a0\u03bcM during the peak of stimulation and to a slightly lesser value at the end of the hypotonicity challenge phase. Addition of the same volume of isotonic medium (a \u201cvolume addition\u201d control) increased ATP secretion slightly from 0.4\u00a0to 0.5\u00a0\u03bcM (data not shown). Uniformly, rescued cilium-competent cell monolayers responded more vigorously to the hypotonic stimulus. This stimulus again followed the ionomycin treatment. ATP release increased from approximately 2\u00a0to 5\u201310\u00a0\u03bcM at the peak of the response and to a lesser sustained level at the end of the hypotonic challenge phase. In volume addition control experiments, ATP release was triggered that was only transient and increased from 2\u00a0to 4\u00a0\u03bcM on average (data not shown). Taken together, these data show that rescued cell monolayers respond more vigorously to hypotonic challenge than mutant cell monolayers with regard to ATP secretion, a signal required for cell volume regulation. Moreover, increases in cell Ca2+ prime swelling-induced ATP release and allow a larger ATP secretion signal to visualize.\nFig.\u00a03Hypotonic cell swelling-induced ATP release across the apical cell surface is markedly attenuated in mutant cilium-deficient versus genetically rescued cilium-competent orpk kidney cell monolayers. Cell monolayers were assessed at left for the summary data derived from the 6.5-mm diameter filter support preparation. Hypotonic challenge was induced by adding an equal volume of distilled water (200\u00a0\u03bcl with 1\u00a0mg\/ml L:L reagent) down the wall of the apical chamber into the apical medium. The fold difference in three other preparations is graphed at right and was relatively constant again (n\u2009=\u20096\u20139). Statistics are shown like in Fig.\u00a02\nMechanically-induced ATP release is more robust in rescued versus mutant cell monolayers Harsh and fast pipetting of an 50\u00a0\u03bcl volume of isotonic medium directly down on the center of the cilium-competent monolayers (the \u201cmechanical stimulus\u201d) in three repetitions triggered an immediate ATP secretion that had transient and sustained phases like the swelling-induced ATP release from 2\u00a0to 3\u20134\u00a0\u03bcM in rescued monolayers. This response was greatly attenuated in the mutant monolayers where ATP release was augmented only slightly from 0.4 to 0.5\u00a0\u03bcM. These experiments were performed after ionomycin challenge and the summary data are presented in Fig.\u00a04b. In the absence of ionomycin pretreatment (Fig.\u00a04a), mutant monolayers failed to respond to this stimulus altogether, whereas rescued monolayers responded significantly with an ATP release transient. Again, it is noted that increases in cell Ca2+ also appear to prime this mechanically sensitive ATP release mechanism. Taken together, these data suggest that the presence of a well-formed apical central monocilium is a prerequisite for robust ATP secretion to provide a meaningful autocrine ATP signal on the luminal surface of cortical collecting duct principal cells.\nFig.\u00a04Mechanically induced ATP release across the apical cell surface is markedly attenuated in mutant cilium-deficient versus genetically rescued cilium-competent orpk kidney cell monolayers. In a where no ionomycin pretreatment was performed, four mutant and four rescued cell monolayers were compared. In b where mechanical stimulation was performed after ionomycin challenge, six mutant and six rescued cell monolayers were compared. Without ionomycin, there was no response from the mutant cell monolayers. With ionomycin, there was a response from mutant monolayers that was barely significant. In contrast, rescued cell monolayers responded robustly to the addition of 50\u00a0\u03bcl of Opti-MEM 1 medium with detection reagent pipetted vigorously onto the center of the monolayer. In two additional repetitions, 50\u00a0\u03bcl of apical medium was drawn up and ejected onto the center of the monolayer before luminescence readings continued. Figure\u00a05 shows the nature of the response versus the hypotonic challenge and the ionomycin stimulation. Statistics are shown like in Fig.\u00a02\nFigures\u00a05, 6, 7, and 8 provide typical real-time courses showing the luminescence data (in arbitrary light units) for mutant and rescued monolayers illustrating the summary data presented in Figs.\u00a01, 2, 3, and 4. With the exception of basal ATP release, all phases of ATP secretion are markedly more robust in cilium-competent models versus cilium-deficient models across the apical cell surface. These data also revealed that there may be shared and different pools of \u201creleasable\u201d ATP within the cell. These data also began to reveal that increases in cell Ca2+ influence ATP secretion markedly.\nFig.\u00a05Typical real-time course of mechanically induced ATP release across the apical cell surface in mutant versus rescued cell monolayers. In this and three subsequent figures, the green (closed or solid) balls reflect data from cilium-competent cell monolayers and the white (open) balls reflect data from cilium-deficient cell monolayers. Lines do not connect the points because there are delays of a few seconds between readings. One can observe that there are clear ATP release transients induced in rescued cell monolayers by the mechanical insults. The signals observed from mutant cell monolayers are greatly attenuatedFig.\u00a06Typical real-time course of ionomycin and mechanically induced ATP release across the apical cell surface in mutant versus rescued cell monolayers. Ionomycin induces a slow monophasic sustained increase in ATP secretion that is three- to fivefold more robust in rescued cell monolayers. In the presence of ionomycin stimulation, the mechanical insults produce even more pronounced ATP secretion that is more sustained in nature when cell Ca2+ is elevated. Nevertheless, the signals observed from mutant cell monolayers remain greatly attenuatedFig.\u00a07Typical real-time course of hypotonic challenge-induced ATP release across the apical cell surface in mutant versus rescued cell monolayers. Hypotonicity induced a marked increase in ATP release that was fivefold more robust in rescued cell monolayers versus mutant cell monolayers. Of interest, however, mechanical stimulation via repeated pipetting near the center of the cell monolayers had no effect on ATP release after hypotonic cell swelling. These data suggest that both stimuli are indeed mechanical in nature and that the same pool of \u201creleasable\u201d ATP is being affected by each stimulus. The slight drop in signal with the pipetting stimulus is the addition of 50\u00a0\u03bcl of isotonic medium to the 400\u00a0\u03bcl of 50% diluted medium and the resultant change in osmotic strength at the apical cell surface. The hypotonicity-induced signal observed from mutant cell monolayers is again greatly attenuatedFig.\u00a08Typical real-time course of all three stimuli given in an order where each stimulation can be observed in mutant versus rescued cell monolayers. Although the large scale diminished the mechanically induced signal and data, an ATP release transient is observed in rescued cell monolayers and not in mutant cell monolayers. The monophasic ionomycin response is then observed, again more robust in rescued cell monolayers. Then, hypotonic challenge is performed in the presence of ionomycin. Here, the largest values of secreted ATP are observed that approach \u223c15\u201320\u00a0\u03bcM in the apical medium bathing cilium-competent cell monolayers. In these plots, the effects of the broad specificity anion transport inhibitor, DIDS, are shown. DIDS diminishes ATP release in both types of monolayers and in other epithelial cell monolayer preparations to different degrees. The inhibition is partial and DIDS does not affect the detection reagent. Hexokinase is added at the end of each experiment to eliminate secreted ATP and to diminish the luminescence signal to low levels\nAn original physiological role for the apical central monocilium was that of a \u201cmechanosensor.\u201d Figure\u00a05 validates this idea using ATP secretion as the biological endpoint. Repeated pipetting of isotonic medium containing the same amount of detection reagent revealed repeated stimulation of ATP release transients in cilium-competent monolayers. The responses in mutant cilium-deficient cell monolayers were greatly attenuated. This comparison was performed on the same day with the same preparation of luciferase:luciferin detection reagent. Figure\u00a06 shows a similar experiment but with ionomycin pretreatment. Here, the classic ionomycin-induced ATP secretion phenotype is shown. It is a slow and monophasic increase in secreted ATP that is robust; however, the response of rescued monolayers is fourfold greater than mutant monolayers. Subsequent mechanical stimulation via repeated pipetting showed a more vigorous response in the cilium-competent monolayers versus the cilium-deficient monolayers. Figure\u00a07 provides multiple compelling illustrations. First, the response to hypotonic challenge is shown and it is much more robust in rescued versus mutant cell monolayers. Interestingly, however, the repeated harsh pipetting stimulus is without effect in both rescued and mutant cells after the hypotonic challenge. In fact, the luminescence decreases modestly. These data suggest that these two mechanical stimuli may mobilize the same \u201creleasable\u201d pool of ATP. Figure\u00a08 shows the relative effects of the three stimuli used in this study on rescued cilium-competent cell monolayers and on mutant cilium-deficient cell monolayers. Although greatly diminished because of the Y-axis scale, the mechanical stimulus triggered a fourfold greater ATP secretion transient in the rescued cell monolayers versus the mutant cell monolayers. Ionomycin also produced a fourfold greater sustained ATP release in rescued versus mutant monolayers. Hypotonic challenge also triggered a more robust response in the mutant cell monolayers. A large degree of the stimulated ATP release was inhibited by the broad specificity anion transport inhibitor, DIDS, suggesting that an ATP transport process may be mediating the release of ATP from intracellular pools. In the presence of DIDS inhibition, release is inhibited while degradation of released ATP proceeds unabated, leading to a sharp decline in the signal. In contrast, ionomycin-stimulated ATP release is attenuated by performing the luminescence experiment at 4\u00b0C (data not shown), suggesting a vesicular mechanism of release. Hexokinase is added at the end of every protocol to scavenge the ATP and abolish the luminescence signal.\nTaken together, these data suggest that cell Ca2+ is critically important for priming the ATP release machinery. These data also suggest that the \u201creleasable\u201d pools of ATP are present in cilium-competent cell monolayers beneath the apical cell surface, while they may be impaired or missing in cilium-deficient cell monolayers. These ideas will be revisited and discussed below.\nFlow-induced calcium signals are attenuated in cilium-deficient mutant monolayers versus cilium-competent monolayers An original physiological role for the apical central monocilium was that of a mediator of flow- or touch-induced Ca2+ signals in MDCK cells by Praetorius and Spring [17\u201319]. Subsequent studies in heterologous cells, renal collecting duct cell models, and renal collecting ducts showed that an intact cilium is required for the flow-induced Ca2+ signal [20\u201330]. To be faithful to a polarized epithelial cell monolayer system used routinely for this study and other studies [32, 34\u201340], we devised a flow chamber where the apical versus basolateral sides of the monolayer could be perfused independently and at different flow rates. The 12-mm diameter Snapwell transwell filter can then be inserted into this chamber for selective perfusion and fluorescence imaging. With a constant low rate of perfusion of the basolateral surface of the monolayers, low versus high rates of perfusion were performed on the apical surface in rescued versus mutant monolayers.\nFigure\u00a09 shows the response to modifications in saline Na and Mg and to high flow (5\u00a0ml\/min) versus low flow (1\u00a0ml\/min). When Na and Mg are lowered to mimic the amount left in collecting duct tubular fluid after the bulk of each cation is reabsorbed along the nephron (10\u00a0and 0.1\u00a0mM, respectively), an increase in cell Ca2+ is observed that is due to Ca2+ entry from extracellular stores. The modifications in extracellular saline have augmented Ca2+ entry and cytosolic free Ca2+ in previous experience [39, 40, 47]. Ca2+ competes with Na+ through all Ca2+ entry channels that are, by definition, Ca2+-permeable nonselective cation channels [39, 40, 47]. Mg2+ blocks or impedes the pore of many different Ca2+ entry channels [39, 40, 47]. The response was greater in cilium-competent cell monolayers versus cilium-deficient cell monolayers to this Na+ and Mg2+ removal. When flow is increased, a flow-induced calcium signal is observed in cilium-competent monolayers that is reminiscent of such signals seen in isolated perfused renal collecting duct [20] and in other systems [21\u201329]. In cilium-deficient monolayers, this signal is present but impaired markedly. Taken together, these data show that cilium-competent cell monolayers are more able to respond to flow stimuli than cilium-deficient cell monolayers.\nFig.\u00a09Typical real-time courses of flow-induced Ca2+ transient signals across the apical cell surface in mutant versus rescued cell monolayers. Fluorescence ratio values were shown for Fura-2. It was very difficult to perform ionomycin-based calibrations to derive real cytosolic free Ca2+ values in this monolayer preparation that could be trusted. Moreover, the ionomycin used contaminated the perfusion lines for subsequent experiments. The flow-induced responses are observed in the center of each trace and the flow effect was reversed by returning to low flow. The use of the low Na+, low Mg2+ Ringer allowed us to view this transient more easily; it was more robust and long-lived under these ionic conditions where the Ca2+ entry channel inhibitors, Na+ and Mg2+, were minimized\nSummary data are also shown in Fig.\u00a010 that shows the responses under different saline conditions alone and responses to high flow in different conditions. Several nuances emerge from these data that agree with previous work in which our laboratory and our collaborators have been involved [20, 38]. It is important to note that our main intent was not to reproduce previously published data but to link cilium-derived Ca2+ signals to ATP secretion studied above. First, basal cell Ca2+ is indeed significantly higher in mutant cells versus rescued cells in this monolayer imaging preparation (Fig.\u00a010a). These results agree with those performed by another collaborator in a study by Siroky et al. [38]. Second and curiously, however, the basal cell Ca2+ phenotype switches in experiments performed in the absence of extracellular Ca2+ (Fig.\u00a010b). In nominally Ca2+-free solutions, mutant cell Ca2+ is driven quite low, while rescued cell Ca2+ is maintained if not slightly augmented. These data with regard to resting cell Ca2+ indeed suggest that Ca2+ entry under basal or unstimulated conditions is actually heightened in mutant cell monolayers versus rescued cell monolayers due to a deregulated Ca2+ entry pathway [38]. A valid explanation for the reversal in phenotype under Ca2+-free conditions may result from the loss of ER cisternae immediately beneath apical central monocilia, causing mutant cell Ca2+ to decrease markedly under these conditions. In contrast, cilium-competent monolayers may have robust intracellular Ca2+ stores tied immediately beneath the monocilia to maintain basal cell Ca2+ in the absence of Ca2+ entry.\nFig.\u00a010Summary data under different experimental conditions for flow-induced Ca2+ transient signals across the apical cell surface in mutant versus rescued cell monolayers: requirement for autocrine secreted nucleotides. a Summary data from the experiments shown in Fig.\u00a09. b Responses to flow in the absence of extracellular calcium. A flow-induced Ca2+ signal is still present in the cilium-competent cell monolayers. It is as robust in the peak values of Ca2+ increase (the tip of the spike is used for the summary data); however, the shoulder is absent from the transient in the absence of extracellular Ca2+. In the cilium-deficient cell monolayers, there is a slow increase in Ca2+ that is not significant and appears unrelated to flow. The asterisks in a and b refer to a P\u2009<\u20090.05 significant change from the previous stage of the experiment by paired Student\u2019s t-test. The crosses show significant difference by ANOVA between mutant and rescued cell monolayer Ca2+ levels. c The asterisks refer to a P\u2009<\u20090.05 significant change from the previous stage of the experiment by paired Student\u2019s t-test. Only data from rescued cell monolayers are shown where the Ca2+ signal is much more robust. Gadolinium and apyrase blocked the flow-induced Ca2+ transient signal, suggesting that Ca2+ entry channels and an autocrine extracellular ATP signal are required to observe the Ca2+ signal\nIn the presence of extracellular Ca2+, modification of the Ringer to mimic collecting duct tubular fluid and to disinhibit Ca2+ entry channels augmented cell Ca2+ significantly only in cilium-competent cell monolayers (Fig.\u00a010a). A change in the flow rate from 1\u00a0ml\/min to 5\u00a0ml\/min again only augmented cell Ca2+ in the form of a brief Ca2+ transient with a \u201cspike\u201d and a \u201cshoulder\u201d (Fig.\u00a010a). A typical phenotype is shown in Fig.\u00a09. Mutant cell monolayers failed to respond to flow with a significant change in cell Ca2+ (Fig.\u00a09 shows the typical degree of response to flow). In the absence of extracellular Ca2+, the same magnitude and type of Ca2+ entry \u201cspike\u201d was observed in rescued cilium-competent cell monolayers (Fig.\u00a010b). However, the sustained \u201cshoulder\u201d of this response was missing in the absence of extracellular Ca2+. Any response from the mutant cell monolayers was insignificant and sluggish. In this light, the rescued cell monolayers showed a complete reversal when flow was slowed; however, the mutant cell monolayers did not. The reason for this lack of reversal is unclear, but we speculate that the cells can no longer \u201csense\u201d flow and, therefore, display deregulation with regard to cell Ca2+ in this manner as well. In Fig.\u00a010c, responses in the rescued cell monolayers were assessed in the presence of apical gadolinium chloride, a broad spectrum inhibitor of mechanosensitive ion channels and Ca2+ entry channels [30, 38]. Infusion of gadolinium chloride during the course of the apical perfusion in the presence of extracellular calcium and in low and high flow also quieted the flow-induced calcium signal (Fig.\u00a010c). The rise in cell Ca2+ in response to the reduction of Na+ and Mg2+ was not blocked, suggesting that this Ca2+ entry mechanism is insensitive to gadolinium. These data suggest that a mechanosensitive Ca2+ entry channel is sensitive to gadolinium and plays a role in flow-induced Ca2+ entry. These data agree with the literature [17\u201330].\nMany studies, however, have also implicated autocrine and paracrine purinergic signaling as a major player in mechanically induced Ca2+ sparks and waves in monolayer and tissue preparations [33, 48\u201354]. Indeed, in immature and mature MDCK cells lacking or bearing monocilia, a pressure pulse-induced Ca2+ signal was observed in each preparation and the signal was abolished by antagonists to purinergic signaling [33]. To test whether cilium-conferred autocrine ATP release and signaling was important for the cilium-dependent Ca2+ signal, we performed a similar apical flow protocol in the presence of the ATPase\/ADPase, apyrase (Fig.\u00a010c). Apyrase blocked the flow-induced Ca2+ signal. Apyrase also attenuated the rise in cell Ca2+ induced by lowering apical Na+ and Mg2+. Taken together, these data suggest that an autocrine ATP signal, released by the monolayer itself, contributes directly to the flow-induced and cilium-derived Ca2+ signal. With the more robust stimulated ATP release phenotypes and the flow-induced Ca2+ signals in the rescued monolayers versus mutant monolayers, we speculate that each may require the apical central monocilium as an integrating sensory organelle.\nDiscussion\nFigure\u00a011 provides a cartoon that illustrates our current working hypothesis concerning the requirement of an autocrine ATP signal for monocilium-specific signaling. Our data suggest that the secreted ATP near the apical surface of principal cells of the renal collecting duct with well-formed monocilia is robust enough to engage cell surface P2X receptor channels or P2Y G protein-coupled receptors. In cilium-deficient mutant cell monolayers, the amount of ATP secreted under basal conditions or in response to stimuli may be insufficient. P2Y and P2X receptors may be present on or near the monocilium to transduce this autocrine ATP signal. Recently, in a P2X4-deficient mouse [55], flow- or shear stress-induced Ca2+ signals and resultant nitric oxide production were attenuated markedly in vascular endothelial cells versus wild-type controls. These transgenic mice also have blood pressure regulation problems [55]. Ando and colleagues had implicated ATP release and P2X4 previously in endothelial cell Ca2+ entry signaling [56\u201358]. Our laboratory has emerging data that P2X4 is expressed on the basal half or just beneath motile respiratory cilia in human respiratory tissues (L. Liang, Z. Bebok, and EM Schwiebert, unpublished observations). Silberberg and colleagues have long postulated that a \u201cP2X cilia\u201d is critical in mediating extracellular ATP regulation of ciliary beat in freshly isolated epithelial cells from airway and esophagus [59\u201361]. Our laboratory has found that P2X4, P2X5, and P2X6 have shared and robust expression in epithelia from the respiratory tract, gastrointestinal (GI) tract and renal system [35, 36], and from vascular endothelia from multiple blood vessels [37]. Work in progress is examining the expression of P2X and P2Y purinergic receptors on cilia.\nFig.\u00a011Working model: the monocilium is required for a mature ATP secretion apparatus at the apical cell surface and both the monocilium and an autocrine ATP signal are required for flow-induced Ca2+ signals\nAs introduced, it is becoming abundantly clear that the monocilium in particular and cilia and flagella in general are sensory organelles [1\u20135]. In tissues where high flow is present (large airways, proximal tubule, arteries, arterioles, etc.), the monocilium or cilia may be flow sensors for the cell on which they are present. In tissues where low flow is present (bile duct, pancreatic duct, renal collecting duct, small airways, capillaries, venules, veins, etc.), the monocilium may be a chemosensor or an osmosensor. Our data suggest that it may both influence ATP secretion as well as be a chemosensor for the secreted ATP. Arguably, secreted nucleotides and nucleosides are most potent in a local microenvironment within a cellular or tissue microenvironment. Burnstock and colleagues have described this concept elegantly in past review where they described purinergic signaling as being potent and essential in the lumen of tubules and ducts of kidney and gut [62, 63]. A semiclosed system such as the lumen of a renal tubule or duct is ideal in this regard.\nAlong these lines, the effect of changes in flow has been studied extensively and elegantly. Touch, flow, shear stress, and cell swelling are potent regulators of ion transport [64\u201367]. Ca2+ entry as well as Na+, K+, and Cl\u2212 transport are governed by mechanical stimuli [17\u201330, 64\u201367]. Mechanical stimuli are also well-known to trigger ATP release in many cell and tissue systems [48\u201354, 68]. The propagation of Ca2+ sparks and waves triggered by mechanical stimuli are thought to be mediated by at least two cellular mechanisms: (1) Ca2+-permeable gap junctions that link the cells together and (2) paracrine extracellular purinergic signaling that allows cells to communicate in a local environment [68]. In fact, our data show that an increase in cell Ca2+ primes the \u201creleasable\u201d pools of ATP that are mobilized by hypotonic cell swelling and other mechanical stimuli. However, one can still observe both pipetting- and hypotonicity-induced ATP release without ionomycin pretreatment that is more robust in cilium-competent versus cilium-deficient cell monolayers. Moreover, we also found that we could not observe a mechanically induced ATP release signal after hypotonic cell swelling. This finding suggests that these two \u201cmechanical\u201d stimuli (albeit different) affect the same pool of releasable ATP. Unfortunately, we still need better tools to define each ATP release mechanism and pool. However, our work with low temperature inhibition of vesicle traffic and inhibition of anion transport properties with the broad specificity inhibitor, DIDS, suggests that both ATP transport mechanisms and exocytosis of ATP-filled vesicles contribute to secreted ATP phenotypes (EM Schwiebert et al., unpublished observations).\nRecently, we found that ENaC-mediated Na+ absorption is upregulated markedly in cilium-deficient CCD PC monolayers derived from the Tg737orpk mouse [32]. Of many hypotheses put forward to explain this pathophysiological phenotype, one prominent postulate was that the malformed cilium caused the loss of key inhibitory signals that are normally cilium-derived that limit ENaC activity. Indeed, in several different cellular systems, there is agreement that purinergic signaling inhibits ENaC function [69\u201374]. Modulation of purinergic receptor-driven cell Ca2+ signaling may be a future putative therapeutic target (along with ENaC itself) to control hypertension that occurs in the majority of human ARPKD patients, especially the children that escape respiratory insufficiency soon after birth [6].\nFinally, it is our hope that this work connects the seemingly disparate conclusions of Satlin and coworkers [20] and those of Leipziger, Praetorius, and colleagues [33]. Taken together, our data suggest that a well-formed monocilium central to the apical membrane of a collecting duct principal cell is essential for a mature ATP secretion apparatus. One could conceive of this apparatus as a well-formed pool of ATP poised to be secreted in response to different stimuli or the appropriate presence of ATP release machinery (ATP-filled vesicles and\/or ATP transport mechanisms). Our studies also suggest that the cilium-derived Ca2+ transient, induced by flow in our study or by other modes in other studies, requires an underlying autocrine ATP signal that is likely transduced by P2X and P2Y ATP receptors on or near the monocilium. Without a well-formed cilium at the apical surface, autocrine purinergic signaling, cilium-derived signaling, and modulation of downstream effectors become disrupted.","keyphrases":["cilia","atp secretion","kidney","purinergic signaling","renal collecting duct","nucleotide secretion"],"prmu":["P","P","P","P","P","R"]}
{"id":"Antonie_Van_Leeuwenhoek-3-1-2140094","title":"New taxa of Neosartorya and Aspergillus in Aspergillus section Fumigati\n","text":"Three new species of Neosartorya and one new Aspergillus of section Fumigati are proposed using a polyphasic approach based on morphology, extrolite production and partial \u03b2-tubulin, calmodulin, and actin gene sequences. The phylogenetic analyses using the three genes clearly show that the taxa grouped separately from the known species and confirmed the phenotypic differences. Neosartorya denticulata is characterized by its unique denticulate ascospores with a prominent equatorial furrow; N. assulata by well developed flaps on the convex surface of the ascospores which in addition have two distinct equatorial crests and N. galapagensis by a funiculose colony morphology, short and narrow conidiophores and ascospores with two wide equatorial crests with a microtuberculate convex surface. Aspergillus turcosus can be distinguished by velvety, gray turquoise colonies and short, loosely columnar conidial heads. The four new taxa also have unique extrolite profiles, which contain the mycotoxins gliotoxin and viriditoxin in N. denticulate; apolar compounds provisionally named NEPS in N. assulata and gregatins in N. galapagensis. A. turcosus produced kotanins. N.denticulata sp. nov., N. assulata sp. nov., N. galapagensis sp. nov., and A. turcosus sp. nov. are described and illustrated.\nIntroduction\nAspergillus section Fumigati and its teleomorph Neosartorya include many species which are important because they can be pathogenic or allergenic to man (Brakhage and Langfelder 2002), cause food spoilage and produce mycotoxins (Cole and Cox 1981). Certain species also produce interesting bioactive extrolites that are potential drug candidates (Turner and Aldridge 1983). Section Fumigati currently includes now 26 Neosartorya species and nine anamorph species (Pitt et\u00a0al. 2000; Samson 2000; Horie et\u00a0al. 2003; Hong et\u00a0al. 2005, 2006).\nDuring a survey of Aspergillus and Penicillium species from Korea, many isolates belonging to section Fumigati were isolated. These isolates were compared to known taxa and those present at the CBS and IBT culture collections which were atypical or unidentified, using a polyphasic approach (Frisvad and Samson 2004). We have examined the macro- and micromorphology, extrolite profiles and \u03b2-tubulin, calmodulin, and actin gene sequences of the isolates, and based on the above data, here we describe four new species in Aspergillus section Fumigati.\nMaterials and methods\nMorphological examinations\nFor macro-morphological observations, isolates were cultivated on Czapek yeast autolysate (CYA), malt extract agar (MEA), CZ agar (CZA), and oatmeal agar (OA) (Samson et\u00a0al. 2004). The isolates were inoculated at three points on each plate of each medium and incubated at 25\u00b0C in the dark for 7\u00a0days, and additionally at 37\u00b0C on CYA. For microscopic observations, mounts were made in lactic acid from MEA colonies; a drop of alcohol was added to remove air bubbles and excess conidia. Scanning Electron Microscopy (SEM) was performed using a Hitachi S570 electron microscope. For SEM, mature cleistothecia were transferred to aluminum stubs with double sided adhesive tape. A small drop of 10\u00a0mM ACES buffer containing 0.05% Tween-80 was added and the cleistothecia crushed. The suspension was air dried and coated with platinum.\nDNA analyses\nIsolates used for sequence analyses are listed in Table\u00a01. Genomic DNA was extracted according to the procedure described by Lee and Taylor (1990). The 5\u2032 portion of the \u03b2-tubulin gene (benA) was amplified using primers bt2a and bt2b (Glass and Donaldson 1995). Amplifications of the partial calmodulin and actin genes were set up as described previously (Hong et\u00a0al. 2005). The amplified DNA fragments were purified by QIAquick PCR purification kits (Qiagene, Hilden, Germany). DNA sequences were determined using BigDye Terminator v3.1 Cycle Sequencing kit (ABI 0401041, Foster, CA, USA) and ABI 3100 DNA sequencer. Both strands of each fragment were sequenced using the same primers.\nTable\u00a01Aspergillus section Fumigati isolates used in this studySpeciesIsolate numberGenBank accession numberSource\u03b2-tubulinCalmodulinActinA. brevipesCBS 118.53TAF057311Soil, AustraliaA. duricaulisCBS 481.65TAF057313Soil, Buenos Aires, ArgentinaA. fumigatiaffinisIBT12703TDQ094885Soil, USAA. fumigatusCBS 133.61AY685150Chicken lung, USAA. fumisynnematusIFM 42277TAB248076Soil, VenezuelaA. lentulusCBS 117887AY738513Man, USAA. novofumigatusIBT 16806TDQ094886Soil, EcuadorA. unilateralisCBS 126.56TAF057316AY689366DQ094847Rhizosphere, AustraliaA. viridinutansCBS 127.56TAF134779Rabbit dung, AustraliaA. turcosus sp. nov.KACC 42090\u00a0=\u00a0IBT 27920DQ534142DQ534147DQ534178Air conditioner, Inchen, South KoreaKACC 42091T\u00a0=\u00a0IBT 27921DQ534143DQ534148DQ534179Air conditioner, Seoul, South KoreaKACC 41955\u00a0=\u00a0CBS 117265\u00a0=\u00a0IBT 3016DQ534144DQ534149DQ534180Car air conditioner, Seoul, South KoreaN. assulata sp. nov.KACC 41691TDQ114123DQ114131DQ534189Tomato soil, Buyeo, North KoreaN. aurataCBS 466.65TAF057318AY870685DQ534112Jungle soil, BruneiN. aureolaCBS 105.55TAF057319Soil, Tafo, GhanaN. coreanaKACC 41659TAY870758Tomato soil, Buyeo, North KoreaN. denticulata sp.nov.CBS 652.73T\u00a0=\u00a0KACC 41183DQ114125DQ114133DQ534181Soil under Elaeis guineensis, SurinameCBS 290.74\u00a0=\u00a0KACC 41175DQ114126DQ114134DQ534182Acer pseudoplatanus, NetherlandsN. fennelliaeCBS 598.74TDQ114127DQ114135DQ534121Eye ball of Oryctolagus cuniculus, USAN. fischeriCBS 544.65TAF057322Canned applesN. galapagensis sp. nov. CBS 117522T\u00a0=\u00a0IBT 16756\u00a0=\u00a0KACC 41935DQ534145DQ534151DQ534190Soil, EcuadorCBS 117521\u00a0=\u00a0IBT 16763\u00a0=\u00a0KACC 41936DQ534146DQ534152DQ534191Soil, EcuadorN. glabraCBS 111.55TAY870734AY870693DQ534183Rubber scrab from old tire, Iowa, USAN. hiratsukaeCBS 294.93TAF057324AY870699DQ534184Aloe juice, Tokyo, JapanN. laciniosaKACC 41657TAY870756Tomato soil, Buyeo, North KoreaN. multiplicataCBS 646.95TDQ114129DQ114137DQ534185Soil, Mouli, TaiwanN. nishimuraeIFM 54133AB201360Forest soil, KenyaN. nishimuraeCBS 116047DQ534075DQ534150DQ534186Cardboard, NetherlandsN. pseudofischeriCBS 208.92TAY870742AY870702DQ534187Human vertebrate, USAN. quadricinctaCBS 135.52TAF057326Cardboard, York, UKN. spathulataCBS 408.89TAF057320Soil under Alocasia macrorrhiza, TaiwanN. spinosaCBS 483.65TAF057329Soil, NicaraguaN. strameniaCBS 498.65TAY870766AY870726DQ534188Soil from maple-ash-elm forest, Wisconsin, USAN. tatenoiCBS 407.93TDQ114130DQ114139Soil of sugarcane, Timbauba, BrazilN. udagawaeCBS 114217TAF132226Soil, BrazilCBS Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands, IBT Institute for Biotechnology, Lyngby, Technical University of Denmark, IFM Institute for Food Microbiology (at present, the Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University), Chiba, Japan, KACC Korean Agricultural Culture Collection, Suwon, South Korea, T type strain\nDNA sequences were edited with the DNASTAR computer package. Sequence alignments were performed by using CLUSTAL W (Thompson et\u00a0al. 1994) and improved manually. The neighbor-joining (NJ) method was used for the phylogenetic analysis. Evolutionary distances between the sequences were calculated by Kimura\u2019s formula (Kimura 1980) using the program DNADIST of the PHYLIP program package (Felsenstein 1995). Phylogenetic trees were prepared by the NJ method (Saitou and Nei 1987) using the program NEIGHBOR of the PHYLIP package. Bootstrap values were calculated from 1,000 replications of the bootstrap procedure using programs SEQBOOT, DNADIST, NEIGHBOR, and CONSENSE of the package (Felsenstein 1995).\nFor parsimony analysis, the PAUP* Version 4.0 software was used (Swofford 2002). Alignment gaps were treated as a fifth character state and all characters were unordered and of equal weight. Maximum parsimony analysis was performed for all data sets using the heuristic search option with 100 random taxa additions and tree bisection and reconstruction (TBR) as the branch-swapping algorithm. Branches of zero length were collapsed and all multiple, equally parsimonious trees were saved. The robustness of the trees obtained was evaluated by 1,000 bootstrap replications (Hillis and Bull 1993). An Aspergillusclavatus isolate was used as outgroup in these experiments.\nThe \u03b2-tubulin, calmodulin and actin gene sequences, determined in this study, have been deposited in GenBank and the accession numbers are listed in Table\u00a01.\nAnalysis for extrolites\nThe isolates were grown at 25\u00b0C for 1\u00a0week on CYA and YES in the dark and extracted according to Smedsgaard (1997). Extrolites were analyzed by HPLC using alkylphenone retention indices and diode array UV-VIS detection as described by Frisvad and Thrane (1987), as modified by Smedsgaard (1997).\nResults and discussion\nMolecular studies\nFor the phylogenetic analysis of \u03b2-tubulin sequences, most accepted species in section Fumigati except Neosartorya indohii, N. sublevispora, and N. tsurutae were included to determine the phylogenetic positions of the putatively new species (Fig.\u00a01). For the calmodulin and actin datasets only sequences of closely related species were included (Suppl. Figs 1, 2). 468 nucleotides of the \u03b2-tubulin gene were analyzed. Among the 225 polymorphic sites, 126 were found to be phylogenetically informative. The topology of the NJ tree is the same as one of the 28 most parsimonious trees inferred by the PAUP program (length: 441 steps, consistency index: 0.7143, and retention index: 0.6993). The calmodulin data set included 538 characters, with 87 parsimony informative characters (tree length: 272, consistency index: 0.8051, and retention index: 0.7706). The actin data set included 394 characters, with 66 parsimony informative characters (tree length: 200, consistency index: 0.7900, and retention index: 0.7801).\nFig.\u00a01Taxonomic position of some new species in Aspergillus section Fumigati inferred from Neighbor-Joining analysis of partial \u03b2-tubulin gene sequences. The numbers above\/below the nodes represent bootstrap values of >60% (out of 1,000 bootstrap replications). The number of nucleotide changes is represented by branch length\nThe cladograms based on \u03b2-tubulin, calmodulin, and actin gene sequences revealed that isolates CBS 652.73 and CBS 290.74, which had identical sequences at each loci, were related to the heterothallic species N. fennelliae, but the similarity between this species and the two isolates was quite low (96.5% in the \u03b2-tubulin gene partition and 97.8\u201398.4% in the calmodulin gene partition). These two strains had unique ascospore ornamentations, with denticulate convex surfaces and a prominent equatorial furrow (Fig.\u00a02) and could be easily microscopically differentiated from any other Neosartorya species (Samson et al. 1990; Horie et\u00a0al. 2003). Both isolates produced gliotoxin, while CBS 652.73 also produced viriditoxin. Gliotoxin is also produced by A. fumigatus and N. pseudofischeri, but there were several differences in the profile of extrolites in N. pseudofischeri and these isolates (data not shown). A. fumigatus and N. pseudofischeri are among the most divergent species in the group (Geiser et\u00a0al. 1998; Horie et\u00a0al. 2003; Hong et\u00a0al. 2005, 2006; Varga et\u00a0al. 2000), yet they share the production of this mycotoxin. Here we describe CBS 652.73 and CBS 290.74 as N. denticulata sp. nov.\nFig.\u00a02Neosartoryadenticulata sp. nov. (A) colonies on OA after 28\u00a0days of incubation, (B) aspergillum, (C) ascoma, (D) ascospores under a light microscope, (E) and (F) ascospores by SEM\nIsolate KACC 41691 did not show a clear relationship to any species in the \u03b2-tubulin phylogeny, but was closest to CBS 116047 based on calmodulin and actin sequence data (Suppl. Figs 1, 2). CBS 116047 is best accommodated as N. nishimurae. However, isolate KACC 41691 is homothallic, whereas N. nishimurae is heterothallic. This isolate has similar morphological characteristics to N. pseudofischeri, but our genotypic analyses indicate that they are phylogenetically distinct. KACC 41691 produces ascospores with several large flaps and two distinct equatorial crests (Fig.\u00a03). These characteristics are similar to those of N. pseudofischeri in which the ascospores also have triangular flaps on a convex surface (Peterson 1992), but in KACC 41691 the flaps are more pronounced. Furthermore, KACC 41691 differs from N. pseudofischeri by its growth rates on MEA and CZA (after 7\u00a0days at 25\u00b0C colonies were 49\u201358 and 24\u201342\u00a0mm, respectively, for KACC 41691, and 90, and 60\u201370\u00a0mm for N. pseudofischeri). The ascomatal initials in N. pseudofischeri are characterized by many coiled hyphae whereas the initial in KACC 41691 is simpler. Ascospores are larger (5.1\u20136.0\u00a0\u03bcm in KACC 41691, while 4.5\u20135\u00a0\u03bcm in N. pseudofischeri). We could not detect any known extrolites in KACC 41691 but it produced partially characterized apolar compounds in common with several Neosartorya species. Here we propose the name N.assulata sp. nov. for isolate KACC 41691.\nFig.\u00a03Neosartoryaassulata sp. nov. (A) colonies on CYA after 7\u00a0days of incubation, (B) aspergillum, (C) ascoma, (D) ascospores under a light microscope, (E) and (F) ascospores by SEM\nIsolates CBS 117522 and CBS 117521, both isolated from soil from the Galapagos Islands, were phylogenetically distinct from all other species within section Fumigati (Fig.\u00a01, Suppl. Figs 1, 2). The colony texture of these two isolates is funiculose with conidiophores which arise from bundles of aerial hyphae. These conidiophore structures resemble as described by Horie et\u00a0al. (1993) for A. fumisynnematus, but in this species the conidophores are longer, up to 210\u00a0\u03bcm, with larger vesicles (16\u201320(25) \u03bcm in diam.). Ascomata were produced in 2\u00a0weeks-old colonies and ascospores were released after about 3\u00a0weeks. Ascospores resemble those of N. glabra and N. laciniosa, and have two conspicuous equatorial crests with a microtuberculate convex surface (Fig.\u00a04). Isolates CBS 117522 and 117521 produced gregatins and several other extrolites not yet found in other Neosartorya or Aspergillus species, and appeared to be chemically unique. Gregatins have previously been found in A. panamensis in section Sparsi (Anke et\u00a0al. 1980, 1988; Peterson 2000).\nFig.\u00a04Neosartoryagalapagensis sp. nov. (A) colonies on CYA after 7\u00a0days of incubation, (B) ascoma, (C) and (E) stipes and conidial heads arisen from hyphal bundle, (D) conidia under a light microscope, (F) ascospores under a light microscope,  (G) ascospores by SEM\nHere we describe isolates CBS 117522 and CBS 117521 as N.galapagensis sp. nov.\nIsolates KACC 42090, KACC 42091, and KACC 41955 also showed a distinct taxonomic position within section Fumigati in the three gene phylogenies. The closest taxon to these three isolates in the \u03b2-tubulin and calmodulin gene phylogenies was the heterothallic species N.nishimurae (Fig.\u00a01, Suppl. Fig.\u00a01). However, the similarity of \u03b2-tubulin sequences between the two species was only 96.9% which is close to that observed between N. fischeri and N. spinosa (data not shown). Although A. fumigatus, A. lentulus, A. viridinutans, A. fumigatiaffinis, and A. novofumigatus share similar morphological characteristics with these three isolates, these species showed comparatively low \u03b2-tubulin gene sequence similarities of 89.8, 91.6, 93.6, 92.3, and 92.7%, respectively. Isolates KACC 42090, 42091, and 41955 did not produce any teleomorph structure after incubation for 28\u00a0days on CYA, MEA, CZA, and OA at 25\u00b0C. During mating experiments, all of the pairings with N.fennelliae, N.nishimurae, N. spathulata, N. udagawae, and between conidial strains failed to yield cleistothecia. Some conidiophore characters suggest a similarity to A. fumigatus and A. lentulus, but these isolates are different from A. fumigatus by the vesicles which are fertile over the upper two-thirds and has short, loosely columnar conidial heads. These isolates are different from A. lentulus by their velvety and gray turquoise colonies. These isolates grow at 10 and 50\u00b0C on MEA and CZA. On the contrary, A. fumigatus does not grow at 10\u00b0C, while A. lentulus, A. fumigatiaffinis, and A. novofumigatus are unable to grow at 50\u00b0C (Hong et\u00a0al. 2005). Isolates KACC 42091, 42090, and 41955 were also chemically unique. The extrolites produced by the isolates described here are typical for Aspergillus section Fumigati (data not shown). The three isolates also produced kotanins, previously found in species in less obviously related groups of Aspergillus, such as A. niger from section Nigri and A. clavatus from section Clavati (Turner and Aldridge 1983). Here we describe isolates KACC 42090, KACC 42091, and KACC 41955 as A. turcosus sp. nov.\nThe list of 26 known species of Neosartorya and nine anamorph species from the section Fumigati (Horie et\u00a0al. 2003; Hong et\u00a0al. 2005, 2006) is still expanding. With the species proposed here, there are now 29 Neosartorya species and 10 Aspergillus species in this group, 39 species in total. Unfortunately, some of the recently described species are not available for the scientific community, such as N. indohii, N. nishimurae, N. otanii, N. sublevispora, N. takakii, and N. tsurutae.\nTaxonomy\nNeosartoryadenticulata Samson, S.B. Hong and Frisvad. sp. nov. (Fig.\u00a02).\nSpecies homothallica; ascomata superficialia, luteo-alba vel dilute lutea, globosa vel subglobosa, 140\u2013230\u00a0\u03bcm in diam., hyphis hyalinis vel luteolis laxe textis circumdata. Asci 8-spori, globosi vel subglobosi, 12\u201314\u00a0\u03bcm diam. Ascosporae lenticulares, 4\u20135\u00a0\u03bcm diam, denticulatae. Conidiophora ex hyphis aeriis oriunda, 3\u20134.5\u00a0\u03bcm lata; conidiorum capitula columnaria sed brevia, uniseriata, vesiculae spathulatae vel subclavatae, 7\u201312\u00a0\u03bcm diam; phialides 7.5\u20139\u00a0\u00d7\u00a02\u20133\u00a0\u03bcm, didimidium superius vesiculae occupantes. Conidia subglobosa vel late ellipsoidea, levia, 2\u20133\u00a0\u03bcm diam.\nHolotype of N.denticulata, here designated as CBS 652.73T (dried culture), isolated from soil in Suriname.\nHomothallic, cleistothecia superficial, yellowish white to pale yellow, globose to subglobose, 140\u2013230\u00a0\u03bcm in diam., surrounded by a loose covering of hyaline to yellowish white hyphae. Asci 8-spored, globose to subglobose 12\u201314\u00a0\u03bcm, evanescent at maturity. Ascospores, 4\u20135\u00a0\u03bcm, denticulate with a prominent equatorial furrow. Mycelium composed of hyaline, branched, septate, smooth-walled hyphae. Conidial heads short, columnar. Conidiophores arising from aerial hyphae, uniseriate, stipes 3\u20134.5\u00a0\u03bcm wide; vesicles spathulate to subclavate, 7\u201312\u00a0\u03bcm in diam.; phialides 7.5\u20139\u00a0\u00d7\u00a02\u20133\u00a0\u03bcm, covering the upper half of vesicle. Conidia subglobose to broadly elliptical, smooth, 2\u20133\u00a0\u03bcm. Colonies on MEA growing rapidly, 35\u201340\u00a0mm in 7\u00a0days at 25\u00b0C, white. Conidial heads produced only in colony margins. Colonies on CYA, 22\u201324\u00a0mm in 7\u00a0days at 25\u00b0C, 35\u201338\u00a0mm in 7\u00a0days at 37\u00b0C, white, loosely overgrown by aerial hyphae in center, weakly sulcate in marginal area. Conidial heads few in number. Reverse yellowish white to pale yellow (12A23) (Kornerup and Wanscher 1978).\nExtrolites: The two isolates produced the mycotoxin gliotoxin. CBS 652.73 was a particularly strong producer, and also produced the mycotoxin viriditoxin. Furthermore, the two isolates produced some unique, yet unelucidated secondary metabolites.\nAdditional isolates: CBS 290.74\u00a0=\u00a0KACC41175, from Acer pseudoplatanus, The Netherlands.\nDistinguishing features: Denticulate ascospores with a prominent equatorial furrow and the production of gliotoxin.\nNeosartorya assulata S.B. Hong, Frisvad and Samson. sp. nov. (Fig.\u00a03).\nSpecies homothallica; ascomata superficialia, alba vel luteo-alba, globosa vel subglobosa, 150\u2013250\u00a0\u03bcm diam, hyphis hyalinis vel luteolis laxe textis circumdata. Asci 8-spori, globosi vel subglobosi, 14\u201316\u00a0\u03bcm diam. Ascosporae lenticulares, 5\u20136\u00a0\u03bcm diam, duabus cristis distantibus praeditae, valvis nonnullis distinctis longis intumescentii ornamentatae. Conidiophora hyalina, 3\u20137.5\u00a0\u03bcm lata; conidiorum capitula columnaira, brevia, uniseriata; vesicula subclavata, 10\u201318\u00a0\u03bcm diam. Phialides 7\u20139\u00a0\u00d7\u00a02\u20133\u00a0\u03bcm, didimidium superius vesiculae occupantes. Conidia subglobosa vel late ellipsoidea, levia, 2\u20133\u00a0\u03bcm diam.\nHolotype of N. assulata, here designated as KACC 41691T (dried culture), isolated from soil, tomato field, Buyeo, North Korea.\nHomothallic, cleistothecia superficial, white to yellowish white, globose to subglobose, 150\u2013250\u00a0\u03bcm in diam. Asci 8-spored, globose to subglobose 14\u201316\u00a0\u03bcm, evanescent at maturity. Ascospores lenticular, spore body 5.0\u20136.0\u00a0\u03bcm, with two well-separated equatorial crests and convex surface decorated with several large, round flaps. Mycelium composed of hyaline, branched, septate, smooth-walled hyphae. Conidial heads short, columnar. Conidiophores arising from aerial hyphae and substrate, 3\u20137.5\u00a0\u03bcm wide; vesicles subclavate, 10\u201318\u00a0\u03bcm in diam., uniseriate, phialides 7\u20139\u00a0\u03bcm, covering the upper half of vesicles. Conidia, subglobose to broadly elliptical, ovoid, smooth, 2\u20133\u00a0\u03bcm. Colonies on MEA, 49\u201358\u00a0mm in 7\u00a0days at 25\u00b0C, white, radially weak sulcate. Conidial heads aerial, numerous. Colonies white on CYA, 37\u201341\u00a0mm at 25\u00b0C, 64\u201368\u00a0mm at 37\u00b0C in 7\u00a0days. Radially and roundly sulcate, with some clear exudates. Conidial heads aerial, abundant. Reverse yellowish white (1A2) to pale yellow (1A3) (Kornerup and Wanscher 1978).\nExtrolite profile: This species is characterized by relatively weak production of secondary metabolites. It does produce some indole alkaloids and some apolar metabolites.\nDistinguishing features: Large, round flaps on convex surface of ascospores with two distinct equatorial crests.\nNeosartorya galapagensis Frisvad, S.B Hong and Samson. sp. nov. (Fig.\u00a04).\nSpecies homothallica; ascomata luteo-alba, globosa vel subglobosa, 90\u2013200\u00a0\u03bcm diam,. hyphis hyalinis vel luteolis laxe textis circumdata. Asci 8-spori, globosi vel subglobosi, 12\u201315\u00a0\u03bcm diam; ascosporae late lenticulates, ca. 5\u00a0\u03bcm diam, duabus cristis distantibus 1\u20132\u00a0\u03bcm latis praeditae, valvis exigue tuberculatis. Conidiophora singula vel funiculosa, levia, 2\u20134\u00a0\u03bcm lata; conidiorum capitula columnaria, brevia, uniseriata; vesiculae subclavatae, 4\u201311\u00a0\u03bcm diam. Phialides lageniformes, 5\u20137\u00a0\u00d7\u00a02\u20133\u00a0\u03bcm, dimidium superius vesiculae occupantes. Conidia globosa vel subglobosa, levia vel exigue asperulata, 2.3\u20133.0\u00a0\u03bcm diam.\nHolotype of N.galapagensis, here designated as CBS 117522T (IBT 16756\u00a0=\u00a0KACC 41935) (dried culture), isolated from soil, Galapagos Islands, Ecuador, D. Mahooney.\nColonies on MEA 28\u201335\u00a0mm in diam. after 7\u00a0days at 25\u00b0C and more than 70\u00a0mm after 7\u00a0days at 37\u00b0C, funiculose in texture, yellowish white (3A12). Conidiophores sparse, cleistothecial initials produced after ca. 10\u00a0days of incubation. Colonies on CYA 27\u201340\u00a0mm in diam. after 7\u00a0days at 25\u00b0C and 61\u201365\u00a0mm after 7\u00a0days at 37\u00b0C, strongly funiculose in texture and white with a golden yellow (5B78) reverse without diffusible pigment. Conidial heads columnar. Conidiophores arising from bundles of aerial hyphae or the basal mycelium, smooth walled, up to 100\u00a0\u03bcm long, 2\u20134\u00a0\u03bcm in width; vesicles 4\u201311\u00a0\u03bcm (sub)clavate with 5\u20137\u00a0\u03bcm flask-shaped phialides which are fertile on the upper half to two-thirds of the surface. Conidia 2.3\u20133.0\u00a0\u03bcm, globose to subglobose and the surface usually smooth. Cleistothecia yellowish white (4A2), globose to subglobose, 90\u2013200\u00a0\u03bcm in diam., surrounded by a loose covering of aerial hyphae. Peridium consisting of angular cells, 3\u20138\u00a0\u03bcm in diam.; asci 8-spored, globose to subglobose, 12\u201315\u00a0\u03bcm in diam.; ascospores broadly lenticular, spore body ca. 5\u00a0\u03bcm in diam. with two distinct equatorial crests 1\u20132\u00a0\u03bcm wide, convex surface of ascospores microtuberculate.\nExtrolite profile: All isolates examined in the species produce several gregatins and several partially characterized secondary metabolites. This species is chemically very distinct and different from the other species in section Fumigati or Neosartorya species. The gregatins have also been found in A. panamensis (Anke et\u00a0al. 1980, 1988). The latter species was placed in Aspergillus section Usti by Raper and Fennell (1965).\nAdditional isolates: CBS 117521\u00a0=\u00a0IBT 16763\u00a0=\u00a0KACC 41936, ex soil, Galapagos Islands, Ecuador.\nDistinguishing features: The Aspergillus anamorph arising in bundles of aerial hyphae and the ascospores with two wide conspicuous equatorial crests and microtuberculate convex surface.\nAspergillus turcosus S.B. Hong, Frisvad and Samson. sp. nov. (Fig.\u00a05).\nFig.\u00a05Aspergillus turcosus sp. nov. (A) and (B) colonies on MEA (A) and CYA (B) after 7\u00a0days at 25\u00b0C, (C) conidia under a light microscope, (D) conidial heads\nColoniae in agaro maltoso ad 42\u201351\u00a0mm diam post-seven dies 25\u00b0C, 70\u00a0mm diam 37\u00b0C. Coloniae velutinae, griseo-glaucae vel griseo-virides, plerumque planae; reversum luteo-aurantium vel griseo-aurantium. Conidiophora levia, 4\u20137\u00a0\u03bcm lata. Conidiorum capitula columnaria, brevia, uniseriata; vesiculae globosae vel subclavatae, 15\u201325\u00a0\u03bcm diam. Phialides lageniformes, 6\u20138\u00a0\u00d7\u00a02\u20133\u00a0\u03bcm, duo tertia superiora vesiculae occupantes. Conidia subglobosa vel ovoidea, levia, 2.5\u20133.5\u00a0\u03bcm diam.\nHolotype of A. turcosus, here designated as KACC 42091T (=IBT 27921) (dried culture) isolated from home air conditioner, Seoul, South Korea.\nColonies on MEA 42\u201351\u00a0mm in diam. after 7\u00a0days at 25\u00b0C and more than 70\u00a0mm after 7\u00a0days at 37\u00b0C. Colony texture velvety, gray-turquoise to gray-green (24-25B3-5) and usually plane. In reverse, colonies are yellowish orange (4B6) to grayish orange (5B6). Colonies on CYA attain a diam. of 38\u201341\u00a0mm after 7\u00a0days at 25\u00b0C and more than 70\u00a0mm after 7\u00a0days at 37\u00b0C. Colony texture is velvety. Colony texture and color similar to that on MEA. In reverse, colonies are deep yellow (4A78). Conidial heads loose and short columnar. Conidiophores smooth-walled, up to 80\u00a0\u03bcm long and 4\u20137\u00a0\u03bcm wide. Vesicles are 15\u201325\u00a0\u03bcm in diam., flask-shaped to globose, bearing 6\u20138\u00a0\u03bcm flask-shaped phialides over two-thirds of the surface. Conidia are subglobose, ovoid and smooth, 2.5\u20133.5\u00a0\u03bcm in diam.\nExtrolite profile: Kotanins and several unique compounds but not yet elucidated secondary metabolites.\nAdditional isolates: KACC 42090\u00a0=\u00a0IBT 27920, KACC 41955\u00a0=\u00a0IBT 3016.\nDistinguishing features: Velvety colony, gray-turquoise (green) color on CYA, phialides over two-third of the vesicle and growth at 10 and 50\u00b0C are distinctive characteristics of the species.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\n(DOC 19 kb)","keyphrases":["extrolites","\u03b2-tubulin","calmodulin","actin","neosartorya denticulata","n. assulata","n. galapagensis","aspergillus turcosus","dna sequencing"],"prmu":["P","P","P","P","P","P","P","P","P"]}
{"id":"Ann_Surg_Oncol-4-1-2277449","title":"Isolated Hypoxic Hepatic Perfusion with Retrograde Outflow in Patients with Irresectable Liver Metastases; A New Simplified Technique in Isolated Hepatic Perfusion\n","text":"Background Isolated hepatic perfusion with high-dose chemotherapy is a treatment option for patients with irresectable metastases confined to the liver. Prolonged local control and impact on survival have been claimed. Major drawbacks are magnitude and costs of the procedure. We developed an isolated hypoxic hepatic perfusion (IHHP) with retrograde outflow without the need for a heart-lung machine.\nApproximately 50\u201360% of colorectal cancer patients will develop liver metastases during follow-up. In nearly a quarter of these patients the liver is the only site of disease.1 If hepatic metastases of colorectal cancer are resectable, 5-year survival rates are reported between 25% and 45% depending on several prognostic factors.2 Patients with irresectable hepatic metastases have a 0\u20132% 5-year survival rate and a median survival of 10\u00a0months without treatment.3 Therefore, aggressive, selective treatment of the liver seems justified since control of hepatic metastases translates into improved overall survival. Despite the increasing response rates with systemic chemotherapeutic agents such as oxaliplatin and irinotecan in combination with 5-FU and the promising results with the addition of angiogenesis inhibitors,4 overall survival remains poor.5,6 In order to improve responses and survival, locoregional chemotherapeutic regiments have been developed. These therapies include hepatic arterial embolization, intratumoral injections of ethanol, acetic acid, biological agents, stereotactic or intra-arterial radiotherapy, intralesional laser therapy, cryotherapy, radiofrequency ablation, and regional infusion or perfusion of chemotherapeutic drugs. The best approach for regional infusion of chemotherapeutics in the liver is unknown. Hepatic artery infusion (HAI), hepatic artery ligation with hepatic artery and portal vein infusion, or portal vein infusion have all been attempted. Of these modalities, HAI is the most widely applied form. A number of studies have been conducted comparing systemic chemotherapy with HAI, and a modest but significant improvement of survival was demonstrated by HAI in a meta-analysis.7\u20139\nIn a leakage-free perfusion setting, isolated hepatic perfusion (IHP) shields the systemic compartment to drug exposure, and in combination with a washout procedure it protects against systemic toxicity. Clinical studies using melphalan with or without tumor necrosis factor alpha (TNF) have shown promising results.10\u201317 However, IHP is a major, complex, expensive, and time-consuming operation. These features in combination with hepatic and systemic toxicity are major drawbacks toward wide clinical application. Our aim was to develop an IHP that is easy to perform, with limited morbidity or mortality, not time consuming, and with minimal costs of the operation without affecting the antitumor efficacy. We started an isolated hypoxic hepatic perfusion (IHHP) technique with percutaneously placed balloon catheters and retrograde outflow without a heart-lung machine and extracorporeal venovenous bypass in pigs.18 In a recent study, we demonstrated the feasibility of this technique (retrograde outflow of the liver) in a clinical setting.17 A major drawback of the technique was 38% systemic leakage rate. On the basis of these results, we modified the technique by applying an \u201copen\u201d surgical procedure; balloon catheters are replaced by clamps and all tributaries to the vena cava such as the adrenal, lumbar, and diaphragmatic veins are dissected and ligated. Here we present the results of the first 24 patients with irresectable liver metastases who underwent an IHHP with melphalan. The major difference between this new technique and the classic IHP is that there is no veno-veno bypass and heart-lung machine involved and the drainage of the liver is retrograde through the portal vein.\nMATERIALS AND METHODS\nPatient Selection Criteria\nInclusion criteria were as described previously.17 In all patients a radical resection of the primary tumor was performed prior to entering the study protocol except for one patient with hepatic metastasis of unknown primary. The liver metastases were considered irresectable, on the basis of multiple lesions in multiple segments of the liver and\/or location near vascular structures. Tumor involvement had to be less than 50% of the total liver volume to prevent massive necrosis in case of a response. Absence of extra hepatic tumor growth was evaluated by computed tomography (CT) scan of thorax and abdomen. All patients had a Karnofsky performance score of at least 90, liver enzymes (ALAT, ASAT and AF) not higher than five times the normal values, and bilirubin not higher than two times the normal values. Exclusion criteria included: age younger than 18 or older than 70, portal hypertension, significant central nervous system disease, significant cardiovascular, pulmonary or renal disease, uncontrolled infections, presence of organ grafts, and chemotherapy or radiation therapy within 4\u00a0weeks prior to the IHHP. Routinely, an angiography was performed to exclude aberrant hepatic arteries or to visualize other anatomic anomalies. The study protocol was approved by the Medical Ethical Committee of the Erasmus University Medical Centre, and written informed consent was obtained from all patients.\nPerfusion Circuit\nPerfusion sets (PfM, GmbH, Cologne, Germany) consisted of a tubing set with a volume of 220\u00a0mL, containing a bubble trap. All IHHPs were performed with inflow via the hepatic artery and outflow via portal vein. In the perfusion circuit, flow was maintained by a roller pump and pressure was measured via a sideline.\nDrugs\nA dosage of 1\u00a0mg\/kg melphalan (L-Pam, Alkeran, Wellcome Ltd. London, UK) was used in 20 of the 24 patients and infused through a sideline into the perfusion circuit. Two times 1.5\u00a0mg\/kg melphalan and two times 2.0\u00a0mg\/kg was used. In the first eight patients, a bolus infusion was used. In the last 16 patients, a 10-minute pump infusion was used. The total perfusion (10-minute infusion included) with melphalan was conducted for 20 to 30\u00a0minutes.\nSurgical Procedure of the Retrograde Flow IHHP\nA right subcostal incision was performed, and the liver was mobilized from its retroperitoneal and diaphragmatic attachments. A prophylactic cholecystectomy was not performed routinely. Tributaries to the vena cava such as the adrenal, lumbar, and diaphragmatic veins were dissected and ligated. The vena cava was isolated and clamped above and below the liver, respectively, to prevent venous leakage. The portal vein, proper hepatic artery, and gastroduodenal artery were dissected, and the hepatic artery cannulated via the gastroduodenal artery with an 8 F catheter for inflow of the perfusate. In two patients, an aberrant left hepatic artery, coming from the left gastric artery was cannulated together with the proper hepatic artery for inflow. The portal vein was cannulated with a 14 F catheter for outflow. Patients subsequently received 2\u00a0mg\/kg heparin. The hepatic artery catheter and the portal vein catheter were connected to the perfusion circuit primed with 220\u00a0mL Haemaccel (Behring Pharma, Amsterdam, The Netherlands). After clamping of the caval vein superior and inferior of the liver, clamping of the aorta just beneath the diaphragm, clamping the portal vein just above the pancreas, clamping the proper hepatic artery just proximal of the gastroduodenal branch, and clamping of the common bile duct with its surrounding tissue, the retrograde isolated perfusion was performed. The retrograde perfusion setup is depicted in Fig.\u00a01.\nFIG.\u00a01.The retrograde perfusion setup.\nThe perfusate was circulated by a constant flow. Stable perfusion was monitored by pressure measurement and the perfusate level in the bubble trap. Hereafter, melphalan was infused into the circuit and the perfusion was conducted for 20 to 30\u00a0minutes. Thereafter, a washout procedure was performed by 1\u00a0L of Haemaccel collecting the venous effluent. Total liver ischemia time never exceeded 60\u00a0minutes.\nThe isolation was terminated by relief of the clamps on the caval vein followed by the relief of the aortic clamp, controlling systemic blood pressure. The catheter in the hepatic artery could be removed and the gastroduodenal artery could be ligated followed by decanulation and closing the venotomy of the portal vein. In case of an aberrant left hepatic artery, this artery was ligated after removal of the catheter.\nLeakage Monitoring\nDuring IHHP, potential leakage of drugs was monitored using a radioactive tracer. A small calibration dose of human serum albumin radiolabeled with 131I was injected into the systemic circulation prior to the perfusion and 10-fold higher dose of the same isotope was injected into the isolated hepatic perfusion circuit. Continuous monitoring was performed with a precordial scintillation probe. Systemic leakage is expressed quantitatively as a percentage (100% leakage representing a homogeneous distribution of the isotope in the body).14\nBlood Sampling\nBefore, during, and after the perfusion blood samples were taken and collected to study pharmacokinetics of melphalan and hematological, renal, hepatic, and gastrointestinal toxic side effects. Toxicity is graded according to the standardized WHO common toxicity criteria.19\nMeasurement of Melphalan Concentrations\nMelphalan was measured in plasma by gas chromatography\u2013mass spectrometry (GC-MS). P-[Bis(2-chloroethyl)amino]-phenylacetic acid methyl ester was used as an internal standard. Samples were extracted over trifunctional C18 silica columns. After elution with methanol and evaporation, the compounds were derivatized with trifluoroacetic anhydride and diazomethane in ether. The stable derivates were separated on a methyl phenyl siloxane GC capillary column and measured selectively by single ion monitoring GC-MS in the positive EI mode described earlier by Tjaden et al.20\nAssessment of Tumor Response\nThis was done by comparing pre-perfusion CT and\/or MRI scans of the liver with scans made at 8\u201310\u00a0weeks after IHHP. The tumor marker carcinoembryonic antigen (CEA) was monitored (when indicated) preoperatively and 8\u201310\u00a0weeks postperfusion, but was not used for response assessment. Clinical responses are assessed by standardized WHO criteria.19\nRESULTS\nPatient Characteristics (Table\u00a01)\nIn a total of 24 consecutive patients, 13 males and 11 females with a median age of 57\u00a0years (range, 41\u201370) were included in the protocol. Eighteen patients had irresectable metastases of colorectal origin, four ocular melanomas, one sarcomas, and one unknown primary hepatic metastases.\nTABLE\u00a01.Characteristics of 24 patients with irresectable liver metastases treated by IHHP with retrograde outflow via portal vein with melphalanPTTumor primaryProgressive on chemotherapyToxicityResponse after perfusionTime to progr. (months)StatusMonths after perfusionHepatoBloodRenal1CRCNo100CR10Alive462CRCNo100PR24Dead383CRCYes140SD12Dead424CRCNo100PR6Dead295CRCNo130PR20Dead326EMNo100PR5Dead97SARCNo130PR13Dead178CRCNo200PR9Dead99CRCYes100SD12Dead3710CRCYes100PD2Dead1011CRCNo100PR9Dead2912CRCNo100PD9Dead1813CRCNo100PD2Dead814CRCYes100PR4Dead1815EMNo100PR5Dead616CRCNo120PR9Alive2917UPYes130PD2Dead518EMNo100PR9Dead2019CRCNo220PR4Dead620CRCNo101PR5Dead521EMNo120PR13Dead1522CRCYes100PD4Dead623CRCYes100SD5Dead624CRCYes100PD9Alive11PT, patient number; CRC, colorectal cancer; CR, complete response; SARC, sarcoma; PR, partial response; EM, eye melanoma; SD, stable disease; UP, unknown primary; PD, progressive disease.\nOperation Characteristics\nMedian operation time was 240\u00a0minutes (150\u2013300\u00a0minutes), including perfusion time. Median blood and fluid loss was 900\u00a0mL (300\u20133200\u00a0mL). In the first eight patients, melphalan was administered as a bolus into the isolated liver circuit. The last 16 patients received a 10-minute pump infusion of melphalan. The perfusion (10-minute infusion included) with melphalan was conducted for 20 to 30\u00a0minutes. Perioperative mortality (<90\u00a0days) was 0%. Median hospital stay was 9\u00a0days (5\u201329\u00a0days).\nLeakage Control\nAll IHHPs were leakage free during the perfusion except in one patient, where 2% systemic leakage was measured. When this perfusion was finished, it appeared that in this patient there was an insufficient clamping of the suprahepatic caval vein. After washout of the isolated circuit with Haemaccell, a median of 7% (4\u201310%) leakage into the systemic circuit was demonstrated by continued monitoring until the end of the operation.\nToxicity Study (Table\u00a01)\nHepatic toxicity consisted mainly of a transient rise of liver enzymes during the first week after IHHP. WHO grade I occurred in 92% of the patients (22 of 24) and WHO grade II in 8% of the patients (2 of 24). Three patients experienced bile duct necrosis (discussed in detail in the section \u201cComplications\u201d). No other hepatic toxicities occurred (no coagulopathy was observed). Systemic toxicity was mainly leucopenia, WHO grade I\u2013III in 25% (6 of 24), and severe grade IV leucopenia in one patient (4%) after perfusion. Two of these patients received G-CSF (Neupogen, Amgen B.V., Breda, The Netherlands). One patient received prophylactic G-CSF. Eighteen patients (71%) did not develop leucopenia. No renal (except for one patient grade 1) or gastrointestinal toxicity was observed.\nMelphalan Pharmacokinetics\nFigure\u00a02 shows a drug concentration vs time curve in the isolated circuit in a patient with a pump infusion. The area under the concentration versus time curve (AUC) calculation showed a regional concentration of 2382 (\u03bcg\u00a0\u00d7\u00a0min\/mL) versus undetectable systemic that makes the ratio AUC regional\/systemic infinite (Fig.\u00a0II). The median peak regional melphalan concentration was 68.04 \u03bcg\/mL (range, 42.3\u2013256.99) and negligibly low to undetectable systemic concentrations. After washout and subsequent releasing of the clamps, median peak systemic 4.2 \u03bcg\/mL (range, 3.00\u201318.33) melphalan concentrations were observed. The AUC calculation showed a systemic concentration of 275 \u03bcg\u00a0\u00d7\u00a0min\/mL after washout. Thus, intrahepatic melphalan concentrations during the IHHP are >9 fold higher than the post-IHHP systemic melphalan concentrations.\nFIG.\u00a02.Drug-concentration versus time curve.\nComplications\nIn three patients, bile duct necrosis occurred (Fig.\u00a03). One of these patients was asymptomatic, and bile duct necrosis was found on routine CT scan during follow-up. Two of the patients with bile duct necrosis developed fever. No other explanation could be found for the fever, besides the intrahepatic \u201cbilomas,\u201d and therefore one patient was treated with percutaneous drainage and endoscopic stent placement in ductus choledochus. The other patient with fever received antibiotics. Both patients recovered, and slight elevations of bilirubin persisted, without complaints. In the first eight patients melphalan was administered as a bolus into the isolated liver circuit. After four patients melphalan dose was increased to 1.5\u00a0mg\/kg in two patients and to 2\u00a0mg\/kg melphalan in another two patients. Three of these patients (37.5%) developed bile duct necrosis, which is why the other 16 patients were treated with 1\u00a0mg\/kg melphalan using a 10-minute pump infusion into the circuit. Bile duct necrosis did not occur in these patients. Postoperative chemical cholecystitis was not demonstrated.\nFIG.\u00a03.Intra-hepatic bile duct necrosis after IHHP\nTumor Response and Patient Survival (Table\u00a01)\nIn one patient a complete response was observed (4%). Partial response (PR) was seen in 14 patients (58%), and stable disease (SD) was demonstrated in three patients (13%). Overall, a clinical response rate of 62% and a clinical benefit of nonprogression in 75% were observed. Six patients developed progressive disease (25%). Progressive disease occurred with median interval after IHHP of 9\u00a0months (range 2\u201324\u00a0months). Sixteen patients had progression of the liver tumors during follow-up. Fourteen patients developed extrahepatic disease during follow-up. To analyze the response rates and survival, it may be more opportune to include only the 18 patients with colorectal liver metastases. Median follow-up was 18\u00a0months. Median patient survival for patients with colorectal liver metastases (n\u00a0=\u00a018) was 18\u00a0months (range, 5\u201346\u00a0months). Median time to progression was 9\u00a0months (2\u201316\u00a0months). Seven of these 18 patients experienced failures of systemic chemotherapy. Two patients (PtNo 3 and 23) were progressive on the combination of 5-fluorouracil (5FU) and oxaliplatin (FOLFOX) (10 and 6 cycles, respectively), two patients (PtNo 9 and 10) were progressive on 5-FU and leucovorine (22 and 16 cycles, respectively), one patient (PtNo 14) was progressive after Xeloda (four cycles) and irinotecan (four cycles), one patient (PtNo 24) after six cycles of FOLFOX and four cycles of irinotecan, and one patient (PtNo 22) was progressive after Xeloda (six cycles), irinotecan (six cycles), and combination of Xeloda and oxaliplatin (XELOX) (eight cycles). Median survival for patients with colorectal liver metastases, not treated with chemotherapy before IHP (n\u00a0=\u00a011), was 29\u00a0months (range, 5\u201346\u00a0months).\nDISCUSSION\nIsolated liver perfusions are only performed by a few centers worldwide, demonstrating promising antitumor effects; however, the technique is a major, complex, expensive, and time-consuming operation. In the present study we describe an alternative technique using a retrograde hepatic flow in an isolated hypoxic hepatic perfusion (IHHP) system. The technique was performed in 24 patients and demonstrated to be safe, with minimal morbidity and no morbidity. Overall response rate was 62% with no progression in 75%, and median time to progression of disease was 9\u00a0months.\nThe magnitude of the standard isolated liver perfusion technique is well demonstrated in two recently published series by the centers with the largest experience in IHP (NCI, Bethesda, MD, USA, and LUMC, Leiden, The Netherlands, respectively).11,15 Median IHP operation time was reported to be more than 8\u00a0hours (range, 5.8\u201314.7\u00a0hours). In the study of Bartlett et al.11 the estimated blood loss was 2.2\u00a0L (range, 0.8\u20134.5\u00a0L), and Rothbarth et al.15 reported a range of 0.9\u201310\u00a0L (median not given, older series mean of 4.4\u00a0L). Mortality (<30\u00a0days) occurred in both series with 5.6% in the Leiden series (n\u00a0=\u00a071) and 4% in the NCI series (n\u00a0=\u00a0124). Classical IHP uses a venovenous bypass and a heart-lung machine, which is a time-consuming procedure necessitating a specialized perfusion team. In the present study a modification was developed, allowing a safe perfusion in a shorter operation time (median duration, 4\u00a0hour), with less blood loss (median, 900\u00a0cc), reducing morbidity and without mortality, without the need of a heart-lung machine or venovenous bypass.\nThe absence of mortality (<90\u00a0days) in the present study can be explained by the smaller number of patients and\/or patient selection. However, it may well be related to the smaller magnitude of the operation and the corresponding decreased operation time and blood loss. Median hospital stay was 9\u00a0days (range, 5\u201329), which was also shorter than the 12 and 11\u00a0days in the aforementioned studies.11,15\nHepatic toxicity is remarkably low in the present series except for three patients who developed bile duct necrosis (Table\u00a01). High peak concentrations of melphalan in the isolated circuit might explain the bile duct necrosis that was observed in patients who were perfused with higher melphalan concentrations. In a preclinical study, the occurrence of severe cholangiofibrosis was strongly dependent on the melphalan concentration.21 The study also demonstrated that tumor response was not affected by melphalan peak concentration, but by the total dose of melphalan. Therefore, to prevent high peak concentrations keeping the same concentration of melphalan, bolus infusions were replaced by a 10-minute pump infusion. This resulted in the absence of bile duct necrosis in the last 16 perfused patients.\nThe maximum period of 60-minute hypoxia did not seem to result in extrahepatic and\/or systemic toxicity. Moreover, clamping of the aorta just underneath the diaphragm for a maximum of 60\u00a0minutes did not lead to renal and\/or gastrointestinal morbidity.\nTo overcome the use of a venovenous bypass and heart-lung machine, we performed a hypoxic perfusion. The use of hypoxia might also enhance the antitumor effect. Hypoxia renders tumor cells more sensitive to cytostatic agents in general and enhances the antitumor effects of drugs such as melphalan.22,23 Changing the perfusion direction may reduce liver toxicity, without affecting the antitumor efficacy as described by Rothbarth et al.24 An animal study demonstrated an unaffected tumor uptake and a reduced liver uptake by 80% in an IHP model with arterial inflow and retrograde portal outflow.25 This may explain our remarkably low hepatic toxicity in the present series using retrograde outflow in a clinical setting.\nIn the large IHP series of patients with colorectal liver metastasis, response rates of 74% and 59%,11,15 respectively, are reported, which is in line with the response rate in the present study. The median time to progression in these IHP studies was 14.5 and 7.7\u00a0months, respectively. The time to tumor progression of 9\u00a0months after IHHP seems to be comparable to these numbers. Alexander et al. published their results in patients with progressive disease after irinotecan-based therapies, and still a response rate of 60% was obtained after IHP with melphalan.16 The 2-year survival was 28% with a 12-month median survival. Although interpretation and comparison of the results from these different studies are difficult, the results of the retrograde IHHP technique described here are similar. In the past 10\u00a0years, new anticancer agents (oxaliplatin and irinotecan) have increased the response and survival rates. The addition of targeted therapies (cetuximab and bevacuzimab) may increase the response rates more than 70%. Especially in this era of exciting developments, tailored treatment to increase individual survival rates while keeping the toxic effects to a minimum is challenging. The role of IHP in colorectal liver metastases has been discussed in recent reviews.24,26,27 The promising results of the IHP studies in the past years cannot be disregarded. It is conceivable to combine IHP with adjuvant treatment strategies to reduce the recurrence rate, which is the major determinant of the survival rate after IHP. Continuing locoregional treatment by HAI after an IHP procedure is technically feasible and appears to prolong the duration of the response and survival in patients with colorectal hepatic metastases.11 Of the 18 patients with colorectal liver metastases in the present series, 10 developed extrahepatic disease, suggesting the need for additional systemic treatment. Duration of tumor control needs to be improved. To achieve this, the addition of vasoactive drugs, identified in our laboratory to have synergistic activity in combination with melphalan by significantly increasing drug uptake in the metastases, will be explored in further studies.28,29 At this time, IHHP should be reserved for those patients who are progressive on standard chemotherapy protocols. When responses in future trials are improved, this technique might be optional for patients who prefer a single procedure, which could potentially lead to similar results as multiple courses of systemic or intra-arterial chemotherapy. Leaving behind at the end of the IHHP a port-a-cath system for subsequent HAI in case of (re)progression should also be considered as a strategy to further improve prolonged locoregional tumor control.\nIn ocular melanoma, the liver is the only organ site involved in 70\u201380% of patients and is usually responsible for death.30,31 The prognosis of a patient with hepatic metastases from uveal melanoma is extremely poor, with a median survival between 2 and 5\u00a0months.32 Partial liver resection is seldom possible. The therapeutic options are not very effective with systemic chemotherapy, providing 9% partial responses and no survival benefit.33,34 There is limited experience with IHP for hepatic metastases from uveal melanoma, but the response rates are promising.35\u201337 Despite the fact that the experience with hepatic metastases from uveal melanoma is small in the present study (n\u00a0=\u00a04), IHP seems to be an effective treatment. All four patients died because of progressive tumor growth and consequent liver failure after 6, 9, 15, and 20\u00a0months. Therefore, especially in this group of patients, local tumor control is likely to result in a survival benefit.\nCONCLUSIONS\nIsolated hypoxic hepatic perfusion with retrograde outflow via portal vein is a promising technique that is a relatively simple procedure with reduced blood loss, no mortality, very limited toxicity, and response rates comparable to classic, extensive IHP. The operation time is reduced to 4\u00a0hours, without the need of a heart-lung machine and perfusionists, which reduces the costs.","keyphrases":["hypoxic","retrograde outflow","isolated hepatic perfusion","melphalan","metastasis"],"prmu":["P","P","P","P","P"]}
{"id":"Pharm_World_Sci-3-1-2082656","title":"When pharmacotherapeutic recommendations may lead to the reverse effect on physician decision-making\n","text":"For long the medical literature has shown that patients do not always receive appropriate care, including pharmacotherapeutic treatment. To achieve improved patient care, a number of physician-oriented interventions are being delivered internationally in an attempt to implement evidence based medicine in routine daily practice of medical practitioners. The pharmacy profession has taken an active role in the delivery of intervention strategies aimed at promoting evidence based prescribing and improved quality and safety of medicine use. However, the medical literature also supports the notion that valid clinical care recommendations do not always have the desired impact on physician behaviour. We argue that the well-established theory of psychological reactance might at least partially explain instances when physicians do not act upon such recommendations. Reactance theory suggests that when recommended to take a certain action, a motivational state compels us to react in a way that affirms our freedom to choose. Often we choose to do the opposite of what the recommendation is proposing that we do or we just become entrenched in our initial position. The basic concepts of psychological reactance are universal and likely to be applicable to the provision of recommendations to physicians. Making recommendations regarding clinical care, including pharmacotherapy, may carry with it implied threats, as it can be perceived as an attempt to restrict one\u2019s freedom of choice potentially generating reactance and efforts to avoid them. By identifying and taking into account factors likely to promote reactance, physician-oriented interventions could become more effective.\nIt is well known that the use of medicines often results in avoidable complications or adverse events. In the United States a study showed that approximately 60% of iatrogenically related admissions to intensive care units were drug related and 75% were preventable [1]. In Australia, adverse medicine events are responsible for 1.6% of all hospital admissions [2].\nTo achieve improved quality and safety of medicine use, a number of physician-oriented interventions are being delivered internationally in an attempt to improve evidence based prescribing in routine daily practice of medical practitioners\u2014e.g., distribution of educational materials, decision support systems, outreach visits, medication reviews, etc.\nThe pharmacy profession has taken an active role in the delivery of intervention strategies related to pharmacotherapy. For example, in medication review programmes, pharmacists interview patients, conduct assessments of information gathered on patients\u2019 use of medicines and prepare reports for physicians, which may contain pharmacotherapeutic recommendations [3]. Pharmacists also are typically involved in educational outreach programmes. In such programmes it is common for pharmacists to take up the role of educator, visiting physicians in their office to provide educational interventions on specific pharmacotherapeutic topics. As pharmacists and physicians share expertise in pharmacotherapy, their involvement in intervention strategies aimed at promoting quality and safety of medicine use makes sense. Indeed, the literature has reported positive results on patient health outcomes arising from such interventions [4, 5].\nHowever, despite the delivery of interventions designed to foster quality and safety of medicine use, the literature provides support for the notion that, at times, despite physicians agreeing to the value of evidence based clinical recommendations, intervention strategies do not always have an impact on physician behaviour. For example, in a study in which both physicians and community pharmacists had negotiated actions based on medication reviews, the researchers were only able to document a rate of implementation of 42% of the proposed actions [6]. These findings are supported by our own experience working with community pharmacists involved in medication management programmes in Australia. Some of these pharmacists report that sometimes their recommendations have the desired impact on physician choice of pharmacotherapy, but at times they have no impact at all and in some instances they even cause physicians to respond negatively.\nAs expected, failure to change physician behaviour is not restricted to the prescribing area. Indeed, non-compliance to evidence based recommendation is a consistent finding in clinical care research [7]. For example, Grol and Grimshaw [7] note that hospital-acquired infections affect one in every 11 patients with 13% mortality rate and that 15\u201330% of infections are considered to be preventable by handwashing [8]. Still, compliance with handwashing by health care providers, in particular medical practitioners, is known to be poor [7]. Indeed, with surprising frequency, and to the considerable dismay of interventionists, physician-oriented interventions often fail to improve handwashing behaviour [7, 9]. Without a doubt while there are certainly circumstances that may lead physicians to justifiably question clinical care recommendations, in general non-compliance to evidence based recommendations proves costly to patients and society alike.\nCurrently, there is a lack of theoretical framework for understanding instances when valid recommendations do not lead to the desired impact on physician behaviour. The key question is: What are the barriers that impede physician acceptance of valid recommendations? We argue that the theory of psychological reactance [10] might at least partially explain instances when evidence based recommendations fail to impact on physician behaviour.\nPsychological reactance\nPsychological reactance is a tendency to resist perceived attempts by others to control the individual\u2019s behaviour [10]. Most of us like to perceive ourselves as being in control; we value our sense of freedom and autonomy and like to project an image of being in control [11]. In general, when we perceive that our freedom of choice is being restricted by the action of others, we react in a way that affirms our ability to choose. Often we choose to do the opposite of what the recommendation is proposing that we do or we just become entrenched in our initial position. This phenomenon is called psychological reactance [10]. Reactance is directed towards reattaining the restricted freedom; a motivational state compels us to reassert that freedom [10]. In many circumstances, reactance results in an increase in the attractiveness of the constrained behaviour and an increased sense of confidence in the ultimate decision made [10]. Particular noteworthy is the finding that reactance appears to be a biological tendency, an inherent human characteristic. At the very least, it can be said that reactance manifests itself at a very young age [12].\nFor the last four decades the theory of psychological reactance has been widely tested and applied with reactance being widely recognised as a powerful determinant of behaviour. To date, studies across a vast array of behaviours have consistently demonstrated that recommending that individuals take a certain course of action often produces the opposite effect, consistent with this tendency to resist perceived attempts by others to restrict freedom of choice. For example: in the marketing area research indicates that expert recommendations are desirable as long as they are consistent with the choice tendency of individuals. When expert recommendations contradict the consumer\u2019s choice tendencies, a reactant state is activated on the part of the decision maker leading to a behavioral \u201cbacklash\u201d that results not only in consumers ignoring the recommendations but in intentionally contradicting them [13]. Not only there is an increase in choice of the non-recommended option, but also a significant increase in confidence in the non-recommended option [13].\nIn the healthcare literature, psychological reactance has been primarily used to explain patient non-compliance, i.e., why some patients refuse to cooperate with medical treatment [14, 15]. Fogarty and Young [15] conducted research that indicates that individual variability in compliance to a medication regimen is related to individual variability in reactance as a personality trait [15]. Psychological reactance has also for long been observed in the natural setting in response to perceived attempts to restrict one\u2019s freedom of choice. A classic example of psychological reactance in the everyday life took place when a city in the United States banned the possession and use of laundry detergents containing phosphates for environmental reasons. It was found that residents stockpiled an enormous quantity of detergents containing phosphates before the ban went into effect. Others organised carpools to neighbouring counties to purchase detergents containing phosphates [16, 17]. As a result of the ban, the perceived value of the prohibited products was increased; compared with residents in neighbouring counties, who were free to choose whatever detergent they liked, residents subjected to the ban rated phosphate detergents as gentler, easier to pour, better whiteners and stain removers, and more effective in cold water. It is noted that phosphates have no impact whatsoever on the cleaning effectiveness of detergents.\nPsychological reactance and physician-oriented interventions\nThe long history of successful support for reactance theory in diverse settings and areas of behaviour may also have implications for physician-oriented interventions in which evidence based clinical recommendations are provided, as health care providers may not be immune to the phenomenon of psychological reactance. It is true that the basic concepts of psychological reactance are universal and likely also to be applicable to the provision of evidence based clinical recommendations to physicians.\nIn the context of physician-oriented interventions, the theory and research on psychological reactance would predict that at times when opposing recommendations are provided, physicians\u2019 perceptions of threats to their freedom to select an appropriate course of action could produce the opposite reaction leading them to become more likely to ignore the recommendation or to choose the non-recommended option. This would be the case even in circumstances where physicians may agree, in essence, to the value of the recommendation....to the extent that it is perceived by the individual that the communicator is trying to make him change, his freedom to decide for himself will be threatened and he will experience reactance... Information and arguments can be quite helpful to the individual and may result in positive influence, but the perception that the communicator is attempting to influence will tend to be seen as a threat to one\u2019s freedom to decide for oneself ([10], p. 94 cited in [14]).Indeed, making recommendations regarding clinical care, including pharmacotherapy, may carry with it implied threats, as it can be perceived as an attempt to restrict one\u2019s freedom of choice generating reactance and efforts to avoid them. Furthermore, the inherent \u201cveto\u201d power that physicians have over the treatment regimen may facilitate manifestation of reactant-style responses making non-compliance to evidence based recommendations more likely to occur.\nIt is also important to recognise that even in circumstances where physicians agree to participate in interventions, they may not necessarily expect their practice to be challenged. For example, there are several possible reasons for a physicians agreeing to a medication review. In some cases, physicians may wish to monitor the individual patient\u2019s self-medication habits or they may be concerned about possible patient non-compliance with the medication regimen.\nIt is also true that physicians have for long subscribed to a body of ethical rules created above all for the benefit of the patient [18]. Professional behaviour of medical practitioners is guided by these ethical rules, which dictate what is morally approved and disapproved conduct specifying what ought to be done [18]. It is reasonable to expect medical ethics to have a considerable impact on physician decision-making in situations that involve potential harm to the patient. For example, the use of a non-steroidal anti-inflammatory drug (NSAID) in a patient taking the anticoagulant warfarin puts the patient at an increased risk of bleeding. In such situations, which involve potential harm to the patient, recommendations arising from intervention strategies most certainly will be recognised by the physician as an injunctive professional norm (a norm of \u201cwhat ought to be done\u201d) and most certainly will be implemented. However, it is possible that providing physicians with valid opposing clinical care recommendations that do not involve potential harm to the patient may at times be counterproductive. For example, in a patient with low gastrointestinal and cardiovascular risk, recommendation to use a non-selective NSAID if the patient is responding well to a COX-2 selective NSAID, may be perceived as an unwarranted attempt to restrict one\u2019s action, increasing the likelihood of a reactant-style response.\nAlthough no study appears to have specifically addressed reactance from a health care providers\u2019 perspective, a recent qualitative study has provided some indication that reactance might play a role in physician decision-making in response to pharmacotherapeutic recommendations [19]. This study investigated determinants of successful collaboration between community pharmacists and physicians in medication review. Participating pharmacists discussed the need to be diplomatic in their recommendations, as they believed physicians\u2019 misperception that they were \u201ctelling them what to do\u201d could induce dismissal of their recommendations.\nPossible determinants of reactance\nIt is possible that reactant responses may be more likely to occur in situations where the physician has not requested the information\/recommendation. Experiments outside the medicines area have demonstrated that unsolicited information can evoke psychological reactance even when recommendations provide a substantial amount of information beyond what the decision maker knows [20]. Interestingly enough, in situations where the recommended action went contrary to the individual\u2019s choice tendency, this reactance to unsolicited information was significantly more likely to occur when recommendations came from a source perceived to be an expert [20]. The authors suggest that unsolicited recommendations may be perceived as more of a threat or an intrusion when they come from credible sources. It is noted that although unsolicited information is typically provided to physicians through written materials and computerised decision support systems, in some programmes medicines use review can be initiated by the pharmacist and report sent to the patient\u2019s physician in an unsolicited fashion [21].\nIt is also possible that the perception that the particular interventionist is directing recommendations specifically to the particular physician, as opposed to the treatment of the patient\u2019s condition in general, also could make reactance more likely to occur. Brehm [10] demonstrated that reactant responses are more likely to occur when individuals perceive the constraint as being specifically directed at them as opposed to when it is perceived to be impersonal [10]. If indeed, this is found also to apply to physician-oriented interventions, it may have implications for how pharmacists and other health care professionals provide recommendations to physicians.\nSense of medical professionalism might also be implicated in reactant responses. We do value our sense of freedom and autonomy and we like to project an image of being in control [11]. Indeed, physicians are trained to make sound decisions with regard to treatment regimen and it is reasonable to expect them to believe that they are in control and have the knowledge and skills required to make the best possible decisions. The acceptance of a contrary recommendation to what is being used to treat the patient\u2019s illness could undermine the physician\u2019s own sense of competence, reducing the cognitive basis of professionalism. On the other hand, dismissal of the recommended option could have the opposite effect reinforcing physician\u2019s ability to choose the most appropriate treatment.\nWe also know that once aroused, reactance may heighten individual\u2019s sensitisation to additional threats to freedom of control [22]. It is also reasonable to believe that at a time when governments are encouraging greater involvement of allied healthcare professionals in patient care, issues related to professional territoriality may sensitise physicians to reactance in response to recommendations from allied healthcare professionals, including the pharmacist. Although the literature reports positive medical profession\u2019s attitudes to pharmacists and their clinical role, some sentiments of encroachment are supported by studies that found that not all physicians view favourably greater involvement of pharmacists in patient care [23\u201325]. Thus, the physician-pharmacist interaction in the intervention process could be burdened with social and personal pressures, which can increase the possibility of a reactant response. Indeed, psychological reactance may offer a theoretical framework that could enhance our understanding of the, at times, difficult relationship between pharmacists and physicians.\nIt is also possible that the nature of the physician relationship with the allied healthcare professional is also a determinant of reactance. For example, it has been argued that the most successful pharmacist interventions have been those in which the pharmacist works in close liaison with the physician [26]. Indeed, in the United States much of the evidence supporting pharmacist involvement in collaborative drug therapy management is derived from the ambulatory care setting [27] where routine contact with physicians may allow for the development of the trusting relationship needed for interprofessional collaboration [28].\nFinally, another factor that may determine reactant responses to recommendations include individual differences, as some people are more likely to have a reactant response in general to any perceived constraint [29]. Individual variations in susceptibility to reactance can explain why identical situations may lead to markedly different perceptions of threat and magnitudes of reactant responses in different persons. Indeed, scales developed to measure reactance as a trait demonstrate that individual reactance levels lie along a continuum from very low to very high [29]. Naturally, in the context of physician-oriented interventions, the nature and intensity of the reactant response is also likely to be a function of how much the particular physician values his\/her freedom to select the treatment option him\/herself. Again, a recent qualitative study provides some support to the notion that some physicians are more prone to psychological reactance than others [19]. In this study, personality of medical practitioners was discussed as a factor influencing the outcome of medication review. Participating pharmacists distinguished between physicians who they thought were amenable to discussing their prescribing decisions and those who were not. These results indicate that interventionists do assess the reactance potential of the individual physician although it is unlikely that they label the assessment as such. It is noteworthy that when interventionists sense reactance in their interaction with the physician, they may be faced with a difficult dilemma, as they are aware of the possibility that the provision of evidence based clinical recommendation may trigger the opposite response. What can the interventionist do in such situation?\nDealing with reactance\nA phenomenon like psychological reactance, which is likely to impede the desired outcome of physician-oriented interventions, needs to be adequately addressed when training healthcare professionals to deliver such interventions. We must be mindful that the process of delivering physician-oriented interventions can be difficult as psychological reactance is not a reasonable or rational response; it can cause people to become highly emotional and motivated to defend their behaviour.\nPrior research on reactance suggests that individuals\u2019 ability to exercise some choice (freedom) in relation to recommendations may impact on compliance to the advice provided. That is, maximising the individual\u2019s perception of free choice can minimise reactance [22]. It is possible that the degree to which the physician is incorporated in the selection of evidence based clinical care practices may affect the extent to which the intervention will induce reactance. Indeed, it is possible that intervention strategies which give physicians the impression they have little or no say on their own clinical practices will be less likely to be effective, as they are perceived as more threatening, compared to those where physicians perceive themselves as exercising some choice.\nWe are now training pharmacists on how to deal with psychological reaction in the context of medication review. We also expect such training to enhance commitment to the pharmacy lead intervention from the interventionist\u2019s perspective, as after being equipped with tools for dealing with reactance, pharmacists may experience a greater sense of self-efficacy in dealing with all types of physicians, including those who are highly reactant. In addition, awareness of reactant responses and its causes may assist pharmacists and other healthcare professionals in altering their emotional response to reactant behaviour avoiding a decreased sense of self-efficacy.\nConcluding remarks\nThe current paper addressed an intrinsic human characteristic, psychological reactance, as a factor that may influence the outcome of physician-oriented intervention strategies, including those typically delivered by pharmacists. Although psychological reactance has not been tested from a healthcare provider\u2019s perspective, there is enough research to suggest the basic cognitive process represented is universal and also valid in the context of clinical care recommendations in the healthcare setting. Indeed, reactance theory fits well as it offers some explanation of why at times valid recommendations fail to have the desired impact on physician behaviour.\nIt is possible that as human beings, even healthcare professionals are subjected to the same human emotions and weaknesses as humans in general. However, a role for reactance in clinical care recommendations needs to be investigated empirically. If this role is established, factors likely to increase the potential for reactance may need to be taken into account when implementing intervention strategies that attempt to influence physician behaviour.\nIt also is acknowledged that physician non-compliance to valid recommendations is more likely to be a complex, multi-faceted phenomenon. Physicians may not adhere to evidence based recommendations for a number of reasons unique to the individual patient, and the circumstance. It is most likely that reactance, although a powerful determinant of behaviour in general, is only one of the many factors that influence decision-making in relation to clinical care\/pharmacotherapeutic recommendations.","keyphrases":["physician","interventions","psychological reactance","decision support systems","medication review","pharmacist","professional behavior change","continuing medical education","health psychology"],"prmu":["P","P","P","P","P","P","R","M","R"]}
{"id":"Oecologia-4-1-2270359","title":"Impacts of savanna trees on forage quality for a large African herbivore\n","text":"Recently, cover of large trees in African savannas has rapidly declined due to elephant pressure, frequent fires and charcoal production. The reduction in large trees could have consequences for large herbivores through a change in forage quality. In Tarangire National Park, in Northern Tanzania, we studied the impact of large savanna trees on forage quality for wildebeest by collecting samples of dominant grass species in open grassland and under and around large Acacia tortilis trees. Grasses growing under trees had a much higher forage quality than grasses from the open field indicated by a more favourable leaf\/stem ratio and higher protein and lower fibre concentrations. Analysing the grass leaf data with a linear programming model indicated that large savanna trees could be essential for the survival of wildebeest, the dominant herbivore in Tarangire. Due to the high fibre content and low nutrient and protein concentrations of grasses from the open field, maximum fibre intake is reached before nutrient requirements are satisfied. All requirements can only be satisfied by combining forage from open grassland with either forage from under or around tree canopies. Forage quality was also higher around dead trees than in the open field. So forage quality does not reduce immediately after trees die which explains why negative effects of reduced tree numbers probably go initially unnoticed. In conclusion our results suggest that continued destruction of large trees could affect future numbers of large herbivores in African savannas and better protection of large trees is probably necessary to sustain high animal densities in these ecosystems.\nIntroduction\nSavanna ecosystems are characterized by a continuous grass layer interspersed with trees and shrubs. The density and diversity of large herbivores is very high in African savannas and many pastoral communities depend on semi-natural savannas through livestock grazing (Prins and Olff 1998; Olff et al. 2002). The effects savanna trees have on understorey grass productivity have been a focus of research for decades, and both negative and positive effects have been described (Belsky et al. 1989; 1993; Mordelet and Menaut 1995; Ludwig et al. 2001; 2004a). Trees reduce grass growth by competing with grasses for water, light and nutrients (Scholes and Archer 1997; Anderson et al. 2001; Ludwig et al. 2001, 2004b), but trees can also improve grass production through hydraulic lift (Caldwell et al. 1998; Ludwig et al. 2003), reduced evapotranspiration (Amundson et al. 1995: Ludwig et al. 2001) and increased nutrient availability (Scholes and Archer 1997; Ludwig et al. 2004a). Although total plant production affects animal biomass, forage quality is of much more importance for understanding herbivore performance and food limitation (Van Soest 1994) and the interplay between forage quality and quantity determines to a large extent the composition of the herbivore assemblage (Prins and Olff 1998; Olff et al. 2002). Two previous studies which compared forage quality under trees and in the open field both report higher forage quality under Acacia (Belsky 1992) and Eucalyptus (Jacskon and Ash 1998) trees. Increased forage quality is probably caused by higher soil nutrient concentrations under tree canopies (Kellman 1979; Belsky et al. 1989; Scholes and Archer 1997). Also water competition and shade could increase forage quality because nutrients taken up by grasses are less diluted due to a lower biomass production.\nCurrently, large tree cover is decreasing in several African savannas due to high elephant pressure and frequent fires (Eckhardt et al. 2000). In Tarangire National Park (NP) (northern Tanzania) the cover of large trees declined from 20% in 1970, to only 5% in 1996 (Van de Vijver et al. 1999). A recent study in Namibia showed that the number of large trees was reduced by 50% over the last 36\u00a0years while the extent of shrubland dramatically increased (Mosugelo et al. 2002). Also in Kruger NP (South Africa) and in the Serengeti-Masai Mara ecosystem (Tanzania and Kenya) the number of large trees in savannas has decreased over the last decades (Ruess and Halter 1990; Eckhardt et al. 2000). Outside protected areas the situation is often even more dramatic, with most of the large trees being removed by local people for production of charcoal (Kituyi et al. 2001; Luoga et al. 2002). Reductions in tree cover could have serious consequences if trees have a positive effect on herbivore food quality and availability.\nThe objective of this study was to investigate the impact of trees on forage quality and how a reduction in the number of large trees could affect herbivore populations in African savannas. Specifically, we focussed on the impact of the dominant East African savanna tree, Acacia tortilis, on nutrient concentrations, protein content and digestibility of grasses. We included small, large and dead trees in our study to be able to determine how large tree destruction affects forage quality through time. A linear programming model was used to test the hypothesis that forage growing under trees is necessary for the long-term survival of wildebeest, the dominant large herbivore in East African savannas.\nMaterials and methods\nSite description\nData were collected in Tarangire NP (4\u00b0S, 37\u00b0E, 1,200\u00a0m above sea level) which is located at the eastern side of the Great Rift Valley in northern Tanzania and encompasses an area of about 2,600\u00a0km2. Mean rainfall is 650\u00a0mm\/year (Van de Vijver 1999). The wet season is from November until May with most of the rain typically falling during March and April. The Tarangire River runs through the park, and in the dry season this river is the main permanent fresh water supply within the entire 35,000\u00a0km2 Masai ecosystem (Prins 1987).\nNorthern Tarangire NP is in the dry season range of large migratory herds of Burchell\u2019s zebra (Equus burchelli) and wildebeest (Connochaetes taurinus) and to a lesser extent Cape buffalo (Syncerus caffer). Towards the end of the dry season Northern Tarangire has one of the highest concentrations of large herbivores in the world. At the beginning of the wet season wildebeest and zebra leave Tarangire NP to graze on the Simanjiro Plains. During most years both species give birth and lactate on the Simanjiro plains and return to Tarangire NP in the dry season (Voeten and Prins 1999). The park is also famous for its large herds of elephants (Loxodontaafricana) which primarily reside in the park during the dry season and sometimes disperse to surrounding areas in the wet season. The number of large elephants has increased sharply over the last 20\u00a0years due to destruction of habitat outside the park which forced more elephants into Tarangire NP.\nTwo wooded savanna types dominate the park: Acacia savanna, which can be found in the riverine area with lacustrine soils in which A. tortilis is the dominant tree species, and the deciduous savanna, which is concentrated on the ridges and upper slopes on Precambrian well drained red loamy soils (see Van de Vijver et al. 1999). Here, the dominating trees are Combretum and Commiphora species. Most of the herbivores concentrate in the Acacia savanna (Tarangire Conservation Project 1997, Voeten 1999), presumably because of the higher forage quality, so we focused our work on this savanna type. As A. tortilis accounts for 90% of the large trees in this savanna type (Van de Vijver et al. 1999) we focused our study on grass vegetation growing under and around this tree species. Total woody cover, including bushes, is about 20%, and especially the cover of large trees has declined over the last 30\u00a0years (Van de Vijver et al. 1999).\nGrass sample collection and chemical analyses\nA study site of 2\u00a0\u00d7\u00a02\u00a0km was selected with a vegetation which was representative of the Acacia savanna in Tarangire NP. The study site was located between the main entrance of the Tarangire NP and the park headquarters. For this study the same four large trees were used as described in Ludwig et al. (2004a). Trees were selected on the basis that they were separated from other large trees by at least 100\u00a0m and 50\u00a0m from any bushes or small trees. Trees close to termite hills were avoided. Measurements and calculations by Prins and Van der Jeugd (1993) suggest that these trees are ca. 100\u00a0years old. Trees have a canopy diameter between 15 and 25\u00a0m. A previous study showed that around large trees, three different vegetation zones of the herbaceous layer can be distinguished, namely: (1) under large tree canopies, (2) just outside tree canopy projections (called \u201caround tree canopies\u201d), and (3) in open patches more than 50\u00a0m from any tree (Ludwig et al. 2004a).\nSpecies composition was recorded at peak standing biomass during the early dry season in June 2000 in 20 different 2\u00a0\u00d7\u00a02-m plots in each of the three different communities. For each plot the cover of all grass species was visually estimated. Nomenclature followed Clayton and Renvoize (1982). Under tree canopies, plots were located in between the tree stem and canopy edge. Plots around tree canopies were selected at twice the distance from the stem, as the edge of the tree crown. For example, if the canopy edge was at 10\u00a0m from the trunk the centre of the plot under the canopy was located at 5\u00a0m from the trunk and the plot \u201caround canopies\u201d at 20\u00a0m from the trunk. Open grassland plots were at least 50\u00a0m from any tree.\nTo analyse grass nutrient concentration, grass samples were collected in the early dry season in June 2000, because this is the time of year when migratory herds of wildebeest and zebra return to Tarangire NP and start grazing at the study site. At each of the three different vegetation zones around large trees, samples of the most abundant grass species were collected. Different species were sampled in the three zones because of differences in species compositions. See Table\u00a01 for species sampled in each zone. Three Cynodon species, Cynodon dactylon, Cynodon nlemfluensis and Cynodon plectostachius, were treated as one group because they are very similar, rarely flower and it is very hard to distinguish between the species without uprooting them and when they are not flowering (see Clayton and Renvoize 1982).\nTable\u00a01Average (SD in parentheses, n\u00a0=\u00a04) vegetation cover, live and dead stems and leaves and leaf\/stem ratio of grass species growing under and around canopies of large Acacia tortilis trees and in open grassland patchesLocationSpeciesCover of vegetation at specific zone (%)Live stem (%)Live leaf (%)Dead stem (%)Dead leaf (%)Leaf\/stem ratioDataUnder tree canopyPanicum maximum933.1 (5)51.1 (13)4.5 (6)11.3 (4)1.81 (0.73)Cynodon spp.6247.4 (7)42.0 (8)4.4 (2)6.2 (3)0.95 (0.24)Cenchrus ciliaris940.8 (15)28.3 (9)6.8 (3)24.0 (13)1.22 (0.54)Around tree canopyCynodon spp.750.4 (6)25.4 (3)5.4 (3)18.8 (7)0.80 (0.14)Cenchrus ciliaris640.9 (19)27.2 (3)15.4 (10)16.5 (10)0.83 (0.37)Digitaria macroblephera1439.0 (15)25.2 (8)7.9 (15)27.8 (3)0.70 (0.24)Chloris virgata943.7 (7)30.7 (8)9.0 (6)16.6 (6)1.14 (0.12)Urochloa mosambicencis2152.4 (3)34.7 (11)4.9 (5)8.0 (10)0.91 (0.20)Heteropogon contortis1743.8 (6)27.2 (9)15.9 (4)13.1 (3)0.77 (0.25)Open grasslandH. contortis3848.3 (4)32.9 (7)8.4 (5)10.4 (5)0.77 (0.11)Sehima nervosum3650.1 (12)29.2 (9)12.0 (7)8.7 (14)0.62 (0.16)StatisticsadfLocationF2 ,381.766.57**1.861.848.60***Tree (block)F3, 38 1.600.421.180.081.15**\u00a0P\u00a0<\u00a00.01, ***\u00a0P\u00a0<\u00a00.001aVariance of data was analysed with a general linear model (GLM) with grass species and location (open grassland and under and around canopies) as fixed factors\nGrass samples were collected following the \u201chand plucking method\u201d (Wallis de Vries 1995). Samples were plucked between thumb and backward-bent index finger to simulate large herbivore grazing as closely as possible. One sample of every species was collected within each zone. One sample consisted of between 15 and 25 pickings collected in small mono-specific stands at different spots within the same zone. Samples were dried in the sun and sorted into leaves and stems and live and dead material.\nAfter drying at 70\u00b0C for at least 48\u00a0hours, live leaves were analysed for total N, P, K, Ca, Mg and N using a modified Kjeldahl procedure with Se as a catalyst (Novozamski et al. 1983). After digestion, N and P concentrations were measured colorimetrically with a continuous flow analyser (Skalar SA-4000; The Netherlands) and K, Ca, Mg and Na were analysed with an atomic absorption spectrophotometer (Varian Spectra AA-600; The Netherlands). Neutral detergent fibre (NDF) was determined according to Goering and Van Soest (1970) and digestibility of organic matter (DOM) according to Tilley and Terry (1963). Crude protein was calculated as 6.25\u00a0\u00d7\u00a0total N concentration. DOM, NDF and protein concentration of the vegetation per zone were calculated by multiplying the nutrient concentration in a specific species by its relative abundance within the vegetation.\nDead and smaller trees have a different effect on the understorey vegetation than large trees or open grassland (Ludwig et al. 2004a). To study how large-tree removal affects forage quality through time, grass samples were also collected around dead and smaller A. tortilis trees. Samples of the dominant grass species were collected under bushes, small trees and around dead trees. Well-developed \u201cbushes\u201d of A. tortilis were ca. 4\u00a0m high and had no signs of tree or umbrella shape development. \u201cSmall trees\u201d had a clear tree form and were about 10\u00a0m tall and had started to form an umbrella shape. \u201cDead trees\u201d were large trees which had died 4\u20138\u00a0years ago. For detailed description of tree stages see Ludwig et al. (2004a). Samples were collected as described above and analysed for protein, NDF and DOM.\nLinear model\nThe problem of having to comply with several different requirements can be analysed with a linear programming model (Belovsky 1978; Voeten 1999). This is an optimization model in which one variable is maximized or minimized, while subjected to different constraints. Here, it is used to study whether herbivores can meet all their nutritional and energy requirements by selecting either food from under or around trees or from open grassland or a combination of food sources. As a basis the model described by Voeten (1999) is used. We used wildebeest as model species because it is the most abundant herbivore in Tarangire NP (Tarangire Conservation Project 1997) and nutrient and energy constraints of this species have partly been described by Murray (1993, 1995). It would be ideal to include more herbivore species but only data for wildebeest are available. Each constraint was formulated as a general linear equation: where C is a constraint value which stands for either nutrient, energy or fibre intake. We formulated minimum requirements for nutrient, protein and energy intake, and a maximum value for fibre intake. Iiis the amount of food consumed by class i; in this study grass leaves from under or around tree canopies or open grassland. The parameter ciconverts Ii into the same unit as C and is based on the nutrient, energy and fibre content measured in the grass leaves of the different zones.\nMurray (1995) calculated, from a feeding trial in the Serengeti, northern Tanzania, that wildebeest need an energy intake of 22.32\u00a0MJ\/day for maintenance. This was based on an average body mass of 143\u00a0kg. The metabolic energy of grasses equals the digestible energy multiplied by 0.82 (Van Soest 1994). The digestible energy can be calculated from the DOM multiplied by the gross energy of grass. The energy content of grasses averages 19\u00a0MJ\/kg DOM (Crampton and Harris 1969), and this number was used for all grass species. Thus the constraint equation for energy intake is: where DOM is the digestibility of organic matter as determined in vegetation. I is the intake rate [kg\u00a0dry weight (DW)\/day] and i stands for the foraging zone.\nDigestible protein (DP) requirements at maintenance for ruminants can be calculated as (Voeten 1999): where W stands for metabolic weight (kg). We used an average weight of wildebeest of 143\u00a0kg (Murray 1993) which means they need 130\u00a0g DP per day for maintenance. DP for tropical grasses can be calculated from crude protein using a formula proposed by Prins (1987): \nThe second constraint equation used in the model is: For ruminants the daily intake rates are often constrained by rate of digestion and passage through the rumen (Voeten 1999). The digestibility rate of food is often correlated with the cell wall content, measured in the vegetation as NDF (Van Soest 1994). Reid et al. (1988) calculated from a feeding trial of cattle on a C4 grass diet that maximum daily NDF intake can be calculated as: \nFor wildebeest of 143\u00a0kg, the maximum intake is thus 2.76\u00a0kg NDF so the third constraint equation is: \nIn addition, two equations were defined based on requirements of the two most important nutrients for wildebeest as determined by Murray (1995). The constraint equation for maintenance levels of Ca and P are: where P and Ca are the concentrations measured in the vegetation.\nNa is not considered in this model because wildebeest can satisfy their daily requirements through drinking water from the Tarangire River (Voeten 1999). The different constraints were used to calculate whether wildebeest can meet all nutritional and energy requirements for maintenance (to maintain a stable body weight) by selecting forage from different foraging zones. As an input value for each of the three zones a weighted average, depending on plant cover, of the dominant grass species was used. Plant cover instead of biomass was used because only plant cover data were available.\nStatistical analysis\nPercentages of dead and live leaves and stems, nutrient concentrations, NDF and DOM of grass samples were analysed with a general linear model (GLM) with vegetation zone (under and around tree canopies, and open grassland) as fixed factors and trees as a random factor. Differences in NDF, DOM and protein concentrations of grasses from different tree stages (open grassland, bushes and small, large and dead trees) were analysed with a GLM with tree stage as a fixed factor. All data were statistically analysed with SPSS 11.0.\nResults\nCynodon species covered 62% of the vegetation under tree canopies (Table\u00a01). The other two abundant species under trees were Panicum maximum and Cenchrus ciliaris. The remaining 20% was covered mainly by herb species. Around tree canopies the vegetation was more species rich with six more or less abundant grass species (see Table\u00a01). In open grassland, the vegetation consisted almost exclusively of the grass species Heteropogon contortis and Sehima nervosum. Percentage of live and dead stem and dead leaves was similar under and outside trees. Percentage of live leaves, however, was higher under trees compared to around trees and open grassland.\nForage quality was much higher under large tree canopies than in open grassland. This was indicated by higher leaf\/stem ratios, higher concentrations of protein and DOM and lower NDF concentrations (Table\u00a02, Fig.\u00a01). For example, digestibility of green leaf organic matter (DOM) was highest in Cynodon grasses growing under the canopy where 70% of green leaves was digestible (Table\u00a01) and DOM was lowest in H. contortis growing in open grassland. For NDF a reversed pattern was observed with the lowest values for Cynodon spp. and highest for H. contortis and S. nervosum. Protein contents of grasses were highest under tree canopies and Cynodon spp. and Cenchrus ciliaris showed a higher protein content under than around tree canopies. Forage quality of grasses growing around tree canopies was also higher than in grasses from open field but lower than under canopies. Leaf\/stem ratios, protein concentration and DOM were all higher around trees canopies than in open field. P concentrations showed a different pattern: concentrations were higher in grasses growing around canopies than under canopies and in open grassland (Table\u00a01).\nTable\u00a02Average (SD in parentheses) of digestibility of organic matter (DOM), neutral detergent fibre (NDF), protein and nutrient concentrations of grass species growing under and around canopies of large A. tortilis trees and in open grassland patchesLocationSpeciesDOM (%)NDF (%)Protein (mg\/g)P (mg\/g)K (mg\/g)Ca (mg\/g)Mg (mg\/g)DataUnder tree canopyP. maximum65.7 (6.5)70.6 (3.3)141 (19.7)1.77 (0.17)40.6 (2.2)4.12 (0.26)3.25 (0.79)Cynodon spp.70.3 (7.2)61.5 (4.9)165 (8.4)1.80 (0.32)38.4 (2.4)6.91 (0.71)2.44 (0.57)Cenchrus ciliaris59.8 (3.2)70.1 (5.0)137 (5.0)1.74 (0.18)44.8 (3.8)4.34 (2.19)1.95 (0.43)Around tree canopyCynodon spp.65.1 (12.4)66.5 (8.6)127 (26.2)2.38 (0.42)36.1 (11.2)6.70 (2.18)2.58 (0.38)Cenchrus ciliaris58.0 (9.0)71.5 (6.1)106 (15.4)2.43 (0.07)41.9 (7.0)3.30 (0.65)1.87 (0.38)D. macroblephera62.6 (8.0)72.9 (6.2)75 (13.5)2.85 (0.52)38.6 (1.5)3.75 (0.76)2.26 (0.55)Chloris virgata66.8 (5.9)70.4 (4.1)105 (5.0)2.59 (0.61)37.6 (1.8)4.91 (0.30)3.03 (0.45)U. mosambicencis69.1 (5.4)64.8 (3.5)104 (10.3)3.59 (0.78)44.3 (3.9)5.28 (0.56)4.00 (0.40)H. contortis57.0 (4.8)70.2 (3.0)82 (11.8)1.46 (0.13)17.2 (0.5)3.30 (0.3)1.64 (0.18)Open grasslandH. contortis51.7 (2.2)74.0 (2.2)68 (9.3)1.65 (0.19)15.1 (0.7)3.31 (0.31)1.22 (0.15)S. nervosum54.7 (1.0)74.6 (1.0)61 (9.7)1.46 (0.13)12.0 (0.8)4.67 (0.36)1.47 (0.06)StatisticsaLocationF10.4***5.55**49.9***11.7***28.2***1.347.77**Tree (block)F7.08***5.45***1.361.071.360.640.71 **\u00a0P\u00a0<\u00a00.01, ***\u00a0P\u00a0<\u00a00.001aVariance of data was analysed with a GLM with grass species and location (open grassland and under and around canopies) as fixed factorsFig.\u00a01Indicators of forage quality of grasses growing under different sized Acacia tortilis trees, dead trees and in open grassland. Mean (+SD) protein, digestibility of organic matter and neutral detergent fibre (NDF) concentrations of green grass leaves. Bars with the same letter are not significantly different (LSD test, P\u00a0>\u00a00.05)\nProtein concentration, NDF and DOM differed significantly between different tree stages (P\u00a0<\u00a00.05). Protein concentrations were highest under large trees and lowest in open grassland (Fig.\u00a01). Smaller Acacia (bushes and small trees) showed intermediate values. Under dead trees grass protein concentrations were higher than in open grassland but lower than under large trees. DOM percentages showed a similar trend to protein concentrations; under small Acacia trees and bushes DOM of grasses was higher than in open grassland but lower than under large trees. NDF values showed the opposite trend with lowest values under large trees and highest values in open grassland (Fig.\u00a01). NDF values of grasses growing under bushes and small and dead trees were lower than in open grassland but not significantly different from those of grasses growing under large trees. In general, these patterns show that forage quality increased with tree age and size. Under dead trees forage quality was higher than in open grassland but lower than under large trees.\nResults from the linear programming model show that by selecting forage from under large tree canopies wildebeest can meet all their energy, nutrient and protein requirements before reaching their maximum intake determined by the grass fibre concentration (Fig.\u00a02). Wildebeest can also satisfy their requirements for nutrients, protein and energy by selecting grass around tree canopies. However, the results of the linear programming model indicate they cannot do so by selecting food exclusively from open grassland. Due to the relatively high fibre content in forage from open grassland the maximum intake for wildebeest is about 3,700\u00a0g\/day. Due to the low nutrient and protein concentrations in grasses from the open field, this amount is not enough to satisfy all the dietary requirements for wildebeest. All requirements can be satisfied by combining forage from open grassland with either forage from under or around tree canopies. Especially if forage from open grassland is combined with grass from under large tree canopies only a relatively small amount (10\u201320%) of forage is required from under trees and the rest of the forage can be selected from open grassland (Fig.\u00a02).\nFig.\u00a02Results of a linear programming model, predicting whether wildebeest can meet their daily requirements for energy, nutrients and protein by selecting forage from under tree canopies, around tree canopies, or in open grassland patches. Within this model the minimum daily amount of energy, protein, P and Ca needed by wildebeest is defined. The lines indicate the minimum food intake needed to meet these requirements for a combination of two of the three food sources available. For fibre the maximum food intake is shown. The maximum and minimum intakes rates are calculated using Eqs. 2, 5, 7, 8 and 9. The shaded part indicates all possible combinations of food sources which meet nutrient, energy and protein requirements without exceeding maximum intake rates determined by the fibre content. DW Dry weight\nDiscussion\nOur results show that the forage quality of the herbaceous layer is much higher under trees than in open grassland. Grasses growing under tree canopies contained a lower fibre content and the highest concentrations of protein and Ca and had a higher digestible organic matter content. The higher forage quality under trees is partly caused by the different species which dominate the vegetation under large trees. However, Cynodon spp. growing under trees also had higher protein concentrations and DOM contents than the same species growing around tree canopies. This shows that a higher below-crown forage quality it is not only a species effect but also due to a different environment under trees.\nThe positive effect of trees on forage quality did not stop at the canopy edge. Grasses around tree canopies still had higher nutrient concentrations and leaf\/stem ratios and a higher DOM than the two species dominating open grassland. The grasses we sampled around tree canopies were growing about 7\u201310\u00a0m from the canopy edge. So one mature Acacia tree with a canopy diameter of 15\u00a0m can potentially increase forage quality over an area of more than 600\u00a0m2.\nOur results could have been affected by the fact that we only sampled once. However plant samples taken in 2 previous years during different periods within the season show much higher nutrient concentration in grasses under trees compared to open grassland (Ludwig et al. 2001, 2004a). So it is likely that forage quality is higher under trees throughout the season.\nIncreased forage quality under savanna trees can be caused by a range of different effects because savanna trees influence the availability of all major resources used by grasses. Trees reduce light availability through shade, often increase soil nutrient concentrations, and compete with grasses for belowground resources, especially water (Belsky 1994; Scholes and Archer 1997; Anderson et al. 2001). Shade alone can already increase forage quality (Cruz et al.1999). For example, two independent studies showed that N concentrations were increased in P. maximum growing under artificial shade (Deinum et al.1996; Durr and Rangel 2000). However, probably most important is the higher soil nutrient availability under savanna trees. In a previous study, under the same Acacia trees, we found that concentrations of all major nutrients were higher under trees compared to open grassland (Ludwig et al. 2004a). Higher soil nutrient concentrations increase plant nutrient concentrations which improve the forage quality. Higher soil nutrient concentrations usually also increase grass productivity which would result in reduced plant nutrient concentrations due to dilution. However, grass productivity under these Acacia trees is mainly limited by water. Previous studies showed that soils under Acacia trees had lower soil moisture contents and soil water potentials than soils beyond the edge of canopies (Ludwig et al. 2003, 2004a). Higher forage quality under these savanna trees is probably caused by a combination of reduced soil moisture and increased nutrient availability.\nThe results of the linear model show the importance of the higher forage quality from under and around trees for large herbivores. When wildebeest forage only in open grassland, they cannot satisfy all their nutritional and energy requirements to maintain a stable body weight. The grass species growing in open grassland have a high fibre content which limits the maximum daily intake. Due to this limited intake insufficient amounts of protein and P can be extracted. Most of the available data on energy and nutrient requirements originate from agricultural studies and still few data are available for wild herbivores. For our model, we adjusted daily intake requirement for wildebeest from agricultural data in combination with experimental work on wildebeest (Murray 1993). These adjustments could have resulted in some errors in the model. For example the maximum NDF intake used originated from cattle and could be different for wildebeest. Also only one standard value for energy contents of grasses was used because no other data were available. Due to the large differences in forage quality of grasses from under trees compared to the open field, it is unlikely that a small change in model parameters or input values will significantly affect the conclusion that wildebeest need at least some forage from under trees. For example, wildebeest would have to increase their fibre intake by more than 35% to satisfy their protein needs in open grassland, and only if their protein needs were 30% less than assumed in our model could wildebeest satisfy their needs without grazing under or around trees. Another indication that large herbivores would need to forage under trees is that the crude protein concentration is below 7% in grasses from open grassland. At these protein concentrations the digestion rate is severely limited for all herbivores (Van Soest 1994) which suggests that large herbivores cannot survive when foraging from open grassland only. These results show the importance of trees for the functioning of savanna ecosystems and many large grazers probably depend indirectly on large trees for their long-term survival.\nTrees are not the only source of spatial heterogeneity in forage quality. For example also termite hills, faeces concentrations and rivers can increase nutrient availability and thus improve forage quality (Grant and Scholes 2006). So in the absence of trees, large herbivores could forage in these \u201chot spots\u201d to satisfy their nutrient requirements. However, trees cover a much larger part of the landscape than other sources of spatial heterogeneity and are thus particularly important in improving forage quality. In the absence of trees, herbivores can increase forage quality through positive feedbacks of grazing (Augustine and McNaughton 2006). For example through the formation of grazing lawns (McNaughton 1985) However, these positive feedbacks are mainly observed in nutrient-rich savannas and grasslands such as the Serengeti (Grant and Scholes 2006).\nForage quality of grasses growing around dead trees was lower than under large trees but still higher than in open grassland. Grass protein concentrations, for example, were almost twice as high around dead trees as in open grassland. So, forage quality does not dramatically decline immediately after trees die. This might explain why negative effects of tree removal have largely been ignored. In a previous study, we also showed that grass biomass production is higher around dead trees than in open grassland and under large trees due to the lingering positive effect of trees (increased soil nutrient availability persists for some years), combined with the removal of negative effects such as shade and competition for water (Ludwig et al.2004a). Thus, the first impression after trees are cut down\/killed is that trees were suppressing grass production.\nWhen the positive effect of higher soil nutrient concentrations around dead trees has disappeared, grass forage quality and productivity will be as currently found in open grassland, where both productivity and quality are lower than under trees that died recently. So only after a time lag of at least 8\u00a0years (Ludwig et al. 2004a), when the effect of trees on forage quality has disappeared, does it become clear that tree felling only temporarily increases grass biomass and ultimately reduces forage quality, thereby jeopardising animal productivity. Large trees killed by elephants or human pressure can be replaced by regenerating small trees or bushes; however, their impact on grass quality and availability is different from that of large trees. Bushes tend to reduce grass production (Ludwig et al. 2004a) and have a smaller positive effect on forage quality than large trees. Grass forage quality under the small trees was also still significantly lower than under large trees. The small trees we used for our study were about 20\u00a0years old so it will take more than two decades before forage quality will be as good as before clearing. Another problem is that the contradictory effects of different sized trees on grass quality and production can encourage bush-clearing. Increased grass production after large tree removal suggests that trees negatively affect grass production. After clearing the large trees, regenerating bushes start to repress grass production which again encourages removal of all woody species. Bush-clearing is, however, ultimately counterproductive because it will result in savannas with few or no large trees and thus a much lower forage quality.\nOver the last 20\u00a0years, numbers of elephants have increased in several NPs in Africa (Van de Vijver et al.1999; Eckhardt et al.2000; Skarpe et al.2004). This increase can have serious effects on other large herbivores. For example, the increase in the number of African elephants in Kruger NP which took place over the last 50\u00a0years, and the consequent reduction in the number of large trees (Eckhardt et al.2000) could have caused the demise of rare selective antelopes such as the roan. Roan largely depend on forage from P. maximum (Heitkonig 1994), a grass species which dominates the vegetation under trees in southern African savannas (Ruess and Halter 1990; Scholes and Walker 1993; Scholes and Archer 1997; Eckhardt et al.2000). As the effects of tree removal only become clear years after trees die, impacts of increased elephant pressure might not be clear at the moment but could become a serious problem in the near future.\nIn conclusion, our results presented here show that savanna trees have an important role in affecting the quality of forage available for wildebeest in Tarangire NP. Some recent work shows that forage quality is also higher under trees in other African savannas and more future work should show how general our results are (Treydte et al.2007). As trees increase food quality for herbivores the reduction in the number of large trees observed in savannas across Africa (Van de Vijver et al.1999; Eckhardt et al.2000; Mosugelo et al.2002) can have serious consequences for the survival of large herbivores. These negative effects of tree removal are probably usually not recognised because forage quality only slowly reduces after trees die. However, better protection of large trees in savanna ecosystems could be necessary to sustain current numbers of large herbivores.","keyphrases":["wildebeest","acacia tortilis","nutrients","plant\u2013animal interactions","tree\u2013grass interactions"],"prmu":["P","P","P","U","U"]}
{"id":"Clin_Oral_Investig-4-1-2238790","title":"A survey of failed post-retained restorations\n","text":"Survival of endodontically treated, post-restored teeth depends on a multitude of factors, all of which are practically impossible to include in a randomized, controlled clinical study. The purpose of this survey was to characterize and analyze reported failures of post-retained restorations to identify factors critical to failure and to type of failure. A questionnaire was mailed to private practitioners in Denmark with a request to complete the questionnaire whenever a patient presented with a failed post-retained restoration. Information was gathered on factors related to the patient, the tooth, the restorative materials, and the techniques. Two-hundred and sixty questionnaires were collected from 171 practitioners over a 3-year period. Functioning time until failure varied between 3 months and 38 years. Mean survival time until failure was 11 years. Of the failed restorations, 61% had functioned for 10 years or less. Fracture of the tooth was the most common type of failure reported, followed by loosening of the post and fracture of the post. Tapered posts implied an increased risk of tooth fracture compared to loosening or fracture of the post, and the relative risk of tooth fracture increased with the functioning time until failure. Fracture of the post was more common among male than female patients. On the basis of this survey of failed post-retained restorations, it was concluded that tapered posts were associated with a higher risk of tooth fracture than were parallel-sided posts.\nIntroduction\nEndodontically treated teeth have been reported to have shorter survival time than vital teeth [14, 20, 29, 33, 35]. This is believed to reflect that endodontically treated teeth are often structurally compromised because of a series of events: caries or trauma, endodontic therapy, and preparation for restorative procedures [12, 19, 32, 34, 40, 41, 47\u201349]. Thus, to minimize the negative influence on longevity, the restorative procedure of endodontically treated teeth calls for careful consideration of treatment alternatives.\nIn the case of minimal-remaining tooth substance, the retention of a crown has traditionally been provided by a core and the retention of the core by a post. Two fundamentally different techniques find widespread use for the construction of a post and core: (1) a post and core cast as a single unit and (2) a prefabricated post upon which a core is directly produced in, e.g., amalgam or resin composite. The first prefabricated posts on the market were made of stainless steel or gold-plated brass. Later, posts of titanium alloys became popular, and recent developments have introduced posts made of fiber-reinforced resin composite or ceramic. Simultaneously, adhesive dentistry, offering reliable bonding to tooth substance and to restorative materials via various pretreatments, has markedly progressed to widen the possibilities also for post and core treatment.\nAs mentioned, the post serves to retain the restoration. However, the post should also serve to protect the remaining tooth structure. These two functions of endodontic posts may be evaluated by determining the retentive ability of the posts and the fracture resistance of endodontically treated teeth provided with post-retained restorations. The latter may also be indirectly determined by analysis of the stress-distributing characteristics of the posts. Post-related factors that have been found to exert influence on retention of posts and protection of tooth structure include: shape, length, diameter, surface design, and stiffness of the post as well as the type of luting cement used.\nAs regard to post shape, parallel-sided posts provide better retention and less stress formation and increased fracture resistance than do tapered posts [2, 5, 18, 28, 37, 38, 42, 46]. An increase in post length has a similar, positive effect [2, 5, 7, 8, 17, 42]. However, some restrictions apply to post length. First, the apical seal of the endodontic restoration must be ensured through maintenance of 5\u20136\u00a0mm of apical gutta-percha [1, 24, 53]. Second, dentin in the apical third of teeth is often very thin. This not only increases the risk of parietal perforation, but it also weakens the tooth to a relatively high degree, thereby increasing the risk of root fracture. Little evidence is available as regard to the influence of post diameter. Consensus regarding retention seems to be that an increase in post diameter cannot be relied on as a measure to improve retention [5]. A study based on finite element analysis found that stress formation in the dentin decreased when post diameter was increased [2]. Conversely, another study showed that thinner posts provided increased resistance to fracture [48]. A consensus regarding fracture resistance seems to be that the root canal should not be uncritically enlarged, as decreasing the bulk of dentin weakens the tooth and reduces the resistance to fracture [5, 12, 30]. As regard to surface design, serrated posts provide better retention than smooth-sided posts, and threaded posts provide even better retention [5, 7, 37]. However, the superior retention of threaded posts, obtained by engaging in dentin, simultaneously is accompanied by increased stress formation within the root [36, 43] and thus by an increased risk of root fracture [5, 10, 12, 43]. Post stiffness, which is a combination of post diameter and elastic modulus of the post material, is another important characteristic, but an unsettled discussion prevails as to optimal stiffness. On the one hand, insufficient stiffness will allow excessive distortion of the restoration at the margins during function, leading to breakdown of cement and risk of secondary caries. Moreover, a decrease in the elastic modulus of the post material has been found to increase stress formation in the root and to decrease fracture strength of the restored tooth [2, 23, 38, 39]. Thus, low-modulus posts fail sooner or at lower stress values than do high-modulus posts. On the other hand, several studies have found that low-modulus posts display failures that cause little damage to the remaining tooth structure (loss of marginal seal, loss of retention, core fracture, and post fracture), while high-modulus posts are associated with a higher incidence of root fractures when they finally fail; that is, they cause more damage to the remaining tooth structure and often result in the extraction of the tooth involved [22, 23, 39, 47]. Retention of posts in the root canal is primarily sought through the use of a luting agent. Zinc phosphate cement has been the luting agent of choice for many years. The progress within adhesive dentistry and the advent of ceramic- and fiber-reinforced resin composite posts have prompted questions of the usefulness of resin cement for the luting of posts. Zinc phosphate cement provides retention by mechanical interlocking in irregularities in the surface of the root canal on one side and in the surface of the post on the other. After appropriate pretreatment of the respective surfaces, resin cements provide retention not only by mechanical interlocking but also by micromechanical and chemical adhesion. In vitro studies have found improved retention, less and more favorable stress formation, and improved fracture resistance with resin cements than with zinc phosphate cement [2, 6, 25, 31, 51].\nOther factors from those related to the post are of importance for success of post-retained restorations. Thus, researchers agree that the extent of remaining tooth structure is a key issue for fracture prognosis [9, 12, 30, 42, 47]. Another paramount factor is the ferrule effect: Teeth that allow preparation of a 1.5\u20132-mm ferrule show superior fracture resistance [44, 45, 54].\nThe foregoing has shown that numerous factors influence the longevity of endodontically treated teeth provided with post-retained restorations and that it would be practically impossible to carry out a randomized controlled clinical trial that includes all relevant variables. Previous studies have shown annual failure rates related to post and core treatment of up to 5% [4, 9, 11, 15, 41, 46]. The purpose of this survey of failed post-retained restorations was to analyze reported failures in an attempt to identify factors that are critical to failure and to type of failure.\nMaterials and methods\nA questionnaire was developed and piloted, and in June 2000, it was posted, in five copies, to the members of the Danish Dental Association who worked in private practice (3,444 practitioners). The practitioners were asked to complete the questionnaire in case a patient presented with a failed post-retained restoration. Questions were asked about age and gender of the patient, tooth involved, number of posts, type, design, length, and diameter of the post(s), type of core, type of luting agent, type of restoration, height of any ferrule, degree of tooth destruction, functioning time until failure, and type of failure.\nAfter 3\u00a0years, 260 questionnaires had been received, and the collection of questionnaires was terminated. Data were transferred to a computer and analyzed by use of the Statistical Analyzing System (SAS). Basic descriptive statistics including frequency distributions were performed. Selected data were analyzed by chi-squared tests and Fischer\u2019s exact probability tests (\u03b1\u2009=\u20090.05). Cross tabulations were carried out to further characterize the data and to identify codeterminants of functioning time until failure and codeterminants of type of failure.\nResults\nThe 260 questionnaires had been completed by 171 practitioners. The response rate per question varied between 26 and 98%. Of the 260 patients, slightly more were female (58%) than male (42%). The age of the patients varied between 18 and 91\u00a0years, and the age distribution was statistically similar among female and male patients. The sample involved nearly three times as many maxillary teeth (74%) than mandibular teeth (26%). In the maxilla, there were fewer molars (15%) than premolars (38%) and incisors and canines (47%), and in the mandible, there were fewer incisors and canines (8%). Table\u00a01 shows the distribution of the types of posts and cores: Slightly fewer teeth had been restored with cast post and cores than with prefabricated posts and direct cores. Of the 114 cast post and cores, 57% had been cast in a gold alloy, 25% in a silver\u2013palladium alloy, and 18% were unaccounted for. Of the 130 prefabricated posts used in combination with a direct core, 51% were titanium posts, 20% were stainless steel posts, 8% were carbon fiber-reinforced resin composite posts, and 21% posts were either of an alternative or unknown material.\nTable\u00a01Distribution of type of post and cores among failed restorationsType of post and coreNumberCast post and core114Prefabricated post with direct core130Prefabricated post with cast core5One-piece post crown7Total256\nOf the 260 failed post-retained restorations, 54% comprised tapered posts, 22% comprised parallel-sided posts, 20% comprised posts of a combined tapered-parallel design, and 4% comprised posts with a design that was not accounted for. The majority of the cast posts (76%) and of the stainless steel prefabricated posts (73%) were tapered. The titanium posts were equally distributed between the three designs (38, 31, and 31%), and the carbon fiber-reinforced resin composite posts were either parallel sided (50%) or of a combined design (50%).\nOf the 176 failed post-retained restorations that were specified as regard to the maximum diameter of the post, 68% comprised posts with a diameter equal to or less than one third of the diameter of the root, and 32% comprised posts with a diameter of more than one third. Of the 237 failed post-retained restorations that were specified as regard to length of post, 76% comprised posts with a length of more than 50% of the total length of the root. Table\u00a02 shows the distribution of the posts as regard to type, material, and length of posts: For all posts, except those of stainless steel, the majority of the posts were of a length that was equal to or more than half the length of the root. For the stainless steel posts, 50% were shorter than half the length of the root.\nTable\u00a02Distribution of posts of failed restorations as regards post type, post material, and post length in percent of root lengthTypeMaterialLengthTotal15\u201349%50\u201374%75\u201390%CastGold9391260Silver\u2013palladium318829PrefabricatedTitanium14301761Stainless steel1311125Carbon fiber0729Total3910540184\nOf the 130 teeth with a failed restoration that had been treated with a prefabricated post and a direct core, 29% had a core made in amalgam, 59% a core made in resin composite, 3% a core made in glass ionomer cement, and 9% a core made in a material that was not accounted for. Amalgam had been used more often than resin composite for stainless steel posts (70 vs 30%), whereas resin composite had been used more often than amalgam for titanium posts (84 vs 16%) and, in all cases, for carbon fiber-reinforced resin composite posts.\nOf the 260 failed post-retained restorations, 40% had been luted with zinc phosphate cement, 17% with glass ionomer cement, 10% with resin cement, and 33% posts with an agent that was not accounted for. The majority of the cast posts (68%) and of the stainless steel prefabricated posts (93%) had been luted with zinc phosphate cement. Most titanium posts had been luted with zinc phosphate cement (48%) or glass ionomer cement (37%). Of the carbon fiber-reinforced resin composite posts, 90% had been luted with resin cement and 10% with glass ionomer cement.\nOf the 260 failed restorations 69% had a ferrule, 29% did not have a ferrule, and 2% were not accounted for. As regards the degree of tooth destruction before luting of the now failed post-retained restoration, only 25% restored teeth out of 260 were accounted for. In 47 cases, the whole clinical crown had been missing at the time of restoration, and in 18 cases, between one half and three fourths of the clinical crown had been missing.\nTable\u00a03 shows for how long the restorations had functioned in the mouth at the time of failure. The time of functioning of the 178 restorations that were accounted for varied between 3\u00a0months and 38\u00a0years. The mean survival time until failure was 11\u00a0years, and the median survival time was 8\u00a0years. Of the 178 restorations, 61% had functioned 10\u00a0years or less at the time of failure. Of the 260 restorations, 51% displayed fracture of the tooth, 30% displayed loosening of the post, 17% displayed fracture of the post, and 2% displayed a type of failure that was not accounted for.\nTable\u00a03Distribution of failed post-retained restorations as regards functioning time until failureFunctioning time (years)Number0\u20131152\u20135506\u2013104311\u2013204321\u20133827Total178\nAmong the post-retained restorations that failed after a relatively short functioning time (0\u201310\u00a0years) as compared to a relatively long functioning time (11\u201338\u00a0years) were: (1) significantly more parallel-sided and combined tapered-parallel posts than tapered posts, (2) significantly more carbon fiber-reinforced resin composite prefabricated posts than titanium and stainless steel prefabricated posts, (3) significantly more cast silver\u2013palladium posts than cast gold posts, (4) significantly more resin composite cores than amalgam cores, and finally (5) significantly more posts luted with resin cement or with glass ionomer cement than with zinc phosphate cement. These results are unexpected, and as the \u201cDiscussion\u201d will argue, the results do not truly reflect factors critical to functioning time.\nSeveral factors were significantly related to type of failure. (1) The frequency of tooth fracture was similar for female and male patients (51 vs 50%) as was the frequency of loosened posts (36 vs 25%), but the frequency of fractured posts was higher for male than for female patients (24 vs 13%). (2) The shorter the post, the lower the frequency of post fracture (Table\u00a04). (3) Tapered posts more often than parallel-sided and combined tapered-parallel posts displayed fracture of tooth (61 vs 39 and 41%) and less often fracture of post (9 vs 30 and 31%). (4) Stainless steel prefabricated posts more often than titanium prefabricated posts displayed fracture of the tooth (81 vs 36%). (5) Teeth restored with amalgam cores displayed mainly tooth fractures (70%), teeth restored with cast gold cores displayed mainly tooth fractures (51%) or loosening of post (35%), and teeth restored with resin composite cores displayed all three types of failure with almost similar frequency (35, 25, and 40%). (6) More teeth in which the post had been luted with zinc phosphate cement displayed tooth fracture than teeth for which glass ionomer cement or resin cement had been used (54 vs 34 and 36%). (7) Compared with the frequencies of post fracture and loosening of the post, the frequency of tooth fracture increased with increasing functioning time until failure, and correspondingly, restorations that failed early on showed an equal frequency of the three types of failure (Table\u00a05).\nTable\u00a04Distribution of failed post-retained restorations as regards length of post and type of failureLength (mm)FailureTotalFracture of toothFracture of postLoosening of post2\u2013520211336\u20131077204714411\u2013201212731Total1093465208Table\u00a05Distribution of failed post-retained restorations as regards functioning time of restoration and type of failureFunctioning time (year)FailureTotalFracture of toothFracture of postLoosening of post0\u20131546152\u20135141511406\u201310188174311\u201320272144321\u201338183526Total823253167\nDiscussion\nFive percent of the private practitioners that had been invited to participate in the survey did so. Possible explanations for this low response rate include the open invitation design, the relative extensive questionnaire, and the fact that the questionnaire could not be completed once and for all but had to be procured whenever a patient presented with a failed post-retained restoration. Considering that prosthodontics is not a registered specialty in Denmark, there is no reason to believe that the treatments provided by the participating practitioners were of another type or quality than those provided by private practitioners in general. The fact that the questionnaires were filled out by individual practitioners in their everyday environment and not by calibrated investigators in an optimal research facility explains that the response rate to each question was not 100% and also implies some uncertainty as to the validity of the answers, especially those that included an element of estimation, e.g., of post diameter. The abovementioned factors call for some caution when interpreting the results of the survey.\nAs regards age and gender, the patient sample is not only representative of the private practice patient population in Denmark as such but also similar to patient samples in previously published clinical studies of post-retained restorations [9, 27, 46]. These clinical studies also supply explanations for the present finding that more maxillary teeth, especially incisors and canines, than mandibular teeth presented with failed post-retained restorations: Not only are more maxillary teeth than mandibular teeth treated with posts [9, 11, 26, 46], but post-retained restorations in maxillary teeth have also been reported to fail relatively more frequently than similar restorations in mandibular teeth [46]. In particular, post-retained restorations in anterior maxillary teeth have been found to show high failure rates, presumably because of higher functional horizontal forces acting on the anterior teeth than on posterior teeth [13, 26, 27, 46].\nThe types of posts, cores, and cements used for the failed restorations, not only reflect that preferences vary among practitioners but also that the age of the restorations vary widely, as did the materials and treatments available at the time of restoration. Given the design of this survey, i.e., we have only considered restorations that have failed, we have no knowledge of the entire population of post-retained restorations. Thus, the survey does not allow distinction between an over-representation of a given material or treatment caused by outspread use and over-representation caused by poor longevity.\nThe length of time that the post-retained restorations had functioned before failure varied from 3\u00a0months to 38\u00a0years with a mean survival time until fracture of 11\u00a0years. In agreement with the findings of previous surveys of failed post-retained crowns [21, 50], there was a tendency to a relatively high initial failure rate. However, the proportion of restorations that failed during the first 10\u00a0years was lower in the present survey (61%) than in the survey by Lewis and Smith (94%) [21]. In the survey of Turner [50], only 17% had survived more than 5\u00a0years. The longer lifespan found in the present survey may reflect that the samples of the previous surveys included posts that were generally shorter and more often smooth and\/or tapered than the posts of the present survey. These features are likely to have increased the prevalence of loosened posts at an early stage.\nA number of specific types of posts or materials were found to have significantly high prevalence in the group of post-retained restorations that failed after a relatively short functioning time: carbon fiber-reinforced resin composite posts, posts cast in silver\u2013palladium, parallel-sided and combined tapered-parallel posts, resin composite cores, and resin cement and glass ionomer cement. Some of these posts and materials are probably found in this group of restorations that failed after a short functioning time, not because they in fact are associated with short functioning time but because they have been on the market for a relatively short time compared with the alternatives and thus are over-represented in the total group of restorations with a short functioning time.\nThe most commonly reported type of failure was tooth fracture followed by loosening of the post and, finally, fracture of the post. This relative distribution is at odds with that of previous, retrospective studies, which found loosening of the post to be the most common type of failure [3, 21, 46, 50]. Failures in the form of loss of retention are most often restorable, whereas tooth fractures are most often nonrestorable and lead to extraction of the tooth. It is possible that it was easier for the practitioner to remember to submit a questionnaire when a more drastic tooth fracture occurred than when a post had lost retention and had to be recemented. Such a situation would tend to underestimate the proportion of loosened posts. An alternative explanation is that the distribution between types of failure has indeed changed to reflect a decrease in the use of smooth and tapered posts.\nSeveral factors were found to significantly influence the distribution between types of failures. One factor was gender of the patient: Whereas there were, relatively speaking, equally many male and female patients in the group of post-retained restorations that displayed tooth fracture or loosening of post, there were more male patients in the group of restorations that displayed post fracture. It may be that the greater bite forces exerted by men [16] not only increase the failure rate of post-retained restorations as such [46] but also predispose for fracture of the post. Another factor was post length: Failed shorter posts were associated with a lower prevalence of post fracture than were longer posts. This finding is not readily explained on the basis of stress analyses but may reflect that there was an over-representation of stainless steel posts and\/or of tapered posts in the group of short posts: Stainless steel has a higher elastic modulus and is a stronger material than gold alloys and titanium alloys [52] and may thus be expected to withstand higher stresses. Tapered posts were associated with a higher prevalence of tooth fracture than were parallel-sided and combined tapered-parallel posts and with a correspondingly lower prevalence of post fracture. This significant tendency to fail because of tooth fracture confirms results of previous studies [42, 46]. Likewise, failed stainless steel posts were associated with a higher prevalence of tooth fracture than were titanium posts. This could be an indirect result of most stainless steel posts being short, having high elastic modulus, and having a tapered shape in contrast to most titanium posts being parallel sided. Teeth that failed and had been restored with amalgam cores displayed significantly more tooth fractures than teeth restored with cores of resin composite and cast gold post and cores. Because of intermediate mechanical properties, e.g. elastic modulus, of amalgam as compared to resin composite and gold alloy, mechanical properties cannot easily explain the high prevalence of tooth fracture. Instead, it is likely to be caused not by the amalgam core itself but by other factors that characterize the posts with which amalgam was used. Amalgam was the preferred core material for stainless steel posts, whereas resin composite was the preferred core material for titanium posts. As just mentioned, stainless steel posts were more often than other posts tapered, a factor that has also been shown to predispose for tooth fracture. The fact that zinc phosphate cement also seemed to predispose for tooth fracture may similarly be explained, not by inferiority of the material itself but by the posts with which it was used: A relatively higher percentage of posts cast in silver\u2013palladium and of prefabricated stainless steel posts had been luted with zinc phosphate cement than with any other cement, and these two types of posts were associated with a high prevalence of tooth fracture. The final factor that was found to be significantly associated with type of failure was the length of time that the post-retained restorations had functioned until failure: The prevalence of tooth fracture increased with increasing functioning time until failure. This finding is in contrast to that of previous surveys of failed post-retained crowns in which no correlation between type of failure and length of functioning time was found [21, 50]. The discrepancy can be explained by the fact that the present survey included teeth of a much wider range of functioning time until failure and higher mean age at failure.\nThis survey did not confirm the generally accepted, paramount influence on fracture prognosis of extent of remaining tooth structure and of a ferrule [9, 12, 30, 42, 44, 45, 47, 54]. With regard to extent of remaining tooth structure, the reason is that an exceptionally low response rate did not allow statistical analysis of this parameter on a possible influence on functioning time and type of failure. The fact that the survey also did not find a significant influence of the existence of a ferrule calls for the following comments. First, the fact that most restorations were reported to have a ferrule reduced the probability that the statistical analysis would find a significant effect. Second, it is very positive to note that clinicians are aware of the importance of including a ferrule in the preparation. Third, the fact that restorations failed despite that the crowns had a ferrule points to the relevance of optimizing other elements in the restoration of an endodontically treated tooth.\nConclusions\nThe following are the conclusions derived from the study:\nFracture of the tooth was the most common type of failure among the failed post-retained restorations followed by loosening of the post and fracture of the post.Compared with parallel-sided posts, tapered posts more often displayed fracture of the tooth and less often fracture of the post.Post-retained restorations that had functioned for a long time before failure showed an increased risk of tooth fracture compared to loosening of the post and fracture of the post.Fracture of the post was more common among male patients than among female patients.","keyphrases":["post","type of failures","questionnaire","dowel","fixed prosthodontics"],"prmu":["P","P","P","U","M"]}
{"id":"Int_J_Cardiovasc_Imaging-3-1-2121118","title":"Alterations in aortic elasticity in noncompaction cardiomyopathy\n","text":"Background Noncompaction cardiomyopathy (NCCM) is a recently recognized disorder frequently associated with systolic and diastolic heart failures. This study was designed to examine aortic stiffness in NCCM patients and to compare these results to age- and gender-matched controls.\nIntroduction\nNoncompaction cardiomyopathy (NCCM) is a recently recognized rare disorder [1, 2]. It is characterized by prominent myocardial trabecularizations, and deep intertrabecular recesses leading to the spongy appearance of the myocardium [3]. The disease is frequently associated with systolic and diastolic heart failure (HF), ventricular arrhythmias, and systemic embolization [4].\nAlterations in arterial function have been demonstrated in patients with HF [5, 6]. With two-dimensional transthoracic echocardiography (TTE), ascending aortic diameter changes during a heart cycle can be measured. When blood pressure data are also available, aortic elasticity can be characterized [7, 8]. This study was designed to examine aortic stiffness in NCCM patients and to compare these results to age- and gender-matched controls.\nMethods\nStudy population\nA total of 20 patients with typical echocardiographic features of NCCM were investigated [3, 9]. Their clinical and demographic data are presented in Table\u00a01. Clinical assessment included medical and family history, physical examination, electrocardiography, two-dimensional echocardiography, and, in most cases, contrast echocardiography. Their results were compared to 20 age- and gender-matched controls without apparent cardiovascular disease. Informed consent was obtained from each patient, and the study was approved by the institutional review board and complied with the Declaration of Helsinki.\nTable\u00a01.Clinical and demographic data of NCCM patientsNCCM patientsAge (years)38\u00a0\u00b1\u00a016Male (%)8 (40)Diabetes (%)1 (5)Index eventArrhythmia (%)6 (30)Heart failure (%)8 (40)Screening (%)6 (30)ElectrocardiogramAtrial fibrillation (%)2 (10)LV hypertrophy (%)3 (15)Left bundle branch block (%)6 (30)NCCM noncompaction cardiomyopathy\nDiagnostic criteria for NCCM\nPreviously proposed echocardiographic diagnostic criteria for NCCM by Jenni et al. [3] were used: (1) absence of coexisting cardiac anomalies, (2) segmental, excessive thickening of the left ventricular (LV) wall with a two-layered structure: a thin, compacted epicardial layer and a much thicker, noncompacted layer with the characteristic appearance of numerous, prominent trabeculations (meshwork) and deep intertrabecular recesses, (3) color Doppler evidence of deeply perfused intertrabecular recesses, and (4) predominant localization of thickening in the LV apical, midlateral, and midinferior walls. Hypertensive heart disease was excluded by clinical and echocardiographic examinations (septal thickness\u00a0<\u00a013\u00a0mm).\nBlood pressure measurement\nSystolic and diastolic blood pressures (SBP and DBP, respectively) were measured in the supine position with an automatic mercury cuff sphygmomanometer from the left arm after 10\u00a0min of rest. The first and the fifth Koratkoff sounds were taken for the SBP and DBP. Blood pressure values were averaged from three consecutive measurements. None of the patients or controls used coffee or tea within 1\u00a0h before blood pressure measurements.\nTransthoracic echocardiography\nAll patients underwent a complete two-dimensional TTE and Doppler study using a Philips Sonos 7500 echocardiography equipment (Philips, Best, The Netherlands) in the left lateral decubitus position from multiple windows. The LV wall segments were analyzed according to the 9-segment model as described by Jenni et al. [3]. Systolic and diastolic ascending aortic diameters (SD and DD, respectively) were recorded in M-mode at a level of 3\u00a0cm above the aortic valve from a parasternal long-axis view (Fig.\u00a01). Gain, depth, and sector angles were individualized for the best measurement. The SD and DD were measured at the time of maximum anterior motion of the aorta and at the peak of the QRS complex, respectively.\nFig.\u00a01Measurements of systolic (SD) and diastolic (DD) diameters of the ascending aorta are shown on the M-mode tracing obtained at a level 3\u00a0cm above the aortic valve\nEvaluation of aortic stiffness\nAortic stiffness index (\u03b2) was defined as ln(SBP\/DBP)\/[(SD\u00a0-\u00a0DD)\/DD], where ln is the natural logarithm.\nStatistical analysis\nData are reported as means\u00a0\u00b1\u00a0standard deviation. For variables, Student\u2019s t test and analysis of variance (ANOVA) test were used where needed. A value of p\u00a0<\u00a00.05 was considered statistically significant. SPSS 12.0 software was used for statistical calculations.\nReproducibility of echocardiographic measurements\nThe reproducibility of the aortic diameter measurements was tested in all NCCM patients at both systole and diastole by two independent, blinded observers. Agreement between the two observers was verified using the Bland\u2013Altman method [10].\nResults\nPatient population\nThe presenting symptoms were HF in eight (40%) and arrhythmias in six (30%) patients. The remaining six (30%) asymptomatic patients were NCCM relatives and were diagnosed after family screening (Table\u00a01). Three HF patients were in NYHA-class III HF and the other five were in NYHA-class II. Cardiac medication used in NCCM patients were: angiotensin-converting enzyme inhibitors in 13 (65%), beta-blockers in 11 (55%), oral anticoagulant therapy in 10 (50%), diuretics in 8 (40%), digitalis in 2 (10%), and nitrate in 1 (5%). None of the controls received any cardiac medication.\nTransthoracic echocardiography\nThe number of noncompacted segments in the NCCM patients was 4.6\u00a0\u00b1\u00a02.0 in the NCCM group and (as seen in Table\u00a02) these patients had significantly increased LV dimensions and reduced LV ejection fraction. Aortic stiffness index (\u03b2) was significantly increased in NCCM patients compared to controls. For controls, NCCM patients with moderate HF, and NCCM patients with severe HF, \u03b2 values were 3.5\u00a0\u00b1\u00a01.1, 7.9\u00a0\u00b1\u00a05.5, and 10.4\u00a0\u00b1\u00a01.8, respectively (p\u00a0<\u00a00.001). Aortic stiffness indices of all individual patients and controls are presented in Fig.\u00a02.\nTable\u00a02.Transthoracic echocardiographic and blood pressure data in NCCM patients and normal subjectsGroup 1 (normal subjects)Group 2 (NCCM patients)LV end-diastolic diameter (mm)47.4\u00a0\u00b1\u00a03.661.0\u00a0\u00b1\u00a010.9*LV end-systolic diameter (mm)29.9\u00a0\u00b1\u00a02.948.2\u00a0\u00b1\u00a012.3*LV ejection fraction (%)67.5\u00a0\u00b1\u00a05.936.0\u00a0\u00b1\u00a017.6*Systolic aortic diameter (mm)26.7\u00a0\u00b1\u00a04.126.6\u00a0\u00b1\u00a04.4Diastolic aortic diameter (mm)23.3\u00a0\u00b1\u00a03.824.6\u00a0\u00b1\u00a04.3Pulsatile change in aortic diameter (mm)3.4\u00a0\u00b1\u00a01.12.0\u00a0\u00b1\u00a01.2*Systolic blood pressure (mmHg)125.2\u00a0\u00b1\u00a012.9120.4\u00a0\u00b1\u00a017.4Diastolic blood pressure (mmHg)77.8\u00a0\u00b1\u00a08.574.1\u00a0\u00b1\u00a09.6Aortic pulse pressure (mmHg)47.4\u00a0\u00b1\u00a08.746.3\u00a0\u00b1\u00a011.7Aortic stiffness index (\u03b2)3.5\u00a0\u00b1\u00a01.18.3\u00a0\u00b1\u00a05.2*Continuous variables are given as mean\u00a0\u00b1\u00a0standard deviationLV left ventricular, NCCM noncompaction cardiomyopathy*p\u00a0<\u00a00.001Fig.\u00a02Individual \u03b2 indices of NCCM patients and control subjects. NCCM noncompaction cardiomyopathy\nReproducibility\nThe mean\u00a0\u00b1\u00a0standard deviation difference in values obtained by two observers for the measurements of aortic diameter at systole was \u22120.7\u00a0\u00b1\u00a02.2\u00a0mm, with a correlation coefficient between these independent measurements of 0.88 (p\u00a0<\u00a00.01) (Figs.\u00a03a and 4a). At diastole, the difference between these observations was 0.05\u00a0\u00b1\u00a01.95\u00a0mm, with a correlation coefficient between observations of 0.9 (p\u00a0<\u00a00.01). The difference in values that were detected by observers was within twofold of the standard deviation of the mean (Figs.\u00a03b and 4b).\nFig.\u00a03a Interobserver correlation (r\u00a0=\u00a00.88, p\u00a0<\u00a00.01) between aortic systolic diameters in NCCM patients. b Interobserver correlation (r\u00a0=\u00a00.9, p\u00a0<\u00a00.01) between aortic diastolic diameters in NCCM patients. NCCM noncompaction cardiomyopathyFig.\u00a04a Reproducibility of systolic aortic measurements in NCCM patients. b Reproducibility of diastolic aortic measurements in NCCM patients. NCCM noncompaction cardiomyopathy\nDiscussion\nNCCM is a recently recognized disorder characterized by a loosened, spongy myocardium associated with a high incidence of progressive systolic and diastolic HF. In recent studies, alterations have been demonstrated in arterial function in patients with chronic HF [11\u201314]. To the best of authors\u2019s knowledge, this is the first time that the aortic distensibility in a series of NCCM patients was examined. In this study, increased aortic stiffness index (\u03b2) was found in NCCM patients compared to age- and gender-matched controls.\nIn prior studies, it has been shown that pulsatile changes in ascending aortic vessel diameter can be indirectly measured during routine TTE. Stefanadis et al. [7] found that noninvasive measurements of aortic distensibility were as accurate as invasive methods. Aortic stiffness index (\u03b2) relies on aortic and blood pressure data and is one of the most frequently used parameters to characterize arterial stiffness [7, 8].\nIncreased aortic stiffness is an independent risk factor, predictor of cardiovascular mortality, and a contributor to LV afterload [15, 16]. Increased aortic stiffness occurs early during the development of pacing-induced congestive HF in animals [17, 18] and has also been described in clinical patients with HF [5, 6], with comparable changes in arterial distensibility in ischemic and idiopathic dilated cardiomyopathy [19]. Rerkpattanapipat et al. [11] demonstrated that the distensibility of the proximal aorta is markedly reduced in older patients with HF due to LV systolic dysfunction beyond the changes from the aging process. They also described a correlation between exercise intolerance and reduced aortic distensibility. Giannattasio et al. [12] described that arterial compliance is impaired in congestive HF, and although more marked in severe congestive HF, the impairment is manifested in mild congestive HF as well [12]. Nakamura et al. [13] described vascular hypertrophic remodeling and endothelial dysfunction-associated alterations in vascular wall elastic properties in limb muscle conduit arteries in patients with congestive HF. Poelzl et al. [14] described an intriguing relationship between phenotype changes and functional impairment in peripheral conduit arteries of HF patients. This process is very similar to what is known for ventricular remodeling in HF.\nThere are several potential factors affecting aortic function in patients with HF [11]. Neurohormonal changes associated with HF include an increase in sympathetic drive and an activated renin\u2013angiotensin system resulting in increased plasma norepinephrine levels causing vasoconstriction and sodium retention [20\u201323]. Angiotensin II has a hypertrophic effect on smooth muscle cells of the vascular wall [24]. Early atheromatous changes and endothelial dysfunction can also be associated with HF [25], and can affect vascular elasticity. However, there is also an interaction between aortic stiffening and HF. Aortic stiffening can increase LV load causing LV stiffening with increased wall tension, early impairment in diastolic LV relaxation, and contractility inducing LV hypertrophy and fibrosis [26, 27]. These results suggest that aortic stiffness may contribute to the progression of systolic and diastolic LV dysfunctions.\nStudy limitations\nDuring this study, brachial cuff pressure measurement instead of a direct assessment of aortic pulse pressure by catheter was used. However, previous studies demonstrated an excellent correlation in aortic distensibility measured by invasive and noninvasive methods [7]. Coronary artery disease is correlated with increased aortic stiffness and was not excluded in most but not in all our patients by coronary angiography. The aortic elastic properties of control patients were somewhat larger than those described in the literature. The reason for this can be the larger body mass index (29.0\u00a0\u00b1\u00a05.1\u00a0kg\/m2) in our controls. However, a relatively higher \u03b2 in the normal subjects in our study only strengthens the abnormal findings in the NCCM patients.\nConclusions\nIncreased aortic stiffness can be observed in patients with NCCM with moderate to severe HF. These alterations may be due to HF-induced neurohormonal changes.","keyphrases":["noncompaction","aortic stiffness","echocardiography"],"prmu":["P","P","P"]}
{"id":"Surg_Endosc-4-1-2292805","title":"The effects of laparoscopic cholecystectomy, hysterectomy, and appendectomy on nosocomial infection risks\n","text":"Background Recent reviews of the literature have concluded that additional, well-defined studies are required to clarify the superiority of laparoscopic or open surgery. This paper presents precise estimates of nosocomial infection risks associated with laparoscopic as compared to open surgery in three procedures: cholecystectomy, appendectomy, and hysterectomy.\nWith the realization of smaller incisions, better cosmesis, less postoperative pain, same-day surgery, speedier postoperative recovery, and the potential for reduced complications, laparoscopic approaches have all but replaced the traditional laparotomic alternatives for certain commonly performed surgical procedures. However, the widespread adoption of laparoscopic techniques into the overall surgical armamentarium has been slowed by a variety of factors, including the learning curves required to integrate new levels of depth perception and fine dexterity, longer operating times without commensurate economic reward, and the nullification of savings from earlier hospital discharge by the cost of disposable surgical instrumentation. Nevertheless, the laparoscopic approach is now widely accepted as the gold standard for cholecystectomy and the surgical treatment of gastroesophageal reflux [1, 2].\nLaparoscopic appendectomy has been controversial since its introduction in the early 1980s, particularly for cases of complicated appendicitis. Although laparoscopic appendectomies now account for almost half of appendectomies in the United States [3], several recent reviews of the literature have concluded that additional, well-defined studies will be required to clarify the superiority of laparoscopic or open approaches [4\u20136].\nDespite the demonstration that abdominal hysterectomy is associated with higher morbidity and worse outcomes when compared to the vaginal or laparoscopic approach, the majority of hysterectomies worldwide are still performed in this fashion. For the remaining cases, laparoscopic hysterectomy is least apt to be performed [7, 8]. Although laparoscopy facilitates vaginal hysterectomy for the larger uterus, allows for the concurrent diagnosis and treatment of benign pelvic conditions such as endometriosis or pelvic adhesions, permits concomitant adnexal surgery, and provides the ability to secure and reaffirm intraperitoneal hemostasis at the end of the procedure [9], a meta-analysis of randomized controlled trials comparing different types of hysterectomy published by the Cochrane Collaboration failed to clearly demonstrate the superiority of laparoscopic hysterectomy over vaginal hysterectomy [10].\nSome studies have reported that laparoscopic approaches are associated with lower risks of surgical site infections than their open counterparts [11\u201313], but the effects of laparoscopic surgery on overall nosocomial (hospital-acquired) infection risks have not been established. Since a significant number of nosocomial infections in surgical patients occur after discharge [14, 15], it is likely that comparisons of laparoscopic and open surgeries have underestimated risks.\nNosocomial infections are a leading cause of death in the United States, affecting two to three million patients annually [16]. Starting in 2009, Centers for Medicare and Medicaid Services (CMS) will stop reimbursing hospitals for certain complications including surgical-site infections, catheter-associated urinary tract infections, and central-line associated bloodstream infections [17]. Therefore, the establishment of nosocomial infection risks in general is important.\nThe objective of this study is to obtain more precise estimates of nosocomial infection risks associated with laparoscopic and open approaches for cholecystectomy, appendectomy, and hysterectomy. We hypothesize that laparoscopic surgery will reduce the risk of nosocomial infections for each of these surgical modalities. To test these hypotheses we performed a retrospective analysis of more than 11,000 admissions, each with one of the procedures of interest, from 22 hospitals that have implemented a nosocomial infection monitoring system that can detect nosocomial infections up to 30 days post discharge.\nMethods\nThe Nosocomial Infection Marker (NIM)\nThe Nosocomial Infection Marker (NIM, patent pending, Cardinal Health) monitors and tracks nosocomial infection rates for hospitals and communities. Cardinal Health extracts data from client facilities on an ongoing basis\u00a0using a secure, Health Insurance Portability and Accoutability Act- (HIPAA) compliant method. Data are cleaned and mapped in real time as they arrive at the Cardinal Health data center by proprietary software systems. Rare exceptions that are not electronically modeled are modeled by technical and clinical experts, processed and loaded. The new models are then used by the systems to process like data in the future.\nThe Nosocomial Infection Marker is a computer algorithm that identifies the existence of nosocomial infections at the microbiological level. Specifically, the NIM algorithm distinguishes likely pathogens from contaminants, identifies duplicate isolates, and temporally determines hospital versus community-acquired pathogen acquisition [18].\nIn a multihospital study using comprehensive medical records review and gold-standard infectious disease physician discrepancy resolution, the NIM algorithm identified nosocomial infections with 86% sensitivity and 98.5% specificity hospital-wide, statistically outperforming Centers for Disease Control (CDC) case finding methods [18]. Unlike the NIM, traditional CDC case finding methods are subjectively applied with inconsistent results and are only used for certain types of infections, mostly in ICUs [18, 19]. Like the NIM, the performance characteristics of CDC methods have only been formally evaluated in one study [19].\nData\nThe Nosocomial Infection Marker (NIM) was used to identify nosocomial infections during hospitalization and post discharge. Data were extracted from the Cardinal Health (CAH) data repository for the period September 1, 2004 through December 31, 2006 from 22 hospitals in 15 states. Hospitals with matching International Statistical Classification of Diseases and Related Health Problems (ICD-9) procedure codes and more than 100 admissions with a primary ICD-9 procedure for cholecystectomy, appendectomy, or hysterectomy were eligible. These hospitals had a median number of beds of 359, with an interquartile range from 191 to 483 beds; one hospital exceeded 1,000 beds and two had fewer than 150. Mean hospital CMI was 1.56 with a standard deviation of 0.22. Admissions with primary Diagnosis Related Group (DRGs) listed in Table\u00a01 comprised more than 95% of eligible admissions. When these admissions were restricted to adults 18\u00a0years and older for cholecystectomy and hysterectomy, and patients 2\u00a0years and older for appendectomy, 11,662 admissions were available for analysis.\nTable\u00a01DRGs included in the analysis% admissions NIM rate% laparoscopySimple presentations (complexity\u00a0=\u00a00)166Appendectomy W\/O complicated Principal Diag W Cc3.162.7167.21167Appendectomy W\/O Complicated Principal Diag W\/O Cc14.531.1271.66195Cholecystectomy W C.D.E. W Cc0.5214.7516.39196Cholecystectomy W C.D.E. W\/O Cc0.210.0025.00197Cholecystectomy Except By Laparoscope W\/O C.D.E. W Cc3.649.4311.79198Cholecystectomy Except By Laparoscope W\/O C.D.E. W\/O Cc2.012.5640.60358Uterine & Adnexa Proc For Non-Malignancy W Cc11.433.3036.53359Uterine & Adnexa Proc For Non-Malignancy W\/O Cc25.721.2047.92493Laparoscopic Cholecystectomy W\/O C.D.E. W Cc14.864.15100494Laparoscopic Cholecystectomy W\/O C.D.E. W\/O Cc11.420.68100Complex presentations (complexity\u00a0=\u00a01)164Appendectomy W Complicated Principal Diag W Cc2.969.2847.25165Appendectomy W Complicated Principal Diag W\/O Cc3.393.0454.43354Uterine, Adnexa Proc For Non-Ovarian\/Adnexal Malig W Cc2.248.055.36355Uterine, Adnexa Proc For Non-Ovarian\/Adnexal Malig W\/O Cc2.042.1019.33357Uterine & Adnexa Proc For Ovarian Or Adnexal Malignancy1.8910.005.00Diag = Diagnosis; W\/O = Without; C.D.E. = Common Duct Exploration; Proc = Procedure; Malig = Malignancy; W = With; Cc = Complication and comorbidities\nData elements included NIMs, age, gender, insurance type (Medicaid, Medicare, private, other), hospital case mix index (CMI), primary DRG, whether or not the admission was through emergency department (ED), and ICD-9 procedure codes. The primary ICD-9 procedure code was used to identify both procedure (cholecystectomy, appendectomy, or hysterectomy) and type of approach (open or laparoscopic) and the primary DRG was used to differentiate simple from complex presentations in an attempt to account for intrinsic infection risks and biases towards open approaches. DRGs indicating malignancy or other complex presentations were assigned a complexity value of 1, as shown in Table\u00a01. Hospital CMI was used to control for differences between and clustering within hospitals.\nStatistical Analysis\nSingle and multiple logistic regression analyses were performed to quantify the associations between NIM rate and procedure, approach, patient age, gender, insurance type, complexity of presentation, ED admission status, and hospital CMI. The first model pooled all three procedures and included binary variables to adjust for the influence of each procedure on the acquisition of NIMs. Then separate models for cholecystectomy, appendectomy, and hysterectomy were constructed. Finally models were constructed for procedure and approach for wound, urinary tract, bloodstream, and respiratory tract NIMs.\nResults\nHysterectomies comprised 43.3% of all procedures, cholecystectomies 32.7%, and appendectomies 24.0%. The percentage of cholecystectomies, appendectomies, and hysterectomies that were laparoscopic was 84.7%, 65.6%, and 39.5%, respectively. Unsurprisingly, fewer than one-quarter of all patients were male. Approximately 19.3% of admissions were covered by Medicare, 7% by Medicaid, 58.8% by private health insurance, and the remaining 14.8% by other types of insurance.\nNIM rates were defined as the number of admissions with at least one NIM divided by the total number of admissions. Of the 11,662 admissions, 337 (2.89%) had at least one NIM (Table\u00a02). Overall, NIM rates were higher for open approaches (4.09%) than laparoscopic ones (2.11%). NIM rates were highest for cholecystectomy (3.57%), followed by appendectomy (2.60%), then hysterectomy (2.53%).\nTable\u00a02Nosocomial infection rates by approach and procedureAdmissionsAdmissions with \u22651 NIMRate (%)11,6623372.89Approach\u00a0\u00a0Laparoscopic70611492.11\u00a0\u00a0Open46011884.09Procedure\u00a0\u00a0Cholecystectomy38081363.57\u00a0\u00a0Appendectomy2803732.60\u00a0\u00a0Hysterectomy50511282.53Approach by procedure\u00a0\u00a0Laparoscopic\u00a0\u00a0\u00a0\u00a0Cholecystectomy3226842.60\u00a0\u00a0\u00a0\u00a0Appendectomy1840422.28\u00a0\u00a0\u00a0\u00a0Hysterectomy1995231.15\u00a0\u00a0Open\u00a0\u00a0\u00a0\u00a0Cholecystectomy582528.93\u00a0\u00a0\u00a0\u00a0Appendectomy963313.21\u00a0\u00a0\u00a0\u00a0Hysterectomy30561053.44\nThere were 399 NIMs identified in 337 admissions. Of all NIMs identified, 118 (30%) were from surgical wounds, 122 (31%) were from the urinary tract, 37 (9%) were from the blood, 29 (7%) were from the respiratory tract, and 93 (23%) were from other sources.\nAt least one post-discharge NIM was identified in 136 admissions, accounting for 40% of all admissions with a NIM. Of the 147 post-discharge NIMs, 39% were from surgical wounds, 31% were from the urinary tract, 7% were from blood, and 22% were from other sources. Of the 136 total admissions with at least one post-discharge NIM, 92 patients had NIM-associated readmissions.\nUnivariate Analyses\nSimple logistic regressions examined associations between NIM rates and surgical approaches (laparoscopic or open), and the following covariates: gender (male, female), age (<18\u00a0y, 18\u201334\u00a0y, 35\u201349\u00a0y, 50\u201364\u00a0y, 65\u201374\u00a0y, \u226575\u00a0y), surgical procedure (cholecystectomy, appendectomy, hysterectomy), type of insurance (private, Medicare, Medicaid, other), complexity of admission on presentation (0\/1), admitted through ED (0\/1), and hospital CMI. The results, summarized in Table\u00a03, show significantly higher NIM rates for males, adults aged \u226565\u00a0years, patients undergoing cholecystectomy, complex admissions, and admissions covered by Medicare; and significantly lower NIM rates for laparoscopic surgery, females, adults 18\u201349\u00a0years old, patients undergoing hysterectomy, and those covered by private insurance. NIM rates were positively correlated with hospital CMI, but were unaffected by ED admission status.\nTable\u00a03Univariate analyses of factors associated with NIMVariable CategoryNIM rate (%)OR95% CIGenderMale3.841.501.19\u20131.90Female2.59Age<18\u00a0years2.410.820.49\u20131.3918\u201334\u00a0years1.500.460.32\u20130.6535\u201349\u00a0years2.200.660.52\u20130.8450\u201364\u00a0years3.411.250.97\u20131.6165\u201374\u00a0years3.891.411.02\u20131.95\u226575\u00a0years7.102.962.25\u20133.90InsurancePrivate2.110.510.42\u20130.65Medicare5.242.321.85\u20132.92Medicaid3.191.120.74\u20131.68Other2.770.950.70\u20131.29ApproachLaparoscopic2.110.320.21\u20130.52Open4.09ProcedureCholecystectomy3.571.411.13\u20131.76Appendectomy2.600.870.67\u20131.13Hysterectomy2.530.790.64\u20130.97CMI2.281.59\u20133.27ComplexityComplex6.312.742.14\u20133.50Not complex2.40Emergency department admissionEmergent3.031.060.72\u20131.55Nonemergent2.88CI: confidence interval; NIM: nosocomial infection marker; OR: odds ratio\nMultivariable analyses \nSince ED admission status was insignificant in the univariate analysis, it was excluded from the multivariable analyses. Pairwise correlations of all remaining covariates were performed, and all pairs were reasonably uncorrelated. Therefore, all were included in the multivariable analyses.\nResults of multiple logistic regression models, which controlled for gender (male, female), age (<18\u00a0yr, 18\u201334\u00a0yr, 35\u201349\u00a0yr, 50\u201364\u00a0yr, 65\u201374\u00a0yr, \u226575\u00a0yr), type of insurance (private, Medicare, Medicaid, other), complexity of admission on presentation (0\/1), and hospital CMI, show that laparoscopic procedures reduced the odds of acquiring a nosocomial infections by half, but that the effect is entirely attributable to reduced infection risks in laparoscopic cholecystectomy and hysterectomy with odds ratios (ORs) of 0.34 (p\u00a0<\u00a00.01) and 0.48 (p\u00a0<\u00a00.01), respectively. No change in nosocomial infection risk was found for laparoscopic appendectomy. Estimates for individual procedures are shown in Table\u00a04.\nTable\u00a04Multivariable logistic regression analyses of factors associated with NIMVariableOdds ratio for NIMPooled (n\u00a0=\u00a011,662)Cholecystectomy (n\u00a0=\u00a03,808)Appendectomy (n\u00a0=\u00a02,803)Hysterectomy (n\u00a0=\u00a05,051)Laparoscopy0.48**0.34**0.970.48**Type of procedure\u00a0\u00a0\u00a0\u00a0Cholecystectomy1.87**\u2013\u2013\u2013\u00a0\u00a0\u00a0\u00a0Hysterectomy1.05 \u2013\u2013\u2013Age\u00a0\u00a0\u00a0\u00a0<18\u00a0years0.83\u20130.90\u2013\u00a0\u00a0\u00a0\u00a018\u201334\u00a0years0.64*0.470.840.72\u00a0\u00a0\u00a0\u00a050\u201364\u00a0years1.222.13*1.000.96\u00a0\u00a0\u00a0\u00a065\u201374\u00a0years1.02*2.21*0.580.51\u00a0\u00a0\u00a0\u00a0\u226575\u00a0years1.92**4.04**3.310.61Male1.4*1.111.89*\u2013Type of insurance\u00a0\u00a0\u00a0\u00a0Medicare1.421.331.122.09*\u00a0\u00a0\u00a0\u00a0Medicaid1.453.47**1.110.79\u00a0\u00a0\u00a0\u00a0Others1.291.531.221.32\u00a0\u00a0\u00a0\u00a0CMI1.63*1.091.312.88*\u00a0\u00a0\u00a0\u00a0Complexity2.45**NS3.952.54**** Statistically significant at the 1% level* Statistically significant at the 5% level\nConsistent with results of the univariate analyses, multivariable regression showed a significantly higher risk of NIMs for males, patients aged \u226565\u00a0years, and complexity of presentation at the time of admission. CMI was found to be  significant in both univariate and multivariable analyses [OR\u00a0=\u00a01.63 (CI95 1.11\u20132.40), p\u00a0<\u00a00.05], and this risk was significantly higher for hysterectomy patients, for whom the odds of acquiring a NIM were as high as 2.88 times that of not acquiring NIM.\nAnalysis of the same dataset by source of infection (urinary tract, wounds, respiratory tract, bloodstream, and others) revealed that the overall infection rates at each of these sites were all statistically significantly lower for laparoscopic approaches, as summarized in Table\u00a05. The odds of acquiring a site-specific infection were statistically significantly lower for all sites in laparoscopic cholecystectomy and for wound sites in hysterectomy.\nTable\u00a05Odds ratios by sourceUrinary tractWound Respiratory tractBloodstreamOthersOverall OR (95% CI)0.61 (0.38\u20130.96)0.41 (0.27\u20130.62)0.20 (0.08\u20130.49)0.31 (0.14\u20130.65)0.52 (0.33\u20130.82)By procedure\u00a0\u00a0\u00a0\u00a0Cholecystectomy0.48 (0.24\u20130.97)0.20 (0.11\u20130.39)0.17 (0.06\u20130.45)0.23 (0.10\u20130.55)0.34 (0.18\u20130.64)\u00a0\u00a0\u00a0\u00a0Appendectomy0.83 (NS)1.06 (NS)0.27 (NS)Too few NIMs0.91 (NS)\u00a0\u00a0\u00a0\u00a0Hysterectomy0.76 (NS)0.27 (0.09\u20130.79)Too few NIMs 0.48 (NS)0.62 (NS)OR: odds ratio, CI: confidence interval, NS: not significant\nThere were no significant differences between laparoscopic versus open appendectomy for all sources of nosocomial infections (urine, blood, wound, respiratory, and other). However, laparoscopic appendectomy is associated with a statistically significantly higher risk of abscess\u00a0(p\u00a0<\u00a00.05), a finding consistent with the literature [12].\nForty-one percent of patients with a nosocomial infection had at least one post-discharge nosocomial infection, and 58 of 115 (50%) of surgical wound infections were post discharge. There were 118 readmissions associated with at least one post-discharge NIM, and post-discharge NIMs associated with readmission were significantly lower for laparoscopic approaches (p\u00a0<\u00a00.001). Excluding appendectomy, the odds ratio for laparoscopic versus open NIM-associated readmission was 0.346 (CI95 0.19\u20130.63).\nDiscussion\nThis study demonstrates that laparoscopic cholecystectomy and hysterectomy reduced the overall odds of acquiring nosocomial infections from all sources by more than 50% and reduced the odds of readmission with nosocomial infection by two-thirds. Laparoscopic appendectomy showed no differences in overall nosocomial infection risks compared to open surgery. The findings for wound infections are consistent with results from randomized trials, which have reported statistically significantly lower surgical site infection rates for laparoscopic approaches [10\u201312, 20].\nThis study also demonstrates statistically significant differences in source-based infection risks by procedure and approach. Specifically, wound, bloodstream, respiratory tract, urinary tract, and other nosocomial infections were all statistically significantly less likely to occur in association with laparoscopic cholecystectomy. Risks of wound infections in laparoscopic hysterectomy were also significantly lower than in open procedures. However, no differences in infection risks were found between laparoscopic and open appendectomy.\nIt was important to stratify admission by complexity of presentation to avoid biases associated with complex presentations, higher intrinsic infection risks, and surgical approaches; for example, primary DRGs indicating malignancy or complex presentations (Table\u00a01) were associated with open surgical approaches in 69% of admissions, whereas simple presentations were associated with open approaches in only 35% of admissions. Complex presentations are also commonly believed to be at higher risk of infections, an association that is also demonstrated in this analysis. Therefore, by controlling for presentation complexity, this analysis accounts for some of the intrinsic risk of infection as well as a bias towards open surgical approaches.\nThe difference in patient severity between hospitals was accounted for by using CMI, and indeed CMI is significant in the univariate and multivariable models, with CMI contributing to nosocomial infection risks. Interestingly, admission through the emergency department was not significant in determining differences in nosocomial infection risks. One possible explanation is that emergency department use is a crude measure of patient severity because it may also be related to other factors such as time of day of admission and socioeconomic status. However, other variables associated with comorbidity, like age and certain payer types, were significant.\nLimitations\nWhile a variety of confounders were controlled for, this study is limited by the absence of certain data; for example, antibiotic use, anesthesia scores, wound class, body mass index, prior hospitalization, and certain comorbidities (i.e., cardiovascular status, diabetes mellitus, and immunodeficiency) were unavailable. These variables could explain additional NIM risk. Although omitted-variable bias is often a concern in multivariable modeling using retrospective databases, the similarity of findings in our univariate and multivariable analyses suggest that these results are robust.\nThis study documents for the first time that laparoscopic hysterectomy and cholecystectomy are associated with statistically significantly lower overall risks of nosocomial infections (p\u00a0<\u00a00.01). Laparoscopic hysterectomy and cholecystectomy were also associated with statistically significantly fewer readmissions with nosocomial infections (p\u00a0<\u00a00.01). Differences in infection risks between laparoscopic and open appendectomy were not found to be statistically significant, suggesting that these differences are likely small or nonexistent. Where differences in risks have been demonstrated, future studies should be performed to quantify their effects on health care costs and length of stay.\nOther important directions for future research include controlling for potentially important confounders to test the robustness of our results and extending the analysis to examine the effect of laparoscopic versus open surgery on the risk of nosocomial infections for additional surgical procedures. Another interesting avenue for future research would be to examine the effect of hospital volume on the rate of nosocomial infections.","keyphrases":["laparoscopic","cholecystectomy","hysterectomy","appendectomy","nosocomial"],"prmu":["P","P","P","P","P"]}
{"id":"Anal_Bioanal_Chem-3-1-2117336","title":"Development of an open-tubular trypsin reactor for on-line digestion of proteins\n","text":"A study was initiated to construct a micro-reactor for protein digestion based on trypsin-coated fused-silica capillaries. Initially, surface plasmon resonance was used both for optimization of the surface chemistry applied in the preparation and for monitoring the amount of enzyme that was immobilized. The highest amount of trypsin was immobilized on dextran-coated SPR surfaces which allowed the covalent coupling of 11 ng mm\u22122 trypsin. Fused-silica capillaries were modified in a similar manner and the resulting open-tubular trypsin-reactors having a pH optimum of pH 8.5, display a high activity when operated at 37 \u00b0C and are stable for at least two weeks when used continuously. Trypsin auto-digestion fragments, sample carry-over, and loss of signal due to adsorption of the protein were not observed. On-line digestion without prior protein denaturation, followed by micro-LC separation and photodiode array detection, was tested with horse-heart cytochrome C and horse skeletal-muscle myoglobin. The complete digestion of 20 pmol \u03bcL\u22121 horse cytochrome C was observed when the average residence time of the protein sample in a 140 cm \u00d750 \u03bcm capillary immobilized enzyme reactor (IMER) was 165 s. Mass spectrometric identification of the injected protein on the basis of the tryptic peptides proved possible. Protein digestion was favorable with respect to reaction time and fragments formed when compared with other on-line and off-line procedures. These results and the easy preparation of this micro-reactor provide possibilities for miniaturized enzyme-reactors for on-line peptide mapping and inhibitor screening.\nIntroduction\nA demand for smaller enzyme reactors has emerged in recent years, as a consequence of ongoing miniaturization in the biochemical and analytical sciences. These micro-reactors have been used in biocatalysis and biosensing. In the field of proteomics the reactors are a tool in peptide mapping, in which proteins are identified via peptide fragment identification after proteolysis. Currently, in spite of its limitations, most of these analyses are conducted by means of 2D gel electrophoresis followed by digestion of the proteins, liquid chromatographic (LC) separation, and mass spectrometric (MS) identification of the peptides [1\u20133]. The most time-consuming step in this procedure is digestion of the protein using a protease. In general, every protein to be investigated is individually incubated with the protease at a concentration of approximately 1\u20132% protein weight for 2 to 18\u00a0h at an elevated temperature (typically 37\u00a0\u00b0C). In addition to the long incubation time needed, a certain level of auto-digestion of the protease can be expected. To reduce sample handling, digestion time, and the risk of sample contamination, methods for the on-line digestion of proteins have been developed that use proteases immobilized on a solid support.\nImmobilized enzyme reactors have been developed and used over the years for several industrial and analytical purposes [4\u20136]. An obvious benefit for immobilizing biocatalysts is the fact that the enzyme can be used in several catalytic cycles and that both catalyst and reaction mixture can easily be separated. Moreover, immobilized enzymes generally show an improved stability even at more extreme reaction conditions. Several procedures have been developed for immobilization of enzymes, e.g. adsorption or encapsulation in a matrix or membrane. Alternatively, and more often used, is the covalent attachment of biocatalysts to carrier materials, which allows the immobilization of a large amount of enzyme for a high activity per surface area. Generally, particulate large-pore carrier materials are used, such as controlled-pore glass [7, 8], silica [9], or polymers like the commercially available poroszyme [10\u201312]. Current research in the production of immobilized enzymes is focused on the use of monolithic materials, as they enable efficient fragmentation of proteins [13\u201317]. Although both commercially available and self-prepared reversed-phase capillary monolithic columns have successfully passed reproducibility assessment [18, 19], synthesis of monoliths suitable for small-scale enzyme reactors can still be troublesome. Materials suitable for the fabrication of larger-scale enzyme reactors are commercially available from BIA Separations (Ljubljana, Slovenia).\nAlthough it is possible to apply an immobilized enzyme reactor (IMER) positioned after the separation column [20], most papers dealing with on-line digestion of protein samples position the IMER upstream of the separation column. In these cases the sample is first digested and the resulting peptide fragments are separated and identified by LC\u2013MS. This approach is often employed in multi-dimensional LC methods [13, 21, 22], and has also found application in peptide mapping using capillary electrophoresis [23, 24]. Alternatively, as recently shown by Zhao et al. [25] and Krenkova et al. [26], who covalently coupled trypsin to the wall of fused-silica nanoelectrospray emitters, a protein sample can be analyzed by direct infusion into a mass spectrometer.\nThis paper describes the development of trypsin-modified open-tubular micro-reactors. The chemistry was controlled and optimized using surface plasmon resonance (SPR), a technique allowing sensitive and real-time monitoring of surface reactions such as protein binding [27]. The surface modification resulting in the highest enzyme immobilization yield, was used to covalently immobilize the trypsin on the inside wall of a fused-silica capillary. The constructed trypsin micro-reactor, which is compatible with micro- and nano-LC, was further characterized. The influence of reaction time, pH, temperature, and reactor stability were investigated with the model substrate insulin B-chain. The reactor was also applied to digestion of the proteins cytochrome C and myoglobin. The produced peptides were analyzed by liquid chromatography\u2013mass spectrometry.\nExperimental\nMaterials\nThe SPR equipment used was from IBIS Technologies (currently available from Eco Chemie, Utrecht, The Netherlands) equipped with a 200-\u03bcL polycarbonate cuvet. The gold-sensor disks, purchased from SSENS (Hengelo, The Netherlands), were positioned on the IBIS-prism using index-matching oil from R.P. Cargille Laboratories (Cedar Grove, USA). PEEK nuts, unions, tubing, and loops were from Upchurch (Santa Monica, USA). Manual injections during the preparation of the reactors were performed using a Rheodyne 7010 injector (Inacom, Veenendaal, The Netherlands) equipped with a 1-mL PEEK loop. Model 10ADvp HPLC pumps from Shimadzu (Kyoto, Japan) were used for reactor preparation and activity determinations. The water used for washing and to prepare buffers was produced by a Sartorius Arium 611 ultrapure water system (Nieuwegein, The Netherlands; conductivity >18.2\u00a0M\u03a9 cm). The model ABS759A UV absorbance detector was equipped with a capillary flow-cell (75-\u03bcm i.d.) and was obtained from Applied Biosystems (Nieuwerkerk a\/d IJssel, The Netherlands).\nOn-line digestion experiments with micro-HPLC separations were conducted using LC-Packings instruments and columns (Amsterdam, The Netherlands). The equipment consisted of an injector (Famos), nanovalve column switcher (Switchos), nanopump (Ultimate), and a photodiode-array detector (PDA) equipped with a micro flow cell (45\u00a0nL). The reversed-phase pre-columns were 5\u2009\u00d7\u20090.3\u00a0mm with 5-\u03bcm 100 \u00c5 C18 particles. The 150\u2009\u00d7\u20090.3\u00a0mm reversed-phase micro-column contained 3-\u03bcm 100 \u00c5 C18 PepMap particles. The mass spectrometer was an Agilent LC\/MSD XCT ion trap (Amstelveen, Netherlands).\nAcetic acid, boric acid, calcium chloride (CaCl2), ethanol, ethanolamine (EA), hydrochloric acid (HCl), hydrogen peroxide, sodium dihydrogen phosphate, sodium chloride, sodium hydroxide (NaOH), and sulfuric acid were purchased from Merck (Darmstadt, Germany). HPLC grade acetonitrile and ethanol were from Biosolve (Valkenswaard, The Netherlands). 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), tris-hydroxymethylaminoethane (TRIS), urea, and acetone were purchased from Acros Organics (Geel, Belgium). Benzamidin, N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), benzoyl-arginine ethyl ester (BAEE), iodoacetamide, N-hydroxysuccinimide (NHS), and carboxyl-modified dextran (CMD) were purchased from Fluka (Buchs, Switzerland). Horse-heart cytochrome C, insulin B (oxidized), polyoxyethylenesorbitan monolaurate (Tween 20), and porcine pancreas trypsin were purchased from Sigma (St Louis, USA). The specific activity of trypsin was determined according to the method of Schwert and Takenaka [28] using BAEE as substrate and turned out to be 14700\u00a0U mg\u22121. Aminopropyltriethoxysilane (APTES), carbonyl diimidazole (CDI), glycidoxypropyltrimethoxysilane (GOPS), and mercaptoethanol (ME) were from Aldrich Chemical Company (Milwaukee, USA). Amino-modified dextran (AMD) was from Unavera ChemLab (Mittenwald, Germany). Fused-silica capillaries were purchased from Bester (Mijdrecht, The Netherlands).\nPiranha solution was prepared by mixing 1 part 30% hydrogen peroxide and 6 parts concentrated sulfuric acid (caution, aggressive solution). TRIS digestion buffer consisted of 50\u00a0mmol L\u22121 TRIS and 1\u00a0mmol L\u22121 CaCl2 adjusted to pH 8.2 by use of 1\u00a0mol L\u22121 HCl solution.\nMethods\nPreparation of dextran-coated SPR sensor disks (Fig.\u00a01)\nFig.\u00a01\nSurface modifications used to immobilize the enzyme in fused-silica capillaries. a, GOPS-modified; b, GOPS\/AMD-modified; c, APTES\/CMD\/AMD-modified surface. In the last case the CDI activation and enzyme (H2N\u2013enzyme) coupling are as in b\nThe SPR sensor disks were extensively cleaned in freshly prepared piranha solution. After 1\u00a0h the disks were thoroughly rinsed with water, dried in a stream of nitrogen gas, and immediately incubated for 6\u00a0h in a 10\u00a0mmol L\u22121 solution of ME in ethanol in order to produce a self-assembled monolayer (SAM) containing hydroxyl functionalities. After SAM formation, the disks were washed with ethanol and water, and dried with nitrogen gas. The resulting ME disks were incubated for 1\u00a0h with a 10% GOPS solution in 98% ethanol after which the disks were washed with ethanol, dried with nitrogen gas and stored overnight at 50\u00a0\u00b0C.\nDextran-modified sensors were prepared by incubating GOPS-modified sensor disks for 20\u00a0h at room temperature with a 10% AMD solution in a 50\u00a0mmol L\u22121 borate buffer, pH 9.5. After removing the solution the disks were washed with water, ethanol, and water, dried in a gentle flow of nitrogen gas, and stored at room temperature in a closed box until use.\nAlternatively, ME disks were incubated for 1\u00a0h with a 10% APTES solution in acetone. After washing, drying, and storing overnight in the oven, similar to the GOPS disks, the surfaces were incubated for 1\u00a0h with a solution containing 5% CMD in water containing 200\u00a0mmol L\u22121 EDC and 50\u00a0mmol L\u22121 NHS. After removing the solution, the disks were washed with water and ethanol, and incubated for 15\u00a0min in a solution containing 100\u00a0mmol L\u22121 CDI in acetone. These activated surfaces were washed with ethanol, blown to dryness with nitrogen, and incubated overnight with a solution of 10% AMD in water. After removing the solution the disks were washed with water, ethanol, and water, dried in a gentle flow of nitrogen gas, and stored at room temperature in a closed box until use.\nSPR experiments\nThe GOPS-modified SPR sensor disks were incubated overnight with 200\u00a0\u03bcL of a solution containing 2.5\u00a0mg mL\u22121 trypsin and 50\u00a0\u03bcg mL\u22121 benzamidin in 50\u00a0mmol L\u22121 borate buffer, pH 9.5 (Fig.\u00a01a). Both types of dextran-modified sensor disk were activated for 60\u00a0min with 200\u00a0\u03bcL 100\u00a0mmol L\u22121 CDI in dry acetone. After washing the surface with water to remove the last traces of acetone, the sensors were incubated overnight with 200\u00a0\u03bcL 2.5\u00a0mg mL\u22121 trypsin and 50\u00a0\u03bcg mL\u22121 benzamidin in 50\u00a0mmol L\u22121 borate buffer, pH 8.5 (Fig.\u00a01b and c, respectively). The remaining esters were inactivated by incubating the disks for 10\u00a0min with 1\u00a0mol L\u22121 EA in 50\u00a0mmol L\u22121 borate buffer, pH 8.5. The successive steps in the immobilization were monitored with SPR and the amount of covalently coupled enzyme was calculated from the recorded angle shift. During the experiments the SPR system was thermostatted at 25.0\u00a0\u00b0C.\nPreparation of dextran-coated fused-silica capillaries\nIn order to generate a proper surface for silanization, fused-silica capillaries were cleaned for 30\u00a0min with 2\u00a0mol L\u22121 NaOH solution at a flow-rate of 5\u00a0\u03bcL min\u22121. The capillary was then washed for 30\u00a0min with 0.1\u00a0mol L\u22121 HCl, for 5\u00a0min with water, and finally for 5\u00a0min with ethanol. In order to prepare dextran-coated capillaries, the capillaries were flushed for 60\u00a0min with a 10% GOPS solution in ethanol. After this step, the capillaries were closed with silicon plugs and dried overnight at 50\u00a0\u00b0C. After silanization, the capillaries were flushed with methanol at a flow-rate of 10\u00a0\u03bcL min\u22121 after which the capillaries were chemically modified in flow, using injections with a Rheodyne 7010 manual injector equipped with a 1-mL PEEK loop, in a way similar to that described above in the SPR section. Washing steps were conducted at flow rates of 10\u00a0\u03bcL min\u22121, overnight incubations at a flow rate of 1\u00a0\u03bcL min\u22121. All chemistries are outlined in Fig.\u00a01.\nEnzyme activity determination\nInsulin B chain was used as a substrate to determine the activity of trypsin. Injections of 1\u00a0\u03bcL of a concentration of 20\u00a0\u03bcmol L\u22121 in digestion buffer were introduced into the reactor that was kept at the indicated temperature using a water bath. To determine the enzyme activity in solution, trypsin was incubated in these solutions also (final trypsin concentration 0.4\u00a0\u03bcg mL\u22121). At time intervals from 0.5 to 30\u00a0min, 100-\u03bcL samples were taken and the activity was stopped by the addition of 5\u00a0\u03bcL 20% TFA in water. Buffers that were used to determine the effect of pH on activity were MES (pH 5.5 to 6.5), MOPS (pH 6.5 to 7.8), TRIS (pH 7.5 to 9), and CHES (pH 8.6 to 10.1). The buffers were adjusted to pH by use of 1\u00a0mol L\u22121 NaOH and were prepared at a 50\u00a0mmol L\u22121 concentration, also containing 5\u00a0mmol L\u22121 CaCl2. To determine the effect of temperature, the temperature during incubation was varied between 10\u00a0\u00b0C and 60\u00a0\u00b0C. Both the on-line and off-line samples were analyzed using micro-HPLC with PDA detection, as outlined below. The insulin B conversion of both immobilized trypsin and the enzyme in solution was calculated from the peak areas of substrate and products.\nOn-line protein digestion in micro-HPLC\nFor the on-line peptide and protein digestion experiments the trypsin-modified capillaries were positioned between the LC injector and a 10-port valve, as shown in Fig.\u00a02, and were immersed in a thermostatted water bath set at 37\u00a0\u00b0C unless mentioned otherwise. Protein samples were prepared in digestion buffer and were transported through the capillary towards a 10-port valve using a 5% acetonitrile solution containing 0.05% TFA. The peptide fragments formed during digestion were concentrated on an RP trapping column (TC) and salts and other buffer components present in the sample were removed. By switching the valve the trapping column is in series with the RP micro column and an acetonitrile gradient started. The gradient was composed of two solutions: (A) 5% acetonitrile in water containing 0.05% TFA and (B) 80% acetonitrile in water containing 0.04% TFA. In 30\u00a0min the gradient changed linearly from 0 to 50% B, followed by 10\u00a0min at 90% B and 20\u00a0min at 0% B. The eluent was monitored with the PDA detector in the range from 200 to 595\u00a0nm.\nFig.\u00a02Set-up used for on-line protein digestion using a trypsin-modified fused-silica capillary. For detection a PDA or an ion-trap MS was used\nESI-MS was conducted in the positive-ion mode with the capillary voltage set at 3500\u00a0V. The flow rate and temperature of the nitrogen drying gas were 5\u00a0L min\u22121 and 325\u00a0\u00b0C, respectively. The sequence of the peptide fragments was determined by using the mass spectrometer in auto-MS\u2013MS mode fragmenting the two peptides that were most abundantly present when the signal reached threshold. The MS\u2013MS result was analyzed by a Mascot database search (http:\/\/www.matrixscience.com).\nResults and discussion\nSurface chemistry and enzyme immobilization\nAs shown before [29], modification of an SPR sensor surface with a dextran hydrogel leads to less non-specific adsorption of proteins compared with unmodified surfaces. The presence of such a layer also enhances the immobilization capacity of biomolecules compared with monolayer-based coatings. Due to the flexible nature of the dextran chains, the accessibility also is often improved compared with molecules immobilized on a flat surface. As the amount of protein present in a capillary after immobilization cannot be determined easily, SPR sensors were used as a model to investigate the effect of the different surface modifications on the amount of trypsin that could be attached covalently. Hydroxyl functionalities necessary to enable silanization are introduced using mercaptoethanol (ME), but all other surface modifications are carried out in exactly the same way, both on the SPR sensor surfaces and in the fused-silica capillaries.\nTrypsin immobilization after silanization with GOPS by reaction of the trypsin primary amines and the glycidyl function of GOPS results in an SPR angle shift of 350\u2009\u00b1\u200920\u00a0m\u00b0 (n\u2009=\u20093), which equals an amount of trypsin of 2.9\u2009\u00b1\u20090.2\u00a0ng mm\u20132 covalently attached to the surface, which is close to monolayer coverage of trypsin. When the GOPS-silanized surface is modified with AMD, resulting in a dextran hydrogel, the amount of trypsin that can be immobilized after CDI activation increases to 9.5\u2009\u00b1\u20090.3\u00a0ng mm\u22122 (n\u2009=\u20093). To further increase the amount of trypsin, an intermediate dextran layer was added. Therefore, fused-silica capillaries were APTES silanized and modified with CMD and AMD, subsequently. The amount of trypsin that could covalently be attached to these layers was determined with SPR and proved to be 11.1\u2009\u00b1\u20090.5\u00a0ng mm\u22122 (n\u2009=\u20092). As this amount is significantly more than obtained with the GOPS\/AMD surface, all further experiments have been conducted with fused-silica capillaries the surface of which is modified with dextran in this way.\nAssuming that the investigated surface modification allows a similar amount of trypsin to be immobilized per surface area in fused-silica capillaries, the quantity of enzyme immobilized on the dextran-modified capillaries is more than five times larger per surface area compared with other open-tubular, trypsin micro-reactors described in the literature [23, 30\u201332]. These reactors, first described by Amankwa and Kuhr [30], are based on immobilized avidin and, at saturation, 6.5\u00a0pmol biotinylated trypsin was immobilized in a 50-cm long capillary of 50-\u03bcm i.d.. This amount equals 2\u00a0ng trypsin mm\u22122 capillary surface, which is less than monolayer coverage.\nCharacterization of the enzyme reactor\nFused-silica capillaries were modified with dextran as described above and the activity and characteristics of the trypsin micro-reactor were assessed with the oligopeptide insulin B chain, a 30 amino acids long insulin fragment. When insulin B is exposed to the enzyme present in the reactor, hydrolysis at the arginine present at position 22 and, to a lesser extent, the lysine at position 29 is observed. Using the aforementioned substrate insulin B, the pH optimum was determined for the enzyme in solution and the immobilized enzyme. As can be observed in Fig.\u00a03a, the optimum pH value for both free and immobilized trypsin is pH 8.5. Therefore, all further experiments are conducted at this pH value.\nFig.\u00a03Effect of (a) pH and (b) temperature on the relative activities of trypsin in solution (circles) and trypsin immobilized on an APTES\/CMD\/AMD-modified fused-silica surface (triangles). In b the maximum insulin B conversion rate determined under experimental conditions is 4.41\u2009\u00b1\u20090.02\u00a0pmol min\u22121\u00a0\u03bcg\u22121 for the enzyme in solution and 12.32\u2009\u00b1\u20090.35\u00a0pmol min\u22121\u00a0\u03bcg\u22121 for a 3-\u03bcL, 50-\u03bcm i.d. microreactor operated at a flow rate of 1\u00a0\u03bcL min\u22121\nAlso the effect of temperature on the activity of both the immobilized trypsin and the enzyme in solution was determined in the range 10\u00a0\u00b0C to 60\u00a0\u00b0C. As can be observed in Fig.\u00a03b, the activity of trypsin increases with temperature to reach a maximum around 40\u00a0\u00b0C and decreases at higher temperatures. The immobilized enzyme shows a higher activity compared with the enzyme in solution, which may be due to an often observed higher stability of immobilized enzymes, also under more extreme conditions. From the data the activation energy can be determined. For the immobilized enzyme a value of 12.2\u2009\u00b1\u20090.3\u00a0kJ mol\u22121 can be calculated and for the enzyme in solution a value of 17.2\u2009\u00b1\u20090.5\u00a0kJ mol\u22121. This means that for the immobilized enzyme the temperature has less effect on the activity than for the enzyme in solution. As the highest activity is observed at 37\u00a0\u00b0C, all further experiments are conducted at that temperature.\nFor the enzyme in solution a specific activity of 4.41\u2009\u00b1\u20090.02\u00a0pmol min\u22121 \u03bcg\u22121 is determined at a concentration of 20\u00a0pmol \u03bcL\u22121 insulin B and pH 8.5 and 37\u00a0\u00b0C. Under similar conditions, in a microreactor of 3\u00a0\u03bcL with a 50\u00a0\u03bcm i.d. and operated at a flow rate of 1\u00a0\u03bcL min\u22121, an amount of 12.32\u2009\u00b1\u20090.35\u00a0pmol insulin B is converted. In such a reactor a total of 2.67\u00a0\u03bcg trypsin might be present taking the earlier SPR observations into account, which means that the immobilized enzyme is both accessible and still active after immobilization. The specific activity after immobilization is higher than observed in recent papers for the protease pepsin immobilized on beaded chitosan [33, 34] and trypsin photoimmobilized in a fused-silica capillary [34].\nThe stability during operation of the open-tubular reactor was tested at 37\u00a0\u00b0C and pH 8.5. Activity tests using the model substrate show that the activity of a reactor that is continuously in operation, is constant for at least two weeks. The enzyme trypsin dissolved in digestion buffer and incubated for 24\u00a0h at 37\u00a0\u00b0C loses 60% of its activity, and after three days no activity is measured.\nOn-line digestion of proteins\nThe on-line digestion of horse cytochrome C is accomplished as described in the Methods section. Samples are submitted to on-line digestion in a 22-cm long trypsin-modified APTES\/CMD\/AMD-coated capillary with an i.d. of 75\u00a0\u03bcm (total volume 1\u00a0\u03bcL) or with an i.d. of 50\u00a0\u03bcm and a length of 51\u00a0cm (1\u00a0\u03bcL) or 140\u00a0cm (2.75\u00a0\u03bcL). When the protein is not reduced, the heme-moiety will remain covalently attached to the peptides containing the protein sulfhydryls. By monitoring the heme-containing peptides at 395\u00a0nm, the progress of the digestion and the amount of undigested protein can be determined. The results are summarized in Table\u00a01.\nTable\u00a01Summary of the on-line digestion experiments for cytochrome C. The experiments were conducted at pH 8.5 and 37\u00b0C (n\u2009=\u20093)capillary ID (\u03bcm)reactor volume (\u03bcL)amount injecteddigestion time (s)% undigested protein (SD)(\u03bcg\u00a0mL\u22121)(pmol)751248206065.9 (4.8)751124106035.8 (3.475112.4106023.6 (1.3)501248206014.0 (1.9)50112410602.3 (2.0)502.75248201650502.75124101650502.7512.4101650The experiments were conducted at pH 8.5 and 37\u00a0\u00b0C (n\u2009=\u20093)\nAs expected when a limited amount of enzyme activity is present in a reactor (22\u00a0cm \u2009\u00d7 \u200975\u00a0\u03bcm), for increasing concentrations cytochrome C a larger amount of protein is undigested. Nevertheless, many tryptic peptides are still generated. As can be expected, an increase in exposure time of the substrate with the immobilized enzyme will result in an improved digestion yield. A longer contact time is achieved by increasing the reactor volume by using a longer enzyme-modified capillary. Additionally, the enzyme-to-substrate ratio is increased, which is accomplished by changing the surface-to-volume ratio by using a capillary with a smaller internal diameter. As can be observed in Table\u00a01, decreasing the i.d. of the capillary leads to improved digestion for a reactor of equal volume due to a higher surface-to-volume ratio and hence a higher amount of enzyme. The use of a 140\u00a0cm \u00d750\u00a0\u03bcm capillary allows the complete digestion of up to 20\u00a0pmol (248\u00a0\u03bcg mL\u22121) of cytochrome C in less than 5\u00a0min including the sample concentration and removal of salts by the trapping column.\nChromatograms for the on-line digestions obtained with capillaries with an i.d. of 50\u00a0\u03bcm are shown in Fig.\u00a04. Figure\u00a04a shows a blank run in a capillary containing no enzyme and Fig.\u00a04b and c present on-line digestions for a micro-reactor of 2.75\u00a0\u03bcL operated at 5\u00a0\u03bcL min\u22121 and 1\u00a0\u03bcL min\u22121, respectively. As discussed above, incomplete digestion of the injected cytochrome C will lead to the presence of multiple peptide fragments containing the heme group, as is visible in Fig.\u00a04b. The digestion is complete when the sample exposure time is 165\u00a0s (flow rate 1\u00a0\u03bcL min\u22121) as both intermediate products and the undigested protein (retention time 44\u00a0min in Fig.\u00a04a), which are visible at a wavelength of 395\u00a0nm as outlined above, are no longer observed. An injection of off-line-digested cytochrome C showed a similar chromatogram as is shown in Fig.\u00a04c.\nFig.\u00a04Chromatograms obtained from injection of 10\u00a0pmol horse cytochrome C in capillary digestion systems monitored at 214\u00a0nm and 395\u00a0nm. The experiments were conducted with an APTES-CMD-AMD derivatised fused-silica capillary of (a) 510\u2009\u00d7\u20090.050\u00a0mm, not containing trypsin, operated at 1\u00a0\u03bcL min\u22121 (blank); (b) 1400\u2009\u00d7\u20090.050\u00a0mm, trypsin-modified, operated at 5\u00a0\u03bcL min\u22121 (average sample residence time 33\u00a0s); (c) as (b) but operated at 1\u00a0\u03bcL min\u22121 (average sample residence time 165\u00a0s). For clarity the beginning of the chromatogram displaying the 214\u00a0nm signal is offset as indicated\nThe on-line digestion of horse cytochrome C is also monitored with mass spectrometry. The effect of flow rate and hence incubation time on the digestion of the protein and the number of peptides identified with a Mascot database search is determined and summarized in Table\u00a02. In this table the undigested amounts of protein which have been determined using the PDA detector are also shown. At a flow rate of 1\u00a0\u03bcL min\u22121 protein digestion is complete and many peptides are matched, resulting in high sequence coverage and Mascot score. With higher flow, and hence decreasing incubation time, the amount of protein that remains undigested increases and consequently fewer peptides are produced and observed. However, even at relatively high flow rates an adequate amount of peptides is still formed and the protein can be identified on the basis of the fragments present. Nevertheless, the ion intensity is low and some peptides are not retrieved as they are below the threshold for auto-MS\u2013MS. Table\u00a03 summarizes the peptides observed and matched using the MS\u2013MS data and a Mascot.database search of proteins digested at a flow rate of 1\u00a0\u03bcL min\u22121. A base-peak chromatogram (BPC) of the digestion of cytochrome C under these conditions is shown in Fig.\u00a05a. The MS\u2013MS fragmentation of one of the peptides is presented in Fig.\u00a05b.\nTable\u00a02General overview of the on-line digestion experiments for cytochrome C (n\u2009=\u20093)flow rate (\u03bcL min-1)digestion time (s)sequence coverage (%)total no peptides matchedMascot scoreundigested protein (%)1017661134760.3533662150131.8283662364918.5116587278330Table\u00a03Peptide fragments observed by MS in the on-line digestion of cytochrome C and myoglobin The experiments were conducted at pH 8.5 and 37\u00a0\u00b0C at a flow rate of 1\u00a0\u03bcL min\u22121 using a 2.75-\u03bcL trypsin reactorPeptide sequenceTheoretical m\/zExperimental m\/zPositionMissed cleavageCytochrome C (sequence coverage 87%)1....IFVQKCAQCHTVEK1632.8816.9 (2+)9\u20132212....HKTGPNLHGLFGR1432.8717.1 (2+)26\u20133813....TGPNLHGLFGR1167.6584.6 (2+)28\u20133804....TGPNLHGLFGRK1295.7648.6 (2+)28\u20133915....KTGQAPGFTYTDANK1597.8533.4 (3+)39\u20135316....TGQAPGFTYTDANK1469.7735.6 (2+)40\u20135307....TGQAPGFTYTDANKNK1711.8571.4 (3+)40\u20135518....GITWKEETLMEYLENPKK2208.1736.8 (3+)56\u20137329....EETLMEYLENPK1494.7748.0 (2+)61\u201372010  EETLMEYLENPKK1622.8812.1 (2+)61\u201373111...YIPGTK678.4678.1 (1+)74\u201379012...MIFAGIKK906.5454.0 (2+)80\u201387113...KTEREDLIAYLKK1477.8739.6 (2+)88\u201399214...TEREDLIAYLKK1349.7675.5 (2+)89\u201399115...EDLIAYLK963.5482.6 (2+)92\u201399016...EDLIAYLKK1091.6546.6 (2+)92\u20131001Myoglobin (sequence coverage 88%)1....GLSDGEWQQVLNVWGK1814.9908.7 (2+)1\u20131602....VEADIAGHGQEVLIR1605.8803.6 (2+)17\u20133103....VEADIAGHGQEVLIR1605.8536.1 (3+)17\u20133104....LFTGHPETLEK1270.7636.0 (2+)32\u20134205....HLKTEAEMK1085.6543.5 (2+)48\u20135616....HGTVVLTALGGILK1377.8689.7 (2+)64\u20137707....HGTVVLTALGGILKK1505.9502.7 (3+)64\u20137818....KKGHHEAELKPLAQSHATK2109.1703.7 (3+)78\u20139629....KGHHEAELKPLAQSHATK1981.0661.1 (3+)79\u201396110  GHHEAELKPLAQSHATK1853.0618.8 (3+)80\u201396011...YLEFISDAIIHVLHSK1884.0628.8 (3+)103\u2013118012...HPGNFGADAQGAMTK1500.7751.5 (2+)119\u2013133013...ALELFRNDIAAK1359.8680.6 (2+)134\u2013145114...YKELGFQG940.5471.0 (2+)146\u20131531Fig.\u00a05On-line digestion of 10\u00a0pmol cytochrome C at a flow rate of 1\u00a0\u03bcL min\u22121: (a) base peak chromatogram, the numbers correspond to the matched peptides in Table\u00a03, and (b) MS\u2013MS fragmentation of the peptide TGPNLHGLFGR with m\/z 584.9 showing several of the matched fragment ions\nIn further experiments horse myoglobin was digested on-line. This protein is generally regarded as difficult to digest [15, 35]. When 1\u00a0\u03bcL of a 10\u00a0\u03bcmol L\u22121 solution in buffer is injected at a flow rate of 1\u00a0\u03bcL min\u22121 (165\u00a0s exposure time), the injected protein is completely digested, as was observed with UV detection (data not shown). Using mass spectrometric analysis, 13 different peptides are observed and matched using the MS\u2013MS data and a Mascot database search, resulting in a sequence coverage of 88%. The matched peptides are summarized in Table\u00a03 and a BPC of the on-line digestion of myoglobin is shown in Fig.\u00a06a, with an MS\u2013MS spectrum of one of the tryptic peptides. Both the degree of digestion and the sequence coverage are adequate compared with other systems that often use a high percentage of modifier to enhance digestion, as the absence of denaturing agents during digestion leads to little or no digestion of myoglobin [35]. Therefore these reactors are generally used for direct infusion into MS or off-line protein digestion as the presence of high concentrations of methanol or acetonitrile in the digestion buffer will seriously impede on-line protein digestion in combination with RP-LC.\nFig.\u00a06Results from on-line digestion of 10\u00a0pmol myoglobin at a flow rate of 1\u00a0\u03bcL min\u22121: (a) base peak chromatogram, the numbers correspond with the matched peptides in Table\u00a03, and (b) the MS\u2013MS spectrum of the peptide HGTVVLTALGGILK with m\/z 689.7\nAmankwa and Kuhr [30] reported that proteins and peptides adsorb to the capillary surface of their trypsin reactor. Reactor fouling, and as a result sample carry-over, is also observed when trypsin-modified particulate polymers or monoliths are used [36]. Adsorption of proteins and peptides to these relatively hydrophobic columns is prevented by the addition of up to 20% methanol to the digestion buffer. For dextran-coated surfaces, it is known that the non-specific adsorption of protein is minimal and depends on the character of the oligosaccharide layer [29, 37, 38]. During protein-digestion experiments on SPR sensor surfaces modified identically with the capillary reactors no adsorption during incubation is observed (data not shown). Similarly, during on-line digestion using the dextran coated capillaries, sample adsorption and carry-over are not observed, as repeated sample injections result in identical chromatograms and blank buffer injections do not show peptide fragments. On basis of the latter result and the long lifetime of the reactor, trypsin auto-digestion is probably absent.\nThe developed IMER has been applied for the digestion of horse cytochrome C and myoglobin in buffer without prior pretreatment followed by separation and MS\u2013MS sequencing. The time needed to obtain complete protein digestion and sample trapping is 5\u00a0min with an average sample residence time of 165\u00a0s. The total analysis time, digestion and LC separation, is 65\u00a0min. The developed micro-reactor is competitive when compared with other systems used for on-line protein digestion described in literature [13, 14, 39\u201341]. In the capillary IMERs described by Amankwa and Kuhr [30] and Bossi et al. [34] the complete digestion of native proteins was only obtained when the protein resided in the trypsin-coated capillary for 15\u201325\u00a0min. The peptide separation was carried out with CE in these latter cases.\nMany reactors use a protein denaturation step to make the protein more susceptible to proteolytic action. The digestion efficiency can be enhanced by partial denaturation by addition of 35\u201345% acetonitrile to the buffer [11, 15, 35, 36] or by sonication [42, 43]. However, the latter has not yet been used in an automated set-up and the presence of high amounts of modifier will make reversed-phase chromatography in a subsequent step for the separation of the fragments produced during protein digestion very difficult. The described reactors were efficient in terms of time needed for digestion and the digestion result, but were, as a rule, used for direct infusion into the MS or used for off-line digestion.\nAlternatively, protein denaturation is accomplished using chemicals such as SDS, guanidine HCl or urea. A recent example is the miniaturized on-line proteolysis\u2013capillary LC system as described by Samskog et al. [12], who employed a 10-\u03bcL column packed with poroszyme. The sample contained guanidine.HCl for protein denaturation, and was diluted with buffer, also containing methanol, prior to injection. The total time needed for digestion, trapping, and removal of the high concentration of salt was 15\u00a0min. The system needed periodic regeneration to counteract the effects of residual salts in the analyte.\nConclusions\nTo study the immobilization of the protein trypsin in a fused-silica capillary, a number of surface modifications was tested. In order to investigate the amount of protein that can be covalently attached to these surfaces, SPR sensor disks are modified with these coatings to mimic the capillary surface. The SPR measurements show that the best results were obtained using a dextran coating with an intermediate layer. The resulting open-tubular trypsin reactors having a pH optimum of pH 8.5 display a high activity when operated at 37\u00a0\u00b0C and are stable for at least two weeks when used continuously.\nThe capillary reactors show flow-dependent catalysis. For a capillary IMER, the conversion of the insulin B chain increases with decreasing flow and hence a longer residence time. The same is observed for the digestion of horse cytochrome C. The complete digestion of 20\u00a0pmol \u03bcL\u22121 horse cytochrome C is observed without the need of protein denaturation, reduction, or alkylation when the average residence time of the protein sample in a 140\u00a0cm \u2009\u00d7\u2009 50\u00a0\u03bcm capillary IMER is 165\u00a0s. For the proteins used in this study trypsin-reactors were described that were capable of faster digestion using denaturing agents like acetonitrile to enhance the digestion process. However, the presence of such agents would seriously hamper direct analysis of the peptides formed using RPLC and MS. Identification of the proteins cytochrome C and myoglobin is possible by the tryptic peptides that are produced on-line, separated by micro-RPLC, and analyzed using mass spectrometry with auto MS\u2013MS.\nThe open-tubular reactor can be produced easily, reproducibly, and inexpensively, and can be used for other applications such as enzyme-inhibitor studies. Future research will focus on the development of miniaturized multi-dimensional analysis systems employing on-line digestion using these IMERs. The capillary enzyme reactors show no backpressure and seem promising for coupling to other analytical techniques such as capillary electrophoresis and surface plasmon resonance.","keyphrases":["trypsin reactor","on-line digestion","surface plasmon resonance","dextran hydrogel","liquid chromatography"],"prmu":["P","P","P","P","R"]}
{"id":"J_Gastrointest_Surg-3-1-1852378","title":"Management of Hepatic Angiomyolipoma\n","text":"Preoperative diagnosis of hepatic angiomyolipoma is difficult, and the treatment for it remains controversial. The aim of this study is to review our experience in the treatment of hepatic angiomyolipoma and to propose a treatment strategy for this disease. We retrospectively collected the clinical, imaging, and pathological features of patients with hepatic angiomyolipoma. Immunohistochemical studies with antibodies for HMB-45, actin, S-100, cytokeratin, vimentin, and c-kit were performed. Treatment experience and long-term follow-up results are summarized. During a period of 9 years, 10 patients with hepatic angiomyolipoma were treated at our hospital. There was marked female predominance (nine patients). Nine patients received surgical resection without complications. One patient received nonoperative management with biopsy and follow-up. One patient died 11 months after surgery because of recurrent disease. We propose all symptomatic patients should receive surgical resection for hepatic angiomyolipoma. Conservative management with close follow-up is suggested in patients with asymptomatic tumors and meet the following criteria: (1) tumor size smaller than 5 cm, (2) angiomyolipoma proved through fine needle aspiration biopsy, (3) patients with good compliance, and (4) not a hepatitis virus carrier.\nIntroduction\nHepatic angiomyolipoma (AML) is a rare mesenchymal tumor of the liver composed of smooth muscle cells, adipose tissue, and proliferating blood vessels. Since its first description by Ishak in 1976, approximately 200 cases have been reported in the English literature.1 This type of tumor is usually seen in kidneys associated with tuberous sclerosis.2 Definite pathologic diagnosis is made by identification of the three different components and HMB-45 positive staining.3\nIn the past, this tumor has been considered an entirely benign and slow-growing lesion without the possibility of malignant transformation. Therefore, several authors have suggested that this disease can be managed with conservative treatment.4\u20137 However, since 2000, several reports have revealed that this kind of tumor can be malignant with evidence of recurrence.8\u201310 Although the combination of ultrasonography, computed tomography (CT), magnetic resonance (MR) imaging, and angiography increases the accuracy in diagnosis of hepatic AML, the correct preoperative diagnostic rate of imaging studies has been reported to be less than 50%.6,10\u201314 Even the postoperative pathologic diagnosis has been easily mistaken as hepatocellular carcinoma (HCC).14,15 Many patients have been treated with surgical resection of the tumor. Therefore, the proper treatment of hepatic AML has remained controversial.\nThe purpose of this study is to retrospectively review the clinical, imaging, and pathological features of patients with hepatic AML treated at our hospital and to summarize our experience in the diagnosis and treatment of this disease. We also review the literature to highlight the important questions concerning hepatic AML: (1) Is hepatic AML a pure benign tumor? (2) What is the natural course of this tumor? Does the tumor size enlarge frequently during observation? (3) What difficulties exist in preoperative diagnosis with imaging studies and fine needle aspiration biopsy (FNAB)? (4) Is it proper for a hepatitis-carrier patient with hepatic AML to be treated with conservative management? (5) What are the criteria for patients with hepatic AML to be treated with surgical resection or conservative management?\nMaterials and Methods\nThe clinical, imaging, and pathological features of 10 patients with hepatic AML treated at the authors\u2019 institute were retrospectively reviewed. The follow-up information was obtained in each case. All tumor tissue was paraffin-embedded for routine hematoxylin and eosin (H&E) staining. Immunohistochemical assays were performed using a three-step indirect peroxidase complex technique with the following antibodies: HMB-45 (DAKO, dilution 1:40), actin (DAKO, dilution 1:50), S-100 (DAKO, dilution 1:800), cytokeratin (Biogenix, dilution 1:80), vimentin (DAKO, dilution 1:50), and c-kit (MBL, dilution 1:200).\nResults\nPatients and Clinical Data\nTen patients with hepatic angiomyolipoma were diagnosed at National Taiwan University Hospital from July 1995 to June 2004. There was marked female predominance (9\/10). The median age was 44\u00a0years old with a range from 34 to 64\u00a0years. Most patients (60%) presented no symptoms and were detected incidentally by health check-ups or during medical exams for other diseases. Four of 10 patients had symptoms caused by the space-occupying effect of the tumors such as abdominal pain, abdominal fullness, and palpable mass, or other nonspecific symptoms such as fever, general malaise, or body weight loss (Tables\u00a01 and 2). None of them had a history of renal AML or tuberous sclerosis. Two patients were hepatitis B-virus (HBV) carriers. The plasma levels of \u03b1-FP and CEA were within normal limits in all patients. \nTable\u00a01Clinical Presentation of Hepatic AngiomyolipomaClinical FeatureNo. of PatientsAge34\u201364\u00a0years (median 44\u00a0years)Gender (female: male)9:1Symptoms\u00a0No symptom6\u00a0Abdominal pain2\u00a0Abdominal fullness2\u00a0Palpable mass1\u00a0Body weight loss2\u00a0Malaise1\u00a0Fever2Tumor location\u00a0Right lobe5\u00a0Left lobe4\u00a0Caudate1Tumor size (cm)\u00a0<53\u00a05\u2013101\u00a0>106Preoperative diagnosisAngiomyolipoma4(40%)\u00a0Based on radiological images2\u00a0Based on tumor biopsy2Hepatocellular carcinoma3(30%)Angiosarcoma1(10%)Hemangioma1(10%)Metastasis1(10%)Associated liver diseaseHBV carrier2Table\u00a02Clinical Profile of Patients with Hepatic AngiomyolipomaCaseSex\/AgeTumor Size (cm)\/lobeSymtoms\/SignsIncidental FindingTreatmentOutcome\/F\/U Months1F\/3418\/RNilH\/CAtypical hepatectomyWell\/39 mon2F\/3410\/REpigastralgiaRight lobectomyWell\/59 mon3F\/3713\/LPalpable mass, abdominal fullness, BW loss, feverExtended left lobectomyDead\/14 mon recurrent, liver and lung mets4F\/4020\/REpigastralgiaRight lobectomyWell\/109 mon5F\/427\/RNilH\/CFNAB and F\/ULost F\/U\/6mon6F\/4611\/LAbdominal fullness, malaise, BW loss, feverLeft lateral segmentectomyWell\/40 mon7F\/4915\/RNilExam of appendicitisS56 segmentectomyWell\/37 mon8F\/513\/CNilH\/CCaudate lobectomyWell\/40 mon9F\/532.5\/LNilF\/U echo due to colon cancer s\/pLeft lateral segmentectomyWell\/33 mon10M\/644\/LNilH\/CLeft lobectomyWell\/32monH\/C\u2009=\u2009health check-up, BW\u2009=\u2009body weight, F\/U\u2009=\u2009follow-up, FNAB\u2009=\u2009fine needle aspiration biopsy, mon\u2009=\u2009month, mets\u2009=\u2009metastasis, s\/p\u2009=\u2009postoperation\nImaging Studies\nBased on the combined imaging studies of abdominal ultrasonography, CT, MR imaging, and angiography, the diagnostic accuracy of hepatic AML in this series was only 20% (Table\u00a01). Other preoperative imaging impressions included hepatocellular carcinoma, angiosarcoma, hemangioma, and metastatic lesions.\nTwo other cases were diagnosed by fine needle aspiration biopsy (FNAB). The accurate preoperative diagnostic rate was 40% (4\/10) after imaging studies and FNAB (Table\u00a01).\nPathologic Study\nAll 10 patients had a single tumor. Five tumors were in the right lobe of the liver and four were in the left lobe. One tumor was located in the caudate lobe. Most tumor sizes were larger than 5\u00a0cm (70%). The median tumor size was 10.5\u00a0cm, ranging from 2.5\u00a0cm to 20\u00a0cm (Tables\u00a01 and 2).\nGross pathology identified all tumors as a well-circumscribed, nonencapsulated tumor masses consisting of soft to elastic tissue. The cut surface in tumors varied from yellow to dark brown.\nHistopathologic studies of these 10 tumors showed a picture of hepatic angiomyolipoma composed of myoid and vascular components with a variant content of fatty tissue. Hematopoiesis was noted in two cases. Immunohistochemical studies were performed in all patients except one (case 5). Most tumors were found positive for HMB-45 (10\/10), SMA (4\/9), S-100 (7\/9), Vimentin (6\/9), but negative for cytokeratin (0\/9). Only three tumors were found positive for c-kit (Table\u00a03). \nTable\u00a03Immunohistochemical StudyCaseHMB-45ActinS-100CytokeratinVimentinc-kit1++\u2212++\u2212\u2212+2+++++\u2212++3++\u2212++\u2212+\u22124++++\u2212\u2212+\u22125++6+++++\u2212+\u22127++\u2212++\u2212+\u22128++\u2212\u2212\u2212+\u22129++\u2212++\u2212\u2212\u221210++++\u2212++++: strongly staining, >30% positivity; +: weakly staining, 10\u223c30% positivity; \u2212 no staining, or <10% positivity\nTreatment and Follow-up\nOne patient (case 5) was confirmed with AML through fine needle aspiration biopsy. Nonoperative management with close follow-up was performed. However, this patient was lost after 6\u00a0months of follow-up. The other nine patients underwent hepatectomy with tumor resection. These nine patients, except for one patient (case 3), had no postoperative complications or disease recurrence, and were regularly followed up at our outpatient department, follow-up ranging from 32 to 109\u00a0months (Table\u00a02). The very unusual patient (case 3) was a 37-year-old woman with a 13\u2009\u00d7\u20099\u2009\u00d7\u20099\u00a0cm, large tumor at the left lobe of the liver, receiving extended left lobectomy (Fig.\u00a01a,b). Pathology revealed a picture of hepatic AML (Fig.\u00a02a,b).\nFigure\u00a01A 37-year-old woman (case 3) presented with fever and palpable abdominal mass. (a) The axial view of contrast-enhanced CT scans on portal venous phase shows a huge hepatic tumor at the left hepatic lobe with heterogeneous enhancement. Notice the engorged vessels within the tumor are vividly identified (arrow). (b) The MR coronal Tru FISP, fast imaging with steady-state precession. (TR\/TE\/FA\u2009=\u20094.3\/2.1\/72\u00b0) shows engorged vessels in the tumor. The right portal vein (arrow) is displaced by the tumor. (c) After 6\u00a0months of extended left lobectomy, the abdominal ultrasonography reveals a huge recurrent tumor (arrows) in the previous location of left hepatic lobe, and numerous smaller tumors in the right lobe. (d) Celiac angiography also demonstrates the recurrent huge tumor and other multiple smaller ones in the right lobe of liver. Note the early drainage vein (arrow).Figure\u00a02Microscopic appearance of the hepatic angiomyolipoma in case 3. (a) The primary tumor is composed of polygonal to spindle cells arranged in solid sheets or trabecular pattern with endothelial lining. Some of the tumor cells have eosinophilic cytoplasm, and some have large fat vacuoles. Some of the nuclei are bizarre, and some have large eosinophilic nucleoli (H&E stain, original magnification\u2009\u00d7100). (b) The tumor cells are strongly immunoreactive for HMB-45 (original magnification\u2009\u00d7100). Recurrent tumor was noted 6\u00a0months later, and the patient received fine needle aspiration biopsy. (c) Microscopically, it shows tumor cells with clear to ample eosinophilic cytoplasm arranged in trabecular pattern (H&E stain, original magnification\u2009\u00d740). (d) Immunohistochemical staining shows the tumor cells are also positive for HMB-45 (original magnification\u2009\u00d7200).\nUnfortunately, 6\u00a0months later, ultrasonography showed recurrent hepatic lesions at the right lobe of the liver (Fig.\u00a01c). MRI also confirmed a large tumor in the caudate lobe and numerous smaller nodules in the right lobe of the liver. Angiography also revealed multiple tumor stains (Fig.\u00a01d). Fine needle aspiration biopsy was performed. The biopsy specimen was immunoreactive to HMB-45 antibody (Fig.\u00a02c,d). The clinical and histologic picture demonstrated recurrent malignant hepatic angiomyolipoma. At the 11th postoperative month, chest CT scans revealed multiple metastatic nodules. Three months later, the woman died due to hepatic failure and renal failure.\nDiscussion\nIn the past, hepatic AML has been considered as a \u201cbenign\u201d mesenchymal tumor. However, in 2000, Dalle reported the first case of malignant hepatic AML with vascular invasion and recurrence with multiple liver metastases and suspected portal vein thrombosis 5\u00a0months after primary tumor resection.8 Another two cases have been reported with hepatic recurrence after operation. One case was a 16-year-old girl with hepatic AML, receiving left lobectomy, with late recurrence noted 6\u00a0years after operation.9 The other one was in the Flemming\u2019s report. Recurrent hepatic tumors were noted 3\u00a0years after operation.10 Flemming also suggested that a proliferation index exceeding 3% and multicentric growth indicate a propensity for recurrence.\nIn this study, we reported a 37-year-old woman with left hepatic AML. A recurrent hepatic mass was noted 6\u00a0months after tumor resection, and multiple lung metastases were noted later. The patient died 14\u00a0months after diagnosis. To our knowledge, this case is the fourth reported case of recurrence in the literature, and the tumor in this case behaved as the most malignant one.8\u201310 Therefore, hepatic AML should not be considered as an entirely benign tumor; at least, it has malignant transformation potential. Accordingly, conservative treatment should be performed carefully, especially for patients with poor compliance, who are unable to undergo a strict follow-up regimen.\nThere were few reports concerning the growth velocity of hepatic AML in long-term follow-up. In one retrospective study of 26 patients, there were six patients who were followed up for more than 1\u00a0year and finally decided to receive operation because of the enlargement of the lesions. In that study, the tumor size of one patient increased from 4 to 10\u00a0cm during the 5-year follow-up. Another patient had a tumor increasing from 1.5 to 5\u00a0cm in 13\u00a0years follow-up.13 In another case report of a 38-year-old patient, the tumor size enlarged from 8 to 14.4\u00a0cm over a 3-year follow-up period.16 Irie also reported that a 40-year-old woman had hepatic AML with tumor size increasing in size from 4\u00a0cm to 7\u00a0cm during a 14-month follow-up period.16\nAlthough hepatic AML seems slow-growing, the probability of tumor enlargement and hence an induced mass-compression effect is not uncommon in the long-term follow-up period. In the present series, the median age of patients was 44\u00a0years old, and 70% of patients were below 50\u00a0years. If all of these patients had received nonoperative management, the mass effect of tumor enlargement might have been presented during a long-term follow-up period, especially in younger patient groups with longer remaining years of life. Moreover, the difficulties and complications of operation at later years would increase when the tumor enlarges, especially for those patients with an original larger tumor (>5\u00a0cm).\nSimilar to the patients presented in this series, most patients with hepatic AML are not symptomatic.12\u201314 Usually, these patients are diagnosed during health check-ups. Most symptoms are mass-compression effects including upper abdominal pain, abdominal fullness, and palpable mass. There are also some vague symptoms such as body weight loss, general malaise, and fever. In one review article with a collection of 52 patients, the incidence of symptoms or signs dramatically increased when tumor size was larger than 5\u00a0cm.11 Twenty-one percent (4\/19) of patients with a tumor smaller than 5\u00a0cm present symptoms\/signs; however, the incidence increases to 64% (7\/11) when tumor size is between 5 and 10\u00a0cm. The incidence increases to 89% when tumor size is larger than 10\u00a0cm. In our series, 40% (4\/10) of patients were symptomatic, and all four of these patients had a tumor larger than 10\u00a0cm. Accordingly, we suggested that patients with tumor larger than 5\u00a0cm should receive tumor resection, because most patients in this group were predisposed toward being symptomatic.\nThe typical findings in imaging studies of hepatic AML are as follows: (1) heterogeneously hyperechoic mass in US, (2) heterogeneously low density with low attenuation value (less than \u221220 HU) in plain CT, (3) high intensity on T1 and T2 weighted MRI, and (4) hypervascularity and tumor stain on angiography.12 Although a combination of US, CT, MRI, and angiography is able to increase the accuracy in preoperative diagnosis, hepatic AML usually shows various patterns in imaging studies. The differences in imaging studies occur because the relative proportions of vessels, muscles, and fatty tissue vary widely from one tumor to another. Consequently, hepatic AML is sometimes difficult to diagnose based on imaging studies.17 Therefore, fine needle aspiration biopsy has been reported to be useful in the preoperative diagnosis of this tumor.4,5,17,18\nHowever, more attention should be paid to the tumor\u2019s various morphologic appearances when minute samples are interpreted. With the combined tools of imaging studies and FNAB, the preoperative diagnostic accuracy has been smaller than 32% (ranging from 0 to 32%) in larger series.6,10\u201315 In a collaborative study reported by Tsui, including 30 cases from nine international hepatology centers, 50% were primarily misdiagnosed as carcinoma or sarcoma, either by imaging studies or by needle biopsy.15 In Flemming\u2019s series, only one preoperative case was diagnosed correctly.10 In the present series, only four preoperative cases (40%) were correctly diagnosed by combined imaging studies and FNAB.\nDefinite pathologic diagnosis of this tumor is usually made by identification of the three different components of smooth muscle cells, adipose tissue, and blood vessels. HMB-45 positive staining of myoid cells has been used as a pathologic characteristic of hepatic AML.3,19 Because of the rarity and pleomorphism of the histological features of hepatic AML, histologic diagnosis may be difficult, especially with needle biopsy. Many features in AML can mislead the unwary pathologist to a diagnosis of hepatocellular carcinoma: polygonal cells in trabecular arrangement, peliosis, nuclear pleomorphism, prominent eosinophilic nucleoli, deficient reticulin framework, presence of glycogen, eosinophilic globules, and tumor necrosis.14 In Zhong\u2019s series of 2000, none of the 14 cases were correctly diagnosed before operation. Furthermore, five cases were misdiagnosed as hepatocellular carcinoma or sarcoma by pathologists, even after operation. Therefore, we should be cautious when using FNAB as a diagnostic tool.\nIn an endemic area of hepatocellular carcinoma such as Taiwan,20 conservative management is risky because cases of fat-rich minute hepatocellular carcinoma will make the differential diagnosis more difficult. Furthermore, Chang reported one case with hepatic AML and concomitant hepatocellular carcinoma.21 In this series, two patients were carriers of hepatitis B virus with a high risk for hepatoma formation. Not only would these hepatitis-carrier patients bear more risk, but physicians would also bear more risk and psychological pressure during a long-term follow-up period if conservative management were adopted.\nBecause of the small patient number, we could not get definitely conclusive management suggestions solely from the results of this retrospective study. But a combination of our experience and a review of the literature, we suggest all symptomatic patients should receive surgical resection for hepatic angiomyolipoma. Conservative management with close follow-up is suggested in patients with asymptomatic tumors and meet the following criteria: (1) tumor size smaller than 5\u00a0cm, (2) angiomyolipoma proved through fine needle aspiration biopsy, (3) patients with good compliance, and (4) not a hepatitis-virus carrier.","keyphrases":["hepatic angiomyolipoma","angiomyolipoma","liver tumor"],"prmu":["P","P","R"]}
{"id":"Appl_Microbiol_Biotechnol-4-1-2271084","title":"Competition and coexistence of sulfate-reducing bacteria, acetogens and methanogens in a lab-scale anaerobic bioreactor as affected by changing substrate to sulfate ratio\n","text":"The microbial population structure and function of natural anaerobic communities maintained in lab-scale continuously stirred tank reactors at different lactate to sulfate ratios and in the absence of sulfate were analyzed using an integrated approach of molecular techniques and chemical analysis. The population structure, determined by denaturing gradient gel electrophoresis and by the use of oligonucleotide probes, was linked to the functional changes in the reactors. At the influent lactate to sulfate molar ratio of 0.35 mol mol\u22121, i.e., electron donor limitation, lactate oxidation was mainly carried out by incompletely oxidizing sulfate-reducing bacteria, which formed 80\u201385% of the total bacterial population. Desulfomicrobium- and Desulfovibrio-like species were the most abundant sulfate-reducing bacteria. Acetogens and methanogenic Archaea were mostly outcompeted, although less than 2% of an acetogenic population could still be observed at this limiting concentration of lactate. In the near absence of sulfate (i.e., at very high lactate\/sulfate ratio), acetogens and methanogenic Archaea were the dominant microbial communities. Acetogenic bacteria represented by Dendrosporobacter quercicolus-like species formed more than 70% of the population, while methanogenic bacteria related to uncultured Archaea comprising about 10\u201315% of the microbial community. At an influent lactate to sulfate molar ratio of 2 mol mol\u22121, i.e., under sulfate-limiting conditions, a different metabolic route was followed by the mixed anaerobic community. Apparently, lactate was fermented to acetate and propionate, while the majority of sulfidogenesis and methanogenesis were dependent on these fermentation products. This was consistent with the presence of significant levels (40\u201345% of total bacteria) of D. quercicolus-like heteroacetogens and a corresponding increase of propionate-oxidizing Desulfobulbus-like sulfate-reducing bacteria (20% of the total bacteria). Methanogenic Archaea accounted for 10% of the total microbial community.\nIntroduction\nThe anaerobic digestion of organic material is accomplished by the concerted action of various trophic groups of bacteria (Schink et al. 1992). The soluble organic compounds are degraded to CH4, CO2, and H2S via a syntropic interaction of fermentative and acetogenic bacteria with methanogens or sulfate-reducing bacteria (SRB). The electrons for CO2 reduction to methane or acetate, or sulfate reduction to sulfide are derived from the fermentative metabolism. The fermentative metabolism may include homoacetogenic or heteroacetogenic microorganisms depending upon whether acetic acid is the only major metabolic product (Drake 1994; Thauer et al. 1977) or other organic acids, such as propionic acid, in addition to acetic acid are produced (Seeliger et al. 2002). The products of fermentative acetogenesis and CO2-dependent homoacetogenesis can serve as substrates for acetate- and propionate-oxidizing SRB or acetoclastic methanogens depending upon the presence or absence of sulfate as electron acceptor. SRB are believed to outcompete methanogens in the presence of nonlimiting sulfate concentrations because they compete better for common substrates. This is explained on the basis of their kinetic properties (Ks and \u03bcmax) and given the favourable thermodynamic conditions (Ward and Winfrey 1985; Widdel 1988). In contrast, methanogens tend to dominate in low-sulfate environments (Stams 1994).\nIn most anaerobic environments, hydrogen is present as an intermediate for which SRB, hydrogenotrophic methanogens and homoacetogens will compete. Under standard conditions, sulfate reduction and methanogenesis are thermodynamically more favourable than homoacetogenesis. However, due to the interspecies transfer of fermentation products, such as hydrogen, the real thermodynamic values may be far from standard conditions and even less to reverse reactions. Studies with the sludge from the bioreactors have indicated that hydrogen is mainly consumed by SRB when H2 is limiting and sufficient sulfate is present (Isa et al. 1986). However, when H2\/CO2 was added as the sole substrate, a coexistence of heterotrophic SRB and homoacetogens was reported by Weijma et al. (2002), suggesting that when SRB are dependent for growth on homoacetogens, it is likely that the growth kinetics of homoacetogens determines the competition (Stams et al. 2005).\nThe composition of the influent chemical oxygen demand (COD; electron donor) to  ratio tends to have a significant effect on the competition for electron flow between different microbial communities (McCartney and Oleszkiewics 1993). The theoretical  mass ratio needed to achieve complete removal of organic matter is 0.67, assuming that eight electrons are transferred per molecule of sulfate (Lens et al. 1998). In general, low and high  mass ratios should favor sulfidogenesis and methanogenesis, respectively. However, values reported in the literature vary considerably (Annachhatre and Suktrakoolvait 2001; Choi and Rim 1991; Mizuno et al. 1994). Although the effect on the major processes like sulfidogenesis and methanogenesis on the whole has been well studied (McCartney and Oleszkiewicz 1993), our understanding of its influence on the diversity and dynamics of the microbial communities is still limited. This is partially due to the limitations of the traditional microbiological techniques, including pure culture isolations, most probable number estimation, selective enrichments, etc. Fortunately, molecular techniques, like denaturing gradient gel electrophoresis (DGGE; Muyzer et al. 1993) and fluorescence in situ hybridization (FISH; Amann et al. 1995), have provided alternative approaches to overcome the limitations associated with culture-based analyses.\nDGGE profiling of 16S rRNA gene fragments amplified from both genomic DNA and reverse-transcribed RNA, i.e., cDNA not only determines the presence and distribution but also the actively growing members in the mixed microbial communities (Dar et al. 2007a). FISH, on the other hand, allows for the identification and quantification at the level of populations and even single cells (Amann et al. 1995).\nIn the present study, the structure and function of anaerobic communities maintained in lab-scale continuously stirred tank reactors (CSTR) under sulfidogenic and nonsulfidogenic conditions was investigated. An integrated approach of molecular techniques including DGGE and FISH together with chemical analysis was used to link the microbial population dynamics with changes in the lactate (electron donor) concentration and sulfate availability. The focus of our study was the competition and interaction between the dominant microbial communities of SRB, acetogens and methane-producing Archaea.\nMaterials and methods\nReactor operation\nThe schematic representation of the experimental design is given in Fig.\u00a01. The start-up reactor (R-0) was inoculated with sludge from a methanogenic wastewater treatment plant treating lactate-containing wastewater. The reactor was operated in a continuous mode for about five to six volume changes at a dilution rate of 0.02\u00a0h\u22121 with lactate as the only electron donor and maintained under excess of sulfate. The lactate (12 electron) to sulfate (8 electron) molar ratio (r) was 0.65\u00a0mol mol\u22121. The culture in the reactor R-0 was used as an inoculum for the reactors, R1 and R2. The reactors R1 and R2 were operated at 30\u00b0C with a working volume of 2\u00a0l and at two different concentrations of lactate, 3.5 and 20\u00a0mM, respectively. Sulfate was fed to the reactors at a concentration of 10.32\u00a0mM, i.e., at ratios far below and far above the theoretical ratio of 0.67. Nonsulfidogenic conditions were applied to reactor R2 by further reducing influent sulfate feed and reducing the lactate concentration to 6.7\u00a0mM (r\u2009=\u200920.9). The composition of influent mineral medium was as follows (concentration in mM): KH2PO4, 3.6; Na2SO4, 10 or 0; NH4Cl, 6; MgSO4\u00b77H2O, 0.3; CaCl2\u00b72H2O, 0.4; FeSO4\u00b77H2O, 0.02; a stock trace element solution, 1\u00a0ml\/l; a stock selenite\u2013tungstate solution, 1\u00a0ml\/l; and a stock vitamin solution, 1\u00a0ml\/l. The stock trace element solution contained (mg\/l): H3BO3, 30; MnCl2\u00b74H2O, 100; CoCl2\u00b76H2O, 190; NiCl2\u00b76H2O, 24; CuCl2\u00b72H2O, 2; ZnCl2, 140; Na2MoO4\u00b72H2O, 36. The stock selenite\u2013tungstate solution contained (mg\/l): Na2SeO3\u00b75H2O, 6 and Na2WO4\u00b72H2O, 8. The stock vitamin solution was composed of (mg\/l): biotin, 10; vitamin B12, p-aminobenzoic acid, and calcium D (+) pantothenate, 50; thiamine, pyridoxine\u2013HCl, and nicotinic acid, 100. The medium was autoclaved, and the dissolved oxygen removed from the hot media by sparging it with oxygen-free nitrogen. The medium was maintained in an oxygen-free atmosphere by continuously flushing it with nitrogen. The pH in the reactors was maintained at 7.5 by automatic titration with 1\u00a0M NaOH and 1\u00a0M HCl. An automatic level controller maintained the working volume of 2\u00a0l inside the reactor vessels. The cultures were continuously sparged with oxygen-free nitrogen to flush the gaseous H2S. Sulfide precipitated as ZnS after letting the gaseous H2S through a zinc acetate trap connected to the gas exhaust of the reactor. Cultures were run at D\u2009=\u20090.02\u00a0h\u22121 for at least five to six volume changes (one volume change is 50\u00a0h) and until chemical analysis of effluent showed constancy over at least two consecutive days.\nFig.\u00a01Schematic representation of the experiment. Reactor R-0 was the start-up reactor. The culture in R-0 was used as an inoculum for the reactors R1 and R2a. The cultures were run at a dilution rate of 0.02\u00a0h\u22121 for five to six volume changes with each volume change of 50\u00a0h\nChemical analysis\nAcetate and other volatile fatty acids were analyzed either by gas chromatography (GC) or by high-performance liquid chromatography (HPLC). Sulfate was analyzed by ion chromatography as described previously (Scholten and Stams 1995). Sulfide was measured quantitatively by a colorimetric assay (Cline 1969). As sulfide was stripped from the culture liquid with nitrogen to reduce sulfide toxicity, the sulfide produced was calculated from the sulfate consumed. Hence, the given sulfide values indicate minimum values.\nNucleic acid extraction, reverse transcription of RNA, and PCR amplification\nNucleic acid extraction was carried out as described previously (Dar et al. 2007b). One microliter (80\u2013100\u00a0ng) of RNA template was used to carry out the reverse transcription (RT) of the isolated RNA into cDNA. Reverse transcription was performed using the iScript cDNA Synthesis Kit (BioRad, California, USA) according to the manufacturer\u2019s protocol. Amplification of the 16S rRNA gene fragment was performed using the primer pair 341F-GC and 907R and the protocol as described previously (Muyzer et al. 1995). Nested amplification with group-specific primers for SRB was also performed as described previously (Dar et al. 2005). We used 1\u00a0\u03bcl of genomic DNA and 2\u00a0\u03bcl of cDNA as template for the amplification reactions. The quality of the PCR products was examined on 1% (w\/v) agarose gel, and the yield was quantified by absorption spectrophotometry using the Nanodrop ND-1000 TM (NanoDrop Technologies, Delaware, USA).\nDGGE of 16S rRNA gene fragments and phylogenetic analysis\nDGGE was performed as described by Sch\u00e4fer and Muyzer (2001) using the D-Code system (BioRad Laboratories, California, USA). After electrophoresis, the gels were incubated for 30\u00a0min in a solution containing ethidium bromide (0.5\u00a0\u03bcg\/ml), rinsed for 20\u00a0min in Milli-Q water, and photographed using a BioRad GelDoc station (BioRad, California, USA). Individual bands were excised, resuspended in 20\u00a0\u03bcl of Milli-Q water, and stored overnight at 4\u00b0C. A volume of 3 to 5\u00a0\u03bcl of the supernatant was used for reamplification with the original primer sets. The reamplified PCR products were run again on a denaturing gradient gel to check their purity. Before sequencing, the PCR products were purified using the Qiaquick PCR purification kit (QIAGEN, Hilden, Germany).\nThe obtained 16S rRNA gene sequences were first compared to the sequences stored in publicly accessible database using the NCBI BLAST search tool (http:\/\/www.ncbi.nlm.nih.gov\/BLAST; McGinnis and Madden 2004). Subsequently, the sequences were imported into the ARB software program (Ludwig et al. 2004) and aligned using the automatic aligner function. The alignment was further corrected manually, and an optimized tree was calculated using the neighbor-joining algorithm with Felsenstein correction.\nDesign of oligonucleotide probes\nSpecific oligonucleotide probes for the 16S rRNA of the dominant anaerobic communities in the reactors were designed using the probe design tool of the ARB software package (Ludwig et al. 2004) as described previously (Dar et al. 2007a). The oligonucleotides used for in situ hybridisation are given in Table\u00a01.\nTable\u00a01Oligonucleotides used in this studyProbe nameTarget organismProbe sequence (5\u2032\u20133\u2032)ReferenceEUB338IMost bacteriaGCT GCC TCC CGT AGG AGTAmann et al. 1990EUB338IIPhylum PlanctomycetesGCA GCC ACC CGT AGG TGTDaims et al. 1999EUB338IIIPhylum VerrucomicrobiaGCT GCC ACC CGT AGG TGTDaims et al. 1999ARCH915ArchaeaGTG CTC CCC CGC CAA TTCStahl and Amann 1991SPS770Genus SporomusaATC CCG TTC ACT CCC CTGThis studySRB385Most DeltaproteobacteriaCGC GTC GCT GCG TCA GGAmann et al. 1990SRB385DbSome DeltaproteobacteriaCGG CGT TGC TGC GTC AGGRabus et al. 1996DSR660Genus DesulfobulbusGAA TTC CAC TTT CCC CTC TGDevereux et al. 1992DSM1265Genus DesulfomicrobiumAGA TTC GCT CGA CCT CGCThis studyDSV139Desulfovibrio sp. strain L7CGC TGT TAT CCC GAT CACDar et al. 2007aDSCOC814Desulfococcus groupACC TAG TGA TCA ACG TTTDevereux et al. 1992\nFluorescence in situ hybridization\nFixed cell samples from the reactors were immobilized on Teflon-coated multiwell microscopic slides as described previously (Dar et al. 2007b). Hybridization was carried out according to the protocol as described previously (Manz et al. 1992) using a formamide concentration of 35% (v\/v). Quantification of the hybridized cells was performed as described previously (Neef et al. 1996). The hybridized cells were analyzed by two independent observers for determining the fraction of positive signal from each probe relative to the signal visualized with general probes for bacteria (EUB338 I, II, and III), sulfate-reducing bacteria (SRB385 and SRB385Db), or with the general DNA stain DAPI (4\u2032,6\u2032-diamidino-2-phenylindole). In addition, a general probe specific for members of the domain Archaea (ARC915) was used. The hybridization experiments were done in duplicate, using different fluorochromes for each probe. Different microscopic fields on each slide were analyzed to confirm the results.\nHybridization stringencies of the newly designed probes were determined by performing hybridizations with increasing formamide concentrations as described previously (Manz et al. 1992) using target organism(s) and nontarget organism displaying three mismatches within the target region.\nSequence accession numbers\nThe sequences determined in this study were submitted to GenBank under accession numbers EU276620\u2013EU276626.\nResults\nMicrobial community dynamics at different lactate\/sulfate ratio\nFigure 2 summarizes the effluent composition in the bioreactors, after a minimum selection period of five to six volume changes, as a function of the different lactate to sulfate ratios imposed. The start-up reactor R-0 had a lactate\/sulfate ratio (r\u2009=\u20090.65\u00a0mol mol\u22121) theoretically sufficient to allow full lactate oxidation to CO2 with sulfate (also see Table\u00a02). The results for R1 with excess sulfate (r\u2009=\u20090.34\u00a0mo\/mol) indicate that after five to six volume changes only sulfate, sulfide, and acetate, and no other organic acids were detected in the effluent of the bioreactor (Fig.\u00a02). Nearly all lactate (3.5\u00a0mM) had been converted to acetate (3.2\u00a0mM) with the equivalent conversion of sulfate to sulfide. These measurements indicate partial oxidation of lactate to acetate, coupled to sulfate reduction was an important reaction in R1 (reaction 2, Table\u00a02). At a high lactate to sulfate ratio (r\u2009=\u20091.94\u00a0mol mol\u22121) in the reactor R2a, propionate was detected in the effluent besides acetate (Fig.\u00a02), demonstrating that lactate was partially fermented to a propionate and acetate mixture (reaction 3, Table\u00a02). In this sulfate-limited reactor, nearly all sulfate had been converted to sulfide. At a very low sulfate concentration (r\u2009=\u200920.9), lactate fermentation to acetate and propionate was observed as a dominant reaction (reactor R2b). The shift in the microbial population structure due to different lactate to sulfate ratios after five to six volume changes was recorded by DGGE profiling of 16S rRNA gene fragments, which were amplified either from genomic DNA or from reverse-transcribed RNA, i.e., cDNA.\nFig.\u00a02Effluent concentrations of sulfide, sulfate, acetate, and propionate in reactors R1, R2a, and R2b. The sulfide data are minimum numbers due to N2 strippingTable\u00a02Stoichiometry of anaerobic degradation reactions relevant to this studyNo.Reaction\u0394G01 kJ reaction\u221211\u2212128.52\u221280.83\u221255.74\u221237.75\u221285.46\u221231.1\nThe comparative analysis of the profiles based on the DNA and RNA of the same sample reflect not only the presence but also the actively growing populations. Figure 3 illustrates the results of the bacterial 16S rRNA\u2013DGGE reflecting the shift in the predominant populations with the changing lactate to sulfate ratios. For the three lactate to sulfate ratios used (i.e., r\u2009=\u20090.34, 1.94, and 20.9), the DGGE profiles based on 16S rRNA were not identical to the 16S rDNA-based profiles of the same sample. The phylogenetic affiliation of 16S rRNA gene sequences is presented in Fig.\u00a04. A neighbor-joining tree was generated using the sequences of the DNA fragments excised from the denaturing gel. Band B3, representing the most persistent species of all the microbial communities in the DNA based profiles, showed high sequence similarity to the Sporomusa\u2013Pectinatus\u2013Selenomonas group with the closest relative being Dendrosporobacter quercicolus. The DGGE profile of the samples taken from R2a to R2b showed an increase in the intensity of fragment B3. Fragment B5 was abundant in reactors R1 and R2a but with varying degrees of intensity. The fragment was not only persistently present but also belonged to an actively growing population as suggested by its simultaneous presence in the RNA-based profiles. However, the fragment was not present in the DGGE profile when the sulfate feed to the reactor R2b was brought down (r\u2009=\u200920.9). Comparative sequence analysis showed that band B5 clustered with the genus Desulfovibrio with Desulfovibrio mexicoense as the closest relative among the cultured representatives. Fragment B6, which was also significantly present, especially in the RNA-based profiles of R1 and R2a, was related to the members of genus Desulfobulbus with the closest relative being Desulfobulbus rhabdoformis. Band B4 was observed only in R1 maintained at a limiting lactate concentration (r\u2009=\u20090.34\u00a0mol mol\u22121). The fragment was found closely related to Desulfomicrobium apsheronum. Two other fragments excised, band B1 and band B2, although present, did not form a significant proportion of the active microbial communities in R1 and R2 as indicated by the RNA-based profiles. The two fragments were affiliated to an uncultured group of Cytophaga and Spirochetes, respectively. Nested amplification with group-specific primers (results not shown) resulted in the identification of an acetate-utilizing SRB represented by band B7. The sequence of the fragment was found closely related to Desulfococcus biacutus.\nFig.\u00a03DGGE analysis of 16S rRNA gene fragments using DNA and RNA samples from reactors R-0, R1, R2a, and R2b. Lanes 1 (DNA) and 2 (RNA) sample from reactor R-0 (influent lactate 6.7\u00a0mM); lanes 3 (DNA) and 4 (RNA) sample from reactor R1 (influent lactate 3.5\u00a0mM); lanes 5 (DNA) and 6 (RNA) from reactor R2a (influent lactate 20\u00a0mM); lanes 7 (DNA) and 8 (RNA) from reactor R2b (in the near absence of sulfate). Bands indicated with a dot were excised and sequencedFig.\u00a04Phylogenetic tree based on 16S rRNA gene sequences obtained from the DGGE bands (see Fig. 3). Sequences determined in this study are in boldface. Black dots on the nodes indicate bootstrap values of 90% and higher (1,000 replicates). The scale bar indicates 10% sequence difference\nThe archaeal 16S rRNA gene-targeted DGGE indicated that the diversity was limited to a single species (results not shown) and it was found at all lactate to sulfate ratios except the lowest ratio (r\u2009=\u20090.34) with 3.5\u00a0mM lactate. Sequence analysis of the excised fragment indicated its close relatedness to a group of uncultured Archaea distantly related to Methanosaeta sp.\nMicrobial community dynamics using FISH\nThe FISH results were obtained using oligonucleotide probes that targeted dominant microbial communities present under different substrate concentrations. The results are presented as relative abundances of populations targeted by specific probes and are expressed as the percentage of the total positive signal obtained by general probes for bacteria (EUB338 I, II, and III) or with the general DNA stain DAPI (4\u2032,6\u2032-diamidino-2-phenylindole). In addition, the general probe specific to Archaea (ARC915) gave a measure of Archaea. The percentage abundance of the specific probes relative to the general probes is summarized in Fig.\u00a05a and b. In general, probe EUB338 stained more than 90% of all the cells in the samples. Probe SPS770 specifically targeted the species that was closely related to D. quercicolus. At the influent lactate concentration of 3.5\u00a0mM (reactor R1), the relative abundance of this organism was <2% of the total bacterial population (Fig.\u00a05a and 6b). When compared to the start-up reactor R-0, the relative abundance of the cells targeted by probe SPS770 was 8\u201310% of the total bacterial cells (Fig.\u00a05a and 6a). The relative percentage of cells that hybridized with this probe was significantly higher (40\u201345% of the total bacterial cells), in samples taken from reactor R2a (lactate excess, 20\u00a0mM; Fig.\u00a05a and 6c) and increasing even higher to more than 70% (Fig.\u00a05a and 6d), when the sulfate feed to reactor R2b was minimized. Probes DSR660, DSV139, and DSM1265 were used to target different sulfate-reducing communities (Table\u00a01) that were identified in the reactor samples using DGGE. Desulfomicrobium-like SRB targeted by probe DSM1265 were present in high proportions at excess sulfate (reactor R1, r\u2009=\u20090.34\u00a0mol mol\u22121). The percentage abundance relative to the total bacteria was more than 65% compared to 15\u201320% in the inoculum (R-0; Fig.\u00a05b). The numbers were about 5\u20138% at limiting sulfate concentrations in reactor R2a (r\u2009=\u20091.94) and decreased further to undetectable levels in the virtual absence of sulfate in reactor R2b (r\u2009=\u200920.9). Desulfovibrio-like SRB targeted by probe DSV139 were the most resilient of all the SRB. Their numbers did not change significantly, fluctuating between 10% and 7% of the total bacteria in the two reactors R1 and R2-a, respectively (Fig.\u00a05b). They were reduced to undetectable levels when the sulfate supply to reactor R2-b was virtually stopped.\nFig.\u00a05a Relative abundance of Archaea, sulfate-reducing bacteria, and acetogenic bacteria. b Relative abundance of Desulfomicrobium-, Desulfovibrio-, Desulfobulbus-, and Dendrosporobacter-like bacteria. The percentage abundance is relative to the signal obtained with probe EUBmixFig.\u00a06Whole-cell hybridization of samples from reactors R-0, R1, R2a, and R2b probe EUBmix labeled with Cy5 (blue), probe SRB385 and 385Db labeled with Fluos (green), and probe SPS770 labeled with Cy3 (red). a Reactor R-0 (influent lactate 6.7\u00a0mM); b reactor R1 (influent lactate 3.5\u00a0mM); c reactor R2a (influent lactate 20\u00a0mM); d reactor R2b (in the near absence of sulfate)\nThe relative abundance of Desulfobulbus-like SRB targeted by probe DSR660 showed the most significant increase among all SRB detected, responding to an increase in lactate concentration combined with sulfate limitation. The percentage abundance increased from about 10\u201312% in the inoculum (reactor R-0; Fig.\u00a05b) to about 20% of the total bacteria in reactor R2a maintained at limiting sulfate concentration (r\u2009=\u20091.94). In reactor R1 (i.e., at sulfate excess conditions), the Desulfobulbus-like SRB were <5% of the total bacteria (Fig.\u00a05b).\nThe change to a low sulfate concentration in the reactor resulted in a considerable increase in the methanogenic population. FISH with probe ARCH915 indicated an increase of the methanogenic population from 5% in the inoculum (R-0) to more than 10\u201315% under limiting sulfate (R2-a) and nonsulfidogenic (R2-b) conditions (Fig.\u00a05a). The relative abundance of the methanogenic population decreased to undetectable levels after the shift from R-0 to the lactate-limited reactor R1.\nDiscussion\nThis study evaluates the shift in microbial structure in lab-scale CSTR reactors as affected by changing lactate to sulfate ratios using a combined approach of molecular methods (PCR\u2013DGGE and FISH) and chemical analysis. The combination of different methods allowed the establishment of a link between the population structure and function of the anaerobic communities in the reactors under sulfidogenic and nonsulfidogenic conditions.\nPCR\u2013DGGE profiles produced using DNA templates may not reflect the dynamic changes in community structure. Metabolically active cells have a much higher ratio of rRNA to DNA than dormant cells (Weller and Ward 1989). Therefore, the use of rRNA templates may emphasize the actively growing populations in the community as opposed to those that are relatively inactive but persistent in the community. Because PCR-based approaches for the analysis of microbial diversity in mixed populations can be influenced by several constraints (von Wintzingerode et al. 1997), our results based on PCR\u2013DGGE do not necessarily reflect the abundance of target sequences in the reactor samples. We, therefore, tried to confirm the relevance of the sequence data by whole-cell hybridization using fluorescently labeled oligonucleotide probes.\nDepending on different lactate to sulfate ratios, theoretically, various scenarios involving SRB in the degradation of lactate are possible as shown in Table\u00a02.\nComplete oxidation of lactate to CO2 and sulfide by SRB (reaction 1, Table\u00a02).Incomplete oxidation of lactate to acetate by SRB (reaction 2, Table\u00a02).Fermentative degradation of lactate to propionate and acetate and the utilization of fermentation products by SRB and acetoclastic methanogens (reactions 3, 4, 5, and 6, Table\u00a02).In the absence of sulfate the fermentative degradation of lactate coupled to acetoclastic methanogens (reactions 3 and 6, Table\u00a02).\nThe net metabolic pathway of the degradation of lactate by a mixed culture inoculum used in this study suggests its dependence upon the relative quantities of lactate and sulfate. Theoretically, the complete oxidation of lactate to inorganic carbon requires a minimum lactate to sulfate ratio of 0.67\u00a0mol mol\u22121 (reaction 1, Table\u00a02) and if the ratio is below 0.67, additional electron donor is required for complete sulfate reduction. At lower molar ratio of lactate to sulfate (0.34\u00a0mol mol\u22121, reactor R1), only sulfate, sulfide, and acetate were detected in the effluent. These results directly show the quantitative oxidation of lactate to acetate coupled to sulfate reduction as a dominant reaction (reaction 2, Table\u00a02). This is consistent with high percentage abundance of Desulfomicrobium- and Desulfovibrio-like SRB (known, incompletely oxidizing, SRB capable of performing reaction 2, Table\u00a02; Figs.\u00a05a and 6b; Widdel 1988). Complete lactate oxidation to carbon dioxide with sulfate as electron acceptor would be energetically favorable (reaction 1, Table\u00a02). However, the kinetics of this complete oxidation may not enable efficient competition with the incomplete lactate oxidizers. This is evident from the observation that in batch culture in the presence of excess sulfate, incompletely oxidizing SRB outcompete completely oxidizing species due to their superior maximum specific growth rate on fermentation intermediates like lactate, ethanol, propionate, etc. (McCartney and Oleszkiewicz 1993; Widdel 1988). The actual competition between partial and complete lactate oxidizing SRBs will be determined by the affinity for lactate (\u03bcmax\/Ks).\nGiven the fact that the nested amplification suggested that the numbers of the complete oxidizers were very low in the inoculum, a long period of time would have been required before such SRB would have grown to a significant number. Therefore, we cannot rule out that eventually the complete oxidizers might have become dominant under lactate-limiting conditions in R1.\nIn the near absence of sulfate, lactate fermentation to acetate and propionate (reaction 3, Table\u00a02) was observed as a dominant reaction in the reactor R2b. This was reflected by a significant increase in the relative abundance of Dendrosporobacter-like species and a near washout of SRB species (Fig.\u00a05a and 6d). D. quercicolus is an obligate anaerobe that clusters with the Sporomusa\u2013Pectinatus\u2013Selenomonas group (Strompl et al. 2000). The members of this group are known to perform acetogenic and propionic acid fermentation (reaction 3, Table\u00a02; Biebl et al. 2000). Major fermentation products of D. quercicolus and other closely-related members of the group are acetate and propionate. Theoretically (reaction 3, Table\u00a02), the fermentation of 6.7\u00a0mM of lactate should yield approximately 4\u00a0mM of propionate and 2\u00a0mM of acetate assuming that 0.5\u00a0mM is assimilated into the biomass. However, the acetate concentration in the effluent of the reactor R2b (Fig.\u00a02) indicated a net loss of 15% of the electron donor. The plausible explanation for this loss can be acetoclastic methanogenesis (reaction 6, Table\u00a02) that can be assumed from an increase in the methanogenic population to 15% under nonsulfidogenic conditions (Fig.\u00a05a).\nThe lactate to sulfate molar ratio in reactor R2a (1.94\u00a0mol mol\u22121) was increased by a factor of three compared to the inoculum, reactor R-0 (0.67\u00a0mol mol\u22121) and a factor of about six compared to reactor R1 (0.34\u00a0mol mol\u22121). The conditions in reactor R2a were changed to sulfate limitation with a substantial increase in influent lactate concentrations. Indeed, the sulfate concentration had dropped to <0.2\u00a0mM, showing that the SRBs could no longer compete for the remainder of the substrates. The products in the effluent included propionate and acetate besides sulfide, suggesting that lactate was partially fermented to a propionate and acetate mixture (reaction 3, Table\u00a02). This indicates a change in the net metabolic pathway for the degradation of lactate compared to the reactor R1. This change may be attributed to the lactate fermenting bacteria catalyzing reaction 3 (Table\u00a02). The same is suggested by a significant increase in the population of D. quercicolus-like species, 5 times increase relative to reactor R-0 and even 20\u201325 times relative to reactor R1 (Fig.\u00a05a and 6c). This is in agreement with the previous studies performed (McCartney and Oleszkiewicz 1993; Mendez et al. 1989; Pichon et al. 1988; Ueki et al. 1986). In all these studies, an increase in the buildup of propionate was observed with increasing organic substrate to sulfate ratio. McCartney and Oleszkiewicz (1993) attributed this change to the net metabolism of lactate, the combined influence of the increased lactate to sulfate ratio, and the time for which the culture was exposed to excess sulfate. This is clearly also the case in our experiments: if a steady state has been reached, the DNA and RNA bands in the DGGE are expected to be similar, if not identical. As demonstrated in Fig.\u00a03, this was not the case. Again, if growth rates would have been only marginally different, a true steady state might have taken several weeks to become established, and hence, we cannot rule out that we have looked at a snapshot during a shift in the population. In this study, the exposure of the original inoculum from the anaerobic wastewater plant to excess sulfate was approximately 13\u00a0days during start-up reactor, R-0, before an increase in the lactate to sulfate ratio was imposed in reactor R2a (1.94\u00a0mol mol\u22121). This exposure time was enough to enrich SRB in sufficiently high numbers for the development of substantial sulfidogenesis leading to sulfate depletion. As pointed out above, the consequence was that the culture runs into sulfate limitation and, under this condition, acetogens and methanogens experience no competition for the remaining substrate. It is interesting to note that the buildup of propionate in reactor R2a was concurrent to the increase in D. rhabdoformis-like SRB (Fig.\u00a05b), which are known to degrade propionate (reaction 4, Table\u00a02). This increase in the relative abundance of D. rhabdoformis-like SRB, suggesting a synergistic association between heteroacetogenic bacteria and SRB, is a feasible scenario in the presence of limiting sulfate.\nAn overall electron balance over the system was established, assuming a biomass yield of 0.07\u00b0C-mol C-mol\u22121. In the experiment, at a lactate to sulfate ratio of 0.34\u00a0mol mol\u22121 (reactor R1), the electron balance was nearly 95%. However, in the other experiments with R2, a net loss of reduction equivalents between 10% and 50% is observed. This may be attributed to methane or H2 formations, which were stripped out with the sulfide. Assuming the dominance of partial lactate oxidation coupled to sulfate reduction in reactor R1 (0.35\u00a0mol mol\u22121) and a partial lactate fermentation to propionate and acetate in reactor R2-a (1.94\u00a0mol mol\u22121), both H2 and acetate are the most plausible precursor for methane formation. However, the observation that relatively high concentrations of acetate were found in the reactor effluent (1\u20134\u00a0mM) and the detection of methanogenic Archaea which clustered with Methanoseata sp. suggests that, given the dilution rate of 0.02\u00a0h\u22121, only a low affinity acetoclastic methanogen catalyzing reaction (reaction 6, Table\u00a02) could be maintained in the system. These results were consistent with the increase in the percentage abundance of methanogenic Archaea to more than 10\u201315% of the total DAPI-stained cells. Because the conditions in the reactors did not favor the growth of nonmethanogenic Archaea (extreme halophiles, thermoacidophiles, the Archaeoglobales, and thermophiles placed in Crenarchaeota kingdom; Woese 1987), cells targeted by probe ARCH915 can be assumed to be the total methanogenic representation in this environment.\nIn conclusion, this study points to different metabolic routes being followed by a mixed anaerobic community, even in the mineralization of simple substrates like lactate. We could establish a link in microbial population dynamics to major perturbations caused by changing influent lactate to sulfate ratios. This study demonstrated that at low lactate to sulfate molar ratios in the influent, SRB had a competitive advantage over acetogens and methanogens and that, in the near absence of sulfate, heteroacetogens formed a syntropic association with methanogens. Higher lactate to sulfate ratio resulted in a pathway that had propionate and acetate as products, and the majority of sulfidogenesis and methanogenesis was dependent on the fermentation products. All these results were substantiated by corresponding shifts in relative abundance of the microbial communities present as analyzed by DGGE and FISH.","keyphrases":["sulfate-reducing bacteria","acetogens","methanogens","dgge","fish","anaerobic consortia"],"prmu":["P","P","P","P","P","M"]}
{"id":"Arch_Dermatol_Res-3-1-2064945","title":"Experimental models for the autoimmune and inflammatory blistering disease, Bullous pemphigoid\n","text":"Bullous pemphigoid (BP) is a subepidermal skin blistering disease characterized immunohistologically by dermal-epidermal junction (DEJ) separation, an inflammatory cell infiltrate in the upper dermis, and autoantibodies targeted toward the hemidesmosomal proteins BP230 and BP180. Development of an IgG passive transfer mouse model of BP that reproduces these key features of human BP has demonstrated that subepidermal blistering is initiated by anti-BP180 antibodies and mediated by complement activation, mast cell degranulation, neutrophil infiltration, and proteinase secretion. This model is not compatible with study of human pathogenic antibodies, as the human and murine antigenic epitopes are not cross-reactive. The development of two novel humanized mouse models for the first time has enabled study of disease mechanisms caused by BP autoantibodies, and presents an ideal in vivo system to test novel therapeutic strategies for disease management.\nEtiology of bullous pemphigoid\nIn 1953, Lever [28] described bullous pemphigoid (BP) as a subepidermal blistering disorder primarily seen in the elderly. Lesional\/perilesional skin of BP patients exhibits detachment of the basal keratinocytes of the epidermis from the dermis at the level of the lamina lucida [55], resulting in tense, fluid-filled vesicles. BP is both an inflammatory disease and an autoimmune disease, characterized by an inflammatory infiltrate at the site of the dermal\u2013epidermal junction separation and by the deposition of autoantibodies and complement components along the basement membrane zone (BMZ).\nA number of inflammatory cells are present in the upper dermis and bullous cavity, including eosinophils (the predominant cell type), neutrophils, lymphocytes, and monocytes\/macrophages. Both intact and degranulating eosinophils, neutrophils, and mast cells (MC) are found in the dermis. Local activation of these cells may occur via the multiple inflammatory mediators present in the lesional skin and\/or blister fluids, including (a) granular proteins derived from degranulated leukocytes, such as eosinophil cationic protein (ECP), eosinophil major basic protein (MBP), and neutrophil-derived myeloperoxidase (MPO) [1, 4, 8] and (b) chemoattractants and cytokines, such as C5a fragments, histamine, leukotriene B4, interleukin-1, -2, -4, 5, -6, -8, -15, TNF-\u03b1, IFN-\u03b3, RANTES, and eotaxin [9, 10, 21, 22, 46, 47, 48, 58, 62]. Additionally, several proteinases are found in BP blister fluid, including plasmin, collagenase, elastase, and 92-kDa gelatinase [2, 14, 24, 27, 44, 45, 52, 57]. These proteolytic enzymes may play a crucial role subepidermal blister formation in BP via their ability to degrade extracellular matrix proteins.\nBP patients generate a polyclonal repertoire of autoantibodies that bind to the BMZ and activate complement, as well as circulating autoantibodies [20]. These autoantibodies target two major hemidesmosomal antigens of 230\u00a0kD (BP230 or BPAG1) and 180\u00a0kD (BP180, BPAG2, or type XVII collagen) [25, 40, 56, 57]. BP230, a component of the hemidesmosomal plaque, is an intracellular protein, while BP180 is a type II transmembrane protein [19, 23, 56]. Like BP230, BP180\u2019s amino-terminal portion localizes to the intracellular hemidesmosomal plaque [15, 18, 19]. Its carboxyl-terminal region extends into the extracellular milieu of the BMZ, making it the preferred target for pathogenic BP autoantibodies. This antigenic extracellular region consists of 15 collagen domains separated from one another by non-collagen sequences. The largest of these non-collagen domains is referred to as NC16A. Epitope mapping studies indicate that BP autoantibodies of IgE and IgG isotypes and IgG1 and IgG4 subclasses recognize multiple epitopes that cluster within BP180 NC16A [3, 11, 16, 26, 63]. Serum levels of these autoantibodies are correlated with disease severity [11, 17, 49].\nMost BP patients elicit a cell mediated autoimmune response in addition to the humoral response described. Autoreactive CD4+ T lymphocytes recognize epitopes within the extracellular region of BP180, primarily in the NC16A domain [5, 29]. These T cells express memory cell surface markers and exhibit a Th1\/Th2 mixed cytokine profile. These studies suggest that BP is a T and B cell-dependent and antibody-mediated skin autoimmune disease.\nDevelopment of murine IgG passive transfer model of BP\nThe strong correlation between BP disease severity and serum BP180-specific autoantibody levels suggests that BP blister formation is mediated by autoantibodies. Early attempts to demonstrate the pathogenicity of patient autoantibodies via a passive transfer mouse model were unsuccessful because BP autoantibodies that react with an immunodominant and potentially pathogenic epitope in BP180-NC16A fail to cross-react with the murine form of this autoantigen (mBP180 NC14A) [30]. In 1993, Liu et al. [30] devised a strategy to overcome this difficulty and generated rabbit polyclonal antibodies raised against a cloned segment of mBP180 NC14A and passively transferred the purified rabbit anti-mBP180 IgG into neonatal BALB\/c mice. The injected animals developed a disease that exhibited the following hallmarks of human BP: (a) clinical skin lesions; (b) in vivo deposition of rabbit IgG and mouse C3 at the basement membrane by direct IF; (c) dermal-epidermal separation and an extensive inflammatory cell infiltration by H&E staining [30]. This infiltrate includes neutrophils, lymphocytes, and monocytes\/macrophages, with neutrophils being the predominant cells [7, 30].\nImmunopathogenesis of experimental BP in the murine model\nDevelopment of an in vivo system to study an experimental BP model has allowed for great progress in defining the etiopathogenesis of disease. Specifically, the roles of pathogenic antibodies, the complement system, inflammatory cells, and proteolytic enzymes have all been elucidated in the context of the murine IgG passive transfer model.\nInjection of anti-mBP180 IgG initiated subepidermal blister formation, and the levels of circulating anti-mBP180 antibodies completely determine disease onset and severity [30, 34]. Epitope mapping studies demonstrated that pathogenic anti-BP180 antibodies recognize a 9\u201312 amino acid stretch within the murine BP180 NC14A region of the antigen [31]. This epitope overlaps the region of the human BP180 NC16A that contains the immunodominant epitopes recognized by human BP autoantibodies, supporting the relevance of this murine system as a model for human disease.\nUsing mice deficient in different inflammatory cell types, it has been demonstrated that mast cells, macrophages, and neutrophils, but not T and B lymphocytes, play a direct role in subepidermal blistering in experimental BP [7]. Anti-mBP180 autoantibodies trigger skin blistering in wild-type mice and mice deficient in T, B, or both T and B cells, but fail to induce lesions in mice deficient in mast cells, macrophages, or neutrophils. Quantification of disease severity in these mice demonstrated that mast cells and neutrophils play a key role in experimental BP.\nAutoantibodies bind to basement membrane antigens and activate complement in human BP. In the rabbit anti-mBP180 IgG-induced BP, complement activation is absolutely required. Both BALB\/c mice depleted of complement (by pretreatment with cobra venom) and C5-deficient mice are resistant to experimental BP, and reconstitution of the C5-deficient mice with C5a restores susceptibility [32]. F(ab\u2019)2 fragments generated from the pathogenic anti-mBP180 IgG cannot induce subepidermal blisters in C5-sufficient mice [32]. Mice deficient in complement component C4 (specific for the classical and lectin pathways), but not in factor B (specific for the alternative pathway), are resistant to BP [41]. Wild-type mice depleted of complement component C1q (specific for the classical pathway) fail to develop BP skin lesions when injected with pathogenic IgG [41]. These data suggest that the classical pathway of the complement activation plays a major role in development of disease, namely through the generation of C5a that in turn activates MCs [6].\nMice injected with pathogenic anti-mBP180 antibodies exhibit extensive MC degranulation in the lesional skin, similar to that observed in human BP [6, 59]. MC-deficient mice are resistant to experimental BP, but reconstitution of these mice with MCs restores their susceptibility to disease. MC activation precedes neutrophil infiltration, and either the absence of MCs or the inhibition of MC degranulation prevents neutrophil infiltration and blister formation. However, MC-deficient mice reconstituted locally with neutrophils, or injected locally with neutrophil chemoattractants IL-8 or TNF-\u03b1 form blisters in response to anti-mBP180 IgG. These results suggest that mast cells release proinflammatory cytokines critical for neutrophil recruitment [6].\nExperimental BP requires neutrophil infiltration, and there is a direct correlation between disease severity and number of infiltrating neutrophils [33]. Depletion of neutrophils, or disruption of the events upstream of neutrophil infiltration (complement activation and MC degranulation), renders mice resistant to BP blister formation. However, complement system and MC deficiencies only confer protection from the pathogenic effects of BP180 autoantibodies if neutrophil infiltration is blocked as a consequence; that is, complement activation and MC degranulation are only critical to disease progression in their roles of neutrophil recruitment. This suggests that infiltrating neutrophils are the cells that directly cause tissue injury in the dermal\u2013epidermal junction, leading to BP skin blisters.\nUpon activation through molecular interactions between the Fc\u03b3IIIR on the neutrophil cell surface and the Fc portion of pathogenic anti-mBP180 IgG [61], infiltrating neutrophils secrete proteolytic enzymes known to degrade the extracellular matrix, including neutrophil elastase (NE) and gelatinase B (GB). A deficit of either of these two enzymes blocks blister formation in mice [35, 36]. In vitro, although both GB and NE are capable of degrading the recombinant BP180 protein, only NE produces DEJ separation when incubated with skin sections [36, 52]. In vivo, the degradation of BP180 depends on NE activity. Taken together, these findings suggest that GB acts upstream of NE. Specifically, GB proteolytically inactivates the physiological inhibitor of NE (\u03b11-proteinase inhibitor), which allows for unmitigated NE cleavage of extracellular matrix proteins (including BP180), resulting in DEJ separation [37].\nDevelopment of the humanized murine passive transfer model\nWhile the rabbit anti-mBP180 IgG passive transfer model has provided invaluable insight to the key steps in BP disease development, it does not allow for experimentation with BP autoantibodies isolated from human clinical samples. A second animal model, in which rabbit antibodies directed against the extracellular domain of hamster BP180 (NC16A region) are transferred into neonatal hamsters, also lacks cross-reactivity with antibodies directed against human BP180 [59]. To assess the pathogenicity of human anti-BP180 autoantibodies, we generated a novel mouse strain in which the murine BP180NC14A was replaced with the homologous human BP180NC16A epitope cluster region [38]. The humanized NC16A (NC16+\/+) mice injected with anti-BP180NC16A autoantibodies develop BP, and the disease development depends on complement, mast cells and neutrophils (Fig.\u00a01).\nFig.\u00a01Proposed mechanism of subepidermal blister formation in humanized NC16A mouse model of BP. Subepidermal blistering is an inflammatory process involved in following steps: a anti-BP180 autoantibodies are injected into NC16A mice; b pathogenic antibodies bind to the pathogenic epitope of BP180 antigen in basal keratinocytes (BK); c the molecular interaction between BP180 antigen and anti-BP180 IgG activates the complement system (C\u2032) as evidenced by BMZ deposition of mouse C3; d C\u2032 activation products C3a and C5a cause mast cells (MC) to degranulate; e MC activation leads to neutrophil (PMN) recruitment; f infiltrating PMNs bind to the BP180-anti-BP180 immune complex via the molecular interaction between Fc\u03b3 receptors on neutrophils and the Fc domain of anti-BP180 IgG. Activated PMNs release proteolytic enzymes; g proteolytic enzymes degrade BP180 and other extracellular matrix proteins, leading to dermal\u2013epidermal junction separation; h pathogenic antibody-injected mice develop clinical blisters\nUsing the NC16A+\/+ mice and BP patients\u2019 sera, we mapped the pathogenic epitope of BP180 in vivo [39]. IgG passive transfer experiments identified one pathogenic epitope (referred to as BP180NC16A2.5). BP180NC16A2.5-specific autoantibodies induced subepidermal blisters in the NC16A+\/+ mice, and these blisters were blocked by pretreatment with recombinant BP180NC16A2.5. We also found that the NC16A2.5 pretreated mice had significantly reduced levels of BMZ-bound and circulating pathogenic antibodies and showed reduced complement activation, mast cell degranulation and neutrophil infiltration. These results suggest that targeting the pathogenic epitope specifically could be a new therapeutic strategy to treat BP.\nA second humanized animal model has been developed by Nishie et al. [42, 43] that replaces the mouse BP180 (COL17) with the human analogue. Upon injection of human anti-BP180 autoantibodies, these mice reproduce the DEJ separation at the lamina lucida, the deposition of human IgG along the DEJ, and an inflammatory cell infiltrate consisting of neutrophils and eosinophils seen in human disease. The pathogenic effects of the autoantibody injection are ablated by pretreatment with a 77-amino acid peptide fragment of COL17 NC16A, referred to as R1. These studies further support the therapeutic potential of employing decoy peptides to block the pathogenic epitope.\nRelevance of murine and humanized passive transfer models to human BP\nBoth in vitro and in vivo data demonstrate that BP180 is the target for pathogenic autoantibodies in BP (Table\u00a01). While clinical human BP and experimental murine and humanized murine BP closely mimic each other at the clinical, histological, and immunological levels, the IgG passive transfer models do not reflect the large number of eosinophils typically found in the inflammatory infiltrate of human BP lesional skin. Some patients do exhibit neutrophil-rich pemphigoid, indicating that the neutrophil-mediated blistering observed in these mouse models may be one of several disease mechanisms that contribute to the formation and persistence of subepidermal blisters. The presence of a neutrophil-rich inflammatory infiltrate in the two humanized mouse models, as well as in the aforementioned hamster model, lend credence to a neutrophil-mediated disease mechanism. Furthermore, findings that in vitro DEJ separation induced by human BP autoantibodies specific for BP180NC16A depends on neutrophils [51] and neutrophil-derived elastase and gelatinase B [50], and that BP180 degradation by human BP blister fluid depends on neutrophil elastase activity [57] support this theory. Taken together, these findings strongly suggest that like the IgG passive transfer model of BP, neutrophils may be responsible for subepidermal blister formation in human BP, at least in those patients who show neutrophil infiltration in their lesional\/perilesional skin. The humanized mouse models do not yet offer insight to an eosinophil-mediated mechanism of disease progression, leaving the role of these inflammatory cells, if there is any, yet to be discovered. It is possible that these established mouse models, without additional experimental manipulations, may not be able to fully duplicate human BP pathology related to eosinophil infiltration; therefore, they may not be appropriate to study the role of eosinophils in BP.\nTable\u00a01In vitro and in vivo evidence of pathogenicity of anti-BP180 antibodiesSystemAntibodies usedReferenceIn vitro\u00a0Human skin sectionBP sera[13]Anti-BP180NC16A autoantibodies[51]Rabbit anti-BP180NC16A IgGIn vivo\u00a0Wild-type miceRabbit anti-murine BP180 IgG[30]\u00a0HamsterRabbit anti-hamster BP180 IgG[60]\u00a0Humanized BP180 miceAnti-BP180NC16A autoantibodies[38, 39]\u00a0Humanized NC16A miceAnti-BP180NC16A autoantibodies[42, 43]\nZone et al. [64] reported that IgE hybridoma to LABD97 antigen, a component of the shed ectodomain of BP180, when injected subcutaneously in SCID mice with engrafted human skin, induced eosinophil infiltration and histological blisters in engrafted human skin. Fairley et al. [12] also reported that total IgE isolated from BP sera, when injected into human skin grafted onto athymic nude mice, triggered a dermal infiltrate composed of neutrophils, eosinophils, and histological separation of the epidermis from the dermis. These findings implicate eosinophils in BP; however, a direct link between eosinophil recruitment and blistering in these human skin graft models needs to be established.\nConclusions\nThe murine IgG passive transfer model of BP developed by Liu et al. [30] has provided an invaluable tool for the dissection of the mechanism of disease progression. The development of humanized murine models of BP now provides unprecedented insight to the pathogenic role of the autoantibodies that mediate disease. Initial observations with these humanized models indicate that humanized mouse BP exhibits phenotypic and immunological similarities with murine and human BP. Additionally, these humanized models enabled the first identification of pathogenic epitopes in human disease. Blocking of the pathogenic epitope with decoy peptides, if they do not enhance the autoimmune responses, presents a novel clinical therapeutic strategy that may be very effective in abolishing the pathogenic effects of disease.","keyphrases":["hemidesmosome","autoimmune disease","basement membrane","humanized animal model","inflammation"],"prmu":["P","P","P","P","U"]}
{"id":"J_Med_Internet_Res-5-4-1550574","title":"Generic Design of Web-Based Clinical Databases\n","text":"Background The complexity and the rapid evolution and expansion of the domain of clinical information make development and maintenance of clinical databases difficult. Whenever new data types are introduced or existing types are modified in a conventional relational database system, the physical design of the database must be changed accordingly. For this reason, it is desirable that a clinical database be flexible and allow for modifications and for addition of new types of data without having to change the physical database schema. The ideal clinical database would therefore implement a highly-detailed logical database schema in a completely-generic physical schema that stores the wide variety of clinical data in a small and constant number of tables.\nIntroduction\nClinical databases may contain a large variety of data from different domains, eg, patient visits, test results, laboratory reports, diagnoses, therapy, medication, and procedures. Clinical databases may have different purposes, eg, patient management, electronic patient records, clinical research, and quality control. Clinical databases usually have a large number of users with different requirements for views of the database. The administrator does not want to view data per patient, while the nurse must be able to lookup current medication for a specific patient. The researcher may want to do data mining on clinical information for thousands or millions of patients, and the clinician should be able to see his or her ambulatory schedule. Most clinical databases comprise only a part of these functionalities, but these examples illustrate the challenge that designers of clinical databases face. Furthermore, in contrast to schemas from many other domains (eg, finance and public administration) the logical data schemas of clinical data are always incomplete and developing.\nIn databases, an entity is a single person, place, or thing (eg, patient or diagnostic test) about which data can be stored. In conventional relational database design, each entity is mapped to one or more tables using values of one or more rows to uniquely identify each record. That means that for each entity there exists at least one table. This strategy works well for most databases even if the number of concepts involved in a domain may be high. As long as the domain of interest remains relatively unchanged, the table layout (ie, the physical schema) should work well for many years. The domain of clinical science in particular (and biology in general) is, however, under constant development as new concepts appear and old concepts are modified or deferred.\nIn a conventional database (that is, in a conventional relational database), new tables must be created to record new concepts. To give users access to the new tables, new forms must be designed and links to these forms must be provided in the user interface. If a table that is already in the database needs to be modified care must be taken not to destroy existing data and not to break any constraints. Accordingly, user-interface forms must be redesigned to reflect changes (eg, fields that have been added or removed) in existing tables.\nThe complexity and the rapid evolution and expansion of the domain of clinical information thus require a large maintenance overhead if data are laid out using a conventional design. For this reason, it is desirable that a clinical database be flexible and allow for modifications and for addition of new types of data without having to change the physical database schema. The ideal clinical database would therefore implement a highly-detailed logical database schema in a completely-generic physical schema that stores the wide variety of clinical data in a small (and constant) number of tables.\nThe aim of this project was to provide an overview of techniques and problems in generic design of Web-based clinical databases.\nMethods\nMedline was searched through PubMed [1]. Searching was done by trial-and-error using combinations of keywords to get the best match of articles covering the problem. Furthermore a search strategy was devised for Google [2] using a similar trial-and-error strategy.\nResults\nThe final PubMed search was done on July 11, 2003 using the search term:\n(generic database design clinical) OR (entity attribute value).\nThis term was translated by PubMed into:\n(((entity[All Fields] AND attribute[All Fields]) AND value[All Fields]) OR (((generic[All Fields] AND \n(\"databases\"[MeSH Terms] OR database[Text Word])) AND design[All Fields]) AND clinical[All Fields])).\nThirty-three papers were found and 13 were selected based on their title. Of these, 7 were selected based on their abstract and the full-text papers [3- 9] were either downloaded or ordered from the Danish National Library of Science and Medicine.\nGoogle was searched on the same day using the search term:\nclinical database generic design.\nThe search was restricted to the first 30 hits. One additional paper [10] and 1 Web resource [11] of interest were found.\nThe 9 resources were all from either of 2 research groups: Department of Medical Informatics, Columbia University, New York, NY and Center for Medical Informatics, Yale University, New Haven, Conn. Three production databases were the basis of the 2 group's research: The Clinical Data Repository at Columbia-Presbyterian Medical Center (CPMC), the Adaptable Clinical Trials DataBase (ACT\/DB), and SENSELAB.\nCPMC [8- 10] is a large clinical repository for millions of patients dating back to the beginning of the nineteen nineties. Several front-end applications offer access to the database giving different views for health care professionals, administrators and researchers.\nACT\/DB [3,4,6,7,11] is a clinical-trials database built upon the same design principles as CPMC. Nadkarni et al introduce the term \"entity-attribute-value (EAV) design\" for generic structuring of data in a relational database [7]. The database is accessible through a generic Web-based interface (WebEAV) [4]. Web forms for displaying and editing data are generated automatically during run time from metadata stored in the database.\nSENSELAB [5] is a database for heterogeneous neuronal data. As such it is not a clinical database. However, the SENSELAB architecture uses an object-oriented approach to the EAV model by defining classes and relations (EAV\/CR). The EAV\/CR architecture is useful for scientific data in general, but it is of special interest for clinical databases.\nThe principles and design issues involved in these databases are the focus of the remainder of this paper. I will not go into details about the specific implementations of these systems, rather I will present techniques involved in the design of generic database systems. For design details about the 3 database systems the reader is encouraged to consult the references.\nEntity-Attribute-Value Design\nIn conventional database design, each parameter of interest is represented in a separate column in a table. As new kinds of data need to be managed, the number of columns and\/or tables needs to grow.\nTo add a new attribute for patient description (eg, phone number) to a conventional relational database design (Table 1), another column has to be added to the table.\nTable 1\nConventional relational database design (example)\nPatientID\nName\nDate of Birth\n1\nJens Hansen\n1956-Aug-01\n2\nHans Jensen\n1974-Sept-04\nIn EAV design, however, data may be stored in a single table with (conceptually) 3 columns: 1 column for entity identification, 1 for attribute, and 1 for the value of the attribute (Table 2).\nTable 2\nEAV (Entity-Attribute-Value) database design\nPatientID\nAttribute\nValue\n1\nName\nJens Hansen\n1\nDateOfBirth\n1956-Aug-01\n2\nName\nHans Jensen\n2\nDateOfBirth\n1974-Sept-04\nTo add a phone number attribute in the EAV table (Table 2), all that is required is to define a new code for phone number to be stored in the attribute column. No change to the table schema is needed. Theoretically, most of the facts that are stored in a database can be stored in a single EAV table.\nThe EAV design has several advantages:\nFlexibility: There are no limits to the number of attributes per entity. The logical database schema can grow without affecting the physical schema.\nStorage: In a clinical database thousands of parameters are available while only a few may be recorded for each patient. In a conventional design this may lead to empty (NULL) fields. The EAV design does not need to reserve space for attributes with NULL values.\nEfficient entity-centered queries: If, for example, all information for a single patient is needed, it is necessary to query all data tables looking for information about this patient. In a conventional database this may be a time-consuming task that requires looking through hundreds of tables each of which may or may not have information for this patient. As the number of tables and columns grow, the query must be reprogrammed. In an EAV database only 1 table needs to be queried, no joins are necessary, and no change of code is required as the domain evolves. (A join combines data from 2 or more tables based upon a common attribute.)\nThe EAV design has, however, some drawbacks:\nData display: As discussed later, the user naturally regards data as being organized conventionally in tables and columns regardless of the physical layout of data. Consequently it may be necessary to transform (\"pivot\") EAV data into a conventional layout when displaying data. This and other tasks that a conventional database would do automatically (eg, referential integrity checking or form-to-subform linkage) require considerable front-end programming in EAV designs. (Referential integrity checking is checking that values in one table that are intended to be used as keys to another table are indeed found in the second table.)\nLess-efficient attribute-centered queries: In contrast to entity-centered queries, complex attribute-centered queries, which are based on attribute values, are significantly less efficient and technically more difficult in an EAV database than in a conventional database. The query \"show me all patients whose name starts with J and whose date of birth is earlier than 1970\" is straightforward in a conventional database. To achieve the same result in an EAV database, set operations (for example, INTERSECT) or joins on multiple versions of the EAV table would have to be performed. (INTERSECT is an operation that compares 2 queries to identify records that are found in both.) Set operations and joins are considerably slower than simple select operations. As the number of attributes increase the execution time increases exponentially. Querying EAV data will be discussed in greater detail later.\nConstraint checking: In a well-designed conventional database, constraint checking is either unnecessary or trivial. For example, in a conventional table non-null constraints may be placed on columns to prevent incomplete records from being saved. An incomplete record would appear if, for example, the user forgets to fill in a field on a form. In an EAV table a missing attribute-value pair would normally result in a missing record. For example, if no record for one patient's last name is saved in the EAV table this will\u2014from a logical point of view\u2014lead to data that is inconsistent, in the sense that the data for this patient will not be similar to the data for other patients. To prevent this from happening in an EAV database, checking of such constraints should be programmed into the user interface.\nMetadata\nEAV design is a way of simplifying the physical schema of the database, making it domain-independent. Regardless of the physical schema, the user naturally perceives the data as conventionally structured in tables and columns. The logical schema of the database reflects the user's perception of the data. In an EAV database the logical schema differs greatly from the physical schema. In a conventional database the two are similar. Therefore, an EAV system must have some means of translating the physical schema into a logical schema that reflects the user's understanding of data. This is achieved through metadata (or dictionary) tables whose content defines the semantics of the domain being modeled. An example of a metadata table could be a table listing the attributes available to the data in Table 2. In this example the metadata table would have 2 records, Name and DateOfBirth. If it is necessary to record further information about patients, eg, sex and phone number, that information should simply be added to the metadata table. Thus, in this case, metadata represent what would be the column names of a conventional data table. The metadata model may be enhanced considerably by, eg, adding more descriptive attributes to the metadata table. These attributes may have several purposes\u2014eg, definition of an attribute's data type, constraints, or display layout (text field, select box, etc). These issues will be discussed in greater detail in the next section.\nEvolution of the EAV Model\nIn the following sections, I give examples of different EAV schemas going from the most-simple, least-flexible to the most-advanced, most-flexible schema. The term \"simple\" is not to be interpreted as inadequate. The simple solution may be the right solution for a specific task.\nThe examples reflect the systems described in the literature but are simplified for pedagogical reasons.\nA Simple EAV Model\nA simple EAV schema for a clinical database is outlined in Figure 1.\nFigure 1\nSimple EAV schema for a clinical database. (The crows-foot symbol\u20143 small lines at the end of a relationship line\u2014illustrates a one-to-many relationship between patient and data, and between attribute and data. Text in each ellipse identifies table type.)\nTable 3 shows the database tables depicted in the schema of Figure 1. Data have been arranged in a conventional table for patient demographics, an EAV table for clinical events, and a metadata table defining the attributes available to the EAV table. Table 3 represents the patient from Table 1 after a course of influenza that started July 1, 2003 and ended July 11, 2003:\nThe entity part of the Data table is defined by the combination of patientID and date. The attributeID column holds a reference to the Attribute table, which defines the name and type of available attributes. In a real-world production database there would probably be another table to hold the definition of data types.\nValues may of course be of any type, for example, text, number, or Boolean (true\/false). In the example in Table 3, the Value field of the Data table is text type. Such a design achieves simplicity by storing all simple types as text values. This approach has, however, some drawbacks. First, not all data types will fit into a text field. Binary objects, eg, x-ray pictures or ECG (electrocardiogram) curves, are too large as are long texts (memo-fields). Second, queries based on values will be less efficient for nontextual values. The text \"12\" is less than the text \"2\" even though it is numerically greater, because text is sorted character by character, from left to right.\nTable 3\nDatabase tables for the simple EAV schema in Figure 1\nPatient table*\npatientID\nname\nDate of Birth\ngender\n1\nJens Hansen\n1956-08-01\nMale\nData table\n\u2020\npatientID\ndate\nattributeID\nValue\n1\n2003-07-01\n1\nInfluenza\n1\n2003-07-01\n2\n2003-07-11\nAttribute table\n\u2021\nattributeID\nattributeName\ndataType\n1\nDiagnosis\nText\n2\nEndDate\nDate\n* Conventional table for patient demographics.\n\u2020 EAV table for clinical events (data).\n\u2021 Metadata table defining attributes available to the EAV table.\nDifferent strategies have been used to store binary data and to increase the efficiency of value-based queries. The simple solution is to ignore the problem and accept that all values be stored as text. This approach may be fully acceptable if it is not necessary to store binary data and if fast value-based queries of large data sets are not required. Another approach is to add a column to the Data table for each data type necessary. For each record, only 1 value-field will be filled in (Table 4).\nTable 4\nData table with a column for each data type, as a strategy for storing binary objects\npatientID\ndate\nattributeID\ntextValue\nnumericValue\nlongValue\ndateValue\n1\n2003-07-01\n1\nInfluenza\n1\n2003-07-01\n2\n2003-07-11\nThis approach, of course, does not comply with rules for good database design as empty fields are recorded for each record. It may, however, be acceptable in small \"quick-and-dirty\" applications [12].\nThe most solid and, from a database designer's perspective, correct solution is to segregate the data table into a number of tables based on the data type of the attribute (Table 5).\nTable 5\nData table segregated into multiple tables based on the data type of the attribute, as a strategy for storing binary objects\nData table\npatientID\ndate\ndataID\n1\n2003-07-01\n1\n1\n2003-07-01\n2\nDataDate table\ndataID\nattributeID\nvalue\n1\n2\n2003-07-11\nDataText table\ndataID\nattributeID\nvalue\n2\n1\nInfluenza\nThis approach is used in CPMC, ACT\/DB, and SENSELAB. For simplicity I chose to show only 1 data table in the illustrations.\nThe modeling of patient demographic data in a separate conventional table rather than in the EAV table is deliberate (although not necessary). For a schema that is not expected to change often, as is the case with patient demographics, the advantages of an EAV layout do not exceed its disadvantages; and conventional tables and EAV tables can coexist happily together. Furthermore, this design makes it easy to model the one-to-many relation between patient and clinical events. Relations between entities in an EAV table are complicated to model in the simple EAV design. In an electronic patient-record system, for example, it should be possible to record relationships between clinical events (eg, infection leads to a course of penicillin or myocardial infarction leads to death). The enhancement of the EAV design to handle complex relationships between classes will be described later with the EAV\/CR schema.\nFor a simple application intended mainly for data entry, the simple EAV schema may suffice. With the need for a more-advanced user interface for data-display and input purposes, however, some means of grouping attributes becomes necessary. With the simple EAV schema, grouping attributes together on display forms may be done only by entity (patientID and date) or attribute. The application has no way of telling how EAV data records are related and should be displayed together\u2014eg, multiple values from the same blood chemistry panel.\nEnhancing the EAV Model\nGrouping related attributes for display purposes may be accomplished in several ways. One or more descriptive columns may be added to the \"entity part\" of the Data table, or the metadata schema may be enhanced. An example of a combination of both methods is shown in Figure 2.\nFigure 2\nEnhanced EAV schema with grouping of attributes for form display. (Text in each ellipse identifies table type.)\nA group table and a form table have been added to the metadata schema. Attributes may now be grouped and attribute groups may be part of forms. To the entity part of the Data table a new field, formID, has been added telling the application to which form a data record belongs. Now any medical event recorded in the Data table belongs to a form and then may be displayed together with all the other attributes on that form. Furthermore, this design facilitates reuse of attribute groups on different forms.\nDepending on the domain being modeled and the requirements of the users, other metadata schemas may be suitable.\nThe simple and the enhanced EAV schemas discussed above are examples of the use of generic EAV tables in clinical database applications. Although to some degree generic, the proposed schemas will need adjustment to the actual domain in question. To achieve total domain-independence more refined models must be created.\nAn Object-oriented Approach to EAV Modeling\nThe EAV\/CR model adds an object-oriented framework to the EAV model by definition of classes and relations. The EAV\/CR model was developed for scientific data in general but is useful for clinical data [5].\nFigure 3 shows a simplified example of the EAV\/CR table layout used in the SENSELAB database. The class and the attribute tables hold the definitions of classes and their fields. The ClassHierachy table records relations between classes. In this example a subclass can have any number of superclasses, and a superclass can have any number of subclasses. The attribute table records the class to which the attribute belongs and the type of attribute. An attribute can be of any simple type and may even be of class type. Class instances (objects) are recorded in the Object table and instance fields are recorded in the Data table, which is similar to the data table in the simple EAV models.\nFigure 3\nEAV schema with classes and relations (EAV\/CR). Simplified from Nadkarni et al [5]. (Text in each ellipse identifies table type.)\nThe example in Table 6 depicts 2 classes, patient and doctor, which are subtypes of a common person class. The patient class has an attribute of object type referring to the patient's responsible doctor. For readability IDs are presented as names instead of numbers.\nThis example illustrates the use of inheritance and composition in database design. Inheritance and composition are two important concepts in object-oriented programming. Inheritance can be regarded as an \"is-a\" relationship between objects\u2014a patient is a person, and a doctor is a person. Composition is often referred to as a \"has-a\" relationship\u2014a patient has a doctor.\nThus, with this simple layout with (conceptually) just 5 tables, any real-world object can be recorded. Furthermore, objects may be part of other objects; and objects may be related through inheritance. Ad hoc relations between objects (eg, penicillin leads to rash) may be recorded as objects themselves. For this purpose, a class, ObjectRelation, could be defined with 2 attributes, objectID and relatedObjectID. More descriptive attributes may be added to this class if required\u2014eg, causality.\nObviously, considerable up-front programming is required to drive an ergonomic user interface for the EAV\/CR model in a real-life production environment. On the other hand, this is a one-time-only job. Another drawback of the EAV\/CR design is that the system administrator must have a solid understanding of the object-oriented framework in order to design useful classes. An EAV\/CR database is therefore hardly an end-user tool for the average clinician or researcher. As always, flexibility comes with a price.\nTable 6\nDatabase tables as an example of the EAV schema with classes and relations (EAV\/CR) in Figure 3\nClass table\nclassName\nPerson\nPatient\nDoctor\nClassHierachy table\nsuperClassID\nsubClassID\nPerson\nPatient\nPerson\nDoctor\nAttribute table\nclassID\nattributeName\ndataType\nPerson\nName\nText\nPerson\nDate-of-birth\nDate\nPatient\nDoctor\nClass: Doctor\nPatient\nGender\nText\nDoctor\nPosition\nText\nObject table\nobjectName\nclassID\nPatient01\nPatient\nDoctor01\nDoctor\nData table\nobjectID\nattributeID\nvalue\nPatient01\nName\nJens Hansen\nPatient01\nDate-of-birth\n1956-08-01\nPatient01\nDoctor\nDoctor01\nPatient01\nGender\nMale\nDoctor01\nName\nDoc\nDoctor01\nDate-of-birth\n1960-03-12\nDoctor01\nPosition\nHead\nQuerying EAV Data\nFrom a database perspective, querying EAV data is not different from querying conventional data. As mentioned earlier, however, in an EAV database, the physical layout differs greatly from the logical layout, and the user generally wants to see data displayed in a conventional format.\nAs an example, querying Table 1 for facts about patients whose names start with Jens and who were born before 1970 is straightforward:\nSELECT * \nFROM table1 \nWHERE name LIKE 'Jens%' \nAND dob < '1970';\nTo achieve the same result from querying Table 2 requires executing a rather-complex SQL (Structured Query Language) statement:\nSELECT d1.patientID AS patientID, \nd1.value AS name, \nd2.value AS dob \nFROM table2 AS d1 INNER JOIN table2 AS d2 \nUSING (patientID) \nWHERE d1.attribute='name' \nAND d1.value LIKE 'Jens%' \nAND d2.attribute = 'dob' \nAND d2.value < '1970';\nThe same result may be obtained in several ways, but in any case the query must include set operations (INTERSECT) or as in this example a self join for each attribute. (A self join is a join of a table with itself.) Aside from being complex and out of reach for most end users, these operations are far slower than simple select statements.\nI did an experiment using data for one million patients described by 3 attributes: name, date of birth, and gender. These facts were duplicated in a conventional table and in an EAV table in a MySQL database. Three queries were performed on each table with 1, 2, and 3 attributes respectively. Execution time was approximately 2 seconds for the conventional table irrespective of the number of attributes. For the EAV table execution time was 7, 14, and 24 seconds respectively. Thus execution time increases linearly with the number of rows (1 million in the conventional table and 3 million in the EAV table) and\u2014in the EAV table\u2014with the number of joins involved in a query. In the conventional table, however, the number of joins did not affect query time.\nSome strategies have been suggested to deal with this problem:\nThere may not be a problem. Attribute-centered queries are important for research questions; their performance is not critical for the care of individual patients. If the need for cross-patient data is infrequent the advantages of EAV design probably exceeds the disadvantages.\nAny need for regular cross-patient data access could be met by making backups of the production database and restoring them onto separate hardware. Resource-intensive queries run on the backup data will not affect the production server. Additionally, the EAV data schema could be transformed into numerous conventional tables after backup thus easing query design by end users with modest SQL skills [6].\nIf complex, attribute-centered, user-defined, ad hoc queries are important to an application, steps should be taken to facilitate this. First, a user interface, whether graphical or not, should be built to help users retrieve data. The user should be able to freely select any combination of attributes and criteria. The interface should then translate user requests into semantically-valid and syntactically-valid SQL statements; and from the user's point of view, it should not matter whether data are stored in conventional tables or EAV tables. This approach was taken by Nadkarni and Brandt in the development of the ACT\/DB Query Kernel [6].\nOptimization of queries may increase the efficiency considerably. Breakdown of complex SQL statements into smaller parts run sequentially may increase query speed. Each part accesses 1 or 2 tables to create a temporary table (or view). These (smaller) temporary tables are then joined [3]. Depending on the ability of the database engine to devise an efficient search strategy, the overall query speed may benefit from creating and joining smaller temporary tables compared to self-joining the full EAV table. An efficient database engine should, however, itself be able to optimize the original query, so that little is gained from this approach. In the MySQL database described above, the creation of a single temporary table took longer (more than 30 seconds) than the execution of the full 3-attribute search (24 seconds).\nJohnson et al [10] suggest an extension to the SQL-query language to facilitate \"pivoting\" of attribute-centered data into a conventional layout\u2014the Extended Multi-Feature (EMF) SQL. Extended Multi-Feature SQL processing time is linearly proportional to number of attributes.\nIn summary, querying EAV data is a more complex task than querying data in a conventional layout; and attribute-centered queries are less efficient with EAV data compared to conventional data.\nGraphical User Interface\nThe challenge for the user-interface designer of an EAV database is to display data and to let the user manipulate data simulating a conventional layout irrespective of the physical layout\u2014in other words: to bridge the physical and the logical schemas.\nThe World Wide Web offers an opportunity to simplify database deployment and maintenance. In a typical Web database application, the user's browser requests data from a remote Web server, which sends the request to a database server. After receiving data back from the database server, the Web server formats it into a Web page and sends it to the client browser.\nThere are several advantages of Web deployment:\nProblems of form deployment are eliminated since all forms reside on the Web server.\nDeployment costs are reduced because Web browsers are available free. Also, hardware costs are reduced since browsers usually have smaller hardware requirements than desktop database-management systems do.\nThe form-rendering model of Web pages is simpler and smarter than that of traditional software platforms. Objects on a Web page can be automatically reformatted when the browser win\u00addow is resized or the user changes the font size. Traditional software developers must put much effort into physical screen size issues. This is not necessary with Web forms.\nWeb browsers use clever caching algorithms. That means that when the browser visits a particular page, its contents are cached on the client. On revisit, only components that have changed are downloaded again. This reduces download time and network load.\nFor these reasons, Web deployment is becoming more and more popular for multi-user applications. However, Web database applications are significantly more complex to develop than traditional database applications for several reasons:\nWeb-development tools are less mature than tools for traditional software development; and development of Web database applications still requires much \"coding-by-hand.\" As an example, simple errors such as misspelled variable names, which would be trapped at edit or compile time in a traditional environment, will not be detected until runtime in a Web application.\nBrowser-server communication is inherently stateless; when the server has sent a Web page to the client, it \"forgets\" about the client. Tracking information (eg, user authentication) through several Web pages therefore involves extra programming. To maintain information, the developer must store data either in (hidden) form fields on Web pages or in session variables, which can be accessed as long as the session lasts. Both approaches complicate development and may compromise security because other users (or processes) may gain access to these data intentionally or accidentally.\nDesigning Web forms requires much more programming than does designing forms in traditional client-server environments. Web form fields are typeless and input masks for formatting user inputs are not inherent parts of Web forms. (In typeless fields the user may accidentally enter numbers in text-only fields or accidentally enter text in numbers-only fields.) This puts pressure on the programmer to put much effort into both client-side and server-side data validation. In a traditional environment, form fields may be typed; thus, eg, the programmer does not need to worry about users entering letters in number fields or invalid dates in date fields. In a Web form, all validation procedures must be hand coded. Finally, population of select boxes (drop-down menus) and radio buttons (option buttons) with dynamic data is usually much easier in a traditional environment than on a Web form.\nProgramming Web forms is tedious and error prone, and automation is highly recommended. Nadkarni et al have studied a generic framework for automatic generation of Web forms for display and manipulation of EAV data (WebEAV) [4]. The main objective was to automate the generation of Web forms based on metadata in an EAV database. When details about an event are requested, a form is generated from the metadata of the attributes involved. Each form field has a unique name, which is constructed such that the field name contains its own metadata. When data is sent back to the server, the server creates the correct SQL statements by parsing field names, and data are updated accordingly.\nWebEAV makes extensive use of client-side validation of data. Standard validation code in the form of JavaScript is built into the Web page. Validation relies on the use of form field events (eg, OnChange, OnFocus, and OnBlur) and metadata for the attributes in the form (eg, data type, maximum and minimum bounds, and non-null requirements).\nDiscussion\nBased on searching the literature, it appeared that the Entity-Attribute-Value model is useful for generic design of clinical databases. The most advanced model uses an object-oriented approach and gives tremendous flexibility, allowing the designer to model any type of concept and any relation between concepts in the domain of interest without ever having to worry about changing the table layout or maintaining the user interface. With the ever changing and evolving domain of clinical information, generic design is of special interest for clinical databases, because changes to the logical schema will not affect the physical schema. However, database designers from other areas (eg, biology or literature) may also find the EAV approach useful.\nHistorically, EAV was introduced into clinical databases in TMR (The Medical Record), built at Duke in the 1970s [13,14]. In addition to the ones mentioned in this paper, production databases using EAV components include TrialDB [14], the HELP system [15], the Cerner and 3M repositories, ClinTrial, and Oracle Clinical.\nPros and Cons of EAV Design\nThe advantages of generic design are obvious. The disadvantages, however, may be less obvious and depend on the objectives of the specific application in question.\nFrom a performance point of view, the strength of the EAV design lies in effective entity-centered queries since no joins are necessary to retrieve all facts about entities (eg, patients or medical events) as would be the case in a conventional design with facts spread over hundreds of tables. The drawback lies in inefficient attribute-centered queries, since a (self) join is necessary for each attribute that is requested.\nPerformance of EAV tables may not be an issue for small databases, but for large clinical repositories with hundreds of concurrent users, query time may be a critical factor. Also, the need for complex attribute-centered data retrieval differs greatly between applications. An electronic patient-record system, for example, is usually aimed at displaying patient-centered (ie, entity-centered) facts, while a research database usually must have some means of aggregating data across a large number of patients. In the latter, however, query efficiency may not be a problem, since data summaries are retrieved only intermittently and may be stored on separate hardware.\nThese issues warrant careful design of the database schema and cautious decisions about when to use conventional tables in place of generic EAV tables. As a rule of thumb, conventional table design is appropriate for entities whose schemas are not expected to change often (eg, people or institutions).\nMetadata Preserves Information\nThe simplicity and flexibility of the database schema also increases the complexity of collecting and displaying information from data. The user needs to see and enter related data on the same form. Often single values do not make sense unless coupled with other values. Take as an example a body weight of 176 lb (80 kg). This would be perfectly normal (and desirable for some of us) for an adult male with a height of 5 ft, 10 in (182 cm). For a 10-year-old girl, 176 lb would be highly disturbing. Using a simple EAV data table layout, relations between data are lost unless steps are taken to store these as well. This is the whole idea of metadata\u2014to conserve information about relationships between atomic data values. The metadata schema is the only thing that differs between the different EAV models presented in Figure 1, Figure 2, and Figure 3 and between the actual implementations of the EAV model presented in the articles. The data parts are for practical purposes the same.\nIt appears that metadata schemas themselves may be more or less generic depending on how closely related they are to the actual domain being modeled. The more specific the metadata schema is, the less flexible it will be. On the other hand, a specific metadata schema will require less programming to drive the user interface than a highly generic one.\nTo summarize this part, the choice of model depends on the domain and the requirements for flexibility. The object-oriented approach is by far the most flexible solution and in many ways an elegant solution. On the other hand, the complexity introduced by this model may not be justified unless the domain requires the fine-grained control over objects and relations. A simple model may well be the right solution for a simple job.\nDatabases and Objects\nMuch effort has been put into generalizing clinical databases. The most flexible and generic models take an object-oriented approach to data modeling the mapping of objects to tables in a relational database. There is no doubt that object-oriented design is \"hot\" in the medical area. But porting of object-oriented generic databases from traditional relational databases to produce object-oriented database management systems (OODBMSs) does not seem to be just around the corner. One reason for this may of course be that object-oriented database management systems are still lagging behind relational database management systems with respect to efficiency and availability, although extensive research is going on in this field. Furthermore, object orientation is still a new concept to most clinicians who design databases. But even with modest skills in an object-oriented programming language such as Java, the similarities between object-oriented programming and object-oriented data management seem striking.\nObject-oriented databases come in two flavors [16]:\nSystems that provide object-oriented extensions to relational systems by adding composite attributes, class hierarchies, and extensions to a data manipulation language such as SQL. These systems are called object-relational systems.\nSystems that extend an existing object-oriented programming language like C++ or Java to deal with databases. Such languages are called persistent programming languages. The term \"persistent\" refers to the fact that the programming language must devise some means of storing objects even when the program is not running. Databases built upon persistent programming languages are called object-oriented databases.\nThe former approach has similarities to the approaches described in this project in that these build upon conventional relational database management systems. The SENSELAB database allows for composition and inheritance, and CPMC has explored the extension of SQL to facilitate attribute-centered querying EAV data.\nThe latter approach to generic database design has to my knowledge not been described in the medical literature. The idea of encapsulating all data and functionality relevant to an object within each object opens up a plethora of possibilities of interest for the developer and manager of clinical information systems:\nThe object-oriented paradigm (\"everything is an object\") is a means of describing real-world concepts, and objects may be easier to understand for a clinician than complex relationship sets in a relational database. One could say that object-oriented design brings together the logical and the physical schema. Even if this may not be completely true, the user should not have to worry about how to design tables for storing of objects. The database will take care of this.\nObject-oriented languages handle complex attributes and inheritance much more elegantly than do even the most cleverly-designed relational database. When referring to an object in an object-oriented programming language, the object's fields and methods are available to the user immediately, through the object's interface. To mimic an object in a relational database, the database must be queried for all attributes of interest, and each value must be accessed separately.\nObjects may contain methods. For example, a person object may contain a print() method, which outputs all information related to the objects in a suitable format. The client programmer, who builds the user interface, does not have to worry how this information is gathered. This programmer only needs to grab the information and present it in a nice layout on a form. Furthermore, different subtypes of the person class, eg, patient or doctor, may have different implementations of the print() method. This is an example of polymorphism and is one of the most powerful features of object-oriented programming languages.\nClasses may be reused. If a class has been designed, it may be reused in other applications; and if a class is redesigned (eg, to improve execution speed) the client programmer does not need to know this, as long as the class' interface is unchanged.\nA detailed discussion of object-oriented programming is outside the scope of this article. However, the power of object-oriented programming may be summarized in the terms encapsulation and polymorphism. Encapsulation means that an object knows all about itself and that it interacts with the surroundings only through a well-defined interface. Encapsulation facilitates reuse and safe programming. Polymorphism means \"having many forms.\" A polymorphic reference is one that can refer to objects of different (sub) types at different times, which is exactly what we need in a generic database.\nIt is obvious that these (and other) facilities of object-oriented programming languages would be of immense value in the creation of generic clinical databases. It is, however, important to realize that a database management system, whether object-oriented or not, comprises much more than a programming and query language\u2014important issues being storage management, transaction management and concurrency control\u2014and these issues are still under development in object-oriented database management systems. (Concurrency control involves locking parts of the database to prevent unintentional overwriting of data.)\nConclusions\nThe objective of generic database design is to provide a robust physical database schema that does not need to change as the domain evolves. Generic databases are of special interest for clinical information systems, and several approaches to generic design have been exercised. They have in common the use of Entity-Attribute-Value tables for storing data and a number of metadata tables to describe the semantics and the relations between data. An object-oriented approach to generic modeling of metadata is by far the most flexible and domain-independent approach. However, the overhead in taking this approach may not be justified for less-advanced applications.\nFurther studies regarding the implementation of object-oriented database management systems for the purpose of generic clinical databases are suggested.","keyphrases":["databases","medical informatics applications","software design","internet"],"prmu":["P","R","R","U"]}
{"id":"Osteoporos_Int-4-1-2277446","title":"Efficacy of different doses and time intervals of oral vitamin D supplementation with or without calcium in elderly nursing home residents\n","text":"Summary The effect of equivalent oral doses of vitamin D3 600 IU\/day, 4200 IU\/week and 18,000 IU\/month on vitamin D status was compared in a randomized clinical trial in nursing home residents. A daily dose was more effective than a weekly dose, and a monthly dose was the least effective.\nIntroduction\nVitamin D deficiency is common in older persons, in particular in residents of homes for the elderly and nursing homes and in patients with hip fracture [1\u20133]. In these groups the prevalence of vitamin D deficiency, defined at that time as serum 25(OH)D\u2009<\u200930\u00a0nmol\/L based on values in healthy blood donors, was reported to be 75% [3]. This is mainly explained by the fact that older persons do not often go outside in the sunshine and dietary vitamin D intake is low. Vitamin D deficiency causes secondary hyperparathyroidism, which leads to cortical bone loss, osteoporosis and fractures [4]. It may also cause fatigue, muscle weakness, increased body sway and falls [5, 6] . Vitamin D supplementation in vitamin D deficient elderly increases the serum concentration of 25-hydroxyvitamin D (25(OH)D) and decreases the serum concentration of parathyroid hormone (PTH) [3]. It also decreases wintertime bone loss from the lumbar spine [7] and increases bone mineral density of the femoral neck [8]. Vitamin D supplementation combined with calcium decreased body sway and falls in a German study [5] and decreased hip as well as other non-vertebral fractures in French nursing home residents [9], whereas the results in more healthy elderly, living independently in the community were equivocal [10\u201314].\nVitamin D status in the elderly may be improved by ultraviolet irradiation [15] or by vitamin D supplementation [3, 7, 8\u201314]. Some controversy exists on the required serum 25(OH)D level, but most investigators agree that the level should be at least 50 or even 75\u00a0nmol\/l [16, 17].\nThe Dutch Health Council advises vitamin D 600\u00a0IU daily for elderly of 70\u00a0years and older who do not come outside in the sunshine [18]. Oral vitamin D3 can be taken once a day but also with longer intervals because of its long half life, being around 25\u00a0days. It is not known whether equivalent doses once a week or once a month are equally effective.\nA low calcium intake aggravates vitamin D deficiency by increasing the turnover of vitamin D metabolites by secondary hyperparathyroidism [4]. On the other side, a high calcium intake does not completely protect against secondary hyperparathyroidism, and thus cannot compensate for vitamin D insufficiency [19]. The calcium requirement for skeletal maintenance is raising with age whereas the capacity for compensating a low calcium intake declines with age [20]. In the Netherlands the mean daily calcium intake of independently living elderly in homes and apartments of the elderly is about 900\u00a0mg [2]. In the guidelines of the Dutch Health Council, the advised daily amount of calcium for elderly 70\u00a0years and older is 1200\u00a0mg [18].\nThe aim of the present study was to investigate, in a Dutch nursing home population, whether there is a difference in efficacy of different doses and intervals of oral vitamin D3 supplementation with the same total dose. A second aim was to assess the additional effect of calcium supplementation following vitamin D supplementation on serum PTH and markers of bone turnover.\nSubjects and methods\nSubjects\nTen somatic and psychogeriatric nursing homes participated and 1,006 subjects were invited. Of these, 146 did not respond, 386 refused to participate and 136 did not meet inclusion criteria. Participants were 338 (76 male and 262 female) patients of 70\u00a0years or older with a mean age of 84\u00a0years (SD 6.2). Exclusion criteria were going outside in the sunshine more than once a week, the use of vitamin D or calcium supplementation, the use of more than one vitamin D-fortified food or drink per day, complete immobilisation and a very poor life expectancy. Poor cognition was not an exclusion criterion. This did not affect adherence. Nursing homes were enrolled in the study throughout the year. Participants living together in the same nursing home started the study during the same season. The dietary vitamin D intake was estimated at about 100\u00a0IU\/day, based on fish and margarine consumption. In the Netherlands only margarine is fortified with vitamin D3 (3\u00a0IU\/g) and the diet does not contain vitamin D2. Written informed consent was obtained from participants or their proxies. The protocol as well as the patient information letters were approved by the Ethical Review Board of the VU University medical centre.\nRandomisation\nParticipants were randomised in blocks of six, to receive, during the study period of four and a half months, either oral vitamin D3 600\u00a0IU\/day (one tablet) or placebo, 4200\u00a0IU\/week (seven tablets once a week) or placebo or 18,000\u00a0IU\/month (one powder once a month) or placebo. (Solvay Pharmaceuticals, Weesp, Netherlands). After four months, participants in every group were randomised again to receive during 14\u00a0days either calcium carbonate or placebo. The first 156 participants who were randomised received 800\u00a0mg calcium carbonate (320\u00a0mg Ca2+) or placebo, the subsequent 120 participants received 1,600\u00a0mg calcium carbonate (640\u00a0mg Ca2+) or placebo. The study medication was centrally distributed to ensure compliance. The study was completed by 269 patients.\nMeasurements\nAt baseline co-medication was registered and a questionnaire for dietary calcium intake was used to calculate the mean daily calcium intake from dairy products, underestimating calcium intake by 200\u2013300\u00a0mg\/day [2, 21].\nThe ability of standing and walking was assessed by a standing score, ranging from 1 (cannot stand alone) to 5 (can easily get up and remain standing without help) and a walking score ranging from 1 (cannot do one active step) to 5 (completely independent walking). Both scores have previously been described [22].\nDuring the study all falls and fractures were registered by the nursing staff on special forms and checked with the routine incident registration. At the end of each study period in a nursing home, every ward was asked to complete a questionnaire on the opinion of the nursing staff about the suitability of each distribution form, compliance, the risk of making mistakes, time investment and preferences.\nRandom samples of the returned medication were counted in order to verify compliance.\nAdequate compliance was defined to exist when more than 80% of the study medication was ingested. Twice a quality check was made on the research medication by taking random samples for determining the vitamin D3 content of tablets and powders. Fasting blood samples were obtained at baseline, at two and four months.\nSerum 25(OH)D was measured by radioimmunoassay (Diasorin, Stillwater, MN) with an inter-assay coefficient of variation (CV) of 10% at 30\u00a0nmol\/L. Serum PTH was measured by radioimmunoassay (Incstar, San Juan Capistrano, CA) with an inter-assay CV of 10% at 3.5\u00a0pmol\/L. Serum carboxy-terminal collagen crosslinks or CTX, a marker for bone resorption, was measured by immuno-assay (CrossLaps, (Roche) with an interassay CV of 5%.\nFor these parameters the sera of a single participant were all measured within the same run to decrease variation. Serum calcium, phosphate, albumin, creatinine and alkaline phosphatase (APh) were measured using standard laboratory procedures, immediately after obtaining the blood samples. Serum calcium was corrected for serum albumin using the formula:\nStatistical analysis\nStatistical analysis was performed using SPSS 12.0.1. Data are presented as means (and standard deviation [SD]) or \u2013 in case of skewed distributions \u2013 as medians (and interquartile range [IQR]). Associations between baseline serum 25(OH)D and PTH, PTH and AF, and AF and CTX were examined by means of the Pearson correlation coefficient or \u2013 when one or both outcome variables had a skewed distribution \u2013 the Spearman rank order correlation coefficient. Baseline characteristics of dropouts and completers were compared by logistic regression analysis. Linear multilevel analysis with SPSS Mixed Models was used to investigate: (1) the effect of vitamin D supplementation on change (from baseline (t0) to 4\u00a0months (t2)) in biochemical outcome variables (serum 25(OH)D, serum PTH, bone turnover markers) and (2) the effect of additional calcium supplementation on change (from 4\u00a0months (t2) to 4.5\u00a0months (t3)) in biochemical outcome variables, adjusting for possible clustering of observations. The included levels were repeated measures (i.e., time), respondent, and nursing home. Nursing home was included in the final analyses only in case of a change of the effect size of more than 10%. Separate models were created with 25(OH)D, phosphate, corrected calcium, CTX, PTH, and APh as the respective dependent variables. We examined the potential confounding effect of season, age, sex, mean daily calcium intake, creatinine, standing and walking score at t0. For PTH and APh, logarithmic transformations were performed to normalize variance to allow parametric tests. For these log-transformed outcome variables, the estimated mean difference between two intervention groups was transformed back using an antilog transformation. The resulting estimate is the ratio of the geometric means of the outcome variable in both intervention groups. The geometric mean resembles the median. The level of significance was set at P\u2009<\u20090.05.\nResults\nBaseline characteristics\nTable\u00a01 shows the baseline characteristics of the 338 participants enrolled in the study. These were very similar for the different intervention groups. In the total group, baseline mean serum 25(OH)D was 25.0\u00a0nmol\/L (SD 10.9). In 55% of the participants, serum 25(OH)D was lower than 25\u00a0nmol\/L while 77% had levels below 30\u00a0nmol\/L and 98% below 50\u00a0nmol\/L (data not shown). Baseline median serum PTH was 7.2\u00a0pmol\/L (IQR 5.1\u201310.5) (ref. values: 1\u201311\u00a0pmol\/L). There were statistically significant correlations at baseline between serum 25(OH)D and serum PTH values (r\u2009=\u2009\u22120.25; P\u2009<\u20090.001), serum PTH and serum APh values (r\u2009=\u20090.16; P\u2009<\u20090.01), and serum APh and serum CTX values (r\u2009=\u20090.23; P\u2009<\u20090.001) (data not shown). The median daily calcium intake from dairy products was 750\u00a0mg (IQR 560\u20131035).\nTable\u00a01Characteristics of 338 participants at baseline (t0) by intervention group at t0 (Pl D = placebo vitamine D, D = vitamin D) and t2 (Pl Ca = placebo calcium, Ca = calcium)\u00a0TotalD totalD dailyD weekyD monthlyPl D totalCabPl Cab(\u2009=\u2009338)(\u2009=\u2009166)(\u2009=\u200955)(\u2009=\u200954)(\u2009=\u200957)(\u2009=\u2009172)(\u2009=\u200968)(\u2009=\u200971)% female77.576.583.672.273.778.576.578.9VariableMean (SD)Mean (SD)Mean (SD)Mean (SD)Mean (SD)Mean (SD)Mean (SD)Mean (SD)Age (yr)84.2 (6.2)84.2 (6.5)84.3 (6.3)84.3 (6.4)83.9 (6.9)84.2 (5.9)83.3 (6.2)84.5 (6.8)25(OH)D (nmol\/L)25.0 (10.9)24.9 (10.1)24.0 (8.6)26.7 (12.6)24.1 (8.8)25.0 (11.7)25.3 (10.6)24.1 (9.6)Calcium corrected (mmol\/L)2.42 (0.10)2.42 (0.09)2.42 (0.09)2.41 (0.08)2.42 (0.10)2.43 (0.10)2.41 (0.08)2.42 (0.10)Phosphate (mmol\/L)1.03 (0.14)1.02 (0.14)1.02 (0.13)1.02 (0.16)1.02 (0.13)1.04 (0.14)1.01 (0.14)1.03 (0.13)CTX (ng\/L)592 (277)571 (274)594 (274)626 (311)496 (218)613 (280)552 (288)565 (255)Albumin (g\/L)33.4 (3.3)33.4 (3.3)33.0 (3.5)33.1 (3.4)34.2 (2.8)33.4 (3.2)34.0 (3.2)33.0 (3.4)Standing score (1\u20135)3.3 (1.6)3.3 (1.6)3.5 (1.6)3.1 (1.7)3.5 (1.6)3.3 (1.6)3.2 (1.7)3.5 (1.6)Walking score (1\u20135)3.0 (1.4)3.0 (1.4)3.0 (1.4)3.0 (1.6)3.0 (1.3)3.0 (1.4)2.9 (1.4)3.1 (1.4)Skewed variableMedian (IQR)Median (IQR)Median (IQR)Median (IQR)Median (IQR)Median (IQR)Median (IQR)Median (IQR)Calcium intake (mg\/day)b750 (560\u20131035)750 (594\u20131015)725 (623\u20131039)755 (550\u20131028)788 (583\u2013955)750 (550\u20131053)773 (614\u20131024)730 (565\u20131003)PTH (pmol\/L)7.2 (5.1\u201310.5)7.2 (5.2\u201310.4)7.4 (5.2\u201310.4)6.7 (5.3\u20139.9)7.2 (5.1\u201310.5)7.1 (5.1\u201311.1)6.5 (5.2\u20139.4)7.3 (5.1\u201310.9)AF (U\/L)85 (69\u2013102)86 (71\u2013104)82 (73\u2013100)90 (68\u2013106)87 (67\u2013105)85 (69\u201399)89 (68\u2013105)82 (67\u201398)Creatinine (\u03bcmol\/L)93 (81\u2013103)92 (81\u2013103)87 (80\u201395)96 (85\u2013104)94 (81\u2013105)93 (82\u2013105)91 (80\u2013102)92 (81\u2013102)IQR = interquartile rangeaFrom dairy productsbWithin participants completing the vitamin D intervention (\u2009=\u2009276) treated with vitamin D (\u2009=\u2009139)\nTrial schedule\nFigure\u00a01 shows the trial schedule as well as the results of the randomisation procedure of both the vitamin D and the calcium intervention. Of the 341 participants originally enrolled, three were enrolled incorrectly because of hypercalcemia (corrected serum calcium: 2.69; 2.83; and 2.85), leaving 338 participants eligible for the study.\nFig.\u00a01Flow diagram of progress through the randomized clinical trial of vitamin D supplementation followed by the randomized clinical trial of calcium supplementation\nVitamin D intervention\nThe 338 enrolled participants were randomised to treatment with vitamin D3 one tablet of 600\u00a0IU each day (n\u2009=\u200955), a placebo in the form of one tablet each day (n\u2009=\u200957), vitamin D3 in the form of seven tablets (4200\u00a0IU) once a week (n\u2009=\u200954), a placebo in the form of seven tablets once a week (n\u2009=\u200958), vitamin D3 in the form of one powder once a month (n\u2009=\u200957), or a placebo in the form of one powder of 1,800\u00a0IU once a month (n\u2009=\u200957). The treatment period of four months was completed by 276 participants. Of the 62 drop-outs, 41 died during the study period and there were 21 withdrawals: nine participants became terminally ill; five participants became uncooperative to donate a blood sample; one participant showed signs of discomfort during blood sampling; one participant became immobile; 3 participants were moved elsewhere; and one participant dropped out for unknown reasons. Finally, one participant was excluded from the analyses due to extremely high levels of alkaline phosphatase (278, 1025, 2661\u00a0U\/L at, respectively, t0, t1, t2) for unknown reasons (further analysis was refused by the patient). The number of drop-outs did not differ significantly between the placebo (n\u2009=\u200935) and the vitamin D group (n\u2009=\u200927). Dropouts were similar to completers with respect to most baseline characteristics (sex, age, 25(OH)D, corrected calcium, phosphate, albumin, standing score, walking score, mean daily calcium intake, and creatinine), but had higher serum levels of CTX, and APh (P\u2009<\u20090.05) (data not shown).\nCalcium intervention\nThe 276 participants who completed the vitamin D intervention study were randomised to treatment with calcium one tablet each day (n\u2009=\u2009138), or placebo one tablet each day (n\u2009=\u2009138). The treatment period of 14\u00a0days was completed by 269 participants. Of the seven drop-outs, three died, three became terminally ill and one participant dropped out for unknown reasons. Only those treated with vitamin D were included in the analysis of the calcium intervention (n\u2009=\u200968 for calcium; n\u2009=\u200971 for placebo); there was only one drop-out in each group.\nEffectiveness of Vitamin D supplementation\nNursing home and potential confounding variables at baseline were not included in the final models since the effect sizes were hardly influenced by adding these variables.\nSerum 25-hydroxyvitamin D\nEffects of vitamin D supplementation on serum 25(OH)D in the various treatment groups are shown in Table\u00a02 and Fig.\u00a02. The mean difference in increase of serum 25(OH)D was 38.5\u00a0nmol\/L (95% confidence interval (CI) 25.6\u201341.5) in favour of vitamin D when compared to placebo. Daily, weekly and monthly administration of vitamin D resulted in increase of serum 25(OH)D when compared to placebo (P\u2009<\u20090.001). The mean difference in increase of serum 25(OH)D was highest after 4\u00a0months with daily administration of vitamin D (mean 47.2\u00a0nmol\/l) when compared to weekly (mean 40.7\u00a0nmol\/l, P\u2009<\u20090.01) and monthly (mean 27.6\u00a0nmol\/l, P\u2009<\u20090.001) administration. Weekly administration of vitamin D resulted in a greater increase of serum 25(OH)D than monthly administration (P\u2009<\u20090.001). The percentage of patients with serum 25(OH)D below cut offs of 25\u00a0nmol\/l, 50\u00a0nmol\/l and 75\u00a0nmol\/l is shown in Table\u00a03. At 4\u00a0months, the percentage of patients with serum 25(OH)D\u2009<\u200950\u00a0nmol\/l was 10.9, 10.6 and 36.4% in the daily, weekly and monthly groups of vitamin D supplementation respectively.\nTable\u00a02Mean and standard deviation (SD) or median and interquartile range (IQR) for biochemical measurements at baseline (t0), after 2\u00a0months (t1) and after 4\u00a0months intervention (t2) with placebo (Pl) or vitamin D (D) daily, weekly, or monthly in 276 participants\u00a0Mean (SD) or median (IQR)GroupsaMean differenceb or ratio geometric meansc (95% CI)P valuebGroupsaMean differenceb or ratio geometric meansc (95% CI)P valuebt0t1t2t0\u2192t2t0\u2192t2t0\u2192t2t0\u2192t2Serum concentration25(OH)D (nmol\/L)Pl25.2 (12.1)24.3 (11.2)25.5 (12.0)D\/Pl38.5 (35.7\u201341.3)0.000D daily23.0 (8.3)59.9 (16.5)69.9 (17.8)Dd\/Pld47.2 (42.3\u201352.1)0.000Dd\/Dw6.6 (1.7\u201311.4)0.009D weekly27.3 (12.7)58.8 (12.8)67.2 (14.0)Dw\/Plw40.7 (35.8\u201345.6)0.000Dw\/Dm11.2 (6.3\u201316.1)0.000D monthly23.8 (8.0)44.8 (14.1)53.1 (15.9)Dm\/Plm27.6 (22.8\u201332.5)0.000Dm\/Dd\u221217.8 (\u221222.7\u2013\u221212.8)0.000Phosphate (mmol\/L)Pl1.04 (0.12)1.05 (0.14)1.01 (0.14)D\/Pl0.057 (0.025\u20130.088)0.001D daily1.01 (0.14)1.07 (0.12)1.05 (0.11)Dd\/Pld0.088 (0.033\u20130.143)0.002Dd\/Dw0.022 (\u22120.033\u20130.076)0.434D weekly1.03 (0.15)1.06 (0.13)1.04 (0.14)Dw\/Plw0.065 (0.01\u20130.12)0.022Dw\/Dm\u22120.001 (\u22120.055\u20130.054)0.981D monthly1.02 (0.13)1.07 (0.16)1.04 (0.12)Dm\/Plm0.017 (\u22120.037\u20130.072)0.533Dm\/Dd\u22120.021 (\u22120.076\u20130.034)0.454Calcium corrected (mmol\/L)Pl2.42 (0.10)2.40 (0.09)2.42 (0.09)D\/Pl0.029 (0.008\u20130.05)0.007D daily2.42 (0.10)2.42 (0.10)2.45 (0.10)Dd\/Pld0.036 (0\u20130.071)0.050Dd\/Dw0.006 (\u22120.03\u20130.042)0.736D weekly2.41 (0.08)2.41 (0.10)2.43 (0.10)Dw\/Plw0.019 (\u22120.018\u20130.055)0.307Dw\/Dm\u22120.001 (\u22120.036\u20130.035)0.974D monthly2.42 (0.09)2.42 (0.09)2.44 (0.10)Dm\/Plm0.033 (\u22120.003\u20130.068)0.072Dm\/Dd\u22120.005 (\u22120.041\u20130.03)0.762PTH (pmol\/L)cPl7.2 (5.0\u201311.8)7.8 (5.6\u201310.8)7.5 (5.1\u201311.0)D\/Pl0.77 (0.7\u20130.85)0.000D daily7.3 (5.0\u201310.3)5.7 (4.3\u20137.45.1 (3.7\u20137.7)Dd\/Pld0.66 (0.56\u20130.78)0.000Dd\/Dw0.83 (0.7\u20130.99)0.037D weekly6.5 (5.3\u20139.5)6.5 (4.6\u20138.7)5.9 (5.2\u20137.6)Dw\/Plw0.85 (0.72\u20131.01)0.067Dw\/Dm1.02 (0.86\u20131.22)0.773D monthly7.2 (5.1\u201310.9)6.6 (4.4\u20139.3)5.6 (4.3\u20138.9)Dm\/Plm0.81 (0.68\u20130.96)0.019Dm\/Dd1.17 (0.99\u20131.39)0.061CTX (ng\/L)Pl598 (270)612 (274)624 (256)D\/Pl\u221214 (\u221257\u201329)0.491D daily578 (267)574 (265)579 (279)Dd\/Pld\u221234 (\u2212109\u201341)0.341Dd\/Dw1 (\u221273\u201375)0.976D weekly606 (316)599 (314)595 (304)Dw\/Plw\u221227 (\u2212102\u201348)0.445Dw\/Dm\u221236 (\u2212111\u201339)0.314D monthly490 (207)528 (229)523 (230)Dm\/Plm19 (\u221256\u201393)0.589Dm\/Dd35 (\u221240\u2013110)0.329AF (U\/L)cPl84 (67\u201399)82 (69\u201399)82 (67\u2013101)D\/Pl0.97 (0.92\u20131.03)0.363D daily82 (72\u201399)88 (69\u2013101)82 (67\u201397)Dd\/Pld0.96 (0.87\u20131.06)0.412Dd\/Dw0.96 (0.87\u20131.06)0.394D weekly88 (66\u2013107)81 (64\u2013105)80 (69\u2013104)Dw\/Plw1.03 (0.93\u20131.14)0.553Dw\/Dm1.1 (1\u20131.21)0.057D monthly86 (67\u2013103)80 (64\u201396)74 (63\u2013100)Dm\/Plm0.94 (0.85\u20131.03)0.180Dm\/Dd0.95 (0.86\u20131.05)0.270aDifferences in mean change between following groups: D\/Pl = vitamin D versus placebo; Dd\/Pld = vitamin D daily versus placebo daily; Dw\/Plw = vitamin D weekly versus placebo weekly; Dm\/Plm = vitamin D monthly versus placebo monthly; Dd\/Dw = vitamin D daily versus weekly; Dw\/Dm = vitamin D weekly versus monthly; Dm\/Dd = vitamin D monthly versus dailybMean difference of for example 38.5 for 25(OH)D (D\/Pl) means that the mean increase of 25(OH)D over 4\u00a0months was 38.5 in the vitamin D group compared to zero (set as a reference) in the placebo groupcFor PTH and AF the ratio of geometric means (resembling the ratio of medians) is presented in stead of the mean difference. Ratio of geometric means of 0.77 for PTH (D\/Pl) means that the median PTH decreases with a ratio of 0.77 over 4\u00a0months in the vitamin D group compared to a ratio of 1.00 (set as a reference) in the placebo groupFig.\u00a02Mean (\u00b1 1.96\u2009\u00d7\u2009SD) serum 25(OH)D concentrations at baseline, 2 and 4\u00a0months during treatment with vitamin D daily, weekly, or monthly, or placeboTable\u00a03Percentage of participants with 25(OH)D levels below a certain cut-off point at baseline and after vitamin D or placebo supplementationGroup:TotalPlaceboVitamin D totalVitamin D dailyVitamin D weeklyVitamin D monthly25(OH)D\u2009<\u200925\u00a0nmol\/LBaseline (t0)55.356.953.660.948.951.12\u00a0months (t1)30.157.82.92.20.06.84\u00a0months (t2)27.452.62.22.20.04.525(OH)D\u2009<\u200950\u00a0nmol\/LBaseline (t0)97.596.498.6100.095.7100.02\u00a0months (t1)65.494.836.526.119.165.94\u00a0months (t2)58.097.119.010.910.636.425(OH)D\u2009<\u200975\u00a0nmol\/LBaseline (t0)99.699.3100.0100.0100.0100.02\u00a0months (t1)95.299.391.287.089.497.74\u00a0months (t2)88.3100.076.663.072.395.5\nSecondary outcome measures: serum phosphate and corrected calcium\nSerum phosphate and corrected serum calcium values increased significantly more in the vitamin D group than in the placebo group. However, no differences between daily, weekly, or monthly administration were found (Table\u00a02).\nSerum parathyroid hormone and bone turnover markers\nEffects of vitamin D supplementation on serum PTH, serum APh and CTX in the various treatment groups are shown in Table\u00a02. Serum PTH (P\u2009<\u20090.000) decreased in the vitamin D group from 7.2 to 5.5\u00a0pmol\/l when compared to placebo, which is a decrease of 23% (ratio 0.77, 95% CI 0.70\u20130.85, \u2009<\u20090.001). The decrease of serum PTH was greater with daily administration of vitamin D when compared to weekly (P\u2009<\u20090.05) and monthly (P\u2009<\u20090.10) administration. The difference between weekly and monthly administration of vitamin D was not significant. The serum concentrations of alkaline phosphatase and CTX did not change following vitamin D supplementation.\nEffectiveness of calcium supplementation\nThere was no effect of calcium supplementation on any of the six biochemical outcome variables when compared to placebo. Also after stratification by administration of vitamin D (daily, weekly or monthly), an effect of calcium supplementation was not observed except for corrected calcium levels which increased more in the calcium group than in the placebo group in the daily dose vitamin D group only (P\u2009<\u20090.05). No effect was found of calcium doses (800\u00a0mg vs 1,600\u00a0mg calcium carbonate). Adding nursing home and other potential confounding variables at baseline did not influence the results.\nFractures\nThe number of falls and fractures did not differ between the intervention groups and the control groups, which was expected given the short study period of four and a half months.\nCompliance\nThe compliance assessed within 96 random samples of the returned medication was good. In the daily administration group, all 33 participants were compliant\u2013i.e., used at least 80% of the tablets\u2013(median\u2009=\u200997.0; IQR 94.5\u2013100). For weekly administration, 80% of the 35 participants were compliant\u2013i.e., used at least 80% of the tablets (median\u2009=\u200998.5; IQR 84.0\u2013100). For monthly administration, 93% of the 28 participants were compliant\u2013i.e., used at least four out of five powders (median\u2009=\u2009100; IQR 85.0\u2013100).\nSurvey nursing staff\nA survey among the nursing staffs of the participating nursing home wards showed a distinct preference (72%) for daily administration compared to weekly and monthly. Thirty-ning percent of the nursing staffs reported the impression that fewer mistakes were made using daily administration instead of weekly or monthly administration.\nDiscussion\nThe results of this study confirm the poor vitamin D status often observed in institutionalised elderly. In this study, baseline serum 25(OH)D levels in these nursing home residents was comparable to those observed in other studies in institutionalized elderly in the Netherlands [2, 3], resulting in median serum PTH levels in the upper normal range. A negative correlation between serum PTH and serum 25(OH)D was observed, confirming other studies [4].\nIn all treatment groups oral vitamin D supplementation appeared to be effective, resulting in increasing serum 25(OH)D levels and decreasing serum PTH levels as observed in other studies [3, 4, 23]. However, no effect was seen on serum alkaline phosphatase and CTX levels.\nDaily administration of vitamin D3 was significantly more effective than weekly and monthly administration. This could be due to more regular absorption in the gut or better compliance. The percentage of participants with serum 25(OH)D\u2009<\u200950\u00a0nmol\/l after four months of supplementation was about 10% in the daily and weekly groups, but was more than 35% in the monthly group. An option would be to increase the dose when vitamin D is supplemented only once per month. The dose of 600 IU\/day was chosen according to the Dutch and US recommendations [4, 18]. In order to attain a higher percentage of people with serum 25(OH)D\u2009>\u200950\u00a0nmol\/l, the dose of vitamin D should be 700\u2013800\u00a0IU\/day as was recommended for patients with osteoporosis in a recent review [24].\nThe overall decrease of serum PTH was 23% with vitamin D supplementation, which corresponds to our previous vitamin D supplementation study in a nursing home [15] and which is a larger decrease than that observed in healthy independently living elderly women where the decrease of serum PTH was 15% [8]. This is consistent with the more severe vitamin D deficiency and the greater degree of secondary hyperparathyroidism observed in these, mainly psychogeriatric, nursing home residents. Improvement of vitamin D status and suppression of PTH secretion may reduce bone turnover and bone loss, increase bone mineralization and thereby reducing fracture risk, although this was not the subject of this study.\nFor calcium supplementation, calcium citrate, lactate or carbonate can be used. In this study, calcium carbonate was used based upon bioavailability, cost and clinical efficacy [25]. The absorption of calcium from dairy products is about similar to that from calcium carbonate [26, 27]. Because of the side effects of calcium carbonate (gastrointestinal irritation, constipation, belches), possibly more pronounced in a population of frail elderly with substantial comorbidity and comedication, one should not choose a too high supplementation dose. Given the expected dietary calcium intake of about 900\u00a0mg per day, two supplementation doses 800\u00a0mg calcium carbonate (320\u00a0mg Ca2+) and 1,600\u00a0mg calcium carbonate (640\u00a0mg Ca2+), respectively) were used.\nThe median calcium intake in these Dutch nursing home residents (750\u00a0mg per day from dairy products, total estimated dietary intake 950\u20131,000\u00a0mg per day) was slightly lower than the guidelines recommend and relatively high compared to institutionalised elderly in other countries, probably due to a higher dairy intake. This was expected since every Dutch nursing home has its own dietician. Calcium supplementation combined with vitamin D in the last part of the study did not lead to a decrease of biochemical markers of bone turnover. An explanation might be that immobility is a cause of high bone turnover, which is not suppressed by calcium supplementation. The nursing staff\u2019s preference for daily supplementation of vitamin D is probably due to the fact that it fits better in a regular distribution routine, is less time consuming and less susceptible for making mistakes.\nIn conclusion, 98% of the participants had a baseline serum 25(OH)D lower than 50\u00a0nmol\/L. Oral vitamin D3 supplementation, administered daily was more effective than weekly doses in nursing home residents, while monthly administration was the least effective, 35% still having a serum 25(OH)D\u2009<\u200950\u00a0nmol\/l after 4\u00a0months treatment in this group. Calcium supplementation did not augment the effect of vitamin D supplementation.","keyphrases":["vitamin d supplementation","vitamin d deficiency","secondary hyperparathyroidism","calcium supplementation"],"prmu":["P","P","P","P"]}
{"id":"Qual_Life_Res-4-1-2275305","title":"Quantification of the level descriptors for the standard EQ-5D three-level system and a five-level version according to two methods\n","text":"Objectives Our aim was to compare the quantitative position of the level descriptors of the standard EQ-5D three-level system (3L) and a newly developed, experimental five-level version (5L) using a direct and a vignette-based indirect method.\nIntroduction\nThe EQ-5D is a widely used instrument to describe and value generic health (status) in terms of five dimensions: mobility, self-care, usual activities, pain\/discomfort, and anxiety\/depression. Each dimension comprises three levels, indicating no problems, some or moderate problems, and extreme problems, resulting in a total of 243 (35) unique health states [1].\nThe condensed format of the EQ-5D has undoubtedly contributed to its global dissemination, as it is easy to include in existing surveys by questionnaire designers, easy to fill out by respondents, and easy to report by analysts. However, compared with other generic preference based instruments such as the Health Utilities Index Mark 2 and Mark 3 (HUI2 and HUI3) and the Short Form 6D (SF-6D), which define respectively 24,000, 972,000, and 18,000 unique health states, the EQ-5D is lacking descriptive richness [2\u20135]. Although the EQ-5D descriptive system has demonstrated strong psychometric properties in general, its restricted ability to discriminate (clinically relevant) small to moderate differences in health status between individuals or within individuals over time is recognized [6\u20139]. Moreover, several studies have reported on the ceiling effect of the EQ-5D in the general population as well as in patient populations [10\u201315].\nA straightforward way of improving the discriminatory potential of the EQ-5D descriptive system is to increase the number of response options. In most health-status classification systems, the response options are ordered in terms of severity along a hypothetical measurement continuum. Since the exact position of the response options defines the discriminatory abilities of the descriptive system [16, 17], it is important to know where on the measurement continuum the level descriptors are quantitatively positioned.\nPrevious research in which a five-level (5L) version of EQ-5D was compared with the standard three-level (3L) EQ-5D demonstrated increased discriminatory power, increased reliability, and satisfactory validity [18, 19]. This paper presents a head-to-head comparison of the quantitative positioning of the level descriptors of the standard 3L EQ-5D descriptive system versus a newly developed, experimental 5L system, which covers 3,125 unique health states (55). Two independent methods were used. The first method directly compared the nonextreme level descriptors (for 3L: the level two midcategory; for 5L: the level two, level three, and level four categories) for each dimension separately on a visual analogue scale (VAS). The second, indirect, method required respondents to score complete health scenarios (vignettes) on dimension-specific VAS scales and subsequently to classify the same vignettes on the two EQ-5D instruments (3L and 5L).\nMethods\nInstruments\nThree instruments were used in this study: the standard EQ-5D3L version, an adapted Dutch 5L version developed in 1993 [20], and a set of five dimension-specific VAS scales. The version of the 5L EQ-5D used in this study was an experimental version, since at the time of this study, no official five-level version had been advocated by the EuroQol Group. We chose to test a five-level EQ-5D system, even though we also could have chosen four or six levels. An increase in the number of levels is always an increase of discriminatory potential at the cost of a more complex descriptive system (which might compromise the robustness of the value function). Five levels appears to be an optimal number of response options concerning reliability [21, 22]. Furthermore, Preston et al. (2000) investigated feasibility for 11 different rating formats (ranging from 2 to 11 and a 101 point scale) and found that feasibility peaked at five levels [23]. We chose to add two in-between levels to the existing 3L descriptive system (between levels 1 and 2 and levels 2 and 3) because we considered this the most obvious option in regard to the objective of refining the EQ-5D instrument. In any preference-based instrument, level descriptors are practically required for valuation research in which generic profiles are to be valued. A small focus group was assigned to determine the wording of the level descriptors. The level descriptors presented here were translated from Dutch. The one-, three-, and five-level descriptors in 5L were the same as the one-, two-, and three--level descriptors in the standard EQ-5D3L. The grading terms that were used for the intermediate levels two and four in the 5L-system were \u201ca little\u201d for level 2 (5L-2) in Anxiety\/Depression and \u201cmild problems\u201d for the remaining dimensions; and \u201csevere\u201d for level 4 (5L-4) in Pain\/Discomfort, \u201cvery\u201d for Anxiety\/Depression, and \u201cmany problems\u201d for the remaining dimensions. One further alteration was made to both the 3L and 5L systems: the most severe response category in Mobility was changed from \u201cconfined to bed\u201d to \u201cunable to walk about\u201d, so it would be analogous to the extreme response categories of the other dimensions. Table\u00a01 displays the exact wording of the descriptors in the 3L and 5L systems, respectively.\nTable\u00a01Direct quantification of three- and five- level (3L, 5L) descriptorsNumberMeanMedian95% CI3LMobility\u00a0\u00a0\u00a0\u00a0No problems in walking abouta\u2013\u2013\u2013\u2013\u00a0\u00a0\u00a0\u00a0Some problems in walking about7426.702222.82\u221230.59\u00a0\u00a0\u00a0\u00a0Unable to walk abouta\u2013\u2013\u2013\u2013Self-care\u00a0\u00a0\u00a0\u00a0No problems with self-carea\u2013\u2013\u2013\u2013\u00a0\u00a0\u00a0\u00a0Some problems washing or dressing self7430.182826.10\u221234.25\u00a0\u00a0\u00a0\u00a0Unable to wash or dress selfa\u2013\u2013\u2013\u2013Usual activities\u00a0\u00a0\u00a0\u00a0No problems with performing usual activitiesa\u2013\u2013\u2013\u2013\u00a0\u00a0\u00a0\u00a0Some problems with performing usual activities7729.742525.95\u221233.53\u00a0\u00a0\u00a0\u00a0Unable to perform usual activitiesa\u2013\u2013\u2013\u2013Pain\/Discomfort\u00a0\u00a0\u00a0\u00a0No pain or discomforta\u2013\u2013\u2013\u2013\u00a0\u00a0\u00a0\u00a0Moderate pain or discomfort6632.333128.56\u221236.10\u00a0\u00a0\u00a0\u00a0Extreme pain or discomfort6686.368983.75\u221288.98\u00a0\u00a0\u00a0\u00a0Worst imaginable pain or discomforta\u2013\u2013\u2013\u2013Anxiety\/Depression\u00a0\u00a0\u00a0\u00a0Not anxious or depresseda\u2013\u2013\u2013\u2013\u00a0\u00a0\u00a0\u00a0Moderately anxious or depressed6733.943429.89\u221237.99\u00a0\u00a0\u00a0\u00a0Extremely anxious or depresseda6788.829086.88\u221290.77\u00a0\u00a0\u00a0\u00a0Worst imaginable anxiety or depressiona\u2013\u2013\u2013\u20135LMobility\u00a0\u00a0\u00a0\u00a0No problems in walking abouta\u2013\u2013\u2013\u2013\u00a0\u00a0\u00a0\u00a0Mild problems in walking about7511.31119.73\u201312.88\u00a0\u00a0\u00a0\u00a0Some problems in walking about7538.394035.39\u201341.39\u00a0\u00a0\u00a0\u00a0Many problems in walking about7579.808276.81\u201382.79\u00a0\u00a0\u00a0\u00a0Unable to walk abouta\u2013\u2013\u2013\u2013Self-care\u00a0\u00a0\u00a0\u00a0No problems with self-carea\u2013\u2013\u2013\u2013\u00a0\u00a0\u00a0\u00a0Mild problems washing or dressing self7611.24109.72\u201312.76\u00a0\u00a0\u00a0\u00a0Some problems washing or dressing self7637.143834.14\u201340.15\u00a0\u00a0\u00a0\u00a0Many problems washing or dressing self7680.618177.81\u201383.40\u00a0\u00a0\u00a0\u00a0Unable to wash or dress selfa\u2013\u2013\u2013\u2013Usual activities\u00a0\u00a0\u00a0\u00a0No problems with performing usual activitiesa\u2013\u2013\u2013\u2013\u00a0\u00a0\u00a0\u00a0Mild problems with performing usual activities7711.08109.29\u201312.87\u00a0\u00a0\u00a0\u00a0Some problems with performing usual activities7739.014036.12\u201341.90\u00a0\u00a0\u00a0\u00a0Many problems with performing usual activities7780.818377.70\u201383.91\u00a0\u00a0\u00a0\u00a0Unable to perform usual activitiesa\u2013\u2013\u2013\u2013Pain\/Discomfort\u00a0\u00a0\u00a0\u00a0No pain or discomforta\u2013\u2013\u2013\u2013\u00a0\u00a0\u00a0\u00a0Mild pain or discomfort538.8587.43\u201310.26\u00a0\u00a0\u00a0\u00a0Moderate pain or discomfort5332.323129.58\u201335.06\u00a0\u00a0\u00a0\u00a0Severe pain or discomfort5367.946864.98\u201370.90\u00a0\u00a0\u00a0\u00a0Extreme pain or discomfort5391.269488.96\u201393.57\u00a0\u00a0\u00a0\u00a0Worst imaginable pain or discomforta\u2013\u2013\u2013\u2013Anxiety\/Depression\u00a0\u00a0\u00a0\u00a0Not anxious or depresseda\u2013\u2013\u2013\u2013\u00a0\u00a0\u00a0\u00a0A little anxious or depressed599.4687.97\u201310.94\u00a0\u00a0\u00a0\u00a0Moderately anxious or depressed5932.563330.01\u201335.11\u00a0\u00a0\u00a0\u00a0Very anxious or depressed5967.376664.55\u201370.20\u00a0\u00a0\u00a0\u00a0Extremely anxious or depressed5991.349289.42\u201393.25\u00a0\u00a0\u00a0\u00a0Worst imaginable anxiety or depressiona\u2013\u2013\u2013\u2013CI confidence intervalaLevel descriptor used as anchor in visual analog scale\nTo obtain quantitative values for each level descriptor of 3L and 5L, the VAS was used. We used five VAS scales, one for each EQ-5D dimension. Each VAS consisted of a horizontal hashmarked line without corresponding numbers, with the extreme-level descriptors belonging to that dimension as anchors. Respondents were asked to indicate their score on the VAS by marking the line. For the most severe category of Pain\/Discomfort and Anxiety\/Depression, the original descriptor was labeled \u201cextreme\u201d. Because the study was part of a larger process of choosing the definite level descriptors for the official five-level version of the EQ-5D, we decided to use the entire continuum of disability (extreme included), and used \u201cworst imaginable\u201d as upper VAS anchor for these two dimensions. This is analogous to the other three dimensions, which ranged from \u201cno problems\u201d to \u201cunable to\u201d.\nStudy design\nData collection took place in the form of one of two panel sessions and a follow-up postal survey 2 weeks later. A convenience sample of 82 laypeople from an existing general population panel (N\u00a0=\u00a0560) participated. All participants were familiar with the vignette presentation form used in the indirect method.\nAll participants completed both the direct and the indirect quantification task. For the direct method, all 3L answers were obtained during the panel sessions and all 5L answers as part of the postal survey to avoid memory effects. For the indirect method, participants scored ten health states in the panel sessions (acute pharyngitis, exacerbation of eczema, hip fracture, cerebrovascular accident\/stroke with moderate impairments, moderate gastritis, low spinal cord lesion, mild depression, back and neck pain, severe dementia, and acute multiple injury) and the remaining five in the survey (otitis externa, severe stable brain injury, irritable bowel syndrome, acute large burn, and posttraumatic stress disorder), because we expected that more than ten health states within one session could lead to concentration problems. The two sets of health states were balanced according to severity and duration. Following this design, the indirect method provided 225 responses for each respondent: 15 diseases\u00a0\u00d7\u00a05 dimensions\u00a0\u00d7\u00a03 response scales.\nDirect quantification of level descriptors\nIn the direct method, respondents were asked to project the 3L and the 5L descriptors on the VAS scales for each dimension separately. As the extreme levels were used as anchors of the VAS, for 3L only, the midcategory (3L-2) level descriptor needed to be scored, except for Pain\/Discomfort and Anxiety\/Depression, which needed additional scoring of 3L-3 (extreme). Similarly, the midcategories 5L-2, 5L-3, and 5L-4 descriptors were scored for each dimension, except for Pain\/Discomfort and Anxiety\/Depression, which included the scoring of 5L-5.\nIndirect quantification of level descriptors\nAs an alternative to the direct method, we developed an indirect method that we believe lies closer to the actual use of the EQ-5D instrument, as it uses a (hypothetical) health state as a calibrator or medium to derive a VAS score. In contrast to the direct method, the object of measurement in the indirect method is not a 3L or 5L descriptor but a complete health scenario (vignette). Each vignette was scored with the 3L and 5L descriptors and on a VAS, one for each separate dimension, independently. Consequently, an indirect head-to-head comparison of 3L and 5L scores could be made, calibrated via the common VAS score.\nFigure\u00a01 shows one of the vignettes. Each vignette was designed to present a disease as close to clinical reality as possible, therefore also including information on disease duration. All 15 diseases were presented on a standardized sheet (vignette) that contained (1) a disease label with a naturalistic description of the disease; (2) the course of the disease over a 1-year period using a calendar (the grey scales represent the duration of the disease); (3) the location of the disease with, if relevant, a visual representation; and (4) the EQ-5D dimensions, of which the levels were left unspecified, as the respondents were invited to select the appropriate EQ-5D level (according to his or her own view) for each dimension. Respondents were asked to read each vignette carefully and to select the level of each dimension of the EQ-5D descriptive system that best described the presented health state in their view using three response scales: the standard 3L response scale, the new 5L scale, and the VAS scale (similar to the VAS used in the direct method).\nFig.\u00a01Disease vignette with empty EQ-5D descriptive system\nThe 5L and 3L response scales were presented on the left and the right side of one page (per dimension), respectively. The respondents were first invited to score the 5L descriptors for all dimensions and all vignettes while covering the right side of the page that showed the 3L descriptors. Next, they were instructed to return to the first vignette, asked to cover the left side with the 5L scores, and provide the 3L response for all vignettes. Pilot testing revealed that when respondents scored 3L first, there was a tendency to avoid the in-between levels 2 and 4 of 5L, and for this reason, all respondents were asked to score 5L first. Adequate instruction was critical, stressing that 3L and 5L were two independent ways of scoring (in the postal survey, these instructions were repeated in writing). Subsequently, VAS scores were obtained on a separate form without respondents having access to the 3L and 5L scores. The demanding task of first providing 5L classifications on all five dimensions of all 15 vignettes minimized possible memory effects when the participants were instructed to return to the first vignette to score the 3L classifications while covering the 5L responses.\nAnalysis\nResults of the direct and indirect methods are presented with conventional descriptive statistics. Results of the indirect method were derived by grouping 3L-VAS pairs and 5L-VAS pairs for each respondent per vignette and subsequently by calculating level means over all vignettes and all respondents combined. For each respondent, scorings were removed for the combined 3L, 5L, and VAS scores if at least one of the 3L, 5L, or VAS scores was missing, equalizing the number of VAS observations between 3L and 5L.\nCharacteristics\nFor both the direct and indirect methods, the 3L\u20135L extension of EQ-5D was investigated in terms of three characteristics. First, equidistance addresses the degree to which 3L and 5L level descriptors are distributed evenly over the VAS continuum, either without or with transformation. Equidistance is determined for each dimension and each instrument (3L and 5L) separately. Untransformed equidistance implies that level descriptors are distributed according to VAS ratings of 0\u201350\u2013100 for 3L and 0\u201325\u201350\u201375\u2013100 for 5L. There is evidence that the precision of the VAS might be illusory, as respondents mentally divide the VAS continuum in a smaller number of segments, which is nine or ten at maximum [23, 24]. Therefore, we defined a deviation of 5 VAS points as the maximum acceptable deviation (which makes a segment of 10 VAS points, as the deviation can be either way). Furthermore, a deviation of 5 VAS points has been used before [16]. If untransformed equidistance is rejected, equidistance using power [y\u00a0\u00a0=\u00a0(ax)b] transformation is considered. A power relation of, e.g., y\u00a0=\u00a0(5.38*x)1.5 for 5L would result in a VAS rating distribution of 0\u201312\u201335\u201365\u2013100. Note that transformation is only possible for 5L, as there is only one 3L observation apart from the anchors.\nPart of the evaluation of equidistance is analysis of the position of the extreme levels according to the indirect method: are the VAS ratings for the extreme level descriptors close to the supposed anchor values for the indirect method? Ideally, 3L-1 and 5L-1 scores would equal 0 and 3L-3 and 5L-5 scores would equal 100, except for Pain\/Discomfort and Anxiety\/Depression in which the 3L and 5L extreme level descriptors were not identical to the VAS anchors.\nSecond, isoformity is the degree to which the positions of 3L-2 and 5L-3 level descriptors (and also 3L-3 versus 5L-5 for Pain\/Discomfort and Anxiety\/Depression) are similar. Isoformity directly compares the 3L and 5L descriptive systems for each separate dimension between instruments. For the indirect method, all 3L level means, including 3L-1 and 3L-3, can be compared with 5L. Analysis of isoformity is based on paired 3L\u20135L response means for each dimension separately. For the direct method, isoformity was tested with a paired t test between the 3L and 5L scorings. For the indirect method, a deviation of 5 VAS points was defined as the maximum acceptable deviation.\nFinally, consistency between dimensions is the degree to which the positions of the same level descriptors differ across dimensions. Consistency, between dimensions was tested for each instrument (3L, 5L) separately. The first three dimensions (Mobility, Self-Care, and Usual Activities) were distinguished from the last two (Pain\/Discomfort and Anxiety\/Depression), as these\u2014in Dutch\u2014share identical level descriptors, e.g., some problems for Mobility, Self-Care, and Usual Activities. For the direct method, analysis of variance (ANOVA) was used for each identical level descriptor for the first three dimensions combined (one comparison for 3L and three for 5L) and Pain\/Discomfort and Anxiety\/Depression combined (two comparisons for 3L and four for 5L), resulting in a total of ten comparisons . For the indirect method, consistency is tested with a generalizability study (G-study). In a G-study, one is able to separate multiple sources of error variance [25]. Generalizability coefficients (G-coefficients) can be constructed as functions of the estimated variance components, expressing consistency on a 0\u20131 scale, with 1 expressing perfect consistency [26, 27]. We used a variance components analysis based on the restricted maximum likelihood method and identified four possible sources of variance: label, vignette, dimension, and respondent. Four separate G-studies were conducted, one on the first three dimensions and one on the remaining two dimensions, for each instrument (3L, 5L) separately. A G-coefficient expressing consistency between dimensions was calculated on the basis of these variance components (\u201cAppendix A\u201d).\nWe regarded transformed or untransformed equidistance to be a desirable characteristic for the new 5L system as opposed to no systematic relation between the quantitative position of the level descriptors at all. Consistency between identical-level descriptors across dimensions was also regarded as a desirable property because this expresses that respondents have a consistent conceptualization of the grading terms used over different dimensions of health. When consistency is achieved, this does not imply that utility values would also be expected to be consistent over dimensions, because utility values are an expression of an entire EQ-5D profile, whereas we investigated VAS scores within each dimension separately. Furthermore, a choice-based method presumably leads to different results than the dimension-specific VAS scales we used. We investigated isoformity to see whether the new 5L system was a refinement or a new system, and whether isoformity was achieved or not does not tell us anything about the 5L system in itself.\nResults\nThe mean age of the participants was 53.6 years, with 42.7% being men. Of the 82 respondents who attended in the panel sessions, 81 returned the survey. Three respondents (4%) were of Turkish nationality, two (2%) were of Moroccan nationality, and the remaining 75 (94%) were of Dutch origin. In the Pain\/Discomfort and Anxiety\/Depression dimensions, respondents often failed to score the extreme-level descriptor when using the direct method (8 and 9 for 3L, respectively, and 22 and 16 for 5L, respectively). For these respondents, the remaining scorings were deleted for that dimension because of possible context effects (i.e., spreading out the VAS scores of the remaining 3L descriptors over the VAS scale). For the direct method, missing responses for 3L ranged from 6.1% (Usual Activities) to 19.5% (Pain\/Discomfort) and for 5L from 4.9% (Usual Activities) to 34.6% (Pain\/Discomfort). For the indirect method, missing responses ranged from 1.1% (Usual Activities) to 2.5% (Pain\/Discomfort) for the three response scales (3L, 5L, and VAS) combined.\nCharacteristics: direct method\nResults for the direct method are shown in Table\u00a01 and Fig.\u00a02. Untransformed equidistance was rejected for all level descriptors except 5L-4 in Mobility (80), although Self-Care and Usual Activities were only 1 VAS point away for Mobility. Regardless of dimension, level descriptors were positioned systematically lower than the expected value for equidistance for 3L-2 (16\u201323 VAS points lower), 5L-2 (14\u201316 points lower), and 5L-3 level (11\u201318 points lower), whereas 5L-4 was sometimes higher (4\u20135 points) and sometimes lower (7\u20138 points). Transformed equidistance (power function) provided an excellent fit for all dimensions of 5L (R2\u00a0\u2265\u00a00.99).\nFig.\u00a02Direct quantification of the three- and five-level (3L, 5L) descriptors. Visual analog scale (VAS) means by dimension\nIsoformity could not be established except for the middle-level descriptors (3L-2 vs. 5L-3) for Pain\/Discomfort and Anxiety\/Depression (Table\u00a02). Relatively large gaps appeared between 3L-2 and 5L-3 for Mobility (11), Self-Care (8), and Usual Activities (9), with 5L-3 showing systematically higher values. Although there was a statistically significant difference between the extreme level descriptors (3L-3 vs. 5L-5) for Anxiety\/Depression, the absolute difference was 3 VAS points.\nTable\u00a02Isoformity of identical three-and five-level (3L, 5L) descriptors for the direct quantification methodDimensionComparisonMean differenceP valueMobility3L-25L-3\u221211.4<0.001Self-care3L-25L-3\u22128.00.002Usual activities3L-25L-3\u22129.4<0.001Pain\/Discomfort3L-25L-3\u22121.40.501Pain\/Discomfort3L-35L-5\u22124.90.012Anxiety\/Depression3L-25L-32.80.276Anxiety\/Depression3L-35L-5\u22123.00.025\nConsistency between dimensions gives supportive results for both 3L and 5L, as none of the ten comparisons (ANOVA) showed significant differences (see Fig.\u00a02). Generally, VAS means are similar among the first three dimensions as well as among Pain\/Discomfort and Anxiety\/Depression.\nCharacteristics: indirect method\nResults of the indirect method are shown in Table\u00a03 and Fig.\u00a03. Untransformed equidistance of 3L-2 was rejected for all dimensions (systematically 7\u201314 VAS points too low) as well as for 5L-2 (systematically 8\u201313 points lower) and 5L-3 (systematically 8\u201317 points lower). Untransformed equidistance was achieved only for the 5L-4 level for all dimensions (systematically 1\u20135 points lower), with VAS scores ranging from 70 (Mobility and Usual Activities) to 74 (Anxiety\/Depression). Transformed equidistance (power function) provided an excellent fit for all dimensions of 5L (R2\u00a0\u2265\u00a00.99).\nTable\u00a03Indirect quantification of three- and five-level (3L, 5L) descriptorsNumberMeanMedianCI3L Mobility\u00a0\u00a0\u00a0\u00a0No problems in walking about5991.6901.31\u20132.07\u00a0\u00a0\u00a0\u00a0Some problems in walking about40342.944040.24\u201345.64\u00a0\u00a0\u00a0\u00a0Unable to walk about18091.709989.28\u201394.12Self-care\u00a0\u00a0\u00a0\u00a0No problems with self-care4823.2402.40\u20134.08\u00a0\u00a0\u00a0\u00a0Some problems washing or dressing self43539.183436.58\u201341.78\u00a0\u00a0\u00a0\u00a0Unable to wash or dress self27385.479582.77\u201388.16Usual activities\u00a0\u00a0\u00a0\u00a0No problems with performing usual activities2354.5023.49\u20135.51\u00a0\u00a0\u00a0\u00a0Some problems with performing usual activities58236.553034.40\u201338.71\u00a0\u00a0\u00a0\u00a0Unable to perform usual activities37888.549586.87\u201390.22Pain\/Discomfort\u00a0\u00a0\u00a0\u00a0No pain or discomfort24612.6449.94\u201315.34\u00a0\u00a0\u00a0\u00a0Moderate pain or discomfort64335.763133.92\u201337.60\u00a0\u00a0\u00a0\u00a0Extreme pain or discomfort27583.218980.82\u201385.61Anxiety\/Depression\u00a0\u00a0\u00a0\u00a0Not anxious or depressed4336.2915.01\u20137.57\u00a0\u00a0\u00a0\u00a0Moderately anxious or depressed47842.454040.26\u201344.63\u00a0\u00a0\u00a0\u00a0Extremely anxious or depressed27084.809082.73\u201386.865LMobility\u00a0\u00a0\u00a0\u00a0No problems in walking about5471.3000.92\u20131.69\u00a0\u00a0\u00a0\u00a0Mild problems in walking about14715.331112.64\u201318.02\u00a0\u00a0\u00a0\u00a0Some problems in walking about15936.483133.00\u201339.97\u00a0\u00a0\u00a0\u00a0Many problems in walking about21769.827666.72\u201372.92\u00a0\u00a0\u00a0\u00a0Unable to walk about11297.3610095.24\u201399.48Self-care\u00a0\u00a0\u00a0\u00a0No problems with self-care3982.4501.43\u20133.48\u00a0\u00a0\u00a0\u00a0Mild problems washing or dressing self20412.70910.76\u201314.64\u00a0\u00a0\u00a0\u00a0Some problems washing or dressing self18436.093333.00\u201339.17\u00a0\u00a0\u00a0\u00a0Many problems washing or dressing self25771.207868.33\u201374.06\u00a0\u00a0\u00a0\u00a0Unable to wash or dress self14791.379987.80\u201394.94Usual activities\u00a0\u00a0\u00a0\u00a0No problems with performing usual activities1363.2201.49\u20134.95\u00a0\u00a0\u00a0\u00a0Mild problems with performing usual activities26812.39910.68\u201314.10\u00a0\u00a0\u00a0\u00a0Some problems with performing usual activities22832.533029.97\u201335.09\u00a0\u00a0\u00a0\u00a0Many problems with performing usual activities35169.547567.18\u201371.90\u00a0\u00a0\u00a0\u00a0Unable to perform usual activities21295.3510093.74\u201396.96Pain\/Discomfort\u00a0\u00a0\u00a0\u00a0No pain or discomfort1458.3405.32\u201311.37\u00a0\u00a0\u00a0\u00a0Mild pain or discomfort27417.271215.13\u201319.41\u00a0\u00a0\u00a0\u00a0Moderate pain or discomfort36736.833534.91\u201338.76\u00a0\u00a0\u00a0\u00a0Severe pain or discomfort26371.727969.05\u201374.39\u00a0\u00a0\u00a0\u00a0Extreme pain or discomfort11592.769889.86\u201395.65Anxiety\/Depression\u00a0\u00a0\u00a0\u00a0Not anxious or depressed3054.7503.07\u20136.43\u00a0\u00a0\u00a0\u00a0A little anxious or depressed24116.481014.33\u201318.63\u00a0\u00a0\u00a0\u00a0Moderately anxious or depressed27141.984139.72\u201344.25\u00a0\u00a0\u00a0\u00a0Very anxious or depressed24874.198071.69\u201376.70\u00a0\u00a0\u00a0\u00a0Extremely anxious or depressed11692.339789.61\u201395.04CI confidence intervalFig.\u00a03Indirect quantification of the three- and five-level (3L, 5L) descriptors. Visual analog scale (VAS) means by dimension\nVAS results for the extreme-level descriptors show that the lower extreme is close to 0, except for Pain\/Discomfort (3L-1\u00a0=\u00a013; 5L-1\u00a0=\u00a08). VAS results for the upper extreme values are systematically higher for 5L than for 3L (range of difference: 6\u201310). Noticeable are large deviations in Self-Care (3L-3\u00a0=\u00a085; 5L-5\u00a0=\u00a091) and Usual Activities (3L-3\u00a0=\u00a089). Isoformity was accepted for 3L-1 versus 5L-1 for all dimensions and for 3L-2 vs. 5L-3 for all dimensions except Mobility (showing a gap of 7 points). Isoformity was rejected for the upper extreme comparison 3L-3 versus 5L-5 for all dimensions. Consistency between dimensions gave supportive results for both 3L and 5L. Table\u00a04 shows the G-study results. Most variance is attributed to the label component, whereas less than 2% of variance is attributed to the components including dimension, which is reflected in high G-coefficients for all comparisons. Consistency for 5L is somewhat higher (0.87; 0.86) than for 3L (0.86; 0.81).\nTable\u00a04Consistency between dimensions for the indirect quantification method. Variance components estimates (percentages) and generalizability coefficients (G-coefficients) for comparable dimensions of three- and five-level (3L, 5L) instruments3L5LMobility\/Self-care\/Usual activitiesLabel66.12Label71.52Vignette8.05Vignette6.35Dimension0.26Dimension0.04Respondent0.33Respondent0.79Label\u00a0\u00d7\u00a0vignette5.60Label\u00a0\u00d7\u00a0vignette2.91Label\u00a0\u00d7\u00a0dimension0.22Label\u00a0\u00d7\u00a0dimension0.12Label\u00a0\u00d7\u00a0respondent2.20Label\u00a0\u00d7\u00a0respondent2.59Vignette\u00a0\u00d7\u00a0dimension0.60Vignette\u00a0\u00d7\u00a0dimension0.17Vignette\u00a0\u00d7\u00a0respondent3.77Vignette\u00a0\u00d7\u00a0respondent2.57Dimension\u00a0\u00d7\u00a0respondent0.76Dimension\u00a0\u00d7\u00a0respondent0.60Residual12.09Residual12.34G-coefficient0.86G-coefficient0.87Pain\/Discomfort; Anxiety\/DepressionLabel65.25Label73.58Vignette4.95Vignette2.73Dimension0.00Dimension0.00Respondent0.65Respondent0.77Label\u00a0\u00d7\u00a0vignette1.91Label\u00a0\u00d7\u00a0vignette1.02Label\u00a0\u00d7\u00a0dimension0.04Label\u00a0\u00d7\u00a0dimension0.00Label\u00a0\u00d7\u00a0respondent2.96Label\u00a0\u00d7\u00a0respondent3.36Vignette\u00a0\u00d7\u00a0dimension1.06Vignette\u00a0\u00d7\u00a0dimension0.17Vignette\u00a0\u00d7\u00a0respondent5.52Vignette\u00a0\u00d7\u00a0respondent4.50Dimension\u00a0\u00d7\u00a0respondent0.88Dimension\u00a0\u00d7\u00a0respondent0.45Residual16.78Residual13.42G-coefficient0.81G-coefficient0.86\nDiscussion\nIn this study, we compared the quantitative position of the level descriptors of the standard EQ-5D3L and a new five-level version using two independent methods. The study showed that the extension of the EQ-5D3L to a five-level version by inserting two extra levels, leaving the existing descriptors unaltered, is not a simple refinement but a redesign. The inserted levels pushed the extreme levels closer to the anchors, which indicates that 5L makes better use of the measurement continuum, contributing to superior descriptive power of the 5L version. In both the 3L and 5L versions, the position of the 3L or 5L descriptors, reassuringly, was independent of dimension.\nEquidistance was not achieved for both systems, in most cases showing values lower than the equidistant values. Both methods revealed a large gap between the 5L-3 and 5L-4 levels, regardless of dimension. This could be caused by the wording of 5L-3 [some and moderate(ly)] being interpreted as fairly mild.\nIn Pain\/Discomfort, respondents tended to avoid the lower anchor of the scale, indicating some pain or discomfort on VAS while scoring no problems on 3L and 5L. This indicates that respondents preferred a more refined response scale for scoring pain or discomfort, maybe a scale with even more than five response options (as is the case of, e.g., the HUI3 or SF-36). Also noticeable were the gaps observed for the upper extreme in Self-Care, for which we cannot provide an explanation.\nIsoformity between 3L and 5L showed mixed results. The 3L-1 vs. 5L-1 descriptors showed isoformity (indirect method only), as expected, as these both indicated the upper ceiling (no problems). Isoformity was also established for the middle level descriptors of Pain\/Discomfort and Anxiety\/Depression for both methods. This could be due to the wording of the middle level descriptors, as the descriptor some problems represented a wider range and hence more potential variation, than moderate(ly), as used in Pain\/Discomfort and Anxiety\/Depression. Assuming that the descriptor some problems was a well-considered choice in the development of the original EQ-5D3L system in order to cover the entire range between the two extremes, it is questionable whether that descriptor is still suitable in a 5L version.\nDirect quantification is a well-known method of estimating the magnitude of level descriptors or response labels [16, 17, 28, 29]. This approach, however, ignores the fact that the VAS values expressed for the level descriptors did not necessarily reflect the self-report use of such descriptors (and the use in subsequent valuation studies) in a similar way, because the valuation of an abstract level descriptor might lead to different results than self-reported health. The indirect method is novel: to our knowledge, this is the first time a quantification of level descriptors is estimated with this method. The indirect method has several advantages. First, we believe it is a better representation of the hypothesized measurement continuum of EQ-5D, as the medium of the vignette (disease) was used to calibrate 3L and 5L descriptors on a VAS scale. Second, it is closer to the general use of the EQ-5D instrument as a self-report health status assessment measure and is therefore likely to be more valid. Classifying a vignette can be regarded similarly to a health status classification by proxy assessment. Other advantages of the indirect method are analytical: values can be calculated for all level descriptors, including the anchors, and it is possible to investigate explained variance for various components (G-study). Furthermore, the indirect method proved to be much more feasible than the direct method, considering the lower number of missing responses. Disadvantages are that no direct comparison (e.g., paired t test) between 3L and 5L is possible, as there is only one VAS value for each 3L\u20135L response pair, and that the indirect method is more time consuming.\nA potential weakness of the study procedure is that 3L and 5L were presented on one sheet, and panelists were asked to score 5L dimensions first while covering 3L and vice versa. We cannot be sure that respondents actually complied to the blinding procedure in the follow-up measurement. Also, there might have been an order effect, as 5L always preceded 3L.\nThe 5L instrument presented here obviously improves the discriminatory potential of the EQ-5D descriptive system, as the level descriptors generally capture a larger part of the measurement continuum and broaden the measurement space. Furthermore, 5L showed slightly better consistency between levels. In a previous study, we demonstrated increased discriminatory power of the same 5L version of EQ-5D, as well as superior reliability (interobserver and test\u2013retest) and face validity when compared with the standard EQ-5D3L [18]. Awaiting a valuation study for an official version of 5L, a set of preference weights was developed for this 5L version of EQ-5D using item response theory (IRT) methodology [30]. An officially sanctioned five-level descriptive system will become available within a short period [31] and is expected to be in use alongside the standard three-level EQ-5D.\nThe experimental five-level EQ-5D version presented here is likely to demonstrate a less severe ceiling effect. Assuming that milder states are more common in the general population, we expect increased benefit in the detection of mild problems and in measuring and monitoring general population health, although the extra 5L-4 level is expected to also lead to better differentiation and detection of more severe health states. The methodology presented here can be of use in the development of generic or disease-specific health status measures.","keyphrases":["eq-5d","psychometrics","health status","methodology","health-related quality of life"],"prmu":["P","P","P","P","M"]}
{"id":"Surg_Endosc-4-1-2262145","title":"Virtual reality training for endoscopic surgery: voluntary or obligatory?\n","text":"Introduction Virtual reality (VR) simulators have been developed to train basic endoscopic surgical skills outside of the operating room. An important issue is how to create optimal conditions for integration of these types of simulators into the surgical training curriculum. The willingness of surgical residents to train these skills on a voluntary basis was surveyed.\nEndoscopic surgery requires dedicated skills such as three-dimensional orientation in a two-dimensional representation of the operating field and complex instrument handling [8, 7, 5]. Training of these skills in the operating room (OR) is under pressure due to planning issues and ethical considerations. Virtual reality (VR) simulators have been developed to train basic endoscopic surgical skills outside of the OR. Several simulators have been validated and found adequate for the transfer of skills from the simulator to the OR [2, 6, 9, 13, 14]. However, discussion arises on how to integrate these simulation-based training modalities in the surgical training curriculum.\nA questionnaire was distributed to 245 Dutch surgical residents to explore the perspective of the trainee on this issue. Approximately 75% of residents felt that endoscopic skills training outside the OR is useful [12]. In another study, sixty Dutch gynaecology residents responded positively (3.9 on a five-point Likert scale) with regard to training in laparoscopic skills before real surgery [10]. Fifty-five percent of these 60 residents did not have the opportunity to train laparoscopic skills. However, those that did appeared to train only once or twice a year and 33% did not use available skills trainers voluntarily at all. We hypothesized that insufficient simulator access might be the reason for this contradiction. Therefore, we investigated the willingness of surgical residents to train in endoscopic skills on a voluntary basis when VR simulators were indeed readily available. We also evaluated the effect of competitive incentives on the frequency and duration of simulator training.\nMaterials and methods\nEquipment, tasks and scoring system\nThis study is performed with the LapSim virtual reality simulator, which uses the Virtual Laparoscopic Interface (VLI) hardware, (Immersion Inc., San Jose, CA, USA) The VLI has an interface with a 2600 MHz hyperthreading processor Pentium IV computer running Windows XP and is equipped with 256 random-access memory (RAM), a GeForce graphics card and an 18-inch thin-film transistor (TFT) monitor. The systems feature LapSim Basic Skills 3.0 software (Surgical Science Ltd, G\u00f6teburg, Sweden), from the LapSim Basic Skills package, consisting of nine tasks.\nA training program was designed that included all nine tasks: camera navigation, instrument navigation, coordination, grasping, lifting and grasping, cutting, clipping and cutting, suturing and fine dissection [4].\nThe computer stores and displays between seven and eleven parameters of performance per task. These parameters are related to time, errors or efficiency of handling.\nTasks can be adjusted to different levels of difficulty. The training program for this study was set at an advanced level with thresholds that are based on the performance of 30 experienced endoscopic surgeons (more than 100 endoscopic procedures).\nThe scoring system is two-tiered. First, for any given parameter the system determines whether or not the participant passes or fails the test. Secondly, if a participant passes, a score of between 0% and 100% is attached to his or her performance on that particular parameter. The overall score per task is determined by the sum score of the parameters, divided by their number. Hence, an overall score of 100% can only be obtained by scoring 100% on each of the individual parameters measured during performance of the particular task. An outcome score of 100 points is given to those participants who score a 100% on the task performed. Logically, a score of 85% thus translates into 85 points. A maximum overall score of 900 could be obtained (i.e., 100 points on each of the nine tasks measured).\nParticipants\nTwenty-one surgical residents, ranging from postgraduate year (PGY) -one level to PGY 6 level, with different endoscopic surgical experience, were given unlimited access to the simulator. Seven residents were at the beginning of their surgical educational program (PGY 1 and PGY 2) and therefore inexperienced in endoscopic surgery. Seven residents were in the middle (PGY 3 and PGY 4) and eight residents were at the end (PGY 5 and PGY 6) of their surgical educational program.\nSetting and incentives\nIn the period May 2005 to January 2006 a simulator was placed in the general room for surgical residents at the surgical ward of the University Medical Centre in Utrecht. Before the study, residents were instructed on how to operate the simulator, and allocated a personal login number for the simulator. By placing the simulator in the general residents\u2019 room, it was readily and easily accessible 24 hours a day. The room is secured by a code-locked door and accessible by residents only.\nDuring the first four months, there were no additional incentives other than the permanent (24-hour) accessibility to the residents for training on the simulator. After these four months, a competitive element was introduced in which the frequency of training was also rewarded (bi-weekly). The overall end-score was calculated every other week by adding this frequency bonus to the highest scores for each task. These overall end-scores for each resident were publicly announced to the complete department of surgery and the winner (the resident with the highest score) was awarded a prize.\nQuestionnaire\nAfter eight months all residents were requested to fill out a questionnaire.\nTen questions were presented on a five-point Likert scale, concerning their perception of their own experience level in endoscopic procedures, their opinion of the possibility to develop and train endoscopic skills within the current surgical curriculum, and their opinion about the application of virtual reality as a means to training endoscopic skills. Value 1 was assigned to \u201ctotally agree\u201d, value 5 to \u201ctotally disagree\u201d. In addition, the residents were asked about their frequency of usage of the simulator. If a participant indicated little usage, he or she was questioned why, and what could motivate increased usage.\nResults\nIn the first four months only two of the 21 residents (10%) trained on the simulator, for a total of 163 minutes. One resident was a PGY 2, the other one a PGY 5. In the second period of four months the number of trainees increased to seven residents (33%, two PGY 2, two PGY 3, one PGY 5 and two PGY 6). The duration of training increased to 738 minutes, thereby constituting an average increase of 23.9 minutes per subject. Fifty-eight percent of training was performed during night shifts.\nAll 22 residents (100%) replied to the questionnaire. The total training time, as an accumulation of estimation on individual training time, was 4140 minutes. The actual cumulated training time for all residents was 901 minutes (22%). Thirteen out of 15 residents who did not train at all (86%) stated that this was due to a lack of time during the day. One resident (7%) stated he had been not interested enough to train and indicated that he had alternative priorities. Another resident (7%) stated that she was fully occupied due to an intensive-care traineeship and maternity leave.\nResidents suggested that the use of the VR trainers could be enhanced by incorporating a mandatory VR training into the surgical curriculum (9x\u00a0=\u00a0people agreeing), to oblige certain skills level on VR simulator before starting endoscopic surgery in the OR (3x), to implement competitive training with coaching (2x), to diminish working pressure (2x), to have more-advanced exercises available on the simulator (3x), to place the VR simulator in a location other than in the residents\u2019 room (1x). Only two stated that more initiative of residents was required to improve outcome.\nFigure\u00a01 refers to the perception of residents\u2019 own experience level in endoscopic procedures, their opinion of the possibility to develop and train endoscopic skills within the current surgical curriculum and their opinion about the application of virtual reality as a means to training endoscopic skills.\nFig.\u00a01.Results of questionnaire\nIn general, the opinion of the residents on their own experience level, on the possibility to develop and train endoscopic skills during their training and on the role of virtual reality varies considerably [standard deviation (SD) 0.44 to 1.39]. Their opinion on obligation of VR training to improve endoscopic skills and having VR training as a mandatory part of the basic skills training is most uniform. Residents do not have a marked positive, nor a marked negative opinion on the presented statements on receiving enough training for acquiring basic skills (mean 2.63, SD 1.19), on receiving sufficient training time in the OR to train in endoscopic skills (mean 3.47 SD 1.17) and on acquiring a satisfactory level of basic psychomotor skills (mean 2.42, SD 1.16). The same applies to their opinion on the representation of their training results on the simulator (mean 2.98, SD 0.83); as well as on the statement that thresholds should be reached before training in the OR is allowed (mean 2.47, SD 1.39). There is one statement they do not agree with; I will not train unless it is obligatory (mean 4.26, SD 1.10).\nDiscussion\nVirtual reality training has the potential to improve and professionalize the training in endoscopic basic psychomotor skills [2, 6, 9, 13, 14]. Training results can be shown instantly to demonstrate objective performance and progress of performance assessment. However, one of the main concerns in acquisition of expensive equipment for educational purposes is its effectiveness. While initial enthusiasm about new innovative equipment is usually high among the surgical community, actual usage tends to be disappointing [3]. This study was undertaken to evaluate the aptitude for training on a voluntary basis when a VR simulator was readily available. Free unlimited access to a VR simulator without obligation or assessment in our setting did not seem to motivate surgical residents to use the simulator for improvement of their psychomotor endoscopic skills level. The addition of a competitive element and a desirable prize had only a marginal effect on the frequency and duration of training. We believe that the effort required to provide this incentive is disproportionate to its marginal effect.\nThe majority of residents (86%) stated that \u2018lack of time due to high working pressure\u2019 is the most important reason for not using the simulator. Following a recent European guideline, as set by the European Commission, a working week for a resident in training is being reduced from 70 to 48 hours [1, 11]. This may have led to an increase in pressure during working hours, with little time available for voluntary training. However, spare time has increased vastly compared to the former curriculum. Residents did not use personal free time for VR simulator training to improve their skills.\nThe perception of their own experience level in endoscopic procedures and the possibility of developing and training in endoscopic skills within the current surgical curriculum was in general neutral. Therefore no conclusions can be drawn from this. There is favorable, uniform opinion on the desirability of integration of skills training into the curriculum. In addition, residents believe skills training ought to be mandatory for marked improvement of their psychomotor skills. Interestingly, the disagreement on the statement of not training unless it is obligatory (mean 4.26, SD 1.10) appears to have no bearing in reality, because our study shows very limited use of the simulator. This incongruence might be caused by political correctness or by a discrepancy between intentions and actions.\nIt must be said that our result reflects the quantity of training on a voluntary basis of 22 residents in a single institute only, and might therefore not represent the attitude of national or international surgical residents.\nIn conclusion, the acquisition of expensive devices to train basic psychomotor skills for endoscopic surgery is probably only effective when it is a mandatory part of the curriculum.","keyphrases":["endoscopic surgery","surgical training","virtual reality simulation","curriculum design","resident participation"],"prmu":["P","P","P","R","R"]}
{"id":"Pflugers_Arch-3-1-1915642","title":"Carbohydrate supplementation during prolonged cycling exercise spares muscle glycogen but does not affect intramyocellular lipid use\n","text":"Using contemporary stable-isotope methodology and fluorescence microscopy, we assessed the impact of carbohydrate supplementation on whole-body and fiber-type-specific intramyocellular triacylglycerol (IMTG) and glycogen use during prolonged endurance exercise. Ten endurance-trained male subjects were studied twice during 3 h of cycling at 63 \u00b1 4% of maximal O2 uptake with either glucose ingestion (CHO trial; 0.7 g CHO kg\u22121 h\u22121) or without (CON placebo trial; water only). Continuous infusions with [U-13C] palmitate and [6,6-2H2] glucose were applied to quantify plasma free fatty acids (FFA) and glucose oxidation rates and to estimate intramyocellular lipid and glycogen use. Before and after exercise, muscle biopsy samples were taken to quantify fiber-type-specific IMTG and glycogen content. Plasma glucose rate of appearance (Ra) and carbohydrate oxidation rates were substantially greater in the CHO vs CON trial. Carbohydrate supplementation resulted in a lower muscle glycogen use during the first hour of exercise in the CHO vs CON trial, resulting in a 38 \u00b1 19 and 57 \u00b1 22% decreased utilization in type I and II muscle-fiber glycogen content, respectively. In the CHO trial, both plasma FFA Ra and subsequent plasma FFA concentrations were lower, resulting in a 34 \u00b1 12% reduction in plasma FFA oxidation rates during exercise (P < 0.05). Carbohydrate intake did not augment IMTG utilization, as fluorescence microscopy revealed a 76 \u00b1 21 and 78 \u00b1 22% reduction in type I muscle-fiber lipid content in the CHO and CON trial, respectively. We conclude that carbohydrate supplementation during prolonged cycling exercise does not modulate IMTG use but spares muscle glycogen use during the initial stages of exercise in endurance-trained men.\nIntroduction\nDuring exercise, there is a complex interaction between carbohydrate and fat metabolism to provide the required adenosine triphosphate (ATP) to sustain the necessary power output. The relative utilization of endogenous fat and carbohydrate stores during exercise can vary enormously and strongly depends on exercise intensity [28, 38], duration [28, 41, 45], and training status [19, 36]. During moderate intensity exercise, the oxidation of plasma-derived free fatty acids (FFA) in conjunction with other fat sources (muscle- and\/or lipoprotein-derived triacylglycerol) contributes approximately half of the total energy required for contracting skeletal muscle. As the exercise duration lengthens, there is an increasing contribution of fat oxidation to total energy expenditure, which is primarily attributed to an increase in the release, uptake, and oxidation of plasma FFA [28, 41, 45]. Studies utilizing more prolonged (\u22652\u00a0h) moderate-intensity exercise protocols have consistently shown that the progressive increase in plasma-FFA delivery to working skeletal muscle late in the exercise coincides with an inhibition of intramyocellular lipid (intramyocellular triacylglycerol [IMTG]) mobilization and\/or oxidation [28, 41, 45]. In accordance, pharmacological inhibition of adipose tissue lipolysis has been shown to stimulate the use of intramyocellular lipids during the latter stages of prolonged exercise [42, 44, 47]. Carbohydrate supplementation can also serve to decrease FFA delivery and lower whole-body fat-oxidation rates during exercise [9, 10]. Therefore, it could be hypothesized that the reduced FFA availability via carbohydrate supplementation stimulates IMTG use during the latter stages of prolonged exercise. In contrast, other studies have reported an inhibitory effect of glucose ingestion on hormone-sensitive lipase activity [48], which would actually reduce IMTG hydrolysis [11]. It therefore remains to be established how carbohydrate supplementation during exercise modulates IMTG utilization.\nCarbohydrate supplementation during exercise has been reported to improve endurance performance by maintaining euglycemia during the late stages of prolonged exercise and\/or by sparing the muscle glycogen stores [9]. However, the latter remains equivocal, as most studies have failed to report muscle glycogen sparing after carbohydrate supplementation during prolonged cycling exercise [5, 9, 13, 17, 24, 25]. The inconsistent findings of the effects of carbohydrate supplementation on subsequent muscle glycogen sparing may likely be attributed to differences in the applied research design, with specific reference to the mode of exercise (running vs cycling) and\/or specific muscle group and\/or fiber type recruitment [33].\nThe present study applies continuous infusions of [U-13C] palmitate and [6,6-2H2] glucose with muscle biopsy sampling before and after exercise to quantify both whole-body and muscle-fiber-type-specific IMTG and glycogen use. The purpose of this study was to assess the impact of carbohydrate supplementation on skeletal muscle glycogen and intramyocellular lipid use during prolonged cycling exercise in endurance-trained men and, as such, to determine whether carbohydrate supplementation reduces muscle glycogen use and\/or augments the use of the IMTG pool as a substrate source.\nMaterials and methods\nSubjects\nTen endurance-trained male cyclists were selected to participate in this study. Subjects\u2019 characteristics are provided in Table\u00a01. Subjects were informed about the nature and risks of the experimental procedures before their written informed consent were obtained. This study was approved by the local Medical Ethical Committee of the Academic Hospital Maastricht. \nTable\u00a01Subjects\u2019 characteristics (n\u2009=\u200910)Characteristics\u00a0Age (years)23\u2009\u00b1\u20093Height (m)1.83\u2009\u00b1\u20090.04Body mass (kg)72.5\u2009\u00b1\u20099.6BMI (kg m\u22122)21.7\u2009\u00b1\u20091.9Body fat percentage (%)9.8\u2009\u00b1\u20092.4Fat free mass (kg)65.3\u2009\u00b1\u20097.9Basal plasma glucose (mmol l\u22121)5.1\u2009\u00b1\u20090.3Plasma glucose120\u00a0min (mmol l\u22121)3.9\u2009\u00b1\u20091.1Basal plasma insulin (mU l\u22121)9.0\u2009\u00b1\u20092.6HbA1c (%)5.2\u2009\u00b1\u20090.2VO2max (ml kg\u22121 min\u22121)59.8\u2009\u00b1\u20095.6Wmax (W)398\u2009\u00b1\u200941Values are expressed as means \u00b1 SD. Body mass index (BMI) is calculated by dividing body mass by the square of the height.Plasma glucose120\u00a0min Plasma glucose concentration 120\u00a0min after ingesting 75 glucose in the oral glucose tolerance test\nPretesting\nSubjects initially performed a continuous incremental cycling test on a cycle ergometer (LODE Instrument, Groningen, The Netherlands) to exhaustion to determine peak pulmonary oxygen uptake (VO2max; Oxycon-\u03b2, Mijnhart, The Netherlands) and maximal workload capacity (Wmax). After an overnight fast, body composition was assessed using the hydrostatic weighing method, with corrections made for residual lung volume via the helium dilution technique (Volugraph 2000, Mijnhart, Bunnik, Netherlands). Body-fat percentage was calculated using Siri\u2019s [31] equation. To assess whole-body insulin sensitivity an oral glucose tolerance test was performed in each subject according to the World Health Organization criteria [2]. In addition, insulin resistance was estimated using the homeostasis model assessment for insulin resistance or HOMA-IR index [23].\nDiet and activity before testing\nAll subjects were instructed to maintain their usual dietary and physical activity patterns throughout the entire experimental period. In addition, they filled out a food intake diary for 2\u00a0days before the first exercise trial to keep their dietary intake as identical as possible before the other trials. Subjects refrained from heavy physical labor and exercise training for 3\u00a0days before each trial. Dietary analyses showed no differences in energy intake during the days before each of the trials with an average energy intake of 12.7\u2009\u00b1\u20092.4\u00a0MJ, with 57\u2009\u00b1\u20097, 28\u2009\u00b1\u20095, and 15\u2009\u00b1\u20093% of the energy intake (energy%) derived from carbohydrate, fat, and protein, respectively. The evening before each trial, subjects received the same standardized meal (41.2\u00a0kJ kg bw\u22121; containing 72, 11, and 17 energy% carbohydrate, fat, and protein, respectively).\nExperimental trials\nEach subject performed three trials, separated by at least 1\u00a0week: two experimental trials (CON: control vs CHO: carbohydrate) and an acetate correction trial (Fig.\u00a01). Each trial consisted of 60\u00a0min of resting measurements, followed by 180\u00a0min of cycling at 50%Wmax. In the main trials, an [U-13C] palmitate and [6,6-2H2] glucose tracer were infused continuously at rest and during exercise with breath and blood samples collected at regular intervals. Muscle biopsy samples were collected before and immediately after exercise. In the carbohydrate supplementation trial (CHO), plasma FFA availability was reduced, and plasma glucose availability was augmented through carbohydrate ingestion. To allow for a continuous supply of glucose from the gut, subjects ingested an 8% CHO solution every 20\u00a0min during exercise at the rate of 0.7\u00a0g CHO kg\u22121 h\u22121, as recommended by the American College of Sports Medicine [1]. In the control trial, a placebo water drink was provided (CON). In a third trial, [1,2-13C] acetate was infused continuously at rest and during exercise, and only breath samples were collected. Both the CON and acetate trials were performed in an overnight fasted state. The acetate recovery factor was used to accurately correct [U-13C] palmitate oxidation rates for carbon label retention in the bicarbonate pool(s) and by way of isotopic exchange reactions in the TCA-cycle for each subject [40].\nFig.\u00a01Schematic of study protocol. After an initial resting blood, breath, and muscle biopsy sample, a continuous infusion with [U-13C] palmitate and 6,6-2H2 glucose was started for 240\u00a0min. At rest and during 3\u00a0h of cycling at 50% of Wmax substrate use was assessed with (CHO trial) and without (CON trial) carbohydrate supplementation\nProtocol\nAfter an overnight fast, subjects arrived at the laboratory at 8:00\u00a0a.m. by car or public transportation. After 30\u00a0min of supine rest, a percutaneous muscle biopsy was taken from the vastus lateralis muscle [3]. A Teflon catheter (Baxter, Utrecht, The Netherlands) was inserted into an antecubital vein of one arm for blood sampling, another catheter was inserted in the antecubital vein of the contralateral arm for isotope infusion. Thereafter, a resting blood sample was taken and expired breath samples were collected into vacutainer tubes. Subsequently, subjects were administered a single intravenous dose of NaH13CO3 (0.06375\u00a0mg kg\u22121), to prime the bicarbonate pool(s), followed by a [6,6-2H2] glucose prime (13.5\u00a0\u03bcmol kg\u22121). Thereafter, a continuous infusion of [6,6-2H2] glucose (0.3\u00a0\u03bcmol kg\u22121 min\u22121) and [U-13C] palmitate (0.01\u00a0\u03bcmol kg\u22121 min\u22121; or [1,2-13C] acetate in the acetate recovery trial) was started (t\u2009=\u20090\u00a0min) via a calibrated IVAC pump (IVAC 560, San Diego, CA) and continued for 4\u00a0h. At t\u2009=\u200960\u00a0min, an initial drink bolus (7\u00a0ml kg\u22121) was given, and then nine more drink boluses (2.5\u00a0ml kg\u22121) were given at t\u2009=\u200980, 100, 120, 140, 160, 180, 200, 220, and 240\u00a0min of either an 8% carbohydrate solution or a placebo. At t\u2009=\u200960\u00a0min, subjects started to exercise on a cycle ergometer at a workload of 50%Wmax for 3\u00a0h. Expired VO2 and VCO2 were measured (Oxycon-\u03b2) at rest and throughout exercise for 5\u00a0min every 20\u00a0min before sampling of blood and expired breath collection. Immediately after cessation of exercise, a second muscle biopsy was taken (t\u2009=\u2009240\u00a0min). Breath and blood samples were collected at t\u2009=\u20090, 30, and 60\u00a0min (during rest) and at t\u2009=\u200980, 100, 120, 140, 160, 180, 200, 220, and 240\u00a0min (during exercise).\nBeverages\nIn the CHO trial, subjects received a beverage volume of 2.5\u00a0ml kg\u22121 every 20\u00a0min to ensure a given dose of 0.7\u00a0g CHO kg\u22121 (50% as glucose and 50% as maltodextrin) every hour. Repeated boluses were administered to enable a continuous supply of glucose in the circulation, preventing perturbations in [6,6-2H2] glucose enrichment. In the CON trial, a sweetened placebo drink, which contained no carbohydrate, was provided. Glucose and maltodextrin were obtained from AVEBE (Veendam, The Netherlands). To make the taste comparable in all experiments, beverages were uniformly flavored by adding 0.2\u00a0g sodium\u2013saccharine solution (25% w\/w), 1.8\u00a0g citric acid solution (50% w\/w), and 5\u00a0g of cream vanilla flavor (Numico Research, Wageningen, The Netherlands) for each liter of beverage. Experiments were performed in a randomized order, with test drinks provided in a double-blind fashion.\nTracer infusion\nInfusion rates of [U-13C] palmitate and [6,6-2H2] glucose averaged 7.3\u2009\u00b1\u20090.8 and 817.5\u2009\u00b1\u200930.4\u00a0nmol kg\u22121 min\u22121, respectively, in the CON and CHO trial. At the onset of exercise, [U-13C] palmitate infusion rates were doubled (14.6\u2009\u00b1\u20091.6\u00a0nmol kg\u22121 min\u22121). In the acetate recovery trial, a corresponding amount of 13C was infused, resulting in an average [1,2-13C] acetate infusion rate of 61.6\u2009\u00b1\u20092.4 and 123\u2009\u00b1\u20095\u00a0nmol kg\u22121 min\u22121 at rest and during exercise, respectively. Palmitate, glucose, and acetate tracer concentrations in the infusates averaged 1.03\u2009\u00b1\u20090.07, 64.8\u2009\u00b1\u20097.2, and 4.74\u2009\u00b1\u20090.18\u00a0mmol l\u22121, respectively.\nBlood- and breath-sample analysis\nBlood samples (7\u00a0ml) were collected in ethylenediamine tetraacetic acid (EDTA)-containing tubes and centrifuged at 1,000\u00d7g for 10\u00a0min at 4\u00b0C. Aliquots of plasma were frozen immediately in liquid nitrogen and stored at \u221280\u00b0C. Plasma glucose (Uni Kit III, Roche, Basel, Switzerland), lactate [16], FFA (NEFA-C, Wako Chemicals, Neuss, Germany), free glycerol (148270, Roche Diagnostics, Indianapolis, IN), and triglyceride (TG; GPO-trinder 337B, Sigma Diagnostics, St. Louis, MO) concentrations were analyzed with a COBAS semi-automatic analyzer (Roche).\nExpired breath samples were analyzed for 13C\/12C ratio by gas chromatography continuous flow isotope ratio mass spectrometry (GC-IRMS; Finnigan MAT 252, Bremen, Germany). For determination of plasma palmitate and FFA kinetics, FFA were extracted from plasma, isolated by thin-layer chromatography, and derivatized to their methyl esters. Palmitate concentration was determined on an analytical gas chromatograph with flame ionization detection using heptadecanoic acid as an internal standard and on average comprised 26\u2009\u00b1\u20092 and 24\u2009\u00b1\u20093% of total FFA for the CON and CHO trials, respectively. Isotope tracer\/tracee ratio of [U-13C] palmitate was determined using GC-combustion IRMS (Finnigan MAT 252). Plasma glucose was first extracted with chloroform\u2013methanol\u2013water and derivatization was performed with butylboronic acid and acetic anhydride as described previously [27]. After derivatization, plasma [6,6-2H2] glucose enrichment was determined by electron ionization GC-MS (Finnigan INCOS-XL). Glucose (Uni Kit III, Roche) and acetate (Kit 148261, Boehringer) concentrations in the infusates were determined with the COBAS FARA.\nCalculations\nFrom respiratory measurements, total fat and carbohydrate oxidation rates were calculated using the nonprotein respiratory quotient [26]. \nwith VO2 and VCO2 in liters per minute (l min\u22121) and oxidation rates in grams per minute (g min\u22121). Breath and plasma enrichments are expressed as tracer\/tracee ratios (TTR); \nin which sa indicates sample and bk indicates background value. Rate of palmitate and glucose appearance (Ra) and disappearance (Rd) were calculated using the single-pool nonsteady-state Steele [32] equations adapted for stable isotope methodology as described elsewhere [49]. As such, plasma palmitate and glucose Rd were calculated by correcting the Ra for the time-dependent changes in plasma metabolite concentration. \nwhere F is the infusion rate (\u03bcmol kg\u22121 min\u22121); V is the distribution volume for palmitate or glucose (40 and 160\u00a0ml kg\u22121, respectively); C1 and C2 are the palmitate or glucose concentrations (mmol l\u22121) at time 1(t1) and 2(t2), respectively, and E2 and E1 are the plasma palmitate or glucose enrichments (TTR) at time 1 and 2, respectively. 13CO2 production (Pr13CO2; mol min\u22121) from the infused palmitate tracer was calculated as: \nwhere TTRCO2 is the breath 13C\/12C ratio at a given time point, VCO2 is the carbon dioxide production (l min\u22121), k is the volume of 1\u00a0mol of CO2 (22.4\u00a0l mol\u22121), and Ar is the fractional 13C label recovery in breath CO2 observed after the infusion of labeled acetate [30] and calculated as: \nwhere F is the infusion rate of [1,2-13C] acetate (mol min\u22121). Plasma palmitate oxidation (Rox; mol min\u22121) can subsequently be calculated as: \nwhere Rd palmitate is the rate of palmitate disappearance (mol min\u22121); F is the palmitate infusion rate (mol min\u22121), and 16 is the number of carbon atoms in palmitate. Total plasma FFA oxidation was calculated by dividing palmitate oxidation rates by the fractional contribution of plasma palmitate to total plasma FFA concentration. Muscle derived TG use was estimated by subtracting plasma FFA oxidation from total fat oxidation. However, it should be noted that the indirect stable isotope methodology does not differentiate between muscle- and lipoprotein-derived TG use. However, the contribution of lipoprotein-derived TG oxidation to total energy expenditure is assumed to be of relative minor quantitative importance, especially in an overnight-fasted state [37].\nIn a previous study, where we applied both an [U-13C] and [6,6-2H2] glucose tracer [18] during moderate intensity exercise, it was shown that the percentage of plasma glucose Rd that was oxidized varied between 96\u2013100%. Therefore, plasma glucose oxidation rate during exercise was calculated as: \nTherefore, muscle glycogen oxidation was calculated by subtracting plasma glucose oxidation from total carbohydrate oxidation.\nMuscle-sample analyses\nMuscle tissue samples were freed from any visible nonmuscle material and rapidly frozen in liquid nitrogen. About 15\u00a0mg of each muscle sample was frozen in liquid nitrogen-cooled isopentane and embedded in Tissue-Tek (Sakura Finetek, Zoeterwoude, The Netherlands). Multiple serial sections (5\u00a0\u03bcm) from all biopsy samples were thaw mounted together per subject on uncoated, precleaned glass slides and stained with either acid-Schiff (PAS) [29] or immunolabelled oil red O [21] to measure fiber-type-specific glycogen or intramyocellular lipid content, respectively. To determine the muscle-fiber typing, (type I vs type II), we performed a myosin adenosine 5\u2032-triphosphatase stain [22]. As previously described, the applied fluorescence and bright field microscopy techniques represent semiquantitative methods that can be used to compare fiber-type-specific IMTG and glycogen content [39, 41, 43, 44]. Fiber-type-specific IMTG content was expressed as the fraction of the measured area that was stained with oil red O. For IMTG quantification, average lipid-droplet size was calculated by dividing the total number of lipid aggregates by the total area measured. Mixed muscle-lipid content, lipid-droplet size, and lipid-droplet density were determined by calculating the average value in the type I and type II muscle fibers, with a correction for the relative area occupied by each fiber type within each field of view of each muscle cross-section, within each individual subject. The oil red O epifluorescence signal was recorded for each muscle fiber resulting, on average, in a total of 71\u2009\u00b1\u200919 muscle fibers analyzed for each muscle cross-section (45\u2009\u00b1\u200914 type I, 26\u2009\u00b1\u200910 type II) per subject. The PAS bright-field images were converted post hoc into eight-bit grayscale values to quantify glycogen. The mean optical density of the PAS-stained muscle fibers were determined by averaging the optical density measured in every pixel in the cell, corrected for the mean optical density of the background stain, containing no muscle fibers. For PAS analysis, on average per subject, a total of 151\u2009\u00b1\u200961 muscle fibers were analyzed for each muscle cross-section (92\u2009\u00b1\u200932 type I, 59\u2009\u00b1\u200926 type II).\nStatistics\nAll data are expressed as mean \u00b1 SD. Before statistical analysis, data normality was confirmed at P\u2009>\u20090.05 using the Kolmogorov\u2013Smirnov test on log-transformed raw data. To compare tracer kinetics, substrate utilization rates, IMTG contents, and\/or plasma metabolite concentrations over time between trials, a two-way repeated measures analysis of variance was applied. When a significant F ratio was obtained, post hoc analyses were completed using a Student\u2013Newman\u2013Keuls test. For nontime-dependent variables, a Student\u2019s t test for paired observations was used. Statistical significance was accepted at P\u2009<\u20090.05.\nResults\nPlasma metabolite and hormone concentrations\nPlasma FFA, triacylglycerol (TG), glycerol, glucose, lactate, and insulin concentrations over time during exercise are shown in Fig.\u00a02. There were no differences in plasma FFA and glycerol between trials at rest or during the first 40\u00a0min of exercise (Fig.\u00a02a,c). However, at all time points after 60\u00a0min, plasma FFA and glycerol concentrations increased over time in both trials, but levels remained significantly lower in the CHO vs CON trial (P\u2009<\u20090.01). Plasma TG levels declined throughout exercise in both trials but were significantly higher in the CHO vs the CON trial (P\u2009<\u20090.05; Fig.\u00a02b). Plasma glucose concentrations increased in the CHO trial at the initiation of carbohydrate ingestion and were significantly higher in the CHO vs CON trial (P\u2009<\u20090.05; Fig.\u00a02d). Over the last hour of exercise, plasma glucose steadily declined in both trials but remained significantly higher in the CHO vs CON trial. Plasma insulin levels in the CHO trial significantly increased during the initial 90\u00a0min of exercise as compared to CON (P\u2009<\u20090.05; Fig.\u00a02f). Thereafter, plasma insulin steadily declined in both trials. Plasma lactate concentrations increased above baseline levels, with plasma lactate concentrations being greater in the CHO vs CON trial (Fig.\u00a02e). In contrast, during the latter stages of exercise, significantly higher plasma lactate levels were observed in the CON vs CHO trial. Plasma norepinephrine concentrations increased threefold within the first 20\u00a0min of exercise and continued to rise throughout the exercise period in both trials, with no significant differences between trials (Fig.\u00a03). Plasma epinephrine levels increased gradually in both trials during the first 2\u00a0h of exercise, after which, levels increased substantially (P\u2009<\u20090.001; Fig.\u00a03). However, in the CHO trial, there was an attenuated increase in epinephrine, resulting in significantly (P\u2009<\u20090.05) lower concentrations vs CON.\nFig.\u00a02Plasma metabolite concentrations during 3\u00a0h of cycling at 50% Wmax in CON and CHO trials. Values are means \u00b1 SD, n\u2009=\u200910. Crosses, significantly different than CON trial (P\u2009<\u20090.05)Fig.\u00a03Plasma catecholamine concentrations during 3\u00a0h of cycling at 50% Wmax in CON and CHO trials. Values are means \u00b1 SD, n\u2009=\u200910. Crosses, significantly different from the CON trial (P\u2009<\u20090.05)\nPlasma tracer kinetics\nAs plasma FFA and glucose concentrations varied over time in both trials (Fig.\u00a02a,d), nonsteady-state Steele [32] equations were applied to calculate tracer kinetics. At rest, tracer kinetics did not differ between trials as carbohydrate supplementation did not start until the onset of exercise. At rest, plasma glucose Ra and Rd averaged 14.9\u2009\u00b1\u20091.6 and 15.0\u2009\u00b1\u20091.8\u00a0\u03bcmol kg\u22121 min\u22121, respectively. Resting palmitate Ra, Rd, and Rox averaged 1.92\u2009\u00b1\u20090.43, 1.89\u2009\u00b1\u20090.55, and 0.74\u2009\u00b1\u20090.22\u00a0\u03bcmol kg\u22121 min\u22121, respectively, with 38\u2009\u00b1\u20094% of palmitate Ra being oxidized. The onset of exercise significantly elevated plasma glucose and palmitate Ra, Rd, and\/or Rox (Table\u00a02). Plasma glucose Ra and Rd, and palmitate Ra, Rd, and Rox increased continuously during the exercise in both trials (Table\u00a02). During the entire exercise period, average glucose Ra and Rd was 49\u2009\u00b1\u20099% greater in the CHO compared with the CON trial (P\u2009<\u20090.05; Table\u00a02). In contrast, plasma palmitate Ra, Rd, and Rox was 41\u2009\u00b1\u200912% lower during exercise in the CHO vs CON trial (P\u2009<\u20090.05; Table\u00a02). Acetate label recovery maintained a constant value during exercise and averaged 86\u2009\u00b1\u20094% of the acetate infusion rate. In a pilot experiment, we confirmed that carbohydrate supplementation does not affect acetate label recovery during exercise, which was in accordance to previous findings [44]. \nTable\u00a02Tracer kinetics and respiratory measures during exercise\u00a0First hourSecond hourThird hourExercise averageCONCHOCONCHOCONCHOCONCHOGlucose\u00a0Ra (\u03bcmol kg\u22121 min\u22121)21.1\u2009\u00b1\u20092.746.8\u2009\u00b1\u20099.6a28.8\u2009\u00b1\u20094.855.8\u2009\u00b1\u20096.8a38.1\u2009\u00b1\u20097.572.9\u2009\u00b1\u20098.2a30.4\u2009\u00b1\u20094.7 59.9\u2009\u00b1\u20097.2a\u00a0Rd (\u03bcmol kg\u22121 min\u22121)21.9\u2009\u00b1\u20093.148.3\u2009\u00b1\u200912.3a30.3\u2009\u00b1\u20097.960.0\u2009\u00b1\u200911.2a39.5\u2009\u00b1\u20097.272.9\u2009\u00b1\u20098.5a31.7\u2009\u00b1\u20094.7 61.9\u2009\u00b1\u20097.3aPalmitate\u00a0Ra (\u03bcmol kg\u22121 min\u22121)2.49\u2009\u00b1\u20090.802.03\u2009\u00b1\u20090.40a3.55\u2009\u00b1\u20090.781.97\u2009\u00b1\u20090.57a6.43\u2009\u00b1\u20090.893.60\u2009\u00b1\u20091.92a4.36\u2009\u00b1\u20090.712.59\u2009\u00b1\u20090.70a\u00a0Rd (\u03bcmol kg\u22121 min\u22121)2.48\u2009\u00b1\u20090.792.06\u2009\u00b1\u20090.40a3.50\u2009\u00b1\u20090.791.96\u2009\u00b1\u20090.56a6.36\u2009\u00b1\u20090.883.57\u2009\u00b1\u20091.34a4.32\u2009\u00b1\u20090.702.58\u2009\u00b1\u20090.69a\u00a0Rox (\u03bcmol kg\u22121 min\u22121)2.28\u2009\u00b1\u20090.731.84\u2009\u00b1\u20090.42a3.49\u2009\u00b1\u20090.721.91\u2009\u00b1\u20090.68a6.35\u2009\u00b1\u20090.733.62\u2009\u00b1\u20091.38a4.26\u2009\u00b1\u20090.592.53\u2009\u00b1\u20090.77a\u00a0%Ra ox91.5\u2009\u00b1\u20095.990.9\u2009\u00b1\u20098.698.7\u2009\u00b1\u20095.8395.6\u2009\u00b1\u20098.899.4\u2009\u00b1\u20097.899.8\u2009\u00b1\u20097.097.2\u2009\u00b1\u20096.296.0\u2009\u00b1\u20096.8\u00a0Ac recovery81.7\u2009\u00b1\u20095.7\u201386.5\u2009\u00b1\u20094.7\u201386.2\u2009\u00b1\u20094.1\u201385.8\u2009\u00b1\u20093.9\u2013Respiratory measures\u00a0VO2 (ml)2,666\u2009\u00b1\u2009912,690\u2009\u00b1\u2009902,713\u2009\u00b1\u2009882,709\u2009\u00b1\u2009982,773\u2009\u00b1\u2009932,772\u2009\u00b1\u20091092,717\u2009\u00b1\u2009892,724\u2009\u00b1\u200997\u00a0VCO2 (ml)2,361\u2009\u00b1\u2009812,412\u2009\u00b1\u2009762,336\u2009\u00b1\u2009762,424\u2009\u00b1\u200984a2,327\u2009\u00b1\u2009882,453\u2009\u00b1\u200994a2,342\u2009\u00b1\u2009792,430\u2009\u00b1\u200983a\u00a0RER0.89\u2009\u00b1\u20090.010.90\u2009\u00b1\u20090.010.86\u2009\u00b1\u20090.010.90\u2009\u00b1\u20090.01a0.84\u2009\u00b1\u20090.010.89\u2009\u00b1\u20090.01a0.86\u2009\u00b1\u20090.010.89\u2009\u00b1\u20090.01aValues are expressed as means \u00b1 SD, n\u2009=\u200910. Tracer kinetics during each hour of exercise and averaged during 3\u00a0h of cycling at 50% Wmax in the CON and CHO trial.Ra Rate of appearance; Rd rate of disappearance; Rox rate of oxidation; %Raox %Ra palmitate oxidized; Ac recovery fraction 1,2-13C acetate label recovery in expired CO2aSignificantly different compared with the CON trial (P\u2009<\u20090.05)\nSubstrate source utilization at rest\nThere were no differences in total resting energy expenditure (5.37\u2009\u00b1\u20090.52 vs 5.32\u2009\u00b1\u20090.53\u00a0kJ min\u22121 in the CON and CHO trial, respectively) or substrate source utilization rates between trials. Total fat oxidation averaged 0.08\u2009\u00b1\u20090.01\u00a0g min\u22121, contributing 58\u2009\u00b1\u20096% to total energy expenditure. Plasma FFA oxidation rates averaged 0.061\u2009\u00b1\u20090.011\u00a0g min\u22121, contributing 53\u2009\u00b1\u20097 to total energy expenditure at rest. The use of muscle- and\/or lipoprotein-derived TG sources averaged 0.016\u2009\u00b1\u20090.008\u00a0g min\u22121 and contributed 7\u2009\u00b1\u200920% to total energy expenditure. Total carbohydrate oxidation rates averaged 0.13\u2009\u00b1\u20090.03\u00a0g min\u22121, contributing 42\u2009\u00b1\u20096% to total energy expenditure.\nSubstrate source utilization during exercise\nThe applied 50% Wmax workload resulted in subjects cycling at 199\u2009\u00b1\u200921\u00a0W, corresponding with a relative workload intensity of 63\u2009\u00b1\u20091% VO2max From rest to exercise, energy expenditure increased tenfold. No differences in energy expenditure were observed over time or between trials (57.3\u2009\u00b1\u20095.9 and 57.8\u2009\u00b1\u20096.4\u00a0kJ min\u22121 in the CON and CHO trial, respectively; Fig.\u00a05). Total carbohydrate oxidation rates were significantly higher in the CHO vs the CON trial (2.35\u2009\u00b1\u20090.37 vs 1.97\u2009\u00b1\u20090.33\u00a0g min\u22121, respectively; P\u2009<\u20090.001). Concomitantly, total fat oxidation rates were higher in the CON vs CHO trial (0.62\u2009\u00b1\u20090.12 vs 0.48\u2009\u00b1\u20090.16\u00a0g min\u22121, respectively; P\u2009<\u20090.001). In the CHO trial, carbohydrate and fat oxidation contributed 66\u2009\u00b1\u20096 and 34\u2009\u00b1\u20095% to total energy, respectively. In the CON trial, carbohydrate and fat oxidation contributed 56\u2009\u00b1\u20095 and 44\u2009\u00b1\u20097% to total energy expenditure during exercise.\nThe use of specific substrate sources over time during exercise is illustrated in Fig.\u00a04. The calculations for substrate source utilization in Figs.\u00a04 and 5 commences at 40\u00a0min because of the nonsteady-state transition from rest to exercise. Plasma FFA and glucose oxidation rates increased over time in both trials (P\u2009<\u20090.01; Fig.\u00a04a,c). In contrast, the respective use of muscle- (and lipoprotein) derived TG and glycogen declined over time (P\u2009<\u20090.01; Fig.\u00a04b,d). Plasma glucose oxidation was significantly greater in the CHO vs CON trial and averaged 13.2\u2009\u00b1\u20092.1 vs 6.7\u2009\u00b1\u20091.1\u00a0kJ min\u22121, respectively (P\u2009<\u20090.001: Fig.\u00a04c). Plasma FFA oxidation rates were substantially greater in the CON vs CHO trial and averaged 13.8\u2009\u00b1\u20092.9 vs 9.1\u2009\u00b1\u20092.8\u00a0kJ min\u22121, respectively (P\u2009<\u20090.001; Fig.\u00a04a). No significant differences were observed in the use of muscle (and lipoprotein-derived) TG and glycogen over time between trials.\nFig.\u00a04a Plasma FFA, b other fat sources, c plasma glucose, and d muscle glycogen rate of oxidation (kJ min\u22121) during 3\u00a0h of cycling at 50% Wmax in CON and CHO trials. Values are means \u00b1 SD, n\u2009=\u200910. Crosses, significantly different from CON trial (P\u2009<\u20090.05)Fig.\u00a05Whole-body substrate source utilization (kJ min\u22121) during the first, second, and third hour of cycling and averaged over the entire 3\u00a0h, at 50% Wmax in the CON and CHO trial. Crosses, plasma FFA, other fat sources, total carbohydrate, and\/or plasma glucose and\/or glycogen use significantly different from the CON trial (P\u2009<\u20090.05)\nFigure\u00a05 illustrates average substrate source utilization rates calculated over each consecutive hour of exercise. During the first hour of exercise, there were no differences in endogenous fat source utilization between trials. In contrast, plasma glucose oxidation rates were higher in the CHO trial, resulting in a 10\u2009\u00b1\u20096% lower glycogen use compared with the CON trial (P\u2009<\u20090.05; Fig.\u00a05). During the latter stages of exercise, plasma glucose oxidation rates remained higher in the CHO vs CON trial, with no differences in muscle glycogen use between trials (Fig.\u00a05). In the first hour, fat contributed nearly the same amount of energy in the CON and CHO trial, 36\u2009\u00b1\u20096 and 33\u2009\u00b1\u20097%, respectively. In contrast, during the second and third hour of exercise, total fat oxidation rates increased substantially, with greater fat use in the CON vs CHO trial. By the third hour, fat oxidation contributed 51\u2009\u00b1\u20098% to total energy expenditure in the CON trial, whereas fat oxidation contributed only 36\u2009\u00b1\u20099% in the CHO trial (P\u2009<\u20090.01; Fig.\u00a05). In the CHO trial, the attenuated increase in fat oxidation rate during exercise was offset by a significant increase in total carbohydrate oxidation rate. Total carbohydrate oxidation rates during exercise averaged 1.97\u2009\u00b1\u20090.33 and 2.35\u2009\u00b1\u20090.37\u00a0g min\u22121 in the CON and CHO trial, respectively (P\u2009<\u20090.01). As there were no differences in the average rate of glycogen utilization between treatments during the second and third\u00a0hour of exercise (Figs.\u00a04d and 5), the increased carbohydrate use in the CHO trial was fully attributed to increases in plasma glucose oxidation over the last 2\u00a0h of cycling (Figs.\u00a04c and 5). Muscle (and lipoprotein-derived) TG and glycogen use declined over time but did not differ between trials during the latter stages of exercise (Fig.\u00a05).\nSkeletal muscle lipid content\nPreexercise fiber-type-specific muscle IMTG was not different between conditions and averaged 0.067\u2009\u00b1\u20090.025 and 0.018\u2009\u00b1\u20090.010 arbitrary units (AU) in type I and II muscle fibers, respectively. There was a significantly greater net decline in intramyocellular lipid content in the type I vs type II muscle fibers after exercise (Fig.\u00a06a). No differences in the net decline in myocellular lipid content were observed between trials. Exercise resulted in a 76\u2009\u00b1\u200921 and 78\u2009\u00b1\u200922% reduction in type I muscle-fiber lipid content in the CHO and CON trial, respectively (P\u2009<\u20090.01). Type II muscle-fiber lipid content was not significantly reduced after exercise and showed a net decline of 0.003\u2009\u00b1\u20090.014 and 0.008\u2009\u00b1\u20090.0012\u00a0AU in the CHO and CON trials, respectively (P\u2009=\u2009NS). The observed decrease in type I muscle intramyocellular lipid content was attributed to significant decreases in both lipid-droplet size (preexercise, 0.560\u2009\u00b1\u20090.150; vs postexercise, 0.359\u2009\u00b1\u20090.152\u00a0\u03bcm2) and lipid-droplet density (preexercise, 0.119\u2009\u00b1\u20090.031; vs postexercise, 0.041\u2009\u00b1\u20090.022 droplets \u03bcm\u22122).\nFig.\u00a06Fiber-type-specific net intramyocellular lipid and glycogen utilization (expressed as mean arbitrary units \u00b1 SD, n\u2009=\u200910) during 3\u00a0h of cycling at 50% Wmax in CON and CHO trials. Asterisk, type II significantly different than type I (P\u2009<\u20090.05); Crosses, significantly different from the CON trial (P\u2009<\u20090.05)\nSkeletal muscle glycogen content\nAt rest, glycogen contents did not differ between trials (0.087\u2009\u00b1\u20090.018 vs 0.103\u2009\u00b1\u20090.021\u00a0AU in the CHO vs CON trial, respectively; P\u2009>\u20090.05) or between fiber types (0.095\u2009\u00b1\u20090.019 vs 0.094\u2009\u00b1\u20090.021\u00a0AU in the type I vs type II muscle fibers, respectively; P\u2009>\u20090.05). Exercise resulted in a significantly greater net reduction in muscle glycogen content in the type I vs the type II muscle fibers (P\u2009<\u20090.05; Fig.\u00a06b). The net decline in muscle glycogen content after exercise was significantly greater in the CON vs the CHO trial (38\u2009\u00b1\u200919 and 57\u2009\u00b1\u200922% greater in the type I and II muscle fibers, respectively; P\u2009<\u20090.01)\nDiscussion\nThe aim of this study was to examine substrate source utilization with (CHO) or without (CON) carbohydrate supplementation on subsequent glycogen and IMTG use during prolonged cycling exercise in endurance-trained male cyclists. Carbohydrate ingestion increased total carbohydrate oxidation, which was entirely attributed to a greater plasma glucose appearance (Ra) and disappearance (Rd) rate. The greater glucose availability in the CHO trial lowered muscle glycogen use during the first hour of exercise, resulting in significant muscle glycogen sparing in both type I and II muscle fibers after 3\u00a0h of exercise. Carbohydrate ingestion inhibited adipose tissue lipolysis, resulting in lower plasma FFA Ra, Rd, and Rox. The lower total fat oxidation rate was entirely attributed to reduced plasma FFA oxidation, as muscle IMTG use was not affected by carbohydrate supplementation.\nIMTG use during prolonged exercise\nThe importance of the IMTG pool as a substrate source during exercise has been disputed. Many of the inconsistencies in the literature regarding IMTG use seem to be explained by the methodological limitations associated with the biochemical mixed-muscle TG extraction analyses [46]. More recent studies using either stable isotope tracers, 1H-magnetic-resonance spectroscopy, electron microscopy, and\/or immunofluorescence microscopy on ORO stained muscle cross-sections have now established that IMTG is indeed a viable substrate source during exercise in healthy humans [37]. In accordance, substantial net reductions in type I muscle-fiber lipid content as assessed by immunohistochemical oil red O staining of muscle cross-sections have been observed after both endurance and resistance type exercise [11, 20, 41, 44]. Furthermore, our laboratory has found a significant correlation between IMTG contents assessed by biochemical TG extraction analyses and immunofluorescence microscopy on ORO stained muscle cross-sections (unpublished observations). In the present study, we observed a significant 20\u2009\u00b1\u20093 and 19\u2009\u00b1\u20092% contribution from muscle- and\/or lipoprotein-derived TG sources use during exercise in the CHO and CON trial, respectively (Fig.\u00a05). Concomitantly, muscle biopsy analyses revealed a 76\u2009\u00b1\u200921 and 78\u2009\u00b1\u200922% net decline in type I muscle-fiber IMTG content in the CHO and CON trial, respectively (Fig.\u00a06a). This translated in a 67\u2009\u00b1\u200919 and 70\u2009\u00b1\u200920% decline in mixed-muscle intramycellular lipid content in the CHO and CON trial, respectively.\nIMTG utilization during prolonged exercise with carbohydrate supplementation\nEarlier estimations [28] and more recent findings [41, 45] show that the progressive increase in plasma FFA delivery during the latter stages of prolonged exercise suppresses IMTG mobilization and\/or oxidation. In agreement, pharmacological inhibition of adipose tissue lipolysis has been shown to reduce plasma FFA availability and augments IMTG use during exercise [42, 44, 47]. This possibly suggests that during situations of decreased FFA availability there is a compensatory increase in IMTG utilization in vivo in humans. Carbohydrate supplementation during exercise also inhibits adipose tissue lipolysis and reduces plasma FFA availability [9, 10]. Therefore, it could be speculated that carbohydrate supplementation could augment IMTG use during the latter stages of prolonged exercise. In the present study, carbohydrate supplementation resulted in a 23\u2009\u00b1\u200917% reduction in whole-body fat oxidation because of the concerted reductions in adipose tissue lipolysis with a concomitant 41\u2009\u00b1\u200912% lower plasma palmitate Ra, Rd, and Rox when compared with the CON trial. Despite the attenuated increase in plasma FFA availability in the CHO trial, no differences in estimated IMTG use were observed between trials (Figs.\u00a04b and 5). In agreement, after exercise, type I muscle-fiber lipid content was similarly reduced by 76\u2009\u00b1\u200921 and 78\u2009\u00b1\u200922% in the CHO and CON trial, respectively (Fig.\u00a06a).\nIn the present study, we did not observe any differences in IMTG use after carbohydrate ingestion. This might be because of the metabolic effects of increased glucose availability in the CHO trial, as we observed a 49\u2009\u00b1\u20099% greater glucose Ra (Table\u00a02) and a subsequent 48\u2009\u00b1\u20099% higher plasma glucose oxidation rate throughout exercise (Fig.\u00a04c). Furthermore, carbohydrate ingestion elevated plasma insulin concentrations by nearly 60% in the first hour of exercise vs CON (Fig.\u00a02f). Elevated plasma insulin is a potent inhibitor of adipose tissue lipolysis [6], resulting in a substantially lower FFA availability in the CHO vs CON trial (Table\u00a02). Although reduced plasma FFA availability could stimulate IMTG hydrolysis by reducing the allosteric inhibition of hormone-sensitive lipase (HSL) [44], we did not observe greater IMTG use in the CHO trial (Figs.\u00a04b and 6a). However, this elevated plasma insulin could also serve to inhibit HSL activity, as a recent study showed blunted HSL activity during exercise when glucose is ingested [48]. Watt et al. [48] suggested that the decreased HSLa was most likely mediated via increased insulin and decreased epinephrine concentrations, and that even in situations when local intramuscular mediators of HSL are altered (i.e., muscle contraction, substrates\/products), the concurrent hormonal milieu may play a greater role in the activation of HSL. This previous study also found no effect of glucose ingestion on estimated IMTG oxidation, despite a decrease in plasma FFA availability and HSL activity [48].\nOur present findings are not in line with de Bock et al.[11], who reported attenuated IMTG use after carbohydrate supplementation during a more intense exercise session in less-well-trained subjects. As such, we conclude that the proposed stimulating effects of reduced plasma FFA availability on IMTG mobilization and\/or oxidation are offset by the inhibitory effects of carbohydrate supplementation on IMTG hydrolysis. The latter likely includes the greater glycolytic flux (Table\u00a02), concomitantly higher circulating insulin (Fig.\u00a02f), and decreased epinephrine levels (Fig.\u00a03), all of which have been associated with reduced HSL activity in muscle tissue [48].\nEffects of carbohydrate supplementation on muscle glycogen use\nIt has been established that prolonged moderate-intensity exercise results in greater glycogen depletion in type I vs type II muscle fibers [8, 14]. In accordance, we observed a 73\u2009\u00b1\u200925 and 42\u2009\u00b1\u200918% greater net utilization of type I vs type II muscle-fiber glycogen content in the CHO and CON trial, respectively (Fig.\u00a06b). This is the first study to show glycogen sparing during prolonged cycling exercise in both type I and II muscle fibers (Fig.\u00a06b). This finding seems to be in contrast to most previous studies that failed to observed muscle glycogen sparing after carbohydrate supplementation during cycling exercise [5, 9, 13, 17, 24, 25]. In line with our findings, some studies have also reported muscle glycogen sparing after carbohydrate supplementation during running exercise [34, 35]. However, these studies reported muscle glycogen sparing to be restricted to the type I muscle fibers only [34, 35]. The apparent discrepancy with our findings is likely explained by the differences in the type (running vs cycling) and duration (1\u20132 vs 3\u00a0h) of the exercise that was implemented, as with increasing exercise duration, muscle-fiber-type recruitment shifts to include more type II fiber activation as the type I fibers become glycogen depleted [15].\nOur findings of muscle glycogen sparing with carbohydrate supplementation during prolonged constant intensity cycling are in line with some [4, 12], but definitely not with the majority of studies [5, 9, 13, 17, 24, 25]. The apparent discrepancy between the current study and prior work that failed to observe muscle glycogen sparing with carbohydrate supplementation during cycling might be explained by differences in training status of the subjects, the intensity and duration of the exercise, the amount and timing of the ingested carbohydrate, the absence of the time-dependent assessment of muscle glycogen use, and the lack of muscle-fiber-type-specific glycogen analyses.\nGlycogen sparing occurs early in exercise with carbohydrate supplementation\nIn agreement with our findings of skeletal muscle glycogen sparing, whole-body muscle glycogen use was lower during the initial hour of exercise in the CHO vs the CON trial (Fig.\u00a05). In accordance to the hypothesis set forth by Coggan and Coyle [7], we observed a greater dependence on muscle glycogen as a substrate source during the early stages of exercise in the CON trial (Fig.\u00a05). As exercise duration lengthened, the contribution of muscle glycogen decreased over time as muscle glycogen stores became depleted, while blood glucose uptake and oxidation rates increased progressively (Fig.\u00a04). In line with this hypothesis, we observed lower muscle glycogen use during the initial hour of exercise in the CHO trial. At the onset of exercise, carbohydrate administration caused a 49\u2009\u00b1\u20099% greater plasma glucose Ra and Rd in the CHO compared with the CON trial (Table\u00a02), which paralleled a 60% increase in plasma insulin concentrations as compared to CON during the first hour of exercise (Fig.\u00a02). Concomitantly, these changes because of the augmented glucose availability at exercise onset decreased the reliance of muscle glycogen as a fuel source during the first hour of exercise. The latter is in accordance with a previous work reporting significant glycogen sparing after carbohydrate supplementation after 60\u00a0min of running exercise [34]. We conclude that the majority of glycogen sparing because of carbohydrate supplementation occurs during the initial stages (<1\u00a0h) of prolonged exercise, resulting in lowered glycogen utilization in type I and II muscle fibers. As our findings show that muscle glycogen sparing after carbohydrate supplementation occurs early in exercise, endurance athletes should be recommended to initiate sports-drink consumption immediately at the onset of exercise to maximize the potential for glycogen sparing, along with maintaining euglycemia and high blood glucose oxidation rates late in exercise.\nConclusions\nThis study provides compelling evidence that carbohydrate supplementation spares muscle glycogen utilization in a time-dependent manner during prolonged exercise in endurance-trained cyclists. Carbohydrate supplementation augments plasma glucose uptake and oxidation rate, while reducing muscle glycogen use during the early stages of exercise, thereby sparing type I and II muscle fiber glycogen stores. Carbohydrate supplementation during exercise lowers plasma FFA rate of appearance, uptake, and oxidation, but does not modulate the use of IMTG as a substrate source during cycling exercise.","keyphrases":["exercise","glycogen","isotopes","intramyocellular triacylglycerol","imtg","fat metabolism","sports drinks"],"prmu":["P","P","P","P","P","P","R"]}
{"id":"Mol_Neurobiol-3-1-2039847","title":"Heat Shock Proteins and Amateur Chaperones in Amyloid-Beta Accumulation and Clearance in Alzheimer\u2019s Disease\n","text":"The pathologic lesions of Alzheimer\u2019s disease (AD) are characterized by accumulation of protein aggregates consisting of intracellular or extracellular misfolded proteins. The amyloid-\u03b2 (A\u03b2) protein accumulates extracellularly in senile plaques and cerebral amyloid angiopathy, whereas the hyperphosphorylated tau protein accumulates intracellularly as neurofibrillary tangles. \u201cProfessional chaperones\u201d, such as the heat shock protein family, have a function in the prevention of protein misfolding and subsequent aggregation. \u201cAmateur\u201d chaperones, such as apolipoproteins and heparan sulfate proteoglycans, bind amyloidogenic proteins and may affect their aggregation process. Professional and amateur chaperones not only colocalize with the pathological lesions of AD, but may also be involved in conformational changes of A\u03b2, and in the clearance of A\u03b2 from the brain via phagocytosis or active transport across the blood\u2013brain barrier. Thus, both professional and amateur chaperones may be involved in the aggregation, accumulation, persistence, and clearance of A\u03b2 and tau and in other A\u03b2-associated reactions such as inflammation associated with AD lesions, and may, therefore, serve as potential targets for therapeutic intervention.\nIntroduction\nPathological lesions consisting of intra- and\/or extracellular accumulations of misfolded proteins are characteristic for neurodegenerative diseases such as Alzheimer\u2019s disease (AD). AD is characterized by three distinct pathological lesions: senile plaques (SPs), neurofibrillary tangles (NFTs), and cerebrovascular amyloid angiopathy (CAA) [1]. Both SPs and CAA are formed by extracellular deposition of aggregated amyloid-beta protein (A\u03b2), whereas NFTs consist of intracellular aggregates of hyperphosphorylated tau protein in the cytoplasm of neurons [2, 3]. The A\u03b2 protein is a 4-kDa proteolytic cleavage product [2] of the transmembrane amyloid-\u03b2 precursor protein (APP). The two major forms of A\u03b2 in human brain are A\u03b21-40 and A\u03b21-42, differing from each other only by two amino acids. Cerebral production of A\u03b2 is balanced by clearance from the brain either via active transport across the blood\u2013brain barrier (BBB) or via uptake and degradation of A\u03b2 by microglial cells and astrocytes [4\u20136]. Active transport of A\u03b2 is mediated by A\u03b2 receptors that are capable of transporting A\u03b2, or A\u03b2 in complex with other proteins, across the BBB [7]. In contrast to normal brain, the cerebral A\u03b2 balance is disturbed in AD brains, resulting in accumulation and aggregation of A\u03b2.\nA\u03b2 aggregation includes the formation of A\u03b2 oligomers, protofibrils, and eventually, mature fibrils. Both A\u03b2 oligomers and protofibrils are considered the most toxic forms of A\u03b2 that initiate degeneration of neurons and of cells within the vasculature, such as smooth muscle cells and pericytes [8, 9]. A\u03b2 aggregates do not clear from the brain as efficiently as soluble A\u03b2, and thus, directly lead to increased levels of A\u03b2 in the brain [10]. Furthermore, deposition of A\u03b2 in SPs is accompanied by attraction and activation of both microglial cells and astrocytes [11\u201313]. Activation of these cell types results in increased secretion of pro-inflammatory cytokines as part of a neuro-inflammatory reaction.\nChaperones can be defined as proteins that: (1) have a role in the intracellular handling of misfolded proteins, (2) induce conformational changes of proteins, (3) act as transporter of proteins. \u201cProfessional\u201d chaperones, such as the heat shock protein family (Hsp), are defined as proteins that have a specific function in facilitating normal folding of proteins and intracellular handling of misfolded proteins. Members of the Hsp family recognize misfolded proteins and transport them to the proteasome for degradation. Therefore, this protein family acts as the first line of defense against toxicity induced by misfolded proteins such as A\u03b2 and tau. In contrast to professional chaperones, \u201camateur\u201d chaperones can be defined as proteins that bind to other proteins and induce conformational changes or, alternatively, serve as transporter proteins. Examples of putative amateur chaperones are apolipoprotein E (ApoE), heparan sulfate proteoglycans (HSPGs), and complement factors such as C1q. They have, in contrast to the professional chaperones, primarly an extracellular function. In this paper, we will review the role of both amateur and professional chaperones in the pathogenesis of AD.\nA\u03b2-Binding Proteins in Extracellular Interaction with A\u03b2\nApolipoproteins\nThe apolipoprotein family consists of proteins that conjugate with lipids to form different classes of lipoprotein particles. In human brain, several members of this protein family are expressed, such as apolipoprotein E (ApoE), apolipoprotein J (ApoJ), and apolipoprotein D (apoD).\nApoE is a major determinant of lipid transport and metabolism and is expressed in brain by astrocytes, microglia, pericytes, and smooth muscle cells [14\u201318]. In human, three common isoforms are expressed: apoE2, apoE3, and apoE4 which are all products of alleles at a single gene locus [19, 20].\nThe \u025b4 allele of ApoE is the major genetic risk factor for AD, whereas the \u025b2 allele appears to be protective against AD [21\u201324]. As ApoE immunoreactivity was found in extracellular amyloid deposits in subjects with AD, it has been suggested that it affects amyloidogenesis [25, 26]. In vitro studies provided evidence for a direct interaction of ApoE with A\u03b2 and the formation of stable complexes [27, 28]. Binding of ApoE to A\u03b2 is, however, ApoE isoform-dependent (\u025b2>\u025b3>>\u025b4) [29, 30] and depends on the degree of lipidation [29]. Lipidation of ApoE also seems a major factor in its effect on A\u03b2-mediated cellular toxicity [18]. In addition, ApoE4 promotes the conversion of soluble A\u03b2 into \u03b2-sheet-rich amyloid more than ApoE3 [31\u201333].\nIn contrast to its effect on A\u03b2 in vitro where a consistent accelerating effect of ApoE on A\u03b2 aggregation is observed, the effect of ApoE on A\u03b2 deposition in transgenic (Tg) mice studies is less equivocal. In early studies, both A\u03b2 immunoreactivity and amyloid formation were reduced in ApoE knockout mice [31, 32]. In addition, CAA and associated microhemorrhages were also suppressed in ApoE knockout mice [34]. This effect might be due to the absence of ApoE\/A\u03b2 complexes [35]. In contrast to the effects of murine ApoE in the early studies, both human ApoE3 and ApoE4 suppressed A\u03b2 deposition in Tg mice [36]. In addition, when these mice aged, ApoE4 induced a tenfold higher deposition of fibrillar A\u03b2 than ApoE3 [37]. Consistent with these latter results, human ApoE4 accelerated A\u03b2 deposition in APPSwe Tg mice relative to human ApoE3 [38]. In addition, when human ApoE3 or ApoE4 were knocked in in Tg mice, A\u03b2 deposition was reduced compared to mice carrying endogenous ApoE at 9\u00a0months, and at 15\u00a0months, substantial CAA was observed in mice with human ApoE4, but not with human ApoE3, and, in either case, parenchymal A\u03b2 was sparse [39]. Thus isoform- and species-specific differences in ApoE direct the aggregation or clearance of A\u03b2. Furthermore, it is suggested that the presence of ApoE facilitates internalization and degradation of A\u03b2 from brain parenchyma by astrocytes [40] and human ApoE may reduce A\u03b2 deposition in mouse brain by facilitating A\u03b2 transport across the BBB [36]. Although these Tg mice studies seem contradictional, ApoE clearly affects conformational changes of A\u03b2 and functions as an A\u03b2-transporter protein.\nBesides colocalization of ApoE with A\u03b2 in AD brains, ApoE is also found within neurons containing NFTs [25] where it is able to interact directly with tau protein [41]. Furthermore, ApoE has an isoform-dependent effect on tau phosphorylation, as ApoE3 binds to tau in vitro, whereas ApoE4 fails to bind tau [42]. In addition, an ApoE4-dependent increase in phosphorylated tau has been observed [43\u201345].\nNeuroinflammation in AD comprises both activation of microglial cells and astrocytes and activation of the complement system. A\u03b2 deposits in brain are associated with activated microglia and astrocytes, but also with elevated levels of complement [5, 6, 46]. ApoE may have an anti-inflammatory effect by suppressing microglial and astrocytic activation [47\u201350]. ApoE-deficient mice demonstrate increased levels of IL-6 and TNF\u03b1 after LPS stimulation, suggesting a role of ApoE in inflammatory gene regulation [51]. In addition, ApoE isoform-dependent (\u025b2<\u025b3<\u025b4) differences in nitric oxide (NO) levels have been observed in microglia cells [52]. Transgenic mice expressing the ApoE4 protein isoform show a greater NO production than mice expressing the ApoE3 protein isoform. These data indicate that ApoE4 has a less efficient anti-inflammatory affect, and thus, may accelerate the development of AD.\nApolipoprotein J, also known as clusterin or SP-40\/40, is a highly conserved heterodimeric secreted glycoprotein expressed in brain by epithelial and neuronal cells [53]. ApoJ colocalizes with fibrillar A\u03b2 deposits, and it is suggested that it prevents misfolding and aggregation of soluble A\u03b2 [54\u201356]. ApoD is a glycoprotein associated with high-density lipoproteins in human plasma and also has a high expression level in human brain [57], but neither its physiological role nor its ligand has been identified. ApoD levels are increased in the hippocampus of AD patients and in ApoE-deficient mice [58, 59].\nIn conclusion, ApoE and ApoJ can be regarded as amateur chaperones that regulate A\u03b2 aggregation in vitro. By accelerating the A\u03b2 aggregation process towards mature fibril formation, (human) ApoE prevents formation of toxic A\u03b2 intermediates such as oligomers and protofibrils, and thus, may have a protective function towards development of AD. Moreover, ApoE protects against the development of AD by suppressing the inflammatory reactions associated with AD lesions. Besides its role in inducing conformational changes in A\u03b2, ApoE facilitates A\u03b2 clearance from brain by serving as a transporter molecule of A\u03b2, which will be discussed in paragraph 4.\nHeparan Sulfate Proteoglycans\nProteoglycans are members of a large family of macromolecules with a wide variety of functions ranging from simple physical support to various effects on cell adhesion, motility, proliferation, differentiation, and even tissue morphogenesis. They are composed of linear sulfated polysaccharides (glycosaminoglycans, GAGs), consisting of disaccharide units, covalently bound to a core protein. One of the members of this superfamily is the heparan sulfate proteoglycan (HSPG) family characterized by polymers of repeating disaccharides, N-acetylglucosamine and glucuronic acid, which can be subsequently modified by sulfatation [60, 61]. HSPGs can be subdivided into a family of extracellular matrix proteins, including perlecan, agrin, and collagen XVIII, and a family of cell surface proteins, including syndecans and glypicans [60, 62].\nEver since GAGs were demonstrated in amyloid deposits, the proteoglycans became of interest in amyloidogenesis. The presence of HSPGs in SPs, CAA, and NFTs in AD brains was already demonstrated in the late 1980s [63\u201365]. Only when antibodies became available that could identify the various individual HSPG species was it described that perlecan colocalized with all three lesions characteristic of AD brains [65\u201367]. However, we were not able to confirm these findings [68, 69]. Furthermore, it was shown that in both diffuse and classic SPs, several other HSPGs were found, such as agrin, glypican 1, and syndecan 1-3, whereas collagen XVIII is only present in classic SPs and CAA [69\u201372].\nThese data suggest that HSPGs interact with A\u03b2, thereby contributing to development or persistence of SPs or CAA. HSPGs isolated from Engelbreth\u2013Holm\u2013Swarm tumor prevented proteolytic breakdown of aggregated A\u03b2 [73]. In addition, both agrin and perlecan directly interacted with A\u03b2 and promoted conversion of non-fibrillar A\u03b2 into fibrillar A\u03b2 [70, 74\u201376]. Although the interaction between HSPGs and A\u03b2 is likely mediated predominantly by the sulfate moieties of the GAGs, a role for the protein backbone in A\u03b2 aggregation could not be excluded [77, 78]. As sulfated GAGs were also demonstrated in NFTs in AD brains [79], these macromolecules may also play a role in tangle development. Indeed, sulfated GAGs may induce the formation of paired helical filaments by stimulating tau phosphorylation [80].\nAs heparan sulfates bind to A\u03b2 and interfere with its fibrillogenesis, they are interesting candidates for therapeutic intervention [81]. GAG mimetics are able to inhibit this binding and may block the formation of \u03b2-pleated sheets and adherence of A\u03b2 to the cell surface [82]. The use of GAG mimetics has already been explored in mouse models where they reduced progression of inflammation-associated amyloidosis [83]. The efficacy of one of these compounds is currently being tested in a human phase III trial.\nAs exemplified by ApoE, A\u03b2-binding proteins may play a role in the inflammatory reactions in AD brains. Recently, it was demonstrated that the semi-synthetic proteoglycan analogue dextran sulfate blocks activation of the complement cascade [84]. In addition, chondroitin sulfate proteoglycans are also known to bind to C1q and prevent the formation of the C1 complex in vitro [85]. By doing so, chondroitin sulfate proteoglycans inhibit normal complement function. Furthermore, heparin has long been regarded as a potential complement inhibitor [86].\nIn conclusion, HSPGs do not only colocalize with A\u03b2 and tau, but they also contribute to the development of these lesions. The role of HSPGs in A\u03b2 aggregation might even be a protective one. HSPGs prevent the persistence of toxic A\u03b2 forms, e.g., oligomers or protofibrils, and transform them into more harmless aggregates, i.e., the classic senile plaques containing mature A\u03b2 fibrils that are less toxic than the intermediate aggregates. In addition, HSPGs might play a role in the development of AD lesions by inhibiting complement activation. According to the definitions, HSPGs can therefore be regarded as amateur chaperones. Their ability to recognize a variety of proteins may originate from the heterogeneous structure of the heparan sulfate chains. The negatively charged HS chains are structurally heterogeneous and bind a diverse repertoire of proteins, such as amyloid A, protease-resistant prion protein, \u03b1-synuclein, and tau, providing HSPGs with the ability to interact with a wide range of intracellular and extracellular amyloidogenic proteins [61].\nComplement Factors\nThe complement system is an ancient host defense mechanism which is involved in boosting antibody activity. The system consists of a group of soluble serum proteins C1\u2013C9 and is activated either by immunoglobulin M or G bound to a foreign particle or directly by microorganisms. Proteins such as Hageman factor, C4 binding protein, CDS46, CD59, and C1 inhibitor regulate the complement system. In AD, the complement system is overexpressed and activated [46]. The A\u03b2 protein itself activates this system, and complement factor concentrations are increased in AD brains [87\u201389]. A\u03b2 induces C3 and C4 in AD, and elevated levels of the membrane attack complex (MAC) composed of C5\u2013C9 have been observed [90, 91]. In addition, factors such as Hageman factor, C1q, C3, and C5\u20139 are commonly found in SPs and NFTs [87, 92, 93]. C1q is associated with A\u03b2 deposits and directly binds fibrillar A\u03b2 which activates the complement cascade [94]. In contrast, the complement inhibitor (C1 INH) is downregulated in AD [95, 96]. Thus, an activated complement system is a general feature observed in AD. However, the contribution of complement to the pathogenesis of AD is controversial.\nOn the one hand, it is suggested that complement activation protects against A\u03b2-induced toxicity and even contributes to reducing the accumulation of A\u03b2 in SPs [97]. Transgenic mice expressing complement inhibitors develop increased AD-pathology, whereas increased complement C3 production was associated with a reduction of A\u03b2 deposition [97]. Thus, the complement activation in the brain may be beneficial in AD and possibly also other neurodegenerative diseases [98\u2013100].\nHowever, complement activation may lead to accelerated neurodegeneration as well. Activation of complement in an antibody-independent fashion is achieved by binding of aggregated, but not soluble, A\u03b2 to C1q [12, 90, 101, 102]. This latter finding suggests that in AD, aggregated A\u03b2 induces chronic complement activation. Thus, C1 binding to fibrillar A\u03b2 deposits may precede microglial activation. Both A\u03b2 and pro-inflammatory stimuli are able to activate microglia, which results in increased A\u03b2 and cytokine production [103]. Furthermore, cultured human microglial cells show an increase in cytokine production after co-stimulation of A\u03b2 with C1q and serum amyloid P (SAP) [104]. This suggests that microglia may get triggered by both A\u03b2- and SP-associated factors such as C1q, which results in the secretion of pro-inflammatory cytokines and A\u03b2, both accelerating neurodegeneration.\nAlthough none of the complement factors directly regulate conformational changes of A\u03b2, complement activation as a whole plays a role in the A\u03b2 aggregation in vivo. Therefore, complement factors might act as amateur chaperones, although their exact role in A\u03b2 aggregation remains to be elucidated.\nProfessional Chaperones\nHeat shock proteins (Hsp) are professional chaperones. They are highly conserved proteins constitutively expressed in most cells under normal conditions where they play a role in cellular metabolism and help normal folding processes [105]. In addition, during cell stress, they bind unfolded proteins to keep them in their native state [105]. Heat shock proteins can be divided into two different families based on size and function: classic Hsps such as Hsp100, Hsp90, Hsp70, Hsp60, and the small heat shock proteins (sHsps). Hsps with a molecular weight of 60\u00a0kD or more possess an ATP-binding site and are actively involved in the process of refolding of misfolded proteins [106]. Small Hsps, with a molecular weight of 40\u00a0kD or less, lack this ATP-binding site and assist the Hsps in their refolding function [107]. The role of Hsps in misfolded protein recognition and refolding is illustrated in Fig.\u00a01.\nFig.\u00a01The role of heat shock proteins (Hsp) and small heat shock proteins (sHsps) in recognition and refolding of unfolded and misfolded proteins. Unfolded or misfolded proteins are recognized by Hsps and sHsps. Together with these unfolded or misfolded proteins, Hsps and sHsps form a complex. In addition, Hsps recover unfolded or misfolded proteins back to their native form using ATP. If unfolded or misfolded protein are not recognized by the Hsp\/sHsps, these unfolded or misfolded proteins are capable of forming aggregates\nSmall Heat Shock Proteins\nSmall Hsps function as molecular chaperones that can prevent proteins from adopting an incorrect conformation [108]. The sHsp family is characterized by the presence of an \u03b1-crystallin domain, a stretch of 80\u2013100 amino acids in the C terminal half of the proteins [109]. So far, the sHsp family comprises ten sHsps, including \u03b1B-crystallin, Hsp27, Hsp20, HspB8, and HspB2\/B3 [110]. Although sHsps are predominantly expressed in muscle cells, several family members are also found in human brain.\nIn AD, \u03b1B-crystallin and Hsp27 are upregulated and expressed by astrocytes surrounding SPs and NFTs [111\u2013114], whereas Hsp20, HspB2, and HspB8 colocalize with A\u03b2 in SPs and CAA [115, 116]. Although \u03b1B-crystallin or Hsp27 do not colocalize with A\u03b2 in SPs, direct interaction between A\u03b2 and these sHsps in addition to Hsp20 and HspB8 has been demonstrated [111, 117\u2013119]. In addition, high-affinity binding of \u03b1B-crystallin and A\u03b2 has been observed in eye lenses from AD patients [120]. Furthermore, \u03b1B-crystallin is able to prevent mature A\u03b2 fibril formation, retaining it in a non-fibrillar, but likely a protofibrillar state, which is highly toxic to neurons [121]. Recently, we demonstrated that \u03b1B-crystallin, Hsp20, and HspB8 inhibit A\u03b2-mediated toxicity towards cerebrovascular cells probably by preventing aggregation of A\u03b2 at the cell surface [116, 117]. Others showed that Hsp27 directly binds to hyperphosphorylated tau, thereby protecting against cell death [122].\nHsps are involved in the formation and persistence of misfolded protein aggregates. They are upregulated in several neurodegenerative diseases, such as AD, Creutzfeldt\u2013Jakob disease, and Parkinson\u2019s disease probably as a reaction to the formation of misfolded proteins [113, 123\u2013126]. However, despite of increased intracellular levels, they are unable to prevent accumulation of A\u03b2 in AD possibly because of decreased chaperone activity. In aged rats, this was illustrated by a significant decrease of Hsp90 function [127], resulting in diminished hepatic chaperone capacity. Furthermore, the increasing amount of damaged or misfolded proteins as a result of defects in protein degradation might lead to a total decrease in chaperone activity in aged cells [128]. Thus, the state of misfolded protein recognition and repair systems, such as the (s)Hsp system, might be of great importance in the development of neurodegenerative diseases.\nMiscellaneous Proteins\nApart from the above-described proteins, several other molecules are also associated with the pathological lesions of AD, and some of these can be regarded as amateur chaperones. Acute phase proteins, such as \u03b11-antichymotrypsin (ACT), \u03b12-macroglobulin (\u03b12M), and SAP, are all associated with A\u03b2 deposition [129\u2013132]. ACT is a serine protease inhibitor of the serpin family, and in AD, ACT levels are upregulated, and binding of ACT with A\u03b2 induces A\u03b2 fibrillogenesis [133\u2013135]. Furthermore, when ACT is overexpressed in transgenic mice, an increased plaque load in the brains of these mice and impaired spatial learning is observed [134, 135]. \u03b12M also binds A\u03b2, although in contrast to ACT, this binding prevents A\u03b2 fibril formation and fibril-associated neurotoxicity [136, 137]. \u03b12M promotes the protease-mediated degradation of \u03b12M\/A\u03b2 complexes and contributes to clearance of A\u03b2 from the brain (discussed in paragraph 4) [138, 139]. The glycoprotein SAP belongs to the pentraxin family and is a common component of all known types of amyloid fibrils. SAP is upregulated in AD and protects amyloid fibrils from proteolysis in vitro [140, 141]. SAP not only colocalizes with SPs and interacts with aggregated A\u03b2; SAP oligomers also bind and activate C1 [142]. Both C1 and SAP may bind to fibrillar A\u03b2 deposits in vivo and induce microglial activation, as cultured human microglial cells show an increase in cytokine production after co-stimulation of A\u03b2 with C1q and SAP [104]. These observations further strengthen the above-noted suggestion that not only A\u03b2, but also several A\u03b2-binding proteins, are capable of activating the complement system, and thus, contribute to neuroinflammation in AD. In addition, both \u03b12M and ACT, in contrast to SAP, can be regarded as amateur chaperones, as they regulate conformational changes of A\u03b2.\nTissue-type plasminogen activator (tPA) regulates activation of plasminogen into plasmin and is expressed in various regions of the brain especially in the hippocampus [143]. Several reports suggested an important role for tPA in AD, as the tPA system is involved in A\u03b2 turnover [144, 145]. Fibrillar forms of A\u03b2 stimulate tPA activity in vitro, whereas in AD patients, a reduction of tPA activity is observed in the affected areas [144, 146]. Although tPA has no effect on conformational changes of A\u03b2, it might play a role in the clearance of A\u03b2 from the brain (paragraph 4].\nThe actin-regulatory protein gelsolin is found both intracellularly and in plasma [147, 148]. Plasma gelsolin can be considered an amateur chaperone, as it binds A\u03b2 and not only inhibits its A\u03b2 fibrillization but is also capable of degrading preformed A\u03b2 fibrils [149, 150]. Furthermore, gelsolin inhibits A\u03b2-mediated neurotoxicity [151].\nOne of the major gangliosides in the brain is GM1. Soluble A\u03b2 binds GM1 and the formed complexes accelerate A\u03b2 fibrillogenesis by acting as a seed for A\u03b2 [152]. In the presence of GM1, A\u03b2 is more neurotoxic than A\u03b2 alone, and cholesterol-rich membranes demonstrate accelerated A\u03b2 binding due to the formation of GM1 clusters [153, 154]. As GM1 is a major component of lipid rafts and recent studies suggest that A\u03b2 accumulation in these lipid rafts is an early event in AD development, GM1 might play an important role in the early steps in AD pathogenesis [155, 156].\nIn summary, several proteins are associated with A\u03b2 aggregates in the AD brain and contribute to the aggregation of A\u03b2 and should, therefore, be considered as amateur chaperones. In addition, they might play a role in triggering inflammation.\nA\u03b2-Binding Proteins and Intracellular Interactions with A\u03b2\nIntracellular accumulation of A\u03b2 already starts in the ER or in the Golgi apparatus of the cell [157\u2013159]. Intracellular A\u03b2 is associated with neuronal damage [160, 161], and intraneuronal accumulation of A\u03b2 in transgenic mice was correlated with impairments in synaptic plasticity [162]. Intraneuronal accumulation of A\u03b2 in those brain areas affected earliest in AD suggests a possible relation between intracellular A\u03b2 and development of AD [160].\nA few proteins that interact with intracellular A\u03b2 and affect its intracellular fate have been identified. The endoplasmic reticulum amyloid beta-peptide-binding protein binds intracellular A\u03b2 and mediates neurotoxicity in neuronal cells by forming an intracellular target for A\u03b2 [163]. In addition, the mitochondrial enzyme amyloid-\u03b2 alcohol dehydrogenase also binds A\u03b2 inside neurons, resulting in the production of free radicals [164]. However, whether these intracellular A\u03b2-binding proteins affect aggregation of A\u03b2 within the cells remains unknown. Therefore, both these proteins cannot, for the time being, be defined as amateur chaperones of A\u03b2.\nThe first lines of defense against misfolded and aggregated proteins are the professional chaperones, which counteract these processes and are able to stimulate clearance of misfolded proteins by proteosomal degradation. Newly synthesized proteins are folded by several other proteins, such as immunoglobulin-binding protein (BiP)\/glucose-regulated protein (GRP78), and calnexin. GRP78 is a member of the Hsp70 protein family and interacts with intracellular APP. GRP78 regulates APP and A\u03b2 secretion by intervening between APP and \u03b2-\/\u03b3-secretases within the cell [165].\nIt is not surprising that in AD, where misfolded protein molecules accumulate, both Hsp90 and Hsp70 synthesis is increased. Several members of the Hsp family directly interact with intracellular A\u03b2, but only recently, Hsp70 was identified as a protector against intracellular A\u03b2 accumulation [166, 167]. Besides, immunoreactivity of both Hsp90, 70, and Hsp60 is found in SPs [132], which suggests that these professional chaperones may not only interact with misfolded protein in the cell interior [168\u2013171]. In addition, it has also been postulated that up-regulation of Hsp90 and Hsp70 may suppress the formation of NFTs by partitioning tau into a productive folding pathway and thereby preventing its aggregation [172]. Recently, it was demonstrated that the chaperone CHIP\u2013Hsc70 complex conjugates ubiquitin to hyperphosphorylated tau, which enhances cell survival by elimination of soluble hyperphosphorylated tau [173]. These data suggest that the cell increases production of the Hsps to cope with the presence of misfolded proteins such as hyperphosphorylated tau and accumulating A\u03b2. At some point, this protective mechanism seems to fail, however. In line with this hypothesis, it was shown that the actin and tubulin specific chaperone Hsp60 is decreased in AD, resulting in a decrease of cytoskeletal proteins in AD-affected neurons [174]. Thus, both production and function of Hsps seems to be disturbed in AD, which might result in the accumulation of misfolded proteins. The role of other Hsps in regulating intracellular A\u03b2 or tau folding remains to be investigated (Table\u00a01).\nTable\u00a01Summary of the expression of chaperones in AD brains and their interaction and effects on A\u03b2 and tau\u00a0SP\/CAANFTDirect interactionEffects on A\u03b2 or tau in generalApolipoproteins\u00a0ApoE++A\u03b2\/tau\u2191 Fibrillar A\u03b2 \/\u2193 hyperph. Tau\u00a0ApoJ+?A\u03b2\u2193 A\u03b2 aggregationHSPGs\u00a0Perlecan\u00b1\u00b1A\u03b2HSPGs:\u00a0Agrin+\u2212A\u03b2\u2193 Proteolytic breakdown A\u03b2\u00a0Glypican 1+\u2212?\u2191 Non-fibrillar \u2192 fibrillar A\u03b2\u00a0Syndecan 1\u20133+\u2212?\u2191 Phosphorylation tau\u00a0Collagen XVIII+\u2212?\u00a0GAGs++A\u03b2\/tauComplement factors\u00a0Hageman Factor++?A\u03b2 activates complement in AD\u00a0C1q++A\u03b2C3 \u2193 A\u03b2 deposition\u00a0C3\/C4++A\u03b2\u00a0C5-9++?Heat shock proteins\u00a0Hsp90+?Tau\u2193 Tau aggregation\u00a0Hsp70+?A\u03b2\/tau\u2193 Tau aggregationSmall Hsps\u00a0\u03b1B-crystallin\u2212\u2212A\u03b2\u2193 A\u03b2 fibril formation\u00a0Hsp27\u2212\u00b1A\u03b2\/tau\u2193 A\u03b2 fibril formation\u00a0Hsp20+\u2212A\u03b2\u2193 A\u03b2 fibril formation\u00a0HspB2\/B3++\u2013No effect\u00a0HspB8+\u2212A\u03b2\u2193 A\u03b2 fibril formationAcute phase proteins\u00a0\u03b11-antichymotrypsin+\u2212A\u03b2\u2191 A\u03b2 fibrillization\u00a0\u03b12-macroglobulin+\u2212A\u03b2\u00a0serum amyloid P++A\u03b2\u2191 A\u03b2 fibrillizationMiscellaneous compounds\u00a0tPA\u2212\u2212A\u03b2\u2193 A\u03b2 fibril formation\u00a0Gelsolin\u2212\u2212A\u03b2\u2191 A\u03b2 fibrillizationExpression of chaperones in a specific lesion is illustrated as follows: present (+), by conflicting reports (\u00b1), absence (\u2212), and unknown (?); \u2193 = inhibition or down-regulation, \u2191 = induction or up-regulationSP Senile plaques, CAA cerebral amyloid angiopathy, NFT neurofibrillary tangles, HSPGs heparan sulphate proteoglycans, A\u03b2 amyloid-beta, Hsp heat shock proteins, Apo apolipoproteins, SAP serum amyloid P, tPA tissue-type plasminogen activator, GAGs glycosaminoglycans, LDLR low-density lipoprotein receptor, LRP-1 LDL receptor protein-1, BBB blood\u2013brain barrier\nA\u03b2-Binding Proteins and A\u03b2 Clearance\nA\u03b2-binding proteins, amateur chaperones, play a role in the clearance of A\u03b2 from brain by functioning as a transporter molecule. Two major pathways govern A\u03b2 clearance. By the first pathway, A\u03b2 is removed from brain to blood via active transport across the BBB. This active transport is performed by specialized transporters, so-called \u201cA\u03b2-receptors\u201d, expressed by endothelial cells. Second, A\u03b2 is removed from brain via phagocytosis by both microglial cells and astrocytes. In both pathways, interaction of A\u03b2 with cell surface A\u03b2-receptors is crucial; therefore, the expression levels of A\u03b2-binding proteins might contribute to A\u03b2 clearance by regulating its binding with A\u03b2 receptors.\nThe low-density lipoprotein receptor-related protein-1 (LRP-1) binds A\u03b2 in a complex with ApoE at the abluminal side of the endothelium and internalizes these ApoE\/A\u03b2 complexes followed by degradation in lysosomes or transport into the plasma [4, 175]. However, LRP-1 also mediates transport of free A\u03b2 across the BBB [10]. In contrast to LRP-1, the receptor for advanced glycation end products (RAGE) transports A\u03b2 from the circulation into the central nervous system [176]. Similar to RAGE, the A\u03b2 receptor megalin is also involved in the transport of A\u03b2 from blood to brain, although megalin probably plays only a minor role in A\u03b2 transport. Furthermore, megalin binds A\u03b2\/ApoE complexes rather than free A\u03b2 [177, 178]. Clearance of A\u03b2\/ApoE complexes from brain might be ApoE isoform-dependent. ApoE4 forms less stable complexes with A\u03b2 than ApoE3 or ApoE2; therefore, ApoE4 reduces A\u03b2 transport efficiency across the BBB. Additionally, as described above (paragraph 2), ApoE4 enhances A\u03b2 aggregation more efficiently than ApoE3, which also inhibits clearance. On the other hand, the LDL receptor shows a marked preference for the ApoE3 and ApoE4 isoforms and binds the ApoE2 isoform poorly [179]. Given the similarity between the LDL receptor family, other LDL receptors, such as the LRP-1 receptor, might display similar specificities towards the ApoE isoforms, but this has not been reported yet. Moreover, lipidation of ApoE also affects clearance of ApoE and ApoE\/A\u03b2 complexes from brain, as LRP preferentially binds lipid-rich forms of ApoE [179]. These data indicate that A\u03b2-binding proteins, especially ApoE, and possibly, ApoJ, play an important role in transport of A\u03b2 across the BBB and that both the ApoE isoform and the ApoE lipidation state affect A\u03b2 clearance. In addition to ApoE and ApoJ, the A\u03b2-binding protein \u03b12M also forms complexes with A\u03b2. As \u03b12M is a ligand for LRP-1, these \u03b12M\/A\u03b2 complexes might undergo LRP-1-mediated endocytosis and degradation or translocation into the plasma [7, 139].\nStimulation of the transport of A\u03b2 across the BBB demonstrated to be an effective therapeutic approach in AD, as several studies demonstrated elevated levels of A\u03b2 in the plasma of mice after passive immunization with anti-A\u03b2 antibodies or Fab fragments [180\u2013182], and decline in cognitive performance was arrested in patients that received vaccination [183]. However, the occurrence of severe meningoencephalitis in human patients after active immunization with A\u03b2 hampered widespread application of this type of therapy. Administration of A\u03b2-binding proteins that demonstrate similar positive effects, but possibly, without the severe immune reactions associated with antibody therapy, might provide an alternative strategy. An interesting example of such an A\u03b2-binding protein is gelsolin. This protein has high affinity for A\u03b2 and reduces A\u03b2 levels in a transgenic mouse model of AD [184]. Furthermore, administration of gelsolin or GM1 in PS\/APP mice resulted in decreased A\u03b2 aggregation in the brains [184]. Both gelsolin and GM1 act as a \u201cperipheral sink\u201d for A\u03b2. Although both compounds did not enter the brain, they lowered soluble A\u03b2 concentrations in the blood, shifted the balance between blood and cerebral A\u03b2 concentrations, and accordingly, stimulated A\u03b2 transport over the BBB. Therefore, other A\u03b2-binding proteins administered in the circulation might also act as \u201cperipheral sinks\u201d [181, 184].\nBoth activated microglial cells and activated astrocytes are associated with A\u03b2 deposition and may internalize A\u03b2 fragments via phagocytosis [185\u2013187]. Activation of the complement system is, among others, achieved by Hsps such as Hsp60 and Hsp70, which are able to induce phagocytosis by microglia, and thus, clearance of A\u03b2 [132, 188, 189]. In addition, the absence of ApoE reduces internalization and degradation of A\u03b2 by astrocytes in the brain, demonstrating that ApoE is directly involved in the clearance of A\u03b2 from brain via phagocytosis by microglial cells and astrocytes [40]. tPA might also contribute to clearance of A\u03b2, as it accelerates A\u03b2 clearance from transgenic mouse brains [146]. Thus, as A\u03b2-chaperones contribute to activation of the complement system or activation of microglial cells and astrocytes, these proteins might contribute to the clearance of A\u03b2 from the brain via phagocytosis.\nConcluding Remarks\nProfessional chaperones, such as the heat shock protein family, and amateur chaperones, such as apolipoproteins and HSPGs and several other proteins, have a role in the intracellular handling of misfolded proteins, induce conformational changes of proteins, or act as transporter of proteins (Fig.\u00a02). This suggests that these chaperones form interesting therapeutic targets in the prevention and treatment of neurodegenerative diseases.\nFig.\u00a02The putative role of chaperones in amyloid-\u03b2 (A\u03b2) fibril formation, proteolytic breakdown, and clearance from the brain. In Alzheimer\u2019s disease, soluble A\u03b2, predominantly produced in neurons, is converted into \u03b2-sheet rich protofibrils and eventually forms mature A\u03b2 fibrils. The conversion from soluble A\u03b2 to protofibrils and fibrils, which accumulate in senile plaques and cerebral amyloid angiopathy, is enhanced by chaperones as apolipoprotein E (ApoE), Gelsolin, \u03b11-antichymotrypsin (ACT) and several heparan sulphate proteoglycans (HSPGs), which function as catalysts. In contrast, the heat shock protein family, tissue-type plasminogen activator (tPA) and complement factors prevent the transition of soluble A\u03b2 into protofibrils and mature fibrils. Furthermore, heat shock proteins and tPA stimulate the proteolytic breakdown of (proto)fibrils, whereas HSPGs prevent this breakdown. Finally, the clearance of A\u03b2 from the brain across the blood\u2013brain barrier is stimulated by ApoE, ApoJ, and \u03b12-macroglobulin (\u03b12M), whereas complement factors stimulate phagocytosis-mediated clearance of A\u03b2 by activated microglia and astrocytes\nIn the process of clearance of A\u03b2 from the brain, A\u03b2-binding partners might play important roles by acting as A\u03b2 transporter proteins in both the receptor-mediated clearance of A\u03b2 across the BBB but also as a \u201cperipheral sink\u201d for A\u03b2. Both ApoE isotype and local concentrations in the brain might regulate A\u03b2 transport across the BBB, but as this transport is receptor-mediated, other A\u03b2-binding proteins might also fulfill such a role. In addition, transport of aggregated A\u03b2 across the BBB is less efficient than soluble A\u03b2. Thus, by preventing self-aggregation of A\u03b2, A\u03b2-binding proteins contribute to the clearance of A\u03b2 from the brain. As a therapeutic strategy, A\u03b2-binding proteins serving as a \u201cperipheral sink\u201d, such as gelsolin, seem promising [184].\nOverexpression of professional chaperones, such as the Hsps, to prevent aggregation of misfolded proteins will have to be evaluated carefully, as they also interact with other chaperones and are dependent on this interaction to fulfill some of their functions. This strategy may therefore result in instability of the cell-stress mechanism, which may cause the system to collapse. A solution may be found in the overexpression of several chaperones, which may be required to achieve an impact on the progression of the disease.\nAnother pitfall in the use of professional chaperones as therapeutic agents is their ability to bind misfolded proteins and keep them in an intermediate conformation. This type of conformation might even be more toxic than the aggregated state. As an example, co-incubations of \u03b1B-cystallin with A\u03b2 are more toxic to neurons than A\u03b2 alone [121]. Furthermore, Hsps are most likely to be involved in early development of neurodegenerative diseases, given their natural function. Yet, the role of this protein family in maturation of the neurodegenerative lesions remains to be elucidated.\nIn conclusion, studying the role of chaperones, both professional and amateur, in the pathophysiology of AD will provide us with a better understanding of the mechanisms underlying the formation and accumulation of toxic aggregates in AD, which, eventually, will lead to the design of more effective therapeutic strategies.","keyphrases":["heat shock proteins","chaperones","amyloid","alzheimer\u2019s disease","apolipoproteins","heparan sulfate proteoglycans","amyloid beta-protein"],"prmu":["P","P","P","P","P","P","M"]}
{"id":"Eur_J_Clin_Pharmacol-3-1-2071966","title":"Evaluation of patients\u2019 experiences with antidepressants reported by means of a medicine reporting system\n","text":"Objective To assess experiences related to antidepressant use reported to an internet-based medicine reporting system and to compare the nature of the side effects reported by patients with those reported by health care professionals (HCPs).\nIntroduction\nNon-adherence to prescribed medication regimens is a persistent problem in the treatment of depression with antidepressants. Premature discontinuation, dosing lapses and partial non-adherence often occur [1\u20133]. The illness itself as well as physician-, patient- and treatment-related factors have been suggested to contribute to antidepressant non-adherence, but the ability of health care professionals (HCP) to predict who discontinues medication remains poor [4\u20136]. Earlier studies provide scant information on how patients in clinical practice perceive their treatment with antidepressants, thus insight and an understanding of the patients\u2019 perspectives on non-adherence and discontinuation are limited. Side effects and ineffectiveness are the major reasons cited for the discontinuation of antidepressant therapy [7, 8]. However, the information available on side effects is mostly based on controlled clinical trials, which standardly do not evaluate how bothersome side effects are to the individual patient. Moreover, both the side effects and the effectiveness of the antidepressant therapy observed in clinical trials may not reflect the experiences of the patients in real world settings. Many interventions have been developed to improve adherence to antidepressants, but to date these have failed to demonstrate a clear benefit [9, 10]. Treatment non-adherence remains one of the least understood health-related behaviours. Information received directly from the patient may improve our understanding of the relative importance of antidepressant treatment issues and, consequently, help to explain patients\u2019 behaviour towards antidepressant use.\nReporting systems have shown to be a useful tool in collecting experiences and identifying issues related to the daily use of medicines [11]. In 2004, an internet-based medicine reporting system was established in The Netherlands where users of medicines are able to report all types of experiences with medicines \u2013 side effects as well as experiences with effectiveness and practical and reimbursement issues. We extracted data from this medicine reporting system in order to (1) assess the type of experiences related to antidepressant use reported by patients, (2) assess the relevance of these experiences and (3) compare the nature of the suspected side effects reported by patients with those reported by HCPs.\nMethods\nSetting\nReports were obtained from an internet-based medicine reporting system launched on 11 May 2004 in The Netherlands. This reporting system was initiated by DGV, The Dutch Institute for the Proper Use of Medicine, the Science Shop for Medicines and patient and consumer organisations. Individuals are able to report their experiences with medicines anonymously by completing a report form downloaded by accessing a website (http:\/\/www.meldpuntmedicijnen.nl). Users of the medicine themselves as well as their relatives, acquaintances or (professional) carers, can report an experience. All reports submitted between 11 May 2004 and 13 May 2005 related to the use of antidepressants were reviewed for the clarity and completeness of the description of the experience and checked for multiple reports from the same IP-address. One duplicate report had to be excluded. All other reports were included in the study. The data were stored in an Access database.\nReport form\nThe report form contained items which requested information on age, gender, type of medicine used, nature of the experience, a description of the experience and the relevance of the experience. Individuals filling in the report had to indicate whether the experience was related to either effectiveness, a side effect, a practical issue (e.g. difficulties with swallowing tablets) or a reimbursement issue. More than one experience related to the same drug could be submitted. Experiences related to effectiveness had to be further specified by the individual by selecting one of the following categories: ineffectiveness, a positive effect or a different type of effect (e.g. drug interactions). Experiences related to side effects had to be further specified by selecting one of the following categories: negative side effects, positive side effects (e.g. less side effects as compared with previous medication) or the absence of side effects. Relevance of the experience was assessed by asking the individual filling in the report to indicate both the impact of the experience on a 5-point scale (ranging from very negative to very positive) and whether the experience caused a change in antidepressant use. To specify the change in antidepressant use, those reporting had to select one of the following categories: discontinuation, switching to other treatment, adjustment of dosage or administration, any other action taken or no action taken. In the case of discontinuation of the antidepressant, the report also asked whether the HCP has been informed.\nSide effects\nAll reported side effects (including experiences on ineffectiveness) were coded by one of the authors (SvdW) and checked by a second author (EvG), using preferred terms of the World Health Organisation Adverse Reaction Terminology (WHO-ART) [12]. Preferred terms were combined into groups of similar side effects. Spontaneous reports of adverse drug reactions on antidepressants received from HCPs from May 2004 to May 2005 were included for comparison. These reports were received by The Netherlands Pharmacovigilance Centre Lareb either on paper forms or electronically via http:\/\/www.lareb.nl. Coding and assessment were carried out by qualified assessors from Lareb [13].\nData analysis\nThe relevance of the side effects was assessed by comparing the impact of the (grouped) side effects as a proportion of the side effects that were perceived as \u201cvery negative\u201d. Relevance was also assessed by comparing the proportion of the side effects that caused discontinuation of initial antidepressant use, including the categories \u201cdiscontinuation\u201d and \u201cswitching to other treatment\u201d. The impact and discontinuation proportions of the different groups of side effects were compared using the chi-square test.\nFor the comparison between patients and HCPs, the groups of side effects were compared and expressed as an Odds Ratio (OR) and 95% confidence interval (CI). The 15 most frequently reported (groups of) side effects by patients as well as the 15 most frequently reported (groups of) side effects by HCPs were included in the analysis.\nResults\nCharacteristics\nIn total 2232 individuals submitted a report to the medicine reporting system during the study period, of whom 258 (12%) submitted a report on antidepressants. Of these 258 individuals, 248 (96%) reported for themselves and ten reported for a relative or acquaintance.\nThe mean (\u00b1SD) age of the of the antidepressant users was 42.8 (\u00b113.5) years and 72% were female. The majority of those reporting on antidepressants (63%) were reporting on a serotonin-reuptake inhibitor (SSRI); 12% were reporting on a tricyclic antidepressant (TCA) and 25% on another type of antidepressant. The antidepressants most frequently reported on were paroxetine (35% of total), venlafaxine (15%), citalopram (10%) and mirtazapine (8%). The use of a benzodiazepine as a concomitant drug was reported by 19% of those reporting on antidepressant use.\nNature of experiences\nThe 258 individuals reporting on antidepressant use described 327 experiences. Table\u00a01 shows the nature of the experiences described. Ninety-two individuals (36%) described an experience with effectiveness, of whom 40 (16%) described an experience with ineffectiveness. Four individuals claimed generic substitution as the reason for ineffectiveness, and 45 (17%) reported a positive experience with the effectiveness of an antidepressant. \u201cOther type of effects\u201d in terms of effectiveness that were submitted by seven (3%) individuals included experiences on suspected drug interactions.\nTable\u00a01Nature of the reported experiences related to antidepressant use reported to a medicine reporting system (http:\/\/www.meldpuntmedicijnen.nl)aIssueProportion of total number of individuals (n\u2009=\u2009258) reporting on antidepressant useNature of issueProportion individuals reporting on a specific issueEffectiveness36% (n\u2009=\u200992)Ineffectiveness16% (n\u2009=\u200940)Positive effect17% (n\u2009=\u200945)Other3% (n\u2009=\u20097)Side effects84% (n\u2009=\u2009217)Negative side effect78% (n\u2009=\u2009202)Positive side effect1% (n\u2009=\u20093)Absence of side effect5% (n\u2009=\u200912)Practical issues5% (n\u2009=\u200914)Reimbursement and availability issues2% (n\u2009=\u20094)aA total of 258 reporters submitted 327 experiences. The reported experiences are expressed as a percentage of the total number of individuals submitting reports\nIn total, 217 of the 258 individuals (84%) reporting on antidepressant use submitted a report on side effects. Of these, 202 (78%) described a total of 630 side effects that were experienced as negative. The number of reported side effects ranged from 1 to 11 per reporter experiencing a negative side effect. Twelve individuals (5%) reported that they had not experienced any side effect at all, and three (1%) submitted a positive experience with side effects, reporting that their current antidepressant therapy caused fewer side effects than their previous medication.\nFourteen individuals (5%) reported a practical issue (such as problems with swallowing tablets or bad taste), and four (2%) reported a reimbursement or availability issue (receiving treatment for a relative short duration).\nBox\u00a01 shows a number of the reported experiences.\nRelevance of side effects\nTable\u00a02 shows the relevance, impact and discontinuation rates of the most frequently reported side effects. Of all side effects, 48% resulted in discontinuation of the initial antidepressant therapy. The proportion of side effects that caused discontinuation of the initial antidepressant did not differ significantly between the different side effects. Of those individuals who simultaneously reported an experience on side effects and one on ineffectiveness, 59% (17\/29) reported that their experience resulted in discontinuation of the initial antidepressant therapy. Of all individuals who reported discontinuation of therapy, 29% did not inform their HCP.\nTable\u00a02Relevance of the most frequently reported side effects related to antidepressant useSide effectNumber of reported experiences (n)Impact (% perceived as \u201cvery negative\u201d)Discontinuation of antidepressant use (%)Sleep disorder684750\u00a0Somnolence, drowsiness, fatigue395159\u00a0Insomnia, sleeplessness294138Weight increase484656Sexual problems434433Discontinuation symptoms404350Ineffectiveness403553Apathy284654Excessive sweating234339Nausea, gagging233930Dizziness, fainting236535Headache226850Dry mouth185061Suicidal attempt, thought or tendencya97878aSuicidal attempts, thoughts or tendency is also included, although the number of reported experiences was less than 15.\nOf all side effects, 52% were perceived as \u201cvery negative\u201d. The impact of the side effects differed almost significantly between the groups of side effects (p\u2009=\u20090.052), with headache, dizziness and fainting perceived as most negative.\nSide effects compared between patients and HCPs\nTable\u00a03 presents those side effects most frequently reported by patients and HCPs. Patients and HCPs differed in the nature of reported side effects. Compared with HCPs, patients reported significantly more events such as apathy, excessive sweating, ineffectiveness, discontinuation symptoms, somnolence, insomnia, sexual problems and weight increase. HCPs reported significantly more rash, pruritis, laboratory abnormalities, muscle and joint complaints, congenital disorders, eye disorders, extrapyramidal disorders and menstrual disorders than patients.\nTable\u00a03Reported side effects on antidepressants by patients compared with reported side effects on antidepressants by health care professionals (HCPs)Side effectNumber of reported side effects by patientsPercentage of total number of side effects (n\u2009=\u2009670)Number of reported side effects by HCPsPercentage of total number of side effects (n\u2009=\u2009471)Odds ratio (95%\u00a0CI)aTop 15 most frequently reported side effects by patients\u00a0\u00a0Weight increase487.261.35.98 (2.54\u201314.09)\u00a0\u00a0Sexual problems436.461.35.31 (2.24\u201312.59)\u00a0\u00a0Discontinuation symptoms406.030.614.14 (4.35\u201345.93)\u00a0\u00a0Ineffectiveness406.030.614.14 (4.35\u201345.93)\u00a0\u00a0Somnolence, drowsiness, fatigue395.881.73.58 (1.66\u20137.73)\u00a0\u00a0Insomnia, sleeplessness294.340.85.28 (1.84\u201315.13)\u00a0\u00a0Apathy284.200\u2013\u00a0\u00a0Excessive sweating233.400\u2013\u00a0\u00a0Nausea, gaggingb233.481.72.05 (0.91\u20134.64)\u00a0\u00a0Dizziness, faintingb233.481.72.05 (0.91\u20134.64)\u00a0\u00a0Headacheb223.3112.31.42 (0.68\u20132.96)\u00a0\u00a0Dry mouth182.730.64.31 (1.26\u201314.71)\u00a0\u00a0Abdominal pain142.130.63.33 (0.95\u201311.65)\u00a0\u00a0Anxiety131.920.44.64 (1.04\u201320.66)\u00a0\u00a0Depressed mood111.630.62.60 (0.72\u20139.39)Top 15 most frequently reported side effects by HCPs\u00a0\u00a0Rash, urticaria and pruritis30.4234.90.09 (0.03\u20130.29)\u00a0\u00a0Laboratory abnormalities00224.7\u2013\u00a0\u00a0Muscle and joint complaints30.4204.20.10 (0.03\u20130.34)\u00a0\u00a0Congenital disorders00153.2\u2013\u00a0\u00a0Eye and vision disorders60.9153.20.27 (0.11\u20130.71)\u00a0\u00a0Paraesthesia81.2122.50.46 (0.19\u20131.14)\u00a0\u00a0Headacheb223.3112.31.42 (0.68\u20132.96)\u00a0\u00a0Extrapyramidal disorders, Parkinsonism10.1102.10.07 (0.01\u20130.54)\u00a0\u00a0Menstrual disorders, vaginal bleedings30.4102.10.21 (0.06\u20130.76)\u00a0\u00a0\u00a0Heart rhythm problems71.0102.10.49 (0.18\u20131.29)\u00a0\u00a0Convulsions, epilepsy0091.9\u2013\u00a0\u00a0Drug substitution problems71.081.70.61 (0.22\u20131.70)\u00a0\u00a0Serotonin syndrome0081.7\u2013\u00a0\u00a0Nausea, gaggingb233.481.72.06 (0.91\u20134.64)\u00a0\u00a0Dizziness, faintingb233.481.72.06 (0.91\u20134.64)95% CI, 95% confidence intervalaOR\u2009>\u20091: Patients were more likely to report the side effect than the HCPs; OR\u2009<\u20091: HCPs were more likely to report the side effect than the patientsbSide effects that appear in both the patients\u2019 and HCPs\u2019 list\nDiscussion\nBoth side effects and a lack of effectiveness appear to be important treatment issues for patients who reported on antidepressants in our study. Most of the individuals reporting on antidepressant use described one or more side effects, of which the most frequent were weight increase, sexual problems, somnolence, insomnia and apathy. Ineffectiveness was also reported by a considerable number of individuals. Ineffectiveness is not often reported in literature as an adverse effect of treatment [14]. However, our study reveals that it is a relevant issue for patients during antidepressant therapy. The fact that side effects tend to occur before the therapeutic effect of the antidepressant is perceived may play an important part in explaining early discontinuation of the therapy. Ineffectiveness should therefore receive attention from HCPs in order to prevent early discontinuation of antidepressants.\nOne of the unique features of this medicine reporting system is the possibility to gather information on the relevance of the experiences \u2013 that is the impact of the experience and the change in initial treatment. Overall, half of the experiences were perceived as very negative. Experience of one or more bothersome side effects means an individual is threefold more likely to stop taking antidepressants [7]. Our results show that one half of the side effects resulted in discontinuation of the initial antidepressant therapy. Moreover, of all those individuals who reported discontinuation of therapy, 29% did not even inform their HCP. This supports the concept that this medicine reporting system provides data of which HCPs are often not aware, but which are of crucial importance to any understanding of patients\u2019 behaviour related to the use of antidepressants.\nPatients and HCPs differed in the nature of the side effects reported. Patients were found to report more frequently those events which may be less tangible and visible to HCPs, such as sleeping problems and apathy. In addition, HCPs may consider symptoms reported by patients with psychiatric disorders as a symptom of the disease rather than as one related to the medication. Other notable differences between patients and HCPs in terms of the frequency of reported side effects were those of weight increase, sexual problems, discontinuation symptoms and excessive sweating. The differences in the nature of the reported side effects show that patients and HCPs differ in which type of side effects can be considered to be bothersome and\/or relevant to report. The burden of side effects is clearly underestimated by HCPs [15]. Although HCPs are knowledgeable on the side effects related to SSRI use, they underestimate the frequency of these side effects and how bothersome they are to patients [16]. In addition, HCPs may be reticent in reporting a side effect to a reporting system. More than patients, HCPs evaluate the side effect reported by the patient according to perceived relevance and causality related to the medication. Side effects which are considered by the HCP as well-known or not related to the medication consequently get lost to the health care system [17].\nPatient reporting is not yet widely accepted, and the number of systems collecting experiences from patients is still limited. In the UK, the Prescription-Event Monitoring (PEM) system seeks to identify adverse events recorded following the use of newly marketed drugs selected for monitoring on the first 20,000\u201350,000 patients given the new drug [18]. Since the middle of 2003, the Danish Medicines Agency and the Netherlands Pharmacovigilance Centre Lareb also accept reports on adverse drug reactions from patients [19]. In Sweden, the KILEN Consumer Institute for Medicines and Health started a consumer database in 1997 that collects experiences related mainly to dependence and side effects of benzodiazepine and antidepressant use [19]. However, these systems focus only on adverse events, while other aspects of medicine use, including experiences on ineffectiveness, practical and reimbursement issues, have also been shown to be relevant to patients.\nA limitation of this internet-based reporting system is that the reporters are anonymous and, therefore, further contact and feedback are not possible. Consequently, a thorough assessment of causality between the side effect and the antidepressant was not always possible. The Netherlands Pharmacovigilance Centre Lareb, on the other hand, whose primary aim is the early detection of new adverse drug reactions, has the facilities to request further medical information by contacting the patient and\/or his\/her HCP [20]. However, we believe this does not detract from our conclusion. The individuals reporting through the internet-based reporting system proved to be capable of providing clear descriptions of their experiences and of balancing the benefits and burden of treatment. The patient experiences provide important information on how patients in clinical practice perceive treatment with antidepressants. Such patients experience sleep disorders, weight increase, sexual problems, apathy and ineffectiveness as events which have a negative impact and which frequently lead to discontinuation of the antidepressant therapy. Because adherence decisions are mostly a rational balance of perceived benefits versus burden [21], an assessment of patient experiences may improve the understanding of patients\u2019 behaviour towards antidepressant use. This information can be used in the development of more targeted adherence-enhancing strategies that may lead to optimisation of antidepressant treatment from the perspective of both HCPs and patients.","keyphrases":["antidepressants","discontinuation","patient reporting","adverse events"],"prmu":["P","P","P","P"]}
{"id":"Int_J_Hematol-4-1-2330060","title":"Differential expression of HOX genes upon activation of leukocyte sub-populations\n","text":"The HOX genes are key determinants of cellular identity both in early development and in the renewal and differentiation of adult blood cells. Although a number of studies have examined the expression of individual HOX genes in defined blood cell lineages, we have undertaken a comprehensive analysis of HOX gene expression in resting and activated lymphocytic and monocytic subpopulations. This has revealed distinct patterns of expression between different cell types and resting and activated states. (Main category A: Erythrocytes, Leukocytes and Hematopoiesis, subcategory: 8: Lymphocytes).\nIntroduction\nThe HOX genes are a family of homeodomain-containing transcription factors that define specific positional identities, both in vertebrate and invertebrate embryos [4]. They are also involved in the regulation of haemopoiesis (reviewed by [13]), and blocking HOX gene function by the use of antisense oligos or gene knock out perturbs a number of different haemopoietic events. Thus for example myeloid, erythroid and lymphoid haemopoiesis are all defective in HoxA9 knock out mice [8], and the antisense ablation of HoxB5, HoxB6, HoxB7 [15], or HoxA5 [5] blocks erythroid differentiation. Forced expression of HOXA10 in CD34+ progenitor cells purified from cord blood results in a significant reduction in the number of B cells and natural killer cells but a marked increase in monocytes [14]. There are also examples of specific regulatory functions for HOX genes, including the modulation of globin transcription by HOXB6 [12]. In addition, HOX genes can also act to regulate the proliferation of haemopoietic stem cells (HSCs). Hence HOXB4 is a strong, positive regulator of HSC self renewal (reviewed by [10]), and an estrogen-dependant version of HOXB8 can be used to drive the production of neutrophils and macrophages from their respective progenitor cells [16].\nDespite these studies, relatively little is still known about the expression of HOX genes in the mature blood lineages (reviewed by [6]). In order to address this we purified different cell populations from human peripheral blood on the basis of their expression of the surface markers CD4, CD8, CD14 and CD19 that are present on T-lymphocytes (CD4 and CD8), monocytes, and B-lymphocytes respectively. HOX expression was compared in \u2018resting\u2019 cells and cells that had been activated by challenging with specific antigenic stimuli\u2014antibodies against CD2, CD3 and CD28 for CD4+ and CD8+ cells, Lipopolysaccharide for CD14+ cells and anti-IgG for CD19+ cells. Each population of cells underwent proliferation in response to this treatment, as measured by [3H]thymidine incorporation (Fig.\u00a01). RNA was extracted from each cell type and the expression of each of the 39 HOX genes was assed by quantitative PCR (QPCR), relative to the amount of Beta-actin transcript present (Fig.\u00a02).\nFig.\u00a01Proliferation of activated lymphocyte populations. Proliferation was measured by [3H]thymidine incorporation 4\u00a0h after activation. Values shown are the fold increase in incorporation compared to resting cells. Error bars: SEM from triplicate experimentsFig.\u00a02HOX gene expression in resting (a) and activated (b) after 6\u00a0h, (c) after 24\u00a0h) lymphocytic and monocytic populations. Expression was assessed by quantitative PCR and is shown as a relative value to the number of beta-actin transcripts (\u2018Relative expression\u2019). HOX genes are abbreviated to show only their family and paralogue position, hence HOXA2 is shown as \u2018A2\u2019. HOX genes for which no expression was detected are not shown in the figure. Each value shown is the mean from three experiments. Significant changes in expression levels (p\u00a0<\u00a00.05) 6 and 24\u00a0h after activation are marked (*). Error bars: SEM\nThe highest level of expression is generally shown by the HOXA and HOXC genes, with HOXD genes generally exhibiting a tenfold lower expression level and HOXB genes a 100 fold lower expression level, a feature that is also true for the other cell populations examined. For all of the HOX genes expressed in CD4+ cells there is a significant reduction in HOX expression upon activation. This is particularly striking for the most posterior genes of the HOXA, HOXC and HOXD clusters. A similar pattern is also observed in CD8+ cells, although there is a general reduction in HOX expression with every HOX gene except HOXC10 being present at lower levels in these cells.\nThere are a number of notable differences between the HOX expression profile of CD14+ cells and the CD4+\/CD8+ populations. HOXC4, HOXC6 and HOXC8 are not expressed. The most striking difference though is the change of HOX expression upon activation\u2014in contrast to CD4+\/CD8+ cells, CD14+ cells show a large increase in HOXA2, HOXB13, HOXC10, HOXC13, HOXD1 and HOXD9. Other HOX genes such as HOXB4 and HOXA10 show no significant change in expression upon activation, which again contrasts with the response of CD4+\/CD8+ cells.\nCD19+ cells show a broadly similar pattern of HOX expression to CD14+ cells prior to activation, but have a distinct response to activation. Like CD4+\/CD8+ cells, CD19+ cells exhibit a large reduction in expression of most HOX genes with the exception of HOXD10, HOXD11 and HOXD12 that show a very strong increase in expression upon activation.\nTo our knowledge this is the first comprehensive analysis of HOX gene expression in these leukocyte populations. Taking the data as a whole, each population shows a unique pattern of HOX expression that may define cellular identity. Generally the expression levels are in agreement with those previously reported for individual HOX genes in other studies [6, 7, 9]. However the changes of HOX gene expression upon cellular activation have not previously been reported. The magnitude of these changes is striking, with the activated T cell subsets exhibiting an almost complete loss of expression of many HOX genes especially the more posterior members of the groups (HOXA10, HOXC13, and HOXD12). Conversely, activated monocytic and B-lymphocyte cells show a large increase in expression of the same genes. Given the key regulatory roles of HOX genes it is likely that these changes in expression facilitate the cellular changes associated with activation. One of these is increased proliferation, a response that is known to be mediated by HOX genes in a number of hematopoietic lineages (reviewed by [1]). Indeed the antisense mediated ablation of HOXB2 or HOXB4 in T-lymphocytes was previously shown to block the proliferation of activated cells (Care et al. 1995). Furthermore HOXA10 deficiency causes a severe immunological disturbance in uterine tissues characterised by rapid T cell proliferation and a failure of progesterone-mediated immunosuppression [17]. The HOX genes are also likely to regulate lineage-specific transcription upon activation, and in this context the contrasting expression levels of these genes presumably reflect the dramatically different response of each cell type.\nMaterials and methods\nCD4+, CD8+, CD14+ and CD19+ cells were isolated using the Dynabead system (Invitrogen) and cultured as previously described [2]. Quantitative PCR was performed as previously described [11]. [3H]thymidine incorporation was evaluated by a standard procedure. Briefly, 2\u00a0\u03bcCi of [3H]thymidine (20\u00a0Ci\/mmol) was added for 4\u00a0h to each well. Cells were then recovered, washed, and processed for the determination of TCA precipitable radioactivity. Each evaluation was performed in triplicate. T cell activation was achieved using anti-CD2, anti-CD3 and anti-CD28 antibodies as supplied in the T cell activation\/expansion kit (Miltenyi Biotec).","keyphrases":["hox","activation","monocytes","t-lymphocytes","b-lymphocytes"],"prmu":["P","P","P","P","P"]}
{"id":"Childs_Nerv_Syst-3-1-2092440","title":"Deliberate termination of life of newborns with spina bifida, a critical reappraisal\n","text":"Objects Deliberate termination of life of newborns (involuntary euthanasia) with meningomyelocele (MMC) is practiced openly only in the Netherlands. \u2018Unbearable and hopeless suffering\u2019 is the single most cited criterion for this termination, together with the notion that \u2018there are no other proper medical means to alleviate this suffering\u2019. In this paper, both (and other) statements are questioned, also by putting them in a broader perspective.\n\u201cAn infant with spina bifida cannot \u2018suffer unbearably\u2019. Infants might be able to experience unbearable pain, but spina bifida does not cause it.\u201d (Chervenak 2006)\nIntroduction and historical overview of the treatment of newborns with MMC\nBefore the 1960s, most newborns with meningomyelocele (MMC) were not treated given the consideration that it was in the best interests of the child and family, in most cases resulting in the death of the child. They died from sepsis, meningitis, hydrocephalus, or renal failure. Contraindications to operation included \u201chydrocephalus, irreparable deformities, paralysis of the sphincters, complete paraplegia or any ulcerative process in the region of the spina bifida\u201d [22]. The influence of the surgical approaches since the 1960s allowed gradually effective early treatment of the complicating hydrocephalus, resulting in many patients surviving into childhood. Early treatment of the spinal lesion and hydrocephalus resulted in decreased mortality and morbidity [66]. \u2018Wait and see\u2019 changed into active treatment. Lorber (1971) published the first large series of patients treated between 1959 and 1969 [44]. He found that only 7% of the survivors had less than \u201ccrippling disability\u201d, \u201cmost had a quality of life inconsistent with self-respect, learning capacity, happiness, and even marriage\u201d. To \u201cspare children and families prolonged suffering\u201d he proposed the well-known \u2018Lorber selection criteria\u2019. These four major adverse criteria are: severe paraplegia, gross enlargement of the head, severe kyphosis or scoliosis and associated gross congenital anomalies (such as heart disease) or major birth injuries. No active treatment was advised for children who had one or any combination of these criteria. The result of this proposed selection was to treat only those with lesions on lumbar and sacral level without any other major complication. Others would, according to Lorber, \u201cdie quickly\u201d.\nSince the Lorber era there have been tremendous successes in the treatment of patients with MMC [48, 50]. Over the last 30\u00a0years, continual progress has not only been made in the neurosurgical (closure of the back defect, shunting, or endoscopic techniques for the complicating hydrocephalus, treatment of tethered spinal cord and Chiari II malformation), orthopedic (treatment of scoliosis, correction of club feet), urological (preserving normal renal function, securing social continence), and psychosocial treatment of patients with MMC, but also in support for independence by modern rehabilitation programs.\nNevertheless, early decision making (for restriction) and selective treatment of newborns with MMC is still propagated [27, 69], resulting in approvals but also in critiques [7, 23, 41, 42, 49, 79].\nWhat is considered ethical at one time, or in one country, can be considered unethical or unacceptable at\/in another. The ethical debate on treating children with MMC is unique in the sense that these children will\/can have a life of substantial and sometimes severe physical disability but often with normal mentation, this in contrast to severely psycho-motor handicapped children. Children and adults with MMC can be aware of their deficits.\nLorber (1971) stated that untreated children will die quickly [44]. However, he delivered the children anticonvulsants to prevent seizures, opioids and chloral hydrate to prevent pain [25]. One pediatrician has said that these untreated children received the same care and attention as others, being fed and loved, but they also received 60\u00a0mg\/kg body weight of chloral hydrate, four times a day. This was being administered for pain, but the children who were operated on did not receive this regimen. Another physician just said: we don\u2019t feed them [79]. Consequently, the duration of life seems to depend on the attitude of the responsible doctor.\nIn the United States, untreated children were not given morphine or anticonvulsant drugs and were fed every 4\u00a0h, resulting in many \u2018survivors\u2019 with severe handicaps. On the contrary, in the United Kingdom the children died quickly. However, the question remains if these children were really in pain or suffered from seizures; perhaps not.\nNot all physicians did follow the \u2018Lorber criteria\u2019, but treated all children unselectively, regardless of the level of the lesion. This approach resulted in multidisciplinary MMC clinics and MMC teams. The nonselective treatment of newborns with MMC started in the early 1970s and gives us a picture of long-term outcome of newborns with MMC [4, 9, 32, 48, 52] and also of the quality of life and health status of adults with spina bifida [58, 63]. Some physicians became bitter after treating children with MMC for years [2].\nIn the early 1980s, the \u2018Baby Jane Doe (\u2018Stony Brook baby\u2019) case resulted in the United States in an ethical debate on the prospect physicians make about the future life of handicapped children born with MMC. This girl was born with MMC, complicated with hydrocephalus (although diagnosed by the attending physician as microcephalic). The team of physicians told the parents that the child would spend her life \u201clying in bed, bottle fed....She would experience no joy, sadness or any such emotion except response to pain and would develop no cognitive skills. She might survive for 20\u00a0years\u201d [51].\nBased on the given information, the parents agreed not to treat their child. However, the physicians turned out to be wrong. Although the child developed meningitis, sepsis, and hydrocephalus (which were only treated to some extent), she survived all these complications, was taken home, became ambulating and communicating on an acceptable level of intelligence, all this despite the partial treatment [24, 25].\nAfter the introduction of alpha feto-protein (AFP) screening in the late 1980s (followed by amniocentesis) and ultrasound diagnosis, many fetus with MMC can now be detected early in pregnancy. Nowadays, after being counseled, most pregnant couples decide to abort a fetus with MMC (in many Western countries resulting in an abortion rate of 95%, resulting in fewer children with MMC born [13, 14, 53]), seen by many as \u2018prevention\u2019 of MMC. But what advice should neonatologists, neurologists, or neurosurgeons give the parents when a full-term child with MMC is born? Is the advice not to treat, or to terminate the life of the newborn deliberately (involuntary euthanasia) an ethically defendable one?\nDeliberate and active termination of life in newborns with MMC is only discussed and practiced in an open way in the Netherlands [35, 38_40, 71_76, 78]. The judgment \u201cunbearable and hopeless suffering\u201d of the newborn and \u201cthe prospect of unbearable and hopeless suffering\u201d in the future, together with the notion that there is \u201cno other proper medical means to alleviate the unbearable suffering\u201d form the basis for this decision [73]. Because these judgments are questioned, and to quantify the level of discomfort in these neonates, the \u201cRotterdam Prospective Study on Discomfort in Newborns with Spina Bifida\u201d was started in 2005 in the Erasmus Medical Center (EMC)\u2013Sophia Children\u2019s Hospital (Rotterdam, the Netherlands). Although in due time the results of this study will be published, some important aspects will already be addressed in this paper by elaborating on the case of a newborn with a very severe form of MMC and hydrocephalus.\nDeliberate termination of life in newborns with MMC in The Netherlands\nThe Dutch neurosurgeon De Lange analyzed the selection criteria for treatment of newborns with MMC in the Netherlands already in the 1970s [41, 42]. It became clear from an enquiry among Dutch neurosurgeons that a number of them advised against treatment on the basis of physical handicaps alone. Others regarded the expected mental abilities as an overruling factor. When basic communication skills could be expected, most children were treated. At the time, deliberate termination of life was never practiced. De Lange (1970) also concluded \u201cIf we were to decide that in our opinion patients with spina bifida who are below a certain level of physical or mental ability should not be kept alive, it should be illogical not to apply the same rules to other categories of handicap.\u201d [41]\nIn the 1980s, the Dutch ethicist Hertogh (1988) published his paper \u201cEthical considerations on the dilemmas caused by the selective treatment of newborns with MMC\u201d in the leading Dutch medical Journal [30]. He elaborated on the philosophical and ethical aspects of the Lorber selection criteria: \u201cThe objective medical fact of a physical shortcoming is something completely different than the subjective reality of \u2018being-handicapped\u2019 itself. Studies have shown that living as \u2018being-handicapped\u2019 does not correlate directly to the severity of physical shortcomings. The question as to what can be considered as an acceptable handicap transgresses, in other words, the scope of what can be ascertained in a medical-scientific way and this, therefore, refers inevitably to the subjective experience. It has to be questioned whether the quantification of qualitative aspects of medical treatment perhaps offers nothing more than pretence-rationality, caused by the used decisive terminology. In conclusion: up to date the criterion \u2018quality of life\u2019 appears to be more deceptive than clarifying in the medical decision about the possible continued existence of a newborn with spina bifida.\u201d [30] He also referred to the ethics of the philosopher Levinas: \u201cEthos cannot be founded in scientific facts; it is founded in the human relationship itself. Mutual dependency, which is characteristic for \u2018human being\u2019, makes us responsible just by itself and therefore defines \u2018the disposition of ethos\u2019 as a rational fact. This relationship always precedes facts themselves.\u201d[30] In other words: human beings, just because of the fact that they are human beings, have an intrinsic and mutual responsibility for the well-being of others, this responsibility itself preceding physical qualities. This thoughtful paper, however, was not given much attention in the Dutch medical press.\nIn 1993, baby Rianne was born in a small town in the Netherlands with a MMC at level of the first and second lumbar vertebrae complicated by hydrocephalus. The lower limbs were deformed and paralyzed. Computed tomography (CT) scan of the head confirmed the diagnosis of severe hydrocephalus. On the first day of her life, the decision was made by the pediatrician and the gynecologist in consultation with a neurologist, neurosurgeon, and pastor not to operate the back lesion and the hydrocephalus. On the fourth day of her life, at the request of the parents, the gynecologist Prins terminated the life of the baby by a lethal injection. In an interview, Prins stated [37]: \u201cClosing the defect and fighting infections with maximal treatment could enable the child to survive for years. No one knows how long\u201d. On the question of what an operation could have accomplished, Prins answered: \u201cThe defect would have been closed. The possibility of secondary infection, which was an immediate threat to her life, would have been diminished. Her brain pressure would rise, however, and it would have been necessary to implant a shunt\u201d. On the question of how he would calculate the benefit of treatment, he answered: \u201cIt (the operation) would have prolonged her life without treating the symptoms and consequences of the defect because the operation would not have alleviated them. The operation would only have minimized the possibility of infection, but it would have created the necessity for repeated operations involving the shunt and the urological system because there were obstructions in the passage between the kidney and the bladder. Her brain damage was so extensive and the possibility of movement so limited. She would never have been able to walk or sit unsupported; she would have been incontinent in her bladder and bowel. There was sensory damage as well; she could not feel sensation in her lower body. She would have started life with repeated operations, need for persistent medical attention, no possibility for self-support, and very low communicative ability\u201d [37]. As a definition of a severe handicap he stated: \u201cThis can relate to the estimated possibility of becoming happy\u201d [37], and he further referred to the Dutch Association of Paediatrics who developed criteria, including the lack of capacity to communicate, lack of capacity for self-maintenance, persistent need for medical assistance, and the existence of pain. All these criteria grouped together gave a picture of the baby\u2019s quality of life. Because most of the criteria were satisfied, the baby was considered to be severely handicapped, resulting in a very poor expected quality of life; the child was not operated on because of the severity of the expected handicaps.\nOn the necessity of life-termination, Prins (1997) explained: \u201cThe baby was in a great deal of pain and the normal analgesics did not work well. So it would have been necessary to use narcotic analgesics\u2014morphine-like drugs. And that would have caused depression of consciousness. It is clear to me that the use of such drugs would have depressed normal functioning\u2014behavior, expressions, and sensibility. Using morphine-like drugs would have caused a situation of floating between heaven and earth. It would have been dishonest and should be considered as unethical and unjust. There is no moral difference between killing and withholding useless medical treatment. There is an emotional difference. And there should not be a legal difference\u201d [37].\nAlthough elements of the offense of murder were proven, the physician was acquitted based on the unavoidable conflict of duties between his duty to prolong life and his duty to alleviate unbearable suffering [26].\nIn 1996, three Dutch child neurologists and one neurosurgeon questioned in the leading Dutch medical journal the active termination of life of newborns with MMC [59]. They defended the opinion that (severe) handicapped life could also be meaningful. Deliberate termination of life was not considered appropriate; sedation and the delivery of opioids were mandatory in the case of suffering. Not treating a child with MMC should not result in a more severe handicapped life; the decision to withhold treatment was only made in the case of severe pulmonary or cardiac complications with the prospect of early death. The possible suffering of the parents should not be an indication for deliberate termination of the life of a newborn with MMC. They referred to the study of Steinbok et al. (1992) [68] for their argument that the life of children and adults with (severe) MMC can be meaningful [59].\nIn 2003, the Dutch physician\/medical ethicist Van de Vathorst stated that whenever it is decided not to treat a defective newborn, deliberate termination of the life of this newborn should be regarded as a moral demand, based on the principle of \u2018mercy\u2019 [72]. She illustrated her arguments with a case of a newborn with a thoracic MMC. Interestingly enough, the life of this child was not terminated. It died by itself, a few days old, because of pulmonary insufficiency. Although she received a national award for this paper, it resulted in several disapproving letters to the editor.\nIn January 2005, two pediatricians, one physician, and one pediatric neurologist reported on the deliberate termination of life of 22 newborns in the Netherlands between 1997 and 2004 [73]. Explaining the fact that all 22 cases were newborns with MMC and hydrocephalus, Verhagen et.al. referred to the \u2018baby Rianne case\u2019, stating that \u201cher life was dominated by unbearable suffering\u201d and \u201csince this case it is established that newborns with a severe form of spina bifida do suffer severely\u201d [73].\nAccording to the authors, the deliberate termination of life was necessary because of the presence of \u201cunbearable and hopeless suffering, acutely and long-term, with no other proper medical means of alleviating the suffering\u201d [73]. All parents consented to the proposal of termination of life; in four cases, they explicitly requested for it. For the public prosecutor, the termination of life was acceptable if four requirements were properly fulfilled: (1) the presence of hopeless and unbearable suffering, (2) consent of the parents to termination of life, (3) consultation of another physician or of a multidisciplinary MMC team, and (4) a proper and careful execution of the termination. These four criteria eventually became the cornerstone of the so-called \u2018Groningen Protocol\u2019, a protocol meant to insure that the decision for life termination, the executing itself, together with reporting to the legal authorities, are all conducted in a proper way. This protocol was approved and adopted by the Dutch Association of Paediatrics in 2005.\nThe motivation for the judgement of \u2018unbearable and hopeless suffering\u2019 in the 22 cases of spina bifida was [73]:\n\u2018suffering\u2019 (acute and chronic pain, physical pain) (100% of the 22 cases);the \u2018lack on ability to live or do things independently\u2019 as the result of \u2018severely disturbed sensomotoric development\u2019 (100% of the 22 cases);the \u2018lack of possibility to verbal and non-verbal communication\u2019 (82% of the 22 cases);\u2018the prospect to dependency to the medical circuit as the result of frequent hospital admissions and operations\u2019 (77% of the 22 cases)the \u2018life expectation\u2019 (\u2018the burden of severe suffering increases by a longer life span\u2019) (in 59% of the 22 cases).\nNone of the cases led to prosecution; all cases were found to be in accordance with good medical practice.\nThis article of Verhagen et al. (2005a) gave rise to only a few reactions in the Netherlands. Kompanje et al. (2005) [38] questioned the validity of \u2018unbearable suffering\u2019 as a useful criterion. They also stated that with the use of modern palliative care, \u2018suffering\u2019 always can be dealt with in an adequate way, as did Laane (2005) [39, 40]. De Jong (2006) [35] made a plea for \u2018letting them die\u2019 instead of \u2018making them die\u2019 in the case of an untreated newborn with MMC.\nOn request of the editor of the New England Journal of Medicine, Verhagen and Sauer (2005) published another article concerning the Groningen protocol and the 22 cases [75] and a similar article appeared in Pediatrics [76]. The reactions in the international medical press were mostly negative. In his article \u201cControl of suffering on the slippery slope of care\u201d in the Lancet, Feudtner (2005) also addressed the poor quality and the insufficiency of the offered palliative care and questioned whether legalization of active termination of life might not lead to the abuse of it [21]. Saugstad (2005) stated in the Acta Paediatrica: \u201cNeonatologists must be extremely careful not to start on the slippery slope ending in the Dutch practice of euthanasia of newborns. I would strongly warn against this attitude, which I find non-compatible with Western humanistic traditions\u201d [61]. Jotkowitz and Glick (2006), in the Journal of Medical Ethics, were worried: \u201cWe feel an important line has been crossed if the international medical community consents to the active euthanasia of severely ill infants and are concerned about the extension of the policy to other at risk groups\u201d [36]. Bondi et. al. (2006), in Pediatrics, made a plea for an open discussion: \u201cRather than performing an in-depth examination of the philosophical, ethical, and moral implications of this protocol, the authors focus on its technical details and acceptability within the European medical community. The Groningen Protocol centers not on patient self-determination, but instead on the perception of the patient\u2019s current and future suffering. Although the authors note that an open discussion of these issues is \u2018extremely important,\u201d they fail to initiate it. We hope that thoughtful ethicists and legislators will initiate the open discussion that the authors advocate\u201d [8]. In their article \u2018Why the Groningen Protocol should be rejected\u2019, Chervenak et al. (2006) concluded that: \u201cThe justification they offer is woefully inadequate. It relies on appeals to the concepts of \u201chopeless and unbearable suffering,\u201d the \u201cbest interests of the patient,\u201d and \u201cmedical-ethical values,\u201d but it never explains these concepts, and it gives no ethical argument for their clinical application. In fact, not only is no argument for the Protocol given, none can hope to succeed. The Groningen Protocol should therefore be rejected as the basis for obstetric and neonatal practice in The Netherlands and throughout the world.\u201d (11)\nManninen (2006a), finally, made an important contribution with her article \u201cA case for justified non-voluntary active euthanasia: exploring the ethics of the Groningen Protocol\u201d in the Journal of Medical Ethics [45]. She made a thoughtful plea for the ethics of the Groningen Protocol: \u201cWhat the Groningen Protocol proposes to do is both ethical and also the most human alternative for these suffering and dying infants.\u201d Newborns eligible for this protocol \u201cdo face no future at all\u2014that is, the infants must be terminally ill\u201d, \u201cwill die soon no matter what actions doctors undertake, modern medicine can do nothing to save them\u201d, \u201chave no prospects, have crossed the threshold into a comparatively valueless state of existence\u201d, \u201cwill spend the remainder of their lives in a chronic state of nothing but pain and suffering\u201d [45]. Interestingly enough, on November 23 an additional letter of herself, \u201cRegarding the Groningen Protocol\u201d [46], was published in the same journal. Some key passages of this letter: \u201cIt has recently been brought to my attention that the physicians of the Groningen Hospital who originally proposed the Groningen Protocol have proposed to violate the first two conditions of the original Groningen Protocol: (1) The suffering must be so severe that the infant has no prospects for a future; (2) There is no possibility that the infant can be cured or alleviated of her affliction with medication or surgery. For example, Dr. Verhagen has maintained that the Protocol could apply to infants that are not necessarily terminally ill, but nevertheless may face a compromising future. In essence, the Groningen Protocol has begun to make quality of life judgments. If the Groningen physicians have commenced making quality of life judgments, they have strayed away from the original purpose of the Protocol, and this, I believe, may have concerning ethical implications. As I write in my paper: \u201c...the Groningen Protocol does not make quality of life judgments, and it is because of this very important requirement that the Protocol strikes me as humane and morally permissible, for it seems utterly vicious to extend the life of a suffering infant with no prospects for a future. If the infant did have a possible future ahead of her, I would be more hesitant to condone a legal practice that begins to make quality of life judgments, for such judgments can be, and have been, subject to error.\u201d [46] In her opinion, in these end-of-life decisions \u2018quality of life judgments\u2019 should not be made and, in a way, with this letter she also highlights the inconsistencies in the reports by Verhagen et al. Being of crucial importance, the subject of \u2018quality of life judgments\u2019 will be discussed more extensively in paragraphs 4 and 5.\nFinally, in 2006, in The Netherlands a national multidisciplinary expert review committee has been appointed. In the case of active termination of the life of a newborn, the responsible physician is supposed to inform this committee, which then has to determine whether the criteria for life termination have been met. Subsequently this committee has to advise the Public Persecutor\u2019s Office (OM) whether the physician should be prosecuted or not. Although such a committee seems to guarantee more certainty, it can be doubted, however, if this \u2018checking on afterhand\u2019 is of much value for the newborn in question. It seems to be more sensible to change this procedure to a \u2018checking on beforehand\u2019. Legally, this might perhaps be difficult to establish, but it seems not justifiable that legal procedures themselves do dominate the question of appropriateness of life termination; errors in judgment being unacceptable (cf. the level of certainty required for a death penalty in crime in, e.g., the USA).\nAnalysis of the mentioned criteria for non-treatment and deliberate termination of life\nSuffering\nThe actual presence of acute unbearable and hopeless suffering of the newborn and the prospect of future\/chronic unbearable and hopeless suffering as the result of handicaps, pain, and discomfort is the most given argument for deliberate termination of life in neonates with MMC. In the recent Dutch series, this suffering was stated to be present in 100% of the 22 cases [73, 75, 76].\nAcute pain Not any convincing evidence can be found in the literature that newborns with MMC actually do suffer from untreatable pain in the days after birth caused by their back lesion or by other complications. A pediatric surgeon who devoted much of his professional life of more than 30\u00a0years to the treatment of those who have been born with spina bifida stated: \u201cI personally have seen little evidence that the babies have pain in the newborn period, nor have I found them unable to sleep.\u201d [79] Delight and Goodall (1988; 1990) [17, 18] studied the experiences of parents of 44 children born with MMC, all managed without any surgery. All 44 children died before their first birthday. Only a quarter of them did require some medication such as analgesics and anticonvulsants; half of them were given drugs in the dying period. Analgesics were given in a low dose simply to enhance their quality of life [17, 18].\nChronic pain The literature on chronic pain in MMC is scarce. The first systematic examinations of the nature and prevalence of pain in children with MMC was published in 2005 [12]. Headache is not uncommon in adults with MMC and is associated with hydrocephalus. In this series, 56% of 68 patients reported experiencing pain once a week or more frequent. Of the shunted children, 88% reported headaches compared to 79% of the children without a shunt [12]. Children in this study reported frequent and sometimes intense pain at multiple locations associated with the physiological sequelae, equipment usage, and medical management of the disability. The authors concluded that children with spina bifida frequently report clinically significant, under-recognized and untreated pain, and they propagated proper care and adequate medical management of these children. In another series, 55% of 42 patients underwent investigations for one or more episodes of chronic headache due to shunt malfunction, ventriculostomy failure, or symptomatic Arnold\u2013Chiari malformation [20]. Although all these problems sometimes can be fairly serious, the notion of \u2018unbearable and hopeless suffering\u2019 is never mentioned.\nThe \u2018lack of ability to live or do things independently\u2019 as the result of \u2018severely disturbed senso-motoric development\u2019\nThe second argument for unbearable and hopeless suffering is the prospect of lack of ability to live or do things independently as the result of severely disturbed senso-motoric development experienced by the patient in childhood, puberty, and adult life [73].\nMcLone reported in 1982 the results of the unselective treatment of 100 consecutive newborns with MMC [48]. After a follow-up period of 3.5 to 7\u00a0years there were 86 survivors. He concluded that the total percentage of children who could be independent and competitive is approaching 70\u201380%. Hunt and Oakeshott (2003) [32] and Oakeshott and Hunt (2003) [52] studied the outcome in 117 unselected and actively treated patients with MMC at age 35. The patients were treated between 1963 and 1971 without any attempt at selection. Forty-two patients had a severe lesion at a sensory level above Th11, 30 of whom died, most before their first birthday. They died mostly of cardio-respiratory or renal failure, hydrocephalus, or CNS infection. Two of the 12 survivors live independently, drive cars, and have an open employment; six have an IQ >80. In another study [62], 29% out of 60 adolescents with spina bifida had the lesion at a high lumbar or thoracic level. The authors studied the quality of life in these patients. They concluded that there were no significant relationships between the level of the lesion or spina bifida severity and the overall quality of life, which appeared to be 70 to 80 on a 100-point scale; both patients and their parents view the overall quality of life positively [62].\nBesides these results, criteria such as \u2018living independently\u2019 or \u2018doing things independently\u2019 can be confusing when deciding about the future life of vital newborns. Most people who do live a meaningful life are dependent on others or interdependent on each other. In fact, interdependency and especially the willingness to care for other (perhaps dependent) people can be considered as a criterion of a truly civilized society, certainly so when this means to offer \u2018something for nothing\u2019 (this being real mercy).\nThe \u2018lack of possibility of verbal and non-verbal communication\u2019\nIn 18 of the 22 studied Dutch cases, it was predicted \u201c(future) communication of and with the child would not be possible, neither verbally nor nonverbally\u201d [73]. In other words, these babies were predicted to be or to become in a state of deep coma or in a persistent vegetative state. Neither of these states of very severe disturbed consciousness, however, is applicable to a newborn with MMC and hydrocephalus. Furthermore, several publications concerning the level of mental functioning of adults with MMC have been published [3, 4, 9, 32, 33, 47, 67, 68, 70]. Intellectual development varies between normal IQ (this being the case in 60% [9, 68] to 70% [32] of the studied population) and severe mental disability, but the predicted complete absence of the possibility to communicate is never mentioned. On the contrary: \u201cVirtually all infants with spina bifida are capable of meaningful human relationships, independent of the level of the lesion. Indeed, most are of normal intelligence\u201d [23]. Aside from this incorrect medical description there are some other important aspects that need to be highlighted. When all communication is claimed to be impossible, one wonders by what methods at all (the amount of) suffering can be established. Furthermore, when a person is in such a state of deep coma or in a persistent vegetative state, there simply is no cognitive experience whatsoever, also not of (unbearable) suffering. This was confirmed in a recent conviction of the Dutch Medical Disciplinary Committee against a general practitioner who euthanized a comatose patient; the ground of the verdict being \u201cbecause of the comatose state there was no longer any question of unbearable suffering nor any question of euthanasia\u201d [15].\n\u2018The prospect of dependency to the medical circuit as the result of frequent hospital admissions and operations\u2019\nIt seems hardly possible to quantify this criterion in such a way that it can be used to justify life termination of vital newborns. When compared to persons with other congenital malformations or congenital diseases or some acquired diseases, patients with MMC do not appear to need substantially more medical care than, e.g., patients with intracranial tumors or with chronic diseases such as severe pulmonary, cardiac, or neurological disorders. Furthermore, the claim of Verhagen (2004) that \u201cthis child would have to undergo at least 60 operations in the course of a year to temporarily alleviate its problems\u201d [71] again cannot be substantiated: a retrospective survey of treated newborns with MMC over 2\u00a0years in the Sophia Children Hospital showed that three to four operations were needed in their first year of life.\n\u2018Life expectation\u2019 (\u2018the burden of severe suffering increases by a longer life span\u2019)\nIt is clear that the longer a patient lives, the more medical care will be needed in a quantitative way. Whether this means that this will affect the experienced quality of life in a negative way remains totally unclear and cannot be predicted. Furthermore, patients with MMC do require relatively much of their medical care in their first two decades, this steadily decreasing when they grow older and when disabilities and coping with these disabilities are gradually stabilized.\nCan \u2018suffering\u2019 be applied as a reasonable criterion in newborns?\nSuffering, especially the \u2018unbearable and hopeless\u2019 aspects of it, is a complex psychosocial phenomenon and by definition strictly individualized: only the individual itself can experience the existence, the intensity, and hopelessness of it. Others (parents, physicians, nurses) can only to some degree make an estimation of it. It is therefore tentative whether \u2018suffering\u2019 can be applied to newborns at all. What is possible, however, is to establish quite precisely the level of \u2018(dis)comfort\u2019 of a newborn by making use of standardized score charts such as the Comfort score and the Visual Analogue Scale (VAS) (see below).\nThe Dutch physician Beijk(1998) stated it as follows: \u201csuffering will be interpreted by every patient, by every doctor, and by every other involved person in a different way and therefore can lead to completely different conclusions. The bearableness is especially dependent on personal and environmental factors; it is all about the subjective experience of the individual itself who is suffering, about the durableness of the suffering, and about the wish to die\u201d [6]. In 2005, in his Ph.D. thesis, also Hamburg (physician and jurist) concluded \u201cfor the criterion \u2018suffering\u2019 there are no objective or scientifically validated criteria available, with the consequence of decisions of life-termination being dependent of the subjective judgment of the doctor, leading to inconsistent decisions\u201d [29]. Therefore, according to Hamburg, \u201cin a time of evidence-based medicine \u2018suffering\u2019 is not acceptable anymore\u201d [29], as also was suggested by Kompanje et al. (2005) [38]. Although Beijk and Hamburg are referring to the practice of euthanasia (which in the Netherlands is strictly referring to competent patients wishing to die), their statements are the more applicable to newborns, as in these cases predictions are being made about future suffering of others. According to Chervenak et al. (2006): \u201cAn infant with spina bifida cannot have and therefore cannot have lost the ability to realize intentions, desires, and hopes for the future; it can feel pain but cannot \u2018suffer\u2019 as a psychosocial phenomenon\u201d [11]. Laane (2005a) came to the same conclusion: \u201chaving no biographical consciousness, \u2018suffering\u2019 cannot be applied to newborns [39]. To date, it is unclear why this improper use of \u2018suffering\u2019 still continues in this debate.\nCan physicians predict quality of life?\nCounseling the parents\nIn a time in which most fetuses with MMC will be detected by AFP and amniocentesis or by ultrasound diagnosis (in most cases being followed by iatrogenic abortion), the birth of a newborn with MMC is increasingly becoming a rare phenomenon [14]. The decision to terminate a pregnancy in the case of a fetus with MMC is usually made shortly after the diagnosis. It can be questioned whether there is enough time for proper counseling about the prospect of living with spina bifida, as a patient and also as parents. Freeman (1998) stated: \u201cDecisions are made by the parents without knowledge of the child\u2019s future; without knowing whether the child will have minimal deficit and walk with only short leg braces or walk with no braces at all, and without awareness of whether this child will have a thoracic lesion with high paraplegia and spend his or her life in a wheelchair. Few parents at that stage in the pregnancy know much about spina bifida or about what life will be like for that child and for that family. In other words, these rarely are informed decisions\u201d [25]. He points in the same direction concerning physician experience: \u201cToday, few younger physicians have experience with the decision-making process for spina bifida either before or after birth. Thus, physician advisors will lack experience with the joys of caring for a child, even a handicapped child, as well as experience with the pains and problems. They will have little experience with the feelings of the handicapped children themselves. With the lack of experience, how should the physician counsel? On what basis can the inexperienced physician advise uninformed parents? What decision should the parents make?\u201d [25]\nIn their article \u201cTell the truth about spina bifida,\u201d Bruner and Tulipan(2004) also showed their worries about the quality of counseling: \u201cUnfortunately, many healthcare professionals are equally ignorant of the current prognosis of children with spina bifida who have ready access to comprehensive care in a modern multidisciplinary clinic. As a result, much of the information initially provided to couples with a newly diagnosed fetus is biased and misleading. As medical ethicists Bliton and Zaner at Vanderbilt University Medical Center observed, \u201cTo date we have met and held intense conversations with more than 150 pregnant women and their partners. Many times, couples admitted, the initial counseling they received from their obstetrician was slanted\u2014both against disability and toward termination of pregnancy. What they remembered was how the initial obstetric consultation portrayed as grim a picture as possible about their future child\u2019s prognosis.\u201d [10]\nThe statements of Verhagen et al. (2005) mentioned earlier, together with other incorrect predictions (\u201cthese newborns have no prospect of a future and no chance to survive at all; are barely able to breathe\u201d, \u201cthese children face a life of agonizing pain that cannot be alleviated by any proper medical means\u201d, \u201cthe child would suffer such unbearable pain that it has to be constantly anaesthetized\u201d [71], that \u201cthey all have spina bifida of the most severe form at level of the neck and all have non-functioning kidneys\u201d [78]), unfortunately seem to confirm these worries, also in counseling the parents of a newborn with MMC. To date, it is not clear why such incorrect information is presented to parents, colleagues, and society as a whole. When parents are told that communication with and of their child never will be possible, that their child will face a life of agonizing pain and will suffer unbearably, that this suffering cannot be alleviated by any means, that annually at least 60 operations will be needed to offer some (but only temporary) relief, then these parents are left without any hope and their consent\/decision to non-treatment, or to active termination of life, almost can be predicted. However, in such situations the fundamental concept of \u2018informed decisions\u2019 is obviously violated.\nDoes the level of the lesion matter?\nDoes the level of the lesion determine the amount and severity of disturbances? In general, the higher up the spine the lesion is, the more likely are severe neurological disabilities to be found. Cervical lesions often do not contain neural tissue and are simple meningoceles. At lower levels (thoracic and especially lumbar), the converse is true: the infant nearly always being (partially) paralyzed below the level of the lesion, the cord showing the classical features of dysplasia [66]. However, the extent of handicaps that a person with MMC experiences is determined not by the site of the lesion, but almost entirely by the neurological deficit. Verhoef (2005) concluded that young adults with a mild form of spina bifida (and subsequently with few disabilities), did experience their disabilities more often as problematic than contemporaries with a serious form of spina bifida [77]. Likewise, in the study of Seller (1990) [65], eight patients ended up with minimal handicaps, having bony lesions in the thoracic (one patient), lumbar (two patients), and lumbosacral (five patients) spine. Three others with lumbosacral bony lesions were among the most gravely handicapped [65]. According to Hetherington (2005), low lesions do not necessarily indicate a mild course; physical well-being was not related to the level of the spinal lesion [31]. Although persons with sacral lesions showed less impairment compared to persons with higher spinal lesions, Padua et al. (2002) did not find a correlation between quality of life (both physical and mental) and the site of the lesion [54]. According to Pit-ten Cate et al. (2002), lesion level, type of spina bifida, and gender were not significantly related to the quality of life in children with spina bifida [55]. This is in agreement with the results of the study of Sawin et al. (2002) [62]: no significant relationship with lesion level, spina bifida severity or shunt status and quality of life was found in 60 adolescents. Schoenmakers (2003) [64] concluded that being independent in mobility contributes more to quality of life than other functional abilities such as whether or not being wheelchair-dependent. And, although children with the most severe forms of MMC have significantly more and severe disabilities than children with milder forms of MMC, scores about self-esteem did not differ significantly between the two groups [64].\nIn conclusion, the level of the lesion does not correlate with the actual experience of the handicaps themselves.\nIs the diagnosis \u2018extensive brain damage\u2019 easy to make?\nThere is little doubt that surgical procedures are unjustified in hydrocephalic newborns with little or no potential for independent survival, the decision as to what constitutes as irreversible and severe brain damage may however be difficult to make. Ventricular size alone cannot be used as a conclusive predictive criterion. It has been repeatedly demonstrated that there is no consistent correlation between the thickness of the cortex and the eventual psychomotor development. Barf et. al. (2003) [3] concluded that cognitive status was especially negatively influenced by multiple shunt revisions and by other pathologies associated with hydrocephalus. Amacher and Wellington (1984) [1] treated 170 children with hydrocephalus in 636 operative procedures and concluded that \u201cthere is no difference in results based upon measurement of the initial thickness of the cerebral mantle.\u201d They found normal intelligence in 63% of the 5-year survivors. In a recent study also, Beeker et al. (2006) described the difficulty in making a reliable prognosis of the intellectual development based on ventricular size [5].\n\u2018Quality of life\u2019, to conclude\nIt seems impossible to predict with certainty the future quality of life of newborns with MMC. One also has to be aware of the fact that the self-reported quality of life of children with handicaps does not differ from that of children without disabilities, and health care professionals are known to underestimate disabled persons\u2019 quality of life as compared to self-reports [11]. Generally speaking, prognostic judgments about quality of life are conceptually plausible; their failing is simply that, given the available evidence, they do not appear to be reliable [11]. Finally, and perhaps the most important, quality of life judgements are considered as being ethically unacceptable in end-of-life decisions [45, 46].\nNon-discrimination principle\nIn his Ph.D. thesis, Dorscheidt (2006) raises the issue as to whether the deliberate termination of the life of disabled newborns is compatible with the \u2018non-discrimination principle\u2019, in particular the legal prohibition of discrimination on the ground of disability [19]. Deliberate termination of the life of newborns with spina bifida is always preceded by a non-treatment decision (as in the baby Doe case and the baby Rianne case) and according to Dorscheidt, the decision of non-treatment should be questioned with regard to the non-discrimination principle as well. He elaborates on two fundamental rights of the child, being the child\u2019s right to life and its right to health(care), mentioned in international human rights instruments such as the Universal Declaration of Human Rights (UDHR), The United Nations Convention on the Rights of the Child, and the European Convention of Human Rights, and on the authoritative views of international human rights bodies such as the United Nations Children\u2019s Rights Committee (UNCRC) and the Committee on Economic, Social and Cultural Rights (CESCR).\nSome examples: Article 25, UDHR: \u201cEveryone has the right to a standard of living adequate for the health and well-being of himself, including medical care and the right to security in the event of disability. All children shall enjoy the same social protection\u201d. CESCR, par. 26: According to standard Rules: \u201dStates should ensure that persons with disabilities, particularly infants and children, are provided with the same level of medical care within the same system as other members of society.\u201d UNCRC: \u201cIn its examination of States parties reports, the Committee should commit itself to highlighting the situation of disabled children and the need for concrete measures to ensure recognition of their rights, in particular the right to live, survival and development\u201d. UN, General Counsel: \u201cWe will take all measures to ensure the full and equal enjoyment of all human rights and fundamental freedoms, including equal access to health, education and recreational services, by children with disabilities and children with special needs, to ensure the recognition of their dignity, to promote their self-reliance and to facilitate their active participation in the community\u201d. Proposal text of the American delegacy for the UN Convention to Promote and Protect the Rights and Dignity of Persons with Disabilities: \u201dStates Parties reaffirm the inherent right to life of all persons with disabilities, shall take all necessary measures to ensure its effective enjoyment by them, and shall ensure that disability, or perceived quality of life, shall not serve as a basis for infringement of the right to life.\u201d [19]\nThese international political statements and agreements are also reflected in the resolution of the International Federation for Spina Bifida and Hydrocephalus (IF) as formulated at the 12th International Conference (\u201cThe Right to be Different\u201d) for Hydrocephalus and Spina Bifida in Toulouse [34]:\nPeople with spina bifida and hydrocephalus can live a full life with equal value to that of any other citizen and they should not be seen as a medical condition. Their views should be sought and heard by governments and health professionals, who should acknowledge the right of people with spina bifida and hydrocephalus to speak for themselves.People with disabilities have the right to aim for lives as rewarding as those of their peers who have what is regarded as a normal life. Supportive systems must be in place when they are needed.Experience over the past 30\u00a0years has improved considerably the medical outcome and the quality of life of people with spina bifida and hydrocephalus.Most adults with spina bifida underline that their quality of life is not automatically\u2014and should not be given as\u2014a reason for abortion [34].\nAs a recognition of the importance of non-discrimination on the basis of disability (that is to say, present as well as expected disability) or (expected) quality of life in deciding whether or not a newborn child with, e.g., spina bifida should be treated or whether or not it is justified to deliberately terminate such a child\u2019s life, Dorscheidt (2006) recommends to involve the legal concept of \u2018objective justification\u2019 in the medical-decision making process. By using a particular questionnaire, the physician in charge may take advantage of this concept when considering (and deliberating on) how to act properly and professionally. Through this, the physician can ensure the disabled newborn child\u2019s rightful claim to equal protection of its right to life and its right to health(care). Doing so would offer possibilities to replace the usual doctor-centered approach of decision making by a more patient-centered approach as well.\nTo summarize from another perspective: \u201cProminent theories of justice emphasize equality of opportunity for human experience and development; if society has an obligation to provide citizens this very broad equality of opportunity, then it has an obligation to shoulder the burden of care associated with citizens who have spina bifida\u201d [11].\nPalliative care for untreated newborns with MMC\nWhen a newborn with spina bifida is not treated, discomfort can be expected in the nearby future caused by the lesion itself (e.g., by becoming infected), by a subsequent meningitis\/ventriculitis, or by raised intracranial pressure caused by an untreated hydrocephalus. Therefore, the decision not to treat should always be accompanied by offering proper palliative care. It is becoming increasingly evident that, taken modern palliative techniques in newborns seriously, pain and discomfort can always be dealt with in an adequate way [43, 57]. In their article \u201cStrong opioids in pediatric palliative medicine\u201d Hain et al. (2005) also stated that \u201cclinical evidence is accumulating that strong opioids can be used safely and effectively; they should be used as part of a rational approach to the management of pain\u201d [28]. This use of strong pain killers is also highlighted in the recent Oxford Textbook of Palliative Care for Children [16]. In the highly unlikely event of insufficient effect of modern pain medication, terminal palliative sedation should be the next step in the palliative approach [16]. With palliative sedation, the possible experience itself of discomfort can always be dealt with adequately. In the \u201cConsensus guidelines on sedation and analgesia in critically ill children\u201d by Playfor et al. (2006) in Intensive Care Medicine, some 20 guidelines are mentioned by which pain and discomfort in critically ill children can be treated in an efficient way; life termination not being mentioned at all [56].\nWhen it is decided that surgical treatment is not a feasible option in a newborn with spina bifida, it is regarded by some to be an act of \u2018mercy\u2019 to terminate the life of the newborn [72]. This notion, however, is a misconception: they are not terminally ill because of the MMC and\/or hydrocephalus per se; they are \u2018terminally ill\u2019 because of the decision not to treat their congenital malformations. The needs of these untreated newborns, however, are still the same as the needs of all newborns: proper care, feeding, comfort, respect and love; till death (and apparently not death itself). The \u2018need\u2019 for life termination in these patients can only be regarded as an indication of insufficient palliative care. Life termination as an act of mercy can only be applied to hopeless situations that can occur, e.g., on battlegrounds or in mountaineering, when there are no proper means indeed to alleviate real unbearable suffering. This is obviously not the case in a modern equipped hospital (at least, it should not be) with experience in palliative care and pain management.\nThis is all confirmed in the paper of Sauer in 2001, \u201cEthical dilemmas in neonatology: recommendations of the Ethics Working Group of the CESP (Confederation of European Specialists in Pediatrics)\u201d in the European Journal of Pediatrics [60]. In this paper, some ethical principles, applicable to every newborn, are being formulated, like: \u201cdecisions to withhold or withdraw treatment should always be accompanied by optimal palliative therapy and dignified and comforting care\u201d and \u201cevery form of intentional killing should be rejected in paediatrics\u201d [60]. This is followed by: \u201cHowever, giving medication to relieve suffering in hopeless situations which may, as a side effect, accelerate death, can be justified\u201d.\nTo date, it is not clear why these recommendations should not hold for cases of untreated spina bifida and hydrocephalus in the Netherlands and why the Dutch Association of Paediatrics does not adopt these recommendations.\nIllustrative case\nAfter an uneventful pregnancy, a full-term female child was delivered spontaneously, Apgar scores being 4 and 5. Physical examination revealed a huge MMC at high thoracic level (Fig.\u00a01), a very large head with a large but soft fontanel, a marked deformation of the thorax with scoliosis, and clubfeet. After counseling the parents, they persisted in their wish to treat the child, if this probably could save her life. The MMC team decided to respect the parents will and on day 3 the back lesion was closed by the neurosurgeon in collaboration with the plastic surgeon, making use of extensive rotational flaps together with free skin grafting (Fig.\u00a02). Shunt placement could be postponed till day 9; a medium pressure valve being used. Because postoperatively the fontanel did not soften adequately, 2\u00a0weeks later the medium pressure valve was changed in a low-pressure valve, now with satisfying clinical results.\nFig.\u00a01Photograph of the newborn, showing the extensive MMC, extending from +\/\u2212 the 4th thoracic vertebra till the midlumbar regionFig.\u00a02Photograph at the end of the surgical procedure. The MMC was closed by making use of extensive rotational flaps together with free skin grafting\nAll period long, from birth till 1\u00a0week after the shunt revision, the well-being of the baby was estimated by making use of the Comfort score and the VAS and also medication used for treatment of pain or discomfort (paracetamol and morphine) was recorded (Fig.\u00a03a,b).\nFig.\u00a03a Comfort scores during the first month of life (min\u2009=\u20095: no discomfort; max\u2009=\u200935: extreme discomfort). Green bars: only paracetamol (intermittently) being used. Green\/black bars: low-dose morphine together with paracetamol being used. b The same as a, but now with the VAS (min\u2009=\u20090: no pain; max\u2009=\u200910: most severe pain) Note: The combination of a Comfort score of at least 17 and a VAS of at least 4 indicates that the well-being of the newborn is in jeopardy. In such a situation, the attending nurse is expected to determine the cause of the discomfort and to take adequate measures (changing a full nappy, feeding a hungry child, emptying a full bladder, giving adequate painkillers if appropriate, etc.) to secure the child\u2019s well-being\nStudying these data and particularly the amount of medication needed, several conclusions can be made:\nWhen untreated (the first 3\u00a0days), the child was not discomfortable.Closing of the back lesion, in this case certainly a major procedure, did not contribute significantly (and only temporarily) to more discomfort. In other words, closing of the open spine can be qualified as a proportional treatment modality.The same holds true for placing a ventricular shunt.Treating an active hydrocephalus adequately contributes to the well-being of the patient.In 1\u00a0month\u2019s time, this active treatment resulted in a stable situation of overall well-being in this patient.Only paracetamol and low-dose morphine were necessary in the treatment of actual discomfort.\nIn summary, this case not only demonstrates that there is no such thing as \u2018suffering\u2019 in these newborns, but also the incorrectness of the statement of Verhagen et al. (2005a) [73] that \u201cthe suffering of these newborns cannot be alleviated by any means in a proper medical way\u201d (Fig.\u00a04). The overall impression of this case is in accordance with the preliminary results of the Rotterdam Prospective Study on Discomfort in Newborns with MMC: there is no \u201cacutely unbearable suffering\u201d in these newborns (Figs.\u00a05a,b)\nFig.\u00a04The same child at 7\u00a0months old; fixating and following with the eyes, vocalizing, normal movements of the arms, moving both legs vigorously (perhaps nonvoluntary), still being partially fed by a nasogastric tubeFig.\u00a05a and b Preliminary results of the Rotterdam Prospective Study on Discomfort in Newborns with MMC (n\u2009=\u200913)\nAlthough in an interview Verhagen himself reported that \u201call 22 cases had a lesion of the most severe form, at level of the neck\u201d [78], in their published articles Verhagen et al. [73_76] do not quantify the level of the lesion. However, it seems very unlikely that his statement is correct or that the levels of lesion in these 22 cases are of the same level and\/or magnitude as in the case described.\nSynthesis and conclusion\nThere is no evidence that newborns with MMC and hydrocephalus do either \u2018suffer\u2019 unbearably or hopelessly and certainly not without the prospect to relieve this suffering by standard care. \u2018Suffering\u2019 itself is a nonconclusive, and in newborns, inapplicable denominator that should not be used anymore in this debate. Although they will in their future life be confronted with handicaps, sometimes very severe, their future prospects and their actual experienced quality of life cannot be predicted with such certainty at birth that their lives can be regarded as hopeless or meaningless (\u2018quality of life judgments\u2019 as such being unacceptable in this decision making). Possible discomfort in these newborns can easily be treated in a straightforward way by active treatment (closure of the defect and shunting the hydrocephalus) and, when necessary, by the use of a professional pain\/symptom protocol.\nThe decision not to treat such a newborn, when based on expected handicaps, possibly violates the \u2018non-discrimination\u2019 principle (Dorscheidt, 2006). When not being treated, they are not terminally ill because of the MMC and\/or hydrocephalus per se; they are \u2018terminally ill\u2019 because of this nontreatment decision. Not being terminally ill, it is not \u2018humane\u2019 or \u2018merciful\u2019 to terminate their life, this also being not in accordance with international legislation and international medical recommendations.\nWhen untreated and when it is the intention to alleviate actual discomfort (this question of intent being crucial to a moral and legal analysis of end-of-life decisions including active termination of life), this can always be achieved in an effective way by using one of the widely accepted palliative protocols. Such a child can and should be cared for in a respectful and dignified way, providing all its actual needs (which apparently is not death itself). This being the case, there is no indication whatsoever for the deliberate termination of the life of children born with MMC.\nWhile Verhagen et al. (2005, 2006) must be credited for bringing the discussion about deliberate termination of newborns in the open, to date, there does not seem to be much in their qualifications that can be quantified properly, especially not in cases of MMC, which is prone to quality of life judgments (and thereby neglecting the needs and rights of the newborn). To date, the Groningen protocol, therefore, cannot be regarded as very useful. Being a tool, it seems to have been changed in a means in itself. To gain more usefulness, at least it has to be extended in a way as proposed by Dorscheidt (2006), by adding charts such as the Comfort Score and the VAS and by adding palliative medication charts. Thus, it should be changed from a doctor-centered protocol to a patient-centered protocol.\nTo throw more light on these end-of-life decisions, Verhagen et al. should consider to extend their retrospective study, focusing on the medical aspects of the 22 cases. Thus, it must be possible to get a basic understanding of the extent of the malformations themselves, of possible co-morbidities, whether or not quality of life judgments were made, how \u2018unbearable suffering\u2019 was ascertained, how palliative care was offered, and why this failed. Such a study would contribute significantly to the open discussion they propagate. Verhagen is undoubtedly correct indeed in his appeal: \u201cIt\u2019s time to be honest about the unbearable suffering endured by newborns with no hope of a future\u201d. [71]\nFinally, to date it remains unclear on what grounds the Dutch Association of Paediatrics has adopted the Groningen protocol and why international legislation, international human rights instruments, and international medical recommendations appear not to hold for the Netherlands, especially not in newborns with MMC.","keyphrases":["termination of life","newborn","meningomyelocele","suffering","pain","quality of life","groningen protocol","palliative care"],"prmu":["P","P","P","P","P","P","P","P"]}
{"id":"Acta_Neuropathol-4-1-2270355","title":"Applicability of current staging\/categorization of \u03b1-synuclein pathology and their clinical relevance\n","text":"In Parkinson\u2019s disease (PD) and dementia with Lewy bodies (DLB) \u03b1-synuclein (\u03b1S) pathology is seen that displays a predictable topographic distribution. There are two staging\/categorization systems, i.e. Braak\u2019s and McKeith\u2019s, currently in use for the assessment of \u03b1S pathology. The aim of these diagnostic strategies in pathology is, in addition to assess the stage\/severity of pathology, to assess the probabilities of the related clinical symptomatology i.e. dementia and extrapyramidal symptoms (EPS). Herein, we assessed the applicability of these two staging\/categorization systems and the frequency of dementia and EPS in a cohort of 226 \u03b1S-positive-subjects. These subject were selected from a large autopsy sample (n = 1,720), irrespective of the clinical presentation, based on the detection of \u03b1S-immunoreactivity (IR) in one of the most vulnerable nuclei; in the dorsal motor nucleus of vagus, substantia nigra and basal forebrain. The frequency of \u03b1S-IR lesions in this large cohort was 14% (248 out of 1,720). If applicable, each of the 226 subjects with all required material available was assigned a neuropathological stage\/category of PD\/DLB and finally the neuropathological data was analyzed in relation to dementia and EPS. 83% of subjects showed a distribution pattern of \u03b1S-IR that was compatible with the current staging\/categorization systems. Around 55% of subjects with widespread \u03b1S pathology (Braak\u2019s PD stages 5\u20136) lacked clinical signs of dementia or EPS. Similarly, in respect to those subjects that fulfilled the McKeith criteria for diffuse neocortical category and displaying only mild concomitant Alzheimer\u2019s disease-related pathology, only 48% were demented and 54% displayed EPS. It is noteworthy that some subjects (17%) deviated from the suggested caudo-rostral propagation suggesting alternative routes of progression, perhaps due to concomitant diseases and genetic predisposition. In conclusion, our results do indeed confirm that current staging\/categorization systems can readily be applied to most of the subjects with \u03b1S pathology. However, finding that around half of the subjects with abundant \u03b1S pathology remain neurologically intact is intriguing and raises the question whether we do assess the actual disease process.\nIntroduction\nThere are two staging\/categorization systems commonly in use for the assessment of the progressive regional distribution of the pathology seen in Parkinson\u2019s disease (PD) and dementia with Lewy bodies (DLB). Both of these staging\/categorization systems are based on the assessment of misfolded \u03b1-synuclein (\u03b1S) protein within selectively vulnerable neuronal populations which is considered to be either directly responsible or at least intimately linked to the neuronal dysfunction seen in PD and DLB. In this respect, \u03b1S-immunoreactive (IR)-inclusions in the brainstem have been claimed to be responsible for the extrapyramidal symptoms (EPS), whereas dementia has been attributed to the limbic and neocortical spread of these lesions. Thus, PD and DLB are thought to form a clinico-pathologic continuum wherein the clinical manifestation of EPS and\/or dementia depends on the anatomical distribution and the load of \u03b1S pathology [4, 17, 19, 25, 28, 30].\nIn 2003, Braak and colleagues reported that the \u03b1S pathology begins in clearly defined induction sites and advances, not in a random, but in a predictable sequence with increasing severity throughout the brain [4, 11]. Based on the analysis of the regional distribution of \u03b1S-IR inclusions in a cohort including both neurologically unimpaired subjects and patients with PD, a staging system was devised whereby \u03b1S pathology was divided into six successive stages. In the central nervous system, the proposed sequence begins in the dorsal motor nucleus of vagus (dmV), and then proceeds with an upward progression via locus coeruleus (LC) (stage 2) to the substantia nigra (SN) (stage 3), and then to the basal foreberain (BFB) and transentorhinal region (stage 4) until it finally reaches the neocortex (stages 5\u20136).\nAlready in 1996, the consortium on DLB international workshop proposed their consensus guidelines for the clinical and pathologic diagnosis of DLB [27] that later, in 2005 were somewhat revised [28]. These consensus criteria of DLB subdivide subjects into three different neuropathological categories; brainstem predominant, limbic\/transitional and diffuse neocortical depending on the anatomical distribution of the \u03b1S-IR structures [28]. These criteria also include the semiquantitative grading of lesion density, although the pattern of regional involvement has been assumed to be more important than the actual count of inclusions. It is noteworthy that in the revised recommendations by McKeith et al from 2005 [28], it is emphasized that the concomitant pathologies should be taken into account when assessing the causative relationships between pathologies and symptoms. Thus, the most common pathology seen in aged demented individuals, i.e. Alzheimer\u2019s disease (AD)-related pathology, should be evaluated while assessing the likelihood of causation, i.e. that the \u03b1S pathology is associated with a DLB clinical syndrome.\nHerein, we assess the applicability of these two current staging\/categorization systems of synucleinopathies in a large autopsy material collected, not on the basis of clinical presentation, but by \u03b1S immunoreactivity in some of the most vulnerable nuclei; dmV, SN and BFB. Thus, the selection of material was entirely based on the presence of \u03b1S pathology irrespective of clinical phenotype. All subjects, if applicable, were assigned a stage following in detail Braak staging recommendations and a McKeith neuropathological category following in detail recommendations by the consortium on DLB international workshop [4, 28]. The frequency of dementia and EPS was assessed in each stage and the likelihood that dementia was due to AD-related pathology or \u03b1S pathology was also examined.\nMaterials and methods\nSelection of subjects and the clinical assessment\nThe flowchart delineates the logistics of this study (Fig.\u00a01). A total of 1,720 elderly individuals (age at death >40\u00a0years) that had undergone an autopsy together with an examination of the brain during the years 1996\u20132005 in the Kuopio University Hospital were included in this study. From this large autopsy sample, we selected 248 (14%) subjects that displayed \u03b1S-IR structures in the SN and\/or BFB nuclei: nucleus basalis of Meynert (nbM) and amygdaloid complex (AC). Screening of dmV was also carried out in 1996\u20132000 and revealed that in 24 subjects out of 904 (3%) \u03b1S-IR-structures were restricted to the lower brain stem nuclei (dmV and\/or LC) [33]. Five subjects were excluded from the analysis because they had received a pathological diagnosis of MSA. Seventeen subjects were excluded due to lack of clinical information or inadequate amount of brain material required for the classification as recommended by Mc Keith and Braak [4, 28], and thus ultimately, this study examined 226 subjects.\nFig.\u00a01The flowchart delineating the logistics of this study. a The \u03b1S-immunoreactive inclusions were screened in substantia nigra, amygdaloid complex and dorsal motor nucleus of vagus. b Schematic presentation of both Braak staging and McKeith categorization [4, 28] and c schematic presentation of assessment of likelihood that \u03b1S-immunoreactive inclusion are associated with dementia with Lewy bodies [28]\nThe diagnosis of dementia was based on the DSM-III criteria and the criteria of the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer\u2019s Disease and Related Disorders Association (NINCDS-ADRDA) [29]. The diagnosis of PD followed the criteria established by the United Kingdom Parkinson\u2019s Disease Society Brain Bank whereby PD was considered present if the patient had at least two of the four cardinal symptoms (resting tremor, rigidity, hypokinesia and postural instability) and exhibited a positive response to levodopa [9]. The initial screening for both dementia and EPS had taken place in a primary health care centre. With respects to dementia, all patients scoring 26 or less in minimental State Examination (MMSE) had been referred to a neurologist for further evaluation and all patients scoring >26 in MMSE but displaying signs of memory impairment had been referred to a CERAD test. Consequently, many subjects had been examined by a neurologist and all had at least visited a general physician (within a maximum of 1\u00a0year before death). It is noteworthy that many of the subjects included in this study had been under continuous clinical follow-up due to some chronic disease. Admittedly, some subjects may have developed subtle extrapyramidal signs or mild cognitive impairment between the last clinical examination and death, but it is most unlikely that the presence of moderate or full-blown parkinsonian syndrome\/dementia would have been overlooked.\nNeuropathological assessment\nAccording to the dissection protocol used in the Kuopio University Hospital, the brains were weighed, evaluated for grossly detectable lesions and vessel abnormalities, perfused with and immersed in 10% buffered formalin for at least one week and cut in coronal slices of 1\u00a0cm thickness. Brain specimens were embedded in paraffin and cut into 7\u00a0\u03bcm-thick sections. Immohistochemical (IHC) methodology was used to visualize the expression of \u03b1S and hyperphosphorylated \u03c4 (HP\u03c4). The sections were deparaffinized and rehydrated according to a routine procedure. For \u03b1S IHC, the sections were autoclaved (120\u00b0C) in citrate buffer for 10\u00a0min and pretreated with 80% formic acid at room temperature for 5\u00a0min. Monoclonal antibodies to human \u03b1S1\u2013140 (Novocastra, Newcastle upon Tyne, UK) at a dilution of 1:1,000 and to human HP\u03c4 at a dilution of 1:500 (Innogenetics, Ghent, Belgium) were applied. For detection, Histostain SP kit (Zymed, San Francisco, CA) was used with Romulin AEC chromogen (Biocare Medical, Walnut Creek, CA). The expression of \u03b1S was assessed in 10 brain regions: (1) medulla with dmV; (2) pons with LC and raphe nucleus (RN); (3) midbrain with SN; (4) BFB including nbM, AC, and transentorhinal cortex; (5) posterior hippocampus at the level of geniculate nucleus including the CA2 region of the hippocampus and temporo-occipital gyrus; (6) insular cortex; (7) anterior cingulate gyrus; (8) superior temporal gyrus (Broadman area 22); (9) frontal cortex (Broadman area and (10) parietal cortex (Broadman areas 39, 40). The selection of regions was based on the currently used staging systems [4, 28] (See Fig.\u00a01). HP\u03c4 IHC was carried out on sections from hippocampus, temporal and occipital cortices and the regional distribution of AD-related neurofibrillary pathology was subdivided into 6 stages (I-VI) as has been described previously [6].\nThe number of \u03b1S-IR inclusions was counted within the microscopic field at \u00d7200 magnification (diameter of 1\u00a0mm) and assessed semiquantitatively in all brain areas examined. The total thickness of the cortical grey matter and AC were assessed according to the established pathological guidelines [28] and rated as follows: 1\u00a0=\u00a0mild (sparse inclusions at \u00d7100); 2\u00a0=\u00a0moderate (>1 inclusion in low power field at \u00d7200 magnification); 3\u00a0=\u00a0severe (\u22654 inclusions in low power field at \u00d7200 magnification); 4\u00a0=\u00a0very severe (numerous inclusions). In the nbM and all subcortical regions, \u03b1S-IR inclusions were counted unilaterally within entire nuclei and assessed following an arbitrary grading system: in nbM and SN,\u00a0+\u00a0=mild (<25 inclusions); ++ = moderate (25\u201350 inclusions); +++\u00a0=\u00a0severe (>50 inclusions), in LC and dmV,\u00a0+\u00a0= mild (1\u20132 inclusions); ++\u00a0=\u00a0moderate (2\u201310 inclusion), +++\u00a0=\u00a0severe (>10 inclusions). Several inclusions within one neuron were counted as a single inclusion. The \u03b1S-IR structures were designated as being present (+) or not (0) in raphe nucleus and CA2 region of the hippocampus. If no \u03b1S-IR inclusions were identified in the sections of medulla, pons, midbrain or BFB, the result was verified in at least 3\u20134 consecutive sections i.e. the subsequent 5th, 10th, 15th and 20th sections were stained. This was done in order to increase the likelihood of capturing incipient neurons with \u03b1S-IR inclusions.\nResults\nAssessment of clinical data\nThe 226 \u03b1S-positive subjects examined included 114 (50%) patients with a clinical diagnosis of a neurodegenerative disorder, 15 (7%) patients with other neurological disorders and 97 (43%) individuals in whom the clinical information indicated that they had no neurological impairment. The mean age at death was 77\u00a0\u00b1\u00a00.6\u00a0years, ranging from 44 to 98\u00a0years and the gender was rather evenly distributed (108 females\/118 males).\nFrequency of \u03b1S-immunoreactivity in the most vulnerable anatomical regions\nThe most frequently affected regions in the 226 \u03b1S-positive subjects were dmV (197\/223) and SN (197\/225) where the \u03b1S-IR was seen in 88% of the analyzed subjects. The LC was affected in 81% (181\/223) of subjects, making this area the second most vulnerable nuclei. The large neurons in the nbM were \u03b1S-immunopositive in 78% of cases (171\/219), whereas the involvement of AC, particularly the cortico-medial nuclear group, was seen in 73% (162\/223) of subjects.\nStaging of Parkinson disease-related \u03b1S pathology according to Braak and the frequency of dementia and extrapyramidal symptoms\nOut of the 226 \u03b1S-positive subjects, 187 (83%) displayed a distribution pattern of \u03b1S-IR that was compatible with the current staging systems of PD\/DLB-related synucleinopathies [4, 28]. Braak stage 1\u20132 was applicable for 22 subjects, 3\u20134 for 38 subjects and 5\u20136 for 127 subjects (Table\u00a01). When all subjects with severe AD-related neurofibrillary pathology, i.e. Braak\u2019s AD stage V-VI, were excluded, 168 cases of the 187 subjects remained. Notably, only 25\u201330% of the subjects with PD-related Braak stage 5 and 50% of subjects with PD-related Braak stage 6 were demented and\/or had EPS. It is noteworthy that when only demented subjects were included in the analysis (53 out of 168), 91% (48\/53) were assigned to PD-related Braak stages5\u20136. Similarly, when only subjects with EPS were included in the analysis (52 out of 168), 94% (49\/52) were in the PD-related Braak stages 5\u20136.Table\u00a01Applicability of Braak staging and the incidence of dementia and extrapyramidal signs (EPS) in each stage\nCategorization of the distribution of \u03b1S pathology following the recommendations by the consortium on DLB international workshop, i.e. McKeith\u2019s categorization, and the frequency of dementia and extrapyramidal symptoms\nSixty-six subjects fulfilled Mc Keith\u2019s criteria for the brainstem predominant form, 30 for the limbic (transitional) and 91 for the diffuse neocortical form of DLB (Table\u00a02).\nTable 2Applicability of neuropathological categorization as recommended by the consortium on DLB international workshop, i.e. McKeiths categorization and the incidence of dementia and extrapyramidal signs (EPS) in each category\nFifty-seven percent of subjects with the diffuse neocortical form of DLB were demented and 47% displayed EPS (Table\u00a02). When AD-related pathology was taken into account as suggested by McKeith and colleagues [28] (Table\u00a03), the percentage of demented in the high likelihood category varied from 24 to 60%. Irrespective of the \u03b1S pathology, in Braak\u2019s AD stage V-VI, all subjects were demented. Notably, when only demented subjects were included in the analysis and the subject with severe AD-related pathology (Braak\u2019s AD stage V-VI) were excluded, 85% (45\/53) were assigned to a high likelihood category of DLB.\nTable\u00a03Assessment of likelihood that the pathological findings are associated with a DLB clinical syndromeCategory of Lewy body type pathologyAlzheimer type pathologyBraak stage 0-IIBraak stage III-IVBraak stage V-VIBrainstem predominant n\u00a0=\u00a066Low n\u00a0=\u00a058 5 demented (9%) 5 with EPS (9%)Low n\u00a0=\u00a07 2 demented (29%) 0 with EPS (0%)Low n\u00a0=\u00a01 1 demented (100%) 0 with EPS (0%)Limbic transitional n\u00a0=\u00a030High n\u00a0=\u00a025 6 demented (24%) 9 with EPS (36%)Intermediate n\u00a0=\u00a01 1 demented (100%) 0 with EPS (0%)Low n\u00a0=\u00a04 4 demented (100%) 1 with EPS (25%)Diffuse neocortical n\u00a0=\u00a091High n\u00a0=\u00a063 30 demented (48%) 34 with EPS (54%)High n\u00a0=\u00a015 9 demented (60%) 4 with EPS (29%)Intermediate n\u00a0=\u00a013 13 demented (100%) 5 with EPS (36%)\nAtypical cases i.e. cases that could not be classified as recommended\nThirty-nine cases were not classifiable as recommended and thus were assigned as being atypical i.e. neither Braak staging nor McKeith categorization systems could be applied. The deviating topographical distribution of \u03b1S-IR inclusions of these subjects is shown in Table\u00a04. In four subjects, the BFB nuclei (AC predominant) were severely affected (cases 1\u20134) together with some cortical inclusions, whereas the brainstem had been preserved. Five subjects displayed isolated minor involvement of SN without any \u03b1S-IR inclusions in the lower brainstem nuclei (cases 5\u20139). In six subjects (cases 10\u201315) minor involvement of both SN and BFB nuclei was detected (cases 10\u201315). In addition to these two areas, three cases exhibited severe \u03b1S pathology in AC (cases 16\u201318, AC predominant). In seven subjects, some inclusions were seen in the BFB, SN and LC, but not in the dmV (cases 19\u201325). In the final 14 subjects (cases 26\u201339), inclusions were seen in the dmV and SN, but not in the LC, together with variable affection of BFB and other cortical regions. In only minority of these cases EPS symptoms were seen (13%), whereas dementia was more common (54%). In 14 cases dementia was due to AD-related neurofibrillary pathology (Braak stage V-VI), in six cases numerous multifocal microscopic infarcts and concomitant severe white matter rarefaction was considered as the causative agent regarding dementia and in one cases neuropathological lesions consistent with frontotemporal dementia with ubiquitin only lesions were seen.\nTable\u00a04Topographic distribution of \u03b1S immunoreactive lesions in the 39 atypical cases, i.e. not classifiable following current recommendationsIDAgeSexEPSDemAD stageTopographical distribution of \u03b1S-immunoreactive structuresdmVRNLCSNnbMACTrEnt CxCA2Temp-OccIns CxCGTCxFCxPCx179FNoYes400000410000000262FNoYes600000420100000374FNoYes20000+420201100479FYesYes60000+300011100586FNoNo4000+0000000000644MNoNo0000+0000000000768MNoNo1000+0000000000883MNoNo2000+0000000100983MNoNo10+0+00000000001075FNoNo0000++0000000001176MNoNo1000++0000002001270MNoNo0000++0000000001377FNoNo2000++2100000001473MYesYes2000+++0000001001571FNoYes6000++++0001111001679FNoYes5000+03300100001787FNoYes2000++4nana0000001874FNoYes6000+na4na+1000001962MYesYes000++00000000002079MNoYes000+++0000000002184FNoYes200++++0000000002285FNoNo000+++11nana000002382FNoYes300++++na200100002458MNoNo000++++++0000000002584MNoNo300+++++3201020002676MNoNo1+00+00000000002787FNoNo1+00++0000na00002878MNoNo1+00++2200000002984FNoYes5+00+01na+1000003075FYesYes5++00+01000000003160FNoNo0+++00+00000000003280MNoYes6+00++00000010003369MNoNo1+++0++++1100010003480MYesYes5++00+++32+1110003586FNoYes6++0+044+3001003664FNoYes6+00+na4na+na001003774FNoYes6+00++440na012003878MNoYes5+00++33+1211003989MNoNo2+00++220001110EPS extrapyramidal signs, Dem clinical signs of dementia, AD stage the stage of Alzheimer\u2019s disease related neurofibrillary pathology stages as recommended by Braak from 0 to VI., dmV dorsal motor nucleus of vagus, RN raphe nucleus, LC locus coeruleus, SN substantia nigra, nbM nucleus basalis of Meynert, AC amygdaloid complex, TrEntCx transentorhinal cortex, CA2 CA2 region of the Cornu Ammonis of the hippocampus, Temp-Occ temporo-occipital gyrus, Ins Cx insular cortex, CG cingulate gyrus, TCx temporal cortex, FCx frontal cortex, PCx parietal cortex; na not available. The \u03b1S-immunoreactive inclusions were examined in the total thickness of the cortical grey matter and AC and rated as follows: 1 mild (sparse inclusions in \u00d7100), 2 moderate (>1 inclusion in \u00d7200 magnification), 3 severe (\u22654 inclusions in \u00d7200 magnification), 4 very severe (numerous inclusions), in the nbM and subcortical regions, \u03b1S- immunoreactive inclusions were counted unilaterally within entire nuclei and assessed following an arbitrary grading system: in nbM and SN,\u00a0+ mild (<25 inclusions), ++ moderate (25\u201350 inclusions), +++ severe (>50 inclusions), and in LC and dmV, + mild (1\u20132 inclusions), ++ moderate (2\u201310 inclusion), +++ severe (>10 inclusions). In Raphe nucleus and CA2 region of the hippocampus, \u03b1S-immunoreactive structures (inclusion and\/or neurites) were marked to be present (+) or not (0)\nDiscussion\nMost of our (83%) \u03b1S-positive cases could be assigned to one of the six PD stages as described by Braak and also into the brainstem, limbic or diffuse neocortical neuropathological category as recommended by McKeith and colleagues [4, 28]. Braak and colleagues depicted the topographical distribution of \u03b1S-IR structures by assessing 110 \u03b1S-positive subjects (69 incidental and 41 symptomatic PD patients) [4]. The initial intent of Braak and colleagues was not to correlate the designated neuropathological stages with clinical symptoms, however this was later contemplated [5]. Stages 1 and 2, i.e. stages where \u03b1S pathology is confined to the dmV and\/or LC, are considered to be presymptomatic, whereas EPS appear and the cognitive decline increases with each stage. In stage 3, when SN is affected, EPS appear and subsequently in stage 4 when amygdaloid complex, transentorhinal region and temporo-occipital gyrus become involved, moderate cognitive impairment is observed (MMSE scores 21\u201324) and finally in stage 5 and 6 when the neocortex succumbs to the pathology, severe cognitive impairment is evident (MMSE scores 11\u201320 and 0\u201310, respectively) [5]. Our results also suggest that the risk of EPS increases with disease progression though not to the same extent as earlier reported. In our study, we found one subject with EPS already in stage 2, whereas none of our cases in stage 4 displayed EPS, and more importantly no EPS had been reported in 55% of subjects who exhibited widespread pathology (Braak stages 5\u20136), i.e. this being compared with the 14% previously reported by Braak and colleagues [4].\nThe initial decline in cognition was postulated to occur already during stages 3 and 4 i.e. around the same time when the initial manifestation of somatosensor dysfunction start to appear. When assessing 88 subjects, Braak and colleagues reported, that 36% of their subjects in stage 3, 67% in stage 4, 94% in stage 5 and 100% in stage 6 were demented [5]. This is clearly in odds with our results where the percentage of demented increased from none to 50% between stages 3\u20136. It is noteworthy that when only demented subjects were included, 91% were assigned to PD-related Braak stages 5\u20136 and when only subjects with EPS were included, 94% were in the PD-related Braak stages 5\u20136. Thus the key difference between our study when compared to most other clinico-pathological correlation studies that have reported good correlation between risk of disease and progression of pathology is the study design [5]. Consequently, when we only included subjects with clinical signs in our analysis the correlation between stage\/severity of \u03b1S pathology and EPS\/dementia was excellent in line with previous reports.\nPD and DLB are distinguished as separate clinical entities and in 1996 the consortium of the DLB international workshop subdivided the neuropathological features of DLB into three categories: brainstem predominant, limbic and diffuse neocortical type [27]. The foundation of these three categories is also based on the progressive propagation of \u03b1S pathology along a caudo-striatal axis. Similarly to the Braak staging [4], when applying this categorization it is presumed that 100% of subjects with widespread \u03b1S pathology, i.e. in the diffuse neocortical stage all will be demented and display EPS. When we followed the classification strategy proposed by McKeith and colleagues [28], our results also differed from those expected, i.e. only a subset of our subjects who were classified to be in the diffuse neocortical category displayed dementia and\/or EPS (57 and 47%, respectively). In line with the above, when only demented subjects were included in the analysis the correlation between \u03b1S pathology and dementia was close to excellent (85%).\nThe clinical relevance of cortical \u03b1S pathology in relation to dementia is a matter of intense debate. Some authors have emphasized their key causative role [1, 18, 21, 26], whereas others have reported that there may be abundant cortical pathology in non-demented PD patients [8] as well as in neurologically unimpaired subjects [10, 24, 32]. It is noteworthy that the current study differs significantly from most other studies since it is based on neuropathologiacal findings rather than on clinical presentation. Our results emphasize that abundant pathology may be detected in many subjects without notable signs of dementia (MMSE >26) (43%), if it is sought. This has one unexpected consequence, i.e. a detailed regional assessment of \u03b1S pathology cannot reliably predict the clinical status observed premortem [33].\nThere has been much discussion concerning the significance and influence of concomitant AD-related pathology, particularly as this is quite frequently seen in aged subjects. Therefore, the revised consensus criteria have recommended taking AD-related pathology into account while assessing subjects with suspected DLB [28]. It was presumed that this would increase the diagnostic specificity since it was believed that the pathological substrate behind DLB was indeed \u03b1S pathology. When we assessed our unique material, we found, that within the neuropathological high likelihood categories of DLB, i.e. those cases where limbic\/diffuse neocortical \u03b1S pathology is combined with mild\/moderate AD-related changes, 56% of subjects remained cognitively intact. However, when we examined only demented subjects without severe AD-related pathology (Braak\u2019s AD stage V-VI), 85% were assigned to a high likelihood category of DLB. This shows that when \u03b1S pathology is examined in clinically demented subjects, the correlation received between particular pathologic change and dementia is good. It is noteworthy that with respect of AD-related neurofibrillary pathology, all cases in the neocortical stage (Braak stage V-VI) were indeed demented.\nOne important issue with respect to the pathogenesis of synucleinopathies is not only to understand the molecular mechanisms behind the intracytoplasmic aggregation of \u03b1S, but also to appreciate where this process first appears and how it may progress through the brain. Thus, many recent studies have attempted to localize the most vulnerable neuronal populations. In this study, dmV and SN were found to be equally susceptible nuclei, but even earlier affected structures have been reported to appear in the spinal cord, dmV, olfactory bulb and AC [2, 4, 11, 16, 22, 35]. Thus, mapping out the \u201ctrigger site\u201d for \u03b1S pathology appears to depend on the screening process i.e. if one screens medulla alone, those cases where lesions are restricted to other areas (e.g. SN) are not found and vice versa.\nAt variance to the studies of Del Tredici and Braak [4, 11] we identified a number of subjects where dmV and\/or LC were not affected but \u03b1S-IR inclusions were found in the SN, BFB and or other cortical regions, and thus, the distribution of \u03b1S pathology did not strictly follow the caudo-rostral propagation pattern described by Braak and colleagues [4]. Thus, the proposed ascending pathway is not the only possible route and our results indicate that pathology can emerge simultaneously in subcortical and cortical regions. Jellinger has also reported subjects with multiple \u03b1S-IR inclusions but with preservation of medullary nuclei [20]. Furthermore, in some subjects we found the AC to be devoid of pathology although \u03b1S-IR structures were detected elsewhere in the neocortex. This refutes the proposal that in order to have neocortical involvement then the subcortical lesions have to expand through the basal forebrain nuclei. In addition, according to Braak and colleagues [4], the \u03b1S pathology in previously involved regions should become exacerbated with disease progression. It is difficult to evaluate this proposal when one takes into account the increasing neuronal loss. If these two pathological hallmarks are linked in a causative chain, the load of \u03b1S-IR structures should show an inverted u-shape distribution over time where the number of inclusions would increases with the progression of the disease until the neurons start to die [12].\nWe observed some cases with severe \u03b1S pathology in AC and in that situation this structure was either only involved region or was affected together with BFB and SN. All these subjects were demented and exhibited coexistent severe AD-related pathology (Braak AD stage V-VI [6]). In one case, the dementia was considered to be vascular in origin. This is in line with the results of Uchikado and colleagues who have reported the AC predominant form to be common finding among patients with AD [35].\nIn conclusion, our results confirm that the current staging\/categorization systems can readily be applied to most of the subjects when assessing regional distribution of \u03b1S-pathology. It is noteworthy, however, that outliers do exist and in these cases the presumed distribution may have been modified by other coexisting pathologies or genetic factors [23, 35]. It is intriguing that around every second subject displaying abundant pathology did remain neurologically intact. It is noteworthy that these results were seen when a rather unselected sample of cases was investigated. It has been suggested that the key lesions begin to develop a considerable time prior to the appearance of clinical symptoms [13], but based on our results there do seem to be some subjects who can tolerate substantial amounts of pathology. As only 50% of subjects with widespread \u03b1S pathology were demented (MMSE\u00a0<\u00a026) and displayed EPS, the clinical relevance of \u03b1S-IR inclusions as such still remains to be resolved. Hitherto, the aggregation of \u03b1S has been thought to lead to neuronal death but recent evidence has suggested that the formation of large inclusions may actually represent a protective process [3, 14, 34]. Many biophysical studies have suggested that it is a protofibrillar form of \u03b1S rather than the \u201cmature\u201d fibrils that are responsible for the cell death [7, 15, 36], and moreover, the fibrillar form that is typically observed at autopsy may actually be a sign of a well functioning neuroprotection [31, 34]. Thus, when we are assessing regional distribution of \u03b1S pathology, the question arises if we are really evaluating a stage of degeneration or conversely monitoring the level of functional neuroprotection.","keyphrases":["\u03b1-synuclein","dementia","extrapyramidal symptoms","lewy body dementia","parkinson disease"],"prmu":["P","P","P","R","R"]}
{"id":"Anal_Bioanal_Chem-3-1-1802726","title":"Vibrational microscopy and imaging of skin: from single cells to intact tissue\n","text":"Vibrational microscopy and imaging offer several advantages for a variety of dermatological applications, ranging from studies of isolated single cells (corneocytes) to characterization of endogenous components in intact tissue. Two applications are described to illustrate the power of these techniques for skin research. First, the feasibility of tracking structural alterations in the components of individual corneocytes is demonstrated. Two solvents, DMSO and chloroform\/methanol, commonly used in dermatological research, are shown to induce large reversible alterations (\u03b1-helix to \u03b2-sheet) in the secondary structure of keratin in isolated corneocytes. Second, factor analysis of image planes acquired with confocal Raman microscopy to a depth of 70 \u03bcm in intact pigskin, demonstrates the delineation of specific skin regions. Two particular components that are difficult to identify by other means were observed in the epidermis. One small region was formed from a conformationally ordered lipid phase containing cholesterol. In addition, the presence of nucleated cells in the tissue (most likely keratinocytes) was revealed by the spectral signatures of the phosphodiester and cytosine moieties of cellular DNA.\nIntroduction\nTechnological developments in infrared and Raman microscopic imaging have permitted several new types of biomedical applications. As demonstrated elsewhere in this volume, the diagnosis of pathological states in a wide range of tissues is now feasible, and is being pursued vigorously in laboratories around the world. The vast volume of spectral data generated from imaging experiments permits sophisticated multivariate statistical analysis of data and enables the experimentalist to discern tissue regions where spectral signatures from pathological states differ from those of healthy tissue.\nAn important attribute of vibrational spectroscopy is the availability of spectra\u2013structure correlations from many tissue components. This extensive background information can provide a useful supplement for biomedical diagnostics. By way of example, Boskey and her collaborators have characterized a new form of brittle bone disease (osteoporosis) based on structural changes in the collagen component of mineralized tissue [1]. This pathological state differs from the more widely known forms of osteoporosis, which are characterized by altered hydroxyapatite levels\/structure. The origin of the spectral changes in the collagen component of pathological tissue biopsies was traced to increased levels of collagen cross links and alterations in their spatial distribution. Evidently, the availability of such information facilitates the development of strategies for tracing the origin (i.e., genetic predisposition, external factors, etc.) of the pathology.\nSkin is another tissue where the availability of spectra\u2013structure correlations as well as the ability to detect spectral signals from exogenous constituents opens up new possibilities for vibrational microscopic imaging experiments relevant to pharmacology, physiology, and biochemistry [2\u20134]. In this article, we provide two examples depicting the interplay of vibrational microscopic imaging with spectra\/structure correlations.\nThe first example uses both IR and Raman microscopy to evaluate structural changes in the keratin of single cells (corneocytes) induced by treatment with dimethyl sulfoxide (DMSO) or chloroform\/methanol (C\/M). These solvents have been widely used in dermatology for studies of permeation enhancement and for extracting lipid from the stratum corneum (SC), respectively. The characterization of DMSO-induced structural changes in keratin will aid in the determination of DMSO permeation pathways and in the evaluation of SC barrier recovery. Assessing C\/M\u2019s effect on keratin in isolated corneocytes including the reversibility of potential structural changes, will facilitate the delineation of solvent treatment effects from that of lipid removal.\nIn the second example, we demonstrate that confocal Raman microscopy along with multivariate (factor) analysis provides the detection of particular skin regions (stratum corneum, epidermis, dermis) as well as endogenous sub-areas (keratinocytes, lipid inclusions) in the epidermis. The availability of spectra\u2013structure correlations permits molecular structure information to be deduced from either the spectra or from the factor loadings.\nMaterials and methods\nPreparation of corneocytes\nThe Rutgers University Internal Review Board has approved all protocols. Corneocytes were collected by tape stripping (Sellotape, 3M Scotchguard) human forearm skin. The forearm was flushed with water for several minutes prior to tape stripping. The first two strips were discarded and corneocytes from the third tape strip were isolated as follows. Corneocytes were flushed from the tape with HPLC-grade hexane. The hexane\/corneocyte suspension was sonicated for approximately 5\u00a0min to break up desmosomes and lipid cohesion between individual corneocytes. Corneocytes were then isolated using nitrocellulose membrane filters with a pore size of 0.22\u00a0\u03bcm (GE Osmonics Labstore, Minnetonka, MN) and resuspended with fresh hexane. This procedure was repeated three times. A small aliquot of this suspension was separately deposited on either CaF2 windows for transmission IR or gold-coated silicon substrates for Raman measurements. Samples were dried overnight under house vacuum prior to spectroscopic measurements. The remaining corneocytes were dried under N2 gas prior to treatment with DMSO or C\/M.\nCorneocytes were placed on substrates that were directly dipped in pure DMSO for about 1\u00a0min. Separate aliquots of harvested corneocytes were transferred to glass vials using 5\u00a0mL C\/M (2:1 v\/v). Non-covalently bound lipids were extracted for 68\u00a0h while stirring at room temperature in sealed vials. Corneocytes, collected by centrifugation, were further washed 3 times using fresh C\/M before the final suspension was placed on the appropriate substrate. Samples were dried overnight under vacuum prior to spectroscopic measurements. No vibrational bands from residual solvent were observed in corneocyte spectra. Subsequently, corneocytes from both DMSO and C\/M treatment were rehydrated on the solid substrates by exposure to 100% relative humidity overnight before a final set of spectra were acquired.\nIntact porcine skin preparation\nSkin biopsies from Yucatan white, hairless pigs were purchased from Sinclair Research Center, Inc (Columbia, MO). The stratum corneum of the biopsies was washed with water and samples were cut to a size of approximately 1\u2009\u00d7\u20091\u00a0cm2, 0.5-cm-thick sections. Sections were placed, SC side up, into a milled brass cell, covered, and sealed with a microscope coverslip for confocal Raman measurements.\nIR microspectroscopy of corneocytes\nIR spectra were acquired with the PerkinElmer Spotlight system described elsewhere in detail [4]. The instrument has imaging capabilities, but in this study the point mode was used. Since the typical diameter of corneocytes is about 40\u201360\u00a0\u03bcm, an aperture size of 40\u2009\u00d7\u200940\u00a0\u03bcm2 was chosen to optimize the spectral quality. A total of 256 scans was acquired for each spectrum using 4\u00a0cm\u22121 spectral resolution. One level of zero-filling yielded data encoded every 2\u00a0cm\u22121. Approximately 50 corneocytes were examined from each sample preparation.\nRaman microspectroscopy\nRaman spectra were acquired with a Kaiser Optical Systems Raman Microprobe. The instrument has been described in detail elsewhere [5, 6]. A 785-nm diode laser generates approximately 7\u201312\u00a0mW of single mode laser power at the sample with a spot size of about 2\u00a0\u03bcm using a \u00d7100 objective. The backscattered radiation illuminates a near-IR CCD (ANDOR Technology, Model DU 401-BR-DD). Spectral coverage is from 100 to 3,450\u00a0cm\u22121 at spectral resolution of 4\u00a0cm\u22121. Following linearization, data are encoded every 0.3\u00a0cm\u22121. Spectra were acquired from 10\u201315 corneocytes from each sample preparation using an 80-s exposure time, 4 accumulations, and cosmic ray correction. Optical images of corneocytes were acquired with a Leica microscope (Model DMLP) coupled to the Kaiser system with a \u00d7100 objective.\nConfocal Raman spectra of intact pigskin were acquired using a 60-s exposure time, 2 accumulations, and cosmic ray correction. A \u00d7100 oil immersion objective was used for the intact pigskin samples. To evaluate axial characteristics of the optical set-up used in these experiments, the following observations were made with the oil immersion lens using protocols previously published [5]. The test sample consisted of five layers of a polymer laminate which incorporated two chemically identical layers (out of the five), each with a thickness of 7.5\u00a0\u03bcm separated by an intermediate layer of thickness 15\u00a0\u03bcm. This provides a center-to-center distance of 22.5\u00a0\u03bcm between the identical layers. Raman intensity profiles show a baseline-resolved width for each of the chemically identical layers of approximately 12\u201313\u00a0\u03bcm, a half-width of the intensity profile for each peak of about 8\u00a0\u03bcm, and a peak-to-peak distance of about 25\u00a0\u03bcm, in good agreement with the known test sample physical characteristics. The actual axial spatial resolution in the skin samples cannot be directly ascertained.\nData analysis\nGrams\/32 AI software version 6.0 (Thermo Galactic, Salem, NH) was used for processing individual Raman and IR spectra. Raman spectra were Fourier smoothed (80\u201390%) and 2-point baselines were applied to spectral regions of interest. This degree of Fourier smoothing could be applied without inducing spectral lineshape distortions. Confocal Raman spectra were analyzed (e.g., factor analysis) and Raman image planes were generated using ISys software version 3.1 (Spectral Dimensions, Inc. Olney, MD). Factor analysis was performed using the ISys score segregation routine. The analysis seeks to detect simple patterns in the relationships between observed variables in order to reduce the dimensionality of the data. Score segregation begins by normalizing PCA scores which may then be sharpened by raising them to a power specified by the acceleration parameter. The default value of 10 was used. Factor loadings are then calculated according to (S\u2032S)\u22121*S\u2032*X, unless S\u2032S is not invertible, in which case iteration stops, and a factor is deleted before continuing.\nResults and discussion\nSolvent-induced conformational changes in the keratin of single cells\nAlthough its mechanism of action is not well understood, DMSO has often been utilized as an efficient permeation enhancer for a wide variety of drugs, including antiviral agents, steroids, and antibiotics [7]. It has been suggested to alter protein conformation in the stratum corneum (SC), the outermost layer of skin and the main barrier to permeability. Anigbogu et al. [8] monitored the protein (keratin) \u03b1-helix to \u03b2-sheet transition in hydrated SC. In addition, Caspers et al. [3] monitored protein structure following DMSO penetration into human SC in vivo by confocal Raman spectroscopy. A broadening of the Amide I contour was noted, although major new spectral features were not resolved. It is noted that DMSO concentrations differed in the two experiments.\nIR (1,200\u20133,600\u00a0cm\u22121) and Raman (800\u20131,720\u00a0cm\u22121) spectra, shown in Fig.\u00a01a and b, respectively, demonstrate the quality of data obtained from untreated human corneocytes. Several strong bands characteristic of the protein and lipid constituents are observed in each spectrum. The Amide I and Amide II bands observed at approximately 1,650 and 1,550\u00a0cm\u22121 in the IR spectra, respectively, are of particular interest. The Amide I band is also one of the strongest bands in the Raman spectrum. In both types of spectra, the measured Amide I frequency reflects the predominantly helical secondary structure found in epidermal keratin. Absorbance in the Amide I region due to ceramides and other components, some containing amide groups and others C=C, is expected to be low compared to keratin absorbance in corneocytes isolated from the topmost layers of the stratum corneum [6]. Raman spectral quality is very good considering the thickness of an individual corneocyte (0.5\u20130.8\u00a0\u03bcm) and is comparable to spectra obtained from much thicker stratum corneum samples and intact skin [2, 5, 8]. It is likely that the observed signal has been enhanced due to a surface-enhanced Raman scattering (SERS) effect between the corneocytes and gold-coated substrate. Raman band assignments pertinent to stratum corneum samples have been listed previously [5, 9, 10].\nFig.\u00a01Vibrational microscopy of human corneocytes. a IR point mode spectrum (1,150\u20133,600\u00a0cm\u22121 region) acquired using a 40\u00a0\u03bcm2 aperture of a single corneocyte isolated from the third sequential tape strip applied to human forearm skin. The inset shows the optical image of the corneocyte (bar\u2009=\u200910\u00a0\u03bcm). b Raman spectrum (800\u20131,720\u00a0cm\u22121 region) of a similarly isolated corneocyte displaying bands characteristic of the lipid and protein (keratin) components\nIR and Raman spectra of isolated corneocytes prior to and following immersion in DMSO, and after an overnight rehydration period (top to bottom trace) are displayed in Fig.\u00a02a and b, respectively. A similar series of IR and Raman spectra acquired from corneocytes treated with C\/M are shown in Fig.\u00a02c and d. Both solvents induce several alterations in the spectra. The most significant change in the IR spectra is the appearance of an intense low frequency Amide I component observed for the DMSO-treated sample at 1,626\u00a0cm\u22121 in conjunction with a weak high frequency shoulder at approximately 1,695\u00a0cm\u22121. This pattern is diagnostic for formation of antiparallel \u03b2-sheet structure [11]. A redistribution of the maximum intensity in the IR Amide II band from 1,550 to 1,517\u00a0cm\u22121 for the same two spectra is also consistent with \u03b2-sheet formation [11].\nFig.\u00a02Solvent-induced conformational changes in the keratin of single corneocytes. a and b IR and Raman spectra, respectively, of an isolated corneocyte prior to and following immersion in DMSO, and after an overnight rehydration period (top to bottom, respectively). c and d IR and Raman spectra, respectively, of a corneocyte prior to and following C\/M treatment, and after an overnight rehydration period (top to bottom, respectively). In the IR spectra (a and c), \u03b1-helical and \u03b2-sheet components of the Amide I (ca. 1,650\u00a0cm\u22121) and II (ca. 1,500\u20131,550\u00a0cm\u22121) bands are noted. In the Raman spectra (b and d), \u03b1-helical and \u03b2-sheet components of the Amide I are marked along with disordered constituents at 1,685\u00a0cm\u22121. One component of the Amide III band is marked at 1,240\u00a0cm\u22121\nHelix:sheet transitions are well documented for different types of keratin, and have been detected by IR spectroscopy. In particular, Oertel [12] used IR to observe similar DMSO-induced changes in human SC. The current work extends observation of this conformational transition to isolated human corneocytes. It is also noted that, based on the IR Amide I and II band contours, a fair amount of helical structure remains subsequent to DMSO treatment.\nThe Raman spectrum (Fig.\u00a02b, middle trace) corroborates the \u03b2-sheet assignment with the appearance of a high frequency Amide I component at about 1,669\u00a0cm\u22121 and a strong Amide III component at approximately 1,240\u00a0cm\u22121. Both features are within frequency ranges assigned to antiparallel \u03b2-sheets [13, 14]. The observed changes in the Amide I region of the Raman spectrum are consistent with a previous report detailing the effects of a series of aqueous DMSO solutions on human SC [8]. Comparisons between individual cells and the SC in the Amide III region are difficult due to overlapped bands from lipid components in the SC sample. In the current work, a second slightly weaker band is also observed for the DMSO-treated cells at approximately 1,270\u00a0cm\u22121. Bands in this region are usually assigned to \u03b1-helical structure [13, 14]. Generally, caution is required in associating characteristic frequency ranges with conformation for the Amide III mode given that the NH in-plane bend internal coordinate contributes to a number of modes in the 1,200\u20131,400\u00a0cm\u22121 region [15] and secondary-structure-dependent coupling occurs between the Amide III and the C\u03b1\u2013H bending vibration [16, 17]. This complexity, in addition to the inherent heterogeneity of corneocytes and variable lipid content of the samples examined herein, precludes an in-depth analysis of the Amide III contour in the current context.\nThe majority of the DMSO-induced secondary structure change is reversed upon rehydration of the corneocytes as demonstrated by comparison of the top and bottom spectra shown in Fig.\u00a02a and b. Some residual broadening remains on the low frequency side of the Amide I band in the IR spectrum. The average full width at half height for the Amide I band in the IR spectra increased from 44\u00a0cm\u22121 for untreated corneocytes to 52\u00a0cm\u22121 following DMSO treatment and rehydration, indicating that some residual sheet structure may be present. An accurate measure of the Amide I bandwidth in the Raman spectra is precluded by the presence of a weak side chain vibration at approximately 1,608\u00a0cm\u22121. The general reversibility of the conformational change upon hydration was also observed by Oertel [12] for isolated SC.\nC\/M solutions are commonly used to extract lipids from isolated SC and intact skin samples, but solvent effects on the structure of the remaining constituents have not been detailed. The utility of vibrational spectroscopy for evaluation of keratin secondary structure after lipid removal is somewhat hampered by the native ceramide contribution to the Amide I and II band prior to solvent exposure. In SC samples it is therefore difficult to accurately access the contribution ceramides make to Amide I and II band intensities and position. In the current work, this concern is mostly overcome by acquiring spectra of isolated corneocytes in which the ceramide contribution to the Amide modes is substantially reduced.\nSeveral changes are apparent in the IR and Raman spectra of corneocytes upon exposure to C\/M (Fig.\u00a02c and d, compare top to middle traces). Some of the solvent-induced alterations are similar to those just discussed for the DMSO-treated cells, e.g., the low (high) frequency Amide I component in the IR (Raman) spectrum at 1,626\u00a0cm\u22121 (1,670\u00a0cm\u22121) and the appearance of an Amide III band at 1,240\u00a0cm\u22121 in the Raman spectrum. These features provide strong evidence that C\/M also induces antiparallel \u03b2-sheet formation. Differences are also evident when spectra from the two treatments are compared. Broadening on the high frequency side of the Amide I band in the Raman spectra is much more intense after C\/M treatment vs. DMSO (compare the middle trace in Fig.\u00a02d to b). Amide I Raman intensity in this region (ca. 1,685\u00a0cm\u22121) has been assigned to non-hydrogen-bonded disordered structures [13]. The broadening in the Raman Amide I band is not completely reversible upon rehydration of the sample that had been treated with C\/M, even though the intensity of the \u03b2-sheet component at about 1,670\u00a0cm\u22121 has diminished (Fig.\u00a02d, bottom trace). In addition, upon rehydration, intensity remains in the Amide III band at approximately 1,240\u00a0cm\u22121, although this band also broadens. Finally, measurements of the Amide I bandwidth in IR spectra upon rehydration of several C\/M-treated corneocytes are essentially the same (\u00b12\u00a0cm\u22121) as widths measured for untreated cells. The close proximity of Amide I bands arising from helical and random coil structures makes it difficult to differentiate between these two forms. Considering both the Raman and IR spectral data after rehydration, it seems safe to conclude that the majority of keratin exists in a helical form with some disordered structure remaining.\nThe extent of the C\/M-induced protein structural changes has been observed to vary among corneocytes. IR spectra of some cells with significant lipid bands (i.e., those characterized by relatively intense methylene stretching modes near 2,850 and 2,918\u00a0cm\u22121) still exhibit notable protein conformation changes (data not shown), while some cells from which the lipid has been essentially completely extracted do not show significant changes in protein secondary structure. It thus appears that the extent of lipid extraction is not directly related to C\/M-induced protein conformational changes. Similar results have been noted in IR studies of DMSO-induced protein conformation changes in SC samples [12]. Additional experiments under conditions of controlled hydration are needed to further explore possible origins of these conformational changes. One explanation for the \u03b1-helix to \u03b2-sheet structure change in keratin is that DMSO displaces bound water necessary for maintaining secondary structure [12]. DMSO is known to form an association complex with water stronger than that formed between water molecules alone [8]. It may be that C\/M destabilizes keratin\u2019s native secondary structure in a manner similar to DMSO, since the majority of the native structure is restored after both solvent treatments when the corneocytes are exposed to high relative humidity.\nThe importance of the current experiments lies in the fact, that in studies of supposedly native protein structures in the SC, C\/M is often used to remove lipid components. As shown in the current work, this procedure is not innocuous, but can induce irreversible changes in protein structure in corneocytes.\nConfocal Raman microscopy: delineation of skin regions from factor analysis\nAs demonstrated above, Raman microscopy complements the information available from IR. Although Raman scattering is inherently weaker than IR absorbance, the technique offers two major advantages for tissue studies. First, the spatial resolution of the method is inherently better than IR. For an excitation wavelength of 785\u00a0nm, Rayleigh\u2019s rule for spatial resolution (\u0394Z\u2009=\u20091.22\u03bb\/2(NA)) predicts a spatial resolution of approximately 0.5\u00a0\u03bcm for a wavelength (\u03bb) of 785\u00a0nm and a numerical aperture (NA) of the \u00d7100 objective of 0.95. In practice, as has been discussed in Materials and methods and elsewhere, the apparent depth and related measured optical properties are degraded by refractive index variations in the tissue [5, 18, 19]. Second, and equally important, spectra may be acquired in a confocal manner. Thus, physical sectioning of samples is not required to obtain molecular structure information from within the tissue. This evidently opens up the possibility for in vivo examination of skin, especially at the low laser powers (ca. 10\u00a0mW) used in these experiments. Puppels\u2019 group has demonstrated the utility of confocal Raman determination of water levels in vivo in skin [20].\nThe benefit of using both a multivariate approach and existing spectra\u2013structure correlations to characterize the microanatomy of skin is demonstrated in the next few figures. An optical micrograph of a 5-\u03bcm-thick pigskin section, shown in Fig.\u00a03a, allows delineation of different skin regions. The outermost layered stratum corneum (SC) region appears to be ca. 15-\u03bcm thick with the underlying epidermis (ca. 50-\u03bcm thick) and dermis discernible. The physical sampling used in the confocal Raman measurement precludes the acquisition of a meaningful micrograph from the same sample; however to allow for comparison, confocal Raman spectra were acquired from a similar pigskin biopsy using approximately the same dimensions. Spectra were acquired from an untreated piece of pigskin, in steps of 2.5\u00a0\u03bcm in the z direction and 5\u00a0\u03bcm in the x (or y) direction. The area sampled was 75\u00d740\u00a0\u03bcm so that 240 spectra (30\u2009\u00d7\u20098) were acquired during the experiment. These were analyzed with factor analysis as described in Materials and methods and as applied to liposome-treated pigskin in a previous report [21]. In the current work, five significant factors were determined as shown in Fig.\u00a03b, while score images for each factor are depicted in Fig.\u00a03c. Three of the score images and corresponding factor loadings in Fig.\u00a03 may be directly correlated with the known regions of skin as follows: Factor 1 corresponds to the SC, Factor 4 to the viable epidermis, and Factor 5 to the dermis. Thus, delineation of the skin regions based on the factor score images is wholly consistent with the optical image. The two remaining factor score images (Factors 2 and 3 ) highlight particular features within the viable epidermis. Examination of the factor loadings or averaged spectra corresponding to those pixels where the respective scores are high provides an indication of the molecular origin of the components. In general, factor plots tend to offer somewhat higher S\/N ratios, since they represent a substantial condensation of data. However, it is important to remember that factors include all sources of variance in the data. For example, baseline distortions may appear as \u201cfeatures\u201d in the factor loadings, which however are not related to vibrational modes in the sample. Thus, it is useful to routinely compare factors with appropriately selected spectra. In Fig.\u00a04 an average spectrum of the stratum corneum is overlaid with the average localized to the small spatial region highlighted in Fig.\u00a03c, labeled Factor 2. The average spectrum labeled \u201cordered lipid\u201d has strong bands near 1,058, 1,130, 1,296, 2,880, and 2,850\u00a0cm\u22121 (Fig.\u00a04b and c). These bands are readily assigned [9, 22] to lipid C\u2013C stretching vibrations (1,058, 1,130\u00a0cm\u22121) of a conformationally ordered (all-trans) chain, lipid CH2 twist (1,296\u00a0cm\u22121) and lipid CH2 stretch (2,850 and 2,880\u00a0cm\u22121). In addition, the bands observed at 605 and 700\u00a0cm\u22121 (Fig.\u00a04a) are indicative of cholesterol [23]. Thus, the small pocket in Fig.\u00a03c (Factor 2) likely contains an ordered lipid phase. The fact that the same bands are present in the average stratum corneum spectrum (Fig.\u00a04, top), albeit at lower intensity, strengthens the assignment. Highly ordered lipid phases (ceramides, fatty acids, and cholesterol) comprise the inter-corneocyte matrix of the stratum corneum. In addition, infrared spectroscopic evidence exists (D.J. Moore, unpublished results) for ordered lipid regions with relatively low protein content in appendages (i.e., pores and hair follicles) well below the stratum corneum. It is possible that the confocal Raman image planes shown in Fig.\u00a03c cut through such an appendage.\nFig.\u00a03a An optical micrograph of a microtomed 5-\u03bcm-thick pigskin section acquired using the PerkinElmer Spotlight (bar\u2009=\u200910\u00a0\u03bcm). The results of factor analysis in the 800\u20131,150\u00a0cm\u22121 region conducted on a confocal Raman map acquired from intact pigskin. b The five distinct factor loadings generated by the ISys score segregation algorithm (see Materials and methods) are offset and labeled Factor 1\u20135. c The spatial distribution of factor scores for each of the loadings as marked. Dark blue indicates the lowest score with green, yellow, orange, and red indicative of progressively higher scores. Factor loadings and score images have been assigned to different micro regions in skin as described in the textFig.\u00a04Averaged Raman spectra from within the stratum corneum (top) and lipid (bottom) regions as noted in Fig.\u00a03b and c as Factor 1 and 2, respectively. a The 590\u2013790\u00a0cm\u22121 region with bands assigned to cholesterol marked at 605 and 700\u00a0cm\u22121. b The 800\u20131,380\u00a0cm\u22121 region displays several bands (1,058, 1,130, and 1,296\u00a0cm\u22121) characteristic for ordered (all-trans) lipid acyl chains. c The CH stretching region (2,800\u20133,030\u00a0cm\u22121) with methylene symmetric (2,850\u00a0cm\u22121) and asymmetric (2,880\u00a0cm\u22121) stretching modes noted. The frequencies and relative intensity of the bands are consistent with well-ordered lipid acyl chains\nFinally, Factor 3 (Fig.\u00a03b), whose score image reveals several small-localized pockets in the epidermis, contains one broad spectral feature centered at approximately 1,090\u00a0cm\u22121 that is not present in the other factor loadings. Based on both the position of this particular band and the spatial distribution of the factor score, the band is tentatively assigned to the phosphodiester mode of DNA [24, 25]. Since the identification of DNA is of interest, as it would reveal the location of nuclei within keratinocytes or other cells in the tissue, an additional strategy was employed to acquire better spectra from the epidermis.\nThe experiment, whose results are depicted in Fig.\u00a05, was carried out as follows. The top \u224820\u00a0\u03bcm of a pigskin section, including most of the SC, were removed by tape stripping prior to confocal Raman imaging. This allows for the acquisition of higher quality spectra in deeper regions of the viable epidermis. The Raman experiment was also carried out with more data points\/unit area (2-\u03bcm step size) providing a closer examination of the tissue compared to the sample presented in Fig.\u00a03. The presence of cell nuclei was revealed by factor analysis conducted over two wavenumber regions, each separate analysis restricted to compute only two factor loadings. An additional DNA band assigned to cytosine is detected in the factor loadings shown in Fig.\u00a05b, which also strengthens the previously identified phosphodiester DNA band assignment at about 1,090\u00a0cm\u22121 [24, 25]. As expected, in the tape stripped sample, the phosphodiester band is again observed (Fig.\u00a05c). The score image shown in Fig.\u00a05a is derived from the factor with the strong cytosine band and is essentially the same as the score image generated from the phosphodiester factor (not shown). Thus, the image shown in Fig.\u00a05a, depicting keratinocyte or other epidermal cell (e.g., melanocyte) nuclei, demonstrate the ability of confocal Raman to detect DNA in intact skin tissue.\nFig.\u00a05The results of factor analysis conducted in two separate spectral regions for a confocal Raman map of pigskin after stratum corneum removal by tape stripping. Both analyses were performed allowing the generation of only two factors. In each case, one factor contained a spectral feature specific to DNA and the other mapped in a fairly uniform way throughout the epidermal region. a Factor score image for the factor-labeled nuclei shown in (b) with red corresponding to the highest score and dark blue to the lowest. b Factor loadings for the 600\u2013820\u00a0cm\u22121 region with a distinctive vibrational band at approximately 785\u00a0cm\u22121 assigned to cytosine. c Factor loadings for the 800\u20131,150\u00a0\u22121 region with a second characteristic DNA vibrational band due to the phosphodiester backbone stretching mode, at about 1,090\u00a0cm\u22121 noted\nConclusions\nThe current experiments highlight some unique advantages of vibrational microscopic imaging. An understanding of the spectroscopy of the various tissue components permits insight into the molecular origins of the images generated from factor analysis. Thus, identification of the lipid pocket in Fig.\u00a03c (Factor 2) and of the cell nuclei (as detected from the DNA spectral signatures) in Fig.\u00a03c (Factor 3) and Fig.\u00a05a were greatly facilitated by the availability of Raman spectra of lipids and of DNA, respectively. In addition, the availability of correlations between lipid spectra and chain conformation, permitted assignment of the lipid inclusions as having arisen from ordered lipid phases. The biological relevance of this observation remains to be determined. From a pharmacological perspective, the detection of cell nuclei (most likely keratinocytes) within intact skin permits co-localization experiments to begin to be designed. For example, for classes of drugs targeted to these cells, confocal Raman measurements will permit determination of whether the putative therapeutic agent reaches its intended target.\nIn addition, although all microscopic methods can obviously readily detect single cells, the ability of IR- or Raman-based methods to detect conformational changes within a particular component of a single isolated cell is unique. The example we have chosen in Fig.\u00a02 is of practical importance in skin research, since solvents such as DMSO or chloroform\/methanol are used for permeation enhancement and lipid extraction, respectively. In each case these solvents were found to be far from innocuous. Each induced large conformational changes in the cellular keratin. This fact had been known previously from spectra of intact SC, but two aspects of the current measurements are novel. The observation of IR data from single cells provides a sharper spectral assignment of the bands. We note for example that ceramides, a major component of the SC, have strong bands arising from the amide bond located in these lipids. These modes contribute significantly to the spectra of the intact SC and overlap protein Amide I and II vibrations; however, they are much reduced in relative intensity in spectra of cells. Thus, studies of the reversibility of the solvent-induced conformational changes benefit from the reduced intensity of ceramide interference.\nFinally, it is feasible to begin to examine biochemical heterogeneity at a single cellular level. Issues such as the relationship of protein structure (either native or solvent-modified) to hydration levels or to the levels of natural moisturizing factors, in single cells may be profitably probed. In addition, changes in the spectra of cells from pathological states may be examined.","keyphrases":["corneocytes","confocal raman","epidermis","protein secondary structure","infrared imaging"],"prmu":["P","P","P","P","R"]}
{"id":"Apoptosis-4-1-2217618","title":"RGD-avidin\u2013biotin pretargeting to \u03b1v\u03b23 integrin enhances the proapoptotic activity of TNF\u03b1 related apoptosis inducing ligand (TRAIL)\n","text":"Recombinant TNF-related apoptosis-inducing ligand (TRAIL) is considered a powerful and selective inducer of tumor cell death. We hypothesize that TRAIL\u2019s potential as anticancer agent can be enhanced further by promoting its accumulation in tumor tissue. For this purpose, we developed TRAIL complexes that bind to angiogenic endothelial cells. We employed an avidin\u2013biotin pretargeting approach, in which biotinylated TRAIL interacted with RGD-equipped avidin. The assembled complexes killed tumor cells (Jurkat T cells) via apoptosis induction. Furthermore, we demonstrated that the association of the RGD-avidin-TRAIL complex onto endothelial cells enhanced the tumor cell killing activity. Endothelial cells were not killed by TRAIL nor its derived complexes. Our approach can facilitate the enrichment of TRAIL onto angiogenic blood vessels, which may enhance intratumoral accumulation. Furthermore, it offers a versatile technology for the complexation of targeting ligands with therapeutic recombinant proteins and by this a novel way to enhance their specificity and activity.\nIntroduction\nTNF ligands have strong potential as anti-tumor agents, acting in different ways on either tumor cells or tumor vasculature, or by promoting the induction of tumor-directed immune responses [1]. The broad clinical use of TNF ligands is prohibited however due to lack of tumor selectivity, causing various side effects. A recently identified TNF family member is TNF related apoptosis-inducing ligand (TRAIL) [2]. Many tumor types are sensitive to TRAIL or become sensitive after treatment with chemotherapeutic drugs [3]. Soluble TRAIL variants showed no cytotoxic effects on most primary human cells and were well tolerated in mice and chimpanzees [4, 5]. However, the selectivity of TRAIL for tumor cells over normal cells is lost when TRAIL forms multimeric complexes. For example, it has been observed that cross-linked TRAIL induces apoptosis more efficiently than non-aggregated trimeric TRAIL [6], but such cross-linked TRAIL also caused cell death in normal hepatic cells [7]. It seems therefore attractive to design clustered TRAIL variants with enhanced specificity for tumor tissue. The increased tissue retention of cellular targeted TRAIL variants may counterbalance or prevent side effects elsewhere in the body. In the present paper, we investigated the development of TRAIL complexes equipped with targeting ligands directed to the tumor vasculature. Enhanced retention of TRAIL complexes at the tumor vasculature will enhance their accumulation in the tumor tissue, thereby favoring the desired therapeutic activity and reducing potential toxicity.\nSeveral recent papers described the development of recombinant TNF family members with enhanced target specificity. One approach is to modify the intrinsic specificity of TRAIL for its target Death-receptor 5 specific by substituting amino-acids located in the receptor binding interface of TRAIL [8]. Development of chimaeras is another successful approach. Linkage of TRAIL to scFv antibody fragments yielded constructs which bind to additional surface-exposed receptors on tumor cell types [9\u201311]. Similarly, other TNF family members like Fas-ligand and TNF\u03b1 (have been conjugated to tumor-cell directed targeting ligands, yielding chimeric proteins with full or even enhanced therapeutic activity [11\u201315].\nAlthough the recombinant approach has been applied successfully for the production of chimeric proteins, low production yields may hamper the feasibility of such a strategy. Especially when multiple targeting ligands are introduced into a protein subunit, which is preferred for high avidity binding, aberrant folding of chimeric proteins can occur. In the current study we therefore pursued a chemical approach, in which TRAIL was biotinylated and complexed to avidin equipped with multiple targeting ligands. Avidin\u2013biotin complexation is a widely accepted approach in diagnostic applications that employ immunodetection steps, and is also considered an attractive strategy for the preparation of tumor targeted complexes, especially in so-called pretargeting approaches in which the complexes are assembled in vivo at the cell surface of tumor cells. Commonly, such strategies employ biotinylated tumor-specific antibodies and a therapeutic or diagnostic cargo coupled to avidin [16, 17].\nThe approach presented in this paper differs to the above-discussed strategies in several aspects. First, we now equipped TRAIL with targeting ligands directed to angiogenic endothelium rather than to tumor cells, allowing an enhanced binding to the tumor blood vessels. The vascular wall is in direct contact with the systemic circulation, which makes it an easily attainable target for therapeutic proteins. Furthermore, intratumoral pressure, which hampers the penetration of macromolecules into tumor tissue will not affect the homing to of RGD-equipped complexes to the angiogenic endothelium.\nSecond, we have prepared a different type of targeted avidin that can interact directly via covalently attached RGD-groups with tumor endothelial cells. Thus, the developed avidin\u2013biotin-TRAIL complexes will home to angiogenic endothelium without the need of complexation with a biotinylated antibody, thereby leaving its biotin-binding sites available for complexation with the biotinylated TRAIL. The functionalization of avidin with RGD-homing ligands was carried out using a cyclic RGD-peptide with high affinity and specificity for \u03b1v\u03b23 integrin on angiogenic endothelium [18]. We furthermore applied a polyethylenglycol (PEG) linker for coupling of the RGD-ligand, which brings several advantages, such as a reduced non-specific binding of the avidin and reduced immunogenicity [19]. The now developed RGDPEG-avidin conjugates contain the RGD homing ligand attached to the distal end of the PEG groups.\nIn order to minimize loss of functional TRAIL activity, we have compared two different biotinylation strategies that react at different functional groups in the protein. We used a conventional Biotin-NHS reagent that binds to primary amino groups in the protein, and we employed the Biotin-ULS (ULS: universal linkage system) reagent, which reacts at methionine residues. Figure\u00a01 shows a schematic representation of the developed complexes and their interaction with target cells. The products were evaluated for their association with \u03b1v\u03b23 integrin expressing endothelial cells and for their antitumor activity in both endothelium and cultured tumor cells. Our results demonstrate that recombinant TRAIL can be complexed with endothelial specific targeting ligands in such a way that it maintains its extensive cell killing activity either in solution or when displayed on the surface of endothelial cells.\nFig.\u00a01Schematic representation of RGD-avidin:TRAIL complexes. Soluble recombinant human TRAIL was modified with biotin groups via two different reagents that either react at lysine or methionine residues. Biotinylated TRAIL was complexed to an avidin-based targeting device equipped with approximately 5 RGD-peptide ligands per avidin molecule. A hydrophilic PEG-linker was used to append the RGD-peptides to avidin. The assembled complexes can bind to tumor blood vessels via RGD\/\/\u03b1v\u03b23-integrin interactions, while apoptosis can be induced via TRAIL\/DR ligation\nMaterials and methods\nPreparation and characterization of RGDPEG-avidin\nAvidin was modified with heterobifunctional PEG groups that can react with primary amino groups in avidin, after which RGD-peptide groups can be conjugated to their distal ends. The hydrophilic PEG groups of 3.4\u00a0kDa will furthermore reduce non-specific protein interactions.\nBriefly, avidin (2\u00a0mg, 32\u00a0nmol, Sigma) was dissolved in PBS at 10\u00a0mg\/ml and treated with a 50-fold molar excess of the heterobifunctional vinylsulfone-polyethylenglycol-N-hydroxysuccinimide ester (1\u00a0mg, 292\u00a0nmol, VNS-PEG-NHS; Nektar, Alabama, USA), which had been dissolved in water at a concentration of  20\u00a0mg\/ml. The PEG linker was added drop wise. The reaction mixture was protected from light with tin foil and incubated for 1\u00a0h at room temperature on a spiramix rollerbank. Meanwhile, the RGD peptide c(RGDf(\u03b5-S-acetylthioacetyl)K) (Ansynth Service, Roosendaal, The Netherlands) was dissolved at 10\u00a0mg\/ml in a 1:4 acetonitrile\/water mixture. The peptide was added drop wise to the reaction mixture at a molar ratio of 55:1, after which freshly prepared hydroxylamine was added to a final concentration of 50\u00a0mM. The reaction was carried out overnight at room temperature while protected from light. Remaining VNS groups were quenched by addition of cysteine (55:1 molar excess over the amount of avidin), after which the product was purified by size exclusion chromatography on a Highload superdex 200 column (Amersham Biosciences), pre-equilibrated in PBS. The final product RGDPEG-avidin was stored in PBS at \u221220\u00b0C.\nRGDPEG-avidin conjugates were characterized for protein content by BCA protein assay (Pierce). Coupling of RGDPEG groups to avidin was demonstrated by SDS-PAGE performed on a mini-Protean II system (Bio-Rad) using 12% Ready Gels (Bio-Rad, The Netherlands). Duplicate gels were either stained for protein (Coomassie Brilliant Blue staining) or blotted on a PVDF membrane (Roche, Mannheim, Germany). The membrane was blocked with BSA and subsequently incubated with an in-house raised anti-RGD polyclonal rabbit antiserum specific for the conjugated RGD-peptide [20]. Blots were further developed with goat-anti-rabbit peroxidase (GARPO)\/DAB detection. The grade of RGD-pegylation was furthermore determined by MALDI-TOF analysis as described previously [21].\nExpression and purification of recombinant human His-tagged TRAIL\nA cDNA corresponding to human soluble TRAIL (amino acids 114\u2013281) was cloned downstream of the hexahistidine (His) tag sequence in pET15B (Novagen) using NdeI and BamHI restriction sites. Escherichia coli BL21 (DE3) (Invitrogen) was subsequently transformed with the His-TRAIL construct. The transformed host was grown in shake flasks at four liter scale as described previously [22]. The culture was centrifuged at 2,500\u00a0rpm and the supernatant was discarded. The isolated pellet was resuspended in three volumes of extraction buffer (20\u00a0mM phosphate buffer pH 8 containing 200\u00a0mM NaCl, 10% (v\/v) glycerol and 7\u00a0mM \u03b2-mercaptoethanol).\nCells were disrupted using sonication and extracts were clarified by centrifugation at 40,000 g. Subsequently, the supernatant was loaded on a cobalt-charged IMAC TALON column (Clontech) and His-TRAIL was eluted with a step gradient of extraction buffer containing 0.5\u00a0M imidazole. Trimeric His-TRAIL was purified by size-exclusion chromatography on a Hiload Superdex 75 column (Amersham Biosciences), to remove impurities. The final product was flash frozen and stored at \u221280\u00b0C in storage buffer (phosphate buffer pH 7.4 containing 150\u00a0mM NaCl, 3.5\u00a0mM DTT and 20\u00a0\u03bcM ZnSO4). Purified protein was >95% pure as determined by colloidal Coomassie Brilliant Blue-stained SDS\u2013PAGE gel.\nPreparation and characterization of bio-NHS-TRAIL and bio-ULS-TRAIL\nWe studied two different linkage strategies to introduce biotin into His-TRAIL, by applying either a conventional biotinylation reagent that reacts with primary amino groups in the protein (biotin-NHS), or one that reacts to methionine residues (biotin-ULS\u2122, ULS\u00a0=\u00a0universal linkage system). Such an alternative strategy may prove less harmful to the therapeutic activity of TRAIL, depending on the extent to which side chain residues in the active domains are modified. Biotin-ULS was provided by Kreatech Biotechnology (Amsterdam, The Netherlands). All syntheses were performed in a 10:1 molar ratio of biotinylation reagent over TRAIL.\nHis-TRAIL in storage buffer was extensively dialyzed against PBS containing 10% glycerol at 4\u00b0C using Slide-A-Lyzer dialysis cassettes (10,000 MWCO, Pierce) to remove DTT and other components of the storage buffer that can interfere in the reactions. Typically, the purified His-TRAIL (500\u00a0ug, 7.6\u00a0nmol) at 0.7\u00a0mg\/ml was mixed either with Sulfo-NHS biotin (Pierce) (10\u00a0mg\/ml in DMF) or with biotin-ULS (10\u00a0mg\/ml in 20\u00a0mM NaCl), after which the mixtures were incubated for 4\u00a0h at 37\u00b0C. Unreacted biotinylation reagent was removed by dialysis against PBS at 4\u00b0C. The products were sterilized by filtration via disposable 0.2\u00a0\u03bcm filters and stored at \u221220\u00b0C.\nBiotin-TRAIL conjugates were characterized for protein content using BCA protein assay (Pierce). The relative number of coupled biotin molecules was determined by anti-biotin ELISA. Wells were coated with serial dilutions of the biotinylated proteins for 1\u00a0h at room temperature, washed with PBS containing 0.05% Tween20 and incubated for 1\u00a0h at room temperature with streptavidin-peroxidase complex (Dako, 1:2500 in PBS), followed by a standard incubation with OPD. The protein concentration at which 50% of the maximum absorbance was measured (EC50) was calculated by nonlinear regression (Graphpad Prism), and used to calculate relative biotin:protein ratios in each conjugate. The grade of biotinylation was furthermore assayed by MALDI-TOF analysis as described above for RGDPEG-avidin.\nCells\nJurkat T cells were cultured in RPMI 1640 culture medium (containing 25\u00a0mM HEPES and l-glutamine), supplemented with 10% heat-inactivated FCS (Bodinco, Alkmaar, The Netherlands), 1\u00a0mM sodium pyruvate, 2\u00a0mM l-glutamine, 0.5\u00a0mM 2-ME, and 0.1\u00a0mg\/ml gentamycin sulfate obtained from BioWhittaker, and 0.02\u00a0\u03bcg\/ml fungizone. (Bristol-Meyers, Woerden, The Netherlands)\nHUVEC were isolated by the UMCG Endothelial Cell Facility by previously described methods [23]. Primary isolates were cultured in 1% gelatin-coated tissue culture flasks (Corning, Costar, The Netherlands) at 37\u00b0C under 5% CO2\/95% air. The culture medium, hereafter referred to as EC medium, consisted of RPMI 1640 (BioWhittaker, Verviers, Belgium) supplemented with 20% heat inactivated fetal calf serum (Integro, Zaandam, The Netherlands), 2\u00a0mM l-glutamine (Invitrogen, Breda, The Netherlands), 5\u00a0U\/ml heparin (Leo Pharmaceutical Products, Weesp, The Netherlands), 100\u00a0U\/ml penicillin (Yamanouchi Pharma, Leiderdorp, The Netherlands, 100\u00a0\u03bcg\/ml streptomycin (Radiumfarma-Fisiopharma, Italy), and 50\u00a0\u03bcg\/ml endothelial cell growth factor supplement extracted from bovine brain. After attaining confluence, cells were detached from the surface by trypsin EDTA (0.5\/0.2\u00a0mg\/ml in PBS) treatment and split in 1:3 ratio. Cells were used up to passage four.\nBinding of RGDPEG-avidin to HUVEC\nBinding affinity of RGDPEG-avidin to \u03b1v\u03b23-integrin expressed on the surface of HUVEC was determined by competitive displacement of 125I-labeled Echistatin, which is a known ligand for \u03b1v\u03b23 [24]. Confluent monolayers of HUVEC in 24-well plates (Costar) were incubated with 100,000 cpm 125I-Echistatin either in the presence of excess of RGDPEG-avidin, free RGD-peptide or avidin as competitors (competitors all in a concentration of 10\u00a0\u03bcM), or a serial dilution of RGDPEG-avidin and free RGD-peptide. Competition experiments were carried out with divalent cation containing binding buffer (50\u00a0mM Tris-HCl, pH 7.4, 150\u00a0mM NaCl, 1\u00a0mM CaCl2, 1\u00a0mM MgCl2, 1\u00a0mM MnCl2 and 1% BSA) at 4\u00b0C for 4\u00a0h. Cells were washed three times with binding buffer and lysed with 1\u00a0M NaOH. Radioactivity was counted in a Packard RIASTAR multiwell gamma counter (GMI, Minnesota, USA). Data were analyzed by non-linear regression using the GraphPad Prism program.\nComplexation of biotinylated TRAIL with RGDPEG-avidin\nWe studied the complexation of biotinylated TRAIL with RGDPEG-avidin in an ELISA-like experimental setup. Wells were coated with 50\u00a0ng of avidin or RGDPEG-avidin overnight at room temperature, blocked with BSA (1% in PBS) and incubated with 10\u00a0ng of biotinylated TRAIL for 2\u00a0h at room temperature. After the wells were washed with PBS\/0.05% Tween20, the amount of TRAIL associated with the avidin was detected by anti-TRAIL immunodetection (anti-TRAIL antibody 2E5, Alexis, Breda, The Netherlands) for 1\u00a0h, followed by standard detection with GARPO\/OPD.\nApoptotic activity of biotinylated TRAIL\nThe apoptosis inducing activity of the biotinylated products was evaluated on Jurkat T cells and on endothelial cells, using a cell viability assay and a caspase activity assay. Experiments were conducted with the single cell types, Jurkat tumor cells or HUVEC, or with a combination of tumor and endothelial cells.\nAssessment of TRAIL activity by MTS cell viability assay\nBriefly, cells were seeded in flat-bottom 96-well plates at a density of 3\u00a0\u00d7\u00a0104cells\/well (Jurkat) or 1\u00a0\u00d7\u00a0104cells\/well (HUVEC) in 100\u00a0\u03bcl medium supplemented with the indicated compounds. Concentrations of TRAIL, bio-NHS-TRAIL and Bio-ULS-TRAIL ranging from 1\u00a0\u03bcg\/ml to 1\u00a0ng\/ml were tested on Jurkat. Only one concentration (100\u00a0ng\/ml) was tested on HUVEC. The MTS cell viability assay (Promega) was performed after 48\u00a0h of incubation, according to the manufacturer\u2019s recommendations. Experiments were performed in triplicate.\nAssessment of TRAIL activity by caspase 3\/7 assay\nWe investigated the induction of apoptosis in Jurkat T cells using a caspase 3\/7 activity assay (Promega) employing luminescence detection. Different experimental settings were evaluated either reflecting direct incubation of the products with tumor cells, or settings in which biotinylated TRAIL was complexed to RGDPEG-avidin and\/or anchored onto endothelial cells.\nFirst, we determined pro-apoptotic effects of non-complexed TRAIL. About 5\u00a0\u00d7\u00a0103 Jurkat cells were incubated with biotinylated TRAIL at a concentration of 100\u00a0ng\/ml. Experiments were carried in 96-well plates in 50\u00a0\u03bcl medium supplemented with the indicated compounds for 4\u00a0h, after which 50\u00a0\u03bcl of caspase 3\/7 substrate was added to the cells, followed by luminescence detection with a microplate luminometer according to the provider\u2019s instructions.\nNext, we determined proapoptotic effects of RGDPEG-avidin:TRAIL complexes in Jurkat cells. These experiments were performed in the presence of endothelial cells, to allow adherence of the RGD-equipped complexes to \u03b1v\u03b23 integrin expressing cell layers. HUVEC were preincubated with 10\u00a0\u03bcg\/ml RGDPEG-avidin for 2\u00a0h at room temperature after which the excess of non-cell associated RGDPEG-avidin was removed by washing with PBS. Control experiments included RGDPEG-avidin treated HUVEC that were not washed and cells to which no RGDPEG-avidin was added. All wells were supplemented with biotinylated TRAIL (100\u00a0ng\/ml in culture medium), after which Jurkat cells (5\u00a0\u00d7\u00a0103 cells in 50\u00a0\u03bcl of medium) were added. Cells were incubated for 4\u00a0h at 37\u00b0C, after which caspase activity was assayed as described above. Experiments were conducted in triplicate.\nStatistical analysis\nStatistical analysis was performed using Student\u2019s two-tailed t-test, assuming equal variances. Difference were considered to be significant when P\u00a0<\u00a00.05 unless otherwise stated.\nResults\nPreparation and characterization of RGDPEG-avidin\nWe modified avidin with PEG-linked RGD-peptides to obtain an avidin molecule which could bind to angiogenic endothelial cells. Successful synthesis of RGDPEG-avidin was proven by several analytical techniques. SDS-PAGE analysis (Fig.\u00a02a) showed a single band for avidin of approximately 15\u00a0kDa, corresponding to the weight of one of its four subunits. Different protein bands were observed for RGDPEG-avidin with molecular weights corresponding to the unmodified subunit and to subunits modified with one or multiple RGDPEG moieties. Second, Western blot of the product showed positive staining of these bands by an anti-RGD-peptide antiserum. Due to the size heterogeneity of the PEG polymer, the RGD-band stained in a broad streak rather than a discrete band (Fig.\u00a02b). Of note, the tetrameric band of avidin was not observed due to the denaturating conditions of SDS-PAGE. Third, we analyzed the product by MALDI-TOF mass spectrometry (Fig.\u00a02c), which showed peaks corresponding to avidin subunits modified with RGDPEG to a different degree. From the average molecular weights (Mn) we calculated a relative derivatization ratio of 1.4 RGDPEG groups per avidin subunit, or a total of 5.6 RGDPEG groups per tetrameric avidin.\nFig.\u00a02Characterization of RGDPEG-avidin. Panel A and B: SDS-PAGE separation followed by CBB protein staining (a) or Western blotting and anti-RGD detection  (b). lane 1: MW marker, lane 2: avidin, lane 3: RGDPEG-avidin. Unmodified avidin was separated into its 15\u00a0kDa subunits, while RGDPEG-avidin was disassembled into different bands corresponding to subunits with 0, 1, 2 or 3 attached RGDPEG groups. The intrinsic heterogeneity of PEG polymers can be observed in the broadly stained band of the RGDPEG-avidin Western blot. Panel C: MALDI-TOF analysis of RGDPEG-avidin. Upper line: RGDPEG-avidin, bottom line: avidin. During ionization, the tetrameric proteins have disassembled into subunits, yielding a single peak for avidin and multiple peaks for RGDPEG-avidin corresponding to modification degree 0, 1, 2, 3, 4\nNext, we investigated the interaction of RGDPEG-avidin with HUVEC, which express \u03b1v\u03b23-integrin when being at a subconfluent state. To determine binding affinity of RGDPEG-avidin for \u03b1v\u03b23-integrin, we studied its capability to compete with the \u03b1v\u03b23-integrin ligand 125I-echistatin. While RGDPEG-avidin completely displaced 125I-echistatin from the cells, similar to the free RGD-peptide, avidin did not affect binding of the radiolabeled peptide (Fig.\u00a03a). The sigmoid displacement curve with RGDPEG-avidin (Fig.\u00a03b) demonstrated a 2.9-fold increase in affinity (IC50\u00a0=\u00a0134\u00a0nM) as compared to the single RGD-peptide (IC50\u00a0=\u00a0383\u00a0nM).\nFig.\u00a03Association of RGDPEG-avidin with endothelial cells. Subconfluent HUVEC were incubated with radiolabeled echistatin, which is a known ligand for \u03b1v\u03b23 integrin, and RGDPEG-avidin, free RGDpeptide or unmodified avidin. Panel A: Binding specificity. Echistatin was displaced by excess of RGDPEG-avidin or free RGD-peptide (both at 10\u00a0\u03bcM) but not by excess of avidin. *P\u00a0<\u00a00.05. Panel B: Affinity of binding. Echistatin binding was displaced by different concentrations of the added competing ligands, showing the increased affinity of RGPEG-avidin over the single RGDpeptide ligand\nPreparation and characterization of biotinylated TRAIL\nSince the modification of side-chain residues may diminish the activity of a therapeutic protein, we studied two different biotinylation strategies that target chemical modifications at different amino acid residues, i.e., at lysine groups (biotin-NHS) or methionine residues (biotin-ULS). Such different labeling positions at the surface of the protein may correlate to differences in pharmacological activity of the biotinylated proteins. Anti-biotin ELISA (Fig.\u00a04a) demonstrated a higher biotin\/TRAIL modification of biotin-NHS over biotin-ULS, which is in line with the lower abundance of methionine residues as compared to the number of lysine residues. We tried to confirm these data by analysis of the biotinylation extent by MALDI-TOF mass spectrometry, which provided reliable data on the average number of incorporated RGDPEG units in avidin. Although this approach succeeded for biotin-NHS-TRAIL (Fig.\u00a04b), demonstrating the incorporation of about 1 biotin group per subunit of TRAIL, MALDI-TOF analysis failed to detect biotin-ULS modification of TRAIL. Most likely, the mass ionization step induced the dissociation of biotin-ULS from the protein. From the collective results, we concluded that both strategies had yielded biotinylated TRAIL with approximately 1\u20133 biotin groups per protein (summarized in Table\u00a01).\nFig.\u00a04Characterization of biotinylated TRAILs. Panel A: Anti-biotin ELISA with streptavidin-HRP. Signals were corrected for background and expressed relative to the maximum detected intensity. Panel B: MALDI-TOF analysis of TRAIL and biotin-NHS-TRAILTable\u00a01Characteristics of biotinylated TRAIL variantsRelative biotin content (Biotin\/TRAIL)aActivity in cell viability assay (IC50 in ng\/ml)bActivity in caspase 3\/7 assayc (%)TRAIL\u201340100Bio-NHS-TRAIL1.612335Bio-ULS-TRAIL0.860126aExpressed as mole biotin bound per mole of trimeric TRAIL. Relative biotin content was determined by MALDI-TOF analysis (bio-NHS-TRAIL) or ELISA (BIO-ULS-TRAIL)bAs determined after 48\u00a0h of incubation with Jurkat cellscAs determined after 4\u00a0h of incubation at 10\u00a0ng\/ml with Jurkat cells\nWe studied complexation of biotinylated TRAIL with RGDPEG-avidin that had been coated in 96-well plates. Highest binding was observed for Bio-NHS-TRAIL, probably due to its higher biotin content, while bio-ULS-TRAIL showed approximately 40% less binding (Fig.\u00a05). Unmodified His-TRAIL showed non-specific binding to RGDPEG-avidin as well, despite the absence of biotin groups in this product. Non-specific binding of TRAIL to RGDPEG-avidin was however far lower than binding to unmodified avidin, most likely due to the PEGylation (data not shown).\nFig.\u00a05Association of biotinylated TRAIL with RGDPEG-avidin. 96-well plates were coated overnight with 50\u00a0ng RGDPEG-avidin and incubated with indicated compounds for 2\u00a0h. TRAIL binding was assessed by anti-TRAIL immunodetection and expressed relative to the highest signal obtained with biotin-NHS-TRAIL. Experiments were performed in triplicate\nCytotoxic and proapoptotic activity of TRAIL complexes\nTo test whether our products were functionally active, i.e., capable of inducing cell death and apoptosis, we assessed their activity on Jurkat T leukemic cells and HUVEC endothelial cells. Figure\u00a06a shows the dose-dependent cytotoxic effects at Jurkat cells after treatment for 48\u00a0h. Both His-TRAIL, Bio-NHS-TRAIL and Bio-ULS-TRAIL effectively killed Jurkat cells with IC50 values between 40\u00a0ng\/ml and 123\u00a0ng\/ml (Table\u00a01). While the Bio-NHS modification resulted in a considerable reduction in activity compared to the unmodified protein, Bio-ULS-TRAIL displayed almost similar activity as unmodified TRAIL. The pro-apoptotic activity of His-TRAIL and biotinylated TRAIL derivatives in Jurkat cells was furthermore determined by analyzing caspase 3\/7 activity, as shown in Fig.\u00a06b. These experiments confirmed the relative higher activity of bio-ULS-TRAIL versus bio-NHS-TRAIL, and the absence of caspase induction by RGDPEG-avidin (data not shown). In contrast to Jurkat cells, no cytotoxic effects were observed when HUVEC were incubated with the products (Fig.\u00a06c).\nFig.\u00a06TRAIL activity assays. Panel A: Cell viability of Jurkat T cells after 48\u00a0h of incubation with indicated compounds. Cell viability was assessed by MTS assay. Experiments were performed in triplicate. Panel B: Caspase 3\/7 activity in Jurkat T cells after 4\u00a0h of incubation with 100\u00a0ng\/ml of the indicated TRAIL derivatives. RLU: relative light units. Panel C: Cell viability (MTS assay) of HUVEC after 48\u00a0h of incubation with 100\u00a0ng\/ml of indicated compounds\nFinally, we investigated the proapoptotic effects of RGDPEG-avidin:TRAIL complexes in Jurkat cells. These experiments were performed in the presence of endothelial cells, to allow adherence of the RGD-equipped complexes to \u03b1v\u03b23 integrin expressing cell layers. First, we determined the effects of avidin:TRAIL complexes formed in the presence of an excess of RGDPEG-avidin. As can be observed in Fig.\u00a07, such complexes  with both types of biotinylated TRAIL showed similar activity to the non-complexed biotinylated TRAIL derivatives (striped bars versus white bars, respectively). Furthermore, in agreement with Fig.\u00a06b, Bio-NHS-TRAIL showed a reduced activity as compared to unmodified TRAIL, while Bio-ULS-TRAIL retained its activity.\nFig.\u00a07Proapoptotic activity of RGDPEG-avidin:TRAIL complexes. Assays were performed in 96-well plates seeded with HUVEC that were subsequently incubated at room temperature with RGDPEG-avidin (10\u00a0\u03bcg\/ml)  and biotinylated TRAIL (100\u00a0ng\/ml), to allow assembly of the complexes. Next, Jurkat T cells were added followed by 4\u00a0h of incubation at 37\u00b0C, after which the activation of caspase 3\/7 was determined by a luminescence based assay. Experiments were carried out in the absence of RGDPEG-avidin (control experiment, white bars), in the presence of excess of RGDPEG-avidin (striped bars, 100-fold molar excess over biotinylated TRAIL) or in wells incubated with RGDPEG-avidin that had been washed before addition of biotin-TRAIL and Jurkat cells (black bars). *P\u00a0<\u00a00.05 versus TRAIL; #P\u00a0<\u00a00.05 versus biotin-TRAIL in the absence of RGDPEG-avidin\nA striking result was observed when the excess of RGDPEG-avidin was removed before addition of TRAIL and Jurkat cells, leaving only endothelial-associated RGDPEG-avidin available for complexation with biotinylated TRAIL. This procedure significantly potentiated the proapoptotic effect for both Bio-NHS-TRAIL and Bio-ULS-TRAIL, the latter even beyond the activity of unmodified TRAIL.\nDiscussion\nIn the present study we describe the development and functional evaluation of RGD-avidin:TRAIL complexes. These targeted complexes of TRAIL showed enhanced binding to endothelial cells and maintained their pro-apoptotic activity. As a result of the binding to angiogenic endothelial cells of these complexes, an improved tumor accumulation and enhanced residence within tumor tissue can be expected. Such an improvement in the pharmacokinetic profile of TRAIL may further enhance its therapeutic efficacy and enable a reduction in dosing frequency.\n\u03b1v\u03b23 integrin is a validated target for drug delivery and imaging approaches directed to angiogenic endothelium. One of the most popular ligands for \u03b1v\u03b23 integrin is the cyclic RGD-peptide cyclo(RGDfK). Due to the special constrained conformation of the cyclic RGD-peptide and the d-phenylalanine amino acid, its binding motif adopts a high affinity conformation for \u03b1v-integrins [25]. The cyclo(RGDfK) peptide has been used successfully for the delivery of a wide range of drug or gene carrying systems (e.g., liposomes, polymers, adenoviruses) [18]. Since the cyclopeptide is an unnatural peptide, it can not be encoded by a DNA sequence and requires chemical synthesis and hence chemical coupling techniques to prepare multivalent targeting devices. The present paper describes a versatile approach in which multivalent RGD-avidin is complexed with biotinylated TRAIL. The RGD-peptides are linked to avidin via bifunctional PEG linkers, thereby surrounding the avidin by a hydrophilic PEG shell tethered with targeting ligands. PEGylation is a widely accepted methodology for improvement of therapeutic proteins, and affects both the immunological properties as well as the renal filtration of proteins [26]. The functionalization of avidin with RGD-PEG groups thus will combine several advantageous properties. RGDPEG-avidin displayed nanomolar affinity for \u03b1v\u03b23 integrin expressing endothelial cells, which is in good agreement with the data obtained with other RGD-equipped proteins prepared in our group [27].\nWe have compared two different biotinylation procedures, which react at different amino acid side chain residues. While biotin-NHS couples at lysine residues, biotin-ULS binds at methionines residues in TRAIL. The methionines in TRAIL are not solvent exposed, as can be derived from the crystal structure of TRAIL [28]. Therefore, the most likely binding site for biotin-ULS is the methionine in the HIS-tag of the recombinant protein. In contrast, most lysine residues are readily accessible, including ones at positions that will interface with the Death receptor. This could explain why Biotin-ULS-TRAIL better maintained its proapoptotic activity than biotin-NHS derivatized TRAIL. Whether the greater loss in activity of biotin-NHS-TRAIL is due to the relative higher extent of derivatization or to modifications of lysine residues in the activity domains of protein can not be concluded from our data.\nUpon complexation of biotinylated TRAIL with RGDPEG-avidin, a bulky structure has been introduced onto the surface of the therapeutic protein. The assembled complexes, however, fully maintained their TRAIL activity indicating that steric hindrance did not interfere in the TRAIL-death receptor ligation. Figure\u00a07 showed similar proapoptotic activities for either biotinylated TRAIL or the complexes formed in the presence of excess of RGDPEG-avidin. Remarkably, we observed an enhancement of caspase activity when the excess of RGDPEG-avidin was removed, leaving only surface-exposed RGDPEG-avidin at the endothelial cell layer. One of the explanations that may account for this is the assembly of multiple TRAIL molecules onto a single avidin, thereby affording a \u2018mega\u2019TRAIL complex with enhanced death-inducing activity, as was also observed for multimeric forms of Fas-ligand [29]. Such types of complexes would facilitate the close assembly of multiple death receptors, thereby potentiating the induction of death signaling. The presence of an excess of RGDPEG-avidin would avoid the formation of such complexes, as both components were added in a 100:1 molar ratio of avidin:TRAIL. Gently washing of the endothelial cells after RGDPEG-avidin incubation  removed the excess of unbound RGDPEG-avidin, leaving only cell-associated avidin after incubation at room termperature. These conditions therefore provided an excess of biotinylated TRAIL over avidin, now favoring the formation of multimeric TRAIL complexes. Alternatively, the exposure of TRAIL via RGD-PEG-avidin onto the surface of endothelial cells may explain its increased activity, as has been observed for chimeric TRAIL variants directed to tumor receptors [10].\nHUVEC were not killed by TRAIL or its derivatives, as has been described by others [30]. On the other hand, although TRAIL does not induce apoptosis in endothelial cells, it activates other processes such as production of NO and reactive oxygen species which may alter the tumor responsiveness to TRAIL [31]. This can  not explain the results shown in Fig.\u00a07, since endothelial cells were preincubated with RGDPEG-avidin only and preincubations were performed at room temperature rather than at 37\u00b0C. Such effects of RGD-equipped TRAIL may occur however in vivo. In addition, tumor derived growth factors might sensitize endothelial cells to TRAIL\u2019s apoptosis inducing capacity, similar to the findings reported for TNF-sensitivity [32].\nThe complexation of biotinylated TRAIL with RGDPEG-avidin and its association with angiogenic endothelial cells may afford an increased accumulation into the tumor blood vessels, similar to the enhanced tumor accumulation of other RGD-modified proteins [18]. An unresolved issue is whether RGD-avidin\/TRAIL complexes bound to the tumor vasculature can interact with tumor cells behind the endothelial lining in vivo. To interact with tumor cells and induce tumor apoptosis, TRAIL complexes bound to the tumor vasculature should dissociate and penetrate into the tumor tissue. Several considerations may be important in this respect, such as the expression of \u03b1v\u03b23 integrin on both luminal and abluminal side of the endothelium and the fact that many tumor cells express \u03b1v\u03b23 integrin [18]. Both of these will favor the interaction of RGD-avidin complexed TRAIL with tumor cells. Furthermore, RGD-avidin binding to endothelial cells is less tight than the typical antibody binding. This may favor redistribution of the complexes upon their initial binding to \u03b1v\u03b23 integrin. Homing to the tumor vasculature will provide elevated concentrations within the tumor microenvironment, which may subsequently provide elevated intratumoral concentrations. Together with the observed enhancement of TRAIL activity, this may favor a more efficient induction of apoptosis in tumor cells.\nTo summarize, we have prepared a novel type of RGD-targeted TRAIL with binding specificity for angiogenic endothelium and maintenance of strong cytotoxic activity. Whether in vivo application of this product  will lead to an enhanced tumor accumulation still needs to be investigated. We now succeeded in coupling the well known homing device RGD to the TRAIL molecule, without losing the apoptosis-inducing activity of TRAIL or the receptor-binding properties of RDG peptides. An enhanced activity could even be demonstrated in vitro. This may, in combination with an improved pharmacokinetic profile induced by the PEG and RGD moieties, lead to improved effects of TRAIL in vivo. Lastly, the followed synthetic approach offers opportunities for the complexation with alternative targeting ligands that bind directly to tumor cells, such as tumor-specific antibodies or peptide ligands.","keyphrases":["apoptosis","therapeutic proteins","angiogenesis","drug targeting"],"prmu":["P","P","U","R"]}
{"id":"Matern_Child_Health_J-2-2-1592161","title":"Promising Practices in Preconception Care for Women at Risk for Poor Health and Pregnancy Outcomes\n","text":"Objectives: Two programs targeting urban African-American women are presented as promising models for preconception care, which includes interconception care. Methods: The Grady Memorial Hospital Interpregnancy Care (IPC) Program in Atlanta, Georgia, and the Magnolia Project in Jacksonville, Florida, are described. The IPC program aims to investigate whether IPC can improve the health status, pregnancy planning and child spacing of women at risk of recurrent very low birthweight (VLBW). The Magnolia Project aims to reduce key risks in women of childbearing age, such as lack of family planning and repeat sexually transmitted diseases (STDs), through its case management activities. Results: Seven out of 21 women in the IPC were identified as having a previously unrecognized or poorly managed chronic disease. 21\/21 women developed a reproductive plan for themselves, and none of the 21 women became pregnant within nine months following the birth of their VLBW baby. The Magnolia Project had a success rate of greater than 70% in resolving the key risks (lack of family planning, repeat STDs) among case management participants. The black to white infant mortality (IM) ratio was better for the babies born to women managed in the Magnolia Project compared to the same ration for the United States. Conclusions: Preconception care targeted to African-American women at risk for poor birth outcomes appears to be effective when specific risk factors are identified and interventions are appropriate. Outreach to women at risk and case management can be effective in optimizing the woman's health and subsequent reproductive health outcomes.\nIntroduction\nCurrent national level statistics demonstrate the potential for preconception care to improve the health of women and impact their reproductive health outcomes in terms of achieving optimally spaced, planned pregnancies and averting adverse birth outcomes. Each year in the United States, approximately 6 million pregnancies result in over 4 million live births. Almost half of all pregnancies are unintended, despite the fact that 62% of the 62 million women of reproductive age use a contraceptive method [1]. Chronic diseases and adverse health behavior known to affect pregnancy outcomes are prevalent among women of reproductive age (15\u201344 years). In 2002, 9.3% of women of reproductive age had diabetes, and 11.4% of pregnant women smoked tobacco and 10.1% of pregnant women consumed alcohol [2].\nThe U.S. Public Health Service has designated preconception care as a critical component of prenatal care [3]. Initiating interventions to address a woman's health risks upon her entry into prenatal care is often too late to impact the outcome of pregnancy. For example, if a woman has her first prenatal visit after the first month of pregnancy, it is too late to prescribe folic acid supplementation to help prevent neural tube defects as the neural tube closes by the 28th day of gestation. Likewise, women who are advised to discontinue their use of potentially teratogenic medications at the first prenatal visit may have already sustained damage to the fetus, as most organogenesis is complete by day 56 following conception [4].\nPreconception care is the care of women of reproductive age before a first pregnancy or between pregnancies (commonly known as interconception care) to ensure that conditions and behaviors which may pose a risk to mothers and infants are identified and managed. A specific element of interconception care is the identification and reduction of risks indicated by a prior adverse pregnancy outcome. The major components of preconception care are risk assessment (identification of conditions that may be amenable to intervention that adversely affect reproductive health outcomes), education and health promotion, and medical and psychosocial interventions.\nThere are varying concepts of preconception care ranging from a pre-pregnancy planning visit in the months prior to conception to a component of each health encounter for women. Ideally, preconception care would be an integral component of primary and preventive care for women of reproductive age. Presently, the status of preconception care in the United States is far from ideal. Approximately one of six obstetrician\/gynecologists or family physicians had provided preconception care to the majority of the women for whom they provided prenatal care [5].\nGiven the diversity of women of reproductive age, the numerous health and psychosocial issues that impact reproductive health outcomes and the varied settings for delivery of health care, a single \u2018best\u2019 model of preconception care for all women is unlikely. Rather, tailoring the delivery of preconception care interventions to women with specific circumstances and in specific care settings will be necessary. This paper describes two programs for delivering preconception care for women at-risk for poor health and pregnancy outcomes. One targets women whose risk status is defined by African-American ethnicity and delivery of a very low birth weight (VLBW; <1500\u00a0g) infant and, thus, specifically involves the provision of an interconception intervention. The other program targets women whose risk status is defined by African-American ethnicity and residence in Jacksonville-Duvall County, Florida, and involves the delivery of an intervention with preconception and interconception components.\nThe grady memorial hospital Interpregnancy Care (IPC) program\nBackground\nGeorgia ranks among the 10 states with the highest feto-infant mortality rate. The largest contributor to Georgia's comparatively high feto-infant mortality is the delivery of VLBW infants. VLBW delivery comprises only 2% of births in Georgia, yet accounts for approximately 50% of feto-infant mortality. African-American women in Georgia have 3\u20134 times the rate of VLBW delivery compared to whites. Approximately 66% of the observed racial disparity in feto-infant mortality rates between African-Americans and whites can be attributed to the higher rate of VLBW delivery among African-American women [6].\nThere is a close link between the poor health status of women and VLBW delivery [7\u201317]. The poor health status of women in Georgia is estimated to account for approximately 60% of Georgia's excessive infant mortality [18]. With each subsequent VLBW delivery, there is an increased chance that the woman's next pregnancy will result in recurrence of VLBW delivery [19]. In fact, the best predictor of whether a woman will have a VLBW delivery is her history of a previous VLBW delivery, with the rates of recurrence after a single VLBW delivery being approximately 1.5 times higher for African-American compared to white women (13.4% vs. 8.2%, respectively, for non-teen mothers; 26.8% vs. 16.4%, respectively, for teen mothers). The reason for recurrence of VLBW delivery is likely that aspects of the woman's pre-existing health status\u2014including untreated or poorly managed medical problems and unaddressed nutritional, social, and behavioral risk factors\u2014that may have contributed to delivery of the VLBW delivery persist after delivery and in subsequent pregnancies.\nIn 1998 the Georgia Task Force on Perinatal Care was convened to make recommendations for reducing Georgia's overall feto-infant mortality rate and racial disparities in feto-infant mortality. From its findings as outlined above, the Task Force specifically recommended that interpregnancy care be initiated and evaluated for women at risk for having recurrence of VLBW delivery. The Grady Memorial Hospital Interpregnancy Care (IPC) Program was initiated in response to the recommendation of the Task Force.\nTarget population\nAfrican-American women residing in Fulton or DeKalb counties, Georgia who qualify for county-supported indigent care services and who deliver liveborn or stillborn VLBW infants at Grady Memorial Hospital in Atlanta are considered eligible for the IPC program.\nProgram description\nThe Grady Memorial Hospital IPC program provides 24 months of integrated primary health care and dental services through enhanced nurse case management and community outreach via a Resource Mother. The Resource Mother is a layperson who is trained by the Fulton County Health Department using a specific set of objectives to facilitate life skills and health education acquisition to support high-risk women. The nurse case manager offers all women who deliver a VLBW (stillborn or liveborn) infant at Grady Memorial Hospital enrollment in the program during or soon after their delivery admission. Women who choose to enroll in the IPC program have their initial home visit with the Resource Mother within 1\u20132 weeks of discharge from the delivery admission; they are scheduled for their initial IPC clinical evaluation at a clinic within Grady Memorial Hospital at 4\u20136 weeks postpartum.\nAt the initial IPC clinical evaluation, the IPC program's family physician or nurse- midwife performs a comprehensive survey of medical, obstetrical, nutritional, psychological, and social issues (using standardized assessment tools); a thorough physical examination including pelvic exam; and laboratory evaluations to screen for anemia, nutritional deficiencies, sexually transmitted diseases (STDs) and reproductive tract infections. Standard postpartum care issues are addressed in the initial IPC clinical evaluation, thus eliminating a separate postpartum appointment.\nAs part of the evaluation, the participant and provider explicitly discuss a care plan for the 24-month period of the program. For each woman, the care plan addresses the following seven areas epidemiologically linked to LBW delivery: 1) Pregnancy intendedness and child-spacing through the provision of health education concerning the importance of achieving at least a 9-month (and preferably an 18-month) interpregnancy interval, assisting the woman to articulate her own reproductive plan and select a corresponding contraceptive method; 2) Management of chronic disease (if present) through the promotion of self-care and adherence to scheduled appointments in Grady Health System that are facilitated by nurse case management; 3) Screening and treatment for nutritional deficiencies; 4) Prevention, screening, and treatment for STDs and reproductive tract infections; 5) Treatment and referral for substance abuse (if present) including linkage with rehabilitation programs for illicit substance abuse, and support in and linkage with existing programs for tobacco and alcohol abuse; 6) Screening and treatment or support for depression, psychosocial stressors, and domestic violence; 7) Prevention, screening and treatment for periodontal disease.\nSubsequent visits to the IPC clinic are offered every 1\u20133 months, depending upon the extent and severity of the woman's health and social issues. Elements of the care plan are addressed at each subsequent visit to the IPC clinic. Peer group learning experiences are integrated with IPC health care visits and are modeled on the Centering Pregnancy concept of group prenatal care [20].\nResource Mother support services are focused on identification and management of psychosocial stressors, and life skills enhancement, including parenthood preparedness, safe housing, skills training, employment acquisition, and relationship issues. Resource Mother support is offered at least twice monthly in the form of home visits and telephone contact. Participants are able to contact the Resource Mother via her cell phone to request support, if needed.\nProviders\nPrimary care and outreach services are delivered by a multidisciplinary team comprised of a family physician, an advanced practice nurse trained as both a nurse midwife and a family nurse practitioner, a periodontist, a nurse case manager, and a Resource Mother. Social workers affiliated with Grady Health System also support the IPC program and interface with the IPC team.\nOutcome objectives\nThe purpose of the Grady Memorial Hospital IPC program is to investigate whether interpregnancy care can improve the health status, pregnancy planning and child spacing of women at risk of recurrent VLBW delivery. Findings will contribute to the field of primary health care of reproductive age women in several important ways: (a) the content of a successful IPC package for improving the health of high-risk women will be explored; (b) the concept of IPC will be tested as a means of improving attainment of desirable interpregnancy intervals and decreasing the occurrence of subsequent adverse pregnancy outcomes for high-risk women; (c) the cost of providing IPC to high-risk women will be studied.\nFunding\nThe health care services rendered to IPC participants were provided through the services of the Grady Health System. Funding for the research and evaluation components of the IPC Program, including Vasser-Wooley Foundation, Healthcare Georgia Foundation, Centers for Disease Control and Prevention (CDC), Rockdale Foundation, and March of Dimes.\nResults from the first year of the IPC program\nEnrollment\nDuring November 2003 through March 2004, the feasibility phase of the IPC program was initiated by enrolling the first sequential 29 eligible women who gave their informed consent for participation. During the enrollment period, a total of 47 women delivered VLBW infants at Grady Memorial Hospital. Of these 47 women, nine were not African-American or did not reside in Fulton or DeKalb counties, four declined enrollment, three who wished to be contacted afterdischarge from the hospital were unable to be contacted, and two were discharged from the hospital within 24\u00a0h and before being offered enrollment.\nParticipation\nTwenty-one of 29 enrolled women (72.4%) successfully completed the initial 12 months of the planned 24 months of follow-up. During the first 12 months, eight women became disenrolled from the program two moved out of state (and informed us of their move), three electively disenrolled (two before prior to the initial home visit and the initial IPC clinical evaluation; one after a single IPC clinic visit), and three became lost to follow-up (two before the initial IPC clinical evaluation; one after a single IPC clinic visit and four home visits) all of whom who had noted problems with cocaine abuse in their medical records during their pregnancy.\nOutcomes\nA detailed program description and final results for all evaluated outcomes for the full 24 months of follow-up will be published when all follow-up and outcome ascertainment is complete (anticipated June 2006). Important findings related to the health status of women in the IPC intervention cohort who had at least two visits during the first 12 months of IPC are given below:Chronic diseases that were previously unrecognized or poorly managed were identified for 7\/21 participants, and include valvular heart disease (1\/21), sickle cell anemia (1\/21), hypertension (2\/21), diabetes (1\/21), asthma (1\/21), systemic lupus erythematosus (1\/21), prolactinoma (1\/21), panic attacks (1\/21), and generalized anxiety disorder (1\/21);Reproductive tract infections were diagnosed and treated for 15\/21 participants;Iron-deficiency anemia was diagnosed and treated for 5\/21 participants;Concerns about finances, employment, and needs of the child are almost universal stressors;Average annual outpatient charges per participant for IPC are $1,801 (average 4.6 visits, $389 per visit).\nImportant findings related to the reproductive outcomes during the first 12 months of IPC are as follows:With extensive case management and patient education, 21\/21 women who had at least two visits during their first 12 months of IPC, stated a reproductive plan for themselves and initiated a contraceptive plan in accordance with their stated reproductive plan. Despite a stated desire to either avoid or delay a pregnancy (21\/21 women), a significant number of barriers to effective contraception existed and were dealt with, including misinformation about contraceptive methods and reproduction, concerns about side effects of contraceptive methods, and perceptions of partners\u2019 desires regarding child bearing and contraception.All of the women who had at least two visits during their first 12 months of IPC (21\/21) achieved at least a nine-month interpregnancy interval (i.e., none have become pregnancy within nine months of the index VLBW delivery). For the 21 actively participating women, pregnancy ascertainment involved ongoing contact with the women and reviewing the Grady Healthy System medical record system for any evidence of health care visits or laboratory tests in which a pregnancy was diagnosed, incidentally noted, or treated. No pregnancies are known to have occurred within nine months of the index VLBW delivery for the eight women who became disenrolled, for which pregnancy ascertainment involved telephone follow-up, where possible (two women), and review of the Grady Health System medical record system. It is possible that those women with whom contact was lost have had a pregnancy for which no care was sought or care was sought outside of Grady Health System.\nBarriers\nIn the United States, a major barrier to studying the impact of interpregnancy care on the health status and reproductive outcomes of high-risk women has been the lack of financial coverage for the health care component of the IPC intervention. For the IPC program in Atlanta, the Grady Health System has provided the two years of primary health care and treatment of all identified diseases for all enrollees.\nSpecific barriers exist for retaining participation in the IPC program. Women with substance abuse problems who do not enroll in formal substance abuse treatment programs are difficult to maintain in the IPC program. These women seem to have poor insight into the effects that substance abuse has on their repeated poor pregnancy outcomes. For some enrolled women, receiving health care services is less of a priority than securing employment, which negatively influences their health care seeking behaviors.\nNext steps\nUpon conclusion of the planned 24 months of follow-up, the final results for all evaluated outcome objectives from the feasibility phase will be reported. The IPC program is currently seeking grant support to conduct a randomized clinical trial to test the hypothesis of whether IPC can decrease the occurrence of subsequent adverse pregnancy outcomes for pregnancies conceived within 18 months of a VLBW delivery at Grady Memorial Hospital. The project plans to enhance participant retention by offering more opportunities for life skills enhancement, job training, and delivery of health care services via the community-based Grady Neighborhood Health Centers.\nThe magnolia project\nBackground\nIn 1998, infant mortality rates in Jacksonville-Duval County, Florida were increasing while statewide rates were declining. The Northeast Florida Healthy Start Coalition, Duval County Health Department and other community partners undertook a community assessment and identified racial disparities as a major factor contributing to the city's high infant mortality. The Coalition utilized the Perinatal Periods of Risk (PPOR) and findings from its Fetal & Infant Mortality Review (FIMR) project to analyze linked birth and death data. The results of this analysis indicated the need for interventions that focused on the health of women, particularly African-American women, before conception. The Coalition used this information to apply for federal Healthy Start funding under a 1999 initiative to address racial disparities in birth outcomes. This funding was used to establish The Magnolia Project, which adapts selected Healthy Start program models to at-risk African-American women of childbearing age who are not pregnant, but sexually active and likely to become pregnant. The project is implemented in a five-zipcode area of Jacksonville-Duval County that accounts for over half of all African-American infant mortality and about 30% of African-American births.\nTarget population\nThe Magnolia Project targets African-American women ages 15\u201344 living in a socioeconomic high-risk area of Jacksonville-Duval County. The project focuses on women of childbearing age who are likely to become pregnant and have some identified risk factor associated with poor birth outcomes. The project also maintains a small caseload of pregnant women (<10% of patients).\nProgram description\nThe Magnolia Project provides outreach, case management, risk reduction, support, well-woman prenatal care, health education, and community development. The project engages high-risk women through an empowerment model that promotes improved wellness and health, rather than future childbearing. Clinical services and health education are available to all eligible women in the target area. The project offers intensive case management to a subset of women who have specific risk factors identified through FIMR, including previous fetal or infant death or delivery of a LBW infant; repeated STDs; lack of family planning; substance abuse; first pregnancy before age 15; and lack of access to health care.\nThe project's intensive case management is a collaborative team approach that supports participants based on a comprehensive assessment and a client care and goal plan. The plan is developed in collaboration with each participant and reflects her choices, preferences, and goals for coordination of services and activities that address identified risk factors associated with infant mortality. Case management services include:A comprehensive assessment of each participant's abilities and needs at program entry. This assessment includes completion of a problem checklist; screening for depression and stress; and evaluation of self-esteem. Participants are re-assessed periodically based on need and progress to determine resolution or recurrence of problems and risks, achievement of personal goals, and improvements in coping skills and self-esteem.Participant care and goal plan. This plan addresses the needs and preferences of each participant. The written plan is a collaborative process involving the participant and the case manager. It addresses health and social risks and outlines specific steps that the participant must follow to improve her health and well-being and to meet her goals for social, economic, and personal development.Ongoing monitoring and service coordination. The case management team monitors services to ensure that quality care is being provided, participants are addressing their care plans, and participants who are referred to another agency follow through and receive appropriate services. The risk profile (problem checklist) and other measures (stress, self-esteem) are updated at regular intervals (at 6 and 9 months).Anticipatory guidance, health education and advocacy. Case management staff addresses participant risk factors, ensures compliance with services, and promotes achievement of personal goals. Educational support groups are organized monthly. Women receive an incentive ($50 gift certificate) for each six months that they remain in the program, provided they have made progress in addressing their goals.\nProviders\nThe clinical component of the program is staffed by a nurse-midwife and related nursing, lab and support staff. The program has a full-time health educator who provides a brief counseling intervention on key issues (e.g., use of multivitamins, nutrition, douching, and safe sex) to every woman who comes into the clinic. The clinic serves approximately 800 women annually. A team that includes a nurse, a social worker and two specially trained paraprofessionals provides intensive case management to about 75 women a year. Community development and outreach staff, as well as members of the Magnolia Project Community Council, who are indigenous neighborhood leaders, conduct outreach and community awareness activities. The project uses a collaborative, multi-agency staffing model. The Northeast Florida Healthy Start Coalition is the grantee and project administrator. Subcontractors include the Duval County Health Department (clinical care and case management) and Shands Jacksonville, a tertiary care and teaching hospital (case management and outreach). Staff is co-located at a storefront community site within walking distance of six public housing complexes. The University of North Florida Center for Community Initiatives serves as project evaluator.\nOutcome objectives\nThe project addresses all the outcome objectives required by the Maternal and Child Health Bureau of the Health Resources and Services Administration (HRSA) regarding birth outcomes (LBW, VLBW, infant mortality, adequacy of prenatal care, etc.). However, because the project primarily focuses on women who are not pregnant, it has developed specific outcome objectives that examine its success in reducing key risks associated with poor outcomes through its case management activities. These include lack of family planning and STDs. Specific outcome objectives have been developed to measure the program's success in addressing participant risks identified at program entry and closure.\nFunding\nThe Magnolia Project is primarily funded through a grant from the federal Healthy Start program ($925,000). Additional funding is provided through Medicaid and other third- party reimbursement for prenatal, family planning and STD services; state categorical funding through the health department (about $250,000 a year); and smaller community grants for special projects. Total 2004\u20132005 budget was just over $1 million.\nResults to date\nFrom 2001 to 2005, Magnolia Project clients were predominantly African-American (83%) and single (82%). The average age of participants was 25 years old. More than half (55%) had less than a high school education. Based on patient history and assessment, case management participants had an average of 7.9 risk factors or problems (current or history of) that could potentially impact a pregnancy. Seventy-two percent of all current problems were social problems (alcohol use, drug abuse, depression, family planning, housing, stress, and domestic violence). The remainder were medical problems (anemia, diabetes, hypertension, poor nutrition, previous poor outcome, STDs). The most common risk factors addressed in case management were family planning issues, education\/training, bacterial vaginosis, repeated STDs, stress, and poor nutrition. Table 1 identifies frequency of participant risks\/problems.Table 1Frequency of risks\/problems, case management participants, The Magnolia Project, July 2001\u2013May 2005\u2007Risk factor\/problemPercentage with history (n=1214)Percentage with current (n=1727)Social problems\u2003Family planning issues2643.5\u2003Job placement8.233.0\u2003Education\/training11.327.3\u2003Stress8.721.7\u2003Housing4.115.7\u2003Domestic violence7.211.3\u2003Depression8.211.1\u2003No source of care6.78.7\u2003Lack of exercise7.08.7\u2003Injury prevention\/safety2.88.0\u2003Drug abuse10.06.7\u2003Transportation4.45.9\u2003Sexual abuse7.04.4\u2003Alcohol abuse6.44.1Medical problems\u2003Bacterial vaginosis12.930.2\u2003Poor nutrition17.819.3\u2003Repeated STDs12.416.5\u2003Tobacco use13.215.5\u2003Breast health3.911.3\u2003Douching8.510.8\u2003Abnormal pap9.59.5\u2003Overweight11.69.5Source: Magnolia Project database; compiled by UNF Center for Community Initiatives, August 2005.\nTwo-hundred-forty-seven participants received at least three months of case management and were closed to care because they completed their care plans, voluntarily withdrew or were lost to service. For key risk factors, case management participants were most successful in resolving or managing issues related to domestic violence (68%) and poor nutrition (63%). The project was least successful in addressing substance abuse by participants (31%).\nIn October 2004, the HRSA Office of Performance Review conducted a site visit and assessment of Magnolia Project outcomes and activities. Its report noted that participants receiving case management services from 2001 to 2003 successfully addressed two priority risks: 86% of participants with family planning issues were consistently using a method at closure; 74% of participants with repeated STDs had no recurrent STDs at closure.\nBarriers\nInsufficient funding has limited expansion of the Magnolia Project. Existing funding streams focus primarily on pregnancy, not women's health. The Magnolia Project has successfully cobbled together categorical funding and Medicaid reimbursement for most clinical services, however, in Duval county, the delivery of most categorically funded services remains siloed (e.g., STDs, primary care, prenatal care).\nAdditionally, case management\/risk assessment is not universally accepted or valued in the medical model of health care, even though many of the risk factors associated with poor outcomes are social, rather than medical, in nature. More longitudinal evaluations are needed to determine the impact of preconception intervention on pregnancy outcomes. Longitudinal evaluations are not easily incorporated into service delivery projects that rely on short-term outcomes for continued funding. Finally, although outcomes have improved in the target area since the implementation of the Magnolia Project, infant mortality rates in the overall community remain high. The project is unlikely to affect these rates because of its comparatively small reach.\nNext steps\nThe Magnolia Project is aggressively seeking funding for a longitudinal evaluation. Current program evaluation is limited to an assessment of how well the project is achieving short-term objectives and objectives set by the funding agency (primarily focusing on birth outcomes for the small number of pregnant women served by the project). The CDC recently provided support to the Duval County Health Department for the initial design and piloting of tools for a longitudinal assessment of case management participants served by the project. Additionally, the project is working to interest local providers in replicating the intervention in other at-risk areas in Jacksonville-Duval County. Funding for replication is a significant challenge.\nDiscussion\nThe Grady IPC program and the Magnolia project use a classic public health model of care. They identify women at known risk for an adverse outcome and attempt to alter the woman's risk status to improve outcomes. These programs deliver aspects of preconception care to women at risk for poor health and pregnancy outcomes. The programs do, however, target different categories of high-risk African-American women and utilize different sites for contacting and interfacing with them. The IPC program identified women at risk based on race\/ethnicity, qualification for charity care (based on financial status and geographic residence in two counties of metropolitan Atlanta) through Grady Memorial Hospital, and a prior poor birth outcome (VLBW delivery). The IPC program enrolled women soon after their VLBW delivery and provided interconception care in the clinical setting of the hospital (with community outreach). The Magnolia project serves women at risk based on race\/ethnicity and residence in Jacksonville-Duval County, Florida. It provides preconception care in a community-based storefront setting with enhanced interconception care for women with a previous LBW delivery or a previous fetal or infant death. Access to the Magnolia project is enhanced by offering evening clinics and walk-in Wednesdays.\nBoth programs deliver services via a multidisciplinary team approach, though there are differences in the exact composition of the team. Inherent in the team approach of each program is attention to participants\u2019 physical and psychological health and social well-being.\nThe programs emphasize several comparable intervention strategies, including community outreach via lay persons; psychosocial support; group education and health promotion modeled on the Centering Pregnancy philosophy of care; and provision of health services. Both programs also emphasize the provision of family planning services for helping women achieve intendedness of any subsequently conceived pregnancy.\nEach program offers unique strategies as part of the interconception intervention. Because of the link between periodontal disease and preterm delivery (almost all VLBW deliveries are preterm), the IPC program incorporates the services of a periodontist to screen and treat women. To specifically enhance particpants\u2019 life skills, the IPC program utilizes a layperson as a Resource Mother who has undergone a tailored training program through the local department of health. A unique feature of the Magnolia Project's approach is its reliance on indigenous neighborhood leaders (e.g., Magnolia Project Community Council) and community-based organizations to provide outreach, community awareness, and education. The Magnolia Project also has established a partnership with the Ryan White III program for STD\/HIV\/AIDS screening and treatment.\nBoth programs have had success in accessing and providing services to the specific target group of women. Women who choose not to avail themselves of the services seem to have greater socioeconomic issues, such as substance abuse and lack of housing, jobs, and childcare. Due to the magnitude of some of the problems, maintaining contact is challenging and care often is not continuous.\nExisting literature supports the strategy of addressing family planning to improve pregnancy outcomes. Preterm delivery, the underlying factor responsible for most LBW and VLBW deliveries, is the leading cause of neonatal mortality and among the leading causes of infant mortality. Haas and colleagues examined whether a woman's health status and risk factors before pregnancy are associated with her risk of preterm birth, independent of risk factors that occur during pregnancy. They found that, after adjusting for sociodemographic characteristics and prepregnancy and pregnancy risk factors, women who reported poor physical function during the month before conception were nearly twice as likely to deliver preterm. The authors concluded that a broader focus on the health of women before pregnancy might improve rates of preterm birth [21].\nIn a related editorial, Goldenberg suggests that reducing smoking, depression, and stress; treating periodontal disease and genital tract infections; and reaching a normal weight might all be necessary to substantially reduce the risk of preterm birth [22]. Goldenberg advocates for a Medicaid mandate that women who have had an adverse pregnancy outcome be eligible for continued medical coverage in the postpartum period to reduce risk factors and improve health status before the next pregnancy.\nData from the 1999 PRAMS survey indicate that the prevalence of unintended pregnancies in the United States ranges from 34 to 52% with 27 to 36% of these pregnancies being mistimed, and 6 to14% being unwanted [23]. In the United States, a clear causal association between unintended pregnancy and poor pregnancy outcomes has not been established. However, women with unwanted pregnancies are at greater risk for poor pregnancy outcomes than women with wanted pregnancies. Hogue asserts that one of the key approaches to prevention of high-risk pregnancy is assuring that the pregnancy is consciously desired, with adequate attention to preconception care [24].\nBoth programs demonstrate several components essential to any successful preconception care program: identification of risk factors amenable to change by the target population; tailored interventions for the target population; integration of preconception care into existing services; incorporation of family planning counseling and clinical services, health education, and community outreach. Preconception care aims to promote the health of women of reproductive age before conception and thereby improve pregnancy-related outcomes. Both the IPC program and the Magnolia Project show promise in achieving this goal for their target audiences.","keyphrases":["preconception","interconception","family planning","birth outcomes","risk reduction"],"prmu":["P","P","P","P","P"]}
{"id":"J_Membr_Biol-2-2-1764598","title":"Structure and Function of the Hair Cell Ribbon Synapse\n","text":"Faithful information transfer at the hair cell afferent synapse requires synaptic transmission to be both reliable and temporally precise. The release of neurotransmitter must exhibit both rapid on and off kinetics to accurately follow acoustic stimuli with a periodicity of 1 ms or less. To ensure such remarkable temporal fidelity, the cochlear hair cell afferent synapse undoubtedly relies on unique cellular and molecular specializations. While the electron microscopy hallmark of the hair cell afferent synapse \u2014 the electron-dense synaptic ribbon or synaptic body \u2014 has been recognized for decades, dissection of the synapse\u2019s molecular make-up has only just begun. Recent cell physiology studies have added important insights into the synaptic mechanisms underlying fidelity and reliability of sound coding. The presence of the synaptic ribbon links afferent synapses of cochlear and vestibular hair cells to photoreceptors and bipolar neurons of the retina. This review focuses on major advances in understanding the hair cell afferent synapse molecular anatomy and function that have been achieved during the past years.\nIntroduction\nHearing relies on faithful synaptic transmission at the ribbon synapse of auditory hair cells. Figure\u00a01 shows an electron micrograph of a typical hair cell ribbon synapse. The synapse is characterized by the presence of a large presynaptic organelle, the synaptic ribbon or dense body, that tethers synaptic vesicles and is itself anchored to the active zone. The performance of this synapse is remarkable in several aspects.\nFigure\u00a01Hair cell ribbon synapse. Electron micrograph of an inner hair cell afferent synapse of an 8-week-old mouse. The ribbon (rib) is anchored to the presynaptic plasma membrane and faces the postsynaptic density (PSD) of the auditory nerve fibers. The arrowhead and the star indicate a ribbon-associated synaptic vesicle and a free cytoplasmic coated vesicle, respectively.\nPHASIC RELEASE PROPERTIES\nThe synapse\u2019s precision to code the temporal fine structure of acoustic stimuli is unparalleled. For example, our capacity to locate sound sources in space depends on interaural time differences in the arrival of sound of only tens to hundreds of microseconds. Unlike a conventional synapse that is driven by action potentials, the hair cell afferent synapse responds to graded changes in membrane potential. While the action potential results in a strong all-or-none stimulus-secretion coupling, the hair cell afferent synapse is faced with the challenge of encoding sounds of extremely different intensities and, hence, must rely on mechanisms that allow for variable stimulus-secretion coupling. At the same time, the hair cell afferent synapse must also maintain temporal fidelity. For example, the firing of an auditory nerve fiber can follow at a constant phase relationship the periodicity of tonal stimuli into the low kHz range of frequencies, and we infer that the hair cell neurotransmitter release must comply with this extraordinary timing. Such temporal precision in synaptic transmission is even observed with sub-threshold stimuli, where the temporal pattern of spike firing regularizes already before there is an overall increase in the discharge rate.\nTONIC RELEASE PROPERTIES\nAs we all can tell from our own experience, the cochlear hair cell afferent synapse must be able to encode prolonged, ever-present sounds. For example, despite adaptation, the constant background noise of the room\u2019s air handling system is faithfully reported by the peripheral auditory system. To meet this requirement of audition, sustained neurotransmitter release must be maintained and implicates the need for extensive synaptic vesicle cycling (both, vesicle exo- and endocytosis). The hair cell afferent synapse\u2019s capacity for sustained signal has often been attributed to the presence of the synaptic ribbon, and apparent depot of synaptic vesicles. However, recent morphological and physiological studies suggest additional\/different roles for the hair cell synaptic ribbon, and have fuelled the structure-function debate at the ribbon synapse.\nMolecular Anatomy and Physiology of the Hair Cell Synapse\nWe are still far from a comprehensive understanding of the molecular composition of the presynaptic active zone and the postsynaptic density of the hair cell afferent synapse. Homologies between retinal and inner ear sensory ribbon-type active zones are evident (Safieddine & Wenthold, 1999). Given the larger number of receptor cells in the retina (millions of photoreceptors vs. thousands of coding hair cells) and relative accessibility of the visual end organ, a more advanced molecular description is available presently for retinal ribbon synapses (e.g., tom Dieck et al., 2005) and promises to guide our understanding of auditory and vestibular ribbon synapses. For example, ribbons in both organs contain RIBEYE (Schmitz, Konigstorfer & Sudhof, 2000; Zenisek et al., 2003; Khimich et al., 2005), Bassoon and Piccolo (Khimich et al., 2005; tom Dieck et al., 2005). RIBEYE is thought to be a structural component of the ribbon but may also have enzymatic activity at the synapse (Schmitz et al., 2000). The photoreceptor ribbon also contains the close RIBEYE homologue and transcriptional co-repressor C-terminal binding protein 1 (tom Dieck et al., 2005). A putative role for this enzyme\u2019s lysophosphatidic acid acyl-CoA transferase activity in membrane fission has been proposed (Valente et al., 2005, but see for an opposing view Gallop, Butler & McMahon, 2005). Some hint for a ribbon function in membrane fission may be seen in the accumulation of cisternal membrane profiles in ribbon-deficient inner hair cells (IHCs) (Khimich et al., 2005). However, any hypothesized enzymatic role for this ribbon constituent in synaptic transmission will require further investigation.\nBassoon, like Piccolo, is a large cytomatrix molecule found at the active zone, and most likely contributes to the anchoring of the ribbon at the active zone (Dick et al., 2001; Dick et al., 2003; Khimich et al., 2005). Deletion of a major part of the Bassoon gene (Altrock et al., 2003) results in the lack of synapse-anchored ribbons at most active zones of mouse IHCs. Moreover, the amplitude of a readily releasable pool of IHC synaptic vesicles is decreased, and both synchronous afferent synaptic transmission and hearing are impaired (Khimich et al., 2005). In addition, a minor reduction in the IHC Ca2+ current is observed, potentially reflecting impaired insertion of Ca2+ channels at the active zone due to the Bassoon and\/or ribbon deficiency. Although less severe than the complete synaptic dysfunction resulting from the lack of CaV1.3 channels (Brandt, Striessnig & Moser, 2003), the loss of ribbons causes a synaptopathic hearing impairment with the previously described audiological signature of an auditory neuropathy (preserved cochlear amplification but impaired auditory evoked potentials). Interestingly, hearing was found intact in a Piccolo mutant (Sendin and Moser, unpublished data), but some immumoactivity was observed in the Synapses of the mutants. Perhaps the failure of the Piccolo mutation to impair hearing reflects a sufficient abundance of residual functional Piccolo protein in the mutant mouse or the action of some unknown compensatory mechanism.\nHair cells express the SNARE proteins syntaxin 1, SNAP-25 and synaptobrevin 1 (Safieddine & Wenthold, 1999). These proteins are thought to build the key exocytic machinery, as their cleavage by clostridial neurotoxins shuts off exocytosis in other systems (e.g., Xu et al., 1998; Sakaba et al., 2005). The effects of clostridial neurotoxin action on hair cell afferent synaptic transmission remain to be investigated. Hair cell afferent synapses, like photoreceptor synapses, lack synapsins (Favre et al., 1986; Safieddine & Wenthold, 1999), a class of proteins that are involved in synaptic vesicle mobilization. Moreover, synaptotagmins 1 and 2, which are considered the calcium sensors of vesicle fusion in the central nervous system and neuroendocrine cells (Sudhof, 2004), have not been observed in hair cells (Safieddine & Wenthold, 1999). Genetic ablation of Synaptotagmin 7 in mice, which is expressed in IHCs (Safieddine & Wenthold, 1999) and involved in exocytosis of hippocampal neurons (Virmani et al., 2003), did not result in a hearing impairment (Nouvian and Moser, unpublished data). Thus, taken together, these findings suggest that either other synaptotagmin isoforms or other proteins altogether may act as the calcium sensor at the hair cell afferent synapse.\nDespite some homologies, IHC ribbon synapses differ molecularly from retinal ribbon synapses. Most prominently, IHC transmitter release and sound coding are controlled by CaV1.3 Ca2+ channels (Platzer et al., 2000; Brandt et al., 2003; Brandt, Khimich & Moser, 2005), while visual signalling relies on stimulus-secretion coupling by retinal CaV1.4 channels (Bech-Hansen et al., 1998; Strom et al., 1998; Mansergh et al., 2005). Some vestibular hair cells, in addition, may involve non-L-type Ca2+ channels (Rodriguez-Contreras & Yamoah, 2001). Evidence obtained by various techniques in various species all suggests that CaV1.3 channels cluster at the ribbon-type active zones of the hair cell. These include loose-patch recordings (Roberts, Jacobs & Hudspeth, 1990), cell-attached recordings (Rodriguez-Contreras & Yamoah, 2001), Ca2+ imaging (e.g., Issa & Hudspeth, 1994; Tucker & Fettiplace, 1995; Zenisek et al., 2003) and immunohistochemistry (Sidi et al., 2004; Brandt et al., 2005). Estimates of the total number of Ca2+ channels per active zone have been obtained in frog saccular hair cells (\u223c90 channels, Roberts et al., 1990; but see alsoRodriguez-Contreras & Yamoah, 2001) and mouse cochlear IHCs (\u223c80, Brandt et al., 2005) by ensemble fluctuation analysis. This similarity in channel number is unexpected given the size differences between the active zones of the two species (see below and Table\u00a01) and highlights how much more we have to learn about this intriguing synapse.\nTable\u00a01Size and kinetics of release components related to the synaptic ribbon and to morphological vessel pools associated from different species\nClassical work by Robert\u2019s and Fettiplace\u2019s groups on intracellular calcium dynamics at the plasmalemma face of hair cells advocated the view that calcium domains were micromolar in concentration and micrometer in dimension. This work was based largely on computational models and studies of calcium-activated potassium channel gating, but was assumed to be also applicable to hair cell afferent synapse exocytosis. However, more recent experiments on the stimulus-secretion coupling in mouse IHCs suggested that exocytic fusion of a given readily releasable vesicle is controlled only by few nearby Ca2+ channels (Brandt et al., 2005). These authors argued that such a \u201cnanodomain\u201d control would support the temporal precision of coding also for weak sounds. Additional experiments using complimentary techniques are needed to further test this provocative hypothesis.\nRim binding protein, interacting with the active zone protein RIM and the CaV1.3 channel, is currently the best characterized candidate for a molecular link of channel and release site of a given vesicle (Hibino et al., 2002). However, functional evidence is still lacking and it remains possible that other proteins, e.g., scaffolding proteins of the cytomatrix of active zones like Bassoon or Piccolo, contribute. It is also possible that the synaptic ribbon itself is responsible for positioning of the CaV1.3 channel and readily releasable vesicles (Khimich et al., 2005). Clearly, molecular work, physiological analysis of mutants and particular immunoelectronmicroscopy are required to characterize the molecular interactions and the ultrastructural topography at the active zone. Figure\u00a02 illustrates the subcellular localization of a few synaptic proteins.\nFigure\u00a02Molecular composition of the hair cell synapse. (A) Montage of a Nomarski image (note the hair cell bundles) and a confocal reconstruction of the mouse organ of Corti with immunolabeled ribbons (stained for RIBEYE\/CtBP2, red) and postsynaptic transmitter receptor clusters (GluR2\/3 glutamate receptor subtypes, green). Presumptive hair cell border (white) and postsynaptic fibers (orange) contacting an IHC were drawn illustration. (B) Previously identified molecular determinants of the hair cell ribbon synapse. 1Safieddine & Wenthold, 1999; 2Furness & Lawton, 2003; 3Eybalin et al., 2002; 4Schmitz et al., 2006; 5Khimich et al., 2005; 6Platzer et al., 2000; 7Brandt, Striessnig & Moser, 2003; 8Brandt, Khimich & Moser, 2005; 9Matsubara et al., 1996; 10Eybalin et al., 2004.\nBesides a molecular coupling, other mechanisms contribute to localized Ca2+ signalling. The presence of the mobile, proteinaceous Ca2+ buffers calretinin, calbindin and parvalbumin has now been documented in a variety of cochlear and vestibular hair cells (e.g., Edmonds et al., 2000; Heller et al., 2002; Hackney et al., 2005). Their relevance for presynaptic Ca2+ signalling has been inferred from recordings of Ca2+-activated large-conductance K+ channels (Fettiplace, 1992; Roberts, 1993; Tucker & Fettiplace, 1996; Edmonds et al., 2000), simulations (Roberts, 1994) and capacitance measurements (Moser & Beutner, 2000; Spassova et al., 2004). Most likely they spatiotemporally restrict the presynaptic Ca2+ domains and, hence, improve the timing of synaptic transmission. However, discrepancies regarding both the amount and kinetic properties of buffers in different hair cells (Roberts, 1993; Edmonds et al., 2000 vs Moser & Beutner, 2000) suggest that our understanding of calcium buffers at the hair cell active zone synapse is far from complete. Examination of hair cells from mutant mice lacking the major proteinaceous Ca2+ buffers promises further insight into this important question.\nIn addition to synchronous synaptic transmission, the molecular machinery needs to be suited for long-lasting transmitter release. By yet unknown mechanisms CaV1.3 currents in hair cells are much less inactivating than in heterologous expression systems (Koschak et al., 2001). Most likely this is due to interactions with a special combination of auxiliary subunits and proteins, such as the class of Ca2+ binding proteins (Haeseleer et al., 2004). None of these auxiliary determinants of hair cell Ca2+ current are known so far.\nOnce the neurotransmitter is released into the synaptic cleft, it activates an AMPA-like receptor residing within a receptor cluster at the postsynaptic density of afferent auditory nerve fibers. In a classical study, Ottersen and his colleagues showed the abundance of the glutamate receptor subtypes (GluR) 2, 3 and 4 by immunogold labelling (Matsubara et al., 1996). Interestingly, the distribution of the glutamate receptor was not homogenous in the postsynaptic density (PSD): the receptor numbers increase at the border of the PSD. Using patch-clamp recordings from the postsynaptic endings contacting rat IHCs, Glowatzki & Fuchs (2002) showed that excitatory postsynaptic currents of spiral ganglion neuron afferent fibres are mediated by AMPA receptors. They failed to detect NMDA or Kainate currents. Recently, Eybalin and colleagues studied the developmental expression changes of postsynaptic glutamate receptors using western blotting, RT-PCR and immunohistochemistry (Eybalin et al., 2004). They showed that GluR2 becomes expressed only around the onset of hearing, while GluR3 and 4 are present before. Little is known about the other molecular determinants of the PSD. Davies et al. (2001) report the presence of PSD-95 and PSD-93 in the PSD of auditory nerve fibres.\nTo avoid saturation and excitotoxicity due to an excess of glutamate in the synaptic cleft, glutamate is taken up by glutamate transporters (EAAT) into supporting cell transporters (Furness & Lehre, 1997; Furness & Lawton, 2003; Rebillard et al., 2003). Interestingly, glutamate-aspartate transporters (GLA ST) are more abundant on the pillar IHC side, where high spontaneous rate fibers are located, compared to the modiolar cell side, where low spontaneous rate fibres are located (Liberman, 1982; Furness & Lawton, 2003).\nRelating Structural and Functional Vesicle Populations\nMORPHOLOGY\nSynaptic Ribbons\nThe active zone of the hair cell is characterized by an electron dense structure called synaptic body or ribbon. Most synaptic ribbons found in mature hair cells are anchored to the plasma membrane, one ribbon per active zone, and oppose a postsynaptic terminal. A small number of \u201cfloating\u201d ribbons (<5 %) are observed, and probably reflect the turnover of these subcellular organelles (Zenisek et al., 2004; Khimich et al., 2005). Shape, size and number of the ribbons differ (see Table\u00a01) between species as well as along the tonotopic axis, during development (Shnerson, Devigne & Pujol, 1981; Sobkowicz et al., 1982; Khimich et al., 2005) and even among different active zones within a single hair cell (Merchan-Perez & Liberman, 1996). For example, frog saccular hair cells display about 20 large, synaptic bodies irrespective of the cells\u2019 location in the end organ (Roberts et al., 1990; Lenzi et al., 1999, 2002). Whereas in the chick cochlea, the number of synaptic bodies is constant in tall hair cells (functional homologue of the mammalian IHCs), the synaptic body diameter increases with increasing characteristic frequency (Martinez-Dunst, Michaels & Fuchs, 1997). Conversely, the number of ribbons varies along the tonotopic axis in many other species including turtle (plus small size differences: Sneary, 1988; Schnee et al., 2005), cat (Liberman, Dodds & Pierce, 1990), gerbil (Slepecky et al., 2000) and guinea pig (Hashimoto, Kimura & Takasaka, 1990).\nBoth the number and shape of the synaptic ribbons can also change with the onset of hearing. Mouse IHCs contain a large number (>20 in the apical cochlea, Nemzou et al., unpublished data) of small synaptic bodies (Shnerson et al., 1981; Sobkowicz et al., 1982) before the onset of hearing. After the onset of hearing only fewer but larger ellipsoid ribbons are observed (Shnerson et al., 1981; Sobkowicz et al., 1982; Francis et al., 2004; Khimich et al., 2005). An interesting feature described for cat IHCs is the spatial segregation of synapses with fibres of different spontaneous rates and thresholds. Thus, low-spontaneous-rate, high-threshold fibres contact the IHCs on their modiolar side, whereas high-spontaneous-rate, low-threshold fibres synapse on the pillar IHC side. EM analysis showed that the ribbon size and accordingly the number of ribbon-associated synaptic vesicles tend to increase with decreasing spontaneous rate of the contacted fibres (Liberman, 1980; Liberman et al., 1990; Merchan-Perez & Liberman, 1996).\nSynaptic vesicles\nRibbons tether a monolayer of synaptic vesicles at a packing density that is \u223c55% of maximum by 20 nm long filaments (Lenzi et al., 1999). These ribbon-associated vesicles represent only a tiny fraction of the huge number of synaptic vesicles contained in a hair cell. At least three vesicle populations can be morphologically distinguished at the hair cell ribbon synapse as illustrated by Figure\u00a03:Docked vesicles (no discernible space between vesicle and plasma membranes)Ribbon-associated vesiclesFree cytosolic synaptic vesicles.\nThese vesicle populations have been quantified for hair cells of various species (Table\u00a01). A variety of approaches have been used to assess the vesicle populations and accordingly, varying degrees of certainty surround these vesicle counts. The different approaches have included: precise three-dimensional EM counts based on few synapses (EM tomography: Fig\u00a03, Lenzi et al., 1999; Lenzi et al., 2002; reconstructions based on ultrathin serial sections: Schnee et al., 2005); extrapolations from random ultrathin EM sections of tens of synapses (Khimich et al., 2005) and approximations based on ribbon surface areas and vesicle packing density estimates (Spassova et al., 2004; Khimich et al., 2005: more than 200 ribbons analyzed).\nFigure\u00a03Morphology of synaptic ribbons. (a, b and c) Electron micrographs of inner hair cell ribbon synapses of 8-week-old mouse. Ribbons are attached to the plasma membrane and surrounded by a monolayer of synaptic vesicles. Colored dots in (b) illustrate the morphologically defined classes of synaptic vesicles: red dots indicate the docked vesicle associated with the ribbon; yellow marks ribbon- associated SV which are not docked and green indicates outlying cytoplasmic vesicles. (d) Slice through an osmium-stained frog saccular hair cell synapse, reconstructed by electron tomography. Note the round shape of the synaptic body in contrast to the elipsoid one of the mouse. (e) Three-dimensional structure of presynaptic organelles from the same reconstruction shown in (d). The synaptic body (blue) is open and lies adjacent to the hair cell\u2019s plasma membrane in red. Synaptic body-associated vesicles (yellow) surround the SB; outlying vesicles (green) lie further out in the cytoplasm. Also visible are coated vesicles (gold) and cisterns (purple).\nDocked vesicles at a conventional synapse are viewed as a homogeneous anatomical pool. However, at the hair cell ribbon synapse docked vesicles are found in a variety of anatomical states; this includes underneath the ribbon (docked and ribbon-associated), at the active zone without contact to the ribbon, and outside the active zone. Lenzi et al., (1999) termed these last two classes of docked vesicles as \u201coutlying\u201d. The functional implications of the different classes of docked vesicles are poorly understood. Obviously, proximity to a calcium channel increases the likelihood that a docked vesicle is of functional significance. Accordingly, Lenzi et al., (1999) attempted to correlate the positions of docked vesicles and Ca2+ channels at the active zone. While the \u201coutlying\u201d vesicles are substantial in number, they are located further from the presumed calcium channel containing presynaptic densities than are other docked vesicles. \u201cOutliers\u201d has been alternatively used in evanescent wave microscopic studies (Zenisek, Steyer & Almers, 2000) to describe the \u201cdocking\u201d and fusion of synaptic vesicles outside of functionally defined active zones.\nPHYSIOLOGY\nOne fruitful way to investigate the compact afferent presynaptic terminal of the inner ear is to monitor exocytic fusion and endocytic retrieval of synaptic vesicle membrane as changes of the membrane capacitance (Cm) using the patch-clamp technique (for review seeNeher, 1998). During the fusion of the synaptic vesicle to the plasma membrane the cell surface and consequently Cm increase. This then is usually followed by slower fission accompanying Cm decrease. This technique has become increasingly popular to study the presynaptic function of hair cells in various species (Table\u00a01). Upon depolarization, calcium influx, mainly through CaV1.3 L-type Ca2+ channels, triggers exocytosis of readily releasable synaptic vesicles. Depending on the stimulus intensity and duration, the resulting Cm increase may report exocytosis occurring at all active zones as well as the fusion of synaptic vesicles outside of active zones or even the fusion of non-synaptic membrane organelles (Coorssen, Schmitt & Almers, 1996). This lack of specificity associated with capacitance measurements can complicate its interpretation.\nAt least two kinetic components of exocytosis are commonly discriminated in the recordings of hair cell membrane capacitance following stimuli thought to trigger exocytosis. Such findings from a variety of species are also summarized in Table\u00a01.\nThe first kinetic component of these membrane capacitance increases mediates exocytosis at extremely high rates but saturates within a few milliseconds. Hence, it is generally agreed that this component represents exocytosis of a small, finite pool of vesicles. Because it is the fastest discernible and exhaustible component of exocytosis several authors denominated it the readily releasable vesicle pool (RRP, Moser & Beutner, 2000; Spassova et al., 2004; Rutherford & Roberts, 2006) following the classical terminology of functional vesicle pools (Liley & North, 1953; Birks & MacIntosh 1961; Elmqvist & Quastel, 1965). Three major findings indicate that the first kinetic component is, indeed, involved in synaptic sound-coding by hair cells:Correlation between the RRP size and the number of afferent synapses made by a hair cell as it varies along the tonotopic axis of the cochlea (Schnee et al., 2005).Correlation between the RRP size and the number of synapse-anchored ribbons (Khimich et al., 2005).Correlation between the RRP size and the amplitude of the spiral ganglion compound action potential (reflecting synchronous activation of spiral ganglion neurons) (Khimich et al., 2005).\nSeveral other lines of evidence support the notion that the first kinetic component of exocytosis represents a synaptic relative pool of vesicles. Resistance of the readily releasable pool to inhibition by exogenous slow calcium buffers infers a co-localization of this functional pool with Ca2+ channels (Moser & Beutner, 2000; Spassova et al., 2004). Calcium channels are widely believed to be concentrated at hair cell active zones (e.g., Roberts et al., 1990; Issa & Hudspeth, 1994; Tucker & Fettiplace, 1995; Zenisek et al., 2003; Sidi et al., 2004; Brandt et al., 2005). Nanodomain control of synaptic vesicle exocytosis (Brandt et al., 2005) requires an intimate spatial relationship between the readily releasable pool and calcium channels. There is also good correspondence between the quantal content predicted by measurement of action potential-driven exocytic Cm changes in immature IHCs (Beutner & Moser, 2001; Johnson, Marcotti & Kros, 2005) and direct measures of postsynaptic currents from the afferent fiber (Glowatzki & Fuchs, 2002). Finally, additional supportive evidence comes from comparison of the timing of peripheral auditory adaptation and the kinetics of RRP depletion and recovery.\nAdaptation is a common property of sensory systems. It reduces encoding redundancy by decreasing the sensitivity to constant stimulation, is proposed to optimize a sensory system\u2019s operating range (Koutalos & Yau, 1996) and maximize its information transfer (Brenner, Bialek & de Ruyter van Steveninck, 2000). In the peripheral auditory system, adaptation is likely localized to the cochlear hair cell afferent synapse because hair cell receptor potentials do not adapt to maintained acoustic stimulation (Holton & Weiss, 1983; Russell & Sellick, 1978). Previous work (Furukawa & Matsuura, 1978; Moser & Beutner, 2000) suggested that the exhaustion and replenishment of the readily releasable pool of vesicles in the hair cell contribute to a fast form of adaptation and recovery from it in the auditory nerve (Westerman & Smith, 1984; Yates, Robertson & Johnstone, 1985).\nParsons and colleagues (Spassova et al., 2004) employed both in vitro patch-clamp recordings of hair cell synaptic vesicle fusion and in vivo single-unit recording of cochlear nerve activity at the same synapse to further examine this hypothesis and probe the possible auditory significance of the hair cell ribbon synapse structure-function relationship. Similar to other hair cell preparations, exocytosis of the chick cochlear tall cell\u2019s readily releasable pool is fast, saturating in < 50 ms, and its recovery is also rapid, regaining 95% of its initial amplitude following a 200 ms period of repolarization. Interestingly, sound-evoked afferent synaptic activity also adapted and recovered with similar time courses as readily releasable pool exhaustion and recovery. This work supports the view that the fast exocytic component is of synaptic relevance by providing evidence that exhaustion and replenishment of the readily releasable pool mediates behaviorally relevant phenomena \u2014 namely short-term auditory nerve adaptation and its recovery from adaptation.\nIn an elegant two-photon imaging study Ashmore and colleagues utilized the turnover of the membrane dye FM1-43 to track the exocytosis of synaptic vesicles at hair cell active zones. Their findings on the readily releasable pool of the adult guinea pig IHCs were similar to descriptions provided in other species by capacitance measurements. The significance of these findings is that the imaging experiments, unlike the capacitance measurements, are spatially resolved and lend credence to previous interpretations of the first exocytic component. Optically recorded exocytosis was extremely fast at the onset of a depolarization (initial rate of 3,000 vesicles per second) and saturated with a time constant of 30 ms in mature guinea pig hair cells (Griesinger, Richards & Ashmore, 2005). This intial rate is comparable to the initial rate of exocytosis evoked at one ribbon synapse of mature mouse IHCs by maximal stimulation amounting to \u223c6,000 vesicles per second (after Cm conversion into vesicle numbers using 28 aF\/vesicle). Moreover, estimates of the time course of recovery from depletion obtained by the two methods were quite similar, too (Table\u00a01).\nBoth the amplitude and kinetics of the first kinetic component in mature mouse and guinea-pig hair cells were found to depend on the stimulus strength (Brandt et al., 2005; Griesinger et al., 2005). This is in contrast to a study on frog saccular hair cell exocytosis where the kinetics, but not amplitude of exocytosis, varied with stimulus amplitude (Edmonds, Gregory & Schweizer, 2004). The reasons for this discrepancy among the different experiments are currently unclear. Moser and colleagues have suggested the Ca2+ nanodomain control of RRP exocytosis in mouse IHCs (Brandt et al., 2005). This hypothesis predicts that an increase in intensity of stimuli would recruit more calcium channel-release site units, presumably increasing the amplitude of the RRP without major changes of release kinetics. Classical quantal analysis at the goldfish hair cell synapse (Furukawa, Kuno & Matsuura, 1982) also revealed intensity-dependent increases in the number of available vesicles (as opposed to changes in the release probability). While the synaptic relevance of the first kinetic component is indisputable, clearly a detailed mechanistic understanding of it awaits further experimentation.\nOur current knowledge about the further kinetic component (s) is much more limited. This is mainly due to the lack of spatial resolution of Cm recordings of exocytosis, which report fusion to the plasma membrane no matter whether it happens at the synapse or elsewhere. The slower kinetic components are typically triggered by prolonged, strong depolarizing steps (tens of ms to s in duration) and have the potential to drive calcium levels within the cell to levels that could not be achieved with more physiological time-varying, smaller-amplitude stimuli. The relative resistance of the CaV1.3 calcium channel to inactivation allows for the influx of large numbers of calcium ions during prolonged depolarizations that may be sufficient to saturate cellular calcium buffers. Clearly, aphysiologic calcium gradients within the hair cell promise to further compound the limitations of membrane capacitance measurements and cloud the interpretation of secondary, slower exocytic components.\nSince the first Cm measurements of hair cell exocytosis (Parsons et al., 1994), we have been astonished and puzzled by the huge amount of membrane turnover during prolonged depolarization. During the first second of depolarization thousands of vesicles are released (see Table\u00a01). A membrane turnover of more than one equivalent of the initial hair cell\u2019s surface has been reported for maximal stimulation over several seconds (Schnee et al., 2005). In this respect, the hair cell\u2019s exocytic capacity exceeds that of retinal photoreceptors and bipolar neurons. This difference between retinal cells and hair cells is most dramatic when examining the fast exocytosis evoked by Ca2+ uncaging. A uniform step increase in calcium concentration through the cell\u2019s cytoplasm triggers the exocytosis of all fusion-competent vesicles. Flash photolysis of caged calcium in the goldfish bipolar nerve terminal results in an average capacitance increase of \u223c150 fF (Heidelberger et al., 1994), whereas a similar stimulus in the mouse IHC drives a 10-fold larger increase in membrane capacitance (Beutner et al., 2001). This discrepancy in the amount of exocytosis is even more remarkable given that a hair cell contains nearly 4 times fewer, but similar-sized and vesicle-populated synaptic ribbon active zones as the retinal bipolar terminal (von Gersdorff et al., 1996; Khimich et al., 2005). Hence we have to either assume that the turnover at a hair cell ribbon synapse is 40 times faster than that of a bipolar neuron or to conclude that hair cells contain an extremely large pool of fusion-competent vesicles that reside outside of the active zone. Spatially-resolved exocytic measurements, such as imaging or conventional post-synaptic recordings, will be required to ascertain the contribution of the hair cell\u2019s large pool of fusigenic vesicles to synaptic relevant exocytosis.\nIs it Possible at all to Relate Anatomically and Physiologically Defined Pools of Synaptic Vesicles?\nIt would be a significant breakthrough in our understanding of the hair cell ribbon synapse if we managed to arrive at a joint anatomical and physiological definition of synaptic vesicle populations. This goal requires relating physiologically derived capacitance or fluorescence changes to anatomically defined synaptic vesicle numbers. Unfortunately, several technical issues limit the accuracy and certainty of this pursuit. Estimations of single-vesicle capacitance are required to determine the equivalent number of vesicles associated with a given Cm increase. Surface calculations based on the vesicle diameter in fixed tissue (possibly correcting for a potential shrinkage bias) and an assumption of the specific membrane capacitance (usually 10\u00a0\u03bcF\/\u03bcm2, Breckenridge & Almers, 1987) are commonly employed. Both are prone to errors complicating this structure-function approach. The inherent problems with the fixation artefacts are highlighted by the factor of 2 discrepancy between \u201cin vitro\u201d single-vesicle capacitance values derived from anatomical studies (37 aF, Lenzi et al., 1999 and 28 aF, Khimich et al., 2005) and the more direct \u201cin vivo\u201d estimates obtained by electrophysiology in other systems (\u223c50 aF, Klyachko & Jackson, 2002 and 65 aF, Sun, Wu & Wu, 2002).\nThe physiological approach to pool definition also faces technical limitations. The rate of the first or fastest kinetic component is limited by the bandwidth and signal-to-noise ratio of the recording equipment. Much confusion in the retinal bipolar terminal literature stems from the initial definition of a readily releasable pool with a time constant of \u223c100 ms (von Gersdorff & Matthews, 1994) prior to the subsequent discovery of a 5-fold smaller, but nearly 50-fold faster kinetic component of exocytosis (Mennerick & Matthews, 1996; Neves & Lagnado, 1999). At the chick hair cell synapse, which features some of the smallest-numbered and -sized synaptic ribbons, the total number of docked vesicles is \u223c100. Exocytosis of these vesicles would generate a capacitance increase perhaps as small as 3 fF, at or below the resolution of whole-cell capacitance measures and likely obscuring the observation of this pool that is readily measured in cells with large ribbon structures.\nWhat Is the Anatomical Substrate of the Fast Kinetic Component in Hair Cell Exocytosis?\nSeveral caveats described above constrain our ability to answer this question. However, recognizing these limitations and uncertainties, it remains an important and interesting question underlying our understanding of the hair cell afferent synapse. The recent literature provides two alternative hypotheses: it may preferentially draw on readily releasable vesicles docked at the plasma membrane of the active zone (Moser & Beutner, 2000; Khimich et al., 2005; Schnee et al., 2005; Rutherford & Roberts, 2006) or involve release of all ribbon-associated vesicles (Edmonds et al., 2004; Spassova et al., 2004). Table\u00a01 highlights these views. The first hypothesis is consistent with independent fusion of the individual vesicles, similar to what is expected for docked vesicles at the active zone of conventional synapses. The second hypothesis requires the rapid recruitment and fusion of non-docked ribbon-associated vesicles. As active vesicle transport seems too slow (see discussion in Edmonds et al., 2004), different modes of exocytosis, such as compound and \u201cpiggy back\u201d (cumulative) fusion, have been considered (Parsons & Sterling, 2003; Edmonds et al., 2004; Spassova et al., 2004). Functional evidence for multivesicular release at the hair cell ribbon synapse was first obtained by Glowatzki and Fuchs (2002). Using direct post-synaptic recordings at rat afferent synapses, they observed large variations in the amplitude of kinetically quite homogeneous postsynaptic currents, as well as clear composite events characterized by multiple quanta (Glowatzki & Fuchs, 2002). Clearly, the exact presynaptic release mechanism of vesicle release is likely to have great impact on the transfer characteristics of the hair cell synapse and remains an extremely important problem for continued study.\nThis review cannot solve the debate over the anatomical basis of the hair cell fast exocytic component. However, we aim to fuel the on-going discussion with some selected comparisons of data complied from various studies and summarized in Table\u00a01. Any considerations of structure-function comparisons are complicated by the fact that the physiological pool size estimates obtained by different groups can vary substantially even for hair cells of the same species (Edmonds et al., 2004; Rutherford & Roberts, 2006, see discussion in Rutherford\u2019 paper). Moreover, the morphological vesicle counts or estimations vary dramatically in their degree of precision (from direct counts in electron microscopical 3-D reconstructions to approximations based on ribbon size, see Morphology section above and Table\u00a01). In addition, we need to remember that our capacitance-based approximations probably overestimate the number of vesicles due to underestimation of single-vesicle capacitances (due to the shrinkage during chemical fixation, see above). With these caveats in mind, studies on hair cells of the frog (work from the Roberts lab) and the mouse (work from the Moser lab) provide the most compelling structure-function correlations, in part because both the anatomy and physiology were carried out in the same laboratory. In each case, their observations support the hypothesis that for these particular hair cells RRP is defined by the fusion of docked vesicles (Table\u00a01). Correcting the conversion factor for shrinkage would tend to still improve the match between RRP size and docked vesicle number.\nThe question still lingers as to whether non-docked vesicles can be released rapidly at ribbon synapses. Zenisek et al. (2000), using evanescent wave microscopy of single FM1-43 stained vesicles in retinal bipolar nerve terminals, provide some of the most direct evidence addressing this question. They reported that the first component of exocytosis was dominated by \u201cresident\u201d (probably docked) vesicles at the hot-spots of exocytosis, whereas arrivals or \u201cnewcomers\u201d contributed only marginally within the first milliseconds. This argues against a major contribution of non-docked, ribbon-associated vesicles. On the other hand, Griesinger et al. (2005) concluded from two-photon imaging of basolateral hot-spots of FM1-43 fluorescence that rapid exocytosis of non-docked vesicles does, indeed, occur at hair cell ribbon synapses. A certain degree of caution, however, is required in interpreting either of these studies, as the best resolution of an optical microscope (200 nm) is comparable to the size of most synaptic ribbons.\nThe apparent differences between the behavior of ribbon-tethered vesicles in these two types of ribbon synapses highlight the fact that it could be an oversimplification to assume that a common anatomical substrate underlies the first\/fastest recorded kinetic component of hair cell exocytosis. The physical dimensions of hair cell active zones, shapes of synaptic ribbons, best stimulation frequency, and possibly endogenous calcium buffering vary dramatically across end organ, species and in some cases even within the same end organ of a given species. Thus, subsequent studies may reveal that different hair cells use different anatomical substrates for different functional pools of synaptic vesicles to meet their respective synaptic demands.\nWhat about the Anatomical Substrate of Subsequent, Slower Kinetic Components of Exocytosis?\nThings are still more complicated for the further kinetic component(s) of exocytosis reported by capacitance measurements. Most studies concur that replenishment of readily releasable vesicles occurs at high rates, exceeding those of conventional synapses and even retinal ribbon synapses (Moser & Beutner, 2000; Spassova et al., 2004; Edmonds et al., 2004; Griesinger et al., 2005). However, whether the slow kinetic components of exocytosis simply represent the serial refilling of the readily releasable pool and or parallel exocytosis at \u201cectopic\u201d fusion sites is debatable. Schnee et al. (2005) proposed a purely serial and synaptic model to explain the three kinetic components of the depolarization-induced exocytic Cm changes in turtle hair cells. They inferred that the second component reflected exocytic turnover of the ribbon-associated vesicle population, while vesicle recruitment to the ribbon would become rate-limiting thereafter. Not surprisingly, a similar model was appropriate to describe destaining of FM1-43 hotspots and recovery of staining in the study of Griesinger and colleagues. However, these optical studies differ from the capacitance recordings in the turtle hair cell (Schnee et al., 2005), as they are spatially resolved and focus on membrane turnover at individual active zones.\nFindings that argue for a significant contribution of ribbon-independent and at least in part parallel extrasynaptic exocytosis to sustained exocytic Cm changes include:Robust sustained exocytosis in hair cells that lack 90% of their synapse-anchored ribbons (Khimich et al., 2005)Presence of docked synaptic vesicles at extrasynaptic stretches of the plasmamembrane (Lenzi et al., 1999, 2002)The extensive fast exocytosis during flash photollysis, which by far exceeds fusion of the readily releasable pool (Beutner et al., 2001), potentially arguing for the presence of many fusion-competent vesicles outside the active zone.\nIn addition, exocytosis of such \u201coutlying\u201d vesicles has been described in retinal bipolar nerve terminals by evanescent microscopy (Zenisek et al., 2000). Hence, taken together, present evidence supports the interpretation that sustained hair cell exocytosis reported by Cm tracking represents both the serial re-supply of vesicles to the active zones and the parallel extrasynaptic turnover of synaptic vesicles. However, the quantitative contribution of the slow exocytic components to synaptically relevant neurotransmitter release remains to be determined.\nConclusion and Outlook\nThe multidisciplinary approach to the hair cell ribbon synapse has for the first time quantitatively described important aspects of the synapse\u2019s structure and function. Most importantly, despite all limiting uncertainty, we begin to relate molecules, structure and function. Although still debated, the concept of a readily releasable pool has been substantiated for the ribbon synapse of several hair cells. We have gained a few insights into the mechanisms underlying the incredible temporal precision of synapses that participate in the coding of sound. Moreover, in addition to learning about the molecular physiology, the availability of mouse mutants with defined synaptic lesions provided the possibility to study the properties of synaptopathic hearing impairment in greater detail. However, we are far from a comprehensive molecularly defined model of ribbon structure and function. The molecular dissection of the hair cell synapse is technically challenging due to the low amount of tissue and will require much more time and effort. Ear-specific genetic deletion also will be helpful to investigate synaptic protein function in hair cell sound coding. A more precise and direct biophysical analysis of single hair cell synapses will require combined pre- and postsynaptic recordings as well as optical measurements, such as evanescent wave microscopy and confocal techniques. The optical approach will be strongly facilitated by the generation of genetically targeted fluorescent vesicle tags.","keyphrases":["hair cell","hair cell","ribbon synapse","synaptic ribbon","exocytosis","calcium","synaptic protein","synaptic vesicle pool"],"prmu":["P","P","P","P","P","P","P","R"]}
{"id":"Soc_Psychiatry_Psychiatr_Epidemiol-2-2-1764203","title":"Characteristics associated with involuntary versus voluntary legal status at admission and discharge among psychiatric inpatients\n","text":"Background The objective of this analysis was to determine the ways in which patients\u2019 legal statuses at hospital admission and discharge are associated with select sociodemographic and clinical variables. This study specifically investigated differences between patients who were voluntary during both admission and discharge, patients who were involuntary on admission but voluntary on discharge (having converted to voluntary status during hospitalization), and patients who were involuntary during both admission and discharge.\nIntroduction\nIn the United States, criteria for involuntary psychiatric hospitalization generally require imminent dangerousness to oneself or others, or complete inability to care for oneself [1, 2], though specific criteria vary between states [3]. An important rationale for involuntary psychiatric hospitalization is that patients admitted for evaluation often have severe deficits in their capacities to make treatment decisions [4, 5]. Patients often revise their beliefs about the need for treatment during or after hospitalization. A study conducted by Gardner et al. [6] found that slightly more than half of patients who initially stated (upon admission) that they did not need hospitalization later admitted they indeed had needed treatment when re-interviewed several weeks after discharge.\nIt has been suggested that involuntary treatment may reduce the likelihood that patients will voluntarily seek care in the future [7, 8]. Subsequent aversion to psychiatric services may be due to a distrust of clinicians arising from coercive tactics or a lack of procedural justice (e.g., the patient is treated unfairly, disrespected, ignored, or excluded from the decision-making process) encountered during hospitalization [1, 8, 9]. On the other hand, involuntary hospitalization may provide patients with needed treatment, even if involuntarily, that results in improved outcomes and treatment adherence [10]. Some research indicates that the presence or absence of coercion is accurately reflected by the patient\u2019s legal status, but other studies demonstrate that coercive pressures and perceived coercion do not necessarily correlate well with legal status in all settings [8, 11].\nEvidence associating legal status with sociodemographic and clinical variables is sparse and inconsistent. One study found that higher re-admission rates and more unstable discharge living arrangements were associated with involuntary legal status on hospital admission [12]. A meta-analytic review conducted using studies from the 1950s through the early 1980s found that the only significant outcome measure correlated with admission legal status was length of stay [13]. Studies have reached opposing conclusions regarding whether or not male gender is significantly associated with involuntary legal status, especially when controlling for severity of illness [7, 12, 14]. With regard to clinical characteristics, psychotic disorder diagnoses, greater illness severity, and more frequent preadmission assaultive behaviors may be associated with involuntary admission [13]. Even fewer studies have examined factors associated with converting from involuntary to voluntary legal status during hospitalization [14\u201316]. Cuffel [14] identified greater clinical improvement, less severe diagnoses, and non-minority ethnicity as factors predictive of converting to voluntary status early in the hospitalization. Later during hospitalization, the only factor associated with legal status conversion was having discharge living arrangements involving family and friends.\nStudies that find significant associations between legal status and outcomes may influence policies relating to resource allocation during hospitalization, discharge planning, and outpatient follow-up [12, 15]. For instance, research characterizing those at risk for very short involuntary hospitalization (i.e., discharge upon expiration of short-term commitment) addresses the need for more thorough and engaged evaluation and treatment while hospitalized. One may be able to enhance treatment adherence with a more focused treatment and evaluation plan, in conjunction with closer follow-up after discharge [12]. Though legal status is often overlooked in studies in which it would be particularly relevant, it is clearly an important variable for further research.\nThe purpose of the present study was to compare three groups of hospitalized psychiatric patients: (1) patients admitted and discharged voluntarily (VOL-VOL), (2) patients admitted involuntarily who later signed-in voluntarily and were discharged voluntarily (INV-VOL), and (3) patients admitted and discharged on an involuntary legal status (INV-INV). As such, this trichotomous criterion variable addressed the change (or lack thereof) in patients\u2019 legal status during hospitalization, allowing an examination of whether or not certain sociodemographic and clinical factors are associated with differences in admission-to-discharge legal status. Treating legal status as a trichotomous variable (rather than solely considering either admission or discharge legal status) may yield meaningful results that add to the current body of research on correlates of patients\u2019 legal status.\nMethods\nSubjects and setting\nThis analysis was part of a study aimed at determining predictors of non-adherence with the first scheduled community mental health outpatient appointment following hospitalization [17]. Inclusion in the study was restricted to consecutively discharged patients with a scheduled follow-up appointment at any of 12 participating community mental health settings in a single urban county. Patients re-hospitalized during the course of data collection were not included a second time, in order to maintain independence of observations. This analysis included data that were collected on 227 patients from December 2003 to July 2004.\nAll patients were admitted to the 22-bed inpatient unit or the 8-bed crisis stabilization unit of a large, public-sector hospital in the southeastern United States. Both the longer-stay inpatient unit and the crisis stabilization unit admit patients primarily for evaluation and treatment of first episodes of illness or exacerbations of severe and persistent mental illnesses (primarily schizophrenia and other psychotic disorders and severe affective disorders). This county hospital serves a predominantly low-income, urban, African American population.\nIn the state of Georgia, commitment for involuntary evaluation can be initiated by a licensed physician, psychologist, clinical social worker, or psychiatric clinical nurse specialist. Taking custody for transportation to an emergency evaluation facility also can be carried out by a law enforcement officer. In either case, this initial commitment allows for the individual to be taken to and held at a locked evaluation facility. Commitment criteria include the fact that the person appears to have a mental illness, and either (1) presents a substantial risk of imminent harm to self or others as manifested by recent overt acts or threats of violence presenting a probability of physical injury, or (2) is so unable to care for his\/her own physical health and safety as to create an imminently life-endangering crisis [3]. Within 48\u00a0h of admission, if the individual continues to meet commitment criteria, a physician may sign for commitment for further evaluation and treatment of up to five business days in duration. During this period of time, some patients will sign-in as voluntary patients. If the patient does not sign-in voluntarily, then he or she is typically discharged after the 5\u00a0days, unless commitment criteria clearly continue to be present. In that case, a court hearing is scheduled to determine whether or not the legal standard is met for longer-term involuntary hospitalization. However, in this setting extended commitment is used relatively rarely, and only in circumstances in which the threat to self or others is very apparent.\nProcedures\nThe majority of data collected were abstracted from patients\u2019 medical charts. Subjectively rated variables (e.g., treatment plan adherence during hospitalization, the treatment team\u2019s opinion of likelihood of follow-up) were obtained by consulting with the social worker, the psychiatry resident or psychology intern, and the attending psychiatrist caring for the patient during hospitalization. As described in two recently published reports, basic demographic, socioeconomic, psychosocial, and clinical data were recorded in a systematic fashion using a standardized data collection instrument [17, 18]. The criterion variable of interest in this analysis was a three-level variable representing the patient\u2019s legal status at admission and at discharge: VOL-VOL, INV-VOL, and INV-INV. The research was approved by the university\u2019s institutional review board and the hospital\u2019s research oversight committee.\nAnalysis\nBivariate analyses were performed using a variety of independent variables in relation to the criterion variable. Continuous variables were analyzed using one-way analysis of variance and categorical variables were analyzed using chi-square tests of association. These bivariate analyses were conducted to inform the selection of variables for inclusion in the subsequent multivariate regression analysis.\nPolytomous logistic regression modeling was required due to the trichotomous nature of the admission\/discharge legal status criterion variable. Those patients admitted and discharged voluntarily (VOL-VOL) were designated as the referent group in the regression model. Entering variables into the logistic model was based on significant bivariate test results (P\u00a0<\u00a00.05). A thorough assessment of confounding was conducted, and relevant confounding variables were controlled for in the model. The backward elimination method was used to identify variables that were independently significantly associated (P\u00a0<\u00a00.05) with legal status. Appropriate modeling diagnostics were conducted to ensure the fit of the model to the data. Adjusted odds ratios and 95% confidence intervals were computed to determine effect estimates for independent variables that were significantly associated with legal status, controlling for all other covariates in the model. The SAS version 9.1 software (SAS Inc., Cary, North Carolina, USA) was used for all statistical analyses.\nResults\nBasic sociodemographic and clinical descriptive statistics for the overall sample are shown in Table\u00a01. Slightly over half (55.5%) of patients were female. The racial composition of the sample is consistent with that of the population served by the two inpatient units, and by the hospital in general. Specifically, 84.9% of patients were Black\/African American. At the time of admission, 94.2% of patients were unemployed, and 25.6% reported being homeless. The majority of patients were single\/never married (67.8%). The majority of patients had been previously hospitalized for psychiatric treatment (75.1%). An even greater percentage of patients were reported as having psychotic symptoms at the time of admission (85.8%). The mean age was 38.7\u00a0\u00b1\u00a012.4\u00a0years, and the mean educational level was 12.1\u00a0\u00b1\u00a02.3\u00a0years. The mean Global Assessment of Functioning (GAF) scale score on admission was 30.3\u00a0\u00b1\u00a08.2, ranging from 10 to 55.\nTable\u00a01Basic sociodemographic and clinical descriptive statistics for the overall sample (n\u00a0=\u00a0227)VariableFrequencyGender, female126 (55.5%)Race\u00a0\u00a0\u00a0\u00a0Black\/African American191 (84.9%)\u00a0\u00a0\u00a0\u00a0Hispanic\/Latino4 (1.8%)\u00a0\u00a0\u00a0\u00a0White\/Caucasian\/European American 28 (12.4%)\u00a0\u00a0\u00a0\u00a0Other2 (0.9%)Employment status, unemployed213 (94.2%)Homeless on admission 58 (25.6%)Marital status\u00a0\u00a0\u00a0\u00a0Single\/never married154 (67.8%)\u00a0\u00a0\u00a0\u00a0Married\/living as married 19 (8.4%)\u00a0\u00a0\u00a0\u00a0Separated\/divorced\/widowed 54 (23.8%)Prior psychiatric hospitalization169 (75.1%)Psychotic symptoms present on admission194 (85.8%)VariableMean\u00a0\u00b1\u00a0SDMedianRangeAge 38.7\u00a0\u00b1\u00a012.44017\u201376Years of education 12.1\u00a0\u00b1\u00a02.312 4\u201318GAF score on admission 30.3\u00a0\u00b1\u00a08.23010\u201355\nAmong the 227 patients, 56 (24.7%) were in the VOL-VOL group, 81 (35.7%) were in the INV-VOL group, and 90 (39.6%) were in the INV-INV group. As shown in Table\u00a02, select demographic and social variables were examined across the three groups. The difference in percentage of patients documented as having Axis IV psychosocial problems in the area of housing was statistically significant (\u03c72\u00a0=\u00a06.43, df\u00a0=\u00a02, P\u00a0=\u00a00.04), with patients admitted and discharged voluntarily most likely to have housing problems (60.7%). The percentage of patients who were receiving disability payments\u2014Supplemental Security Income (SSI) or Social Security Disability Insurance (SSDI)\u2014over the past 3\u00a0months also differed significantly across the three legal status groups (\u03c72\u00a0=\u00a06.82, df\u00a0=\u00a02, P\u00a0=\u00a00.03), with patients admitted and discharged involuntarily most likely to be receiving disability payments (61.1%).\nTable\u00a02Sociodemographic characteristics of 227 hospitalized patientsVariableLegal status on admission and dischargeaTest statistic (df)P-valueVOL-VOL (n\u00a0=\u00a056)INV-VOL (n\u00a0=\u00a081)INV-INV (n\u00a0=\u00a090)Gender, female32 (57.1%)48 (59.3%)46 (51.1%)1.23 (2)0.54Race, Black\/African American45 (81.8%)66 (81.5%)80 (89.9%)2.87 (2)0.24Employed part-time or full-time2 (3.64%)7 (8.64%)4 (4.44%)1.99 (2)0.37Homeless on admission17 (30.4%)23 (28.4%)18 (20.0%)2.48 (2)0.29Current marital\u00a0\u00a0\u00a0\u00a0Single\/never married32 (57.1%)56 (69.1%)66 (73.3%)4.32 (4)0.36\u00a0\u00a0\u00a0\u00a0Married\/living as married6 (10.7%)7 (8.6%)6 (6.7%)\u00a0\u00a0\u00a0\u00a0Separated\/divorced\/widowed18 (32.1%)18 (22.2%)18 (20.0%)Age on admission (Mean\u00a0\u00b1\u00a0SD)41.0 \u00b1\u00a011.537.0 \u00b1\u00a013.238.8 \u00b1\u00a012.11.73 (222)0.18Axis IV housing problems34 (60.7%)35 (43.2%)36 (40.0%)6.43 (2)0.04Axis IV economic problems32 (57.1%)51 (63.0%)41 (45.6%)5.40 (2)0.07Receiving disability payments over past 3\u00a0months (SSI or SSDI)22 (39.3%)40 (49.4%)55 (61.1%)6.82 (2)0.03aLegal status on admission and discharge: VOL-VOL, Patients having voluntary legal status on both admission and discharge; INV-VOL, Patients having involuntary status on admission and voluntary status on discharge; INV-INV, Patients having involuntary legal status on both admission and discharge\nTable\u00a03 shows select clinical and diagnostic characteristics. The three legal status groups differed significantly on 13 of these 20 variables. The INV-INV group was most likely to have had previous psychiatric hospitalizations (84.3%), followed by 71.6% among the INV-VOL group, and 65.5% among the VOL-VOL group (\u03c72\u00a0=\u00a07.27, df\u00a0=\u00a02, P\u00a0=\u00a00.03). Compared to the VOL-VOL group, patients who were admitted and discharged involuntarily were more likely to have a primary diagnosis of a psychotic disorder, to have a change in GAF score from admission to discharge of <20 points, and to have psychotic symptoms on discharge. Patients admitted and discharged involuntarily were less likely to have documentation of medical problems requiring medications at discharge, to be taking >2 psychiatric medications at discharge, and to have good treatment plan adherence during hospitalization compared to the VOL-VOL group. Not unexpectedly, length of stay in days (F\u00a0=\u00a020.45, df\u00a0=\u00a0224, P\u00a0<\u00a00.0001) and the inpatient unit to which patients were admitted (\u03c72\u00a0=\u00a032.84, df\u00a0=\u00a02, P\u00a0<\u00a00.0001) were highly significantly different across the groups.\nTable\u00a03Clinical and diagnostic characteristics of 227 hospitalized patientsVariableLegal status on admission and dischargeaTest statistic (df)P-valueVOL-VOL (n\u00a0=\u00a056)INV-VOL (n\u00a0=\u00a081)INV-INV (n\u00a0=\u00a090)Prior psychiatric hospitalization36 (65.5%)58 (71.6%)75 (84.3%)7.27 (2)0.03Psychotic symptoms on admission43 (76.8%)71 (87.7%)80 (89.9%)5.20 (2)0.07Primary diagnosis, schizophrenia or other psychotic disorder26 (47.3%)56 (69.1%)63 (70.0%)9.03 (2)0.01Comorbid substance use disorder diagnosis26 (46.4%)34 (42.5%)40 (44.4%)0.21 (2)0.90Comorbid personality disorder diagnosis10 (17.9%)13 (16.1%)12 (13.3%)0.58 (2)0.75Change of <20 in GAF score (Discharge\u2013Admission)23 (42.6%)26 (32.9%)47 (53.4%)7.14 (2)0.03Depressive symptoms at discharge23 (41.1%)23 (28.4%)22 (24.4%)4.69 (2)0.10Anxious symptoms at discharge23 (41.1%)43 (51.1%)28 (31.1%)8.49 (2)0.01Psychotic symptoms at discharge27 (48.2%)51 (63.0%)67 (74.4%)10.34 (2)<0.01Documented medical problems requiring medication at discharge35 (62.5%)41 (50.6%)32 (35.6%)10.52 (2)<0.01>2 Psychiatric medications at discharge20 (36.4%)13 (16.1%)14 (15.9%)10.40 (2)<0.01Experiencing side effects at discharge7 (12.5%)23 (28.4%)13 (14.4%)7.41 (2)0.02Required seclusion5 (8.9%)11 (13.6%)18 (20.0%)3.52 (2)0.17Required restraints4 (7.1%)8 (9.9%)8 (8.9%)0.31 (2)0.86Required PRN medicine14 (25.0%)30 (37.0%)38 (42.2%)4.48 (2)0.11Patient has an established outpatient clinician15 (26.8%)20 (25.0%)38 (42.2%)6.78 (2)0.03Good treatment plan adherence during week prior to discharge41 (73.2%)60 (74.1%)50 (55.6%)8.06 (2)0.02Treatment team\u2019s opinion of likelihood of follow-up\u00a0\u00a0\u00a0\u00a0Poor15 (26.8%)16 (20.0%)35 (38.9%)15.02 (4)<0.01\u00a0\u00a0\u00a0\u00a0Fair19 (34.0%)29 (36.3%)38 (42.2%)\u00a0\u00a0\u00a0\u00a0Good22 (39.3%)35 (43.8%)17 (18.9%)Length of stay in days (Mean\u00a0\u00b1\u00a0SD)11.3 \u00b1\u00a07.515.5 \u00b1\u00a08.48.4 \u00b1\u00a06.020.45 (224)<0.0001Unit, longer-stay inpatient unit36 (64.3%)67 (83.8%)37 (41.1%)32.84 (2)<0.0001a Legal status on admission and discharge: VOL-VOL, Patients having voluntary legal status on both admission and discharge; INV-VOL, Patients having involuntary status on admission and voluntary status on discharge; INV-INV, Patients having involuntary legal status on both admission and discharge\nThe average lengths of hospital stay (in days) across the three groups of patients are displayed in Fig.\u00a01. Patients in the INV-INV group had an average length of hospitalization of 8.4\u00a0days, whereas patients in the VOL-VOL group had an average length of stay of 11.3\u00a0days. Patients in the INV-VOL group had the longest average length of hospital stay, at 15.5\u00a0days. Based on post-hoc tests using the Tukey approach, the difference between mean lengths of stay was significant for the INV-VOL group compared to the VOL-VOL group and for the INV-VOL group compared to the INV-INV group.\nFig.\u00a01Average lengths of hospital stay (days) in the three groups of patients\nFigure\u00a02 illustrates the differences among the three groups in terms of the percentage of patients adhering with the first scheduled community mental health appointment following hospitalization. The average time interval between hospital discharge and the first scheduled community mental health appointment was 14\u00a0days and ranged from 0 (i.e., the appointment was scheduled to occur on same day as discharge) to 59\u00a0days. (Follow-up was assessed by a single phone call to the community mental health center where the discharged patient had been scheduled for the first outpatient appointment. The follow-up phone call was made a few days after the appointment, and further outpatient adherence was not tracked [17].) Patients in the INV-VOL group were the most likely to adhere with the first follow-up appointment (52.0%), whereas patients in the VOL-VOL group (30.9%) and the INV-INV group (27.1%) were far less likely to adhere (\u03c72\u00a0=\u00a011.75, df\u00a0=\u00a02, P\u00a0<\u00a00.01).\nFig.\u00a02Percentage of patients adhering with the first scheduled community mental health appointment following hospitalization\nBased on the significant bivariate test results and a thorough consideration of relevant confounding variables, 18 factors were entered into the polytomous logistic regression model. In this model, three of these factors were independently associated with legal status: psychotic symptoms at discharge, documented medical problems requiring medication, and number of psychiatric medications. Psychiatric inpatient unit, length of hospital stay, prior psychiatric hospitalization, and receiving disability payments (SSI\/SSDI) were deemed confounding variables and were, therefore, controlled for in the final model. Because of sparse missing values, modeling results were generated using data from 221 individuals. Table\u00a04 shows model coefficients, standard errors, adjusted odds ratios, and 95% confidence intervals for each of the three significant variables, by legal status category.\nTable\u00a04Independent variables significantly associated with admission and discharge legal status among 221 hospitalized patientsVariableLegal status categoryaBSEaOR95% CIPsychotic symptoms at dischargeINV-VOL\u22120.020.440.980.42, 2.32INV-INV1.490.464.421.80, 10.86Documented medical problems requiring medication at dischargeINV-VOL\u22120.480.400.620.28, 1.35INV-INV\u22121.510.430.220.10, 0.51Number of psychiatric medications at discharge (>2)INV-VOL\u22121.800.520.170.06, 0.46INV-INV\u22120.880.510.410.15, 1.12The model controls for psychiatric hospital unit, length of hospital stay, prior psychiatric hospitalization, and receiving disability payments (SSI\/SSDI)a VOL-VOL (voluntary legal status on both admission and discharge) is the referent group for all comparisons; INV-VOL, involuntary legal status on admission, voluntary status on discharge; INV-INV, involuntary legal status on both admission and dischargeB, Coefficient value from the final regression model; SE, standard error; aOR, adjusted odds ratio; CI, confidence interval\nPatients who were involuntary on admission and discharge (INV-INV) were approximately 4.4 times more likely to have psychotic symptoms at discharge, relative to the VOL-VOL group. Patients in the INV-INV group were also about 4.5 times less likely (or equivalently, 0.22 times as likely) to have documented medical problems requiring medications at discharge, relative to those in the VOL-VOL group. Patients who were involuntary on admission and voluntary on discharge (INV-VOL) were approximately 6 times less likely (0.17 times as likely) to be prescribed more than two psychiatric medications at discharge.\nDiscussion\nMultivariate logistic regression modeling yielded three clinical variables that were independently significantly associated with admission\/discharge legal status, even after controlling for psychiatric unit, length of hospital stay, prior psychiatric hospitalization, and whether or not the patient received disability payments. As described below, the associations between legal status and each of these three significant variables potentially may be explained by the lower overall treatment engagement common among involuntary patients.\nFirst, patients in the INV-INV group were much more likely to be experiencing psychotic symptoms at discharge, compared to the VOL-VOL group. The most plausible explanation for this finding relates to a difference in the level of acceptance of treatment between the two groups. Involuntary legal status is generally an indicator of a lack of treatment engagement, resistance to treatment, and\/or impaired insight. Bivariate test results for \u201ctreatment plan adherence during the week prior to discharge\u201d support the idea that involuntary patients generally had poorer overall treatment adherence.\nWhen patients refuse to sign-in voluntarily and no longer meet dangerousness criteria, they may have to leave the hospital before resolution of psychotic symptoms. The abbreviated hospitalization may not have allowed for sufficient time for stabilization and adjustments to psychiatric medications. Due to the laws governing involuntary inpatient commitment in this state, individuals who have been hospitalized for the duration of the short-term commitment and who have refused to convert to voluntary status, must be discharged unless they clearly meet further commitment criteria [3]. On the other hand, patients with voluntary legal status are generally discharged based on clinical, rather than legal, criteria\u2014they are released from the hospital when symptoms have sufficiently resolved and the patient is stable enough to return to the community.\nA significant difference in psychotic symptoms at discharge was not found between the INV-VOL group and the VOL-VOL group. This is not surprising given the fact that these two groups were virtually equally as likely to be rated as having good treatment adherence during the week prior to discharge. Furthermore, these patients were discharged after sufficient resolution of symptoms rather than legal expiration. From a clinical vantage point, it appears that INV-INV patients are being discharged \u201ctoo soon\u201d due to legal constraints.\nAn alternate explanation for the difference in psychotic symptoms at discharge among the three groups is that INV-INV patients were more likely to have psychotic symptoms at the time of admission compared to the other two groups. However, the bivariate test revealed no statistically significant difference between the three groups in terms of psychotic symptoms present on admission.\nSecond, patients admitted and discharged involuntarily (INV-INV) were much less likely to have documented medical problems requiring medications at the time of discharge, relative to the VOL-VOL group. Because length of stay was controlled for in the model, the most likely reason for the difference in medical problems at discharge is a generalized lack of treatment engagement among involuntary patients. That is, poor treatment engagement is associated with a decreased likelihood of medical problems being reported and diagnosed. Involuntary patients are more likely to refuse medical history taking, physical examination, vital sign checks, diagnostic blood work, and other diagnostic procedures during hospitalization. Thus, clinicians may not have sufficient opportunities or information to detect common medical problems, such as hypertension, diabetes mellitus, and anemia.\nIt is notable that a significant difference in documented medical problems requiring medication was not found between the INV-VOL and VOL-VOL groups. As noted earlier, these two groups had similar treatment adherence during the week prior to discharge. These results support the idea that converting to voluntary status is associated with better treatment engagement, affording more opportunities for medical problems to be detected and medication to be prescribed. Future research is needed to determine whether a general lack of treatment engagement alone, or some additional factors in combination with poor treatment engagement, accounts for the difference in documented medical problems requiring medication.\nSome alternate explanations as to why the INV-INV patients had fewer medical problems requiring medication at discharge merit consideration. For example, it is conceivable that even when clinicians detect a medical problem, they may be biased toward not prescribing medication to involuntary patients who are highly unlikely to agree to take it. However, in this particular study setting, when medical problems are detected in involuntary patients, clinicians typically prescribe the indicated medication, regardless of the likelihood that the patient will comply with taking it. Another possible explanation is that patients in the INV-INV group are generally healthier than the other two groups. However, because the three legal status groups were very similar with respect to age, gender, and race, it is highly unlikely that one group of patients would have been healthier than the other two.\nThird, though the INV-VOL and VOL-VOL groups did not differ with respect to psychotic symptoms and medical problems requiring medication at discharge, these two groups did differ in the number of psychiatric medications at time of discharge. Those patients in the INV-VOL group were much less likely to have been prescribed more than two psychiatric medications at discharge, relative to the VOL-VOL group. The difference in number of psychiatric medications between the INV-INV and VOL-VOL groups was not significant, though the effect for the INV-INV group was in the same direction as with the INV-VOL group. The most plausible explanation for this finding may be related to both a lower overall treatment engagement among involuntary patients and differences in clinician prescribing behavior for involuntarily admitted patients compared to voluntary patients. That is, perhaps clinicians err on the side of caution with patients recently converting to voluntary status by limiting the number of psychiatric medications used to treat symptoms. Clinicians may limit the number of prescribed medications, especially if psychiatric symptoms are resolving, in order to simplify the medication regimen and sustain good treatment adherence. Interestingly, a much higher percentage of patients in the INV-VOL group adhered with the first community mental health appointment compared to the VOL-VOL group and the INV-INV group. It is conceivable that a simpler treatment regimen (one or two psychiatric medications) may contribute to higher rates of adherence with initial community mental health follow-up.\nSeveral methodological limitations of this study merit consideration. First, the fact that patients are admitted voluntarily or involuntarily is a legal distinction, which may not accurately reflect the patient\u2019s level of treatment engagement [11, 12, 19, 20]. Patients admitted voluntarily do not necessarily always have the motivation and adherence behaviors of individuals truly pursuing treatment for mental illness of their own accord [11\u201313]. For example, a voluntary patient who is pressured or coerced by family members to seek inpatient treatment may have motivation, adherence, and insight more similar to patients admitted involuntarily than to those voluntarily seeking treatment [11, 12]. Likewise, some involuntary patients may be more similar to voluntary patients than to other involuntary patients. This might occur when patients who would willingly have signed-in voluntarily are admitted involuntarily in order to overcome obstacles associated with voluntary admission procedures in certain mental health systems [11]. Because legal status may not be an appropriate surrogate for measuring perceived coercion or treatment engagement [5, 8, 11], future research should consider utilizing MacArthur\u2019s Perceived Coercion Scale [21] and a continuous scale to assess treatment engagement.\nA second methodological limitation is that commitment statutes \u201ccensor\u201d the number of patients that convert to voluntary status, since non-converters must be discharged upon expiration of the commitment. Therefore, the true difference between the INV-VOL and INV-INV groups is somewhat obscured because of the limited timeframe in which patients have to convert to voluntary status. One cannot assume that any given INV-INV patient would never have converted to voluntary status; rather one can only infer that he or she had not done so by time of discharge. In other words, caution must be exercised in assigning ontological status to the INV-INV group. A third limitation is that patients re-admitted during the data collection period were excluded from the analysis. Future research might consider focusing on INV-INV patients with multiple hospitalizations. Another limitation with regard to data collection was that clinical and diagnostic variables (some of which were subjectively rated) were based on clinicians\u2019 reports or information in the hospital chart, without using rating scales administered directly to patients. However, given the goals of this study, the researchers deemed patient assessments, beyond that done as part of routine evaluation and treatment, unnecessary. Also, involuntary patients would have been less likely to participate in formal research evaluations compared to voluntary patients, thus restricting the ability of the analyses to examine the issues of interest. Lastly, the findings from this study may have limited generalizability, given the specific sociodemographic and diagnostic characteristics of the sample. However, the population of interest to the researchers was that of minority individuals with severe psychiatric illnesses being treated in a large, urban, public-sector hospital.\nDespite the methodological limitations inherent in the study design, there were also several strengths in the methodology and data analysis process. First, an extremely thorough assessment of confounding was conducted. The final model controlled for four variables that were considered likely confounders. Second, alternate modeling techniques were conducted, all of which resulted in the same findings. Lastly, a concerted effort was made to minimize missing values during data collection, and to prevent biases, the few variables with significant missing values were excluded from the analysis.\nConclusions\nResearch on associations between patients\u2019 legal status and sociodemographic and clinical variables can directly and indirectly affect psychiatric treatment during and after hospitalization. For instance, patients in the INV-INV group have the shortest average length of hospitalization, which is a direct result of the commitment law dictating that patients cannot be involuntarily hospitalized for more than 5\u00a0days unless they continue to meet dangerousness criteria [3], even if symptoms have not yet resolved. In addition to being more likely to have psychotic symptoms at discharge, patients in the INV-INV group are less likely to have documented medical problems requiring medication at discharge. Thus, involuntary patients frequently may be discharged from the hospital with unresolved psychotic symptoms and undetected, untreated medical problems. The poor outpatient treatment follow-up rate among INV-INV patients suggests that this is a particularly vulnerable group needing focused attention by the mental health system.\nDuring hospitalization, clinicians should be attentive to the manner in which involuntary patients are treated because this can greatly influence patients\u2019 perceptions of coercion. It has been suggested that a concerted effort to reduce the level of coercive practices used during the admission process could mitigate some of the negative effects of coercion, such as future non-adherence with treatment and outpatient follow-up [8]. After discharge from the hospital, involuntary outpatient commitment can be employed to stabilize patients in the community, though this form of coercive treatment is also controversial. Recent research has suggested efficacy of involuntary outpatient commitment in enhancing treatment compliance and reducing re-admission rates among persons with severe psychiatric illnesses [22, 23]. Increased resource allocation for involuntary patients during the admission process and throughout hospitalization, discharge planning, and outpatient follow-up may have the downstream effect of preventing future hospital admissions and worsening of medical comorbidity.","keyphrases":["involuntary","voluntary","commitment","compulsory"],"prmu":["P","P","P","U"]}
{"id":"J_Gen_Intern_Med-3-1-2150633","title":"Performance of a Web-Based Clinical Diagnosis Support System for Internists\n","text":"BACKGROUND Clinical decision support systems can improve medical diagnosis and reduce diagnostic errors. Older systems, however, were cumbersome to use and had limited success in identifying the correct diagnosis in complicated cases.\nINTRODUCTION\nThe best clinicians excel in their ability to discern the correct diagnosis in perplexing cases. This skill requires an extensive knowledge base, keen interviewing and examination skills, and the ability to synthesize coherently all of the available information. Unfortunately, the level of expertise varies among clinicians, and even the most expert can sometimes fail. There is also a growing appreciation that diagnostic errors can be made just as easily in simple cases as in the most complex. Given this dilemma and the fact that diagnostic error rates are not trivial, clinicians are well-advised to explore tools that can help them establish correct diagnoses.\nClinical diagnosis support systems (CDSS) can direct physicians to the correct diagnosis and have the potential to reduce the rate of diagnostic errors in medicine.1,2 The first-generation computer-based products (e.g., QMR\u2014First Databank, Inc, CA; Iliad\u2014University of Utah; DXplain\u2014Massachusetts General Hospital, Boston, MA) used precompiled knowledge bases of syndromes and diseases with their characteristic symptoms, signs, and laboratory findings. The user would enter findings from their own patients selected from a menu of choices, and the programs would use Bayesian logic or pattern-matching algorithms to suggest diagnostic possibilities. Typically, the suggestions were judged to be helpful in clinical settings, even when used by expert clinicians.3 These diagnosis support systems were also useful in teaching clinical reasoning.4,5 Surprisingly and despite their demonstrated utility in experimental settings, none of these earlier systems gained widespread acceptance for clinical use, apparently related to the considerable time needed to input clinical data and their somewhat limited sensitivity and specificity.3,6 A study of Iliad and QMR in an emergency department setting, for example, found that the final impression of the attending physician was found among the suggested diagnoses only 72% and 52% of the time, respectively, and data input required 20 to 40 minutes for each case.7\nIn this study, we evaluated the clinical performance of \u201cIsabel\u201d (Isabel Healthcare Inc, USA), a new, second generation, web-based CDSS that accepts either key findings or whole-text entry and uses a novel search strategy to identify candidate diagnoses from the clinical findings. The clinician first enters the key findings from the case using free-text entry (see Fig\u00a01). There is no limit on the number of terms entered, although excellent results are typically obtained with entering just a few key findings. The program includes a thesaurus that facilitates recognition of terms. The program then uses natural language processing and search algorithms to compare these terms to those used in a selected reference library. For Internal Medicine cases, the library includes 6 key textbooks anchored around the Oxford Textbooks of Medicine, 4th Edition (2003) and the Oxford Textbook of Geriatric Medicine and 46 major journals in general and subspecialty medicine and toxicology. The search domain and results are filtered to take into account the patient\u2019s age, sex, geographic location, pregnancy status, and other clinical parameters that are either preselected by the clinician or automatically entered if the system is integrated with the clinician\u2019s electronic medical record. The system then displays a total of 30 suggested diagnoses, with 10 diagnoses presented on each web page (see Fig.\u00a02). The order of listing reflects an indication of the matching between the findings selected and the reference materials searched but is not meant to suggest a ranked order of clinical probabilities. As in the first generation systems, more detailed information on each diagnosis can be obtained by links to authoritative texts.\nFigure\u00a01The data-entry screen of the Isabel diagnosis support software. The clinician manually enters the age, gender, locality, and specialty. The query terms can be entered manually from the key findings or findings can be pasted from an existing write-up. This patient was ultimately found to have a B-cell lymphoma secreting a cryo-paraproteinFigure\u00a02The first page of results of the Isabel diagnosis support software. Additional diagnoses are presented by selecting the \u2018more diagnoses\u2019 box\nThe Isabel CDSS was originally developed for use in pediatrics. In an initial evaluation, 13 clinicians (trainees and staff) at St Mary\u2019s Hospital, London submitted a total of 99 case scenarios of hypothetical case presentations for different diagnoses, and Isabel displayed the expected diagnosis in 91% of these cases.8 Out of 100 real case scenarios gathered from 4 major teaching hospitals in the UK, Isabel suggested the correct diagnosis in 83 of 87 cases (95%). In a separate evaluation of 24 case scenarios in which the gold standard differential diagnosis was established by two senior academic pediatricians, Isabel decreased the chance of clinicians (a mix of trainees and staff clinicians) omitting a key diagnosis by suggesting a highly relevant new diagnosis in 1 of every 8 cases. In this study, the time to enter data and obtain diagnostic suggestions averaged less than 1 minute.9\nThe Isabel clinical diagnosis support system has now been adapted for adult medicine. The goal of this study was to evaluate the speed and accuracy of this product in suggesting the correct diagnosis in a series of complex cases in Internal Medicine.\nMETHOD\nWe considered 61 consecutive \u201cCase Records of Massachusetts General Hospital\u201d (New England Journal of Medicine, vol. 350:166\u2013176, 2004 and 353:189\u2013198, 2005). Each case had an anatomical or final diagnosis, which was considered to be correct by the discussants. We excluded 11 cases (patients under the age of 10 and cases that focused solely on management issues). The 50 remaining case histories were copied and pasted into the Isabel data-entry field. The pasted material typically included the history, physical examination findings, and laboratory test results, but data from tables and figures were not submitted. Beyond entering the patient\u2019s age, sex, and nationality, the investigators did not attempt to otherwise tailor the search strategy. Findings were compared to the recommended (but slightly more time consuming) strategy of entering discrete key findings, as compiled by a senior internist (MLG). Because the correct diagnosis is presented at the end of each case, data entry was not blinded.\nRESULTS\nUsing the recommended method of manually entering key findings, the list of diagnoses suggested by Isabel contained the correct diagnosis in 48 of the 50 cases (96%). Typically 3\u20136 key findings from each case were used. The 2 diagnoses that were not suggested (progressive multifocal encephalopathy and nephrogenic fibrosing dermopathy) were not included in the Isabel database at the time of the study; thus, these 2 cases would never have been suggested, even with different keywords.\nUsing the copy\/paste method for entering the whole text, the list of diagnoses suggested by Isabel contained the correct diagnosis in 37 of the 50 cases (76%). Isabel presented 10 diagnoses on the first web page and 10 additional diagnoses on subsequent pages up to a total of 30 diagnoses. Because users may tend to disregard suggestions not shown on later web pages, we tracked this parameter for the copy\/paste method of data entry: The correct diagnosis was presented on the first page in 19 of the 37 cases (51%) or first two pages in 28 of the 37 cases (77%). Similar data were not collected for manual data entry because the order of presentation depended on which key findings were entered.\nBoth data entry approaches were fast: Manually entering data and obtaining diagnostic suggestions typically required less than 1 minute per case, and the copy\/paste method typically required less than 5 seconds.\nDISCUSSION\nDiagnostic errors are an underappreciated cause of medical error,10 and any intervention that has the potential to produce correct and timely medical diagnosis is worthy of serious consideration. Our recent analysis of diagnostic errors in Internal Medicine found that clinicians often stop thinking after arriving at a preliminary diagnosis that explains all the key findings, leading to context errors and \u2018premature closure\u2019, where further possibilities are not considered.11 These and other errors contribute to diagnoses that are wrong or delayed, causing substantial harm in the patients affected. Systems that help clinicians explore a more complete range of diagnostic possibilities could conceivably reduce these types of error.\nMany different CDSSs have been developed over the years, and these typically matched the manually entered features of the case in question to a database of key findings abstracted from experts or the clinical literature. The sensitivity of these systems was in the range of 50%\u201360%, and the time needed to access and query the database was often several minutes.3 More recently, the possibility of using Google to search for clinical diagnoses has been suggested. However, a formal evaluation of this approach on a subset of the same \u201cCase Records\u201d cases used in our study found a sensitivity of 58%,12 in the range of the first-generation CDSSs and unacceptably low for clinical use.\nThe findings of our study indicate that CDSS products have evolved substantially. Using the Isabel CDSS, we found that data entry takes under 1 minute, and the sensitivity in a series of highly complex cases approached 100% using entry of key findings. Entry of entire case histories using copy\/paste functionality allowed even faster data entry but reduced sensitivity. The loss of sensitivity seemed primarily related to negative findings included in the pasted history and physical (e.g., \u201cthe patient denies chest pain\u201d), which are treated as positive findings (chest pain) by the search algorithm.\nThere are several relevant limitations of this study that make it difficult to predict how Isabel might perform as a diagnostic aid in clinical practice. First, the results obtained here reflect the theoretical upper limit of performance, given that an investigator who was aware of the correct diagnosis entered the key findings. Further, clinicians in real life seldom have the wealth of reliable and organized information that is presented in the Case Records or the time needed to use a CDSS in every case. To the extent that Isabel functions as a \u2018learned intermediary\u2019,13 success in using the program will also clearly depend on the clinical expertise of the user and their facility in working with Isabel. A serious existential concern is whether presenting a clinician with dozens of diagnostic suggestions might be a distraction or lead to unnecessary testing. We have previously identified these trade-offs as an unavoidable cost of improving patient safety: The price of improving the odds of reaching a correct diagnosis is the extra time and resources consumed in using the CDSS and considering alternative diagnoses that might turn out to be irrelevant.14\nIn summary, the Isabel CDSS performed quickly and accurately in suggesting correct diagnoses for complex adult medicine cases. However, the test setting was artificial, and the CDSS should be evaluated in more natural environments for its potential to support clinical diagnosis and reduce the rate of diagnostic error in medicine.","keyphrases":["clinical diagnosis support systems","decision support","diagnostic error","isabel","internal medicine","google"],"prmu":["P","P","P","P","P","P"]}
{"id":"Soc_Psychiatry_Psychiatr_Epidemiol-2-2-1764202","title":"Help-seeking pathways in early psychosis\n","text":"Introduction Understanding the help-seeking pathways of patients with a putative risk of developing psychosis helps improving development of specialised care services. This study aimed at obtaining information about: type of health professionals contacted by patients at putative risk for psychosis on their help-seeking pathways; number of contacts; type of symptoms leading to contacts with health professionals; interval between initial contact and referral to a specialised outpatient service.\nIntroduction\nIn recent years, the aim of intervening in the early phases of psychosis has drawn great interest. Early intervention may shorten duration of untreated psychosis or even prevent onset of psychosis. Exact knowledge about the help-seeking pathways of patients is pivotal in order to provide early intervention and, thus, supply specialised and focussed health care. One study has investigated the patterns of referral in patients known to be at risk for developing a psychotic illness [1]. Further studies investigating these trajectories in first-episode patients [2\u20135] were able to show the important role of GPs. GPs were reported to be commonly contacted by patients who later develop psychosis. In particular, GPs were often reported to be contacted early along the help-seeking pathways and were shown to commonly be the first contacted professional group [3]. It can thus be hypothesised that a considerable part of GPs are contacted by patients who may still be presenting more insidious features such as functional disability or social withdrawal, given that these insidious features have been found to be highly prevalent in the early course of psychosis [6]. It must therefore be expected that studies of help-seeking pathways which, in addition to first-episode patients, further include patients in presumed at-risk states for psychosis would be able to corroborate the importance of GPs as early contacted professional groups. Up to date, only one other such study has been reported on patients who are considered to be at-risk for psychosis, but who have not yet developed psychosis [1]. This study is the first to compare the help-seeking behaviour of at-risk patients, patients with first psychotic episodes and of patients neither meeting clinical at-risk nor first episode criteria, but who are concerned about their mental health and thus were assessed in a prodromal service. To the best of our knowledge, it is also the first study to assess symptoms in patients at risk for psychosis at their first presentation to a prodromal service. We expected GPs to be highly represented among the early help-seeking contacts of at-risk patients.\nMethod\nRecruitment strategy\nIn August 2002, a specialised, low-threshold outpatient service for the assessment of patients considered at-risk for psychosis was established in a semi-urban catchment area of North Western Switzerland (population = 300.000). This \u201cprodromal clinic\u201d is part of the only general psychiatric outpatient clinic of the catchment area and is associated with a public psychiatric hospital. As is the case all over Switzerland, patients can refer themselves directly to any public or private psychiatric facility and do not require referral to mental health systems via gate-keeping GPs. Patients can also be referred by any other source such as GPs, school counsellors, paediatricians or social workers.\nBetween August and December 2002, the majority of GPs (n = 240), private psychiatrists and psychologists (n = 130) of the catchment area were educated either individually or in small groups about the early warning signs of emerging psychosis and manifest psychotic features. Education strategies were based on the findings from a large, nation-wide survey among 1089 GPs [7] that showed that GPs often underidentified the insidious features of beginning psychosis, such as functional decline. In keeping with these findings, education strategies primarily focussed on the importance of the insidious features of beginning psychosis such as sustained functional decline and social withdrawal as important early warning signs. With the inclusion of the adolescent psychiatric service into our prodromal clinic in January 2004, all private youth psychiatrists as well as paediatricians were equally enrolled in this campaign so as to ensure additional referrals of patients aged 14\u201317 years, as symptom onset in this young age group has been associated with less favourable outcomes [8].\nIn our study, sensitised primary and secondary health care providers were encouraged to contact the outpatient clinic whenever one of their patients showed sustained decline in social functioning in a still relatively asymptomatic state; if they showed attenuated or brief intermittent positive psychotic symptoms; or if they suspected that \u201csomething odd had been going on\u201d for some time in their patients.\nThe research protocol was approved by the \u201cEthikkommission beider Basel\u201d. It was designed to obtain data on help-seeking pathways and to collect longitudinal information about progression of symptoms and social as well as neuropsychological functioning, and was not a treatment trial.\nPatient sample\nAs of January 1st 2003, patients could be referred to our prodromal clinic. Consenting patients were included in the study. Each individual was fully informed about the research protocol. Written informed consent was obtained from all patients, and additionally from the parents if under 18. Patients were considered ineligible for further assessment within the study if they presented a history of a past psychotic episode, traumatic brain injury, epilepsy or other known neurological disorder; other significant medical conditions considered to affect cognitive performance and self-perception; an IQ of below 70; or patients below the age of 18 in the first year and below the age of 14 years in the second year of the present study.\nSymptom ratings\nWe assessed the entire range of potential symptoms from early prodrome to first psychotic episode using the following instruments: potential early prodromes were assessed using the Schizophrenia Prediction Instrument\u2014Adult Version (SPI-A) [9], potential late prodromes using the Scale of Prodromal Symptoms (SOPS) with its companion interview manual (Structured Interview for Prodromal Symptoms, SIPS) [10, 11]. For the assessment of potentially manifest psychosis, we used the Positive and Negative Syndrome Scale (PANSS; 12). Ratings were performed by an experienced consultant research psychiatrist (A.S.) or by a trained masters-level psychologist (D.D.). Interrater reliability was established by extensive training by one of the authors of the SPI-A and by repeated training sessions involving all raters. However, formal assessment of interrater reliability was not conducted.\nThe SPI-A has been developed from a hierarchical cluster analysis of the BSABS [13] and includes those 10 basic symptoms for which a good positive predictive value for later schizophrenia has previously been reported [14, 15]. These basic symptoms include: thought interferences; thought perseveration; thought pressure; thought blockages; disturbances of receptive language; decreased ability to discriminate between ideas and perception, phantasy and true memory; unstable ideas of reference; derealisation; visual and acoustic perception disturbances.\nThe structure of the SIPS manual and the SOPS rating scale as well as its high validity and reliability have been reported elsewhere [10, 11]. In summary, these authors classified patients with either attenuated positive psychotic symptoms, brief limited intermittent positive psychotic symptoms or with a combined functional decline and genetic high-risk as ultra high-risk (UHR).\nGroup assignment\nDepending on symptom severity, patients were assigned to one of the three following groups: (1) the First Episode (FE) group was constituted by patients scoring 6 on any of the five SOPS positive symptom items for more than 1 week, which is identical with a minimum score of 4 on any of the PANSS positive items for the same time period. (2) The at-risk (AR) group included: patients meeting severity, but not duration criteria of the FE group; patients meeting scores of 3\u20135 on any SOPS positive psychotic symptom item at least once a week over the past month; patients with a first degree relative with any psychotic disorder and\/or patient meeting DSM-IV Schizotypal Personality Disorder criteria and a 30% or greater drop in the GAF [16] score during the last month compared to 12 months ago; patients scoring a minimum of 3 on any of the 10 predictive basic symptoms according to the SPI-A. (3) Help-seeking patients who were referred to the prodromal clinic for risk assessment, but who did neither meet FE nor AR criteria, were assigned to the Patient Control (PCo) group.\nAssessment of help-seeking pathways and of symptoms at prior contacts\nAfter symptom assessment, patients were asked which other professional groups they had previously contacted for similar problems. Further, timing and number of contacts as well as type of symptom leading to each single prior contact were recorded and, whenever possible, corroborated with information from family members. These data were assessed using a semi-structured interview that was designed for this study. However, no information was collected whether referrals to mental health services were voluntary or involuntary.\nAnalysis\nData on patients who were enrolled between January 2003 and December 2004 were analysed using the computer package SPSS Version 11.04. We compared number of contacts and professionals contacted along the help-seeking pathways between groups. Similarly, symptoms that patients had presented during their previous help-seeking contacts and the interval between initial contact and referral to our prodromal service were compared between groups. Continuous variables were compared with t-tests or ANOVA, categorical variables with \u03c72 tests or, if ranked, with Kruskal\u2013Wallis and Mann\u2013Whitney-U tests. An alpha level of 0.05 was considered significant.\nResults\nSample characteristics\nOf the 104 patients included into this study, 28 were female and 76 male. The mean age was 23.2 years (SD: \u00b15.43; range: 16\u201338) in the first year of the study, and 20.0 years (SD: \u00b16.08; range: 14\u201340) in the second year. 28 patients met criteria for the FE group, 50 for the AR group, and 26 patients were assigned to the PCo group. A summary of the socio-demographic characteristics across these study groups is shown in Table\u00a01.\nTable\u00a01Socio-demographic characteristicsFirst episodeAt-riskControlsTotalSignificance valuesNumber of patients285026104Mean age23.42122.122\u03c7\u00b2\u00a0=\u00a03.744, df\u00a0=\u00a02, P\u00a0=\u00a00.153Gender\u00a0\u00a0\u00a0\u00a0m (%)22 (79)31 (62)23 (88)76 (73)\u03c7\u00b2\u00a0=\u00a06.676, df\u00a0=\u00a02, P\u00a0=\u00a00.036\u00a0\u00a0\u00a0\u00a0f (%)6 (21)19 (38)3 (12)28 (27)Marriage status\u00a0\u00a0\u00a0\u00a0Unmarried (%)26 (93)45 (90)25 (96)96 (92)\u03c7\u00b2\u00a0=\u00a00.929, df\u00a0=\u00a02, P\u00a0=\u00a00.629Children\u00a0\u00a0\u00a0\u00a0No children (%)28 (100)48 (96)25 (96)101 (97)\u03c7\u00b2\u00a0=\u00a01.140, df\u00a0=\u00a02, P\u00a0=\u00a00.566Living situation\u00a0\u00a0\u00a0\u00a0Alone (%)5 (18)7 (14)6 (23)18 (17)\u03c7\u00b2\u00a0=\u00a08.412, df\u00a0=\u00a04, P\u00a0=\u00a00.078\u00a0\u00a0\u00a0\u00a0With partner or friends (%)5 (18)5 (10)8 (31)18 (17)\u00a0\u00a0\u00a0\u00a0With parents or relatives (%)18 (64)36 (72)10 (39)64 (62)City size\u00a0\u00a0\u00a0\u00a0>200,000 (%)5 (18)5 (10)4 (15)14 (14)\u03c7\u00b2\u00a0=\u00a03.337, df\u00a0=\u00a06, P\u00a0=\u00a00.766\u00a0\u00a0\u00a0\u00a010,000\u2013200,000 (%)7 (25)12 (24)6 (23)25 (24)\u00a0\u00a0\u00a0\u00a0<10,000 (%)16 (57)33 (66)16 (62)59 (62)School education\u00a0\u00a0\u00a0\u00a0None completed (%)1 (4)1 (2)2 (8)4 (4)\u03c7\u00b2\u00a0=\u00a04.688, df\u00a0=\u00a06, P\u00a0=\u00a00.584\u00a0\u00a0\u00a0\u00a0Obligatory school (%)19 (68)36 (72)18 (69)73 (70)\u00a0\u00a0\u00a0\u00a0Maturity (%)5 (18)4 (8)4 (15)13 (13)\u00a0\u00a0\u00a0\u00a0Still in education (%)3 (11)9 (18)2 (8)14 (13)Professional training\u00a0\u00a0\u00a0\u00a0None (%)9 (32)22 (44)8 (31)39 (38)\u03c7\u00b2\u00a0=\u00a010.429, df\u00a0=\u00a06, P\u00a0=\u00a00.108\u00a0\u00a0\u00a0\u00a0Apprenticeship (%)12 (42)18 (36)12 (46)42 (40)\u00a0\u00a0\u00a0\u00a0Academic education (%)5 (18)1 (2)1 (4)7 (7)\u00a0\u00a0\u00a0\u00a0Still in training (%)2 (7)9 (18)5 (19)16 (15)Work situation\u00a0\u00a0\u00a0\u00a0No occupation (%)12 (43)19 (38)10 (39)41 (39)\u03c7\u00b2\u00a0=\u00a04.097, df\u00a0=\u00a06, P\u00a0=\u00a00.664\u00a0\u00a0\u00a0\u00a0At work (%)10 (36)11 (22)6 (22)27 (26)\u00a0\u00a0\u00a0\u00a0In training (%)6 (21)20 (40)10 (39)36 (35)\nNumber of contacts and professionals contacted\nThe mean number of contacts over all three patient groups was 2.38 (SD: \u00b11.42; median: 3.0; range: 1\u20138) with no significant between-group differences (\u03c72\u00a0=\u00a0208.375, df\u00a0=\u00a02, P\u00a0=\u00a00.605). The overall patient sample reported a total of 247 contacts prior to their referral to our prodromal clinic. Table\u00a02 shows the type of professional groups contacted by the patients across the three study groups along their help-seeking trajectories. FE patients more often presented themselves to mental health professionals (psychiatrists, psychologists as well as psychiatric out- and in-patient services) than AR and PCo patients (71.2% vs. 57.6% vs. 53.5% of contacts); however, this comparison did not reach significance (\u03c72\u00a0=\u00a04.724, df\u00a0=\u00a02, P\u00a0=\u00a00.094). When comparing FE patients with the combined non-psychotic AR and PCo patients, FE patients significantly more often presented themselves to mental health professionals (\u03c72\u00a0=\u00a04.461, df\u00a0=\u00a01, P\u00a0=\u00a00.024). Furthermore, when comparing the numbers of contacts with mental health professionals (149\/247\u00a0=\u00a060.3%) with those to non-mental health professionals (98\/247\u00a0=\u00a039.7%), FE patients more often contacted in-patient services, whereas PCo and AR patients more often attended GPs and \u201cother\u201d professionals such as alternative medical practitioners, non-medical counselling services and non-specified professionals (\u03c72\u00a0=\u00a020.189, df\u00a0=\u00a08, P = 0.010). Of the overall sample, 51.0% (53\/104) contacted a GP at some point in time, whereas 83.7% had at least once visited a mental health professional.\nTable\u00a02Distribution of professionals contactedFEARPCoTotal(n) % of total(66) 26.7(125) 50.6(56) 22.7(247) 100General practitioners21.2% (14)21.6% (27)37.5% (21)25.1% (62)Private Psychiatrists\/Psychologists21.2% (14)24.8% (31)21.4% (12)23.1% (57)Psychiatric out-patient services30.3% (20)25.6% (32)16.1% (9)24.7% (61)Psychiatric in-patient services19.7% (13)7.2% (9)16.1% (9)12.6% (31)Other professionals7.6% (5)20.8% (26)8.9% (5)14.6% (36)Percentages (and absolute numbers) of contacts made with professional groups at any stage of the help-seeking pathway\nHowever, as shown in Fig.\u00a01, there was a trend for progression from non-psychiatric towards psychiatric services. Contacts with GPs were often at an earlier stage on the help-seeking pathways. Moreover, GPs were the most often first-contacted professional groups, whereas psychiatric in-patient services were most commonly the last contacted professional group (\u03c72\u00a0=\u00a015.816, df\u00a0=\u00a04, P\u00a0=\u00a00.003). However, differences among study groups did not reach significance (first contacts: \u03c72\u00a0=\u00a013.102, df\u00a0=\u00a08, P\u00a0=\u00a00.108; last contacts: \u03c72\u00a0=\u00a09.699, df\u00a0=\u00a08, P\u00a0=\u00a00.287).\nFig.\u00a01Progression to specialized services. Distribution of first (n\u00a0=\u00a0104) and last contacts (n\u00a0=\u00a0104) in percent made to different helper groups. Contacts representing the only help-seeking attempt by a subject appear in both first and last contacts\nSymptoms at prior presentations\nThe complete set of signs and symptoms which led patients or their relatives to seek help at each of the recorded contacts was obtained from 100 of the total patient sample (96.2%). The frequencies of the most commonly presented symptoms are shown in Table\u00a03. In the AR and PCo groups, the trias depression, social decline and social withdrawal were presented most frequently. In the FE group, unusual thought content was the most frequent symptom, followed by the above-mentioned triad.\nTable\u00a03Frequency of presented symptomsFEARPCoTotalNumber of contacts6612556247Positive symptoms\u00a0\u00a0Ideas of reference (%)15 (23.1)2 (<5.0)1 (<5.0)18 (7.7)\u00a0\u00a0Unusual contents of thought (%)36 (55.4)18 (14.8)1 (<5.0)55 (23.5)\u00a0\u00a0Hallucinations (%)13 (20.0)11 (9.0)2 (<5.0)26 (11.1)\u00a0\u00a0Perceptual disturbances (%)5 (7.7)21 (17.2)026 (11.1)\u00a0\u00a0Alienation or derealisation (%)16 (24.6)29 (23.8)045 (19.2)Negative symptoms\u00a0\u00a0Deterioration of social functioning (%)32 (49.2)54 (44.3)16 (34.0)102 (43.6)\u00a0\u00a0Social withdrawal (%)32 (49.2)42 (34.4)14 (29.8)88 (37.6)\u00a0\u00a0Avolition (%)9 (13.8)26 (21.3)12 (25.5)47 (20.1)Cognitive symptoms\u00a0\u00a0Impaired concentration (%)13 (20.0)29 (23.8)1 (<5.0)43 (18.4)\u00a0\u00a0Impaired attention (%)7 (10.8)12 (9.8)1 (<5.0)20 (8.5)\u00a0\u00a0Impaired memory (%)10 (15.4)5 (<5)2 (<5.0)17 (7.3)\u00a0\u00a0Formal thought disorders (%)17 (26.2)25 (20.5)042 (17.9)Other symptoms\u00a0\u00a0Depression (%)24 (36.9)74 (60.7)38 (80.9)136 (58.1)\u00a0\u00a0Anxiety (%)14 (21.5)27 (22.1)9 (19.1)50 (21.4)\u00a0\u00a0Lack of impulse-control (%)2 (<5.0)14 (<5.0)11 (23.4)27 (11.5)Absolute numbers (percentages) of presented symptoms. Percentage values do not add to 100% due to multiple symptoms recorded for most contacts\nFE patients had presented at least one positive symptom in 80.3% of their prior contacts, AR patients in 56.6% and PCo patients in 5.9% of their prior contacts (\u03c72\u00a0=\u00a065.680, df\u00a0=\u00a02, P\u00a0<\u00a00.001). Similarly, when comparing numbers of patients rather than numbers of contacts, 89% FE, 65% AR and 8% PCo patients had reported positive symptoms at any stage along their help-seeking pathway (\u03c72\u00a0=\u00a035.483, df\u00a0=\u00a02, P\u00a0<\u00a00.001). Of patients presenting positive symptoms along their help-seeking pathways, 81.2% already did so during their first contact. Positive symptoms were significantly more often reported in first help-seeking contacts with psychiatric out-patient (63.6%) and in-patient (100%) services as compared to GPs (37.1%) and private psychiatrists\/psychologists (31.8%) (\u03c72\u00a0=\u00a013.425, df\u00a0=\u00a04, P\u00a0=\u00a00.009).\nTable\u00a04 shows the relationship between positive and non-positive symptoms and the professional groups to whom they were presented. Positive symptoms were significantly more often reported in contacts with both psychiatric out- and in-patient services (67.2%, 70.0%) as compared to GPs, who were contacted for positive symptoms only in 37.3% recorded contacts. Consequently, GPs were contacted with more unspecific symptoms (62.7%) such as depression and negative symptoms (\u03c72\u00a0=\u00a018.046, df\u00a0=\u00a04, P\u00a0=\u00a00.01).\nTable\u00a04Positive vs. non-positive symptoms presented to professional groupsGeneral practitionersPsychiatrists psychologistsOut-patient servicesIn-patient servicesOther xprofessionalsTotalNumber of contacts5955613034239Contacts made with positive symptoms37.3% (22)40% (22)67.2% (41)70% (21)55.9% (19)(125)Contacts made with non-positive symptoms62.7% (37)60% (33)32.8% (20)30.0% (9)44.1% (15)(114)Percentages (absolute numbers) of symptoms presented to professional groups. Percentage values do not add to 100% due to multiple symptoms recorded for most contactsPositive symptoms included items according to Table\u00a03, non-positive symptoms included items according to \u201cnegative\u201d, \u201ccognitive\u201d or \u201cother\u201d symptoms in Table\u00a03\nDuration from initial contact to referral\nThe median interval from initial contact to time of referral to our prodromal clinic was 36\u00a0weeks (range: 1\u00a0day to 7.6\u00a0years; mean: 124.0\u00a0weeks; \u00b1SD 217.1). About a third (33.3%) of the patients whose initial contact was over 18\u00a0months before referral to the prodromal clinic accounted for the relatively long mean help-seeking duration. Two-thirds (66.6%), however, were referred within 18\u00a0months, with one-third (35.4%) being referred within 2\u00a0months. The median help-seeking duration of the FE group (12.5\u00a0weeks) was shorter than for the AR (42.5\u00a0weeks) and PCo groups (46.5\u00a0weeks), however, between-group comparisons dropped just below level of significance (z\u00a0=\u00a0\u22121.879, P\u00a0=\u00a00.06). More significantly, patients presenting with positive symptoms at some stage along their help-seeking trajectories (n\u00a0=\u00a056) showed shorter pathway durations (median\u00a0=\u00a024.5\u00a0weeks) than patients presenting only with negative, affective or \u201cunspecific\u201d symptoms, i.e. patients never presenting positive symptoms (n\u00a0=\u00a044; median\u00a0=\u00a046.0\u00a0weeks) (z\u00a0=\u00a0\u22122.072, P\u00a0=\u00a00.038).\nWe further calculated the median intervals from any of the prior contacts to the time of referral and compared these \u201cdelays to referral\u201d for the different professional groups that were contacted. Median \u201creferral delay\u201d across all three-study groups was 28\u00a0weeks with no between-group differences (z\u00a0=\u00a0\u22121.260, P\u00a0=\u00a00.208). Median \u201cdelays\u201d were 15\u00a0weeks for contacts with GPs, 68.5\u00a0weeks for private psychiatrists and psychologists, each 13.0\u00a0weeks for psychiatric in- and out-patient services, and 52.2\u00a0weeks for contacts made to \u201cothers\u201d (z\u00a0=\u00a0\u22123.554, P\u00a0<\u00a00.001). For 165 of the 247 recorded contacts, we were able to obtain information on whether a continuous treatment of three or more consecutive sessions was provided. The significantly longer \u201cdelays to referral\u201d following contacts with private psychiatrists and psychologists was associated with the finding that in 58% of all contacts made with these groups, three or more sessions were administered. In comparison, in the vast majority of the cases (83.7%) contacts with GPs were single consultations.\nDiscussion\nTo the best of our knowledge, this is the first study to investigate the help-seeking pathways of a patient cohort, which was referred to a prodromal clinic for a suspected at-risk state for psychosis. We were able to confirm the important role of GPs along the help-seeking pathways of patients with emerging psychosis. Furthermore, GPs were contacted in particular by those patients who presented insidious features. It was these patients that showed the longest delays in referral to our specialised outpatient service.\nThe importance of GPs along the early pathways\nThis study was able to confirm recent reports from Australia [1] and from Germany [17, 18] that a substantial number of contacts made along the help-seeking pathways were with mental health care professionals. As the gatekeeping model applies for Australia, but not for Germany or Switzerland, mental health care professionals seem to play an important role independent of a particular health system. At the same time, our study also confirms earlier reports [2\u20135] about the pivotal role of GPs in the early pathways to care of help-seeking individuals that were referred to our prodromal clinic for an assessment of a potential psychotic at-risk state. While they were solicited in half of all contacts, GPs even constituted the most frequently first-contacted professional group (34.6%). It is noteworthy that in Switzerland patients can refer themselves to GPs without a \u201cgate-keeping\u201d GP. It can be thus assumed that in health systems with \u201cgate-keeping\u201d GPs [19], the present results may be even more representative.\nThe challenge of detecting the insidious symptoms\nIn addition, our results revealed that patients in less symptomatic states more commonly seek help with their GPs. Patients with manifest psychotic symptoms, however, more often contacted specialists. These results are paralleled by the finding that a shift from contacts made with primary carers to contacts with more specialised professional groups is taking place along the pathways (as shown in Fig.\u00a01). Similar findings were reported by Lincoln et al. [3]. We assume that this process may also be an expression of the symptom progression along the course of early psychosis [20, 21].\nAs we thus had expected, GPs are faced with the difficult task to detect potential at-risk states in patients that do not yet present psychotic, but the unspecific insidious features. This finding is of particular relevance as a recent comprehensive survey among 1089 Swiss GPs was able to show that GPs commonly under-identified the insidious features of emerging psychosis [7]. Preliminary results of an international replication study (IGPS) of the Swiss survey across 10 countries were able to confirm these findings [22]. Although features such as functional disability or social isolation may not necessarily lead to overt psychosis and may either be expression of another psychiatrically relevant process or remit after time, it is the detection of the earliest signs and symptoms of emerging psychosis that has become the aim of preventive efforts. Similarly, depression has been shown to be highly prevalent in emerging psychosis [6]. While this triad\u2014functional disability, social isolation and depression\u2014was found to be highly prevalent across all groups in the present study, it is also a characteristic of the deficit syndrome of schizophrenia [23]. If such symptoms are true-positive precursors of later psychosis, the non-detection of such patients contributes to a substantial delay on their way to specialised services and adequate treatment, which in turn has been shown to negatively affect the outcome of patients with first-episode psychosis [24]. Accordingly, our study confirmed earlier reports that patients with more insidious features showed longer pathway durations than patients with predominant positive symptoms [25]. Deficit syndrome patients, however, may show lower adherence to treatment and are per se characterised by worse outcomes [23].\nDelays in referral\nInterestingly, when compared to specialists and other professional groups, GPs referred patients more rapidly to other professional groups once they were contacted. In contrast, more contacts per patient were found with private psychiatrists and psychologists before final referral to the prodromal clinic was established. Partially, this finding may be explained by our large and repetitious sensitisation of GPs about the insidious features of early psychosis. However, some of the sampled patients had contacted a GP before the sensitisation had taken place, suggesting that GPs tend to refer such patients more rapidly. This would be in line with the findings from both the Swiss survey [7] and its international replication study [22], in which GPs indicated that they wished to rapidly refer patients in whom they suspected a beginning psychosis to specialised outpatient services. Given their degree of specialty, private psychiatrists and psychologists, in contrast, seem not to engage into rapid referral of these patients.\nGiven the considerable potential for recall bias, our study did not assess type of treatment that was provided to patients at each of their contacts. Thus, no conclusions can be drawn about the adequacy of applied treatment strategies. It may be the psychiatrists and psychologists that are seeing the more symptomatic and \u201cdifficult-to-treat\u201d patients who require a stable and continuous treatment setting. However, all patients included into this study were finally referred to our prodromal clinic, which may have either taken place for diagnostic assessment or for optimising treatment. It may thus be suggested that referrals of patients could have ideally been initiated already at an earlier stage along their help-seeking pathways. Interestingly, in two German studies on the pathways to care of patients with first-episode psychosis, contacts with private psychiatrists and psychologist were associated with longer duration of untreated psychosis as compared to GPs and general casualty services [17, 18]. Taken together, these findings suggest that education may not only need to include primary carers such as GPs, but also other professional groups as well as secondary carers such as private psychiatrists and psychologists.\nNumber of contacts and duration of help-seeking pathways\nFinally, the comparatively low mean number of contacts (2.38) on the pathways to care over all patient groups may express the effect of the large sensitisation of professional groups that was conducted when our prodromal clinic was established. This finding is identical with the Phillips et al. study [1] who reported a mean number of contacts of 2.36 in their at-risk sample. Additionally, the median duration from initial contact to referral to our prodromal service was very similar to the one reported by Phillips et al. [1] (42.5 vs. 41.4\u00a0weeks). In those studies assessing help-seeking pathways of first-episode cohorts, only one other study reported lower mean contacts (1.7; range 1\u20134) and also was conducted as part of an early psychosis service [5]. In comparison, number of contacts of patient samples not treated in specialised services were reported to be higher. A mean number of contact of 4.9 was reported by Johnstone et al. [2], a finding which was identically reported in a study that included patients treated before and after the establishment of an early psychosis service [3]. These findings point to the importance of specialised early psychosis services.\nWeaknesses of this paper\nFinally, a few weaknesses of this survey should be acknowledged. First, this study, like any retrospective research, is limited by an inherent potential for recall bias. Given the considerable length of pathway duration in the case of some patients, information on the exact number and on timing of contacts may be subject to errors. Ideally, all contacted professional groups would need to be contacted and interviewed in order to validate the information obtained by patients and their relatives. Second, pathways were studied on a patient sample, which was mainly constituted by subjects without manifest psychosis. Contacted professional groups may therefore not have been alarmed to refer their patients, and referral to our prodromal clinic may have only occurred in order to exclude an at-risk state for psychosis once the prodromal clinic was established. It can, however, be suggested that referrals would only take place if there is particular need for further assessment. Moreover, referrals may also have been initiated by patients themselves, although we did not control for that aspect in our study. Third, we cannot exclude that pathways may differ in patients that are never referred to our prodromal clinic. It may well be possible that an unknown number of patients in presumed at-risk states are treated by private specialists and undergo complete remission. It may therefore be a per se selection of more impaired patients that will be referred to a prodromal clinic. Although the at-risk criteria applied in the current study are now well-established in the early psychosis research community [26], it may well be possible that not all true at-risk patients meet these criteria. Thus, our study may have only captured a fraction of the individuals developing psychosis [27]. Fourth, we did not control for patient-related factors that may in a large part contribute to referral delays. These factors may include lack of insight, poor social adjustment, paranoid thoughts or avolition. Finally, the training and role of GPs in the health care system may vary across countries; thus, not all of the findings of our study may be generalisable to other settings. However, our findings emerge from a health system where specialists may be contacted without the referral of the \u201cgate-keeping\u201d GPs. Thus, in health systems with gate-keeping models our findings that GPs are more commonly contacted by individuals with insidious features may warrant appropriate education efforts even stronger.\nConclusions\nOur study confirms earlier reports of the GPs\u2019 important role on the help-seeking pathways of psychotic patients. Furthermore, our study revealed that GPs are often contacted by patients with suspected at-risk states for psychosis early on their pathways to care. The insidious nature of the symptoms presented by these patients in the early stages of their pathways imposes an important challenge for GPs, as insidious features of early psychotic stages are difficult to detect. Our results thus support the need for appropriate education of GPs on these insidious features and for rapidly accessible specialised outpatient services. In a few countries, such specialized outpatient services have been established in recent years [28]. For example, in England, under the National Health Service Plan, 50 early intervention teams have been established at a cost of \u00a370\u00a0million [29]. It can thus be expected that help-seeking pathways of patients in early phases of psychosis may be reduced, thus maybe improving the overall outcome of patients.","keyphrases":["help-seeking pathways","early psychosis","first episode","prodrome","schizophrenia"],"prmu":["P","P","P","P","P"]}
{"id":"Health_Qual_Life_Outcomes-2-_-395842","title":"A new instrument to describe indicators of well-being in old-old patients with severe dementia \u2013 The Vienna List\n","text":"Background In patients with very severe dementia self-rating of quality of life usually is not possible and appropriate instruments for proxy-ratings are not available. The aim of this project is to develop an instrument of clinical proxy-ratings for this population.\nBackground\nIn industrial societies the proportions of old people and of people suffering from dementia are steadily increasing. Consequently, the number of people depending on different types of institutional care is growing. The care is provided in general hospitals, geriatric hospitals, nursing homes, private homes, and senior residences and within the families. Obviously, there are large differences in costs and efficacy of these different types of care. In general, quality of life data have been increasingly appreciated as the key outcome measure for the assessment of therapeutic interventions and for the usefulness of various treatment facilities. Quality of life is defined by WHO as \"an individual's perception of their position in life, in the context of the culture and value systems in which they live, and in relation to their goals, expectations, standards, and concerns. It is a broad ranging concept, effected in a complex way by the person's physical health, psychological state, level of independence, social relationships and their relationships to salient features of their environment\" [1]. As concerns people with dementia, many of the instruments in use represent brief global measures, which cannot be applied to patients with severe dementia.\nThe most important reason for this slow growth of empirical data in this area probably relates to the obvious methodological problems of obtaining reliable subjective accounts of individuals with severe dementia who have compromised cognitive abilities, frequently with concurrent impaired communicative skills.\nDue to these impairments in this group of patients most of the available instruments are not applicable for assessing quality of life related issues by means of traditional measures such as questionnaires or interviews which require a highly complex procedure of introspection and evaluation, involving several components of cognition including implicit and explicit memory [2].\nFacing these problems, attempts have been made to develop direct observational methods [3] measuring positive affects such as pleasure, interest, and contentment as well as negative affects such as sadness, anxiety\/fear, and anger according to operationalized criteria during a series of 10-minutes direct observation. An alternative approach was chosen by the Bradford Research Group in the UK [4] with the Dementia Care Mapping (DCM) based on the psychosocial model of \"person-centred care\" which provides detailed observational ratings covering aspects of articulation, feeding, social withdrawal, passive engagement, walking and a number of indicators of well-being.\nLiterature searches (-2003) of Medline, Embase, Psyclit, Cinahl using the keywords dementia and well-being and quality of life were conducted. Lawton et al. [5] developed the Minimum Data Set (MDS) comprising cognition, activities of daily living, time use, depression, and problem behaviours. He proposed to include observations of demented patients' emotional behaviours [6]. Most of the measurements are derived from existing instruments and are confined to few of the following dimensions: affect, activity, enjoyment, self-esteem, and social interactions [7]. Ready and Ott concluded from their review [7] that the psychometric properties of most of the available instruments have to be regarded as preliminary.\nAs mentioned above, there were no quality of life assessment tools for patients with very severe dementia as representative of our population. Unfortunately, the instruments developed for demented patients were not found applicable to our patients who were in much more advanced stages of dementia (e.g. the Mini Mental State Examination could not be completed by any of our patients). For this reason a project was launched in 1998 by the department of Palliative Geriatrics (Geriatriezentrum am Wienerwald), to develop a new instrument based on observations made by the staff (physicians, nurses and physiotherapists) completely independent on the patients' cooperation.\nMethods\nPatients\nIn the present study 217 consecutive patients (44 males, 173 females) were included. The average age was 84 years (range 61\u2013105 years). The majority of patients suffered from severe dementia according to ICD-10 (34,5 % F00 \u2013 Dementia in Alzheimer's Disease; 61,5 % F01 \u2013 Vascular Dementia; 4,0 & F02 \u2013 other). It appeared that more women (38 %) had been diagnosed as suffering from Alzheimer's disease compared with men (23 % \u2013 \u03c72 (df = 2) = 6,05; p = 0,049). As concerns age, the patients with Alzheimer's disease (87,3 \u00b1 5,7 years) and vascular dementia (86,3 \u00b1 7,0 years) were significantly older than those with other diagnoses (78,5 \u00b1 15,6 years \u2013 t = 3,27; p = 0,002 and t = 2,74; p = 0,007 respectively).\nDevelopment of the item-pool\nThirteen staff members comprising doctor, nurses, and therapists from the department of Palliative Geriatrics at the Geriatriezentrum am Wienerwald in Vienna had observed severely demented patients during a one year period (May 1998 to April 1999). The patient's behaviour was documented at one of the wards (32 beds). Based on this documentation, 65 items for the description of behaviour in demented inpatients were derived and allocated to categories, supposedly reflecting relevant aspects of their well-being such as voice, language, mood, eye contact, acceptance of body contact, gait, muscular tension, hand movement, sleep, activities, communication, independence concerning food intake. This approach is different from prevailing approaches, which mainly are based on the use of items from existing instruments measuring specific aspects.\nSubsequently, by means of this item-list, 771 assessments of 217 in-patients in various situations such as eating, dressing, grooming had been obtained between June 1999 and September 2000 by physicians and nurses. Each of the original 65 items was scored on a 5-point Likert scale from 0 = never to 4 = always.\nFurther assessments\nPatients were diagnosed according to ICD-10. They were rated by means of the Brief Cognitive Rating Scale (BCRS) [8], the Global Deterioration Scale (GDS) [9], the Barthel-Index [10], and the Spitzer Index [11].\nThe BCRS describes the severity of cognitive impairment providing five main axes (concentration, short term memory, long term memory, orientation, and self-care ability) and five co-axes (language, psychomotoric, mood and behaviour, drawing skills, calculating skills) each rated on a 7-step scale. The GDS is a proxy rating scale to assess the severity of dementia in elderly people on a seven point Likert-type scale (1 = no impairment; 7 = most severe impairment).\nThe Barthel-Index was used to assess the activity of daily living in 10 areas (feeding, transfers bed to chair and back, grooming, toilet use, bathing, mobility, climbing stairs, dressing, stool control, bladder control).\nThe Spitzer Index is a global quality of life measure covering five areas (activity, daily life, health, social relations, future) with a maximum score of 10 points.\nThe BCRS, GDS, and Spitzer Index had been rated by physicians and the Barthel index by nurses. All ratings including the 65 items list were made on the same occasion. Data collection was carried out using electronic questionnaires implemented through the Quality-of-Life-Recorder technology [12].\nStatistical analysis\nDescriptive statistics were generated for demographic data and diagnostic categories and for the BCRS and GDS scales, for the Barthel and Spitzer Indices, as well as for the newly developed instrument.\nA factor analysis (principle component analysis, oblimin with Kaiser normalisation as rotation method), based on these 771 assessments was performed. The number of interpretable factors was determined by interpretation of a Scree plot. The consistency of the factors was tested by Cronbach's alpha coefficients. To improve the consistency of the scales, items have been deleted based on the criteria of changes in magnitude of the Cronbach's alpha coefficients and on the fit of the item with the content of the core items of the factors. To test the stability of the factor structure, we conducted separate analyses for doctors and nurses. For testing construct validity, we used the two external criteria, Brief Cognitive Rating Scale and the Barthel-Index.\nTo test for inter-rater reliability Spearman rank correlation coefficients were calculated. We included only data in which the electronic recording confirmed that it was obtained at exactly the same time.\nResults\nMost patients suffered from severe dementia as indicated by the results of the BCRS (mean \u00b1 SD: 57 \u00b1 8.8) and GDS (mean \u00b1 SD: 5.7 \u00b1 1.1) and the low level of activity of daily living (mean \u00b1 SD of Barthel Index: 26.8 \u00b1 29.7) in the present sample also reflected by the distribution of diagnoses according to ICD-10.\nOf the 771 assessments 386 had been performed by nurses and 385 assessments by physicians. By means of the electronic recordings we identified 22 pairs of assessments made at the same time by a nurse and a physician. A planned feasibility analysis after 120 assessments resulted in the exclusion of the Spitzer Index because of a general floor effect (mean score <3).\nThe factor analysis suggested five factors based on the interpretation of the Scree plot. The results of the five-factor solution are given in Table 1 with high Eigenvalues and an explained variance of more than 60 per cent.\nTable 1\nEigenvalues and explained amounts of variance for the 5-factor solution\nEigenvalues\n% of Variance\nCumulative %\nFactor\nnurse\nphysician\nnurse\nphysician\nnurse\nphysician\n1\n10.0\n10.7\n24.4\n26.0\n24.4\n26.0\n2\n5.7\n6.1\n13.8\n15.0\n38.2\n41.0\n3\n4.9\n4.8\n11.9\n11.8\n50.0\n52.1\n4\n2.5\n2.1\n6.1\n5.0\n56.1\n58.0\n5\n2.0\n2.5\n4.9\n6.1\n61.0\n63.9\nExtraction Method: Principal Component Analysis.\nTo improve the consistency of the five factors, 18 of the original 65 items were deleted based on the criteria of changes in magnitude of the Cronbach alpha coefficients and on the fit of the item with the content of the core items of the factors. As a next step, eight further items were excluded due to different factor-loadings between raters and of content considerations resulting in the following five factors: communication (15 items), negative affect (10 items), bodily contact (5 items), aggression (4 items) and mobility (6 items). The factor structure matrix including the single items for physicians and nurses is shown in table 2 and demonstrates a high congruency of the factor structure between both groups of raters on the item level.\nTable 2\nStructure Matrix\nFactor\n1\n2\n3\n4\n5\nNurse\nPhysician\nNurse\nPhysician\nNurse\nPhysician\nNurse\nPhysician\nNurse\nPhysician\nCommunication\nITEM 59\n0.82\n0.83\n-0.19\n-0.14\n0.04\n-0.32\n0.29\n0.05\n0.06\n0.25\nITEM 62\n0.81\n0.64\n0.02\n0.16\n0.08\n-0.39\n0.28\n0.22\n-0.09\n0.34\nITEM 6\n0.79\n0.78\n-0.26\n0.13\n-0.07\n-0.29\n0.18\n0.08\n0.10\n0.17\nITEM 61\n0.79\n0.67\n-0.23\n-0.13\n0.10\n-0.56\n0.22\n0.09\n0.08\n0.25\nITEM 8\n0.78\n0.81\n-0.20\n0.08\n-0.16\n-0.25\n0.14\n0.12\n0.12\n0.18\nITEM 65\n0.73\n0.77\n-0.27\n-0.13\n-0.05\n-0.05\n0.52\n0.10\n0.01\n0.48\nITEM 56\n0.70\n0.69\n-0.08\n0.08\n-0.15\n0.07\n0.07\n-0.06\n0.05\n0.10\nITEM 14\n0.70\n0.85\n-0.11\n-0.11\n0.02\n-0.10\n0.44\n-0.06\n0.15\n0.26\nITEM 32\n0.66\n0.64\n-0.14\n-0.06\n0.35\n-0.48\n0.03\n-0.11\n0.15\n0.22\nITEM 31\n0.65\n0.65\n-0.12\n0.01\n0.32\n-0.54\n0.08\n0.00\n0.15\n0.19\nITEM 13\n0.64\n0.76\n-0.24\n-0.04\n0.06\n-0.42\n0.11\n0.13\n0.12\n0.20\nITEM 50\n0.64\n0.77\n-0.40\n-0.19\n-0.04\n-0.17\n0.46\n-0.03\n0.19\n0.35\nITEM 64\n0.62\n0.61\n0.05\n0.13\n0.06\n-0.17\n0.49\n-0.21\n0.03\n0.40\nITEM 60\n0.59\n0.69\n0.12\n0.17\n-0.27\n0.03\n0.01\n0.28\n-0.11\n0.07\nITEM 15\n0.45\n0.73\n0.03\n-0.13\n-0.08\n0.19\n0.34\n-0.19\n0.12\n0.06\nNegative Affect\nITEM 18\n-0.08\n0.04\n0.83\n0.87\n-0.03\n-0.14\n-0.08\n0.11\n-0.28\n-0.02\nITEM 22\n-0.04\n0.09\n0.80\n0.74\n0.11\n-0.34\n-0.10\n0.08\n-0.20\n0.00\nITEM 3\n-0.07\n0.02\n0.75\n0.82\n0.16\n-0.18\n-0.13\n0.17\n-0.11\n-0.06\nITEM 24\n-0.09\n-0.01\n0.74\n0.76\n-0.05\n-0.18\n-0.05\n0.12\n-0.37\n0.08\nITEM 27\n0.04\n0.02\n0.66\n0.77\n-0.13\n-0.10\n-0.17\n0.45\n-0.45\n-0.05\nITEM 17\n-0.28\n-0.17\n0.66\n0.71\n-0.08\n0.14\n-0.16\n0.38\n-0.58\n-0.12\nITEM 25\n-0.30\n0.17\n0.65\n0.63\n0.03\n-0.02\n-0.29\n-0.02\n-0.08\n-0.07\nITEM 54\n-0.20\n-0.20\n0.62\n0.58\n-0.09\n0.10\n0.19\n0.28\n-0.34\n0.05\nITEM 53\n-0.10\n-0.01\n0.60\n0.50\n-0.07\n0.23\n0.14\n0.17\n-0.28\n0.01\nITEM 47\n-0.28\n-0.26\n0.53\n0.61\n0.00\n0.11\n-0.26\n0.37\n-0.45\n-0.11\nBodily contact\nITEM 35\n0.00\n0.17\n0.02\n-0.03\n0.93\n-0.76\n-0.08\n-0.13\n0.11\n-0.11\nITEM 34\n0.01\n0.27\n0.00\n0.07\n0.93\n-0.81\n-0.09\n-0.12\n0.12\n-0.09\nITEM 33\n0.03\n0.37\n0.00\n0.14\n0.90\n-0.77\n-0.09\n-0.06\n0.12\n0.00\nITEM 36\n-0.06\n-0.21\n0.01\n0.10\n0.89\n-0.81\n-0.13\n-0.15\n0.16\n-0.08\nITEM 37\n-0.06\n-0.27\n0.01\n0.15\n0.86\n-0.76\n-0.12\n-0.16\n0.17\n-0.06\nAggression\nITEM 4\n-0.14\n0.01\n0.25\n0.27\n-0.19\n0.08\n0.13\n0.91\n-0.88\n0.08\nITEM 19\n-0.24\n-0.05\n0.31\n0.18\n-0.22\n0.16\n0.13\n0.84\n-0.83\n0.09\nITEM 1\n0.05\n0.20\n0.28\n0.20\n-0.07\n0.12\n0.01\n0.71\n-0.78\n0.08\nITEM 29\n0.22\n0.11\n0.20\n0.21\n-0.10\n0.05\n0.03\n0.84\n-0.77\n0.15\nMobility\nITEM 40\n0.42\n0.56\n-0.31\n-0.23\n-0.20\n0.08\n0.79\n-0.14\n0.22\n0.80\nITEM 41\n0.49\n0.64\n-0.28\n-0.27\n-0.23\n0.07\n0.71\n-0.19\n0.23\n0.63\nITEM 57\n0.01\n0.01\n0.01\n0.08\n-0.05\n0.00\n0.68\n0.14\n-0.18\n0.70\nITEM 42\n0.04\n0.07\n0.07\n0.06\n-0.02\n0.05\n0.68\n0.20\n-0.18\n0.80\nITEM 43\n0.43\n0.63\n-0.43\n-0.15\n-0.08\n-0.09\n0.66\n0.00\n0.22\n0.64\nITEM 55\n0.41\n0.46\n-0.14\n-0.07\n-0.10\n0.04\n0.42\n-0.23\n0.03\n0.34\nExtraction Method: Principal Component Analysis. Rotation Method: Oblimin with Kaiser Normalization.\nIn addition, it appeared that the factors were generally unrelated to each other except for significant correlations between the factors 'communication' and 'bodily contact' (r = 0.25; p < 0.001 each) and of 'mobility' with 'negative affect' (physicians: r = 0.22; p = 0.001; nurses: r = -0.33; p < 0.01). The Cronbach alpha coefficients as a measure of internal consistency were high for both nurses and physicians (table 3). The congruence of nurses' and physician's ratings is further demonstrated by similar item severity (relative ratings) and selectivity of the single factors (table 4).\nTable 3\nCronbach alpha coefficients\nFactor\nNumber of Items\nNurse\nPhysician\n1 \u2013 Communication\n15\n0.93\n0.94\n2 \u2013 Negative Affect\n10\n0.88\n0.89\n3 \u2013 Bodily Contact\n5\n0.90\n0.90\n4 \u2013 Aggression\n4\n0.86\n0.87\n5 \u2013 Mobility\n6\n0.81\n0.82\nTable 4\nItem severity and Item selectivity\nItem severity\nItem selectivity\nCommunication\nNurse\nPhysician\nNurse\nPhysician\n59 Responding to distant calls\n0.57\n0.63\n0.82\n0.85\n62 Looking for contact\n0.53\n0.56\n0.81\n0.71\n06 Speaks comprehensibly\n0.64\n0.69\n0.79\n0.81\n61 Contact possible\n0.83\n0.90\n0.79\n0.74\n08 Speaks meaningful groups of words\n0.59\n0.68\n0.77\n0.82\n65 Eats and drinks by him-\/herself\n0.66\n0.68\n0.79\n0.78\n56 Reads newspaper\n0.24\n0.19\n0.69\n0.64\n14 Carries out simple orders\n0.39\n0.64\n0.73\n0.84\n32 Maintains visual contact\n0.72\n0.74\n0.65\n0.71\n31 Visual contact possible\n0.80\n0.85\n0.63\n0.72\n13 Comprehends single words\n0.89\n0.88\n0.63\n0.81\n50 Uses both hands intentionally\n0.62\n0.67\n0.70\n0.79\n64 Worries about others\n0.25\n0.23\n0.65\n0.63\n60 Rings the bell\n0.29\n0.31\n0.58\n0.67\n15 Carries out complicated orders\n0.08\n0.24\n0.47\n0.64\nNegative Affect\n18 Full of despair\n0.40\n0.43\n0.82\n0.86\n22 Sad\/crying\n0.32\n0.36\n0.77\n0.75\n03 Whining voice\n0.33\n0.32\n0.72\n0.83\n24 Nervous\/anxious\n0.37\n0.40\n0.76\n0.75\n27 Wailing\n0.30\n0.31\n0.69\n0.80\n17 Tensed\n0.36\n0.43\n0.73\n0.72\n25 Resignated\n0.27\n0.38\n0.65\n0.63\n54 Restless\/confused\n0.25\n0.31\n0.62\n0.59\n53 Problems to fall asleep\n0.25\n0.36\n0.59\n0.49\n47 Muscular tension\n0.37\n0.43\n0.60\n0.62\nBodily Contact\n35 Bodily contact possible at shoulders\n0.77\n0.87\n0.92\n0.78\n34 Bodily contact possible at arms\n0.81\n0.91\n0.90\n0.80\n33 Bodily contact possible at hands\n0.82\n0.94\n0.88\n0.73\n36 Bodily contact possible at the head\n0.63\n0.63\n0.91\n0.88\n37 Bodily contact possible in the face\n0.56\n0.55\n0.88\n0.84\nAggression\n04 Aggressive voice\n0.27\n0.27\n0.89\n0.90\n19 Aggressive acts\n0.21\n0.19\n0.85\n0.84\n01 Loud voice\n0.32\n0.33\n0.80\n0.80\n29 Insulting others\n0.23\n0.22\n0.81\n0.88\nMobility\n40 Walking upright\n0.37\n0.40\n0.92\n0.95\n41 Walking straightup to\n0.33\n0.36\n0.87\n0.87\n57 Departs from ward\n0.07\n0.05\n0.53\n0.46\n42 Wanders around\n0.16\n0.17\n0.53\n0.62\n43 Sitting upright\n0.57\n0.57\n0.80\n0.81\n55 Ready to help on ward\n0.13\n0.09\n0.55\n0.54\nFor testing construct validity, we used two external criteria, the Brief Cognitive Rating Scale (BCRS) used by physicians and the Barthel-Index used by nurses (table 5). The correlation coefficients between the various areas of the BCRS and the two relevant scales of the new instrument (communication and mobility) point to a satisfactory validity. Concerning the second criteria, the Barthel-Index (a measure of activities of daily living), it was significantly correlated with the scales \"communication\" and \"mobility\" of our instrument. Furthermore, the latter was correlated with the scale \"negative affect\" and \"acceptance of body contact\" in the expected direction.\nTable 5\nCorrelations with BCRS scores and Barthel Index\nCommunication\nNegative Affect\nBodily contact\nAggression\nMobility\nPhysicians\nBCRS 1 \u2013 concentration\n-0.71*\n0.05\n0.07\n-0.02\n-0.45*\nBCRS 2 \u2013 short time memory\n-0.67*\n0.02\n0.11\n-0.01\n-0.42*\nBCRS 3 \u2013 long time memory\n-0.68*\n0.14\n0.09\n0.08\n-0.46*\nBCRS 4 \u2013 orientation\n-0.65*\n0.12\n0.12\n0.11\n-0.40*\nBCRS 5 \u2013 everyday life competency\n-0.47*\n-0.04\n-0.04\n-0.10\n-0.44*\nBCRS 6 \u2013 language\n-0.71*\n-0.02\n0.02\n-0.09\n-0.37*\nBCRS 7 \u2013 psychomotorics\n-0.41*\n-0.01\n0.06\n-0.10\n-0.59*\nBCRS 8 \u2013 mood and behaviour\n-0.60*\n0.10\n-0.02\n0.03\n-0.34*\nBCRS 9 \u2013 constructive skills\n-0.55*\n0.03\n0.02\n-0.06\n-0.34*\nBCRS 10 \u2013 calculation skills\n-0.59*\n0.17*\n0.09\n0.09\n-0.35*\nMain axis\n-0.73*\n0.07\n0.09\n0.02\n-0.49*\nCo-axis\n-0.71*\n0.07\n0.04\n-0.03\n-0.49*\nBCRS total score\n-0.74*\n0.07\n0.06\n-0.01\n-0.50*\nNurses\nBarthel Item 1 \u2013 feeding\n0.70*\n-0.21*\n-0.10\n0.02\n0.63*\nBarthel Item 2 \u2013 transfer\n0.46*\n-0.27*\n-0.17*\n-0.08\n0.83*\nBarthel Item 3 \u2013 personal care\n0.41*\n-0.17*\n-0.02\n-0.12\n0.36*\nBarthel Item 4 \u2013 toilet use\n0.47*\n-0.25*\n-0.20*\n-0.12\n0.67*\nBarthel Item 5 \u2013 bathing\n0.08\n-0.07\n0.04\n-0.06\n0.09\nBarthel Item 6 \u2013 moving\n0.43*\n-0.28*\n-0.18*\n-0.09\n0.83*\nBarthel Item 7 \u2013 stairs\n0.32*\n-0.23*\n-0.14\n-0.03\n0.72*\nBarthel Item 8 \u2013 dressing\n0.51*\n-0.23*\n-0.22*\n-0.09\n0.67*\nBarthel Item 9 \u2013 bowel\n0.48*\n-0.20*\n-0.16*\n-0.15*\n0.57*\nBarthel Item 10 \u2013 bladder control\n0.44*\n-0.20*\n-0.22*\n0.14\n0.56*\nBarthel Index\n0.56*\n-0.28*\n-0.20*\n-0.10\n0.83*\n* p < 0.001\nWhen testing for gender differences concerning the factors, we found significant differences for all but one factor (table 6).\nTable 6\nFactor Scores of Observations by Gender of the Patients\nFactor\nObservations of Males (x \u00b1 SD) N = 123\nObservations of Females (x \u00b1 SD) N = 648\nt-score\np value\n1\u00a0\u2013\u00a0Communication\n34.8 \u00b1 13.2\n33.8 \u00b1 11.8\n0.82\n0.415\n2\u00a0\u2013\u00a0Negative\u00a0Affect\n9.5 \u00b1 6.7\n14.8 \u00b1 6.7\n-8.37\n<0.001\n3 \u2013 Bodily Contact\n14.9 \u00b1 4.3\n16.7 \u00b1 4.9\n-4.18\n<0.001\n4 \u2013 Aggression\n3.1 \u00b1 3.0\n4.3 \u00b1 3.5\n-3.21\n0.001\n5 \u2013 Mobility\n8.1 \u00b1 5.5\n6.3 \u00b1 5.5\n3.38\n0.001\nThe interrater-reliability between sub-samples of physicians and nurses proved to be satisfactory (table 7).\nTable 7\nPaired sample test and Spearman Rank correlation coefficients between nurses and physicians related to the same patient at the same day (22 pairs)\nFactor\nNurse (x \u00b1 SD)\nPhysician (x \u00b1 SD)\nt-score\/p\nR\/p\n1\u00a0\u2013\u00a0Communication\n25.8 \u00b1 10.5\n26.4 \u00b1 8.9\n-0.35\/0.727\n0.71\/<0.001\n2\u00a0\u2013\u00a0Negative\u00a0Affect\n11.9 \u00b1 7.6\n8.6 \u00b1 5.0\n2.46\/0.023\n0.57\/0.006\n3 \u2013 Bodily contact\n15.9 \u00b1 5.7\n18.4 \u00b1 2.9\n-2.22\/0.038\n0.53\/0.011\n4 \u2013 Aggression\n4.3 \u00b1 3.0\n2.2 \u00b1 2.1\n3.69\/0.001\n0.35\/0.112\n5 \u2013 Mobility\n6.5 \u00b1 5.7\n5.1 \u00b1 5.8\n2.03\/0.056\n0.81\/<0.001\nDiscussion\nThe special problem in the assessment of well-being in patients with severe dementia is their lack of competence which is compromising the reliability of their reports. Consequently, observer ratings are the only alternative for such self-ratings. However, observer ratings inherit the potential risk of overrating the well-being of patients if the provider and rater of health care services are identical. We have controlled for this risk by semi-quantitatively describing the frequency of distinct behaviour patterns in demented patients.\nThe results of this study demonstrate that the behaviour of old-old patients with severe dementia can be described by five factors of the Vienna List. By explaining more than 60% of the total variance these five factors obviously cover a considerable part of the possible spectrum of behaviour in these patients.\nSince nurses and physicians have different intensity of contact and corresponding different perspectives, it was surprising that their assessments were highly correlated in three of the five factors. The two factors, aggression and mobility, yielded higher scores among the nurses as compared to doctors.\nAs concerns aggression, there are mainly two explanations for this difference. Firstly, nurses spend more time and have closer contact with the patients and consequently have a higher risk to induce aggressive behaviour in the patients. In addition, the extended period of contact increases the chance to experience an episode of aggressive behaviour. Secondly, patients normally behave differently towards nurses and doctors due to differences in role expectation and familiarity related to the frequency of contact. However, we consider this later explanation as unlikely in these patients due to their cognitive impairment.\nRegarding mobility it is plausible that the doctors report lower scores for mobility of the patients as the doctor mainly sees the patient under certain circumstances, i.e. during the rounds where the ward routines limit the mobility of the patient.\nSince these five factors encompass most of the behavioural repertoire of demented old-old patients we assume that these factors can be regarded as a useful approach to describe the well-being in these patients.","keyphrases":["well-being","old-old","severe dementia","quality of life"],"prmu":["P","P","P","P"]}
{"id":"Acta_Neuropathol_(Berl)-3-1-2039821","title":"Detection of amyloid beta aggregates in the brain of BALB\/c mice after Chlamydia pneumoniae infection\n","text":"Neuroinflammation, initiated by cerebral infection, is increasingly postulated as an aetiological factor in neurodegenerative diseases such as Alzheimer\u2019s disease (AD). We investigated whether Chlamydia pneumoniae (Cpn) infection results in extracellular aggregation of amyloid beta (A\u03b2) in BALB\/c mice. At 1 week post intranasal infection (p.i.), Cpn DNA was detected predominantly in the olfactory bulbs by PCR, whereas brains at 1 and 3 months p.i. were Cpn negative. At 1 and 3 months p.i., extracellular A\u03b2 immunoreactivity was detected in the brain of Cpn-infected mice but also in the brain of mock-infected mice and mice that were neither Cpn infected nor mock infected. However, these extracellular A\u03b2 aggregates showed morphological differences compared to extracellular A\u03b2 aggregates detected in the brain of transgenic APP751SL\/PS1M146L mice. These data do not unequivocally support the hypothesis that Cpn infection induces the formation of AD-like A\u03b2 plaques in the brain of BALB\/c mice, as suggested before. However, future studies are required to resolve these differences and to investigate whether Cpn is indeed an etiological factor in AD pathogenesis.\nIntroduction\nThe most common cause of dementia is Alzheimer\u2019s disease (AD), which manifests in a familial and a sporadic form. The neuropathology in both conditions is characterized by neuritic or senile plaques, consisting mainly of amyloid beta protein (A\u03b2) and neurofibrillary tangles with hyperphosphorylated tau protein as its key components. Mutations in the amyloid precursor protein (APP), presenilin-1 (PS-1) or -2 (PS-2) genes have been associated with the familial form of AD [27]. However, the most common type of this disease, sporadic AD, is not due to these mutations, and although risk factors have been identified, the initiating trigger is still unknown [18]. The potential importance of infections in the development of sporadic AD has been recently recognized [1, 21, 31]. Specifically, not only various viruses like the human immunodeficiency virus, herpes simplex virus and cytomegalovirus but also bacteria like Borrelia burgdorferi, have been linked to the process of neurodegeneration [6, 12, 13, 17, 20]. Some data also suggest that Chlamydia pneumoniae (Cpn) should be added to the list of pathogens possibly involved in the pathogenesis of AD. Cpn is a widespread, obligatory intracellular pathogen, often causing acute respiratory infections including pneumonia, sinusitis and bronchitis [10, 11, 15]. This microorganism has also been implicated in a variety of severe, chronic diseases including some disorders of the central nervous system [7, 14, 30, 32]. In an initial study, Balin et al. demonstrated the presence of Cpn in brains of 17 out of 19 patients with sporadic AD, using a variety of techniques, including PCR, electron microscopy and immunohistochemistry, while the pathogen could only be demonstrated in 1 out of 19 controls [2]. The presence of Cpn was demonstrated in various cell types like microglia, astrocytes, perivascular macrophages and monocytes, and predominantly in brain areas linked with AD pathology. Recently, these observations were confirmed [8]. On the contrary, others were unable to detect Cpn in AD patient tissues [9, 22, 23, 28]. The only animal study so far supporting a role for this pathogen in AD demonstrated an AD-like pathology in the brain of BALB\/c mice post Cpn infection [19]. Three-month-old mice were inoculated intranasally with Cpn and the authors claimed that this resulted in the deposition of A\u03b2 in the brain up to 3\u00a0months post infection with increasing pathology as the infection progressed. Based on these results, it was concluded that in na\u00efve BALB\/c mice, AD-like pathology could be induced by Cpn infection. In spite of these results, doubts remain concerning the amyloid-like plaques as being only unrelated elevations in amyloid [24]. Because of the possible implications of this study and the need for confirmation, it was our aim to repeat these experiments under quite corresponding conditions. Moreover, we compared our findings with the pathology found in a transgenic mouse model of AD.\nMaterials and methods\nChlamydia pneumoniae\nThe Cpn strain, TWAR 2043 (ATCC) was cultured and continuously propagated on Hep2 cells as previously described [25]. Bacterial titres were determined by titration in Hep2 cells [7] and staining with a monoclonal Chlamydia LPS-specific antibody followed by a FITC-conjugated secondary antibody (RR-402; DAKO, Glostrup, Denmark). Titres were expressed as the number of inclusion forming units (IFU) per ml.\nAnimals and infection protocol\nA total of 43 female, specific pathogen-free inbred BALB\/c mice were obtained from Charles River (Someren, The Netherlands). Animals were kept under standard housing and care conditions at the Central Animal Facilities of Maastricht University (Maastricht, The Netherlands). All experimental procedures were approved by the Ethical Committee for the Use of Experimental Animals of Maastricht University.\nAt 3\u00a0months of age n\u00a0=\u00a024 mice were inoculated intranasally with 107 IFU Cpn in 10\u00a0\u03bcl (5\u00a0\u03bcl per nostril), and were analyzed at 1\u00a0week, 1\u00a0month or 3\u00a0months p.i., respectively, as shown in Table\u00a01. Pilot experiments revealed that this dosage ensures both survival of the mice and optimal dissemination throughout the body. Another n\u00a0=\u00a015 mice were mock infected with a sucrose-phosphate-glucose solution (SPG, solution for storage of Cpn) at 3\u00a0months of age and were also analyzed at 1\u00a0week, 1\u00a0month or 3\u00a0months after mock infection, as shown in Table\u00a01. In addition, two mice, which did not receive any treatment, were investigated at 4\u00a0months of age, as well as two mice at 6\u00a0months of age (Table\u00a01).\nTable\u00a01Numbers of animals per group investigated in the present studyMouse strain; analysisInfection with CpnMock infectionNo treatment1w1m3m1w1m3mM4M6M17BALB\/c; PCR444111\u2013\u2013\u2013BALB\/c; IHC\u201366\u20136622\u2013APP751SL\/PS1M146L\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u20131CpnChlamydia pneumoniae, IHC immunohistochemistry, 1w\/1m\/3m, analysis 1\u00a0week (or 1\u00a0month or 3\u00a0months, respectively) after Cpn infection or mock infection at 3\u00a0months of age. M4\/M6\/M17, 4, 6 or 17\u00a0months of age, respectively\nIn addition, a 17-month-old transgenic mouse expressing human mutant APP751 (carrying the Swedish and London mutations KM670\/671NL and V717I, under the Thy1 promoter) and human mutant presenilin-1 (PS-1 M146L, under the HMG promoter) was used (APP751SL\/PS1M146L mouse; for a detailed description see refs [4] and [24]) (Table\u00a01). This APP751SL\/PS1M146L mouse was bred on a CBA (12.5%)\u00a0\u00d7\u00a0C57Bl6 (87.5%) background.\nDetection of Cpn DNA in brain tissue with real-time PCR\nAs shown in Table\u00a01, 15 mice were used to detect Cpn DNA in brain tissue with real-time PCR at different time points after infection with Cpn or mock infection. To this end the animals received a weight-adjusted dose of pentobarbital (Nembutal; Sanofi Sante B.V., Maassluis, The Netherlands) and were then perfused with sterile phosphate buffered saline. Brains were removed quickly and dissected into five different regions, i.e., olfactory bulbs, cerebellum, hippocampus, subventricular zone and neocortex. The isolated regions were snap-frozen in liquid nitrogen and stored at \u221280\u00b0C until further processing.\nDNA was isolated from brain material using methods previously described [5]. The Nanodrop\u00ae ND-1000, a full-spectrum spectrophotometer (220\u2013750\u00a0nm) was used to calculate the DNA content of each sample. Finally, the Cpn DNA copy number\/\u03bcg DNA in the collected brains was determined by real-time PCR (ABI Prism 7000; Applied Biosystems, Foster City, CA, USA) using the following primers and probe; forward (5\u2032TCCGCATTGCTCAGCC3\u2032), reverse (5\u2032AAACAATTTGCATGAAGTCTGAGAA3\u2032) and probe (5\u2032-FAM-TAAACTTAACTGCATGGAACCCTTCTTTACTAGG3\u2032-TAMRA) as described previously [5, 29]. All PCR runs were performed in duplo. Mean Cpn DNA copy numbers\/\u03bcg brain DNA of three independent experiments (\u00b1SEM) was calculated. Then, comparisons between all groups of mice, analyzed for the presence of Cpn DNA in brain tissue, were performed with three-way ANOVA with Treatment (Cpn infection or mock infection), Time-point of analysis (1\u00a0week, 1\u00a0month or 3\u00a0months p.i.) and Brain region (olfactory bulbs, cerebellum, hippocampus, subventricular zone or neocortex) as fixed factors. P values smaller than 0.05 were considered statistically significant. Calculations were performed with SPSS v. 12.0.1 for Windows (SPSS, Chicago, IL, USA).\nDetection of A\u00df aggregates in brain tissue with immunohistochemistry\nAs also shown in Table\u00a01, n\u00a0=\u00a029 mice were used to detect A\u03b2 aggregates in brain tissue with immunohistochemistry at different time points after infection with Cpn, mock infection or without any treatment, respectively. To this end, the animals received a weight-adjusted dose of pentobarbital (Nembutal; Sanofi Sante B.V., Maassluis, The Netherlands) and were then subjected to intracardial perfusion fixation, as previously described [26]. In case of the APP751SL\/PS1M146L mouse, anaesthesia was performed with chloral hydrate (10% aqueous solution, 0.005\u00a0ml\/g body weight, i.p.). Brains were removed rapidly and post-fixed for 2\u00a0h at 4\u00b0C in the same fixative, omitting the glutaraldehyde. In case of the APP751SL\/PS1M146L mouse, the brains were halved in the midsagittal line. Brain tissues were then cryoprotected by immersion in 30% sucrose in Tris-buffered saline at 4\u00b0C overnight. Afterwards, brains (or brain halves, respectively) were quickly frozen and stored at \u221280\u00b0C until further processing.\nFor immunohistochemical analysis, the entire brains (or the right brain half in case of the APP751SL\/PS1M146L mouse) were cut into entire series of 30-\u03bcm-thick frontal sections on a cryostat (Leica CM 3050 S; Leica, Nussloch, Germany). After cutting, sections were again stored at \u221280\u00b0C until further processing.\nImmunohistochemical detection of A\u03b2 (as well as of glial fibrillary acidic protein) was performed with standard immunofluorescence procedures using a first layer, consisting of a combination of mouse anti-human amyloid 17\u201324 (4G8 clone; 1:1,200 in TBS-T; Sigma-Aldrich, St Louis, USA) [3, 16, 33] and polyclonal rabbit anti-mouse GFAP (1:1,600 in TBS-T; DAKO, Glostrup, Denmark). The corresponding secondary antibodies were donkey anti-mouse Alexa 488 (1:100 in TBS-T; Molecular Probes) and donkey anti-rabbit Alexa 594 (1:100 in TBS-T; Molecular Probes). Finally, counterstaining was performed with Hoechst (1:500 in TBS; Sigma, St Louis, MO, USA) and sections were mounted on gelatine-coated glasses. As a negative control, sections were only incubated with the secondary antibodies.\nDetection of extracellular A\u00df aggregates in brain tissue with thioflavin-S\nFor the detection of extracellular, fibrillary A\u03b2, a standard thioflavin-S (Sigma) staining was performed. Finally, the sections were counterstained with Hoechst and mounted as described above.\nMicroscopy and photography\nMicroscopic inspection of sections was performed with a MBF Bioscience Stereo Investigator Confocal Spinning Disk (SI-SD) system (MBF Bioscience; Williston, VT, USA), consisting of a modified Olympus BX51 fluorescence microscope (Olympus, Tokyo, Japan) with UPlanSApo objectives 10\u00a0\u00d7\u00a0(N.A.\u00a0=\u00a00.4), 40\u00a0\u00d7\u00a0(N.A\u00a0=\u00a00.9) and 100\u00a0\u00d7\u00a0(oil; N.A\u00a0=\u00a01.4); Olympus, customized spinning disk unit (DSU; Olympus), computer-controlled excitation and emission filter wheels (Olympus), three-axis high-accuracy computer-controlled stepping motor specimen stage (4\u00a0\u00d7\u00a04 Grid Encoded Stage; Ludl Electronic Products, Hawthorne, NY, USA), linear z-axis position encoder (Ludl), ultra-high sensitivity monochrome electron multiplier CCD camera (1,000\u00a0\u00d7\u00a01,000 pixels, C9100-02; Hamamatsu Photonics, Hamamatsu City, Japan) and controlling software (MBF Bioscience). In case of sections processed for the detection of A\u00df and GFAP, digital RGB image stacks showing the same microscopic field in 50 consecutive focal planes with a distance of 0.5\u00a0\u03bcm between the focal planes were generated with the 40\u00d7 objective. In the case of sections stained with thioflavin-S, digital image stacks showing the same microscopic field at 150 consecutive RGB focal planes with a distance of 0.2\u00a0\u03bcm between the focal planes were generated with the 100\u00d7 objective. From these image stacks three-dimensional reconstructions (maximum intensity projections) were produced with Imaris software (Version 4.0; Bitplane, Zurich, Switzerland). In addition, low-power photomicrographs (10\u00d7) were taken from sections stained with thioflavin-S. Final figures were constructed using Corel Photo-Paint v.11 and Corel Draw v.11 (Corel, Ottawa, Canada). Only minor adjustments of contrast and brightness were made, without altering the appearance of the original materials. No deconvolution was performed on images obtained by confocal microscopy.\nResults\nPresence of Cpn in the brain of BALB\/c mice infected with Cpn\nAt 1\u00a0week p.i., the mean Cpn DNA copy number per \u03bcg DNA was 310.97\u00a0\u00b1\u00a061.6 (mean\u00a0\u00b1\u00a0SEM) in the olfactory bulbs, 2.6\u00a0\u00b1\u00a00.8 in the cerebellum and 7.9\u00a0\u00b1\u00a00.8 in the hippocampus, while no Cpn DNA was detected in the subventricular zone and the neocortex (Table\u00a02). No Cpn DNA was detected in any brain region of the Cpn infected mice at 1 and 3\u00a0months p.i.. Furthermore, Cpn DNA was not detected in the brain of mock-infected BALB\/c mice at any time point. Statistical analysis revealed significant differences among the groups of mice with respect to Treatment (T) (P\u00a0=\u00a00.024), Time point of analysis (Tpoa) (P\u00a0=\u00a00.008) and Brain region (Br) (P\u00a0=\u00a00.002), as well as with respect to the combinations T\u2013Tpoa (P\u00a0=\u00a00.008), T\u2013Br (P\u00a0=\u00a00.002), Tpoa\u2013Br (P\u00a0<\u00a00.001) and T\u2013Tpoa\u2013Br (P\u00a0<\u00a00.001).\nTable\u00a02Detection of Cpn DNA in various brain regions (olfactory bulbs, cerebellum, hippocampus, subventricular zone and neocortex) with real-time PCR at 1\u00a0week, 1\u00a0month and 3\u00a0months post infection1\u00a0week p.i.1\u00a0month p.i.3\u00a0months p.i.Olfactory bulbs310.97\u00a0\u00b1\u00a061.600Cerebellum2.6\u00a0\u00b1\u00a00.800Hippocampus7.9\u00a0\u00b1\u00a00.800Subventricular zone000Neocortex000Data were expressed as mean values of three independent experiments \u00b1SEM\nDetection of A\u03b2 and GFAP in the brain of BALB\/c and APP751SL\/PS1M146L mice\nAmyloid beta immunoreactivity was found in the brains of all mice, i.e. in Cpn-infected BALB\/c mice at both 1 and 3\u00a0months p.i. (Fig.\u00a01a), mock-infected BALB\/c mice at both 1 and 3\u00a0months after mock infection (Fig.\u00a01b), not inoculated BALB\/c mice (Fig.\u00a01c) and in APP751SL\/PS1M146L mice (Fig.\u00a01d). However, A\u03b2-immunopositive aggregates were found only occasionally in the brains of the BALB\/c mice (on average one to two aggregates per section without a preference for a certain brain region), whereas they were found abundantly in hippocampus and neocortex of APP751SL\/PS1M146L mice (see also Fig.\u00a02H in [24]). Furthermore, there were several qualitative differences between the APP751SL\/PS1M146L and the BALB\/c mice. First, in the brain of APP751SL\/PS1M146L mice, the materials immunopositive for A\u03b2 formed a dense core, devoid of Hoechst-positive structures (i.e. cell nuclei). In contrast, the materials immunopositive for A\u03b2 in the brain of BALB\/c mice did not present with a dense core, and Hoechst-positive structures were scattered within these materials. Second, the astrocytic reaction in the brain of APP751SL\/PS1M146L mice, surrounding the A\u03b2-immunopositive materials, was much stronger than in the brain of BALB\/c mice (arrowheads in Fig.\u00a01a\u2013d). Third, in the brain of APP751SL\/PS1M146L mice, cells with intracellular deposits of A\u03b2 were found (asterisk in Fig.\u00a01d), which was not observed in the brain of BALB\/c mice, irrespective of treatment.\nFig.\u00a01Immunohistochemical detection of A\u03b2 (green) and GFAP (red) in 30\u00a0\u03bcm-thick cryostat sections counterstained with Hoechst (blue) from the brain of a 6-month-old Cpn-infected BALB\/c mouse 3\u00a0months post infection (a), a 6-month-old mock infected BALB\/c mouse 3\u00a0months after mock infection (b), a 6-month-old BALB\/c mouse neither infected with Cpn nor mock infected (c) and a 17-month-old APP751SL\/PS1M146L transgenic mouse (d). Note that material immunopositive for A\u03b2 was found in the brain of all mice (arrows). However, there were differences between the APP751SL\/PS1M146L mouse and the BALB\/c mice: (1) in the brain of the APP751SL\/PS1M146L mouse the material immunopositive for A\u03b2 formed a dense core devoid of Hoechst-positive structures (i.e. cell nuclei). In contrast, the material immunopositive for A\u03b2 in the brain of the BALB\/c mice did not present with a dense core, and Hoechst-positive structures were scattered within it. (2) The astrocytic reaction in the brain of the APP751SL\/PS1M146L mouse surrounding the material immunopositive for A\u03b2 was much stronger than in the brain of the BALB\/c mice (arrowheads). (3) In the brain of the APP751SL\/PS1M146L mouse cells with intracellular deposits of material immunopositive for A\u00df were found (asterisk in d). This was not observed in the brain of the BALB\/c mice. Furthermore, extracellular thioflavin-S-positive structures in the brain of the 17-month-old APP751SL\/PS1M146L mouse are shown [e (arrowheads) and f, resembling fibrillary A\u03b2 deposits. Scale bar\u00a0=\u00a025\u00a0\u03bcm in a\u2013d, 100\u00a0\u03bcm in e and 10\u00a0\u03bcm in f\nDetection of thioflavin-S-positive structures in the brain of APP751SL\/PS1M146L but not in the brain of BALB\/c mice\nIn the brain of APP751SL\/PS1M146L mice, extracellular thioflavin-S-positive structures were found (Fig.\u00a01e,\u00a0f) as also previously reported [4, 26], resembling fibrillary A\u03b2 deposits. Such extracellular thioflavin-S-positive structures were not found in the brain of BALB\/c mice.\nDiscussion\nThe role of inflammatory factors, predominantly caused by infections, in the initiation or acceleration of neurodegenerative diseases is increasingly recognized. Next to several viruses [6, 12, 13, 17], the obligatory intracellular bacterium Cpn has also been linked to a variety of neurodegenerative diseases such as AD. In 1998, the presence of this pathogen was described in postmortem brains from patients with AD [2]. That Cpn may contribute to neurodegenerative processes was recently supported by data from our laboratory, as we demonstrated that Cpn is able to infect neurons, ultimately resulting in neuronal cell death in vitro [5]. However, to the best of our knowledge only one experimental in vivo study has been published so far supporting a role for Cpn in AD [19]. In this study, na\u00efve BALB\/c mice were inoculated intranasally with Cpn at an age of 3\u00a0months. Brains were recovered at 1\u20133\u00a0months p.i., revealing the presence of amyloid-like plaques and astrocyte reactivity. Due to the possible impact of this study, it was our goal to confirm these data by repeating these experiments. Focusing on our results of Cpn-inoculated mice at 3\u00a0months p.i., material immunopositive for A\u03b2 was indeed detected in the brain. Accordingly, at first glance, our results are in line with the study of Little et al. [19]. Comparing the morphological appearance of this material immunopositive for A\u03b2 with corresponding material in the brain of APP751SL\/PS1M146L mice, however, some significant and important differences were observed. First, one characteristic of neuritic (AD) plaques is the presence of filamentous, star-shaped aggregates of A\u03b2 fibrils with a thioflavin-S-positive dense core. However, neither such star-shaped aggregates of A\u03b2 fibrils nor extracellular thioflavin-S-positive structures were found in any of the brains of Cpn infected or mock infected BALB\/c mice, whereas both lesions were abundant in the brain of APP751SL\/PS1M146L mice. Second, no damage was found in the tissue surrounding the material immunopositive for A\u03b2. Rather Hoechst-positive structures (i.e. cell nuclei) could be detected within the material immunopositive for A\u03b2, a feature that was not seen in the brain of APP751SL\/PS1M146L mice. Finally, although astrocytes were found in the vicinity of the material immunopositive for A\u03b2 in the brain of Cpn infected or mock infected BALB\/c mice, they were not intimately associated with the A\u03b2 immunoreactivity, as seen in the brain of APP751SL\/PS1M146L mice. In summary, except that A\u03b2 plaques were detected in most brains, our data suggest that at 3\u00a0months after Cpn infection most characteristic features of AD pathology could not be detected in 6-month-old na\u00efve, non-transgenic BALB\/c mice, whereas these were prominent in the brain of the \u201ccontrol\u201d APP751SL\/PS1M146L mice. It may still be argued that the material immunopositive for A\u03b2 in the brain of the Cpn-infected mice represented early precursors of neuritic plaques. To our surprise, however, nearly identical patterns of A\u03b2 immunoreactivity were detected in mock-infected BALB\/c mice as well as in BALB\/c mice, neither Cpn infected nor mock infected. This suggests that the observed immunoreactivity for A\u03b2 in the brain of BALB\/c mice does not directly result from Cpn infection but might represent unrelated elevations in amyloid. Positive staining for A\u03b2 in the brain of mock-infected mice was also observed by Little et al. [19], who found that the amyloid deposition load and size were significantly enhanced in Cpn-infected mice compared to mock infected ones. As outlined above, this was not found in the present study. Furthermore, Little et al. also demonstrated the presence of a small subset of thioflavin-S positive plaques, which were not found in our mice. These discrepancies between the study by Little et al. [19] and the present study could be due to the fact that Little et al. [19] used a Cpn strain (C. pneumoniae, 96-41, isolated from the postmortem brain of an AD patient) that was rather different from the Cpn strain used in the present study (TWAR 2043, ATCC). Notably, Little et al. [19], were able to detect Cpn in olfactory tissues of infected mice up to 3\u00a0months p.i., by both light and electron microscopy. In the present study, however, we were only able to detect Cpn DNA in the olfactory bulb of infected BALB\/c mice at 1\u00a0week p.i. but not at 1 or 3\u00a0months p.i., despite the use of a more sensitive detection method (real-time PCR). As it is well established that the clinical isolates of pathogens are often more infectious than their laboratory counterparts, this could partially explain the discrepancy between both studies. Furthermore, it cannot be excluded that differences in solutions used for mock infection (Hanks balanced salt solution in the study by Little et al. [19] vs. SPG solution in the present study) may have affected the final outcome. Yet, in the present study, the immunosignal in mice that were neither Cpn infected nor mock infected appeared somewhat weaker than in the brain of the BALB\/c mice that were either Cpn- or mock-infected (a detailed quantitative analysis was not performed). This suggests that intranasal application of Cpn or other infectious agents may trigger the production and deposition of A\u03b2 in the brain of BALB\/c mice. For the time being, however, the impact of this remains unclear.\nIn conclusion, our data do not unequivocally support the hypothesis that Cpn infection induces the formation of AD like A\u03b2 plaques in the brain of BALB\/c mice, as suggested by Little et al. [19]. Accordingly, future studies are required to investigate whether Cpn infections may aggravate AD pathology by stimulating the formation of A\u03b2 plaques as has been suggested in atherosclerosis, a disease that is also characterized by pronounced local inflammation.","keyphrases":["chlamydia pneumoniae","neuroinflammation","amyloid-beta"],"prmu":["P","P","U"]}
{"id":"J_Gastrointest_Surg-3-1-1852383","title":"Liver Transplantation Across Rh Blood Group Barriers Increases the Risk of Biliary Complications\n","text":"Background Cold ischemia time and the presence of postoperative hepatic arterial thrombosis have been associated with biliary complications (BC) after liver transplantation. An ABO-incompatible blood group has also been suggested as a factor for predisposal towards BC. However, the influence of Rh nonidentity has not been studied previously.\nIntroduction\nBiliary complications (BC) after liver transplantation (LT) are still a significant cause of morbidity and mortality.1,2 Hepatic arterial thrombosis, long ischemia time, inadequate exposure of the biliary epithelium to the preservation solution, and chronic rejection have been associated with BC.3\u20135 However, complications may also occur in the absence of these pathogenic factors. The expression of donor ABH antigen in the vascular and biliary epithelium of hepatic allografts has been linked to higher risk of biliary and arterial complications, especially in ABO-incompatible grafts.6 However, the influence of Rh nonidentity in LT has not been analyzed until now.\nThe aim of this study is to analyze the incidence of BC after LT, to define their etiological risk factors, and to study the influence of Rh nonidentity on presentation of BC after LT.\nMaterials\nThree hundred and fifty-six (n\u2009=\u2009356) liver transplants were performed at our hospital between January 1995 and November 2000. Eleven were excluded from the study because important data were missing (n\u2009=\u2009345). The study was closed on May 2001 with a 6-month-minimum graft follow-up. The procurement procedure was based on the rapid flush technique.7 After reperfusion of the allograft, cholecystectomy was performed, the donor bile duct being divided just above the cystic duct junction in most cases. Biliary anastomosis was performed with interrupted stitches of 6-0 Polydioxanone suture Ethicon\u00ae (Johnson & Johnson, Brussels, Belgium). The biliary reconstruction technique was end-to-end anastomosis in the majority of LTs (315 patients, 91%). T-tubes were used in four patients due to diameter discrepancy between the biliary ducts of the donor and the recipient. If the primary disease affected the biliary tract, or if technical factors made end-to-end anastomosis difficult, Roux-en-Y-hepaticojejunostomy was performed (25 out of 345, 7%). The outcome was assessed in terms of biliary and arterial complications and patient status (alive, retransplanted, or dead). BCs were studied routinely with abdominal ultrasonography at day\u00a01 post LT, weekly before discharge from hospital and monthly thereafter or when there was clinical or biochemical suspicion of BC, and this was confirmed with magnetic resonance cholangiography, percutaneous transhepatic cholangiography, or endoscopic retrograde cholangio-pancreatography when necessary. The immunosuppressive regimen was based on quadruple sequential therapy with antilymphocyte globulin, Sandimmune Neoral\u00ae (Novartis, Basel, Switzerland), steroids, and azathioprine in most patients, as reported elsewhere.8 Cellular rejection was diagnosed according to histological criteria.9\nStatistical analysis Chi-squared analysis was used to compare dichotomous variables and the presence of BC. Variables that were statistically significant in the univariate analysis were introduced in a multivariate logistic regression analysis. Other known risk factors for developing BC were introduced into the model as covariates: donor age, acute rejection, and chronic rejection. Variables with P\u2009>\u20090.05 were excluded from the final equation. Kaplan\u2013Meier estimates of the rate of BC for both groups and the results were compared with a log-rank test.\nResults\nPerioperative data and surgical details The preservation solutions used were the University of Wisconsin solution in 332 (97%), Celsior solution in 5 (1%), and histidine-tryptophan-ketoglutarate solution in 8 (2%). Twenty grafts (6%) were shipped by another team. The study of donor risk factors demonstrated that there was no hemodynamic instability (systolic pressure lower than 60\u00a0mmHg for more than 1\u00a0h) in 265 cases (77%), and that the main cause of death was traumatic head injury (129\/36%). Donor Rh blood group was positive in 301 (87%) and negative in 44 cases (13%). Recipient Rh blood group was positive in 284 (82%) patients, and negative in 61 (18%). Rh blood groups of donor and recipient were positive to positive in 255 cases (74%), positive to negative in 46 (13%), negative to positive in 30 (9%), and negative to negative in 14 (4%). Donors and recipients had identical Rh in 269 cases (78%) and nonidentical in 76 (22%). The donor ABO blood group was A in 157 (46%), B in 29 (8%), AB in 19 (5%), and O in 140 (41%), while recipients were A in 161 cases (46%), B in 34 (10%), AB in 20 (6%) and O in 130 (38%). The ABO groups of transplanted grafts and hosts were identical in 97% of cases (335 patients) and compatible in 10 cases. There were no cases of incompatible grafts.During postoperative development, 25 LTs presented arterial thrombosis (7%). Eight grafts presented primary nonfunction and were retransplantated (2.2%), and 43 presented initial poor function that recovered spontaneously (12%).3 Biopsy-proven acute rejection was diagnosed in 66 grafts (19%) after transplantation, with development to chronic ductopenic rejection in 8 (2%).\nBCs and Rh mismatch BCs appeared in 70 of the 345 patients (20%). Cross-tabs were built to analyze differences between the grafts that suffered BCs and the rest. Both groups were similar in terms of donor preoperative evaluation and support, recipient descriptive data, and surgical terms. The incidence of BC in nonidentical Rh graft\u2013host cases (23\/76; 30%) was significantly higher than in cases of identical Rh graft\u2013host (47\/269; 17%, P\u2009=\u20090.01). Cases of arterial thrombosis after LTs and ischemia time longer than 430\u00a0min were also associated with a higher incidence of BC (Table\u00a01). \nTable\u00a01Demographics and Major Complications Occurring in Both Groups Studied, Chi-square Test\u00a0BCs (n\u2009=\u200970)No BCs (n\u2009=\u2009275)Chi-squareDonor dataABO blood group0.2\u00a0A25\/157 (16%)132\/157 (84%)\u00a0B6\/29 (21%)23\/29(79%)\u00a0AB6\/19 (32%)13\/19(68%)\u00a0O33\/140 (24%)107\/140 (76%)Rh blood group0.4\u00a0Positive59\/301 (19%)242\/301 (81%)\u00a0Negative11\/44 (25%)33\/44 (75%)Sex0.4\u00a0Male51\/233 (22%)182\/233 (75%)\u00a0Female19\/110 (17%)91\/110 (83%)Donor age0.4\u00a0\u226470\u00a0years66\/320 (20%)254\/320 (80%)\u00a0>70\u00a0years4\/25 (16%)21\/25 (84%)Recipient dataRh blood group0.2\u00a0Positive54\/284 (19%)230\/284 (81%)\u00a0Negative16\/61 (26%)45\/61 (74%)Rh D-R crossing0.1\u00a0Positive\u2013positive45\/255 (18%)210\/255 (82%)\u00a0Positive\u2013negative14\/46 (30%)32\/46 (70%)\u00a0Negative\u2013positive9\/30 (30%)21\/30 (70%)\u00a0Negative\u2013negative2\/14 (14%)12\/14 (86%)Rh D-R identity0.01\u00a0Identical47\/269 (17%)222\/269 (83%)\u00a0Nonidentical23\/76 (30%)53\/76 (70%)ABO blood group0.4\u00a0A27\/161 (17%)134\/161 (83%)\u00a0B7\/34 (20%)27\/34(80%)\u00a0O30\/130 (23%)100\/130(77%)\u00a0AB6\/20 (30%)14\/20 (70%)ABO D-R identity0.4\u00a0Identical67\/335 (20%)268\/335 (80%)\u00a0Nonidentical3\/10 (30%)7\/10 (70%)Sex of recipient0.4\u00a0Male48\/214(22%)166\/214 (78%)\u00a0Female22\/130(17%)108\/130(83%)Recipient age0.2\u00a0<60\u00a0years41\/221 (18%)180\/221 (82%)\u00a0\u226560\u00a0years29\/124 (24%)95\/124 (76%)Diagnosis0.3\u00a0Choleostasis2\/14(15%)12\/14(85%)\u00a0Cirrhosis32\/162 (20%)130\/162(80%)\u00a0Hepatocarcinoma27\/103(26%)76\/103(74%)\u00a0Other etiology1\/18(5%)17\/18(95%)\u00a0Re-OLT7\/39(18%)32\/39 (89%)\u00a0Other tumors1\/9(11%)8\/9 (89%)Surgical dataCold ischemic time0.01\u00a0\u2264430\u00a0min26\/178 (15%)148\/178 (85%)\u00a0>430\u00a0min44\/171 (26%)127\/171 (74%)Type of anastomosis0.6\u00a0Termino-terminal64\/315(20%)251\/315(80%)Graft evolution dataArterial thrombosis0.03\u00a0Yes9\/25 (36%)16\/25(64%)\u00a0No60\/319 (19%)259\/319(81%)Initial poor function0.6\u00a0Yes10\/43 (23%)33\/43 (77%)\u00a0No60\/302 (20%)242\/302 (80%)Primary nonfunction0.1\u00a0Yes08\/8 (100%)\u00a0No70\/336 (20%)266\/336 (80%)Acute rejection0.4\u00a0Yes11\/66(17%)55\/66(83%)\u00a0No59\/279(21%)220\/279(79%)Chronic rejection0.5\u00a0Yes1\/8(12%)7\/8(88%)\u00a0No69\/337 (20%)268\/337(80%)OLT orthotopic liver transplantation\nMultivariate Analysis of Risk Factors for BCs\nIn multivariate analysis, arterial thrombosis presented an adjusted relative risk (aRR)\u2009=\u20092.6, CI 95%\u2009=\u2009(1.1\u20136.4) (P\u2009=\u20090.02), cold ischemia time aRR\u2009=\u20091.8 (CI 95% \u2009=\u20091\u20133.2) (P\u2009=\u20090.02), and Rh graft\u2013host nonidentity aRR\u2009=\u20092 (CI 95%\u2009=\u20091.1\u20133) (P\u2009=\u20090.02) were confirmed as independent risk factors for BC. Other variables included in the initial analysis were nonsignificant (Table\u00a02). Kaplan\u2013Meier estimator and log-rank test confirmed these findings (P\u2009=\u20090.01, Fig.\u00a01). To discard the possible association between the Rh match and the two other risk factors, we demonstrated that arterial thrombosis had similar incidence in Rh-nonidentical grafts (3\/75, 4%) and in Rh-identical grafts (22\/269, 8%; P\u2009=\u20090.1). Moreover, grafts with long ischemia times (>430\u00a0min) had similar incidence in the Rh-nonidentical (42\/76, 55%) group and in Rh-identical patients (129\/269, 48%; P\u2009=\u20090.1). \nTable\u00a02Biliary Complications\u00a0Univariate logistic regressionMultivariate logistic regressionDonor age >70\u00a0years0.50.7Rh D-R identity identical nonidentical0.01; 2(1.1\u20133.6)0.02; 2 (1.1\u20133.6)Cold ischemic time >430\u00a0min0.01; 1.9(1.1\u20133.3)0.02; 1.8(1\u20133.2)Arterial thrombosis (yes)0.04; 2.4(1\u20135.7)0.02; 2.6 (1.1\u20136.4)Acute rejection (yes)0.40.4Chronic rejection (yes)0.50.5Univariate and multivariate logistic regression.Figure\u00a01Kaplan\u2013Meier estimates for the onset of BCs for nonidentical Rh graft\u2013host (P\u2009=\u20090.01).\nType and Management of BCs\nBCs were diagnosed in 70 patients. Biliary duct anastomosis stricture was the main complication, presented clinically, with (7\/10%) or without postoperative leak (21\/30%). Solitary leaks (17\/24%), ischemic-type BCs (ITBC) with arterial thrombosis (6\/9%), ITBC without arterial thrombosis (10\/14%), and lithiasis (9\/13%) were also related complications. The therapeutic approach was surgical in 23 patients (33%), endoscopic in 20 (28%), retransplantation in 11 (16%), and conservative treatment in 16 (23%).\nChi-square test was performed to analyze differences between the type of BC and the three risk factors found. As would be expected, arterial thrombosis was identified in all the cases of ITBC with thrombosis, in a higher percentage than the other BCs (P\u2009<\u20090.001). However, the different types of BCs were not associated with long ischemia time (P\u2009=\u20090.2) or Rh-identity (P\u2009=\u20090.4).\nFinally, chi-square test was also performed to analyze differences among the three BC risk factors and medical management. The therapeutic approach regarding Rh-mismatch was similar in both groups (P\u2009=\u20090.3). Retransplantation was a frequent approach in arterial thrombosis management (4\/9, 40%; P\u2009=\u20090.04). Interestingly, when the BC was presented in grafts with short ischemic time (<430\u00a0min), management by surgery (10\/26, 38%) or an endoscopic approach (10\/26, 38%) were sufficient, and it was not necessary to retransplant. However, when the BC arose in a graft with a long ischemic time (>430\u00a0min) the management was more aggressive, with 29% of patients (13\/44) needing surgery and 25% (11\/44) needing retransplantation.\nDiscussion\nEtiopathogenesis of BC\nCurrently, orthotopic liver transplants are performed with good results at several centers without taking the donor\u2013recipient Rh relationship into account. In fact, no prior work has shown greater morbidity or mortality after the usage of Rh-incompatible liver grafts. However, it was in studying the causes of BC in our grafts in prior studies that we began to suspect the existence of a possible relationship between BC and Rh incompatibility. First, we observed a greater rate of BC in the presence of preservation lesions in postreperfusion biopsies.10 Thus, we found that the biliary epithelium is very sensitive to changes during preservation. Second, the description of lesions like ITBC,11 to which surgical technique does not contribute as a primary cause, led us to suspect possible immunological pathogenesis of the BC. Lastly, it is paradoxical to find that, while surgical technique is improving and satisfactory results are achieved in liver transplants, BCs are still a problem, leading us to suspect that there are unidentified factors that cause them. The arterial thrombosis and long cold ischemia time were independent risk factors for developing BC, as was expected and reported by others.11,12 In our study we have demonstrated that even though BC in grafts with short ischemia can be resolved with surgery or endoscopy, the prognosis of BC was worse in cases of long ischemic grafts requiring retransplantation.\nRelationship Between ABO and Rh in Liver Transplants\nAs for the donor\u2013recipient ABO relationship, the usage of ABO-incompatible grafts has been discouraged due to the high rate of BC and poor graft survival.6,13,14 In the Sanchez-Urdazpal study,6 82% of the 18 ABO-incompatible grafts presented BC in comparison with 6% of the control group, while Farges,14 published slightly better results several years later with BC of 54%. Therefore, these grafts are used in extremely urgent cases when there is no other possible alternative. Immunological phenomena, such as rejection,3 may also lead to biliary strictures. In the same way, the ABO system was shown to cause more BC and worse graft survival in LT.6,13\u201315 However, Rh nonidentity seems to have better tolerance and is not a cause of graft refusal when a donor appears. Surprisingly, our study demonstrated a higher incidence of BC in the Rh-nonidentical group.\nSome authors16,17 reported low rates of alloimmunization in Rh-negative recipients of Rh-incompatible transfusion after LT. It was suspected that immunosuppressant drugs modified the immunosuppressive response.16 However, debate still exists as to whether the D barrier can be crossed in LT. Previous studies of ABO barrier and BC suggested the hypothesis of an immunological injury to the bile duct epithelium, and the expression of ABH antigens in the donor 150\u00a0days after transplantation.6 However, the D antigen is only expressed in erythrocytes.\nThe nonidentical Rh group has two mismatch possibilities: positive donor to a negative recipient or negative donor to positive recipient. In the first case (positive to negative) the immunologic mechanism is easy to understand because the humoral anti-D (Rh) response may be responsible for the graft injury.\nThe other subgroup (negative to positive) may have a different pathogenic explanation. Bryan et al.17 hypothesized that two mechanisms could be involved in the same process in kidney transplantation: other Rh antigenic loci (C and E) and histocompatibility antigenic crossings. A negative liver graft in a positive recipient with lymphatic cells and tissues predisposes to cellular response against it. Finally, the biliary tract can probably suffer immunological damage, and thus further BC. Therefore, while the results of the study lead to suspicion of an immunological pathogenesis, the mechanism is still unclear.\nIn conclusion, Rh-nonidentical LT involves a higher rate of BCs. Future studies should examine the influence of Rh donor and blood group on graft development. Finally, our results suggest that there is a summation effect of BC risk factors. In our opinion, Rh-nonidentical liver grafts should not undergo a very long ischemia time.","keyphrases":["liver transplantation","biliary complications","rh nonidentity"],"prmu":["P","P","P"]}
{"id":"Appl_Microbiol_Biotechnol-3-1-1914237","title":"Genomotyping of Pseudomonas putida strains using P. putida KT2440-based high-density DNA microarrays: implications for transcriptomics studies\n","text":"Pseudomonas putida KT2440 is the only fully sequenced P. putida strain. Thus, for transcriptomics and proteomics studies with other P. putida strains, the P. putida KT2440 genomic database serves as standard reference. The utility of KT2440 whole-genome, high-density oligonucleotide microarrays for transcriptomics studies of other Pseudomonas strains was investigated. To this end, microarray hybridizations were performed with genomic DNAs of subcultures of P. putida KT2440 (DSM6125), the type strain (DSM291T), plasmid pWW0-containing KT2440-derivative strain mt-2 (DSM3931), the solvent-tolerant P. putida S12, and several other Pseudomonas strains. Depending on the strain tested, 22 to 99% of all genetic elements were identified in the genomic DNAs. The efficacy of these microarrays to study cellular function was determined for all strains included in the study. The vast majority of DSM6125 genes encoding proteins of primary metabolism and genes involved in the catabolism of aromatic compounds were identified in the genomic DNA of strain S12: a prerequisite for reliable transcriptomics analyses. The genomotypic comparisons between Pseudomonas strains were used to construct highly discriminative phylogenetic relationships. DSM6125 and DSM3931 were indistinguishable and clustered together with strain S12 in a separate group, distinct from DSM291T. Pseudomonas monteilii (DSM14164) clustered well with P. putida strains.\nIntroduction\nThe sequencing and annotation of the Pseudomonas putida KT2440 genome (Nelson et al. 2002) has greatly catalyzed research on this strain and other academically and biotechnologically relevant but non-sequenced P. putida strains. A number of key scientific advances (both pure and applied) have been made via proteome and transcriptome analysis of P. putida strains (Dominguez-Cuevas et al. 2006; Hallsworth et al. 2003; Morales et al. 2006; Reva et al. 2006; Santos et al. 2004; Segura et al. 2005; Volkers et al. 2006; Yuste et al. 2006). In our laboratories, we have studied both P. putida KT2440 (for recent publications: Hallsworth et al. 2003; Martins dos Santos et al. 2004; Nelson et al. 2002; Timmis 2002) and the solvent-tolerant P. putida S12 (De Bont 1998; Hartmans et al. 1990; Wery and De Bont 2004). Unlike most pseudomonads, solvent-tolerant strains possess an extraordinary tolerance to a broad variety of toxic solvents (for reviews: De Bont 1998; Kieboom and De Bont 2000; Ramos et al. 2002). A large number of high-added value bioconversions involve toxic, generally apolar, products (aromatics, aliphatic alcohols, epoxides, etc.). Especially for bioprocesses involving such products, the use of solvent-tolerant P. putida strains renders advantages in terms of productivity and the application of multiphase media for product recovery (Ramos-Gonzalez et al. 2003; Rojas et al. 2004; Wery and De Bont 2004; Wery et al. 2000; Wierckx et al. 2005). Despite their biotechnological potential, the genomes of S12 and other Pseudomonas strains have not been sequenced. So, recent studies of their metabolic potential have been limited to comparative proteomics (Segura et al. 2005; Volkers et al. 2006) or transcriptomics based on the database information for P. putida KT2440.\nThe genus Pseudomonas is very heterogeneous (Anzai et al. 2000); even strains of one species tend to vary greatly in terms of both phenotypic (Grimont et al. 1996) and genotypic characteristics (Brosch et al. 1996). Strains belonging to the species P. putida can be categorized into biovar A and B: the former grouping (A) is the more heterogeneous (Brosch et al. 1996) and the phylogenetic and metabolic diversity of biovar A strains has yet to be fully characterized and industrially exploited.\nComparative transcriptomics-based approaches have played a pivotal role in recent investigations of complex cellular responses of P. putida strains (Dominguez-Cuevas et al. 2006; Duque et al. 2007; Yuste et al. 2006). As transcriptome profiling is based on the highly-sensitive detection of DNA\u2013cDNA hybridization, DNA sequence similarity determines the validity of these analyses. The inherent heterogeneity of the P. putida grouping provides additional cause for concern that microarray platforms derived from strain KT2440 may provide a valid framework for the study of nonsequenced P. putida strains.\nWe therefore carried out this study to assess the utility of KT2440-based high-density DNA microarrays for transcriptomics studies of DSM 6125 (subculture of KT2440), DSM 3931 (subculture of mt-2), DSM 291T (the P. putida type strain), the solvent-tolerant S12, and other nonsequenced Pseudomonas strains. In addition, the use of such microarrays to establish highly discriminative phylogenetic relationships between these strains was demonstrated.\nMaterials and methods\nStrains, cultivation conditions, and DNA extraction\nSingle colonies from each Pseudomonas strain studied (see Table\u00a01) grown on Pseudomonas Isolation Agar (Difco) were used to inoculate Luria\u2013Bertani broth (LB) (Sambrook et al. 1982) in duplicate. After cultures were grown overnight at 30\u00b0C, genomic DNAs were prepared from 5\u00a0ml of culture (approximately 3\u2009\u00d7\u2009108 cells\/ml) using the Genomic DNA 100\/G Kit (Qiagen, Germany) according to the manufacturer\u2019s instructions. DNA concentrations were determined at 260\u00a0nm using ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA), and purity was confirmed by agarose (1% w\/v) gel electrophoresis.\nTable\u00a01Source and designations of Pseudomonas strains used in this studySpecies and strain numberaSource or referencebOther designationscP. putida DSM 6125DSMZKT2440, ATCC 47054; NCIMB 11950P. putida DSM 3931DSMZmt-2, ATCC 23973; ATCC 33015; JCM 6156; NCIMB 12182P. putida S12Hartmans et al. 1990ATCC 700801P. putida DSM 291TDSMZDSM 50202T; ATCC 12633T; ICPB 2963T; NCTC 10936T; CCUG 12690T; LMG 2257TP. putida DSM 50198DSMZATCC 17453; ICPB 2563-77; JCM 6157; NCIMB 10007P. putida DSM 50208DSMZATCC 17485; ICPB 2789-111; JCM 6158; NCIMB 12092P. monteilii DSM 14164DSMZATCC 700476; CCUG 38736; LMG 21609; CIP 104883P. fluorescens DSM 50090TDSMZATCC 13525T; ICPB 3200T; NCIMB 9046T; NCTC 10038T; CCUG 1253T; LMG 1794TaStrain designations used in this studybDSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen, GermanycSubcultures of the used strains available in other culture collections. ATCC American Type Culture Collection, ICPB International Collection of Phytopathogenic Bacteria, USA, NCIMB National Collections of Industrial and Marine Bacteria, UK, NCTC National Collection of Type Cultures, UK, JCM Japan Collection of Microorganisms, Japan, CIP Collection bacteri\u00e8nne de l\u2019Institut Pasteur, France, CCUG Culture Collection University G\u00f6teborg, Sweden, LMG BCCM\/LMG Bacteria Collection, Belgium.\nPreparation of biotin-labelled fragmented genomic DNAs\nApproximately 17\u00a0\u03bcg of heat-denatured genomic DNA was fragmented using 0.3\u00a0U DNaseI (Pharmacia)\/\u03bcg DNA at 37\u00b0C for 20\u00a0min (Wolfgang et al. 2003). DNaseI was inactivated by immediate heat treatment at 95\u00b0C for 10\u00a0min, followed by cooling on ice. An aliquot of fragmented DNA was analyzed by agarose (4% w\u2009\/\u2009v in TAE buffer) gel electrophoresis to confirm the purity and establish the mean size of DNA fragments (20\u201350\u00a0bp). Approximately 7\u00a0\u03bcg of fragmented DNA was labelled with biotin according to the Affymetrix \u201cExpression Analysis Technical Manual for Prokaryotic Samples\u201d.\nDesign of high-density oligonucleotide microarrays and hybridization with genomic DNAs\nHigh-density oligonucleotide microarrays based on the annotated genome of P. putida KT2440 (NC 002947.3) were constructed according to Affymetrix specifications (http:\/\/www.affymetrix.com\/support\/technical\/other\/custom_design manual. pdf ) with the pair-wise configuration of 13 perfect match (PM) and mismatch (MM) 25-mer oligonucleotides per gene (probe set). The microarrays included 7,781 probe sets: 5,338 representing 5,350 annotated genes or open reading frames (orfs), and 2,443 targeting intergenic regions. Hybridization and washing of the microarrays were performed on a GeneChip\u00ae Node system (Hybridization temperature 49\u00b0C; Hybridization oven 640, Fluidics station 450; Affymetrix, Santa Clara, CA, USA) following the supplier\u2019s instructions. Scanning was carried out by ServiceXS (Leiden, The Netherlands) on a high resolution Gene Chip\u00ae Scanner 3000 7G system with autoloader (Affymetrix, Santa Clara, CA, USA) using the default analysis settings of the manufacturer (filter: 570\u00a0nm; pixel size: 2.5\u00a0\u03bcm). Hybridization intensity data were extracted from the scanned array images, and the designations for presence or absence were calculated with Affymetrix Microarray Suite (MAS) 5.0 software. A one-sided Wilcoxon\u2019s Signed Rank was used to calculate p-values reflecting the statistical significance of differences between PM and MM of a probe set. The significance levels for detection calls of probe sets were p-value\u2009<\u20090.05 for present and p-value\u2009\u2265\u20090.05 for absent. Differences between PM and MM were considered insignificant and removed from further consideration by comparisons of the discrimination score [(PM\u2212MM)\/(PM+MM)] with the defined discrimination threshold \u03c4 (discrimination score<0.015). Comparisons of present\/absent designations for genes in the different strains were made with GeneSpring version 7.2 (Agilent). Replicate assays of biological duplicates were performed for P. putida DSM 6125 and S12. The deviation caused by present\/absent designations of genes in only one of both replicates was 0.8% for DSM 6,125 and 4.2% for S12 regarding all probe sets for putative genes and intergenic regions (Table\u00a02).\nTable\u00a02Similarity indices based on designations of presence or absence (stated in brackets) from hybridization signals of genomic DNA fragments from different Pseudomonas members on P. putida KT2440-based microarraysaStrainPresent (absent) in percent (%)Putative genes and intergenic regions (7,781 probe sets)Putative genes (5,338 probe sets)Putative genes with assigned function (3,670 probe sets)P. putida DSM 6125b97.6 (1.4)99.6 (0.0)99.8 (0.0)P. putida DSM 393198.6 (1.4)99.9 (0.1)100.0 (0.0)P. putida S12b78.0 (16.9)81.8 (13.9)86.9 (9.7)P. putida DSM 291T60.5 (39.5)64.2 (35.8)69.3 (30.7)P. putida DSM 5019856.2 (43.8)60.7 (39.3)66.5 (33.5)P. putida DSM 5020858.8 (41.2)62.6 (37.4)67.8 (32.2)P. monteilii DSM 1416457.7 (42.3)61.7 (38.3)66.8 (33.2)P. fluorescens DSM 50090T22.0 (78.0)27.2 (72.8)30.7 (69.3)aPresent\/absent designations (present: p\u2009<\u20090.05; absent: p\u2009\u2265\u20090.05; \u03c4\u2009=\u20090.015) derived from a decision matrix in Affymetrix MAS 5.0bReplicate assays were performed for P. putida DSM 6125 and S12 and the values listed include only genes that were designated present or absent in both replicates.\nCalculation of dendrograms\nSimple matching similarity matrix based on the present\/absent detection calls in microarray analysis of different pseudomonads (SM = m\/n; m, number of matching probe sets; n, total number probe sets), and the microarray genotyping dendrogram were calculated using UPGMA parameters (unweighted pair group method, arithmetic average) and agglomerative hierarchical clustering with XLSTAT version 7.5.3 (Addinsoft, Paris, France).\nAFLP analysis\nIn this paper, we used amplified fragment length polymorphism (AFLP) (Janssen et al. 1996; Savelkoul et al. 1999; Vos et al. 1995) to analyze different strains of P. putida a P. monteilii and a Pseudomonas fluorescens. AFLP is based on selective amplification of restriction fragments from totally digested genomic DNA. AFLP fingerprints were performed by KeyGene (Wageningen, The Netherlands). Genomic DNA of the different pseudomonads was digested using restriction enzyme combination NlaIII (Westburg, Leusden, The Netherlands) and Csp6I (Fermentas, St. Leon-Rot, Germany) according to manufacturers instructions. Each restriction enzyme was combined with the ligation of specific linker oligonucleotide pairs (NlaIII: 5-GACGATGAGTCCTGAG-3\/5-TGTACGCAGTCTAC-3; Csp6I: 5-GACGAT GAGTCCTGAG-3\/5-TACTCAGGACTCAT-3). For each of these linker combinations, AFLP was performed using nine N\/C AFLP primer combinations, which were selected using KT2440 genome sequence as reference and software package REcomb (Keygene) for prediction analysis. These primers were extended with a 3\u2032 terminal dinucleotide (+ 2) and the extensions were CA\/AC, CA\/CA, CA\/CC, CA\/GG, CA\/TT, CT\/TT, CT\/CA, CT\/CT, and CT\/TC. For further detail, please refer to Van den Braak et al. (2004). PCRs were performed in the presence of radioactive nucleotides, and the amplimers obtained were separated on 50\u2009\u00d7\u200920\u00a0cm polyacrylamide slabgels. Using phosphor-imaging, the individual presence\/absence in a total of 757 markers (DNA bands) per strain was analyzed.\nThe total marker score data table (presence\/absence of individual DNA bands) was subjected to genetic distance analysis using simple matching similarity matrix (SM = m\/n; m, the number of matched scores; n the total sample size), consisting of similarity indices for all combinations of AFLP-banding patterns. Simple matching coefficients were calculated using NTSYSpc-software version 2.2 (Exeter Software, Setauket, NY, USA). To visualize the relationship between the strains, a dendrogram was generated using Sequential Agglomerative Hierarchical Nested (SAHN) cluster analysis with the use of UPGMA parameters.\nStandard PCR and sequencing\nDiagnostic PCR of selected putative orfs (PP1265, PP5224) that were called absent in microarray analyses of P. putida DSM 3931 genomic DNA was performed using proofreading enzyme mixture High Fidelity PCR master (Roche Diagnostics, The Netherlands) and specific primers (PP1265: forward: 5\u2032-CTGCTGCACCAGGCCTAT-3\u2032, reversed: 5\u2032-TTGGTCACATAGCCGTCAAC-3\u2032; PP5224: forward: 5\u2032-CAACGGCTAAACCTTTGCAT -3\u2032, reversed: 5\u2032-AGGATCGAGACCTTGCCTTC-3\u2032). Yielded PCR-amplicons of expected sizes, 1,108 and 1,062\u00a0bp, respectively, were sent to Baseclear (Leiden, The Netherlands) for sequencing according to Sanger et al. (1977) using nested sequencing primers (PP1265: 5\u2032-CCAGGCAATCCGTGTCAT-3\u2032; PP5224: 5\u2032-GGTGTCCTGACCGTCAAGTT-3\u2032), and the resulting sequences were used for sequence alignments.\nBiological function-derived phylogenetic analysis\nThe concept of Clusters of Orthologous Genes (COG; see Tatusov et al. 1997, 2003) was used during analysis of the genomic content of nonsequenced Pseudomonas strains by linkage to primary biological function (Table\u00a03). A COG consists of individual proteins or groups of paralogs from at least three lineages and thus corresponds to an ancient conserved domain (Tatusov et al. 1997, 2003). In the NCBI-COG database, 4,497 proteins of 5,350 putative orfs in P. putida KT2440 were identified as COGs and were arranged in functional categories (see http:\/\/www.ncbi.nlm.nih.gov\/sutils\/coxik.cgi?gi=266 ). Because of the limited number of COGs in the functional categories (A) RNA processing and modification, (B) chromatin structure\/dynamics, and (W) extracellular structures (1, 2, and 3, respectively), these COGs were collectively grouped together with general function prediction COGs (Category R) under the designation R\u2032(see Table\u00a03).\nTable\u00a03Primary functional designationa of genes identified in P. putida S12 genomic DNACodebDescriptionCOGs in KT2440cCOGs KT2440 in S12FNucleotide transport and metabolism9387 (93.5%)JTranslation194178 (91.8%)HCoenzyme transport and metabolism183166 (90.7%)TSignal transduction mechanisms427387 (90.6%)ILipid transport and metabolism194174 (89.7%)NCell motility130116 (89.2%)GCarbohydrate transport and metabolism264232 (87.9%)PInorganic ion transport and metabolism368322 (87.5%)UIntracellular trafficking and secretion119104 (87.4%)EAmino acid transport and metabolism630550 (87.3%)OPosttranslational modification, protein turnover, chaperones194169 (87.1%)CEnergy production and conversion321276 (86.0%)QSecondary metabolites biosynthesis, transport and catabolism161138 (85.7%)KTranscription499426 (85.4%)VDefense mechanisms6656 (84.8%)R\u2032General function prediction only733620 (84.6%)SFunction unknown442365 (82.6%)MCell membrane biogenesis288227 (78.8%)DCell cycle control5641 (73.2%)\u2013Not in COGs853544 (63.8%)LReplication, recombination and repair269158 (58.7%)aBased on the Clusters of Orthologous Groups (COG) system, also see Materials and methods section)bCodes of functional categories of COGcProteins, 4,497 of 5,350 putative orfs in P. putida KT2440 can be found in COG database (http:\/\/www.ncbi.nlm.nih.gov\/ COG\/)\nResults\nGenomic DNA hybridizations with P. putida KT2440-based high-density DNA microarrays\nTotal genomic DNA from P. putida strains, and other nonsequenced Pseudomonas members (see Table\u00a01), was hybridized to custom KT2440-based high-density oligonucleotide microarrays. Presence or absence designations for each probe set (designed for specific genes and intergenic regions) were calculated by the Affymetrix MAS 5.0 algorithm from the significant difference (see Materials and methods section) in hybridization intensities between the corresponding perfect match and mismatch oligonucleotides (Table\u00a02). Absence designation is a synonym for the absence of significant signal values and stands for divergent DNA still coding a similar biological function or for the complete absence of the specific DNA. Replicate array hybridizations were performed for DSM 6125 DNA and S12 DNA only. For these DNAs the values given in Table\u00a02 represent only probe sets designated present or absent in both replicates. As expected, the DSM 6125 DNA yielded an almost perfect score: 97.6% for probe sets corresponding to all genetic elements (including the intergenic regions) and 99.8% for probe sets designed for genes with an assigned function. Strain DSM 3931 (subculture of P. putida mt-2: Teruko, 2002) is a TOL plasmid (pWW0)-containing variant of DSM 6125 and was used as an additional control for the accuracy of the microarray experiments. As expected, both strains were virtually indistinguishable in the array hybridization study (Table\u00a02). Nonetheless four orfs were indicated absent in DSM 3931. These orfs were found to be called absent in only one of the DSM 6125 replicates. We therefore used diagnostic PCR to investigate the presence of two of them (PP1265, PP5224) in DSM 3931; the other two appeared less important due to their limited size (~90\u00a0bp). In both cases, PCR products of the expected sizes (1,108 and 1,062\u00a0bp) were obtained that, after sequencing, proved to be identical to the KT2440 homologs.\nApart from DSM 3931, the solvent-tolerant S12 showed the highest genomic similarity to KT2440. Nearly 3,188 of 3,670 (86.9%) genes with assigned functions in the KT2440 genome were identified in the genomic DNA of S12 (Table\u00a02). Approximately 71% of 1,668 (putative) genes without an assigned function, and 70% of the intergenic regions were found to be present in the S12 (data not shown).\nMicroarray-based genomotyping\nA dendrogram was constructed (Fig.\u00a01a) based on genomic similarity of all 7,781 genetic elements of KT2440 in the tested genomic DNAs except for the control strain DSM 3931 (Table\u00a02). DSM 6125, DSM 3931 (not shown), and S12 clustered in a group separate from the other P. putida strains (Fig.\u00a01a). The nonsequenced P. fluorescens (DSM 50090T), that was included as an out-group, did not cluster with any other strain. By contrast, Pseudomonas monteilii DSM 3931 that was included as a non-putida member clustered with other P. putida strains suggesting a closer relationship with the P. putida taxonomic grouping (Fig.\u00a01). To assess the validity of these microarray-derived phylogenetic relationships, AFLP DNA fingerprinting was used to obtain an independent phylogenetic classification of strains (Janssen et al. 1996; Savelkoul et al. 1999; Vos et al. 1995). The phylogenetic tree constructed following AFLP analyses showed an identical pattern in terms of strain grouping. (Fig.\u00a01b).\nFig.\u00a01Genome similarity between different Pseudomonas strains. a High-density microarray genotyping tree based on absent\/present designations generated by Affymetrix MAS 5.0 algorithm (Table\u00a02) using simple matching similarity coefficient, UPGMA, and agglomerative hierarchical clustering. b AFLP-genotyping. Tree is based on the score of 757 AFLP markers using the simple matching similarity coefficient and Sahn cluster analysis\nAn inventory was made of genes encoding proteins belonging to COGs (Tatusov et al. 1997, 2003) that were identified in the different genomic DNAs (Fig.\u00a02). Of 5,350 orfs in the KT2440 genomic DNA sequence, 4,497 encode proteins that have been categorized into classes of primary biological function based on the COG system (http:\/\/www.ncbi.nlm.nih.gov\/ COG\/). It was found that, after DSM 3931, P. putida S12 showed the highest present score in all functional classes (Fig.\u00a02). The \u201cpresent\u201d designations for S12 genes encoding COG members ranged from 58.7 to 93.5%, depending on their primary biological function (Table\u00a03, Fig.\u00a02). The unequal distribution of the present designations over the different functional classes was also characteristic of other Pseudomonas strains (Fig.\u00a02). Strikingly, all pseudomonads other than DSM 6125, DSM 3931, S12 and the out-group DSM50090T showed a comparable distribution pattern of identified genes over the different COGs.\nFig.\u00a02Distribution of genes encoding COG proteins over different functional categories as identified in Pseudomonas strains. Functional categories were adopted from the COG database for P. putida KT2440 (http:\/\/www.ncbi.nlm.nih.gov\/sutils\/coxik.cgi?gi=266 ). The categories of biological function corresponding to COG codes are given in Table\u00a03.\nP. putida strains are well-known for their broad metabolic potential regarding aromatic compounds (Jimenez et al. 2002; Wackett 2003, for reviews). Several pseudomonads, including P. putida KT2440, have been reported to degrade and\/or transform a variety of aromatics. Among these are compounds of industrial importance, and there is an interest in studying these metabolic pathways on the level of gene expression and regulation thereof. The utility of KT2440-based microarrays in transcriptomics studies of aromatics metabolism of the pseudomonads under investigation was appraised. The presence of 70 genes of central and peripheral metabolic routes of aromatics were chosen based on reports by Jimenez et al. (2002) and Wackett (2003) (Table\u00a04). All genes (100%) were detected in DSM 6125, S12 and in DSM 3931. Higher divergence was found for P. putida DSM 291T (82.9% detectable), DSM 50198 and DSM 50208 (both 75.7% detectable), and DSM 14164 (74.3% detectable). DSM 50090T again behaved as an out-group with only 25.7% of the 70 genes detectable. In the P. putida strains used in the present study and DSM 14164, almost all genes for degradation of benzoate (ben), homogentisate (hmg, mai), phenylalanine\/tyrosine (phh, tyrB), and catAB were detected. In DSM 291, 50198, 50208 and 14164 putative regulatory genes of different pathways (e.g. catR, pcaQ, pobR, phaNM) and some isoenzymes (e.g., catA2) were not found (Table\u00a04). Interestingly, in all these strains, the muconolactone isomerase (catC), and in the catabolism of phenylpropenoid compounds, vanillin dehydrogenase (vdh), putative conifer aldehyde dehydrogenase (calB; except DSM 50198), and feruloyl-CoA synthetase (fcs; except for DSM 50208) were not identified and can thus not be studied in KT2440-based microarray-based transcriptome analysis.\nTable\u00a04Comparison of the presence\/absence designations of genes encoding metabolic pathways of aromatic compounds in different pseudomonads derived from Affymetrix decision matrix MAS 5.0StrainPresent (%)aGenes of aromatic pathways not identified in hybridizations with genomic DNAP. putida DSM 6125100.0P. putida DSM 3931100.0P. putida S12100.0P. putida DSM 291T82.9catC; fadAxB2x; fcs; pcaQ; phaDHMN; vdh; calB; catA2P. putida DSM 5019875.7benE-1; catCR; ech; fadAxB2x; fcs; pcaDQ; phaGHMN; pobAR; vdh; catA2P. putida DSM 5020875.7catCR; ech; fadAxB2x; pcaBCQ; phaDHJMN; phhR; pobR; vdh; calB; catA2P. monteilii DSM 1416474.3benE-1; catCR; ech; fadB2x; fcs; pcaBDQ; phaEGHMN; pobAR; vdh; calB; catA2P. fluorescens DSM 50090T25.7aat; acdA; benACDE-1E-2FKR; catABCR; fadAxBB1xB2xDx; fcs; hpd; pcaCDHIJKQT; pcm; phaAB-EG-ILMN; phhBR; pobAR; tyrB-2; vanAB; vdh; calB; ferR; catA2aSelected 70 genes (100%) involved in catabolism of aromatic compounds annotated in P. putida DSM 6125: protocatechuate (pcaBCDFGHIJKQRT), catechol branches \u03b2-ketoadipate pathway (catABCRA2), homogentisate pathway (hmgA, mai), central meta-cleavage (pcm), p-hydroxybenzoate (pobAR), benzoate (benABCDE-1E-2FKR), phenylacetate pathway (phaABCDEFGHIJKLMNJ1), n-phenylalkanoic acids (fadABDHAxB1xB2xD2Dx), phenylpropenoid compounds (vanAB, vdh, calB, ferR, fcs, ech, aat, acdA), and phenylalanine\/tyrosine (phhABR, tyrB-1B-2, hpd, pcm).\nDiscussion\nA major advantage of microarray-based comparisons of species is the ability to pinpoint differences in individual genes and intergenic regions. Through these comparisons, detailed insight was gained in the utility of P. putida KT2440-based microarrays in transcriptomics studies of different pseudomonads at the level of specific categories of biological function. It was found that genes involved in, e.g., \u201ctranslation\u201d and \u201cnucleotide transport and metabolism\u201d could be identified at a high frequency (>80%) in all P. putida strains tested, in contrast to other functional groups where the frequency of gene identification dropped below 60%. Whereas P. fluorescens DSM 50090T behaved as a typical out-group in these functional studies, it was clear that, depending on the functional category, up to 45% of the genes of this strain could still be identified.\nThere is a biotechnological requirement for P. putida biocatalysts that can function at high solvent concentrations, such as strain S12, and that can be swiftly optimized for different bio-based production processes. In our group, studies have focused on the construction of strains that are able to convert renewable substrates, such as sugars, into aromatics of interest via central metabolites (Nijkamp et al. 2005, 2006; Wierckx et al. 2005). These conversions take place via multiple metabolic pathways each consisting of several enzymatic steps with regulatory mechanisms that are being investigated using S12 as a model system. A comparative transcriptomics approach is invaluable to gain detailed insights into the complex cellular systems of the metabolically versatile pseudomonads. The employment of the high-density KT2440 microarrays would enable highly sensitive and reproducible transcriptome analyses that are compatible with those used for model species such as P. aeruginosa (Ochsner et al. 2002; Wagner et al. 2003; Whiteley et al. 2001) and Escherichia coli (Woo et al. 2004).\nIn the present study, we showed that the use of KT2440-based microarrays would enable reliable transcriptomics analysis of P. putida S12. Significantly, we found that of all pseudomonads tested, the genomic content of P. putida S12 showed the highest similarity to that of P. putida DSM 6125 (KT2440). The vast majority of KT2440 genes coding for proteins involved in primary metabolism, including biosynthesis of important intermediates such as amino acids, and the genes involved in the conversion of aromatic compounds were shown to be sufficiently similar to those of S12.\nThe high resolution achieved by comparative genomotyping enabled the identification of minute genotypic differences between tested strains, making a meaningful analysis of phylogenetic relationships feasible. For example, the genomic DNA of P. putida DSM 6125 was shown to be virtually identical to that of the control strain DSM 3931, and this is consistent with the origin of strain KT2440 as a derivative of strain mt-2 (Regenhardt et al. 2002).\nThe relationship between the P. putida DSM 291T and KT2440 has been an issue of controversy. Based on 16S rRNA gene comparisons (99% identity), both strains appeared closely related; however, a DNA\u2013DNA hybridization experiment indicated only 50.5% genome relatedness between both strains (Regenhardt et al. 2002). The results lead to doubts about the classification of both strains as part of the same Pseudomonas species, given the widely accepted recommendation that strains of the same species shall have genome similarities higher than 70% (Stackebrandt and Goebel 1994). In the same study, an appreciable distance between DSM 291T and KT2440 was established by REP-PCR genomic fingerprint patterns and Biolog GN metabolic profiling. In our genomotyping approach, 69.3% of the genes with an assigned function, 60.5% of all genetic elements (including intergenic regions), and 52.2% of the intergenic regions (not shown) were identified in the genomic DNA of P. putida DSM 291T. These differences, which are supported by the AFLP analysis, indicate a considerable phylogenetic distance between DSM 291T and KT2440.\nThe diversity within the species P. putida was previously reflected in studies concerning genomic DNA ribotyping (Brosch et al. 1996), whole cell protein electrophoretic fingerprinting (Vacanneyt et al. 1996) and Biolog\/Biotype-100 experiments (Grimont et al. 1996). In our genomotyping studies, strains of P. monteilii and P. fluorescens were included as out-groups. P. fluorescens DSM 50090T was shown to be distantly related to the other Pseudomonas members tested, which supports its classification as a separate species. However, P. monteilii DSM 14164 clustered well with P. putida DSM 50198 and to a lesser extent with DSM 291T and P. putida DSM 50208. AFLP analysis showed a comparable result and confirmed the close relation between P. putida and P. monteilii. The present study thus suggests that DSM 14164 should more accurately be classified as a P. putida.\nOther studies based on classification of P. monteilii by classical, well established taxonomic methods do not support our findings. DNA\u2013DNA hybridizations among P. monteilii CFML 90-60T, DSM 291T, and DSM 50208 generated relative bindings of genomic DNA of 40 and 48%, with \u0394Tm values of 9.2 and 7.9, respectively (Elomari et al. 1997). Pyoverdine typing (siderotyping) analyzing the excreted siderophores during iron starvation of P. monteilii CFML 90-60T and DSM 291T produced different patterns for both strains (Dabboussi et al. 2002). Phenotypically, however, P. monteilii was previously shown to be highly similar to P. putida and could only be differentiated by assimilation experiments of the substrates inositol, \u03b1-aminobutyrate, and o-\/m-hydroxybenzoate (Dabboussi et al. 2002; Elomari et al. 1997).\nIn conclusion, the genomotyping of different pseudomonads using KT2440-based DNA microarrays yielded novel insights in their phylogenetic relationships and the underlying identification of genes and their distribution over different primary and secondary biological functions. This revealed the utility of KT2440-based microarrays in transcriptomics and classification studies of these strains.","keyphrases":["genomotyping","pseudomonas putida","transcriptomics","solvent-tolerant bacteria"],"prmu":["P","P","P","R"]}
{"id":"Diabetologia-4-1-2292424","title":"Prediction of outcome in individuals with diabetic foot ulcers: focus on the differences between individuals with and without peripheral arterial disease. The EURODIALE Study\n","text":"Aims\/hypothesis Outcome data on individuals with diabetic foot ulcers are scarce, especially in those with peripheral arterial disease (PAD). We therefore examined the clinical characteristics that best predict poor outcome in a large population of diabetic foot ulcer patients and examined whether such predictors differ between patients with and without PAD.\nIntroduction\nDiabetic foot ulcers are a common and much feared complication of diabetes, with recent studies suggesting that the lifetime risk of developing a foot ulcer in diabetic patients may be as high as 25% [1]. Foot ulceration requires long and intensive treatment, has important effects on quality of life of both patients and care-givers [2] and is associated with major healthcare costs [3\u20135]. Although in recent years much effort has been put into the development of international guidelines in order to stimulate the delivery of uniform and structured care [6], prospective data on outcomes and predictors of outcome in patients with diabetic foot ulcers are limited.\nThe population of diabetic patients who present with foot ulceration is heterogeneous: although most patients have peripheral polyneuropathy, there are several other characteristics that may vary among patients, such as the presence of peripheral arterial disease (PAD), infection and co-morbidities. PAD is present in approximately one-half of all patients with foot ulcers [7] and is considered an important predictor of outcome [8, 9]. Therefore, outcome data on this important subgroup of patients with diabetic foot disease are needed. Such a requirement is underlined by the fact that although diabetic foot ulcers are usually reported and analysed as one clinical entity, marked differences in patient, foot and ulcer characteristics can exist between patients with and without PAD [7]. These observations raise the question of whether predictors of outcome in patients with and without PAD may differ.\nThe aim of the present study was therefore: (1) to obtain prospective data on outcome of individuals presenting with a new diabetic foot ulcer, including patients both with and without PAD; (2) to assess clinical characteristics that best predict poor outcome (i.e. non-healing of the foot ulcer) from this large set of patients; and (3) to examine whether such predictors differ between patients with and without PAD.\nMethods\nStudy design and population\nThe EURODIALE consortium is an international collaborative network that was created to stimulate further research in the field of diabetic foot disease. Its main objective was to assess outcome and the major predictors of clinical outcome in a large sample of European patients with diabetic foot ulcers. The design and rationale of this study have been described in detail elsewhere [10].\nBriefly, between 1 September 2003 and 1 October 2004, 1,232 patients with a new foot ulcer were included in 14 diabetic foot centres in ten European countries. The mean (range) number of included patients per centre was 88 (40\u2013125). All participating centres have a longstanding expertise in the field of diabetic foot disease. Patients included were those presenting for the first time with a new foot ulcer within a period of 12\u00a0months, either at the outpatient or inpatient clinics of participating centres. Excluded patients were those who had been treated at the participating centres for an ulcer on the ipsilateral foot during the previous 12\u00a0months and those with a life expectancy of less than 1\u00a0year. Participants attended follow-up visits on a monthly basis. At baseline and during all follow-up visits, data were collected and recorded on standardised case record forms. This was done by dedicated investigators in each centre who were trained during plenary meetings and on-site visits. Recorded data included demographics, data on co-morbidities and foot and ulcer characteristics, as well as management. The local ethics committees of the 14 hospitals approved the study protocol and all patients gave written informed consent.\nManagement of diabetic foot ulcer\nAll patients were treated according to protocols based on the International Consensus on the Diabetic Foot [11], which include off-loading, diagnosis and treatment of infection, assessment of vascular status, treatment of PAD and regular wound debridement.\nPotential predictive factors\nPotential determinants of healing were chosen on the basis of (1) current literature; (2) expert opinion after extensive discussions during EURODIALE meetings; and (3) suitability for use in daily clinical practice. In addition to sex, age at baseline and duration of diabetes, several disease-specific characteristics and co-morbidities were investigated [10].\nUlcer characteristics All patients underwent a standardised examination according to the PEDIS system. This was developed by the International Consensus on the Diabetic Foot to enable classification of patients for clinical research purposes [11, 12] and classifies foot ulcers according to five categories: perfusion, extent, depth, infection and sensation.Perfusion assessment included evaluation of the presence of pedal pulses and measurement of the ankle\u2013brachial pressure index (ABPI) using a handheld Doppler device; PAD was considered to be present if ABPI was <0.9 and\/or two foot pulses were absent.Extent (i.e. size) was determined by multiplying the largest by the second largest diameter perpendicular to the first and divided into three categories: <1\u00a0cm2, 1\u20135\u00a0cm2 and >5\u00a0cm2.Depth was described as either deep or superficial if a full thickness lesion of the skin was or was not extending through the subcutis, respectively.Infection was diagnosed if two or more of the following signs were present: frank purulence, local warmth, erythema, lymphangitis, oedema, pain, fever and foul smell. The term infection covers both soft tissue infection and bone infection.Evaluation of sensation (peripheral neuropathy [PNP]) included pressure sensation (10\u00a0g monofilament on plantar aspect of hallux, metatarsophalangeal joints 1 and 5), tactile sensation (cotton wisp on dorsum of foot), vibration sensation (128\u00a0Hz tuning fork on dorsum of the hallux) and blunt\/sharp discrimination (dorsum of foot). PNP was diagnosed if the results of two or more of the aforementioned tests were abnormal.In addition, the location of the ulcer was divided into plantar (on the plantar toes, plantar mid- or forefoot and plantar hind foot) and non-plantar (on the dorsal or interdigital part of the toes, on the dorsal or lateral aspect of the foot and heel ulcers). Ulcer duration was divided into three categories: <1\u00a0week, between 1\u00a0week and 3\u00a0months, and >3\u00a0months.\nCo-morbidities The following disabling co-morbidities were assessed: presence of severe visual impairment (defined as the inability to read a newspaper after correction), end-stage renal disease (ESRD) (defined as dependency on haemodialysis or peritoneal dialysis or a previous renal transplant procedure), heart failure (New York Heart Association [NYHA] classification III or IV), any neurological disorder (excluding diabetic polyneuropathy) resulting in loss of motor or sensory function (e.g. stroke) and inability to stand or walk without help.\nStudy main outcome\nMain outcome was complete healing (with or without minor amputation) of the foot, within the maximum follow-up period of 1\u00a0year. Healing was defined as healing (intact skin) of the whole foot at two consecutive visits. If more than one ulcer was present, the foot was defined as healed once all ulcers were healed. Outcome information was not obtained in 144 patients (11.7% of the patients included) who dropped out of the study and were therefore excluded from the analyses. Reasons for dropout were non-compliance (n\u2009=\u200924), inability to follow the patient (lack of transportation, no social support, too sick to attend; n\u2009=\u200925) or if care had been taken over by other specialists (n\u2009=\u200929); in 66 patients the reason for dropout could not be discovered. At baseline these participants were slightly older and had a higher incidence of heart failure, deeper ulcers and ulcers of longer duration than those included in the analyses (n\u2009=\u20091,088; Table\u00a01).\nTable\u00a01Baseline characteristics of participants included and those excluded (dropouts) from the present studyVariableIncluded (n\u2009=\u20091,088)Dropouts (n\u2009=\u2009144)p valueAge (years)64.7\u2009\u00b1\u200912.568.0\u2009\u00b1\u200911.60.003Male sex, n (%)a703 (64.6)85 (59.0)0.189Duration of diabetes, n (%)a0.418<5\u00a0years148 (14.1)19 (13.5)5\u201310\u00a0years169 (16.1)17 (12.1)>10\u00a0years731 (69.8)105 (74.5)Deep ulcer, n (%)a476 (43.8)80 (55.6)0.007Size of ulcer, n (%)a0.843<1\u00a0cm2403 (37.2)50 (35.0)1\u20135\u00a0cm2563 (52.0)76 (53.1)>5\u00a0cm2117 (10.8)17 (11.9)Duration of ulcer, n (%)a<0.001<1\u00a0week184 (17.0)10 (7.0)1\u00a0week\u20133\u00a0months627 (58.1)68 (47.6)>3\u00a0months269 (24.9)65 (45.5)Plantar location, n (%)a493 (48.2)62 (46.3)0.675Pretibial oedema, n (%)a197 (18.2)29 (20.3)0.538Heart failure NYHA III\u2013IV, n (%)a117 (10.9)23 (16.1)0.065Neurological disorder, n (%)a70 (6.5)9 (6.3)0.918Inability to stand or walk without help, n (%)a107 (9.9)15 (10.4)0.843Visual impairment, n (%)a164 (15.3)19 (13.2)0.507ESRD, n (%)a63 (5.8)7 (4.9)0.639Polyneuropathy, n (%)a826 (78.5)105 (76.1)0.515Infection, n (%)a591 (57.2)82 (61.2)0.380PAD, n (%)a505 (47.5)78 (56.1)0.056Unless otherwise stated, data are mean values\u2009\u00b1\u2009SD.aPercentages may not sum to 100 due to missing information\nStatistical analyses\nAll statistical analyses were carried using the STATA software package version 9.2 (STATA, College Station, TX, USA). Comparisons between groups\u2019 characteristics were made with \u03c72 tests (frequency data) or Student\u2019s t test (continuous data).\nMultiple imputation of missing values of predictor variables Values for one (n\u2009=\u2009188), two (n\u2009=\u200935) or three (n\u2009=\u200913) predictor variables were not available for 236 participants; the number of missing values per predictor ranged from 0 to 6%. In order to decrease bias and increase power of the analyses [13], we used multiple imputation chained equations (procedure \u2018ICE\u2019 in STATA) to impute those missing values (1.7% of all required values) rather than performing complete case analyses [14, 15]. With ICE the imputation model of a single variable uses all the other variables as predictors by appropriate regression models (i.e. linear, logistic or multinomial if imputed variable is continuous, dichotomous or categorical). We generated five imputed datasets that were used to fit the regression models of interest (in each dataset and in the final, i.e. the combined dataset). Parameter estimates and standard errors were combined across the five replicates according to the procedure described by Rubin [16] and Carlin et al. [17] (procedure \u2018micombine\u2019 in STATA).\nDevelopment of predictive models First, univariable logistic regression analyses were performed for all potential predictor variables with the outcome of interest (non-healing), with values presented as univariable odds ratios (ORs) along with the respective 95% CI. Second, all potential predictors were entered simultaneously in a multivariable logistic regression model that was reduced to a most parsimonious model using a backward selection method based on Akaike\u2019s Information Criterion. These models yielded a set of variables that best predict (and can be regarded as independent predictors of) outcome.\nResults\nClinical outcome\nWithin the 1\u00a0year follow-up, 77% of the 1,088 patients healed, 12% were still undergoing treatment, 5% underwent a major (i.e. above the ankle level) amputation and 6% died (before healing of the foot ulcer). Among the patients who healed, 17% underwent a minor amputation; this rate was similar to that in those patients who did not heal (20%, p\u2009=\u20090.425).\nWhen stratifying patients according to the presence or absence of PAD, significantly (p\u2009<\u20090.001) worse healing rates were observed in patients with than in those without PAD (69% vs 84%, respectively). Major amputation and mortality rates were also higher in patients with (8% and 9%, respectively) than in patients without PAD (2% and 3% respectively; p\u2009<\u20090.001). Baseline characteristics of patients with PAD compared with those without PAD are provided in Table\u00a02.\nTable\u00a02Patients\u2019 baseline characteristics according to their PAD statusVariablePatients with PAD (n\u2009=\u2009505)Patients without PAD (n\u2009=\u2009558)p valueAge (years)69.1\u2009\u00b1\u200911.260.5\u2009\u00b1\u200912.3<0.001Male sex, n (%)a321 (65.6)366 (63.6)0.490Duration of diabetes, n (%)a0.265<5\u00a0years63 (12.9)80 (14.9)5\u201310\u00a0years72 (14.7)93 (17.4)>10\u00a0years354 (72.4)363 (67.7)Deep ulcer, n (%)a266 (52.7)200 (35.8)<0.001Size of ulcer, n (%)a0.002<1\u00a0cm2173 (34.4)219 (39.5)1\u20135\u00a0cm2259 (51.5)294 (53.0)>5\u00a0cm271 (14.2)42 (7.5)Duration of ulcer, n (%)a<0.001<1\u00a0week58 (11.5)120 (21.7)1\u00a0week\u20133\u00a0months296 (58.0)318 (57.5)>3\u00a0months148 (29.5)115 (20.8)Plantar location, n (%)a197 (40.9)284 (55.0)<0.001Pretibial oedema, n (%)a111 (22.0)83 (14.9)0.002Heart failure NYHA III\u2013IV, n (%)a64 (12.7)47 (8.5)0.027Neurological disorder, n (%)a40 (8.0)27 (4.9)0.039Inability to stand or walk without help, n (%)a65 (12.9)36 (6.5)<0.001Visual impairment, n (%)a89 (17.9)66 (12.0)0.007ESRD, n (%)a35 (7.0)25 (4.5)0.082Polyneuropathy, n (%)a383 (77.2)424 (79.3)0.429Infection, n (%)a293 (60.9)282 (53.4)0.016Unless otherwise stated, data are mean values\u2009\u00b1\u2009SDaPercentages may not sum to 100 due to missing information\nPredictors of healing\nTable\u00a03 shows the univariable associations of the potential predictors of non-healing in the overall population and Table\u00a04 presents the variables retained in the predictive models after backward selection in the combined imputed datasets. The estimates were similar to those obtained in the complete cases dataset (n\u2009=\u2009854) indicating that missing values were non-selective (data not shown). These include the following eight characteristics, all of which predict lower probabilities of healing: older age, male sex, larger ulcer size, heart failure, inability to stand or walk without help, ESRD, PNP and PAD. These variables were consistently identified in all five imputed datasets.\nTable\u00a03Association of each potential predictor with non-healing in the overall population (n\u2009=\u20091,088)Predictor variablesOutcome: healingOR95% CIp valueAge, per 10\u00a0year increase1.321.17\u20131.49<0.001Sex, men vs women1.501.07\u20131.970.018Duration of diabetes0.712\u00a05\u201310 vs <5\u00a0yearsa0.960.56\u20131.65\u00a0>10 vs <5\u00a0yearsa1.050.69\u20131.60Depth of ulcer, deep vs superficial1.661.25\u20132.20<0.001Size of ulcer<0.001\u00a01\u20135 vs <1\u00a0cm2a2.251.60\u20133.17\u00a0>5 vs <1\u00a0cm2a4.222.64\u20136.72Duration of ulcer<0.001\u00a01\u00a0week to 3\u00a0months vs <1\u00a0weeka1.811.15\u20132.85\u00a0>3\u00a0months vs <1\u00a0weeka2.611.60\u20134.27Location, plantar vs non-plantar0.730.55\u20130.980.035Pretibial oedema, yes vs no1.791.27\u20132.510.001Heart failure (NYHA III\u2013IV), yes vs no2.031.35\u20133.050.001Neurological disorder, yes vs no1.440.85\u20132.460.176Inability to stand or walk without help, yes vs no2.501.62\u20133.79<0.001Visual impairment, yes vs no1.360.94\u20131.980.105ESRD, yes vs no2.201.30\u20133.730.004Polyneuropathy, yes vs no1.410.98\u20132.040.065Infection, yes vs no1.471.09\u20132.000.012PAD, yes vs no2.311.72\u20133.10<0.001aReference categoryTable\u00a04Multivariable models with independent predictors of non-healing in the whole study population and in patients with and without PADVariableAll patientsPatients with PADPatients without PADOR95% CIp valueOR95% CIp valueOR95% CIp valueAge, per 10\u00a0year increase1.281.11\u20131.470.0011.420.17\u20131.73<0.0011.550.91\u20132.630.105Sex, men vs women1.721.23\u20132.400.0021.971.25\u20133.110.003\u2013\u2013\u2013Size of ulcer<0.001<0.0010.008\u00a01\u20135 vs <1\u00a0cm2a2.261.58\u20133.223.221.95\u20135.321.250.74\u20132.12\u00a0>5 vs <1\u00a0cm2a3.882.37\u20136.343.841.97\u20137.483.481.62\u20137.46Duration of ulcer\u2013\u20130.086\u00a01\u00a0week to 3\u00a0months vs <1\u00a0weeka\u2013\u2013\u2013\u20132.141.05\u20134.36\u00a0>3\u00a0months vs <1\u00a0weeka\u2013\u2013\u00a0\u2013\u20132.180.98\u20134.84Heart failure (NYHA III\u2013IV), yes vs no1.550.99\u20132.430.0541.540.87\u20132.740.141\u2013\u2013\u2013Inability to stand or walk without help, yes vs no2.001.27\u20133.140.0032.361.34\u20134.170.0031.910.86\u20134.240.112ESRD, yes vs no2.511.41\u20134.480.0023.041.38\u20136.700.0062.000.76\u20135.250.161Polyneuropathy, yes vs no1.420.96\u20132.080.078\u2013\u2013\u20131.700.89\u20133.250.108Infection, yes vs no\u2013\u2013\u20131.631.03\u20132.580.036\u2013\u2013\u2013PAD, yes vs no1.711.23\u20132.370.001N\/AN\/AaReference categoryN\/A, not applicable\nSince we hypothesised that, from an aetiological point of view, predictors of non-healing would differ between patients with and those without PAD, predictive models were also fitted for these two groups separately (Table\u00a04). In patients with PAD almost all of the predictors identified in the whole study population, with the exception of PNP, were again found to be independent predictors of healing. In addition, the presence of infection emerged as an additional independent predictor of non-healing. In patients without PAD, older age, larger ulcer size, inability to stand or walk without help, ESRD, PNP and, in addition, longer ulcer duration were independent predictors of poorer healing.\nThe observed interaction between infection and PAD status partly supports the classification of foot ulcer disease into four stages as suggested by Armstrong et al. (University of Texas classification system) [9]. Accordingly, upon analysis of the odds of non-healing per PAD \u00d7 infection status, it was only in those patients with both PAD and infection that the odds of non-healing were markedly increased compared with those without PAD or infection: OR 2.82, CI 1.88\u20134.22, p\u2009<\u20090.001 in unadjusted analyses (Fig.\u00a01) vs OR 1.87, CI 1.20\u20132.91, p\u2009<\u20090.001 after adjustments for the other variables included in the predictive model.\nFig.\u00a01ORs of healing per PAD and infection (Inf) status\nDiscussion\nThe EURODIALE study is one of the few large prospective, international studies on outcome and determinants of outcome in diabetic foot disease. Despite the severity of the underlying disease and the important co-morbidity [7], clinical outcome of this population within a 1\u00a0year follow-up can be considered favourable. In our cohort, 77% of the patients healed (with or without a minor amputation), 5% underwent a major amputation and 6% died. However, healing rates in patients with PAD were considerably worse. In addition, predictors of healing also differed between the groups with and without PAD. The presence of infection, which is generally regarded as an important predictor of healing, was only predictive in individuals with PAD.\nWith regard to the overall outcome in our cohort, two recent studies found relatively comparable outcomes. Jeffcoate et al. [18] reported healing (excluding minor amputations) rates of 66% and an amputation rate of 5% with a similar prevalence of PAD. In a German cohort, Beckert et al. [19] found healing rates between 57% and 93% and major amputation rates of 3%, although the data as presented in that report cannot be easily compared because of their unique classification system. Oyibo et al. [20] also found similar rates of major amputation in their cohort (5%).\nOur study shows that the combination of PAD and infection has a major impact on healing rates (Fig.\u00a01); this significant interaction between PAD and infection is, in our opinion, one of the major findings of this study. In the patients without PAD we did not observe an association between infection and non-healing, which suggests that in these patients current antibiotic regimens and surgical techniques seem adequate to save a limb with adequate perfusion. However, within the total population of individuals with diabetic foot disease in developed countries, the group of infected and ischaemic ulcers accounted for almost one-third of all patients in our earlier report [7]. In a recent study, a significant relation between PAD, infection and poor outcome was also observed: in that study\u2019s large cohort of outpatients with type 2 diabetes, PAD was an independent predictor of infection-related mortality [21]. Unfortunately, there is very little insight into the pathophysiology and treatment of infection in individuals with PAD. Currently it is not clear why infection is more prevalent and more difficult to treat in individuals with PAD. Remarkably, very few patients with PAD were included in most of the randomised trials on antibiotic therapy in diabetic foot infections [22, 23]. It has previously been demonstrated that lower limb tissue levels of antibiotics can be markedly decreased as a result of impaired perfusion in PAD [24]. It is open to speculation whether aggressive revascularisation will improve control of infection in these patients.\nAlthough some earlier studies have examined the impact of co-morbidities on ulcer healing [25], no studies, to our knowledge, have systematically assessed in a multivariable analysis the effects on ulcer healing of patient characteristics including co-morbidities, as well as foot and ulcer characteristics at baseline. In our study, older age, ulcer size and several co-morbidities were independent predictors of non-healing in patients with and without PAD. Recently a number of larger studies reported data on determinants of outcome in diabetic foot disease such as the single-centre study by Beckert in Germany and the UK multi-centre study initiated by Jeffcoate [19, 26]. The former focused on wound-based characteristics and also found that the presence of PAD (defined as absence of pedal pulses) was an independent predictor of outcome (healing), while infection was not. In the recent UK multi-centre study, ulcer area was a strong predictor of outcome, as was the presence of PAD; co-morbidities were not taken into account in the regression analyses. Surprisingly, depth of the ulcer was not associated with outcome in our multivariable model, a finding also shown by Ince et al. [26]. In a retrospective study, Miyajima et al. [27] reported on patient characteristics that determined major lower extremity amputation and found that haemodialysis was an independent predictor of major amputation; in this study wound characteristics were not part of the regression analyses. The poor prognosis of foot ulcers for individuals in our study with ESRD is in line with earlier reports, in which amputation rates of 57% in individuals on haemodialysis were observed [25]. Although in our study ESRD was a predictor of non-healing in patients with and without PAD, it seemed to have a particularly negative effect in the latter patient group. PAD is frequently diagnosed and associated with adverse outcomes in haemodialysis patients [28]. PAD in ESRD patients is more severe and is accompanied by diffuse vascular calcifications, involvement of both distal infrapopliteal and foot arteries, and by impaired microcirculatory perfusion [29\u201331]. The severity of PAD in ESRD may explain the importance of ESRD in our healing models; additional mechanisms are probably impaired host defences in chronic renal failure and uraemia, or the presence of more resistant micro-organisms [32, 33].\nThe outcome of patients without PAD in our study was relatively favourable: 84% of the patients healed with or without minor amputation, 2% underwent a major amputation and 3% died. In our multivariate models, loss of sensation was associated with a poorer outcome in these patients, suggesting that loss of protective sensation is not only a key factor in the development of an ulcer, but also affects its outcome. This may be related to the preserved mechanism of off-loading the ulcer in individuals with intact protective sensation. However, neuropathy may also have direct effects on wound healing. Although data on the effect of neuropathy on wound healing in humans are scarce, some animal studies suggest that denervation may contribute to impaired wound healing in diabetes [34, 35]. A large dataset on individuals with neuropathic ulcers comes from retrospective database analyses in which healing rates of 47% were observed [36]. Although this study reported on healing at 20\u00a0weeks (whereas the current one examined healing rates at 1\u00a0year), the different results compared with our study are striking and may be related to an increased awareness of the importance of adequate off-loading, as a result of publication of international guidelines and reports on casting techniques [37\u201339].\nThere are several limitations to our study. Individuals who were lost to follow-up were excluded from the analyses as healing status could not be obtained; these individuals were slightly older and had a greater incidence of heart failure, deep ulcers or ulcers with a longer duration and PAD at baseline. In addition, we excluded patients who had had a previous ulcer within 12\u00a0months prior to presentation (i.e. we probably excluded patients with recurrent ulcers). Also we excluded patients with a life expectancy shorter than 1 year because of anticipated problems with follow-up. The estimates obtained in our models may therefore have underestimated the probability of non-healing in patients with a recurrent foot ulcer, although in one earlier study healing rate of neuropathic foot ulcers did not decrease in patients with multiple recurrences [40]. Since our study was embedded in daily clinical practice, limitations had to be set with regard to the number and type of data collected. It was therefore not possible to record more characteristics of these patients such as medication and extensive documentation of all complications. Moreover, to facilitate data collection, some continuous data (e.g. ulcer size) had to be transformed into a limited set of categories. Nevertheless, the set of potential predictors used in the present study do cover relevant patient and disease-specific aspects that can be easily assessed and used for patient risk estimation in clinical practice. Finally, our predictive model is based on outcomes that can be obtained in developed countries with access to the necessary resources such as antibiotic treatment and revascularisation; our results, therefore, are most relevant for diabetic foot ulcer patients in developed countries.\nIn conclusion, the results of this study have several implications. Both ulcer characteristics and several patient-related characteristics affected the outcome of diabetic foot ulcers. Therefore, a holistic approach by healthcare professionals who are familiar with the treatment of complicated diabetic patients is essential in order to identify the high-risk patient and start appropriate treatment. We found that patients with and without PAD differ in clinical characteristics, outcome and predictors of outcome. Taking into account these findings and the different pathophysiology and treatment of PAD and non-PAD ulcers, we feel that that diabetic foot ulcer with and without PAD should be defined as two separate disease states. The prevalent combination of PAD and infection is a unique entity; an important challenge lies in the development of evidence-based strategies to improve the poor outcome of these patients. Both studies comparing different antibiotic regimens in PAD, and studies evaluating the effects of early revascularisation on control of infection are urgently needed.","keyphrases":["outcome","diabetes","foot ulcer","peripheral arterial disease","infection","co-morbidities","non-healing","predictive model"],"prmu":["P","P","P","P","P","P","P","P"]}
{"id":"Naunyn_Schmiedebergs_Arch_Pharmacol-4-1-2323035","title":"Dipeptidyl peptidase IV inhibitors in diabetes: more than inhibition of glucagon-like peptide-1 metabolism?\n","text":"Inhibitors of the protease dipeptidyl peptidase IV (DPP-IV) are promising new drugs for the treatment of type 2 diabetes. They are thought to act by inhibiting the breakdown of glucagon-like peptide-1 and, thereby, selectively enhancing insulin release under conditions when it is physiologically required. These drugs are selective for DPP-IV, but the enzyme itself has a broad range of substrates other than glucagon-like peptide-1. Other high affinity substrates of DPP-IV including peptide YY may also play a role in the regulation of energy homeostasis. Moreover, DPP-IV is also known as CD26 and considered to be a moonlighting protein because it has a wide range of other functions unrelated to energy homeostasis, e.g. in immunity. The potential role of DPP-IV inhibition on substrates other than glucagon-like peptide-1 in diabetes patients remains to be elucidated.\nGlucagon-like peptide-1 (GLP-1) is a hormone which is released following meals and stimulates insulin release from the pancreas. Its effects are terminated by breakdown by the enzyme dipeptidyl peptidase IV (DPP-IV). Therefore, inhibition of DPP-IV increases GLP-1 levels in the circulation and, hence, insulin release under conditions when it is needed, i.e. after a meal but not during fasting. Consequently, inhibition of GLP-1 inactivation is an insulinotropic principle which is unlikely to cause hypoglycaemia between meals. The lower risk for hypoglycaemic events as compared with other insulinotropic or insulin-sensitising agents makes DPP-IV inhibitors very promising candidates for a more physiological treatment of type 2 diabetes (Combettes and Kargar 2008).\nIn recent years, a number of selective DPP-IV inhibitors such as vildagliptin and sitagliptin have been evaluated in clinical trials (Hermansen et al. 2007; Utzschneider et al. 2008) and may have a future role in the treatment of type 2 diabetes (Combettes and Kargar 2008). In this issue of the journal, a novel DPP-IV inhibitor, ASP8497, is being introduced, which is highly selective for DPP-IV as compared to other peptidases (Someya et al. 2008). However, DPP-IV itself is not selective for GLP-1 but has a wide range of other natural substrates (Boonacker and Van Noorden 2003). Therefore, we wish to highlight potential implications of this lack of selectivity of DPP-IV for the use of DPP-IV inhibitors in diabetes treatment.\nAnother high affinity substrate of DPP-IV is peptide YY (PYY; Mentlein et al. 1993). Whereas cleavage of GLP-1 by DPP-IV causes inactivation, cleavage of PYY yields the long C-terminal fragment PYY3\u201336, which is inactive at some but active at other subtypes of PYY receptors. Specifically, it converts the non-subtype-selective agonist PYY into a selective agonist at Y2 and Y5 receptors (Michel et al. 1998). Thus, DPP-IV does not inactivate PYY but, rather, qualitatively alters its biological activity.\nIndeed it has been reported that peripherally administered PYY3\u201336 inhibits food intake in rats, whereas PYY is a potent central stimulator of food intake (Batterham et al. 2002). Given the role of obesity in type 2 diabetes, prevention of the formation of such an endogenous food intake inhibitor by a DPP-IV inhibitor may be undesirable. It has been proposed that the effects of peripherally administered PYY3\u201336 on central nervous functions such as food intake may be mediated by excitation of afferent vagal fibres (Koda et al. 2005). However, the majority of subsequent rodent studies did not confirm inhibition of food intake by PYY3\u201336, particularly not following chronic administration (Boggiano et al. 2005). On the other hand, recent studies in non-rodents such as pigs (Ito et al. 2006) or humans (Degen et al. 2005; Sloth et al. 2007a; Sloth et al. 2007b) have reported reduced food intake upon peripheral administration of PYY3\u201336 but typically, these effects were found only at relatively high concentrations. Moreover, the effect of PYY3\u201336 on food intake was biphasic, depending on the duration of its administration (Parkinson et al. 2008). Two additional findings deserve consideration. Firstly, PYY3\u201336 was reported to promote fat oxidation and ameliorate insulin resistance in mice even under conditions of chronic administration where it did not reduce food intake (van den Hoek et al. 2006). Secondly, PYY3\u201336 was reported to lower plasma glucose levels even in the absence of alterations in circulating insulin levels (Bischoff and Michel 1998).\nTaken together, the presently available data on PYY3\u201336 on food intake and metabolic parameters are not yet conclusive. However, it is clear that PYY3\u201336 is largely formed by DPP-IV, raising the possibility that selective DPP-IV inhibitors may exert part of their effects by modulating the PYY\/PYY3\u201336 ratios. Therefore, it remains to be explored how possible effects on PYY cleavage contribute to metabolic effects of DPP-IV inhibitors in diabetic patients.\nMoreover, DPP-IV is not only a protease for substrates relevant to energy homeostasis, but it also has a range of additional functions (Boonacker and Van Noorden 2003). Therefore, it is considered to be a moonlighting protein. As a protease, it has several other substrates, and it also acts as a receptor and costimulatory protein in the immune system. In this regard, CD26 is considered to be an important regulator of T-cell function (Reinhold et al. 2008). These pleiotropic effects of DPP-IV or CD26 lead to numerous potential uses of its inhibitors other than type 2 diabetes including inflammatory diseases (Ohnuma et al. 2006; Reinhold et al. 2007; Thielitz et al. 2008; Thompson et al. 2007) and, perhaps, certain types of cancers (Kikkawa et al. 2005; Thompson et al. 2007). Some of these effects may manifest as useful secondary actions when being used for the treatment of diabetic patients whereas others may manifest as adverse events. Most of these potential additional effects may not yet have manifested in the currently published diabetes literature, but it appears prudent to keep an eye on them.","keyphrases":["dipeptidyl peptidase iv","diabetes","glucagon-like peptide-1","peptide yy","cd26"],"prmu":["P","P","P","P","P"]}
{"id":"Graefes_Arch_Clin_Exp_Ophthalmol-4-1-2413123","title":"Refractive properties of the healthy human eye during acute hyperglycemia\n","text":"Purpose To measure the refractive properties of the healthy human eye during acute hyperglycemia by means of Scheimpflug imaging and Hartmann-Shack aberrometry.\nTransient refractive changes, due to a variation in blood glucose levels, are well-known complications of diabetes mellitus (DM). Both myopic shifts [2, 6, 10, 23, 31, 36] and hyperopic shifts [9, 13, 18, 26, 32, 37] have been reported in patients with DM after several days or weeks of hyperglycemia. It has been suggested that the predominant cause of the refractive changes is a change in the thickness of the lens [16, 17, 19, 25, 29] or shape of the lens [21], and\/or a change in its refractive index [24, 27, 29, 33]. Two studies have been conducted in which hyperglycemia was induced under controlled circumstances to investigate refractive changes during hyperglycemia. Firstly, Gwinup et al. [15] administered glucose intravenously to ten patients with DM. A myopic shift of maximal -0.75\u00a0D was measured with autorefractometry. Secondly, Furushima et al. [11] induced hyperglycemia in seven healthy, young subjects through somatostatin injections and an oral glucose load. By means of autorefractometry and ultrasound biometry a large change in ocular refraction (-2.0 diopters (D)) and thickness of the lens (1.0\u00a0mm) was measured within 150 minutes after administration of the glucose load (maximal hyperglycemia: 13.9\u00a0mmol\/l). The results of these two studies indicated that induced hyperglycemia can cause changes in refraction and that these changes appeared to be larger in healthy subjects.\nIn the study of Furushima et al. [11], only the thickness of the lens was measured, and there is no information about the change in the shape and the refractive index of the lens due to acute hyperglycemia. Therefore, the aim of our study was to induce hyperglycemia in healthy subjects under controlled circumstances, and accurately measure ocular geometry and refraction by means of corrected Scheimpflug imaging and Hartmann-Shack aberrometry, in order to investigate the mechanism underlying refractive changes during hyperglycemia. From the ocular geometry and refraction, in combination with the measurement of the axial length of the eye, it is also possible to calculate the equivalent refractive index of the lens [4, 5].\nSubjects and methods\nFive healthy Caucasian subjects (two males and three females) were recruited for this study. Mean age was 24.8\u00a0years (SD \u00b14.6 and range 21.2\u201332.6) and mean body mass index (BMI) was 24.2\u00a0kg\/m2 (SD \u00b13.2 and range 21.4 - 29.7). The Medical Ethics Committee of the VU University Medical Center in Amsterdam approved the study protocol, and written informed consent was obtained from all subjects after the nature of the study had been explained. Subjects with a history of diabetes mellitus (or a fasting plasma glucose >5.5\u00a0mmol\/l), a BMI of >30\u00a0kg\/m2, visual acuity of <0.5 (Snellen), or a history of ocular pathology were excluded from the study.\nProcedure to induce hyperglycemia\nAfter a 10-hour overnight fast, the subjects received a subcutaneous injection of 100\u00a0\u00b5g synthetic somatostatin (Sandostatin, Novartis, Basel, Switzerland) in order to suppress the endogenous insulin secretion during glucose loading. Each subject had an oral glucose tolerance test (OGTT) (glucose 75\u00a0g) 30 minutes after the somatostatin injection. Blood glucose levels were measured with a blood glucose analyzer (HemoCue Diagnostics BV, Oisterwijk, the Netherlands). Endogenous insulin levels were determined with immunometric assays (Luminescence, Bayer Diagnostics, Mijdrecht, the Netherlands) in the Laboratory of Endocrinology of the Department of Clinical Chemistry in the VU University Medical Center. The subjects remained in a fasting state during the entire procedure.\nMeasurement of ocular parameters\nBefore and 120 minutes after the OGTT, 1.0% cyclopentolate and 5.0% phenylephrine eye-drops were administered to the right eye of the subjects. Hartmann-Shack aberrometry was performed with an IRX3 aberrometer (Imagine Eye Optics, Paris, France) and Scheimpflug imaging was performed with a Topcon SL-45 Scheimpflug camera, in which the film was replaced by a charge-coupled device (CCD) camera (St-9XE, SBIG astronomical instruments) with a range of 16 bits of grey values (512\u2009\u00d7\u2009512 pixels, pixel size 20\u2009\u00d7\u200920\u00a0\u00b5m, magnification: 1\u00d7). Before and every 30 minutes after the OGTT, three measurements were made with each apparatus. To obtain accurate measurements of the shape of the lens, ray-tracing was performed to correct the Scheimpflug images for the distortion that is inherent to this technique [4, 5]. By combining the measurements of the corneal thickness (d1), the depth of the anterior chamber (ACD), the anterior (R1) and posterior (R2) radius of the cornea, the lens thickness (d3) and the anterior (R3) and posterior (R4) radius of the lens, the axial length of the eye, and ocular refraction, it was possible to calculate the equivalent refractive index of the lens (n lens) by means of an iterative process [5]. In these calculations it was assumed that the refractive indices of the cornea, aqueous humor, and vitreous do not change, because in vitro experiments have shown that large changes in glucose levels induce negligible changes in the refractive indices of the ocular media [8, 22]. The axial length of the eye was measured with an IOL-master (Carl Zeiss Inc., North America). The equivalent refractive error (ERE) was calculated as: ERE = sphere + (cylinder\/2). Furthermore, the higher order aberrations (HOA) of each eye were analyzed for a pupil size of 5.7\u00a0mm and they were summarized in root mean square errors (RMS), including the third up to the eighth Zernike orders [34]. Furthermore, the HOA, including the spherical aberrations, were analyzed separately.\nMeasurements at baseline and during hyperglycemia were compared in the whole group by means of Wilcoxon signed rank sum tests. A refractive change of more than 0.2\u00a0D and a change in HOA of more than 0.025\u00a0\u00b5m were considered to be meaningful, according to the precision (defined as 95% confidence interval) for measuring the ERE and HOA of the aberrometer [3, 30]. Error analysis indicated that a change in R1, R2, d1, ACD, d3, R3, R4, and n lens of more than 0.05\u00a0mm, 0.05\u00a0mm, 0.02\u00a0mm, 0.14\u00a0mm, 0.15\u00a0mm, 0.30\u00a0mm and 0.40\u00a0mm, and 0.007 respectively, could be considered as significant, according to the precision of corrected Scheimpflug imaging [4]. In each subject individually, the significance of a change was determined from the precision of the measurements and the difference in the ocular parameters at baseline and during hyperglycemia. P values \u22640.05 were considered statistically significant.\nResults\nFigure\u00a01 shows the changes in blood glucose after the OGTT. In all subjects the mean blood glucose levels rose from 4.0\u00a0mmol\/l (range 3.4\u20134.5\u00a0mmol\/l) to 18.4\u00a0mmol\/l (range 16.1\u201322.0\u00a0mmol\/l) in 126 minutes (range 90\u2013210 minutes) after the OGTT. Subject 1 had a delayed elevation of blood glucose level, and therefore this subject received a second 75\u00a0g oral glucose load at time\u2009=\u00a030 minutes. Furthermore, venous blood samples of subject 1 were taken from the antecubital vein, which was kept open with 0.9% saline (100\u00a0cl). Endogenous insulin was suppressed by the subcutaneous injection of somatostatin during the glucose load to a mean value of 2.1\u00a0pmol\/l (range 0.4\u20134.5\u00a0pmol\/l), and remained below basal secretion level (<110.0\u00a0pmol\/l) for 147 minutes (range 75\u2013270 minutes).\nFig.\u00a01Changes in blood glucose (BG) levels in the five subjects after the administration of somatostatin and glucose; the oral glucose load (75\u00a0g) was administered at time\u2009=\u20090 minutes\nNo significant change in ERE was found in the whole group or in four of the subjects individually. In subject 1, a hyperopic shift in ERE of 0.4\u00a0D and an increase in the spherical aberration (\u00b1SE) from 0.03\u00a0\u00b1\u00a00.01 \u00b5m at T\u2009=\u20090 to 0.11\u00a0\u00b1\u00a00.01 \u00b5m during hyperglycemia was measured (p\u2009<\u20090.001). The other HOA did not change in subject 1. Furthermore, there was no significant change in the HOA separately or the RMS values in the group or in any of the other subjects individually. No changes in corneal thickness (d1), corneal shape (R1 and R2), anterior chamber depth (ACD), lens thickness (d3), or posterior lens shape (R4) were found in the group or in any of the subjects individually. In the whole group and in four subjects, no changes were found in the anterior radius of the lens (R3) or refractive index (n lens). However, in subject 01, the R3 and n lens changed significantly during hyperglycemia, compared to normal conditions; R3 decreased from 11.65 to 9.69\u00a0mm (mean decrease R3\u2009=\u20091.96\u00a0mm; p\u2009<\u20090.001) and n lens decreased from 1.436 to 1.422 (mean decrease n lens\u2009=\u20090.014; p\u2009=\u20090.003). Figure\u00a02 presents graphs of the normalized ERE, ACD, d3, R3, R4 and n lens of the five subjects. Figure\u00a03 shows two corrected Scheimpflug images and a schematic drawing of the lens of subject 1, in order to illustrate the differences in lens geometry during hyperglycemia compared to baseline. In all subjects, both hematological and ocular parameters normalized within 6 hours after the OGTT.\nFig.\u00a02Graphs of the normalized equivalent refractive error (ERE), lens thickness (d3), anterior chamber depth (ACD), anterior (R3) and posterior (R4) radius of the lens, and the refractive index of the lens (n lens) of the five subjects. Data are normalized by subtracting the value at baseline from the measured value in each subject. In subject 01 a hyperopic shift of 0.4\u00a0D, an increase in R3 and decrease in n lens were found during hyperglycemia at T\u2009=\u2009240 minutes (p\u2009<\u20090.01). The oral glucose load was administered at T\u2009=\u20090Fig.\u00a03Corrected Scheimpflug images of the right eye of subject 1. a At baseline time\u2009=\u20090 minutes, the shape of the cornea and lens are indicated with a solid line. b During hyperglycemia at time\u2009=\u2009240 minutes after the first oral glucose load, the shape of the cornea and lens are indicated with a line of dashes. c A drawing of the changes in the shape of the lens in hyperglycemic condition (line of dashes) compared to the normal condition (solid line)\nDiscussion\nRefractive changes occur frequently in patients with DM. The underlying mechanism is still unclear, and therefore the aim of our study was to measure ocular refraction and geometry during hyperglycemia, in an attempt to identify a possible explanation for these refractive changes. The effect of reproducible hyperglycemia was studied in healthy subjects without the systemic effects of DM. An OGTT in combination with a somatostatin injection was used to induce hyperglycemia. In an earlier study, this was shown to induce large changes in the refractive error and lens thickness [11]. Somatostatin inhibits insulin secretion [7], and to our knowledge there are no reports of refractive errors or changes in the lens due to this agent.\nIn general, and in four of the five subjects individually, no changes in ocular refraction or geometry were found during hyperglycemia. It could be that a more prolonged and severe hyperglycemia is needed to induce changes in refractive error or geometry of the eye. Glucose within the lens is metabolized via the sorbitol pathway, which consists of two enzymes that catalyze the conversion of glucose into its sugar alcohol sorbitol and the further conversion of sorbitol to fructose. These sugar alcohols tend to accumulate within the lens, because they penetrate cell membranes poorly. This accumulation of sugar alcohols causes the lens to swell [12]. This process might have taken longer than the observation period of the present study. Furthermore, the subjects measured in our study were young, and it could be that their lenses, which still have a fast metabolic reaction capacity, tolerate short hyperglycemic stress without swelling.\nThe results of our study are not in accordance with the findings of Furushima et al. [11], who observed a large myopic shift (-2\u00a0D) and a large increase in the thickness of the lens (1\u00a0mm) during hyperglycemia in healthy subjects. It must be noted that in our study the mean increase in the blood glucose level was even higher than that of the study of Furushima et al.. One main difference between our study and the study by Furushima et al. is the methods that were used to measure ocular refraction (aberrometry versus autorefractometry) and ocular geometry (corrected Scheimpflug imaging versus ultrasound biometry). However, the precision of aberrometry and autorefractometry are comparable for the measurement of defocus, astigmatism and, consequently, the equivalent refractive error [3, 30, 35]. Furthermore, the precision of corrected Scheimpflug imaging and ultrasound biometry are comparable as well. It could be that the difference in ethnicity (Caucasian subjects in the present study versus Asian subjects in the study by Furushima et al.) caused the inconsistency between the two studies, since Asian people generally have a more myopic ocular refraction than Caucasian subjects [14, 38].\nIn our study, small but significant changes in ocular refraction and lens geometry were found in one subject. A hyperopic shift of 0.4\u00a0D was found, in combination with an increase in anterior convexity of the lens. A combination of hyperopia and an increase in lens thickness during hyperglycemia has been described by Kluxen et al. [21], who found a 6\u00a0D hyperopic shift and a 0.4 mm increase in lens thickness in a diabetic patient with hyperglycemia. Saito et al. [29] reported hyperopic shifts (1.1 to 4.9\u00a0D) and an increase of approximately 0.2\u00a0mm in the thickness of the lens in five diabetic subjects during hyperglycemia. Therefore, it has been suggested that hyperglycemia causes a change in the refractive index of the lens [21, 24, 27, 29, 33]. The results of our study support this hypothesis; in subject 1, a decrease in the equivalent refractive index of the lens was calculated during hyperglycemia. It could be suggested that if the change in the shape of the lens is small, hyperopia will predominate during hyperglycemia due to a decrease in the refractive index of the lens. Alternatively, if the change in the shape of the lens is large in comparison to the decrease in the refractive index of the lens, the overall refractive error will result in myopia. The controversy in the literature with regard to refractive changes during hyperglycemia could be explained by this underlying mechanism of a balance between changes in the shape or the refractive index of the lens, which eventually determine the overall refractive outcome [20, 23, 28, 37].\nIt is surprising that a change in refraction and ocular parameters could be determined in only one subject. It must be noted that the procedures for inducing hyperglycemia and monitoring blood glucose were, to some extent, different for subject 1 compared to the other subjects. Because of a delayed elevation in blood glucose level, a second oral glucose load (150\u00a0g instead of 75\u00a0g glucose) was administered. Nevertheless, the maximum blood glucose value of subject 1 did not exceed that of the other subjects, and the endogenous insulin level was adequately suppressed during the glucose loading. Furthermore, in order to obtain sufficient blood samples, 0.9% saline had to be administered to keep the antecubital vein open. Therefore, it could not be excluded that the administration of saline contributed to the refractive change and alterations in the lens of subject 1. However, no studies have yet reported any refractive change due to saline administration.\nThe change in refractive error in patients with hyperglycemia could also be caused by a change in the shape of the cornea. However, previous research has shown that hyperglycemia has no influence on the shape of the anterior corneal surface [10, 24, 29, 32]. The results of our study also agree with these findings. No change in the anterior or posterior corneal radius was found during hyperglycemia. Therefore, the cornea does not seem to play a role in the explanation of refractive changes during hyperglycemia. This also applies for the higher order aberrations. Applegate et al. [1] reported that an increase in the higher order aberrations could cause a decrease in visual acuity. However, no changes in the higher order aberrations were found in any of the subjects in our study. Therefore, it can be assumed that blurred vision in hyperglycemia cannot be explained by changes in the higher order aberrations of the eye.\nIn sum, the results of this study show that induced hyperglycemia generally does not cause changes in the refractive properties of the healthy human eye. However, there were interindividual variations, as illustrated by subject 1, who had a hyperopic shift of refraction and a change in shape and equivalent refractive index of the lens during hyperglycemia. This could provide an explanation for the mechanism underlying the refractive changes often experienced by patients with DM and hyperglycemia.","keyphrases":["hyperglycemia","scheimpflug imaging","hartmann-shack aberrometry","diabetes mellitus","lens","refractive errors"],"prmu":["P","P","P","P","P","P"]}
{"id":"Histochem_Cell_Biol-4-1-2413109","title":"Organization of multiprotein complexes at cell\u2013cell junctions\n","text":"The formation of stable cell\u2013cell contacts is required for the generation of barrier-forming sheets of epithelial and endothelial cells. During various physiological processes like tissue development, wound healing or tumorigenesis, cellular junctions are reorganized to allow the release or the incorporation of individual cells. Cell\u2013cell contact formation is regulated by multiprotein complexes which are localized at specific structures along the lateral cell junctions like the tight junctions and adherens junctions and which are targeted to these site through their association with cell adhesion molecules. Recent evidence indicates that several major protein complexes exist which have distinct functions during junction formation. However, this evidence also indicates that their composition is dynamic and subject to changes depending on the state of junction maturation. Thus, cell\u2013cell contact formation and integrity is regulated by a complex network of protein complexes. Imbalancing this network by oncogenic proteins or pathogens results in barrier breakdown and eventually in cancer. Here, I will review the molecular organization of the major multiprotein complexes at junctions of epithelial cells and discuss their function in cell\u2013cell contact formation and maintenance.\nIntroduction\nIn multicellular organisms, cell\u2013cell adhesion is involved in most developmental processes. It is necessary for example for the assembly of coherent sheets of barrier-forming epithelial and endothelial cells which line the inner and outer surfaces of the organism like those of the intestine, the skin or the blood vessels. However, also in adult tissues cell\u2013cell contacts are far from being static structures which maintain the barriers by simply holding cells together. During the turnover of growing tissues such as the intestine or the skin, they are constantly remodeled to allow the extrusion of \u201cold\u201d cells and the incorporation of \u201cyoung\u201d cells derived from stem cells without a concomitant loss of the barrier function (Fuchs et al. 2004). Similarly, during leukocyte extravasation in secondary lymphoid organs or at sites of an ongoing immune response, the homotypic interactions between endothelial cells must be altered to allow the passage of the leukocytes without affecting the barrier properties of the endothelium (Ley et al. 2007). Finally, during wound healing and tissue repair, cells undergo a coordinated movement, proliferate and establish new cell\u2013cell contacts once they encounter cells from the opposing site of the wound (Perez-Moreno and Fuchs 2006). These different demands in different cell types and different physiological situations require a sophisticated regulatory network which enables a partial dismantling and re-establishment of cell\u2013cell contacts while simultaneously preventing the loss of an epithelial phenotype which in adult tissues frequently correlates with tumor progression and metastasis (Thiery 2002).\nNot surprisingly, the organization of cell\u2013cell contacts of epithelial has attracted a great deal of attention. Due to the easy accessibility of cultured epithelial cells many discoveries have been made in epithelial cells. Epithelial cell\u2013cell contacts contain three major adhesive structures which can be identified at the ultrastructural level, the tight junctions (TJs), the adherens junctions (AJs), and the desmosomes. In polarized epithelial cells of certain tissues like the intestine, TJs and AJs are asymmetrically distributed at the apical region of the lateral cell contact forming the apical junctional complex (AJC) which encircles the apex of the cells and demarkates the border between the apical and the basolateral membrane domains (Nelson 2003). Common to all three types of structures is the presence of several adhesion molecules that link the neighbouring cells through homophilic and heterophilic adhesive interactions, and the presence of cytoplasmic scaffolding proteins that organize signaling complexes and which provide a mechanical link to the actin cytoskeleton (or intermediate filaments in the case of desmosomes). The scaffolding proteins might also link different protein complexes\u2014at least temporarily\u2014and thus organize supramolecular protein complexes. It should be noted that these protein complexes are dynamic, and that their composition is subject to regulation depending on junctional maturation and integrity. During the last few years, a rapid progress has been made in identifying new proteins at cell\u2013cell contacts and in particular in deciphering the molecular composition of the TJs. Among the most exciting findings was probably the discovery of protein complexes at TJs which are highly conserved through evolution and which regulate cellular polarization in different organisms and various cell types. Here, I will review the major multiprotein complexes present at cell\u2013cell contacts of vertebrate epithelial cells and highlight the most recent advances in the understanding of their role in the organization and functions of epithelial cell\u2013cell contacts in vertebrates.\nAdherens junctions\nThe function of adherens junctions\nA main function of AJs is to connect cells to regulate tissue formation and morphogenesis during development as well as the maintenance of solid tissues in the adult organism (Gumbiner 1996). The major cell adhesion molecules at AJs, the classical cadherins, connect adjacent cells through homophilic interactions and are linked to the cytoskeleton through proteins associated with their cytoplasmic tail, the catenins. This link generates a transcellular network of actin filaments running through the entire sheet of cells with the cadherin\u2013catenin complexes serving as connectors of the actin filaments bundles at the intercellular space. During morphogenetic events, for example during neural tube formation, mechanical forces can thus be applied to the whole cellular sheet. In adult tissues, cadherin-mediated cell adhesion is absolutely required for cell\u2013cell adhesion to be maintained as a loss of cadherin adhesion by Ca2+-depletion results in a loss of cell\u2013cell interaction and rounding up of the cells despite a number of other adhesion molecules (which are not Ca2+-dependent) at cell\u2013cell contacts (Takeichi 1977).\nCadherins and catenins\nSince the cadherin\u2013catenin complex has been the subject of a number of reviews, I will only summarize the central interactions and refer the reader to recent comprehensive reviews (Gumbiner 2005; Halbleib and Nelson 2006; Perez-Moreno and Fuchs 2006; Perez-Moreno et al. 2003; Pokutta and Weis 2007). As mentioned above, the cytoplasmic tail of classical cadherins like E-cadherin forms a multiprotein complex with \u03b2-catenin, \u03b3-catenin and p120 catenin (p120ctn), members of the armadillo repeat domain-containing family of proteins. In this complex, \u03b2-catenin and p120ctn are directly associated with the membrane-distal and membrane-proximal region, respectively (Fig.\u00a01). Both regions are highly conserved among classical cadherins underscoring the importance of these interactions. Under conditions where \u03b2-catenin is limiting, its binding site in E-cadherin can be occupied by the \u03b2-catenin-related plakoglobin\/\u03b3-catenin which otherwise associates preferentially with desmosomal cadherins (Zhurinsky et al. 2000). The interaction with \u03b2-catenin is required for the transport of E-cadherin from the ER to the basolateral cell surface (Chen et al. 1999). It is also required for the adhesive function of E-cadherin as post-translational modifications of \u03b2-catenin which alter its affinity towards E-cadherin alter the strength of E-cadherin adhesive activity (Perez-Moreno et al. 2003; Pokutta and Weis 2007). The adhesive activity is thus dependent on its association with \u03b2-catenin. Conversely, this association is also necessary to stabilize \u03b2-catenin which is otherwise rapidly degraded by the ubiquitin-proteasome pathway (Nelson and Nusse 2004).\nFig.\u00a01Major protein complexes at adherens junctions. Two major protein complexes exist at AJs of epithelial cells. The cadherin\u2013catenin complex consist of the Ca2+-dependent adhesion molecule E-cadherin and the armadillo repeat proteins p120ctn and \u03b2\/\u03b3-catenin which directly bind to the cytoplasmic domain of E-cadherin. \u03b1-catenin directly associates with \u03b2-catenin but not simultaneously with F-actin. The nectin\u2013afadin complex consists of the Ca2+-independent adhesion molecule nectin and the PDZ protein afadin. Afadin contains a F-actin-binding domain and thus can link the nectin\u2013afadin system to the actin cytoskeleton. The two adhesion complexes can be linked through several molecular interactions. Afadin can directly interact with \u03b1-catenin. It also interacts with ponsin\/SH3P12 which can interact with the F-actin-binding protein vinculin, and it interacts with LMO7 and ADIP which both can interact with the F-actin binding protein \u03b1-actinin. The nature of the link between the cadherin\u2013catenin complex and the actin cytoskeleton is still unclear. Double arrows indicate direct interactions, the question mark symbolizes the missing link\nThe association of E-cadherin with p120ctn is subject to a similar reciprocal regulation of cell\u2013cell contact localization: The cadherin molecule is necessary to localize p120ctn at the cell contact, and p120ctn is required for the stable localization of the cadherin molecule at AJs (Reynolds and Roczniak-Ferguson 2004). In contrast to \u03b2-catenin, p120ctn stabilizes cadherin which is constitutively endocytosed (Bryant and Stow 2004) by regulating its turnover rate at the surface (Davis et al. 2003). Besides its additional role in regulating transcription by interacting with transcription factors (van Roy and McCrea 2005), p120ctn has a also a function in regulating the activity of Rho small GTPases. The formation of early cell\u2013cell contacts correlates with the activation of Cdc42 and Rac1 and the inhibition of RhoA (Noren et al. 2001), and these changes in activities of the small GTPases are at least in part regulated by p120ctn which interacts with the Rac activator Vav2 (Noren et al. 2000) and the RhoA inhibitor p190RhoGAP (Wildenberg et al. 2006). The activation of Cdc42\/Rac1 and the downregulation of RhoA activity serves to facilitate new cell adhesion by increasing the cell surface interacting with membranes of neighbouring cells through lamellipodia and filopodia formation and simultaneously inhibiting cell migration by inhibiting stress fiber formation, respectively (Perez-Moreno and Fuchs 2006).\nThe role of \u03b1-catenin is less clear. For a long time considered to bridge the AJs to the actin cytoskeleton through direct interactions with both \u03b2-catenin and F-actin, this role has been challenged by the observations that its binding to F-actin (occurs only as homodimer) and its heterodimeric association with \u03b2-catenin are mutually exclusive (Drees et al. 2005; Yamada et al. 2005). Therefore, the physical bridge between the cadherin\u2013catenin complex and the actin cytoskeleton remains to be identified. It is possible that \u03b1-catenin present in the cadherin-associated heterodimeric \u03b2-catenin\u2013\u03b1-catenin complex is able to bind to other proteins which associate with F-actin. The putative linker is required to fulfill two critera: to bind directly to \u03b1-catenin and simultaneously\u2013either indirectly or directly\u2014to F-actin. From several proteins which fulfill these requirements, vinculin and \u03b1-actinin turned out not to mediate actin binding to the cadherin\u2013catenin complex excluding them from the list of possible candidates (Yamada et al. 2005). Other candidate proteins including AF-6\/afadin (Pokutta et al. 2002), ZO-1 (Itoh et al. 1997), formin (Kobielak et al. 2004), spectrin (Pradhan et al. 2001) or the LIM protein ajuba (Marie et al. 2003) remain to be tested.\nNectins and afadin\nThe second major adhesive protein complex at AJs consists of members of the nectin family of adhesion molecules and a scaffolding protein that is directly associated with the cytoplasmic domain of nectins named AF-6\/afadin (Takai and Nakanishi 2003). Nectins are immunoglobulin-like proteins and comprise a family consisting of four members (nectin-1 to -4), which are localized at AJs of epithelial cells (Reymond et al. 2001; Sakisaka and Takai 2004). Unlike classical cadherins which undergo only homophilic interactions in trans, nectins undergo both trans-homophilic and trans-heterophilic interactions. The major heterophilic binding partners are other members of the nectin family as well as members of nectin-related adhesion molecules Nectin-like (Necl)-1 to -5 (Sakisaka et al. 2007). Nectins are \u201ctrue\u201d adhesion molecules as they support cell aggregation when ectopically expressed in cells (Aoki et al. 1997; Lopez et al. 1998; Satoh-Horikawa et al. 2000; Takahashi et al. 1999). They also seem to influence the E-cadherin-mediated adhesion (Martinez-Rico et al. 2005; Sato et al. 2006) suggesting that they are contributing to the overall strength of cell\u2013cell adhesion. Like E-cadherin, nectin-2 appears very early at cell\u2013cell contacts during junction formation and is present at so-called primordial, spot-like AJs (pAJs) or puncta (Asakura et al. 1999), which are formed at the tips of protrusions of two contacting cells (Perez-Moreno and Fuchs 2006). An important function of nectins which is similar to that of cadherins is their ability to activate Cdc42 and Rac1 small GTPases. Trans-interaction of nectins results in the recruitment and activation of c-Src, followed by the activation of the two guanine-nucleotide exchange factors \u201cFGD-1-related Cdc42 GEF\u201d (FRG) and Vav2, which are specific for Cdc42 and Rac1, respectively (Fukuhara et al. 2004; Kawakatsu et al. 2002, 2005). As pointed out above, the activation of these small GTPases is probably required to facilitate junction formation suggesting that nectins cooperate with cadherins in the regulation of the actin cytoskeleton at sites of cell adhesion. However, in addition they might also help to regulate the formation of tight junctions and their physical separation from AJs during junctional maturation (see below).\nAll nectins directly associate with afadin (Reymond et al. 2001; Takahashi et al. 1999). L-Afadin, the longer version of two afadin isoforms with a F-actin-binding domain, is a scaffolding protein which interacts with both nectins and F-actin through independent domains suggesting that it directly links nectin-based adhesion sites to the actin cytoskeleton (Mandai et al. 1997) (Fig.\u00a01). However, through additional protein interactions, it might also establish an indirect link to the actin cytoskeleton as well as to the cadherin\u2013catenin complex by its interaction with \u201cclassical\u201d AJ-associated proteins. Afadin directly associates with \u03b1-catenin (Pokutta et al. 2002; Tachibana et al. 2000), with vinculin through its association with ponsin\/SH3P12 (Mandai et al. 1999), and with \u03b1-actinin through its association with \u201cafadin Dil domain-interacting protein\u201d (ADIP) and \u201cLim domain only 7\u201d (LMO7) (Asada et al. 2003; Ooshio et al. 2004) (Fig.\u00a01). It should be mentioned that some of these protein interactions might occur specifically in certain tissues, and that the molecular mechanisms underlying these interactions are not revealed in detail. Nevertheless, it is likely that through these multiple interactions the two major protein complexes at AJs are physically linked and that they influence each other in their localization and activity (Sakisaka et al. 2007).\nTight junctions\nThe function of TJs\nIn polarized epithelial cells, the TJs forms a belt-like structure at the apical region of the cellular junction and represent a boundary between the apical and the basolateral membrane domains which differ in the composition of lipids and proteins (Tsukita et al. 2001). At the TJ area, two structures can be distinguished by ultrathin electron microscopy: sites where the intercellular space is basically obliterated and where the outer leaflets of the adjacent membranes appear to be in direct contact, and regions where the membranes of the two adjacent cells are separated by intercellular space. By freeze-fracture electron microscopy, the TJ area appears as a branched network of strands where the strands reflect the sites of direct membrane contacts (Tsukita et al. 2001). Two major functions are attributed to TJs: First, the regulation of the paracellular permeability of the epithelial sheet for ions and small solutes, which is an organ-specific function and which varies for different epithelia depending on the specific requirements of the organ (Furuse and Tsukitas 2006; Van Itallie and Anderson 2006); second, the formation of a physical barrier to prevent intramembrane diffusion of lipids and proteins, a rather cell-autonomous function which is necessary for a cell to maintain an asymmetric distribution of membrane components and to develop membrane polarity (Tsukita et al. 2001). In the recent years it has become clear that the molecular basis of the TJ strands are claudins (Fig.\u00a02), a family of integral membrane proteins at TJs which are constituents of the TJ strands and which induce the formation of TJ strands upon ectopic expression in fibroblasts (Furuse et al. 1998). Claudins do not just create TJ strands, but within the strands form a selective permeability barrier by forming size- and charge-selective aqueous pores (Tsukita and Furuse 2000; Van Itallie and Anderson 2006). Surprisingly, the TJ strands do not seem to be the basis for the second major function of TJs, the diffusion barrier for intramembrane particles (Umeda et al. 2006). It has also become clear that the TJs harbor peripheral membrane proteins which regulate cell polarity and membrane asymmetry suggesting that these proteins are involved in the role of TJs in regulating apico-basal polarity.\nFig.\u00a02Integral membrane proteins at tight junctions of epithelial and endothelial can be grouped into three classes based on their overall organizations. The first class is characterized by two extracellular loops, four transmembrane regions, and two cytoplasmic tails (occludin, claudins, tricellulin). The second class consists of Ig-SF members which all contain two Ig-like domains. The third class (contains only one member, CRB3) is characterized by a short extracellular domain (36 AA), a single transmembrane domain and a short cytoplasmic tail. In contrast to the other integral membrane proteins, the function of the extracellular domain of CRB3 is not clear\nIntegral membrane proteins at TJs\nThree different classes of integral membrane proteins have been identified at TJs (Fig.\u00a02). One class comprises occludin, claudins and tricellulin, which all contain four transmembrane domains, two extracellular loops, and the N-terminal and C-terminal ends are localized in the cytoplasm. Twenty-four claudins have been identified in humans. The major role of claudins is to form paired strands through homophilic and heterophilic cis and trans interactions. The large number of claudins, their ability to undergo heterophilic interactions and their ion selectivity allows for the formation of TJ strands with specific permeability properties depending on the needs of a specific tissue (Furuse and Tsukita 2006). Occludin is incorporated into TJ strands but its ectopic expression does not induce strand formation (Furuse et al. 1998) suggesting that it has rather an accessory role in TJ strand formation (Yu et al. 2005). Tricellulin differs from claudins and occludin in its specific enrichment at tricellular contact sites (Ikenouchi et al. 2005). The second class of integral membrane proteins comprises members of the CTX subfamily of the immunoglobulin superfamily (Fig.\u00a02) which is characterized by one V-type and one C2-type Ig-like domain (Williams and Barclay 1988). Based on sequence homology, the length of their cytoplasmic tails and the type of the C-terminal PDZ domain motifs, the Ig-SF proteins at TJs can be further subdivided into a group consisting of JAM-A, JAM-B and JAM-C, and in a second group consisting of CAR, CLMP, ESAM, and JAM4 (Ebnet et al. 2004). Except for JAM-A, there is only little information on the role of these proteins in the development or the function of tight junctions. All but CLMP interact with TJ-associated scaffolding proteins like ZO-1 (CAR, JAM-A, -B, -C (Cohen et al. 2001; Ebnet et al. 2000, 2003), PAR-3 (JAM-A, -B, -C (Ebnet et al. 2001, 2003) or MAGI-1 (ESAM, JAM4; (Hirabayashi et al. 2003; Wegmann et al. 2004) (Fig.\u00a03), and in some cases, their ectopic expression increases the transepithelial electrical resistance (TER) or decreases the paracellular permeability suggesting a regulatory role in TJ formation (Cohen et al. 2001; Hirabayashi et al. 2003; Mandicourt et al. 2007; Raschperger et al. 2003). For JAM-A, there is good evidence that it regulates TJ formation through its direct association with the scaffolding protein PAR-3 (Ebnet et al. 2001; Itoh et al. 2001). JAM-A localizes very early at sites of cell adhesion during cell\u2013cell contact formation (Ebnet et al. 2001; Suzuki et al. 2002), and this localization probably serves to recruit PAR-3 to these site to initiate the polarization of the lateral membrane resulting in TJ formation (see below for details). Both RNA interference-mediated downregulation of JAM-A and ectopic expression of a JAM-A dominant-negative mutant that mislocalizes PAR-3 result in decreased TER, increased paracellular permeability and a defect in the development of membrane asymmetry (Mandell et al. 2005; Rehder et al. 2006) pointing to a general defect in the formation of functional TJs. The third class of integral membrane proteins comprises only one protein, Crumbs3 (CRB3), a homologue of the Drosophila Crumbs protein with a very short extracellular domain of only 36 AA and a short cytoplasmic domain of 41 AA (Makarova et al. 2003; Medina et al. 2002). CRB3 directly associates with two peripheral membrane proteins, Pals1 (Roh et al. 2002b) and PAR-6 (Lemmers et al. 2004), which are components of the two major cell polarity protein complexes localized at TJs, i.e. the Pals1\u2013PATJ complex and the PAR-3\u2013aPKC\u2013PAR-6 complex (Fig.\u00a03, see also below). Its overexpression delays TJ formation (Lemmers et al. 2004; Roh et al. 2003), and its ectopic expression in a cell line that expresses only little endogenous CRB3 results in the development of functional TJs (Fogg et al. 2005). These effects can most likely be attributed to its association with cell polarity proteins and the regulation of their subcellular localization (see below).\nFig.\u00a03Major protein complexes and functional classes of molecules at tight junctions. The TJs contain three major multi-protein complexes consisting largely of scaffolding proteins, the ZO protein complex, the CRB3\u2013Pals1\u2013PATJ complex and the PAR-3\u2013aPKC\u2013PAR-6 complex. Besides these three protein complexes which seem to be constitutively associated at TJs, a number of proteins with different functions has been identified at TJs. These include additional scaffolding proteins like MUPP1 and MAGI-1, adaptor proteins, transcription regulators and RNA processing factors, regulatory proteins like small GTPases and G-proteins, kinases and phosphatases, and heat shock proteins. Double arrows indicate direct interactions. Not all direct interactions that have been identified are depicted\nMultiprotein complexes at TJs\nSince the discovery of ZO-1 as the first protein at TJs and its molecular cloning (Itoh et al. 1993; Stevenson et al. 1986), the number of proteins that are localized at TJs has steadily increased. These proteins comprise scaffolding and adapter proteins, regulatory proteins like small GTPases, G-proteins, kinases and phosphatases, as well as transcription factors or factors regulating RNA processing (Fig.\u00a03). The large number and the functional diversity of these proteins suggest that TJs are a focus of incoming and outgoing signals and that their composition is dynamic. In accordance with this, many proteins identified at TJs are found at other compartments of the cell as well including the nucleus or the cytoskeleton and are actively shuttling between these compartments and the TJs (Matter and Balda 2007). The organization of these networks is regulated by proteins containing multiple protein\u2013protein interaction domains such as PDZ domains, GuK domains, SH2 or SH3 domains (Pawson and Nash 2003). At the TJs three major protein complexes exist which involve one or several scaffolding proteins, the ZO protein complex, the Pals1\u2013PATJ complex, and the PAR-3\u2013aPKC\u2013PAR-6 complex (Fig.\u00a03).\nThe ZO protein complex\nZO-1 is a classical scaffolding protein of the MAGUK family with three PDZ domains, one SH3 domain and one GuK domain (Funke et al. 2005). It can directly associate with several integral membrane proteins at TJs including occludin, claudins, JAMs and CAR through independent domains (Fig.\u00a04), and it probably serves to cluster these proteins at the TJs. It also interacts with other cytoplasmic proteins including its homologues ZO-2 and ZO-3, and in addition with the actin cytoskeleton (Fanning et al. 1998; Fanning et al. 2002; Wittchen et al. 1999). The exact composition of the ZO complex is not completely understood, yet. ZO-1 forms independent complexes with ZO-2 and ZO-3 (Wittchen et al. 1999), and both ZO-2 and ZO-3 can also interact with F-actin and share with ZO-1 some of the integral membrane proteins at TJs like occludin and claudins (Itoh et al. 1999; Wittchen et al. 1999). Alltogether, the ZO complex provides the major link to the actin cytoskeleton at the TJs (Fig.\u00a04). However, regarding the role of the individual ZO proteins, some redundancy might exist. In agreement with this, the absence of ZO-1 results in a slight delay in TJ formation but does not impair the formation of functional TJs in two epithelial cell lines (McNeil et al. 2006; Umeda et al. 2004). Only when all three ZO proteins are absent the formation of TJs is blocked as indicated by the absence of TJ strands, the lack of other TJ proteins like occludin, claudin-3 and JAM-A, and the complete loss of the barrier function (Umeda et al. 2006). Together, these findings indicate a critical role for the ZO proteins for the development of TJ strands, probably by forming the physical scaffold for the strand-forming proteins like claudins and occludin. As will be discussed below, ZO proteins also form a platform for signaling proteins to regulate epithelial proliferation and morphogenesis.\nFig.\u00a04Organization of the tight junctional plaque. The major protein complexes at TJs interact with specific transmembrane proteins. CRB3 recruits the Pals1\u2013PATJ complex to TJs. CRB3 interacts with the PDZ domain of Pals1, Pals1 interacts with PATJ through a heterodimeric L27 domain interaction. CRB3 is also localized at the apical membrane domain of epithelial cells (Makarova et al. 2003). The CRB3\u2013Pals1\u2013PATJ complex regulates TJ formation but the mechanism is largely unknown. The ZO complex is associated with the membrane through multiple interactions of ZO-1 with various integral membrane proteins including occludin, claudins, JAM-A and CAR. ZO-2 and ZO-3 can interact with both ZO-1 and also with claudins. The ZO protein complex probably serves to link TJs to the cytoskeleton as all three ZO proteins directly interact with F-actin. The PAR-3\u2013aPKC\u2013PAR-6 complex is associated with the membrane through the interaction of PAR-3 with JAM-A. PAR-3 interacts with aPKC through its aPKC-interacting domain, PAR-6 interacts with aPKC through a PB1\u2013PB1 domain interaction. A direct interaction between the PDZ domain of PAR-6 and PDZ domain 1 of PAR-3 has also been described. The PAR complex regulates the formation of TJs and apico-basal polarity. The JAM-A-related Ig-SF member JAM4 interacts directly with MAGI-1; the role of this protein complex is not clear. It should be noted that this drawing is incomplete as it does not depict interactions among the various protein complexes which have been described (e.g. PAR-6 can also associate with CRB3 and Pals1, ZO-3 can associate with PATJ). Also, the multiple PDZ domain protein MUPP1 (not depicted in this Figure, see Fig.\u00a03 for a schematic representation) associates with claudins, JAM-A and CAR, as well as with angiomotin family members (Coyne et al. 2004; Hamazaki et al. 2002; Sugihara-Mizuno et al. 2007). Double arrows with solid lines indicate direct protein\u2013protein interactions, double arrows with broken lines indicate interactions with F-actin\nThe CRB3\u2013Pals1\u2013PATJ complex\nThe CRB3\u2013Pals1\u2013PATJ complex has originally been described in Drosophila as a protein complex (the Crumbs\u2013Stardust\u2013Discs lost complex; this complex is now called Crumbs\u2013Stardust (Sdt)\u2013dPATJ complex (Pielage et al. 2003)) involved in the regulation of apico-basal polarization (Tepass et al. 2001). In Drosophila epithelial cells, this complex localizes to the apical region of the lateral membrane domain, called subapical region (SAR) or marginal zone (Knust and Bossinger 2002), that is positionally analogous to the TJs in vertebrate epithelial cells. The localization of the CRB3\u2013Pals1\u2013PATJ complex at the TJ is mediated through a direct and PDZ domain-dependent interaction of Pals1 with the C-terminal PDZ domain motif in CRB3, and a direct interaction between Pals1 and PATJ involving the L27N domain of Pals1 and the L27 domain present at the NH2-terminal region of PATJ (Fig.\u00a03) (Roh et al. 2002b). RNA interference-mediated knockdown of Pals1 leads to defects in the formation of TJs as well as in the development of lumen-containing epithelial cysts (an assay system for apico-basal polarity development (O\u2019Brien et al. 2002)), and is accompagnied by a loss of PATJ protein expression (Straight et al. 2004). Conversely, knockdown of PATJ impairs the barrier function of TJs and results in a loss of Pals1 at TJs, an internalization of CRB3, and a redistribution of other TJ components like occludin and ZO-3 (Michel et al. 2005; Shin et al. 2005). These observations strongly suggest that the CRB3\u2013Pals1\u2013PATJ complex is important for the development of functional TJs in vertebrate epithelial cells.\nThe PAR-3\u2013aPKC\u2013PAR-6 complex\nIn evolutionary terms, the PAR-3\u2013aPKC\u2013PAR-6 complex is the most ancient among the three major protein complexes at TJs. As opposed to the ZO protein complex and the CRB3\u2013Pals1\u2013PATJ complex, all three proteins of this complex exist in C.elegans where they cooperate to regulate the development of membrane asymmetry in the zygote (Kemphues 2000). The acronym Par stands for partitioning-defective and reflects the lack of partition of cytoplasmic P granules in C.elegans mutant embryos in response to sperm entry (Kemphues et al. 1988). The initial screen identified six par genes, and their molecular characterization revealed that they encoded proteins of different structures and functions which include scaffolding\/adapter proteins with several protein\u2013protein interaction domains (PAR-3, PAR-6), serine\/threonine kinases (PAR-1, PAR-4), a protein containing a RING finger domain typical for E3 ubiquitin ligases (PAR-2) and a member of the 14-3-3 family of signaling proteins (PAR-5) (reviewed in (Goldstein and Macara 2007; Suzuki and Ohno 2006)). With the exception of PAR-2, all PAR proteins exist in Drosophila and vertebrates. Two PAR proteins, PAR-3 and PAR-6, form a functional unit with aPKC, the PAR-3\u2013aPKC\u2013PAR-6 complex (Ohno 2001). In this complex, PAR-3 and PAR-6 undergo direct interactions with aPKC (Fig.\u00a03). The interaction of PAR-6 with aPKC is mediated by a heterotypic PB1\u2013PB1 domain interaction, the interaction of PAR-3 with aPKC by the CR3 domain of PAR-3 and the kinase domain of aPKC (Ohno 2001). The interactions of the two scaffolding proteins PAR-3 and PAR-6 with aPKC are assumed to regulate the localization and the activity of aPKC, respectively. Among all integral membrane proteins tested, PAR-3 binds specifically to JAM-A, -B, and -C (Ebnet et al. 2001, 2003), and the interaction with JAM-A might serve to anchor the PAR\u2013aPKC complex to TJs. The interaction with PAR-6 is assumed to regulate the activity of aPKC (Lin et al. 2000). In the absence of small GTPases like Cdc42 or Rac1 aPKC is inactive. The binding of active Cdc42 or Rac1 to the CRIB domain of PAR-6 activates aPKC, probably by inducing a conformational change of PAR-6 which allows aPKC to become active (Yamanaka et al. 2001).\nWhat is the function of the PAR\u2013aPKC complex in TJ physiology? A large body of evidence indicates a critical role of the PAR complex in TJ formation rather than in TJ maintenance (Chen and Macara 2005, 2006; Gao et al. 2002; Hirose et al. 2002; Joberty et al. 2000; Mizuno et al. 2003; Nagai-Tamai et al. 2002; Ooshio et al. 2007; Suzuki et al. 2001, 2002; Yamanaka et al. 2001). Many of these studies applied dominant-negative mutants of either PAR-3 or PAR-6 or aPKC. The negative effects on TJ formation were only observed when these mutants were expressed during the process of cell\u2013cell contact formation but not when expressed in fully polarized epithelial cells where TJ formation had already been completed (Gao et al. 2002; Nagai-Tamai et al. 2002; Suzuki et al. 2001, 2002; Yamanaka et al. 2001). This strongly suggests that the PAR\u2013aPKC complex develops its polarizing activity at an early stage of cell\u2013cell contact formation and that it is critical for the formation of TJs rather than for their maintenance.\nRegulation of membrane asymmetry and TJ formation by the PAR-3\u2013aPKC\u2013PAR-6 complex\nThe formation of cell\u2013cell contacts and the development of intercellular junctions with distinct structures like AJs and TJs is a step-wise process (Fig.\u00a05). In the absence of cell\u2013cell contacts, cells form thin protrusions filled by axial actin filaments which upon encountering protrusions of other cells form multiple transient contacts which are subsequently stabilized (McNeill et al. 1993). The first sites of cell\u2013cell contact formation are called \u201cprimordial, spot-like AJs\u201d (pAJs) or \u201cpuncta\u201d (Adams et al. 1996; Yonemura et al. 1995). The formation of multiple pAJs between the protrusions of adjacent cells results in a zipper-like appearance of the early cell contact sites (McNeill et al. 1993) (Fig.\u00a05). During maturation of cell\u2013cell contacts, the pAJs gradually fuse to form a linear contact region, the cells start to polarize and eventually develop cell junctions with AJs and TJs.\nFig.\u00a05A step-wise recruitment of proteins to cell\u2013cell contacts. The earliest sites of stable physical interaction during cell\u2013cell contact formation are primordial, spot-like AJs (pAJs) or puncta at the tips of cellular protrusions. During junctional maturation, the cellular protrusions of adjacent cells interdigitate, and multiple puncta are formed along the sides of protrusions. These puncta gradually fuse to form linear arrangements of cell\u2013cell contacts sites thus generating a zipper-like appearance. During further maturation, cell\u2013cell contacts are formed along the entire lateral cell surface, and the zipper-like cell\u2013cell structure disappears. Cell contact-associated proteins are recruited in a step-wise manner. The pAJs\/puncta are positive for integral membrane proteins (E-cadherin, JAM-A, nectin-2), but also peripheral membrane proteins (ZO-1, \u03b1-catenin, afadin) and contain proteins associated with AJs as well as TJs in polarized cells. During the formation of zipper-like cell contacts, occludin is recruited, probably through its interaction with ZO-1. Thereafter, PAR-3 is recruited by JAM-A and\/or nectin-2, and claudins are recruited, probably through interaction with ZO-1. Indirect evidence suggests that aPKC and PAR-6 appear slightly later than PAR-3. The vertical bar reflects the increase in the contacting membrane area during cell\u2013cell contact formation\nThe pAJs are positive for typical AJ proteins like E-cadherin, \u03b1-catenin, \u03b2-catenin, nectin-2, AF-6\/afadin and ponsin but also for typical TJ proteins like ZO-1 and JAM-A (Adams et al. 1996; Asakura et al. 1999; Ebnet et al. 2001; Suzuki et al. 2002; Yonemura et al. 1995). During maturation, occludin is recruited to these sites, and during further maturation, claudin-1, PAR-3 and aPKC appear (Suzuki et al. 2002) (Fig.\u00a05). Although direct comparison has not been performed yet, it is likely that aPKC together with PAR-6 appear slightly later than PAR-3 (Suzuki et al. 2002). The formation of cadherin-based pAJs marks the early sites of cell\u2013cell adhesion and probably serves as a \u201clandmark\u201d or \u201cpositional cues\u201d for membrane growth and for the recruitment of other integral and peripheral membrane proteins (Yeaman et al. 1999). After the localization of the first set of proteins at pAJs other proteins can be recruited through direct physical interactions with those already present. For example, \u03b1-catenin-associated ZO-1 could serve to recruit occludin and claudins, afadin could serve to recruit JAM-A and nectin-2 (or vice versa), and JAM-A or nectin-2 could recruit PAR-3 which serves as scaffold to assemble the PAR-3\u2013aPKC\u2013PAR-6 complex.\nOnce the PAR-3\u2013aPKC\u2013PAR-6 complex has been recruited to nascent cell\u2013cell contacts aPKC has to be activated as suggested by the observation that ectopic expression of a kinase-dead, dominant-negative mutant of aPKC prevents the maturation of pAJs into belt-like AJs and TJs (Suzuki et al. 2001, 2002). The activation occurs most likely by the Rho GTPases Cdc42 and Rac1 which bind to the Crib domain of PAR-6 thereby inducing a conformational change which leads to the activation of PAR-6-associated aPKC (Garrard et al. 2003; Ohno 2001; Peterson et al. 2004; Yamanaka et al. 2001). Both E-cadherin and nectin-2 could be responsible for the activation of Cdc42 and Rac1. Whereas E-cadherin seems to activate Rac1 but not Cdc42 (Betson et al. 2002; Kovacs et al. 2002; Nakagawa et al. 2001; Noren et al. 2001; Yamada and Nelson 2007), nectin-2 induces the activation of both Cdc42 and Rac1 after ectopic expression in cultured epithelial cells (Fukuhara et al. 2003, 2004; Fukuyama et al. 2005; Kawakatsu et al. 2002, 2005). The association of the Rac1 GEF Tiam1 with PAR-3 (Chen and Macara 2005; Mertens et al. 2005) could regulate a locally restricted activation of Rac1 specifically at those sites where cell\u2013cell adhesion has occured and where the activity of aPKC is required to promote the maturation of cell\u2013cell contacts and the development of TJs from pAJs.\nThe exact mechanism how the maturation of cell\u2013cell contacts is regulated by aPKC is not clear. One could imagine that aPKC phosphorylates components of TJs and thereby regulates their specific localization or their specific functions at the TJs. Phosphorylation of occludin, claudin-1 and ZO-1 by aPKC\u03b6 has been found in vitro (Nunbhakdi-Craig et al. 2002). However, a physiological relevance of these phosphorylations has not been demonstrated, yet. Alternatively, aPKC could regulate TJ formation indirectly by regulating the development of membrane asymmetry along the lateral cell\u2013cell contacts. The following example might serve to illustrate an example for this activity. In polarized epithelial cells, aPKC and PAR-1, another Ser\/Thr kinase, are separately localized along the apico-basal axis: aPKC localizes to TJs whereas PAR-1 localizes to the basolateral membrane domain (Bohm et al. 1997). PAR-1 is a substrate for aPKC, and aPKC-mediated phosphorylation of PAR-1 leads to its dissociation from the membrane into the cytoplasm (Hurov et al. 2004; Suzuki et al. 2004). As a result, PAR-1 is absent from aPKC-containing membrane domains. A reciprocal mechanism has been described in Drosphila epithelial cells (Benton and St Johnston 2003). Drosphila PAR-1 phosphorylates PAR-3\/Bazooka thereby inhibiting its dimerization and blocking its ability to assemble a functional PAR-3\u2013aPKC\u2013PAR-6 complex. As a result, the PAR-3\u2013aPKC\u2013PAR-6 complex is absent from PAR-1-containing membrane domains. Through these reciprocal inhibitory interactions two distinct membrane domains are generated characterized by the mutual exclusion of aPKC and PAR-1. Thus, by regulating the formation of a specific membrane domain from which certain proteins are actively excluded, aPKC could indirectly promote the formation of TJs. It is not clear, yet, if the abilities of the PAR-3\u2013aPKC\u2013PAR-6 complex to regulate TJ formation and to regulate membrane asymmetry are mechanistically linked.\nRegulation of TJ maintenance by the Rich1\u2013Amot complex\nRecently, a new protein complex has been identified which has been suggested to regulate the maintenance rather than the formation of TJs. The functional core of this complex is a set of two proteins which regulate the activity of Cdc42, the Rich1 and angiomotin (Amot) proteins (Wells et al. 2006) (Fig.\u00a06). Rich1 is a Rho GTPase activating protein (RhoGAP) for Cdc42 and Rac1 with a Cdc42-selective activity in epithelial cells; it contains a BAR domain and a RhoGAP domain (Richnau and Aspenstrom 2001; Richnau et al. 2004; Wells et al. 2006). Amot is a scaffolding\/adapator protein with a coiled-coil domain region and a C-terminal PDZ domain-binding motif (Bratt et al. 2002). The Rich1\u2013Amot complex is targeted to TJs through a PDZ domain-dependent interaction of Amot with PATJ (Fig.\u00a06). Both overexpression of Amot and partial downregulation of Rich1 by RNA interference affect the barrier function of TJs. More importantly, the loss of the barrier function in response to Ca2+-removal is accelerated after partial Rich1 downregulation (Wells et al. 2006) suggesting that the Rich1\u2013Amot complex is important for the maintenance of functional TJs. This function of the Rich1\u2013Amot complex probably resides in regulating the cycling of Cdc42 and maintaining the pool of active Cdc42 at TJs at a low level.\nFig.\u00a06The Rich1\u2013Amot complex at TJs. Rich1 is a RhoGAP with specificity for Cdc42 in epithelial cells. Rich1 interacts with Amot through a reciprocal BAR domain-dependent interaction, and Amot binding regulates Rich1 activity. Rich1 associates with Cdc42 through its GAP domain. Amot directly interacts with one (or several) of the PDZ domains 3\u201310 of PATJ that in turn associates with CRB3\u2013Pals1. Surprisingly, Rich1 is directly or indirectly associated with PAR-3 and aPKC, and this PAR complex is distinct form the PAR-3\u2013aPKC\u2013PAR-6 complex because it does not contain PAR-6 and because aPKC cannot be associated with the 100 kDa isoform of PAR-3 which lacks the aPKC-binding domain. All three Amot-like proteins (Amot, Amotl1, Amotl2) form Rich1-independent protein complexes with MUPP1 and PATJ. Double arrows with solid lines indicate direct protein\u2013protein interactions, arrows with broken lines indicate the presence of the two proteins in the same complex but the nature of the interaction has not been characterized in detail, yet\nBesides Amot, PATJ and Pals1, the Rich1 immunoprecipitates contain PAR-3 and aPKC (Wells et al. 2006). Surprisingly, the Rich1 immunoprecipitates do not contain PAR-6, and they contain the 100 kDa isoform of PAR-3 which lacks the aPKC-interacting domain and thus cannot directly associate with aPKC (Lin et al. 2000). This suggests that PAR-3 and aPKC can undergo interactions with the Rich1\u2013Amot complex which are independent of the PAR-3\u2013aPKC\u2013PAR-6 interaction with the Pals1\u2013PATJ complex (Hurd et al. 2003) (Fig.\u00a06). The functional relevance of this interaction is not clear. Two Amot-like (Amotl) proteins\u2014Amotl1\/JEAP and Amotl2\/MASCOT\u2014which have been described earlier as TJ components (Nishimura et al. 2002; Patrie 2005) are present in Amot but not Rich1-containing protein complexes. All three Amot proteins directly interact with the scaffolding protein MUPP1 and its paralogue PATJ (Sugihara-Mizuno et al. 2007) (Fig.\u00a06). These findings suggest that additional Amot protein-containing complexes exist with functions different from regulating Cdc42 activity.\nSignaling from TJs\nIn addition to the relatively stable protein complexes described so far (Fig.\u00a04), many protein complexes at TJs assemble only transiently. In addition, some proteins are not exclusively associated with TJ but shuttle between the TJ and other compartments in the cell. The identification of such proteins has revealed that TJ proteins are engaged in receiving signals but also in delivering signals to the cell interiour and thereby regulate epithelial proliferation and differentiation (Matter and Balda 2003). The mechanism by which TJ proteins influence for example gene expression is most likely indirect through binding and sequestration of regulatory molecules at the TJs as exemplified by ZO-1.\nZO-1 associates with ZONAB\/DbpA, a transcription factor which promotes proliferation of epithelial cells, in part by interacting with the cell division kinase CDK4 and also by regulating the expression of genes involved in proliferation like cyclin D1 and PCNA (Balda et al. 2003; Balda and Matter 2000; Sourisseau et al. 2006). In proliferating cells, which have little ZO-1, ZONAB\/DbpA expression is high and ZONAB\/DbpA protein is localized in the nucleus. When cells reach confluence and develop intercellular junctions, ZO-1 is accumulating at cell\u2013cell contacts and recruits ZONAB\/DbpA to the junctions thus sequestering it away from the nucleus (Balda and Matter 2000). Interestingly, during cellular stress ZONAB\/DbpA associated with ZO-1 can be re-activated. The heat shock protein Apg-2 that is distributed in the cytoplasm under normal conditions is recruited to cell\u2013cell contacts in response to heat shock where it binds directly to ZO-1 using the same binding interface like ZONAB\/DbpA, i.e. the SH3 domain of ZO-1 (Tsapara et al. 2006). This leads to a loss of ZONAB\/DbpA from cell junctions and in activation of the transcriptional activity of ZONAB\/DbpA (Tsapara et al. 2006). Thus, ZO-1 influences gene expression and cell cycle progression in a cell density-dependent manner, and this function can be regulated during cellular stress.\nZO-2 is another scaffolding proteins at TJs which is involved in signaling. In contrast to ZO-1, however, ZO-2 seems to actively shuttle between TJs and the nucleus. ZO-2 contains functional nuclear localization and nuclear export signals (Gonzalez-Mariscal et al. 2006; Jaramillo et al. 2004) and interacts with various proteins that have nuclear functions including the transcription factors AP-1 and C\/EBP (Betanzos et al. 2004), the DNA-binding protein SAF-B (Traweger et al. 2003), and the p120ctn family member ARVCF (Kausalya et al. 2004). ZO-2 probably inhibits the activity of the transcription factors AP-1 and C\/EBP by regulating their export from the nucleus which is consistent with the predominant nuclear localization of ZO-2 in sparse cells and the localization of ZO-2 as well as AP-1 and C\/EBP at TJs in confluent cells (Betanzos et al. 2004). In the case of ARVCF, ZO-2 regulates its nuclear import (Kausalya et al. 2004) where it might regulate transcription similar to other p120ctn family members (Hatzfeld 2005). In summary, the identification of protein complexes formed by typical TJ proteins and typical nuclear proteins involved in the regulation of transcription indicates that the TJs participate in the regulation of proliferation and differentiation.\nA protein complex at the lateral membrane which regulates TJ formation: the Scribble\u2013Discs Large\u2013Lethal Giant Larvae complex\nThe Scribble complex comprises the proteins Scribble (Scrib), Discs Large (Dlg), and Lethal Giant Larvae (Lgl) (Fig.\u00a07) which have originally been identified in Drosophila as tumor suppressor proteins (Bilder 2004). Mutations in the scrib, dlg or lgl genes result in overgrowth of certain tissues ultimately leading to a \u201cgiant larvae\u201d phenotype and also in a disruption of apico-basal polarity (Bilder 2004). All three proteins are membrane-associated in Drosophila epithelial cells. Scrib and Dlg localize to the Septate Junctions (SJ), a structure that is a functional homologue of vertebrate TJs but which is localized basally of the AJs; Lgl localizes along the lateral membrane domain and is excluded from the SAR (Tepass et al. 2001). Importantly, the Scrib complex genetically interacts with the PAR-3\u2013aPKC\u2013PAR-6 and the Crumbs\u2013Sdt\u2013PATJ complexes to regulate apico-basal polarity (Bilder et al. 2003; Tanentzapf and Tepass, 2003). All three proteins are conserved and exist in vertebrates. In mammals, one homologue of Drosophila Scrib, four homologues of Dlg (Dlg1\u2013Dlg4) and two homologues of Lgl (Lgl1, Lgl2) exist, and the proteins localize to the basolateral membrane domain of epithelial cells (Dow and Humbert 2007; Humbert et al. 2003). As opposed to the PAR-3\u2013aPKC\u2013PAR-6 and the CRB3\u2013Pals1\u2013PATJ complexes, Scrib, Dlg and Lgl do not seem to form a ternary complex through direct interactions. Recent evidence indicates that Scrib exists in a complex with Lgl2 (Kallay et al. 2006), but it is not clear if this interaction is direct or indirect as described in Drosophila (Mathew et al. 2002). The role of the Srib protein complex in regulating apico-basal polarity and cell\u2013cell contact formation seems to be conserved in vertebrates. Scrib knockdown results in a delayed TJ formation (Qin et al. 2005), and knockdown of Dlg1 disturbs TJ formation after Ca2+-switch-induced new cell\u2013cell contact formation (Stucke et al. 2007). Furthermore, Lgl has been described to regulate cell\u2013cell contact and apico-basal polarity formation by forming a complex with PAR-6 and aPKC (Plant et al. 2003; Yamanaka et al. 2003) (Fig.\u00a07). The interaction of Lgl with PAR-6 and aPKC precludes the binding of PAR-3 to PAR-6 and aPKC. Also, the Lgl\u2013aPKC\u2013PAR-6 complex counteracts the activity of the PAR-3\u2013aPKC\u2013PAR-6 complex by suppressing not only its formation but also its activation by Cdc42 (Yamanaka et al. 2006). According to the current model (Fig.\u00a07), Lgl forms a complex with aPKC and PAR-6 early during cell\u2013cell contact formation and blocks the formation of the PAR-3\u2013aPKC\u2013PAR-6 complex at this stage. Upon phosphorylation by aPKC, Lgl dissociates from the complex thus allowing for the formation and activation of the PAR-3\u2013aPKC\u2013PAR-6 complex and the development of apical TJs harboring the PAR\u2013aPKC complex and a basolateral domain harboring Lgl (Yamanaka et al. 2003, 2006).\nFig.\u00a07The Scribble (Scrb), Discs large (Dlg) and Lethal giant larvae (Lgl) proteins localize to the basolateral membrane domain in polarized epithelial cells. Scrb and Lgl exist in a complex but it is not clear if the interaction is direct. Inset: During the process of cell\u2013cell contact formation, Lgl forms a transient complex with aPKC and PAR-6 from which PAR-3 is excluded. After aPKC-induced dissociation of Lgl from the complex, PAR-3 associates with PAR-6 and aPKC which promotes TJ formation and the development of apical and basolateral membrane domains. Double arrows with solid lines indicate direct protein\u2013protein interactions, double arrows with broken lines indicate the presence of the two proteins in the same complex but the nature of the interaction has not been characterized in detail, yet\nProtein complexes at cell junctions and cancer\nGiven the important role of protein complexes localized at cell junctions of epithelial cells in regulating cell\u2013cell contact formation, cell polarity and cell proliferation, it is not surprising that altering the compositions of these protein complexes will result in changes in cell\u2013cell adhesion, a loss in cell\u2013cell contact integrity and eventually in uncontrolled proliferation and cancer. A loss of cell polarity is frequently associated with cancer (Bissell and Radisky 2001; Wodarz and Nathke 2007). As pointed out above, many tumor suppressor genes identified in Drosophila including dlg, scrib and lgl turned out to encode proteins which regulate epithelial cell polarity (Bilder 2004), and loss of their expression correlates with more invasive and aggressive cancers in mammalian cells (Dow and Humbert 2007). Recent evidence identified the PAR-3\u2013aPKC\u2013PAR-6 complex at TJs as a target for the oncogenic receptor tyrosine kinase ErbB2. When cultured on reconstituted basement membrane, MCF-10A mammary epithelial cells form three-dimensional spheroids that resemble glandular structures (acini) with a single-layered, polarized epithelium surrounding a luminal space (Debnath and Brugge 2005). ErbB2 signaling leads to multiacinar structures as a result of hyperproliferation and to filling of the luminal space in individual acini due to a block of apoptosis of the inner cells. The latter of these two effects turned out to be regulated by the PAR-3\u2013aPKC\u2013PAR-6 complex (Aranda et al. 2006). Although the molecular mechanism has not been revealed in detail, the findings indicate that in response to ErbB2 activation ErbB2 physically interacts with the PAR-3\u2013aPKC\u2013PAR-6 complex which leads to a removal of PAR-3 and the formation of a ErbB2\u2013PAR-6\u2013aPKC complex (Fig.\u00a08). The newly formed ErbB2\u2013PAR-6\u2013aPKC complex disrupts apico-basal polarity and blocks apoptosis of inner acinar cells through aPKC activity (Aranda et al. 2006). A role of PAR-6\u2013aPKC in regulating apoptosis of inner cells has been observed in MDCK cysts as well (Kim et al. 2007). Thus, the ErbB2 oncogene exploits the PAR\u2013aPKC system to regulate survival of the ErbB2-transformed cells.\nFig.\u00a08The ErbB2 oncogene targets the PAR complex. In normal cells, PAR-3, aPKC and PAR-6 form a stable complex at TJs of epithelial cells (left panel). This complex is required for the fomation of TJs and the development of apico-basal polarity. ErbB2 activation triggers the association of the ErbB2 homodimer with PAR-6 and aPKC thereby disrupting the PAR-3\u2013aPKC\u2013PAR-6 complex. As a consequence, the development of apico-basal polarity is inhibited, and inner cells do not undergo apoptosis (right panel)\nBesides increased proliferation and reduced apoptosis, epithelial-mesenchymal transition (EMT) is another hallmark of tumor progression (Thiery 2002). During EMT, polarized epithelial cells adopt a mesenchymal or fibroblastoid, highly motile phenotype, and this is required during phases of embryonic development when epithelial cells leave a primitive epithelium to migrate to a distinct site in the embryo in order to induce new organ formation. Not surprisingly, typical characteristics of EMT are transcriptional repression of E-cadherin expression, profound changes in the cytoskeleton concomitant with a loss of apico-basal polarity (Thiery 2002). When this developmentally regulated programme is re-activated in the adult organism, it easily contributes to tumor progression by facilitating invasion and metastasis. Among the various physiological inducers of EMT, TGF\u03b2 signaling turned to be critically involved in EMT through cooperation with receptor tyrosine kinases (RTKs) and oncogenic Ras (Huber et al. 2005). Recent evidence indicates that TGF\u03b2 signaling affects not only the integrity of AJs through its known effect on E-cadherin expression but also the integrity of TJs by targeting the PAR-3\u2013aPKC\u2013PAR-6 complex. TGF\u03b2 signaling is mediated by two TGF\u03b2 receptors, the Ser\/Thr kinases TGF\u03b2-receptor I (T\u03b2RI) and T\u03b2RII. In polarized NMuMG cells, TGF\u03b2 induces EMT by recruiting T\u03b2RII to TJs which results in TJ dissolution. T\u03b2RI localizes to TJ through a direct interaction with occludin (Barrios-Rodiles et al. 2005). Interestingly, T\u03b2RI interacts also directly with PAR-6 (Ozdamar et al. 2005) (Fig.\u00a09). A TGF\u03b2 signal triggers heterodimeric complex formation between the two TGF\u03b2 receptors bringing T\u03b2RII in close vicinity of the PAR complex. PAR-6 is then phosphorylated by T\u03b2RII, and this leads to the recruitement of the ubiquitin ligase Smurf1 which in turn mediates ubiquitination of RhoA. RhoA, however, is critical for junctional integrity (Sahai and Marshall 2002), and its localized degradation might thus lead to TJ dissolution. TGF\u03b2 does not only induce RhoA degradation via PAR-6 and Smurf1 but also induces the downregulation of PAR-3 (Wang et al. 2007) (Fig.\u00a09). Although the fate of aPKC during this process has not been clarified, these findings suggest that the PAR-3\u2013aPKC\u2013PAR-6 complex is a major target of TGF\u03b2 signaling at TJs during TGF\u03b2-induced EMT.\nFig.\u00a09TGF\u03b2 signaling targets the PAR complex at TJs. Left panel: Under normal conditions, TGF\u03b2 receptor I (T\u03b2RI) localizes to TJ through direct interactions with occludin and PAR-6. The cells maintain a polarized morrphology. Middle panel: TGF\u03b2 induces heterodimer formation of the two TGF\u03b2 receptors T\u03b2RI and T\u03b2R2 leading to activation of T\u03b2RII folllowed by T\u03b2RII-mediated phosphorylation of PAR-6 at Ser345. Right panel: Ser345-phosphorylated PAR-6 recruits Smurf1 leading to ubiquitination and degradation of the local pool of RhoA. As a consequence, the integrity of TJs is disturbed, the polarized morphology can not be maintained and the development of a fibroblastoid morphology is facilitated. In addition to PAR-6 phosphorylation, TGF\u03b2 signaling also induces downregulation of PAR-3. By targeting the PAR-3\u2013aPKC\u2013PAR-6 complex at TJs, TGF\u03b2 impairs the ability of cells to maintain a polarized morphology\nProtein complexes as targets for pathogens\nMany pathogens need to overcome epithelial and endothelial barriers to invade the host and establish infection. For this purpose, various strategies have evolved to disrupt the barrier and allow the pathogen the passage into tissues. These strategies include the release of proteolytic enzymes that cleave adhesion molecules like occludin, E-cadherin or desmoglein (Hanakawa et al. 2004; Pentecost et al. 2006; Wu et al. 1998, 2000), or the release of toxins that act via cell surface receptors to induce intracellular changes (e.g. of the actin cytoskeleton) which eventually lead to alterations of the barrier (Hopkins et al. 2003; Nusrat et al. 2001). More \u201cadvanced\u201d strategies involve the delivery of pathogen-derived proteins via secretion systems into the host cell cytoplasm where these proteins directly associate with host cell proteins to influence their function. One example is the Helicobacter pylori (H.pylori) effector protein \u201cCytotoxin-associated gene A antigen\u201d (CagA). H.pylori induces morphological changes of epithelial cells, alterations of the composition of the apical junctional complex as well as a breakdown of the epithelial barrier function (Amieva et al. 2003; Bagnoli et al. 2005), and H.pylori infections can result in mucosal damage (ulceration), inflammation (gastritis) and cancer (gastric carcinoma) (Peek and Blaser 2002). The CagA protein is involved in many of these processes through multiple interactions with a number of host proteins. After the translocation into the host cell, CagA is phosphorylated by src-family kinases and recruits the phosphotyrosine phosphatase SHP-2 (Higashi et al. 2002). In addition, CagA associates with several proteins involved in the regulation of TJs and in the formation of apico-basal polarity. First, it recruits and thereby mislocalizes the TJ proteins ZO-1 and JAM-A to the site of bacterial attachment (Amieva et al. 2003). Second, it directly interacts with the serine\/threonine kinase PAR-1 (Saadat et al. 2007). Under normal conditions, PAR-1 cooperates with the PAR-3\u2013aPKC\u2013PAR-6 complex through reciprocal phosphorylations to regulate the formation of distinct membrane domains (Hurov et al. 2004; Suzuki et al. 2004) (see also above). The binding of CagA to PAR-1 blocks the kinase activity of PAR-1 thus preventing the phosphorylation of PAR-3; at the same time, it prevents the phosphorylation of itself by aPKC. As a result, the integrity of cell\u2013cell contacts is disturbed and cells are extruded from the monolayer (Saadat et al. 2007). Thus, through its multiple interactions with signaling molecules, scaffolding proteins and cell polarity proteins, CagA disregulates critical cellular functions to enter the sub-epithelial tissues which also leads to inflammation and eventually to cancer (Hatakeyama 2004).\nConclusions and perspectives\nThe last decade has witnessed a steady increase in the number of new proteins localized at cell\u2013cell contacts of epithelial cells. The identification of claudins at TJs has strongly increased the understanding of the molecular basis of TJ function. The identification of cell polarity protein complexes like the PAR-3\u2013aPKC\u2013PAR-6 complex and the CRB3\u2013Pals1\u2013PATJ complex at TJs has added new aspects on the mechanisms underlying the development of TJs. The identification of the nectin\u2013afadin system provided evidence for a second major adhesive system besides the cadherin\u2013catenin system at AJs. It also became evident that TJs and AJs are signaling centers which are actively engaged in regulating proliferation and differentiation through feed-back mechanisms with the cytoskeleton and the nucleus.\nMeanwhile, many of the proteins that regulate formation and maintenance of cell\u2013cell contacts in epithelial cells have been found in other cellular systems as well suggesting a general function in cell\u2013cell contact regulation. Claudins, nectins, and JAMs are used by cells of the male reproductive system to mediate homotypic Sertoli\u2013Sertoli cell as well as heterotypic Sertoli cell\u2013spermatid interactions (Gliki et al. 2004; Gow et al. 1999; Takai and Nakanishi 2003), and similar functions are performed by these molecules as those proposed in epithelial cells, i.e. formation of TJ strands, regulation of the actin cytoskeleton and regulation of cellular polarization, respectively (Gliki et al. 2004; Gow et al. 1999; Ozaki-Kuroda et al. 2002). In the peripheral nervous system (PNS), a large number of proteins typically associated with TJs or AJs was found to mediate autotypic Schwann cell interactions within the myelin sheath as well as heterotypic Schwann cell\u2013axon interactions. For example, different claudins, JAM-C, Necl-1, Necl-2, Necl-4, and various scaffolding proteins like MUPP1, PATJ, ZO-1, ZO-2, PAR-3 and MAGI-2 are localized at areas of non-compact myelin including Schmidt-Lanterman-incisures, paranodal loops, and mesaxons, and some proteins preferentially localize to some but not other areas (Maurel et al. 2007; Poliak et al. 2002; Spiegel et al. 2007) (Fig.\u00a010). The absence of claudin-19 or JAM-C in mice results in defective nerve conduction indicating that both are critically important for the proper functioning of the PNS (Miyamoto et al. 2005; Scheiermann et al. 2007). Necl-1 and Necl-4 mediate heterotypic interactions between axons and Schwann cells during myelination (Maurel et al. 2007; Spiegel et al. 2007). And PAR-3 has recently been identified at the interface between pre-myelinating Schwann cells and axons along the internodal region (Chan et al. 2006). PAR-3 is expressed by the Schwann cells, not by the axon, and recruits the p75 neurotrophin receptor to the glial\u2013axon junction to regulate myelination of the axon.\nFig.\u00a010Proteins at autotypic and heterotypic cell\u2013cell contacts in the peripheral nervous system (PNS). A multitude of proteins localized at TJs of polarized epithelial cells localizes to autotypic glial\u2013glial cell contacts and heteroytpic glial cell\u2013axon contacts. Abbreviations: JXP juxtaparanodal region, MV microvilli, PNL paranodal loops, PNJ paranodal junction, SLI Schmidt-Lanterman incisure\nIt is not clear if these proteins form the same complexes like in epithelial cells, and it is likely that differences exist in the composition of protein complexes to regulate the specific requirements in the context of the given cell or tissue. As one example, JAM-C deficiency in mice leads to a mislocalization of PAR-6, aPKC and PATJ bot not PAR-3 in spermatids (Gliki et al. 2004) suggesting that PAR-3 is not part of the JAM-C-associated polarity complex in spermatids despite its ability to interact with PAR-6, aPKC and also directly with JAM-C (Ebnet et al. 2003; Suzuki and Ohno 2006). As another example, in endothelial cells two PAR protein complexes have been identified, a \u201cconventional\u201d PAR-3\u2013aPKC\u2013PAR-6 complex and a second PAR complex in which PAR-3 and PAR-6 are independently associated with VE-cadherin and which lacks aPKC (Iden et al. 2006). Nevertheless, the use of a set of conserved proteins by morphologically diverse cell types to regulate cell\u2013cell contact formation highlights both the importance of the proteins for cellular function as well as their versatility that allows the regulation of similar aspects in different cell types.\nMany open questions remain. For example, what is the molecular nature of the intramembrane diffusion barrier (fence function) of the TJs? The absence of TJ strands in cells lacking all three ZO proteins results in a complete loss of the barrier function of the epithelial sheet but, unexpectedly, the fence function which has been attributed to the presence of TJ strands is retained (Umeda et al. 2006). Membrane diffusion barriers exist also in other cells even in the absence of a physical cell\u2013cell contact, for example at the axonal hillock of neurons to separate somatodendritic and axonal membrane domains (Winckler et al. 1999). It has been suggested that in these cells the accumulation of integral membrane proteins that are anchored to the submembranous cytoskeleton function as rows of pickets which prevent the free diffusion of even small molecules (Nakada et al. 2003). A second unresolved issue is if the activity of the PAR-3\u2013aPKC\u2013PAR-6 complex to regulate membrane asymmetry is mechanistically linked to its role in TJ formation. Finally, the functional interrelationship of the three major polarity complexes at TJs (depicted in Figs\u00a03, 4) has not been resolved, yet. Members of individual complexes interact with each other. For example, CRB3 can also interact with PAR-6 (Lemmers et al. 2004), PAR-6 can also interact with Pals1 (Hurd et al. 2003), and ZO-3 can interact with PATJ (Roh et al. 2002a). Genetic evidence in Drosophila suggests a functional hierarchy among the protein complexes (Johnson and Wodarz 2003) but it is unclear if a similar hierarchy exists in vertebrate epithelial cells and which aspects of cell\u2013cell contact formation are regulated by these interactions. Most likely, many of these interactions are dynamically regulated and occur in a temporally and spatially restricted manner. The large number of scaffolding and signaling molecules identified at cell\u2013cell contacts and the multitude of physical interactions described so far among these proteins indicates that cell\u2013cell contact formation, the development of TJs from pAJs and the aquisition of membrane polarity is a highly dynamic process the complexity of which is still far from being completely understood.","keyphrases":["protein complexes","tight junction","adherens junction","cell\u2013cell adhesion","cell polarity","jam","par proteins"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Matern_Child_Health_J-2-2-1592140","title":"Ensuring the Safe and Effective Use of Medications During Pregnancy: Planning and Prevention Through Preconception Care\n","text":"Introduction\nWhether to use a medication during pregnancy can be a concern for both women and health care providers. It has been estimated that up to 10% of congenital anomalies may be caused by environmental exposures\u2013that is, exposures to medications, alcohol, or other exogenous factors that have adverse effects on the developing embryo or fetus [1]. Avoiding all potentially adverse exposures can prevent these adverse effects. However, it is not always possible to avoid taking medications during pregnancy. Women who are pregnant continue to experience short-term and long-term health conditions that must be managed. Discontinuing treatment of a serious condition when a woman becomes pregnant can have profound, long-term implications both for her health and that of her baby. In a retrospective study from eight health maintenance organizations, researchers estimated that approximately 59% of pregnant women were prescribed a medication other than a vitamin or mineral supplement at some time during pregnancy [2]. Use of over-the-counter medications during pregnancy may be even higher, and many women take a dietary or herbal supplement other than multivitamins or folic acid while pregnant [3, 4].\nPreconception care provides the opportunity to optimize a woman\u2019s use of medications in preparation for pregnancy. Such care includes identifying patterns of medication use before pregnancy occurs; adjusting those patterns to avoid the use of nonessential medications; minimizing exposure to medications known to be harmful to the embryo or fetus; and adjusting the dose, route of administration, and timing of essential treatments to optimize maternal health at each stage of pregnancy while safeguarding the embryo, fetus, and infant. In this paper, we summarize the basic principles of teratology and the current state of knowledge about the effects of medication use during pregnancy; outline basic components of preconception care that can help minimize the risk of birth defects; and provide examples of approaches to planning for the safe and effective use of medications during pregnancy through preconception care.\nPrinciples of teratology\nThe term \u201cteratogen\u201d is sometimes used to describe an agent that can produce structural or functional abnormalities in a developing embryo or fetus. Although this implies that an agent is inherently either teratogenic or not, teratogenicity is actually a property of the exposure taken as whole\u2014not only the physical and chemical nature of the agent, but also the dose, route of administration, and timing in gestation, as well as concurrent exposure to other agents and biological susceptibility of the mother and embryo or fetus.\nThe developmental stage of the embryo or fetus at the time of exposure is critical. During conception and for about 2 weeks thereafter, most cells of the conceptus are not yet committed to a specific developmental program. One damaged cell can be replaced by another, and normal development will usually ensue, although the embryo will not survive if too many cells are damaged or killed. This is known as the \u201call-or-nothing\u201d period, but even during this period some exposures can adversely affect the development of surviving embryos [5]. The subsequent period of organogenesis, from 18 to 60 days after conception (about 4.5\u201311 weeks after the start of the last normal menstrual period) is the time of greatest sensitivity to most teratogenic exposures. Fetal exposure later in gestation usually does not produce gross structural abnormalities, although there are exceptions. Adverse exposures during the fetal period more often result in growth restriction or functional disorders of the central nervous system, kidneys, or other organs.\nDose is a critical feature of any teratogenic exposure. Teratogenic effects occur only when the dose of an agent exceeds a certain threshold [1]. Medications that are generally considered safe during pregnancy can have adverse effects on the embryo or fetus if the mother takes them in doses that are so high that they cause maternal toxicity. Chronic exposure is usually of more concern than a single exposure, if the doses are similar. The route of exposure is also important. For example, risk is unlikely from the use of dermal agents that lack substantial systemic absorption. The teratogenicity of an exposure is also influenced by both the maternal and fetal genotypes, which can result in differences in cell sensitivity, placental transport, metabolism, receptor binding, or drug distribution. Some medications are metabolized extensively by the mother; their teratogenicity depends upon whether a toxic form reaches the embryo or fetus in sufficient quantities to produce adverse effects.\nCurrent state of knowledge about the effects of medication use during pregnancy\nMaternal effects\nMany women begin pregnancy with medical conditions that require ongoing or episodic treatment. Examples include asthma, epilepsy, and hypertension. In addition, other medical problems, such as migraine headache or auto-immune disorders, may be exacerbated by pregnancy. When planning for the management of maternal conditions during pregnancy, it is important to distinguish conditions for which withholding treatment could be harmful to the mother, embryo, or fetus, from those for which cessation of treatment is unlikely to pose significant risk. For example, women with major depression who discontinue antidepressant medication before conception are at high risk of relapse and consequent self-injurious or even suicidal behavior [6]. In contrast, cessation of treatment for moderate hypercholesterolemia with a statin drug while a woman is pregnant is unlikely to increase her cardiovascular morbidity or mortality significantly.\nPhysiologic changes occur during pregnancy that can alter the effective dose of medications a woman is taking. Some changes occur abruptly, while others evolve slowly. Most begin during the first trimester and peak during the second trimester of pregnancy [7]. It may be necessary to adjust the dose and\/or frequency of medication use repeatedly during pregnancy. Physiologic changes that can affect the pharmacokinetics and\/or pharmacodynamics of medications during pregnancy include:Changes in total body weight and body fat composition.Delayed gastric emptying, prolonged gastrointestinal transit time, and decreased gastric acid secretion, all of which can affect the bioavailability of drugs [8\u201310].Expanded plasma volume and significantly increased extracellular fluid space and total body water content. These vary with the patient\u2019s weight and can affect the volume of distribution of drugs [11].Increased cardiac output, stroke volume, heart rate, and blood flow to the uterus, kidneys, skin, and mammary glands. The percentage of cardiac output attributed to hepatic blood flow is lower during pregnancy [12].Decreased concentration of plasma albumin, which can reduce the protein binding of some drugs [13].Increased glomerular filtration rate early in pregnancy, with a continued rise throughout pregnancy [14].Changes in the activity of hepatic enzymes, including the cytochrome P450 enzymes, xanthine oxidase, and N-acetyltransferase [15, 16].\nUnfortunately, there are relatively few studies of drug pharmacokinetics during pregnancy. The dose of medications usually prescribed during pregnancy is the same used in nonpregnant adults, but this may result in substantial under- or over-dosage during pregnancy. When blood or serum concentrations of medications can be measured and the most effective level is known, they should be monitored throughout pregnancy and appropriate dosage adjustments made as needed. Further well-designed and well-conducted pharmacokinetic and pharmacodynamic studies of medications during pregnancy are needed.\nFetal effects\nMaternal treatment with conventional doses of some medications during a susceptible period of pregnancy is known to be harmful to the developing embryo. Thalidomide and isotretinoin are the most notable examples, but there are others [17]. In contrast, taking some other medications or dietary supplements such as folic acid helps to prevent adverse pregnancy outcomes [18]. Because pregnant women are traditionally excluded from clinical trials for ethical reasons and because premarketing animal studies do not necessarily predict the effects of treatment in human pregnancy, little information about the teratogenic risks or safety of most drugs is available at the time they are marketed. Animal teratology studies are not routinely conducted for non-prescription drugs, vitamins, and dietary and herbal supplements, although these substances may produce pharmacological or toxic effects in the fetus. Moreover, there is no standard requirement for studies of adverse effects among children of women who took a drug during pregnancy after it has been approved by the FDA. In a review conducted in 2001, researchers found that there was not enough information to assess the teratogenic risk or safety during human pregnancy of more than 90% of prescription medications approved by the FDA in the previous 20 years [19].\nEven when available, studies addressing fetal effects of maternal medication use during pregnancy may provide conflicting results or insufficient information to assess all potential outcomes or levels of risk. The concept of safety implies the absence of risk, which is impossible to demonstrate conclusively with any kind of study. Thus, it can be difficult for women and health care providers to decide whether to use a medication during pregnancy. The balance of risk, benefit, and efficacy of treatment for both mother and fetus is not always clear and must be individualized for different women under different circumstances.\nBasic components of preconception care that can minimize the risk of birth defects\nSerious congenital anomalies, including chromosome abnormalities and Mendelian disorders, can be identified in about 2% of infants at birth [52]. However, some anomalies do not become apparent until later in life [20]. While most birth defects are not preventable, some can be avoided through appropriate planning and medical interventions. The following components of preconception care can help minimize the risk of birth defects:Optimize health before conception occurs. This includes counseling women to avoid smoking, use of excessive alcohol and illicit drugs, and exposure to potentially toxic environmental or occupational hazards before they are pregnant.Establish effective treatment for chronic conditions before conception occurs.Carefully manage all chronic conditions and intercurrent illnesses throughout pregnancy.Counsel women to avoid the use of nonessential medications, including prescription and over-the-counter medications and dietary or herbal supplements.Avoid the use of medications with high teratogenic risk when equally effective treatments with lower risks are available.Limit the use of essential medications to the smallest number of drugs possible that will effectively treat maternal disease without compromising the health of the woman or her fetus.Limit each essential medication to the smallest dose that can be used to effectively treat maternal disease without compromising the health of the woman or her fetus.Recommend that all women who are capable of becoming pregnant take a vitamin supplement or eat fortified foods to assure consumption of 0.4\u00a0mg (400 micrograms) of folic acid per day.\nEffective pregnancy management in women with chronic conditions requires careful planning, close medical supervision before and during pregnancy, and continuous communication between the pregnant woman and her health care providers.\nExamples of approaches to preconception planning for the use of medications during pregnancy in different clinical settings\nExactly how the components of preconception planning for the use of medications during pregnancy are implemented depends on the nature of the condition requiring treatment, the known risks and safety of use of the specific drugs during pregnancy, and the woman\u2019s individual circumstances, among other factors. In this section, we provide three examples of approaches to planning for the safe and effective use of medications in clinical settings where these factors vary\nAvoiding teratogenic treatments for non life-threatening maternal conditions\u2014isotretinoin\nIsotretinoin is indicated for the treatment of severe nodular cystic acne unresponsive to other therapy but is also used to treat non-nodular, but scarring, acne. A single course of therapy typically lasts 15\u201320 weeks and can result in complete and prolonged remission of the acne in many patients. However, isotretinoin treatment in the first trimester of pregnancy is teratogenic. Exposed infants can have craniofacial, cardiac, thymic, and central nervous system malformations [21]. Research has also shown a high incidence of developmental delay in children whose mothers used isotretinoin early in the first trimester, regardless of whether the children had structural malformations [22].\nIsotretinoin is indicated for use only in men and nonpregnant women. It should never be used during pregnancy. However, because approximately half of pregnancies in the United States are unintended, some women use isotretinoin in the early weeks of gestation before realizing they are pregnant [23]. Teratogenic outcomes have been reported after only one dose of isotretinoin during pregnancy [24]. The half-life of isotretinoin is approximately 24\u00a0h, but about 2 weeks are required to eliminate 99% of the drug from the body after cessation of use.\nSeveral risk management strategies have been implemented to prevent the use of isotretinoin during pregnancy, but pregnancy exposures continue to occur [25]. An enhanced risk management program called iPLEDGE became fully operational in March 2006 [26, 27]. iPLEDGE is a single, mandatory program for all marketed isotretinoin products. It requires that wholesalers, pharmacies, doctors, and patients register with the program in order to obtain the drug. Female patients who are capable of having children must obtain counseling about the risks of isotretinoin treatment and the requirements for its safe use; complete an informed consent form; have two negative pregnancy tests documented before starting isotretinoin therapy; use two different forms of contraception simultaneously or agree not to engage in heterosexual intercourse for one month prior to starting isotretinoin, throughout the course of treatment, and for one month after completing treatment; and have a negative pregnancy test documented every month during treatment and one month after completing treatment. More information about the iPLEDGE program can be found at www.ipledgeprogram.com.\nPreconception care provides a unique and critical opportunity to prevent exposure to teratogens such as isotretinoin. Any woman who is considering pregnancy should be asked whether she is taking any medications including any preparation containing isotretinoin. If so, her health care provider should emphasize the risks of isotretinoin use during pregnancy, reevaluate the need for and duration of treatment, assess thoroughly the use and effectiveness of contraceptive measures, and reinforce the key elements of the iPLEDGE program. Women should be informed that conception cannot be attempted without risk of teratogenicity until one full month after the last dose of isotretinoin was taken. Educational information and materials from the iPLEDGE program can also be helpful in the setting of preconception care.\nManaging maternal conditions that require continuous treatment\u2014epilepsy\nAt least one in every 250 pregnant women, or about 0.4%, takes an anticonvulsant drug [28]. Approximately half take the drugs to prevent seizures, but anticonvulsants are also used to manage mood disorders, migraine headaches, and chronic pain. Anticonvulsant drugs have several different modes of action, such as targeting a specific receptor or enzyme [29]. As a result, anticonvulsant medications vary in their effectiveness for specific types of epilepsy. Individuals with epilepsy may respond differently to particular medications, reflecting genetic differences such as polymorphisms in the cytochrome P450 enzymes. It is not yet possible to screen for pharmacogenetic differences that would help select the appropriate anticonvulsant for individual use. Physiologic changes during pregnancy also can affect the disposition of anticonvulsant drugs and the dose needed to prevent seizures [30].\nWhile not all anticonvulsant drugs have been studied in pregnancy, a number of adverse effects have been identified in infants and children of women treated with these medications during pregnancy. Major malformations, midface and digit hypoplasia, microcephaly, growth restriction, and deficits in IQ are sometimes seen, although the pattern of abnormalities and specific effects vary for individual drugs [28, 31\u201333]. For example, spina bifida occurs in approximately 1% of fetuses exposed to carbamazepine and in 2 to 5% of fetuses exposed to valproic acid [34, 35]; microcephaly and growth restriction are observed more frequently in infants whose mothers took two or more anticonvulsant drugs simultaneously during pregnancy [28]; and subtle effects on intelligence have been identified in some studies of children exposed to carbamazepine, phenytoin, or phenobarbital in utero, although developmental delay and deficits in cognitive function are much more frequent with exposure to valproic acid [36\u201338]. While some infants exposed to an anticonvulsant drug in utero have abnormalities, others do not. Genetic differences in the fetal response to medications probably play a role.\nIt has been hypothesized that maternal epilepsy might cause fetal abnormalities independent of any drug effect, but several studies suggest that anticonvulsant treatments are usually responsible [39]. However, it seems likely that repeated or prolonged maternal seizures, such as occur in status epilepticus, can be devastating to the fetus [40]. Discontinuation or inadequate treatment of maternal epilepsy may be more dangerous to the fetus than effective anticonvulsant therapy.\nPreconception care provides an opportunity to choose a plan of anticonvulsant treatment that will pose the least risk to the fetus while appropriately managing maternal symptoms. New data about the effects on the fetus of anticonvulsant medications are emerging steadily, and the latest information should always be sought. The treatment plan must be individualized for each woman in collaboration with her neurologist, psychiatrist, or other specialist. Primary considerations include assessing whether taking an anticonvulsant drug is essential to the mother\u2019s health; using the fewest number of anticonvulsant drugs possible; using the lowest dose for each drug that will effectively treat maternal symptoms [41]; establishing the most effective blood level of each drug before conception; and monitoring drug levels throughout pregnancy. For carbamazepine, phenobarbital, phenytoin, primidone, and valproic acid, levels of the non-protein bound, or \u201cfree,\u201d concentration should be measured. Because abrupt cessation of a medication may result in increased seizures or exacerbation of other symptoms, the number and dose of medications should be adjusted, and the woman\u2019s response stabilized, over a period of time before attempting conception.\nIn addition, preconception care offers the opportunity to consider whether additional measures, such as the use of a higher daily dose of folic acid, might be beneficial. Only limited and somewhat conflicting information is available about whether periconceptional supplementation with folic acid at levels higher than 0.4\u00a0mg per day decreases the risk for neural tube defects associated with anticonvulsant drug exposure in utero [33, 42]. Currently, many women who require anticonvulsant drug therapy during pregnancy take 4 or 5\u00a0mg of folic acid daily.\nManaging maternal conditions with intermittent symptoms\u2014asthma\nAsthma is a chronic condition with intermittent symptoms, for which treatment during pregnancy is essential to safeguard the health and well-being of both the mother and fetus. Abrupt cessation or undertreatment of asthma during pregnancy can endanger both. Studies suggest that maternal asthma during pregnancy can increase the risk for perinatal mortality, preeclampsia, preterm delivery, and low birth weight [43, 44]. Maternal asthma can lead to alkalosis with decreased blood flow to the uterus, decreased venous return, and a leftward shift of the oxyhemoglobin dissociation curve, all of which may contribute to fetal hypoxia. In the extreme, maternal hypoxia can result in decreased umbilical blood flow, increased systemic and pulmonary vascular resistance in the fetus, and decreased fetal cardiac output. Fetal compromise may occur well before maternal symptoms become severe [45]. Additional pathophysiologic mechanisms that could contribute to these adverse outcomes include hyperactivity of uterine and bronchial smooth muscle and the release of bioactive mediators during symptomatic asthma [43]. For these reasons, it is considered safer for pregnant women to be treated with asthma medications than to experience asthma symptoms and exacerbations [46].\nA variety of medications are available to treat acute and chronic asthma. They include beta2-agonoists, corticosteroids, cromolyn, leukotriene modifiers, theophylline, and anticholinergics. These drugs have different mechanisms of action and thus potentially different effects on the fetus. Before attempting conception, it is important to maximize asthma control using medications that can also be used to manage asthma symptoms during pregnancy. This will ensure maximum oxygen delivery to the developing embryo and fetus while avoiding unintended medication exposures. A period of time may be needed to wean patients off some medications and to stabilize symptoms using others before attempting conception. In addition, because an estimated one third of pregnant women with asthma experience an increase in the frequency or severity of their symptoms during pregnancy, it is important to develop a plan before conception occurs for regular monitoring of asthma symptoms and pulmonary function during pregnancy and for early recognition and prompt treatment of exacerbations [47, 48]. This plan should include a review of the proper use of medications, including the proper technique for using inhalers; education about self-monitoring and self-management at home; and specific instructions about when to contact a health professional for additional care.\nThe National Asthma Education and Prevention Program Working Group outlines a step-wise approach for managing asthma during pregnancy in its report Managing Asthma During Pregnancy: Recommendations for Pharmaceutical Treatment\u2014Update 2004. This report was developed after a systematic review of the current evidence from safety studies of asthma medications during pregnancy [46]. The approach is centered on the use of beta2-agonoists and corticosteroids, by inhalation when possible to minimize systemic absorption. The dose, frequency, and number of medications are decreased when possible but increased as needed, and pulmonary function is measured regularly (e.g., by monthly spirometry testing) during pregnancy. The approach also emphasizes the need for a treatment plan tailored to each patient\u2019s needs and circumstances, the frequency and severity of her symptoms, and her individual response to treatment. Regardless of the specifics, the benefits and risks of asthma treatment and of uncontrolled asthma during pregnancy for both the mother and fetus should be discussed during preconception planning so that a fully informed plan for asthma management can be agreed upon.\nAdditional measures can be instituted preconceptionally to minimize the severity or frequency of asthma symptoms during pregnancy, and thus decrease reliance on, and fetal exposure to, medications. Identifying, reducing, or eliminating exposure to allergens such as animal dander, house-dust mites, cockroaches, pollen, and indoor mold; to irritants such as perfumes, sprays, and cleaning agents; and to smoke, including primary and secondhand tobacco smoke, wood burning stoves, or fireplaces, may decrease asthma symptoms. Preconception counseling also provides an opportunity to discuss the use of over-the-counter medications for symptoms such as rhinitis, congestion, and esophageal reflux that are often associated with asthma. While over-the-counter medications can be purchased without a prescription, they contain pharmacologically active ingredients that may affect the fetus. For example, studies have suggested that use of pseudoephedrine or aspirin in the first trimester may increase the risk of gastroschisis, a rare abdominal wall defect [49\u201351]. It has been theorized that gastroschisis could result from vascular disruption. Both aspirin and pseudoephedrine can have vasoactive effects, although the causal pathway has not been established for either drug, and the absolute risk of their use is very small. Most over-the-counter medications have not been adequately studied in pregnancy.\nConclusion\nAlthough it is always better to avoid unnecessary medical treatment during pregnancy, some women with chronic conditions may not be able to become pregnant without appropriate therapy. In many other cases, proper treatment of a chronic condition during pregnancy may be safer for both the woman and her baby than stopping this treatment. It is important that women who are planning a pregnancy talk with their health care provider before beginning a new medication or making changes in current medications for the management of acute or chronic conditions. Continuous communication between a pregnant woman and her health care providers, careful preconceptional planning, effective management of conditions prior to pregnancy, and close medical supervision during pregnancy can help assure the best possible outcome for every woman and baby.\nNew information about the effects of medication use and the optimal management of maternal conditions during pregnancy continually becomes available. The following are regularly updated sources of such information and advice:The Organization of Teratology Information Specialists (OTIS) provides medical consultation, usually by phone, to individuals and health care providers about the reproductive risks of prenatal exposures.Phone: 866-626-6847Website: http:\/\/otispregnancy.orgREPROTOX\u00ae is an information source for laboratory scientists, practicing physicians, and government agencies that contains commentaries on the effects of chemicals and physical agents on human pregnancy, reproduction, and development.Website: www.reprotox.orgThe Teratogen Information System (TERIS) is a computerized database of summaries of individual agents designed to assist health care professionals in assessing the risks of exposures in pregnant women. Each summary is based on a review of the published literature and includes a risk assessment derived by consensus of an advisory board of authorities in clinical teratology.Website: http:\/\/depts.washington.edu\/\u223cterisweb\/teris\nInformation about the results of premarketing studies of individual medications, including animal reproductive studies and human clinical trials, is usually available from the drug manufacturer.\nA list of many of the existing postmarketing pregnancy registries that seek to monitor exposure to specific medications during pregnancy is maintained by the Office of Women\u2019s Health, U.S. Food and Drug Administration at www.fda.gov\/womens\/registries.\nAdditional information is available from the March of Dimes at www.marchofdimes.com\/pnhec\/173_1453.asp.","keyphrases":["medications","pregnancy","preconception care","asthma","isotretinoin","anticonvulsants"],"prmu":["P","P","P","P","P","P"]}
{"id":"springeropen-4-1-2200676","title":"Transcriptome analysis of the response to chronic constant hypoxia in zebrafish hearts\n","text":"Insufficient blood supply during acute infarction and chronic ischemia leads to tissue hypoxia which can significantly alter gene expression patterns in the heart. In contrast to most mammals, some teleost fishes are able to adapt to extremely low oxygen levels. We describe here that chronic constant hypoxia (CCH) leads to a smaller ventricular outflow tract, reduced lacunae within the central ventricular cavity and around the trabeculae and an increase in the number of cardiac myocyte nuclei per area in the hearts of two teleost species, zebrafish (Danio rerio) and cichlids (Haplochromis piceatus). In order to identify the molecular basis for the adaptations to CCH, we profiled the gene expression changes in the hearts of adult zebrafish. We have analyzed over 15,000 different transcripts and found 376 differentially regulated genes, of which 260 genes showed increased and 116 genes decreased expression levels. Two notch receptors (notch-2 and notch-3) as well as regulatory genes linked to cell proliferation were transcriptionally upregulated in hypoxic hearts. We observed a simultaneous increase in expression of IGF-2 and IGFbp1 and upregulation of several genes important for the protection against reactive oxygen species (ROS). We have identified here many novel genes involved in the response to CCH in the heart, which may have potential clinical implications in the future.\nIntroduction\nLow oxygen levels (hypoxia) play important roles in clinical conditions such as stroke and heart failure. Insufficient blood supply leads to tissue hypoxia in the heart during acute infarction and chronic ischemia (Semenza 2001).\nEffective protection of the heart against ischemia\/reperfusion injury is one of the most important goals of experimental and clinical research in cardiology. Besides ischemic preconditioning as a powerful temporal protective phenomenon, adaptation to chronic hypoxia also increases cardiac tolerance to all major deleterious consequences of acute oxygen deprivation such as myocardial infarction, contractile dysfunction and ventricular arrhythmias (Kolar and Ostadal 2003). Although many factors have been proposed to play potential roles, the detailed mechanism of this long-term protection remains poorly understood. Some of the molecular mechanisms of cardiac protection by adaptation to chronic hypoxia and chronic high-altitude hypoxia have recently been reviewed (Kolar and Ostadal 2003; Ostadal and Kolar 2007). KATP channels, PKC\u03b4 as well as the different MAPK pathways were shown to be involved in the mechanism of increased tolerance of chronically hypoxic hearts and further the controversial role of ROS in hypoxia tolerance is discussed (Kolar and Ostadal 2003). Furthermore, a recent study has profiled the gene expression changes induced by chronic constant hypoxia (CCH) and chronic intermittent hypoxia (CIH) in newborn mice (Fan et al. 2005).\nIn contrast to most mammals (with the exception of some marine mammals), some teleosts, have developed the ability to withstand extreme chronic hypoxia (Stecyk et al. 2004). It is well assumed that these vertebrate species possess unique adaptations in order to survive short and long term oxygen deprivation. However, the molecular basis of these adaptations in fish has so far not been extensively investigated.\nSeveral studies have profiled gene expression changes in teleosts exposed to hypoxia. Gracey et al. showed in adults of the euryoxic gobiid fish Gillichthys mirabilis (the long-jawed mudsucker), that 5\u00a0days of hypoxia induced a complex transcriptional response, including a shut down of energy requiring pathways like protein synthesis and locomotion, and an induction of genes needed for anaerobic ATP production in different tissues (Gracey et al. 2001). Recently, we described phenotypic and behavioral adaptations to long-term hypoxia and described the gene expression changes induced by chronic constant hypoxia in the gills of adult zebrafish (van der Meer et al. 2005).\nTon et al. identified global gene expression changes in zebrafish embryos. Zebrafish embryos at 48\u00a0h post fertilization were exposed to water with 5% oxygen content for 24\u00a0h. The authors identified 138 genes responsive to short-term hypoxia and could also show that transcriptional changes indicated metabolic depression, a switch from aerobic to anaerobic metabolism and energy conservation (Ton et al. 2003).\nIn this study, we have identified CCH-induced gene expression changes in the zebrafish heart by looking at over half of the zebrafish genome. We have compared several of these novel changes described in other species and tissues. We have here identified the heart-specific molecular adaptations to CCH. Future functional experiments are warranted to determine whether some of the findings can be used to better adapt mammalian hearts to CCH.\nMaterial and methods\nAnimal handling\nAdult wild-type zebrafish (Danio rerio) around 3\u00a0month of age, were obtained from a local pet store. Cichlids (Haplochromis piceatus) have been collected in the Mwanza Gulf of Lake Victoria in 1984 and were bred in our laboratory for about 20 generations. All animals were handled in compliance with animal care regulations. Our animal protocols were approved by the review board of Leiden University in accordance with the requirements of the Dutch government. Zebrafish were kept at 25\u00b0C in aquaria with day\/night light cycles (12\u00a0h dark vs. 12\u00a0h light). Cichlids were kept at 25\u00b0C with the same day\/night light cycles.\nHypoxia treatment\nFor gradual hypoxia treatment, oxygen levels were gradually decreased in 4\u00a0days from 100% air saturated water to 40% (day 1), 30% (day 2), 20% (day 3) and the final 10% air saturation (day 4). After day 4, the fish were kept for an additional 21\u00a0days at 10% air saturation. (at 100% air saturation and 28\u00b0C the O2 concentration is 8\u00a0mg\/l and pO2 is 15\u00a0Torr). In parallel, a control group was kept at 100% air saturated water. Both groups were kept in identical aquaria of 100\u00a0l. The oxygen level on the hypoxia group was kept constant by a controller (Applikon Biotechnology, The Netherlands) connected to an O2-electrode and solenoid valve in line with an air diffuser. The oxygen level in the tank was kept constant by adding oxygen via the diffuser and thereby compensating the oxygen consumption of the fish. In case of immediate hypoxia exposure, tanks were pre-equilibrated to the respective pO2 concentration and fish were then directly set in the equilibrated aquaria.\nPerfusion of cichlid hearts\nIn order to minimize blood clotting, a perfusion protocol was developed in which the whole blood volume of clinically dead animals was initially replaced with isotonic buffer and with a fixative solution secondarily.\nHeart dissection\nThe fish were killed with an overdose of anesthetic (MS-222; Tricaine Methanesulfonate from Argent Chemical Laboratories, USA). Hearts were dissected from the fish immediately after the anesthetic worked. For RNA preparation the hearts were immediately shock-freezed in liquid nitrogen. For histology and microscopy, the hearts were left intact and fixed immediately in Karnovsky fixative (4% paraformaldehyde (PFA) and 2.5% glutaraldehyde in 0.1\u00a0M phosphate buffer, pH 7.2) for 4\u00a0h at 4\u00b0C. After three washes in 0.1\u00a0M phosphate buffer, pH 7.2, they were transferred to 70% ethanol.\nHistology of adult fish hearts, statistical analysis and scanning electron microscopy\nHearts from zebrafish and cichlids raised either under normoxic or hypoxic conditions, were dissected from the fish and fixed for 24\u00a0h in 4% PFA in PBS. After fixation, hearts were washed with 1\u00a0\u00d7\u00a0PBS, cut in halfs (sagittal through the midline) and then dehydrated through ethanol series starting at 70% ethanol, followed by 80, 90, 96 and 100% ethanol, each step was done once for 1\u00a0h except the last one which was done twice. After dehydration the hearts were embedded in increasing gradients of Historesin (Technovit 7100, Heraus Kulzer, Germany) (25, 50, 75 and 100% Historesin in ethanol, for 2\u00a0h at room temperature; 100% Historesin, 24\u00a0h at 4\u00b0C). Afterwards the plastic with the hearts was polymerized at 40\u00b0C (overnight). A 5\u00a0\u03bcm sagittal sections were made using the Ultramicrotome (Reichter-Jung) and a glass knife. Approximately 500 sections on each side of the midline were visually analyzed per heart (1,000 sections in total per heart). Sections were left to dry and later stained with hematoxylin\u2013eosin staining. Pictures were taken with Axioplan 2 imaging, (Carl Zeiss, Jena). Azan staining of histological sections were done as follows. Paraffin sections of the hearts were prepared as described before (van der Meer et al. 2006). Sections were incubated with Azokarmine solution, for 30\u00a0min at 60\u00b0C. Afterwards they were washed in water and differentiated in 0.2% Anilin alcohol. They were then rinsed in 1% acetic acid in 95% alcohol, followed by 45\u00a0min incubation period, in 5% phosphotungstic acid. After that, sections were rinsed in distilled water and incubated for 45\u00a0min with aniline blue. Finally, they were rinsed with distilled water, differentiated and dehydrated in 95% alcohol followed by absolute, cleared in xylene and mounted in Entallan. The protocols used here for SEM had been described before (van der Meer et al. 2005). The statistical analysis of cardiac myocyte nuclei per section was done using Statistica by performing an independent t test. A P value of less than 0.05 was considered significant.\nImmunohistochemistry and statistical analysis\nZebrafish, raised either under normoxic or hypoxic conditions, were killed with an overdose of anesthetic MS-222 and frozen in liquid nitrogen. Subsequently, transversal cross-sections (10\u00a0\u03bcm thick) of the body, were cut using a cryostat at \u221220\u00b0C and mounted on glass slides coated with Vectabond (Vector Laboratories, Burlingame, USA). Sections were fixed in 4% formaldehyde in Tris-buffered saline (TBS; 50\u00a0mM Tris and 150\u00a0mM NaCl, pH 7.5) for 10\u00a0min and subsequently washed in TBS with 0.05% Tween-20 (TBST) (Sigma-Aldrich, Zwijndrecht, The Netherlands). Subsequently, sections were incubated for 10\u00a0min with 10% normal swine serum (Vector laboratories) in TBST after which sections were incubated for 24\u00a0h at 4\u00b0C with anti-phospho Akt polyclonal antibody (Santa-Cruz Biotechnology, USA) diluted 1:50 in TBST. After incubation with primary antibody, the slides were washed in TBST and subsequently placed in 0.25% (v\/v) acetic anhydride in 0.1\u00a0M triethanolamine buffer (pH 8) for 10\u00a0min followed by rinsing in TBST. After this, sections were incubated for 60\u00a0min at 20\u00b0C with secondary anti-rabbit immunoglobulin G (IgG) antibody covalently coupled to alkaline phosphatase (Vector Laboaratories) diluted 1:100 and washed in TBST. After this, sections were incubated for 5\u00a0min with alkaline phosphatase (AP) buffer (0.1\u00a0M NaCl, 0.1\u00a0M Tris, 50\u00a0mM MgCl2 and 0.1% Tween-20, pH 9.5) followed by incubation with BM Purple AP substrate (Roche Applied Sciences, Almere, The Netherlands) for 30, 45 or 60\u00a0min which was followed by rinsing in TBST. All sections were mounted in glycerine-gelatin and stored at 4\u00b0C in the dark until staining intensity was measured. The absorbance values of the BM Purple in the sections were determined using a Leica DMRB microscope (Wetzlar, Germany) fitted with calibrated gray filters using different interference filters. Absorbances for BM Purple were determined at 550\u00a0nm. Images were recorded with a \u00d720 objective and a Sony XC-77CE camera (Towada, Japan) connected to a LG-3 frame grabber (Scion; Frederick, MD) in an Apple Power Macintosh computer. Recorded images were analysed with the public domain program NIH-Image V1.61 (US National Institutes of Health, available at http:\/\/rsb.info.nih.gov\/nih-image\/). Gray values were converted to absorbance values per pixel using the gray filters and a third-degree polynomial fit in the calibrate option of NIH-image programme. Morphometry was calibrated using a slide micrometer and the set scale option in NIH-image, taking the pixel-aspect ration into account. An independent t test was used to test for differences in phospho-Akt levels in cardiac myocytes of normoxic and hypoxic fish. A P value of less than 0.05 was considered significant. Values are means\u00a0\u00b1\u00a0S.E.M.\nRNA preparation and biological sampling\nAfter dissection hearts were homogenized in a Dounce homogenizer using 1\u00a0ml Trizol solution (GibcoBrl, Life technologies). The whole heart was used and for each biological sample hearts were pooled from five different animals. After Trizol extraction, total RNA was further purified using RNAeasy columns (Qiagen). RNA samples were analyzed for quality control by Lab-on-a-chip analysis (Agilent) and on agarose gels. For the array experiment five arrays were done for normoxic and 5 arrays for the hypoxic condition. Biological samples (BS) came from two independent experiments and one technical replicate (TR) was included (2BS\u00a0+\u00a02BS\u00a0+\u00a0TR for normoxia and 2BS\u00a0+\u00a02BS\u00a0+\u00a0TR for hypoxia). As mentioned above for each BS, hearts from five different animals were pooled.\nMicroarray analysis\nThe Affymetrix GeneChip\u00ae Zebrafish Genome Arrays containing 15,509 Danio rerio gene transcripts were used. Probe sets on the arrays were designed with 16 oligonucleotide pairs to detect each transcript and procedures were in full support of MIAME standards. Labeling and microarray hybridization were performed by ServiceXS (Leiden, The Netherlands), including prior a standard round of RNA amplification according to standard Affymetrix protocols. The criteria used for differential expression were greater or equal than 2-fold induced or reduced and P\u00a0\u2264\u00a00.02. Data analysis was done using Rosetta Resolver. All expression data was submitted to the NCBI Gene Expression Omnibus (http:\/\/www.ncbi.nlm.nih.gov\/geo). The series entry number is GSE4989 and the following 10 accession numbers were assigned: GSM112796 and GSM112798-GSM112806. A complete list of differential regulated genes is shown in Supplemental Table\u00a01.\nTable\u00a01Statistical analysis of histological sections (5 \u03bcm) from zebrafish (Danio rerio) and cichlid (Haplochromis piceatus) heartsNumber of cardiomyocyte nuclei per sectionZebrafish (nuclei per 900\u00a0\u03bcm2)Cichlid (nuclei per 10,000\u00a0\u03bcm2)NormoxiaHypoxiaNormoxiaHypoxiaMean9.8113.6714.724SD0.300.390.500.68P value9.9\u00a0\u00d7\u00a010\u2212177.6\u00a0\u00d7\u00a010\u221212Midline sections of zebrafish and cichlids raised under normoxic and hypoxic conditions were chosen and subdivided in smaller areas. In the case of D. rerio, each subarea of the section was 900\u00a0\u03bcm2 whereas in the case of the H. piceatus it was 10,000\u00a0\u03bcm2. Then, subareas were randomly picked and the amount of cardiac myocyte nuclei were counted. The vast majority of cardiac myocytes were mononucleated cells. Hundred subareas per section were counted per specimen. Three sections per heart were visually analyzed (with a magnification of 20\u00d7). In total 6 different zebrafish hearts (from three independent experiments) were investigated for hypoxic conditions and 6 for normoxic condition. In total 1,800 subareas were quantified per heart and condition used. The same was done for the cichlids (for which bigger subareas were counted). A two-tailed t test was applied and a significant difference (P\u00a0<\u00a00.001) in the amount of nuclei present in the hearts of normoxia versus hypoxia groups was observed for both species\nGene ontology analysis\nThe Gene ontology analysis was performed using eGOn and the database of the Norwegian Microarray Consortium (http:\/\/www.genetools.microarray.ntnu.no\/egon\/) and the most recent updated Unigene numbers for zebrafish (May 2007). 13414 Unigene numbers were annotated to the Affymetrix gene chip used. eGOn Enrichment analysis was used for the entire Affymetrix set versus the identified 376 gene set based on the new Unigene numbers. A master target test was performed (using Fishers exact test) and the cut off for positives was set to P\u00a0<\u00a00.05. The available three categories, molecular function, biological process and cellular component were all tested and are shown in this dataset (Supplemental Data 1). The fold enrichment was based on the percentage found within the differentially expressed genes divided by the percentage on the total chip. A list of all Unigene numbers found in the different categories is given in Supplemental Data 2.\nReal-time quantitative RT-PCR\nFor verification of gene expression data, we used quantitative real-time RT-PCR. Biological RNA samples were obtained from a third independent experiment and as mentioned above for the microarrays, for each BS hearts from five different animals were pooled. The Roche Master SYBR Green kit was used for the RT-PCR reactions. The annealing and synthesis temperature was 55\u00b0C alternating with 96\u00b0C for 45\u00a0cycles. Dissociation protocols were used to measure melting curves and control for unspecific signals from the primers. A measure of 100\u00a0ng of total RNA was used per reaction. A standard curve for \u03b2-actin using 1, 5, 10, 100 and 500\u00a0ng of total RNA was used for normalization. Samples were measured in the Roche LightCycler. The primer3 software (http:\/\/frodo.wi.mit.edu\/cgi-bin\/primer3\/primer3_www.cgi) was used to design primers for short amplicons between 50 and 100 bases. The primers used are shown in Supplemental Table 2.\nResults\nWe describe here the survival rates of adult zebrafish upon immediate and gradual exposure to different pO2 concentrations. Immediate exposure to pO2 of 15\u00a0Torr (O2 concentration of 0.8\u00a0mg\/l or 10% air saturated water) was lethal for the adult zebrafish and none survived for longer than 72\u00a0h (Fig.\u00a01). If lowered gradually (see experimental procedures for regimen in Sect. \u201cHypoxia treatment\u201d) zebrafish were able to grow and gain weight at O2 levels of 10% air saturation. The zebrafish were able to survive for longer than 6\u00a0months and no mortality was observed, demonstrating that they can well adapt to these conditions (data not shown). For the experiments with zebrafish and cichlids, the pO2 concentration was gradually lowered to 10% air saturation (0.8\u00a0mg\/l) (see \u201cMaterial and methods\u201d) and fishes were kept under 10% air saturation for 3\u00a0weeks. Control groups were always kept in parallel under normoxic conditions.\nFig.\u00a01Survival of zebrafish embryos after immediate exposure to hypoxia. Zebrafish were immediately exposed to hypoxia (15\u00a0Torr; 0.8\u00a0mg\/ml; 10% air saturated water). Results are derived from three independent experiments with n\u00a0=\u00a030 in each experiment (adding to a total of 90 fish tested). After 24, 48 and 72\u00a0h, dead and alive fish were counted. Shown here is the percentage of dead fish at the respective time points and the standard deviation. At 24\u00a0h 53.3%(\u00b14.4) of fish were dead, at 48\u00a0hours 85.3% (\u00b15.1) and at 72\u00a0hours 100% (\u00b10). Control groups (n\u00a0=\u00a030), which were in parallel exposed to normoxic conditions showed no mortality (not shown). None of the zebrafish survived the immediate exposure to an O2 concentration of 0.8\u00a0mg\/l (10% air saturation) over the 3-day period. In contrast, fishes gradually exposed to hypoxia showed no induced mortality for even the 25\u00a0day time periods used in our experiments\nPhenotypic changes in the heart of adult teleosts under chronic constant hypoxia (CCH)\nIn comparison to the normoxic control groups, we observed a significantly smaller ventricular outflow tract and reduced lacunae within the central ventricular cavity and reduced lacunae around the trabeculae in midline sections of hearts of both zebrafish (Danio rerio) (Fig.\u00a02a) and cichlids (Haplochromis piceatus) (Fig.\u00a02b) exposed to CCH. In addition to the midline sections, none of the sections investigated from hypoxia treated fishes showed a ventricular outflow tract in comparable size to the normoxic controls (data not shown). In addition to the midline sections, none of the lateral sections (to both sides of the midline) investigated from hypoxia treated fishes showed a ventricular outflow tract as well as lacunae in a comparable size to the normoxic controls (data not shown). The larger cichlid hearts were also perfused and midline sectioned and showed similar results with a smaller ventricular outflow tract and reduced lacunae (Fig.\u00a02b-H). This might represent ventricular hypertrophy or hyperplasia in both walls and trabeculae, which could lead to the observed cavity obliteration in these sections. We quantified the number of cardiac myocyte nuclei per area in the midline sections of both zebrafish and cichlids under normoxic, as well as hypoxic conditions. A significant difference in both species was observed and showed that hypoxia led to a 1.4- and 1.6-fold increase in the number of cardiac myocyte nuclei per area in zebrafish and cichlid hearts, respectively (Table\u00a01). Cardiac myocyte nuclei in sections were clearly distinguishable from nuclei of other cells like erythrocytes and fibroblasts and only centralized nuclei in cardiac myocytes (which are more elongated than nuclei from erythrocytes) were counted. Furthermore, scanning electron microscopy (SEM) was used to confirm these findings in the smaller zebrafish hearts (Fig.\u00a03). Future research is warranted in order to assess further how the cardiac myocytes adapt to CCH in the fish heart.\nFig.\u00a02Histological changes of zebrafish and cichlid hearts after exposure to chronic constant hypoxia. a shows zebrafish hearts that were dissected, sectioned and stained with a hematoxylin\u2013eosin staining with A, B and E representing normoxic and C, D and F hypoxic conditions. Cell nuclei are seen in dark (dark blue in online version) and cell cytoplasm in light (pink in online version). Pictures A, C, E and F have the same magnification (10\u00d7). Images B and D represent a 20\u00d7 magnification of cardiac muscle (D). Abbreviations used are: a atrium; v ventricle; vo ventricular outflow tract and ca conus arteriosus. b (A\u2013F) corresponds to sections of cichlid hearts, which were treated the same way as the ones above from zebrafish and G and H show cichlid hearts which have been perfused prior to dissection and were stained with either hematoxylin\u2013eosin (A\u2013F) or Azan blue (G, H). In A, B, C and G pictures of normoxic conditions are shown and D, E, F and H represent the corresponding hypoxic conditions. Similar results for both the zebrafish and the cichlid hearts were observed in three independent experimentsFig.\u00a03Morphological changes of zebrafish and cichlid hearts after exposure to chronic constant hypoxia. Scanning electron microscopy pictures of hearts from normoxia control zebrafish (a, c and d) and hypoxia-treated zebrafish (b, e and f). The bigger images show half of a heart ventricle, sectioned longitudinally. The smaller images represent a higher magnification of cardiac muscle. The scale is given at the bottom of each picture. Abbreviations used are: v ventricle and vo ventricular outflow tract\nGene expression changes in the heart of adult zebrafish under chronic constant hypoxia (CCH)\nIn this study, we used microarrays for the transcriptional profiling of up and downregulated genes in response to hypoxia in the zebrafish heart. We identified 376 genes that were differentially expressed under hypoxic conditions, out of which 116 genes showed a decrease in gene expression (30.9%) in comparison to 260 genes which showed increased expression levels (69.1%).\nAll 376 differentially expressed genes, including the ones with oligo sequences which could not be annotated so far (referred therein either as transcribed locus or zebrafish clone) are shown in the complete file (Supplemental Table 1). Functional groups are color coded and if possible, gene functions are briefly summarized and OMIM links given.\nFunctional groups of differentially expressed genes in the heart\nWe have clustered the differential expressed genes according to known functions (Table\u00a02). Genes can have more than one particular function assigned, so some genes can appear in more than one group. In addition, a gene ontology analysis was performed using eGOn to determine gene enrichment and overrepresentation in the three categories of molecular function, biological processes and cellular components (Supplemental Data 1 and 2).\nTable\u00a02Functional groups of differentially expressed genesUniGeneGeneBankFoldGene nameUpregulated genesAngiogenesisDr.11575NM_1732442.3T-cell acute lymphocytic leukemia 1; TAL1Dr.845BG7290132.8Fibrinogen alpha\/alpha-E chain Dr.4907BC0458684.2Fibrinogen, gamma polypeptideApoptosisDr.15862AF4939872.1BCL2adenovirus E1b 19 interacting protein3DrAffx.1.39AF3027892.3Death receptorDr.20106AI7222772.8Apoptosis inhibitor 5Dr.4039BQ48068821.8BAX inhibitor 1Cell adhesionDr.6007NM_1318202.9Cadherin 1Dr.25140BQ2628023.3Tumor-associated calcium signal transducer Dr.4409BC0490364.4CD9 antigenDr.25140BQ2628027.7Tumor-associated calcium signal transducer glycoproteinDevelopmentDr.11575NM_17322.3T-cell acute lymphocyte leukemia 1 (tal 1)Dr.23348BE2016532.6Bone morphogenetic protein 3b; (bmp 3)Dr25405BC0139232.8SOX2 SRY-box 2Dr.6382AW1650532.9Hedgehog-interacting proteinDr.10879U976693.0NOTCH3 Notch homolog 3 (Drosophila)Dr.15055BC0501723.6Chemokine receptor 4aDr.6787BI5334265.5NoelinDr.16720BI9808476.3notch 2Disease relatedDr.6349AW1166682.4Eparin cofactor IIDr.21064BC0460754.54hydroxyphenylpyruvate dioxygenase HPDDr.12584NM_1312115.4Gata binding protein 3 (GATA3)Dr.3530AI49754579.3Prion protein (prp) geneGrowth regulationDr.8145NM_131432.2Insulin like growth factor 2 (IGF-2)Dr.7609BI4758572.4Prolactin receptorDr.8285NM_131362.4Mad homolog 2 Dr.8947CD5947352.5Spint 2Dr.822BM1841272.5Spint 2Dr.3563CD0144882.8Tetraspan membrane protein IL-TMPDr.8587NM_173282.9Insulin-like growth factor binding protein 1 Dr.2596BM3429013.2Cyclin IDr.8587AL9108223.4Insulin-like growth factor binding protein 1Dr.26458BC0532065.6m-rasHeart relatedDr.15088BM1817494.3Lectin galactoside-binding soluble 1; (galectin10-like 3)Dr.4867AI4968405.5HaptoglobinDr.3585AY0497316.6Angiotensinogen Dr.2452BQ2848484.3Complement component C9 Dr.18453BC0445254.8Uridine phosphorylaseDr.3025BG7382042.7Alpha-2 macro-globulin; A2MGInflammationDr.12491BI6721682.1Complement C4\u20132Dr.4047NM_1316272.3Small inducible cytokine A (scyba)Dr.5053NM_1317232.3Kruppel-like factor 4 Dr25207X064652.5Complement component 8, gamma polypeptideDr.6845K027652.9C3 complement component 3Dr.5741BU7104823.2Complement component b fbDr.7722BI8784143.5Complement C3-H1Dr.22244AW0197813.6Complement C1sDr.22133AW0767683.7c1rs-A and clrs-BDr.5528AI4972124.2Complement component C9Dr.2452BQ2848484.3Complement component C9 Dr.1730AI7215284.8cfI-B complement control protein factor I-B Dr.2452BM7780025.8Complement component C9Dr.20291BM0363896.5Complement C3-SDr.190NM_1313387.9Complement component factor B Dr.1192AB0716012.Lipocalin-type prostaglandin D synthase-like proteinMetabolismDr.9492BI8822442.0Sulfide dehydrogenase likeDr.15574BM5714672.1Hypoxanthine _hosphor-ribosyltransferase 1Dr.3332AI9430532.2Angiopoietin 5 Dr.16130CD0148982.3Alcohol dehydrogenase 8\u00a0bDr.3959BI430012.55\u2032-nucleotidaseDr.22205AW0194772.6OxidoreductaseDr.1699AI6672492.7Pyruvate kinaseDr.5504BI8795503.2Cystathionine-beta-synthaseDr.1202AJ2454913.9Apolipoprotein A-IDr.4111BC0532674.2Fructose-1,6-bisphosphatase 1Dr.18834AW0193214.2Urate oxidaseDr.19224BC0501674.3Aldolase bDr.4938NM_1316454.4Fatty acid desaturase 2Dr.12654BC04690114.8ELOVL family member 6,Dr.5488AI54559317.3Apolipoprotein A-IV Muscle relatedDr.3585AY0497316.6AngiotensinogenDr.2452BQ2848484.3Complement component C9ProteolysisDr.20934AF5419522.6Trypsin precursorDr.3025BG7382042.7Alpha-2-macroglobulin Dr.22139AW0189653.0Alpha-1-antitrypsinDr.25331AI6580724.1Alpha-2-macroglobulin-2Dr.12602NM_1391804.3LysozymeDr.1605BM1853884.4Protease inhibitor 1Dr.17459CD5868374.8Inter-alpha-trypsin inhibitor heavy chain H3 Dr.3073AI5850305.0Serine protease inhibitor alpha 1Dr.26371AI6676765.4ProstasinDr.3025BM5304275.6Alpha-2-macroglobulin-1Dr.3025BM3168676.5Alpha-2-macroglobulin-2Dr.2960X670553.5ITIH3 pre-alpha (globulin) inhibitor, H3 polypeptideDr.25379BI3267836.7Alpha-2-macroglobulinDr.4797AI9595347.826\u201329\u00a0kD-Proteinase proteinROS protectionDr.20068NM_1310752.1Metallothionein (mt)Dr.5399AI9577652.3Biliverdin I Beta ReductaseDr.14058CD0153513.5Glutathione S-transferase theta 1Dr.25160BC0494755.9Metallothionein 2Dr.3613BC0480376.0CerulopasminDr.4905.1BC0454646.5Uncoupling protein 4Signal transductionDr.9852AW8264252.1CAM kinase 1Dr.8591BM1865082.9Rho guanine nucleotide exchange factor 10Dr.6236AW1159733.1Rho guanine nucleotide exchange factor 5Dr.1267BC0511573.4Phospholipase C deltaDr.22129BC0166683.9RRAGC Rag C (Ras-related GTP binding C)Dr.7255AW1164794.4Protein phosphatase 1,Dr.4453BC0444215.8Phosphoprotein phosphataseTranslationDr.13234BM0364712.0Ribonuclease P Dr.382CB3638302.1NucleolinDr.6949AW0781162.1RNA 3\u2032-terminal phosphate cyclase-like protein (HSPC338)Dr.13563BI8907292.3Methionyl aminopeptidase 2Dr.26328AL7236962.3Eukaryotic translation initiation factor 4A,Dr.17693BQ0782853.740\u00a0S ribosomal protein S6Dr.20270BI6740505.9Ribosomal protein L12 Dr.25224CD01533020.4Ribosomal protein L12 Dr.12439BM53384817.5Heterogeneous nuclear ribonucleoprotein K Dr.12439BM53384824.2Heterogeneous nuclear ribonucleoprotein KDr.14821BM07171433.8Heterogeneous nuclear ribonucleoprotein K Dr.12502BQ28468640.7Heterogeneous nuclear ribonucleoprotein K Dr.12439. BM53443240.9Heterogeneous nuclear ribonucleoprotein KDr.12439BQ61693045.4Heterogeneous nuclear ribonucleoprotein KTransportDr.1084BQ1097723.0Clathrin coat assembly protein AP19Dr.5562X045063.0APOB apolipoprotein B (including Ag(x) antigen)Dr.13231BM7786464.2Solute carrier family 22Dr.30444AY3296294.3Embryonic globin beta e2Dr.24250AF4891052.0Uroporphyrinogen III synthaseDr.10343NM_1316874.7Na+K+ transporting, alpha 1a.2 polypeptideDr.7634AW11575711.3HemopexinDownregulated genesAngiogenesisDr.26411BQ783571\u22128.9Fast muscle troponin IDr.15501BM316040\u22122.1Similar to CYR6 HUMAN CYR61 protein precursor, Insulin-like growth factor-binding protein 10Cell adhesionDr.251BQ285646\u22122.3Cadherin 11Disease relatedDr.22774AW280206\u22125.7ras-like GTP-binding protein RAB27ADr.1816AL720262\u22124.4Ataxin 2-binding proteinDr.9893BM036473\u22122.3FibrillarinDr.16726BI429372\u22122.0netrin G1Growth regulationDr.12986CA787334\u22125.3v-fosDr.12986BI881979\u22125.0v-fosDr.12986BM957279\u22124.5v-fosDr.1221AW510198\u22124.3Pmx-1b (PHOX-1)Dr.12986BI881979\u22124.2v-fosDr.12410NM_131826\u22122.4Sprouty homolog 4 Dr.6431BC049326\u22122.3Suppressors of cytokine signaling 3Dr.6511NM_130922\u22122.2B-cell translocation gene 2Dr.5365AI601685\u22122.2Dual specificity phosphatase 5Dr.12062BC047814\u22122.1Epidermal growth factor receptor kinase substrate EPS8Dr.17286BM777144\u22122.0Hormone-regulated proliferation-associated 20\u00a0kDa proteinDr.9448BM156058\u22122.0TGF-beta-inducible early growth response protein 2Heart relatedDr.20010BQ826502\u22127.0ATPase, Ca++ transporting, cardiac muscle (ATP2A1)Dr.1448AL717344\u22123.5Fast skeletal myosin light chain 1aDr.20990AY033829 AY081167\u22122.4\u20132.1TitinMetabolismDr.24950BC053305\u22124.1Creatine kinase CKM3Dr.9528BC045993\u22123.5Pyruvate dehydrogenase kinaseDr.146AI477401\u22122.9Carnitine O-palmitoyltransferase IIDr.21501AI667180\u22122.4Short-chain acyl-CoA dehydrogenase Dr.19643AL918850\u22122.4FabG beta-ketoacyl -reductaseDr.15059BM530407\u22122.2Elongation of very long chain fatty acids (Cig30)Dr.21040BC045479\u22122.1Glucose-6-phosphatase, transport protein 1Dr.988AW154697\u22122.1Dodecenoyl-coenzyme A delta isomerase Dr.11971BG727588\u22122.0Carnitine O-acetyl-transferaseDr.4777AW420997\u22122.0Succinate-CoA ligaseDr.11252BC047826\u22122.0Creatine kinase, mitochondrial 1 Muscle relatedDr.21800AI883923\u22125.0Myosin binding protein CDr.5066AF524840\u22123.4Alpha-actinin 3Dr.24260NM_131619\u22123.0Myosin, light polypeptide 3Dr.2914BC045520\u22122.5Myosin light polypeptide 2; mylz2Dr.20990AY033829 AY081167\u22122.4\u20132.1TitinDr.1435AI353817\u22122.0Caveolin 3Dr.18657BQ479700\u22122.1Carbonic anhydrase IIDr.26411BQ783571\u22128.9Troponin IProteolysisDr.3581BM101561\u22128.3Chymotrypsinogen B1 Dr.3581BM101561\u22127.5Chymotrypsinogen B1Signal transductionDr.22841AI641080\u22122.4Serum deprivation response protein (SDPR)TranslationDr.7939AW281840\u22122.7Mitochondrial elongation factor G1Dr.1286BM036808\u22122.2Mitochondrial ribosomal protein L48Dr.18218AL909921\u22122.1Mitochondrial 28\u00a0S ribosomal protein S12 TransportDr.676BC050956\u22124.4ADT2, ADP,ATP carrier proteinDr.25199CD014403\u22122.1Calcium-binding mitochondrial carrier protein Aralar2 (Citrin) Dr.2784AI942949\u22122.0Solute carrier family 25 Dr.11127BG306498\u22123.7Synaptotagmin IDr.11127AW826278\u22123.2Synaptotagmin I Dr.13273BI885460\u22122.3GTP-binding protein rab15 Dr.22748AW280026\u22122.7trpn1Dr.11302BG306530\u22122.1ATPase (Ca++ transporting plasma membrane 2)\nProteinbiosynthesis (Translation): In the group linked to proteinbiosynthesis, only three genes showed decreased expression under hypoxic conditions. All three were found to be part of the mitochondrial translational machinery. In contrast, 9 non mitochondrial genes linked to proteinbiosynthesis showed all increased expression (Table\u00a02).\nMetabolism: The group with metabolic genes contains 11 repressed and 28 genes with enhanced expression (Table\u00a02). The repression majorly involves metabolic genes linked to \u03b2-oxidation and lipid metabolism. Among the metabolic genes with enhanced expression, are pyruvate kinase and aldolase which both are key enzymes for glycolysis indicating a shift from aerobic to anaerobic metabolism induced by hypoxia.\nProtection against reactive oxygen species (ROS): The group of genes important for protection against ROS contains 6 genes which are all enhanced under hypoxia.\nApoptosis: We found four genes linked to programmed cell death to be enhanced by hypoxia. Two of these, the death receptor 5 (DR5) and the BNIP3 homologue, are considered to be pro-apoptotic, whereas apoptosis inhibitor 5 and Bax inhibitor have been shown to have anti-apoptotic properties (Tewari et al. 1997; Xu and Reed 1998).\nGrowth regulation: In the group of genes linked to growth regulation, we found 9 genes to be repressed and 8 genes with enhanced expression. Within the group of 8 repressed genes, we found 5 antiproliferative genes: spry4 and dual specificity phosphatase 5 both inhibit mitogen-activated kinases (MAPK), SOCS3 binds and inhibits Janus kinases (JAK) and thereby prevents STAT3 activation. BTG2 which is important in the G1\/S transition and TIEG2 is a transcriptional repressor with antiproliferative functions. Although some genes involved in cell proliferation, like the transcription factor c-fos were repressed by hypoxia (see also \u201cDiscussion\u201d), the regulation of the vast majority (13 of 17) of identified genes in this group suggests stimulation of proliferation (Table\u00a02).\nInflammation: 14 genes involved in the inflammatory response were identified to be differentially regulated and all showed increased expression upon hypoxia treatment.\nHeart-related function: Several genes linked to cardiac hypertrophy, cardiomyopathy (disease of the heart muscle) and cardiac infarction were identified in this study (see Table\u00a02 and \"Discussion\").\nMuscle-related function: Several sarcomeric genes linked to hypertrophy showed decreased expression under hypoxic conditions (Table\u00a02 and \u201cDiscussion\u201d).\nDevelopment: The genes in this group were all found to be upregulated by hypoxia among them the gene for notch-2 and notch-3. Notch receptors are transmembrane receptors with essential roles in development including heart development.\nTransport (cellular and vascular): A heterologous group containing the gene for embryonic hemoglobin beta e2, important for oxygen transport and hemopexin which is important for heme and iron transport and was found to be upregulated.\nAngiogenesis: It is well known that hypoxia via the hypoxia inducible factor 1\u03b1 (HIF1\u03b1) pathway leads to angiogenesis. In the zebrafish heart under CCH, we observed increased expression of HIF1\u03b1 as well as fibrinogen-\u03b1 and -\u03b3. Fibrinogen-\u03b1 has been shown to stimulate HIF1\u03b1 and VEGF expression and thereby induces angiogenesis (Shiose et al. 2004).\nExpression changes of known hypoxia responsive genes\nExamples for regulation of known hypoxia responsive genes are HIF1\u03b1, insulin growth factor-2 (IGF-2), insulin growth factor binding protein 1 (IGFbp1) and caveolin 3. The transcription factor Hif1\u03b1 is pivotal in the cellular response to hypoxic stress (Semenza 1999). We further observed increased expression of the egl nine homolog, a gene which was shown to be induced by hypoxia through the Hif1\u03b1 pathway (Pescador et al. 2005). IGF-2 gene- and protein expression had been shown to be upregulated by hypoxia (Beilharz et al. 1995). It was shown that IGFbp1 also is a hypoxia-inducible gene in zebrafish embryos and it mediates hypoxia-induced embryonic growth-inhibition and developmental-retardation (Kajimura et al. 2005a). Both IGF-2 and IGFbp1 were found upregulated in our study. We observed decreased expression of caveolin 3 in the hearts of zebrafish exposed to CCH, earlier findings showed that chronic myocardial hypoxia led to decreased caveolin-3 protein expression in rabbit hearts (Shi et al. 2000). These findings indicate that the hypoxic conditions used lead to hypoxic stress in the fish heart.\nEvaluation of microarray results by quantitative real-time RT-PCR\nTo further verify our results, we used quantitative real-time PCR for 10 of the transcripts. We confirmed the gene expression changes found in the microarray studies for these 5 up- and 5 downregulated transcripts by this independent method (Fig.\u00a04). The downregulated zebrafish genes tested were: c-fos, phox1, creatine kinase (ckm3), nebulin, titin, and the upregulated genes tested were: metallothionein, pyruvate kinase, apoptosis inhibitor 5, igfbp1 and notch-2. The fold induction values were not always directly comparable to the array data but in all cases induction or reduction was confirmed. Quantitative differences between array data and qPCR results have been reported before (Meijer et al. 2005; Ton et al. 2003; van der Meer et al. 2005).\nFig.\u00a04Verification of gene expression changes by quantitative real-time PCR. 10. selected genes, which were found to be differentially expressed on the microarrays, were further analyzed by quantitative real-time RT-PCR. Relative expression is given based on normalization to \u03b2-actin. A standard curve for \u03b2-actin was included in each experiment and data represents three independent experiments each done in triplicates. The primers used are given in Supplemental Table 2\nAssessment of microarray results for the IGF\/PI3K\/Akt pathway by comparing phospho-Akt levels in cardiac myocytes of normoxic versus hypoxic zebrafish hearts\nThe IGF\/PI3K\/Akt pathway is activated by IGFs, which are antagonized by the IGFbp1. To test the effects of the upregulation of both IGF-2 and IGFbp1, we assayed phospo-Akt levels in cardiac myocytes and showed that phospho-Akt levels were not different between normoxic and hypoxic cells. Figure\u00a05 shows cytoplasmic immunohistochemical staining of phospho-Akt. The antibody recognizes phosphorylated and detects the phospho-Akt1\/2\/3 forms. The incubation times for the primary and secondary antibody as well as the BM Purple were optimized to obtain a good signal to noise ratio. Absorbances were linearly related to the time of incubation with the primary and secondary antibodies as well as that of BM Purple. Figure\u00a05d shows for normoxic fish the absorbance values of the phospho-Akt staining in cardiac myocytes as well as skeletal muscle fibers from the tail as a function of the incubation time with BM Purple AP substrate. The absorbance values for the heart are considerably higher than for the skeletal muscle fibers. However, for both cardiac and skeletal muscle the absorbances are linearly related with the incubation time with BM Purple AP substrate and increase at the same relative rate. This implicates that the absorbance of BM Purple after 45\u00a0min incubation with BM Purple AP substrate did not reach saturation and therefore provides a semi-quantitative estimate of the phospho-Akt content in the cardiac myocytes. Absorbances of staining for phospho-Akt in normoxic and hypoxic cardiac myocytes were not shown to be significantly different (Fig.\u00a05E, P\u00a0<\u00a00.48), which indicates that hypoxia did not change the activation of the Akt pathway in the cardiac myocytes.\nFig.\u00a05Effects of chronic constant hypoxia of the activation of Akt in zebrafish cardiac myocytes. Immunohistochemical staining of phospho-Akt in cardiac myocytes of zebrafish raised under hypoxic a or normoxic b conditions. Specificity is shown by the control sections obtained from normoxic fish which were not incubated with primary antibody against phospho-Akt c. For both cardiac myocytes and skeletal muscle fibers, the absorbance of the BM Purple is linearly increasing at the same relative rate with the incubation time with BM Purple AP substrate at the same relative rate d. Mean absorbances of phospho-Akt staining (+S.E.M.) from normoxic and hypoxic cardiac myocytes was not different e)\nDiscussion\nIn the aquatic environment, oxygen concentrations can often vary, and being able to adapt to changes in oxygen levels can be advantageous for the survival of aquatic animals. This might be in part the reason why some teleosts have developed the ability to withstand extreme hypoxic conditions.\nIn this study, we have focused on the long-term response to hypoxia in the fish heart. The hypothesis is that the zebrafish heart, in contrast to most mammalian hearts, which are characterized by relative intolerance to injury or the lack of oxygen, are able to adapt to extreme hypoxic conditions.\nWe showed that chronic hypoxia of zebrafish caused a smaller ventricular outflow tract, reduced lacunae and increased cardiac myocyte densities in the heart. These findings suggest that hypoxia induced an increase of the cardiac myocyte volume or at least did not result in a loss of cardiac myocytes. This is in contrast to mammals where tissue hypoxia in chronic heart failure leads to apoptosis and considerable losses of cardiac myocytes (see for review, Sabbah et al. 2000a). In mammals, compensation for this loss of cardiac myocytes occurs mainly by hypertrophy of the remaining cardiac myocytes (Ostadal and Kolar 2007), although regeneration of myocardium by proliferation of cardiac myocytes may occur also but to a limited extend (Beltrami et al. 2001). Our assay for phosho-Akt did not show any enhancement of Akt activity in response to the CCH, suggesting a lack of hypertrophic signaling via the phosphotadilinositol 3 kinase pathway. However, the density of cardiac myocyte nuclei increased by 50% during CCH, which indicates substantial proliferation of cardiac myocytes and\/or nuclear hyperplasia. Recently, it has been shown that the zebrafish heart has the ability to regenerate from mechanical cardiac injury by proliferation of cardiac myocytes (Poss et al. 2002). If the zebrafish heart responds to chronic hypoxia in a similar way as to mechanical dissection, this will be beneficial in preventing apoptosis of cardiac myocytes as the diffusion distance for oxygen are not increased as during hypertrophy, which will help to prevent the development of anoxic cores in the cardiac myocytes (Des Tombe et al. 2002; van der Laarse et al. 2005). In the case that the increase in cardiac myocyte nuclear density during chronic hypoxia was due to nuclear hyperplasia and nuclear ploidy, the observed smaller ventricular outflow tract and reduced lacunae may have been the result of hypertrophy of the existing cardiac myocytes. The histological assay in this study does not clearly distinguish between hyperplasia and hypertrophy of cardiac myocytes. Future investigation of the mechanisms underlying the general morphological adaptations of teleost fish heart in response to CCH requires a nuclear stain in combination with a clear membrane stain, which allows determination of cell sizes in addition to counts of cardiac myocyte nuclei.\nWe were interested in the underlying gene expression changes of these adaptations. We found gene regulations in a transcriptional network of the serum response element (SRE), which are opposed to the ones described in mammals. In the zebrafish heart hypoxia repressed c-fos and phox1 expression. Both genes are important in the same transcriptional network, phox1 can transduce serum-responsive transcriptional activity to the c-fos (SRE) by interacting with serum response factor (SRF) (Simon et al. 1997). In mammals many studies showed increased expression of c-fos by hypoxia, e.g. in rats hypoxia induces c-fos expression in the LV and RV (Deindl et al. 2003) and the same held true in tissue culture cells (Webster et al. 1993). Interestingly, repression of c-fos by hypoxia has also been shown for the short-term response to anoxia in anoxia tolerant turtles (Greenway and Storey 2000). Is it possible that repression of c-fos and phox-1 are important adaptations in hypoxia tolerant animals?\nSeveral novel gene expression changes induced by CCH have been identified in this study (Table\u00a02 and Supplemental Table 1). An example is the two Notch receptors, notch-2 and notch-3, whose expression were induced by CCH. Notch receptors have been shown to be important for heart patterning and differentiation (Armstrong and Bischoff 2004), cell fate determination and self renewal of stem cells (Androutsellis-Theotokis et al. 2006; Silvia Bianchi 2006) but so far have not been linked to the hypoxic response.\nSeveral genes with links to human heart pathology were found to be upregulated in this study. Among these were two markers for myocardial infarction, complement component C9 and haptoglobin. C9 is used as a marker for myocardial infarction (Doran et al. 1996) and a polymorphism for haptoglobin predicts 30-day mortality and heart failure in patients with diabetes and acute myocardial infarction (Levy et al. 2002). We also observed increased expression of fetuin-\u03b1, a circulating calcium-regulatory glycoprotein that inhibits vascular calcification. Low fetuin-\u03b1 levels have been associated with heart failure in mice (Merx et al. 2005). Upregulation of fetuin-\u03b1 in the zebrafish heart could help to better tolerate CCH. Selenoprotein P (SEPP1) is a heparin-binding protein that appears to be associated with endothelial cells and has been implicated as an oxidant defense in the extracellular space. Human populations that are selenium deficient are susceptible to the development of Keshan disease, a potentially fatal form of cardiomyopathy (Nezelof et al. 2002). SEPP1 expression was found to be increased in our study indicating a potential protective mechanism against oxidative stress in the heart. This was further supported by the increased expression observed for several genes important for the protection against reactive oxygen species (ROS) (Table\u00a02). Among them were metallothionein and glutathione S-transferase, which are both known ROS scavengers. Metallothionein has further been described to be protective against hypoxia-induced apoptosis when overexpressed (Wang et al. 2001). Our findings suggest ROS protection as an important adaptation to CCH in the fish heart.\nFurthermore, our data show for the first time that CCH simultaneously induced upregulation of IGF-2 and the insulin-like growth factor binding protein 1 (IGFbp1). In isolated cardiomyoblasts, angiotensin II stimulates IGF-2 expression which is involved in the induction of apoptotic signaling in rat hearts (Lee et al. 2006). We have observed here both angiotensin and IGF-2 upregulation (Table\u00a02). The upregulation of IGFbp1 seems to be a general response to hypoxia in zebrafish embryos, where it mediates hypoxia-induced embryonic growth retardation and developmental delay (Kajimura et al. 2005b). IGFbp1 is a secreted protein, which binds to IGFs in the extracellular environment and prevents receptor activation (Florini et al. 1996; Stewart and Rotwein 1996). Here, IGFbp1 by binding IGF-2, may have prevented both cardioprotective as well as apoptotic effects of enhanced IGF-2 expression. To test the effects of the upregulation of both IGF-2 and IGFbp1, we assayed phospo-Akt levels in cardiac myocytes and showed that phospho-Akt levels were not different between normoxic and hypoxic cells. This suggests that either hypoxia-induced changes in mRNA expression did not occur at the protein level or the effects of increased IGF-2 expression on the IGF-1 receptor (which can be activated by IGF-2) were blunted by the upregulation of the IGFbp1. The fact that we did not observe increased phospho-Akt levels suggests a lack of hypertrophic signaling as enhanced phospho-Akt is required for cardiac hypertrophy (DeBosch et al. 2006). This may be beneficial for the heart as hypertrophy of cardiac myocytes implicates an increase in the diffusion distance for oxygen which may cause the development of anoxic cores (Des Tombe et al. 2002), release of cytochrome c (van Beek-Harmsen and van der Laarse 2005) and production of ROS (Lee et al. 2006; Powers et al. 2007) and eventually causing apoptosis of cardiac myocytes. The importance of the IGF-2\/IGFbp1 signaling in the protection of the zebrafish heart and its underlying mechanisms remain to be determined.\nThe gene expression changes we observed for the specific response to CCH in the fish heart were very different from the responses we observed in an earlier study in the gills (van der Meer et al. 2005). Under the same criteria as used in this study, we found that the majority of differentially regulated genes in the gills showed a decrease in gene expression (68.1% or 250 genes in total) in comparison to genes, which showed increased expression levels (31.9% or 117 genes in total). This is opposed to the heart where 69.1% of differentially regulated genes (260 genes in total) showed increased and 30.9% (116 in total) decreased expression levels. Many genes linked to protein synthesis showed a similar trend, and were downregulated in the gills (van der Meer et al. 2005) and upregulated in the heart (Table\u00a02). The major differences observed in gene regulation between heart and gills point to very tissue specific responses to CCH. A list of genes identified in both studies, as identified by a direct comparison based on Accession numbers and old Unigenes numbers are shown in Supplemental Data 3.\nTeleosts have developed specific phenotypic adaptations to low oxygen, due to the natural occurrence of hypoxia in the water environment. Here, we identified the changes in gene expression as well as associated morphological changes of the fish heart to hypoxia. We observed repression of c-fos, which differs compared to the described increase in expression in mammals (Deindl et al. 2003). Other changes found like upregulation of the two Notch receptors have not been described before to the best of our knowledge. Similarly, the simultaneous increase in expression of IGF-2 and IGFbp1 has not been shown. The changes identified here can contribute to the ability of teleosts to adapt to severe hypoxia (for example, the upregulation of fetuin-\u03b1 and sepp1 levels). Future functional studies are warranted to validate the role of the identified genes in cardiac protection to hypoxia.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\n(XLS 76 kb)\n(DOC 33 kb)\n(DOC 57 kb)\n(XLS 52 kb)","keyphrases":["hypoxia","zebrafish","heart","gene expression","hyperplasia"],"prmu":["P","P","P","P","P"]}
{"id":"Arch_Microbiol-3-1-2111041","title":"Structural characterization of diabolic acid-based tetraester, tetraether and mixed ether\/ester, membrane-spanning lipids of bacteria from the order Thermotogales\n","text":"The distribution of core lipids in the membranes of nine different species of the order Thermotogales, one of the early and deep branching lineages in the Bacteria, were examined by HPLC\/MS and demonstrated to consist of membrane-spanning diglycerol lipids comprised of diabolic acid-derived alkyl moieties. In the Thermotoga species the core membrane lipids are characterized by the presence of both ester and ether bonds, whereas in the phylogenetically more distinct Thermosipho and Fervidobacterium spp. only ester bonds occur. A tentative biosynthetic route for the biosynthesis of these membrane-spanning lipids is proposed. Since species of the order Thermotogales are assumed to have occurred early during the evolution of life on Earth, as suggested by its position in the phylogenetic tree of life, these data suggest that the ability to produce both ether and ester glycerol membrane lipids developed relatively early during microbial evolution.\nIntroduction\nThe group of anaerobic bacteria falling in the order Thermotogales represents a very deep phylogenetic branch within the phylogenetic tree of life based upon the ribosomal RNA gene (Woese 1987). Within this order, members of the genus Thermotoga have an upper growth limit of 90\u00b0C and represent, together with species in the order Aquificales, cultivated bacteria with the highest known growth temperatures. Bacteria belonging to the Thermotogales occur widespread in the environment: they thrive within continental solfatara springs of low salinity, shallow and deep-sea marine hydrothermal systems and high-temperature marine and continental oil fields (see Huber and Hannig 2005 for a recent review). Their strict organotrophic way of life makes them consumers of microbial organic matter within high temperature ecosystems. Within the order Thermotogales, the genera Thermotoga, Thermosipho, Fervidobacterium, Geotoga, Petrotoga and Marinitoga have been described, all belonging to the single family Thermotogaceae (Huber and Hannig 2005). The type species T. maritima for the genus Thermotoga, also the first identified member of the Thermotogales, was isolated from a geothermally heated, shallow marine sediment in Italy (Huber et al. 1986). Subsequently, quite a number of phylogenetically closely related (T. neapolitana, T. petrophila, T. naphtophila) and slightly more distant (T. thermarum, T. elfii, T. subterranea, T. hypogea, T. lettingae) species were isolated, mostly from marine settings. Thermotoga-related organisms have been detected in oil reservoirs (Huber and Hannig 2005), whilst members of the genus Fervidobacterium seem to be restricted to hydrothermal settings with low salinity such as hot springs. For example, Fervidobacterium pennivorans was firstly isolated from a hot spring on San Miguel (the Azores) (Friedrich and Antranikian 1996). Members of the genus Thermosipho have been isolated from a wide variety of high temperature environments such as marine hydrothermal springs (e.g. T. africanus) and deep sea hydrothermal vents (e.g. T. melanesiensis) (Antoine et al. 1997; Huber et al. 1989).\nTo be able to grow at high temperatures, the members of the Thermotogales possess a variety of thermostable enzymes and proteins, which are also of biotechnological interest. In addition, the cell membranes of these organisms must be able to cope with high temperatures. Indeed, a variety of unusual membrane lipids has been reported for members of the Thermotogales. Unusual, long-chain dicarboxylic (diabolic) acids are present in the core lipids of the genus Thermotoga (Jeanthon et al. 1995; Manca et al. 1992; Windberger et al. 1989; Carballeira et al. 1997; DeRosa et al. 1988; Huber et al. 1986). About 50% of the total polar lipids of T. maritima are two amphipathic monopolar glycolipids with a very rare \u03b1-(1\u20134) diglucosyl structure (Manca et al. 1992).\nIn the present paper, we describe in detail the structures of these and other lipids in a suite of bacteria belonging to the Thermotogales and discuss their distributions. We applied an HPLC\/MS method, which for the first time enabled to characterize the full membrane-spanning core lipids.\nMaterials and methods\nCultures\nCells were grown in 500-ml batch cultures in 1.2-l-bottles at temperatures of 55\u201380\u00b0C and with either medium A or B (see Table\u00a01). Medium A was composed (per liter demineralized water) of KCl 0.335\u00a0g; MgCl2\u00b76H2O 4.0\u00a0g; MgSO4\u00b77H2O 3.45\u00a0g; NH4Cl 0.25\u00a0g; K2HPO4 0.14\u00a0g; NaCl 10.0\u00a0g; glucose 3.8\u00a0g; yeast extract 0.5\u00a0g; peptone 0.5\u00a0g; NaHCO3,\u00b72.0\u00a0g; Na2S.7-9H2O, 0.40\u00a0g; trace element solution, 10\u00a0ml and vitamin solution, 10\u00a0ml, which were based on medium 141 of DSM (http:\/\/www.dsmz.de); CaCl2\u00b72H2O, 0.140\u00a0g; resazurin, 0.5\u00a0mg. The medium was anaerobically dispensed into serum bottles and a gas phase of 180\u00a0kPa N2\/CO2 (80\/20 v\/v) was applied. NaHCO3, Na2S.7-9H2O, glucose, CaCl2\u00b72H2O and vitamin solution were added after sterilization. The pH was 7.0\u20137.2. Medium B is a modified DSM medium 640 and composed (per liter demineralized water) of NH4Cl 0.90\u00a0g; NaCl 0.90\u00a0g; MgCl2\u00b76H2O 0.40\u00a0g; KH2PO4 0.75\u00a0g; K2HPO4 1.50\u00a0g; peptone 0.50\u00a0g; yeast extract 0.50\u00a0g; trace element solution SL-10 (see DSM medium 320), 1.00\u00a0ml; FeCl3\u00b76H2O 2.50\u00a0mg; glucose 3.8\u00a0g; cysteine\u2013HCl\u00b7 H2O, 0.75\u00a0g; resazurin 0.5\u00a0mg. The medium was anaerobically dispensed into serum bottles and a gas phase of 180\u00a0kPa N2 (100%) was applied. Glucose and cysteine\u2013HCl\u00b7H2O were added after sterilization. The pH was adjusted to pH 7.2. The cultures were inoculated with cells from the DSM collection of microorganisms (see Table\u00a01 for strain number) and were harvested in their exponential growth phase. Two different batches of T. lettingae were grown and analysed separately.\nTable\u00a01Bacteria from the order Thermotogales with some background information and the culture conditions used in this studyBacterial speciesSource for the isolateOptimal growth temperature (\u00b0C)ReferenceDSM strain numberCulture conditionsMediumaT (\u00b0C) Thermotoga maritimaAnaerobic marine sediment, Italy 80Huber et al. (1986)3109A80, 55T. neapolitanaSubmarine hot spring, Italy 85Jannasch et al. (1988)4359A80T. elfiiOil-producing well, Africa 65Ravot et al. (1995)9442A65T. lettingaeSulfate-reducing bioreactor, The Netherlands 65Balk et al. (2002)14385A65T. hypogeaOil-producing well, Africa 70Fardeau et al. (1997)11164B70Thermosipho sp.Spring, New Zealand 656568B65T. africanusMarine hydrothermal spring, Obock 75Huber et al. (1989)5309B75T. melanesiensisDeep sea hydrothermal vent, Pacific Ocean 70Antoine et al. (1997)12029B70Fervidobacterium pennivoransHot mud of spa, Italy 65Koch et al. (1997)9078B65a\u00a0See Materials and method sections for a description of the media used\nLipid analysis\nLyophilized cells were repeatedly (\u00d75) ultrasonically extracted with dichloromethane (DCM)\/ methanol (MeOH) (2:1 v\/v). The solvent of the combined extracts was removed by rotary evaporation at reduced pressure. The extract was taken up in DCM and weighed in a small vial by removing the solvent under a gentle stream of nitrogen. An aliquot (ca. 1\u00a0mg) of the extract to which a known amount of the internal standard, 2-methylheneicosane, was added, was methylated with BF3\/MeOH, filtered over a small pipette filled with silica using ethyl acetate as the eluent, and subsequently silylated with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) in pyridine at 60\u00b0C for 15\u00a0min. These so-called total lipid fractions (TLF-1) were analysed with gas chromatography (GC) and gas chromatography\u2013mass spectrometry (GC\u2013MS). In selected cases, acetylation instead of silylation of the eluate obtained after chromatography over silica was performed following a procedure described elsewhere (Cox et al. 1972).\nThe cell residue after solvent extraction was, after addition of a known amount of the internal standard, 2-methylheneicosane, hydrolysed with 1\u00a0N KOH\/MeOH (96%) by refluxing for 1\u00a0h. The hydrolysate was neutralized with 2\u00a0N HCl\/MeOH and after addition of water extracted with DCM. This fraction was methylated, chromatographed over silica and silylated as described above and the resulting TLF-2 fraction was analyzed by GC and GC\u2013MS. In specific cases alkaline hydrolysis was also performed with an aliquot of the solvent extract following the same procedure as described above. This resulted in a fraction named TLF-1-OH which was also analyzed by GC and GC\u2013MS.\nFor analysis of core lipids by high performance liquid chromatography\/atmospheric pressure chemical ionization mass spectrometry (HPLC\/APCI\u2013MS) aliquots of the solvent extracts (ca. 2\u00a0mg) were separated over a small Al2O3 column using hexane\/DCM (1:1 v\/v) and DCM\/MeOH as eluent to obtain apolar and polar fractions, respectively. The polar fraction was dissolved in hexane\/propanol (99:1 v\/v), filtered over an 0.4\u00a0\u03bcm PTFE filter, and analysed by HPLC\/APCI\u2013MS.\nGC and GC\u2013MS\nGC was performed using a Fisons GC8000 instrument, equipped with an on-column injector and a flame ionization detector. A fused silica capillary column (25\u00a0m\u00a0\u00d7\u00a00.32\u00a0mm) coated with CP Sil-5 (df 0.12\u00a0um) was used with helium as carrier gas. The samples were injected at 70\u00b0C and the oven temperature was programmed to 130\u00b0C at 20\u00b0C\/min and then at 4\u00b0C\/min to 320\u00b0C, at which it was held for 10\u00a0min. GC\u2013MS was performed on a HP 5890 gas chromatograph interfaced with a VG Autospec Ultima mass spectrometer operated at 70\u00a0eV with a mass range of m\/z 40\u2013800 and a cycle time of 1.7\u00a0s (resolution 1,000). The gas chromatograph was equipped with a fused silica capillary column as described for GC. The carrier gas was helium. The same temperature program as for GC was used.\nHPLC\/APCI\u2013MS\nMembrane-spanning core lipids were analysed by HPLC\/APCI\u2013MS according to Hopmans et al. (2000) with minor modifications. Analyses were performed on an Agilent 1100 series HPLC\/MS instrument, equipped with an auto-injector and HP Chemstation software. Separation was achieved on an Alltech Prevail Cyano column (150\u00a0mm\u00a0\u00d7\u00a02.1\u00a0mm; 3\u00a0\u03bcm). Flow rate of the hexane:propanol 99:1 (v\/v) eluent was 0.2\u00a0ml\u00a0min\u22121, isocratically for the first 5\u00a0min, thereafter with a linear gradient to 1.8% propanol in 45\u00a0min, subsequently to 10% propanol in 20\u00a0min, and subsequently isocratically for the last 20\u00a0min. Injection volume of the samples was usually 10\u00a0\u03bcl.\nResults\nNine species of bacteria belonging to the order Thermotogales were analysed for their lipid composition; five of them are Thermotoga species (T. maritima, T. lettingae, T. elfii, T. neapolitana, T. hypogea), three are Thermosipho species (T. africanus, T. melanesiensis, T. sp.) and one is a Fervidobacterium species (F. pennivorans) (Table\u00a01).\nComposition of total lipid fractions\nFigure\u00a01a shows a typical gas chromatogram of a total lipid fraction (TLF-1) for T. maritima. In addition to straight-chain fatty acids (predominantly n-C16 and n-C14), a number of more unusual lipids were detected. The first group comprises long-chain dicarboxylic acids; 15,16-dimethyltriacontanedioic acid (or diabolic acid 1; see Fig.\u00a02 for structures) and 13,14-dimethyloctacosanedioic acid (2), identified on basis of mass spectral data reported previously (Carballeira et al. 1997; Klein et al. 1979). The mass spectra of 1 and 2 as methyl ester derivatives are shown in Fig.\u00a03. The relative abundance of the C30 and C32 diabolic acids varied substantially between different species (Table\u00a02). F. pennivorans also contained small amounts of a tentatively identified C34 diabolic acid. In some species, isomers of these diabolic acids, with virtually an identical mass spectrum but with a different retention time, were present in varying quantities. These probably represent stereoisomers, possibly related to the presence of two chiral centres in the middle of the carbon chain of the diabolic acids. The second group of unusual components comprises glycerol monoethers. 1-O-hexadecyl glycerol (3) and 1-O-tetradecyl glycerol (4) were identified by mass spectral data (m\/z 205 base peak and appropriate molecular ion) and a co-elution experiment with an authentic standard. The higher molecular weight monoethers are 15,16-dimethyl-30-glyceryloxytriacontanoic acid (5) and 13,14-dimethyl-28-glyceryloxyoctadecanoic acid (6). Glycerol monoether 5 has been identified previously and the mass spectrum of 5 as acetyl derivative was reported (DeRosa et al. 1988). For identification purposes we used acetylation in addition to silylation to be able to compare the mass spectrum of the derivative of 5 with that reported in the literature (DeRosa et al. 1988). The mass spectrum of 5 as TMS derivative is shown in Fig.\u00a03. Component 6 was tentatively identified by interpretation of its mass spectrum in comparison with that of 5 (Fig.\u00a03). The relative amounts of monoethers 5 and 6 in the TLF-1 vary widely between the examined species of the Thermotogales (Table\u00a02). As observed for the diabolic acids 1 and 2, two additional minor isomers with similar mass spectrometric characteristics but different retention times are observed. The third group of lipids are glycerol dialkyl diesters and glycerol mixed ester\/ethers. They were predominantly found in the TLF-1 of T. lettingae (Fig.\u00a04a) and are composed of 1,2- and 1,3-diacyl glycerols (e.g. 7 and 8) and 1-alkyl, 2-acyl (e.g. 9) and 1-alkyl, 3-acyl (e.g. 10) glycerols. They were identified by comparison with mass spectra reported in the literature (Wood et al. 1980). Some Thermotoga species contained small amounts of 1,2-di-O-alkyl glycerols.\nFig.\u00a01Gas chromatograms of the total lipid fractions (TLF-1) (a\u2013b) and these fractions after alkaline hydrolysis of the residue after extraction (TLF-2) (c\u2013d) of Thermotoga maritima (grown at 55\u00b0C) and F. pennivorans pennivorans, respectively. Fatty acids and alcohols were derivatized to the corresponding methyl esters and trimethylsilyl ethers prior to gas chromatographic analysis. Numbers refer to structures indicated in Fig.\u00a02. FA Fatty acid, std internal standardFig.\u00a02Structures of lipids encountered in species from the order ThermotogalesFig.\u00a03Mass spectra (subtracted for background) of derivatised diabolic acids and diabolic acid glycerol ethersTable\u00a02Relative distribution (in % of total quantified) of lipids in different species of the order ThermotogalesBacterial speciesFractionaLipidsbC14:0 FAC16:0 FAC18:0 FA123456Thermotoga maritima (80\u00b0C)TLF-14.244.21.121.31.79.15.110.62.7TLF-21.725.90.938.74.22.21.017.67.8T. maritima (55\u00b0C)TLF-17.554.70.514.32.311.12.56.61.2TLF-20.715.00.448.44.60.70.223.86.2T. neapolitanaTLF-14.661.32.817.31.33.70.77.11.2TLF-21.946.52.826.42.10.615.83.9T. elfiiTLF-13.561.04.028.22.60.50.10.2TLF-21.220.01.270.76.40.20.3T. lettingae (1st batch)TLF-13.037.25.924.65.26.93.98.35.0TLF-20.811.61.048.37.82.20.918.78.7T. lettingae (2nd batch)TLF-1-OH6.326.41.132.68.84.85.88.55.6TLF-20.57.40.357.614.10.40.211.87.7T. hypogeaTLF-14.854.52.314.12.37.43.58.13.0TLF-20.54.51.047.41.50.60.133.311.1Thermosipho sp.TLF-17.951.528.012.6TLF-22.616.05.066.89.6T. africanusTLF-14.457.632.52.7TLF-22.561.88.125.42.2T. melanesiensisTLF-15.562.130.81.6TLF-25.684.77.12.6Fervidobacterium. pennivoransTLF-11.054.19.535.10.3TLF-20.213.53.082.31.0a\u00a0\u00a0TLF-1 Total lipid fraction, TLF-2 total lipid fraction obtained after alkaline hydrolysis of the residue after extraction;TLF-OH Total lipid fraction after alkaline hydrolysis of the extractb\u00a0\u00a0Numbers refer to Fig.\u00a02. Blank entries indicate that the lipid was not detectedFig.\u00a04Gas chromatograms of the total lipid fraction (TLF-1) (a), this fraction after alkaline hydrolysis (TLF-1-OH) (b) and this fraction obtained after alkaline hydrolysis of the residue after extraction (TLF-2) (c) of Thermotoga lettingae (2nd batch). Fatty acids and alcohols were derivatized to the corresponding methyl esters and trimethylsilyl ethers prior to gas chromatographic analysis. Numbers refer to structures indicated in Fig.\u00a02. FA Fatty acid, std internal standard\nThe TLF after alkaline hydrolysis of the cell residue after extraction (TLF-2) contained a similar suite of lipids as the TLF-1 (Table\u00a02), albeit that some other components (e.g. unsaturated C16:1 and C18:1 fatty acids) were also present but only in the examined Thermosipho species. Most striking was that the characteristic long-chain diacids 1 and 2 and the complex monoethers 5 and 6 were relatively much more abundant in TLF-2 (Figs.\u00a01c,d and 4b; Table\u00a02), suggesting that they are predominantly formed by alkaline hydrolysis from larger lipid structures. A similar distribution was observed when the extract of T. lettingae (2nd batch) was subjected to alkaline hydrolysis (TLF-1-OH): the long-chain diacids 1 and 2 and the monoethers 5 and 6, which were not observed in the TLF-1 (Fig.\u00a04a), are one of the most abundant lipids in the TLF-1-OH fraction (Fig.\u00a04b). This indicates that these lipids are building blocks of larger, more complex lipids not amenable to GC.\nThe absolute amount of the most abundant lipids (in mg\u00a0g\u22121 dry weight) was for a number of species measured in both the TLF-1 and TLF-2 (Table\u00a03). This showed that the amounts released by alkaline hydrolysis from the residue after extraction are still substantial. It also showed that the highest concentrations occur in the Thermotoga species and in F. pennivorans, while concentrations in the Thermosipho species are substantially lower. Quantification of the lipids in the TLF-1-OH fraction of T. lettingae (2nd batch) showed that the concentration increases substantially after alkaline hydrolysis of the extract.\nTable\u00a03Absolute concentrations (in mg\u00a0g\u22121 dry weight) of diabolic acids and glycerol monoether acids in different species of the order ThermotogalesBacterial speciesFractionaLipidsb1256Thermotoga maritima (55\u00b0C)TLF-15.60.512.70.50TLF-24.10.402.00.52T. lettingae (2nd batch)TLF-1TLF-1-OH7.11.91.81.2TLF-21.90.460.380.25T. hypogeaTLF-11.00.170.600.22TLF-20.700.020.490.16Thermosipho sp.TLF-10.05TLF-20.260.04T. africanusTLF-10.01TLF-20.180.02T. melanesiensisTLF-1TLF-20.01FervidobacteriumpennivoransTLF-17.90.07TLF-23.50.04a\u00a0TLF-1 Total lipid fraction, TLF-2 total lipid fraction obtained after alkaline hydrolysis of the residue after extraction, TLF-1-OH\u00a0total lipid fraction after base hydrolysis of the extractb\u00a0\u00a0Numbers refer to Fig.\u00a02. Blank entries indicate that the lipid was not detected\nDistribution of membrane-spanning core lipids in T. maritima\nTo characterize the membrane-spanning core lipids of the species belonging to the Thermotogales, the polar fraction of the solvent extract was analysed by an HPLC\/APCI\u2013MS method (Hopmans et al. 2000) slightly adjusted to enable the elution of more polar components than glycerol dialkyl glycerol tetraethers (GDGTs) from the LC column (see Materials and methods for details). Analysis of the polar lipids of Thermotogamaritima revealed a complex composition exemplified by the base peak chromatogram (Fig.\u00a05a). The first eluting, small peak with a retention time of 29.0\u00a0min (labelled 11a in Fig.\u00a05b) showed an APCI mass spectrum (Fig.\u00a06a) characterised by peaks at [M\u00a0+\u00a0H]+, [M\u00a0+\u00a0H]+-18 (loss of water) and [M\u00a0+\u00a0H]+-74 (loss of glycerol). This characteristic pattern is well known from archaeal GDGTs (Hopmans et al. 2000) and non-isoprenoidal GDGTs (Sinninghe Damst\u00e9 et al. 2000), suggesting a tetraether structure. Indeed, upon alkaline hydrolysis of this fraction and re-analysis by HPLC\/APCI\u2013MS, this minor peak was now the most abundant peak in the base peak chromatogram. These data indicate a GDGT structure for the major peak comprised of two glycerol units connected by two C32 alkyl chains, resulting in a molecular mass of 1077 (C70H140O6). Since 15,16-dimethyltriacontanedioic acid (1) and 15,16-dimethyl-30-glyceryloxytriacontanoic acid (5) are abundant lipids in the TLF-1 of T. maritima, it seems likely that the alkyl chains in this GDGT are the same, resulting in structure 11a. At the flank of peak 11a, at slightly higher retention time, a similar mass spectrum was observed with all ions shifted by 28\u00a0Da to lower m\/z values. This compound is likely a GDGT with a C32 and C30 dimethyl alkyl chains (12a), consistent with the presence of small amounts of 13,14-dimethyloctacosanedioic acid (2) and 13,14-dimethyl-28-glyceryloxyoctadecanoic acid (6) in the TLF-1 (Table\u00a02).\nFig.\u00a05HPLC\/APCI\u2013MS base peak chromatogram (a) and specific mass chromatograms (b\u2013i) of polar lipids of Thermotoga maritima grown at 55\u00b0C. The mass chromatograms, indicative for specific components, are m\/z 1,078 (b [M\u00a0+\u00a0H]+ C70 tetraether), m\/z 1,074 (c [M\u00a0+\u00a0H]+-18 C70 triether\/monoester), m\/z 1,046 (d [M\u00a0+\u00a0H]+-18 C68 triether\/monoester), m\/z 1,088 (e [M\u00a0+\u00a0H]+-18 C70 diether\/diester), m\/z 1,060 (f [M\u00a0+\u00a0H]+-18 C68 diether\/diester), m\/z 1,102 (g [M\u00a0+\u00a0H]+-18 C70 monoether\/triester), m\/z 1,074 (h [M\u00a0+\u00a0H]+-18 C68 monoether\/triester), m\/z 1,116 (i [M\u00a0+\u00a0H]+-18 C70 tetraester), m\/z 1,088 (j [M\u00a0+\u00a0H]+-18 C68 tetraester). Filled and blocked peaks indicate membrane-spanning lipids based on C32\/C32 and on C32\/C30 branched alkyl chains, respectively. Note that some mass chromatograms are shown twice as they reveal the presence of different componentsFig.\u00a06Partial APCI mass spectra (corrected for background) of (a) the C70 tetraether 11a, (b) the C70 triether\/monoester 11b or 11c, (c) the C70 diether\/diester 11d, (d) the C70 ether\/triester 11e, (e) the C70 diester 11f, (f) the C70 diether\/diester 13d\nThe first larger peak in the base peak chromatogram at a retention time of 33.0\u00a0min (labelled 11b or 11c in Fig.\u00a05c) shows an APCI mass spectrum (Fig.\u00a06b) in which all ions are shifted by 14\u00a0Da to higher m\/z values relative to those in the mass spectrum of GDGT 11a (Fig.\u00a06a). In addition, the [M\u00a0+\u00a0H]+-18 peak is substantially increased relative to the [M\u00a0+\u00a0H]+ peak and an [M\u00a0+\u00a0H]+-74 peak is also observed. Since this peak disappears when the fraction is subjected to alkaline hydrolysis, this component is likely to contain an ester moiety. This information, together with its molecular mass, indicates that this component is structurally related to GDGT 11a but with one of the ether bonds replaced by an ester bond: a glycerol dialkyl glycerol triether\/monoester with two C32 dimethyl alkyl chains (11b), resulting in a molecular mass of 1091 (C70H138O7). The presence of the ester moiety likely results in the enhanced [M\u00a0+\u00a0H]+-18 peak in its APCI mass spectrum. Mass chromatography of m\/z 1,074 (Fig.\u00a05c), the abundant [M\u00a0+\u00a0H]+-18 peak, reveals that there is an additional, much later eluting isomer with an identical mass spectrum. This could relate to the position of the ester moiety in this type of components: with the parallel configuration of the glycerol groups, as indicated in 11, there are two possible isomers (11b and 11c). However, it is at present unclear why these positional isomers would differ to a large degree in polarity (i.e. the second isomer elutes much later and is, thus, much more polar than the first one). At the flank of the peak of the triether\/monoester a component elutes with a mass spectrum similar to that of 11b but with all the major ions shifted by 28\u00a0Da to lower m\/z values. This component is the triether\/monoester with C32 and C30 dimethyl alkyl chains (12b). Mass chromatography of m\/z 1,046 (Fig.\u00a05d), the abundant [M\u00a0+\u00a0H]+-18 peak of this component, reveals a second, much later eluting isomer for this component as well.\nThe most abundant peak in the base peak ion chromatogram at a retention time of 37.0\u00a0min (Fig.\u00a05a) shows a mass spectrum similar to that of the triether\/monoester 11b but in which again all major ions are shifted by 14\u00a0Da to higher m\/z values (cf. Fig.\u00a06b and c). This component likely represents a glycerol dialkyl glycerol diether\/diester with two C32 dimethyl alkyl chains (11d), resulting in a molecular mass of m\/z 1,105 (C70H136O8) and an [M\u00a0+\u00a0H]+-18 of m\/z 1,088. This component is composed of two esterified 15,16-dimethyl-30-glyceryloxytriacontanoic acid (5) units. Moreover, mass chromatography of m\/z 1,060 (Fig.\u00a05f) reveals the presence of a diester\/diether with C32 and C30 building blocks (12d). Both the mass chromatograms of m\/z 1,088 (Fig.\u00a05e) and m\/z\u00a01,060 (Fig.\u00a05f) reveal the presence of a much later eluting isomer with similar mass spectra. This latter cluster of peaks contains co-eluting components with a mass spectrum with peaks at m\/z 1,120, 1,106, 1,046 and 1,028, presumably the corresponding glycerol dialkyl glycerol monoether\/triester with two C32 dimethyl alkyl chains (11e). Mass chromatograms of m\/z 1,074 (Fig.\u00a05h) and m\/z 1,102 (Fig.\u00a05g) show the presence of a monoether\/triester with two C32 dimethyl alkyl moieties (11e) and C32 and C30 building blocks (12e) and much later eluting isomers for both components. These isomers possess identical mass spectra (e.g. Fig.\u00a06d). Finally, small amounts of tetraesters with two C32 dimethyl alkyl moieties (11f) and a C32 and a C30 dimethyl alkyl moiety were detected by mass chromatography of m\/z 1,116 (Fig.\u00a05i) and m\/z 1,088 (Fig.\u00a05j), respectively. A typical mass spectrum is shown in Fig.\u00a06e. In these traces three distinct isomers were observed.\nThe distribution of membrane-spanning lipids in T. maritima is not depending on the growth temperature; cells grown at 80 and 55\u00b0C showed almost the same distribution.\nDistribution of membrane-spanning core lipids in T. lettingae\nThe distribution of the membrane-spanning lipids in T. lettingae was quite different (Fig.\u00a07) from that observed in T. maritima: it was dominated by the diether\/diesters with the components with two C32 building blocks (11d; m\/z 1,088; Fig.\u00a07b) and those with a C32 and a C30 dimethyl alkyl chain (12d; m\/z 1,060; Fig.\u00a07c) in approximately equal abundance. Diether\/diesters with two C30 building blocks are present in relatively small amounts as revealed by the m\/z 1,032 mass chromatogram (Fig.\u00a07d). This is consistent with the substantially higher abundance of the C30 diabolic acid (2) relative to the C32 diabolic acid (1) and the C33 glycerol monoether 6 relative to the C35 glycerol monoether 5 in the TLF-2 fraction of T. lettingae when compared to T. maritima (Table\u00a02). The relatively high abundance of lipids with a C32 and a C30 dimethyl and, to a lesser extent, with two C30 dimethyl alkyl chains is also observed for the monoether\/triesters (Fig.\u00a07e\u2013g) and tetraesters (Figs.\u00a07h\u2013j). Tetraethers (e.g. 11a), which were present in small quantities in T. maritima, were not encountered in T. lettingae.\nFig.\u00a07HPLC\/APCI\u2013MS base peak chromatogram (a) and specific mass chromatograms (b\u2013j) of polar lipids of Thermotoga lettingae. The mass chromatograms, indicative for specific components, are m\/z 1,088 (b [M\u00a0+\u00a0H]+-18 C70 diether\/diester), m\/z 1,060 (c [M\u00a0+\u00a0H]+-18 C68 diether\/diester), m\/z 1,032 (d [M\u00a0+\u00a0H]+-18 C66 diether\/diester), m\/z 1,102 (e [M\u00a0+\u00a0H]+-18 C70 monoether\/triester), m\/z 1,074 (f [M\u00a0+\u00a0H]+-18 C68 monoether\/triester), m\/z 1,046 (g [M\u00a0+\u00a0H]+-18 C66 monoether\/triester), m\/z 1,116 (h [M\u00a0+\u00a0H]+-18 C70 tetraester), m\/z 1,088 (i [M\u00a0+\u00a0H]+-18 C68 tetraester) and m\/z 1,060 (j [M\u00a0+\u00a0H]+-18 C66 tetraester). Filled, blocked and grey peaks indicate membrane-spanning lipids based on C32\/C32, C32\/C30 and C30\/C30 branched alkyl chains, respectively. Note that some mass chromatograms are shown twice as they reveal the presence of different components\nAn additional cluster of early eluting peaks not observed in the HPLC\/MS analysis of the extract of T. maritima is present in the base peak chromatogram of the polar fraction of T. lettingae (Fig.\u00a08a). The mass spectra of the peaks comprising this cluster are characterized by one dominant fragment ion which is 2\u00a0Da higher than that of the [M\u00a0+\u00a0H]+-18 peak of the diether\/diesters (cf. Fig.\u00a06f, c). Mass chromatography of m\/z 1,090, 1,062 and 1,034 (Fig.\u00a08b\u2013d) reveals that this cluster of peaks is build up in a similar way as the clusters reflecting the diether\/diesters, ether\/triesters and tetraesters (cf. Fig.\u00a07). These mass chromatograms also reveal that these compounds are accompanied by a later eluting isomer which possess similar mass spectra. These components likely represent glycerol trialkyl glycerol diether\/diesters (13d) comprising one long-chain C32 or a C30 dimethyl alkyl chain and two shorter linear (predominantly C16) alkyl chains. It is well known from archaeal GDGT analyses that so-called \u201copen\u201d or acyclic (in comparison to the macrocyclic components described so far) GDGTs elute substantially earlier under these LC conditions (and are, thus, less polar) and possess APCI mass spectra that do not show an [M\u00a0+\u00a0H]+-74 ion resulting from the loss of a glycerol moiety (Hopmans et al. 2000). Instead, they easily loose one of the shorter alkyl chains upon APCI. Indeed, when the polar fraction of T. lettingae was analysed using an extended mass range (m\/z 100\u20131,400), an abundant ion at m\/z 792 was observed, which is likely formed by loss of a C16 moiety. Mass chromatography of ions 2\u00a0Da higher than used to detect the monoether\/triesters and tetraesters reveals the presence of acyclic monoether\/triesters (Fig.\u00a08e\u2013g) and tetraesters (Fig.\u00a08h\u2013j).\nFig.\u00a08HPLC\/APCI\u2013MS base peak chromatogram (a) and specific mass chromatograms (b\u2013j) of polar lipids of Thermotoga lettingae. The mass chromatograms, indicative for specific components, are m\/z 1,090 (b [M\u00a0+\u00a0H]+-18 C70 \u201copen\u201d diether\/diester), m\/z 1,062 (c [M\u00a0+\u00a0H]+-18 C68 \u201copen\u201d diether\/diester), m\/z 1,034 (d [M\u00a0+\u00a0H]+-18 C66 \u201copen\u201d diether\/diester), m\/z 1,104 (e [M\u00a0+\u00a0H]+-18 C70 \u201copen\u201d monoether\/triester), m\/z 1,076 (f [M\u00a0+\u00a0H]+-18 C68 \u201copen\u201d monoether\/triester), m\/z 1,048 (g [M\u00a0+\u00a0H]+-18 C66 monoether\/triester), m\/z 1,118 (h [M\u00a0+\u00a0H]+-18 C70 \u201copen\u201d tetraester), m\/z 1,090 (i [M\u00a0+\u00a0H]+-18 C68 \u201copen\u201d tetraester) and m\/z 1,062 (j [M\u00a0+\u00a0H]+-18 C66 \u201copen\u201d tetraester) for the polar fraction of Thermotoga lettingae. Black, blocked and grey peaks indicate membrane-spanning lipids based on C32\/C32, C32\/C30 and C30\/C30 branched alkyl chains, respectively. Note that some mass chromatograms are shown twice as they reveal the presence of different components\nDistribution of membrane-spanning core lipids in F. pennivorans\nMembrane-spanning lipids were also detected in F. pennivorans (data not shown). In this case the distribution was relatively simple: only tetraesters with predominantly two C32 dimethyl alkyl chains (11f) were detected in a distribution similar to those observed in T. maritima (Fig.\u00a05i) and T. lettingae (Fig.\u00a07h), i.e. dominated by three isomers. Smaller amounts of tetraesters with a C32 and a C34 dimethyl alkyl chain were also detected. In addition, the TLF-1 also contained a glycerol esterified with two 15,16-dimethyltriacontanedioic acid moieties or 15,16-dimethyltriacontanedioic acid and 15,16-dimethyldotriacontanedioic acid.\nDiscussion\nOur results indicate that the diabolic acids and their ether derivatives previously identified in members of the Thermotogales (DeRosa et al. 1988; Windberger et al. 1989; Jeanthon et al. 1995; Huber et al. 1986) are synthesized to produce membrane-spanning lipids and probably represent biosynthetic intermediates. The relative concentration of the diabolic acids and their ether derivatives is substantially higher in the extract obtained after alkaline hydrolysis of the extracted cell residue (TLF-2) (Table\u00a02). The HPLC\/APCI\u2013MS results clearly revealed the presence and, in some cases, the structural complexity of the membrane-spanning core lipids in species of the order Thermotogales for the first time. Previous investigators (Clarke et al. 1980) have suggested that diabolic acids may act as linkers between two glycerol moieties in polar membrane lipids of Butyrivibrio spp. It is generally thought that the production of membrane-spanning lipids in prokaryotes is an adaptation to high growth temperature, although Butyrivibrio is a mesophile. The ubiquitous presence of diabolic acid-based membrane-spanning lipids in members of the Thermotogales, which are all (hyper)thermophiles, is in line with this general idea (Table\u00a02). However, the distribution of membrane-spanning lipids in T. maritima is not directly affected by the temperature of growth: at both 80 and 55\u00b0C diabolic acid-based membrane-spanning lipids were produced.\nDespite the overall similarity in the lipid composition, there is a clear dichotomy: all members of the genus Thermotoga produce ether lipids (i.e. 3\u20136, 9\u201311e, 12a\u201312e, 13a\u201313e) in addition to ester lipids, whereas ether lipids are absent in the genera Thermosipho and Fervidobacterium (Table\u00a02). For example, the HPLC\/APCI\u2013MS results showed only the presence of tetraesters in F. pennivorans, whereas the Thermotoga species investigated showed a wide variety of mixed ester\/ether membrane spanning core lipids (Figs.\u00a05, 7, 8). This suggests that only Thermotoga species possess enzymes to produce ether linkages. This classification based on lipid biochemistry is in good agreement with the phylogenetic classification based on the sequence of the 16S rRNA gene (Fig.\u00a09), which reveals that the genera Thermosipho and Fervidobacterium are quite distinct from the genus Thermotoga, although the phylogenetic diversity within the Thermotoga cluster is also substantial.\nFig.\u00a09Phylogenetic tree based on the 16S rRNA gene outlining the relationship between molecular phylogeny and lipid composition for the studied species of the Thermotogales. The neighbor-joining tree was reconstructed from distance matrices and Escherichia coli served as outgroup. The bar indicates an evolutionary distance of 0.10. Since no 16S rRNA gene sequence is available for F. penniforans, two other Fervidobacterium species were used in the construction of the tree. Gene Bank accession numbers are given in parentheses\nThe composition of the low-molecular-weight lipids (i.e. fatty acids, glycerol monoethers 3 and 4), higher molecular-weight lipids (1\u20132, 5\u20136) and the membrane-spanning lipids suggest a biosynthetic relationship between these different lipid classes although the relatively high concentrations of lipids 1\u20136 also suggest a direct functional role. It has been demonstrated that diabolic acids are produced from condensation reactions of fatty acids at the \u03c9-1 positions (Fitz and Arigoni 1992). These authors showed by the use of [16-2H3]palmitic acid and [14-2H2]palmitic acid that unsaturated fatty acids are involved in this condensation reaction and that it may be mediated by a free radical reaction. In good agreement with the proposed biosynthetic pathway for diabolic acids is that in all species investigated in our study the C16:0 fatty acid is the major fatty acid and 15,16-dimethyltriacontanedioic acid (1), composed of two condensed C16:0 fatty acid moieties, is the major diabolic acid (Table\u00a02). In cases where the C34 and C30 (2) diabolic acids are higher in concentration (e.g. F. pennivorans, and T. lettingae and Thermosipho sp., respectively) also a concomitant increase of the required monomer, i.e. the C18:0 and C14:0 fatty acids, respectively, is observed (Table\u00a02). This is also seen in the presumed biosynthetic end-products, i.e. the membrane-spanning core lipids. For example, T. lettingae contains relatively more of these lipids based on C30 (2) diabolic acids, whereas in F. pennivorans the C34 diabolic acid is a more important building block. This is consistent with the published observation that during growth of T. maritima in batch culture the concentration of the C16:0 fatty acid decreased with a concomitant increase of tentatively identified diabolic acid (Zhang et al. 2002). This was interpreted to reflect the result of synthesis of diabolic acid using the C16:0 fatty acid. The same observations can be made for the ether lipids only encountered in the Thermotoga species. The presence of mono alkyl glycerol ethers in all Thermotoga species (Table\u00a02) and mixed glycerol ether\/esters in T. lettingae (2nd batch) (Fig.\u00a04a) suggests that formation of the ether bond may also occur before the condensation reaction, resulting in the synthesis of membrane-spanning core lipids, takes place. In archaea, GDGTs are also thought to be produced from (partial) condensation of two glycerol diethers (Nishihara et al. 1989; Kon et al. 2002; Koga et al. 1993).\nGenerally, the distribution of the membrane-spanning lipids is quite complex in the species from the Thermotogales. It is noteworthy that several isomers with a quite different polarity were detected in the HPLC traces. The structural composition of the diacyl glycerols and the mixed glycerol ester\/ethers in T. lettingae perhaps provides a clue to the observed complex composition of the membrane-spanning core lipids. Our analyses revealed that the ester moiety in these lipids can be at both at position 2 and 3 of the glycerol unit when the ether or ester moiety is at position 1 (Fig.\u00a04a). It is quite likely (although this remains to be proven) that this also holds for membrane-spanning core lipids. This introduces a wide variety of structural isomers, which may explain why more than one peak was detected in the various HPLC mass chromatograms (Figs.\u00a05, 6 and 8).\nOur data show that some members of the Thermotogales (i.e. the Thermotoga genus) produce membrane-spanning lipids which are comprised of mixed glycerol ether\/ester derivatives, whereas all examined species produce membrane-spanning diabolic acids. Mixed glycerol ether\/ester lipids have been increasingly recognized in the Bacterial Kingdom, e.g. in Planctomycetes capable of anaerobic ammonium oxidation (Sinninghe Damst\u00e9 et al. 2002; Sinninghe Damst\u00e9 et al. 2005), in deep-branching thermophilic bacteria such as Aquifex (Huber et al. 1992) and in two mesophilic sulfate-reducing bacteria (R\u00fctters et al. 2001). The tetraethers 11a and 12a, which occur in low amounts in T. maritima, are, to the best of our knowledge, the first example of this kind of lipid in bacteria. Non-isoprenoidal GDGTs have been detected before in peats (Sinninghe Damst\u00e9 et al. 2000) but their biological origin is as yet unknown. Membrane spanning lipids are quite common in the Kingdom Archaea (Koga et al. 1993) but have also been identified in a very limited number of bacteria, e.g. in Butyrivibrio spp. (Clarke et al. 1980), and in Thermoanaerobacter species (Jung et al. 1994). It is tempting to associate the peculiar membrane biochemistry of the order Thermotogales to its position in the phylogenetic tree of life, i.e. close to the root of the tree (Woese et al. 1990). The unusually numerous archaeal-like genes (almost 25%) found in the Thermotoga maritima genome (Nelson et al. 1999) reflect that these are deep-branching bacteria. If species from the order Thermotogales occurred early during the evolution of life on Earth, as suggested by its position in the phylogenetic tree of life, our results indicate that the ability to produce both ether and ester glycerol membrane lipids developed relatively early during evolution. All Archaea produce ether membrane lipids, whereas Bacteria and Eukaryotes typically produce glycerol esters. Some members of the Thermotogales, thus, represent a hybrid in this way.","keyphrases":["diabolic acid","thermotoga","thermosipho","fervidobacterium","ether lipids","ester lipids"],"prmu":["P","P","P","P","P","P"]}
{"id":"Exp_Brain_Res-3-1-2039806","title":"Judging surface slant for placing objects: a role for motion parallax\n","text":"People have a variety of sources of information (cues) about surface slant at their disposal. We used a simple placing task to evaluate the relative importance of three such cues (motion parallax, binocular disparity and texture) within the space in which people normally manipulate objects. To do so, we projected a stimulus onto a rotatable screen. This allowed us to manipulate texture cues independently of binocular disparity and motion parallax. We asked people to stand in front of the screen and place a cylinder on the screen. We analysed the cylinder\u2019s orientation just before contact. Participants mainly relied on binocular cues (weight between 50 and 90%), in accordance with binocular cues being known to be reliable when the stimulus surface is nearby and almost frontal. Texture cues contributed between 2 and 18% to the estimated slant. Motion parallax was given a weight between 1 and 9%, despite the fact that it only provided information when the head began to move, which was just before the arm did. Thus motion parallax is used to judge surface slant, even when one is under the impression of standing still.\nIntroduction\nIt is often important to accurately judge the slant of surfaces in our nearby environment. Whether placing our foot on the ground when we walk or climb stairs, or our fingers on an object when we grasp it and place it elsewhere, the interaction always involves making contact with surfaces. In order to interact successfully, we need to know the orientation of these surfaces. We have many ways to judge a surface\u2019s orientation, including ones based on texture gradients, binocular disparity and motion parallax.\nOne important cue that contributes to most people\u2019s slant perception is binocular disparity (see Howard and Rogers 1995 for an extensive review of the literature on binocular vision). The small differences between the images in the two eyes suffice to obtain information about the slant in depth. From the literature on grasping it could be inferred that binocular cues normally dominate our actions. For example, Servos and Goodale (1994) claim that binocular vision is the principal source of information for reaching and grasping movements. However, binocular information does not guarantee correct grasping (Hibbard and Bradshaw 2003), so there is reason to expect other cues to also play a role in guiding our actions.\nA second cue that contributes to slant perception is the deformation of any surface texture and of shapes\u2019 outlines as a result of perspective. We will refer to the combined information from all such sources as the texture cue. This is the cue that allows us to have a powerful and striking impression of surface slant from a flat image, as exemplified in Fig.\u00a01. It is well known that surface texture provides valuable information on slant perception (Gibson 1950; Stevens 1981; Buckley et al. 1996; Landy and Graham 2004) and motor control (Knill 1998a; Watt and Bradshaw 2003).\nFig.\u00a01The deformation of the regular texture of a chess board provides a profound impression that the surface is slanted relative to the plane of the 2D picture\nRetrieving the slant of a surface from texture cues is based on the assumption that the distribution of texture elements over the surface is more or less even and that shapes are more or less symmetrical, see Rosenholtz and Malik (1997). This assumption holds for a wide variety of natural and artificial objects.\nThe third cue that we will consider is motion parallax (Rogers and Collett 1989; Rogers and Graham 1979, 1982; Ono and Steinbach 1990; Gillam and Rogers 1991; Ujike and Ono 2001). For a review of the widespread use of motion parallax in the animal kingdom, see Kral (2003). Movement of the head relative to a surface generates changes in the surface\u2019s retinal image over time. These changes depend on the motion of the head relative to the items in the surrounding, and on the items\u2019 relative distances. The latter dependency can be used to obtain information about depth and slant. Watt and Bradshaw (2003) have shown that motion parallax can guide human movements when binocular cues are not available.\nWhen more than one cue is available the cues are combined. It is generally accepted that the estimated slant is a weighted average of the slants indicated by various cues, and that the weight of a cue is related to its accuracy, although some details of the mechanism are still under debate (Landy et\u00a0al. 1995; Hillis et\u00a0al. 2002; Hogervorst and Brenner 2004; Rosas et\u00a0al. 2005; Muller et\u00a0al. 2007). The relative contribution of binocular cues and texture cues depends on the distance (Hillis et al. 2004) and surface orientation (Knill 1998b; Buckley and Frisby 1993; Ryan and Gillam 1994), because binocular vision is better nearby and texture gradients change least rapidly with the angle of slant for near-frontal surfaces (for purely geometrical reasons). Knill (2005) measured how binocular cues and texture cues to surface orientation are combined to guide motor behaviour. Cue weights were found to be dependent on surface slant and also on the task: more weight was given to binocular cues for controlling hand movements than for making perceptual judgements. Knill used cue-consistent and cue-conflict stimuli in a virtual reality environment.\nWe wanted to find out whether motion parallax contributes to judgement of slant in the presence of other cues (such as binocular and texture cues) under more or less natural conditions. To do so, we used a setup in which the physical slant of a surface could be manipulated independently of the slant indicated by a pronounced texture. Thus a conflict between the texture cue and all the other cues was created by violating the assumption that the distribution of texture elements is homogeneous. The judged surface slant was determined by asking participants to swiftly place a flat cylindrical probe on the slanted surface (as in Knill 2005). Our main interest was in the extent to which head movements and the resulting motion parallax contribute to the perceived surface slant. So, conditions in which the head could move freely were compared with one in which a head restraint was used. We compared conditions with and without binocular vision in order to be able to evaluate the importance of motion parallax in relation to this cue.\nMethods\nParticipants\nFive people, four of whom were male, participated in the experiment. All participants gave their informed consent prior to their inclusion in the study. The experiment was part of an ongoing research program that was approved by the local ethics committee. All participants were right-handed, had normal or corrected-to-normal vision and good binocular vision (stereo acuity <40\u00a0arcseconds).\nExperimental setup\nParticipants were standing upright in front of a large rotatable screen. A sketch of the setup is given in Fig.\u00a02. The screen was a plexiglass plate covered with projection foil. Images were projected from below by an Hitachi cp-x325 LCD projector with a resolution of 1024\u00a0\u00d7\u00a0768\u00a0pixels. The screen and the projector could be rotated as a whole. Participants wore computer-controlled PLATO shutter-glasses, with which we could alternate between monocular and binocular vision, see Milgram (1987). A chessboard pattern was projected onto the screen (see Fig.\u00a03). Slants were defined relative to the gravity-defined horizontal. A grey ring, 104\u00a0mm in diameter, indicated the target position for the probe. Two positions of the ring, one near the participant (the target\u2019s centre 100\u00a0mm below the stimulus centre) and one further away (100\u00a0mm above the stimulus centre) were displayed in random order to make sure that participants did not simply repeat the previous movement. Figure\u00a03 shows the stimuli with 0\u00b0 and 10\u00b0 texture slant relative to the surface\u2019s physical slant. The geometry of the stimulus was calculated by projecting the texture-defined slant on the physically rotated surface. The projection is calculated from the point of the observer\u2019s eyes. The 0\u00b0 stimulus was a 40\u00a0cm square, with 40\u00a0cm corresponding to a visual angle of about 27\u00b0. A dark grey rim was drawn around the stimulus to mask any real edges that could become visible due to reflections within the set-up. To avoid illuminating objects around the set-up, the luminance of the image at the position of the eyes was limited to 0.4\u00a0Cd\u00a0m\u22122. This was achieved by placing filters in front of the projector.\nFig.\u00a02Participants were standing upright, facing the screen. They moved a probe from a starting position 50\u00a0cm to the right of the surface midline to a target position on the surface (indicated by the grey ring). Participants wore PLATO glasses with which we could switch between no, monocular and binocular vision. During the experiment, only the slanted surface was visibleFig.\u00a03Examples of consistent (left) and conflict (right) images. The left panel shows the 0\u00b0 texture slant on a 0\u00b0 surface slant as seen from above. The right panel shows the 10\u00b0 texture slant on the 0\u00b0 surface slant. The deformation of the stimulus is consistent with the actual viewing geometry but for clarity the image is presented as seen from above. The target (grey ring) indicates the position at which participants have to place the probe. The targets are shown at the \u2018near\u2019 position. The dark grey rim\u2019s shape is in accordance with the texture cue\nThe probe was a flat cylinder (diameter\u00a0=\u00a0104\u00a0mm, height\u00a0=\u00a022\u00a0mm, mass\u00a0=\u00a00.2\u00a0kg). Movements of the probe were registered by an optotrak\u00a03020 system (northern digital inc., Waterloo, ON, Canada). This system tracks the position of active infrared markers with an accuracy better than 0.5\u00a0mm. The 3D-positions of five markers on the probe were tracked at a rate of 200\u00a0Hz. The position, orientation and velocity of the probe were calculated from these data.\nProcedure\nExperiments were performed in a completely dark room. The dark environment and the low intensity of the stimuli ensured that there was no visible external reference frame. Participants were instructed to place the probe at the indicated target position on the surface. They were to start moving as soon as the target was visible. Stimuli were shown for 2.5\u00a0s. All movements were completed well within this interval.\nIn order to avoid dark adaptation a bright lamp was turned on for 5\u00a0s immediately after each trial. During this period participants placed the probe at the starting position, 50\u00a0cm to the right of the midline of the screen. Then the light was turned off for about 5\u00a0s, during which time the experimenter adjusted the orientation of the screen in preparation for the next trial.\nFigure\u00a04 shows the six combinations of surface slant and texture slant that were used in the experiment. The different combinations could be viewed monocularly or binocularly. The four consistent combinations (on the diagonal in Fig.\u00a04) were used as a standard. The physical surface slant (see Fig.\u00a02) was \u221210\u00b0, 0\u00b0, 10\u00b0 or 20\u00b0, and the image on the surface was as shown on the left in Fig.\u00a03. The two conflict combinations either involved presenting the image shown on the right in Fig.\u00a03 on a horizontal surface (surface slant 0\u00b0; texture slant 10\u00b0) or presenting a similarly transformed image (slanted in the opposite direction) on a surface with 10\u00b0 slant. All six stimuli were presented under various conditions. In total there were four experimental conditions, each consisting of 192\u00a0trials, divided into 3 blocks of 64 trials. Within every condition 75% of the trials were without conflict and 25% involved a conflict between texture and the other cues present in that condition.\nFig.\u00a04The six combinations of physical surface slant (continuous lines) and slant suggested by texture (dashed lines) that were used in the experiment. In four cases there was no conflict between the cues (solid disks). In two cases there was a conflict (open disks). The slants have been exaggerated for clarity\nWe used four conditions in which different combinations of the available cues were presented. The choice of conditions will become clear when we describe the data analysis. We chose three conditions with which we could calculate the five parameters of our model, and one condition to test one of our assumptions.\nIn the \u2018binocular\u2019 condition viewing was binocular and head-free in both conflict and consistent trials. In this condition all cues to slant perception were available. In the \u2018monocular\u2019 condition the conflict and consistent trials were both presented monocularly and head-free. Stimuli were viewed with the left or right eye in random order. No binocular cues were available, but all other cues were present. In the \u2018biteboard\u2019 condition the head was fixed in combination with monocular viewing. The biteboards were made individually with an impression of the participant\u2019s teeth. The biteboard severely limits head movements, removing information from motion parallax.\nIn the consistent trials of the \u2018mixed\u2019 condition the screen was viewed monocularly (75% of all trials), but in the conflict trials (25%) it was viewed binocularly. This condition was included to evaluate whether participants adapt their strategy at the level of a session rather than per trial. In the \u2018binocular\u2019 condition binocular information was always reliable, so participants could have learnt to use this cue. In the \u2018mixed\u2019 condition, in contrast, binocular cues were absent in the majority of trials, so participants could have learnt to use texture or motion parallax. Note that the 25% binocular, conflict trials in the \u2018mixed\u2019 condition are identical to the conflict trials in the \u2018binocular condition\u2019, whereas the 75% consistent trials are identical to the consistent trials in the \u2018monocular\u2019 condition. Thus the \u2018mixed\u2019 condition serves as a control condition to test whether the weight given to the cues stays about the same under changing viewing conditions on other trials.\nAnalysis\nDuring some moments of some trials the participant\u2019s fingers or hand occluded one or more of the five markers. The position of each marker relative to the centre of the probe is known, so the position and orientation of the probe can be calculated from any set of at least three markers. Frames in which fewer than three marker positions were known were not analysed. Figure\u00a05 shows a schematic side-view of the path followed by the probe. The end of the movement was defined as the first sample at which the centre of the probe was less than 2\u00a0mm from the screen. The probe orientation was averaged over all samples at which the centre of the probe was between 100 and 20\u00a0mm from its position at the end of the movement (the grey line segments in Fig.\u00a05). The last 20\u00a0mm of the path were excluded to avoid considering moments at which the edge of the probe could be in contact with the real surface of the projection screen.\nFig.\u00a05The upper left panel gives a schematic side view of the probe\u2019s path (curved thin line) towards the slanted surface (straight thick line). The position and orientation of the probe were measured by the optotrak system. The upper right panel shows a side view of a few paths towards the \u2018far\u2019 target position. The average probe orientation is determined during the last 100 to 20\u00a0mm before the end of the movement (indicated schematically by the dark-grey line segments). The lower panels show examples of the probe orientation with time intervals of 25\u00a0ms\nTo understand our method for determining the cue weights, consider the two extreme hypothetical outcomes shown in Fig.\u00a06. At the one extreme, if information from the texture cue is not used at all, probe orientations will always follow the slant of the physical surface (left panel). At the other extreme, if the observer only relies on texture cues the orientation of the probe will follow the texture-defined slant (right panel). In the latter case the line connecting the cue-conflict conditions has approximately the opposite slope of that for consistent conditions. Note that the probe orientations in the consistent trials may differ from the physical surface slant (grey line). Some flattening may arise because the hand may still be rotating towards the surface slant during the last 100\u00a0mm of its trajectory. Participants may also rely to some extent on previous slopes that they encountered during the experiment. We will model these effects as a prior for a single surface slant for all conditions.\nFig.\u00a06A schematic depiction of possible results for two extreme cases: probe orientation is not affected by texture at all (left panel) or only depends on the texture slant (right panel)\nWe assume that the weights that participants give to the different cues are the same in all conditions. This would for instance be so if observers base the weights on the reliability, as in optimal cue combination (Landy et\u00a0al. 1995). We can therefore model the estimated slant (S) as a weighted average of the slant estimated from each of the available cues (si), which are all assumed to give veridical estimates except for the prior for a fixed slant (as mentioned in the previous paragraph):where wi is the weight (in arbitrary units) given to each available cue, with i\u00a0\u2208\u00a0{b, m, t, r, p} indicating binocular vision, motion parallax, texture, a rest category containing any other valid cues, and the prior. Note that we predict that the cue weights (wi) of all available cues will be the same in all conditions, although the relative weight given to a cue (wi\/\u2211wi) will differ between conditions because it depends on the cues that are available in that condition.\nThe slant of the prior is a constant. Its value is likely to be near the mean of the slants in all previous conditions, but this is not essential for our analysis: \nIf s denotes the simulated slant, then the slants indicated by all other available cues are: except for the conflict trials, for which the texture differs 10\u00b0 from all other available cues (see Fig.\u00a04):  Combining these equations gives: and \nThe sums in Eqs.\u00a05 and 6 are only over cues that were available in each condition. The slopes (\u03b2) of the regression lines in Fig.\u00a06 are given by the first derivatives of the estimated slant:  Combining Eqs.\u00a07 and 8 yields: and \nEquations\u00a09 and 10 apply to all experimental conditions as modified for cue availability, except for the \u2018mixed\u2019 condition. In the \u2018binocular\u2019 condition all cues yield information about the surface\u2019s slant: \nSimilar equations can be written for the other conditions. Binocular information is not available in the \u2018monocular\u2019 condition, so wB does not occur in the equation: \nSimilarly, motion parallax ceases to contribute to the estimated slant in the \u2018biteboard\u2019 condition, giving: \nSince the weights are in arbitrary units, we are free to define them in such a way that the sum of all weights is one: \nFirst we determine the weight of the prior relative to that of texture (wP\/wT) for each condition, using Eq.\u00a010. Then, the weighted average of these ratios is calculated for each participant. We found no clear evidence that the assumption that wP\/wT is the same across conditions was not justified. Next, determining the ratios between the slopes in the \u2018binocular\u2019, \u2018monocular\u2019 and \u2018biteboard\u2019 conditions from our data allows us to use Eqs.\u00a011 to 14 to determine the values of the weights (wB, wT, wM and wR).\nThe data of the \u2018mixed\u2019 condition was analysed by comparing the slopes (\u03b2conflict and \u03b2consistent) with the matching slopes in the \u2018binocular\u2019 and the \u2018monocular\u2019 conditions. Data for \u2018near\u2019 and \u2018far\u2019 target positions were pooled before calculating the slopes. The weights of the cues were calculated for individual participants. In addition, we also pooled the data of all participants before calculating the slopes, which yields the weights for \u2018All\u2019 participants.\nResults\nWe determined average probe orientations for each participant and condition. The slopes of probe orientation as a function of surface orientation for conflict and consistent conditions enable us to calculate the cue weights, as explained in the \u2018methods\u2019 section.\nConditions\nThe upper panel of Fig.\u00a07 shows the probe orientations in the binocular condition. Conflict (open symbols) and consistent (closed symbols) probe orientations are almost the same. The difference between the slopes is small but significant (P\u00a0<\u00a00.05). The second panel of Fig.\u00a07 shows the probe orientations in the monocular condition. The slopes clearly differ between the conflict and the consistent trials (P\u00a0<\u00a00.01). In the \u2018biteboard\u2019 condition (third panel of Fig.\u00a07) the conflict trials have almost the opposite slope than the consistent trials. The difference between the slopes is significant (P\u00a0<\u00a00.01). The cue weights were calculated using Eqs.\u00a010 to 14 and the values of the regression slopes.\nFig.\u00a07Pooled data for all participants. Each panel is for one of the four experimental conditions. Average probe orientations are shown for surfaces with (open symbols) and without (closed symbols) conflicts between real slant and texture slant. The error bars show the overall standard deviation. Correct probe orientations for the consistent trials (grey lines), linear regression (black lines) and regression slopes, \u03b2 (numbers in lower right corner) are also shown. Asterisks indicate whether the difference between the regression slopes is significant (*P\u00a0<\u00a00.05; **P\u00a0<\u00a00.01)\nThe \u2018mixed\u2019 condition was included as a control to ascertain that the cue weights do not depend on the viewing conditions on other trials. We compared the monocular consistent trials with the identical trials in the \u2018monocular\u2019 condition, and the binocular conflict trials with the identical trials in the \u2018binocular\u2019 condition. The regression slope for monocular, consistent trials (\u03b2consistent\u00a0=\u00a00.72) is not significantly different (P\u00a0>\u00a00.05) from the same trials in the \u2018monocular\u2019 condition (\u03b2consistent\u00a0=\u00a00.76). The slope of the (binocular) conflict trials in the \u2018mixed\u2019 condition (\u03b2conflict\u00a0=\u00a00.61) is slightly but significantly lower (P\u00a0=\u00a00.04) than for conflict trials in the \u2018binocular\u2019 condition (\u03b2conflict\u00a0=\u00a00.72). Thus the weights may not be completely independent of the conditions. The difference was small enough to accept the calculation of the weights on the basis of the assumption that the condition is irrelevant. Our analysis may however underestimate the weight given to the texture cue.\nCue weights\nThe weight of the binocular cue lies between 50 and 90%, for individual participants (see Fig.\u00a08). The average standard error is 11%. The errors in the cue weights are calculated by the method of propagation of errors based on the errors in the regression slopes. If we determine the slopes across all participants, the binocular weight is 71\u00a0\u00b1\u00a06%. The weight of the texture cue lies between 2 and 18% with an average standard error of 3% for individual participants. The weight given to the texture cue is 8\u00a0\u00b1\u00a01% across all participants. The weight given to motion parallax lies between 1 and 9% for individual participants with an average standard error of 6%. The weight given to motion parallax is 8\u00a0\u00b1\u00a03% across all participants. The weight attributed to the rest category of cues was only 3\u00a0\u00b1\u00a02% across all participants. The prior contributed between 6 and 23% for individual participants with an average standard error of 3%. The weight of the prior varies between 7 and 13% across conditions. Across all participants the weight of the prior is 10\u00a0\u00b1\u00a02%.\nFig.\u00a08The weights given to the binocular, texture, motion parallax, other cues and to the prior, for each participant (horizontal axis). The weights are obtained by substituting the slopes of conflict data and consistent data in Eqs. 10\u00a0to\u00a014. For \u2018All\u2019, first all participant\u2019s data in each condition was pooled and then the weights were determined, which are all significantly different from zero. For individual participants the texture cue was not significantly different from zero in one case, the motion cue in four cases and the rest category in three cases\nHead movements\nHead movements were measured in the monocular viewing condition for three of the participants. Participants move their head considerably when placing the cylinder: EB moved on average 103\u00a0mm, JG 53\u00a0mm and DdG 37\u00a0mm in the lateral direction. Interestingly, the head movement only started just before the arm movement. Shortly before (100\u00a0ms) the onset of arm movement (when the probe was 10\u00a0mm from the starting position), the head had only moved 10\u00a0mm (EB), 4\u00a0mm (JG) or 6\u00a0mm (DdG). Thus the information from motion parallax is mainly picked up during the arm movement. None of the participants were aware of having made head movements.\nDiscussion\nWe used a physically rotatable screen as a surface. The projected stimulus was viewed in a completely dark environment within a space in which objects are normally manipulated. In our analysis systematic deformations that affect a single cue (like depth compression resulting from an erroneous depth estimate) were not considered. Moreover, we assume that the cue weights are the same in all conditions, so that their contributions to the percept only depend on which cues are available. We determined the contributions of binocular disparity, texture cues, motion parallax, a rest category and a prior. Under these conditions and based on these assumptions we conclude that participants mainly relied on binocular information (between 50 and 90%). Texture cues contributed between 2 and 18% to the estimated slant. Motion parallax contributed up to 9%. The prior contributed between 6 and 23%. Residual cues may account for up to 9%.\nComparing conditions with and without head movement revealed that motion parallax plays a role in slant perception. This is evident from the weights (Fig.\u00a08) but also from a comparison of performance in the \u2018monocular\u2019 and \u2018biteboard\u2019 conditions (Fig.\u00a07). It is not unusual to move the whole body, including the head, when making large arm movements. Beside mechanical reasons for doing so we here show that it may also have perceptual advantages.\nWe included in our analysis a rest category of cues that might contain information about slant that was not manipulated. The results suggest that this category indeed includes cues that yield some information about slant. Accommodation, or the rate at which the image becomes blurred with distance from fixation, might provide such information (Mather 1997; Watt et\u00a0al. 2005). However, artefacts of our setup such as the possible visible micro texture (fibres in the projection foil or pixels on the screen) and the angular distribution of the light scattered from the surface could play a role too. Taken together in a rest category such cues contribute only a few percent to the estimated slant.\nThe probe orientations in the consistent trials in Fig.\u00a07 are not equal to the physical surface slant. We incorporated a prior in our model to take into account behaviour that is not related to the instantaneous information, like visual or haptic information from previous trials. The slant indicated by the prior cue is a constant; i.e. it does not depend on the stimulus. Flatter slopes indicate that the prior plays a relatively large role. The weight of the prior is about as large as the weight of the texture cue. One component of the prior could be that the hand orientation is still changing towards its final value at the moment that we sample, which is slightly before contact (Cuijpers et\u00a0al. 2004). Any biases towards a certain orientation of the hand or towards a certain perceived slant will also contribute to the weight of the prior.\nThe purpose of having the \u2018mixed\u2019 viewing condition was to check whether the weights change under different conditions. Ernst et al. (2000) have shown that haptic feedback can make more weight be given to a visual slant cue that is consistent with the feedback. In our study the haptic feedback was always consistent with the physical slant of the surface, so only the texture cue was sometimes unreliable. So, with a conflict between surface slant and texture the haptic feedback may yield a bias towards other cues than texture. Our results show a small difference between the \u2018mixed\u2019 condition and the comparable trials in the \u2018monocular\u2019 and \u2018binocular\u2019 condition. Thus here too the extent to which cues are used does probably depend to some extent on experience in previous trials. This indicates that the estimated slant does not only depend on the accuracy of the presented cues, as is often assumed in theories of optimal cue combination (Landy et\u00a0al. 1995; Hillis et\u00a0al. 2002; Muller et\u00a0al. 2007 ). It is however possible that the difference arises from less use of motion parallax when binocular information was always available, perhaps because participants move less when there is enough information from other sources than motion parallax. We do not know whether this is the case because we did not measure head movements in all conditions. However, these differences are all too small to be taken seriously without further research.\nIn our study binocular disparity is given most weight, which is in accordance with binocular cues being known to be reliable when the stimulus surface is nearby and almost frontal. Because experimental conditions were all in favour of binocular disparity, the role of motion parallax and texture cues is probably smaller here than in natural viewing conditions. Motion parallax and texture cues both contribute to a small but significant extent to slant perception, although marked differences between participants were observed. Motion parallax was available only shortly and began relatively late, as the head began to move only just before onset of the arm movement. From animal studies it is known that a range of animals gain depth information by moving from side to side just before the performance of an action, see Kral (2003). We have shown that humans are able to use motion parallax during an action. It was known that monocular depth information can be used to guide our actions (Marotta et al. 1998; Dijkerman et al. 1999; Watt and Bradshaw 2003). It was not known, however, that motion parallax plays a role under conditions where other cues are dominantly available and without actively moving one\u2019s head before starting the action. We conclude that motion parallax is used as a cue to manipulate objects in our nearby environment, even when one is under the impression of holding one\u2019s head still. Motion parallax should therefore not be ignored in a \u2018static\u2019 task unless the head is really fixated.","keyphrases":["motion parallax","binocular","texture","cue integration","cue conflict"],"prmu":["P","P","P","M","R"]}
{"id":"Arch_Dermatol_Res-4-1-2254657","title":"Manifestation of palmoplantar pustulosis during or after infliximab therapy for plaque-type psoriasis: report on five cases\n","text":"Infliximab is a monoclonal antibody directed against TNF-\u03b1. It has been approved for use in rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, psoriatic arthritis and plaque-type psoriasis. In case reports, positive effects on pustular variants of psoriasis have also been reported. However, paradoxically, manifestation of pustular psoriasis and plaque-type psoriasis has been reported in patients treated with TNF antagonists including infliximab for other indications. Here, we report on 5 patients with chronic plaque-type psoriasis who developed palmoplantar pustulosis during or after discontinuation of infliximab therapy. In two of the five cases, manifestation of palmoplantar pustulosis was not accompanied by worsening of plaque-type psoriasis. Possibly, site-specific factors or a differential contribution of immunological processes modulated by TNF inhibitors to palmoplantar pustulosis and plaque-type psoriasis may have played a role.\nIntroduction\nIncreased expression of TNF-\u03b1 has been identified as an important pathophysiological mechanism in different types of chronic inflammation including psoriasis and psoriatic arthritis. The knowledge about the central role of TNF-\u03b1 in certain diseases has successfully been converted to the therapeutic level; over the last years TNF antagonizing agents such as the anti-TNF directed antibodies infliximab and adalimumab and the fusion protein etanercept have substantially improved the treatment of some of the most common chronic inflammatory conditions such as rheumatoid arthritis (RA). With 80% of the patients reaching at least a 75% reduction of their baseline skin symptoms after 10\u00a0weeks of therapy [14], infliximab is regarded as one of the most potent agents available at present for the treatment of psoriasis vulgaris. Other variants of psoriasis, such as pustular psoriasis have not been formally tested in larger clinical trials. However, in individual reports, positive effects of infliximab on generalized pustular psoriasis (GPP) with dramatic improvements in some cases have been reported [3, 13, 15]. Because this type of pustular psoriasis is believed to correspond to an extreme activation of psoriatic disease mechanisms, the high bioavailability of infliximab and its rapid onset of action following intravenous infusion have been used to explain the surprisingly fast decrease of pustule formation in patients treated with the agent. Paradoxically, on the other hand, manifestation of pustular psoriasis has been reported in patients treated with TNF antagonists including infliximab for other indications.\nPalmoplantar pustulosis (PPP) has long been regarded as a localized variant of pustular psoriasis, although more recent epidemiological and genetic findings argue against this concept [2, 12]. Here, we report on five patients with chronic plaque-type psoriasis who developed PPP during or after discontinuation of infliximab therapy.\nCases and discussion\nRelevant aspects of the five patients with chronic plaque-type psoriasis who developed PPP during or after discontinuation of infliximab therapy are presented in the Table\u00a01 and Fig.\u00a01.\nTable\u00a01Description of casesCase12345SexMaleMaleFemaleFemaleMaleAge3742673041Disease duration (years)17 2830Unknown15Psoriasis typePlaquePlaquePlaquePlaquePlaquePrior pustular psoriasisNoNoNoNoNoPsoriatic arthritisYesNoNoNoYesFamiliy history of psoriasisNegativePositiveNegativeUnknownNegativePrior UV-therapyYesYesYesYesYesPrior systemic therapiesMethotrexateLeflunomideEtanerceptPrednisoloneFumaric acid estersCyclosporineFumaric acid estersFumaric acid estersCyclosporineRetinoidsMethotrexateFumaric acid estersCyclosporineMethotrexateInitial response to infliximabExcellentcPASI 75fPASI 75PASI 75PASI 75Time of manifestation of PPPWeek 38d8\u00a0weeks after end of infliximab treatmentWeek 3dWeek 22dWeek 40dConcomitant worsening of plaque psoriasisaYesYesNoNoYesDevelopment of GPP in addition to PPPYesNoNoNoNoPotential trigger factors of PPP\u00a0Infection prior to PPPYesNoYesNoNo\u00a0Present smokingNoNoYes (36 packyears)UnknownUnknownDiscontinuation of infliximabYesYesYesNoYesSystemic therapy with sufficient control of PV and PPPbAdalimumab 40\u00a0mg e.o.wEtanercept 25\u00a0mg BIW plus methotrexate 7.5\u00a0mg\/week orallyPUVA-therapy of palms and soles(Additional topical therapy)Etanercept 50\u00a0mg BIWSystemic therapy that failed to control PV or PPPEtanercept 50\u00a0mg BIWe plus methotrexate 15\u00a0mg\/week orallyEtanercept 25\u00a0mg BIWPPP Pustulosis palmoplantaris, GPP Generalized pustular psoriasis, PV Psoriasis vulgaris, e.o.w Every other weeka\u00a0Loss of >50% of maximum PASI response or increase of physician\u2019s global assessment (PGA) by \u2265\u00a02b\u00a0All patients received additional topical therapy with glucocorticosteroids and Vitamin D analoguesc\u00a0Improvement rated by PGA with \u201calmost clear\u201d (PGA\u00a0=\u00a01)d\u00a0Week of infliximab treatmente\u00a0BIW\u00a0=\u00a0twice weeklyf\u00a0PASI 75\u00a0=\u00a0Reduction in the psoriasis area and severity index (PASI) by \u226575%Fig.\u00a01Clinical picture of pustulosis palmoplantaris in patient 3 with pustules in different stages of evolution on a sharply delineated erythematous lesion on the left sole (a) and yellowish pustules on the left palm (b). Histological examination showing intraepidermal vesiculopustular dermatitis (c, H.E. stain of a biopsy from the left plantar arch) with intraepidermal accumulation of neutrophils and subcorneal pustule formation (d)\nTo the best of our knowledge, the development of PPP during the treatment of plaque-type psoriasis with infliximab has not yet been reported. The occurrence of pustular skin lesions usually resembling GPP or palmoplantar pustular psoriasis has occasionally been observed in patients treated with infliximab for other indications [1, 6, 11, 16\u201319]. Induction of pustular skin lesions seems not to be limited to infliximab therapy, but has also been described in association with the use of the TNF-antagonists etanercept and adalimumab, including the use in one patient with plaque-psoriasis treated with etanercept [4, 8\u201310, 16]. One patient with seropositive RA developed GPP as well as PPP during treatment with infliximab [11]. This patient later experienced a relapse of PPP when treatment with etanercept was initiated, which also suggests that a class effect of TNF-antagonists may play a role.\nIn two of the three cases in whom an exacerbation of plaque-psoriasis occurred parallel to the manifestation of PPP, typical trigger factors for active psoriasis could be identified such as an infection (case 3) and the abrupt termination of anti-psoriatic treatment (case 2). These two cases are compatible with the existence of common trigger factors for plaque psoriasis and PPP. What are other factors that might contribute to the development of PPP during treatment of psoriasis vulgaris? While the exact etiology of PPP remains to be established, a history of smoking is the most important known risk factor for PPP. However, only one out of the three patients in whom a smoking history had been obtained was a smoker at the time of onset of pustular psoriasis (case 3). Streptococcal infection, a known risk factor for psoriasis vulgaris, has not been established as a risk factor for PPP and probably plays a minor role there. However, in the cases presented here, one patient (case 3) suffered an upper respiratory tract infection a few days before manifestation of PPP, while another patient (case 1) had suffered from a persistent cold 6\u00a0weeks before manifestation of pustules. In the former patient, the close temporal relationship between infectious symptoms and manifestation of PPP may point to a possible contribution of the infection to triggering PPP, and a modulation of the immune response to infliximab appears possible.\nIt is likely that beyond the contribution of known risk factors, other, immunological mechanisms may be involved in the manifestation of PPP under infliximab therapy. Interferon (IFN)-\u03b1 has been suggested as a cytokine mediating the manifestation of psoriasiform lesions in patients treated with TNF-inhibitors as a consequence of crosstalk of TNF-\u03b1 and IFN-\u03b1: TNF-\u03b1 is known to suppress the generation of plasmacytoid dendritic cells that are very potent producers of IFN-\u03b1. Appearance of plasmacytoid dendritic cells (and IFN-\u03b1) in ths skin is considered to be an early and crucial step in the pathogenesis of psoriasis (reviewed in [7]). Thus, in patients treated with TNF-antagonists, the inhibition of TNF-\u03b1 might induce an increase of IFN-\u03b1 in the skin favoring the manifestation of psoriasiform dermatitis. In fact, an increase of IFN-\u03b1 signaling has been shown in biopsy specimens from psoriatic plaques induced by TNF-inhibitors compared with traditional psoriatic plaques [6]. The relevance of IFN-\u03b1 for PPP and TNF-inhibitor induced PPP, however, still needs to be determined. The observation of an improvement of pre-existing psoriasis plaques parallel to the first manifestation of PPP in two of the cases described here supports the concept that immunological mechanisms and\/or local factors are not identical in the pathogenesis of plaque-type psoriasis and PPP. Differences in pathogenesis between plaque psoriasis and PPP are also supported by their different genetic background, with plaque psoriasis, but not PPP being linked to PSORS1, the major susceptibility locus for plaque-type psoriasis located on 6p21 [2]. The localized nature of the pustules on palms and soles occurring in association with infliximab therapy in some patients suggests a contribution of site-specific factors. Possibly, eccrine sweat glands that are numerous in the palmoplantar location are involved in these processes. Changes secondary to binding of infliximab to TNF-\u03b1, that has been shown to be expressed in eccrine sweat glands, have been suggested as a possible mechanism [11].\nWithdrawal or dose reduction of systemic cyclosporine and systemic or topical glucocorticosteroids given for treatment of psoriasis vulgaris have been associated with manifestation of GPP, but not with PPP. To the best of our knowledge, no reports on manifestation of GPP or PPP associated with treatment with methotrexate, another drug frequently used in the therapy of psoriasis, exist. However, in the clinical trials with the CD11a-antagonist efalizumab in the indication plaque psoriasis, development of PPP has also been observed in 0.2% of 1,620 patients during the first 12\u00a0weeks of treatment with efalizumab, compared to none of the 715 patients in the placebo groups [5].\nWhether therapy with TNF-antagonists may induce a more severe form of plaque psoriasis or psoriatic arthritis in some patients treated for these indications is difficult to determine, as the natural course of both plaque psoriasis and psoriatic arthritis is variable and a loss of effect is not easily differentiated from an induction of a more severe form of the respective disease. However, as a manifestation of plaque psoriasis has been noted in a number of patients treated with TNF-antagonists for other indications without a personal or family history of psoriasis [6, 16], it is likely that TNF-antagonists may in some individuals and probably in conjunction with environmental factors favor manifestation or worsening of psoriatic skin and\/or bone disease.\nThe treatment of patients with plaque-psoriasis developing PPP should be decided on an individual basis. In case plaque-psoriasis remains controlled under infliximab therapy, addition of topical therapy may be sufficient for treatment of PPP in some instances (case 4). However, in some cases, additional topical therapy may not be sufficient and UV-therapy or another systemic therapy may be necessary in addition to or as replacement of infliximab therapy (case 3). When manifestation of PPP is accompanied by worsening of plaque-psoriasis, discontinuation of infliximab therapy is advisable and initiation of other systemic antipsoriatic agents, such as cyclosporine or a different TNF-antagonist, may be necessary (cases 1 and 5).\nIn summary, pustular psoriasis may show a good response to treatment with TNF-antagonists such as infliximab. However, pustular psoriasis may also manifest during treatment of rheumatological diseases, and, as described here, also in patients with plaque-type psoriasis under treatment with infliximab. Manifestation of PPP under infliximab is not necessarily accompanied by worsening of pre-existing plaque psoriasis. Management of the pustular skin lesions has to be decided on an individual basis. In a subgroup of patients, therapy with the TNF-antagonist has to be discontinued and another systemic therapy (including a different TNF-antagonist) will probably be necessary to sufficiently control plaque-psoriasis and PPP.","keyphrases":["pustulosis","infliximab","psoriasis","tnf","antagonist"],"prmu":["P","P","P","P","P"]}
{"id":"Fam_Cancer-4-1-2253650","title":"Hereditary diffuse gastric cancer: association with lobular breast cancer\n","text":"Hereditary diffuse gastric cancer (HDGC) has been shown to be caused by germline mutations in the gene CDH1 located at 16q22.1, which encodes the cell\u2013cell adhesion molecule, E-cadherin. Not only does loss of expression of E-cadherin account for the morphologic differences between intestinal and diffuse gastric cancer (DGC) variants, but it also appears to lead to distinct cellular features which appear to be common amongst related cancers that have been seen in the syndrome. As in most hereditary cancer syndromes, multiple organ sites may be commonly affected by cancer, in HDGC, lobular carcinoma of the breast (LBC) and possibly other organ sites have been shown to be associated with the familial cancer syndrome. Given the complexity of HDGC, not only with regard to the management of the DGC risk, but also with regard to the risk for other related cancers, such as LBC, a multi-disciplinary approach is needed for the management of individuals with known CDH1 mutations.\nIntroduction\nDespite an overall decrease in the global incidence of gastric cancer (GC) [1], the incidence of the subtype, diffuse gastric cancer (DGC) has remained stable and may even be increasing [2]. Within the past ten years, germline mutations in CDH1, which encodes E-cadherin, have been found [3] in over 50% of hereditary diffuse gastric cancer (HDGC) families with at least two cases of GC, with one diagnosed as DGC before the age of 50\u00a0years [4]. Within these HDGC families, we and others have noted an overrepresentation of lobular breast cancer (LBC) [4\u20138]. This observation has led to efforts to determine whether or not CDH1 is a breast cancer susceptibility gene, distinct from its gastric cancer risk. Recently our group has reported a novel germline CDH1 truncating mutation (517insA) in an LBC family with no known history of GC [9]. Within this review we report a germline CDH1 mutation in a second family in which breast cancer is the predominant cancer diagnosis. The management of HDGC in all patients with a particular focus on the management of the breast cancer risk associated with germline CDH1 mutations will be discussed.\nMethods\nThe described family was referred to the ongoing HDGC study at the British Columbia Cancer Agency from a cancer genetics clinic in Seattle, WA, USA. Informed consent was obtained from the proband by the referring genetic counselor following ascertainment of a detailed cancer family history and appropriate genetic counseling prior to germline mutation testing. Our laboratory carried out the molecular genetic testing for the CDH1 mutation on a research basis. Approval for the HDGC study is by the clinical research ethics board of the University of British Columbia.\nThe proband (IV-4) was diagnosed with widely metastatic lobular breast cancer at age 53\u00a0years (Fig.\u00a01a). Her family, of European ancestry, had a history of breast cancer diagnoses occurring in an autosomal dominant fashion on the maternal side of the family where her mother, aunt, and first cousin developed breast cancer in their 50\u2019s. Due to her high-risk pedigree BRCA1, BRCA2, and PTEN genetic testing was undertaken and all were negative. CDH1 testing was also pursued.\nFig.\u00a01(a) Pedigree of family reported showing a predominance of breast cancer. (b) Sequence from family carrying 1565\u00a0+\u00a01G\u2192A mutation\nResults\nAll 16 exons were amplified for DHPLC analysis [6]. For exon 10 of CDH1, the initial amplicon failed and was therefore analyzed by direct sequencing and thus revealed a donor splice site mutation, 1565\u00a0+\u00a01G\u00a0>\u00a0A (Fig.\u00a01b). Due to its position at a donor splice site, this mutation is regarded as pathogenic [10].\nThe proband\u2019s sisters (IV-2 and IV-6) participated in all aspects of the proband\u2019s genetic consultation. They were appropriately concerned about their risk of breast cancer, but had not thought much about the possibility of getting gastric cancer until the CDH1 mutation was found. IV-2 and IV-6 had predictive genetic testing for the CDH1 mutation testing and both were found to be negative. Other family members are being informed about the availability of predictive genetic testing.\nDiscussion\nE-cadherin\nCDH1 (OMIM *192090), located on chromosome 16q22.1 encodes, E-cadherin, an epithelial transmembrane cell\u2013cell adhesion molecule and member of the cadherin superfamily of glycoproteins. In a zipper-like fashion, its extracellular domain forms calcium-dependent homodimers between the E-cadherin molecules of adjacent epithelial cells, to act as the primary mediator of epithelial cell adhesion at the adherens junction complex [11]. Through interactions of its cytoplasmic tail with multiple signalling and structural molecules, such as the catenins, E-cadherin, maintains cellular adhesion and epithelial architecture with this link to the cytoskeleton. The cytoplasmic tail of E-cadherin directly associates with \u03b2-catenin and \u03b3-catenin which in turn binds to the f-actin microfilaments of the cytoskeleton, directly or through \u03b1-catenin [12]. p120-catenin also associates with E-cadherin\u2019s cytoplasmic tail at a different site, the juxtamembrane domain, and acts to both strengthen the adhesion between cells and regulate cadherin membrane trafficking and degradation [13, 14]. E-cadherin is considered to have an invasion suppressor role, where decreased expression permits cells to dissociate from each other in order to migrate and invade [15]. In cancers, this manifests as increased infiltrative and metastatic potential [16]. E-cadherin is also thought to act as a tumour suppressor, potentially through its interaction with the multipurpose \u03b2 -catenin molecule which is an effector of the WNT signalling pathway [17]. Loss of E-cadherin can result in \u03b2-catenin release from the membrane and translocation to the nucleus where it complexes with Tcf\/Lef-1 transcription factors to initiate transcription of WNT responsive genes [18]. Activation of these genes have been implicated in tumourigenesis through the WNT signalling pathway as seen in adenomatous polyposis coli (APC) [19]. In support of the role of E-cadherin as a tumour suppressor is the observation of abnormal or absent E-cadherin expression in precursor lesions of DGC and LBC, where the phenomenon is seen in in\u00a0situ signet ring cell carcinomas found in prophylactic gastrectomy specimens from germline CDH1 mutation carriers [20] and the lobular carcinoma in\u00a0situ lesions seen adjacent to invasive lobular breast cancers [21]. These examples suggest that loss of E-cadherin is an early or even tumour-initiating event, however the actual molecular basis of such a potential role of E-cadherin in such cases is unknown.\nInactivating CDH1 mutations are found in 50% of sporadic DGCs [22, 23] and cluster between exons seven and nine [11], in contrast with the low percentage of mutations seen in sporadic intestinal type GCs [23]. Decreased expression of E-cadherin in DGCs may account for morphologic differences between intestinal and DGC variants [24]. Unlike somatic CDH1 mutations, germline mutations associated with DGC are distributed throughout the gene [7] (Fig.\u00a02). In the cancers from individuals with CDH1 mutations, CDH1 acts as a classic tumour suppressor gene with loss of expression of the wildtype allele [25, 26]. In a single study of 6 hereditary DGC cancers, inactivation of the wild-type allele could be attributed to promoter hypermethylation in 5 (83%) of cases [26]. This finding warrants verification in a larger cohort as abnormal promoter methylation in early cancers could potentially form the basis of a screening test.\nFig.\u00a02DGC and LBC associated CDH1 germline mutations. Mutations shown above CDH1 gene schematic occur in families with DGC history and those below CDH1 occur in families with an additional or exclusive LBC history. In addition to the known CDH1 germline mutations compiled by Kaurah et\u00a0al. [4], the recent mutation in an LBC family [9] and novel mutation from this paper are shown and identified below the symbol denoting mutation type. * Denotes the halfway point of the CDH1 coding sequence (1324 or the start of exon 10)\nLobular breast cancer and diffuse gastric cancer: loss of E-cadherin\nCurrently germline mutations in single genes account for approximately 5\u201310% of breast cancer [27]. High penetrance genes such as BRCA1 and 2 account for 3\u20138%, and TP53 and PTEN as seen in Li-Fraumeni and Cowden syndrome together only account for <0.1% of breast cancer diagnoses [28]. Other medium and low penetrance genes such as CHK2, BRIP1, PALB2 and ATM [29\u201332] have been identified, however, there still remains a proportion of hereditary breast cancer not yet determined. LBC accounts for approximately 10% of all breast cancers compared to the other major histologic subtype, invasive ductal carcinoma (IDC) [33]. Several factors suggest that LBC has a stronger hereditary basis relative to IDC, such as the higher frequency of bilateral disease [33], and also where excess familiality of LBC has been observed in population studies [34]. LBCs compose only 3% and 9% of the breast cancer tumour types seen in germline BRCA 1 and 2 mutation carriers, respectively [35], illustrating that the genetic risk factors for the majority of cases are unaccounted for by these genes.\nThe histology of LBC is characterized by infiltrative cancer cells which are isolated, highly dispersive and demonstrate a growth pattern with scattered and single files of tumor cells dispersed in stromal tissue [36]. This pathologic appearance is remarkably similar to DGCs and both LBC and DGC demonstrate characteristic mucinous, signet ring cells. This is not unexpected as E-cadherin staining is absent in 85% of sporadic invasive LBC [37] and somatic CDH1 mutations have been identified in 56% of sporadic LBCs [38]. Furthermore, in IDC, somatic CDH1 mutations are not found [38] and complete loss of E-cadherin expression is an uncommon feature. As loss of E-cadherin expression is a distinctive trait of both LBCs and DGCs, it likely contributes to the unique histopathologic features shared by the two cancers.\nThere are some differences with regard to the nature of the mutations seen in LBC and DGC. Generally mutations associated with sporadic LBC have been found to be nonsense or frameshift mutations [39] which encode truncated, non-functional proteins, whereas in sporadic DGC, mutations have generally been found to be splice site and in-frame mutations [11]. In sporadic LBC, mutations in CDH1 are spread throughout the gene [11] compared with the mutations seen in sporadic DGC which tend to cluster. Germline CDH1 mutations associated with DGC and\/or LBC occur throughout the gene (Fig.\u00a02). However, when the DGC and LBC associated CDH1 mutations are tabulated and compared based on their 3\u2032 or 5\u2032 positions relative to the halfway point of the CDH1 coding sequence (1324 or the start of exon 10), LBC associated mutations show a statistically significant trend towards clustering at the 3\u2032 end (Fisher\u2019s exact test, two-tailed P-value equals 0.0467) (Fig.\u00a02). As this association is of weak statistical significance, it is unlikely to impact clinical testing strategies. Future analyses of novel germline LBC-associated CDH1 mutations should help to confirm this observation. Another difference between the molecular genetics of the two types of cancers, is that in sporadic LBC, silencing of E-cadherin expression is generally accomplished by a mutation in one allele in combination with loss of heterozygosity (LOH) or promoter hypermethylation in the remaining allele [40]. This is in contrast to sporadic DGC, where biallellic inactivation is achieved by mutations in one allele in concert with promoter hypermethylation in the other [41].\nWe have recently identified a truncating germline CDH1 mutation in an LBC family where analysis of the tumour was suggestive of partial LOH in the WT allele [9]. Our current case demonstrates a germline CDH1 mutation (1565\u00a0+\u00a01G\u00a0>\u00a0A) in a predominantly breast cancer family, which is predicted to disrupt splicing. The mutation is in the same conserved position as a previously reported mutation (1565\u00a0+\u00a01G\u00a0>\u00a0T) which was found in an Arabian HDGC family with no recorded history of breast cancer [42]. Moreover, a previous study reported a germline missense mutation in a proband with LBC but did not detail family history, or functionally characterize the missense mutation [43]. These examples demonstrate the need for further studies of germline mutations in LBC families in order to determine the mutation frequency and potential genotype-phenotype correlations.\nLobular breast cancer and HDGC\nBreast cancer has been observed in HDGC kindreds to the extent where clustering of LBCs within HDGC families has led to the misclassification of families as breast cancer kindreds who test negative for BRCA1\/2 mutations [4]. In 1998, Keller described the first case of histologically defined LBC in association with HDGC [5]. Since then, several more HDGC families with associated breast cancer were reported where it was observed, that these cases were LBCs when pathology was available [4, 6\u20138].\nPrior to establishment of the association between HDGC and LBC, several efforts to determine whether CDH1 was a breast cancer susceptibility gene were attempted in view of the well-recognised phenotype of loss of E-cadherin expression displayed by the breast cancer subtype. For various reasons these studies failed to demonstrate the link. Rahman et\u00a0al. examined 65 cases of lobular carcinoma in\u00a0situ, however did not pre-screen the cases based on family history and included a wide age range, from 26 to 71\u00a0years, not necessarily in keeping with the usual age of onset seen in hereditary cancer syndromes [44]. Salashor examined 19 breast cancer tumours exhibiting LOH at the CDH1 locus, however of those, only 3 were confirmed to be pure LBC or mixed LBC\/IDC pathology [45]. Lei examined 13 familial LBC cases and found no mutations, however did not define the extent of the family history [46].\nPenetrance data based on 11 HDGC families, estimated the cumulative risk for LBC for female mutation carriers to be 39% (95% CI, 12\u201384%) by 80\u00a0years of age [47]. More recently we have published an estimated cumulative risk for breast cancer for females by the age of 75\u00a0years as being 52% (95% CI, 29\u201394%) from analysis of 4 predominantly gastric cancer pedigrees from Newfoundland with the 2398delC CDH1 founder mutation [4]. This is with the caveat that LBC risk for CDH1 mutation carriers has been assessed within high risk HDGC families, leading to a potential ascertainment bias and underestimation of the role of CDH1 mutations in LBC development. To accommodate for this we have begun analysis of CDH1 mutations within familial lobular breast cancer families or those families ascertained through a relatively young index case with confirmed LBC and have found germline CDH1 mutations in these kindreds [9].\nClinical implications of CDH1 associated LBC risk\nAt this time, it seems reasonable to conclude that at least four groups of women are at increased risk for LBC: women with LBC and a family history of breast cancer, women with a known CDH1 mutation, women from families with diffuse gastric cancer in whom no CDH1 mutation has yet been identified; and women with a germline BRCA2 mutation. Since there has not yet been a large population based study of the prevalence of CDH1 mutations among women with lobular breast cancer, it is premature to recommend genetic evaluation to women with a family history of breast cancer unless, at the very least, one of the breast cancers can be shown to have been lobular. Additional research can be expected to provide better guidance for these families.\nAlthough there are not yet definitive data available on surveillance or risk reduction programs for women with known CDH1 mutations or untested women from CDH1-positive families, the high lifetime risk of LBC (39\u201352%) [4, 47] that these women face mandates their careful management. We suggest that they follow the recommendations for other high-risk women with hereditary breast cancer predisposition. This subgroup should be advised to practice breast self-examination; and to have annual mammograms, and semiannual clinical breast examination, beginning at least by age 30. There is certainly interest in regular bilateral breast MRI, as lobular breast cancer are known to frequently elude mammographic detection because they do not form masses or develop calcifications. These women can also be counseled to consider hormonal chemoprevention, since most LBCs are estrogen receptor positive [33], and both tamoxifen and raloxifene reduce the risk of estrogen receptor positive [48, 49] breast cancers in randomized trials. In addition, the risk reduction was greatest with both agents in women with lobular carcinoma in\u00a0situ [50].\nProphylactic mastectomy may also be considered an option by some CDH1-positive women, particularly those who have been previously diagnosed with breast cancer in one breast or those who have had to undergo multiple biopsies for abnormal clinical findings. Several studies have reported a 90% reduction in breast cancer incidence with prophylactic mastectomy among women with a strong family history or with a germline BRCA1 or BRCA2 mutation [51, 52]. The published series include some lobular breast cancers, but not at numbers sufficient to permit meaningful subset analysis at this time.\nManagement of hereditary diffuse gastric cancer\nPenetrance studies examining data from HDGC families, have estimated the lifetime risk of developing gastric cancer by age 75 and 80 respectively, to be from 40\u201367% in men, to 63\u201383% in women [4, 47]. Although identification of germline CDH1 mutations has enabled a significant proportion of HDGC families to utilise predictive testing to determine their individual risks of GC within CDH1 mutation positive pedigrees, unfortunately screening for DGC is ineffective and the current recommendation is for consideration of prophylactic gastrectomy in mutation positive individuals. Positron emission tomography [53] and chromoendoscopic-directed biopsies [54] have been proposed over basic endoscopy as more sensitive means of screening carriers, however screening methods have been consistently undermined by the recurrent discovery of multifocal DGC lesions underlying normal mucosa in prophylactic gastrectomy specimens of individuals with recent negative screening [4, 55, 56]. Regardless of the current limitations of screening, it is currently recommended that consideration for genetic testing and screening begin in at risk individuals in the late teens or early twenties [4] and that prophylactic total gastrectomy be considered in the early twenties for mutation carriers. Female mutation carriers will need specialized counseling to the potential nutritional effects on pregnancy following gastrectomy [57]. Further studies are currently underway to examine the quality of life impact of prophylactic gastrectomies. In the case report herein, although there was a GC in the maternal grandfather, the family history was more striking for the large number of breast cancer cases. This highlights the particular challenges we currently face with regard to counseling these families which appear to be mainly breast cancer, as it is unknown if the penetrance of DGC in this family is as high as it is in other HDGC pedigrees.\nConclusion\nHDGC is one of a number of hereditary cancer syndromes that feature both an increased breast and gastric cancer risk (Table\u00a01). In general, a lack of shared genetic risks for most breast and GI cancers was suggested through a recent study of 13,023 genes in 11 breast and 11 colon cancer cell lines in which the only commonly mutated gene between these two cancer types is p53 [58]. This likely reflects underlying differences in the biology of these diseases, however also highlights the unique nature of germline mutations in the CDH1 gene which are strongly associated with specific histologically defined subtypes of breast and GI cancer, namely LBC and DGC which are both part of the HDGC syndrome.\nTable\u00a01Other syndromes with familial susceptibility to breast and gastric cancersSyndromeMode of inheritanceAssociated gene(s)Sites of primary cancer(s)Evidence for association with the syndrome BRCA2 Hereditary Breast\/Ovarian CancerADBRCA2BreastOvaryLarynxProstateBC is considered an integral tumor of the syndrome with an average cumulative risk in carriers by age 70 years of 45% (95% confidence interval (CI) 31\u201356%) [59]Familial aggregations of both BC and GC have been reported [60]In a study of 173 families, relative risk for GC was 2.6 [61]Among Ashkenazi Jewish GC patients, the frequency of 617delT mutation is five times that of the general Ashkenazi Jewish population frequency [62] Among other cancers, GC occurred in first degree relatives when mutations were located in the ovarian cancer cluster region of exon 11 of BRCA2 [63] BRCA1 Hereditary Breast\/Ovarian CancerADBRCA1BreastOvary ProstateBC is considered an integral tumor of the syndrome with an average cumulative risk in carriers by age 70 years of 72.8% (95% confidence interval [CI]\u00a0=\u00a067.9% to 77.7%) [64]There is a 4 times increased risk for GC [64]Peutz-Jeghers SyndromeADSTK 11Gastrointestinal (GI) tractGC is considered an integral tumor of the syndrome with a relative risk of 213 (95% confidence interval 96\u2013368) [65] BC is considered an integral tumor of the syndrome with a relative risk of 15.2 (95% CI 7.6\u201327) [65] Cowden SyndromeADPTENBreastThyroidEndometriumBC is considered an integral tumor of the syndrome with an incidence of 22\u201350% [66, 67]GC in\u00a0situ has been reported in a patient with Cowden Syndrome [68]Li-Fraumeni SyndromeADTP53CHK2BreastAdrenal cortexConnective tissueKidneyNervous systemPancreas White blood cellsBC is frequently found in families with this cancer susceptibility syndrome [69]Chompret expanded the spectrum of cancers to include GC [70]Germline TP53 mutations have been found in GC families without CDH1 mutations [71\u201373]The 1100delC CHK2 allele confers a 2.2 fold risk of BC to carriers [29], however germline mutations in GC kindreds have not been identified [74]Familial Adenomatous PolyposisADAPCColon and rectumDuodenumThyroidPancreasLiterature review by Shimoyama et\u00a0al. totalled 30 reported cases of GC and FAP [76]47% to 49% of primary BCs had promoter hypermethylation at the APC locus [77, 78]23% of LBCs have been shown to have LOH of APC [43]Lynch Syndrome (Hereditary Nonpolyposis Colon Cancer (HNPCC))ADhMSH2hMLH1hMSH6hPMS1hPMS2Colon and rectumEndometriumStomach Small intestineUrotheliumKidneyOvarySkinPancreasBrainWhite blood cells Biliary tractGastric cancer accounted for 5% of cancers in families harboring MLH1 or MSH2 mutations [76]hMLH1 mutations in large kindred segregated with BCs exhibiting microsatellite instability (MSI) [79]A slight increased incidence of BC was seen in hMLH1 mutation carriers [80]Germline hMSH2 mutation carrier with BC exhibited LOH for hMSH2 in tumors analyzed [81]Analysis of primary invasive BCs demonstrated that 25% of tumours were immunonegative for MSH2 staining [82]\u00a0Ataxia-telangiectasia (AT)ARATMWhite blood cellsMutations causing AT in homozygotes, confer susceptibility to BC in heterozygotes, where women with ATM mutations have a \u223c2-fold risk of BC and \u223c15% of these women will develop the disease [32]GC has been reported in association with the syndrome [83\u201385]There is evidence of excess risks of GC in heterozygotes (RR\u00a0=\u00a03.39, 95% CI\u00a0=\u00a00.86 to 13.4) [86]Xeroderma pigmentosumARXPAERCC3 (XPB)XPCERCC2(XPD)DDB2(XPE)ERCC4(XPF)ERCC5(XPG)POLH(XP-V)SkinEyesBC and GC have both independently been reported with the syndrome [87,88]Werner SyndromeARWRNConnective tissue SkinThyroidGC has been reported in association with the syndrome [89] There are no reports of BC in association with Werner syndrome. Although, there is evidence supporting WRN as a low-penetrance familial BC susceptibility gene, where patients harboring both WRN Cys1367Arg or TP53 MspI variants had an increased BC risk (OR\u00a0=\u00a03.39, 95% CI 1.19\u20139.71) [90] AD\u00a0=\u00a0autosomal dominant, AR\u00a0=\u00a0autosomal recessive\nWith the recent demonstration of a CDH1 mutation in a family ascertained through an index case of LBC and in view of the additional new mutation in a predominantly breast cancer family that we have described here, the evidence for establishing LBC as part of the HDGC syndrome is strong. There now is a need for establishing the prevalence of CDH1 mutations in LBC families to avoid the ascertainment bias generated from only looking at cases from families identified because of their family history of GC. It is not currently known what the risk of GC is in these families which present predominantly as having a susceptibility to breast cancer and therefore identification of CDH1 as a true susceptibility gene for LBC could result in CDH1 screening and effective risk reduction strategies for selected breast cancer families and further studies examining their risk for gastric and other cancers.\nMost hereditary cancer syndromes are associated with cancer risk involving multiple organs. Here we have discussed germline CDH1 mutations and the risks with regard to DGC and LBC, however as the recognised spectrum of related cancers broadens, more affected families will be identified and successfully managed with regard to avoidance of specific cancer risks. Longer life expectancy in individuals with penetrant mutations could potentially lead to the development of different, later onset disease as yet to be identified in these kindreds. This represents a particular challenge in hereditary cancer practice as the clinical community tends to be segregated into organ specific specialties where as the cancer risks and the risk reduction strategies for germline mutation carriers require a variety of expertise. The medical needs of the HDGC families are therefore best served through an engaged multidisciplinary team.","keyphrases":["diffuse gastric cancer","lobular breast cancer","hereditary diffuse gastric cancer (hdgc)","cdh1 mutation","screening","prophylactic total gastrectomy","e-cadherin mutation"],"prmu":["P","P","P","P","P","P","R"]}
{"id":"J_Abnorm_Child_Psychol-3-1-1915590","title":"Associations of Maternal Prenatal Smoking with Early Childhood Physical Aggression, Hyperactivity-Impulsivity, and Their Co-Occurrence\n","text":"This study investigated associations between maternal prenatal smoking and physical aggression (PA), hyperactivity-impulsivity (HI) and co-occurring PA and HI between ages 17 and 42 months in a population sample of children born in Qu\u00e9bec (Canada) in 1997\/1998 (N=1745). Trajectory model estimation showed three distinct developmental patterns for PA and four for HI. Multinomial regression analyses showed that prenatal smoking significantly predicted children\u2019s likelihood to follow different PA trajectories beyond the effects of other perinatal factors, parental psychopathology, family functioning and parenting, and socio-economic factors. However, prenatal smoking was not a significant predictor of HI in a model with the same control variables. Further multinomial regression analyses showed that, together with gender, presence of siblings and maternal hostile reactive parenting, prenatal smoking independently predicted co-occurring high PA and high HI compared to low levels of both behaviors, to high PA alone, and to high HI alone. These results show that maternal prenatal smoking predicts multiple behavior regulation problems in early childhood.\n\u00a0\nThe literature on maternal prenatal smoking and externalizing behavior problems is clearly separated in two developmental periods. The bulk of the current literature on maternal prenatal smoking and externalizing behavior problems covers either childhood and adolescence or the preschool years. However, the rates of common childhood psychiatric disorders such as conduct disorder (CD) and attention deficit hyperactivity disorder (ADHD) and the patterns of comorbidity among them in early childhood are similar to those seen in later childhood (Egger & Angold, 2006). Limitations in current diagnostic criteria for early childhood psychopathology have resulted in a research focus on specific behaviors such as physical aggression (PA) and hyperactivity-impulsivity (HI) rather than clinical disorders in this age group. Both physical aggression and hyperactivity during early childhood appear to be typical precursors of full-blown CD and ADHD during the school years and beyond (S\u00e9guin, Nagin, Assaad, & Tremblay, 2004). A substantial number of studies show continuity between early and late childhood externalizing behavior problems (e.g. Campbell, Breaux, Ewing, & Szumowski, 1986; Keenan & Wakschlag, 2000) or have identified consistent developmental trajectories for PA and HI that start as early as age 1 1\/2 years (e.g. C\u00f4t\u00e9, Vaillancourt, LeBlanc, Nagin, & Tremblay, 2006; NICHD Early Child Care Research Network, 2004; Romano, Tremblay, Farhat, & C\u00f4t\u00e9, 2006; Shaw, Lacourse, & Nagin, 2005).\nIn reviewing childhood and adolescence studies, maternal prenatal smoking has consistently been associated with CD- and ADHD- symptoms (for reviews, see Linnet et\u00a0al., 2003; Wakschlag, Pickett, Cook, Benowitz, & Leventhal, 2002). The associations have been observed in clinical samples (e.g. Mick, Biederman, Faraone, Sayer, & Kleinman, 2002; Milberger, Biederman, Faraone, Chen, & Jones, 1996), \u2018at-risk\u2019 samples (e.g. Wakschlag & Hans, 2002) and large population-based samples (e.g. Braun, Kahn, Froehlich, Auinger, & Lanphaer, 2006; Kotimaa et\u00a0al., 2003). Whereas these studies generally controlled for familial psychopathology and numerous environmental risk factors, there are also indications from studies using (large) twin samples that maternal prenatal smoking predicts children\u2019s CD- and ADHD-symptoms beyond the effects of heritable risk (e.g. Button, Thapar, & McGuffin, 2005; Maughan, Taylor, Caspi, & Moffitt, 2004; Silberg et\u00a0al., 2003; Thapar et\u00a0al., 2003). Studies showing associations between maternal prenatal smoking and CD-symptoms and delinquency during adolescence and adulthood (Brennan, Grekin, Mortensen, & Mednick, 2002; Fergusson, Woodward, & Horwood, 1998; R\u00e4s\u00e4nen et\u00a0al., 1999) and studies showing behavior problems in children of mothers who smoked during pregnancy at different time points (e.g. Maughan et\u00a0al., 2004; Wakschlag & Hans, 2002; Wakschlag, Pickett, Kasza, & Loeber, 2006) suggest long-lasting effects.\nAlthough there are indications that maternal prenatal smoking is more strongly or even exclusively related to CD symptoms (Wakschlag & Hans, 2002; Wakschlag, Leventhal, Pine, Pickett, & Carter, 2006; Wakschlag, Pickett et\u00a0al., 2006), only few studies tested for specificity of the relations between maternal prenatal smoking and CD- or ADHD-symptoms (Button et\u00a0al., 2005; Mick et\u00a0al., 2002; Thapar et\u00a0al., 2003). This is an important matter considering the high comorbidity of these behavior problems (e.g. Jensen, Martin, & Cantwell, 1997). Mick et\u00a0al. (2002) found a robust link with ADHD-symptoms after controlling for CD-symptoms in a clinical population. In that study, children aged 6 to 17 years with ADHD and non-ADHD controls were compared after statistical control for CD. In contrast, Thapar et\u00a0al.\u2019s (2003) and Button et\u00a0al.\u2019s (2005) population-based twin study using the Greater Manchester Twin Register did not select on either CD- or ADHD-symptoms. Whereas Thapar et\u00a0al. showed that prenatal maternal smoking predicted a unique proportion of the variance in ADHD-symptoms after control for CD-symptoms in children between 5 and 16 years, Button et\u00a0al. (2005) tested a more complex model showing unique variance for both CD- and ADHD-symptoms in children 5 to 18 years of age. Although the aforementioned studies used some form of statistical control for the symptoms associated with the \u2018other\u2019 disorder, only one study to date examined a relation between prenatal maternal smoking and co-occurring behavior problems (Wakschlag, Pickett et\u00a0al., 2006). In that study maternal prenatal smoking predicted the co-occurrence of Oppositional Defiant Disorder (ODD) and ADHD when high levels of both were contrasted with absence of both. This suggests that maternal prenatal smoking, to the extent that it is a causal factor, may affect behavior much more seriously than initially shown in specificity studies. However, such a conclusion may be premature because that analysis did not inform us if maternal prenatal smoking was related specifically to the co-occurrence of both behavior problems or whether it was driven by its association with either behavior problem because we do not know if the combined group differed from the ODD-only or ADHD-only groups. Thus whether maternal prenatal smoking is related to the co-occurrence of behavior problems remains an open question in this literature.\nIn contrast to childhood and adolescence studies, there is a more limited number of early childhood studies and they tend to focus on symptoms rather than diagnoses. Tremblay et\u00a0al. (2004) reported an association with early childhood trajectories of PA across time, and Romano et\u00a0al. (2006) found an association between maternal prenatal smoking and hyperactive symptoms from age 2 to 7, but both studies focused on one externalizing behavior problem. Other studies focused on multiple behaviors. Orlebeke, Knol, and Verhulst (1997) and Williams et\u00a0al. (1998), using the Child Behavior Checklist (CBCL) in large cohort studies of 3-year-old and 4 to 6-year old children, respectively, found associations with externalizing behavior problems (aggressive, overactive, oppositional) but not with internalizing behavior problems (withdrawn, anxious, depressed). Day, Richardson, Goldschmidt, and Cornelius (2000) reported significant associations of third trimester exposure with scores on each of the subscales of the Toddler Behavior Checklist (Physical Aggression, Oppositional Behavior, Immaturity, and Emotionality) and Activity level assessed with the Routh Activity Scale. Wakschlag, Leventhal et\u00a0al. (2006) noted a persistent association between maternal prenatal smoking and disruptive behaviors during early childhood. None of these multiple behavior studies examined co-occurrence. Thus there also remains a need in the early childhood literature to examine whether maternal prenatal smoking is specifically associated with the co-occurrence of problem behaviors.\nFrom a developmental perspective, early onset of externalizing behavior has been associated with the poorest outcomes (Brame, Nagin, & Tremblay, 2001; Lacourse et\u00a0al., 2006; Moffitt, 1993; Woodward, Fergusson, & Horwood, 2002). In addition, co-occurring externalizing behaviors during middle childhood have been associated with the poorest behavioral outcomes during adolescence and adulthood (Lacourse et\u00a0al., 2006; Lahey, McBurnett, & Loeber, 2000; S\u00e9guin et\u00a0al., 2004). However, co-occurring behavior problems may be etiologically different from individual behavior problems, with their own social, sociodemographic and biological precursors (Waschbusch, 2002). Yet, we know very little about the prenatal markers of co-occurring behavior problems in general. Given that maternal prenatal smoking has been clearly associated with several single externalizing problems across development, in the present study, we test the hypothesis that maternal prenatal smoking is associated with co-occurring externalizing problems. Specifically we predict that maternal prenatal smoking will be associated to the co-occurrence of PA and HI in contrast with the absence of both, PA-only, and HI-only in a large early childhood sample.\nMethods\nParticipants\nThe children of this study were born in 1997\/1998 in the province of Qu\u00e9bec, Canada and participate in the Qu\u00e9bec Longitudinal Study of Children\u2019s Development. This sample excluded very remote regions of the province populated mainly by aboriginal people (2.1% of live births), babies for whom gestational age could not be computed (1.3%), babies born in a different territory but whose parents reside in Qu\u00e9bec (4.5%), and very premature babies (<24 weeks) and babies for whom there were delays in filing the birth records in the Master Birth Registry on time for the first assessment, i.e. babies born after 42\u00a0weeks gestation (0.1%; for full details, see Jett\u00e9 & Des Groseilliers, 2000). A total of 2940 infants met inclusion criteria and were selected through a region-based stratification procedure (Jett\u00e9 & Des Groseilliers, 2000). Of this original selection, a number of families could not be included in the initial 5 month-assessment for the following reasons: (1) Families not found on time (incorrect address\/tel no.) (n=172, 5.9%); (2) Families excluded (total n=93, 3.2%) because of death of the baby (n=5), because of participation in other longitudinal studies (n=5), because they had no command of either English or French language (n=81), or because the instruments of the study were not designed to adequately measure development of children with severe physical or mental handicaps (n=2); (3) Families that could not be reached (n=14, 0.5%); (4) Families who declined participating (n=438, 16.4%). 2223 Families (75.6%) took part in the first assessment, which took place when the infants were 5 months old. Demographic characteristics of this Qu\u00e9bec sample were comparable to those of a large Canadian sample consisting of 13,439 households which contains sufficient samples from each of the 10 Canadian provinces (Human Resources Development Canada, 1996: National Longitudinal Survey of Children and Youth, 1994\u20131995). Assessments relevant to this study took place at 17, 30 and 42 months. Parental informed consent was obtained before every assessment. 478 participants had either dropped out since the initial assessment at 5 months or had missing values for PA, HI or one or more predictor variables. Thus, the final study sample consisted of 1745 children (78.5% of families enrolled at 5 months). Table 1 indicates that, despite the fact there was a slight tendency for less advantaged families to drop out or have missing values on relevant variables, demographic characteristics were largely similar for in- and excluded families.Table 1Demographic characteristics of families enrolled in the Qu\u00e9bec Longitudinal Study of Children\u2019s Development (QLSCD 1) (n\u00a0max=2223) and families included in the present study (QLSCD 2: n=1745)QLSCD 1QLSCD 2\nn\n%\nn\n%Sex of child\u2003Male1,12351.288450.7\u2003Female1,06948.886149.3Age of mother at birth of first child\u200321 years or less47122.237221.3\u2003Older than 21 years164877.8137378.7Mother graduated from high school\u2003Yes186584.0150486.2\u2003No35516.024113.8Family status\u2003Two parents161779.2140780.6\u2003One parent42420.833819.4Number of siblings in the home\u2003None83040.671340.9\u2003One or more121559.4103259.1Family income\u2003Less than 30,000$63929.346826.8\u200330,000$\u201350,000$62528.752730.2\u200350,000$\u201380,000$61128.049928.6\u2003More than 80,000$30714.125114.4Data courtesy of the Institut de la Statistique du Qu\u00e9bec\nMeasurements\nMaternal prenatal smoking\nWhen the child was 5 months of age, mothers filled out a number of questionnaires. One set of questions concerned substance use (alcohol, tobacco, and illegal drugs) during pregnancy. The questions assessing smoking behavior during pregnancy were straightforward, \u2018Did you smoke during pregnancy?\u2019 and \u2018How many cigarettes\/day did you smoke whilst pregnant?\u2019 We also asked when during pregnancy the mother had smoked, i.e. (only) in the first, second or third trimester or throughout pregnancy. These questions are similar to those found in most other assessment instruments (e.g., the Centers for Disease Control and Prevention (CDC) Pregnancy Risk Assessment Monitoring System (PRAMS) Questionnaire, see Beck et\u00a0al., 2002) and like those found in most other studies (particularly those assessing large samples, e.g. Button et\u00a0al., 2005; Fergusson et\u00a0al., 1998; Maughan et\u00a0al., 2004; Thapar et\u00a0al., 2003; Wakschlag et\u00a0al., 1997; Wakschlag & Hans, 2002). Because amount of cigarettes reportedly smoked tends to be a \u2018rounded\u2019 number, e.g. 5, 10, 15, etc, mothers were classified into 1 of 4 groups (0, 1\u20139, 10\u201319, or \u226520 cigarettes\/day). This or similar classifications have been used in most other studies investigating smoking during pregnancy (e.g. Button et\u00a0al., 2005; Fergusson et\u00a0al., 1998; Maughan et\u00a0al., 2004; Thapar et\u00a0al., 2003; Wakschlag et\u00a0al., 1997; Wakschlag & Hans, 2002). Although there is a risk for a social desirability bias, several studies have indicated a relatively strong association between retrospective self-report and blood\/urine cotinine-levels (i.e. the main nicotine metabolite) (e.g. Law et\u00a0al., 2003; Pickett, Rathouz, Kasza, Wakschlag, & Wright, 2005). Further reliance on self-report measures may be inferred from the strong relation between the amount reportedly smoked during pregnancy and birth weight (e.g. Huijbregts et\u00a0al., 2006; Kramer et\u00a0al., 2001). Other studies have shown that whereas for some prenatal exposures (e.g. alcohol, drugs) higher levels were reported postnatally than antenatally, this was not the case for tobacco exposure, which might indicate that smoking is considered a more habitual behavior that is more reliably recalled (e.g. Jacobsen, Chiodo, Sokol, & Jacobson, 2002). For the current analyses, 1307 (74.9%) mothers reported not to have smoked during pregnancy, 202 (11.6%) reported to have smoked 1\u20139 cigarettes\/day, 174 (10%) between 10 and 19, and 62 (3.6%) reported to have smoked 20 or more cigarettes\/day during pregnancy.\nPhysical aggression and hyperactivity-impulsivity\nMaternal ratings of child behavior were obtained with the use of an early childhood behavior scale from the Canadian National Longitudinal Study of Children and Youth (Statistics Canada, 1995), which incorporates items from the Child Behavior Checklist for Ages 2\u20133 (Achenbach, Edelbrock, & Howell, 1987), the Ontario Child Health Study Scales (Offord, Boyle, & Racine, 1989), and the Preschool Behavior Questionnaire (Tremblay, Desmarais-Gervais, Gagnon, & Charlebois, 1987). In order to assess PA mothers were asked at 17, 30 and 42 months to indicate whether the child: (1) hits, bites, kicks; (2) fights; and (3) bullies others. The items for HI were: (1) can\u2019t sit still, is restless (or hyperactive), (2) fidgets, (3) cannot settle down to do anything for more than a few moments, (4) is impulsive, acts without thinking, and (5) has difficulty waiting for turn in games. The first three representing hyperactivity, and the latter two representing impulsivity. The items were scored as follows: never or not true (score = 0), sometimes or somewhat true (score = 1), or often or very true (score = 2). The items were summed to obtain PA (range = 0 to 6) and HI scores (range = 0 to 10). The internal consistency values (Cronbach\u2019s \u03b1) for PA were 0.80 at 17 months, 0.82 at 30 months, and 0.72 at 42 months. For HI they were 0.75, 0.75, and 0.71, respectively. The scales were shown to have good discriminant validity in the prediction of different types of adolescent criminal behaviors (Broidy et\u00a0al., 2003; Nagin & Tremblay, 1999), and although correlated, the PA and HI scales were related in predictable ways with mother and child reports of CD and ADHD, respectively (S\u00e9guin et\u00a0al., 2004). Their validity in early childhood is also well established (Tremblay et\u00a0al., 2004; Romano et\u00a0al., 2006).\nControl variables\nPotential confounders of associations between maternal prenatal smoking and early behavior problems were selected from four key domains: demographic factors (age at birth of first child, family status (separation\/divorce: yes\/no), presence of siblings, family income, maternal education); perinatal factors (alcohol and drug exposure during pregnancy, birth weight); family functioning and parenting (hostile reactive parenting, responsiveness, involvement), and parental background and mental health (mother\u2019s and father\u2019s history of antisocial behavior, maternal depression). Mothers provided information on most of these factors when their child was 5 months of age and again when their child was 17\u00a0months of age.\nFamily income was indicated on a 7-point scale (1 = less than $10,000 (Canadian) to 8 = more than $80,000, as was maternal education (1 = no high school diploma to 7 = university degree). The poverty line in Canada is situated at around $15,000 per capita (Statistics Canada, 2006).\nPerinatal factors included exposure to illegal drugs (yes\/no) and alcohol (7-point scale ranging from \u2018never\u2019 to \u2018daily\u2019) during pregnancy and birth weight. Birth weight and gestational age were derived from birth records. Birth weight for gestational age was standardized within gender for each week of gestation using Canadian norms (Kramer et\u00a0al., 2001).\nParenting measures were obtained by observing the mother-child dyad during home visits at 5 and 17 months using the Home Observation for Measurement of the Environment (HOME)-Infant version (Caldwell & Bradley, 1984). The observations were carried out by trained observers who spent about 3 hours in the home to complete a battery of questionnaires and tests. These observers received a one-week training session and were closely supervised during the data collection. Three scales were obtained: hostile reactive parenting (e.g. \u201ctalks negatively about her child,\u201d \u201cshouts at her child,\u201d \u201chits or physically punishes her child\u201d; \u03b1=0.43 at 5 months and 0.73 at 17 months), responsiveness (e.g. \u201cresponds verbally to child\u2019s vocalizations or verbalizations,\u201d \u201ctells child name of object or person during the visit,\u201d \u201cspontaneously praises the child at least twice\u201d; \u03b1=0.85 at 5 months and 0.83 at 17 months), and involvement (e.g. \u201cprovides toys that challenge child to develop new skills,\u201d \u201cstructures child\u2019s play periods\u201d; \u03b1=0.85 at 5 months and 0.88 at 17 months). Scores for each item on each scale ranged from 1 (never) to 5 (all the time). Only the scale score for hostile reactive parenting at 17 months was used because of the low internal reliability of this scale at 5 months. Mean scores across the 5 and 17 months assessments were used for the responsiveness and involvement scales.\nFamily functioning (at 5 and 17 months) was assessed with a scale containing 12 items measuring communication, problem resolution, control of disruptive behavior, and showing and receiving affection (Statistics Canada, 1995). Scores per item could be 0 (\u2018never\u2019), 1 (\u2018sometimes\u2019), or 2 (\u2018often\u2019), thus ranging from 0 to 36 on the scale. The Cronbach \u03b1\u2019s were 0.86 (5 months) and 0.98 (17 months).\nIn order to assess history of antisocial behavior, both parents completed a questionnaire at the 5-month assessment. The questionnaire included items related to childhood\/adolescence (i.e. the period before the end of high school) and items related to adulthood (Zoccolillo, 2000; see also Tremblay et\u00a0al., 2004), and was largely derived from the NIMH-Diagnostic Interview Schedule (Robins, Helzer, Croughan, & Radcliff, 1981). Childhood\/adolescence items included \u2018starting fights,\u2019 theft, involvement with youth protection or police, expulsion or suspension from school, truancy, and running away from home. Adult items included arrests (other than for traffic violations), being fired from a job (excluding layoffs for lack of work), trouble at work, with family, or with the police due to drug or alcohol abuse, \u2018starting fights\u2019 (fathers), and \u2018hitting or throwing things at the spouse or partner\u2019 (mothers). Adolescent and adult scores (0 = no, 1 = yes) were summed for mothers and for fathers. Internal reliability for mother\u2019s items was 0.54 (Cronbach\u2019s \u03b1) and 0.59 for father\u2019s items. Latent class analysis identified 3-class models for both mothers ((1) not antisocial, (2) moderately antisocial, (3) antisocial) and fathers ((1) not antisocial, (2) antisocial as an adolescent but not as an adult, (3) moderately antisocial as an adolescent, antisocial as an adult). The Center for Epidemiological Studies-Depression Scale (CES-D; Radloff, 1977) was used for report of symptoms associated with depression (at 5 and 17 months).\nStudies have shown that all these factors are associated with increased risk for physical aggression, hyperactivity, CD- and ADHD-symptoms and with prenatal maternal smoking (e.g. Biederman, Milberger, & Faraone, 1995; Campbell, Shaw, & Gilliom, 2000; Huijbregts et\u00a0al., 2006; Linnet et\u00a0al., 2003; Maughan et\u00a0al., 2004; NICHD ECCRN, 2004; Tremblay et\u00a0al., 2004; Nagin & Tremblay, 2001; Romano et\u00a0al., 2006; Shaw et\u00a0al., 2005). Apart from the dichotomous variables (i.e. gender, presence of siblings, drug use during pregnancy, family status), all scores were standardized for statistical analyses.\nData analyses\nAssignment to trajectories\nScores from the three assessment points were analyzed to identify distinctive behavioral trajectories across time (Nagin, 1999, 2005; Nagin & Tremblay, 2001). Rather than to assume that all children follow the same developmental pattern, this methodology identifies different groups of individuals who tend follow similar patterns over time. For example, some children may never show a given problem behavior (intercept model or zero order polynomial), others may show constant high levels (also intercept model), and others may increase or decrease over time (e.g., linear \u2013 1st, quadratic \u2013 2nd, or cubic \u2013 3rd order polynomials). The methodology can also be adapted to accommodate various data distributions (i.e., binary, censored normal, zero-inflated Poisson, and count data). The trajectory methodology uses all available developmental data points and assigns individuals to trajectories on the basis of a posterior probability rule. Resulting groups are meant to represent approximations of an underlying continuous process. In order to identify the model that best represents development of a specific behavior during a given time frame, models with a varying number of trajectories are estimated. Model selection is dependent on a combination of statistical and investigator-guided concerns. Besides a need to determine the best model for the data distribution, key decisions are also based on Bayesian fit indices for model selection in accordance to procedures described by Nagin (2005), e.g. the higher the Bayesian Information Criterion (BIC), the better. The optimal model is thus also determined by adding trajectories to the model until the BIC ceases to improve. The investigator would then have enough information to determine the best model.\nA key output of model estimation is the posterior probability of group membership. For each trajectory group this probability measures the likelihood of an individual of belonging to that trajectory group based on observations across assessments. In other words, 100% accuracy in classification is not assumed nor required. For example, in the case of an individual who scores high on hyperactivity at all assessment periods, the posterior probability of membership to the chronic group would be high whereas the probability of membership to the low trajectory group would be near 0. Participants can be assigned to the trajectory group for which they show the highest probability of belonging. Ideally, the posterior membership probability should be near 1 for this trajectory group. Further, when trajectories are joined, conditional and joint probabilities can be used to further describe the relationship between the joint factors. Posterior probabilities will then be applied to weight PA, HI and their combination when they enter further analyses (see S\u00e9guin et\u00a0al., 2004).\nPrediction of physical aggression, hyperactivity\/impulsivity, and their co-occurrence\nWeighted multinomial regressions were conducted with PROC CATMOD in SAS v.8.2. (SAS Institute Inc., 2001). First, weighted multinomial regressions were conducted separately on PA and HI between ages 17 and 42 months for descriptive purposes. The influence of maternal prenatal smoking on these behaviors was investigated in analyses with and without control variables.\nFor the second set of analyses predicting PA and HI between ages 17 and 42 months, we contrasted, in weighted multinomial regression, those children who were high on both PA and HI (combined group), to those who were only high on PA (PA only group), to those only high on HI (HI only group) and to the remainder of the sample.\nResults\nAssignment to trajectories\nModels with between 2 to 5 trajectories and varied shapes for each trajectory were compared using BIC for both PA and HI. Three trajectories were modeled for PA between ages 17 and 42 months using a zero-inflated Poisson distribution: a consistently low, a moderate and rising, and a high and rising trajectory, each representing respectively 25, 50, and 25% of the sample, all of these were best modeled using a linear trend except for the low group which was best represented by a constant term (Fig. 1, left panel). The shape and level of the PA trajectories were very similar to those we identified in Tremblay et\u00a0al., 2004, in a smaller sample using the same measures. It reveals that most of the children show an increase of physical aggression over time, which is consistent with other longitudinal and cross sectional studies of early childhood.\nFour trajectories were modeled for HI using a censored normal distribution: consistently low, low to moderate, moderate to high and chronic HI, representing respectively, 12, 45, 37, and 6% of the sample. All were best modeled by a constant term except for the low to moderate group that was best modeled with a quadratic trend (Fig. 1, right panel).Thus in contrast to PA, HI in early childhood appears to be more stable in level across time, with very few children being atypically high.Fig. 1Observed and predicted means for developmental trajectories of physical aggression (PA) and Hyperactivity-impulsivity (HI) between ages 17 and 42 months. *Physical aggression: H-R = high-rising; M-R = moderate-rising; C-L = consistently low; Hyperactivity-Impulsivity: H-C = high-chronic; M-H = moderate-high; L-M = low-moderate; C-L = consistently low. Data courtesy of the Institut de la Statistique du Qu\u00e9becTable 2Maternal prenatal smoking and physical aggression (PA), hyperactivity-impulsivity (HI) and joint PA+HI trajectories between ages 17 and 42 monthsMLE 1 (SE)MLE 2 (SE)MLE 3 (SE)\n\u03c7\n2\nP\nPhysical aggression\u2212.40 (.09)\u2212.24 (.07)NA23.6<.001Physical aggression adjusted for control variables\u2212.29 (.10)\u2212.15 (.08)NA8.4.015Hyperactivity\u2212.56 (.15)\u2212.41 (.11)\u2212.22 (.11)22.8<.001Hyperactivity adjusted for control variables\u2212.32 (.17)\u2212.24 (.13)\u2212.13 (.13)5.8.121Physical aggression and hyperactivity\u2212.65 (.14)\u2212.57 (.22)\u2212.41 (.14)31.2<.001Physical aggression and hyperactivity adjusted for control variables\u2212.46 (.16)\u2212.55 (.25)\u2212.33 (.17)10.5.015Note. MLE = Maximum Likelihood Estimate, Reference categories for PA, HI and PA+HI are high PA, high HI and high PA + high HI, respectively. NA = Not Applicable. (MLE) 1 = consistently-low PA; consistently-low HI; and low PA + low HI; (MLE) 2 = moderate-rising PA; low-moderate HI; and low PA + high HI; (MLE) 3 = moderate-high HI; and high PA + low HI. DF = 2 for PA, DF = 3 for HI and for PA+HI. Data courtesy of the Institut de la Statistique du Qu\u00e9bec\nIn the current models for PA and HI the average posterior probability for the assigned trajectory group ranged between .75 and .83, thereby indicating good fit (Nagin, 1999, 2005). Further, a close match of predicted and observed means shown in Fig. 1 also illustrates this good fit. Models with three trajectories for PA and four trajectories for HI have also produced the best fit in studies using other samples that included children in early childhood (e.g. Tremblay et\u00a0al., 2004; C\u00f4t\u00e9 et\u00a0al., 2006; Romano et\u00a0al., 2006; Shaw et\u00a0al., 2005).\nPhysical aggression\nA first multinomial regression revealed that maternal prenatal smoking significantly predicted PA. In an analysis without control variables, both the odds ratio (OR) contrasts between high and low PA [OR=1.49 (95% CI: 1.25 to 1.75), , p<.001] and between high and moderate PA [OR=1.27 (95% CI: 1.11 to 1.45), , p<.001] were significantly predicted by maternal prenatal smoking (, p<.001; see also Table 2). The ORs represent one categorical increase in maternal prenatal smoking, i.e. from \u20180 cigarettes\/day\u2019 to \u20181\u20139,\u2019 \u20181\u20139\u2019 to \u201810\u201319,\u2019 and from \u201810\u201319\u2019 to \u226520 cigarettes\/day.\nMaternal prenatal smoking was also a significant predictor of PA in a multinomial regression with control factors [, p=.015]. The ORs were reduced to 1.33 (95% CI: 1.10 to 1.61) [, p=.004] for the contrast between high and low PA, and to 1.16 (95% CI: 0.99 to 1.35) [, p=.057] for the contrast between high and moderate PA. Other significant predictors of PA between ages 17 and 42 months were gender [, p<.001], presence of siblings [, p<.001], and hostile reactive parenting (by the mother) [, p<.001].\nHyperactivity-impulsivity\nA first multinomial regression predicting HI without control variables revealed significant contrasts between high and low HI [OR=1.75 (95% CI: 1.32 to 2.33), , p<.001], between high and low-moderate HI [OR=1.49 (95% CI: 1.20 to 1.85), , p<.001] and high and moderate-high HI [OR=1.25 (95% CI: 1.01 to 1.56), , p=.043], resulting in an overall significant effect (, p<.001). In a second multinomial regression with control variables the association of maternal prenatal smoking with HI was strongly attenuated, although the overall effect was still not too far from significance [, p=.12] (see also Table 2). This attenuation can also be seen in the contrasts between high and low HI [OR=1.38 (95% CI: 0.99 to 1.91), , p=.052], between high and low-moderate HI [OR=1.27 (95% CI: 0.98 to 1.63),, p=.070] and high and moderate-high HI [OR=1.13 (95% CI: 0.88 to 1.46), , p=.327]. The remaining significant predictors of HI between ages 17 and 42 months were gender [, p<.001], hostile reactive parenting (by the mother) [, p<.001], maternal depression [, p=.006], and age of the mother when she had her first child [, p=.012].\nCombining physical aggression and hyperactivity-impulsivity\nAlthough distinct, the trajectories of PA and HI were moderately but significantly related (, p<.001; Spearman r=.245, p<.001). The conditional probabilities for PA and HI in Table 3 show that, with higher PA or HI, there is also a higher probability of HI and PA, respectively. The conditional probabilities further reveal an asymmetry in the association in that most high HI children were likely to be high PA, but that most high PA children are not likely to be high HI. Table 3 also shows the joint probabilities of displaying particular levels of PA and HI simultaneously. There are 12 cells (3 PA\u00a0\u00d7\u00a04 HI). In order to test the contrasts relevant to our research question, i.e. that maternal prenatal smoking might specifically predict co-occurring PA and HI as opposed to PA only or HI only, these 12 groups were collapsed into 4 groups representing the combined group (PA3HI4), the high PA only group (PA3HI1\u2009+\u2009PA3HI2\u2009+\u2009PA3HI3), the high HI only group (PA1HI4\u2009+\u2009PA2HI4), and the remainder of the sample (PA1HI1\u2009+\u2009PA1HI2\u2009+\u2009PA1HI3\u2009+\u2009PA2HI1\u2009+\u2009PA2HI2\u2009+PA2HI3) (Table 4). For the next analysis, these four groups will constitute our dependent variable.Table 3Co-occurrence of physical aggression (PA) and hyperactivity-impulsivity (HI)Probability of PA conditional on HISumLow PAModerate PAHigh PALow HI.40.49.111.0Low-Moderate (L-M) HI.28.54.181.0Moderate-High (M-H) HI.18.49.331.0Chronic-High (C-H) HI.12.39.491.0Probability of HI conditional on PALow HIL-M HIM-H HIC-H HILow PA.19.51.27.031.0Moderate PA.12.48.36.041.0High PA.05.34.50.111.0Joint probability of PA and HILow HIL-M HIM-H HIC-H HISum\/PALow PA.05.13.07.01.25Moderate PA.06.24.18.02.51High PA.01.08.12.03.24Sum\/HI.12.45.37.061.0Data courtesy of the Institut de la Statistique du Qu\u00e9becTable 4Co-occurrence of physical aggression (PA) (2 levels) and hyperactivity-impulsivity (HI) (2 levels)Probability of PA conditional on HISumLow PAHigh PALow HI.77.231.0High HI.51.491.0Probability of HI conditional on PALow HIHigh HILow PA.96.041.0High PA.89.111.0Joint probability of PA and HILow HIHigh HISum\/PALow PA.73.03.76High PA.22.03.24Sum\/HI.95.051.0Data courtesy of the Institut de la Statistique du Qu\u00e9bec\nIn order to be able to conclude that prenatal smoking specifically predicts co-occurring PA and HI the a priori contrasts of all groups to the combined group should be significant. A multinomial regression with the control variables that had featured in the previous separate analyses of PA and HI showed that this was indeed the case. The overall effect of maternal prenatal smoking [, p=.015], and the predictions by prenatal smoking of the contrasts of the combined group with the PA only group [OR=1.39, 95% CI to 1.01 to 1.92, , p=.047], with HI only group [OR=1.72, 95% CI 1.06 to 2.86, , p=.029], and with the remainder of the sample [OR=1.59, 95% CI 1.16 to 2.17, , p=.004], were all significant (see also Table 2). Although additional contrasts were not significant, the contrast between the PA-only group and \u2018the remainder of the sample\u2019- group approached significance [, p=.08]. Other significant and independent predictors of the combined group were gender [, p<.001], presence of siblings [, p<.001], and hostile reactive parenting (by the mother) [, p<.001].\nDiscussion\nThe results of this study of early childhood show that maternal prenatal smoking was associated with PA but not with HI in covariate regression analyses when PA and HI were examined separately. When PA and HI were combined, a contrast between the PA-only group and the remainder of the sample, which was neither high on PA or HI, fell short of significance. However, maternal prenatal smoking predicted co-occurring elevated levels of PA and HI. Therefore the key finding of this study is that maternal prenatal smoking predicted high PA, but largely when it is co-occurring with high HI. Had we just examined PA and HI separately, as has often been done in studies of specificity, we would have missed the fact that HI is also sensitive to maternal prenatal smoking, even after controlling for several confounding variables, but only when it is combined with PA.\nThe first implication of these findings is that maternal prenatal smoking may be associated with the most severe forms of PA and HI, i.e. their combination, in early childhood. Second, the fact that maternal prenatal smoking predicts co-occurring PA and HI may be considered an important finding in light of evidence indicating that multiple co-occurring behavior problems at an early age are associated with a higher risk of persistent antisocial problems (Brame et\u00a0al., 2001; Broidy et\u00a0al., 2003; Nagin & Tremblay, 1999; Lacourse et\u00a0al., 2006; Lahey et\u00a0al., 2000; Moffitt, 1993; Moffitt, Caspi, Dickson, Silva, & Stanton, 1996), as well as with several other functional impairments (Waschbusch, 2002; S\u00e9guin et\u00a0al., 2004). Finally, the risk factors identified for these combined behavior problems, are largely modifiable.\nOur study replicates the reports from Orlebeke et\u00a0al. (1997), Williams et\u00a0al. (1998), Day et\u00a0al. (2000), and Tremblay et\u00a0al. (2004), based largely on early childhood behavior problems. However, we extend these findings because none of those early childhood studies had tested for the prediction of specific behavior problems or for the specificity of their combination. Tremblay et\u00a0al. (2004) covered the same developmental period and used the exact same PA scale as we did here on another sample. We both found that the association between maternal prenatal smoking and PA was not explained by control variables. However, our results for HI appear to contrast slightly with those of Romano et\u00a0al. (2006) who reported that the association between maternal prenatal smoking and hyperactivity was not explained by control variables. There are a number of key differences between the current study and Romano et\u00a0al. (2006). Besides using a different sample, we included impulsivity along with hyperactivity items, and focused exclusively on behavior during early childhood. Thus, in the current study the effects of maternal prenatal smoking on HI were largely explained by control variables. It is possible that control variables have a greater effect on the impulsivity component of HI than on the hyperactivity component. We note that our results appear to be consistent with those of Wakschlag, Leventhal et\u00a0al. (2006), who also failed to find a relation of maternal prenatal smoking but with ADHD, which includes impulsivity. Because of our goal for a strict test for specificity of effects on PA, it was more conservative to have a combination of impulsivity and hyperactivity items in our scale. Alternately, by having information extending beyond the early childhood period to estimate trajectories Romano et\u00a0al. (2006) may have more accurately identified children for whom maternal prenatal smoking matters beyond the effects of control variables. Despite this limitation to early childhood, and despite the fact that we cannot claim to have accounted for all possible control variables (examples include exposure to environmental smoking postnatally and forms of parental psychopathology other than maternal depression and parental history of antisocial behavior such as parental ADHD), this study replicates and extends all previous studies by providing an empirical basis for how the association of prenatal smoking to both PA and HI may manifest itself. It is now much more clear that studies need to examine co-occurrence of behavior problems.\nThere is a leap in drawing parallels between early childhood symptoms to DSM-based disorder, i.e., from PA to CD and from HI to ADHD. CD is characterized by aggression to people and\/or animals, deceitfulness or theft, vandalism and serious rule violations whereas ADHD is characterized by hyperactivity, behavior disinhibition (or impulsivity), and inattention and distractibility (American Psychiatric Association, 1994). It is not yet clear to what extent behavior problems during the early childhood years map onto ADHD and CD symptoms during later childhood and (antisocial) behavior problems during adolescence and adulthood, although we have shown that childhood PA trajectories and hyperactivity trajectories were respectively related to CD and ADHD measured in adolescence (S\u00e9guin et\u00a0al., 2004). But manifestation of PA and HI symptoms are often not sufficient to warrant diagnoses. We also note that behavior problems during the early childhood years tend to be more common and typically decline over time (C\u00f4t\u00e9 et\u00a0al., 2006; Romano et\u00a0al., 2006; Bongers, Koot, Van Der Ende, & Verhulst, 2003).\nA number of pioneering studies showed that behavior problems later in life (ranging from school-age to adulthood) could already be identified during early childhood (e.g. Campbell et\u00a0al., 1986; Keenan & Wakschlag, 2000), but only recently a number of studies have started to show, particularly with respect to HI, a significant consistency in the likelihood to display (very) high levels from age 1 1\/2 years onwards (e.g. Romano et\u00a0al., 2006; Shaw et\u00a0al., 2005). With respect to PA, there is a larger group of very young children showing rather high levels, but most of them will use less PA from early school age onwards (e.g. C\u00f4t\u00e9 et\u00a0al., 2006; NICHD Early Child Care Research Network, 2004; Shaw et\u00a0al., 2005). Such general trends might also underlie differences in number of groups identified by the trajectory methodology in samples with different age ranges. Whereas our three PA-groups were consistent with the three groups identified by some authors (e.g. C\u00f4t\u00e9 et\u00a0al., 2006) covering early and middle childhood, other authors identified more than three groups for this period (NICHD Early Child Care Research Network, 2004; Shaw et\u00a0al., 2005). However, most children will not yet have finished learning alternative strategies (i.e. have undergone \u2018socialization of aggression,\u2019 Tremblay, 2003) between 17 and 42 months, so the expected \u2018desisting\u2019 patterns of PA will not yet be evident. One thing the abovementioned studies have in common is that they show a degree of consistency between early and middle childhood conduct problems, i.e. children with chronic PA during middle childhood generally had high PA levels during early childhood as well. This, in turn, emphasizes the importance of searching for predictors of co-occurring early childhood behavior problems: children most at risk of subsequent behavior problems might be those who display both high PA and high HI at this stage.\nThe combination of conduct problems and hyperactivity-impulsivity-attention problems has been distinguished from their pure forms at a number of levels (Waschbusch, 2002). Although research has again mainly focused on older children with diagnosed disorders rather than the combination of behaviors such as PA and HI (but see S\u00e9guin, Arseneault, Boulerice, Harden, & Tremblay, 2002; S\u00e9guin et\u00a0al., 2004), evidence has been provided for a number of etiological and developmental differences between the comorbid and individual conditions. For example, greater behavioral and autonomic nervous system reactivity in response to provocation has been shown in the comorbid compared to the separate conditions (Waschbusch, 2002). Children with CD+ADHD were also shown to have lower baseline sympathetic arousal than children with the separate disorders (specifically those with ADHD only; Herpertz et\u00a0al., 2001). There is also evidence for differences in patterns of brain activity between CD+ADHD and the separate conditions (Banaschewski et\u00a0al., 2003) and for specific heritable risk of CD+ADHD (Dick, Viken, Kaprio, Pulkkinen, & Rose, 2005; Thapar, Harrington, & McGuffin, 2001). Several studies have shown that specific family psychosocial characteristics (e.g. parent-child conflict; parental psychopathology) specifically predict CD+ADHD or predict CD+ADHD more strongly than the separate conditions (e.g. Burt, Krueger, McGue, & Iacono, 2003; Pfiffner, McBurnett, Rathouz, & Judice, 2005). Thus, although extrapolation to CD and ADHD must be done with caution, the results of the present study support an etiological basis for the combined behavior problems in an early childhood sample.\nFinally, in addition to prenatal maternal smoking, we found that maternal hostile- reactive parenting was also associated with co-occurring PA and HI beyond its effects on the separate behaviors. Hostile-reactive parenting and similar \u2018negative\u2019 parenting behaviors have been associated with children\u2019s PA and HI levels in earlier studies (e.g. C\u00f4t\u00e9 et\u00a0al., 2006; Romano et\u00a0al., 2006; Shaw et\u00a0al., 2005; Tremblay et\u00a0al., 2004), Negative parenting has also been related to the failure of training programs aimed at improving child conduct problems (Webster-Stratton, Reid, & Hammond, 2001). This association could be seen as resulting from the impact of maternal behavior on their child\u2019s behavior. However, they may also reflect, at least in part, parent\u2019s reactions to their child\u2019s behavior. For instance, when faced with conduct problems, mothers of preschoolers at risk for ADHD and Oppositional Defiant Disorder tend to resort to more negative parenting strategies (Cunningham & Boyle, 2002). Furthermore, mothers hostile-reactive parenting toward their 5 month-old infants has been found to be partly driven by their infant\u2019s difficult temperament (Boivin et\u00a0al., 2005).\nOur finding that maternal hostile-reactive parenting is associated with an increased risk of co-occurring PA and HI may thus reflect the increased challenge of parenting when a child displays multiple behavior problems (cf. Seipp & Johnston, 2005). This pattern could lead to the establishment of a family coercive process and the further learning of anti-social behaviors by the child. However, the state of evidence precludes any definite conclusions at this point, and there is a clear need to better document the early dynamics of negative parenting and externalizing problems in early childhood.\nTo the extent that they may be involved in causation, the good news is that prenatal maternal smoking and hostile-reactive parenting could be significantly attenuated in prevention and intervention programs. The fact that the effects of smoking during pregnancy on both PA and PA+HI were robust, added to the evidence that PA+HI increases the risk for continued serious behavior problems, emphasizes the importance of smoking cessation during pregnancy (for a review on effectiveness of cessation programs, see Lumley, Oliver, Chamberlain, & Oakley, 2004). Considering the high prevalence of prenatal smoking (around 25% in Western countries), it will be important to further refine the identification of families most at risk of having children with a prepotency to develop co-occurring PA and HI. Our results further confirm that hostile-reactive parenting should be targeted in preventive intervention aimed at children with early multiple behavior problems. However, because parenting interventions often are the least effective in multiple-risk families (for a review, see Hutchings & Lane, 2005), including those characterized by prenatal smoking (Vuijk, van Lier, Huizink, Verhulst, & Crijnen, 2006), cessation programs targeting the most vulnerable families should be emphasized, intensified, and given appropriate means to succeed.","keyphrases":["prenatal smoking","physical aggression","hyperactivity","impulsivity","comorbidity"],"prmu":["P","P","P","P","P"]}
{"id":"Breast_Cancer_Res_Treat-3-1-2001220","title":"Femara\u00ae and the future: tailoring treatment and combination therapies with Femara\n","text":"Long-term estrogen deprivation treatment for breast cancer can, in some patients, lead to the activation of alternate cellular pathways, resulting in the re-emergence of the disease. This is a distressing scenario for oncologists and patients, but recent intensive molecular and biochemical studies are beginning to unravel these pathways, revealing opportunities for new targeted treatments. Far from making present therapies redundant, these new discoveries open the door to novel combination therapies that promise to provide enhanced efficacy or overcome treatment resistance. Letrozole, one of the most potent aromatase inhibitors, is the ideal candidate for combination therapy; indeed, it is one of the most intensively studied aromatase inhibitors in the evolving combinatorial setting. Complementary to the use of combination therapy is the development of molecular tools to identify patients who will benefit the most from these new treatments. Microarray gene profiling studies, designed to detect letrozole-responsive targets, are currently under way to understand how the use of the drug can be tailored more efficiently to specific patient needs.\nIntroduction\nThe proliferative, invasive, and metastatic potential of breast tumors may be largely predetermined at an early stage in the course of disease, whereas genetic alterations that accumulate during progression from in situ to metastatic disease are unpredictable and result in specific phenotypic changes and loss of sensitivity to treatments [1]; for example, although the estrogen receptor-positive (ER+) phenotype may be largely stable over time [2], hormone therapy-sensitive breast tumors may develop resistance and progress to a hormone-independent state [3]. In addition, progression to hormone independence may be associated with alterations in the expression of other regulatory genes, such as human epidermal growth factor receptor 2 (HER2) [4]. In the clinical setting, a quantitative decrease in ER expression was found in over 900 patients with primary breast cancer when HER2 was amplified [5].\nRecent advances in translational research studies have highlighted the complexity of ER signaling, including differential roles for the ER\u03b1 and ER\u03b2 subtypes [6], and multiple regulatory interactions between steroid hormone, growth factor, and other tyrosine kinase signaling pathways [7\u201310]. Greater understanding of tumor biology is beginning to help physicians to individualize treatment selection based on clinical, pathologic, molecular, and genetic profiling, and to rationally design novel combinations to improve efficacy and safety.\nThis article reviews novel approaches with the more potent third-generation aromatase inhibitor (AI) letrozole (Femara\u00ae; Novartis Pharmaceuticals) in combination strategies with agents targeting other growth factor pathways. The review explores the hypothesis that combining letrozole with specifically targeted therapies may delay or overcome endocrine therapy resistance in postmenopausal women with hormone receptor-positive (HR+) breast cancer.\nEndocrine therapy resistance\nIt is well established that breast cancer cells can adapt to low concentrations of estrogen by becoming hypersensitive to estradiol [11]. Long-term exposure to tamoxifen induces hypersensitivity to estradiol [12] and this adaptive change can result in resistance to endocrine therapy [13]. It has been postulated that tamoxifen is more susceptible than AIs to this phenomenon because of its intrinsic agonist properties [12]. Moreover, it has been suggested that highly potent AIs are required to block estrogen synthesis when breast cancer tumors are hypersensitive to small amounts of estradiol [13].\nRecent research has provided a compelling explanation for the development of resistance [13, 14]. Experiments using MCF-7 and other breast cancer model systems have identified alternative intracellular signaling pathways used by breast tumors to enhance and activate ER signaling, thus allowing cells to escape from the inhibitory effects of endocrine therapies [15, 16]. It has been shown that long-term estrogen deprivation upregulates ER\u03b1 and growth factor signaling pathways such as mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), and the mammalian target of rapamycin (mTOR) pathways [14\u201319]. Of note, Jeng et al. [15] reported that a specific inhibitor of MAPK (PD98059) could block the elevation of activated MAPK observed in MCF-7 cells exposed to long-term estrogen deprivation. Furthermore, studies in both wild-type and long-term estrogen-deprived MCF-7 cells suggested that mTOR has a key role in breast cancer cell proliferation and showed that mTOR inhibition by farnesylthiosalicylic acid (FTS) can reduce proliferation and induce apoptosis [19].\nGrowth factor pathways\nThe role of nongenomic pathways has been highlighted in resistance to antiestrogen therapy [20]. Classically, estrogens bind to nuclear ER to enhance transcription of genes important in breast cancer proliferation and survival (genomic pathway) [21]; however, estrogen may also act through ER located in or near the cell membrane [22]. Nongenomic actions include activation of various growth signaling pathways, including MAPK [15]. In addition, ER may indirectly activate epidermal growth factor receptors (EGFR) via coactivators, including src, leading to activation of EGFR [23\u201325]. Subsequently, dimerization of activated EGFR with other HER family receptors, particularly HER2, activates intracellular signaling pathways, which in turn may enhance nuclear ER signaling [4], thus completing a vicious cycle of events. Cross-talk between ER and HER2 pathways has been implicated in clinical resistance to tamoxifen [4, 7]. Shou et al. [4] reported that tamoxifen behaves as an agonist in MCF-7 breast cancer cells that express high levels of the coactivator AIB1 (src3) and HER2, resulting in de novo resistance. Interestingly, addition of an anti-EGFR tyrsosine kinase inhibitor eliminated cross-talk and restored tamoxifen\u2019s antitumor activity [4].\nIt has been postulated that AIs may be more effective than selective estrogen-receptor modulators (SERMs) [26] because they can block genomic and nongenomic activities of ER [27]. Elucidation of ER biology and interactions with growth factor signaling pathways will help to identify potential therapeutic targets for HR+ breast cancer [4, 28].\nCombination therapy\nSeveral strategies to inhibit growth factor signaling and signal transduction in breast cancer have been tested in the preclinical setting (see Fig.\u00a01). The humanized monoclonal antibody trastuzumab specifically targets the extracellular domain of HER2 [29, 30]. Amplification of HER2 occurs in approximately 25% of breast tumors and is associated with more aggressive disease and a poor prognosis [31]. Trastuzumab has been shown to restore sensitivity to estrogen and tamoxifen in estrogen-independent HER2-transduced MCF-7 cells [32]. Furthermore, trastuzumab blocked HER2 heterodimer formation and phosphorylation, reduced ERK1\/2 activity, and strongly inhibited cell growth in MCF-7 cells overexpressing EGFR and HER2 and resistant to tamoxifen [18]. Of note, synergistic inhibition of the BT474 breast cancer cell line was observed with trastuzumab and the HER dimerization inhibitor pertuzumab [33], which targets a different domain of HER2 [34]. The combination of tamoxifen and trastuzumab in ER+, HER2+ BT-474 cells resulted in synergistic growth inhibition due to the enhancement of cell accumulation in the G0\u2013G1 phase of the cell cycle, and a decreased number of cells in S phase [35].\nFig.\u00a01Cross-talk between signal transduction pathways and ER signaling in endocrine resistant breast cancer, with opportunities for targeted intervention. Estrogen (E2)-liganded ER activates E2-regulated genes in classical pathway (thick black arrow), but following long-term tamoxifen therapy resistance can develop with bidirectional cross-talk (gray arrows) between ER and growth factor receptors, with association of membrane bound ER with growth factor receptors, and\/or IGFR or EGFR\/HER2 activation of ER phosphorylation. Stars show various targeted therapies. AI aromatase inhibitor, SERD selective estrogen receptor down-regulator, MoAb monoclonal antibodies, TKI tyrosine kinase inhibitor, FTI farnesyltransferase inhibitor, CCI cell cycle inhibitor. Reprinted from [28] with permission from the American Association for Cancer Research\nHER signaling pathways can also be targeted by inhibiting receptor phosphorylation by intracellular tyrosine kinases [36]. Gefitinib and erlotinib both specifically inhibit the EGFR tyrosine kinase and have demonstrated inhibitory activity in models of hormone-resistant breast cancer [37, 38]. Interestingly, pretreatment of HER2+, hormone-resistant MCF-7 cells with gefitinib eliminated the agonist effects of tamoxifen and restored its antitumor activity [4]. In ER+ breast tumors, targeted therapy with single-agent gefitinib was found to be less effective than endocrine therapy [39]; however, tamoxifen plus gefitinib had greater activity than tamoxifen alone in vivo in hormone-sensitive cells [39]. Data also support the use of EGFR tyrosine kinase inhibitors in combination with HER2 antibodies, such as trastuzumab, against breast tumors that express EGFR and high levels of HER2 [37]. Alternatively, the tyrosine kinase inhibitor lapatinib provides single-agent targeting of both EGFR and HER2 [40, 41]. Using a panel of 31 human breast tumors, Konecny et al. [41] showed that response to lapatinib was significantly correlated with HER2 expression and its ability to inhibit HER2, Raf, Akt, and ERK phosphorylation. In vivo studies showed that lapatinib had a sustained antitumor effect that was further prolonged by combination with trastuzumab [41]. Another study showed that the combination of AEE788 (an EGFR\/HER2 tyrosine kinase inhibitor) with letrozole enhanced antiproliferative effects of these agents by 20\u201330% in MCF-7 and ZR75.1 cell lines and by 60\u201370% in the BT474 cell line [42]. In a model system of acquired resistance to letrozole, AEE788 partially restored sensitivity to letrozole, whereas rapamycin was not effective, suggesting that letrozole resistance and mTOR activation may not be connected in this model [43]. The authors concluded that inhibition of both HER2-mediated signaling and mTOR-dependent translation may restore responsiveness to letrozole in breast cancer refractory to this AI [43].\nStrategies to inhibit downstream signal pathways have also been developed. Farnesyl transferase inhibitors block the first and most important step in the activation of Ras signaling pathways [44]. Aberrant function of the Ras signal transduction pathway is common in breast cancer as a result of upstream activation via HER2 or EGFR [45]. The farnesyl transferase inhibitor R11577 (tipifarnib) was found to have antitumor activity against MCF-7 xenografts [44]. Another interesting therapeutic target is mTOR, a central regulator of G1 cell cycle protein synthesis, that precedes commitment to normal cellular replication [46]. Treatment of MCF-7 Arom-1 cells with letrozole and the mTOR inhibitor RAD001 resulted in a further 50% reduction in proliferation compared with letrozole alone [47]. Another set of experiments, developed to test the hypothesis that Akt kinase confers resistance to endocrine therapy through suppression of ASK1\/JNK pathway, showed that combining RAD001 with letrozole restored activation of the ASK\/JNK pathway and increased the sensitivity of MCF-7 cells with constitutively active Akt to endocrine therapy [48]. Studies have also targeted the selective estrogen receptor down-regulator (SERD) fulvestrant, which indirectly inhibits growth factor pathways by down-regulating ER [3]. In MCF-7Ca xenografts, combined treatment with fulvestrant and letrozole prevented increases in HER2 and activation of MAPK and inhibited tumor growth [49].\nThese preclinical models (see Table\u00a01) suggest that treatments to reduce growth factor signaling pathways may be useful in the treatment of human breast cancer [52]. Specifically, novel combination strategies may be developed to prevent or delay the development of endocrine therapy resistance [3, 39], to restore sensitivity to endocrine therapy [53], and to treat hormone-resistant tumors [18, 46]. Anti-vascular endothelial growth factor (VEGF) therapy with bevacizumab may be able to overcome resistance to endocrine therapy and improve efficacy in HR+ metastatic breast cancer [54], and preclinical models have shown that the estrogen-induced increase in VEGF expression may be counteracted by aromatase inhibition. Inhibition of growth factor signaling and angiogenesis pathways may be rationally combined with conventional endocrine strategies for breast cancer [4, 35, 50, 55, 56].\nTable\u00a01Summary of letrozole in combination with growth factor signaling inhibitors in preclinical modelsTarget for growth factor inhibitorCombination regimenSummary of key findingsReferencesEGFR\/HER2Letrozole\u00a0+\u00a0AEE78Combination enhanced antiproliferative effects in MCF-7 (ER+ HER2\u2212), ZR75.1 (ER+ HER2+), and BT474 (ER+ HER2+) cell lines[42]Partial restoration of growth inhibitory effects of letrozole in refractory cell lines (LTLT-Ca; long-term letrozole treated)[43]mTORLetrozole\u00a0+\u00a0RAD001 (everolimus)Letrozole\u00a0+\u00a0RAD001 significantly increased apoptosis compared with either agent alone[50]Co-treatment increased sensitivity to letrozole in resistant MCF-7 cells with constitutively active Akt[48]RAD001 increased antiproliferative effects of letrozole in MCF-7 Arom 1 cell line[47]IGFBPLetrozole\u00a0+\u00a0rhIGFBP-3rhIGFBP-3 Enhanced letrozole activity in MCF-7-Ca cells in vitro and in vivo[51]rhIGFBP recombinant human insulin-like growth factor binding protein\nThe activity of inhibitors of growth factor signaling depends on the presence of specific cellular aberrations, such as overexpression of HER2 [57] or mutations of EGFR [58]. Consequently, targeted therapies may have limitations as single agents because the target is active in a restricted subset of patients, and breast tumors may undergo adaptive changes to render the target redundant. Since tamoxifen exhibits agonist effects on breast cancer cells exposed to long-term estrogen deprivation [12], it may be better to combine AIs with inhibitors of growth factor signaling. Letrozole is one of the most potent AIs and is one of the most extensively studied AIs in combination with new agents (see Table\u00a01).\nClinical trials of letrozole in combination with inhibitors of growth factor signaling pathways\nBased on results from preclinical studies, several clinical trials of novel combinations are under way, with the aim of improving efficacy and safety of endocrine therapy with letrozole (see Table\u00a02). Many of these trials are being conducted in patients with locally advanced or metastatic breast cancer who have failed prior tamoxifen or have a suboptimal response to letrozole. This represents a high-risk, difficult-to-treat population who are candidates for cytotoxic chemotherapy. Preliminary results have shown that letrozole can be safely combined with trastuzumab, lapatinib, everolimus, tipifarnib, bevacizumab, and imatinib. It is too early, however, to make definitive conclusions about efficacy and clinical benefits with these novel combinations.\nTable\u00a02Clinical studies of letrozole and inhibitors of growth factor signaling pathwaysTarget for growth factor inhibitorCombination regimenStudy type and patient populationSummary of key findingsReferencesHER2Letrozole\u00a0+\u00a0trastuzumabPhase IIMetastatic BC, postmenopausal, ER+ and\/or PR+, HER2+ (n\u00a0=\u00a031)ORR 26%; median TTP 5.8\u00a0months[59]EGFR\/HER2Letrozole\u00a0+\u00a0lapatinibPhase IAdvanced BC (ER+ or PR+) or other tumors (n\u00a0=\u00a036)Letrozole\u00a0+\u00a0lapatinib safely combined at recommended single agent doses[60]Phase IIIAdvanced\/metastatic BC (n\u00a0=\u00a01,200 target accrual)Ongoing trial; primary end point TTP[61]mTORLetrozole\u00a0+\u00a0RAD-001 (everolimus)Phase IbAdvanced BC pts with suboptimal response to letrozole (n\u00a0=\u00a06)RAD001 pharmacokinetics not altered by letrozole[62]Phase IIPresurgical therapy in patients with newly diagnosed ER+ BC (n\u00a0=\u00a0255 planned)Ongoing trial of efficacy and biomarkers[63]Letrozole\u00a0+\u00a0CCI-779 (temsirolimus)Phase IIAdvanced or metastatic BC (n\u00a0=\u00a092)No difference in ORR, but trend to longer PFS with letrozole\u00a0+\u00a0temsirolimus (30\u00a0mg)[64]Phase IIIAdvanced or metastatic breast cancer (n\u00a0=\u00a01,236 planned)Terminated[65]Farnesyl transferaseLetrozole\u00a0+\u00a0tipifarnib Randomized phase IIAdvanced or metastatic BC that has progressed on tamoxifen (n\u00a0=\u00a0121)ORR 38% for letrozole and 26% for letrozole\u00a0+\u00a0tipifarnib (NS)[66]Randomized, placebo-controlled phase IIAdvanced or metastatic BC that has progressed on antiestrogen therapy (n\u00a0=\u00a0120)No longer recruiting[67]VEGFLetrozole\u00a0+\u00a0bevacizumab (anti-VEGF monoclonal antibody)Phase IIMetastatic BC, postmenopausal, candidates for AI (n\u00a0=\u00a028)Letrozole\u00a0+\u00a0bevacizumab is well-tolerated[54]Endocrine therapy (tamoxifen or aromatase inhibitor)\u00a0+\u00a0bevacizumabPhase III placebo-controlledFirst-line therapy in ER+\/PR+ Metastatic BC (n\u00a0=\u00a0TBC)Planned trialPlanned CALGB trial [54]Bcr-ablLetrozole\u00a0+\u00a0imatinibPhase IIMetastatic BC, postmenopausal ER+ and\/or PR+ (n\u00a0=\u00a015)Letrozole\u00a0+\u00a0imatinib is feasible[68]BC breast cancer, ORR overall response rate, TTP time to progression, PFS progression-free survival, AI aromatase inhibitor, TBC to be confirmed\nIn a clinical trial [69] designed to test whether combination therapy with letrozole and bevacizumab was possible, patients with ER+ or progesterone receptor-positive (PR+) metastatic or locally advanced breast cancer were treated with letrozole (2.5\u00a0mg daily) and bevacizumab (15\u00a0mg\/kg IV every 3\u00a0weeks) [54]. The majority of patients had received prior therapy with a nonsteroidal AI. The combination of bevacizumab and letrozole was found to be well-tolerated. Common drug-related toxicities reported were hypertension, fatigue, headache, and joint pain. Median progression-free survival was reported to be 10\u00a0months, and this compares favorably with the published data on median time to progression with first-line letrozole (9.4\u00a0months) [70]. However, analysis of efficacy and biomarker data was confounded by the long duration of prestudy aromatase inhibition [54, 71]. Nevertheless, when the data were corrected for duration of previous AI therapy, the study did determine that changes in circulating endothelial cell (CEC) levels may be a biomarker of response or progression on anti-angiogenic therapy [71]. Based on these findings, a randomized, double-blind, placebo-controlled trial of bevacizumab combined with endocrine therapy in patients with ER+ or PR+ metastatic breast cancer has been initiated by the Cancer and Leukemia Group B (CALGB) (see Fig.\u00a02) [54]. The primary end point of the trial is progression-free survival.\nFig.\u00a02Planned CALGB trial of first-line endocrine therapy (tamoxifen or aromatase inhibitor) with or without bevacizumab\nLetrozole plus trastuzumab produced durable responses in about one in four patients with ER\/PR+, HER2+ metastatic breast cancer, but early progression occurred in one in two patients [59]. This suggests that common resistance pathways may be responsible for relapse [59]. Targeting multiple pathways may reduce the risk of resistance. The combination of letrozole and the dual EGFR\/HER2 inhibitor lapatinib was found to be feasible and well-tolerated in a phase I study [60], and this regimen is currently being compared with letrozole plus placebo in a phase III study in women with ER\/PR+ advanced or metastatic breast cancer [61] (see Fig.\u00a03). Future studies will focus on finding the right combination or sequence of agents for different patients in specific treatment settings. Furthermore, studies in the neoadjuvant setting in patients with locally advanced breast cancer will allow correlative biomarker assessment, such as the proliferation marker Ki-67, to determine the efficacy of combination therapies. For example, everolimus and letrozole are being studied as preoperative therapy of primary breast cancer in postmenopausal women [63]. In this phase II trial, patients are randomized to receive letrozole in combination with everolimus or placebo, an adaptive design strategy, so that identification of biomarkers can be used to optimize patient selection for a future phase III trial of first-line combination therapy in patients with advanced breast cancer. Even if a biomarker is not identified, the trial is adequately powered to demonstrate a statistically significant difference in treatment effect of the combination in the overall population.\nFig.\u00a03Letrozole and lapatinib phase III trial design. Target recruitment: 1,280 patients\nCombination strategies may also change the conventional approach for treatment selection based on HR status. Interestingly, it has been suggested that treatment of HER2+, ER\u2212 metastatic breast cancer with trastuzumab may transform the tumor phenotype to become hormone responsive [72]. The hypothesis-generating study found that three of ten patients demonstrated ER overexpression at 9, 12, and 37\u00a0weeks, respectively, following the initiation of trastuzumab. Two of these patients were subsequently treated with endocrine therapy alone and one received letrozole for 3\u00a0years without evidence of progression. Further trials of letrozole used in combination with trastuzumab or sequentially in patients with HER2+, ER\u2212 tumors appear warranted.\nMicroarray\/gene profiling studies and optimization of treatment with letrozole\nThe growing importance of biomarkers in oncology has been reflected in the US Food and Drug Administration (FDA), the National Cancer Institute (NCI), and the Centers for Medicare and Medicaid Services (CMS) Oncology Biomarker Qualification Initiative (OBQI), designed to improve the development of cancer therapies and the outcomes for cancer patients through biomarker development and evaluation [73]. Not only are biomarkers potentially useful as prognostic and predictive factors but they also serve as surrogate end points for long-term outcomes. For example, Dowsett et al. found that changes in Ki-67 in the neoadjuvant setting may be used to predict likely benefit (improved disease-free and overall survival) of AIs in the adjuvant setting [74], potentially expediting clinical development. Therefore, identifying biomarkers that predict endocrine resistance is essential for the optimal use of letrozole. In addition, biomarker studies that help to delineate the oncogenic pathways may be particularly useful for the development of novel combination therapies with letrozole.\nGene expression analysis of tumors represents a novel approach for biomarker development that promises to increase the understanding of breast cancer heterogeneity and facilitate the development of individualized treatment strategies. Microarray analysis, such as the Affymetrix Human Genome Gene Chips, is an exciting development in breast cancer diagnostics that allows the expression of genes in tumors to be quantified using RNA retrieved from breast cancer biopsies. Using cDNA microarray and unsupervised clustering analysis, ER+ breast cancer can be subdivided into at least two subtypes, luminal A and luminal B, with distinct gene expression patterns and clinical outcomes [75\u201377]. Supervised analysis, comparing gene expression patterns between tumors that relapsed and those that did not, generated a \u201cgene signature\u201d as an independent prognostic parameter for lymph-node negative disease [78, 79]. The National Surgical Adjuvant Breast and Bowel Project (NSABP) has developed a multigene profiling assay, using reverse transcriptase polymerase chain reaction (RT-PCR) to quantify the expression of 21 genes [80]. The 21-gene assay (Oncotype DX, Genomic Health) can predict risk of recurrence in patients with HR+ early breast cancer receiving tamoxifen [80, 81] or chemotherapy [82]. Naderi et al. [83] have also reported validation of gene expression signatures that may have predictive value in the clinic. Furthermore, newly identified interactions between ER and other signaling pathways have been studied using microarray analysis [84, 85]. For example, Bex1 and Bex2 genes have been identified as novel breast cancer-related genes and identify a subtype of ER+ tumors associated with estrogen response and nuclear factor kappa B (NF-\u03baB) pathways [84]. Advances in gene profiling will provide further insights into tumor biology and improve prediction of likely response to specific therapies in the clinic.\nMicroarray analysis has shown to be valuable in predicting response to neoadjuvant endocrine therapy and was used in a study of neoadjuvant letrozole 2.5\u00a0mg\/day administered to postmenopausal women with large operable or locally advanced breast cancers for 3\u00a0months [86, 87]. Changes in patterns of gene expression were assessed from tissue samples taken at diagnosis, 14\u00a0days, and 3\u00a0months (N\u00a0=\u00a058). Changes in gene expression level with treatment were identified as early as 14\u00a0days and involved classical markers of estrogen action (trefoil factors 1 and 3, LIV-1, KIAA0101) as well as tumor proliferation (cyclin D1, cyclin B2, CSK2, cell division cycle 2). The objective clinical response to neoadjuvant letrozole was 71% (N\u00a0=\u00a052 assessable). Of note, the observed changes in gene expression, when clustered, were predictive of response in all cases except one, whereas classical markers of estrogen action were not predictive [87]. In another study, postmenopausal women with primary operable breast cancer were randomized to 2\u00a0weeks of presurgical treatment with letrozole 2.5\u00a0mg\/day or anastrozole 1\u00a0mg\/day [88]. Microarray gene expression profiling (Breakthrough Centre cDNA chips) of biopsies taken before and during treatment identified differences in gene expression patterns between the two AIs, although the clinical significance of these preliminary findings remains to be clarified. Itoh et al. [89] also found differences between letrozole, anastrozole, and tamoxifen using microarray analysis on MCF-7 cells stably transfected with the aromatase gene (MCF-7Aro). Gene expression patterns revealed a high correlation between the AIs (letrozole and anastrozole) and a clear difference between AIs and tamoxifen [89].\nEmerging data therefore suggest that estrogen-responsive genes are candidate biomarkers [90] and may be useful in the clinic as predictive factors of benefit from AI treatment [89]. The value of gene expression profiling is now being evaluated in prospective studies. MINDACT (Microarray In Node negative Disease may Avoid ChemoTherapy), an ongoing three-part randomized trial of 6,000 patients with node-negative breast cancer, is comparing the efficacy of selection of breast cancer patients for adjuvant chemotherapy based on either clinical criteria or the 70-gene microarray prognosis profile [91]. In one part of the trial, 3,500 node-negative, HR+ positive patients will be randomized to receive either 7\u00a0years of letrozole or 2\u00a0years of tamoxifen followed by 5\u00a0years of letrozole. In the US, the TAILORx Trial [92] is comparing hormone therapy with or without combination chemotherapy as adjuvant therapy for node-negative, ER+ breast cancer. The objective of this randomized phase III trial is to determine the best individual therapy using Oncotype DX gene profiling.\nOne of the more recent advances in transcriptional profiling addresses its potential application as an identification tool for \u201concogenic pathway signatures\u201d that could be used to guide targeted therapy. Oncogenic pathway signatures, developed in cell-line models, have been shown to predict sensitivity to therapeutic agents in vitro [93]. This offers an opportunity to identify pathway-specific drugs in endocrine therapy-resistant tumors and creates an opportunity for the rational design of combination therapy with letrozole. In addition to the RNA-based gene expression profiling approach, high throughput analysis at the DNA level, such as genome-wide microarray comparative genomic hybridization (aCGH) and DNA sequencing, has made it possible to decipher the genetic anomalies that drive a particular tumor phenotype. The use of these molecular approaches for biomarker development is still in its infancy but is now feasible with recent advances in genomic technologies.\nConclusions\nHormone-sensitive breast cancer can be regarded as a chronic disease with a persistent risk of escape from effective endocrine control. Activation of growth factor signaling pathways has been implicated in progression of HR+ breast cancer to an estrogen-independent phenotype and the development of resistance to endocrine therapy, particularly tamoxifen. It has been hypothesized that combining endocrine therapy with targeted signal transduction inhibitors may circumvent hormone-independent signaling pathways, so that patients may experience prolonged disease control.\nLetrozole is one of the most potent AIs. As such, it may be more effective than tamoxifen for patients with tumor profiles associated with a high risk of developing hormone resistance (e.g., tumors with HER2 gene amplification) and represents an ideal combination partner for agents that inhibit growth signaling pathways implicated in hormone resistance. The efficacy of letrozole is currently being investigated with a variety of signal transduction inhibitors with different mechanisms of action, including monoclonal antibodies against HER family receptors, receptor tyrosine kinase inhibitors, and downstream signaling pathway inhibitors.\nGene expression profiling has been validated as a useful new tool to predict risk of relapse in patients treated with hormone therapy and chemotherapy. This approach will help physicians to identify which patient will likely benefit from specific therapies, such as letrozole. Tailoring therapy to individual patient profiles (clinical, histologic, pathologic, and genetic) will become more sophisticated in the future, helping to maximize the benefits of endocrine therapy throughout the breast cancer continuum; letrozole will undoubtedly become an integral part of the next generation of tailored combination regimens for the treatment of breast cancer.","keyphrases":["combination therapy","breast cancer","letrozole","aromatase inhibitors","postmenopausal","adjuvant therapy","mechanism of resistance"],"prmu":["P","P","P","P","P","P","R"]}
{"id":"Mol_Biochem_Parasitol-1-5-1906846","title":"Characterisation of exogenous folate transport in Plasmodium falciparum\n","text":"Folate salvage by Plasmodium falciparum is an important source of key cofactors, but little is known about the underlying mechanism. Using synchronised parasite cultures, we observed that uptake of this dianionic species against the negative-inward electrochemical gradient is highly dependent upon cell-cycle stage, temperature and pH, but not on mono- or divalent metal ions. Energy dependence was tested with different sugars; glucose was necessary for folate import, although fructose was also able to function in this role, unlike sugars that cannot be processed through the glycolytic pathway. Import into both infected erythrocytes and free parasites was strongly inhibited by the anion-channel blockers probenecid and furosemide, which are likely to be acting predominantly on specific folate transporters in both cases. Import was not affected by high concentrations of the antifolate drugs pyrimethamine and sulfadoxine, but was inhibited by the close folate analogue methotrexate. The pH optimum for folate uptake into infected erythrocytes was 6.5\u20137.0. Dinitrophenol and nigericin, which strongly facilitate the equilibration of H+ ions across biological membranes and thus abolish or substantially reduce the proton gradient, inhibited folate uptake profoundly. The ATPase inhibitor concanamycin A also greatly reduced folate uptake, further demonstrating a link to ATP-powered proton transport. These data strongly suggest that the principal folate uptake pathway in P. falciparum is specific, highly regulated, dependent upon the proton gradient across the parasite plasma membrane, and is likely to be mediated by one or more proton symporters.\n1\nIntroduction\nReduced folates are essential cofactors for one-carbon transfer reactions, including the conversion of dUMP to dTMP, which is a prerequisite for DNA synthesis. Because of this, the folate pathway has long been a target for drugs deployed against rapidly reproducing cells such as cancers and a range of microbial pathogens. Whereas most microorganisms can synthesise the folates they need from the simple precursors GTP, p-aminobenzoic acid (pABA) and glutamate, higher animals (but not plants) have lost the ability to do this and depend on dietary intake of pre-formed folate as an essential nutrient. The human malaria parasite Plasmodium falciparum is able to exploit both of these routes [1\u20134]. Thus, it can utilise folate provided in culture medium in vitro or salvaged from the host plasma in vivo on the one hand, or convert the above precursors de novo into folate derivatives on the other, a characteristic also shared by the related apicomplexan parasite Toxoplasma gondii\n[5,6]. The relative importance of the biosynthetic and salvage pathways across the complete life cycle in vivo and the interplay between them is poorly understood, although existing data support the view that both are necessary for healthy propagation of the parasite, at least in the erythrocytic stages [7].\nAn important aspect of exogenous folate utilisation is the machinery and mechanism(s) by which folate is imported into the parasite. The highly polar nature of folate derivatives suggests that salvage must employ some kind of mediated transport process, as diffusion alone across the membrane is likely to be far too inefficient. Moreover, folate molecules are dianionic at physiological pH and must be imported into the parasite against an inwardly negative electropotential that has been measured as ca. \u221295\u00a0mV in P. falciparum\n[8]. Although the transport of other key molecules, such as pantothenate, lactate, glucose and choline, has been investigated [9\u201313], there has been no detailed study to date of this aspect of folate metabolism in P. falciparum, a better understanding of which might lead to new ways of inhibiting parasite growth. A preliminary approach to this end has been taken by demonstrating that the anti-gout drug, probenecid, which among other things inhibits folate transport in mammalian cells [14], can increase the sensitivity of P. falciparum to antifolate inhibitors [15]. Here, we demonstrate that folate uptake by this parasite is a regulated process that is critically dependent upon provision of glucose or another sugar that can proceed through the glycolytic pathway, that the existence of a pH gradient across the plasma membrane is also required for efficient transport, and that folate is transported principally by a proton-symport mechanism.\n2\nMaterials and methods\n2.1\nChemicals\n[3\u2032,5\u2032,7,9-3H]folic acid, 24\u00a0Ci\u00a0mmol\u22121, 1\u00a0mCi\u00a0ml\u22121 was from Amersham, UK, [3\u2032,5\u2032,7,9-3H]folinic acid (26\u00a0Ci\u00a0mmol\u22121, 1\u00a0mCi\u00a0ml\u22121) and [3\u2032,5\u2032,7,9-3H]5-methyltetrahydrofolic acid (44\u00a0Ci\u00a0mmol\u22121, 1\u00a0mCi\u00a0ml\u22121) were both from Moravek, California. Folic acid, folinic acid, 5-methyltetrahydrofolic acid, 2,4-dinitrophenol, concanamycin A, d- and l-glucose, d-fructose, d-xylose, d-galactose, 6-deoxy-d-glucose, probenecid and furosemide were all purchased from Sigma, UK. Note that we use the term \u2018folate\u2019 generically to indicate derivatives of the folate family of molecules regardless of their oxidation state, modifications at the 5 and 10 positions or polyglutamation status.\n2.2\nParasite culture\nP. falciparum was routinely cultured under 1% O2, 3% CO2, 96% N2 in RPMI 1640 medium, supplemented with d-glucose (22\u00a0mM final concentration), hypoxanthine (36\u00a0mM), HEPES (25\u00a0mM), gentamicin sulfate (50\u00a0\u03bcg\/ml) and 0.5% Albumax II (Invitrogen). The cultures were synchronised by haemolysis of mature, late trophozoite-stage parasitised erythrocytes by suspension in 9 volumes of a 5% sorbitol solution at room temperature for 5\u00a0min. Cells surviving the treatment were used to set up new cultures and the process repeated where necessary to achieve a tighter synchronisation [16]. Given that synchrony is never perfect, time zero for the erythrocytic cycle was taken as the point where ca. 90% of infected erythrocytes were ring form, with the remainder as very late schizonts. To obtain free parasites, synchronised cultures, normally in the late (mature) trophozoite stages (ca. 30\u00a0h into the cycle), were quickly lysed with 0.05% (w\/v) saponin at room temperature, permeabilising the red cell and parasitophorous membranes and leading to haemolysis [17,18]. Any remaining unlysed red cells were further treated with a PBS wash containing the same amount of saponin. The freed parasites were then resuspended in the appropriate buffer with or without glucose, depending upon the purpose of the assays. Saponin-freed parasites that are glucose-replete maintain intracellular levels of ATP for periods of at least 30\u00a0min and do not show evidence of leakage [19].\n2.3\nUptake assay of radiolabelled folates\nFolate uptake\/export assays were performed on free parasites, parasitised red cells or uninfected red cells, as appropriate. Cell numbers were estimated with a haemocytometer. Normally preparations containing 107\u2013108 parasites were used for each assay point (or ca. 107\u2013108 uninfected red cells). Prior to uptake assays by naked parasites, infected cells were lysed in 0.05% saponin and the freed parasites washed either in PBS or folate\/pABA depleted RPMI 1640, depending upon the experiment. Wash steps were also performed with different buffered or non-buffered isotonic salt solutions where necessary as indicated in the relevant text.\nAll cells, washed extensively to remove folate present in the culture medium, were mixed with or without glucose (20\u00a0mM final concentration) and inhibitors as appropriate, together with radiolabel (normally 1\u00a0\u03bcCi\/ml, equivalent to 38\u00a0nM, unless otherwise stated) to make up a total volume of 100\u00a0\u03bcl with PBS or alternative buffer. All the components were prewarmed to 37\u00a0\u00b0C before the start of the assay, the reaction mix incubated at 37\u00a0\u00b0C normally for 30\u00a0min, and stopped by addition of 1\u00a0ml ice-cold PBS.\nWhen assaying for folate uptake by whole parasitised red cells, these were spun down after the uptake period and the pellet washed with 1\u00a0ml ice-cold PBS at least three times to bring extracellular label down to the background level. The washed parasite pellet obtained by subsequent saponin treatment was then lysed with 0.02% SDS before counting. Uptake by naked parasites was determined in the same way. Unless otherwise indicated, the uptake values represent labelled folate that has been imported into synchronised trophozoite stage parasites. All assays were routinely performed in triplicate on different batches of parasites unless otherwise indicated, and the data expressed as the mean\u00a0\u00b1\u00a0S.D.\n2.4\nExtraction and affinity purification of folate derivatives\nFor characterisation of labelled folates, parasite pellets were washed three times in 1\u00a0ml PBS to remove any extracellular label and resuspended in 1\u00a0ml extraction buffer (0.1\u00a0M Tris\u2013HCl, 2% ascorbic acid, pH 7.5), heated in a boiling water bath for 10\u00a0min, centrifuged at 10,000\u00a0\u00d7\u00a0g for 10\u00a0min and supernatants stored at \u221220\u00a0\u00b0C until affinity purification and HPLC analysis, performed as described [20]. Unlabelled folic acid, folinic acid and PteG2 to PteG5 of the polyglutamated forms of folic acid were added into the extracted folates as internal standards. An identical aliquot of sample was also spiked with 3H-folinic acid in a second run to confirm the identity of the radioactive peak of folinic acid that had been extracted from the parasites.\n2.5\npH measurements\n3H-folinic acid uptake as a function of pH was measured in a combination HEPES-MES buffer system containing 20\u00a0mM of each component, and the salt concentration made up to 154\u00a0mM with NaCl. By taking the temperature coefficients of the buffers into account, pH values were adjusted at room temperature such that the apparent pH value at the assay temperature (37\u00a0\u00b0C) was exactly the pH required. Glucose was added to 20\u00a0mM as required. The internal pH of isolated parasites was measured as described [21] using the pH-sensitive fluorescent indicator BCECF-AM. Loading was achieved by incubating isolated parasites suspended at a cell density of 0.9\u20132.1\u00a0\u00d7\u00a0108\u00a0cells\/ml in folate and pABA depleted RPMI 1640 without Albumax II, and containing 10\u00a0\u03bcM BCECF-AM, for 10\u00a0min at 37\u00a0\u00b0C. The cells were then washed by centrifugation and resuspended in culture medium and used immediately. An aliquot of cells was transferred to an appropriate buffer in a 2\u00a0ml cuvette in the temperature-controlled chamber of a spectrofluorometer, maintained at 37\u00a0\u00b0C. The sample was measured at 520\u00a0nm after successive excitations at 440 and 495\u00a0nm. The ratio of the fluorescence emissions (excitation at 495\u00a0nm\/excitation at 440\u00a0nm) was used to calculate the internal pH of the cells. A standard curve was prepared with parasites resuspended in 130\u00a0mM KCl, 10\u00a0mM NaCl, 1\u00a0mM MgCl2 and 40\u00a0mM of MES-HEPES buffer from pH 5.5 to pH 8 and 30\u00a0\u03bcM nigericin [21].\n3\nResults\n3.1\nUptake of folates over the asexual erythrocytic cycle\nTo monitor the ability of P. falciparum to salvage folate at different stages of the erythrocytic cycle, synchronised parasite cultures were incubated with different forms of radiolabelled folate. 5-Methyltetrahydrofolate (5-MeTHF) is the fully reduced form most abundant in human plasma [22] and thus the principal derivative available to the parasite in vivo. Folinic acid (5-formylTHF), although not known to be a normal physiological substrate for the parasite, is a more stable member of the fully reduced (active) forms that closely resembles 5-MeTHF and is used clinically (leucovorin). The capacity of ring stages to take up both of these compounds was very low but increased markedly ca. 15\u00a0h into the cycle as the parasites entered the trophozoite stages, peaking at around 30\u201335\u00a0h, before falling to very low levels again in mature schizonts (Fig. 1a). The numbers of parasites at the different stages were monitored throughout such time-courses and showed that uptake of folate increased as the parasites proceeded through the trophozoite stages against a near-constant number of infected cells, as expected for a synchronous culture. At around 40\u00a0h, some schizonts began to release merozoites, leading to a sharp rise in the total number of infected cells, but the uptake from this population continued to diminish, consistent with lower levels of uptake for both rings and schizonts, relative to trophozoites (Fig. 1a). To check that the marked reduction of uptake in the latter stages of the cell-cycle (beyond 40\u00a0h) was not due to parasites becoming unhealthy or inviable, the phenomenon was also monitored through a subsequent cycle, where the same pattern was repeated (data not shown). Only minor differences were seen between parasite clones of different lineages, and labelled folic acid, the stable oxidised form extensively used experimentally to study folate metabolism and a common dietary supplement, gave qualitatively similar results to the reduced folates. This pattern strongly suggested that folate uptake is a process regulated by P. falciparum, likely to involve a transport mechanism more complex than facilitated diffusion. On the basis of the data above, parasites at the late trophozoite stage of synchronous cultures were used to investigate the characteristics of uptake in subsequent experiments, using 3H-folinic acid as the label, as this is considerably more stable than 5-MeTHF, is more readily available in labelled form, and has been shown in earlier studies to provide a better source of cofactor than equimolar amounts of folic acid [23].\nUptake of folinic acid into the parasite was also markedly dependent upon temperature. Only a very low level of transport was observed between 10 and 25\u00a0\u00b0C, over which the parasites are viable, but metabolically retarded. The rate of uptake increased dramatically above ca. 30\u00a0\u00b0C, peaking quite sharply between 37 and 39\u00a0\u00b0C, but was abolished entirely above 45\u00a0\u00b0C, a temperature at which the parasites are no longer viable. Data for free parasites are shown in Fig. 1b; very similar profiles were obtained for infected erythrocytes with the same optimum temperature (data not shown). This demonstrated that active parasite metabolism in live organisms at their normal growth temperature is required for folate to be imported to a significant degree.\n3.2\nKinetics of folate uptake\nAssays of the time-dependence of folate uptake into trophozoites showed that it was linear (R2\u00a0\u2265\u00a00.98) over a period of at least 80\u00a0min (Fig. 2a). In subsequent experiments, unless otherwise indicated, a standard incubation time of 30\u00a0min was therefore adopted to ensure that adequate counts for accurate measurements were obtained, whilst remaining well within the window of linearity. Uptake rates of folinic acid into parallel samples of free parasites and infected red cells in equal numbers were comparable, with somewhat higher levels measured in the latter (Fig. 2b), indicating that the rate-limiting step is not passage across the erythrocytic membrane, but rather across the parasite plasma membrane. In free parasite cells, the folate uptake process exhibited saturable and non-saturable components (Fig. 2c), indicative of more than one mechanism. In low concentrations of the substrate (in the region of 0 to 15\u00a0\u03bcM, which encompasses the normal range of human plasma folate, 30\u201340\u00a0nM [24]), folinic acid uptake is non-linear, consistent with the operation of a carrier-mediated process (Fig. 2c). At higher concentrations up to mM levels, uptake became linear, a phenomenon also observed in other folate transport systems [25,26], including that of Toxoplasma\n[6]. Subtraction of the linear component yielded Km values for the saturable process of 3.4\u00a0\u00b1\u00a00.8 and 2.4\u00a0\u00b1\u00a00.3\u00a0\u03bcM, respectively, for FCB and K1 parasite strains (Fig. 2d). Interestingly, while their Km values were closely similar, the  observed for K1 (34.7\u00a0\u00b1\u00a02.6\u00a0pmol\/107\u00a0parasites\/h) was ca. 10-fold higher than that for FCB (3.3\u00a0\u00b1\u00a00.3\u00a0pmol\/107\u00a0parasites\/h).\n3.3\nExport of folinic acid\nExport of folinic acid from free parasites was compared with that from uninfected cells. Uptake was first allowed to proceed into either infected or uninfected erythrocytes for 1\u00a0h in PBS plus 20\u00a0mM glucose at 37\u00a0\u00b0C with labelled folinic acid. After incubation and washing, cells were resuspended in the same buffer without label and incubated for different times (Fig. 3). Cells were then spun down and the radioactivity in the supernatant measured. To measure label exported from free parasites, they were first released from erythrocytes with 0.05% saponin and then washed and treated in a similar manner as for the uninfected red cells. The level of label in the supernatant of the red cell aliquots tripled over the 80\u00a0min incubation time, indicative of facile export. However, export of the label from free parasites was much less rapid, only increasing by \u223c20% over the same period.\nIn the case of Lactobacillus salivarius, folate internalised over a similar period cannot be released from the cells and is recovered in cell extracts primarily in polyglutamated forms that play an important role in the retention and accumulation of intracellular folate [27,28], and previous studies using P. falciparum have demonstrated the presence of polyglutamated forms of folate produced from labelled monoglutamated precursors [2,23,20]. To examine whether the slow rate of export seen above was due to trapping by polyglutamation, the folinic acid internalised after 1\u00a0h was then extracted and analysed by HPLC on a C18 column [23,20]. The elution profiles indicated that the imported folinic acid had not been significantly modified by this stage, as no labelled polyglutamated folates could be detected in the cell extracts after this relatively short period of incubation, nor was there evidence of any conversion of label to another form of folate (data not shown). These results, together with our earlier data, where overnight incubations were required to detect polyglutamated forms [23,20], suggest that although these forms of folate probably play an important role in ultimately retaining the folates in malaria parasites and modulating the affinity of different enzymes for them, conversion to such forms is not rapid and appears not to play any immediate role in the salvage process. The low rate of net export from the parasite is however consistent with the operation of a transport system that actively imports folates into the parasite, where they are efficiently retained (see below).\n3.4\nThe dependence of folate uptake on carbon source\nTo investigate possible metabolic requirements for folate uptake, its dependence on glucose and other sugars was studied in parasitised red cells. In the absence of glucose or at concentrations <1\u00a0mM, only a very low level of import was measurable. As the glucose concentration was raised, a marked increase in imported folinic acid was observed, peaking around 20\u00a0mM (Fig. 4a). This corresponds closely to the level of glucose in standard P. falciparum culture medium (22\u00a0mM), empirically determined historically to give optimal levels of growth. Time course experiments also showed that the higher the glucose concentration, the longer the uptake process could be sustained by the parasites (Fig. 4b).\nd-Glucose could be replaced by the alternative hexose sugar d-fructose, which can also be utilised in the glycolytic pathway of P. falciparum\n[29,30], but substitution by sugars that cannot, i.e. l-glucose, d-galactose, d-xylose or 6-deoxy-d-glucose, almost completely abolished uptake (Fig. 4a). Cellular energy in P. falciparum is predominantly provided by glycolysis, with the parasite using ca. 100 times more glucose than uninfected erythrocytes to supply their considerable energy needs [31], and uptake of folate is thus clearly dependent upon the operation of normal levels of intracellular energy metabolism.\n3.5\nEffect of pH on folate uptake\nIn the presence of glucose or other sugar susceptible to glycolysis, as in the above experiments, the malaria parasite has a higher cytoplasmic pH (7.2\u20137.4) and a lower parasitophorous vacuole pH (6.9), produced by proton pumping activity on their plasma membrane [21,32]. Such pumping is necessary to prevent the build-up of intracellular acidity, and the higher proton concentration thus created outside the cell can be utilised for the purposes of nutrient transport [21]. This prompted us to investigate the effect of pH on the uptake of folinic acid. For this, a HEPES-MES buffer system was used that provided a wider range of buffering capacity than either buffer alone. The HEPES-MES system contained 20\u00a0mM of each component and the salt concentration was made up to 154\u00a0mM with NaCl. Initially, glucose (20\u00a0mM) was included in all wash steps and assay reactions. For parasitised red cells under these conditions, the optimal pH was 6.5\u20137.0, uptake then decreasing linearly with increasing pH between pH 7.0 and 8.0 (Fig. 5a), with a similar pattern being observed for free parasites over this range (Fig. 5b). However, the transmembrane pH gradient generated in this way is dynamic, subject to change by normal cellular metabolism. To avoid this potentially confounding effect, similar experiments were also conducted in the absence of glucose, thus cutting off the energy supply for the proton pump(s), with the desired buffering and osmotic requirements maintained by NaCl, HEPES and MES. A series of artificially created, constant transmembrane proton gradients were established in this way for the uptake assay. Saponin-released parasites were washed thrice with 154\u00a0mM NaCl to remove folate and other nutrients and to effect exhaustion of the glucose, thereby bringing all parasites in a given sample to the same internal pH. The results showed that under these conditions, the uptake of folinic acid is positively correlated with the transmembrane pH gradient over a wider range (Fig. 5c). The lower the pH of the resuspension buffer, the greater the pH gradient thereby provided, which in turn yielded a higher uptake of folinic acid. Above pH 6.5, the pH response curve flattened out and exhibited a relatively constant, low level of uptake. This inflection point may correlate with the internal pH of the parasites when depleted of glucose under the above conditions [21]. In some cases this was confirmed by measuring the internal pH value with BCECF-AM as described in Section 2. Attempts to directly measure a change in pH as a result of folate import in the absence of glucose and using a minimal buffer concentration were unsuccessful, possibly because of the very small quantities that are taken up and the relatively slow rates involved.\n3.6\nEffect of perturbing the plasma membrane proton gradient on folate uptake\n2,4-Dinitrophenol (DNP) is a widely used ionophore in the uncoupling of oxidative phosphorylation from electron transport on the mitochondrial membrane, as well as in studies of transport across the plasma membrane. Mechanistically, it provides an efficient by-pass route for protons that dissipates their gradient and hence the proton motive force. Here it was found that it also inhibits folinic acid uptake into parasite cells, with an IC50 of ca. 26\u00a0\u03bcM (Fig. 6a and Table 1), again suggesting that this uptake is intimately coupled with the energy metabolism of the parasites. Similarly, the K+\/H+ antiporter nigericin had a profound effect on the uptake of label, which decreased in a dose-dependent manner, with a marked drop in the 100\u00a0pM range. Interestingly, this effect levelled off at around 1\u00a0nM to about 75% inhibition and showed little further drop up to 1\u00a0\u03bcM nigericin (Fig. 6b), suggesting that the major, but possibly not sole, factor determining the uptake of folinic acid is the magnitude of the pH gradient. A similar phenomenon was observed when the V-type ATPase inhibitor concanamycin A [33] was employed. Such an enzyme has been identified as the principal source of proton pumping in P. falciparum\n[21,32]. This inhibitor was effective at sub-micromolar concentrations (IC50 ca. 16\u00a0nM; Fig. 6c), but only abolished ca. 60% of folate uptake, with no further effect beyond about 100\u00a0nM.\n3.7\nMetal ion dependency of folate uptake\nTo investigate whether folate uptake was affected by other cations, in addition to its dependence on the proton gradient demonstrated above, parasitised cells were first washed with 100 volumes of buffer containing either (i) PBS (the buffer in which other uptake experiments were conducted), (ii) a near-physiological concentration of Na+ (130\u00a0mM NaCl, 20\u00a0mM Tris\u2013HCl, pH 7.0), (iii) an equivalent concentration of K+ (130\u00a0mM KCl, 20\u00a0mM Tris\u2013HCl), (iv) the non-ionic polyol mannitol (130\u00a0mM mannitol, 20\u00a0mM Tris\u2013HCl), or (v) Tris alone (150\u00a0mM Tris\u2013HCl). Washes were repeated thrice to reduce the contribution of pre-existing salts to an insignificant level compared to those in the assay buffer. Uptake rates of folinic acid from all five of these solutions were similar (<\u00b120% from the mean). Also, the presence of EDTA up to 5\u00a0mM or Mg2+ up to 10\u00a0mM had only a minor effect (data not shown). It thus appears unlikely that sodium, potassium or divalent metal ions are critically involved in the co-transport of folate into the malaria parasite.\n3.8\nEffect of antifolate drugs and other inhibitors on folate uptake\nA range of other potential inhibitors and competitors were tested for their ability to affect transport of folate into the parasite (Table 1). Antifolate drugs exert their effect principally by binding to target enzymes (DHFR and DHPS) in the folate pathway, lowering pools of reduced folate and thus compromising key metabolic steps, especially DNA synthesis. However, in principle, they might also exhibit an inhibitory capability by interfering with the uptake of exogenous folate [4]. To investigate this possibility, uptake of labelled folinic acid into free parasites was monitored in the presence of the DHFR inhibitors pyrimethamine (PYR) and methotrexate (MTX), and the DHPS inhibitor sulfadoxine (SDX). Over a range of 0.4\u00a0nM to 40\u00a0\u03bcM for PYR and 0.3\u00a0nM to 322\u00a0\u03bcM for SDX, no inhibitory effect on folate uptake could be detected (the highest concentrations are ca. 900 and 500 times greater than the respective IC50 values for these drugs measured for growth inhibition). However, MTX did show inhibition of folinic acid uptake, consistent with its much closer structural relationship to folate than either Pyr or SDX. This was seen clearly when MTX (IC50 of ca. 13\u00a0\u03bcM) was compared to unlabelled folinic acid (2.6\u00a0\u03bcM), 5-MeTHF (3.5\u00a0\u03bcM) and folic acid (19.8\u00a0\u03bcM), where the drug competed with uptake of the label to a similar degree. The specificity of the transport process across the parasite plasma membrane suggested by the above results is consistent with a complete lack of competition with pantothenate (Table 1), the precursor of coenzyme A and an essential parasite nutrient [21], which is also a small but unrelated organic acid with a molecular weight about half that of folate.\nTo further explore the nature of the transporters involved in folate uptake, we employed two known channel blockers, probenecid and furosemide. Probenecid is known to inhibit folate uptake via an anion carrier in mammalian cells at mM levels [34,35], and is able to sensitise parasites to antifolate drugs [15]. Furosemide has been extensively used in nutrient transport studies of the malaria parasite, as it efficiently inhibits the so-called new permeability pathways (NPP) [36\u201338], which first appear in the erythrocyte membrane of parasitised red cells about 12\u201315\u00a0h into their intraerythrocytic development. The NPP show characteristics of anion-selective channels, but are of broad specificity and increase erythrocyte permeability to a range of small molecular weight anions, cations and other species [39\u201342], including essential nutrients such as pantothenate [11]. In experiments conducted on free parasites, where the NPP are not relevant, probenecid strongly inhibited folinic acid uptake with an IC50 of ca. 46\u00a0\u03bcM, and interestingly, furosemide was just as effective (IC50 of ca. 39\u00a0\u03bcM) (Fig. 7a and Table 1). When parasitised erythrocytes were used in the assay, furosemide blocked uptake effectively with an IC50 value of ca. 21\u00a0\u03bcM, similar to that for the free parasite, whereas probenecid was now less effective (IC50 of ca. 187\u00a0\u03bcM). The two drugs were assayed individually and also in combination under identical conditions, but no synergy was apparent between them (Fig. 7b). Indeed, the apparent absence even of an additive effect showed that when both drugs are present in equimolar amounts, the overall effect is equivalent to that seen with the drug of higher potency, i.e. furosemide. This suggests that the two drugs are likely to be acting on the same target. Given this and the above results with free parasites, where the target involved is shown by our prior experiments to behave as a specific folate transporter, it seems probable that the major effect of these two drugs on infected erythrocytes is also at folate transporters on the erythrocyte membrane, rather than the NPP, although we cannot exclude transport via the latter acting to supplement the endogenous capacity of the erythrocyte to import the higher levels of folate required by the parasite.\n4\nDiscussion\nAlthough P. falciparum possesses the biosynthetic machinery required to produce folate derivatives de novo, folate salvage is also an important aspect of the parasite's metabolism [7]. We present several lines of evidence to demonstrate that transport of exogenous folate into P. falciparum is primarily a carrier-mediated process dependent upon cellular energy and the trans-plasma membrane proton gradient. Uptake over the erythrocytic cycle is clearly regulated, with the highest capacity occurring at the late trophozoite stage. This coincides with a period of intense metabolic activity and the onset of multiple rounds of DNA replication, for which folate cofactors are essential, and resembles the pattern seen in the folate auxotroph Leishmania, a parasite that also imports folates maximally in the logarithmic growth phase [43\u201345]. The exogenous folate requirement of P. falciparum can be satisfied in vitro by folic acid, 5-formylTHF (folinic acid) and 5-MeTHF, similar to that of the folate-requiring bacterium Lactobacillus casei, but not L. salivarius, which cannot use the last of these [27]. The process also exhibits a marked temperature dependence, characteristic of a membrane carrier system, with significant uptake only occurring close to the parasite's optimum growth temperature of 37\u00a0\u00b0C. Once imported, net loss in the reverse direction appears to be a slow process, but over the short periods monitored here, did not involve a detectable level of polyglutamation that would trap the folate within the cell, although a significant portion of imported folate is ultimately modified in this way [2,23,20]. This may imply that the role of polyglutamation in this case may be more related to enzyme-substrate recognition, rather than intracellular trapping.\nThe Km values of ca. 2\u20133\u00a0\u03bcM that we measure for the saturable binding component for folinic acid of two strains of P. falciparum are higher than those of 0.4 and 42\u00a0nM, respectively, for folic acid uptake into Lactobacillus\n[27], Xenopus oocytes [46], and of 250\u2013700\u00a0nM for such uptake into Leishmania parasites, depending upon the species [47]. This may reflect the absolute dependence of these three organisms on folate salvage, whereas P. falciparum also possesses an active folate biosynthesis pathway. The  values we observed are of the same order as the rate of uptake of the vitamin pantothenic acid into the parasite [11] but considerably slower than that of glucose [48,49,10], presumably reflecting the great difference between reusable cofactors required in limited quantities and the enormous consumption of the latter as the principal source of energy. The more rapid uptake of folate into the antifolate-resistant strain of K1 compared to the sensitive FCB strain, as indicated by their quite different  values, could result from the selection of parasites better able to cope with drug-mediated inhibition of folate metabolism by increasing their salvage efficiency, in addition to the familiar mutation events in genes encoding folate pathway enzymes that reduce drug binding to the latter [50]. Moreover, if the mutant enzymes (DHFR-TS and PPPK-DHPS) from such parasites process their substrates in vivo with a lower efficiency than in the wild-type, compensation by an increased flux through the salvage pathway might be important for healthy growth, even in the absence of drug. These observations merit extension to other parasite strains with differing responses to antifolate drugs, to further test this hypothesis.\nIn principle, import of folates into the infected erythrocyte could occur via the new permeation pathway(s) (NPP) induced in the host cell membrane by the growing parasite [39,41,42]. However, mammalian cells also exhibit two types of endogenous folate transport system, the reduced folate carriers (RFC), which possess multiple transmembrane domains and have a greater affinity for the reduced folates than for folic acid, and the folate receptors (FR), which are GPI-anchored external folate binding proteins that mediate transport of both folic acid and 5-MeTHF with high specificity [51\u201353]. In mature erythrocytes, the FRs lose functionality with age [51] and the RFC is thought then to be the major transporter type [25,53]. Whatever the exact nature of the relevant transporters on the erythrocyte membrane, they appear not to represent a bottleneck for folate uptake into the parasite, as indicated by our rate experiments. Nor would the parasitophorous vacuolar membrane, which is highly porous and freely permeable to small molecules [54]. The rate-limiting step is therefore passage through the parasite plasma membrane, a process that is strongly inhibited in free parasites both by probenecid, a proven inhibitor of folate transport in mammalian cells [34] and furosemide. Given the similarity in these anion channel blockers (both are benzoic acid derivatives of comparable size with aminosulfonyl substituents on the benzene ring) and the evidence that they target the same sites on infected erythrocytes, we conclude that their major effect with respect to folate uptake at the erythrocyte membrane is likely to be at endogenous folate transporters rather than the NPP. Consistent with the view that the transporters involved in the parasite plasma membrane also have a high degree of specificity for folate, we note that the antifolate drugs PYR and SDX, which have some structural features in common with folate, do not compete with uptake of folinic acid even at very high concentrations. In contrast, the anticancer antifolate MTX, which is a much closer structural analogue to folic acid than either PYR or SDX, does inhibit uptake to a similar extent to the unlabelled folates themselves (\u03bcM range) in competition experiments. We note that the lack of inhibition of folate uptake by PYR that we report here rules out our earlier hypothesis [4] that such inhibition might contribute to the synergy observed between PYR and SDX.\nWe have demonstrated that folate uptake into P. falciparum is dependent upon the presence of d-glucose or other sugar that can be metabolised via glycolysis, a process that is essential to maintain the proton and electrochemical gradients across the parasite plasma membrane [21,8]. d-Fructose can be phosphorylated by hexokinase to fructose 6-phosphate, the substrate of the third glycolytic enzyme, phosphofructokinase-1. Interestingly, although fructose is only about half as efficient as glucose in maintaining overall growth of the parasite [30], it was as efficient as glucose in supporting folate import on a molar basis in our experiments. This may reflect the fact that only a small fraction of the glycolytic flux is likely to be necessary to support import of the relatively low amounts of exogenous folate required by the parasite. Of the other sugars we tested, which were all unable to support uptake, l-glucose is stereochemically excluded, while 6-deoxy-d-glucose and d-xylose can bind to hexokinase but cannot be phosphorylated. d-Galactose, derived principally from lactose hydrolysis, can be converted to glucose 6-phosphate in mammals via a four-step mechanism involving uridine diphosphate derivatives, but its inability to support folate import here would be consistent with an apparent lack of galactokinase and the other enzymes required for this conversion in the predicted proteome of P. falciparum.\nThe dramatic reduction of folate transport in the absence of a metabolisable sugar mirrors the rapid decline of intracellular ATP concentration and drop in the internal pH of the parasite seen in the same circumstances [21]. This suggested that inward movement of folates depends upon maintenance of a pH (interior alkaline) or electrochemical gradient (interior negative), or both, across the plasma membrane. Such gradients can be generated by membrane-bound ATPases, and a range of organic anions move into bacterial cells using proton symport. To differentiate between movement of folate via symport with protons or another cationic species, we examined the influence of the latter, but saw only minor changes in uptake rates in the presence or absence of Na+, K+, Mg2+ or EDTA. This differs from the folate transport system in Lactobacillus, which although similarly dependent upon glucose, also has a requirement for divalent cations [55,27].\nThe pH optimum that we observe for folate import (6.5\u20137.0) corresponds well with that (pH 6.9) measured just outside of the parasite in infected erythrocytes [32], 0.4 units lower than the pH of 7.3 in the parasite cytoplasm [21,32]. The primary mechanism for exporting protons in P. falciparum is thought to be via a V-type H+ ATPase on the plasma membrane [21,32], and consistent with this, we found that concanamycin A, a specific inhibitor of such ATPases [56], inhibited folate uptake in the low nM range. Genes encoding the A and B subunits of a V-ATPase have been identified in P. falciparum\n[57,58] and immunological studies indicate that this enzyme is expressed throughout the erythrocytic cycle [32]. Interestingly though, a significant acidification of the extracellular space just outside of the parasitophorous vacuole could only be measured in trophozoites [32], the stage of the life-cycle at which we observe the highest levels of exogenous folate uptake. The substantial proton gradient that is generated via the V-ATPase is the major contributor to an inwardly negative membrane potential of ca. \u221295\u00a0mV [8], but protons are also extruded in a symport system together with the large amounts of lactate generated by parasite glycolysis [12]. This may explain why we did not observe 100% inhibition of uptake with concanamycin, even at \u03bcM concentrations, as it blocks only the major portion of the total proton export.\nThe importance of the proton gradient across the plasma membrane was emphasised by the sensitivity of folate uptake to the ionophores DNP and nigericin, both of which strongly perturb this gradient. In particular, we find that folate uptake is exquisitely sensitive to nigericin, with substantial inhibition at sub-nM levels. Whereas DNP collapses the pH gradient completely by enabling free passage of protons in both directions, the K+\/H+ antiporter mechanism of nigericin is presumably limited by the degree to which the K+ gradient can be perturbed, which may explain why we also did not observe 100% inhibition of uptake with this compound. Although the profiles observed for nigericin and concanamycin A can be explained in principle by assuming that neither abolishes the pH gradient completely, we cannot exclude the possibility that there is also a component of folate transport independent of this gradient. This scenario is suggested by the fact that a low level of transport persists even when the external pH is set at the same, or a higher, level than the internal pH of parasites deprived of glucose, and would also be consistent with the two-phase nature of the concentration dependence of uptake. At first sight, this picture is difficult to reconcile with the data for DNP, which by insertion into the membrane, permits rapid equilibration of proton concentrations on each side, but we note that complete abolition of uptake only occurs at ca. 1\u00a0mM, a concentration at which quite profound changes to the membrane might be expected, possibly impacting on any proton gradient-independent component.\nA plausible model encompassing our observations would be that folate uptake at the parasite plasma membrane is mediated by a transport complex or multi-binding site protein possessing a binding domain specific for folate recognition and a probenecid\/furosemide-sensitive proton symport channel through which the folate is internalised. To date, no folate transporters have been functionally identified in P. falciparum, but bioinformatics studies [59] predict the existence of 3 candidate proteins (gene loci MAL8P1.13, PF11_0172 and PF10_0215 in PlasmoDB; http:\/\/www.plasmodb.org) that are related to biochemically verified molecules in Leishmania\n[47,45], Synechocystis and Arabidopsis\n[60]. Related putative candidates have also been identified in Toxoplasma gondii\n[6]. This sub-family of folate-biopterin transporters (FBT) are thought on bioinformatic grounds to function as proton symporters [59], and on this basis, the data we present here would be consistent with one or more of these proteins providing the machinery for folate uptake in P. falciparum, which might ultimately present a novel target type for parasite inhibition. We are currently investigating the genes encoding these candidate molecules in knockout studies to better understand the relationship between the phenomena reported here and the parasite proteins involved.","keyphrases":["transporters","folate salvage","proton symport","malaria parasites","folate metabolism","bcecf-am, 2\u2032,7\u2032-bis-(2-carboxyethyl)-5,6-carboxylfluorescein acetoxymethyl ester","dhfr, dihydrofolate reductase","dhps, dihydropteroate synthase","dnp, 2,4-dinitrophenol","paba, p-aminobenzoic acid","mtx, methotrexate","npp, new permeability pathway(s)","pbs, phosphate-buffered saline","pppk, 6-hydroxymethylpterin pyrophosphokinase","pyr, pyrimethamine","sdx, sulfadoxine","thf, tetrahydrofolate","ts, thymidylate synthase","metabolic inhibitors"],"prmu":["P","P","P","P","P","M","M","M","R","R","R","R","M","M","R","R","M","M","R"]}
{"id":"DNA_Repair_(Amst)-1-5-2077361","title":"Mrc1 protects uncapped budding yeast telomeres from exonuclease EXO1\n","text":"Mrc1 (Mediator of Replication Checkpoint 1) is a component of the DNA replication fork machinery and is necessary for checkpoint activation after replication stress. In this study, we addressed the role of Mrc1 at uncapped telomeres. Our experiments show that Mrc1 contributes to the vitality of both cdc13-1 and yku70\u0394 telomere capping mutants. Cells with telomere capping defects containing MRC1 or mrc1AQ, a checkpoint defective allele, exhibit similar growth, suggesting growth defects of cdc13-1 mrc1\u0394 are not due to checkpoint defects. This is in accordance with Mrc1-independent Rad53 activation after telomere uncapping. Poor growth of cdc13-1 mutants in the absence of Mrc1 is a result of enhanced single stranded DNA accumulation at uncapped telomeres. Consistent with this, deletion of EXO1, encoding a nuclease that contributes to single stranded DNA accumulation after telomere uncapping, improves growth of cdc13-1 mrc1\u0394 strains and decreases ssDNA production. Our observations show that Mrc1, a core component of the replication fork, plays an important role in telomere capping, protecting from nucleases and checkpoint pathways.\n1\nIntroduction\nTelomeres are specialized DNA\u2013protein complexes at the end of eukaryotic chromosomes. Proper telomere structure is essential for chromosome integrity and genome stability because telomeres protect natural chromosome ends from degradation and end-to-end fusion and because they ensure complete genome replication. Telomeres differ from Double Strand Breaks (DSBs) in that they normally fail to activate DNA repair or DNA damage checkpoint pathways. If that was the case, then they would undergo recombination and chromosomal fusions and dicentric chromosomes would be generated [1\u20134].\nMany proteins associate with telomeric DNA. These proteins ensure that telomeres behave differently from DSB ends and help maintain chromosomal stability. Some telomeric proteins bind specifically to dsDNA and others show higher affinity to ssDNA. In budding yeast, there are numerous proteins with affinity for telomeric dsDNA, such as Rap1, Sir2, Sir3, Sir4, Rif1 and Yku70\/Yku80 [5]. The budding yeast telomeric ssDNA ends are thought to be protected by three essential proteins, Cdc13, Stn1 and Ten1 [6\u201311].\nIf telomeres become uncapped, they activate a DNA damage response pathway leading to cell cycle arrest [12\u201314]. Moreover, recently it has been suggested that telomeres trigger a transient DNA damage response in each S phase in order to complete DNA replication and cap chromosome ends [15]. DNA damage response pathways are complex networks which include \u2013 among other components \u2013 mediators. Mediators facilitate the transmission of the DNA damage signal from sensors to downstream effectors; activation of the latter affecting cell cycle progression [16]. Mrc1 (Mediator of Replication Checkpoint 1) appears to take the mediator role in Rad53 activation under replication stress [17]. However, parallel pathways exist because in mrc1\u0394 mutants Rad53 activation occurs through Rad9 (another mediator protein), presumably because the accumulation of \u201cDNA damage\u201d rather than \u201creplication defects\u201d in mrc1\u0394 mutants leads to activation of Rad9 and thereby activation of Rad53 [17].\nMrc1 also appears to be directly involved in DNA replication and, because of this, mrc1\u0394 cells display a slow S phase [18]. Mrc1 is an active component of the replication machinery, loaded onto DNA shortly after replication initiates, and moving with other components of replication forks [19\u201321]. In the presence of hydroxyurea, a type of replication stress, Mrc1 appears to form a stable replication-pausing complex preventing the uncoupling of the replication machinery from DNA synthesis [20\u201322]. According to this model, Mrc1 mediates activation of Rad53 under conditions of replication stress so that subsequent DNA repair events occur and cell replication resumes normal function [22]. However, recent experiments suggest that the role of Mrc1 at stalled replication forks is more than activating Rad53, since mrc1AQ cells, defective in Rad53 activation, are not defective in replication fork initiation or progression [18,20,21]. mrc1AQ is a mutant allele in which SQ\/TQ residues have been substituted with AQ, resulting in its inability to mediate phosphorylation and activation of Rad53 [18].\nAlthough Mrc1 is involved in the DNA replication checkpoint, it has been shown that it is not required for the DNA damage checkpoint, since cdc13-1 mrc1\u0394 double mutants arrest in G2 at non-permissive temperatures [17]. It has been reported that activation of Rad53 in response to telomere shortening still occurs in the absence of Rad9 and that Mrc1 is responsible for this activation in telomerase-deficient cells, in which telomeres continually shorten until they activate a checkpoint [23]. Surprisingly, though, tlc1\u0394 mrc1\u0394 double mutants arrest cell division, suggesting that Mrc1 is not required for cell cycle arrest in telomerase negative cells. In contrast, after cdc13-1 induced telomere uncapping, Rad53 activation is entirely Rad9-dependent and Mrc1-independent [23].\nHere we investigated the role of Mrc1 at uncapped telomeres, using the temperature sensitive cdc13-1 and yku70\u0394 mutations to uncap telomeres. Our experiments indicate that Mrc1 protects telomeres from the DNA damage response and that the role of Mrc1 in DNA replication forks, rather than in checkpoint activation, is important for protection of telomeres.\n2\nMaterials and methods\n2.1\nYeast strains and plasmids\nAll strains in the W303 background are RAD5 and they contain an ade2-1 mutation (Supplemental Table 1); therefore yeast extract\/peptone\/dextrose (YEPD) was supplemented with adenine at 50\u00a0mg\/l. Strains 3393\u20133402 are in the S288C background (Supplemental Table 1) and they were generated by mating a single gene deletion mutant array [24] with a cdc13-1 query strain [25]. To construct strains, standard genetic procedures of transformation and tetrad analysis were used [26]. pMRC1 and pmrc1AQ, also carrying the URA3 gene were a gift from Steven Elledge [17,18].\nMRC1 was disrupted in two different ways. Firstly, the MRC1 ORF was substituted with KanMX6, with a PCR based method [27]. Primers 5\u2032-tcgttattcgcttttgaacttatcaccaaatattttagtgCGGATCCCCGGGTTAATTAA-3\u2032 (#878) and 5\u2032-ctggagttcaatcaacttcttcggaaaagataaaaaaccaGAATTCGAGCTCGTTTAAAC-3\u2032 (#881), which contain homology to upstream and downstream sequences of MRC1 (bases in lowercase), were used to amplify a 1559\u00a0bp KanMX6 sequence (pFA6a-kanMX6; [27]). The PCR product was transformed into yeast and candidate colonies were selected for G418 resistance. Integration of the KanMX6 marker into the MRC1 locus was confirmed by PCR, using two sets of primers: (i) forward 5\u2032-CCAAGAACAGACAAACAACTAAGGA-3\u2032 (#876) with reverse primer 5\u2032-TCAGCATCCATGTTGGAATT-3\u2032 (#81) and (ii) forward 5\u2032-CCATCCTATGGAACTGCCTC-3\u2032(#82) with reverse 5\u2032-CCTAGACTCGGGTGCCATCT-3\u2032 (#880). Disruption of MRC1 was also confirmed by Southern blot (data not shown). Alternatively, MRC1 was substituted with URA3 using a restriction enzyme digest approach. First, pMRC1 was digested with XhoI and a 5008\u00a0bp fragment containing the full MRC1 gene was cloned into XhoI digested pIC19H vector (2.7\u00a0kb) to create pAT1065. A correct clone was identified by restriction digests. pAT1065 was digested with SpeI to remove a 2.31\u00a0kb DNA fragment containing the bulk of MRC1, which was replaced with a 1.3\u00a0kb BamHI URA3 gene fragment from pDL349 (pBSB\u00a0+\u00a0KS containing a BamHI fragment containing the URA3 gene) by blunt cloning following treatment with DNA polymerase I Large (Klenow) fragment (New England Biolabs). Positive clones were selected by restriction enzyme digests to identify the disruption of the bulk of MRC1 with URA3 (pAT1066). Disruption of MRC1 was also confirmed by Southern blot (data not shown). To disrupt MRC1, pAT1066 was digested with XhoI prior to transformation of yeast.\n2.2\nSpot tests\nSingle colonies were inoculated into 2\u00a0ml YEPDextrose (YEPD) and incubated overnight, with aeration, at 23\u00a0\u00b0C. The following day, 200\u00a0\u03bcl of each culture was inoculated into 2\u00a0ml of fresh YEPD and returned to 23\u00a0\u00b0C. Cells were grown for three more hours, and cell numbers were determined in a haemocytometer. The cells were then centrifuged (13,000\u00a0rpm for 10\u00a0s in a microcentrifuge), washed twice with sterile water and resuspended in water to a final concentration of 1.5\u00a0\u00d7\u00a0107\u00a0cells\/ml. A five-fold dilution series of each of the cultures was prepared using sterile water in a 96 well plate and 3\u20135\u00a0\u03bcl spotted onto plates using a 48-prong replica plating device. Plates were incubated at various temperatures for 2\u20133 days before being photographed. For spot tests with strains containing pMRC1, pmrc1AQ or pRS416 the steps were as described above, but strains were grown on selective medium (-URA). All strains shown as if on a single agar plate were grown on the same plate, although in some cases their positions were moved using Adobe Photoshop and Adobe Illustrator CS. Unless otherwise stated, at least two different strains of the same genotype were spot tested and representative examples are shown.\n2.3\nYeast transformation\nHigh efficiency transformations needed for gene disruptions were performed according to Gietz et al. [28]. For plasmid transformations a more rapid method was used [29].\n2.4\nWestern blots\nProtein extracts were prepared by glass bead breakage in TCA, essentially as previously described [30,31]. Bio-Rad gels (7.5% Tris\u2013HCl), Schleicher and Schuell Protan Nitrocellulose membranes and the Pierce Supersignal West Pico Chemiluminescent Substrate detection kit were used in a standard Western blot procedure. Rabbit anti-Rad53 polyclonal antibody (AbDL50, 1:1000 dilution, a gift from Dan Durocher [32]) was a primary antibody used with an anti-rabbit-HRP (AbDL7, 1:10,000 dilution, Dako P0448) as a secondary antibody used. Mouse anti-tubulin (TAT-1, AbDL42, 1:2000 dilution, a gift from Keith Gull [33] and anti-mouse-HRP (AbDL6, 1:10,000 dilution, Dako P0447) were used for tubulin loading controls.\n2.5\nTelomere length measurement by Southern blot\nStrains were grown to saturation in liquid YEPD at 23\u00a0\u00b0C. DNA from each strain was subjected to XhoI cut. The digested DNA was loaded on a 0.8% agarose gel, run at low voltage overnight, transferred to a Magna nylon membrane and UV cross-linked. The membrane was then hybridised with a Y\u2032-TG probe [34]. A non-radioactive detection kit was used for the detection of the hybridisation (Amersham, Arlington Heights, IL).\n2.6\nSynchronous cultures\ncdc13-1 cdc15-2 bar1\u0394 strains with additional mutations (see Supplemental Table 1) were grown in YEPD at 23\u00a0\u00b0C overnight. In the morning, cells were adjusted to a concentration of 8\u00a0\u00d7\u00a0106\u00a0buds\/ml in 250\u00a0ml. Cultures were grown for three more hours, then arrested with 20\u00a0nM \u03b1-factor for a further 2.5\u00a0h. The cultures were then released from G1 arrest by centrifugation and washed twice in YEPD and placed at 36\u00a0\u00b0C, 40\u00a0min after the culture was first centrifuged. Cell cycle position was monitored as previously described [13]. DNA was prepared and the fraction of single stranded DNA (ssDNA) was measured as previously described [35].\n2.7\nAsynchronous cultures\ncdc13-1 strains with additional mutations indicated were grown in YEPD at 23\u00a0\u00b0C overnight. In the morning, cells were adjusted to a concentration of 1\u00a0\u00d7\u00a0107\u00a0cells\/ml and temperature was raised to 27.3\u00a0\u00b0C. Every 90\u00a0min samples were taken for cell cycle position and cell density was re-adjusted to 1\u00a0\u00d7\u00a0107\u00a0cells\/ml. Cell numbers were determined with a haemocytometer. Samples for Western blots were collected from exponentially growing cultures 2\u00a0h after the temperature was raised from 23\u00a0\u00b0C to 36\u00a0\u00b0C.\n2.8\nCell cycle position determination\nA 1\u00a0ml sample of culture was centrifuged for 8\u201310\u00a0s at high speed, the supernatant was aspirated, and cells were fixed at 70% ethanol overnight. The fixed cells were washed twice in water before being resuspended in 4,6-diamidino-2-phenylindole (DAPI, 0.2\u00a0\u03bcg\/ml). Cell cycle distribution was monitored by DAPI staining of nuclei and fluorescence microscopy. For DAPI staining, 100 cells for each sample were counted and classified as: (1) unbudded, single DAPI-stained body; (2) small budded, single DAPI-stained body, with the bud <50% of the diameter of the mother cell; (3) medial nuclear division, single DAPI-stained body, with bud >50% diameter of mother cell, the cdc13-1 arrest point; (4) late nuclear division, two buds, and two DAPI-stained bodies, the cdc15-2 arrest point [36].\n3\nResults\n3.1\nMrc1 contributes to the vitality of cdc13-1 and yku70\u0394 mutants\nSince Mrc1 plays a role in the checkpoint response to stalled replication, we wondered if it also plays a role at uncapped telomeres. The temperature sensitive cdc13-1 mutation causes a defect in Cdc13, a telomere binding protein, and cells containing this mutation accumulate large amounts of ssDNA at telomeres at non-permissive temperatures [12,37,38]. Interestingly, deletion of checkpoint proteins, like Chk1, Mec1, Mec3, Rad9, Rad17, Rad24 and Rad53 improves growth of cdc13-1 strains at semi-permissive temperatures [39\u201341]. This is presumably because checkpoint pathways inhibit cell division by responding to low levels of ssDNA that accumulates at telomeres at semi-permissive temperatures. Deletion of other checkpoint proteins, like the MRX complex, which appears to play a role in telomere capping, worsens the growth of cdc13-1 strains [42]. Therefore, we wanted to investigate whether Mrc1 plays a role at uncapped telomeres and, if so, whether it behaved like Rad9 or MRX. Fig. 1A shows that deletion of MRC1 dramatically reduces the growth of cdc13-1 mutant strains at 25\u00a0\u00b0C. The effect of Mrc1 is not as profound as that of the MRX complex, as cdc13-1 mre11\u0394 and cdc13-1 rad50\u0394 display more severe growth defects than cdc13-1 mrc1\u0394 even at 23\u00a0\u00b0C (Supplementary Fig. 1). Thus, Mrc1, like MRX, but unlike the majority of checkpoint proteins, contributes to the vitality of cdc13-1 strains.\nWe next wanted to investigate whether analogous growth defects of cdc13-1 mrc1\u0394 strains occur in yku70\u0394 strains. Yku70 is a telomere capping protein which is also involved in dsDNA damage repair and in Non-Homologous End Joining (reviewed in Ref. [43]). Deletion of YKU70 results in a temperature sensitive phenotype at 37\u00a0\u00b0C, due to telomere uncapping, which activates a Chk1-dependent cell cycle arrest [36]. Fig. 1B demonstrates that deletion of MRC1 results in a severe growth defect of yku70\u0394 mutant strains at 35\u00a0\u00b0C and 36\u00a0\u00b0C. Thus, Mrc1 contributes to the vitality of yku70\u0394 strains. We conclude that Mrc1 contributes to the vitality of two cell types defective in telomere capping.\nMrc1, Tof1 and Csm3 are three proteins that play similar, although distinct roles in DNA replication [19\u201321,44]. Therefore, we wished to address whether Tof1 and Csm3, like Mrc1, contributed to the vitality of cdc13-1 mutants. Spot test analysis showed that although deletion of TOF1 or CSM3 also confers some growth defects on cdc13-1 mutants, deletion of MRC1 has a stronger phenotype (Fig. 1C). Therefore, we decided to focus on understanding the role of MRC1 at uncapped telomeres.\n3.2\nGrowth defects of cdc13-1 mrc1\u0394 cells are not due to checkpoint defects\nIn budding yeast, two independent roles have been previously reported for Mrc1. One role implicates Mrc1 as a mediator of checkpoint activation under replication stress and the other role is as part of the replication machinery [17\u201321]. Therefore, we investigated whether the heightened temperature sensitivity phenotype of cdc13-1 mrc1\u0394 mutant strains is a result of a replication defect, a checkpoint defect or both.\ncdc13-1 mrc1\u0394 mutants were complemented with either wild type pMRC1, pmrc1AQ or an empty vector (pRS416) and strains were grown at various temperatures. Fig. 2 shows that at 26.2\u00a0\u00b0C, complementation of mrc1\u0394 cdc13-1 mutant strains, with either pMRC1 or pmrc1AQ allele improves growth compared to the empty vector control (compare rows 1\u20133). Thus, we conclude that the checkpoint role of Mrc1 is not important for the vitality of cdc13-1 strains.\nAt higher temperature we noticed an increased growth of cdc13-1 mrc1\u0394 cells carrying the mrc1AQ allele, compared to pMRC1. However, this phenotype was observed even in the presence of the wild type MRC1 allele, suggesting that this effect is due to some type of dominant effect of mrc1AQ (Fig. 2, compare rows 2 and 11).\n3.3\nExo1 inhibits growth of cdc13-1 mrc1\u0394 and yku70\u0394mrc1\u0394 mutants\nEXO1 encodes a nuclease known to contribute to ssDNA production at uncapped telomeres of cdc13-1 and yku70\u0394 strains [36,39]. If Exo1-dependent ssDNA production at uncapped telomeres is responsible for the poor growth of cdc13-1 mrc1\u0394 and yku70\u0394 mrc1\u0394 mutants, then removing Exo1 should suppress their poor growth. Fig. 3A demonstrates that at 25\u00a0\u00b0C, cdc13-1 mrc1\u0394 exo1\u0394 triple mutants exhibit better growth than cdc13-1 mrc1\u0394 strains, showing that Exo1 contributes to the growth defects observed in cdc13-1 mrc1\u0394 strains. Importantly, deleting EXO1 also reverses the growth defect of mrc1\u0394 yku70\u0394 mutants (Fig. 3B). These data suggest that Mrc1 protects uncapped telomeres from Exo1.\n3.4\nEffects of checkpoint mutations on cdc13-1 mrc1\u0394 and yku70\u0394 mrc1\u0394 growth\nTo understand if checkpoint pathways are activated in mrc1\u0394 strains after telomere uncapping, we wanted to combine cdc13-1 mrc1\u0394 and yku70\u0394 mrc1\u0394 strains with checkpoint mutations. A genetic screen has revealed that mrc1\u0394 is synthetically lethal with rad9\u0394, rad17\u0394 or rad24\u0394 checkpoint mutations [24]. Consistent with these results we were unable to recover viable offspring carrying mrc1\u0394 in combinations of any of these checkpoint genes (data not shown). However, we were able to combine mrc1\u0394 with rad53\u0394 and chk1\u0394, encoding two downstream checkpoint kinases (analogues of human Chk2 and Chk1, respectively).\nWhen cdc13-1 mutants grow at non-permissive temperatures Rad53 and Chk1 dependent parallel pathways are activated [36,45,46]. We deleted RAD53 or CHK1 and examined the effects in cdc13-1 mrc1\u0394 mutants. Deletion of RAD53 requires simultaneous deletion of SML1 to obtain viable spores. Sml1 is a small protein that inhibits the activity of ribonucleotide reductase (RNR) which catalyzes the rate-limiting step of de novo dNTP synthesis [47]. Normally Sml1 is degraded in a Rad53-dependent manner during S phase [48]. We found that removal of RAD53 and SML1 improved the growth of cdc13-1 mrc1\u0394 strains (Fig. 4A). However, deletion of SML1 alone (cdc13-1 mrc1\u0394 sml1\u0394) also rescued the growth defects associated with MRC1 deletion (Fig. 4A). Thus, we were unable to observe any strong role for Rad53, in maintaining vitality of cdc13-1 mrc1\u0394 mutants, other than in degrading Sml1. We suggest that the reason that deleting Sml1 improves the growth of cdc13-1 mrc1\u0394 strains is that increased ribonucleotide reductase activity may stabilise the replication forks.\nRemoval of Chk1, like removal of Rad53, does not rescue growth of cdc13-1 mrc1\u0394 strains, indicating that Chk1 does not inhibit growth of these mutants (Fig. 4B). Therefore, we find no evidence that inactivating DNA damage checkpoint pathways improves growth of cdc13-1 mrc1\u0394 mutants.\nA CHK1 deletion strongly rescues growth of yku70\u0394 mrc1\u0394 mutants at restrictive temperatures (Fig. 4C) similarly to its effect in yku70\u0394 mutants [36]. Thus, mrc1\u0394 yku70\u0394 uncapped telomeres qualitatively behave like yku70\u0394 uncapped mutants. The effects of chk1\u0394 in yku70\u0394 mrc1\u0394 and cdc13-1 mrc1\u0394 strains are consistent with earlier findings showing that the Chk1-dependent pathway is more important in yku70\u0394 mutants [36,45].\n3.5\nMrc1 is not required for the cell cycle arrest after cdc13-1 uncapping\nTo directly test whether Mrc1 plays a checkpoint role in cdc13-1 strains, cdc13-1 strains with additional mutations were first grown at the semi-permissive temperature 27.3\u00a0\u00b0C. Strains defective in telomere capping (cdc13-1) arrest at medial nuclear division before entry to anaphase after 3\u00a0h at 27.3\u00a0\u00b0C (Fig. 5A). As expected, when the checkpoint is compromised in cdc13-1 chk1\u0394, cdc13-1 rad9\u0394 or cdc13-1 rad53\u0394 cells, no accumulation at medial nuclear division is observed over 9\u00a0h (Fig. 5A). In contrast, cdc13-1 and cdc13-1 mrc1\u0394 strains rapidly accumulate at medial nuclear division and within 3\u00a0h more than 90% of cells are arrested at this point. Consistent with these conclusions cell numbers stopped increasing in cdc13-1 and cdc13-1 mrc1\u0394 strains, but continued to increase in the other strains (Fig. 5B). We conclude that Mrc1 is not required for the checkpoint response to cdc13-1 dependent telomere uncapping.\nRad53 and Chk1 are components of parallel checkpoint pathways that respond to cdc13-1 induced telomere uncapping [45,46]. It appears that the Rad53 pathway is more important for arrest of cdc13-1 mrc1\u0394 mutants because 80% of cdc13-1 mrc1\u0394 chk1\u0394 cells have arrested at medial nuclear division by 7.5\u00a0h, whereas there is no arrest of cdc13-1 mrc1\u0394 rad53\u0394 cells.\nTo test the role of Mrc1 in checkpoint control in a single cell cycle we combined mrc1\u0394 with cdc13-1 cdc15-2 and bar1 mutations. Over many years we and others have used these mutations to determine the effects of checkpoint proteins in responding to telomere uncapping [13,39,40,45]. Bar1 encodes a protease that degrades the mating pheromone \u03b1-factor. Cells bearing the bar1 mutation can efficiently arrest in G1 phase of the cell cycle with low levels of \u03b1-factor. Cdc15 is necessary for mitotic exit. At 36\u00a0\u00b0C, cdc13-1 cdc15-2 bar1 control strains, released from alpha factor arrest, accumulate at medial nuclear division due to cdc13-1-dependent telomere uncapping. However, if cells have escaped the G2\/M checkpoint, like cdc13-1 rad9\u0394 cdc15-2 bar1 strains, they arrest at late nuclear division due to cdc15-2 and they are unable to proceed to the next cycle. The cdc15-2 dependent cell cycle arrest helps in two ways, it ensures that DNA damage checkpoint defects are easily quantified because cells with checkpoint defects accumulate at a later stage of the cell cycle and that DNA damage caused by cdc13-1 is not amplified during new rounds of DNA replication.\ncdc13-1 cdc15-2 bar1 strains with additional mutations were arrested with \u03b1-factor, then released from G1 and transferred to a non-permissive temperature to induce telomere uncapping and the cell cycle position was monitored. Fig. 5C shows that in contrast to cdc13-1 rad9\u0394 strains cdc13-1 and cdc13-1 mrc1\u0394 strains arrest at medial nuclear division with similar kinetics at 36\u00a0\u00b0C, supporting the idea that Mrc1 does not play a role in checkpoint activation after cdc13-1 telomere uncapping. Interestingly, cdc13-1 mrc1\u0394 exo1\u0394 remain arrested at medial nuclear division (Fig. 5C and D) which contrasts to the behaviour of cdc13-1 exo1\u0394 that begin to escape arrest during the 4-h period in analogous experiments [39]. This difference most likely reflects the fact that cdc13-1 mrc1\u0394 exo1\u0394 mutants have more severe telomere capping defect than cdc13-1 exo1\u0394 mutants and therefore activate a stronger checkpoint signal.\nIn response to both replication stress and DNA damage, activation of the checkpoint machinery induces phosphorylation and activation of Rad53 kinase. Therefore, we addressed the role of Mrc1 in Rad53 phosphorylation after telomere uncapping. cdc13-1 strains were exposed to 36\u00a0\u00b0C for 2\u00a0h and Rad53 phosphorylation was measured by Western blot. Rad53 phosphorylation is observed in cdc13-1 mrc1\u0394 strains but not in cdc13-1 rad9\u0394 mutants (Fig. 5F), confirming a previous study [23]. We conclude that activation of Rad53 after cdc13-1 dependent uncapping at non-permissive temperatures is Rad9-dependent but Mrc1-independent.\n3.6\nMrc1 contributes to telomere length regulation\nIf Mrc1 plays a protective role at telomeres, this predicts that strains lacking Mrc1 may have short telomeres. Fig. 6A shows that absence of Mrc1 results in shorter telomeres, compared to the wild type. However, the telomere length defects of mrc1\u0394 mutants are not as severe as in rad50\u0394 or yku70\u0394 mutants, and this may help explain why cdc13-1 mrc1\u0394 cells grow better than cdc13-1 rad50\u0394 cells (Supplementary Fig. 1). Our experiment is consistent with replication proteins having an important role in telomere length regulation [49\u201352].\n3.7\nMrc1 protects telomeres from extended ssDNA accumulation\nAll our data suggest an important role of Mrc1 in telomere capping but no role in cell cycle arrest. To directly test the role of Mrc1 in telomere capping, we measured ssDNA accumulation on the 3\u2032 TG strand, at PDA1, a single copy locus approximately 30\u00a0kb away from the right end of chromosome V in cdc13-1 strains (Fig. 6B). cdc13-1 strains were synchronised, as in Fig. 5C and D in order to follow the effects of Mrc1 on ssDNA accumulation at non-permissive temperatures within a single cell cycle. We find that cdc13-1 mrc1\u0394 mutants, like cdc13-1 rad9\u0394 strains, accumulate more 3\u2032 TG ssDNA at PDA1, 30\u00a0kb from the uncapped telomere, compared to cdc13-1 strains (Fig. 6C) [39]. This shows that Mrc1, like Rad9, protects cdc13-1 mutants from ssDNA production. Consistent with our conclusion, increased ssDNA accumulation, closer to the telomere, in telomere repeats, was recently reported in cdc13-1 mrc1\u0394 and yku70\u0394 mrc1\u0394 mutants using both in gel and dot blot analyses [53]. Importantly, ssDNA production is reduced in cdc13-1 mrc1\u0394 exo1\u0394 strains in comparison to cdc13-1 mrc1\u0394 strains showing that Mrc1 protects uncapped telomeres from Exo1-dependent nuclease action. This ssDNA data is consistent with our finding that Exo1 contributes to the poor growth of cdc13-1 mrc1\u0394 and yku70\u0394 mrc1\u0394 mutants (Fig. 3). Taken together, we conclude that Mrc1 inhibits accumulation of Exo1-dependent ssDNA accumulation after telomere uncapping and, by this criterion, contributes to telomere capping.\n4\nDiscussion\nOur experiments demonstrate that Mrc1 contributes to the vitality of budding yeast cells with uncapped telomeres. Therefore, Mrc1 behaves differently from many other known checkpoint proteins such as Chk1, Mec1, Rad9, Rad17, Rad24 or Rad53, deletion of which improves the growth of cdc13-1 mutants at semi-permissive temperatures [39,40]. The effect of Mrc1 is more similar to that of the MRX complex, another checkpoint complex with roles in telomere capping [36,42,54,55]. It seems that it is the role of Mrc1 at the replication fork that contributes to the vitality of telomere capping mutants, rather than its role in checkpoint activation. Our findings are in accordance with recent work that demonstrated a protective role of Mrc1 in cells with cdc13-1 or yku70\u0394 uncapped telomeres or in telomerase deficient cells [53]. Additionally, our work demonstrates that the growth defects of cdc13-1 mrc1\u0394 and yku70\u0394 mrc1\u0394 mutants and enhanced ssDNA levels of cdc13-1 mrc1\u0394 strains are suppressed when the nuclease encoded by EXO1 is deleted. Therefore, Mrc1 protects uncapped telomeres from Exo1.\nMrc1 is recruited to the replication machinery as DNA replication initiates and is required for normal rates of replication fork progression [17,18,22]. The biochemical role of Mrc1 in replication fork progression is unclear which makes it difficult to know its precise role in telomere capping. Mrc1 is also part of a replication-pausing complex formed when replication is arrested by the S phase poison hydroxyurea (HU), and required for replication fork restart after HU. However, this restart role for Mrc1 is not universal, since Mrc1 plays no role in replication restart after cells are treated with the alkylating agent MMS [22].\nIt is interesting that there is evidence from budding yeast, fission yeast and human cells that telomeric sequences contain DNA regions that slow or stall replication forks [15,56,57]. From this it seems plausible that telomeric DNA may be more dependent on proteins like Mrc1, which contribute to fork stability and restart, than other chromosomal regions. That is because the replication fork struggles to reach the end of the chromosome in mrc1\u0394 mutants where a telomere capping defect is observed.\nNumerous studies on budding yeast mutants with DNA replication defects have demonstrated interactions between DNA replication and telomere structure. For example both cdc17\/pol1 and cdc44\/rfc1 (large subunit of replication factor C) mutants affect telomere length [49]. Here we show that budding yeast mrc1\u0394 mutants have short telomeres. In S. cerevisiae, cdc17\/pol1 mutants, encoding temperature sensitive DNA polymerase \u03b1, exhibit very long telomeres, high levels of telomeric ssDNA and elevated recombination at telomeres [50,51]. Interestingly, the B subunit of DNA polymerase \u03b1 physically interacts with Stn1, which in turn interacts with Cdc13 [10,52]. This shows there is a very direct interaction between budding yeast telomere capping proteins and the replication fork machinery, and suggests that telomere capping is intimately linked with DNA replication. In this regard it is, perhaps, relevant that the 5\u2032\u20133\u2032 exonuclease Exo1 is involved in generating single stranded DNA at uncapped telomeres [36,39] and in processing stalled replication forks [58] and highlights the similarities between uncapped telomeres and stalled replication forks.\ncdc13-1 cells maintain a functional telomere cap (low levels of telomeric ssDNA), when released from G1 arrest into the S phase poison hydroxyurea. HU stalls replication forks and stops late origins of replication from firing. However, if the same cdc13-1 cells are permitted to complete DNA replication, by removing the S phase poison HU, telomere uncapping occurs and high levels of ssDNA are observed [59]. Therefore, Cdc13-dependent telomere capping may depend on a coordinated interaction between the chromosome end, Cdc13\/Stn1\/Ten1 and the DNA replication fork. Further studies examining the interactions between telomeric DNA, the telomere cap and the replication fork will be necessary to better understand these interactions.","keyphrases":["mrc1","telomere","exo1","cdc13","yku70","ssdna"],"prmu":["P","P","P","P","P","P"]}
{"id":"Pediatr_Radiol-4-1-2292494","title":"Gadolinium and nephrogenic systemic fibrosis: time to tighten practice\n","text":"Nephrogenic systemic fibrosis (NSF) is a relatively new entity, first described in 1997. Few cases have been reported, but the disease has high morbidity and mortality. To date it has been seen exclusively in patients with renal dysfunction. There is an emerging link with intravenous injection of gadolinium contrast agents, which has been suggested as a main triggering factor, with a lag time of days to weeks. Risk factors include the severity of renal impairment, major surgery, vascular events and other proinflammatory conditions. There is no reason to believe that children have an altered risk compared to the adult population. It is important that the paediatric radiologist acknowledges emerging information on NSF but at the same time considers the risk:benefit ratio prior to embarking on alternative investigations, as children with chronic kidney disease require high-quality diagnostic imaging.\nIntroduction\nNephrogenic systemic fibrosis (NSF; formerly named nephrogenic fibrosing dermopathy) is a systemic disease that was first described in 1997 and recognized as a separate clinical entity in 2000 [1\u20134]. Until now, all confirmed cases of NSF have been reported in patients with chronic kidney disease (CKD) with estimated glomerular filtration rates (eGFR) <60\u00a0ml\/min per 1.73\u00a0m2 or end-stage renal failure requiring renal replacement therapy. NSF affects both sexes with a reported age range of 8\u201387\u00a0years and has no race predilection [5, 6]. Relatively few paediatric cases have been reported, which may reflect the lower incidence of CKD in children.\nPatients present with swelling, induration and tightening of the skin, often with a peau d\u2019orange appearance or textured plaques, papules or nodules, usually limited to the extremities (from ankles to mid-thighs and from wrists to mid-upper arms). The skin lesions sometimes involve the trunk, but spare the neck and face. The condition develops over days to weeks and may ultimately result in severe contractures and disability. Patients may experience burning, itching or sharp pains in involved areas. Deep pain has also been described in the hips and ribs. Radiography may reveal calcifications of the soft tissue [1, 5\u20139]. Systemic involvement may be fatal with fibrosis of the lungs, skeletal muscles, pleura, pericardium, myocardium, kidneys, muscle, bone, testes and dura [2\u20134, 10]. Clinical differential diagnoses include scleroderma, scleromyxoedema, systemic sclerosis\/morphoea, eosinophilic fasciitis and eosinophilia-myalgia syndrome, and the condition may mimic cellulitis, panniculitis or drug reactions [6\u20138, 11, 12].\nThe severity of CKD appears to be the main determinant of risk. Also, several contributing risk factors have been suggested: hypercoagulability and thrombotic events; cardiomyopathy; hepatic disease; idiopathic pulmonary fibrosis; systemic lupus erythematosus; brain tumours; recent surgical procedures, especially those with a major vascular component; and other proinflammatory conditions, including liver transplantation [5, 6, 8, 13, 14].\nA specific aetiology is still unknown. The only common factor in all patients is the underlying kidney disease, with most patients dialysis-dependent. However, NSF has also been reported in patients who have never been dialysed [1, 5, 6, 8]. The physiopathological mechanism for fibrosis is not yet known. Histopathologically, biopsies from the skin lesions show a proliferation of fibrocytes (which may extend into subcutaneous tissue), together with thick collagen bundles and surrounding clefts with a variable amount of mucin and elastic fibres. Fibrocytes have a specific immunophenotype and express CD34, CD11b, CD45, HLA-DR, CD71, CD80, CD86, which implies that they are recruited from the circulation and do not arise as a result of proliferation in situ. Inflammatory cells are not usually evident, although small clusters of perivascular mononuclear cells may be seen [11, 15, 16].\nTherapeutic options in NSF are limited. Improvements have been reported with extracorporeal photopheresis, plasmapheresis, ultraviolet phototherapy, intravenous immunoglobulin and renal transplantation [17\u201321].\nLink with gadolinium-based contrast agents\nThe main concern for the radiologist is a growing body of evidence that clinical use of gadolinium-based contrast agents may trigger the disease. A number of patients with NSF seem not to have had any previous gadolinium exposure, but these are still under investigation. Grobner [22] was the first to report this association in January 2006. Grobner described nine patients with end-stage renal failure on dialysis who underwent MR angiography (MRA) with gadodiamide (Omniscan; GE Healthcare, Chalfont St Giles, UK); five (mean age 57.2\u00b110.7\u00a0years) developed NSF after 2\u20134\u00a0weeks. All patients who developed NSF had confirmed metabolic acidosis, whilst all patients who did not develop NSF had normal pH and bicarbonate level at the time of the MRA. The average contrast agent volume used was 35\u00a0ml.\nMarckmann et al. [23] reviewed case notes from their own nephrology department from August 2005 to May 2006 and found 13 patients with confirmed NSF, all exposed to gadodiamide. All patients were adults (33\u201366\u00a0years of age, mean 50\u00a0years), developed NSF 2\u201375\u00a0days (mean 25\u00a0days) after gadolinium exposure and had an eGFR <8\u00a0ml\/min. From this cohort of renal patients the authors estimated an odds ratio between 32.5 and 47.6 for developing NSF following gadolinium exposure.\nThe link between gadolinium exposure and NSF has been further strengthened by two reports of gadolinium deposits in tissue samples of patients with NSF. High et al. [24] detected gadolinium in 4 out of 13 skin and soft-tissue biopsies from seven patients with documented NSF. All patients with NSF received gadolinium-based contrast medium prior to disease and the interval between dosing and biopsy could be determined with certainty only for two of four patients (age range 4\u201311\u00a0months). The same group reported in a research letter following quantitative analysis of the four tissue samples in which gadolinium was detected that the amount of gadolinium in the affected tissue of patients with NSF was approximately 35- to 150-fold the level of gadolinium in the bone of healthy volunteers with normal renal function. In a case report, Boyd et al. [25] found gadolinium deposits in areas of calcium phosphate deposition in blood vessels in a 68-year-old patient with NSF who had been previously exposed to gadodiamide.\nSadowski et al. [26] retrospectively reviewed data from 13 patients with confirmed NSF (eight males, five females, age 17\u201369\u00a0years), all exposed to gadodiamide. The median time between onset of NSF symptoms and last contrast-enhanced MRI examination was 11.5\u00a0days. The dose of contrast agent administered varied from 0.1 to 0.31\u00a0mmol\/kg (actual) body weight. At the time of the contrast-enhanced MRI all 13 patients had CKD (eGFR <60\u00a0ml\/min per 1.73\u00a0m2) and were hospitalized for a proinflammatory event (major surgery, infection or vascular event). The group with NSF had significantly lower eGFR, more proinflammatory events, and more contrast-enhanced MR examinations per patient (P<0.002) than did the control group. The incidence of NSF in this study and institution was 4.6% per year.\nA review of six further NSF cases that were also related in time to gadodiamide administration has recently been published [27]. Patients were aged 23\u201371\u00a0years and the onset of symptoms consistent with NSF was between 19\u00a0days and 2\u00a0months after gadodiamide exposure. All patients had severe renal failure and were exposed to a gadodiamide dose ranging from 0.11 to 0.36\u00a0mmol\/kg body weight. Despite having normal serum bicarbonate, five of the six patients had an elevated anion gap metabolic acidosis.\nBroome et al. [28] have recently reported a retrospective study of 12 patients with NSF, all of whom received a double dose (0.2\u00a0mmol\/kg body weight) of gadodiamide 2\u201311\u00a0weeks before the development of skin fibrosis. This group included eight men and four women (age 26\u201364\u00a0years) and all had renal insufficiency at the time of gadodiamide administration. The odds ratio for development of NSF in the gadodiamide-exposed patients compared with the gadodiamide-unexposed patients was 22.3 and the prevalence of NSF among gadodiamide-exposed dialysis patients was 4.0%. The odds ratio for development of NSF with double-dose compared to single-dose gadodiamide administration was 12.1. Ten patients were dialysed within 2\u00a0days of gadodiamide administration, but this failed to prevent the development of NSF.\nAlthough the overwhelming majority (around 90%) of patients with gadolinium-associated NSF had been exposed to gadodiamide, there are now several reports of exposure to other gadolinium contrast agents. In a recent editorial in Radiology, Kuo et al. [29] reported 57 patients with NSF investigated by the United States Food and Drug Administration (FDA). In 43 of these the NSF was linked to gadodiamide; in 6 to gadopentetate dimeglumine (Magnevist; Berlex Imaging, Montville, N.J.); in 2 to gadoversetamide (OptiMARK; Mallinckrodt, St. Louis, Mo.); and in 3 to gadodiamide plus gadoversetamide; in 3 other patients, the associated specific gadolinium-based MR contrast agent was not definitively identified. Sadowski et al. [26] also reported one patient with confirmed NSF associated with both gadodiamide and gadobenate dimeglumine (MultiHance, Bracco Diagnostics, Milan, Italy). NSF related to specific gadolinium-based contrast agents is also monitored by the manufacturers, who have recently published several reports [30\u201332]. In a retrospective study in Scotland of 1,826 haemodialysis patients, Collidge et al. [33] found a positive association between the cumulative dose of gadodiamide and the development of NSF. Patients who developed NSF had received a higher median cumulative dose of gadodiamide (0.39\u00a0mmol\/kg vs. 0.23\u00a0mmol\/kg in patients without NSF).\nA case-control study by Marckmann et al. [34] conducted in 19 patients with histologically verified NSF and 19 matched controls found that increasing cumulative gadodiamide exposure, high-dose epoietin-b treatment and higher serum concentrations of ionized calcium and phosphate increase the risk of gadodiamide-related NSF in patients with renal failure.\nPathophysiology\nAlthough not completely understood, several factors are known to contribute to the physiopathological mechanism in NSF. Gadolinium chelates are mostly eliminated through the kidneys. Renal impairment will therefore prolong the presence of these agents in the body, facilitating the release of the toxic free gadolinium ion (Gd3+) by dissociation from its chelate (chemical imbalance, acidosis, anion gap, inflammation) and its subsequent binding to endogenous ions. This chemical process, known as transmetallation, may be promoted by acidosis and anion gap, e.g. under inflammatory conditions [35\u201340]. It has therefore been suggested that both the chemical stability of a particular chelate (cyclic compounds are more stable than linear ones in vitro) and the dose administered are risk factors for triggering NSF [28, 41]. Once released Gd3+ forms hydroxides and phosphates (insoluble at a pH >6.2) and these are probably engulfed by phagocytic cells [42]. This might affect the reticuloendothelial system, inhibiting the activity of certain enzymes with consequent foreign body and fibrous reactions [41, 43\u201345]. Gadodiamide, the agent involved in NSF in most patients to date, is the least stable in terms of susceptibility to transmetallation in vitro, being an open-chain compound [46]. Free gadolinium ions may remain in the body for weeks to months, allowing more time for toxic effects [47]. Gadolinium is a well-known inorganic calcium channel blocker and its acute toxicity may also partly be explained via this mechanism [48, 49]. A review of the properties of commercial gadolinium contrast agents and their relation to NSF is presented in Table\u00a01.\nTable\u00a01Properties and approval status of extracellular gadolinium contrast agents (CNS central nervous system, MRA magnetic resonance angiography, N\/A not available)Chemical nameTrade nameManufacturerCharge and chemical structureConditional stability at pH 7.4 (log)Excess chelate (mg\/ml)Thermodynamic stability constant (log)Kinetic stabilityaBody region(s) approvedApproval statusApproved doses for imaging (mmol\/kg)Approved doses for children (mmol\/kg)NSF related to gadoliniumGadodiamideOmniscanGE HealthcareNonionic linear14.91216.935\u00a0sCNS, whole bodyUSA, EU, JapanBody 0.1\u20130.3, CNS 0.1\u20130.3, MRA 0.1\u20130.3From 6\u00a0months: 0.1Yes (180 cases worldwide)Gadopentetate dimeglumineMagnevistBayer Schering PharmaIonic linear17.70.422.110\u00a0minCNS, whole bodyUSA, EU, JapanBody 0.1, CNS 0.1\u20130.2, MRA 0.1\u20130.30.1 (doses of 0.2 may be used if necessary for children older than 2\u00a0years)Yes (78 cases worldwide)Gadobenate dimeglumineMultiHanceBraccoIonic linear16.9None22.6N\/ACNS, liverUSA, EULiver 0.05, CNS 0.1, MRA not approvedNot approved <18\u00a0yearsYes (1 case in a patient coadministered Omniscan)GadoversetamideOptiMARKTycoNonionic linear15.028.416.6N\/ACNS, liverUSABody 0.1, CNS 0.1, MRA not approvedNot approved <18\u00a0yearsYesGadoterate meglumineDotaremGuerbetIonic cyclic18.8None25.8>1 monthCNS, whole bodyEUBody 0.1, CNS 0.1\u20130.3, MRA 0.20.1NoGadoteridolProHanceBraccoNonionic cyclic17.10.2323.83\u00a0hCNS, whole bodyUSA, EU, JapanBody 0.1\u20130.3, CNS 0.1\u20130.3, MRA not approved0.1 from 2\u00a0years, caution 6\u00a0months to 2\u00a0years, contraindicated <6\u00a0monthsNoGadobutrolGadovistBayer Schering PharmaNon-ionic cyclicN\/AN\/A21.85\u00a0minCNS, MRAEU, CanadaBody not approved, CNS 0.1\u20130.3, MRA (imaging 1 FOV) 0.1\u20130.15, MRA (imaging >1 FOV) 0.2\u20130.3Not approved <18\u00a0yearsNoGadoxetic acidPrimovistBayer Schering PharmaIonic linearN\/AN\/A23.5N\/ALiverUSA, EUBody 25\u00a0\u03bcmol\/kg or 0.1\u00a0ml\/kg, CNS not approved, MRA not approvedNot approved <18\u00a0yearsNoGadofosvesetVasovistBayer Schering PharmaIonic linearN\/AN\/AN\/AN\/AAbdominal and limb vesselsEUMRA 0.03Not approved <18\u00a0yearsNoaKinetic stability: dissociation half-life at pH 1.0.Table modified from Bellin MF (2006) Eur J Radiol 60:314\u2013323.\nNSF in children\nNSF in children has also been reported. Five patients under 18\u00a0years of age have thus far been reported (discussed below) out of a total of ten in the central database of the International Center for Nephrogenic Fibrosing Dermopathy Research (Dr. S. Cowper, personal communication). However, 13 possible patients have been reported to an online paediatric nephrology forum in June 2007 (https:\/\/listhost.uchicago.edu\/mailman\/private\/pedneph\/) and these are still being investigated in relation to previous gadolinium exposure.\nIn 2003 Jan et al. [50] reported two paediatric patients with nephrogenic fibrosing dermopathy: a 16-year-old female patient with a life-long history of kidney failure and an 8-year-old boy with a 9-month history of membranoproliferative glomerulonephritis type II. Both patients had the clinical and histopathological features of NSF and no contrast-enhanced MRI procedures were known to be associated with these patients. However, this was before the publication of a possible link with gadolinium.\nJain et al. [51] reported two more patients with NSF in 2004: a 9-year-old boy on peritoneal dialysis and a 19-year-old male on haemodialysis with a 10-year history of previous failed renal transplants, both diagnosed with NSF on skin biopsies. The 9-year-old boy had a solitary MRA performed in June 2002, approximately 2\u00a0months prior to development of NFD. The history of the 19-year-old male was more complicated as he underwent 13 separate MRA procedures, all with gadolinium contrast agents, between April 2000 and August 2004. At least nine of the MRAs were performed prior to the NFD diagnosis in January 2003 [52]. Also, both patients had persistent metabolic acidosis in the months preceding the development of NSF, which may have been a contributing factor [22].\nDiCarlo et al. [53] described another patient with histopathologically proven NSF. This patient was a 17-year-old male renal transplant recipient (15\u00a0years following transplant) who had recently begun peritoneal dialysis due to graft failure. No relationship to MRI investigations was mentioned.\nRecently, Auron et al. [54] reported two patients with NSF. One of the patients was a 13-year-old boy previously reported at the age of 8\u00a0years by Jan et al. [50] in 2003 (discussed above). The second patient was a 20-year-old male who at the age 14\u00a0years was diagnosed with acute monoblastic leukaemia and 2\u00a0years later underwent bone marrow transplant and later developed kidney failure. No references to contrast-enhanced MRI are available from these reports.\nOur own hospital has a large paediatric renal unit with up to 30 renal transplants per year. We have actively used MRI both for concurrent conditions and complications to CKD, as well as for providing a presurgical vascular road-map, using contrast-enhanced MRA (Gd-DTPA, Magnevist, Schering, Germany, 0.1\u20130.3\u00a0mmol\/kg, i.v.). A review of all contrast-enhanced scans in children referred via our nephrology service (March 2002 to March 2007) showed that 75 nephrology patients (neonate to 18\u00a0years of age; median 9.6\u00a0years) had had 93 MRI scans with a follow-up of at least 6\u00a0months. There were no patients with NSF in this high-risk cohort.\nRegulatory advice\nRegulatory advice can be obtained from the websites listed in Table\u00a02. The FDA has issued a general warning for all gadolinium-based contrast agents and advise against their use in patients with eGFR <30\u00a0ml\/min per 1.73\u00a0m2 and in all patients with acute renal insufficiency \u201cunless the diagnostic information is essential and not available with non-contrast enhanced magnetic resonance imaging\u201d. At the FDA\u2019s request the manufacturers of gadolinium-based contrast agents have revised the product labels, which now include a new boxed warning and a new warnings section that describes the risk of NSF. The European Medicines Agency has so far not published any update of general contraindications, and is not in a position to do so since all but one gadolinium-based agent are licensed by individual member states\u2019 regulatory bodies. However, the issue was discussed at a meeting of its pharmacovigilance group in June and May 2007, and issued specific advice as follows (http:\/\/www.mhra.gov.uk):The use of Omniscan\u00ae and Magnevist\u00ae is contraindicated in patients with severe renal impairment (i.e. GFR or eGFR <30\u00a0mL\/min\/1\u00b773\u00a0m2). Omniscan is also contraindicated in patients with renal dysfunction who have had, or who are awaiting, liver transplantation. For patients with moderate renal impairment (GFR or eGFR 30\u201359\u00a0ml\/min\/1\u00b773\u00a0m2) or neonates and infants up to 1\u00a0year of age, Omniscan and Magnevist should be used only after careful consideration. All patients should be screened for renal dysfunction by obtaining a history and\/or laboratory tests, especially before Omniscan or Magnevist are used. Haemodialysis shortly after administration of a gadolinium-containing MRI contrast agent in patients currently under haemodialysis may be useful for removal of contrast agent from the body. However, there is no evidence to suggest that haemodialysis can prevent or treat development of NSF. Careful consideration should also be given to the use of the other gadolinium-containing MRI contrast agents in patients with severe renal impairment.Table\u00a02Websites for regulatory updates and for information and registration of cases (accessed on 20 September 2007)OrganizationInformation providedURLEuropean Medicines AgencyRegulatory informationhttp:\/\/www.emea.europa.eu\/US Food and Drug AdministrationRegulatory informationhttp:\/\/www.fda.gov\/cder\/drug\/infopage\/gcca\/default.htmInformation for healthcare professionalshttp:\/\/www.fda.gov\/cder\/drug\/InfoSheets\/HCP\/gcca_200705.htmInternational Center for Nephrogenic Fibrosing Dermopathy ResearchUpdated information and contact details for registration of caseshttp:\/\/www.icnfdr.org\/\nConsequences for paediatric practice\nThere is an associated high morbidity and mortality with CKD, and imaging aids clinical management, such as assessment of secondary vasculopathies, infections etc, and surgical planning prior to renal transplantation. Patients at risk also include children with known or suspected CKD, as well as newborns and infants with renal immaturity or congenital cardiopathy. It seems plausible that impaired excretion, dissociation of the gadolinium chelate, and prolonged tissue retention of gadolinium is a trigger for NSF in patients with CKD. This risk increases with cumulative dose of gadolinium, with decreasing GFR <60\u00a0ml\/min per 1.73\u00a0m2, and in the presence of acidosis and proinflammatory conditions. The scarcity of reported children with NSF may well be due to the lower prevalence of CKD and secondary conditions in children. It is therefore imperative that paediatric practitioners are prudent and immediately tighten their local policies for use of intravenous gadolinium-based contrast agents.\nIt is difficult at present to suggest general guidelines, because:\nThe absolute risk of gadolinium exposure, as well as the risk associated with the suspected contributing clinical and biochemical factors, is unknown. Each case must therefore be assessed individually.The risk attributable to the kinetic stability of different gadolinium-based agents is not clear. The majority of cases have been reported in association with a less stable agent (gadodiamide); however, we are still in an early stage of recording cases, and also the market share and the actual use of different agents in this patient population is not fully known. Thus, it seems reasonable to discontinue the use of gadodiamide in at-risk patients.The relationship to dose is not completely known. Since there is no indication that we are dealing with a side effect not related to dose, it seems reasonable to reduce the dose of gadolinium if possible, and also to monitor the cumulative dose in at-risk patients.It is unclear whether acidosis correction before gadolinium administration and haemodialysis immediately after gadolinium administration can prevent the development of, or treat, NSF, but such actions should be considered in children already on haemodialysis. This underlines the necessity for good communication between radiologists and nephrologists, both for issuing local guidelines and for management of the individual child.\nIt is often the case that MRI is requested after an inconclusive US examination. In our own experience, more meticulous patient preparation and dedicated rescanning of the patient with US can often give diagnostically adequate results. In patients with CKD not yet on dialysis CT is not a desirable alternative due to ionizing radiation and the nephrotoxicity of iodinated contrast agents. However, CT may provide an option in patients who are already on dialysis. MRI without intravenous contrast agent administration has not yet been validated for vascular anatomy prior to renal transplantation, but several techniques may be valuable, such as 3-D-balanced steady-state free precession imaging and time-of-flight angiography.\nPatients with renal impairment may be referred for MRI from non-nephrology units and a crucial question is how to identify them. Our approach would be to include a mandatory tick box on the referral form requiring the referring clinician\u2019s statement about renal function, and also to include a question about any known renal problems on the patient\/guardian prescan checklist. This is a pragmatic approach, which will need revision after an initial phase.\nConclusion\nWe would advocate prudence, and in practice suggest:\nInformation collection from clinicians and patients\/guardians on renal dysfunction and active collaboration with the local nephrology unit.\nA case-by-case assessment in terms of the necessity for gadolinium-enhanced MRI, and whether an alternative test (repeat US, noncontrast-enhanced MRI, CT) would be acceptable.\nCumulative dose recording, reduction of the dose of any gadolinium-based contrast agent, and acting accordingly to guidance published in the literature.\nLonger follow-up, especially in high-risk children.\nGFR measurement or estimation should be part of a systematic prescan investigation in children with known or suspected renal dysfunction; however, a general screening is probably not justified.\nTo minimize the risks, we should take advantage of the current knowledge, and would suggest using cyclic nonionic compounds as they are more stable in vitro and therefore theoretically less likely to undergo transmetallation.","keyphrases":["gadolinium","nephrogenic systemic fibrosis","children","chronic kidney disease"],"prmu":["P","P","P","P"]}
{"id":"Graefes_Arch_Clin_Exp_Ophthalmol-4-1-2413124","title":"One-year results of a multicenter controlled clinical trial of triamcinolone in pars plana vitrectomy\n","text":"Purpose To evaluate the 1-year results of using triamcinolone acetonide (TA) in pars plana vitrectomy (PPV).\nIntroduction\nRecently, vitrectomy has been used to treat various vitreoretinal diseases. It still remains a demanding procedure requiring a skilled and experienced surgeon, despite advances in surgical instrumentation and techniques. One major difficulty with this procedure is the transparency of the vitreous. Intra-operative triamcinolone acetonide (TA) has been used to address this problem, as it can be injected into the vitreous to visualize the posterior hyaloid or the internal limiting membrane (ILM) [1, 2]. Several previous reports have demonstrated the usefulness of TA as an adjunct to pars plana vitrectomy (PPV) [3\u201313]. Although their findings supported the use of TA-assisted PPV, these studies involved relatively small numbers of eyes and were not randomized. In addition, more recent reports on the use of intravitreous TA in patients not undergoing vitrectomy raised concerns about the risks of elevated intra-ocular pressure (IOP) [14\u201319].\nWe therefore undertook a multicenter prospective controlled study of TA-assisted pars plana vitrectomy (PPV). Considering the enduring nature of the effect of PPV on ocular tissue, it is important to investigate the long-term results in order to evaluate TA-assisted PPV. Moreover, the crucial issue of the effects on patients\u2019 vision must be explored before TA-assisted PPV can be recommended. The current report presents the 1-year results of the use of TA in PPV, and evaluates the procedure in light of the aforementioned issues.\nMethods\nStudy design, randomization and sample size\nThe prospective controlled clinical trial took place at eight hospitals in Japan as previously described (see Acknowledgements below) [13].\nThe patients were assigned to the two groups using a single-blind quasi-randomization approach within the participating clinical centers. Our preliminary study showed that the most important factor influencing the surgical outcome was not the nature of the disease for which PPV was used, but rather the hospital in which the surgery was performed (data not shown). To minimize this bias, equal numbers of patients from each hospital were assigned to each of the study groups. Thus, the patients were assigned by a single-blind quasi-randomization within the participating clinical centers. Briefly, after the patients passed the inclusion\/exclusion criteria and gave consent for the study, they were numbered serially at each hospital, and alternate numbers were assigned to the TA-assisted and conventional PPV groups, respectively, as previously described [13]. Data analysis was performed by an intent-to-treat analysis method. To conceal the allocations at the participating hospitals, the surgeons were not informed of the group to which a patient had been assigned until immediately before surgery.\nThis is the second report of controlled clinical trial of intraocular TA in PPV. Primary outcome was whether the use of TA might reduce the incidence of intraoperative complications. The sample size was determined based upon our preliminary study, and the method has been described elsewhere [13]. Briefly, the incidence of intraoperative retinal breaks was found to be approximately 15% in the participating hospitals. The sample size calculation was based on our pilot study data, and was aimed to detect a difference in short-term postoperative complications as reported previously [13]\nEligibility criteria and surgery\nInstitutional review board (IRB)\/ethics committee approval was obtained from all of the participating clinical establishments, such that all patients undergoing PPV at these hospitals were eligible to enroll in the study. Each patient was fully informed about the nature of the treatment, and provided written consent. The following groups were excluded: (1) patients with diagnoses that included macular holes; patients with uveitis, (2) patients with phakic lenses, (3) patients who were known to be steroid responders, to have glaucoma, or to have apparent infectious endophthalmitis, and (4) patients who were undergoing a second PPV. For ethical reasons, if at the time of surgery a surgeon felt that TA was necessary to achieve a successful PPV outcome, it was included in the procedure; for the purposes of the intent-to-treat analysis, these cases were included within the conventional PPV group.\nThe details of the surgical protocols varied between the hospitals, but the basic procedure was similar. PPV was performed according to a previously reported methodology, either with or without the use of a TA suspension prepared as described elsewhere [2, 13]. The recording of the operations, control of surgical quality, use of antibiotics, and termination of the study have been described elsewhere [13].\nDuring the follow-up period, additional surgeries were performed at each hospital as and when they were needed. For example, filtering surgeries were performed when the intra-ocular pressure (IOP) remained at an uncontrollably high level (25\u00a0mmHg or greater) even with full medication. Full medication included the use of all latanoprost, beta-blocker, and dorzolamide hydrochloride eyedrops and acetazolamide medicine.\nData collection and documentation were performed as previously described [13]. For the purposes of this study, the adverse events related to surgery were defined as follows: retinal breaks, macular pucker, retinal fibrous membrane formation, retinal detachment, vitreous hemorrhage (VH), rubeosis iridis, optic-disc damage, corneal diseases, and after cataract. The serious adverse events related to surgery were defined as follows: infectious endophthalmitis, retinal degeneration, unexplained optic-disc damage, and unexplained deterioration of vision.\nMasking and outcome assessment\nThe study records were collected by the principal investigator (TS) up until November 2006. After checking the records for harmful events, the data were sent to the controllers (YN) at Kyushu University. The remaining investigators were not permitted access to any information on the outcomes before completion of the analysis. Visual acuity was measured by ophthalmic technicians using the Snellen high-contrast acuity test. IOP was also measured by the ophthalmic technicians, and the findings were reviewed by the physicians. IOP was measured with pneumotonometer. The ophthalmic technicians were not informed of the purpose of the study or the assignment schedule. Anti-glaucoma eye drops and\/or acetazolamide were used when an IOP of 22\u00a0mmHg or higher persisted for 3\u00a0days.\nStatistical analysis\nThe visual acuity was converted to the logarithm of the minimum angle of resolution (LogMAR), and the baseline value was compared with those at 1\u00a0week, 1\u00a0month, 3\u00a0months, 6\u00a0months, and 1\u00a0year. The eyes were categorized into the following three groups: (1) the improved group, in which patients showed a 0.3 logMAR or greater improvement of visual acuity at the final examination compared with baseline, (2) the deteriorated group, in which patients showed a 0.3 logMAR or worse deterioration of visual acuity at the final examination compared with baseline, and (3) the unchanged group, in which patients did not meet the criteria of either the improved or the deteriorated group.\nA Kaplan-Meier survival analysis was used to estimate the probability of improved visual acuity with time after surgery. The improvement or deterioration was analyzed by the log rank test. The earliest time point after surgery at which the events in each category were reported was designated as the time point of each event.\nWe also estimated the multiple-adjusted odds ratio (OR) and the 95% confidence interval (95%CI) both for TA use and for the other variables, using a logistic regression model. The parameters that were adjusted for included age, gender, and disease diagnosis, which was categorized into the following five groups: proliferative diabetic retinopathy (PDR), diabetic macular edema (DME), rhegmatogenous retinal detachment (RRD), retinal vein occlusion (RVO), and others. A diagnosis of PDR was used as a reference. Even when a single eye received multiple additional surgeries, it was counted as one eye. All statistical analyses were performed using SAS\u00ae (Proprietary Software Release 8.2; SAS Institute Inc., Cary, NC, USA). The differences in diagnosis or surgical intervention between the two groups were compared using a two-tailed Chi-squared test with the Yates\u2019 correction. P values\u2009<\u20090.05 were considered statistically significant.\nResults\nNumber of eyes\nIn total, 920 patients were initially registered for PPV during this period. Among these, 146 eyes were not included in the study because they did not meet the eligibility criteria or because the patient declined to participate. In total, 774 eyes were randomized, with 391 eyes (from 216 male and 175 female patients) assigned to the TA-assisted PPV group and 383 eyes (201 male and 182 female patients) assigned to the conventional PPV group. The mean ages of the patients were 63.5\u2009\u00b1\u200912.1\u00a0years in the TA-assisted PPV group and 63.0\u2009\u00b1\u200911.3\u00a0years in the conventional PPV group. In total, 19 eyes that were originally assigned to the conventional PPV group received unplanned TA based on surgical judgment. The 1-year post-operative follow-up was completed for a total of 305 eyes in the TA-assisted PPV group (78.0%) and 295 eyes in the conventional PPV group (77.0%). The overall follow-up rate was 77.5% (Fig.\u00a01).\nFig.\u00a01Schematic diagram of the study. TA: triamcinolone acetonide, PPV: pars plana vitrectomy\nDiagnoses\nThe diagnoses for each group are shown in Table\u00a01. In both groups, PDR accounted for the largest number of cases, followed by RRD. In both groups, the pattern at the 1-year follow-up was similar to that at the baseline, and the different diagnoses had significantly different distributions within the two groups: the TA-assisted PPV group had a relatively larger number of patients with DME (P\u2009=\u20090.018 at baseline, P\u2009=\u20090.006 at 1-year follow-up) and a smaller number of patients with epiretinal membrane (ERM) formation (P\u2009=\u20090.031 at baseline, P\u2009=\u20090.008 at 1-year follow-up).\nTable\u00a01Diseases of each group at baseline and 1\u00a0year after surgery\u00a0TA-assisted PPVConventional PPVBaseline1\u00a0yearbaseline1\u00a0yearN (%)N (%)N (%)N (%)AMD7 (1.8%)3 (1.0%)3 (0.8%)3 (1.0%)BRVO50 (12.8%)41 (13.4%)37 (9.7%)29 (9.8%)CRVO11 (2.8%)9 (3.0%)7 (1.8%)6 (2.0%)DME67 (17.1%)60 (19.7%)42 (11.0%)34 (11.5%)ERM24 (6.1%)19 (6.2%)41 (10.7%)37 (12.5%)Lens luxation16 (4.1%)11 (3.6%)16 (4.2%)12 (4.1%)Macroaneurysm10 (2.6%)8 (2.6%)9 (2.3%)4 (1.4%)PDR113 (28.9%)85 (27.9%)131 (34.2%)101 (34.2%)RRD80 (20.5%)59 (19.3%)77 (20.1%)54 (18.3%)VH6 (1.5%)6 (2.0%)14 (3.7%)11 (3.7%)Others7 (1.8%)4 (1.3%)6 (1.6%)4 (1.4%)total383 (100%)305 (100%)391 (100%)295 (100%)TA; triamcinolone acetonide: PPV; pars plana vitrectomy: AMD; age-related macular degeneration: BRVO;branch retinal vein occlusion: CRVO: central retinal vein occlusion: DME; diabetic macular edema: ERM; epiretinal membrane: PDR; proliferative diabetic retinopathy: RRD; rhegmatogenous retinal detachment: VH; vitreous hemorrhage.\nThere were no significant differences between the two groups in the usage of surgical methods, such as cataract surgery (phacoemulsification P\u2009=\u20090.724 and pars plana lensectomy P\u2009=\u20090.272), ILM peeling (P\u2009=\u20090.075) and endolaser-photocoagulation (P\u2009=\u20090.413), or in the use of post-operative tamponade (sulfur hexafluoride gas P\u2009=\u20090.810, octa-fluorepropane gas P\u2009=\u20090.756, room air P\u2009>\u20090.999, and silicone oil P\u2009=\u20090.831) [13].\nVisual acuity\nThe visual acuity in both groups improved over time. In the TA-assisted PPV group, 322 eyes were categorized as improved, 21 were categorized as unchanged, and 48 were categorized as deteriorated. In the conventional PPV group, 312 eyes were categorized as improved, 45 eyes were categorized as unchanged, and 26 eyes were categorized as deteriorated. The log-rank test revealed no significant difference between the two groups regarding the improvement (P\u2009=\u20090.98) or deterioration (P\u2009=\u20090.26) of visual acuity. The logistic regression model also showed that the intra-operative use of TA was not a significant factor for the improvement of visual acuity (P\u2009=\u20090.91) after adjusting for age, gender, and diagnosis (Table\u00a02). Neither gender (P\u2009=\u20090.54) nor age (P\u2009=\u20090.31) was a significant factor for the improvement of visual acuity. However, PDR diagnosis was a significant factor for the improvement of visual acuity compared with DME (OR\u2009=\u20090.63, 95%CI\u2009=\u20090.478\u20130.817, P\u2009=\u20090.0006; Table\u00a02).\nTable\u00a02Factors affecting improvement of visual acuityVariable (no. of improved eyes\/total eyes)Odds ratio (95%CI)P-valueTA-assisted PPV (322\/391) vs conventional PPV (312\/383)1.10 (0.860\u20131.183)0.91Age1.00 (0.991\u20131.005)0.54Gender (male vs female)0.92 (0.785\u20131.080)0.31DiseasesRD vs PDR1.04 (0.834\u20131.340)0.71DME vs PDR0.63 (0.478\u20130.817)0.0006*RVO vs PDR1.21 (0.938\u20131.561)0.14ERM vs PDR0.78 (0.568\u20131.061)0.11Others vs PDR1.21 (0.924\u20131.579)0.17CI; confidence interval: TA; triamcinolone acetonide: PPV; pars plana vitrectomy: RVO; branch retinal vein occlusion + central retinal vein occlusion: DME; diabetic macular edema: ERM; epiretinal membrane: PDR; proliferative diabetic retinopathy: RRD; rhegmatogenous retinal detachment. * statistically significant\nPost-operative complications\nSubgroup analysis\nIn terms of post-operative complications, there was not any statistically significant difference. Fibrous ERM was observed in five eyes (1.4%) in the conventional PPV group, but in only one eye (0.3%) in the TA-assisted PPV group. A subgroup analysis showed that this difference was not significant (P\u2009=\u20090.10). Among the other complications, an IOP increase was found more frequently in the TA-assisted PPV group (23 eyes, 5.9%) than in the conventional PPV group (13 eyes, 3.4%), although this difference was not statistically significant (P\u2009=\u20090.10). There were no significant differences in the other complications between the two groups (Table\u00a03).\nTable\u00a03Post-operative complications in each group\u00a0TA-assisted PPV (total 391 eyes): n (%)Conventional PPV (total 383 eyes): n (%)P-value*After cataract18 (4.6%)17 (4.4%)0.91Macular pucker23 (5.9%)22 (5.7%)0.93IOP increase23 (5.9%)13 (3.4%)0.10Retinal detachment9 (2.3%)9 (2.3%)0.96Vitreous hemorrhage22 (5.6%)29 (7.8%)0.28Fibrous membrane1 (0.3%)5 (1.4%)0.10Iris synechiae6 (1.5%)4 (1.0%)0.55Others8 (2.0%)12 (3.1%)0.34TA; triamcinolone acetonide: PPV; pars plana vitrectomy: PPL; pas plana lensectomy: IOP; intraocular pressure: * Chi-square test with the Yates\u2019 correction.\nAdditional surgery\nAlthough the filtering surgery was more performed in TA-assisted PPV and the removal of vitreous hemorrhage was more performed in conventional PPV, there was not any statistically significant difference between the two groups with respect to causes of additional surgery after PPV. In the TA-assisted PPV group, 22 surgeries were performed (excluding filtering surgery). The most common reasons for additional surgery were removal of silicone oil (11 eyes), followed by retinal detachment (eight eyes), VH (six eyes), and others (seven eyes). In the conventional PPV group, 26 surgeries were performed (excluding filtering surgery). The most common reasons were VH (12 eyes), followed by retinal detachment (nine eyes), removal of silicone oil (seven eyes), and others (six eyes). Although there was a tendency for VH to be less common in TA-assisted PPV eyes, no significant difference was found in comparison to conventional PPV (P\u2009=\u20090.13; Table\u00a04). Filtering surgery was more frequently performed in the TA-assisted PPV group (15 cases, 3.8%) than the conventional PPV group (seven cases, 1.8%), although this difference was not significant (P\u2009=\u20090.10). Ten eyes received multiple additional surgeries in TA-assisted PPV group and seven eyes in conventional PPV group.\nTable\u00a04Causes of additional surgery after PPV\u00a0TA-assisted PPV (total 391 eyes): n (%)Conventional PPV (total 383 eyes) : n (%)P-value*Silicone removal11 (2.8%)7 (1.8%)0.36Retinal detachment8 (2.0%)9 (2.3%)0.77Vitreous hemorrhage6 (1.5%)12 (3.1%)0.14Filtering surgery15 (3.8%)7 (1.8%)0.10Others7 (1.8%)6 (1.6%)0.81Total37 (9.5%)\u271d32 (8.6%)\u271d\u271d0.68TA; triamcinolone acetonide: PPV; pars plana vitrectomy: * Chi-square test with the Yates\u2019 correction: \u271d Ten eyes received multiple additional surgeries: \u271d\u271d Seven eyes received multiple additional surgeries.\nThe survival curve produced by the log-rank test showed that the incidence of additional surgery was not significantly different between the two groups (P\u2009=\u20090.45). The logistic regression test also showed that the use of TA was not a significant factor for additional surgery after adjustments for gender, age, and diagnosis. However, a diagnosis of PDR was a significant factor for additional surgery in comparison to DME (P\u2009=\u20090.005), RVO (P\u2009=\u20090.006), and ERM (P\u2009=\u20090.02; Table\u00a05). None of the variables were related to the requirement for filtering surgery (Table\u00a06).\nTable\u00a05Factors affecting additional surgeryVariable (no. of additional surgery\/total)Odds ratio (95%CI)P-valueTA-assisted PPV (37\/391) vs conventional PPV (32\/383)1.23 (0.797\u20131.911)0.35Age0.97 (0.950\u20130.984)0.0001*Gender (male vs female)1.15 (0.733\u20131.797)0.55DiseasesRD vs PDR0.60 (0.345\u20131.029)0.06DME vs PDR0.29 (0.122\u20130.681)0.005*RVO vs PDR0.23 (0.084\u20130.657)0.006*ERM vs PDR0.087 (0.012\u20130.631)0.02*Others vs PDR0.64 (0.299\u20131.374)0.25CI; confidence interval: RVO; branch retinal vein occlusion + central retinal vein occlusion: DME; diabetic macular edema: ERM; epiretinal membrane: PDR; proliferative diabetic retinopathy: RRD; rhegmatogenous retinal detachment. * statistically significantTable\u00a06Factors affecting filtering surgeryVariable (no. of filtering surgery\/total)Odds ratio (95%CI)P-valueTA-assisted PPV (12\/391) vs conventional PPV (5\/383)1.74 (0.708\u20134.265)0.23Age0.98 (0.941\u20131.018)0.28Gender (male vs female)1.81 (0.696\u20134.728)0.22DiseasesRRD vs PDR\u20130.99DME vs PDR\u20130.99RVO vs PDR\u20130.99ERM vs PDR\u20130.99Others vs PDR0.83 (0.266\u20132.573)0.74CI; confidence interval: TA; triamcinlone acetonide: PPV; pars plana vitrectomy: RVO; branch retinal vein occlusion + central retinal vein occlusion: DME; diabetic macular edema: ERM; epiretinal membrane: PDR; proliferative diabetic retinopathy: RRD; rhegmatogenous retinal detachment\nSerious adverse events\nNone of the serious adverse events related to surgery defined in this study (such as retinal degeneration, endophthalmitis, an unexplained decrease in visual acuity, or optic-disc atrophy) were observed in either group during the 1-year follow-up.\nDiscussion\nThe first report on this study showed that the intra-operative use of TA significantly decreased the incidence of intra-operative complications associated with PPV, and no adverse events related to surgery were found over a 3-month observation period [13]. A decreased incidence of intra-operative complications is beneficial; however, the critical issue is whether the surgery improves the patients\u2019 vision. We therefore followed up the visual acuity, which is a crucial factor influencing vision, for 1\u00a0year after surgery. Our results revealed no significant difference in visual acuity between the TA-assisted PPV group and the conventional PPV group after 1\u00a0year. As described in the earlier report, which analyzed data from the same eyes, intra-operative retinal breaks and retinal detachment were significantly less frequent in the TA-assisted PPV group than in the conventional PPV group. Nonetheless, the decreased incidence of intra-operative complications did not significantly affect the visual acuity after 1\u00a0year. This finding can be explained by the following facts. First, surgical skill is the most important factor for surgical success. As all of the operations in this study were performed by qualified and experienced surgeons, the intra-operative complications were managed so as not to cause serious post-operative complications. Thus, the influence of intra-operative complications on visual acuity might have been minimal. Second, a patient\u2019s vision after vitrectomy might be more strongly affected by a disease diagnosis than by intra-operative complications. Indeed, eyes with PDR were significantly more likely to show improved visual acuity compared with baseline than eyes with DME in the current study. As our present case series included various diseases, the impact of intra-operative complications on post-operative visual acuity might have been masked by another strong factor, such as disease diagnosis, even after adjustments. Third, the eyes in the current study were primarily chosen for the detection of intra-operative retinal breaks, based on the hypothesis that the intra-operative use of TA might decrease their incidence from 15% to 8% [13]. Thus, the sample size might not have been large enough to detect the superiority or inferiority of TA-assisted PPV. For example, in order to detect the difference of post-operative improvement of vision in TA-assisted PPV and conventional PPV based upon the present results, approximately 29,315 cases in each group are necessary with \u03b1 power of 0.05 and 1-\u03b2 of 0.8. However, considering the relative stability of the visual acuity observed here, we are confident that the intra-operative use of TA does not have a positive or negative effect on postoperative visual acuity for 1\u00a0year.\nAdditional surgeries carry associated risks and can be costly; they are thus important when justifying the intra-operative use of TA in PPV. The incidence of additional surgery after vitrectomy was found to be equivalent using both methods. Considering the IOP increase after surgery, the opportunity to use anti-glaucoma eye drops was high in the TA-assisted PPV group [13], and the number of filtering surgeries performed during 1\u00a0year after TA-assisted PPV was also high; however, the incidence of additional surgeries was not statistically different between the groups. Several previous reports showed that intravitreous TA injection required filtering surgery, and its frequency was less than 1% of all treated eyes [18, 19]. In contrast to intravitreous TA injection, most of the TA was removed from the eye at the end of the surgery. Thus, it was plausible that the necessity of filtering surgery in TA-assisted PPV was not significant. The other causes of additional surgery occurred at similar frequencies, and there were no significant differences between the groups. A diagnosis of PDR was a significant factor for the requirement for additional surgery, as it was for the improvement of visual acuity. Furthermore, in our earlier report on the 3-month follow-up study, a diagnosis of PDR was found to be a significant risk factor for the occurrence of intra-operative complications. PDR eyes thus appear to have several poor prognostic factors, consistent with the present results.\nWith regard to the post-operative complications, pre-retinal fibrosis appeared to be less common in the TA-assisted PPV group than in the conventional PPV group, although this was not statistically significant. This advantage of TA-assisted PPV has been described in previous reports [3, 5\u20139]. The intra-operative use of TA visualizes the posterior hyaloid clearly, which facilitates its removal [6, 7]; as the residual posterior hyaloid can act as a scaffold for pre-retinal fibrous-membrane formation, its removal could reduce the incidence of post-operative pre-retinal fibrosis [5\u20137]. In the current study group, the removal of the residual posterior hyaloid was carried out according to the surgeons\u2019 preference\u2014some surgeons removed it completely, while others did not. This might have obscured any potential advantage of the intra-operative use of TA in inhibiting post-operative pre-retinal fibrosis.\nThere were clear limitations to our analysis. The number of cases in each study group was primarily chosen to allow the detection of intra-operative complications; thus, the significance of the intra-operative use of TA might only become clear when studying larger numbers of patients. In addition, the sample size was too small to detect the risk of endophthalmitis. The incidence of endophthalmitis related to PPV was 0.046% in the conventional PPV group and 0.053% in the TA-assisted PPV group [20, 21]. An analysis of a greater number of cases will thus be necessary to evaluate the risk of endophthalmitis related to TA-assisted PPV. However, the present sample size was large enough to detect several factors that affected post-operative visual acuity or the need for additional surgery. Another limitation was that the post-operative visual acuity was strongly influenced by the diagnosis of disease. Eyes with simple VH clearly had a greater chance of surgery improving vision above the baseline level than did eyes with complicated PDR. In order to evaluate the influence of the intra-operative use of TA on visual acuity more precisely, we are currently performing a study on eyes with the same disease diagnosis. Furthermore, the subgroup of DME or ERM in each group was not completely matched. The interpretation of the results should be done carefully. Additionally, caution must be applied when making generalizations on the basis of our current results, and their application to clinical practice must be undertaken with care. Other factors, such as invasiveness and cost, are also important in seeking justification for this treatment. The success of surgery depends upon various factors, and surgical skill is critical. When considering the likelihood of retinal breaks and their recovery without damaging vision, comparisons of the hazard ratios of TA-assisted PPV and conventional PPV would be valid only if made by individuals who qualify as PPV specialists.\nIn summary, the intra-operative use of TA in PPV did not affect visual acuity over 1\u00a0year. As negative or adverse events were not observed in this case series, a more detailed study is warranted to establish the value of TA in PPV.","keyphrases":["steroid","retinal detachment","diabetic retinopathy","proliferative vitreoretinopathy","adjuvant"],"prmu":["P","P","P","M","U"]}
{"id":"Clin_Oral_Investig-4-1-2238784","title":"The evolution of tooth wear indices\n","text":"Tooth wear\u2014attrition, erosion and abrasion\u2014is perceived internationally as an ever-increasing problem. Clinical and epidemiological studies, however, are difficult to interpret and compare due to differences in terminology and the large number of indices that have been developed for diagnosing, grading and monitoring dental hard tissue loss. These indices have been designed to identify increasing severity and are usually numerical. Some record lesions on an aetiological basis (e.g. erosion indices), others record lesions irrespective of aetiology (tooth wear indices); none have universal acceptance, complicating the evaluation of the true increase in prevalence reported. This article considers the ideal requirements for an erosion index. It reviews the literature to consider how current indices have evolved and discusses if these indices meet the clinical and research needs of the dental profession.\nIntroduction\nThere is both a clinical and a scientific need to be able to measure tooth wear, and the literature abounds with many methods which can be broadly divided into quantitative and qualitative in nature. Quantitative methods tend to rely on objective physical measurements, such as depth of groove, area of facet or height of crown. Qualitative methods, which rely on clinical descriptions, can be more subjective if appropriate training and calibration are not carried out but which, with correct safeguards, can be valuable epidemiological tools. In a clinical intra-oral examination, there will be an inclination towards descriptive assessment measures, such as mild, moderate or severe, rather than quantitative measurement, which is easier to perform reliably on a model or in the laboratory. Such methods tend to be more sensitive but do not lend themselves readily to clinical use\u2014especially in epidemiology, where fieldwork data collection is often carried out in an environment lacking sophisticated equipment.\nQuantitative and qualitative methods typically utilise grading or scoring systems designed to identify increasing severity or progression of a condition; these are described as indices and are usually numerical. An ideal index should be simple to understand and use, clear in its scoring criteria and be demonstrably reproducible. Its application should be useful for research into the aetiology, prevention and monitoring of a condition, essentially being an epidemiological and clinical tool.\nReview of the literature reveals the fact that many different tooth wear indices have been developed for clinical and laboratory use all over the world. Unfortunately, the production of so many indices does not allow for ready comparison of results between different working groups, and this is especially important in epidemiology when trying to define the prevalence of a condition. Confusion is further generated in the literature as the majority of researchers, in their attempts to quantify the amount of tooth tissue loss due to tooth wear, have historically concentrated on one aetiology only, and these indices tend to be surface limited. Often, the wear patterns described do not appear to reflect the aetiology suggested, and this relates to lack of uniformity with tooth wear terminology and translation errors. Many diagnostic indices do not properly reflect the morphological defects, and there is little international standardisation. All of these factors complicate the comparison of data and evaluation of the efficacy of preventive and therapeutic measures.\nThe literature identifies separate indices for use in clinical and laboratory situations and specific indices for attrition, abrasion, erosion and multifactorial tooth wear. There are common threads to all of the indices, such as descriptive diagnostic criteria and criteria for quantifying the amount of hard tissue loss. These generally consider the size of the affected area\u2014as a proportion of a sound surface and\/or the depth of tissue loss\u2014often expressed as a degree of dentine exposure.\nThe clinical measurement of erosion\nThe earliest indices shared common, arbitrary criteria, relying on descriptive terms such as slight, mild, moderate, severe and extensive. Restarski et al. [26] developed a six-point grading system to evaluate the severity of erosive destruction observed on the lingual surfaces of rat and puppy molars, but concerns were raised with regards to reproducibility. With vague criteria definitions, variability in recording is expected. Each animal was allocated a total score, calculated by summing the mean molar quadrant scores. Whilst producing simple data for analysis, it is acknowledged that averaging scores in this manner leads to the loss of much data. If the number of teeth severely affected is small, the erosion score will be low; but this could mask a significant, localised clinical problem [27].\nEccles [9] originally classified lesions broadly as early, small and advanced, with no strict criteria definitions, thus allowing wide interpretation. Later, the index was refined and expanded, with greater emphasis on the descriptive criteria [10]. It was presented as a comprehensive qualitative index, grading both severity and site of erosion due to non-industrial causes, and is considered as one of the cardinal indices from which others have evolved. In essence, it breaks down into three classes of erosion, denoting the type of lesion, assigned to four surfaces, representing the surface where erosion was detected (Table\u00a01).\nTable\u00a01Eccles index for dental erosion of non-industrial origin [10]ClassSurfaceCriteriaClass I\u00a0Early stages of erosion, absence of developmental ridges, smooth, glazed surface occurring mainly on labial surfaces of maxillary incisors and caninesClass IIFacialDentine involved for less than one third surface; two typesType 1 (commonest): ovoid\u2013crescentic in outline, concave in cross section at cervical region of surface. Must differentiate from wedge shaped abrasion lesionsType 2: irregular lesion entirely within crown. Punched out appearance, where enamel is absent from floorClass IIIaFacialMore extensive destruction of dentine, affecting anterior teeth particularly. Majority of lesions affect a large part of the surface, but some are localised and hollowed outClass IIIbLingual or palatalDentine eroded for more than one third of the surface area. Gingival and proximal enamel margins have white, etched appearance. Incisal edges translucent due to loss of dentine. Dentine is smooth and anteriorly is flat or hollowed out, often extending into secondary dentineClass IIIcIncisal or occlusalSurfaces involved into dentine, appearing flattened or with cupping. Incisal edges appear translucent due to undermined enamel; restorations are raised above surrounding tooth surfaceClass IIIdAllSeverely affected teeth, where both labial and lingual surfaces are extensively involved. Proximal surfaces may be affected; teeth are shortened\nGreater accuracy was introduced by Xhonga and Valdmanis [33] who divided erosions into four levels by measurement with a periodontal probe: none, minor (less than 2\u00a0mm), moderate (up to 3\u00a0mm) and severe (greater than 3\u00a0mm). They further differentiated types of erosion by morphological descriptions, such as wedge, saucer, groove and atypical. They did not address the problem of inter- or intra-examiner variability.\nThe clinical measurement of tooth wear\nIt is perhaps significant that the earliest index documented by Broca [4] and used as a foundation for the development of further indices graded horizontal or oblique patterns of occlusal wear without presupposing the aetiology. Smith and Knight [30] introduced the more general concept of measuring tooth wear per se, irrespective of the cause, and since then more recent indices have been developed or modified from Smith and Knight that do not rely on a prior diagnosis and are more clinically relevant. Most of these stress the importance of user training sessions and calibration exercises.\nSmith and Knight [30] took Eccles\u2019 ideas a stage further, producing the tooth wear index (TWI), a comprehensive system whereby all four visible surfaces (buccal, cervical, lingual and occlusal\u2013incisal) of all teeth present are scored for wear, irrespective of how it occurred (Table\u00a02). This avoids the confusion associated with terminology and translation or differences in opinion for diagnosis of aetiology based on clinical findings. Guidelines for using the criteria were produced in a booklet by the authors to aid training and standardisation with other investigators; in cases of doubt, the lowest score is given. Complete enamel loss (score 4) may, however, be misleading, as there is almost always a rim of enamel at the worn surface margins\u2014the colloquial \u201cenamel halo.\u201d\nTable\u00a02Smith and Knight tooth wear index [30]ScoreSurfaceCriteria0B\/L\/O\/INo loss of enamel surface characteristicsCNo loss of contour1B\/L\/O\/ILoss of enamel surface characteristicsCMinimal loss of contour2B\/L\/OLoss of enamel exposing dentine for less than one third of surfaceILoss of enamel just exposing dentineCDefect less than 1\u00a0mm deep3B\/L\/OLoss of enamel exposing dentine for more than one third of surfaceILoss of enamel and substantial loss of dentineCDefect less than 1\u20132\u00a0mm deep4B\/L\/OComplete enamel loss\u2013pulp exposure\u2013secondary dentine exposureIPulp exposure or exposure of secondary dentineCDefect more than 2\u00a0mm deep\u2013pulp exposure\u2013secondary dentine exposure\nThis index was the first one designed to measure and monitor multifactorial tooth wear; a further pioneering feature was the ability to distinguish acceptable and pathological levels of wear, by comparison with threshold normal values for the age groups studied. Tooth wear was defined as pathological if the teeth became so worn that they do not function effectively or seriously mar the appearance\u2014before they are lost through other causes\u2014or the patient dies. Results from inter- and intra-reproducibility exercises were within a range regarded as acceptable for epidemiological purposes, and the index appears simple to use clinically\u2014intra-orally or from models and photographs. However, some problems have been identified with the TWI, including the time necessary to apply to a whole dentition, amount of data generated and the comparisons with threshold levels for each age group; the thresholds proposed were high, erring towards understatement rather than exaggerations of pathological wear. Full use of the index as a research tool is not feasible without computer assistance.\nA sign of professional confidence in this index is its adopted use by a number of different investigators in the UK looking at tooth wear prevalence and severity [3, 20, 25, 27], aetiology and risk [1, 21]. Other researchers have used it with modifications pertaining to the particular age group being studied. Millward et al. [18] made adjustments to study erosion in the primary and secondary dentitions, excluding cervical surfaces. Subjects were then grouped together to produce three classifications: no or mild erosion\u2014no score >1, moderate\u2014at least one tooth in dentition score 2, severe\u2014at least one tooth in dentition score 3 or 4.\nIn a survey of elderly people, Steele et al. [31] combined low wear scores representing small losses of enamel and used the worst surface score per tooth as an overall tooth score. Donachie and Walls [7, 8] outlined various flaws in the tooth wear index as an epidemiological tool in the ageing population and suggested a need to increase the sensitivity of TWI at extremes of tooth wear, to take account of the capacity of the elderly to have adequate function in cases of significant wear. They suggested altering threshold values, amplifying scoring criteria and creating a sixth point to distinguish between exposure of secondary dentine and frank pulp exposure.\nDevelopment of indices\nMany other indices have been proposed for measuring erosive tooth wear [2, 14, 15, 17, 23] which have their roots in the indices of Eccles [10] and Smith and Knight [30]. Linkosalo and Markkanen [15] utilised a qualitative index with listed diagnostic criteria to confirm lesions as erosive and a four-scale grading of severity, relating to involvement of dentine. Their scoring system was modified by Lussi et al. [17] to create an erosive index that has been used widely by European workers to score the facial, lingual and occlusal surfaces of all teeth except the third molars (Table\u00a03).\nTable\u00a03Erosion index according to Lussi [16, 17]SurfaceScoreCriteriaFacial0No erosion. Surface with a smooth, silky glazed appearance, possible absence of developmental ridges1Loss of surface enamel. Intact enamel cervical to the erosive lesion; concavity on enamel where breadth clearly exceeds depth, thus distinguishing it from toothbrush abrasion. Undulating borders of the lesion are possible and dentine is not involved2Involvement of dentine for less than half of tooth surface3Involvement of dentine for more than half of tooth surfaceOcclusal\/lingual0No erosion. Surface with a smooth, silky glazed appearance, possible absence of developmental ridges1Slight erosion, rounded cusps, edges of restorations rising above the level of adjacent tooth surface, grooves on occlusal aspects. Loss of surface enamel. Dentine is not involved2Severe erosions, more pronounced signs than in grade 1. Dentine is involved\nO\u2019Sullivan [23] proposed a new index for the measurement of erosion specifically in children. The index was qualitative with a broad attempt at quantification noting whether less or more than half of the surface was affected. Every tooth was examined and assigned a three-digit score relating to the site of erosion, severity (grade 0\u20135) and area of surface affected. O\u2019Brien [22] reported the use of a partial recording system for measuring erosion in children in UK children\u2019s dental health surveys, where only the facial and lingual surfaces of the primary and permanent maxillary incisor teeth were scored for erosion. Again, the criteria were mostly qualitative and descriptive, with a broad attempt made to quantify the area involved.\nBardsley et al. [2] pioneered a new, simplified version of TWI [30] when carrying out epidemiological studies on large numbers of adolescents in North West England (Table\u00a04). Tooth wear scoring was essentially dichotomised into the presence or absence of dentine, with even cupping of dentine scoring one. A partial recording system was used, collecting data from 40 surfaces including occlusal surfaces of the four first molar teeth and the labial, incisal and lingual\u2013palatal surfaces of the six upper and lower anterior teeth.\nTable\u00a04Simplified scoring criteria for TWI [2]ScoreCriteria0No wear into dentine1Dentine just visible (including cupping) or dentine exposed for less than 1\/3 of surface2Dentine exposure greater than 1\/3 of surface3Exposure of pulp or secondary dentine\nHowever, despite calibration and training, difficulties were experienced diagnosing dentine exposure in the epidemiological field and there is some debate as to the significance of dentinal cupping when exposed dentine does not relate to significant amounts of tissue loss [12]. In a recent study of Ganss et al. [12], teeth were visually and histologically examined for presence of exposed dentine, and the correlation in accuracy between the two examinations was poor. The diagnosis of exposed dentine is obviously important but may not be accurate from visual examination alone.\nOilo et al. [24] concentrated on a different type of scoring system, with criteria based on treatment need. They criticised the use of indices that used a nonlinear scoring method, claiming calculated mean wear scores can be misleading. Their index was based on Ryge and Snyder\u2019s [28] system for evaluating the quality of restorations and had five categories divided into two broad camps; Romeo, Sierra and Mike were satisfactory, whilst Tango and Victor indicated unacceptable levels of wear requiring treatment. All groups except Romeo were subdivided according to degree of dentine exposed and clinical findings such as pain, sensitivity and fracture of restorations, giving the impression of a cumbersome system. Dahl et al. [6] modified it with the introduction of even more categories, with an aim to establish subjective dental criteria for present and future evaluations of tooth wear and the need for treatment. In practice, these indices require experience for reliable use; individuals with differing clinical backgrounds will not get consistent, objective results.\nIndices that attempt to visualise, measure and monitor the amount of worn enamel or exposed dentine by difficult direct clinical interpretation [13] and indirectly on accurate, serial study casts which must be retained [5, 11, 29, 32] have been described. Larsen et al. [14] recommended a new clinical index based on a combination of clinical examination, photographs and study casts, with complicated qualitative and quantitative criteria. Plaque-free teeth were clinically examined and photographed prior to taking silicone impressions for epoxy resin casts. They considered clinical and photographic data to be supplemental with final wear classification based on visual inspection of casts at \u00d710 magnification. Each tooth surface was scored, with six grades of erosion severity modelled from Smith and Knight [30]; the index and its criteria are complicated and time consuming.\nConclusion\nReview of the literature on indices for tooth wear (or erosion) is confusing; there are too many indices proposed and used, with lack of standardisation in terminology. There are many epidemiological studies reported, but it is difficult to quantify the increases in prevalence reported internationally, as results are not easily comparable. It is doubtful whether any of the indices described are sensitive enough to monitor all but the most severe changes in tooth wear clinically and these cannot be used to measure a rate of wear [19]. It is a challenge to try to develop a simple index that can be used clinically to assess progression of wear.\nTo date, there is not one ideal index that can be used for epidemiological prevalence studies, clinical staging and monitoring, and it may be necessary to accept that one simple index does not yet exist to meet all requirements of both clinical and research teams. There should, however, be an aim for indices that can be relevant to both fields and can be used internationally in order to strengthen knowledge of dental erosion.","keyphrases":["tooth wear","tooth wear indices"],"prmu":["P","P"]}
{"id":"FEBS_Lett-1-5-1942071","title":"Phosphoinositide-dependent protein kinase-1 (PDK1)-independent activation of the protein kinase C substrate, protein kinase D\n","text":"Phosphoinoisitide dependent kinase l (PDK1) is proposed to phosphorylate a key threonine residue within the catalytic domain of the protein kinase C (PKC) superfamily that controls the stability and catalytic competence of these kinases. Hence, in PDK1-null embryonic stem cells intracellular levels of PKC\u03b1, PKC\u03b21, PKC\u03b3, and PKC\u03b5 are strikingly reduced. Although PDK1-null cells have reduced endogenous PKC levels they are not completely devoid of PKCs and the integrity of downstream PKC effector pathways in the absence of PDK1 has not been determined. In the present report, the PDK1 requirement for controlling the phosphorylation and activity of a well characterised substrate for PKCs, the serine kinase protein kinase D, has been examined. The data show that in embryonic stem cells and thymocytes loss of PDK1 does not prevent PKC-mediated phosphorylation and activation of protein kinase D. These results reveal that loss of PDK1 does not functionally inactivate all PKC-mediated signal transduction.\n1\nIntroduction\nPhosphoinoisitide dependent kinase l (PDK1) phosphorylates a key threonine residue within the catalytic domain of a number of AGC family kinases, including diacylglycerol-regulated kinases of the protein kinase C (PKC) superfamily and phosphatidyl inositol-3 kinase (PI3K)-controlled serine kinases such as Akt\/PKB and the 70-kilodalton ribosomal S6 kinase 1 (S6K1) [1]. PDK1-mediated phosphorylation of many of these AGC kinases is essential for their activation. Accordingly, deletion of PDK1 by homologous recombination removes one of the rate-limiting regulators of multiple serine kinases and causes embryonic lethality [2]. To circumvent this prenatal death and explore the role of PDK1 in different tissues, mice expressing PDK1 alleles flanked with the loxP Cre excision sequence (PDK1fl\u0394neo\/fl\u0394neo mice) have been generated. These were first used to achieve muscle specific deletion of PDK1, producing mice that die early with severe defects in cardiac function [3]. The importance of PDK1 for T lymphocyte biology has also been explored by looking at T cell development following T lineage specific deletion of PDK1. These studies showed that deletion of PDK1 in T cell progenitors in the thymus results in a block in T cell differentiation, indicating that PDK1 has essential functions in T lymphocyte development [4].\nThe importance of PDK1 for T cell development in the thymus focuses attention on the identification of the PDK1 substrates directly involved in mediating the unique and non-redundant functions of this kinase in vivo. In this context, studies of mouse embryonic stem cells in which both PDK1 alleles have been deleted have shown that PDK1 is necessary for T-loop phosphorylation, and catalytic activity, of the AGC kinases Akt\/PKB, S6K1 and p90 ribosomal S6 kinase (p90RSK) [5]. However, PDK1 is not required for the phosphorylation and activation of other AGC kinases such as PKA or AMPK [5].\nOne very important subfamily of AGC kinases that can be regulated by PDK1 are the protein kinase C enzymes. There are multiple isoforms of PKC: the classical PKCs (\u03b1, \u03b2I, \u03b2II and \u03b3) which are regulated by calcium, diacylglycerol (DAG) and phospholipids; novel PKCs (\u03b4, \u03b5, \u03b7 and \u03b8) which are regulated by DAG and phospholipids; and the atypical PKCs (\u03b6 and \u03bb) which are insensitive to both calcium and DAG. The catalytic domain of PKCs have a conserved T-loop motif that is a substrate for PDK1, and it has been proposed that PDK1 is responsible for T-loop phosphorylation of the various PKC isoforms [6\u20138]. PDK1-mediated T-loop phosphorylation of PKCs is proposed to stabilize protein expression by acting as a priming signal that enables PKCs to achieve catalytic maturity [6,7]. Here, PDK1 docks to a hydrophobic motif in the C-terminus of newly synthesised PKC enzymes, enabling it to phosphorylate their T-loop sites. Following T-loop phosphorylation, PDK1 disengages enabling PKCs to autophosphorylate their C-terminal hydrophobic motifs and achieve catalytic maturity [9\u201311]. Once mature, PKCs remain inactive due to autoinhibition from their pseudosubstrate motifs and are only activated following the generation of second messengers, such as diacylglycerol and calcium.\nThe release of PDK1 from PKCs is rate limiting and so PDK1 is responsible for controlling the amount of mature\/catalytically competent PKCs expressed in cells [11]. Hence, in the absence of PDK1, intracellular levels of PKC\u03b1, PKC\u03b21, PKC\u03b3, and PKC\u03b5 are strikingly reduced [6,12]. PKC\u03be levels are similar in wild-type and PDK1-null embryonic stem cell (ES cells) although PKC\u03be is not phosphorylated on its T-loop Thr410 site in the absence of PDK1 [6,13]. PKC\u03b4 expression is also reduced in PDK1-null cells [6,12] although there was still phosphorylation of the PKC\u03b4 T-loop site Thr505 in the residual pool of PKC\u03b4 still present in PDK1-null cells [13]. Moreover, there is some evidence in the literature that T-loop phosphorylation is not absolutely critical for PKC\u03b4 catalytic activity, since a bacterially-expressed Thr505A PKC\u03b4 mutant protein retains some activity in vitro [14]. Of note, a glutamic acid residue at position 500 within the PKC\u03b4 activation loop, which is important for catalytic activity [15], could potentially compensate for the lack of PKC\u03b4 T-loop phosphorylation in PDK1-null cells.\nThese studies support the hypothesis that PDK1 has an important role in regulating PKC phosphorylation and stability. However, it has not been shown unequivocally that the reduced levels of PKCs in PDK1-null cells abrogate PKC-mediated cellular responses. In this context, it has been shown that in the absence of PDK1 there is loss of Akt\/PKB and S6K1 T-loop phosphorylation and a corresponding inability of cells to regulate phosphorylation of Akt\/PKB or S6K1 substrates [5]. There is thus confidence that Akt\/PKB and S6K1 function is defective in PDK1-null cells. This has not been determined for the PKCs because although PDK1-null cells have reduced endogenous PKC levels the integrity of downstream PKC effector pathways in PDK1-null cells has not been determined.\nOne well characterised target for novel PKCs is the serine kinase protein kinase D (PKD). Activation of PKD requires the phosphorylation of two key serine residues within the catalytic domain [16,17]. These residues are absolutely conserved through evolution from C. elegans to man and are substrates for novel PKCs. The activation of PKCs is thus required for PKD activation in a variety of cell lineages in response to a number of different stimuli [16,17]. For example, it has been demonstrated that PKD activity in lymphocytes is regulated by PKC-mediated phosphorylation of its catalytic domain [18,19] and phosphorylation of the PKD catalytic domain is reduced in PKC\u03b2-deficient spleenocytes [20]. It would therefore be expected that PKD would remain inactive if PKCs cannot achieve catalytic maturity in PDK1-null cells. In support of this hypothesis, previous work has established that co-expression of PDK1 and the novel PKC\u03b5 isoform, together with PKD, enhances PKD activity [21]. The hypothesis that PKD activation is dependent on PDK1 has not been fully investigated and is important to address as it is one way to assess whether there is residual functional PKC activity in PDK1-null cells. Accordingly, the objective of the present study was to examine whether loss of PDK1 results in loss of the PKC signalling pathway that regulates PKD.\n2\nResults and discussion\n2.1\nActivation of protein kinase D in PDK1\u2212\/\u2212 thymocytes\nTo investigate the impact of PDK1 loss on protein kinase D activation in PDK1 deficient T lymphocytes, mice expressing PDK1 alleles flanked with the loxP Cre excision sequence (PDK1fl\u0394neo\/fl\u0394neo) were bred with transgenic mice expressing Cre recombinase under the control of the proximal p56lck promoter which induces expression of Cre in T cell progenitors in the thymus. It has been shown previously that in LckCre+PDK1fl\u0394neo\/fl\u0394neo mice PDK1 is deleted in pre-T cells, preventing normal T cell development [4]. To explore the role of PDK1 in the regulation of PKD activity, wild-type or PDK1\u2212\/\u2212 pre-T cells were isolated and left unstimulated, stimulated with a crosslinking \u03b1-CD3 antibody (to activate preTCR signalling) or were stimulated with the phorbol ester PdBu (a diacylglycerol mimetic which activates classical\/novel PKCs), as indicated. PKD catalytic activity was monitored using an antisera that recognises PKD molecules that are autophosphorylated on Ser916 [22]. The data (Fig. 1) show that in quiescent wild-type thymocytes PKD is not autophosphorylated on Ser916 whereas \u03b1-CD3 or PdBu stimulation strongly induced PKD catalytic activity, as shown by increased Ser916 autophosphorylation. The results also show that both \u03b1-CD3 and PdBu treatment induce strong PKD Ser916 autophosphorylation in PDK1\u2212\/\u2212 pre-T cells (Fig. 1). PKD activation is dependent on PKC-mediated phosphorylation of two key serine residues (Ser744\/S748) within the PKD catalytic domain and as shown in Fig. 1, PKD is phosphorylated on Ser744\/S748 following \u03b1-CD3 or PdBu activation in both wild-type and PDK1\u2212\/\u2212 pre-T cells.\nThe phosphorylation and activation of PKD in PDK1\u2212\/\u2212 pre-T cells argues that these cells still contain a functional, catalytically competent PKC(s) that is able to respond to diacylglycerol\/phorbol ester signals. RNAi experiments have demonstrated that PKC\u03b5 in Swiss 3T3 cells or PKC\u03b4 in HeLa cells are the PKC isoforms responsible for serine phosphorylation (Ser744\/S748) within the PKD activation loop [23,24]. PKC\u03b4 has been shown to be residually phosphorylated on its T-loop Thr505 site in PDK1-null ES cells [13]. We therefore examined the phosphorylation status of the PKC\u03b4 T-loop site, Thr505, in PDK1\u2212\/\u2212 pre-T cells. The data in Fig. 1 show basal phosphorylation of PKC\u03b4 Thr505 in wild-type pre-T cells and also show that significant phosphorylation of PKC\u03b4 on Thr505 in retained in PDK1\u2212\/\u2212 pre-T cells. In contrast, as previously demonstrated, PDK1\u2212\/\u2212 pre-T cells have lost activity of the PKB\/S6K1 signalling pathway that controls the phosphorylation of the ribosomal S6 subunit on Ser235\/236 [4,25]. The data in Fig. 1 also explore the impact of PDK1 loss on the T-loop phosphorylation of p90RSK1 and PRK1. The data show Western blot analysis of wild type and PDK1 null pre-T cells with a phospho-specific antibody against Ser227 in p90RSK1 and show that this site is basally phosphorylated in wild-type pre-T cells but is not phosphorylated in PDK1-null pre-T cells. Similarly, basal phosphorylation of PRK1 on its Thr774 T-loop site is substantially reduced in PDK1-null pre-T cells. Thus, PDK1-null pre-T cells show defective activation of some, but not all, AGC family kinases: regulation of a PKC\u2013PKD signalling pathway remains intact in these cells.\n2.2\nActivation of protein kinase D in PDK1\u2212\/\u2212 ES cells\nThe experiments in Fig. 1 show that PDK1 is not essential for PKD activation in thymocytes. To probe further the involvement of PDK1 in the regulation of PKC\u2013PKD signalling, experiments in wild-type and PDK1\u2212\/\u2212 mouse embryonic stem cells were performed. Western blot analysis of wild-type and PDK1\u2212\/\u2212 ES cells with a PKC\u03b4 Thr505 phosphospecific antisera confirm that PKC\u03b4 Thr505 phosphorylation is detectable, albeit slightly reduced, in PDK1-null versus wild-type ES cells (Fig. 2A\u2013C). To probe PKD activity, cells were left unstimulated or activated by exposure to serum or phorbol ester as indicated and PKD catalytic activity monitored using the pSer916 antisera. In quiescent, wild-type ES cells, PKD was not autophosphorylated on Ser916 but both serum and PdBu stimulation strongly and rapidly induced PKD activity as measured by increased Ser916 autophosphorylation (Fig. 2A and B). Serum or PdBu stimulation also induced PKD activity in the PDK1\u2212\/\u2212 ES cells (Fig. 2A and B) whereas p90RSK1 T-loop phosphorylation (Ser227) was basally detectable in wild-type ES cells but not in the PDK1\u2212\/\u2212 ES cells (Fig. 3). S6K1 signalling was also abrogated, as expected, in the PDK1\u2212\/\u2212 ES cells, as shown by the lack of phosphorylation of its substrate, the S6 ribosomal protein subunit (Fig. 2B).\nThe ability of serum and phorbol esters to activate PKD in PDK1-null ES cells is an indication that there is residual, catalytically competent PKC in these cells. PKC-mediated activation of PKD can be prevented by the PKC inhibitor GF109203X [17]. Therefore, the effects of GF109203X, on PKD activation in wild-type or PDK1\u2212\/\u2212 ES cells was examined. The data (Fig. 3) show that PKD was phosphorylated on Ser916 in activated PDK1+\/+ and PDK1\u2212\/\u2212 cells but that PKD autophosphorylation on Ser916 was prevented by prior treatment of both cell types with GF109203X.\n3\nConcluding remarks\nLoss of PDK1 in ES cells causes a reduction in levels of PKC\u03b1, \u03b2I, \u03b3, \u03b4, and \u03b5 [6,12] but it was not resolved whether the residual PKC in these cells is functional. As discussed above, there is a large body of evidence that PKD activation is mediated by novel PKCs such as PKC\u03b4 and \u03b5 and it has previously been proposed that PDK1 is required for activation of PKD [21], reflecting the requirement of PDK1 for phosphorylation of the key priming T-loop phosphorylation sites in PKCs. However, the present study shows that the hypothesis that PKD activation is dependent on PDK1 is incorrect. The reason this model fails is that there is not a complete loss of PKC activity in the absence of PDK1. We show that in PDK1-null pre-T cells PKC\u03b4 is still phosphorylated on its T-loop site Thr505; hence activation of pre-T cells with phorbol esters can still induce phosphorylation of PKD on its PKC substrate sequences Ser744\/S748, inducing PKD activation and subsequent autophosphorylation on Ser916. In addition, the present data show that PKD can also be activated by phorbol esters or serum stimulation of PDK1-null ES cells in a PKC-regulated manner. Thus, the present results showing activation of PKD in PDK1\u2212\/\u2212 cells is evidence that at least one PKC-mediated signalling pathway is intact in the absence of PDK1.\nWhile loss of PDK1 does not prevent the activation of PKD, the issue of whether the efficiency of PKD activation is affected in PDK1-null cells requires further experimentation. While robust activation of PKD signalling is observed in PDK1-null ES cells and pre-T cells in response to phorbol esters or CD3 crosslinking we cannot exclude the possibility that PKD activation may be compromised in PDK1-null mature peripheral T cells during physiological peptide:MHC stimulation, however these experiments await the development of genetic models to test this.\nIn summary, PDK1 is essential for pre-T cell development and its loss is associated with loss of PKB\/S6K1-mediated signal transduction [4,25]. The present results now show loss of p90RSK and PRK1 T-loop phosphorylation in PDK1\u2212\/\u2212 pre-T cells but reveal that the PKC\u03b4 T-loop site Thr505 is still phosphorylated in PDK1-null pre-T cells. It has been proposed that the Thr505 T-loop site in PKC\u03b4 (as well as the corresponding T-loop site in PKC\u03b8, [26]) can be autophosphorylated, which may explain why PKC\u03b4 Thr505 is still phosphorylated in the PDK1-null cells. Alternatively the present results could argue for the existence of another PKC\u03b4 priming kinase in developing thymocytes. Whatever the molecular basis for the continued phosphorylation of PKC\u03b4 following the loss of PDK1, the present results show that there is still residual functional PKC activity once PDK1 is lost. Hence, the importance of PDK1 for T cell development must reflect the requirement for PDK1 mediated phosphorylation of other members of the AGC serine kinase family, such as Akt\/PKB, S6K1 or p90RSK, since phosphorylation and activity of these kinases is strictly dependent on PDK1 expression.\n4\nMaterials and methods\n4.1\nCell preparation and stimulation\nPDK1+\/+ and PDK1\u2212\/\u2212 murine embryonic stem cells [5] were grown on gelatinised tissue culture plates in Knockout DMEM supplemented with 10% knockout serum replacement, 15% foetal bovine serum (Hyclone), 0.1\u00a0mM non-essential amino acids, antibiotics (100\u00a0U\/ml penicillin G, 100\u00a0\u03bcg\/ml streptomycin and 1\u00a0\u03bcg\/ml ciprofloxacin [Bayer Pharmaceuticals]), 2\u00a0mM l-glutamine, 1\u00a0mM sodium pyruvate, 0.1\u00a0mM \u03b2-mercaptoethanol and 25\u00a0ng\/ml murine leukaemia inhibitory factor (LIF).\n4.2\nMice\nAll mice used were between 5 and 7 weeks of age. Mice were bred and maintained in specific pathogen-free conditions and animal experimentation was approved by Home Office project license PPL60\/3116. T-PDK1\u2212\/\u2212 mice were generated by crossing mice with PDK1 floxed alleles (PDK1fl\u0394neo\/fl\u0394neo) with mice expressing Cre recombinase under the control of the p56lck promoter to ablate expression of PDK1 in T cells [2,4]. Control mice used for analyses of T-PDK1\u2212\/\u2212 mice were age-matched phenotypically normal littermates.\n4.3\nPurification of thymocyte subpopulations\nAntibodies conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC) and biotin were obtained from PharMingen. Cells were stained for surface expression of the following markers: CD4, CD8, CD25, CD44, CD3\u03b5, \u03b3\u03b4, B220, and Thy1. The DN4 subpopulation were defined by their cell surface marker expression and subsequently sorted using a Vantage cell sorter (Becton Dickinson) or AutoMacs magnetic cell sorter (Miltenyi Biotec).\n4.4\nCell lysis and Western blot analysis\nCells were lysed for 10\u00a0min at 4\u00a0\u00b0C (20\u00a0\u00d7\u00a0106\u00a0cells\/ml) using buffer: 100\u00a0mM HEPES, pH 7.4, 150\u00a0mM NaCl, 1% NP40, 20\u00a0mM sodium fluoride, 20\u00a0mM iodoacetamide, 2\u00a0\u03bcM EDTA, 1\u00a0mM sodium orthovanadate, 2\u00a0\u03bcg\/ml pepstatin A, 2\u00a0\u03bcg\/ml leupeptin, 2\u00a0\u03bcg\/ml chymostatin, 2\u00a0\u03bcg\/ml antipain, 40\u00a0mM \u03b2-glycerophosphate and 1\u00a0mM phenylmethylsulfonylflouride. Soluble proteins were concentrated by precipitation with 1.5\u00a0vol. of acetone and separated by 8% sodium dodecyl sulfate\u2013polyacrylamide gel electrophoresis (SDS\u2013PAGE), transferred to polyvinylidene difluoride membranes and detected by Western blot analysis with the indicated antibodies (Cell Signalling Technology).","keyphrases":["pdk1","pkc","pkd","es cell, embryonic stem cell","p90rsk, p90 ribosomal s6 kinase","pdk1, 3\u2032-phosphoinositide-dependent protein kinase-1","pkc, protein kinase c","pkd, protein kinase d","s6k1, 70-kilodalton ribosomal s6 kinase 1"],"prmu":["P","P","P","R","R","M","R","R","R"]}
{"id":"Environ_Health_Perspect-116-2-2235228","title":"Testicular Dysgenesis Syndrome and the Estrogen Hypothesis: A Quantitative Meta-Analysis\n","text":"Background Male reproductive tract abnormalities such as hypospadias and cryptorchidism, and testicular cancer have been proposed to comprise a common syndrome together with impaired spermatogenesis with a common etiology resulting from the disruption of gonadal development during fetal life, the testicular dysgenesis syndrome (TDS). The hypothesis that in utero exposure to estrogenic agents could induce these disorders was first proposed in 1993. The only quantitative summary estimate of the association between prenatal exposure to estrogenic agents and testicular cancer was published over 10 years ago, and other systematic reviews of the association between estrogenic compounds, other than the potent pharmaceutical estrogen diethylstilbestrol (DES), and TDS end points have remained inconclusive.\nImpaired spermatogenesis, male reproductive tract abnormalities such as hypospadias and cryptorchidism, and testicular cancer have been proposed to comprise a common underlying syndrome with a common aetiology resulting from the disruption of embryonic programming and gonadal development during fetal life, termed the testicular dysgenesis syndrome (TDS) (Sharpe and Skakkebaek 2003; Skakkebaek et al. 2001). A hormonal etiology most likely underlies this syndrome, although it is believed to have more than one cause, possibly including other than endocrine disruption. Some common causes of endocrine disruption include infection, diet and body weight, lifestyle, genetics, and environmental exposure, but endocrine-disrupting chemicals (EDCs), particularly those with estrogen-like properties, have received the most scientific attention.\nThe synthetic estrogenic drug diethylstilbestrol (DES) was prescribed to more than 5 million pregnant women from the late 1940s to the early 1970s to prevent abortions and pregnancy-related complications (Palmlund et al. 1993). Evidence later showed that maternal ingestion of DES during early pregnancy increased the risk of vaginal clear cell adenocarcinoma in female offspring (Herbst et al. 1971) and resulted in an increased incidence of malformations of the testes, the development of epididymal cysts, and impaired sperm quality in male offspring (Bibbo et al. 1977). During pregnancy, maternal estrogen levels are significantly elevated. However, more than 90% of maternal endogenous estrogens are effectively sequestered via binding to sex hormone binding globulin (SHBG), and thus the fetus is relatively protected (Joffe 2001; Vidaeff and Sever 2005). On the other hand, DES and ethinylestradiol do not bind well to SHBG, having a higher biopotency if ingested (Sharpe and Skakkebaek 2003; Vidaeff and Sever 2005). Additionally, transgenerational exposure is also possible when lipophilic xenoestrogens are mobilized during pregnancy and lactation (Colborn et al. 1993).\nPrevious systematic reviews of studies in which pregnant women were exposed to estrogens other than DES have failed to find evidence of an increased risk of urogenital abnormalities in the male offspring (Raman-Wilms et al. 1995; Storgaard et al. 2006; Toppari et al. 1996; Vidaeff and Sever 2005), and have raised the possibility that nonestrogenic or atypical estrogenic effects of DES exposure in utero induce male reproductive abnormalities. However, none of the effects of DES exposure on either male or female offspring of exposed wild-type pregnant mice were induced when administered to ERKO (ER-\u03b1 knockout) mice (Couse et al. 2001), strongly suggesting an ER-\u03b1\u2013mediated mechanism. There is, however, a body of experimental data that is consistent with an effect of antiandrogenic industrial chemicals on male sexual differentiation (Gray et al. 1999, 2000). Moreover, mechanisms other than endocrine disruption may be involved in testicular toxicity; for example, the nematocide dibromochloropropane, an alkylating agent, is one of the most potent known testicular toxins in adults (Joffe 2001). In this review we focus on the estrogen hypothesis of TDS.\nAlthough several systematic reviews of the literature on the association between estrogenic agents and the disorders thought to belong to the TDS have been published, they are predominantly qualitative and the only quantitative summary estimate of the association between prenatal exposure to estrogenic agents and testicular cancer was published over 10 years ago (Toppari et al. 1996). The primary objective of a quantitative meta-analysis is to combine the results of previous studies examining a specific research question to arrive at a summary conclusion about a body of research. It has been found particularly useful when individual studies are too small to yield a valid conclusion, but it cannot, however, correct for bias and confounding. When applied to observational studies, subset analysis can be a useful tool to explore the reasons for discrepancies among the results of different studies.\nThe objectives of this research were therefore to carry out a quantitative meta-analysis of the association between three of the end points related to TDS and prenatal exposure to estrogenic agents that would account for both the size and quality of the studies included and yield updated summary estimates in light of the body of research carried out since the formulation of the estrogen hypothesis. Inclusion in this analysis was based on mechanistic criteria, and the plausibility of an ER-\u03b1\u2013mediated mode of action was specifically explored. Moreover, subset analysis has been applied to categories of compounds with estrogenic potencies differing by several orders of magnitude in an attempt to detect the existence of any potency\u2013response trend. Most of the studies of sperm quantity or quality have been concerned with time trends rather than etiology, and this end point was not considered further here.\nMaterial and Methods\nIdentification and selection of literature\nA computerized search was conducted using the databases PubMed (National Center for Biotechnology Information 2007) and Web of Science (ISI Web of Knowledge 2007) for the period 1970 to April 2007. The general search keywords were \u201cestrogen,\u201d \u201crisk,\u201d \u201cdose,\u201d and either \u201chypospadias,\u201d \u201ccryptorchidism,\u201d or \u201ctesticular cancer.\u201d A preliminary identification was performed by screening the titles and, if relevant, the abstracts of retrieved literature. The next stage was to check the citations and references of selected studies. This was an iterative process, repeated until no new study could be identified. A set of both inclusion and exclusion criteria was defined, and all relevant literature was then checked for eligibility. The inclusion criteria considered were a) study design, namely, either a case\u2013control, cohort, or clinical trial; b) written in English; c) exposure to one or a mixture of known estrogenic compounds; and d) sufficient data reported to be used in meta-analysis.\nThe following exclusion criteria were used:\nExposure to a group of compounds (suspected endocrine disruptors) for which mode of action was unspecified, for example, pesticides.\nStudies of exposure to phytoestrogens. Some phytoestrogens have been found to have a greater binding affinity for ER-\u03b2 than for ER-\u03b1 and can result in agonistic or antagonistic effects (Mueller et al. 2004).\nStudies of maternal endogenous hormones.\nStudies of the same cohort as this would bias the results towards the particular studies.\nIncomplete data.\nData extraction and quality rating\nIn addition to the number of exposed and nonexposed cases and controls, and risk ratios (RRs) with their confidence intervals (CIs), information regarding the study design, estrogenic agent, geographic location of the study, and year of publication were extracted from the selected literature to allow subset analysis to be carried out. When more than one RR was reported, the following priorities were set for choice:\nAdjusted RRs were used, except when the study provided only unadjusted estimates.\nWhen multiple estimates were given, the RR estimator on which the authors had relied for their assessment of causal association was used.\nOverall RRs were chosen instead of those derived from further stratifications. If an overall estimate was not provided, the RRs of the maximum duration of exposure or the maximum exposure concentration were chosen.\nSeveral aspects of the quality of each study were also recorded according to a rating scheme adapted from those previously described (Altman 1991; Rushton 2000). Every criterion was assessed on a scale of 0 to 2, 0 suggesting that it was not present, 1 when it was unclear, and 2 when that criterion was satisfied. A maximum score of 50 and 52 could be assigned for retrospective (case\u2013control) and prospective (cohort and clinical trials) studies, respectively. This enabled a quality sensitivity analysis to be performed to check the influence of studies with low quality on the pooled estimate.\nData analysis\nGraphical representation\nThe RRs and CIs were plotted against the year of publication to determine whether any positive or negative trends in reporting RRs had occurred over time. Similarly, quality scores were plotted against the year of publication to investigate whether the quality of studies improved over time. To assess publication bias, a funnel plot (SE vs. RR) was produced based on the assumption that smaller studies are less precise in their RRs and thus have less weight and larger SE and should scatter more widely at the lower end of the graph, whereas larger studies will tend to be closer together (Sterne et al. 2001). Forest plots present the RRs against the reference of the study and help check homogeneity visually.\nStatistical pooling\nPooled estimates and 95% CIs were calculated using both a fixed-effects model (Mantel\u2013Haenszel method) and a random-effects model (DerSimonian\u2013Laird method), allowing evaluation of the dependence of the conclusions of the analysis on the model assumptions. A summary estimate is considered statistically significant at the 0.05 level if its CI does not include unity.\nThe Mantel\u2013Haenszel pooled effect estimate was used in a chi-square statistical test of homogeneity to assess the between-study variance. The magnitude of the test statistics depends on the weight of each study. When the number of studies is low or the studies themselves are small, the test statistic Q tends to be small. Tests of heterogeneity in meta-analyses are generally low in their power to reject the null hypothesis of homogeneity. For this reason, the chi-square statistical test of homogeneity was carried out at both 0.05 and 0.1 significance levels. Additionally, pooled estimates calculated using fixed effect and random effect models differ only if there is lack of homogeneity between studies. The estimates obtained by both methods were therefore compared to better assess potential heterogeneity between studies, in which case a single summary estimate of effect may be considered inappropriate.\nSubset and sensitivity analyses\nTo investigate potential sources of heterogeneity between studies, we performed subset analyses for the study design, estrogenic agent, and geographic location.\nSome studies exploring the influence of hormonal treatment during pregnancy did not specify the type of hormone. From what is known of the hormonal treatment of common conditions occurring during pregnancy, it was deemed reasonable to assume that they would have been likely to include estrogens, and these studies were included in the analysis. The validity of this assumption was tested by applying stricter criteria and calculating a summary estimate of effect excluding any study in which the hormone used had not been specified. Further sensitivity analysis was performed by excluding low-quality studies and extremes (exclusion of the studies with the largest and smallest RR estimators and exclusion of the studies with the largest and smallest weights) to verify that either the quality of the studies or one particular study did not have an excessive influence on the pooled estimate.\nResults\nA total of 50 studies were identified for the association between in utero exposure to estrogenic agents and hypospadias and\/or cryptorchidism, including 16 that had not been included in previous systematic reviews. Sixteen studies, of which 8 were new studies, were included in the calculation of a summary estimate of effect for either or both end points (Table 1). Studies predating the formulation of the TDS hypothesis often were designed to explore the association of in utero exposure to a range of pharmaceuticals with birth malformations. Other than 2 recent studies for which pesticide exposure was determined by chemical analysis of specific compounds, assessment of exposure to pesticides is generally derived from the occupation of the mother and specific agents are not identified.\nOf the 12 studies identified for the association with testicular cancer, only 3 were excluded from the calculation of a summary estimate of effect (Table 2).\nHypospadias\nThe data from studies included in the meta-analysis for hypospadias are summarized in Table 3. Three extreme values, two greater than and one lower than unity, can be identified visually from the forest plot of the RRs and their CIs (Figure 1). These extremes correspond to studies with larger SEs, and the shape given to the funnel plot (Figure 2) by those smaller positive studies would be consistent with publication bias. These two extreme positive risk ratios were, however, reported after what is commonly referred to as \u201cthird-generation exposure\u201d to DES, when the mother herself had been exposed to DES prenatally. It was recognized that the inclusion of such studies in the meta-analysis could have introduced heterogeneity, and the influence of this choice was investigated in the subset analysis. Plots of the quality score and RRs versus year of publication did not suggest any significant trends in quality of the studies or estimates of effect over time (not shown).\nThe pooled estimates of effect by both the Mantel\u2013Haenszel and DerSimonian\u2013Laird methods are very close to unity, and no relationship between in utero exposure to estrogenic agents and hypospadias could be detected (Table 4). None of the chi-square tests allowed the rejection of the null hypothesis of homogeneity between the studies at the 0.05 or 0.1 level of statistical significance. The subsets of studies in which exposure to DES and pharmaceutical estrogens were investigated yielded statistically significant risk ratios with both models, although the modest discrepancy between the fixed-effects and random-effects estimates suggests heterogeneity. Summary estimates for the latter subset were no longer significant when studies that included DES exposure were excluded. Although these results were based on four studies that all addressed in utero exposure to oral contraceptives, some heterogeneity between studies remained. Excluding the studies of third-generation exposure to DES, values for the summary estimate of effect were found to be 1.33 (95% CI, 0.63\u20132.83) by the Mantel\u2013Haenszel method and 1.31 (95% CI, 0.52\u20133.26) by the DerSimonian\u2013Laird method, a very modest and nonsignificant increase in risk. Excluding third-generation exposure from the DES subset yielded estimates of 2.02 (95% CI, 1.12\u20133.65) by the Mantel\u2013Haenszel method and 2.00 (95% CI, 0.97\u20134.15) by the DerSimonian\u2013Laird method, on the basis of two studies investigating exposure to any estrogenic drug during the first trimester of pregnancy. The difference between the results obtained by the two models for studies of third-generation exposure to DES was reduced only slightly by excluding the study by Klip et al. (2002); the Mantel\u2013Haenszel method yielded an estimate of 2.46 (95% CI, 0.91\u20136.67) and the DerSimonian\u2013Laird method of 2.18 (95% CI, 0.64\u20137.46). The latter study\u2019s cohort had been recruited in a fertility clinic, and whether results obtained with subfertile women are generalizable to all women exposed to DES in utero has been questioned (Hernandez-Diaz 2002).\nAlthough the equality of the results obtained by both methods for the environmental estrogens subset suggests those results are robust, the influence of the weight of the study by Vrijheid et al. (2003) cannot be underestimated, as shown by the sensitivity analysis. Exclusion of this study from the analysis yielded a statistically significant Mantel\u2013Haenszel estimate but a lower and not statistically significant DerSimonian\u2013Laird estimate, revealing heterogeneity. A statistically significant estimate was obtained for prospective studies by the Mantel\u2013Haenszel method, but the wide difference with the estimate using the random effect model was suggestive of heterogeneity. Geographic subsets point to a higher risk in Latin America, although the pooled estimates for this location were based on only two studies and did not reach statistical significance.\nIn addition to the results of the sensitivity analysis presented in Table 4, a pooled estimate of effect was calculated when a stricter inclusion criterion was applied, namely, excluding results from the study by Monteleone-Neto et al. (1981). This had little influence on the overall result, generating summary estimates of 0.97 (95% CI, 0.83\u20131.13) for the fixed effect model or 0.93 (95% CI, 0.80\u20131.09) for the random effect model.\nCryptorchidism\nData for the six studies included in the meta-analysis for cryptorchidism can be found in Table 5. The results of only two studies significantly differ from unity, as illustrated by the forest plot (Figure 3). The small number of eligible studies renders analysis of the funnel plot and potential for publication bias difficult (Figure 4). The SEs do, however, illustrate well that the studies were all relatively small. No time trends for the estimate of effect or the quality of studies could be detected (not shown).\nAs presented in Table 6, the pooled estimates of effect by both the Mantel\u2013Haenszel and DerSimonian\u2013Laird methods are marginally superior to unity, and their relative divergence implies there may be sources of heterogeneity. Chi-square tests did not, however, detect that any of the subsets were significantly heterogeneous. Excluding studies in which DES exposure was examined, either exclusively or along with hormonal therapeutics, yielded summary estimates consistent with no relationship. Statistical pooling of the studies including DES exposure generated a statistically significant estimate by the Mantel\u2013Haenszel method, suggesting a doubling of the risk of cryptorchidism after in utero exposure to DES. The same estimate by the DerSimonian\u2013Laird method did not, however, reach statistical significance and the difference relative to the fixed effect model is indicative of heterogeneity. The heterogeneity introduced by the DES subset of studies can again be observed by comparing the results obtained for all pharmaceutical estrogens with those obtained by pooling the two studies of accidental use of oral contraceptives during pregnancy. Study design also appeared to be a source of heterogeneity. If case\u2013control studies are prone to recall bias, this subset also included the study with the highest estimate, itself a source of heterogeneity, as shown by the sensitivity analysis. Excluding the study by Depue (1988) reduced the difference between estimates by both models, the Mantel\u2013Haenszel estimate then calculated as 1.29 (95% CI, 0.87\u20131.91) and that by the DerSimonian\u2013Laird method as 1.23 (95% CI, 0.81\u20131.86). This was also observed for the American subset of studies. When the Depue (1988) study is omitted, the Mantel\u2013Haenszel method yielded a no longer statistically significant estimate of 1.34 (95% CI, 0.84\u20132.14) and the DerSimonian\u2013Laird method an estimate of 1.27 (95% CI, 0.72\u20132.23).\nApplying a stricter exclusion criterion to studies examining hormonal treatment did not affect which studies were included in the meta-analysis of cryptorchidism. The study with the highest weight appears to lower the overall estimates, whereas increasing quality seems to reduce heterogeneity and lower the estimate of effect toward unity. These variations did not, however, influence the overall conclusion that aside from the DES studies subset, summary estimates did not detect any association between in utero exposure to estrogenic substances and cryptorchidism.\nTesticular cancer\nNine studies were included in the meta-analysis of testicular cancer and the data used are summarized in Table 7. Of these, 4 had not been included in the summary estimate previously calculated by Toppari et al. (1996). The lack of homogeneity between studies is evident from the forest plot (Figure 5). Further, the funnel plot (Figure 6) also illustrates the relatively small size of the included studies. Although a positive trend over time was found for the quality of the included studies (Figure 7), no significant time trend could be detected for the effect size (not shown).\nBoth the fixed and random effect models yield a statistically significant estimate; however, the discrepancy between the two results is suggestive of heterogeneity despite the result from the chi-square test (Table 8). Conversely, the subset analysis was limited by the similarity of the question addressed by the studies included. Eight of the nine studies were interested in hormonal exposure and were conducted in the United States. Despite this, statistically significant heterogeneity between the studies was detected at the 0.1 level. Pooling the two studies examining DES exposure specifically produced a raised but statistically nonsignificant result. Despite the unexplained heterogeneity, all estimates that were calculated point to a doubling of the risk of developing testicular cancer after exposure to estrogenic agents in utero. The work on chlorinated biphenyls (PCBs) by Hardell et al. (2004) was the only study examining environmental estrogens. Its size was relatively small, and it did not detect such an effect.\nApplying a stricter exclusion criterion to studies examining hormonal treatment excluded four studies from the meta-analysis; namely, Brown et al. (1986), Gershman and Stolley (1988), Henderson et al. (1979), and Weir et al. (2000). This resulted in a slightly lower Mantel\u2013Haenszel estimate of 1.98 (95% CI, 1.23\u20133.18) and if the DerSimonian\u2013Laird estimate remained equal to 1.59, because of the wider confidence interval (95% CI, 0.93\u20132.69), statistical significance was no longer achieved. The sensitivity analysis is consistent with some heterogeneity between the studies, the estimates obtained being relatively sensitive to the exclusion of particular studies varying above and below a risk estimate of 2. The quality of the studies seemed to explain at least some of this heterogeneity.\nDiscussion\nWhile it is clear that hypospadias, cryptorchidism, and testicular cancer are all positively associated with prenatal exposure to DES, this meta-analysis was unable to produce evidence that such effects were associated with environmental estrogens or even accidental use of oral contraceptives during pregnancy. This is consistent with the results obtained in earlier meta-analyses (Raman-Wilms et al. 1995; Toppari et al. 1996).\nThe main limitations of meta-analysis are a) the susceptibility of its summary results to publication bias, b) the influence of the quality of studies, c) the possibility of including multiple results from the same study, and finally, d) heterogeneity between studies that could lead to invalid conclusions. The methodology employed in this present review attempts to address these issues. Additionally, the importance of carrying out and reporting a sensitivity analysis was illustrated by the case of hypospadias where the weight attributed to one particularly large study had a nonnegligible influence on the results. In this particular case, the study by Vrijheid et al. (2003) inferred exposure to phthalates from registry data about occupation, and although such an approach can allow the analysis of a great number of cases, assessment of exposure is much more likely to be prone to confounding. The number of studies included in meta-analyses lies typically between 5 and 15, and the results presented here also fall within this range. The size of the homogeneity test statistic depends on both the number and size of individual studies. The funnel plots offer a good visual representation of the precision and size of individual studies, and it is clear that most studies published on the association between estrogenic agents and the probable end points of a TDS were found to be relatively small. The chi-square tests had, therefore, a relatively low power to detect heterogeneity. However, in the absence of statistical heterogeneity, the results of the fixed effect and random effect models should be virtually identical, and the comparison of results obtained by applying both the Mantel\u2013Haenszel and DerSimonian\u2013Laird models enabled the exploration of sources of heterogeneity despite this low statistical power.\nIf the quality of the studies was found to explain some of the heterogeneity observed, particularly in the case of testicular cancer, the remaining heterogeneity could not be explained solely by the fact that environmental, and therefore generally much weaker, estrogens were included in the analysis. The systematic review of published literature yielded relatively few studies examining the association of male urogenital abnormalities or testicular cancer with environmental estrogens specifically; a number of studies concerned with an association with broad categories of putative endocrine disruptor, most often pesticides, were excluded from the meta-analyses. This illustrates the difficulties associated with assessment of exposure, pesticide exposure often being inferred from parental occupation rather than direct measurement. Furthermore, there is increasing evidence that, in accordance with pharmacokinetic theory, the effects of xenobiotics acting via the same mechanism can be predicted fairly accurately by concentration addition (Zhu et al. 2006). Accurately accounting for combined exposure or adjusting for the confounding introduced by environmental exposures will probably require the development of mechanism-specific biomarkers of exposure.\nWhen DES is excluded, there is no conclusive evidence of an effect of pharmaceutical estrogens. Exposure to such estrogens is related mainly to the accidental use of oral contraceptives during pregnancy or hormonal pregnancy tests. Such estrogenic pharmaceuticals often are given in combination with progestagens, and it is legitimate to question whether unopposed estrogens would have the same effects as opposed estrogens. This also highlights another difficulty associated with exposure assessment, that of critically sensitive periods of development and the ascertainment of whether exposure took place during a \u201cwindow\u201d of susceptibility to hormone disruption. Nonetheless, studies in which maternal levels of hormones were measured in the first and third trimester of pregnancy do not support an association with elevated estrogen levels but rather indicate that a lower estrogen\/androgen ratio and\/or higher levels of \u03b1-fetoproteins may be beneficial (McGlynn et al. 2005; Zhang et al. 2005). If in animals both estrogenic and antiandrogenic compounds have been associated with end points consistent with those of human TDS (Fisher 2004; Veeramachaneni 2000), epidemiologic evidence remains elusive. Alternatively, the doubling of the risk estimates of all three effects associated with DES exposure would be consistent with a shared etiology and the TDS hypothesis. It does not constitute conclusive evidence of an estrogenic mode of action, however, as common etiologic factors could be related to the underlying condition for which DES was prescribed. Furthermore hypospadias, cryptorchidism, and testicular cancer have all been found to be associated with low birth weight, suggesting a potential association with an underlying placental defect.\nThe understanding of the importance of endogenous estrogens in normal adult testicular function is becoming clearer. Their roles during fetal life, however, remain relatively unclear, but those mediated by the ER-\u03b1or ER-\u03b2 have been shown to differ (Habert et al. 2006). Interestingly, DES has been found to have similar affinity for both receptors, whereas estradiol has only a slightly stronger affinity for ER-\u03b1compared with ER-\u03b2 (Mueller et al. 2004). ER-\u03b1 has been detected in undifferentiated gonads as early as 10 days postconception in the mouse and found to be localized in the Leydig cells of fetal testis in rodents (Habert et al. 2006). Studies of the expression of ER-\u03b1 and ER-\u03b2 in human and nonhuman primates have so far yielded inconsistent results. Gaskell et al. (2003) reported that ER-\u03b1 could not be detected in human fetal testes between weeks 12\u201319 of gestation, whereas Shapiro et al. (2005) found that ER-\u03b1 was apparent by week 12, its levels peaked at 16 weeks before diminishing, and it was localized in Leydig cells. Current research focus has shifted to the role played by testosterone, anti-M\u00fcllerian hormone and insulin-like factor 3 produced by the fetal testes during masculinization. In the male rat, exposure to high levels of estrogens has been shown not only to suppress testosterone production but also to downregulate the expression of the androgen receptor protein in reproductive target tissues including the testes, Wolffian duct, and prostate (Sharpe 2006). Further research in this area may help shed light on possible mechanisms of injury or relevance of the rodent model.\nThe subset analyses did not generate many clues to explain the heterogeneity of the collected data. This is, however, consistent with the wide geographic variability in the incidence of the conditions of interest (Boisen et al. 2004; Richiardi et al. 2004). Interactions between genetic susceptibility and the environment have been the focus of research in this area (Martin et al. 2007), and advances in genomics have allowed the identification of polymorphisms associated with hypospadias, cryptorchidism, and testicular cancer (Beleza-Meireles et al. 2006; Kurahashi et al. 2005; Starr et al. 2005; Yoshida et al. 2005). Such discoveries may, however, give rise to as many questions as they offer to answer. This is well illustrated by the recent identification of the association of a variant of the gene for the ER-\u03b1 with hypospadias and cryptorchidism in Japanese cohorts (Watanabe et al. 2007; Yoshida et al. 2005) that has now been found to be associated with a decreased incidence of hypospadias in a European cohort (Galan et al. 2007).\nConclusion\nThe modest increase in risk for all three end points associated with DES exposure is consistent with a shared etiology and the TDS hypothesis, whereas the results of the subset analyses suggest the existence of yet unidentified sources of heterogeneity between studies or within the study populations. Although 10 years of further research on the potential effects of endocrine disruptors on male reproductive health have provided some clues regarding the etiology and mechanism of conditions such as hypospadias, cryptorchidism, and testicular cancer, there is still no conclusive evidence of the role played by environmental estrogens.","keyphrases":["testicular dysgenesis","estrogen","meta-analysis","hypospadias","cryptorchidism","testicular cancer","diethylstilbestrol","endocrine disruption","environment","oral contraceptives"],"prmu":["P","P","P","P","P","P","P","P","P","P"]}
{"id":"Osteoporos_Int-4-1-2277453","title":"Effects of risedronate on fracture risk in postmenopausal women with osteopenia\n","text":"Summary This posthoc analysis of four trials studied the efficacy of risedronate to reduce fragility fractures in postmenopausal women with osteopenia (i.e., T-scores between \u22121 and \u22122.5). Risedronate reduced the fracture risk by 73% ( = 0.023) in this population of women with low femoral neck bone mass and no prevalent vertebral fractures.\nIntroduction\nLow bone mass, defined by bone mineral density (BMD) values in the osteopenic range, can progress to osteoporosis if left untreated. Additionally, it has recently become apparent that osteopenia is a significant risk factor for fragility fractures in older women. Studies using peripheral and central dual x-ray absorptiometry (DXA) to determine BMD have demonstrated that approximately half of all fragility fractures occur in women with osteopenic BMD [1\u20135]. As the number of women with low bone mass is projected to increase to 30.4 million in 2020, this condition presents a significant medical problem and socioeconomic burden [6]. Prevalence projections for 2020 are significantly higher than current rates (21.8 million women in 2002) due to expected population growth and aging [7].\nBased on BMD testing, the World Health Organization (WHO) clinically defines women with osteopenia as those whose BMD T-score is between 1 and 2.5 standard deviations (SD) below the mean of peak bone mass in healthy, young normal women (i.e., \u22121 to \u22122.5 SD); women with osteoporosis include all those at or below \u22122.5 SD [8\u201310]. However, these definitions provide a diagnostic categorization of low bone mass rather than an intervention threshold that could be used to determine the need for the initiation of treatment to reduce fracture risk. Physicians are often faced with making treatment decisions for postmenopausal patients with T-scores in the osteopenic range, increasing the need for evidence-based guidance to define populations at high risk for fracture [11].\nThe National Osteoporosis Foundation (NOF) currently recommends an algorithm for initiation of therapy to reduce the risk of fracture [12]. According to this algorithm, treatment should be initiated in osteopenic women with T-scores <\u22122.0 by central DXA with no risk factors and in women with T-scores <\u22121.5 by central DXA with one or more risk factors [9, 10, 12, 13].\nDespite the existence of NOF guidelines for the treatment of postmenopausal women with low bone mass, limited clinical data exist as a foundation for supporting therapeutic intervention in this group. Although it is possible to identify osteopenic women at higher or lower short-term risk for fracture [11], there are limited data regarding the effectiveness of osteoporosis treatments to lower fracture risk in these women. This is in part due to the inherent difficulty in collecting fracture data from osteoporosis trials, which usually exclude women with higher T-scores who may fall into the osteopenic range. The assembly of large data sets is, therefore, difficult and the feasibility of prospective osteopenic fracture trials is limited due the lower incidence of fracture in this population compared with patients with osteoporosis [14]. The use of post hoc analysis is one way to overcome some of these obstacles and provide information about effectiveness of treatments in non-osteoporotic populations.\nWe have analyzed data from four Phase III risedronate trials: BMD Multinational (BMD-MN) [15], BMD-North America (NA) [16], Vertebral Efficacy with Risedronate Therapy-Multinational (VERT-MN) [17], and Vertebral Efficacy with Risedronate Therapy-North America (VERT-NA) [18] in which efficacy and safety of risedronate in the prevention and treatment of postmenopausal osteoporosis have been demonstrated. Using data only from osteopenic women included in these trials, this study evaluated the effect of risedronate in reducing the risk of fragility fractures in women with femoral neck T-scores in the osteopenic range and without prevalent vertebral fracture.\nMethods\nStudy design and patients\nThis retrospective analysis included the subsets of postmenopausal women with osteopenia in four risedronate placebo-controlled osteoporosis trials [15\u201318] of 1.5 to 3\u00a0years\u2019 duration.\nPatients included in the current analysis were postmenopausal women who had no radiographic vertebral fractures at baseline and had a femoral neck T-score between \u22121 and \u22122.5 SD as measured by DXA (NHANES III). These selection criteria were similar to those in previously published studies looking at similar populations [19, 20].\nAlthough inclusion criteria for the VERT trials required patients to have prevalent vertebral fractures, re-adjudication conducted by central radiographic assessment after enrollment showed that between 10% and 15% of enrolled patients had no prevalent vertebral fracture and could, therefore, be included in the present study. Our analysis was based on BMD assessment at the femoral neck rather than the lumbar spine, due to its greater reliability and predictive value and wide acceptance internationally for clinical diagnosis [2, 13].\nPatients treated with estrogen and estrogen-related drugs within 3\u00a0months of study entry or for more than 1\u00a0month within 6\u00a0months of study entry were excluded from the original studies. All patients received 1,000\u00a0mg calcium and, if needed, 500 IU vitamin D daily.\nAdverse event information was collected at the study visits made every 3\u00a0months during the trials. The investigator recorded adverse events reported by the patients, as well as adverse events observed on examination of the patient.\nIdentification of fragility fractures\nFragility fractures were defined as a composite of a patient\u2019s incident morphometric vertebral and osteoporosis-related nonvertebral fractures (i.e., six fracture types including clavicle, humerus, wrist, pelvis, hip or leg fractures). This endpoint was chosen to include all radiographically confirmed fractures.\nWith respect to the nonvertebral fractures, the selection of the nonvertebral fracture sites (clavicle, humerus, wrist, pelvis, hip or leg) followed the pre-specified definition of osteoporosis-related nonvertebral fractures of the risedronate trial program, which has been reported in all regulatory and previous scientific reports from this program. All osteoporosis related nonvertebral fractures required radiographic confirmation. The term \u201cleg\u201d fracture as used in the risedronate trials is a summary term, including the treatment emergent adverse event codes for fibula, tibia, subtrochanteric fracture, and leg, but not those for ankle or distal tibia fracture, the terms by which ankle fractures are usually reported.\nStatistical analyses\nThe primary analysis focused on investigating the effect of risedronate to reduce fragility fractures (both vertebral and nonvertebral) in postmenopausal women who had no radiographic vertebral fractures at baseline and were considered osteopenic based on a femoral neck T-score ranging between \u22121 SD and \u22122.5 SD.\nFor consistency with previous risedronate studies, the statistical analysis used time-to-first-event methodology. Cumulative fracture incidence was estimated using the Kaplan\u2013Meier method, and a Cox proportional hazards regression model was used to estimate the relative fracture risk reduction, via the hazard ratio (HR), between the risedronate 5\u00a0mg and placebo groups. The regression model was stratified for each trial to allow for separate underlying hazard functions. In order to determine the acceptability of combining data from all trials, potential heterogeneity assessments were performed using the Cox regression model to assess the trial-by-treatment, age-by-treatment and baseline femoral neck BMD-by-treatment interactions. Fracture efficacy for nonvertebral osteoporosis-related fractures and new vertebral fractures were also investigated. A sensitivity analysis was conducted to investigate whether the exclusion of patients with lumbar spine T-score <\u22122.5 SD would influence the outcome of the primary analyses. As the inclusion criteria for the primary analysis were low bone mass at the femoral neck (BMD T-score between \u22121 and \u22122.5 SD), we felt it was important to test whether the exclusion of patients with an osteoporotic BMD at another site would significantly affect the results.\nAdverse events, serious adverse events, withdrawals, deaths, and upper gastrointestinal adverse events were summarized by treatment group. Differences in the proportions of patients between the treatment groups were tested using the Fisher\u2019s exact test.\nResults\nPatients\nA total of 620 postmenopausal women met the criteria for low bone mass and were included in the current analysis. Patients received either placebo (\u2009=\u2009309) or risedronate 5\u00a0mg (\u2009=\u2009311). Baseline characteristics were similar for each patient group (Table\u00a01). The mean age was 64\u00a0years for both groups and mean baseline femoral neck T-scores were \u22121.84 in the placebo group and \u22121.85 in the risedronate group, respectively.\nTable\u00a01Baseline characteristics of patients with a femoral neck T-Score \u2265 \u22122.5 SD and \u2264 \u22121 SD without prevalent vertebral fractures\u00a0All patientsExcluding lumbar spine T-score <\u22122.5 SDPlacebon n\u2009=\u2009309Risedronate 5\u00a0mg n\u2009=\u2009311Placebon n\u2009=\u2009146Risedronate 5\u00a0mg n\u2009=\u2009147Age (years)a64 (7.5)64 (7.5)63 (8.3)65 (7.0)Femoral neck T-scorea, b\u22121.84 (0.396)\u22121.85 (0.406)\u22121.77 (0.418)\u22121.76 (0.398)Height (cm)a159.8 (6.92)159.8 (6.05)160.6 (6.14)160.4 (6.32)Weight (kg)a65.9 (11.92)66.8 (11.19)69.8 (12.91)69.6 (12.27)BMI (kg\/m2)a25.9 (4.54)26.2 (4.36)27.2 (5.1)27.1 (4.78)Patients with prevalent OP-related nonvertebral fractures56 (18%)59 (19%)34 (23%)31 (21%)a Mean (standard deviation)b Based on National Health and Nutrition Examination Survey III reference database\nPotential heterogeneity assessments revealed that there were no significant interactions for trial-by-treatment (\u2009=\u20090.495), age-by-treatment (\u2009=\u20090.981) or baseline femoral neck BMD-by-treatment (\u2009=\u20090.188)\nEffect on fractures\nRisedronate treatment was found to significantly reduce the risk of combined morphometric vertebral and nonvertebral fractures by 73% in women with a femoral neck T-score between \u22121 and \u22122.5 and no prevalent vertebral fracture; HR\u2009=\u20090.27, 95%CI\u2009=\u20090.09\u20130.83, \u2009=\u20090.023 (Fig.\u00a01). The cumulative fragility fracture incidence was 6.9% and 2.2% respectively for placebo and risedronate.\nFig.\u00a01Reduction of fragility fracture risk in patients with femoral neck T-score between \u22121 and \u22122.5 SD and no prevalent vertebral fractures\nWhen separated according to nonvertebral and vertebral fractures, the cumulative nonvertebral fracture incidence was 5.4% and 0.4%, respectively, for placebo and risedronate (HR\u2009=\u20090.09, 95%CI\u2009=\u20090.01\u20130.71 (\u2009=\u20090.022), and the cumulative vertebral fracture incidence was 4.2% and 1.8%, respectively, for placebo and risedronate (HR\u2009=\u20090.44, 95%CI\u2009=\u20090.11\u20131.78 (\u2009=\u20090.249).\nSensitivity analyses\nWhen patients with baseline LS spine T-scores below \u22122.5 SD were excluded, results consistent with those from the primary analysis population were observed, although the reduced sample size widened the confidence intervals and led to a loss of statistical significance. For the 293 patients who had both femoral neck and LS spine T-scores between \u22122.5 and \u22121 SD, the hazard ratio for fragility fractures was 0.22 (95%CI\u2009=\u20090.03\u20132.02, \u2009=\u20090.182), a magnitude of effect similar to that in the primary analysis population.\nSafety\nIn the subgroup investigated for this analysis, no statistically significant differences in adverse events between the risedronate treated and the placebo group were recorded during the trial (Table\u00a02). The observation in the subgroup analysis reported here is consistent with the findings in the overall population of the original trials [15\u201318].\nTable\u00a02Summary of adverse events (AEs) for patients with a femoral neck T-score \u2265 \u22122.5 SD and \u2264 \u22121 SD and noprevalent vertebral fractures (BMD and VERT Trials)CategoryNumber of patients (%)-valuePlacebo (\u2009=\u2009309)Risedronate 5 mg (\u2009=\u2009311)AEs290 (93.9%)293 (94.2%)0.87Serious AEs40 (12.9%)56 (18.0%)0.10AEs associated with death 2 (0.6%)0 (0.0%)0.25UGI AEs 77 (24.9%)81 (26.0%)0.78Withdrawn due to AEs 38 (12.3%)26 (8.4%)0.11-values were from Fisher\u2019s exact test.UGI: upper gastrointestinal\nDiscussion\nOsteopenia is common in postmenopausal women and contributes to the increased risk of fracture in this population. There is a need for clinical data examining both fracture risk in women with T-scores in the osteopenic range and the evidence for an effect of treatment on reducing that risk. Data from the National Osteoporosis Risk Assessment (NORA) study reported that more than half of women experiencing self-reported fractures of the hip, vertebrae, rib and wrist\/forearm at one year fell into the osteopenic category based on peripheral BMD T-scores between \u22121 and \u22122.5 [21]. Results from the present study show that risedronate treatment significantly reduced the overall risk of fragility fractures by 73% over 3\u00a0years in patients with a femoral neck T-score between \u22121 and \u22122.5 and no prevalent vertebral fracture. A similar reduction in risk \u2014 that did not reach statistical significance due to low numbers \u2014 was observed when patients with lumbar spine T-scores below \u22122.5 were excluded from the analysis.\nPrevious studies in women with osteoporosis have shown that risedronate reduces the risk of morphometric vertebral fractures by up to 65% in the first year of treatment [17, 18], with significant reductions in clinical vertebral and nonvertebral fractures as early as 6\u00a0months [22, 23]. Moreover, a study in osteoporotic women without prevalent fracture demonstrated that treatment with risedronate reduced the risk of first vertebral fracture by 75% (i.e., a relative risk of 0.25) [24].\nPreviously reported studies have examined the efficacy of alendronate [20, 25] and raloxifene [19] on fracture risk reduction in osteopenic populations. A retrospective re-analysis of data from the Fracture Intervention Trial (FIT) showed a 60% reduction of vertebral fractures [RR 0.40 (0.19\u20130.76 95% CI)] in osteopenic women defined as having a femoral neck BMD T-score between \u22121.6 and \u22122.5 [20]. The overall analysis included patients with or without prevalent vertebral fractures, however, and since prevalent vertebral fractures are an important risk factor for further fractures and a criterion for severe osteoporosis [8, 13], classifying such women as osteopenic is subject to debate. When a subset of this population, those without prevalent fractures, was analyzed separately the results were similar, but did not reach statistical significance [RR 0.46 (0.16\u22121.17 95% CI)]. Results in patients with both hip and spine BMD in the osteopenic range are not reported in this paper. A re-analysis of the Multiple Outcomes of Raloxifene Evaluation (MORE) study reported similar rates of vertebral fracture risk reduction in raloxifene-treated women with osteopenia \u2013 defined as a total hip T-score >\u22122.5 without a prevalent vertebral fracture - compared with those with osteoporosis at 3\u00a0years [19]. The relative risk reduction for vertebral fractures with raloxifene compared with placebo was 0.53 (0.32\u20130.88, 95% CI) in osteopenic women; the relative risk for clinical vertebral fractures in osteopenic women was 0.25 (0.04\u20130.63). Information about reduction of nonvertebral fractures has not been provided in either the alendronate or raloxifene analyses. Conjugated equine estrogens significantly reduced the risk of clinical vertebral, hip and total fractures in postmenopausal women in the Women\u2019s Health Initiative, the vast majority of whom did not have bone density testing but who were not selected based on having diagnosed osteoporosis [26]. Finally, a study of women \u226575\u00a0years of age in the UK reported that oral daily clodronate 800\u00a0mg for 3\u00a0years reduced any clinical fracture by 20%, but not hip fractures, in women unselected for having osteoporosis [27]. This reduction was independent of baseline BMD, but the number needed to treat was greater in the non-osteoporotic subjects.\nThe fracture benefit observed with risedronate in patients with low bone mass in our analysis may result in part from the effect of this compound on the preservation of bone microarchitecture. In previous studies of both early postmenopausal women and osteoporotic patients risedronate was observed to preserve trabecular architecture, including trabecular number, thickness and separation, compared to placebo [28, 29], which is likely to contribute to a maintenance of bone strength [30]. This beneficial effect on trabecular architecture has also been observed with long-term treatment with risedronate, as shown by recent data from a study in postmenopausal osteoporotic women [31]. Bone microarchitecture, assessed by three-dimensional micro-computed tomography, was maintained at similar levels over a five-year treatment period [31]. The sustained preservation of bone microarchitecture equates to a long-term beneficial effect on bone strength and may contribute to the decrease in fracture risk observed with risedronate treatment.\nLimitations of the current study include the selection of initial populations in the VERT and BMD trials based on the presence of risk factors for osteoporosis (including low BMD and prevalent vertebral fractures). Because the original trials were designed to examine the efficacy of osteoporosis treatments in an osteoporotic population, rather than to determine representative incidence of fracture in a general osteopenic population, the data provided in the present analysis may reflect an overestimation of the fracture risk in the general postmenopausal population.\nIt is likely that in the relatively near future fracture risk assessment using a combination of femoral neck BMD, age, and other risk fractures will be utilized to determine absolute fracture risk and also serve as a basis for determining treatment intervention thresholds. In many older postmenopausal women, osteopenia will be an important risk factor for fracture, placing some of them at high absolute risk of a fracture event over a 10\u00a0year interval [32], and treatment with a pharmacologic agent will be indicated to lower risk. The data presented here demonstrate that in addition to the established efficacy in preventing fracture in osteoporotic women with or without prevalent fractures, risedronate significantly reduces the risk of fragility fractures in this population of postmenopausal women with osteopenic BMD values at the femoral neck and no prevalent vertebral fractures.","keyphrases":["risedronate","osteopenia","fragility fracture","vertebral fracture","nonvertebral fracture","bisphosphonates"],"prmu":["P","P","P","P","P","U"]}
{"id":"Cancer_Causes_Control-2-2-1764594","title":"Meeting the mammography screening needs of underserved women: the performance of the National Breast and Cervical Cancer Early Detection Program in 2002\u20132003 (United States)\n","text":"Objective To examine the extent to which the National Breast and Cervical Cancer Early Detection Program (Program) has helped to meet the mammography screening needs of underserved women.\nIntroduction\nBreast cancer is the leading cause of cancer-related death among Hispanic women in the United States and the second-leading cause of cancer-related death among non-Hispanic women, causing more than 41,000 deaths in 2002 [1]. Breast cancer mortality is higher in poorer areas of the country than in wealthier areas [2]. Although timely breast cancer screening with mammography reduces mortality [3], in the United States, screening rates are low among low-income women who lack insurance coverage for mammography [4, 5]. In 1992, among women aged 40\u00a0years and older, 60% of women with health insurance and 22% of women without health insurance had received a mammogram within the previous 2\u00a0years. Among those women with family incomes greater than or equal to 200% of the federal poverty level, 64% had received a mammogram within the previous 2\u00a0years, but only 41% of women from families with incomes less than 200% of the poverty level had received this service (Robert Uhler, MS, Personal Communication, April 2006). The annual income level for a family of four at 200% of poverty in 1992 was approximately $28,000 [6, 7].\nIn this context, to help low-income, uninsured women gain access to mammography screening services, the U.S. Congress passed the Breast and Cervical Cancer Mortality Prevention Act of 1990 (Public Law 101-354), authorizing the Centers for Disease Control and Prevention (CDC) to establish the National Breast and Cervical Cancer Early Detection Program (NBCCEDP) [8] referred to henceforth as the Program. The Program provides services through cooperative agreements, which are in place in all 50 states, the District of Columbia (DC), 4 U.S. territories, and 13 American Indian\/Alaskan Native organizations; for a total of 68 grantees. Services provided by an American Indian\/Alaska Native organization were consolidated with services provided by the state-based program where the American Indian\/Alaska Native organization participant resides. In this work, \u201cstate(s)\u201d refers to the 51 geopolitical units (50 states and the District of Columbia (DC)).\nThe Program provides both breast and cervical cancer screening services to low-income uninsured women aged 18\u201364. From 1991 through June 2005, the Program screened 2.6\u00a0million women for breast and cervical cancer, provided 3\u00a0million mammograms to 1.7\u00a0million women and diagnosed 5,309 cases of in situ breast cancers and 15,493 cases of invasive breast cancers. The Program provides screening mammography to women ages 40 and older. In 1996, Program policy prioritized mammography screening of older women to allow for best use of limited resources. Of women receiving their initial screening mammogram through the Program in 2000\u20132004, 2.4% were under 40\u00a0years of age; 28.0% were 40\u201349\u00a0years old; 66.4% were between the ages of 50 and 64; and 3.2% were over 64\u00a0years of age. Fifty-two percent of the women were non-Hispanic Whites, 14% were non-Hispanic Blacks, 6% were non-Hispanic Asian\/Pacific Islanders, 4% were non-Hispanic American Indian\/Alaska Natives, 31% were Hispanics, and 3% were of unknown race\/ethnicity [9]. The Program grew from eight grantees funded at $30\u00a0million in Fiscal Year 1991 to 68 grantees that received $204\u00a0million in 2005. Sixty percent of federal funds received by the grantees are spent on clinical services. The remaining 40% are used to fund other Program components including program management, data collection, quality assurance and improvement, partnership development, professional education, recruitment and evaluation. Treatment is covered by state Medicaid funding and other non-Program sources. A detailed description of the history of the Program is provided elsewhere [8]. Earlier research [10] found that in 1994\u20131996 the Program provided either breast or cervical cancer screening services to about 12\u201315% of eligible women aged 50\u201364, but that study did not estimate the proportion of women who were provided mammography screening specifically or provide estimates by race\/ethnicity or for states.\nThe objective of the current analysis was to examine the extent to which the Program has helped to meet the mammography screening needs of this underserved population. Specifically, we estimated the numbers and percentages of women aged 40\u201364 who in 2002\u20132003 were eligible for breast cancer screening through the Program at both the state and national levels and the percentage of these eligible women who received Program-funded mammography. Finally, because disparities by race and ethnicity in the provision of screening services have commonly been reported in the U.S. [5, 11\u201313], we examined the extent to which the Program at the national level provided services to women of different racial and ethnic backgrounds.\nMaterials and methods\nWe examined eligibility using the Annual Social and Economic Supplement (ASEC) to the Current Population Survey (CPS) conducted by the U.S. Census Bureau, and screening service delivery using Program data. We used data from 2002 and 2003 because Program data were complete for those years [8].\nEligibility for NBCCEDP breast cancer screening services\nWomen aged 40 and older who do not have health insurance or whose insurance does not cover mammography screening are eligible for free breast cancer screening through the Program if their family incomes are below 250% of the federal poverty level. Twenty-two states and Washington, DC set eligibility criteria at lower poverty level. The annual income levels for a family of four at 200% and 250% of poverty in 2003 were approximately $37,000 and $46,000, respectively [7, 14]. Because 95.8% of women age 65 and over were covered by Medicare [15] and were therefore not served by the Program, we included only women aged 40\u201364 in this analysis.\nData sources\nCPS ASEC\nQuestions about age, family size, sex, race, and Hispanic origin were included in the basic CPS, which is a monthly national survey undertaken primarily to determine the characteristics of the labor force of the U.S. civilian non-institutional population. Respondents were asked to identify their race by selecting one or more options from a list. Hispanic origin was asked separately from race. About 78,000 interviewed households in the CPS were asked a set of supplementary questions (the ASEC) about health insurance coverage, income received, and place of residence during the previous year [16]. The methods used to collect and report CPS ASEC data have been described previously [17].\nNBCCEDP\nData for the number of women screened during 2002\u20132003 were obtained from Program service records. Grantees routinely collect income, family size, and insurance information to determine eligibility and collect screening information on each woman in the Program. These data include screening location, demographic characteristics, service dates, and outcomes. Demographic data are self-reported. Reporting of race and Hispanic origin is optional. The structure of the Program and methods for collecting and reporting Program data have been described elsewhere [8].\nData analysis\nCPS ASEC\nWe estimated the number of women eligible for the Program by race and ethnicity (Hispanic origin) at the national level by extrapolating the respondents\u2019 answers to the general U.S. population using standard Census Bureau methods [15\u201319]. Given the CPS sample size, it was impossible to produce meaningful estimates by race or ethnicity for individual states.\nWe categorized women who reported they were of Hispanic origin as Hispanic regardless of race. We categorized the remaining women, who were non-Hispanic, into one of the following racial groups: White, Black, American Indian\/Alaska Native, Asian\/Native Hawaiian\/other Pacific Islander, or multiracial (which is henceforth defined synonymously with Two or More Races). In the CPS, if a respondent did not report his or her race, the respondent\u2019s race was allocated using the race of another member of the household, or failing that, the previous record on the CPS file [17]. People were considered uninsured if they were not covered by any type of private or government health insurance for the entire previous year [18]. Poverty was computed by comparing total family income (or the person\u2019s own income if she did not live with family members) with 1 of 48 dollar amounts called poverty thresholds [18, 19]. Each person\u2019s poverty threshold was based on family size and number of children younger than 18\u00a0years [14]. The method of computing confidence intervals (CI) for the estimates of the eligible population is provided in Appendices 1 and 2. We use two-tailed 90-percent CI to be consistent with the Census Bureau\u2019s practices for reporting poverty and health insurance data.\nNBCCEDP\nIn counting the total number of women who received at least one Program-funded mammogram in 2002 and 2003, women were classified into the race\/ethnic categories used to estimate Program eligibility. About 2.4% of women who were screened did not claim any race or Hispanic origin. These women (12,653) were counted in the total number of women screened, but not in the specific race\/ethnicity categories.\nNBCCEDP screening rates\nBased on the number of women screened and estimates of the numbers of women aged 40\u201364 in both the U.S. population and the Program-eligible population we estimated the percentage of all U.S. women aged 40\u201364 and the percentage of Program-eligible women who received a Program-funded mammogram at least once in 2002 or 2003. We estimated provision of mammography screening over a 2-year period because the U.S. Preventive Services Task Force recommends that women aged 40 and older be provided with screening mammography every 1\u20132\u00a0years [3]. We further examined the distribution of mammography screening among women from different racial and ethnic backgrounds. The method of computing CI for the estimated screening rates is provided in Appendix 3.\nResults\nDuring 2002\u20132003, approximately 47\u00a0million women aged 40\u201364 resided in the United States (Table\u00a01). Of those women, approximately 4.0\u00a0million (90% CI, 3.8\u00a0million\u20134.2\u00a0million) or 8.5% (CI, 8.1\u20139.0%) were estimated to be eligible for a Program-funded mammogram. Non-Hispanic White women constituted the largest number of women eligible for the Program: 2.0\u00a0million (CI, 1.8\u20132.1\u00a0million). However, the percentage of non-Hispanic White women who were eligible for the Program (5.7%; CI, 5.3\u20136.1%) was smaller than that of other racial groups except for multiracial women. The percentage of multiracial women who were eligible, 8.9%, was lower than the percentage for Hispanic women and for non-Hispanic American Indian\/Alaska Native women, but was not statistically distinguishable from that of the other groups. The percentage eligible among Hispanic women (23.1%) was not statistically distinguishable from non-Hispanic American Indian\/Alaska Native women (19.8%) but was higher than the percentage eligible among the other racial and ethnic groups.\nTable\u00a01Number and percentage of U.S. women aged 40\u201364\u00a0years who were eligible for the National Breast and Cervical Cancer Early Detection Program (NBCCEDP), 2002\u20132003Race\/ethnicityAll women age 40\u201364Women eligiblea for NBCCEDP mammography screeningNumber (thousand)Percent distributionbNumber (thousand)90% CIPercentc90% CITotal46,8991004,0073,806\u20134,2088.58.1\u20139.0Non-Hispanic42,50490.62,9912,817\u20133,1667.06.6\u20137.4\u00a0\u00a0\u00a0\u00a0White34,40373.41,9721,835\u20132,1095.75.3\u20136.1\u00a0\u00a0\u00a0\u00a0Black5,43911.6714629\u201379913.111.6\u201314.6\u00a0\u00a0\u00a0\u00a0AI\/AN2250.54523\u20136619.811.3\u201328.4\u00a0\u00a0\u00a0\u00a0A\/NH\/OPI1,9774.2221173\u201326811.28.9\u201313.4\u00a0\u00a0\u00a0\u00a0Multiracial4601.04120\u2013618.94.6\u201313.2Hispanic4,3959.41,016909\u20131,12223.120.9\u201325.3AI\/AN: American Indian and Alaska Native; A\/NH\/OPI: Asian and\/or Native Hawaiian and other Pacific Islanders; Multiracial: two or more racesa\u00a0Women eligible for NBBCCEDP-funded mammography screening include women aged 40\u201364 with family incomes below 250% of federal poverty level, who are uninsured for mammography. The number of eligible women could be an underestimate because it excludes women who have insurance but whose insurance does not cover mammography screening. See Sect. Methods for detailsb\u00a0Percent of all U.S. women aged 40\u201364c\u00a0Percent of all U.S. women aged 40\u201364 in a given racial or ethnic group who were eligible for NBCCEDP funded mammography servicesSource: Authors\u2019 tabulations of data from the U.S. Census Bureau, Current Population Survey, 2003\u20132004 Annual Social and Economic Supplements\nDuring 2002\u20132003, 528,622 women aged 40\u201364 received at least one mammogram through the Program, among whom 12,653 (2.4%) were of unknown race\/ethnicity (Table\u00a02). Of all women in the United States aged 40\u201364, the Program provided mammography screening to approximately 1.1%. The rates of screening with mammography in the past 2\u00a0years (2002\u20132003), referred to henceforth as the screening rates, varied substantially by race\/ethnicity. Approximately 9.7% of all American Indian\/Alaska Native women were screened and approximately 0.3% of all multiracial women were screened.\nTable\u00a02Number and percentage of women eligible for the National Breast and Cervical Cancer Early Detection Program (NBCCEDP) and U.S. women provided with mammography screening services, at least once, through NBCCEDP, between 2002 and 2003Race\/ethnicityNumber of women screenedPercentage of U.S. women screenedaPercentage of NBCCEDP-eligible women screenedb%90% CITotal528,6221.113.212.5\u201313.9Non-Hispanic349,6550.811.711.0\u201312.4\u00a0\u00a0\u00a0\u00a0White221,4330.611.210.4\u201312.0\u00a0\u00a0\u00a0\u00a0Black74,2591.410.49.2\u201311.6\u00a0\u00a0\u00a0\u00a0AI\/AN21,8829.749.225.5\u201372.9\u00a0\u00a0\u00a0\u00a0A\/NH\/OPI30,6871.613.910.9\u201316.9\u00a0\u00a0\u00a0\u00a0Multiracial1,3940.33.41.7\u20135.2Hispanic166,3143.816.414.7\u201318.1Unknown Race\/ethnicity12,653\u2013\u2013\u2013AI\/AN: American Indian and Alaska Native; A\/NH\/OPI: Asian and\/or Native Hawaiian and other Pacific Islanders; Multiracial: two or more racesNBCCEDP mammography eligible women include: uninsured women aged 40\u201364 with family incomes below 250% of federal poverty levela\u00a0Percent of all U.S. women in a given racial and ethnic group who were provided mammograms funded by NBCCEDPb\u00a0Percent of all U.S. women in a given racial and ethnic group who are eligible and who were provided with NBCCEDP funded mammogramsSource: Authors\u2019 tabulations of data from the U.S. Census Bureau, Current Population Survey, 2003\u20132004 Annual Social and Economic Supplements, and from NBCCEDP April 2005 data\nAmong all women eligible for the Program, approximately 13.2% (CI, 12.5\u201313.9%) were screened one or more times (Table\u00a02). This estimate is based on the national Program eligibility limit of 250% of poverty. Using state-specific poverty level criteria, we estimated the screening rate for all states combined to be 14.7% (CI, 13.8\u201315.6%), reflecting the lower eligibility limits used in many states (data not shown).\nThe percentage of all eligible women who were screened in the Program varied by race\/ethnicity (Table\u00a02). The screening rates for non-Hispanic White and non-Hispanic Black women were not statistically distinguishable. Hispanic women had a higher screening rate than other women except for non-Hispanic American Indian\/Alaska Native women, who had the highest screening rate, with an estimated 49.2% (CI, 25.5\u201372.9%) screened. The screening rate for non-Hispanic multiracial women was lowest (3.4%, CI, 1.7\u20135.2%).\nThe estimated number and percentage of women in each state who were eligible for the Program and state-specific poverty levels used as eligibility criteria are shown in Table\u00a03. The estimated numbers of eligible women were greater in heavily populated states such as California, Texas, New York and Florida than in the other states. The estimated percentage of women eligible for the Program was highest in New Mexico (15.6%; CI 10.7\u201320.5%), although that percentage was not statistically distinguishable from the percentage for Louisiana, Mississippi, and five other states. Similarly, while Minnesota\u2019s estimated percent eligible appeared lowest (2.8%; CI 1.2\u20134.5%), it was not statistically distinguishable from Delaware, Massachusetts, and 11 other states.\nTable\u00a03Number of women aged 40\u201364 and estimated number of women eligible for the National Breast and Cervical Cancer Early Detection Program (NBCCEDP), breast cancer screening, by state: 2-Year averages; 2002\u20132003U.S. populationEligible womenTotalPoverty criterionaNo. (thousands)90% CI (thousands)% Of totalb90% CI (%)U.S.46,899200\/2503,5943,403\u20133,7857.77.27\u20138.06Alabama7482005734\u2013807.64.6\u201310.6Alaska10325095\u2013128.55.5\u201311.6Arizona8072507241\u20131028.95.3\u201312.4Arkansas4232003923\u2013559.25.7\u201312.8California5,506200476386\u20135658.67.1\u201310.2Colorado7142505535\u2013757.75.0\u201310.3Connecticut5852002312\u2013344.02.0\u20135.9Delaware14325052\u201383.71.7\u20135.8District of Columbia9225063\u201396.63.5\u20139.7Florida2,805200262206\u20133189.37.4\u201311.2Georgia1,4072009555\u20131356.84.0\u20139.5Hawaii19625094\u2013144.52.1\u20136.9Idaho213200169\u2013237.24.1\u201310.4Illinois1,93620012385\u20131606.34.5\u20138.2Indiana1,0112006036\u2013835.93.6\u20138.2Iowa4802502413\u2013355.02.7\u20137.3Kansas4292502413\u2013355.63.1\u20138.0Kentucky6692508054\u201310612.08.4\u201315.6Louisiana71225010875\u201314215.210.9\u201319.5Maine219250148\u2013196.23.9\u20138.6Maryland9332505431\u2013765.73.4\u20138.0Massachusetts1,0612504021\u2013603.82.0\u20135.6Michigan1,65525011076\u20131436.64.7\u20138.6Minnesota8612502410\u2013392.81.2\u20134.5Mississippi4712506140\u20138213.08.8\u201317.1Missouri8942005128\u2013735.73.3\u20138.2Montana159200159\u2013219.25.7\u201312.8Nebraska276225157\u2013225.32.8\u20137.9Nevada3412502817\u2013388.15.2\u201311.0New Hampshire225250116\u2013164.92.7\u20137.0New Jersey1,4582509666\u20131266.64.6\u20138.6New Mexico2952504631\u20136215.610.7\u201320.5New York3,212250262209\u20133158.26.6\u20139.7North Carolina1,33020013797\u201317610.37.5\u201313.1North Dakota10520074\u201396.33.7\u20139.0Ohio1,96820011579\u20131515.84.1\u20137.6Oklahoma5252004023\u2013577.64.5\u201310.7Oregon6152504527\u2013627.24.5\u201310.0Pennsylvania2,07025011177\u20131455.43.8\u20137.0Rhode Island18125095\u2013134.92.9\u20137.0South Carolina6712004828\u2013687.24.3\u201310.0South Dakota11620085\u2013116.74.1\u20139.4Tennessee1,0252507040\u20131006.84.0\u20139.7Texas3,205200396318\u201347412.410.1\u201314.7Utah3022501910\u2013296.43.3\u20139.4Vermont11525052\u201374.12.1\u20136.1Virginia1,2452006233\u2013915.02.7\u20137.2Washington1,0732007041\u2013996.53.9\u20139.1West Virginia3112003222\u20134210.27.1\u201313.4Wisconsin9202504526\u2013654.92.8\u20137.0Wyoming8525096\u20131210.57.1\u201313.9a\u00a028 States and DC set income eligibility at 250% of poverty, 21 states at 200% of poverty and 1 state at 225% of poverty. The estimated number of eligible women for the U.S. is based on the eligibility criteria used in each stateb\u00a0Eligible women as percentage of all women aged 40\u201364\u00a0years in that stateSource: Authors\u2019 tabulations of data from the U.S. Census Bureau, Current Population Survey, 2003\u20132004. Annual Social and Economic Supplements.\nThe percentages of eligible women who were screened through the Program varied greatly across states (Fig.\u00a01). The percentage of eligible women screened by in individual state ranged from about 2.2% (CI, 1.5\u20132.8%) to approximately 79% (CI, 49.4\u2013108.9%). The median percentage screened among the states was 18.2% (CI, 11.7\u201324.7%). The tenth highest percentage estimate was 28.5 (CI, 23.2\u201333.9%) and the tenth lowest percentage estimate was 10.71 (CI, 7.4\u201314.1%).\nFig.\u00a01Percentages of NBCCEDP eligible women screened for breast cancer, by State and District of Columbia, compared with the National Aggregate Percentage, 2002\u20132003. Note: The boxes show the percentage of eligible women screened by each state. The whiskers in each box, show the variation in the screening rate (the upper and lower CI). NBCCEDP referes to National Breast and Cervical Cancer Early Detection Program. *States includes DC, sorted by percentage of eligible women screened. Source: Authors\u2019 tabulations of data from the U.S. Census Bureau, Current Population Survey, 2003\u20132004. Annual Social and Economic Supplements, and from NBCCEDP April 2005 data\nDiscussion\nWe found that a large number of women\u20144\u00a0million women or 8.5% of all U.S. women aged 40 to 64\u2014were uninsured during 2002\u20132003 and had incomes below 250% of the federal poverty level, meeting Program eligibility requirements. Of these, only about 13.2% received mammograms funded through the Program. Although many of the estimates for groups of women classified by race\/ethnicity were imprecise, as indicated by wide CI, there was clearly wide variation in numbers and percentages of women eligible for the Program and in the percentages of eligible women who were screened. The percentage of eligible women screened was highest among American Indians and Alaska Natives. Although the estimates of eligible women in each state were also imprecise, the findings clearly showed wide variability. The percentage of eligible women screened in each state ranged from approximately 2% to approximately 79%.\nThe Program is an important source of mammography screening services for low-income, uninsured women, but neither NBCCEDP nor other providers that serve this population are able to meet the current needs. The Program has grown since 1991\/1992 when 38,476 women were screened in 12 states [8] to screen over 500,000 women during 2002\/2003 in all states. A large number of federally funded community health centers, hospitals, clinics, and voluntary associations provide mammography screening services to underserved women. The numbers of women screened by these programs are not readily available. However, we know from the 2003 National Health Interview Survey (NHIS) that only 42.3% of women with no health insurance and family incomes less than 250% of the poverty level reported having had a mammogram during the previous two years (Robert Uhler, MS, Personal Communication, February 2006). Of the 4\u00a0million women we have identified in this study as eligible for the Program, the NHIS results indicate that about 1.7\u00a0million women were screened, meaning that approximately 1.2\u00a0million received screening outside of the Program. However, this leaves about 2.3\u00a0million low-income, uninsured women who did not obtain those services from either the Program or other sources.\nThe largest numbers of women eligible for the Program were non-Hispanic White women. In contrast, the largest percentages of women eligible for the Program were from minority groups, except for women of Two or More Races. Hispanic and non-Hispanic American Indian\/Alaska Native groups had the highest percentages of eligible women. We were limited in our ability to assess the extent to which the Program met the mammography screening needs of women by race\/ethnicity because about 12,000 women screened in the Program were of unknown race\/ethnicity. If we had been able to correctly allocate these women to their appropriate race\/ethnic groups, the percentages of women screened would have differed from those shown, potentially by an important margin. However, the findings indicate that the Program was most successful in meeting the needs of American Indian\/Alaska Native women, approximately 49% of whom were screened. A possible reason for this success may be that these populations are the focus of health services through which the Program operates. In 1993, Congress amended the Breast and Cervical Mortality Prevention Act, Public Law 108-183, to authorize funding for American Indian\/Alaska Native organizations and provided the opportunity to direct resources to these populations, specifically four grantees in Alaska and another nine geographically distributed across the contiguous United States.\nThe number and percentage of women who were eligible for the Program varied greatly from state to state, because of differences in population size, age, and sex distributions, as well as differences in income and insurance coverage, including Medicaid [18]. In addition to the aforementioned factors, reasons for variations in the percentage of eligible women screened include differences in income eligibility criteria, presence of American Indian\/Alaska Native grantee, CDC funding levels, other sources of funding, and organization and efficiency of the screening Programs. The upper and lower bounds of the CI indicate that some estimates were not precise, but were useful nevertheless. These estimates have been made available to each state for their use in Program planning to better understand the need for screening services in their states.\nOur study is subject to a number of limitations. As already noted, many of the estimates are imprecise because the numbers of women in the CPS ASEC sample who are eligible for the Program are relatively small. In addition, health insurance coverage could be underreported in the CPS ASEC given that the survey uses annual retrospective questions and respondents may have difficulty recalling the information [18]. Also, some women are eligible for the Program even if they have health insurance, but are underinsured, meaning the insurance does not cover mammography screening services, or there is a high copayment. Since CPS ASEC insurance questions do not measure covered services, these underinsured eligible women are not included in the denominators of our screening percentages. It is uncertain how many low-income women in the United States population are underinsured. Finally, our inability to define the race or ethnicity of some women in the study could result in an underestimate of the participation rate for any given race or ethnic group.\nWe suggest two strategies to improve screening rates: increasing efficiencies of the Programs and improving their collaboration with other organizations. First, the Program must seek ways of increasing its efficiency to serve more women with existing resources. A study of Program costs found that the average cost of screening a woman through the Program was lower for grantees screening greater numbers of women because of economies of scale, that is, average cost decreased as number of women screened increased [20]. States with small populations in larger geographical areas may have limited opportunities to achieve such economies of scale. CDC has recently initiated a cost-effectiveness evaluation of the Program and is developing methods to better collect and analyze information on resources and how they might be used more efficiently. A variety of means to increase efficiency will need to be pursued. For example, many women in the Program are screened annually. The U.S. Preventive Services Task Force recommends screening every 1\u20132\u00a0years because it has found little evidence that annual screening is more effective than biennial screening [3]. Many European programs provide screening every 2\u20133\u00a0years [21]. The Program may need to evaluate the potential balance of health benefits from adopting a biennial schedule that could serve more women.\nSecond, the Program needs to improve collaboration and coordination with other providers that serve a similar client population. The Program already coordinates substantially with private and nonprofit organizations, businesses, and other groups involved in breast cancer screening, but that coordination needs to be increased to recruit the women who are not currently being served. For example, in addition to providing screening services, the Program provides diagnostic services for eligible women screened by organizations outside of the Program. Alternative sources of diagnostic services may need to be pursued to free resources for increased screening of eligible women.\nAlthough greater efficiency and improved coordination with other screening providers could better meet the needs of underserved women, they are unlikely to be enough. Given that about 2.3\u00a0million low-income uninsured women did not obtain recommended breast cancer screening services in 2003 and that the Program provided those services to about 500,000 women; increased efficiency and coordination alone will be insufficient to meet the needs of the eligible population.\nIn 2000, when Healthy People 2010 first set out its objectives of eliminating health disparities and increasing the proportion of women aged 40 and older who have received a mammogram within the previous two years to 70% [12], the greatest disparities in breast cancer screening were for women who had no health insurance, those who had no usual source of care, and recent immigrants [5]. Although progress has been made since 1987 in increasing mammography screening among low-income and uninsured women, the increases for low-income women are less than those for higher-income women, and screening among the uninsured lags far behind screening among women with private or public health insurance [5]. The Program contributes substantially to the effort to provide breast cancer screening services to those women by serving 13.2% of those eligible. However, the Healthy People 2010 objectives are still far from being met.","keyphrases":["mammography screening","breast cancer","screening rates","medically underserved"],"prmu":["P","P","P","M"]}
{"id":"J_Comput_Aided_Mol_Des-3-1-2039848","title":"Construction of 3D models of the CYP11B family as a tool to predict ligand binding characteristics\n","text":"Aldosterone is synthesised by aldosterone synthase (CYP11B2). CYP11B2 has a highly homologous isoform, steroid 11\u03b2-hydroxylase (CYP11B1), which is responsible for the biosynthesis of aldosterone precursors and glucocorticoids. To investigate aldosterone biosynthesis and facilitate the search for selective CYP11B2 inhibitors, we constructed three-dimensional models for CYP11B1 and CYP11B2 for both human and rat. The models were constructed based on the crystal structure of Pseudomonas Putida CYP101 and Oryctolagus Cuniculus CYP2C5. Small steric active site differences between the isoforms were found to be the most important determinants for the regioselective steroid synthesis. A possible explanation for these steric differences for the selective synthesis of aldosterone by CYP11B2 is presented. The activities of the known CYP11B inhibitors metyrapone, R-etomidate, R-fadrazole and S-fadrazole were determined using assays of V79MZ cells that express human CYP11B1 and CYP11B2, respectively. By investigating the inhibitors in the human CYP11B models using molecular docking and molecular dynamics simulations we were able to predict a similar trend in potency for the inhibitors as found in the in vitro assays. Importantly, based on the docking and dynamics simulations it is possible to understand the enantioselectivity of the human enzymes for the inhibitor fadrazole, the R-enantiomer being selective for CYP11B2 and the S-enantiomer being selective for CYP11B1.\nIntroduction\nAldosterone is a member of the renin angiotensin aldosterone system (RAAS) that plays an important physiological role in the regulation of electrolyte homeostasis and thereby blood pressure. This endogenous mineralocorticoid exerts its function by binding to the mineralocorticoid receptor. Upon ligand binding, the protein-ligand complex is translocated to the cell nucleus, where it modulates the gene expression of proteins involved in electrolyte homeostasis [1]. Aldosterone is produced predominantly in the adrenal cortex and is derived from cholesterol through various steps involving a number of dehydrogenases and cytochrome P450 enzymes (CYPs). This includes aldosterone synthase (CYP11B2), which catalyses the final steps of aldosterone biosynthesis.\nRecently, various studies on the pathophysiology of heart failure have revealed that aldosterone plays a role in the formation of myocardial hypertrophy, reactive myocardial fibrosis, vascular remodelling and electrolyte imbalance. This may contribute to the development of arrhythmias, hypertension and congestive heart failure [2, 3]. Although great therapeutic successes have been achieved by treating heart failure patients with blockers of the RAAS (CONSENSUS trial [4], SOLVD trial [5]), the mortality due to heart failure is still high. Therefore, exploring new therapeutic possibilities is highly desirable. Recently, it became clear that aldosterone is also a key player in heart failure. In the RALES study [6], blocking the action of aldosterone using the mineralocorticoid receptor antagonist spironolactone, proved to greatly reduce mortality, hospitalisation numbers and hospitalisation time in patients with severe heart failure. Later, similar encouraging results were found for eplerenone, another mineralocorticoid receptor antagonist (EPHESUS trial [7, 8]), in patients with post myocardial infarction. From this it can be derived that reducing aldosterone action seems highly beneficial in the treatment of heart-failure and that it is particularly worthwhile to find new pharmacological manners to interfere with this hormone.\nAn alternative manner to reduce aldosterone action would be not to block the mineralocorticoid receptor, but to prevent formation of the hormone itself by inhibiting its biosynthesis [9]. Although the net outcome of aldosterone receptor inhibition versus aldosterone synthesis inhibition apparently is identical, a number of potential advantages for the latter approach exists: reduction of side effects as particularly observed for the antagonist spironolactone [6, 10], prevention of compensatory aldosterone synthesis inherent to mineralocorticoid receptor blockade (of which long term effects are unknown) [11, 12], and possible circumvention of inter-individual variations regarding pharmacodynamics and pharmacokinetics that are as observed for mineralocorticoid receptor antagonists [13]. The clinical use of inhibitors of aromatase (CYP19, which converts androgens into estrogens) next to estrogen receptor antagonists in the treatment of breast cancer, is one of the examples that shows that the approach of ligand synthesis inhibitors clinically can be of great use, despite the existence of receptor blockers for that ligand [14, 15].\nThe last steps of the biosynthesis of aldosterone are mediated by the mitochondrial cytochrome P450 11B family (CYP11B) (Fig.\u00a01). Members of this protein family contain a heme prosthetic group in the core of the active site with which they catalyse (subsequent) oxidation reactions on C11, C18 and C19 on the \u03b2-side of the steroid skeleton (for enumeration see Fig.\u00a01). In bovine [16], pig [17] and frog [18], aldosterone synthesis is performed by only one cytochrome, CYP11B, but in human [19] and mouse [20] the synthesis involves two isoforms, CYP11B1 (steroid 11\u03b2-hydroxylase) and CYP11B2 (aldosterone synthase). Rat possesses four isoforms for which CYP11B1 and CYP11B2 are the most important ones. CYP11B3 is only expressed in neonatal rat and carries the same activity as CYP11B2, and CYP11B4 encodes a pseudogene [21].\nFig.\u00a01Biosynthesis of Aldosterone by the CYP11B family. Indicated with arrows are the possible substrate conversions performed by human CYP11B1 and CYP11B2 [19]. Rat CYP11B1 and CYP11B2 possess the same activities as the human isoforms, except that rat CYP11B1 can also oxidise 11-deoxycorticosterone on C19 [21] \nMost remarkable is the substrate specificity of the different isoforms. In both man and rat, only the CYP11B2 isoform can perform the final oxidation of C18 to produce aldosterone [19, 21]. For the CYP11B1 isoform, C19 hydroxylation has been reported for rat but not yet for man [21], and the CYP11B1 isoform in general is known to play an important role in the biosynthesis of glucocorticoids. If an inhibitor of aldosterone synthesis is to be designed, the biosynthesis of glucocorticoids should remain unaffected, indicating that the inhibition must be CYP11B2 selective.\nWe developed three-dimensional in\u00a0silico models as a decision making tool to facilitate the selection of potential CYP11B2 inhibitors for synthesis and in\u00a0vitro testing. In order to obtain specific CYP11B2 inhibitors, we also investigated its highly homologous family member CYP11B1.\nThe aim of this report is (1) to deduce by homology\/comparative modelling the architecture of CYP11B1 and CYP11B2 around their active site, (2) to investigate protein-substrate interactions and propose a mechanism for substrate regioselectivity and (3) to validate the homology models by correlating the in\u00a0vitro activity of four known inhibitors to in\u00a0silico data. The inhibitors we have chosen are metyrapone [22], R-etomidate [23], R-fadrazole and S-fadrazole [24] (Fig.\u00a02). The in\u00a0silico models not only represent an important tool in modern drug discovery but will also help in elucidating molecular mechanisms and (substrate binding) preferences of the substrate conversion of the enzymes of interest.\nFig.\u00a02Chemical structures of the known CYP11B inhibitors, metyrapone, R-etomidate, R-fadrazole and S-fadrazole\nModelling considerations\nHomology modelling has been widely applied to the family of cytochrome P450 enzymes and is mainly oriented towards enzyme inhibition. Before the first mammalian cytochrome P450 structures became available, modelling attempts were classically performed with crystal structures possessing sequence identities lower than 25% because no realistic alternatives were available. Homology models were often based on a template of bacterial CYP101 [25\u201327], but the introduction of bacterial CYP102 allowed for modelling the functional properties of eukaryotic class II P450s [28]. Although these homology models contain a low sequence identity with their template structures and are intuitively suboptimal, it has been shown that they can describe key features of protein-ligand interactions [27, 28]. For example, features observed for inhibitor binding in aromatase models have provided important insights for the development of drugs [27, 29]. Current models often still feature bacterial P450s as template [30, 31], but methods involving the usage of multiple crystal structures for model construction may prove to be the future trend [32, 33]. A model based on the structure of several known enzymes would be more accurate since every additional segment will improve similarity or spatial coordination of protein regions. However, structural flaws can be expected at locations where the template structures are joined and if these regions are within the active site, they need to be thoroughly refined.\nSome modelling work on CYP11B1 and CYP11B2 has already been performed earlier by Belkina et\u00a0al. [34] and Ulmschneider et\u00a0al. [35]. The models of Belkina et\u00a0al. discuss the potential spatial arrangement of the amino acids in the active site and hypothesise the hydrogen-bonding network involved in heme stabilisation. Furthermore, the effect of several amino acid mutations have been detailed. The models of Ulmschneider et\u00a0al. focus on describing protein-inhibitor interactions and structure activity relations of their developed inhibitors. The so far published models were thoroughly characterised for those specific purposes, however, the goal of our model construction work was to investigate the regioselectivity of the natural ligands within the enzymes and to detail potential protein-ligand interactions.\nFor validation of our own three-dimensional models, the in\u00a0silico data are presented in the form of molecular docking and molecular dynamics simulations. These methods are regularly used to investigate protein-ligand interactions. Because the only difference in the activity of the two isoforms CYP11B1 and CYP11B2 is the formation of aldosterone by the latter, successful 3D modelling of the isoforms relies on a careful analysis of the specific substrate conversion activities that exists between these two isoforms.\nBecause of this we reviewed an experimental mutation study by Bottner et\u00a0al. [36] on the human CYP11B1 and CYP11B2 proteins, performed in a similar manner as by Belkina and Ulmschneider for the currently published models [34, 35]. The study by Bottner et\u00a0al. showed that mutation of three residues outside the active site (L301P, E302D, A320V) is sufficient to convert the catalytic activity of CYP11B2 into that of CYP11B1, suggesting that remote steric aspects play a more important role in the substrate binding and substrate conversion than the presence of different amino acids in the active sites of both isoforms. This led us to postulate that the difference in substrate conversion is caused by a difference in the relative positioning of the substrate above the heme in the active site. To be more specific, we postulate that there is a correlation between substrate selectivity and the substrate hydroxylation distance, the distance between the heme iron and the substrate carbon. In other words, the binding mode of the natural substrate dictates which carbon atom is oxidised first, with conversion taking place on the carbon atom which is in closest proximity to the iron-oxygen complex.\nFor human CYP11B1 this means that C11 and C18 are to be in close proximity to the catalytic iron atom, with C11 closest to the iron. Rat CYP11B1 possesses a similar binding mode, but we expect that it also presents C19 in a position allowing oxidation. Explaining the preference for C18-hydroxylation, human and rat CYP11B2 would bind with C18 closest to the iron atom and C11 at a correct distance for oxidation.\nTo substantiate this hypothesis, the three dimensional architectures of the human and rat CYP11B enzymes were constructed using comparative modelling. For reasons of relevance only the CYP11B1 and CYP11B2 isoforms were investigated. We intend to show how knowledge of these various hydroxylation patterns of aldosterone precursors can result in working models for the substrate selective activity of the two isoforms. From here on, the human isoforms will be noted as hCYP11B1 and hCYP11B2, whereas the rat isoforms will be noted as rCYP11B1 and rCYP11B2.\nAs stated above, another aim was to validate the in\u00a0silico models with in\u00a0vitro activity data of four known inhibitors. These inhibitors were chosen for the following reasons. Metyrapone is a known inhibitor of CYP11B1 and is clinically used in the diagnosis of Cushing Syndrome [22, 37]. R-etomidate is clinically used as anaesthetic, but it is known to be a highly potent suppressor of the CYP11B family [23, 38]. Racemic fadrazole was designed for the selective inhibition of aromatase, a cytochrome P450 enzyme which is closely related to the CYP11B family because it oxidises steroids on C19. Next to aromatase inhibition, racemic fadrazole also shows considerable inhibition of members of the CYP11B family [24, 39].\nMethods\nHomology modelling\nThe amino acid sequences of the CYP11B family were taken from Swissprot [40] (hCYP11B1 accession P15538, hCYP11B2 accession P19099, rCYP11B1 accession P15393, rCYP11B2 accession P30099, rCYP11B3 accession P30100) and the determination of the secondary structures of the CYP11B family members was performed using the secondary structure prediction program JPred [41]. The alignment used for homology modelling was obtained with MOE-Align [42] by performing a topological alignment of the cytochrome P450 enzymes for which a crystal structure is publicly available (Fig.\u00a03). The three dimensional architecture of cytochrome P450 enzymes consists of 12 alpha-helices annotated from A to L, as well as six beta-sheets, despite having a sequence identity among P450s less than 20%. Additionally, several short helices are present in various cytochrome P450 enzymes (annotated B\u2032, F\u2032, G\u2032, J\u2032, K\u2032) whilst being absent in others. The structural core of all cytochrome P450 enzymes consists of a four-helix bundle composed of alpha-helices D, E, I and L, and the two alpha-helices J and K. The variable structural features involved in ligand binding and substrate specificity are situated in alpha-helices A, B, B\u2032, F, G and their adjacent loops [43\u201345].\nFig.\u00a03Topology alignment of human and rat CYP11B isoforms to related cytochrome P450 enzymes of which a three dimensional structure has been elucidated. Indicated with a & are the Arg123 in alpha-helix B\u2032 and Glu310 in alpha-helix I. Indicated with a * is the triple mutant L301P, E302D, A320V in alpha-helix I. Indicated with a ^ is the catalytic Thr318 in alpha-helix I. Indicated with a # is the conserved Glu459 in alpha-helix L\nThe predicted secondary structures for the CYP11B family members were aligned to these topological features of the crystal structures, as well as by preserving several characteristic CYP features [46]: W(R\/K)XXR (X indicates any amino acid) in helix C connecting and stabilising the heme prosthetic group in the active site, EXXR in helix K and (W\/F)XXPXX(F\/Y)XPX(H\/R)(W\/F) following helix K\u2032 comprising the typical meander region, and finally XXF(G\/S)XGX(H\/R)XCXGXX(L\/F)AXXE before helix L which contains the cysteine residue by which the heme prosthetic group is bound to the enzyme. Furthermore, a glutamic acid was aligned that is thought to be involved in the formation of a catalytically important water channel [47] (Glu366 in CYP101, Glu459 in the CYP11B family), and the entire helix I was aligned for its catalytic threonine (Thr252 in CYP101, Thr318 in CYP11B family) [47]. As a result of small variations in loop regions, some insertions and deletions were introduced in segments outside the active site. These segments did not correspond to any of the key secondary structures.\nClearly, CYP11B1 and CYP11B2 portray a high degree of homology possessing a pair wise sequence identity percentage as high as 94% for human and 83% for rat (Table\u00a01, highlighted). This emphasises the difficulty of modelling the difference between the two isoenzymes and the challenge of reaching the level of modelling accuracy that is required. For P450 enzymes for which a crystal structure is publicly available, the overall pair wise sequence identity with the CYP11B family is found to be less than 20%, whereas it is around 30% for active site residues. This low degree of homology indicates that none of the reviewed cytochromes can be used as a sole representative for modelling the CYP11B family.\nTable\u00a01Generic pair wise sequence identity (in percentages) between the human and rat CYP11B isoforms and cytochrome P450 enzymes for which a three dimensional structure has been elucidatedChains10110210710811955512B42C52C82C92D63A4h11B1h11B2r11B1r11B2r11B3CYP101\u201317.330.237.739.638.523.521.223.130.823.111.519.626.424.524.526.426.42CPPCYP10216.3\u201324.528.339.619.229.426.934.630.832.714.847.126.426.424.524.524.51BU7CYP10720.012.3\u201328.350.936.521.619.221.221.225.014.321.620.818.920.818.918.91JINCYP10823.215.822.1\u201335.828.829.425.026.928.825.017.929.426.426.428.328.328.31CPTCYP11918.816.025.620.8\u201334.627.528.828.832.728.825.029.426.426.428.326.426.41F4UCYP5521.211.228.524.724.3\u201319.617.319.221.223.118.519.618.917.017.017.017.01ROMCYP5112.318.019.117.214.716.0\u201323.128.825.025.011.135.320.820.817.020.820.81EA1CYP2B414.616.716.414.015.016.016.7\u201357.765.459.633.333.332.132.132.130.230.21SUOCYP2C516.817.816.614.816.915.014.951.0\u201369.278.844.433.332.132.128.332.132.11NR6CYP2C815.817.616.415.515.815.813.453.873.6\u201369.244.431.430.230.230.230.230.21PQ2CYP2C915.618.017.415.016.615.314.351.077.378.4\u201340.733.332.132.128.330.230.21OG2CYP2D613.116.914.914.314.716.016.739.640.040.638.5\u201314.825.025.025.021.421.42F9QCYP3A414.122.019.614.517.214.816.522.822.022.921.917.9\u201332.132.130.230.230.21W0Eh11B116.016.314.615.516.112.014.917.417.615.817.314.917.1\u201398.181.186.884.9\u2013h11B215.316.913.614.815.812.014.517.417.616.217.715.517.593.6\u201383.088.786.8\u2013r11B112.315.413.213.815.311.013.417.416.716.617.714.417.363.663.6\u201388.786.8\u2013r11B215.315.814.414.516.613.014.715.916.215.817.113.516.868.268.882.6\u201398.1\u2013r11B315.415.814.414.516.613.014.715.916.215.817.113.816.868.669.283.097.3\u2013\u2013The bottom triangle indicates the pair wise sequence identity of the whole protein, the top triangle indicates the pair wise sequence identity of the residues within 5.0\u00a0\u00c5 from the surface of the active site cavity of CYP101. Species information: Pseudomonas-Putida 2CPP, Bacillus Megaterium 1BU7, Saccharopolyspora-Erythreaea 1JIN, Pseudomonas-SP 1CPT, Archaeon Sulfolobus Solfataricus 1F4U, Fusarium-Oxysporum 1ROM, Mycobacterium Tuberculosis 1EA1, Oryctolagus Cuniculus 1SUO and 1NR6, Homo Sapiens 1PQ2, 1OG2, 1W0E and 2F9Q\nBecause of the low sequence identity of the CYP11B family, we have chosen to create a hybrid template for hCYP11B2 using MOE-Homology [42], constructed from the crystal structures of CYP101 (pdb code: 2CPP) and CYP2C5 (pdb code: 1NR6). Our criteria for using CYP101 and CYP2C5 involve similarity in functionality of both the cytochrome P450 reduction system and ligand characteristics, but importantly also involves the spatial positioning of active site regions.\nThus far, all modelling attempts on cytochrome P450 family 11 have included the usage of microsomal P450s such as CYP102 [30, 34] and CYP2C9 [35]. However, the CYP11B family belongs to the bacterial\/mitochondrial cytochrome P450 class which obtains electrons from the ferredoxin reductase family in the electron transfer chain [48]. Using CYP101 for the modelling of mitochondrial P450s is therefore more intuitive and has been successfully applied to other mitochondrial P450s [33, 49].\nThe natural ligands of the CYP11B family are steroids, and steroids can be substrates for hepatic cytochromes that belong to the microsomal cytochrome P450 class. In CYP2C5 and CYP2D6 steroids are oxidised on the \u03b2-side of the steroid skeleton at carbon atoms close to C11 and C18 [50], and their crystal structures may possess the necessary interaction features for model construction. However, investigation of the crystal structure of CYP2D6 (PDB code: 2F9Q) raised doubt on the appropriateness for its use as a template structure. The crystal structure is resolved at low resolution (3.0\u00a0\u00c5) in a tetramer complex. In this complex numerous amino acids participate in an extensive interaction network between the monomers and the structure was therefore excluded from our modelling work. Of the other hepatic P450 enzymes, CYP3A4 can also oxidise steroids, but its oxidation sites are on the opposite side of the steroid skeleton or on the different steroid rings. This means that the steroids bind with a rotated or reversed orientation in the active site [51], which may provide different active site conformation than the CYP11B family. Furthermore, many CYP2D6 substrates are characterised by a basic nitrogen [52], and the CYP2C9 substrates are mostly weakly acidic [53]. These properties are found to be less desirable for modelling of the CYP11B family than the functionally similar properties of the cyclic alkane substrates of CYP101. The structures of CYP2C9 also do not provide information about the molecular basis of regioselectivity of the substrate, since a conformational change of the active site is required to allow the substrate hydroxylation sites to contact the heme [54]. Because of the different ligand properties, the structures of hepatic CYP2D6, CYP2C9 and CYP3A4 were found to be less suitable as template structure.\nAn important decision criterion to use CYP101 as the template is also its spatial properties. When comparing its active site cavity to that of the mammalian P450s, we found that the steroidal ligands would not be able to fit into the active site cavity of the mammalian P450s with C11 and C18 oriented to the heme, unless additional space is introduced near helix K. Because the most important interactions between protein and ligand take place near the heme, the regions lining the active site must be modelled with the highest accuracy. From the multiple sequence alignment with the CYP11B family one can see that beta-sheet 6-1 following helix K possesses a 1 amino acid insertion in the CYP101 structure and a 1 amino acid deletion in the mammalian structures (Fig.\u00a03). Although it is possible to model this site through a deletion in the mammalian structures, the resulting active site cavity would become strained. Since the structure of CYP101 is elongated, modelling a 1 amino acid insertion and relaxing the fold will create a better definition of this active site region. Taking into account that its substrate and reduction system are similar to that of the CYP11B family, CYP101 is found to possess the best structural core for the modelling of the CYP11B isoforms.\nBecause the structural core of the cytochrome P450 enzyme structure is the four-helix bundle comprising the helices D, E, I and L, and the two helices J and K, they should all be taken from one and the same template structure [43\u201345]. Hence, when taking the helices I and K from CYP101, the other regions must be used as well. Within the topological alignment of these regions, the mammalian P450s contain insertions and deletions in the connectivity between helix C and D, which is an additional reason why we have decided to construct the CYP11B models using the CYP101 structure.\nUsing the main structural features of CYP101, the remaining variant regions of the P450 fold (helices B\u2032, F, G, J\u2032 and the meaner region) were modelled with CYP2C5. CYP2C5 was chosen because after aligning the desired structures, it was found to possess slightly better spatial alignment with CYP101 than the other mammalian crystal structures, and additionally, CYP2C5 is a steroid synthase itself. Special attention was given to the modelling of helix B\u2032 by CYP2C5 and its connection to the helices F and G. This three helical complex makes up the entrance of the active site cavity and closes the pocket like a lid. The helix B\u2032 is predicted to be 3 turns long in the CYP11B family and there is an apparent insertion of 3 residues compared to that of the other cytochrome structures (Fig.\u00a03). In the CYP101 structure, this helix is positioned too high in the active site cavity, which is probably a reason why it is regularly seen as inappropriate to model on. We chose the structure of CYP2C5 where we extended its helix from a 2 turn length to a 3 turn length.\nThe first 50\u00a0N-terminal residues corresponding to the membrane binding region of the human cytochrome 11B family were omitted from the models because no complementary sequence is present in the crystal structures of either CYP101 or CYP2C5. Some manual adjustments were made to the hCYP11B2 model to compensate for large steric hindrances and the model was refined by energy minimisation using a MOE succession method of steepest descent, conjugate gradient and truncated newton until an RMS gradient of 0.1\u00a0kcal\/(mol\u00a0\u00c5) was reached. The Charmm22 forcefield [55] was used with a dielectric constant of 4 and all backbone atoms were tethered with a force constant of 100\u00a0kcal\/(mol\u00a0\u00c52) to prevent large movements.\nTo investigate the influence of the triple mutant introduced in the hCYP11B2 protein by Bottner et\u00a0al. [36], a hCYP11B2-triple mutant homology model (hCYP11B2-TM) was created. The three dimensional architecture of this structure should turn out to be similar to the active site of hCYP11B1. Models for hCYP11B1, rCYP11B1 and rCYP11B2 were also constructed by amino acid replacement in the model of hCYP11B2, followed by a structural relaxation of the fold with MOE.\nThe four ligands 11-deoxycorticosterone (DOC), corticosterone (B), 18-hydroxy-11-deoxycorticosterone (18OH-DOC) and 18-hydroxycorticosterone (18OH-B) were fit in the active sites of all CYP11B models matching all three CYP11B hydroxylation sites (C11, C18, C19) to the oxygen atom occupying the 6th ligating position of the heme iron. First the steroids were docked into the active site using automated docking to get an impression of the relative positioning of the C11, C18 and C19 above the heme. Next, alternate orientations of the steroids were investigated by visual inspection, taking into account alternate folding of amino acid side chains. A distance of 2.5\u00a0\u00c5 was chosen as a starting distance between the oxygen and carbon atoms, which is a near-optimal distance for hydrogen atom abstraction during hydroxylation [56]. A threshold of 5.0\u00a0\u00c5 was estimated to be the representative maximal distance for hydroxylation. The models were then equilibrated with MOE without tethering the backbone atoms, allowing the model active sites to diverge.\nLigand docking\nThe docking of all ligands was performed using GOLD v3.0 [57], in order to investigate the protein-ligand interactions and investigate the application of the models for inhibitor potency prediction. The docking parameters used were taken from the default 1 GOLD GA settings. These settings were used for all docking runs presented in this study. Each ligand was docked 5\u00a0times for 100 poses each for which the GOLD Fitness score was obtained using the standard Goldscore function. The amount of docking runs and poses was taken to ensure an appropriate sampling of the ligand conformations in the active site of the protein. For the inhibitors, the fitness score was averaged for the top 10 ranking poses if the conformation was within an RMSD of 1.0\u00a0\u00c5 from the best ranking pose (results Table\u00a05). Subsequently, to get an impression of the correlation of the docking with the in\u00a0vitro data, the Goldscore measure of binding affinity was calculated by the method reported by Verdonk et\u00a0al. [57] per \u0394Gbinding\u00a0=\u00a0\u22120.1075 * Goldscore\u22122.2665 (R2\u00a0=\u00a00.5529, N\u00a0=\u00a060, \u0394Gbinding expressed in kcal\/mol). The average poses were also rescored with the Chemscore scoring function [58].\nDocking of steroids was performed in the presence of an iron-bound oxygen atom and their conformations were afterwards checked to investigate alternate orientations of the steroid in the active site cavity. The protein structures used for docking the substrates were the unequilibrated structures, whereas for docking the inhibitors, the hCYP11B1 and hCYP11B2 models were used after they were equilibrated with the ligand 18-hydroxycorticosterone. All docking runs were performed in the absence of water molecules. For each inhibitor, the best ranked pose was used as input for the molecular dynamics study.\nMolecular dynamics of inhibitors\nSeveral molecular dynamics simulations were performed to investigate the stability of the enzyme models of hCYP11B1 and hCYP11B2, and the dynamic behaviour of the three inhibitors inside the respective active sites. The simulations were performed with the NAMD package [59] using the Charmm22 forcefield [55]. The protein models of hCYP11B1 and hCYP11B2 were solvated in a 80\u00a0\u00d7\u00a080\u00a0\u00d7\u00a080\u00a0\u00c5 equilibrated water box, removing any overlapping water. Counter-ions were added to ensure an overall net charge of zero. The protein-ligand complexes were slightly equilibrated for 25\u00a0ps at a temperature of 100\u00a0K and were subsequently simulated for 1ns at a temperature of 310\u00a0K in an NPT ensemble. All simulations were carried out using periodic boundary conditions. To calculate the electrostatic interactions we chose the implementation of NAMDs Particle Mesh Ewald [60]. To accommodate the filling of the active site cavity volume by the three ligands, three and four explicit water molecules were positioned in the active sites of hCYP11B1 and hCYP11B2 respectively (volumes are hCYP11B1 360\u00a0\u00c53, hCYP11B2 334\u00a0\u00c53, etomidate 269.7\u00a0\u00c53, fadrazole 241.9\u00a0\u00c53, metyrapone 247.7\u00a0\u00c53, water 15.5\u00a0\u00c53). The positioned water molecules were optimised in the equilibration step.\nCellular assay for measuring inhibitor in\u00a0vitro activity\nFor determining inhibitor potencies for CYP11B1 and CYP11B2, an assay was used in which cells overexpressing CYP11B1 or CYP11B2 convert the used steroid substrates for these cytochromes into their products. Product formation rates were assessed by HPLC. V79 cells overexpressing CYP11B1 (stably transfected with a pcDNA3.1 vector, carrying a hygromycin resistance box) were constructed at NV Organon (Oss, the Netherlands). These cells were constructed in the following manner. The full length cDNA for human CYP11B1 was obtained by PCR from human adrenal cDNA as described by Kawamoto et\u00a0al [61] and cloned into pPCR SCRIPT (Stratagene, La Jolla, USA). After digestion with Xho I and Not I, the obtained cDNA was inserted into a Xho I\/Not I digested pcDNA3.1 vector (InVitrogen, Breda, Netherlands), carrying a hygromycin resistance box. Following transfection to the V79 cells, positive cells were selected based on their hygromycin resistance. The presence of the CYP11B1 gene was confirmed by PCR and the presence of 11\u03b2-hydroxylase activity (i.e., the ability to produce corticosterone or cortisol from 11-deoxycorticosterone or 11-deoxycortisol respectively). Although no electron-transporting proteins were co-transfected, the CYP11B1 expressing V79 cells showed abundant 11\u03b2-hydroxylase activity, as was previously also shown by Denner et\u00a0al. for both CYP11B1 and CYP11B2 [62]. In normal (non transfected) V79 cells, no detectable 11\u03b2-hydroxylase activity or PCR signals were found. V79 cells stably (over)expressing CYP11B2 were developed in the laboratory of Prof. R. Bernhardt, Institute of Biochemistry, Saarland University, Saarbr\u00fccken, Germany [62\u201364].\nCells were cultured under standard conditions in DMEM\/FK12 medium (Gibco, Gaitersburg, USA) supplemented with 10% foetal calf serum (Hyclone, Logan, USA), penicillin\/streptomycin (100\u00a0U\/mL and 100\u00a0\u03bcg\/mL, respectively, Gibco), inside a humid environment of 37\u00a0\u00b0C, and 5%CO2 atmosphere. For assessing inhibitor potencies, cells were transferred to 12 well plates and grown until they were confluent. Next, cells were incubated for 1\u00a0h in serum-free medium with cumulative inhibitor concentrations, followed by addition of 500\u00a0nM (0.5\u00a0times Km) 11-deoxycorticosterone (Sigma-Aldrich, St Louis, USA) as substrate. Finally, V79 CYP11B1 cells were incubated for another 1\u00a0h and V79 CYP11B2 cells for another 3\u00a0h. Medium was removed and the (produced) steroids were extracted with 5\u00a0mL diethylether from 1\u00a0mL medium aliquots, mixed with 1\u00a0mL 1\u00a0M sodium-glycine buffer (pH 10.5) containing 500\u00a0nM methylprednisolone (Sigma-Aldrich) as internal standard. Extracts were dried under nitrogen and dissolved in mobile phase for automated HPLC analysis, using a stationary phase consisting of an MR column (4.6\u00a0\u00d7\u00a050\u00a0mm, particle size 2.5\u00a0\u03bcm) and a mobile phase consisting of a mixture of 680\/320\/1 (v\/v\/v) milliQ water, acetonitrile and trifluoro-acetic acid. Detection of UV absorption was performed at 243\u00a0nm (Shimadzu, Tokyo, Japan).\nResults and discussion\nModel quality\nOne of the theorems often applied to the quality assessment of a protein model is the Ramachandran Plot, which is an indicative measure for the correctness of the residue torsion angles. The plot is a graphic display of torsion angle \u03c6 (Cn\u22121\u2013Nn\u2013C\u03b1,n\u2013Cn) versus torsion angle \u03c8 (Nn\u2013C\u03b1,n\u2013Cn\u2013Nn+1) for each residue of the protein of which the secondary structure character of the residue can be extracted. The alpha-helix character of a protein backbone is located roughly in the region where \u221260\u00b0\u00a0<\u00a0\u03c6\u00a0<\u00a0\u221230\u00b0 and \u2212120\u00b0\u00a0<\u00a0\u03c8\u00a0<\u00a0\u221230\u00b0, and the beta-sheet character is located roughly in the region where \u2212180\u00b0\u00a0<\u00a0\u03c6\u00a0<\u00a0\u221260\u00b0 and 90\u00b0\u00a0<\u00a0\u03c8\u00a0<\u00a0180\u00b0 [65]. In Table\u00a02, the results of the Ramachandran Plot are summarised for both hCYP11B1 and hCYP11B2 models after they were equilibrated with the ligand 18-hydroxycorticosterone.\nTable\u00a02Validation results for the lowest energy models of CYP11B1 and CYP11B2 and the crystal structures which were used for the template, part IRamachandran Plot (core regions) (%)Ramachandran Plot (favourable regions) (%)Errat2 (quality factor, %)aVerify 3D (total score)bTemplate (PDB, resolution)CYP101 (2CPP, 1.63\u00a0\u00c5)92.1100.096.0197CYP2C5 (1NR6, 2.10\u00a0\u00c5)87.899.293.6195ModelhCYP11B178.894.784.1126hCYP11B278.794.787.5125hCYP11B2-TripMut80.696.581.1117rCYP11B179.796.580.2113rCYP11B282.496.580.1114a\u00a0\u00a0Outliers are positioned in the loop regions before alpha-helix F where the two template structures connect and at the end of alpha-helix D where a large insertion was introducedb\u00a0\u00a0Additional outliers are positioned at the end of alpha-helix G\nFor both hCYP11B1 and hCYP11B2 around 95% of the residues are positioned in the favoured and core regions of the Ramachandran Plot, indicating that for hybrid models, the structures are of acceptable quality. Due to the high quantity of alpha-helices and beta-sheets, the majority of residues is positioned in the expected regions. The residues which are situated in disallowed and unfavoured regions of the plot, are located in loop regions outside the active site. In total, 9 residues in the hCYP11B1 model are situated in the disallowed regions and 15 residues in the unfavoured regions. For the hCYP11B2 model, 10 residues are situated in disallowed regions and 14 residues in the unfavoured regions. The causes for these disparities are several insertions or deletions introduced in the models for which the structural minimisation was not sufficiently adequate to correct the backbone dihedrals. In particular, these regions are a relatively large insertion between alpha-helix D and beta-sheet 3-1, and an insertion between helix G and H.\nAdditionally, the amino acid environment of the models was evaluated with Errat [66] and Verify3D [67] (Table\u00a02). By comparing the results, it can be seen that the models all score equally well but are less accurate than the template structures. An Errat quality factor of 95% is expected for crystal structures resolved at a resolution of 2.5\u00a0\u00c5. Using the Errat score per amino acid we were able to locate the deviations in the loop regions at the end of helix E where our two template structures connect and at the end of helix D where a large insertion was introduced. It is likely that in the minimisation protocol with tethered heavy atoms, the connectivity between template structures has not been fully optimised. Investigation of the amino acid environment with Verify3D resulted in similar conclusions as found with Errat and the Ramachandran data. The low scoring of Verify3D can be attributed to a bad folding of the regions around the helices D and F, and additional potential errors were located at the end of helix G, where again the two templates have been connected to each other.\nFinally, the stereochemistry of the models was analysed with MOE (Table\u00a03 [68]). Except for the already determined structural flaws in the regions around helices D, F and G, no additional flaws were found using these measurements, indicating that the overall fold of the protein is of acceptable quality.\nTable\u00a03Validation results for the lowest energy models of CYP11B1 and CYP11B2 and the crystal structures which were used for the template, part IIMOE-protein reportObserved CYP101 (2CPP)Observed CYP2C5 (1NR6)Observed CYP11B1 modelObserved CYP11B2 modelReference valuesaParameterMeanS.D.MeanS.D.MeanS.D.MeanS.D.MeanS.D.Trans-Omega176.62.7178.90.9172.07.7172.07.7180.05.8C-alpha chirality32.83.634.31.730.811.130.810.833.84.2Chi1-gauche minus\u221263.017.4\u221263.314.8\u221262.321.7\u221262.622.1\u221266.715.0Chi1-gauche plus55.420.756.316.451.526.753.028.364.115.7Chi1-trans185.313.3184.212.9186.321.9186.820.3183.616.8Helix phi\u221265.211.9\u221267.415.5\u221260.719.7\u221261.119.8\u221265.311.9Helix psi\u221241.216.5\u221237.617.9\u221242.525.1\u221241.925.2\u221239.411.3Chi1-pooled S.D.15.513.822.521.615.7Proline phi\u221265.811.3\u221261.99.5\u221265.319.6\u221267.620.5\u221265.411.2Dihedral outliers041517Bond angle outliers0034Bond length outliers0001Results were generated with the MOE module: protein Eval. The thresholds were chosen to be 5 for the Z-Score and 70 for the vanderWaals contactsa\u00a0\u00a0Reference values were published in a statistical survey of the high-resolution data in the Protein Data Bank [68]\nNone of the structural flaws are located at residues in the active site or at residues lining the active site. The occurrence of Ramachandran errors and problems with modelling external loop regions seems an inevitable circumstance in homology modelling [69, 70] and will probably not pose a problem for modelling protein-ligand interactions. All the abovementioned errors are expected to be alleviated during further investigation of protein-ligand interactions using molecular dynamics simulations. Therefore the current models have been selected as appropriate starting points for further analysis.\nProtein-substrate interactions\nAfter quality assessment of the created protein models encompassing the various ligands, we investigated the steroid binding mode. All docking results from GOLD favoured the \u03b2-side of the steroid oriented to the heme with the C3-carbonyl pointing towards alpha-helix B\u2032 (Fig.\u00a04). Visual inspection revealed that in case of reverse orientation of the steroid, unfavourable clashes of the C3-carbonyl in the opposite side of the pocket would occur. After docking the steroids into the active site, the protein\u2013ligand complexes were subjected to MOE minimisation as described in the homology modelling section. After minimisation, the distance between the heme iron atom and the different substrate hydroxylation sites (C11, C18 and C19) was measured. The results are summarised in Table\u00a04.\nFig.\u00a04Hypothetical binding of 18-hydroxycorticosterone (18OH-B) inside the CYP11B2 active site for the synthesis of aldosterone. The heme prosthetic group contains a bound oxygen atom needed for catalytic function. The CYP11B2 model possesses Arg123 in alpha-helix B\u2032 which is stabilised by Glu310 in alpha-helix I. 18OH-B possesses several hydrogen bonds: one internal hydrogen bond between the C18-hydroxyl and the C20-carbonyl, two hydrogen bonds between the C21-hydroxyl and the backbone carbonyls of Gly379 and Phe381, and finally a hydrogen bond between the C3-carbonyl and Arg123Table\u00a04Hydroxylation distance table (iron atom\u2013carbon atom) after minimisation with MOE (distances in Angstrom)hCYP11B2hCYP11B2-TripMutHCYP11B1rCYP11B1rCYP11B2C11C18C19C11C18C19C11C18C19C11C18C19C11C18C19DOC4.724.305.614.374.655.324.304.565.484.304.754.834.704.245.5418OH-DOC4.334.305.42a4.314.515.21b4.314.605.19b4.304.685.17b4.324.315.39aB5.394.065.465.374.405.225.434.395.285.334.494.945.284.215.2018OH-B4.864.215.50a5.424.645.29c5.384.625.26d5.474.625.28d5.294.355.29aa\u00a0\u00a0Ligand C18-hydroxyl group forms a hydrogen bond with the C20-ketone group of the ligandb\u00a0\u00a0Ligand C18-hydroxyl group forms a hydrogen bond with the iron-oxygen of the proteinc\u00a0\u00a0Ligand C18-hydroxyl group forms a hydrogen bond with the C11-hydroxyl group of the ligandd\u00a0\u00a0Ligand C11-hydroxyl group forms a hydrogen bond with the C18-hydroxyl group of the ligand\nAll ligands showed two very distinct interactions in the modelled active site cavities. Firstly, the ligands possess a steric fit for the C20-carbonyl and the C21-hydroxyl in a small cavity created between helix K and beta-sheet 6-1. Inside this cavity, the C21-hydroxyl group possesses two hydrogen bonding interactions with the protein backbones of Gly379 and Phe381 (Fig.\u00a04). The presence of these amino acids in the active site cavity coincides with the models of Belkina et\u00a0al., but for those models, no interactions between protein and ligand were discussed [34].\nSecondly, the ligands possess an interaction between the C3-carbonyl and active site residue Arg123 in helix B\u2032. Arg123 is stabilised by Glu310 in helix I, which has further stabilising interactions with the protein backbone. Glu310 coincides with an aspartic acid in the CYP2 family which from visual inspection of the crystal structures of CYP2C5 (pdb-code 1NR6) and CYP2C9 (pdb-code 1OG5) seems to play a specific stabilising role in the active site structure; it is expected that Glu310 has the same stabilising role. The difference in chain length between an aspartic acid and a glutamic acid also determines the flexibility of helix B\u2032. It can move 1.5\u00a0\u00c5 further out of the active site cavity in all CYP11B models allowing the steroid to fit parallel to the heme. The ligands also possess many hydrophobic interactions in this region, particularly with Phe130 (Fig.\u00a04). Due to the close interactions with the A and B rings of the steroid skeleton, this amino acid might play an important role in substrate stabilisation. The presence of Phe130 in our models coincides with the models of Ulmschneider et\u00a0al. [35] where it is seemingly involved in ring stacking with their inhibitors.\nThere are also two striking differences between the CYP11B1 and CYP11B2 models. Firstly, the active site cavity near beta-sheet 6-1 is smaller in both CYP11B2 models compared to the CYP11B1 models, with rCYP11B1 being the largest. This small cavity is formed by the loop region between helix K and beta-sheet 6-1 (Fig.\u00a05). Comparing this cavity in both the hCYP11B1 and hCYP11B2 models, we find that in hCYP11B1 this cavity has a 9\u201314% larger volume, dependent on amino acid flexibility as well as the probe radius chosen for calculation of the volume (differing from 1.0 to 1.5\u00a0\u00c5). This difference in size is caused by the folding of Leu407 which induces a 1.0\u00a0\u00c5 outward shift of the loop region. A comparison of the models with the crystal structures of the CYP2 family reveals that the loop region in the CYP11B models is in closer proximity to helix I (by 1.5\u00a0\u00c5). This contact is defined by two amino acids having relatively smaller sidechains (Pro322 and Val378 in CYP11B) than observed in the CYP2C family (generally Thr\/Val and Leu\/Ile). Changes in helix I such as the mutant A320V between hCYP11B1 and hCYP11B2 are likely to have a direct influence on the folding of this region.\nFig.\u00a05Active site volume difference between the hCYP11B1 (white) and hCYP11B2 models (orange). Shown for both hCYP11B1 and hCYP11B2 are the active site volume and the backbone trace. R-fadrazole and S-fadrazole are indicated with blue and purple respectively. It is clear to see that hCYP11B1 contains a larger active site between helix I and sheet 6-1. This cavity allows S-fadrazole to fit the cavity, but not R-fadrazole (black arrow). On the other side of the active site near Helix B\u2032 and Arg123, hCYP11B2 contains the larger cavity, which might rationalise the better fit of R-fadrazole in the cavity\nSecondly, helix B\u2032 is shifted outward by 1.1\u00a0\u00c5 in both CYP11B2 models (the backbone RMSD of helix B\u2032 of hCYP11B1 and hCYP11B2 is 2.13\u00a0\u00c5). The change in spatial positioning of this helix is probably caused by the mutants L301P and E302D in helix I, but because this region is on the surface of the protein, the exact cause is less clear. In the hCYP11B2 model, Glu302 contacts helix B\u2032 at Lys127, which forms the counter charge for the C-terminal end of this helix. Hasemann et\u00a0al have posed that movement of helices B\u2032, F and G is involved in the opening of the active site cavity [71]. Changing the stabilising environment of helix B\u2032 has a likely effect on the structural stability of the helix and may result in a loss of activity.\nIntroducing the triple mutant investigated by Bottner et\u00a0al. [36] by means of the hCYP11B2-TripMut model showed that the hydroxylation distance pattern of the substrates shifts as expected from hCYP11B2 to that of hCYP11B1. Both hCYP11B1 and hCYP11B2-TM display similar active site cavities near beta-sheet 6-1 (backbone RMSD 1.43\u00a0\u00c5) with only slight deviations in the hydroxylation distances for the ligands (Table\u00a04). This confirms the behaviour of the triple mutant in enzymatic activity found by the study of Bottner et\u00a0al. To speculate further, Bottner et\u00a0al have shown that the A320V mutant alone or the L301P\/E302D mutant alone, is not enough to significantly change aldosterone synthesis [36]. Only mutation in both regions results in an almost complete loss of the aldosterone synthesis capabilities of hCYP11B2. It is likely that the subtle changes on both sides of the active site go hand-in-hand to (nearly) completely convert the activity of hCYP11B2 into that of hCYP11B1.\nFor DOC, our results indicate that hCYP11B1 and rCYP11B1 preferentially catalyse C11-hydroxylation, whereas hCYP11B2 and rCYP11B2 preferentially catalyse C18-hydroxylation. For both CYP11B2 models, the shifted alpha-helix B\u2032 causes DOC to present its C18 closest to the iron as it is repositioned by the strong interaction of the C3-carbonyl group with Arg123 in alpha-helix B\u2032. Additionally, the larger active site of rCYP11B1 around beta-sheet 6-1 allows DOC to fit further into the niche presenting C19 into hydroxylation range (distance less than 5.0\u00a0\u00c5). A point of argument against a preferred C18-hydroxylation of DOC by CYP11B2 is that in\u00a0vitro measurements indicate higher levels of the C11-hydroxylated product B [19, 21]. However, both C11- and C18-hydroxylated products of DOC can be promptly consumed as a substrate for the production of 18OH-B and subsequently aldosterone, in in\u00a0vitro [19, 21]. This apparent discrepancy between observed and predicted regioselectivity of DOC hydroxylation by CYP11B2 may possibly indicate that other factors than hydroxylation distances are involved in the formation of the actual products.\nFor 18OH-DOC, the C18-hydroxyl group forms a hydrogen bond with the iron\u2013oxygen limiting hydroxylation to C11 which is in closest proximity to the iron. In the CYP11B2 models both C11 and C18 are in approximately equal distance to the iron and an internal hydrogen bond is formed by the substrate between the C18-hydroxyl group and the C20-carbonyl. Although both C11- and C18-hydroxylation are shown to be possible in such a complex, oxidation on the unsubstituted C11 is likely to form a more stable reaction intermediate.\nIn all models, the two C11-hydroxylated ligands B and 18OH-B only portray C18 in close proximity to the iron, as the C11-hydroxyl group blocks access of C11 to the heme iron. The positioning of the C18-hydroxyl group of 18OH-B in the active site cavity appears to determine conversion into aldosterone. In both CYP11B2 models, the natural substrate is shifted above the heme, which creates a slightly larger active site cavity near beta-sheet 6-1 for the C18-hydroxyl group to rotate in. This difference in size allows room for an internal hydrogen bond between the C18-hydroxyl group and the C20-carbonyl for both 18OH-DOC and 18OH-B. In rCYP11B1 and hCYP11B1, the C18-hydroxyl group forms a hydrogen bond with the heme oxygen atom which blocks C18 for hydroxylation (Fig.\u00a04). Interestingly, in the hCYP11B2-TripMut, the C18-hydroxyl group forms an internal hydrogen bond with the C11-hydroxyl group, and thereby also blocks the C18 for hydroxylation.\nIn conclusion, we propose that the immediate folding of the active site around the substrate C18-hydroxyl group may be the key difference between the two isoforms leading to the production of aldosterone by CYP11B2 and not by CYP11B1. To speculate further on the mechanism of the enzymatic formation of aldosterone from 18OH-B, we hypothesise that a stable internal hydrogen bond for the C18-hydroxyl group of the ligand may be essential to stabilise its C18 against further oxidation. A second oxygen atom can then be inserted between C18 and one of its hydrogen atoms, forming a C18-gem-diol intermediate which forms aldosterone by elimination of water. Although the formation of a C18-gem-diol has been proposed by Johnston et\u00a0al. [72], the precise mechanism of oxidation is still unknown. This proposed method of substrate stabilisation may explain the regioselectivity of the two CYP11B isoforms and can be attributed to the subtle changes observed on two sides of the active site cavity; the cavity near helix K and the positioning of helix B\u2032.\nProtein-inhibitor interactions\nThe non-steroidal inhibitors, metyrapone, R-etomidate, R-fadrazole and S-fadrazole were docked flexibly into the active site of the human CYP11B models where their aromatic nitrogen atom forms a strong interaction with the sixth ligating position of the heme iron atom. The accessible electron lone pair of the heterocyclic nitrogen atom is required for heme iron complexation, a well-known interaction for non-steroidal CYP inhibitors [73, 74].\nThe GOLD docking results showed all ligands to bind in one favourable conformation. The enantiomers R-fadrazole and S-fadrazole mainly favoured one particular orientation in the active site cavities of hCYP11B2 and hCYP11B1 respectively. The higher affinity of S-fadrazole to hCYP11B1 and R-fadrazole to hCYP11B2 can be attributed to the active site difference near beta-sheet 6-1 and helix I (Fig.\u00a05). The difference in affinity is determined by the steric aspects of the active site cavity allowing only S-fadrazole to fit in the active site of hCYP11B1, where R-fadrazole possesses a steric clash (black arrow). In hCYP11B2, it is less clear why R-fadrazole fits the active site best, but this is mainly determined by steric effects near helix B\u2032. Due to the presence of rotatable bonds, R-etomidate was able to dock in different orientations, but favoured one particular conformation with its phenyl ring pointing into the direction of helix B\u2032. The main flexibility observed was caused by the ethyl-ester group and the docking score of those conformations was very similar. Metyrapone was able to dock with both pyridine rings to the iron, but mainly favoured a conformation with the carbonyl facing Arg110. Since all ligands favoured only one orientation, it was this orientation that was investigated by MD.\nOur in\u00a0vitro test results show a striking enantioselectivity of fadrazole binding for the CYP11B family. We find R-fadrazole to be the most active enantiomer for CYP11B2 as well as being selective (Table\u00a05). In contrast, for CYP11B1 it is S-fadrazole which appears to be the most potent enantiomer. A similar stereoselectivity as for CYP11B1 has also been described for aromatase, wwith the S-enantiomer being the better aromatase inhibitor [75]. The docking results from both Goldscore and Chemscore predict the same enantioselectivity for fadrazole in the CYP11B family as observed in\u00a0vitro, indicating that the three dimensional models contain promising accuracy for the valid prediction of enantiomer selectivity. Docking results of the two other inhibitors metyrapone and R-etomidate confirm the trend of inhibitor potency as determined by the in\u00a0vitro experiments (Table\u00a05).\nTable\u00a05Correlation of docking and molecular dynamics results to in\u00a0vitro data for both human CYP11B1 and CYP11B2 modelsIC50 (nM)Goldscore\u0394G Goldscore (kcal\/mol)\u0394G Chemscore (kcal\/mol)Unon-bonded (kcal\/mol)hCYP11B1Metyrapone46.4\u00a0\u00b1\u00a010.457.33\u22128.43\u22128.73\u221248.4\u00a0\u00b1\u00a03.7R-Etomidate0.5\u00a0\u00b1\u00a00.266.21\u22129.38\u22129.25\u221256.0\u00a0\u00b1\u00a02.4R-Fadrazole118.6\u00a0\u00b1\u00a08.954.01\u22128.07\u22128.14\u221238.4\u00a0\u00b1\u00a02.5S-Fadrazole39.5\u00a0\u00b1\u00a04.456.67\u22128.36\u22128.77\u221256.3\u00a0\u00b1\u00a03.4hCYP11B2Metyrapone207.8\u00a0\u00b1\u00a04.549.99\u22127.64\u22127.95\u221236.2\u00a0\u00b1\u00a07.5R-Etomidate1.7\u00a0\u00b1\u00a00.965.21\u22129.28\u22129.21\u221254.4\u00a0\u00b1\u00a02.9R-Fadrazole6.0\u00a0\u00b1\u00a01.963.20\u22129.06\u22129.38\u221255.9\u00a0\u00b1\u00a03.3S-Fadrazole171.2\u00a0\u00b1\u00a051.753.81\u22128.05\u22128.12\u221244.3\u00a0\u00b1\u00a01.8Indicated are the Goldscore and the extracted Goldscore binding free energy as well as the Chemscore binding free energy. Unon-bonded indicates the total non-bonded energies between the protein and the ligand for the molecular dynamics simulations\nOur human CYP11B1 and CYP11B2 models are able to rationalise the (inverse) enantioselectivity of CYP11B1 and CYP11B2 inhibition by fadrazole as observed in the in\u00a0vitro tests. The enantioselectivity can be attributed to the small cavity size differences of the models. Both enantiomers bind with a steric fit comparable to the A, B and C rings of the endogenous substrates, where the cyanobenzyl moiety forms a \u03c0\u2013\u03c0 stacking interaction with Phe130. In addition to the steric interactions, both enantiomers possess a strong polar interaction between their cyanide moiety and Arg123, mimicking the interaction that the C3-carbonyl of the natural substrates possesses with this amino acid.\nMetyrapone was not found to possess any hydrogen bonding or polar interaction with the active site other than the aromatic nitrogen. Instead it possesses a pronounced steric fit with its second aromatic ring overlapping the space occupied by the A-ring in case of the steroidal substrate. R-etomidate, which was not designed as a CYP11B specific inhibitor, does possess a hydrogen bonding interaction. The hydrogen bond is made between its ester moiety and the catalytic Thr318, and is present in both hCYP11B1 and hCYP11B2. This may explain the non-selective inhibitory action of R-etomidate on both isoforms. Its aromatic phenyl ring also possesses strong steric interactions, overlapping the same space of the steroidal A-ring like metyrapone.\nMolecular dynamics\nGiven the promising prediction of the models for substrate conversion, substrate binding, inhibitor binding and the satisfying Ramachandran Plots, we conducted molecular dynamics simulations to obtain a better understanding of the dynamic behaviour of the inhibitors in the active site of the enzyme models. To investigate the structural integrity change during the simulation, we calculated the relative root mean square deviation (RMSD) over all heavy atoms (Fig.\u00a06).\nFig.\u00a06RMSD for the molecular dynamics simulations of the different protein-ligand complexes of the hCYP11B1 and hCYP11B2 models. Metyrapone is coloured black, R-etomidate green, R-fadrazole red, and S-fadrazole blue\nDuring the first 500\u00a0ps, the RMSD increased and the protein still adapted towards its optimal conformation. After this point in time hardly any change in the three dimensional structures of the proteins was observed. The largest fluctuations of the protein were found in the flexible regions with peak values located in the structures around alpha-helix D (not shown). In the random coil following alpha-helix D we introduced a large insertion of seven amino acids, which elongates the alpha-helix by one turn before it connects to the following beta-sheet. Inside the water box, this region is found to protrude into the water without any stabilising protein interactions and unfolds due to interaction with water. In all the simulations we observed an opening of the active site and the continuous flow of water molecules in and out of the active site cavity. Several water molecules retained key positions, such as the water molecules that make up the channel towards the conserved Glu459 (not shown).\nAll the inhibitors maintained the same interactions as observed in the docking study. In Fig.\u00a07, the resulting poses of the inhibitors metyrapone and R-etomidate are displayed. Both possess a ring stacking with Phe130 in the active site, and R-etomidate also possesses a hydrogen bond with Thr318. Arg110 stabilises the heme in the active site. In Fig.\u00a08, the poses of R-fadrazole in the CYP11B2 active site and S-fadrazole in the CYP11B1 active site are compared. Here it can be seen that in the CYP11B2 model, alpha-helix B\u2032 is moved further out of the active site. In the CYP11B2 model, Arg123 possesses a polar interaction with Glu310, whereas Phe130 provides a horizontal ring stacking for R-fadrazole. In the CYP11B1 model, S-fadrazole possesses the same interactions with the protein, although the ring stacking with Phe130 is vertical rather than horizontal. Importantly, the interaction points of the fadrazole enantiomers and those of the natural substrate coincide very well.\nFig.\u00a07CYP11B2 active site model with metyrapone (purple) and R-etomidate (blue) in the active site. Indicated are the stabilising interaction between Arg110 and the heme, as well as Phe130 which accommodates a ring stacking with the ligands. The ester group of R-etomidate possesses a hydrogen bond with Thr318Fig.\u00a08CYP11B1 model (orange) containing S-fadrazole (purple) in the active site and CYP11B2 model (white) containing R-fadrazole (blue) in the active site. Indicated, the stabilising interaction between Arg110 and the heme, as well as the stabilising hydrogen bonds between Arg123 and Glu310. Phe130 accommodates a ring stacking with the fadrazole enantiomers; horizontally for the R-enantiomer and vertically for the S-enantiomer\nR-fadrazole and S-fadrazole displayed different behaviour in the other protein models. R-fadrazole lost its direct interaction with the heme iron atom in the hCYP11B1 model, exchanged it for a water molecule and drifted to the top of the active site. S-fadrazole displayed a similar behaviour in the hCYP11B2 model. Repeated simulations did not change either scenario, indicating that unlike the eutomers, the distomers do not comfortably fit our starting models.\nBecause the complex was stable over the last 500 ps, we sampled the non-bonded interactions between the protein and the ligand for this time period (Table\u00a05). These interactions contribute to the binding free energy, although the solvation effects of free ligand still need to be subtracted [76]. These energies follow the same generic trend which emphasise the strong interactions for R-etomidate in both hCYP11B models and the strong interactions of S-fadrazole in hCYP11B1 and R-fadrazole in hCYP11B2 respectively.\nConclusion\nWe have constructed homology models of the two isoforms of the human CYP11B family, as well as promising starting models for the rat isoforms. These models are based on the knowledge of substrate specificity, which were defined as the differences in hydroxylation distances between the active site and the C11, C18 and C19 of the steroid skeleton. As both hCYP11B1 and hCYP11B2 possess virtually similar active sites, the steric fit of the different steroidal ligands seems to be one of the strongest determinants for substrate specificity.\nWe have found that within the active site of the hCYP11B2 model, the endogenous ligand 18-hydroxycorticosterone forms a stabilising internal hydrogen bond. This is not observed for the hCYP11B1 model, which might rationalise why 18-hydroxycorticosterone is solely a substrate for CYP11B2 to yield aldosterone. In addition, the interactions of the C3-carbonyl with Arg123 in alpha-helix B\u2032, the interaction of the C21-hydroxyl with the backbones of Gly379 and Phe381, and the presence of Phe130 are also important for the stabilisation of the ligand in the protein active site. Determinant for the interaction of Arg123 is the presence of Glu310 in alpha-helix I, which stabilises Arg123 and alpha-helix B\u2032 in the active site.\nThe known non-steroidal CYP11B inhibitors metyrapone, R-etomidate, R-fadrazole and S-fadrazole were postulated to occupy the same space in the active site as the endogenous substrates. Conclusions from both molecular docking and molecular dynamics simulations corroborate the measured activity data from in\u00a0vitro experiments, supporting the validity of the constructed models for these ligands. Importantly, these models rationalise the enantioselectivity of fadrazole, with the R-enantiomer being most potent on CYP11B2 and the S-enantiomer being most potent in CYP11B1.\nThe constructed models are useful tools in trying to understand some of the molecular mechanisms involved in ligand binding and substrate conversion for the CYP11B family. As such, these models might also be appropriate tools for more detailed protein-inhibitor modelling studies as well as for ligand design or database screening, following further model optimisation and model tuning.","keyphrases":["cyp11b2","cyp11b1","fadrazole","molecular docking","molecular dynamics","aldosterone synthesis","homology modelling"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Crit_Care-8-1-420054","title":"Clinical review: Outreach \u2013 a strategy for improving the care of the acutely ill hospitalized patient\n","text":"We examined the literature relating to the safe care of acutely ill hospitalized patients, and found that there are substantial opportunities for improvement. Recent research suggests substantial benefit may be obtained by systems of outreach care that facilitate better integration, co-ordination, collaboration and continuity of multidisciplinary care. Herein we review the various approaches that are being adopted, and suggest the need for continuing evaluation of these systems as they are introduced into different health care systems.\nIntroduction\nHealth care providers in many countries are increasingly aware that quality of care can be improved by measures to reduce errors. Evidence suggests that the greatest opportunities for improvement may be in the management of the acutely ill hospitalized patient, where uncertainty, urgency and lack of integration substantially increase the risk of errors leadinf to adverse outcomes. Different approaches to this problem have evolved, including hospitalists in the USA, medical emergency teams in Australia, and outreach care in the UK. Critical care has a central role to play in all three developments, which share the common aim of improving the safe care of acutely ill patients as they travel through the health care system. We will review the background, methods, roles and benefits of these various systems which we group under the heading of 'outreach' care.\nMethod\nWe focused on identifying research publications that examined ways of improving the integration of critical care with acute care services as a means of improving the safe care of acutely ill hospitalized patients. Publications were initially identified by an electronic search of Medline and Cinahl, and the cited references provided additional material. The initial date range searched was 1995\u20132003 to ensure that current research and up to date literature was reviewed. However, this produced only a limited number of references, and the time period was therefore extended to 1990. Keywords searched singularly and in combination were 'acute pain team', 'suboptimal care', 'patient at risk', 'critical illness' and 'cardiopulmonary resuscitation'. Only English language references were included in the analysis.\nBackground\nTrends in hospital care\nHospitals are becoming increasingly complex environments because of developments in medical technology, more potent treatments, and an ageing and dependent population of patients. The proportion of emergency admissions continues to rise in most countries [1], whereas the stock of hospital beds has fallen. Combined with political demands for cost containment, this has resulted in shorter hospital stays, and increased bed occupancy and throughput [2]. At the same time there has been a trend toward greater transparency in decision making, rising public expectations, and improved public access to information about quality of care and outcomes from medical interventions. These changes have coincided with increasing difficulty with the recruitment and retention of trained nurses [3], and constraints on doctors' hours of work. The implementation of the European Working Time Directive [4], and similar trends in the USA [5], means that proportionately fewer staff are available to manage this increased workload, particularly the out-of-hours and emergency elements. Shorter training times reduce the expertise acquired through apprenticeship. The net effect is that sicker patients are receiving care from fewer and less experienced staff.\nError and quality in health care\nThese pressures have been accompanied by a growing awareness of the problem of error in health care and an exponential increase in litigation. The much quoted Institute of Medicine report 'To Err is Human' [6] estimated that between 44 000 and 98 000 patients die each year in the USA as a result of preventable clinical errors. Studies in Australia and the UK suggest that around 10\u201316% of hospitalized patients experience an adverse event related to clinical care, with a mortality rate in these patients of 5\u20138%, and overall financial costs in the range US $4.7\u201329 billion [7-9]. Clinical error is now widely recognized as a systems problem; that is, adverse events affecting individual patients are usually caused by a sequence of events in the macro- and micro-environment that involve deficiencies in the structure and organization of health care, and are not simply a consequence of human error by the responsible clinician who is the terminal link in the chain. To improve safety and quality in health care requires a systems approach in which all participants \u2013 politicians, administrators and health care professionals \u2013 assume responsibility for patient outcomes. This ambitious task requires focusing of efforts on those patients most at risk.\nIdentifying at-risk populations\nAcutely ill patients in general\nThe risk of error and adverse outcomes would appear to be higher in the context of acute and emergency care, for example in emergency departments [7] or in general medical (rather than elective surgical) admissions [8]. The elderly are more susceptible [7]; they are also more likely to be admitted as emergencies and exposed to emergency surgery [10]. Inexperienced clinicians and unsupervised trainees (who often deliver first-line care out-of-hours) have a higher error rate [11,12]. The risk for an adverse event increases by approximately 6% per day for patients admitted with emergency conditions [13], and is much increased in severely ill patients who undergo life-saving invasive interventions [14]. Discontinuities in care [15] created by shift working or poor information transfer contribute to error by failing to recognize trends in a deteriorating patient's condition.\nPostoperative patients\nIn the UK the 1993 National Confidential Enquiry into Perioperative Deaths showed that two thirds of perioperative deaths occurred 3 or more days after surgery when the patient had been returned to the ward. The majority of these deaths were from cardiorespiratory complications, and many were considered preventable by earlier identification and treatment [16]. The 1999 report [17] analyzed deaths within 30 days of a surgical procedure in elderly patients (>90 years), and found that suboptimal fluid management was a major cause of serious postoperative morbidity and mortality in this group. It recommended more accurate monitoring and recording of fluid balance and earlier recognition and correction of problems as a means of reducing the incidence of postoperative complications. The report also identified deficiencies in multidisciplinary care, despite the high incidence of comorbid disease in these elderly patients, and recommended more collaborative working between surgeons, anaesthetists and physicians with expertise in the care of the elderly.\nThis type of audit based on a large observational database is essential for identifying current practice and opportunities for improvement. However, recommendations were based on peer review and data from questionnaires provided by assessors who were unblinded to clinical outcomes, examining only those patients who died \u2013 there are no denominator data.\nCardiopulmonary arrest\nHospitalized patients who undergo cardiopulmonary resuscitation commonly exhibit premonitory signs and symptoms many hours before the cardiac arrest [18-21]. Schein and coworkers [18] studied 64 patients following cardiac arrest, and reported that 84% of vital signs charts showed an acute deterioration in the patient's condition prior to arrest. Franklin and Mathew [20] examined the case notes of 150 consecutive patients who had suffered a cardiac arrest on a general ward, and found documented prior clinical deterioration in 99 (66%). A common finding was the failure of the nurse to notify a physician of deterioration in the patient's condition.\nThe interface with critical care\nCritical illness increases the opportunity for clinical error [22,23], and this is related at least in part to the complexity of diseases, the multiplicity of therapies, frequent invasive interventions and, within the intensive care unit (ICU), the intensity of monitoring and observation, which may paradoxically increase the apparent error rate in this environment simply by improved detection. Iatrogenic complications are a common cause for ICU admission [24], and suboptimal care before referral to intensive care is associated with a markedly increased mortality [25]. Premature discharge from intensive care of patients recovering from critical illness is also associated with a markedly increased hospital mortality [26], supporting the view that organizational aspects of clinical care profoundly influence patient outcomes [27]. Common errors include lack of attention to detail, poor communication, failures of organization, lack of knowledge, failure to appreciate clinical urgency, insufficient supervision and failure to seek advice.\nTreatment limitation decisions, futility, and end of life care\nStudies in the USA and Europe have demonstrated considerable diversity of practice and opportunities for improving autonomy, patient centred decision making, and quality of end-of-life care [28,29]. The Study to Understand Prognosis and Preferences for Outcomes and Risks of Treatment in the USA [29] demonstrated that 31% of the cohort of patients admitted to intensive care would have preferred not to be resuscitated, but that clinicians were aware of this preference in only 47% of the subset and in half it had not been documented. Aarons and Beeching [30] surveyed the use of 'do not resuscitate' orders in a community hospital in the UK and suggested that end-of-life care of dying patients and their families in hospital could be substantially improved in terms of symptom relief, communication and respect for the patients' wishes. They also concluded that poor decision making by health care workers could be improved by education. One consequence of a lack of communication between staff and a reluctance to discuss these difficult issues with patients and families is the inappropriate resuscitation and subsequent admission to the ICU of patients for whom further intervention would be futile and add to the burden of suffering.\nA more appropriate approach to management of acutely ill patients at risk for critical illness or cardiac arrest would be to prevent these complications, or agree treatment goals and limitations, by earlier recognition of simple warning signs. We consider the various approaches that are being adopted below.\nSystems for earlier recognition and management of patients at risk\nThe problems described above require a systems approach to improving the safe care of the acutely ill hospitalized patient. The USA, the UK, Australia, Canada and Denmark are instituting national systems for improving patient safety [31-33]. Within this general framework, three countries have specifically identified the need to improve the care of acutely ill patients. In the USA the Leapfrog Group [34] has recommended that ICUs be managed by intensivists \u2013 doctors specifically accredited in critical care medicine \u2013 while in a parallel development many hospitals are appointing 'specialist generalists' ('hospitalists') to provide inpatient care on the wards [35]. Australia has promoted the establishment of medical emergency teams (METs) led by doctors as an alternative to cardiac arrest teams [36,37]; and the UK has implemented the recommendation of the expert group report 'Comprehensive Critical Care' [38] to establish multidisciplinary outreach care. All these developments are based on the concept of earlier intervention by people with appropriate knowledge and skills in managing acutely ill patients. Is this concept valid, and which model is the best?\nStructures and processes for early intervention\nIn the UK a survey of intensive care facilities found that, in 94% of the units questioned, staff regularly visited wards in response to requests for advice from medical and nursing members of the admitting team [39], demonstrating that this is clearly an important role for intensive care staff. However, there is little information describing the experiences and perspectives of ward based staff who care for acutely ill patients outside the intensive care environment. Gibson [40] found that ward staff often lacked confidence and felt ill prepared to deal with acutely ill unstable patients, and that they experienced increased stress and anxiety. Such emotions are unlikely to enhance staff retention or reduce sickness rates. It therefore makes sense to put in place systems that improve the support not only of sick patients but also of the staff responsible for their care, and that reduce discontinuities in clinical care. The key to this is empowerment through adequate resourcing and training, and simple methods of clinical monitoring. Three models are currently employed: hospitalists in the USA, the MET in Australia, and outreach care in the UK.\nHospitalists\nHospitalists are internists who specialize in acute hospital medicine. This new speciality has appeared in the USA during the past 8 years in response to perceived difficulties with primary care clinicians maintaining continuity of care for their patients admitted to hospital. Currently at around 5000, their numbers are expected to increase rapidly. Hospitalists are usually salaried employees of managed care organizations, which favour their development as a means of reducing costs and duration of hospital stay [41,42]. The training of hospitalists is rooted primarily in internal medicine, but there is no national core curriculum. They do not appear to have responsibility for surgical patients, and therefore cannot be considered generalists in the sense of dealing with all acutely ill patients. The relationship between hospitalists and intensivists has not been defined [43], although there clearly are opportunities for interaction. Hospitalists occupy a role that is close to that of the general physician in UK hospitals, except that the latter group often have a subspeciality.\nThis model is suited to the system of care in the USA, where primary care physicians often have continuing responsibility for inpatient care. It might also be of value to other health care systems in which increasing specialization is creating a need for generalist acute care clinicians in hospital practice.\nAcute pain teams\nA joint expert committee report from a Royal College of Surgeons and College of Anaesthetists working party [44] reviewed the evidence related to postoperative pain management and recommended the establishment of an acute pain service in all major hospitals. However, the Audit Commission reported in 1997 [45] that only 57% of hospitals in the UK had established a pain service. The situation had improved by 2000 when the Clinical Standards Advisory Group found that 88% of responding UK hospitals had set up an acute pain service, although in some cases this was only a token service. The 1990 working party did not describe a model for the ideal acute pain team [44], and this has led to many inconsistencies in the provision of pain management throughout the UK. Acute pain teams ensure adequate postoperative pain relief by supporting and educating ward staff [46]. Concerns have been expressed that they may de-skill ward nurses [47], although there is no research evidence to support this view.\nBecause more complicated surgery is performed in older patients, who have more comorbidities, we can expect to see an increase in the number of life-threatening postoperative complications [16]. Surgical patients make up 60\u201370% of the workload of ICUs in the UK [48,49]. It has been suggested that the role of acute pain teams should be extended to identify nonpain problems in patients, and liaise with other specialties to manage them [46]. Some acute pain services have already extended their role from pain management to include fluid balance, oxygen therapy, management of nausea and vomiting, and anticoagulant prophylaxis [50]. This role extension requires proper training in the management of acutely ill patients and an understanding of the interface between acute medicine and intensive care.\nMedical emergency teams\nThe concept of a better integrated, multidisciplinary approach to postoperative care [51,52] has been extended to other groups of patients in the form of specific groups or teams of clinicians, usually centred on the ICU. Examples of this evolution include postoperative care teams [53], patient at risk teams [54] and METs [36,48,52]. All suggest that the introduction of such teams to identify and manage complications on the ward might prevent unnecessary admissions to the ICU and reduce morbidity and mortality.\nThe team based approach uses calling criteria based on abnormal clinical or physiological variables, specific conditions, or patients causing concern to ward staff. The research evidence to support the validity of these criteria is limited by the practical and ethical difficulties of conducting randomized controlled trials in the clinical environment. Although postoperative care teams provide additional support, expertise and equipment for postoperative patients [54], and similar approaches could improve suboptimal ward care in general [25], there are no a priori definitions of inadequate care that have been prospectively calibrated against outcome in this context. However, pragmatic studies of the impact of METs indicate a reduction in cardiac arrest rates and in the use of intensive care resources for cardiac arrest survivors [55,56].\nCritical care outreach\n'Outreach' care is a systems approach for identifying and managing patients at risk of critical illness through collaborative care and education. Rather than providing a service through an external group, it aims to empower ward staff by offering them regular support, usually led by critical care trained nurses visiting the wards, with the facility to call on more expert assistance if required. Currently, critical care is seen as occurring within a defined environment (the ICU) and patients must achieve a certain level of severity of illness to merit admission. Outreach services facilitate a more flexible approach that is based on the needs of the patients and the skills and abilities of the ward staff.\nBoth the Audit Commission [57] and the Department of Health working party report 'Comprehensive Critical Care' [55] supported the development of outreach care as a means of improving the care of acutely ill patients in hospital wards. The latter report identified three goals for a critical care outreach team. The first was to identify patients at risk for critical illness and either prevent their admission to ICU by timely interventions at a ward level or ensure early appropriate admission to ICU. The second was to facilitate timely and safe discharge from intensive care by following up patients discharged to the ward. The third was to share ICU skills with ward staff. Given the wide variations in hospital size, specialties, staff expertise and skills, the Department of Health report did not prescribe a standard structure for achieving these three goals. The development of outreach services should not be taken in isolation from other critical care initiatives and should be part of an integrated, multidisciplinary, hospital-wide delivery of critical care services that improves liaison between intensive, high dependency and ward care [56].\nThe majority of UK centres have nurse led outreach systems, supported by critical care doctors with sessional recognition for this service. However, the aim of outreach is to diffuse skills across many disciplines, and to enhance collaborative care. Many professional groups can therefore contribute to this approach, including physiotherapists and nutritionists as well as physicians. Studies are needed to evaluate the efficacy of this development, but current experience indicates a strongly favourable response from ward based staff. Standards for the development of outreach care are now available [58].\nIdentifying the patient at risk \u2013 scoring systems for decision support\nTraditionally, the process of identifying critically ill and deteriorating patients has relied on the clinical intuition of staff. The value of experienced clinical judgement is well recognized [36,54,59,60], but given the trend toward shorter training times and reduced hours of work in the clinical environment, objective systems are becoming increasingly important because inexperienced practitioners may fail to recognize impending critical illness and the need for assistance [61].\nThere are several such systems that utilize combinations of physiological variables as indicators of risk [36,54,56,62-66]. A summary of component variables is given in Table 1. Hodgetts and coworkers [67] analyzed factors that predicted risk for cardiac arrest and could therefore be used as triggers to call for help ('activation criteria'). Variables with predictive capacity included chest pain, staff concern, systolic blood pressure, oximetry, pulse and respiratory rate, and temperature. The choice of vital signs is necessarily constrained to those that are easy to record in the ward environment. More finesse may be achieved with the inclusion of laboratory tests such as serum electrolytes, blood sugar and acid-base analysis, or the inclusion of diagnosis, but the problem with this approach is that recording vital signs is often omitted or inaccurate [17,54], and the greater the degree of complexity, the more likely it is that errors or omissions will occur. Scoring systems for use in ordinary wards must be simple, and should direct attention to patients who need more intensive observation; they should be a trigger for investigation, not a precise tool for predicting individual patient outcomes. Thus, although physiological abnormalities may be unreliable predictors of the need for intensive care admission, they remain important stimuli for empowering staff to call for assistance in improving simple aspects of care of acutely ill patients.\nDoes earlier intervention improve outcome?\nIt may seem unnecessarily argumentative to suggest that something that is so evidently 'good' requires evaluation. Is it not self-evident that the earlier a life-threatening disease process is identified and treated, the better? This may be so, but complex systems \u2013 of which the acutely ill patient is one example \u2013 demonstrate sensitivity to initial conditions; that is, the outcome may be more difficult to predict with earlier application of an intervention. The outcome from cardiopulmonary resuscitation is well defined \u2013 a survival rate of around 10\u201315%, with death usually occurring when resuscitation attempts are discontinued. Is it not possible that earlier intervention that prevents cardiac arrest might result in delayed death following prolonged organ system support in the ICU, with the attendant suffering that this may cause? What is the cost\u2013benefit of implementing the different models of care? Who will manage the process of discussing treatment goals or limitations with patients, and what do patients themselves feel about it?\nPhysiological goals\nThe literature relating to preoperative optimization of systemic oxygen delivery in high-risk surgical patients suggests that early intervention is beneficial [68-72]. However, a recent large-scale study [73] conducted in surgical patients did not confirm this in terms of benefit from goal-directed therapy guided by pulmonary artery catheterization, perhaps because patients in the control group were already being optimally managed (and experienced a low overall mortality) as a result of improvements in clinical practice derived from earlier research.\nIn critically ill septic patients, early intervention to optimize oxygen delivery with fluid resuscitation, vasoactive drugs and respiratory support appears to reduce mortality [74], whereas similar interventions applied later in the course of illness do not [75-77]. It seems reasonable to conclude that using fluids and supplemental oxygen to optimize circulating volume, cardiac output and systemic oxygen delivery as early as possible in acutely ill hospitalized patients will tend to reduce the incidence and severity of organ dysfunction related to a systemic oxygen debt. These simple measures may need to be supplemented later by more complex interventions, but the ability to manage the initial phase of preventive care should be within the ability of most health care staff.\nClinical outcomes\nEarly intervention may reduce morbidity and mortality, but it is also possible that the earlier application of supportive treatment could contribute additional burdens. Buist and colleagues [62] demonstrated that the introduction of a MET contributed to a reduction in ward cardiac arrest rates, but this was also associated with an increase in emergency admissions to intensive care with no significant change in ICU mortality rates. This suggests that for some patients earlier intervention may have the effect of shifting the burden of mortality from the ward to the ICU, replacing a 'cheap' death with an expensive one. More recently, however, Bellomo and colleagues [63] also demonstrated a marked reduction in cardiac arrest rates associated with the introduction of a MET, and a parallel reduction in the use of intensive care resources on cardiac arrest survivors. It seems likely, therefore, that hospitals with high ward based cardiac arrest rates may well benefit from the introduction of systems aimed at identifying and managing sick patients earlier. Further multicentre studies from Australia are awaited.\nAutonomy and treatment limitation decisions\nIn an ideal world we would all possess clear advance directives giving guidance on how we would wish to be treated given differing circumstances. However, treatment preferences will not be static over time and are likely to be influenced by circumstances and the provision of information. How do patients respond to opportunities to discuss treatment preferences? Studies conducted in elective or out-patient settings demonstrate that many patients wish to be involved in decisions about treatment goals and intensity. They also demonstrate that the accuracy of information substantially alters preferences for resuscitation [78]. In the acute care context, however, there is considerable variability in clinical practice; moreover, many patients may not wish to discuss their preferences [79], even though this results in inappropriate treatment decisions [80]. It is clear that discussions about treatment preferences must be conducted with sensitivity by staff with appropriate experience and training, who have had an opportunity to develop a relationship of trust with the patient. This mandates a collaborative approach between the various medical and nursing teams, the patient and the family. Early intervention may buy time for these complex discussions to take place and an appropriate decision to be made.\nChanging behaviour through education and training\nThe key to improving safe care of acutely ill hospitalized patients is through team working and education, combined with improvements in resources for integrated delivery of care. In accident and emergency care, team based working reduces clinical error rates and enhances overall quality of care [81,82], and the principles of crew resource management drawn from aviation have important messages for clinical practice in this respect, by empowering all members of the team to make contributions to safety [83]. To achieve this we must incorporate team based attitudes in medical education, starting at undergraduate level and following this through into speciality training so that there is more overlap between disciplines than is currently the case. A competency based core curriculum for acute care that incorporates education in patient safety is essential. This process has started in intensive care medicine [84], and it is hoped that other disciplines will follow.\nPersonal responsibility and continuity of care are important features of quality care, and one of the challenges of implementing outreach is that it may encourage the attitude that someone else is responsible for the patient, and thus disem-power and de-skill ward staff [85]. Team working and continuity of care may be secured through the development of collaboratively produced guidelines or protocols that seek to support and guide the interaction between ward and outreach staff. Outreach staff should also recognize the limitations of their own expertise. Education is a key element in outreach activities, and is a two-way process that requires sharing of expertise, collaborative support, and blurring of traditional boundaries [86]. Important attitudinal attributes of outreach staff must therefore include the capacity to teach, learn from, and support other clinicians, sometimes under difficult circumstances.\nConclusion\nThere are substantial opportunities for improving the safety and quality of care delivered to acutely ill hospitalized patients. The methods which are adopted will vary according to local circumstance, but common elements include the need for better integration of care across disciplines and systems for earlier identification of patients at risk, and we refer to these as 'outreach' care. We do not yet know which of these various approaches will best improve patient outcomes, and there is a need for prospective studies in this area which take into account the difficulties of using randomization and controls, and which employ long-term follow-up. METs appear to reduce the incidence of cardiac arrests in ordinary wards, and consequential use of scarce intensive care resources. Outreach-based systems which support and educate ward-based staff in delivering clinical care appear to have achieved a high degree of acceptance in the UK. Health care managers need to work closely with clinicians to introduce these methods of team-working into hospital practice, while evaluating their effectiveness.\nCompeting interests\nNone declared.\nAbbreviations\nICU = intensive care unit; MET = medical emergency team.","keyphrases":["critical care","acute pain team","suboptimal care","patient at risk","critical illness","cardiopulmonary resuscitation"],"prmu":["P","P","P","P","P","P"]}
{"id":"Neurogenetics-2-2-1513515","title":"Investigation of autism and GABA receptor subunit genes in multiple ethnic groups\n","text":"Autism is a neurodevelopmental disorder of complex genetics, characterized by impairment in social interaction and communication, as well as repetitive behavior. Multiple lines of evidence, including alterations in levels of GABA and GABA receptors in autistic patients, indicate that the GABAergic system, which is responsible for synaptic inhibition in the adult brain, may be involved in autism. Previous studies in our lab indicated association of noncoding single nucleotide polymorphisms (SNPs) within a GABA receptor subunit gene on chromosome 4, GABRA4, and interaction between SNPs in GABRA4 and GABRB1 (also on chromosome 4), within Caucasian autism patients. Studies of genetic variation in African-American autism families are rare. Analysis of 557 Caucasian and an independent population of 54 African-American families with 35 SNPs within GABRB1 and GABRA4 strengthened the evidence for involvement of GABRA4 in autism risk in Caucasians (rs17599165, p=0.0015; rs1912960, p=0.0073; and rs17599416, p=0.0040) and gave evidence of significant association in African-Americans (rs2280073, p=0.0287 and rs16859788, p=0.0253). The GABRA4 and GABRB1 interaction was also confirmed in the Caucasian dataset (most significant pair, rs1912960 and rs2351299; p=0.004). Analysis of the subset of families with a positive history of seizure activity in at least one autism patient revealed no association to GABRA4; however, three SNPs within GABRB1 showed significant allelic association; rs2351299 (p=0.0163), rs4482737 (p=0.0339), and rs3832300 (p=0.0253). These results confirmed our earlier findings, indicating GABRA4 and GABRB1 as genes contributing to autism susceptibility, extending the effect to multiple ethnic groups and suggesting seizures as a stratifying phenotype.\nIntroduction\nAutism is a neurodevelopmental disorder characterized by severe impairment in social interaction and communication, as well as repetitive behavior. Autism is part of a spectrum of disorders denoted autism spectrum disorders (ASD), which in addition to autism, include Asperger syndrome, childhood disintegrative disorder, and pervasive developmental disorder not otherwise specified (PDD-NOS). Onset of this disorder is typically before 3\u00a0years of age. The incidence of autism is estimated at approximately one in 1,000 individuals, with an increased incidence in males [1\u20133]. Incidences of approximately two to three in 1,000 are reported when a broader diagnosis includes the entire spectrum of pervasive developmental disorders [2, 4].\nEvidence indicates that this disease has a strong genetic component. Twin studies have shown that monozygote twins have a higher recurrence rate than dizygotic twins. While the actual percentages have varied, recurrence rates have been measured as high as 95% in monozygote twins and 23% in dizygotic twins [5\u20137]. Despite this strong evidence for a genetic component, the inheritance appears to be complex, with estimates that more than 15 genes may be involved in its inheritance [8, 9], with different families carrying different combinations of contributing genes. The genes may act independently or interactively, adding to the complexity of determining the genetic contribution to this disorder.\nThere are two approaches to identifying genetic contributors to disease. The first is a genome wide search in which linkage or association analysis is used to identify regions of the genome that may contain autism susceptibility genes. The second is the candidate gene approach, which investigates a specific gene or genes for involvement in autism risk. In the candidate gene approach, genes are chosen for study based on either what is known about the gene\u2019s function, its location (for example in a recognized linkage peak), or a combination of both. Several candidates are hypothesized to be involved in autism; however, no single candidate gene has consistently emerged as involved in autism risk.\nOne candidate pathway that is hypothesized to be involved in autism is the GABAergic system. Hussman [10] suggested that autism is the result of an imbalance of the excitatory glutamatergic and inhibitory GABAergic pathways, resulting in overstimulation in the brain and inability to filter out excess stimuli from environmental and intrinsic sources. This theory is supported by multiple lines of evidence. First, histological, biochemical, and molecular approaches have demonstrated altered levels and distribution of GABA and GABA receptors in peripheral blood and plasma, as well as in the brain, including decreased GABA-A receptors and benzodiazepine binding sites in the hippocampal formation [11\u201313]. There are also reported alterations in GABAergic neurons, as demonstrated by the increased packing density of GABAergic interneurons in the CA3 and CA1 subfields, and by the decreased numbers and reduced size of cerebellar GABAergic Purkinje cells [14, 15]. In addition, duplications and isodicentric chromosomes in the region containing the three clustered GABA receptor subunits GABRB3, GABRA5, and GABRG3 on chromosome 15q have been associated with autism [16, 17]. As well, evidence for both linkage and allelic association have been reported for this same GABA gene cluster, although the findings have not been consistent across datasets [18\u201322]. Investigation of association of GABA receptor subunits outside of the chromosome 15 region has been limited [23]. Lastly, mutations have been reported in multiple GABA receptor genes in families with epilepsy [24]. Given the high comorbidity of autism with epilepsy and seizures, these data suggest that a similar molecular etiology could exist between the disorders.\nSignaling in the GABAergic system is mediated by receptors for the neurotransmitter GABA. There are 19 known GABA receptor subunits arranged in clusters throughout the genome. Functional pentamers formed by various combinations of these subunits result in receptors of varying properties and sensitivities. The amounts and functional capabilities of individual receptor subunits that form a specific pentamer can affect the amount and quality of signaling in different parts of the brain.\nPreviously published work from our laboratory analyzed 14 autosomal GABA receptor genes on four different chromosomes, using 70 SNPs in a Caucasian dataset of 470 families. This analysis revealed allelic association to rs1912960 (p=0.01) within gamma-aminobutyric acid (GABA) A receptor, alpha-4 (GABRA4) on chromosome 4 [23] and a significant interaction with rs2351299 within the neighboring gene gamma-aminobutyric acid (GABA) A receptor, beta-1 (GABRB1).\nDespite similar prevalence rates between Caucasian and African-Americans [25, 26], autism studies in African-Americans are rare. Risk alleles may be different between ethnic groups or the same risk alleles may have differential effects in each ethnic group, warranting studies in multiple groups. Evidence that phenotypic factors, including indicators of language development, may be more severe in African-Americans, compared to non-Hispanic Caucasians, [27] is consistent with these possibilities and underscores the need to investigate autism in different ethnic groups. In this study, we present a validation of our previous report in an independent dataset of 54 African-American families, as well as confirmation of our previous results in an expanded Caucasian sample of 557 autism families.\nMaterials and methods\nSamples\nAll families were drawn from a large multisite study of autism genetics conducted in the southeastern United States. These families are recruited through the Center for Human Genetics (CHG) at Duke University Medical Center (DUMC), the University of South Carolina, and the Center for Human Genetic Research at Vanderbilt (N=54 African-American and 557 Non-Hispanic Caucasian families) through support groups, advertisements, and clinical and educational settings. All sites recruited, enrolled, and sampled individuals with autism and family members, per study protocols approved by their respective institutional review boards (IRBs). Written informed consent was obtained from parents and from children who were able to give informed consent. Families were enrolled based on probands meeting the following core inclusion criteria of: (1) probands ranging from three to 21\u00a0years of age; (2) a presumptive clinical diagnosis of autism; and (3) an expert clinical diagnosis of autism using DSM-IV criteria [28], supported by the Autism Diagnostic Interview-Revised (ADI-R) [29] and in some cases, the Autism Diagnostic Observation Schedule (ADOS) [30]. Our original ascertainment protocol relied on clinical expertise and the ADI-R. The ADOS was subsequently added as a required diagnostic method, and families that were missing ADOS assessments were updated as available. To assure valid ADI-R results, all participants who met current diagnostic criteria for autism were included only if they had a minimal developmental level of 18\u00a0months, as extrapolated from the Vineland Adaptive Behavior Scale score [31], or had an IQ equivalent greater than 35 based on scores from a standardized measure of cognitive ability such as an the Wechsler Scale of Intelligence for Children\u20144th edition [32], Differential Abilities Scale [33], Mullen Scales of Early Learning [34] or Leiter Intelligence Scale-Revised [35]. IQ data was derived from medical records or direct assessment. Exclusion criteria for participation in the larger genetics study included: severe sensory problems (e.g., visual impairment or hearing loss), significant motor impairments (e.g., failure to sit by 12\u00a0months, or walk by 24\u00a0months), or identified metabolic, genetic, or progressive neurological disorders, based on screening by clinical staff. Additional samples are from the Autism Genetic Research Exchange (AGRE). These individuals were qualified using similar methods including ADI-R, ADOS, VABS, and a standardized measure of IQ.\nThirty-nine African-American families were used in an initial GABA receptor screen. Follow-up analysis of significant findings was performed in 54 African-American families. Analysis of the extended Caucasian dataset included 557 non-Hispanic Caucasian families. One-hundred and five new non-Hispanic Caucasian families were added to the analysis (18 families previously analyzed by Ma et al. [23] were newly identified as Hispanic, and were omitted from the current study in an effort decrease heterogeneity in the Caucasian dataset).\nClassification of history of seizure activity in autism patients was based on question 92 from the ADI-R, which queries for both current and lifetime presence of convulsions, seizures, and epilepsy. Caregiver responses to question 92 are coded to indicate no seizure activity, seizure activity with no definitive diagnosis of epilepsy, and seizures with a definite diagnosis of epilepsy. Using lifetime ratings, two groups of families were defined: those in which no seizure activity was reported, and those in which seizure activity was present in at least one autism patient. In addition, question 92 allows for coding of febrile seizures. Families with only febrile seizures were classified as negative for seizure activity and not included in the seizure subset analysis. Both families with positive and negative history of seizure activity were included in our overall dataset. This resulted in a dataset of 41 Caucasian families with a positive history for seizures.\nMolecular analyses and genotyping\nThe analysis of 14 GABA receptor subunit genes was performed in 39 African-American families as previously described [23]. Briefly, between three and seven intronic, UTR and synonymous coding SNPs within each gene were identified from Applied Biosystems (ABI, Foster City, CA, USA) Assay on Demand (AoD) products, resulting in 70 SNPs within 14 GABA receptor genes on four autosomes. Genes analyzed were: GABRA1, GABRA6, GABRB2, GABRG2, and GABRP from chromosome 5; GABRA2, GABRA4, GABRB1, and GABRG1 from chromosome 4; GABRB3, GABRA5, and GABRG3 from chromosome 15; and GABRR1 and GABRR2 from chromosome 6.\nAdditional SNPs within GABRA4 and GABRB1 were analyzed in the extended African-American (N=54) and Caucasian (N=557) datasets to expand the coverage of variation across this region. Thirty-five SNPs, representative of different linkage disequilibrium (LD) blocks across the two genes (20 in GABRA4 and 15 in GABRB1), were genotyped (Fig.\u00a01). SNP\u2019s for genotyping were selected from online databases (University of California Santa Cruz http:\/\/genome.ucsc.edu and NCBI dbSNP http:\/\/www.ncbi.nlm.nih.gov\/entrez\/query.fcgi?db=snp) and from resequencing of exons and surrounding areas of both GABRB1 and GABRA4 genes.\nFig.\u00a01Approximate locations of SNPs for extended analysis within GABRA4 and GABRB1. Horizontal arrows represent transcriptional direction. Vertical arrows represent SNPs, as numbered and identified below the diagram. Pter indicates the P-terminal end of the chromosome and cen indicates the centromere\nSNP genotyping was performed using Taqman allelic discrimination assays (Applied Biosystems). DNA was extracted from whole blood according to established protocols [36], and 3\u00a0ng of genomic DNA was used per reaction. Amplification was performed on GeneAmp PCR Systems 9700 thermocyclers, with cycling conditions as recommended by Applied Biosystems. Fluorescence was measured using Applied Biosystem\u2019s 7900. Genotype discrimination was conducted using ABI Prism SDS 2.1 software. Quality control, to ensure accurate genotyping, involved two different CEPH DNAs in quadruplicate on each 384 well plate, as well as the presence of samples which were replicated elsewhere in the sample list. Additionally, \u226595% genotyping efficiency is required.\nStatistical analysis\nTo ensure genotyping quality, Pedcheck was run for detection of Mendelian inheritance inconsistency. One affected and one unaffected individual from each family were selected randomly for tests of Hardy\u2013Weinberg equilibrium (HWE), which was assessed using exact tests from the Genetic Data Analysis program [37]. Pairwise LD between markers was calculated using graphical overview of linkage disequilibrium (GOLD) [38] in the parents of autism cases for both the African-American and Caucasian samples. LD was evaluated in parents to increase the available sample size for analysis and comparison between the two ethnic groups. The pedigree disequilibrium test (PDT) and its extension the genotypic pedigree disequilibrium test (genoPDT) [39, 40] were used to test for association to autism susceptibility.\nThe EMDR [23, 41], an extension of the MDR [42, 43], was used to test for potential gene\u2013gene interaction, to identify specific locus combinations of interest for further investigation and validation of previous results. EMDR analysis was performed using seven SNPs, the four in GABRA4 found to show significant allelic or genotypic association in the Caucasian sample-set, and the three in GABRB1 found to be significant in the seizure subgroup. One-, two-, and three-way analysis was performed on the Caucasian dataset. For case-control pairs used in EMDR, the proband (or most completely genotyped affected child) from each multiplex and triad family was selected (n=470 total) as a case. Controls were generated using the untransmitted alleles from parental genotypes. In this study, a cross-validation option was not utilized. The sample size of the African-American dataset is too small to provide power for EMDR analysis, not allowing us to test for validation of the interaction seen in the paper of Ma et al. within the African-American dataset.\nThe haplotype family-based association test (HBAT; [44]) was used for haplotype association analysis using the significant SNPs in GABRA4.\nResults\nAllelic association studies of 70 SNPs across the 14 GABA receptor subunit genes in the 39 African-American screen set of families, revealed association in rs2280073 (GABRA4; p=0.0053) and hcv2119841 (GABRB1; p=0.0343), the same two genes identified through allelic association and interaction analysis in the Caucasian dataset [23]. Genotypic association analysis revealed the same GABRA4 SNP, rs2280073 (p=0.0262), and marginal significance within GABRP, rs1862242 (p=0.0471). The remaining SNPs showed no significant association (data not shown).\nAnalysis of the screening SNPs and newly identified SNPs within GABRA4 and GABRB1 in the Caucasian population (N=557), and within the extended African-American population (N=54) (Table\u00a01), revealed new SNPs with significant association. In the Caucasian dataset, rs1912960, also significant in the study of Ma et al. (p=0.012), showed significant allelic association (p=0.0073). Additional significant SNPs were identified in GABRA4 as well, rs17599165 (p=0.0015) and rs17599416 (p=0.0040). Genotypic association was also seen in these SNPs (p=0.0046, 0.0009, and 0.0043, respectively), as well as in a fourth SNP, also in GABRA4 (rs7660336, p=0.0368). In the African-American dataset, rs2280073 (p=0.0287), identified in the smaller African-American dataset above, and rs16859788 (p=0.0253), were found to be associated with the allele based test. Genotypic association was also identified in rs16859788 (p=0.0412). No SNPs within GABRB1 were found to be associated with autism in either ethnic group. \nTable\u00a01Analysis of GABRA4 and GABRB1 in extended Caucasian and African-American datasetsGABRA4GABRB1SNPCaucasianAfrican-AmericanSNPCaucasianAfrican-AmericanPDTaGeno PDTaPDTaGeno PDTaPDTGeno PDTPDTGeno PDTRS76783380.93500.99230.22300.4190RS18669890.30710.38600.55530.7892RS64475170.88260.70340.5050.3930RS23512990.45290.08220.57750.8614RS175991020.80550.89130.75180.8805RS100163880.15850.26600.23670.4259RS76603360.08330.0368(G\/G)0.51640.7410RS13724960.23620.37400.24820.3715RS15121360.90520.98690.28880.3575RS31140840.09340.19420.20590.3281RS175991650.0015(T)0.0009(T\/T)0.65470.4304RS44827370.14950.25041.00001.0000HCV15925450.77980.94270.22300.5313HCV113535240.19590.31171.00001.0000RS76855531.00000.84190.49130.6044RS37755340.18930.18310.49130.4913RS19129600.0073(C)0.0046(C\/C)0.41110.5110HCV21198410.33520.08380.22780.4111RS20559430.96710.94340.49270.7607RS62870.40450.35710.61710.6984RS22800730.14040.09550.0287(G)0.1100RS62890.93490.55540.86580.9220RS168597880.31730.31730.0253(A)0.0412(A\/A)RS62900.42850.19730.31730.4594RS175994160.0040(A)0.0043(A\/A)0.80840.80844P04131.00001.00000.73890.7389RS37922081.00000.49800.17970.1797RS100289450.32720.45841.00000.8179RS105171740.94840.08940.71500.8903RS38323000.40940.63520.65470.6547RS76940350.43370.62660.36570.3657RS37922110.90570.83820.65470.2895RS22299400.78730.93750.54850.7866RS131517590.75290.93260.23670.5759RS131517690.47400.74360.18240.4768Bold indicates significant valuesaAssociated allele\/genotype shown in parenthesis\nThe majority of pairwise r2 values between the significant SNPs were less than 0.3, in both ethnic groups (Table\u00a02). However, a few SNPs have values between 0.3 and 0.35. SNPs, rs17599165, and rs17599416 have r2 values of 0.709 in African-Americans and 0.853 in Caucasians, and rs7660336 and rs2280073 have a pairwise r2 of 0.907 in Caucasians and 0.905 in African-Americans. Allele frequencies are similar, yet not identical between the two groups. One SNP, however, did have large differences in minor allele frequencies (MAFs) between the two groups, with a MAF of 0.24 in African-Americans but only 0.001 in Caucasians (Table\u00a02). Haplotype analysis, using the four SNPs with significant allelic or genotypic association in the Caucasian families, revealed a significant global test (p=0.014) in the Caucasian population, further supporting the involvement of these SNPs or another variant on the haplotype background. The haplotype composed of all risk alleles trends towards significance with autism as a risk haplotype (p=0.0763), while the haplotype of all protective alleles resulted in being highly significantly protective (p=0.0006; Table\u00a03) \nTable\u00a02Minor allele frequencies and linkage disequilibrium in Caucasian and African-American datasetsBold indicates significant valuesaMAF minor allele frequencyTable\u00a03Haplotype frequency and association in Caucasian populationRS7660336RS17599165RS1912960RS17599416Haplotype frequencyP-valueGTCA0.5030.0763CTCA0.2770.9214CTGA0.1240.6094CAGG0.0820.0006Bold indicates significant values\nSubsetting of the GABRA4 and GABRB1 data to analyze all families with positive history for seizures revealed no association to GABRA4. However, three SNPs within GABRB1 were found to be both allelically and genotypically associated with autism: rs2351299 (p=0.0163 and p=0.0189 for PDT and genoPDT, respectively), rs4482737 (p=0.0339 and p=0.0339), and rs3832300 (p=0.0253 and p=0.0357). These three SNPs all had pairwise r2 values less than 0.1 (data not shown).\nIn the Caucasian population, EMDR verified the single locus effect identified through PDT analysis in rs1912960 (p=0.024), and identified two different significant two-locus gene\u2013gene effects between GABRA4 and GABRB1, rs1912960 with rs2351299 (p=0.004), and rs17599416 with rs2351299 (p=0.014). Several three locus effects were also significant [rs7660336, rs1912960, and rs2351299 (p=0.012); rs17599165, rs1912960, and rs2351299 (p=0.012); rs1912960, rs17599416, and rs2351299 (p=0.038); and rs7660336, rs17599416, and rs2351299 (p=0.047)] (Table\u00a04). \nTable\u00a04EMDR results in Caucasian dataset between GABRA4 and GABRB1Input SNPsSignificant interactionsGeneSNP numberSNPSNPsP-valuesGABRA41RS7660336One-way30.0242RS17599165Two-way3, 50.0043RS19129604, 50.0144RS17599416Three-way1, 3, 50.012GABRB15RS23512992, 3, 50.0126RS44827373, 4, 50.0387RS38323001, 4, 50.047\nDiscussion\nWe have confirmed the involvement of GABRA4 in autism through identification of significantly associated SNPs within an independent African-American population. Furthermore, we have strengthened our original findings, including identification of additional associated SNPs and a significant interaction between GABRA4 and GABRB1 and in an extended dataset (N=557) of Caucasian autism families. The AA dataset contains only 54 families, and in general power can be a problem in small sample sets if an effect is not seen. However, for the GABRA4 gene, we did find significant results in the AA dataset. Furthermore, studies in small datasets such as the AA dataset can also be less robust than in larger datasets. However, the fact that we find the same gene, GABRA4, significant in two different ethnic groups is supportive of the role of GABRA4 in autism risk.\nThe identification of two different two-way interactions between GABRA4 and GABRB1 provides additional evidence of the complex interaction of these two genes in autism. The rs1912960 (GABRA4) with rs2351299 (GABRB1) interaction is between the same two SNPs previously reported in the study of Ma et al., and is still significant in our larger dataset. A second significant two-way interaction was found, also including rs2351299 in GABRB1 as well as rs17599416 in GABRA4, further supporting that interactions between these two genes are involved in autism risk. Although rs2351299 does not have a significant PDT result, it is found in both significant interaction pairs, and in each of the significant three-way interactions. The MDR approach used in these analyses is specifically designed to detect interaction effects both in the presence and absence of main effects.\nIt is possible that both of the two-way interactions are being identified due to LD between the two GABRA4 SNPs (rs1912960 and rs17599416). There is significant correlation between the two SNPs, although the r2 value (Table\u00a02) between them is not large (0.32). Given that these do not appear to be causative variants, it is likely that the true variant, yet to be identified, is in LD with these GABRA4 SNPs. Examination of interaction in the independent dataset of African-American families was not possible due to the limited sample size.\nWe also identified variants within GABRB1 as associated within the autistic population with seizures. One of these variants, rs2351299, also shows a significant interaction with SNPs in GABRA4 (Table\u00a04). While no effect was seen in GABRA4 for the seizure subset, the sample size may be too small to conclusively determine its role in seizure status in autism. However, the enhanced findings in GABRB1 in the seizure subset implicate GABRB1 as a contributor to genetic risk in these patients.\nDespite the identification of GABRA4 in both ethnic groups, different SNPs were found to be associated. The identification of distinct SNPs within these populations may indicate differences in allele frequency and linkage disequilibrium within the two racial groups, differences in the haplotypic background in which identical causative variations originated, or differences in the causative variation. SNP rs16859788 for example, which is significant in the African-American group, shows little variation in the Caucasian dataset, therefore, providing no power for detection of an effect in this group. Other SNPs, however, show similar allele frequencies between the two populations.\nSome differences in LD do exist between the two ethnic groups as well; however, the majority of the differences are small. The largest differences in LD are in pairwise values with rs16859788, which appear to mostly be due to the fact that the SNP is practically monoallelic in the Caucasian population. The Caucasian dataset suggests that there is a significant association of SNP haplotypes with autism risk, while the African-American set does not. However, this difference may be due to the small size of the African-American dataset. Therefore, while it appears that minor allele frequency differences can account for the lack of association of rs16859788 in the Caucasian dataset, additional studies are needed to determine whether the other differences in results between the two ethnic groups are due to sample size differences, differential LD with the causative variation in the two populations, or are population-specific risk alleles.\nWhile we have identified several associated SNPs, we do not predict any of the ones in GABRA4 to have functional consequences; therefore, it is unlikely that these are primary variants leading to the autism susceptibility. One of the SNPs identified in GABRB1 in the seizure subset, however, is in the 3\u2032 untranslated region (UTR). Given that multiple GABA receptor subunits combine in varying combinations to form a functional GABA receptor, even minor changes in levels of a particular subunit may alter the makeup of receptors within a particular cell type, and alter the GABAergic signaling. Therefore, variations within potential regulatory regions, such as untranslated regions and promoters, could play an important role. It will be important to look at potential changes that may result from this and other potential GABRB1 UTR variations, as well as sequence coding and potential regulatory regions to identify the primary variation, or variations leading to altered autism susceptibility.\nIn summary, these data show that the GABA receptors are implicated in the etiology of autism in two different ethnic populations and suggest seizures as a stratifying phenotype. Furthermore, these results support our earlier findings, indicating GABRA4 and GABRB1 as genes contributing to autism susceptibility, independently, in the case of GABRA4, and through complex interactions with each other.","keyphrases":["autism","gaba receptors","ethnicity","association","snps"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_Arch_Otorhinolaryngol-4-1-2254466","title":"Management and prognostic factors of recurrent pleomorphic adenoma of the parotid gland: personal experience and review of the literature\n","text":"The aim of this study was to investigate the management and prognostic determinants of recurrent pleomorphic adenoma (RPA). A retrospective analysis was performed to examine the clinical features, the prevalence of surgical complications, and new recurrences of RPA. Tumor recurrence rate was estimated by the Kaplan\u2013Meier method, and the prognostic value of some of the variables was tested by univariate analysis using the log rank test. The study focused on 33 patients, 18 female (54.5%) and 15 male (45.5%), aged 12\u201371 years (median 41). A total or extended total parotidectomy was performed in 16 cases (48.5%), a superficial parotidectomy in 10 cases (30.3%), and a local excision in 7 cases (21.2%). In ten patients (30.3%), a branch or the trunk of the facial nerve was deliberately sacrificed. Major complications included one unexpected definitive paralysis of the marginal mandibular branch of the facial nerve and 14 cases of Frey syndrome. Follow-up varied from 2 to 25 years (median 10.5 years), and there were 11 new recurrences (33.3%) within a period varying from 1 to 16 years (median 6 years). The estimated tumor recurrence rates were 14.1 \u00b1 6.6% at 5 years, 31.4 \u00b1 9.4% at 10 years, 43.0 \u00b1 10.8% at 15 years, and 57.2 \u00b1 14.8% at 20 years. Presence of a multinodular lesion and the type of intervention performed were significantly associated with a higher probability of recurrence. RPAs are prone to new recurrences, especially when multinodular and treated with a local excision. Surgical treatment should include facial nerve resection in selected cases. Follow-up for the patient\u2019s lifetime is warranted.\nIntroduction\nPleomorphic adenoma is the most common neoplasm of the parotid gland. It is a benign tumor composed of epithelial and myoepithelial cells arranged in various morphological patterns. Thinning or absence of the pseudocapsule and the presence of fingerlike projections of the tumor have been observed in all histologic subtypes of pleomorphic adenoma, in particular the myxoid type [1]. Even though many hypotheses for recurrences of parotid gland pleomorphic adenoma have been advanced, including cell biological and genetic factors [2, 3], obvious or underestimated tumor spillage, incomplete excision, and violation of the pseudocapsule of the tumor are considered the only proven reasons contributing to recurrent disease [4].\nThis is why the abandonment of enucleation techniques in favor of more extended surgical procedures, which require the tumor to be excised with the surrounding normal tissue and the facial nerve to be identified and preserved, have dramatically reduced the recurrence rate of pleomorphic adenomas of the parotid gland from 20\u201345% to less than 4% in the last decades [5\u20137].\nHowever, pure en bloc excision in parotid benign tumor disease has been considered impossible by most surgeons when the pleomorphic adenoma is lying on the facial nerve, and so partial enucleation is often the only surgical option in those cases [5, 7], thus explaining the possibility of recurrence even after proper surgery.\nMost of the recent literature on recurrent pleomorphic adenoma (RPA) of the parotid gland has attempted to analyze the causes of primary failure and discusses other possible risk factors for recurrence [8\u201312]; however, there are very few papers focused on the prognostic factors of RPA and retreatment policies [13\u201318].\nThe present study was undertaken to address these important issues based on a follow-up period of up to 20\u00a0years. Here, we present our findings and review some of the more recent literature.\nMaterials and methods\nA retrospective analysis of the charts of patients treated for benign parotid neoplasm between 1983 and 2004 was performed to identify those patients operated on for RPA. We collected information on patient demographics and clinical history, clinical features of the lesions, diagnostic work-up, the type of surgery, operative findings, facial nerve management, and adjuvant treatment.\nThe study analyzed the prevalence of surgical complications and new recurrences. Statistical analysis was performed using the SPSS statistical package. Tumor recurrence rate was estimated by the Kaplan\u2013Meier method: the entry point was the date of surgery for a recurrent tumor seen for the first time in our department, and the end point was the date of a new recurrence or the date of last consultation for censored observations.\nThe prognostic value of certain categorical variables (age, gender, number of previous operations, type of previous operation, lobe of origin, type of surgery performed for RPA, maximum diameter, facial nerve resection, presence of a pseudocapsule at the surgical margins, rupture of the pseudocapsule, number of neoplastic nodules, postoperative radiotherapy) was tested by univariate analysis using the log rank test.\nResults\nAmong 524 patients treated for a benign parotid neoplasm between 1983 and 2004, 33 underwent surgical treatment for RPA. Eighteen (54.5%) patients were female and 15 (45.5%) male; their ages ranged from 12 to 71\u00a0years (median 41). None had been previously affected by a malignant neoplasm or submitted to radiation treatment. Seven patients were previously operated on in our department (with a prevalence of recurrence after first treatment of 1.3%) and 26 patients had previous surgical treatment(s) performed elsewhere. A summary of the patient\u2019s previous clinical history is reported in Table\u00a01. A swelling in the parotid region was the most significant complaint for 87.9% (29\/33) of patients when they came to our attention for the recurrence; pain or facial nerve paresis was associated with the swelling in two cases. In four (12.1%) cases, the relapse was recognized by routine postoperative follow-up examinations. Other complaints, present from previous surgeries, were facial nerve paralysis in four patients and Frey syndrome in one.\nTable\u00a01Summary of clinical history of patients treated for the first time at the Department of Otolaryngology of the University of Brescia for RPA of the parotid glandPrevious recurrences\u00a0\u00a0One63.6% (21)\u00a0\u00a0Two 18.2% (6)\u00a0\u00a0Three 12.1% (4)\u00a0\u00a0Four 6.1% (2)Median time to recurrence\u00a0\u00a0First recurrence (33) 6\u00a0years (range 1\u201323)\u00a0\u00a0Second recurrence (12) 5\u00a0years (range 2\u201328)\u00a0\u00a0Third recurrence (6) 5.5\u00a0years (range 3\u201319)\u00a0\u00a0Fourth recurrence (2) 8\u00a0years (both patients)Last operation\u00a0\u00a0Subtotal parotidectomy 33.3% (11)\u00a0\u00a0Local excision 33.3% (11)\u00a0\u00a0Superficial parotidectomy 21.2% (7)\u00a0\u00a0Not reported 12.1% (4)Number of patients in parentheses\nUltrasonography was the most frequently used diagnostic tool (23 patients, 69.7%), followed by fine needle aspiration biopsy in 21 (63.6%) patients, magnetic resonance imaging in 14 (42.4%), computed tomography in 3 (9.1%), and sialography in 2 (6.1%). The different types of diagnostic procedures employed reflect the multi-decade period analyzed. At present, after ultrasonography, which is routinely adopted for follow-up, magnetic resonance imaging is used to delineate the extension of the recurrent lesions.\nIn 12 (36.4%) patients, the lesion involved the superficial lobe of the parotid gland, in 9 (27.3%) both lobes, and in 12 (36.4%) only the deep lobe was involved. In 5 (15.2%) patients the lesion also extended to the parapharyngeal space. Information on multinodularity of the lesion could be obtained for 31 patients: multiple lesions were observed in 24 (77.4%) of them.\nSurgical treatment was tailored to the single patient in an attempt to obtain a margin of healthy tissue (Table\u00a02). Postoperative complications are listed in Table\u00a03. Nine (27.3%) patients were submitted to postoperative radiotherapy. There were no standard criteria for postoperative radiotherapy, but it was performed when the excision did not guarantee sufficient free margins of healthy tissue based on the judgment of the surgeon.\nTable\u00a02Surgical treatment for RPA of the parotid gland at the Department of Otolaryngology of the University of BresciaTotal or extended total parotidectomy 48.5% (16)Superficial parotidectomy 30.3% (10)Local excision 21.2% (7)Partial or total facial nerve resection30.3% (10)Number of patients in parenthesesTable\u00a03Postoperative complications of patients treated for RPA of the parotid gland at the Department of Otolaryngology of the University of BresciaFrey syndrome 43.7% (14\/32)Temporary paresis of a branch or of the entire facial nerve30.4% (7\/23)Definitive paralysis of the marginal mandibular branch of the facial nerve4.3% (1\/23)Salivary fistula3.0% (1\/33)Keloid3.0% (1\/33)\nThe patients were followed by yearly ultrasonography. Follow-up varied from 2 to 25\u00a0years (median 10.5\u00a0years). Three patients died from a metachronous malignant neoplasm, one of them after a new recurrence. The other patients are still alive. Overall, 11 (33.3%) new recurrences were observed. No patient with a single node had a recurrence. Four of the new recurrences had been operated on again and one had a further recurrence. Seven patients with small (less than 1\u00a0cm) and stable new recurrences are still on follow-up. The time for new recurrence varied from 1 to 16\u00a0years (median 6\u00a0years). There were 2 cases of focal atypical cells but no malignant transformation in the new recurrences. Distribution of the recurrences in relation to the different variables under analysis is shown in Table\u00a04. The estimated tumor recurrence rates were 14.1\u00a0\u00b1\u00a06.6% at 5\u00a0years, 31.4\u00a0\u00b1\u00a09.4% at 10\u00a0years, 43.0\u00a0\u00b1\u00a010.8% at 15\u00a0years, and 57.2\u00a0\u00b1\u00a014.8% at 20\u00a0years (Fig.\u00a01). Among the variables evaluated with the log-rank test, only the presence of a multinodular recurrence and the type of intervention performed were significantly associated with a higher probability of a new recurrence (Table\u00a05). The high variation in tumor recurrence rates observed after 20\u00a0years of follow-up (Table\u00a05) is probably related to the low number of patients with longer follow-up times.\nTable\u00a04Distribution of the new recurrences after treatment for RPA of the parotid gland at the Department of Otolaryngology of the University of Brescia (No.\u00a0=\u00a011; 33.3%)Variable%P-valueAge\u226430\u00a0years (3\/10)30.01>30\u00a0years (8\/23)34.8GenderFemale (4\/18)22.20.1Male (7\/15)46.7Number of previous operations1 (7\/21)33.31>1 (4\/12)33.3Type of previous operationsEnucleation (2\/11)18.20.2Parotidectomy (8\/18)44.4Lobe of originSuperficial (5\/21)23.80.1Deep (6\/12)50.0Intervention performedParotidectomy (6\/26)23.10.02Other (5\/7)71.4Maximum diameter\u22642\u00a0cm (7\/16)43.80.3>2\u00a0cm (3\/15)20.0Facial nerve resectionNo (7\/23)30.40.7Yes (4\/10)40.0Involvement of surgical marginsNo (8\/23)34.80.5Yes (2\/8)25.0Capsular ruptureNo (6\/23)26.10.2Yes (4\/8)50.0Multiple nodulesNo (0\/7)00.04Yes (10\/24)41.7Postoperative radiotherapyNo (9\/24)37.50.3Yes (2\/9)22.2Fig.\u00a01Estimated recurrence rate after treatment for recurrent pleomorphic adenoma of the parotid gland at the Department of Otolaryngology of the University of Brescia (Kaplan\u2013Meier method)Table\u00a05Estimated tumor recurrence rates after treatment for RPA of the parotid gland at the Department of Otolaryngology of the University of Brescia (log-rank test)VariableEstimated tumor recurrence rates (%)P-value5-year10-year15-year20-yearAge\u226430\u00a0years (10)12.5\u00a0\u00b1\u00a011.741.7\u00a0\u00b1\u00a018.6\u2013\u20130.8> 30\u00a0years (23)14.7\u00a0\u00b1\u00a07.926.9\u00a0\u00b1\u00a010.542.4\u00a0\u00b1\u00a012.856.8\u00a0\u00b1\u00a015.7GenderFemale (18)13.4\u00a0\u00b1\u00a09.030.7\u00a0\u00b1\u00a013.130.7\u00a0\u00b1\u00a013.130.7\u00a0\u00b1\u00a013.10.4Male (15)14.3\u00a0\u00b1\u00a09.430.7\u00a0\u00b1\u00a013.056.7\u00a0\u00b1\u00a015.171.1\u00a0\u00b1\u00a016.3Number of previous operations1 (21)11.8\u00a0\u00b1\u00a07.925.4\u00a0\u00b1\u00a011.145.6\u00a0\u00b1\u00a014.763.7\u00a0\u00b1\u00a017.80.9>1 (12)18.5\u00a0\u00b1\u00a011.941.8\u00a0\u00b1\u00a016.341.8\u00a0\u00b1\u00a016.341.8\u00a0\u00b1\u00a016.3Type of last previous operationEnucleation (11)9.1\u00a0\u00b1\u00a08.724.2\u00a0\u00b1\u00a015.624.2\u00a0\u00b1\u00a015.624.2\u00a0\u00b1\u00a015.60.2Parotidectomy (18)20.0\u00a0\u00b1\u00a010.345.1\u00a0\u00b1\u00a014.058.9\u00a0\u00b1\u00a015.9100Lobe of originSuperficial (21)013.8\u00a0\u00b1\u00a09.132.1\u00a0\u00b1\u00a013.654.8\u00a0\u00b1\u00a020.60.07Deep (12)35.8\u00a0\u00b1\u00a014.459.9\u00a0\u00b1\u00a016.459.9\u00a0\u00b1\u00a016.459.9\u00a0\u00b1\u00a016.4Intervention performedParotidectomy (26)5.3\u00a0\u00b1\u00a05.116.4\u00a0\u00b1\u00a08.730.5\u00a0\u00b1\u00a011.747.9\u00a0\u00b1\u00a017.40.001Other (7)42.9\u00a0\u00b1\u00a018.7\u2013\u2013\u2013Maximum diameter\u22642\u00a0cm (16)19.6\u00a0\u00b1\u00a010.226.9\u00a0\u00b1\u00a011.644.3\u00a0\u00b1\u00a014.072.2\u00a0\u00b1\u00a020.90.4>2\u00a0cm (15)10.0\u00a0\u00b1\u00a09.532.5\u00a0\u00b1\u00a015.532.5\u00a0\u00b1\u00a015.532.5\u00a0\u00b1\u00a015.5Facial nerve resectionNo (23)9.4\u00a0\u00b1\u00a06.327.2\u00a0\u00b1\u00a010.635.3\u00a0\u00b1\u00a012.151.4\u00a0\u00b1\u00a016.70.4Yes (10)25.0\u00a0\u00b1\u00a015.340.0\u00a0\u00b1\u00a018.2\u2013\u2013Involvement of surgical marginsNo (23)14.0\u00a0\u00b1\u00a07.524.7\u00a0\u00b1\u00a09.737.9\u00a0\u00b1\u00a011.753.4\u00a0\u00b1\u00a016.00.3Yes (8)25.0\u00a0\u00b1\u00a021.762.5\u00a0\u00b1\u00a028.6\u2013\u2013Capsular ruptureNo (23)16.4\u00a0\u00b1\u00a08.838.7\u00a0\u00b1\u00a012.838.7\u00a0\u00b1\u00a012.838.7\u00a0\u00b1\u00a012.80.8Yes (8)12.5\u00a0\u00b1\u00a011.712.5\u00a0\u00b1\u00a011.740.0\u00a0\u00b1\u00a018.2100Multiple nodulesNo (7)00000.02Yes (24)19.6\u00a0\u00b1\u00a08.936.8\u00a0\u00b1\u00a011.253.2\u00a0\u00b1\u00a013.4100Postoperative radiotherapyNo (24)14.2\u00a0\u00b1\u00a07.734.3\u00a0\u00b1\u00a011.848.9\u00a0\u00b1\u00a012.965.9\u00a0\u00b1\u00a016.40.3Yes (9)12.5\u00a0\u00b1\u00a011.625.0\u00a0\u00b1\u00a015.325.0\u00a0\u00b1\u00a015.325.0\u00a0\u00b1\u00a015.3\nDiscussion\nIt is generally accepted that the standard treatment of pleomorphic adenoma of the parotid gland is a formal parotidectomy or at least the removal of the tumor with a surrounding cuff of normal parotid tissue after identification, dissection and preservation of the facial nerve [7]. Unfortunately, if the tumor is in contact with the facial nerve, even a total parotidectomy can not guarantee the presence of a cuff of normal tissue, because the surgeon must carry on the dissection between the tumor and the branches of the nerve with the risk of leaving microscopic disease behind or causing microscopic spillage of the tumor. This accounts for 1\u20134% probability of recurrence [5, 7]. These low percentages, together with the increasing number of institutions that treat parotid neoplasms, have reduced the number of cases of RPA being referred to a single institution; this, in turn, has contributed to a lack of experience in managing these tumors. Moreover, with the number of reported patients usually varying from less than 15 to about 50 [6, 8, 9, 11, 12, 14\u201317, 19\u201324], and only a few studies reporting about a hundred patients [10, 13, 18, 25], there has been relatively little discussion of the prognostic factors for RPA [13\u201318].\nFollow-up of parotidectomies is currently performed by ultrasonography, but most patients do not avail themselves of regular follow-up and only 12.1% of our patients with an RPA were diagnosed by routine ultrasonography, while the majority came to our observation for a swelling in the parotid region. In 77.4% of our patients, there was a multinodular lesion (in Stennert et al. [4], 90% were multinodular at pathologic examination), reinforcing the pathogenetic hypothesis of incomplete excision, violation of the pseudocapsule of the tumor, or obvious or underestimated tumor spillage during the first surgery due to the impossibility of obtaining healthy free margins when the lesion is adjacent to the facial nerve.\nDelineation of the extension of the RPA is better depicted by MRI, which better demonstrates the multinodular nature of the disease [24] and potential deep-lobe or parapharyngeal extension. Many patients that we treated came from other institutions without clear documentation of their previous treatment(s); in such cases, MRI can also help in determining the amount of parotid gland still present after any previous surgery. The signal and enhancement characteristics of RPA of the parotid gland are nonspecific [26]. The lesions are usually round; they are of low intensity on T1-weighted images and of high intensity on T2-weighted MR images. With contrast administration, the lesions show mild enhancement [26]. However, MRI can also be inadequate for identifying all nodules, and frequently the surgeon\u2019s microscope or the pathologist\u2019s microscope reveals many more nodules than suspected from clinical assessment [4].\nManagement options for RPA include observation only, localized resection, resection with facial nerve dissection, wide-field resection with facial nerve sacrifice, and radiotherapy [2, 4, 24]. Only observation has been suggested for the elderly or medically infirm patient [2, 4, 24]. When surgical excision is considered, it should be tailored to the individual patient [19, 23]. In general, it is recommended to resect the scars of any previous surgery. Localized resection of the tumor has been employed after multiple recurrences or after previous total parotidectomy when it is the only option to preserve the facial nerve [10, 14]. Superficial or total parotidectomy, depending on the location of the recurrence, has been suggested when the previous operation was a simple local excision [6, 8\u201315, 17, 18, 20, 23\u201325]. Radical and extended parotidectomies have been considered for patients with infiltration of branches or the main trunk of the facial nerve, and in cases of multinodular recurrences [9, 11\u201313, 15, 18, 23\u201325]. In particular, facial nerve resection has been suggested for patients with a history of multiple recurrences or failed radiotherapy [24]. In 30.3% of our patients, the resection of at least a branch of the facial nerve was performed (Table\u00a02). Facial nerve dissection and preservation were performed in all cases with still healthy parotid tissue between the nerve and the tumor, and in the case of facial nerve encasement when a patient refused facial nerve resection. The observation of many cases with unexpected multinodularity persuaded Stennert et al. [4] to routinely perform a total parotidectomy for all RPAs. However, such an aggressive policy of treatment does not prevent leaving microscopic residuals; indeed, a recent study by the same group reported a 52% probability of new recurrences [18].\nThe role of radiotherapy remains questionable [9, 13, 16, 17, 24, 27]. Some authors suggest that radiotherapy should be reserved only for malignant tumors since it carries, especially in young people, a risk of malignant transformation that increases over time [17]. Nonetheless, its use is not infrequent [6, 9, 13\u201316, 19, 20, 27]. Jackson et al. [9] used postoperative radiotherapy in patients in whom histology showed that resection margins of the specimen were not free of tumor or in those with intraoperative spillage of tumor. Renehan et al. [13] recommended postoperative radiotherapy in multinodular recurrences. Douglas et al. [16] reported their experience of fast neutron radiotherapy and recommended radiotherapy for selected cases: when complete extirpation is not possible, when sacrifice of the facial nerve is necessary, or after multiple recurrences. Glas et al. [17] suggested postoperative radiotherapy for difficult cases in which further surgical treatment of recurrent disease is not recommended for technical reasons. Kamida et al. [27] suggested the use of stereotactic radiosurgery for difficult selected cases, such as the patient they treated for skull base invasion with no regrowth after 5\u00a0years. Our experience, including only nine patients, is too limited to draw any conclusion about indications for radiation therapy.\nComplications of treatment of RPA are similar to those for primary surgery for parotid pleomorphic adenoma. The main difference is the higher risk of facial nerve damage in the former, with unexpected permanent facial nerve paralysis in more than 30% of cases [24].\nEven though we did not have cases of malignant transformation, a high incidence is expected, especially after multiple recurrences, reaching up to 15% in the experience of Mercante et al. [22].\nThe main purpose of our study was to analyze risk factors for new recurrence after treatment for RPA. The percentage of new recurrences varies from 10 to 58% [8, 10, 12\u201315, 17\u201320, 23\u201325]. In two surveys of the literature with a 10-year follow-up, the prevalence of a new recurrence was 43 and 45%, respectively [12, 25]. Wittekindt et al. [18] reported a recurrence rate increasing from 42% at 5\u00a0years to 75% at 15\u00a0years. We observed a prevalence of 33.3% with an estimation of recurrence rate increasing from 14.1\u00a0\u00b1\u00a06.6% at 5\u00a0years to 31.4\u00a0\u00b1\u00a09.4% at 10\u00a0years, 43.0\u00a0\u00b1\u00a010.8% at 15\u00a0years, and 57.2\u00a0\u00b1\u00a014.8% at 20\u00a0years (Fig.\u00a01).\nAmong the variables tested with the log-rank test against the estimated new recurrence rate (Table\u00a05), just two were significantly associated with the likelihood of developing a new recurrence: local excision of the RPA instead of a formal parotidectomy and the presence of multinodular disease. To date, no new recurrences have been observed for uninodular recurrences. There was a trend toward increased risk of a new recurrence for deep-lobe localization, although it was not statistically significant.\nFew papers have analyzed prognostic factors for RPA [13\u201318]. Renehan et al. [13] studied 114 patients with first-recurrence RPA and reported significantly better, new recurrence-free survival for patients treated with postoperative radiotherapy when the RPA was multinodular. Age, gender, time to first recurrence, and the type of prior treatment were all not significant predisposing factors for a new recurrence [13]. Yugueros et al. [14] followed 39 patients treated for RPA at their institution: they found no significant difference in development of another recurrence in relation to gender, time after the previous treatment, size of the tumor, type of resection, and postoperative radiotherapy. Carew et al. [15] analyzed 31 patients treated for RPA and concluded that the extent of the first parotid operation was the only factor with a statistically significant impact on tumor control after resection of the recurrence. Local control was achieved in all patients whose initial procedures involved local excision. Superficial lobe location and postoperative radiotherapy showed better results but were not significant [15]. Fast neutron radiotherapy of 16 patients with residuals of RPA stopped progression of the tumor in patients without gross residuals, without multifocal disease and with an interval from diagnosis of less than 25\u00a0years; however, the results were not statistically significant [16]. Glas et al. [17] analyzed 52 patients with RPA, treated after one or more recurrences: no significant influence was seen in the recurrence rate with respect to gender, age at initial treatment, time of first recurrence, and intraoperative tumor spillage. Wittekindt et al. [18], in a group of 108 patients, observed a significantly higher rate of new recurrences in female patients, in younger patients, and in patients treated with a simple enucleation.\nConclusion\nThe management of RPA is a major challenge for the clinician. RPAs, particularly multinodular tumors, are prone to new recurrences especially when treatment of the initial tumor is performed according to currently accepted standards. MRI is considered to be the best tool to delineate the extension of the lesion, although it can miss microscopic nodules. Options for management include pure observation, not only for the elderly or infirm, but also in cases of small lesions until they begin to grow. When surgery is elected, it should be tailored to the single patient, because even if on one side a limited local excision is considered acceptable, on the other side a total or extended parotidectomy may be inadequate to control an RPA adjacent to the nerve. In these cases, facial nerve resection and reconstruction must also be considered and discussed with the patient in the preoperative counseling. Postoperative radiotherapy is an option, particularly for older patients for whom the risk of inducing other malignancies is considered to be low. Follow-up for patients treated for RPA should be done with regular ultrasonography for the lifetime of the patient and with magnetic resonance in selected cases with deep lobe or parapharyngeal involvement. All patients should be informed about the possibility of the need for multiple treatment procedures.","keyphrases":["pleomorphic adenoma","tumor recurrence","parotid gland neoplasms"],"prmu":["P","P","R"]}
{"id":"Clin_Oral_Investig-4-1-2238792","title":"Methodological considerations concerning the development of oral dental erosion indexes: literature survey, validity and reliability\n","text":"Within the context of preventing non-communicable diseases, the World Health Report (2002) and the WHO Global Oral Health Program (2003) put forward a new strategy of disease prevention and health promotion. Greater emphasis is placed on developing global policies in oral health promotion and oral disease prevention. The Decayed, Missing, Filled Teeth (DMFT) index does not meet new challenges in the field of oral health. Dental erosion seems to be a growing problem, and in some countries, an increase in erosion of teeth is associated with an increase in the consumption of beverages containing acids. Therefore, within a revision of the WHO Oral Health Surveys Basic Methods, new oral disease patterns, e.g. dental erosion, have to be taken into account. Within the last 20 years, many studies on dental erosion have been carried out and published. There has been a rapid growth in the number of indexes quantifying dental erosion process in different age groups. However, these indexes are not comparable. This article discusses quality criteria which an index intended for assessing tooth erosion should possess.\nIntroduction\nWithin the context of preventing non-communicable diseases, the World Health Report (2002) and the WHO Global Oral Health Program (2003) put forward a new strategy of disease prevention and health promotion [52]. The main orientation of the WHO Oral Health Program (2003) is that oral health is integral, essential and interrelated to general health and a determinant factor for quality of life. Proper oral health care reduce the burden of disease as well as premature mortality. Risk factors for oral diseases are common with cardiovascular diseases, diabetes, cancer and chronic obstructive pulmonary diseases.\nThe actual WHO Global Oral Health Program focuses on priority action areas as: \u201cDiet, nutrition and oral health\u201d, \u201cOral health and fluorides\u201d, \u201cTobacco and oral health\u201d, \u201cSchool children\u201d, \u201cElderly people\u201d and \u201cOral health services\u201d. Nutrition affects oral health in many ways, e.g. dental erosion. To minimize the occurrence of dental erosion which is particularly related to acidic beverages, the WHO\/FAO (Food and Agricultural Organization of the UN) recommends (within their recently published Global Strategy on Diet, Physical Activity and Health) a reduction in the amount and frequency of intake of soft drinks and juices. WHO recommends that the Ministries of Health should ensure that the mechanisms for intersectorial collaboration are strengthened. Strategies include taxation and pricing, food labeling, school lunch policies and nutrition programs. This WHO Programme is elaborated for all countries of the World. The Regional Offices of WHO play an important role in the improvement of oral health within risk factor approach in disease prevention and health promotion. Therefore, the Global Oral Health Programme will help to achieve greater equity in oral health. But to evaluate the Global Oral Health Programme, we need suitable and validated indicators and indexes [52]. There is currently only one global oral health index to measure oral health. It is the Decayed, Missing, Filled Teeth (DMFT) index. According to the WHO Oral Health Data Bank in the year 2000 from 184 countries, 68% had a DMFT index less than 3 [52]. Up to now, there is only the DMFT index for 12-year-old children in the Health for all 21 Database (www.euro.who.int: HFA-21 DB, downloaded June 13, 2007).\nThe European Commission launched from 2003 to 2005 the EU-Project: European Global Oral Health Indicators coordinated by the Universit\u00e9 Claude Bernard de Lyon, to support European Member States in their efforts to reduce the toll of morbidity, disability related to oral health diseases and especially [18]:\nTo identify indicators of oral health, of critical oral health care, its quality of care and of essential health resourcesTo strengthen the ability at the local, national and regional levels to measure, compare and determine the effects of oral health services and use of resourcesTo identify indicators of oral health (problems, determinants and risk factors related to lifestyle) of critical oral health careTo identify the types of data generation and management problems within the Health Information SystemTo identify principles for guiding the selection and use of oral health indicatorsTo identify a set of core indicators for oral healthTo review the recent oral health-indicator selection efforts\nIn total, 66 indicators are proposed, but no indicator for dental erosion is considered [18]. Therefore up to now, the only international reference in the area of oral health is the DMFT index. The European Commission assesses the quality of oral health data as inadequate for planning, implementation, management and evaluation. The number of internationally recommended indicators complicates the national selection of indicators and the comparability of indicators and indexes [18]. The statements of the European Global Oral Health Indicators Report coincide with the experience in the field of development and using of different erosion indexes. Therefore, it would be necessary to strengthen the international alliance of dentists, epidemiologists, statisticians and other scientists and politicians, to develop an adequate oral health indicators set, i.e. as a subset or user-window of the ECHI-Database (European Community Health Indicators-Database) and within the Oral Databank of the WHO and to develop an internationally agreed and accepted erosion index.\nSocieties around the world today are increasingly witnessing significant changes in diets and lifestyles which have an impact on oral health. Dental erosion seems to be a growing problem, and in some countries, an increase in erosion of teeth is associated with a higher consumption of beverages containing acids [52]. Within a revision of the WHO Oral Health Surveys Basic Methods, new oral disease patterns, e.g. dental erosion, have to be taken into account [52]. The WHO urges the development of methodologies and approaches for evaluating the effectiveness of community oral health programs focusing on health promotion and disease prevention. Additionally, the formulation of new WHO oral health goals up to the year 2020 have been initiated [52].\nMost indexes use different clinical examination standards for measuring tooth erosion especially in preschool- and schoolchildren [25]. Such examination standards could be:\n\u2013The full mouth or partial recording\u2013The examination of primary and\/or of only permanent teeth\u2013The examination of all surfaces or partial recording of surfaces\nOn this basis, a lot of indexes for the clinical diagnosis of erosive tooth wear have been proposed, which are more or less modifications of combinations of the index published by Eccles [17] or Smith and Knight [48]. The most cited examples of Erosion Indexes developed during the last 20\u00a0years are [adopted from 20]:\n\u2013The Smith and Knight Tooth Wear Index (TWI) (1984) [48]\u2013The Eccle\u2019s Index (1979) [17]\u2013UK National Survey of Children\u2019s Dental Health Index (1999\/2003) [35]\u2013Erosion Index according to Lussi (1996) [33]\u2013Modified scoring system of Linkosalo and Markkanen (1985) [19]\u2013Aine Index 1993 [1]\u2013The Larsen and Westergaard Index (2000) [30]\u2013The O\u2019Sullivan Index (2000) [39]\nRecently, the question has arisen how reliable and valid current diagnostic criteria and data on erosion are. The indexes developed and used during the last 20\u00a0years are not comparable; a gold standard does not exist, and validation studies have not had the effect of identifying an index that could be used as a standard for assessing tooth erosion. The following questions should be answered to advance the definition and assessment of tooth erosion:\n\u2013Which erosion indexes are mostly used in the scientific literature?\u2013Which quality criteria should indexes possess?\u2013What differences exist between individual- and population-based erosion indexes?\nFinally in the discussion, we will debate whether it is possible to involve erosion indexes into Health Indicator Sets and into International Classification of Diseases (ICD; e.g. ECHI, HFA21, country databases on oral Health, ICD-11).\nErosion index in the scientific literature\nA literature analysis with regard to erosion indexes for the period 2000\u20132006 was carried out. For this purpose, a Medline research was done considering mesh terms and keywords \u201ctooth erosion\u201d and \u201cdental erosion\u201d in connection with \u201cindex\u201d. All human studies published in English found by this search strategy were analyzed for the erosion indexes used. In the literature analyses, a total of 1,380 articles were found with regard to the mesh terms and keywords \u201ctooth erosion\u201d or \u201cdental erosion\u201d. Approximately 326,112 articles were found with regard to the mesh term and keyword \u201cindex\u201d. The conjunction of both revealed a total of 91 articles. From these articles, 40 were excluded as they were published before the year 2000, three were excluded because they were not English-language publications, eight studies were not included as no humans were involved, and one review was excluded. In total, 40 publications were considered in the literature review. Of 22 publications, 14 cross sectional studies were found in children, and 18 publications\/cross sectional studies were found in adults. The erosion indexes used are presented in Tables\u00a01 and 2 for children and adults, respectively. In children, the UK children dental health survey index [35] was used in four studies, the O\u2019Sullivan Index [39] in three, an index developed in 1993 by Aine et al. [1] was used in two studies, and the Smith and Knight TWI [48] in one study. However, in most studies (n\u2009=\u20094), differently designed erosion indexes were considered. In adults, the Smith and Knight TWI [48] was used in most studies (n\u2009=\u200910) followed by the Eccle\u2019s Index [17] (n\u2009=\u20092), Lussi Index [33] (n\u2009=\u20091), UK adult dental health survey index (n\u2009=\u20091) and an index developed by Schweizer-Hirt et al. 1978 [47]. In one study, the method for estimating the tooth erosion was not mentioned.\nTable\u00a01Used dental erosion indexes in human cross-sectional studies for children found in Medline from the years 2000\u20132006Aim of the studynStudy populationReferred indexSourceErosive tooth wear463Kindergarten, 3- to 5-year-old childrenO\u2019Sullivan IndexWiegand et al. [54]Dental erosion15311-year-oldsO\u2019Sullivan IndexCaglar et al. [10]Prevalence of dental erosion499School setting; 12-year-old childrenO\u2019Sullivan IndexPeres et al. [43]Prevalence of dental erosion1,949Preschool children; 3- to 5-year-old childrenUK Children dental health survey indexLuo et al. [32]Prevalence of dental erosion8326- and 12-year-old school childrenOwn erosion indexTruin et al. [50]SES and ethnicity and oral health1,753Random sample of 12- and 14-year-old childrenUK Children dental health survey indexDugmore and Rock [16]Accuracy and reproducibility of school dental screening570Primary-school childrenNew index was developedHetherington and White [24]Prevalence of and risk factors for dental erosion95Children and adultsOwn erosion indexJohansson et al. [28]Oral health and gastro-oesophageal reflux52Children with gastro-oesophageal reflux diseaseAine IndexLinnett et al. [31]Asthma and dental erosion418Random Sample of 14-year-old childrenSmith and Knight TWIAl-Dlaigan et al. [2]Prevalence of dental erosionOral health and gastro-oesophageal reflux37Children with gastro-oesophageal reflux diseaseAine IndexDahshan et al. [15]Prevalence of dental erosions987Preschool children, 2- to 5-year-old childrenUK Children dental health survey indexAl-Malik et al. [4]Oral health of children with clefts914-, 8-, and 12-year-old childrenUK Children dental health survey indexChapple and Nunn [11]Dental erosion and consumption of oranges1,01012-year-old childrenOwn erosion indexK\u00fcnzel et al. [29]Table\u00a02Used dental erosion indexes in human cross-sectional studies for adults found in Medline from the years 2000\u20132006Aim of the studynStudy populationRefered indexSourceTooth survey (surface?) check! loss155Patients attending a dental hospitalUK Adult dental health survey indexRafeek et al. [44]Tooth wear among psychiatric patients143Psychiatric patientsSmith and Knight TWIAl-Hiyasat et al. [3]Tooth wear in elderly690Local survey, elderlyEccles IndexTaiwo et al. [49]Tooth surface in winemakers36WinemakersSchweizer-Hirt et al. 1978Chikte et al. [12]Workplace and dental erosion20Silicon workersOwn erosion indexJohansson et al. [27]Oral and dental health34Inpatients in treatment of alcohol disordersNo information givenAraujo et al. [6]Dental erosion and gastro-oesophageal reflux?Patients with gastro-oesophageal reflux diseaseSmith and Knight TWIMoazzez et al. [36]Prevalence of dental erosion18,555Permanent dentitionSmith and Knight TWIBorcic et al. [9]Dental erosion and gastro-oesophageal reflux253Patients with gastro-oesophageal reflux diseaseEccles IndexMunoz et al. [37]Monitoring of tooth wear500Patients referred for a variety of restorative procecduresSmith and Knight TWIBartlett [7]Risk factors of tooth wear506Patients attending a dental hospitalSmith and Knight TWIChuajedong et al. [13]Sport drinks and dental erosion304AthletesLussi IndexMathew et al. [34]Prevalence of tooth wear126Patients attending a dental hospitalSmith and Knight TWIOginni and Olusile [41]Recreational drug and tooth surface loss13Undergraduate studentsSmith and Knight TWINixon et al. [38]Risk factors for dental erosion10Male military Saudi inducteesOwn erosion indexJohansson et al. [28]Oral health status of workers68Workers exposed to acid fumesSmith and Knight TWIAmin et al. [5]Methamphetamine and tooth wear43Methamphetamine usersSmith and Knight TWIRichards and Brofeldt [45]Dental erosion and gastro-oesophageal reflux20Patients with gastro-oesophageal reflux diseaseSmith and Knight TWIGregory-Head et al. [21]\nQuality criteria of indexes for measuring tooth erosion\nInstruments or indexes for measuring a construct of interest\u2014quality of life or intelligence, mental health status or tooth erosion\u2014should possess certain quality characteristics. Otherwise, the scientific value of the obtained results is questionable [8]. Important quality characteristics are validity and reliability as well as sensitivity and specificity, which will be explained in the following paragraphs.\nThe validity of an instrument indicates to what extent it measures what it is supposed to measure. There are different\u2014partly overlapping\u2014types of validity which emphasize different aspects [42]. The most important types are content, construct, and criterion validity which will be explained in the following paragraphs.\nContent validity describes whether all aspects, which are relevant to grasp the construct of interest, have been considered adequately. For example, a test that is supposed to measure the ability to calculate should not be restricted only to addition and subtraction but include all basic arithmetic operations. In our context, e. g. the erosion of surfaces of all teeth has to be considered, not only the erosion of incisors. The extent to which an instrument possesses content validity cannot be determined numerically. It is solely based upon the subjective meaning of established experts [8, 23]. Therefore, a major step in developing a new instrument is a thorough discussion of which aspects should be included.\nThe optimal method to validate a newly developed instrument is the comparison with a so-called gold standard, which measures the same construct. A gold standard is \u201cthe method, procedure or measurement that is widely accepted as being the best available\u201d [46]. The so-called criterion validity is high if the results of the new instrument and the gold standard are highly correlated. For example, a thermometer might serve as a gold standard for self-reported temperature [23].\nHowever, because a gold standard is seldom available, construct validity is of major importance [8]. Construct validity is subdivided into convergent and discriminant validity. There is convergent validity if the results obtained by the new instrument are correlated with the results of an established instruments that measures similar aspects. If, for example, results of a new instrument for measuring physical health are correlated with the results of an established instrument which measures activity of daily living, convergent validity is indicated [8]. Or in our context: if the results of a tooth-wear index is correlated with tooth-erosion index, convergent validity is indicated. On the other hand, an instrument possesses discriminant validity if the results of this instrument are not too highly correlated with the results of an established instrument that measures a different construct. For example, results of an instrument for measuring mental health should not be too highly correlated with an instrument for measuring physical function [23]. With regard to teeth, a high correlation between tooth erosion and wedge-shaped defects might indicate insufficient discriminant validity. Both convergent and discriminant validity have to be given for a complete confirmation of construct validity.\nThe reliability of an instrument indicates how precise it is able to measure, independent of whether it really measures what it is supposed to (this is a question of validity) [42]. There are different aspects of reliability. For our purposes, inter-examiner reliability and intra-examiner reliability are important.\nInter-examiner reliability can be determined if two or more dentists (the \u201cexaminer\u201d) assess dental erosion of a number of patients independently of each other [14]. Roughly spoken, the more often the examiners agree in their assessment, the higher the inter-examiner reliability. Intra-examiner reliability or test\u2013retest reliability can be determined if each dentist rates dental erosion of a group of patients twice [23]. The period of time between the two assessments should be fairly long, otherwise the examiners may be overly consistent because they remember their former ratings. On the other hand, if the time interval between the two assessments is too long, changes in the erosion status might bias the reliability estimates. The more often the assessments at the two points in time concerning each examiner are identical, the higher the intra-examiner reliability. A widespread index to calculate the degree of agreement is Cohen\u2019s Kappa [14], which \u201cis now fairly well disseminated as one of the standard summary statistics used in the medical literature\u201d [22]. The calculation of Kappa is based on the percentage of agreement which is adjusted for agreement expected by chance.\nThe sensitivity of an instrument indicates its ability to detect\u2014in our case\u2014dental erosion. In contrast, an instrument with high specificity is able to indicate no dental erosion if dental erosion is not present. Both assessment of sensitivity and specificity require the comparison with a gold standard. Comparing a new instrument that is supposed to detect dental erosion with an instrument that is known to be able to detect dental erosion, i.e. a gold-standard instrument, can yield one of the four following outcomes: (1) dental erosion is indicated by the new instrument if there is in fact dental erosion (correct positive), (2) dental erosion is indicated if there is no dental erosion (false positive), (3) dental erosion is not indicated if there is dental erosion (false negative) and (4) dental erosion is not indicated if there is no dental erosion (correct negative). The degree of sensitivity is calculated by dividing the frequency of correct positive results by the sum of correct positive and false negative results, while the degree of specificity is determined by dividing the frequency of correct negative results by the sum of correct negative and false positive results [46].\nIndividual versus population-based erosion indexes\nAn individual-based index should fulfill other criteria than a population-based index. The first should allow assessment of the full truth of the construct \u201ctooth erosion\u201d. Therefore, a very good individual-based erosion index should nearly be a gold-standard instrument. Using a population-based erosion index, the individual assessment should take less time to allow an assessment in big population samples. Therefore, for practical reasons, a population-based erosion index is often a short form of an individual-based index. However, a good population-based erosion index should possess very high validity as well as high reliability.\nDiscussion\nThere is no common sense in the usage of the above-mentioned erosion indexes. A wide range of different forms and indexes was used in the last 6\u00a0years. Therefore, comparability between different studies was not given, and meta-analyses were not possible. If anything, it can be stated that for adults, the Smith and Knight TWI [48] has been widely used in most Medline-cited cross-sectional studies. For children, there is no index that is most used. However, the literature analysis is limited. Only one search strategy was used. Some articles which do not use the mentioned key words or mesh headings might not be included in the analysis. However, the results presented help to get a first view on the different indexes on tooth erosion cited in the last 6\u00a0years.\nIn all these indexes, the criteria to record the grading for erosion differed [26]. Most of the indexes used are based on the clinical severity of erosion, focusing on accessible teeth but not all [29]. For example, the structure of the Smith and Knight TWI is shown in Table\u00a03. However, this index is made for scoring tooth wear in general irrespective of its predominant aetiology. It is only a system for quantifying and grading the amount of tissue loss but does not include diagnostic criteria for erosion. The UK National Survey of Children\u2019s Dental Health Index was a modified version of the Smith and Knight TWI assessing only buccal or palatal surfaces of maxillary incisors and added erosion criteria. However, while using the first index, the conclusion was drawn that the figures might not be completely accurate [40]. The next version focused on erosion of the palatal surface of at least one maxillary anterior tooth with the majority of surfaces having dentin involvement [29]. To date, literature assessing the prevalence of erosion has been published from different countries. Erosion was recorded using various indexes. In addition, selection criteria of study populations, sampling techniques and considered age groups differed. The prevalence of dental erosion of different studies is therefore difficult and nearly impossible to compare [29].\nTable\u00a03Smith and Knight Tooth Wear Index [48]ScoreSurfaceCriterion0B\/L\/O\/I\/CNo loss of surface characteristic, no loss of contour1B\/L\/O\/I\/CLoss of enamel surface characteristics, minimal loss of contour2B\/L\/OLoss of enamel exposing dentine for less than one third of the surfaceILoss of enamel just exposing dentineCDefect less then 1\u00a0mm deep3B\/L\/OLoss of enamel exposing dentine for more than one third of surfaceILoss of enamel and substantial loss of dentine not exposing secondary dentine or pulpCDefect 1\u20132\u00a0mm deep4B\/L\/OComplete loss of enamel, or pulp exposure, or exposure of secondary dentineIPulp exposure or exposure of secondary dentineCDefect more than 2\u00a0mm deep, or pulp exposure, or exposure of secondary dentineB Buccal or labial, L lingual or palatal, O occlusal, I incisal, C cervical\nA good index is characterized by a conducted validation study. For those indexes for which a validation study has not been conducted so far, that should be made up to check whether they possess the quality criteria as described above. In a validation study, it is not only the validity of an index that is examined but also aspects of reliability as well. Intra-examiner and inter-examiner reliability should be checked. That is done quite easily as described in \u201cQuality criteria of indexes for measuring tooth erosion\u201d. At the beginning of the study, it has to be considered how many patients and how many examiners respectively have to be involved. This is because the number of patients and examiners should be as small as possible for practical and economic reasons. On the other hand, the number of patients and examiners has to be sufficiently high because otherwise a certain degree of reliability, which actually exists, might be overlooked. Therefore, solid sample-size calculations should be conducted with the help of an experienced statistician.\nSo far, there is no consensus concerning a gold-standard instrument. However, a gold standard is important for comparison. Otherwise, criterion validity cannot be checked. If one attempts to develop a gold-standard instrument, the first step would be a thorough discussion of which aspects of the construct \u201ctooth erosion\u201d have to be included. This should be conducted by established experts to ensure content validity. When the instrument is constructed, inter-examiner reliability and intra-examiner reliability can be examined as described above. However, this should be done not only in a national but in an international context.\nA gold-standard instrument would explain the full truth of the construct \u201ctooth erosion\u201d. Nevertheless, the measurement procedure in general takes a long time. It is not possible to deal with such a comprehensive assessment instrument in a population-based study design. Therefore, more simple assessment instruments should be available. These assessment instruments should fulfill the following qualifications: (a) a validation study should be available where all quality criteria of the assessment instrument were tested and (b) the instrument should be used in other scientific studies, too. If the population-based short version of an assessment instrument and the gold-standard instrument are measuring the same construct, and when both conditions are fulfilled, calibration studies might be possible. Calibration refers to a process in which values from one method are quantitatively related to values from a superior, gold-standard method. The aim of such an analysis is to ensure that the measurement uncertainty is known and is consistent with the required measurement capability. In a so-called calibration study, known data on the observed relationship between an independent variable of the population-based short version of the assessment instrument and the dependent variable of the gold-standard instrument are used to make estimates of other \u201ctrue\u201d values of the independent variable from new observations of the gold standard.\nOne of the prospective targets ought to be the inclusion of indexes on dental health prevention especially of an internationally agreed \u201cerosion index\u201d into the existing Health Indicator Sets. These would be the Health for All Database of the WHO (HFA21), The European Community Health Indicator Set (ECHI), Country Databases on Oral Health and the inclusion of Dental Erosion into the International Classification of Diseases and Health-Related Problems.\nIn April 2007, the World Health Organization announced plans to revise the ICD-10, in other words, to prepare for ICD-11. Within the actual ICD-10, there exists two codes for Dental Diseases: \u201cK02\u201d for Caries and \u201cK03\u201d for \u201cOther diseases of hard tissues of teeth\u201d including with \u201cK03.2\u2014Erosion of teeth.\u201d On the four-digit level, we find the following different diagnoses presented in Table\u00a04.\nTable\u00a04Code description for K03.2\u2014Erosion of teeth (ICD 10 code) [51]ICD 10 codeDescriptionK03.2Erosion of teethK03.20Occupational erosion of teethK03.21Erosion of teeth due to persistent regurgitating or vomitingK03.22Erosion of teeth due to dietK03.23Erosion of teeth due to drugs and medicamentsK03.24Idiopathic erosion of teethK03.28Other specified erosion of teethK03.29Erosion of teeth, unspecified\nThe Updating and Revision Committee proposes major changes like addition of new codes or deletion of codes and developed submission guidelines. Therefore, it would be possible to check critically if the codes for dentistry within the Application of the International Classification of Diseases to Dentistry and Stomatology are adequate to future developments or have to be replaced or completed [53].\nConclusion\nFurther efforts have to be made in the development of an internationally agreed index which is able to assess dental erosion with as much reliability and validity as possible. Current recommendations of the WHO and the European Union and health task forces within countries and Dental Associations should be used to develop and to discuss the concept of tooth erosion and the development of a unified erosion index.","keyphrases":["index","validity","reliability","tooth erosion","gold standard"],"prmu":["P","P","P","P","P"]}
{"id":"Appl_Microbiol_Biotechnol-3-1-1914249","title":"Co-existence of physiologically similar sulfate-reducing bacteria in a full-scale sulfidogenic bioreactor fed with a single organic electron donor\n","text":"A combination of culture-dependent and independent methods was used to study the co-existence of different sulfate-reducing bacteria (SRB) in an upflow anaerobic sludge bed reactor treating sulfate-rich wastewater. The wastewater was fed with ethanol as an external electron donor. Twenty six strains of SRB were randomly picked and isolated from the highest serial dilution that showed growth (i.e. 108). Repetitive enterobacterial palindromic polymerase chain reaction and whole cell protein profiling revealed a low genetic diversity, with only two genotypes among the 26 strains obtained in the pure culture. The low genetic diversity suggests the absence of micro-niches within the reactor, which might be due to a low spatial and temporal micro-heterogeneity. The total 16S rDNA sequencing of two representative strains L3 and L7 indicated a close relatedness to the genus Desulfovibrio. The two strains differed in as many as five physiological traits, which might allow them to occupy distinct niches and thus co-exist within the same habitat. Whole cell hybridisation with fluorescently labeled oligonucleotide probes was performed to characterise the SRB community in the reactor. The isolated strains Desulfovibrio L3 and Desulfovibrio L7 were the most dominant SRB, representing 30\u201335% and 25\u201335%, respectively, of the total SRB community. Desulfobulbus-like bacteria contributed for 20\u201325%, and the Desulfobacca acetoxidans-specific probe targeted approximately 15\u201320% of the total SRB. The whole cell hybridisation results thus revealed a consortium of four different species of SRB that can be enriched and maintained on a single energy source in a full-scale sulfidogenic reactor.\nIntroduction\nMany industrial processes, such as metal smelting, flue gas scrubbing and mining generate sulfate-rich wastewater (Lens et al. 1998). These wastewaters usually do not contain any organic carbon, and the addition of external electron donor is required for their treatment. These waste streams cause a range of problems associated with the now well-established anaerobic treatment of these wastewaters, such as a decrease in methane production (Colleran et al. 1995), sulfide toxicity (O\u2019Flaherty and Colleran 2000), foul smell (Lens et al. 2001) and corrosion (Vincke et al. 2001). Of late, bioreactor processes have been developed for treating these sulfate and metal-rich wastewaters. This technology developed by the company Paques BV in The Netherlands (Pol et al. 2001) makes use of the dissimilatory sulfate-reducing capacity of sulfate-reducing bacteria (SRB) to simultaneously remove sulfate and metals in the form of metal sulfides. A typical application is a two-step process that includes dissimilatory sulfate reduction to sulfide (Gibson 1990) as the first step. Subsequently, the sulfide will be bound by heavy metals, such as cobalt, lead, nickel and zinc and precipitated as metal sulfide (Colleran et al. 1995). The second step is the biological oxidation of the remaining sulfide to insoluble elemental sulfur, which is either recovered by sedimentation or a small portion converted to H2SO4 and re-used to neutralise the alkalinity generated because of sulfide production in the first step.\nSeveral full-scale sulfate removal bioreactors are currently in operation. The dominant sulfidogenic communities in these bioreactors are heterotrophic SRB belonging to the family Desulfovibrionaceae (Kaksonen et al. 2004; van Houten et al. 2006). The genus Desulfovibrio represents a phylogenetically coherent group; all species incompletely oxidise lactate to acetate but can utilise hydrogen, formate and ethanol as well (Widdel and Bak 1992).\nIn an engineered system fed with a single nutritional source and kept under constant operational parameters such as pH, temperature and salinity, competition among species might tend to reduce the species and sub-species diversity according to the principle of competitive exclusion (Gause 1934). However, physiologically competing species can co-exist if they occupy different niches, whereby each species uses distinct parts of the resource base. In the present study, we investigated the co-existence of physiologically similar hydrogenotrophic SRB in a full-scale sulfidogenic bioreactor treating sulfate-rich wastewater using a combination of cultivation and molecular techniques. The reactor was fed with ethanol as carbon and energy source. SRB isolated in pure culture were characterised genetically and physiologically. Micro-diversity among the dominant culturable isolates was assessed by repetitive enterobacterial palindromic polymerase chain reaction (rep-PCR) (Versalovic et al. 1994). Whole-cell hybridisation with fluorescently labelled general and specific probes was used for SRB community characterisation and for the estimation of the relative abundance of the different SRB populations in the reactor.\nMaterials and methods\nSludge source\nGranular sludge was obtained from an upflow anaerobic sludge bed (UASB) reactor, treating sulfate-rich wastewater from a chemical plant located in Emmen, The Netherlands. The wastewater fed to the reactor did not contain any organic compound, so ethanol was added as an external electron donor and carbon source. The reactor had a volume of 300\u00a0m3 and a feed rate of 60\u201375\u00a0m3 h\u22121 The ratio between the amount of electron donor added and the amount of sulfate in the reactor was around 0.4\u00a0kg\/kg. Sulfide produced in the reactor was either converted to elemental sulfur through biological sulfide oxidation or precipitated with toxic metals. The reactor was operated at a temperature of 30\u00b0C and a pH of 7.0\u20137.5. The concentration of sulfate in the wastewater was approximately 1,500\u00a0mg\/l and ca. 100\u00a0mg\/l in the effluent.\nCulture media and isolation of SRB\nA basal bicarbonate-buffered and sulfide-reduced medium was used for the enumeration and isolation procedures. The mineral medium contained per litre of distilled water: 0.2\u00a0g KH2PO4, 0.25\u00a0g NH4Cl, 0.5\u00a0g KCl, 0.1\u00a0g CaCl2\u00b72H2O, 0.4\u00a0g MgCl2\u00b76H2O, 1.0\u00a0g NaCl and 0.5\u00a0ml of a resazurin solution (0.5\u00a0mg ml\u22121) as a redox indicator. The medium was supplemented with (per litre) the following: 30\u00a0ml 1\u00a0M NaHCO3 solution, 1\u00a0ml of a vitamin solution, 1\u00a0ml of trace element solution with ethylenediamine tetraacetic acid (EDTA; Widdel and Bak 1992) and 0.1\u00a0g of yeast extract. As a reducing agent, 7.5\u00a0ml l\u22121 of 0.2\u00a0M Na2S\u00b79H20 was added. Either lactate or ethanol (20\u00a0mM) was used as an electron donor and sulfate (10\u00a0mM) as electron acceptor. Enumeration of the potentially dominant heterotrophic sulfate reducers was performed by serial dilutions in Hungate tubes. The Hungate tubes were incubated at 30\u00b0C in the dark for 7\u00a0weeks. The highest positive dilution tubes that showed growth were selected for further isolation. Growth was assayed by measuring sulfide production photometrically (Cord-Ruwisch 1985).\nPure cultures were obtained by repeated transfer in agar shake tubes (Widdel and Pfennig 1984). Purity of the isolates was checked by microscopic observation and further confirmed by denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA gene fragments (Teske et al. 1996).\nPhenotypic characterisation\nSubstrate utilisation by the isolated strains was determined in duplicate in the same bicarbonate-buffered medium as used for enumeration and isolation but without yeast extract. Growth was tested in 100-ml serum bottles closed with butyl rubber stoppers and aluminium crimp seals. The inoculum size was 1% (v\/v). The cultures were incubated at 30\u00b0C for 5\u00a0weeks in the dark. Growth was determined by substrate consumption or product formation as well as by checking for increase in optical density at 660\u00a0nm (OD660). The following substrates were tested as electron donors in the presence of 10\u00a0mM sulfate: 10\u00a0mM each of pyruvate, fumarate, butyrate, formate, propanol, ethanol, methanol, serine and cysteine; 5\u00a0mM each of malate, glycolate and glycerol; 2.5\u00a0mM of benzoate; and 0.5% (w\/v) of casamino acids. Sulfite, thiosulfate and 2% (w\/v) elemental sulfur were tested as electron acceptors in the presence of lactate as electron donors.\nFor measuring catalase activity, the fully grown isolates were centrifuged at 13,000\u2009\u00d7\u2009g. The cell pellets were re-suspended on glass slides with a drop of 3% (v\/v) H2O2, bubbles indicated the presence of catalase. Detection of desulfoviridin was performed according to Postgate (1959). Gram staining was performed as previously described (Bartholomew 1962).\nAnalytical methods\nAcetate and other volatile fatty acids were analysed either by gas chromatography (GC) or high performance liquid chromatography (HPLC). For GC, a Chromopack 9001, equipped with a flame ionisation detector and a fused-silica capillary column 15\u2009\u00d7\u20090.53\u00a0mm HP-Innowax, was used. The column temperature was 120\u00b0C. The temperature of the injector and detector were 180 and 200\u00b0C, respectively. Helium was used as a carrier gas. An Aminex HPX-87H column from Bio-Rad (T\u2009=\u200960\u00b0C) coupled to a UV and a RI detector was used for HPLC; phosphoric acid (0.05\u00a0M) was used as an eluent. Sulfide was measured quantitatively by a colorimetric assay (Cline 1969).\nDNA extraction\nGenomic DNA was isolated from the bacterial cultures using the Ultra Clean Soil DNA extraction kit (MOBIO Laboratories, California) according to the manufacturer\u2019s protocol. The quality of the extracted DNA was examined on a 1% (w\/v) agarose gel and the amount quantified by absorption spectrophotometry using the Nanodrop ND-1000 TM (NanoDrop Technologies, Delaware). Extracted DNA was stored at \u221220\u00b0C until subsequent use in different PCR reactions.\nPCR amplification and DGGE of 16S rRNA genes\nFor DGGE analysis, amplification of partial 16S rRNA gene was carried out using the primers 341F-GC and 907R as described by (Sch\u00e4fer and Muyzer 2001), while primers GM3 and GM4 (Muyzer et al. 1995) were used to amplify the nearly complete 16S rRNA gene for sequencing and subsequent phylogenetic analysis. PCR amplification and DGGE was performed as described previously (Sch\u00e4fer and Muyzer 2001). The quality of the PCR products was examined on 1% (w\/v) agarose gel, and the yield was quantified by absorption spectrophotometry using the Nanodrop ND-1000 TM (NanoDrop Technologies).\nDNA sequencing and phylogenetic analysis\nThe nearly complete 16S rRNA gene fragments, obtained from the strains L3 and L7, were purified using the Qiaquick Gel Extraction Kit (Qiagen, Hilden, Germany). Purified PCR products were sequenced by the company BaseClear (Leiden, The Netherlands). The DNA sequences of about 1,400\u00a0bp were first compared to the sequences deposited in public databases using the NCBI BLAST search tool (http:\/\/www.ncbi.nlm.nih.gov\/BLAST; McGinnis and Madden 2004). Subsequently, the sequences were imported into the ARB software programme (Ludwig et al. 2004) and aligned using the automatic aligner function. The alignment was further corrected manually, and a phylogenetic tree was constructed using the neighbour-joining algorithm with Felsenstein correction.\nrep-PCR fingerprinting\nThe genetic diversity of the isolates was analysed by rep-PCR (Versalovic et al. 1994) using the primer GTG5 (5O\u2032-gTggTggTggTggTg-3\u2032). The amplification reaction was performed as previously described (Foti et al. 2006). A 1-kb size marker and 600\u00a0ng of the PCR product were loaded onto a 1.5% (w\/v) agarose gel containing 0.5\u2009\u00d7\u2009TAE-buffer (200\u00a0mM Tris-acetate, 0.5\u00a0mM EDTA, pH\u00a08). The electrophoresis was performed for 14\u00a0h in a cold room at a constant voltage of 65\u00a0V. The gel was subsequently stained with ethidium bromide (0.5\u00a0\u03bcg\/ml) and photographed under UV illumination using the GelDoc UV Transilluminator (Bio-Rad, Hercules, CA).\nSodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis\nDenaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell protein (in cell-free extract obtained by sonication) was performed on 12% polyacrylamide gels according to Laemmli (1970). The gels were stained with Coomassie Brilliant Blue and photographed using visible light.\nDesign of oligonucleotide probes\nSpecific oligonucleotide probes for the 16S rRNA of the two strains were designed using the Probe Design tool of the ARB software package (Ludwig et al. 2004). The requirements for designing the probes included high specificity, with no organism outside the intended target group having 100% similarity within the target sequence, and the target sequence being located within the high accessibility region of 16S rRNA molecule as suggested previously (Behrens et al. 2003). The probes were named with a number that indicates the position of the first base in the target sequence (by Escherichia coli numbering). The oligonucleotides used for in situ hybridisation are given in Table\u00a01.\nTable\u00a0116S rRNA-targeted oligonucleotide probes used in this studyProbe nameaTarget organismsProbe sequence (5\u2032\u20133\u2032)ReferenceEUB338_IMost bacteriaGCT GCC TCC CGT AGG AGTAmann et al. (1990)EUB338_IIPhylum PlanctomycetesGCA GCC ACC CGT AGG TGTDaims et al. (1999)EUB338_IIIPhylum VerrucomicrobiaGCT GCC ACC CGT AGG TGTDaims et al. (1999)ARCH915ArchaeaGTG CTC CCC CGC CAA TTCStahl and Amann (1991)SRB385Most DeltaproteobacteriaCGG CGT CGC TGC GTC AGGAmann et al. (1990)SRB385DbSome DeltaproteobacteriaCGG CGT TGC TGC GTC AGGRabus et al. (1996)DSR660Genus DesulfobulbusGAA TTC CAC TTT CCC CTC TGDevereux et al. (1992)DSBA1017Desulfobacca acetoxidansGTT GCC AGG CAC CCC CATDar et al. (2007)DSV119Desulfovibrio strain L3GGC AGA TCA TCC ACG CGTThis studyDSV139Desulfovibrio strain L7CGC TGT TAT CCC GAT CACThis studyaEUB338 is a combination of EUB338_I, EUB338_II and EUB338_III.\nWhole cell hybridisation\nCells from strains L3 and L7 and from the original reactor were fixed, washed and spotted onto Teflon-coated multi-well microscopic slides as described previously (Dar et al. 2007). Hybridisation was carried out according to the protocol described by Manz et al. (1992) using a formamide concentration of 35% (v\/v). Quantification of the hybridised cells was performed as described previously (Neef et al. 1996). The hybridised cells were analysed by two independent observers for determining the fraction of positive signal from each probe relative to the signal visualised with general probes for bacteria (EUB338 I, II and III), SRB (SRB385 and SRB385Db) or with the general DNA stain DAPI (4\u2032, 6\u2032 -diamidino-2-phenylindole). In addition, a general probe specific for members of the domain Archaea (ARCH915) was used. The hybridisation experiments were done in duplicate using different fluorochromes for each probe. Different microscopic fields on each slide were analysed to confirm the results. Hybridisation stringencies of the newly designed probes were determined by performing hybridisations with increasing formamide concentrations as described previously (Manz et al. 1992) using target organism(s) and non-target organism displaying three mismatches within the target region.\nSequence accession numbers\nThe nearly complete rRNA gene sequences of strains L3 and L7 have been deposited in GenBank under accession nos. EF055876 and EF055877, respectively.\nResults\nReactor performance\nThe amount of ethanol dosed to the reactor was continuous and limited to the amount required for sulfate reduction according to the net stoichiometric reaction for sulfidogenic oxidation of ethanol and its major degradation intermediate, acetate. The equations are as follows:\nThe sulfate removal efficiency at the time of sampling was more than 93%. Less than 1\u00a0mg of acetate was estimated in the effluent and no significant biogas (CH4) production from the oxidation of organic source was observed at the time of sampling.\nEnumeration and isolation of SRB\nThe maximum number of culturable SRB observed with lactate or ethanol as substrate in the serial dilution tubes was in the order of 108 cells ml\u22121. The tubes with growth in the highest dilution were used for isolation in agar shake tubes. In total, 26 strains were obtained in pure culture, 17 on lactate and nine on ethanol as substrate. Sub-cultivation of the isolated strains was carried out on lactate. Apart from microscopic observation, the purity of the isolated strains was confirmed by single bands obtained from the DGGE of partial 16S rRNA genes amplified from the strains. The DGGE results of the 26 isolated strains identified two melting types (results not shown).\nGenomic fingerprinting\nThe isolated strains were subjected to genomic fingerprinting, i.e. rep-PCR, to resolve any higher degree of genetic diversity among the strains. The rep-PCR profiles of the strains (Fig.\u00a01) revealed the presence of two distinct genotypes with no micro-diversity among the strains. Furthermore, the whole cell protein profile of representative strains of the two groups, grown on the same substrate (i.e. lactate and sulfate) and under similar conditions of pH and temperature, gave only two distinct profiles (Fig.\u00a02), confirming the presence of two genotypes. Two isolates, strain L3 and L7, were chosen as representatives of the two genotypes and were subjected to a more detailed phylogenetic and phenotypic analysis.\nFig.\u00a01rep-PCR patterns of different strains isolated from the full-scale sulfidogenic bioreactor. M is the molecular weight markerFig.\u00a02Whole cell protein profile of different strains isolated from the full-scale sulfidogenic bioreactor. M is the molecular weight marker. The arrows depict protein bands that are expressed in one or the other genotype\nPhylogenetic analysis\nA similarity check using the NCBI BLAST search tool of the nearly complete 16S rRNA gene sequences obtained from strain L3 and L7 indicated 98% sequence similarity to Desulfovibrio strain SB1 and 99% sequence similarity to Desulfovibrio mexicoense, respectively. The phylogenetic affiliation of the obtained sequences is presented in Fig.\u00a03. A neighbour-joining tree based on nearly complete 16S rRNA gene sequences was generated, confirming the close affiliation of the isolated strains L3 and L7 to Desulfovibrio strain SB1 and Desulfovibrio mexicoense, respectively.\nFig.\u00a03Neighbour-joining tree based on nearly complete 16S rRNA gene sequences showing the phylogenetic affiliation of the two isolated SRB, strain L3 and L7. The sequence of Desulfobacter postgatei was used as an outgroup but was pruned from the tree. Dots on the nodes indicate bootstrap values of 90% or higher (1,000 replicates).The bar indicates 1% sequence difference\nPhenotypic characterisation of the strains\nAll isolates had a vibrio to spiral cell morphology. Two distinct groups could be identified based on size and motility. One group of cells related to Desulfovibrio strain SB1 was motile, and their size ranged from 4\u20137\u00a0\u03bcm in length and 1\u00a0\u03bcm in width. The other group was non-motile, and the size was 1\u20132\u00a0\u03bcm in length and 0.5\u00a0\u03bcm in width.\nTable\u00a02 shows the substrate utilisation patterns of the two isolates. Both strains used lactate, ethanol, pyruvate, glycerol and casamino acids as electron donors. Hydrogen and formate could only be used as substrates in the presence of acetate as carbon source. The organic substrates were incompletely oxidised to acetate. Acetate, propionate, butyrate, glycolate and methanol could not be used as electron donors by both strains. Fumarate and malate were used by strain L3 but not by strain L7, while serine and cysteine were utilised by strain L7 but not by strain L3. Sulfate, thiosulfate and sulfite were used as electron acceptors by both strains. In addition, strain L7 could also use elemental sulfur as an electron acceptor.\nTable\u00a02Phenotypic characterisation of strains L3 and L7CharacteristicsStrain L3Strain L7Cell morphologyVibrioVibrioMotility+\u2212Desulfoviridin++Catalase++Gram staining\u2212\u2212Electron donors\u00a0H2 plus acetate++\u00a0Pyruvate++\u00a0Lactate++\u00a0Acetate\u2212\u2212\u00a0Propionate\u2212\u2212\u00a0Fumarate+\u2212\u00a0Butyrate\u2212\u2212\u00a0Formate++\u00a0Propanol\u2212\u2212\u00a0Ethanol++\u00a0Methanol\u2212\u2212\u00a0Serine\u2212+\u00a0Cysteine\u2212+\u00a0Malate+\u2212\u00a0Glycolate\u2212\u2212\u00a0Glycerol++\u00a0Benzoate\u2212\u2212\u00a0Casamino acids++Electron acceptors\u00a0Thiosulfate++\u00a0Sulfite++\u00a0Elemental sulfur\u2212+\nWhole cell hybridisation\nAfter isolation of the most abundant culturable SRB, whole cell hybridisation with specific oligonucleotide probes was performed to estimate the abundance of strains L3 and L7 in the original sludge sample. The specificity of the designed oligonucleotide probes (DSV119 and DSV139) was verified using growing cells of strain L3 and Desulfovibrio gigas for probe DSV119 and cells of strain L7 and Desulfovibrio mexicoense for probe DSV139. Strain L3 served as a non-target species for L7 and vice versa. A formamide concentration of 35% (v\/v) was found stringent enough to discriminate between the two strains (Fig.\u00a04a). In addition, probes specific for cells of Desulfobulbus (i.e. DSR660) and Desulfobacca acetoxidans (i.e. DSBA1017), which we had detected previously in a similar sulfidogenic reactor (Dar et al. 2007), were also used.\nFig.\u00a04a Mixture of cells of strain L3 and L7 hybridised with probe Dsv139 labelled with Cy5 (blue), probe Dsv119 labelled with Cy3 (red) and probe SRB385 labelled with Fluos (green). b Sludge sample from a full-scale sulfidogenic bioreactor hybridised with probe Dsv139 labelled with Cy3 (red), probe Dsv119 labelled with Cy5 (blue) and SRB385 labelled with Fluos (green). c Sludge sample hybridised with probe Dsv139 labelled with Cy5 (blue), probe Dsv119 labelled with Cy3 (red) and EUB338 labelled with Fluos (green). d Sludge sample hybridised with probe DSR660 labelled with Fluos (green), probe SRB385 labelled with Cy3 (red) and EUB338 labelled with Cy5 (blue). e Sludge sample hybridised with probe SRB385 labelled with Fluos (green), probe DSBA1017 labelled with Cy3 (red) and probe EUB338 labelled with Cy5 (blue). f Sludge sample hybridised with probe Arch915 labelled with Fluos (green), probe SRB385 labelled with Cy3 (red) and EUB338 labelled with Cy5 (blue). Bar is 20\u03bcm\nThe relative percentage of cells that hybridised with probe DSV119, which is specific for strain L3, was 30\u201335% of the total SRB385-positive cells and 15\u201320% of the EUB338-positive cells (Table\u00a03; Fig.\u00a04b,c). The probe specific for strain L7, i.e. DSV139, was detected between 25\u201335% of the SRB385-positive cell and 10\u201320% of the EUB338-positive cells (Table\u00a03; Fig.\u00a04b,c). The Desulfobulbus and Desulfobacca-specific probes also gave a positive signal, but these SRB were somewhat less abundant than Desulfovibrio positive cells. The Desulfobulbus-specific probe, DSR660, targeted approximately 20\u201325% of the total SRB cells and 10\u201315% of the total bacterial cells (Table\u00a03; Fig.\u00a04d). The Desulfobacca acetoxidans-specific probe DSBA1017 targeted approximately 15\u201320% of the total SRB cells and 8\u201310% of bacterial cells (Table\u00a03; Fig.\u00a04e). The percentage of cells that hybridised with probe specific for Archaea (probe ARCH915) was less than 0.1% of the total DAPI stained cells (Table\u00a03; Fig.\u00a04f).\nTable\u00a03Relative abundance of SRB and ArchaeaaProbeSRB385EUB338DAPIDSV11930\u20133515\u20132012\u201316DSV13925\u20133510\u2013208\u201316DSR66020\u20132510\u2013158\u201312DSBA101715\u2013208\u2013106\u20138ARCH915\u2013\u2013<0.1aPercentage of positive cells relative to those detected by a mixture of probes SRB385 and SRB385Db (SRB385), a mixture of probes EUB338 I, II and III (EUB338) and to cells stained with the DNA stain DAPI.\nDiscussion\nBased on the observed sulfate removal efficiency (>93%) and the amount of ethanol dosed to the reactor, it may be assumed that a major part of electron flow from the substrate is scavenged by the SRB. The presence of less than a milligram of acetate in the effluent stream with no significant production of CH4 points to the oxidation of ethanol mainly through sulfate reduction, although fermentation of ethanol through acetogenesis cannot be ruled out. The metabolic products of fermentation and acetogenesis, mainly acetate and hydrogen, may serve as substrates for methanogens and sulfate reducers; however, under high sulfate concentrations in the reactor, hydrogen and acetate would be more readily used by hydrogenotrophic and acetate-utilising sulfate reducers, respectively, because of more favourable substrate affinity (Ks) values of the SRB for these substrates (Stams et al. 2005). Fermentation of ethanol with propionate as the major reduced end product has also been described in the literature (Schink 1984; Tholozan et al. 1992); and under such fermentation, propionate will serve as an energy source for the members of the genus Desulfobulbus among the SRB.\nGenetic diversity among the dominant culturable isolates\nCompared to high micro-diversity among phylogenetically similar strains detected in sediments (Sass et al. 1998; Wieringa et al. 2000; Klepac-Ceraj et al. 2004), no such micro-diversity was observed within the 26 strains isolated from the bioreactor. The chemical complexity of sediments with steep gradients of substrate concentrations, redox potential and pH may give rise to a number of physico-chemical and depth-defined micro-niches, resulting in the evolution of co-existing but genetically distinct sub-populations (Gray et al. 1999). In addition, Torsvik et al. (2002) suggested structural complexity of sediments being an important factor that allows nutritional partitioning, creating numerous niches that, in turn, results in genetic diversification of populations. From the 26 strains isolated, the ones that had identical 16S rRNA gene sequences were also identical by genomic fingerprinting (Fig.\u00a01) and whole cell protein electrophoresis (Fig.\u00a02). Based on the rep- and protein profiles, two genotypes could be identified among the 26 strains isolated. The absence of micro-diversity among the dominant culturable Desulfovibrio populations suggests the existence of a few ecological niches in the reactor. The long-term operation of the reactor under stable operational parameters, like constant temperature, pH, salinity etc., combined with the selection pressure because of nutritional limitation (i.e. the use of a single energy source), might be the reason for absence of micro-diversity. In addition, the upward flow of wastewater through the reactor results in frequent mixing, which, in turn, might prevent the creation of micro-habitats that are assumed to be important for the evolution of genetically distinct sub-populations (Torsvik et al. 2002).\nAlthough the viable cell count of SRB observed (108 cell ml\u22121) was comparable to the previous studies (Vester and Ingvorsen 1998; Oude Elferink et al. 1999; Roest et al. 2005; van Houten 2006), it cannot be ruled out that potential biases associated with culture-based enumeration techniques might have underestimated the overall SRB population diversity.\nPhenotypic and phylogenetic characterisation of the dominant culturable isolates\nThe physiological characteristics (Table\u00a02) of strain L3 and L7 indicated that they are sulfate reducers. The two isolates oxidised lactate and ethanol incompletely to acetate in the presence of sulfate as electron acceptor. Both used H2 and formate in the presence of acetate as carbon source. These metabolic traits, in addition to the presence of desulfoviridin and a typical vibrio-shaped morphology, suggested that they are the members of the genus Desulfovibrio (Widdel and Bak 1992). Desulfovibrio are Gram-negative sulfate reducers, most of which oxidise their substrates incompletely to acetate (Widdel and Bak 1992). Previous studies have demonstrated the dominance of Desulfovibrio species in freshwater sediments (Sass et al. 1998), in oil wells (Voordouw et al. 1996) and in several wastewater treatment plants (Santegoeds et al. 1998; Dar et al. 2005). 16S rRNA sequence analysis of the two strains, L3 and L7, confirmed their affiliation to other members of the genus Desulfovibrio (Fig.\u00a03),with Desulfovibrio strain SB1 and Desulfovibrio mexicoense as closest relatives, respectively. Desulfovibrio strain SB1, a mesophilic, Gram-negative SRB was isolated from anaerobic sludge of a gas lift reactor-treating sulfate and zinc-rich wastewater (van Houten 2006); while Desulfovibrio mexicoense was isolated from a UASB digester-treating wastewater from a cheese-manufacturing factory in Mexico (Hernandez-Eugenio et al. 2000).\nCo-existing SRB populations in the reactor\nAfter isolation of the most abundant culturable SRB, i.e. the motile strain L3 and non-motile strain L7, whole cell hybridisation using fluorescently labelled oligonucleotide probes was performed to search for their specific signals in the fixed sludge sample. Hybridisation results not only confirmed the presence of the two isolates but also gave an estimate of their abundance relative to the total SRB population and to the overall bacterial community present (Table\u00a03; Fig.\u00a04). Fluorescence in situ hybridisation (FISH) results indicated that the two isolates indeed made up a major part of the hydrogenotrophic SRB community present in the reactor. Cells detected by probe Dsv119, specific for the motile strain L3, appeared as single cells or as chains of three to four cells, while cells detected by probe Dsv139, specific for the non-motile strain L7, appeared as individual cells or as loose aggregates.\nAccording to the ecological principle of competitive exclusion, co-existence of physiologically related populations in the same habitat can be understood if they occupy distinct ecological niches (Gause 1934). Although a number of similarities can be drawn between the strains, L3 and L7, from their substrate utilisation profiles (Table\u00a02), the phenotypic differences together with differences in the range of substrates used by the two isolates may allow them to adapt to slightly different niches within the reactor. The two isolates differed in as many as five physiological properties, which are the use of malate, formate, serine and cysteine as energy sources and sulfur as an electron acceptor (Table\u00a02). The use of elemental sulfur in particular by strain L7 will give it a selective advantage over strain L3 in those places in the reactor where elemental sulfur might be available because of the re-circulation fluid from the aerobic reactor; whereas the motility of strain L3 will confer a competitive advantage to it over strain L7. Potential benefits of motility may include increased efficiency of nutrient acquisition and avoidance of toxic substances (An et al. 2006). The presence of Desulfobulbus-like SRB besides the members of Desulfovibrio in the same habitat could be explained because of the ability of Desulfobulbus to use the fermentation product propionate as an energy source as well; furthermore, under limiting sulfate concentrations, Desulfobulbus competes more successfully for ethanol than other sulfate reducers by its ability to ferment ethanol (Laanbroek et al. 1982). The simultaneous presence of Desulfovibrio and Desulfobulbus in wastewater treatment systems has often been reported in the literature (Nanninga and Gottschal 1987; Raskin et al. 1995; Okabe et al. 2003).\nAmong the complete oxidisers, the probe specific to Desulfobacca acetoxidans gave a positive signal, suggesting their dominance among the acetotrophic sulfate reducers. Desulfobacca acetoxidans, first isolated from a sulfidogenic bioreactor (Oude Elferink et al. 1999), is a Gram-negative SRB that can utilise acetate as the only source of organic carbon and electron donor. Kaksonen et al. (2004) also found Desulfobacca acetoxidans-like SRB in their lab-scale fluidised-bed reactors that were fed with a single electron donor, i.e. lactate or ethanol.\nIn summary, this study demonstrated the presence of a consortium of four sulfate-reducing populations in the reactor maintained on a single substrate (ethanol). This is in contrast to the findings of Kaksonen et al. (2004) who reported a relatively more diverse consortium of SRB in a fluidised-bed reactor fed with ethanol or lactate as the only energy source, using clone libraries and DGGE as molecular methods for microbial characterisation. The observed difference in the extent of diversity might be due to the difference in the molecular methods employed. The general probes like DSR660, used during FISH analysis, cannot discriminate among the different species, and the probes like DSV119, DSV139 and DSBA1017 target only the specific populations. A likely complete oxidation of ethanol through sulfate reduction might thus be assumed to be taking place through a combined effort of hydrogenotrophic sulfate reducers (i.e. the motile strain L3, the non-motile strain L7 and Desulfobulbus spp.) oxidising ethanol incompletely to acetate and Desulfobacca acetoxidans oxidising acetate completely to CO2. Genetic diversity analysis of the most dominant culturable Desulfovibrio populations suggests that long-term stable operation of a reactor with constant operational parameters and frequent mixing might result in the absence of micro-diversity.","keyphrases":["sulfate-reducing bacteria","ecological niches","microbial ecology"],"prmu":["P","P","R"]}
{"id":"Neurochem_Res-3-1-1794624","title":"The Role of Citrullinated Proteins Suggests a Novel Mechanism in the Pathogenesis of Multiple Sclerosis\n","text":"The pathogenesis of MS is unknown. In our studies, we have demonstrated an important role for citrullinated myelin basic protein (MBP). The accompanying loss of positive charge compromises the ability of MBP to interact with the lipid bilayer. The conversion of arginine to citrulline in brain is carried out by an enzyme peptidyl arginine deiminase (PAD) 2. The amount of PAD 2 in brain was increased in MS normal-appearing white matter. The mechanism responsible for this increase involved hypomethylation of the promoter region in the PAD 2 gene in MS, but no change (compared to normal) was found in thymus tissue DNA from the same MS patients. In addition, no change was observed in other neurological diseases, including Alzheimer\u2019s, Parkinson\u2019s, and Huntington\u2019s. We propose that citrullinated MBP, resulting from elevated levels of PAD 2 represents an important biochemical pathway in the pathogenesis of MS.\nIntroduction\nDemyelinating diseases, such as multiple sclerosis (MS), the major demyelinating disease of humans affects not only young adults, but also children. It is the leading cause of neurological deficit in North America and Western Europe [1]. It is characterized by the patchy destruction of the myelin sheath around the axon and is, therefore, classified as a primary demyelinating disease [2]. The reason for this patchy destruction is not known. The MS plaque is characterized by a focal area of myelin destruction associated with astroglial scar formation. These lesions are scattered throughout the CNS with a predilection for optic nerves, brain stem, spinal cord, and periventricular white matter [3]. Perivascular lymphocytic infiltration with macrophages, microglial cells and astrocytes is a common feature. More recently, it has been recognized that extensive oligodendrocyte apoptosis and myelin macrophages are found in MS tissue, suggesting a primary neurodegenerative mechanism, followed by the autoimmune response as a secondary event [4]. In this type of apoptosis, the cardinal feature is damage to the most distal part of the oligodendiocyte process in the periaxonal region [5]. The chemical changes in myelin that precede this destruction are under intensive investigation. In the present overview, we suggest that enzymatic changes induced in myelin basic protein (MBP) may represent an important factor.\nThe cause of MS is unknown. Two major theories have emerged: an autoimmune or a neurodegenerative cause. The autoimmune etiology is considered to arise from sensitization of T-cells in the periphery by a mechanism of molecular mimicry, usually involving a viral antigen with peptide sequences similar to those found in myelin proteins, especially MBP, a candidate autoantigen in MS. The neurodegenerative hypothesis involves metabolic changes in the myelin constituents, which destabilize the membrane architecture, resulting in myelin degradation. Since the myelin proteins are primarily responsible for maintaining the structure of the membrane we focused our attention on myelin proteins, in particular, modifications that are found on MBP.\nMyelin basic protein is an unusual protein. It is a single protein chain consisting of 170 amino acids. Since it does not contain cysteine, it cannot form disulfide bonds. It is a flexible structure, which adapts to environmental conditions, by changing conformation. It is a member of a group of proteins considered to be intrinsically disordered and, therefore, have considerable conformational flexibility (reviewed extensively in the contribution of Harauz and Musse in this issue). A further unusual feature of MBP is the large number and variety of post-translational modifications, such as N-terminal acylation, GTP- and ADP-ribose binding sites, deamidation, methylated arginine, methionine sulphoxide, phosphorylation, and deimination of arginyl residues. Citrullination occurs on six sites in the 18.5\u00a0kDa MBP from humans, while phosphorylation is found on ten sites at least. These are summarized in Fig.\u00a01.\nFig.\u00a01Post-translational modification of MBP. All post-translational modifications, except methylation of Arg107 (human sequence), decrease the positive charge. The known modifications are acylation of the N-terminus, GTP binding, ADP-ribosylation (if R25 is not citrullinated), deamidation, methionine sulphoxide, mono, and dimethylation of Arg107, phosphorylation at Ser\/Thr residues and Tyr and deimination of arginyl residues (six sites in the 18.5\u00a0kDa MBP from normal brain). The structure, shown as a hairpin, occurs at the tri-proline region at the site of the bend. The figure was adapted and modified from Moscarello (1990, Myelin basic protein: a dynamically changing structure. In: Dynamic interactions of myelin proteins, George Hashim and Mario Moscarello (eds) Prog Clin Bio Res, vol 336)\nOne of the modifications, in which we are particularly interested, involves the conversion of peptide-bound arginine to peptide bound citrulline, an enzymatic reaction called deimination. The conversion of arginine to citrulline involves the release of ammonia. For each arginine converted to citrulline, one positive charge is lost from the protein. We first demonstrated that citrulline was present in a crude MBP fraction from myelin [6]. We then purified the citrulline containing MBP on CM52 cation exchange columns essentially as described earlier [7\u20139]. From these columns, we obtained a number of fractions, all of which were MBP. Fractionation of the various MBPs was shown to be dependent on the net charge of the highly modified MBP, resulting from phosphorylation, deamidation, C-terminal arginine loss, and others (Fig.\u00a01). The citrullinated MBP was recovered in the flow-through volume and required further purification by high-performance liquid chromatography (HPLC). The purified protein was sequenced and the arginyl residues that were deiminated were identified. These were R25, R31, R122, R130, R159, and R169 [10]. This decreased positive charge affected the ability of the protein to interact with and organize lipid vesicles. Using liquid X-ray diffraction, we were able to show that the citrullinated MBP (termed \u201cC-8\u201d for convenience) was unable to organize lipid bilayers into compact multilayers, which was readily achieved by the non-citrullinated charge isomer (termed C-1, so-called because it bound most tightly to the CM52 column) [11]. At this time, we speculated that this failure to organize bilayers into compact multilayers, may contribute to myelin instability as seen in MS. These data demonstrated that the citrullinated MBP had a limited ability to organize and compact the lipid bilayers.\nThe citrullinated MBP was isolated from white matter of MS brain and compared to that isolated from normal brain. We reported that the relative amount of the citrullinated MBP was increased in MBP isolated from MS brain. Whereas, it accounted for 20% of the MBP isolated from normal brain, a threefold increase was found in chronic MS. Thus, the ratio of citrullinated MBP\/non-citrullinated MBP was 0.82 in the normal and it was 2.45 in the MS MBP [12]. In a single case of Marburg\u2019s disease (a fulminating form of MS), 80\u201390% of the MBP was citrullinated, with a citrullinated MBP\/non-citrullinated MBP ratio of 6.7 [13, 14]. Therefore, MBP in Marburg\u2019s type of MS was almost totally deiminated. Citrullination of MBP has been reported to diminish charge density, which diminishes its interaction with lipids, making it more vulnerable to proteolytic attack [15].\nThe effects of deimination on protein structure and protein-lipid interactions in myelin are diverse and have been thoroughly reviewed [16]. Briefly, deimination of MBP alters the 3D structure of MBP, producing a more open conformation. The more open conformation of MBP increases its susceptibility to proteases such as cathepsin D, a myelin associated protease. MBP containing six citrullinyl residues is digested four times faster than non-deiminated MBP. The MBP containing 18 citrullinyl residues was digested 45 times faster than MBP from normal brain. These data support the view that deimination of MBP not only decreases the positive charge on the molecule, but also creates a more open structure [17].\nThese perturbations in structure affect the interactions of the protein with the lipid bilayer and decrease the compaction of the multilayer structure of myelin. The deiminated protein had a reduced ability to bind and aggregate lipid vesicles and the highly deiminated form of MBP caused fragmentation of lipid vesicles [18], a process which may have implications for myelin breakdown in MS.\nElectrostatic interactions between the negatively charged phospholipids and the positively charged amino acids (Arg and Lys) are the origin of attraction of MBP to the lipid. MBP is postulated to work as a glue for adjacent bilayers [19, 20] by binding to the polar head groups. This mechanism of interaction is interfered with by deimination of arginyl residues, since the resulting citrulline carries a neutral charge.\nThe conversion of arginine\u2192citrulline in proteins is carried out by the enzyme peptidyl arginine deiminase (PAD). Of the five known PAD enzymes, PAD 2 is the one involved in the deimination of MBP. Since our studies on the deiminated MBP in humans was obtained from autopsy material, the question of the origins of the citrullinated protein, as possibly artefactual, was addressed. To resolve this issue, we explored the temporal relationship between the appearance of the citrullinated protein and the up-regulation of the PAD 2 enzyme, which could only be done in a relevant animal model. Such a model was available to us. It is a transgenic model, containing 70 copies of the transgene for the myelin proteolipid protein DM20. This mouse is normal for the first 3\u00a0months of its life and then demyelinates spontaneously. We measured PAD enzyme and citrullinated MBP. We found that PAD 2 was up-regulated at 2\u00a0months of age and citrullinated MBP at 3\u00a0months of age, suggesting a precursor-product type relationship [21]. We postulated that up-regulation of PAD 2 was the primary response. To substantiate this hypothesis, a thorough study was carried out on PAD 2 regulation.\nIn this regard, we have reported that paclitaxel (Taxol) inhibited the activity of purified PAD 2 isolated from bovine brain in an in vitro assay [22]. In in vivo studies, we demonstrated that paclitaxel attenuated clinical disease in the spontaneously demyelinating mouse model described above [23]. In a recently submitted manuscript, we extended our Taxol studies further by demonstrating that the addition of the methyl donor, vitamin B12, suppressed both immune and non-immune demyelinating disease in mice (F. G. Mastronardi et al. 2006, submitted data), better than paclitaxel alone. These data suggest that drugs, which target PAD 2, in the presence of a methyl donor, such as vitamin B12, may be beneficial in demyelinating disease.\nAnother PAD enzyme, PAD 4, has been implicated in the pathogenesis of psoriasis [24], rheumatoid arthritis [25, 26] and MS (F. G. Mastronardi et al. 2006, submitted data). PAD 4 is different from other PAD enzymes, because the gene contains a nuclear localization signal (PPAKKKST) [27], not present in any of the other PAD enzymes. Translocation of the enzyme from the cytoplasm to the nucleus, has been shown to involve the cytokine tumor necrosis factor \u03b1 (TNF\u03b1). The substrate for PAD 4 in the nucleus is histone (F. G. Mastronardi et al. 2006, submitted data). Deimination of arginyl residues of histones decreases the positive charge on histone, which compromises its ability to interact with DNA, possibly resulting in apoptosis of oligodendrocytes. On the other hand, deacetylation of histones would increase the positive charge on histone. Therefore, these two processes, the deimination of arginyl residues and the deacetylation of lysyl residues of histones affect the positive charge on histone in opposite ways, and these two processes may have a regulatory function.\nSince we determined that up-regulation of PAD 2, preceded demyelination in the animal model, we searched for the mechanism responsible. An examination of the nucleotide sequence of the PAD 2 promoter revealed that it was high in CpG sequences. CpG sequences are important, because cytosine is a target for methylation by DNA methyltransferase, which produces 5-methylcytosine. Methylation of the promoter region of a gene silences the gene, whereas demethylation increases transcription.\nAccordingly, we isolated DNA from white matter obtained from normal individuals and patients who died with MS. The DNA was treated with bisulphite to convert cytosine into uracil. The methylcytosine is not affected by bisulphite. We found that methylcytosine accounted for less than 4% of the cytosines in the PAD 2 promoter in MS (n\u00a0=\u00a017), whereas in the normal, the methylcytosine in the PAD 2 promoter was 12% (n\u00a0=\u00a08). Therefore, the PAD 2 promoter was hypomethylated in the DNA from MS white matter samples. DNA was also obtained from thymus of the same MS patients. The amount of methylcytosine was the same in both MS and normal samples. Therefore, the hypomethylation found in MS appeared to be tissue specific. When we examined other neurological diseases, including Alzheimer\u2019s, Parkinson\u2019s, and Huntington\u2019s, the amounts of methylcytosine were the same as normal (Mastronardi et al. 2006 in preparation). Therefore, the hypomethylation of the PAD 2 promoter was unique to MS brain. This hypomethylation of the promoter of PAD 2 may account for the increase in PAD 2 in MS brain (summarized in Fig.\u00a02).\nFig.\u00a02Deimination pathway in the pathogenesis of demyelinating disease. Normal myelin is shown on the left. The promoter of the PAD 2 gene contains methylated cytosines (closed lollipops) along its CpG island, which regulates its transcription. Some MBPcit is made under normal conditions by the deimination of arginyl residues \u201cC\u201d on MBP. The amount of MBPcit is not enough to destabilize the bilayer. During demyelination, the promoter of PAD 2 is demethylated (open lollipops) at cytosines in the CpG island. This demethylated promoter increases transcription of PAD 2, which increases the amount of MBPcit in myelin with subsequent destabilization of the myelin. Degradation of myelin protein fragments, in particular MBPcit fragments, become available to sensitize T-cells in the periphery, initiating the immune response\nThe removal of methyl groups from methylcytosine in CpG sequences is carried out by a DNA demethylase. We proposed that up-regulation of the demethylase may account for the hypomethylation state of the PAD 2 promoter in MS. When we measured the total demethylase activity in brain homogenates from normal and MS white matter, using a synthetic methylcytosine-guanosine (CpG) substrate [28] we demonstrated that the enzyme in NAWM from MS brain was increased twofold over normal, suggesting the DNA demethylase may have an important role in the hypomethylation of the PAD 2 promoter. Therefore, the hypomethylated state of the PAD 2 promoter may involve up-regulation of the DNA demethylase at least in part.\nAs a result of the work summarized in this overview, we postulate the following sequence of events during the pathogenesis of MS. The citrullinated MBP, because of its decreased positive charge, prevents proper compaction of the bilayer, so that the multilamellar structure is less stable. Since PAD 2 has been shown to be present in myelin by immunogold electron microscopy (unpublished results), the conversion of non-citrullinated to citrullinated MBP can occur locally in myelin. Preliminary results have shown that immunogold labeling of PAD 2 in mouse optic nerve was not random, but was found in clusters in the axon itself and in the myelin sheath. In the latter, the clusters were found mainly at the junction of the myelin sheath and the axon (periaxonal) with some gold particles distributed in the myelin sheath. The presence of the PAD 2 enzyme at these sites suggests that the citrullinated MBP would be formed in these clusters in myelin, eventually resulting in plaques.\nWhen we examined the extent of labeling in the optic nerve of the spontaneously demyelinating transgenic mouse, we found a threefold increase in the number of gold particles. This increase in PAD 2 labeling demonstrated that increased amounts of PAD 2 were present in the transgenic mouse, accounting for an increase in the citrullinated MBP. Since citrullinated MBP is present in the normal myelin sheath (20% of the total MBP), citrullinated MBP can be tolerated to this extent. However, increased amounts as found in chronic MS (threefold), and in fulminating MS (six to sevenfold), cannot be accommodated in a compact myelin sheath, resulting in destabilization.\nPeptidyl arginine deiminase 2 has been reported recently to be elevated in primary open-angle glaucoma, where it was considered responsible for the deimination of several optic nerve proteins, in addition to MBP. Concomitant with this protein deimination, a decrease in arginine methylation was also observed [29]. Therefore, the PAD 2 enzymes have important roles in several nervous system diseases.\nOligodendrocyte apoptosis has been reported in MS white matter in the absence of lymphocytes or myelin phagocytes [4]. The major feature of this type of injury is damage to the most distal part of the oligodendrocyte process (periaxonal), which results in clumping of proteins [5], followed by apoptosis. The possible role of the PAD 2 enzyme in this process has not yet been demonstrated. If it can be established that protein deimination at sites of PAD 2 clusters does occur, it may suggest that demyelination begins in the periaxonal myelin sheath and subsequently moves outward. Although this hypothesis is speculative, it warrants investigation. The increased amount of PAD 2 in MS white matter probably reflects a greater production of the enzyme. Since the PAD 2 promoter is rich in CpG islands, we showed that the promoter was hypomethylated. These studies are ongoing in our laboratory. They suggest immediately that the reversal of the hypomethylated state may be a therapeutic target in MS.\nThe importance of methylation in a therapeutic strategy of MS has been suggested by us recently [(F. G. Mastronardi et al. 2006, submitted data), 30]. In these studies, we demonstrated that vitamin B12 in combination with either interferon \u03b2 or paclitaxel, effectively ameliorated demyelinating disease in both EAE and non-immune demyelinating disease. In a detailed mass spectrophotometric study of the various components of MBP, we showed arginine methylation was decreased in most of the components of the MBPs from MS material [31], supporting the view that defects in methylation may involve protein methylation as well. The methylation process requires Vitamin B12, which transfers its methyl group to homocyteine synthesizing methionine, which is then converted to S-adenosytmethionine, the methyl donor in all biological methylation reactions, including methylation of cytosine in DNA to methylcytosine. These studies with the preliminary studies reported above suggest an important role for methylation\/demethylation reactions in the pathogenesis of MS.","keyphrases":["citrulline","multiple sclerosis","myelin","peptidyl arginine deiminase","demyelination","apoptosis"],"prmu":["P","P","P","P","P","P"]}
{"id":"Matern_Child_Health_J-2-2-1592156","title":"Toxoplasmosis, Cytomegalovirus, Listeriosis, and Preconception Care\n","text":"\u00a0\nToxoplasma gondii (T. gondii), cytomegalovirus (CMV), and Listeria monocytogenes (L. monocytogenes) can all negatively affect pregnancy outcomes. Preconception counseling about such effects can reduce the risks posed by these pathogens. Informing women of childbearing age about these pathogens and how to prevent their negative effects can help women make informed decisions about prevention. This brief summarizes some basic information about these infections and provides some web sites and articles for further information about how to prevent them within the context of preconception care.\nToxoplasmosis is a disease caused by Toxoplasma gondii (T. gondii), a protozoan parasite mainly transmitted to humans via three routes: a) ingestion of raw or undercooked contaminated meat; b) exposure to T. gondii oocysts (a form of the organism passed in cat feces), through cat litter or soil (e.g., from gardening or unwashed fruits or vegetables), or contaminated water; and c) congenital in which maternal infection is passed transplacentally via blood to the fetus [1]. Congenital infection leads to stillbirth and severe neurological illness in some instances, although the majority of infected newborns are asymptomatic at birth and some develop sequelae such as mental retardation, blindness, and epilepsy later in life [2]. Extrapolation from regional studies suggests that \u223c400\u20134,000 cases of congenital toxoplasmosis occur each year in the United States [2].\nAdults with normal immune function who are infected with T. gondii are usually asymptomatic or have self-limited symptoms (e.g., fever, malaise, and lymphadenopathy) [1]. Once infected, these individuals usually develop an immune response against toxoplasmosis [3, 4]. A recent study based on the National Health and Nutrition Survey conducted from 1988\u20131994 (NHANES III) reported that, among women aged 15\u201344 years, seroprevalence of T. gondii antibodies was 15%, suggesting that \u223c85% of women of childbearing age are susceptible to T. gondii infection [5].\nThree principal interventions are presently used to reduce morbidity and mortality from congenital toxoplasmosis: a) education about how to prevent infection (especially during pregnancy); b) prenatal and newborn screening to identify and treat congenital infection; and c) animal rearing and production methods designed to reduce T. gondii contamination of meat. Of the three, education about how to prevent infection is applicable to adolescents and women in the preconception period. Education programs during pregnancy have been associated with improved knowledge and behavior and a reduction in infection rates [6\u20139]. Toxoplasma infection can be prevented by one or more of the following: a) cooking meat to a sufficient temperature to kill Toxoplasma; b) peeling or thoroughly washing fruits and vegetables before eating; c) cleaning cooking surfaces and utensils after they have contacted raw meat, poultry, or unwashed fruits or vegetables; d) pregnant women avoiding changing cat litter or using gloves, then washing hands thoroughly; and e) not feeding raw or undercooked meat to cats and keeping cats inside to prevent acquisition of Toxoplasma by eating infected prey [2].\nAlthough prenatal and newborn screening programs have been evaluated, they are controversial because of the lack of proven efficacy of treatment, side effects of treatment, and potential complications of invasive procedures such as amniocentesis to evaluate fetal infection [10\u201323]. Animal rearing and production methods to reduce T. gondii contamination of meat are effective and have been associated with a reduction in the prevalence of T. gondii in important meat sources such as pork [24]; however the need for improvement in producers\u2019 knowledge and production practices is acknowledged [25]. Education about toxoplasmosis is an important component of preconception care that can be integrated with information about other diseases that affect women.\nHuman cytomegalovirus (CMV) is the largest DNA virus belonging to the herpesvirus family. Humans are the only reservoirs for the human herpesviruses, and they can transmit these agents through direct contact with infected blood, tissues, bodily fluids, feces, and fomites [26]. A pregnant woman infected with CMV can transmit this virus to her unborn fetus, which can cause damage to the central nervous system, hematopoietic system, kidneys, endocrine glands, gastrointestinal tract, lungs, and liver. Long-term sequelae include cerebral palsy, mental retardation, and hearing loss [26, 27]. The birth prevalence rate of congenital CMV infection varies between \u223c0.6\u20131.5% [28\u201330] in the United States, making it the most commonly transmitted virus in utero [26, 31] and a major cause of cerebral palsy, mental retardation, and hearing loss among children [27, 31].\nCMV infects almost all humans at some point in their lives. Adults with normal immune function infected with CMV are usually asymptomatic or might experience mild flu-like symptoms, or even mononucleosis with symptoms such as malaise, persistent fever, myalgia, and cervical lympadenopathy [26, 31]. Once a human is infected, the virus passes into a latent state [26]. Although the virus can be reactivated, it is usually kept under control, because adults with normal immune function usually retain lifelong immunity against CMV.\nPrimary CMV infection of women during pregnancy or periconceptionally results in transmission to the fetus transplacentally in \u223c30\u201340% of maternal infections [26, 32\u201334]. Preexisting maternal immunity strongly reduces the risk of transmission to the fetus [34]. However, approximately 10\u201315% of newborns with congenital CMV will be symptomatic and from \u223c6\u201325% of those born without symptoms will develop late sequelae [35].\nThe most common means for women to be infected with CMV is by exposure to toddlers who shed large amounts of the virus in their saliva and urine for many months following their first (usually asymptomatic) infection [26, 36\u201340]. Daycare providers and pregnant women who have a toddler of their own are at high risk for infection [41\u201346]. Sexual transmission, blood transfusion, and organ transplantation are other means by which CMV is transmitted [26]. It is important to counsel all women about safe sex practices.\nCurrently, there are no vaccines available for preventing CMV infection, although some promising advances have been made [47, 48]. It is, therefore, extremely important to provide women of childbearing age with information about how they can prevent CMV infection before conception. The most effective means for preventing CMV infection is handwashing [26, 49\u201353]. Education about careful hygiene and frequent handwashing, especially after contact with the saliva and urine of young children, and careful disposal of diapers, tissues, and other contaminated items can reduce the transmission of CMV. Avoiding sharing drinking glasses and eating utensils with young children can also prevent transmission. This is especially true for women who work in daycare settings and for those who are pregnant [26, 49\u201354].\nAs some studies suggest that CMV infection prior to conception can result in congenital CMV infection [26, 30, 55], counseling all women of childbearing age about how to prevent transmission preconceptionally could significantly reduce the incidence of congenital CMV infection.\nListeriosis is an invasive foodborne infection caused by the motile, gram-positive bacterium Listeria monocytogenes (L. monocytogenes). The disease affects primarily pregnant women, newborns, and adults with weakened immune systems [56]. Maternal infection during pregnancy is usually a self-limited, nonspecific acute febrile illness of the third trimester. In contrast to the maternal illness, fetal and neonatal infection is severe and frequently fatal. Infection in the fetus and newborn occurs by transplacental transmission or possibly from exposure to L. monocytogenes in the perinatal period. The effects of intrauterine infection on the fetus and neonate include preterm labor, amnionitis, spontaneous abortion, still birth, and early-onset of the neonatal sepsis syndrome, evident at birth or shortly thereafter. Late-onset neonatal disease is likely due to infection at or around the time of birth, and presents as meningitis at one to several weeks of age [57].\nListeriosis is a rare disease; the incidence rate in 10 states participating in the Foodborne Diseases Active Surveillance Network (FoodNet) was 2.7 cases per 1,000,000 population in the year 2004 [58]. However, the incidence rate in susceptible subgroups is much higher. The rate among neonates younger than 28 days of age in FoodNet sites was 52.8 per 100,000 population in the year 2000 (CDC unpublished data). More importantly, listeriosis has a very high case fatality rate (20\u201330% in neonates) and is responsible for an estimated 500 deaths each year in the United States [59].\nThe food items implicated in outbreaks of listeriosis include ready-to-eat meats, such as turkey delicatessen meat [60], meat pat\u00e9 [61], pork tongue in jelly [62], hot dogs [63], and dairy products made from unpasteurized milk, in particular fresh soft cheeses [64].\nPrimary prevention for listeriosis focuses on improvements in food processing and on consumer education. Substantial efforts by the food industry and food regulatory agencies have been directed toward reducing the likelihood that high risk foods will be contaminated with L. monocytogenes [65]. Despite this, pregnant women, immunocompromised persons, and the elderly should be advised to avoid pat\u00e9, fresh soft cheeses made from unpasteurized milk and to cook ready-to-eat foods such as hotdogs, delicatessen meats, and left over foods until steaming [66]. Information regarding these foods at high risk of contamination with Listeria can be incorporated into preconception care dietary recommendations.\nFor more information\nCDC web sites\nToxoplasmosis: http:\/\/www.cdc.gov\/ncidod\/dpd\/parasites\/ toxoplasmosis\/default.htm\nCMV: http:\/\/www.cdc.gov\/cmv\nListeria: http:\/\/www.cdc.gov\/ncidod\/dbmd\/diseaseinfo\/ listeriosis_g.htm\nPublications for practitioners\nDisclaimer: These references are included for information only. CDC has no control over the information in these articles. Views and opinions of these organizations are not necessarily those of CDC, the Department of Health and Human Services (HHS), or the U.S. Public Health Service (PHS).\nAmerican College of Obstetricians and Gynecologists. Perinatal viral and parasitic infections. ACOG Practice Bulletin 2000;20.\nBrundage, SC. Preconception health care. American Family Physician. 2002 June; 65(12):2507\u201314.\nHanlin RB. Congenital infections and preconception counseling. J S C Med Assoc. 2002 Oct;98(6):277\u201380.","keyphrases":["toxoplasmosis","cytomegalovirus","listeriosis","preconception care","listeria","infection"],"prmu":["P","P","P","P","P","P"]}
{"id":"J_Interv_Card_Electrophysiol-4-1-2292475","title":"Acute success and short-term follow-up of catheter ablation of isthmus-dependent atrial flutter; a comparison of 8 mm tip radiofrequency and cryothermy catheters\n","text":"Objectives To compare the acute success and short-term follow-up of ablation of atrial flutter using 8 mm tip radiofrequency (RF) and cryocatheters.\nIntroduction\nAtrial flutter is a common arrhythmia [1], difficult to suppress with medication [2], and is associated with significant symptoms. Since it was first proposed [3\u20135], ablation for atrial flutter has increasingly been used for its therapy, especially since induction of bidirectional cavo-tricuspid isthmus block was shown to be associated with better immediate outcomes and lower recurrence rates [6\u201311]. Failure to successfully ablate atrial flutter in the long-term may be due to particular anatomic problems [12\u201314], poor catheter stability in this region, and incorrect interpretation of isthmus block. Important developments in the field of atrial flutter ablation have been a better understanding of the anatomy of the isthmus, refinements of the definition of bidirectional isthmus block, and the arrival of new catheter technology.\nNumerous studies have compared different energy types, different catheter tip sizes and different energy settings [15\u201319], as well as the use of advanced cardiac mapping systems [20]. The preferred tools now are 8\u00a0mm, or irrigated tip catheters [21]. From a recent meta-analysis it would appear that these two technologies are equally effective with acute success quoted at 84% and 85% for the primary catheter chosen before changeover, whereafter final success rates of up to 99% can be achieved [22]. Not all studies have this high success rate and the preponderance of studies on ablation of atrial flutter suggest that these high success rates are not repeated in all centers [23]. Also of importance is a clinical recurrence rate, in the face of acute success, of at least 5 to 12% when using radiofrequency [19, 24, 25]. Disadvantages of radiofrequency (RF) energy include pain, overheating with \u201cpopping\u201d, char formation and risk to coronary arteries.\nCryoablation is a relatively recent addition to the transvenous ablation armamentarium and has been shown to be comparable to RF for some arrhythmias. It may have some advantages over RF, especially as regards discomfort during ablations [17, 26]. The development of 6 and 8 mm tip cryocatheters has increased the interest in this technology for atrial flutter ablation. As far as we are aware there have been no randomized comparative studies published comparing 8\u00a0mm tip cryocatheters with 8\u00a0mm tip RF catheters.\nMethods\nPatient population\nConsecutive patients with ongoing symptoms and documented atrial flutter with or without fibrillation were included. At least one recent episode of atrial flutter (within the last 6\u00a0months) was documented on a 12 lead ECG and was suggestive for isthmus dependency. Patients with drug-induced flutter (and prior AF) could be included, and had their drug therapy continued after ablation. Patients were excluded if they had undergone a previous flutter ablation, if thrombus was present in the atria, or after previous cardiac surgery for valvular or congenital heart disease. Baseline investigations included a standard echocardiogram, a simple questionnaire asking about clinical wellbeing and a subjective assessment of the arrhythmia burden both in terms of duration and frequency. The study was approved by the ethics committee of our institution. All patients signed written informed consent.\nAssessment before ablation\nAntiarrhythmic drugs, except for AV nodal slowing agents, and amiodarone, were discontinued at least five half-lives prior to ablation. Patients were studied in the fasting, post-absorptive state. A coronary sinus (CS) catheter was inserted through the left subclavian vein, and a multipolar circular right atrial catheter with alternating 2\u201310\u20132\u00a0mm interelectrode distance was positioned from the right groin, with the tip positioned immediately lateral to the planned position of the isthmus line and anterior to the crista terminalis. Heparin 100\u00a0U\/kg was given and a further 5\u00a0U\/kg given if the procedure lasted longer than 180\u00a0min.\nIf the patient was in sinus rhythm, isthmus conduction was confirmed by pacing. If flutter was present, entrainment was performed to confirm isthmus dependence, and the patient left in flutter. If AF was present, the patient was cardioverted after a transoesophageal echocardiogram, and then isthmus conduction confirmed. Absence of isthmus conduction or non-isthmus dependence was not seen in the selected patients. No induction of arrhythmia was attempted if patients had sinus rhythm.\nRadiological assessment of the right atrial isthmus was made in a right anterior oblique (RAO) 30\u00b0, and a left anterior oblique (LAO) 45\u00b0 view (each with 40\u00a0cc at 18\u00a0cc\/s). Angiograms were acquired digitally to allow for post-hoc analysis. The treating physician was able to view the angiographic findings to optimize the planned ablation line. The isthmus length was assessed from the inferior hinge point of the tricuspid valve to the IVC at the end of atrial diastole (the frame before opening of the tricuspid valve) [14]. Morphology was assessed visually as to the presence of a Eustachian valve or a recess, as well as to the general shape i.e. flat or concave.\nAblation procedure\nThe catheters were a 9Fr 8\u00a0mm tip catheter (FreezorMax, Cryocath Technologies Inc, Kirkland, Canada) with a cryoconsole for the cryoablation group, and a 7Fr 8\u00a0mm tip single sensor catheter (EPT Blazer II, Boston Scientific, Natick, MA, USA) with an EPT-1000XP generator for the radiofrequency group. A large curve was initially selected in both groups, with change out of catheter curve during the study only as necessary. Applications of \u221275\u00b0, for 4\u00a0min were given with cryothermy, and applications of 60 Watt, for 60\u00a0s, targeted at 60\u00b0C for RF. Lines were made with discrete applications between the tricuspid valve and the inferior vena cava at an approximately 6 o\u2019clock position in LAO 45\u00b0, unless otherwise dictated. If termination of atrial flutter occurred, or if the patient was in sinus rhythm, continuous pacing from the proximal coronary sinus was employed to continually assess isthmus conduction. After the first line, a new assessment of conduction was performed. If conduction over the isthmus remained present, gaps were sought. If there was still isthmus conduction, a slightly more medial or lateral line was made. In no patient was an attempt made to perform a septal ablation line. Final assessment of acute block was confirmed after 30\u00a0min waiting.\nThe end point for successful ablation was induction of complete bidirectional isthmus block, defined as the presence of reversal of activation on the lateral and septal wall when pacing the CS os and low lateral RA, the presence of widely split potentials along the isthmus line, by activation mapping across the isthmus, and by differential pacing. All 4 were required before calling the ablation successful. In the case where bidirectional block was not achieved, ablation was stopped when no large, sharp signals could be identified over a broad area of the isthmus.\nAs pain perception was assessed, sedation was standardized. Before venous puncture 5\u00a0mg of diazepam was given intravenously, and repeated at the patient\u2019s request. Fentanyl 50\u00a0\u03bcg intravenously was given when the patient requested pain control and the physician considered this necessary. This was repeated as needed. Dosages of both diazepam and fentanyl were recorded.\nIn the initial 40 patients creatine kinase (CK) and CK-MB were taken before the procedure, 2 and 24\u00a0h after the start of the procedure. For the final 22 patients the laboratory had changed the measurement to CK mass. We then modified the protocol to measure CK-MB mass, Troponin T, and Myoglobin at 4 and 24\u00a0h after the start of the procedure.\nNo crossover, other than in catheter curve, was allowed in an attempt to remove any possible bias. Change over to an irrigated tip ablation catheter was also not allowed. In patients in whom no block could be induced, a repeat procedure was scheduled not earlier than 6\u00a0weeks after the initial ablation, at the physicians\u2019 discretion. The choice of energy source at that time was at the physicians\u2019 discretion.\nPatients were all questioned with regard to pain perception using a visual analogue score, where patients are shown a line from 0 to 10, where 0 is no pain, and 10 is the highest pain level imaginable, and were asked to point to the position on the line where their pain level during ablation was.\nFollow-up\nAll patients received an event-recorder for the first 6\u00a0weeks after the procedure and were requested to send at least daily strips as well as strips made during symptoms. Patients visited the outpatient clinic 6\u00a0weeks after the procedure. After this period all patients were asked to report symptoms and if these were present were given a further event monitor until documentation of symptoms was obtained. If at all possible a 12 lead ECG was also obtained. A second assessment with a questionnaire was performed after 3\u00a0months, again asking a question about general clinical well being and also symptom burden in regard to duration and frequency. Clinical files were followed up after 9\u00a0months.\nStatistical analysis\nFor patients in whom another ablation was performed (AVNRT in two, and pulmonary vein ablation in six others) procedure and fluoroscopy times were limited to the flutter approach, which was done first, including 30\u00a0min waiting time. Biomarker assessment was not done in these patients. Continuous variables are expressed as mean \u00b1 standard deviation, with medians as necessary. Parametric and non-parametric tests were used where appropriate. A p-value of <\u20090.05 was considered significant.\nResults\nPatient data\n62 patients were included as planned, with clinical characteristics as outlined in Table 1. There were no significant differences in any of the parameters between the group assigned to radiofrequency (RF group) versus that assigned to cryotherapy (cryo group). The large number of patients with prior AF is due to the fact that we had initially taken a decision to perform isthmus ablation first in all patients with AF who had shown typical atrial flutter on any 12-lead ECG, prior to performing a left atrial procedure, initially in a separate procedure and only later in the same session.\nTable\u00a01Demographics\u00a0AllCryoRFp valueNumber623230Age (years\u2009\u00b1\u2009SD)56\u2009\u00b1\u20091055\u2009\u00b1\u20091156\u2009\u00b1\u20099NSMale\/female27\/527\/528\/2NSAtrial fibrillation history47 (76%)25 (78%)22 (73%)NSCryo Cryoablation; NS not significant; RF radiofrequency; SD standard deviation\nAngiographic data\nRight atrial angiography was not performed in 4 patients because of mild renal dysfunction or allergy to contrast material. The angiogram was of insufficient quality in eight others.\nThe mean isthmus length was 35.2\u2009\u00b1\u200914.6\u00a0mm and its topography was assessed as being flat or only mildly concave in 28, markedly concave in 19 and showed a pit or aneurysm in 10. A clear Eustachian valve was seen on six angiograms.\nAblation data\nAssessment of acute results showed bidirectional isthmus block, using the criteria mentioned, in 47 of 62 patients (76%). This was in 25 of 30 patients (83%) of the RF group and in 22 of 32 (69%) of the cryo group (NS). In 1 patient in the RF group the procedure was terminated because of recurrent AF with early recurrence and inability to assess isthmus block. This patient was taken as a failure which was confirmed at the time of a subsequent AF ablation. Procedural data for all patients are given in Table 2.\nTable\u00a02Procedure data and recurrent arrhythmias\u00a0AllCryoRFp valueNumber623230Application number22\u2009\u00b1\u20091318\u2009\u00b1\u20091025\u2009\u00b1\u2009160.05Ablation time (s)2,742\u2009\u00b1\u20091,9303,792\u2009\u00b1\u20091,9001,459\u2009\u00b1\u2009950<0.001Acute success47 (76%)22 (69%)25 (83%)NSSingle line47 (76%)25 (78%)22 (73%)NS2 lines9 (15%)2 (6%)7 (24%)0.0733 lines4 (6%)3 (9%)1 (3%)NSReversal of block during application7 (11%)6 (19%)1 (3%)0,091Reversal of block during 30 min6 (10%)1 (3%)5 (7%)NSIsthmus length (mm)35\u2009\u00b1\u20091535\u2009\u00b1\u20091736\u2009\u00b1\u200911NSSheath usage7 (11%)0 (0%)7 (23%)<0.005Recurrent arrhythmiasFlutter (typical)\u00a0After success1 (2%)0 (0%)1 (4%)NS\u00a0After failure11 (73%)7 (70%)4 (50%)NSFlutter (atypical)1 (2%)0 (0%)1(3%)NSAtrial tachycardia5 (8%)2 (6%)3 (10%)NSAtrial fibrillation28 (45%)13 (41%)15 (50%)NSThe numbers are given with the standard deviation.Cryo Cryoablation; NS not significant; RF radiofrequency\nIn the successful patients the number of applications to ensure block in the whole group was 17\u2009\u00b1\u200911. It was 23\u2009\u00b1\u200913 in the RF group and 12\u2009\u00b1\u20096 in the cryo group (p\u2009<\u20090.005). Total ablation time was 1,283\u2009\u00b1\u2009777\u00a0s and 2,905\u2009\u00b1\u20091,245\u00a0s (p\u2009=\u20090.0001). In those in whom bidirectional block could not be achieved, the total number of applications was 33\u2009\u00b1\u200914. For RF and cryo the values were 39\u2009\u00b1\u200921 applications with a total time of 2,724\u2009\u00b1\u20091,102\u00a0s vs 29\u2009\u00b1\u20098 applications with a time of 5,873\u2009\u00b1\u20091,337\u00a0s (p\u2009<\u20090.03 and <\u20090.0011, respectively).\nIn 22 of 30 in the RF group and 25 of 32 in the cryo group, a single line at approximately 6 o\u2019clock was drawn. In the RF group two lines were drawn in seven, and three in one. In the cryo group two lines were made in four, and three lines in three. The need to draw more than one line was associated with failure of the procedure in four of eight in the RF group and five of seven in the cryo group (NS).\nShort lived block, either recurring during the application or immediately thereafter, occurred in one patient in the RF group and in six in the cryo group, with a trend to statistical significance (p\u2009=\u20090.091), with it being a predictor of failure if it occurred more than three times. In five patients in the RF group and one patient in the cryo group, conduction recurred later during a waiting period (with a median of 15\u00a0min) requiring further applications (NS). In only one patient in the RF group was late recurrence associated with failure to induce bidirectional block. In no patient in whom bidirectional block was present at the end of the 30\u00a0min waiting period did isoprenaline change this. The average power applied in the RF group was 52\u2009\u00b1\u20096\u00a0W.\nIn the RF group the signal was significantly diminished at the end of each application whether isthmus block was present or not, while there tended to be preservation of signals on the cryoablation catheter after ablation across the isthmus until block occurred. Only then were low voltage signals seen.\nFailures were not significantly associated with length of the isthmus (39.2\u2009\u00b1\u200923.5 vs 34\u2009\u00b1\u20099.0 mm in success, although there was a trend to this (p\u2009=\u20090.12) There was no significant difference in anatomy between the two groups.\nProcedure data and complications\nThe overall procedure time was 160\u2009\u00b1\u200949\u00a0min, with no difference between 170\u2009\u00b1\u200948\u00a0min in the RF group and 151\u2009\u00b1\u200949\u00a0min in the cryo group. Overall fluoroscopy times were 28\u2009\u00b1\u200914\u00a0min, with a difference between 33\u2009\u00b1\u200915\u00a0min in the RF group and 23\u2009\u00b1\u200911\u00a0min in the cryo group (p\u2009<\u20090.02).\nChange of catheter curve occurred in one patient in the RF group from large to standard curve. An SL1 sheath (Daig, Minnetonka, MN, USA) was used in seven patients in the RF group for stability, while no sheath usage occurred in the cryo group (p\u2009=\u20090.005). During the procedure, six patients required cardioversion for induced AF, four in the RF group and two in the cryo group (NS). There were two small pericardial effusions seen on echocardiography without further significance (one in each group).\nFor the initial 40 patients there was a significantly higher peak CK and CK-MB after cryo (Table\u00a03). This remained so after 24\u00a0h. For the last 22 patients we observed the same for CK-MB mass, but not for Troponin T (Table\u00a03).\nTable\u00a03Biomarkers after catheter ablation\u00a0AllCryoRFp valueProcedure valuesCK (U\/l)141\u2009\u00b1\u200996184\u2009\u00b1\u200910296\u2009\u00b1\u2009600.02CK-MB (U\/l)27\u2009\u00b1\u20091636\u2009\u00b1\u20091718\u2009\u00b1\u200980.011CK-MB mass (\u03bcg\/l)18\u2009\u00b1\u20092133\u2009\u00b1\u2009244.4\u2009\u00b1\u20091.20.004Troponin T (\u03bcg\/l)0.49\u2009\u00b1\u20090.320.54\u2009\u00b1\u20090.380.39\u2009\u00b1\u20090.27NSValues after 24 hrsCK (U\/l)264\u2009\u00b1\u2009245289\u2009\u00b1\u2009173136\u2009\u00b1\u2009740.022CK-MB (U\/l)37\u2009\u00b1\u20092851\u2009\u00b1\u20093018\u2009\u00b1\u200960.011CK-MB mass (\u03bcg\/l)8.4\u2009\u00b1\u20097.312\u2009\u00b1\u2009105.16\u2009\u00b1\u20093.30.07Troponin T (\u03bcg\/l)0.44\u2009\u00b1\u20090.300.54\u2009\u00b1\u20090.360.38\u2009\u00b1\u20090.29NSThe numbers are given with the standard deviation.CK Creatine kinase; Cryo cryoablation; NS not significant; RF radiofrequency\nIn assessing the level of comfort during the procedure and the pain experienced by patients, similar numbers from both groups assessed sedation as adequate (67% for RF and 63% for cryo). The pain scores given at the end of the procedure were not significantly different (42.9\u2009\u00b1\u200924.0 for RF and 43.7\u2009\u00b1\u200915.8 for cryo). Diazepam was given as standard at the beginning of the procedure and as necessary thereafter for discomfort related to having to lie still for protracted periods. The usage of diazepam was statistically similar in both groups (7.4\u2009\u00b1\u20093.4\u00a0mg in the RF group and 8.0\u2009\u00b1\u20093.1\u00a0mg in the cryo group). However, a significantly higher usage of fentanyl in the RF group was observed (70.0\u2009\u00b1\u200944.9\u00a0\u03bcg vs 10.0\u2009\u00b1\u200922.1\u00a0\u03bcg; p\u2009<\u20090.01).\nFollow-up results\nPatients were followed up for between 90 and 411\u00a0days (138\u2009\u00b1\u200981\u00a0days, median 90\u00a0days), which was similar in both groups (p\u2009=\u2009ns).\nA total of 6 patients were taking no antiarrhythmic medication prior to ablation and this increased to 13 post isthmus ablation.\nRecurrent arrhythmias were frequent in both groups of patients. ECG documented recurrent flutter occurred only in one patient from the RF group. A further procedure confirmed recovery of isthmus conduction. This recurrence was seen at 14\u00a0months post ablation, whereas he had previously had monthly episodes of flutter. One other patient had symptoms with an apparently non-isthmus dependent flutter documented on ECG on day\u00a01 post ablation and not since, and elected not to have a further procedure. We cannot exclude asymptomatic arrhythmias but at least during the first 6\u00a0weeks, patients sent in daily event monitor transmissions, which one would hope would have captured at least some asymptomatic recurrences, particularly of atrial flutter.\nIn 11 of 15 patients in whom the initial procedure failed, a redo procedure was performed after the elected period of 6\u00a0weeks because of documented recurrent flutter. The redo procedure required a small number of point touch ups in five patients (three in the RF group and two in the cryo group), while more extensive diffuse isthmus applications were required in six (one in the RF group and five in the cryo group). In those undergoing repeat ablation, success was achieved in all and during follow up no recurrent atrial flutter was noted.\nPreviously undocumented atrial tachycardias were also seen in both groups (three in the RF group, and two in the cryo group). ECG documented AF recurrence occurred in 28 patients, at a similar rate in both groups (15 in the RF group and 13 in the cryo group). The likelihood of asymptomatic recurrences is probably higher for atrial fibrillation but was not the primary focus of this study.\nDiscussion\nAcute success\nThis study showed an acute success rate of 83% for an 8\u00a0mm tip RF catheter vs. 69% for an 8\u00a0mm tip cryocatheter. This non significant difference is clearly concerning. However, the cryoablation group required significantly less applications to achieve success, with a similar procedural duration, significantly lower fluoroscopy time, and with a much lower requirement for analgesia with fentanyl. Arrhythmia recurrences in the initially successful patients were similar with a very low flutter recurrence rate.\nThe acute success rate with 8\u00a0mm tip RF compares favorably with that found in the meta-analysis of Da Costa et al. [22]. However, the acute results for cryotherapy are lower than in previously published studies. The shorter fluoroscopy time using cryotherapy is related to cryoadherence during applications [26]. With the catheter firmly attached, no fluoroscopy to check position is required. This is also reflected in the fact that no long sheaths were necessary. A lower requirement for analgesia has previously been described and this study confirms this finding [17, 26].\nDifficulties during ablation\nRecurrence during or shortly after ablation occurring more commonly in the cryo group probably relates to a reversible cooling effect at the periphery of the ice ball which recovers during or shortly after termination of ablation, while the central lesion acutely formed is more permanent. The more common recurrence during the waiting period after RF ablation probably relates to a longer reversal time of the acute RF effects (either edema or temperature effect).\nCryoablation for flutter\nCryoablation may have some advantages over RF in addition to those mentioned above such as less thrombogenicity [27], and maintenance of tissue architecture with homogenous well delineated lesions [28]. A 10Fr, 6.5\u00a0mm tip catheter showed acute success of 94\u2013100% and 6\u00a0month recurrence rates of 0\u201325% [17, 29, 30]. A system with 7Fr, 6 mm tip catheters showed success in 87% to 88% using a septal line, without symptomatic recurrences, but with resumption of isthmus conduction at repeat study at 6\u00a0months in 30\u201335% [31, 32]. Using a 9Fr, 8\u00a0mm tip increased success rates to100% with symptomatic recurrence of 0% to 10% and recurrent conduction at 1 to 3\u00a0months in 19% to 32% [32\u201334]. There is some discussion as to whether 3, 4 or 8\u00a0min of ablation are needed for adequate lesion formation [33, 35].\nHigher peak CK levels, confirmed by the high CK-mass in the last patients, suggest that the damage caused by cryotherapy may be more extensive despite the lower number of applications. Several explanations are possible. The first is that the CK levels may be underestimated after RF, which denatures proteins in another way than cryotherapy. Troponin T levels are more accurate in estimation of myocardial damage, but they also tended to be higher, be it not significantly. This may suggest that lesions are equal with both approaches.\nFuture developments\nNewer data suggest that ablation per point guided by the maximal voltage may be a useful technique, but this has not been confirmed for cryotherapy [36].\nStudy limitations\nThis study was a single centre, largely single operator study involving a relatively limited number of patients with a high population of AF patients, making follow-up rather complicated. In present practice and later in this series, patients with the combination are often treated in one session, which still leaves physicians the choice whether cryo should be used for flutter, following its use for the pulmonary veins [37]. We only studied the acute and short-term success, while flutter recurrence may happen at more than 4\u00a0months of follow-up. While we attempted to exclude asymptomatic recurrences especially in the first 6\u00a0weeks, we cannot exclude the possibility of these, especially of AF and also late after the ablation. Isthmus conduction recovery is also possible in asymptomatic cases. In the RF group we limited the power to 60\u00a0W to ensure safety and this may be too conservative. Further, in our practice routine use of long sheaths was discouraged. At the time of this study use of an 8\u00a0mm tip with RF was considered as effective as irrigated tip ablation, so we elected to use this approach for the RF group. More recent studies have shown higher success rates than we obtained and have also suggested alternatives in the approach. These include the use of irrigated tip catheters and long sheaths as well as other technical issues and newer techniques such as maximal voltage guided ablation. A higher success rate for RF would clearly have been even more prejudicial to cryotherapy in this study. We were also extremely critical in our assessment of isthmus conduction, using multiple criteria. Angiograms were also performed in fixed views and not based on other catheter positions as has been suggested by others [14].\nConclusion\nAcute success with cryotherapy for atrial flutter ablation, while non-statistically less effective in this study, requires fewer applications and is associated with a significantly lower requirement for pain relief. While our acute results for both cryotherapy and RF may not be as high as those in some comparable studies the recurrence rate was only 2.5% in the RF group, with no clinical recurrence in the cryo group. While cryotherapy cannot perhaps be advocated as first line therapy, it may be useful in certain circumstances. Certainly, if cryoablation is performed for AF, and if the isthmus needs to be ablated, cryotherapy might be used as well [37].","keyphrases":["catheter ablation","atrial flutter","radiofrequency","cryothermy","arrhythmia","atrial fibrillation"],"prmu":["P","P","P","P","P","P"]}
{"id":"Histochem_Cell_Biol-4-1-2248607","title":"Discovery and progress in our understanding of the regulated secretory pathway in neuroendocrine cells\n","text":"In this review we start with a historical perspective beginning with the early morphological work done almost 50 years ago. The importance of these pioneering studies is underscored by our brief summary of the key questions addressed by subsequent research into the mechanism of secretion. We then highlight important advances in our understanding of the formation and maturation of neuroendocrine secretory granules, first using in vitro reconstitution systems, then most recently biochemical approaches, and finally genetic manipulations in vitro and in vivo.\nMorphological era of discovery (1950s and 1960s)\nVisualization of the cell interior by electron microscopy catalysed both morphologists and biochemists to initiate experiments and make observations to identify, define and understand the complex compartmentalization of specialized cells, most notably including the morphologists G.E. Palade, C. DeDuve, D.W. Fawcett, K.R. Porter, J. Rhodin and F.S. Sjostrand. The exocrine pancreatic acinar was a specialized cell type favoured by GE Palade, and it was with this cell type that the first observations on cells with specialized granule compartments were made.\nThe complex morphology of the cell compartments, and the desire to link a morphological observation with basic biochemical functions and pathways known at that time initiated the monumental effort to couple a biochemical approach with EM observations. Indeed in 1956, Palade observed intracisternal granules in the endoplasmic reticulum (ER) which resembled zymogen granules, and postulated that the material in the intracisternal granules was possibly the same as the zymogen granules, and these granules were related to the granules in the cells in the endocrine pancreas, \u03b2-cell granules (Palade 1956). Using the pancreatic acinar cell model, experiments by Siekevitz and Palade between the late 1950s and early 1960s defined the role of the ER and other subcellular compartments in the synthesis of zymogen granule proteins, and resulted in a series of papers published in the newly established Journal of Biophysical and Biochemical Cytology, soon to be renamed Journal of Cell Biology (Siekevitz and Palade 1958, 1962). The concept that proteins are transported from their site of synthesis, the ER, through the Golgi complex was also being recognized and visualized using EM autoradiography in the exocrine pancreas (Caro and Palade 1964), first at a low temporal level of resolution. Direct evidence for transport from ER through Golgi to zymogen granules was then obtained using more precise pulse-chase protocols on tissue slices by Jamieson and Palade (Jamieson and Palade 1967a, b). This technical advance was further exploited by Jamieson who applied subcellular fractionation techniques to pulse-labelled slices. Separation of the rough and smooth ER from condensing vacuoles and zymogen granules allowed a quantitative kinetic analysis and a direct demonstration that proteins are transferred from the ER to zymogen granules via condensing vacuoles (Jamieson and Palade 1967a, b).\nParallel studies in the anterior pituitary gland showed that discrete, small individual granules, called immature secretory granules (ISG) in mammotroph cells originated from the Golgi cisternae. These small ISGs coalesced into aggregates surrounded by a single membrane, finally becoming mature secretory granules (MSGs) (Smith and Farquhar 1966). Additional information about the secretory process in mammotrophs was obtained using high resolution EM autoradiography. ISGs (20\u2013100\u00a0nm structures) were found to be maximally labelled after 30\u00a0min pulse with [3H]-leucine, and mature over the following 2\u00a0h to MSGs. This kinetic of maturation correlated well with the observations in the exocrine pancreas which demonstrated the condensing vacuoles were labelled after 37\u00a0min (Jamieson and Palade 1967a). The complexity of the secretory granule compartment was revealed in subsequent studies on dispersed pituitary cells where four types of secretory granules were detected (Salpeter and Farquhar 1981). After 15\u201355\u00a0min of chase the labelled protein was found in small (Type I) ISGs, and subsequently in type II and III polymorphic granules, and then larger Type IV MSGs after 55\u2013185\u00a0min of chase (see Fig.\u00a01, and Farquhar et al. 1978). A higher degree of resolution was obtained using newly developed fine-grain emulsion which allowed an accurate detection of small structures (20\u2013100\u00a0nm), which combined with a more sophisticated analysis of silver grain distribution, led to the discovery that concentration of secretory protein in the ISG compartment was 200 times that of the adjacent Golgi cisternae (Salpeter and Farquhar 1981).\nFig.\u00a01Mammotroph cell from the anterior pituitary gland of a lactating rat. This micrograph illustrates the morphological complexity of the regulated secretory pathway, and the different types of ISGs. ER endoplasmic reticulum; CM cell membrane; SG secretory granule; SV smooth vesicles; VE vesicle; LB lytic body. Reproduced from the J Cell Biol, 1966, 31:319\u2013347. Copyright 1966 The Rockefeller University Press\nDuring this period Tartakoff and colleagues outlined the concept that secretory cells may have different ways to secrete newly synthesized proteins; cells such as plasma cells, fibroblasts, and macrophages secrete in a \u201cnon-regulated\u201d fashion in contrast to \u201cregulated\u201d cells such as exocrine pancreas, the hallmark of which is the storage of the secretory proteins (Tartakoff and Vassalli 1978). The nature of the secretory pathways in such \u201cnon-storage\u201d secretory cells was also explored by the use of drugs that perturbed secretion, such as monensin, a Na+\/K+ ionophore which causes a neutralization of acidic intracellular compartments, or drugs that alter the energy status of the cell (Tartakoff et al. 1977). The common requirement for energy, cyclic nucleotides, Ca+, and cytoplasmic Na+\/K+, in both non-regulated and regulated cells, gave rise to the consensus that there is a common secretory pathway originating in the RER, through the Golgi complex (for review see Palade 1975).\nMany of the observations made were obtained using careful morphological approaches and expanded using biochemical techniques, and subcellular fractionation. However, these early biochemical approaches were limited by the inability to manipulate the cell systems in use, typically tissues from mice or rats. The arrival of molecular tools and genetic manipulation provided the next wave of advances bringing the field to our current level of understanding.\nCell line model systems, the molecular age and the sorting problem\nRegulated secretory cells, as well as other cell types such as liver cells, have specialized plasma membrane domains, which by definition have a unique composition. Therefore, in addition to classification of regulated versus non-regulated secretion (also called constitutive secretion (Kelly 1985), it was recognized that there must be multiple ways to reach distinct domains from the Golgi complex, in particular for membrane proteins. The first direct demonstration of multiple routes to the plasma membrane was from the work of Gumbiner and Kelly in 1982. Their observations using the AtT20 cell line, a mouse pituitary cell line, showed that the regulated secretory hormone ACTH was secreted with different kinetics from a viral model membrane protein, gp70, a glycoprotein of the endogenous murine leukaemia virus. Surprisingly, the kinetics of the secretion of a proportion of the ACTH precursor, POMC, which escaped the activity of pro-hormone processing enzymes present in the MSG was the same as gp70 (Gumbiner and Kelly 1982). These experiments solidified \u201cthe two pathways hypothesis\u201d (Kelly 1985) which defined distinct post-Golgi pathways for proteins targeted for regulated and constitutive secretion. At this time it was also recognized that the mechanism for segregation of cargo proteins (either soluble or membrane associated), or sorting into a particular pathway was an important issue, as was the identification of the precise location for the initiation of the sorting process. A key technological advance in elucidating both sorting and location was the development of immunoelectron microscopy using thin, frozen sections (Tokuyasu 1980) which allowed the identification of proteins within subcellular compartments.\nTGN and post-TGN sorting and processing in neuroendocrine cells\nIt was recognized in mid-1980s that the trans-Golgi network (TGN) might be the key exit point for proteins destined to the plasma membrane, constitutive secretory vesicles (CSV), endosomes, and ISGs (Griffiths and Simons 1986). It was proposed that sorting receptors might function in this compartment to segregate different cargos into different vesicles (Burgess and Kelly 1987). It also became apparent from studies on the mannose-6-phosphate receptor (M6PR), which with its bound lysosomal enzyme is sorted to endosomes, that sorting to endosomes from the TGN utilized clathrin and clathrin-coated vesicles (Geuze et al. 1985). At this time it was also proposed that CSVs were formed without the aid of clathrin coats (Griffiths et al. 1985). However, clathrin-coated regions were detected on the surface of \u03b2-cell secretory granules associated with the TGN after monensin treatment (Orci et al. 1984) or on ACTH-containing ISGs in AtT20 cells (Tooze and Tooze 1986) raising the possibility that 1) ISGs formation is through clathrin-coated regions of the TGN, or 2) that the biogenesis of ISGs involved a clathrin-coated vesicle dependent pathway.\nAcidification of the TGN and post-TGN secretory compartments was increasingly recognized to be important for both transport (see Tartakoff et al. 1977) and sorting. A key contribution at this time was the direct demonstration, using DAMP labelling (Anderson et al. 1984), that the TGN and post-TGN compartments were acidic, and there was a gradient of acidification in the secretory pathway. It was proposed that acidification could play role in receptor-ligand uncoupling and recycling, thereby providing directionality to transport (Anderson and Pathak 1985). Although the concentration of DAMP, was thought to be proportional to the extent of acidification, later studies provided accurate measures of pH of the TGN and ISG (see review Moore et al. 2002).\nNeutralization of acidic compartments caused mis-sorting of regulated secretory proteins to the constitutive pathway (Moore et al. 1983), a result which gave rise to the possibility that regulated secretory proteins may have sorting signals, and could be sorted by a receptor-ligand interaction in the TGN, or in a post-TGN compartment. The most controversial observation in support of this hypothesis was the proposed role for carboxypeptidase E as the sorting receptor (Cool et al. 1997), although this was immediately refuted (Irminger et al. 1997). Evidence in support for a sorting signal was the demonstration that regulated secretory proteins have transferable sorting signals, the first being in 1986 (Moore and Kelly 1986). An alternative hypothesis was that aggregation of regulated secretory proteins, best demonstrated for the Granins but also shown for a variety of other regulated secretory proteins, and favoured by low pH on the TGN, drives lumenal segregation of regulated secretory proteins. The segregation of the regulated secretory proteins maybe further enhanced by a homophilic interaction with membrane associated population of Granins (Gerdes et al. 1989). Furthermore, evidence for formation of multiple secretory granule populations with different hormone content supports the idea that aggregation drives sorting (Hashimoto et al. 1987). Some of these results form the basis for the \u201csorting for entry\u201d hypothesis, which proposes that regulated secretory proteins are sorted into ISGs in the TGN, while constitutively secreted proteins enter CSVs (see review Tooze 1998).\nAnother model to explain sorting in the regulated pathway was developed from experiments in \u03b2-cells, where it was shown that insulin-containing secretory granule formation is driven by the hexamerization and condensation of insulin in the ISGs (Arvan et al. 1991). These studies revealed an essential difference between neuroendocrine cells and \u03b2-cell insulin granules, giving rise to the \u201csorting by retention\u201d hypothesis (see review Arvan and Castle 1998) which proposes that rather than an active sorting in the TGN, the crucial sorting step occurs in the post-TGN ISGs. Both the \u201csorting for entry\u201d and \u201csorting by retention\u201d hypothesis agree on that non-regulated (possibly mis-sorted) secretory proteins are removed by ISG-specific clathrin-coated vesicles in a pathway called \u201cconstitutive-like secretion\u201d, after secretory granules have formed from the TGN.\nMost, if not all of the soluble regulated secretory proteins are processed by the endopeptidases, the pro-hormone convertases (PCs). Their discovery and final molecular characterization was driven by the original pioneering work of Steiner and colleagues working in the \u03b2-cell (Steiner et al. 1974), and reviewed by (Seidah et al. 1993). These enzymes are present in ISGs and MSGs, and are subjected to the same sorting machinery as are proteins such as the Granins. As their activity is pH-dependent, a large number of studies used the kinetics of their activation as monitors for studying where and when the sorting of regulated proteins occurred (see also below). A direct demonstration that the site of prohomone processing of proinsulin was the clathrin-coated ISG was obtained by immunocytochemistry (Orci et al. 1985b), which were subsequently shown to be acidic (Orci et al. 1986).\nThe progress from the 1950s until the early 1990s was the result of many researchers\u2019 effort, most of which have not been covered in this brief review. The reader is directed to comprehensive reviews from the period (Burgess and Kelly 1987; Mains et al. 1987), and more recently (Borgonovo et al. 2006; Dannies 2001; Kim et al. 2006; Meldolesi et al. 2004; Solimena and Gerdes 2003). The main questions which we focus on in the next section are the molecular machinery of secretory granule formation and maturation, as addressed by the work started in the laboratory of W. Huttner and continued in the author\u2019s laboratory which is largely based on in vitro reconstitution assays using isolated TGN, and post-TGN subcellular compartments, in particular ISGs as first developed using Golgi membranes by Rothman and colleagues (Fries and Rothman 1980).\nIn vitro reconstitution and biochemical analysis of secretory granule biogenesis\nCell-free reconstitution of ISG budding from TGN\nWhile MSGs from a variety of tissues had been extensively characterized morphologically as well as biochemically, little was known about ISGs beyond their morphological appearance (Smith and Farquhar 1966). The cell-free reconstitution of secretory granule budding from the TGN brought new insights to understanding the formation of ISGs (Tooze and Huttner 1990). This assay was based on (1) the high fidelity sorting properties of PC12 cells which, unlike AtT20 cells, target greater than 90% of their regulated secretory protein to ISGs and MSGs, (2) the selective labelling of a regulated secretory marker, secretogranin II (SgII), and a constitutively secreted protein, heparan sulfate proteoglycan (hsPG) with radioative sulfate by sulfotransferases present only in the TGN, and (3) the ability to separate the TGN, ISGs and CSVs from one another by sequential velocity and equilibrium gradients centrifugations. This assay demonstrated that SgII and hsPG were present in two vesicle populations, SgII-containing regulated secretory vesicles (ISGs), and hsPG-containing CSVs. These results provided the first demonstration that regulated secretory proteins and constitutive secretory proteins were sorted into two distinct vesicle populations directly upon exit from the TGN, in support of the \u201csorting for entry\u201d hypothesis. The formation of both ISGs and CSVs required GTP-binding proteins (Tooze et al. 1990), possibly heterotrimeric G-proteins (Leyte et al. 1992). The rate of formation of the ISGs was indistinguishable from the CSVs, occurring with a t1\/2 of 15\u00a0min.\nIn addition, the subcellular fractionation protocol developed to distinguish ISGs and MSGs allowed the biochemical and morphological characterization of ISGs, in comparison to MSGs, isolated from PC12 cells (Tooze et al. 1991). ISGs were shown to have several components of the clathrin-coat machinery, including AP-1 (Ditti\u00e9 et al. 1996), and ARF1 (Austin et al. 2000), in addition to non-granule proteins such as the M6PR and furin (Ditti\u00e9 et al. 1997, 1999). In addition, it was possible to determine the size of ISGs and MSGs, results which provided direct support for the early EM autoradiography (Smith and Farquhar 1966) and became the basis for the experiments directed towards a molecular understanding of the change in size of the ISG through homotypic fusion and subsequent remodelling of the ISG through clathrin coats (see Fig.\u00a02).\nFig.\u00a02Key steps in secretory granule biogenesis. Model based on data discussed above, in addition to contributions from many researchers which have not been mentioned, but whose contributions are noted. Step 1 depicts the formation of ISGs from the TGN, Step 2 the homotypic fusion event between ISGs, and Step 3 the clathrin-mediated membrane remodelling event. See text for abbreviations\nDetermination of the pH of ISGs using an in vitro approach\nWhile it was known from earlier studies that in endocrine cells MSGs are acidified it was not known what the precise pH of the ISG was, and if the pH of the ISG differed from the TGN. The establishment of a PC12 stable cell line expressing the PC2 allowed us to characterize the endopeptidase PC2 activity during granule maturation (Ditti\u00e9 and Tooze 1995). In this cell line, PC2 is correctly targeted to the ISGs and co-sedimented with SgII in fractions containing ISGs and MSGs. [35S] sulfate labelling demonstrated that SgII was proteolytically processed by PC2 in ISGs after approximately 30\u00a0min of chase into several lower-molecular-mass proteins, the major ones being a 18 and 28\u00a0kDa sulphated fragment.\nAs the efficiency of processing of SgII by PC2 was pH dependent, we used the ability to quantitate the extent of processing at different pHs to determine the pH in the ISGs (Urb\u00e9 et al. 1997). Isolated ISGs and TGN, containing [35S] sulfate pulse-labelled SgII were incubated in presence of ATP at physiological pH and the extent of SgII processing in these compartments was compared to a standard curve prepared using ISGs equilibrated at a defined set of pHs. This allowed us to determine that the ISGs intra-granular pH was 6.3, similar to the TGN pH and clearly higher than the pH of MSGs (pH 5.5\u20135.0). Interestingly, no processing of SgII could be observed in the membrane fraction highly enriched in TGN under conditions for which processing was readily obtained in isolated ISGs. This data represent further evidence that the ISG is indeed a functionally distinct organelle from the TGN. Furthermore, the rate of SgII processing was strongly dependent on the intragranular pH, demonstrating that processing of SgII can be used as a pH indicator for granule interior.\nISG-ISG homotypic fusion\nAs shown originally by Farquhar (Smith and Farquhar 1966) in the anterior pituitary, and more recently in PC12 cells (Tooze et al. 1991), ISGs increase in size during maturation, and the increase in size was proposed to reflect homotypic fusion of ISG. To demonstrate that ISG\u2013ISG fusion occurred, we developed an assay that provided the first biochemical evidence for such a fusion event and allowed us to dissect the molecular requirements of this process.\nThe cell-free assay to reconstitute homotypic fusion was performed by mixing two populations of ISGs, one containing the pro-hormone convertase PC2 and the other containing [35S]sulfate-labelled secretogranin II (SgII) (Urb\u00e9 et al. 1998). The fusion was then measured by quantification of the 18\u00a0kDa PC2 cleavage product of SgII. This in vitro reconstitution of ISG-ISG fusion revealed that homotypic fusion is dependent on NSF (Urb\u00e9 et al. 1998), \u03b1-SNAP and on the SNARE Syntaxin 6 but not on Syntaxin 1 or SNAP-25 (Wendler et al. 2001). More recently, Synaptotagmin IV (Syt IV), a member of the Synaptotagmin family of proteins involved in membrane fusion, has also been shown to be required for this step of granule maturation (Ahras et al. 2006).\nISG membrane remodelling\nIn addition to ISG-ISG homotypic fusion, ISG content and membrane remodelling is another important step during granule maturation. This remodelling is performed via budding of clathrin-coated vesicle from the maturing granule membrane, and is the pathway for non-regulated constitutive-like secretion, or possibly sorting to endosomes. This clathrin-mediated remodelling step is a common feature in neuroendocrine and endocrine secretory granules, and is thought to provide a mechanism for proof-reading the content and membrane composition of the maturing ISG to ensure the production of MSGs which contain biologically active hormones and can undergo efficient exocytosis.\nISGs in endocrine and neuroendocrine cells were shown by morphological techniques to be partially clathrin coated (Orci et al. 1985a; Tooze and Tooze 1986) but the recruitment mechanism and the composition of this clathrin coat were unknown until a biochemical approach was used. Our laboratory developed a fourth cell-free assay whereby the recruitment of the clathrin adaptor complex AP-1 to ISGs was reconstituted by the addition of bovine brain cytosol (Ditti\u00e9 et al. 1996). These experiments showed that AP-1 recruitment to ISGs was ATP-independent but GTP- and, ARF1-dependent. This study demonstrated that it is the AP-1 complex that is involved in the clathrin binding to ISGs. Cross-linking experiments demonstrated the direct interaction of AP-1 and ARF1 on the ISG (Austin et al. 2000). The M6PR and furin are likely part of the cargo targeted for removal from the ISGs by clathrin-coats as they possess sequences in their cytoplasmic domains which interact with AP-1, and are present on ISGs, but absent from MSGs (Ditti\u00e9 et al. 1997, 1999).\nFurther investigation into cargo sorting from the ISG showed that VAMP4, a SNARE involved in endosome to TGN vesicle trafficking, which is present on ISGs but removed from the maturing granule membrane, is also able to bind AP-1. The recruitment of AP-1 by VAMP4 is dependent on phosphorylation of the cytoplasmic domain of VAMP4 by the kinase CKII. CKII phosphorylation of VAMP4 allows the recruitment of PACS1 to ISGs enhancing the AP-1 dependent sorting event (Hinners et al. 2003). These data suggest that sorting non-regulated secretory proteins from ISGs, including the SNARE proteins, can be mediated by the recruitment of clathrin coats and may be essential for maturation of ISGs.\nMore recently, our laboratory demonstrated that the GGAs proteins are also involved in this clathrin-mediated membrane-remodelling step of secretory granule maturation. GGAs (Golgi-associated, \u03b3-ear-containing, ADP-ribosylation factor-binding protein) are involved in recruitment of AP-1 and clathrin coats to membranes (for review see Robinson and Bonifacino 2001). siRNA mediated knock-down of GGA3 and over-expression of dominant negative form of GGA proteins resulted in a retention of the SNAREs Syntaxin 6 and VAMP4 in the MSGs. In addition, an alteration of the PC2 activity was also detected, suggesting that inhibition or alteration of clathrin-coat remodelling inhibited or altered intra-lumenal acidification (Kakhlon et al. 2006). Similar effects on PC2 activity were seen after siRNA mediated knock-down of Syt IV, suggesting that inhibition of either homotypic fusion or remodelling have similar detrimental effects on acidification of ISGs.\nIt was proposed that secretory granules, like other membranes have lipid micro-domains, or rafts (Tooze et al. 2001), and that the lipid composition of the secretory granule membrane is important for protein sorting during clathrin-coat mediated membrane remodelling. In fact, cholesterol was shown to be an important component in secretory granule formation (Wang et al. 2000). Recent work by Katsumata et al. (2007) has shown that the secretory granule membrane is composed of two kinds of micro-domains and that the proteins expected to be removed during the membrane remodelling (VAMP4 and Syntaxin6) are clustered together in the same micro-domain and separated from the proteins (VAMP2) that stay on the MSG membrane. These experiments validate our observations (see above), as well as the work by H.P. Moore in AtT20 cells (Eaton et al. 2000) and provide new information that opens new avenues for research into the role of lipids in sorting from ISGs.\nRecent developments in secretory granule formation and maturation\nIn this last section we summarize the most recent developments in our understanding of secretory granule biogenesis obtained from biochemical screens, in vivo experiments based on genetic manipulation of cell in culture as well as in vivo transgenic mouse models. Only a small number of recent results are highlighted and readers are directed to recent reviews (Borgonovo et al. 2006; Dannies 2001; Kim et al. 2006; Meldolesi et al. 2004; Solimena and Gerdes 2003). The experiments we highlight show the direction that the field has taken, and the way less tractable questions can be addressed by new biochemical screens and transgenetic mouse models.\nRole of cholesterol in secretory granule biogenesis\nSecretory granule formation is dependent on lipid raft microdomains that are enriched in cholesterols (Wang et al. 2000). A recent investigation of the role in vivo of cholesterol in secretory granule biogenesis was done using Smith\u2013Lemli\u2013Opitz Syndrome (SLOS) and lathosterolis mouse models (Gondre-Lewis et al. 2006). Both diseases are a result of inborn errors in cholesterol synthesis. SLOS is a disease caused by a defect in the function of the enzyme, 7-dehydrocholesterol reductase (DHCR7), required for the final step of cholesterol biosynthesis. Patients with lathosterolosis lack lathosterol-5-desaturase (SC5D), the enzyme that catalyse the next-to-last step in cholesterol synthesis. Data obtained from DHCR7 and SC5D knock-out mice demonstrated that impairment of the cholesterol biosynthesis results in a significant decrease in secretory granules in pancreas, pituitary and adrenal glands. In addition, abberant zymogen granules were found in the exocrine pancreas, in which there was also observed a decrease in regulated secretion which could be rescued with exogenous cholesterol. The authors conclude that the presence of an elevated quantity of other sterols, 7-DHC and lathosterol, in Dhcr7-\/- and Sc5d-\/- mice, respectively, could not replace cholesterol in the regulated secretory pathway. They speculate that the abnormal properties of the secretory granule in these mice models may be attributed to the reduced rigidity of membranes containing these sterols instead of cholesterol. Importantly, this study demonstrates that cholesterol is essential during secretory granule biogenesis in vivo.\nRegulation of secretory granule maturation\nPeptidylglycine \u03b1-amidating monooxygenase (PAM) is an essential enzyme in the processing of many bioactive peptides and hormones (Prigge et al. 2000). PAM is targeted to the regulated secretory pathway in neurons and neuroendocrine cells. It is a type I membrane protein containing two enzymatic domains within the lumen of the secretory granule, a transmembrane domain, and a cytosolic domain containing sorting signals. PAM catalyses one of the final steps in peptide biosynthesis, and is retrieved from ISGs and the plasma membrane for re-utilization in newly forming secretory granules (Ferraro et al. 2005). Among the proteins able to interact with PAM are two Rho guanine nucleotide exchange factors (GEFs), Kalirin and Trio, identified by yeast two-hybrid screens using the cytosolic domain of PAM as bait (Alam et al. 1996; Xin et al. 2004).\nIn a recent study, Kalirin and Trio have been shown to be involved in the maturation of secretory granules (Ferraro et al. 2007). Overexpression of their N-terminal GEF domain enhances secretion from ISGs, reducing regulated secretory protein storage through constitutive-like secretion, in the absence of secretagogue stimulation of regulated exocytosis. Conversely, when GEF activity is inhibited the constitutive-like release is inhibited, resulting in the accumulation of cargo in MSGs above even normal levels. These results indicate that these two Rho-GEFs regulate sorting into secretory granules, constitutive-like secretion from ISGs, and perhaps constitutive secretion, and provide a novel mechanism for the regulation of secretory granule maturation.\nRole of Chromogranin A in secretory granule formation\nChromogranin A (CgA) and B (CgB), members of the Granin family, have long been proposed to control the secretory granule biogenesis because of their pH-, calcium- and catecholamine-dependent aggregation properties, and widespread expression pattern. Recent results supports this hypothesis, however other recent results suggest that the regulated phenotype (Day and Gorr 2003; Meldolesi et al. 2004) is not simply conferred by expression of CgA.\nIn support of the hypothesis, in 2001, Kim et al. demonstrated that specific depletion of CgA, but not of CgB, by siRNA impaired secretory granule production in PC12 cells. In contrast, over-expression of CgA in 6T3 cells lacking CgA and the regulated secretory pathway, or fibroblastic CV-1 cells, not only triggered recovery of secretory granules but also regulated secretion (Kim et al. 2001). However, Malosio et al.(2004) refuted the hypothesis by demonstrating with very similar approaches that the \u201cnewly formed\u201d vesicles which contained the CgA were in fact lysosomes, and overexpression of CgA did not induce the appearance of secretory granules, or alter the number of secretory granules.\nThree groups used mouse models to determine the role of Chromogranin A in vivo by targeted ablation of the Chromogranin A gene. Mahapatra et al.(2005) confirmed the putative role of CgA in secretory granule biogenesis, in particular a decrease of chromaffin granule size and number. In addition, defects were observed in neurotransmitter storage and release and regulation of blood pressure. Similar results were obtained using anti-sense vectors specific for CgA in transgenic animals which resulted in a reduction of the CgA levels (Kim et al. 2005). In the adrenal medulla of these mice there was a large decrease in the number of secretory granules; in addition it was noted that the secretory granules present appeared to be swollen. This defect may be related to a need to maintain a proportional catecholamine and CgA concentrations.\nLastly, Hendy et al. (2006) found that the CgA null mutant mice had elevated secretion of epinephrine, norepinephrine and dopamine and that mRNA and protein level of other secretory granule proteins were up-regulated. No obvious abnormalities in development or neuronal and endocrine functions have been noticed. The authors suggested then that the increased expression of the other Granin family members is likely to compensate for the CgA deficiency.\nRole of prohormone convertases in secretory granule maturation\nBoth in vitro and ex vivo the activity of the PC enzymes (notably PC1\/3 and PC2 which will be the focus of the discussion here) have been extensively studied. PC null mice models have confirmed the in vivo function of the PCs and extended the characterization of their specific substrates and action in different tissues.\nThe first PC transgenic mice model obtained was the PC2 null mouse (Furuta et al. 1997). Mice lacking PC2 activity develop normally and are fertile, but exhibit a variety of neuroendocrine processing abnormalities in the brain and in pancreatic islets. These mice show elevated levels of pro-insulin (Furuta et al. 1998) and pancreatic islet pro-glucagon processing is completely blocked. Other defects in PC2 null animals include lack of production of \u03b1-MSH associated with accumulation of ACTH in the pituitary intermediate lobe (Laurent et al. 2002).\n7B2, also known as SgV or SGNE-1, is a small acidic protein exclusively localized to neuroendocrine tissues. This protein has been shown to be associated to PC2 and it functions as a chaperone for PC2 (Braks and Martens 1994), and for review see Mbikay et al. (2001). A 7B2 null transgenic mice model has shown that 7B2 is required for PC2 activation in vivo, having in addition important functions in regulating pituitary hormone secretion (Westphal et al. 1999). 7B2 null mice have no demonstrable PC2 activity, and in general agreement with the PC2 null mouse (Furuta et al. 1997), are deficient in processing islet hormones and display hypoglycemia, hyperinsulinemia, and hypoglucanemia, with generalized islet cell expansion and altered islet cell morphology. However, the most important impairment of PC2-mediated peptide processing in 7B2 nulls involved the synthesis of the corticotrophin ACTH. ACTH is produced by cleavage of POMC through the action of PC1\/PC3, further internal cleavage of ACTH occurs by the action of PC2 specifically in the neurointermediate lobe. In 7B2 nulls, ACTH remains intact in the intermediate lobe, resulting in extremely high levels of ACTH in this lobe (Westphal et al. 1999). The overall phenotype suggests that 7B2 may be involved in PC2 activity, as well as secretory granule biogenesis.\nFinally, the role of the PC1\/3 endopeptidase has also been investigated in a transgenic mouse model. Disruption of gene-encoding mouse PC1\/PC3 results in a syndrome of severe post-natal growth impairment and multiple defects in processing many hormone precursors, including hypothalamic growth hormone-releasing hormone (GHRH), pituitary proopiomelanocortin to adrenocorticotropic hormone, islet proinsulin to insulin and intestinal proglucagon to glucagon-like peptide-1 and -2 (Zhu et al. 2002). In summary, the analysis of PC2, 7B2 and PC1\/3 null mice has confirmed that PCs play a crucial role in the processing of many hormone precursors and the highlight the importance of PC2 and PC1\/3 in neuroendocrine tissues, and regulated secretion.\nRole of Rab3D in secretory granule maturation\nRab proteins are small GTPases that belong to the Ras protein superfamily and that function in membrane traffic. Rab3A is one of the best-characterized Rab protein. In mammals, three additional isoforms are expressed which are referred to as Rab3B, Rab3C and Rab3D. Rab3A, Rab3B and Rab3C are predominantly expressed in the nervous system, where they are localized to synaptic vesicles (Fischer von Mollard et al. 1994; Martelli et al. 2000). Rab3A-deficient mice studies suggested that Rab3A has a function intimately associated with fusion more than a function in vesicle tethering at the synapse (Geppert et al. 1997).\nIn contrast to Rab3A, Rab3B and Rab3C, Rab3D is predominantly expressed outside the nervous system, in peripheral tissues where the other isoforms either are expressed at low levels or are lacking. Originally identified in fat cells, Rab3D is present in several additional cell types including secretory cells such as pancreatic and parotid acinar cells, mast cells and peptide-secreting cell lines. In secretory cells Rab3D appears to be predominantly localized to secretory granules, thus mirroring the distribution of Rab3A in neurons and neuroendocrine cells. It was hypothesised that Rab3D could have the same function as Rab3A but in secretory cells.\nRiedel et al. (2002) investigated the role of this Rab3 isoform by knocking out the Rab3D gene in mice. Rab3D-deficient mice are viable and fertile and showed no obvious defect. In particular, both kinetics and dose response of secretagogue-induced enzyme secretion of the pancreas were normal. However, these mice show a substantially increased size of secretory granules in both exocrine pancreas and the parotid gland with the volume being doubled, while the intragranular protein concentration appears to be unchanged. These data suggest that Rab3D functions in granule maturation but not in exocytic membrane fusion.\nConclusions and future perspectives\nThe early morphological and biochemical characterization of the regulated secretory pathway provided key insights to the secretory process. More recent experiments have provided a more detailed understanding of the functional properties, and the regulation of the maturation of neuroendocrine secretory granules. Many important issues need to be resolved, for example how the cytoplasmic events, such as membrane remodelling are coupled with the intralumenal biochemical changes such as PC activation, prohormone cleavage, and acidification. In addition, the biological relevance of the maturation process, as it affects hormone processing and secretion, remains to be determined in light of the differences between endocrine and neuroendocrine granules.","keyphrases":["immature secretory granule","mature secretory granule","secretogranin","chromogranin","prohormone convertase"],"prmu":["P","P","P","P","P"]}
{"id":"Invert_Neurosci-4-1-2257991","title":"The cys-loop ligand-gated ion channel gene superfamily of the nematode, Caenorhabditis elegans\n","text":"The nematode, Caenorhabditis elegans, possesses the most extensive known superfamily of cys-loop ligand-gated ion channels (cys-loop LGICs) consisting of 102 subunit-encoding genes. Less than half of these genes have been functionally characterised which include cation-permeable channels gated by acetylcholine (ACh) and \u03b3-aminobutyric acid (GABA) as well as anion-selective channels gated by ACh, GABA, glutamate and serotonin. Following the guidelines set for genetic nomenclature for C. elegans, we have designated unnamed subunits as lgc genes (ligand-gated ion channels of the cys-loop superfamily). Phylogenetic analysis shows that several of these lgc subunits form distinct groups which may represent novel cys-loop LGIC subtypes.\nIntroduction\nMembers of the cysteine-loop ligand-gated ion channel (cys-loop LGIC) superfamily are neurotransmitter receptors that mediate synaptic transmission in vertebrates and invertebrates. Cys-loop LGICs are made up of five homologous subunits arranged around a central ion channel (Sine and Engel 2006). The characteristic cys-loop motif is situated in the N-terminal extracellular ligand-binding region of each subunit and consists of two disulphide-bond forming cysteines separated by 13 amino acid residues, several of which are highly conserved to form a signature sequence (Gene Ontology ID GO:0005230). The cys-loop plays roles in receptor assembly (Green and Wanamaker 1997) and gating of the ion channel (Grutter et al. 2005).\nThe subunit composition determines the functional and pharmacological properties of the cys-loop LGIC, thus receptor diversity is generated by multiple subunit-encoding genes. For instance, the human cys-loop LGIC superfamily consists of 45 genes encoding cation-permeable nicotinic acetylcholine receptors (nAChRs) (Kalamida et al. 2007), cation-permeable serotonin or 5-hydroxytryptamine type 3 (5-HT3) receptors (Reeves and Lummis 2002), anion-permeable \u03b3-aminobutyric acid (GABA) type A and C (GABAA and GABAC) receptors (Bormann 2000; Darlison et al. 2005) and anion-permeable glycine receptors (Kirsch 2006). As demonstrated in the fruit fly (Drosophila melanogaster), honey bee (Apis mellifera) and red flour beetle (Tribolium castaneum), insect cys-loop LGIC superfamilies are more compact, consisting of just over 20 subunits (Jones and Sattelle 2006; Jones and Sattelle 2007; Littleton and Ganetzky 2000). Approximately 15 of these genes are known to encode cation-permeable nAChRs (Sattelle et al. 2005), anion and possibly cation channels gated by GABA (Buckingham et al. 2005), glutamate-gated anion channels (Vassilatis et al. 1997) and histamine-gated anion channels (Gisselmann et al. 2002; Zheng et al. 2002). The largest known cys-loop LGIC superfamily belongs to the simple nematode, Caenorhabditis elegans, where an early analysis of its completed genome revealed 90 ligand-gated ion channel genes (Bargmann 1998). In common with vertebrates, members of this large cys-loop LGIC superfamily include cation-permeable nAChRs (Jones and Sattelle 2004) and anion-permeable GABA receptors (Schuske et al. 2004). As is the case for insects, C. elegans has glutamate-gated anion channels (Wolstenholme and Rogers 2005) but so far no nematode histamine-gated ion channels have been found. However, C. elegans possesses cys-loop LGIC receptors not identified in vertebrates and insects which include anion channels gated by acetylcholine (Putrenko et al. 2005) and serotonin (Ranganathan et al. 2000). The considerable diversity of receptor subtypes in C. elegans may be broadened further as many cys-loop LGIC subunits have yet to be characterised. Here, we present an update on the genes present in the C. elegans cys-loop LGIC superfamily.\nMethods\nAll C. elegans protein sequences bearing motifs particular to cys-loop LGICs were assembled from WormBase (http:\/\/www.wormbase.org release WS185 Dec 23 2007). These motifs and their source databases are neurotransmitter-gated ion-channel ligand-binding INTERPRO:IPR006202, neurotransmitter-gated ion-channel INTERPRO:IPR006201, neurotransmitter-gated ion-channel ligand binding domain PFAM:PF02931, neurotransmitter-gated ion-channel transmembrane region PFAM:PF02932 and ligand-gated ion channel KOG3644. Cys-loop and TM2 sequences were identified from an alignment of protein sequences of whole subunits constructed with ClustalX (Thompson et al. 1997) using the slow-accurate mode with a gap opening penalty of 10 and a gap extension penalty of 0.1 as well as applying the Gonnet 250 protein weight matrix (Benner et al. 1994). The protein alignment was viewed using GeneDoc (http:\/\/www.nrbsc.org\/gfx\/genedoc\/index.html). The neighbour-joining method (Saitou and Nei 1987), available with the ClustalX program, was used to construct a phylogenetic tree, which was then displayed using the TreeView application (Page 1996).\nResults and discussion\nOur analysis of the C. elegans genome revealed the largest cys-loop LGIC superfamily so far characterised consisting of 102 genes. As we have previously reported the C. elegans nAChR and orphan subunits (Brown et al. 2006; Jones et al. 2007; Jones and Sattelle 2004; Mongan et al. 1998), we concentrate here on the remaining cys-loop LGIC genes. For unnamed subunit genes, we continued to use the nomenclature first used for orphan nAChR subunits (Jones et al. 2007). Thus, 24 genes were designated lgc for ligand-gated ion channel of the cys-loop LGIC superfamily (Table\u00a01). As shown in Fig.\u00a01, the C. elegans cys-loop LGIC subunits were grouped according to sequence homology. As with the C. elegans nAChR subunits (Mongan et al. 1998), each group was named, if possible, after a well characterised subunit. For a detailed comparison of C. elegans cys-loop LGIC subunits with those of other organisms, see (Dent 2006).\nTable\u00a01C. elegans cys-loop LGIC subunits that have been given names in this report. Amino acid residues preceding TM2 that are important for determining the charge selectivity of the ion channel (Jensen et al. 2005) are given. The cys-loop sequence is shown with the cysteine residues highlighted in black shading while gray shading indicates conserved residues. The presence of a putative second cys-loop (Dent 2006) is also notedFig.\u00a01Tree showing the cys-loop LGIC gene superfamily of C. elegans. This figure focuses on cys-loop LGIC subunits other than nAChRs although representative subunits of the nAChR groups are shown. For the complete C. elegans nAChR gene family, see (Jones et al. 2007). Based on protein sequence homology, the subunits are divided into eight groups with three highly divergent subunits, LGC-32, LGC-33 and LGC-34, not belonging to any group\nUngrouped subunits\nThree subunits, LGC-32, LGC-33 and LGC-34, were not placed into any groups as they are highly divergent (Fig.\u00a01), only showing up to 10, 10 and 15% identity respectively with other C. elegans, human and D. melanogaster cys-loop LGICs. These subunits have not been functionally characterised so it is unknown what ligands act on them. The sequence of LGC-34 and a close homologue in the parasitic nematode, Dirofilaria immitis, has been previously described where it has been noted that even though the subunit possesses features common to cys-loop LGICs, such as conserved residues in the N-terminal extracellular region and four transmembrane domains, the cys-loop is absent (Yates and Wolstenholme 2004). Interestingly, a bacterial proton-gated ion channel (Glvi) resembling cys-loop LGICs but also lacking the cys-loop has been recently reported (Bocquet et al. 2007; Tasneem et al. 2005). LGC-34 may thus represent more ancestral members of the cys-loop LGIC superfamily. Unlike Glvi, LGC-34 possesses two cysteine residues which are conserved in glutamate-, histamine- and vertebrate glycine-gated anion channels (Dent 2006; Yates and Wolstenholme 2004). Known as the second cys-loop, these two cysteines flank loop C which is a region that contributes to ligand binding (Corringer et al. 2000).\nThe EXP-1 Group\nThis group consists of only two subunits, one of which is EXP-1, a GABA-gated cation channel that mediates enteric muscle contraction (Beg and Jorgensen 2003). While EXP-1 overall resembles more closely anion permeable cys-loop LGICs such as UNC-49 as opposed to cation-permeable nAChRs, it lacks the PAR motif preceding the second transmembrane domain (TM2) which is important for anion selectivity (Jensen et al. 2005). Instead it possesses the residues ETE where the presence of glutamic acid residues has been implicated in determining cation selectivity (Jensen et al. 2005; Wotring and Weiss 2008). The other subunit in this group, LGC-35, also has a glutamate residue preceding TM2 (Table\u00a01) and thus may be a cation channel.\nThe AVR-14 Group\nThis group consists of six subunits (Fig.\u00a01) that make up glutamate-gated chloride channels that are targeted by the avermectin and milbemycin anthelmintics (Wolstenholme and Rogers 2005). Examples of GluCl function include AVR-15 and GLC-2 in the inhibition of pharyngeal pumping (Dent et al. 1997; Laughton et al. 1997; Pemberton et al. 2001) and AVR-14, AVR-15, GLC-1 and GLC-3 in regulating locomotion (Cook et al. 2006). Recently, it has been shown GLC-3 plays a role in olfactory behaviour (Chalasani et al. 2007).\nThe UNC-49 Group\nProducts of the unc-49 gene form a GABA-gated anion channel that mediates body muscle inhibition during locomotion (Bamber et al. 1999; Richmond and Jorgensen 1999). There are four other subunits included in this group (Fig.\u00a01). With regard to other species, members of the UNC-49 group are clearly most closely related to mammalian and insect GABA-gated anion channels (Dent 2006). GAB-1 forms functional GABA receptors when coexpressed in Xenopus laevis oocytes with either HG1A or HG1E, which are putative GABA receptor subunits from the parasitic nematode Haemonchus contortus (Feng et al. 2002). The remaining subunits have yet to be characterised but it is worth noting that LGC-36 possesses the amino acids AER instead of PAR before TM2 (Table\u00a01). The presence of a glutamic acid residue at this position and the absence of a proline may result in a cation-selective channel (Jensen et al. 2005).\nThe GGR-1 Group\nThis group includes six subunits (Fig.\u00a01), the functional characterisation of which has so far not been published. The neurotransmitters\/ligands to which GGR-1 group subunits respond to cannot be easily inferred through phylogenetic analysis. For example, when comparing with human cys-loop LGICs they bear closest resemblance to glycine \u03b1 subunits with approximately 25% identity while LGC-42 is slightly more similar to histamine-gated anion channels with 26% identity when comparing with insect cys-loop LGICs. Members of the GGR-1 group have been previously denoted as \u201cce_Group I\u201d subunits which most closely resemble insect histamine-gated anion channels (Dent 2006). It would be of interest to determine if members of the GGR-1 group respond to glycine, histamine or other ligands not so far known to act on C. elegans cys-loop LGICs. The subunits of the GGR-1 group are likely to be anion-selective as they all possess the PAR motif before TM2 (Table\u00a01) which is important for anion selectivity (Jensen et al. 2005). The only exception is GGR-1 which instead has the amino acid residues PGR. With the proline still present and an absence of an acidic residue (particularly glutamic acid) in this region, it is likely that GGR-1 will also be anion selective.\nThe LGC-45 Group\nThis group consists of three subunits (Fig.\u00a01), the functional characterisation of which has so far not been published. As is the case for members of the GGR-1 group, LGC-43, LGC-44 and LGC-45 most closely resemble glycine \u03b1 subunits and histamine-gated anion channels when compared with human and insect cys-loop LGICs respectively. However, unlike the GGR-1 group, the LGC-45 group subunits are likely to be cation selective as they lack the PAR motif before TM2 (Table\u00a01) which is important for anion selectivity (Jensen et al. 2005). In particular, LGC-44 and LGC-45 possess glutamic acid instead of the proline residue which is likely to result in a cation-selective channel (Wotring and Weiss 2008). While LGC-44 shows notable sequence identity with other cys-loop LGIC subunits (up to 37% with LGC-45), it lacks the cys-loop (Table\u00a01), thus it may represent an ancestral cys-loop LGIC subunit as has been noted for LGC-34.\nThe ACC-1 Group\nThis group includes ACC-1, ACC-2, ACC-3 and ACC-4 which are acetylcholine-gated anion channels (Putrenko et al. 2005). Acetylcholine-gated anion channels have also been characterised from the snail, Lymnae stagnalis, which appear to have evolved from cation channels through amino acid substitutions in the ion channel pore (van Nierop et al. 2005). In contrast, acetylcholine-gated anion channels of the ACC-1 group may have arisen from substitutions in the ligand-binding domain of anion channels (Putrenko et al. 2005; van Nierop et al. 2005).\nThe MOD-1 Group\nMOD-1 modulates locomotory behaviour and is the only known invertebrate cys-loop LGIC to be gated by serotonin (Ranganathan et al. 2000). Unlike mammals which possess cation-permeable serotonin receptors (Reeves and Lummis 2002), MOD-1 is anion selective. One other subunit, LGC-50, is closely related to MOD-1 (Fig.\u00a01).\nThe GGR-3 Group\nThis group includes six subunits (Fig.\u00a01), the functional characterisation of which has so far not been published. All six subunits are likely to form anion-selective channels as they have the PAR amino acid motif before TM2 (Table\u00a01) (Jensen et al. 2005). When comparing with human cys-loop LGICs they bear closest resemblance to GABA \u03b1, \u03b3 and \u03b5 subunits with approximately 22% identity while they most closely resemble histamine-gated chloride channels (21% identity) when comparing with insect cys-loop LGICs. As with the GGR-1 group, it would be of interest to determine if the GGR-3 group consists of ion channels responding to ligands not so far known to act on C. elegans cys-loop LGICs.\nCys-loop LGIC superfamilies of other nematode species\nGenome sequencing projects are allowing the comparison of cys-loop LGIC superfamilies from different nematode species. For instance, Caenorhabditis briggsae, which is seemingly identical to C. elegans (Gupta et al. 2007; Stein et al. 2003), also has an extensive cys-loop LGIC gene superfamily although there are a few exceptions where there appears to be gene expansions in C. elegans, mainly within the nAChR orphan group. For example, lgc-23, lgc-24 and lgc-28 in C. elegans (Jones et al. 2007) may be paralogues resulting from gene duplication in the elegans species lineage since only one homologue (Cbr-lgc-28) is observed in C. briggsae. In line with this, lgc-23 and lgc-24 are situated close together within 6\u00a0kb in the C. elegans genome which may reflect a recent gene duplication event. Likewise, lgc-11 and lgc-12, as well as lgc-25 and lgc-26 have single homologues in C. briggsae, Cbr-lgc-11 and Cbr-lgc-26 respectively. Six C. elegans cys-loop LGIC subunits, lgc-13 to lgc-18, are tightly clustered within 20\u00a0kb of the T01H10 cosmid whereas in C. briggsae there are seven similar genes (CBG01219, CBG01221, CBG02706, CBG16212, CBG16226, CBG16227 and CBG16229), all of which are not clustered together. Outside of the nAChR orphan group, avr-15 and glc-1 may be paralogues arising in the elegans lineage as there is only a single homologue (CBG06688) in C. briggsae.\nThe cys-loop LGIC gene families of the parasitic nematodes, Brugia malyai and Trichinella spiralis, are much smaller than those of C. elegans and C. briggsae consisting of 30 and 19 subunits respectively (Williamson et al. 2007). The cys-loop LGIC subunits of both parasites have clear orthologous relationships with those of C. elegans with one exception, a nAChR subunit (ACR-26) which so far appears particular to B. malayi. As discussed by Williamson and colleagues, the striking difference in the cys-loop LGIC gene family sizes may reflect a free-living versus a parasitic lifestyle. Thus, the large complement of subunits in C. elegans and C. briggsae may be required for a small nervous system to be able to respond to many environmental cues which may be more restricted in the specific environmental niche occupied by parasitic nematodes. This shows that the extensive cys-loop LGIC gene superfamily observed for C. elegans is not a feature of all nematodes.","keyphrases":["ion channel","caenorhabditis elegans","acetylcholine","gaba","glutamate","serotonin"],"prmu":["P","P","P","P","P","P"]}
{"id":"Evid_Based_Complement_Alternat_Med-4-1-1810371","title":"Does the Consumption of Green Tea Reduce the Risk of Lung Cancer Among Smokers?\n","text":"Experimental and epidemiological studies were reviewed to assess whether the consumption of green tea could reduce the risk of lung cancer in smokers. Articles published since 1990 were located by searching electronic databases PubMed, Ovid and Science Direct, using keywords \u2018lung cancer\u2019, \u2018tea\u2019 and \u2018smoking\u2019 without any restriction on language. After relevant articles had been located, further papers were obtained from their reference lists. Evidence from experimental studies (in vitro animal and human trials) suggested that regular intake of green tea may be protective against tobacco carcinogens. However, the mechanism behind the protective effect is only partly understood. In most of the epidemiological studies reviewed, the green tea exposure was within 5 years of the interview or follow-up, which would coincide with the induction period and latent period of lung cancer. Longer term studies are thus needed to further quantify the cancer risk. There is some evidence suggesting regular intake of green tea at high level (>3 cups per day) may reduce the risk of smokers developing lung cancer. Improvement in measuring green tea intake is required in order to confirm the evidence from epidemiological studies.\nIntroduction\nLung cancer has become one of the leading causes of death in both developed and developing countries (1,2). Most of the deaths from lung cancer are due to smoking tobacco products. Worldwide in 2000, tobacco smoking contributed 77% of the lung cancer deaths in males aged 30\u201369 years, and 82% in males over 70 years (1,2). In 2003, for example, the prevalence of smoking in China was estimated to be 58.9% among adult males, while Chinese smokers are four times more likely to die from lung cancer than non-smokers (3).\nAlthough elimination of tobacco smoke exposure is the obvious strategy to control lung cancer, no country has been completely successful in implementing an effective intervention. The efficacy of smoking cessation programs may be enhanced if they are conducted in conjunction with other complementary therapies. Epidemiological and experimental studies have suggested that green tea may be protective against cancer in different sites (4\u201321). In particular, recent evidence from in vitro, animal and human trial studies indicated the possibility that the consumption of green tea may reduce the risk of lung cancer among smokers (2,22\u201335), yet findings from epidemiological studies remained inconsistent.\nThe aim of this review is to assess evidence from experimental and epidemiological studies whether green tea is a risk modifier for those exposed to tobacco smoke. The focus is on the measurement of green tea exposure undertaken in these studies.\nMethods\nArticles published since 1990 were located by searching electronic databases PubMed, Ovid and Science Direct, using keywords \u2018lung cancer\u2019, \u2018tea\u2019 and \u2018smoking\u2019 without any restriction on language. After relevant articles had been located, further papers were obtained from their reference lists. A total of 78 relevant articles were eventually found.\nResults and Discussion\nIn vitro and Animal Studies\nBoth in vitro and animal studies have demonstrated that green tea or tea polyphenols have a chemopreventive effect against carcinogens from tobacco and is dependent on both the period and dose of the exposure.\nPeriod of exposure\nThe period of exposure to green tea or tea polyphenols and the period of exposure to carcinogens must overlap. In one in vitro study, green tea polyphenols were introduced once into the living cell environment 2 h before exposing the cells to a smoke solution for 30 min. Result showed that green tea polyphenols can reduce DNA strand breakage induced by cigarette smoke in cultured human bronchial cells A549 (32). In another in vitro study, human lymphoid cells were exposed to one single dose of green tea extracting solution 2 h before adding benzo(a)pyrene-diol-epoxide (BPDE)\u2014a metabolic production of benzo(a)pyrene (B(a)P), which is a major component of tobacco. The results demonstrated that green tea can significantly reduce DNA damage caused by BPDE (22). Similarly in four other animal studies (23,29,33,34), mice were given green tea prior to or within 48 h of exposure to tobacco carcinogens, and the green tea was continuously administered for 16 weeks or 9 months. Other experimental conditions such as diet, humidity, temperature and dark\/light cycle were controlled. Three out of the four studies found a reduction in the incidence and the size of lung cancer tumors due to drinking green tea (23,33,34).\nDose of exposure\nThe preventive effect of green tea is likely to be related to the concentration of green tea as well as the dose of carcinogens. In experimental studies both cells and animals were often treated with relatively high doses of carcinogens and green tea. For example, in a study which showed a protective effect of green tea, the concentration of tobacco carcinogens used was equivalent to the amount contained in the sidestream smoke from one cigarette dissolved in 24 ml of water, while the concentration of green tea polyphenol was set at 250 \u03bcg ml\u22121, corresponding to 116.5 \u03bcg ml\u22121 of epigallocatechin-3-gallate (EGCG) (32). Moreover, the bioavailability of different green tea polyphenols has been a concern in animal and human studies. Green tea polyphenols are not usually stable, specifically, in water, EGCG can oxidize more than 50% within 12 h, yet green tea used in experimental studies was typically prepared every 48 h (33,34) to every 7 days (29).\nIn some studies where mice were exposed to both NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone] and green tea, the NNK dosage administered was usually equal to that experienced by a smoker in a lifetime (36). In one experiment mice were randomized into different groups and given green tea solution at concentrations of 0.2%, 0.4% and 0.6% as drinking fluid 24 h after treating with NNK. Comparing with the control group that drank only water, the protective effect of green tea was only evident at the 0.6% concentration level (33). In another study (29), mice were exposed to high levels of environmental tobacco smoke for 5 months, while being given 1.25% green tea (2.5 g tea leave mixed with 200 ml boiling water). The results indicated green tea had little effect on reducing tumor incidence or multiplicity of the tumors. However, none of the animals in the green tea treatment group experienced early death while >10% of the mice in the control group experienced early deaths, probably due to an overdose of carcinogens exposure (29). Findings from these experiments suggest that green tea may not be effective to inhibit the formation or growth of lung tumor under high level of carcinogens exposure and over a long duration.\nHuman Trials\nShort-term effect\nBecause a single dose of green tea intake only produces a short-term antioxidant effect, regular consumption seems necessary. In a human study (37), 21 non-smokers consumed 300 ml of green tea solution or about three cups. Ferric reducing ability of plasma (FRAP-assay) was used to determine the antioxidant activity in plasma. The results of this study showed that green tea consumption produced a significant increase in antioxidant activity within 30\u2013120 min of exposure, but after 120 min the antioxidant activity appeared to decrease. Another human trial with 10 healthy subjects similarly found a short-term increase of plasma antioxidant induced by drinking two to three cups of green tea at one time (38). Such a short-term increase seemed to correspond with the relatively short half-life of tea polyphenols (30). In one pharmacokinetic study (39), 30 healthy participants ingested 200 mg of EGCG equivalent to two cups of green tea. Blood samples were collected at 0.5, 1, 2, 4, 6, 8 and 24 h after the EGCG administration. This experiment found that the mean half-life of EGCG in plasma was only 118 min.\nProtective effect from regular consumption\nRegular green tea consumption could be protective over an extended period. In one pilot study (35), three non-smokers and three smokers (consumed more than 10 cigarettes per day) drank two cups of green tea in the morning, two cups in the afternoon and one cup at night daily for 4 weeks. Each cup of green tea contained 400\u2013500 mg of green tea extract. Oral keratinocytes were harvested from each subject for analysis. Outcomes included a reduction in smoking-induced DNA damage, increased apoptosis and a further decrease in the number of cells carrying damaged DNA (35). In a randomized control trial (27), 133 smokers who smoked at least 10 cigarettes per day during the past year were randomized into three groups consuming at least four cups daily of either (i) decaffeinated green tea, (ii) decaffeinated black tea, or (iii) water. After 4 months of intervention, smokers who drank decaffeinated green tea had a significant decrease in their urinary 8-hydroxydeoxyguanosine (8-OHdG) level, but no significant changes were observed in both water and black tea groups. Similar reduction in urinary 8-OHdG level was observed among HbsAg positive volunteers who took daily capsules containing green tea polyphenols equivalent to two to four cups of tea for 3 months (40). In an intensive observational study (25), the frequencies of sister-chromatid exchange (SCE) in peripheral lymphocytes were measured as markers of smoking-induced DNA damage. The frequencies of SCE were significantly higher among smokers who were non-tea drinkers, than those of non-smokers and smokers who consumed green tea at least two to three cups per day during the past 6 months. The differences between the latter two groups, however, were not significant. It is possible that consuming high levels of green tea over a long period may reduce the DNA damage caused by tobacco smoking.\nLung tumor treatment\nOnly one clinical trial used green tea for treatment of lung cancer (41). In this Phase I study, 17 advanced lung cancer patients were given green tea extract at a single dose of 3 g m\u22122 per day for 4 weeks. The dosage equates to 20 cups of green tea, which has been found to be the maximum tolerated dose. Unfortunately, no effect of the treatment was observed. Therefore, green tea is unlikely to be an effective cytotoxic agent against existing tumor.\nLimitation of human trials\nExcept two studies (27,40), most human intervention studies involved small sample sizes generally less than 50 subjects. In addition to ethical concerns, there were neither direct measurement on the concentration of green tea polyphenols in lung tissue nor on the change of lung tissue after green tea exposure. Instead, the effects of green tea were measured by the change in DNA damage and apoptosis of oral cells (35), changes in biomarkers of DNA damage such as 8-OHdG and SCE (25,27,40), and changes in plasma antioxidant capacity (30,37,38). Trials with sufficient sample size and longer period of follow-up are needed to confirm the protective effect of green tea.\nMechanisms Based on Experimental Studies\nAs shown in Fig. 1, carcinogens contained in cigarette smoke are activated by Phase I enzymes in the body, leading to the formation of DNA adducts (36). If DNA adducts escape cellular repair mechanisms, it may result in miscoding and eventually a stable mutation. Blocking any step in the pathway would reduce the incidence of lung cancer among smokers (36). Table 1 provides the possible mechanisms of chemoprevention effect of green tea. The evidence suggested that catechins in green tea, especially EGCG, may prevent the formation of a mutated cell.\nFigure 1.Mechanism of tobacco carcinogens inducing lung cancer.\nTable 1.Possible mechanism of actionPossible mechanism of chemoprevention effect of green teaReferencesActing as antioxidant to prevent DNA damage and reduce the formation of DNA adducts(11,22,25,27,35,40)Increasing apoptosis and preventing the persistence of miscoding DNA (possibly by inducing gene regulation)(11,26,31,33,35,40,43,44)Activating Phase II detoxifying enzymes, and increasing the excretion of carcinogens (possibly by inducing gene regulation)(2,8,28,31)\nIn both animal and in vitro studies, green tea was found to increase the activities of Phase II enzymes (2,8,28,31). Phase II enzymes transform carcinogens into non-toxic molecules for subsequent excretion (42). The amount of carcinogens that may be further activated, as well as DNA adducts, are thus reduced. The antioxidant properties of green tea have also been demonstrated in trials involving smokers. It reduced the urine concentration of 8-OHdG, a strong biomarker of DNA damage (27). Moreover, green tea extract could inhibit the formation of human DNA adducts (11,22,35).\nOnce carcinogen DNA adducts have formed, persistent mutation is likely unless cells with DNA adducts are removed by apoptosis (36). Human (35), animal (11,33) and in vitro studies (26,43,44) have shown that green tea can induce apoptosis among injured cells exposed to carcinogens from tobacco smoke. However, smoking may be regarded as a chronic exposure, since long duration of tobacco smoking has greater impact on lung caner risk than the amount smoked per day (45). Therefore, a long exposure to green tea is needed in order to reduce the damage caused by tobacco carcinogens.\nEpidemiological Studies\nInduction time and latent period of lung carcinoma\nLatent period refers to the time between disease occurrence and detection, while tumor induction time commences when the effect of the exposure occurs until all of the component causes involved in one sufficient cause are satisfied\u2014the time of disease occurrence (46). Exposure within the induction and latent periods is irrelevant to the formation of a tumor (47). Moreover, the incidence of lung cancer does not appear to be lower among ex-smokers who quit smoking within 5 years than current smokers (48,49). This indicates that the sum of induction period and latent period of lung cancer caused by tobacco smoking may be longer than 5 years. Logically, no reduction in incidence of lung carcinoma is expected among current smokers who drink green tea for 5 years prior to the calculation of incidence, regardless of their consumption level.\nMeasurement of green tea exposure and other limitations\nTable 2 summarizes epidemiological studies on tea and lung cancer. Three case\u2013control studies (50\u201352) found a significantly lower risk of lung cancer among non-smokers with a high consumption of green or black tea, but no protective effect was evident among smokers (50,51). However, the period of tea consumption referred to 5 years or less before the diagnosis or interview. Although the subjects might have been drinking tea for a long time, the duration and quantity of tea consumption was not assessed, so the effect of green tea could not be fully justified. Cohort studies, on the other hand, were based on information of tea consumption at the beginning of the study (53\u201355) and suggested that tea consumption increased with age (54,55). In particular, the Japanese study (53) with a long follow-up period of 14 years showed that people consuming green tea two to four times per day had a relative risk of 0.78 (95% CI = 0.6\u20131.0), when compared with those drinking once or less per day and after adjustment for potential confounders. But for subjects drinking green tea more than five times per day, the relative risk was only 0.79 (95% CI = 0.59\u20131.1), the non-significance result probably due to insufficient sample size at this high level of consumption (53).\nTable 2.Epidemiology studies on tea and lung cancerCountryStudy designSample sizeTea type and highest consumption levelReference period of intakeSmoking status of subjectsResultsChina (50)Population-based case\u2013control studyCases: 649 Controls: 675Green tea: >1500 g tea leaves per year5 years before interviewSmokers and non-smokersOR = 0.65; 95% CI = 0.45\u20130.93 for non-smokers OR = 0.94; 95% CI = 0.40\u20132.22 for smokersCanada (52)Population-based case\u2013control studyCases: 582 Controls: 582Type unspecified: >7 cups per day2 years before studyNon-smokersOR = 0.4; 95% CI = 02\u20130.7USA (51)Population based case\u2013control studyCases: 161 Controls 483Green tea: >1 cup per day1 year before diagnosis or interviewSmokers and non-smokersOR = 0.9; 95% CI = 0.5\u20131.6Japan (53)Cohort study (1980\u201394)Subjects: 38 540 Person-years at risk: unspecified Cases: 436Green tea: >5 times per dayUnspecified; survey taken at start of studySmokers and non-smokersRR = 0.78; 95% CI = 0.6\u20131.0 for people drinking tea 2\u20134 times per dayJapan (54)Cohort study (1986\u201395)Subjects: 8552 Person-years at risk: 71 248.5 Cases: 384Green tea: >10 cups per dayUnspecified; survey taken at start of studySmokers and non-smokersCancer in all sites RR = 0.68 for males; 95% CI = 0.39\u20131.21 RR = 0.57 for females; 95% CI = 0.33\u20130.98OR, odds ratio; RR, relative risk; CI, confidence interval.\nIn summary, there is only limited evidence from epidemiological studies that green tea is protective against lung cancer among smokers. Moreover, green tea exposure was generally taken to be within 5 years of interview or follow-up, which would have little impact on the incidence of lung cancer.\nConclusion\nOur review suggests that regular intake of green tea at high levels (>3 cups per day) may offer protection against tobacco carcinogens for smokers, provided that the duration of green tea consumption is sufficiently long to cover the smoking period. For epidemiology studies, improvement in measuring green tea intake is required in order to confirm the chemopreventive effect of green tea observed in in vitro animal and human trials.","keyphrases":["smoking","tobacco","polyphenols","carcinoma"],"prmu":["P","P","P","P"]}
{"id":"Eur_Spine_J-2-2-1602186","title":"Adjacent level discitis after anterior cervical discectomy and fusion (ACDF): a case report\n","text":"This report describes a case of spondylodiscitis occurring adjacent to levels at which anterior cervical discectomy and fusion was performed. The objective is to describe a rare cause of spondylodiscitis and discuss its successful management. Post-operative discitis involving the same level is a known occurrence. We report an interesting case of spondylodiscitis occurring at the adjacent level of fusion, and to our knowledge this is the first such case reported in literature. A two-level decompression and fusion was performed at C5\u20136 and C6\u20137 levels with PEEK cages and anterior cervical plating in a middle-aged gentleman for persistent axial neck pain and left-sided radiculopathy involving C6 and C7 distribution. After 6 weeks, the patient presented to us with complaints of mild paresthesia in the abdomen and extremities. Radiological investigations including plain radiographs and MRI revealed a surprising finding of discitis at C4\u20135 level with an associated epidural abscess. In view of the patient\u2019s myelopathic symptoms, surgical debridement and decompression of the spinal cord was performed. The plate and screws were removed, the cages were left intact, and the C4\u20135 disc level was reconstructed with tricortical iliac crest autograft. No further instrumentation was performed. The biopsy specimen from the disc at C4\u20135 level grew Serratia marcescens. It was contemplated that C4\u20135 discitis was initiated by inoculation of bacteria at the superior endplate of C5 by contaminated vertebral pins\/drill-bit or screws. Adjacent level discitis is a rare but potentially serious complication of anterior cervical fusion. A high index of suspicion of infection is necessary if the patient complains of new symptoms after anterior cervical fusion. Thorough assessment and aggressive treatment is necessary for successful management.\nIntroduction\nPost-operative discitis occurring at the operated level is a known complication of spinal surgery. The most common organisms implicated in the pathogenesis are Staphylococcus aureus and Staphylococcus epidermidis. Degeneration occurring at the adjacent level is also a well-established phenomenon after spinal fusion. We present a unique case of discitis occurring at a level adjacent to the levels that underwent anterior cervical fusion. The case becomes interesting because of numerous factors e.g. the site of involvement, the likely etiology, the organism concerned, the subtle nature of the symptoms, problems with diagnosis and its management.\nCase report\nA healthy 56-year-old male patient presented with neck pain of 6\u00a0months duration and associated radicular pain and numbness in the left upper-limb. The history was negative for fever, night pain or constitutional symptoms. MRI revealed left-sided foraminal stenosis at C5\u20136 and C6\u20137 levels secondary to disc herniation. A decision to perform surgical decompression and stabilization was taken after a failure of conservative trial of 12\u00a0weeks. A standard anterior cervical discectomy and fusion (ACDF) was performed at both levels using a right-sided approach. Based on anatomical landmarks and radiological guidance, adequate care was taken to expose only the presumed C5\u20136 and C6\u20137 disc levels and avoid inadvertent injury to other discs. The Casper distractor system was subsequently utilized throughout the surgery by obtaining anchorage of the Casper vertebral pins in the C5 and C7 vertebral bodies. Iliac crest autograft-packed PEEK (Solis, Stryker, Allendale, NJ, USA) cages were placed at both levels and cervical plating (Orion, Medtronic-Sofamor-Danek, Memphis, TN, USA) was performed. Prophylactic intravenous antiobiotics (cefazolin) was administered 1\u00a0h before surgery and continued for 48\u00a0h post-surgery. The immediate post-operative period was uneventful and the patient had good recovery from his neurological symptoms.\nThe patient presented 6\u00a0weeks later with mild neck and bilateral shoulder pain associated with paresthesia in his upper limbs, left lower limb and abdomen. On examination, the patient was afebrile and there was no warmth or tenderness in the region of the neck. The neck movements were terminally restricted, but there was no motor weakness. Hyperreflexia could be elicited in both lower limbs, Babinski\u2019s sign was negative and there were no objective sensory deficits. Hematological investigations revealed a mild increase in ESR (30\u00a0mm\/h), normal CRP (1.1\u00a0\u03bcg\/ml) and a mild increase in total WBC count (8.18\u00d7109\/l). Radiographs revealed persistently enlarged pre-vertebral soft-tissue shadow, decreased disc height at C4\u20135 level with erosion of end-plates and segmental C4\u20135 kyphosis, apart from the presence of halo around the screws as shown in Fig.\u00a01. MRI revealed features of discitis at C4\u20135 level with associated pre-vertebral and epidural abscesses from C4 to C6 levels as well as artifacts secondary to instrumentation and cages as shown in Fig.\u00a02a and b.\nFig.\u00a01Lateral X-ray of cervical spine at 6\u00a0weeks after index surgery. Persistence of pre-vertebral soft-tissue shadow, erosion and irregularity of C4 and C5 end plates apart from segmental kyphosis and loosening of screws can be seenFig.\u00a02a Sagittal T-2 weighted MRI image showing hyperintensity signal at C4\u20135 disc space. Anteriorly located epidural abscess at the same level can be appreciated. b Axial T-2 weighted MRI image displaying anterior compression of the spinal cord by the epidural abscess\nThe patient underwent an aggressive surgical debridement and stabilization through a left-sided approach. The loose screws and the plate were removed. A thorough decompression of the epidural abscess along with surgical debridement of C4\u20135 disc space was performed. No pus or evidence of infection was seen at the C5\u20136 and C6\u20137 levels and the cages were left behind as they appeared stable and secure. Tricortical iliac-crest autograft was impacted at the prepared C4\u20135 disc space and thorough lavage with antibiotic solution (cloxacillin and gentamycin) was carried out. The wound was then closed over a drain and an empirical course of intravenous antibiotic (cefazolin) was initiated. The drain was taken out after 3\u00a0days and the culture specimen grew Serratia marcescens, sensitive to ceftriaxone but resistant to the more commonly used antibiotics including cefazolin. Intravenous ceftriaxone was initiated for the first 3\u00a0weeks, and then the patient was discharged on oral antibiotics for another 3\u00a0weeks. The patient\u2019s condition improved dramatically with complete neurological recovery within the first week. At the 8-month mark, he is asymptomatic and the X-rays performed after 5\u00a0months reveal solid fusion at all levels and obliteration of the pre-vertebral soft-tissue shadow as shown in Fig.\u00a03.Fig.\u00a03Lateral X-ray at 5\u00a0months after the revision surgery. Solid fusion at all levels and obliteration of the pre-vertebral soft-tissue shadow can be seen\nDiscussion\nPost-operative discitis in cervical spine is not a frequent occurrence due to the rich vascularity and the routine use of antibiotic prophylaxis [5]. The prevalence ranges from 0 to 1.1%. The use of metal implants may increase the possibility of infection [4]. Discitis is a known complication of cervical discography [2, 11]. Occasional cases of cervical discitis secondary to a fishbone lodged in the throat have been reported [4, 7].\nA case of post-operative discitis adjacent to the operated level has not been previously reported. The potential causes of adjacent level discitis at C4\u20135 were scrutinized. One of the possibilities was an accidental inoculation of bacteria into the C4\u20135 disc space intra-operatively by a contaminated spinal needle that was used as a radiological marker. When we reviewed the intra-operative localizing X-ray, it was evident that the needle was placed into the C5\u20136 intervertebral disc and hence this possibility was ruled out. The possibility of discitis secondary to an esophageal perforation was ruled out since the patient neither had dysphagia nor the severe systemic symptoms that usually accompany esophageal injury in the post-operative period. Additionally, no esophageal tear was noticed during the second surgery. Discitis secondary to annular injury of C4\u20135 intrevertebral disc was ruled out since great care was taken to avoid inadvertent exposure of uninvolved levels during surgery. Hematogenous spread is another possibility but the only positive cultures came from the C4\u20135 intervertebral disc during the operation. Other samples, e.g. blood, sputum, and urine cultures demonstrated no growth and chest X-ray appeared normal. The only other possibility could be inoculation of bacteria into the superior endplate of C5 and initiation of an infective process in the C4\u20135 peridiscal region. This could be through the contaminated Caspar pins or the drill-bit utilized to create holes for the screws used for anterior instrumentation. This is a possibility since the organism that was cultured (S. marcescens) is known to contaminate and breed in normal saline solution [3, 6], and the operative instruments in question are generally placed in seemingly sterile large bowls filled with normal saline during surgery.\nPost-operative spinal infection due to S. marcescens is very rare. In a review of 2,391 consecutive index procedures on the spine, 46 cases of wound infection were identified [8]. Amongst them, only one case of Serratia infection was recognized and that was a part of mixed infection. Serratia spondylodiscitis has been reported recently involving the lumbar spine [3]. One of the patients had discitis at the same level that underwent a micro-discectomy. The second case was interesting in that her discitis occurred at the same level that had earlier undergone a posterior decompression (laminotomies and foraminotomies) but the disc was untouched. These two patients presented acutely within a week of index surgery with a fiery presentation, described as life threatening by the authors. The ESR, WBC counts and CRP levels were elevated and the blood culture grew S. marcescens. However, our patient had a subacute course and was afebrile throughout. The only abnormal features were subtle axial neck pain with subjective paresthesia in the extremities and abdomen. The ESR and WBC count were modestly raised but the CRP was normal. A high index of suspicion along with reliance on radiological investigations was essential to diagnose the condition and institute appropriate treatment. Persistence of pre-vertebral soft-tissue shadow apart from reduction in the C4\u20135 disc height, segmental kyphosis and irregularity of the endplates at C4\u20135 pointed towards a diagnosis of C4\u20135 discitis and prompted us to perform MRI in order to confirm the diagnosis and decide on the further management.\nThe literature is not very clear with respect to the duration of antibiotics to be given for spinal fusions. The protocols recommended range from no antibiotics for anterior spinal surgery [9] to 3\u00a0days of antibiotics for all spinal surgeries [10]. A meta-analysis performed to identify the efficacy of prophylactic antibiotics in spinal surgery found that there was no difference in the efficacies of various antibiotic regimens, provided at least one dose of pre-operative antibiotic with gram-positive coverage was administered [1]. Our protocol for antibiotic prophylaxis for spinal surgeries consists of a pre-operative dose followed by 2-day course of intravenous cefazolin. The protocol followed in our division is based on the fact that the drainage tube as well as the urinary catheter is removed by 48\u00a0h and hence the antibiotics are usually continued till then. S. marcescens is highly resistant to cefazolin and sensitive to third-generation cephalosporins [3], and this was evident in the culture sensitivity reports of our patient.\nAn aggressive approach to debride the C4\u20135 disc space as well as to decompress the epidural space was necessary in the wake of myelopathic symptoms and in order to isolate the organism. The spine was approached from the opposite side (left side) for the second surgery since inadvertent esophageal or neurovascular injury as a result of post-operative scarring was a possibility on the right side. The issue of leaving the PEEK cages at C5\u20136 and C6\u20137 levels behind during the revision surgery is debatable. We did not replace them because of a number of reasons. The infection was principally arising from the C4\u20135 disc level, the cages were absolutely stable and we wanted to minimize graft site morbidity. Most importantly, we felt that infection would not thrive in the presence of stability. This approach has been advocated by other authors too [8]. In the second surgery, a plate was not utilized for instrumentation for the following reasons. Firstly, the bones were soft as a result of inflammatory edema resulting from infection and hence were not ideal for screw implantation. Secondly, since we were also not sure what the organism was and as most of the times it is Staphylococcus aureus, the risk of persistent infection as a result of glycocalyx membrane formation is high. Additionally, the construct appeared stable after the C4\u20135 level was impacted with the autologous iliac crest graft and the lower two levels were already stable as aforementioned.\nConclusions\nThe possibility of infection should be considered in patients with an alteration in the nature of their symptoms after index surgery. Unusual presentations can be a challenge to diagnose and treat and a high index of suspicion and aggressive approach are prudent for a successful outcome.","keyphrases":["adjacent level discitis","epidural abscess","serratia marcescens","cervical spine","cervical spondylodiscitis"],"prmu":["P","P","P","P","R"]}
{"id":"Graefes_Arch_Clin_Exp_Ophthalmol-4-1-2206251","title":"Incidence and visual outcome of endophthalmitis associated with intraocular foreign bodies\n","text":"Purpose To determine the risk factors and visual outcome of endophthalmitis associated with traumatic intraocular foreign body (IOFB) removal and its allied management.\nIntroduction\nIntraocular foreign body (IOFB) caused ocular trauma is a significant and unique type of trauma that requires skillful investigation and an early intervention. Endophthalmitis is an uncommon but potentially catastrophic complication of penetrating ocular injury with retained intraocular foreign bodies (IOFB) [1]. Studies have reported incidence of endophthalmitis ranging from none to as high as 13.5% [1\u20135]. Management of endophthalmitis associated with retained IOFB is a challenging subject. With advancement in vitreous surgery, a large number of eyes with ocular trauma and IOFB are being saved. Visual prognosis, however, is affected by the complexity of the confluent factors surrounding the IOFB, which include the size, site, material, trajectory, reactivity of foreign body, inflammatory response, degree and type of tissue damage, length of time since injury and any associated endophthalmitis [2]. Limited information is available regarding ocular trauma and IOFB-associated endophthalmitis outside of the United States and other developed countries [6\u201310]. In particular, very limited information on this entity is available from the Middle East. The current study was undertaken to analyze and report our experience at a large tertiary eye care referral center in the Middle East regarding the management of eyes with IOFBs and associated endophthalmitis. We identify the risk factors for the development of clinical endophthalmitis and visual outcome of such eyes at a major referral center in the Middle East.\nMethods\nA detailed retrospective review was conducted of all patients who presented to King Khaled Eye Specialist Hospital, a JCIA (Joint Commission International Accreditation, USA) accredited tertiary eye care referral center in Riyadh, Saudi Arabia, from January 1983 to August 2004 with penetrating ocular trauma and retained intraocular foreign bodies (IOFB). Methods of IOFB extraction has varied during these years. For simplicity, the study period was divided into the first decade from 1983 to 1993 and the second decade from 1994 to 2004. For the past decade pars plana vitrectomy has been used more often for the posteriorly located IOFBs. Prior to removal of the foreign body (FB) all adhesions around the FB were released and it was freed from encapsulation where indicated. All IOFBs were removed by using IOFB forceps. Where necessary the sclerotomy was enlarged to facilitate easy removal of the FB. Endophotocoagulation was applied to the retina adjacent to the site of the IOFB. After removal of the FB, vitrectomy was utilized to remove any remains of FB capsule or fibrous tissue with the vitrectomy cutter. Only those eyes which showed any clinical evidence of endophthalmitis after trauma and retained IOFB were included in the study. Patients demographic studied included, age at presentation, sex, place of trauma, occupation, mode of injury and time between injury and repair. Other parameters included initial and final best corrected Snellen visual acuity, entry and location of IOFB and associated cataract, vitreous hemorrhage, retinal detachment, development of endophthalmitis, diagnostic studies performed, treatment rendered, type and size of IOFB. Complications such as cataract, retinal detachment and secondary procedures performed were also noted. Causes of visual loss such as corneal scarring, cataract, retinal detachment, retinal scars and loss of an eye were also investigated. In particular, all eyes with a retained IOFB and associated endophthalmitis were investigated and analyzed in detail.\nStatistical analysis was carried out using SPSS version 10 (SPSS Inc., Chicago, IL, USA). We studied the association between the variables using the chi-square test because the data was categorical. Further multiple logistic regression analysis was conducted to predict the factors that were associated with favorable or poor visual outcome. A P\u2009<\u20090.05 was taken as a level of statistical significance.\nResults\nAmong the 589 eyes of 565 patients (90.3% males; 9.7% females; 24 bilateral) with retained intraocular foreign bodies (IOFB) after trauma, 44 eyes (7.5%) had evidence of clinical endophthalmitis at the time of initial evaluation or subsequent to removal of the IOFB (Table\u00a01). The setting of the injury resulting in IOFB varied, with the majority occurring at the place of employment in younger patients. The size of the IOFBs recovered ranged from 0.5\u00a0mm to 18\u00a0mm (average 4.2\u00a0mm). At presentation visual acuity (VA) of 20\/200 or better was found in 8 eyes (18.1%), 20\/400 to counting fingers in 6 (13.6%) and hand motion (HM) to light perception (LP) in 30 eyes (68.2%) (Table\u00a02). Delay in treatment was mostly due to late patient referral after the trauma or lack of understanding on the part of the patient about the urgency in presentation to the primary care center. Endophthalmitis was recognized preoperatively in 32 eyes (72.7%) and post-operatively in 12 eyes (27.3%). In general, the size of the IOFB posed no significant risk of causing endophthalmitis except in cases where larger IOFB was associated with a significant trauma to the eye. The most important predictive factor of developing endophthalmitis was delayed repair of the globe and removal of IOFB. A delay in intervention of more than 24-hours was associated with a risk of clinical endophthalmitis (Fig.\u00a01). In fact, only 11 eyes (25%) which developed endophthalmitis were repaired and IOFB removed within 24 hours after their trauma compared with 33 eyes (75%) that were repaired and IOFB removed more than 24 hours after trauma (range 2\u201342 days). In other words, a delay of more than 2 days in the repair of the traumatic globe and removal of IOFB was associated with a significant risk of endophthalmitis development (P\u2009<\u20090.05). The composition of IOFB had no significant effect on the development of clinical endophthalmitis. In particular, eyes with wood IOFB did not appear to be associated with increased risk of endophthalmitis compared to eyes with metallic IOFBs. The age of the patient had no bearing on the development of clinical endophthalmitis. However, when age and delayed repair of the trauma and removal of IOFB was correlated, an association with increased evidence of endophthalmitis was observed. The risk of endophthalmitis development and poor visual outcome was less common in eyes where the location of IOFB was in the anterior chamber or lens compared to the eyes having IOFB in the vitreous or retina. Among the 10 eyes where the final vision of no light perception (NLP) was recorded after the treatment of IOFB trauma and associated endophthalmitis, only 2 eyes had anterior location of foreign bodies compared with 8 eyes where IOFB were found in the posterior chamber, the difference being significant (P\u2009<\u20090.05). There were 24 cases of endophthalmitis during the first decade (1983\u20131993) and 20 cases of endophthalmitis during the second decade (1994\u20132004) of the study; the difference however was not statistically different.\nTable\u00a01Reported incidence of endophthalmitis with IOFBStudiesEyes with endophthalmitis (total eyes)PercentBrinton et al. [11]11 (103)10.7Khan et al. [12]10 (198)5.1Williams et al. [3]14 (105)13.3Behrens-Baumann and Praetorius [5]14 (297)4.7Thompson et al. [1]34 (492)6.9El-Asrar et al. [4]13 (96)13.5Present study (Chaudhry et al.)44 (589)7.5Table\u00a02Presenting and final visual acuity of patients with IOFB and endophthalmitisVisual acuity (VA)PresentingFinal20\/20\u201320\/6031120\/80\u201320\/2005920\/400\u2013CF64HM\u2013LP3010NLP010VA visual acuity, CF count finger, HM hand motion, LP light perception, NLP no light perceptionFig.\u00a01Patient with endophthalmitis associated with retained intraocular foreign body\nPositive cultures were obtained from 17 eyes (38.6%) with clinical signs of endophthalmitis (Fig.\u00a01). In addition, 10 eyes showed evidence of microorganisms on the Gram stain but no growth was observed in cultures. Staphylococcus and Streptococcus species were most often recovered (9 eyes); the other species included Hemophilus, Bacillus, Pseudomonas, Eikenella, Corynebacterium, Propionebacterium acnea and Escherichea coli. None of the eyes with Bacillus, Pseudomonas or Corynebacterium attained any useful vision. All eyes suspected of endophthalmitis were administered intravitreal antibiotics and in some cases the treatment was repeated to obtain resolution of the signs. Most common intraocular antibiotics administered included vancomycin, ceftazidime and amikacin. Systemic preoperative antibiotics were administered in eyes with signs of endophthalmitis at approximately the same frequency as eyes without any signs of clinical endophthalmitis. However, systemic antibiotics were continued postoperatively for longer duration in eyes with signs of infectious endophthalmitis.\nThirty-one eyes (70%) underwent primary pars plana vitrectomy and 20 eyes (45%) had pars plana lensectomy at the time of IOFB removal and primary repair. Secondary procedures included scleral buckle around 8 globes, endolaser or external cryotherapy in 10 eyes and gas tamponade using SF6\/C3F8 in 10 eyes for retinal breaks or retinal detachments. In addition, 11 eyes required cataract surgery, 12 eyes developed retinal detachment requiring additional treatment after the removal of IOFB and treatment of endophthalmitis. Vitreous hemorrhage was noted in 24 eyes that were associated with posteriorly located IOFBs but none among the eyes with IOFBs located in the anterior part of the eye. The final VA was 20\/200 or better (defined as a good visual outcome) in 20 eyes (45%). Twenty-four eyes (55%) had final VA of less then 20\/200 (defined as poor visual outcome). Twenty eyes (45%) had a final VA of hand motion or worse (Fig.\u00a02). The causes of final vision of HM or worse in 20 eyes were phthisis (Fig.\u00a03) or enucleation in 10 eyes, retinal detachment with proliferative vitroretinopathy in 5 eyes and corneal or macular scar in 5 eyes. Overall, 22 eyes (50%) that developed endophthalmitis due to trauma and retained IOFB had improvement in their VA. Ten eyes (22.7%) with endophthalmitis became NLP compared to 58 eyes (10.6%) from the larger group which became NLP but had no evidence of endophthalmitis, the difference being significant (P\u2009<\u20090.05). Among the 10 eyes with endophthalmitis where the final vision of NLP was recorded after the treatment of IOFBs and associated endophthalmitis, only 2 eyes had anterior location of FBs compared to 8 eyes where FBs were found in the posterior chamber. Among the patients with endophthalmitis, 4 eyes (12.9%) from the vitrectomy group (31 eyes) had final vision of NLP, while from the eyes without vitrectomy (11 eyes), 5 eyes (45.4%) had final vision of NLP, the difference between these two groups being significant (P\u2009<\u20090.05).\nFig.\u00a02Patient with light perception vision after treatment of endophthalmitis and removal of intraocular foreign bodyFig.\u00a03Patient with phthisical left eye and no light perception after treatment of endophthalmitis and removal of intraocular foreign body\nDiscussion\nOcular trauma associated with retained intraocular foreign bodies (IOFB) constitutes a significant proportion (18\u201340%) of all ocular injuries requiring surgical management [1, 2]. Despite advances in vitreoretinal and microsurgical techniques, the management of these injuries remains a challenge. The risk of endophthalmitis developing after penetrating ocular injury with retained IOFB is relatively low with the current techniques of surgical repair [1]. The incidence of infectious endophthalmitis after retaining an IOFB has been reported to be as low as 0% to as high as 13.5% [3\u20135, 13], the mean incidence being 6.8% (Table\u00a01). The results from the present study are in agreement with the above studies of endophthalmitis associated with retained IOFB. A delay in management of more than 24 hours was found to increase the risk of infectious endophthalmitis in the present study. From a large study of the National Eye Trauma System Registry, 91% of the eyes in which infectious endophthalmitis developed had such evidence at the time of initial evaluation [1]. Delay in primary repair at our tertiary care eye hospital in the Middle East was either due to the patient presenting late or being referred to our facility later in the course of trauma and retained IOFB. Despite methods of improved transportation, early diagnosis and referral to properly trained eye care professionals, morbidity due to IOFB associated trauma has not decreased over the last decade.\nThe risk of an infectious endophthalmitis developing with IOFB has been reported to increase with age [1]; although, no such trend was observed in the present study. However, when age and delayed repair of the trauma and removal of IOFB was correlated, an association with increased evidence of endophthalmitis in our group of patients was observed. These results are in agreement with previous observations that older patients with delayed primary repair do appear to have an increased susceptibility to developing infectious endophthalmitis compared with younger patients [1].\nVisual acuity (VA) appears to be significantly affected in the eyes with IOFB that subsequently developed endophthalmitis. From a previous large study of retained IOFB, Roper-Hall [14] reported 555 cases over a 19\u00a0year period, where there were no bilateral cases: in 60 eyes IOFB was not removed, size of IOFB was recorded in only 89 eyes and the enucleation rate was 20%. From a more recent report of 96 cases of posterior segment IOFBs studied over a 14\u00a0year period with 8.6\u00a0months of average follow-up, endophthalmitis was recorded in 13.5% of cases [4]. The overall rate of 7.5% cases with endophthalmitis in the current study are within the reported range of larger series [1, 3\u20135, 14, 12].\nSeveral preoperative and operative factors have been found to have prognostic value in the final visual outcome of the traumatized eye with retained IOFB and endophthalmitis. Initial VA is an indicator of final VA and is a well recognized predictor of visual outcome in most of the previously published studies on IOFBs [4, 15]. In our study, poor initial VA of HM or light perception was significant in predicting a poor visual outcome (Table\u00a02). The risk of endophthalmitis development and poor visual outcome appeared to be significantly increased when the IOFBs were recovered from the posterior chamber (vitreous or retina), compared to the IOFBs recovered from the anterior chamber. Among the 10 eyes with endophthalmitis where the final vision of NLP was recorded after the treatment of IOFBs and associated endophthalmitis, only 2 eyes had anterior location of foreign bodies compared to 8 eyes where foreign bodies were found in the posterior chamber.\nIn our study, positive cultures were obtained in slightly more then one-third of the eyes with clinical signs of endophthalmitis. In general, the culture is often positive in clinically diagnosed endophthalmitis secondary to trauma than due to intraocular surgery. However, considering that our hospital is a tertiary eye care referral center, most of these patients were on topical as well as systemic antibiotics prior to their referral and obtaining cultures at the time of IOFB removal. This view is corroborated by the observations that 10 additional patients who had evidence of microorganisms on Gram stain showed no growth in culture. The microorganisms isolated from the eyes in the present study were similar to those reported in previous studies on the development of infectious endophthalmitis after IOFB trauma [1, 4]. Although, from many eyes suspected of infectious endophthalmitis, no definite organism was recovered. The fact that these eyes showed clinical improvement after intravitreal injection of antibiotics supports the notion that an infectious process was involved. In a recent study, reasonable visual outcomes with immediate intravitreal injection of antibiotics and delayed vitrectomy in patients with clinical features of endophthalmitis and retained IOFB have been achieved [16].\nThe role of vitrectomy in the treatment of penetrating ocular trauma has been considered a major advance in the last two decades [17]. Early vitrectomy has helped in attaining useful functional vision in 25\u201351% of the eyes with posterior segment trauma [17, 13], and in some series it has helped in significant survival of eyes without improvement of final visual outcome [17]. In our hospital, during the last decade, pars plana vitrectomy had been used more frequently for the posteriorly located IOFBs. Although there were fewer cases of IOFB-associated endophthalmitis during the second decade of the study period, statistically we did not find a difference between the two decades. In our study of endophthalmitis, only 12.9% of the eyes in which vitrectomy had been performed initially did the vision later become NLP compared to 38.5% in which the vision became NLP in the eyes which had not undergone vitrectomy as an initial procedure at the time of IOFB removal and repair of ocular trauma. Vitrectomy removes the vitreous and blood clot scaffold that provides a framework to the visual localization of IOFB; it helps to find retinal breaks and detachments which may not be visible in the setting of vitreous hemorrhage. Our results corroborate previous reports of beneficial effects of early vitrectomy in the setting of ocular trauma associated with IOFBs [13]. Removal of lens was not necessary in all cases with corneal entry wound because the lens injury was localized without interfering with view for vitrectomy. Removal of posterior segment IOFB by pars plana vitrectomy has been advocated because it provides direct viewing and controlled removal of the IOFB [3, 18]. With the advent of advanced instrumentation and viewing systems used in vitrectomy, most vitrectomies in our hospital were performed in the second decade compared to the first.\nThe size of IOFB has been found to be a significant predictive factor of poor visual outcome in the previous studies of IOFB removal [18]. A large IOFB is more likely to inflict severe damage at the time of entry because of its higher kinetic energy, leading to a poor visual prognosis [18]. However, when considering similar sized IOFBs, no particular association between the visual outcome and the size of IOFB in eyes which developed endophthalmitis was found in our study.\nA preoperative retinal detachment may be present in 5\u201321% of the eyes with IOFB and has been reported to be an important risk factor for poor visual outcome [3, 19]. The timing of surgery in these eyes for the removal of IOFBs has been found to be an important prognostic factor for better visual outcome. Without delay, removal of IOFB has been found to reduce the chances of endophthalmitis [15, 19, 20]. In the absence of endophthalmitis, many studies have found no significant difference on the visual outcome when IOFB removal was delayed for several weeks [3, 18, 21]. Ahmadieh et al. reported a delay in IOFB removal of more then 4 weeks in 85% of their patients because a majority of their patients had war related IOFBs [22]. Lack of suspicion of IOFBs by primary care physicians has led to the delay in the treatment of up to 44% of patients resulting in a delay of up to 3\u00a0years in the diagnosis, treatment, or both, of IOFB [18, 21].\nIn conclusion, the data from this retrospective study reiterate the importance of prompt recognition of retained IOFBs and early repair of related injuries to prevent endophthalmitis and associated complications of visual loss. Final vision appears to be considerably determined by the presenting VA and severity of injury. Eyes with IOFBs in the anterior segment appear to have better prognosis compared to eyes having IOFBs in the posterior segment. Early vitrectomy at the time of IOFB removal may be beneficial for overall visual outcome of the operated eyes. Owing to the retrospective nature of our study and most of the reported studies and the various types of injuries in different settings, results from these studies are difficult to compare. It is hoped that with the advent of modern instruments such as wide-angle viewing systems and high-speed cutters, the chances of complications such as retinal detachment may be greatly reduced. A multi-center prospective study may be required to address some of the confounding factors in the management of IOFB and associated endophthalmitis.","keyphrases":["endophthalmitis","foreign bodies","risk factors","trauma","visual acuity"],"prmu":["P","P","P","P","P"]}
{"id":"Neurosci_Lett-1-5-1885995","title":"The Notch-1 intracellular domain is found in sub-nuclear bodies in SH-SY5Y neuroblastomas and in primary cortical neurons\n","text":"Notch signalling affects most aspects of development, not least the determination of neural stem cell fate. Here, we describe the presence of the Notch-1 intracellular domain (N1ICD) in sub-nuclear bodies in SH-SY5Y neuroblastomas and in primary rat cortical neurons as well as several other mammalian cell lines. We also demonstrate that these N1ICD-positive sub-nuclear bodies are distinct from premyelocytic leukaemia (PML) and SC35 bodies. Furthermore, using Notch deletion constructs we determined that a region C-terminal of amino acid 2094 is involved in targeting the N1ICD into sub-nuclear bodies. These findings have ramifications for nuclear architecture and gene transcription.\nNotch signalling regulates many aspects of invertebrate and vertebrate development ranging from somite formation [7] to epithelial\u2013mesenchymal interactions [20] and the determination of neural stem cell fate [23]. Notch signalling is also active in adult tissues including the brain where it is involved in synaptic plasticity and memory [3,19]. There are four mammalian Notch receptors (Notch 1\u20134), which undergo a series of sequential proteolytic cleavages. The first cleavage (S1) occurs in the Golgi by a furin-like convertase generating two fragments that remain non-covalently attached to form the mature Notch receptor at the cell surface. Ligands of the Jagged and Delta families, expressed on neighbouring cells, bind to the Notch receptor leading to the second cleavage (S2) by tumor necrosis factor alpha converting enzyme (TACE), which results in the shedding of the extracellular domain. The second cleavage is followed by a constitutive cleavage (S3) within the transmembrane domain of Notch by \u03b3-secretase. This cleavage releases the Notch intracellular domain (NICD), which migrates to the nucleus [20,14,15] where it interacts with the C-promoter binding factor-1 (CBF-1), the mammalian homologue of Suppressor of hairless (Drosophila) and Lag-1 (C. elegans), also known as RBP-J\u03ba [16]. In the absence of NICD, CBF-1 forms a complex with repressor proteins, resulting in the suppression of gene transcription. Interaction with NICD displaces the repressor proteins and allows the entry of transcriptional activators such as p300\/CBP and Mastermind, which convert the CBF-1 repressor complex into an activator of gene transcription [8]. Notch\/CSL target genes include the hairy and enhancer of split (HES) family and the HES related proteins (HERP\/Hey) that encode basic helix-loop-helix transcription factors, which suppress differentiation by antagonising the expression of down-stream lineage-specifying genes. In this report, we demonstrate the presence of the N1ICD in sub-nuclear bodies in SH-SY5Y neuroblastomas and in primary rat cortical neurons.\nTo undertake this study the following cell lines, antibodies and plasmid constructs were used: SH-SY5Y neuroblastoma cells and chinese hamster ovary cells (CHO) were obtained from ECACC (UK) and human embryonic kidney 293a cells (HEK293a; an adherent clone of HEK293 cells) were purchased from Quantum Biotechnologies (Canada). Mouse monoclonal anti-splicing factor SC-35 antibody and rabbit polyclonal anti-FLAG tag antibody were from Sigma (UK). Mouse monoclonal anti-myc antibody was from Cell Signalling Technology (UK). Mouse monoclonal anti-promyelocytic leukaemia protein antibody, goat anti-Notch-1, goat anti-mouse IgG and donkey anti-goat IgG were from Santa Cruz Biotechnology (USA). Alexa Fluor 488 and 594 goat anti-mouse and donkey anti-goat IgG conjugates were from Molecular Probes (UK). C-terminally tagged myc-His human N1ICD, \u0394TAD-N1ICD (lacking the transactivation domain TAD; the OPA domain and the proline (P), glutamate (E), serine (S), threonine (T) rich sequence\u2014PEST) and \u0394RA-N1ICD (lacking the RAM domain and the Ankyrin repeats) were gifts from Dr. Tom Kadesch (University of Pennsylvania, USA) [13]. Enhanced green fluorescent protein (EGFP) C-terminally tagged human N1ICD (N1ICD-EGFP) was from Dr. Allan Levey (Emory University School of Medicine, USA) [11]. The Notch-CBF-1 reporter, 4xwt-CBF-Luc, which contains four tandem repeats of the consensus CBF-1 DNA binding sequence, GTGGGAA and N-terminally tagged FLAG CBF-1 were generous gifts from Dr. Diane Hayward (Johns Hopkins University School of Medicine, USA) [5].\nCells were maintained in 5% CO2 in air in a humidified incubator at 37\u00a0\u00b0C. All culture media and supplements were from Invitrogen, UK, unless otherwise stated. SH-SY5Y neuroblastomas were grown in a 50\/50 mix of F12\/Eagle's minimal essential medium (EMEM) supplemented with 15% (v\/v) fetal bovine serum (FBS; Autogen Bioclear, UK), 2\u00a0mM l-glutamine, non-essential amino acids (Sigma), 100 IU penicillin and 100\u00a0mg\/ml streptomycin. HEK293a were cultured in low glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% (v\/v) FBS, 2\u00a0mM l-glutamine, 100 IU penicillin and 100\u00a0mg\/ml streptomycin. CHO cells were grown in F12 media supplemented as described above for HEK293a cells. Primary cortical neurons were prepared and cultured in Neurobasal media plus B27 supplement as previously described from E19 rat embryos [17].\nCells on coverslips were transfected with 500\u00a0ng of N1ICD-EGFP or 500\u00a0ng of myc-His N1ICD using FuGene 6 according to the manufacturer's instructions. Cells were fixed in ice-cold methanol 24\u00a0h after transfection then stained according to standard protocols. In brief, cells were incubated with goat anti-Notch-1 antibody (1:100), mouse monoclonal anti-promyelocytic leukaemia protein antibody (1:100) or mouse monoclonal anti-splicing factor SC-35 antibody (1:250) before being incubated with the appropriate fluorescent secondary antibody (1:200). Nuclei were stained with Hoescht 33342. Immunofluorescence was visualized and captured using a Zeiss LSM510 meta confocal microscope. Images were processed using LSM5 image examiner software (Zeiss). All experiments were performed in triplicate. Figures shown are representative images from a single experiment.\nFor luciferase reporter gene assays SH-SY5Y neuroblastomas were transfected with 400\u00a0ng of CBF-Luc (firefly luciferase based reporter DNA) and 500\u00a0ng of N1ICD-EGFP or 500\u00a0ng of myc-His N1ICD using FuGene 6 according to the manufacturer's instructions (Roche, UK). To control for transfection efficiency 50\u00a0ng of phTK-Renilla luciferase (Promega, UK) was also included in transfections. Empty vector DNA was included where necessary to maintain constant DNA concentrations. Twenty-four hours post-transfection the cells were lysed and firefly and Renilla luciferase activities were sequentially measured using Dual-Glo reagents (Promega) in a Wallac Trilux 1450 Luminometer (Perkin-Elmer, UK). Values were normalized by dividing firefly values by the Renilla value from the same well. Data for each set of four replica transfections was averaged, the control in each set normalized to 1 and data presented as fold increases over control. All experiments were performed in triplicate.\nEndogenous S3 cleaved, nuclear, NICDs are expressed at very low levels and are difficult to detect in cell culture experiments [9], therefore we expressed exogenous N1ICD-EGFP in SH-SY5Y neuroblastomas to model N1ICD in vitro. In these cells N1ICD-EGFP exhibited a diffuse nuclear staining pattern with intense sub-nuclear bodies (Fig. 1A and B). In contrast, expression of exogenous p53 or \u03b2-catenin resulted in diffuse nuclear staining (data not shown), providing evidence that the N1ICD-positive sub-nuclear bodies are specific. The sub-nuclear bodies were not attributable to the EGFP protein since SH-SY5Y cells transfected with the EGFP expression vector containing no insert did not display a speckled nuclear staining pattern (data not shown). Furthermore, a myc-His tagged form of N1ICD was found in sub-nuclear bodies in SH-SY5Y neuroblastoma cells (Fig. 1C) as demonstrated using an anti-Notch-1 antibody, providing additional evidence that the observed speckled staining pattern is specific and not due to the presence of EGFP. Control cultures that were not exposed to primary antibody did not exhibit any positively stained cells (data not shown). To ensure the presence of nuclear bodies was not an artefact of the fixation method used (ice-cold methanol), SH-SY5Y cells were transfected with both EGFP and myc-His tagged Notch constructs and fixed with either 3.7% formaldehyde or 4% paraformaldehyde. With both methods very similar nuclear bodies were seen with both constructs (data not shown). Of note, both N1ICD-EGFP and myc-His tagged N1ICD were functionally active as demonstrated using CBF-Luc (Fig. 1D) and we have previously shown that exogenous N1ICD expression promotes the inhibition of retinoic acid-induced differentiation in SH-SY5Y neuroblastomas [4].\nN1ICD-positive sub-nuclear bodies were not cell-type specific. We observed identical sub-nuclear structures in primary rat cortical neurones (Fig. 2A), HEK293a cells (Fig. 2B) and in CHO cells (Fig. 2C) with N1ICD-EGFP and myc-His tagged N1ICD (data not shown). As the punctate structures were detected with both constructs in a variety of cell lines and in primary neurons they are unlikely to be an artefact of tagging or a result of cellular transformation. Furthermore, we found that exogenous CBF-1 exhibited a diffuse nuclear staining pattern in SH-SY5Y neuroblastomas (Fig. 2D), but in the presence of N1ICD CBF-1 re-located into sub-nuclear bodies (Fig. 2E), indicative of functional gene transcription within these nuclear domains.\nSeveral sub-nuclear structures, similar to those shown here, have been previously described including premyelocytic leukaemia (PML) bodies and SC35 bodies. These foci are believed to be areas of high transcriptional activity [2] and pre-mRNA splicing, respectively [10]. We therefore transfected SH-SY5Y cells with N1ICD-EGFP and used antibodies to detect endogenous PML and SC35 bodies. Both PML (Fig. 3A, mid left panel) and SC35 reactive bodies (Fig. 3B, mid left panel) were detected in SH-SY5Y cells. However, in both cases these formations were distinct from the N1ICD-EGFP positive foci (Fig. 3A and B, far right panels). The PML and SC35 bodies were more consistent in size and typically smaller and more numerous than the formations in which N1ICD was detected.\nNext we examined which region of N1ICD was involved in targeting the protein to sub-nuclear bodies by using two myc-tagged N1ICD deletion constructs, \u0394TAD-N1ICD and \u0394RA-N1ICD. \u0394TAD-N1ICD runs from a.a. 1760 to 2093 and lacks the transactivation domain, the OPA domain and the proline (P), glutamate (E), serine (S), threonine (T) rich sequence (PEST). \u0394RA-N1ICD extends from a.a. 2094 to 2556 and lacks the RAM domain and the Ankyrin repeats (Fig. 4A). \u0394TAD-N1ICD was detected diffusely throughout the nucleus in SH-SY5Y neuroblastomas (Fig. 4B, left panel), while \u0394RA-N1ICD was located in sub-nuclear bodies very similar in appearance to those seen with N1ICD (Fig. 4B, right panel). This indicates that a region C-terminal of a.a. 2094, lying within the TAD, the OPA domain or the PEST domain or an unidentified motif within the intervening regions, is involved in targeting N1ICD into sub-nuclear bodies.\nIn summary, we demonstrate that exogenous N1ICD is expressed in sub-nuclear bodies in SH-SY5Y neuroblastomas, primary rat cortical neurons as well as other mammalian cell lines and these sub-nuclear structures were distinct from SC35 or PML bodies. N1ICD foci were detectable as early as 5\u00a0h after transfection (cells were routinely fixed 24\u00a0h post-transfection in all other experiments described in this report), which suggests that the nuclear inclusions are not merely a consequence of protein aggregation with time (data not shown). The presence of the N1ICD in sub-nuclear bodies has previously been demonstrated when associated with the Epstein-Barr virus derived protein, RPMS [24] and the Mastermind family of Notch\/CBF-1 co-activators [18,21,22]. Here, we report that the N1ICD appeared in foci independently of other exogenously expressed proteins. A number of endogenous Notch associated proteins, including CBF-1, have recently been observed in similar nuclear foci in primary hippocampal neurons [12], although the presence of NICDs was not examined. Interestingly, we found that exogenously expressed CBF-1 exhibited a diffuse nuclear staining pattern, however in the presence of exogenous N1ICD CBF-1 was found in sub-nuclear bodies. A number of previous reports [1,6] concerning Notch cytochemistry contain figures in which N1ICD appears to be in punctate formations, which with greater resolution could have been interpreted as distinct sub-nuclear bodies. It is feasible then that the N1ICD-positive bodies form part of a physiologically relevant, multimeric, protein complex involved in gene transcription. Moreover, the presence of such sub-nuclear bodies provides evidence that the nucleus contains defined domains and is not merely a homogenous pool of nucleic acids and proteins.","keyphrases":["notch","sub-nuclear bodies","transcription","cbf-1","luciferase"],"prmu":["P","P","P","P","P"]}
{"id":"Purinergic_Signal-1-2-2096532","title":"Guanosine stimulates neurite outgrowth in PC12 cells via activation of heme oxygenase and cyclic GMP\n","text":"Undifferentiated rat pheochromocytoma (PC12) cells extend neurites when cultured in the presence of nerve growth factor (NGF). Extracellular guanosine synergistically enhances NGF-dependent neurite outgrowth. We investigated the mechanism by which guanosine enhances NGF-dependent neurite outgrowth. Guanosine administration to PC12 cells significantly increased guanosine 3-5-cyclic monophosphate (cGMP) within the first 24 h whereas addition of soluble guanylate cyclase (sGC) inhibitors abolished guanosine-induced enhancement of NGF-dependent neurite outgrowth. sGC may be activated either by nitric oxide (NO) or by carbon monoxide (CO). -Nitro-l-arginine methyl ester (l-NAME), a non-isozyme selective inhibitor of nitric oxide synthase (NOS), had no effect on neurite outgrowth induced by guanosine. Neither nNOS (the constitutive isoform), nor iNOS (the inducible isoform) were expressed in undifferentiated PC12 cells, or under these treatment conditions. These data imply that NO does not mediate the neuritogenic effect of guanosine. Zinc protoporphyrin-IX, an inhibitor of heme oxygenase (HO), reduced guanosine-dependent neurite outgrowth but did not attenuate the effect of NGF. The addition of guanosine plus NGF significantly increased the expression of HO-1, the inducible isozyme of HO, after 12 h. These data demonstrate that guanosine enhances NGF-dependent neurite outgrowth by first activating the constitutive isozyme HO-2, and then by inducing the expression of HO-1, the enzymes responsible for CO synthesis, thus stimulating sGC and increasing intracellular cGMP.\nIntroduction\nThe roles of extracellular purine nucleosides and nucleotides as neurotransmitters and modulators are well documented [1]. Extracellular purines also exert trophic effects on cells; influencing growth, division [2, 3] differentiation, and even apoptosis [4\u20136]. Purines are released from cells under physiological conditions, acting as neurotransmitters and neuromodulators [7\u20139]. Trauma and other insults to the central nervous system are also potent stimuli that cause the release of purines [3], most of which are converted by ectoenzymes to adenosine and guanosine [3, 5]. After insults to cells, more guanine-based than adenine-based purines are released [10]. Furthermore, the extracellular concentration of guanosine remains elevated for prolonged periods in vitro [10] and for up to a week after central nervous system (CNS) injury [11], implying that extracellular guanosine may exert trophic effects in vivo.\nSprouting of neurites, which later become axons and dendrites, is an important change associated with neural development and differentiation [12, 13]. Neurite sprouting through regeneration or collateral sprouting also plays an important role in the functional recovery following injury to the central or peripheral nervous systems [14]. Amongst its trophic effects, guanosine stimulates neurite outgrowth and enhances NGF-dependent neurite outgrowth [3, 15].\nPC12 cells serve as a useful model for studying cell signaling [16]. They respond to many growth factors, neurotrophins and hormones, which initiate multiple signaling pathways [17\u201320]. The specific cellular targets of these signaling pathways mediate the distinct responses of differentiation, proliferation and survival, all of which can be assessed [16]. The addition of NGF to PC12 cells causes a sustained activation of ERK, a mitogen-activated protein kinase, through activation of the TrkA receptor [16, 19]. This leads to the development of many phenotypic characteristics in PC12 cells, which are also associated with mature sympathetic neurons [13, 21].\nExtracellular guanosine not only stimulates neurite outgrowth from primary cultures of rat hippocampal neurons [3], and PC12 cells [15] but also enhances the neuritogenic effects of NGF on PC12 cells [15, 22, 23]. Both in astrocytes [24] and in PC12 cells [22], guanosine increases intracellular adenosine 3\u2032,5\u2032-cyclic monophosphate (cAMP). Some of the neuritogenic effects of guanosine in PC12 cells appeared to be mediated through this mechanism [22]. However, guanosine also has cAMP-independent effects, synergistic with NGF, because its effects are not abolished by an adenylate cyclase inhibitor [22] and are synergistic with substances that increase intracellular cAMP [15].\nThe cAMP-independent component of the signal transduction mechanism through which guanosine synergistically enhances NGF-dependent neurite outgrowth is unknown. However, several lines of evidence indicate a role for guanosine 3\u2032,5\u2032-cyclic monophosphate (cGMP). First, extracellular guanosine increases intracellular cGMP in rat mesenteric artery [25]. Second, nitric oxide (NO) donors enhance NGF-dependent PC12 cell neurite outgrowth via a cGMP-dependent mechanism; and thirdly, the neuritogenic effects of the NO donors are abolished by guanylyl cyclase inhibitors and mimicked by cGMP analogs [26]. The mechanism by which cGMP enhances neurite outgrowth is currently unclear.\nNitric oxide synthases are a family of enzymes, which synthesize NO through the catalytic conversion of l-arginine to l-citrulline. The constitutively expressed forms are the endothelial (eNOS) or neuronal (nNOS), which are regulated by the cytosolic concentration of Ca2+ [27]. The inducible isoform (iNOS) is widely distributed, and becomes active only hours after an inducing event [28]. NO is involved in cell communication and signal transduction in many systems including the CNS [29\u201331]. NGF induced different isoforms of NOS in PC12 cells after 4 days in vitro [32, 33]. Peunova and Enikolopov [32] reported that the NO produced preceding the development of the differentiated phenotype is due predominantly to iNOS.\nThe diffusible gas carbon monoxide (CO) is a putative neurotransmitter [34]. Heme oxygenase (HO) synthesizes CO from the biologically active substrate biliverdin, which is rapidly reduced to bilirubin, and iron from intracellular heme [34]. CO can modulate activities attributed to cGMP in the nervous system [35\u201338]. It has been proposed that CO production is responsible for baseline cGMP levels in the hippocampus [36]. In cerebellar granule cell cultures, CO produced by HO affects intracellular cGMP concentrations by modulating the NO-soluble guanylate cyclase signaling system [39]. Conversely, NO, synthesized by NOS may induce the expression of HO-1 [40\u201342], indicating a close and reciprocal interaction between the NOS-NO and the HO-CO signaling systems [35, 36]. Based on these findings it has been proposed that a possible role for HO-1 is to counteract NO toxicity [43].\nTogether, these data led us to question whether guanosine enhanced NGF-dependent neurite outgrowth is through a mechanism involving cGMP, and if so, whether it was attributable to the stimulation of either NO or CO synthesis.\nMaterials and methods\nCell culture and treatments\nTissue culture supplies were from Life Technologies. All other supplies were obtained from Sigma RBI unless otherwise stated. 2.5S NGF was a generous gift from Dr. M. Coughlin, Department of Medicine, McMaster University, 6-(phenylamino)-5,8-quinolinedione (LY83583) was obtained from (Calbiochem), copper protoporphyrin from (Porphyrin Products), and zinc protoporphyrin IX from (Research Biochemical).\nPC12 cells were maintained in either RPMI 1640 medium supplemented with 5% heat-inactivated (HI) fetal calf serum (FCS), 5% HI-horse serum (HS) and 1% antibiotic-antimycotic (Anti-Anti; 10,000 units of penicillin, 10,000 \u00b5g of streptomycin, 25 \u00b5g amphotericin B\/ml in 0.85% saline) [15] or F-12K (Kaighn's Modification) medium supplemented with 15% HI-HS, 2.5% HI-FCS, and 1% Anti-Anti at 37 \u00b0C in a 5% CO2 environment.\nNeurite outgrowth assay\nTo evaluate the effect of test compounds on neurite outgrowth in PC12 cells, they were added to cultures for 48 h as previously described [15]. Briefly, PC12 cells were plated onto poly-d,l-ornithine (PORN)-coated 24-well plates at a density of 2.5 \u00d7 104 cells\/well. Cells were cultured in RPMI 1640 supplemented with 1.5% HI-HS, 1.5% HI-FCS and 1% Anti-Anti. Guanosine was dissolved in 10% sodium hydroxide (1N NaOH) and when added to the culture medium the final concentration was 0.01% sodium hydroxide. In experiments in which guanosine (300 \u00b5M) was added to each well first, followed within 15 min by the addition of 2.5S NGF (40 ng\/ml). Methylene blue, LY83583, hemoglobin, N\u03c9-nitro-l-arginine methyl ester (l-NAME), copper protoporphyrin-IX (CuPP) and zinc protoporphyrin-IX (ZnPP), were added 15 min prior to the addition of guanosine. All test substances remained in the medium throughout the 48-h test period. Cells were maintained at 37 \u00b0C in a humidified atmosphere of 95% air, 5% CO2. After 48 h, cells were fixed with 10% formalin in phosphate buffered saline (PBS), pH 7.4 for 10 min and stored at 4 \u00b0C in PBS (pH 7.4) containing sodium azide (0.1%, w\/v) until counted (usually within 2 weeks). Total cell number was determined by counting two randomly selected areas in each well. The observer was blind to the treatments the cells had received. At least 50 cells per well were counted, and the number of cells bearing one or more neurites determined using a NIKON Diaphot microscope equipped with phase contrast optics. Neurites were defined as processes extending at least one cell body diameter from the cell with growth cones at their tips [44].\nWestern immunoblot analysis\nThe expression of HO-1, HO-2, iNOS, nNOS, and \u03b2-actin in PC12 cells was determined by Western immunoblot analysis. PC12 cells were seeded onto PORN-coated plates at a density of 1 \u00d7 106 cells\/plate and maintained at 37\u00b0 for 48 h prior to treatments. Cells were placed in a medium containing 3% HI-FCS and 3% HI-HS 12 h before the start of treatments. Cells were then treated with various agents for 0, 6, 12, 24, or 48 h. At the end of the treatment period cells were washed once with PBS and then harvested at 4 \u00b0C using a lysis buffer (25 \u00b5M Tris\/HCl pH 7.4, 10 \u00b5M NaCl, 10 \u00b5M EDTA, 100 \u00b5l\/10 ml Tween 20, 10 \u00b5M sodium pyrophosphate decahydrate, 10 mM sodium orthovanadate, 5 \u00b5g\/ml leupeptin, 10 mM glycerophosphate). Cells were disrupted by sonication and aliquots (25 \u00b5l) were removed for the determination of protein concentrations using the bicinchoninic acid (BCA) protein assay (Pierce, Illinois, USA). Cells lysates were separated on 12% SDS-polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes (PALL, Michigan, USA). Membranes were incubated with a specific primary antibody overnight at 4 \u00b0C then were exposed to a secondary antibody for 1 h at room temperature. Positive control peptides for nNOS and iNOS (obtained from Cayman Chemical, Ann Arbor. MI) were used as electrophoresis standard. The following antibodies were used: monoclonal antibody to brain NOS (Sigma, dilution: 1:1,000), monoclonal antibody to the inducible NOS (Sigma, dilution: 1:1,000), anti-iNOS\/bNOS, goat anti-mouse IgG-HRP (StressGen Biotechnologies, dilution 1:100,000), monoclonal antibody to \u03b2-actin (abCAM, dilution: 1:20,000), monoclonal anti-\u03b2-actin, goat anti-mouse IgG-HRP (Novus Biological, dilution 1:100,000), monoclonal antibody to HO-1 (StressGen Biotechnologies, dilution 1:2,000), anti-HO-1, goat anti-mouse IgG-HRP (StressGen Biotechnologies, dilution 1:200,000), rabbit polyclonal antibody to HO-2\/HO-1 (StressGen Biotechnologies, dilution 1:1,000), anti HO-2\/HO-1, goat anti-rabbit IgG-HRP (StressGen Biotechnologies, dilution 1:200,000). Immunocomplexes were then visualized using a chemiluminescence substrate (Sigma-Aldrich). Immunoblots were quantified by densitometric analyses, using the Northern Eclipse program (EPIX). All bands on the immunoblots were normalized to their corresponding \u03b2-actin bands prior to statistical analysis.\nDetermination of cyclic GMP\nPC12 cells were seeded onto PORN-coated 12-well plates at a density of 5 \u00d7 105 cells\/well and maintained at 37 \u00b0C for 48 h prior to treatments. Cells were placed in a medium containing 3% HI-FCS and 3% HI-HS 12 h before the start of treatments. Cells were then treated with various agents for different times (0, 6, 12, 24, or 48 h). At the end of the treatment period cGMP was extracted and analyzed using a cGMP enzyme immunoassay (EIA) kit (Amersham Biosciences). The assay is based on a competition between unlabeled cGMP in the sample or (standard) and a fixed quantity of peroxidase-labeled cGMP for a limited number of binding sites on a cGMP specific antibody. With fixed amounts of antibody and cGMP-peroxidase conjugate, the amount of bound cGMP-peroxidase conjugate is inversely proportional to the concentration of unlabeled cGMP.\nStatistical analysis\nStatistical analysis was carried out using a two-way ANCOVA, when applicable, or a two-way ANOVA followed by Fischer's LSD test for multiple comparisons.\nResults\nGuanosine enhances NGF-dependent neurite outgrowth via activation of soluble guanylate cyclase\nAs we have reported previously, we have evaluated the effect of guanosine on NGF-dependent neurite outgrowth during the first 48 h, as guanosine most likely exerted its effects within the first 1\u20132 h after treatment [23]. Therefore, all experiments reported in this manuscript were performed during the first 48 h of PC12 cell treatment with various agents.\nPC12 cells contain both soluble, and particulate isoforms of guanylate cyclase (GC) [26, 45]. To determine whether activation of GC was necessary for the neuritogenic effect of guanosine, inhibitors of GC were added for 48 h to PC12 cells treated with NGF plus guanosine. Methylene blue at concentrations ranging from 0.1 to 1 mM was added to PC12 cell cultures that contained NGF alone (40 ng\/ml), or guanosine (300 mM) plus NGF (40 ng\/ml). Cultures were then evaluated for neurite outgrowth. At the concentrations used in these experiments, methylene blue inhibits sGC but not particulate GC [46, 47]. Methylene blue had no effect on the outgrowth of neurites induced by NGF alone (Figure 1). However, in cultures treated with both guanosine and NGF, the addition of methylene blue reduced significantly (P < 0.01) the proportion of neurite-bearing cells in a concentration-dependent manner (Figure 1). LY83583 inhibits both, particulate GC and sGC [48]. When LY83583 (10 nM) was added to PC12 cell cultures, it inhibited neurite outgrowth elicited by guanosine plus NGF but had no effect on neurite outgrowth in cultures treated with NGF alone (data not shown). These data support the hypothesis that cGMP plays a role in enhancing the effect of guanosine on NGF-mediated neurite outgrowth. Moreover, since methylene blue at the concentrations used inhibits soluble but not particulate GC, these data imply that guanosine activates sGC.\nFigure 1\nMethylene blue attenuates guanosine-enhanced nerve growth factor-dependent neurite outgrowth in PC12 cells. PC12 cells were cultured in RPMI 1640 medium supplemented with 5% heat-inactivated fetal calf serum, 5% heat-inactivated horse serum and 1% antibiotic-antimycotic for 48 h. Cultures were then treated with NGF (40 ng\/ml) or NGF (40 ng\/ml) plus guanosine (300 \u00b5M) and with increasing concentrations of methylene blue (0\u20131 \u00b5M). After 48 h, the total cell number and number of cells bearing one or more neurites were determined by counting two random areas in each well. The mean proportion of neurite-bearing cells in cultures treated with NGF was approximately 25%\u201335%. Because this value varied slightly between experiments, all experimental values are expressed relative to the NGF treated cultures, which was defined as 100%. Open bars: NGF treatment, closed bars: NGF plus guanosine treatment. Cultures treated with guanosine plus NGF had a significantly (P < 0.01, two-way independent ANOVA) greater proportion of neurite-bearing cells than those treated with NGF alone. Methylene blue (0.1 to 1.0 \u00b5M) had no effect on the proportion of neurite-bearing cells in cultures treated with NGF alone, but at concentrations from 0.1 to 1.0 \u00b5M, it significantly (**P < 0.01, two-way independent ANOVA) reduced the proportion of neurite-bearing cells in cultures treated with guanosine plus NGF. Data represent the mean \u00b1 SEM of 12 determinations from two replicate experiments.\nGuanosine may activate sGC either directly, or indirectly by stimulating the formation of NO, CO or hydroxyl radicals, which are all physiological activators of sGC [49]. To distinguish between these possibilities we added hemoglobin (100 nM) to cultures of PC12 cells. Extracellular hemoglobin directly scavenges NO and CO because these substances avidly bind to the heme moiety, thereby inhibiting their GC activating activity [46]. Hemoglobin reduced the synergistic effects of guanosine on NGF-mediated neurite outgrowth but had no effect on neurite outgrowth elicited by NGF alone (data not shown). Although these data imply that guanosine stimulates the synthesis of a sGC-activating factor, they do not show whether this factor is NO, CO or hydroxyl radical.\nInhibition of nitric oxide synthase (NOS) has no effect on guanosine-enhanced NGF-dependent neurite outgrowth\nIn the rat mesenteric artery, guanosine stimulates NO formation [25]. Therefore, we investigated if guanosine could also stimulate NO synthesis in PC12 cells. To examine whether NO was involved in the signal transduction pathway that mediated the enhancement of neurite outgrowth by guanosine through sGC, we inhibited the enzyme NOS, which catalyzes the conversion of l-arginine to l-citrulline plus NO. The l-arginine analog, N\u03c9-nitro-l-arginine methyl ester hydrochloride (l-NAME) [50] inhibits NOS competitively [25, 51]. In this experiment, we reduced the concentration of l-arginine in the culture medium from 1 mM to 80 \u00b5M, thus permitting more effective competition of the NOS inhibitor with l-arginine. Under these conditions, L-NAME inhibits NOS at 10 \u00b5M [25, 51]. When added to the culture medium of PC12 cells at 10 \u00b5M l-NAME did not inhibit NGF-dependent neurite outgrowth, and had no effect on the guanosine-mediated enhancement of NGF-dependent neurite outgrowth (Figure 2a). To confirm that NO was not involved in the signal transduction pathway that mediates the enhancement of neurite outgrowth by guanosine, we determined the expression of two subtypes of NOS isoenzymes: iNOS, the inducible isoform (Figure 2b), and nNOS, the constitutive isoform (Figure 2c). PC12 cultures were exposed to guanosine alone (300 \u00b5M), or NGF alone (40 ng\/ml), or to the combination of guanosine plus NGF for 48 h, and NOS expression was determined by Western immunoblot analysis. We analyzed various protein concentrations (5\u201350 \u00b5g\/ml) and determined \u03b2-actin expression on the same Western immunoblots. Using protein concentrations as high as 50 \u00b5g\/ml neither nNOS nor iNOS expression could be detected in PC12 cells treated with guanosine, or NGF, or the combination of the two agents. These data indicate that since NOS is not expressed during the first 48 h of treatment, thus NO cannot mediate the effect of guanosine on neurite outgrowth.\nFigure 2\n(a) Inhibition of nitric oxide synthase has no effect on the proportion of neurite-bearing PC12 cells cultured for 48 h in the presence of NGF, or NGF plus guanosine. PC12 cells were cultured with NGF (40 ng\/ml) or NGF (40 ng\/ml) plus guanosine (300 \u00b5M) as described in Figure 1. Cultures were treated with the general nitric oxide synthase inhibitor, l-NAME (0.1\u201310 mM) for 48 h and the number of cells bearing one or more neurites were determined as described in Figure 1. Open bars: NGF treatment, closed bars: NGF plus guanosine treatment. Neurite outgrowth in cultures treated with guanosine plus NGF was significantly (P < 0.01, two-way independent ANOVA) greater than in cultures treated with NGF alone. l-NAME (0.1\u201310 \u00b5M) had no effect on the proportion of neurite bearing cells in cultures treated with NGF alone, or in combination with guanosine. Data represent the mean \u00b1 SEM of 12 determinations from two replicate experiments. (b) Inducible nitric oxide synthase is not expressed in PC12 cells cultured for 48 h in the presence of guanosine, or NGF, or guanosine plus NGF. PC12 cells were cultured on plates coated with poly-d,l-ornithine for 72 h. Cells were then grown in serum-reduced medium (3% heat-inactivated fetal calf serum and 3% heat-inactivated horse serum) for 12 h, and were treated with guanosine (G, 300 \u00b5M) or NGF (N, 40 ng\/ml) or guanosine (300 \u00b5M) plus NGF (40 ng\/ml) (G + N), or with no added treatments (C). The expression of inducible nitric oxide synthase was determined at various time points (6, 12, 24 and 48 h) by Western immunoblot analysis. Recombinant inducible nitric oxide synthase protein (50 ng) was used as a positive control (P). Immunoblots were quantified by densitometric analysis and were normalized to the corresponding \u03b2-actin bands as described in the Materials and methods. Statistical analysis was performed using a two-way ANCOVA followed by Fischer's LSD post-hoc comparison test. Data presented are representative of at least three independent experiment. (c) Neuronal nitric oxide synthase is not expressed in PC12 cells cultured for 48 h in the presence of guanosine, or NGF, or guanosine plus NGF. PC12 cells were cultured as described in panel (b), and the expression of neuronal nitric oxide synthase was determined at various time points (6, 12, 24 and 48 h) by Western immunoblot analysis. Recombinant neuronal nitric oxide synthase protein (50 ng) was used as a positive control (P). Immunoblots were quantified by densitometric analysis and were normalized to the corresponding \u03b2-actin bands as described in the Materials and methods. Statistical analysis was performed using a two-way ANCOVA followed by Fischer's LSD post-hoc comparison test. Data presented are representative of at least three independent experiment.\nInhibition of heme oxygenase (HO) attenuates guanosine-enhanced NGF-dependent neurite outgrowth\nSince NO apparently did not mediate the effects of guanosine, we examined the possibility that guanosine stimulated CO synthesis. Heme oxygenase (HO), the enzyme which synthesizes CO, has been detected in rat adrenal glands [52]. Zinc protoporphyrin-IX (ZnPP) inhibits both the constitutive HO (HO-2) and the inducible HO (HO-1) isoforms of this enzyme [52, 53]. We, therefore, tested the effect of increasing concentrations of ZnPP on the ability of NGF or, NGF plus guanosine to induce neurite outgrowth. Addition of ZnPP (0.01, 0.1, 1 \u00b5M) to PC12 cell cultures did not significantly reduce outgrowth of neurites induced by NGF (Figure 3), when compared to control cultures. In contrast, ZnPP attenuated the ability of guanosine to enhance NGF-stimulated neurite outgrowth (Figure 3). As a control for non-specific effects of metalloporphyrins we used copper protoporphyrin-IX (CuPP), which does not inhibit heme oxygenase [54]. The addition of CuPP to PC12 cells did not inhibit either NGF-dependent neurite outgrowth or the ability of guanosine to enhance NGF-dependent neurite outgrowth (data not shown).\nFigure 3\nInhibition of heme oxygenase attenuates guanosine-enhanced NGF-dependent neurite outgrowth in PC12 cells. PC12 cells were cultured with NGF (40 ng\/ml) or NGF (40 ng\/ml) plus guanosine (300 \u00b5M) as described in Figure 1. Some cultures were treated with the selective inhibitor of heme oxygenase zinc protoporphyrin-IX (0.01Y1 \u00b5M) for 48 h and the number of cells bearing one or more neurites was determined as described in Figure 1. Open bars: NGF treatment, closed bars: NGF plus guanosine treatment. Cultures treated with guanosine plus NGF had a significantly (P < 0.01 two-way independent ANOVA) greater proportion of neurite-bearing cells than cultures treated with NGF alone. Zinc protoporphyrin-IX significantly decreased (**P < 0.01) the neurite growth from cultures treated with guanosine plus NGF, but had no significant effect on neurite outgrowth in cultures treated with NGF alone. Data represent the mean \u00b1 SEM of 12 determinations from two replicate experiments.\nGuanosine induces heme oxygenase-1 (HO-1) expression\nPC12 cells have been previously shown to express both HO-1 and HO-2 proteins under basal conditions [55]. Since the addition of ZnPP to PC12 cultures decreased NGF-dependent neurite outgrowth, we attempted to establish whether guanosine treatment had any effect on the expression of the HO-1 protein during the first 48 h of this process. We treated PC12 cultures with guanosine alone (300 \u00b5M), or with NGF alone (40 ng\/ml), or with a combination of guanosine plus NGF for 48 h, and determined HO-1 expression by Western immunoblot analysis. We used the monoclonal antibody directed against rat HO-1, which recognizes a single protein band, at 32 kDa, the reported molecular weight for this enzyme [36] (Figure 4b). Western immunoblots were quantified and normalized against their corresponding \u03b2-actin band. Statistical analysis revealed no differences in \u03b2-actin expression for any of the treatments at the different time points (data not shown); therefore, this was used as a control for normalizing HO-1 protein expression. There was no significant change in HO-1 expression in untreated PC12 cells during the 48-h period. In cells exposed to guanosine alone HO-1 expression, unexpectedly, was significantly reduced at 6 h compared to untreated cells at 0 time (P < 0.05), and at 6 h (P < 0.01) (Figure 4). A possible explanation for this result may stem from the observation that HO-1 gene expression is suppressed by elevated intracellular calcium concentrations [56] and guanosine has been shown to increase intracellular calcium concentrations in astrocytes [57]. Since increases in calcium are rapid and transient, this may account for the reduced HO-1 expression at 6 h. The addition of NGF, however, is sufficient to override this effect and so at later time points HO-1 expression is up-regulated by the combination of NGF and guanosine. The addition of guanosine plus NGF significantly increased HO-1 expression at 12 h (P < 0.05), compared to that of control cells at 0 time (Figure 4). After 24 h all three treatments (guanosine alone, or NGF alone or guanosine plus NGF) led to a significant increase in HO-1 expression compared to control cells at 0 time (P < 0.01) (Figure 4). In cells exposed to guanosine alone the enhanced HO-1 expression at 24 h was also significantly different from that detected in untreated cells at 24 h (P < 0.05). Whereas in cells treated with NGF alone, or with the combination of NGF plus guanosine this difference was significant at P < 0.01 (Figure 4). After 48 h, HO-1 expression was elevated significantly only in cells exposed to NGF alone when compared to untreated cells at 0 time, and at 48 h (P < 0.05) for both comparisons. In cells exposed to guanosine alone or to NGF plus guanosine HO-1 expression by 48 h declined and was similar to untreated control values at 0 time and at 48 h.\nFigure 4\nGuanosine induces the expression of heme oxygenase-1 protein in PC12 cells. PC12 cells were cultured on plates coated with poly-d,l-ornithine for 72 h. Cells were then grown in serum-reduced medium (3% heat-inactivated fetal calf serum and 3% heat-inactivated horse serum) for 12 h, and were treated with guanosine (G, 300 \u00b5M) or NGF (N, 40 ng\/ml) or guanosine (300 \u00b5M) plus NGF (40 ng\/ml) (G + N), or with no added treatments (C) as described in Figure 2b. The expression of heme oxygenase-1 was determined at various time points (6, 12, 24 and 48 h) by Western immunoblot analysis. Immunoblots were quantified by densitometric analysis and were normalized to the corresponding \u03b2-actin bands as described in the Materials and methods. Open bars: untreated controls (C), closed bars: guanosine plus NGF (G + N) treatment, stippled bars: NGF (N) treatment, hatched bars: guanosine (G) treatment. Statistical analysis was performed using a two-way ANCOVA followed by Fischer's LSD post-hoc comparison test (\u25cb P < 0.05 compared with control time point 0); (\u25cb\u25cb P < 0.01 compared with time point 0); (* P < 0.05 compared with control at same time point); (** P < 0.01 compared with control at same time point). (a) Data represent the mean optical density \u00b1 SEM obtained in six independent experiments. (b) Results are representative Western immunoblots obtained in these experiments.\nIn parallel experiments, we determined whether guanosine had an effect on the expression of the constitutive isoform, HO-2 in these cells. We treated PC12 cultures with guanosine alone (300 \u00b5M), or NGF alone (40 ng\/ml), or in combination guanosine plus NGF for 48 h and determined HO-2 expression by Western immunoblot analysis. Using the monoclonal antibody directed against rat HO-2 we detected a single protein band, of molecular weight 36 kDa, as described for HO-1 [36] (Figure 5b). Western immunoblots were quantified and normalized against their corresponding \u03b2-actin band. Again, statistical analysis revealed no differences in \u03b2-actin expression for any of the treatments at the different time points (data not shown), therefore, this was used as a control for normalizing HO-2 protein bands. HO-2 expression was detectable at 0 time in untreated PC12 cells, but none of the treatments had any significant effect on its expression at any time point determined. In the guanosine-treated cells HO-2 expression was elevated slightly at 12 h, but this was not statistically significant.\nFigure 5\nGuanosine has no effect on heme-oxygenase-2 protein expression in PC12 cells. PC12 cells were cultured on plates coated with poly-d,l-ornithine for 72 h. Cells were then grown in serum-reduced medium (3% heat-inactivated fetal calf serum and 3% heat-inactivated horse serum) for 12 h, and were treated with guanosine (G, 300 \u00b5M) or NGF (N, 40 ng\/ml) or guanosine (300 \u00b5M) plus NGF (40 ng\/ml) (G + N), or with no added treatments (C) as described in Figure 2b. The expression of heme oxygenase-2 was determined at various time points (6, 12, 24 and 48 h) by Western immunoblot analysis. Immunoblots were quantified by densitometric analysis and were normalized to the corresponding \u03b2-actin bands as described in the Materials and methods. Open bars: untreated controls (C), closed bars: guanosine plus NGF (G + N) treatment, stippled bars: NGF (N) treatment, hatched bars: guanosine (G) treatment. Statistical analysis was performed using a two-way ANOVA. (a) Data represent the mean optical density T SEM obtained in three independent experiments. (b) Results are representative Western immunoblots obtained in these experiments.\nGuanosine increases intracellular cGMP concentrations in PC12 cells during guanosine-enhanced NGF-dependent neurite outgrowth\nCO is a known activator of sGC [36]. Since we have shown that HO-1 expression is elevated in PC12 cells exposed to guanosine, we next investigated whether this increased HO-1 expression is accompanied by elevation in intracellular cGMP concentrations. PC12 cell cultures were treated with guanosine alone (300 \u00b5M), or NGF alone (40 ng\/ml), or in combination guanosine plus NGF for 48 h. Intracellular cGMP concentrations were determined after 6, 12, 24 and 48 h of treatment. In cells exposed to guanosine alone, cGMP concentrations increased significantly after 6 h compared to untreated cells at this time point (P < 0.05). In PC12 cells treated with guanosine plus NGF, cGMP concentrations increased significantly after 6 h compared to untreated cells at 0 time (P < 0.05), remained significantly elevated at 12 h (P < 0.05) and at 24 h (P < 0.05), and declined to values comparable to control by 48 h.\nInhibition of heme oxygenase (HO) attenuates intracellular cGMP concentrations in PC12 cells during guanosine enhanced NGF-dependent neurite outgrowth\nPrevious experiments have demonstrated that the HO inhibitor, ZnPP, blocked guanosine enhanced NGF-dependent neurite outgrowth of PC12 cells (Figure 3). We have also shown that HO-1 expression and cGMP concentrations were elevated during the first 12\u201324 h of this neurite outgrowth process (Figures 4 and 6, respectively). We therefore examined whether pre-treatment of cells with the HO inhibitor ZnPP had any effect on cGMP concentrations. Although metalloporphrins, such as ZnPP-IX have been used widely to demonstrate the role for this enzyme in numerous physiological situations, their selectivity for HO has been questioned; as they may also inhibit haemoproteins such sGC and NOS [58]. However, metalloporphrins used at a concentration below 10 \u00b5M are more selective for HO [59]. Since we found that ZnPP, at 100 nM concentration significantly attenuated guanosine-enhanced neurite outgrowth (Figure 3), we used this concentration of ZnPP to test its effect on intracellular cGMP concentration. PC12 cell cultures were exposed to guanosine alone (300 \u00b5M), or NGF alone (40 ng\/ml), or in combination guanosine plus NGF for 12 h. ZnPP (100 nM) was added to some cultures prior to the addition of guanosine, or NGF, or NGF plus guanosine, and intracellular cGMP concentrations were determined. In cells exposed to guanosine alone, or to guanosine plus NGF, cGMP concentrations were significantly elevated compared to untreated cells (P < 0.01) and to cells treated with NGF alone (P < 0.01) (Figure 7). In cultures pretreated with ZnPP, cGMP concentrations were significantly reduced in cells exposed to guanosine alone (P < 0.05), or guanosine plus NGF (P < 0.05) compared to the corresponding treatments without the inhibitor. Treatment of PC12 cells with NGF did not increase intracellular cGMP concentrations, and the addition of ZnPP had no effect on cGMP concentrations.\nFigure 6\nGuanosine increases cGMP concentrations during guanosine-enhanced NGF-dependent neurite outgrowth in PC12 cells. PC12 cells were cultured on plates coated with poly-d,l-ornithine for 72 h. Cells were then grown in serum-reduced medium (3% heat-inactivated fetal calf serum and 3% heat-inactivated horse serum) for 12 h, and were treated with guanosine (G, 300 \u00b5M) or NGF (N, 40 ng\/ml) or guanosine (300 \u00b5M) plus NGF (40 ng\/ml) (G + N), or with no added treatments (C) as described in Figure 2b. Cells were lysed at time points 0, 6, 12, 24, and 48 h and cGMP concentrations were determined by a competitive enzyme immunoassay. Open bars: untreated controls (C), closed bars: guanosine plus NGF (G + N) treatment, stippled bars: NGF (N) treatment, hatched bars: guanosine (G) treatment. Statistical analysis was performed using a two-way ANCOVA followed by Fischer's LSD post-hoc comparison test (\u25cb P < 0.05, compared to time point 0); (* P < 0.05, relative to control); (** P < 0.01, relative to control); (\u0394 P < 0.05, relative to NGF). Data presented represent the mean relative optical density T SEM obtained in six independent experiments.\nFigure 7\nInhibition of heme oxygenase attenuates cGMP concentrations during guanosine enhanced NGF-dependent neurite outgrowth in PC12 cells. PC12 cells were cultured on plates coated with poly-d,l-ornithine for 72 h. Cells were then grown in serum-reduced medium (3% heat-inactivated) fetal calf serum and 3% heat-inactivated horse serum) for 12 h, and were treated with guanosine (G, 300 \u00b5M) or NGF (N, 40 ng\/ml) or guanosine (300 \u00b5M) plus NGF (40 ng\/ml) (G + N), or with no added treatments (C) as described in Figure 2b. The selective inhibitor of heme oxygenase, zinc protoporphyrin-IX (100 nM), was added to some cultures prior to the addition of guanosine, or NGF, or guanosine plus NGF. Cells were lysed after 12 h and cGMP concentrations were determined by a competitive enzyme immunoassay. Open bars: no zinc protoporphyrin-IX added, closed bars: zinc protoporphyrin-IX added. Statistical analysis was performed using a one-way ANOVA followed by Fischer's LSD post-hoc comparison test (** P < 0.01, relative to control); (\u0394\u0394 P < 0.01, relative to NGF); (\u25cb P < 0.05, relative to treatment without zinc protoporphyrin-IX). Data presented represent the mean relative optical density T SEM obtained in six independent experiments.\nDiscussion\nAddition of guanosine to cultures of undifferentiated, NGF-na\u00efve PC12 cells modestly increases the proportion of cells with neurites after 48 h and, in cultures treated with maximally effective concentrations of NGF, addition of guanosine produces a significant further increase in the proportion of neurite-bearing cells [15]. This implied that the effects of NGF and guanosine are mediated through distinct signaling systems. We reported previously that, whereas some of the neuritogenic effects of guanosine were mediated by increases in intracellular cAMP [22, 23], there was also a cAMP-independent component [22]. Several points led us to consider that a signaling pathway involving cGMP may be responsible for the cAMP-independent component. Vuorinen et al. [25] had shown that extracellular guanosine increases intracellular cGMP in rat mesenteric artery through a NO-dependent mechanism; nitric oxide donors enhance NGF-dependent PC12 cell neurite outgrowth via a cGMP-dependent mechanism [26]; and the neuritogenic effects of the NO donors are abolished by guanylyl cyclase inhibitors and mimicked by cGMP analogs [26]. Our data indicated that indeed cGMP is involved in the neuritogenic effects of guanosine in PC12 cells, but, surprisingly, that NO is not.\nIncreases in intracellular cGMP are often due to activation of GC by NO [35, 45]. Schulick et al. [60] demonstrated that activation of GC by NO donors or atrial natriuretic peptide enhanced NGF-dependent neurite outgrowth in PC12 cells. Further, in PC12 cells NGF upregulates NOS expression, and NO plays an important role in their cellular differentiation [32]. However, NGF-induced differentiation of undifferentiated NGF-na\u00efve PC12 cells could not be abolished by inhibitors of sGC, and NO alone is not sufficient to induce PC12 cell differentiation [26, 33].\nIn the experiments reported here, pre-treating undifferentiated, NGF-na\u00efve PC12 cells with the NOS inhibitor l-NAME, had no effect on the guanosine-enhanced NGF-induced neurite outgrowth. Moreover, like others [61, 62], we found that neither nNOS nor iNOS isoforms were expressed during the first 48 h of cellular differentiation of PC12 cells. Therefore, NO cannot mediate the neuritogenic effects of guanosine on PC 12 cells during the first 48 h.\nRather than NO, our data indicated that CO might activate sGC in response to guanosine in undifferentiated PC12 cells. The heme oxygenase inhibitor, ZnPP attenuated both the ability of guanosine to enhance NGF-dependent neurite outgrowth, and its ability to increase intracellular cGMP, alone or in the presence of NGF. This led us to conclude that guanosine either activated constitutive HO-2 or induced the expression of either HO-1 and\/or the HO-2 isoforms of this enzyme.\nOur data indicated that, as others have reported [55], both HO-1 and HO-2 were expressed constitutively in PC12 cells under basal cell culture conditions. Guanosine induced a significant increase in the expression of HO-1 protein at 24 h in undifferentiated PC12 cells, but had no significant effect on HO-2 expression. Guanosine, in the presence of NGF, produced an even more rapid and pronounced enhancement of HO-1 expression, which was detectable after 12 h. Treatment of PC12 cells with NGF alone also increased the expression of HO-1. Several authors reported recently that NGF promotes HO-1 gene transcription in PC12 cells [63, 64] via activation of the MEK pathway. HO-1 mediates the anti-oxidative and antiapoptotic effects of NGF in serum-deprived PC12 cells [63]. In PC12 cells exposed to oxidative stress induced by 6-hydroxy dopamine, NGF promotes HO-1 upregulation, in this case via the PI3K\/Akt\/PKB pathway [64].\nOthers [63, 64] have reported increased HO-1 protein by 6\u20139 h after exposure to NGF, whereas in our study we observed no change in HO-1 protein concentration until 24 h after NGF addition. This discrepancy may be due to a difference in the PC12 cell line, or to the experimental conditions to which the cells were exposed.\nWe did not expect to find inhibition of HO-1 protein expression by guanosine at 6 h. It has been reported however, that the HO-1 gene expression is suppressed by the elevation of intracellular calcium concentration [56]. Since we have found that guanosine increases intracellular calcium concentrations in astrocytes [57], similar mechanisms may downregulate HO-1 expression in PC12 cells at the 6-h time point. As the guanosine-stimulated calcium increases are rapid and transient, at later time points (12\u201324 h) other intracellular pathways, such as the MAPK and PI-3 kinase pathways are activated, and these are known to promote HO-1 gene expression. Most likely, by 48 h these pathways are no longer activated.\nIn contrast to HO-1, neither guanosine nor NGF, alone or in combination with guanosine, affected HO-2 expression. This confirms the results of others who have found that HO-2 is not regulated by NGF [63, 64]. The HO-1 isozyme is induced by diverse intracellular signals (reviews: Maines [36], Immenshchuh and Ramadori [65]), that include some of the pathways which activate protein kinases, such as PKA [66], or PKC [67, 68], or the second messenger cGMP [42] most likely via PKG [69]. These pathways likely converge on nuclear effectors such as the nuclear factor-\u03baB (NF-\u03baB), activator protein-1 (AP-1) and heat shock factor, to enhance HO-1 gene transcription by interacting with the regulatory elements in the promoter region the gene [36, 65].\nWe reported previously that inhibition of transport of guanosine into PC12 cells did not affect its neuritogenic activity [23], raising the possibility that its effects are mediated by activation of a cell surface, membrane-bound receptor. Indeed, recently we have identified and pharmacologically characterized a specific binding site for guanosine in rat brain membranes [70], in cultured astrocytes [71] and in PC12 cells (F. Caciagli et al., unpublished observations), with characteristics of a Gi-protein coupled receptor [5, 70]. Occupation of this putative receptor by guanosine activates the MAPK pathway [5] and the PI3K\/Akt\/PKB pathway [72, 73]. These effects are most likely mediated by the \u03b2,\u03b3-subunit of the Gi-protein, coupled to the guanosine receptor [74\u201376]. Guanosine has also been shown to increase intracellular cAMP concentrations in several cell types [22\u201324] and phosphorylate the cAMP binding protein, CREB (F. Caciagli et al., unpublished observations).\nIn PC12 cells NGF-promoted induction of HO-1 expression requires the activation of the MEK\/ERK pathway [63] and the PI3K\/Akt\/PKB pathway [64]. Since guanosine, via its putative receptor may also stimulate these intracellular pathways, HO-1 expression in PC12 cells may be regulated by similar mechanisms.\nGuanosine plus NGF in combination induce HO-1 expression to a similar extent than NGF alone at 6, 12 and 24 h. At 48 h, however the effect of guanosine plus NGF on HO-1 expression is comparable to that of control and lower than NGF alone. Since the mechanisms that induce HO-1 expression are complex, the roles played by guanosine and NGF, and the time course of these effects are unclear. We have shown, however that guanosine alone is insufficient to cause major neurite outgrowth. It does however enhance the NGF-induced neuritogenesis. Neuritogenesis is not solely due to HO-1 expression. Either HO-1 or HO-2 could be responsible for these early events since ZnPP, a non-selective HO inhibitor reduces the concentration of cGMP elicited by guanosine or guanosine plus NGF by 12 h.\nNGF activates a number of signalling pathways in PC12 cells, including the MAP kinase and PI3-kinase pathway [18] and the c-JUN N-terminal kinases (JNKs.) [20]. Induction of the HO-1 gene by these pathways seems to override the inhibitory effect of guanosine on HO-1 expression. HO-1 expression at 12 and 24 h is upregulated to a similar extent by guanosine alone, or by NGF alone or by guanosine plus NGF, so there is no additive effect of the combination treatment. As the mechanisms involved in HO-1 gene induction are complex and not clearly defined, our results suggest that the activation of these processes by the three treatments is comparable.\nAt 48 h HO-1 expression in cells treated with guanosine alone, or guanosine plus NGF is similar to control. In the NGF-treated cells, however HO-1 expression is still significantly upregulated compared to control. We attribute this to a difference in the time course of HO-1 gene activation by guanosine, and NGF. Since NGF-activated intracellular signalling pathways are complex and manifold [18, 20] these effects may persist for a longer time. These results are different from those we obtained at 6 h. At this time point, the only significant effect on HO-1 expression is its inhibition by guanosine alone, and this may be due to downregulation of HO-1 expression by guanoisne-stimulated elevations in intracellular Ca2+ concentrations as described above.\nIn several types of cells, HO-1 expression is regulated by the NO-cGMP pathway (reviewed by Maines [36]). In the case of guanosine this mechanism is not likely to play a significant role, as neither iNOS, nor nNOS was detected in NGF-treated PC12 cells during the first 48 h of guanosine-promoted neurite outgrowth.\nIn contrast, upregulation of HO-1 expression by activation of the constitutive HO-2 via the transcription factor NF-\u03baB, is a very plausible alternative mechanism [36]. HO-2 may be activated by PKC, which in turn is stimulated by increased intracellular calcium concentrations [77]. PKC may also activate sGC directly, by phosphorylation, thus providing a receptor-mediated elevation of cGMP concentrations in PC12 cells [78]. We have shown that guanosine increases intracellular calcium concentrations under certain conditions [57], and this in turn may activate PKC. Thus, guanosine may activate HO-2 in a calcium-dependent manner. The time course of intracellular cGMP elevation elicited by guanosine, or by guanosine plus NGF, is consistent with this hypothesis. In PC12 cells exposed to guanosine alone, or to guanosine and NGF, cGMP concentrations were significantly elevated by 6 h and until 12 h in cells exposed to guanosine plus NGF. Although the addition of NGF to PC12 cells induces HO-1 expression at 24 and 48 h, this treatment does not increase cGMP concentrations. Initially we were also puzzled by these data, as we expected a parallel increase in the cGMP concentrations with this treatment. It has been reported by Liu et al. [79] that NGF treatment of PC12 cells rapidly decreases sGC mRNA and protein in a transcription- and translation-dependent manner. So our interpretation of these results is that although HO-1 protein is upregulated by NGF, there is a concurrent suppression of sGC expression, so no increase in cGMP is detected under these conditions. In addition, NGF may also inhibit the activities of some phosphodiesterase isoforms, and thus decrease intracellular cGMP concentrations [80].\nSince HO-1 expression in the guanosine treated cells was not observed until 24 h, and since neither iNOS nor nNOS was detectable at these times, our data is most parsimoniously explained by activation of HO-2.\nAlthough we have not determined HO-2 activity, we have shown, using Western immunoblot analysis that this isozyme is expressed in PC12 cells constitutively, throughout the time course of the experiment (from 0 to 48 h) during all treatment conditions.\nCO synthesized by HO-1 and HO-2 will bind and activate sGC, increasing intracellular cGMP [35\u201338] and this in turn may activate protein kinase G (PKG) [81, 82] and regulate phosphodiesterases (PDE) [81, 82]. Our data are compatible with the neuritogenic effects of guanosine being mediated by cGMP, possibly from early activation of HO-2, and later the induction of HO-1, leading to CO generation, and activation of sGC. But how these molecular events enhance neuritogenesis is unknown. CO itself may directly activate several intracellular signaling pathways, including the MAPK pathway [83] and p38 MAPK [84], the transcription factor NF-\u03baB [85] and induce the transcription of immediate early genes [37, 38].\nRecently, Xiang et al. [86] proposed that navigation of growing axons in response to guidance cues, is mediated by Gi protein coupled receptors, via elevation of cytosolic cGMP [86]. This is another mechanism by which guanosine may promote differentiation of PC12 cells.\nIn conclusion, we have demonstrated the importance of HO, CO and cGMP in mediating the neuritogenic effect of guanosine. The relationship of this signaling mechanism to the putative Gi-protein-linked receptor for guanosine is currently the subject of active investigation in our laboratories.","keyphrases":["guanosine","neurite outgrowth","pc12 cells","heme oxygenase","cyclic gmp","ngf","carbon monoxide","nitric oxide synthase"],"prmu":["P","P","P","P","P","P","P","P"]}
{"id":"J_Gastrointest_Surg-3-1-1852372","title":"Duodeno-Gastric-Esophageal Reflux\u2014What is Pathologic? Comparison of Patients with Barrett\u2019s Esophagus and Age-Matched Volunteers\n","text":"Introduction The aim of the study was to analyse pH- and bile-monitoring data in patients with Barrett\u2019s esophagus and in age- and gender-matched controls.\nIntroduction\nThe incidence rates for adenocarcinoma (AC) of the esophagus and gastric cardia have risen rapidly in Western industrialized countries.1 Besides nicotine and alcohol abuse, nutritional factors, high body mass index, acidic gastric reflux, and Barrett\u2019s esophagus are believed to be critical factors of carcinogenesis.2\u20134 Recent studies have shown that the presence of biliary reflux in combination with acidic gastric reflux damages the esophageal mucosa and causes complications of gastro-esophageal reflux disease (GERD), e.g., development of Barrett\u2019s mucosa (BM).5,6 Duodeno-gastric reflux into the esophagus (DGER), in particular, appears to be important to the pathogenesis of Barrett\u2019s esophagus.7 Prolonged esophageal aspiration studies have documented increased bile acids in patients with severe esophagitis and Barrett\u2019s esophagus.8 Eighty percent of patients with Barrett\u2019s esophagus on proton-pump inhibitors show a normal esophageal pH profile, but 60% show abnormal esophageal exposure to bile as measured by Bilitec 2000.9\nIn the past, direct and prolonged quantification of duodenoesophageal reflux has been difficult to achieve. Now, bilirubin concentration can be directly measured by spectrophotometry, based on the specific absorption at a wavelength of 453\u00a0nm. Biliary reflux can be measured with a transnasally passed, ambulatory fiberoptic probe (Bilitec 2000), which records bile absorption. A number of papers have already been published on the exposure of the esophagus10\u201313 and stomach14\u201316 using this technique. However, in these studies, the control patients were between 25 and 35\u00a0years old. In clinical practice, patients with Barrett\u2019s mucosa tend to be older. In addition, the authors of each study used varying reference values to measure biliary reflux in the esophagus, making comparison of the measured values difficult.\nThe aim of the present study was to analyze data of pH and bile monitoring in a collective of healthy age- and gender-matched controls and patients with Barrett\u2019s esophagus.\nSubjects and Methods\nSubjects\nSelection of controls was carried out according to a strict protocol. Healthy volunteers treated from 1999 to 2000 between the ages of 40 and 60\u00a0years were included in the study. None of the controls were on acid suppressing or gut motility medications, had a history of upper gastrointestinal disease, had undergone upper or major abdominal surgery, or had had therapeutic endoscopic procedures of the upper gastrointestinal tract. Diagnostic endoscopy and barium swallows were not performed, but gallstone disease was excluded by ultrasound scan.\nFrom 1999 to 2002, 24 patients with histologically confirmed Barrett\u2019s mucosa were included in the study. For additional comparison, we include a group of patients with esophagitis (stage I to III according Savary and Miller) without Barrett\u2019s esophagus, which had the same diagnostic procedures before planned laparoscopic fundoplication. During the aforementioned time span, 21 patients age older than 40\u00a0years were available for this study. Exclusion criteria were history of esophageal, gastric, or biliary surgery, history of abdominal or thoracic radiotherapy, or presence of peptic ulcer disease, active gastrointestinal bleeding, esophageal or fundic varices, esophageal or upper small intestine chronic disease, or neoplastic disease. All drugs potentially affecting gastrointestinal motility and secretion were discontinued at least 1\u00a0week before the study.\nUpper Gastrointestinal Endoscopy\nAll patients underwent classical upper gastrointestinal endoscopy. If sedation was necessary, intravenous administration of propofol (up to 200\u00a0mg) was normally used, or occasionally, midazolam (up to 5\u00a0mg) was used. During endoscopy, the presence and extent of esophagitis, Barrett\u2019s esophagus, and hiatal hernia was noted. Biopsies were taken from the Barrett\u2019s mucosa.\nAmbulatory Esophageal\/Gastric pH and Bile Monitoring\nAll groups underwent esophageal manometry and 24-h pH and simultaneous bile monitoring using a standardized protocol. Ambulatory pH monitoring was performed using a transnasally inserted antimony pH electrode with a separate skin reference electrode (Synetics Medical, Stockholm, Sweden). The data were stored on a portable digital recorder (Digitrapper MkIII, Synectics Medical Stockholm, Sweden). Before each study, the pH probe was calibrated in buffer solutions of pH\u00a07 and 1. An episode of acid reflux was defined as a decrease in esophageal pH to less than 4 for more than 10\u00a0s.\nTo quantify duodeno-esophageal reflux, a transnasally passed, ambulatory fiberoptic spectrophotometer (Bilitec 2000, Synectics, Sweden) was used. The system consists of a miniaturized probe of 1.5-mm diameter that carries light signals into the esophagus and back via a plastic fiberoptic bundle. Before each study, the probe was calibrated in water. Corresponding to the current literature, a bilirubin absorption >0.25 was used as a reference for noxious biliary reflux.14\nThe bile and pH probes were taped together and passed transnasally into the esophagus and stomach, as described in detail elsewhere.17 The upper tips of the probes were positioned 5\u00a0cm above the upper border of the lower esophageal sphincter as defined by esophageal manometry. The distal pH electrode and fiberoptic sensor were placed in the fundus of the stomach, 10\u00a0cm distal to the lower esophageal sphincter (Fig.\u00a01). Controls and patients were asked to follow a strict protocol of three meals per day, with no liquids between meals. Recumbent phases of recording were permitted only at night. Patients were asked to keep a diary recording of the exact nature of meals, the supine and erect phases of measurement, and the sensations of heartburn and regurgitation.\nFigure\u00a01Position of the pH- and bilirubin-probes in the stomach and in the esophagus. (UES\u2009=\u2009upper esophageal sphincter; LES\u2009=\u2009lower esophageal sphincter).\nThe simultaneous biliary and pH monitoring was done with administration of a colorless \u201cwhite diet\u201d (WD) including liquid and solid foods with a maximum in vitro bile absorbance of 0.25 [absorbance scale ranging from 0 (plain water) to 1 (total screen)]. The meals included water, milk, toast, potatoes, chicken, dry biscuits, and fish.\nData Acquisition and Interpretation\nAfter completion of the measurements, probes were withdrawn from the patients, and data were stored via interface on an IBM-compatible computer equipped with Polygram\u00ae software (Medtronic). The data of each second of the 24-h measurements were used for analysis. To assess the presence of gastric or esophageal biliary reflux, the percentage of time when absorbance was greater than 0.25 was calculated for the following periods: total supine, upright, and postprandial. The postprandial period was defined as 2\u00a0h after the end of meals. The percentage of time with esophageal pH lower than 4 and median gastric pH and the percentage of time with gastric pH measuring 1, 2, 3\u20137, and >7 was also calculated for the above periods. The mean duration of the ambulatory pH and Bilitec monitoring study was 22\u00a0h, 40\u00a0min in patients and 23\u00a0h, 44\u00a0min in the controls.\nStatistical Analysis\nThe SPSS (version 11.0, Chicago, Illinois) program was used to analyze the results. For graphical presentation, we used the program MedCalc for Windows, (Version 9.0, MedCalc Software, Belgium). Median, interquartile range (IQR or 25th to 75th percentile) values were established. The nonparametric tests (Mann\u2013Whitney and Kruskal\u2013Wallis analysis) were used to assess the relationship between variables. Box and Whisker plots were used to present some of the data. In these plots, the box represents the IQR, and the Whiskers represent the highest and lowest values. Outliers are also plotted, defined as more than 1.5 times the IQR from the 75th centile. Extreme values were defined as more than three times the IQR from the 75th centile.\nA receiver operator characteristic (ROC) curve was used to find a cutoff value for optimal sensitivity and specificity according Zweig and Campbell.18 The area under the curve (AUC) as a measurement of diagnostic performance of the test was used. The results are given as point with the 95% confidence interval (95% CI) and graphically for presentation of all data. As the positive group, we used the patients with Barrett\u2019s mucosa, and the negative test group was defined by the healthy volunteers. A nonparametric distribution of the area under the curve was assumpted.\nThe assumptions for calculation of the required sample size were alpha\u2009=\u20090.05, beta\u2009=\u20090.80, and that a test is only valid for daily use if less than 20% of the healthy controls and at least 80% of the patients have positive test results. The calculated sample size for each group was 20.\nEthics\nThe study protocol was approved by the ethics committee of the University of Cologne. Each subject gave written informed consent.\nResults\nTwenty-four patients with Barrett\u2019s esophagus (mean age: 58\u00a0years), 21 patients with esophagitis (mean age: 57\u00a0years), and 19 healthy controls with a mean age of 51\u00a0years were included in the study. Patients with BM showed esophagitis grade 0 (4 cases), grade I (12 cases), and grade II (8 cases). The control group of patients with esophagitis showed nine cases with grade I, eight cases with grade II, and three cases with grade III. Demographics of patients and volunteers are displayed in Table\u00a01 (the data of one volunteer was not usable due to technical problems). \nTable\u00a01Demographic Data of Patients with Barrett-Mucosa or Esophagitis and Healthy VolunteersParametersPatients with Barrett\u2019s Esophagus (n\u2009=\u200924)Patients with Esophagitis (n\u2009=\u200921)Controls (n\u2009=\u200919)Significance Pat. with Barrett vs ControlsAge (median) 57\u00a0years58\u00a0years51\u00a0years\u2013Min\u2013max29\u201375\u00a0years42\u201377\u00a0years39\u201362\u00a0yearsGender m:f16:811:1011:8n.s.BMI (median) min\u2013max27.0\u00a0kg\/m2 (18.6\u201333.1)26.9\u00a0kg\/m2 (17.9\u201331.5)24.1\u00a0kg\/m2 (19.62\u201327.34)p\u2009=\u20090.003Smokers (%)n\u2009=\u20095 (20.8)n\u2009=\u20094 (19.0)n\u2009=\u20096 (31.6)n.s.No alcohol %n\u2009=\u20093 (12.5)n\u2009=\u20095 (23.8)n\u2009=\u20095 (26.3)n.s.BMI Body mass index\nAcidic Gastric Reflux (AGR)\nPatients with Barrett\u2019s esophagus, 19 of 24 (79%) and 20 of 21 control patients with esophagitis (95%) had pathologic AGR [pH\u2009<\u20094 in >5% of total measuring time (TMT)], but also 6 of the 19 healthy controls (32%) showed pathologic AGR without any symptoms (p\u2009=\u20090.002). During the TMT, the median AGR was 10.6% for Barrett patients and 3.2% for controls (p\u2009<\u20090.01). In particular, measurements of long acid reflux (LAR), defined as reflux pH\u2009<\u20094 lasting longer than 5\u00a0min, showed significant differences between patients and controls. Pathologic AGR was found in patients during both the supine and upright fasting measuring periods. In contrast, pathologic AGR in healthy controls occurred only in the upright position (Table\u00a02). \nTable\u00a02Median of Acidic Gastric Reflux into the Esophagus in Patients with BM or with Esophagitis and in Healthy ControlsParametersPatients with Barrett\u2019s Esophagus (n\u2009=\u200924) [median (LQ-UQ)]Patients with Esophagitis (n\u2009=\u200921) [median (LQ-UQ)]Controls (n\u2009=\u200919) [median (LQ-UQ)]Significance Pat. with Barrett vs ControlsPercentage of total measuring time pH\u2009<\u20094 (%)10.6 (6.2\u201338.3)19.9 (1.6\u201371.7)3.2 (0.9\u20135.5)p\u2009=\u20090.01Percentage of upright measuring time pH\u2009<\u20094 (%)11.7 (6.03\u20136.4)18.9 (8.7\u201360.8)2.4 (0.9\u20136.1)p\u2009<\u20090.05Percentage of supine measuring time pH\u2009<\u20094 (%)10.9 (0.4\u201327.1)6.3 (0.0\u201313.3)0.3 (0.0\u20134.2)p\u2009=\u20090.004LQ Lower quartile, UQ upper quartile\n24-h Intragastric pH and Bile Monitoring\nGastric pH monitoring showed no significant differences between patients and controls for all measuring periods (Table\u00a03). Gastric bilirubin exposure, indicating biliary reflux, was significantly more frequent in patients than in controls during all measuring periods (Table\u00a03). Biliary exposure in the supine position typically occurred during the early hours of the morning during sleep, represented by increased absorbance over 2\u20133\u00a0h, with a rapid return to baseline values around the time the subject resumed the upright position. Over the same time period, gastric pH monitoring showed increased pH levels to greater than 2 (Fig.\u00a02). \nTable\u00a03Results of 24-H Intragastric pH and Bile Monitoring in Patients with Barrett Esophagus and Healthy ControlsParametersPatients (n\u2009=\u200924) [Median (LQ\u2013UQ)]Controls (n\u2009=\u200919) [Median (LQ\u2013UQ)]SignificanceMedian of intragastric pH during TMT1.3 (1.0\u20131.4)1.4 (1.1\u20131.7)n.s.Bilirubin exposure percentage (%) of TMT 7.8 (1.6\u201317.8)0.0 (0.0\u20131.0)p\u2009=\u20090.001Bilirubin exposure percentage (%) of upright time6.9 (0.1\u201312.9)0.0 (0.0\u20131.3)p\u2009<\u20090.01Bilirubin exposure percentage (%) of supine time2.0 (0.0\u201328.6)0.0 (0.0\u20130.0)p\u2009=\u20090.001LQ Lower quartile, UQ upper quartile, TMT total measuring timeFigure\u00a0224h intragastric pH- and bile monitoring in a patient with Barrett\u2019s esophagus demonstrating the duodenogastric reflux in the early morning. a. Bilitec\u00ae-monitoring, b. pH-monitoring.\nBilirubin Exposure of the Esophagus\nOver the TMT, the median of esophageal biliary reflux was 7.8% for patients with Barrett\u2019s esophagus (LQ\u2013UQ\u2009=\u20091.6\u201317.8%) and 3.5 (LQ\u2013UQ\u2009=\u20090.1\u201313.5) for control patients, in contrast to 0% for the controls (LQ\u2013UQ\u2009=\u20090\u20131.0%), p\u2009=\u20090.001). Figure\u00a03 shows that esophageal bile monitoring in patients with Barrett\u2019s esophagus and healthy controls varied during the total measuring and supine periods.\nFigure\u00a03Results of the esophageal bile-monitoring in 24 patients with Barrett\u2019s esophagus, 21 patients with esophagitis and 19 healthy controls a) total measuring period (Kruskal-Wallis Test\u2009=\u20090.01) b) supine period (Kruskal-Wallis Test p\u2009=\u20090.01).\nThe receiver operating curve, plotting the true positive rate (patients with Barrett\u2019s esophagus identified by bilirubin exposure) in function of the false positive rate (healthy controls with high bilirubin exposure) is shown in Fig.\u00a04. With an area under the curve of 0.78 (95% CI\u2009=\u20090.56\u20130.89), the ROC analysis of biliary monitoring showed the optimal value for patients at 1% of the TMT [75% sensitivity (95% CI\u2009=\u200953\u201390%), 84% specificity (95% CI\u2009=\u200960\u201396%)]. Therefore, the cutoff value to distinguish normal vs pathologic biliary reflux using 24-h biliary monitoring in the esophagus (absorbance threshold >0.25) should be fixed at 1% of TMT.\nFigure\u00a04ROC-curve with 95% confidence intervals for pathologic bile-monitoring in patients with Barrett esophagus compared to age and sex matched healthy controls.\nBarrett patients, 18 of 24 (75%), 15 of 21 control patients with esophagitis (71%), and 3 of 19 controls (16%) showed biliry reflux into the esophagus more than 1.1% of the TMT (p\u2009<\u20090.001). Using this cutoff value, none of the controls, 10 of the control patients (48%), and 11 of 24 Barrett\u2019s patients (46%) had pathologic bilirubin exposure during sleep.\nDiscussion\nThe results of our study confirm that patients with Barrett\u2019s esophagus have significantly more frequent duodenogastric reflux into the esophagus than age- and sex-matched healthy controls. In addition, this reflux, measured by acid and bilirubin exposure, remains longer in the esophagus, especially during sleep.\nThe role of acid and nonacid reflux into the esophagus as a causative factor of symptoms and mucosal lesions has been addressed in a number of studies. Not only the duration, but possibly the composition of the reflux, is instrumental in the development of such lesions.13 Twenty-four-hour intragastric bile monitoring has provided the clinician with unequivocal evidence of excessive duodenogastric reflux (DGR) in 41% of patients with endoscopic esophagitis, gastroesophageal reflux (GER) symptoms, and gastric symptoms suggestive of DGR.19 Reflux of duodenal contents into the stomach, especially postprandially, is a physiological event;20 however, biliary reflux is a large contributor to mucosal lesions in the whole stomach.21\nIn our study, the control group of patients with different grades of esophagitis showed no significantly different measurements of acidic or bile reflux into the stomach or the esophagus compared to Barrett\u2019s patients. This may be caused by selection of patients with esophagitis, which were candidates for fundoplication, but both groups of patients differed significantly compared to healthy controls. Therefore, our results are of great clinical relevance especially for preoperative diagnostic.\nMarshall et al. compared healthy controls to patients with different grades of reflux-esophagitis and Barrett\u2019s esophagus with regard to bile measurements in the stomach.22 In this study, the average age of the control patients was 25\u00a0years, and that of the patients in Groups I, II, and III was 42, 50, and 60\u00a0years, respectively. The bilitec-probe was positioned 10\u00a0cm below the lower esophageal sphincter (LES). The threshold of bilirubin absorbance was 0.14, and although no difference was found between groups over the TMT, gastric bilirubin exposure was higher in the supine than in the upright position. In the current study, the control group was older than that of the Marshall study. More duodeno-gastric reflux was recorded in both study and control patients during all periods of measurement. These findings may be due to improved study conditions.\nWe used an esophageal threshold of 0.25 for bilirubin absorbance. Fein et al.,14 in an in vitro study of absorption of different white meals, showed that the least food interference during bile monitoring was measured with an absorbance > 0.25.\nTack et al. reported the influence of meal consistency on Bilitec measurement results in healthy subjects.23 They compared two groups of young controls. The subjects took either liquid meals only, not absorbing light of the same wavelength as bilirubin, or solid food, avoiding diets that interfere with bilirubin absorbance. The authors found significant differences between the two groups using a bilirubin absorbance threshold >0.14 with a median percentage (interquartile range) over the TMT of 10.9 % (6.7\u201319.3) for solid meals and 0.3% (0.0\u20132.8) for liquid meals. Major meal artifacts were present in two-solid-meal (10%) and no-liquid-meal subjects. In our study, we found such a meal artifact in one patient and one control, but the values of bilirubin absorbance were lower than 0.25, and therefore, not relevant to our results.\nIt is not unusual for gastro-esophageal reflux to contain bile, duodenal, and pancreatic secretions. Utilization of the Bilitec spectrophotometric probe has demonstrated a higher prevalence of abnormal esophageal bilirubin exposure in patients with Barrett\u2019s metaplasia when compared to those with erosive injury or without signs of esophagitis.6,8,10,13 In those studies, patients were consistently older than volunteers included in the control group. However, other studies have shown an increased prevalence of gastroesophageal reflux with age.24 For these reasons, we studied age and sex matched healthy controls and patients with Barrett\u2019s esophagus or with esophagitis. We found pathologic acid and biliary reflux of the esophagus in one-third of the controls. Perhaps, this may be caused by artefacts or by violation of the protocol by the volunteers. But in a previous published study, we could show that younger healthy controls had no such pathologic reflux.17 Possibly, these phenomena are caused by relaxations which occur more often in older people. In contrast, nearly all patients with Barrett\u2019s esophagus (87%) and all patients with esophagitis (100%) showed pathologic acidic reflux and\/or bile reflux measured with combined pH and bile monitoring. Bile reflux into the esophagus during sleep, in particular, was only found in patients with BM or with esophagitis.\nIn our study, we measured the intragastric pH and the bile reflux from the duodenum into the stomach (DGR). The median of the intragastric pH was similar in both groups. But patients with Barrett\u2019s esophagus had significantly longer duodenogastric reflux during the 24-h measuring period than controls. More DGR was demonstrated at night than during the day in both groups of study patients and in healthy controls. This could be associated with an alkaline shift in the pH, according to previously published studies.25\u201327 The precise mechanism by which nocturnal DGR occurs and the roles posture plays remain unclear.\nBowrey et al. were unable to establish either gastro-esophageal or duodenogastric reflux as the predominant cause of inflammation in gastric cardiac mucosa with use of the Bilitec 2000 device.16 This is understandable, as the amount of reflux into the stomach (DGR) does not necessarily correlate with DGER into the esophagus. In this study, the authors demonstrated more DGR in females during the supine period, while males presented more DGER. At the same time, there was no correlation between bile levels in the stomach and esophagus. The controls were, however, much younger than the patients. We found significant differences in bile measurements of the stomach and esophagus between BM patients and controls. In contrast to Bowrey et al., we saw more DGER in females during the supine period and more DGR in male patients.\nIn contrast, Banki reported similar esophageal exposure to refluxed acid and bilirubin in females and in males with Barrett\u2019s mucosa.28 Pfaffenbach et al.29 studied esophageal bile and acid reflux in patients with long segment Barrett\u2019s esophagus (LSBE), short segment Barrett\u2019s esophagus (SSBE) and patients with gastro-esophageal reflux disease (GERD). Subjects underwent esophageal manometry and simultaneous 24-h pH and bile monitoring (Bilitec 2000) with an absorbance value >0.2 for 10.9% of the total period. GER did not differ between the groups. However, DGER differed between patients with LSBE (14.7%), SSBE (2.1%), and GERD (2.1%).\nIn summary, the analysis of reference values of esophageal acid and bile-reflux measurements in a collective of healthy, age- and gender-matched controls compared to patients with BM led to the following conclusions: \nAlthough about 30% of the healthy controls showed acid reflux in pH monitoring, patients with BM had significantly more acid reflux during all measured periods.Healthy controls did not have relevant duodeno-gastric-esophageal reflux measured by bilirubin absorbance. Especially during the supine period, there was no bile reflux.The optimal threshold for pathological bile reflux is 1.1 % (bile monitoring with an absorbance of 0.25).","keyphrases":["esophagus","barrett\u2019s mucosa","spectrophotometry","acid reflux","bile reflux","reference value bilitec"],"prmu":["P","P","P","P","P","R"]}
{"id":"J_Interv_Card_Electrophysiol-4-1-2292477","title":"Long term outcome of cavotricuspid isthmus cryoablation for the treatment of common atrial flutter in 180 patients: A single center experience\n","text":"Objective Recent literature has shown that common type atrial flutter (AFL) can recur late after cavotricuspid isthmus (CTI) catheter ablation using radiofrequency energy (RF). We report the long term outcome of a large group of patients undergoing CTI ablation using cryothermy for AFL in a single center.\nIntroduction\nAblation of the cavotricuspid isthmus (CTI) for the treatment of atrial flutter (AFL) has become standard practice. Most of the procedures are performed using radiofrequency energy (RF) [1\u20135]. High chronic success rates are described but the majority of the data comes from a relatively short term follow-up [1\u201313]. A recent study by Chinitz et al. [14] reported some interesting data regarding the long term follow-up of 80 patients with common type AFL who underwent CTI ablation using RF. They found a 12.5% (ten patients) recurrence rate at an average of 21\u00a0months after the procedure with most patients having a recurrence after the first year post ablation. Our prior experience using cryothermy in a limited number of patients [15] also showed that AFL may recur 1\u00a0year after CTI cryoablation.\nThe purpose of this study was to evaluate the long term outcome of CTI cryoablation in a large patient population with common type AFL in a single center.\nMethods\nOne hundred and eighty patients with sustained symptomatic common type AFL referred for CTI ablation were enrolled prospectively from July 2001 to July 2006 in our institution. Signed written consent, approved by the local ethics committee, was obtained from all participants.\nBefore CTI cryoablation, anticoagulation with warfarin aiming a therapeutic international normalized ratio of 2 to 3 was kept for at least 3\u00a0weeks. Antiarrhythmic drugs were not discontinued before ablation.\nElectrophysiologic study and ablation\nWe focused our study on the clinical aspects and long term follow-up of patients with AFL who were submitted to CTI cryoablation.\nOur CTI cryoablation protocol has been already reported [15]. Briefly, our methodology was as follows: under local anesthesia and via the femoral route, a decapolar catheter is positioned in the distal coronary sinus (for evaluation of left atrial activation), a duodecapolar catheter (2-mm interelectrode spacing, Halo catheter, Biosense Webster, Baldwin Park, CA) for mapping the right atrial lateral wall and a quadripolar catheter in the His bundle area. A deflectable, 10.5\u00a0F, 6.5\u00a0mm tip cryoablation catheter (CryoCor Inc., San Diego, CA) is inserted into the right atrium through a 12F, 65-cm-long sheath (DAIG, St. Jude Medical Inc., St. Paul, MN) [15\u201319].\nEntrainment to confirm the isthmus dependence of the AFL circuit was performed in every patient in whom AFL was present or could be induced at the start of the procedure, according to previously published techniques [2, 10, 15, 16, 20, 21]. If AF, requiring cardioversion, was present or developed during stimulation or if we were unable, even under isoproterenol infusion, to induce AFL, CTI ablation was performed during sinus rhythm. Linear lesions were created by use of a point-by-point technique with gradual pullback of the cryocatheter in a ventricular atrial fashion. The first application was delivered at the ventricular insertion of the isthmus and applications were continued for an average of 3\u00a0min. In patients in whom ablation of the posterior isthmus proved insufficient, an attempt was made to ablate the septal isthmus (four patients). After documentation of bidirectional isthmus conduction block, the atrial pacing (from the proximal coronary sinus) protocol (up to three atrial extrastimuli at three pacing cycle lengths and incremental atrial pacing) was performed without and with isoproterenol infusion (1 to 3\u00a0\u03bcg\/min). Acute success was defined as bidirectional isthmus conduction block, 30\u00a0min after the last application without and with isoproterenol infusion [22].\nAll patients were studied in the fasting state without sedation. Those presenting in AF while on the catheterization table were converted to sinus rhythm by internal or external cardioversion. During the procedure intravenous heparin was given as a 100-IU\/kg bolus dose after the venous sheaths were inserted. The 12-lead ECG and intracardiac electrograms were recorded and stored by the BARD Labsystem PRO.\nPost ablation management\nAll patients were monitored in hospital for 24\u00a0h and oral anticoagulation was started the day of ablation. Antiarrhythmic drugs (AAD) were stopped after the procedure in patients without a history of AF; in those with AF\/AFL the same AAD were continued. All patients had anticoagulation for at least 1\u00a0month. Subsequently, the need for chronic anticoagulation was assessed by the amount of recurrences of AFL\/AF and the presence of risk factors for thromboembolic events.\nAll patients had a 12-lead ECG and a 24\u00a0h Holter recording at discharge and during each clinic visit (1\u00a0month, 3, 6, 9, 12\u00a0months and yearly thereafter) or earlier if they had symptoms.\nDue to the logistics of the Maastricht area\u2014and also the presence of a dedicated research nurse (S. P.) who was available to address patients\u2019 concerns and questions at any time\u2014we were able to follow every patient on an individual basis.\nStatistical analysis\nContinuous variables are presented as mean\u2009\u00b1\u2009SD, where appropriate. In cases of a non-Gaussian distribution, medians and quartiles are given. Categorical variables are expressed as numbers and percentages of patients.\nStatistical analysis was performed using the Student t test for unpaired data. All values were considered significant at P\u2009<\u20090.05.\nThe authors had full access to the data and take responsibility for its integrity.\nResults\nOf the 180 enrolled patients, 39 patients (22%) were women with a mean age of 58 (from 18 to 80) years. More than half of the patients (92 patients, 52%) had structural heart disease: arterial hypertension: 57 patients, coronary artery disease: 22 patients, valvular heart disease: 13 patients, congenital heart disease: 11 patients, idiopathic cardiomyopathy: 18 patients. Counterclockwise AFL was documented in 91% (164) of the patients and clockwise AFL in 9% (16 patients).\nThe mean left atrial diameter and the mean left ventricular ejection fraction were 44\u2009\u00b1\u20097\u00a0mm and 57\u2009\u00b1\u20097%, respectively. A prior history of AF was present in 123 (69%) of the patients. The clinical characteristics of the patients, related to the presence or absence of AF before ablation, are described in Table\u00a01. Note that AF during follow-up is significantly higher in the group with a prior history of this arrhythmia.\nTable\u00a01Characteristics of the 180 patients with atrial flutter referred for CTI cryoablation related to the presence or absence of atrial fibrillation AF before ablation\u00a0AF\/AFL patients (123 patients), 69%AFL only (57 patients), 31%p valueAge (year)57\u2009\u00b1\u20091358\u2009\u00b1\u200913nsWomen19% (23 patients)28% (16 patients)nsNo SHD55% (68 patients)32% (18 patients)< 0.05LAd (cm)4.44.5nsLVEF (%)5855nsAcute failuresa5% (6 patients)5% (3 patients)nsAF in long term follow up69% (85 patients)35% (20 patients)< 0.05aPatients in whom CTI cryoablation did not result in bidirectional block (failed procedure).AF Atrial fibrillation, AFL atrial flutter, CTI cavotricuspid isthmus, Lad left atrium diameter, LVEF left ventricular ejection fraction, SHD structural heart disease\nTotal fluoroscopic\u2014mean of 30\u2009\u00b1\u200918\u00a0min (range, 12 to 152\u00a0min)\u2014and procedure times\u2014mean of 2.6\u2009\u00b1\u20091.1\u00a0h (range, 1 to 6.5\u00a0h)\u2014decreased throughout our study with a long duration of a procedure and fluoroscopy being attributed mainly to the learning curve of a new technology. An average of 7 (3 to 20) applications per patient were delivered with a mean temperature and duration of \u221288\u00b0C and 3\u00a0min, respectively.\nThe acute success rate for cryoablation of the CTI was 95% (171 patients). There were no complications. Of the nine patients in whom bidirectional CTI block was not achieved, three underwent a successful reablation. The other six patients had much improvement of their symptoms (despite an incomplete line) and preferred not to have another procedure.\nAfter a mean follow-up of 27\u2009\u00b1\u200917 (range from 12 to 60) months, recurrences of symptomatic AFL occurred in 15 patients (9%) resulting in a 91% chronic success rate. Those recurrences occurred in six patients within the first 3\u00a0months post ablation, in four patients from 3 to 6\u00a0months post ablation, in four patients from 6 to 9\u00a0months post ablation and in one patient at 14\u00a0months post ablation (Fig.\u00a01). Ten of those 15 patients underwent a second successful cryoablation of the CTI. The other five had not only reablation of the isthmus but also pulmonary vein cryoisolation (PVI) for AF during the second procedure.\nFig.\u00a01Percentage of patients (171 successfully ablated) free of common type atrial flutter over time\nDespite the success as far as AFL was concerned, AF was still present in 85 patients (69%) with a prior history of this arrhythmia. Those patients were treated by AAD (69 patients, 81%), PVI (14 patients, 16%) or AV nodal ablation with pacemaker implantation (two patients, 3%). New episodes of AF developed in 20 (35%) of those 57 patients without documented AF prior to CTI cryoablation and were all controlled by AAD.\nDiscussion\nMain findings\nOur current study showed a 91% chronic success rate of CTI cryoablation in a large population (180 patients) with AFL followed for a long period of time (1 to 5\u00a0years, mean of 27\u00a0months).\nAccording to the most recent ACC\/AHA\/ESC guidelines for the management of supraventricular arrhythmias CTI ablation is the only therapy with a class I indication for the long term management of AFL [2]. The majority of those ablations are done using RF energy. Concerning the follow-up length of those procedures, most of the literature available reports on a relatively short period, the majority of them being less than 2\u00a0years. However Chinitz et al. [14] published a study of 80 patients with AFL submitted to CTI ablation using RF that had up to 6\u00a0years follow-up. Interestingly, they found a 12.5% recurrence of AFL that occurred on an average of 21\u00a0months post ablation, ratifying the need of data with a longer follow-up. It is important to keep in mind though, that arrhythmias after ablation do not always correlate with symptoms [23, 24] and one could question if those patients with such a late recurrence had those episodes much earlier than what is reported. That might be one reason why our results, where most of our recurrences happened within 1\u00a0year, are discrepant with those from their study. The intrinsic characteristics of our hospital, our clinical follow-up and the population of Maastricht could be responsible for our ability to find those recurrences earlier.\nRegarding cryothermy as energy source, a 9\u00a0months follow-up study in 39 patients undergoing CTI cryoablation with a different system was reported [25]. They achieved a chronic success rate of 100% in the cryoablated group, despite documenting reconduction through the isthmus in 31% of patients during a 3\u00a0month follow-up electrophysiologic study. The CRAAFT trial presented the results in 32 patients with AFL submitted to either cryo- or RF ablation of the CTI [26]. They report an 84% success rate after a follow-up of 14\u00a0months. Those two small studies, with a relatively short follow-up, reported similar success rates with RF and cryothermy for AFL ablation. The results from our study\u2014which included a significant larger patient population (180) with a longer follow-up (1 to 5\u00a0years, mean of 27\u00a0months)\u2014reinforces the effectiveness of cryothermy for the treatment of AFL, being the outcomes comparable to those reported in the literature using RF (where most outcome data also comes from non invasive follow-up) [1, 4\u20137, 11, 12, 27, 28]. Therefore, cryothermy can be considered as an efficient energy source for the treatment of common type AFL.\nThe relation of AF with AFL\nThe close relation between AF and AFL is well described [1, 5\u201314, 21, 29\u201339]. Our data showed a high prevalence of AF (123 out of 180 patients, 69%) in patients with predominant AFL referred for CTI ablation. A recent study by Ellis and colleagues [40] strengthened even more this association. They followed 363 patients with lone AFL who underwent CTI RF ablation\u2014during a mean follow-up of 39\u00a0months\u2014and reported an 82% incidence of drug refractory AF in their patient population.\nThe new development of AF in our patient population without a prior history of AF preceding AFL ablation may be a sign of an already present electrical and morphological change in the right and left atria. If in addition, a functional or anatomical line of block between the two venae cavae (or elsewhere) occurs, atypical AFL(s) may develop either in the right (because the CTI is already ablated) or in the left atrium [11, 14, 21, 31, 32, 37\u201339].\nStudy limitations\nOur recurrence data rely mostly on the subjective assessment by the patient, like the great majority of RF data [1, 4, 6\u20138, 12]. Only an objective measurement (such as a repeat electrophysiological study with documented bidirectional block) will determine the long lasting effect of CTI ablation for the treatment of common type AFL.\nConclusions\nIn this prospectively studied large population of patients with common type AFL, cryoablation of the CTI has a 91% chronic success rate during long term follow-up (range 1 to 5\u00a0years, mean of 27\u00a0months). These results are similar to those obtained with RF, validating cryothermy as an efficient alternative energy source. We also were able to ratify the frequent association of AF with AFL.","keyphrases":["cavotricuspid isthmus","cryoablation","atrial flutter","long term follow-up","atrial fibrillation"],"prmu":["P","P","P","P","P"]}
{"id":"Pediatr_Nephrol-4-1-2259256","title":"Neurogenic bladder: etiology and assessment\n","text":"A review of the various causes of neurologic impairment to the lower urinary tract in children was the aim of this presentation. The emphasis was on diagnosis, pathophysiology, and treatment that strive to maintain as normal a function as possible in order to achieve eventual urinary continence and health of the upper urinary tract. The latest principles based on the most up to date evidence are promulgated but with an eye towards historical prospective. The reader should gain an adequate understanding of various disorders that comprise this condition and feel comfortable with proposing options for management when faced with the responsibility of caring for an affected child.\nIntroduction\nNeurogenic bladder dysfunction in children is an ever-evolving condition. The expansion of its understanding and treatment over the past 50\u00a0years has been just remarkable. In the mid 1950s there were few insights and minimal alternatives to the child\u2019s being in diapers or wearing an appliance over an abdominal wall stoma. Starting with the development of adequate X-ray assessment and reliable urodynamic investigation, the advent of clean intermittent catheterization, artificial sphincter implantation, continent urinary conduits and a plethora of drug therapies that modulate lower urinary tract function, we have learned a great deal about the pathophysiology, pathogenesis and treatment of this disorder and the evidence specific ways to manage it. With the promise of tissue engineering and stem cell therapy, new vistas for treatment seem to be on the horizon.\nThe most common cause of neurogenic bladder dysfunction in children is neurospinal dysraphism, primarily an open back lesion, but an occult or closed dysraphic state is being diagnosed with more frequency as neonatal spinal ultrasound and magnetic resonance imaging (MRI) are used with increasing regularity to visualize any lower midline spinal cutaneous or gluteal cleft malformation. It was thought that folic acid deficiency was a cause of this disease and that its replacement in women of childbearing age would practically eliminate the condition, but this has not happened. There has been a definite decrease in its incidence; however, in parts of the USA its prevalence has not diminished at all [1\u20134].\nOther causes of neurogenic dysfunction involving the spine include sacral agenesis, tethered spinal cord associated with imperforate anus, cloacal malformations, and spinal cord injuries from sporting injuries and motor vehicle accidents. Central nervous system abnormalities include spastic diplegia (cerebral palsy) and learning disabilities, i.e. attention deficit hyperactivity disorder (ADHD) or attention deficit disorder (ADD). Terminology used throughout this manuscript will conform to the standardization document recently published in the Journal of Urology [5].\nUrodynamic studies\nA urodynamic study consists of the following components. The child is catheterized with a triple-lumen urodynamic catheter after a small amount of liquid lidocaine (1%) has been injected into the urethra and held in place for a moment or two. First, intravesical pressure is recorded; then, the bladder is drained and the residual urine carefully measured, yielding a pressure at residual volume (this helps determine detrusor compliance at natural filling and is more accurate than cystometric compliance measured during even slow filling of the bladder). A small balloon catheter is passed into the rectum to measure intra-abdominal pressure during the cystometrogram to identify artifacts of motion and monitor increases in abdominal pressure during the filling and emptying phases of the study. The side-hole port of the urethral pressure channel is positioned at the highest point of resistance in the urethra and kept in place, measuring this resistance throughout bladder filling and emptying to determine the leak point pressure. External urethral sphincter electromyography (EMG) is performed using a 24-gauge concentric needle electrode inserted perineally in boys or para-urethrally in girls and advanced into the skeletal muscle component of the sphincter until individual motor unit action potentials are seen or heard on a standard EMG recorder. The characteristics of the individual motor unit potentials at rest, in response to various sacral reflexes (i.e. bulbocavernosus, anocutaneous, Valsalva and Cred\u00e9 maneuvers) and bladder filling and emptying are recorded to detect degrees of denervation. Next, the bladder is filled through the second port while intravesical pressure is monitored via the third port of the tri-lumen urodynamic catheter. The rate of filling is set at 10% of expected capacity for age [age (in years) +30\u2009\u00d7\u200930\u2009=\u2009expected capacity in milliliters] [6]. Detrusor pressure measurements are continuously recorded throughout filling to calculate compliance, and during voiding or leaking to denote emptying pressure. Detrusor overactivity is defined as any short-lived pressure rise of >15\u00a0cm H2O from baseline before capacity is reached [6]. Sometimes, the urodynamics study is combined with fluoroscopic video-imaging using a dilute radio-opaque contrast agent to visualize the appearance of the bladder wall and bladder neck or to detect the presence of vesicoureteral reflux during the test. Alternatively, a radionuclide agent is instilled, with the patient lying above a nuclear camera, to determine at what pressure reflux occurs, when it is known to be present beforehand. The study is not considered complete until the child actually urinates or leaks and the \u2018voiding\u2019 pressure is measured. The small size of the urodynamic catheter does not seem to affect the voiding pressure adversely, even in very young children. The normal end filling pressure should be <10\u00a0cm H2O, while the normal voiding pressure varies from 55\u00a0cm to 80\u00a0cm H2O in boys and from 30\u00a0cm to 65\u00a0cm H2O in girls [5]. Detrusor overactivity is considered an abnormal finding at any time [6].\nThe examination findings are considered normal when there is an appropriate capacity, good compliant bladder, with no overactivity, and normal innervation of the sphincter with normal sacral reflexes, and an increase in sphincter activity during filling and complete silencing during emptying. An upper motor neuron lesion is present when there is detrusor overactivity and\/or hyperactive EMG responses to sacral reflexes and\/or a failure of the sphincter muscle, on EMG, to relax (either partially or completely) with a bladder contraction or leaking at capacity. A lower motor neuron lesion is noted when there are no contractions of the detrusor muscle and\/or there is a degree of denervation, either partial or complete, in the sphincter muscle, with characteristic EMG changes in the motor units or no motor unit activity at all, respectively, and little or no response in the sphincter to sacral reflexes and\/or bladder filling or emptying [7].\nNeurospinal dysraphism\nMyelomeningocele\nWith regard to embryology, the developing spinal canal begins on the 18th day of gestation and is completed by day\u00a035, closing in a caudad direction from the cephalic end of the body. Failure of mesodermal in-growth over the developing spinal cord results in an open lesion, most commonly seen in the lumbo-sacral area and, with decreasing regularity, in the thoracic and cervical areas (Table\u00a01). The exposed spinal cord and its nerve roots, some of which may protrude into the meningocele sac, and tension on the spinal cord as the cord \u2018rises up\u2019 the canal with elongation of the fetus (from L2, L3 in mid- to late fetal life, to L1 at birth), contribute to a variable picture of neural injury to the lower urinary tract and lower extremities [8]. Coupled with obstruction of the aqueduct to the fourth ventricle (Chiari malformation), with possible herniation of the brainstem and the center for micturition coordination (the pontine mesencephalic center), additional layers of dysfunction are added to those nerve pathways already affected.\nTable\u00a01Spinal bony level of myelomeningocele (uppermost vertebral abnormality)LocationIncidence (%)Cervical-high thoracic2Low thoracic5Lumbar26Lumbosacral47Sacral20\nThe over-riding issue at birth is whether or not the child has detrusor external urethral sphincter dyssynergy and whether the infant can empty the bladder completely at low pressure. All newborns with radiologic abnormalities at birth (5\u201310% with hydronephrosis, reflux) have active obstruction of the bladder outlet that had caused these changes to occur in utero [9]. The presence of elevated detrusor filling pressure, bladder sphincter dyssynergy or high voiding or leaking pressures (above 40\u00a0cm H2O) at capacity, which can result in upper urinary tract deterioration in as many as 63% of children [9], warrants early intervention with clean intermittent catheterization (CIC) and anticholinergic drugs [10\u201313]. Thus, the investigation of these children when newborn includes a renal and bladder ultrasound, a catheterized measurement of urine residual after voiding or leaking, a determination of serum creatinine concentration after 7\u00a0days of life and a urodynamic study that incorporates both detrusor pressure measurements and urethral sphincter electromyography. Voiding cystography is undertaken when hydronephrosis is present and\/or urodynamic studies indicate bladder outlet obstruction with either increased pressure at capacity or bladder sphincter dyssynergy. The incidence of reflux when there is functional obstruction of the bladder outlet can range as high as 50% [14, 15].\nAlthough some clinicians still preach watchful waiting if the renal ultrasound findings are normal, and do not recommend urodynamic studies in the newborn period, opting instead for instituting CIC and drug therapy only with the first sign of ureteral or renal pelvic dilation [16, 17], most centers within the USA now advocate full investigation of the lower urinary tract and initiate prophylactic treatment if there are signs of outlet obstruction and\/or elevated bladder filling or voiding pressure [18\u201320]. The incidence of urinary tract deterioration can be greater than 50% [11, 12, 20] when children with the potential for deterioration are followed with expectant and not preemptive therapy. Even though the \u2018watchful waiters\u2019 demonstrate that they can reduce the presence of hydronephrosis and, possibly reflux, the changes in detrusor dynamics are not as easily reversed, and the need for subsequent aggressive management of the bladder to control incontinence and a poorly compliant bladder is commonplace (Table\u00a02). Therefore, CIC is begun when detrusor sphincter dyssynergy, elevated leak point pressures greater than 40\u00a0cm H2O, and\/or reflux grade\u00a03 or higher (on a scale of 1 to 5) are present. Instituting CIC and anticholinergic therapy in infancy has revealed many advantages over time [19\u201321]: the parents and the child adapt to the routine of CIC much easier than they would have if it were to be begun when the child is older; the bladder often remains very compliant, expanding as the child grows and maintaining appropriate wall thickness as noted on bladder echography; hydronephrosis and vesicoureteral reflux develop in fewer than 10%; continence is readily achieved in greater than 50% with no additional maneuvers; and the need for augmentation cystoplasty to maintain a reasonable organ for storage is markedly reduced from almost 60% to 16% when compared with that in children followed expectantly [22].\nTable\u00a02Surveillance in infants with myelodysplasia (until age 5\u00a0years) (IVP intravenous pyelogram, ECHO sonogram, UDS urodynamic study, VCUG voiding cystourethrogram, RNC radionuclide cystogram)Sphincter activityRecommended testsFrequencyIntact-synergicPost-void residual volumeEvery 4\u00a0monthsIVP or renal ECHOEvery 12\u00a0monthsUDSEvery 12\u00a0monthsIntact-dyssynergicaIVP or renal ECHOEvery 12\u00a0monthsUDSEvery 12\u00a0monthsVCUG or RNCbEvery 12\u00a0monthsPartial denervationPost-void residual volumeEvery 4\u00a0monthsIVP or renal ECHOEvery 12\u00a0monthsUDScEvery 12\u00a0monthsVCUG or RNCbEvery 12\u00a0monthsComplete denervationPost-void residual volumeEvery 6\u00a0monthsRenal ECHOEvery 12\u00a0monthsaPatients receiving intermittent catheterization and anticholinergic agentsbIf detrusor hypertonicity or reflux is already presentcDepending on degree of denervation\nWhen vesicoureteral reflux is present, CIC effectively lowers the intravesical emptying pressure when the bladder is drained. In addition, anticholinergic medication can be added to lower detrusor filling pressure, increasing compliance without fear of causing urinary retention when combined with CIC. The lowered filling and emptying pressures has proven to be very beneficial; in 30\u201350% of children reflux is resolved within 2\u20133\u00a0years of its discovery and initiation of therapy.\nThe disadvantages are few and include a higher rate of bacteriuria (60\u201370% versus 30%) but a lower rate of symptomatic urinary tract infection (20% versus 40%) during childhood than in children followed expectantly [23, 24]. Because the subsequent risk of reflux is lower, the effect on renal function and the development of scarring is reduced when these prophylactically treated children are compared to those monitored with watchful observation [16, 25].\nCred\u00e9 voiding is not an efficacious form of bladder emptying in children with myelodysplasia, especially if the urethral sphincter is partially or fully innervated. Because most children have intact motor function above L1, any increase in abdominal pressure from a Cred\u00e9 maneuver can lead to a reflexive increase in urethral sphincter activity, thus producing an increase in bladder outlet resistance resulting in \u201chigh voiding pressure\u201d. This can be particularly noxious in children with moderate or severe grades of reflux. In addition, as the child grows, the bladder resides more in the pelvis and not intra-abdominally, further reducing the effectiveness of the Cred\u00e9 maneuver.\nThe key to a stable bladder and, consequently, renal function is the maintenance of a good capacity, highly compliant, detrusor muscle, combined with periodic and complete emptying of the bladder at low pressure. Anticholinergic medication (primarily oxybutynin, tolterodine, glycopyrrolate, hyoscyamine or trospium) and CIC achieve that in a majority of children and provide an added benefit of continence if the child has reasonable bladder outlet resistance [9]. Several alpha sympathomimetic agents, such as phenylpropanolamine, ephedrine or pseudoephedrine, are used to increase bladder outlet resistance when it is not sufficient to maintain continence between CICs. A variety of surgeries has been devised when these conditions cannot be met. A detailed description of the surgical treatment is beyond the scope of this discussion, but, suffice it to say, bowel augmentation onto the bladder has been a common practice to lower pressure and increase capacity in order to make the bladder a useful organ for storage. Disadvantages abound, including problematic mucus production, recurrent urinary infection, electrolyte imbalance, stone formation and the recently documented risk for the late occurrence of cancer in the augmented segment [26].\nA plethora of surgical procedures has been designed to increase bladder outlet resistance in those children with a level that is insufficient to maintain continence between catheterizations, that include implantation of an artificial urinary sphincter, bladder neck tightening, using adjacent tissue, a fascial sling, and various bulking agents. All can provide more resistance, but no one procedure is ideally suited for every patient.\nCreating a catheterizable urinary stoma has become fashionable in those children with intractable urethral incontinence (with obliteration of the bladder neck) or inability to catheterize their urethra easily due to obesity, poor eye\u2013hand coordination or caretaker issues surrounding genital organ privacy. Long-term success has been achieved that provides the individual with a degree of independence, but problems with stomal stenosis can occur.\nOccult spinal dysraphism\nOccult spinal dysraphism has been diagnosed with increasing frequency since the advent of spinal ultrasound and MR imaging [27]. An intraspinal lipoma or lipomeningocele, a diastematomyelia, a fatty filum with tethering, or a dermal sinus tract make up the extent of these disorders. Most pediatricians now will image the back of any newborn with a cutaneous lower midline back lesion, which can be detected in 90% of affected individuals [28]. These lesions include a subcutaneous mass, dermal vascular malformation, hypertrichosis, a midline dimple or sinus tract, a skin tag or an asymmetric gluteal cleft. These lesions often signify an underlying bony and\/or spinal cord malformation. Ultrasound within the first 3\u00a0months of the infant\u2019s life can easily visualize the intraspinal space. After the infant has reached that age MR imaging is needed to diagnose and\/or confirm the presence of a dysraphic state.\nMost infants have no other manifestation of this disease (other than the cutaneous lesion) because lower extremity neurologic function is normal. In the past, before intraspinal imaging was feasible, these lesions often went undetected until urinary and\/or fecal incontinence became problematic or lower extremity difficulties became evident [28\u201331]. This frequently occurred around the time of a pubertal growth spurt, when increased traction on the spinal cord took place [32]. As the imaging techniques evolved and urodynamic studies in children were performed at an early age, it became clear that most babies had minimal neurologic impairment at first but that the neurologic lesion progressed with advancing age [33].\nThe pathophysiology involves apparent tension on the lower end of the spinal cord as the child grows. Normally, the conus medullaris ends at L1, L2 at birth but \u2018rises\u2019 cephalad to T12, L1 at puberty. The differential growth rate between the spinal cord and the vertebral bodies stretches the lower cord and cauda equina due to fixation of the filum terminale to the bottom of the vertebral canal, or the nerves roots emanating from the cord become compressed by an expanding intraspinal lipoma. With time, this stretching and\/or compression affects the oxidative process of the neural tissue that then leads to impaired function of the lower extremities and\/or lower urinary tract [34, 35].\nInitial investigation of the affected child includes a urodynamics study and a renal and bladder ultrasound. The findings of urodynamics studies in children less than 1\u00a0year old are invariably normal, but when abnormal they are not coupled with abnormalities in the lower extremities [33, 36]. When findings are abnormal, partial denervation in the urethral sphincter muscle or failure of the sphincter to relax during a detrusor contraction are the most common findings in infancy, while extensive denervation of the sphincter and\/or an acontractile detrusor combined with changes in lower extremity function are the most common abnormalities in an older child [37]. As noted previously for myelomeningocele in children, a voiding cystourethrogram is warranted only when the urodynamics parameters suggest risk to the upper urinary tract from increased bladder outlet resistance or poor detrusor compliance. Vesicoureteral reflux, hydronephrosis and urinary incontinence are all managed in the same fashion as one would treat children with similar neurologic impairment due to an open spinal abnormality.\nThe abnormal condition tends to improve following spinal cord de-tethering in the infant, but this normalization is unlikely when an older child is operated on [33, 37\u201339]. Therefore, specific urologic therapy is not instituted following delineation of an abnormality in infants until urodynamics studies have been repeated 3\u00a0months after the condition has been repaired. If the findings have not changed, or the child is older (when the chance that the neurologic abnormality will not improve), treatment based on principles outlined in the section on open spinal lesions is instituted.\nThirty percent of children will have secondary spinal cord tethering over time, some as late as puberty or just beyond it, when the last growth spurt occurs [28, 37, 39]. Therefore, careful surveillance and repeated assessment at the first sign of incontinence or changing lower extremity function is warranted. No child is considered risk free until he or she has reached full adult height.\nSacral agenesis\nPartial or complete absence of the lowermost vertebral bodies is labeled sacral agenesis. The condition can range from the absence of just the last two or three sacral bodies to the absence of sacral and several lumbar bones as well (sirenomyelia). This can be seen in offspring of insulin-dependent diabetic mothers (1%) [40], but it may be part of a genetic disorder due to a deletion of part of chromosome\u00a07 (7q36), leading to absence of an important transcription factor that plays a role in the development of the caudal end of the spinal cord and vertebral column [41]. In familial cases of sacral agenesis associated with the Currarino triad syndrome (presacral mass, sacral agenesis and anorectal malformation), deletions in chromosome\u00a07 (7q) resulting in HLXB9 genetic mutations have been found [42]. A mutation in HLXB9, a homeodomain gene of a 403 amino acid protein, which appears to be responsible for neural plate infolding, has been identified in 20 of 21 patients with familial Currarino triad syndrome and in two of seven sporadic cases of this syndrome [43, 44]. Heterozygote carriers within these families have also been identified [45]. Thus, sacral agenesis may represent one point on a spectrum of abnormalities that encompass a sacral meningocele and ano-rectal malformations [46].\nIn the newborn period (and even afterwards) these infants appear normal, with no lower extremity abnormality. Unless it is thought of during the examination of a newborn of a diabetic mother, such babies often go undiagnosed. With time, as they have difficulty in toilet training or have urinary infection, it becomes evident that there is a problem [40, 47]. The pathognomic sign is absence of the upper end of the gluteal cleft, with flattened buttocks. When the diagnosis is considered a lateral spine film (or a spinal ultrasound in infants) will confirm the abnormality. A spinal MR reveals a sharp cut off to the cord at about T-12, with nerve roots streaming from it. Approximately 90% of children develop neurogenic bladder dysfunction [48].\nAn equal number of children have an overactive detrusor with sphincter dyssynergy or an acontractile detrusor with complete denervation in the urethral sphincter [47, 49]. The former is often associated with recurrent urinary infection and vesicoureteral reflux, whereas the latter produces continuous incontinence. The type of neurologic impairment affecting the lower urinary tract cannot be predicted from the level of absent or abnormal vertebral bones [47]. Obviously, management depends on the type of dysfunction present. CIC, anticholinergic medication and antibiotics are instituted in those with an upper motor neuron type lesion, while surgical measures with CIC are needed in those with an incompetent sphincter mechanism.\nAssociated conditions\nImperforate anus\nFailure of the lowermost portion of the developing colon in the fetus to canalize fully, resulting in a closed rectum that does not open onto the anal skin verge, is a rare anomaly, but one that needs immediate attention after delivery. In boys there is a fistula from the end of the rectal canal to the posterior urethra, whereas a stenotic, anteriorly placed anal canal that ends at the posterior aspect of the vestibule, may be noted in girls (Table\u00a03). Usually, a colostomy is performed, or, more recently, definitive repair is undertaken in the first 24\u00a0h of life in boys, while anal dilatation is begun in girls.\nTable\u00a03Wingspread classification of anorectal malformationsFemaleMaleHighHigh\u00a0Anorectal agenesis\u00a0Anorectal agenesis\u00a0\u00a0With rectovaginal fistula\u00a0\u00a0With rectourethral (prostatic) fistula\u00a0\u00a0Without fistula\u00a0Without fistula\u00a0Rectal atresia\u00a0Rectal atresiaIntermediateIntermediate\u00a0\u00a0Rectovestibular fistula\u00a0\u00a0Rectovestibular urethral fistula\u00a0\u00a0Rectovaginal fistula\u00a0Anal agenesis without fistula\u00a0\u00a0Anal agenesis without fistulaLowLow\u00a0Anovestibular fistula\u00a0Anocutaneous fistula\u00a0Anocutaneous fistula\u00a0Anal stenosis\u00a0Anal stenosis\u00a0Rare malformationCloacal malformationRare malformation\nFrom a urologic standpoint, this condition is often part of a constellation of abnormalities known as the VATER or VACTERL association (the mnemonic denotes all affected organs: V vertebral; A anal; C cardiac; TE tracheoesophageal fistula; R renal; L limb). Unilateral renal agenesis, vesicoureteral reflux and spinal cord tethering are the most common abnormalities affecting the urinary tract [50]. Urinary, and fecal, incontinence issues that become prominent as the child matures, due to progressive denervation of the lowermost nerves modulating bladder and urethral and anal muscle function, are likely clinical scenarios. This is most commonly seen in children where the rectum has ended above the levator ani muscle (50%), but it is also seen when the rectum ends below that pelvic floor muscle (18%) [51]. A spinal vertebral bony image is not a reliable sign that spinal cord abnormality is present [52]. A spinal ultrasound in newborns, and MRI of the spine in older children, are a mandatory part of the investigation in these children. An upper motor neuron lesion with detrusor overactivity and\/or detrusor sphincter dyssynergy are most likely to develop, but an acontractile detrusor and sphincter denervation are also seen as a result of spinal cord tethering [49, 53]. It has been shown that the earlier neurosurgical intervention is undertaken, the better the individual\u2019s chances of having normal sacral spinal cord and lower urinary tract function [54]. Therefore, early detection of this condition is necessary to improve the child\u2019s chance of maintaining healthy kidneys and becoming continent.\nCentral nervous system disorders\nCerebral palsy\nThis condition results from a non-progressive injury to the brain, occurring in the perinatal period, that produces a neuromuscular disability or a specific symptom complex of cerebral dysfunction [55]. It is caused by perinatal infection or a period of anoxia (or hypoxia) affecting the tissues of the central nervous system [56] (Table\u00a04). It appears in babies who are less than 2\u00a0kg at birth, have had intraventricular hemorrhage, experienced a neonatal seizure or received mechanical ventilation for a prolonged period of time in the postnatal period [57]. The incidence of cerebral palsy is increasing, as more severely premature infants are surviving and going home.\nTable\u00a04Perinatal risk factors in cerebral palsy. Adapted from [61] used with permission. UMN upper motor neuron lesion, LMN lower motor neuron lesionFactorUMN (no. of patients)LMN (no. of patients)Prematurity101Respiratory distress\/arrest\/apnea92Neonatal seizures5\u2013Infection51Traumatic birth5\u2013Congenital hydrocephalus3\u2013Placenta previa\/abruption22Hypoglycemia seizures2\u2013Intracranial hemorrhage2\u2013Cyanosis at birth13No specific factor noted15\u2013\nAffected children have delayed gross motor development, abnormal fine motor performance, altered muscle tone, abnormal stress gait, tight heel cords and exaggerated deep tendon reflexes [58]. These findings can vary substantially, from being quite obvious to very subtle, unless a careful neurologic examination is performed. These abnormalities may not be manifest in the early postnatal period, but become evident over time, because myelination of axons and maturation of neurons in the basal ganglia are required before spasticity, dystonia, and athetosis become apparent. Some less affected children have even milder forms of the disease, with only learning disabilities, attention deficit, or attention deficit hyperactivity disorders being seen.\nMost children with cerebral palsy develop total urinary control, albeit at an age later than most unaffected children. Incontinence is a feature in some (24% of surveyed families in one study) [59], but the exact incidence has never been truly determined. The presence of incontinence is often related to the extent of the physical impairment, primarily because the handicap prevents the child from reaching the bathroom on time, causing an episode of wetting [60]. Urinary infection and vesicoureteral reflux are not features of this disease, and the kidneys are invariably normal on ultrasonic imaging.\nAn upper motor neuron type of bladder dysfunction with detrusor overactivity (80%), but not necessarily with detrusor sphincter dyssynergy (5%), is invariably present, but lower motor neuron injury with sphincter denervation due to spinal cord involvement can be seen as well (11%) [61] (Tables\u00a05 and 6). Volitional control over sphincter function is present, and affected children have some ability to prevent leaking from an overactive detrusor by tightening the muscle for a variable period of time. Children with milder forms of dysfunction, with just learning disabilities without spasticity, have an overactive detrusor leading to either urgency (with or without incontinence) and nocturia, or day and night wetting. Therefore, modulating the overactivity with anticholinergic medication is the treatment of choice. However, this must be done judiciously, with careful monitoring of residual urine to prevent the development of retention.\nTable\u00a05Lower urinary tract function in cerebral palsyTypeNumberUpper motor neuron lesion49Mixed upper + lower motor neuron lesion5Incomplete lower motor neuron lesion1No urodynamic lesion2Table\u00a06Urodynamics findings in cerebral palsy (some patients had more than one finding)Type of LesionNo. of PatientsUpper motor neuron (detrusor or sphincter)Detrusor overactivity35Detrusor sphincter dyssynergy7Overactive sacral reflexes6No voluntary control3Smaller than expected bladder capacity2Poorly compliant2Lower motor neuron (abnormal motor unit potentials)Excessive polyphagia of sphincter5\u2191 Amplitude + \u2191 duration potentials4\nTrauma\nTraumatic injuries to the spine\nFortunately, spinal cord injuries in children are rare (2.6 per million) [62]. The incidence tends to increase geometrically with age [63]. When an injury does occur, it is more likely to happen in a boy than in a girl, and it is usually the result of a motor vehicle or bicycle accident (24\u201352%), a fall from a high place, a gunshot wound, or a diving or sports incident. Injuries may also occur iatrogenically after surgery to correct scoliosis, kyphosis or other intraspinal processes, congenital aortic anomalies, or patent ductus arteriosus operations [62, 64\u201367]. Newborns are particularly prone to a hyperextension injury during high forceps delivery [68].\nSpinal cord injuries in children are intrinsically different from those in adults, owing to a variety of factors, including the mechanism of injury and the difference in configuration of the cord in children compared with that in adults. In addition, the horizontal versus vertical orientation of the facet joints in vertebral bodies that predisposes to anteroposterior subluxation in children, the delayed supportive effect of the paraspinous musculature and ligaments, and the relative heaviness of the head, which causes a fulcrum of maximal flexion of the upper cervical region in infants and young children, all contribute to a high degree of hypermobility that places the child\u2019s spinal cord at risk for ischemic necrosis [65].\nThe lower urinary tract dysfunction that ensues is not likely to be an isolated event but is usually associated with loss of sensation and paralysis of the lower limbs. Radiologic investigation of the spine may not reveal any bony abnormality, although momentary subluxation of osseous structures resulting from the elasticity of the vertebral ligaments can result in a neurologic injury. This condition has been seen only in children (usually younger than 8\u00a0years old) and has been labeled SCIWORA (spinal cord injury without radiologic abnormality) [69]. Overall, SCIWORA can account for up to 38% of spinal cord injuries in children [70]. Often, what appears to be a permanent lesion initially turns out to be a transient phenomenon with time. Although sensation and motor function of the lower extremities may be restored relatively quickly, the dysfunction involving the bladder and rectum may persist considerably longer.\nDuring the acute phase of the injury, the bladder is often acontractile and the urethral sphincter nonreactive, although normal-appearing bioelectric potentials can be recorded on sphincter EMG (spinal shock). Over a variable but unpredictable period of time, detrusor contractility and sphincter reactivity return as spinal cord edema subsides. With this return of function, an overactive detrusor and bladder-sphincter dyssynergy may develop if the lateral reticulospinal cord pathways to and from the brainstem have been disrupted. When the lesion affects the cauda equina, there is probably little to no return of bladder or sphincter function. Sacral sensation and peripheral reflexes are not good indicators of ultimate lower urinary tract function [71]. Over time, the predominant urodynamic pattern in patients with a thoracic-level lesion is an overactive detrusor with sphincter dyssynergy, high voiding pressures, eventual hydronephrosis and vesicoureteral reflux. Patients with an upper thoracic or cervical lesion are likely to exhibit autonomic dysreflexia with a spontaneous discharge of \u03b11 stimulants during bladder filling and with contractions of the detrusor that require careful monitoring of their blood pressure during any investigational studies of the lower urinary tract [72, 73].\nFoley catheter draining in the immediate post-injury phase is needed, but CIC should be begun as soon as feasible after that time [74\u201376]. When a child starts volitional voiding, CIC can be tapered and stopped as residual urines are measured and found to be insignificant (less that 5\u00a0ml). Urodynamics studies should be undertaken no earlier than 6\u00a0weeks after the injury, to allow for the manifestation of the extent of the neurologic injury [77]. Periodic reassessment of bladder and sphincter function is appropriate up to 2\u00a0years after the injury, due to the potential for change during that time. Renal ultrasonography should be part of the assessment, but voiding cystography is only needed when there are signs of potential risk (i.e. sphincter dyssynergy or poor detrusor compliance). Low detrusor filling and voiding pressures with complete emptying are the goal [78]. When this is not present during testing, starting or continuing CIC and adding anticholinergic medication is paramount to insuring the long-term health of the lower and upper urinary tract [79].\nConclusions\nNeurogenic bladder dysfunction in children takes in a very wide spectrum of conditions that include congenitally acquired conditions that may even be preventable today, conditions that are associated with specific anatomic abnormalities, and acquired conditions that may occur perinatally or from accidents or sports or motor vehicle related injuries. Despite the etiology, the guiding principles for management are similar; insuring and maintaining an adequate sized, normally compliant, reservoir that evacuates urine completely, at a relatively low pressure, is the key to maintaining a healthy environment for the kidneys. A plethora of methods has come into existence, especially since the advent of clean intermittent catheterization and the advancement of pharmacologic understanding and manipulation. Future prospects look bright for affected children, with the overall health of the individual the most paramount goal to be achieved.","keyphrases":["myelodysplasia","pediatric neurogenic bladder","diagnosis and evaluation"],"prmu":["P","M","M"]}
{"id":"Plant_Mol_Biol-3-1-1805041","title":"A large-scale collection of phenotypic data describing an insertional mutant population to facilitate functional analysis of rice genes\n","text":"In order to facilitate the functional analysis of rice genes, we produced about 50,000 insertion lines with the endogenous retrotransposon Tos17. Phenotypes of these lines in the M2 generation were observed in the field and characterized based on 53 phenotype descriptors. Nearly half of the lines showed more than one mutant phenotype. The most frequently observed phenotype was low fertility, followed by dwarfism. Phenotype data with photographs of each line are stored in the Tos17 mutant panel web-based database with a dataset of sequences flanking Tos17 insertion points in the rice genome (http:\/\/tos.nias.affrc.go.jp\/). This combination of phenotypic and flanking sequence data will stimulate the functional analysis of rice genes.\nIntroduction\nRice is the most important staple crop for half the world\u2019s population. Improvements in rice yield and quality beyond the benefits of the green revolution of 30\u00a0years ago are required to meet the demands of an increasing global population. At the beginning of the 21st century, with the hope of finding creative solutions to the problems of food production, nutrition and transportation, nearly the entire nucleotide sequence of the rice genome, Oryza sativa L. cv. Nipponbare, was determined through a world-wide collaborative effort (International Rice Genome Sequencing Project 2005). The sequence data provide an important resource for promoting the discovery of important genes for crop improvement. Currently, a Rice Annotation Project (RAP) using nucleotide sequences of full-length cDNAs (The Rice Full-Length cDNA Consortium 2003) is in progress to position functional genes on the rice genome map (Ohyanagi et al. 2006); however, many genes remain \u201cunknown\u201d due to lack of experimental evidence or sufficient similarity with characterized genes from other organisms. The next great challenge after completion of genome sequencing is the functional characterization of these genes and discovery of genes that affect vital developmental, agronomic or biochemical plant functions (Hirochika et al. 2004).\nGene annotation is mostly based on the sequence similarity to known genes from other species. The limitations of this method are that every organism may have unique genes that do not have homologues even in closely related species, and assignment of a protein\u2019s function based on similarity may only give a partial description. For example, a gene may contain a domain that is conserved among protein kinases, but the actual substrate of the enzyme would be difficult or impossible to determine without experimental evidence. Categorization using cladistic associations (i.e. a phylogenetic tree) is more sensitive and is able to detect BLAST mis-hits or false positives (Sjolander 2004). However, characteristics supported by experimental data, e.g., correlation between the phenotype and the function of the protein kinase, are indispensable to the exact annotation, because all of the associative algorithms ultimately depend on experimental data.\nMutational analysis through gene disruption is one of the most efficient methods for identifying gene function. The related approaches of interrupting gene expression with RNAi (Miki and Shimamoto 2004), and overexpression are also effective because the functions of genes can be determined by the correlation of disrupted genes and their associated phenotypes. Currently, more than 130,000 T-DNA insertion lines of Arabidopsis thaliana have been created and are publicly available (Alonso et al. 2003). Phenotyping of Ds insertion lines in Arabidopsis is also in progress (Kuromori et al. 2006). For characterizing monocot genomes, we have produced more than 50,000 disruption lines of Oryza sativa cv. Nipponbare (Japonica), using the endogenous retrotransposon Tos17, which has a \u201ccopy and paste\u201d type of transposition activity (Miyao et al. 2003). There are two native copies of Tos17 in the Nipponbare genome that are activated specifically in cultured cells (Hirochika et al. 1996). On average, ten new copies of Tos17 are transposed in each cell during 5\u00a0months in culture. When plants have been regenerated from the cultured cells, Tos17 retrotransposition is immediately inactivated, and Tos17 copies become fixed and segregate in a Mendelian fashion in the next generation.\nThere are many advantages to the Tos17 disruption system for mutational analyses. Because the flanking regions of Tos17 insertion points are easily amplified by TAIL-PCR or a suppression PCR method using a 3\u2032-end primer of Tos17, insertion sites in the rice genome can be easily determined. Furthermore, mutants can be screened by PCR using the Tos17 end primer and a primer from any desired genomic sequence. The distribution of transposed Tos17s is not random, and a large number of transpositional \u201chot spots\u201d are detected throughout the rice genome. The insertion frequency of Tos17 into genic regions is three-fold higher than that into non-coding regions. Owing to this polarization, Tos17 insertion lines have great advantages for the functional analysis of rice genes (Miyao et al. 2003). Because Tos17 is an endogenous retrotransposon, regenerated lines can be grown in the field, and seeds can be exported without regulations associated with genetically modified organisms such as rice lines that have undergone \u201ctransgenic-\u201d insertion of T-DNA (Jeon et al. 2000; Sallaud et al. 2003; Wu et al. 2003), Ds (Greco et al. 2003; Kim et al. 2004), or En\/Spm (Kumar et al. 2005).\nMutant lines generated by Tos17 retrotransposition have already been used for the functional analysis of rice genes. For example, Tos17 insertion mutants with a dwarf or viviparous phenotype were used to identify and analyze genes for gibberellin and abscisic acid metabolism (Sakamoto et al. 2004; Agrawal et al. 2001). Phenotypic characteristics of most of the Tos17 insertion lines, however, remain to be described. A large number of plant scientists working with rice or other monocotyledonous species could benefit from a systematic phenotypic analysis of many Tos17 insertion lines and the creation of a public database. To promote the functional analysis of rice genes, the phenotypes of all of our mutant lines have been observed in rice fields through the collaboration of seven laboratories. Collected phenotypic data are useful for predicting the function of a disrupted gene. In this paper, we report the phenotypic statistics of a Tos17 insertion mutant population for the discovery of agronomically important genes.\nMaterials and methods\nPlant materials\nNipponbare calli derived from embryos were grown in N6 liquid medium (Otsuki 1990) containing 1\u00a0mg\/l 2,4-D for 5\u00a0months. In total, about 50,000 plants were regenerated. Seeds of the M2 generation were harvested from each M1 plant. To check the activity of Tos17 under various hormone conditions, N6 liquid media containing 1, 2, 5, 10 or 20\u00a0mg\/l 2,4-D with or without 0.1\u00a0mg\/l BA were used.\nPhenotyping\nTen to twenty-five seeds were planted per line. Germination rates and seedling phenotypes were observed in the nursery. After 1\u00a0month from seeding, the seedlings were transferred to the paddy field. Phenotypes in the field were observed at the vegetative stage, near the heading stage, at the seed maturation stage, and at harvest. Lines segregating abnormal plants at about 25% frequency were digitally photographed and assigned a phenotype ID.\nDatabase\nAll phenotype data were stored into a relational database on the PostgreSQL relational database managing system (http:\/\/www.postgresql.org\/) with a FreeBSD 5.5 operating system (http:\/\/www.freebsd.org\/). FreeBSD 6.1 was used for the statistical analysis. Tabular structure for phenotypic description consists of the line name, plant serial number, observation date, observing person, a link to the photograph file, phenotype ID, and detailed descriptions. Additional data for each line are inserted as a new row. Data can be modified only by the observing person. With this table structure, observation logs for each line by many persons over the course of many years can be stored without conflict. For counting lines of each mutant phenotype, Perl script, which can connect with the PostgreSQL server with a Pg.pm module, reads the phenotype table, stores line names and phenotype IDs into an associative array (\u201chash\u201d), to convert from a redundant number of phenotype IDs to a unique number for each line. The number of lines showing each phenotype ID in the hash was counted. Flanking sequence data of Tos17 insertion sites are also stored in the same database. Loci of Tos17 insertions are determined by BLASTN searches against rice genome sequences of the International Rice Genome Sequencing Project (IRGSP) Build3. Loci of annotated genes are also stored in the relational database.\nSOM analysis\nSoftware for SOM analysis was downloaded from the Neural Networks Research Center of Helsinki University of Technology (http:\/\/www.cis.hut.fi\/research\/som_pak\/). For the initialization program, randinit, parameters: 24 for x dimension, 16 for y dimension, hexa for topology, bubble for neighborhood function, and 123 for seeds were used. For the map training program, vsom, parameters for first learning: 1000 for learning length in training, 0.05 for initial learning rate, 10 for initial radius of the training area in som-algorithm, parameters for second learning: 10000 for learning length, 0.02 for initial learning rate, 3 for initial radius. The analyzed data were visualized using the umat program.\nResults\nFlow of phenotypic analysis\nNipponbare calli from 92 seeds were independently cultured for 5\u00a0months, and about 50,000 plants were regenerated. Seeds (M2 generation) from the regenerated plants were independently harvested and labeled with a line name. Ten to twenty-five M2 plants of each line were observed over a full developmental cycle in the nursery and paddy.\nEach line was designated by \u201cN\u201d for \u201cNipponbare\u201d followed by a letter A-G that indicates the yearly lot, and four figures, e.g. NA1234. Lines named with \u201cNA\u201d and \u201cNB\u201d were used in a small-scale pilot study and \u201cNC\u201d to \u201cNG\u201d were used in large-scale studies.\nTo evaluate the effect of hormone (auxin and cytokinin) concentration in the medium on Tos17 retrotransposition activity, the NC line was subcultured in media containing 1, 2, 5, 10, or 20\u00a0mg\/l of 2,4-dichlorophenoxyacetic acid (2,4-D) with or without 0.1\u00a0mg\/l benzyl adenine (BA). There was no significant difference in observed transposition events among lines derived from the subcultures. In lots, ND, NE, NF, and NG, calli were induced in a medium containing 2\u00a0mg\/l 2,4-D, and maintained in a medium supplemented with 1\u00a0mg\/l 2,4-D for 5\u00a0months. The cultural conditions for each line are available with the phenotypic description list from the mutant panel database.\nCo-segregation of mutant phenotypes with Tos17 insertion can be detected by DNA blot hybridization, which will help the further analysis of gene function. If co-segregation is detected, the flanking region of co-segregated Tos17 can be isolated by TAIL-PCR or suppression PCR (Miyao et al. 1998).\nClassification of phenotypes\nTo provide a classification system and a database useful for most rice scientists, phenotype scoring was limited to 53 phenotype descriptors belonging to 12 classes. Because environmental conditions such as day length, temperature and soil conditions differ significantly among the seven fields in which this project was conducted, some variability in traits such as heading date or leaf color may have occurred between different fields. Thus, some of the present data may vary with environmental conditions, although most of mutant phenotypes are stably expressed. Phenotype classifications and a summary of observations are shown in Table\u00a01. Each subclass has a phenotype ID code to enable data entry as a barcode with a portable recording device and to enable data compilation from all seven laboratories.Germination. This trait was evaluated by measuring germination rate under defined conditions. Since wild type (cv. Nipponbare) showed a germination rate higher than 95%, lines that showed germination rates less than 75% were recorded in the present project. Since several laboratories measured germination rates of all lines, all primary data are also stored in the database. In total, 3489 lines showed a low germination rate, and 525 lines showed germination rates less than 50%. Some of the lines with low germination rates may be embryo mutants.Growth. Growth was observed at the seedling stage in the rice nursery or at an early stage after transfer to the field. \u201cWeak\u201d refers to mutants that formed slim seedlings with retarded growth, probably caused by a deficiency in some housekeeping gene product (Fig.\u00a01, NG0352). Some of the \u201cWeak\u201d lines were reclassified eventually to \u201cLethal\u201d. An example of \u201cAbnormal shoot\u201d is shown in Fig.\u00a01 (NG0356). Another example is represented by NE3024. This seedling is smaller than wild type and its leaves are short and wide, resembling mutants defective in genes associated with gibberellin biosynthesis or signaling pathways (Sakamoto et al. 2004; Uegchi-Tanaka et al. 2005).Leaf color. Frequently appearing pigmentation phenotypes are \u201cAlbino\u201d (Fig.\u00a01, NG1048) and \u201cVirescent\u201d (Fig.\u00a01, NE1517). Completely white or yellow (Fig.\u00a01, NG1469) seedlings died within 3\u00a0weeks after seeding. If green and white segments coexisted on leaves, a condition called virescent, the seedlings survived in the field. Zebra mutants that show repetition of a white or a pale green band and a green band in the longitudinal direction were also observed (Fig.\u00a01, NF6044). However, in most lines, the \u201cZebra\u201d phenotype was limited to young stages. The \u201cStripe\u201d phenotype (Fig.\u00a01, NE4001) often showed extremely biased segregation, e.g., only one plant in 25, and often was not stably inherited.Leaf shape. Many different kinds of abnormally shaped leaves were observed. \u201cShort Leaf\u201d and some \u201cWide leaf\u201d phenotypes were eventually reclassified as \u201cDwarf\u201d or \u201cSeverely dwarf\u201d phenotypes (Fig.\u00a01, NE8114). The \u201cShort leaf\u201d phenotype is similar to mutants defective in gibberellin biosynthesis or signal transduction. The line NE5022 could not develop a normal, flat leaf blade and eventually died (Fig.\u00a01). Several lines showed pleiotropic phenotypes of the shoot. The line NE8329 did not develop tillers, showed dwarfism and formed rolled or twisted leaves (Fig.\u00a01). Since these phenotypes are difficult to characterize exactly by only phenotype codes, additional remarks were presented in the comment column of the database. Frequency distribution of the leaf width mutants was biased toward the narrow type (Fig.\u00a01, NG0754), rather than the wide type.Culm shape. Dwarfism is the most abundant mutant phenotype, along with sterility. The \u201cSemi-dwarf\u201d condition is characterized by plant heights that are 70\u201380% of wild type values. \u201cDwarf\u201d describes plants with heights smaller than \u201cSemi-dwarf\u201d but larger than \u201cSeverely dwarf\u201d. Plants are classified \u201cSeverely dwarf\u201d when the plant height is smaller than 30\u00a0cm at maturity. Some dwarf phenotypes often co-segregated with other abnormal phenotypes such as \u201cFine leaf\u201d, \u201cWide leaf\u201d, \u201cSpiral leaf\u201d, or \u201cAbnormal panicle shape\u201d. The dwarf mutants accompanying other shoot\/panicle abnormalities are expected to be involved in hormonal signaling or synthesis pathways. Other culm phenotypes such as \u201cThick culm\u201d (Fig.\u00a01, NG9874) appeared infrequently.Spotted leaf\/lesion mimic. Various types of \u201cSpotted leaf\/lesion mimic\u201d phenotypes, e.g., small and scattered spots, large and dispersed spots, were observed. Heavy lesions caused the early death of leaves (Fig.\u00a01, NG0752). Mutants with large white lesions were also observed. Brown spots were more frequently observed than white ones.Tillering. In the tillering class, \u201cLow tillering\u201d mutants were abundant. The \u201cHigh tillering\u201d phenotype tends to co-segregate with the \u201cFine leaf\u201d phenotype. The \u201cLazy\u201d (Fig.\u00a01, NG0667) mutants have open and recumbent tillers. A quite unusual phenotype among \u201cHigh tillering\u201d lines is NG2552 that produced tillers reiteratively from upper internodes and failed to produce panicles.Heading date. In this category, lines whose heading date deviated more than 7\u00a0days from normal were classified as heading date mutants. This category includes \u201cEarly heading\u201d, \u201cLate heading\u201d, and \u201cNon-heading?\u201d mutants. A small number of lines segregated as \u201cNon-heading?\u201d mutants that did not form panicles even at harvest (five to 6\u00a0months after sowing). The number of \u201cEarly heading\u201d lines (1797) is a little larger than that of \u201cLate heading\u201d lines (1244).Spikelet. Each spikelet of rice is composed of two rudimentary glumes, two empty glumes and one floret comprised of one lemma, one palea and three kinds of floral organs (two lodicules, six stamens and one pistil). \u201cAbnormal hull\u201d refers to any glume abnormality and \u201cAbnormal floral organ\u201d refers to any abnormality in floral organs (e.g. an abnormal pistil). In the \u201cAbnormal hull\u201d class, there is a mutant that failed to close hulls after flower opening. On the other hand, mutants that could not open flowers due to underdeveloped stamens were often re-classified as \u201cCompletely sterile\u201d mutants. Another mutant produced an extra glume. Abnormal flower phenotypes showing shoots growing from floral organs as in Fig.\u00a01 (NC7672), were also detected at relatively low frequency.Panicle. The most frequently observed abnormal panicle phenotype was the precocious germination of seeds while still attached to the maturing panicle, or \u201cViviparous\u201d. As for panicle shape, \u201cDense panicle\u201d and \u201cShort panicle\u201d mutants (Fig.\u00a01, NE6778) were also frequently observed, and \u201cLong panicle\u201d and \u201cLax panicle\u201d were somewhat rare. Incomplete emergence of the panicle from the flag leaf sheath was categorized as \u201cNeck leaf\u201d.Sterility. \u201cSterile\u201d is the third most frequent mutant in this Tos17 insertion mutant population after low seed fertility and dwarf mutants. The \u201cSterile\u201d and \u201cLow fertility\u201d conditions correspond to lines with less than 2% and ca. 50% seed fertility, respectively.Seed. Only \u201cLarge grain\u201d, \u201cSmall grain\u201d, and \u201cSlender grain\u201d were distinguished in this category. The frequency of \u201cSmall grain\u201d was two and four times higher than the frequency of \u201cLarge grain\u201d and \u201cSlender grain\u201d, respectively. Among the \u201cOthers\u201d, white or dull kernel phenotypes were often observed.Table\u00a01Summary of phenotype dataClassPhenotypeID codeNCNDNENFNGTotal1GerminationLow germination rate13275311326100530034892GrowthLethal21353006892712341629Abnormal shoot31891722198233841787Weak411420434155639016053Leaf colorAlbino112643842302752541407Yellow129613918631771809Dark green 1329528535458691061Pale green 142704293722653951731Virescent1535278206131184834Stripe16464064132102384Zebra171215133929108Others18915232216854Leaf shapeWide leaf21382851421142Narrow leaf221653244322042511376Long leaf2333144529Short leaf2424258140Drooping leaf258140351231226Rolled leaf2631521532383342Spiral leaf27102355318109Brittle leaf\/culm28514405310122Abnormal lamina joint angle29142713142896Withering3011227025188147868Others31375446113933435Culm shapeSemi-dwarf416458226616339003661Dwarf428031550135188211235709Severely dwarf432484113551432171374Long culm44258217506883676Fine culm45116118Thick culm46312421462Others47114286Spotted leaf\/lesion mimicSpotted leaf\/lesion mimic5111521119726930210947TilleringHigh tillering611127252127111Low tillering624186786966094212822Lazy63701155380563748Heading dateEarly heading6535297699297651789Late heading661115612472151101244Non-heading?6722423218969SpikeletAbnormal hull71412683652111508Abnormal floral organ721212843111721110PanicleLong panicle8113193329Short panicle82517942692104752Lax panicle831816152332104Dense panicle844118976543264Viviparous85854732671421381105Shattering861326Neck leaf873851481024171Abnormal panicle shape882143265792543311SterilitySterile9144110268186588823825Low fertility9228773127935299126121254212SeedLarge grain101557833425195Small grain10247959614544427Slender grain1032028182114101Others10497911178831433473469Lots NC through NG were harvested in 1997 through 2001, respectively. Numbers of mutant lines with the corresponding phenotypes are listedFig.\u00a01Representative phenotypes of Tos17 insertional mutants. Phenotype and line designation are indicated on each photograph\nCorrelation between phenotypes\nTo understand how the 53 phenotypic descriptors are related, lines showing two or more abnormal phenotypes were selected, and a matrix with 53 rows and 53 columns of phenotypes with a number of lines showing each pair of phenotypes was devised. Values for the number of respective phenotypes were changed to \u201cx\u201d in the matrix, (\u201cx\u201d is ignored by the SOM program), and data were subjected to self-organizing map (SOM) analysis (Kohonen 1995). The SOM algorithm is used for visualization of multidimensional complex data using an unsupervised learning method based on a grid of artificial neurons. The 53-dimensional correlation data of phenotypes was reflected in a two-dimensional map (Fig.\u00a02).\nFig.\u00a02Self-organizing map for correlation of phenotypes. Lines that showed two or more abnormal phenotypes were analyzed by SOM. Distances between phenotypes indicate the magnitude of correlation of phenotypes. Col_Oth., Color Others; Lg_lf, Long leaf; L_joint, Abnormal lamina joint angle; Lg_pa, Long panicle; Ab_floral_organ, Abnormal floral organ; Sl_grain, Slender grain; Thick_cm, Thick culm. Grayscale levels of each node represents the distance between references\nTopologies (not distances) of phenotypes on the SOM coordinates coincide with the correlation between phenotypes. Distance between two phenotypes is indicated by grayscale color on the SOM. For example, assume that phenotype A, B, and C appeared in 5000, 50, and 10 lines, respectively. Seven lines showed phenotype A and C. Three-lines showed phenotype B and C. The color between phenotype A and C should be darker than the color between phenotype B and C, because the line number of phenotype A lines is much greater than that of phenotype B lines, although the phenotype correlation of C with A is stronger than that with B. It is difficult to explain multidimensional data completely on a two dimensional map, but a SOM map indicates generally that neighboring phenotypes have relatively tight correlations. The SOM analysis in Fig.\u00a02 shows that there are many pairs of phenotypes that are apparently correlated. That is, among 53 phenotypes, several phenotypes have a high probability to emerge simultaneously with other specific phenotypes. This phenotypic correlation could be caused by pleitropic expression of a single gene, or could reflect developmental causality of the two abnormalities. For example, \u201cLong leaf\u201d and \u201cLong panicle\u201d (lg_lf, lg_pa) located in the upper left corner on the SOM map in Fig.\u00a02 have a very strong correlation. This result suggests that the gene involved in leaf length also affects panicle length. On the contrary, long leaf and dwarfism, at the opposite corner of the map, do not have any correlation. Both dwarf and semi-dwarf phenotypes have a relatively strong correlation with sterile and low fertile phenotypes. Since this correlation is not so strong, a part of \u201cDwarf\u201d and \u201cSemi-dwarf\u201d mutants do not affect seed fertility, but other \u201cDwarf\u201d and \u201cSemi-dwarf\u201d mutants may be regulated by genes associated with some housekeeping functions.\nCorrelations between mutant phenotype and Tos17 insertion\nIf allelic insertion lines show the same phenotype, it is highly probable that the mutant phenotype is caused by disruption of the target gene by a Tos17 insertion. We searched for genes that were disrupted by Tos17 in at least two lines. A total of 391 genes were detected based on the Rice Annotation Project (Ohyanagi et al. 2006). Table\u00a02 is a partial list of such loci and their mutant phenotypes. For example, five lines have insertions of Tos17 in the Os06g027500 gene (Fig.\u00a03A), of which two lines have the Tos17 insertions in exons and show an early heading phenotype. On the other hand, the other three lines have Tos17 insertions in introns and are not early heading. (see http:\/\/tos.nias.affrc.go.jp\/). The Os06g027500 gene is Hd1, a key gene for determining heading date (Yano et al. 2000). Another example is the magnesium chelatase subunit gene, Os07g0656500 in Fig.\u00a03B (Jung et al. 2003). Tos17 insertion into this gene was correlated with an albino phenotype, because mutants defective in this gene lack active chlorophyll and eventually become albino. All correlations between phenotypes and disrupted genes by Tos17 insertion are displayed on the mutant panel database website (http:\/\/tos.nias.affrc.go.jp\/).\nTable\u00a02List of loci that have Tos17 insertions in exons in at least two linesLocus nameDescriptionPhenotypesOs01g0113200LRK14Pale green leaf, low fertilityOs01g0113300Receptor-like kinase ARK1ASDwarf, spotted leaf\/lesion mimicOs01g0147800Protein of unknown function DUF547 domain containing proteinPale green leaf, semi-dwarf, long culm, short panicle, sterile, low fertileOs01g068590065\u00a0kD Microtubule associated proteinNarrow leaf, semi-dwarfOs02g05526008-Oxoguanine DNA glycosylaseLethalOs04g0464200Betaine-aldehyde dehydrogenase (EC 1.2.1.8) (BADH)Low fertilityOs04g0680400Allantoinase (EC 3.5.2.5)Dwarf, low fertilityOs05g0318600Protein kinase domain containing proteinNarrow leafOs05g0548900Phosphoethanolamine methyltransferaseEarly headingOs05g0552400Zn-finger, RING domain containing proteinEarly headingOs06g01768002OG-Fe(II) oxygenase domain containing proteinDark green leaf, dwarf, severely dwarfOs06g0275000Hd1Dwarf, early headingOs06g0680500Glutamate receptor 3.1 precursor (Ligand-gated ion channel 3.1) (AtGLR2)Low fertilityOs07g0197100HexokinaseDwarf, sterileOs07g0646500SWIM Zn-finger domain containing proteinLate headingOs07g0656500Protoporphyrin IX Mg-chelatase subunit precursorLethal, albino, dwarfOs09g0278300Phosphatidylinositol-4-phosphate 5-kinase family proteinSemi-dwarf, dwarf, low tillering, sterile, low fertilityOs10g0567100Chlorophyll b synthase (Fragment)Dark green leaf, pale green leaf, withering, semi-dwarf, severely dwarf, early heading, late heading, low fertilityOs12g0127600WRKY transcription factor 57Sterile, low fertilityOs12g0566000HCO3-Transporter domain containing proteinSeverely dwarfOs12g0572500Protein of unknown function XH domain containing proteinEarly headingLocus name and description are from RAP dataFig.\u00a03Examples of genes that have insertions of Tos17. (A) Os06g0275000, Hd1 gene. (B) Os07g0656500, Magnesium chelatase subunit gene. The structure and insertion points of these genes are drawn on demand from our web site (http:\/\/tos.nias.affrc.go.jp\/). Coding regions are indicated with broad lines. Positions of Tos17 insertions are indicated by flags. The direction of the flag is the direction of the Tos17 insertion. Detailed descriptions of insertion points, sequence names, accession numbers, and line names will appear on the web page. When a sequence name is clicked, the figure will be re-drawn with a flag labeling the sequence name to distinguish closely inserted Tos17s\nA large-scale phenotypic characterization of an M2 population generated by Tos17 retrotransposition has revealed that this population contains a large number of mutants covering many easily scored phenotypes. In addition, linkage between the mutant phenotype and a specific Tos17 insertion facilitates greatly the isolation of the causal genes and the elucidation of the gene functions.\nDiscussion\nThree-years of collaboration among seven laboratories has led to a large-scale phenotypic characterization of about 50,000 M2 plants generated by Tos17 retrotransposition. We have examined 53 kinds of abnormal phenotypes in rice from the seedling to harvest stages that are easily evaluated in the field. This project has revealed that this population contains a large number of mutants covering a wide range of phenotypes. Although several mutant phenotypes may be environment-sensitive and their expression may be unstable, most of the mutant phenotypes were stable. We also deposited additional information about certain phenotypes in a comment field within the phenotype description table, if this information was provided. A text search box on the phenotype list web page is envisioned to facilitate use of the resource.\nBecause M2 plants were observed, inserted Tos17s segregate among individuals. An average of 10 new copies of Tos17 are inserted into each regenerated plant. If the phenotype co-segregates with an insertion of Tos17, the phenotype is likely to be caused by disruption of the target gene. Of course, there are lines whose mutant phenotypes are not correlated with Tos17 insertion. These mutants might be caused by insertions of other native transposons, by chromosomal aberrations, or by other mutations during tissue culture. In this case, their causal genes could be isolated by usual positional cloning methods.\nThere were some difficulties in clustering phenotypes using standard relational algorithms, because each of the 53 phenotypes has as many as 52 kinds of frequencies against other phenotypes, respectively. SOM analysis is a kind of clustering method suitable for such multi-dimensional non-linear data (Kohonen 1995). SOM analysis confirmed some associations due to hormonal or developmental constraint, but some additional intriguing correlations were also detected, e.g., between leaf color\/shape and heading date, between dwarfism and sterility. In Fig.\u00a02, the \u201cLong leaf\u201d phenotype is strongly correlated with the \u201cLong panicle\u201d phenotype. The \u201cWeak\u201d phenotype at the seedling stage has a relatively strong correlation with \u201cLethal\u201d, \u201cAbnormal shoot\u201d, \u201cPale green\u201d Leaf and \u201cSpotted leaf\/lesion mimic\u201d phenotypes, indicating that the \u201cWeak\u201d plants at the seedling stage likely show \u201cLethal\u201d, \u201cAbnormal shoot\u201d, \u201cPale green\u201d and\/or \u201cSpotted leaf\/lesion mimic\u201d phenotypes at later stages. \u201cDwarf\u201d, \u201cSemi-dwarf\u201d, \u201cSterile\u201d, \u201cLow fertile\u201d and \u201cLow tillering\u201d phenotypes showed relatively high correlations. The SOM map is quite useful for the overview of phenotype correlations. This method of analysis will be useful not only for considering the major and side-effects of gene disruption but also for re-categorization of phenotypes. The advantage of SOM analysis is the ability to analyze many different kinds of data simultaneously. Currently, metabolome data have been obtained from Arabidopsis (Hirai et al. 2004), tomato (Schauer et al. 2006) and other plants. It is reasonable to predict that the combinatorial analysis of phenotype and metabolome data of rice will reveal correlations between metabolic pathways and phenotypes without additional genetic information.\nPhenotype databases of rice have been developed in many countries, e.g., Oryzabase (Kurata and Yamazaki 2006), RMD (Zhang et al. 2006), and IRRS (Wu et al. 2005). Our system includes both phenotypic and insertion data on the rice genome in a relational database. The number of photographs on file is more than 58,000, and the number of phenotype description records is more than 158,000. Our database structure enables direct access of Perl script to the database and the extraction of many kinds of correlations. Flanking sequences of insertion points and phenotypes of the insertion lines provide a provisional assignment of the function of a disrupted gene. We have already sequenced more than 25,000 flanking sequences from 20% of the insertion lines, and the number of flanking sequences is still increasing. Of the 27,448 total annotated loci based on RAP1\/IRGSP Build3, 391 loci have more than two insertions in exons. When two or more lines have Tos17 insertions in a common gene, they usually exhibited similar phenotypes. These data are a strong indication of the function of a disrupted gene. However, several phenotypes such as \u201cDwarf\u201d and \u201cLow fertile\u201d were observed in many lines. It might be difficult to assign a gene function based solely on correlation for these frequently observed phenotypes, and a complementation test would be required to confirm the correlation. If flanking sequence data from all insertion lines can be obtained, this correlation will be more useful for the annotation of genes. We are continuing to sequence the flanking regions of all our mutant lines.\nIn this study, we collected a large amount of phenotypic data in seven fields under natural conditions. If phenotypes are observed under other conditions such as drought or temperature stress conditions or under pathogen pressure, phenotypic description of this population would be much more enriched. In addition, the present study evaluated only a limited number of traits easily scored on above-ground organs. Thus, other traits such as roots and seed storage composition remain to be investigated in the future. Expansion of this study to include new traits would enable investigators to find new correlations with disrupted genes. Furthermore, integration of phenotype data with those of microarray experiments, metabolic profiling, and other approaches will be a powerful tool for revealing new aspects of plant physiology.\nAll phenotype and flanking sequence data can be obtained via http:\/\/tos.nias.affrc.go.jp\/. At this site, all annotated rice genes and locations of Tos17 insertions are shown on a clickable chromosome map. Details containing illustrated gene structures with insertion points, phenotypes of corresponding lines, nucleotide sequences flanking the disrupted region, candidate primer sequences for segregation analysis, and annotation from RAP data corresponding to the selected position are displayed. Mutant seeds are available for scientific use from the Genome Resource Center at NIAS (http:\/\/www.rgrc.dna.affrc.go.jp\/).","keyphrases":["phenotyping","retrotransposon","database","oryza sativa","insertion mutagenesis"],"prmu":["P","P","P","P","M"]}
{"id":"Pituitary-3-1-2045692","title":"Acromegaly caused by growth hormone-releasing hormone-producing tumors: long-term observational studies in three patients\n","text":"We report on three newly diagnosed patients with extracranial ectopic GHRH-associated acromegaly with long-term follow-up after surgery of the primary tumor. One patient with a pancreatic tumor and two parathyroid adenomas was the index case of a large kindred of MEN-I syndrome. The other two patients had a large bronchial carcinoid. The first patient is still in remission now almost 22 years after surgery. In the two other patients GHRH did not normalize completely after surgery and they are now treated with slow-release octreotide. IGF-I normalized in all patients. During medical treatment basal GH secretion remained (slightly) elevated and secretory regularity was decreased in 24 h blood sampling studies. We did not observe development of tachyphylaxis towards the drug or radiological evidence of (growing) metastases. We propose life-long suppressive therapy with somatostatin analogs in cases with persisting elevated serum GHRH concentrations after removal of the primary tumor. Independent parameters of residual disease are elevated basal (nonpulsatile) GH secretion and decreased GH secretory regularity.\nIntroduction\nAcromegaly caused by ectopic extracranial growth hormone-releasing hormone (GHRH) secretion is a very rare disorder occurring probably in less than 1 % of the acromegalic patients [1]. The majority of the GHRH-secreting tumors are bronchial carcinoids. Other GHRH-secreting tumors in decreasing order of occurrence are pancreatic adenomas, gastro-intestinal tumors, thymic tumors, and tumors associated with the MEN-I syndrome [2].\nHere we report our clinical experience in three patients with the ectopic GHRH-syndrome derived from a population of over 200 acromegalic patients, diagnosed and treated at the Leiden University Medical Center from 1976 till 2002. Long-term follow-up studies in these patients and results of medical therapy with somatostatin analogs are scarce. The purpose of this report is to expand our knowledge of this rare clinical entity. In addition, we report detailed results of diurnal GH secretion, before and after removal of the GHRH-producing source in order to investigate whether pulsatile and basal GH secretion due to GHRH overproduction differs from that of a primary pituitary somatotropinoma.\nMethods\nBasal concentrations of hormones, including prolactin, free thyroxin, triiodothyronine, cortisol, testosterone, estradiol, progesterone, IGF-I, and IGFBP3 were measured. In addition the following tests were performed: Oral glucose loading test (75\u00a0g), TRH test (200\u00a0\u03bcg i.v. ), and a GHRH test (50\u00a0\u03bcg i.v. ) and the following hormones were measured: glucose tolerance test: GH, insulin and glucose at 0, 30, 60, 90 and 120\u00a0min, TRH test: TSH, prolactin and GH at \u221215, 0, 15, 20, 30 45, 60, 90 and 120\u00a0min; GHRH test: GH and prolactin at 0, 20, 30, 45, 60, and 90\u00a0min. For the 24-h GH secretion profile the patients were hospitalized, and an indwelling i.v. cannula was inserted in a forearm vein, and blood samples were withdrawn at 10-min intervals. The patients were free to move around, but not to sleep during daytime. Meals were served at 0800, 1230 and 1730\u00a0h. Lights were turned off between 2200 and 2400\u00a0h.\nAssays\nPlasma GH was measured with a sensitive time-resolved fluoro-immunoassay (Wallac Oy, Turku, Finland). The assay is specific for the 22\u00a0kDa GH. The standard was biosynthetic recombinant human GH (Genotropin, Pharmacia & Upjohn, Uppsala, Sweden), and was calibrated against the WHO First International Reference Preparation 80\/505 (to convert \u03bcg\/l to mU\/l multiply by 2.6). The limit of detection of this assay (defined as the value 2\u00a0SD above the mean value of the zero standard) was 0.01\u00a0mU\/l (0.0038\u00a0ng\/ml). The intraassay coefficient of variation varied between 1.6% and 8.4% in the range from 0.01\u00a0\u03bcg\/l to 18\u00a0\u03bcg\/l and interassay coefficient of variation was 2.0\u20139.0% in the same range.\nTotal IGF-I was determined by RIA (Incstar, Stillwater, MN) after extraction and purification on ODS-silica columns. The intraassay coefficient of variation was less than 11%. The detection limit was 1.5\u00a0nmol\/l. Age-related normal data were determined in the same laboratory. The measurement of IGFBP3 was performed by RIA (Nichols Institute Diagnostics, San Juan Capistrano, CA). The limit of detection of this assay was 0.08\u00a0mg\/l, and the interassay coefficient variation was below 6.8%.\nDeconvolution analysis\nA multiparameter deconvolution technique was used to estimate relevant measures of GH secretion from the 24-h serum GH concentration profiles, as described previously [3]. Initial estimates of basal GH secretion rate were calculated to approximate the lowest 5% of all plasma GH concentrations in the time series. Peak detection entailed application of 95% statistical confidence intervals to two thirds of all GH secretory peaks considered jointly and individual 95% statistical confidence intervals to the remaining one third smaller pulses, as validated in simulations [4]. The following four secretory and clearance measures of interest were estimated: (1) the number and locations of secretory events; (2) the amplitudes of secretory bursts; (3) the durations of randomly dispersed GH secretory bursts; and (4) the endogenous single component subject specific plasma half-life of GH. It was assumed the GH distribution volume and half-life were time and concentration invariant. The following parameters were calculated: Half-duration of secretory bursts (duration of the secretory burst at half-maximal amplitude), hormone half-life, burst frequency, amplitude of the secretory burst (maximal secretory rate attained within a burst), mass secreted per burst, basal secretion rate, pulsatile secretion rate (product of burst frequency and mean burst mass) and total secretion (sum of basal and pulsatile).\nApproximate entropy\nThe univariate approximate entropy (ApEn) statistic was developed to quantify the degree of irregularity, or disorderliness, of a time series [5]. Technically, ApEn quantifies the summed logarithmic likelihood that templates (of length m) of patterns in the data that are similar (within r), remain similar (within the same tolerance r) on next incremental comparison and has been formally defined elsewhere [6]. The ApEn calculation provides a single non-negative number, which is an ensemble estimate of relative process randomness, wherein larger ApEn values denote greater irregularity, as observed for ACTH in Cushing\u2019s disease, GH in acromegaly, and PRL in prolactinomas [7\u20139]. In the present analysis, we calculated ApEn with r\u00a0=\u00a020% of the SD of the individual time-series and m\u00a0=\u00a01. This choice of parameters affords sensitive, valid and statistically well-replicated ApEn metrics for assessing hormone time-series of this length. ApEn results are reported as absolute values or as the ratio of the absolute value to that of the mean of 1,000 randomly shuffled data series. Ratio values that approach 1.0 thus denote mean empirical randomness.\nCopulsatility\nCopulsatility between the GHRH and GH time-series was quantified by the hypergeometric (joint binomial) distribution [10]. This program calculates the probability that hormone pulses in time-series occur randomly. We used a time-window of 20\u00a0min, with GHRH as leading hormone series. Because details of the secretion characteristics of GHRH are not well established, we estimated significant pulses in both time-series with Cluster, which is largely model-free [11].\nClinical findings at diagnosis and initial treatment\nCase 1. A 50-year-old male was referred in 1982 because of recurrent kidney stones, hypercalciuria (24\u00a0h urinary calcium excretion between 14.5\u00a0mmol and 16.3\u00a0mmol, normal upper value 6\u00a0mmol\/24\u00a0h) and hypercalcaemia (serum Ca between 2.86\u00a0mmol\/l and 2.95\u00a0mmol\/l, normal values between 2.25\u00a0mmol\/l and 2.60\u00a0mmol\/l). The referring internist suspected the patient of having mild acromegaly, because of the coarse facial features. The diagnosis primary hyperparathyroidism was confirmed and the patient underwent parathyroid surgery, and two large adenomas were removed, after which the patient became normocalcaemic until now. The patient had noted increase in size of his feet and hands since several years, but otherwise he had no complaints. Glucose loading decreased GH from 12\u00a0mU\/l to below 0.5\u00a0mU\/l (normal value during glucose suppression is below 2.5\u00a0mU\/l with RIA). Intravenous administration of 200\u00a0\u03bcg TRH, however, increased GH from 3.5\u00a0mU\/l to 58.0\u00a0mU\/l and PRL increased from 7.0\u00a0\u03bcg\/l to 13\u00a0\u03bcg\/l. CT scanning of the pituitary gland with the first generation CT-scanner (in 1983) did not show abnormalities, and CT scanning of the thorax and abdomen also failed to show the presence of a tumor. Because of progressive complaints of fatigue after cure for hyperparathyroidism and strong clinical suspicion of acromegaly, the patient underwent transsphenoidal pituitary exploration. At surgery a small suspect lesion was removed with part of the surrounding pituitary gland tissue. Histology of the lesion was compatible with somatotrope hyperplasia. After the patient had recovered from surgery, CT, MRI and arteriographic studies were repeated, and serum samples were sent to St. Bartholomew\u2019s Hospital, London, UK (Dr L.H. Rees) for GHRH measurement. The abdominal CT-scan showed a 4\u00a0cm mass in the middle section of the pancreas (Fig.\u00a01) and the mass was also visible with selective arteriography of the superior mesenterial artery (Fig.\u00a01). The fasting GHRH concentration amounted to 3,810\u00a0pg\/ml (normal range 10\u201360\u00a0pg\/ml). GHRH concentration in the arterial supply to the tumor was 12,470\u00a0pg\/ml, and in the venous tumor outflow 31,120\u00a0pg\/ml, while in the systemic venous system the concentration was 8,900\u00a0pg\/ml. After removal of the pancreatic tumor, peripheral GHRH concentration decreased to normal values of 16\u201333\u00a0pg\/ml. In retrospect, the first abdominal CT-scan, 2\u00a0years before, already showed the pancreatic tumor with a similar size.\nFig.\u00a01Upper panel shows the abdominal CT at the level of the pancreas and the lower panel the selective arteriography of the superior mesenterial artery\nThe patient was the index case of a large kindred affected by MEN-I syndrome. Later, a gene mutation was revealed in exon 2 of chromosome 11q13. During long-term follow-up GH and IGF-I concentrations remained normal.\nSix years later, in 1988, the patient developed diabetes mellitus, initially treated with oral hypoglycemic drugs and with insulin from 1992 onwards. The patient also had mild bilateral nodular adrenal hyperplasia since 1988, with no evidence of growth during the last recent 10\u00a0years. No excess of adrenal (cortex and medulla) hormones or precursors was demonstrable during follow-up. During the last 3\u00a0years the plasma level of pancreatic polypeptide (PP) increased to 350\u00a0nmol\/l (normal\u00a0<\u00a0100\u00a0nmol\/l). Repeat CT and MRI scanning of the upper abdomen failed to reveal the presence of a pancreatic tumor thus far.\nRandom serum GH during follow-up ranged from 0.16\u00a0mU\/l to 1.81\u00a0mU\/l and IGF-I ranged from 12\u00a0nmol\/l to 17\u00a0nmol\/l from 1994 till now (see Fig.\u00a02). All these values are perfectly normal for his age and gender. The last serum GHRH measurement in 2006 was normal with 32\u00a0pg\/ml.\nFig.\u00a02Follow-up of GH and IGF-I concentration in patient 1 after removal of the pancreatic source of GHRH. Fasting serum GH concentrations are depicted as circles, and IGF-I as triangles\nCase 2. A 27-year-old acromegalic female patient was referred in 1993 to our center for octreotide treatment. She had a 5-year history of hyperhydrosis, fatigue, paraesthesias, and acral enlargement. After delivery of a healthy daughter in 1992 she had persisting amenorrhea and galactorrhea. Physical examination revealed mild, although characteristic features of acromegaly. Serum GH concentration was elevated at 99\u00a0mU\/l and decreased insufficiently to 55\u00a0mU\/l following oral glucose administration. IGF-I concentration amounted to 86\u00a0nmol\/l (normal upper level for her age 32\u00a0nmol\/l) Thyroid and adrenal functions were normal, but prolactin concentration was elevated to 20\u00a0\u03bcg\/l (normal upper limit for females 12\u00a0\u03bcg\/l). Other investigations performed (TRH test, GHRH test, and the i.v. octreotide test) are summarized in Fig.\u00a03. TRH bolus injection (200\u00a0\u03bcg iv) caused a \u223c8-fold increase of GH and a 3.5-fold increase of PRL. Intravenous injection of 50\u00a0\u03bcg GHRH (1-40) caused a decrease of GH while PRL concentrations remained unchanged. Octreotide (50\u00a0\u03bcg i.v.) caused a 98% inhibition of serum GH but no effect on PRL. MRI scanning of the pituitary gland showed global enlargement without signs of an adenoma (Fig.\u00a04). Further investigations revealed a large tumor in the right lower lobe of the lung (Fig.\u00a05). The tumor and the pituitary gland were positive on 111In-labeled-octreotide scintigraphy (Fig.\u00a06). The plasma GHRH concentration was increased 50-fold by 2,519\u00a0pg\/ml (normal values <50\u00a0pg\/ml).\nFig.\u00a03Dynamic GH (circles) and PRL (triangles) tests in two patients with a GHRH-secreting lung carcinoid before (closed symbols) and after surgery (open symbols). Note the GH increase after TRH (200\u00a0\u03bcg) administration in both patients, the decrease in serum GH concentration in patient 2 after GHRH (50\u00a0\u03bcg), and the moderate GH decrease after i.v. octreotide (50\u00a0\u03bcg)Fig.\u00a04MRI scans of the pituitary gland of patient 2 (left panel) and of patient 3 (right panel) before treatmentFig.\u00a05Chest X-rays of patient 2 (left panel) and patient 3 (right panel), showing the large bronchial carcinoidFig.\u00a06Octreoscans of the patients 2 and 3. In the female patient (left panel) the tumor is seen in the right lower lobe of the lung and also the positive staining of the pituitary gland. The male patient (right panel) had a large tumor in the left lung, without pituitary staining\nUnder the diagnosis of ectopic GHRH-producing lung tumor (carcinoid) the patient underwent thoracotomy and the tumor was resected completely. The plasma GHRH disappearance profile after removal of the tumor is shown in Fig.\u00a07, but the GHRH concentration did not normalize. GHRH concentrations in simultaneously withdrawn blood samples from the arterial supply to the tumor, venous tumor outflow and the systemic venous system were 1,890, 2,180, and 1,680\u00a0pg\/ml, respectively.\nFig.\u00a07Decrease in serum GHRH concentration in two patients before and immediately following removal of the lung tumor. Patient 2 is shown by triangles and patient 3 by circles. Note that the GHRH concentration is shown on a logarithmic scale. The GHRH concentration did not normalize in the patients after 2 and 3\u00a0days, respectively. Upper normal GHRH is 50\u00a0pg\/ml\nTwo weeks after surgery, endocrine investigations revealed a persisting paradoxical increase (9-fold) of GH to TRH, insufficient GH suppression after oral glucose loading (52\u201316\u00a0mU\/l) and a GH increase after GHRH injection. Octreotide caused a 98% decrease of GH from 41\u00a0mU\/l to 0.9\u00a0mU\/l (see Fig.\u00a03). Treatment with octreotide was started in 1994, about 8\u00a0months after surgery, because of persisting elevated GHRH concentration (between 360\u00a0pg\/ml and 488\u00a0pg\/ml), increased GH( 31\u201342\u00a0mU\/l), insufficient suppression by glucose loading (minimum GH concentration 9.13\u00a0mU\/l), an increased IGF-I concentration ( 40\u201343\u00a0nmol\/l), and lesions in the liver on repeat CT scans, suspect for metastases (one lesion in segments 2 and 8, and two lesions in segment 7).\nCase 3. A 27-year-old male was referred in 1997 to our center. He had a 9-year history of hyperhydrosis, acral enlargement and headaches. Examination revealed moderately advanced features of acromegaly. The circulating GH concentration was elevated at 110\u00a0mU\/l and decreased insufficiently to 52\u00a0mU\/l following glucose administration, and IGF-I was elevated to 63\u00a0nmol\/l (normal upper level at this age 32\u00a0nmol\/l). Thyroid and adrenal functions were normal, but prolactin concentration was elevated to 21\u00a0\u03bcg\/l (normal upper limit for males 6\u00a0\u03bcg\/l). Other investigations performed (TRH test and the i.v. octreotide test) are summarized in Fig.\u00a03. TRH bolus injection (200\u00a0\u03bcg i.v.) caused a \u223c8-fold increase of GH and a 2-fold increase of PRL. Octreotide (50\u00a0\u03bcg i.v) caused a 94% inhibition of GH (Fig.\u00a03). MRI scanning of the pituitary gland showed a macroadenoma II AE (Hardy classification, modified by Wilson [12, 13] see Fig.\u00a04). He was treated for 6\u00a0months with sc octreotide (100\u00a0\u03bcg tid). The size of the adenoma decreased slightly, but because of the moderate clinical response to medical treatment and failure of normalization of GH and IGF-I, pituitary surgery was advised. One day before surgery a chest X-ray was taken, showing a large parahilar lesion (Fig.\u00a05). CT scanning of this lesion was classified by the consultant pulmonologist and radiologist as a bronchial cyst and apparently unrelated to acromegaly. During pituitary surgery the adenoma was removed completely, but the neurosurgeon remarked that the consistency of the adenoma was firmer than normal. Since we suspected that the lung tumor was potentially a GHRH-producing source, postoperative investigations were focused on this possibility. After surgery, MRI scanning of the pituitary region did not show residual tumor, GH concentrations decreased considerably and PRL concentrations became normal. GH dynamic tests did not completely normalize: GH increased after TRH injection from 4.63\u00a0mU\/l to 22.1\u00a0mU\/l and after glucose loading GH decreased from 4.33\u00a0mU\/l to 2.56\u00a0mU\/l (normal value below 1\u00a0mU\/l). During an i.v. octreotide test GH decreased from 6.21\u00a0mU\/l to 0.85\u00a0mU\/l, but treatment with this drug was not reinstituted. On 111In-labelled-octreotide scanning the tumor was positive (see Fig.\u00a06).\nUnder the diagnosis of ectopic GHRH-producing lung tumor (carcinoid) the patient underwent thoracotomy and the tumor was removed completely. GHRH concentrations in the arterial supply to the tumor were 48,290\u00a0pg\/ml, in the venous outflow 94,000\u00a0pg\/ml and in the systemic venous system 49,000\u00a0pg\/ml. After removal of the tumor GHRH concentrations remained slightly elevated, as shown in Fig.\u00a07.\nHistopathological studies\nPituitary gland\nThe removed part of the anterior pituitary gland of patient 1 consisted of hyperplastic cells, immunostaining positively for GH. The removed tissue of the third patient consisted of a mixture of hyperplasia and adenoma formation. The cells stained positively for both GH and PRL.\nGHRH-producing tumors\nPatient 1. The pancreatic tumor had a diameter of 5\u00a0cm. Amorphous material was present between the cells, staining as amyloid. On electronmicroscopy, the cells contained neurosecretory granules with a diameter between 100\u00a0nm and 200\u00a0nm. The tumor stained positively, but sparsely for somatostatin, insulin and glucagon and negatively for cytokeratine, vimentine, neurofilaments, desmine and GH. In the removed part of the pancreas three additional small adenomas with identical staining characteristics were present.\nPatient 2. The diameter of the removed lung tumor was 5\u00a0cm, and contained centrally calcified material. The cells were layered in nests, slightly polymorphic, but without mitotic figures. The tumor cells stained positively for keratine, vimentin, synaptophysin, SCCL (N-CAM), leu 7, and chromogranin and negatively for calcitonin, GH, pancreatic polypeptide, insulin, prolactin, somatostatin, gastrin, ACTH, CEA, and neurofilaments.\nPatient 3. The dimensions of the tumor were 8\u00a0\u00d7\u00a07\u00d77\u00a0cm3. The tumor showed clear proliferation of neuroendocrine cells with three mitotic figures per high power field, staining positively for NSE, CD56, and synaptophysin and negatively for keratine, chromogranin, serotonin, somatostatin, prolactin, insulin, glucagons, gastrin, ACTH, GH, and insulin.\nSomatostatin analog therapy\nAfter removal of the lung carcinoid, patients 2 and 3 received long-term treatment with octreotide, because GHRH was not normalized. Patient 2 received the medication via chronic sc infusion (300\u00a0\u03bcg\/24\u00a0h) till the end of 1998. Her complaints quickly disappeared and the menstrual cycle was restored. GH and IGF-I concentrations normalized and are detailed in the left panel of Fig.\u00a09. The size of the pituitary gland decreased markedly (Fig.\u00a08). PRL concentration also normalized from 15.2\u00a0\u00b1\u00a00.2\u00a0\u03bcg\/l to 6.8\u00a0\u00b1\u00a00.2\u00a0\u03bcg\/l. From 1999 onwards the medication was changed to octreotide long-acting repeatable (Sandostatin LAR), 20\u00a0mg in 4-weekly i.m. injections. Growth hormone and IGF-I concentrations remained unchanged (see Fig.\u00a09). During chronic treatment with the short-acting octreotide formulation treatment was withheld several times for GHRH measurement. Invariably, the concentration was (slightly) elevated (range 116\u2013363\u00a0ng\/ml) so that in combination with the radiological suspicion for liver metastases, treatment is continued until now. During treatment with octreotide long-acting repeatable (Sandostatin LAR) GHRH concentration ranged from 63\u00a0ng\/ml to 108\u00a0ng\/ml. Repeat CT scanning of the liver during the successive years demonstrated the stabilization of size and number of lesions until now.\nFig.\u00a08MRI of the pituitary gland of patient 2 during therapy with octreotide. These pictures were taken after 12\u00a0months treatment with 300\u00a0\u03bcg octreotide given as a continuous subcutaneous infusionFig.\u00a09GH (circles) and IGF-I (triangles) concentrations during long-term treatment with octreotide. Patient 3 received only the long-acting repeatable form, but patient 2 was treated initially with chronic sc octreotide infusion. The time of change into the slow-release formulation is indicated by the arrow. Normal values for IGF-I for this age: <32\u00a0nmol\/l. Normal value for random GH\u00a0<\u00a05\u00a0mU\/l\nAfter removal of the carcinoid tumor in patient 3, GHRH, GH, and IGF-I concentrations remained elevated, and therefore he was also treated with the long-acting repeatable octreotide (20\u00a0mg\/4\u00a0weeks). GH and IGF-I concentrations are shown in Fig.\u00a09, right panel, showing clinically normal values. Repeat investigations with CT, 111In-labeled octreotide and 131I-MIBG however, did not reveal suspect (liver) metastases. At the end of 2001, GHRH was elevated to 1,725\u00a0ng\/ml, so that thereafter the dose of Sandostatin was increased to 30\u00a0mg at 4-weekly intervals, which normalized IGF-I concentrations. Last year the patient stopped medication. Subsequently, IGF-I levels increased, as shown in Fig.\u00a09. GHRH concentration became larger than 2,000\u00a0pg\/ml. Detailed localizing studies with octreotide and CT revealed a small metastasis in the superior anterior mediastinum for which surgery is scheduled.\nFig.\u00a010Serum GH concentrations obtained by 10\u00a0min blood sampling for 24\u00a0h. Patient 2 was studied before therapy and after surgical removal of the lung tumor. Note that GH concentration decreased more than 10-fold and that the secretion pattern became more regular, but basal GH concentration remained slightly elevated. The left lower panel represent the profile of patient 3 after pituitary surgery, but before removal of the carcinoid tumor. Nadir values were clearly increased. After thoracic surgery and under octreotide treatment GH secretion pattern visually normalized\nGH secretory profiles\nDetailed GH secretory profiles were obtained before removal of the GHRH-producing bronchial carcinoids (Fig.\u00a010). The secretory patterns were irregular, showing increased burst frequency and increased basal concentrations. The GH secretory parameters as estimated by multiparameter deconvolution are listed in Table\u00a01 with normal values obtained in healthy adults of comparable age. The distinct and persisting abnormality in both patients after removal of the carcinoid and while on octreotide treatment was the increased basal (nonpulsatile) GH secretion.\nTable\u00a01Deconvolution of the 24\u00a0hour serum GH profiles in patients with ectopic GHRH syndrome and controlsPatient 2 before surg.Patient 2 after surg.Female controlsPatient 3 before surg. Patient 3 after surg. Male controlsJaffe\u2019s patientVance\u2019s patientPulse frequency (no\/24\u00a0h)301617 (14\u201321)302412 (7\u201314)3021Half-life (min)15.515.912.9 (12.0\u201315.5)15.615.717.5 (15.2\u201319.7)24.616.2Pulse half-duration (min)20.932.927.7 (22.7\u201329.8)25.323.225.3 (19.5\u201334.4)30.541.3Pulse height (mU\/l \/min)4.920.4420.387 (0.198\u20130.881)0.2270.1600.141 (0.087\u20130.422)6.01.87Pulse mass (mU\/l)10114.410.2 (7.3\u201318.5)5.663.684.44 (2.42\u20139.55)18176.5Basal secretion(mU\/l \/24\u00a0h)2,26130.29.1 (5.5\u201317.6)18.014.93.4(1.4\u20135.3)3,9004,60Pulsatile secretion (mU\/l \/24\u00a0h)3,047230173 (122\u2013312)1708843.5 (17.8\u2013103)5,4001,600Total secretion(mU\/l \/24\u00a0h)5,309260182 (132\u2013325)18810347 (19.7\u2013107)9,3002,060Blood samples were taken at 10-min intervals for 24-h and analyzed by multiparameter deconvolution. The female patient (no. 2) was studied before surgical removal of the GHRH-secreting bronchus carcinoid and repeat sampling study was done after thoracic surgery under octreotide LAR. The male patient (no. 3) was studied first after adenomectomy of the pituitary tumor, but before thoracic surgery. The second sampling study was performed after removal of the bronchial carcinoid during octreotide-LAR treatment. The serum profiles of the patients reported in literature were digitized and deconvoluted with the assay precision according to the authors. The GH data were subsequently transformed from \u03bcg-mass units into mU using the conversion factor 2.0. Reference values were obtained in nine males and 10 females healthy controls. Values shown are medians and 95% confidence intervals between brackets\nIn addition, the secretory regularity was quantified with the approximate entropy statistic, ApEn. In patient 2, ApEn was 1.256 before removal of the carcinoid, and after surgery and under somatostatin analog treatment ApEn was still increased to 0.686 (median normal for women 0.400, 95% confidence interval 0.300\u20130.440). In patient 3 ApEn also remained abnormal: preoperative 1.256 and after surgery 0.687, median normal for males 0.240, 95% confidence interval 0.160\u20130.350 (see Table 2). In addition, the serum GH profiles of two patients reported in literature were digitized and analyzed in a similar way [14, 15]. The results of these analyses are also displayed in Table\u00a01. In these male patients basal GH secretion was much higher than in our healthy controls and pulsatile secretion was augmented via increased pulse frequency and pulse amplitude. ApEn of GH secretion was 1.533 in Jaffe\u2019s patient and 1.248 in the patient reported by Vance (increased SD scores by 8- and 6-fold, respectively). ApEn for the serum GHRH-time series were 1.759 and 1.223, respectively. Copulsatility of the GH and GHRH hormone series was highly significant in both patients (P\u00a0<\u00a00.0001).Table 2Approximate entropy of GH secretion in ectopic GHRH syndromePatientBefore removal of the ectopic GHRH sourceAfter surgery and during octreotide treatmentReference values, Median and 95% CINo. 2 (female)1.2560.6860.400 (0.300\u20130.440)No. 3 (male)0.8420.5610.240 (0.166\u20130.350)Jaffe\u2019s patient1.5330.240 (0.166\u20130.350)Vance\u2019s patient1.2480.240 (0.166\u20130.350)Calculations were performed on GH data series consisting of 145 samples obtained at 10\u00a0min intervals during 24\u00a0h. Normal values were derived from nine males and 10 females healthy controls\nDiscussion\nIn this study we described in detail the clinical and biochemical characteristics of three patients with ectopic extracranial GHRH secretion diagnosed in a series of about 200 acromegalics investigated and treated in our center during the last 25\u00a0years. The incidence in the present series agrees with that mentioned in literature [1, 16]. Faglia summarized the clinical findings of 39 reported acromegalic patients with proven ectopic GHRH secretion [17]. Subsequently, van den Bruel and colleagues [18] mentioned 52 reported patients in 1999, including their own patient and since then 14 other patients have been reported, bringing the total number reported to 66 patients [19\u201332]. From a conservative estimation of the total number of newly diagnosed acromegalic patients in Europe, Japan and the USA, it is evident that most patients with ectopic GHRH syndrome are either not reported or remain undiagnosed.\nThe criteria for demonstration of ectopic extracranial GHRH-induced acromegaly are summarized by Losa and von Werder [2], and include the presence of high circulating concentration GHRH by specific radioimmunoassays, the presence of GHRH in the tumor, the presence of mRNA for GHRH by in situ hybridization and\/or a significant arterio-venous gradient across the ectopic source. The second requirement to be fulfilled is the reversibility of acromegaly after complete removal of the ectopic-hormone producing tumor. All our patients met at least two criteria, although the tumors were not investigated for the presence of GHRH.\nThe clinical symptomatology in ectopic GHRH-induced acromegaly is not different from that of the primary pituitary adenomatous form. However, symptoms due to the underlying neoplasm or cosecretion of other substances by the tumor might suggest the ectopic origin of acromegaly [33]. Specific dynamical tests for GH excess do not allow classification with certainty in either category, although most patients with ectopic GHRH syndrome exhibit a paradoxical increase of GH after TRH and glucose (i.e. >50%) and a blunted GH rise (<100%) after exogenous GHRH injection [33]. In addition, most patients also exhibit a moderate increase in serum prolactin concentration, which regresses after removal of the ectopic GHRH source, supporting the notion that GHRH is directly responsible for the hyperprolactinemia, a finding also present in hGHRH-transgenic mice [34]. The patients reported here all exhibited GH increase after TRH, and two had mild hyperprolactinemia. After surgery, hyperprolactinemia normalized, but GH still increased after TRH administration, suggesting that GHRH was still being produced by tumor remnants or metastases in two patients (nos. 2 and 3).\nThe most frequent source of ectopic GHRH is the bronchial carcinoid, followed by pancreatic islet tumors [17], as we also found in the present evaluation of our patient series. The pancreatic tumor in the first patient was found by CT-scanning and MRI. The large bronchial carcinoids were easily seen by chest X-ray examination, and showed uptake of radio-labeled octreotide, thus demonstrating in vivo the presence of somatostatin receptors. Although positive somatostatin receptor-scintigraphy is usually found in carcinoids in general, its demonstration in ectopic GHRH tumors is limited. Nevertheless, this examination could be particularly useful in the diagnostic phase of patients with suspected, but not yet proven, ectopic GHRH secretion.\nHistological investigation of surgically removed pituitary tissue revealed pituitary hyperplasia in most cases (i.e. with an intact reticulin fiber network), but in other patients adenomatous transformation can be found [35]. Indeed, the histological findings in hGHRH transgenic mice resemble those in the human: in young animals diffuse hyperplasia was found, while those of older mice showed adenomas [34, 36]. The pathological changes in the pituitary gland are the result of GHRH per se and not of GH, since hyperplasia and tumor formation still occur in the absence of GH signaling [37]. A hypothalamic GHRH-producing tumor (gangliocytoma) is generally associated with a pituitary adenoma rather than with pituitary hyperplasia and is attributed to a much higher (assumed) local (pituitary) concentration of GHRH or the presence of other growth stimulating factors [38]. In addition, the combination of an intrasellar GHRH-containing gangliocytoma and a somatotropinoma has been described in rare patients [39, 40]. In two of our patients pituitary histology was available: one patient (no. 1) showed hyperplasia in the absence of an enlarged gland, while histology in the other patient (no. 3) showed a mixture of hyperplasia and adenomatous transformation.\nNeuroimaging studies of the pituitary gland are variable: in about half of the patients no tumor or a slight enlargement of the sella is detected, while in other patients intrasellar adenomas or macroadenomas with suprasellar extension are found [18]. The CT-scan of patient 1 was normal and MRI scanning in patient 2 showed diffuse enlargement of the pituitary gland without a clear adenoma and with diffuse uptake of gadolinium DTPA. In patient 3 asymmetric enlargement was present, suggestive of a macroadenoma, but uptake of gadolinium DTPA throughout the whole pituitary gland was diffuse, illustrating the variability of imaging results. Although the pituitary MRI studies in the ectopic GHRH syndrome are non-specific, in the absence of a clear adenoma, ectopic GHRH secretion should be considered and appropriate investigations installed to prevent unnecessary pituitary surgery. On the other hand, primary medical treatment for acromegaly in these patients might obscure the real cause.\nCarcinoids, especially of the lung and gastrointestinal tract constitute about two-thirds of all cases reported so far, followed by pancreatic islet cell tumors [17, 33]. Metastases of carcinoid tumors were present in about 30%, a frequency similar to lung carcinoids in general [35]. Two of our patients had a large primary lung carcinoid, but had no histological proven metastases. Because serum GHRH concentration did not normalize after removal of the GHRH source we assumed (liver) metastases.\nImmunochemistry of the GHRH-secreting tumors has regularly demonstrated cosecretion of other hormones, including gastrin, gastrin-releasing peptide, calcitonin, pancreatic polypeptide, VIP, glucagon, insulin, and somatostatin, pointing to the multihormonal nature of these tumors. Particularly, cosecretion of somatostatin may modify the clinical picture and severity [31\u201344].\nOnly few patients with a GHRH-producing pancreatic islet cell tumor and with MEN I syndrome have been described [41, 43, 44]. In agreement with our patient 1, they had multiple tumors; however, the pancreatic tumors in our patient showed an identical immunohistochemical profile in contrast with the other reported patients. The tumor stained positively for somatostatin, which might explain the only moderately increased GH concentration and possibly also the normal GH response to glucose loading, as alluded to above. In the follow-up of more than 20\u00a0years, no recurrence of acromegaly occurred, but the (modest) increase of pancreatic polypeptide could point to the development of a new pancreatic adenoma.\nGH secretory characteristics of two patients were investigated before removal of the GHRH source. We established that pulsatile and non-pulsatile (basal) secretion, pulse frequency and secretory regularity (ApEn) were comparable to untreated acromegalic patients with primary pituitary adenomas [8, 45]. Because of the highly significant copulsatility of GHRH and GH in two other patients reported in literature, it is reasonable to postulate that pulsatile GH secretion in these patients was generated via episodic GHRH secretion of the primary tumor, while in patients with a pituitary GH-secreting adenoma pulsatile secretion is probably a tumorous feature [8].\nApproximate entropy calculations of serum GHRH profiles disclosed high values, suggesting highly irregular secretion, a feature common with many other hormone-secreting tumors [7\u20139]. It is reasonable to assume that the irregular secretion of GH in the four patients who were investigated till now was mainly the result of the irregular GHRH-input signal, and analogous to the irregular cortisol secretion driven by ACTH in pituitary-dependent Cushing\u2019s syndrome [7, 46]. Nevertheless, part of the irregularity of GH secretion might also be due to increased GHRH per se, since GHRH infusion in healthy subjects augments irregular GH secretion [47]. After surgery and on effective octreotide therapy aimed at normalizing IGF-I, GH secretion became almost normal. GH secretion remained however irregular (increased ApEn), notwithstanding the increased feedback signal of octreotide, and the diminished forward input signal of GHRH by octreotide [48, 49]. Similarly, octreotide treatment in (classical) acromegaly represses secretory-burst mass and non-pulsatile secretion but does not restore event frequency or orderly GH secretion [50]. Collectively, these results suggest that even a modestly increased GHRH concentration can maintain irregular GH secretion in ectopic acromegaly.\nTherapy\nSurgical removal of the GHRH source is obviously the treatment of choice and results in the resolution of acromegaly when no metastases are present, as we observed in the first patient with more than 20\u00a0year follow-up. In the presence of metastases octreotide treatment is often, but not invariable, successful in suppressing GH and GHRH, because of its dual action on the pituitary gland and the GHRH-secreting tumor [51]. Most published reports concerned subcutaneously administered octreotide mostly by three daily injections and in a minority by chronic subcutaneous infusion [48, 52] and summarized by van den Bruel et al. [18]. In the majority of patients GH decreased substantially, but IGF-I remained elevated in eight of 13 patients in whom IGF-I was measured. The serum concentration of GHRH generally remained elevated [2], as we found in patient 3. Publications of treatment results with the slow release formulation of octreotide and lanreotide are scarce and follow-up short-term. We could find only four cases in literature: three patients were treated with lanreotide and one with octreotide-long acting repeatable (octreotide LAR) [19, 21, 24, 25]. In the present study we reported the long-term effect of octreotide-LAR treatment in two patients, which resulted in suppressed GH secretion and normalized IGF-I concentration in both patients, but with still slightly elevated GHRH concentrations, as long as the patients continued suppressive therapy. The latter finding is indeed often mentioned in other reports as well, as discussed above. Other treatment modalities applied in single cases are the use of a GHRH-receptor antagonist and chemotherapy in a patient with severely metastasized disease [15, 53].\nDuring octreotide treatment no visible growth of (eventual) metastases was noted for many years with high-sensitivity liver CT scanning and CT scanning of the thorax in our patients, which suggests that in selected cases octreotide can inhibit growth of metastases from a carcinoid tumor. In addition, pituitary hyperplasia, the likely cause of the preoperative enlargement in patient 2, slowly disappeared completely, as previously described in other patients under medical therapy or after complete surgical removal of the GHRH source [27, 54]. Nevertheless, in patient 3 GHRH levels increased slowly, suggesting growing metastasis under octreotide restraint. Recently, we could localize a suspect lesion in the anterior mediastinum.\nGHRH and its receptor are normally present in several organs, including the ovary and testis, playing a role in the regulation of steroidogenesis, and the pancreas [55\u201357]. It has been found that some GHRH-producing carcinoids also express GHRH-receptors, which may stimulate tumor growth via an autocrine mechanism [22]. Particularly interesting was the description of a single acromegalic patient whose large pituitary adenoma co-expressed GHRH-receptor and GHRH together with a 100-fold increased plasma GHRH concentration. After pituitary adenomectomy plasma GHRH normalized. It is likely that the high local concentration of GHRH contributed to the growth of the adenoma [58].\nGHRH and its receptor are also expressed in cancers of the breast, ovary, and endometrium and small-cell lung cancer, and may function as a paracrine\/autocrine growth factor in regulating local IGF-I and\/or IGF-II secretion. Other studies have shown that GHRH receptor-antagonists inhibit the growth of colorectal, prostatic, mammary, lung, and pancreatic cancers, partly by direct suppression of IGF-I and IGF-II production, or acting directly without influencing these growth factors [59\u201363]. When more potent GHRH-receptor antagonists will become available for clinical application patients with advanced disease may benefit from this new approach, because they respond at best to somatostatin analogue therapy by suppressing GH secretion and partly that of GHRH, but the tumor and metastases will usually grow with fatal outcome.","keyphrases":["acromegaly","ectopic ghrh","octreotide","gh secretion","approximate entropy"],"prmu":["P","P","P","P","P"]}
{"id":"Semin_Immunopathol-4-1-2315691","title":"The role of complement in ocular pathology\n","text":"Functionally active complement system and complement regulatory proteins are present in the normal human and rodent eye. Complement activation and its regulation by ocular complement regulatory proteins contribute to the pathology of various ocular diseases including keratitis, uveitis and age-related macular degeneration. Furthermore, a strong relationship between age-related macular degeneration and polymorphism in the genes of certain complement components\/complement regulatory proteins is now well established. Recombinant forms of the naturally occurring complement regulatory proteins have been exploited in the animal models for treatment of these ocular diseases. It is hoped that in the future recombinant complement regulatory proteins will be used as novel therapeutic agents in the clinic for the treatment of keratitis, uveitis, and age-related macular degeneration.\nComplement system\nComplement has long been recognized as a critical component of the innate immune system [1\u20136]. It comprises of soluble and surface proteins that play a central role in host defense against infection and in the modulation of antigen-specific immune and inflammatory responses [4, 5]. Thus, complement also serves as a bridge between innate and adaptive immunity. The complement system can be activated by three proteolytic cascades namely, the classical, the alternative, and the lectin pathways (Fig.\u00a01). Although the initial signal for the activation of each cascade differs, complement activation via these pathways triggers a sequence of biologic reactions in which each component is activated by the upstream component [3, 5]. This sequential activation of complement components by all three pathways leads to the formation of membrane attack complex (MAC). Under normal conditions, the activation of the complement system is kept under tight control by the coordinated action of soluble and membrane-associated complement regulatory proteins (CRegs). However, interference with the function and\/or expression of CRegs leads to unwanted host tissue damage and tissue pathology due to unregulated complement activation [7, 8].\nFig.\u00a01Complement system and complement regulatory proteins (CRegs). The complement system can be activated through three pathways and complement activation triggers a sequence of biological reactions which result in the generation of effector molecules (e.g., C3a, C3b, C5a, MAC) with potent immunological activities. The classical pathway can be activated by antigen-antibody complexes or by substances such a C-reactive protein. C1, C2, C4, and C3 are the components of the classical pathway. Activation of the alternative pathway does not require the presence of an antibody; C3, factor B, factor D, and properdin are the components of the alternative pathway. Like the alternative pathway, the activation of the lectin pathway is also antibody independent and can be achieved by interaction of serum lectins (e.g., mannose-binding protein, MBL), with sugar residues present on the bacterial cell wall. All three pathways converge on a common reaction that results in the formation of an enzyme\u2014C3 convertase. This enzyme cleaves C3 into C3a and C3b. C3b forms C5 convertase that ultimately leads to the formation of membrane attack complex (MAC). The complement cascade is regulated at different check points (shown with black blocks) by CRegs. The soluble CRegs are shown in white boxes and the membrane-bound CRegs are shown in gray boxes\nCRegs can be broadly categorized into two classes\u2014membrane-bound CRegs and soluble CRegs (Fig.\u00a01). Membrane-bound CRegs (Fig.\u00a01) include decay-accelerating factor (DAF, CD55), membrane co-factor protein (MCP, CD46), complement receptor 1 (CR1, CD35), and membrane inhibitor of reactive lysis (MIRL, CD59). DAF, a glycosylphosphatidylinositol (GPI)-anchored protein prevents the activation of the complement system by inhibiting the formation of C3 and C5 convertases of both the classical and alternative pathways and accelerating the decay of these convertases [9, 10]. However, DAF does not irreversibly inactivate C3b and C4b and, after interaction with DAF, these molecules still possess cytolytic activity. MCP a transmembrane glycoprotein regulates the complement cascade by serving as a co-factor for serine protease factor I, which irreversibly inactivates the hemolytic potential of C3b and C4b [11, 12]. CR1 has been reported to possess both decay-accelerating and co-factor activities [13]. Like DAF, CD59 is also attached to the cell surface by GPI anchors. CD59 acts at the terminal step of complement activation and prevents the formation of membrane attack complex (MAC, C5b-9 complex), the final activation product of all three complement cascades by blocking the incorporation of C9 [14, 15]. Crry (5I2 antigen) is a C3 convertase inhibitor that possesses both decay-accelerating and membrane co-factor activities and present only in rodents [16, 17]. However, DAF, MCP, and CD59 have also been identified and characterized in rodents [18\u201323].\nC1 inhibitor (C1INH), C4 binding protein (C4bp), complement factor H (CFH), complement factor I (CFI), S-protein and clustrin, are the examples of important soluble CRegs (Fig.\u00a01). C1INH regulates activation of the classical complement pathway by inactivating the protease function of activated C1 complex. It also prevents spontaneous activation of C1 and binding to the zymogen forms of C1r and C1s [24]. C4bp acts as a co-factor for CFI, catalyzing the cleavage and inactivation of C4b [25]. CFH inhibits C3 activation by binding to C3b and acting as a co-factor for factor I-mediated cleavage of C3b and also has decay-accelerating activity for the alternative pathway C3 convertase, C3bBb. It also competes with factor B for surface-bound C3b [26]. Both clustrin and S-protein prevent the formation of MAC [6]. Soluble forms of MCP, DAF, CR1, CD59, and Crry have been reported to be present in various biological fluids in both humans and rodents [27\u201330]. Thus, a wide variety of CRegs (both membrane bound and soluble) have evolved to create efficient checkpoints at critical steps of complement cascades so that the host could be protected from the inadvertent activation of complement on its own tissue (Fig.\u00a01).\nSince complement is a powerful cytotoxic defense system, regulation of complement activation is crucial for host tissue damage control during inflammation especially in a sensitive organ like the eye. The eye is an immunologically privileged site and ocular immune privilege does not refer to the absence of immune system but, rather its fine regulation [31]. The studies discussed in this article highlight the role of complement activation and regulation in the protection of the normal eye. In addition, the studies discussed below demonstrate the importance of interplay between complement activation and complement regulation in the development of vision-threatening complications such as keratitis, uveitis, and macular degeneration.\nRole of complement in the protection of the normal eye\nIt is important that we discuss the role of complement and CRegs in ocular protection under normal conditions before discussing their importance in ocular pathology. As mentioned above, the eye is an immune-privileged organ [31] and is highly vulnerable to various immunological insults. Therefore, it is not surprising that the complement system, which represents the first line of defense, plays an important part not only in protecting the eye from immunological insults but also in maintaining the immune-privileged state of the eye. Our results and several other independent studies have demonstrated the presence of various components of the complement system in the normal eye [2, 32\u201337]. We have further shown that ocular complement components are not just silent bystanders but are chronically active at a low level in normal eye [32]. We reported that both iC3b (activation product of C3) and MAC (end product of complement activation) are present in normal rat eye indicating that the complement system is continuously activated at a low level in the normal eye [32]. In 2003, we documented that the interaction between complement activation product iC3b and its receptor is vital for the creation of immunosuppressive environment that leads to the induction and the maintenance of ocular immune privilege and protection of intraocular structures that are critical for vision [38]. The presence and expression pattern of CRegs in the normal human eye was first described by us [33]. We reported that membrane-bound CRegs\u2014MCP, DAF, and CD59\u2014are differentially expressed in normal human and eyes [33]. Our studies along with other independent studies that were performed later demonstrated that Crry and CD59 (membrane bound and soluble) are also present in the normal rodent eye [32, 34, 35]. We have further shown that ocular CRegs are functionally active and tightly regulate the activation of intraocular complement [27, 32]. Functional inhibition of ocular CRegs using specific antibodies resulted in unregulated complement activation leading to severe intraocular inflammation in normal animals [32]. Furthermore, complement inhibitory activity in normal human intraocular fluid was blocked by inhibiting the function of MCP, DAF, and CD59 [27].\nThus, our results clearly established that within the normal eye the complement system seems to perform two important functions. First, the chronic low level of complement activation serves as a primary defense mechanism of the eye against pathogenic infection and is finely regulated by the soluble and membrane-bound intraocular complement regulatory proteins. This enables destruction of the putative pathogen without inadvertent damage of ocular tissue, which is vital for the maintenance of vision. Second, complement activation products (such as iC3b) generated as a result of the low level of complement activation results in the selective suppression of harmful T cell responses. In this role complement protects the eye from innocent bystander destruction associated with the T cell response to the invading pathogens. Complement components and CRegs present in normal tears have also been reported to be functionally active and have been proposed to serve as first line of defense in protecting the eye [36]. However, additional studies are required to determine the role of the complement system in normal tears.\nRole of complement in corneal disease\nThe cornea is constantly exposed to various physical as well as immunological insults. Indeed the normal cornea is well equipped to protect itself from these insults and has been shown to possess functional complement activity [39\u201346]. In addition, low levels of C3, C3 split products, and MAC were detected in the normal cornea [32, 39, 46]. This observation further proves that low level of complement activation is taking place in the normal cornea since complement activation is necessary for the generation of these products. Studies reported in the literature suggest that complement cascade can be aggressively activated in the cornea during an immune and inflammatory reaction [45, 46]. Two reports by Mondino and colleagues described interesting sets of experiments [45, 46]. In these experiments, the normal cornea from human donors was exposed to lipopolysaccharide (LPS), ribitol teichoic acid immune complex, acid (HCl), or alkali (NaOH) separately and the generation of anaphylatoxins\u2014C3a, C4a, and C5a\u2014as well as MAC in the corneas was monitored. It was reported that anaphylatoxins\u2014C3a, C4a, and C5a\u2014could be generated when the cornea was injured with LPS, immune complexes, acid, or alkali. Interestingly MAC could only be generated when the cornea was exposed to LPS or immune complexes. Cornea failed to generate MAC when insulted with acid or alkali. The immune response mounted to LPS or immune complex is similar to that generated against infectious agents like gram-negative bacteria. Indeed the complement system has been shown to play a critical role in protection against Pseudomonas aeruginosa infection that causes keratitis [47, 48]. Additionally, complement activation is believed to play an important role in ulceration of human cornea induced by gram-negative bacteria [49].\nThe aforementioned observations have important clinical implications. If in future anti-complement agents were to be considered for the treatment of corneal pathology, anaphylatoxins would be an ideal target for the patient presenting with chemical injury such as exposure to acid or alkali. On the other hand, in the case of bacterial infection both anti-anaphylatoxin and anti-MAC therapy should be considered. Although as discussed below the cornea possesses the ability to express different CRegs to protect itself from complement-mediated damage, it may not be able to upregulate various CRegs to an appropriate level during an acute episode of complement activation as observed during chemical insults or acute bacterial infection. In such cases, topical application of recombinant CRegs could be beneficial and serve as a better alternative for intervention.\nEnhanced complement activation can cause damage to the autologous corneal tissue during an infection or inflammation. Membrane-bound CRegs such as MCP, DAF, Crry (Fig.\u00a02a), and CD59 (Fig.\u00a02b) are expressed in the different layers of the cornea to protect this tissue from complement-mediated damage [27, 33, 50]. Interestingly, compared to corneal stroma and corneal endothelium, various CRegs are very strongly expressed on the corneal epithelium [33] (Fig. 2a and b). This high expression of CRegs on the corneal epithelium is crucial for the protection of the cornea because the cornea is continually exposed to various pathogens including bacteria. These bacteria produce phospholipase and other enzymes which can remove GPI-anchored DAF and CD59 from the corneal surface [51]. Thus, in the absence of a very high expression of CRegs on the corneal epithelium, bacterially induced loss of DAF and CD59 could be deleterious to the cornea and lead to vision loss after the putative pathogen has been destroyed by the complement system. To investigate if a deficiency or abnormality in the expression of CRegs may play a role in ocular surface disease, systemic examination of corneal tissue from patients with different diseases such as pseudophakic bullous keratopathy, HSV-1 keratitis, and herpes zoster scleritis is needed.\nFig.\u00a02Complement regulatory proteins in normal cornea. The figure shows immunofluorescent staining for Crry (a) and CD59 (b) in the cornea of naive Lewis rat. Objective magnification \u00d720. Epi Epithelium, S stroma, Endo endothelium, AC anterior chamber\nRole of complement in autoimmune uveitis\nUveitis, the inflammation of the uveal tract, can be classified anatomically as anterior, intermediate, posterior, or diffuse (panuveitis) depending on the segment of the eye that is affected. Each year, approximately 17% of active uveitis patients experience some degree of vision loss [52]. Uveitis may be idiopathic, associated with systemic diseases such as Beh\u00e7et\u2019s disease, and Vogt\u2013Koyanagi\u2013Harada disease or resulting from a variety of infectious agents. Inflammation resulting from uveitis may lead to conditions like cataract, glaucoma, and cystoid macular edema that may cause irreversible vision loss. Anterior uveitis is the most common form of uveitis, and the most common form of anterior uveitis is of unknown (i.e., idiopathic) etiology [52].\nAlthough the exact cause of the disease is still unknown, a wide array of studies using different animal models have established that the inflammation in uveitis is due to an autoimmune response to various ocular antigens [53\u201356]. Unfortunately, almost all of the studies in the past three decades have focused on the role of T cells in the pathogenesis of uveitis [54, 57, 58]. Very few laboratories have investigated the role of the complement system in the development of autoimmune uveitis [32, 58\u201366].\nThrough our recent studies, the critical role of complement and complement regulatory proteins (CRegs) in the pathogenesis of idiopathic anterior uveitis was addressed and recognized. Our laboratory has used experimental autoimmune anterior uveitis (EAAU)\u2014an animal model of human autoimmune anterior uveitis\u2014to investigate the role of the complement system, a significant and previously neglected facet of uveitis [53, 62, 63]. EAAU is induced by injecting bovine melanin-associated antigen emulsified in Freund\u2019s complete adjuvant, in the foot pad of Lewis rats [53]. The onset of the disease (days 12\u201314 post immunization) is marked by inflammation of the iris\/ciliary body followed by massive infiltration of the inflammatory cells in the anterior segment of the eye during the peak (days 16\u201319 post immunization) of EAAU (Fig.\u00a03). The inflammation resolves after approximately 3\u00a0weeks, and the eye returns to the normal state with no apparent sign of any tissue damage (Fig.\u00a03). Interestingly, studies from our laboratory have demonstrated that the activation of the complement system is critical for the development of EAAU [62]. During EAAU, the complement system is activated (detected by Western blot analysis for iC3b) in the eye, and the kinetics of complement activation follows a pattern that is similar to the clinical course of the disease. Furthermore, depletion of the complement system of the host resulted in complete inhibition of EAAU. In complement-depleted rats, the levels of IFN-\u03b3, IP-10, ICAM-1, and LECAM-1 were extremely low compared to the complement-sufficient rats during EAAU. Our observations suggest that the activation of the local complement system plays a critical role in the development of inflammation during EAAU and suppression of intraocular complement system, thus may provide a successful strategy for uveitis therapy. At present, the exact mechanism and pathways involved in local complement activation during uveitis are not known. It is also not known if the increase in complement components in the anterior segment of the eye is due to the upregulation of their synthesis by resident ocular cells or is a result of spillover from the systemic complement system. Previous studies from our laboratory established that a functionally active complement system is present in the anterior segment of the eye because when a well-known activator of complement such as zymosan was injected in the anterior chamber, severe anterior uveitis was induced. This zymosan-induced uveitis was due to the activation of the complement system because anterior uveitis was completely inhibited when the complement system of the host was depleted [32]. Furthermore, we and others have reported the presence of various complement components in aqueous humor as well as other parts of the normal eye [32, 39, 41, 59, 61]. These observations taken together suggest that ocular complement is activated during the course of EAAU and the inhibition of the intraocular complement system may lead to more effective management and\/or treatment of autoimmune uveitis.\nFig.\u00a03Clinical and histologic pictures of rat eye during different stages of experimental autoimmune anterior uveitis (EAAU). No inflammation could be detected in the iris (I) and the ciliary body (CB) after clinical (a) and histologic (d) examination at day 8 post immunization (before the onset of EAAU). At the peak of disease (day 19 post immunization) the anterior chamber (AC) appeared cloudy and opaque due to the presence of proteinaceous material and inflammatory cells (b). Histopathologic picture of rat eye at the peak of EAAU (e) revealed severe inflammation of the iris (I) and ciliary body (CB). Heavy infiltration in the anterior chamber (AC) with spillover into the anterior vitreous was also observed at this time point. The eye appeared normal both clinically (c) and histologically (f) after the resolution of EAAU (day 30 post immunization). Objective magnification \u00d710\nWhen considering anti-complement therapy, we cannot ignore the role of CRegs in the resolution of autoimmune uveitis. We have demonstrated that the functionally active CRegs downregulate intraocular complement activation and are critical to protect the ocular tissues from complement-mediated damage during EAAU [63]. We observed that various CRegs were upregulated during the course of EAAU on the resident ocular tissue and this upregulation was not due to the infiltrating inflammatory cells because the levels of CRegs remained upregulated after the resolution of the disease when all the infiltrating cells were cleared from the anterior segment of the eye [63]. Furthermore, when the function or expression of CRegs was inhibited in vivo, more severe EAAU with early onset and delayed resolution was observed. These observations demonstrate that ocular tissue can protect itself by upregulating various CRegs during uveitis and provide strong evidence for the use of recombinant CRegs as therapeutic agents in the treatment of autoimmune uveitis.\nActivation of the complement system has also been reported in other animal models of anterior uveitis such as endotoxin-induced uveitis (EIU) [64, 65]. In EIU, it was demonstrated that the complement system is critical for the development of inflammation, and there is a local activation of the complement system as evident by accumulation of complement components (C1q, C3, and C4) in various parts of the eye during inflammation [66]. Activation of the complement system was also implicated in experimental allergic uveitis [67]. Using experimental autoimmune uveoretinitis (EAU) animal model, the complement system has been reported to play an important role in the pathogenesis posterior uveitis [68]. Recently, Read and co-workers demonstrated that the transgenic mice that express soluble Crry in the retina had decreased incidence and severity of EAU [68]. In these transgenic mice, EAU was not completely suppressed. This may be due to relatively low levels of Crry expressed compared with the levels that are required for the complete inhibition of the complement system. It is possible that a higher dose of exogenous Crry or similar CReg may result in complete inhibition of EAU.\nThus, these studies provide strong evidence that the complement system and CRegs play an important role in the pathogenesis of autoimmune uveitis and provide alternative approaches for the development of effective therapy. More research in the future will present us enough information to develop anti-complement therapy for uveitis. Hopefully, in the future, complement inhibitors might be used as novel anti-uveitic agents in the clinic for the treatment of this important form of human ocular disease.\nRole of complement in age-related macular degeneration\nAge-related macular degeneration (AMD) is the most common cause of legal blindness worldwide among the elderly over the age of 50. Approximately 1.8 million people in the United States alone have AMD and it is projected that by 2020, roughly 2.9 million people will develop this disease [69\u201371]. In AMD, there is a progressive destruction of the macula leading to the loss of central vision. AMD is classified into non-exudative (dry type) and exudative (wet type) [69\u201371]. In the dry type of AMD, drusen\u2014small yellow deposits\u2014are formed between the retinal-pigmented epithelium (RPE) and Bruch\u2019s membrane [72]. In wet-type AMD, abnormal vessel growth takes place from choroid, termed choroidal neovascularization (CNV), under the retinal pigment epithelium [71, 72]. Although several risk factors are associated with AMD, the exact pathogenesis still remains unknown. The last few years have witnessed an amazing series of research establishing the central role of the complement system in the pathogenesis of AMD both in humans and in experimental animals. Various studies in the literature have indicated a potential role for complement in drusen formation in the non-exudative form of AMD in humans [73\u201375]. Complement components, complement activation products (C3a, C5a, MAC), and complement regulatory proteins (CD46, Vitronectin) have been localized in drusen in patients with AMD [73\u201375].\nThe discovery of allelic variants of complement factor H (CFH) as a major risk factor for AMD has become a landmark study [76\u201379]. The polymorphism that gives rise to this allelic variant of CFH results from a tyrosine to histidine replacement at position 402. The \u201chigh risk\u201d variant, i.e., homozygous for histidine residue at position 402 is associated with five- to sevenfold increased risk of AMD [76\u201379]. CFH consists of 20 short consensus repeats (SCRs) and the polymorphic site 402 resides in SCR7. SCR7 contains the binding site for C3b, glycosaminoglycans (GAG) and C-reactive protein (CRP). The polymorphism at position 402 in SCR7 reduces the binding affinity to CRP [80] and GAG [81]. Binding of CFH to CRP and GAG plays an important role in the regulation of the alternative pathway. Thus the polymorphic form of CFH with histidine at position 402 may have reduced ability to regulate the activation of the alternative pathway. This may lead to uncontrolled activation of alternative pathway which may result in the development of AMD. The loss of regulatory activity of CFH due to polymorphism can have a direct effect on the predisposition of the eye to AMD as it has been shown recently that CFH is expressed locally in the optic nerve, retina, and retinal pigment epithelium [82]. The importance of CFH in AMD was further demonstrated in a recent study. It was reported that the aged mice genetically deficient in CFH (cfh\u2212\/\u2212) develop spontaneous changes in retina (e.g., photoreceptor dysfunction, increased deposition of C3 in retina, thinning of Bruch\u2019s membrane) which is consistent with those seen in AMD [83].\nPolymorphic variants of other complement components have also been shown to be associated with AMD. In a recent study, Gold et al. reported that complement components\u2014factor B (CFB) and complement component 2 (C2)\u2014are also associated with AMD [84]. In this report, the investigators identified high risk allele and two protective alleles for CFB and C2 that were associated with AMD. An independent study further established a similar association between CFB, C2, and AMD [85]. More recently, investigators compared 847 patients with AMD with 701 unaffected people and observed that a variant in the complement C3 gene affected the risk of developing AMD [86].\nThe aforementioned studies establish that the polymorphism in the genes of certain complement components and regulators genetically predisposes some individuals to AMD. However, we should also pay attention to the fact that other risk factors for AMD such as alcohol consumption and smoking nicotine can directly affect the level of complement activation and CRegs. For instance, chronic ethanol consumption upregulates CFB but downregulates CFH in mice [87]. Thus, association of alcohol consumption with AMD may be due to its direct effect on the complement system. Indeed we have reported that chronic alcohol consumption increases the size of CNV complex in rat model of laser-induced CNV [88]. Similarly, cigarette smoking has been shown to decrease the affinity of CFH for C3 and activate the complement system [89].\nIn our laboratory, we use a mouse model of laser-induced CNV to study the role of complement in the pathogenesis of wet-type AMD. A reliable way to produce CNV in animals is to rupture Bruch\u2019s membrane with laser photocoagulation [90\u201392]. We and others have found that choroidal neovascularization induced in rodents by laser photocoagulation is useful to gain insights into the pathogenesis of new vessel growth from the choroid [90\u201396]. In 2005, we demonstrated for the first time using this animal model that complement activation and generation of MAC is critical for the development of laser-induced CNV. In this study, C3\u2212\/\u2212 mice and anti-C6-treated mice failed to develop CNV [93]. As mentioned above, the importance of C3 in the pathogenesis of AMD was recently confirmed by Yates et al., who established an association of C3 polymorphism with AMD [86]. We further observed that in the absence of MAC deposition, release of angiogenic factors such as vascular endothelial growth factor (VEGF), TGF-\u03b22, and \u03b2-FGF was inhibited. This is a crucial observation as it suggests that the complement system is a potential therapeutic target for the treatment of CNV. Subsequently, we reported that the activation of complement by alternative pathway is responsible for MAC formation in laser-induced CNV. This alternative pathway activation was due to the decrease in CFH levels and increase in CFB levels in the RPE\/choroid after laser treatment [94]. In a separate study, we used recombinant-soluble CD59-Fc chimeric protein to inhibit the formation of MAC after laser photocoagulation [95]. Treatment of mice with recombinant-soluble CD59-Fc inhibited the development of choroidal angiogenesis in the laser-induced mouse model by blocking MAC formation which in turn blocked the induction\/release of angiogenic growth factors such as VEGF, \u03b2-FGF, and TGF-\u03b22 (Fig.\u00a04). Thus, our studies suggest that the recombinant forms of CD59 could be used as a potential drug in the future to treat CNV. Another potential target could be anaphylatoxins\u2014C3a and C5a. It was reported that C3a and C5a are present not only in human drusen but are also localized in the choroid of mice with laser-induced CNV [97].\nFig.\u00a04Role of CD59 in laser-induced choroidal neovascularization (CNV). Laser photocoagulation results in complement activation and MAC formation in the posterior segment of the eye. MAC is a complex assembly of C5b to C9 components with multimers of C9 forming a pore in the cell membrane. This causes increased release of growth factors\u2014VEGF, TGF\u03b22, and \u03b2-FGF\u2014which in turn leads to the development of CNV. The membrane-bound as well as recombinant-soluble (rs) CD59, bind to C9 and C8 and inhibit the polymerization of C9 which is prerequisite for MAC formation. In the absence of MAC, release of angiogenic factors is downregulated. This inhibits the growth of new vessel from the choroid (CNV)\nUsing the mouse model of laser-induced CNV, we observed that the CH50 levels in the serum of lasered mice varied when compared to non-lasered mice [93, 94]. Another study demonstrated that the plasma complement C3a des Arg levels increased in AMD patients compared to controls [98]. This increase in plasma C3a des Arg concentration reflects systemic complement activation. Thus systemic activation of the complement system may also contribute to the pathogenesis of AMD.\nCurrently, several different approaches are being used to treat wet AMD in humans [99]. Unfortunately, these treatment options have limitations due to their short-term and serious side effects. Although, photodynamic therapy (PDT) reduces the rate of vision loss in most patients, it does not lead to significant improvement in vision. Furthermore, repeated PDT can cause severe damage to the posterior segment of the eye and is not cost effective. Two drugs Lucentis and Macugen are approved by Federal Drug Administration for the treatment of AMD and both drugs inhibit vascular endothelial growth factor. One of the major drawbacks of Lucentis or Macugen is that repeated injections of these drugs are needed to treat AMD. Repeated injections can cause some serious side effects including hemorrhage of the eye membrane, eye pain, and vitreous floaters. Therefore, alternative therapeutic strategies with minimum side effects are required to better treat AMD patients. We believe that the results derived from the studies reviewed above would open up the window of opportunities for the development of anti-complement therapy in the treatment of AMD.\nIn view of important developments relative to the role of the complement system in AMD during the past several years, we believe that the next decade would be critical for such studies. We hope that the interest will continue to grow in the future so that an alternative therapy could be developed by inhibiting complement activation. Since untreated CNV leads to the irreversible loss of central vision, it is important for the pharmaceutical companies to utilize the knowledge available to them from different research laboratories and should make every effort to transfer this knowledge to develop new drugs and therapies for AMD patients.\nConclusions and clinical implications\nDuring the last decade, a lot of progress has been made to understand the pathophysiology of corneal diseases, uveitis, and macular degeneration. Specifically, the studies defining the role of the complement system in ocular diseases have drawn a lot of attention. It is an exciting area for future investigations and the basic questions such as what triggers the complement activation and which specific pathway is involved in corneal diseases, uveitis and macular degeneration need to be addressed. This understanding will help us to develop better therapies for ocular diseases in the future so that blindness could be prevented.\nThe evidence derived from the studies reviewed here supports the concept that recombinant complement regulatory proteins may be therapeutically useful in the prevention of ocular tissue damage and in the treatment of various ocular diseases. Agents that specifically inhibit the complement system or complement activation products have been proven beneficial in the treatment of both human and experimental diseases over the past few years. Thus, the therapy based on complement inhibition has great potential in the future for the treatment of various ocular diseases such as keratitis, uveitis, and macular degeneration.","keyphrases":["complement system","complement regulatory proteins","eye","uveitis","age-related macular degeneration","corneal inflammation"],"prmu":["P","P","P","P","P","R"]}
{"id":"Int_J_Cardiovasc_Imaging-4-1-2233707","title":"Evaluation of left atrial systolic function in noncompaction cardiomyopathy by real-time three-dimensional echocardiography\n","text":"Background Noncompaction cardiomyopathy (NCCM) is a rare disorder with persistance of the embryonic pattern of myoarchitecture. NCCM is characterized by loosened, spongy myocardium associated with a high incidence of systolic and diastolic left ventricular (LV) dysfunction and heart failure (HF). It is known that LV dysfunction contributes to elevated left atrial (LA) and pulmonary vascular pressures, however atrial function has not been examined in NCCM. The objective of the present study was to assess LA systolic function characterized by LA ejection force (LAEF) in NCCM patients using real-time three-dimensional echocardiography (RT3DE) and to compare to control subjects. Methods The study comprised 17 patients with an established diagnosis of NCCM and their results were compared to 17 healthy age-matched controls with no evidence of cardiovascular disease. Forty-one percent of NCCM patients were in NYHA functional class II \/ III HF. Previously proposed echocardiographic diagnostic criteria for NCCM were used. All patients underwent conventional two-dimensional echocardiography and RT3DE. LAEF was measured based on MA annulus diameter (LAEF3D-MAD) and area (LAEF3D-MAA) using RT3DE. Results The presence and severity of mitral regurgitation were more frequent in NCCM patients than in control subjects. LV diameters and mitral annulus were significantly increased in NCCM patients. Compared with control subjects, both LAEF3D-MAD (3.8 \u00b1 2.2 vs 2.3 \u00b1 1.0 kdyne, P < 0.05) and LAEF3D-MAA (12.7 \u00b1 7.6 vs 4.9 \u00b1 2.1 kdyne, P < 0.01) were significantly increased in NCCM patients. Conclusions LAEF as a characteristic of LA systolic function is increased in NCCM patients compared to normal individuals. These results can suggest compensating left atrial work against the dysfunctional LV in NCCM patients.\nIntroduction\nNoncompaction cardiomyopathy (NCCM) is a rare disorder with persistance of the embryonic pattern of myoarchitecture, and has been assumed to occur as a consequence to intrauterine arrest of compaction [1]. NCCM is characterized by loosened, spongy myocardium associated with a high incidence of systolic and diastolic left ventricular (LV) dysfunction and heart failure. It is known that LV dysfunction contributes to elevated left atrial (LA) and pulmonary vascular pressures, however atrial function has not been examined in NCCM. It is difficult to assess non-invasively LA contraction, but Manning et\u00a0al. reported a non-invasive method for the evaluation of LA ejection force (LAEF) as a characteristic of LA systolic function [2]. Anwar et\u00a0al. demonstrated the usefulness of real-time three-dimensional echocardiography (RT3DE) for the evaluation of LAEF [3]. The objective of the present study was to assess LAEF in NCCM patients using RT3DE and to compare to control subjects.\nMethods\nPatient populations\nThe study comprised 17 patients with an established diagnosis of NCCM and their results were compared to 17 healthy age-matched controls with no evidence of cardiovascular disease. All patients and control subjects were in sinus rhytm and all of them were examined by two-dimensional echocardiography (2DE) and RT3DE.\nDiagnostic criteria for NCCM\nPreviously proposed echocardiographic diagnostic criteria for NCCM by Jenni et\u00a0al. were used to include patients into the present study from the Outpatient Clinic of Erasmus Medical Center, Rotterdam during 3\u00a0years [4]: (1) absence of coexisting cardiac anomalies, (2) segmental, excessive thickening of the LV wall with a two-layered structure: a thin, compacted epicardial layer and a much thicker, noncompacted layer with the characteristic appearance of numerous, prominent trabeculations (meshwork) and deep intertrabecular recesses, (3) colour Doppler evidence of deeply perfused intertrabecular recesses, and (4) predominant localization of thickening in the LV apical, midlateral, and midinferior walls. Hypertensive heart disease was excluded by clinical and echocardiographic examinations (septal thickness\u00a0<\u00a013\u00a0mm).\n2DE examination\nThe 2DE was undertaken with the patient lying in the left lateral decubitus position using both apical and parasternal views. The 2DE studies were performed using a 3.5-MHz transducer and a commercially available ultrasound system (Sonos 7500, Philips, Best, The Netherlands). The following measures were obtained: (1) LV end-diastolic diameter (LV-EDD) and LV end-systolic diameter (LV-ESD) by M-mode echocardiography; (2) LV fractional shortening (LV-FS); (3) peak velocities of transmitral E and A waves with pulsed Doppler and (4) mitral regurgitation graded as mild (jet area\u00a0<\u00a04\u00a0cm2), moderate (jet area 4\u20138\u00a0cm2), and severe (jet area\u00a0>\u00a08\u00a0cm2) according to maximum jet area by color Doppler.\nRT3DE examination\nRT3DE was performed using the same ultrasound system, and performed with a X-4 matrix transducer capable of providing real-time B-mode and colour Doppler. The 3D images were collected within 5\u20137\u00a0s of breath holding in full-volume mode. The 3D data were transferred to an offline analysis system (TomTec Inc., Munich, Germany). Data were stored digitally and subsequently evaluated by two echocardiographers. Data analysis of RT3DE imaging was based on a two-dimensional approach relying on the echocardiographic images obtained from the apical views and on manual tracing of inner border of the mitral annulus (MA). Once this was completed the surface area was automatically delineated and could be visualized from different points of views. Manual modification was done to correct any image if necessary. The following measures were obtained: (1) MA diameter (MAD3D) and area (MAA3D) measured at end-diastole (just before mitral valve closure) and (2) LAEF3D-MAD and LAEF3D-MAA calculated by formula as 0.5\u00a0\u00d7\u00a01.06\u00a0\u00d7\u00a0(MAD3D or MAA3D)\u00a0\u00d7\u00a0V2, where V is the peak velocity of A wave, 1.06\u00a0gm\/cm3 is blood viscosity and 0.5 is a coefficient factor [2] (Fig.\u00a01).\nFig.\u00a01Automatically cropped two-dimensional image from real-time three-dimensional echocardiographic dataset demonstrating left ventricular cavity in a patient with noncompaction cardiomyopathy (A). Measurement of mitral annulus area and mitral inflow A wave velocity can help calculation of left atrial ejection force as a characteristic of atrial systole. Real-time three-dimensional echocardiography allows the `en-face` evaluation of mitral annulus helping exact measurement of mitral annulus diameter and area (B). Abbreviations: MA: mitral annulus, MAD: mitral annulus diameter, E and A: mitral inflow E and A wave velocity measured by pulsed Doppler\nStatistical analysis\nAll data obtained by 2DE or RT3DE were presented as mean\u00a0\u00b1\u00a0SD. Data analysis was performed using statistical software (SPSS, Version 12.1, SPSS Inc, Chicago, USA). Independent sample t test was performed to compare between means of variables of groups to determine the statistical significance of difference. The level of significance was set to p\u00a0<\u00a00.05. In our department, an excellent correlation (r\u00a0=\u00a00.82, P\u00a0<\u00a00.0001) has been described between MAD3D and MRI measurements of MAD [5]. There was no significant difference between MAD3D and MADMRI. Similarly, there was no difference between MAA3D and MAAMRI.\nResults\nClinical and 2D echocardiographic data\nClinical and echocardiographic data of NCCM patients and controls are shown in Tables\u00a01 and 2. The presence and severity of mitral regurgitation were more frequent in NCCM patients than in control subjects. LV diameters were significantly increased, while LV-FS was significantly decreased in NCCM patients. The number of noncompacted segments was 5.4\u00a0\u00b1\u00a02.9 in NCCM patients, which did not show any correlation with any echocardiographic parameters.\nTable\u00a01Demographic and clinical dataParameterNCCM patients (n\u00a0=\u00a017)Controls (n\u00a0=\u00a017)Age (years)48\u00a0\u00b1\u00a02043\u00a0\u00b1\u00a010Male sex (%)8 (47)11 (65)Diabetes mellitus (%)9 (53)10 (59)Hypercholesterolaemia (%)10 (59)11 (65)NYHA-class II\/III HF (%)3 (18)\/4 (24)0 (0)Abbreviations: NCCM: noncompaction cardiomyopathy, NYHA-class II\/III HF: New York Heart Association functional class II\/III heart failureTable\u00a02Echocardiographic dataParameterNCCM patients (n\u00a0=\u00a017)Controls (n\u00a0=\u00a017)2D echocardiography\u00a0\u00a0\u00a0\u00a0LV-EDD (mm)60.6\u00a0\u00b1\u00a011.0 **51.2\u00a0\u00b1\u00a06.6\u00a0\u00a0\u00a0\u00a0LV-ESD (mm)48.1\u00a0\u00b1\u00a013.0 **33.0\u00a0\u00b1\u00a06.7\u00a0\u00a0\u00a0\u00a0LV-FS (%)22.1\u00a0\u00b1\u00a017.1 **35.7\u00a0\u00b1\u00a06.4\u00a0\u00a0\u00a0\u00a0Mitral inflow A wave velocity (cm\/s)50.1\u00a0\u00b1\u00a016.2 *41.8\u00a0\u00b1\u00a09.2Mitral regurgitation\u00a0\u00a0\u00a0\u00a0No8 (47) *15 (88)\u00a0\u00a0\u00a0\u00a0Mild6 (35) *2 (12) \u00a0\u00a0\u00a0\u00a0Moderate-severe3 (18) *0 (0)RT3DE examination\u00a0\u00a0\u00a0\u00a0MAD3D (cm)3.5\u00a0\u00b1\u00a00.5 *3.2\u00a0\u00b1\u00a00.3\u00a0\u00a0\u00a0\u00a0MAA3D (cm2)12.0\u00a0\u00b1\u00a02.8 *7.9\u00a0\u00b1\u00a01.4\u00a0\u00a0\u00a0\u00a0LA-EF3D-MAD (kdyne)3.8\u00a0\u00b1\u00a02.2 *2.3\u00a0\u00b1\u00a01.0\u00a0\u00a0\u00a0\u00a0LA-EF3D-MAA (kdyne)12.7\u00a0\u00b1\u00a07.6 **4.9\u00a0\u00b1\u00a02.1*\u00a0P\u00a0<\u00a00.05 vs controls**P\u00a0<\u00a00.01 vs controlsAbbreviations: 2D: two-dimensional, 3D: three-dimensional, A: mitral inflow A wave velocity measured by pulsed Doppler, D: diastolic, EDD: enddiastolic diameter, ESD: endsystolic diameter, FS: fractional shortening, NYHA-class II\/III HF: New York Heart Association functional class II\/III heart failure, LA-EF: left atrial ejection force, LV: left ventricular, MAA: mitral annulus area, MAD: mitral annulus diameter, NCCM: noncompaction cardiomyopathy\nRT3DE data\nAcquisition and postprocessing of RT3DE data were performed successfully with clear delineation of the MA in all patients. Compared with control subjects, MAD3D and MAA3D were significantly larger, while LAEF3D-MAD and LAEF3D-MAA were significantly increased in NCCM patients (P\u00a0<\u00a00.05) (Table\u00a02).\nDiscussion\nLeft atrial ejection force (LAEF), based on classic Newtonian mechanics, provides a physiological assessment of atrial systolic function and is a potentially useful index for assessing atrial contribution to diastolic performance. To the best of authors` knowledge this is the first time to demonstrate increased LAEF during RT3DE indicating high LA workload in NCCM patients. NCCM is known to be a relatively new clinicopathologic entity characterized by a pattern of prominent trabecular meshwork and deep intertrabecular recesses communicating with the LV cavity [1]. Results of recent studies confirmed the hypothesis that isolated ventricular noncompaction can be a part of a more widespread cardiomyopathy, involving both the morphologically normal and dysmorphic LV myocardial segments [6\u20139]. However, the pathophysiological mechanisms of LV dysfunction remained unclear and, moreover little is known about LA function in NCCM.\nManning et\u00a0al. surmised that LAEF is equal to left ejection volume times acceleration of LA flow according to Newton\u2019s law of motion (force equals mass times acceleration) [2]. Anwar et\u00a0al. confirmed that RT3DE is a useful method for the evaluation of LAEF3D-MAA [3]. In recent studies MAA3D has been confirmed to be traced successfully with good interobserver agreement and comparable to magnetic resonance imaging [5]. RT3DE allowed exact \u2018en-face\u2019 measurement of MAA3D suggesting overstretching and dilatation in dilated [10], hypertrophic [3, 10] and noncompaction cardiomyopathy (present study). LAEF was higher in hypertrophic cardiomyopathy (HCM) patients compared with control subjects, with the highest value found in patients with obstructive HCM due to higher LV resistance [3]. LAEF3D-MAA calculated by the MAA3D-derived formula was higher in HCM patients than by the \u2018mitral valve area\u2019-derived formula either by 2D echocardiography or RT3DE [3].\nIn the present study, 41% of NCCM patients were in NYHA functional class II \/ III HF. Recently, Triposkiadis et\u00a0al. demonstrated that in patients with HF, LAEF and LA kinetic energy are higher than in normal controls [11]. This is most likely due to LA dilation, which compensates for the depressed LA systolic shortening and increases LA active emptying volume and LA contribution to LV stroke volume, maintaining LV stroke volume and cardiac output [11, 12]. However, it should be noted that other authors found decreased LA kinetic energy in patients with advanced HF (NYHA functional class III HF) [13, 14]. It has been concluded that LA systolic failure contributes to the transition from moderate to advanced HF.\nLimitations\nDuring evaluation of LA systolic function, consideration of other factors affecting LAEF is important. According to recent findings, age considerably influences LAEF [15]. This fact could also affect our results, but no significant differences were found in age between NCCM patients and controls. LA systolic function is deteriorated in patients with sick sinus syndrome and in those with paroxysmal atrial fibrillation [16]. However, all of our patients were in sinus rhythm. Cioffi et\u00a0al. demonstrated that LV geometry can also influence LA size and function [17]. The concentric LV pattern was associated with the greatest LA volumes and with the strongest LAEF, suggesting that chronic pressure overload more than volume overload stimulates the LA in increasing its workload. Left atrial dilation can be resulted from chronic mitral regurgitation, which can also affect LA systolic function. Another important limitation is that left atrial contraction produces both the forward blood flow toward the LV through the mitral valve and backward flow towards the pulmonary veins. Unfortunately, only the forward blood flow was examined during the present study.\nConclusions\nLAEF is increased in NCCM patients compared to normal individuals. These results can suggest compensating left atrial work against the dysfunctional LV in NCCM patients. However, further studies are warranted evaluating LA function by RT3DE in patients with different stages of NCCM.","keyphrases":["noncompaction","three-dimensional echocardiography","atrial function"],"prmu":["P","P","P"]}
{"id":"Virchows_Arch-4-1-2329735","title":"Mixed-type gastric carcinomas exhibit more aggressive features and indicate the histogenesis of carcinomas\n","text":"To investigate the pathobiological behaviors of gastric mixed-type (MT) carcinomas and gastric carcinogenesis, the clinicopathological characteristics of MT carcinomas were analyzed and compared with intestinal-type (IT) and diffuse-type (DT) carcinomas. The expression of Ki-67, caspase-3, p53, fragile histine triad (FHIT), maspin, extracellular matrix metalloproteinase inducer (EMMPRIN), vascular growth factor (VEGF), MUC-2, 4, 5AC and 6, CD44, E-cadherin, \u03b2-catenin, and phosphorylated glycogen synthase kinase 3\u03b2-ser9 (P-GSK3\u03b2-ser9) was examined on tissue microarrays using immunohistochemistry. It was found that MT carcinomas exhibited large size, deep invasion, frequent local invasion, and lymph node metastasis in comparison with IT and DT carcinomas (p < 0.05). All the markers except MUC-5AC showed higher expression in IT than DT carcinomas (p < 0.05). The expression of maspin, EMMPRIN, VEGF, MUC-4, and membrane E-cadherin was stronger in MT intestinal than diffuse component (p < 0.05). Immunoreactivities to Ki-67, EMMPRIN, and VEGF were weaker in IT carcinoma than in the MT intestinal portion (p < 0.05), while the opposite was true for CD44, MUC-2, and MUC-6 (p < 0.05). The MT diffuse component displayed a higher expression of FHIT, VEGF, and P-GSK3\u03b2-ser9 than DT carcinoma (p < 0.05). The accumulative survival rate of the IT carcinoma patients was higher than the other types (p < 0.05). The invasive depth, venous invasion, lymph node, peritoneal or liver metastasis, and Lauren's classification were independent prognostic factors for gastric carcinomas (p < 0.05). These findings suggested that MT carcinomas were also indicated to be more aggressive than IT and DT carcinomas. Significant differences were observed in the proliferation, apoptosis, angiogenesis, mucin secretion, and cell adhesion between IT and DT carcinomas, whereas only a few of these characteristics showed differences between the MT intestinal and diffuse parts, thus suggesting that both the MT components might originate from the stem cells with similar genetic traits, but follow different histogenic pathways.\nIntroduction\nGastric carcinoma is the second leading cause of cancer-related death behind lung carcinoma despite a worldwide decline in both its incidence and mortality since the late half of the twentieth century [11]. It continues to be a major health problem because of the slow decrease in incidence in Asia and high mortality of diagnosed gastric carcinoma in the West [23]. Generally, the prognosis of patients with gastric carcinoma is somewhat dependent on its histological type, such as intestinal type (IT) or diffuse type (DT) as defined by Lauren [16].\nAlthough gastric carcinoma is a malignant tumor originating from the same gastric epithelium, its morphological features vary substantially among individual patients. Histologically, IT carcinoma principally includes papillary, well-differentiated, and moderately-differentiated or mucinous adenocarcinoma without signet ring carcinoma (SRC) cells, whereas DT mainly consists of the SRC, poorly differentiated and undifferentiated adenocarcinoma of WHO classification [7, 23]. Although Kushima and Hattori [12] firstly proposed an entity of gastric-type carcinoma and terminologically seemed homologous to the IT one, the carcinoma cells of this type closely resemble normal foveolar cells on the basis of tissue morphology and mucin properties and are considered to have been derived from foveolar hyperplasia or pyloric gland adenoma [12, 13]. Recently, our group also focused on Lauren's classification and found that IT carcinoma frequently occurred in old men, while DT was more frequent in comparatively young women. The latter group was more inclined to invasion into the muscularis propria, lymphatic invasion, and lymph node metastasis, and belonged to a higher TNM staging, in comparison to its IT counterparts. Further analysis demonstrated that IT gastric carcinomas with a more favorable prognosis were prone to high levels of proliferation and apoptosis and also always accompanied by a strong expression of the fragile histine triad (FHIT), phosphatase and tensin homology deleted from human chromosome 10 (PTEN), p53, and extracellular matrix metalloproteinase inducer (EMMPRIN) [30]. Additionally, many genetic and epigenetic changes, such as mutation, deletion, loss of heterozygosity, methylation, and microsatellite instability were differentially observed in both histological types of gastric carcinoma [27, 36]. These data indicate that different carcinogenic pathways exist for gastric IT and DT carcinomas. Although carcinogenesis is a multistage process which consists of a multi-factorial etiology resulting from gene\u2013environment interaction, the intestinal type is frequently related to environmental factors like diet and predominates in areas with a high incidence of this disease. In contrast, the diffuse type is thought to be of genetic origin and evenly distributed worldwide [30, 24]. It is commonly believed that Lauren's classification is valuable for both the epidemiological studies and gastric carcinogenesis.\nHowever, a small number of gastric carcinomas remain characterized as unclassified or mixed-type (MT) ones, which are comprised histologically of non-homogenous mixtures of IT and DT carcinomas. Stelzner and Emmrich [26] found that 28 MT carcinomas showed a deep infiltration of the gastric wall, frequent regional lymph node metastasis, and high staging in comparison to the other two types. Machado et al. [21] reported that inactivating E-cadherin mutations were exclusively observed in the diffuse component of the tumors, thus suggesting that MT carcinomas displayed phenotypic divergence. A recent study showed that bone-marrow-derived cells (BMDCs) could progress through metaplasia and dysplasia to intraepithelial cancer under the induction of the carcinogen, Helicobacter pylori [9]. Therefore, the analysis of distinct MT carcinoma components can be of remarkable pathogenic significance if it would be true that carcinoma cells originate from BMDCs. In the present study, 814 cases of gastric carcinomas were collected to further clarify the pathological characteristics of MT carcinoma and the gastric carcinogenesis pathways using a combination of tissue microarray (TMA) and immunohistochemical techniques.\nMaterials and methods\nSubjects\nA total of 814 gastric carcinomas were collected from surgical resection in Kouseiren Takaoka Hospital between 1998 and 2006. The patients with carcinomas included 573 men and 241 women (29\u201391\u00a0years, mean\u2009=\u200965.7\u00a0years). Among them, 312 cases were demonstrated with lymph node metastasis and 24 with liver metastasis. None of these cases underwent either chemotherapy or radiotherapy before surgery. All patients gave their informed consent for the use of tumor tissue specimens for clinical research and the University Ethical Committee approved the research protocol. All patients were followed up by consulting their case documents and through telephone interviews.\nPathology\nAll tissue specimens were fixed in 4% neutralized formaldehyde, embedded in paraffin, and cut into 4-\u03bcm sections. These sections were stained by hematoxylin and eosin (HE) to confirm their histological diagnosis and other microscopic characteristics. The staging for each gastric carcinoma was evaluated according to the TNM system of the Internationale Contre le Cancer (UICC) indicating the extent of tumor spread [25]. The histomorphological architecture of the tumors was expressed according to Lauren's classification [7, 16, 30]. The growth patterns were divided into five groups based on a modification of Emmrich's method [26]. Briefly, group I showed a combination of two components with diffuse distribution. Group II displayed both components with the border clearly visible. Group III represented some signet cells in the intestinal and diffuse components. Group IV were mucinous carcinomas with signet cells. Group V exhibited separately intestinal and diffuse components in the gastric wall. In addition, the depth of invasion, lymphatic and venous invasion, and peritoneal dissemination were all determined.\nTissue microarray\nIT and DT carcinomas, as well as both components of the mixed type, were identified in HE stained sections of the selected tumor cases and a 2-mm-diameter tissue core of each donor block was punched out and transferred to a recipient block with a maximum of 48 cores using a Tissue Microarrayer (AZUMAYA KIN-1, Tokyo, Japan). Four-micrometer-thick sections were cut from the recipient block and transferred to poly-lysine-coated glass slides. HE staining was performed on TMA for confirmation of the tumor tissue (Fig.\u00a01).\nFig.\u00a01HE staining of TMA of gastric carcinomas\nImmunohistochemistry\nConsecutive sections were deparaffinized with xylene, dehydrated with alcohol, and subjected to antigen retrieval by irradiating in target retrieval solution (TRS, DAKO, Carpinteria, UAS) for 15\u00a0min in a microwave oven (Oriental rotor Lmt. Co. Tokyo, Japan). Five percent bovine serum albumin (BSA) was then applied for 15\u00a0min to prevent non-specific binding. The sections were incubated with primary antibodies for 15\u00a0min, then treated with the anti-mouse or anti-rabbit Envison-PO (DAKO, USA) antibodies for 15\u00a0min. All incubations were performed in the microwave oven for intermittent irradiation as described previously [15]. After each treatment, the slides were washed with TBST (10\u00a0mM Tris\u2013HCl, 150\u00a0mM NaCl, 0.1% Tween 20) three times for 1\u00a0min. The primary antibodies are summarized in Table 1. All immunostaining was visualized with 3, 3\u2032-diaminobenzidine (DAB) with 5-min reaction and counterstained with Mayer's hematoxylin. Omission of the primary antibody was used as a negative control.\nTable\u00a01 Primary antibodies used in this studyNamesSourceCompanyDilutionKi-67RabbitDAKO, Carpinteria, USA1:25Caspase-3RabbitDAKO, USA1:150p53MouseDAKO, USA1:100FHITRabbitNeomarkers, Fremont, USA1:200MaspinMouseNovocastra, Newcastle upon Tyne, UKRead-to-useEMMPRINMouseNovocastra, UK1:100VEGFRabbitLabvision, Fremont, USA1:50P-GSK3\u03b2-ser9RabbitSAT, USA1:300MUC-2MouseNovocastra, UK1:100MUC-4MouseNovocastra, UK1:100MUC-5ACMouseNovocastra, UK1:100MUC-6MouseNovocastra, UK1:100CD44MouseDAKO, USA1:50E-cadherinMouseTakara, Otsu, Japan1:100\u03b2-CateninMouseCalbiochem, CA, USA1:200\nThe immunoreactivity to Ki-67 and p53 was localized in the nucleus; FHIT, caspase-3, vascular growth factor (VEGF), maspin, phosphorylated glycogen synthase kinase 3\u03b2-ser9 (P-GSK3\u03b2-ser9), MUC-2, MUC-5AC, and MUC-6 in the cytoplasm; EMMPRIN and MUC-4 in the cytoplasm and membrane; CD44 and E-cadherin in the membrane; and \u03b2-catenin in the nucleus, cytoplasm, and membrane (Fig.\u00a02). All evaluations were performed blindly by two independent observers (Takano Y and Zheng HC).\nFig.\u00a02Immunostaining of TMA of gastric carcinomas. The immunoreactivity to Ki-67 (a) and p53 (c) was localized in the nucleus; FHIT (d), caspase-3 (b), VEGF (g), maspin (e), P-GSK3\u03b2-ser9 (h), MUC-2 (i), MUC-5AC (k), MUC-6 (l) were seen in the cytoplasm, EMMPRIN (f) and MUC-4 (j) were seen in the cytoplasm and the plasma membrane, CD44 (m), and E-cadherin (n) in the plasma membrane; and \u03b2-catenin (o) in the nucleus, cytoplasm, and plasma membrane. These molecules were strongly expressed in the intestinal-type gastric carcinomas\nStatistical analysis\nThe statistical evaluation was performed using Fisher's exact possibility to differentiate the rates and the Mann\u2013Whitney U test to differentiate the means of the different groups. Kaplan\u2013Meier survival plots were generated and comparisons between the survival curves were made with the log-rank statistic. The Cox proportional hazards model was employed for multivariate analysis. p\u2009<\u20090.05 was considered to represent a statistically significant difference. The SPSS 10.0 software program was employed to analyze all data.\nResults\nClinicopathological characteristics of gastric IT, DT, and MT carcinomas\nAs summarized in Table\u00a02, 415 IT cases were identified (51.0%), 221 DT (27.1%), and 178 MT (21.9%) among the 814 gastric carcinomas in this study. Patients with IT carcinoma were found to be significantly older than those with DT (p\u2009<\u20090.05). The male and female ratios in the carcinoma cases gradually decreased from IT to DT through the MT groups (p\u2009<\u20090.05). It was the same for lower TNM staging in the three groups (p\u2009<\u20090.05). The positive rate of peritoneal spread was higher in the DT and MT cases than IT (p\u2009<\u20090.05). MT carcinomas showed aggressive characteristics, such as large size, deep invasion, frequent local invasion, and lymph node metastasis, compared to the IT and DT (p\u2009<\u20090.05). There was no significance between the three types of gastric carcinomas in the incidence of the liver metastasis (p\u2009>\u20090.05).\nTable\u00a02 Comparison of the clinicopathological features in gastric IT, DT, and MT carcinomasClinicopathological featuresIntestinal-type carcinomaDiffuse-type carcinomaMixed-type carcinomaCase number415 (51.0%)221 (27.1%)178 (21.9%)Age (mean \u00b1 SD, years)67.09\u2009\u00b1\u200910.7362.03\u2009\u00b1\u200911.3*65.80\u2009\u00b1\u200913.28Sex (male:female)324:91125:96124:54**Tumor size (mean \u00b1 SD, cm)3.82\u2009\u00b1\u20093.215.50\u2009\u00b1\u20096.955.56\u2009\u00b1\u20093.08**Invasion into muscularis propria153 (36.9%)121 (54.8%)123 (69.1%)**Lymphatic invasion (+)117 (28.2%)83 (37.6%)99 (55.6%)**Venous invasion (+)49 (11.8%)32 (14.5%)39 (21.9%)**Lymph node metastasis (+)113 (27.2%)94 (42.5%)105 (59.0%)**Liver metastasis (+)10 (2.4%)8 (3.6%)6 (3.4%)Peritoneal spread (+)14 (3.4%)***22 (10.0%)21 (11.8%)TNM staging (O, I)302 (72.8%)69 (31.2%)112 (63.0%)***p\u2009<\u20090.05 compared with intestinal- and mixed-type carcinomas**p\u2009<\u20090.05 compared with intestinal- and diffuse-type carcinomas with both analyzed***p\u2009<\u20090.05 compared with diffuse- and mixed-type carcinomas\nPathological behaviors of gastric MT carcinomas with different growth patterns or histological distribution of components\nAccording to the growth pattern, the intestinal and diffuse components diffusely distributed without absolute border in group I of the MT carcinomas, which occurred more frequently (60.0%, 106\/178) than the other four groups. As indicated in Table\u00a03, most groups III and V gastric carcinomas were generally of early stage with less local invasion, infrequent lymph node metastasis and low TNM staging. In this study, only four advanced gastric carcinomas belonged to group IV, among which three cases were women and all were accompanied with lymphangiogenic invasion, lymph node metastasis, and high TNM staging. Although the main histological component was different from that in lymph node metastasis, the statistical data revealed that there was histological consistency between primary and lymph node metastasis foci (p\u2009<\u20090.05). There were no remarkable differences in the pathological behaviors of MT carcinomas with different ratios of intestinal and diffuse portions (p\u2009>\u20090.05).\nTable\u00a03 Pathological behaviors of gastric MT carcinomasClinicopathological featuresnSex (male)Invasion into MPLymphatic invasionVenous invasionLymph node metastasisStagingnI>DI=DI<D(O, I)Growth pattern\u00a0I10673 (68.9%)82 (77.4%)64 (60.4%)26 (24.5%)69 (65.1%)32142335 (33.0%)\u00a0II2518 (78.3%)21 (84.0%)16 (64.0%)7 (28.0%)16 (64.0%)5567 (28.0%)\u00a0III2922 (75.9%)11 (37.9%)9 (31.0%)3 (10.3%)10 (34.5%)43318 (62.1%)\u00a0IV41 (25.0%)4 (100.0%)4 (100.0%)1 (25.0%)4 (100.0%)0220 (0.0%)\u00a0V1410 (71.4%)5 (35.7%)6 (42.9%)2 (14.3%)6 (42.9%)2049 (64.3%)Histological appearance\u00a0Intestinal>diffuse8059 (73.8%)54 (67.5%)43 (53.8%)20 (25.0%)48 (60.0%)3081033 (41.3%)\u00a0Intestinal=diffuse2015 (75.0%)12 (60.0%)9 (45.0%)5 (25.0%)10 (50.0%)3528 (40.0%)\u00a0Intestinal<diffuse7850 (64.1%)57 (73.1%)47 (60.3%)14 (18.0%)47 (60.3%)10112628 (35.9%)MP Muscularis propria; in lymph node metastatic foci: I>D intestinal>diffuse, I<D intestinal<diffuse, I=D intestinal=diffuse\nImmunohistochemical analysis of intestinal or diffuse components from three types of gastric carcinomas\nTable\u00a04 demonstrates that all the markers except MUC-5AC were expressed at higher levels in IT carcinomas than in MT ones (p\u2009<\u20090.05). The expression of maspin, EMMPRIN, VEGF, MUC-4, and E-cadherin was stronger in the intestinal component of MT carcinomas than in their diffuse counterpart (p\u2009<\u20090.05). Immunoreactivities to Ki-67, EMMPRIN, and VEGF were weaker in the intestinal-type carcinomas than in the intestinal component of the MT (p\u2009<\u20090.05), while it was the opposite for CD44, MUC-2, and MUC-6 (p\u2009<\u20090.05). In contrast, the diffuse components of the MT carcinomas showed greater expression of Ki-67, FHIT, VEGF, and P-GSK3\u03b2-ser9 than DT carcinomas (p\u2009<\u20090.05).\nTable\u00a04 Immunohistochemical analysis in gastric IT, DT, and MT carcinomasBiological markersIntestinal-type carcinomaMixed-type carcinomaDiffuse-type carcinomaIntestinal partDiffuse partKi-6788\/146 (60.3%)*,**81\/110 (73.6%)78\/110 (70.9%)*57\/119 (47.9%)Caspase-384\/152 (55.3%)*52\/119 (43.7%)37\/119 (31.1%)24\/115 (20.9%)p5376\/151 (50.3%)*37\/118 (31.4%)30\/118 (25.4%)25\/120 (20.8%)FHIT90\/150 (60.0%)*63\/113 (55.8%)48\/113 (42.5%)*27\/116 (23.3%)Maspin71\/150 (47.3%)*55\/112 (49.1%)***34\/112 (30.4%)38\/120 (31.7%)EMMPRIN87\/147 (59.2%)*,**77\/112 (68.8%)***43\/112 (38.4%)32\/117 (26.5%)VEGF95\/151 (62.9%)*,**90\/114 (78.9%)***61\/114 (53.5%)*31\/121 (25.6%)P-GSK3\u03b2-ser988\/144 (61.1%)*74\/113 (65.5%)67\/113 (59.3%)*48\/116 (41.4%)MUC-255\/145 (37.9%)*,**31\/118 (26.3%)27\/118 (22.9%)23\/117 (19.7%)MUC-458\/153 (37.9%)*39\/118 (33.1%)***21\/118 (17.8%)18\/118 (15.3%)MUC-5AC84\/148 (56.8%)57\/106 (53.8%)50\/106 (47.2%)54\/113 (47.8%)MUC-660\/146 (41.1%)*,**26\/117 (22.2%)18\/117 (15.4%)9\/123 (7.3%)CD4464\/152 (42.1%)*,**32\/114 (28.1%)31\/114 (27.2%)32\/120 (26.7%)E-cadherin91\/142 (64.1%)*58\/112 (51.8%)***38\/112 (33.9%)43\/119 (36.1%)Membrane \u03b2-catenin57\/144 (39.6%)*36\/117 (30.8%)26\/117 (22.2%)26\/118 (22.0%)*p\u2009<\u20090.05 compared with the diffuse-type carcinomas**p\u2009<\u20090.05 compared with intestinal-part carcinomas***p\u2009<\u20090.05 compared with diffuse-part carcinomas\nPatients' outcome with different gastric carcinomas\nFollow-up information for 500 carcinoma patients was used before 2002 for a period ranging from 5\u00a0days to 9.15\u00a0years (mean\u2009=\u200949.4\u00a0months). Figure 3 shows the survival curves stratified according to Lauren's classification. Kaplan\u2013Meier analysis indicated that the patients with IT carcinoma had a higher cumulative survival rate than those with the DT and MT lesions (p\u2009<\u20090.05). Although the MT carcinoma patients' survival rate was comparatively lower than the DT ones, there was no statistical significance (p\u2009>\u20090.05). Multivariate analysis demonstrated that invasive depth, venous invasion, lymph node, peritoneal or liver metastasis, and Lauren's classification were independent factors for the poor prognosis of gastric carcinoma patients (p\u2009<\u20090.05; Table 5).\nFig.\u00a03Relationship between prognosis of carcinoma patients' and Lauren's classification Kaplan\u2013Meier curves of cumulative survival rate for the patients with gastric carcinoma according to Lauren's classification. IT Intestinal type, DT diffuse type, MT mixed typeTable\u00a05 Multivariate analysis of the clinicopathological variables of gastric carcinomasClinicopathological parametersRelative risk (95% CI)p ValueAge (\u226565\u00a0years)1.224 (0.849\u20131.764)>0.05Sex (male:female)1.255 (0.827\u20131.904)>0.05Tumor size (\u22654\u00a0cm)1.466 (0.862\u20132.493)>0.05Invasive depth (Tis,1\/T2,3)2.945 (1.533\u20135.657)<0.05Lymphatic invasion (\u2212\/+)1.394 (0.865\u20132.247)>0.05Venous invasion (\u2212\/+)1.692 (1.097\u20132.608)<0.05Lymph node metastasis (\u2212\/+)1.983 (1.111\u20133.537)<0.05Peritoneal spread (\u2212\/+)3.197 (2.031\u20135.031)<0.05Liver metastasis (\u2212\/+)5.248 (2.575\u201310.697)<0.05Lauren's classification (intestinal\/diffuse\/mixed)1.351 (1.084\u20131.683)<0.05CI Confidence interval\nDiscussion\nHistologically, Lauren's classification of gastric carcinomas is based on the morphological appearances. IT carcinomas are characterized by cohesive carcinoma cells forming gland-like tubular structures with an expanding or infiltrative growth pattern, like a papillary and well-differentiated adenocarcinoma. However, the cell cohesion is less apparent or absent in DT carcinomas, and cancer cells diffusely spread in the gastric wall as poorly differentiated adenocarcinomas, SRC, or undifferentiated carcinomas [23, 30]. However, there are two types of histological distribution in the MT carcinoma, as described by Lauren [16]. In this study, carcinoma diagnosed as MT made up 21.9% of the whole series in this population, more than that reported by Borch et al. (13%), Lauren (14%), and Stelzner and Emmrich (14.1%), and less than that reported by Carneiro et al. (38.5%) [2, 5, 16, 26]. This discrepancy might be due to the differences in diagnosis criteria, geographical regions, human races, living habits, and so forth. The age and sex distribution of the patients with MT carcinoma was intermediate although IT carcinomas frequently occurred in old men, compared with DT carcinomas.\nMorphologically, there are both intestinal and diffuse components in MT carcinomas, and they were found to be significantly larger, more advanced, and more frequently spread into local lymphatic and venous vessels or regional lymph nodes, compared with IT and DT carcinomas in the present study, consistent with other reports [6, 16, 30]. Kozuki et al. [14] found that prominent lymphatic permeation and lymph node metastasis were more frequently observed in MT than in the pure type of gastric carcinomas. It was previously documented that the MT carcinomas showed a deeper infiltration of the gastric wall, a higher metastatic rate to the regional lymph nodes, and the need for higher staging with the TNM system of UICC classification, in contrast to IT and MT carcinomas [26]. These findings suggest that the pathobiological characteristics of the MT carcinomas were more aggressive than the other two types, which accounted for their comparatively poor prognosis observed in the present study although there was no statistical significance. Among the MT carcinomas, five groups were classified according to their growth pattern. Half of the present cases belonged to group I, which means there was diffuse distribution of the intestinal and diffuse component without clear borders in most of the MT cases. In general, groups III and IV of MT carcinomas did not exhibit deep invasion, comparatively high staging or frequent spreading, compared with other types. However, group IV of carcinomas with mucin and signet cells was more advanced with wide spreading, including local vessels and lymph node metastasis. Therefore, surgeons should carefully treat the patients with MT carcinomas in clinical practice because of their aggressive characteristics and poor prognosis, especially those classified as group IV. Furthermore, it was found that the major component in the MT primary foci morphologically paralleled to that in the corresponding lymph node metastasis although the converse situation also appeared. This inconsistent phenomenon could be explained by sampling bias or the metastatic potential of individual carcinoma cells.\nCompared with IT carcinomas, the MT counterpart always exhibited more severe characteristics, including invasion into muscularis propria, lymphatic invasion and lymph node metastasis, and high TNM staging in the present study, as observed by Lauren and other studies [16, 26, 30]. To clarify the pheno\/genotypes of gastric carcinoma, the expression of Ki-67, caspase-3, p53, FHIT, maspin, VEGF, EMMPRIN, P-GSK3\u03b2-ser9, MUC-2, MUC-4, MUC-5AC, MUC-6, E-cadherin, and \u03b2-catenin was examined by immunostaining. p53, FHIT, and maspin as tumor suppressor genes play important roles in regulating the balance between the proliferation and apoptosis of cancer cells [25, 30, 31, 34, 35]. Ki-67 antigen is present in the nuclei of cells undergoing proliferation and should be regarded as a good marker for cell proliferation [34]. Caspase-3 is responsible for the cleavage of poly (ADP-ribose) polymerase, actin, and sterol regulatory element binding protein and reflects the apoptotic level as a key protease in the cascade reaction of the apoptotic pathway [28]. In vivo and vitro evidences indicated that EMMPRIN and VEGF are involved in angiogenic processes in malignancies [29, 32]. Glycogen synthase kinase-3\u03b2 (GSK3\u03b2) belongs to the serine\/threonine protein kinase family and is also involved in regulating the balance between proliferation and apoptosis and can be inactivated via ser-9 phosphorylation by p70 S6 kinase, p90Rsk, Akt, certain isoforms of proteins kinase, and cyclic AMP-dependent protein kinase [1]. MUC-2, MUC-5AC, and MUC-6 are markers for intestinal goblet cells, superficial epithelium, and gastric pyloric gland cells, respectively, which can reflect the mucin secretion and variations in gastrointestinal malignancies. MUC-4 is a heterodimeric glycoprotein complex and expressed in several human epithelial carcinomas [3, 33]. CD44 is a cell surface glycoprotein involved in cell\/cell and cell\/matrix interactions. CD44 overexpression has been linked to the growth and spread of a range of different types of malignancies [8]. The E-cadherin can interact with \u03b2-catenin to form a complex, which is closely linked to cell adhesion and differentiation [19, 27, 36]. In the present study, all of the markers except MUC-5AC showed greater expression in the IT than in the DT carcinomas. Although some evidences indicate that MUC-5AC was strongly expressed in gastric DT carcinoma, in comparison with IT ones, there was no statistical difference in line with our finding [20, 22]. These data suggested that there were significant differences in the proliferation, apoptosis, angiogenesis, mucin production, and cell adhesion between the IT and DT carcinomas and these molecules mechanistically contributed to the molecular distinction in the morphological, behavioral, and histogenic aspects between both types of gastric carcinomas.\nIn general, DT carcinoma is believed to derive de novo from the peripheral stem cells of gastric gland neck proliferative zone without a recognizable precursor lesion except hereditary diffuse gastric cancer because in situ carcinoma or globoid dysplasia is its precancerous lesion, respectively, according to the Chinese and Western pathologists' opinions [4, 5, 9, 18,30]. When long-standing gastric inflammation causes tissue injury and stem cell failure with time, BMDCs are recruited and engrafted into the tissue stem cell niche, where BMDCs can behave in a way indistinguishable from endogenous tissue stem cells. With continued inflammation and injury, they can undergo sustained proliferation and malignant transformation into IT carcinomas, passing through precancerous stages of metaplasia and dysplasia when genetic defects, such as mutation, deletion, or rearrangement, are accumulated resulting in corruption of the balance between proliferation and apoptosis [17]. Likewise, dedifferentiation of intestinal to diffuse carcinoma had been identified as another histogenic pathway according to the histological heterogeneity of tumor cells, especially in the carcinomas containing moderately and poorly differentiated components with similar morphological appearance and diffuse distribution. If so, it is possible that the distinct components in MT carcinomas arise from the stem cells with common genetic traits and follow different carcinogenic pathways. Conversely, diffuse carcinoma could be derived from heterogeneity of the intestinal counterpart. According to our present data, no differences in the expression of Ki-67, caspase-3, FHIT, CD44, P-GSK3\u03b2-ser9, MUC-2, MUC-5AC, MUC-6, and membrane \u03b2-catenin were observed between the intestinal and diffuse components of MT carcinomas, which supports the possibility of similar origin or dedifferentiation. However, increased expression of EMMPRIN, VEGF, MUC-4, and E-cadherin in the intestinal component compared to the diffuse counterpart also provided evidence that the original carcinoma cells might undergo distinct carcinogenic routes resulting in the morphological distinction of both components. Both the intestinal and diffuse components in MT carcinomas had increased expression of Ki-67, EMMPRIN, and VEGF and reduced E-cadherin. Serum VEGF levels were found to be significantly higher in patients with MT gastric carcinomas than those with pure lesions [10]. It was proposed that MT carcinomas biologically displayed more aggressive behaviors than other types, including decreased cell adhesion, increased proliferation, and angiogenesis.\nMost of the evidences in the present data indicated that the pathological behaviors of MT carcinomas were more aggressive than the other types, which was closely linked to the prognosis. It was previously documented that the MT patients' survival is significantly worse than those with IT or DT carcinomas regardless of their location [5]. However, this study demonstrated that there was no significance between the MT and DT carcinoma patients' survival rates although the former was lower than the latter. Additionally, both survival rates were lower than that with IT carcinomas. To avoid sampling bias from a shorter follow-up time, the older cases were chosen for the survival analysis, but they yielded consistent results. Furthermore, Cox's hazard proportional analysis indicated that the invasive depth, venous invasion, lymph node, peritoneal or liver metastasis, and Lauren's classification were independent prognostic factors for gastric carcinomas.\nIn summary, IT carcinoma, which is positively correlated with favorable prognosis, frequently displayed high levels of proliferation, apoptosis, angiogenesis, mucin production, and cell adhesion. Gastric MT carcinoma showed more aggressive behaviors than IT and DT ones. There was a significant difference in the proliferation, apoptosis, angiogenesis, mucin secretion, and cell adhesion between the IT and DT carcinomas, whereas only a few characteristics were differentially detected in the intestinal and diffuse component of the mixed-type carcinoma, suggesting that different components of MT carcinoma might originate from common stem cells, but follow distinct histogenic pathways. Furthermore, these results confirm that Lauren's classification is significant regarding the histopathogenesis and differentiation and considered as a guide to the clinical treatment of gastric carcinoma.","keyphrases":["gastric carcinoma","pathobiological behaviors","carcinogenesis","lauren's classification","mixed type"],"prmu":["P","P","P","P","P"]}
{"id":"Biochim_Biophys_Acta-2-1-2323027","title":"Translational control by cytoplasmic polyadenylation in Xenopus oocytes\n","text":"Elongation of the poly(A) tails of specific mRNAs in the cytoplasm is a crucial regulatory step in oogenesis and early development of many animal species. The best studied example is the regulation of translation by cytoplasmic polyadenylation elements (CPEs) in the 3\u2032 untranslated region of mRNAs involved in Xenopus oocyte maturation. In this review we discuss the mechanism of translational control by the CPE binding protein (CPEB) in Xenopus oocytes as follows:1.The cytoplasmic polyadenylation machinery such as CPEB, the subunits of cleavage and polyadenylation specificity factor (CPSF), symplekin, Gld-2 and poly(A) polymerase (PAP).2.The signal transduction that leads to the activation of CPE-mediated polyadenylation during oocyte maturation, including the potential roles of kinases such as MAPK, Aurora A, CamKII, cdk1\/Ringo and cdk1\/cyclin B.3.The role of deadenylation and translational repression, including the potential involvement of PARN, CCR4\/NOT, maskin, pumilio, Xp54 (Ddx6, Rck), other P-body components and isoforms of the cap binding initiation factor eIF4E.\n1\nIntroduction\nThe regulation of translation of mRNAs by cytoplasmic elongation of the poly(A) tail was discovered some twenty years ago in the oocytes and early embryos of clams, worms, frogs and mice [1\u20136]. These maternal mRNAs are stored in the growing oocyte with a short poly(A) tail of 20 to 40 nucleotides and are translationally repressed (masked). Upon oocyte maturation or after fertilisation, the poly(A) tail of masked mRNAs is elongated to 80\u2013250 residues and the mRNAs are translationally activated. A long poly(A) tail is thought to be stimulatory to translation through the binding of cytoplasmic poly(A) binding proteins, which recruit initiation factors and form a closed-loop complex through their association with the translation initiation factor eIF-4G [7]. Cytoplasmic polyadenylation is essential for the meiotic maturation of the oocyte in both Xenopus and mouse, as it mediates the translational activation of the mRNAs encoding c-Mos kinase and mitotic cyclins [8]. In addition, cytoplasmic polyadenylation has been implicated in the cell cycle, in cellular senescence and in the formation of memory through synaptic plasticity [9]. In this review we will discuss the current knowledge of the mechanism of translational control by cytoplasmic polyadenylation, with an emphasis on Xenopus oocyte maturation and CPEB, the cytoplasmic polyadenylation element binding protein. A review of the role of cytoplasmic polyadenylation in mammalian gametogenesis is provided elsewhere in this volume (Tadashi Baba).\n2\nmRNA elements and RNA binding proteins\nIn order for mRNAs to be singled out for cytoplasmic polyadenylation, they have to be recognised by the polyadenylation machinery. In oocytes and early embryos of the African clawed frog, Xenopus, cytoplasmic polyadenylation can be conferred by several discrete elements in the 3\u2032 untranslated region (3\u2032 UTR) of the mRNA which are recognised by specific RNA binding proteins. At least 4 different cytoplasmic polyadenylation elements have been identified in Xenopus. The C-rich cytoplasmic polyadenylation element and the U-rich embryonic cytoplasmic polyadenylation element normally mediate cytoplasmic polyadenylation in the zygote and early embryo [10,11]. The putative cytoplasmic polyadenylation factors binding to these elements are poly(rC) binding protein 2 (PCBP2) and Elav related protein A, the ortholog of HuR, respectively [10,12\u201314]. A polyadenylation element that functions early in oocyte maturation, the polyadenylation response element (PRE), was predicted computationally [15,16]. The RNA binding protein Musashi was shown to bind to some, but not all PREs and mutating Musashi or its known consensus binding site blocked cytoplasmic polyadenylation, indicating that it is likely to be a cytoplasmic polyadenylation factor [17]. These data suggest that the collection of cytoplasmic polyadenylation elements that have been called PREs may represent a mixture of Musashi binding sites and at least one more uncharacterised cytoplasmic polyadenylation element.\nBy far the best characterised cytoplasmic polyadenylation element is the CPE, which is required for the cytoplasmic polyadenylation of a number of mRNAs, including cyclin B1 mRNA, during oocyte maturation and during the embryonic cell cycle [18\u201320]. The consensus CPE is often described as U5AU, but variations such as U4AU (c-mos), and U4\u20135A2U (cyclin B1) have also been shown to be active [5,6,21,22]. The CPEs in the mRNA encoding mouse c-Mos are active in mature Xenopus oocytes, but only conform to U5A [23,24]. Strongly polyadenylating mRNAs, however, generally adhere to the U5A1\u20132U consensus. In addition, examination of the cases in the existing literature suggests that CPEs that mediate significant polyadenylation tend to be close to the polyadenylation signal, from downstream to overlapping and up to approximately 60\u00a0nt upstream [21,25\u201329].\nThe CPE binds cytoplasmic polyadenylation element binding protein (CPEB), an RNA binding protein containing two RNA recognition motifs and a zinc finger region, all of which are required for recognition of the CPE [21,30,31]. In addition to the fact that many mRNAs bearing a CPE undergo cytoplasmic polyadenylation, a wealth of evidence indicates that CPEB is required for cytoplasmic polyadenylation. Depletion of CPEB from an egg extract abolishes polyadenylation and injection of a CPEB antibody blocks it in oocytes and embryos [20,21,31,32]. N-terminal deletions and phosphorylation site mutants of CPEB act as dominant negative blockers of cytoplasmic polyadenylation [20,21,31,32]. In mice, knock out or knock down of Cpeb1 causes poly(A) tail changes in target mRNAs in early oogenesis and oocyte maturation [33,34]. CPEB is therefore the only well-established mRNA specificity factor for cytoplasmic polyadenylation in vertebrates.\nIn addition to an mRNA specific element, the nuclear poly(A) signal (AAUAAA or AUUAAA) is absolutely required for cytoplasmic polyadenylation [5,6,11,22,28]. The poly(A) signal is thought to recruit a variant of the cleavage and polyadenylation specificity factor (CPSF), a four subunit complex that binds this element and mediates polyadenylation in the nucleus [35,36]. The 160\u00a0kDa subunit is the RNA binding protein that recognises the poly(A) signal [37], while the 73\u00a0kDa subunit is the endonuclease that mediates the formation of the 3\u2032 ends of all mRNAs [38\u201341]. The 100\u00a0kDa subunit resembles the 73\u00a0kDa subunit but does not appear to have nuclease activity and its precise function in 3\u2032 end processing is unknown. The 30\u00a0kDa subunit is a zinc finger protein that has been reported to have some affinity for RNA and it potentially has endonuclease activity. It was recently shown to mediate interactions between CPSF and the body of RNA polymerase II [42\u201345]. An additional CPSF associated factor, Fip1, has been shown to mediate the interaction with poly(A) polymerase and have U-rich RNA binding activity [46]. It has probably been present in most of the previously utilised preparations of CPSF and is thought of as a fifth subunit by many in the field [40,46].\nThe 100 and 30\u00a0kDa CPSF subunits are present in the Xenopus oocyte cytoplasm, and the 160\u00a0kDa subunit has been inferred to be present from its binding activity, while the 73\u00a0kDa endonuclease is absent from the cytoplasmic CPSF complex [47]. It is unknown whether Fip1 is present in the oocyte cytoplasm. Immunodepletion of the 100\u00a0kDa subunit from egg extracts or expression of a viral protein that blocks nuclear polyadenylation by binding to the 30\u00a0kDa subunit also abolishes cytoplasmic polyadenylation, demonstrating that CPSF is required for this process [47]. In addition, CPEB is coimmunoprecipitated with the 100\u00a0kDa CPSF subunit from oocyte extracts (but not with the 73\u00a0kDa subunit) and it binds to the 160\u00a0kDa subunit in reticulocyte lysate [47,48]. Whether these interactions are direct or mediated by other proteins present in both oocyte extract and reticulocyte lysate is not yet clear. Curiously, the binding of CPSF to the poly(A) signal is stimulated by a CPE even in the absence of CPEB, a phenomenon that could be mediated by the RNA binding activity of the 30\u00a0kDa subunit or by Fip1 [46,49]. However, CPEB strongly stimulates polyadenylation of a CPE containing RNA in a pure reaction system containing nuclear CPSF and poly(A) polymerase [48]. It is therefore likely that CPEB improves the recruitment of CPSF to the mRNA by binding directly to both.\n3\nSymplekin\nSymplekin is a protein found in nuclear complexes containing CPSF and other processing factors and is thought of as a scaffold protein involved in 3\u2032 end RNA processing [50\u201352]. In yeast and plants, homologues of symplekin are required for proper cleavage and polyadenylation, and this is possibly also the case in vertebrates [53\u201355]. In vertebrate somatic cells symplekin is predominantly nuclear and in epithelial cells also localised at tight junctions [50,56]. In the Xenopus oocyte, however, symplekin is also found in the cytoplasm in complexes with the 100\u00a0kDa subunit of CPSF [50]. A symplekin antibody also precipitates CPEB from oocyte extracts, indicating that the protein is in cytoplasmic polyadenylation complexes [57]. Moreover, the symplekin antibody inhibits cytoplasmic polyadenylation in oocytes and extracts [57]. These data strongly suggest that symplekin is involved in cytoplasmic polyadenylation.\n4\nThe cytoplasmic poly(A) polymerase\nInitially, a form of one of the classical nuclear poly(A) polymerases was thought to be responsible for cytoplasmic polyadenylation too. In vitro reconstitution experiments indicated that the poly(A) polymerase (PAP) isolated from bovine thymus can mediate CPE and poly(A) signal dependent polyadenylation [47\u201349]. Cytoplasmic poly(A) polymerase species were detected in Xenopus oocytes and an isoform was cloned that was 87% identical to bovine PAP but lacked the nuclear localisation signal [58,59]. Three different polyclonal antibodies against this protein inhibited polyadenylation in egg extracts [58]. However, after these findings the work on the cytoplasmic poly(A) polymerase stagnated. No native complex containing CPEB and a PAP isoform was ever reported and studies on the regulation of PAP activity indicated that the enzyme is inactivated by phosphorylation during oocyte maturation, when cytoplasmic polyadenylation is actually induced [60].\nIn 2002, a Caenorhabditis elegans germline determinant called gld-2 was cloned and shown to encode a cytoplasmic poly(A) polymerase that is recruited to certain mRNAs by an RNA binding protein [61]. The Gld-2 protein belongs to the same large family of DNA polymerase \u03b2 nucleotidyl transferases, but only has limited additional homology to the classical poly(A) polymerases and it lacks the RNA binding domain. It soon transpired that this protein is widely conserved, having homologues in fission yeast and mammalians [62,63].\nThe Xenopus Gld-2 protein (xGld-2) coimmunoprecipitated very efficiently with symplekin in both mature and immature oocytes. In addition, tagged xGld-2 was shown to bind the 160\u00a0kDa CPSF subunit as well as CPEB synthesised in reticulocyte lysate [57]. This indicates that xGld-2 can form complexes with symplekin, CPSF and CPEB, although the direct interactions are as yet unclear. Gld-2 overexpressing oocytes and extracts supplemented with Gld-2 displayed an increase in their CPE-dependent polyadenylation and symplekin depleted extracts regained some polyadenylation activity if they were replenished with recombinant CPEB, symplekin and Gld-2 [57].\nThe less than optimal reconstitution of cytoplasmic polyadenylation by Gld-2 may indicate that cytoplasmic PAP or an unknown additional cytoplasmic polyadenylation factor is being depleted with symplekin. However, it is also very plausible that not all the supplemented proteins are completely active. Especially CPEB is notoriously difficult to obtain in a soluble form and isolation procedures usually include a denaturing step [30]. On the whole, the evidence seems to indicate that xGld-2 is an authentic cytoplasmic poly(A) polymerase in Xenopus oocytes, but involvement of the cytoplasmic PAP can as yet not be excluded. In Drosophila, PAP has been implicated in cytoplasmic polyadenylation in embryos [64], and although initial experiments implicated mouse Gld-2 in cytoplasmic polyadenylation in oocytes, polyadenylation appeared unimpaired in a knock out mouse [65,66]. The cytoplasmic polyadenylation mechanisms in mammalian germ cells are discussed in more detail by T. Baba elsewhere in this volume.\nWhat part of the cytoplasmic polyadenylation complex recruits either PAP or Gld-2 is also still an open question. It is possible that CPEB replaces Fip1 in cytoplasmic polyadenylation, or that another specific recruitment factor exists for Gld-2. Also, the nuclear cleavage stimulation factor CstF77 has been reported to be present in the cytoplasmic polyadenylation complex in Xenopus oocytes, but appears to have an as yet undefined role in translational repression, rather than in cytoplasmic polyadenylation [67].\nIn nuclear polyadenylation, binding of the nuclear poly(A) binding protein, PABP2, is required to limit the length of the poly(A) tail. It has been suggested that such a function is required in cytoplasmic polyadenylation, as symplekin immunoprecipitates from polyadenylating egg extracts add abnormally long poly(A) tails [57]. It is unknown if the length of the poly(A) tail added is controlled in cytoplasmic polyadenylation, but it is worth noting in this context that the main poly(A) binding protein in oocytes is not the somatic cytoplasmic PABP, but an embryo specific protein, ePABP, which may have a specific function in this context [68,69].\nRecently, it was shown that ePAB and somatic PABP both can bind to CPEB, and that this interaction is reduced by phosphorylation of CPEB during oocyte maturation [65,70]. The ePABP\u2013CPEB interaction has been proposed to stimulate cytoplasmic polyadenylation and translational activation, as demonstrated by depletion experiments [65]. ePABP may therefore be a cytoplasmic polyadenylation factor. However, the data presented appear to show that symplekin is partially depleted with ePABP, which could explain the reduction in polyadenylation. The add-back experiment does unfortunately only show a minimal restoration of polyadenylation. Depletion of CPEB with ePABP is described for experiments described in the discussion section of this paper, adding to these concerns. In addition, because most panels in this paper do not show direct comparisons between CPE containing and CPE lacking RNA substrates, the intriguing effects of ePABP depletion could perhaps also be explained by a general effect of enhanced deadenylation and reduced translation of all polyadenylated mRNAs, which are predicted effects of low ePABP levels [68,69]. For the moment, it therefore appears prudent to reserve judgement on the function of the CPEB\u2013ePABP interaction.\n5\nThe activation of cytoplasmic polyadenylation\nIn Xenopus oocytes, meiotic maturation is induced by steroid hormones, probably through the action of both classical nuclear steroid receptors and a G protein coupled transmembrane receptor [71\u201373]. A rapid drop in cyclic AMP leads to the inactivation of protein kinase A, which is necessary for the induction of cytoplasmic polyadenylation. In recent years, progress has been made in various aspects of the activation mechanisms of cytoplasmic polyadenylation, although the picture is far from complete.\nTethering of mammalian Gld-2 to an mRNA using a fusion with an RNA binding domain and an mRNA with a binding site causes strong polyadenylation in immature Xenopus oocytes, indicating that the protein is constitutively active and only needs to be recruited to the mRNA to elongate the poly(A) tail [62]. However, as described above, Gld-2 is already associated with the polyadenylation factors in immature oocytes [57]. In addition, Gld-2 overexpression also leads to robust polyadenylation of CPE containing RNA in immature oocytes, which implies that the protein is probably inactivated by factors that are titrated by the overexpressed protein [57]. Since the action of Gld-2 requires recruitment to the RNA for activity, the titrated factor is unlikely to be CPEB itself. An intriguing possibility is that the protein depleted by Gld-2 is the cytoplasmic isoform of poly(A) ribonuclease (PARN). A 64\u00a0kDa catalytically active fragment of PARN has been found to coimmunoprecipitate with symplekin, CPEB and Gld-2 in immature but not in mature oocytes and binds to CPEB and Gld-2 synthesised in reticulocyte lysate [74]. The full length PARN is not abundantly found in this complex. The 64\u00a0kDa PARN isoform is thought to be responsible for the short poly(A) tails of CPE containing mRNAs, as a PARN antibody inhibited CPE-mediated deadenylation in immature oocytes [74]. In addition, catalytically inactive PARN can induce polyadenylation of CPE containing RNAs in immature oocytes and overexpression of wild type PARN represses the polyadenylation induced by Gld-2 overexpression [74]. These data indicate that PARN is the repressive factor depleted by Gld-2 injection and that it is the ejection of PARN from the polyadenylation complex that induces cytoplasmic polyadenylation [74]. There are two possibilities to explain the action of PARN on CPE containing mRNAs, either Gld-2 continuously adenylates while PARN takes these nucleotides off, or the presence of PARN blocks Gld-2 activity, for instance by preventing access to the 3\u2032 end of the mRNA.\nSince not all mRNAs are activated by cytoplasmic polyadenylation at the same time, the activation of the complexes must depend at least in part on mRNA specificity factors. In maturing Xenopus oocytes, cytoplasmic polyadenylation has been classed into 2 types, early (class I) and late (class II). Early polyadenylating mRNAs gain a poly(A) tail well before the activation of cdk1\/cyclin B and the breakdown of the nuclear envelope (germinal vesicle breakdown, GVBD). Early polyadenylation is independent of the synthesis of the kinase c-Mos (which is itself encoded by a class I mRNA), while late polyadenylating mRNAs are dependent on c-Mos synthesis and the subsequent cdk1\/cyclin B activation and gain their tails after GVBD [16,75,76]. The less well characterised polyadenylation response element (PRE, see above) and its binding factor Musashi have been shown to be responsible for some of the early polyadenylation events, despite the fact that these mRNAs also contain CPEs [15,17]. However, there is a clear correlation between the placement of the CPEs in an mRNA and the timing of its polyadenylation [15,77,78]. In addition, some late polyadenylating mRNAs also contain PREs\/Musashi binding sites and the histone B4 mRNA, which contains both a PRE and a CPE, has been reported to be polyadenylated early, but has been shown to be Mos-dependent, indicating that the current classification in early and late perhaps oversimplifies the situation [15,17,75,78].\nThe evidence for which factor mediates early polyadenylation rests primarily on the use of dominant negative Musashi and CPEB mutants, which block translation and polyadenylation of c-Mos mRNA and GVBD [15,17,48]. Charlesworth et al. compared Musashi and CPEB mutants, and came to the conclusion that Musashi alone mediates the early polyadenylation, in contrast to earlier findings by Mendez et al., which implicated CPEB in the early polyadenylation [17,32]. The CPEB phosphorylation mutant used by Charlesworth et al. is a rather weak dominant negative (L.E. Hake, Boston, USA, unpublished observation), so it is possible that CPEB-dependent early polyadenylation is not completely repressed by it in all cases. Differences in expression levels and examination of endogenous or injected RNAs could also explain the discrepancies between the two studies. In addition, either or both dominant negative proteins could be titrating other cytoplasmic polyadenylation factors and inhibit polyadenylation on mRNAs to which they do not normally control, or they could fail to enter a specific mRNA\u2013protein complex due to low exchange rates. Importantly, injection of a CPEB antibody prevented GVBD and gave strong repression of polyadenylation of injected early RNAs, including a Mos 3\u2032 UTR construct, indicating that CPEB-mediated early polyadenylation exists [21]. In addition, interference with pathways that are known to affect CPEB phosphorylation and activation leads to a reduction in pre-GVBD c-Mos mRNA polyadenylation and a delay or block in oocyte maturation, including in those papers that claim that early polyadenylation is CPEB-independent [15,16,32,79,80]. However, there remains the possibility that the same signal transduction pathways ultimately activate Musashi and CPEB, which would make it impossible to distinguish between the effects of the two proteins in these experiments, with the possible exception of the CPEB antibody injection. With the limitations of the currently available evidence, it seems safest to assume that Musashi and CPEB both contribute to the polyadenylation of early mRNAs and that some mRNAs may be exclusively dependent on one or the other. Further work is required to resolve this issue definitively.\nThe induction of Xenopus oocyte maturation and early cytoplasmic polyadenylation by progesterone is dependent on a drop in cAMP levels and the resulting inhibition of protein kinase A [17,81]. Downstream of this event is the phosphorylation of CPEB on Ser174, which happens early in oogenesis. Although CPEB, CPSF and Gld-2 are already in a complex in immature oocytes, this phosphorylation appears to induce a stronger association of CPEB with CPSF and Gld-2 [32,48,82]. Importantly, Ser174 phosphorylation also induces the ejection of PARN from the polyadenylation complex [74]. Mutation of Ser174 to alanine results in a dominant negative protein that inhibits oocyte maturation and early polyadenylation partially or fully and late polyadenylation completely [15,32,83]. In addition, injection of a peptide containing the phosphorylation site delays GVBD considerably, suggesting that it is inhibiting an early polyadenylation event [32]. All evidence therefore indicates that phosphorylation of Ser174 is a crucial event in the activation of cytoplasmic polyadenylation.\nThere is some controversy surrounding the kinase mediating the early phosphorylation of CPEB on Ser174. Initially, it was demonstrated that Aurora A kinase (Eg2) can phosphorylate this site and that this requires the N-terminus of CPEB, in addition to the phosphorylation site itself [32]. This suggested that the N-terminally deleted CPEB may owe its dominant negative properties to the lack of a binding site for Aurora A. In addition, in vitro polyadenylation reactions containing recombinant CPEB, purified CPSF and nuclear PAP are strongly stimulated by the addition of Aurora A [48]. Glycogen synthase kinase 3 (GSK3) is thought to negatively regulate Aurora A by phosphorylation and this block appears to be relieved during oocyte maturation [83]. Consistent with a role in early oocyte maturation, GSK3 overexpression inhibited GVBD in Xenopus oocytes [83]. However, studies from three independent groups have failed to detect Aurora A activation early in oocyte maturation [79,84\u201386]. In addition, Aurora A inhibitors do not affect oocyte maturation or CPEB phosphorylation and depletion of Aurora A from oocyte extracts does not inhibit early CPEB phosphorylation either [79]. There is therefore sufficient evidence to throw serious doubt on the role of Aurora A in the activation of CPE-mediated cytoplasmic polyadenylation early in oogenesis. In mouse neurons, calmodulin dependent kinase II (CamKII) can phosphorylate the corresponding site on mouse CPEB, Thr171 [87]. CamKII plays a role in the induction of mouse oocyte maturation and is known to be present in Xenopus eggs, and could therefore be an alternative candidate kinase for mediating the early phosphorylation [88\u201391]. Because CamKII can be activated independently of calcium, the absence of a calcium flux in early oocyte maturation is not necessarily an obstacle for this hypothesis [92]. Evidence for a requirement of CamKII activity early in oocyte maturation would be necessary to make this more than speculation. However, Aurora A is very active later in oogenesis and it is undeniably capable of activating CPEB. It therefore appears likely that at the very least the maintenance of CPEB phosphorylation on Ser174 is carried out by this kinase.\nMitogen activated protein kinase (MAP kinase) has been implicated in the activation of early CPE-mediated, but not PRE-mediated, cytoplasmic polyadenylation [16,17,79,93]. An early, c-Mos independent activation of MAP kinase has been detected during oocyte maturation and CPEB is phosphorylated by MAP kinase on multiple sites [32,79,94]. MAP kinase does not phosphorylate Ser174, but has been suggested to either prime CPEB for Ser174 phosphorylation or to activate the Ser174 kinase [79]. As the early MAPK activation is dependent on protein synthesis, translational activation of another mRNA is probably required [94]. One potential candidate is RINGO\/Speedy, a Cdk1 interacting and activating protein that is transiently expressed after progesterone treatment and required for oocyte maturation and CPEB phosphorylation [65,95\u201398].\nA guanine nucleotide exchange factor for the Rho family of GTPases was found to interact with the N-terminal half of CPEB in a yeast 2 hybrid screen and called XGef [80]. This interaction was confirmed in oocytes and has been shown to be direct [80,99]. There appear to be 2 binding sites for CPEB on XGef [99]. Antibodies against XGef block oocyte maturation and prevent the polyadenylation and translation of c-Mos mRNA [80,99]. Conversely, overexpression of XGef accelerates oocyte maturation and c-Mos polyadenylation, independent of the production of c-Mos protein, indicating that it is upstream of this kinase in the signal cascade of meiotic maturation [80]. XGef enhances the early phosphorylation of CPEB and the DH domain associated with nucleotide exchange activity is required for its effects of on cytoplasmic polyadenylation [80,99]. In addition, a mutant of XGef that retains nucleotide exchange activity but has impaired binding to CPEB reduced early CPEB phosphorylation and delayed oocyte maturation [99]. However, a broad spectrum Rho GTPase inhibitor did not affect oocyte maturation or CPEB phosphorylation, indicating that the DH domain may not function to activate a Rho family GTPase, but is required in another capacity [79]. Strikingly, XGef immunoprecipitates were found to contain MAPK in both immature and mature oocytes and it therefore may be required to bring CPEB to the signalling complexes involved in its phosphorylation [79].\nIn another yeast 2 hybrid study, mouse CPEB was found to bind the small intracellular domain (ICD) of the transmembrane protein amyloid precursor like protein 1 (APLP1) and its relatives [100]. In Xenopus oocytes, Gld-2, the CPSF 100\u00a0kDa subunit and symplekin were predominantly associated with the plasma membrane in the same fractions as overexpressed APLP1 and immunoprecipitation of CPEB from these fractions indicates that it can associate with APLP1 [100]. Immunogold electron microscopy confirmed that CPEB and CPE containing RNA is associated with membranes. Overexpression of full length APLP1 induced some cytoplasmic polyadenylation in untreated oocytes and enhanced the effect of low concentrations of progesterone. The ICD alone had even stronger effects, indicating that the action of APLP1 is not dependent on membrane localisation. After treatment with low concentrations of progesterone, APLP1 stimulated CPEB phosphorylation on Ser174 [100]. While the association with amyloid precursor proteins may have great significance for the role of CPEB and cytoplasmic polyadenylation in neurons, it is as yet unclear whether APLP1 or its relatives are required for cytoplasmic polyadenylation in oocytes and whether it mediates the membrane association of the cytoplasmic polyadenylation complexes.\nLate cytoplasmic polyadenylation is required for progression from meiosis I to meiosis II during oocyte maturation [101]. mRNAs that undergo late polyadenylation often have a CPE overlapping with their poly(A) signal, e.g. UUUUAAUAAA [15,77,78]. The late polyadenylation during oocyte maturation is dependent on the activation of the mitotic kinase cdk1 and its regulatory cyclin subunits [16,77,78,93]. Cdk1 does phosphorylate CPEB leading to degradation of most of the oocyte CPEB via ubiquitin-mediated degradation [101\u2013103]. This degradation is required for late polyadenylation to occur. Remarkably, symplekin bound CPEB appears to represent the stable fraction of CPEB, as symplekin immunoprecipitates do not show a difference in CPEB content between immature and mature oocytes [57]. This implicates that CPEB in polyadenylation complexes is stable during oocyte maturation, consistent with the polyadenylation activity observed. A model for late cytoplasmic polyadenylation can be devised in which the abundant \u2018free\u2019 CPEB (i.e. CPEB in a different complex than symplekin, CPSF and the poly(A) polymerase) binds to the CPE overlapping the poly(A) signal in late polyadenylating mRNAs and prevents the recruitment of CPSF and symplekin by the polyadenylation signal early in oocyte maturation. After cdk1 activation, most free CPEB is depleted and a complex containing CPEB, symplekin and CPSF binds both the CPE and the poly(A) signal [101].\nA current view of CPE-mediated cytoplasmic polyadenylation in oocyte maturation is depicted in Figs. 1 and 2 and can be summarised as follows:1.The activation of CPE-mediated cytoplasmic polyadenylation during progesterone induced oocyte maturation is induced by a drop in protein kinase A activity and requires an early translation event, perhaps translation of RINGO\/Speedy.2.This induces the early activation of MAP kinase, which is associated with the polyadenylation complex through XGef and phosphorylates CPEB on multiple sites, but not on Ser174.3.CPEB is phosphorylated on Ser174 by an as yet not fully confirmed kinase, possibly Aurora A or CamKII, which is required for the induction of CPE-mediated polyadenylation and causes an increase in the binding between Gld-2, CPSF and CPEB, causing the ejection of PARN from the complex and allowing Gld-2 to elongate the poly(A) tail of the mRNA.4.After GVBD, CPEB is phosphorylated by cdk1 and the free CPEB is mostly degraded, allowing the CPEB in polyadenylation complexes (now probably phosphorylated on Ser174 by Aurora A) to activate cytoplasmic polyadenylation on mRNAs which contain a CPE overlapping with the poly(A) signal.\nA surprising number of proteins involved in the activation of polyadenylation are themselves upregulated by this process. This should create positive feedback loops which amplify the signal and contribute to the progression of meiotic maturation. As discussed above, the mRNA for the serine\/threonine kinase c-Mos is one of the early targets of cytoplasmic polyadenylation [16,75,76]. It is an activator of the MAPK kinase MEK, and its synthesis leads to further activation of MAP kinase, activation of cdk1 and phosphorylation of CPEB [71]. The mitotic cyclins, activators of cdk1, are induced by cytoplasmic polyadenylation, as is Aurora A [19,26]. Similarly, CamKII is regulated by cytoplasmic polyadenylation in neurons [104]. Finally, Gld-2 targets its own mRNA, which contains CPEs in its 3\u2032 UTR[105]. A system with so much positive feedback will require strong brakes, and it appears that at least in some cases this is provided by deadenylation factors, as discussed below.\n6\nDeadenylation and translational repression\nThe maternal mRNAs that are stored in the oocyte in an untranslated state have short poly(A) tails, but have been reported to have normal poly(A) tail addition in the nucleus in both mouse and frog [74,106]. In both organisms, the deadenylation of RNA injected into the cytoplasm requires CPEs, indicating that the CPEB\u2013PARN complex, discussed above, mediates this process [74,106]. However, RNA substrates containing CPEs alone have not been tested in these experiments and it is therefore not clear if a CPE is sufficient for deadenylation or if other sequence elements are required. In fact almost all CPE containing mRNAs have putative binding sites for other deadenylation factors.\nFirstly, the deadenylation factor Pumilio binds directly to CPEB, and therefore could contribute to the deadenylation of all CPE containing mRNAs [107,108]. Many CPE containing mRNAs also contain Pumilio binding sites, including cyclin B1 and Gld-2 [97,107\u2013109], indicating that the recruitment of Pumilio maybe both by protein\u2013protein and RNA\u2013protein associations. Pumilio is a member of a highly conserved family of RNA binding proteins called the Puf family. Members of this family can mediate translational repression and mRNA destabilisation in organisms from yeast to vertebrates [110,111]. In yeast, the Puf family protein Mpt5 interacts with the conserved deadenylase complex CCR4\u2013Pop2\u2013Not, by binding to Pop2\/Caf1 [112\u2013114]. This association was also confirmed for the human and worm proteins [113]. In addition, Pumilio is known to aid the recruitment of the translational repressor Nanos, both in fly and frog [107,115]. Nanos can recruit the CCR4\u2013Pop2\u2013Not complex by binding to Not4 and contributes to the translational repression of fly cyclin B [116]. So far, the role of the CCR4\u2013Pop2\u2013Not complex in poly(A) tail metabolism has not been studied in Xenopus oocytes and embryos, but the conservation of both the recruitment proteins and one of the target mRNAs (cyclin B) seems to indicate that it is very likely to play a role.\nA subset of mRNAs that are activated by CPE-mediated cytoplasmic polyadenylation during oocyte maturation loose their poly(A) tails after fertilisation [19,117,118]. In most cases, this is mediated by an embryonic deadenylation element (EDEN) in the 3\u2032 UTR of the mRNA, which binds the deadenylation factor EDEN-BP [119]. Aurora A, cyclin B1 and c-Mos encoding mRNAs all bind EDEN-BP and the 3\u2032 UTR of Gld-2 mRNA contains good consensus binding sites, making EDEN-BP a good candidate repressor of the cytoplasmic polyadenylation-mediated positive feedback loops during early embryogenesis [105,119\u2013121]. The mammalian ortholog of EDEN-BP, CUGBP1, can recruit PARN [122]. A similar association of EDEN-BP and PARN in the Xenopus embryo could explain its role in deadenylation, but so far this complex has not been reported. It can therefore not be excluded that EDEN-BP mediates its deadenylation activity in Xenopus embryos through another deadenylase, for instance the CCR4\u2013Pop2\u2013Not complex. No direct contacts between CPEB containing and EDEN containing complexes has been described so far. Because CPEs, EDEN sequences, Musashi and Pumilio binding sites often occur in the same 3\u2032 UTR, their associated complexes can be expected to compete for the end of the mRNA to mediate deadenylation or polyadenylation.\nAlthough a short poly(A) tail will lead to inefficient translation of an mRNA, it is not sufficient for complete translational repression, as illustrated by the partial polysomal association of histone B4 mRNA and the accumulation of B4 protein in Xenopus oocytes [78,123,124]. Many of the other stored maternal mRNAs have to be strongly translationally repressed to enable the subsequent stages of growth, maturation and fertilisation of the oocyte [71]. CPEs, Musashi and Pumilio binding sites can repress translation of specific mRNAs, often in the absence of a poly(A) tail or in the absence of deadenylation, indicating that they have an additional function in blocking translation [75,105,107\u2013109,125\u2013127]. Several detailed models exist to explain the translational repression by CPEs, some of which are presented in Fig. 3.\nIn full grown oocytes, a CPEB binding factor called maskin was found to associate with the cap binding initiation factor eIF4E in a manner that should preclude recruitment of eIF4G and thus inhibit translation [128]. Maskin is a homologue of the transforming acidic coiled-coil domain protein 3 (TACC3) and these proteins play a vital role in the formation of the mitotic spindle in multiple organisms [129\u2013134]. The binding between maskin and eIF4E is weak but detectable and this association is abrogated by binding of PABP to the poly(A) tail elongation and by phosphorylation of maskin in both oocytes and embryos [129,135\u2013137]. As the eIF4E binding site in maskin appears not to be present in other organisms, it is not clear how widespread this regulation is [138]. A more conserved CPEB associated eIF4E binding protein is the neuronal protein neuroguidin, which has been proposed to function in a similar manner to maskin in repressing translation in the nervous system [139].\nThe deadenylase PARN is a well characterised cap binding protein and a 5\u2032 cap structure is required for deadenylation by PARN [140\u2013142]. CPEB can recruit PARN to the mRNA, but deadenylation still requires a cap structure [74]. This leads to a conflict between the maskin model and the opposing polymerase-deadenylase model [74,128]. Either the maskin model is true and eIF4E\/maskin is stably bound to the cap and PARN is not continuously active, or the opposing polymerase-deadenylase model is correct and CPEB bound PARN is stably bound to the cap and continuously active, excluding eIF4E from the cap. In fact, PARN could very well mediate both the deadenylation and the translational repression of mRNAs to which it is recruited by excluding eIF4E from the mRNA [120].\nBoth maskin and PARN are absent in early oocytes, so other repression mechanisms must exist to mediate translational repression in these cells. Recently, a large CPEB containing RNP complex was found in early oocytes [143]. This complex contains several proteins found in P-bodies and related RNP granules that have been implicated in both translational control and mRNA degradation [144]. In mammalian cells, CPEB can also be found in large RNP granules including P-bodies [145]. The early oocyte CPEB complex contains the eIF4E variant eIF4E1b, the eIF4E binding protein 4E-T, the RNA helicase Xp54 (Ddx6, Rck, Me31B, Dhh1) and the P-body components p100 (Pat1) and Rap55 (Scd6, CAR-1). It does not contain PARN, maskin or the canonical cap binding initiation factor eIF4E1a. Rap55 and Xp54 can mediate translational repression in Xenopus oocytes when tethered to an mRNA [146\u2013148]. In addition, the yeast homologues of both proteins, as well as of p100, are involved in general repression of translation [149]. Strikingly, the yeast Pumilio homologue Mpt5 recruits Dhh1, a homologue of the Xp54 RNA helicase, indicating that Pumilio-mediated translational repression may involve a similar complex [113]. The mechanism of this highly conserved type of translational repression is not entirely clear, but it is likely to involve assembly of large RNP particles that exclude translation initiation factors and\/or ribosomes. In addition, eIF4E1b was shown to be defective in eIF4G binding and to bind 4E-T independently of the canonical eIF4E binding site. This implies that the mRNAs in this complex are translationally repressed through sequestration of the cap by eIF4E1b, which would exclude eIF4E1a\/eIF4G from the mRNA. Indeed, tethering of 4E-T causes translational repression and injection of an eIF41b antibody enhances oocyte maturation [143]. It is as yet unclear which proteins in the early complex bind directly to CPEB and therefore are likely to confer specific repression of CPE containing mRNAs. As eIF4E1b is also the predominant CPEB associated eIF4E in fully grown oocytes, this protein could in principle convey CPE-mediated translational repression throughout oogenesis [67,143].\nAt present it is difficult to choose amongst the multitude of models for translational repression by CPEs. Some of the complexes detected by pulldown and immunoprecipitation may not contain the majority of the repressed mRNA, even though presence of some mRNA was demonstrated by RT-PCR. Alternatively, it may well be that every one of these models is correct at a particular stage of oogenesis or embryogenesis or that the repression is different for specific mRNAs, depending on binding sites for other proteins such as Pumilio and EDEN-BP. An attractive option is that sequestration to P-body like large complexes is the result of translational repression and acts as an enhancer of translational repression, while smaller, more mRNA specific translational repression complexes are formed during the movement of mRNAs in and out of P-bodies. To distinguish between these models, it will be important to characterise the cap binding proteins and P-body components present on specific mRNAs at different developmental stages.\n7\nCytoplasmic polyadenylation and translational activation\nThe simplest explanation for why cytoplasmic polyadenylation leads to translational activation is that the poly(A) tail recruits translation initiation factors through its association with PABP and that these mediate release of the mRNA from the repression complexes, either by exchanging the cap binding complex for eIF4E1a\/eIF4G or by somehow extracting the mRNA from a P-body-like RNP complex, a function which has been ascribed to PABP in yeast [150]. In support of this hypothesis, the translational repression of the histone B4 and cyclin B1 3\u2032 UTRs on mRNAs injected into Xenopus oocytes can be abolished by the addition of a poly(A) tail, at least until that tail is deadenylated [29,74]. However other mRNAs (G10, Cl2) appear to be still repressed even if a long poly(A) tail is added and only become translated if the mRNA is actively polyadenylated in the maturing oocyte [6,13]. In a seminal experiment, Barkoff et al. cleaved the endogenous c-Mos mRNA with an oligonucleotide, removing the polyadenylation signal and CPE. As expected, this abrogated c-Mos mRNA polyadenylation and translation in oocytes exposed to progesterone. A \u2018prosthetic\u2019 poly(A) tail was tethered to the remaining 3\u2032 UTR using basepairing. This did not lead to c-Mos synthesis in unstimulated oocytes, but restored the synthesis of c-Mos in response to progesterone [76]. This suggests that, at least for the translation of c-Mos mRNA, both the presence of a poly(A) tail and an additional, polyadenylation independent, modification of the mRNP are required for translational activation.\n8\nDiscussion\nSince the discovery of CPEB ten years ago, our knowledge of the mechanisms of CPE-mediated cytoplasmic polyadenylation has made great strides, especially in Xenopus oocytes. However, there are still quite a few questions that need to be resolved, for instance:1.A more systematic investigation of the consensus CPE sequence and its maximum distance to the poly(A) signal would enable a more reliable bioinformatic prediction of the targets of CPE-mediated cytoplasmic polyadenylation. Are all mRNAs containing these sequences polyadenylated during oocyte maturation?2.The relative roles of Gld-2 and PAP in cytoplasmic polyadenylation need to be further evaluated. Can they substitute for each other? What are their contact points in the cytoplasmic polyadenylation complex? A cytoplasmic polyadenylation system reconstituted from pure components would be ideal to resolve these questions.3.The kinase(s) responsible for the early activating phosphorylation on Ser174 of CPEB should be identified unequivocally. If Aurora A is responsible, why do inhibitors of its activity not block oocyte maturation and can no early activation of this kinase be detected? Do inhibitors of CamKII affect early polyadenylation? What is the signal transduction cascade leading to induction of the activating kinase?4.What are the relative roles of Musashi and CPEB in the polyadenylation of c-Mos mRNA and other substrates? How do dominant negative mutants of CPEB and Musashi achieve their repressive functions? May there be off target effects through titration of common polyadenylation factors or signal transduction machinery?5.What are the direct interaction between proteins and RNA elements in the cytoplasmic polyadenylation complex? Much of the work discussed here has been performed either in oocyte or reticulocyte lysates, where other interacting proteins may be present. It appears necessary that these interactions are mapped in detail in vitro or in a 2 hybrid system, as this could lead to predictions for the functions of dominant negative mutants and potentially clarify how different complexes are assembled.6.Are the complexes on cytoplasmic polyadenylation sequences other than the CPE similar to the CPEB associated complexes? For instance, do Gld2 or PAP associate with the RNA binding proteins that recognise them?7.Which models for translational repression and deadenylation of CPE containing mRNAs are correct in what stage of oogenesis? Again, a more intimate knowledge of the direct interactions involved in assembling the repression complexes is likely to yield important new investigative tools for resolving this question. In addition, the study of mRNP complexes assembled in vivo could yield some more conclusive answers.8.Are the polyadenylation and translation repression complexes identical for each CPE containing mRNA or are there mRNA specific differences in the complexes? Immunoprecipitation followed by RT-PCR and RNA affinity chromatography can partially answer this question, but affinity purification methods that target specific mRNPs are likely to be crucial to obtain a full answer.\nSome of the controversies discussed here are a result of the experimental restrictions of the Xenopus oocyte and egg systems. These systems are very good for biochemical assays both in injected oocytes and egg extracts and they provide ample material for purification and identification. However, the high levels of stable maternal proteins make clean knock down or knock out experiments impossible in most cases, which has led to a heavy reliance on dominant negative approaches. To resolve some of the outstanding issues we are therefore likely to have to look to vertebrate genetic systems such as mouse and\/or to develop tissue culture based cytoplasmic polyadenylation systems that are amenable to siRNA knock down, for instance using neuronal cells or cells synchronised in the cell cycle.\nThe advent of high throughput screens will strongly impact the field of cytoplasmic polyadenylation in the near future as the results of RNP immunoprecipitation microarray experiments and poly(A) tail profiling studies will increase our knowledge of the range of mRNA substrates [9,151\u2013153]. Such screens may also identify new cytoplasmic polyadenylation elements as well as novel cell types and biological processes in which this form of regulation is involved. Only one cytoplasmic polyadenylation element and its binding protein have so far been studied in detail, predominantly in oocyte maturation. As outlined above, a large number of important questions are still unresolved, even in this one system. With at least three other cytoplasmic polyadenylation sequences in existence in oocytes alone and the evidence that cytoplasmic polyadenylation also is involved in neuronal events and the mitotic cell cycle [9], there appears ample scope for expansion of the field in the near future.","keyphrases":["translational control","cytoplasmic polyadenylation","oocyte","deadenylation","meiotic maturation"],"prmu":["P","P","P","P","P"]}
{"id":"Acta_Neuropathol_(Berl)-3-1-1794627","title":"Hippocampal sclerosis dementia differs from hippocampal sclerosis in frontal lobe degeneration\n","text":"Hippocampal sclerosis (HS) is characterized by selective neuronal loss and gliosis in CA1 and the subiculum and has been associated with several disorders, including Alzheimer\u2019s disease, frontotemporal lobar degeneration with ubiquitin immunoreactive inclusions (FTLD-U), vascular dementia and some tauopathies. In some cases, HS is not associated with other degenerative pathologies. Such cases are sometimes referred to as HS dementia (HSD). Differences between HSD and HS in the setting of FTLD-U have not been systematically investigated. To this end, eight cases of HSD and ten cases of HS associated with FTLD-U were studied with Nissl and periodic acid-Schiff stains to assess neuronal loss and corpora amylacea, respectively. Sections were immunostained with antibodies to glial fibrillary acidic protein, HLA-DR and synaptophysin and immunoreactivity was measured with image analysis in CA1 and the subiculum of each case. Additionally, sections were immunostained with antibodies to 4-R tau to determine the presence of argyrophilic grains. HSD was different from HS associated with FTLD-U. Specifically, it was more common in the elderly, and it was associated with more marked neuronal and synaptic loss and with greater reactive gliosis. Corpora amylacea tended to be more frequent in HSD than in FTLD-U, but there was no difference in frequency of argyrophilic grains.\nIntroduction\nHippocampal sclerosis (HS) is defined as selective neuronal loss and gliosis of CA1 and the subiculum of the hippocampus. Its prevalence in demented patients ranges from 2.8 to 13% [4\u20136, 15, 25, 26]. It is associated with several other disorders, including Alzheimer\u2019s disease (AD), frontotemporal lobar degeneration with ubiquitin immunoreactive inclusions (FTLD-U), vascular dementia, dementia with Lewy bodies and some tauopathies. Occasionally, HS has been reported as an independent pathologic explanation for dementia, the so-called HS dementia (HSD). The reported prevalence of HSD ranges from 0.4 to 2% [2, 4, 15, 27].\nThe etiology of HS is uncertain in most cases, but it is hypothesized to be due to either hypoxic-ischemic injury or to neurodegeneration of selectively vulnerable neuronal populations in CA1 and the subiculum [4, 36]. An hypoxic-ischemic origin is supported by the known deficient hippocampal vascular supply [8] and the fact that CA1 hippocampal neurons have a higher susceptibility to hypoxia due to their concentration of glutamate receptors, which may be involved in excitotoxic cell death [30]. Further arguments in favor of this theory are the association of hypoxic-ischemic episodes in patients with HS [12, 34, 37] and the increased prevalence of cardiac [3, 10, 29] and cerebrovascular disease [11, 12, 26, 35] in cases of HS of presumed hypoxic-ischemic origin.\nSince many cases of HS do not have histories of hypoxia or ischemia [2, 10, 12, 15, 27], cardiac disease [2, 11, 12, 27] or cerebrovascular accidents [10, 27], a degenerative cause is also proposed. This hypothesis is supported by the frequent association of HS with degenerative dementias like AD [3, 4, 12, 15, 16, 27, 29] and FTLD-U [3, 4, 13, 15, 19, 22, 24, 29] and the less frequently with tauopathies, mainly argyrophilic grain disease (AGD) and FTDP-17 [5, 12, 17].\nThe aim of this study was to compare the pathological characteristics of HSD versus HS in the setting of FTD-U.\nMaterials and methods\nThe Mayo Clinic Jacksonville brain bank database between 1998 and 2005 of individuals with dementia or degenerative neuropathology (N\u00a0=\u00a01,487) was screened for specimens with HS that had complete neuropathologic evaluations. This evaluation included description of gross and microscopic findings, as well as quantitative information about Alzheimer type pathology. Specifically, the reports include counts of senile plaques and neurofibrillary tangles in six cortical sections, four sectors of the hippocampus, two regions of the amygdala, as well as subcortical regions with thioflavin-S fluorescent microscopy. It also involves immunohistochemistry for tau, \u03b1-synuclein and ubiquitin, as appropriate, as previously described [4]. A Braak neurofibrillary tangle stage was assigned to all cases based upon the distribution of neurofibrillary tangles with thioflavin-S fluorescent microscopy, as previously described [4, 14, 21, 31, 33].\nHS was detected in 103 cases; in 95 cases (92%) it was associated with a primary degenerative disease process, mostly AD (36; 18 men and 18 women; 85\u00a0\u00b1\u00a05\u00a0years of age) and FTLD-U (44; 23 men and 21 women; 75\u00a0\u00b1\u00a012 years of age; P\u00a0<\u00a00.01 compared to AD). HS was detected in about 5% of AD cases (36 of 637) and 75% of FTLD-U cases (44 of 59). The remainder of cases included HS in the setting of a wide range of pathologies, including Lewy body disease, progressive supranuclear palsy, corticobasal degeneration and multiple system atrophy. In eight cases, HS was not associated with degenerative pathology.\nFor this study, 18 cases were selected for further study and placed in one of two groups. The first group of eight cases was operationally termed HSD; most HSD cases (75%) were associated with evidence of cerebrovascular disease. When compared to a control group of 30 cases from the same brain bank matched for age, sex and Braak stage, cerebrovascular disease was detected in 80% of the controls, a frequency similar to that in HSD. Therefore, we could not necessarily assume that the HS was due to hypoxic-ischemic causes. In all HSD cases, FTLD-U was ruled out with ubiquitin immunohistochemistry. The second group (\u201cFTLD-U HS\u201d) included 10 cases of HS associated to FTLD-U. None of the FTLD-U HS cases had pathologic evidence of cerebrovascular disease or other pathologic processes, and HS was assumed to be related to FTLD.\nIn all 18 cases other pathologic processes, such as progressive supranuclear palsy, corticobasal degeneration or Lewy body disease were absent. Alzheimer type pathology was minimal. None of the cases in either the HSD or FTLD-U HS group had a Braak neurofibrillary tangle stage greater than IV. In none of the cases could neuronal loss in the hippocampus be attributed to neurofibrillary tangles, since none had many extracellular (\u201cghost\u201d) neurofibrillary tangles. Medial temporal tauopathy due to AGD was not a basis for exclusion, and all cases were screened for this age-related pathology with immunohistochemistry using isotype-specific tau antibodies, as previously reported [17].\nCoronal sections of the posterior hippocampus at the level of the lateral geniculate nucleus were cut at a thickness of 5\u00a0\u03bcm. Sections were stained with Nissl and periodic acid-Schiff (PAS) stains. Sections were immunostained on a DAKO Autostainer with antibodies to HLA-DR, glial fibrillary acidic protein (GFAP), 4R tau and synaptophysin using procedures similar to those reported previously [23]. The following primary antibodies were used: anti-GFAP (GA-5, monoclonal, 1:1,000, Biogenex, San Ramon, CA), anti-HLA-DR (LN3, monoclonal, 1:100, eBioscience, San Diego, CA), anti-4R tau (ET3, monoclonal, 1:25, Dr. Peter Davies, Albert Einstein College of Medicine, Bronx, NY) and anti-synaptophysin (EP10, monoclonal, 1: 10, Dr. Peter Davies, Albert Einstein College of Medicine, Bronx, NY). For antigen retrieval, slides were steamed in distilled water for 30 min. For ET3 immunostaining, which has been shown to be a sensitive and specific method for detecting AGD [17, 32], slides were also pretreated in 99% formic acid for 30\u00a0min.\nThe degree of neuronal loss in the subiculum, CA1, CA2\/3, CA4 (end plate) and dentate gyrus was evaluated on Nissl stained sections using a three-point rating scale: 0, absent; 1, mild-to-moderate; and 2, marked. PAS staining was used to assess the density of corpora amylacea. Corpora amylacea were defined as round, PAS-positive structures in the neuropil, perivascular, subpial and subependymal areas [7]. Presence of corpora amylacea throughout the hippocampus was graded on a four-point scale: 0, absent; 1, sparse; 2, moderate; and 3, many.\nTo assess astrocytic, microglial and synaptic immunoreactivity, sections immunostained for GFAP, HLA-DR and synaptophysin were used. Standardized, non-overlapping, digital images of the pyramidal layer of CA1 and subiculum were taken under a 40\u00d7 objective on a light microscope. Immunoreactivity was quantified as a percentage area using MetaMorph software, version 6.3r0 (Molecular Devices, Sunnyvale, CA).\nFour-R (4R) tau immunostained slides were used to determine the presence of AGD. Cases were considered to have AGD if they had small dot- or comma-like argyrophilic lesions in neuronal processes of the pyramidal layer and the entorhinal cortex, as well as coiled bodies in temporal white matter, which are the same criteria used in other studies [31].\nClinical and neuropathologic records were reviewed to collect data on age, sex, Braak neurofibrillary tangle stage, brain weight and macroscopic and microscopic neuropathology findings at autopsy. The recorded gross findings at autopsy were lobar, hippocampal and mammillary body atrophy. The remarkable microscopic findings were vascular pathology, ischemic brain injury and white matter disease.\nDifferences between the two groups in age, Braak stage, brain weight, corpora amylacea, neuronal loss and glial and synaptic immunoreactivity pathology were analyzed with unpaired t test and Mann\u2013Whitney U tests depending upon the variable. Fisher\u2019s exact test was used to compare sex, presence of AGD and remarkable pathologic findings. The statistical analyses were performed using Sigma Stat for Windows, version 3.11 (Systat Software, Richmond, CA) and the significance levels were set at P\u00a0<\u00a00.05.\nResults\nA summary of the demographic and neuropathologic findings of the 18 cases included in this study is presented in Table\u00a01. A comparison of the two groups is presented in Table\u00a02. The cases with HSD had an older age of death than those with FTLD-U HS (84 vs. 65, P\u00a0=\u00a00.001). In the HSD group, six of eight cases were men, while four of six cases in the FTLD-U HS group were men, but this difference was not significant.\nTable\u00a01Demographic and neuropathologic features of HS casesAgeSexNFT stageNeuronal lossVascular pathologyCerebrovascular pathologyWMSubCA1CA2CA4DGAtherosclerosisArteriosclerosisAmyloid angiopathyLacunar infarctsMicroscopic infarctsIschemic gliosisP187 MI2200050\u201375% (PCA)PV WM, BG, Th\u2013\u2013F, M, WSAThPVP284 FII22000MinimalBG\u2013BG, ThO\u2013PV, DCP386 M02200050\u201375% (ICA, MCA)WM, BGF, P, OBG, pons\u2013WM, BG, ThPV, DCP476 MIV02202Minimal\u2013F, O\u2013\u2013\u2013\u2013P579 MIII1100050\u201375% (MCA, PCA, ACA)BG, CBO\u2013BGMPVP679 FIII02111\u2013\u2013\u2013\u2013Cortical WSACortical WSA, ThPVP787 MII22002Minimal\u2013O\u2013\u2013\u2013PVP890 MIII21000<50%WM, BG\u2013PV WM, BG, amygdala\u2013ThPV, DCF183 FI22NA00\u2013\u2013\u2013\u2013\u2013\u2013F, T subcorticalF256 M022000\u2013\u2013\u2013\u2013\u2013\u2013F, P subcorticalF356 F021000\u2013\u2013\u2013\u2013\u2013\u2013F, T, P subcorticalF441 FI22002\u2013\u2013\u2013\u2013\u2013\u2013F, T subcorticalF570 MI22000Minimal\u2013\u2013\u2013\u2013\u2013F, subcorticalF670 M022000\u2013\u2013\u2013\u2013\u2013\u2013F, T subcorticalF762 F010000Minimal\u2013\u2013\u2013\u2013\u2013F, T subcortical and PVF872 FIII10002\u2013\u2013\u2013\u2013\u2013\u2013F, T subcortical and PVF966 F022002Minimal\u2013\u2013\u2013\u2013\u2013F, P subcorticalF1073 M022002NA\u2013\u2013\u2013\u2013\u2013F, T, P subcorticalSub subiculum, CA cornu ammonis, EP endplate, DG dentate gyrus, F frontal, T temporal, P parietal, O occipital, M motor, ICA internal carotid artery, ACA anterior cerebral artery, MCA middle cerebral artery, PCA posterior cerebral artery, VA vertebral artery, BA basilar artery, PV periventricular, DC deep cerebral, WM white matter, BG basal ganglia, Th thalamus, CB cerebellum, WSA watershed area, NA not available, P #  represent HSD cases; F# represent FTLD-U HS casesTable\u00a02Comparison of HSD and FTLD-U HSHSD (n\u00a0=\u00a08)FTLD-U HS (n\u00a0=\u00a010)Age at death (years), mean\u00a0\u00b1\u00a0Std. Dev.84\u00a0\u00b1\u00a0565\u00a0\u00b1\u00a012\u2021Sex (% of cases)Male7540Female2560Braak NFT stage, median (25%-tile, 75%-tile)2.25 (1.5, 3)0 (0, 1)\u2020Brain weight (g), mean\u00a0\u00b1\u00a0Std. Dev.1130\u00a0\u00b1\u00a0100868\u00a0\u00b1\u00a0130\u2021Gross atrophy (% of cases)Frontal lobe25100\u2020Medial temporal lobe1378\u2020Lateral temporal lobe3367Parietal lobe1767Occipital lobe1311Mammillary bodies100100Hippocampal formation8990Neuronal loss (score), median (25%-tile, 75%-tile)Subiculum1 (0.25, 1)1 (1, 1)CA11 (0.75, 1)1 (0.5, 1)CA2\/30 (0, 0.25)0 (0, 0)EP0 (0, 0)0 (0, 0)DG0 (0, 1)0 (0, 1)Synaptophysin (% area), mean\u00a0\u00b1\u00a0SEMSubiculum 23.4\u00a0\u00b1\u00a012.336.4\u00a0\u00b1\u00a021.3CA126.5\u00a0\u00b1\u00a024.451.7\u00a0\u00b1\u00a020.8\u2020GFAP (% area), mean\u00a0\u00b1\u00a0SEMSubiculum 9.5\u00a0\u00b1\u00a07.56.2\u00a0\u00b1\u00a02.9 CA114.9\u00a0\u00b1\u00a012.59.2\u00a0\u00b1\u00a07.8HLA-DR (%area), mean\u00a0\u00b1\u00a0SEMSubiculum0.80\u00a0\u00b1\u00a00.770.56\u00a0\u00b1\u00a00.50CA10.59\u00a0\u00b1\u00a00.750.19\u00a0\u00b1\u00a00.18Corpora amylacea (score), median (25%-tile, 75%-tile)1 (0.5, 1.75)0.5 (0, 2)Argyrophilic grain disease (% of cases)1320Fisher exact test was used for categorical variables, t test for continuous variables and Mann\u2013Whitney Rank Sum test for discontinuous variables\u2020P\u00a0<\u00a00.05\u2021P\u00a0<\u00a00.001\nNeuropathologic findings\nThe Braak neurofibrillary tangle stage was significantly greater in HSD than in FTLD-U HS. The brain weight was also greater in HSD than in FTLD-U HS. Cortical atrophy was more frequent in FTLD-U HS than HSD in frontal (100% vs. 25%), medial temporal (78% vs. 13%), lateral temporal (67% vs. 33%) and parietal lobes (67% vs. 17%). The difference reached statistical significance in frontal and medial temporal regions. Both groups showed marked gross atrophy of the hippocampal formation and mammillary body.\nIn this comparative study, cerebrovascular pathology was virtually limited to the HSD group. Atherosclerosis of large vessels at the base of the brain was present in seven of eight HSD cases. Three had minimal, 1 had mild (occluding less than 50% of the lumen) and 3 had moderate atherosclerosis (occluding between 50% and 75% of the lumen). Arteriosclerosis defined by presence of hyalinosis of the media and adventitial fibrosis of small (<100\u00a0\u03bcm diameter) vessels was present in 5 (63%) cases and cerebral amyloid angiopathy (CAA) was present in 4 (50%) of 8 HSD. Three (30%) cases of FTLD-U HS had minimal atherosclerosis. None of the cases of this group had significant arteriolosclerosis or evidence of CAA.\nIschemic brain injury was present in six of eight HSD cases, but in none of the FTLD-U HS cases. The eight HSD cases included 3 (38%) with lacunar infarcts, 4 (50%) with microscopic infarcts and 5 (63%) with microscopic foci of ischemic gliosis.\nMost cases, independent of their group, showed white matter pathology defined by rarefaction and attenuation of white matter, associated with dilated perivascular spaces, loss of myelinated fibers and gliosis. These findings were present in the periventricular white matter in seven of eight HSD cases. In three of these cases more diffuse white matter pathology was also present, involving the centrum semiovale. All of the cases with FTLD-U HS showed white matter pathology that followed the distribution of the cortical atrophy.\nTwo of the HSD cases (P4 and P7) had only minimal atherosclerosis and CAA, which was probably insufficient to cause hippocampal hypoperfusion, but both cases had a history of coronary artery disease and congestive heart failure, which might account for hypoxic-ischemic injury to the hippocampus, although this remains purely speculative, since these risk factors are common in individuals of this age without HS [12].\nAnother case of HSD (P6) with minimal atherosclerosis, arteriosclerosis and CAA had a history of epilepsy, as well as laminar ischemic gliosis in the watershed cortices and cortical cystic infarcts and thalamic ischemic gliosis. Epilepsy in this patient was probably secondary to a porencephalic cyst that was present in the temporal pole. Given the absence of degenerative pathology, this case was included in the HSD group, but presence of extensive cerebrovascular disease raises the possibility that HS in this case was related to hypoxic-ischemic injury.\nQuantitative and semi-quantitative analyses\nBoth HSD and FTLD-U HS had severe neuronal loss in the pyramidal layer of CA1 and the subiculum. No significant differences were found in the severity of neuronal loss between the two regions; nevertheless, differences in the distribution of neuronal loss were noted. Most cases had neuronal loss in both CA1 and the subiculum, except for two cases of HSD that had isolated CA1 involvement and two cases of FTLD-U HS that had isolated subicular involvement.\nThe CA2\/3 region and the endplate were not affected in most cases. Only cases of HSD occasionally had neuronal loss in these regions, with involvement of CA2\/3 in P4 and involvement of CA2\/3 and endplate in P6. The dentate was involved in half of the cases of both groups.\nSynaptic loss assessed by image analysis of density of synaptophysin immunoreactivity was significantly less in CA1 region in HSD compared to FTLD-U HS (Fig.\u00a01). A similar trend was noted in the subiculum. Gliosis as assessed by image analysis with GFAP and HLA-DR immunostains tended to be greater in HSD compared with FTLD-U HS (Fig.\u00a01), although it did not reach statistical significance probably because of small sample size. Both GFAP and HLA-DR immunoreactivity were greater in HSD than in FTLD-U HS in both CA1 and the subiculum.\nFig.\u00a01Comparison of CA1 region of hippocampus in HS associated with FTLD-U (a, c, e) and HSD (b, d, f) immunostained for synaptophysin (a, b), GFAP (c, d) and HLA-DR (e, f). Note better preservation of neuropil and less gliosis in FTLD-U HS compared to HSD (All figures are \u00d7200)\nThere tended to be more corpora amylacea in HSD than in FTLD-U HS, but this did not reach statistical significance. A concomitant diagnosis of AGD was made in two cases of FTLD HS and in one case of pure HS (P6).\nDiscussion\nThe present study confirmed the characteristic findings of neuronal loss and gliosis in CA1 and the subiculum in HS regardless of etiology; however, there were some significant differences between HSD and HS that occurs in FTLD-U. In addition, there were subjective differences that did not meet statistical significance probably related to the small sample size in this study. The results support the hypothesis that there are differences in underlying disease mechanisms in HSD compared to HS in FTLD-U.\nFrom a demographic perspective, demented patients with HS have been shown to be older than demented patients without HS [11, 12, 27, 29], although there are exceptions [2, 10]. Data concerning the age distribution among patients with HS has to our knowledge not been reported. In the present study, we show HSD to mainly affect the elderly (>80\u00a0years of age) as opposed to HS in FTLD-U. The younger age of the FTLD-U may account for the lower frequency of concurrent cerebrovascular disease, since an age-, sex- and Braak neurofibrillary tangle stage-matched cohort had comparable frequency of cerebrovascular disease.\nMicroscopically, there were three important differences in the pathology in HSD compared with HS in FTLD-U. First, HSD had lower synaptophysin immunoreactivity, suggesting a more destructive process resulting in more severe synaptic loss, while in FTLD-U there is selective neuronal loss, but relative preservation of the neuropil, presumably from preserved afferent and efferent projections to sectors that show neuronal loss. Second, the difference in the distribution of neuronal loss suggested subicular involvement to be characteristic of HS in FTLD-U. Even though the differences in distribution of neuronal loss did not reach statistical significance, other observations support this idea [20]. Third, HSD was associated with greater astrocytic and microglial reaction compared to HS in FTLD-U. Glial activation has been proposed to be caused by neuronal damage, which is present in both, ischemic and degenerative pathology, but may be exacerbated in HSD due to more extensive tissue damage affecting not only neurons, but also the neuropil.\nMacroscopically, FTLD-U HS had more severe cortical atrophy, a not surprising finding, since it correlates with FTLD [25]. HSD on the other hand showed a pattern of atrophy often localized to the hippocampal formation, arguing against this pathology being associated with more widespread cortical degeneration. This fits with results from previous studies of HSD that showed that loss of synaptophysin immunoreactivity in the hippocampus was not accompanied with similar loss in the cortex or basal ganglia [12].\nCorpora amylacea have been proposed as a possible marker for hypoxic-ischemic HS, since they may be numerous in brains of patients exposed to repetitive hypoxic episodes [1, 6]. They have also been proposed as a useful marker for evaluating HS in surgical specimens removed for treatment of temporal lobe epilepsy [9]. Although there was a trend for more corpora amylacea in HSD than in FTLD-U HS, it did not reach statistical significance, which may be further evidence against the argument that HSD is due to hypoxic-ischemic injury.\nEven though we did not find a difference in the frequency of AGD in HSD and HS associated with FTLD-U, the presence of AGD in 3 of the 18 cases, may suggest an association of HS with AGD as reported in previous studies [5, 17].\nThis study included two cases of HSD (P4 and P7) in which there was not only no evidence of degenerative pathology, but also no evidence of cerebrovascular pathology. Cases in which HS is the only pathologic explanation for dementia are rare. Hattanpa found some cases of HS to be false negative cases for FTLD-U after further studying them with ubiquitin immunohistochemistry [13]. Since we analyzed our cases with ubiquitin, false negatives were excluded, giving a probable explanation for the lower prevalence compared to the 0.48, 0.53 and 2% previously reported by others [2, 15, 27].\nThe two cases of HSD with neither degenerative nor cerebrovascular pathology had cardiovascular risk factors that could link HS to an ischemic condition of systemic origin. They are similar to the ones that have been reported by others [34, 37]. On the other hand, in a systematic survey of cardiovascular risk factors, including electrocardiograph abnormalities, congestive heart failure and cardiomegaly by imaging and autopsy studies, there was no increased frequency of these findings in HSD compared to age-matched controls without HS [12].\nThe present study confirms the finding that HS in the setting of dementia in most cases should be regarded as a feature linked to a concurrent degenerative disease process, particularly FTLD-U [13, 18, 19, 28]. In other cases, HS may be linked to a systemic condition (e.g., cardiorespiratory failure) or cerebrovascular disease [8, 36]. In rare cases, the etiology is unknown. The present study suggests that HSD is a disorder of the elderly that is associated with more marked neuronal and synaptic loss and greater reactive gliosis than that seen in HS associated with FTLD-U. This argues for a different pathogenesis","keyphrases":["hippocampal sclerosis dementia","image analysis","immunohistochemistry","frontotemporal degeneration"],"prmu":["P","P","P","R"]}
{"id":"Surg_Endosc-4-1-2292804","title":"Retracting and seeking movements during laparoscopic goal-oriented movements. Is the shortest path length optimal?\n","text":"Aims Minimally invasive surgery (MIS) requires a high degree of eye\u2013hand coordination from the surgeon. To facilitate the learning process, objective assessment systems based on analysis of the instruments\u2019 motion are being developed. To investigate the influence of performance on motion characteristics, we examined goal-oriented movements in a box trainer. In general, goal-oriented movements consist of a retracting and a seeking phase, and are, however, not performed via the shortest path length. Therefore, we hypothesized that the shortest path is not an optimal concept in MIS.\nCurrently, there are various virtual-reality (VR) trainers available to train MIS skills [1, 2]. These trainers have the advantage over box trainers of providing objective feedback about the performance of the user (performance parameters), which motivates residents to learn [3\u20135]. The most common parameter used to measure basic MIS skills objectively is task completion time. However, this parameter alone does not adequately evaluate the task performance [6]. Therefore, the time for completion is often used in conjunction with other parameters [1, 7\u201312]. One of the task-independent parameters that is used in each VR trainer is path length \u2013 the length of the curve described by the tip of the instrument over time [13]. Intuitively, a longer path length indicates a less-efficient movement and is often interpreted as lack of experience.\nIn MIS, goal-oriented movements (point-to-point movements) are very common (e.g., during grasping, placing a clip on the vessel, or while using diathermy). In general, a simple strategy to perform such a movement is to move along a straight line between an initial position and a specified target position [14, 15]. However, it is hardly possible to make such a movement during MIS. Movements of MIS instruments are done through the incision point, which acts as a pivot point. Therefore, point-to-point aiming movements in MIS may simply result in a curved workspace path (Fig.\u00a01). Both these ways of performing point-to-point movements are very efficient. In general, however, goal-oriented movements are not performed via the shortest path; a pull-back movement is performed before the specified target position is approached [16, 17]. Therefore, we hypothesized that the shortest path, as presently used to indicate more-efficient performance in MIS, is not a suitable reference.\nFig.\u00a01Three strategies of performing a goal-oriented movement in MIS. (a) a movement along the straight line between the initial and the end position; (b) a movement along a curved path between the initial and the end position, which is the result of the movement of the MIS instrument through the incision point; and (c) a realistic movement performed by the surgeon during the goal-oriented task. A \u2013 the initial position; B \u2013 the end position; P \u2013 incision (pivoting) point\nThe objective of this study was to examine goal-oriented movements during training of the basic MIS skills. The study consisted of two parts: first, we investigate whether goal-oriented movements can be split into two phases: a retracting phase and a seeking phase; and, subsequently, we investigate whether movements of the MIS instrument in these two phases depend on the surgeon\u2019s experience. The performance of the participants was analyzed and compared using normalized path lengths.\nMethods\nParticipants\nExperienced gynaecologists (with the experience of more than 100 MIS procedures), their residents (experience of 10\u2013100 MIS procedures), and novices (medical students with no previous experience in MIS) were invited to participate in this study. Each participant, voluntarily enrolled in this study, was asked to complete a short questionnaire detailing demographic information and prior experience in laparoscopy.\nTask\nThe hypothesis was tested using a simple one-hand positioning task, which requires touching the top of eight cylinders with the tip of the MIS instrument (Fig.\u00a02). All cylinders were situated in a box trainer in various three-dimensional (3D) positions. Every correctly touched cylinder resulted in the lighting of the lamp corresponding to this cylinder. The start\/end point and the order of touching cylinders (indicated by the numbers located next to the cylinders) were the same for each participant.\nFig.\u00a02The positioning task. The task requires touching the top of the eight cylinders (of varying 3D position) with the tip of the MIS instrument. A correctly touched cylinder results in lighting up a lamp (above on the picture) corresponding to this cylinder. The start\/end point and the order of touching the cylinders are indicated by the numbers located next to the cylinders\nTo mimic the in vivo gynaecological MIS situation, all participants performed the task with their left hand, while the camera was held in the right hand. To provide the same conditions for each participant, the position of the task and the incision points for the camera and the MIS instrument were standardized in the box trainer. The image of a 0\u00b0 laparoscope was presented on a monitor.\nBefore the test, all participants were instructed how to perform the positioning task. Furthermore, they were allowed to make one trial before testing.\nData analysis\nMovements of the MIS instrument were recorded with our newly developed TrEndo tracking system with a sample frequency of 100 Hz [18]. The data of the eight simple goal-oriented movements performed by each participant during the task were analyzed. The first goal-oriented movement was defined by the movement between the start point and first cylinder. Each successive goal-oriented movement was defined by the movement between two consecutive cylinders (Fig.\u00a02).\nThe retracting phase describes the pull-back movement of the MIS instrument in the direction of the pivoting point. Therefore, we analyzed the projected movements of the instrument in the ABP plane (Fig.\u00a03), which passes through the begin point of the movement (point A), the end point of the movement (point B), and the pivoting point (point P). To examine the deviation of the movement from the plane ABP, we also analyzed the movements projected in the plane AB, which is perpendicular to the plane ABP, and which passes through the points A and B (Fig.\u00a03). Point M, defined as the point of the movement most distant from the line AB, was used to make a distinction between the retracting phase AM (from point A to point M) and the seeking phase MB (from point M to point B). The analysis of each of the two phases was done using a normalized path length, which was derived as follows:  where PLreal is the real path length (between A and M for retracting, and M and B for seeking phase), and PL0 is the distance (length of the straight line) between A and M and M and B (for retracting, and seeking phases, respectively). The normalized path lengths of all eight successive goal-oriented movements were averaged per participant and analyzed.\nFig.\u00a03The ABP (described by the points A, B, and P) and AB (that passes through points A and B, and is perpendicular to the plane ABP) planes presented in an orthographic projection: (a) the ABP and AB planes, and the goal-oriented movement presented in the three-dimensional space; (b) front view: projection of the goal-oriented movement in the ABP plane; (c) top view: projection of the goal-oriented movement in the AB plane; and (d) side view: the ABP and AB planes, and the goal-oriented movement presented in a side view. A \u2013 the initial position; B \u2013 the end position; M \u2013 a point, which is used to make a distinction between the retracting and the seeking phases; P \u2013 incision (pivoting) point\nStatistics\nData were analyzed using the Statistics Toolbox of MATLAB 7. Statistical analysis was performed using one-way analysis of variance (ANOVA), and Wilcoxon tests. A probability p\u00a0<\u00a00.05 was considered to be statistically significant.\nResults\nParticipants\nThirty-eight participants completed the task and the questionnaire. The group of experts consisted of 9 experts (age 38\u201359 years). The group of residents consisted of 17 gynaecological residents (age 29\u201341 years). The group of novices consisted of 12 medical students (age 23\u201328 years). All participants were right-handed.\nRetracting and seeking phases\nTypical trajectories for an expert and a novice performing the goal-oriented movement are presented in Fig.\u00a04. The figure shows that the retracting and seeking phases can easily be distinguished from each other. No significant difference between the experts, residents, and novices was observed in the distance between point M and the line AB.\nFig.\u00a04The typical trajectories for an expert (left) and a novice (right) performing a goal-oriented movement. Top: projection of the goal-oriented movement in the plane ABP. Bottom: projection of the goal-oriented movement in the plane AB. A \u2013 the initial position; B \u2013 the end position; M \u2013 a point, which is used to make a distinction between the retracting and the seeking phases\nNormalized path lengths of the two phases are presented in Fig.\u00a05. In 3D space, the experts\u2019 normalized path length was shorter during the retracting phase [median (range) %: 152 (129\u2013178)] than during the seeking phase [180 (172\u2013247)]. Similarly, the residents\u2019 and novices\u2019 path lengths were shorter during the retracting phase [164 (126\u2013250), and 168 (136\u2013268)] than during the seeking phase [201 (163\u2013287), and 290 (244\u2013469), respectively].\nFig.\u00a05Normalized path lengths of the retracting and the seeking phases for experts, residents, and novices. Upper: normalized path length in 3D space. Middle: normalized path length in the ABP plane. Lower: normalized path length in the AB plane. The results are presented as notched box-and-whisker plots, where every box has a line at every quartile, median, and upper quartile values. The whiskers are presented as lines that extend from each end of the box in order to show the extent of the rest of the data. The notches represent the 95% confidence interval for the median. Boxes whose notches do not overlap are significantly different (p\u00a0<\u00a00.05). **p\u00a0<\u00a00.01; ***p\u00a0<\u00a00.001; E \u2013 experts; R \u2013 residents; N \u2013 novices; 1 \u2013 retracting phase; 2 \u2013 seeking phase\nIn the ABP plane, the experts\u2019 normalized path length in the retracting phase [132 (120\u2013194)] was significantly shorter than in the seeking phase [199 (154\u2013234)]. The normalized path lengths of residents and novices were also significantly shorter in the retracting phase [133 (108\u2013492), and 180 (118\u2013287)] than in the seeking phase [208 (162\u2013286), and 310 (252\u2013469)].\nIn the AB plane, the experts\u2019 normalized path length was significantly shorter in the retracting phase [122 (100\u2013200)] than in the seeking phase [183 (141\u2013209)]. Normalized path lengths of residents and novices were also significantly shorter during the retracting phase [147 (107\u2013264), and 207 (128\u2013271)] than during the seeking phase [199 (162\u2013285), and 310 (252\u2013469)].\nInfluence of the experience\nThe experts\u2019, residents\u2019, and novices\u2019 normalized path lengths during the retracting and the seeking phases are presented in Fig.\u00a06. The data in Fig.\u00a06 are the same as the data in Fig.\u00a05, but presented per phase for all groups. In 3D space, there was no significant difference between experts, residents, and novices for the retracting phase. The normalized path lengths of experts and residents were significantly shorter than the path length of the novices during the seeking phase. No significant difference between experts and residents was observed in that phase.\nFig.\u00a06The influence of the experience on the performance of the retracting and the seeking phases. The data presented in this figure are the same as the data in Fig. 7, but presented per phase. *p\u00a0<\u00a00.05; ***p\u00a0<\u00a00.001; E \u2013 experts; R \u2013 residents; N \u2013 novices; 1 \u2013 retracting phase; 2 \u2013 seeking phase\nIn the ABP plane, we found no significant difference between experts, residents, and novices for the retracting phase. In the seeking phase, the normalized path lengths of experts and residents were significantly shorter than the path length of novices. No significant difference between experts and residents was observed in that phase.\nIn the AB plane, a significant difference between experts, residents, and novices was found for both phases. In both the retracting and the seeking phases, the normalized path lengths of the experts and residents were significantly shorter than the path length of the novices. No significant difference between experts and residents was observed for both phases.\nDiscussion\nOur findings indicate that goal-oriented movements are not performed via the shortest path. The movements clearly distinguish a retracting and a seeking phase. The results show that the normalized path length during the retracting phase is significantly shorter than during the seeking phase. Furthermore, experience in MIS does only influence the seeking phase. Experts\u2019 and residents\u2019 normalized path lengths were significantly shorter than the path lengths of the novices. The shorter path length in the seeking phase implies better performance; therefore, the seeking phase is characteristic of the differences in performance. This finding confirms that the path length is an important measure in the assessment of basic MIS skills, in which the seeking phase is the only component that can distinguish between novice and expert.\nThe retracting phase is essential in MIS, because it improves safety of the patient by avoiding unpredicted contact with the tissue. It seems that the best strategy to perform this phase safely is to pull back the instrument along its axis (in the direction of the pivoting point) and avoid any movements in the AB plane, which can cause unpredicted contacts with the tissue. The results of this study confirm this assumption; experts\u2019 and residents\u2019 normalized path lengths are smaller in the AB plane than the path length of the novices. Movements in the AB plane can result in unintended tissue contact and be dangerous for the patient; therefore, it would be beneficial to let novices learn how to perform a more-precise retracting movement.\nThe experience in MIS does not influence the retracting phase in the ABP plane. Moreover, a longer retraction of the MIS instrument in the direction of the pivoting point does not denote worse or less-efficient performance. In contrast, it may be a sign of the safer (for the patient) use of the MIS instruments. This finding is important, because it shows that the shorter path length, as presently used to indicate more-efficient performance in MIS, is not a correct concept for analyzing optimal movements in laparoscopy.\nTo investigate whether the retracting phase really represents safety, it is necessary to design a different study. For example, one possibility would be to include obstacles which, upon touching represent an error. With such a study design, it would be feasible to determine how much retraction is necessary and whether the experts have learnt the optimal retraction amount. The present study cannot be used to decide on these aspects.\nThe results of this study indicate that the comparison of the novices\u2019 path length to the experts\u2019 path length is an important and valid component of the overall criterion-based assessment of basic MIS skills. It is necessary to realize that this study was not designed to determine whether expert performance actually results in the optimal path length and, therefore, the results of this study cannot be used to indicate that experts\u2019 movements are optimal.\nIn the literature, there is a tendency to assess MIS performance using very basic terms (e.g., time, path length, penalty points), which do not show \u201cthe size and the nature of the gap between expert and novice performance\u201d [13]. The results of this study show that the analysis of the separate phases of the movement in various planes can give such details. For example, movements of the MIS instrument along its axis (in and out the trocar) during the seeking phase characterize the surgeon\u2019s level of depth perception. Movements of the instrument in the AB plane may indicate eye\u2013hand coordination problems (e.g., orientation). Since every phase of the movement analyzed in different planes provides details about different limitations, it is crucial to analyze the movements in MIS separately for each phase and in both planes. Only then will the feedback about the performance and the nature of the limitations lead to goal-oriented training curricula.\nThis study shows for the first time the importance of proper analysis of the instruments\u2019 movements during training of MIS skills. The clinical impact of such extended analysis is that only in this way is it possible to implement a correct objective score that will measure and certify the competence of surgeons\u2019 basic psychomotor MIS skills in addition to the existing criteria for the assessment of MIS performance. Moreover, this extended motion analysis can result in improvement of the training of basic MIS skills, since it will identify the differences between the experts\u2019 and novices\u2019 performance, and which areas require more training.\nConclusions\nMovements during laparoscopic tasks are not performed via the shortest path. Therefore, the shortest path length, as presently used for the assessment of basic MIS skills, may be not a proper concept for analyzing optimal movements and therefore needs to be revised. Goal-oriented movements should be split into two phases: a retracting and a seeking phase. Novices are less efficient in the seeking phase. This finding confirms that path length is an important measure in the assessment of basic MIS skills, but that the seeking phase is the only component that distinguishes between novice and expert. Furthermore, the retracting phase is very important in MIS, since it improves safety by avoiding intermediate tissue contact. Analyzing motions in the separate phases provides greater insight into the nature of the gap between expert and novice performance. Motion analysis in MIS should be seen as an addition to the existing criteria in assessing performance.","keyphrases":["goal-oriented movement","path length","minimally invasive surgery","objective assessment","training","motion analysis"],"prmu":["P","P","P","P","P","P"]}
{"id":"J_Med_Internet_Res-5-4-1550578","title":"Searching for Cancer Information on the Internet: Analyzing Natural Language Search Queries\n","text":"Background Searching for health information is one of the most-common tasks performed by Internet users. Many users begin searching on popular search engines rather than on prominent health information sites. We know that many visitors to our (National Cancer Institute) Web site, cancer.gov, arrive via links in search engine result.\nIntroduction\nFor members of the general public who use the Internet, many seek medical information [1- 6]. According to a recent systematic review of 24 peer-reviewed publications describing the proportions of Internet users among various populations of cancer patients in the developed world, about 39% of cancer patients are using the Internet directly, and in addition, 15% to 20% of persons with cancer use the Internet \"indirectly\" through family and friends [7]. Studies have evaluated information-seeking behavior on the Internet by cancer patients generally [8- 10], their companions [11,12], and patients with the following common specific cancer diagnoses: breast [13- 16], prostate [17,18], lung [19], and gastrointestinal cancers [20]. Studies have also evaluated information gathering by cancer patients undergoing radiotherapy [21] and chemotherapy [22], and those from centers outside of North America [23,24]. Individuals from certain disadvantaged groups have been shown to seek medical information online less frequently and with more difficulty [7,25,26].\nEysenbach and Kohler found that general consumers search for medical content using search engines rather than medical portals or sites of medical societies or libraries [27]. Newly-diagnosed cancer patients and their families often start their searches as users less sophisticated in Web and medical terminology. They too commonly begin searching on popular search engines rather than on prominent cancer-information sites. We know that many visitors to our own Web site [28] arrive via search engine result links.\nTo better understand users' needs this research aimed to establish what lay users really want to know when they search online for cancer information. To do this we evaluated data from Ask.com [29], a popular natural-language-processing (NLP) search engine. Natural-language-processing search engines allow users to create queries using whole phrases and sentences of any length, rather than just key words.\nEarlier reports of this project have been published in abstract form only. The abstracts reported a brief project summary [30], and data specific for breast cancer [31] and gastrointestinal cancer [20]. This is the first comprehensive report of the entire project.\nMethods\nThe National Cancer Institute (NCI) partnered with AskJeeves, Inc to develop a methodology to capture, sample, and analyze 3 months of cancer-related queries on the Ask.com Web site, a prominent US natural-language-processing consumer search engine. At the time of the project, Ask.com was receiving over 35 million queries per month.\nSearch Terms\nAn NCI oncologist (JLB) developed a benchmark set of 500 terms and word roots that were matched against actual AskJeeves user queries. Most terms and word roots were from the NCI dictionary on the NCI Web site [32]. NCI also suggested additional terms not included in the dictionary. These terms related to anatomy, organ systems, treatments, pharmaceuticals, treatment and diagnostic procedures, genetics, epidemiology, and pathology.\nTable 1\nTop 37 search terms and roots with \u2265 5 queries per week during test week\nTerm\nActual Queries During Test Week\n% of Total Queries\n*cancer*\n9765\n56.75\n*tumor*\n1396\n8.11\n*carcino*\n656\n3.81\n*leukemia*\n635\n3.69\nlymphom*\n419\n2.43\nchemotherapy\n378\n2.20\nbiopsy\/biopsies\n375\n2.18\n*melano*\n348\n2.02\n*sarcoma*\n294\n1.71\n*dysplasia*\n255\n1.48\nhodgkin*\n245\n1.42\nMRI\n214\n1.24\nclinical trial\n187\n1.09\nmammogr\n175\n1.02\nmaligna*\n170\n0.99\n*metasta*\n155\n0.90\n\"*\" is a placeholder for the part of the search term before or after the root.\nThe test sample of these 500 words and roots was used to filter cancer queries from the Ask.com Web site for 1 week in August 2001. From these 500 terms, only 37 appeared \u2265 5 times per day over the trial week. The list of 37 terms (plus common misspellings) yielded 17208 queries for the test week. The frequency of each term is shown in Table 1. Queries with common misspellings, (eg, prostate and prostrate, biopsy and biopsey, leukemia and lukemia, chemotherapy and chemothereapy) were captured and analyzed. It was felt that the cut off of 5 times per day (\u2265 35 times per week) would capture the key queries and include any common query topic, since even with a frequency of 35 queries a week, the majority of these terms accounted for less than 1% of the total population. Of the original 500 terms supplied by NCI, only 7% (35\/500) appeared in the logs at a high frequency, but this 7% accounts for over 37% of user queries identified as cancer related on Ask.com during the study period.\nCollecting Queries and Sampling\nThe process used for collecting and sampling queries is outlined in Figure 1. Using the 37 terms to search the Ask.com query logs, 204165 instances of cancer-related queries were found for June, July, and August 2001. Of these queries, 7500 individual user questions were randomly selected by AskJeeves for detailed analysis (see Appendix 1).\nFigure 1\nProcessing of cancer queries on Ask.com\nVery often there were multiples of the same questions. Thus, these 7500 queries actually represented 76077 queries that were entered into Ask.com, about 37% (76077\/204164) of all queries identified as cancer-related from the 3 month period of log analysis. For example, a user question might be \"Where can I find information about breast cancer?\" This individual example represents 1 user question, but might have been queried by more than 100 people on any given day. Each query was counted only once.\nSampling Issues\nThe random sample of 7500 individual queries provides a confidence interval of 1.11% at a confidence level of 95%. This means that even if more samples were taken from 204165 queries, 95% of those samples should not be off by more than 1.1%. While this means that the samples themselves would not vary more than 1.1% over 95% of the samples taken, as the data are categorized and classified, in effect smaller and smaller samples are taken. Therefore, to offset this problem additional queries were examined, even though a smaller sample would still provide a high degree of confidence in the results.\nIn other words, although broad generalizations\u2014such as \"breast cancer accounts for 25% of all cancer queries\"\u2014can be easily presented, a large sample size is required to break down data far enough to conclude that when users ask about breast cancer, they are most often asking about specific types of treatments.\nHighest-Level Categories for Queries\nUser queries were assigned to a set of 6 highest-level categories (as shown in Table 2):\nCancer (ie, specifically mentioning a cancer type)\nGeneral Research\nTreatment\nDiagnosis and Testing\nCause\/Risk\/Link\nCoping\nTable 2\nHighest-level categories for queries\nHighest-Level Category\nNumber of Queries\nPercent of All Sampled Queries\n*\nCancer \u2020\n59619\n78.37\nGeneral Research\n7808\n10.26\nTreatment\n3832\n5.04\nDiagnosis and Testing\n3315\n4.36\nCause\/Risk\/Link\n1249\n1.64\nCoping\n254\n0.33\nTotal\n76077\n* Percentages do not sum to 100% due to rounding.\n\u2020 ie, specifically mentioning a cancer type.\nHighest-level categories were created in a collaborative effort between the AskJeeves data-analysis team and NCI staff before the study period began, but the final category titles were revised as the actual queries were analyzed. The initial categories were based on user queries entered into Ask.com and a variety of online sources, such as NCI's online dictionary [32] and NCI's Physician Data Query (PDQ) [33].\nThe highest-level categories were populated using proprietary AskJeeves filters and automated-analysis tools that sorted queries according to specific types of cancers, or\u2014in the absence of mentioning a specific cancer type\u2014whether the query asked about other areas such as Treatment or Coping. (AskJeeves did not share the filters and automated analysis tools with the authors.) Queries that could not be sorted by the filters and automated-analysis tools were placed in a temporarily-uncategorized category; they were categorized during the next step (reading and analysis).\nReading and analyzing each individual query not only verified the automated process, but also helped to refine existing categories and create new categories and subcategories, as appropriate. For example, without this type of analysis, the query \"Where can I find a Web site with information on using high protein food to fight Breast cancer?\" might have been left under Breast Cancer > Media and Organizations > Web sites (where \">\" indicates a change in category level). This would not be correct, as the true user intent was to inquire about Alternative Treatments. As a result, under the category Breast Cancer > Treatment, \"Alternative\" was added to the Breast Cancer > Treatment category analysis as a subtopic. (Treatment\u2014without a specific cancer site designated\u2014is both a highest-level category and a subcategory under Breast Cancer and under most cancer types.)\nApproximately 78% of all categorized queries from the sample referenced a particular type of Cancer, and were placed in the highest-level category Cancer. An example of this kind of query would be \"Where can I find information about Breast Cancer?\" (This query would be classified as Cancer > Breast Cancer > General Information.) Any query that did not mention a specific kind of Cancer, even though the question was about cancer, was placed on 1 of the 5 other highest-level categories. An example of this type of query would be \"Where can I find information on cancer treatment with radiation?\" This query was assigned to the Radiation subcategory in the highest-level category Treatment (ie, it was classified as Treatment > Radiation).\nQueries that did not relate to a specific Cancer type were placed in 1 of the 5 other highest-level categories: General Research, Treatment, Diagnosis and Testing, Cause\/Risk\/Link, or Coping. For example the query \"How does smoking cause cancer?\" would be placed in the Cause\/Risk\/Link category, as it did not refer to any specific type of cancer.\n\"Cancer\" Queries (Related to Specific Cancer Types)\nAs shown in Table 3, there were 14 cancer types (N = 59619 queries) selected as subcategories of the Cancer highest-level category. For cancer types with the most-frequent queries, like Digestive\/Gastrointestinal\/Bowel (D\/G\/B), Breast, and Genitourinary, there were enough queries to populate standard subcategories like General Information, Treatment, Symptoms, Diagnosis and Testing, and Cause\/Risk\/Link. These common cancer types often warranted the creation of customized subcategories, like Breast > Media and Organizations > Web sites. For the less-common cancer type queries, like Bile (duct) in D\/G\/B, few queries were received and only those in General Information are shown.\nTable 3\nCancer types\nType within Top-Level Cancer Category\nNumber of Queries\n% Queries in Cancer Category\n*\n\u2020\n% Queries in This Report\n*\n\u2021\nDigestive\/Gastrointestinal\/Bowel (D\/G\/B)\n8959\n15.0\n11.8\nBreast\n6953\n11.7\n9.1\nSkin\n6709\n11.3\n8.8\nGenitourinary\n6250\n10.5\n8.2\nHematologic\/Blood\n5448\n9.2\n7.2\nGynecological\n5344\n9.0\n7.0\nLung\n4630\n7.8\n6.1\nSoft Tissue\/Muscle\n3954\n6.6\n5.2\nLymphoma\n3333\n5.6\n4.4\nHead and Neck\n2522\n4.2\n3.3\nBrain\/Neurological\n1852\n3.1\n2.4\nMiscellaneous Cancer\n1633\n2.7\n2.1\nBone\n1429\n2.4\n1.9\nPediatric\n603\n1.0\n0.8\n* Percentages do not sum to 100% due to rounding.\n\u2020 Denominator (N = 59619) was the total number of queries about specific types in the Cancer category.\n\u2021 Denominator (N = 76077) was the total number of queries analyzed in this report.\nPrivacy Issues\nAlthough NCI helped create the search terms and the categories into which the analyzed data was placed, NCI did not have access to: the raw query logs at AskJeeves, any information about what AskJeeves users did with the searches generated on the AskJeeves Web site (ie, what links they picked), or the identities of any users of the Ask.com Web site. NCI did not require permission from the Institutional Review Board.\nResults\nFrequency of Top-Level Categories\nAs shown in Table 2, The 6 highest-level categories in order of decreasing frequency of queries were:\nCancer (N = 59619, 78.37%)\nGeneral Research (N = 7808, 10.26%)\nTreatment (N = 3832, 5.04%)\nDiagnosis and Testing (N = 3315, 4.36%)\nCause\/Risk\/Link (N = 1249, 1.64%)\nCoping (N = 254, 0.33%)\nThe data in Table 2 indicate that the great majority of users asked for information about specific types of cancers, but rarely asked about a Treatment option or Diagnosis and Testing procedure without specifying the particular cancer about which they were concerned. Similarly, users asked few queries about general Symptoms of cancer unrelated to a specific type of cancer (see Diagnosis and Testing > Symptoms, N = 473, 14.27%). An example would be \"what are some symptoms of cancer?\"\nSubdividing Cancer Queries\nTable 3 breaks down the highest-level category Cancer queries (N= 59619) into more specific cancer types. In order of decreasing frequency within the Cancer category, the 14 subcategories were:\nDigestive\/Gastrointestinal\/Bowel (D\/G\/B) (N = 8959, 15.0%)\nBreast (N = 6953, 11.7%)\nSkin (N = 6709, 11.3%)\nGenitourinary (N = 6250, 10.5%)\nHematologic\/Blood (N = 5448, 9.2%)\nGynecological (N = 5344, 9.0%)\nLung (N = 4630, 7.8%)\nSoft Tissue\/Muscle (N = 3954, 6.6%)\nLymphoma (N = 3333, 5.6%)\nHead and Neck (N = 2522, 4.2%)\nBrain and Neurological (N = 1852, 3.1%)\nMiscellaneous (N = 1633, 2.7%)\nBone (N = 1429, 2.4%)\nPediatric (N = 603, 1.0%)\nAny query specifically mentioning a cancer type by name, was assigned to that subcategory. For example, questions about Breast-Cancer-specific Treatment, Diagnosis and Testing, Causes, and Coping are found in the Cancer > Breast Cancer category, within 1 of the 10 subcategories displaying Breast Cancer information. All questions about Leukemia or Myeloma would be found in Hematologic\/Blood, Hodgkin's Disease queries in Lymphoma, and Esophageal cancer questions in D\/G\/B.\nThe number of subcategories assigned to each of the 14 different cancer types varied somewhat and was driven by the nature and number of the specific queries in those cancer types.\nDetailed Analysis of Queries\nThe detailed categorizations and verbatim display of examples of sampled queries are shown in Appendix 1. There is a breakdown of all the 14 cancer types within the highest-level category Cancer as well as a breakdown of queries within the 5 other highest-level categories not referencing any particular cancer type. These 19 are arranged alphabetically in the Appendix.\nMajor observations about the 19 categories and subcategories are noted below, in the order they appear in the Appendix. Our comments emphasize issues related to requested cancer content more than technology issues related to the natural language processing.\n1.0 Bone Cancer\nAs shown in Appendix 1, there were 1429 queries about Bone Cancer. The vast majority of Bone Cancer queries asked for General Information (N = 1107, 78%). An example of this category would be: \"Where is information on bone cancer?\" Users asked questions about Bone Cancers linked to various sites of Anatomy as well as certain Histologies. There were some questions related to Bone Cancers in teenagers that were assigned to this category, rather than the Pediatric category. There were more questions about Diagnosis and Testing (N = 64, 4.48%) and Symptoms (N = 135, 9.45%) than Treatment (N = 26, 1.82%).\n2.0 Brain and Neurological Cancer\nOf the 1852 Brain and Neurological Cancers queries, General Information accounted for the vast majority (N = 1323, 71.44%). There were 427 (23.1%) questions about specific cancer types in this category. Some cancer types queries asked about Medulloblastoma, which is typically but not always a Pediatric tumor. As with Bone Cancer above, some questions could have been meaningfully assigned to more than 1 top-level Cancer site category. In this category there were more queries about Symptoms (N = 259, 13.98%) than Treatment (N = 112, 6.05%).\n3.0 Breast Cancer\nAs shown in Appendix 1, Breast Cancer was one of the simpler cancer types, from a data-display standpoint. There was only 1 anatomic-cancer type and all of the individual queries for that cancer type were assigned into 1 of 10 subcategories.\nThe 10 top-level Breast Cancer subcategories were:\nGeneral Information (N = 3423, 49.23%)\nSymptoms (N = 889, 12.79%)\nTreatment (N = 570, 8.20%)\nMedia\/Organization (N = 428, 6.16%)\nCause\/Risk\/Link (N = 393, 5.65%)\nDiagnosis and Testing (N = 376, 5.41%)\nStatistics (N = 274, 3.94%)\nPictures (N = 225, 3.24%)\nType (N = 217, 3.12%)\nDefinition (N = 158, 2.27%)\nNine of the 10 Breast Cancer subcategories were analyzed in detail in Appendix 1. The tenth, Pictures, did not require further analysis. Most queries asked for General Information.\nThere were more frequent queries about Breast Cancer (N = 6953) than any other cancer type. This may not be apparent from Table 3, which appears to show more D\/G\/B cancers (N = 8959). However, D\/G\/B overall is actually composed of 10 cancer types. The most frequently queried cancer type in D\/G\/B was Colorectal (N = 4,801) which had fewer queries than Breast.\nEven though other cancer types may have been assigned more subcategories than the 10 for Breast, the detail and the medical specificity and technical vocabulary of Breast queries appear to be the most complex than other Cancer sites, probably reflecting the sophistication of basic research and clinical data on this topic and the relative sophistication of the breast cancer information seekers.\n4.0 Cause and Risk\nThere were 1249 queries in this highest-level category. Without mentioning a specific cancer by name, there were N = 1115 (89.27%) queries about Causes and Links but only N = 134 (10.73%) about Prevention. Among the 1115 queries in the Causes and Links subcategory, the following topics were noted:\nDrugs (N = 287, 25.74%)\nUnspecified (N = 247, 22.15%) (eg, \"What is cause a cancer?\" [sic])\nRadiation (N = 247, 22.15%)\nPersonal (N = 116, 10.40) (eg, \"Can anti-persperant [sic] deodorant cause cancer?\")\nChemical\/Plastics (N = 74, 6.64%)\nEnvironmental (N = 70, 6.28%)\nFood Supplement (N = 64, 5.74%)\nGenetic Mutation\/Virus (N = 10, 0.90%)\nSmoking was not in this list, probably because most queries about smoking were included under a query about a specific type of cancer, like Lung or Head and Neck.\n5.0 Coping\nThere were only 254 queries about Coping. The queries referenced Support Groups (N = 127, 50%), Pain (N= 98, 38.58%), and Depression (N = 29, 11.42%). Even though there were few questions in this highest-level category, the issue was of specific interest to NCI, which asked for this category to be created and analyzed separately.\n6.0 Diagnosis and Testing\nThere were 3315 queries in this highest-level category, which did not mention a specific cancer by name. Most were queries about specific Testing (N = 2842, 85.73%). The others (N = 473, 14.27%) were queries about Symptoms. Among Testing queries, CAT\/CT scan (Computerized Axial Tomography\/Computed Tomography scan) (N = 1509, 53.10%) and MRI (N = 587, 20.65%) were the most-common Testing topics, followed by Biopsy (N = 502, 17.66%).\n7.0 Digestive\/Gastrointestinal\/Bowel (D\/G\/B)\nThe presentation of data queries for D\/G\/B in Appendix 1 is complex because, there were 7 top-level subcategories, including General Information and 10 cancer types identified in the General Information subcategory\nAs shown in Appendix 1, 8959 queries for D\/G\/B sites were broken down into 7 subcategories:\nGeneral Information (N = 5568, 62.15%)\nSymptoms (N = 1506, 16.81%)\nDiagnosis and Testing (N = 1125, 12.56%)\nTreatment (N = 294, 3.28%)\nStatistics (N = 184, 2.05%)\nDefinition (N = 163, 1.82%)\nCause\/Risk\/Link (N = 119, 1.33%)\nMost queries asked for General Information. Examples of General Information queries would be \"Where can I learn about the cancer esophageal cancer?\"? and \"Where can I find information on Stomach cancer\"?\nA breakdown of all D\/G\/B queries by cancer type is shown in the list below. The absolute numbers and percentages (of all D\/G\/B queries) in the list below differ from the pie diagram in Appendix 1 because the list below includes organ-type queries from General Information plus the 6 other subcategories in D\/G\/B.\nColorectal (N = 4801, 53.59)\nLiver (N = 1413, 15.77%)\nGastrointestinal (stomach) (N = 1094, 12.21%)\nPancreas (N = 965, 10.77%)\nBowel (N = 273, 3.05%)\nEsophagus (N = 260, 2.90%)\nOther (N = 153, 1.7%)\nThe organ subsites in Other include Gall Bladder, Bile Duct, Anal, and Abdominal.\nAs noted in Appendix 1, for D\/G\/B there were far more questions about Symptoms (N = 1506, 16.81%) than Treatment (N = 294, 3.28%) possibly reflecting the fact that (1) users of Ask.com were just beginning their D\/G\/B information seeking and (2) there is less complexity in the published Treatment data for D\/G\/B compared to some other cancer types, like Breast Cancer.\nThe terms Bowel, Gastrointestinal, Stomach, and Abdominal may have been used interchangeably by users. They appear not to recognize that queries for sigmoid, rectum, cecum, appendix, transverse colon, small bowel, and stomach (gastric) cancer would provide much more useful information.\nFor D\/G\/B, some queries about Liver Metastases were included with queries about primary Liver Cancers.\n8.0 General Research\nThere were 7808 queries assigned to the highest-level category General Research, a topic not linked to a specific cancer type. In this category the 5 most-common subcategories were:\nResearch (N = 2819, 36.10%)\nOrganization (N = 1656, 21.21%)\nClinical Trials (N = 1272, 16.29%)\nConcerns (N = 1201, 15.38%)\nPictures (N = 559, 7.16%)\nAmong the queries about Organization, there were 1065 queries about the American Cancer Society (ACS) and 223 about the National Cancer Institute (NCI).\nAmong the 1272 queries about Clinical Trials, the most-common 3 questions\/topics were:\nWhat are ... (N = 634, 49.84%) eg, \"What are clinical trials?\"\nLatest ... (N = 260, 20.44%) eg, \"latest cancer clinical trial research\"\nTypes of ... (N = 111, 8.73%) eg, \"types of cancer trials\"\n9.0 Genitourinary Cancers\nIn decreasing order, the frequency of Genitourinary organ-type queries (N = 6250) in all 12 Genitourinary subcategories including General Information was:\nProstate (N = 3141, 50.26%)\nTesticular (N = 1772, 28.35%)\nBladder (N = 708, 11.33%)\nKidney (N = 496, 7.94%)\nOther (N = 133, 2.12%)\nAlthough it has been estimated that there were 198100 new cases of Prostate Cancer diagnosed in the US in 2001 and only 7200 cases of Testis Cancer [34], the relative frequency of Testis Cancer queries was quite high. One possible reason might be that males diagnosed with Testis Cancer are generally much younger than those diagnosed with Prostate Cancer, and those younger individuals might be more-frequent information seekers on the Internet. It may also reflect the fact that the 2001 Tour de France bicycle race won by Lance Armstrong, a Testis Cancer survivor, was held during July, coinciding with the study period for this project.\nAs with most sites, the most-common Prostate Cancer questions were General Information (N = 1715, 54.6%). For Prostate Cancer, there were more questions about Treatment (N = 460, 14.65%) than Symptoms (N = 364, 11.59%). This may reflect major medical controversies about treatment options and the typically asymptomatic presentation of the disease.\nFor the Genitourinary category as a whole, there were more questions about Symptoms (N = 854, 13.66%) than Treatment (N = 604, 9.66%).\nExpected misspellings of prostate (prostrate) were noted.\n10.0 Gynecological Cancers\nThere were 5344 queries overall. The breakdown of subcategories in decreasing frequency was:\nGeneral Information (N = 3409, 63.79%)\nSymptoms (N = 939, 17.57%)\nDiagnosis and Testing (N = 452, 8.46%)\nTreatment (N = 247, 4.62%)\nDefinition (N = 158, 2.96%)\nCause\/Risk (N = 83, 1.55%)\nStatistics (N = 42, 0.79%)\nPrevention (N = 14, 0.26%)\nIn decreasing order of frequency, the cancer types queried in all 8 Gynecological subcategories included the following:\nOvarian (N = 2031, 38.00%)\nCervical (N = 1924, 36.00%)\nUterine (N = 606, 11.34%)\nEndometrial (N = 225, 4.21%)\nVulvar (N = 166, 3.11%)\nVaginal (N = 219, 4.09%)\nOther or not specified (N = 173, 3.24%)\nThere were nearly as many questions about Cervical Cancer as Ovarian Cancer despite the fact that in the United States in 2001 the estimated incidence of new Ovarian Cancers was about twice that of invasive Cervical Cancer [34].\nThere were questions about Endometrial cancer as well as Uterine cancer. These data suggest that Web site information needs to be provided using both labels.\n11.0 Head and Neck\nThere were 2522 queries overall. Most queries asked for General Information (N = 1485, 58.88%). The vocabulary used to ask about specific cancer types within General Information was:\nThroat\nMouth\nOral\nTongue\nHead\nNeck\nThe vocabulary confirms the need to offer health information with words that are not technical like larynx, glottis, pharynx, or nasopharynx. There were 59 questions asking about Definitions of Head and Neck cancer. Specifics about cancer anatomy of this cancer type may be less familiar to the general public than other sites.\nThere were 422 queries asking for Pictures of Head and Neck Cancer. There were only 47 questions (1.86%) asking about Cause\/Risk\/Link issues, despite the fact that there is a great deal known about the Causes and Prevention of Head and Neck Cancer. There were 418 questions (16.57%) about Symptoms and but only 52 (2.06%) about Treatment.\n12.0 Hematologic and Blood Cancers\nAmong 5448 queries in this category, the 5 most common of the 12 subcategories were: General Information (N = 3781, 69.40%), Definition (N = 701, 12.96%), Symptoms (N = 539, 9.89%), Treatments (N = 175, 3.21%), and Organizations (N = 102, 187%). Within General Information users asked about Leukemia (N = 2895, 76.57%), Myeloma (N = 592, 15.66%), Bone Marrow (N = 148, 3.91%), and Blood Cancers (N = 146, 3.86%). Various misspellings of Leukemia were noted and nontechnical terms such as Blood Cancer and Bone Marrow Cancer were frequent.\n13.0 Lung Cancer\nLung Cancer (N = 4630) accounted for 8% of organ-type specific queries within the highest-level Cancer category. This is a disproportionately-low percentage given the relative incidence of Lung Cancer in the United States in 2001 [32]. There were more queries about Gynecological and Hematologic\/Blood cancers, even though the US incidence for these is far lower.\nAmong Lung Cancer queries, the queries were classified as follows:\nGeneral Information (N = 3223, 69.61%)\nSymptoms (N = 530, 11.45%)\nCause\/Risk\/Link (N = 305, 6.59%)\nTreatment (N = 219, 4.73%)\nDefinition (N = 150, 3.24%)\nStatistics (N = 113, 2.44%)\nDiagnosis and Testing (N = 90, 1.94%)\nIn the Cause\/Risk\/Link category of Lung Cancer, there were only N = 180 queries (59.02%) that asked generally about Causes of Lung Cancer and N = 102 queries (33.44%) that asked specifically about Smoking. There were N = 23 queries (7.54%) asking if Marijuana caused Lung Cancer.\nOnly N = 255 (7.91%) queries within General Information asked about Lung Cancer by (histologic cell) Type, despite the fact that this is a major determinant of triage for treatment.\nFor Lung Cancer > Treatment, there were 219 queries (4.73%). Most Treatment queries were Unspecified (N = 118, 53.88%), eg, \"What are treatments for lung cancer?\" There were 26 Treatment questions about Cure (11.87%). There were few specific questions about Medications (chemotherapy) (N = 21, 9.59%), Radiation (N = 19, 8.68%), or Surgery (N = 10, 4.57%). Although all numbers were small, there were more questions about Alternative Treatment (N = 13, 5.94%) than Surgery (N = 10, 4.57%). There were only 4 Treatment questions (1.83%) about palliative care, despite the grave prognosis for most Lung Cancers. Clearly the questions about Lung Cancer, the most-common lethal cancer, were far less sophisticated than the questions about either Breast Cancer or Prostate Cancer.\n14.0 Lymphomas\nAmong the 3333 queries about Lymphoma (including both Hodgkin's Disease and Non-Hodgkin's Lymphoma), General Information (N = 2391, 71.74%) questions were the most common. Unlike many cancer types, there was frequent mention of histologic types, as is appropriate, given the wide variety of clinically-different prognoses and treatments in this subcategory. There were many different spellings of Hodgkin's Disease.\n15.0 Miscellaneous Cancers\nThere were 1633 queries assigned to this Cancer subcategory. The Miscellaneous Cancers were:\nEndocrine (N = 901, 55.17%)\nNeoplasm (N = 272, 16.66%)\nKaposis (N = 262, 16.04%)\nOcular (N = 179, 10.96%)\nGerm Cell (N = 19, 1.16%)\nSeveral of the Ocular queries, eg, Ocular Melanoma and Retinoblastoma, could have been considered for other subcategories, such as Skin and Pediatric respectively. Germ cell tumors could also have been placed in either Genitourinary or Gynecological subcategories. These ambiguities illustrate the difficulty in categorizing precise user information needs despite the use of natural language processing.\n16.0 Pediatric\nThere were only 603 Pediatric queries, and most asked about a specific cancer type (N= 403, 66.83%). There were relatively few General Information queries (N = 81, 13.43%) eg, \"where can I find information on children's cancers?\" Since patients with Pediatric cancers in the US are usually managed generally by pediatric oncology specialists at major regional medical centers, those seeking Pediatric cancer information are probably directed to specialized Web sites rather than general sites like Ask.com.\nOf 403 queries for cancer types, the most common were Hematologic\/Blood (N = 137, 34%), Neuroblastoma (N = 133, 33%), and Rhabdomyosarcoma (N = 68, 16.87%). There were only 4 questions referring to pediatric Brain and Neurological cancers. Since this is such a common Pediatric tumor type, it is possible that some Pediatric neurological tumor questions were assigned to the Brain and Neurologic Cancer category even though the questions were really meant to target a Pediatric issue.\n17.0 Skin Cancers\nAmong 6709 queries in this Cancer subcategory, 3596 (53.60%) asked for General Information. Like Lymphoma, there was frequent mention of specific Skin Cancer types (N = 2157, 32.15%), probably because of the significantly-different clinical prognoses and treatments.\nOnly 169 queries (2.52%) asked about Cause\/Risk\/Link, and 60 queries (0.89%) asked about Prevention despite the fact that so much is known about these topics and Skin Cancer.\nAmong Skin Cancers queried by histologic cancer type (N = 2157, 32.15%), Melanoma was the most common (N = 1707, 79.14%), even though it is far-less common than Basal Cell Skin Cancers (N = 322, 14.93%) [10]. Frequent mention of Melanoma probably reflects its more-serious prognosis and more-complicated clinical triage.\n18.0 Soft Tissue Cancers\nThere were 3954 queries in this Cancer subcategory. Although most appropriately refer to sarcomas of various types, there was a minority of misplaced queries. Some queries appear to reference conditions that are probably benign (Ganglion, Fibroid, Dysplasia, and Lipoma) and others should have been placed in different Cancer subcategories eg, Brain and Neurological (Oligodendroglioma and Glioma) These will be corrected on later analyses.\n19.0 Treatment\nIn the 3832 highest-level category queries about Treatment, most questions were about a specific Treatment Type (N = 3223. 84.11%), even though no specific cancer was mentioned. Within Treatment > Treatment Type there were many general queries about Chemotherapy (N = 2275, 70.59%). There were questions about general Radiation Therapy (N = 534, 16.57%), and few about specialized Radiation Therapy treatments like Gamma Knife, Laser, and Protons. There were more general questions about Alternative Therapies (N = 239, 7.42%) than Surgery (N = 127, 3.94%) Many Alternative Therapy questions also appear in specific organ-type subcategories, particularly Breast.\nQuery Frequency Relative to US Incidence of Cancer Types\nTable 4 compares the incidence of selected cancers in the United States (US) in the year 2001 with the frequency of selected site-specific cancer queries in this report. It has been estimated that there were 1268000 new cancer cases in the US in 2001 [34]. The sites in Table 4 were selected specifically because they were easiest to compare directly.\nThe relative percentage of specific organ-type queries exceeds the percentage of annual incidence only for rarer cancers. The difficulty of finding useful information on prominent cancer portals or with standard search engines may be one explanation, although there are others. The comparison is not meant to be definitive as there are clearly issues with validity of this comparison:\nCancer prevalencemight be a better benchmark than incidence\nUS incidence data exclude cases of in situ breast and cervix cancers as well as the very-common basal cell and squamous cell skin cancers\nQueries could have come from anywhere in the world, not just the United States\nQuery total may include those who accessed the site more than once\nQueries could have come from individuals who are not newly-diagnosed patients\nTable 4\nComparing relative annual US incidence of selected cancers and query frequency\nCancer Site\nEstimated Number of New US Cancers Diagnosed in 2001\n*\n% of Estimated New US Cancers in 2001\n*\n\u2020\nNumber of Cancer Site-Specific Queries in This Report\n% Queries in Cancer Category\n\u2020\n\u2021\nDigestive (D\/G\/B)\n235700\n18.6\n8959\n15.0\nProstate\n198000\n15.6\n3141\n5.3\nBreast\n193700\n15.3\n6953\n11.7\nLung\n169000\n13.3\n4630\n7.8\nLymphoma\n63600\n5.0\n3333\n5.6\nBladder\n54300\n4.3\n708\n1.2\nUterus\/Endometrial\n38300\n3.0\n931\n1.6\nHead and Neck\n30100\n2.3\n2522\n4.2\nOvary\n23400\n1.9\n2031\n3.4\nBrain and Neurological\n17200\n1.4\n1852\n3.1\nCervix\n12900\n1.0\n1924\n3.2\nSoft Tissue\n8 700\n0.69\n3954\n6.6\nTestis\n7200\n0.57\n1772\n3.0\n* Data from 2001 Estimated Annual US Cancer Incidence Figures (N = 1268000) [10].\n\u2020 Percentages in columns 3 and 5 do not add up to 100% because only selected cancers were included in this chart.\n\u2021 Only selected Cancers were included in this chart. Denominator (N = 59619) was the total number of queries about specific subsites in the Cancer category.\nOther Observations\nThe query analysis reveals that online users generally seek information about Symptoms and Treatment for specific cancers, rather than about cancers generally. In addition, Symptom queries showed a frequency between 2 and 5 times that of Treatment queries, for most cancers.\nFor this study we did not specifically target queries about Adult Immune Deficiency Syndrome (AIDS), even though AIDS can often be associated with Cancer. There were 262 questions about Kaposi's Sarcoma in the Miscellaneous Cancers category.\nDiscussion\nGeneral Information was the largest category for almost all cancers, probably reflecting the nature of the Ask.com consumer search engine. It is a consumer-oriented Web-wide search engine where users tend to seek general information that can help them learn either how or where they should further pursue their inquiries. It is likely the users are just starting their Web searches on Ask.com and they are not yet interested in or they do not yet know enough information to ask more-sophisticated questions. This behavior may not reflect that of users who go directly to a known cancer-information portal with a predetermined need for detailed information.\nWe attempted to capture and analyze all cancer-related queries, including those with correct and incorrect spellings. Misspellings were noted relatively frequently, but we have no data on the number of misspellings, as we did not target this in advance as an endpoint, and we did not have direct access to the raw data logs. Appendix 1 shows verbatim queries with examples of the misspellings. Automating help for users who enter misspelled words is a major issue for search engines in order to optimize query results. Other researchers have noted the search difficulties related to spelling of cancer search terms correctly [35].\nAsk.com users entered both keyword searches and sentence-style queries, despite the fact that this is a natural-language-processing search engine. We recognize that even if users typed in a long query it was still sometimes difficult to discern absolutely what specific information the user needed, particularly since we did not have access to the links users picked.\nThe vocabulary employed by users of Ask.com ranged from unsophisticated to very sophisticated. This suggests that allowing users to employ less-technical language on cancer Web sites would significantly help them find the information they seek.\nThe queries captured for this study undoubtedly reflect the news and research studies in the public arena during the time period from June to August 2001. A different time period would certainly reflect a different distribution. Examples of the kinds of events that could affect the results include the diagnosis or death of a celebrity with cancer, the publication of a major trial about bone marrow transplantation for breast cancer, or the Food and Drug Administration approval of an important new drug.\nThe presence of a search engine with natural language processing on a Web site, while potentially valuable to users, does not obviate the need for good user-centered Web site design and information architecture [36]. It has been shown that searching via search engine can be minimized and user satisfaction maximized if information architecture and link titles follow appropriate guidelines [37]. Nevertheless, for less-sophisticated users, a natural-language-processing search engine can be helpful in finding the information users seek and provide enhanced success in searching.\nAn October 30, 2003 search of the PubMed Web site [38] of the National Library of Medicine [39] yielded 458 search results from a query for \"Natural Language Processing.\" Most citations were from publications within the last 3 years, attesting to the currency of natural language processing as an important research topic cutting across a wide variety of research disciplines. Potential data-mining applications of this tool in medicine extend far beyond the use described in this paper.\nEysenbach and Kohler have recently developed a novel methodology, similar to the method used in this study, to estimate the actual volume and prevalence of health-related searches on the Web in relation to the total number of searches conducted daily on the Internet [40]. They collected queries from 2 search engines, Metacrawler (a search engine of search engines) [41] and Ask.com [29] (the same natural-language-processing search engine used for this report). These 2 search engines were selected because they allowed \"peeking\" at actual user search-query topics. They concluded that 4.5% of all searches on the Web might be health related. The queries were collected from Metacrawler between February 2001 and April 2002, and from Ask.com between February 2001 and April 2001. The first date range overlapped our study dates and the second occurred just before data collection for our study.\nIn summary, natural-language-processing tools such as the one used for this study are able to filter and subset raw query data into useful analysis categories. Retrieval and analysis of these data can be used to better understand the actual content users want and the level of understanding and sophistication they have when they come to the Web site. Using the information on a continuing basis can form the basis for updating content on Web sites based on the most-current user needs. If a natural-language search engine were offered on a health-information portal, for example, it could improve customer access to desired information, particularly for those users with less sophistication about content or language. Additional analyses of query results are planned for the future. Consideration has been given to piloting the use of natural language processing on subsites of our Web portal.","keyphrases":["cancer","internet","search engines","natural language processing"],"prmu":["P","P","P","P"]}
{"id":"Intensive_Care_Med-3-1-1915613","title":"Pediatric delirium in critical illness: phenomenology, clinical correlates and treatment response in 40 cases in the pediatric intensive care unit\n","text":"Objective To study the phenomenology, clinical correlates, and response to treatment of delirium in critically ill children in the pediatric intensive care unit (PICU).\nIntroduction\nDelirium is a\u00a0neuropsychiatric disorder secondary to a\u00a0general medical condition, and must be considered a\u00a0serious complication of the underlying disease or its treatment. In the revised fourth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV-TR\u00ae), delirium is defined by four concurrent diagnostic criteria: (1) acute onset and fluctuation of (2) a\u00a0disturbance of consciousness with reduced ability to focus, shift or maintain attention and (3) a\u00a0change of cognition with memory deficit, disorientation, language disturbance, perceptual disturbances or hallucinations, (4) caused by the direct physiological consequences of a\u00a0general medical condition\u00a0[1]. It is frequently seen in critically ill adult and geriatric patients\u00a0[2\u20135] and is associated with a\u00a0poor prognosis, reflected by longer hospital stay, worse functional and cognitive outcome, and a\u00a0higher mortality rate after discharge from hospital\u00a0[3]. In mechanically ventilated critically ill adults, delirium is an independent predictor of elevated 6-month mortality and a\u00a0longer hospital stay\u00a0[6]. If appropriate diagnostic tools validated for bedside use by non-psychiatrists [e.g. Delirium Rating Scale (DRS), Confusion Assessment Instrument for the Intensive Care Unit (CAM-ICU)] are used, delirium is diagnosed in over 80% of critically ill adult patients\u00a0[7]. Thus, systematic monitoring for delirium and appropriate treatment with haloperidol in critically ill adult patients were included in the recently published clinical practice guidelines for sedatives and analgesia of the Society of Critical Care Medicine\u00a0[8]. However, the optimal management of patients with delirium and the effects of the pharmacological treatment on the outcome are still key concerns for today\u00a0[9]. Given lack of age-appropriate diagnostic criteria and assessment tools in children, even less is known about the incidence, clinical presentation, response to treatment and consequences of childhood delirium in general, and in critically ill children in particular\u00a0[10\u201312]. The few available published data on childhood delirium suggest that morbidity and mortality are higher in children with than in children without delirium\u00a0[13]. Therefore, delirium in children should be considered a\u00a0serious complication and be treated accordingly. Unfortunately, while there are comprehensive guidelines on the diagnosis and treatment of delirium in adults, clinical guidelines for delirium in children are nonexistent.\nThe aim of this study was to investigate the incidence, patient and population characteristics, clinical presentation and response to treatment of delirium in a\u00a0cohort of critically ill children admitted to a\u00a0tertiary pediatric intensive care unit (PICU). Given the necessarily multidisciplinary approach to assessment and treatment of these children, the input of four disciplines \u2013 child psychiatry, pediatric intensive care medicine, child neurology and adult neuropsychiatry \u2013 was used.\nMethods\nDesign, setting and patients\nA\u00a0descriptive study was carried out over a\u00a04-year period (January 2002 to December 2005) in an eight-bed tertiary PICU. This PICU is a\u00a0tertiary referral center for both general and surgically critically ill children in the southeastern region of the Netherlands (population 1.4 million, 350 annual admissions). Critically ill children, acutely, non-electively and consecutively admitted, were prospectively sampled. Both mechanically ventilated and non-ventilated patients were included.\nDiagnostic approach\nAll children with (1) confusion, agitation, anxiety, moaning, discomfort, or behavioral disturbances with no acceptable medical explanation or (2) failure of standard analgosedative treatment were systematically assessed for the presence of delirium in a\u00a0two-step diagnostic approach. The standard analgosedative treatment can be summarized as follows: children who required analgosedation because of obvious or expected pain or because of stress related to their underlying disease or treatment received adequate doses of opioids and\/or benzodiazepines according to internationally published guidelines for analgesia and sedation in critically ill children\u00a0[14]. Drug doses were individually tailored to achieve optimal patient comfort and were slowly reduced in order to avoid a\u00a0withdrawal syndrome. In the event that a\u00a0withdrawal syndrome was suspected, based on clinical observation or the revised Finnegan score, specific treatment with long-acting benzodiazepines (e.g. lorazepam) or opioids (e.g. methadone) was started according to internationally published guidelines\u00a0[15, 16].\nAt the time the assessment for delirium was initiated, none of the patients had signs of imminent life-threatening respiratory, circulatory or neurological failure, while ongoing asphyxia, respiratory acidosis, metabolic disturbances, fighting the ventilator due to inappropriate ventilator settings and withdrawal syndrome all had been excluded systematically as an explanation for the observed behavior.\nThe first step of the diagnostic approach was a\u00a0systematic assessment by a\u00a0child neuropsychiatrist (J.S.) using DSM-IV criteria for delirium. Criteria were evaluated on the basis of (1) hetero-anamnestic information from parents, nurses, intensivists, and child neurologists about behavior and behavioral changes and (2) child psychiatric examination. Based on the findings, patients were categorized as having a\u00a0(probable) delirium or not. In a\u00a0second step, the provisional diagnosis of delirium was further tested in a\u00a0daily multidisciplinary consensus meeting. The team consisted of the child neuropsychiatrist, the attending pediatric intensivist, and occasionally a\u00a0geriatric neuropsychiatrist specialized in delirium in geriatric patients and\/or a\u00a0child neurologist. If this team agreed that alternative explanations for a\u00a0child's behavior were unlikely, the consensus diagnosis was delirium.\nBased on the dominant clinical presentation, cases of delirium were classified as \u201chyperactive\u201d when psychomotor agitation was present, and as \u201chypoactive\u201d when retardation and\/or inhibition was present. A\u00a0number of children presented with cognitive and\/or attentional disturbances in the context of severe anxiety states, often accompanied with moaning and restlessness, but without clear agitation or retardation. This latter group was classified as \u201cemerging\u201d or \u201cveiled\u201d delirium\u00a0[17], in reference to the way delirious syndromes have been described as \u201cpartial delirium\u201d in adult ICU patients or as \u201csubsyndromal delirium\u201d in elderly medical patients\u00a0[7, 18, 19]. The different presentation forms were not always clear-cut, and some fluctuated dramatically over time. The severity of illness was scored according to the Pediatric Index of Mortality (PIM) and Pediatric Risk of Mortality (PRISM)\u00a0[20].\nTherapeutic approach\nWhenever delirium was identified or suspected, a\u00a0two-track treatment approach consisting of both psychosocial and pharmacological interventions was implemented. Psychosocial interventions \u2013 the parents' presence and comforting throughout the day (and night), familiar music, favorite toys, pictures of home and pets, friends, school, lighting schedules, sometimes even fragrances \u2013 are standard in the PICU. The parents also received an information leaflet on childhood delirium\u00a0[21]. All patients were also treated with antipsychotic medication after the referring pediatric intensivist had agreed and the parents, because of the off-label use, had given informed consent, which was never refused. In children with psychomotor agitation that was acutely threatening to their health status, haloperidol at a\u00a0loading dosage of 0.15\u20130.25 mg i.v. was used, given slowly over a\u00a0period of 30\u201345 min, followed by a\u00a0maintenance dose of 0.05\u20130.5 mg\/kg\/24\u202fh i.v.\u00a0[11, 22, 23]. In less acute situations, and when oral medication was possible, risperidone at a\u00a0loading dose of 0.1\u20130.2 mg p.o. was used, followed by a\u00a0maintenance dose of 0.2\u20132.0 mg\/24\u202fh p.o. as the treatment of choice. Clinical response and side effects were recorded by the child neuropsychiatrist and the pediatric intensivists. In order to tailor the treatment for delirium, daily discussions were held with the multidisciplinary team. Adjustment of treatment was based on the clinical observations and judgements of the parents, nurses, intensivists and child psychiatrist.\nChildren were followed up for 6 weeks after discharge from the hospital either at the outpatient clinic or by contacting the parents by telephone.\nAs the study solely involved the structured recording of routine clinical practice, under Dutch law no institutional review board approval was required.\nResults\nFrom January 2002 to December 2005, there were 877 acute, non-elective admissions to the PICU. Distribution of age and gender are shown in Table\u202f1. In 61 cases (7%), a\u00a0systematic assessment by a\u00a0child neuropsychiatrist was requested, usually for agitation, anxiety, moaning, discomfort, behavioral disturbance or problematic analgosedation.\nTable\u00a01Number and incidence of delirium in the total sample* by age and genderAgeTotal sample*Patients with deliriumIncidence (%)0\u20132.99 years51314 2.7Male310 9 2.9Female203 5 2.53\u20135.99 years106 4 3.8Male 56 3 5.4Female 50 1 2.06\u20138.99 years 80 6 7.5Male 46 1 2.2Female 34 514.79\u201311.99 years 77 3 3.9Male 61 3 4.9Female 16 0 012\u201314.99 years 70 710Male 35 514.3Female 35 2 5.715\u201318 years 31 619.4Male 13 430.8Female 18 211.1Total87740 4.6Male52125 4.8Female35615 4.2*Critically ill children, acutely, non-electively and consecutively admitted\nduring a 4-year period\nOf these 61 patients, 40 (61%) were diagnosed with delirium, yielding a\u00a0cumulative incidence of 5% (boys 5%; girls 4%). Age-specific incidences increased from 3% in the 0\u20133 years age group (boys 3%; girls 3%), to 19% in the 16\u201318 years age group (boys 31%; girls 11%) (Table\u202f1, Fig.\u202f1).\nFig.\u00a01Incidence of delirium in the sub-groups by age and gender\nThe child psychiatric disorders diagnosed in the 61 referrals are summarized in Table\u202f2.\nTable\u00a02Child psychiatric diagnosis at the first consultation (n\u202f=\u202f61)nDiagnosis40delirium 5adjustment disorders with anxiety and depressed mood, post operative 4psychological\u2013psychiatric factors affecting the medical condition 3anxiety disorder 3emotional and behavioral problems during chronic ventilation 2adjustment disorders with depressed mood 1mood disorder 1adjustment disorder with anxiety 1sleeping problem 1feeding problem\nTable\u202f3 summarizes the population characteristics of the sample diagnosed with delirium, while Table\u202f4 lists the underlying somatic disease or causative pharmacological treatment.\nTable\u00a03Population characteristics of the 40 PICU cases with delirium, 2002\u20132005CharacteristicsFrequency (total n\u202f=\u202f40)Age (mean\u202f\u00b1\u202fSD) 7.6\u202f\u00b1\u202f5.9Malen25 age (mean\u202f\u00b1\u202fSD) 7.6\u202f\u00b1\u202f6.1Femalen15 age (mean\u202f\u00b1\u202fSD) 7.6\u202f\u00b1\u202f5.8Ethnicity White36 (90%) African 3 (7.5%) Asian 1 (2.5%)Mechanical ventilation34 (85%)PIM score (mean\u202f\u00b1\u202fSD)9.96\u202f\u00b1\u202f16.20PRISM score (mean\u202f\u00b1\u202fSD)23.54\u202f\u00b1\u202f24.94Major somatic pharmacological features Recent increase or decrease of analgosedative medication22 (55%) Neurological disorders21 (52%) Infectious disorders20 (50%) Respiratory disorders12 (30%)PIM, Pediatric Index of Mortality; PRISM, Pediatric Risk of MortalityTable\u00a04Patient characteristics of the 40 PICU cases with delirium 2002\u20132005No.SexAgePrimary diagnosis on admission PICUMechanical ventilationDelirium typeTreatment 1M 3 monthsMultiple congenital malformations+EmergingHaloperidol 2F 4 monthsMeningococcal septic shock+EmergingRisperidone 3M 4.5 monthsSevere CLD+EmergingRisperidone 4F10 monthsNear drowning+EmergingHaloperidol 5F 1 yearPneumonia+EmergingHaloperidol 6M 1 yearSepsis due to perforated appendicitis+EmergingHaloperidol 7M 1 yearSubarachnoid bleeding+EmergingHaloperidol 8M 1 yearPericardial effusion with pretamponade+HyperactiveHaloperidol 9M 1 yearMultiple dysmorphia, upper airway obstruction\u2013EmergingHaloperidol10F 2 yearsMeningococcal septic shock+HyperactiveHaloperidol11M 2 yearsADEM+HyperactiveRisperidone12M 2 yearsCervical mass, upper airway obstruction+EmergingHaloperidol13M 2 yearsAspiration and pneumothorax+Hypoactive\u201314F 2 yearsMeningococcal meningitis with sepsis and DIC+HyperactiveHaloperidol15F 3 yearsCystic fibrosis and pneumonia+HypoactiveHaloperidol16M 4 yearsIntracerebral hemorrhage, Marfan syndrome+HypoactiveHaloperidol17M 5 yearsMedulloblastoma post surgery+EmergingRisperidone18M 5 yearsUpper respiratory tract infection+HyperactiveHaloperidol19M 6 yearsMultiple trauma+EmergingRisperidone20F 8 yearsMeningo-encephalitis+HyperactiveRisperidone21F 8 yearsViral encephalitis+Hyperactive\u201322F 9 yearsStatus asthmaticus+HyperactiveRisperidone23F 9 yearsTBI, gunshot wound+HypoactiveRisperidone24M 9 yearsStatus asthmaticus+HyperactiveHaloperidol25M 9 yearsNeural tube defect and drain dysfunction\u2013Emergingfirst Haloperidol, then Risperidone26M11 yearsHypovolemic shock, typhus abdominalis\u2013HyperactiveHaloperidol27F12 yearsTBI+HypoactiveHaloperidol28M12 yearsTBI and fracture of lower leg+EmergingHaloperidol29M13 yearsSepsis, paronychia\u2013HyperactiveHaloperidol30M13 yearsStatus epilepticus+EmergingHaloperidol31F14 yearsTBI+HyperactiveHaloperidol32F15 yearsPost TBI+HypoactiveHaloperidol33M15 yearsPostoperative state+HypoactiveRisperidone34M15 yearsAcute lymphoblastic leukemia\u2013HypoactiveHaloperidol35M15 yearsTBI+EmergingHaloperidol36F15 yearsStatus asthmaticus+HyperactiveHaloperidol37M16 yearsMultiple trauma\u2013HypoactiveHaloperidol38F16 yearsBacterial meningitis+HyperactiveRisperidone39M16 yearsRespiratory failure, Duchenne disease+EmergingHaloperidol40M17 yearsSeptic shock+EmergingHaloperidolCLD, chronic lung disease; ADEM, acute disseminated encephalomyelitis; DIC, diffuse intravascular coagulation; TBI, traumatic brain injury\nThe underlying features were: recent increase or decrease in analgosedative medication (n\u202f=\u202f22), neurological disorders (n\u202f=\u202f21), infections (n\u202f=\u202f20) and respiratory disorders (n\u202f=\u202f12). Usually, a\u00a0combination of these existed.\nAll but two patients were treated with an antipsychotic drug. Twenty-seven children were given haloperidol, 10 risperidone, and 1 child received both drugs in succession. In most cases, the beneficial results were observed rapidly, especially in the hyperactive forms, sometimes even after a\u00a0single dose\u00a0[11]. Sometimes it took a\u00a0few hours or days. Two patients experienced acute dystonia as a\u00a0likely side effect of the haloperidol, responding well to biperidene. Two children received no medication: one because of lack of consensus in our expert team, and one because of an endotracheal intubation at the time that medication was indicated. In most cases, the medication was stopped or tapered off successfully during hospitalization or afterwards in an outpatient setting. Five children (12.5%) with delirium died of their underlying disease; the mean PIM was 10% and the mean PRISM 24% (Table\u202f3).\nDiscussion\nThis is the first systematic multidisciplinary study of the phenomenology and treatment of delirium in 40 critically ill children in a\u00a0PICU context. The low cumulative incidence of 5% is mainly the result of the low incidence in the younger age groups (<\u202f9 years old), this segment constituting the majority of the total sample (80%). A\u00a0clearly higher incidence is seen in the older age groups. However, in critically ill adult patients substantially higher incidences have been reported, ranging from 10\u201330% in general hospital settings to 50% in postoperative patients and up to 80% in the terminally ill\u00a0[2].\nThere are several possible explanations for this difference. First, the incidence of delirium in young critically ill children may be truly low: differences in age-related resilience and underlying conditions may contribute to true differences in the incidence between the very young and the very old. However this explanation seems unlikely, given the tendency in the very young to develop delirium under even much less severe circumstances\u00a0[1, 11, 24]. A\u00a0second explanation may relate to the fact that extensive psychosocial interventions are provided as a\u00a0routine in Dutch PICU settings, with a\u00a0possibly preventive effect on delirium in much the same way as observed in geriatric patients\u00a0[25]. A\u00a0third factor may be an anti-delirium effect of the routinely used analgosedative medication, although especially benzodiazepines may have excitatory side effects in children. A\u00a0fourth factor may relate to reluctance on the part of the intensivists and\/or child neurologists to request psychiatric evaluation in the case of behavioral changes for fear of adding stigmatization to an already burdened system. Perhaps the fifth, most likely and important explanation is that parents, nurses, pediatric intensivists and child neurologists do not easily recognize delirium, because the medical condition of these critically ill PICU children is so complex and changeable. It is possible that a\u00a0psychiatric consultation was readily requested only in cases of anxiety and\/or hyperactive delirium, not in the hypoactive and or veiled ones.\nThe differential diagnosis of pediatric delirium consists of acute stress reactions, acute anxiety states, adjustment disorders with mixed emotions, dissociative and\/or regressive states and childhood-onset psychosis (see also Table\u202f2). However, differentiating delirium from extreme stress and agitation due to acute and life-threatening conditions is not only impossible, it is also unwanted, because it is usually irrelevant at that point of time in the process of medical care. Causative treatment, if possible, is always the first step to be taken. Furthermore, the diagnosis of delirium in children is complicated by the fact that the criteria for adult delirium are not always easily applicable to children because of important differences in age and developmental levels. The DSM-IV criteria for delirium are not always useful in the case of pediatric delirium, especially in a\u00a0PICU context. This is a\u00a0source of concern. Moreover, delirium is not mentioned in the DSM-IV section on child psychiatry. DSM-IV describes as an essential feature of delirium the \u201cdisturbance of consciousness\u201d leading to \u201cimpairment of the ability to focus, shift and sustain attention\u201d. This, however, is of little relevance in the critically ill in a\u00a0PICU context, where disturbance of attention is routinely present due to the disorder(s) itself. In fact, attention is often the first \u201cto go\u201d\u00a0[17, 26]. In addition, patients almost always require treatment with opioids and benzodiazepines, which also have a\u00a0strong impact on attention. Furthermore, it has been hypothesized that a\u00a0disturbance of consciousness is not a\u00a0discriminating characteristic of delirium in an ICU setting\u00a0[27].\nOur case series suggests that in addition to the hyper- and hypoactive forms of delirium, a\u00a0third form may be characterized by anxiety, moaning, and\/or restlessness. This was referred to as an \u201cemerging\u201d or veiled delirium, as described previously\u00a0[17]. In the PICU population this state did not develop into a\u00a0frank hyper- or hypoactive form, but appeared to exist in its own right, accompanied by disturbances of consciousness and cognition.\nAlthough atypical presentations of disorders are often not captured in classification systems, the high prevalence of \u201cemerging\u201d delirium in our sample (17\/40) stresses the importance of further phenomenological study. Adhering too strongly to DSM-IV criteria for adult delirium, for clinical use in a\u00a0PICU context, may result in persistent under-diagnosis. On the other hand, the incidence of delirium may be overestimated by using the CAM-ICU, so most important is the issue of what constitutes delirium in critical illness\u00a0[28].\nThere are accumulating indications that pediatric delirium can be subtle and accompanied or even dominated by other neuropsychiatric signs such as: reduction of awareness of the caregiver and\/or the surrounding environment, purposeless actions, restlessness, inconsolability, signs of autonomic dysregulation and other subtle higher cortical dysfunctions\u00a0[29\u201332]. Parents and nurses have a\u00a0way of discriminating patterns in their children that may be diagnostically important. Thus, parents sometimes state: \u201cThis is not my child anymore.\u201d Pediatric delirium therefore may have various subtle presentations and can be considered a\u00a0spectrum disorder, which makes it sometimes difficult to diagnose\u00a0[33]. Neither haloperidol nor risperidone is registered for the treatment of childhood delirium, although both are mentioned as the treatment of choice for adults\u00a0[1]. Moreover, haloperidol is not registered for i.v. administration, even though it is used in this way in many places. We prefer risperidone in non-acute situations because of the theoretically lower risk of extrapyramidal side effects. Haloperidol and risperidone have been used for other indications in young children as well, such as childhood psychosis\u00a0[34] or aggression in autism\u00a0[35]. There are two limitations regarding our observations of treatment response. First, no severity scale for pediatric delirium was used, because none exists for this PICU population. Second, because no studies on the natural course of childhood delirium exist that have established the rate of spontaneous remission, spontaneous remissions may have been misclassified as response to treatment. In our opinion, however, the time frame of response points towards a\u00a0medication effect.\nMost medications employed in pediatrics and child psychiatry are used off-label\u00a0[36], which means that special attention should be paid to information and informed consent procedures. Given the relatively high incidence of extrapyramidal symptoms with haloperidol, the \u201coff-label\u201d use needs further study\u00a0[37].\nThis study has several limitations. First, it was a\u00a0study of routine clinical practice. Observations were based on referrals emerging from care as usual. Although the focus on delirium may have altered referral paths and rates, we did not actively advocate any change, nor did we screen all admissions for delirium. In the absence of a\u00a0hard clinical indication, no routine blood level measurements were performed to rule out persistent high levels of sedatives as a\u00a0possible explanation for any neuropsychiatric symptom. Next, the lack of DSM-IV criteria for pediatric delirium and the disputed relevance of its main criterion in a\u00a0(P)ICU setting make a\u00a0standardized diagnosis difficult. Finally, treatment was provided in an open setting and based on consensus, especially among child psychiatrist, pediatric intensivist and child neurologist.\nIn conclusion, critically ill children in a\u00a0PICU can develop delirium, with a\u00a0hyperactive, hypoactive or veiled presentation, despite adequate analgosedation and intensive psychosocial interventions. The approach using an algorithmic structuring and an intensifying of daily clinical care, including the use of multidisciplinary daily consensus meetings, appeared effective in assessing, diagnosing and treating childhood delirium at the PICU. The findings suggest that the incidence is much lower than in adults, but a\u00a0likely explanation is that delirious states requiring child psychiatric referral are still frequently under-diagnosed. There is also still a\u00a0great need for developing delirium criteria in critically ill patients, children and adults alike, in a\u00a0(P)ICU setting. Treatment with haloperidol or risperidone was successful in all patients. Future research is necessary to identify the risk factors for pediatric delirium in a\u00a0multivariate prospective approach, to develop \u201ceasy to use\u201d bedside tools for non-psychiatric trained team members for the early detection of delirium in all pediatric PICU patients, and to study the effects of interventions in a\u00a0double-blind and ideally placebo-controlled fashion.\nDisclosure\nJim van Os is a\u00a0speaker or member of the advisory board for Lilly, BMS, Janssen-Cilag and AstraZeneca. He received grant or research support from Lilly, GSK, BMS and AstraZeneca. Albert Leentjens participated in research for Boehringer Ingelheim and is on the advisory board of a\u00a0study related to Parkinson's disease by the same company. The other authors have no financial relationships to disclose.","keyphrases":["delirium","critically ill children","pediatric intensive care unit (picu)","haloperidol","consensus meetings","risperidone"],"prmu":["P","P","P","P","P","P"]}
{"id":"Diabetologia-3-1-1849422","title":"Impact of the population at risk of diabetes on projections of diabetes burden in the United States: an epidemic on the way\n","text":"Aims\/hypothesis The aim of this study was to make projections of the future diabetes burden for the adult US population based in part on the prevalence of individuals at high risk of developing diabetes.\nIntroduction\nConsiderable evidence has been presented on the rise in diabetes prevalence in the United States and the United Kingdom [1, 2]. The prevalence of diabetes has become so large that it has been termed an epidemic [1, 3]. This rise is particularly important for healthcare needs in the US because almost 30% of individuals with diabetes are currently undiagnosed and diabetes is disproportionately represented in minority populations [4].\nProjection of future disease prevalence helps to plan for healthcare needs. An understanding of the population at risk of developing the disease is critical when projecting future disease burden. Several studies have projected future diagnosed diabetes prevalence for the US and other countries [2, 5\u20139]. These studies, however, did not consider that not all individuals are equally at risk of developing diabetes, thereby possibly distorting estimates of downstream prevalence. For example, some risk factors for diabetes, e.g. obesity, have increased substantially in the population [10\u201312]. Moreover, many of these projections are based on estimates of diagnosed diabetes and exclude estimates of total diabetes (diagnosed and undiagnosed), which may lead to serious underestimation of the diabetes burden in the population.\nMajor risk factors for diabetes have been identified and are currently used by the American Diabetes Association to guide screening strategies. Although there are various measures for assessing the risk of having undiagnosed diabetes [13\u201317], few measures are available for assessing the risk of developing diabetes [18, 19]. Moreover, accounting for changes in the proportion of high-risk individuals, particularly as assessed through clinical indicators, has not been incorporated into previous projections of future diabetes burden.\nThe purpose of this study was to project the prevalence of diabetes for the adult US population up to 2031, using models based on data contained in the nationally representative National Health and Nutrition Examination Survey (NHANES) II mortality survey (1976\u20131992), NHANES III (1988\u20131994) and NHANES 1999\u20132002.\nMaterials and methods\nDiabetes prevalence model\nThe model for diabetes prevalence used in this study was created using data from the NHANES III (1988\u20131994), and then fitted to data from the NHANES 1999\u20132002 as a validity check of the accuracy of the model\u2019s projections. The resulting model was then used to project the number of individuals with diabetes in the US in 10-year increments into the future. We evaluated 10-year age classes at each 10-year interval. Our model has the following components: \nNumber of individuals with diabetesTime 2\u2009=\u2009\u2211 (number of individuals with diabetesTime 1i\u2009+\u2009incident casesi\u2009\u2212\u2009mortalityi), where i equals each 10-year age group, and incident cases consist of: (1) persons converting from a disease-free state to having diabetes; (2) diabetic patients immigrating to the United States; and (3) persons with diabetes moving into the 20 to 29-year-old age class.The percentage of persons with diabetes, which is calculated thus:percentage diabetesTime 2\u2009=\u2009(number of individuals with diabetesTime 2\u2009\/\u2009total populationTime 2)\u2009\u00d7\u2009100.The estimate of future diabetes is therefore based on this equation, including the number of individuals with diabetes in the previous time period, conversion to diabetes, migration, and mortality, rather than being a linear extrapolation of the change in diabetes prevalence from the known values of 1991 and 2001.\nData sets\nThe NHANES is a programme of surveys conducted by the National Center for Health Statistics and designed to assess the health and nutritional status of adults and children in the United States. The survey is unique on a national level in that it combines interviews and physical examinations. The NHANES uses a complex multistage sampling design, making it representative of the non-institutionalised US population and allowing weighted estimates to be computed.\nFor this study we used several of the NHANES data sets. Specifically, we used the NHANES III (1988\u20131994) (unweighted n\u2009=\u20094,950) and the NHANES 1999\u20132002 (unweighted n\u2009=\u20093,804) to estimate among individuals of 20\u00a0years of age and older the prevalence of diagnosed diabetes, the total diabetes burden (diagnosed and undiagnosed diabetes), and the proportion of the population at risk of developing diabetes. Since mortality within the population affects future prevalence [20], we also used the cohort from the NHANES II mortality survey (1976\u20131992) (unweighted n\u2009=\u20093,916) to provide estimates of diabetes mortality. Computation of all analyses using the NHANES data sets to provide nationally representative estimates for the models was designed to account for the complex survey design and the appropriate sample weights. All analyses were conducted using SUDAAN software (Research Triangle Institute, Research Triangle Park, NC, USA).\nVariables used in models\nPrevalence of diabetes\nDiabetes burden was assessed as diagnosed diabetes plus undiagnosed diabetes. Because of the substantial proportion of people with undetected diabetes, we focused on this formula for total diabetes, rather than using diagnosed diabetes to indicate diabetes burden in the population. Moreover, by focusing on diabetes as diagnosed and undiagnosed disease, we minimised the possible impact on future diabetes prevalence of changes in screening practices for diagnosing diabetes during an ensuing time period.\nDiagnosed diabetes was assessed as individuals who answered yes to a question of whether a doctor had told them they had diabetes. Undiagnosed diabetes was estimated on the basis of individuals who said they had not had a previous diagnosis of diabetes, but who had fasting plasma glucose (FPG)\u2009 >\u20097.0\u00a0mmol\/l. Although, the diagnostic criteria for diabetes during the time between the NHANES II and the NHANES III changed from FPG\u2009 >\u20097.8\u00a0mmol\/l to FPG\u2009 >\u20097.0\u00a0mmol\/l, we used the newer criteria to gain an awareness of the total diabetes burden at each point in time using the same criteria [21].\nPersons converting from a disease-free state to having diabetes\nAlthough a variety of diabetes risk scores exist, most have been created from cross-sectional studies and have as their aim the identification of individuals with undiagnosed diabetes. Their ability, therefore, to make predictions on development of diabetes is unknown [13, 14, 16]. The risk score used in this study is based on one developed for the Atherosclerosis Risk in Communities (ARIC) cohort study [18]. Among individuals without diagnosed diabetes or FPG\u2009 >\u20097.0\u00a0mmol\/l, we used a scoring strategy which includes: high waist circumference (>102\u00a0cm in men, >88\u00a0cm for women), raised blood pressure (>130\/85\u00a0mmHg or antihypertensive medications), low HDL-cholesterol (<1.03\u00a0mmol\/l for men, <1.29\u00a0mmol\/l for women), high triacylglycerol (>1.7\u00a0mmol\/l), BMI\u2009>\u200930\u00a0kg\/m2, and hyperglycaemia. Each of the characteristics is worth 1 point except for hyperglycaemia, which can be worth 2 points if FPG is >\u20095.6\u00a0mmol\/l or 5 points when FPG is >6.1\u00a0mmol\/l. A score of >4 puts an individual at high risk of developing diabetes, whether diagnosed or undiagnosed. A score of <4 indicates that a person has a low risk of developing diabetes.\nThis particular risk score was chosen for several reasons. First, it has moderate sensitivity (68%) and specificity (75%). Second, it is computed in a reasonably straightforward manner without having to use coefficients from the ARIC cohort that may be specific to that cohort. Third, data and results provided in the study by Schmidt et al. [18] allowed for computation of the rate of development of diabetes in both the high-risk group and the low-risk group. The ratio of development of diabetes in the high-risk group versus the low-risk group was 4.5:1. Variables needed to compute this diabetes risk score are available only in the NHANES III and the NHANES 1999\u20132002.\nAlthough the ARIC diabetes risk score did not specifically consider race or age in the computation [18], we computed conversion rates for 10-year age classes for three race\/ethnic groups (non-Hispanic Whites, non-Hispanic Blacks and Hispanic individuals) by fitting age categories for the data from 1991 to 2001 and then fitting race\/ethnicity on to the same time change. We did not compute specific sex-specific conversion rates because sex was already differentiated in several of the variables in the ARIC diabetes risk score [18].\nMigration of persons with diabetes\nMigration of individuals with or without diabetes into the population can also affect future diabetes prevalence. Recent projections have included migration within their models [5]. Because we are looking at changes in diabetes prevalence among adults, migration of adults, particularly from ethnic minorities, could substantially affect the 10-year projections. We used data from the NHANES III to estimate migration of persons with diabetes in the 20\u00a0years and older age groups. The NHANES III measured how many years foreign-born immigrants had been in the US. Thus, we estimated the number of foreign-born individuals who had been in the country for 9\u00a0years or less for the total population as well as for different racial\/ethnic groups. The NHANES III data allowed us to make estimates for non-Hispanic Whites, non-Hispanic Blacks and Hispanic individuals.\nPersons with diabetes moving into the 20 to 29-year-old age class\nFor 2011, 2021 and 2031 the total number of persons with diabetes in the 20 to 29-year-old age class was estimated using a linear projection of the NHANES III and NHANES 1999\u20132002 data. The proportion of 20 to 29-year-olds with diabetes in each race\/ethnic group was held constant at the proportions found in the NHANES 1999\u20132002 data at the later time intervals.\nMortality among individuals with diabetes\nDiabetes mortality for the total population was based on data from the NHANES II mortality survey (1976\u20131992). This population-based cohort study was used to provide estimates of diabetes mortality, since mortality within the population affects future prevalence [20]. Diabetes mortality was estimated as all-cause mortality among individuals with diabetes (either diagnosed or undiagnosed) at baseline, rather than as mortality with diabetes listed as the cause of death. This definition is more consistent with the potential impact of diabetes on future prevalence. Mortality estimates were computed separately for the total population by age classes.\nThe NHANES II mortality cohort is based on a sample of individuals aged 30 to 75, whereas we made diabetes estimates on individuals aged 20\u00a0years and older. Consequently, we assumed no deaths due to diabetes in the 20 to 29-year-old age group over the 10-year period.\nPopulation estimates\nTotal population of 10-year age classes was estimated using data from NHANES III for 1991, NHANES 1999\u20132002 for 2001, and US Census Bureau, Middle Series projections for 2011, 2021 and 2031 [22]. Total population of race\/ethnic groups was also determined by 10-year age classes using the same sources of information.\nAnalysis\nIn an effort to provide an estimate of future trends in diabetes and the population at high risk of developing diabetes, we employed the following procedure. We used the NHANES III data to fit a model to predict total diabetes in the NHANES 1999\u20132002. We used this strategy prior to making future projections, because it allowed us to develop and fit the model to an existing national estimate of diabetes prevalence. Because both the NHANES III and the NHANES 1999\u20132002 are based on multi-year data collection, we estimated a mid-point of 1991 and 2001 for the two surveys.\nThe number of persons with diabetes 10\u00a0years post-baseline was calculated for 10-year age classes by first adding baseline prevalence and incidence (the number of low-risk and number of high-risk persons who developed diabetes over the 10-year interval), then adding persons with diabetes who immigrated to the United States, and persons with diabetes who moved into the 20 to 29-year-old age class, and finally subtracting the number of diabetic subjects who died. Percentage of persons with diabetes was estimated for each time period by taking the total number of persons with diabetes and dividing by the expected total population, then multiplying by 100.\nVarying model assumptions\nOur initial predictions of future diabetes burden were based on the assumption of a constant proportion of individuals at high risk of diabetes at the levels present in the NHANES 1999\u20132002. To account for potential changes in the proportion of persons at high risk of diabetes, we also evaluated increases in the proportion of persons at high risk by 10, 20 and 30%, as well as estimates based on decreases in the proportion of persons at high risk by 10, 20 and 30%. Theoretically, it is unlikely that the proportion of persons at high risk will remain stable, because from NHANES III to NHANES 1999\u20132002 the proportion at high risk was seen to increase. Also, a major risk factor for diabetes, obesity, has increased substantially over a 40-year time period [10, 12]. We evaluated the effect of decreasing proportions at high risk, to account for the possibility that interventions to improve lifestyle of adults in the US may be effective.\nIn addition, to address the potential impact on mortality of healthcare interventions in management of diabetes, we examined potential reductions of 10, 20 and 30% in mortality among individuals with diabetes. Finally, we computed a model examining a combination of effects, assuming that lifestyle interventions would yield a 10% decrease of persons at high risk and healthcare interventions would yield a 10% decrease in mortality of persons with diabetes.\nResults\nTable\u00a01 shows estimates of the total diabetes burden from the NHANES III and the NHANES 1999\u20132002 and the future 10-year projections for 2011 through to 2031. The number of individuals at high risk of diabetes based on the multivariable diabetes risk score was 38.4 million in 1991 and 49.9 million in 2001. Using our model to predict the known diabetes prevalence in 2001 from the 1991 data, results were satisfactory and within 0.2% of the actual population prevalence of total diabetes. If the proportion of individuals at high risk within the adult population remains stable at 2001 levels, we could expect 55.8 million in 2011, 60.9 million in 2021, and 66.1 million in 2031. As can be seen, the prevalence of diabetes is projected to increase. The diabetes prevalence of 6.3% in 1991 and 8.8% in 2001 is projected to increase to 14.5% in 2031 with 37.7 million adults having diagnosed or undiagnosed diabetes. Assuming stability in the population proportion of individuals at high risk of developing diabetes, the rate of increase in the number of individuals with diabetes and the proportion with diabetes tends to slow over time. Among individuals aged 30 to 39\u00a0years who are not currently targeted for screening according to age, the prevalence of diabetes is expected to rise from 3.7% in 2001 to 5.2% in 2031. \nTable\u00a01Number of people (in millions of persons) with and prevalence of diabetes by year and age categoryAge (in years)NHANES IIINHANES 1999\u2013200220112021203120\u201329Number0.20.40.50.70.9Percentage0.51.01.31.72.030\u201339Number0.61.61.82.12.3Percentage1.43.74.75.05.240\u201349Number1.53.54.34.34.9Percentage4.58.110.311.111.250\u201359Number2.53.96.67.26.9Percentage11.412.215.617.718.260\u201369Number3.14.16.59.810.2Percentage15.319.822.025.126.6>70Number3.24.15.78.412.4Percentage16.217.420.522.623.9TotalNumber11.117.525.432.637.7Percentage6.38.811.513.514.5NHANES National Health and Nutrition Examination Survey\nThe results shown in Electronic supplementary material (ESM) Table 1 show the projected prevalence of diabetes according to different racial\/ethnic groups. Non-Hispanic White adults are projected to continue to have a lower prevalence of diabetes than both non-Hispanic Black and Hispanic individuals. By 2031, the Hispanic community will have an overwhelming diabetes burden, with more than 20 percent of the adult population having diabetes.\nThe projections in ESM Table 2 are based on different assumptions regarding changes in the number of individuals who are at high risk of developing diabetes and changes in mortality among individuals with diabetes. As might be expected, as mortality decreases the prevalence of diabetes increases in the subsequent 10\u00a0years. The estimate for 2031 indicates that potential decreases in mortality and a potential decrease in individuals at high risk of developing diabetes yields a prevalence similar to that achieved if the proportion at high risk is kept stable from 2001. All of these estimates indicate a larger diabetes burden among Hispanics.\nDiscussion\nThis national projection of diabetes prevalence for the US is the first to model the projection on the number of individuals at high risk of developing diabetes using a multivariable risk assessment. Projections suggest a rising and substantial diabetes burden for the population. Hispanic adults will be most affected, with estimates suggesting that by 2031 more than 20% of the adult Hispanic community will have diabetes. These results are particularly worrisome for this community in light of recent evidence that the gap in healthcare quality between Hispanic and non-Hispanic White individuals has continued to widen [23].\nMany previous diabetes projections have been limited to estimates of diagnosed diabetes and thus have lower estimates of projected diabetes burden, and have not incorporated an evaluation of the population at high risk of diabetes, with clinical indicators, into their models [5, 9]. Our estimates will be less likely to be affected by changes in screening strategies. Additionally, they incorporate potential changes in the level of risk for diabetes in the US population, a change which is likely given national trends in obesity [3]. Moreover, recent data have suggested that individuals with undiagnosed diabetes are similar to those with diagnosed diabetes with regard to the development of complications; thus our estimates are more robust in describing the burden of disease in the population [24].\nComparing our projections with those from other studies, we note that an estimate, published in 2006, for diagnosed diabetes in the US among individuals aged 20 to 64\u00a0years in 2030 is 16.8 million [25]. Our estimates are based both on diagnosed and undiagnosed diabetes, and our projection of total diabetes among that age group for 2031 is higher, namely 19 million. It is possible that estimates based solely on diagnosed diabetes could become more consistent with our estimates, if greater vigilance were shown for screening for undiagnosed diabetes. However, not accounting for the at-risk population in the estimates is likely to lead to inaccurate estimates. A comparison of our estimates of total diabetes with those of another study [7], which projected total diabetes but did not account for the population at high risk of developing diabetes, reveals that the latter\u2019s projections are most probably underestimates. Using data from 1993, the investigators projected a population prevalence estimate of total diabetes in the US among individuals aged 20\u00a0years and older for the year 2000 to be 7.6%, while the NHANES 1999\u20132002 yielded a prevalence of 8.8%. For 2025 the same team [7] projected a prevalence of 8.9% versus 13.5% for 2021 in our study.\nThe results have several implications for the delivery of healthcare and healthcare financing.\nFirst, we estimated our models under several assumptions for the number of individuals at high risk of diabetes in the population. Regardless of these assumptions, the US will have a substantial number of individuals at high risk of diabetes in 2011, 2021 and 2031. Interventions to modify lifestyle are critical to decrease the number of individuals at high risk, and consequently to lower the expected increase in diabetes in the future. Although some of the diabetes estimates suggest seemingly small decreases in future prevalence, based on decreases in the population at risk, the actual numbers are substantial. For example, a one-percentage point drop in the US population estimate of diabetes among individuals aged 20 and older in 2031 is quite substantial and would account for a decrease in prevalence of diabetes equivalent to 2,600,000 people.\nSecond, the projection that a substantial proportion of the population will have diabetes indicates greater spending will be necessary to manage the disease. This will include spending on drugs, ongoing monitoring, and treating of complications including nephropathy, retinopathy, and cardiovascular disease.\nThird, the disproportionate impact of diabetes on minorities, particularly Hispanics, demands new intervention strategies to decrease the number of individuals at high risk and to deliver care to individuals who have historically had poor access to care. Additionally, with the projected increase in diabetes prevalence among 30 to 39-year-olds, a population not currently targeted for screening, a re-examination of current public health policy and screening strategies may be warranted [26].\nThere are several strengths to the design of this study. One is that the study utilised multiple NHANES data sets, which have the advantage of allowing for nationally representative population estimates. Thus, the initial data used to fit the model as well as to make mortality estimates of diabetes, both diagnosed and undiagnosed, are nationally representative. Another strength is that this study is the first to make a nationally representative assessment of the at-risk population for development of diabetes and then use that assessment to model the future prevalence of diabetes. The assessment of risk used, moreover, is based on the ARIC diabetes risk score [18], a multivariable risk score that used clinical indicators.\nWhen interpreting our results, however, several limitations need to be considered. Thus, although this is the first study to use a validated diabetes risk score to assess the high-risk population for the development of diabetes for the entire US population, potential limitations exist with regard to the diabetes risk score. The ARIC diabetes risk score [18] was based on a cohort of individuals aged 45 to 64\u00a0years at baseline and may therefore be limited when estimating diabetes development among individuals aged 20\u00a0years and older. However, we estimated diabetes prevalence in 10-year age increments. Moreover, the risk score\u2019s moderate sensitivity and specificity may cause the model to under- or potentially overestimate future prevalence projections. Another possible limitation is that estimates of future disease burden are based on assumptions about the number at risk of disease and about mortality within the population. We have attempted to address this limitation by presenting the results of a sensitivity analysis, which includes variations in the proportion of the population at risk and in mortality. The third limitation is the diagnosis of diabetes in the NHANES data on the basis of a single FPG value. This strategy, although common in epidemiological studies, could potentially underestimate the prevalence of diabetes associated with isolated post-challenge hyperglycaemia, which occurs more commonly in women, the elderly, and in lean populations. It could also overestimate diabetes prevalence, because a clinical diagnosis of diabetes in asymptomatic patients requires two abnormal fasting glucose levels.\nIn summary, a continued focus on effective interventions for lifestyle modifications to decrease diabetes risk, as well as vigilant ascertainment of diabetes, appears crucial if the future prevalence and burden of diabetes in the US population are to be adequately addressed. This is especially important for minority populations, particularly the Hispanic community, which is projected to have an overwhelming future diabetes burden. Considering that minorities have historically had limited access to healthcare, these findings emphasise the importance of interventions targeting these populations.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nTable 1\nProjections of non-Hispanic White, non-Hispanic Black and Hispanic persons with diabetesa and prevalence in 2011, 2021, and 2031 (43\u00a0kb)\nTable 2\nProjections, using different assumptions, of diabetes prevalence among individuals aged >20\u00a0years in 2031 (32\u00a0kb)","keyphrases":["diabetes","projection","epidemiology"],"prmu":["P","P","P"]}
{"id":"Skeletal_Radiol-4-1-2257990","title":"Distinguishing benign notochordal cell tumors from vertebral chordoma\n","text":"Objective The objective was to characterize imaging findings of benign notochordal cell tumors (BNCTs).\nIntroduction\nThe giant notochordal rest and notochordal hamartoma are terms that have been used to describe a benign notochordal cell tumor (BNCT) that may be mistaken for a chordoma [1, 2]. This recently discovered intraosseous benign lesion of notochordal cell origin may be a potential precursor of chordoma [3\u20136], but based on the few cases with long-term follow-up (those previously published and our present series) none has clinically grown to become a chordoma. The anatomical predilection is identical to that of chordoma. The most common locations are the sacro-coccygeal region and the base of the skull, followed by cervical and lumbar vertebrae. Most tumors are usually small and asymptomatic and are often found at autopsy. The occasional lesion may become large and can be detected on advanced imaging. Our working group collected 7 cases of BNCT that were detected on imaging. We report on their management, outcomes, and histology. Five patients underwent surgical management based on a misdiagnosis of chordoma and failure to recognize the entity of BNCT. This paper focuses on the clinical, radiological, and histological features of these cases, which get overtreated when misdiagnosed.\nMaterials and methods\nThe data came from 7 patients with BNCTs that were found clinically. Clinical records included patient sex, age, presenting symptoms, and anatomic site involved. All formalin-fixed specimens were decalcified with formic acid solution and then routinely processed for hematoxylin-eosin and periodic acid Schiff (PAS) staining. For immunohistochemical study, paraffin-embedded specimens were cut into 3- to 5-\u03bcm-thick sections and then mounted on glass slides coated with 3-aminopropyltriethoxysilane. The sections were stained by the streptavidin-biotin-peroxidase method (Nichirei, Tokyo, Japan). The antibodies used are listed in Table\u00a01. Microwave antigen retrieval was carried out prior to immunostaining with vimentin, AE1\/AE3, CAM 5.2, and cytokeratin (CK) 18. To determine the specificity of immunostaining, we included known positive and negative tissues as controls.\nTable\u00a01Antibodies used in the studyAntibodiesSourceClonalityDilutionEpithelial membrane antigenDAKO Cytomation, Glostrup, DenmarkM1:100Cytokeratin (AE1\/AE3)DAKO Cytomation, Glostrup, DenmarkM1:100Cytokeratin (CAM5.2)Becton Dickinson, San Jose, CA, USAM1:1Cytokeratin 18DAKO Cytomation, Glostrup, DenmarkM1:20VimentinDAKO Cytomation, Glostrup, DenmarkM1:100S-100 proteinNichirei, Tokyo, JapanP1:200M, monoclonal; P, polyclonal\nResults\nTable\u00a02 summarizes ages, symptoms, treatment, and outcomes of the 7 patients with BNCTs that were found clinically (Figs.\u00a01, 2). Two lesions were documented as possible vertebral osteonecrosis without collapse; however, recent histological re-evaluation indicated BNCTs [7, 8]. The other cases were originally diagnosed as chordoma histologically. The patient group consisted of 3 men and 4 women ranging in age from 22 to 55\u00a0years (mean, 44\u00a0years). One patient was found to have two lesions at different sites, evident in the fifth lumbar vertebra and the second sacral vertebra (Fig.\u00a03). The latter was not examined microscopically. A small lesion was found incidentally in another patient in the far distal coccyx and was resected. Of the 9 lesions, 4 were in the cervical vertebra; 2 each in the lumbar vertebra and sacrum; and 1 in the coccyx. The most common symptom was pain in 5 patients; however, 1 patient complained of pain radiating to the fingers and numbness that possibly was caused by known ossification of the posterior longitudinal ligament (OPLL) at the sixth to seventh cervical vertebrae. Another patient was found to have cervical OPLL on radiographs. The pain was usually indolent except for that caused by OPLL. Lesions were found incidentally in 2 patients on MR images when they were examined for unrelated symptoms due to traffic accidents. A needle or open biopsy was done for 5 lesions. Four lesions were diagnosed as chordoma and 1 as osteonecrosis. The remaining 2 lesions were clinically diagnosed as osteonecrosis and an intraosseous benign condition respectively. The patients whose lesions were diagnosed as osteonecrosis and chordoma respectively underwent curettage. Three patients underwent vertebrectomy after the diagnosis of chordoma. One patient underwent wide resection following a preceding curettage. One patient who did not undergo a surgical procedure was followed because the diagnosis of osteonecrosis was made at biopsy. Another patient did not undergo any surgical management in spite of the histological diagnosis of chordoma at biopsy and was followed because the clinician doubted the histological diagnosis of chordoma based on unusual radiological findings for chordoma. The prognosis of all patients but 1 was excellent. Four patients who underwent surgical management were well without recurrent or metastatic disease for follow-up periods between 17 and 84\u00a0months (mean 54.5\u00a0months). Two patients who did not undergo surgery were alive without progressive disease, as judged by imaging, for 13\u00a0months and 44\u00a0months after biopsy respectively (Fig.\u00a04). One patient who underwent vertebrectomy died of hemostasis after surgery because she would not accept a blood transfusion on religious grounds.\nTable\u00a02Summary of benign notochordal cell tumors found clinicallyPatientAge (years)GenderLocationSymptomsTreatmentOriginal diagnosisFollow-up information130MaleL4Low back painVertebrectomy following biopsy and curettageChordomaAlive with no evidence of disease, 60\u00a0months253FemaleC6Numbness and pain of the fingers bilaterally, probably due to OPLL at C6, 7Vertebrectomy following biopsyChordomaAlive with no evidence of disease, 57\u00a0months355FemaleC5Neck pain, shoulder stiffnessBiopsyChordomaAlive with no progressive disease, 13\u00a0months450MaleC3Car accident injury (incidentally found on MRI)CurettageOsteonecrosisAlive with no evidence of disease, 84\u00a0months552MaleC5Mild upper back pain OPLL, focalNeedle biopsyOsteonecrosisAlive with no progressive disease, 44\u00a0months622FemaleS4, Cox (microscopic)Buttock painWide resection following curettageChordomaAlive with no evidence of disease, 17\u00a0months743FemaleL5, S2Car accident injury (incidentally found on MRI)Vertebrectomy following biopsy at L5ChordomaDied of postoperative complicationsL, lumbar vertebra; C, cervical vertebra; S, sacrum; Cox, coccyx; OPLL, ossification of posterior longitudinal ligamentFig.\u00a01A 30-year-old man who complained of low back pain (patient 1). a Lateral radiograph of the lumbar spine reveals vague sclerosis in the fourth lumbar vertebra. b Axial computed tomography (CT) scan of the fourth lumbar vertebra demonstrates significant sclerosis in the center of the body, partly extending to the cortex. c Sagittal T1-weighted spin echo magnetic resonance (MR) image reveals a large intraosseous lesion with low signal intensity. The normal bone marrow signal is preserved in the anterior and posterior portions of the body. d Sagittal T2-weighted MR image shows slightly bright signal intensity intermingled with intermediate signal. e Sagittal gadolinium-DTPA-enhanced MR image does not demonstrate any enhancement. No soft tissue mass is recognizedFig.\u00a02A 53-year-old woman who was examined for ossification of the posterior longitudinal ligament at the sixth to seventh cervical vertebrae (patient 2). A lesion was found incidentally. a Lateral radiograph reveals faint or vague osteosclerosis in the 6th cervical vertebral body. The physiological lordosis of the cervical spine is decreased. b Axial CT scan reveals diffuse sclerosis within the vertebral body. c Sagittal T1-weighted spin echo MR image reveals low signal intensity in almost the entire marrow space of the sixth cervical vertebra. d Sagittal T2-weighted MR image reveals intensely high signal replacing the entire marrow space. No soft tissue mass is evidentFig.\u00a03A 43-year-old woman who was found to have two separate abnormalities in the lower spine during an imaging study for a traffic accident (patient 7). a Lateral radiograph of the lumbar spine and sacrum reveals intense sclerosis of the entire fifth lumbar vertebral body and mild sclerosis in the cephalad portion of the sacrum. b Sagittal T1-weighted MR image reveals homogeneous low signal intensity in both the fifth lumbar and second sacral vertebraeFig.\u00a04A 52-year-old man who complained of mild upper back pain (patient 5). a Sagittal T2-weighted MR image reveals an intraosseous lesion with high signal intensity in the fifth cervical vertebra at initial presentation. b Sagittal T2-weighted MR image demonstrates no progressive disease 14\u00a0months after needle biopsy. No extraosseous tumor extension or enlargement is recognized\nImaging findings\nClinical images reviewed included conventional radiographs (n\u2009=\u20099), bone scintigrams (n\u2009=\u20092), CT scan (n\u2009=\u20097), and MR imaging (n\u2009=\u20098). Conventional radiographs revealed ill-defined, vague sclerosis within the vertebral body in 5 lesions (Figs.\u00a01, 2) and diffuse prominent sclerosis presenting as ivory vertebra in 1 (Fig.\u00a03). The remaining 3 lesions were not identified on radiographs. In all 4 lesions involving the cervical spine, physiological lordosis was decreased. Bone scintigrams did not reveal any abnormal uptake.\nAll lesions were sclerotic on CT. One was mildly sclerotic in the center of the vertebral body. The others were moderately to markedly sclerotic (Figs.\u00a01, 2). Among them, 5 lesions that were located in the central part of the vertebral body extended partly to the cortex. Another lesion had diffuse sclerosis replacing the entire bone marrow space. No cortical disruption or bone destruction was evident in any of the lesions.\nT1-weighted MR images revealed an intraosseous lesion with homogeneous low signal intensity (Figs.\u00a01\u20133). T2-weighted MR images revealed homogeneous intermediate to bright signal intensity. Gadolinium-DTPA-enhanced T1-weighted MR images were available for 3 lesions and did not show any significantly increased intensity (Fig.\u00a01). Three lesions occupied the entire marrow space and the other 5 lesions were surrounded by residual bone marrow tissue. No extraosseous disease was demonstrated.\nHistological findings\nEight lesions were histologically examined. All lesions were located within the vertebral bodies. The lesions were unencapsulated and composed of solid sheets of adipocyte-like vacuolated chordoid cells mixed with eosinophilic, less vacuolated cells of various degrees (Fig.\u00a05a). The adipocyte-like cells had clear cytoplasm and eccentrically or centrally located round or polygonal nuclei (Fig.\u00a05b). Some tumor cells were multi-vacuolated and their nuclei were mildly polymorphic or hyperchromatic (Fig.\u00a05c). No mitotic figures were recognized in any of the lesions. No myxoid background was evident. In some cases, cystic spaces were found in the lesions that contained eosinophilic colloid-like material. The lesions lacked any apparent vascular network. Islands of hematopoietic bone marrow were seen within the lesions. The entrapped bone trabeculae were sclerotic because of appositional or reactive new bone formation (Fig.\u00a05a,b). PAS staining demonstrated intracytoplasmic glycogen granules. In all 7 lesions examined immunohistochemically, the tumor cells stained intensely positive for vimentin, S-100 protein, EMA, CAM5.2, AE1\/AE3, and CK18 (Fig.\u00a05d,e). The appearance in each of the cases was consistent with benign notochordal cell tumor [4, 5]. There were no features to support the diagnosis of chordoma. Patient 7 was presented at the Members Meeting of the International Skeletal Society in 1999.\nFig.\u00a05Photomicrographs of benign notochordal cell tumors. a Low power magnification (patient 1) reveals solid sheets of adipocyte-like vacuolated cells. The affected bone trabeculae are sclerotic. Some islands of non-neoplastic hematopoietic bone marrow are seen in the lesion (hematoxylin-eosin [HE] stain). b Higher power magnification (patient 1) reveals a solid sheet of vacuolated tumor cells of various sizes. The nuclei are oval and appear bland (HE stain). c Some tumor cells (patient 1) are multivacuolated and centrally located nuclei are mildly polymorphic. They are reminiscent of lipoblasts (HE stain). d Biopsy specimen (patient 5) reveals sheets of vacuolated cells with pyknotic nuclei between bone trabeculae. The tumor cells may be mistaken for degenerative fatty marrow (HE stain). e The tumor cells (patient 5) stain immunohistochemically positive for cytokeratin (AE1\/AE3) indicating notochordal cell origin (SAB immunohistochemical stain)\nDiscussion\nSix lesions consistent with BNCT have been documented previously by others [1, 2, 9\u201311]. Darby and coauthors [1] first described a case in 1999, which was surgically excised. A 39-year-old man who complained of a 7-week history of persistent low back pain was found to have an intraosseous lesion replacing almost the entire fifth lumbar vertebra. Radiographs and scintigraphy appeared normal. The lesion was well visualized by MRI, but showed only mild sclerosis on a CT scan. Histological findings were identical to those of our cases presented here. They were not typical for chordoma; however, the authors suspected that the lesion might be an intraosseous chordoma rather than a giant notochordal rest. Mirra and Brien [2] reported 2 cases under the name of giant notochordal hamartoma of intraosseous origin. A 42-year-old woman complained of low back pain after falling down the stairs and was found to have an intraosseous lesion involving almost the entire sixth thoracic vertebral body on MRI. The other case was a 41-year-old man who had suffered from intermittent stiffness and mild pain in the neck. MR images demonstrated an intraosseous lesion involving about 98% of the fifth cervical vertebral body. Neither of the lesions was visible on radiographs. CT scans revealed osteosclerosis in the center of the affected vertebral bodies. The lesions showed low signal intensity on T1-weighted MR images and intermediate to high signal intensity on T2-weighted images and no extraosseous disease. No abnormal uptake was detected on bone scintigrams in either case. The patients did not undergo a surgical procedure after the biopsies. The follow-up examinations after 10\u00a0years and 38\u00a0months respectively demonstrated no progressive disease and indicated their benign nature. Kyriakos et al. [9] reported a case that involved the fourth lumbar vertebra. An 11-year-old boy with Tourette\u2019s syndrome had a 2.5-year history of repeated low back pain after a playground injury. Radiographs, CT scan, and single photon emission computed tomographic bone scan (SPECT) were normal. MRI revealed an intraosseous lesion with low T1- and high T2-weighted signal intensities within the fourth lumbar vertebral body. Cell block material obtained by a fine needle aspiration biopsy led to the diagnosis of chordoma, and the patient underwent L4 vertebrectomy. On subsequent evaluation, they concluded that the lesion was likely a giant notochordal rest rather than chordoma. The most recently documented case reported by Chauvel et al. [10] was an intraosseous lesion in the fifth lumbar vertebral body. The patient was a 45-year-old woman who had complained of low back pain for 2\u00a0years. Radiographs and bone scan were normal; however, CT scan revealed fine sclerosis in the L5 vertebra. MRI demonstrated an intraosseous lesion with homogenous low signal on T1-weighted image and high on T2-weighted image. The patient underwent L5 vertebrectomy because the lesion was histologically diagnosed as a chordoma on biopsy. The histological features from the L5 vertebra were similar to those of previously documented cases reported by Darby et al. [1], Mirra and Brien [2], and Kyriakos et al. [9]. A further case was illustrated in the textbook by Dorfman and Czerniak [11]. The lesion entirely occupied the third lumbar vertebra.\nRecent studies have discovered unique benign notochordal cell lesions within axial bone that may be precursors of chordoma [3\u20136, 12]. The new term \u201cbenign notochordal cell tumor\u201d was proposed for these lesions [4, 5]. Careful autopsy examination can identify the lesions in approximately 20% of adult cadavers [4, 5]. They are usually tiny or small and found most frequently in both ends of the axial skeleton followed by the mobile spine. Their histological features are different from those of classic chordoma or notochordal vestiges in fetal intervertebral disks (Table\u00a03). BNCTs are characterized histologically by intraosseous sheets of adipocyte-like vacuolated chordoid cells intermingled with less vacuolated eosinophilic cells of various degrees. The lesions were not lobulated. Some colloid-like material containing cystic spaces are seen in the lesions; however, the lesions lack any myxoid background. The nuclei are usually bland, but sometimes appear mildly atypical. No mitotic figures are recognized. The affected bone trabeculae are sclerotic because of appositional or reactive new bone formation. These histological features are completely identical to those of the lesions reported by Darby et al. [1], Mirra and Brien [2], Kyriakos et al. [9], Chauvel et al. [10], and Dorfman and Czerniak [11]. These reported cases are considered by us to represent BNCTs that grew and replaced almost the entire vertebrae.\nTable\u00a03Imaging and microscopic features of benign notochordal cell tumorsEvaluation method\u00a0FeaturesRadiograph (n\u2009=\u20099)Vague sclerosis (n\u2009=\u20095)Marked sclerosis (n\u2009=\u20091)Invisible (n\u2009=\u20093)Bone scintigram (n\u2009=\u20092)No abnormal uptake (n\u2009=\u20092)CT scan (n\u2009=\u20097)Osteosclerosis in vertebral body (n\u2009=\u20097)No bone destruction or cortical disruption (n\u2009=\u20097)MRI (n\u2009=\u20098)T1-WI (n\u2009=\u20098): low signal intensityT2-WI (n\u2009=\u20098): intermediate\u2013high signal intensityGd-DTPA T1-WI (n\u2009=\u20093): no enhancementNo invasive soft tissue mass (n\u2009=\u20098)Histology (n\u2009=\u20098)Solid sheets of adipocyte-like vacuolated cells combined with less vacuolated eosinophilic cells of various degreesRound or polygonal pyknotic nuclei occasionally with polymorphismNo lobular configurationNo extracellular myxoid matrixSome cystic spaces containing eosinophilic colloid-like materialNo mitotic figuresPoor vascular networkEntrapped islands of hematopoietic bone marrowSclerotic bone trabeculae affectedPositive immunostaining for vimentin, cytokeratin (AE1\/AE3, CK18), EMA, and S-100 proteinCT, computed tomography; MR, magnetic resonance; WI, weighted image; Gd; gadolinium; CK, cytokeratin; EMa, epithelial membrane antigen.\nBenign notochordal cell tumors are usually asymptomatic or indolent. Most lesions are overlooked, even at autopsy, because of their anatomical location or size. Only one such lesion had been documented with a name of ecchordosis physaliphora vertebralis in 1982 [13]. Enlarged BNCTs may be detected on CT or MR images, or rarely by conventional radiography. The imaging findings of our cases are somewhat variable, but distinctive in the absence of trabecular destruction on CT or of a soft tissue mass on either CT or MR images (Table\u00a03). Gadolinium enhancement was absent in the 3 patients in whom it was administered. Radiographs may reveal ill-defined, vague sclerosis in the vertebral body and occasionally show diffuse sclerosis presenting as an ivory vertebra (Fig.\u00a03). Often the lesions are invisible on radiographs. CT scans may reveal unequivocal sclerosis, which is much more obvious than on radiographs. No bone destruction or cortical disruption is recognized. T1-weighted MR images reveal relatively well-demarcated intraosseous lesions with homogeneous low signal intensity. T2-weighted MR images show homogeneous high signal intensity. Gadolinium-DTPA enhanced T1-weighted MR images do not show enhancement. Bone scintigraphy did not show any abnormal uptake.\nDiagnosis of the cases presented was based upon biopsy or preoperative imaging; the diagnosis was chordoma in 5 cases and osteonecrosis in 2. Hence, 5 patients underwent vertebrectomy, wide resection, or curettage. All patients but 1 are well without recurrent or metastatic disease. Two patients who underwent only biopsy had no progressive disease, as judged by radiography, CT, or MR images, for 13\u00a0months and 44\u00a0months respectively. As described by Mirra and Brien, the follow-up information was consistent with the benign nature of the tumors.\nThe terminology of the lesions is controversial. Dorfman and Czerniak [11], Kyriakos et al. [9] and Chauvel and coauthors [10] used the term giant notochordal rest. Mirra and Brien [2] proposed the term giant notochordal hamartoma of intraosseous origin. In addition, the International Skeletal Society has used the term benign chordoma on the brochure of the Pathology Refresher Course since 2003. However, we prefer the term \u201cBNCT\u201d to the others. BNCTs show histological and immunohistochemical features that are different from those of notochordal vestiges in fetal intervertebral disks. BNCTs lack any myxoid background and show positive immunoreactivity for cytokeratin 18 although notochordal vestiges in the fetal intervertebral disks are associated with myxoid matrix and are negative for cytokeratin 18. Notochordal vestiges are often found in the intervertebral disks of neonates and finally disappear by the age of 1 to 3\u00a0years. In our personal autopsy experience, we have never seen any notochordal tissue in the vertebral bodies in fetuses and babies who had notochordal vestiges in their intervertebral disks. In contrast, BNCTs are found in 20% of the axial skeleton dissected from adult cadavers. Therefore, BNCTs are considered to develop after birth. The term of hamartoma is not proper for the lesions, as mentioned by Kyriakos et al. [9] and one of the present authors [3]. The term of benign chordoma is oxymoronic because \u201cchordoma\u201d has been used to describe a malignant notochordal cell tumor [14].\nIn 15 clinically identified BNCTs, including our cases and those reported by others, 6 tumors were found in the lumbar spine, 5 in the cervical spine, 2 in the sacrum, and 1 each in the thoracic spine and coccyx. This anatomical distribution is different from that found at autopsy [2]. We believe the reason for this discrepancy is that the cervical and lumbar regions often are examined with MRI for neck pain and low back pain. Lesions in the sacrum and coccyx may be overlooked because they are less frequently imaged.\nThe most important disease that should be distinguished from BNCT is chordoma. Microscopically, chordoma is composed of cords or strands of atypical chordoid cells with a myxoid matrix of various degrees [11, 15, 16]. Chordomas are associated with a large soft tissue component. However, differential diagnosis may be difficult because cellular atypia of BNCT and chordoma may overlap. They also share the same immunohistochemical profile [3]. Imaging is critical in separating the benign lesion from the chordoma. Chordomas are osteolytic tumors. In contrast, BNCTs may show osteosclerotic reaction and do not exhibit osteolytic change. Chordomas are not intracompartmental lesions and are associated with a soft tissue mass. In those cases with histological overlap, with our present understanding of these diseases, we would suggest that the absence of a soft tissue mass should favor a BNCT and the presence of a soft tissue mass should indicate a chordoma.\nBiopsy specimens of BNCTs may be easily mistaken for fatty marrow because of their morphological similarity to that tissue. Indeed, 2 lesions among our cases were overlooked as normal fat marrow on biopsy sections. Careful observation can distinguish BNCTs from fatty marrow. Furthermore, immunohistochemical study can help to distinguish BNCTs from fatty marrow. BNCTs stain positive for epithelial markers whereas fat cells do not (Fig.\u00a05). Differential diagnosis also includes metastatic carcinoma, particularly originating from clear cell carcinoma of the kidney. Metastatic clear cell carcinoma usually is osteolytic on radiographs and CT scans. Gadolinium-DTPA enhanced T1-weighted MR images have increased signal intensity. Histologically, clear cell carcinoma has an alveolar pattern and vascular fibrous septa. Clear cell carcinoma rarely exhibits positive immunoreaction for S-100 protein [17].\nLarge BNCTs may be found during routine clinical examination. They may have the potential of malignant transformation to chordoma; however, the transformation, as we know from the collected cases, is a rare happening [6]. We, however, are not aware of any cases of what we term BNCT growing beyond the confines of bone and displaying features of chordoma, and therefore they should be managed as benign lesions and carefully followed by thin section CT and MRI for signs of extraosseous disease. More extensive follow-up by cross-sectional imaging over a long period of time would be required before firmly determining the biological behavior of BNCTs. BNCTs do not require any surgical management until they undergo malignant transformation. BNCTs should be recognized by radiologists, pathologists, and orthopedic surgeons to prevent unnecessary radical surgery and carefully followed by advanced imaging.","keyphrases":["benign notochordal cell tumor","chordoma","notochordal rest","notochordal hamartoma","vertebra","mri","spine"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Ann_Surg_Oncol-4-1-2190341","title":"Second-Look Operation for Unresectable Pancreatic Ductal Adenocarcinoma at a High-Volume Center\n","text":"Background The value of re-exploration for pancreatic ductal adenocarcinoma after the initial diagnosis of unresectability is unclear.\nSurgical resection is the only curative approach available for patients suffering from pancreatic cancer.1 However, resection rates remain low, not only because advanced disease is present in the majority of patients, but also because nonspecialized centers often have insufficient experience with radical surgery.2\u20134 Resection rates in tertiary referral centers are increasing due to technical progress, improved perioperative management, increasing emphasis on a more-radical approach, such as resection of tumor-infiltrated portal vein, and improved experience in judging resectability.3,5\u20138 This holds particularly true for evaluating vascular infiltration, which may preclude resection. In these cases, extensive mobilization and careful dissection are necessary to distinguish accompanying inflammatory adhesions from tumor infiltration of retroperitoneal vessels. However, determination of resectability remains challenging, and thus in selected cases less-experienced surgeons are potentially more prone to mistakenly regard a tumor as unresectable.\nSince randomized trials on the value of neoadjuvant therapy have not yet been published, it also remains unclear whether\u2014particularly in younger patients\u2014neoadjuvant treatment (or reoperation after initially palliative chemotherapy) may be beneficial, at least in some patients, and may thus lead to resectability of an initially unresectable tumor.\nReoperation of patients who were deemed unresectable may be a further option to increase resection rates.9\u201318 The value of re-exploration has only been analyzed by a few groups: 10 studies have addressed this question, with the first study concluding that an \u201cappreciable salvage rate\u201d is feasible on re-evaluation.9 Subsequent studies confirmed these results and demonstrated that a second-look operation can be performed with low morbidity and mortality rates, and that this approach confers a survival benefit for the then resectable patients.10\u201318 Calculations of statistically significant differences in survival of resected and unresected patients at reoperation are hampered by insufficient power of the individual studies due to small patient groups. However, there is a general trend showing that re-exploration may be an option in carefully selected patients at specialized centers.\nIn this study, we retrospectively analyzed our single-center experience with a group of patients who had initially been diagnosed as unresectable and who were subsequently referred to us for re-exploration. In addition, a literature review of the available (retrospective) studies was performed to more precisely define the value of re-exploration.\nPATIENTS AND METHODS\nFrom our database we identified 33 patients with ductal adenocarcinoma of the pancreas who had initially been diagnosed as unresectable (at other institutions) but were re-explored in the course of their disease. Patient characteristics, initial procedures, subsequent palliative or neoadjuvant therapies, operative details of the re-exploration, morbidity, and mortality (death within 30 days after surgery) were prospectively recorded. Patients were followed up until February 2007. At the time of analysis, two out of 14 patients in the group of unresectable patients and 11 out of 19 patients in the resection group were alive.\nDuring the second-look operation, resectability was defined using previously reported criteria.1 If resection was impossible, a tumor biopsy was taken from those patients in whom the initially performed palliative operation was sufficient. In the case of an obstruction, a gastroenterostomy, hepaticojejunostomy or a double bypass procedure was performed. In resectable patients, a pylorus-preserving pancreaticoduodenectomy (ppPD) was the standard operation. However, if the tumor involved the pyloric region, a classical PD was carried out. To achieve tumor-free resection margins, a total pancreatectomy had to be performed in two cases. A distal pancreatectomy was performed for one tumor of the tail of the pancreas.\nPancreatic tumors other than ductal pancreatic adenocarcinoma were excluded. The specimens from resectable (at reoperation) patients were classified according to the tumor, node, metastasis (TNM) classification.19\nFor the preoperative imaging of operable patients, computed tomography of the abdomen or magnetic resonance imaging\/magnetic resonance cholangiopancreaticography (MRI\/MRCP) was the standard.\nStatistical Analysis\nThe Kaplan\u2013Meier method was used for presentation of the survival curve, and differences in survival were evaluated with the log-rank test. A P value of\u00a0<\u00a00.05 was regarded as statistically significant.\nLiterature Review\nA literature search was performed independently by CWM, JK, and JB as described previously.20 The last search was carried out on 30 January 2007. Available manuscripts were cross-searched to identify studies that had not initially been found. The search strategy revealed 10 retrospective studies on re-exploration after initial diagnosis of unresectability. No stratification was performed because only retrospective analyses have been published. Data collection was conducted as previously described.20\nRESULTS\nIn this study, 33 patients (median age 60 years) from our prospectively maintained database who had been re-explored after having initially been defined as unresectable (operation between 2001 and 2005) were included. At the time of the initial operation, an exploratory laparotomy was performed in 51% of the patients, while in the other 49% a bypass operation or a biopsy was carried out (Table\u00a01). The main reason for the definition of unresectability at the initial operation was vascular infiltration (six arterial infiltrations, nine venous infiltrations). In some patients, metastatic disease was present (Table\u00a02). After the first operation, most patients received palliative\/neoadjuvant therapy. The types of treatment varied considerably: 13 patients received chemoradiation, 12 patients were treated with chemotherapy, and eight patients received no therapy. There were no standardized treatment protocols for palliative\/neoadjuvant therapy, but patients were treated according to local guidelines at the institution where the initial diagnosis had been made.\nTable\u00a01.Patient characteristicsUnresectable patients (n\u00a0=\u00a015)Resectable patients (n\u00a0=\u00a018)Age60 (41\u201368)61 (33\u201371)Female34Male1214Initial operation\u00a0\u00a0Explorative laparotomy48\u00a0\u00a0Gastroenterostomy50\u00a0\u00a0Double bypass35\u00a0\u00a0Lymph node biopsy10\u00a0\u00a0Resection of abdominal wall tumor10\u00a0\u00a0Laparoscopy02\u00a0\u00a0Hepaticojejunostomy13Interval between surgeries (median)88 days101 daysPreoperative tumor markers\u00a0\u00a0CA19-9 (median)608 U\/l117 U\/l\u00a0\u00a0CEA (median)39143 U\/l136 U\/lDiabetes mellitus36Neoadjuvant therapy1213Table\u00a02.Criteria for initial inoperabilityUnresectable patients (n\u00a0=\u00a015)Resectable patients (n\u00a0=\u00a018)Peritoneal metastasis40Vascular infiltration69Liver metastasis31Duodenal infiltration11Lymph node infiltration14Other\/unknown03\nUpon referral to our department, all patients presented in adequate general condition (ASA score\u00a0\u2264\u00a03; American Society of Anesthesiologists). At the second operation, 18 patients were found to have a resectable tumor, while in 15 patients the primary tumor could not be resected. The median time from initial surgery to re-exploration was 88 days in the unresectable group and 101 days in the group of patients in whom a resection could be performed. Except for one patient with a large tumor infiltrating the retroperitoneal vessels, metastatic disease (not detectable in the abdominal CT scan) was present in all the unresectable patients, precluding a surgical resection (Table\u00a04).\nIn the group of patients who were found to be resectable, 15 pancreaticoduodenectomies, two total pancreatectomies and one distal pancreatectomy were performed (Table\u00a03). Portal vein\/superior mesenteric vein (SMV) resections had to be carried out in four patients. There was one patient with a liver metastasis at the initial operation (Table\u00a02) who was resected at the second-look surgery. The patient had no liver metastases at the second look and was therefore considered resectable. This patient is alive at 1241 days after the initial surgery. Four out of five patients without neoadjuvant treatment who were resected (Table\u00a01) received adjuvant chemotherapy. One patient refused adjuvant treatment.\nTable\u00a03.Surgical procedures at second operation, morbidity and mortalityUnresectable patients (n\u00a0=\u00a015)Resectable patients (n\u00a0=\u00a018)Exploration with biopsy7\u2013Double bypass3\u2013Gastroenterostomy3\u2013Hepaticojejunostomy2\u2013Pancreaticoduodenectomy\u20135Pylorus-preserving pancreaticoduodenectomy\u201310Total pancreatectomy\u20132Left resection\u20131Duration of operation (min; median)105440Blood loss (ml; median)100500Morbidity23Delayed gastric emptying10Bilioma10Wound dehiscence01Cholangitis01Lymph fistula01Mortality01Length of stay (days; median)1112Table\u00a04.Criteria for unresectability at reoperationUnresectable patients (n\u00a0=\u00a015)Peritoneal metastasis6Liver metastasis5Omental metastasis1Peritoneal and liver metastasis2Retroperitoneal infiltration1Table\u00a05.Review of studies on re-exploration for pancreatic cancerAuthorYearInstitutionPatientsResection rateMoosa1979University of Chicago2417\/24 (71%)Jones1985University of Toronto50N\/AHashimi1989Bradford Royal Infirmary2611\/26 (42%)McGuire1991Johns Hopkins Medical Institutions5533\/55 (60%)Tyler1994M.D. Anderson Cancer Center1914\/19 (74%)Robinson1996M.D. Anderson Cancer Center2929\/29 (100%)Johnstone1996Naval Medical Center, San Diego2916\/29 (55%)Sohn1999Johns Hopkins Medical Institutions7852\/78 (67%)Chao2000Fox Chase Cancer Center4022\/40 (55%)Shukla2005Tata Memorial Hospital, India1515\/15 (100%)This series2007University of Heidelberg3318\/33 (55%)\nIn the group of unresectable patients, bypass procedures were carried out in eight patients, while seven patients underwent exploration with tumor biopsy (Table\u00a03). The postoperative course of the patients was mostly uneventful, with two morbidities in the inoperable group and five in the resected patients (Table\u00a03). One patient who was resected died in the immediate postoperative period (mortality 1\/18). The duration of the operation, amount of blood loss, and length of stay were increased in the resection group (Table\u00a03).\nPathology reporting revealed that a T3 tumor was present in most of the cases. Only one patient was found with a T2 tumor. Nine pancreatic specimens (50%) were node positive, with an average of 21.5 harvested lymph nodes. A grade 1 tumor was found in only two patients, whereas a grade 2 tumor was present in nine patients and a grade 3 pancreatic cancer was present in seven patients.\nThe Kaplan\u2013Meier survival curves revealed a median survival of 439 days for unresectable patients versus 934 days for resected patients (after the second operation). A log-rank test demonstrated that this difference was statistically significant (P\u00a0=\u00a00.013; data not shown). To better judge the influence of neoadjuvant\/palliative treatment on survival, a Kaplan\u2013Meier curve was calculated for the survival after the initial operation. This analysis showed a significantly different median survival of 547 versus 1078 days for unresectable versus resectable patients (P\u00a0=\u00a00.018; Fig.\u00a01).\nFIG.\u00a01.Kaplan\u2013Meier survival curve of re-explored patients after initial operation. Comparison of the survival curves of re-explored, resected patients (n\u00a0=\u00a017; one excluded due to postoperative mortality; red) and re-explored, unresectable patients (n\u00a0=\u00a015; black). A log-rank test demonstrated significantly increased survival in the resected patients (P\u00a0=\u00a00.018).\nTo judge whether resection at reoperation influences the outcome, we compared re-resected (18) and non-re-resected (15) patients with a cohort of 554 patients who were resected at the initial operation at our center and with a cohort of 250 patients who could not be resected. The Kaplan\u2013Meier survival curve followed by a log-rank test revealed no differences in survival (P\u00a0=\u00a00.2514; Fig.\u00a02) when comparing initially resected patients and patients resected at the second operation. Furthermore, there were also no differences in comparison of the unresectable patients, irrespective of the second surgery (P\u00a0=\u00a00.45; Fig.\u00a03).\nFIG\u00a02.Survival curves of re-explored, resected patients and 572 primarily resected pancreatic ductal adenocarcinoma (PDAC) patients. Comparison of the survival curves of re-explored, resected patients (n\u00a0=\u00a018; red) and a control cohort of 572 patients (black) who were initially resected revealed no differences regarding survival (P\u00a0=\u00a00.2514).FIG.\u00a03.Overall survival of re-explored, unresectable patients and 256 unresectable patients. The survival of a control cohort of 256 PDAC patients who were unresectable (n\u00a0=\u00a0265) was compared with re-explored, unresectable patients (n\u00a0=\u00a015; red), revealing no differences in survival (P\u00a0=\u00a00.45).\nSubsequently, we performed a retrospective review of the causes of initial unresectability. This revealed that only 11 of 33 patients had initially been treated at a high-volume center (defined as hospitals with a case load of\u00a0>16 pancreatic resections per year7). Except for four cases with metastasized disease, infiltration of local structures was the main reason for initial unresectability. A detailed analysis of these causes of unresectability showed that 14 of 33 patients would probably have been resected if they had initially been referred to our center. In these cases, mainly venous (SMV\/PV) infiltration was present, which may not be considered a contraindication for a resection.\nReview of Retrospective Studies\nTen studies on the value of re-exploration to potentially achieve resection have been published so far.9\u201318 In the first study, by Moosa,9 17 patients were re-evaluated after having initially been diagnosed as unresectable. Of these, nine patients underwent total pancreatectomy, while in two patients no tumor was detectable. The survival of the resected cohort was 1.5\u20136 years, which was higher than the mean survival in the group of patients with inoperable disease (7\u201312 months). Similar results were reported by Hashimi et al., who performed a second-look operation on 30 patients, revealing that nine were resectable, while in two patients no tumor was found despite several biopsies.11 In the study by Jones and co-authors, preliminary surgery (nonresective) had been carried out before referral in 51% of the patients, leading to the correct diagnosis in only 44% of these.10 However, outcomes for this patient cohort are not specifically described. Two publications from the Johns Hopkins Institutions also extensively describe results obtained in re-exploratory surgery of two patient cohorts (recruited at different time periods: from 1979 to 1990 and from 1991 to 1997). In the first publication,12 the resection rate for pancreatic cancer at re-exploration was 58%, with subsequent mean survival periods of 20.5 months (pancreatic adenocarcinoma) and 33 months (nonpancreatic periampullary malignancies), which were comparable to the cohort of initially resectable patients. Sohn et al. subsequently described results obtained from re-exploration of a second cohort of patients that included all periampullary tumors.16 The resection rate at reoperation was 67%. There was a striking survival difference when comparing the unresectable and the resectable groups (at re-exploration), with a median survival of 7 versus 23 months, respectively. Comparison of the patients who were resected at re-exploration with a control group who had initially been resected revealed significantly increased survival rates in the reoperative group (Kaplan\u2013Meier survival curve generated from the time of initial operation: 20 months in the control group versus 33 months in the reoperative group, P\u00a0=\u00a00.02). In the study by Tyler et al. from 1994, reoperation rates were comparable with the earlier published studies (14 out of 19 patients who underwent re-laparotomy were resected), as were the (low) complication rates (three out 14 resected patients).13 At the time of median follow-up of 26 months, four of ten patients with pancreatic adenocarcinoma were alive, suggesting survival rates comparable to previously published results. Similarly, Johnstone and Sindelar showed a resection rate of 55% at re-exploration and significantly improved disease-specific survival rates in the resected group (P\u00a0<\u00a00.01) 14. Robinson and co-workers demonstrated an exceptionally high resection rate at reoperation of 100%.15 In a study by Chao and co-workers, a resection rate at the second look operation of 55% was shown.18 Furthermore, they could demonstrate that resection at the second operation led to \u201ca median survival comparable with that of patients who did not undergo previous exploration\u201d. In the most recently published study, by Shukla et al., all patients who were reoperated could be resected.17 Confirming the previously published studies, low morbidity and mortality rates were demonstrated.\nDISCUSSION\nThis single-center experience shows that re-exploration of patients initially deemed unresectable may be an option for the treatment of pancreatic cancer. In concordance with previously published studies (which were all retrospectively conducted), re-exploration can yield resectability rates greater than 50% and can be performed with low morbidity and mortality. However, whether reoperation confers a survival benefit is still a matter of discussion.\nAlthough diagnosis of resectability has been facilitated by the use of multidetector, high-resolution computed-tomography exploration often remains necessary.21,22 Particularly when neoadjuvant therapy has been applied, fibrosis and an inflammatory reaction frequently hamper exact assessment of tumor size and (potential) metastatic disease. A number of studies have demonstrated that both the complication rate and mortality after pancreatic surgery are significantly higher in hospitals that perform few operations.3,7,8 We found that, upon re-exploration, more than half of the tumors were resectable, and that 42% of the patients initially deemed unresectable could have been resected at high-volume centers. These results support the concept that patients with pancreatic cancer should be treated with an interdisciplinary approach at large tertiary referral centers.\nFurthermore, resection in a second-look operation was shown to potentially confer a survival benefit, although it remains unclear whether this is due to resection or to favorable tumor biology in the patients in whom a regression of the initially metastasized or infiltrating tumor was achieved. Altogether, there was significantly increased survival in the reoperated group of patients compared with a control cohort of patients who were not reoperated (resected and unresectable). These results suggest that there might be a generally favorable tumor biology in this group of reoperated patients. However, the differences in survival conferred by resection at reoperation confirm the notion that in a subgroup of patients in good condition, re-laparotomy (and potentially resection) should be attempted. Furthermore, reoperated but not resected patients do not fare worse than those who were not resected initially and who were not explored again after chemotherapy or chemoradiation. Therefore, we again conclude that in a selected cohort of patients with a tendency towards remission, re-laparotomy may be a valid option. This holds particularly true when patients have initially been considered unresectable at smaller hospitals with less experience in pancreatic surgery and especially with less experience in judging whether a tumor is resectable. This is supported by many studies on reoperative pancreaticoduodenectomy for pancreatic cancer.12,13,15\u201317 As stated by Robinson and co-authors, \u201cdetailed preoperative imaging and a clearly defined operative plan would have allowed successful resection at the initial operation\u201d in the majority of patients. However, accompanying inflammatory changes allow judgment of resectability only during the surgical procedure itself in many cases. Therefore, not only preoperative assessment but also the extension of resectability criteria (as present in high-volume centers) is a key factor for improving the outcome in patients with pancreatic cancer. We strongly suggest that all patients suffering from pancreatic cancer should be referred to high-volume centers. Furthermore, patients in generally good condition but locally unresectable disease should receive neoadjuvant chemoradiation, preferably within randomized controlled clinical trials.23 However, we do not consider infiltration of the SMV\/portal vein (in contrast to superior mesenteric artery (SMA)\/celiac trunk involvement) a contraindication for resection. Following the last cycle of chemoradiation, high-resolution CT will allow judging resectability in many cases and in the absence of metastatic disease, surgical exploration remains the only option to determine resectability.\nIn conclusion, reoperation for pancreatic cancer after initial classification of unresectability revealed resectability in half of patients. Since the majority had received chemotherapy\/chemoradiation after the initial operation, the concept of selecting patients by neoadjuvant therapy may be supported. Furthermore, a subgroup analysis revealed that a large number of patients who were initially deemed unresectable at smaller hospitals would have been resectable at a large tertiary referral center, which again promotes the concept of patient centralization in pancreatic surgery. A survival benefit for the resected patients underlines the efficacy of a surgical resection even in a situation in which the initial findings preclude a potentially curative approach.","keyphrases":["unresectability","pancreatic cancer","reoperation","second look"],"prmu":["P","P","P","P"]}
{"id":"Ann_Biomed_Eng-2-2-1705516","title":"An Ex Vivo Study of the Biological Properties of Porcine Aortic Valves in Response to Circumferential Cyclic Stretch\n","text":"Normal physiological mechanical forces cause constant tissue renewal in aortic valve leaflets (AVL) while altered mechanical forces incite changes in their structural and biological properties. The current study aims at characterizing the remodeling properties of AVL subjected to cyclic circumferential stretch in a sterile ex vivo bioreactor. The leaflets cultured were stretched at a maximum rate of 300%s\u22121 corresponding to a 15% strain for 48 h. Collagen, sulfated glycosaminoglycan (sGAG), and elastin contents of the stretched, fresh, and statically incubated leaflets were measured. Cusp morphology and cell phenotype were also examined. AVLs exposed to cyclic stretch showed a significant increase in collagen content (p < 0.05) when compared to fresh and statically incubated AVLs. sGAG content was significantly reduced in the stretched AVLs (p < 0.05) when compared to the fresh leaflets and was comparable between stretched and statically incubated AVLs. There was no statistically significant change in elastin content in all the three groups of AVLs (p > 0.05). Native aortic valve morphology was well preserved in stretched leaflets. Immunohistochemistry and immunoblotting studies showed an increased expression of \u03b1-smooth muscle actin (\u03b1-SMA) in stretched leaflets while \u03b1-SMA expression was reduced in statically incubated AVLs when compared to the fresh leaflets. To conclude, circumferential cyclic stretch altered the extracellular matrix remodeling activity of valvular cells, and consequently the extracellular matrix composition of the AVLs. Most interestingly, the contractile and fibrotic phenotypic expression of valve interstitial cells was enhanced. These results show that circumferential cyclic stretch is a possible mediator for AVL remodeling activity.\nIntroduction\nCardiac valves play a crucial role in controlling blood flow through the body.6 Of particular interest in this study is the aortic valve, which at its most basic level controls blood flow between the left ventricle and the systemic circulation. In the United States, aortic valve disease has emerged as the third most common cardiovascular disease,17 with the number of aortic valve replacement surgeries steadily increasing over the past 20 years.33\nThe aortic valve is subjected to 30\u201340\u00a0million cycles of opening and closing a year, which translates to approximately 3\u00a0billion cardiac cycles in a lifetime.26 During each cardiac cycle, the normal functional aortic valve interacts closely with the surrounding hemodynamic environment and is exposed to a myriad of mechanical forces such as transvalvular pressure, axial, shear and bending stresses, and cyclic flexure.26 Normal hemodynamic forces have been shown to cause constant tissue renewal in aortic valves,19 while altered mechanical forces are believed to induce changes in aortic valve biology possibly leading to valvular disease.16,30 However, the cellular and molecular events involved in these processes are not well characterized. Studies indicate that abnormal hemodynamics, especially hypertension, experienced by the valve leaflets cause tissue inflammation, which can lead to calcification, stenosis and ultimately valve failure1,15,23Knowledge of the adaptive mechanisms of the aortic valves to altered hemodynamics will therefore improve our understanding on mechanotransduction of valvular disease progression and valve failure.\nMechanical forces modulate cell physiology, and have been shown to affect the biosynthetic activity of cells in tissue matrices. Previous studies on aortic valve mechanobiology have shown that porcine aortic valves cultured under normal hemodynamic conditions in a sterile ex vivo flow loop undergo constant renewal to maintain their native phenotypes.13 Porcine aortic valve endothelial cells exposed to 20\u00a0dyn\u00a0cm\u22122 of steady laminar shear stress showed an organization of focal adhesion complexes and a cell alignment different than in static endothelial cell culture.4 Aortic valve leaflets exposed to hypertensive cyclic pressure showed increased collagen and sulfated glycosaminoglycan (sGAG) synthesis which varied in a magnitude and frequency-dependent manner.31 Studies exposing native porcine aortic valve leaflets to the isolated effects of flow, laminar steady shear stress, and constant static and cyclic pressure resulted in a reduced level of expression of \u03b1-smooth muscle actin (\u03b1-SMA).29,31,32 This last finding might indicate the involvement of other forces in maintaining and regulating the contractile phenotype of aortic valve interstitial cells. To date the effect of cyclic stretch on aortic valves has not been studied.\nThe mechanical mode of cyclic stretching is important for normal aortic valve functionality as it allows, among other things, the valve leaflets to extend and form a coaptive seal with each other. However, it was seen that the valve leaflet tissue becomes less extensible and loses its ability to stretch rapidly with increasing age.15 This is mainly because collagen fibrillogenesis proceeds to maturity by increasing the diameter of some of the constituent collagen fibrils that require greater force to produce the same leaflet extension.8 Although the valve leaflet undergoes biaxial stretching in vivo, studies that investigate the effects of separate uniaxial (circumferential and radial) as well as biaxial cyclic stretch on the aortic leaflets would provide useful information on valve functionality or failure.\nWe hypothesize that the regulation of extracellular matrix remodeling as well as the expression of the contractile phenotype of aortic valve cells depend on the loading forces due to cyclic stretch. Here, we examined this hypothesis with a focus on the effect of physiological uniaxial circumferential cyclic stretch on the biosynthetic response of aortic valve leaflets using porcine aortic valve leaflets and an ex vivo tensile stretch bioreactor. Results from the current study will improve our understanding of the biological responses of native valves to circumferential stretching and will provide a good basis of comparison for conducting studies on radial cyclic stretching.\nMaterial and Methods\nTissue Harvest\nFresh porcine aortic valves were obtained from a local abattoir (Holifield Farms, Covington, GA) following on-site dissection of the hearts within 30\u00a0min of slaughter. The valves were then transported to the laboratory in sterile, ice-cold Dulbecco\u2019s Phosphate Buffered Saline (DPBS; Sigma, St. Louis, MO).\nUpon arrival to the laboratory, the three leaflets were excised from each valve and a rectangular section of tissue was isolated from the center of each leaflet. This tissue section was further divided into three rectangular sections having an aspect ratio of 12\u00a0\u00d7\u00a05\u00a0mm and oriented in the circumferential direction from the basal region. These three sections from each leaflet were randomly selected as fresh control, static control, and stretched leaflets, respectively (Fig.\u00a01). Leaflet sections from the fresh control group were analyzed within 4\u00a0h following valve dissection. Leaflet sections from the static control group were statically incubated in Dulbecco\u2019s Modified Eagle Medium (DMEM; Sigma) at 37\u00b0C for the duration of the experiment (48\u00a0h). Leaflet sections from the stretched group had loops sutured to the 5\u00a0mm ends and were suspended in DMEM in a tensile stretch bioreactor.10 These samples were then cyclically stretched at a frequency of 1.167\u00a0Hz (equivalent to 70 beats per minute) to 15% strain at a maximum strain rate of 300%s\u22121 at 37\u00b0C. The temporal strain waveform imposed (Fig.\u00a02a) in the circumferential direction was derived from the variations of the leaflet length in the circumferential direction over one cardiac cycle.25,26 The extension of the leaflet corresponded to diastole (2\/3 of the cardiac cycle), while the unstretched state of the leaflet corresponded to systole (1\/3 of the cardiac cycle). The gradients of the extension and relaxation approximated those experienced in vivo.25 The above waveform resulted in a tissue extension of 1.8\u00a0mm at a maximum rate of extension of 36\u00a0mm\u00a0s\u22121. Each experiment was run for 48\u00a0h, with 12\u201315 leaflet sections for each experimental run.\nFigure\u00a01.Preparation of aortic valve leaflets for the experiment. Three sections were cut out in the circumferential direction from the base, belly and tip regions of the aortic valve leaflet. These sections were randomly chosen as stretched, fresh and static leaflet groups. Fresh sections were processed immediately. Static control sections were incubated for 48\u00a0h in DMEM. Stretched sections were stretched for 48\u00a0h.Figure\u00a02.(a) Loading curve used in this study. A near physiologic loading curve 25 was input to the actuator to stretch the leaflets. (b) Ex vivo tensile stretch bioreactor used in this study. A magnified image of the tissue chamber is shown on the right showing eight tissue wells, with aortic valve leaflets in four of the wells.\nStretch Bioreactor System and Validation\nThe stretch bioreactor used in this study (Fig.\u00a02b) was developed by Engelmayr et al. 10The device consists of two tissue chambers coupled via a cross-arm to a centrally positioned linear actuator (Ultra Motion LLC, Mattituck, NY). Each chamber consists of eight culture wells within which two \u201cstationary posts\u201d are positioned. The linear motion of the actuator is translated into uniaxial motion of two \u201cmoving posts\u201d via the cross-arm which bifurcates to two moving arms. These moving arms then enter each individual bioreactor chamber and terminate at each culture well with the two \u201cmoving posts\u201d mentioned above. The interior of the chamber is protected from external contamination by a neoprene-lined lid. The bioreactor was constructed from polysulfone, which is thermally and chemically stable, while the lids were constructed using acrylic. The device was assembled using 18-8 stainless steel screws (McMaster Carr, Cleveland, OH) and fabricated by Apollo Precision Inc. (Plymouth, MN). The motor input waveform (Fig.\u00a02a) was downloaded to the actuator controller card using the Si-Programmer software (Ultra Motion LLC).\nAfter assembly, the bioreactor was validated for uniformity of strains on the leaflet sections in all the culture wells. This was achieved by placing dye markers (Shandon tissue marking dye, Thermo Electron Corp, Pittsburgh, PA) on aortic valve leaflets and capturing the distance between the markers under uniaxial cyclic stretch using a high-speed camera capture technique developed in our laboratory.12The bioreactor was validated at input strains of 15 and 25%.\nIn order to determine if the stretch bioreactor induced a shear stress on the leaflet surface due to the relative motion of the leaflet with respect to the surrounding media, a computational fluid dynamics (CFD) model was designed, validated and used to predict the leaflet wall shear stress under the imposed loading curve in the stretch bioreactor.\nFor the experiment, the bioreactor was sterilized using hydrogen peroxide in a gas-plasma sterilizer (Advanced Sterilization Products, Irvine, CA), and assembled aseptically in a sterile laminar flow hood (Fisher Hamilton, Two Rivers, WI). The culture wells were filled with 8\u00a0ml of serum-free DMEM supplemented with 50\u00a0mg\u00a0l\u22121 ascorbic acid, 3.7\u00a0g l\u22121 sodium bicarbonate, 1% (v\/v) nonessential amino acid solution, and 1% (v\/v) antimycotic-antibiotic solution (all reagents from Sigma). Each tissue section was suspended in the medium by the two sutured loops \u2013 one end was looped over the stationary posts while the other end was looped over the moving post. The bioreactor was then placed in an incubator (Fisher Scientific, Hampton, NH) maintained at 37\u00b0C, 5% CO2 atmosphere at normal atmospheric pressure and the leaflets were stretched for 48\u00a0h.\nTissue Processing\nAt the end of the experiment, each leaflet section was further cut into three equal rectangular sections. These resulting samples were dehydrated for 48\u00a0h at room temperature in a vacuum oven (Yamato Scientific America, San Francisco, CA) prior to digestion in pepsin (Sigma), protease (Sigma) and proteinase K (Sigma), for analyzing collagen, sulfated glycosaminoglycan (sGAG), and elastin contents, respectively. All digestions were carried out in a water bath maintained at 37\u00b0C. The total amount of collagen, sGAG and elastin was determined using the SircolTM Collagen assay kit (Biocolor, UK), BlyscanTM sGAG assay kit (Biocolor), and FastinTM Elastin assay kit (Biocolor), respectively. These kits used quantitative dye-binding methods to determine the total quantity of the respective ECM component in the sample.\nValve leaflet samples from additional experiments were isolated for analysis of tissue morphology and cell phenotype. For histological and immunohistochemical analyses, leaflet sections were fixed in 10% neutral buffered formalin (Fisher Scientific, Suwanee, GA) for 24\u00a0h, saturated in 70% ethanol, processed in a tissue processor (Shandon Pathcenter enclosed Tissue Processor) in descending grades of ethanol, embedded in paraffin and cut into 5\u00a0\u03bcm sections.\nMorphological Characterization\nRoutine Hematoxylin and Eosin (H & E) stain was used to examine gross tissue structure and indicate any changes in the structural compositions of the leaflets. H & E staining was done on an autostainer (Leica, Vashaw Scientific, Norcross, GA). Picrosirius red staining was performed to examine collagen fiber structure and morphology. The protocol was as follows. Deparaffinized slides were incubated in Weigert\u2019s hematoxylin (Sigma) followed by picrosirius red solution for one hour. The slides were then washed in acidified water, three changes of 100% ethanol, and xylene before being coverslipped. These slides were viewed at 100\u00d7 using a Nikon imaging microscope (Nikon Inc., Melville, NY). A Q-imaging camera, Retiga 1300C (I-Cube, Glen Burnie, MD) and its bundled software Q-Capture Pro was used to acquire images of the tissue sections. H & E images were recorded using normal white light while picrosirius red images were recorded with circularly polarized light.\nCharacterization of Cell Phenotype\n\u03b1-smooth muscle cell actin (\u03b1-SMA) immunohistochemistry (IHC) was used to detect myofibroblasts and smooth muscle cells. The staining provided information on changes in cell phenotype between the three experimental treatments. The protocol was as follows. Deparaffinized slides were blocked using 1% gelatin\/phosphate buffered saline (PBS; Sigma) for 30\u00a0min. The slides were then incubated in mouse monoclonal anti \u03b1-SMA primary antibody (Sigma) in 1% Bovine Serum Albumin (BSA)\/PBS (Sigma) for one hour. The sections were saturated in biotinylated horse anti-mouse IgG (Vector laboratories) in 1% BSA\/PBS, and 2% normal horse serum (Vector laboratories) for 30\u00a0min. Avidin-D Texas red (Vector Laboratories) fluorochrome was applied to the sections after the secondary antibody was washed off. The sections were then counter stained with 0.25\u00a0\u03bcg\u00a0ml\u22121 4\u2032, 6-Diamidino-2-phenylindole (DAPI; Sigma), coverslipped and stored at 4\u00b0C. Slides were subsequently imaged at 200\u00d7 using a TR-FITC-DAPI triple filter under a mercury lamp.\nImage Analysis\nH & E images were analyzed by measuring the fibrosa: spongiosa length ratio, and the fibrosa: ventricularis length ratio. \u03b1-SMA immunopositive cells appear red with cell nuclei counterstained blue, while the color of collagen fibers changes from green to yellow to orange to red as the fiber thickness (and consequently maturity) increases. The proportion of \u03b1-SMA positive cells were therefore assessed by using the Image J program (NIH, Bethesda, MD) to determine the relative area coverage of \u03b1-SMA positive red staining compared to the area coverage of entire leaflet section. The relative proportion of the different maturity of the collagen fibers in the leaflet section were calculated using published methods.2 Basically, picrosirius red images were first imported into Matlab 7.01 (The Mathworks, Natick, MA) and converted into their hue, saturation and value components. Only the hue components were retained, and a histogram of hue frequency was obtained from the 8-bit hue images that contained 256 possible colors. The colors were defined as follows: red 2\u20139 and 230\u2013256; orange 10\u201338; yellow 39\u201351; and green 52\u2013128. All other hue values corresponded to interstitial space and were confirmed by visual inspection. The number of pixels within each hue range were determined and expressed as a percentage of the total number of collagen pixels and plotted.\nImmunoblotting Analysis\nThe rest of the leaflet sections were processed for analysis by immunoblotting. Leaflets were homogenized and sonicated in ice-cold lysis buffer (in mmol\u00a0l\u22121: Tris\u2013HCl 20 (pH 7.5), EGTA 5, NaCl 150, glycerol-phosphate 20, NaF 10, sodium orthovanadate 1, 1% Triton X\u2212100, 0.1% Tween-20 and protease inhibitors) to extract proteins from the tissue, and then centrifuged at 14,000\u00a0rpm to pellet cellular debris. The supernatant was assayed for protein concentration using the DC Protein Assay (Bio-Rad, Hercules, CA) and equal aliquots of tissue lysates were resolved on a 10 % SDS-PAGE gel and subsequently blotted to a polyvinyldifluoride (PVDF) membrane (Millipore, Billerica, MA). Membranes were incubated with a mouse anti \u03b1-SMA primary antibody (Sigma) at 4\u00b0C overnight (1:1000), and then with a goat anti-mouse alkaline phosphatase-conjugated secondary antibody (Bio-Rad). CDP-Star reagent (NE Biolabs, Ipswich, MA) was added and chemiluminescence of the resulting product was detected using photographic paper. GAPDH antibody (Santa Cruz Biotech, Santa Cruz, CA) was used as a loading control (1:5000). Pixel densities of \u03b1-SMA immunoreactive bands were then analyzed using the Image J program and normalized by the intensities of the GAPDH immunoreactive bands.\nAnalysis of Tissue Culture Medium\nAs a follow-up to determine if sGAGs were leaching out from the leaflet in the DMEM, six samples of the DMEM were analyzed before and after the experiment. The BlyscanTM sGAG assay kit (Biocolor) used in the ECM analysis was used to detect sGAGs in the medium.\nStatistical Analysis\nThe data for collagen, sGAG and elastin contents were expressed as mean plus one standard error of the mean. A total of 27 leaflets were analyzed for the ECM assays, 8 leaflets for the histological and immunohistochemical studies, and 10 leaflets for the Western blotting studies. The quantitative data from the ECM assays, image analysis and immunoblotting were analyzed first using ANOVA followed by pairwise comparisons with the Tukey post-hoc test. A p-value of less than 0.05 was used as a measure of statistical significance. When only two groups were being compared, data were analyzed using student t-tests with a p-value of less than 0.05 indicating statistical significance. All statistical analyses were performed using Minitab (Minitab R14, Minitab Inc. State College, PA).\nResults\nValidation of Stretch Bioreactor\nThe stretch bioreactor produced a uniform output strain across the leaflet of 14.80\u00a0\u00b1\u00a00.53% for 15% input strain, and 25.48\u00a0\u00b1\u00a00.53% for 25% input strain. Performing a student t-test between input and output signals revealed no significant difference (p\u00a0>\u00a00.05, n\u00a0=\u00a016), indicating that the bioreactor could accurately and uniformly reproduce the specified input strain across the entire leaflet section.\nThe peak steady-state wall shear stress experienced by the leaflet as calculated from CFD was 2.5\u00a0dyn\u00a0cm\u22122. This is small compared to the peak 79\u00a0dyn\u00a0cm\u22122 experienced by the leaflet under physiological flow conditions during systole as measured in our laboratory,28 and it can be assumed that stretch is the dominant force acting on the leaflet.\nExtracellular Matrix Content1\nA significant increase in collagen content was observed in stretched leaflets when compared to fresh and static control leaflets (p\u00a0<\u00a00.05, n\u00a0=\u00a027). Stretched leaflets showed a 90% and 161.5% increase in collagen content in comparison to fresh and static controls, respectively. There was no significant difference in collagen content between fresh and static control leaflets (p\u00a0>\u00a00.05, n\u00a0=\u00a027). sGAG content was found to be decreased by 40% in stretched leaflets when compared to fresh valve leaflets (p\u00a0<\u00a00.05, n\u00a0=\u00a027), while sGAG content was comparable between stretched and static control leaflets (p\u00a0>\u00a00.05, n\u00a0=\u00a027). ANOVA showed that there was no significant difference (p\u00a0>\u00a00.05, n\u00a0=\u00a027) between treatment groups for elastin content. The results for the ECM assays are graphically depicted in Fig.\u00a03.\nFigure\u00a03.Cyclic stretch increased collagen, decreased sGAG, while not affecting elastin contents in aortic valve leaflets. The leaflets were exposed to physiologic circumferential cyclic stretch (15% at heart rate of 70\u00a0bpm) or static control conditions for 48\u00a0h. The amounts of collagen, sGAG and elastin in the leaflets exposed to static or stretching were compared to those of fresh leaflets. The data were normalized by tissue dry weight and expressed as a mean value plus one standard error of the mean (* p\u00a0<\u00a00.05, n\u00a0=\u00a027).\nAnalysis of the conditioned medium at the end of experimental duration revealed a statistically significant increase (p\u00a0<\u00a00.05, n\u00a0=\u00a06) in sGAG secreted in to the media (5.83\u00a0\u00b1\u00a00.56\u00a0\u03bcg\u00a0ml\u22121 of DMEM) compared to the fresh media (1.08\u00a0\u00b1\u00a00.30).\nCell Morphology and Phenotype\nH & E staining (Fig.\u00a04a) of the leaflets showed preserved three-layered leaflet architecture in stretched leaflets consistent with fresh and static control leaflets. The cell nuclei were found to be intact with no tears in the tissue section. Image analysis showed no significant difference (p\u00a0>\u00a00.05, n\u00a0=\u00a08) in the fibrosa-spongiosa length ratio as well as the fibrosa-ventricularis length ratio (Fig.\u00a05a) indicating that there was no gross damage to leaflets structure.\nFigure\u00a04.(a) Cyclic stretch did not damage tissue structure and morphology. H & E stained images of fresh, static and stretched aortic valve leaflets are depicted here. Cytoplasm was stained pink and cell nuclei were stained blue. The representative images show the three-layered morphology of the leaflets was intact in all three groups (F \u2013 fibrosa, S \u2013 spongiosa, V \u2013 ventricularis). (b) Cyclic stretch maintained native collagen architecture. Picrosirius red images of fresh, static and stretched aortic valve leaflets are shown. Mature collagen fibers were stained orange-red. Layered collagen morphology of leaflets was observed and crimp was preserved in stretched leaflets (F \u2013 fibrosa). (c) Cyclic stretch increased \u03b1-smooth muscle actin (\u03b1-SMA)-positive cells in the ventricularis side of the aortic valve leaflets. Fresh, static and stretched leaflets were examined by \u03b1-SMA IHC. Actin was stained red and cell nuclei were counterstained blue. Increased expression of \u03b1-SMA was observed in stretched leaflets, and \u03b1-SMA was reduced in static leaflets (V \u2013 ventricularis).Figure\u00a05.(a) Fibrosa-to-spongiosa ratio and fibrosa-to-ventricularis ratio remained unchanged. There was no significant difference (p\u00a0>\u00a00.05, n\u00a0=\u00a08) in the relative thicknesses of the fibrosa, spongiosa and ventricularis layers between fresh, static and stretched leaflets. (b) Static leaflets exhibited reduced levels of newly synthesized collagen fibers. Newly synthesized fiber proportion was reduced in static leaflets signifying reduced levels of collagen synthesis. Newly synthesized fiber proportion was comparable between fresh and stretched leaflets, but the proportion of mature fibers was greater in stretched leaflets. (c) Cyclic stretch increases \u03b1-smooth muscle actin in the aortic valve leaflets. \u03b1-SMA immunopositive staining coverage was increased in stretched leaflets and reduced in static leaflets when compared with fresh leaflets.\nPicrosirius red staining (Fig.\u00a04b) of the leaflets showed that collagen architecture was preserved in fresh, static as well as stretched leaflets. The layered structure of collagen along with the crimp in the collagen fibers could clearly be seen in the stretched leaflets, demonstrating that the stretching motion did not damage valvular collagen. These findings demonstrated that uniaxial cyclic stretch of the leaflets did not damage leaflet morphology and maintained native ECM structure and cellular composition.\nAdditionally, hue analysis of the picrosirius red images (Fig.\u00a05b) revealed that the proportion of newly synthesized (green) fibers in statically incubated leaflet sections was significantly decreased by 36.5% (p\u00a0<\u00a00.05, n\u00a0=\u00a08) compared to fresh leaflets. There was no significant difference in green fibers (p\u00a0>\u00a00.05, n\u00a0=\u00a08) between fresh and stretched leaflets, however, mature (red) collagen fibers significantly increased by 101.4% in stretched leaflets compared to fresh leaflets.\nThe immunohistochemical study with \u03b1-SMA (Fig.\u00a04c), a marker of myofibroblast-like or smooth muscle cell-like valvular interstitial cells2, revealed that cyclic stretch up-regulated the expression of \u03b1-SMA, while static incubation down-regulated the expression of \u03b1-SMA. This was corroborated by image analysis (Fig.\u00a05c) of the images, which showed significantly increased (p\u00a0<\u00a00.05, n\u00a0=\u00a08) \u03b1-SMA immunopositive staining in stretched leaflets compared to fresh leaflets, and significantly reduced (p\u00a0<\u00a00.05, n\u00a0=\u00a08) \u03b1-SMA staining in static leaflets compared to fresh leaflets. Another interesting observation was that the \u03b1-SMA positive cells in the stretched leaflets were predominantly found in the ventricularis region of the aortic valve leaflet while there was little or no \u03b1-SMA expression in the fibrosa. In contrast, \u03b1-SMA positive cells were randomly distributed in all the three regions of the fresh and static leaflets.\nThe above finding was further reinforced by the Western blotting results (Fig.\u00a06). There was a 54.5% increase (p\u00a0<\u00a00.05, n\u00a0=\u00a010) in \u03b1-SMA content of stretched leaflets and 34.9% decrease (p\u00a0<\u00a00.05, n\u00a0=\u00a010) in statically incubated leaflets when compared to fresh leaflets. These results together suggest that cyclic stretching induces phenotypic change of valve interstitial cells toward myofibroblast-like cells in a side-dependent manner.\nFigure\u00a06.Cyclic stretch increased \u03b1-smooth muscle actin in the aortic valve leaflets. Fresh, static and stretched aortic valve leaflets were analyzed by Western blot with \u03b1-SMA antibody and analyzed by densitometry. When compared to fresh leaflets, \u03b1-SMA expression decreased in static leaflets, but increased in stretched leaflets (*p\u00a0<\u00a00.05, n\u00a0=\u00a010). Anti-GAPDH antibody was used as a loading control.\nDiscussion\nEffect of Cyclic Stretch on ECM Components\nThe current study investigated the effects of isolated circumferential cyclic stretch on the biological responses of aortic valve leaflets using a sterile ex vivo stretch bioreactor. Initial validation of the bioreactor showed that strains were uniform across the leaflet section. It was also validated that there was negligible wall shear stress on the leaflet, and therefore cyclic stretch was the only force acting on the leaflet.\nIt was revealed that the collagen content of the aortic valve leaflets stretched to 15% was increased when compared to fresh and static control leaflets, while sGAG content was decreased in stretched leaflets compared to fresh leaflets. Furthermore, elastin content was comparable between the stretched and fresh leaflets. This suggests that exposure to isolated effects of circumferential cyclic stretch at physiological levels has altered the extracellular matrix composition, consequently, altering the valve remodeling.\nCollagen fibers, densely populated in parallel alignment along the circumferential direction of the leaflet, form the load bearing structure of the leaflet during each cardiac cycle.18,26 The increase in collagen suggests that the leaflets adapt to altered mechanical loading due to the effects of isolated cyclic stretch, by either increasing synthesis, or decreasing degradation of collagen. The image analysis of the picrosirius red staining (Fig.\u00a05b) showed comparable proportions of newly synthesized fibers, and hence collagen synthesis in both fresh and stretched leaflets. The increased proportion of mature (red) fibers in stretched leaflets therefore leads us to believe that it is the reduced degradation of collagen that causes the overall total increase in collagen content. The preserved crimp in collagen fibers in the stretched leaflet, as evidenced by picrosirius red staining also implies that the native structure of collagen was not damaged by the stretching motion.\nThis data is in agreement with a study conducted by Ku et al.14 where porcine aortic valve interstitial cells were cultured on a collagen substrate and stretched 48\u00a0h. For cells subjected to 14% cyclic stretch, collagen incorporation in the cell layer was approximately 170% of unstretched cells. No significant changes were observed at time points less than 48\u00a0h. This provides further evidence that 48\u00a0h of culture is a necessary and sufficient time point to observe changes in the valvular ECM.\nThe semi-fluidic nature of the sGAG in the spongiosa layer of the leaflet gives the leaflet considerable plasticity.26 It is therefore believed that the purpose of sGAG might be (i) to help reduce leaflet stresses by lubricating the relative motion of the other two layers as the leaflet deforms, and (ii) to dampen compressive forces during diastole.7 The absence of compressive stresses in this study, with the stretch being carried out at atmospheric pressure, might therefore explain the reduced levels of sGAG observed in the stretched leaflet compared to fresh leaflets. Additionally, analysis of the DMEM at the end of experimental duration showed a statistically significant increase in sGAG content in the media. This suggests that cyclic stretching of the aortic valve leaflets resulted in secreting or leaching of the sGAG from the leaflets into the media, as the various layers of the leaflet rub against each other. A study conducted by Grande-Allen et al.11 also showed a loss of GAG from explanted porcine bioprosthetic valves due to leaching even under normal storage conditions. This suggests that there is a high propensity for sGAGs to be forced out of the native aortic valve either during static incubation or leaflet motion. In contrast, aortic valve leaflets when exposed to normal physiological forces undergo constant sGAG renewal by the interstitial cells.13\nIn the ventricularis layer of the leaflet, the major extracellular component is elastin in the form of fibers that are oriented primarily along the radial direction. Their primary role of elastin fibers is to maintain a circumferential collagen fiber orientation and recoil the collagen fibers to their initial state after the external load has been released.27 Since the valve leaflets were cyclically stretched along the circumferential direction at physiological rates in the current study, it is possible that the elastin fibers were not \u201cactivated\u201d beyond their normal levels and consequently additional synthesis of elastin was not required, explaining why elastin content remained unchanged between fresh and stretched leaflets.\nThe above findings reinforce the fact that valve cells require mechanical stimulation to maintain a balance between synthesis and degradation of ECM components. This occurred without undue damage to the normal tri-layered structure of the aortic valve leaflet as the relative thicknesses of the fibrosa, spongiosa, and ventricularis were maintained (Fig.\u00a05a).\nEffect on Cell Phenotype\nValvular interstitial cells are mainly comprised of fibroblasts, myofibroblasts, and smooth muscle cells that exhibit different phenotypes depending on the microenvironment surrounding the cells. Expression of \u03b1-SMA was associated with the contractile phenotype of smooth muscle cells24 and myofibroblast-like cells,2 and hence was chosen as a marker for examining the interstitial cell phenotype in the present study. Studies have shown that the expression of \u03b1-SMA was decreased by changes in culture conditions and mechanical and biochemical stimulation in cultured vascular cells.21,22 Studies in our laboratory have shown that \u03b1-SMA expression decreased in response to isolated effects of pressure and shear forces.13,29,31 Similar behavior was observed in valvular interstitial cells in 3D static gel culture.5\nThe current study revealed via IHC (Fig.\u00a05c) and Western blotting (Fig.\u00a06) that circumferential cyclic stretch increases the expression of \u03b1-SMA in aortic valve leaflets. It is therefore speculated that cyclic stretch is a regulator of the contractile and fibrotic phenotypes of aortic valve interstitial cells. A reversible equilibrium between F-actin depolymerization and G-actin polymerization exists in the cells of aortic valves. The contractile motion of the leaflets due to stretch may have caused a shift in this equilibrium towards polymerization of G-actin to form F-actin fibrils, which is detected as \u03b1-SMA immunopositive cells.\nThese findings also lead to the possibility that in the normal hemodynamic environment, the up-regulating effect of stretch on \u03b1-SMA expression may be counterbalanced by the down-regulating effects on \u03b1-SMA expression of pressure and shear stress. This provides an additional possible explanation for the fact that aortic valves cultured in normal hemodynamic forces maintain a constant \u03b1-SMA expression.13\nAnother interesting observation was that \u03b1-SMA immunopositive cells occurred preferentially in the ventricularis of the stretched leaflet. This is in contrast to fresh and static leaflets which exhibited an apparent random distribution of \u03b1-SMA through all the three layers of the valve leaflet. This may provide insights into specific regions or \u201chot spots\u201d on the aortic valve leaflet where there is a focal response to mechanical forces. In a study by Simmons et al.,20 spatial heterogeneity of aortic valve endothelial phenotypes was speculated to contribute to the focal susceptibility for calcific aortic valve lesion development, implicating the endothelium as a regulator of valvular calcification. The current study shows the potential focal and side-specific response of the interstitial cells of the aortic valve to mechanical forces. In addition, \u03b1-SMA is a known marker for mesenchymal cells,3, 9 and the expression of \u03b1-SMA could also indicate regions of endothelial cell to mesenchymal cell transdifferentiation. It is clear that further side-specific study is needed in this area as it may provide insights into how valves sense and transmit mechanical forces.\nConclusion\nThe current study demonstrated that circumferential cyclic stretch altered the extracellular matrix remodeling activity of native porcine aortic valve leaflets. These results combined with results from previous ex vivo studies show that the changes in aortic valve leaflet biology depend intimately on the influence of local hemodynamic environmental factors. Further research is needed to understand the mechanistic pathways behind these biological changes. The knowledge gleaned from such studies will ultimately allow for elucidation of mechanisms of aortic valve pathogenesis leading to valve failure.","keyphrases":["aortic valve leaflets","circumferential stretch","contractile phenotype","uniaxial stretch bioreactor"],"prmu":["P","P","P","R"]}
{"id":"Virchows_Arch-3-1-2151131","title":"MicroRNA\u2014implications for cancer\n","text":"MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression post-transcriptionally. After the discovery of the first miRNA in the roundworm Caenorhabditis elegans, these short regulatory RNAs have been found to be an abundant class of RNAs in plants, animals, and DNA viruses. About 3% of human genes encode for miRNAs, and up to 30% of human protein coding genes may be regulated by miRNAs. MicroRNAs play a key role in diverse biological processes, including development, cell proliferation, differentiation, and apoptosis. Accordingly, altered miRNA expression is likely to contribute to human disease, including cancer. This review will summarize the emerging knowledge of the connections between human miRNA biology and different aspects of carcinogenesis. Various techniques available to investigate miRNAs will also be discussed.\nThe \u201csmall RNA revolution\u201d\nSmall ribonucleic acid (RNA) can act as a specific regulator of gene expression. This discovery has been an exciting breakthrough in Biological Sciences of the past decade, culminating in last year\u2019s Nobel Prize in Physiology or Medicine awarded to Andrew Fire and Craig Mello. Building on previous work mainly in plants [50], Fire et al. [23] discovered that exogenous double-stranded RNA can be used to specifically interfere with gene function. This phenomenon was called RNA interference (RNAi). They also speculated that organisms might use double-stranded RNA naturally as a way of silencing genes. It was then shown that RNA interference was mediated by 22 nucleotide single-stranded RNAs termed small interfering RNAs (siRNAs) derived from the longer double-stranded RNA precursors [87]. The small interfering RNAs were found to repress genes by eliminating the corresponding messenger RNA transcripts, and thus, preventing protein synthesis.\nOver the following years, many new small functional RNAs have been found. RNA is usually thought of as messenger RNA that serves as the template for translation of genes into proteins. In contrast, functional or non-coding RNA molecules are transcribed from a DNA sequence, but not translated into protein. The encoding DNA sequence is often referred to as an RNA gene. Functional RNA genes in the human genome include transfer RNA (tRNA), ribosomal RNA (rRNA), and various other small non-coding RNAs. Several hundred genes in our genome encode small functional RNA molecules collectively called microRNAs (miRNAs). Precursors of these miRNA molecules form structures of double-stranded RNA that can activate the RNA interference machinery. MicroRNAs downregulate gene expression either by degradation of messenger RNA through the RNA interference pathway or by inhibiting protein translation.\nThe first miRNA was discovered in 1993 by Victor Ambros and colleagues Rosalind Lee and Rhonda Feinbaum [42]. A genetic screen in the roundworm Caenorhabditis elegans, a millimeter-long animal used as a model organism in biological research, identified genes involved in developmental timing [42]. Surprisingly, one of the genes, termed lin-4, did not encode a protein but instead a novel 22-nucleotide small RNA. Seven years later, Reinhart et al. [70] discovered a second 22-nucleotide small RNA of this type, let-7, a gene also involved in C. elegans developmental timing. The lin-4 and let-7 small regulatory RNAs soon became very exciting for two reasons. Firstly, homologs of the let-7 gene were identified in other animals including humans [65]. The conservation of let-7 across species suggested an important and fundamental biological role for this small RNA. Secondly, the mechanism of RNA interference (RNAi) was discovered at that time, and it became clear that miRNA and RNAi pathways were intricately linked and shared common components. Within the following year, more than 100 additional small regulatory RNAs similar to lin-4 and let-7 were identified in worms, the fruit fly Drosophila, and in humans [38, 40, 41]. These small non-coding RNAs were named microRNAs (miRNAs) [38, 40, 41].\nSubsequently, many more short regulatory RNAs were identified in almost all multicellular organisms, including flowering plants, worms, flies, fish, frogs, mammals [38, 40, 41, 48, 71], and in single cellular algae and DNA viruses [66, 75]. To date, more than 500 human miRNAs have been experimentally identified. Computational predictions of miRNA targets suggest that up to 30% of human protein coding genes may be regulated by miRNAs [46, 68]. This makes miRNAs one of the most abundant classes of regulatory genes in humans. MicroRNAs are now perceived as a key layer of post-transcriptional control within the networks of gene regulation.\nMicroRNAs are sequentially processed from longer precursor molecules that are encoded by the miRNA genes [1] (Fig.\u00a01). MiRNA genes are referred to by the same name (termed mir) written in italics to distinguish them from the corresponding mature miRNA (termed miR) followed by a number, e.g., mir-1 or miR-1. The encoding DNA sequence is much longer than the mature miRNA. Two ribonuclease enzymes, Drosha and Dicer, subsequently process the primary transcripts (or pri-miRNA) to generate mature miRNAs. The primary transcripts contain one or more stem-loop structures of about 70 bases. Stem-loops are double-stranded RNA structures consisting of a nucleotide sequence that can fold back on itself to form a double helix with a region of imperfect base pairing that forms an open loop at the end (Fig.\u00a01a). The ribonuclease Drosha excises the stem-loop structure to form the precursor miRNA (or pre-miRNA) [43]. After export into the cytoplasm, the pre-miRNA is cleaved by the ribonuclease Dicer to generate a short RNA duplex [6, 28]. After untwisting, one RNA strand becomes the mature single-stranded miRNA, while the complementary strand, termed miRNA*, is usually rapidly degraded (Fig.\u00a01b).\nFig.\u00a01The biogenesis and function of miRNAs. a Primary miRNAs (pri-miRNA) are transcribed from longer encoding DNA sequences (miRNA genes). The pri-miRNA contains one or more stem-loop structures of about 70 bases. In the nucleus, the ribonuclease enzyme Drosha excises the stem-loop structure to form the precursor miRNA (pre-miRNA). b After export into the cytoplasm, the pre-miRNA is cleaved by the ribonuclease Dicer to generate a short RNA duplex (miRNA:miRNA*). c The mature single-stranded miRNA is incorporated into the RNA-induced silencing complex (RISC), while the complementary strand (miRNA*) is usually rapidly degraded. The miRNA incorporated into the silencing complex can bind to the target messenger RNA by base pairing, causing inhibition of protein translation and\/or degradation of the target messenger RNA\nMicroRNAs recognize their targets based on sequence complementarity [10]. The mature miRNA is partially complementary to one or more messenger RNAs. In humans, the complementary sites are usually within the 3\u2032-untranslated region of the target messenger RNA. To become effective, the mature miRNA forms a complex with proteins, termed the RNA-induced silencing complex. The miRNA incorporated into the silencing complex can bind to the target messenger RNA by base pairing. This base pairing subsequently causes inhibition of protein translation and\/or degradation of the messenger RNA (Fig.\u00a01c). The potential mechanisms underlying this process were recently reviewed [30, 67]. Protein levels of the target gene are consequently reduced, whereas messenger RNA levels may or may not be decreased. In humans, miRNAs mainly inhibit protein translation of their target genes and only infrequently cause degradation or cleavage of the messenger RNA [1].\nThe biological role and in vivo functions of most mammalian miRNAs are still poorly understood. In invertebrates, miRNAs regulate developmental timing (e.g., lin-4), neuronal differentiation, cell proliferation, growth control, and programmed cell death [9, 33, 42]. In mammals, miRNAs have been found to play a role in embryogenesis and stem cell maintenance [7], hematopoietic cell differentiation [17], and brain development [59, 60]. To date, knowledge of human miRNAs has been primarily descriptive. MicroRNA expression has been found to be deregulated in a wide range of human diseases including cancer. However, it remains uncertain whether altered miRNA expression is a cause or consequence of pathological processes. The underlying mechanisms of why and how miRNAs become deregulated are largely unknown. Although bioinformatics approaches can predict thousands of genes that are potentially targeted and regulated by miRNAs based on sequence complementarity, only very few miRNA target genes have been functionally validated. Our group is currently investigating the role of miRNAs in mammary gland development and breast cancer pathogenesis. A comparison of miRNA and gene expression identified miRNAs that classify molecular breast cancer subtypes [8]. As cancer is ultimately a consequence of disordered gene expression, miRNAs have been suggested to contribute to the development of cancer [11]. This review will focus on the connection between human miRNA biology and different aspects of carcinogenesis. Various techniques available to investigate miRNAs will also be discussed.\nMicroRNAs and cancer\nThree important observations early in the history of miRNAs suggested a potential role in human cancer. Firstly, the earliest miRNAs discovered in the roundworm C. elegans and the fruit fly Drosophila were shown to control cell proliferation and apoptosis [9, 42]. Their deregulation may therefore contribute to proliferative diseases such as cancer. Secondly, when human miRNAs were discovered, it was noticed that many miRNA genes were located at fragile sites in the genome or regions that are commonly amplified or deleted in human cancer [14]. Thirdly, malignant tumors and tumor cell lines were found to have widespread deregulated miRNA expression compared to normal tissues [12, 24, 52]. The question remained whether the altered miRNA expression observed in cancer is a cause or consequence of malignant transformation.\nMicroRNAs as causal cancer genes at genomic breakpoints\nFive years ago, the first direct evidence for an involvement of miRNAs in cancer was reported [13]. Calin et al. studied a well-known deletion on chromosome 13, which is the most frequent chromosomal abnormality in chronic lymphocytic leukemia (CLL). This deletion had long been suspected to contribute to leukemogenesis. However, extensive studies had failed to identify a causal gene. Calin et al. [13] found that two miRNA genes, mir-15 and mir-16, were located within this 30-kb deletion. They subsequently analyzed the expression of miR-15 and miR-16 in blood samples from patients with CLL. Both miRNAs were absent or downregulated in the majority (68%) of cases when compared to normal tissue or lymphocytes. This finding suggested that these two miRNAs were causally involved in the pathogenesis of chronic lymphocytic leukemia.\nIn 2005, three reports provided the first mechanistic insight into how miRNAs might contribute to carcinogenesis. Two independent studies described the relationship between a miRNA cluster, mir-17-92, and the Myc oncogenic pathway [27, 63]. A third report demonstrated an interaction between let-7 miRNA and the RAS proto-oncogene [32].\nThe mir-17-92 cluster\u2014small RNAs with oncogenic potential\nA cluster of six miRNAs, the mir-17-92 cluster, was found to be located within a region on chromosome 13 that is commonly amplified in human B-cell lymphomas [64]. He et al. [27] demonstrated that the miRNAs from the mir-17-92 cluster were overexpressed in lymphoma cell lines carrying this amplification, and expression levels correlated with gene copy number of the mir-17-92 locus [27]. Further, the miR-17-92 primary transcript was found to be overexpressed in tumor samples from lymphoma patients. To test their hypothesis that mir-17-92 actively contributes to lymphomagenesis, the authors took advantage of a mouse model of human B-cell lymphoma. These mice develop lymphomas due to an overexpression of the Myc oncogene. The Myc oncogene encodes the transcription factor c-Myc that regulates cell proliferation, growth, and apoptosis, and overexpression of c-Myc is common in cancer. He et al. [27] demonstrated that additional expression of the mir-17-92 cluster accelerated c-Myc-induced tumorigenesis in mice. The authors therefore suggested that mir-17-92 was the first potential non-coding oncogene, referred to as oncomir-1.\nThe cellular function of miR-17-92 was not identified in these experiments. Nevertheless, the pathology of the tumors indicated lower rates of apoptosis as compared to tumors with Myc overexpression alone. Three recent studies contributed towards our understanding of the oncogenic potential of miR-17-92. Two reports demonstrated an anti-apoptotic effect of miR-17-92 through various pathways that promote cell proliferation and growth [55, 76]. A third study identified mir-17-92 as a mediator of angiogenesis in tumors induced by the oncogene c-Myc [19].\nO\u2019Donnell et al. [63] independently identified the same cluster of miRNAs, mir-17-92, to be regulated by the transcription factor c-Myc. The transcription factor Myc induces expression of E2F1 growth factor. The mir-17-92 cluster which is also induced by c-Myc does, in contrast, inhibit E2F1 expression. The authors therefore suggested a novel regulatory mechanism by which c-Myc fine-tunes gene expression by activating the transcription of target genes and by simultaneously inducing inhibitory miRNAs that reduce their translation.\nThe example of the mir-17-92 cluster highlights that a distinction between oncogenic and tumor suppressor miRNAs is likely to be an oversimplification. The same miRNAs may have oncogenic or tumor suppressor activity depending on the context and the cell type they are expressed in. A single miRNA may regulate various unrelated target genes and thereby control opposing activities such as cellular proliferation and apoptosis. The ultimate function of a miRNA may depend on the tissue type they are expressed in and what target genes are present.\nMicroRNAs with tumor suppressor potential\nThe let-7 family of miRNAs was the first group of miRNAs shown to regulate expression of a proto-oncogene, the RAS protein. RAS proteins are membrane-associated signaling proteins that regulate cell growth and differentiation. A miRNA that controls expression of these potentially oncogenic proteins would be predicted to possess tumor suppressor activity.\nMutations in the RAS oncogene are present in approximately 15\u201330% of all human cancers, and overexpression of the RAS oncogene is common in lung cancer. Johnson et al. [32] showed that overexpression of RAS protein in lung cancer tissue correlated with reduced expression of let-7 miRNA. They experimentally confirmed that let-7 can inhibit RAS expression in human cancer cell lines. Loss or reduction of let-7 in lung cancer leads to RAS overexpression, thus, promoting cellular growth and contributing to tumorigenesis. The authors therefore suggested that let-7 acts as tumor suppressor [32]. Another group independently reported reduced expression of let-7 in lung cancers and found that this correlated with a poor prognosis [77].\nGlobal loss of miRNA expression in cancer\nA global decrease in miRNA levels has been observed in human cancers, indicating that small RNAs may have an intrinsic function in tumor suppression. Lu et al. [52] were the first to show that the expression levels of many miRNAs were significantly reduced in cancers compared to the corresponding normal tissues. They analyzed a total of 217 human and mouse miRNAs across 334 human cancers, cancer cell lines, and normal tissues. Cancers had significantly reduced global miRNA expression. Poorly differentiated tumors had lower miRNA levels compared with more-differentiated tumors. The authors hypothesized that miRNAs can function to drive terminal differentiation and prevent cell division. Global changes in miRNA expression may reflect the degree of cell differentiation [52]. A recent study examined the expression of 241 human miRNAs in a comprehensive panel of human cancer cell lines, the NCI-60 panel, and in normal tissues [24]. The authors confirmed the finding that most miRNAs were expressed at lower levels in human tumor-derived cell lines compared with the corresponding normal tissue [24].\nUntil recently, considerable uncertainty remained as to whether the altered miRNA expression observed in cancer was a cause or consequence of malignant transformation. Earlier this year, a study by Kumar et al. [37] proved for the first time that widespread reduction in miRNA expression does, indeed, promote tumorigenesis. The authors globally reduced the production of mature miRNAs through a knockdown of the miRNA-processing enzymes Drosha and Dicer in cell lines. The mouse and human cancer cells consequently showed decreased steady-state miRNA levels. These cells with global miRNA loss showed enhanced cellular growth in vitro [37]. When injected into nude mice, these cells generated faster growing and more invasive tumors compared to controls. To assess the effect of global miRNA loss in vivo, the authors deleted the miRNA-processing enzyme Dicer in a mouse model of lung cancer. The Dicer mutant mice who had impaired miRNA processing developed an increased tumor burden, with an expansion in tumor number and tumor size, as well as tumors which were less well differentiated compared to controls [37]. Overall, these data clearly suggest that global miRNA loss enhances tumorigenesis. Kumar et al. demonstrated that loss of miRNAs leads to upregulation of proto-oncogenes such as RAS and c-Myc. However, it remains to be elucidated whether loss of all miRNAs is necessary or whether reduction of a subgroup of key tumor suppressor miRNAs, such as let-7, is the event that promotes malignant transformation.\nMicroRNAs in the p53 tumor suppressor network\nTranscriptional networks are often deregulated in cancer cells and may lead to altered transcription of miRNA genes. Two recent studies identified a miRNA, miR-34, to be regulated by the p53 transcription factor [16, 26]. The p53 protein, also called \u201cthe guardian of the genome\u201d, regulates the cellular response to stress and cancer-initiating events such as DNA damage. He et al. [26] found that a miRNA, miR-34, is directly activated by the transcription factor p53 after DNA damage. Expression of miR-34 induces cell cycle arrest and thereby acts together with other effectors of the p53 tumor suppressor network to inhibit inappropriate cell proliferation. Another group independently demonstrated that miR-34 is upregulated by p53 upon DNA damage and promotes apoptosis [16].\nTogether, these data indicate that altered expression of miRNAs is not simply a secondary event that reflects the less differentiated state of cancer cells. In contrast, at least in some cases, miRNA expression is specifically driven by tumor suppressors and oncogenes.\nMicroRNAs with a role in tumor invasion and metastasis\nTranscriptional networks may drive miRNA expression in cancers. Recent work from Ma et al. [54] suggested a model by which a pleiotropic transcription factor, Twist, induces expression of a specific miRNA, which suppresses its direct target and in turn activates a pro-metastatic gene, leading to tumor cell invasion and metastasis. The expression of miR-10b induced by the transcription factor Twist promoted cell migration and invasion in mouse and human breast cancer cells. Furthermore, the expression level of miR-10b in primary human breast carcinomas correlated with clinical progression [54]. These findings, if confirmed, suggest that specific miRNAs may have a role beyond the tumor-initiating event and directly participate in tumor progression and metastasis.\nRegulation of miRNAs in cancer\u2014who regulates the regulators?\nIn few cases, the underlying cause of miRNA deregulation in cancer is clear. As discussed above, the overexpression of miR-17-92 correlates with amplification of its gene locus [27]. Similarly, decreased expression of miR-15 and miR-16 is associated with a corresponding chromosomal deletion [13].\nTranscriptional or epigenetic regulation of miRNAs has been recently reported [53, 73]. The transcription of a miRNA gene, mir-124a, was shown to be inactivated by hypermethylation of its promoter in various human tumors. This process of epigenetic silencing is a well-known mechanism to inactivate protein-coding genes in cancer cells and may similarly apply to miRNAs. The miRNA gene mir-127 is usually expressed in normal cells but not in cancer cells. Saito et al. [73] demonstrated that miR-127 was highly induced in cultured human cancer cells after treatment with demethylating drugs, suggesting that it is subject to epigenetic silencing through promoter hypermethylation.\nA novel mechanism of miRNA regulation was suggested by Mayr et al. [56] and Lee and Dutta [44]. They demonstrated that miRNA function could be regulated through loss of miRNA binding sites in the target gene. Both groups independently demonstrated that chromosomal translocations in a known oncogene, high mobility group A2 (Hmga2), led to loss of the let-7 miRNA binding sites in its messenger RNA. Disrupted repression of Hmga2 by let-7 promoted oncogenic transformation and growth in mammalian cells. These two studies provide the first evidence that disrupting the interaction of a single miRNA and its target can produce an abnormal phenotype in mammalian cells [44, 56].\nIn addition, there is evidence that miRNAs are regulated indirectly through control of their processing enzymes. Thomson et al. [81] showed that a downregulation of miRNAs in human cancer was not associated with reduced levels of the primary miRNA transcripts. The authors therefore suggested regulation of miRNAs during subsequent processing steps, e.g., through altered function of the enzyme Drosha [81].\nMicroRNA profiling\u2014implications for cancer diagnosis\nLu et al. [52] asked the question whether global miRNA expression profiles could classify human cancer. MicroRNA expression profiles clearly differentiated human cancers according to their developmental origin.\nCancers of epithelial and hematopoietic origin had distinct miRNA profiles. A subgroup of gastrointestinal tumors, which arise from endoderm, was distinguished by miRNA expression patterns. Furthermore, tumors within a single cell lineage such as acute lymphoblastic leukemia were further differentiated according to their underlying genetic abnormality into BCR\/ABL-positive tumors, T-cell tumors, and those with MLL gene rearrangement [52]. Finally, the authors applied the miRNA expression profiles they had established to an independent series of 17 poorly differentiated tumors of unknown origin. Based on the differential expression of 217 miRNAs, a correct diagnosis could be established in 12 out of 17 of the tumors. In contrast, gene expression profiling based on \u223c16,000 messenger RNAs did not accurately classify the tumors [52]. This has potential important clinical implications. If miRNAs prove useful for clinical diagnosis, their key advantage might be their high stability. In contrast to most messenger RNAs, they are long-lived in vivo [49] and very stable in vitro [78], which might allow analysis of paraffin-embedded samples for routine diagnostic applications.\nMicroRNAs\u2014novel therapeutic targets?\nRegulatory RNAs may also have therapeutic applications by which disease-causing miRNAs could be antagonized or functional miRNAs restored. The most intuitive choice of molecules to correct altered miRNA\u2013messenger RNA interactions are RNA oligonucleotides. These oligonucleotides need to be chemically modified to allow for stability in serum and cellular uptake. Modified antisense oligonucleotides are already being developed to utilize the intrinsic RNAi pathway for delivery of gene therapy. If the delivery problem can be overcome, then miRNA therapies may also be possible.\nTwo studies have successfully applied 2\u2032-O-Methyl-modified antisense RNAs to inhibit miRNA function in cultured cells [29, 57]. Recent work by Krutzfeldt et al. [36] demonstrated that modified cholesterol-conjugated antisense RNAs designated \u201cantagomirs\u201d could effectively inhibit miRNA function in vivo in the adult mouse. The authors applied three daily intravenous injections of antagomirs and achieved effective inhibition of four miRNAs over a period of weeks in most tissues except brain [36]. A novel approach was recently reported by Ebert et al. [21]. They developed miRNA inhibitors that can be transiently expressed in cultured mammalian cells. These competitive inhibitors termed \u201cmiRNA sponges\u201d derepressed miRNA targets at least as strongly as chemically modified antisense oligonucleotides [21]. A different approach was taken by Tsuda et al. [83]. The authors designed synthetic miRNAs to target overexpressed tumor proteins, such as HER-2 protein. A synthetic miRNA targeting HER-2 messenger RNA successfully inhibited HER-2 protein expression in ovarian cancer cells [83]. Together, these studies hold some promise of miRNAs as future therapeutic targets.\nOne limitation of antisense RNA therapies is the restricted number of cells that can be targeted. Any approach to knock down a particular miRNA with antisense oligonucleotides will only result in partial knockdown. This may represent a limitation for cancer therapies. It remains to be seen whether indirectly mediated bystander effects on cancer cells that have not been directly targeted may partly overcome this limitation. In contrast, a partial effect on function may be of therapeutic value in neurodegenerative diseases, such as Parkinson\u2019s or Alzheimer\u2019s disease. A partial restoration of dopamine production by antisense therapy might result in a significant clinical improvement in Parkinson patients. Similarly, a partial reduction of the disease-causing proteins in Alzheimer\u2019s disease may lead to a clinical improvement and might be achievable by RNA based or miRNA gene therapy.\nTechniques and approaches to study miRNAs\nAll known miRNAs are registered in a public web-based registry, the \u201cmiRBase\u201d database that provides up-to-date information on all published miRNAs [25]. Novel miRNA genes can be discovered by bioinformatics approaches searching for evolutionary conserved stem-loop structures in the genome (reviewed in [3, 5]). Experimentally, miRNAs are discovered by cloning all small RNAs from a certain tissue type or developmental stage and subsequent sequencing to identify the subgroup of small RNAs that fulfill the criteria for miRNAs [38, 51]. Both computational and experimental approaches indicate that many more miRNAs are likely to be identified [4, 5], which is reflected by the rapidly increasing number of annotated miRNAs which increased from less than 300 to more than 4,000 over the past 4\u00a0years [58].\nMicroRNA expression studies\nNorthern blot analysis is a well-established technique for studying messenger RNA expression and was soon adapted to detect miRNAs in cells or tissues [42, 84]. Subsequently, conventional DNA microarray technology was modified to form miRNA microarrays, allowing for the detection of multiple miRNAs simultaneously across various samples [15, 60, 62, 82].\nLu et al. [52] developed a novel microarray strategy to improve probe specificity, which is critical due to the short nature of mature miRNAs. They performed hybridization in solution using polystyrene capture beads that are coupled to oligonucleotide probes complementary to the miRNAs of interest. The solution hybrids are then analyzed using a multicolor flow cytometer measuring bead color, denoting miRNA identity, and labeling intensity, denoting miRNA abundance [52].\nIn parallel to microarray platforms, commercial assays for quantitative reverse transcriptase polymerase chain reaction (RT-PCR) have become available. These allow for the analysis of miRNAs in small tissue samples or even single cells [79], as well as validation of microarray data. In addition to mature miRNAs, these quantitative RT-PCR assays can be applied to analyze miRNA precursors and primary transcripts [31]. In situ hybridization for the detection of mature miRNAs has recently become possible by using special high-affinity locked nucleic acid (LNA)-modified DNA oligonucleotide probes and holds promise for the application on human formalin-fixed and paraffin embedded tissue [35, 61].\nFunctional characterization of miRNAs\nVarious strategies have been used to investigate the function of specific miRNAs. In worms and flies, loss-of-function mutants for specific miRNAs or miRNA families allow us to draw conclusions regarding possible physiological functions of miRNAs from the resulting abnormal phenotype [34, 39, 86]. The knockdown of miRNAs or pre-miRNAs using modified antisense oligonucleotides has proven particularly useful in cell lines [29, 45, 57]. LNA-modified antisense oligonucleotides have been successfully utilized to knock down specific miRNAs in cultured cells [22]. This approach allowed identification of a crucial role for a miRNA, miR-223, in granulocytic differentiation [22]. In addition, the modified antisense RNAs (antagomirs) described by Krutzfeldt et al. [36], which inhibit miRNA function in the adult mouse, may provide a potential research tool to study miRNA function in vivo.\nIn mammals, induced defects in miRNA biogenesis are a useful tool for investigating the biological roles of miRNAs, as loss-of-function mutants are not available for most miRNA genes. Dicer knockout mouse models have revealed essential roles for miRNAs in murine organogenesis [88]. A recent study utilized a combined knockdown of the miRNA-processing enzymes Drosha, Dicer1, and DGCR8 to study the consequences of a global decrease in mature miRNAs in cancer cell lines and in a mouse model for lung cancer [37].\nEarlier this year, four independent groups have, for the first time, deleted genes for single miRNAs in mice [72, 80, 85, 88]. Two of the groups deleted the same DNA sequence for mir-155 and described severe immune defects [72, 80]. Mice lacking miR-155 showed impaired function of B and T lymphocytes and dendritic cells [72]. In particular, T helper cell differentiation and the germinal center reaction to produce a T-cell-dependent antibody response were defective [80]. Together, these two studies demonstrated a key role for miR-155 in normal immune function. The two other groups deleted different miRNAs, miR-1-2 and miR-208, and reported cardiac defects. Mice lacking miR-208 showed inadequate cardiac growth in response to stress [85], while mice lacking miR-1-2 had defects in cardiac morphogenesis and electrical conduction [88].\nMicroRNA target sites\nA validated biochemical strategy for identifying miRNA targets would be highly desirable. Two groups have recently reported promising approaches to experimentally identify miRNA targets. Both approaches apply biochemical methods to purify the effector complexes of miRNAs associated with proteins and bound messenger RNA targets [2, 20].\nAn increasing number of sophisticated bioinformatics approaches are being developed to predict putative miRNA target genes [3, 10, 69, 74]. This is based on the fact that miRNA target recognition is at least partly based on simple sequence complementarity. Interestingly, exact base pairing between miRNAs and their targets commonly appears to be required only in the first six to eight bases from the 5\u2032 end of the miRNA. The short nature of this designated \u201cseed region\u201d allows a single miRNA to act on up to a hundred different target sites, and all human miRNAs together may regulate up to one third of protein coding genes [10, 47]. A different approach to discovering miRNA target genes is to knock out or overexpress a particular miRNA and use conventional microarrays to identify genes that show changes in expression. This approach is based on the observation that some miRNAs can also downregulate messenger RNA levels in addition to downregulating protein levels of their target genes [49]. Experimental validation of miRNA target sites has been limited to date. A common approach has been to express a miRNA in vivo while simultaneously expressing and monitoring the target messenger RNA linked to a reporter gene, i.e., Luciferase [10, 18, 55, 56, 76]. The fact that a single miRNA can regulate multiple targets and a particular target may be regulated by various miRNAs suggests a highly complex network of miRNA-target interactions, which is only beginning to be unraveled.\nConclusions\nOver recent years, miRNAs have emerged as major players in the complex networks of gene regulation and have been implicated in various aspects of human disease. Only 5\u00a0years after the first study reported a direct involvement of miRNAs in cancer, these small RNAs have already significantly improved our understanding of carcinogenesis. In addition to protein-coding oncogenes and tumor suppressor genes, we will have to take into account miRNAs and their regulatory networks if we aim to understand the complex processes underlying malignant transformation.","keyphrases":["microrna","cancer","mirna","development","pathogenesis"],"prmu":["P","P","P","P","P"]}
{"id":"J_Chem_Ecol-4-1-2292484","title":"Increased Terpenoid Accumulation in Cotton (Gossypium hirsutum) Foliage is a General Wound Response\n","text":"The subepidermal pigment glands of cotton accumulate a variety of terpenoid products, including monoterpenes, sesquiterpenes, and terpenoid aldehydes that can act as feeding deterrents against a number of insect herbivore species. We compared the effect of herbivory by Spodoptera littoralis caterpillars, mechanical damage by a fabric pattern wheel, and the application of jasmonic acid on levels of the major representatives of the three structural classes of terpenoids in the leaf foliage of 4-week-old Gossypium hirsutum plants. Terpenoid levels increased successively from control to mechanical damage, herbivory, and jasmonic acid treatments, with E-\u03b2-ocimene and heliocide H1 and H4 showing the highest increases, up to 15-fold. Herbivory or mechanical damage to older leaves led to terpenoid increases in younger leaves. Leaf-by-leaf analysis of terpenes and gland density revealed that higher levels of terpenoids were achieved by two mechanisms: (1) increased filling of existing glands with terpenoids and (2) the production of additional glands, which were found to be dependent on damage intensity. As the relative response of individual terpenoids did not differ substantially among herbivore, mechanical damage, and jasmonic acid treatments, the induction of terpenoids in cotton foliage appears to represent a non-specific wound response mediated by jasmonic acid.\nIntroduction\nPlants produce a broad range of defense chemicals that act as deterrents or toxins against herbivores and pathogens. Several different strategies have evolved concerning the deployment of such compounds. When the synthesis of defense chemicals occurs solely after initial attack, this reduces metabolic costs (Gershenzon 1994), as defenses are produced only when needed. Such induced defenses have ecological advantages (Agrawal and Karban 1999) and might be a useful strategy for plants that are sporadically attacked. However, plants that risk frequent and heavy damage may be better protected by investing in constitutive defense compounds (Wittstock and Gershenzon 2002). The accumulation of constitutive defenses within plants usually conforms to expectations of the \u2018optimal defense theory\u2019 (ODT), which predicts that the highest protection level will be found in parts with the highest fitness value such as young tissues and reproductive organs (McKey 1979; Rhoades 1979). This has been shown for a variety of plant secondary metabolites from different classes such as alkaloids (Hartmann and Zimmer 1986; Baldwin 2001), glucosinolates (Porter et al. 1991. Brown et al. 2003), furanocoumarins (Berenbaum and Zangerl 1999), phenylpropanoids (Harborne 1991; Opitz and Schneider 2002), and terpenes (Gershenzon and Croteau 1991). Under abiotic or biotic stress, plants often exhibit induced responses by enhancing the accumulation of constitutive compounds in certain tissues that increases their deterrence or toxicity to enemies (Baldwin 1988; Wittstock and Gershenzon 2002; Vazquez-Flota et al. 2004). These changes also may be rationalized as a way for plants to reduce the costs of defense by producing high concentrations of constitutive defenses only when and where these are needed. However, more information is required to understand how the levels of constitutive defenses change under different stresses in plants on an organ-by-organ basis.\nAmong the vast number of defensive compounds in plants, terpenoids form the largest group with over 25,000 known structures (Buckingham 1994). Some of the best-studied examples for constitutive plant defenses are terpenoid volatiles such as monoterpenes (C10), which are considered defensive against herbivores and pathogens (Gershenzon and Dudareva 2007). Monoterpenes are frequently constituents of oils and resins that are accumulated in complex secretory structures such as glandular trichomes, secretory cavities, or resin ducts (Fahn 1979). Among the most comprehensively studied systems are monoterpenes in the glandular trichomes of peppermint (Gershenzon et al. 2000; McConkey et al. 2000). However, less is known about the accumulation of other classes of terpenes.\nCotton (Gossypium hirsutum) is particularly suitable for the study of terpenoid accumulation because a large variety of terpenes are produced constitutively in all photosynthetically active parts of the plant and stored in subepidermal glands. Beside monoterpenes, G. hirsutum produces sesquiterpenes (C15; Elzen et al. 1985) and terpenoid aldehydes, such as hemigossypolone (C15) and the heliocides H1 to H4 (C25; Fig.\u00a01; Hedin et al. 1992). All three classes of terpenoids in cotton are biosynthetically related (Stipanovic et al. 1977, 1978a; Davis and Essenberg 1995). The precursor of sesquiterpenes, farnesyl diphosphate, is formed by addition of a C5 isopentenyl diphosphate unit to geranyl diphosphate, the precursor of all monoterpenes. The C25 heliocides are formed from Diels\u2013Alder-type reactions of the C15 hemigossypolone (derived from \u03b4-cadinene) to the monoterpene myrcene for the formation of heliocides H2 and H3 or the monoterpene E-\u03b2-ocimene for the formation of heliocides H1 and H4 (Fig.\u00a02).\nFig.\u00a01Terpenoid products of three structural classes, which are accumulated in glands of the foliage of Gossypium hirsutum. 1 \u03b1-pinene, 2 (E)-\u03b2-ocimene, 3 myrcene, 4 (E)-\u03b2-caryophyllene, 5 \u03b3-bisabolene, 6 \u03b1-humulene, 7 hemigossypolone, 8 heliocide H1, 9 heliocide H2Fig.\u00a02Proposed scheme for the biosynthesis of heliocide H1 in cotton\nThe accumulation of terpenoid aldehydes in cotton leaves was shown to be increased after herbivory (Bezemer et al. 2004), and these compounds have been considered to function as feeding deterrents on generalist herbivores such as Spodoptera exigua (McAuslane et al. 1997). The heliocides specifically have been correlated with antifeedant effects and are locally and systematically induced in response to herbivore attack (Karban and Carey 1984; Croxford et al. 1989; Alborn et al. 1996). However, besides the terpenoid aldehydes, little information is available on the accumulation of other terpenes found in subepidermal glands. In studying the control of terpenoid accumulation in cotton, it is of interest to determine if higher concentrations arise from increases in gland number or from increases in the amount of terpenoids per gland (McAuslane et al. 1997). Increasing numbers of subepidermal glands in cotton leaves were observed after attacks of spider mites and caterpillars (McAuslane et al. 1997; Agrawal and Karban 2000).\nTo learn more about the control of terpenoid accumulation in cotton, we compared the response of plants to herbivore damage, mechanical damage, and the application of jasmonic acid (JA), a ubiquitous mediator of defense responses in the plant kingdom (Browse 2005). Differences in the reaction of plants to these treatments were investigated with respect to plant growth, gland production, and accumulation of major compounds from three classes of terpenoids: terpenoid aldehydes, monoterpenes, and sesquiterpenes at the level of the individual leaf. We also examined how the accumulation was influenced by the number of glands in the total foliage or the relative filling of glands with terpenoids.\nMethods and Material\nPlants G. hirsutum L. of a single variety, \u2018Deltapine acala 90\u2019 (Delta and Pine Company\u2014Hollandala, MS, USA), were reared in a growth chamber under high light intensity [1\u00a0mmol (m2)\u22121 s\u22121 of photosynthetically active radiation], a 15-h light period, 28\u00b0C\/23\u00b0C (day\/night), and 65% relative humidity. Plants were established in pots containing \u2018Seramis\u2019-clay granulate (Masterfood GmbH, Verden\/Germany), which were watered daily and fertilized weekly with a complete fertilizer [1\u00a0g\/l Flory 3 (N\/P\/K, 15:10:15), Euflor\/Germany]. Under these conditions, optimal growth and production of the various terpenoids was obtained.For all experiments, leaves were numbered sequentially such that number 1 refers to the first true leaf initiated right after the cotyledons (Fig.\u00a03). In this study, leaves 1\u20137 were investigated and collectively referred to as total foliage.\nFig.\u00a03Sketch of a Gossypium hirsutum plant at the end of experiment 1, approximately 1-month-old. Leaves are numbered according to the order of development. Grid pattern indicate leaves, which were damaged either mechanically or by caterpillars of Spodoptera littoralis\nHerbivores Eggs of the generalist herbivore, Spodoptera littoralis (Boisd.; Lepidoptera, Noctuidae), Egyptian cotton worm, were obtained from Syngenta (Basel, Switzerland). After hatching, larvae were reared in 2-l plastic boxes containing artificial diet, which consisted of 500\u00a0g of bean flour, 500\u00a0ml water, 9\u00a0g ascorbic acid, 5\u00a0g ethyl-4-benzoic acid, 0.6\u00a0ml \u03b1-tocopherol, 9\u00a0ml Mazola germ oil, 4\u00a0ml 3.7% formaldehyde, and 650\u00a0ml 7.5% agar solution. Fresh diet was applied every third day. Larvae were reared at constant 20\u00b0C, 50\u201360% relative humidity, and ambient light conditions. Two weeks after hatching, larvae reached the third instar and were used for the study.\nExperiment 1\nTreatment of Cotton Plants Three weeks after germination, when plants had developed four to five true leaves, the length of the midrib of leaf 4 was determined as a good measure for growth stage. Next, 24 plants at comparable growth stage were transferred into a second growth chamber (V\u00f6tsch, Germany) with abiotic conditions identical to the chamber where plants were reared. Here, plants were maintained separately in 250-ml plastic pots and watered twice a day to ensure adequate water supply. After 24\u00a0h, plants were divided into four treatment groups of six individuals each. Plants of one group were damaged mechanically three times on leaf 2 on the first day and three times on leaf 3 on the fourth day by using a fabric pattern wheel. In every case, leaves were scratched four times in parallel to the midrib (two times on each side). For a second group, three caterpillars of Spodoptera littoralis were caged on each plant on leaf 2 during the first day and on leaf 3 during the fourth day until larvae consumed at least one third of the available leaf material. Plants of the third group were watered with a 100-\u03bcM JA solution in water (Sigma) during the first day. JA concentrations in this range are commonly used to induce the biosynthesis of secondary products in plants (Koch et al. 1999; van Poecke and Dicke 2002). Afterward, this solution was replaced by pure tap water. A control group of plants was not treated at all. One week after the start of treatments, all true leaves were harvested separately, frozen in liquid nitrogen, and stored at \u221220\u00b0C until further analyses.\nFoliar Glands Just after removal, all leaves were scanned to calculate leaf areas by using the program ImageJ (Wayne Rasband, National Institutes of Health, USA). In this procedure, herbivore-damaged leaves were used as templates to reconstruct the areas of undamaged leaves. In addition, surface view pictures were taken of two circular 24-mm2 sections from the center of each leaf half with a stereomicroscope connected to a digital camera. In doing so, leaves were illuminated from below with a lamp to visualize filled subepidermal glands as pigmented spots. After the number of filled glands was counted, gland densities for 24-mm2 sections were calculated. Based on these sections, gland numbers and gland densities per leaf and per total foliage were extrapolated. These values were slightly underestimated because of higher gland densities at leaf edges (personal observations).\nAnalysis of Monoterpenes and Sesquiterpenes True leaves were separately analyzed for their content of constitutive terpenes. Depending on available material, 30\u2013100\u00a0mg of ground frozen leaf were extracted with 300\u2013500\u00a0\u03bcl pentane\/n-hexane 1:1. Additionally, 2\u00a0\u03bcg of nonylacetate were added as internal standard before extracts were shaken for 3\u00a0h at room temperature. After extracts were cooled to 4\u00b0C, they were filtered through cotton and Na2SO4 to remove remaining water. A subset of extracts was analyzed qualitatively by gas chromatography\u2013mass spectrometry (GC\u2013MS) on a Hewlett-Packard 6890 gas chromatograph (injector temperature 220\u00b0C; 1\u00a0\u03bcl splitless injection) coupled to a Hewlett-Packard 5973 quadrupole mass selective detector. Separations were performed on a DB-5MS column (30\u00a0m\u2009\u00d7\u20090.25\u00a0mm\u2009\u00d7\u20090.25\u00a0\u03bcm film; Agilent Technology) with 2\u00a0ml min\u22121 helium as the carrier gas. The following temperature gradient was used: 40\u00b0C for 3\u00a0min, increased to 90\u00b0C at 5\u00b0C\/min, further increased to 140\u00b0C at 40\u00b0C\/min, followed by an increase to 160\u00b0C at 4\u00b0C\/min, maintained at 160\u00b0C for 3\u00a0min, and a final heating to 300\u00b0C for 3\u00a0min. Mass spectrometry was performed with an ionization potential of 70 electron volts and a scan range of m\/z from 50 to 300. Compound identification was based on comparisons with mass spectra in the Wiley and National Institute of Standards and Technology (NIST) libraries or on direct comparison of mass spectra and retention times with available standards. For quantification, all samples were analyzed on a Hewlet-Packard 6890 gas chromatograph and a flame ionization detector (temperature 250\u00b0C). The separation procedure was identical to the method for GC\u2013MS analysis. Terpenes were quantified by using nonylacetate and corrected with response factors according to Scanlon and Willis (1985). Concentrations and levels of terpenes per subepidermal gland were calculated for single leaves and for total foliage. Eight individual monoterpenes and sesquiterpenes were analyzed, which represent more than 90% of the total terpenes in G. hirsutum.\nAnalysis of Terpenoid Aldehydes True leaves were separately analyzed for their content of terpenoid aldehydes according to Stipanovic et al. (1988). Depending on available material, 30\u2013100\u00a0mg of ground frozen material were extracted \u00d73 with 3\u00a0ml of ethyl acetate\/n-hexane (1:3) and 50\u00a0\u03bcl of 10% HCL. Extracts were shaken at 5\u00b0C for 3\u00a0h. Afterwards, extracts were removed from the leaf material, evaporated with nitrogen, and redissolved in 40% H2O and 60% solvent B (see below) from the following high-performance liquid chromatography (HPLC) procedure. Reversed phase HPLC analysis was performed on a Chromolith LC-18 column (Merck; 5\u00a0\u03bcm; 100\u2009\u00d7\u20094\u00a0mm) with single wavelength detection at 272\u00a0nm. The column was eluted with H2O + 0.05% trifluoracetic acid (solvent A) and EtOH\/MeOH\/MeCN\/EtOAc\/iPrOH, 26.6:7.4:40.5:6.1:19.4 (solvent B), with the following gradient: 50% B (0\u00a0min)\u201350% B (5\u00a0min)\u201361% B (5.1\u00a0min)\u201361% B (20\u00a0min). Flow rate was held constant at 2\u00a0ml min\u22121, and temperature was kept at 25\u00b0C. Five compounds, the hemigossypolone (C15) and the heliocides 1\u20134 (C25), were identified by their retention times according to the literature (Stipanovic et al. 1988) and additionally by liquid chromatography\u2013mass spectrometry (LC\u2013MS) measurements. Quantification was done by using standard curves of purified compounds. Concentrations and levels per subepidermal gland were calculated for single leaves and for the total foliage. Hemigossypolone and the four heliocides represent approximately 90% of the total terpenoid aldehyde content. As a sesquiterpene aldehyde, hemigossypolone was included in our analysis with other terpenoid aldehydes rather than with the sesquiterpenes. In comparison to other cotton varieties (Bezemer et al. 2004), the terpenoid aldehyde gossypol (C30) was found only in traces by LC\u2013MS and was not quantified.\nExperiment 2\nEighteen 2-wk-old plants at the growth stage of two true leaves were chosen and prepared for treatments in a way comparable to the first experiment. Plants were divided into three groups. Plants of one group were damaged mechanically nine times on leaves 1 and 2 during six consecutive days by using a mechanical fabric pattern wheel (procedure see experiment 1). For a second group, two caterpillars of Spodoptera littoralis were caged on each plant on one cotyledon during day\u00a01, on leaf 1 during day\u00a03, and on leaf 2 during day\u00a05 until larvae consumed at least one third of the available leaf material. A control group was not treated at all. One week after the start of treatments, leaf 4 from each plant was harvested. Leaf areas and gland number per leaf were determined according to experiment 1. Afterwards, leaves were frozen in liquid nitrogen and stored at \u221220\u00b0C until further analyses of mono- and sesquiterpenes. Levels of terpene classes were calculated based on the levels of eight individual compounds that represent more than 90% of the accumulated monoterpenes and sesquiterpenes in G. hirsutum.\nStatistics\u2014Experiment 1\nTotal Foliage The effects of mechanical damage, herbivory, and JA application on foliage area, levels of total monoterpenes, sesquiterpenes, and terpenoid aldehydes, as well as on levels of individual major terpenoids, were analyzed with analyses of variance. Data were tested for normality and equal variances. For normally distributed data, one-way analyses of variance (ANOVAs) were used. Differences between treatments and controls were tested for significance by using Dunn\u2019s post hoc tests. Data not normally distributed were analyzed by using Kruskall\u2013Wallis one-way ANOVAs on ranks and Dunn\u2019s post hoc tests. Analyses were performed with Sigma Stat 2.03. The effect of foliage area and treatment on the total number of glands was tested by analysis of codeviance. Generalized linear models (GLM) with the log link as link function were performed. To deal with overdispersion, a quasi-Poisson distribution was used in the models instead of Poisson distribution. The models were simplified by removing non-significant terms and by factor-level reduction (Crawley 2002). These analyses were performed in R, version 2.4.1. All data are presented as mean \u00b1 SE except when it is mentioned otherwise.\nSingle Leaves The effects of leaf position, treatment, and their interactions on levels of terpenoids per gland were tested by using nested two-way ANOVA. For this purpose, terpenoid data were root- or log-transformed to normalize them. For single leaf positions, the effects of the treatments on the leaf area were tested with one-way ANOVAs. The effect of leaf area and treatment on the total number of glands was tested by analysis of codeviance (GLM see above). To analyze the effect of leaf area and treatment on gland density, levels of terpenoids per gland and terpenoid concentrations per milligram fresh leaf material ANCOVAs were used. These analyses were performed in R, version 2.4.1. Changes in per gland concentrations of individual terpenoids at specific leaf positions after treatments were tested with Sigma Stat 2.03. For normally distributed data, one-way ANOVAs were used. Differences between treatments and controls were tested for significance by using Dunn\u2019s post hoc tests. Data not normally distributed were analyzed using Kruskall\u2013Wallis one-way ANOVAs on ranks and Dunn\u2019s post hoc tests.\nStatistics\u2014Experiment 2\nSingle Leaves The effect of the treatments on the areas of leaf 4 was tested with one-way ANOVA. The effect of leaf area and treatment on the total number of glands was tested by analysis of codeviance (GLM see above). To analyze the influence of leaf area and treatments on the accumulation of mono- and sesquiterpenes an ANCOVA was achieved. All analyses were performed in R, version 2.4.1.\nResults\nExperiment 1\nTerpenoid Accumulation in Total Cotton Foliage after Induction The accumulation of terpenoid classes among the four different treatments was first compared on the basis of total foliage (Fig.\u00a04). For monoterpenes, sesquiterpenes, and terpenoid aldehydes, there was a gradual increase in the following order: control, mechanical damage, herbivory, and JA treatment (ANOVA on ranks: monoterpenes, H\u2009\n=\u20099.420, P\u2009\n=\u20090.024; sesquiterpenes, H\u2009\n=\u200914.140, P\u2009\n=\u20090.003; terpenoid aldehydes, H\u2009\n=\u200919.760, P\u2009<\u20090.001). Levels of all three classes were elevated in herbivore-damaged (Dunn\u2019s tests: monoterpenes, Q\u2009\n=\u20092.572; sesquiterpenes, Q\u2009\n=\u20092.490; aldehydes, Q\u2009=\u20092.939; all P\u2009<\u20090.05) and JA-treated plants (Dunn\u2019s tests: monoterpenes, Q\u2009\n=\u20092.735; sesquiterpenes, Q\u2009\n=\u20093.674; aldehydes, Q\u2009\n=\u20094.164; all P\u2009\n<\u20090.05) in comparison to the controls.\nFig.\u00a04Mean (\u00b1SE, N\u2009=\u20096) levels of terpenoids in the total foliage of 4-week-old cotton plants, which were either damaged at leaves 2 and 3 mechanically (Mech), fed upon by larvae of Spodoptera littoralis (Herb), treated with jasmonic acid (JA), or left as untreated controls (Ctrl) 7\u00a0days previously. Asterisks indicate significant differences between treatment and control plants based on Kruskall\u2013Wallis one-way ANOVAs on ranks and Dunn\u2019s post hoc tests. *P\u2009<\u20090.05\nAll individual compounds showed the same pattern that was seen for total terpenoid accumulation: a gradual increase from control to mechanical damage, herbivory, and JA treatment. In 11 out of 13 compounds, this increase was significant (Table\u00a01) for herbivore and JA-treated plants compared to the control plants, while mechanically damaged plants showed tendencies for increased accumulation. Among monoterpenes, considerable differences in accumulation after treatment were shown. (E)-\u03b2-ocimene increased up to eightfold after both herbivory and JA treatment in comparison to controls, whereas myrcene levels slightly but significantly increased after herbivory and JA treatment, and the amounts of \u03b1- and \u03b2-pinene did not show significant differences between treatments and control. Little variation in the induction pattern occurred when comparing individual sesquiterpenes. Levels of all major sesquiterpenes, including \u03b2-caryophyllene, \u03b1-humulene, \u03b3-bisabolene, and \u03b2-bisabolol doubled after herbivory and were 2.0\u20132.6-fold higher after JA treatment in comparison to control plants. Among terpenoid aldehydes, considerable differences appeared. Whereas the amounts of hemigossypolone were doubled, levels of heliocides, especially H1 and H4, increased much more after herbivory and JA treatment compared to the controls. Heliocide H4, for example, showed up to a ninefold increase after herbivory and up to a 15-fold increase after JA treatment in comparison to control plants. For H1, these increases were approximately five- and tenfold, respectively. Meanwhile, the highest levels of H2 and H3 were found in the JA-treated plants, reaching up to 3.3-fold that of control levels.\nTable\u00a01Mean levels (\u00b1SD, N\u2009=\u20096) of terpenoid products in total foliage of 1-month-old G. hirsutum plants that were either mechanically damaged with a fabric pattern wheel, damaged by larvae of Spodoptera littoralis (herbivory), treated with jasmonic acid (JA), or left as untreated controlsCompoundANOVA (df\u2009=\u20093)Levels of terpenoids (mg)F\/HP ValueControlMechanical damageHerbivoryJAMonoterpenes\u03b1-Pinene7.2130.0651.11\u2009\u00b1\u20090.081.26\u2009\u00b1\u20090.111.31\u2009\u00b1\u20090.251.45\u2009\u00b1\u20090.44\u03b2-Pinene7.6470.0540.20\u2009\u00b1\u20090.020.23\u2009\u00b1\u20090.020.25\u2009\u00b1\u20090.050.26\u2009\u00b1\u20090.08Myrcene10.1670.0170.49\u2009\u00b1\u20090.060.68\u2009\u00b1\u20090.040.78**\u2009\u00b1\u20090.150.80**\u2009\u00b1\u20090.33(E)-\u03b2-Ocimene17.487<0.0010.06\u2009\u00b1\u20090.020.25\u2009\u00b1\u20090.060.47**\u2009\u00b1\u20090.110.49**\u2009\u00b1\u20090.26Sesquiterpenes(E)-\u03b2-Caryophyllene16.340<0.0010.50\u2009\u00b1\u20090.070.76\u2009\u00b1\u20090.120.95**\u2009\u00b1\u20090.211.32**\u2009\u00b1\u20090.50\u03b1-Humulene9.509<0.0010.14\u2009\u00b1\u20090.020.22\u2009\u00b1\u20090.030.27 *\u2009\u00b1\u20090.060.36 *\u2009\u00b1\u20090.13\u03b3-Bisabolene18.007<0.0010.23\u2009\u00b1\u20090.040.34\u2009\u00b1\u20090.060.45**\u2009\u00b1\u20090.100.60**\u2009\u00b1\u20090.19\u03b2-Bisabolol14.3400.0020.46\u2009\u00b1\u20090.050.59\u2009\u00b1\u20090.090.82**\u2009\u00b1\u20090.180.91**\u2009\u00b1\u20090.26Terpenoid aldehydesHemigossypolone15.2870.0021.59\u2009\u00b1\u20090.302.16\u2009\u00b1\u20090.443.14**\u2009\u00b1\u20090.403.10**\u2009\u00b1\u20090.83Heliocide 120.487<0.0010.59\u2009\u00b1\u20090.141.65\u2009\u00b1\u20090.373.35**\u2009\u00b1\u20091.115.95**\u2009\u00b1\u20091.93Heliocide 221.031<0.0010.97\u2009\u00b1\u20090.211.45\u2009\u00b1\u20090.421.83 *\u2009\u00b1\u20090.373.06 *\u2009\u00b1\u20090.74Heliocide 323.938<0.0010.37\u2009\u00b1\u20090.070.56\u2009\u00b1\u20090.150.70 *\u2009\u00b1\u20090.151.23 *\u2009\u00b1\u20090.30Heliocide 420.247<0.0010.17\u2009\u00b1\u20090.080.59\u2009\u00b1\u20090.251.47**\u2009\u00b1\u20090.392.60**\u2009\u00b1\u20090.78The effect of treatment on terpenoid levels were tested using one-way ANOVAs. For normally distributed data, one-way ANOVAs were performed (F values). For not normally distributed data, Kruskall\u2013Wallis one-way ANOVAs on ranks were performed (H values).*P\u2009<\u20090.05, significant differences from the control according to Bonferoni post hoc tests**P\u2009<\u20090.05, significant differences from the control according to Dunn\u2019s post hoc tests\nGrowth and Subepidermal Gland Production of Cotton Foliage ANOVA showed an effect of the treatments on the total leaf area (F\u2009=\u200910.44, P\u2009<\u20090.001). Whereas controls, mechanically and herbivore-damaged plants were highly comparable in size (mean, 496\u2013513\u00a0cm2), JA-treated plants (mean\u2009=\u2009306\u00a0cm2) showed a reduction in the total area of foliage down to 60% that of the controls (Bonferoni post hoc test. P\u2009<\u20090.05).\nBecause of significantly smaller leaves in the JA-treated plants, the effect of treatment on the production of subepidermal glands was tested by using a GLM with leaf area as a covariable. For all treatments, this covariable showed an impact on the number of glands (t\u2009=\u20098.113, P\u2009<\u20090.001; Fig.\u00a05). In comparison to the controls, an increase in the number of glands was found for herbivore-damaged plants (t\u2009=\u20094.147, P\u2009<\u20090.001) and for plants that were treated with JA (t\u2009=\u20095.585, P\u2009<\u20090.001). Because there were no differences between mechanically damaged and non-treated plants, the data of both groups were combined in this analysis.\nFig.\u00a05Effect of leaf area and treatment on the number of glands in the total foliage of 4-week-old cotton plants, which were either damaged at leaves 2 and 3 mechanically (Mech), fed upon by larvae of Spodoptera littoralis (Herb), treated with jasmonic acid (JA), or left as untreated controls (Ctrl) 7\u00a0days previously. Regressions were drawn based on transformed data from a generalized linear model (GLM) with a quasi-Poisson error distribution. Data for control and mechanically damaged plants were combined due to factor level reduction\nSingle Leaf Analysis To assess more precisely plant response to the treatments, analyses were carried out at the level of individual leaves. For all treatments, the distribution of leaf areas followed a consistent pattern within plants with the biggest leaves at intermediate positions (Fig.\u00a06a). Whereas no differences in leaf area were evident among mechanically damaged, herbivore-damaged, and control plants, plants treated with JA exhibited significant smaller areas for leaves 3\u20136 in comparison to corresponding control leaves (ESM Tables 3 and 4). Because of the negative impact of JA treatment on the leaf area, the following analyses of the effect of treatments on gland and terpenoid production were performed by using leaf area as covariable (statistical results see ESM Tables 3 and 4).\nFig.\u00a06Mean (\u00b1SE, N\u2009=\u20093\u20136) leaf area, number of glands, gland density, terpenoid level per gland, and terpenoid concentration of 4-week-old cotton plants, which were either damaged at leaves 2 and 3 mechanically (Mech), fed upon by larvae of Spodoptera littoralis (Herb), or treated with jasmonic acid (JA) 7\u00a0days previously. Areas of herbivore-damaged leaves 2 and 3 were extrapolated from remaining leaf material. For each leaf position, asterisks indicate significant differences from corresponding control leaves based on one-way ANOVAs (a), on analyses of codeviance with leaf area as covariable (b), and ANCOVAs with leaf area as covariable (c\u2013e). *P\u2009<\u20090.05\nGland number per leaf revealed a similar distribution pattern among leaves for all treatments: The oldest leaf 1 contained the lowest gland number, which increased gradually toward leaf 5 and again decreased in the expanding leaves 6 and 7 (Fig.\u00a06b). Young leaves (5\u20137) of herbivore-treated plants showed significantly elevated numbers of glands in comparison to corresponding leaves of control plants. Lower but still significant increases in gland numbers were found for leaves 2, 3, and 7 (youngest leaf) of plants that were treated with JA compared to control leaves. Mechanical damage showed nearly no effect on gland number.\nChanges in leaf area and gland numbers after treatment should have an impact on gland densities. Independent of treatment, gland densities increased gradually from the oldest leaf 1 toward the youngest leaf 7 (Fig.\u00a06c). This pattern was enhanced in herbivore-damaged and JA-treated plants because of significantly higher gland densities in young leaves (5, 6, 7) compared to those leaves from the control plants. Mechanical damage did not cause an increased gland density in any leaf.\nTo analyze how terpenoid production was altered by treatment, total terpenoid levels per gland were calculated. Treatments had a considerable effect on the pattern of terpenoids per gland among leaves (Fig.\u00a06d). Control plants showed highest levels in the oldest leaf and lowest levels in the youngest one. In contrast, mechanically and herbivore-damaged plants exhibited highest values in younger leaves, having significantly elevated total terpenoid levels in comparison to those of control leaves. Plants that were treated with JA showed significantly elevated levels of total terpenoids per gland in young and intermediate leaves in comparison to the controls.\nThe increase in total terpenoid and gland production caused by the treatments as well as altered leaf areas had a considerable impact on the pattern of the total terpenoid concentrations in leaves. Total terpenoid concentrations showed a consistent distribution pattern among leaves for all treatments (Fig.\u00a06e). There was a gradual increase starting from the oldest leaf 1 toward the youngest leaves 6 and 7. All treatments enhanced this pattern by causing significantly higher concentrations in intermediate and young leaves in comparison to the leaves of corresponding control plants. The lowest effect was found for mechanically damaged plants, whereas plants treated with JA reached the highest concentration with mean levels of approximately 16\u00a0\u03bcg terpenoids per milligram fresh weight.\nThe analyses of individual terpenoids revealed a significant impact of treatment, leaf position, and interactions between these two factors on levels of all compounds per gland (Table\u00a02). The lowest effects of treatment were found for the monoterpenes \u03b1- and \u03b2-pinene, whereas the biggest changes appeared for (E)-\u03b2-ocimene and heliocides 1 and 4. Differences of single terpenoid levels per gland among leaves appeared to be biggest for (E)-\u03b2-ocimene and heliocides 4, but in case of the latter, this was due to its unusual absence in leaves 2 and 3 (Fig.\u00a07). Significant interactions between treatment and leaf position revealed that changes in levels of total and single terpenoids of induced plants were not consistent in all leaves, which is illustrated in Fig.\u00a07. As with total terpenoid levels per gland (Fig.\u00a06d), mechanical damage and herbivory caused significantly elevated accumulation of individual compounds per gland in the youngest leaves in comparison to control plants. In contrast, JA treatment induced elevated levels in young but also in intermediate leaves compared to control leaves. JA treatment led to the highest increases of most compounds, especially for heliocides, in comparison to those in the corresponding leaves of control plants.\nFig.\u00a07Mean (\u00b1SE, N\u2009=\u20093\u20136) concentrations of terpenoids per gland among true leaves of 4-week-old Gossypium hirsutum plants, which were either damaged at leaves 2 and 3 mechanically (Mech), fed upon by larvae of Spodoptera littoralis (Herb), treated with jasmonic acid (JA), or left as untreated controls (Ctrl) 7\u00a0days previously. Asterisks indicate significant differences (P\u2009<\u20090.05) from corresponding control leaves based on one-way ANOVA\u2019s and Bonferoni post hoc tests. Pound signs indicate significant differences (P\u2009<\u20090.05) from corresponding control leaves based on Kruskall\u2013Wallis one-way ANOVAs on ranks and Dunn\u2019s post hoc testsTable\u00a02Results of nested two-way ANOVA for the effects of treatment (mechanical damage, herbivory, jasmonic acid application), leaf position, and interactions on levels of accumulated terpenoids per gland in plants of G. hirsutumCompoundTreatment (df\u2009=\u20093)Leaf Number (df\u2009=\u20096)Treatment \u00d7 Leaf Number (df\u2009=\u200918)FPFPFPTotal terpenoids37.81<0.00117.45<0.0016.78<0.001Monoterpenes\u03b1-Pinene6.70<0.00139.98<0.0014.02<0.001\u03b2-Pinene4.340.00642.48<0.0013.67<0.001Myrcene10.25<0.00151.60<0.0014.06<0.001(E)-\u03b2-Ocimene106.72<0.00179.24<0.0016.50<0.001Sesquiterpenes(E)-\u03b2-Caryophyllene24.13<0.00113.30<0.0016.59<0.001\u03b1-Humulene20.56<0.0019.66<0.0013.21<0.001\u03b3-Bisabolene37.08<0.00111.33<0.0015.74<0.001\u03b2-Bisabolol23.15<0.00112.48<0.0014.99<0.001Terpenoid aldehydesHemigossypolone10.16<0.00115.48<0.0016.47<0.001Heliocide 164.63<0.00113.78<0.0018.88<0.001Heliocide 227.13<0.00117.65<0.0014.94<0.001Heliocide 330.60<0.00117.66<0.0014.83<0.001Heliocide 455.70<0.001104.05<0.0018.59<0.001\nExperiment 2\nMorphological and Chemical Changes in Young Leaves after Induction In addition to herbivore-treated plants and untreated controls, this supplementary experiment also included mechanically damaged plants that were injured more frequently and over a longer period than in experiment 1. ANOVA showed an effect of the treatments on areas of young leaves (leaf 4; F\u2009=\u20095.97, P\u2009=\u20090.012; Fig.\u00a08a, ESM Table 5). Herbivory reduced the areas of these leaves in comparison to those of the corresponding leaves of the control plants (t\u2009=\u2009\u22123.445, P\u2009=\u20090.004). Therefore, the effect of mechanical damage and herbivory on the production of glands were tested by using a GLM with leaf area as a covariable. For both treatments, leaf area showed an impact on the number of glands (t\u2009=\u20092.256, P\u2009=\u20090.041). In comparison to the control plants, a significant increase in the number of glands was found for mechanically damaged plants (t\u2009=\u20092.599, P\u2009<\u20090.021) and an even more pronounced increase for herbivore-treated plants (t\u2009=\u20093.938, P\u2009=\u20090.002; Fig.\u00a08b).\nFig.\u00a08Mean (\u00b1SE, N\u2009=\u20096) leaf area, number of glands, and monoterpene- and sesquiterpene levels of leaf 4 from 3-week-old Gossypium hirsutum plants, which were either damaged mechanically at leaves 1 and 2 (Mech), fed upon by larvae of Spodoptera littoralis (Herb) on one cotyledon, leaves 1 and 2, or left as untreated controls (Ctrl). Asterisks indicate significant differences from corresponding control leaves based on one-way ANOVAs (a), on analyses of codeviance with leaf area as covariable (b), and ANCOVAs with leaf area as covariable (c, d). *P\u2009<\u20090.05; **P\u2009<\u20090.01; ***P\u2009<\u20090.001\nThe accumulation of mono- and sesquiterpenes in leaf 4 after treatment followed a comparable pattern: a gradual increase from control to mechanical damage and herbivory (ANOVA: monoterpenes, F\u2009=\u20098.13, P\u2009=\u20090.004; sesquiterpenes, F\u2009=\u200927.43, P\u2009<\u20090.001; Fig.\u00a08c,d). Levels of monoterpenes were elevated after herbivory in comparison to the controls (t\u2009=\u20094.029, P\u2009=\u20090.001). Levels of sesquiterpenes were elevated after mechanical damage (t\u2009=\u20095.115, P\u2009<\u20090.001) and herbivory (t\u2009=\u20097.197, P\u2009<\u20090.001) in comparison to the controls.\nDiscussion\nCotton (G. hirsutum) accumulates a large variety of terpenes constitutively, including monoterpenes (C10), sesquiterpenes (C15), and terpenoid aldehydes (C15, C25, and C30). This study is the first to describe the accumulation of all three of these classes after actual or simulated herbivory. Increased levels of terpenoids were observed in the total foliage 1\u00a0week after mechanical damage, feeding by Spodoptera littoralis caterpillars, or treatment with JA, in comparison to the untreated controls. A closer look revealed that, after damage to older leaves (leaves 2 and 3 numbered from the base), the increase was restricted mainly to young leaves (leaves 5\u20137). Similarly, studies on cotton that investigate the effects of herbivory by larvae of S. exigua and the wireworm Agriotes lineatus also showed elevated concentrations of terpenoid aldehydes in young cotton leaves (McAuslane et al. 1997; McAuslane and Alborn 1998; Bezemer et al. 2004). Such an induction pattern is in agreement with ODT, which predicts that an increase in the accumulation of defense chemicals occurs preferentially in plant parts with the highest fitness value such as young tissues or reproductive organs (McKey 1979; Frischknecht et al. 1987; Ohnmeiss and Baldwin 2000).\nAll three classes of cotton terpenoids are stored in subepidermal pigment glands found in leaves and other organs. After treatment, G. hirsutum displayed two ways to achieve elevated terpenoid accumulation: (1) production of additional glands and (2) increased filling of existing glands (Fig.\u00a06). We showed that production of additional glands was restricted to leaves that were still under development or newly formed after treatment. This trend also has been observed after attack by spider mites or larvae of S. exigua on cotton (McAuslane et al. 1997; Agrawal and Karban 2000). In other plants, such as birch and tomato, an elevated number of defense structures like glandular trichomes is also produced in young leaves after induction by herbivores (Boughton et al. 2005; Valkama et al. 2005). Besides the fact that, according to ODT, plants have been selected to increase their resistance to herbivores especially in young tissue, the formation of subepidermal pigment glands in cotton might be necessarily restricted to newly developing leaves because of developmental constraints.\nIn counting the number of cotton leaf glands under a stereomicroscope, we may have overestimated the production of new glands after treatment if herbivory triggered the filling of pre-existing glands that were unpigmented (H. T. Alborn, personal communication). However, by using several microscopic techniques, we did not find any evidence for unfilled glands in the leaves analyzed. Therefore, we assume that actual or simulated herbivory does indeed induce increased numbers of glands in cotton.\nThe increased filling of existing glands with terpenoids was shown in the youngest leaves of induced plants for all major terpenoid classes. However, considerable differences in the magnitude of this additional accumulation were observed among individual terpenoid compounds. Among terpenoid aldehydes, levels of heliocides H1 and H4, showed the highest increase after treatments. Other studies on cotton species also have demonstrated that these two heliocides increase more than H2, H3, and hemigossypolone (HGQ) after herbivory by Spodoptera species (McAuslane et al. 1997; McAuslane and Alborn 1998; Agrell et al. 2004; Bezemer et al. 2004). Here, we showed that levels of (E)-\u03b2-ocimene increased in a magnitude similar to H1 and H4, and that this compound increased most among monoterpenes and sesquiterpenes. Interestingly, (E)-\u03b2-ocimene is a direct precursor in the formation of H1 and H4, combining with hemigossypolone in a Diels\u2013Alder-type reaction (Stipanovic et al. 1978a; Fig.\u00a02). Therefore, in non-treated plants, the biosynthesis of this monoterpene could represent a limiting step in the production of the appropriate heliocides. This proposition is supported by the fact that, in cotyledons of G. hirsutum, which do not contain any heliocides, hemigossypolone is present (S. Opitz, unpublished), but (E)-\u03b2-ocimene is absent. A comparable regulation mechanism that involves myrcene could determine the levels of H2 and H3 (Stipanovic et al. 1977, 1978b).\nOur study compared the terpenoid content of total cotton foliage after three different treatments and showed a gradual increase in terpenoid levels in the following order: mechanical damage with a fabric pattern wheel, herbivory by S. littoralis caterpillars, and application of JA. However, a single incidence of mechanical injury is hard to compare with continuous feeding damage by an insect. Studies on lima bean have demonstrated that both the intensity and frequency of mechanical damage can alter the reaction of a plant (Mith\u00f6fer et al. 2005). This is consistent with the results of experiment 2 where more frequent mechanical damage caused significantly higher numbers of glands (Fig.\u00a08), a response not detectable after the less frequent mechanical damage in experiment 1 (Fig.\u00a05 and 6). However, in both experiments, herbivory induced stronger reactions of plants than mechanical damage. Elicitors found in the regurgitate of herbivores are known to trigger the biosynthesis of defense metabolites (Alborn et al. 1997). If such elicitors play a role in cotton defense reactions, mechanical injury may never result in terpenoid accumulation comparable to that caused by herbivory.\nThe strongest increase in both terpenoid and gland production appeared in plants that were treated with JA, a ubiquitous plant hormone known to mediate defense responses to biotic and abiotic stresses (Browse 2005). A previous study with cotton showed that the application of the methylated derivative, methyl jasmonate, induced the synthesis and emission of volatile terpenes, a response also observed for herbivore-damaged plants (Rodriguez-Saona et al. 2001). Obviously, JA plays an important role in mediating damage-induced signaling in G. hirsutum. Given the effects of exogenous application, herbivory can be assumed to trigger elevated internal levels of JA as in other species (Baldwin et al. 1997; Creelman and Mullet 1997). For example, in tobacco, lima bean, or maize, internal JA levels were determined in ranges of single nanograms per gram fresh weight, showing 20- to 40-fold increases within the first hours after induction (Baldwin et al. 1997; Koch et al. 1999; Schmelz et al. 2003). However, internal JA levels of cotton still need to be investigated. If the irrigation of plants with 100\u00a0\u03bcM JA in our study led to a non-physiological high internal concentration of JA, this could explain why JA treatment induced terpenoid accumulation to a greater extent than herbivory. We observed additionally that JA inhibited leaf development, a response also seen in other plants (Sembdner and Parthier 1993).\nStudies on the induction of chemical defense in plants after herbivory always raise questions about the specificity of such reactions to particular enemies. Certain plants have been shown to induce different spectra of defenses in response to different herbivores (Turlings et al. 1998; Traw and Dawson 2002; Delphia et al. 2007). However, in this study, we did not find substantial differences in the pattern of induction among treatments for the major terpenoids measured (see Table\u00a01). The amounts of single compounds varied in magnitude, leading to changes in the terpenoid profile, but these profile changes tended to be comparable among treatments. The induction of elevated terpenoid accumulation in leaves of G. hirsutum may thus be a non-specific reaction of plants to damage. Similar non-specific reactions to damage are known for terpenes in other species (Banchio et al. 2005), as well as for alkaloids (Frischknecht et al. 1987; Baldwin et al. 1997), phenolics (Cipollini 1997), and glucosinolates (Bodnaryk 1992).\nThe fact that cotton plants react to damage with elevated levels of terpenoids accumulated in the subepidermal pigment glands of their foliage suggests that these compounds function in plant defense. Caterpillar species such as S. exigua or Heliothis virescens prefer feeding on glandless instead of glanded cultivars of G. hirsutum, thus supporting the argument that the gland terpenes play a key role in defense of cotton against herbivores (Montandon et al. 1986; McAuslane and Alborn 2000). Leaf material from plants of glanded cultivar lines that had been induced by herbivory showed increased deterrent or toxic effects on Spodoptera species compared to material from uninduced plants, a finding thought to be due to their elevated contents of terpenoid aldehydes (Alborn et al. 1996; McAuslane et al. 1997; Anderson et al. 2001). Indeed, terpenoid aldehydes such as gossypol, hemigossypolone, and the heliocides H1 and H2 exhibit strong toxicity to caterpillars of H. virescens and Pectinophora gossypiella after addition to artificial diet (Elliger et al., 1978). While the feeding deterrent effects of induced cotton foliage are usually attributed to elevated terpenoid aldehydes contents (Hedin et al. 1992; McAuslane et al. 1997; McAuslane and Alborn 2000), we showed that cotton plants also increase their levels of monoterpenes and sesquiterpenes. Thus, these compounds may also participate in defense against herbivores. A sesquiterpene, caryophyllene oxide has been demonstrated to synergize the negative effect of gossypol, the dominant terpenoid aldehyde in roots and seeds of cotton, on larval development of H. virescens (Gunasena et al. 1988). Additionally, caryophyllene retarded the growth and delayed the time of development of these larvae. Other studies have shown that single sesquiterpenes such as \u03b2-bisabolene impair the development of insect herbivores and deter them from feeding (Bowers et al. 1976; Gonzalez-Coloma et al. 1995; Zipfel 2007).\nBeside their function against herbivores, terpenoids are also considered to play a defensive role against fungi or pathogens. Because plants are exposed especially to infestation at wound sites, the elevated terpenoid levels in damaged leaves might have a critical role in helping plants to cope with pathogens. For cotton, studies have shown that certain terpenoid aldehydes possess antifungal and antipathogen activities (Zhang et al. 1993; Abraham et al. 1999). A good example for the antibiotic activity of terpenoids in other species comes from conifers where the growth and germination of bark beetle associated pathogens is inhibited (Keeling and Bohlmann 2006). In addition, analyses of terpene-rich essential oils from a variety of plant species have shown antibacterial and antifungal activities, indicating the potential role of these substances in plant defense (Oyedeji and Afolayan 2005; Ozer et al. 2007).\nIn summary, our results demonstrate that elevated levels of terpenoids in cotton leaves after real and simulated herbivory represent a general wound response that is mediated by JA. The increase in terpenoids is due to the production of additional glands, in which terpenoids are stored, as well as the increased filling of existing glands. As all three classes of terpenoids (monoterpenes, sesquiterpenes, and terpenoid aldehydes) were elevated after damage, these substances may act synergistically in defense against herbivores or pathogens (Stipanovic et al. 1988).\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nESM 1\n(DOC 183 kb)","keyphrases":["cotton","gossypium hirsutum","pigment glands","monoterpenes","sesquiterpenes","terpenoid aldehydes","spodoptera littoralis","mechanical damage","jasmonic acid","induction","constitutive plant defense"],"prmu":["P","P","P","P","P","P","P","P","P","P","P"]}
{"id":"Appl_Microbiol_Biotechnol-4-1-2266784","title":"Secretion of Streptomyces mobaraensis pro-transglutaminase by coryneform bacteria\n","text":"We previously reported on the secretion of Streptomyces mobaraensis transglutaminase by Corynebacterium glutamicum ATCC13869 (formerly classified as Brevibacterium lactofermentum). In the present work, we investigated whether any other coryneform bacteria showed higher productivity than C. glutamicum ATCC13869. We found that most coryneform species secreted pro-transglutaminase efficiently. Moreover, we confirmed that Corynebacterium ammoniagenes ATCC6872 produced about 2.5 g\/l pro-transglutaminase over a 71-h period in a jar fermentor. Our findings suggest that some other coryneform bacteria, especially C. ammoniagenes ATCC6872, are potential hosts for industrial scale protein production.\nIntroduction\nThe secretion of heterologous proteins into culture medium is a potentially useful method of production (Choi and Lee 2004), although the amounts obtained through this approach are generally small. Thus, there remains a need to develop an efficient secretion system for industrial proteins. Streptomyces mobaraensis transglutaminase (TG) has been used in the food industry to modify proteins (Yokoyama  et al. 2004). It is utilized in binding meat and fish and in gelled food products such as jelly, yogurt, and cheese. Moreover, it has great potential for use in manufacturing the materials found in cosmetics, thermostable microcapsules, and carriers for immobilized enzymes (Yokoyama et al. 2004). However, so far, an efficient production system for TG has been lacking.\nA large number of different coryneform bacteria have been isolated from soil, animals, and plants (Liebl 2005). Some are used in biotechnological production processes; for example, Corynebacterium glutamicum and Corynebacterium ammoniagenes are well-known industrial producers of amino acids and nucleotides, respectively (Liebl 2005). C. glutamicum is a Gram-positive, non-sporulating bacterium with about 53.8% guanine\u2013cytosine (GC) DNA content and its genome has been sequenced (Ikeda and Nakagawa 2003; Kalinowski et al. 2003). This species is extensively used in the industrial production of amino acids, such as glutamate and lysine, which have been applied in human food, animal feed, and pharmaceutical products for several decades (Hermann 2003; Kr\u00e4mer 1994; Liebl 2005). However, there had been only few reports concerning heterologous protein secretion in C. glutamicum (Billman-Jacobe et al. 1995; Liebl et al. 1992; Salim et al. 1997). Recently, we demonstrated that TG could be efficiently secreted in an active form using a signal peptide derived from a cell-surface protein of coryneform bacteria in C. glutamicum ATCC13869 (Date et al. 2003, 2004; Kikuchi et al. 2003). We showed that C. glutamicum ATCC13869 could also secrete an active form of human epidermal growth factor, which consists of 53 amino acid residues, including six cysteine residues that form three disulfide bonds (Date et al. 2006). More recently, it has been shown that the twin-arginine translocation (Tat) pathway, which is a recently detected protein secretion pathway, is active in C. glutamicum ATCC13869 and that the Chryseobacterium proteolyticum pro-protein glutaminase, which is not secreted via the Sec pathway in C. glutamicum ATCC13869, is efficiently secreted by the Tat pathway (Kikuchi et al. 2006, 2007). These results demonstrate that protein production using C. glutamicum might be useful on an industrial scale. However, little is known about heterologous protein production by other species of coryneform bacteria.\nIn this report, we show that most other coryneform bacteria also secrete pro-TG efficiently and that C. ammoniagenes ATCC6872 is the best producer species, accumulating about 2.5\u00a0g\/l pro-TG over a period of 71\u00a0h in a jar fermentor.\nMaterials and methods\nBacterial strains, plasmids, and culture medium\nThe bacterial strains used are shown in Table\u00a01. The plasmids were as follows: pPSPTG1 (Kikuchi et al. 2003), which contains the cspB promoter derived from C. glutamicum ATCC13869; the CspA signal peptide sequence derived from a cell-surface protein of C. ammoniagenes ATCC6872; and the coding region of pro-TG, derived from S. mobaraensis, and pPKPTG1 (Kikuchi et al. 2003), in which the signal peptide for pro-TG secretion is replaced by the CspB signal peptide sequence from C. glutamicum ATCC13869. The coryneform bacteria were aerobically grown in CM2G medium (Kikuchi et al. 2003) at 30\u00b0C in a shaking test tube. Plasmids were introduced by electroporation with a Gene Pulser (Bio-Rad) using the standard protocol for Corynebacterium species. For pro-TG production in a test tube culture, we used MMTG medium containing 60\u00a0g glucose, 1\u00a0g MgSO4\u00b77H2O, 30\u00a0g (NH4)2SO4, 1.5\u00a0g KH2PO4, 0.01\u00a0g FeSO4\u00b77H2O, 0.01\u00a0g MnSO4\u00b74H2O, 450\u00a0\u03bcg thiamine hydrochloride, 450\u00a0\u03bcg biotin, 0.15\u00a0g dl-methionine, and 50\u00a0g CaCO3 per l distilled water, adjusted to pH 7.5 (Kikuchi et al. 2003). Kanamycin (25\u00a0mg\/l) was added to the culture medium when required.\nTable\u00a01Pro-TG accumulation by coryneform bacteria carrying pPSPTG1 in MMTG medium at 30\u00b0C for 72\u00a0hStrainPro-TG (mg\/l)Corynebacterium ammoniagenes ATCC6872454Corynebacterium glutamicum ATCC13032147Brevibacterium taipei ATCC13744201Corynebacterium glutamicum (Micrococcus glutamicus) ATCC1376166Brevibacterium roseum ATCC13825269Corynebacterium glutamicum (Brevibacterium flavum) ATCC13826314Corynebacterium herculis ATCC1386854Corynebacterium glutamicum (Brevibacterium lactofermentum) ATCC13869118Corynebacterium acetoacidophilum ATCC13870369Corynebacterium glutamicum (Brevibacterium divaricatum) ATCC14020391Brevibacterium saccharolyticum ATCC14066238Brevibacterium immariophilium ATCC14068257Microbacterium ammoniaphilum ATCC15354423Corynebacterium glutamicum (Corynebacterium lilium) ATCC15990246Corynebacterium callunae ATCC15991759Brevibacterium thiogenitalis ATCC19240156\nJar fermentation\nFermentor growth was carried out in a 1-l jar fermentor (300\u00a0ml working capacity). Glycerol stock suspensions of bacteria were stored at \u221280\u00b0C in 20\u00a0v\/v glycerol. Glycerol stock suspension (50\u00a0\u03bcl) was inoculated into 50 ml CM2G medium in a 500-ml Sakaguchi flask, and cultured at 27\u00b0C for 24 h. Then, 15\u00a0ml of the culture was inoculated into 300\u00a0ml MMTG-J medium containing 120 g glucose, 1 g MgSO4\u00b77H2O, 30\u00a0g (NH4)2SO4, 1.5\u00a0g KH2PO4, 0.01\u00a0g FeSO4\u00b77H2O, 0.01\u00a0g MnSO4\u00b74H2O, 450\u00a0\u03bcg thiamine hydrochloride, 450\u00a0\u03bcg biotin, 0.15\u00a0g dl-methionine, and 0.2\u00a0g total nitrogen of soybean hydrolyzate per l distilled water, adjusted to pH 7.2. The jar fermentor operating conditions were as follows: agitation rate\u2009=\u2009600\u00a0rpm, airflow rate\u2009=\u20091\/2\u00a0vvm; temperature\u2009=\u200927\u00b0C. The culture was automatically maintained at pH 7.2 by the controlled addition of ammonia gas with airflow. The glucose concentrations were analyzed using an AS-210 glucose analyzer (Sakura Seiki, Tokyo, Japan), and the optical densities were measured at 625\u00a0nm with a DU640 photometer (Beckman Coulter).\nProtein analysis\nSodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a 4\u201320% gradient polyacrylamide gel was carried out as described by Laemmli (1970), with Coomassie brilliant blue R-250 staining. The secreted pro-TG was measured by reverse-phase high-performance liquid chromatography, using purified native TG as a standard (Yokoyama et al. 2002). For processing the pro-TG to convert to an active form, the secreted pro-TG in a culture supernatant was incubated with purified SAM-P45 (Suzuki et al. 1997) at 30\u00b0C for 2\u00a0h, at a 100:1 ratio of pro-PG to SAM-P45. The purified SAM-P45 was a gift from Dr. S. Taguchi. The active-form TG was purified according to the method described previously (Yokoyama et al. 2002). TG activity was measured by the calorimetric hydroxamate procedure using N-carbobenzoxy-L-glutaminylglycine as described previously (Yokoyama et al. 2002). For determining the N-terminal amino acid sequence of the pro-TG, after separation by SDS-PAGE, proteins were transferred to polyvinylidene difluoride membrane by electroblotting. The membrane carrying the pro-TG was directly subjected to a gas phase protein sequencer (model PSQ; Shimadzu, Kyoto, Japan) equipped with an in-line amino acid analyzer (model RF-550; Shimadzu) as described previously (Kikuchi et al. 1997).\nResults and discussion\nSecretion of pro-TG by coryneform bacteria using C. ammoniagenes ATCC6872 CspA signal peptide\nWe transformed the bacteria listed in Table\u00a01 with pPSPTG1, cultured each transformant in MMTG medium at 30\u00b0C for 72\u00a0h, and then subjected the supernatants to SDS-PAGE. As shown in Fig.\u00a01, each transformant could stably secrete the pro-TG, and high levels of pro-TG with the anticipated molecular weight (42\u00a0kDa) were detected in the supernatants for all of the bacteria tested (Table\u00a01). The pro-TG was most efficiently secreted by C. ammoniagenes ATCC6872, Corynebacterium acetoacidophilum ATCC13870, C. glutamicum ATCC14020 (formerly classified as Brevibacterium divaricatum), Microbacterium ammoniaphilum ATCC15354, and Corynebacterium callunae ATCC15991.\nFig.\u00a01SDS-PAGE analysis of pro-TG secretion by coryneform bacteria carrying pPSPTG1. Each track contained a 10-\u03bcl aliquot of supernatant mixed with an equal volume of sample buffer. After electrophoresis, the gel was stained with Coomassie brilliant blue R-250. aLane 1, molecular weight markers; lanes 2 and 3, B. saccharolyticum ATCC14066; lanes 4 and 5, B. roseum ATCC13825; lanes 6 and 7, B. immariophilium ATCC14068; lanes 8 and 9, C. glutamicum ATCC13826; lanes 10 and 11, C. glutamicum ATCC15990; lanes 12 and 13, C. callunae ATCC15991; lanes 14 and 15, C. herculis ATCC13868; lanes 16 and 17, C. acetoacidophilum ATCC13870; lanes 18, C. ammoniagenes ATCC6872. bLanes 1 and 2, C. glutamicum ATCC13761; lane 3, molecular weight markers; lanes 4 and 5, C. glutamicum ATCC14020; lanes 6 and 7, M. ammoniaphilum ATCC15354; lanes 8 and 9, B. taipei ATCC13744; lanes 10 and 11, B. thiogenitalis ATCC19240; lanes 12 and 13, C. glutamicum ATCC13869; lanes 14 and 15, C. glutamicum ATCC13032; lane 16, C. ammoniagenes ATCC6872\nWe tested 16 strains of coryneform bacteria for the secretion of S. mobaraensis pro-TG. As shown in Fig.\u00a01 and Table\u00a01, all of the bacteria secreted pro-TG. We have previously shown that the C. glutamicum system has two advantages for heterologous protein secretion: a lack of proteolytic activity in C. glutamicum culture supernatants and a small amount of protein secretion by the bacteria themselves (Date et al. 2006; Kikuchi et al. 2003). As a result, the purification of secreted heterologous proteins is relatively easy. From the results shown in Fig.\u00a01, it is clear that the other coryneform bacteria share these advantages for heterologous protein secretion: their culture supernatants contain little endogenous protein and no degradation of pro-TG is detected in the supernatants.\nEffect of signal peptide sequence on pro-TG secretion\nNext, to investigate whether the secretion efficiency depended on the signal peptide sequence, we examined the effect of using the CspA signal peptide from C. ammoniagenes ATCC6872 (Fig.\u00a02a) or the CspB signal peptide from C. glutamicum ATCC13869 (Fig.\u00a02a) for the pro-TG secretion. C. ammoniagenes ATCC6872, C. acetoacidophilum ATCC13870, C. glutamicum ATCC14020, M. ammoniaphilum ATCC15354, and C. callunae ATCC15991 could efficiently secrete the pro-TG using the CspA signal peptide from C. ammoniagenes ATCC6872 in this study. These strains and C. glutamicum ATCC13869 were transformed with pPKPTG1 harboring the CspB signal peptide from C. glutamicum ATCC13869. As shown in Fig.\u00a02b, the substitution of the CspB signal peptide had little effect on pro-TG accumulation in C. acetoacidophilum ATCC13870, C. glutamicum ATCC14020, and M. ammoniaphilum ATCC15354. However, in C. callunae ATCC15991, the pro-TG accumulation (403\u00a0mg\/l) was approximately halved (746\u00a0mg\/l) while in C. ammoniagenes ATCC6872, it was about 2.8-fold higher.\nFig.\u00a02Effect of signal peptide sequence on pro-TG secretion in coryneform bacteria. a Amino acid sequence alignment of C. ammoniagenes CspA signal peptide and C. glutamicum CspB signal peptide using Vector NTI software. Identical and similar residues are shown on black and gray backgrounds, respectively. b Accumulation of secreted pro-TG by coryneform bacteria carrying pPSPTG1 or pPKPTG1. The solid bars indicate pro-TG accumulation using the CspA signal peptide from C. ammoniagenes ATCC6872, and the open bars indicate pro-TG accumulation using the CspB signal peptide from C. glutamicum ATCC13869\nThe amount of pro-TG accumulation by C. ammoniagenes ATCC6872 using the CspB signal peptide from C. glutamicum ATCC13869 was about 2.8-fold higher than with its own CspA signal peptide (Fig.\u00a02b). Interestingly, the difference between the effects of the two signal peptides was the opposite in C. glutamicum ATCC13869: the amount of pro-TG accumulation using the CspA signal peptide from C. ammoniagenes ATCC6872 was about 1.5-fold higher than with the CspB signal peptide from C. glutamicum ATCC13869 (Kikuchi et al. 2003). We found the same to be true for all of the heterologous proteins examined so far (data not shown). The reason for the differing effects of these signal peptides is unclear.\nCultivation of C. ammoniagenes ATCC6872 carrying pPKPTG1 in a jar fermentor\nAs C. ammoniagenes ATCC6872 carrying pPKPTG1 was the best producer strain in our study, we subjected it to a fermentation test in a jar fermentor under the conditions described in the \u201cMaterials and methods\u201d section. The MMTG-J medium contained 120\u00a0g\/l glucose; after the consumption of glucose for 71\u00a0h under these conditions, the optical density at 625\u00a0nm reached approximately 140, and the dry cell concentration was approximately 50\u00a0g\/l. During the exponential growth phase, little pro-TG accumulated in the culture supernatant; however, the rate of accumulation increased linearly after entry into the stationary phase, and approximately 2.5\u00a0g\/l pro-TG was produced (Fig.\u00a03). The N-terminal amino acid of the secreted pro-TG was Asp, as in native pro-TG (Kikuchi et al. 2003), demonstrating that the CspB signal peptide had been correctly processed in C. ammoniagenes ATCC6872. We then incubated the culture supernatant with purified SAM-P45, which is a subtilisin-like protease from Streptomyces albogriseolus, for 2\u00a0h at a 100:1 ratio of pro-TG to SAM-P45 in order to process the pro-domain (Kikuchi et al. 2003). The pro-TG secreted by C. ammoniagenes ATCC6872 carrying pPKPTG1 was cleaved by purified SAM-P45 to the C-side of 41-Ser of the pro-domain and converted to an active form and the conversion yield of this enzymatic process was approximately 100%. The specific activity of the purified active-form TG, with additional Phe-Arg-Ala-Pro residues, was similar to that of the native TG (about 23\u00a0U\/mg; Kikuchi et al. 2003).\nFig.\u00a03Jar fermentation experiment employing C. ammoniagenes ATCC6872 carrying pPKPTG1. Growth (optical density at 625\u00a0nm), glucose concentration, and pro-TG accumulation are indicated by open circles, open squares, and closed triangles, respectively\nThe results of the fermentation experiments in the 1-l jar fermentors showed that the pro-TG secreted by C. ammoniagenes ATCC6872 was correctly folded. Thus, protein production using C. ammoniagenes ATCC6872 could be useful for industrial scale protein production.","keyphrases":["transglutaminase","corynebacterium glutamicum","corynebacterium ammoniagenes","protein secretion"],"prmu":["P","P","P","P"]}
{"id":"Bioinformation-1-10-1896059","title":"Functional profiling and gene expression analysis of chromosomal copy number alterations\n","text":"Contrarily to the traditional view in which only one or a few key genes were supposed to be the causative factors of diseases, we discuss the importance of considering groups of functionally related genes in the study of pathologies characterised by chromosomal copy number alterations. Recent observations have reported the existence of regions in higher eukaryotic chromosomes (including humans) containing genes of related function that show a high degree of coregulation. Copy number alterations will consequently affect to clusters of functionally related genes, which will be the final causative agents of the diseased phenotype, in many cases. Therefore, we propose that the functional profiling of the regions affected by copy number alterations must be an important aspect to take into account in the understanding of this type of pathologies. To illustrate this, we present an integrated study of DNA copy number variations, gene expression along with the functional profiling of chromosomal regions in a case of multiple myeloma.\nBackground\nGenomic copy number alterations such as gains or losses of chromosomal regions have been shown to be on the basis of many human pathologies. Classical approaches\nto characterize these genetic aberrations used comparative genomic hybridisation (CGH), in which genomic DNA was hybridised to metaphase\nchromosomes. [1] Recently, the use of different types of microarrays to directly study genomic variations in DNA\ncopy number is becoming more and more popular. Such massive genomic approaches are known as array comparative genomic hybridisation, or Array CGH. [2]\nThese new technologies along with the use of expression arrays allow for a highly accurate characterisation of the dependence of gene expression on alterations in genomic\ncopy number. [3]\nAs in many genome-scale methodologies data analysis and, in particular, the biological interpretation of the results constitutes a well-known bottleneck. Specific\nproblems related to the analysis of Array CGH can be circumscribed mainly to two types: appropriate mapping and visualisation of the data onto the chromosomes, and\nefficient copy number estimation. This last aspect has been the motivation for a number of analytical approaches recently proposed [4], that can be considered\nthe first generation of algorithms for Array CGH analysis. Obviously, copy number variations are expected to have a strong effect on gene expression. [5,6] Nevertheless, the ultimate aim of studies of copy\nnumber chromosomal alterations is to understand what is the effect produced in functional terms. In the classical vision one or a few key genes are the causative factors\nfor the this type of pathologies, and the problem consisted in identifying such genes within the region amplified or deleted. The existence of regions in the\nchromosomes containing coexpressing genes [7] which, in addition, are functionally related has recently been\nreported even in higher eukaryotes. [8] Actually, regional arrangements of genes have found to be\nregulated not only by copy number alterations but also by different mechanisms such as epigenetic modifications. [9] This reinforces \nthe functional role of chromosomal regions including groups of functionally related genes and its possible impact on diseases such as cancer. [10] \nThese observations give credence to a new vision in which chromosomal alterations can be causing effects not by altering single key genes but by acting on\ncomplete molecular sub-systems such as pathways of functionally related genes. Recently, different approaches have focused on the functional aspects of the\nresults of microarray experiments. [11,12] Nevertheless,\nthe possible functional significance at regional level of copy number alterations has been largely ignored. Here we present a combined approach to the study of copynumber\nalterations, gene expression and functional profiling, exemplified in a case of multiple myeloma. [13]\nMethodology\nFunctional profiling of Array-CGH experiments under this new perspective would require of three steps: 1) detection of regions with copy number variations (the\norigin of the disease), 2) detection of regional alterations in gene expression (the causes of the disease) and 3) analysis of enrichment in functional terms in the detected\nregions (the consequences of the alteration or the functional basis of the disease). While copy number alterations can be detected by means of different\nmethods, alterations in the levels of gene expression are not always easy to be detected using the typical methods (t-test or similar) due several factors such as small\nsample sizes. For this reason here we will only use plots to visualize the effect of one variable (copy number) into the other one (expression level). The third step, the functional profiling, becomes then the most important\naspect of the analysis given that it will provide a functional explanation of the molecular basis of the disease caused by copy number alterations.\nDetection of copy number alterations\nWe have used a segmentation method which is a variant of the circular binary segmentation method [14], for copy number change detection (isowindow).\nThe isowindow method tries to identify boundaries between regions with a significant change in the values of intensity of hybridisation of the probes by some\nconsecutive steps. Firstly a t-test is used to determine differences between regions around all possible boundary points. Once all the candidate boundaries have\nbeen selected (a liberal p-value is used at this stage) there are sorted from small to high minimum p-values. In a second step the boundary candidates in the list with\noverlapping neighbourhoods are filtered to obtain a refined list of optimal non-overlapping boundary candidates. All the p-values are recalculated for the\nredefined neighbourhoods and a more stringent threshold is applied here. Finally, regions at both sides of each boundary candidate are again compared with a t-test. If\nthey are not significantly different in their average hybridisation values, then they are merged as a unique region. Otherwise they define two regions with different\ncopy number value. This is a simple and quick procedure that allows for easily changing from fine to coarse resolution by modifying the thresholds for the p-values.\nWe have compared isowindow to other two methods for breakpoint detection, GLAD [15] and circular binary\nsegmentation (CBS) [14], which are among the best performers. [4] \nIn the GLAD method a likelihood function with weights determined adaptively is used to solve the copy number estimation problem locally based\non data smoothed. Then, the algorithm finds, for each probe, the maximal neighbourhood in which the local constant assumption holds. Each of the constant pieces\nof the line define a block of probes with similar copy number among them and different copy number from that of the nearby regions. On the other hand, the CBS\nmethod selects firstly a segment of the data (a group of probes that are all consecutively arranged in the genome or in a chromosome). The copy number measures of the\nprobes in that segment are compared to those in the reminder dataset using a t-statistic. Hence, the method can distinguish whether the segment chosen has a copy\nnumber that is higher or lower than the overall copy number in the data, assumed to be the normal reference. This scheme is iterated exhaustively for all possible\nsegments in the dataset, spotting those that correspond to regions of altered copy number.\nAn approximation to the relative performances of the methods used was obtained by means of simulated data sets. Such datasets were generated by means of a\npiecewise constant function plus random alterations normally distributed with mean value and three different levels for the standard deviation (corresponding to noise\nlevels 0.2, 0.5 and 1). A mean value of 0 would correspond to a normal region, without copy number alterations, while mean values lower and higher would\ncorrespond to deletions or amplifications at different degrees, respectively. Amplified and deleted regions of different sizes are randomly situated within the simulated\nnormal chromosome and the methods have to locate them at different noise levels. The method proposed here performs at least as well as the GLAD and CBS (Table\n1) while being more efficient in terms of runtimes. Isowindow shows a better performance in finding small amplicons.\nFunctional profiling of regions with copy number alterations\nThe final aim of a Array-CGH experiment is to find a molecular explanation for the effects of the detected copy umber alterations. The interpretation of genomescale\ndata is usually performed in two steps: in a first step genes of interest are selected in this case because they are located in the amplified (or lost) region detected.\nIn a second step, the selected genes of interest are compared to the background (here the rest of genes in the chromosome) in order to find enrichment in any\nfunctional category (gene ontology, KEGG pathways, etc.) This comparison to the background is required because otherwise the significance of a proportion (even\nif high) cannot be determined. Different approaches have been developed to this end. [11] Here we will use the\nFatiGO+ (16) program, which uses a Fisher's exact test to determine the enrichment in different functional categories including gene ontology, KEEG pathways,\nInterpro functional motifs, Swissprot keywords and some regulatory elements such as transcription factor binding sites or other regulatory motifs. [17]\nDiscussion\nWe have implemented all the described functionalities in a program, ISACGH (an acronym for In Silico Array CGH), which is used to illustrate the concept of\nfunctional profiling of CGH arrays with an example of multiple myeloma (MM), an incurable form of haematological neoplasia.\nNine MM cell lines were obtained from the DSMZ (Deuche Sammlung von Mikroorganismen und Zelkuturen GmbH, Braunschweig, Germany) and were\ncultured under recommended conditions. DNA and RNA were extracted using supplier's protocols. Microarray assays were performed using the CNIO OncoChip,\nwhich contains 7657 different cDNA clones of cancer related genes. [18] CGH experiments onto cDNA arrays\nand hybridisation were performed as described in [13] and quantified using the GenePix Pro 5.0 software (Axon\nInstruments Inc., Union City, CA). Cy3\/Cy5 ratio values were normalized using the DNMAD tool from the GEPAS [19,20\n,21] and the resulting data were transformed to log2 ratios. Our purpose was to identify\nany possible region that contained copy number gains (amplifications), to study the expression of the genes included in that particular region and to understand the\npossible functional consequences of such alterations.\nUsing the segmentation method as implemented in the ISACGH we could detect a putative amplicon in the chromosome 18 (which remained undetected with both\nGLAD and CBS, because of the low density of the array, although the effect would have been the same in a high density arrays with a small amplicon) The figure shows\nthe region (left) and the slight, although appreciable, differences in gene expression levels within the amplicon (right).\nA unique feature offered by ISACGH is the possibility of obtaining a functional profile of the detected chromosomal regions. When the amplicon is analysed\nthrough the FatiGO+ program [16,17] a number of GO\nterms arise as over-represented in the genes contained in such region. Thus, the GO terms regulation of cellular process (GO:0050794) and regulation of physiological\nprocess (GO:0050791) were significantly overrepresented in the amplicon (FDR adjusted p-value=0.0336). Genes annotated with these terms were: BCL2,\nMALT1, NEDD4L, MBD2, TNFRSF11A and TCF4. Some of them have annotations at more detailed levels in GO, although the number of genes was too small as to\nproduce statistically significant results. For example BCL2 and MALT1 are annotated as negative regulation of programmed cell death (GO:0043069). These\nobservations suggest that some processes altered, that ultimately lead to diseases, are not produced by the deregulation of one unique gene, but are the combined\nresult of simultaneous deregulations of genes involved in a pathway or a particular biological function. In addition, these findings stress the importance of the use of\nfunctional profiling methods for the proper understanding and interpretation of the results of the genome-scale experiments. This unique feature included\nin ISACGH is of extreme importance since growing evidence suggests the existence of clusters of functionally related genes in the chromosomes [8] \nand the possible impact on diseases such as cancer. [10]\nAlthough ISACGH [22] can be used alone, it is tightly integrated in the GEPAS package. [19,\n21,23] GEPAS, that stands for Gene Expression Profile Analysis Suite\n(GEPAS), constitutes one of the most complete resources for microarray data analysis available over the web. GEPAS includes facilities for normalisations, clustering,\ngene selection, predictors and functional profiling. Thus, different operations (including pre-processing or normalization) can directly be performed within the\nsame environment, without the necessity of any file reformatting step.\nConclusion\nDespite a number of applications dealing with the estimation of genomic copy number have been recently published [4], \nthere are different aspects of the analysis of Array CGH data that have been poorly addressed or even ignored. Recent evidences strongly support the\nexistence of regional arrangements of functionally related genes [8], with obvious consequences for the understanding of diseases \ncharacterised by copy number alterations, such as an important number of cancers. [10]\nThis fact reduces the validity to the classical vision, in which one or a few key genes would be the causative factors of the disease, and urges to take into\nconsideration the functional dimension in the interpretation of the effects of copy number alterations. In this new scenario, the deregulation of blocks of\nfunctionally related genes located in the chromosomal regions with copy number alterations would be behind the disease phenotype.\nThe methods for functional profiling have proven in many scenarios its usefulness. An obvious challenge is to increase our knowledge in different aspects of function\nand cooperation between genes in order to be able of applying this methods in a way that allows us to unravel new unknown functional aspects of the biology of the\ncell and their connections to pathologies.","keyphrases":["function","profile","gene expression","chromosomal copy number"],"prmu":["P","P","P","P"]}
{"id":"Acta_Neuropathol-4-1-2270353","title":"Neuronal pentraxin II is highly upregulated in Parkinson\u2019s disease and a novel component of Lewy bodies\n","text":"Neuronal pentraxin II (NPTX2) is the most highly upregulated gene in the Parkinsonian substantia nigra based on our whole genome expression profiling results. We show here that it is a novel component of Lewy bodies and Lewy neurites in sporadic Parkinson\u2019s disease (PD). NPTX2 is also known as the neuronal activity-regulated protein (Narp), which is secreted and involved in long-term neuronal plasticity. Narp further regulates AMPA receptors which have been found to mediate highly selective non-apoptotic cell death of dopaminergic neurons. NPTX2\/Narp is found in close association with alpha-synuclein aggregates in both substantia nigra and cerebral cortex in PD but unlike alpha-synuclein gene expression, which is down-regulated in the Parkinsonian nigra, NPTX2 could represent a driver of the disease process. In view of its profound (>800%) upregulation and its established role in synaptic plasticity as well as dopaminergic nerve cell death, NPTX2 is a very interesting novel player which is likely to be involved in the pathway dysregulation which underlies PD.\nIntroduction\nNeuronal pentraxins constitute a family of proteins that are homologous to C-reactive and acute-phase proteins in the immune system and are thought to be involved in activity-dependent synaptic plasticity [1, 11, 32]. The neuronal pentraxins (NPTX1, NPTX2) and the neuronal pentraxin receptor were identified as synaptic proteins that bind to affinity columns of the snake venom toxin, taipoxin which presynaptically blocks neurotransmission, and the luminal calcium-binding protein TCBP49 [15]. NPTX2 [14] is also known as neuronal activity-regulated pentraxin (Narp), which was first identified as an immediate early gene responding to the induction of seizures in rat hippocampus [31]. The expression of this protein is regulated by synaptic activity and it induces the formation of new excitatory synapses and the regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor clustering at established synapses [7, 22, 23]. Importantly, dopaminergic neurons are selectively susceptible to excitotoxicity mediated via AMPA receptor activation with the resulting cell death being non-apoptotic [5].\nA second line of research that makes NPTX2 a highly interesting gene in the context of Parkinson\u2019s disease (PD) research concerns its expression in hypothalamic nerve cells that co-express hypocretin\/orexin [3] and which are prodigiously affected by the neurodegeneration in PD. These nerve cells have been implicated in the sleep disturbances that are often found in PD patients [9, 30].\nUsing a whole genome expression dataset of the human Parkinsonian nigra [19], we have identified NPTX2 as the most highly upregulated gene in a cohort of sporadic PD cases. Here we demonstrate that NPTX2 labels Lewy bodies in paraffin sections and that there is dysregulation of NPTX2 also in PD frontal cortex.\nMaterials and methods\nTissue samples from human SN and frontal cortex\nSome of the sporadic PD cases used in this study and several controls have been described previously [6, 19, 20]. Information on the cases employed for this project is provided in Supplemental Table. Brain tissue was obtained from the UK Parkinson\u2019s Disease Society Brain Bank at Imperial College London. The diagnostic work was performed in the Department of Neuropathology following international neuropathological consensus criteria for PD (http:\/\/www.ICDNS.org) which require use of alpha-synuclein as a molecular diagnostic marker. Ethics committee approval was available for all human tissue work of this study. For the dissection of the substantia nigra tissue (medial and lateral nigra were prepared separately), the portion of the midbrain containing the substantia nigra was separated from the brainstem distal to the mamillary bodies (45\u00b0 angle cut) at the level of the third cranial nerve and the superior colliculi. A slice approximately 5\u00a0mm in depth was prepared containing both SN and the cross-sectioned red nucleus at the level of its greatest diameter. Then, the SN was dissected from the surrounding nuclei and the pars reticulata. Once the SN was isolated, it was divided into two portions, the medial SN and the lateral SN. In all cases where frontal lobe was used, the superior frontal gyrus was sampled at the same level.\nQuantitative real-time polymerase chain reaction\nTotal RNA was extracted from PD and control cases and transcribed using the RETROscript\u00ae kit (Applied Biosystems, Warrington, UK) following the manufacturer\u2019s protocol. Primers for the internal control genes were as before [19]. Predesigned Taqman primers for NPTX2 (Hs00383983_m1) were purchased (Applied Biosystems) and Taqman mastermix (Applied Biosystems) was used. Each reaction was run in triplicate using an ABI PRISM\u2122 7700 Sequence Detector (Applied Biosystems). For every gene tested, a negative control was run without cDNA template. Data analysis for real-time PCR (DART-PCR Version 1.0; [25]) was used to determine relative differences in NPTX2 mRNA expression between PD cases and controls (also see Supplemental Figure 1).\nIn situ hybridization (ISH)\nISH was performed using radioactive oligonucleotide probes as described previously [18, 29] Oligonucleotides were designed using NetPrimer software (Premier, BioSoft International, http:\/\/www.premierbiosoft.com). Two oligonucleotides complementary to NPTX2 mRNA at nts 899\u2013932 (AGCCACAGGCAGATGGTGAAGGCGTACAGCTCAG\u2014probe A) and nts 1364\u20131397 (TCGACGTTATTGTCCACCCACGG-GATGATGTTGC\u2014probe B) were synthesized (Sigma Genosys, Poole, UK). Oligonucleotides were radioactively end-labelled with 35S-dATP (Perkin-Elmer, UK) using terminal deoxynucleotidyl transferase (Promega, UK).\nSnap frozen, unfixed post-mortem tissue blocks of the frontal cortex and SN were cut on a cryostat (12\u00a0\u03bcm), collected onto Superfrost Plus slides (VWR) and stored at \u221280\u00b0C. Before use, tissue sections were fixed for 5\u00a0min in fresh 4% paraformaldehyde in 0.1\u00a0M phosphate buffer (pH 7.4). Pre-hybridisation treatment included acetylation in 0.25\u00a0M acetic anhydride\/0.1\u00a0M triethanolamine, dehydration in graded alcohol (70\u2013100% ethanols) and delipidation in 100% chloroform. Hybridisation with the oligonucleotide probe was performed overnight at 37\u00b0C. Post-hybridisation sections were washed in standard saline citrate solutions with increasing stringencies. The sections were dehydrated rapidly through graded alcohols (70\u2013100% ethanol), air-dried, dipped in autoradiographic emulsion (LM1, GE Healthcare, Amersham, UK) and exposed for 5\u20136\u00a0weeks. The autoradiographic emulsion was developed, sections were counterstained with toluidine blue (VWR) for Nissl substance and mounted for visualisation using bright field microscopy for the counterstain and epi-polarized illumination for the silver grains of the autoradiographic emulsion. Control slides run to check for specificity of hybridization included competition with an excess of oligonucleotide (250\u00a0times) and the use of two probes to different portions of the mRNA of interest which produced identical patterns of signal. Black and white photographs of emulsion-coated sections were taken using a microscope provided with a Hamamatsu C4742\u201395 digital camera (Herrsching, Germany) and HiPic software (Herrsching) to capture images. Digital colour images were captured with a Retiga 1300 monochrome 12-bit camera using a color option provided by a RGB-HM-S filter (QImaging, Burnaby, BC, Canada) and QCapture 1.1.6 software (QImaging). Adobe Photoshop was used to assemble images.\nImmunohistochemistry\nParaffin embedded tissue sections (8\u00a0\u03bcm) were used for all stainings. No antigen retrieval was required for NPTX2 whereas for alpha-synuclein (SNCA) antibody staining sections were treated with 80% formic acid for 60\u00a0min [24]. Adjacent sections from SN were stained with goat polyclonal anti-NPTX2 antibody (NP2 N-20, sc-12125; Santa Cruz Biotechnology Inc.; 1:25) and mouse monoclonal anti-SNCA (BD Transduction Laboratories, catalogue number 610787; 1:1,000) overnight (4\u00b0C). Secondary antibodies used were gG-HRP (sc-2020; Santa Cruz Biotechnology Inc.; 1:100) and biotinylated horse anti-mouse (B-2000; Vector Laboratories, 1:100), respectively. The ABC kit (Elite 6100, Vector Laboratories) was used for visualisation using 3,3\u2032-diaminobenzidine tetrahydrochloride (0.02%; Sigma, UK). Sections were counter-stained in Mayer\u2019s haemalum.\nFor fluorescence microscopy, anti-SNCA (1:1,000) and anti-NPTX2 (1:25) were added to the sections sequentially (each was incubated overnight at 4\u00b0C). For the detection of the primary antibodies, sections were rinsed in 0.1\u00a0M Tris buffered saline (TBS) and then incubated with either the goat anti-rabbit IgG tagged with Alexa Fluor 546 (red) for 1\u00a0h at room temperature or the biotinylated anti-mouse antibody (Vector Laboratories 1:100) for 1\u00a0hour followed by incubation with streptavidin Alex Fluor 488 (green) for 1\u00a0h at room temperature. Finally, tissue sections were rinsed in TBS and incubated in sudan black for 10\u00a0min before mounting in 80% Tris-buffered glycerol.\nWestern blotting\nSnap frozen midbrain tissue samples containing SN from PD and control cases were homogenized with RIPA buffer (pH 8), heated to 70\u00b0C for 10\u00a0min and subsequently centrifuged (13,000\u00a0rpm, 10\u00a0min). The protein supernatant was removed and the concentration of protein determined (BIO-RAD protein reagent; BIO-RAD, UK) using a spectrophotometer. Denaturing gels were run using 30\u00a0\u03bcg of protein from each sample in 19.5\u00a0\u03bcl RIPA buffer, 3\u00a0\u03bcl \u03b2-mercaptoethanol and 7.5\u00a0\u03bcl lithium dodecyl sulphate sample buffer (Invitrogen, UK). NuPAGE Bis\u2013Tris 4\u201312% polyacrylamide gels (Invitrogen) were run in NuPage SDS running buffer (Invitrogen). Gels were blotted onto nitrocellulose membranes (Invitrogen), immersed in NuPage LDS transfer buffer (Invitrogen) in a cold room (4\u00b0C) overnight applying 30\u00a0V. Successful protein transfer was verified by staining the membrane in Ponceau S (VWR, UK) for 10\u00a0min. Prior to overnight incubation in polyclonal goat antibody to NPTX2 (N-20), membranes were washed in phosphate buffered saline (PBS) containing 0.1% Tween 20 and subsequently rinsed in PBS\/0.1% Tween 20 containing 5% skimmed milk (blocking step) for 30\u00a0min. Incubation with the secondary antibody coupled to horseradish peroxidase (1:5,000; Vector Labs, UK) was carried out for 1\u00a0h at room temperature. Visualization of antibody binding using enhanced chemilluminescence (ECL; Amersham Biosciences, UK) was performed according to the manufacturer\u2019s instructions.\nSemi-quantitative ratings\nThe NPTX2 labelled SN sections were examined by two independent observers blinded to the identity of the respective cases. The occurrence of NPTX2 immunoreactive profiles in the SN was scored for the overall burden of NPTX2 immunoreactive deposits using a seven-point scale ranging from 0\u20130.5 to 3 (absent to severe). Correlation analysis (Spearman; Sigmatstat 2.03) was conducted within the PD cohort for NPTX2 mRNA expression (log\u00a02 microarray data) and the corresponding semi-quantitative immunocytochemical results.\nResults\nNPTX2 is a novel component of Lewy bodies\nWe demonstrate here that NPTX2 protein in the Parkinsonian substantia nigra localizes to Lewy bodies, Lewy neurites and some glial cells. NPTX2 is the most upregulated gene in our microarray data set [19]. It shows the greatest difference between disease and control nigrae (probe 213479_at, P\u00a0=\u00a04.60051E\u221207, differential expression, DElog\u00a02, 3.0036217 which is equivalent to >800%). Many neurons show more than one NPTX2 immunoreactive LB (Fig.\u00a01a). Western blotting confirmed specificity of the antibody revealing a band, which corresponds to the NPTX2 monomer (47\u00a0kDa; Fig.\u00a01b). Very strong upregulation of NPTX2 mRNA expression in PD was confirmed by means of quantitative real time-PCR (Fig.\u00a01c).\nFig.\u00a01Neuronal pentraxin II (NPTX2) is strongly upregulated in substantia nigra and cerebral cortex of PD patients. a NPTX2 immunocytochemistry demonstrating specific staining of Lewy bodies in a dopaminergic neuron. b Western blotting confirms specificity of the anti-NPTX2 antibody. c qRT-PCR shows highly increased levels of NPTX2 mRNA in substantia nigra and, to a lesser extent, in frontal cortex. The arbitrary units along the ordinate represent relative fold changes [25]. The control value is 1; error bars indicate SEM. d, e In situ hybridisation demonstrates NPTX2 mRNA expression in nerve cells as well as glia in both substantia nigra and frontal cortex. Labelling is also found over non-pigmented neurons (arrow in d, SN). The arrows in e mark glial cells (cortex). In addition, there is autoradiographic signal in the neuropil (asterisks) which would be in keeping with the presumed dendritic expression of NPTX2 mRNA. f NPTX2 immunocytochemistry of cortical neuropil reveals occasional intercellular patches of \u201carborised\u201d labelling also possibly suggestive of a dendritic localisation (asterisks). The arrow points to a NPTX2 immunoreactive glial cell. Counterstaining in a and f, haemalum, and toluidine blue in d. Scale bars: 20\u00a0\u03bcm (a, f), 50\u00a0\u03bcm (d), and 100\u00a0\u03bcm (e)\nLocalisation of NPTX2 and SNCA protein\nIn situ hybridisation revealed expression of NPTX2\u00a0mRNA in nerve cells as well as glia in both SN and frontal cortex, respectively (Fig.\u00a01d, e). NPTX2 immunoreactive LBs were also observed in the frontal cortex and there were ramified (\u201carborised\u201d) NPTX2 positive profiles of cell processes in the neuropil (Fig.\u00a01f). Analysis of adjacent tissue sections demonstrated co-localisation of NPTX2 and SNCA in the same nerve cells (Fig.\u00a02a, b). This was confirmed by dual immunofluorescence (Fig.\u00a02c\u2013e). Overall, NPTX2 immunoreactive neuronal deposits mapped very closely to alpha-synuclein aggregates, both cellularly and subcellulary, and NPTX2 was also observed in incidental Lewy bodies (for a summary of cases used see Supplemental Table of Electronic Supplementary Material). In contrast, no NPTX2 immunoreactive intracellular aggregates were observed in two cases of tauopathy, i.e. progressive supranuclear palsy, where alpha-synuclein deposits are also not found. However, not all SNCA immunoreactive profiles were NPTX2 positive, i.e. the number of SNCA labelled nerve cells outnumbered that of NPTX2 stained ones (about 2\u20133:1). In addition, in some nerve cells, SNCA immunopositivity was observed as small granular cytoplasmic deposits (\u201caggresomes\u201d) that lacked NPTX2 immunoreactivity (Fig.\u00a02a, b). However, NPTX2 immunoepitopes were found to decorate the halo of LBs more distinctly than alpha-synuclein (Fig.\u00a03). Strongly NPTX2 immunoreactive Lewy neurites (Fig.\u00a03) were also found. Cases with high levels of NPTX2 protein expression showed a larger alpha-synuclein burden. We estimate that about one-third of the alpha-synuclein immunoreactive protein deposits also stain for NPTX2. Labelling of normal appearing axons by NPTX2 was widespread in both PD (Fig.\u00a03) and control brains. Glial cytoplasmic (Papp-Lantos) inclusions of multiple system atrophy (MSA) were NPTX2 positive (not shown) and in PD a sizeable number of glial cells expressed NPTX2 protein that appeared to be SNCA negative (Fig.\u00a03). Correlation analysis for NPTX2 microarray expression data and the corresponding semi-quantitative immunocytochemical case ratings showed a significant correlation (r\u00a0=\u00a00.970, P\u00a0<\u00a00.001; Table\u00a01). However, there was no association between NPTX2 expression and duration of the disease. \nFig.\u00a02Co-localisation of NPTX2 and alpha-synuclein. a, b NPTX2 and alpha-synuclein immunolabelling of adjacent tissue sections demonstrates significant but not complete overlap between the two proteins. The large arrow in a denotes the same Lewy body-containing dopaminergic nigral neuron. Small granular cytoplasmic deposits showing alpha-synuclein immunoreactivity in some nerve cells (arrows in b) are NPTX2 negative. These aggresomes are sometimes difficult to distinguish from neuromelanin granules based on staining alone but their cytoplasmic distribution is also different from the latter. In contrast, Lewy neurites are double-labelled (arrow head in b). Neuromelanin (asterisks in a) is unstained. c, d, e Dual immunofluorecence demonstrates co-localisation of neuronal pentraxin II (red, c) and alpha-synuclein (green, d). Double-labelled structures (arrows in c) light up in yellow in the overlay (e). Scale bar: 40\u00a0\u03bcm (a, b), and 80\u00a0\u03bcm (c\u2013e), respectivelyFig.\u00a03Morphology of neuronal pentraxin II immunoreactive deposits in the Parkinsonian substantia nigra. a Lewy bodies are labelled which often show a crennelated surface (also see c). Arrows of the inset point to NPTX2 labelled connections\/\u201cbridges\u201d between small Lewy bodies. Cross-sectioned axon bundles in white matter are numerous (longer arrows). b Arrows point to Lewy neurites in the substantia nigra. c NPTX2 is present in pre-Lewy bodies and glial cells. Labelling can be seen inside the cytoplasm of nerve cells in the neighbourhood and often directly attached to mature Lewy bodies (long arrows). Note the crenellated surface of compacted NPTX2 deposits (centre and Lewy body at the bottom of the figure) due to hair-like projections, which radiate into the cytoplasm. NPTX2 immunoreactivity is also present in glial cells. A subset of astrocytes as well as possibly microglia appear to be labelled (small arrows). The short arrow in the lower left points to what appears to be nuclear immunoreactivity. Scale bars: 40 (a), 50 (b) and 30 (c) \u03bcm, respectivelyTable\u00a01Correlation analysis for NPTX2 microarray expression data and the corresponding semi-quantitative immunocytochemical ratings for eight of the PD casesCase numberExpression NPTX2 mRNA (log\u00a02)Semi-quantitative ratings NPTX2 IR in SN27.278105587139.800657274368.7396793641.578.445081081.593.4182034440.5127.9801395781148.9652648212156.4144958231\nDiscussion\nThe most significant finding of this study is the striking presence of NPTX2 in classical Lewy bodies and in Lewy neurites. Experimental studies have demonstrated that Narp, the rat homologue of NPTX2, is important in neuronal development, promoting neuronal migration, synapse formation [1, 8] and neurite outgrowth [31]. Studies in neuronal pentraxin I and neuronal pentraxin II knockout mice have demonstrated a role in early synaptic refinements in the retina and dorsal lateral geniculate nucleus during development [1]. It has also been suggested that Narp\u2019s role in synaptic plasticity may underlie the long-lasting aversive effects of drug withdrawal [10, 26]. These functions of the NPTX2 gene product and its dendritic expression in the cerebral cortex [17] are of special interest in relation to our finding that NPTX2 immunoreactivity was also occasionally present in the cortical neuropil where it had a ramified appearance reminiscent of dendritic arbors.\nThere is limited knowledge on the NPTX2 pathway (Fig.\u00a04). Known physiological functions of NPTX2 include a role in the induction of AMPA receptor clustering [7]. NPTX2, NPTX1 and the GRIA3 subunit of AMPA receptors are all upregulated in our dataset. All other AMPA subunits and the receptor for NPTX2, NPTXR are downregulated. Two additional genes whose functions may be associated with AMPA receptors in dopaminergic neurons are also highly dysregulated in PD, NSF (N-ethylmaleimide sensitive fusion protein; 202395_at, P\u00a0=\u00a01.18252E\u221205, DElog\u00a02\u00a0=\u00a0\u22121.389396766) and Calcyon (dopamine receptor D1 interacting protein, 219896_at, P\u00a0=\u00a04.12902E\u221206, DElog\u00a02\u00a0=\u00a0\u22122.049585553). NSF binds the AMPA receptor GluR2 subunit and acts to disrupt GluR2-PICK1 interactions [28]; it may play a role in AMPA receptor trafficking and stabilization. Calcyon is co-expressed in a number of D1 dopamine receptor-positive neurons in brain and, like D1 dopamine receptors, is found in dendritic spines [16]. We also found NPTX2 in glial cells possibly including microglia. However, staining of glia was only present in a subpopulation of cells while neuronal cell processes appeared to be consistently labelled. Why exactly NPTX2 accumulates in Lewy bodies remains unclear at present. However, it is conceivable that NPTX2 is involved in the uptake of synaptic material. Synaptic dysfunction in PD is very significant as suggested by our microarray experiments (unpublished data). The presence of NPTX2 in only a subset of Lewy bodies and its association with their \u201ccrenellated surface\u201d shown in Fig.\u00a03 as well as NPTX2 immunoreactive \u201cbridges\u201d between Lewy bodies might indicate a role of NPTX2 in Lewy body growth.\nFig.\u00a04NPTX2 pathway obtained by means of the Resnet 5.0 eukaryotic database of molecular interactions (Ariadne) overlayed with microarray expression values. Changes of PD relative to normal are indicated in red (increased in PD) and blue (downregulated), respectively. Abbreviations: AMPA alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; GRIA1 glutamate receptor, ionotropic, AMPA 1; GRIA2 glutamate receptor, ionotropic, AMPA 2; GRIA3 glutamate receptor, ionotrophic, AMPA 3; GRIA4 glutamate receptor, ionotrophic, AMPA 4; NPTX1 neuronal pentraxin I; NPTX2 neuronal pentraxin II; NPTXR neuronal pentraxin receptor. Green line, regulation; purple line, binding. The dotted grey lines connect to a cell process whereas the solid lines connect to a molecule. The grey colour indicates that the effect and\/or mechanism are unknown. References for all protein interactions shown are provided in the Electronic Supplement to this figure\nMany PD patients also show non-motor symptoms such as hallucinations, depression, autonomic dysfunction and sleep-disturbances. All these symptoms are present in narcolepsy where there is loss of orexin (hypocretin) producing nerve cells in the hypothalamus [2, 30]. It is noteworthy therefore that the same population of neurons shows massive cell loss in PD as shown recently [9, 30]. In addition, Thannickal et al. observed a significant correlation with the clinical stage of PD [30]. It is intriguing that orexin neurons of the hypothalamus demonstrate robust expression of Narp in the rat [27].\nThe relationship between NPTX2 and cell death deserves further study. All subunits of the NMDA, AMPA and kainate classes of glutamate receptors and a set of NMDA receptor-associated intracellular proteins are expressed in the human substantia nigra pars compacta, and may play a role in glutamate regulation of dopaminergic activity and\/or release [21]. AMPA in turn has been discovered to be a mediator of selective non-apoptotic cell death in dopaminergic neurons [4]. The latter differs ultrastructurally from both neuronal apoptosis and typical excitotoxicity. Importantly, this mode of cell death is independent of caspase activity and does not have the morphological appearance of classical apoptosis. The findings are thus in keeping with our old postulate that nigral neurons are likely to die of a mechanism other than classical apoptosis and that aposklesis (\u201cwithering\u201d) may be a more appropriate term [12, 13]. The involvement of NPTX2 in cell death mechanisms is additionally supported by studies on the effect of ceramide, which activates TNF-pathways [4]. Therefore, NPTX2\/Narp has to be considered a new important gene in PD dysregulation which may be linked to both motor dysfunction caused by nigral dopaminergic nerve cell death in the midbrain and dementia due to synaptic changes in the cerebral cortex.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nSupplemental Figure 1. Cluster analysis is a method to organize genes in a microarray dataset into groups (clusters) of similar expression values. We have used ArrayAssist 5.5 software (Stratagene) to produce this image which shows that NPTX2 is not the only gene that is significantly up-regulated in the Parkinsonian substantia nigra compared to control and whose expression varies within the cohort. The latter is important because unlike a number of other highly dysregulated genes which show greater similarity across cases and therefore higher p values, NPTX2 fits better with both the clinical and neuromorphological notion of different disease stages in different cases. There was a good correlation between expression values and immunocytochemical results (Table 1). Abbreviations: CON, control; LN, lateral nigra; MN medial nigra; PD, Parkinson\u2019s disease. Codes CON10, CON2, CON3, CON9, PDC1, PD01- PD36 relate to study cases 19, 17, 18, 23, 16, 1-15, respectively (Supplemental Table). The colour range of expression values represents a logarithmic scale. The correlation of expression between different cases, or the distance between rows, i.e. their similarity measure, was determined as follows: a hierarchical clustering algorithm was applied with \"Pearson centred\" as the distance function, the \"centroid\" linkage rule was used, and clustering was performed on the whole nigral transcriptome. (JPG 3.35 mb)\nSupplement to Fig. 4 (PDF 85.7 kb)\nSupplemental Table 1. Cases used in the study (PDF 37.8 kb)","keyphrases":["ampa receptors","cell death","dementia","expresssion profiling","orexin\/hypocretin system"],"prmu":["P","P","P","M","R"]}
{"id":"J_Med_Internet_Res-8-2-1550696","title":"Health Information Literacy and Competencies of Information Age Students: Results From the Interactive Online Research Readiness Self-Assessment (RRSA)\n","text":"Background In an era of easy access to information, university students who will soon enter health professions need to develop their information competencies. The Research Readiness Self-Assessment (RRSA) is based on the Information Literacy Competency Standards for Higher Education, and it measures proficiency in obtaining health information, evaluating the quality of health information, and understanding plagiarism.\nIntroduction\nBackground and Purpose of the Study\nAs society moves toward evidence-based medicine [1], health providers, health educators, and health care consumers must acquire not only basic health information literacy skills but also more advanced competencies [2]. These competencies include evaluation of the quality of health information resources, obtaining health information documents on narrow topics by conducting advanced searches, judging the trustworthiness of health information sources, and understanding the advantages and disadvantages of different media. The last point is of special concern because many individuals have come to rely on the Internet as a main source of health information. This research addresses the Healthy People 2010 Objective 11-2, currently worded as \u201cto improve the health literacy of persons with inadequate or marginal literacy skills,\u201d but which may be expanded to the entire US population instead of only to those with marginal or inadequate literacy skills [3]. In addition, it aims at providing needs assessment information that may aid in accomplishing Objective 11-3, which is related to increasing the proportion of health communication activities that include research and evaluation, and Objective 11-4, set to increase the proportion of health-related websites that disclose information that can be used to assess the quality of the sites.\nRecent reports suggest that over 55% of Americans with Internet access seek health information online [4]. One of the most common complaints about online health information searches is the amount of time required to process the documents that are found [5], but this observation is likely to be related to the general nature of the searches conducted\u2014few information consumers use advanced search features, precisely specify their keywords, or limit their searches in some other way. While Internet search engines help identify a very large number of health-related documents, their use calls for advanced competencies that not all information consumers may possess. For example, the vast majority of documents found on the Internet have not passed a rigorous peer-review process. The ability to conduct one\u2019s own review is clearly an advanced skill. Arguably, health information consumers will be at a greater risk of making health decisions on the basis of noncredible information if they conduct a Google search as opposed to a search in a scholarly library database. This risk will be particularly high for individuals with poor health information competencies. Research comparing clinical evidence to Internet information reveals numerous examples of erroneous and potentially harmful information on such popular topics as cancer rates, smoking cessation methods, and fever management in children [6-8].\nInternet users may tend to underestimate the effort and competence required for obtaining trustworthy health information. A decade ago, communication researchers who compared print and television media described this paradox:\n[Individuals] have learned that print materials, so highly prized in school and elsewhere, are indeed more difficult to process, whereas TV can be processed for pleasure without much effort. However, this argument pertains only to the minimum effort needed for the satisfactory processing of materials; it says nothing about the amount of additional effort one could expend in processing televised material if one aimed at a deeper understanding of it[9].\nAlthough the Internet provides access to a vast number of documents on health-related topics, it is hard to build evidence-based knowledge about a health issue if one cannot determine the credibility of websites and the trustworthiness of the online documents. The minimum effort required for identifying millions of websites on a particular health topic is in sharp contrast with the average effort required to sift through the gigabytes of information in order to sort out the most credible documents, or at least those that appear as such.\nHigher education institutions in the United States provide access to an unprecedented quantity of digital information via library archives, licensed online databases, and the public-access Internet. To differentiate between publicly accessible Web documents and password-protected scholarly databases, which can be accessed by paid members via the Web, we refer to the former as the \u201cthe public-access Internet.\u201d\nOur study explores three basic questions: How proficient are university students at finding and evaluating health-related information? How well do they understand the difference between peer-reviewed scholarly resources and opinion pieces or sales pitches? How aware are they of their own level of health information competencies? The main goal of this project was to identify approaches to building Information Age competencies of young health consumers, specifically a cohort of 18- to 23-year-old students enrolled in higher education programs.\nLiterature Review: Health Information and the Internet\nIn accordance with the Healthy People 2010 health communication objective [3], public health professionals attempt to assist consumers seeking health information via the Internet, for instance, by reinforcing the need for quality standards and widespread criteria for evaluating health information [10-14]. Cline and Haynes [10] note that, while critics are fast to question the quality of online health information, limited empirical research on this topic does not allow any broad conclusions to be drawn. In a study published the same year, Eysenbach and colleagues [15] reported that Internet coverage of health information was often inconsistent, although the accuracy was generally good, and that search engines and simple search terms did not provide efficient access to health information. Crespo [16] reviewed several studies on online health information seekers and concluded that most users seemed to focus on finding information quickly rather than on evaluating the information found. Similarly, Eysenbach and Kohler [17] found that individuals explored only the first few links obtained from a search using a general search engine. Although some Internet users attempted to assess the credibility of sites by, for example, examining their source and professional designs, many people did not read the \u201cabout us\u201d sections of websites, learn about the authors or owners of the sites, or review disclaimers and disclosure statements. Very few Internet users later remembered from which websites they retrieved information or who stood behind the sites [17].\nThus, abundance of health information does not always translate into informed choices. Hibbard and Peters [18] suggest that three factors should be considered in selecting information presentation strategies: (1) the complexity and amount of information; (2) the nature of the choice\u2014degree to which there is a right or best option; and (3) the experience, motivation, and skills of users. The third point, deficient information skills, may prevent members of the public from recognizing that key information is missing, from understanding the difference between biased and unbiased information, from distinguishing evidence-based claims, and from interpreting the information intended for health professionals [10]. Researchers, having observed individuals who, on average, spent about one-half hour looking for health information, concluded that information consumers should have at least a tenth grade reading level to process Web materials. Many websites presented to the participants of this study contained material at a college level [15].\nOnline health care is having a growing cultural impact, affecting the practitioner-patient relationship and opening up the possibility of new roles for social workers and educators in the provision of health services [19]. The increasing use of the Internet draws scientists\u2019 attention to modeling individual behavior, contributing to the development and refinement of individual health theories and models, such as the Theory of Planned Behavior, The Health Belief Model, and The Transtheoretical Model [20]. The theoretical framework for this study is largely based on the information processing theories and concepts discussed below.\nSchneider and Shiffrin [21] distinguish two qualitatively different modes: (1) conscious, intentional processing of information that is capacity limited (controlled processing), and (2) quick and efficient automatic processing of information that has greater capacity, for example, when several tasks can be done at the same time. Automaticity requires less attentional resources than controlled processing, and it is developed through extensive practice under the condition of consistent stimuli and response requirements. When surfing the Internet, for example, health information consumers limit their exposure to inconsistent conditions\u2014they tend to use the same search engines and the same searching methods, such as entering keywords into the nonadvanced search window. The assessment of health information competencies in this study incorporates tasks that call for automatic processing and tasks where stimuli and response requirements of the task are inconsistent with most health information consumers\u2019 information search practices.\nWe also draw upon Anderson\u2019s ACT theory [22,23], which explains skill acquisition. It incorporates research on automaticity and explains the development of cognitive skills important for processing digitized health information from a variety of electronic sources [24]. According to Anderson [22,23], skill development has three stages: (1) the declarative knowledge stage, when knowledge of facts is built, such as facts about reputable sources of health information and general procedures for obtaining information; (2) the knowledge compilation stage, which is characterized by proceduralization and composition; and (3) the procedural stage. To illustrate the second stage, consider a health information consumer who follows a set sequence of specific steps to search for a health-related terms (proceduralization) and reapplies this sequence until sufficient information on a health topic is found (composition). Once at the knowledge compilation stage, a consumer can perform an information search task at a higher speed and with fewer errors than at the declarative knowledge stage. High speed and low error rate are both important markers of skilled performance. However, a disadvantage of knowledge compilation is the rigidity of behavior, when individuals find it increasingly difficult to attend to intermediate feedback (e.g., step-related results) and engage in strategy modification (e.g., by adopting a search strategy that produces a greater number of trustworthy health information resources) [24]. Declarative and procedural knowledge are discussed in greater depth in the Methods section.\nAn Interdisciplinary Research Partnership\nOur research originated from the collaboration of a psychologist, a health educator, and a librarian who set out to understand and improve health information competencies of the Information Age generation. The collaboration enhances our research in several ways. The psychologist contributes expertise in the area of psychometrics and test design, whereas the health educator contributes knowledge of health consumers\u2019 behavior and intervention designs. The librarian contributes expertise in training and enhancing patrons\u2019 health information\u2013seeking skills [25], as well as knowledge about gateways to authoritative consumer health information, for example, Medline Plus [26,27]. Linnan and colleagues [28] believe that library\/public health partnerships are capable of increasing information access, the quality of available health information, and the technological expertise of all community members. Neighborhood libraries often serve the online health information needs of consumers who may not have Internet access at home, such as the elderly, ethnic groups, and low-income and undereducated populations [29,30], whereas university libraries also serve as gateways to scholarly health materials that are not available on the public-access Internet. In addition to public-access health resources available online, this research focuses on scholarly health resources in academic libraries and their use by students who are training to become health professionals.\nMethods\nParticipants\nA sample of 400 college-age students was selected because this cohort is the first Information Age generation that has been exposed, for up to one-half of their lives, to the Internet. Students enrolled in three courses in the College of Health Sciences at a Midwestern university were invited to participate in the study. The first class was a high-enrollment introductory course on the determinants of health. Although only undergraduate students (n = 354) participated in this course, they represented all levels of undergraduates\u2014freshman (59%), sophomores (22%), juniors (9%), and seniors (10%). The second class was an advanced course in health administration in which both undergraduate (n = 19) and graduate students (n = 3) were enrolled. The third class was a mid-level health education course (n = 25) for undergraduate students. All students enrolled in the advanced health administration course and the mid-level health education course were majoring in health professions. About one third of the introductory course students with declared majors were majoring in a health-related discipline, and 31% of students had not made up their minds about a major field of study.\nIntroductory course students completed the assessment for extra credit, while others did it to learn more about their own skills. The instructors emphasized that the purpose of the assessment was to help students become competent consumers of health-related information.\nMeasures\nHealth Information Competencies\nIvanitskaya and Casey developed the Research Readiness Self-Assessment (RRSA) to measure basic research skills based on the Information Literacy Competency Standards for Higher Education developed by the Association of College and Research Libraries [2,31]. The RRSA designers\u2019 original intent was to measure information competencies, both general and discipline specific, of students attending colleges and universities. A health information version of the RRSA is discussed in this paper; it was created to specifically evaluate health information competencies. Competencies are knowledge\/skills sets essential for accomplishing a goal, in this case, finding quality information on a specific health topic. The RRSA measures competencies linked to such college-age health information consumer behaviors as determining possible sources of health information, conducting health information searches, evaluating the quality of documents found, and using those documents appropriately. One of the relevant competencies is knowledge of plagiarism because it can be applied to properly recognize ideas contributed by others and to evaluate health-related documents. The RRSA designers aimed at measuring foundational competencies that are (1) transferable to other knowledge domains (e.g., social sciences in addition to health sciences); (2) applicable to a large number of health information consumers; (3) consistent with typical behaviors or experiences of health information consumers who seek information from electronic sources; and (4) that capture the nature and spirit of critical thinking, life-long learning, and advances in information technology. It is important to note that the RRSA instrument does not measure higher order skills that characterize experienced researchers, such as the design of clinical trials [31]. The word research in the assessment\u2019s title matches the language commonly used by the lay population, as in \u201cgoing to Google to research a health topic,\u201d which is indicative of such behaviors as searching, judging, and making decisions.\nThe RRSA contains the following items: (1) multiple choice or true\/false questions that measure declarative knowledge; (2) interactive, problem-based exercises that measure procedural knowledge; (3) demographic questions; and (4) a question that asks for a self-report about the level of the respondent\u2019s research skills [31].\nDeclarative knowledge, defined as knowledge of facts or verbal knowledge, is a precursor to higher-order learning, which is needed, for example, to complete a sequence of steps to critically analyze a website or to employ elegant information search strategies [32]. Declarative knowledge questions in the RRSA measure knowledge of plagiarism, health information sources, and research-related terminology. For example, the following item is used to measure knowledge of research-related terminology:\nA journal article abstract is\u2026\nan annotated list of references used in the articlea summary of the article\u2019s contenta summary of other research on this topica note or paragraph about the authors of the articlea glossary of abstract concepts included in the researcher\u2019s model\nCompared to declarative knowledge, procedural knowledge is related to skills and problem solving. Essential for reproduction of learned behaviors, procedural knowledge is defined as knowledge of the process used to complete a task (e.g., how an information search process can be sequenced, organized, or controlled) [32]. In the RRSA, problem-based interactive exercises are used to measure procedural knowledge. Procedural knowledge questions include links to websites, library catalogs, and interactive search modules designed specifically for the RRSA. Students demonstrate their database navigation skills by setting up basic and advanced searches. For example, the following item is used to measure skill in conducting a search using Boolean operators (and, or, not):\nYou are interested in gathering information about work stress but are not interested in its medical side effects. Set up a document search in a separate window using the following keywords: stress\nmedical. Click here to begin your search [a hyperlink to an interactive online module similar to searches in health-related library databases, such as Medline, with text fields for entering key words and a choice of Boolean operators]. Report the number of documents you found: a) 255; b) 555; c) 700; d) 1164; e) 55164.\nIn addition, students evaluate the quality of research publications, make judgments about website trustworthiness, and detect plagiarism. For example, the following item is used to measure evaluation of the trustworthiness of websites:\nYou are looking for information on various nutritional supplements. You found three websites. Click on the links below to examine each site and to evaluate its content. Which of these websites is the most trustworthy? a) cognitogenic aids [a hyperlink]; b) dormitogenic aids [a hyperlink]; c) vescorogenic (gustatogenic) aids [a hyperlink].\nInstrument Piloting and Validation\nTo pilot test an earlier version of the RRSA instrument and to gather initial evidence about its validity and reliability, we administered a 60-item assessment to undergraduates (n = 100) and doctoral students (n = 45), as well as professional librarians (n = 5) and health professionals (n = 3). The feedback from librarians and health professionals offered preliminary evidence in support of the instrument\u2019s face validity and content validity. Specifically, the librarians confirmed that the items included in the RRSA assessment conformed to the Information Literacy Competency Standards and addressed knowledge and skills important to health information consumers. The wording of several items, both stems and response options, was revised based on librarians\u2019 recommendations. In addition, the librarians completed the assessment themselves. Their scores were then compared to the scores of students at two academic levels, undergraduate and doctoral. The results indicated that individuals with greater training and experience in managing digital health information performed better than individuals with less experience. Undergraduate students\u2019 overall scores were the lowest (about 66% correct responses), followed by doctoral students\u2019 scores (73%) and librarians\u2019 scores (95%). These results offer preliminary evidence of the assessment\u2019s criterion-related validity. The pilot test indicated an acceptable internal consistency value (Cronbach alpha > .70), although it could be improved (approach .80) if four items were removed. Therefore, four RRSA items that reduced the overall internal consistency were deleted.\nThe revised assessment contains 56 items, including 16 multiple-choice questions and 40 true\/false questions grouped under 7 stems (Multimedia Appendix 1). For example, knowledge of information sources is measured by a stem that states, \u201cWhich of these citations are to journal articles?\u201d The participants then check all that apply from the list of 6 true\/false items (3 references to journal articles, 1 book reference, and 1 book chapter reference). Items are scored as +1 if the answer is a correct positive or a correct negative and +0 if the answer is a false positive or a false negative. Further description of the development of the stimulus materials used in website evaluation appears in the Results section, under Proficiency in Evaluating Health Information.\nThe RRSA assessment was designed to be useable by more than one institution. Its content can be adapted to the needs of various educational programs. Specifically, instructions to participants, the text of individual questions, detailed feedback, links to additional resources, and disclaimers (e.g., about participants\u2019 rights and how the information they provide will be used) can be revised, without help from programmers, using the password-protected online control panel. This has been done by three US universities and one Canadian university that adopted the RRSA for use in their academic programs. For example, all four institutions revised search questions to enable their students to search for documents in their own university\u2019s library catalog. The original RRSA designers provide coaching and training in order to ensure that the changes made to the RRSA do not have a negative impact on its reliability and validity. Ongoing validation studies provide a quality control mechanism and allow the testing of new or revised questions suggested by the partner institutions. The administration of the RRSA to partner institutions is supported through grants, partner donations, and volunteer efforts by the RRSA design team members.\nOther Measures\nWe asked the study participants to share information about their age, gender, and education. Self-reported level of research skills was measured with a single item, \u201cHow do you rate your research skills?\u201d with six response options ranging from 1 (nonexistent) to 6 (excellent).\nProcedures\nThe RRSA instrument was administered online. Each student was issued a unique pass to access RRSA questions. The students had the option of submitting an incomplete survey and then returning to it at a later time to finish the remaining questions. This feature promoted better information processing and relieved the students from the need to rush and finish the entire assessment on their first attempt. The average estimated RRSA completion time was 26 minutes. Upon answering all questions, the students received an individualized results page that summarized their performance in different areas by providing a score, a maximum possible score, and percent attained. In addition to the numerical RRSA results, the Web page displayed individually tailored feedback composed by an experienced librarian. The Web page was programmed to compare, within each performance category, each individual student\u2019s performance to the performance of a norm group. In accordance with the student\u2019s competency level, the feedback provided suggestions for skill improvement and an explanation of factors that may have contributed to low, average, or high performance in each area. Finally, students who completed the RRSA were given the option to request additional materials for remedial learning, such as an explanation of the difference between scholarly and nonscholarly resources. The links to these additional materials were delivered to students via email.\nData Analyses\nDescriptive statistics were used to examine respondents\u2019 performance in four areas\u2014searching for health-related information, understanding plagiarism, evaluating health information, and self-reported skill level. To examine the relationship between self-reported skill level and actual performance, we computed composite scores. A composite overall score, which is indicative of the health information competency level, was created by adding points for 56 items, which were either true\/false or multiple choice. Composite score calculations were preceded by an internal consistency reliability analysis that determined the appropriateness of combining responses from multiple items. We used a Spearman correlation to assess the relationship between the actual skill level (overall score) and self-reported skill level. A multiple regression analysis was used to examine the relationship between actual performance and perceived skill while holding the amount of education (number of credit hours earned) constant.\nResults\nOur research questions were the following: How proficient are university students at searching for and evaluating health-related information? How well do they understand the difference between peer-reviewed scholarly resources and opinion pieces or sales pitches? How aware are they of their own level of health information competencies? The results for each question are presented below, preceded by a sample description.\nRespondent Characteristics\nThe participation rate was 77%. Nonrespondents (n = 92) differed from respondents (n = 308) in terms of their academic level (t\n400 = 2.29, P = .02). Freshmen were slightly more likely not to participate in the RRSA than seniors; the participant group included 7% less freshmen and 10% more seniors than the nonparticipant group. Most respondents were female (77%) and between 18 and 23 years of age (95%). The vast majority of respondents (98%) did not have a bachelor\u2019s degree, and the remaining students were working toward their master\u2019s degrees. Because we administered the RRSA to students in health professions courses, over one third of respondents were majoring in health sciences. Common majors were athletic training and sports medicine, health administration, physical education, pre-physical therapy, and public health promotion. On average, the undergraduates who participated in the study had completed 40 or fewer semester credit hours of university coursework. A quarter of respondents reported earning over 71 credit hours.\nProficiency in Searching for Health Information\nTable 1 summarizes performance in searching for health information. The data indicate that most students recognize common health journal titles and can perform a basic search in a library catalog, for example, by entering an exact book title into the title search. Few students, however, can perform an advanced search for a book when they know the book\u2019s author (with a very common last name), general topic, and publication date. We call this search advanced because imprecise book specifications make it hard to find the book without performing a search that takes into account all or nearly all of the available information.\nThe data also show that two thirds of study participants are unable to understand or apply Boolean operators, such as and, or, and not. Boolean operators are used in most search engines, including those used for navigating the Internet (Google or Yahoo), library databases with scholarly journal articles, and library catalogs. Even though most students (89%) understand that a one-keyword search is likely to return too many documents, few are able to narrow a search by using multiple search categories simultaneously or by employing the Boolean operators. In addition, nearly half of the respondents have trouble discriminating between primary and secondary sources of information, as well as between references to journal articles or other published documents, such as books or book chapters.\nProficiency in Evaluating Health Information\nOne of the most important markers of a competent health information consumer\u2014critical judgment of information\u2014is assessed in two ways: (1) the first set of questions calls for a review of three full-text articles from journals, and (2) the second set of questions calls for a comparison of three health-related websites.\nThe three journal articles are on the topic of job satisfaction, a topic relevant to any profession, and come from a full-text library research database. They include a rigorous empirical study, a case study, and an opinion article. Only the empirical study has a bibliography and an explicit statement about the author\u2019s affiliation. The opinion article, clearly the least authoritative source, makes no mention of the author\u2019s affiliation. As shown in Table 1, most respondents can determine the article publication date; it appears at the top of a full-text article. Many respondents can also identify an opinion article. Fewer respondents know how to determine if an article includes a research review and are able to check for the author\u2019s affiliation.\nThe three Web pages about nutritional supplements are realistic looking interactive screens that appear to be live websites. The content of these mock websites, developed specifically for the RRSA, includes graphics, hyperlinks, and text about nonexistent classes of nutritional supplements\u2014cognitogenics, dormitogenics, and gustatogenics. Each website is dedicated to one class of supplement and explains its purpose (e.g., cognitogenics help people with learning disabilities), prevalence (e.g., \u201cgustatogenic aids have been available in Germany and Canada for over five years\u201d), and safety. Even though the descriptions of nutritional supplements were fictitious, all three websites accurately stated that the US Food and Drug Administration did not evaluate the safety or benefits of these nutritional supplements.\nTable 1\nSearching and evaluating health information: performance on select measures (n = 308)\nRespondents With Correct Answers\nn\n%\nSearching for Health Information\nKnowledge of a scholarly source, Journal of American Medical Association (7)\n293\n95\nDemonstration of a skill in locating a book in a university library catalogue based on its exact title (16)\n286\n93\nUnderstanding that a one-keyword generic search may return too many documents\u2014an overwhelmingly large number of resources on a variety of topics (4)\n275\n89\nUse of a proper research strategy\u2014thinking about a broad topic to identify a sub-area of interest (2)\n268\n87\nAbility to detect a journal citation that is incomplete\u2014lacks a year of publication (17)\n241\n78\nUnderstanding of a term \u201carticle abstract\u201d\u2014a summary of the article\u2019s content (8)\n234\n76\nKnowledge that a journal is a source of scholarly (analytical) information on a narrowly specialized topic (6)\n214\n70\nUnderstanding of a term \u201cbibliography\u201d\u2014a list of references or citations (9)\n213\n69\nIdentification of a primary source of health information: medical record (14)\n195\n63\nIdentification of references to journal articles from a list of references that includes both book references and article references (11)\n187\n61\nKnowledge of a peer-reviewed journal article as an authoritative source of specialized health information (12)\n185\n60\nIdentification of a primary source of health information: hospital annual report (14)\n173\n56\nDemonstration of a skill in locating a book in a university library catalogue based on a non-unique authors\u2019 name and a general topic (15)\n111\n36\nKnowledge of Boolean operators (and, not, or) (3)\n105\n34\nDemonstration of a skill in setting up and performing a search with Boolean operators (and, not, or) (13)\n98\n32\nEvaluation of Information: Full-Text Journal Articles\nEvaluation of journal articles: Identification of an article published prior to year 2000 (22)\n248\n80\nEvaluation of journal articles: Identification of an article based on opinion rather than well-supported evidence (19)\n242\n79\nEvaluation of journal articles: Identification of an article based on a review of existing research (20)\n166\n54\nEvaluation of journal articles: Identification of an article written by an author whose affiliation is unknown (21)\n148\n48\nEvaluation of Information: Websites on Nutritional Supplements\nEvidence-based decision-making: Disagree that \u201call three websites make a good case for taking nutritional supplements\u201d (25)\n187\n61\nEvaluation of health-related websites: Identification of the most trustworthy website (23)\n154\n50\nEvaluation of health-related websites: Ability to identify the purpose of a website\u2014to sell services (24)\n42*\n46\nEvidence-based decision-making: Agree that \u201cnone of the websites makes a good case for taking nutritional supplements\u201d (25)\n67\n22\n*This question was added later, and, therefore, it had a smaller number of respondents (n = 92).\nNote: RRSA question numbers are shown in parentheses; see Multimedia Appendix 1 for exact question wording.\nTo facilitate comparison of the three websites, we built in standard features that provided clues about high or low credibility. The standard features are URLs (two websites were .org and one was .com), links to the authors\u2019 biographies, dates of publication, references, disclaimers, and links to organizations with which the authors are affiliated. These features act as contextual clues that maximize or minimize the trustworthiness of the websites. A review of such features is part of many website evaluation recommendations (for example, in their 1999 publication, Kotecki and Chamness [11] draw evaluators\u2019 attention to a website\u2019s features rather than its text), yet it is unclear if health information consumers are able to compare these features across multiple websites.\nThese standard features, rather than the text content, are intended to differentiate the websites in terms of their credibility. Because all respondents are equally uninformed about the nutritional supplements described in the text, they must attend to other features when making quality-related judgments. This purposeful design was motivated by the desire to avoid the confounding influence of pre-existing knowledge about the subject matter described in the document that is being judged. A good measure of one\u2019s ability to critically evaluate Web pages is being able to disentangle the judgment of a website\u2019s features from the judgment of its content. Study participants may have had preconceived notions about the quality of nutritional supplements depending on their purpose (e.g., cognitogenics are for sleeping disorders and gustatogenics are for appetite suppression). To avoid a possible interaction between the untrustworthy features of a website and the believable description of the nutritional supplement, we asked a group of students (n = 52) to judge the trustworthiness of the supplements\u2019 descriptions presented as Microsoft Word documents rather than as websites. Although the level of trustworthiness was about the same for all nutritional supplement descriptions, the least trusted nutritional supplements were placed on the website with the highest number of untrustworthy features.\nWhen five subject matter experts independently reviewed the three websites and rated their trustworthiness using the Kotecki and Chamness [11] website evaluation tool, they reached 100% agreement regarding the most trustworthy site. In comparison, undergraduates\u2019 performance was much poorer: only 50% of respondents were able to identify the most trustworthy website (see Table 1).\nUnderstanding the Difference Between Scholarly Resources and Sales Pitches\nLess than half of respondents determined the purpose of the least trustworthy website, which was to sell products and services. The visitors to this .com website are charged for reprints of the content, offered discounted products, and provided with multiple prompts (e.g., a running line) to book a consulting appointment with a private nutritionist who has few relevant qualifications. Customer testimonials posted on this site describe fantastic outcomes achieved within an unrealistically short time frame.\nLess than a quarter of study participants reached the correct conclusion that none of the websites made a good case for taking the nutritional supplements, whereas 39% of respondents thought that all three websites made a good case for taking the supplements.\nUnderstanding Plagiarism\nHealth care professionals are expected to share health information with others, for example, by summarizing information from a variety of sources and distributing it to patients and clients. Higher education programs prepare students to apply standard rules for acknowledging contributions by others and referencing idea sources. Because this skill set is expected to become an integral part of their professional ethics, we built the RRSA to include measures of students\u2019 knowledge of plagiarism, their ability to recognize it, and their awareness of its penalties. Our results indicate that the vast majority of students (92%) know that their university may impose a severe penalty for plagiarism, up to and including expulsion. Table 2 and Table 3 display responses to sample questions that measure declarative knowledge of plagiarism. They show that many students are aware that common knowledge can be reproduced without references, whereas words written by others should be enclosed in quotation marks and accompanied by a complete reference. But when presented with more ambiguous examples of plagiarism, some study participants demonstrated misconceptions about what constitutes plagiarism. A surprisingly large number of respondents believed that it is appropriate to present another person\u2019s ideas as their own without citing a specific source, especially if this person is a relative or if the original words have been slightly modified.\nTable 2\nUnderstanding plagiarism: when references are needed (n = 308)\nWhich of the following can be reproduced without proper reference? Check all that apply:\nRespondents With Correct Positive or Negative Answers\nn\n%\nCommon knowledge*\n294\n96\nHospital board member\u2019s point of view\n264\n86\nMy classmate\u2019s ideas\n232\n75\nUnpublished works\n223\n73\nSpoken word\n209\n68\nMy dad\u2019s political opinions\n156\n51\n*Common knowledge can be reproduced without proper reference.\nNote: Items are scored as +1 if the answer is a correct positive or a correct negative and +0 if the answer is a false positive or a false negative.\nTable 3\nDefining plagiarism (n = 308)\nWhich of the following are plagiarism examples? Check all that apply:\nRespondents With Correct Positive or Negative Answers\nn\n%\nSubmitting a free research paper that was downloaded off the Internet.*\n290\n95\nReproducing a sentence that you found quoted in a book without referring to the original source.*\n276\n90\nEnclosing the word-for-word sentence in quotation marks, accompanied by a citation.\n271\n88\nCopying from the source verbatim without any quotation marks but adding a citation.*\n215\n70\nPutting someone\u2019s idea in my own words without citing a specific source.*\n201\n65\nUsing similar sentence structure to express another person\u2019s ideas without referring to the original source.*\n169\n55\n*These items are examples of plagiarism..\nNote: Items are scored as +1 if the answer is a correct positive or a correct negative and +0 if the answer is a false positive or a false negative.\nTo measure procedural knowledge of plagiarism, we ask respondents to compare a sentence from a Health Affairs article by Lapetina and Armstrong [33] to two other sentences that may have been plagiarized (question 20). Over two thirds of respondents (82%, n = 253) detected plagiarism in a sentence that closely follows the original but provides no reference to the original source. The percent of respondents who correctly identified a sentence without plagiarism (89%, n = 275) was comparable to the percent of respondents who knew that they should enclose the word-for-word sentence in quotation marks and cite the source (88%, n = 271, as shown in Table 3).\nAwareness of Personal Health Information Competencies\nWhen asked \u201cHow do you rate your research skills overall?\u201d most respondents (84%) believed that their skills were good, very good, or excellent. To compare self-reported and actual skill levels, we computed an overall health information competency score for each participant. An acceptable level of internal consistency reliability (Cronbach alpha = .78) for 56 right\/wrong items indicates that it is appropriate to calculate the overall score as the sum of points of these 56 items. The overall scores ranged from 20 to 54 with a mean of 37 (SD = 6.35) and did not significantly depart from a normal distribution.\nActual performance was examined by self-reported skill level. The group differences were mostly in the expected direction (see Table 4), but there was a large amount of variation in the overall score within each self-reported skill level. This indicates that the overall health information competency score was high for some students and low for other students, despite the fact that their self-reported competency was the same.\nTable 4\nMeans for health information competency overall score by self-reported skill level\nHow do you rate your research skills?\nn\nMean Overall Score\nSD\nNonexistent\n0\n-\n0\nPoor\n3\n36.33\n4.04\nFair\n47\n34.89\n5.52\nGood\n162\n36.89\n6.29\nVery good\n83\n37.64\n6.89\nExcellent\n13\n36.77\n6.10\nTotal\n308\n36.78\n6.35\nHealth information competencies may vary as a function of education; therefore, we regressed undergraduates\u2019 overall scores on the amount of credit hours earned toward the bachelor\u2019s degree (Step 1) and self-reported skill level (Step 2). The level of education was operationalized as the number of credit hours earned (0-9, 10-24, 25-40, 41-70, and more than 71). The analysis was conducted for 302 undergraduate students (six graduate students were removed from this analysis). Age could not be used as a control variable because most students (95%) fell into the same category of 18 to 23 years of age. The variables entered on Steps 1 and 2 account for 8% of variance in the overall score (R2 = .08). The amount of education significantly predicted the overall score (\u03b2 = .28, P < .001). When credit hours earned were held constant, self-reports of skill fail to explain a significant amount of variance in the overall score (\u03b2 = .08, P = .23). Overall, the results suggest that although students\u2019 self-ratings of research skills tend to increase with the increasing level of education, these self-reports may not be an accurate predictor of students\u2019 actual health information competencies.\nDiscussion\nInterpretation of Findings\nThe present study represents a systematic effort to measure health information competencies using a standardized and reliable measurement tool, the Research Readiness Self-Assessment (RRSA). The data were obtained from a diverse sample of 308 respondents (77% response rate). Nonrespondents (n = 92) differed from respondents (n = 308) in terms of their academic level: freshmen were slightly more likely not to participate in the RRSA than higher-level students. The most likely explanation for nonparticipation is a lack of interest in extra credit rather than the computer-assisted administration of the RRSA. It is possible, of course, that students with particularly poor computer skills found the online administration a barrier. However, a semester after we collected the data reported in this paper, there was a 100% participation rate by 180 undergraduates in two introductory courses where the instructors required RRSA completion. The two course instructors reported no student complaints about not being able to follow emailed instructions on how to complete the assessment.\nThe data indicate that many students lack important competencies that may limit their ability to make informed health choices. We observed deficiencies in the areas of conducting advanced searches, discriminating among different types of information sources, referencing other people\u2019s ideas, and evaluating information from Web pages and journal articles. Our data suggest that undergraduate students are inaccurate judges of their own competencies and hold a very positive view of their ability to do research. This finding may reveal an important barrier to building health information competencies of college-age students.\nWe found that there is a large competency gap between the average and the best information consumer. An average undergraduate in our sample is able to solve only 68% of problems that are solved by the best performing study participant (an average score of 37 versus a maximum score of 54). Health information competencies are applied to transform health-related information into knowledge that is consistent with the most current medical practice. High competence variability is a proxy indicator of students\u2019 varying ability to make evidence-based decisions. In the past, limited access to information may have prevented health information consumers from acquiring knowledge and making informed choices. The new generation of health information consumers has, for the most part, easy access to information; yet it may not be able to take full advantage of this convenient access.\nOur study shows that individuals with limited health information competencies may fail to locate the best available information due to employing poor search strategies. Searches that do not take into account all of the important criteria often produce low-relevancy documents or documents from commercial websites that promote products or services. These sites often present one-sided evidence, which can be detrimental to making a good decision about one\u2019s health. Overall, many students are rather unsophisticated information consumers who rely on basic searchers and the easiest ways of retrieving information.\nWe found that many individuals know little about information sources\u2014primary versus secondary, articles versus books, commercial versus noncommercial websites, and opinion pieces versus empirical studies. Information consumers who do not understand these distinctions are likely to engage in information processing that is shallow and superficial. They may, for example, follow a search path that produces the highest number of documents, rather than a path that produces documents of the highest quality. When the number of documents criterion is applied, Google and Yahoo significantly outperform all scholarly databases available through libraries. For instance, a Google search for the keyword health produces, in less than a second, over 8 million results ordered by popularity (as of June 2005, 25% of these results had .com URLs and 16% had .org or .gov URLs), where a similar search in Medline Plus produces 665 results, organized by health topic. With heavy reliance on public-access Internet search engines, an Information Age generation student may have an inaccurate conception that the Internet is the only place where society stores its best knowledge.\nOnce the plethora of documents is obtained, they need to be critically evaluated. Although health consumers are warned to critically examine websites to determine the document\u2019s purpose, author\u2019s affiliation, date of publication, and other features [11-14], these website evaluation criteria are only useful to those who know how to apply them. Many students in our sample appear not to possess these skills, and this finding is consistent with other observational studies (e.g., [17]). Our website evaluation exercise reveals both poor judgment and readiness to follow the lead, even when the authors of the online documents do not explicitly ask for purchase of their products. Although we measured a behavioral intent, rather than an actual behavior, there is still a significant potential for harm, ranging from financial losses to negative health effects, if only a few individuals execute their intent to take nutritional supplements that can be best described as \u201cfake\u201d or \u201cbogus.\u201d As we designed the most trustworthy website for the RRSA, it was alarming to witness the ease of misrepresenting or even falsifying health information. In designing the trustworthy site, we tried to meet as many website evaluation criteria as possible, and it became very apparent that these criteria do not guarantee information accuracy. Even completely false information about nonexistent food supplements can be made to appear trustworthy, as though it comes from an authoritative source.\nIndeed, there is no substitute for good judgment when it comes to navigating information. Because this good judgment is a product of both critical thinking and extensive knowledge of the subject matter being researched, we believe that higher education programs are uniquely positioned to develop health information competencies. However, initial work on developing Information Age competencies needs to be done at the K-12 level when children are beginning to be exposed to various sources of information, including the Internet.\nIn this study, we reviewed three broad categories of information competencies\u2014obtaining information, evaluating information, and using information. Using information includes such behaviors as reaching evidence-based conclusions and sharing information with others, a behavior guided by one\u2019s understanding of plagiarism. One study of plagiarism revealed that cyberplagiarism, or inappropriate use of phrases and ideas published on the Internet, is prevalent even among scholars [34]. Our findings suggest that, in college students, the plagiarism behavior may originate not only from motivation to cut corners (e.g., to cut-and-paste text without citations) but also from the lack of nuanced knowledge about plagiarism. The information revolution has rapidly intensified the exchange of ideas, but the distinction between plagiarism and proper acknowledgment of others\u2019 ideas continues to be poorly understood. Many students, for example, think that they do not need to provide references for paraphrased sentences or for sentences whose structure they modified. Perhaps these students view plagiarism as a violation of ownership of exact words rather than a violation of ownership of ideas. Similarly, some respondents believe that it is appropriate not to give credit for original ideas that are expressed orally (rather than in writing) or by people whom they know well. If carried into one\u2019s professional life, this misconception can make it difficult to follow ethical norms for recognizing others\u2019 knowledge contributions. Such ethical norms are strong in health professions, and their violation may lead to negative consequences.\nPerhaps the most interesting finding is the fact that participants are so unaware of their own skill deficiencies. It is possible that students make global judgments about their research skills based primarily on their ability to access information. That is, one\u2019s ability to access information may be confused with one\u2019s ability to generate knowledge from the information accessed. But obtaining information is merely the first step of knowledge acquisition. All of our study participants can access the Internet, as demonstrated by completing the RRSA online, but not all may be able to make good use of the information they access. Extending the argument by Solomon and Leigh [9] from television to Internet search engines, we conclude that the effort an individual expends to locate millions of documents in Google is a poor indicator of the true effort needed to process the obtained material \u201cif one aimed at a deeper understanding of it\u201d [9]. The Information Age generation of college students may benefit from this point.\nImplications for Health Promotion Practice\nThe findings of our study have several implications for individuals who practice health promotion for health information consumers. Health educators, librarians, and other professionals who play an active part in promoting health information literacy need to assist health information consumers in becoming more aware of their skill limitations. These professionals should develop their own proficiency in managing modern media and be able to find, evaluate, interpret, and present health-related information to other information consumers. Research on health information competencies of practicing health professionals remains limited, and we do not yet have a complete picture of their preparedness for evidence-based practice. But in one survey study of 1097 registered nurses, it was found that many respondents \u201chad no exposure to the research process in their educational programs, do not appreciate the importance of research to practice, and have great difficulty understanding research articles\u201d [35]. In this study, most registered nurses did not search databases such as Medline or felt skilled to do so. This preliminary evidence suggests that health professionals need to build their health information competencies.\nThe RRSA instrument offers an operational definition of information literacy, which remains an ill-defined concept. Upon examination of 97 Medline articles on the topic of information literacy for health care professionals, Saranto and Hovenga [36] found that the concept of information literacy has not yet been established. It is sometimes used interchangeably with computer literacy and informatics awareness or with the ambiguous term computer experience. The RRSA assessment used in the present study adds to the literature on health literacy by defining basic knowledge and skills needed for managing electronic health information resources.\nAmong the limitations of the present study is the narrowly focused sample, which limits our ability to generalize the study\u2019s findings to the broader population of health information consumers. The students from a Midwestern university may not be completely representative of the entire population of US Information Age students, due to, for example, the relatively homogeneous ethnic composition and possible overrepresentation of individuals raised in rural communities. In our future studies, we intend to broaden the pool of RRSA participants by including multiple educational institutions as well as urban and rural communities located in different geographic regions.\nIn contrast with many health information literacy studies, this research presents the results obtained via direct measure of skills and knowledge rather than via self-reports by health information consumers. While the reliability of the RRSA assessment reaches acceptable levels, it is necessary to further assess its unidimensionality, content validity, and criterion-related validity. A comprehensive validation study of the RRSA instrument is currently under way.\nConclusions\nToday, health consumers are actively seeking information and using it to make health decisions. The ease of accessing information may influence their perceptions of their ability to make informed health decisions. Our study shows that to become savvy information consumers, young people may need assistance in understanding the various health media, building awareness of their own skill sets, and improving their ability to make evidence-based decisions. Individuals with less education and exposure to information-related activities are expected to have even lower health information competencies than our study participants [37]. Health educators must continue to partner with a variety of groups that play an important role in promoting health information literacy, such as librarians and educators.\nThe assessment tool used in the present study is a self-administered instrument that provides a reliable account of health information competencies related to managing electronic health information. Data acquired through this research can be used to suggest curriculum improvements and estimates of the higher end level of skill held by health information consumers. It can also be used to educate health information consumers about their levels of skill necessary for managing health information from electronic sources. RRSA findings suggest that health information competencies of undergraduate students, many of whom will soon enter a variety of health professions, are limited. Health literacy educators can utilize RRSA findings to design educational interventions that impact information consumers\u2019 skills and prepare them for the challenges of living and working in the Information Age.","keyphrases":["health information","electronics","consumer health information","electronic health information","evaluation of electronic resources","telecommunications","patient education","educational status","computer network"],"prmu":["P","P","P","P","R","U","R","M","M"]}
{"id":"Pediatr_Radiol-3-1-1915603","title":"Nonpalpable testes: is there a relationship between ultrasonographic and operative findings?\n","text":"Background Ultrasonography (US) as a diagnostic tool in the work-up of boys with nonpalpable testes (NPT) is still controversial.\nIntroduction\nFailure of testicular descent is a common childhood anomaly, being seen in 0.8\u20132% of full-term and 18\u201330% of premature boys. In undescended testes (UDT) the testis fails to migrate into the processus vaginalis, failing to reach the scrotum at 35\u00a0weeks of gestation. It is unilateral in 60\u201370% of cases. Approximately 20% of UDT are nonpalpable on physical examination [1]. There are four possible explanations for this phenomenon. First, the testis may be situated in the scrotum or inguinal canal, but is too small or there is too much subcutaneous adipose tissue. Second, the testis is located inside an open processus vaginalis in the inguinal canal and may be intermittently inguinal and abdominal in position. Third, it is in a true abdominal position; and fourth, the testis is lacking [2].\nThe literature reports that nearly half of viable nonpalpable testes (NPT) are in an abdominal position and 5% are in the inguinal canal. The remaining 45% are atrophic or absent, mostly as a result of in utero spermatic cord torsion and are located abdominally, inguinally or scrotally [3, 4].\nExperienced paediatric surgeons can accurately diagnose UDT by repeated and meticulous palpation of the scrotal and inguinal region. Since palpation is quite subjective, the diagnosis mostly depends on the examiner\u2019s experience and ability [2]. Currently there is controversy as to the step to take when clinical examination fails to identify a testis. Further exploration might be done by means of laparoscopy or by noninvasive methods such as ultrasonography (US), CT, MRI, venography or arteriography [3, 5, 6]. Of these, US is the imaging method of choice in children because it is noninvasive and does not use ionizing radiation. Furthermore, it is cheap and widely available [7].\nWhen NPT is correctly diagnosed there is consensus on the need for early treatment, as this may decrease the risk of testicular cancer [8\u201312] and fertility problems [13\u201315]. We performed a study aimed at assessing the value of US in the diagnostic work-up of NPT, and to this end evaluated the relationship between preoperative US data and operative findings.\nMaterials and methods\nDuring a 7-year period, 880 boys referred to the Department of Paediatric Surgery, Erasmus MC-Sophia Children\u2019s Hospital, Rotterdam, underwent primary surgery for UDT. US was performed in 137 of these children with 156 NPT. Prior to US the patients were clinically examined by a general practitioner, a paediatrician, a urologist or, in most cases, by a paediatric surgeon. Clinical examination by a paediatric surgeon was with the boy in the supine position, the lower half of the body undressed. The examiner placed the index finger and thumb of the right hand on either side of the inguinal canal, thus preventing testes lying distally from the inguinal canal from withdrawing into the inguinal region during palpation of the scrotum. With the examiner standing on the right side of the patient, the inguinal region was then examined with the fingertips of a warm left hand. If there seemed to be no palpable testis, an attempt was made to empty the inguinal canal by carrying out an ironing movement with the fingertips stroking in the direction of the scrotum. This may reveal a palpable testis at the level of the exit of the inguinal canal, immediately shooting back deep into the inguinal canal. If no testis could be located at all, the perineum, the base of the penis and the thigh were closely examined to exclude an ectopic testis.\nUS was performed using an Ultramark 9 HDI unit (Advanced Technology Laboratories, Bothell, Wash.) with a high-frequency, broadband, 7\u201310-MHz linear array transducer or a Philips ATL HDI 5000 unit, also with a high-frequency, broadband, 5\u201312-MHz linear array transducer. US was performed by eight different paediatric radiologists. After US all boys were clinically re-examined by a paediatric surgeon and again on the operating table when under anaesthesia.\nSubsequently, all boys underwent surgery and testicular position and other operative findings were assessed intraoperatively.\nDepending on the US results for NPT different operative procedures were used. Laparoscopy was usually performed when US could not identify a testis. In the presence of a viable abdominal testis, a one- or two-stage orchiopexy was performed. Anorchia was diagnosed when a blind-ending vas deferens was identified. When the vas deferens and testicular vessels entered the internal inguinal ring the procedure was completed by an inguinal exploration. When a viable testis was found, orchiopexy was performed. When a nubbin (vanished testis) or an atrophic testis was found, this was excised for pathological examination. Inguinal exploration was usually performed when US did show an inguinal testis. When a viable testis was found, orchiopexy was performed. A viable testis was defined as having a volume according to age [16].\nAll US findings were compared with intraoperative findings.\nResults\nEligible for this study were 137 boys with 156 NPT. Excluded from analysis were two with M\u00fcllerian inhibitory factor deficiency syndrome, implying abnormal testis position. The ages of the remaining 135 boys ranged from 4\u00a0weeks to 16.2\u00a0years. Of the remaining 152 NPT, 70 were left-sided, 48 right-sided and 17 bilateral.\nFour patients (four NPT) had been referred to the radiologist by a general practitioner and 24 patients (30 NPT) by a paediatrician. Most patients, 107 with 118 NPT, were first diagnosed by a paediatric surgeon before being referred to the radiologist. All boys were seen by a paediatric surgeon after the US examination for re-examination and to discuss therapy determined by the US findings.\nWith knowledge of the US results, 28% of the NPT previously diagnosed as not palpable now appeared to be palpable. Three of the four NPT (75%) in patients first seen by a general practitioner were palpable when re-examined by a paediatric surgeon, as was the case for 18 of 30 NPT (60%) referred by a paediatrician. Of the NPT first examined by a paediatric surgeon, 18% were palpable on re-examination with knowledge of the US results.\nUS was able to locate 103 of 152 NPT (68%): 16 were found in the abdomen and 87 in an inguinal position; therefore 49 could not be found.\nComparing the US results with the surgical findings, there was a 100% positive predictive value (PPV) for the 16 abdominal testes seen on US; all were indeed found abdominally. Nevertheless, 14 viable abdominally located testes were missed by US. At surgery, all these testes were found in the lower abdomen\u2014on the iliopsoas muscle, in the pelvis, or close to the internal inguinal ring. US located 87 inguinal testes, 84 of which were indeed found in the inguinal region (97% PPV); the other three were viable and found intraabdominally. Thirty-five of the NPT were defined as small, atrophic or vanished at operation.\nUltimately, US failed to locate 49 NPT. Nevertheless, 16 of these at surgery appeared to be viable, with 14 located intraabdominally and two in the inguinal region. Two atrophic testes were found in the abdomen. A further 22 atrophic testes were found inguinally or scrotally. Anorchia was present in nine boys.\nIn summary, 92 (85%) of the 108 NPTs defined as viable at operation were located by US. While nearly all viable inguinal testes (74\/76) were correctly located by US, only half of the viable abdominal testes (16\/33) were seen on US. The relevant data are summarized in Fig.\u00a01.\nFig.\u00a01Ultrasound findings in 152 nonpalpable testes in relation to findings at operative exploration\nDiscussion\nOver the years, a wide range of investigations\u2014from vascular techniques and imaging techniques to laparoscopy\u2014have been used in detecting NPT [17]. Inguinal exploration used to be the standard surgical approach for nonpalpable testes. If the testis was not found at or below the internal inguinal ring, the procedure was extended to the abdomen. Laparoscopy has gained greater acceptance in diagnosing and treating NPT. Cortesi et al. [18] first reported its use in 1976 in an 18-year-old adolescent. While diagnostic laparoscopy is highly sensitive in detecting NPT [1, 2, 19, 20], it carries an approximately 1% risk of major or minor operative complications. Furthermore, the long-term incidence of peritoneal adhesions following laparoscopic procedures in children is approximately 10% [4, 21].\nThe literature is far from unanimous on the usefulness of US in UDT. Some authors recommend US for its feasibility and potential to settle the subsequent operative procedure [7, 21\u201327]. Others are sceptical because they found poor outcome for NPT, and because US does not exclude the necessity for laparoscopy [3, 28\u201330]. Most of the studies included only small groups of patients [24\u201326, 28, 30]. Not all studies investigated only NPT; some included all UDT [26, 27, 30]. Most studies compared US results with findings during the operative procedure [3, 7, 21, 22, 26\u201329], others compared CT and\/or MRI with US [23\u201325, 30] in relation to the operative procedure. The most relevant conclusions of these studies are summarized in Table\u00a01. \nTable\u00a01Review of the literature on the use of US in boys with NPT (n.a. not applicable, because all testes were \u2018true\u2019 NPT)ReferenceStudy designTestes (n)Located by USLocated \u201ctrue\u201d NPTAccuracy of US (%)aPredictive value of US (%)aRe-examinationRemarksAdvice in NPTPositiveNegativeTestes palpablePrevious examiner[22]Retrospective\/medical charts5528\/55 (51%)n.a.8410067n.a.\u2013US[29]Prospective\/cohort6237\/62 (60%)n.a.761000n.a.Vanished not included in accuracy and predictive valuesLaparoscopy[3]Retrospective\/medical charts6612\/66 (18%)0\/21 (0%)18b100b0b45\/66 (68%)Referring physician82% US ordered by referring physician; 93% US performed elsewhereLaparoscopy[23]Retrospective\/medical charts170111\/170 (65%)n.a.72b100b19bn.a.\u2013US[21]Prospective\/cohort7448\/74 (65%)16\/29 (55%)65b100b0b45\/74 (61%)Referring physicianOnly nubbins palpable on re-examinationUS[24]Prospective\/cohort2113\/21 (62%)n.a.769250n.a.US twice and blind to physical examinationUS \u2192 MRI[30]Retrospective\/medical charts225\/12 (42%)3\/7 (43%)581002913\/22 (59%)Referring physician\u2013Laparoscopy[7]Prospective\/cohort4545\/45 (100%)6\/8 (75%)9393\u201336\/45 (80%)Referring physicianPhysical examination and US blind to each otherUS[26]Prospective\/cohort3817\/38 (45%)1\/18 (6%)6188381\/21 (5%)ExpertUS blind to physical examinationUS if obese20\/41 (49%)Referring physician[28]Prospective\/cohort142\/14 (14%)n.a.211008n.aSurgery blind to USLaparoscopy[27]Prospective\/cohort6961\/69 (88%)4\/12 (33%)9910088n.aUS[25]Prospective\/cohort2315\/23 (65%)n.a.919175n.a.US and CT blind to each otherUSaAccuracy and predictive values were (re)calculated: negative surgical exploration\/absent testes were counted as true negative and testicular remnants\/\u201cnubbins\u201d as false negative.bAbsent testes and atrophic testes grouped; number of absent testes not (exactly) mentioned in article (calculated as false negative).\nAn important message emerging from these articles is the very high PPV of US and the importance of thorough examination by a physician with experience in small genital examination. More than two-thirds of testes of referred patients were palpable on examination by experienced physicians. Many US investigations could have been avoided if patients had first been referred to an expert. The (paediatric) radiologist\u2019s experience is also vital and US should be undertaken in the hospital where further treatment will be given.\nUS remains the modality of choice in our centre because we value its noninvasiveness, child-friendliness and cost-effectiveness. It also facilitates planning the correct surgical procedure (Fig.\u00a02) [7, 29].\nFig.\u00a02Flow chart representing the diagnostic and therapeutic approach in boys with NPT\nIn summary, we found 97% sensitivity of US for viable inguinal testes and 48% sensitivity for viable abdominal testes. When US located a testis it was also found at that site during surgery in 97% of NPT (PPV 97%). In our study, only three viable testes were not located correctly, being in the inguinal canal at US and within the abdomen at surgery. This can be explained by the fact that there was a patent internal inguinal ring with a mobile testis. More than two-thirds of NPT could be localized with US. Approximately one-third of NPT that were not found with US appeared to be viable at surgery. Of 34 testes considered nonpalpable by a general practitioner or paediatrician, 21 (62%) were palpable on re-examination by a paediatric surgeon after US. In contrast, of 118 testes considered nonpalpable by a paediatric surgeon, only 21 (18%) were palpable on re-examination.\nConclusion\nWe feel our results underpin the importance of referring boys with a presumed NPT to a physician with expertise in genital examination of small children. This will avoid unnecessary US investigations. Furthermore, we recommend US for all boys with NPT diagnosed by an experienced physician (Fig.\u00a02). The findings would then determine the subsequent operation: diagnostic and\/or therapeutic laparoscopy for all NPT with negative US, or intraabdominal testes located by US. In addition, we recommend inguinal exploration whenever US demonstrates an inguinal testis.","keyphrases":["nonpalpable","testes","children","ultrasound"],"prmu":["P","P","P","U"]}
{"id":"J_Mol_Biol-2-1-2279256","title":"Predicting Protein Function from Structure\u2014The Roles of Short-chain Dehydrogenase\/Reductase Enzymes in Bordetella O-antigen Biosynthesis\n","text":"The pathogenic bacteria Bordetella parapertussis and Bordetella bronchiseptica express a lipopolysaccharide O antigen containing a polymer of 2,3-diacetamido-2,3-dideoxy-l-galacturonic acid. The O-antigen cluster contains three neighbouring genes that encode proteins belonging to the short-chain dehydrogenase\/reductase (SDR) family, wbmF, wbmG and wbmH, and we aimed to elucidate their individual functions. Mutation and complementation implicate each gene in O-antigen expression but, as their putative sugar nucleotide substrates are not currently available, biochemical characterisation of WbmF, WbmG and WbmH is impractical at the present time. SDR family members catalyse a wide range of chemical reactions including oxidation, reduction and epimerisation. Because they typically share low sequence conservation, however, catalytic function cannot be predicted from sequence analysis alone. In this context, structural characterisation of the native proteins, co-crystals and small-molecule soaks enables differentiation of the functions of WbmF, WbmG and WbmH. These proteins exhibit typical SDR architecture and coordinate NAD. In the substrate-binding domain, all three enzymes bind uridyl nucleotides. WbmG contains a typical SDR catalytic TYK triad, which is required for oxidoreductase function, but the active site is devoid of additional acid\u2013base functionality. Similarly, WbmH possesses a TYK triad, but an otherwise feature-poor active site. Consequently, 3,5-epimerase function can probably be ruled out for these enzymes. The WbmF active site contains conserved 3,5-epimerase features, namely, a positionally conserved cysteine (Cys133) and basic side chain (His90 or Asn213), but lacks the serine\/threonine component of the SDR triad and therefore may not act as an oxidoreductase. The data suggest a pathway for synthesis of the O-antigen precursor UDP-2,3-diacetamido-2,3-dideoxy-l-galacturonic acid and illustrate the usefulness of structural data in predicting protein function.\nIntroduction\nThe acceleration of genome sequencing has created a challenge for modern biology, namely, interpretation of the vast and growing amount of genetic information in the databases. Search algorithms such as the basic local alignment search tool (BLAST)1 can efficiently find related genes, but only in a small minority of cases is the precise function of a novel enzyme equivalent to that of the highest scoring hit. In straightforward cases the output is useful to generate hypotheses for the novel enzyme's function, based on common catalytic chemistry, substrates or both, and these postulates are then amenable to direct biochemical investigation. To make full use of genome data it is important that where such standard bioinformatic analysis cannot predict precise roles and biochemical function assays are not possible, tools are developed to differentiate the functions of genes. A good example of such a challenging case is where gene products are annotated as short-chain dehydrogenase\/reductases (SDRs). Members of this family share low sequence identity (typically 15\u201330%) and catalyse a wide range of chemical reactions, including oxidation, reduction, epimerisation, dehydration and decarboxylation (reviewed in Ref. 2).\nBordetella\nparapertussis and Bordetella bronchiseptica are pathogens of mammals. B. bronchiseptica is associated with respiratory tract infections in many animals including acute tracheobronchitis in dogs (kennel cough)3 and atrophic rhinitis in pigs,4 whereas B. parapertussis can cause whooping cough in humans.5 A separate lineage of B. parapertussis infects sheep.6\nThe major component of the outer leaflet of the outer membrane in Gram-negative bacteria is lipopolysaccharide (LPS). The complete structure of this macromolecule in B. bronchiseptica and B. parapertussis has been recently described.7 The lipid A domain of LPS, which consists of a diglucosamine backbone substituted with fatty acyl chains, forms the outer leaflet of the outer membranes of the bacteria. Lipid A is linked to a complex, branching oligosaccharide known as the core. Lipid A-core comprises a proportion of the LPS that is exposed on the cell surface and is known as band B. In B. bronchiseptica, lipid A-core can be substituted with a trisaccharide and this structure is known as band A. Synthesis of the band A trisaccharide requires functions encoded in the wlb gene cluster (previously named bpl).8\nBoth B. bronchiseptica and B. parapertussis also substitute their LPS with an O antigen. This O antigen contains 12 to 15 2,3-diacetamido-2,3-dideoxy-l-galactosaminuronic acid (l-GalNAc3NAcA) residues9 and is required for full virulence in animal and in vitro models of infection.10 O-antigen biosynthesis requires genes in the wbm cluster that is adjacent to the wlb genetic locus. The wbm locus contains three neighbouring SDR genes (wbmF, wbmG and wbmH), all of which have been annotated as nucleotide-sugar epimerases\/dehydratases.\nThe structure of complete O antigen and the homology of wbm genes with genes of known function have led us to propose a pathway for biosynthesis of this polysaccharide and we are currently testing various steps in this scheme as part of an ongoing project to determine the functions of all 24 wbm genes. This report specifically concerns the roles of the SDR genes wbmF, wbmG and wbmH.\nO-antigen residues are synthesised as sugar nucleotide precursors. The probable substrate for wbm locus-encoded biosynthesis of the nucleotide-activated l-GalNAc3NAcA is UDP-2,3-diacetamido-2,3-dideoxy-d-mannuronic acid (UDP-d-ManNAc3NAcA). This compound is related to the l-galacto configuration by inversion of the stereochemistry at the hexose 3 and 5 positions. In the bordetellae, UDP-d-ManNAc3NAcA is formed as a precursor for band A trisaccharide synthesis by 2-epimerisation of UDP-2,3-diacetamido-2,3-dideoxy-d-glucuronic acid, catalysed by WlbD.11 Because there are precedents for either a single12 or multiple13\u201316 SDR enzymes catalysing 3,5-epimerisation conversions of sugar nucleotides, we hypothesise that one or more of WbmF, WbmG and WbmH operate in this biosynthetic pathway to catalyse the conversion of UDP-d-ManNAc3NAcA to UDP-2,3-diacetamido-2,3-dideoxy-l-galacturonic acid (UDP-l-GalNAc3NAcA). Because of the low percentage conversion of the WlbD-catalysed reaction, UDP-d-ManNAc3NAcA is not currently available for biochemical studies and therefore this hypothesis cannot be directly tested at the present time.\nIn this report we demonstrate the involvement of wbmF, wbmG and wbmH in O-antigen expression by mutation of the genes in B. bronchiseptica. Because the putative substrate is unavailable, we elected to probe the catalytic functions of WbmF, WbmG and WbmH by characterising these proteins' three-dimensional structures. With soaking and co-crystallisation experiments, densities corresponding to cofactors and nucleotide portions of substrate analogues can be identified within the active sites. Biochemical and crystallographic studies have defined the structural basis for the catalytic chemistries of a range of other SDR enzymes (reviewed in Ref. 17). Our knowledge of these structure\u2013function relationships enables us to interpret the structures of the wbm SDRs. We have analysed the potential for acid\u2013base chemistry in the active sites to differentiate their potential roles in vivo. Based on the composition of O antigen, the genetic context of these wbm genes and information from the structural studies, we propose roles for these genes in O-antigen biosynthesis. The results demonstrate the usefulness and limitations of X-ray data in elucidating biochemical pathways where catalytic activity cannot be directly measured.\nResults\nMutational analysis of wbmF, wbmG and wbmH\nTo test the hypothesis that wbmF, wbmG and wbmH are involved in O-chain biosynthesis, each gene was disrupted by insertion of a tetracycline-resistance cassette. Mutation of the chromosomal genes was confirmed by Southern hybridisation (data not shown). The effects of these mutations were assessed by silver-stained SDS-PAGE analysis and immunoblotting of mutant LPS (Fig. 1). Mutation of wbmG or wbmH results in apparent abrogation of O-antigen synthesis, as the LPS from these mutants lacks O band as detected by either silver stain or Western blot. O-antigen expression in the wbmF mutant, CNF0a, is dramatically reduced compared with the wild type, but this strain retains its ability to express a small amount of material with the electrophoretic mobility of O-band LPS and which binds the O-antigen-specific monoclonal antibody, D13B11.\nEach of the mutations was complemented by expression of the cognate wild-type allele in trans (Fig. 1). Each coding sequence was cloned into the broad host range vector pBBR1MCS(kan) under the control of the flaA promoter to generate complementation vectors. Complementation of the wbmG and wbmH mutations restored O-antigen expression to near wild-type levels. O-antigen expression in the complemented wbmF mutant was increased relative to the wbmF mutant, but was still considerably less than in wild type. The reason for incomplete complementation of the wbmF mutation is unknown, but is not likely to be due to polar effects of the mutation because, due to the organisation of genes in the wbm locus, wbmF is at the 3\u2032 end of an operon.18 Introduction of the empty complementation vector had no effect on O-antigen expression in any of the mutants.\nThe gene products WbmF, WbmG and WbmH are all in the extended SDR family\nThe SDR family is divided into the classical and extended subfamilies primarily on the basis of protein length and the sequence pattern of the glycine-rich cofactor-binding region close to the N terminus. According to both criteria, the products of wbmF, wbmG and wbmH, which are predicted to have 357, 310 and 313 amino acids, respectively, and all have the GxxGxxG motif near the N terminus, are extended SDRs2 (Fig. 2).\nWbmG and WbmH are closely related to each other, sharing 38% amino acid identity. WbmF, WbmG and WbmH are all homologous to Pfam01370 (NAD-dependent epimerase\/dehydratase family). The most closely related, characterised homologues of these Wbm SDRs in the protein databases are deoxythymidine diphosphate (dTDP)-glucose 4,6-dehydratases from Escherichia coli and Streptomyces venezuelae with 27% or 28% sequence identity (Table 1).19 This level of sequence conservation between members of the SDR family does not imply conservation of either catalytic chemistry or substrate; rather this functional information can be better determined by examination of the residues at key positions in the active sites and binding pockets. Several conserved motifs within the extended SDR subfamily facilitate alignment of sections of the Wbm SDR sequences with homologues (Fig. 2) but many of the critical functional amino acids are located outside of these motifs, and with this low level of conservation, it is not possible to make full sequence alignments with confidence that all of these residues are correctly identified.\nCrystallisation of His6-WbmF, His6-WbmG and His6-WbmH\nThus far the data implicated each of these wbm SDRs in O-antigen synthesis, but did not indicate precise roles for each gene. Therefore, in order to elucidate their individual functions we characterised the gene products by X-ray crystallography. The overexpression, purification and crystallisation of these proteins are described elsewhere.20 The structures discussed in the present work are summarised in Table 2.\nGeneral architecture of His6-WbmF, His6-WbmG and His6-WbmH\nThese proteins all exhibit the typical SDR family architecture (Fig. 3). The structures each comprise two domains. The first of these is the N-terminal Rossmann fold domain in which a central \u03b2 sheet is flanked by two layers of \u03b1 helices. The cofactor-binding motif GxxGxxG is located at the C-terminal edge of this \u03b2 sheet, which has seven parallel \u03b2 strands running in the order 3, 2, 1, 4, 5, 6, 7. The second domain is largely made up of C-terminal sequence and contains all of the residues involved in binding of the nucleotide portion of the substrate. The catalytic sites in SDR enzymes are located at the interface of these two domains where, in sugar-nucleotide-modifying enzymes, the substrate hexose is brought into proximity with the cofactor nicotinamide ring. The main architectural differences between these three proteins lie in their C-terminal regions (Fig. 3). The C terminus of His6-WbmG consists of a loop that stretches out from the C-terminal domain to interact with the Rossmann domain. In His6-WbmH, this loop is not visible in the density, although this difference may not reflect any distinction between the real structures of the two proteins in solution. In WbmF, the last 30 residues form a large bent helix that covers two faces of the C-terminal domain. There is also an insertion of 14 amino acids in WbmF relative to the other structures, including all residues from Gly198 to Arg212. This extra loop extends over the nicotinamide end of the cofactor-binding pocket.\nSDRs typically form dimers or higher oligomers. The crystal packing of these proteins suggests that WbmF, WbmG and WbmH have the conserved four-helix bundle usually found at the dimer interface. Analytical gel filtration chromatography indicated a hydrodynamic radius for each of these proteins consistent with either a monomer or dimer (data not shown). We conclude, therefore, that these proteins are present in solution predominantly in dimer form. In the WbmG and WbmH structures, the dimer interaction buries 1316\u00a0\u00c52 (5.7% of total protein surface) and 1562\u00a0\u00c52 (7.2%) of surface per dimer, respectively. In the WbmF structure the interface region is extended to include a short antiparallel \u03b2 sheet composed of the residues Ile151 to Ser153 from each monomer as well as the loops that connect these strands to the bundle (Leu154 to Pro160); 2131\u00a0\u00c52 is buried in the WbmF dimer interface, which is equivalent to 8.6% of the total protein surface. In all three cases, the majority of the residues buried at these interfaces are hydrophobic or aromatic.\nCofactor binding\nNAD can be modelled into the electron density in all three proteins. His6-WbmG and His6-WbmH copurify with this compound from the E. coli expression host, but in order to stabilise the protein in solution His6-WbmF required the addition of exogenous cofactor,20 and this is the most likely source of this molecule in these His6-WbmF structures. The preference in each case for NAD cofactor as opposed to NADP is indicated by the close interaction (2.7\u00a0\u00c5 hydrogen bonds) of an aspartate side chain with the 2\u2032-hydroxyl of the adenylic ribose. This side chain would disfavour binding of NADP by both steric and electrostatic repulsion of the NADP 2\u2032-phosphate. In some SDR structures [e.g., GDP-4-keto-6-deoxymannose 3,5-epimerase\/reductase (GMER)21 and RmlD22] the bound cofactor has direct access to solvent, indicating a pathway via which spent NAD\/NADP can be replaced from solution. In these structures, the bound cofactor is buried within the core of the protein; in the case of the His6-WbmF structure, the 14-amino-acid insertion supplies an additional occluding loop, further blocking release of cofactor in this conformation.\nBinding of substrate\nSoaking and co-crystallisation experiments with His6-WbmF and His6-WbmG revealed the binding mode for the nucleotide portion of the sugar nucleotide substrates of these enzymes (Fig. 3). The X-ray data from a His6-WbmG crystal soaked with UDP-glucose revealed extra density that could be modelled as far as the \u03b1-phosphate. The location of this molecule is consistent with the nucleotide-binding site for SDR homologue structures and with the hydrogen-bonding chemistry expected to bind uridine. No density was visible corresponding to the hexose portion of the soaked compound nor the second phosphate, suggesting that either this portion of the molecule is disordered within the crystal, or the compound was partially hydrolysed and the smaller uridine monophosphate (UMP) preferentially soaked into the substrate-binding pocket. In either case, the data indicate that the glucose-1-phosphate moiety of UDP-glucose is not strongly bound in a single conformation by WbmG. A similar soaking experiment but with GDP-mannose did not indicate any binding of this sugar nucleotide (data not shown), indicating a possible preference for the uracil base. The hydrogen bonding between the WbmG pocket and UMP suggests a basis for this preference (Fig. 3b). Although we were unable to obtain any complex of WbmH with a substrate analogue bound, the nucleotide-binding domain of WbmH is conserved in sequence and structure with WbmG, implying identical specificity and a common binding mode for the nucleoside portion of their substrates (Fig. 3c). UDP density to the \u03b1-phosphate is also visible in the WbmF\/NAD\/UDP ternary complex. The UMP density in this structure and the chemistry of its interaction with the protein suggests that this protein, like WbmG and WbmH, recognises a UDP-sugar substrate.\nBoth \u03b1 and \u03b2 nucleotide phosphates could be modelled in the data from a UDP soak into a WbmF-NAD+ co-crystal and co-crystallisation of His6-WbmG with UDP. In both structures the \u03b2-phosphate is coordinated by a conserved arginine (Fig. 4a and b), although the precise location of this phosphate is different in the two structures and both are inconsistent with the \u03b2-phosphate positions of bound substrate in the structure of a D128N, E129Q mutant of dTDP-glucose 4,6-dehydratase, DesIV, from S. venezuelae (Fig. 4c).23 In this DesIV structure [Protein Data Bank (PDB) ID 1R6D] the position of the substrate is consistent with the necessary overlap of molecular orbitals required for hydride transfer from the glucose C-4 to the cofactor. It is likely, therefore, that the \u03b2-phosphate positions in WbmF and WbmG structures do not represent the positions of equivalent phosphates when the native substrates for these enzymes are bound in the active site.\nOne common feature of substrate binding in SDR enzymes is pi stacking of the nucleobase with an amino acid side chain. In WbmF, this interaction involves Glu231 (Fig. 3a). Unusually, in WbmG the pi-stacking residue (Phe190) is the one that hydrogen bonds with the uracil N-3 (Fig. 3b). In WbmF, E. coli UDP-galactose 4-epimerase (GalE),24\nS. venezuelae DesIV,23 and Pseudomonas aeruginosa WbpP,25 the hydrophobic contact is provided by the residue in the n\u00a0+\u00a02 position, whose backbone amide hydrogen bonds with the base's O2 group (Fig. 5). This unusual feature of the substrate binding in WbmG could not have been predicted from sequence alignments alone.\nPotential catalytic chemistry\nSDR enzymes have a conserved catalytic triad that is involved in their oxidoreductase activity and that is classically composed of a spatially conserved serine, tyrosine and lysine (SYK) triad, for example, Ser142, Tyr166 and Lys170 in WbpP. The first member of this triad is sometimes found as a threonine (TYK),23 and the tyrosine can be replaced by a methionine (SMK).26 The SDR triad is conserved in WbmG and WbmH as TYK but in WbmF Ala131 superimposes onto the Ser\/Thr position (Fig. 6a).\nIn the context of O-antigen synthesis in Bordetella we are interested in the potential of the wbm SDR enzymes to catalyse secondary reactions of their substrates beyond the oxidoreductase reactions common to all SDRs. Of particular significance is the identification of a potential 3,5-epimerase. Despite the lack of a structure in which a full sugar nucleotide substrate analogue is bound, the cavity into which the substrate hexose binds is easily identifiable by comparison with homologue structures and because it is necessarily interposed between the UMP-binding site and the cofactor nicotinamide ring. The active sites of WbmG and WbmH are devoid of amino acid side chains capable of acid\u2013base chemistry except for their respective TYK triads and, in WbmH, Ser176. SDR enzymes that catalyse 3,5-epimerisation of their substrates have a spatially conserved catalytic cysteine and a basic side chain. In GMER these residues are Cys109 and His179;21 in GDP-mannose 3,5-epimerase (GME) they are Cys145 and Lys217.12 In WbmG and WbmH these amino acids superimpose onto hydrophobic side chains, but in WbmF this catalytic cysteine is conserved (Cys133) and there are two candidates for the basic side chain, Asn213 and His90 (Fig. 6b). Cys133 is disordered in all of our WbmF crystals, indicating that an element of induced fit may be required to bring all of the active-site components into place. His90 has a very unusual backbone conformation, with a cis peptide bond to the following residue. This is strongly suggestive of a functional role for this residue, since such conformations are rarely observed except where they are required to place a key side chain in the active site.\nModelling of the putative substrate into the WbmF active site\nThe analysis of the potential catalytic chemistry of WbmF suggested that its role in O-antigen biosynthesis may be to catalyse the 3,5-epimerisation required in the overall conversion of UDP-d-ManNAc3NAcA to UDP-l-GalNAc3NAcA. The 4-keto derivative of UDP-d-ManNAc3NAcA is therefore a likely substrate or reaction intermediate for WbmF. This compound contains a bulkier sugar than any of the substrates of the characterised sugar-nucleotide-modifying SDRs. An important test of our proposed pathway is that the enzymes must be able to accommodate these unusually large substrates. We modelled 4-keto UDP-d-ManNAc3NAcA into the active site of WbmF (Fig. 7). This showed that it is feasible for the substrate to occupy the active site of WbmF in a manner that is consistent with the experimentally determined binding site for UMP, the proximity of the most likely catalytic site, and good geometry.\nDiscussion\nThe abrogation or reduction of O-antigen expression as a result of wbmF, wbmG or wbmH mutations implicates all three genes in the biosynthesis of this molecule, and is consistent with the hypothesis that these three adjacent SDR genes catalyse the UDP-d-ManNAc3NAcA to UDP-l-GalNAc3NAcA conversion, which is probably required for O-antigen synthesis.\nStructural analysis of these three wbm SDR gene products also enables us to distinguish between them in terms of potential catalytic chemistry. Both WbmG and WbmH have the conserved SDR catalytic triad (TYK in these cases), which suggests that they should be competent to function as oxidoreductases. In both of these enzymes, the pocket that surrounds the active site is hydrophobic and featureless compared with other SDR enzymes; the lack of side chains capable of acid\u2013base chemistry indicates that the functions of these enzymes are likely to be limited to oxidation and\/or reduction and probably rules out specific 3,5-epimerase activity. The hydroxyl of Ser176 in WbmH may be capable of facilitating proton exchange; however, an amino acid pair capable of acid\u2013base chemistry is required for 3,5-epimerase function.\nSome SDRs that catalyse both oxidation and reduction bind cofactor irreversibly and recycle it within the active site.27 In other SDRs, this binding is reversible and the cofactor-binding site is situated at the bottom of a solvent-accessible groove (e.g., in E. coli GMER, PDB ID 1BWS).28 No such groove is visible in the WbmF, WbmG and WbmH structures, where the cofactor-binding sites are almost completely occluded. However, we do not take this observation as being diagnostic of irreversible cofactor binding in these proteins for the following reasons. Firstly, exogenous cofactor is required for WbmF to be stable in solution with respect to precipitation.20 Presumably either cofactor that copurifies with WbmF can be released, resulting in unfolding and aggregation of the protein, or apo-WbmF must bind cofactor supplied from the medium in order to fold correctly. Either case would suggest that NAD can be exchanged between WbmF and solvent. Secondly, human GalE reversibly binds cofactor,29 and yet does not have an open binding site like that of GMER. Finally, a conversion of configuration from d-manno to l-galacto can be catalysed by a single tight NAD+-binding enzyme such as GME,12 but we cannot envisage a pathway in which two or three such enzymes contribute to this transformation. Instead, we suggest that the protein loops that cover the NAD-binding site may be flexible and move to allow release and replacement of spent cofactor.\nWbmF lacks the serine\/threonine of the SDR catalytic triad. The conservation of the classical SDR catalytic triad is not absolute amongst members of the family. For example, in the very unusual SDR enzyme, biliverdin IX\u03b2 reductase (which catalyses the reduction of a range of substrates including some flavins and non-\u03b1-isomers of biliverdin), a histidine occupies the usual place of the catalytic lysine and tyrosine residues.30 The shapes and electron delocalisation exhibited by biliverdin IX\u03b2 reductase substrates imply that compared with the putative substrates of the Wbm SDR enzymes, they will have rather special requirements for binding geometry and reaction intermediate stabilisation during catalysis. In those SDRs that oxidise or reduce sugar nuclotides at the hexose C-4 position, however, the catalytic triad serine\/threonine residue is important because it modifies the pKa of the C-4 hydroxyl by forming a short hydrogen bond.23 Mutation of the triad threonine (Thr134) to alanine in E. coli dTDP-Glc 4,6-dehydratase results in a 200-fold drop in kcat,31 whereas mutation of Ser124 to alanine in E. coli GalE reduces epimerase activity (kcat) almost 3000-fold.32 The incomplete WbmF catalytic triad raises doubts about the competence of this enzyme as a sugar nucleotide oxidoreductase. It is possible that the function of the missing side chain is provided by another active-site residue; this implies a different substrate-binding geometry and further studies will be required to investigate this possibility. Conversely, if a standard substrate-binding orientation is assumed, the active site of WbmF resembles that of 3,5-epimerases and our structures of this protein identify Cys133 and either Asn213 or His90 as potential mediators of proton exchange in the epimerisation reaction\u2014one group abstracts a proton from the sugar while the other donates a proton to the opposite face.\nBy modelling the compound 4-keto UDP-d-ManNAc3NAcA into the WbmF structure we have demonstrated that there is space within this active site to accommodate this bulky sugar nucleotide. Other solutions produced in the modelling process demonstrated alternative plausible substrate positions (data not shown). For this reason, we do not suggest that the solution presented here (Fig. 7) represents the true active conformation. While we did not attempt similar modelling experiments with WbmG and WbmH, visual inspection of these structures indicates that they also have spacious active sites compared with other SDRs and can therefore accommodate diacetamido uronic acids in their hexose-binding pockets.\nWe therefore propose the following biosynthetic pathway (Fig. 8) for the 3,5-epimerisation of UDP-d-ManNAc3NAcA: One of the putative oxidoreductases, WbmG or WbmH, catalyses the initial oxidation at C-4, and the other oxidoreductase is responsible for the final reduction step. Introduction of the keto group lowers the pKa of the C-3 and C-5 hydrogen atoms, allowing WbmF to catalyse proton exchange effecting 3,5-epimerisation. This scheme is the most consistent with all of the available data. The function suggested for WbmF is very unusual in that we suggest that this enzyme does not catalyse oxidation or reduction of its substrate. If this is the case, WbmF would be the first such member of the SDR family, and this would imply that the cofactor in WbmF has a purely structural role, without participating in the catalytic cycle. It has been suggested that the transcriptional regulation protein NmrA from Aspergillus nidulans is a NAD-binding SDR family member that is not an enzyme at all. NmrA appears to function in control of nitrogen metabolism through physical interaction with the GATA family transcription factor AreA, possibly by controlling the rate of entry of AreA into the nucleus,33 and the structural equivalents of the SDR triad tyrosine and serine\/threonine in NmrA are Met127 and Met113, respectively.34 Since the NmrA structure was reported, however, several SDR enzymes with SMK triads have been reported (e.g., WbpM from P. aeruginosa),26 so that the nonconservation of the SDR catalytic triad in NmrA should not rule out an enzyme function as absolutely as first appeared.34 An alternative possibility to the biosynthetic pathway we have outlined (Fig. 8) is that WbmF does in fact catalyse either oxidation or reduction as well as the 3,5-epimerisation, in which case the third SDR may participate in O-antigen expression by performing a function in the synthesis of the complicated sugar residue that adorns the nonreducing terminus of the O-chain.35 The nonredox role we propose for WbmF is consistent, however, with the lack of the conserved SDR triad in this enzyme, and if this protein operates purely as a 3,5-epimerase this may also suggest a mechanism by which the wbmF mutant is able to express O antigen, albeit in greatly reduced amounts. The product of the initial oxidation reaction is activated for proton exchange \u03b1- to the keto group and it may be that racemisation at these positions occurs at a biologically significant rate without an absolute requirement for enzyme catalysis.\nRecent structural studies of the 3,5-epimerase in the dTDP-rhamnose pathway, RmlC,36 suggest that the rate-limiting step in its reaction involves a transition state in which the anomeric linkage is in the disfavoured equatorial position (which is also adopted by the reaction product), and that in order to avoid steric clashes and satisfy the requirement for axial proton abstraction, the reaction mechanism requires a twist-boat conformation such as that proposed in the GME reaction.12 Binding of substrate in a C-1 equatorial conformation may be part of the way that RmlC reduces kinetic barriers to this reaction. For the same reasons, the product of the proposed WbmF-catalysed 3,5-epimerisation will have the nucleotide attached to the l-GalNAcNAcA in the equatorial position in order to avoid 1,3,5-triaxial clashes. This transformation therefore involves considerable kinetic and thermodynamic barriers and will be slow without enzyme catalysis. However, if sufficient starting material is generated by the upstream oxidase, and the 3,5-epimerisation product is efficiently and selectively removed by the downstream reductase, the flux through this bottleneck may be sufficient to allow O-chain synthesis. Furthermore, the slower 3,5-epimerisation rate resulting from mutation of the wbmF gene may make relatively little impact on O-antigen expression levels if this conversion does not usually comprise the rate-limiting step for O-antigen synthesis. A similar effect may operate in the dTDP-l-noviose pathway in Streptomyces spheroides where observation of a naturally occurring l-rhamnoside analogue of novobiocin37 shows that 3,5-epimerisation of the dTDP-noviose precursor (dTDP-6-deoxy-d-xylo-4-hexulose) does occur despite the fact that in vitro, the 3,5-epimerisation catalysed by NovW has a 2000-fold lower kcat than RmlC38 (discussed in Ref. 39).\nIn conclusion, X-ray crystallography of WbmF, WbmG and WbmH enabled definition of these enzymes' NAD-cofactor preference and the substrate nucleotide specificity. We have been able to distinguish the likely function of WbmF from WbmG and WbmH, but cannot differentiate the roles of WbmG and WbmH. In order to achieve this, these proteins must be characterised in vitro. This will require chemical synthesis of UDP-d-ManNAc3NAcA and is beyond the scope of the present study. Subject to future experimental verification of predictions we have made, our data illustrate the usefulness of structural studies for investigating such challenging problems. While they also show why this methodology cannot universally supplant direct in vitro characterisation, this kind of structural analysis will prove essential for researchers to make full use of the gene databases.\nMaterials and Methods\nBacterial strains, plasmids and culture conditions\nBacterial strains used in this study are described in Table 3. Bordetella was grown on Bordet\u2013Gengou agar (Difco) supplemented with 10% defibrinated horse blood (TCS Cellworks Ltd). E. coli was cultured in Luria\u2013Bertani (LB) broth or on LB agar. All strains were incubated at 37\u00a0\u00b0C and ampicillin (100\u00a0\u03bcg ml\u2212\u00a01), kanamycin (50\u00a0\u03bcg ml\u2212\u00a01), tetracycline (10\u00a0\u03bcg ml\u2212\u00a01 for E. coli, 5\u00a0\u03bcg ml\u2212\u00a01 for B. bronchiseptica) or streptomycin (200\u00a0\u03bcg ml\u2212\u00a01) were added where required. Suicide plasmids were based on the host-restricted pEX100T backbone40 and broad host range shuttle vectors were based on a Kmr derivative of pBBR1MCS.41 For preparation of LPS, B. bronchiseptica was grown in medium supplemented with 50\u00a0mM MgSO4, as this maximises O-antigen expression in this strain.\nDNA methods\nStandard methods were used for DNA manipulations. Oligonucleotides were supplied by Sigma-Genosys. PCR was performed with template from boiled bacteria42 and Taq DNA polymerase (Promega) or KOD Hot Start DNA polymerase (Novagen).\nConstruction of wbmF, wbmG and wbmH mutants\nThe multiple cloning site (MCS) from pBluescript II SK+ (Stratagene) was excised using SacI and KpnI, blunt-ended and ligated into SmaI-cut pEX100T, to generate the MCS-containing suicide vector pEXMCS. The wbmF allelic exchange construct was prepared as follows. The wbmF gene in the plasmid Bb540g06 (a clone generated in the B. bronchiseptica RB50 genome sequencing project)43 was disrupted by insertion of a blunt-ended tetracycline-resistance (Tc) cassette into the internal NsiI site. The mutant allele was excised using Acc65I and XbaI, blunt ended, and then cloned into EcoRV-digested pEXMCS. For wbmG, the gene was amplified using primers 5\u2032-ATATCTAGACATATGCGTATTCTGATCACCG-3\u2032 (XbaI site underlined) and 5\u2032-ATAAAGCTTTGATTACTGGCAACTCTTC-3\u2032, and the PCR product was topoisomerase cloned into pCR2.1-TOPO using a TOPO-cloning kit (Invitrogen). wbmG was excised using XbaI and cloned into the XbaI site in pEXMCS. The wbmG gene was then disrupted by cloning a blunt-ended Tc cassette into the internal Acc65I site. The wbmH mutant allele was obtained by in vitro transposon-mediated mutagenesis of the wbm locus-containing cosmid, BbLPS118 (accession number AJ007747) using an EZ-Tn5\u2122 <Tet-1> insertion kit (Epicentre). The <Tet-1> transposon, plus flanking wbmH DNA, was cut out by partial digestion with AluI and ligated into SmaI-cut pEX100T. Allelic exchange constructs were electroporated into E. coli SM10\u03bbpir and transferred to B. bronchiseptica by conjugation with E. coli SM10\u03bbpir as donor.44 Loss of the plasmid-encoded sacB gene in allelic exchange mutagenesis of B. bronchiseptica was selected for by growth on LB agar with reduced salt concentration and supplemented with 10% (w\/v) sucrose.45\nComplementation of mutations\nThe B. bronchiseptica\nflaA promoter was amplified using primers 5\u2032-GCTCTAGATAGGCGCATGCCATGGCC-3\u2032 (XbaI site underlined) and 5\u2032-AAGGATCCCATATGGAGGCTCCCAAGAGAGAA-3\u2032 (BamHI and NdeI sites underlined), and cloned into the XbaI and BamHI sites in pBBR1MCS-kan to generate the vector pCompEmpty. wbmF was amplified using primers 5\u2032-AAAAAAACATATGTTGCCAGTAATCATGAATGC-3\u2032 (NdeI site underlined) and 5\u2032-AAAAAGCTTAGATCTAGTCCGCCGTCTTATTTG-3\u2032 (HindIII site underlined) and cloned into pCompEmpty using NdeI and HindIII to generate the wbmF complementation vector pCompF. wbmG was PCR amplified using primers 5\u2032-AAAAAAACATATGCGTATTCTGATCACC-3\u2032 and 5\u2032-AAAAAGCTTAGATCTGCAACTCTTCAGGTCTTG-3\u2032 and cloned into pCompEmpty using NdeI and HindIII, generating pCompG. wbmH was amplified using 5\u2032-GAGAATTCCATATGAAGAAAGTATTTATTACGG-3\u2032 and 5\u2032-AAAAAGCTTAGATCTTTGTCGATGACCAGGATT-3\u2032 and cloned into pCompEmpty using NdeI and HindIII, generating pCompH. Shuttle vectors were moved into B. bronchiseptica by conjugation with E. coli CC118 as donor46 and trans-acting transfer functions provided by E. coli S17-1 pNJ5000 as helper.44,47\nSDS-PAGE, silver stain and immunoblot analysis of LPS\nLPS was obtained from B. bronchiseptica using a modification of the method of Hitchcock and Brown48 as has been described.49 SDS-PAGE of LPS was performed using Novex precast 16% tricine gels (Invitrogen). LPS was oxidised in-gel with periodic acid50 and visualised with the Silver Stain Plus kit (BioRad). Western blotting was performed as previously reported51 using the monoclonal antibody D13B11 that specifically binds the O-antigen-containing LPS of B. bronchiseptica strain CN7635E.35\nX-ray crystallography\nProtein production, purification, crystallisation and data collection were performed as described.20 Data were processed using DENZO and SCALEPACK (version 1.97).52 Data were truncated and converted to structure factors using TRUNCATE53 from the CCP4 Suite.54 Molecular replacement was carried out using PHASER55 as described.20 For the initial WbmG structure, phases were determined experimentally from selenomethionine-labelled protein: peak, inflection point and high-energy remote data were used to determine initial phases using the PHENIX package.56 Model building was carried out using COOT,57 and refinement was performed with REFMAC version 5.0.58 Initial models were improved using ARP\/wARP version 6.1 where the data quality permitted.59 The CCP4i interface60 was used where appropriate. TLS parameters were assigned (where used) using the TLSMD server.61 CNS62 was used to perform simulated annealing. Structures were validated using COOT, PROCHECK,63 WHATCHECK,64 and RAMPAGE.65\nTable 2 provides a summary of the crystallographic statistics. Protein structures were analysed using Pymol,66 COOT,57 and CCP4MG.67 Surface area calculations were performed using the Richards' Rolling Probe method.68 More extensive details of the crystallographic methods are provided in the Supplementary Data.\nModelling of putative substrate into WbmF structure\nCoordinates and parameters for the 4-keto derivative of UDP-2,3-dideoxy-2,3-diacetamido-d-mannuronic acid were prepared using the CCP4 monomer library sketcher with refinement in REFMAC 5.2.58 An initial structure was prepared by modelling the proposed substrate into the structure of WbmF co-crystallised with UDP in COOT.57 The observed density was used to place the atoms present in UMP, and to place sugar atoms in positions occupied by water or glycerol in the observed structure. Different orientations were used in the two molecules in the asymmetric unit to increase the sampling of conformational space. The structure of WbmF co-crystallised with UDP, with 4-keto UDP-d-ManNAc3NAcA placed in, was truncated to include only protein, NAD and 4-keto UDP-d-ManNAc3NAcA atoms. This was used as an input to MODELLER version 9.2 to determine an optimised structure for the complex with the sugar nucleotide.69 After modelling, the ligand coordinates were regularised in COOT to correct minor distortions. Ten modelling trials were performed with different seeds, and the best was selected by eye on the basis of geometry, agreement with the determined structures and lack of bad interactions.\nProtein Data Bank accession codes\nX-ray amplitudes, phases and the derived atomic coordinates have been deposited with the PDB under the accession codes 2PZJ, 2Q1T, 2Q1U, 2Q1S, 2PZK, 2PZL, 2PZM and 2Q1W.","keyphrases":["short-chain dehydrogenase\/reductase","bordetella","o-antigen biosynthesis","lipopolysaccharide","x-ray crystallography","dtdp, deoxythymidine diphosphate","l-galnac3naca, 2,3-diacetamido-2,3-dideoxy-l-galacturonic acid","gme, gdp-mannose 3,5-epimerase","gmer, gdp-4-keto-6-deoxymannose 3,5-epimerase\/reductase","lps, lipopolysaccharide","pdb, protein data bank","blast, basic local alignment search tool","sdr, short-chain dehydrogenase\/reductase","udp-d-mannac3naca, udp-2,3-diacetamido-2,3-dideoxy-d-mannuronic acid","udp-l-galnac3naca, udp-2,3-diacetamido-2,3-dideoxy-l-galacturonic acid","ump, uridine monophosphate","mcs, multiple cloning site"],"prmu":["P","P","P","P","P","M","M","R","R","R","R","M","R","M","M","R","R"]}
{"id":"Eur_J_Epidemiol-3-1-1914270","title":"Inter-scan reproducibility of coronary calcium measurement using Multi Detector-Row Computed Tomography (MDCT)\n","text":"Purpose To assess inter-scan reproducibility of coronary calcium measurements obtained from Multi Detector-Row CT (MDCT) images and to evaluate whether this reproducibility is affected by different measurement protocols, slice thickness, cardiovascular risk factors and\/or technical variables.\nIntroduction\nA considerable proportion of the western society is at risk of suffering a cardiovascular event during life. Atherosclerosis is one of the main underlying processes. Non-invasive assessment of atherosclerosis is important since it allows studies into the etiology and consequences of early and advanced atherosclerosis in populations at large [1]. The last two decades, measurement of coronary artery calcification (CAC) using computer tomography (CT) has been used to assess coronary atherosclerosis non-invasively. The presence, and more importantly, the quantity of CAC, relates well with the overall severity of the atherosclerotic process [2]. Several studies have demonstrated a strong relation between coronary calcium burden and the incidence of myocardial infarction, a relation which was independent of age [3, 4].\nMost of the evidence on determinants and consequences of coronary calcium is based on data obtained with electron beam CT (EBCT) [5\u20137]. The availability of EBCT scanners is modest, whereas the Multi Detector-Row CT (MDCT) scanners are more widely available and also allow for detection of coronary calcium. Current data suggest that EBCT and MDCT give comparable results [8, 9]. In contrast to EBCT, however, data on reproducibility of CAC measurements using MDCT images is not widely available [10, 11], but information is relevant. Furthermore, due to technical improvement, slice thickness of the images has become smaller which may affect the likelihood of detecting coronary calcium, and hence its reproducibility.\nWe set out to study inter-scan reproducibility of coronary calcium measurements from MDCT images and to evaluate whether reproducibility is affected by different measurement protocols, slice thickness, selected cardiovascular risk factors and technical variables.\nMaterials and methods\nParticipants were recruited from the PROSPECT study [12], cohort of 17,357 healthy breast-cancer screening participants, aged 49\u201370\u00a0years, living in Utrecht and surroundings, enrolled between 1993 and 1997. Between October 2002 and December 2004, a random selection of 1,996 women were invited by mail and 1,000 (50.1%) who were postmenopausal and did not use contraceptives or hormone replacement therapy answered positively. Of these 1,000 women, a random selection of 573 underwent a MDCT examination during a single visit and 76 of them were scanned twice. The Medical Ethical Committee of the University Medical Center Utrecht approved the study and written informed consent was obtained from all participants.\nCurrent cardiovascular drug use (blood pressure lowering, lipid lowering and glucose lowering drugs) was assessed by asking women to bring all packages to the study centre. Smoking behavior, medical history and cardiovascular family history were assessed by a questionnaire. Height and weight were measured and body mass index was calculated as weight divided by height squared (kg\/m2). Waist-to-hip ratio (WHR) was assessed. Systolic and diastolic blood pressures were measured at both arms with an automated and calibrated blood pressure device (DINAMAPTM XL, Critikon, Johnson & Johnson, Tampa, Florida, USA) with the subject in supine position. A venous blood sample was drawn after an overnight fast of at least eight hours. Plasma total cholesterol, plasma triglycerides, and plasma glucose were measured using standard enzymatic procedures. High-density lipoprotein (HDL) cholesterol was measured by the direct method (inhibition, enzymatic). Low-density lipoprotein (LDL) cholesterol was calculated using the Friedewald formula.\nCoronary imaging and calcium measurements \nThe amount of calcium in the coronary arteries was assessed with a Multi Detector-Row CT (MDCT) scanner (Mx 8000 IDT 16, Philips Medical Systems, Best, The Netherlands). Subjects were positioned within the gantry of the MDCT scanner in supine position. During a single breath hold, images of the heart, from the level of the tracheal bifurcation to below the base of the heart, were acquired using prospective ECG triggering at 50\u201380% of the RR-interval, depending on the heart rate. Scan parameters were 16\u00a0\u00d7\u00a01.5\u00a0mm collimation, 205\u00a0mm field of view (FOV), 0.42\u00a0s rotation time, 0.28\u00a0s scan time per table position, 120\u00a0kVp and 40\u201370\u00a0mAs (patient weight <70\u00a0kg: 40\u00a0mAs; 70\u201390\u00a0kg: 55\u00a0mAs; >90\u00a0kg: 70\u00a0mAs). Scan duration was approximately 10\u00a0s, depending on heart rate and patient size. We had the participant get up from the table and lay down again since in studies on change in CAC over one year it is not realistic to assume exactly the same position of the participant at both occasions. Therefore our patients sat up and consequently moved slightly between scans to mimic two separate scan runs.\nFrom the acquired raw data, the whole volume was reconstructed with an intermediate reconstruction algorithm in non-overlapping data sets of 1.5\u00a0mm and 3\u00a0mm thick sections. Quantification of coronary calcium was performed on a separate workstation with software for calcium scoring (Heartbeat-CS, EBW, Philips Medical Systems, Best, The Netherlands). All regions with a density over 130 Hounsfield units were identified as potential calcifications.\nAfter completing a training-program, one scan reader (AR) who was unaware of the scores of the first scan, manually selected the calcifications within one of the coronary arteries (left main, left anterior descending, left circumflex, right coronary artery, and PDA) and scored the second scan of the participants. To reduce the influence of noise, the minimum size of a calcified lesion was set at 0.5\u00a0mm2. The peak density in Hounsfield units and the area in mm2 of each selected region were calculated. The Agatston [13] calcium score was obtained by multiplying the area by a weighting factor that is dependent on the peak signal anywhere in the lesion. The scores of individual lesions were added to obtain the Agatston calcium score for the entire coronary tree. The total calcium volume was calculated by multiplying the area of the calcified lesion (measured in square millimeters) by section thickness (1.5\u00a0mm and 3.0\u00a0mm). The calcium volume for each coronary vessel was computed by summing the volumes of the lesions in that vessel for all sections. Finally, the total volume from all the vessels became the calcium volume for a subject. The mass method uses volumetric, density information and a calibration phantom of hydroxyapatite to calculate the actual mass of the calcified plaques [14].\nIn addition, information on breathing artifact (inconsistency of sternum bone in sagital section in mm), noise (standard deviation of enhancement in fixed cardiac area of 212\u00a0mm2) and mean heart rate (beats\/min) during scan acquisition was collected.\nData analysis\nThe mean and standard deviations (SD) of coronary calcium were calculated for all scoring methods separately. Because of the skewed distribution of scores, medians were also computed. The Intra-class correlation coefficient was estimated for between scans data and for 1.5 and 3.0\u00a0mm slices thicknesses separately. The mean difference in score between scans was calculated as well as the absolute and relative differences.\nTo distinguish between random differences or systematic difference, information on mean and absolute differences is needed. One may assume a priori a non-differential misclassification in the calcium scores, but one has to show that with the results. When the chance of the 2nd result being higher or lower is equal, one would expect a mean difference of zero, with some standard deviation. The absolute difference will not be zero since all differences are \u2018absolutised\u2019, but it is expected that at least the mean difference is much less than the absolute difference. If however the chance of a higher or lower value in the 2nd scan is not equal, the mean difference will be plus or minus a certain value. In addition, the absolute difference will have a value close to that of the mean difference. Therefore we need both parameters.\nTo estimate a weighted kappa as measure of agreement of categorical variables, subjects were divided into four groups according to the mean Agatston score as proposed by Rumberger et\u00a0al. [15]: A: 0\u20139 (absent-minimal), B: 10\u201399 (mild), C: 100\u2013399 (moderate) and D: (400 (severe degree of calcification). This categorization is specifically for the calcium scoring method according to Agatston. Therefore we additionally categorized all scoring methods in their quartiles to calculate kappa as measure of agreement for all scoring methods.\nThe relation between risk factors, technical variables and measurement error was assessed using Spearman correlation coefficients. In a similar manner the relation between calcium level and measurement error was examined. Since logarithms of coronary calcium scores have generally been used in statistical analyses in other papers, we also studied the reproducibility of logarithmic transformed calcium score. Logarithmic analysis of coronary calcium scores was performed by calculating natural log of coronary calcium scores +0.001 (ln (CCS\u00a0+\u00a00.001)) because the logarithm of coronary calcium scores alone excludes all subjects with zero scores [16]. We defined relative difference as absolute difference divided by the mean calcium level multiplied in 100 and expressed in percent. Data analysis was performed with SPSS for windows, version 12.0. A statistically significant difference was assumed when the two-sided P-value was less than 0.05.\nResults\nMean age was 67.3\u00a0\u00b1\u00a05.2\u00a0years. Fifty-five participants (72.4%) had a coronary calcification more than zero in Agatston (1.5\u00a0mm slice thickness). Table\u00a01 shows the general characteristics of the 76 women who had two MDCT scans.\nTable\u00a01Characteristics of studied population (N\u00a0=\u00a076)MeanStd. deviationAge (year)67.35.2BMI (Kg\/m2)26.33.9WHR0.840.06SBP (mmHg)133.918.9DBP (mmHg)71.79.1Total cholesterol (mmol\/l)6.090.86LDL cholesterol (mmol\/l)4.310.97HDL cholesterol (mmol\/l)1.510.36Triglycerides (mmol\/l)1.280.62Glucose (mmol\/l) 4.050.69Heart rate (beat\/minute)7211Current smoking (%)*11Former smoking (%)43Previous CVD (%)1Family history of CAD in either parents (%)10BMI\u00a0=\u00a0Body Mass Index; CAD\u00a0=\u00a0Coronary Artery Diseases; DBP\u00a0=\u00a0Diastolic Blood Pressure; LDL\u00a0=\u00a0Low Density Lipoprotein; HDL\u00a0=\u00a0High Density Lipoprotein; SBP\u00a0=\u00a0Systolic Blood Pressure; WHR\u00a0=\u00a0Waist to Hip Ratio* Percentages have been rounded\nTable\u00a02 presents information on calcium distributions by various scoring techniques and reproducibility results, by slice thickness. Overall, calcium scores were higher when based on the 1.5\u00a0mm slice thickness than based on the 3.0\u00a0mm slice thicknesses. The kappa agreement and Intra-class correlation coefficients between the two scans were high for all scoring methods, indicating that with respect to ranking of subjects all three methods are doing well. In addition, the mean differences in scores were relatively small compared to the absolute differences for all measurements, suggestion no systematic measurement errors. Finally, results for the scans with 1.5\u00a0mm slice thickness were similar to those for the 3.0\u00a0mm slice thickness (Table\u00a02).\nTable\u00a02Characteristics of different coronary calcium scoring methods; effect of slice thickness on inter-scan reproducibilityMass 1st ScanMass 2nd Scan Volume 1st Scan Volume 2nd Scan Agatston 1st Scan Agatston 2nd Scan Slice thickness 1.5\u00a0mmMean32.2131.88154.52149.40170.33163.63Median6.156.0539.9736.5231.8532.00Agreement (k) Rumberger categories0.970.890.87Agreement (k) Quartiles0.840.810.88Mean difference0.35.16.7Absolute difference4.022.324.3Relative difference (%)12.414.614.5ICCC*0.990.990.98Slice thickness 3.0\u00a0mmMean25.5725.45131.45126.98140.06135.82Median4.003.6530.3021.9020.3018.00Agreement (k) Rumberger categories0.920.830.73Agreement (k) Quartiles0.840.840.84Mean difference0.14.44.2Absolute difference3.518.721.3Relative difference (%)13.714.715.4ICCC*0.990.980.98*\u00a0Intra-class correlation coefficient\nTable\u00a03 presents the relation of cardiovascular risk factors with inter-scan mean difference. No consistent relations were found between risk factor levels and measurement error. Importantly, however was the observation that calcium level or the logarithm of the coronary calcium scores were not related to the mean difference between scans, whereas they were significantly related to the absolute and relative differences (Table\u00a04, Figs.\u00a01 and 2). These observations suggest that measurement error increases with increasing CAC levels, yet that this occurs in a random way.\nTable\u00a03Relationship between cardiovascular risk factors and inter-scan mean difference of coronary calcium scoring methods by MDCT (Slice thickness 1.5\u00a0mm)Inter-scan mean differenceCCS methodsMassVolumeAgatstonBiological variablesrP-valuerP-valuerP-valueBMI (Kg\/m2)0.040.730.030.740.020.80Age (year)0.180.100.310.000.280.01Smoking(Categorical)\u22120.000.980.040.710.070.49WHR\u22120.030.730.080.480.130.24SBP (mmHg)0.100.370.160.140.240.03DBP (mmHg)0.160.140.050.610.110.34Cholesterol (mmol\/l)\u22120.270.05\u22120.120.40\u22120.200.17LDL (mmol\/l)\u22120.180.10\u22120.190.09\u22120.090.40HDL (mmol\/l)\u22120.040.72\u22120.160.14\u22120.110.34Triglyceride (mmol\/l)\u22120.020.850.130.240.110.34Glucose (mmol\/l)0.160.24\u22120.000.980.000.98Mean heart rate\u22120.030.77\u22120.030.73\u22120.020.81Technical variablesMean breathing artifact0.010.88\u22120.030.78\u22120.020.87Mean SD of noise0.130.260.080.490.070.52Coronary calciumMean mass score0.000.98Mean volume score0.030.75Mean Agatston score0.020.86Mean log mass score0.000.99Mean log volume score0.030.76Mean log Agatston score0.020.85BMI\u00a0=\u00a0Body Mass Index; DBP\u00a0=\u00a0Diastolic Blood Pressure; LDL\u00a0=\u00a0Low Density Lipoprotein; HDL\u00a0=\u00a0High Density Lipoprotein; r\u00a0=\u00a0spearman correlation coefficient; SBP\u00a0=\u00a0Systolic Blood Pressure; WHR\u00a0=\u00a0Waist to Hip RatioTable\u00a04Relationship between cardiovascular risk factors and inter-scan absolute and relative difference of coronary calcium scoring methods by MDCT (Slice thickness 1.5\u00a0mm)Inter-scan relative differenceCCS methodsMassVolumeAgatstonBiological variablesrP-valuerP-valuerP-valueBMI (Kg\/m2)0.070.530.080.460.090.43Age (year)0.210.060.240.030.l170.12Smoking(Categorical)\u22120.030.73\u22120.070.51-0.140.20WHR0.070.550.050.660.050.66SBP (mmHg)0.060.570.040.680.110.32DBP (mmHg)0.320.0040.310.0050.330.003Cholesterol (mmol\/l)0.130.370.100.500.001.00LDL (mmol\/l)\u22120.140.21\u22120.170.12-0.180.11HDL (mmol\/l)0.070.520.040.670.060.57Triglyceride (mmol\/l)0.030.780.070.490.000.99Glucose (mmol\/l)0.230.090.260.050.240.08Mean heart rate\u22120.010.910.010.930.000.97Technical variablesMean breathing artifact0.100.440.090.490.150.23Mean SD of noise0.190.090.190.090.180.11Coronary calciumMean mass score0.290.009Mean volume score0.330.003Mean Agatston score0.380.001Mean log mass score0.290.010Mean log volume score0.330.003Mean log Agatston score0.370.001Inter-scan absolute differenceTechnical variablesMean breathing artifact0.120.320.120.330.150.22Mean SD of noise0.200.080.190.090.150.17Coronary calciumMean mass score0.86<0.001Mean volume score0.84<0.001Mean Agatston score0.89<0.001Mean log mass score0.86<0.001Mean log volume score0.83<0.001Mean log Agatston score0.89<0.001BMI\u00a0=\u00a0Body Mass Index; DBP\u00a0=\u00a0Diastolic Blood Pressure; LDL\u00a0=\u00a0Low Density Lipoprotein; HDL\u00a0=\u00a0High Density Lipoprotein; r\u00a0=\u00a0spearman correlation coefficient; SBP\u00a0=\u00a0Systolic Blood Pressure; WHR\u00a0=\u00a0Waist to Hip RatioFig.\u00a01Relation between mean calcium score and inter-scan difference in mean calcium scores (Bland-Altman plots)Fig.\u00a02Relation between mean calcium score and inter-scan absolute difference\nDiscussion\nWith respect to ranking of subjects, the inter-scan reproducibility of coronary calcium measurements by MDCT using Agatston, volume and mass scoring algorithms is excellent. The inter-scan reproducibility showed no major differences between scoring methods. The slice thickness did not affect reproducibility, nor did heart rate and technical parameters. Measurement error was related to increased coronary artery calcification, although our findings suggest that the error in the measurements is a random phenomenon.\nOur findings, i.e., no major differences between scoring methods are in contrast with several reports on reproducibility based on EBCT scanning. Direct comparison of the findings of these studies with those of other is difficult since the parameters used to indicate reproducibility differ between studies. Furthermore, potentially the prevalence of CAC and its extent may affect reproducibility, as our findings suggest that measurement error increases with increasing CAC levels. Also the sizes of the studies differ which have undeniable effects on reproducibility results. However, our results are similar to those of by Rumberger and Kaufman [17], who compared these three methods and did not find any one method preferable to another in terms of reproducibility of results from consecutive scans in a patient.\nAlthough the correlation between inter-scan measurements is excellent [18, 19], it still occurs that subjects with small deposits of calcium in scan one may have larger deposits of calcium in the 2nd scan, which leads to proportionally larger error in reproducibility. This has triggered other studies [20] on reproducibility to suggest that \u201cthe variability is partially a function of the absolute calcium score and inversely related to it\u201d, implicating that low coronary calcium scores may not be reproducible. However, our results could not confirm this.\nBesides different algorithms for calcium scoring, slice thickness has been reported to affect the reproducibility of scoring protocols. In our study, the reproducibility of the coronary calcium measurements by MDCT was similar for 1.5\u00a0mm as for 3.0\u00a0mm slice thickness, and equal for Agatston, volume and mass measurements confirming the results by Rumberger and Kaufman [17].\nThe implications of our main findings depend on the research question that is asked in studies using CAC measurements. When the interest is using CAC measurements for prognostic studies our results for kappa and ICCC show that ranking of subjects is adequate based on one CT scan. So the need for duplicate CAC scan is absent. The fact that measurement error increases with increasing CAC values, is in prognostic studies not of major importance since the categorization of individuals seems adequate. When the interest is in etiologic studies using CAC as outcome parameter, our findings show that risk factor relations will be validly estimated since none of the risk factors relates to measurement error. When the interest is in using CAC as risk factor for future events (assessment of relative risks), it is most likely that in analyses with CAC as continues variable the magnitude of association of high CAC levels with events reflects an underestimation of the true magnitude. The direction of the relation will not change since based on our results measurement error is random, leading to random misclassification of the exposure variable. When the interest is in diagnostic value of CAC measurements, which is usually done in categories of CAC, again the relations will be valid given our high kappa coefficients. Although our study was performed in healthy postmenopausal women, we expect that the finding will also be applicable for men.\nOur findings are important in the light of the wider availability of MDCT in countries compared to EBCT. One reason for that is lower equipment cost. Other advantages of MDCT over EBCT have been suggested to be less quantum noise, thinner section thickness, and simultaneous acquisition of four sections (with 16-slice or with 64-slice ), which is reported to reduce misregistration artifact.\nIn conclusion, our findings demonstrate that coronary calcium measurements by MDCT are highly reproducible and are not affected by scoring protocols, slice thicknesses and technical factors.","keyphrases":["reproducibility","multi detector-row ct (mdct)","atherosclerosis","coronary artery calcification","epidemiology"],"prmu":["P","P","P","P","U"]}
{"id":"Biochim_Biophys_Acta-1-5-1906864","title":"Oxidation of chylomicron remnant-like particles inhibits their uptake by THP-1 macrophages by apolipoprotein E-dependent processes\n","text":"The influence of the oxidative state of chylomicron remnants (CMR) on the mechanisms of their uptake and induction of lipid accumulation by macrophages derived from the human monocyte cell line, THP-1, during foam cell formation was investigated using chylomicron-remnant-like particles (CRLPs) at 3 different levels of oxidation. The oxidative state of CRLPs was varied by exposure to CuSO4 (oxCRLPs) or incorporation of the antioxidant, probucol (pCRLPs) into the particles. oxCRLPs caused significantly less accumulation of triacylglycerol in the macrophages than CRLPs, and their rate of uptake was lower, while pCRLPs caused more lipid accumulation and were taken up faster. Uptake of all 3 types of particles was inhibited to a similar extent when entry via the low density lipoprotein (LDL) receptor related protein (80\u201390%), LDL receptor (\u2212 30\u201340%), CD36 (\u2212 40%) and phagocytosis (\u2212 35\u201340%) was blocked using lactoferrin, excess LDL, anti-CD36 and cytochalasin D, respectively, but blocking scavenger receptors-A or -B1 using poly inosinic acid or excess HDL had no effect. These findings show that oxidation of CRLPs lowers their rate of uptake and induction of lipid accumulation in macrophages. However, oxidation does not change the main pathways of internalisation of CRLPs into THP-1 macrophages, which occur mainly via the LRP with some contribution from the LDLr, while CD36 and phagocytosis have only a minor role, regardless of the oxidative state of the particles. Thus, the effects of CMR oxidation on foam cell formation contrast sharply with those of LDL oxidation and this may be important in the role of dietary oxidized lipids and antioxidants in modulating atherosclerosis.\n1\nIntroduction\nChylomicron remnants (CMR) carry lipids of dietary origin from the gut to the liver for processing [1] and there is now a large and growing body of evidence indicating that these lipoproteins are strongly atherogenic. They have been shown to be taken up into the artery wall as efficiently as LDL [2\u20135]; remnant-like particles containing apolipoprotein E (apoE) have been isolated from human aortic intima and atherosclerotic plaque [6,7]; and delayed clearance of CMR from the circulation correlates with lesion development [8,9]. Moreover, we and others have shown that CMR induce extensive lipid accumulation causing foam cell formation in human monocyte-derived macrophages (HMDM) [10] and in human and murine monocyte\/macrophages cell lines [11,12].\nLow density lipoprotein (LDL) plays a major role in atherogenesis and in foam cell generation, but oxidation of the lipoprotein particles, a process which can occur within the artery wall, is necessary before extensive lipid accumulation is induced [13]. In striking contrast, CMR do not require prior oxidation to cause macrophages to form foam cells [10\u201312]. However, our studies have demonstrated that incorporation of lipophilic antioxidants into the particles enhances, rather than inhibits, lipid uptake and accumulation in the cells [14,15], suggesting that the oxidative state of CMRs may play a role in their induction of foam cell formation, but in the opposite way to that of LDL. Oxidized CMR could occur either in the artery wall by the action of the cell-associated lipoxygenase and myeloperoxidase which are believed to oxidize LDL, or in the circulation, because dietary oxidised lipids, which are produced when fat is cooked at high temperatures, have been shown to be transported in these lipoproteins [16,17]. Clearly, therefore, it is important for the understanding of the atherogenicity of CMR to establish how their oxidation influences their uptake and induction of foam cell formation and the pathways by which CMR are internalised by the cells.\nIt has been demonstrated that CMR are taken up by the liver by apolipoprotein E (apoE)-dependent pathways mediated by the LDL receptor (LDLr) and the LDL receptor-like protein (LRP) [1]. The exact mechanisms by which CMR are taken up by macrophages, however, are not yet definitively established, and nothing is known about the effects of oxidation of the particles on the routes by which they are internalised. The LDL receptor (LDLr) appears to play a part [1], but as it is down-regulated by the influx of cholesterol into cells, native LDL does not induce foam cell formation [16], and our studies [18] have suggested that the delivery of cholesterol to macrophages by CMR has a similar effect. Thus, other mechanisms are also likely to be involved, and evidence from experiments using antibodies to the LDLr and animals lacking the LDLr supports this view [10]. Candidates include the LRP [19], the apoB48 triacylglycerol-rich lipoprotein receptor (apoB48r) [20], an as yet unidentified 43\u00a0kDa protein described by Elsegood et al. [21] and scavenger receptors such as scavenger receptor A (SR-A) and CD36 [1]. Phagocytosis has also been suggested as a possible mechanism [22]. Since the route of uptake of LDL by macrophages is profoundly changed after oxidation from the regulated LDLr to the unregulated scavenger receptors [16], it is important to establish whether the oxidative state also alters the mechanisms of uptake of CMR by the cells.\nThe aim of this study is to investigate the effects of the oxidative state of CMR on their uptake by macrophages and on the accumulation of lipid within the cells, and to determine how oxidation affects the pathways involved in the internalisation of the particles. Chylomicron remnant-like particles (CRLPs) at three different levels of oxidation (CRLPs, oxidized CRLPs (oxCRLPs) and CRLPs containing the antioxidant probucol (pCRLPs)) and macrophages derived from the human monocyte cell line THP-1 were used as the experimental model, and the mechanisms of uptake were evaluated using specific inhibitors of the processes believed to be involved. The findings clearly demonstrate that oxidation of CRLPs reduces the rate of their uptake by THP-1 macrophages and decreases lipid accumulation in the cells, and further show that this is due to differential interaction with apoE dependent receptors.\n2\nMaterials and methods\nRPMI 1640 medium, fetal bovine serum (FBS), l-alanyl-l-glutamine (glutamax) penicillin\/streptomycin and \u03b2-mercaptoethanol were obtained from Gibco (Paisley, UK). FBS was heat inactivated (56\u00a0\u00b0C, 30\u00a0min) before use. Trypan blue, fatty acid-free bovine serum albumin (BSA), phospholipids, cholesterol, cholesteryl oleate, phorbol 12-myristate 13-acetate (PMA), Oil red O, probucol, poly inosinic acid (poly I), lactoferrin and cytochalasin D were supplied by Sigma (Poole, UK). 1\u20321\u2032-dioctadecyl-3,3-3\u2032,3\u2032-tetramethylindo-carbocyanide perchlorate (DiI) was from Cambridge Bioscience (Cambridge, UK). The blocking antibody for CD36 was obtained from Immunodiagnostic Systems Ltd (Tyne and Wear, UK) and cholesterol oxidase from Merck Biosciences Ltd (Nottingham, UK). DiI-labelled acetylated LDL (acLDL) was purchased from Molecular Probes (Paisley, UK).\n2.1\nPreparation of lipoproteins\nLDL and high density lipoprotein (HDL) were isolated from human plasma (National Blood Service, London UK) by ultracentrifugation. Plasma was layered under 0.9% NaCl (d\u00a0=\u00a01.006\u00a0g\/ml), centrifuged for 5\u00a0h at 100,000\u00d7g (4\u00a0\u00b0C), and the top fraction discarded. The density of the bottom layer was raised to 1.063\u00a0g\/ml with KBr, layered under KBr (d\u00a0=\u00a01.063\u00a0g\/ml) and centrifuged for 16\u00a0h at 175,000\u00d7g (4\u00a0\u00b0C). LDL was collected from the top fraction by tube slicing. For the preparation of HDL, the bottom fraction was then adjusted to d 1.21\u00a0g\/ml with KBr, layered under KBr (d 1.21\u00a0g\/ml) and centrifuged at 175,000\u00d7g\/ml at 4\u00a0\u00b0C for 18\u00a0h and the top fraction containing HDL was collected by tube slicing. LDL and HDL were dialysed against saline for 48\u201372\u00a0h prior to used.\nCRLPs were prepared by sonication (power setting 22\u201324\u00a0\u03bcm, 20\u00a0min, at 56\u00a0\u00b0C) of a lipid mixture containing 70% trilinolein, 2% cholesterol, 3% cholesteryl ester and 25% phospholipids in 0.9% NaCl (w:v) in Tricine Buffer (20\u00a0mM, pH 7.4) followed by stepwise density gradient (2.5\u00a0ml d 1.065\u00a0g\/ml, 2.5\u00a0ml d 1.020\u00a0g\/ml, 3\u00a0ml d 1.006\u00a0g\/ml) ultracentrifugation as described by Diard et al. [23] at 17,000\u00d7g for 20\u00a0min at 20\u00a0\u00b0C. The upper layer of grossly emulsified lipids was then removed and replaced with an equal volume of NaCl solution (d\u00a0=\u00a01.020\u00a0g\/ml) and tubes were centrifuged at 70,000\u00d7g for 1\u00a0h (20\u00a0\u00b0C). For apoE binding, lipid particles collected from the top layer were incubated with the dialysed d 1.063\u20131.21\u00a0g\/ml fraction of human plasma (National Blood Transfusion Service, North London Centre, UK) prepared as described above at 37\u00a0\u00b0C with shaking for 4\u00a0h (1\u00a0volume of particles: 2\u00a0volumes plasma). The CRLPs containing apoE were then isolated by ultracentrifugation at d\u00a0=\u00a01.006\u00d7g\/ml (120,000\u00d7g for 12\u00a0h at 4\u00a0\u00b0C), harvested from the top layer, purified by a second centrifugation at the same density (202,000\u00d7g for 4\u00a0h at 4\u00a0\u00b0C) and stored at 4\u00a0\u00b0C under argon until required. All preparations were used within 1\u00a0week. For DiI-labelled CRLPs and pCRLPs, probucol (1\u00a0mg) and\/or DiI were added to the lipid mixture prior to sonication. CRLPs were oxidized by incubation with CuSO4 (20\u00a0\u03bcM) with shaking for 5\u00a0h at 37\u00a0\u00b0C and the CuSO4 was then removed by dialysis (0.9% NaCl, 24\u00a0h, 4\u00a0\u00b0C). The oxidation process had no effects on the fluorescent properties of the DiI label.\n2.2\nCulture of THP-1 cells\nTHP-1 monocytes were maintained in RPMI 1650 culture medium containing 10% (v\/v) FBS, glutamax (2\u00a0mM), penicillin\/streptomycin (100\u00a0U\/ml\/100\u00a0\u03bcg\/ml) and \u03b2-mercaptoethanol (20\u00a0\u03bcM) (culture medium). The cells were differentiated into macrophages by incubation with PMA (200\u00a0ng\/ml) for 72\u00a0h at 37\u00a0\u00b0C in 5% CO2\/95% air. The medium containing PMA and any non-adherent cells were then removed and the macrophages were washed with PBS (3\u00a0\u00d7\u00a01\u00a0ml) and incubated with CRLPs or DiI-labelled CRLPs (30\u00a0\u03bcg cholesterol\/ml) in the presence or absence of specific inhibitors as indicated in the text. The inhibitors were added 1\u00a0h prior to the addition of the CRLPs. Cell viability as assessed by Trypan blue exclusion was >\u00a095% and was not affected by any of the CRLP types or conditions used. After the incubation, the macrophages were washed (culture medium 3\u00a0\u00d7\u00a01\u00a0ml) and lipid accumulation was assessed by staining with Oil red O, or harvested for lipid analysis [24]. For studies with DiI-labelled CRLPs, the fluorescence associated with the cells was assessed by viewing with a Zeiss LMS 510 laser -scanning confocal microscope and quantified by absorbance volume analysis, or by fluorescence-activated cell analysis (FACS) using a BD FACS Calibur flow cytometer (BD Biosciences, Oxford, UK). For FACS analysis, cells were harvested in PBS containing EDTA (5\u00a0mM) and lidocaine -HCl (8\u00a0mg\/ml), incubated for 20\u00a0min at 37\u00a0\u00b0C and centrifuged at 10,000\u00d7g (2\u00a0min). The cell pellet was then resuspended and fixed in PBS containing 4% formalin.\n2.3\nAnalytical methods\nFor mRNA analysis, total RNA was extracted from THP-1 macrophages before or after incubation with CRLPs (30\u00a0\u03bcg cholesterol\/ml) using a kit from Sigma (Poole, UK) and first strand synthesis was carried out using a kit supplied by Promega (Southampton, UK) according to the manufacturer's instructions. mRNA levels for the LDLr, LRP and the housekeeping gene glyceraldehyde phosphate dehydrogenase (GAPDH) were determined by real time polymerase chain reaction using a SYBR green quantitative PCR kit (Sigma, Poole, UK) with an Opticon light cycler under the following conditions; denaturation at 94\u00a0\u00b0C for 2\u00a0min followed by 40 cycles of 94\u00a0\u00b0C for 15\u00a0s, 58\u00a0\u00b0C for 1\u00a0min, with a final extension at 72\u00a0\u00b0C for 1\u00a0min. The primers used and the product sizes are shown in Table 1. The Ct values were determined by automated threshold analysis using Opticon Monitor 2 software. Data were normalized with the values obtained for GAPDH and the fold change in mRNA expression in CRLP-treated as compared to untreated macrophages was determined by the method described by Pfaffl [25].\nLipids were extracted from cell samples by the addition of chloroform: methanol (2:1 v:v, 20\u00a0volumes) followed by 0.88% KCl (v:v) (40% total volume). The mixture was shaken and the chloroform layer containing the lipids was collected and dried under nitrogen. The triacylglycerol (TG) content of CRLPs and lipid extracts was determined using a fully enzymatic assay kit (Alpha Laboratories, Eastleigh, Hants, UK) in which glycerol released from triacylglycerol by a lipase is measured using glycerol phosphate oxidase and peroxidase. The total cholesterol (cholesterol\u00a0+\u00a0cholesteryl ester) content of CRLPs and lipid extracts was determined using cholesterol oxidase and cholesteryl esterase (kit supplied by Sigma, Poole, UK), and the cholesterol content of lipid extracts was determined enzymatically using cholesterol oxidase [26]. Cholesteryl ester concentrations were calculated by subtracting the values for cholesterol from those for total cholesterol. The extent of oxidation of CRLPs was determined by measuring the level of thiobarbituric acid-reacting substances (TBARS) in the preparations [27].\nThe apolipoprotein content of CRLPs was assessed by SDS-PAGE using 10% gels [28]. Proteins were visualised by staining with Coomassie blue and the bands corresponding to apoE were quantified by optical density volume analysis.\nSignificance limits were calculated using ANOVA repeated measures or one way ANOVA followed by Student's paired t test for experiments with multiple time points or a single time point, respectively, except where indicated otherwise.\n3\nResults\n3.1\nCharacteristics of CRLPs\nThe lipid composition of CRLPs, oxCRLPs and pCRLPs is shown in Table 2. The concentration of TG and TC in CRLPs and pCRLPs was similar, but that of oxCRLPs was a little lower, reflecting the dilution of the preparations during the oxidation procedure. The mean TG:TC ratio, however, was between 5 and 6 for all 3 CRLP types. Previous work in our laboratory has shown that the phospholipid content of CRLPs, oxCRLPs and pCRLPs prepared by the methods used here are not significantly different [15,29]. Analysis of the TBARS content of the particles showed that there were significant differences in their oxidative states, with values being higher in oxCRLPs as compared to CRLPs, while those for pCRLPs were lower (one way ANOVA, Bonferroni's test post hoc) (Table 2).\nThe apoE content of CRLPs, oxCRLPs and pCRLPs was evaluated by SDS PAGE electrophoresis. The results showed that the particles contained apoE, but no apoE was detected in the top fraction of the d 1.063\u20131.21\u00a0g\/ml fraction of human plasma incubated and centrifuged in the absence of lipid particles (Fig. 1A), indicating that the apoprotein was bound to the CRLPs during the incubation. No apoCs or other apolipoproteins were detectable. There were no significant differences in the apoE content of the CRLPs, oxCRLPs or pCRLPs as assessed by optical density volume analysis (Fig. 1B).\n3.2\nEffect of the oxidative state of CRLPs on the induction of lipid accumulation in macrophages\nIncubation of THP-1 macrophages with CRLPs or oxCRLPs for 5,24 or 48\u00a0h caused a marked increase in the total lipid found in the cells (Fig. 2A) at all time points, but lipid accumulation was greater with CRLPs as compared to oxCRLPs (P\u00a0<\u00a00.001). This effect was mainly due to greater accumulation of TG in the presence of CRLPs (P\u00a0<\u00a00.001) (Fig. 2B), as the trend for an increase in TC in CRLP- as compared to oxCRLP-treated macrophages did not reach significance (Fig. 2C).\n3.3\nEffect of the oxidative state of CRLPs on their uptake by macrophages\nThe uptake of DiI-labelled CRLPs, oxCRLPs and pCRLPs by THP-1 macrophages was assessed by confocal microscopy and FACS analysis (Fig. 3). Examination of cells incubated with DiI-labelled particles for periods up to 24\u00a0h with the confocal microscope showed that the fluorescence associated with the cells increased with time in all cases, but that there was clearly more in pCRLP- and less in oxCRLP-treated as compared to CRLP-treated cells (Fig. 3A). Quantification of the cell-associated fluorescence and analysis by ANOVA repeated measures showed that the rate of uptake of CRLPs over 24\u00a0h was significantly higher than that of oxCRLPs (P\u00a0<\u00a00.01) and significantly lower than that of pCRLPs (P\u00a0<\u00a00.01) (Fig. 3B), and a similar result was obtained using FACS (P\u00a0<\u00a00.05, CRLPs vs. oxCRLPs or pCRLPs (Fig. 3C).\n3.4\nRole of apoE in the uptake of CRLPs of different oxidative states\nTo investigate whether apoE is necessary for uptake of CRLPs, THP-1 macrophages were incubated with DiI-labelled CRLPs or CRLPs without apoE for 2\u00a0h and the fluorescence associated with the cells was determined by FACS analysis. In the absence of apoE, the uptake of CRLPs was reduced by about 90% (fluorescence values: CRLPs, 98.6\u00a0\u00b1\u00a07.1; CRLPs without apoE, 8.4\u00a0\u00b1\u00a00.9, n\u00a0=\u00a03).\nThe potential role of apoE dependent receptors in the uptake of CRLPs by the cells was studied using excess LDL and the LRP ligand, lactoferrin, to block entry via the LDLr and LRP, respectively. The effects of lactoferrin (2\u00a0mg\/ml) on lipid accumulation in macrophages exposed to CRLPs or oxCRLPs as assessed by Oil red O staining are shown in Fig. 4. Lactoferrin had no effect on the lipid content of cells incubated in the absence of CRLPs, but reduced that in macrophages treated with either CRLPs or oxCRLPs (P\u00a0<\u00a00.05, both cases).\nWhen the effects of excess LDL (200\u00a0\u03bcg cholesterol\/ml) and lactoferrin (2\u00a0mg\/ml), on the fluorescence associated with the macrophages after incubation for 1, 4, 16 and 24\u00a0h with CRLPs, oxCRLPs or pCRLPs were assessed by confocal microscopy (Fig. 5A\u2013C), no significant inhibition was observed in the presence of excess LDL, but lactoferrin caused a decrease of >\u00a090% in experiments with CRLPs (Fig. 5A) and oxCRLPs (Fig. 5B) and >\u00a080% with pCRLPs (Fig. 5C) (P\u00a0<\u00a00.0001, all cases). Addition of LDL and lactoferrin together completely abolished detectable uptake of all three types of particles (Fig. 5A\u2013C). FACS analysis of macrophages treated with CRLPs, oxCRLPs or pCRLPs in the presence or absence of lactoferrin (2\u00a0mg\/ml), LDL (300\u00a0\u03bcg cholesterol\/ml) or LDL\u00a0+\u00a0lactoferrin for 2\u00a0h also showed that lactoferrin caused a marked decrease of about 75% in the uptake of all three types of CRLPs (P\u00a0<\u00a00.01), and in this case significant inhibition (\u2212\u00a030\u201340%) was observed in the presence of LDL (P\u00a0<\u00a00.05) (Fig. 5D). Moreover, in a further separate experiment in which the excess of LDL added was increased by lowering the concentration of CRLPs to 10\u00a0\u03bcg cholesterol\/ml and raising that of LDL to 500\u00a0\u03bcg cholesterol\/ml, the uptake of both CRLPs and oxCRLPs was also significantly inhibited (fluorescence values (n\u00a0=\u00a03): CRLPs, 94.8\u00a0\u00b1\u00a06.2; CRLPs\u00a0+\u00a0LDL, 58.7\u00a0\u00b1\u00a05.8 (P\u00a0<\u00a00.05); oxCRLPs 56.7\u00a0\u00b1\u00a01.5; oxCRLPs\u00a0+\u00a0LDL, 29.2\u00a0\u00b1\u00a07.3 (P\u00a0<\u00a00.05)). As observed in the experiments using confocal microscopy, adding both LDL and lactoferrin to the incubations caused a further decrease to similar minimal levels in the uptake of all three types of CRLPs.\nAssessment of the fluorescence associated with THP-1 macrophages after incubation with DiI-labelled acetylated LDL (acLDL) (10\u00a0\u03bcg cholesterol\/ml) for 2\u00a0h showed that acLDL uptake, in contrast to that of CRLPs, was not significantly inhibited by excess LDL (500\u00a0\u03bcg cholesterol\/ml) or lactoferrin (2\u00a0mg\/ml) (Fig. 6).\n3.5\nRole of scavenger receptors and phagocytosis in the uptake of CRLPs of different oxidative states\nThe effects of blocking the class A scavenger receptor SR-A, the class B receptors CD36 and SR-B1 or phagocytosis on the uptake of CRLPs by THP-1 macrophages was investigated using poly-I, a known ligand for SR-A, a blocking antibody to CD36, excess HDL, which binds to SR-B1, and cytochalasin D, which blocks the polymerization of actin microfilaments [30]. Evaluation of the fluorescence associated with the cells after incubation with DiI-labelled CRLPs, oxCRLPs or pCRLPs for 1, 4,16 or 24\u00a0h by confocal microscopy (Fig. 7A\u2013C) show no significant change in the presence of poly-I (5\u00a0\u03bcg\/ml) or excess HDL (300\u00a0\u03bcg\/ml). Significant decreases, however, were observed in the presence of anti-CD36 (1\u00a0\u03bcg\/ml) (\u2212\u00a038% after 24\u00a0h) (P\u00a0<\u00a00.05) and cytochalasin D (10\u00a0\u03bcg\/ml) (\u2212\u00a027% after 24\u00a0h) (P\u00a0<\u00a00.01) in experiments with CRLPs (Fig. 7A), although no change in the uptake of oxCRLPs or pCRLPs was detected with these treatments (Fig. 7B, C).\nFACS determinations after incubation of macrophages with CRLPs, oxCRLPs or pCRLPs for 2\u00a0h confirmed that poly-I and HDL did not inhibit the uptake of the particles (Fig. 7D). In contrast, however, the uptake of DiI-labelled acLDL was decreased by about 90% in the presence of poly-I (50\u00a0\u03bcg\/ml) (Fig. 6). As found in the experiments using confocal microscopy, anti-CD36 and cytochalasin D significantly inhibited the uptake of CRLPs, but in this case effects of a similar magnitude were also seen with oxCRLPs and pCRLPs (After 24\u00a0h:anti-CD36; CRLPs, \u2212\u00a038%, oxCRLPs \u2212\u00a040%, pCRLPs, \u2212\u00a039%: cytochalasin D; CRLPs \u2212\u00a039%, oxCRLPs \u2212\u00a035%, pCRLPs \u2212\u00a038%), although the changes with oxCRLPs did not reach significance (Fig. 7D).\n3.6\nExpression of mRNA for the LDLr and LRP in THP-1 macrophages\nIn THP-1 macrophages exposed to CRLPs (30\u00a0\u03bcg cholesterol\/ml) for 24\u00a0h, LRP mRNA levels were increased in CRLP- treated THP-1 cells (fold change, 2.7\u00a0\u00b1\u00a01.01, n\u00a0=\u00a03), while LDLr mRNA abundance was decreased (0.27\u00a0\u00b1\u00a00.11(range), n\u00a0=\u00a02).\n4\nDiscussion\nSince homogeneous CMR cannot be obtained easily from human blood without contamination with other lipoproteins of a similar density such as chylomicrons and very low density lipoprotein (VLDL), model CRLPs were used in this study. These particles are similar in size, density and lipid composition to physiological remnants [23], and also contained human apoE, thus they differ from physiological CMR only in lacking apoB48. Importantly, extensive previous studies in both humans and experimental animals have demonstrated that chylomicron- and chylomicron remnant-like particles without apoB48 are cleared from the blood and metabolised in a similar way to the corresponding physiological lipoproteins [31\u201334]. In addition, CRLPs lacking apoB48, but containing apoE from the appropriate species, have been found to have effects which mimic those of physiological remnants in rat hepatocytes and pig endothelial cells [23,35], and our earlier work has shown that CRLPs cause extensive lipid accumulation in THP-1 macrophages and human monocyte derived macrophages (HMDM) which is comparable to that found in experiments with physiological CMR from rats and the murine macrophage cell line J774 [24,36]. For the current investigation, the use of CRLPs facilitated the manipulation of the oxidative state of the particles by inclusion of the lipophilic antioxidant probucol or by exposure to oxidising conditions similar to those used extensively in studies with oxLDL [16]. In previous work, we have shown that incorporation of probucol into CRLPs increases their uptake by THP-1 macrophages [15], and that this is due to protection of the particles from oxidation, since lycopene, a chemically unrelated antioxidant, has a similar effect [14]. For the present study, therefore, we used pCRLPs in addition to CRLPs and oxCRLPs, so that 3 different oxidative states were tested. Assessment of TBARS in the particles used showed that the oxidative states of CRLPs, oxCRLPs or pCRLPs were significantly different, and there were no significant differences in their apoE content, thus they provided a convenient and suitable model for the study.\nEarlier work in our laboratory and others has demonstrated that CMR cause the extensive lipid accumulation associated with foam cell formation in HMDM, THP-1 macrophages and murine macrophage cell lines [24,36,37]. In experiments with THP-1 macrophages and HMDM, we found that CRLPs induced a greater increase in the intracellular total lipid content than oxLDL (at the equivalent cholesterol level), and that, as might be expected, this was mainly due to greater accumulation of TG, while TC levels were raised to a comparable extent with both types of lipoprotein [24]. In the current study we compared the effects of oxidized and non-oxidized particles and, although both caused rises in cellular lipid content compared to untreated cells, the increase was clearly smaller with oxCRLPs as compared to CRLPs, mainly because of decreased accumulation of TG (Fig. 2). These results suggest that the uptake of CRLPs by the cells may be inhibited by oxidation, and this was confirmed in confocal microscopy and FACS studies (Fig. 3), which showed that the rate of uptake of CRLPs was decreased by oxidation and increased by incorporation of probucol into the particles to protect them from oxidation (Table 2). These results clearly demonstrate that oxidation of CMR inhibits their uptake by macrophages and attenuates foam cell formation. This is an important new finding, since this effect is strikingly different from that found with LDL, where oxidation is required to induce macrophages to form foam cells.\nCMR are known to be cleared from the circulation by the liver mainly by apoE-dependent pathways involving the LDLr and the LRP, and the present study shows that apoE also plays an important role in the entry of CRLPs into THP-1 macrophages, since a markedly reduced rate of uptake was observed when apoE was not present in the particles. These finding are in agreement with earlier work which has suggested that both the LDLr and the LRP are involved in the uptake of CMR by macrophages [10,19,38]. Our results indicate that both receptors are able to mediate the uptake of CRLPs regardless of their oxidative state, although lactoferrin, a ligand for the LRP, caused a marked reduction in lipid accumulation and uptake after exposure of THP-1 macrophages to all three types of CRLPs (Figs. 4, 5), while the maximum effect of excess LDL on uptake observed was more modest in all cases (Fig. 5). This lesser role of the LDLr may be due to its down-regulation on the influx of lipoprotein into cells. We have shown previously that the expression of mRNA for the LDLr in THP-1 macrophages is decreased by CRLPs while the expression of LRP mRNA is increased [18], and the present study, which shows a decrease of about 73% in LDLr mRNA and a 2.7fold rise in LRP mRNA after exposure of the macrophages to CRLPs, is in agreement with these findings.\nThe differences in rates of uptake of oxCRLPs, CRLPs and pCRLPs were retained in the presence of inhibitors of both the LDLr and the LRP, although when both the LDLr and the LRP were blocked, uptake of oxCRLPs, CRLPs and pCRLPs was reduced to a similar low level (Fig. 5), suggesting that the difference in their rate of uptake is apoE-dependent. Thus, an important novel finding of this study is that oxidation of CRLPs, unlike LDL, does not change their major routes of uptake by macrophages, with apoE-dependent receptors being of major importance and the LRP playing the predominant role, regardless of the oxidative state of the particles. ApoE does not bind to the LDLr family in its lipid-free state, as interaction with lipid is necessary to induce a conformational change which promotes high affinity for the receptors [39]. In addition, apoE has been shown to adopt different conformations when complexed to different lipids. Thus, changes in the lipid composition of lipoproteins such as an increased content of oxidized lipids or the presence of lipophilic compounds such as probucol may alter the conformation of the protein on their surface, and not all apoE molecules on a particular remnant particle may be able to act as ligands [39,40]. The different rates of uptake of CRLPs oxCRLPs and pCRLPs by macrophages demonstrated here, therefore, could be explained by effects on interaction with the LDLr and the LRP caused by differences both in the conformation of apoE and in the number of apoE molecules able to bind to the receptors, even though the total amount of apoE associated with the particles is not changed. Although we cannot rule out the possibility that the faster internalization of pCRLPs is due a specific effect of probucol on the surface structure of the particles, the finding that CRLPs are taken up more slowly after oxidation and more rapidly when the antioxidant is present suggests the changes are more likely to be related to the amounts of oxidized lipid in the particles.\nThe expression of scavenger receptors such as SR-A and the class B receptors CD36 and SR-B1 is a characteristic feature of macrophages, and SR-A and CD36 are know to play a part in the induction of foam cell formation by oxidized or chemically modified LDL [41]. It is possible, therefore, that this type of receptor may also be involved the uptake of oxCMR by macrophages. In the present study, poly I, a ligand for SR-A, reduced the uptake of acLDL by THP-1 cells by about 90% as expected, but had no significant effect on the uptake of CRLPs, irrespective of their oxidative state. In addition, excess HDL, which binds to SR-B1, did not prevent the entry of CRLPs, oxCRLPs or pCRLPs into the cells. In contrast, in the presence of anti-CD36, the uptake of all three CRLP types was inhibited by about 35\u201340% so that the differences in their rates of uptake were retained (Fig. 7). Thus, SR-A and SR-B1 do not appear to play a significant part in the uptake of oxCRLPs by macrophages, although CD36 may have a role which is unaffected by the oxidative state of the particles.\nMamo et al. [22] have reported previously that the phagocytosis may be important in the uptake of CMR by rabbit alveolar macrophages, although in a later electron microscopy study with HMDM they found no evidence for entry via this route [42]. In our experiments with THP-1 macrophages, however, cytochalasin D inhibited the uptake of all CRLPs types tested by 35\u201340%, suggesting that some phagocytosis of CRLPs of all oxidative states does occur in these cells (Fig. 7).\nIt has been suggested that the apoB48r may be involved in the uptake of chylomicron remnants by macrophages [1,20], and Kawakami et al. [43] have reported that it is responsible for the induction of macrophage foam cell formation by remnant lipoproteins from hyperlipidemic patients. Since apoB48 is an integral protein incorporated during the assembly of chylomicrons in intestinal cells, it is not possible to bind it to model CRLPs in a physiological way. The particles used in the present work, therefore, do not contain apoB48 and we were unable to study involvement of this receptor in the uptake of CMR by macrophages. However, antibodies to apoB48 have been found not to inhibit the uptake of chylomicron remnants by rat macrophages [44], and Elsegood et al. [21] who were unable to detect binding of chylomicron remnants to a protein with a molecular weight corresponding to the apoB48 receptor in THP-1 macrophages have suggested that it may be specific for VLDL remnants rather than chylomicron remnants.\nThe results of this study demonstrate that oxidative modification of CMR as compared to LDL has profoundly different effects on the uptake of the particles and the subsequent induction of lipid accumulation in macrophages. Instead of markedly enhancing foam cell formation, oxidation of CMR slows their uptake by macrophages and reduces the amount of lipid subsequently accumulated in the cells. This difference may be due to the different receptor mechanisms involved, since oxidation of LDL shifts the main route of uptake from the regulated LDLr to the unregulated scavenger receptors, while our experiments suggest that CMR are taken up mainly by the LRP with some contribution from the LDLr, with CD36 and phagocytosis playing only minor roles, irrespective of their oxidative state. These findings provide important new information about the way in which oxidation of CMR influences their induction of foam cell formation and the mechanisms involved, and has important implications for the role of dietary factors such as oxidized lipids and antioxidants which are transported in CMR in the promotion of atherosclerosis.","keyphrases":["chylomicron remnants","macrophages","foam cells","atherosclerosis","oxidized lipoproteins"],"prmu":["P","P","P","P","R"]}
{"id":"J_Behav_Med-3-1-2080858","title":"Repression: Finding Our Way in the Maze of Concepts\n","text":"Repression is associated in the literature with terms such as non-expression, emotional control, rationality, anti-emotionality, defensiveness and restraint. Whether these terms are synonymous with repression, indicate a variation, or are essentially different from repression is uncertain. To clarify this obscured view on repression, this paper indicates the similarities and differences between these concepts. Repression is the general term that is used to describe the tendency to inhibit the experience and the expression of negative feelings or unpleasant cognitions in order to prevent one\u2019s positive self-image from being threatened (\u2018repressive coping style\u2019). The terms self-deception versus other-deception, and socially related versus personally related repression refer to what is considered to be different aspects of repression. Defensiveness is a broader concept that includes both anxious defensiveness and repression; the essential difference is whether negative emotions are reported or not. Concepts that are sometimes associated with repression, but which are conceptually different, are also discussed in this paper: The act of suppression, \u2018repressed memories,\u2019 habitual suppression, concealment, type C coping pattern, type D personality, denial, alexithymia and blunting. Consequences for research: (1) When summarizing findings reported in the literature, it is essential to determine which concepts the findings represent. This is rarely made explicit, and failure to do so may lead to drawing the wrong conclusions (2) It is advisable to use scales based on different aspects of repression (3) Whether empirical findings substantiate the similarities and differences between concepts described in this paper will need to be shown.\nIntroduction\nPeople differ in their tendency to openly show, or to hide their negative emotions. This is an important topic in behavioral medicine, since studies have shown that repression is a potential health risk factor for disorders as diverse as chronic pain (Beutler et\u00a0al. 1986) and cancer (Jensen 1987; Weihs et\u00a0al. 2000). Another reason why repression may be considered a relevant topic for research in this field is that the tendency to avoid expressing negative emotions (also labeled \u2018repressive coping style\u2019) is known to distort the assessment of a patient\u2019s distress. As a result, this tendency to repress negative emotions may lead to making false conclusions. For instance, if patients report levels of distress similar to healthy individuals but show more repressive tendencies, they may in fact be more distressed. This repressive tendency may even influence the reporting of somatic symptoms and quality of life (Koller et\u00a0al. 1999).\nThe possible influence of repression on disease development, health behavior and symptom reporting has been investigated in many studies. Summarizing the findings proves problematic, however, as authors use different labels for \u2018repression-like\u2019 concepts, such as repression, suppression, non-expression of negative emotions, emotional control, emotional inhibition, rationality, anti-emotionality, type C response style, defensiveness, restraint, concealment, type D personality, denial, alexithymia and blunting. It is unclear whether this array of terms actually refers to the same concept or altogether different concepts. For instance, is forgetting details of traumatic events (e.g., sexual abuse or war experiences) comparable to not wanting to show one\u2019s emotions because of one\u2019s preference to rationalize? Is the tendency to minimize one\u2019s problems and to emphasize the positive aspects of experiences comparable to non-expression of negative emotions because one is afraid of personal confrontation? And yet in all these instances, the term repression is used.\nThe meanings of the various terms used in this field are defined below and an attempt is made to analyze their relationships on a conceptual level. This treatise does not discuss theories of repression and related constructs, but concerns the meaning of concepts. In our view, confusion exists more on the level of concepts than on the level of theories, and these theories are not very helpful in clarifying the conceptual confusion that exists. For instance, there is no theory that brings to notice that repression and concealment\u2014whereas the literal meaning of these words may suggest overlap\u2014refer to fundamentally different concepts. Nor is there any theory that alerts against the special use in some Behavioral Medicine texts of the term \u2018denial\u2019 in the sense of minimizing the seriousness of a disease and not as denial of negative emotional states in general (Brown et\u00a0al. 2000; Butow et al. 1999, 2000; Greer et al. 1979). It is important to add here, that several terms in this field have been introduced not on the basis of theory, but during the process of developing a measurement method.\nWe make a distinction between those concepts that in our view are related to repression, and other concepts that are sometimes defined as similar to or related to repression, but are clearly different. Figure\u00a01 illustrates the conceptual network.\nFig.\u00a01A conceptual diagram indicating which concepts fall under the headings of repression and anxious defensiveness, respectively, and which concepts are sometimes associated with, but theoretically different from defensiveness (voluntary suppression, repressed memories, denial and alexithymia)\nRepression\nRepression is the general term that is used to describe the tendency to inhibit the experience and the expression of negative feelings or unpleasant cognitions in order to prevent one\u2019s positive self-image from being threatened. A typical example of a person with repressive tendencies would be a sociable and cheerful man who rarely complains about any misfortune including disease, and whose self-image is one of a positive-minded person who is in control of his life. When he encounters someone who discusses an emotional problem, he is inclined to quickly change the subject in an attempt to avoid entering a world of anxiety, sadness or worry, which would imply that he has lost control.\nOther authors have presented comparable definitions, such as \u201cIndividuals who avoid focusing on ego-threatening material are termed repressors\u201d (Ashley and Holtgraves 2003), or \u201crepression can be defined as the avoidance of threatening information\u201d (Baumeister and Cairns 1992). The motive of \u201cpreventing one\u2019s positive self-image from being threatened\u201d is added in order to exclude some forms of non-expression, namely those due to shyness, social phobia and introversion. Shy or social-phobic people fear social situations, which inhibits their emotional expression, whereas repressive people do not fear or avoid social situations. Another difference is that shyness, social phobia and introversion refer to non-expression of both negative and positive feelings, whereas repression refers only to non-expression of negative feelings.\nWe use the term \u2018non-expression of negative emotions\u2019 here as a synonym for repression. This term suggests perhaps a focus on expressive behavior only, but it actually refers to the inhibition of both emotional experiences and behaviors. An explicit restriction is made to negative emotions, rather than emotions in general. This asymmetry, implied in the definition of repression, is mirrored by empirical findings. A repressive tendency appears to be related to downplaying negative aspects rather than to overstating positive aspects of a person (Myers and Brewin 1996), and to a memory deficit for real life events associated with negative emotions but not for events associated with positive emotions (Davis 1987).\nThere is no reason to label repression as either positive or negative on a phenomenological level. Repressive people will generally not bother other people with their problems and may even facilitate social situations by their positive attitude (Furnham et\u00a0al. 2003). Conversely, this coping style may impoverish intimate social interactions and may in the long run have a negative impact on the person\u2019s own functioning, due to a lack of insight into their own psychological functioning, a decrease in the variety of their coping repertoire and overlooking signals that lead to seeking medical help in time. In the long term repression may have negative somatic consequences, including an increased risk of various disorders.\nBelow is first discussed a proposal by Weinberger to make a distinction between repression and anxious defensiveness (Weinberger et al. 1979; Weinberger 1990; Weinberger and Schwartz 1990). This distinction is useful for placing repression-like concepts under one of these two headings.\nDefensiveness\nWhereas most elements in this discussion can be approached on a merely conceptual level, the discussion of the concept of Defensiveness needs to be introduced by mentioning empirical findings. Weinberger (1990) makes a distinction between two types of defensiveness: He defines repression as scoring high on defensiveness but low on anxiety, and anxious defensiveness as scoring high on both defensiveness and distress self-report scales.1 In fact, Weinberger formed six groups, based on a tripartition of restraint scores and a dipartition of distress scores, but the remaining four groups are not relevant to this discussion. In an earlier publication he describes a more familiar division of defensiveness types based on a dipartition of social desirability scores and anxiety scores, which is comparable to his more recent division (Weinberger et\u00a0al. 1979). The two groups appeared to be different on a number of personality variables. Compared to the other groups, the anxious defensive group scored low with respect to assertiveness, ability to express themselves in close relationships, sensitivity to their own needs and feelings, self-esteem and self-control. They also scored high on avoidant personality (shyness), dependency (emotional reliance on others and approval dependence), obsessive worrying, and (minor) physical illnesses. The repressive group, on the other hand, was characterized by high scores for intimacy, self-esteem, self-control (tendency to use self-management techniques), defensiveness and alexithymia2, while low on avoidant personality. These differences in a broad spectrum of personality traits indicate that the division into the two defensiveness groups is more than the product of an arithmetic procedure; it refers to a constellation of essential individual differences (Weinberger and Schwartz 1990).\nDefensiveness, therefore, covers a broader category than repression. Defensiveness concerns different strategies to protect oneself against being hurt psychologically. One strategy is to behave\u2014more or less anxiously\u2014in a socially acceptable way, to be nice in order not to get hurt, and to avoid social confrontations. Another strategy is to inhibit thoughts about negative aspects of oneself, and to consider oneself as the social person one would rather be. The first condition, anxious defensiveness, includes the awareness of negative emotions, whereas the second condition, repression, denies these emotions.\nWeinberger\u2019s division into two forms of defensiveness is highly useful. If high levels of anxiety or other forms of distress are implied in the definition of a repression-like concept, one should place this concept under the heading of anxious defensiveness, rather than under the heading repression. On an empirical level, a low level of distress reporting in repression would be expected (negative relationship between repression and distress), and a relatively high level in anxious defensiveness (positive relation).\n(Un)consciousness\nRepression is a tendency that a person may be (partly) aware of, which in psychodynamic theories is referred to as suppression, or unaware of, referred to as repression in these theories. However, (un)consciousness is often not explicitly included in the definition of repression as a distinctive characteristic. To give some citations: \u201cRepression can be defined as the avoidance of threatening information\u201d (Baumeister and Cairns 1992, p. 853). \u201cRepressors are individuals who habitually and efficiently control their emotions\u201d (Boden and Dale 2001, p. 122). Some authors\u2014on the contrary\u2014stress the role of the unconscious: \u201cRepressive-defensiveness is characterized by a non-conscious avoidance of threatening information\u201d (King et al. 1992, p. 87). Repressive-defensiveness is defined here according to Weinberger\u2019s operational definition of repression (Weinberger et\u00a0al. 1979; see above). Quite confusing is that other authors, using a different term namely repressive coping but referring to the same operational definition of Weinberger, claim the opposite: \u201cRepressive coping is thought to modulate the conscious experience of negative affect following the appraisal of threat\u201d (Newton and Contrada 1992, p. 160). Most contemporary authors describe repression in terms of active cognitive processes, such as selective inattention and motivated forgetting rather than in terms of an unconscious defense mechanisms (Baumeister and Cairns 1992; Newton and Contrada 1992; Mendolia et al. 1996). The most explicit view on this comes from Erdelyi (1993, 2001), who states that empirical findings do not support any distinction between conscious and unconscious forms of emotional inhibition. He also uses an historical argument. Whereas many authors referred to Freud when discussing the difference between repression and suppression, this is\u2014in Erdeleyi\u2019s view\u2014historically unwarranted, given that Freud used these terms interchangeably. The notion that repression must be unconscious is not Sigmund Freud\u2019s, but his daughter\u2019s, Anna Freud (1946).\nIf any distinction is made between suppression and repression, the focus is usually placed on processes, i.e. on time-limited cognitive acts. The distinction between conscious and unconscious forms of inhibition does seem possible and it is perhaps useful when applying it to acts, but this distinction is more difficult to apply to traits. The discussion below is about repression as a trait (\u2018repressiveness\u2019). One may on occasion be aware of one\u2019s tendency to inhibit the experience and expression of negative feelings, but most of the time be vaguely aware, and more often totally unaware of them.\nEmpirical findings indicate that repressors are often unaware, or at least not fully aware of their emotional avoidance style. As indicated below, repressors genuinely perceive themselves as being low in anxiety and are primarily self-deceivers. Another indication for the (mainly) unconscious character of repression is the repressor\u2019s decreased ability to recall personal experiences associated with a negative affect. When memories are recalled by repressors, thus having become conscious, they are not processed more slowly than by the non-repressors (Davis 1987).\nSelf-deception and Other-deception\nExpressing negative emotions may be deliberately avoided as part of the tendency to make a favorable impression on other people. This tendency is called impression management or, originally, other-deception. It is distinguishable from self-deception, in which case the person actually believes his or her positive self-reports (Paulhus 1984; Rogers and Kristjanson 2002).\nImpression management is modestly and negatively related to reports of negative emotions and somatic symptoms, while a self-deceptive response style reduces symptom reporting above the effects of deliberate impression management (Linden et\u00a0al. 1986). Given that underreporting negative emotions is the hallmark of repression, these findings seem to indicate that self-deception is closer to repression than impression management. Another characteristic of repression is a deficiency in the memory for emotional events (Furnham et\u00a0al. 2003; Davis 1987). This deficiency appeared to be related to self-deception, but not to impression management (Ashley and Holtgraves 2003).\nTwo studies found that repressors scored high on both other-deception and self-deception questionnaires (Derakshan and Eysenck 1999; Furnham et al. 2002). The study of Derakshan and Eysenck (1999) also showed that repressors are more self-deceivers than other-deceivers. This was demonstrated with the so-called \u2018bogus pipeline\u2019 method, where participants are connected via electrodes to a piece of apparatus resembling a lie detector, which could allegedly detect whether they are telling the truth. Compared to a control condition, people are generally more willing to report truthfully about their emotional states when subjected to the bogus line condition, even if this report is seen as socially undesirable or embarrassing for that person. Repressors generally did not show any difference in anxiety scores between both conditions. This finding suggests that repressors genuinely perceive themselves as being low in anxiety. They are mainly self-deceivers, though the questionnaire data indicated that they also showed some tendency to present themselves deliberately in a socially desirable light. In another experiment, Baumeister and Cairns (1992) showed that repressors who privately received threatening feedback spent the least amount of time reading it, whereas repressors who received the same feedback publicly spent considerably more time reading it. Non-repressors were unaffected by the favorability of the evaluation (threatening or not) or the private versus public nature of the situation. These findings suggest that repressors abandon self-deceptive strategies in favor of impression management strategies in an attempt to invalidate the socially undesirable information about their personality.\nDoubts have arisen about the validity of self-deception (SD) and other-deception (OD) scales, including the widely used Balanced Inventory of Desirable Responding (BIDR) of Paulhus (1984). Studies have shown that the scores to SD and OD scales can be influenced by instructions (Stober et al. 2002; Pauls and Crost 2004), such as a fake good instruction (e.g. \u201cPresent yourself as much as possible in a favorable light\u201d), a fake being competent instruction (e.g. \u201cPresent yourself as much as possible as being competent and self-confident\u201d), or a fake social harmony instruction (e.g. \u201cPresent yourself as much as possible in an agreeable and conscientious light\u201d). The SD and OD scores changed differently, dependent on the type of instruction. For instance, SD scores were most sensitive to the competence instruction and OD scores were most sensitive to the social harmony instruction (Pauls and Crost 2004). SD and OD were measured in this study with the widely used Balanced Inventory of Desirable Responding. The authors of this study argue that this questionnaire does not so much measure SD and OD, as is generally believed, but more so overconfidence versus need for social harmony. This may imply that the question whether repression is predominantly associated with self-deception or other-deception cannot be answered with so-called SD and OD scales, but only with experimental designs such as applied in the studies of Derakshan and Eysenck (1999) and Baumeister and Cairns (1992).\nWhat is the precise relationship between these two concepts and repression? The character of self-deception, i.e., believing one\u2019s positive self-report, is completely compatible with the definition of repression. There is, however, no complete overlap between repression and impression management. An extreme form of impression management would be consciously trying to project a positive image towards others while being fully aware of one\u2019s negative feelings and negative cognitions. This condition of other-deception without self-deception is not compatible with the description of repression. Because of this conceivable exception, other-deception is depicted in Fig.\u00a01 as only partly overlapping with repression.\nDifferent Aspects of Repression\nCertain explanations of repression seem to emphasize the social aspect, whereas other explanations do not describe the repressive tendency as specifically socially related. For instance, Weinberger and Schwarz, who used the term \u2018self-restraint\u2019, state that it concerns \u2018domains related to socialization and self-control and refers to repression of egoistic desires in the interest of long-term goals and relations with others\u2019. The term \u2018self-restraint\u2019 also encompasses \u2018tendencies to inhibit aggressive behavior, to exercise impulse control, to act responsibly, and to be considerate of others\u2019 (Weinberger and Schwartz 1990, p. 382). To label such tendencies, the term \u2018socially related repression\u2019 would therefore seem appropriate. These tendencies may be part of the broader tendency to behave in a socially acceptable way, which should not be conceived as simply the need to follow external norms, but reflect a self-concept that depends on the approval of other people.\nWe suggest using the label \u2018personally related repression\u2019 for a second aspect of repression, which is not primarily socially related and may be defined as the general tendency to inhibit one\u2019s expression of anxiety, depression, worry and anger, and not to let oneself be influenced by these negative feelings. This heading subsumes several concepts. Watson and Greer (1983) use the term emotional control and describe it as \u2018the extent to which individuals report controlling their reactions when a particular emotion is experienced\u2019, especially anger, anxiety and depressed mood. This description does not specifically suggest social determination. Rationality is another example and is described by Spielberger (1988) as \u2018the extent to which an individual uses reason and logic as a general approach to coping with the environment\u2019.\nEmpirical support for our proposed division was found in a study that applied a secondary factor analysis to investigate the interrelationships between various subscales of two repression questionnaires and three distress questionnaires (Giese-Davis and Spiegel 2001). The scales applied in this study were the three subscales of the Courtauld Emotional Control Scale (CECS) used to measure emotional control, the five subscales of the Weinberger Attitude Inventory (WAI), used to measure self restraint, and three distress questionnaires (POMS, CES-D and IES; four subscales in total). There is no reference to social situations in the items included in the CECS (Watson and Greer 1983). An example item is \u201cWhen I feel afraid or worried, I smother my feelings\u201d. Most of the items of in the WAI subscales (D. A. Weinberger, unpublished ) refer to social situations, such as the item \u201cI think about other people\u2019s feelings before I do something they might not like\u201d. One would expect personally related repression scales (CECS) to load on one dimension and scales assessing socially related repression (WAI) on a second dimension (and the distress scales on a third dimension). The factor analysis yielded a four-factor solution, including (1) subscales of the CECS (2) WAI restraint scales (3) WAI defensiveness scales and (4) the distress scales. We cannot explain why the WAI repression scales loaded on two different factors. However, the finding that the CECS scales clustered in one factor and the WAI repression scales in two other factors, could be seen as support for our division into personally related and socially related repression.\nConcepts Different from Repression\nConcepts that are in our view different from repression are: the act of emotional suppression, repressed memories, habitual suppression, concealment, type C coping pattern, type D personality, denial, alexithymia and blunting. The first concept refers to an \u2018act,\u2019 whereas repression is discussed in this paper as a tendency or coping style. Four of these concepts\u2014habitual suppression, concealment, type C coping style and type D personality should, in our view, be interpreted as types of anxious defensiveness rather than forms of repression. Our motive for discussing these \u2018non-repression\u2019 concepts is that the literature may suggest that these concepts are related to repression.\nThe Act of Voluntary Suppression of Emotionally Charged Material\nIn an experimental context people can be asked to refrain from showing emotional reactions, or to try not to think about an emotional condition. Suppression of emotional behavior or thoughts can also occur spontaneously in everyday life. Given that such acts can be performed incidentally, both by people high or low in repression, the act should be distinguished from the habitual response style. It cannot simply be assumed that the consequences of the act of emotional suppression are similar to the psychological and somatic concomitants of being an habitual repressor. For instance, in the long term emotional disclosure leads to a decrease in reported psychological and somatic symptoms (Smyth 1998). On the other hand, compared to non-repressors, habitual repressors also report less distress (Ward et al. 1988; Weinberger and Schwartz 1990; Weinberger, unpublished ; Swan et\u00a0al. 1992; Tomaka et al. 1992; Bleiker et\u00a0al. 1993). Therefore, the act of emotional expression as well as the response style of non-expression are both associated with low reported distress.\nThere is an area of research that studies the effects of inhibiting emotional behavior (Gross and Levenson 1993) and another area of research that is interested in the effect of thought suppression (Abramowitz et al. 2001). Participants in such studies are asked to refrain from emotional behavior, such as facial expressions, or not to think of a certain image. Gross uses the term \u2018emotional suppression\u2019 in the sense of an act and describes it as the conscious inhibition of behavioral signs of emotion, while being emotionally aroused (Gross and Levenson 1993).\nRepressed Memories\nRepressing memories of traumatic events concerns a complex of cognitions and emotions that is mainly limited to a certain theme or event, such as sexual abuse in childhood. This is different from repression, which concerns the tendency not to express negative emotions in general. Repression of memories is initiated by traumatic events, whereas repression is a habitual style applied in a variety of situations. Although repressing memories of traumatic events could lead to an habitual style of repression, or magnify an existing tendency to repression that does not undo the conceptual difference.\nStrengthening of existing repressive tendencies in response to a traumatic condition was demonstrated in a study among women who were awaiting the outcome of diagnostic tests for breast cancer, which may be conceived as a traumatic event. An increase in the number of repressors was found after the diagnosis of breast cancer was made known to the patients, whereas no increase was found in women who appeared to be free of cancer (Kreitler et al. 1993).\nIn a series of studies, McNally presented evidence that those who believe they were sexually abused as children, but have no memory for these events (\u201crepressed memories\u201d) show a particular style of information processing, which is different from those who have never forgotten their childhood sexual abuse or have never been sexually abused (McNally 2001). Individuals with repressed memories exhibited symptoms of psychological distress, elevated levels of dissociation and absorption, superior forgetting abilities for trauma-related material and memory distortions. Most of these characteristics were also found in individuals who report having recalled long-forgotten episodes of childhood sexual abuse (\u201crecovered memories\u201d). These characteristics may reflect a propensity for repressing traumatic memories, a propensity for forming false memories of trauma, or a consequence of abuse itself (assuming it occurred). Anyhow, this personality profile relates to information processing distortions of trauma-related material, not to emotionally loaded material in general as found in repression. Moreover, the high distress scores of individuals with repressed memories are incompatible with the concept of repression.\nHabitual Suppression\nIn later publications on emotion regulation, Gross shifted his attention from suppression as an act to the habitual use of suppression, which was described as a form of response modulation that involves inhibiting ongoing emotion-expressive behavior (John and Gross 2004). Inhibition of emotional experiences is not assumed, as is the case in the definition of repression. Gross even presumes that using suppression in everyday life might actually be associated with greater negative emotion experience. Acknowledging these characteristics, habitual suppression must come under the heading of anxious defensiveness.\nEmpirical findings indicate that habitual suppression is different from, and in some respects, the opposite of repression. Whereas habitual suppressors experience less positive and more negative emotions than other people (John and Gross 2004), repressors do not differ from other people with respect to the experience of positive emotions and they experience less negative emotions than non-repressors (Furnham et\u00a0al. 2003). Moreover, habitual suppressors have a lower self-esteem and a less optimistic outlook (John and Gross 2004), whereas the opposite was found for repressors (Myers and Reynolds 2000).\nSelf-concealment\nLarson and Chastain (1990) introduced the concept self-concealment as the trait version of the act of inhibition, studied by Pennebaker et\u00a0al. 1990). The authors define self-concealment as a \u201cpredisposition to actively conceal from others personal information that one perceives as distressing or negative,\u201d and they say that \u201cself-concealed personal information is consciously accessible to the individual\u201d (Larson and Chastain 1990, p. 440).\nHow is this concept related to repression? There are three gradual differences with repression: (1) Self-concealment concerns specific distressing secrets, whereas repression concerns negative feelings in general, although it should be said that there is a rather thin line between these two elements; (2) Self-concealment is explicitly a tendency towards voluntary and conscious inhibition, whereas repression is conceptualized as incorporating both unconscious and conscious coping strategies; (3) Self-concealment implies the awareness of distressing thought contents, whereas repression implies the inhibition to become fully aware of such thought contents. Especially this last aspect implies that self-concealment could be better placed under the heading anxious defensiveness, rather than repression.\nEmpirical findings support this supposed conceptual difference. While repression is often negatively related to distress reporting (Weinberger and Schwartz 1990; Weinberger, unpublished ; Swan et\u00a0al. 1992; Tomaka et\u00a0al. 1992; Bleiker et\u00a0al. 1993; Gick et al. 1997; Vetere and Myers 2002), a positive association has been found between self-concealment and depression, anxiety and physical symptoms (Larson and Chastain 1990) and rumination (King et\u00a0al. 1992). A negative relationship between self-concealment and repression (King et\u00a0al. 1992; Ritz and Dahme 1996) has also been reported. In fact, (Ritz and Dahme 1996) found the lowest scores on the Self-Concealment scale (SCS; Larson and Chastain 1990) for repressors and the highest SCS scores among the (truly or defensive) high-anxious persons.\nType C Coping Style\nThe Type C concept was first introduced in 1980 in an abstract presented by Morris and Greer (1980), who considered this coping style as characteristic of cancer patients. The style was described as being emotionally contained, especially in stressful situations. Temoshok independently developed a similar concept, which included several elements (Kneier and Temoshok 1984), and described this coping style as \u2018abrogating one\u2019s own needs in favor of those of others, suppressing negative emotions, and being cooperative, unassertive, appeasing, and accepting. The Type C individual is considered nice, friendly and helpful to others, and rarely gets into arguments or fights... The Type C individual may be seen as chronically hopeless and helpless, even though this is not consciously recognized in the sense that the person basically believes that it is useless to express one\u2019s needs... The Type C individual does not even try to express needs and feelings; these are hidden under a mask of normalcy and self-sufficiency\u2019 (Temoshok 1987, pp. 558\u2013560).\nOne of the repressive types, as distinguished by Weinberger and Schwartz (1990), in our view shows a remarkable resemblance to the Type C coping pattern. The characteristics mentioned for anxious defensiveness resemble the above-mentioned characteristics for individuals who use Type C coping. Both descriptions mention unassertiveness, low sensitivity to one\u2019s own needs and feelings, abrogating one\u2019s own needs in favor of others, emotional reliance on others, being cooperative, appeasing and accepting, and high levels of distress (obsessive worrying\/ helplessness and hopelessness). Because of this similarity and the inclusion of helplessness and hopelessness in the description of this concept, the Type C response pattern seems closer to anxious defensiveness than repression.\nType D Personality\nThe term \u2018Type D personality\u2019 was introduced by Denollet (1997) to describe those people who are distressed, but who also inhibit the expression of emotions. Denollet developed this concept, while working in the field of cardiovascular disorders. It consists of a combination of two factors that seemed predictive for the development of coronary heart disease and hypertension. The first factor was high distress levels (anger, depression, anxiety and vital exhaustion) and the second factor was the inhibition of emotional expression. This second factor was specified as reflecting social inhibition (and introversion).\nIt is important to be aware that according to the description of the Type D person, the negative emotions of anger, anxiety, and depression are experienced consciously. Whereas repression and distress are often negatively related, a high level is implicated in the Type D personality. Type D persons are categorized as scoring high on distress and high on social inhibition (Denollet 2005). Therefore, the Type D personality style is explicitly the anxious defensive type.\nDenial\nDenial is conceived here as denying or minimizing the seriousness of a medical condition, not as denial of emotions or painful events as is commonly the case, and not as an unconscious defense against painful and overwhelming aspects of external reality, as described in psychoanalytic theory. The way denial is described in this paper\u2014denial of diagnosis or denial of impact\u2014is one of the several definitions quoted in the literature (Vos and Haes 2007; Moyer and Levine 1998). Greer et\u00a0al. described denial in breast cancer as \u201capparent active rejection of any evidence about their diagnosis which might have been offered, including the evidence of breast removal, such as \u201cit wasn\u2019t serious, they just took off my breast as a precaution\u201d (Greer et\u00a0al. 1979, p. 786). Minimizing the impact of cancer is a milder and more realistic form of denial, and was measured in studies by Butow. (Butow et\u00a0al. 1999, 2000; Brown et\u00a0al. 2000). Our definition of denial indicates a clear conceptual difference between denial and repression. Repression does not specifically refer to the emotional consequences of a disease, but rather to negative emotions in general. A person might repress these emotions, while not denying the seriousness of the disease. Denial or minimizing can either be an act (an event-driven coping response) or it can reflect a habitual style of minimizing the seriousness of unpleasant events.\nIt is interesting that cancer studies showed opposite consequences of the two phenomena. In studies using a prospective, longitudinal design to investigate the role of psychological factors on the course of cancer, two studies found that repression predicted an unfavorable course (Jensen 1987; Weihs et\u00a0al. 2000). Obversely, in four other studies denial or minimizing was found to predict a favorable course of cancer (Greer et\u00a0al. 1979; Dean and Surtees 1989; Greer et\u00a0al. 1990; Butow et\u00a0al. 1999, 2000).\nAlexithymia\nThe concept of alexithymia is derived from clinical observations of a cluster of specific cognitive characteristics among patients suffering from psychosomatic diseases, substance use disorders, and post-traumatic stress disorders (Nemiah et al. 1976; Bagby et al. 1997). It evolved into a theoretical construct, with the following salient features: (1) difficulty identifying feelings, (2) difficulty describing feelings, and (3) externally oriented thinking (Bagby et\u00a0al. 1997).\nDue to the difficulty of identifying feelings, one might assume that emotions are not expressed either. However, alexithymic persons should not be considered to be emotionally flat. Nemiah et\u00a0al. reported a proneness to sudden outbursts of crying and anger in these persons, though they were unable to connect these behaviors with thoughts and fantasies (Nemiah et\u00a0al. 1976). Corresponding to these observations, Sifneos reported that it was common for his patients to mention anxiety or to complain of depression (Sifneos 1967), although they used a limited vocabulary to describe their emotions. The emotions of alexithymic individuals appear to be rather diffuse, poorly differentiated and not well-represented. Taylor et\u00a0al. concluded that alexithymia should be regarded not as a defense against distressing affects or fantasies, but rather as the reflection of an individual difference in the ability to process and regulate emotions cognitively (Taylor et al. 1997). They suggested that this construct is different from \u2018other emotion-related constructs such as inhibition and the repressive-defensive coping style.\u2019\nThe difference between alexithymia and repression is empirically supported. First, several studies have shown that the Toronto Alexithymia Scale (TAS)\u2014the most widely used and well-validated questionnaire for assessing alexithymic traits\u2014appeared to be unrelated or negatively related with various measures of repression (King et\u00a0al. 1992; Newton and Contrada 1994; Myers 1995; Linden et al. 1996; Taylor et\u00a0al. 1997). Second, the TAS is unrelated (Linden et\u00a0al. 1996), or positively related with self-reported distress (Taylor et\u00a0al. 1997; Verissimo et al. 1998), whereas measures of repression are negatively related with distress.\nBased on psychometric comparisons, alexithymia shows some correspondence to the sensitizing style of high-anxious persons, rather than the avoidant style of repressors. Repressive individuals often report that they are not upset despite objective evidence to the contrary, whereas alexithymic individuals acknowledge that they are upset, but have difficulty in specifying the nature of their distress.\nBlunting\nMiller (1987) distinguishes two types of individual differences in dealing with threatening stimuli. Monitors are those people who tend to seek information when coping with a threat, such as going to the dentist, being taken hostage or flying (information seekers), and blunters tend to avoid information when faced with a threat (distractors). There is some similarity between blunting and repression, as both repressors and high blunters avoid distressing information. Repressors avoid mainly personally relevant, emotionally loaded information. High blunters, on the contrary, avoid material about external conditions that people generally regard threatening. There is no implication in the definition that high blunters avoid expressing negative emotions because that would threaten their self-image, which is explicitly included in the description of repression.\nDiscussion\nThe way in which most people use the term \u2018repression\u2019 in an everyday context indicates that they generally understand what it actually refers to. Whether science was right to introduce the current assortment of subtle differences thus exposing the gross simplicity of society\u2019s everyday use of the term, or whether science has ultimately entangled the term in a maze of unclear terminology is debatable. It is undebatable, however, that there is a lack of consensus about what repression is. The impression is that the many terms used in this field\u2014repression, suppression, non-expression of negative emotions, emotional control, emotional inhibition, rationality, anti-emotionality, defensiveness and restraint\u2014denote something similar to repression, but there is no certainty whether they can be considered synonymous, indicate a subtle variation of repression, or indicate an associated, but essentially different concept. One reason for this confusion is that definitions are rarely given, and that hardly ever is indicated how a new term relates to regularly used terms.\nRepression has been defined as the tendency to inhibit\u2014consciously or unconsciously\u2014the experience and expression of negative emotions or unpleasant cognitions in order to prevent one\u2019s positive self-image from being threatened. The term is used to describe an act, such as avoiding a specific memory, or a tendency or coping style (\u2018repressive coping style\u2019). This paper deals with repression as a tendency or coping style. Terms whose definitions appear to agree with the definition of repression, which can therefore be considered synonyms of repression, are: non-expression of negative emotions, emotional inhibition, emotional control, anti-emotionality, rationality and self-restraint. Although these terms may all be subsumed under the heading repression, their definitions also suggest some differences concerning the motives for repression.\nWe tentatively made a difference between socially related and personally related repression (see Fig.\u00a01). An example of socially related repression is \u2018self-restraint\u2019, which encompasses \u2018tendencies to inhibit aggressive behavior, to exercise impulse control, to act responsibly, and to be considerate of others\u2019 (Weinberger and Schwartz 1990, p. 382). An example of personally related repression is rationality, which is described as \u201cthe extent to which an individual uses reason and logic as a general approach to coping with the environment\u201d (Spielberger 1988). In the first category, the tendency to inhibit the experience and expression of negative emotions in order to prevent one\u2019s positive self-image from being threatened is (more) socially related, which reflects a self-concept that depends (more) on the approval of other people more so than personally related repression. Future research will show whether this distinction is useful or not. A study has indeed provided some empirical evidence for its validity, showing that restraint scales and emotional control scales loaded on different dimensions in a secondary factor analysis (Giese-Davis and Spiegel 2001).\nBy definition, repression implies (some degree of) self-deception, whereas repression may or may not include other-deception. Self-deception implies honestly believing one\u2019s positive self-report. The overlap with repression is evident, given that \u201cthe inhibition of the experience of negative emotions or unpleasant cognitions in order to prevent one\u2019s positive self-image from being threatened\u201d implies self-deception. Other-deception is described as deliberately avoiding expression of negative emotions as part of the tendency to make a favorable impression on other people. Other-deception without self-deception, therefore, seems to be incoherent with the definition of repression.\nDefensiveness is a broader concept than repression. Defensiveness concerns different strategies to protect oneself against being psychologically hurt, which include repression and anxious defensiveness (Weinberger and Schwartz 1990). This distinction was made on the basis of operational criteria. Although high levels of defensiveness characterize both forms, repressors report relatively low distress levels, whereas anxious defensive persons report relatively high distress levels. The division into the two defensiveness groups appears to be more than the product of an arithmetic procedure; it refers to a constellation of essential individual differences (Weinberger 1990). One essential difference concerns the two defensiveness groups\u2019 association with distress.\nWe have also indicated which concepts, although sometimes associated with repression, are basically different from repression: voluntary suppression, repressed memories, habitual suppression, self-concealment, type C coping pattern, type D personality, denial, alexithymia and blunting. The first concept concerns an \u2018act,\u2019 whereas we have discussed repression as a tendency or coping style. Four of these concepts were placed under the heading anxious defensiveness, because their definitions imply experiencing high levels of negative emotions: Habitual suppression, self-concealment, type C coping style and type D personality.\nOur conceptual analysis has motivated us to propose the following recommendations for future research: (1) In studies on the character and the consequences of repression one should ideally include measures of personally related and socially related repression, and\u2014as a contrast\u2014a measure of anxious defensiveness. (2) An acute distinction should be made when summarizing literature findings between repression and concepts that are related to, but essentially different from repression (3) Future research will need to show whether relationships between questionnaires substantiate the similarities and differences between the concepts described in this paper.\nOur objective with this treatise on defensiveness-related concepts is to provide more clarity in this field. The next step in finding our way in the current maze of repression points to a review on defensiveness-related questionnaires.","keyphrases":["repression","emotional control","defensiveness","restraint","self-deception","type c","denial","alexithymia"],"prmu":["P","P","P","P","P","P","P","P"]}
{"id":"J_Biol_Inorg_Chem-3-1-1915633","title":"Crystal structure of the ferritin from the hyperthermophilic archaeal anaerobe Pyrococcus furiosus\n","text":"The crystal structure of the ferritin from the archaeon, hyperthermophile and anaerobe Pyrococcus furiosus (PfFtn) is presented. While many ferritin structures from bacteria to mammals have been reported, until now only one was available from archaea, the ferritin from Archaeoglobus fulgidus (AfFtn). The PfFtn 24-mer exhibits the 432 point-group symmetry that is characteristic of most ferritins, which suggests that the 23 symmetry found in the previously reported AfFtn is not a common feature of archaeal ferritins. Consequently, the four large pores that were found in AfFtn are not present in PfFtn. The structure has been solved by molecular replacement and refined at 2.75-\u00c5 resolution to R = 0.195 and Rfree = 0.247. The ferroxidase center of the aerobically crystallized ferritin contains one iron at site A and shows sites B and C only upon iron or zinc soaking. Electron paramagnetic resonance studies suggest this iron depletion of the native ferroxidase center to be a result of a complexation of iron by the crystallization salt. The extreme thermostability of PfFtn is compared with that of eight structurally similar ferritins and is proposed to originate mostly from the observed high number of intrasubunit hydrogen bonds. A preservation of the monomer fold, rather than the 24-mer assembly, appears to be the most important factor that protects the ferritin from inactivation by heat.\nIntroduction\nFerritin is a protein involved in metal homeostasis by reversible storage of iron and presumably also in protection against oxidative stress by scavenging reactive oxygen species. It is a small protein of approximately 20\u00a0kDa and its main structural motif is a bundle of four parallel \u03b1-helices and a fifth short \u03b1-helix tilted towards the bundle axis and masking the one end of the cylindrical bundle. The functional ferritin molecule is an approximately 500\u00a0kDa hollow spherical assembly of 24 subunits, with outer and inner diameters of 12 and 8\u00a0nm, respectively. The 24-meric ferritin agglomerate may be a homopolymer or, in higher eukaryotes, a heteropolymer consisting of homologous subunits designated as H, M and\/or L. Ferritin subunits, except the L-type subunit, incorporate a ferroxidase center (FC) that consists of a \u03bc-oxo bridged dinuclear iron group and is responsible for iron oxidation. Additionally, a third iron site is observed in the subunits of some bacterial ferritins [1, 2]. The binuclear iron cluster of the FC and the third iron site are referred to as A, B and C iron sites, respectively. Some ferritins, also called bacterioferritins (BFRs), enclose 12 heme groups that are located between homodimeric subunit pairs, and may be involved in electron transfer [3]. The homologous protein DNA protection during starvation (DPS) and also called a \u201csmall ferritin,\u201d is a spherical 12-subunit assembly with smaller dimensions (outer and inner diameters of 9 and 4.5\u00a0nm, respectively) and iron storage capacity (500 iron atoms vs. 3,000 in ferritin) than ferritins [4]. While iron is believed to be the main cationic substrate of ferritins, zinc accumulation in ferritin has also been reported [5].\nFerritins occur in a wide variety of organisms, from prokaryotes to mammals. In humans, their function is related to iron deficiencies or iron-overload disorders such as thalassemia, sickle cell anemia, hemochromatosis and pulmonary hemosiderosis [6\u20139]. Understanding the mechanism of the action of ferritin will be of great value in the treatment of iron-related diseases. Besides its medical relevance, this knowledge is also of fundamental scientific interest, especially in case of anaerobes. To date, an insight into the iron-uptake reaction has only been achieved for aerobic organisms. In these organisms, Fe(II) ions are oxidized, possibly together with phosphate oxoanions, and stored inside a ferritin shell as a soluble Fe(III) mineral, believed to be a ferrihydrite mineral with approximate composition Fe2O3\u00b70.5H2O. Ferric ions from the core can be reduced and released from the ferritin molecule when needed by a cell. Nevertheless, the chemical nature of the physiological reductants for iron release and in the case of anaerobes that of the physiological oxidants for iron uptake have not yet been established.\nCrystal structures of ferritins from bacteria, fungi, plants, insects and vertebrates are available, whereas those from archaea are limited to a ferritin from the sulfate-reducing anaerobe and thermophile Archaeoglobus fulgidus (AfFtn) (Protein Data Bank, PDB, codes 1sq3 and 1s3q) [2] and a DPS protein from Halobacterium salinarum (PDB codes 1tjo, 1tk6, 1tko and 1tkp) [10].\nIn AfFtn, a novel 23 point-group symmetry of the 24-mer assembly was found, which is unusual for ferritins and was only known previously in 12-meric assemblies, such as the DPS proteins [10, 11] and also in the ferritin from the bacterium Listeria innocua [12]. As a result, four outsized triangular pores about 45-\u00c5 wide are present in the protein shell of AfFtn [2].\nPyrococcus furiosus is a strict anaerobe and hyperthermophilic archaeon, living optimally at 100\u00a0\u00b0C. It was isolated from the hot marine springs off the beach of Porto di Levante in Vulcano, Italy. P. furiosus is a fermentative heterotroph that grows on starch, maltose, peptone and yeast extract and produces CO2, acetate, alanine, H2 and H2S. The expression of the native ferritin from P.\u00a0furiosus (PfFtn) has been confirmed by N-terminal sequencing of the purified protein and the recombinant ferritin has been overproduced in Escherichia coli in order to afford extensive biochemical studies [13].\nHerein we present the 3D structure of PfFtn, refined at 2.75-\u00c5 resolution. In contrast to the previously reported first structure of an archaeal AfFtn, the functional PfFtn 24-mer has the typical 432 point-group symmetry observed in other ferritins and BFRs. There are no four large pores such as were found in AfFtn. Instead, the characteristic threefold and fourfold channels are present in PfFtn.\nThe extreme thermostability of PfFtn, which in vitro can withstand 1\u00a0day of incubation at 100\u00a0\u00b0C, has been analyzed at the structural level, in comparison with structurally related ferritins. Although P. furiosus grows at the highest temperature, 100\u00a0\u00b0C, among the organisms used in thermostability analysis, our results suggest that the ferritin from Thermotoga maritima (TmFtn), which has an optimal growth temperature of 80\u00a0\u00b0C, may be even more thermostable than PfFtn. The hyperthermal stability of PfFtn and other ferritins appears to result mainly from the preservation of the monomer fold rather than the 24-mer assembly, owing to a high number of intramolecular hydrogen bonds between main-chain atoms, and between main-chain and side-chain atoms.\nThe FC stability was analyzed by electron paramagnetic resonance (EPR). Without further experiments, the lack of FC iron sites B and C in nonsoaked PfFtn crystals obtained does not allow any conclusions supporting or contradicting the FC as a stable catalytic cofactor.\nMaterials and methods\nProtein production and crystallization \nPfFtn was obtained and purified as previously described [13]. Ferritin iron loading prior to crystallization was performed by adding a freshly and anaerobically prepared aqueous solution of 20\u00a0mM iron sulfate, containing 0.1% HCl, to a solution of apoferritin in 50\u00a0mM N-(2-hydroxyethyl)piperazine-N\u2032-ethanesulfonic acid (Hepes), pH 7, under aerobic conditions. Crystals of PfFtn were produced using the hanging drop vapor diffusion method at room temperature [14]. The crystals used for data collection were grown against 0.5\u00a0ml of 2\u00a0M ammonium sulfate reservoir solution, with drops containing 2\u00a0\u03bcl of 5\u00a0mg\u00a0ml\u22121 protein solution in 50\u00a0mM Hepes, pH 7, and an equal amount of the reservoir solution. The crystals of the as-isolated ferritin, containing approximately 17 Fe per 24-mer, grew in 3\u00a0weeks and those loaded with approximately 1,000 Fe atoms per 24-mer prior to crystallization grew in 4\u00a0months. The crystals exhibited an irregular pyramidal shape, with base side and height between 150 and 250\u00a0\u03bcm. The iron content of as-isolated ferritin was determined with ferrene as previously described [14] by measuring the absorption of the iron\u2013ferrene complex at 593\u00a0nm, \u03b5\u00a0=\u00a035,500\u00a0M\u22121\u00a0cm\u22121.\nIron soaking of ferritin crystals was performed by transferring them to a 10\u201320-\u03bcl drop of a solution with composition 20\u00a0mM FeSO4, 2\u00a0mM Na2S2O4, 25% glycerol and 2\u00a0M ammonium sulfate, for 15\u00a0min. Zinc soaking with ZnCl2 was carried out in a similar fashion, with the exception that dithionite (Na2S2O4) was omitted from the solution.\nX-ray data collection\nCryoprotecting conditions consisted of briefly dipping the crystals into a modified crystallization solution containing 20% glycerol in the case of the as-crystallized and Fe-loaded crystals, and 25% glycerol in the case of the Fe-soaked and Zn-soaked crystals, prior to flash-freezing at \u2212173\u00a0\u00b0C in a nitrogen-gas stream, using an Oxford Cryosystems low-temperature device.\nDiffraction data from an as-crystallized, an Fe-soaked and a Zn-soaked crystal of as-isolated PfFtn, as well as from a loaded PfFtn crystal, with approximately 1,000 Fe per 24-mer, were collected at the ESRF beamline BM-14, from flash-frozen single crystals at \u2212173\u00a0\u00b0C. Two datasets were collected from the Zn-soaked crystal: the first at the Zn K-edge (9.6760\u00a0keV, 1.2810\u00a0\u00c5, Zn peak) as determined from a fluorescence scan, and the second (9.500\u00a0keV, 1.305\u00a0\u00c5, Zn low-E rm) on the low-energy side of the absorption edge. The diffraction images were integrated with the program MOSFLM [15]. The processed data were scaled, merged and converted to structure factors using the CCP4 program suite [16]. Data-processing statistics are summarized in Table\u00a01. Table\u00a01X-ray diffraction data collection and processing statisticsBeamlineESRF BM-14ESRF BM-14ESRF BM-14ESRF BM-14ESRF BM-14DetectorMAR 225 CCDMAR 225 CCDMAR 225 CCDMAR 225 CCDMAR 225 CCDCrystalAs-isolatedFe-loadedFeSO4 soakZnSO4 soak, Zn peakZnSO4 soak, Zn low-E rmWavelength (\u00c5)1.77121.73401.45851.28101.305Space group C2221C2221C2221C2221C2221Unit cell (\u00c5)\u00a0a255.00254.32254.30255.37255.69\u00a0b341.42343.16342.88342.06342.36\u00a0c265.52266.26266.22265.99266.21Resolution range (\u00c5)58.2\u20132.75 (2.90\u20132.75)63.2\u20132.95 (3.11\u20132.95)42.0\u20132.80 (2.95\u20132.80)42.9\u20132.80 (2.95\u20132.80)42.9\u20132.80 (2.95\u20132.80)Observations1,685,078 (151,184)903,956 (85,605)1,011,149 (102,079)1,082,567 (111,068)1,086,719 (111,952)Unique reflections295,335 (42,009)237,583 (32,389)280,407 (38,583)279,537 (37,607)280,474 (37,777)Completeness (overall) (%)99.7 (97.8)98.1 (92.2)99.1 (94.1)98.7 (91.6)98.7 (91.9)Redundancy5.7 (3.6)3.8 (2.6)3.6 (2.6)3.9 (3.0)3.9 (3.0)Rmerge0.085 (0.300)0.113 (0.532)0.081 (0.354)0.075 (0.288)0.077 (0.344)I\/\u03c3(I)16.5 (3.1)10.8 (1.6)8.2 (2.0)8.8 (2.5)8.6 (1.9)Anomalous completeness (%)98.0 (88.3)89.2 (71.1)94.4 (64.8)97.7 (85.4)97.6 (85.5)Anomalous redundancy2.9 (1.9)2.0 (1.5)1.8 (1.6)1.9 (1.5)2.0 (1.5)Ranom0.043 (0.204)0.078 (0.414)0.058 (0.294)0.063 (0.252)0.059 (0.288)Estimated Boverall (\u00c52)53.362.760.554.358.5Values in parentheses refer to the last resolution shell\nStructure determination of as-isolated and Fe-loaded PfFtn\nPreviously [14], the 3D structure of PfFtn was solved by the molecular replacement (MR) method, using the program PHASER [17] and the homologous TmFtn (PDB entry 1vlg) as the search model. The sequence of this 165 amino acid ferritin shares a 51% amino acid sequence identity (88 amino acid residues) with that of PfFtn (174 amino acids), and the only insertions or deletions occur at the termini of the subunit chains. However, despite the success of the MR calculations we believed that, because of the low resolution of the diffraction data, relatively low amino acid sequence identity with the search model, as well as the complexity of the structure, experimental phase information would be needed in order to obtain the best possible structural model from the data. To that end, the first BM-14 dataset was collected at a long wavelength (7.0000\u00a0keV, 1.7712\u00a0\u00c5) from a crystal of as-isolated PfFtn with the intention of using S-SAD in phasing, whereas a second dataset was collected just above the Fe K-absorption edge (7.1500\u00a0keV, 1.7340\u00a0\u00c5) from an Fe-loaded PfFtn crystal, with the aim of using Fe-SAD in phasing and also for better location of the incorporated Fe atoms. However, neither dataset could provide the desired independent phase information, owing to complications that arose from radiation damage, which prevented us from collecting the highly redundant dataset required for application of the S-SAD method to the first dataset, as well as the lack of a sufficiently high ordered Fe content, which limited the usefulness of the anomalous dispersion data obtained from the second dataset.\nTherefore, the 3D structure of PfFtn was solved by repeating the MR calculations previously described [14] with the new \u201cas-isolated\u201d 2.75-\u00c5 dataset, using the standard PHASER protocol as implemented through the CCP4 graphical user interface, with reflection data up to 3.25-\u00c5 resolution. Previously, we had established [14] that the asymmetric unit of the PfFtn crystal structure contained 36 monomers, arranged as 1.5 24-mers. The search model used was half of a TmFtn 24-mer, constructed from the PDB 1vlg coordinates without the Fe sites, and three such models were searched for in the crystal structure of PfFtn. The MR calculations with PHASER gave a single solution. Prior to model rebuilding, electron-density map improvement was carried out both by using Arp\/wArp [18] in atom update and refinement mode as well as by using DM [19] with 36-fold noncrystallographic symmetry (NCS) averaging. In this way, the initial PHASER figure of merit of 0.502 for the full resolution range of the 2.75-\u00c5 dataset was improved to 0.697 (Arp\/wArp) and 0.849 (DM). Rebuilding of the protein chain to take into account the differences in sequence and chain length between TmFtn and PfFtn was carried out with the program TURBO [20] using one monomer and the 36-fold averaged electron-density map obtained from the DM calculations. The coordinates for each of the remaining 35 monomers were obtained from those of the first by applying the appropriate NCS transformations, followed by model correction with TURBO using the electron-density maps obtained from both the DM and the Arp\/wArp calculations. In these maps, peaks corresponding to Fe occupation of the A sites only in the FC were observed, and therefore only 36 iron sites (one per monomer), were included in the model.\nThe structure of the Fe-loaded PfFtn was also determined by MR with PHASER using a similar procedure; however, the |Fo| density map, as well as an anomalous electron density map, calculated with MR phases, showed that only the A site in the FC was occupied by Fe atoms. Since the \u201cas-isolated\u201d dataset had higher resolution and better quality, further work on the \u201cFe-loaded\u201d dataset was abandoned.\nStructure refinement of as-isolated PfFtn\nRefinement was carried out with the program REFMAC [21] using medium main-chain and weak side-chain NCS restraints between the 18 dimers in the asymmetric unit. At a later stage, a translation\u2013libration\u2013screw (TLS) rigid-body motion refinement [22] was made prior to restrained refinement of atomic positions and thermal motion parameters. One rigid body was defined for each of the 36 independent monomers. In the final refinement stages, a total of 832 solvent molecules were located with Arp\/wArp [23]. On the basis of their B factors, observed electron density and interactions with neighboring protein residues, 791 were assigned to water molecules, while the remaining 41 were modeled as sulfate ions. Individual restrained B factors were refined for all non-hydrogen atoms and, for consistency with the structure refinements of the Fe-soaked and Zn-soaked crystals (see below), only tight NCS restraints (0.1\u00a0\u00c5 positional and 1.0\u00a0\u00c52 thermal parameter) were applied to all the protein residues involved in the FC metal coordination, instead of defining the coordination geometry for each individual protein\u2013metal bond as in the earlier stages of refinement.\nThe final values of R and Rfree were 0.195 and 0.247, respectively. Neither TLS rigid-body refinement nor full NCS restraints (i.e., extending over the whole monomer and relating all the chains in the asymmetric unit) were used in the final refinement, as they were seen to lead to higher values of both R and Rfree. Rfree [24] was calculated from a randomly chosen subset of the data, containing approximately 5% of the total number of independent reflections. Throughout the refinement, the model was periodically checked and corrected with TURBO against 2|Fo|\u00a0\u2212\u00a0|Fc| and |Fo|\u00a0\u2212\u00a0|Fc| electron-density maps. The refinement statistics are included in Table\u00a02. The maximum-likelihood estimate of the overall coordinate error, obtained with REFMAC, was 0.20\u00a0\u00c5. For all 36 independent monomers in the PfFtn crystal structure, only the first 168 residues were included in the model. Although some electron density was visible beyond Gly168, it was not possible to build any more C-terminal residues into it, and it is likely that the C-terminal tail is disordered in all the independent PfFtn monomers. On the basis of a B-value comparison with the protein residues directly coordinating the iron atoms, it was realized that at least 17 out of the 36 metal sites were not fully occupied, and were assigned occupation factors of 0.8 (0.7 in one case). In addition, 788 side-chain atoms could not be seen in either 2|Fo|\u00a0\u2212\u00a0|Fc| or |Fo|\u00a0\u2212\u00a0|Fc| electron-density maps and were therefore given zero occupancy during the refinement. Table\u00a02Refinement and structure analysis statistics for the ferritin from Pyrococcus furiosus (PfFtn)CrystalAs-isolated, as-crystallizedFe-soakedZn-soakedResolution (\u00c5)56.8\u20132.7542.0\u20132.8042.8\u20132.80No. of reflections in work set\/test set280,375\/14,914266,173\/14,187265,356\/14,134Rwork\/Rfree0.195\/0.2470.197\/0.2490.201\/0.251Number of atoms\u00a0Protein\u2014total number of atoms49,82449,82449,824\u00a0Protein\u2014atoms with zero occupancy788718720\u00a0Fe\/Zn atoms36108108\u00a0Water molecules (sulfate ions)791 (41)413 (26)162 (44)Mean B factors (\u00c52)\u00a0Protein main chain (side chain)32.6 (34.6)38.0 (39.9)30.9 (32.7)\u00a0Fe\/Zn atoms57.553.236.9\u00a0Water molecules (sulfate ions)26.5 (67.1)29.0 (76.8)21.9 (74.0)Root mean square deviations from ideal values\u00a0Bond lengths (\u00c5)0.0140.0140.015\u00a0Bond angles (\u00b0)1.441.461.48\u00a0Mean positional error (\u00c5)0.200.220.22PROCHECK Ramachandran analysis\u00a0Total no. analyzed residues5,5445,5445,544\u00a0Residues in core regions, no. (%)5,255 (94.79)5,241 (94.53)5,239 (94.50)\u00a0Residues in allowed regions, no. (%)277 (5.00)285 (5.14)289 (5.21)\u00a0Residues in generous regions, no. (%)11 (0.20)10 (0.18)9 (0.16)\u00a0Residues in disallowed regions, no. (%)1 (0.02)8 (0.14)7 (0.13)PROCHECK G factors\u00a0Mean (range) dihedral0.00 (\u22120.13 to 0.07)\u22120.04 (\u22120.23 to 0.03)\u22120.04 (\u22120.21 to 0.09)\u00a0Mean (range) covalent0.38 (0.32\u20130.45)0.38 (0.29\u20130.46)0.36 (0.26\u20130.44)\u00a0Mean (range) overall0.15 (0.07\u20130.19)0.12 (0.00\u20130.18)0.12 (0.01\u20130.17)\nThe structure was analyzed with PROCHECK [25] and its stereochemical quality parameters were within their respective confidence intervals. A Ramachandran [26] \u03d5,\u03c6 plot showed that of the 5,544 non-glycine and non-proline residues, 11 (0.20%) were found to lie in the generously allowed regions and only one (0.02%) outlier was observed. All these residues with more unusual \u03d5,\u03c6 conformations were located in a loop region containing Lys145, for which the electron density was generally poor. The final atomic coordinates were deposited with the PDB [27], to be released upon publication, with the accession code 2jd6.\nStructure determination and refinement of the Fe-soaked and Zn-soaked PfFtn crystals\nIn view of the very small changes in cell parameters observed in comparison with those of the as-isolated crystal, the structures of the Fe-soaked and Zn-soaked crystals of PfFtn were determined directly by using the final refined coordinates of the as-isolated structure, omitting the iron sites and the solvent atoms. Ten cycles of rigid-body refinement were first carried out, followed by a TLS rigid-body motion refinement [22] prior to restrained refinement of atomic positions and thermal motion parameters. At this stage, the 2|Fo| \u2212 |Fc| or |Fo|\u00a0\u2212\u00a0|Fc| electron-density maps were inspected with COOT [28] for inclusion of the FC A, B and C metal sites in the models. Because of the complexity of the metal coordination in the FC, the low resolution of the diffraction data and the realization that some of the FC metal sites were not fully occupied (see \u201cDiscussion\u201d for details), tight NCS restraints (0.1\u00a0\u00c5 positional and 1.0\u00a0\u00c52 thermal parameter) were applied to all the protein residues involved in the FC metal coordination, instead of defining the cordination geometry for each individual protein\u2013metal bond.\nIn the final refinement of the Fe-soaked crystal structure, 423 solvent molecules and 26 sulfate ions were located with Arp\/wArp [23] and included in the model, and individual restrained B factors were refined for all non-hydrogen atoms. On the basis of a B-value comparison with the protein residues directly coordinating the iron atoms, it was realized that not all metal sites were fully occupied: all the B sites and one C site were assigned an occupation of 0.5. The final values of R and Rfree were 0.197 and 0.249. The refinement statistics are listed in Table\u00a02. The structure was analyzed with PROCHECK [25] and its stereochemical quality parameters were within their respective confidence intervals. A Ramachandran [26] \u03d5,\u03c6 plot showed that of the 5,544 non-glycine and non-proline residues, ten (0.18%) were found to lie in the generously allowed regions and only eight (0.14%) outliers were observed. With three exceptions, all these residues with more unusual \u03d5,\u03c6 conformations were located in a loop region containing Lys145, for which the electron density was generally poor.\nIn the final refinement of the Zn-soaked crystal structure, 162 solvent molecules and 44 sulfate ions were located with Arp\/wArp [23] and included in the model, and individual restrained B factors were refined for all non-hydrogen atoms. A B-value comparison with the protein residues directly coordinating the metal atoms, combined with the observation of a mixed iron\/zinc population at the A sites (see below for details) led us to postulate a mixed (50:50 Zn\/Fe) occupancy for the A sites, which was modeled as a Zn atom with 0.9 occupancy. Also, the B sites were modeled as partially occupied by zinc (50%) and the C sites as partially occupied by iron (50%). The final values of R and Rfree were 0.201 and 0.251. The refinement statistics are included in Table\u00a02. The structure was analyzed with PROCHECK [25] and its stereochemical quality parameters were within their respective confidence intervals. A Ramachandran [26] \u03d5,\u03c6 plot showed that of the 5,544 non-glycine and non-proline residues, nine (0.16%) were found to lie in the generously allowed regions and only seven (0.13%) outliers were observed. With two exceptions, all these residues with more unusual \u03d5,\u03c6 conformations were located in a loop region containing Lys145, for which the electron density was generally poor.\nAs for the as-isolated structure, neither TLS rigid-body refinement nor full NCS restraints were used in the final refinement of the Fe-soaked or Zn-soaked structures, as they were seen to lead to higher values of both R and Rfree. Rfree [24] was calculated from a randomly chosen subset of the data, containing approximately 5% of the total number of independent reflections, and with the same h,k,l indices as for the \u201cas-isolated\u201d data. These test-set reflections were not chosen in thin resolution shells. However, while it is true that the 24-mers have very high NCS, in all three refined crystal structures the Rfree\/Rwork ratios are typical for the data resolution and refinement parameters used, [29] and therefore there is no indication that the more simplistic test-set choice may have introduced a bias in the Rfree values. Throughout the refinement, the models were periodically checked and corrected with COOT [28] against 2|Fo|\u00a0\u2212\u00a0|Fc| and |Fo|\u00a0\u2212\u00a0|Fc| electron-density maps. Since the asymmetric unit of both structures contains 36 monomers, and each monomer contains one FC with three metal sites, a total of 108 metal atoms are present in the model. In addition, 718 side-chain atoms could not be seen in either 2|Fo|\u00a0\u2212\u00a0|Fc| or |Fo|\u00a0\u2212\u00a0|Fc| electron-density maps for the Fe-soaked structure (720 for the Zn-soaked structure) and were given zero occupancy during the refinement. The final atomic coordinates were deposited with the PDB [27], to be released upon publication, with the accession codes 2jd7 (Fe-soaked structure) and 2jd8 (Zn-soaked structure).\nResults and discussion\nStructure of the PfFtn monomer\nThe PfFtn monomer exhibits the typical four-helical bundle fold which is encountered in ferritins, BFRs, DPS proteins and rubrerythrins. According to the DSSP algorithm [30] the four helices comprise residues 4\u201334 (helix A), 37\u201364 (helix B), 83\u2013111 (helix C) and 114\u2013144 (helix D). A short, C-terminal \u03b1-helix (helix E, residues 148\u2013160), bordering one of the cylindrical bundle openings, followed by a tail without any secondary structure also occurs in the PfFtn monomer fold (Fig.\u00a01a, b). For convenience of terminology, the two extremities of the cylindrical \u03b1-helical bundle will be further referred to as the open side and the E-helix sides of the monomer. Fig.\u00a01a Topology diagram of the ferritin from Pyrococcus furiosus (PfFtn). b Ribbon diagram of PfFtn showing the location of the ferroxidase center sites A, B and C (orange spheres) near the center of the four-helical bundle. c Superposition of the PfFtn C\u03b1 chain with those of its eight closest homologous structures. In b and c, the molecule colors change from blue to red in the N-terminal to C-terminal direction. b, c Prepared with PyMOL [50]\nThe PfFtn monomer is built of hydrophobic amino acids at its open and E-helix sides and polar and hydrophilic residues in the middle of the subunit, similarly to the subunits from other ferritins. The open side of the monomer contains three leucine residues (Leu10, Leu104 and Leu120), two phenylalanine residues (Phe57 and Phe123), Met6, Tyr60 and Ile101, whereas the E-helix side has three phenylalanine residues (Phe87, Phe31 and Phe39), Trp42 and Met27 (denoted by asterisks in Fig.\u00a02). The residues defining the open and E-helix sides of the monomer are conserved among PfFtn, the ferritin from E.\u00a0coli (EcFtnA), AfFtn and human H chain ferritin (HuHF) in terms of their polarities, apart from Phe123, which in other ferritins is a tyrosine. Fig.\u00a02Amino acid sequence alignment of PfFtn, the ferritin from Archaeoglobus fulgidus (AfFtn), the ferritin from Escherichia\u00a0coli (EcFtnA) and human H chain ferritin (HuHF). Asterisks show the residues around the open and E-helix sides of the monomer; A, B and C denote the residues coordinating the iron sites of the ferroxidase center (FC); red bars highlight the main residues located around the threefold channels and gray bars those around the fourfold channels. The alignment was prepared with ClustalW [51]\nThe central part of the subunit comprises the negatively charged and polar amino acids involved in metal binding. These are Glu17, Glu49, Glu50, Glu94, Glu126, Glu129 and Glu130 and His53, which are shown in Fig.\u00a02 with A, B or C labels to denote the iron site they coordinate (see \u201cThe FC\u201d). In addition, Tyr24 and Gln127, which in EcFtnA [1] indirectly contribute to the A and B sites via hydrogen bonds to Glu94 and Glu17, are also oriented towards the FC in PfFtn.\nIt has been noted [2] that Gly67, Ser84 and Gly147 (in AfFtn numbering) are conserved among AfFtn, EcFtnA and HuHF, and define the BC loop and the end of D helix, respectively. In the case of PfFtn, only Ser84 is conserved, while the two glycine residues are changed into Asn65 and Lys145.\nA surface hydrophobic pocket at the PfFtn dimer interface\nA large surface pocket, largely lined by hydrophobic residues, was located at the PfFtn dimer interface. Interestingly, one of these is Ile51, corresponding to Met 57 in the BFR from Desulfovibrio desulfuricans ATCC 27774 [31], which binds the heme group. Instead of a heme, the pocket appears to be occupied by a disordered string of water molecules (Fig.\u00a03) which form a continuous region of electron density. This feature was observed for all the PfFtn structures investigated. A similar situation was reported by Yu et\u00a0al. [32] for the hydrophobic cavity of human interleukin 1\u03b2. Fig.\u00a03Stereoview of a dimer interface in the as-isolated PfFtn structure, showing a difference electron density feature, drawn at the 2.5 map root mean square (RMS) level, which results from a disordered chain of water molecules trapped within a hydrophobic pocket centered around Ile51. This feature is observed in all dimers of all PfFtn structures investigated. The bulk of the PfFtn dimer is represented by its C\u03b1 trace (blue-gray). The side chains (including C\u03b1 atoms) of the Ile51 residues in both monomers are represented in ball-and-stick mode and are colored gold. The view is down the noncrystallographic twofold symmetry axis of the dimer, looking towards the inside of the PfFtn 24-mer. The figure was prepared with DINO [52]\nSubunit structure comparison\nIn order to compare the PfFtn structure with the structures of other known ferritins, a protein structure comparison against the PDB using the secondary structure matching (SSM) tool at the European Bioinformatics Institute (http:\/\/www.ebi.ac.uk\/msd-srv\/ssm) [33] was performed. This resulted in 377 hits, which, after elimination of duplicate protein chains, were seen to correspond to 63 different protein structures. A multiple structural alignment was then carried out with a subset of eight SSM hits, listed in Table\u00a03 and shown as a superposition of C\u03b1 chains in Fig.\u00a01c. The members of this subset were chosen such that the percentage of their aligned residues was equal to or greater than 25%, all five secondary structure elements (\u03b1-helices) were aligned, the Q score, of their pairwise SSM alignment with PfFtn was greater than 0.5, and considering only one crystal form of each different protein structure from each organism. This subset includes representatives from archaea, bacteria and eukarya. The structural alignment showed PfFtn to be highly similar to TmFtn, followed by AfFtn and EcFtnA (Table\u00a03). The deviations occur mostly in loop regions: AB loop from Glu32 to Leu34, end of the BC loop from Lys78 to Pro83, CD loop and helix D up to Phe123 and DE loop from Lys145 to Leu151 (in PfFtn numbering). Table\u00a03Proteins with known 3D structure most closely homologous to PfFtnPDB IDProtein nameSource organismChain lengthIdentity (%)Identity SS-aligned (%)Q scoreaRMSD (\u00c5)1vlgFerritin (TM1128) Thermotoga maritima1765555.30.920.491s3qFerritinArchaeoglobus fulgidus1735048.20.940.531krqFerritinCampylobacter jejuni1673939.20.811.021eumFerritin ECFTNAEscherichia coli1653838.40.860.901r03Mitochondrial ferritinHomo sapiens 1824030.60.681.552fhaH ferritin K86Q mutantHomo sapiens 1833729.40.621.981mfrM ferritinRana catesbeiana1763629.60.681.671rcgL ferritinRana catesbeiana1733225.00.612.03PDB Protein Data Bank, SS secondary structure, RMSD root mean square deviationaParameter that takes the alignment length and the RMSD between the superimposed atoms into account, and varies from 0 in the case of poor matches to 1 in the case of identical proteins\nStructure of the PfFtn 24-mer and comparison with AfFtn\nA highly conserved quaternary structure appears among all known ferritins\u2014a spherical tetraeicosameric assembly with 432 point-group symmetry. An interesting exception to this rule is the recently reported structure of the AfFtn 24-mer [2], which exhibited 23 point-group symmetry, normally an attribute of the dodecameric DPS proteins. Even though PfFtn and AfFtn are highly similar archaeal ferritins (50 and 70% amino acid sequence identity and similarity, respectively), the PfFtn 24-mer has the canonical 432 point-group symmetry instead of the 23 point-group symmetry found in the AfFtn 24-mer. Although there may be other archaeal tetraeicosameric ferritins with 23 point-group symmetry like AfFtn, our structure clearly suggests that it is not a feature of all archaeal ferritins.\nWhen comparing the two quaternary structures (Fig.\u00a04), it can be seen that the 432 and 23 arrangements in PfFtn and AfFtn result from a different organization of the four fundamental units, hexamers formed by three dimers around a 90\u00b0 vertical turn from a threefold NCS rotation axis. The 23 arrangement leads to the appearance of four outsized triangular pores about 45-\u00c5 wide in the protein shell of AfFtn [2], which are absent in the 432 arrangement of PfFtn and all other known structures of tetraeicosameric bacterial ferritins. Fig.\u00a04Ribbon diagrams of the PfFtn and AfFtn 24-mers. I View down a threefold axis, showing one \u201cfundamental hexamer.\u201d II View rotated by approximately 90\u00b0 about a vertical axis, showing the different hexamer arrangement, which leads to the appearance of large pores in AfFtn. The figure was prepared with PyMOL [50]\nThreefold and fourfold channels\nThe eight threefold channels are formed by helices C and D from three adjacent dimers, in such a way that, with respect to the hollow tetraeicosameric protein shell, the C-terminal ends of helices C define the outer entrance to the channel and the N-termini of helices D define the inner entrance (Fig.\u00a05). The PfFtn threefold channel is lined by a mix of hydrophobic and hydrophilic amino acids Ala106 and Glu109 at the outer side of the channel, Tyr114 and Arg117 in the middle and Ala118 and Glu121 at the inner entrance of the channel (Fig.\u00a05). In general, and similarly to EcFtnA and AfFtn, this channel is less hydrophilic than in vertebrate H and L chain ferritins, suggesting a rather hydrophobic threefold channel in microbial ferritins in comparison with vertebrate ferritins. Nevertheless, when compared with that in EcFtnA and AfFtn, the threefold channel in PfFtn is more similar to that of HuHF, in the sense that is it more negatively charged at its inner entrance and more positively charged in its central region (Fig.\u00a05). Fig.\u00a05Profile view of the threefold and fourfold channels in PfFtn, AfFtn, EcFtnA and HuHF. The exterior of the shell lies on the left side and the inner cavity on the right side of each cartoon as shown schematically in a. The red residues correspond to acidic Glu and Asp residues and the blue ones to positive Lys, Arg and His residues, and highlight the arrangement of positive and negative amino acids along the channels. The figure was prepared with PyMOL [50]\nOn the basis of the crystallographic, kinetic, mutagenesis and electrostatic calculations studies on HuHF [9, 34\u201336], the threefold channels were proposed to be involved in the entry of iron ions into the ferritin molecule. The inhibition of iron uptake in HuHF upon Asp131 and Glu134 substitutions [34] and the coordination of Ca2+ by Asp131 and Glu134 [37] have been observed in this protein. The inner narrow entrance of the threefold channel in PfFtn resembles that of HuHF, suggesting that a similar mechanism is possible in PfFtn. Nevertheless, the mutations of His118 and Cys130 also influenced the iron-binding capacity of HuHF and inhibited the binding of different metals by the channel to different extents. The different amino acid arrangements in the threefold channel of the various ferritins suggests the possibility of different iron incorporation mechanisms.\nThe six fourfold channels, formed around the fourfold symmetry axes of the PfFtn 24-mer, are lined, from the outside to the inside of the protein shell, by Gln149, Met153, Lys156 and Glu157 (Fig.\u00a05). Owing to a nozzlelike shape formed by Gln149, the PfFtn fourfold channel is longer (approximately 16\u00a0\u00c5) than its counterparts in EcFtnA (approximately 14\u00a0\u00c5) and HuHF (approximately 12\u00a0\u00c5). In PfFtn, Lys145 in the DE loop, which forms an outer gate to the fourfold channel, occurs in place of EcFtnA Gly145 or HuHF Gly159, making this gate more polar and charged in PfFtn. In addition, the DE loop and its amino acids are somewhat oriented outwards from the fourfold channel, resulting in a hourglass shape of this channel, which is less strongly developed in EcFtnA and even less so in HuHF, where the pore has an almost cylindrical shape. The outer entrance of the PfFtn fourfold channel is polar and hydrophilic, the region inside the channel is nonpolar and the inner entrance is polar again. The fourfold channels in EcFtnA have a similar arrangement of polar residues at both channel entrances and are nonpolar in the middle, while in HuHF they are mostly apolar with Leu165 and Leu169 and polar His173 on the inner side of the channel (Fig.\u00a05). In electrostatic terms, the PfFtn fourfold channel is similar to that of both EcFtnA and HuHF; the outer entrance of it is uncharged as in HuHF and the inner entrance has a compensated charge from the sequence of positive Lys156 and negative Glu157 as in EcFtnA.\nThe FC\nThe active site responsible for the oxidation of Fe(II) to Fe(III) in ferritins, the FC, is located in the central part of each subunit (Fig.\u00a01b). It is generally believed that in most ferritins the initial stages of the core formation take place via the FC. Subsequently, iron incorporation and removal is thought to occur predominantly through one or the other of the channels formed around the threefold and fourfold symmetry axes of the 24-mer. In this discussion, the nomenclature for the FCs in ferritins and BFRs presented in a recent review [38] will be followed. The FC in PfFtn is of the FtnA (EcFtnA) type, which is strictly conserved in all the closest prokaryote structural homologues of PfFtn besides EcFtnA (Table\u00a04). The EcFtnA FC contains three metal binding sites (A, B and C), in contrast with the HuHF and E. coli BFR FC, which only contain two (A and B). The FC of the as-crystallized PfFtn showed iron bound to site A only. Iron binding to sites B and C was observed only after soaking the PfFtn crystals in Fe(II). Table\u00a04The ferroxidase center in PfFtn and its homologous crystallized ferritinsPDB IDProtein nameSource organismResolution (\u00c5)FC typeMetal site occupation\u2013FerritinPyrococcus furiosus2.75FtnAA (Fe)A, B, C (Fe,Zn)1s3qFerritinArchaeoglobus fulgidus2.10FtnAA, B (Zn)aA, B, C (Fe)1vlgFerritin (TM1128) Thermotoga maritima2.00FtnAA (Fe)1krqFerritinCampylobacter jejuni2.70FtnAA, B (Ow?)b1eumFerritin ECFTNAEscherichia coli2.05FtnAA, B (Zn)cA, B, C (Fe)1r03Mitochondrial ferritinHomo sapiens sapiens1.70HuHF\u20132fhaH ferritin K86Q mutantHomo sapiens sapiens1.90HuHF\u20131mfrM ferritinRana catesbeiana2.80HuHF\u20131rcgL ferritinRana catesbeiana2.20\u2013\u2013FC ferroxidase centeraZn in 1s3q and Fe in 1sq3 [2]b1krq contains four water molecules, two of which are located at the FC, in positions corresponding to A and B sites. They might be low-occupancy metal sites but there is no indication of whether it is a native or a soaked crystalc1eum corresponds to the native structure [1]; these sites were located in the Zn2+ and Fe3+ derivatives, respectively, for which no coordinates were deposited in the PDB\nIn PfFtn, iron site A is coordinated by Glu17, Gly50 and His53, iron site B by Glu50, Glu94 and Glu130, and iron site C by Glu49, Glu126, Glu129 and Glu130. Site B is 3.0\u00a0\u00c5 distant from site A. Site C is located 7.5 and 6.3\u00a0\u00c5 away from sites A and B, respectively (Table\u00a05, Fig.\u00a06). Glu17 and Glu94 appear to be monodentate ligands; therefore, sites A and B are coordinated by side chains from three amino acids, while site C is coordinated by side chains from four amino acids. The final 2|Fo|\u00a0\u2212\u00a0|Fc| electron density around the FC of a selected PfFtn monomer in the as-isolated, Fe-soaked and Zn-soaked crystal structures are represented in Fig.\u00a07a\u2013c, respectively. Table\u00a05Coordination geometry statistics of the ferroxidase center in PfFtnBondsaDistance (\u00c5)As-isolatedbFe-soakedbZn-soakedbMA...Glu17 O\u03b512.24 (0.09) [2.00\u20132.46]2.21 (0.08) [2.00\u20132.47]2.18 (0.07) [2.01\u20132.32]MA...Glu50 O\u03b512.26 (0.11) [2.09\u20132.47]2.24 (0.09) [2.09\u20132.55]2.22 (0.07) [2.02\u20132.38]MA...His53 N\u03b412.12 (0.09) [1.97\u20132.41]2.28 (0.09) [2.12 \u2013 2.47]2.21 (0.08) [2.08 \u2013 2.40]MA...MB3.02 (0.19) [2.62\u20133.63]2.93 (0.18) [2.54\u20133.49]MA ... MC7.41 (0.10) [7.18\u20137.63]7.51 (0.11) [7.30\u20137.77]MB...Glu50 O\u03b522.21 (0.12) [1.93\u20132.45]2.16 (0.12) [1.92\u20132.50]MB...Glu94 O\u03b522.34 (0.14) [2.12 \u2013 2.70]2.40 (0.10) [2.18 \u2013 2.65]MB...Glu130 O\u03b512.87 (0.16) [2.47\u20133.14]2.81 (0.20) [2.41\u20133.21]MB...MC6.29 (0.20) [5.81\u20136.77]6.25 (0.15) [5.93\u20136.55]MC...Glu49 O\u03b512.11 (0.08) [1.92 \u2013 2.36] 2.29 (0.11) [2.13 \u2013 2.56]MC...Glu126 O\u03b512.06 (0.09) [1.87\u20132.26]2.31 (0.16) [1.84\u20132.53]MC...Glu129 O\u03b512.31 (0.10) [2.12\u20132.58]2.02 (0.10) [1.81\u20132.26]MC...Glu130 O\u03b521.89 (0.08) [1.72\u20132.04]1.89 (0.09) [1.66\u20132.07]aM stands for metal (Fe in as-isolated and in Fe-soaked; Zn or Fe in Zn-soaked)bFor each column, the numbers listed are the mean value, calculated from the 36 independent distances in the asymmetric unit, followed by the corresponding standard deviation in parentheses, and finally the minimum and maximum values in the population, enclosed in bracketsFig.\u00a06View of the FC down the PfFtn subunit from the E-helix side. The three metal sites are annotated as A, B and C. Metal occupation of sites B and C is observed only upon crystal soaking in Zn or Fe. The dashed lines correspond to the coordination geometries described in Table\u00a05. The figure was prepared with PyMOL [50]Fig.\u00a07Details of the FC of a selected monomer in the structures of as-isolated, Fe-loaded, Fe-soaked and Zn-soaked PfFtn crystals. a Final 2|Fo|\u00a0\u2212\u00a0|Fc| electron-density map for the as-isolated PfFtn, contoured at 1.2 map RMS. b Final 2|Fo|\u00a0\u2212\u00a0|Fc| electron-density map for the Fe-soaked PfFtn, contoured at 1.2 map RMS. c Final 2|Fo|\u00a0\u2212\u00a0|Fc| electron-density map for the Zn-soaked PfFtn, contoured at 1.2 map RMS. d Anomalous Fourier map for the Fe-loaded PfFtn, contoured at 3.0 map RMS. e Anomalous Fourier map for the Fe-soaked PfFtn, contoured at 3.0 map RMS. f Anomalous Fourier map for the Zn-soaked PfFtn using the peak data, contoured at 3.0 map RMS. g Labeled view of the residues and metal sites in the Fe-soaked FC. h Anomalous Fourier map for the Zn-soaked PfFtn using the low-energy remote data, contoured at 3.0 map RMS. i Dispersive Fourier map for the Zn-soaked PfFtn, contoured at 2.8 map RMS. The anomalous electron-density maps were calculated using as amplitudes the anomalous difference coefficients obtained from each dataset, and the phases (rotated by 90\u00b0) obtained from the respective final structure refinement, except for the \u201cFe-loaded\u201d data, for which the phases were taken from the \u201cas-isolated\u201d refinement. The Zn-dispersive Fourier map was calculated by first scaling together the peak and low-energy remote datasets with CCP4 SCALEIT [16] and then using as coefficients the difference F(low-energy remote) \u00a0\u2212\u00a0F(peak) and the phases from the final \u201cZn-soak\u201d refinement. a\u2013c show that the occupation of site B in the as-isolated crystal is negligible; d shows a small residual occupancy of site B in the Fe-loaded structure and no evidence of site C occupancy; e shows a site B occupancy clearly lower than those of sites A and C in the Fe-soaked structure; f, h and i show that in the Zn-soaked structure, part of the originally present Fe in site A has been displaced to site C by the Zn ions, that site\u00a0B is occupied by Zn only, and that site\u00a0C is very likely occupied by Fe only. All panels were drawn with the same orientation. In all panels except g the bulk of PfFtn monomer is represented as a gray tube onto which the side chains (including C\u03b1 atoms) of the FC residues have been overlaid in ball-and-stick representation (carbon atoms blue-gray, oxygen atoms red, nitrogen atoms blue, iron atoms pink and zinc atoms cyan). In g, only the residues near the FC are represented in ball-and-stick mode, and are labeled for easier identification of the residues mentioned in the text. The figure was prepared with DINO [52]\nFor the Fe-loaded crystal structure, the anomalous difference Fourier map, calculated with the phases obtained from the final \u201cas-isolated\u201d refinement (Fig.\u00a07d), shows a very small occupancy of site B. This map is very similar to that obtained from the MR phases (not shown). In the Fe-soaked crystal, the anomalous difference Fourier map calculated at the end of the refinement showed that sites\u00a0A and C have a clearly higher Fe occupancy than site B (Fig.\u00a07e).\nIn the Zn-soaked crystal, the anomalous difference Fourier maps obtained from the two datasets collected (just above and 176\u00a0eV below the Zn K-absorption edge) clearly show some iron occupation of sites A and C (Fig.\u00a07h), whereas zinc populates sites A and B (Fig.\u00a07f, i). The dispersive Fourier map calculated using the two datasets (Fig.\u00a07i) shows Zn peaks at sites A and B but since no diffraction data were recorded at the inflexion point of the Zn K-absorption edge, this map is noisier and no conclusions can be drawn about any Zn occupation of site C. Since the as-isolated structure only showed Fe occupation of FC site A, we conclude that the Fe occupation of site C in the Zn-soaked structure results from iron initially present in site A, displaced to site C by the Zn soak. This is an interesting new result, since the previous Zn-soaked and Fe-soaked structures reported for AfFtn [2] and EcFtnA [1] were obtained from apoferritin crystals.\nFC stability\nInterestingly, while mutagenesis studies have shown both sites A and B to be important for iron uptake and oxidation in ferritins [38], in the crystallized proteins the occupancy of the both sites was found only in BFR from D. desulfuricans ATCC 27774 [31]. The PfFtn FC followed the common trend, and as mentioned already the occupation of FC sites B and C was only observed in Fe-soaked or Zn-soaked crystals. This contradicted our EPR studies that showed a fully developed dinuclear iron FC EPR signal in the sample prior to crystallization [39]. In order to check the integrity of the PfFtn FC, we loaded three samples of apo PfFtn with iron and incubated them for 1\u00a0day and 2\u00a0months at 4\u00a0\u00b0C in air and, mimicking the crystallization conditions, for 2\u00a0months in a 2\u00a0M ammonium sulfate solution at room temperature. The FC EPR signal from each sample was measured after titration to 130\u00a0mV, which is the potential at which the mixed-valence PfFtn EPR signal is maximum [39]. The amplitude of the FC signal from the sample incubated for 2\u00a0months in air was noticeably but not drastically smaller than that of the sample incubated for 1\u00a0day (Fig.\u00a08). However, the FC EPR signal amplitude of the sample incubated for 2\u00a0months in a 2\u00a0M ammonium sulfate solution was fivefold smaller than that from the sample incubated for 2\u00a0months in air (Fig.\u00a08). The coordination distance from Glu130 to site B (Table\u00a05) is quite long (more than 2.8\u00a0\u00c5); therefore, site B may be rather labile, which could account for its lower occupation in the Fe-soaked and Zn-soaked crystals, as well as for the easy removal of its iron atom by the precipitating agent ammonium sulfate during crystallization of the as-isolated ferritin. Fig.\u00a08The Fe(III)\u2013O\u2013Fe(II) FC electron paramagnetic resonance (EPR) signal of 6\u00a0\u03bcM PfFtn 24-mer, titrated to 130\u00a0mV. The decrease in the amplitude of the signal depending on incubation time and the presence of crystallization solution in the sample illustrates the stability of the center. Prior to EPR measurement, the three samples tested were incubated, respectively, for 1\u00a0day after loading with iron (blue line), for 2\u00a0months (red line) and for 2\u00a0months in the presence of a crystallization solution, 2\u00a0M ammonium sulfate (black line)\nThere is an ongoing discussion in the literature on whether the FC iron sites A and B are transient in the process of iron uptake\u2013oxidation\u2013translocation into the protein core, or whether they form a stable cofactor [38]. In the current experiment, the decrease in the amplitude of the FC EPR signal in the sample incubated in air for 2\u00a0months appears to be too small to be caused by iron translocation into the protein cavity. Instead, as shown by the sample incubated for 2\u00a0months in ammonium sulfate, the most likely cause for the lack of the B iron site in the as-crystallized PfFtn is iron complexation and removal by the crystallization agent.\nThermostability\nThe thermal stability of a protein can often be described as an equilibrium between the protein\u2019s folded and unfolded state, and frequently correlates with the growth temperature of the organism. Analysis of thermostability factors is often made via comparison of the genome\/protein sequences from the hyperthermophiles, thermophiles and mesophiles, or via mutagenesis. In the literature, a number of factors have been proposed to be involved in thermostability. These include salt bridges, hydrogen bonds, van der Waals interactions, distribution of amino acids, higher \u03b2-strand content, more charged amino acids, fewer uncharged polar amino acids, more hydrophobic \u03b2-branched amino acids and others [40\u201346]. However, no single consistent set of determining factors has emerged from the different studies that have been carried out to date; therefore, it appears that protein thermal stability is the cumulative result of various factors.\nFerritins are large and highly symmetrical agglomerates of 24 single subunits. This suggests that these proteins possess forces that keep the big structure together. Horse spleen ferritin has been shown to resist inactivation by up to 10 M urea [47] and temperatures up to 93\u00a0\u00baC [48].\nPfFtn has been shown to be an extremely thermostable protein. No melting temperature could be found with differential scanning calorimetry up to 120\u00a0\u00b0C and the protein could withstand incubation at 100\u00a0\u00b0C for 1\u00a0day or autoclaving at 120\u00a0\u00b0C for half an hour without loss of its iron-uptake activity [13]. There are no data in the literature regarding the thermostability of other thermophilic and hyperthermophilic ferritins.\nIn our analysis of PfFtn thermostability (summarized in Table\u00a06), and given that no thermostability data are available in the literature on ferritins from microorganisms other than P. furiosus, we assumed that it is the most thermostable ferritin among those shown in Table\u00a06, and we used the optimal growth temperature of the organism as a measure of relative thermostability for the other ferritins listed in Table\u00a06. However, it should be noted that the denaturation temperature of a protein may be higher than the optimal growth temperature of the organism where it is produced. Table\u00a06Hydrogen bonds and salt bridges in the PfFtn 24-mer and its closest structural homologues, calculated with HBPLUSPDB IDSource organism aTotal H-bonds bMMMSSSSalt bridges cIntramolecularIntermolecularIntramolecularIntermolecularIntramolecularIntermolecularIntramolecularIntermolecular\u2013Pyrococcus furiosus (100\u00a0\u00b0C)4,6303,3772443972544174172671vlgThermotoga maritima (80\u00a0\u00b0C)4,7673,237305401176332101891201s3qArchaeoglobus fulgidus (80\u00a0\u00b0C)4,4572,78824412804531761141341krqCampylobacter jejuni (42\u00a0\u00b0C)4,6323,360246487238414472721eumEscherichia coli (37\u00a0\u00b0C)4,7203,320248363241296604MM hydrogen bonds between main-chain atoms only, MS hydrogen bonds between main-chain and side-chain atoms, SS hydrogen bonds between side-chain atoms onlyaOptimal growth temperature in parenthesesbCalculated using default program parameterscA subset of SS hydrogen bonds, considering interactions between side chains of negatively charged Asp\/Glu residues with side chains of positively charged Arg\/His\/Lys residues\nThe hydrogen bonds were calculated with HBPLUS [49] using default parameters, and were divided into three classes: between main-chain atoms only (MM); between main-chain and side-chain atoms (MS); and between side-chain atoms only (SS). The salt bridges were determined as a subset of SS hydrogen bonds, considering interactions between side chains of negatively charged Asp\/Glu residues with side chains of positively charged Arg\/His\/Lys residues. Furthermore, a distinction between intramolecular (within the same monomer) and intermolecular (between neighboring monomers) hydrogen bonds was made. The MM bonds are mostly intramolecular and are responsible for the stability of the secondary structure elements (\u03b1-helices). The intramolecular MS and SS hydrogen bonds contribute to stabilize the 3D fold of the monomeric subunits. The intermolecular MS and SS hydrogen bonds are those that contribute most to the stability of the 24-mer.\nA surprising result of this calculation was that the total number of hydrogen bonds in the ferritin 24-mers was quite similar. If we exclude the lowest number (4,457 for AfFtn) which can possibly be attributed to its unusual quaternary structure, PfFtn actually has the lowest total number of hydrogen bonds, two fewer than the ferritin from Campylobacter jejuni (CjFtn), 90 fewer than EcFtnA and 137 fewer than TmFtn. There is no clear single trend in the data listed in Table\u00a06, which only emphasizes the complex nature of the structural factors governing thermostability, even within a group of closely related structures. However, a careful inspection shows some interesting features, which may have a bearing on the structural basis for the different thermostabilities of the different ferritins listed in Table\u00a06. The most striking single feature in Table\u00a06 is the marked increase in intramolecular salt bridges between the ferritins of mesophilic (EcFtnA and CjFtn) and hyperthermophilic (AfFtn, TmFtn and PfFtn) organisms. Indeed, a similar but less clear trend is observed for the SS hydrogen bonds overall. These structural features could explain the higher resistance to thermal inactivation in the latter. However, this trend by itself does not validate our initial assumption that PfFtn is the most thermostable ferritin in the group. A secondary trend can be perceived for the main-chain intramolecular hydrogen bonds in the hyperthermophilic organisms (AfFtn, TmFtn and PfFtn), which is greatest in PfFtn, and may offset the effect of a lower number of intramolecular salt bridges in comparison with TmFtn. Therefore, the most important factor that protects PfFtn and other ferritins from thermal denaturation appears to be the preservation of the monomer fold, rather than the 24-mer assembly.\nConclusions\nIn this study, we reported the crystal structure of PfFtn. High sequence similarity between PfFtn, TmFtn and AfFtn suggests a separate group of thermostable ferritins. PfFtn is the second archaeal ferritin structure to be reported, following that of AfFtn. In spite of the high sequence identity (50%) between these two proteins, their quaternary structures are significantly different. PfFtn exhibited the 432 symmetry commonly found in other known ferritins, whereas AfFtn showed the 23 symmetry similar to dodecameric DPS proteins. The 23 symmetry of the AfFtn 24-mer resulted in four triangular pores with an approximate size of 45\u00a0\u00c5, making large openings in the AfFtn shell. Therefore, the 23 symmetry of the AfFtn 24-mer remains an exception among ferritins, and further research is required to explain such an unusual conformation.\nThree iron sites were observed in the FC of PfFtn monomer. Site A was present in the as-crystallized protein and sites B and C were observed only upon crystal soaking with either Fe or Zn ions. Combining these results with our measurements of the FC EPR signal prior to crystallization, where samples were incubated in air for 1\u00a0day and 2\u00a0months, and for 2\u00a0months in 2\u00a0M ammonium sulfate (the crystallization solution), we conclude that the lack of iron atoms at the B site is very likely due to their complexation by ammonium sulfate, and that the absence of these sites in the as-crystallized protein cannot be taken per se as evidence of a transient nature of the FC, in PfFtn and other ferritins.\nThe possible iron entry and exit routes of PfFtn, the threefold and fourfold channels, are similar to those from bacterial ferritins in the sense that the threefold channel is less hydrophilic and the fourfold channel is more polar than the channels of mammalian H and L chain ferritins.\nThe hyperthermostability of PfFtn was analyzed at the structural level by comparing the number of salt bridges and hydrogen bonds in PfFtn with those in AfFtn, TmFtn, CjFtn and EcFtnA. Although there are no data on the thermal stability of other thermostable ferritins, our results suggest that TmFtn, the ferritin from T. maritima, a bacterium which grows optimally at 80\u00a0\u00b0C, is probably more thermostable than PfFtn. Owing to a high number of intramolecular hydrogen bonds between main-chain atoms, and between main-chain and side-chain atoms, the main factor contributing to the high thermostability of PfFtn as well as the other structurally similar ferritins from hyperthermophilic microorganisms appears to be the preservation of the monomer fold rather than the 24-mer structure.","keyphrases":["ferritin","hyperthermophile","archaeon","ferroxidase center","iron"],"prmu":["P","P","P","P","P"]}
{"id":"Bioprocess_Biosyst_Eng-2-2-1705536","title":"Production of a recombinant polyester-cleaving hydrolase from Thermobifida fusca in Escherichia coli\n","text":"The hydrolase (Thermobifida fusca hydrolase; TfH) from T. fusca was produced in Escherichia coli as fusion protein using the OmpA leader sequence and a His6 tag. Productivity could be raised more than 100-fold. Both batch and fed-batch cultivations yield comparable cell specific productivities whereas volumetric productivities differ largely. In the fed-batch cultivations final rTfH concentrations of 0.5 g L\u22121 could be achieved. In batch cultivations the generated rTfH is translocated to the periplasm wherefrom it is completely released into the extracellular medium. In fed-batch runs most of the produced rTfH remains as soluble protein in the cytoplasm and only a fraction of about 35% is translocated to the periplasm. Migration of periplasmic proteins in the medium is obviously coupled with growth rate and this final transport step possibly plays an important role in product localization and efficacy of the Sec translocation process.\nIntroduction\nSince more than 15\u00a0years, the interest in biodegradable plastics (polymeric materials which can be cleaved by enzymes and assimilated by microorganisms, respectively) is steadily raising and nowadays a number of commercial products are on the market. Polymers containing ester groups, especially polyesters, play a central role as they are easily subject to microbial attack, the degradation rate being largely dependent on their physical structure and chemical composition. First attempts to produce biodegradable plastics were based on natural polymers like poly(\u03b2-hydroxybutyrate) (PHB). However, their material properties proved to be insufficient and could not compete with those of synthetic polyesters. Tokiwa and Suzuki [1] already showed that also synthetic aliphatic polyesters are accessible to a biological degradation. However, aliphatic polyesters exhibit similar disadvantages concerning their material properties (e.g. minor tensile strength, low melting points) and therefore are hardly applicable for every day life products. On the other hand, polyesters such as poly(ethylene-terephthalate) (PET) and poly(butylene-terephthalate) (PBT) exhibit excellent material properties, but were classified as non-biodegradable. Therefore, aliphatic\u2013aromatic copolyesters, which combine both acceptable use properties and sufficient biodegradability [2] were developed. The material properties desired for specific applications can be easily adjusted by the ratio of the aliphatic to the aromatic diacid [3, 4]. Commercial products are available now under the trade name Ecoflex\u00ae (BASF AG, Germany).\nRecently, a thermophilic actinomycete was isolated and identified as Thermobifida fusca (former name: Thermomonospora fusca) exhibiting a remarkable degradation capability for aliphatic\u2013aromatic copolyesters [5, 6]. The extracellular hydrolase (TfH) responsible for the degradation of these copolyesters was purified from the culture broth and classified as a serine hydrolase with the highly conserved G-H-S-M-G motif [7]. The mature hydrolase consists of 261 amino acids, corresponding to a molecular weight of 28.2\u00a0kDa. Maximum activity was found at 65\u201370\u00b0C. The extracellular hydrolase gene was located on an operon containing two nearly identical genes (bta1 and bta2) with an amino acid identity of 92% [6].\nThe enzyme is only expressed by T. fusca if a polyester, such as poly(1,4-butandiole adipate-co-1,4-butandiole terephthalate) (BTA) or poly (\u03b5-caprolactone) (PCL) is present in the medium. Gouda et al. [8] observed, that the amount of enzyme excreted in the medium correlates with the amount of polyester added to the culture. This induction behaviour as well as adsorption and inhibition phenomena [8] complicate a large-scale production of this enzyme (TfH) and therefore, a simpler to handle recombinant expression system for producing TfH, independent of the presence of the polymer was highly desirable, particularly, as recent investigations showed that TfH is capable of degrading even PET [9].\nIn this paper, we report on the heterologous expression of the polyester-degrading hydrolase from T. fusca in E. coli TG1. To this end, the TfH-encoding gene (bta1) was cloned together with a C-terminal His6-affinity tag and the ompA-secretion signal under the control of a thermo-inducible promoter. This strategy should enable translocation of the gene product by the Sec-transport system from the cytoplasm into the periplasmic space, where the properly folded TfH should accumulate after cleavage of the OmpA sequence. Several cultivation strategies and purification routes were considered. The aim of the investigations was to provide sufficient amounts of TfH for larger-scale polyester degradation studies (low purification grade) and a highly purified TfH for mechanistic studies and structural analysis.\nMaterials and methods\nCloning of the recombinant His6-tagged TfH\nEscherichia coli TG1 (DSM 6056) was used for the production of the recombinant hydrolase from Thermobifida fusca (rTfH). TG1 is characterized by the following genetic markers: supE hsd\u03945 thi\u0394(lac-proAB) F\u00b4[traD36 proAB+ lacIq lacZ\u0394M15] [10].\nThe vector pCytexP1-OmpA-bta1 [6] was digested with NdeI and EcoRI to obtain a DNA fragment consisting of 876\u00a0bp. This DNA fragment was used as a template to clone a His6 tag at the C-terminal end using the primer set bta1-rev (5\u2032-TAC TTA GGA TCC TTA TCA GTG GTG GTG GTG GTG GTG CTC GAG GAA CGG GCA GGT GGA GCG-3\u2032) and bta1-for (5\u2032-GTC ATG GGC CAC TCC ATG GGC-3\u2032). The DNA fragment was amplified using VENT DNA polymerase (New England Biolabs, USA), the cycle programme was run at 95\u00b0C for 2\u00a0min, 30 cycles of 30\u00a0s at 95\u00b0C, 30\u00a0s at 50\u00b0C and 2\u00a0min at 55\u00b0C, followed by an extension reaction for 5\u00a0min at 55\u00b0C. The reaction mixture was designed according to the description of the manufacturer. The resulting PCR-fragment rbta1\u2013His6 was purified using the JETQUICK PCR Fragment purification kit (GENOMED GmbH, Germany). rbta1\u2013His6 was ligated to a DNA fragment obtained from the digestion of the origin vector with NcoI and BamHI. The resulting plasmid pCYTEXP1-OmpA-bta1-His6 with the secretion signal of OmpA, the hydrolase from T. fusca (TfH) and the affinity tag was used for the expression of rTfH.\nTransformation of pCYTEXP1-OmpA-bta1-His6 into E. coli TG1 was done by electroporation. Expression of the recombinant TfH (rTfH) was controlled by the strong tandem promoter \u03bbpRpL, which is expressed by the thermolabile repressor cI857.\nMedia\nLouria-Bertani medium (LB medium): 10\u00a0g\u00a0L-1 tryptone, 10\u00a0g\u00a0L\u22121 NaCl 5\u00a0g\u00a0L\u22121, yeast extract and 0.8\u00a0g\u00a0L\u22121 glucose was used for cultivations of E. coli TG1 in the complex medium [11]. The medium was autoclaved at 121\u00b0C for 20\u00a0min. Before cultivation, ampicillin (100\u00a0\u03bcg\u00a0mL\u22121) was added.\nA defined medium as described by Korz et al. [12] was used for batch and fed-batch cultivation of E. coli TG1. The medium contained (per litre): (NH4)2PO4 4\u00a0g, KH2PO4 13.3\u00a0g, citric acid 1.7\u00a0g, glucose 25\u00a0g, MgSO4 1.2\u00a0g, trace elements (Fe(III)citrate\u00b7H2O 100\u00a0mg, CoCl2\u00b76H2O 2.5\u00a0mg, MnCl2\u00b74H2O 15\u00a0mg, CuCl2\u00b72H2O 1.5\u00a0mg, H3BO3 3\u00a0mg, Na2MoO4\u00b72H2O 2.5\u00a0mg, Zn(CH3COO)2\u00b72H2O 13\u00a0mg, EDTA 8.4\u00a0mg) and 100\u00a0\u03bcL antifoam (Sigma Antifoam 204, Sigma-Aldrich, USA). MgSO4 and glucose solution were autoclaved separately from the phosphate buffer and trace elements at 121\u00b0C for 20\u00a0min. pH was adjusted to 6.8 with 5\u00a0M NaOH, before. Ampicillin 100\u00a0mg\u00a0L\u22121 and thiamine hydrochloride 4.5\u00a0mg\u00a0L\u22121 were sterilized by filtration (Sterivex, cut off of 0.2\u00a0\u03bcm, Millipore, USA) and added before starting the cultivation.\nThe feed solution for fed-batch cultivations contained the following components (in 1\u00a0L demineralized water): glucose 300\u00a0g, MgSO4 7.6\u00a0g, trace elements (Fe(III)citrate\u00b7H2O 16.2\u00a0mg, CoCl2\u00b76H2O 1.5\u00a0mg, MnCl2\u00b74H2O 8.9\u00a0mg, CuCl2\u00b72H2O 0.9\u00a0mg, H3BO3 1.9\u00a0mg, Na2MoO4\u00b72H2O 1.5\u00a0mg, Zn(CH3COO)2\u00b72H2O 6.1\u00a0mg, EDTA 4.9\u00a0mg), thiamine hydrochloride 4.5\u00a0mg and ampicillin 100\u00a0mg. The feed solution was sterilized by filtration.\nCultivation\nA bioreactor (Biostat B2 , B. Braun, Melsungen, Germany) with 2\u00a0L working volume connected to an exhaust gas analysis unit (S710, Sick Maihak, Germany) was used for the cultivations. Bioreactor control was done by a computer, operating the MFCS software (B. Braun). Feed and base reservoirs were stored on scales connected to the computer. Starting conditions of the reactor were set to 30\u00b0C, 500\u00a0rpm, pH 6.8 and 1\u00a0L\u00a0min\u22121 airflow. pH was controlled by adding either 25% NH3 (in case of the complex medium: 5\u00a0M NaOH) or 1\u00a0M H3PO4. The minimum level of dissolved oxygen (pO2) was set to 20% and was adjusted automatically by the stirrer speed and aeration rate. If the stirrer speed and aeration rate run at maximum, pure O2 had to be enriched to the inlet airflow. Antifoam was added manually to the cultivation under sterile conditions, if necessary.\nPre-cultures were carried out in the respective medium of the main culture by inoculation with a glycerol stock of E. coli TG1 containing the plasmid pCYTEXP1-OmpA-bta1-His6 at 30\u00b0C and 180\u00a0rpm for 12\u00a0h. Batch and fed-batch cultivation in the bioreactor was started by adding 20\u00a0mL of preculture.\nIn batch cultivation, induction of rTfH production was started at an OD of 0.5\u20130.6 by raising the cultivation temperature from 30 to 39\u00b0C or 42\u00b0C, respectively, within about 15\u00a0min.\nIn the fed-batch cultivation, feed was started after the initial glucose was consumed as indicated by a sharp increase in pO2. An exponential feed profile was used. For a desired specific growth rate \u03bcset (h\u22121), the volumetric feed rate Fglc (L\u00a0h\u22121) was calculated from (Eq.\u00a01) [13]:  where (XV)0 is the biomass (g) at feed start, tf the time (h) after feed start, YX\/S the biomass yield coefficient (0.4\u00a0g\u00a0g\u22121) and S the glucose concentration in the feed (g\u00a0L\u22121). Substrate consumption by maintenance is not considered in Eq.\u00a01. Therefore, the actual growth rate may be less than the set value (\u03bcset\u00a0=\u00a00.12\u00a0h\u22121). Feed rate and volume variations were corrected automatically by the MFCS software. The expression of rTfH was initiated at a biomass level of about 40\u00a0g\u00a0L\u22121 by shifting the temperature to 39\u00b0C, which was found optimal in the preliminary runs. After induction, the feed-rate profiles were kept constant at the value of the induction time or it was continously increased as given by Eq.\u00a01 for \u03bcset\u00a0=\u00a00.12\u00a0h\u22121.\nAnalytical methods\nCell-mass concentration was followed by OD at 600\u00a0nm (Ultrospec 3100 pro, Amersham Biosciences, Sweden). For the determination of the dry cell weight (DCW), 1\u00a0mL of the culture was added in triplicate to pre-weighed tubes and centrifuged for 5\u00a0min at 13,000g and 4\u00b0C. After a subsequent washing step, the pellet was dried until constant mass at 45\u00b0C under vacuum.\nExtracellular glucose and metabolites such as acetate, formate, succinate, propionate and isobutyrate were determined in the supernatant. To this end, the supernatant was heated up to 80\u00b0C for 5\u00a0min and centrifuged (13,000g, 10\u00a0min, 4\u00b0C) to eliminate the precipitated proteins. The supernatant was additionally filtered (pore size 0.2\u00a0\u03bcm) before applied into the HPLC (Shimadzu, Japan) using an Aminex HPX-87H column (Biorad, USA). 5\u00a0mM H2SO4 with a flow rate of 0.6\u00a0mL\u00a0min\u22121 was used as mobile phase at a temperature of 60\u00b0C.\nElectrophoresis\nSDS-PAGE was performed with a Mini Protean\u00ae 3 apparatus (Bio-Rad, USA) according to the manufacturer\u2019s recommendations. The samples were run on 12% polyacrylamide gel. Molecular mass marker was purchased from Bio-Rad. Proteins were stained with Comassie-Blue [11] or silver [14].\nProteins were transferred onto a 0.45-\u03bcm PVDF membrane (Immobilon\u2122-P Transfer Membrane, USA) using a Semi-Dry Transfer Unit (Semiphor\u2122 Hoefer TE\u00ae 70 Series, Amersham Bioscience, Sweden). rTfH was detected by Comassie-Blue staining.\nHydrolase activity\nHydrolase activity was measured by monitoring the cleavage of p-nitrophenylpalmitate (pNPP) as described by Schmidt-Dannert et al. [15]. The released p-nitrophenol was determined spectrophotometrically (Ultrospec 3100 pro, Amersham Biosciences, Sweden). pNPP solution was freshly prepared by mixing 1\u00a0mL pNPP dissolved in 2-propanol at a concentration of 3\u00a0g\u00a0L\u22121 with 9\u00a0mL 20\u00a0mM phosphate buffer (pH 7.1, sodium taurocholate 2.3\u00a0g\u00a0L\u22121, gum arabic 1.1\u00a0g\u00a0L\u22121). Immediately after addition of the enzyme sample the absorption was measured at 400\u00a0nm and 25\u00b0C against a sample without enzyme for 2\u00a0min. One unit was defined as the amount of enzyme that caused the release of 1\u00a0\u03bcmol p-nitrophenol per minute under test conditions. The extinction coefficient of p-nitrophenol is 9.62\u00a0cm2\u00a0\u03bcmol\u22121. To compare the photometrically determined pNPP activity units with a previously used activity test [8] the cleavage of the polyester bonds of PCL (poly(\u03b5-caprolactone)) films in a pHstat titration system was additionally measured as described by Gouda et al. [8]. It was found that one pNPP enzyme unit corresponds to three PCL units.\nLocalization of rTfH\nIn this paper, it is discriminated among the various rTfH fractions located in the extracellular medium, the periplasmic space and the cytoplasm as soluble or insoluble protein. The extracellular fraction (EF) was obtained from the culture supernatant after centrifugation (13,000g, 5\u00a0min, 4\u00b0C). A variation of the protocol of Neu et al. [16] was used to obtain the periplasmic fraction (PF). Biomass (about 37\u00a0mg) corresponding to ten OD was resuspended in 1\u00a0mL ice-cold 30\u00a0mM Tris\u2013HCl (pH 8.0) containing 20% sucrose and 1\u00a0mM EDTA and after incubation for 10\u00a0min at room temperature centrifuged at 13,000g and 4\u00b0C for 10\u00a0min. The supernatant was separated from the cell pellet and stored at an ice bath. The cell pellet was again resuspended in 1\u00a0mL ice-cold 5\u00a0mM MgSO4 solution and was slightly rotated on ice for 10\u00a0min. After centrifugation the supernatant was pooled with the previously obtained supernatant giving the total periplasmic proteins (PF). Each fraction (EF and PF) was stored at \u221220\u00b0C until further use.\nSoluble proteins harboured in the cytoplasm were obtained by resuspending the cells in 275\u00a0\u03bcL PBS buffer [11] [pH 7.2, 10% (v\/v) BugBuster, 0.1% (v\/v) benzonase (both Novagen, USA), 275\u00a0U lysozyme], slightly rotated at room temperature for 20\u00a0min and centrifuged at 13,000g and 4\u00b0C for 25\u00a0min. The supernatant formed the soluble cytoplasmic fraction (sCF). To gain the insoluble cytoplasmic protein fraction (iCF) cells were again resuspended in PBS buffer (10% (v\/v) BugBuster, 27.5\u00a0U lysozyme) and after incubation for 5\u00a0min diluted sixfold with PBS buffer (1% (v\/v) BugBuster). After centrifugation at 5,000g and 4\u00b0C for 15\u00a0min cells were washed twice with 800\u00a0\u03bcL PBS (1% (v\/v) BugBuster). Final centrifugation was done at 13,000g and 4\u00b0C for 25\u00a0min. The pellet was then resuspended in 275\u00a0\u03bcL of 1% (w\/v) SDS.\nPurification of rTfH\nTo gain a rTfH solution of low purification grade 75\u00a0mg biomass was resuspended in 2\u00a0mL ice-cold PBS buffer, pH 7.2 [11]. Cells were disrupted by ultrasonification (Sonopuls HD 2070, Sonotrode UW 2070, Bandelin electronics, Germany) in an ice bath using three intervals each of 30\u00a0s pulse and 2\u00a0min break. Subsequently, disrupted cell were denaturated by heat at 55\u00b0C for 10\u00a0min, and centrifuged (13,000g, 25\u00a0min, 4\u00b0C). The supernatant was stored at \u221220\u00b0C.\nHighly purified rTfH was recovered from the extracellular and the PF (see Localization of rTfH section). Chelating Sepharose\u2122 Fast Flow Gel (Amersham Bioscience) was activated as described by the manufacturer and added to the solution harbouring rTfH (1\u00a0mL chromatographic material was used for 400\u00a0U). The suspension was stirred for 1\u00a0h and filled then into a XK 26\/60-column (Amersham-Bioscience) and after the chromatographic material has settled, the column was connected to the FPLC system (LCC-500 Plus, Amersham Bioscience, Sweden). Column was washed with 3 column volumes (CV) binding buffer containing 20\u00a0mM Tris (pH 7.5), 500\u00a0mM NaCl and 10\u00a0mM imidazole at a flow rate of 4\u00a0mL\u00a0min\u22121. Elution of the bound rTfH was done by a linear increase of the imidazole concentration to 500\u00a0mM within 3 CV. Fractions with activity were pooled and concentrated by ultrafiltration (Amicon Stirred Cell 1050 or 1010, Amicon, USA) using a regenerated cellulose membrane (YM Ultrafiltration Membranes, cut off: 10\u00a0kDa, Amicon, USA). 6\u00b10.5\u00a0mL of the concentrated enzyme was applied manually to the gel filtration column (HiLoad 26\/60 Superdex 75 prep grade, Amersham Bioscience) equilibrated with 0.9% NaCl. The flow rate was set to 2\u00a0mL\u00a0min\u22121 to elute the proteins. Fractions with activity were pooled and stored at \u221220\u00b0C until further use. Whenever necessary, rTfH solution was concentrated by ultrafiltration.\nProtein determination\nProtein was determined by Roti\u00ae-Nanoquant (Roth, Germany) as described by the manufacturer.\nResults \nBatch cultivations\nE. coli TG1 harbouring the plasmid pCYTEXP1-OmpA-bta1-His6 was firstly cultivated batchwise with complex medium in 1\u00a0L scale monitoring OD, DCW, glucose and metabolites in the supernatant. At an OD of about 0.6, cultivation temperature was raised to either 39 or 42\u00b0C to induce rTfH production. After starting induction, generation of rTfH was pursued by the spectrophotometric assay in the extracellular and PFs. Results are shown in Fig.\u00a01.\nFig.\u00a01Expression of rTfH in E. coli TG1 during batch cultivation with LB medium. Cells grew at 30\u00b0C to an OD of 0.6 before starting induction by a temperature shift to 39 or 42\u00b0C, a growth and CO2 generation, b rTfH activity in different locations, extracellular fraction (EF; open square), periplasmic fraction (PF; open circle), sum of all fractions (continuous line), c glucose and acetate in broth\nGlucose present in the complex medium is quickly consumed at about 1.4\u20132\u00a0h after induction (Fig.\u00a01c). Consumption of glucose is accompanied by formation of acetate, its extracellular concentration peaks at 39\u00b0C at 2\u00a0h (3\u00a0h at 42\u00b0C, respectively), which is remetabolized after total glucose consumption. The consumption pattern is reflected by biomass development and CO2 generation (Fig.\u00a01a) which show a two- and three-phase behaviour, respectively. Firstly growth rate is highest as long as glucose is available (induction at 39\u00b0C). The following consumption of the intermediate acetate leads to reduced growth rate. After acetate consumption the growth rate further declines and finally growth ceases, however, CO2 evolution is yet observed in the stationary phase. From the biomass and CO2 evolution, one has to conclude that in all cultivation phases considerable amounts of constituents of the complex fractions of the medium are also consumed. For instance, the 1.5\u00a0g\u00a0L\u22121 biomass (2\u00a0h after induction at 39\u00b0C) cannot be generated alone by glucose consumption. Unfortunately, the complex constituents are not known. Therefore, a carbon balance is not possible.\nThe production of the hydrolase rTfH is shown in Fig.\u00a01b. Both induction profiles, shifting the temperature to 39\u00a0 or 42\u00b0C, revealed similar rTfH production kinetics. Shortly after induction rTfH is only found in the periplasm. However, in the course of the cultivation, rTfH did not accumulate in the PF as expected due to the leader signal OmpA. Surprisingly rTFH was entirely found in the extracellular medium at the end of the cultivation. At an induction of 42\u00b0C the promoter \u03bbpRpL in front of the bta1 gene is stronger than at 39\u00b0C, but expression of rTfH is nearly finished about 6\u00a0h after induction resulting in a lower volumetric activity (only 60%). About 4\u00a0h after induction, a remarkable increase of extracellular rTfH activity was observed which obviously correlates with a high metabolic activity as indicated by the second strong increase in CO2 evolution. The rise in rTfH activity cannot solely originate from translocation but suggest additional secretion from cytoplasm. Apparently expression of rTfH is faster than its transport through the cytoplasmic membrane in the early phase shortly after induction. In shake-flask cultivation, the rTfH production was monitored in the cytoplasm as soluble and insoluble cytoplasmic fractions (iCFs; data not shown). Maximum hydrolase activity was achieved after about 7.5\u00a0h summing up all compartments. Subsequently, the total hydrolase activity was slightly decreased, and finally rTfH was only detected in the supernatant.\nTo study the influence of medium composition on recombinant TfH production and to follow the transport of the enzyme into the medium, batch cultivations with the defined medium containing 23\u00a0g\u00a0L\u22121 glucose were carried out (Fig.\u00a02). rTfH was again induced at low OD. The course of the cultivation with the defined medium is remarkably different from that using the complex medium. Raising the temperature to either 39\u00b0C or 42\u00b0C caused firstly a low growth rate. The consumption of glucose was coupled with acetate formation; 8\u00a0h after induction at 39\u00b0C, growth rate rised to about 0.5\u00a0h\u22121 (0.6\u00a0h\u22121 at an induction at 42\u00b0C) and both glucose and acetate were consumed while a low amount of succinate was generated which was remetabolized after exhaustion of other C-substrates. As expected, growth was directly coupled with CO2 evolution. The respiration coefficient RQ was around 1, the yield coefficient (biomass on glucose) was determined as 0.4\u00a0g g\u22121 for the batch cultivation at 39\u00b0C. Over the entire induction period (about 23\u00a0h), carbon balances of the cultivation with the defined medium were close to  100\u00a0\u00b1\u00a03%.\nFig.\u00a02Expression of rTfH in E. coli TG1 during batch cultivation with the defined medium. Cells grew at 30\u00b0C up to an OD of 0.6 before starting induction by a temperature shift to 39\u00b0C or 42\u00b0C, a growth and CO2 generation, b rTfH activity in different locations, EF (open square), PF (open circle), sum of all fractions (continuous line), c glucose and metabolites in broth\nIn contrast to the cultivation in LB medium, succinate was detected in the extracellular medium under both the induction profiles. Succinate is a typical product of the anaerobic metabolism of E. coli. It is produced during the mixed acid fermentation and the fumarate reduction. Both pathways are strictly regulated by Fnr the global regulator of the anaerobic metabolism [17]. With the applied experimental approach, anaerobiosis can be excluded. The formation of succinate was coupled with the CO2 evolution and hence with a high metabolic activity. This may lead to an imbalance in the citric cycle or reduction equivalents. Unfortunately, other metabolites of the citric cycle were not determined in the supernatant supporting this assumption. Similar observations were made by Korz in batch cultivations using the same defined medium. He correlated the succinate formation with the amount of CO2 raising in the exhaust gas [18].\nWhile secretion of rTfH in the periplasm started immediately after induction, the rate of rTfH release in the cultivation medium correlates with the growth rate, i.e., growth and product transport from the cytosol via the periplasm into the extracellular space are coupled. At the end of the cultivation, almost the entire product was located outside the cells. The final results of the batch cultivations at controlled pH with the complex and the defined medium are summarized in Table\u00a01. For induction in both media, by shifting the temperature to 39\u00b0C gives better results than the increase to 42\u00b0C. While the volumetric activities achieved for the complex and the defined media at the same induction temperature are comparable, the specific activities differ significantly due to the higher biomass obtained with the defined medium. As the induction time is considerably longer for the cultivation in the defined medium, the specific productivities differ even more. These results clearly indicate that production of the rTfH in batch cultivation becomes more attractive when the complex medium is employed.\nTable\u00a01Summary of results on rTfH production in batch cultivationsComplex mediaDefined mediaInduction temperature(\u00b0C)39423942Biomass (gDCW L\u22121)2.22.19.89.6Induction time (h)9.962323.7Volumetric activity (103 U\u00a0L\u22121)8.084.437.52.84Specific activity (103 U\u00a0gDCW\u22121)3.672.110.770.30Volumetric productivity [103 U (L\u00a0h)\u22121]0.820.740.330.12Specific productivity [103 U (gDCW\u00a0h)\u22121]0.3730.3520.0340.013\nDuring the induction phase, the protein content in the culture supernatant was analysed by SDS-PAGE (Fig.\u00a03). About 4\u00a0h after the temperature upshift, the first protein bands can be recognized, particularly, for the cultivation with LB medium, where rTfH and other protein bands appear simultaneously. In cultivations with the defined medium, the pattern looks differently. In the first phase, up to almost 4\u00a0h after induction, a single protein of about 57.4\u00a0kDa is found. By N-terminal sequencing, this band could be identified as the periplasmic located dipeptide binding protein (DBP), which is involved in the transport of dipeptides [19]. In the cultivation with complex medium, leakage of the outer membrane increased significantly 6\u00a0h after induction when C-starvation began, growth was slow but CO2 generation was still high. With the defined medium protein release in the extracellular medium was drastically rising at about 12\u00a0h after induction when all glucose was consumed. The findings shown in Fig.\u00a03 are in accordance with the results reported by Rinas and Hoffmann [20], who studied in detail the leakage of host-cell proteins of recombinant and non-recombinant E. coli. These authors concluded that irrespective of the cell line, proteins of periplasmic origin are predominantly released in the medium, specifically, under stress conditions like temperature increase, nutrient starvation and high cell densities.\nFig.\u00a03SDS-PAGE under denaturating conditions: EFs of different cultivations are shown after increasing the temperature to 39\u00b0C to induce rTfH production, a batch cultivation with complex medium, b batch cultivation with the defined medium, dashed line marks the beginning of glucose starvation\nThe SDS-PAGE shown in Fig.\u00a03 indicates that the further protein bands observed for each media differ only in their intensity but not in their composition. This suggests that protein release in the medium cannot be attributed to cell lysis but is mainly due to an increasing leakage of proteins located and transported to the periplasm from the cytoplasm by the Sec-transport system.\nThis conclusion is also confirmed by analysis of the N-terminal sequence of rTfH, which clearly revealed that the OmpA leader sequence has been cleaved. However, cleavage was not uniform. Only 15% of the secreted rTfH found in the extracellular medium was cleaved at the expected cleavage site (Fig.\u00a04). The remaining rTfH still carried parts of the OmpA leader peptide. The incomplete processing of signal sequences has been reported before [21]. The cleavage motif of a signal petidase is the A-X-A box [21] and fission occurs after the X. The N-terminal analysis of the secreted rTfH revealed that the cleavage motif is kept. These observations may allow the conclusion that the Sec-transportation pathway is overloaded, which might explain the improper cleavage of the signal peptidase.\nFig.\u00a04Amino acid sequence of the signal peptide OmpA (underlined) and the beginning of rTfH; arrows mark the cleavage site and their relative frequency\nFed-batch cultivation\nTo enlarge volumetric rTfH activities, fed-batch cultivations were carried out. The basic assumption is that the volumetric productivity Qp [U\u00a0(L\u00a0h)\u22121] is given by equation  with the specific productivity qp [U (gDCW\u00a0h)\u22121], which is anticipated to be approximately constant when increasing the biomass concentration. As observed in the controlled batch cultivation it was also thought that the target enzyme may be released into the cultivations medium from the periplasmic space under high cell density conditions, thus facilitating down-stream processing [20]. High cell density cultivation (HCDC) for E. coli can be effectively performed by using an appropriate defined medium. After the batch phase with unlimited growth and consumption of glucose, the cultivation is continued by feeding glucose at a feed rate resulting in a reduced growth rate (\u03bcset\u00a0<\u00a0\u03bcmax; Eq. 1). Thus, oxygen starvation can be prevented and the culture grows under glucose limitation in the feed phase. The oxygen partial pressure was kept constant at 20% as described in the Cultivation section. Two typical HCDC runs are shown in Fig.\u00a05.\nFig.\u00a05Expression of rTfH in E. coli TG1 during HCDC. Cells grew at 30\u00b0C before induction by shifting the temperature to 39\u00b0C (dashed line). a After 12.8\u00a0h, the batch phase ended and exponential feed was started and set to a growth rate 0.12\u00a0h\u22121. At the time point of 25.5\u00a0h, feeding was set to a constant rate (12.8\u00a0gglc h\u22121). b Fed batch with an exponential feeding profile with \u03bcset=0.12\u00a0h\u22121 over the entire feed phase including induction\nStarting with an initial glucose concentrations of 24\u00a0g\u00a0L\u22121, the biomass concentration is about 12\u00a0gDCW\u00a0L\u22121 at the end of the batch phase (Fig.\u00a05a, b). Due to the metabolic overflow, acetate was generated in the unlimited growth phase (\u03bcmax = 0.47\u00a0h\u22121), which was remetabolized at the end of the batch phase (Fig.\u00a06). Then the glucose feed was started. The feed rate was adjusted to give a growth rate of about 0.12\u00a0h\u22121 according to Eq. 1, by assuming a yield coefficient YX\/S of 0.4\u00a0g\u00a0g\u22121. The actually measured growth rates were 0.09 (Fig.\u00a05a, b). In Run A, after a feed phase of 12.8\u00a0h, a biomass concentration of 38.5\u00a0gDCW\u00a0L\u22121 was obtained and at this point, the expression of rTfH was induced by shifting the temperature from 30 to 39\u00b0C within 15\u00a0min. The glucose feed rate was not further increased (12.8\u00a0gglc\u00a0h\u22121) but kept constant to the value when the induction was started. Therefore, the growth rate steadily decreased and after about 14\u00a0h, the induction growth ceased. In the first phase after induction (14\u00a0h), an average growth rate of roughly 0.03\u00a0h\u22121 can be estimated from the data. However, over the entire period of 22.4\u00a0h with the constant feed rate, CO2-generation rate was also constant. Therefore, the fed glucose is used only for maintenance in the second part of the induction phase when there was zero growth . In the fed-batch phase of Run B, the same exponentially increasing glucose-feed rate as for Run A was applied, i.e., Fglc was calculated from Eq. 1 for \u03bcset\u00a0=\u00a00.12\u00a0h\u22121 and YX\/S\u00a0=\u00a00.4\u00a0g\u00a0g\u22121. After starting the induction at 42\u00a0g\u00a0L\u22121, DCW exponential glucose feeding was continued for the same \u03bcset (0.12\u00a0h\u22121). In spite of the exponential glucose feeding, growth decreased and ceased about 6\u00a0h after induction. An average growth of 0.07\u00a0h\u22121 can be estimated from the experimental biomass data at the first 6\u00a0h of the induction phase. CO2 generation increased exponentially even when the growth was negligible in the second half of the induction phase. After 11.4\u00a0h induction, a final biomass concentration of 67.8\u00a0gDCW\u00a0L\u22121 was obtained.\nFig.\u00a06Glucose and metabolites formed during HCDC (Run A). cf. Fig. 5\nIn the course of both runs, the respiratory quotient was always about 1 (\u00b1\u00a00.02). This indicates that consumed glucose was exclusively used for ATP production. Indeed, an intermediate formation of fermentative by-products such as acetate, succinate and formate was only observed at low level at the beginning and end of the induction period (Fig.\u00a06). Both during the fed-batch and the induction phase, the glucose concentration was always zero. Carbon balances were checked by the installed computer program with consideration of volume changes by feeding and sampling and found to be close to  100\u00a0\u00b1\u00a02%.\nThe kinetics of the appearance of the heterologous gene product (rTfH) in the periplasm and cultivation broth is shown in Fig.\u00a07 for Run A with a long induction phase (>\u00a020\u00a0h). The activity in the periplasm steadily increased up to a value above 70\u00a0U\u00a0mL\u22121 while in the first 10\u00a0h, no rTfH was released in the supernatant. The final activity in the medium was rather low (12\u00a0U\u00a0mL\u22121). A similar release kinetics as shown in Fig.\u00a07 was also observed for Run B, which differs largely from the behaviour found for batch cultures (Figs.\u00a01, 2). In contrast to the batch cultivations the periplasmic activity does not pass a maximum and, particularly, little product (say less than 15%) is found in the medium.\nFig.\u00a07Production kinetic of rTfH in the fed-batch cultivation (Run A). At a biomass of 38.5\u00a0gDCW L\u22121 rTfH expression was induced by raising the temperature to 39\u00b0C\nHowever, despite the low leakage of rTfH into the medium a SDS-PAGE (Fig.\u00a08) made from the supernatant after the enzyme induction showed that the cells were already leaky at the beginning of the induction, which is proven by the appearance of the periplasmic DBP. During the induction phase, an increase both of DBP and rTFH is observed.\nFig.\u00a08SDS-PAGE: EFs of different cultivations are shown after increasing the temperature to 39\u00b0C to induce rTfH production, HCDC (Run A)\nThe hydrolase activity (pNPP test system) was also measured in the cytoplasm before the induction and at the end of the HCDC. While no activity was found prior to induction, a surprisingly high intracellular activity was detected at the end of the cultivation process. Indeed, about two-thirds of the entire rTfH activity was located in the cytoplasm, apparently in soluble and active form. Any significant inclusion-body formation was not observed. The operational parameters and the results received in the two HCDC runs shown in Fig.\u00a05 are summarized in Table\u00a02. In spite of the different feed rates and the total induction times, the rTfH activities found in the different compartments are in reasonable agreement. The constant feed rate after induction in Run A yields higher specific activities obviously due to lower biomass formation. However, as a result of the long induction time, the productivity is lower for Run A than for Run B. In comparison to the batch runs (Table\u00a01) the accumulation of product in the periplasm and cytoplasm is most noticeable. Thus the total volumetric activity is about 30-fold larger than that achieved in the batch runs. These higher overall product concentrations are advantageously for rTfH recovery and purification.\nTable\u00a02Applied operation parameters and achieved rTfH activities in fed-batch runs (starting glucose concentration 24\u00a0g\u00a0L\u22121, pH 7, T\u00a0=\u00a030\u00b0C, Tind\u00a0=\u00a039\u00b0C)Cultivation Run ARun BDCW at induction (g\u00a0L\u22121)38.542Feed rateConstant (12.8\u00a0gglc\u00a0h\u22121)Exponential (\u03bc\u00a0=\u00a00.12\u00a0h\u22121)Estimated mean growth rate after induction (h\u22121)0.030.07Total induction time (h)22.411.4Final DCW (g\u00a0L\u22121)53.267.8SupernatantVolumetric activity (103\u00a0U\u00a0L\u22121)12.045.16Specific activity (103\u00a0U\u00a0gDCW\u22121)0.2270.076PeriplasmVolumetric activity (103\u00a0U\u00a0L\u22121)74.2251.69Specific activity (103\u00a0U\u00a0gDCW\u22121)1.3950.762Cytoplasm (soluble)Volumetric activity (103\u00a0U\u00a0L\u22121)141.24156.74Specific activity (103\u00a0U\u00a0gDCW\u22121)2.6552.312TotalVolumetric activity (103\u00a0U\u00a0L\u22121)227.5213.59Specific activity (103\u00a0U\u00a0gDCW\u22121)4.283.15Productivity [U (103\u00a0gDCW h)\u22121]a0.190.28aTotal induction time\nPurification\nTwo different strategies were applied to purify rTfH with regard to different uses. To gain rTfH with a high purification grade, the His6-affinity tag cloned at the C-terminal end of the bta1 gene allowed a simple and effective chromatographic procedure. rTfH was purified from the culture broth or the periplasm, which was obtained by an osmotic shock treatment (Localization of rTfH section). In the following, the purification of rTfH is described, which originated from the periplasmic space. About 37\u00a0g of wet biomass (corresponding to ca. 6.5\u00a0gDCW) was subjected to osmotic shock treatment (1\u00a0L scale). After separating the cells by centrifugation, 25\u00a0mL Ni2+ activated sepharose was directly added to the supernatant harbouring the periplasmic protein. After 1\u00a0h incubation, only low amounts of rTfH activity (0.1%) could be detected in the supernatant. The suspension was then filled into a glass column. Particle settling could be improved by connecting a peristaltic pump to the column outlet. After connecting the column to a FPLC system, rTfH was washed with bindung buffer; rTfH was eluted by applying a linear gradient of imidazole (Fig.\u00a09a). rTfH elution was observed at an imidazole concentration of about 200\u00a0mM. In the pooled fractions, a rTfH activity of 6.53\u00a0U\u00a0mL\u22121 could be detected. The His6-affinity step resulted in a 29-fold purification and a loss of about 30% rTfH activity.\nFig.\u00a09Purification of rTfH from the PF. E. coli TG1 cells derived from a fed-batch cultivation (Run A); 37\u00a0g biomass was used for osmotic shock treatment, a Ni2+-affinity chromatography with Chelating Sepharose Fast Flow, b gelfiltration with Superdex 75 prep grade; pooled fraction are marked by a line\nTo further eliminate impurities and to remove high concentrations of NaCl and imidazole from the protein sample, gelfiltration was done using a Hi Load 26\/60 Superdex 75 prep grade column with a 0.9% NaCl buffer (Fig.\u00a09b). Before applying the sample to the column, pooled fractions with rTfH from the Ni2+-affinity chromatography were concentrated by ultrafiltration (concentration factor 7; cut-off 10\u00a0kDa). Gelfiltration assured purification to homogeneity as shown by SDS-PAGE (Fig.\u00a010) although only a minor increase of the specific activity was observed (Table\u00a03). Gel filtration resulted in a further loss of rTfH activity of about 10%. Totally, rTfH could be purified 30-fold with a 57% recovery in activity by the applied purification route. Table\u00a03 gives an overview of the procedure to obtain the highly purified rTfH, which will be used for mechanistic studies (adsorption, kinetic) and structural analysis.\nFig.\u00a010SDS-PAGE: rTfH at different purification steps (silver staining), 37\u00a0g biomass was used for the osmotic shock treatment, cells derived from HCDC (Run A) standard: molecular mass protein standard, 1 supernatant from the periplasm, 2 Ni2+-affinity chromatography, 3 pooled fraction after Superdex 75 gel chromatographyTable\u00a03Summary of two routes used to recover and purify rTfH from HCDC; the highly purified product was obtained from the periplasmic fraction (Run A), the low grade product was recovered from the cytoplasm and periplasm by ultrasonification (Run B)Purification stepTotal units (103\u00a0U\u00a0L\u22121)Total protein (mg\u00a0L\u22121)Specific activity (U mg\u22121)Purification-factorYield (%)High grade rTfH\u00a0Supernatant with periplasmic protein8.57587151100\u00a0Ni2+-affinity chromatography6.53154362971\u00a0Gel filtration4.89114453057Low grade rTfH\u00a0Crude extract26.522,57010.321100\u00a0Heat treatment22.511,47015.31.585\u00a0Ultrasonification + heat treatment28.891,41020.52109aaCrude extract (ultrasonification plus centrifugation)\nContrary to the purification profile of the TfH from the culture broth of T. fusca cultivations, the overall yield could be increased from 14 to 57% [7]. Prior to performing the first purification step, the supernatant containing TfH had to be concentrated by ammonium sulphate precipitation. By this method, a final recovery of only 37% could be achieved because of the limited amounts of the precipitated proteins that could be redissolved [7]. By inserting the His6-affinity tag at the C-terminal end of rTfH, precipitation could be avoided and the recombinant enzyme could be easily obtained from E. coli-culture broths independently of its composition.\nFor industrial processes, purification with Ni2+-affinity chromatography is too costly of course. Therefore, an alternative purification route was developed, which aimed at a rTfH preparation of a low purification grade but sufficient activity and stability for larger-scale investigations on polyester degradation. Activity test of the soluble protein in the cytoplasm obtained during HCDC showed that most of the expressed rTfH was not secreted into the periplasm but remained in the cytoplasm. Hence, the cells were disrupted by ultrasonification to get rTfH both from the cytoplasm and the periplasm. In preliminary experiments, different ultrasonication conditions were tested. Three-fold pulses of 30\u00a0s each with 2\u00a0min break were found as the best treatment for the test conditions of 75\u00a0mg biomass resuspended in 2\u00a0mL PBS buffer. Here, biomass from the HCDC Run B was used. After the separation of cell debris by centrifugation, an activity of 26.52\u00a0U\u00a0mL\u22121 was detected corresponding to a specific activity of 4.1\u00a0U\u00a0gDCW\u22121. Chemical disruption of the cells by a detergent formulation (BugBuster, see Localization of rTfH section) resulted only in a specific activity of 3.07\u00a0U\u00a0gDCW\u22121 implying that cell disruption is more effective than the chemical treatment. Protein concentration of the crude extract was 2.57\u00a0mg\u00a0mL\u22121. Taking the specific activity of the high-grade rTfH into account (445\u00a0U\u00a0mgProtein\u22121), 1\u00a0mg protein harboured 2.3% heterologous produced rTfH. Because rTfH is very stable against higher temperatures [7], heat treatment was carried out at 55\u00b0C for 10\u00a0min. Two routes were tested, namely either heat treatment of the disrupted cells immediately after the ultrasonication or heat treatment after the separation of the cell debris. Activity tests revealed that a heat treatment directly after the cell disruption yielded the best results. Thus the purification factor could be increased from 1.5 to 2 and a higher yield was observed. Table\u00a03 summarized the results in detail.\nDiscussion\nThe recombinant strain E. coli TG1 harbouring the plasmid pCYTEXP1-OmpA-bta1-His6 proved to be an appropriate system for expression, secretion and purification of the hydrolase from T. fusca. In comparison to cultivations with T. fusca and induction of TfH by adding polyesters a 1.5\u20132-fold higher volumetric activity could be obtained in batch cultivations of the recombinant E. coli strain. The volumetric activity could be further improved in fed-batch cultivations with glucose limitation. Considering only the extracellular and PFs, the increase is 20-fold. If one also accounts for the activity found in the cytoplasm, the total volumetric activity is over 50-fold higher than that obtained from T. fusca. The overall volumetric productivity [U\u00a0(L\u00a0h)\u22121] increased by a factor more than 100. Hence, the production of rTfH in recombinant E. coli is an attractive and promising approach for providing hydrolytic activity for polyester cleavage in degradation and recycling processes. This view is supported by the simple recovery route to give a crude and stable rTfH preparation, which did not exhibit activity losses after storage at \u221220\u00b0C for several weeks.\nA further improvement of rTfH production in E. coli is the insertion of the His6 tag at the C-terminal end of the enzyme. Purification of the wild-type TfH proved to be quite ineffective. A three-step purification strategy (precipitation, ion-exchange chromatography and hydrophobic interaction chromatography) resulted in only 14% yield in activity [7]. With the His6 tag, 57% activity yield of rTfH could be obtained in a two-step purification route starting from the supernatant and PFs. A 30-fold purification was achieved yielding a specific activity of 445\u00a0U\u00a0mg\u22121. From this value, the overall concentration of the produced rTfH can be estimated as about 0.5\u00a0g\u00a0L\u22121.\nIn the batch runs with LB and the defined medium, better results were found by induction to only 39\u00b0C. Therefore, fed-batch cultivations were carried out with this induction temperature. While both the media yielded comparable volumetric activities, the specific activities differ significantly (about fivefold in LB). LB medium obviously supplies E. coli with all nutrients to easily synthesize the recombinant protein at high specific productivity and to secrete it across the cell envelope. Increased growth rates and facilitated protein production in LB medium are frequently reported in the literature [22, 23]. In batch cultivations with both media, rTfH expression as well as its transport across the cell membrane in the periplasm and the subsequent release into the cultivation media are seemingly coupled with growth and CO2 generation (Figs.\u00a01, 2).\nIn the fed-batch cultivations with the defined medium, roughly tenfold higher activities could be achieved in the supernatant and periplasm than in batch operation. Surprisingly, the specific activities in the glucose limited fed-batch process are even twofold higher. If one accounts for the soluble cytosolic fraction the activity yields further improve remarkably. The total specific activity of the HCDCs is in the same range as those obtained for the batch process with LB medium. An influence of the His6 tag and the full or truncated OmpA leader sequence on TfH activity can hardly be estimated from the results of this study. However, the high activities of rTfH obtained suggest that small sequences at the C- and N- terminals might have little effect on TfH activity. The fraction of soluble active protein located in the cytoplasm is remarkably high but its amount can be estimated to be less than 2% of the total protein content of the cells (assuming 50% protein of DCW). Therefore, it is understood that inclusion body formation could not be detected.\nAmong the different mechanisms to transport protein from the cytoplasm across the cell membrane to the periplasm, the Sec pathway is the most prominent for prokaryotic organisms [24]. The Sec-translocation process is thought to be generally responsible for the transport of newly synthesized but unfolded proteins out of the cytosol to the periplasm. This process is accompanied by the cleavage of the leader sequence (OmpA) from the pre-protein and proper folding in the periplasm. If we assume that the Sec pathway is also active for the transport of the heterologous rTfH out of the cytoplasm, it is obvious that due to its accumulation in the cytoplasm during the fed-batch runs, the translocation rate is less than the expression rate. Hence, transport into the periplasm apparently presents a limiting step. It might be possible that the entire translocation process is subjected to an equilibrium phenomenon in as far as high protein concentrations in the periplasm reduces the transport rate. This is evident if the rTfH masses (or units) in periplasm and cytoplasm are not referred to cultivation volume as given in Table\u00a02 but to their respective actual volumes. If it is assumed that the volume of periplasm is about 5% of the cell volume then the rTfH concentration in the periplasm is more than tenfold higher than in the cytoplasm. This might explain that no cytoplasmic rTfH activity was found in the batch runs, where the periplasmic protein was entirely released in the extracellular space. In contrast, relatively little protein was transported across the outer cell wall into the medium in the fed-batch runs with high cell densities and higher rTfH activities were found in both the cytoplasm and periplasm.\nAs to the Sec pathway, the rTfH in the cytoplasm should be unfolded and hence is not expected to be in the active form. However, when gaining the cytoplasmic fraction (see Localization of rTfH section) immediate activity was observed. This suggests that either folding takes place spontaneously in the PBS buffer or the cytosol already harbours the active folded rTfH and its translocation follows another mechanism. Unfortunately, our study does not allow to distinguish between the two possibilities.\nThough it is often assumed that E. coli laboratory strains do not secrete proteins across the cell envelope in the extracellular medium at normal growth conditions [25], leakage of the outer cell membrane is a common phenomenon observed for a variety of cultivation conditions as reported in recent papers [20, 25, 26] and review articles [21, 27]. Permeability of the outer cell wall may depend on medium composition, growth rates, cell concentration, changes in cultivation conditions, etc. For instance, Mg2+ and Ca2+ increase leakage of the outer membrane [28]. Rise of temperature causes changes in membrane permeability and fluidity due to alterations in the lipid composition of the cell wall. This aspect was particularly studied in continuous cultures of E. coli [26] and an increased permeability was found at higher growth rates. Rinas and Hoffmann [20] studied in detail the leakage of host-cell proteins during HCDCs of recombinant and non-recombinant E. coli. These authors reported that the proteins found extracellularly are of periplasmic origin except the heat-shock protein DnaK. Various and interrelated stress factors in the temperature-induced HCDC are suggested to be responsible for the migration of periplasmic proteins in the extracellular medium.\nIn all cultivations performed in this study, rTfH could be detected in the medium without applying any additional measures to permeabilize the outer cell membrane. Cell lysis as a possible mechanism of rTfH release can be excluded as the cleavage of the OmpA sequence in the extracellularly found hydrolase indicates transport across the cytoplasmic membrane by the Sec-translocation mechanism. Any significant cell lysis would also release cytoplasmic rTfH still containing the complete signal sequence.\nIn batch cultivations (Figs.\u00a01, 2) with LB and the defined medium, induction of rTfH expression was started at low cell densities. Immediately after the temperature upshift, rTfH appeared in the periplasm but its periplasmic activity passes a maximal value just in the first part of the exponential growth phase. In accordance with the findings of Shokri et al. [26], the migration of the periplasmic rTfH in the medium is obviously growth coupled. This effect may be attributed to changes in cell-wall composition which facilitate transport into the extracellular medium. On the other hand, during cell growth and cell-wall biosynthesis, transient openings of the outer membrane and the murein layer are unavoidable [29], thus the protein of periplasmic origin can migrate into the extracellular medium. Furthermore, vesiculation and fragmentation of the outer membrane may occur as a natural phenomenon during the exponential growth phase [30, 31] which is thought to be caused by an imbalance of the outer-membrane biogenesis and peptidoglucan synthesis. Therefore, several growth-coupled phenomena may contribute to the migration of periplasmic proteins in the medium.\nAt the end of the batch cultivations, the produced rTfH has entirely accumulated in the medium. As can be discerned from the SDS-PAGE shown in Fig.\u00a03 the transport of rTfH across the cell envelope is accompanied simultaneously by release of other periplasmic proteins [20]. The kinetics of rTfH accumulation in the extracellular medium (Figs.\u00a01, 2) agrees qualitatively with the results shown in Fig.\u00a03, particularly, no significant migration is observed at low growth rates after the induction when the culture adapts to the higher temperature. Rinas and Hoffmann [20] found DBP as the most prominent periplasmic protein transported to the medium. This observation and even the release kinetics reported by these authors for high cell-density conditions are in striking agreement with our findings for batch conditions (relatively low cell densities) as observed in the defined medium (Fig.\u00a03b).\nIn contrast to the batch runs, rTfH accumulates in the cytoplasm and periplasm during the glucose-limited fed-batch cultivations after induction at higher cell densities. The relative fractions of rTfH released extracellularly is low. For an induction period of 11\u00a0h, only 5 and 10% of the PF are released for Run A and B, respectively. As in Run B, the growth rate was higher as a result of exponential glucose feeding; this finding underpins the view that the permeability of the outer membrane is related to growth rate. As shown in Fig.\u00a08 the outer membrane is already leaky before the temperature shift from 30 to 39\u00b0C as among other periplasmic proteins the DBP is predominantly found in the extracellular medium as reported previously [20]. Of course, in the induction phase a further increase of DBP is observed (Fig.\u00a08). Though the transport of rTfH in the periplasm as well as its migration into the extracellular medium continued (Fig.\u00a07), the cultivation was terminated to prevent cell lysis after growth ceased. However, in the non-growth phase, glucose does not accumulate in the medium but is consumed as is also exhibited by the monotonous increase of CO2 generation. This points to the increased maintenance and ATP demand for translocation of the heterologous gene product (rTfH) across the inner cell membrane.\nThese mechanisms, which govern the local distribution of rTfH are not yet understood. Though cell-specific productivities are in a comparable range, product distributions differ largely. Our results suggest that the outer membrane permeability is related with the growth rate and hence all rTfH can migrate into the extracellular medium at high growth rates (batch runs). Therefore, final product release across the cell envelope could play an important role in the overall translocation process as product accumulation in the periplasm seemingly reduces the translocation rate from the cytoplasm by the Sec pathway. This hypothesis requires further investigations, which are presently in preparation. Therefrom, new strategies for cultivation and downstream processing with improved product qualities are expected.","keyphrases":["purification","batch culture","sec pathway","recombinant protein expression","fed-batch culture"],"prmu":["P","P","P","R","R"]}
{"id":"Psychopharmacologia-3-1-2082659","title":"Effects of dopaminergic modulation on electrophysiological brain response to affective stimuli\n","text":"Introduction Several theoretical accounts of the role of dopamine suggest that dopamine has an influence on the processing of affective stimuli. There is some indirect evidence for this from studies showing an association between the treatment with dopaminergic agents and self-reported affect.\nIntroduction\nDopaminergic neurotransmission has been suggested to be involved in several functions of the brain that are associated with reward, such as the experience of rewarding feelings (Wise and Bozarth 1985), reward-related reversal learning (Mitul et al. 2001), motivational aspects of reward (Robinson and Berridge 1993), and reward processing (Day and Carelli 2007). In addition, dopamine neurons mediate reward signals in appetitive learning of visual stimuli (Schultz 1997). Recently, Robbins and Everitt (2007) stated that \u201cthe role of enhanced DA activity is to increase responsivity to cues paired with reinforcement\u201d. In addition to these functions, there are suggestions that dopamine is involved in the regulation of affect (Saeedi et al. 2006). Most studies addressing the role of dopamine in affect-regulation have been carried out among animals. Few studies address the role of dopamine in affect among humans. Studies among Parkinson\u2019s disease patients, whose mesencephalic dopamine activity is decreased, show that dopamine is able to modulate emotional responding (Wieser et al. 2006). In addition, it has been demonstrated that dopamine is also involved in the perceptual processing of fearful stimuli in Parkinson disease patients (Tessitore et al. 2002). No studies are known which address whether dopamine is able to modulate the processing of affective stimuli in normal subjects. However, there are some indirect suggestions pointing towards a role of dopamine in affective processing. Takahashi et al. (2005) showed among healthy volunteers watching unpleasant pictures that the dopamine D2 receptor antagonist sultopride decreased responses in the limbic areas (amygdala, hippocampus, anterior cingulate), which are associated with affective processing. In contrast, they found that sultopride produced greater activations in the cortical areas (frontal, temporal, and parietal), and no effect of sultopride on self-reported affect was found, suggesting a complex role of dopamine in affective processing.\nWith regard to psychopathology, what several theoretical accounts of psychopathological behavior have in common is that they state that dopamine may have a function in signaling relevant or salient information (Franken 2003; Kapur 2003; Robinson and Berridge 1993) and suggest that dopamine is involved in the processing of information having motivational relevance. In concordance with this notion, Franken et al. (2004) showed that the attention-capturing properties of heroin-related stimuli in heroin-dependent patients were decreased as result of treatment with the dopamine antagonist haloperidol. Indirect evidence that dopamine is involved in the processing of affective information comes from a study of Taylor et al. (2005) among schizophrenics. They found that structures with extensive dopaminergic innervations, such as the ventral striatum, show blunted responses to emotional salience in schizophrenics.\nIn addition, several theories suggest that dopamine is involved in the experience of self-reported positive affect (Ashby et al. 1999; Burgdorf and Panksepp 2006). In concordance with these notions, several studies examining the effects of dopamine-antagonists, in particular D2 antagonists such as haloperidol, report a blunting of the self-reported affective response (e.g., Mizrahi et al. 2007). In contrast, dopamine agonists result in self-reported enhanced mood (Saeedi et al. 2006; Willner et al. 2005).\nIn the present study, we use event-related potentials (ERPs) to investigate the processing of affective stimuli, in particular reward-associated stimuli, in humans. ERPs measured during the perception of pictures are known to vary with their affective contents (Cacioppo et al. 1994; Cuthbert et al. 2000; Keil et al. 2001; Schupp et al. 2000). Affective pictures, both pleasant and unpleasant, elicit larger late positive potentials (LPP; starting 300\u2013400\u00a0ms after picture onset until picture offset) than neutral pictures. LPPs capture controlled processes and are generated by a diffuse network including anterior areas such as the orbitofrontal cortex (Rule et al. 2002; Vuilleumier 2005) and higher order visual areas such as parietal and occipito-temporal regions (Keil et al. 2002). In addition to these late effects, the viewing of affective pictures results in reduced ERP amplitudes in the early time window (150\u2013300) as compared to neutral stimuli (Codispoti et al. 2007; Schupp et al. 2006). This early ERP effect is sometimes referred to as the early posterior negativity (EPN; Schupp et al. 2003b). The EPN, which is associated with the perceptual encoding of emotional material, may reflect the call for resources in the capacity-limited second stage of processing (Codispoti et al. 2007; Schupp et al. 2006) and is generated in the visual occipito-temporal regions (Schupp et al. 2006).\nIn passive viewing contexts such as in the present study, the EPN and LPP modulation by affective stimuli are similar in respect to the fact that they are both pronounced for high arousing pleasant and unpleasant stimuli (Schupp et al. 2006). However, it seems that the LPP, in contrast to the EPN, is enlarged when allocating top-down attentional resources to emotion, suggesting that the EPN is relatively insensitive to voluntarily attentional processes. Previous studies show that similar designs that study the electrophysiological activation to affective pictures are able to measure the modulation of affective processing by psychopharmacological agents (Franken et al. 2007; Kemp et al. 2004).\nThis study will investigate whether a D2 agonist or a D2 antagonist can modulate the electrophysiological response to affective pictures. Decrease in dopaminergic activity will be induced by a single oral dose of 2\u00a0mg haloperidol. This dose does not produce serious side effects (Franken et al. 2004; Kahkonen et al. 2001), and 2\u00a0mg was shown to be able to induce modulation of the processing of neutral and rewarding information (Franken et al. 2004; Kahkonen et al. 2001). Increase in dopamine will be induced by a single oral dose of 2.5\u00a0mg bromocriptine. Generally spoken, there are several indications that bromocriptine can modulate the response towards rewarding stimuli (Kirsch et al. 2006).\nAs dopamine is associated with an enhanced signalling of reward-related stimuli, it was expected that bromocriptine enhanced the processing of pleasant stimuli and haloperidol decreased the processing of pleasant stimuli. Based on the literature, the hypotheses for unpleasant stimuli could be less clearly formulated. Because some studies show that dopamine only signals stimuli associated with reward while others do find that dopamine also signals punishment-related stimuli, we expected that bromocriptine decreased the processing of unpleasant stimuli and haloperidol enhanced the processing of unpleasant stimuli. Neutral stimuli served as control conditions.\nMaterials and methods\nSubjects and procedure\nA randomized, double-blind, placebo-controlled crossover design was employed. Participants were 32 healthy right-handed male undergraduate psychology students and were recruited by advertisement at the university campus. All participants were non-smokers. Eleven participants could not be included in the analysis because of excessive electroencephalographic (EEG) artifacts in one of the three sessions (n\u2009=\u20093), drop out because of side effects (nausea) in the bromocriptine treatment (n\u2009=\u20096), or drop out because of side effects (low blood pressure, n\u2009=\u20091 and nausea, n\u2009=\u20091) in the placebo treatment. In total, 21 participants completed all three the measurement sessions, and all analyses were conducted using this sample. The mean age of the subjects was 19.8\u00a0years (SD\u2009=\u20091.2, range\u2009=\u200918\u201322).\nTwo weeks before the experiments, participants who were willing to participate were informed about the procedure. One week before the start of the experiments, subjects underwent a clinical psychiatric and physical examination by an experienced psychiatrist (L.P.). Eligible participants were instructed to take the medication at 8:00\u00a0a.m., 10:00\u00a0a.m., or 12:00\u00a0a.m. 4\u00a0h before each session in the laboratory. Further, they were instructed to abstain from coffee, grapefruit, alcohol, and drugs at the days of the experiment. In addition, they were instructed to abstain from alcohol and drugs the day before the experiment. The time between each session was always 1\u00a0week. All participants received a financial compensation of 50 euro. This study was approved by the Medical Ethics Committee of the Erasmus Medical Centre, Rotterdam.\nTreatment\nAll subjects received a single oral dose of placebo (lactose), bromocriptine (2.5\u00a0mg), and haloperidol (2\u00a0mg) in a counterbalanced order. The medication was provided by the pharmacy of the Erasmus Medical Centre in indistinguishable capsules. It has been demonstrated that a 2-mg dose of haloperidol does not produce serious side effects in healthy subjects (Franken et al. 2004; Kahkonen et al. 2001). Further, 2.5\u00a0mg of bromocriptine has been shown to modulate cognitive processes and in absence of side effects. Based on the literature, it was not expected that the 2.5\u00a0mg of bromocriptine would result in serious side effects (Jarvik et al. 2000). However, after several sessions, we noticed nausea occurring in some sessions. Because we excluded all participants with nausea (with reference to the possible interaction between nausea and the viewing of affective pictures), this resulted in relatively high dropout rates. After inclusion of 15 participants, all participants in all sessions were provided with domperidone (10\u00a0mg) to prevent nausea. Participants were instructed to take the domperidone together with the research medication1.\nStimuli and task\nBefore the first session, participants were instructed, and they signed informed consent. Subsequently, participants filled out the Positive and Negative Affect Scales (PANAS) questionnaire and two single-item self-rating scales that were used to assess the subjective effects of the medication (see \u201cQuestionnaires\u201d section). After this, the participants were seated on a comfortable chair in a light and sound-attenuated room. Stimuli were presented on a 21\u2033-monitor 1.5\u00a0m away from the eyes. After attaching the electrodes (approximately 15\u00a0min), they were instructed to pay close attention to the pictures that would be presented. To be sure that they paid attention to the pictures, they were told that questions about the pictures would be asked after the experiment.\nAll pictures were taken from the International Affective Picture System (IAPS; Lang et al. 1999). To reduce test\u2013retest recognition effects, three pictures sets which were closely matched on valence and arousal ratings were used in a counterbalanced order (see Table\u00a01). A one-way analysis of variance (ANOVA) confirmed that there were no differences in arousal and valence ratings between the three sets. Further, a repeated measures ANOVA confirmed that there were significant valence rating differences between the three affective categories, F(2,118)\u2009=\u20091324.2; p\u2009<\u20090.001. As expected, valence ratings for pleasant stimuli were higher than for neutral stimuli, and for neutral stimuli higher than unpleasant stimuli (higher scores indexes more pleasantness). In addition, analysis of the arousal ratings showed also significant differences between the three affective categories, F(2,118)\u2009=\u2009304.5; p\u2009<\u20090.001. Both pleasant and unpleasant pictures had higher arousal ratings than neutral pictures. In addition, pleasant stimuli had slightly higher arousal ratings as compared to the unpleasant stimuli. The neutral pictures consisted mainly of buildings, household objects, and neutral persons. The pleasant pictures consisted mainly of erotic and romantic scenes, while the unpleasant pictures consisted mainly of mutilated bodies and threat scenes.\nTable\u00a01Mean (SD) valence and arousal ratings of the three sets of IAPS pictures according to the normative ratings of the IAPS database (Lang et al. 1999)\u00a0Set 1Set 2Set 3Differences between sets (F values, ns)Neutral valence4.9 (0.3)4.9 (0.4)4.9 (0.2)0.01Arousal2.7 (0.4)2.8 (0.5)2.7 (0.6)0.16Pleasant valence7.2 (0.6)7.2 (0.6)7.2 (0.6)0.06Arousal6.3 (0.9)6.5 (0.7)6.2 (1.1)0.58Unpleasant valence2.5 (0.6)2.6 (0.5)2.6 (0.7)0.05Arousal5.8 (1.0)5.6 (0.9)5.7 (0.9)0.11ns Not significant\nQuestionnaires\nThe PANAS (Watson et al. 1988) were administered as a measure of positive and negative affect. The PANAS is a 20-item bidimensional mood inventory. Positive affect reflects the extent to which a person feels enthusiastic, active, and alert (Watson et al. 1988). Negative affect is a general dimension of subjective distress and unpleasurable engagement that subsumes a variety of aversive mood states including anger, contempt, disgust, guilt, fear, and nervousness (Watson et al. 1988). Psychometric properties of the PANAS scales are good (Boon and Peeters 1999; Watson et al. 1988).\nTwo single-item self-rating scales were used to assess the subjective effects of the medication. A five-point Likert scale (1\u2009=\u2009no effect, 5\u2009=\u2009a very strong effect) was used to assess the magnitude of the effect. A visual analogue scale (0\u2009=\u2009very unpleasant, 100\u2009=\u2009very pleasant) was used to assess the pleasantness of the effect.\nElectroencephalographic recording\nThe electrophysiological signals were recorded through the Active-Two amplifier system (Biosemi, Amsterdam, The Netherlands) from 64 scalp electrodes (10\u201320 system) using Ag\/AgCl electrodes mounted in an elastic cap. Furthermore, six additional electrodes were attached to left and right mastoids, two outer canthi of both eyes (HEOG), infraorbital, and supraorbital regions of the eye (VEOG). Two additional scalp electrodes were used as reference and ground electrodes, respectively. Online signals were recorded from DC to 134\u00a0Hz. All signals were digitized with a sample rate of 512\u00a0Hz and 24-bit A\/D conversion. Data were off-line re-referenced to an average reference. Off-line, EEG and EOG activity was filtered with a bandpass of 0.05\u201330\u00a0Hz (phase shift-free Butterworth filters; 24\u00a0dB\/octave slope). After ocular correction (Gratton et al. 1983), epochs including an EEG signal exceeding \u00b175\u00a0\u03bcV were not included in the average. The mean 200-ms pre-stimulus period served as baseline. After baseline correction, average ERP waves were calculated for artifact-free trials at each scalp site across the three stimulus conditions and across the three treatment conditions. Brainvision Analyzer (Brain Products, M\u00fcnchen, Germany) software was used for all EEG analyses.\nData analysis\nDefinitions of the peaks of interest were based on previous studies using the same IAPS paradigm (Franken et al. 2007; Schupp et al. 2000, 2003a). Inspection of voltage maps of the difference waves (emotional\u2013neutral) revealed that the EPN was maximal present at the following posterior sites: P5\/6, P7\/8, P9\/10, PO3\/4, PO7\/8, and O1\/2 (see Fig.\u00a01). This site is comparable to that used by previous studies (Franken et al. 2007; Schupp et al. 2003b). To reduce the number of covarying variables and to reduce the possible influence of single spurious electrodes, these individual sites were collapsed by averaging the electrodes into one region per hemisphere (Dien and Santuzzi 2005; Schupp et al. 2003b).2 The EPN in this area was defined as the mean value of ERP activities in 150- to 300-ms time windows. Note that the EPN can have positive values: The EPN is a negative going potential showing reduced positivity for emotional stimuli.\nFig.\u00a01Scalp topography of the EPN component (mean value in the 150- to 300-ms time window). Voltages represent difference scores: Pleasant\u2013neutral (left panel) and unpleasant\u2013neutral (right panel). Note that the anterior activity is positive and posterior activity is negative\nInspection of voltage maps of the difference waves (emotional\u2013neutral) revealed that the LPP was maximal at central and centro-parietal sites C5\/6, C3\/4, C1\/2, CP5\/6, CP3\/4, CP1\/2, P5\/6, P3\/4, P1\/2 (see Fig.\u00a02). Again, these individual sites were collapsed by averaging these electrodes into one region per hemisphere. The LPP was defined as mean value of ERP response within 400\u2013700\u00a0ms. The LPP is a positive deflection in the EEG, and emotional stimuli increase the amplitude of this component. In addition, because in previous studies valence effects on the LPP have shown hemispheric differences (e.g., Keil et al. 2001), we exploratively included hemisphere as factor in all ANOVAs.\nFig.\u00a02Scalp topography of the LPP component (mean value in the 400- to 700-ms time window). Voltages represent difference scores: Pleasant\u2013neutral (left panel) and unpleasant\u2013neutral (right panel). Note that the anterior and posterior activities are negative and that the central activity is positive\nStatistical analysis\nTo test the influence of dopaminergic manipulation on the EPN and LPP components, a 3 (medication: placebo, bromocriptine, haloperidol) \u00d7 3 (stimulus category: neutral, pleasant, unpleasant) \u00d7 2 (hemisphere: left, right) repeated measures ANOVA was employed with all variables as within-subjects factors. For the manipulation check, a three-way repeated measures ANOVA was used for each self-reported measure (pleasantness and magnitude of medication effect, positive and negative affect). In all instances, Greenhouse\u2013Geisser adjusted p values were used. In case of a significant ANOVA result, partial Eta squared (\u03b72) as measure of effect-size is reported. Significant ANOVA effects were followed up by pairwise comparisons with Bonferroni adjustment.\nResults\nSelf-reported effects\nThere was no significant effect of treatment on the magnitude of the subjective effects of the medication, F(2,40)\u2009=\u20090.8; p\u2009=\u20090.45. In addition, no differences in experienced pleasantness of the medication between the conditions was observed F(2,40)\u2009=\u20090.0; p\u2009=\u20090.96. Further, no effects of the medication condition on negative, F(2,40)\u2009=\u20090.8; p\u2009=\u20090.45, nor positive, F(2,40)\u2009=\u20090.4; p\u2009=\u20090.66, affect was observed. Summarized, no self-reported effects of medication on magnitude and pleasantness of effects and affect were observed.\nERP data\nEPN component\nTable\u00a02 displays all mean values of the EPN and LPP components, including standard deviations. Figs.\u00a03, 4, 5, and 6 show the difference ERP waves (emotional minus neutral) for affective stimuli at the left and right hemisphere sites. As expected, a significant main effect for stimulus category was observed, F(2, 40)\u2009=\u200981.0, p\u2009<\u20090.001, \u03b72\u2009=\u20090.80. Pairwise follow-up analysis show that both pleasant (p\u2009<\u20090.001) and unpleasant stimuli (p\u2009<\u20090.001) yielded smaller ERP values as compared to neutral stimuli, suggesting an enhanced early perceptual encoding of affective stimuli. However, no significant medication \u00d7 stimulus category interaction effect was observed F(4, 80)\u2009=\u20090.17, ns, suggesting that dopaminergic medication had no influence on this early stage of picture processing. In addition, no other significant main or interaction effects were observed.\nTable\u00a02Mean (SD) values of the EPN and LPP components in left and right hemisphere towards neutral, pleasant, and unpleasant pictures for the placebo, bromocriptine, and haloperidol treatment conditions (n\u2009=\u200921)\u00a0PlaceboBromocriptineHaloperidolEPN neutral left9.4 (3.6)8.4 (3.7)9.4 (4.4)Right8.6 (3.1)8.5 (4.2)8.9 (3.6)EPN pleasant left7.0 (4.3)6.0 (4.0)6.9 (4.2)Right6.7 (3.8)6.3 (4.5)6.8 (3.8)EPN unpleasant left8.6 (4.0)8.0 (3.8)8.6 (4.4)Right8.5 (4.0)8.2 (4.4)8.6 (4.3)LPP neutral left3.5 (1.6)3.2 (1.9)3.4 (1.8)Right3.8 (1.4)3.1 (1.7)3.3 (1.7)LPP pleasant left4.9 (1.9)4.6 (1.8)4.8 (2.1)Right4.6 (2.0)4.2 (1.8)4.2 (1.7)LPP unpleasant left5.4 (1.7)4.7 (1.7)5.5 (2.0)Right4.8 (1.9)4.2 (1.8)4.5 (1.9)Fig.\u00a03Differences waves (pleasant\u2013neutral) for pleasant stimuli at the left and right posterior cluster (P5\/6, P7\/8, P9\/10, PO3\/4, PO7\/8, and O1\/2) for placebo (black line), bromocriptine (gray line), and haloperidol (dotted line) treatment conditions separatelyFig.\u00a04Differences waves (unpleasant\u2013neutral) for unpleasant stimuli at the left and right posterior cluster (P5\/6, P7\/8, P9\/10, PO3\/4, PO7\/8, and O1\/2) for placebo (black line), bromocriptine (gray line), and haloperidol (dotted line) treatment conditions separatelyFig.\u00a05Differences waves (pleasant\u2013neutral) for pleasant stimuli at the left and right central\/centro-parietal clusters (C5\/6, C3\/4, C1\/2, CP5\/6, CP3\/4, CP1\/2, P5\/6, P3\/4, P1\/2) for placebo (black line), bromocriptine (gray line), and haloperidol (dotted line) treatment conditions separatelyFig.\u00a06Differences waves (unpleasant\u2013neutral) for unpleasant stimuli at the left and right central clusters (C5\/6, C3\/4, C1\/2, CP5\/6, CP3\/4, CP1\/2, P5\/6, P3\/4, P1\/2) for placebo (black line), bromocriptine (gray line), and haloperidol (dotted line) treatment conditions separately\nLPP component\nAgain, a significant main effect for stimulus category was observed, F(2, 40)\u2009=\u200948.1, p\u2009<\u20090.001, \u03b72\u2009=\u20090.71. Pairwise follow-up analysis show that both pleasant (p\u2009<\u20090.001) and unpleasant stimuli (p\u2009<\u20090.001) yielded a larger LPP component as compared to neutral stimuli, suggesting an enhanced processing for affective stimuli in the late elaborative phase. In addition, a small but significant main effect of medication was found, F(2, 40)\u2009=\u20095.2, p\u2009<\u20090.05, \u03b72\u2009=\u20090.21. Follow-up analysis indicated that bromocriptine resulted in overall reduced LPP waves as compared to the placebo treatment (p\u2009<\u20090.05), suggesting that bromocriptine results in an overall reduced processing of pictorial stimuli in this stage. Further, a small but significant stimulus-category \u00d7 hemisphere interaction effect was observed, F(2, 40)\u2009=\u20093.7, p\u2009<\u20090.05, \u03b72\u2009=\u20090.16. Because none of the post hoc tests resulted in a significant pairwise comparison, and this interaction had not our major interest, we will not discuss this finding in further detail. Most important for our hypothesis, no significant stimulus category \u00d7 medication interaction effect was found, suggesting that the employed dopamine antagonist and agonist had no selective influence on affective processing.\nDiscussion\nTo our knowledge, this is the first study examining the influence of dopamine on ERP correlates of affective stimulus processing. The present study demonstrates that an acute low dose of bromocriptine resulted in globally reduced LPP waves as compared to the placebo treatment, suggesting that bromocriptine results in decreased cortical processing of pictorial information. Although there are no previous studies using an acute low dose of bromocriptine examining the processing of emotional pictures (as in the present study), these results are in line with a study of Oranje et al. (2004). Although in that study a different paradigm was employed and different components were studied, these authors found a globally reduced ERP response as result of bromocriptine treatment. In contrast to bromocriptine, an acute low dose haloperidol did not result in a non-specific modification of cortical processing of pictorial information. Previous studies addressing information processing using haloperidol and ERP measures yielded mixed results. For example, Kahkonen et al. (2001) did observe a global reduction of ERP waves using 2\u00a0mg (oral) of haloperidol. In contrast, other studies did not observe a general effect of haloperidol on ERP amplitudes (e.g. Ford et al. 1994). Clearly, the effects of haloperidol on ERPs are dependent on dose, ERP component, characteristics of the participants, and experimental paradigms that are used.\nMost importantly, we could not confirm the hypothesis that D2 receptor modulation results in a modulation of cortical processing of affective stimuli. Neither an acute low dose of bromocriptine nor an acute low dose of haloperidol did differentially modulate the brain\u2019s electrophysiological response towards affective stimuli. This finding suggests that dopamine D2 receptors are not involved in the cortical processing of affective information. Because the studied ERP components measured in the present study are associated with attentive motivational processing (Schupp et al. 2006, 2007), it can be concluded that motivational attentive processing is not affected by haloperidol nor bromocriptine. Given that the ERPs in the present study reflect attentive processing, the results are in line with Oranje et al. (2006) who did not find any effects of l-dopa nor bromocriptine on ERP indices of selective attention in healthy volunteers.\nIn addition to the main finding that modulation of the D2 receptor did not result in early (EPN) or late (LPP) effect on ERPs indexing early perceptual encoding of emotional material and subsequent elaborative processing, we did not observe an effect of haloperidol and bromocriptine on the subjective self-reported affect. Neither medication had an effect on positive or negative affect. However, it must be noted that the present study was not specifically designed to measure the subjective effects of the medication. In addition, although the present results show that dopamine is not associated with the perception of reward-associated stimuli per se, it still might be associated with establishing stimulus\u2013reward associations or functions dealing with reward-related motor responses. In addition, it might be that the reward processing is only modulated by dopaminergic agents in persons with a genetically heightened reward sensitivity, for example, carriers of the A1 allele of the dopamine D2 TaqIA gene (Kirsch et al. 2006).\nThe present study suggests that low doses of bromocriptine in Parkinson\u2019s disease patients and haloperidol in schizophrenia patients may have limited influence on affective processing and self-reported affect in patients having these disorders. However, it must be noted that we did only study the effects of an acute dose of these medications; it is not known whether these effects differ from those associated with long term use of medication.\nThe present study has several limitations. First, there was a substantial dropout in the bromocriptine treatment. It may be that this is a specific subgroup of persons with heightened sensitivity for dopamine agonists. Because this group is not included in the analysis, this might have resulted in lowered generalizability of the results. Second, because of risks of unwanted side effects, we employed rather low doses of bromocriptine and haloperidol, 2\u00a0mg and 2.5\u00a0mg, respectively. Third, a possible limitation of this study is that some participants received domperidone and others did not, which may have influenced the results. However, it must be noted that domperidone does not cross the blood\u2013brain barrier but exerts its effect peripherally, and as such, does not have a major influence on the central dopamine system (Barone 1999; Champion et al. 1986). In addition, additional statistical analyses show that domperidone did not influence any of the outcome variables (electrophysiological nor self-reported) used in the present study.\nSummarizing, the present results indicate that neither an acute low dose of bromocriptine nor an acute low dose of haloperidol has selective effect on electrophysiological indices of affective processing and self-reported affect. In contrast, bromocriptine decreased overall processing of all stimulus categories regardless of their affective content. The results indicate that dopaminergic D2 receptors do not seem to play a crucial role in the selective processing of affective visual stimuli.","keyphrases":["dopamine","affective processing","haloperidol","erp","bromocriptine"],"prmu":["P","P","P","P","P"]}
{"id":"J_Biomol_NMR-3-1-1892527","title":"All-atom molecular dynamics simulations using orientational constraints from anisotropic NMR samples\n","text":"Orientational constraints obtained from solid state NMR experiments on anisotropic samples are used here in molecular dynamics (MD) simulations for determining the structure and dynamics of several different membrane-bound molecules. The new MD technique is based on the inclusion of orientation dependent pseudo-forces in the COSMOS-NMR force field. These forces drive molecular rotations and re-orientations in the simulation, such that the motional time-averages of the tensorial NMR properties approach the experimentally measured parameters. The orientational-constraint-driven MD simulations are universally applicable to all NMR interaction tensors, such as chemical shifts, dipolar couplings and quadrupolar interactions. The strategy does not depend on the initial choice of coordinates, and is in principle suitable for any flexible molecule. To test the method on three systems of increasing complexity, we used as constraints some deuterium quadrupolar couplings from the literature on pyrene, cholesterol and an antimicrobial peptide embedded in oriented lipid bilayers. The MD simulations were able to reproduce the NMR parameters within experimental error. The alignment of the three membrane-bound molecules and some aspects of their conformation were thus derived from the NMR data, in good agreement with previous analyses. Furthermore, the new approach yielded for the first time the distribution of segmental orientations with respect to the membrane and the order parameter tensors of all three systems.\nIntroduction\nUsing anisotropic media, such as partially oriented bicelles or macroscopically oriented membranes, NMR investigations can reveal a wealth of information about molecular properties, namely conformation, orientation and dynamics. In many solid state NMR studies of membrane-active peptides and transmembrane proteins, the samples are conveniently prepared with macroscopically oriented bilayers to obtain structural information. The NMR data analysis relies on a uniform alignment of all molecules with respect to the static magnetic field, as it makes use of the orientation dependence of the chemical shift, quadrupolar coupling or dipolar coupling interactions. In contrast to single crystal studies, where the molecules are immobilized in a unique conformation, in the case of lipid membranes and liquid crystalline systems one has to consider a wide distribution of molecular orientations and anisotropic motions. Here, we apply for the first time a new MD strategy to deduce such structural and dynamics information on three representative compounds in biomembranes with increasing complexity: (i) pyrene, (ii) cholesterol, and (iii) the antimicrobial peptide PGLa.\nA convenient nucleus for solid state NMR investigations of oriented membranes is deuterium for several reasons (for reviews see e.g., Vold 1994; Davis 1983; Ulrich and Grage 1998). First, the influence of 2H substitutions on the molecular structure and dynamics is negligible. More importantly, in most biologically relevant cases the quadrupolar interaction tensor of a carbon-bound deuterium is to a good approximation aligned parallel to the 2H\u2013C bond and directly reflects the local molecular orientation and dynamics of the labelled segment. Using 2H-NMR, the alignment and dynamic behaviour of lipids, small organic guest molecules and membrane-active peptides has been studied in numerous examples. Most previous strategies to extract the molecular orientation from NMR data, however, have been restricted to molecules with a rigid conformation, e.g. peptides assuming an ideal \u03b1-helix with fixed backbone torsion angles. In these studies, several individual labels are usually placed into strategic positions on the rigid molecular part. The measured quadrupolar splittings are then compared in a least squares RMSD analysis with the predicted values upon systematically varying the molecular alignment. The best-fit molecular orientation is determined from a 3D error plot as the one yielding minimal differences between the observed and calculated NMR parameters. However, this RMSD (root mean square deviation) analysis can only give preliminary results for flexible molecules such as peptides or proteins with a high degree of internal mobility.\nA proper way to account for motional averaging of the NMR parameters caused by molecular mobility is to run all-atom MD simulations. When applied to biomembranes, however, such simulations pose two closely connected problems: (i) large size of the system and (ii) long simulation times. In particular, water surrounding the lipid bilayer has to be included into the MD simulations, and time-spans have to be covered approaching the NMR time scale. Moreover, the volume of interest has to be surrounded by multiple copies of the central cell to avoid boundary effects. Nevertheless, such simulations have been demonstrated to be feasible for moderately sized membrane segments hosting medium size molecules (see e.g. Tieleman et\u00a0al. 2001). In the present contribution, we propose an alternative strategy, in which the oriented medium is not explicitly considered, but instead the ordering membrane environment is replaced in the MD simulations by pseudo-forces derived from the measured NMR parameters. This way sufficiently long MD runs become possible and allow to calculate the motionally averaged parameters observed in the NMR experiments. As a result, the full information about molecular orientation, order, segmental motions and even aspects of the molecular conformation could be revealed.\nThere is one remarkable advantage in the calculations involving orientational constraints. Since an experimentally observed NMR value usually represents a time average over the motions of the molecules, knowledge of the geometry and strength of these molecular motions is a prerequisite for properly interpreting the NMR data in terms of structural parameters. The order parameter, which is typically used to describe the effect of molecular motion on the NMR spectra, however, is often not known a priori. In the present approach, the molecular motions are intrinsically included and the order parameters are obtained as part of the simulation result.\nTheory\nNMR interaction tensors and coordinate transformations\nThe orientation dependence of the observed frequencies or splittings of an NMR property can be described by a 2nd rank tensor P, whose components are specified in the context of a coordinate system. On the one hand, a description in a molecule-fixed frame of reference is required to follow changes in orientation, but on the other hand the NMR observables are extracted from the interaction tensor in the laboratory frame related to the static magnetic field. We will define the molecule-fixed frames in the following way from the coordinates of the three bonded atoms A\u2013B\u2013C (see Fig.\u00a01):\nFig.\u00a01Local coordinate system used to define a tensorial NMR property on atom A. The bond from A to atom B is taken as the local z-direction, the plane spanned by A, B and C defines the x-direction in-plane, and the y-direction is perpendicular to that plane\nAtom A is selected as the position of the nucleus of interest (for instance 2H), whose property PA is to be calculated. In case there are several possible ways of selecting atoms B and C, they will be defined such that the valence of bond B\u2013C is a maximum (the valence can be estimated from the bond length, see O\u2019Keefe and Brese 1991). This selection ensures that the y-axis will be perpendicular to a \u03c0-system if it exists, and the x-axis will be positioned within the \u03c0-plane.\nOnce we have selected a coordinate system that is fixed to the molecule, we can calculate the columns of the transformation matrix D from the unit vectors along  of this frame, given in coordinates of the laboratory frame.\nMatrix D can be used to transform the tensorial properties from the molecular to the laboratory frame. The tensor components of P in the molecular frame are thus transformed to yield P\u2032 in the laboratory frame according to\nwhere D is the transformation matrix as given in Eq.\u00a02 (the Einstein sum convention is used for Greek subscripts and \u03b1, \u03b2\u00a0=\u00a0{x, y, z}, the second index indicating the unit vector).\nFor most deuterium quadrupolar couplings, as well as vicinal dipolar couplings like 15N\u20131H and 13C\u20131H the tensor PA will be to a good approximation diagonal in this coordinate system, hence we can easily assign the principal values of PA to the three axes. In cases where the chemical shift tensor is to be analysed, the same molecular coordinate system can be used as in those calculations based on the bond polarization theory (BPT, see Sternberg 1988). Therefore, all these types of NMR interactions can be treated within the same formalism.\nIn static NMR experiments the tensor components are observed in a coordinate system of reference whose z-axis is aligned with B0. In this frame the measured frequency or splitting is given by the zz-component of the tensor P. However, in the approach presented here we can incorporate further tensor components in the MD simulations, which are accessible for example by measuring the sample at further tilt angles.\nConstraints and calculation of molecular properties\nTo include experimental constraints in the MD simulations, pseudo-energy terms are added to the molecular energy provided by the force field. These pseudo-energies are defined as functions of the difference between the experimental and a calculated tensor property, Pi:\nwhere k is a force constant which is chosen to adjust the size and unit of the energy. The first sum runs over all observed tensor components and does not need to include all possible elements. In a 1D NMR measurement of a single label in an oriented or monocrystalline sample, only the zz-value of the tensor of this site can be experimentally determined. However, any additional tensor component introduced into the calculations will improve the characterization of the orientation of the system. Because of the nature of the tensor transform (Eq.\u00a03) each principal axis of the tensor can be inverted without changing the observed NMR frequency. Therefore, eight discrete tensor orientations are equivalent, even if all elements of the observed tensor are used as constraints. If, however, only the three principal values of the tensor are known, then in addition to this eightfold degeneracy a continuous set of orientations (instead of discrete possibilities) will be compatible with the experimental data from a single label. Therefore, a larger number of orientational constraints from several labels are necessary to determine the orientation of a rigid molecular segment in an unambiguous way. Obviously, if a molecule is intrinsically flexible, an even higher number of constraints will be required.\nThe experimental NMR values represent an ensemble average over a large number of molecules as well as a time average. The relevant time scale for averaging is given by the inverse of the frequency range that results from the anisotropy of the relevant interaction. When motions are faster than this time scale, a time average of the corresponding NMR parameters is observed. Motions slower than this time scale will result in a distribution of the respective NMR property. In all-atom MD simulations of larger molecules we can only perform calculations for a single molecular system and for time spans on the order of a Larmor period. Assuming that the NMR interactions are already completely averaged within this time, averaged properties can be simulated. This is likely to be the case for motions within the molecule and for the rotational diffusion of small molecules in oriented media such as the lipid bilayer considered here, which are on the time scale of 30\u00a0ps to 100\u00a0ns (see Br\u00fcschweiler 1994). We thus can assume that the time averages over the shorter period accessible for MD simulations do reflect the longer time averages observed by the NMR measurement. For the average of Ptheo we obtain using Eq.\u00a03:\nIf the NMR property does not change with time, we have to perform the time averaging only over the products of the transformation matrix elements . For most deuterium quadrupolar couplings and vicinal dipolar couplings, any conformational influence on the NMR interaction can be neglected. In the case of chemical shift tensors, however, the change of the tensors in the course of the MD simulation has to be considered.\nSince MD simulations are started with random velocities, we have to run many time steps before a thermal equilibrium is reached, and before the kinetic energy is redistributed properly between the different degrees of freedom of our system. During this equilibration phase of the MD the pseudo-energies will be large and unrealistic, since the proper mean values for the interaction tensors are not yet reached. Therefore the time averaging is performed here using an exponential memory function (Torda and van Gunsteren 1991):\nThe memory time constant is denoted with \u03c4, and N is the norm of the integral. This memory function serves to scale down the contributions from the beginning of the MD calculation exponentially. By proper selection of \u03c4 it can be ensured that we average mainly over the late, relevant time span of interest. Since in conventional MD simulations the equations of motion are integrated in finite time steps \u0394t, the integral in Eq.\u00a06 is converted into a discrete sum S. During the MD simulation, the sum Sn+1 of the time step n\u00a0+\u00a01 is calculated from the sum of the previous time step Sn in a recursive manner:\nIn the course of the MD simulation, S will contain the exponentially weighted time average of the property P, and in the case of a tensor the time averaging will be performed with all of its components.\nCalculation of pseudo-forces\nTo include the NMR constraints into the equations of motion, pseudo-forces  have to be calculated from the respective pseudo-energies (Eq.\u00a04). They are obtained as the derivatives of the energies with respect to the coordinates of the atoms. In the case of orientational pseudo-forces, we have to derive the transformation matrices D with respect to the coordinates of the atoms that were used in their definition (see Eq.\u00a01). The pseudo-forces in the Cartesian directions x, y and z (denoted with the Greek index \u03b3) acting on the atoms A, B, and C (denoted with j, see Eq. 1) are then given by\nThe calculation of the orientational pseudo-forces is thus reduced to determining the derivatives of the elements of the transformation matrices D with respect to the Cartesian coordinates of atoms A, B, and C.\nIn the following expressions we will use a short notation for the derivatives, omitting all indices:\nUsing the definitions of Eqs. 1 and 9, these derivatives can be calculated in the following way:\nThe derivatives of the unit vectors  can be used to construct the derivatives of the elements of the transformation matrices .\nBecause of the time dependence of the transformation matrices, the derivatives were calculated continuously during the MD simulation at each time step. The time average is only calculated for the NMR property.\nAs evident from Eq.\u00a08, the pseudo-forces depend linearly on the difference between theory and experiment. To avoid too high pseudo-forces in the beginning of the MD simulation, when the deviation of the simulated and experimental values is still large, the contributions of the pseudo-forces are scaled down. Another possibility to avoid too large pseudo-forces is achieved by multiplying the pseudo-forces by weighting factors , which lead to nearly constant forces if  exceeds a threshold \t\u0394P (see Witter et\u00a0al. 2001):\nFor  this factor behaves similar to the derivative of the original energy expression (4). It is possible to choose different force constants k (Eq. 8) and width parameters \u0394P for each type of NMR property of interest.\nOrder parameter calculation\nBesides determining the structure of a molecule, it is equally important to describe its dynamic behaviour in an oriented medium such as a lipid bilayer. MD simulations provide a unique opportunity to obtain information on the molecular dynamics, which is difficult to determine experimentally. The traditional way to account for motional averages in NMR investigation is to introduce order parameters. To this aim, the time average of the transformation matrices D (Eq. 5) can be used to characterize the influence of restricted molecular motions on the anisotropic properties of a molecule. In a similar way, Saupe (1964) defined the so called order tensor to describe the average orientation of a molecule:\nwhere \u2329\u00a0\u232a denotes the mean value and cos\u0398i are the z components of the unit vectors of a molecule-fixed frame within the laboratory system of reference. To describe the dynamic properties of flexible molecules, however, the Saupe approach is not sufficient, since molecular groups like peptide side chains may exhibit a larger mobility than the rest of the molecule. For this purpose Limmer (1989) introduced a segmental or site-specific order tensor W, which is the tensorial product of the Saupe order tensor and a conformational order tensor.\nBy calculating the NMR properties in the course of the MD simulation it is possible to obtain the segmental order tensor WA of the vicinity of atom A. The principal axis system of the tensor property is used to represent the instantaneous segmental orientation, which is then related to the average tensor P determined by Eq.\u00a05. The transformation matrices DPAS, which link the average P with the principal axis system of P at each time step of the simulation, can then be used to determine W (\u2329\u00a0\u232at denotes the time average):\nThis average is calculated in every step of the MD simulation for all sites A for which the tensorial NMR properties are calculated. In general the complete order tensor has 81 elements, which reduce to nine components if we are interested only in the zz component of a property (Eq.\u00a013). Since the order tensor W (and S as well) is symmetric and traceless, we have to consider only five independent elements. The transformation matrices DPAS in Eq. 13 can be calculated from the eigenvectors corresponding to the principal values of PA. After a full-length MD simulation, the order tensors WA will contain the information describing the time averaged orientation of all sites A.\nTo calculate the molecular Saupe order tensor, a coordinate system has to be fixed to the molecule. In a perfectly rigid molecule any set of three atoms could be selected at will. However, as most molecules are intrinsically flexible, it does not make sense to define the coordinate system that way, as the Saupe order tensor would then depend on this selection. We therefore decided to use the principal axis system of the tensor of inertia to represent the orientation of the entire molecule (see Burnell and De Lange 1980). This way we can calculate a molecular order tensor that is independent of any particular choice of coordinates:\nThe axes of inertia are denoted with a, b and c, corresponding to the moments of inertia in increasing order. For rod-like molecules the long axis will thus be the a-axis of least inertia.\nIn many cases it is convenient to diagonalize the order tensor to introduce a principal frame of order (Low 2002). Within this frame there are only two independent parameters: the order parameter S and a biaxality parameter \u03be:\nBy ordering the principal components as , one gets . If the biaxiality \u03be is zero, the orientational distribution of the molecule is defined by a single axis, which is called director. In the biaxial case with a non-zero \u03be, there exists a second distinct direction orthogonal to the director, and the molecular motions are characterized by two axes. A perfectly axially symmetric molecule that is oriented parallel to the membrane normal will lead to the following order tensor:\nWhen the molecular Saupe tensor is used to interpret the NMR observables, the conformational tensors CA have to be known as well. These tensors mediate between the orientation and order of a site A and the molecular coordinate system. If the transformation matrices leading from the molecular coordinate system to the individual tensor systems of the sites A are known, CA can be calculated the same way as the order tensor (see Limmer 1989). Only in the case of rigid molecules, simple expressions for CA can be derived from the molecular geometry. Since in our MD simulations the segmental order tensors WA and the molecular Saupe order tensor are calculated directly, the conformational order tensors become obsolete.\nMolecular dynamics simulation\nThe integration of the equations of motions is based on Verlet\u2019s algorithm (Verlet 1967), and time steps of 0.5\u00a0fs were employed to sample all high frequency hydrogen atom vibrations. In constrained MD simulations it is generally necessary to control the temperature during the simulation time. This is accomplished by coupling the molecular system to a heat bath which dissipates the heat generated by the pseudo-forces. All prevailing differences between the constraints and their calculated values are sources of heat. To obtain an NTV assemble (with conserved particle number N, temperature T, and volume V), we introduced a proper thermostating procedure (see Evans and Morriss 1990). The coupling to the thermostat is controlled by a coupling time constant \u03b7 which should be much larger than the time step. This time span \u03b7 allows an adjustment of the range of thermal fluctuations in the simulated molecular system.\nTo prevent too large pseudo-forces at the start of the MD simulation the pseudo-forces were gradually increased towards their final values during the MD simulation. To this aim we introduced time dependent scaling factors , which approach the value 1.0 in an exponential fashion. The time constant was set in most cases to 200\u00a0ps, leading to a relatively smooth course of the temperature.\nWhen applying the NMR orientational constraints during an MD run, the resulting pseudo-forces will \u201cheat up\u201d the system and enhance its rotational degrees of freedoms. Because the averaging procedure depends on the molecular re-orientations caused by the NMR constraints, some net rotational motion will prevail up to the end of the simulation. In standard MD simulations any overall molecular rotations and translations are subtracted from the velocities, since these external degrees of freedom are not of interest. In the present orientationally constrained calculations, however, only the net translations of the systems are removed.\nParametrization\nFor NMR spectroscopic considerations it is convenient to introduce the quadrupolar coupling tensor CQ in frequency units:\nwhere Q is the quadrupolar moment of the deuterium nucleus, and V the electric field gradient tensor at the nuclear position. The trace of CQ is zero because of , and in the case of carbon-bonded deuterons the quadrupolar coupling tensor can be considered to a good approximation as diagonal in the coordinate system aligned with the C\u20132H bond. If the electron distribution is axially symmetric there is only one linearly independent tensor component, otherwise there are two. In the general high temperature case we observe two quadrupolar resonances with frequencies symmetric to zero:\nIf the z-axis of the local electric field gradient points into the direction of the magnetic field, the corresponding quadrupolar splitting \u0394 \u03bd is\nIn the case of a C\u20132H bond the deuterium quadrupolar coupling depends only weakly on the molecular surrounding, hence representative values from static solid state NMR measurements of characteristic substances can be used as quadrupolar constants (Table\u00a01). In this selection we have to keep in mind that the quadrupolar coupling constants depend strongly on the hybridization of the carbon, and to some extent also on the polarization of the C\u20132H bonds by partial charges caused by electronegative groups.\nThe coordinate system for the local tensors is defined in Fig.\u00a01. The zz-component is aligned with the C\u20132H bond direction, and the yy-component is perpendicular to any \u03c0 system. For an aromatic C\u20132H (sp2) bond an asymmetry parameter of  with ) was assumed, and the tensor axes were assigned according to calculations of Bailey (1998). The value for the \u2013CD3 group is obtained from the C(sp3)\u20132H value scaled by 1\/3, assuming rapid rotation around the C\u2013CD3 axis.\nProgram implementation\nThe routines to apply the proposed orientational constraints are included into the COSMOS-NMR force field, which has been used in a number of previous applications, but which had so far been restricted to distance and chemical shift constraints (see e.g. Sternberg et\u00a0al. 2003; Witter et\u00a0al. 2002), and for the force field (M\u00f6llhoff and Sternberg 2001; Sternberg et\u00a0al. 2001). These two types of NMR constraints can be combined with the new orientational constraints as will be demonstrated in the example of PGLa below. The authors provide the backend version of the full COSMOS program, containing all computational procedures without the graphics and modelling interface (GUI) (see http:\/\/www.cosmos-software.de). The COSMOS-backend (C++) was compiled for several operation systems including Windows, Unix and Linux.\nApplications\nThe use of NMR orientational constraints is particularly well suited to gain insight into the alignment and dynamics of molecules embedded in biomembranes. In the three examples presented here, we will first demonstrate our new MD approach on pyrene as a simple model compound dissolved in lipid bilayers, then we will apply it to cholesterol as an intrinsic membrane lipid, and finally to the antimicrobial peptide PGLa, which forms an amphiphilic \u03b1-helix in membranes.\nPyrene\nPyrene was used as a first example, not only because of its relatively simple and rigid structure, but also because a full set of data is available from a recent 2H-NMR analysis in oriented lipid bilayers (Hoff et\u00a0al. 2005). The same publication included an all-atom MD simulation of four pyrene molecules in a POPC (1-palmitoyl-2-oleoyl-phosphatedylcholine) membrane. Those previous results will thus allow a direct comparison of our new strategy with established simulation techniques. The present orientational constraint-driven simulation utilizes the experimental 2H-NMR data of deuterium-labelled pyrene (see Fig.\u00a02) embedded at 4\u00a0mol% in POPC bilayers. Spectra had been acquired at two discrete sample orientations, namely with the membrane normal aligned parallel and perpendicular to the B0 field. In both orientations two pairs of narrow lines were observed (corresponding to the sites 2H1 and 2H2\u00a0+\u00a02H3, respectively), whose splittings changed by a factor of 1\/2. This situation can only originate from axially symmetric averaged quadrupolar tensors that are aligned with the membrane normal. This means that the pyrene molecules reorient quickly (on the time scale of the NMR interaction) about the membrane normal. Since the principal axis system of each averaged quadrupolar deuterium coupling tensor coincides with the membrane-fixed coordinate system, the absolute values of all 6 or 5 linearly independent components of the averaged coupling tensor are known and can be included in the present MD simulation. In this case, we could even make use of the signs of the principal quadrupolar components, as they had been deduced from the previous MD simulations mentioned above (Hoff et\u00a0al. 2005).Fig.\u00a02Fully deuterated pyrene molecule with three kinds of labelled sites (2H1, 2H2 and 2H3), illustrating the principal axes of the local deuterium quadrupolar tensors and of the principal axes of inertia a, b and c\nUsing the 2H-NMR data from the literature, we performed two MD simulations on pyrene constrained by the complete tensors. In the first run, only the contributions to the pseudo-energy arising from the three diagonal elements (first sum in Eq.\u00a04) were considered, and in a second run also the three off diagonal values (in this case 0.0) were included. In the first run, 11.4\u00a0ns were simulated, with the MD parameters set as given in Table\u00a02. To study the time behaviour of the pseudo-forces they were not scaled up exponentially as in the other two case studies below. The width parameter was set equal to the experimental error as \u0394P\u00a0=\u00a01\u00a0kHz, and the memory time was \u03c4\u00a0=\u00a0200\u00a0ps. The time development of the simulated tensor components for the first nanosecond can be seen in Fig.\u00a03. The trajectories are shown for the 2H2 site, which possesses an experimental splitting of 40.5\u00a0kHz. After an initial time period of about 250\u00a0ps, the final average coupling tensor components are reached and their values fluctuate only weakly. The residual fluctuations depend on four parameters: (i) the thermal fluctuations controlled by the coupling to the heat bath, (ii) the pseudo-energy, (iii) the width parameter \u0394P given in Eq. 11, and (iv) the memory time \u03c4 of Eq.\u00a06. In the simulated 2H-NMR spectra (Fig.\u00a04) these prevailing small fluctuations translate into line broadening, and the resulting line shapes are indeed very similar to the experimentally observed spectrum (Hoff et\u00a0al. 2005).Table\u00a01Deuterium quadrupolar coupling tensorsGroupCzzQ\/kHzCyyQ\/kHzCxxQ\/kHzSourceC(sp2)\u20132H193\u2212102.29\u221290.71Benzene, naphthalene (Rinn\u00e9 and Depireux 1974)C(sp3)\u20132H175\u221287.5\u221287.5Acenaphthene bridge (Rinn\u00e9 and Depireux 1974)\u2013CD358.33\u221229.17\u221229.17C(sp3)\u20132Haa\u00a0The values of the quadrupolar coupling constant are scaled by 1\/3 to take into account the free methyl rotationTable\u00a02General parameters for the MD simulations with orientational constraintsParameterValueTarget temperature293\u00a0KMD time step0.5\u00a0fsCoupling time \u03b7 to the heat bath0.05\u00a0psPseudo-force width \u0394P (Eq.\u00a011)1\u00a0kHzMemory decay time \u03c4 for the average (Eqs.\u00a06, 7)200\u00a0psTime constant \u03c1 for the exponential rise of pseudo-forces200\u00a0psTotal MD duration1\u00a0nsFig.\u00a03Trajectory of the time development of the averaging for the deuterium tensor components for 2H2 of pyrene. Under the influence of orientational pseudo-forces all components approach their final values after a period of about 250\u00a0ps. The component at about 20\u00a0kHz is experimentally observable in an oriented sample with the membrane normal parallel to B0. The component at \u221210\u00a0kHz would correspond to an experiment with a perpendicular sample alignmentFig.\u00a042H-NMR spectrum from the MD simulation of deuterated pyrene, using orientational constraints from an NMR measurement in oriented POPC membranes (Hoff et\u00a0al. 2005)\nIn this first simulation we had constrained only the three principal components of the quadrupolar tensor. It turned out that in this case small off-diagonal values did not approach zero but remain in the range of \u22121.5 to 2\u00a0kHz up to the end of the simulation (see Fig.\u00a03). At the beginning of the simulation a high pseudo-energy of 31,000\u00a0kJ\/mol was encountered, which dropped to 0.8\u00a0kJ\/mol by the end of the simulation. Because of the large pseudo-energies at the start, we realized that it is preferable to first run a preliminary MD simulation (1\u20134\u00a0ns) with much smaller pseudo-forces, and then to step up the pseudo- forces after some initial averaging has been performed. In the next two cases studies the pseudo-forces were switched on exponentially to avoid long preliminary equilibration periods.\nIn a second MD simulation of pyrene over 7.8\u00a0ns, we also constrained the three off-diagonal tensor components Cxy, Cxz, and Cyz to zero, which are averaged by rotations of the molecule about the membrane normal. The corresponding values calculated from the MD run yielded off-diagonal elements with absolute values smaller than 0.23\u00a0kHz. The pseudo-energy increased by a factor of two, as it now contains contributions from both the diagonal and off-diagonal tensor components. In an attempt to compensate for the higher pseudo-energy, we set the memory time to a higher value of \u03c4\u00a0=\u00a01,000\u00a0ps (see Eq.\u00a06). Altogether we obtained smaller fluctuations of the coupling tensor components and much narrower lines in simulated spectra, which can be most likely attributed to the damping effect of the memory function outweighing the higher pseudo-energies.\nTable\u00a03 summarizes the calculated quadrupolar splittings corresponding to the results of the second simulation, in which all tensor components were constrained. The values were averaged for any sites on pyrene with mutually parallel C\u20132H bond directions, so that the 2H1 splitting is a mean value of two and the others are mean values of four sites each. The MD simulation is seen to reflect the experimental 2H-NMR data very well in several aspects. First of all, the calculated quadrupolar splittings reproduce the experimental values very well. The marginally lower values found for the simulated splittings might be explained by a slightly elevated temperature encountered during the simulation. Namely, the mean temperature of this simulation was 302\u00a0K and the final temperature 299\u00a0K. Since the target temperature of the simulation was 290\u00a0K, the NMR constraints produced a constant temperature increase of about 10\u00a0K. This difference would explain the slightly smaller simulated splittings compared to the experiment. In full agreement with the experimental data, no significant difference was found for the sites 2H2 and 2H3. In the simulations, the difference between their splittings was within the scatter of the individual sites, and this difference could neither be resolved experimentally. Furthermore, also the averaging of the pyrene molecule in the membrane is reflected correctly in the simulation. The values for the tensor components perpendicular to the B0 field direction are negative and half of the values of Table\u00a03, indicating that the simulation does indeed lead to an averaged axially symmetric tensor aligned parallel to the membrane normal.Table\u00a03Calculated quadrupolar splittings in a 7.8\u00a0ns constrained MD simulation of pyrene, compared with the experimental values obtained from a 2H-NMR measurement of the deuterated pyrene in oriented POPC membranesSiteSign of tensor componentaConstrained MD splitting \u0394\u03bd (kHz)NMR experimentalb splitting \u0394 \u03bd (kHz)2H1+91.4 (91.39, 91.4)c93.02H2+39.9 (39.8\u201340.0)c40.52H3+39.7 (39.6\u201339.8)c40.5a\u00a0The sign is given for the orientation of the membrane normal in B0 field directionb\u00a0Hoff et\u00a0al. (2005)c\u00a0Range of simulated values for two or four sites, respectively\nThe experimental 2H-NMR data only reflect dynamic averages of e.g. the C\u20132H bond orientation, but can access only little information on the dynamic processes leading to the observed splittings. Here, the constraint-driven MD simulation provides a way to obtain details on molecular motions that are compatible with the experimental data. In the example of pyrene, the symmetry of the molecule and its motions in the membrane allowed us to derive information on the dynamic behaviour of the molecule from its observed splittings. Since in our case the coupling tensors are diagonal within the bond coordinate system and the segmental order tensor is also diagonal, simple expressions for the quadrupolar splittings are obtained:\nand for the case of an axially symmetric coupling tensor CQ we get\nThe latter expression is especially useful, since the quadrupolar splitting of a site A is directly related to a single segmental order parameter. Assuming that the order tensor is diagonal in the laboratory frame, Eq.\u00a021 applies to dipolar splittings as well, since the static dipolar coupling tensor is always axially symmetric and traceless, and the same holds for a rapidly rotating CD3-group. However, since the coupling tensor CQ for the C(sp2)\u20132H bonds is not axially symmetric (see Table\u00a01), the three principal elements of the order tensors WA contribute to the splitting as in Eq.\u00a020. It is therefore not possible to extract the order parameter tensor components from a single experimental splitting. From the course of our MD simulation, on the other hand, all components of the orders tensors WA (Table\u00a04) are available, and the terms of Eq. 20 can be readily analysed.Table\u00a04Simulated order parameters for pyrene in POPCSiteWxxWyyWzz| Wxy,Wxz,Wyz|2H1+0.021\u22120.342+0.321\u22640.032H2+0.181\u22120.310+0.129\u22640.072H3+0.165\u22120.294+0.129\u22640.03SbbSccSaa|Sab, Sac, Sbc|Moleculea+0.048\u22120.275+0.228\u22640.006a\u00a0The values for the molecular Saupe order tensor are ordered in the columns such that the axes of inertia fit to the corresponding coordinate axes of the 2H1 site (see Fig.\u00a02)\nFor a perfectly rigid pyrene molecule the molecular Saupe order tensor S and the segmental order tensors WA are linked by fixed conformation tensors. With our choice of the molecular coordinate system, S and WA are identical for the site 2H1, and for the other two sites the tensors are related by simple geometric expressions. Comparing the molecular order tensor S (last row in Table\u00a04) and the segmental order tensor W (e.g. of the site 2H1, which is expressed in the same coordinate system, first row in Table\u00a04), it is noticed that most of the molecular S tensor components are slightly smaller than the corresponding values of the segmental order tensor W. This effect is caused by bond vibrations and molecular twists occurring during the MD simulation. However, as these contributions to the dynamics (manifested in the difference between segmental and molecular order) are small, the mobility of the pyrene in the membrane is well described by the molecular order tensor S. Further qualitative conclusions on the behaviour of the pyrene molecule in the membrane can thus be drawn from the order tensor. Because Saa constitutes the largest (signed value) component, it is obvious that the corresponding axis a (in the system of inertia), and with it the long molecular axis of pyrene, shows some preference for an orientation in the direction of the membrane normal. Since Scc and Sbb are different, the molecular motions are not axially symmetric, but display a preference for a second axis, expressed in a biaxiality parameter \u03be\u00a0=\u00a00.11 (see Eq.\u00a016). This pronounced biaxiality is a consequence of the restricted motions of the flat pyrene molecule within the lipid matrix. A further indication for such a restricted motion is the fact that the smallest component of the order tensor S is found along the c-axis direction perpendicular to the aromatic ring system. This direction has thus the smallest average component  along the membrane normal and is most conserved, as expected for the planar shape of the molecule. In their all-atom MD simulation including 128 lipid molecules and water, Hoff et\u00a0al. (2005) obtained essentially the same results as in this work, with the exception that they found a much higher Saupe order component Szz\u00a0=\u00a00.42 (in this work denoted with Scc).\nCholesterol\nCholesterol is a constituent of many biomembranes, and its orientation and motion within the lipid bilayer is of great interest to explain its influence on membrane properties. The behaviour of cholesterol in membranes has been studied extensively, especially by 2H-NMR in view of its sensitivity towards molecular alignment and dynamics (Smith and Oldfield 1984). We have based our MD analysis on a recent study by Marsan et\u00a0al. (1999), who had measured and interpreted 2H-NMR spectra of partially deuterated cholesterol in oriented DMPC (dimyristoylphosphatidylcholine) bilayers. The labelled sites in the sterol ring system are indicated in Fig.\u00a05.Fig.\u00a05Cholesterol molecule with 2H-labelled sites indicated, for which experimental orientational constraints were available. From the MD simulation quadrupolar splittings were calculated for all deuterons in the ring system\nThe authors examined six different models of cholesterol structures proposed in the literature using an RMSD analysis to assign all observed lines in the 2H-NMR spectrum. In this analysis the quadrupolar splittings were calculated from the respective rigid molecular models, treating the molecular orientation and the Saupe tensor as free variables. It turned out that the results depended crucially on the selection of the molecular coordinates, and only the data from a neutron diffraction analysis (McMullan et\u00a0al. 1992) of a cholesterol derivative (20-CH3-methylpregnene-3,20-diol) produced convincing results. This strong dependence on the exact coordinates is regarded as one of the main drawbacks of RMSD analyses of rigid molecular models.\nIn the analysis presented here, we performed MD simulations for both methylpregnenediol and cholesterol, having modelled the latter with COSMOS (see Fig.\u00a05). As constraints for the MD simulations we used the 2H-NMR splittings of the eight experimentally observed deuterium labels of Marsan et\u00a0al. (1999), together with their best assignment (see Table\u00a05). In addition, from the MD simulations we predicted the putative quadrupolar splittings of all other protonated sites of the sterol ring system. This new data from our MD simulations can thus be used in the future to assign further 2H-NMR spectra without the need for any special labelling schemes or particular molecular coordinates. As outlined above, we again used the full quadrupolar tensors as constraints. Two short preliminary MD runs were performed to determine the signs of the coupling constants, since this information is required for the full tensorial constraints. All quadrupolar splittings of cholesterol which had been measured in oriented samples with the membrane normal parallel to B0 yielded negative coupling constants, (giving correspondingly positive values for the perpendicular orientation, see Table\u00a05). As in the example of pyrene, MD simulations over 1,000\u00a0ps were sufficient to reproduce all features of molecular motion. To avoid too large pseudo-forces at the start of the simulation, the pseudo-forces were exponentially increased to their final values during the MD run. The parameters used in the simulations are summarized in Table\u00a02.Table\u00a05Calculated deuterium quadrupolar splittings from a constrained MD simulation compared with the experimental 2H-NMR data (Marsan et\u00a0al. 1999)SiteConstrained MD splitting of methyl-pregnenediol (kHz)Constrained MD splitting of cholesterol (kHz)Experimental NMR splitting (kHz)H2a\u2212101.5\u2212101.4101.68H2e\u221266.7\u221267.767.86H3_1\u2212107.1\u2212106.7107.30H4e\u221263.2\u221262.362.68H4a\u221294.3\u221293.094.98H6\u22127.29\u22126.696.44H7a\u221292.9\u221295.496.12H7e\u221291.6\u221291.391.48C18\u2013CD3\u221233.0\u221233.8C19\u2013CD3\u221237.5\u221234.7H1a\u2212104.2\u221299.2H1e\u221269.4\u221246.1H8_1\u2212107.0\u2212100.3H9_1\u2212112.6\u2212103.0H11a\u2212106.4\u2212102.7H11e\u221250.7\u221274.7H12a\u221246.4\u221213.1H12e\u2212103.1\u2212101.1H14_1\u2212103.4\u2212100.3H15_1\u2212102.1\u221281.8H15_2\u221293.5\u221288.9H16_1\u221217.6\u221240.9H16_2\u221223.1\u221227.6H17_1\u221285.7\u221292.7\nThe final results for the quadrupolar splittings obtained from the constrained MD simulations of both sterols are summarized in Table\u00a05. The corresponding simulated 2H-NMR spectrum of cholesterol is shown in Fig.\u00a06, indicating the peak positions of all ring deuterons. This presentation can be helpful in the future for assigning the signals of fully deuterated cholesterol derivatives. Overall, the theoretical and experimental values agree very well within 1\u00a0kHz (except for 2H6). The only noteworthy deviation is seen for 2H7a, where the calculated splitting of methylpregnenediol differs by 3.2\u00a0kHz from the experiment. Overall, the simulated cholesterol quadrupolar couplings agree slightly better with the experimental values than the respective methylpregnenediol values. This finding is also reflected in the lower mean temperature reached in the simulation of cholesterol (323\u00a0K) than for methylpregnenediol (336\u00a0K). The higher mean temperature of the latter is probably also responsible for the slight differences between the calculated splittings of the two molecules in some of the other sites, too (see Table\u00a05).Fig.\u00a06Calculated 2H-NMR spectrum from the MD simulation of deuterated cholesterol in an oriented membrane, with the membrane normal parallel to B0. Pseudo-forces were applied in the MD simulation to a sub-set of deuterons for which experimental data was available (see Fig.\u00a05). The peak positions of all other deuterons connected to the ring skeleton are also predicted and indicated (see Table\u00a05). The intensity of the CD3-signals has to be multiplied by a factor of three\nThe constrained MD simulation showed that both cholesterol derivatives essentially assume an upright orientation in the membrane and perform rotations mainly around their long molecular a-axis of inertia. This behaviour is reflected in the molecular Saupe order tensor (see Table\u00a06), whose values are not far from the ideal axially symmetric case of Eq.\u00a016 with a low biaxiality of \u03be\u00a0=\u00a00.01. Previous calculations of the order tensor from the NMR data (Marsan et\u00a0al. 1999) had given similar results as the present MD simulation. We find that the preferred orientation of the long a-axis of the molecule displays a small mean tilt angle \u0398 a of 13\u00b0 with respect to the membrane normal. A similar result was obtained by Marsan et\u00a0al. (1999), who reported a tilt of the long molecular axis (as defined in the paper of Dufourc et\u00a0al. 1984) with respect to the membrane normal of 12\u00b0 (provided that in their RMSD data analysis the restriction to a rotationally symmetric order tensor was dropped).Table\u00a06Molecular Saupe order tensor derived from the constrained MD simulation, compared with the values derived from the previous RMSD analysis (Marsan et\u00a0al. 1999)Tensor componentMD simulation of methylpregnenediolMD simulation of cholesterolStatic RMSD analysisSaa0.870.880.94Sbb\u22120.44\u22120.44\u22120.48Scc\u22120.43\u22120.44\u22120.46Sab0.0\u22120.11Sac0.0\u22120.12Sbc0.00.0\nWe note that the axially symmetric averaging of cholesterol does not imply that its motion resembles a smooth rotation about a fixed axis. In the MD simulation we observed frequent jumps of the long axis by 20 to 30\u00b0, accompanied by a change of the conformation of the aliphatic tail of the molecule. This behaviour can be examined more closely by inspecting the trajectory of the angle \u0398 a between the a-axis of inertia and the membrane normal (z-axis, see Fig.\u00a07). Under the influence of the full pseudo-forces after 150\u00a0ps, the long axis of cholesterol frequently jumps off the most preferred orientation of \u0398 a\u00a0=\u00a04\u00b0. As a consequence, the mean orientation and the most frequent orientation do not coincide. Methylpregnenediol behaves clearly different in the MD simulation, since it has only a short aliphatic tail that does not give rise to conformational changes.Fig.\u00a07Trajectory of the time development of the angle \u0398a between the a-axis of inertia and the z-direction (membrane normal). The axis frequently jumps off its most preferred orientation due to conformational changes in the aliphatic tail of the molecule, hence the mean tilt (13\u00b0) and the most preferred tilt (4\u00b0) do not coincide\nPGLa\nIn our third case study, the new MD approach was applied to a membrane bound peptide. PGLa is a 21-residue cationic peptide (GMASKAGAIAGKIAKVALKAL-NH2) from the magainin family of antibiotics present in frog skin, which folds into an amphiphilic \u03b1-helix when bound to lipid bilayers. The mechanism of antimicrobial activity has been addressed in numerous studies and is attributed to the perturbation of bacterial membranes. Solid state NMR has yielded much insight into its structure and dynamic behaviour in model membranes (Bechinger 1999). For our MD simulations we used the constraints from the 2H-NMR investigations of Strandberg et\u00a0al. (2005), who had labelled four native alanine residues (positions 6, 8, 10 and 14) and two isoleucines (positions 9 and 13) one by one with 2H3-alanine. At a peptide-to-lipid ratio of 1:200 in DMPC, it was found from the quadrupolar splittings of these six CD3-groups that the \u03b1-helical PGLa is aligned flat in the plane of the membrane, and the peptide undergoes fast rotational diffusion about the membrane normal at 35\u00b0C. From the same data we now derived the full quadrupolar coupling tensors for the six 2H-labelled sites, including the sign of the tensor elements, and used these as constraints for the MD simulations. For the quadrupolar coupling tensors we used the parameters of the CD3-group (Table\u00a01), both for the native Ala substitutions as well as for the nominal constraint along the C\u03b1\u2013C\u03b2 segment of isoleucine.\nIt is known from previous NMR studies (Strandberg et\u00a0al. 2005) that membrane-bound PGLa forms an \u03b1-helix in the range of the labelled stretch, and further evidence for an \u03b1-helical conformation between residues 6 and 21 is provided by (Bechinger 1999). We therefore started the MD simulations with an idealized \u03b1-helix, and for all backbone hydrogen bonds we introduced 18 additional distance constraints of 1.86\u00a0\u00c5 to keep the molecular model helical. At the end of the simulation, the RMSD of the backbone hydrogen bond lengths from their ideal values was only 0.2\u00a0\u00c5, thus confirming that that the molecule stayed indeed mostly helical during the MD simulation. It also had to be taken into account that the four lysine side chains and N-terminus are positively charged. Since charged NH3+ groups have a strong tendency to form hydrogen bonds, we added a water molecule near each hydrogen atom of a charged group. This way any undesired hydrogen bonds of lysines to the backbone could be prevented, and indeed at the end of the simulation all 15 water molecules were still in contact with the peptide.\nCompared to the two small-molecule case studies above, the NMR data on PGLa provide only few constraints on the large and rather flexible molecule, as the pseudo-forces act only on six amino acids out of 21. Therefore, unrealistically large-pseudo forces at the start of the MD simulation have to be avoided. As a first measure, the pseudo-forces were increased exponentially to their final values during the MD simulation with a time constant of \u03c1\u00a0=\u00a0100\u00a0ps, like in the cholesterol simulation. From the NMR studies it was known that PGLa rotates rapidly (on the NMR time scale) about the membrane normal. This motion opens up a second possibility to scale the pseudo-forces properly. We calculated rotational averages of the quadrupolar tensors in every step of the MD. Only three tensor values rotated by 120\u00b0 around the director axis (in this case the membrane normal oriented parallel to the B0 field directions) are necessary to obtain a mean tensor within the limits of a fast rotation. All off-diagonal values of the simulated tensors thus become zero and only the principal values of the tensors are left as constraints. The mean temperature at the end of the simulation was only 11\u00a0K above the target temperature of 293\u00a0K, indicating that exceedingly high pseudo-forces have been successfully avoided indeed, which would otherwise have lead to additional heat production.\nAs in the other two case studies above, the constraint-driven MD simulations were able to reproduce the experimental 2H-NMR data correctly, as evident from the comparison in Table\u00a07. All calculated frequencies are well within the experimental error margins (as estimated from the experimental line widths). For some additional sites the quadrupolar frequencies were predicted from the MD simulation, and it would be of interest whether future measurements can confirm these values.Table\u00a07Calculated deuterium quadrupolar splittings from a constrained MD simulation of PGLa, compared with the experimental 2H-NMR data (Strandberg et\u00a0al. 2005) in DMPCSiteMD (kHz)NMR (kHz)Ala3\u221222.2Ala6+15.7+15.6Ala8+17.1+17.2Ala10\u221215.0\u221215.0Ala14\u221226.8\u221226.6Ala17+17.1Ala20\u221225.5Ile9\u22124.9\u22125.2Ile13+26.226.4\nThe major aim of the present NMR data analysis was the determination of the time- and ensemble averaged alignment of the helical PGLa backbone in the membrane environment. As illustrated in Fig.\u00a08, the principal axes of inertia offer a suitable molecule-fixed frame of reference to describe the peptide orientation. In the PGLa the a-axis of least inertia points along the helix, and the c-axis of highest inertia is located between the hydrophilic lysine-rich face and the hydrophobic face of the molecule formed by alanines, valines, leucines and isoleucines. Intuitively, we would expect the helix to be in contact with biomembranes just like it is displayed in this view, namely with the intermediate b-axis collinear with the membrane normal.Fig.\u00a08View along the long axis of PGLa modelled as an idealized \u03b1-helix with only the lysine, alanine and isoleucine side chains displayed. The latter two types were labelled by 2H3-alanine, and their 2H-NMR splittings were included into the MD simulation. The principal a-axis of inertia, points along the helix, and the b-axis of inertia points radially through C\u03b1 of lysine 12\nThe outcome of the MD simulation is illustrated most appropriately in Fig.\u00a09, which visualizes the alignment and dynamic behaviour of PGLa in DMPC. Here, the peptide orientations during the MD simulation are presented as a scatter plot of the inertia axes a, b and c on the surface of a unit sphere. A narrow range of orientations is seen for the a-axis representing the peptide helix. In contrast, the broad scatter for b and c indicates that a high degree of motional freedom exists for motions around the helix axis. The time-averaged tilt angle of the a-axis with respect to the membrane normal in found to be 99\u00b0, with a mean deviation of about \u00b14\u00b0. This value is in very good agreement with the tilt angle \u03c4 \u2248 98\u00b0 of PGLa that had been deduced from an RMSD plot of the 2H-NMR by Strandberg et\u00a0al. (2005) assuming a rigid idealized \u03b1-helix. We note, however, that such helix axis definition is not exactly comparable to the a-axis of inertia used in our MD calculations. The authors further described the azimuthal alignment of the amphiphilic peptide by defining an angle \u03c1 between an axis pointing from the centre of the helix radially through the C\u03b1-atom of lysine 12 and the membrane plane. In our case, the b-axis of inertia also happens to be directed to C\u03b1 of lysine 12 (see Fig.\u00a08), but the centre of inertia does not exactly coincide with the helix axis. As can be seen from Fig.\u00a010, the angle \u0398 b between the b-axis and the membrane normal oscillates between 10\u00b0 and 70\u00b0, with a mean value of 33\u00b0. This average value corresponds to an angle \u03c1 of approximately 123\u00b0, which compares well with \u03c1 \u2248 115\u00b0 extracted from the previous analysis (Strandberg et\u00a0al. 2005). In this comparison we have to keep in mind that in our simulations the PGLa molecule was found to be far from rigid and possesses a high degree of mobility around the helix long axis. The lysine side chains are found to spread out much more than in the initial model, and the PGLa helix itself exhibits a small bend.Fig.\u00a09Visualization of the alignment and motional behaviour of the \u03b1-helical peptide PGLa (only lysine side chains shown) in a DMPC membrane, as determined by a 1\u00a0ns MD simulation using experimental 2H-NMR constraints (Strandberg et\u00a0al. 2005) The instantaneous orientation of the three axes of inertia a, b and c is displayed as a scatter plot on a sphere, representing snapshots of these axes orientations every picosecond. The poles of the sphere are oriented parallel to the membrane normal. The helix long axis displays only a small scatter with a mean tilt angle of 99\u00b0, while the two other axes oscillate about this direction producing a large scatterFig.\u00a010Molecular dynamics trajectory of the angle \u0398b between the intermediate b-axis of inertia and the membrane normal\nSimilarly important as the alignment of the peptide are its dynamic properties, which also enter into the observed 2H-NMR data. Table\u00a08 lists the components of the molecular Saupe tensor, which is again used to describe the average alignment a molecule fixed frame (i.e. the principal axes of inertia) with respect to the laboratory frame. However, in contrast to the two previous case studies, this Saupe tensor does not represents the fast reorientation of the molecule around the membrane normal. No pseudo-forces were driving this motion in the simulation, since a rotational mean value was introduced. Here, the order tensor represents only the wobble of the long a-axis and the pronounced oscillations about this molecular axis (see Fig.\u00a010). As expected from the distribution of polar and hydrophobic residues on the different faces of the helix, it is seen that the Saupe tensor is not diagonal Table\u00a08). The biaxiality of \u03be\u00a0=\u00a00.09 (S\u00a0=\u00a00.76, see Eq.\u00a015) is clear evidence that the molecule does not behave like a rod-shaped object, and rotation about its long axis is restricted. In fact, oscillations of about 60\u00b0 around the long axis, but no complete rotation events were observed in the MD simulation (see Fig.\u00a010). These oscillations start after about 100\u00a0ps when the pseudo-forces approach their full strength.Table\u00a08Molecular Saupe order tensor from a constrained MD simulation of PGLaTensor componentMDSaa+0.49Sbb\u22120.28Scc\u22120.46Sab\u22120.46Sac+0.06Sbc\u22120.08\nAt a first glance the rather regular oscillations of PGLa in Fig.\u00a010, or the rotations of cholesterol (Fig.\u00a07), may not seem to reflect reality. However, we have to keep in mind that our MD simulations are performed with single molecules in vacuum, and the temperature is controlled by a continuously acting NTV \u201cthermostat\u201d adjustment. In nature, heat is transferred by stochastic interactions with other molecules, thereby introducing a stochastic behaviour of molecular rotations and re-orientations. This stochastic aspect is not present in our MD simulations, but nonetheless the amplitudes, velocities and directions of the motions adopt realistic values as the NMR constraints have to be satisfied. The molecule in the simulation cannot stop in one preferred orientation, because then no averaging would be performed anymore.\nConclusions\nSolid state NMR is a valuable technique to gain insight into the behaviour of peptides and proteins in oriented media, provided the data can be interpreted in terms of molecular structure and dynamics. In this contribution we developed a new strategy in which all-atom MD simulations and NMR data obtained from oriented samples are combined to obtain such structural and motional information. To this aim, a molecular mechanics force field (in this case COSMOS-NMR) was extended to include pseudo-forces, which drive the molecular dynamics to meet the NMR constraints. They \u201cheat up\u201d molecular rotations or re-orientations, leading to proper averaging of the calculated tensor values such that the calculated tensor values agree with the corresponding experimental observations. The orientational constraints can be further combined with intramolecular constraints such as distances or chemical shifts. This way, similar results can be obtained as in full membrane MD simulations, but without the computational burden of having to perform a detailed simulation of the lipids and surrounding water molecules. Because they are performed in vacuum, the constrained MD simulations can be completed in relatively short simulation times (\u22641\u00a0ns), still reaching a complete averaging of the NMR observables.\nIn three case studies, ranging from a small rigid compound to a 21-residue membrane-active peptide, the MD simulations with orientational NMR constraints succeeded to produce a detailed picture of the molecular motions and orientations in oriented membranes. It could be demonstrated that this new method is not limited to rigid molecules and does not depend on the choice of the initial coordinates. Deuterium quadrupolar splittings from 2H-labelled pyrene, cholesterol and PGLa in oriented lipid bilayers have been used as constraints in the present examples, but the general formalism presented will be applicable to all kinds of tensorial NMR properties.","keyphrases":["molecular dynamics simulations","pyrene","cholesterol","order parameters","2h-nmr","oriented samples","orientational nmr constraints","pgla peptide","force field calculations"],"prmu":["P","P","P","P","P","P","P","R","R"]}
{"id":"Eur_J_Pediatr-3-1-1914268","title":"Long-term cognitive and behavioral consequences of neonatal encephalopathy following perinatal asphyxia: a review\n","text":"Neonatal encephalopathy (NE) following perinatal asphyxia (PA) is considered an important cause of later neurodevelopmental impairment in infants born at term. This review discusses long-term consequences for general cognitive functioning, educational achievement, neuropsychological functioning and behavior. In all areas reviewed, the outcome of children with mild NE is consistently positive and the outcome of children with severe NE consistently negative. However, children with moderate NE form a more heterogeneous group with respect to outcome. On average, intelligence scores are below those of children with mild NE and age-matched peers, but within the normal range. With respect to educational achievement, difficulties have been found in the domains reading, spelling and arithmetic\/mathematics. So far, studies of neuropsychological functioning have yielded ambiguous results in children with moderate NE. A few studies suggest elevated rates of hyperactivity in children with moderate NE and autism in children with moderate and severe NE. Conclusion: Behavioral monitoring is required for all children with NE. In addition, systematic, detailed neuropsychological examination is needed especially for children with moderate NE.\nIntroduction\nIt has been widely accepted that severe NE following PA can lead to serious motor disabilities, mental retardation and seizure disorders [59]. Traditionally, research in this area has focused on developmental consequences at a very young age. In addition, the effects of NE have often been treated as an all-or-nothing phenomenon; patients have been believed to show a completely normal development or become severely disabled. So far, few studies have looked at milder consequences of NE at school age.\nFrom perinatal asphyxia to neonatal encephalopathy\nPA can be defined as impaired respiratory gas exchange accompanied by the development of acidosis [30]. The pathway of PA can have its onset in the antepartum, intrapartum or postpartum period. Estimates of the incidence of PA vary from 1 to 8 per 1,000 live births [4]. This wide range can be largely attributed to problems in selecting indicators to identify children with PA. As a consequence, researchers have used many different sets of inclusion criteria when studying the effects of PA. For this reason, it is difficult to compare results regarding developmental outcome of infants with PA across studies.\nIn the past, single-marker definitions were commonly used to diagnose infants with PA (e.g., low Apgar score). However, these single markers have relatively low sensitivity and specificity for accurate diagnosis [54]. Moreover, they lack predictive power [3, 20, 35\u201337, 40, 51]. Therefore, a shift has been made towards multiple-marker definitions based on combinations of indicators [16, 35]. Among the perinatal indicators are fetal distress (abnormal heart rate or meconium-stained amniotic fluid), delay in onset of spontaneous respiration, low Apgar score (<6 or 7 at 5\u00a0min), need for resuscitation and\/or ventilation, and metabolic acidosis (cord pH\u2009<7.0 or 7.1 and base deficit >12\u00a0mmol\/l). Postnatal indicators of PA include NE, multi-organ failure and abnormal findings on brain imaging. Of the above, the concept of NE is generally accepted as an essential criterion for the diagnosis of PA [4, 30]. NE has been defined as a clinical syndrome of disturbed neurological function in the earliest days of life in term infants, manifested by difficulty with initiating and maintaining respiration, depression of tone and reflexes, subnormal level of consciousness and often by seizures [30].\nRecent studies of the protective effects of head cooling and systemic hypothermia after NE following PA used a combination of clinical and physiological criteria of PA (including a pH of 7.0 or less or a base deficit of 16\u00a0mmol per liter or more) and a neurological examination to determine the severity of NE [22, 52]. These inclusion criteria are closely related to a set of criteria postulated by The American College of Obstetricians and Gynecologists (ACOG). There has been extensive debate about the relationships between PA, NE and serious possible consequences such as CP [25]. Contrary to what was once believed, only a small part of all cases of CP are caused by intrapartum events [10]. Likewise, many cases of NE are not associated with a history of PA [6, 38]. However, NE has to be present in the pathway from PA to subsequent CP [25]. The ACOG and the International Cerebral Palsy Task Force proposed a set of criteria to define an acute intrapartum hypoxic event that could subsequently lead to CP [4, 30]. The presence of moderate or severe NE and metabolic acidosis (pH\u2009<7 and base deficit \u226512\u00a0mmol\/l) are considered to be essential markers for hypoxic ischemic injury. In addition, these criteria underline the importance of excluding other identifiable causes of NE before assuming asphyxia as the underlying etiology.\nBoth in clinical practice and for scientific purposes, patients suffering from NE are often assigned an encephalopathy score. Several classification systems can be identified. Leviton and Nelson provide an overview of qualitative and quantitative grading schemes [27]. In addition, scoring systems were developed that are applicable in low-income settings. Moreover, a new grading system has gained interest in the literature [19, 33]. The classification system of Sarnat and Sarnat developed in 1976 [50] is still widely used and the basis for most modern classification systems. In this review, the Sarnat system is used to grade NE because in most studies that were included this system it had been applied. According to the Sarnat system children are assigned a score of 1, 2 or 3 (1= mild NE; 2= moderate NE; 3= severe NE). Stage 1 lasts less than 24\u00a0h and is characterized by hyperalertness, uninhibited Moro and stretch reflexes, sympathetic effects and a normal EEG. Stage 2 is marked by obtundation, hypotonia, strong distal flexion and multifocal seizures. Infants with the most severe stage of NE (stage 3) are stuporous and flaccid, and brain stem and autonomic functions are suppressed. In addition, the EEG shows abnormal patterns of brain activity.\nGrades of neonatal encephalopathy in relation to developmental outcome\nEncephalopathy scores have often been used to predict neurodevelopmental outcome [43, 44, 46]. Although strong relationships exist between the categories of NE and developmental outcome, diversity of outcome is reported within individual grades [41]. This diversity may be partly explained by individual researchers using different neurological signs for category inclusion [27]. The Sarnat grading system has helped to clarify the NE categories and improved their usefulness in studies of prognosis [45].\nMost outcome studies have focused on neurological functioning and severe deficits in young children (<4\u00a0years). In general, very few children with mild encephalopathy show neurological impairments or have developed severe mental or motor retardation at preschool age. In contrast, children with severe encephalopathy nearly always die or develop severe impairments such as CP, mental retardation, epilepsy and in some cases sensorineural hearing loss or cortical visual impairment [4, 45, 47]. While dyskinetic CP or quadriplegia is more commonly seen following \u201cacute near total type of asphyxia\u201d with MRI abnormalities present in the deep grey nuclei and in the perirolandic cortex, milder forms of CP, such as spastic diplegia, are more common among those with \u201csubacute partial asphyxia\u201d with MRI abnormalities in the watershed areas. Hemiplegia can be seen following \u201cperinatal arterial stroke.\u201d Although this condition will present with NE and seizures, it is less commonly associated with PA [13, 34]. Children who have suffered moderate encephalopathy seem to form a more heterogeneous group; research shows varying rates of infant death and morbidity [16].\nNeed for long-term follow-up and focus on cognitive and behavioral outcomes\nBecause the majority of studies have focused on detecting major developmental abnormalities at a very young age, still little is known about the milder difficulties children may experience in the long run. Recently, however, research has started to focus more on the subtle problems of children without major neurological deficits [8, 23, 32, 35, 43, 44, 46]. Knowledge of the outcome for this group of children is important because these subtle problems can affect later scholastic achievement and behavioral adjustment.\nCognitive and behavioral difficulties can be expected because of the patterns of brain injury that have been associated with NE. The hippocampus and striatum are among the brain structures that can be affected [7, 21, 31, 42, 56]. These structures have been associated with specific cognitive functions such as memory and attention and hypothesized to play a role in the pathogenesis of ADHD, autism and schizophrenia [14\u201316, 28, 57].\nTo gain insight into the long-term effects of NE on development, follow-up is required throughout the school-age period. Specific cognitive functions continue to develop throughout childhood. As it is impossible to examine a function that has not developed yet, short-term follow-up cannot exclude minor cognitive and behavioral difficulties at school age. Moreover, subtle functional deficits usually do not become apparent until a child faces increasing demands to master complex abilities in school.\nThe aim of this review was to discuss the results of previously published studies on the possible effects of NE on general cognitive functioning, educational achievement, neuropsychological functioning and behavior from infancy through adolescence. A literature search was performed using Web of Science. Studies were screened that had been published before October 2006. Search terms were \u2019asphyxia neonatorum,\u2019 \u2018neonatal encephalopathy,\u2019 \u2018newborn encephalopathy,\u2019 \u2018hypoxic-ischemic encephalopathy\u2019 and \u2018perinatal asphyxia\u2019 combined with the terms \u2018cognition\/cognitive\u2019 and \u2018behavior(-al)\u2019. Reference lists of retrieved articles were screened for additional relevant publications. Studies were selected that reported the developmental outcome of NE or HIE following PA of children until the age of 18\u00a0years. The presence of NE was required for selection. When no additional indicators of PA were used, the asphyxial etiology of the NE had to be assumed by the authors. Studies were excluded when NE was partially caused by other conditions than PA, for example [17, 34]. Also, studies were excluded in which outcome was presented in preset categories (e.g., normal versus abnormal), in which several outcome measures were combined, for example [11, 18, 26]. In these cases it was not possible to judge whether a child had been assigned to the category \u2018abnormal\u2019 because of cognitive or behavioral problems or because of other problems, e.g., motor difficulties. A third reason for excluding studies from this review was the selection of children from a larger group of eligible patients. For example, some researchers selected only those children with abnormal findings on MRI in order to examine the correlation with outcome [61]. In this review, outcome is reported according to grade of NE (mild, moderate or severe). Most authors of the included studies used the classification system of Sarnat when determining the severity of the NE. However, in three cases the authors significantly deviated from this system [32, 47, 53]. In order to increase comparability across studies, the patients in these studies were reclassified according to Sarnat criteria. Articles were reviewed that reported general intellectual ability, levels of specific cognitive functions and\/or behavioral problems. Details of the samples, inclusion criteria, outcome measures and results are described in Tables\u00a01, 2, 3, 4, 5, 6, 7 and 8. \nTable\u00a01Griffiths Mental Development Scale* at 1\u00a0year\u00a0CPNo CPCarli et al. (2004) [12]Moderate NE, N=13Moderate NE, N=27\u2022 N=10: <\u22122 SD\u2022 N=4: between \u22122 SD and \u22121 SD\u2022 N=1: between \u22122 SD and \u22121 SD\u2022 N=23: >\u22121 SD\u2022 N=1: >\u22121 SD\u2022 N=1: untestableGray et al. (1993) [24]Moderate NE, N=10Moderate NE, N=9\u2022 N=9: <55Mean =118 (N=1 not assessed)\u2022 N=1: 87Thompson et al. (1997) [55]No grades of NE recorded, N=16No grades of NE recorded, N=24Mean =47, range: 7\u2013101Mean =16, range 94\u2013128Barnett et al. (2004) [9]No grades of NE recorded, N=59Mean =100.34, SD=15.05, range: 55\u2013130Rutherford et al. (1996) [49]Moderate NE, N=8Mild NE, N=4All below normal (cut-off point not reported)All normalSevere NE, N=1Below normal*Griffiths mean of standardization sample =100.18, SD=12.76Table\u00a02IQ at 2\u20136\u00a0years (no CP)\u00a0Age (years)Mild NEModerate NESevere NEBarnett et al. (2004)b [9]2N=45,f Griffiths: mean =103.53, SD=12.33 (range: 81\u2013140)Robertson & Finer (1985)a [43] 3.5N=66, Stanford-Binet: mean IQ =101.5, SD=14.0N=94 (21.3% handicapped) Stanford-Binet2: mean IQ =92.3, SD=23.2N=7, Stanford-Binet2: mean IQ =37.1, SD=26.7Shankaran (1991)cb [53]5N=14,f McCarthy: 64%: >\u22121 SD, 86%: >\u22122 SDBarnett et al. (2002)bd [8] 5.5\u20136.5N=20, WPPSI-R: mean IQ =109.7, SD=14.6 (N=1: 76, rest: >90)N=12, WPPSI-R: (N=1: untestable) N=11: mean IQ =106.2, SD=11.8 (N=1: 84, rest: >94)Barnett et al. (2004)b [9]5\u20136N=53,f WPPSI-R 85%: >\u22121 SD, mean IQ =101.98, SD=16.06, range: 69\u2013139Robertson & Finer (1988)e [44]5.5 N=56, Stanford-Binet: mean IQ =106, SD=12N=71, Stanford-Binet: mean IQ =99, SD=18)Griffiths Mental Developmental Scales: mean of standardization sample =100.18, SD=12.76; Stanford-Binet Intelligence Scales: mean of standardization sample =100, SD=16; McCarthy Scales of Children\u2019s Abilities: mean of standardization sample =100, SD=16; Wechsler Preschool and Primary Scale of Intelligence-revised: mean of standardization sample =100, SD=15. aAll groups are significantly different from the other groups; p=0.001. bNo significance tests performed. cPatients were reclassified by author (MH) according to the classification system of Sarnat [50]. dIn the original article, results are presented for individual cases. Group means and standard deviations have been calculated by one of the authors (MH). eMild NE not significantly below comparison group; moderate NE significantly below mild NE and comparison group. fCombined group of mild, moderate and severe NETable\u00a03IQ at 7\u20139\u00a0years (no CP)\u00a0Age (years)Mild NEModerate NESevere NEMarlow (2005)b [32]7N=65, BAS-II:Group 1 (N=34): GCS=112.3, SD=11.2Group 2d (N=31): GCS=102.7, SD=13.2Robertson et al. (1989)a [46]8N=56, WISC-R: mean IQ =106, SD=13N=66, WISC-R: mean IQ =102, SD=175, WISC-R: mean IQ =36, SD=7Robertson (1997)c [47]9N=64, WISC-R: mean IQ =100, SD=14WISC-R: Wechsler Intelligence Scale for Children-revised: mean of standardization sample =100, SD=15; BAS-II: British Ability Scales, GCS: General Cognitive Score: mean of standardization sample =100, SD=15. aIQ of mild NE is not significantly lower than IQ of comparison group. IQ of moderate and severe NE is significantly lower than IQ of mild NE and comparison group (p\u2009<\u20090.001). bGSC of group 1 is not significantly lower than GSC of comparison group. GCS of group 2 is significantly lower than GCS of comparison group (p\u2009<\u20090.01). cMean IQ is significantly lower than IQ of comparison group. dMarlow refers to this group as \u2018severe NE.\u2019 This group is reclassified by one fo the authors (MH) according to the classification system of Sarnat [50]Table\u00a04Scholastic abilities at 7\u201313\u00a0years (no CP)\u00a0Mild NEModerate NESevere NEReadingRobertson & Finer (1989) [46]13% >1 grade level below expected level for age35% >1 grade level below expected level for agea100% >1 grade level below expected level for agebRobertson (1997) [47]41% >1 grade level below expected level for agedMarlow et al. (2005) [32]Group 1: < control groupcGroup 2f: < control groupdMoster et al. (2002) [35]NE < control groupdNE < control groupdNE < control groupdWritingMarlow et al. (2005) [32]Group 1: = control groupGroup 2f: < control groupeMoster et al. (2002) [35] NE = control groupNE = control groupNE = control groupSpellingRobertson & Finer (1989) [46]2% >1 grade level below expected level for age18% >1 grade level below expected level for agea100% >1 grade level below expected level for agebMarlow et al. (2005) [32]Group 1: < control groupcGroup 2f: < control groupdMoster et al. (2002) [35]NE = control groupNE = control groupNE = control groupMathRobertson & Finer (1989) [46]16% >1 grade level below expected level for age20% >1 grade level below expected level for agea100% >1 grade level below expected level for agebRobertson (1997) [47]39% >1 grade level below expected level for agedMarlow et al. (2005) [32]Group 1: \u2009=\u2009control groupGroup 2f: < control groupdMoster et al. (2002) [35]NE < control groupcNE < control groupcNE < control groupcRobertson & Finer (1989) [46]: age 8\u00a0years; Marlow et al. (2005) [32]: age 7\u00a0years; Moster et al. (2002) [35]: age 8\u201313\u00a0yearsaSignificant difference with mild NE and comparison group, p\u2009<\u20090.01; bstatistical analyses are not given because of small group and low scores; csignificant difference with comparison group, p\u2009<\u20090.05; dsignificant difference with comparison group, p\u2009<\u20090.001; esignificant difference with comparison group, p\u2009<\u20090.01; fMarlow refers to this group as \u2018severe NE\u2019. This group is reclassified by one of the authors (MH) according to the classification system of Sarnat [50]Table\u00a05Neuropsychological functions [43, 44, 46]Tests3.5\u00a0years5.5\u00a0years8\u00a0yearsReceptive vocabularyMild NE: in the mean rangeMild NE = control groupMild NE = control groupModerate NE < mild NEaModerate NE< mild NE+ control groupbModerate NE< mild NE+ control groupaSevere NE < mild + moderate NEaSevere NE: 100% < \u22121 SD cSevere NE < mild NE + control group cVisual- motor integrationMild NE: in the mean rangeMild NE = control groupMild NE = control groupModerate NE < mild NEaModerate NE < mild NE + control groupaModerate NE < mild NE + control groupa Severe NE < mild NE + control group cSevere NE < mild + moderate NEaSevere NE: 100% < \u22121 SD cReceptive vocabulary: Peabody Picture Vocabulary Test, visual-motor integration: Developmental Test of Visual-Motor Integration. ap\u2009<\u20090.001; bp\u2009<\u20090.01; cstatistical analyses are not given because of small group and low scoresTable\u00a06Neuropsychological functions (NEPSY): moderate NE compared to control group [32]Age: 7\u00a0yearsModerate NE group 1Moderate NE group 2*Attention and executiveGroup 1 = control group (p=0.08)Group 2 < control group (p\u2009<\u20090.01)LanguageGroup 1 < control group (p=0.01)Group 2 < control group (p\u2009<\u20090.01)SensorimotorGroup 1 < control group (p=0.04)Group 2 = control group (p=0.10)VisuospatialGroup 1 = control group (p=0.14)Group 2 < control group (p=0.02)Memory and learningGroup 1 = control group (p=0.61)Group 2 < control group (p\u2009<\u20090.01)*Marlow refers to this group as \u2018severe NE.\u2019 This group is reclassified by one of the authors (MH) according to the classification system of Sarnat [50]Table\u00a07Verbal memory (Rey\u2019s Auditory Verbal Learning Test)* [31]Age: 16\u00a0yearsModerate NETotal words learned trial 1\u20135NE: 52.5, SD=8.7Control group: 57, SD=5.8Delayed recall (p=0.034)NE: 11.6, SD=2.2Control group: 13.3, SD=1.6Recognition (p=0.011)NE: 14.2, SD=1.0Control group: 15, SD=0.0*Raw scores (number correct)Table\u00a08Verbal memory (Children\u2019s auditory verbal learning test)* [47]Age: 9\u00a0yearsModerate NELevel of learningaNE: 98, SD=16Control group: 104, SD=15Immediate recallaNE: 96, SD=18Control group: 105, SD=15Delayed recallaNE: 95, SD=15Control group: 103, SD=16Recognition accuracyNE: 22% delayedbControl group: 27% delayedTotal intrusionsNE: 19% delayedbControl group: 20% delayedImmediate attentionNE: 97, SD=15Control group: 100, SD=15*Mean of standardization sample =100, SD=15, ap\u2009<\u20090.01; bDelayed is below the 17th percentile of the standardization sample\nGeneral cognitive development\nInfancy (0\u201324 months)\nEight studies were identified that measured developmental levels between the ages of 12 and 24\u00a0months in children with NE [2, 9, 12, 24, 39, 49, 50, 55]. Two of these were performed in low-income, non-western countries [2, 55]. Standardized testing with the Griffiths Mental Development Scales showed that almost all children with moderate NE who had been diagnosed to have CP at the age of 12\u00a0months were severely developmentally delayed (Table\u00a01). In these studies, the rates of CP among children with moderate NE varied between 23% [24] and 82% [49]. Children with moderate NE without CP at this age performed in the average range or above, although the range of scores varies widely across studies. In addition, other studies in which the Denver Developmental Screening Test or the Bayley Scales of Infant Development were used reported normal development in children with mild NE, none of whom had CP.\nTwo to six years, without CP\nIn all of the selected studies [8, 9, 43, 44, 53], children with mild NE showed average general intellectual abilities that equaled those of a healthy comparison group (Table\u00a02). Although children with moderate NE also performed in the average range, their abilities were significantly below those of children with mild NE and a reference group. The general intellectual abilities of children with severe NE were assessed in only one study. These children showed very low levels of functioning: a mean IQ of 37.1 was found [43]. In two studies, no grades of NE were recorded [9, 53], which hampers interpretation of these data. Rates of average or above-average functioning were 85% and 64%, respectively.\nSeven to nine years, without CP\nAt school age, children with mild NE showed age-appropriate levels of general intellectual ability and performed as well as healthy comprison children on standardized testing [32, 46, 47] (Table\u00a03). Although most children with moderate NE obtained significantly lower scores on intelligence measures than children with mild NE and comparison groups, their ability levels were in the average range.\nEducational achievement\nRelative strengths and weaknesses in cognitive functioning can become visible when children have to acquire specific scholastic abilities in school. A test of school readiness showed that 5.5-year-old children with mild and moderate NE were not significantly behind compared to comparison children [24]. However, children with moderate NE achieved lower scores in all domains, and 42% of them scored more than 1 SD below the average of the standardization group. Children with severe NE showed low school readiness scores at the age of 5.5\u00a0years. Table\u00a04 gives an overview of the results of four studies, which assessed reading, writing, spelling and mathematics\/arithmetic in school-aged children [32, 35, 46, 47]. Survivors of mild NE compared to a comparison group were not significantly more often more than one grade behind in reading, spelling and arithmetic. In these domains, all children with severe NE were more than one grade behind the expected level for their age. The group with moderate NE appeared to have distinct difficulties in reading, spelling and arithmetic\/mathematics. Writing has been found to be less developed in only one subgroup of one of the studies.\nNeuropsychological functions\nStudies of specific neuropsychological functions yielded results that are similar to the findings on general intellectual ability and educational achievement (Tables\u00a05, 6, 7 and 8). Children who suffered from mild NE tended to show normal receptive vocabulary and visual-motor integration at 3.5, 5.5 and 8\u00a0years of age [43, 44, 46]. As is the case in other cognitive domains, the severe group was impaired, and their ability levels were below those of children with mild or moderate NE and comparison groups. The moderate group showed an ambiguous pattern of performance. Deficits were found in receptive vocabulary, language and visual-motor integration. In contrast, attention and executive functions, visuospatial ability, and memory and learning were normal in a subgroup of 7-year-old children [29]. Although memory and learning did not seem to be impaired in this subgroup, these children had difficulties in the sub-domains narrative memory and sentence repetition, compared to intact memory for faces, names, everyday events and intact orientation. In addition, auditory verbal learning and delayed recall was impaired in a group of 9- and a group of 16-year-old children with moderate NE [31, 47]. Tests of other auditory skills in the former showed intact auditory attention and concentration, language and binaural separation, in contrast to impaired binaural integration, and sequencing and labeling [47].\nBehavioral problems\nSo far, few researchers have addressed the issue of behavioral problems in children with a history of NE. Four studies were identified that met the selection criteria. In general, hyperactivity was more often present in children with moderate NE, but not in children with mild NE [32, 35, 44]. In addition, one study that used parent\u2019s observations of their child\u2019s behavior found more problems related to tractability, aggression, passivity and anxiety in a mixed group of children with NE compared to a control group [35]. No differences were found, however, related to attention, habituation and impulsivity. A recent study reported an unexpectedly high proportion of children with moderate and severe NE that developed an autism spectrum disorder [5]. However, the inclusion criteria differed considerably from those of Sarnat. Both groups can be classified as moderate NE when Sarnat\u2019s criteria are applied. In addition, the authors explicitly did not exclude etiologies of NE other than PA.\nDiscussion and conclusion\nIn the introduction section of this review, problems were mentioned with regard to the comparability of different outcome studies of NE. Even after careful selection on the basis of predetermined criteria, comparing studies remained difficult. The main reason for this was the way test results were presented. In many cases, outcome of the children was reported in two or more categories, e.g., normal\/mildly delayed\/abnormal. One of the problems that arise due to such categorization, is the subjectivity involved in determining the cut-off points of the categories. Often, these cut-off points seemed to be chosen arbitrarily or not justified at all. Another issue that adds to this problem is the use of different tests of development, cognitive ability or behavioral problems. One can hardly compare, for example, the proportions of children with developmental delay in two studies when these are represented by a score below 85 on the Bayley Scales of Infant Development in one of the studies and by a score below 70 on the Griffiths Mental Developmental Scales in the other. To enable readers to compare studies, results of significance testing between subgroups of children with NE and comparison groups were added to the tables whenever available.\nChildren with a history of NE who suffer from CP at the age of 12\u00a0months are often also severely developmentally delayed. Estimates vary between 77% [12] and 90% [24]. Of the children without CP, those with mild NE have an excellent prognosis. Their intelligence, educational achievement and neuropsychological functions are comparable to healthy peers at least until middle childhood.\nChildren with severe NE appear to be impaired in every cognitive domain. They are less intelligent and perform worse at school and at neuropsychological tests than both healthy controls and children with mild or moderate NE.\nAs was expected, the children with moderate NE seem to form the most heterogeneous group. In general, these children are significantly less intelligent than children with mild NE and healthy peers, but their scores are still in the average range. And although these children are equally ready to go to school as other children at the age of 5.5\u00a0years, when measured in middle childhood, they perform less well in the domains reading, spelling and mathematics. On the basis of these results, it is possible that children with moderate NE fall behind in school, but do not show clear deficits at standardized testing. Another issue is raised by the relatively broad range of performance of children with moderate NE. Calculating mean IQ scores masks individual differences, which are large in groups of children with moderate NE. Therefore, means do not describe this group well, and it would be more informative to look at the distribution of scores. The description of neuropsychological functions in this review is based on six studies, in which five different tests were used in five different samples of children with moderate NE. These studies do not provide sufficient and comparable data to draw reliable conclusions about patterns of neuropsychological strengths and difficulties. The heterogeneous nature of the group of children with moderate NE raises the question whether these children really form one group. Marlow et al. [32] divided a group of children with moderate NE according to the severity of the symptoms of the NE. The group with the least severe neonatal symptoms showed a few discrete neuropsychological difficulties, but was more intelligent and performed better in school than the group with the most severe neonatal symptoms. Marlow suggested a dose-response effect of hypoxia within this group with moderate NE. This idea of a continuum of casualty with regard to morbidity has already been proposed by Low [29]. He suggested a theory of a critical threshold of asphyxia beyond which brain damage occurs. Beyond this threshold, a spectrum of minor and major deficits would occur. So, although the classification system of Sarnat [50] appears to be very useful for the prediction of the outcome of children with mild or severe NE, in case of moderate NE this qualitative system lacks predictive power. Future research should therefore focus on developing a reliable, more gradual model of prediction.\nAnother issue regarding prediction of outcome after NE also mainly concerns moderately affected children. It is still unclear whether NE has a general effect on intellectual abilities or leads to specific patterns of cognitive strengths and difficulties. This review shows that systematic, detailed research of neuropsychological function is still scarce. A few case reports, which are not included in this review, have shown specific impairments in episodic memory (memory for events), with relatively intact semantic memory (memory for facts) [21, 58]. However, these findings, which were based on small numbers of children, do not justify generalization to all children with NE and should therefore be replicated in larger, well-defined samples.\nThe introduction section of this review referred to possible associations between brain areas that are vulnerable to perinatal hypoxia and psychopathology, such as ADHD and autism. Long-term behavioral outcome of survivors of NE has received very little attention compared to neurological outcome and general intellectual ability. Only a few studies looked at the behavioral consequences of NE. Those studies found elevated rates of hyperactivity and autism in children with moderate NE. Evidence of hyperactivity and impulsive behavior has also been found in animal studies of PA [1, 60]. These findings suggest that, in addition to cognitive impairment, children with NE could be at risk of developing behavioral problems. Because of the clinical implications this would have, future research should include behavioral monitoring of all children with NE.\nLong-term follow-up of children with NE has been advocated before, including the optimal ages of assessment of specific functions [48]. Recommendations include assessment of general cognitive and adaptive behavioral functioning at preschool age, and, in some cases, school achievement tests and a neuropsychological screening at school age. The authors of this review would like to go one step further in recommending standard screening for behavioral problems in all children with NE, regardless of their level of cognitive functioning. In addition, detailed neuropsychological assessment is recommended especially in all children with moderate NE.\nFrom this review it is concluded that general intellectual, educational and neuropsychological outcomes are consistently positive for children with mild NE and negative for severely affected children. However, children with moderate NE form a more heterogeneous group with respect to outcome. On average, intelligence scores are below those of children with mild NE and age-matched peers, but within the normal range. Difficulties have been found in the domains reading, spelling and arithmetic\/mathematics. So far, neuropsychological functioning of children with NE has received relatively little attention. The studies that were selected for this review have yielded ambiguous results in children with moderate NE. A few studies suggest elevated rates of hyperactivity in children with moderate NE and autism in children with moderate and severe NE. Therefore, behavioral monitoring is required for all children with NE. In addition, systematic, detailed neuropsychological examination is needed especially for children with moderate NE.","keyphrases":["neuropsychology","asphyxia neonatorum","behavioral problems","hypoxia, brain","hypoxia-ischemia, brain"],"prmu":["P","P","P","R","M"]}
{"id":"Bioprocess_Biosyst_Eng-4-1-2214824","title":"Feasibility of an in situ measurement device for bubble size and distribution\n","text":"The feasibility of in situ measurement device for bubble size and distribution was explored. A novel in situ probe measurement system, the EnviroCam\u2122, was developed. Where possible, this probe incorporated strengths, and minimized weaknesses of historical and currently available real-time measurement methods for bubbles. The system was based on a digital, high-speed, high resolution, modular camera system, attached to a stainless steel shroud, compatible with standard Ingold ports on fermenters. Still frames and\/or video were produced, capturing bubbles passing through the notch of the shroud. An LED light source was integral with the shroud. Bubbles were analyzed using customized commercially available image analysis software and standard statistical methods. Using this system, bubble sizes were measured as a function of various operating parameters (e.g., agitation rate, aeration rate) and as a function of media properties (e.g., viscosity, antifoam, cottonseed flour, and microbial\/animal cell broths) to demonstrate system performance and its limitations. For selected conditions, mean bubble size changes qualitatively compared favorably with published relationships. Current instrument measurement capabilities were limited primarily to clear solutions that did not contain large numbers of overlapping bubbles.\nIntroduction\nAccurate and representative bubble sizes and distributions are used to characterize biochemical processes containing gas-in-liquid dispersions, specifically processes for industrially important fermentation products. Quantification of bubble sizes and distributions during fermentation is important to establish mass transfer characteristics (based on gas\u2013liquid interfacial area) when oxygen transport to cells across gas\u2013liquid interfaces becomes a limiting factor. In these situations, there is a direct influence of bioreactor parameters that affect bubble size, such as agitation, on culture yields. Thus, measurements of bubble sizes and distributions are useful for biochemical process optimizations.\nDirect application of bubble measurements in fermentation is based on linking bubble size to operational parameters, such as agitation and\/or airflow rate, that influence gas\u2013liquid volumetric mass transfer coefficients [1]. Such measurements can confirm that selected agitation\/aeration conditions do not lead to impeller flooding and can quantify bubble distribution changes for (1) geometrically similar fermenters with scale up and (2) different fermenters of similar scale possessing different operating conditions, geometry, or bioreactor internals. Owing to the automated and fast (<2\u00a0min) nature of the Envirocam\u2122 bubble measurement and analysis, it is a potential control tool for maintaining agitation\/airflow rate set points to obtain desired bubble sizes and distributions for mass transfer, for gas hold up, or for minimizing bubble damage to cells. For example, utility costs for electricity to drive agitators or air compressors might be optimized further for large production scale fermenters.\nHistorical and currently available bubble measurement systems using photographic methods have been previously summarized, and the references cited within describe several applications of bubble analysis in clear solutions [2]. The accuracy, representation, and simplicity of bubble size measurements improve when these measurements are performed on-line and in situ rather than off-line using broth samples. A newly developed novel on-line and in situ bubble measurement device is described and evaluated.\nNovel in situ probe measurement system\nEnviroCam\u2122 probe\nThe EnviroCam\u2122 (Enviroptics; Colmar, PA) probe consisted of a hermetically sealed shroud constructed of 316\u00a0l stainless steel for wetted product-contact parts (Fig.\u00a01). A silicone o-ring was used for the Ingold-style tank port insertion. The camera module itself attached to this shroud, and thus it was readily relocatable to other vessels without disturbing process integrity. There were no cracks or crevices since the shroud was entirely welded and helium leak-tested. The overall length of shroud was set at 6\u00a0in. for consistency with insertion lengths of other commercially available, in situ probes (such as pH, dissolved oxygen, or optical density probes). This length avoided interfering with fermenter internals, such as larger diameter hydrofoil impellers, but extended far enough into the well-mixed fermenter zone. Since bubble sizes and distributions vary according to their distance from high shear impeller zones, measurements were taken at a constant insertion length from the fermenter sidewall. External shroud attachments were minimized to permit location in tight areas, in some cases near platform structural steel supports. The captive retaining nut was obtained from Mettler-Toledo (Ingold; Bedford, MA) to match existing Ingold ports.\nFig.\u00a01Environcam\u2122 shroud diagram\nVarious prototype shrouds and camera modules were developed to evaluate different hardware options. A high-strength sapphire window was integrated into the 316\u00a0l stainless steel shroud via a gold brazed joint rather than epoxy to provide improved robustness. The sapphire window was constructed at a 75\u00b0 angle to offset the 15\u00b0 angle of the fermenter\u2019s Ingold port. This angle resulted in the window being parallel to the vertical tank wall upon insertion. Owing to the low surface tension of sapphire, some small bubbles adhered to the window surface, particularly at the low agitation speeds used for animal cell cultivation. Using the 75\u00b0 vertical orientation, as well as raising agitation speeds, reduced but did not eliminate this accumulation.\nA ceramic disc backscreen provided a measured path length. Initially, light from a 150\u00a0Watt halogen lamp was reflected internally into the shroud backscreen via a high performance liquid light guide, but this arrangement did not result in uniform illumination. Next LEDs (red\u2013orange 640\u2013720\u00a0nm wavelength) were incorporated directly into the shroud backscreen for more uniform illumination, which produced bubble images with improved border definition. To minimize the impact of distortion in the depth and width of the measurement volume, a diffuser was installed in the backscreen. A calibrated reticle, consisting of two intrusion lines (180\u00b0 apart with a gap of 7.5\u00a0mm), emanating from the circumference and heading towards its center, became the standard for in situ calibration. This reticle was located on the sapphire window itself rather than the backscreen to avoid interferences in opaque media.\nWith a backscreen present on the shroud, a defined path length or notch was introduced. Prototype shrouds were constructed to change the notch size between 1\/4 and 1.0\u00a0in., but experiments were conducted with a 1\/2\u00a0in. notch unless otherwise noted. Due to the small width of the support (3\/8\u00a0in.), the orientation of the notch (i.e., up, down, sideways with the opening facing with or against agitator rotation) was deemed not to prevent larger bubbles from entering the measurement field nor influence measured bubble sizes or distributions. The externally located connector for the LED power supply provided confirmation of the internal notch position. Regardless of notch size, the bottom of the notch was located 1\u00a0in. from the insertion end of the shroud (Fig.\u00a01). As notch size decreased, (1) the number of bubbles viewed per frame decreased, resulting in less overlap for high bubble volume broths, and (2) the path length decreased, providing more light for opaque solutions. In contrast, larger notch gaps may be required for lower bubble volume broths, such as animal cell cultivations.\nEnviroCam\u2122 imaging system\nThe EnviroCam\u2122 imaging system is shown in Fig.\u00a02. One imaging system, containing the camera module (about tenfold higher cost than shroud), could be used with multiple shrouds. Two high performance, 1.3\u00a0megapixel, 8\u00a0bit, monochrome cameras were tested, each of which attached directly to the shroud: (1) a monochrome camera (PL-A741, Pixelink; Ottowa, ON), equipped with a CMOS image sensor which operated at a shutter speed of 1\/10,000\u00a0s (1\/10,000\u00a0s\u00a0=\u00a0100\u00a0\u03bcs selected), and (2) a monochrome camera (EC-1380, Prosilica; Burnaby, BC), equipped with a CCD image sensor (IXC-285 Exview, Sony; Tokyo, Japan) which operated at a shutter speed up to 1\/100,000\u00a0s (1\/25,000\u00a0s\u00a0=\u00a040\u00a0\u03bcs selected). Primary magnification of 2\u00d7 (resulting in a system optical magnification of 30\u00d7) was implemented using one additional screw-on lens. The faster shutter speed associated with the CCD camera required additional light, which was sufficiently provided by the backlit LED geometry of the shroud. A partially telecentric 55\u00a0mm lens (Computar; CBC, Commack, New York) was selected for the CCD camera which reduced the viewing angle, but achieved magnification errors of <1% while providing high resolution and contrast with low distortion. The CCD camera became the preferred configuration consistent with the designs of other currently available photographic systems [2].\nFig.\u00a02Environcam\u2122 system diagram\nThe number of pixels [horizontal (H)\u00a0\u00d7\u00a0vertical (V)] was altered using user-defined region of interest (ROI) controls. Higher pixel numbers increased resolution but reduced the number of frames per second (fps). For the selected CCD camera, the hardware was set at 20\u00a0fps using 1,024\u00a0\u00d7\u00a01,024\u00a0pixels with a minimum pixel size of 6.45\u00a0\u03bcm and a pixel depth of 8\u00a0bits (without additional magnification via screw-on lenses). This fps rate of 20 resulted in a time scale of about 25\u00a0s for the initial image scan of 500 frames, which was small relative to expected changes in bubble size characteristics during the time course of a typical fermentation. Bubble residence time through the measurement field (1\/2\u00a0in.\u00a0=\u00a012.5\u00a0mm path length) was quantitatively estimated to be about 1.3\u20133.3\u00a0ms near the impeller blade tip (impeller tip speed of 3.8\u20139.4\u00a0m\/s) and likely was up to an order of magnitude slower away from the impeller. Consequently, it was not necessary to increase the frame speed further above 20\u00a0fps, which corresponded to a characteristic measurement time of 50\u00a0ms\/frame. The bubble residence time in the 1\/2\u00a0in. notch measurement field was qualitatively determined to be 40\u201370\u00a0ms by comparing common features of subsequent frames. Thus, it was necessary to skip at least four to five frames to ensure bubbles were not counted more than once.\nFor the CCD camera, pixel size was set at 20\u00a0\u03bcm\/pixel. With the 2\u00d7 magnification added, and other geometric and positional factors considered, it was reduced to 7.5\u00a0\u03bcm\/pixel. A small annulus of the viewing range (5\u00a0pixels wide) was discarded to reduce grey scale variations near the edges. The measurement area was 81\u00a0mm2, and the measurement volume was 993\u00a0mm3 (5.07\u00a0mm diameter, 5.07\u00a0mm height). A number-based hold-up estimate of 50\u2013150\u00a0bubbles\/cm3 for a bubble size range of 0.5\u20131\u00a0mm was calculated [2]. Thus, for the calculated measurement volume of nearly 1.0\u00a0cm3 about 50\u2013150\u00a0bubbles were expected per frame image. The actual number of bubbles per image ranged up to 1,000 or more when larger numbers of smaller bubbles less than 0.5\u00a0mm were detected.\nIn comparison with these values selected above for the Environcam\u2122 system, prior researchers have used 4\u201330\u00a0\u03bcm\/pixel and 512\u00a0\u00d7\u00a0512\u00a0pixels for gas\/liquid and liquid\/liquid bubble\/drop dispersion measurements [3\u20136]. For cell morphology measurements, 21\u00a0\u03bcm\/pixel was typical [7], and 512\u00a0\u00d7\u00a0512\u00a0pixels was the most common for recent studies [2].\nThe remainder of the Environcam\u2122 hardware system is shown in Fig.\u00a02. The computer itself consisted of an Intel Pentium 4 class notebook PC (XP operating system) with 1\u00a0GB RAM (NEMA rating as required by the facility) and a graphic card minimum display resolution of 1,280\u00a0\u00d7\u00a01,024 (with 32\u00a0bit color). The video output was the Firewire\/IEEE 1394 interface, a universal interface that allowed direct connection of the camera to the PC laptop. A USB port was used for computer control of the external light source supplied by a Nema 4\u00d7 power supply, which was powered only when images were being acquired by the camera. The lens mount was a standard C-mount adapter used for camera installation to lenses and to standard microscopes. A CD\/DVD R\/W drive, USB drive, and\/or some other type of network interface was used for archiving images and\/or data files, preferably with at least 80\u00a0GB of storage.\nThe operating temperature range was limited to 0\u201350\u00b0C for the camera module; consequently, it was not attached to the shroud during vessel sterilization. The shroud LED-operating temperature ranged up to 80\u201390\u00b0C, but its non-operating temperature ranged up to 120\u00b0C. All other shroud components were steam-sterilizable, including the glass diffuser. Thus, the shroud could be sterilized with the vessel if the LEDs remained unpowered. A LED-power supply kill switch based on a bimetallic temperature sensor was installed with a trip value of 90\u00b0C and reset value of 60\u00b0C. When it was powered, typically intermittently for 10\u00a0s per frame measurement cycle or continuously for video stream, the LED was required to be submerged in liquid as a heat sink for adequate cooling. LED lifetime was 100,000\u00a0h assuming the non-operating temperature remained less than 120\u00b0C; raising it a few degrees above this level for effective sterilization might sacrifice some lifetime, however. Shrouds were heat-tested using a 15\u201330\u00a0s temperature ramp from ambient to 130\u00b0C, held for 1\u00a0h, then returned to ambient temperature. No significant degradation, as measured by pixel light output, was observed after 50\u201360 temperature cycles. In addition, an actual sterilization was conducted successfully with the shroud in a pilot scale fermenter (180\u00a0l volume, 122\u00b0C, 40\u00a0min hold time). Based on this performance, shrouds were expected to withstand about 100 sterilizations of 45\u201360\u00a0min hold times for about a 3-year life span, assuming a 2-week batch length.\nThe 1951 USAF resolution target was used to evaluate the pure video resolution of the computer monitor, which was influenced by the quality of the video graphics card. Resolution was measured at 32\u00a0line pairs\/mm, the reciprocal of which resulted in a resolution of 31\u00a0\u03bcm\/pixel, significantly higher than the camera resolution of 7.5\u00a0\u03bcm\/pixel. Thus, the accuracy of the image display did not diminish the accuracy of the photographs obtained.\nThe software platform was customized based on National Instruments\u2019 (Austen, TX) LabVIEW Graphical Development Environment (version 8.2). The system\u2019s main screen is shown in Fig.\u00a03. The image analysis software had the following key features:Measured frames were included in analysis until the number of desired objects to be measured (a user input) was attained. Specifically, for a measurement time of 25\u00a0s (corresponding to 20\u00a0fps) which generated 500 frames, typically every tenth frame was skipped (i.e., measured frames were taken every 0.5\u00a0s) and 50 frames were measured to identify at least 500 bubbles. This number was consistent with other currently available measurement systems described in the literature [2]. This approach also was similar to skipping every 25th frame when measuring floatation cell moving aggregate sizes [8].The background was subtracted from the original image, which avoided repeatedly counting bubbles lodged on the sapphire window and to remove fixed blemishes in the backscreen. (This approach is similar to subtracting a background image without cells from the original image [9]). The background image was calculated by averaging all 500 frames obtained during the measurement period so that moving objects were filtered out to obtain a clear composite background image. This approach was speedy (800\u00a0ms for 500 frames). In addition, the background image was readily reconstructable from stored images if necessary. A user-selectable alternative algorithm was developed to construct a background composed of the brightest pixel of each of the 500 frames for each position, assuming that no pixel should be any brighter than a background pixel. The time to construct the background using either of these methods was similar.Edge enhancement techniques, based on a contrast threshold, were applied to convert grey images to binary black and white images so that the outside perimeters (or diameters) of bubbles were readily identifiable via object recognition.The analyzed image output was prepared using the calibrated reticle (measurement shown in red in Fig.\u00a03) located on the shroud backscreen in the form of a binary mask. In addition, a blue reference grid of 1\u00a0\u00d7\u00a01\u00a0mm squares (Fig.\u00a03) was overlayed to permit the user to approximate bubble sizes on the computer screen.Depending on the application, bubbles of sizes greater than a designated cut-off (e.g., 2\u00a0mm in diameter), between 40 and this cut-off, and below 40\u00a0\u03bcm were identified. Both visual and numerical indications of the classification of objects were developed according to three tiers of predetermined rules:\nGreen circles represented the first level of filtering. Rules in this first tier were: single bubbles of sizes within the target measurement range, circularity cut-off based on a user-selected tolerance above 1.0 value, and discard of bubbles touching the border. The green circle was the best fit \u201ccircle\u201d so there were slight inaccuracies around some of the circumference if the bubbles were not uniformly round. Larger or smaller circular bubbles outside the target measurement range were excluded.Yellow outlines indicated the second level of filtering typically using similar rules as the first level but with more relaxed, user-defined cut-offs. Additional rules also were added in this second tier. Up to three, more or less circular bubbles, that were touching but still individually discernable, were included in the count. Specifically, the ratio cut-off of cluster (i.e., more than one adjacent bubble overlapping) area to the calculated equivalent area for completely separated bubbles of the cluster was based on a user-selected tolerance below 1.0. Bubbles in this category were further differentiable using additional capabilities included in the LabVIEW software.Red outlines comprised a third level that indicated bubbles located by the system that did not pass filtering levels 1 or 2.White outlines comprised a fourth level of classification. This fourth tier contained non-circular blobs and large irregularly shaped or greatly overlapping bubbles. The percentage of viewing area occupied by blobs was estimated and measured frames were omitted based on a user-selected cut-off (e.g., if greater than a target percentage of 75% of the measurement area was comprised of blobs, the frame was discounted).The relevant statistical quantities were calculated (e.g., arithmetic, geometric, and\/or Sauter mean diameters), and the appropriate histograms were displayed [2]. A customized SQL program extracted data into csv files (containing all raw data and analysis calculations). These csv files had a practical limit of including information about 65,000 bubbles, typically an amount greater than required for a single measurement cycle. Data was imported into an Excel template containing graphs and some statistical quantities as part of the measurement cycle (typically requiring 3\u00a0s to 2\u00a0min), and then into other statistical programs (e.g., Sigmaplot, LabView modules) for more advanced analysis should the user desire.The front panel image containing the configuration and user-adjustable controls was archived so that an identical set up could be reproduced in the future, if desired.Tiff files (still frames) and\/or \u201cavi\u201d (video stream) files were archived, but avi files were reconstructable from Tiff files to save storage space. A video playback rate of 5\u00a0fps appeared appropriate. The actual measurement frames utilized for analysis were saved separately for ease of review. Minimum storage requirements for these unanalyzed frames were estimated at about 50\u00a0mB for each 500 frame measurement cycle (versus 4.5\u00a0GB for analyzed frames) with the entire analysis reconstructable from saved data. Thus, the ability to retrieve images and recheck\/reanalyze results readily existed.For measurement frames, a review panel of the analysis sequence was created from archived files (Fig.\u00a04). The analysis progression panels were reviewed manually to confirm accuracy.\nTo obtain an image of acceptable contrast, the user varied the camera shutter speed (length of exposure to light), brightness level (luminescence of image on LCD monitor), aperture (amount of light reaching camera lens), and gain (amplification of signal strength). Several parameters in the software were adjustable to ensure optimal image analysis, and the values selected could be archived. As a first step, threshold was varied automatically by the software to determine the value at which the maximum number of bubbles was identified. Subsequently, various filtering strategies available in the software were examined. FFT (fast Fourier transform) was found to potentially improve accuracy for measurements in water, but it was not implemented for the present work. Localized thresholding [10] was found to improve accuracy for bubble measurements in broths, and it was incorporated. The largest inscribable diameter was taken to calculate the bubble diameter [11].\nFig.\u00a03Example main screen displayFig.\u00a04Analyzed composite for a bubble frame (180\u00a0l DI water with 0.001% P2000, agitation 100\u00a0rpm, air flowrate 300\u00a0rpm) showing a original raw camera image, b binary image output with edge enhancement, c binary mask prior to application to image, d image after binary mask, e analysis output image containing colored circles identifying objects according to the three tiers of rules, and f instrument screen including histogram\nKey features of the EnviroCam\u2122 gas bubble measurement system are summarized in Table\u00a01 in a format readily comparable to those assembled for prior and currently available optically based measurement systems [2].\nTable\u00a01Envirocam\u2122 characteristicsCamera and mode of attachmentMagnification\/calibrationIllumination\/shutter or frame speedImage measurement and analysis methodCCD monochrome camera (Prosilica) probe inserted into an in situ Ingold-fitting shroud; 32\u00a0line pairs\/mm30\u00d7\/internal reticleLEDs in back screen (back lit)\/20\u00a0fps; 1\/100,000\u00a0s (1\/25,000\u00a0=\u00a040\u00a0ms selected)National Instruments\u2019 (Austin, TX) LabVIEW Graphical Development Environment as basisMeasurement time per conditionNumber of objects per measurementMeasurement error\/size rangeNumber of images (pictures\/frames) per measurement5\u201325\u00a0s data acquisition; <2\u00a0min data analysis>500 (typically up to 10,000)<10% for monodisperse beads\/60\u20132,000\u00a0\u03bcm50 (20\u2013200\u00a0bubbles\/picture)\nBead calibration\nA calibration was conducted using beads of a known size distribution in applicable size ranges for bubbles. Calibration beads were measured individually by manually moving the bead past the notch or in a small volume mixed solution contained in a magnetically stirred beaker. Vortexing was minimized by reducing the stirring speed. Settling of the larger glass microsphere beads (e.g., specific gravity of 2.46\u00a0g\/cm3) was reduced by performing the measurements in a perfluoropolyether (1,800\u00a0Da, specific gravity of 1.88\u00a0g\/cm3, Fomblin 06\/6 MFY06\/6BB, Solvay Solexis; Bollate, Italy). In measurement frames, beads had a softer, lighter, less-contrasted outline when compared with bubbles, which slightly reduced the effectiveness of the optical imaging system without additional filters. Thus, measurement of beads was limited to sizes \u226560\u00a0\u03bcm, slightly higher than the lower limit for bubbles.\nFirst, monodisperse spherical polyethylene calibration beads (Baltec; Balzers, Lichtenstein), 0.125\u00a0\u00b1\u00a00.002\u00a0in. (1.2% rsd) or 3.175\u00a0mm in diameter, were measured to evaluate the higher end of the expected Environcam\u2122 measurement range. (Other reported options for calibration beads were expanded polystyrene beads with mean diameters of 5.47 and 3.1\u00a0mm and a density of 30\u00a0kg\/m3 [12], 1.5\u00a0mm ball bearings [13], and red spherical particles of diameters 109 and 644\u00a0\u03bcm [7].) Using the external halogen lamp light source and the CMOS camera system, a value of 3,416\u00a0\u03bcm (single measurement) was obtained which was 6.7% higher than the standard\u2019s value. Using the LED light source installed in the shroud backscreen and the CCD camera system, a value of 3,220\u00a0\u03bcm was obtained which was 3.9% lower than the standard\u2019s value.\nNext, high precision, NIST-tracable, glass micro-spheres (Whitehouse Scientific; Chester, UK) were selected in various monodisperse and polydisperse sizes. Distributions (in the format of count versus bin size) were fit to 4-parameter Sigmoidal or 4-parameter modified Gaussian distributions using Sigmaplot (Systat; San Jose, CA) (Table\u00a02). These distribution equations better fit the bead measurement data than distribution equations used for bubbles [14, 15]. Monodisperse bead sizes, ranging from 20 to 600\u00a0\u03bcm in increments of at least 15\u00a0\u03bcm (corresponding to the instrument resolution), were tested (Table\u00a03). Measured size values agreed reasonably well with the manufacturer\u2019s data values. Measured ranges for 90% of the bead size range typically enveloped the manufacturer\u2019s data, with differences most likely owing to the nature of the fitted distributions. The distribution of polydisperse beads over most of the Environcam\u2122 measurement range (50\u20132,000\u00a0\u03bcm) also was measured and compared with the analyzed manufacturer\u2019s distributions for three size ranges (Table\u00a04). Reasonable agreements in the distribution shape were obtained. The accuracy of these Environcam\u2122 measurements showed that differences in means were observed and standard deviations were lower. In contrast, other researchers using calibration beads for optically based instrument systems reported that means were identical to the standard\u2019s values but standard deviations were higher (manufacturer: 109\u00a0\u00b1\u00a05\u00a0\u03bcm, measured: 109\u00a0\u00b1\u00a010\u00a0\u03bcm; manufacturer: 644\u00a0\u00b1\u00a013\u00a0\u03bcm, measured: 644\u00a0\u00b1\u00a024.8\u00a0\u03bcm) [7].\nTable\u00a02Distributions used to fit calibration bead measurement data (from Sigma plot software)DistributionProbability density functionModified Gaussian 4-parameter (G)Y\u00a0=\u00a0Yo\u00a0+\u00a0a exp[\u22120.5(abs(X\u00a0\u2212\u00a0Xo)\/b)2]Sigmoidal 4-parameter (G)Y\u00a0=\u00a0Yo\u00a0+\u00a0a\/(1\u00a0+\u00a0exp[\u2212(X\u00a0\u2212\u00a0Xo)\/b])Table\u00a03Comparison of manufacturer size (Whitehouse Scientific, Chester, UK) with measured size range for monodisperse beadsManufacturer\u2019s size data (\u03bcm)Manufacturer\u2019s data for 90% of beads within given range (\u03bcm)EnviroCam\u2122 measured size (\u03bcm)EnviroCam\u2122 data for 90% of beads within given range (\u03bcm)22.81\u00a0\u00b1\u00a00.7821.46\u201324.23Not able to be measured38.38\u00a0\u00b1\u00a00.5436.5\u201339.6Not able to be measured59.63\u00a0\u00b1\u00a01.057.1\u201362.266.3\u00a0\u00b1\u00a00.05 (G)58.8\u00a0\u00b1\u00a00.03 (S)53.4\u201364.3 (S)83.43\u00a0\u00b1\u00a00.8779.7\u201387.582.5\u00a0\u00b1\u00a00.11 (G)74.9\u00a0\u00b1\u00a00.09 (S)64.2\u201385.2 (S)98.10\u00a0\u00b1\u00a02.894.4\u2013102.898.28\u00a0\u00b1\u00a00.82 (G)90.64\u00a0\u00b1\u00a00.27 (S)76.0\u2013105.4 (S)155.8\u00a0\u00b1\u00a01.5151.4\u2013163.1163.9\u00a0\u00b1\u00a00.21 (G)156.04\u00a0\u00b1\u00a00.2 (S)143.7\u2013167.4 (S)200.9\u00a0\u00b1\u00a01.9196\u2013206200.69\u00a0\u00b1\u00a00.49 (G)194.15\u00a0\u00b1\u00a00.07 (S)187.6\u2013200.4 (S)258.6\u00a0\u00b1\u00a05.9251.4\u2013265.6259.77\u00a0\u00b1\u00a00.16 (G)253.3\u00a0\u00b1\u00a00.07 (S)246\u2013261 (S)297.9\u00a0\u00b1\u00a03.9289.7\u2013309.3303.1\u00a0\u00b1\u00a00.05 (G)295\u00a0\u00b1\u00a00.07 (S)275.3\u2013316.3 (S)361.6\u00a0\u00b1\u00a09.9344\u2013376366.5\u00a0\u00b1\u00a00.3 (G)359.3\u00a0\u00b1\u00a00.86 (S)337.4\u2013380.8 (S)405.9\u00a0\u00b1\u00a08.7396\u2013419411.7\u00a0\u00b1\u00a00.07 (G)403.9\u00a0\u00b1\u00a00.2 (S)385.9\u2013420.2 (S)589.0\u00a0\u00b1\u00a06572\u2013615586.7\u00a0\u00b1\u00a00.4 (G)578\u00a0\u00b1\u00a00.09 (S)551.9\u2013606.6 (S)Envirocam\u2122 measurements fitted to modified Gaussian 4-parameter equation (G) and sigmoidal 4-parameter equations (S) using Sigmaplot software. All r2 values >0.99 unless otherwise noted. Beads of sizes 22 and 38\u00a0\u03bcm were not measurable owing to blurry edgesTable\u00a04Comparison of manufacturer size (Whitehouse Scientific, Chester, UK) distribution with measured size distribution for polydisperse beadsBead distribution (\u03bcm) Size at fixed percentiles (\u03bcm)1025507590StandardEnviroCam\u2122StandardEnviroCam\u2122StandardEnviroCam\u2122StandardEnviroCam\u2122StandardEnvirocam\u2122500\u20132,000796\u00a0\u00b1\u00a010744.5, 746.8936\u00a0\u00b1\u00a09849.3, 852.01,098\u00a0\u00b1\u00a0111,069.6, 1,074.01,335\u00a0\u00b1\u00a0321,232.0, 1,250.01,618\u00a0\u00b1\u00a0401,442.5, 1,508.0150\u2013650244\u00a0\u00b1\u00a04245.8306\u00a0\u00b1\u00a05304.6362\u00a0\u00b1\u00a05368.2424\u00a0\u00b1\u00a04423.8527\u00a0\u00b1\u00a018493.950\u201335094\u00a0\u00b1\u00a03.5104.7119\u00a0\u00b1\u00a01.2130.9151\u00a0\u00b1\u00a02.5166.5190\u00a0\u00b1\u00a04.8202.8237\u00a0\u00b1\u00a06.0252.8Envirocam\u2122 measurements fitted to sigmoidal 4-parameter equation using Signmaplot software. Duplicate runs executed for 500\u20132,000\u00a0\u03bcm distribution\nGas bubble data analysis\nThe smallest bubble measurable was 30\u00a0\u03bcm in diameter, based on an expected maximum system resolution of 7.5\u00a0\u03bcm\/pixel. A minimum of 2\u00a0pixels were needed to quantify the radii, and radii were used for diameter calculations based on initial ease of programming. Since the radius accuracy was \u00b11\u00a0pixel, the relative standard error for a 30\u00a0\u03bcm bubble was 50%, dropping to 25% for a 60\u00a0\u03bcm bubble. If required for other applications, smaller objects down to 20\u00a0\u03bcm might be measured using (1) two pixels to determine diameter or (2) using a back-calculated diameter based on equivalent surface area since only 2\u00a0pixels were required to define surface area.\nBubble diameter measurements, generated using the image analysis software, were compared with those generated manually for 225 objects from a single frame image taken using the CCD camera system and a 1\/2\u00a0in. gap shroud. Results demonstrated that the greatest percentage error was observed with smaller bubbles in the diameter range of 38\u201357\u00a0\u03bcm. This error generally decreased with larger bubble diameters. Manually measured diameters were slightly longer than image analysis measurements greater than 99.7% of the time, most likely due to a small amount of shadowing around bubble edges. The measurement error was 0.1% for the calibration line itself. Coincidentally, for some actual bubble measurement conditions, the number of objects rose considerably for bubbles 38\u00a0\u03bcm in diameter. This rise may be caused by the greater measurement inaccuracy at this size; thus small changes in size were not detectable.\nSince the minimum hardware resolution was 15\u00a0\u03bcm based on at least a 1\u00a0pixel change in radius, buckets in the distribution possessed 15\u00a0\u03bcm increments. This distribution resolution was consistent with the 25\u201390\u00a0\u03bcm value reported for an optical bubble measurement system with a 20\u00d7 magnification [3, 4] and similar to the 15\u00a0\u03bcm value reported for a stationary flatbed scanner set up [7]. The system\u2019s ability to measure bubble size changes due to typical differences in fermenter operating conditions was estimated based on this resolution. Specifically, an agitation rate increase from 100 to 150\u00a0rpm decreased the size of 300\u00a0\u03bcm bubbles to 184\u00a0\u03bcm, and an agitation rate increase from 100 to 125\u00a0rpm decreased the size of 300\u00a0\u03bcm bubbles to 230\u00a0\u03bcm (D\u00a0\u03b1\u00a0N\u22121.2; [2]). Both of these changes were substantially greater than the 15\u00a0\u03bcm resolution, and thus bubble size differences caused by agitation rate changes were believed detectable using this measurement system. For other operating conditions where the effect on bubble change was less pronounced [2], these differences were not expected to be as readily detectable.\nIncremental and cumulative distributions were plotted, typically as number or cumulative percentage versus bubble size, respectively. Arithmetic, Sauter, and geometric means and standard deviations (as applicable) then were calculated according to published methods [2]. In addition, the 5% largest bubbles and 5% smallest bubbles were discarded and the arithmetic and Sauter means recalculated. Using these 90% cut-offs significantly reduced the skewing and variability impact of smaller numbers of larger bubbles in the size distributions on the arithmetic and Sauter mean diameter calculations (Fig.\u00a05a, b). Owing to the nature of the geometric mean calculation, there was little effect when the 90% cut-off was used (Fig.\u00a05c).\nFig.\u00a05Arithmetic (a), Sauter (b), and geometric (c) means and standard deviations (as applicable) calculated using all the bubbles and for the 90% cutoff. Calculations conducted on 500 frames using every tenth frame for 50 frames and each calculation point covering 50 different frames, starting from successive initial frames\nThe total number of bubbles required to be analyzed per measurement greatly affected the measurement and data acquisition times (MAT and DAT, respectively), plus the data storage requirements. Based on a survey of published techniques [2], the target number was 500 bubbles per measurement condition. This value was confirmed based on determining that means and standard deviations changed only minimally (less than 4%) when bubbles numbers below and beyond 500 were analyzed (specifically 300, 400, 500, 750, 1,000, 1,500 and 2,000), also suggesting that as few as 300 bubbles were sufficient. The desired total bubble number target may be achieved based on a higher number of bubbles per frame (50) and a smaller number of frames (10), or a lower number of bubbles per frame (10) and a larger number of frames (50), with care taken not to count bubbles more than once in successive frames in either case.\nMeasurement reproducibility was evaluated using bubble measurement data from a 15,000\u00a0l fermenter by analyzing every tenth frame for 500 frames starting at the first frame until 2,000 bubbles were obtained, then re-analyzing every tenth frame starting from the second frame, then again, starting from the third through tenth frames. The relative standard deviations of the averages typically were under 7.5% for the 90% cut-off Sauter mean diameter and under 4% for the 90% cut-off arithmetic and geometric means. Thus, the sampling of frames used for analysis was representative of the total number of frames collected.\nDiscarding the blob area from the measurement was not felt to significantly influence bubble diameter means and distributions, nor impact subsequent predictions of volumetric mass transfer coefficients. The overall blob interfacial area per unit volume, a, was relatively low compared to that of the selected measured bubbles (e.g., for 5\u00a0cm blobs, a\u00a0=\u00a01\/500,000\u00a0\u03bcm; for 5\u00a0mm bubbles, a\u00a0=\u00a01\/5,000\u00a0\u03bcm) owing to the large diameters of the blobs. In addition, the percentage of larger bubbles (i.e., 2.5\u20135.0\u00a0mm diameter) was relatively low (<10%) for most typical agitator\/sparger system set points and configurations. Their overall contribution to interfacial area also was low. Similarly, although bubbles below the 40\u00a0\u03bcm limit of detection had a large, collective, interfacial area, their small individual volume resulted in fast oxygen depletion by the broth, making them a less substantial source of oxygen supply [16]. Thus, accuracy for bubble sizes outside the target range of 40\u00a0\u03bcm\u20132\u00a0mm was considered less critical. Consequently, the maximum bubble size cut-off of 2\u00a0mm used for measurement appeared acceptable since larger bubbles tended to be non-spherical as previously described [5], and smaller bubbles (<2\u20132.5\u00a0mm diameter) in aqueous solutions previously were shown to behave as particles with rigid interfaces [17]. Consequently, significant numbers of irregularly shaped bubbles were not obtained within the target measurement range and the use of bubble diameter to approximate size was reasonably accurate.\nTest systems\nWater\/media\/broth\nLiquid test media consisted of deionized water (DIW) and the following solutions, all prepared using DIW: 50\u00a0vol% glycerol (viscosity of 12\u201314\u00a0Pa\u00a0s\u00a0\u00d7\u00a0103 [1, 18] vs. 1.01\u00a0Pa\u00a0s\u00a0\u00d7\u00a0103 for water [19]), 0.01\u20132\u00a0ml\/l (0.001\u20130.2\u00a0vol%) P2000 (polypropylene glycol 2000; Dow, Freeport, TX), 0.01\u20132\u00a0ml\/l (0.001\u20130.2\u00a0vol%) antifoam C (Sigma), mono\/di potassium phosphate (effect of pH at constant ionic strength of 0.037\u00a0M: 5\u00a0g\/l monopotassium phosphate at pH 4.4 versus 6.4\u00a0g\/l dipotassium phosphate at pH 8.8; effect of ionic strength at constant pH of 7.0: 0.037\u00a0M (5\u00a0g\/l) versus 0.0037\u00a0M (0.5\u00a0g\/l) monopotassium phosphate), 0.1\u20132.5\u00a0g\/l (0.01\u20130.25\u00a0wt\/vol%) cottonseed flour (size of 91% of particles <74\u00a0\u03bcm; Pharmamedia, Traders Protein; Memphis, TN), sterility medium [6\u00a0g\/l yeast extract (Biospringer; Milwaukee, WI), 6\u00a0g\/l cerelose (glucose monohydrate), 1\u00a0ml\/l P2000], and low-protein animal cell culture media (LPKM, JRH Biosciences; Lenexa, KS). Varying viscosity by using a glycerol solution varied surface tension by only a few dynes\/cm [20], but antifoam addition varied surface tension without appreciably changing viscosity. These solutions were selected to vary the physical and optical properties of the liquid phase sufficiently to detect changes in bubble size measurement ability. Temperature was 22\u00b0C, except for when its effect on bubble size was examined for set points of 15, 22 and 37\u00b0C (range of 22\u00b0C). Back-pressure was zero except when its effect on bubble size was examined (0.3\u20131.5\u00a0kgf\/cm2).\nTo ensure that contamination did not cause changes in surface active properties [21], test media that supported growth were sterilized if the testing period was expected to be greater than 2\u20133\u00a0h. Also, since vessel cleanliness (i.e., residual surfactant concentrations) affected bubble density and size, all vessels were first rinsed thoroughly with hot water, and then a known amount of antifoam (0.001\u00a0vol% P2000) was added to DIW. These model systems formed the basis for initial tests of actual fermentation broths of microbial (filamentous bacterial culture, Amycolatopsis fastidiosa) and animal cell (suspension-adapted CHO) cultivations.\nFermenters equipped with open pipe\/jet spargers\nWhen performing measurements in agitated fermenters equipped with open pipe\/jet spargers (1\u00a0in. opening at 180, 600, and 15,000\u00a0l scales; 0.7\u00a0in. opening at 1,500\u00a0l scale), it was apparent that under certain conditions the bubble distribution was bimodal. Some images consisted of smaller spherical bubbles along with very large irregularly shaped \u201cblob\u201d bubbles, presumably owing to gas entrainment from vortexing and possible impeller flooding at higher aeration rates relative to agitation rates. Quantification of the discarded blob area from each of these conditions assisted in identifying the onset of flooding conditions, and possibly was directly related to the gas hold up (even under conditions in which individual bubble diameters cannot be discerned).\nBubble size measurements were conducted at the 180, 600, 1,500, 15,000\u00a0l scales, spanning an 80-fold range in scale, all in geometrically similar fermenters. For some combinations of agitation and airflow rates, the bubble density was too high to clearly obtain individual bubble diameter measurements. This limitation to low agitation and airflow rates, and void fractions under 2%, was similar to that observed for other optically based systems [6, 22].\nQualitative pictures of the effect of agitation and airflow rate at the 180\u00a0l scale for a fermenter equipped with a Rushton impeller are shown in Fig.\u00a06. As expected, higher agitation rates and thus power inputs (estimated at 0.24, 0.83, and 1.96\u00a0hp\/1,000\u00a0l; [23]) created larger numbers of smaller bubbles and higher airflow rates (0.55, 1.1 and 1.67\u00a0vvm) increased the number and size of bubbles. Increases in both agitation and airflow rates increased bubble density often to the degree that measurement of individual bubbles was precluded.\nFig.\u00a06Qualitative bubble size as function of agitation and airflow rate for open pipe sparger (1\u00a0in.) at the 180\u00a0l scale (0.001% P2000 in DIW)\nQualitative pictures of the effect of agitation and airflow rate at the 15,000\u00a0l scale for a fermenter equipped with a Rushton impeller are shown in Fig.\u00a07a. Trends were similar to those obtained at the smaller scale, considering vvms (0.067, 0.13 and 0.2\u00a0vvm) and power inputs (estimated at 0.02, 0.12, and 0.36\u00a0hp\/1,000\u00a0l; [23]) were considerably lower. The 90% cut-off Sauter mean diameter is shown by Fig.\u00a07b, following expected trends with agitation and airflow rate. A comparison of the arithmetic, Sauter and geometric mean diameters, both with and without the 90% cut-off, is shown by Fig.\u00a07c with expected trends generally observed. The relationship between bubble size and agitation rate at the 15,000\u00a0l scale was quantified for the 90% cut-off of the Sauter mean diameter as D\u00a0\u03b1\u00a0N\u22120.45, and compared to literature results, D\u00a0\u03b1\u00a0N\u22121.2 [2]. The lower dependence observed on agitation rate likely was due to the fact that the measured power inputs were lower than those used for the published correlation.\nFig.\u00a07Analysis of bubble data at the 15,000\u00a0l scale, containing 0.001% P2000 in DIW, with an open pipe sparger (round 2\u00a0in. ring with four jets of id 1\u00a0in. each): a Qualitative bubble size as a function of agitation and airflow rates. b Bubble Sauter mean diameter (90% cutoff) as a function of agitation and airflow rates. c Arithmetic, Sauter and geometric mean comparisons as function of agitation rate at airflow rate of 2,000\u00a0lpm\nAt the 180\u00a0l scale, 50\u00a0vol% glycerol solution was tested using a fermenter equipped with Rushton impellers at constant agitation and airflow rates. The effect of agitation and airflow rate increases on bubble size and distribution was qualitatively similar between DIW (Fig.\u00a06) and 50\u00a0vol% glycerol (Fig.\u00a08). The number of smaller bubbles qualitatively was somewhat greater for 50\u00a0vol% glycerol, particularly at lower agitation rates, which was inconsistent with the expectation that bubble size increase with solution viscosity. However, at the same 100\u00a0rpm agitation rate, the power per unit volume was likely higher for the glycerol solution, since the impeller Reynold\u2019s number, NRe, was \u223c1.8\u00a0\u00d7\u00a0104 in the laminar flow range for 50\u00a0vol% glycerol and \u223c2.2\u00a0\u00d7\u00a0105 in the turbulent range for DIW.\nFig.\u00a08Comparison of bubble sizes in DIW and 50\u00a0vol% glycerol, containing 0.001% P2000, as a function of agitation and airflow rate at the 180\u00a0l scale\nAt the 180\u00a0l scale, various solutions (differing in ionic strength, pH, temperature, pressure) were tested using a fermenter equipped with Rushton impellers at constant agitation and airflow rates, but notable qualitative changes in bubble distribution were not apparent. As greater amounts of P2000 (beyond 0.001%) were added to DIW, observations became progressively more limited to lower agitation and airflow rates before becoming too dark due to overlapping bubbles. Gas hold up was indirectly measured for aeration and agitation conditions at the 180, 600 and 1,500\u00a0l scales, for conditions, which had high discarded area percentages (Fig.\u00a09a, b). Hold up was estimated using the discarded area for each frame of the measurement and then averaging the results. Discarded areas were not calculated precisely as individual bubble areas, however, and standard deviations of average discard areas were around 50% at lower values, dropping to 10% at higher values. Trends in the values of discarded areas indicated that hold up, as well as bubble residence time, increased with greater agitation rates and airflow rates as observed by others [6]. In addition, as airflow rate increased, the impact of higher agitation rates on discard areas decreased suggesting impeller flooding, which at 0.5\u00a0vvm occurred for the 1,500\u00a0l scale but was not evident at the 600\u00a0l scale owing to lower gassed power draws. Specifically, higher discard areas were evident at the 600\u00a0l scale for hydrofoil versus Rushton impellers, consistent with greater measured gassed power decreases and gas hold ups [23, 24].\nFig.\u00a09Discarded bubble area as a function of a agitation rate at constant airflow rate and b airflow rate at constant agitation rate for 180\u00a0l fermenter containing 0.001% P2000 in DIW\nFermenters equipped with ring spargers\nBubble measurements in fermenters with open pipe spargers exhibited several bubbles in each frame, often in swarms. In contrast, bubble measurements in fermenters with ring spargers represented the opposite extreme in which only a few bubbles were present in each frame. These latter tests were conducted using purified water in 75 and 750\u00a0l geometrically similar bioreactors, spanning a tenfold size range. Fermenters were equipped with A315 impellers and a ring sparger with holes drilled 1\/32\u00a0in. in diameter on the top surface of the sparger ring. Only a few bubbles were observed regardless of notch orientation (left, right, down, top). It was believed that the fewer bubbles observed per frame were due partially to lower gas hold ups, typically 0.02\u00a0vvm, but also the relative spatial placement of the probe at the level of the sparger ring.\nAs the airflow rate was increased to its higher range values, more similarly sized bubbles were observed. In contrast, higher agitation rates caused more surface air entrainment; thus greater numbers of large, irregularly shaped bubbles were present in the frames. Higher airflow rates resulted in bubbles similar in nature to those observed at lower airflow rates, all governed by the holes in the ring sparger. In some cases owing to the slower agitation rates, smaller-sized (geometric mean of 95 vs. 200\u00a0\u03bcm) bubbles collected on surface of the shroud\u2019s sapphire window, and these bubbles needed to be distinguished from the freely moving bubbles. Increases in silicone antifoam from 0.001 to 0.2% did not increase the low number of bubbles observed.\nUse of an in situ bubble measurement system in fermentation\nThe ability of the Envirocam\u2122 to measure in opaque solutions was examined using several model systems:\nFor the 180\u00a0l fermenter equipped with an open pipe sparger, the effect of Pharmamedia on bubble images was investigated. When 20\u00a0g\/l Pharmamedia and 2\u00a0ml\/l P2000 was added to DIW (100\u00a0\u03bcs shutter speed, 20\u00a0gain, 185\u00a0brightness and 8\u00a0aperture), the contrast of bubble edges decreased to an unacceptable level. The particles caused granularity on the screen and blurred bubble edges, making detection difficult using the 1\/2\u00a0in. gap shroud. When 20\u00a0g\/l Pharamedia and 0.5\u00a0ml\/l P2000 was added to DIW using the 1\/4\u00a0in. gap shroud to decrease path length (100\u00a0\u03bcs shutter speed, 0\u00a0brightness, 31\u00a0gain, 8\u00a0aperture), bubble contrast was improved, but the resulting opaque solution appeared still too high for reliable analysis.\nIn addition, there was limited ability to obtain bubbles with edges sufficiently sharp enough for accurate measurements when 50% diluted Amycolatopsis broth (initial dcw of 3\u00a0\u00b1\u00a00.5\u00a0g\/l) was tested, despite raising settings to maximum values. The use of localized thresholding was attempted to sufficiently sharpen the bubble edges by reducing fuzziness for analysis, but it was only partially successful (Fig.\u00a010a). Note that the bubble density is considerably lower for this image taken at 200\u00a0rpm and 300\u00a0lpm, compared with images taken in 0.001% P2000 at 200\u00a0rpm and 100\u00a0lpm (Fig.\u00a06). This comparison demonstrated that the presence of broth potentially improves the agitation and aeration range over which the Envirocam\u2122 can measure bubbles should the optical limitations of the broth on the measurement be mitigated.\nFig.\u00a010Bubble photographs in fermentation broth: aAmycolatopsis fastidiosa broth with 1% P2000, 3\u00a0g\/l dry cell weight diluted 1:1 with DIW, 180\u00a0l scale and open pipe sparger, 200\u00a0rpm, 300\u00a0lpm; b LPKM medium with 0.001% antifoam C, 40\u00a0l scale and ring sparger, 50\u00a0rpm, 4\u00a0lpm\nFor the 75\u00a0l fermenter with the ring sparger, images taken in cell-free LPKM medium (Fig.\u00a010b) were compared with those taken in a 10-day CHO cell culture containing animal protein-free medium (containing 1\u00a0g\/l Pluronic F68 and no antifoam) with about 7\u00a0\u00d7\u00a0106\u00a0cells\/ml at 45% viability. Despite the low cell density of this broth, bubble edges remained fuzzy preventing accurate size analysis.\nSummary and future considerations\nA novel in situ bubble size and distribution measurement device was developed. The bubble measurement instrument design strategy permitted one camera module to be attached to a shroud, and thus one sensor (the camera) was able to be moved to multiple locations without disturbing the fermentation process. The small size and flexibility of the camera attachment permitted it to be readily relocatable. An Ethernet connection for the camera can further reduce the extent of the field hardware, and thus permit one camera system to be even more transportable.\nThe measurement system was tested at the pilot scale, both in clear and opaque model systems, which included fermentation broth. Experimental data for mean bubble size changes versus expected behavior qualitatively compared favorably with published relationships for selected conditions [2]. Quantitative comparisons were more difficult to establish owing to limitations in bubble size measurement capability at the higher agitation and airflow rate ranges at which these published correlations often were established.\nAt this time, application of the EnviroCam\u2122 bubble measurement system appears limited to clear solutions that do not contain large numbers of overlapping bubbles. Different liquids (e.g., water, cottonseed flour, microbial broth, animal cell broth) possess different UV spectrum and light scattering properties, as well as varying surfactant properties, which influence bubble size and hold up. These differences suggest that some adaptation of the measurement system is necessary when moving from system to system. Specifically, agitation and aeration rate combinations which produce too many bubbles for measurement in a model DIW system may produce acceptable amounts of bubbles in a fermentation broth owing to changes in surfactant levels, but the ability to distinguish these bubble edges is diminished. Further expansion of the versatility and range of this instrument is the subject of future efforts, but key approaches being considered involve the further examination of available LabView filtering techniques to process bubble images and the use of smaller notch sizes.","keyphrases":["in situ","measurement","bubbles","size","distribution","fermenter","image analysis"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Intensive_Care_Med-3-1-2092441","title":"High incidence of acute lung injury in children with Down syndrome\n","text":"Objective Acute respiratory tract infection is a common reason for hospitalization in children with Down syndrome (CDS) and is characterized by a high morbidity. The severe course of disease in CDS may be related to a higher incidence of acute lung injury (ALI). This study evaluated the incidence of ALI and acute respiratory distress syndrome (ARDS) in mechanically ventilated CDS.\nIntroduction\nDown syndrome (DS) is the most common chromosomal abnormality and occurs in 1 of 800 live births\u00a0[1]. Although survival beyond the first year of life has increased considerably in recent decades, children with DS (CDS) still have a\u00a0shorter life expectancy than those without DS\u00a0[2]. Acute respiratory tract infection is a\u00a0common reason for hospitalization in CDS and is characterized by high morbidity\u00a0[3]. In children with DS it has been suggested to be related to pharyngeal incoordination predisposing to aspiration in combination with concomitant immunodeficiencies\u00a0[4, 5]. High morbidity in CDS with respiratory disease may also be related to a\u00a0higher incidence of acute lung injury (ALI). The aim of this study was to evaluate the incidence of ALI and acute respiratory distress syndrome (ARDS) in children with DS who were admitted to the pediatric intensive care unit (PICU) and needed mechanical ventilation. Preliminary data from this study were presented at the 35th Congress of the Society of Critical Care Medicine\u00a0[6].\nMethods\nThis study compared the occurrence of ALI\/ARDS in two groups of patients requiring mechanical ventilation because of respiratory insufficiency. The first group consisted of all 24 consecutive children with genetically confirmed DS admitted to our PICU for mechanical ventilation between January 1998 and July 2005. The second group consisted of all 317 mechanically ventilated children without DS admitted to our PICU between January 1998 and January 2001. Baseline characteristics for CDS and the control group are shown in Table\u202f1. The PICU at Emma Children's Hospital is a\u00a016-bed, tertiary, multidisciplinary unit serving the greater Amsterdam area. Data on the incidence rate of ALI\/ARDS in the second group have been published previously\u00a0[7]. In both groups patients who were admitted for postoperative care directly following a\u00a0surgical procedure, and those with a\u00a0cardiac left to right shunt were excluded. Patients were retrospectively evaluated for ALI\/ARDS during the second 24\u202fh after admission according to the American\u2013European Consensus Conference criteria\u00a0[8]. Gas exchange criteria for ALI and ARDS were considered to be met if the PaO2\/FIO2 ratio was less than 40.0\u202fkPa for ALI and less than 26.7\u202fkPa for ARDS in at least two consecutive measurements (>\u202f8\u202fh apart). A\u00a0radiologist blinded to clinical information reviewed the chest radiographs for the presence of bilateral infiltrates. The presence of left heart failure was assessed based on echocardiographic results and\/or clinical information. Disease severity on admission was expressed by the Pediatric Risk of Mortality (PRISM) II score, which depends on diagnosis and clinical parameters in the first 24\u202fh of admission\u00a0[9]. Sepsis was defined as systemic inflammatory response syndrome in the presence of clinical evidence for infection. Lower respiratory tract infection was defined as clinical infection with radiological evidence of alveolar consolidation. Upper respiratory tract infection was defined as infection of the oral and nasal airways, larynx, trachea, and\/or bronchi without signs of lower respiratory tract infection.\nTable\u00a01Baseline patient characteristics and cause for respiratory failureDown syndrome (n\u202f=\u202f24)Control group (n\u202f=\u202f317)pMedian age (months; range)15.2 (0\u2013127.2) 6.1 (0\u2013217.7)0.39 cMales14 (58.3%)199 (62.8%)0.67 dMedian PRISM II score (range)13.5 (0\u201340) 9.0 (0\u201347)0.19 cSepsis 3 (12.5%) 52 (16.4%)0.78 eLower respiratory tract infection18 (75%)130 (41%)0.001 dUpper respiratory tract infection 1 (4.2%) 23 (7.3%)0.96 eCongenital heart disease 1 (4.2%) 22 (6.9%)0.99 eCNS disorders a 0 53 (16.7%)0.04 eOther b 1 (4.2%) 37 (11.7%)0.50 eaCNS disorders: convulsions, infections, asphyxia, contusion, cerebral\nhemorrhage; bOthers, e.\u202fg., trauma, cardiomyopathies, electrolyte disturbances,\nmetabolic disease; c Mann\u2013Whitney test; d Pearson's \u03c72 test; e Fisher's exact test\nThe comparability of patient characteristics for both groups was tested by means of a\u00a0chi-square test or Fisher exact test for dichotomous data and a\u00a0Mann\u2013Whitney U test for continuous data that were not normally distributed. The association between DS and ALI\/ARDS was expressed as an odds ratio (OR) with 95% Confidence Interval (CI). Other well known cause for ALI\/ARDS and thus possible confounders for the incidence of ALI\/ARDS among CDS were identified\u00a0[8, 10] and evaluated with bivariate logistic regression analysis. Statistical significance was set at 5%.\nResults\nThe criteria for ALI were met in 14 of 24 CDS (58.3%) and in 41 of 317 controls (12.9%; odds ratio 9.4, 95% confidence interval 3.9\u201322.6). The criteria for ARDS were met in 11 of 24 CDS (46%) and in 21 of 317 controls (7%; odds ratio 11.9, 95% confidence interval 4.8\u201329.8). There were no deaths in the CDS. In the control group ten patients with ALI died during admission to the PICU. The results of the logistic regression analyses are presented in Table\u202f2. The odds for the incidence of ALI in CDS were hardly altered by adjusting disease severity (PRISM II score), lower respiratory tract infection, or sepsis.\nTable\u00a02Estimated odds ratio (OR) for ALI (Down\/control) after adjustment for possible confounders (CI, confidence interval)Possible confounderOR95% CINone 9.43.9\u201322.6PRISM II score 9.63.9\u201323.6Sepsis12.24.9\u201330.6Lower respiratory tract infection10.84.3\u201326.9\nWe excluded patients who died in the first 24\u202fh after admission. This might have caused a\u00a0selection bias. Therefore PICU deaths during the first 24\u202fh of admission were analyzed for ALI\/ARDS: 36 in the control group, none of whom met the ALI\/ARDS criteria. No CDS died in the first 24\u202fh after admission. Thus the odds of developing ALI in CDS are about 9 times those in controls, and this cannot be attributed to the presence of confounders.\nDiscussion\nThis study found a\u00a0very high incidence of 58% of ALI and 46% of ARDS in mechanically ventilated children with DS. This is significantly higher than the incidence of 13% of ALI and 7% of ARDS in the general pediatric population on mechanical ventilation in our unit. Likewise, Randolph et al.\u00a0[11] reported an incidence of ARDS of almost 8% in a\u00a0mechanically ventilated pediatric population.\nThe results of this study need to be interpreted with caution due to the retrospective design and the relatively low number of CDS. The results need to be confirmed in a\u00a0prospective multicenter study in a\u00a0larger cohort. Due to the small number of CDS admitted annually to our unit we included CDS for a\u00a0longer period of time (1998\u20132005) than the control group (1998\u20132001). This may interfere with the comparability of the study groups. However, medical care on our unit did not change substantially during this extended period. In both study periods respiratory care was based on the same clinical protocols with a\u00a0low tidal volume ventilation strategy. Identical ventilators were used. In addition, the disease severity (expressed as mean PRISM II scores) did not differ between 2001\u20132005 and 1998\u20132001. Therefore we have no reason to assume that the incidence of ALI in mechanically ventilated patients changed substantially during the 2001\u20132005 period.\nDespite the high incidence of ALI\/ARDS we found no mortality in our population of CDS. Others have reported a\u00a0mortality rate of almost 5% in children with ARDS\u00a0[11, 12]. Although this remains speculative, it is possible that CDS are more susceptible to progression to ALI\/ARDS despite less severe underlying conditions, without concomitant mortality. In addition, there were only three patients with sepsis and none with trauma as underlying cause of ALI\/ARDS in the CDS group, conditions associated with a\u00a0high mortality rate. However, ALI\/ARDS is associated with high morbidity in CDS. The need for ventilatory support was 21\u202f\u00b1\u202f21\u202fdays in CDS with ALI vs. 8\u202f\u00b1\u202f5\u202fdays in CDS without ALI (p\u202f=\u202f0.03). Long-term effects of ALI on morbidity in CDS are unknown and remain to be defined by lung function measurements. The high mortality rate among patients with ALI in the control group might be explained by the relatively high number of patients (n\u202f=\u202f4) with severe cerebral damage. This has been shown to be a\u00a0strong predictor for mortality in ALI\u00a0[13].\nThe mechanism leading to the high incidence of ALI in CDS remains uncertain. Initial ventilator settings have recently been shown to be a\u00a0risk factor for the development of ARDS\u00a0[14]. In this single-center study the ventilatory strategies for CDS did not differ from those in other patients and were based on the same low tidal volume protocol. Recently it has been shown that apoptosis or programmed cell death plays a\u00a0pivotal role in the pathogenesis of injurious states of the pulmonary system such as of ALI\/ARDS\u00a0[15, 16]. The higher incidence of ALI\/ARDS may be caused by an elevated rate of apoptosis in CDS. An enhanced level of apoptosis in DS has been shown in several cells including neurons\u00a0[17, 18], thymocytes\u00a0[19], and granulocytes\u00a0[20]. It has been suggested that increased apoptosis in DS cells is related to an inability to deal with oxidative stress, leading to accumulation of reactive oxygen radicals\u00a0[21]. One could speculate whether this is related to an imbalance in the antioxidant\/oxidant status in patients with DS due to increased levels of superoxide dismutase 1, an important enzyme in the antioxidant pathway that is encoded for on chromosome 21\u00a0[22, 23]. To what extent this is associated with an increased susceptibility to develop ALI\/ARDS deserves further investigation. From this point of view our findings are not only of scientific interest but may also be of clinical relevance. For example, ventilatory strategies or oxygen therapy might need to be reevaluated and adjusted for CDS.\nIn conclusion, we found an unexpected high incidence of ALI and ARDS in CDS. The explanation for these findings remains to be elucidated.","keyphrases":["down syndrome","acute respiratory distress syndrome","mechanical ventilation","epidemiology","cohort studies","child"],"prmu":["P","P","P","U","R","U"]}
{"id":"Theor_Appl_Genet-2-2-1544375","title":"Coffee and tomato share common gene repertoires as revealed by deep sequencing of seed and cherry transcripts\n","text":"An EST database has been generated for coffee based on sequences from approximately 47,000 cDNA clones derived from five different stages\/tissues, with a special focus on developing seeds. When computationally assembled, these sequences correspond to 13,175 unigenes, which were analyzed with respect to functional annotation, expression profile and evolution. Compared with Arabidopsis, the coffee unigenes encode a higher proportion of proteins related to protein modification\/turnover and metabolism\u2014an observation that may explain the high diversity of metabolites found in coffee and related species. Several gene families were found to be either expanded or unique to coffee when compared with Arabidopsis. A high proportion of these families encode proteins assigned to functions related to disease resistance. Such families may have expanded and evolved rapidly under the intense pathogen pressure experienced by a tropical, perennial species like coffee. Finally, the coffee gene repertoire was compared with that of Arabidopsis and Solanaceous species (e.g. tomato). Unlike Arabidopsis, tomato has a nearly perfect gene-for-gene match with coffee. These results are consistent with the facts that coffee and tomato have a similar genome size, chromosome karyotype (tomato, n=12; coffee n=11) and chromosome architecture. Moreover, both belong to the Asterid I clade of dicot plant families. Thus, the biology of coffee (family Rubiacaeae) and tomato (family Solanaceae) may be united into one common network of shared discoveries, resources and information.\nIntroduction\nCoffee is an important international commodity, ranking among the five most valuable agricultural exports from developing countries (Food and Agriculture Organization, http:\/\/apps.fao.org). Moreover, production and processing of coffee employs more than 25 million people worldwide (O\u2019Brien and Kinnaird 2003). Despite its economic importance, coffee has received little attention with respect to molecular genetics and genomics research. As of December 2004, only 1,570 nucleotide and 115 protein sequences from coffee had been deposited in GenBank with the majority of those sequences derived from leaf ESTs. Many of the remaining sequences correspond to enzymes in the caffeine biosynthesis pathway\u2014the most extensively studied pathway in coffee (Moisyadi et al. 1998; Ogawa et al. 2001; Mizuno et al. 2003; Uefuji et al. 2003).\nCommercial coffee production relies mainly on two closely related species: Coffea arabica and Coffea canephora, accounting for approximately 70 and 30% of worldwide coffee production, respectively (Herrera et al. 2002). Although C. canephora accounts for a lower total proportion of the coffee market than does C. arabica, it is the main source for soluble coffee, which is consumed widely throughout the world. C. canephora is a diploid (2n=2x=22), outcrossing and highly polymorphic species native to central Africa, but which has expanded, through cultivation, especially to western Africa, Indonesia and Vietnam (Wrigley 1988). In contrast, C. arabica is believed to be a recently derived tetraploid (2n=4x=44) native to a small region of what is now Ethiopia. C. arabica is now grown widely throughout the world.\nThe goal of the current project was to increase the genetic and molecular knowledge of coffee through the generation and annotation of an EST database using high throughput single-pass 5\u2032 sequencing of cDNAs derived from leaf, pericarp and seed tissues from a set of C. canephora varieties. Special emphasis was given to sequencing cDNAs from different stages of seed development, both to shed light on this important, but not well understood aspect of plant development and to capture as many genes as possible involved in determining the final chemical composition of seeds which constitute the commercial product. As a result, the EST database reported herein is, to our knowledge, the largest public database of seed-derived ESTs (White et al. 2000; Suh et al. 2003).\nThe EST database was used to derive a coffee unigene build, which was subsequently subjected to functional annotation. The coffee unigene build was compared with the model species Arabidopsis. Arabidopsis is the only fully sequenced dicot genome and hence provides a complete set of predicted genes against which to compare the EST-derived unigene set of coffee. However, coffee and Arabidopsis belong to different plant families (Rubiaceae and Brassicaceae, respectively) which are distantly related phylogenetically and which diverged from their last common ancestor approximately 94 million years ago (MYA) (Gandolfo et al. 1998; Crepet et al. 2004) (Fig.\u00a01).\nFig.\u00a01Dendrogram depicting phylogenetic relationships of coffee to other higher plant taxa (based on Chase et al. 1993)\nThe plant family most closely related to coffee in which extensive sequencing has been conducted is Solanaceae (Fig.\u00a01). In this family, comprehensive EST databases have been developed for tomato, potato, pepper, eggplant and petunia (http:\/\/www.sgn.cornell.edu\/) (Hoeven et al. 2002; Ronning and Stegalkina 2003; Lee et al. 2004). Both Rubiaceae and Solanaceae belong to the Asterid I clade of dicots, and based on existing fossil evidence, are thought to have diverged from one another approximately 50\u00a0MYA (Gandolfo et al. 1998; Crepet et al. 2004) (Crepet personal communication) (Fig.\u00a01). The closer taxonomic affinities of coffee and Solanaceae (e.g. tomato) are paralleled by a number of striking botanic and genetic similarities, including the production of fleshy berries, a similar genome content (C=950 and 640\u00a0Mb for tomato and coffee, respectively) (Hoeven et al. 2002), similar basic chromosome number (x=12 for tomato and most other Solanaceae; x=11 for coffee) and similar chromosome architecture with highly condensed pericentric heterochromatin and decondensed euchromatin at the pachytene stage of meiosis (Rick 1971; Pinto-Maglio and Cruz 1998). For these reasons, the coffee unigene set was also compared against a series of Solanaceae EST-derived unigene sets.\nMaterials and methods\nLibrary construction\nSource of tissues\nC. canephora was farm-grown in east Java. Plant tissue was frozen in liquid nitrogen, transported on dry ice and stored at \u201380\u00b0C until RNA extraction. The specified tissues (leaf, pericarp, young cherries with seed and pericarp mixed, middle stage seed and late stage seed) were collected from five different varieties Table\u00a01). The maturation period of the C. canephora varieties under study is approximately 9\u201311\u00a0months, from pollination to ripening. The early stage cherry, middle stage seed and late stage seed were collected between 18 to 22\u00a0weeks, 30\u00a0weeks and 42 to 46\u00a0weeks after pollination, respectively.\nTable\u00a01Characteristics of the 5 cDNA libraries used to develop the coffee EST databaseLibrary nameTissueVarietiesAverage insert size, kbGood quality ESTsLeafLeaves, youngBP4091.5\u00b10.68,942PericarpPericarp, all developmental stagesBP358, BP409, BP42, BP961, Q1211.4\u00b10.58,956Early stage cherryWhole cherries, 18 and 22\u00a0week after pollinationBP358, BP409, BP42, Q1211.4\u00b10.39,843Middle stage seedEndosperm and perisperm of seeds, 30\u00a0week after pollinationBP409, BP961, Q1211.4\u00b10.310,077Late stage seedEndosperm and perisperm of seeds, 42 and 46\u00a0week after pollinationBP358, BP409, BP42, BP961, Q1211.4\u00b10.39,096\nRNA and mRNA isolation\nTotal RNA was extracted using phenol\/chloroform (Rogers et al. 1999) and further treated with DNase I (RNase-free) and purified using an RNeasy Kit (Qiagen, Valencia, CA 91355). Messenger RNA was extracted from total RNA with PolyTrack mRNA Isolation Systems (Promega, Madison, WI 53711).\ncDNA libraries\nDirectional cDNA libraries were constructed with 3\u20135\u00a0\u03bcg of mRNA with the ZAP-cDNA Gigapack III Gold Cloning Kit (Stratagene, La Jolla, CA 92037). The average insert length was estimated by PCR in 36 randomly selected clones from each library and ranged 1.2\u20131.5\u00a0kb (Table\u00a01).\nSequencing\nBacteria, containing coffee cDNAs, were cultured in 384-well plates and cDNA inserts subjected to 5\u2032 end sequencing at the BioResource Center at Cornell University (http:\/\/www.brc.cornell.edu). The average size of quality reads was 613\u00a0bp with a maximum of 1,037\u00a0bp.\nSequence quality processing\nEST sequences were base-called and screened for vector sequences using PHRED software (Ewing et al. 1998). The longest stretch of overall high quality (PHRED score over 15 which corresponds to over 98% confidence) of each sequence was identified. PolyA repeats were trimmed to at most 20\u00a0bp and any sequence past the PolyA (mostly low quality sequence) was discarded. After the trimming, the sequences were screened against the E.coli K12 genome to remove any bacteria contamination. The remaining sequences were screened for minimum length (150\u00a0bp) and maximum allowed ambiguity (4%) and low complexity (60% of the sequence are of the same nucleotide, or 80% of same two nucleotides, which indicate error in sequencing).\nUnigene assembly\nUnigene sets were built by combining the sequences from all five coffee cDNA libraries. Clustering was performed using a program developed at the Sol Genomics Network (SGN at http:\/\/www.sgn.cornell.edu), which relied on a custom pre-clustering algorithm, and on the CAP3 program for contig generation (Huang and Madan 1999). The preclustering algorithm clustered sequences using a Smith Waterman type algorithm with initial word matching. The command line settings for CAP3 were as follows: -e 5000 -p 90 -d 10000 -b 60. The -e, -d and -b options are set such that the assembler disregards them or minimizes their effect. The -p option increases the sequence identity necessary on overlaps to 90 from a default of 75, which were found to be not stringent enough. Sequences were also checked for length, complexity and contamination. The builds were uploaded to the database, where each unigene was assigned a unique unigene ID.\nAnnotation\nProtein prediction\nThe most likely coding frame and the corresponding transcribed peptide for each unigene were generated using ESTScan, a program that detects coding regions in EST sequences and corrects some sequencing errors (nucleotide substitution, deletion\/insertion, erroneous stop codon, etc) (Iseli et al. 1999). As a hidden Markov Model (HMM) based program, ESTScan requires a training set of high quality coding sequences to generate a model. However, very few high quality coffee coding sequences are publicly available (less than 50 full length in GenBank). Therefore, we used as a training set of 483 nuclear genes from the closely related species, tomato (http:\/\/www.ebi.ac.uk\/embl). Table\u00a02 compares the coffee unigene assembly and translation (using ESTScan) with that of tomato, using the same training tomato set and parameters. The fact that the two give such similar results, gives confidence that the coffee EST-derived unigenes are being efficiently translated via the ESTScan software using the tomato training set (Table\u00a02).\nTable\u00a02Comparison of the coffee and tomato EST databases derived from use of ESTScan calibrated with the same tomato training set (see Materials and methods for details)TomatoCoffeeTotal unigenes30,57613,175Average unigene length, bp774678Unigenes with coding regions96%95%Average length (bp) of predicated peptides569556Average ESTScan score409346\nBLAST matches between coffee unigenes and other sequence databases\nSequence matches for coffee unigenes against other sequence databases were obtained using BLAST (Altschul et al. 1997) (see Results and discussion section for details of each BLAST analysis). The target databases were:The GenBank non-redundant (NR) protein and dbest dataset (NCBI, http:\/\/www.ncbi.nlm.nih.gov).The predicted Arabidopsis proteome and cDNA databases which were downloaded from the Arabidopsis Information Resource (TAIR, http:\/\/www.arabidopsis.org and ftp:\/\/ftp.arabidopsis.org\/home\/tair\/).The Solanaceae EST-derived unigene sets, including tomato (Solanum. lycopersicum) (184,860 ESTs, 30,576 unigenes), potato (Solanum. tuberosum) (97,425 ESTs, 24,932 unigenes), pepper (Capsicum annuum) (20,738 ESTs, 9,554 unigenes), petunia (Petunia hybrida) (3,181 ESTs, 1,841 unigenes) and eggplant (Solanum melongena) (11,479 ESTs, 5,135 unigenes), all of which can be accessed at SGN (http:\/\/www.sgn.cornell.edu). To estimate ribosomal, chloroplast and mitochondrial contamination, the coffee unigene set was also screened against the Arabidopsis complete mitochondrial genome (NC_001284), tobacco ribosomal rRNA gene (5.8s AJ012367, 18s AJ236016 and 25s S52185) and the complete tobacco chloroplast genome (Z00044) sequences from GenBank.\nFunctional annotation based on predicted peptides\nESTScan-predicted coffee peptides were subjected to InterPro Scan annotation, which integrates the most commonly used protein signature databases (PROSITE, PRINTS, Pfam, ProDom, etc.) together with their associated scanning methods for protein domain analysis (Apweiler et al. 2001; Zdobnov and Apweiler 2001). Based on the domain annotation, GO accession of the unigenes were assigned using interpro2go conversion file from the GO consortium (http:\/\/www.geneontology.org, also available at http:\/\/www.ebi.ac.uk\/interpro).\nFunctional categorization based on gene ontology\nGO annotations were formatted for input into the GOSlim program and the output parsed to count the occurrence of each GO category. GOSlims are \u2018slimmed down\u2019 versions of the ontologies that allow a high-level view of gene functions. The GOSlim file and program were obtained from the Gene Ontology Consortium at http:\/\/www.geneontology.org.\nGene family analysis\nThe predicted protein sequences for the coffee unigene set and the Arabidopsis protein set were combined into a single file, formatted as a blast database using formatdb, and run with BLASTP (protein vs. protein sequence) against itself with option m8 for output. The resulting file was used as the input for the tribeML program. TribeML (Enright et al. 2002) formats the clusters, such that each cluster was given in a tab delimited file, one cluster per line. Simple scripts were used to parse the information to detect the largest gene families, coffee specific families, and families that showed large expansions in coffee.\nResults and discussion\nGeneration of coffee EST database and unigene set\nA total of 62,829 cDNA clones, derived from mRNA from five different tissues, were subjected to 5\u2032 sequencing. After quality evaluation (see Materials and methods for details), the database was reduced to 46,914 high quality ESTs, averaging 613\u00a0bp in length. This corresponded to approximately 9,000 high quality ESTs from each of the five cDNA libraries (Table\u00a01). These high quality ESTs were assembled into 13,175 unigenes with average length of 678\u00a0bp (ranging 150\u20132,714\u00a0bp), among which 7,272 (55%) were singletons and 5,903 (45%) were contigs (Fig.\u00a02). Of the contigs, the majority (87%) was represented by two to ten ESTs while some (13%) were comprised of more than ten ESTs (Fig.\u00a02). All trace files, sequences and derived unigenes\/annotation can be found at the Solanaceae Genomics Network (SGN) website (http:\/\/www.sgn.cornell.edu) and will be submitted to GenBank at the time of publication.\nFig.\u00a02Histogram depicting the distribution of EST content for all coffee unigenes. Numbers above bars equals the number of unigenes represented in each\nDifferentiating between paralogs and alleles\nThe coffee ESTs were derived from five C. canephora varieties (Table\u00a01), however, unigene assembly for EST reads does not automatically distinguish between polymorphic alleles and highly similar paralogs. C. canephora is known to be a highly allogamous, heterozygous and polymorphic species, raising the possibility that some of the EST-derived unigenes might actually represent different allelic forms of the same gene. In order to evaluate how well paralogs were distinguished from allelic polymorphism in the unigene assembly, BLASTN (nucleotide vs. nucleotide sequences) was performed amongst all of the coffee unigenes and the results parsed to identify the best non-self matches. As a control, BLASTN was performed in a similar manner on a complete set of Arabidopsis predicted genes, including both coding and UTR regions obtained from TAIR (http:\/\/www.arabidopsis.org). Since the Arabidopsis database was derived from the complete genome sequence of a single, inbred stock (Columbia), the entire gene complement is present without the complication of allelic variation (initiative). For both coffee and Arabidopsis, pair-wise combinations for which the homology extended over more than 70% of the sequence were recorded. The sequence similarity for each of these combinations was plotted for both coffee and Arabidopsis (Fig.\u00a03).\nFig.\u00a03Plot depicting the sequence identify of the most similar match for each coffee unigene as compared with all other coffee unigenes. As a control, a similar analysis is shown for Arabidopsis genes (see Results for details)\nThe Arabidopsis plot showed two peaks, one with low identity (~87%) and the other with higher identity (over 99%) with 6.4 and 1.6% of genes falling into the low and high identity peaks, respectively. Like Arabidopsis, the coffee plot also showed two peaks, one with lower identity (around 91%) and the other with high identity (over 99%). These two peaks corresponded to 6.2 and 0.8% of the total unigenes, respectively. ESTs corresponding to ten pairs of coffee unigenes, from the >99% peak, were used as probes on genomic southern hybridizations to determine whether the matching pairs were truly duplicated in the coffee genome (paralogs) or rather allelic (single copy). For eight of the ten pairs, the paired ESTs hybridized the same single copy gene on southerns (data not show). Thus, a significant number (approximately 80%) of the unigenes in this peak are likely to be allelic. However, this category represented only a small portion of the coffee unigenes (0.8%). A similar experiment was performed with 11 ESTs from 11 pairs of unigenes in the second, lower homology peak (around 91% identity, see Fig.\u00a03). In this case, the majority (8 out of 11) was determined to represent true paralogs (two or more copies in the genome) (data not shown). Thus for further discussions, it is assumed that the majority coffee EST-derived unigenes do in fact correspond to unique coffee genes.\nFunctional annotation of coffee EST-derived unigenes\nPredicted coffee proteins\nESTScan (see Materials and methods for details) was able to identify protein-coding sequences in 12,534 coffee unigenes (95% of total unigenes), among which 1,515 (12%) were putatively full-length (starting with ATG and ending with a stop codon). Due to the cDNA library construction method, the unigenes were biased for the 3\u2032 end\u201457% of the unigenes covered the 3\u2032 end (ending with a stop codon) while only 36% covered the 5\u2032 end (starting with ATG). Of the 5% of unigenes from which a protein sequence could not be predicted, 81% were singletons and the majority (97%) did not match to any Arabidopsis, GenBank non-redundant (NR) or Solanaceae unigene sequences, suggesting that they are not bona fide gene transcripts.\nProtein domain annotation\nPeptides translated from ESTScan predicted coding sequences were subjected to InterPro Scan (see Materials and methods for details) for protein domain analysis. A total of 1,678 distinct domains were identified in the database, which were present in 4,414 (35%) unigenes. Table\u00a03 lists the 20 most represented InterPro domains found in the coffee genes, as well as comparative statistics for tomato and Arabidopsis genes. Coffee and tomato were very similar with regard to the most frequent protein domains; however, a number of striking differences were noted when comparing coffee and Arabidopsis. Most notably, the following five domains were much more common in coffee encoded proteins than in Arabidopsis: proline-rich regions, tyrosine protein kinases, glucose\/ribitol dehydrogenases, ubiquitins and ubiquitin-conjugating enzymes. The latter two categories, involving ubiquitinization, suggest a more active and\/or complex system for protein turnover in coffee versus Arabidopsis.\nTable\u00a03Twenty most abundant InterPro domains identified in coffee unigene set and comparative statistics for tomato and Arabidopsis genesInterPro accessionDescription% of unigenes (ranking)CoffeeTomato\nArabidopsis\nIPR000719Protein kinase1.61.20 (1)3.0 (1)IPR000694Proline-rich region1.30.91 (4)0.003 (1763)IPR002290Serine\/threonine protein kinase0.851.10 (2)0IPR001245Tyrosine protein kinase0.691.0 (3)0.15 (311)IPR008271Serine\/threonine protein kinase, active site0.610.68 (5)2.6 (2)IPR000504RNA-binding region RNP-1 (RNA recognition motif)0.550.60 (6)0.59 (6)IPR001680G-protein beta WD-40 repeat0.490.51 (8)0.51 (8)IPR001611Leucine-rich repeat0.480.59 (7)0.59 (7)IPR002048Calcium-binding EF-hand0.360.34 (13)0.34 (13)IPR000379Esterase\/lipase\/thioesterase0.330.43 (10)0.43 (10)IPR001806Ras GTPase superfamily0.320.26 (22)0.43 (70)IPR003579Ras small GTPase, Rab type0.290.23 (27)0IPR0051232OG-Fe(II) oxygenase superfamily0.270.26 (21)0.47 (52)IPR000626Ubiquitin0.270.22 (32)0.40 (89)IPR002401E-class P450, group I0.270.46 (8)0.77 (24)IPR002347Glucose\/ribitol dehydrogenase0.260.23 (28)0.33 (110)IPR001005Myb DNA-binding domain0.260.34 (15)1.34 (8)IPR005225Small GTP-binding protein domain0.260.24 (25)0.68 (27)IPR000608Ubiquitin-conjugating enzymes0.260.21 (34)0.19 (221)IPR007090Leucine-rich repeat, plant specific0.250.40 (12)1.07 (11)\nGene ontology annotation\nGene ontology (GO) annotations were made for each coffee unigene based on InterPro domain annotation (see Materials and methods for details). A total of 3,248 unigenes (25% of total unigenes and 74% of unigenes with InterPro domain match) could be assigned a GO annotation. In order to obtain an overview of representation of biological processes by the coffee unigene set, the GO annotations were mapped to the plant GOSlim, a group of selected higher-level categories of the GO ontology (see Materials and methods for details). Figure\u00a04 depicts the biological process GOSlim categories for each of which at least 1% of the coffee unigenes were assigned. Metabolism is the most abundant category, both in terms of number of subcategories (14 subgroups) and number of unigenes in the subcategories (2,541 unigenes and ~80% of the GO annotated unigenes). In addition, signal transduction, cell growth and\/or maintenance, response to stress and response to endogenous stimulus were among the most abundant categories.\nFig.\u00a04Comparison of the gene ontology-based gene annotation categories for the coffee EST-derived unigene set, tomato EST-derived unigene set and the Arabidopsis proteome. Figure contains only categories in which more than 1% of the coffee unigenes were assigned. Categories for which coffee differs most significantly from Arabidopsis are shown in underline bold. (1) Cellular processes other than signal transduction and cell growth and\/or maintenance. (2) Nucleobase\/nucleoside\/nucleotide and nucleic acid metabolism other than DNA metabolism and transcription. (3) Protein metabolism other than protein biosynthesis and protein modification. (4) Metabolism other than amino acid and derivative metabolism, biosynthesis, carbohydrate metabolism, catabolism, electron transport, lipid metabolism, nucleobase\/nucleoside\/nucleotide and nucleic acid metabolism and protein metabolism. (5) Cell growth and\/or maintenance other than cell cycle and cell organization and biogenesis. (6) Physiological processes other than photosynthesis, response to stress, response to endogenous stimulus, response to external stimulus and metabolism\nA comparison was made between the GOSlim biological process of Arabidopsis, tomato and coffee (Fig.\u00a04). For both the tomato and coffee unigene sets, the GO annotations were based on InterProScan results and approximately 25% of both unigene sets were assigned a GO annotation. In Arabidopsis, the genes are of full length, giving a higher chance of finding functional domains. Moreover, extensive experimental research and manual annotation has been carried out in Arabidopsis, resulting in a higher proportion of genes with assigned GO annotation. Therefore 83% of the Arabidopsis genes are assigned GO annotations. No significant differences were observed in the annotated categories for coffee versus tomato\u2014possibly reflecting their close taxonomic affinity. However, for a number of categories, coffee had significantly different proportions of genes than Arabidopsis. The categories with the largest significant differences (P<0.001, based on Chi-square test) are: carbohydrate metabolism, other metabolism, biosynthesis, catabolism, protein biosynthesis, protein modification and energy pathways. In all cases, coffee had a significantly higher proportion of genes in these categories than Arabidopsis (Fig.\u00a04). It is interesting to note that many of these categories center around the synthesis, breakdown or modification of compounds. One of the hallmarks of coffee is its high-level diversity of primary and secondary compounds, which contributes to the sensory quality of brewed coffee beans. The Rubiaceae family in general contains some of the most diverse species with regard to secondary metabolism and is an especially rich source of alkaloids\u2014a number of which have pharmacological\u00a0and\/or psychotropic properties (Kutchan 1995; Facchini 2001). In fact, the most widely used psychotropic drug, caffeine, comes mainly from coffee. One can speculate that this metabolic diversity is reflected in the relatively high proportion of coffee genes with putative functions related to metabolism.\nIn silico analysis of unigene expression\nComplexity and uniqueness of different stages\/tissues\nIn an effort to determine which tissues gave rise to the more redundant or more complex pools of gene expression, three library\/tissue parameters were calculated: (1) the percentage of total coffee unigenes represented by each library\/tissue; (2) the percentage of more highly expressed unigenes (represented by ten or more ESTs from the library) in each library\/tissue; (3) the percentage of unigenes from each tissue\/stage which are unique to that library\/tissue (not found in any other cDNA library). The results from these calculations are depicted in Fig.\u00a05. The pericarp and leaf libraries represented more unigenes, contributed to more unique unigenes, and contained fewer highly abundant unigenes than did the early cherry, middle seed and late seed libraries\u2014perhaps reflecting the overall lower complexity of these latter stages\/tissues (Fig.\u00a05). Among the seed stages, the middle stage of development gave more novel genes than did the early or late stages of seed development (Fig.\u00a05).\nFig.\u00a05Characteristics of each coffee cDNA library in comparison to the entire coffee EST-derived unigene set. The total unigene and highly expressed unigene categories sum to greater 100% since the same unigene may contain ESTs from more than one library\nDifferential expression of genes across stages\/tissues\nThe coffee cDNA libraries were non-normalized and deeply sampled, and thus could be used to compare gene expression amongst the five cDNA libraries using statistical method (Audic and Claverie 1997). In all the pair-wise comparison between the cDNA libraries, only a small proportion of the overall unigenes (approximately 5%) showed significantly different (P<0.05) expression levels between two or more libraries (Table\u00a04). The leaf and the early stage cherry libraries showed the highest divergence in gene expression (752 unigenes which is 5.7% of total unigenes, showed significant differential expression) while the leaf and the pericarp libraries had the lowest number of differentially expressed unigenes (384 unigenes and 2.9% of total). Interestingly, the three stages of seed development showed as much differential expression, relative to each other, as they did to non-seed tissues (leaf and pericarp). These results provide strong evidence that throughout the approximately 11\u00a0months from pollination to mature seed, a continuously changing cascade of molecular events is unfolding in the developing seed.\nTable\u00a04Number of coffee unigenes showing significantly (P<0.05) different expression in pairwise comparisons of cDNA librariesLibraryPericarpEarly stage cherryMiddle stage seedLate stage seedLeaf384752548562Pericarp610458527Early stage cherry602728Middle stage seed585\nHighly expressed genes\nTable\u00a05 lists the blast annotation, library composition and the best Arabidopsis\/Solanaceae gene match for the 20 most highly expressed unigenes from the coffee EST database. Some of these highly expressed genes are common to and highly expressed in all plant species\u2014a good example of this is Unigene 122071, which encodes the small subunit of ribulose bisphosphate carboxylase\u2014a key enzyme in photosynthesis and carbon fixation (Table\u00a05). However, a number of these highly expressed genes are unique to coffee, and many have expression patterns confined to particular tissue\/stages, providing a glimpse into some of the potential unique aspects of coffee biology. A brief description of some of these is given below.\nTable\u00a05The 20 most highly expressed coffee unigenes: functional annotation and most similar Arabidopsis and Solanaceae homologsCoffee unigene#: annotationBest match (e value\/score)EST countArbidopsisSolanaceae Unigene_speciesTotal ESTsLeafPericarpEarly stage cherryMiddle stage seedLate stage seed125230: putative 2s seed storage proteinND243065_tomato (e-103\/238)1,219\n7\n15\n21\n1,037\n139\n120912: 11s seed storage proteinAt5g44120 (1e-88\/324)228376_tomato (0\/802)687\n0\n3\n28\n244\n412\n121707: unknown functionAt1g29050 (1e-139\/489)246695_potato (e-163\/283)324\n2\n3\n1\n149\n169\n120118: unknown functionAt5g59320 (2e-21\/99.8)221585_tomato (e-134\/475)292\n0\n0\n3\n58\n231\n124988: unknown functionNDND20458\n84\n55\n1\n6\n120685: chitinaseAt5g24090 (2e-43\/172)214596_tomato (1e-35\/84.5)202\n99\n40\n58\n0\n5\n124158: photoassimilate-responsive proteinAt3g54040 (2e-36\/149)196924_pepper (2e-39\/138)182\n1\n1\n2\n150\n28119890: unknown functionND204426_pepper (5e-07\/52.8)183\n0\n0\n0\n183\n0123265: ADP-ribosylation factorAt2g47170 (1e-99\/359)238338_tomato (0\/693)18258\n14\n65\n21\n24\n124083: secretory peroxidaseAt4g21960 (e-153\/537)196145_pepper (0\/681)161\n55\n5\n19\n49\n33\n124911: metallothioneinAt5g02380 (0.32\/32.3)207464_petunia (2e-06\/51.0)163\n40\n65\n30\n11\n17\n119817: chitinaseAt3g12500 (e-103\/373)248120_potato (e-148\/521)148\n0\n22\n0\n0\n126\n124815: unknown functionAt3g29240 (1e-87\/320)227940_tomato (e-146\/517)145\n2\n0\n1\n0\n142\n122206: SAM synthaseAt2g36880 (0\/711)270415_petunia (0\/887)142\n1\n9\n130\n0\n2\n119460: WRKY4 transcription factorAt1g80840 (3e-75\/279)237166_tomato (e-137\/487)123\n0\n0\n123\n0\n0\n123045: unknown functionAt3g16000 (0.69\/31.2)218824_tomato (90.36\/33.1)123\n81\n17\n22\n3\n0\n120481: AdoMet synthaseAt4g01850 (0\/723)243236_potato (0\/886)108\n19\n27\n31\n20\n11\n121265: Mobl\/phoceinAt5g45550 (e-119\/425)196814_pepper (e-146\/513)113\n1\n0\n112\n0\n0\n124791: plasmodesmal receptorAt5g15140 (1e-99\/360)203764_pepper (8e-86\/314)105\n0\n0\n3\n26\n76122071: rubiso small subunitAt1g67090 (9e-70\/260)207453_petunia (3e-89\/297)99\n76\n8\n9\n6\n0\nBLAST match values are given in parenthesesBold numbers indicate library for which the highest number of ESTs were observed for each gene. Italic numbers indicate library for which the number of EST is significantly lower (P<0.05) than the highest\nSeed storage protein genes\nUnigene 125230: a putative 2S seed storage protein\nUnigene 125230 is the most highly expressed gene across the entire coffee unigene set (1219 ESTs) and was the dominant transcript in the middle stage seed library, accounting for 10% of the ESTs at this stage (Table\u00a05). This gene shows high homology to a tomato unigene derived from a developing seed cDNA library, but has no detectable homolog in Arabidopsis (Table\u00a05). Other than the match with tomato, weak homology was also detected for 2S seed storage proteins from sesame, sunflower, and Brazil nut (in decreasing order of similarity). It is interesting to note that coffee, tomato, sesame and sunflower are fairly closely related taxonomically. All belong to the Asterid I\/II clade of Eudicots (Fig.\u00a01). This close phylogenetic relationship may explain why Unigene 125230 has homologous matches only in these species. Moreover, since Unigene 125230 shows homology to the 2S seed storage proteins in these related species, we conjecture that unigene 125230, its tomato unigene match, the sesame, Brazil nut and sunflower 2S storage protein gene all encode orthologous 2S seed storage proteins. This is the first time that a 2S seed storage protein has been identified in coffee or any Solanaceae species. Finally, a BLAST search of Unigene 125230 against the coffee unigene set revealed additional putative copies of the 2S seed storage protein. However, on close examination, all appear to be splicing variants or low quality sequences. Moreover, southern hybridization with a 2S cDNA probe on genomic DNA confirmed that the 2S gene is single copy in the coffee genome (data not shown).\nUnigene 120912: 11S seed storage protein\nUnigene 120912 is the second most abundant unigene, containing 687 ESTs (Table\u00a05). This gene is preferentially expressed during middle and late stage seed development and shares high similarity (over 98% identity) with a previously cloned C. arabica 11S seed storage protein (Marraccini et al. 1999; Rogers et al. 1999). This unigene also has a highly significant match to the Arabidopsis 12S storage protein and to a tomato unigene derived from seed ESTs (Pang et al. 1988) (Table\u00a05). Given these results, we conclude that unigene 12912 is allelic with the previously described 11S seed storage protein gene from C. arabica and orthologous to 11S\/12S seed storage proteins in both tomato and Arabidopsis. A BLAST search of Unigene 120912 against the coffee unigene set revealed additional putative copies of the 11S seed storage protein. However, like the 2s seed storage protein (Unigene 124230), all appears to be results of alternative splicing or low sequence quality. Moreover, southern hybridization with an 11S cDNA probe on genomic DNA confirmed that the 11S gene is single copy in the coffee genome (data not shown).\nOther seed-specific genes\nEarly stage seed development\nUnigenes 122206, 119460 and 121265 were all highly expressed and specific to the early cherry stage. The early cherry library was derived from RNA from both pericarp and seed tissue while the pericarp library was derived from RNA coming from all stages of pericarp development. Thus, if the above genes were highly expressed in the pericarp of the early cherry, they should be present in the pericarp library as well. The fact that these genes showed little or no expression outside the early cherry stage, suggests that they are probably specific to early developing seed tissues and not pericarp tissues (Table\u00a05). Unigene 122206 showed high homology to an Arabidopsis gene annotated as encoding the enzyme S-adenosyl-l-methionine (SAM) synthetase (Table\u00a05). This enzyme synthesizes S-adenosyl-l-methionine from l-methionine and ATP and is often represented by multiple isozymes in plant species (Schroder et al. 1997). Thus, Unigene 122206 appears to be an SAM synthetase specific to early seed development (Table\u00a05).\nUnigene 119460 shows high homology to the highly conserved WRKY transcription factor family. The WRKY transcription factor is a large gene family having more than 70 members in the Arabidopsis genome (Dong et al. 2003). Previous studies showed that it is related to wounding, stress, pathogen infection and senescence in many plant species. In some recent studies, the WRKY protein family was found to be involved in sugar signaling in barley and seed development in Arabidopsis (Johnson et al. 2002; Sun et al. 2003). However, the function of the best Arabidopsis match to Unigene 119460 (At1g80840) has not been determined. Hence, understanding the function of this highly expressed, WRKY-like coffee gene awaits further study.\nUnigene 121265 is highly homologous to a gene in Arabidopsis annotated as encoding a Mob1\/phocein protein. Mob1\/phocein proteins are found in virtually all eukaryotes. While they are conjectured to be involved in cell cycle control, there is still little experimental evidence demonstrating biological function (Pon 2004). Thus it seems premature to conjecture what role Unigene 121265 might have that is specific to the early development of coffee seeds.\nMiddle stage seed development\nAs described earlier, Unigene 125230 is a putative 2S seed storage protein with peak expression during middle seed development. Also showing preferential expression during this same stage were Unigenes 121707, 124158 and 119890. Unigene 121707 is a gene of unknown function with high homology matches both in Arabidopsis and Solanaceae EST-derived unigenes (Table\u00a05). Unigene 124158 is homologous to an Arabidopsis gene classified as a photoassimilate-responsive protein, which is related to pathogenesis (Herbers 1995). Finally, Unigene 119890, which is also specific to middle stage seed development, is apparently a gene unique to coffee, which will be discussed more in the following section.\nLate stage seed development\nAs discussed previously, Unigene 120912 corresponds to the 11S seed storage protein, which is largely expressed late in seed development. Other genes with preferential expression in late stage seeds are Unigenes 120118, 119817 and 124791. Unigene 119817 likely encodes a chitinase and is further discussed in the next section. Unigene 120118 shows high homology to genes in both Arabidopsis and Solanaceae EST-derived unigene sets; however, none have known function. Unigene 124791 gives a strong match to an Arabidopsis gene annotated as a plasmodesmatal receptor.\nTwo highly expressed genes with homology to chitinase\nUnigenes 120685 and 119817 show high sequence similarity to a number of genes classified as chitinases in other organisms. Chitinases are a large and diverse class of proteins, some of which have been implicated in resistance to fungi in various plant species, including coffee (Rojas-Herrera 2002; Chen et al. 2003). The two unigenes differ in that Unigene 120685 is expressed in leaves, pericarp and early stage cherries, but not in mid or late stage seed development (Table\u00a05). Unigene 119817, on the other hand, was found to be exclusively expressed in late stage developing seeds and pericarp tissue. As previously mentioned, early stage cherries contained both pericarp and seed tissues. The fact that Unigene 120685 was not found in the middle and late stages of seed development suggests that this gene may not be expressed in seeds, but rather in the maternally derived pericarp and leaf tissues. Based on these results, one can speculate that these two putative chitinase genes may be involved in pathogen defense in developing CHERRIES, with Unigene 120685 being expressed in early developing, post pollination pericarp and leaf tissues and Unigene 119817 being expressed primarily late in seed development, just prior to maturity.\nHighly expressed genes unique to coffee\nUnigene 124988\nThis highly expressed unigene had no significant matches in the Arabidopsis proteome, Solanaceae EST-derived databases, GenBank NR databases, or GenBank dbest. Moreover, the predicted protein encoded by Unigene 124988 has no recognizable domains, which might give clues to its function. ESTs for this unigene were detected in all five libraries, with highest expression being observed in the pericarp (Table\u00a05).\nUnigene 119890\nUnigene 119890 also has no significant match in any of the tested databases, with the possible exception of a very weak match in the Solanaceae unigene sets (the best hit was from pepper with an e value of 5e-7, Table\u00a05). Like Unigene 124988, its predicted protein has no recognizable domains. Unigene 119890 was highly and exclusively expressed in the middle stage of developing seeds (Table\u00a05).\nThe fact that neither Unigene 124988 nor Unigene 119890 have counterparts in any other databases suggests that they may represent coffee-specific genes or genes that have been evolving at such a rapid rate that they no longer bear any recognizable homologies with proteins from other plants, including the closely related Solanaceous plants. We speculate that these genes may be related to chemical or morphological features unique to coffee.\nGene families unique or significantly expanded in coffee\nThe predicted protein sequences for the coffee unigene set and the Arabidopsis proteome were combined and organized into protein families using tribeML software (see Materials and methods for details). In most cases, the number of gene family members was higher in arabidopsis than in coffee (data not shown). However, this is to be expected since the entire Arabidopsis genome has been sequenced (thus revealing all genes), whereas in coffee the EST-derived unigene set is by nature incomplete and unlikely to contain all members of any given gene family. However, despite the incomplete nature of the coffee EST-derived unigene set, several cases was encountered in which coffee has more gene family members than does Arabidopsis. To further investigate this topic, a list was made of the gene families for which coffee has significantly more members than Arabidopsis (Table\u00a06). We refer to these as coffee-expanded gene families. In addition, gene families found in coffee, but for which no counterparts exist in Arabidopsis were also compiled (Table\u00a07). We refer to these as coffee-unique gene families. All gene families listed in both Tables\u00a06 and 7 were subjected to manual examination to remove any members determined to be results of alternative splicing and\/or poor sequence quality\u2014both of which could inflate the number of putative gene family members in coffee. Hence, the differences in copy number reported are likely to be due to true differences in gene copy number and not artifacts of EST-based gene predictions.\nTable\u00a06Gene families expanded in coffee relative to Arabidopsis\nFamily ## Arabidopsis family member# Coffee family memberLongest coffee memberAnnotation266121122330Retrotransposon gag protein, class I180514124952Polygalacturonase isoenzyme 1 beta subunit with BURP domain632112123451Acidic endochitinase386210124158Photoassimilate-responsive protein38248119672Hypersensitive-induced protein, band 7 protein39427122791E-class P45048326120054Bet v I allergen62336119581Root hair defective protein1,18215126674Unknown function69525126974Tyrosine decarboxylase78325122423Unknown function1,11725119449Trypsin inhibitor KunitzTable\u00a07Gene families unique to coffee in comparison to Arabidopsis\nGene family ## Family memberLongest memberSolanaceae hitAnnotation24327122956258190 potatoRetrotransposon gag protein, class II68711120121221585 tomatoThaumatin, pathogenesis related96510119718249401 potatoZn-finger, CCHC type974101202442610402 potatoDisease resistance protein (TIR-NBS-LRR class)8529119638225732 tomatoRetrotransposon gag protein, classs III360812199823671 tomatoDisease resistance protein1,0197124574222350 tomatoLeucine-rich repeat, disease resistance protein1,6077122216noneUnknown function1,6107130519noneUnknown function1,6767126264243065 tomatoUnknown function7086123769236157 tomatoABA\/WDS induced protein1,8525120284213688 tomatoProline-rich region, extension-like protein2,3625122218237314 tomatoUnknown function2,4595124466267984 potatoLeucine-rich repeat, plant specific, receptor-related protein kinase\nCoffee-expanded gene families\nThe most expanded gene family in coffee corresponds to a retrotransposon gag protein (Table\u00a06). This result has two implications. First, the retroelement encoding this gag protein occurs at a higher frequency in coffee compared with Arabidopsis, although we cannot determine whether this difference is due to a true expansion of this element in coffee subsequent to divergence from Arabidopsis, or rather a loss of the element in the Arabidopsis lineage. Second, the fact that this retrotransposon gag protein element was discovered in an EST-database indicates that this particular retroelement is being transcribed in the coffee genome, and hence may represent an active retrotransposon.\nAnother gene family for which coffee has significantly more members than Arabidopsis, encodes proteins annotated in Arabidopsis as acid endochitinases and photoassimilate-responsive proteins (Table\u00a06). As noted earlier, chitinases are associated with fungal resistance and are among the most highly expressed genes in coffee. The fact that chitinases are both highly expressed and represented by an expanded gene family in coffee may reflect a greater need for fungal resistance engendered both by the perennial nature of coffee and the fact that it is a tropical species for which a multiplicity of fungal pathogens is common. The reasons for the putative expansions of the other gene families listed in Table\u00a06 remain for future studies to determine.\nCoffee-unique gene families\nTable\u00a07 lists the top gene families (based on copy number), which occur in coffee, but not in Arabidopsis. Of the 15 gene families listed, four are of unknown function. For those that could be functionally annotated, five (45%) have putative functions related to disease resistance, such as TIR-NBS-LRR disease resistance proteins, LRR proteins and thaumatin pathogenesis-related proteins (Table\u00a07). These finding are consistent with rapid evolution of genes\/gene families related to disease resistance, likely driven by selection pressure from continuously changing pathogens and\/or pathogens unique to the particular environments of a species (Meyers 1998; Michelmore and Meyers 1998). Also included in this list of coffee-unique gene families are two, which encode retrotransposon gag-proteins (Table\u00a07).\nComparison of the coffee gene repertoire with that of Arabidopsis and Solanaceae species\nEach coffee unigene was screened against the Arabidopsis proteome using BLASTX, which compares all six translated frames of the query with the target. In total, 21% of the coffee unigenes had no clear match in Arabidopsis (match score 0\u201330) (Fig.\u00a06). These coffee genes, which lack an Arabidopsis counterpart, may have evolved rapidly since coffee and Arabidopsis diverged from their last common ancestor, and hence their counterparts in each genome are no longer recognizable by standard string searches like BLAST. To investigate this topic further, the 2,853 unigenes with no clear match in Arabidopsis (match score<30, see Fig.\u00a06) were screened against the Solanaceae EST-derived unigene sets using TBLASTX which compares all six translated frames of both query (coffee unigenes) and target (Solanaceae unigenes, see Materials and methods for details) sequences. The family Solanaceae is much more closely related to coffee than is Arabidopsis (Fig.\u00a01), and hence coffee genes without a match in Arabidopsis may well have recognizable orthologs in the Solanaceae. The results show that a large portion (90%) of the coffee genes that lack a match in Arabidopsis do have a match in Solanaceae at the same stringency criteria (match score>30). For many of these cases, however, the coffee-Solanaceae matches are still quite diverged (low match scores) suggesting that the proteins encoded by these genes have been evolving at a rapid rate in dicots.\nFig.\u00a06Histogram showing match scores for each coffee unigene as compared with its best match in the Arabidopsis proteome\nThere were, however, some exceptions to this trend represented by coffee genes having no match in Arabidopsis, yet a strong match in Solanaceae. These latter cases may represent genes that have been under differential selection pressure since the time that the Rosids (which includes Arabidopsis) diverged from the last common ancestor with the Asterids (which includes Solanaceae and coffee) (Fig.\u00a01). Alternatively, these genes may have been lost in the lineage leading to Arabidopsis, but retained in the Asterids. In either case, such genes may have functional roles that are, in part, responsible for the morphological, developmental and chemical characteristics that differentiate Arabidopsis from Rosid species (e.g. coffee, Solanaceae). In an effort to shed more light on this issue, the 20 coffee unigenes having the highest match scores with Solanaceae, but lacking a counterpart in Arabidopsis were parsed and annotated (Table\u00a08). Seven of these coffee genes have no match either in GenBank NR or dbest hence their function is unknown. Another unigene (Unigene 125230, the 2s seed storage protein, see above discussion) had weak match to ESTs of sesame, a species also belonging to the Asterid I clade (Fig.\u00a01). Thus, these eight genes (40%) appear to be specific to the tomato\/coffee\/sesame Asterid I clade (Fig.\u00a01, Table\u00a08). Elucidating their function may shed light on the molecular processes, which diversified early in the evolution of dicot species, especially those species belonging to the Asterid clade of dicots.\nTable\u00a08Coffee genes not found in Arabidopsis, but with conserved counterparts in tomato or other Solanaceous speciesCoffee unigeneSolanaceae EST-derived unigene matchScoreGenBank (non-redundant and dbest) best matchScoreAnnotation124978240871 tomato454Unknown function121324235756 tomato429gblCB686389.1 [Brassica napus]44Unknown function131820213100 tomato426gil50252229.1 [Oryza sativa]73Unknown function121542240321 tomato416refiNP_922676.1 [Oryza sativa]75Unknown function131934219759 tomato377embiCAE05735.1 [Oryza sativa]297TFIIH basal transcription factor p52 subunit121140236347 tomato320Unknown function131445225435 tomato320Unknown function125230243065 tomato238gil13183175 [Seasame indicum]452S albumin131030246364 potato213refINP_524404.1 [Drosophila melanogaster]110Phospyhatidyl inositol transfer protein120120237254 tomato202gbICF349465.1 [Rose]52Unknown function126635237314 tomato185Unknown function126575237314 tomato182Unknown function130675209387 petunia177gbICK093976.1 [Populus tremula]438Unknown function128020237314 tomato167Unknown function123615249253 potato163gbIAAO73272.1 [Oryza sativa]140Unknown function126432240551 tomato163gil34878866 [Rattus norvegicus]56Phosphatidylinositolglycan class N124384197378 pepper156gblCA815435.1 [Vitis vinifera]1,009Unknown function122126239632 tomato153Unknown function131601232010 tomato145gbICK229938.1 [Macaca mulatta]7440S Ribosomal protein S21119644237150 tomato143refINP_921250.1 [Oryza sativa]70HelicaseThe GenBanks Best match exclude those from Solanaceae, Coffea and Hedyotis (both members of the Rubiaceae family). Solanaceae EST-derived\nSurprisingly, 8 (40%) of the 20 coffee genes having no match in Arabidopsis did have matches in species phylogenetically distant from both coffee and Arabidopsis, including two to non-plant species (Drosophila and rat) (Table\u00a08). Five of these matches were to rice genes, which is a monocot and highly divergent from coffee, Arabidopsis and other dicot species (Table\u00a08). The fact that all of these species diverged from Arabidopsis and coffee long before the latter diverged from each other suggests that these genes may have been present in the last common ancestor of Arabidopsis and coffee\/Solanaceae, but subsequently lost in the Arabidopsis lineage.\nCoffee genes share greater similarity to genes in tomato\/Solanaceae than to Arabidopsis\nAs discussed earlier, coffee is much more closely related to the Solanaceae than to Arabidopsis (Fig.\u00a01). Hence, Solanaceae species may be better models for coffee genomics than Arabidopsis. The results for fast-evolving genes, presented above, are consistent with this prediction. To further investigate this assertion, the degree to which each coffee unigene matched Arabidopsis versus Solanaceae was investigated. In doing this analysis, one has to keep in mind that the entire gene repertoire of Arabidopsis is known, whereas the EST-derived unigene sets for Solanaceae do not represent the entire gene repertoire of these species. We estimate that the combined EST-derived unigene sets of Solanaceae species represent as much as three-quarters of the Solanaceae gene content (Hoeven et al. 2002). Moreover, Arabidopsis genes are of full length, while Solanaceae EST-derived unigenes are not necessarily of full length.\nTo mitigate against these factors, each coffee unigene was first screened, via TBLASTX, against the Solanaceae EST-derived unigene databases. Only those coffee unigenes matching a Solanaceae unigene with a match score greater than 100 were compared with the Arabidopsis proteome. Moreover, the match to Arabidopsis was confined to only the portion of the coffee unigene matching a Solanaceae EST-derived unigene. The ratio of the Arabidopsis match score divided by the Solanaceae match score was used as a measure of the degree to which the Solanaceae homolog shows greater similarity to the coffee unigene than to the closest Arabidopsis homolog. The results of this analysis are shown in Fig.\u00a07. The ratio of the Arabidopsis\/Solanaceae match score averaged 0.75, indicating a significantly better match in most cases with Solanaceae versus Arabidopsis. Moreover, in more than 95% of the cases, the ratio was less than one, indicating that, in most instances, coffee contains genes significantly more homologous to Solanaceae than to Arabidopsis. For those cases in which the ratio was greater than 1 (5%), we cannot rule out the possibility that the true ortholog was missing in the Solanaceae EST-derived unigene sets, hence the comparison was between paralogs. These results, combined with the results on fast evolving genes presented earlier, provide clear evidence that the Solanaceae provide a much better model than Arabidopsis for genomic and biological studies in coffee. This is especially relevant as sequencing of the tomato genome is currently underway (http:\/\/www.sgn.cornell.edu\/help\/about\/tomato_sequencing.html).\nFig.\u00a07Ratio of highest Arabidopsis match score to highest Solanaceae match score for individual coffee unigene. The analysis restricted to coffee unigenes with a Solanaceae match score >100\nConclusions\nHerein, we describe the development and analysis of a large EST database for coffee. The resulting 47,000 ESTs correspond to 13,175 unique genes (unigenes), a large portion of which are expressed during seed development\u2014a stage important to coffee as a crop and one for which our understanding of molecular development is still rudimentary. To our knowledge, this is the largest public database for seed-derived ESTs. Hence, this EST database represents a new public resource, which can facilitate a better understanding of seed development, as well as genomic, molecular and breeding research in coffee. By comparisons with Arabidopsis and Solanaceous species, we have identified the two major seed storage proteins of coffee (2S and 11S) and demonstrated that these proteins are expressed at different times during seed development. Through in silico gene expression analysis, we have identified a number of highly expressed genes that show high specificity for different stages of seed development as well as for the pericarp tissue that surrounds the seeds. Many of these highly expressed genes are unique to coffee and\/or the Asterid clade of higher plants. While the functions of most of these highly expressed, tissue\/stage specific genes remain to be determined, the fact that they have been identified points the way to promoters, which can potentially be used to drive gene expression in specific stages\/tissues of the coffee plant. Many of these genes are specific to defined periods of seed and\/or pericarp development\u2014both critically important for insect\/pathogen resistance and in determining the quality of the coffee bean with respect to commercial coffee products.\nCoffee, as a member of the family Rubiaceae, is distantly related to the model species Arabidopsis. A computational comparison of the coffee EST-derived unigene set with the sequence databases for Arabidopsis and Solanaceous species (e.g. tomato, pepper), indicate that the latter are much better genomic models for coffee than is Arabidopsis. These results are consistent with the fact that coffee and solanaceous species share very similar chromosome architecture and are closely related, both belonging to the Asterid I clade of dicot plant family. Moreover, the ability to identify orthologous genes between coffee and tomato opens the door to eventually developing detailed comparative maps for these two species and to the sharing of genomic and biological tools\/discoveries\u2014an outcome that should expedite research in both taxa.","keyphrases":["solanaceae","coffea canephora","seed development","rubiaceae","comparative genomics"],"prmu":["P","P","P","P","R"]}
{"id":"Purinergic_Signal-2-2-2096639","title":"The CD38\/CD157 mammalian gene family: An evolutionary paradigm for other leukocyte surface enzymes\n","text":"Human CD38 is the mammalian prototype of a family of phylogenetically conserved proteins which share structural similarities and enzymatic activities involved in the production of an intracellular second messenger with calcium mobilizing effects. Engagement of CD38 by agonistic monoclonal antibodies and the CD31 ligand initiates a cytoplasmic signaling cascade involving tyrosine phosphorylation of the proto-oncogene c-cbl and of the extracellular regulated kinase 1 of 2 complex. Further requirements for signal transduction include a privileged localization within the cholesterol-rich areas of the plasma membrane and physical association with specialized surface receptors. CD38-mediated signals are crucial in heterotypic cell adhesion and migration as well as in the activation of proliferation\/survival programs by normal and neoplastic cells. Here we review the most recent literature on this complex topic and attempt to formulate a single model reconciling the enzymatic and receptorial activities of CD38.","keyphrases":["calcium mobilization","cell surface enzymes","cell surface receptors","intracellular signaling"],"prmu":["P","R","R","R"]}
{"id":"Neuroradiology-4-1-2275774","title":"Angiographic CT with intravenous administration of contrast medium is a noninvasive option for follow-up after intracranial stenting\n","text":"Intracranial angioplasty and stenting (ICAS) is a therapeutic option in symptomatic intracranial atherosclerotic disease. Adequate follow-up examination is necessary to exclude in-stent restenosis. Conventional intraarterial digital subtraction angiography (ia-DSA) is the current gold standard, but it is an invasive technique and carries the risk of neurological complications. Angiographic CT (ACT) is a new technique that provides a volume dataset of the highest spatial resolution and high contrast resolution derived from a rotational acquisition of a c-arm-mounted flat-panel detector. The feasibility of ACT with intravenous administration of contrast medium (iv-ACT) for follow-up after ICAS is demonstrated. In two patients iv-ACT was performed as a follow-up examination 12 months after ICAS. High-resolution volume data from the rotational acquisitions were processed to provide delineation of the stent lumen as well as imaging of the brain parenchyma and vessels. In both patients the patency of the stent lumen was assessed successfully. In addition, all other brain vessels were displayed in a manner similar to their appearance on CT angiograms. The brain parenchyma was also adequately imaged in a manner similar to its appearance on CT images. We demonstrated the feasibility and diagnostic value of iv-ACT for follow-up imaging after ICAS. This new application has the potential to become the imaging method of choice after ICAS since it not only enables visualization of the patency of the stent lumen but also is minimally invasive and provides additional information about all brain arteries and the brain parenchyma.\nIntroduction\nIn symptomatic intracranial atherosclerotic disease refractive to medical treatment intracranial angioplasty and stenting (ICAS) is a therapeutic option increasingly used in specialized stroke centers. However, restenosis rates ranging from 8% to 30% have been reported, underlining the necessity for regular follow-up examinations to exclude significant restenosis [1\u20134]. CT angiography (CTA) and MR angiography (MRA) are known to suffer from considerable artifacts that often prevent assessment of the stent lumen. Therefore, current follow-up techniques include transcranial Doppler sonography and conventional intraarterial digital subtraction angiography (ia-DSA). However, diagnostic ia-DSA is an invasive technique with a rate of examination-related neurological complications ranging from 0.5% to 0.8% [5, 6]. In addition, ia-DSA is not offered on an outpatient basis at every medical center so patients have to be admitted for at least one night, significantly increasing the treatment costs. Furthermore, patient compliance with follow-up examinations may be a problem because the ia-DSA procedure is not very comfortable.\nThe new angiographic systems equipped with flat-panel detectors which allow rotational acquisition of volume data provide the possibility of CT-like low-contrast imaging [7]. The administration of even highly diluted contrast medium intraarterially gives good delineation of the vessels indicating that intravenous administration of contrast medium could be sufficient. We sought to combine this property with the possibility of nearly artifact-free imaging of small-vessel stents described previously [8].\nMethods\nDSA and angiographic CT (ACT) examinations were performed on an AxiomArtis dBA biplane angiography system equipped with flat panel detectors (Siemens Medical Solutions, Forchheim, Germany). The acquisition of rotational datasets was performed with the following parameters (20s-1k protocol): 20\u00a0s rotation, 538 projections, 220\u00b0 total angle, no zoom (detector size 30\u00d740\u00a0cm), CTDIweighted approximately 22\u00a0mGy (manufacturer\u2019s information). Postprocessing of the image data to a volume dataset (ACT) was performed on a Leonardo workstation (DynaCT, InSpace 3D software, Siemens). The software includes the application of system-specific filter algorithms in order to correct for beam hardening, radiation scatter, truncated projections and ring artifacts. Postprocessing resulted in volume datasets each defined by a batch of about 400 slices in a 512\u00d7512 matrix. Voxel size was about 0.1\u00d70.1\u00d70.1\u00a0mm3. The ACT datasets were further processed to multiplanar reconstructed (MPR) slices with the thin-slice and thick-slice maximum intensity projection (MIP) technique parallel and perpendicular to the stent-supplied section of the vessel, respectively. The contrast medium used in the follow-up ACT examination was Imeron 350 (Bracco ALTANA Pharma, Konstanz, Germany). The contrast medium was injected to a volume of 100\u00a0ml into a cubital vein at a flow rate of 5\u00a0ml\/s, and the start delay for rotational acquisition was 20\u00a0s. The patient was asked to close his or her eyes and to breathe calmly during the examination.\nCTA was performed on a 16-slice CT scanner (Aquilion, Toshiba Medical Systems, Tokyo, Japan) with 0.5-mm slice collimation, intravenous injection of 60\u00a0ml Imeron 350 (Bracco ALTANA Pharma), and a CTDIvol of approximately 50\u00a0mGy [9]. Postprocessing was performed on a Vitrea II workstation (Vital Systems, Minnetonka, Minn.).\nCase illustrations\nCase 1\nA 57-year old man suffering from brainstem infarction and recurring transient ischemic attacks of the vertebrobasilar circulation was admitted to our hospital. Transcranial ultrasonography and MRA revealed a high-grade stenosis of the basilar artery. The symptoms occurred despite double platelet inhibition (aspirin and clopidogrel) and ICAS was chosen as therapy. The procedure was performed under general anesthesia. A balloon-mounted Pharos (3.5\/8\u00a0mm) stent (Micrus Endovascular, Renens, Switzerland) was placed and balloon-inflated to a diameter of 3.2\u00a0mm thereby reconstructing a diameter corresponding to about 90% of the normal vessel diameter (Fig.\u00a01a,b). The successful deployment of the stent was documented by native ACT (Fig.\u00a01c,d). No complications occurred after the intervention and the patient was discharged 3\u00a0days later.\nFig.\u00a01Patient 1. a, b DSA with selective injection of contrast medium into the right vertebral artery shows the eccentric high-grade stenosis of the basilar artery (a). After stent placement (b) the lumen of the basilar artery is well reconstructed and considerable inflow from the contralateral vertebral artery can be seen. c, d Reformatted images in the sagittal plane from an ACT scan directly following the intervention reveal regular deployment of the stent (c MIP, 0.5-mm slice thickness; d MIP, 5-mm slice thickness)\nAn outpatient follow-up CTA was performed 6\u00a0months after the procedure. The stent lumen was not adequately visualized on thin-slice MPR (Fig.\u00a02a). Therefore, admission for follow-up DSA was organized for 2\u00a0months later. The patient refused ia-DSA, so ACT with intravenous administration of contrast medium (iv-ACT) was offered as an alternative. Written informed consent for the examination as a novel application of a rotational acquisition was obtained. The examination was successful. Corresponding to the good low-contrast resolution of ACT described previously, the reconstructed images revealed full patency of the stent lumen (Fig.\u00a02b,c) [10]. This result was highly correlated with the flow-rate measured by transcranial Doppler sonography that had remained normal from directly after the intervention until the 8-month follow-up.\nFig.\u00a02Patient 1. a CTA image obtained at the 6-month follow-up is not of sufficient quality to reveal the stent lumen, and in particular the proximal and distal markers produce substantial artifacts (MIP, 1-mm slice thickness). b, c iv-ACT images obtained at the 8-month follow-up show an unchanged configuration of the stent in comparison to the examination performed directly after the intervention (see Fig.\u00a01c,d). The contrast filling is more faint than on the CTA image but the patency of the stent lumen is visible much more clearly on the sagittal cross sections, and the artifact load is acceptable (b MIP, 0.5-mm slice thickness; c MIP, 5-mm slice thickness)\nCase 2\nAn 82-year-old man was admitted suffering from progressive left-sided hemiparesis and impaired consciousness. CTA revealed a high-grade stenosis of the right middle cerebral artery (MCA) and a second middle-grade stenosis of the basilar artery (data not shown). CT-perfusion (CTP) showed a prolonged mean transit time in the whole right MCA territory but only small infarcted areas level with the basal ganglia, represented by lowered relative cerebral blood volume. Emergency ICAS was performed under general anesthesia. A balloon-mounted Driver (2.5\/8\u00a0mm) stent (Medtronic, Minneapolis, Minn.) was successfully placed in the stenosis of the right M1 segment leading to a good morphological result and immediate restoration of normal blood flow (Fig.\u00a03a,b). Proper stent deployment was documented by native ACT (Fig.\u00a03c,d).\nFig.\u00a03Patient 2. a, b DSA images with selective injection into the right internal carotid artery reveals a high-grade stenosis of the right middle cerebral artery (MCA) with reduced distal blood flow (a). After stent placement the blood flow is normalized (b). c, d Native ACT reformatted images in paracoronal orientation reveal regular deployment of the stent (c MIP, 0.5-mm slice thickness; d MIP, 5-mm slice thickness)\nThe patient was admitted for follow-up DSA 12\u00a0months after the procedure. Transcranial Doppler sonography showed a normal flow velocity in the right MCA. This patient also refused ia-DSA. Written informed consent was obtained in advance to perform iv-ACT and to omit ia-DSA if the iv-ACT was of diagnostic quality. The procedure was performed successfully and the reconstructed images revealed full patency of the stent lumen (Fig.\u00a04a,b). All other intracranial arteries were visible at the highest resolution, revealing an unchanged configuration of the medium-grade stenosis of the basilar artery (Fig.\u00a04c,d). The reconstructed brain parenchyma images showed already known residual infarction (data not shown).\nFig.\u00a04Patient 2. a, b Iv-ACT images obtained 12\u00a0months after the intervention reveal an unchanged stent configuration and full patency of the stent lumen (a MIP, 0.5-mm slice thickness; b MIP, 10-mm slice thickness). Even small perforating arteries originating through the stent struts can be seen (barrow). c A transverse thick-slice (30\u00a0mm) MIP overview image gives a good impression of the insular MCA branches of both sides. d A second thick-slice (15\u00a0mm) MIP image oriented parallel to the basilar artery reveals a medium-grade stenosis (arrow) and an anatomic variant of the origin of the superior cerebellar artery\nDiscussion\nIn patients suffering from intracranial atherosclerosis, ICAS is a promising new therapeutic option [1, 3, 11]. To identify an in-stent restenosis a reliable imaging technique is needed. Here, DSA is the current gold standard, but it carries a 0.5% to 0.8% risk of permanent neurological impairment [5, 6]. Additionally, not every center is able to perform ia-DSA on an outpatient basis. The risk may be less with ia-ACT if the injection is performed in the aortic arch. However, this is still an invasive procedure.\nIn the study reported here we demonstrated that iv-ACT can reliably depict the lumen of small-vessel stents with high spatial resolution. As the low contrast resolution of ACT is comparable to that of conventional CT, a relatively low concentration of intravascular contrast medium is needed. All intracranial vessels can be viewed simultaneously in a high-quality image. CT-like images of the brain parenchyma are provided as well [10]. It therefore represents a new radiological imaging technique between CTA and ia-DSA.\nEven though this new method needs to be evaluated in a larger number of patients, it possibly has many advantages for patients and physicians as a new option in the imaging of cerebrovascular disease: Examinations can be performed on an outpatient basis and the risk of neurological complications is lowered practically to zero. The examination can be performed quickly, in a time similar to that required for a CTA examination. The radiation exposure for the patient from a rotational acquisition is lower than that from a CTA examination (manufacturer\u2019s information) and has been shown to be considerably lower than that from a single biplane DSA series [12].\nPossible drawbacks of the iv-ACT technique first include movement artifacts. The patient has to lie nearly motionless for about 20\u00a0seconds, which requires a high level of compliance and is not always feasible in patients suffering from cerebrovascular disease.\nThe first stent we examined is a relatively new balloon-mounted stent designed for intracranial use (Pharos, Micrus Endovascular, Renens, Switzerland). All dedicated intracranial stents (Neuroform and Wingspan, Boston Scientific, Natick, Mass.; LEO, BALT, Montmorency, France; and Enterprise, Cordis, Miami Lakes, Fl.) have a similar strut size of about 0.06\u00a0mm [13]. In contrast, common coronary stents have thicker struts (e.g. Driver, Medtronic, Minn.: 0.09\u00a0mm) [14]. Our second patient demonstrates that the lumen of these stents can be visualized as well. This issue is not unimportant because until recently dedicated intracranial stents were not available. Therefore, the majority of stents implanted into intracranial vessels are still coronary stents.\nIv-ACT is a promising new technique for minimally invasive follow-up after intracranial stenting. Other possible applications can be envisaged, such as diagnostic and follow-up imaging before and after endovascular treatment of intracranial aneurysms or arteriovenous malformations.","keyphrases":["intracranial stenting","flat-panel","cerebrovascular disease","rotational angiography","angiographic computed tomography"],"prmu":["P","P","P","R","M"]}
{"id":"J_Fluoresc-4-1-2266787","title":"Spectrofluorimetric Determination of Oxamniquine in Dosage Forms and Spiked Human Plasma through Derivatization with 1-dimethylaminonaphthalene-5-sulphonyl chloride\n","text":"A sensitive, simple and selective spectrofluorimetric method was developed for the determination of oxamniquine (OXM) in pharmaceutical formulations and biological fluids. The method is based on the reaction between the drug and 1-dimethylaminonaphthalene-5-sulphonyl chloride (dansyl chloride) in presence of 0.5 M sodium carbonate (pH 10) to yield a highly fluorescent derivative that is measured at 445 nm after excitation at 335 nm. The different experimental parameters affecting the development and stability of the reaction product were carefully studied and optimized. The fluorescence concentration plot was rectilinear over the range of 0.02\u20130.2 \u03bcg ml\u22121 with a lower detection limit (LOD) of 0.007 \u03bcg ml\u22121 and limit of quantitation (LOQ) of 0.02 \u03bcg ml\u22121. The proposed method was successfully applied to the analysis of commercial capsules. The results obtained were in good agreement with those obtained using the official spectrophotometric method. Furthermore, the method was applied for the determination of oxamniquine in spiked human plasma, the mean % recovery (n = 4) is 97.77 \u00b1 1.19. A proposal of the reaction pathway was presented.\nIntroduction\nOxamniquine (OXM), 1,2,3,4-tetrahydro-2[(isopropylamino methyl]-7-nitro-6-quyinoline methanol is an antischistosomal agent that is indicated for the treatment of schistosoma mansoni (intestinal schistosomiasis) infection (Fig.\u00a01). It has been shown to inhibit DNA, RNA and protein synthesis in schistosomes. The oral bioavailability of oxamniquine is good and effective plasma levels are achieved in 1\u20131.5\u00a0h [1].\nFig.\u00a01Structural formula of oxamniquine\nOxamniquine is the subject of a monograph in the USP (XXIII) [2] whereby a spectrophotometric method is recommended for its determination, whether in its pure form or in capsules. Several methods have been published for the determination of OXM, either per se or in pharmaceutical preparations and biological fluids. These methods include: spectrophotometry [3\u20135], non-aqueous titration [6], gas chromatography [7], HPLC [8\u201313], capillary electrophoresis [11], polarography [14], flow injection analysis [15], cyclic voltammetry [16] and fluorimetry [17].\nAll these methods are either insufficiently sensitive or tedious and require highly sophisticated and dedicated instrumentation [8\u201313]. This led us to study the reaction of oxamniquine with dansyl chloride to develop simple and sensitive spectrofluorimetric method for its determination in pharmaceutical preparations and biological fluids. Dansyl chloride is a useful derivatizing agent for primary amines, secondary amines, imidazoles and phenols, etc.. Several pharmaceutical compounds have been determined through this approach [18\u201324].\nThe reported spectrofluorimetric method for oxamniquine [17] is tedious and time consuming. It involves the use of 2-cyanoacetamide, a hazard reagent.\nExperimental\nApparatus\nThe spectrofluorimetric measurements were recorded using ARF-1501 Shimadzu Spectrofluorometer, equipped with Xenon arc lamp.\nMaterials and reagents\nAll reagents and solvents were of analytical reagent grade\nOxamniquine pure sample was kindly provided by Pfizer (Sandwich, UK).Capsules containing 250\u00a0mg of Oxamniquine each (Vansil capsules) were obtained from commercial sources in the local market.1-Dimethyl aminonaphthalene-5-sulphonyl chloride (dansyl chloride), purchased from Sigma (St. Louis, USA). A stock solution containing 0.1% of dansyl chloride was freshly prepared in acetone and was further diluted with the same solvent to obtain 0.001% solution.Sodium carbonate (BDH, UK) 0.5\u00a0M aqueous solution (pH\u00a010).Sodium hydroxide (BDH, UK) 1\u00a0M aqueous solution.Isobutylmethyl ketone (IBMK) (Merck, Darmstadt, Germany).Plasma was kindly provided by Mansoura University Hospital, and kept frozen until assay after gentle thawing.Methanol and diethyl ether (Merck, Darmstadt, Germany).\nStandard solutions\nA stock solution was prepared by dissolving 20.0\u00a0mg of OXM in 20\u00a0ml of acetone and 80\u00a0ml of 0.5\u00a0M sodium carbonate solution. This solution was further diluted with the same solvent mixture as appropriate. The standard solutions were stable for seven days when kept in the refrigerator.\nGeneral procedure\nAliquots of OXM standard solution were transferred into a series of 10\u00a0ml volumetric flasks. 0.7\u2009\u00b1\u20090.1\u00a0ml of 0.001% of dansyl chloride reagent was added, followed by 0.4\u00a0ml of acetone and mixed well. The reaction mixture was left for 30\u00a0min, and then completed to the mark with IBMK. the reaction mixture was allowed to stand for 10\u00a0min. The fluorescence intensity of the reaction product was measured at 445\u00a0nm after excitation at 335\u00a0nm. Blank experiment was carried out simultaneously. The corrected fluorescence intensity was plotted vs the final drug concentration (\u03bcg ml\u22121) to get the calibration graph. Alternatively, the corresponding regression equation was derived.\nApplications\nProcedure for commercial capsules\nThe content of ten capsules were emptied, and mixed well. A weighed quantity of the powder equivalent to 5.0\u00a0mg OXM was transferred into a small conical flask, and extracted with 2\u2009\u00d7\u200910\u00a0ml of methanol. The extract was filtered into 25\u00a0ml volumetric flask. The conical flask was washed with several milliliters of methanol. The washing was passed into the same volumetric flask, and the combined extract was evaporated to dryness on a boiling water bath. The residue was dissolved and diluted to volume with a mixture of 5\u00a0ml of acetone and 20\u00a0ml of 0.5\u00a0M of sodium carbonate solution. Aliquots covering the working concentration range (cited in Table\u00a01) was transferred into 10\u00a0ml volumetric flasks. The \u201cGeneral procedure\u201d was then applied. The nominal content of the capsules was determined either from the calibration curve or using the corresponding regression equation.\nTable\u00a01Performance data of the proposed methodParameterProposed methodConcentration range (\u03bcg ml\u22121).0.02\u20130.2Minimum detection limit, LOD (\u03bcg ml\u22121).0.007Limit of Quantification , LOQ(\u03bcg ml\u22121).0.02Correlation coefficient ( r).0.9999Slope3,473.114Intercept0.574Sy\/x1.273Sa7.733Sb8.965% Error0.28Sy\/x = standard deviation of the residualsSa = standard deviation of the intercept of regression lineSb = standard deviation of the slope of regression line% Error = RSD%\/\u221a n\nProcedure for spiked human plasma\nA stock solution containing 20\u00a0\u03bcg ml\u22121 of OXM was prepared. Control samples of plasma was spiked with different quantities of OXM to give a final drug concentration cited in Table\u00a05. One molar NaOH (0.8\u00a0ml) was added to 1.0\u00a0ml of the spiked plasma and shaken genteelly. The solution was vortexed with 3\u2009\u00d7\u20095\u00a0ml of diethylether for 2\u00a0min, then centrifuged at 2,500\u00a0rpm for 5\u00a0min. The resulting supernatant was evaporated to dryness under nitrogen at ambient temperature. The residue was dissolved and diluted to volume with a mixture of 5\u00a0ml of acetone and 20\u00a0ml of 0.5\u00a0M of sodium carbonate solution. Aliquots covering the working concentration range was transferred into 10\u00a0ml volumetric flasks. The recommended procedures were then applied. The nominal content of the drug was determined using the corresponding regression equation.\nResults and discussion\nDansyl chloride was first introduced for the determination of some primary, secondary amines, imidazoles and phenols [18\u201320].\nIn recent reports. DNS was further used as a fluorogenic reagent for the determination of some pharmaceutical compounds [21\u201324].\nIn the present study, OXM was found to react with Dansyl chloride at pH\u00a010.0 forming a highly yellow fluorescent derivative with \u03bb maximum emission at 445\u00a0nm after excitation at 335\u00a0nm (Fig.\u00a02).\nFig.\u00a02Fluorescence spectra of A and B excitation and emission spectra of blank. Fluorescence spectra of A\u2032 and B\u2032 excitation and emission spectra of oxamniquine (0.2\u00a0\u03bcg ml\u22121) with dansyl chloride at pH\u00a010.0\nOptimization of experimental parameters\nThe spectrofluorimetric properties of the colored product as well as the different experimental parameters affecting the development of the reaction product and its stability were carefully studied and optimized. Such factors were changed individually while the others were kept constant. The factors include pH, concentration of the reagent, type of buffer, temperature, reaction time and dilution time.\nEffect of pH\nThe influence of pH on the fluorescence intensity of the reaction product was studied. Maximum fluorescence intensity was obtained upon using mixture of acetone and 0.5\u00a0M sodium carbonate solution. The pH of the reaction mixture was found to be 10.0. pH\u00a010 was found to be the optimum pH for dansylation because labeling of most amino acids, amines, imidazoles and phenols has been found to be optimal at pH\u00a09.5\u201310.5 [25]. The rate of dansylation process was found to increase with increasing the pH value this is due to an increase in the rate of hydrolysis of dansyl chloride into dansyl hydroxide [25]. The latter shows strong fluorescence and hence interferes seriously in the determination. However, under the proposed chosen conditions and wavelengths used, there was no interference arising from any dansyl hydroxide formed, as indicated by the low fluorescence intensity of the reagent.\nEffect of concentration of dansyl chloride\nThe influence of the concentration of dansyl chloride was studied using different volumes of 0.001% of the reagent solution. It was found that, the reaction of dansyl chloride with OXM started upon using 0.1\u00a0ml of the reagent in the presence of sodium carbonate (pH\u00a010.0). Increasing the volume of the reagent, produces a proportional increase in the fluorescence intensity of the reaction product up to 0.6\u00a0ml and remains constant up to 0.8\u00a0ml. Therefore, 0.7\u2009\u00b1\u20090.1\u00a0ml of 0.001% of dansyl chloride solution was chosen as the optimal volume of the reagent (Fig.\u00a03).\nFig.\u00a03Effect of volume of dansyl chloride (0.001%) on the fluorescence intensity of the reaction product of oxamniquine (0.2\u00a0\u03bcg m\u22121) at pH\u00a010.0\nEffect of temperature\nIncreasing the reaction temperature higher than the room temperature would result in a subsequent decrease in the fluorescence intensity of the reaction product.\nEffect of reaction time\nDifferent time intervals were tested to ascertain the time after which the solution attains its highest fluorescence intensity. It was found that after 30\u00a0min, the reaction product reaches the highest fluorescence intensity (Fig.\u00a04) and remains stable at room temperature for 20\u00a0min.\nFig.\u00a04Effect of reaction time of on the fluorescence intensity of the reaction product of oxamniquine (0.2\u00a0\u03bcg ml\u22121) with dansyl chloride\nEffect of diluting solvent\nDifferent solvents were tried to dilute the reaction mixture through out the study. It was observed that isobutyl methyl ketone gave the highest fluorescence intensity. Dilution with 0.5\u00a0M sodium carbonate solution, water, acetone\u2013water produced almost very week fluorescence and did not reduce the blank fluorescence intensity. While upon using isobutyl methyl ketone, the fluorescence intensity attained its highest value, this was attributed to the low fluorescence value of the reagent.\nEffect of dilution time\nDilution times were tested to ascertain the time after which the solution attains its highest fluorescence. It was found that dilution with IBMK after 10\u00a0min, the reaction product reaches its highest fluorescence intensity (Fig.\u00a05).\nFig.\u00a05Effect of dilution time on the fluorescence intensity of the reaction product of oxamniquine (0.2\u00a0\u03bcg ml\u22121) with dansyl chloride\nAnalytical performance\nValidation of the proposed methods The validity of the method was tested regarding; linearity, specificity, accuracy, repeatability and precision according to ICH Q2B recommendations [26].\nLinearity\nBy using the above procedure, linear regression equation was obtained. The regression plots showed that there was a linear dependence of the fluorescence intensity on the concentration of the drug over the ranges cited in Table\u00a01. Linear regression analysis of the data gave the following equation:\nwhere F is the fluorescence intensity, C is the concentration of the drug in \u03bcg ml\u22121 and r is the correlation coefficient.\nThe limit of quantification (LOQ) was determined by establishing the lowest concentration that can be measured according to ICH Q2B [26]. The results are shown in Table\u00a01. The limits of detection (LOD) were determined by establishing the minimum level at which the analyte can be reliably detected, and the results are also abridged in Table\u00a01.\nLOQ and LOD were calculated according to the following equation [26]:\nWhere \u03c3: is the standard deviation of the intercept of regression line. S: is the slope of the calibration curve.\nThe proposed methods were evaluated for the accuracy as percent relative error (% Er) and the precision as percent relative standard deviation (% RSD) (Tables\u00a01 and 2).\nTable\u00a02Application of the proposed and official methods to the determination of Oxamniquine in pure formParametersSpectrofluorimetric methodOfficial method [2]No. of experiments63Mean found (%)100.13100.09\u00a0\u00b1 SD0.680.87\u00a0% RSD0.680.87Variance0.4620.76Student\u2019s t value0.081 (2.31)Variance ratio F test1.65 (5.41)Figures between parentheses are the tabulated t and F values respectively, at p\u2009=\u20090.05 [27]\nAccuracy\nTo test the validity of the proposed method it was applied to the determination of authentic sample of OXM over the working concentration range. The results obtained were in good agreement with those obtained using official method. Using Student\u2019s t test and variance ratio F test, [27] revealed no significant difference between the performance of the two methods regarding the accuracy and precision, respectively (Table\u00a02).\nThe validity of the methods was proved by statistical evaluation of the regression lines, using the standard deviation of the residuals (Sy\/x), the standard deviation of the intercept (Sa) and standard deviation of the slope (Sb). The results are abridged in Table\u00a01. The small values of the figures point out to the low scattering of the points around the calibration line and high precision.\nPrecision\nRepeatability\nThe repeatability was performed by applying the proposed methods for the determination of two concentrations of OXM in pure form on three successive times, and the results are listed in Table\u00a03.\nIntermediate precision\nIt was performed through repeated analysis of OXM in pure form, using the concentrations showed in Table\u00a03 for a period of three successive days. The results are summarized in Table\u00a03.\nTable\u00a03\nValidation of the proposed method for the determination of oxamniquine in pure form\nSample concentration\n% Recovery (repeatability)\n% Recovery intermediate precision\n0.08\u00a0\u03bcg ml\u22121\n99.64\n101.08\n100.72\n99.28\n101.44\n100.89\n\u00a0X\u2032\n100.60\n100.42\n\u00a0\u00b1 SD\n0.91\n0.99\n\u00a0%RSD\n0.91\n0.99\n\u00a0% Error\n0.53\n0.57\n0.2\u00a0\u03bcg ml\u22121\n99.74\n101.05\n100.44\n100.53\n101.29\n98.95\n\u00a0X\u2032\n100.49\n100.18\n\u00a0\u00b1 SD\n0.78\n1.09\n\u00a0%RSD\n0.78\n1.09\n\u00a0% Error\n0.45\n0.63\nRobustness of the method\nThe robustness of the method adopted is demonstrated by the constancy of the fluorescence intensity with the deliberated minor changes in the experimental parameters such as change in the volume of dansyl chloride (0.001%), 0.7\u2009\u00b1\u20090.1\u00a0ml, the change in reaction time 35\u2009\u00b1\u20095\u00a0min and the change in dilution time 15\u2009\u00b1\u20095\u00a0min. These minor changes that may take place during the experimental operation didn\u2019t affect the fluorescence intensity of the reaction product.\nPharmaceutical applications\nThe proposed methods were then applied to the determination of OXM in its capsules. The methods were tested for linearity, specificity, accuracy, repeatability and precision according to ICH Q2B recommendations.\nSpecificity\nThe specificity of the method was investigated by observing any interference encountered from the common capsule excepients, such as talc, lactose, starch, avisil, gelatine, and magnesium stearate. These excepients did not interfere with the proposed method.\nAccuracy\nThe results of the proposed methods were statistically compared with those obtained using the official method [2]. Statistical analysis [27] of the results, using Student\u2019s t test and variance ratio F test revealed no significant difference between the performance of the proposed and reference methods regarding the accuracy and precision, respectively (Table\u00a04).\nTable\u00a04Application of the proposed methods to the determination of oxamniquine in capsulesPreparations% RecoveryProposed methodOfficial method [2]Vansil capsules (250 mg oxamniquine\/capsule)98.25101.0099.3899.8099.30\u00a0Mean found (%)\u00b1 SD.99.55\u2009\u00b1\u20090.99100.56\u2009\u00b1\u20090.77\u00a0t value1.67\u00a0F value1.66The tabulated values of t and F are (2.37) and (6.59) respectively, at p\u2009=\u20090.05 [27]\nAnalysis of biological fluid\nThe high sensitivity of the proposed method allowed the determination of OXM in spiked human plasma. Oxamniquine is readily absorbed following oral ingestion, and a peak concentration in plasma occurs within about 3\u00a0h. The presence of food significantly delays absorption and limits the concentration achieved in plasma during the first several hours after administration. Urinary excretion is the major route of elimination in man [28]. Oxamniquine is given orally in a dose of 250\u00a0mg three times daily; this leads to a final blood level concentration of about 5\u00a0\u03bcg ml\u22121 i.e. higher than the upper limit of the working concentration range of the proposed method. The high sensitivity of the proposed method allowed the determination of OXM in spiked human plasma. The results are shown in Table\u00a05.\nTable\u00a05Application of the proposed methods to the determination of oxamniquine in spiked human plasmaSampleAmount added (\u03bcg ml\u22121)Amount found (\u03bcg ml\u22121)% Recovery1-a-Plasma (inter-day precision)0.020.0195797.850.040.0385696.400.100.0975497.540.200.1985699.28\u00a0Mean97.77\u00a0\u00b1 SD1.191-b-Plasma (inter-day precision)0.200.191495.700.200.196998.450.200.194897.400.200.195597.750.200.197998.950.200.194597.25\u00a0Mean97.58\u00a0\u00b1 SD1.13Each result is the average of three separate determinations\nThe extraction procedure described by Woolhouse and Wood [7] was adopted here. The results are satisfactorily accurate and precise.\nPrecision The within-day precision was evaluated through replicate analysis of Plasma samples spiked with different concentrations of the drug. The percentage recoveries based on the average of four separate determinations were 97.77\u2009\u00b1\u20091.19, thus indicating the high precision of the method (Table\u00a05).The inter-day precision was also evaluated through replicate analysis of plasma samples spiked with 0.2\u00a0\u03bcg ml\u22121 of drug on four successive days. The percentage recoveries based on the average of four separate determinations were 97.58\u2009\u00b1\u20091.13. The results are shown in Table\u00a05.\nMechanism of the reaction\nThe stoichiometry of the reaction was studied adopting the limiting logarithmic method [29]. The fluorescence intensity of the reaction product was alternatively measured in the presence of excess of either dansyl chloride or OXM. A plot of log fluorescence versus log [dansyl chloride] and log [OXM] gave straight lines, the values of the slopes are 0.901 and 0.984 respectively (Fig.\u00a06). Hence, it is concluded that, the molar reactivity of the reaction is 0.901\/0.984, i.e. the reaction proceeds in the ratio of 1:1. Based on the observed molar reactivity of the reaction, and depending on the presence of secondary amino group and by analogy to similar reports dealing with the reaction of dansyl chloride with compounds containing secondary amino group, the reaction pathway proposed in Scheme\u00a01 is presented.\nFig.\u00a06Limiting logarithmic plots for the molar ratio. a Log F vs. Log [dansyl chloride]. b Log F vs. log [oxamniquine]Scheme\u00a01Proposed reaction pathway between dansyl chloride and oxamniquine\nConclusion\nThe proposed method has the advantage of being simple, sensitive and suitable for routine analysis in quality control laboratory. Also, it is suitable for the determination of oxamniquine in spiked human plasma with minimum detection limit lower than the reported value. In addition, it could be applied to the determination of OXM in its pharmaceutical preparation.","keyphrases":["oxamniquine","dosage forms","spiked human plasma","1-dimethylaminonaphalene-5-sulphonyl chloride"],"prmu":["P","P","P","M"]}
{"id":"Purinergic_Signal-4-2-2377317","title":"Calcium-dependent release of adenosine and uridine nucleotides from A549 cells\n","text":"Extracellular nucleotides play an important role in lung defense, but the release mechanism and relative abundance of different nucleotide species secreted by lung epithelia are not well defined. In this study, to minimize cell surface hydrolysis, we used a low-volume, flow-through chamber and examined adenosine and uridine nucleotide concentrations in perfusate aliquots of human lung A549 cells challenged by 50% hypotonic shock. Adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine (Ado) were quantified in high-performance liquid chromatography (HPLC) analysis of fluorescent etheno derivatives, and uridine triphosphate (UTP) and uridine diphosphate (UDP) were measured using HPLC-coupled radioenzymatic assays. After the onset of hypotonic shock, ATP, ADP, UTP, and UDP in the perfusates increased markedly and peaked at approximately 2.5 min, followed by a gradual decay in the next 15\u201320 min; peak changes in Ado and AMP were relatively minor. The peak concentrations and fold increment (in parentheses) were: 34 \u00b1 13 nM ATP (5.6), 11 \u00b1 5 nM ADP (3.7), 3.3 \u00b1 1.2 nM AMP (1.4), 23 \u00b1 7 nM Ado (2.1), 21 nM UTP (>7), and 11 nM UDP (27). Nucleotide release was almost completely abolished from cells loaded with the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Under isotonic conditions, elevation of intracellular calcium with the calcium ionophore ionomycin (5 \u03bcM, 3 min) also released nucleotides with kinetics and relative abundance as above, albeit less robust. ADP:ATP (1:3) and UDP:UTP (1:2) ratios in perfusates from stimulated cells were markedly higher than the cytosolic ratios of these species, suggesting that a nucleotide diphosphate (NDP)-rich compartment, e.g., the secretory pathway, contributed to nucleotide release. Laser confocal microscopy experiments illustrated increased FM1-43 uptake into the plasma membrane upon hypotonic shock or ionomycin treatment, consistent with enhanced vesicular exocytosis under these conditions. In summary, our results strongly suggest that calcium-dependent exocytosis is responsible, at least in most part, for adenosine and uridine nucleotide release from A549 cells.\nIntroduction\nExtracellular nucleotides control a diverse range of physiological processes by interacting with a large group of cell-surface P2Y and P2X receptor families [1\u20133]. In the lungs, extracellular nucleotides regulate airway surface liquid homeostasis and mucociliary clearance by modulating epithelial ion and fluid transport as well as ciliary beating [4]. In alveoli, adenosine triphosphate (ATP) is a potent secretagogue that stimulates type II cell-surfactant secretion. Nucleotide levels on airway surfaces, measured in vitro and ex vivo, show dynamic changes due to the combination of basal and stimulated release and their rapid metabolism by several groups of membrane-associated and membrane-secreted soluble ectoenzymes. These ectoenzymes extend the signaling potential of ATP by converting it to adenosine (Ado), a ligand of the A1\u20133 family of G-protein-coupled receptors [1, 4\u20137].\nNucleotide release is stimulated by cell mechanical perturbations, such as shear stress, membrane stretch, medium change, hyposmotic swelling, and hypoxia [1, 8]. It is now recognized that mechanosensitive ATP release occurs from healthy cells via physiological processes, which do not involve cell damage. Numerous reports have suggested that the cystic fibrosis transmembrane conductance regulator (CFTR) and other members of the superfamily of ATP-binding cassette-transport proteins serve as a conductive pathway for ATP release or regulate an associated ATP channel. However, other groups using patch-clamp, lipid-bilayer and luminometry techniques have not found any detectable CFTR-mediated or CFTR-regulated ATP release (reviewed in [1]). Volume-regulated anion channels (VRAC) and voltage-dependent anion channels (VDAC, porins or maxi-Cl\u2212 channels) are known to have considerable permeability to cations and large organic anions, and several laboratories have implicated these channels in cell-swelling-induced ATP release and the autocrine regulation of cell volume. However, recent studies have demonstrated that conductive pathways, including VRAC, VDAC, and stretch-activated channels, are not involved in cell-swelling-induced ATP release from A549 cells [1, 9, 10]. Finally, connexin hemichannels also have been proposed to mediate ATP release, but their role in this process in a physiological setting is not always clear [1].\nWhereas conductive release mechanisms are still being debated, exocytotic ATP release is well established in excitatory cells, blood platelets, and chromaffin cells. Furthermore, there is growing experimental evidence supporting such a release mechanism also by epithelial and other nonexcitatory cells. In particular, we recently demonstrated that cell-swelling-induced ATP release from A549 and 16HBE14o\u2212 epithelial cells and 3T3 fibroblasts is tightly correlated with intracellular Ca2+ elevations; it is abolished in cells loaded with the calcium (Ca2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) or by low temperature, strongly suggesting the involvement of Ca2+-dependent exocytosis [10]. Whether release of other nucleotides from epithelial cells also involves Ca2+-dependent exocytosis remains incompletely explored. Such studies, however, are complicated by nucleotide hydrolysis at the airway surface, which makes it difficult to assess the magnitude and relative abundance of different nucleotide species released. Therefore, we used a flow-through chamber to minimize cell surface hydrolysis.\nWe found that hypotonic shock markedly increased ATP, adenosine diphosphate (ADP), uridine triphosphate (UTP), and uridine diphosphate (UDP) concentrations in perfusates, which peaked at approximately 2.5\u00a0min. Nucleotide release was almost completely abolished from cells loaded with BAPTA and, under isotonic conditions, could be evoked by elevation of intracellular calcium with the calcium ionophore ionomycin. High nucleotide diphosphate (NDP) concentrations in perfusates of stimulated cells suggested that an NDP-rich compartment, e.g., the secretory pathway, contributed to this release. Together with real-time FM1-43 fluorescence experiments, our results strongly indicate that calcium-dependent exocytosis is a major mechanism of adenosine and uridine nucleotide release from A549 cells.\nMaterials and methods\nCells\nHuman lung carcinoma A549 cells were grown in Dulbecco\u2019s modified eagles medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2\u00a0mM L-glutamine, 56\u00a0U\/ml penicillin-G and 56\u00a0\u03bcg\/ml streptomycin sulfate. All constituents of the culture media were from GIBCO-BRL (Burlington, ON, Canada). ATP efflux was measured from cell monolayers grown to confluency on 24\u2009\u00d7\u200960-mm glass coverslips. Fura-2 calcium imaging and FM1-43 microscopy experiments were performed on cells grown on circular 15-mm diameter no. 1 glass coverslips.\nNucleotide efflux assay\nTo measure nucleotide efflux during hypotonic challenge with high temporal resolution, we used a custom-designed, low-volume (300\u00a0\u03bcl), flow-through chamber, as described previously [10]. Briefly, 24\u2009\u00d7\u200960-mm glass coverslips with confluent cell monolayers of \u223c500 cells\/mm2 were mounted in the chamber and perfused with warm (37\u00b0C\/in-line heater; Warner Instrument, Hamden, CT, USA) Ringer solution at the rate of 1.3\u00a0ml\/min. The Ringer solution contained (in mM): 110.5 NaCl, 24 NaHCO3, 1.3 KCl, 1 MgCl2, 1 CaCl2, 2.5 Na2HPO4, 2.5 KH2PO4, 1.2 K2HPO4, and 10 glucose; pH 7.4 was maintained by bubbling with 5% CO2. After an equilibration period in isotonic solution (5\u201315\u00a0min), a 50% hypotonic solution was applied, and the perfusate was continuously collected for 30-s intervals during the initial burst of ATP secretion (0\u20135\u00a0min) and during 1\u00a0min elsewhere. Nucleotide concentrations in the samples were evaluated by high-performance liquid chromatography (HPLC0 analysis, as described below. For studies in absence of extracellular calcium, CaCl2 was omitted, and the solutions were supplemented with 0.1\u00a0mM ethyleneglycoltetraacetic acid (EGTA). The 50% hypotonic solution was prepared by appropriate reduction of salt concentration, whereas divalent cation concentrations were kept constant.\nHPLC quantification of adenine and uridine nucleotides\nUTP concentrations were quantified by UDP-glucose pyrophosphorylase-based reaction [11]. Briefly, 100-\u03bcl samples were incubated in the presence of 0.5\u00a0U\/ml UDPglucose pyrophosphorylase, 0.5\u00a0U\/ml inorganic pyrophosphatase, 1.6\u00a0mM CaCl2, 2\u00a0mM MgCl2, 25\u00a0mM hydroxyethylpiperazine ethanesulfonic acid (HEPES) (pH 7.4), and \u223c100,000\u00a0cpm 1\u00a0\u03bcM [14C]glucose-1P. Incubations lasted 1 h at 30\u00b0C. Reactions were terminated by heating the samples at 95\u00b0C for 2\u00a0min. Conversion of [14C]glucose-1P to [14C]UTP was determined by HPLC (Shimadzu) via a Nova Pack C18 column and ion-pairing mobile phase. Radioactivity was measured on-line with a Packard Flo-One detector. Assay sensitivity was 1 pmol (3\u00a0nM in 100-\u03bcl samples).\nUDP was quantitatively phosphorylated in the presence of [\u03b332P]ATP using nucleoside diphosphokinase (NDPK). Briefly, 100-\u03bcl samples containing 0.1\u00a0U\/ml NDPK, 0.1\u00a0\u03bcCi 60\u00a0nM [\u03b332P]ATP, 1.6\u00a0mM CaCl2, 2\u00a0mM MgCl2, and 25\u00a0mM HEPES (pH 7.4) were incubated for 5\u00a0min at 30\u00b0C. Reactions were terminated by heating the samples at 95\u00b0C for 2\u00a0min. The resulting conversion of [\u03b332P]ATP to [\u03b332P]UTP was monitored by HPLC [12]. A calibration curve employing known amounts of UDP was chartered in parallel during each assay. This assay allows UDP quantification with sensitivity of 20 fmol (0.2\u00a0nM in 100-\u03bcl samples).\nFor etheno (\u025b) derivatization, samples (200\u00a0\u03bcl) were derivatized for 30\u00a0min at 72\u00b0C in the presence of 1.0\u00a0M chloroacetaldehyde and 25\u00a0mM Na2HPO4 (pH 4.0). The resulting fluorescent 1,N6-ethenoadenine derivatives were analyzed by HLPC (Waters) in a Hamilton PRP-\u00d7100 anion exchange column, as described previously [4]. \u025b-ATP, \u025b-ADP, \u025b-AMP, and \u025b-Ado were quantified with a sensitivity of 200 fmol (1\u00a0nM in 200-\u03bcl samples).\nFM1-43 studies\nBulk exocytosis was quantified in A549 cells as changes in the fluorescence intensity of FM1-43 incorporated into the plasma membrane [13, 14] and recorded by real-time confocal microscopy. Cells were washed with Hank\u2019s balanced salt solution + 20\u00a0mM HEPES + 2\u00a0mM MgCl2 and 1.6\u00a0mM CaCl2 (HBSSH) or with HBSSH devoid of calcium (HBSSH 0 Ca). They were mounted onto the stage of a Leica SP2 AOBS confocal microscope equipped with HCX Apo L63x NA 0.9 immersion Leica lens and a 488-nm Argon laser. Experiments were initiated by incubation of the cells with 3\u00a0\u03bcM FM1-43. Cells were incubated for at least 10\u00a0min, and hypotonic shock was applied by decreasing salt concentration 33% while maintaining calcium, magnesium, and FM1-43 concentrations constant. Alternatively, cells incubated with FM1-43 in HBSSH or HBSSH 0 Ca were stimulated with 5- to 10-\u03bcM ionomycin. Real-time recording was performed by laser scanning in thexz axes with a galvostage, initially every 10\u00a0s and then every 30\u00a0s for the time periods indicated in the figures. Overall fluorescence intensity changes associated with the plasma membrane were estimated by measuring the intensity value associated with each pixel through time. The entire apical membrane compartment displayed in a confocal plane and five random regions of basolateral and subapical domains were analyzed, normalized to basal values (time = 0), and averaged for each region. Cell swelling was estimated as a change of cell height in thexz plane at different time points, normalized to basal values, and averaged.\nFura-2 calcium measurements\nTo load Fura-2, cells were incubated (1\u00a0h, 37\u00b0C, 5% CO2) in physiological solution containing 25\u00a0\u03bcM Fura-2-AM + 0.02% Pluronic F127 and 2.5\u00a0mM probenecid. This was followed by 30\u00a0min deesterification period in physiological solution containing probenecid. The physiological saline solution consisted of (in mM): 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 glucose and 10 HEPES, pH 7.4, adjusted with sodium hydroxide (NaOH). Fifty percent hypotonic medium was prepared by reducing the salt concentration while keeping divalent cation concentration constant. For calcium imaging, coverslips with Fura-2-loaded cells were mounted in the imaging\/perfusion chamber attached to the heated platform (Warner Instruments) on the stage of an inverted microscope (Nikon TE300). The cells were exposed to alternate (200\u00a0ms) illumination at 340\u00a0nm and 380\u00a0nm with a high-pressure mercury lamp (100\u00a0W) via interference filters (Chroma Technology, Brattleboro, VT, USA) mounted on a filter wheel (Sutter Lambda 10-C, Sutter Instrument, Novato, CA, USA) and a dichroic mirror (510\/540\u00a0nm, Chroma Technology). Fluorescence images were recorded at 15- to 60-s intervals with the digital camera and stored for later analysis.\nChemicals\nFor calcium-imaging experiments, Fura-2-AM was obtained from Molecular Probes, Invitrogen Corp. (Burlington, ON, Canada). Probenicid, Pluronic F127 and all other reagents were from Sigma Aldrich (Oakville, ON, Canada).\nResults\nKinetics of nucleotide release\nFigure\u00a01 shows an example of the time course of nucleotide release induced by 50% hypotonic shock. For clarity, the release of adenine and uridine nucleotides appears on separate graphs: a and b, respectively. The kinetics of release were remarkably similar for all nucleotides. After the onset of hypotonic shock, nucleotide concentration increased rapidly, peaking at \u223c2.5\u00a0min, followed by a gradual decay in the next 10\u201315\u00a0min. The average peak values from several separate experiments are shown in Fig.\u00a01c. Interestingly, ATP was the major species at the peak of stimulated release, whereas for basal release, Ado was the predominant species. The rank order of nucleotide abundance at the peak was: ATP > Ado \u2265 UTP > ADP \u2248 UDP > AMP. Relative increases of nucleotide conconcentrations at the peak were also the highest for nucleotide triphosphates (NTPs) and nucleotide diphosphates (NDPs) (ATP 5.6-fold, ADP 3.7-fold, UTP >7-fold, UDP 27-fold), whereas the increase was smaller for AMP and Ado (1.4-fold and 2.1-fold, respectively).\nFig.\u00a01Transient nucleotide release from A549 cells induced by 50% hypotonic shock. Time course of adenosine (a) and uridine (b) nucleotide release observed in response to 50% hypotonic shock. A representative experiment is shown out of four performed under the same conditions. Hypotonic shock was applied at t\u2009=\u20090\u00a0min and was preceded by 15-min equilibration in isotonic solution. Basal (t\u2009=\u20090\u00a0min) and peak (t\u2009=\u20092.5\u00a0min) nucleotide concentrations detected in perfusates (c). Average values [\u00b1 standard deviation (SD)] are from three to four experiments, such as in a and b, except for uridine triphosphate (UTP) and uridine diphosphate (UDP), which are from a single experiment. * Due to the limited sensitivity of UTP evaluations (\u22483\u00a0nM), the basal level of UTP was found to be below the detection limit and was not shown\nRole of intracellular calcium ([Ca+2]i)\nTo investigate the role of intracellular calcium ([Ca+2]i) in adenine nucleotide release, we tested the effect of the Ca2+ ionophore ionomycin. In the absence of hypotonic shock, exposure to 5\u00a0\u03bcM ionomycin for 3\u00a0min induced transient nucleotide release from A549 cells (Fig.\u00a02a). This release reached a peak at approximately 1.5\u00a0min, i.e., slightly earlier and at somewhat lesser absolute peak amplitude compared with that induced by 50% hypotonic shock. Otherwise, the kinetics and relative nucleotide abundance were similar for both stimuli. Almost complete inhibition of swelling-induced adenine nucleotide release was observed for A549 cells loaded with the Ca2+ chelator BAPTA, compared with control untreated cells of the same batch tested in parallel experiments (Fig.\u00a02b, c, respectively). Fura-2 fluorescence [Ca+2]i measurements confirmed a dramatic reduction of the [Ca+2]i response to 50% hypotonic shock in BAPTA-loaded cells (Fig.\u00a02d). These experiments demonstrate that elevation of [Ca+2]i was required to trigger adenine nucleotide release from A549 epithelial cells.\nFig.\u00a02Effect of intracellular calcium ([Ca2+i]) modulators on nucleotide release. (a). In the absence of hypotonic shock, application of 5\u00a0\u03bcM ionomycin resulted in transient nucleotide release with kinetics similar to that induced by hypotonic shock. An example, out of two similar experiments, is shown. Loading A549 cells with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) almost completely inhibited hypotonic-stress-induced nucleotide release compared with controls that were run in parallel (b and c, respectively) (n\u2009=\u20092). Effect of BAPTA on the [Ca2+]i response (d). The two traces represent changes of the Fura-2 fluorescence ratio at two excitation wavelengths, \u03bb340\/\u03bb380, in response to 50% hypotonic shock, applied at t\u2009=\u20090\u00a0min, in control and BAPTA-loaded cells. Note that the rapid peak of the [Ca2+]i response was abolished in BAPTA-loaded cells. Similar responses were observed in four experiments\nFM1-43 fluorescence changes implicate vesicular exocytosis\nStrong calcium dependence of nucleotide release may indicate the involvement of vesicular exocytosis. To further explore this possibility, bulk exocytosis was examined by real-time confocal microscopy in cells bathed in FM1-43. Fluorescence intensity associated with the plasma membrane increased rapidly by incorporation of the soluble probe into the membrane and remained almost constant after 5\u00a0min in nonstimulated cells. Plasma-membrane-associated fluorescence intensity rose rapidly (\u223c20\u00a0s) with hypotonic shock stimulation. This change was accompanied by increased cell volume seen as increment of cell height (Fig.\u00a03a, b). Cell volume peaked at about 3\u00a0min, followed by a regulatory volume decrease in the next 10\u201315\u00a0min (data not shown). A similar plasma-membrane-associated FM1-43 fluorescence intensity increase was observed in the absence of hypotonic shock when the cells were stimulated with ionomycin in the presence but not in the absence of calcium in the bathing solution (Fig.\u00a04). These data indicate that both hypotonic-shock- and ionomycin-stimulated bulk exocytosis in A549 cells.\nFig.\u00a03Effect of hypotonic shock on FM1-43 surface membrane staining. Confocal FM1-43 fluorescence images of confluent A549 cells after 20\u00a0min of dye loading in isotonic solution, followed by 50% hypotonic shock stimulation (a). The images depict the xz scans of the cell monolayer at different time points. Note the increase in cell size and fluorescence intensity consistent with hypotonic-shock-induced cell swelling and heightened bulk exocytosis. Time course of FM1-43 plasma membrane fluorescence and cell height changes during hypotonic shock (b)Fig.\u00a04Effect of ionomycin on FM1-43 surface-membrane staining. Confocal FM1-43 fluorescence images of confluent A549 cells prior to and after 4-min exposure to 5\u00a0\u03bcM ionomycin in the presence of extracellular calcium. (a). The images show the xz scans of the cell monolayer at different time points. Note the increase in fluorescence intensity after ionomycin stimulation, indicating enhanced exocytosis. Time course of FM1-43 plasma membrane fluorescence changes of unstimulated cells (\u0394 CON) and cells during ionomycin treatment in the presence (\u25aa ION) or absence (\u2022 ION 0 Ca) of extracellular calcium (b)\nDiscussion\nIn this study, we used the flow-through chamber and an etheno derivatization measurement technique to examine the kinetics of nucleotide release from A549 lung epithelial cells induced by hypotonic shock. We demonstrated that ATP, ADP, AMP, Ado, UTP, and UDP appeared in the perfusates with the same kinetics, peaking at \u223c2.5\u00a0min (Fig.\u00a01), which coincided with the peak of [Ca+2]i elevation evoked by hypotonic shock (Fig.\u00a02d). Similar tight temporal correlation between the [Ca+2]i signal and ATP release, measured by luciferase-luciferin luminescence assay, was reported previously for A549, 16HBE14o\u2212 epithelial cells and NIH3T3 fibroblasts [10]. Such a close temporal association suggests that hypotonic-shock-induced elevation of [Ca+2]i is a trigger for the release of nucleotides. This was supported by the strong inhibitory effect of BAPTA-AM and the induction of nucleotide release in the absence of hypotonic shock solely by the rise of [Ca+2]i with ionomycin. These data demonstrate tight Ca2+ dependence of nucleotide release, and point to a Ca2+-dependent exocytotic mechanism. It should be noted that the bulk of [Ca+2]i changes shown in Fig.\u00a02d likely represent superposition of spatially separated, distinct [Ca+2]i responses, one evoked directly by hypotonic shock and the other due to autocrine\/paracrine actions of the released nucleotides on P2Y receptors. The latter effects might be minimized, at least partially, by continuous perfusion, which reduced nucleotide concentration in the bulk of the chamber far below the IC50 of P2Y receptor activation; however, this possibility requires further study.\nConsistent with a Ca2+-dependent exocytotic mechanism, FM1-43 fluorescence studies revealed enhanced incorporation of the probe into the plasma membrane induced by hypotonic shock or ionomycin treatment. Increased plasma membrane-associated FM1-43 fluorescence intensity was a measure of the cumulative amount of membrane added by exocytosis [13]. FM1-43 fluorescence increased rapidly during the first 20\u00a0s of hypotonic shock (Fig.\u00a03). Interestingly, the initial fluorescence increment exceeded the cell-surface increase estimated from xz confocal scans. In our previous study using a dual-image 3D cell reconstruction technique [15], we observed only an 11\u201330% surface increase of single substrate-attached A549 cells swollen in 50% hypotonic solution [10]. This difference could be attributed in part to different methods of cell-height and surface evaluation in that study, as well as differences in swelling responses of single isolated cells compared with confluent cell monolayer. However, a similar discrepancy between FM1-43 fluorescence, cell-surface and membrane electrical capacitance has been also reported by others, e.g., with pituitary lactotrophs, where the dense granules docked into the plasma membrane were intensely stained by FM1-43 in addition to the membrane added to the cell surface [16]. Therefore, the divergence of fluorescence and the cell-surface changes seen in our study with A549 cells may also result in part from FM1-43 staining of the lipophilic content of fused vesicles in addition to the staining of fused vesicular membranes. The FM1-43 dye is often employed to fluorescently marked surfactant-containing lamellar bodies in alveolar type II (ATII) cells [17, 18] and A549 cells, a model of ATII cells, also contain granules enriched in surface-active phospholipids [19]. However, the exact contribution of such granules to increased FM1-43 fluorescence in stimulated A549 cells was out of the scope of the present investigation and will require future direct study.\nVesicle exocytosis may be mechanistically associated with nucleotide release in two ways: nucleotides can be delivered to the extracellular medium as cargo molecules within exocytotic vesicles or, alternatively, secreted from the cytosol via vesicle-associated nucleotide-conducting channels or transporters transiently expressed at the plasma membrane as a consequence of vesicle-plasma membrane fusion. Analysis of the relative abundance of different nucleotide species that appear in the extracellular medium may help to distinguish between these two mechanisms. ATP and UDP sugars accumulate in the lumen of the secretory pathway up to 20- to 50-fold above their cytosolic concentrations, and luminal utilization of these molecules generates ADP and UDP. UDP is the major uridine nucleotide detected in ER\/Golgi fractions [20] and, eventually, a UDP-selective apyrase (UDPase) converts UDP to urodine monophosphate (UMP) [21]. ER\/Golgi ADP and UMP are exchanged for cytosolic ATP and UDP glucose via specific transporters [22\u201325]. As intraluminal nucleotides are not subject to the mechanisms that retrieve resident ER\/Golgi proteins, they are predicted to remain within trafficking vesicles and to be released to the extracellular space from the secretory pathway. Our results suggest that this mechanism likely accounted for UDP release from A549 cells. Figure\u00a01c illustrates that hypotonic stimulation promoted a sharp increase in UDP levels, with a UTP:UDP concentration ratio of 2:1. As the cytosolic UTP:UDP ratio is >10:1 [26], our results suggest, at least in part, a vesicular rather than a cytosolic source of UDP and, as a corollary, of ATP and ADP.\nIn summary, our results provide strong evidence for Ca2+-dependent vesicular exocytosis as a major mechanism of adenosine and uridine nucleotide release from A549 epithelial cells induced by hypotonic stress. Part of this release involves vesicles of the protein secretory pathway. Further investigations are needed to clarify the origin and contribution of other vesicular pools as well as the mechanisms and sources of intracellular Ca2+ elevations that evoke nucleotide secretion.","keyphrases":["hypotonic shock","exocytosis","lung epithelial cells","ca2+ dependence","nucleotide secretion"],"prmu":["P","P","P","P","P"]}
{"id":"Behav_Genet-3-1-2039802","title":"Identification of Genetic and Epigenetic Variations in a Rat Model for Neurodevelopmental Disorders\n","text":"A combination of genetic variations, epimutations and environmental factors may be involved in the etiology of complex neurodevelopmental disorders like schizophrenia. To study such disorders, we use apomorphine-unsusceptible (APO-UNSUS) Wistar rats and their phenotypic counterpart apomorphine-susceptible (APO-SUS) rats that display a complex phenotype remarkably similar to that of schizophrenic patients. As the molecular basis of the APO-SUS\/UNSUS rat model, we recently identified a genomic rearrangement of the Aph-1b gene. Here, we discovered between the two rat lines differences other than the Aph-1b gene defect, including a remarkable cluster of genetic variations, two variants corresponding to topoisomerase II-based recombination hot spots and an epigenetic (DNA methylation) difference in cerebellum and (hypo)thalamic but not hippocampal genomic DNA. Furthermore, genetic variations were found to correlate with the degree of apomorphine susceptibility in unselected Wistar rats. Together, the results show that a number of genetic and epigenetic differences exist between the APO-SUS and -UNSUS rat genomes, raising the possibility that in addition to the Aph-1b gene defect the newly identified variations may also contribute to the complex APO-SUS phenotype.\nIntroduction\nSchizophrenia is a neurodevelopmental disorder affecting nearly 1% of the world\u2019s population (Jablensky et\u00a0al. 1987), and is characterized by positive and negative symptoms (Kay and Opler 1987). The aetiology of schizophrenia and other related disorders, such as schizoaffective and bipolar disorder, is still unclear. Twin, family and adoption studies have suggested that complex interactions at the genetic and environmental level underlie the aetiology of schizophrenia (Gottesman 1991). It is thought that gene variations by themselves do not result in schizophrenia, but they can establish a predisposition status that, when combined with environmental stressors, may lead to schizophrenia pathogenesis. Numerous environmental factors, such as viral infections (Mednick et\u00a0al. 1988), insufficient folate and methionine levels (Regland 2005), or repeated psychological stress (Goldstein 1987), can influence brain development of prenatal or early postnatal individuals with a genetic predisposition for neuropsychiatric disorders. Due to the heterogeneity in genetic and environmental interactions, most of the genes and pathways for schizophrenia and for other complex disorders are still unknown.\nTo get insight into the gene (or genes) that may be involved in schizophrenia pathogenesis, a rat model was developed with schizophrenia-like features. This model was based on the behavioural response of Wistar rats to the dopamine agonist apomorphine (Cools et\u00a0al. 1990). The apomorphine-susceptible (APO-SUS) rat line displayed many features of psychopathology, with similar disturbances at the behavioural, physiological, endocrinological and pharmacological level as seen in schizophrenics (Ellenbroek and Cools 2002). For example, APO-SUS rats have a reduced prepulse inhibition and latent inhibition (Ellenbroek et\u00a0al. 1995), display a higher plasma release of adrenocorticotropin (ACTH) and corticosteroids in response to novelty (Rots et\u00a0al. 1995), are more sensitive to dopamimetic drugs (Ellenbroek et\u00a0al. 2000), and have a higher susceptibility to inflammatory and infectious diseases when compared to apomorphine-unsusceptible (APO-UNSUS) rats (Kavelaars et\u00a0al. 1997). We therefore wondered about the molecular-genetic basis underlying the APO-SUS\/-UNSUS rat model and recently identified a genetic difference between the two rat lines (Coolen et\u00a0al. 2005). Whereas APO-UNSUS rats harbour three gene copies of the \u03b3-secretase component Aph-1b, APO-SUS rats have only one or two copies. This gene-dosage imbalance was due to an unequal crossing over event (nonallelic homologous recombination) between two direct repeats (a segmental duplication) within the Aph-1b locus. In addition, we observed a direct link between the Aph-1b genotypes and a number of phenotypic APO-SUS and -UNSUS characteristics (Coolen et\u00a0al. 2005). Approximately 10\u00a0years after developing the APO-SUS and -UNSUS lines a second, independent breeding procedure was started that resulted in rats with features similar to those displayed by the original APO-SUS and -UNSUS rat lines (Ellenbroek and Cools 2002). Interestingly, the replicated rat lines also resulted in APO-UNSUS rats with three Aph-1b gene copy numbers and APO-SUS rats with only one or two gene copies (Coolen et\u00a0al. 2005).\nIn the present study, we wondered whether genetic variations other than the Aph-1b gene-dosage imbalance may be present between the APO-SUS and -UNSUS rats, and whether epigenetic factors may be involved as well. Epigenetics has been defined as heritable changes in gene expression that do not occur by changes in the DNA sequence, but by modifications in DNA methylation and chromatin remodeling (Wolffe and Matzke 1999), or, in its widest sense, as any change in an organism that is not due to genetic factors (Van de Vijver et\u00a0al. 2002). Increasing evidence suggests that epigenetic modifications play a role in disease susceptibility (reviewed by Jirtle and Skinner 2007). We used the arbitrarily primed-polymerase chain reaction (AP-PCR) fingerprinting technique (Welsh and McClelland 1990) to analyse the genomes and epigenomes (DNA methylation) of the APO-SUS and -UNSUS rats. Comparison of the AP-PCR fingerprints generated from the genomic DNAs of the two rat lines revealed genetic as well as epigenetic alterations and we conclude that, besides in the Aph-1b locus, a number of other variations are present in the APO-SUS and -UNSUS genomes and epigenomes.\nMaterials and methods\nExperimental animals\nThe generation of the APO-SUS and -UNSUS rat lines with a high or low susceptibility for apomorphine, respectively, has been described previously (Cools et\u00a0al. 1990). The present experiments were performed with male APO-SUS and -UNSUS rats belonging to the 32nd (original lines) and 18th (replicate lines) generation. At post-natal day 60 (PND60), APO-SUS and -UNSUS rats were sacrificed and the hippocampus, cerebellum and the combined thalamus\/hypothalamus (further denoted as (hypo)thalamus) were isolated. To establish their apomorphine susceptibility, unselected male Wistar rats of the Nijmegen outbred population (PND60) were injected with apomorphine (1.5\u00a0mg\/kg s.c.) and gnawing scores were measured in a gnawing box for 45\u00a0min, as described previously (Cools et\u00a0al. 1990). Immediately following the measurements, the rats were sacrificed and the same tissues (hippocampus, cerebellum and (hypo)thalamus) were removed. All rats were bred and reared in the Central Animal Facility of the Radboud University Nijmegen under approved animal protocols and in accordance with institutional guidelines.\nArbitrarily primed-PCR\nGenomic DNAs were isolated from hippocampus, cerebellum and (hypo)thalamus using standard procedures involving the use of proteinase K and phenol extraction. Two micrograms of genomic DNA were digested with 20\u00a0units of RsaI, 20\u00a0units RsaI in combination with the methylation-sensitive enzyme HpaII, or 20\u00a0units RsaI and MspI (MBI Fermentas) in a total volume of 40\u00a0\u03bcl at 37\u00b0C for 16\u00a0h. HpaII does not cut DNA if the internal cytosine (CCGG) is methylated, whereas MspI is insensitive to DNA methylation. Using such combinations of methylation-sensitive and -insensitive enzymes allows genome-wide screening for differences at the genetic level (single-nucleotide polymorphisms\u2013\u2013SNPs, duplications, insertions, deletions and recombinations) as well as the epigenetic (DNA methylation) level. Restriction enzymes were heat inactivated by incubating the reactions at 65\u00b0C for 20\u00a0min. Digested DNA (100\u00a0ng) was amplified using AP-PCR (Welsh and McClelland 1990) with a single primer. PCRs were performed in a total volume of 25\u00a0\u03bcl containing 10\u00a0mM Tris-HCl (pH 8.3), 2.5\u00a0mM MgCl2, 50\u00a0mM KCl, 0.001% gelatin, 0.25% Nonidet P-40, 0.25% Tween-20, 200\u00a0\u03bcM each of the four deoxynucleotide triphosphates, \u223c1\u00a0\u03bcCi of [\u03b1-32P]dCTP (3000 Ci\/mmol, Amersham Corp.), 25\u00a0pmol of primer (AP-1: 5\u2032-AACCCTCACCCTAACCCCGG-3\u2032, AP-7: 5\u2032-AACCCTCACCCTAAGGCGCG-3\u2032, AP-777: 5\u2032-CACTCCTCTACAAGGTGCCG-3\u2032 or Topo: 5\u2032-GCCTCCTTGCAGGTCTTT-3\u2032), and 0.8\u00a0units of Taq polymerase (MBI Fermentas). Reactions were carried out in a thermal cycler (Perkin-Elmer) with five cycles of low stringency (94\u00b0C for 30\u00a0s, 40\u00b0C for 60\u00a0s, 72\u00b0C for 1.5\u00a0min), followed by 30 cycles of high stringency (94\u00b0C for 15\u00a0s, 55\u00b0C for 15\u00a0s, 72\u00b0C for 1\u00a0min). Two microliters of the PCR products were analysed on high-resolution 5% polyacrylamide gels under denaturing conditions (7\u00a0M urea) for 4\u20134.5\u00a0h at 70\u00a0W. Gels were dried and radiolabelled DNA was visualized by autoradiography at \u221270\u00b0C (CEA AB, Sweden).\nCloning and sequencing of AP-PCR fragments\nAP-PCR fragments generated from APO-SUS and -UNSUS rat genomic DNAs were excised from the dried gels and incubated in 50\u00a0\u03bcl MilliQ at 80\u00b0C for 10\u00a0min. The eluted DNA (two microliters) was reamplified with the same primer as used for the AP-PCR to generate sufficient amounts of template for subsequent cloning. The reactions were carried out for 40 cycles of 94\u00b0C for 1\u00a0min, 55\u00b0C for 30\u00a0s, 72\u00b0C for 1\u00a0min, under the same conditions as described in the AP-PCR protocol (except that [\u03b1-32P]dCTP was not included). The PCR products were purified, cloned into the pGEM-T easy vector (Promega) and sequenced with a T7 or Sp6 primer according to the manufacturer\u2019s instructions using the ABI310 machine (Applied Biosystems).\nSequencing and genotyping of chromosomal region 9q22\nA 1948-bp fragment that harbours the nucleotide sequence corresponding to product 3 was obtained by PCR on genomic DNA derived from (hypo)thalamic tissue of an APO-UNSUS rat using forward primer 5\u2032-GGGAAGCAACGCATCCTG-3\u2032 and reverse primer 5\u2032-CATATCAAAGCACCAAGTCCACAG-3\u2032. The DNA was subsequently purified and directly sequenced using the ABI310 machine (Applied Biosystems). Genotyping of chromosomal region 9q22 was performed with PCR using primers specific for either the APO-SUS or APO-UNSUS genomic sequence. Briefly, PCRs were performed in a total volume of 20\u00a0\u03bcl containing 50\u00a0ng genomic DNA, 10\u00a0mM Tris-HCl (pH 8.3), 2.5\u00a0mM MgCl2, 50\u00a0mM KCl, 0.001% gelatin, 0.25% Nonidet P-40, 0.25% Tween-20, 200\u00a0\u03bcM each of the four deoxynucleotide triphosphates, 0.6\u00a0\u03bcM of each primer (FW: 5\u2032-AACACTTGGACTCATTCTCACTGG-[G (SUS) or T (UNSUS)]-3\u2032 and RV: 5\u2032-CCTGGATGGAATGTTGACAC-[C (SUS) or T (UNSUS)]-3\u2032), and 0.8 units of Taq polymerase (MBI Fermentas). Reactions were carried out at 94\u00b0C for 60\u00a0s, 58\u00b0C for 60\u00a0s and 72\u00b0C for 60\u00a0s for 35 cycles. Products were analysed on a 1% agarose gel.\nQuantification and statistics\nQuantification of AP-PCR products was performed using the Labworks 4.0 program (UVP BioImaging Systems, Cambridge, UK) and statistical evaluation was performed by means of an unpaired Student\u2019s t-test.\nResults\nAP-PCR DNA fingerprint patterns of the APO-SUS and APO-UNSUS rat genomes and epigenomes\nIn order to identify differences between the genomes and epigenomes of APO-SUS and -UNSUS rats, we performed a comparative analysis of fingerprints of AP-PCR products generated from genomic DNAs of the two rat lines. Initially, genomic DNAs isolated from APO-SUS and -UNSUS (hypo)thalamic tissue and digested with RsaI in combination with the methylation-sensitive restriction enzyme HpaII (CCGG) was analysed using arbitrary primers AP-1, AP-7 or AP-777. These primers were selected from a total set of ten primers because they gave fingerprints with reproducible and discrete products (data not shown). Typical AP-PCR fingerprints obtained with the three selected arbitrary primers are shown in Fig.\u00a01. With each arbitrary primer \u223c30 chromosomal fragments were reproducibly amplified. DNAs digested with RsaI and the methylation-insensitive enzyme MspI served as controls to determine whether the observed differences were due to a differential methylation of the CCGG sequence or a genetic polymorphism in this sequence. AP-PCR analysis with primer AP-1 revealed 16 products corresponding to fragments without an HpaII site (\u201cgenetic fragments\u201d) and 23 products corresponding to fragments containing an HpaII site (\u201cepigenetic fragments\u201d). Analysis with AP-PCR primer AP-7 showed 17 genetic and 11 epigenetic fragments, and with AP-777 primer 22 genetic and 6 epigenetic fragments.\nFig.\u00a01AP-PCR analysis of genomic DNAs from APO-UNSUS (U) and APO-SUS (S) (hypo)thalamus. AP-PCR was performed with primers AP-1, AP-7 and AP-777 using genomic DNAs digested with RsaI and HpaII (H) or RsaI and MspI (M) as templates. Epigenetic products (methylation-sensitive and thus absent in the MspI lanes) are indicated by closed arrows and genetic products (methylation-insensitive and thus present in the MspI lanes) by open arrows\nGenetic variations between the APO-SUS and APO-UNSUS rat genomes\nComparison of the genetic fingerprints generated with primers AP-1, -7 and -777 revealed three reproducible variations between the genomic DNAs from the original (F32) APO-SUS and -UNSUS rats, designated products 1, 2 and 3 (Fig.\u00a02a). Product 1 was less prominent in the APO-SUS than in the APO-UNSUS rats, product 2 was found only in APO-SUS, while the level of product 3 was higher in the APO-SUS than -UNSUS rats. Interestingly, the three products were also present in the replicate (F18) lines and at the same levels, indicating that the replication of the APO-SUS and -UNSUS lines had resulted in a similar genotypic distribution. Next, digestions using RsaI in combination with MspI were used to examine whether the observed differences were due to a genetic or an epigenetic alteration. Following digestion with RsaI and MspI, products 1, 2 and 3 were still found, indicating that the presence of the three AP-PCR products was due to genetic differences (Fig.\u00a02b).\nFig.\u00a02Genetic variations in genomic DNAs from APO-SUS (S) and APO-UNSUS (U) (hypo)thalamus. Rats were from the original (F32) or the replicate (F18) rat lines. (A) Products generated by AP-PCR using primers AP-1 (product 1), AP-7 (product 2) or AP-777 (product 3) on genomic DNAs digested with the methylation-insensitive enzyme RsaI and the methylation-sensitive enzyme HpaII. ND\u00a0=\u00a0not determined. (B) Products 1, 2 and 3 generated by AP-PCR on genomic DNAs digested with RsaI and HpaII (H) or with the two methylation-insensitive enzymes RsaI and MspI (M). MspI products served as controls to determine whether products 1, 2 and 3 were due to differential methylation or to a genetic polymorphism. The fact that products 1, 2 and 3 were still present following MspI digestion indicates that they represent products without an HpaII site (\u201cgenetic fragments\u201d). (C) Products generated by AP-PCR using a topoisomerase II binding site consensus sequence (5\u2032-GCCTCCTTGCAGGTCTTT-3\u2032) on genomic DNAs digested with the methylation-insensitive enzymes EcoRI (product 4) or MboI (product 5). Arrows indicate increased amounts of the AP-PCR products\nWe previously discovered that the Aph-1b gene-dosage imbalance between the APO-SUS and -UNSUS rats is the result of a DNA recombination event between the two Aph-1b genes. Furthermore, we identified the region in which the recombination occurred, namely in a region of 1106 nucleotides that is identical between the two genes and encompasses exon 5 (Coolen et\u00a0al. 2005). In the present study, we decided to examine in detail the site of recombination and found a topoisomerase II binding site (5\u2032-ACCCACCTGCTGGTGTCC-3\u2032) in the DNA region harbouring the recombination site. Topoisomerase II binding sites (with the vertebrate consensus sequence 5\u2032-RNYNNCNNGYNGKTNYNY-3\u2032) (Spitzner and Muller 1988) are known to be hotspots where DNA recombination events occur easily (Craig and Nash 1983). We therefore wondered whether other topoisomerase II binding sites could have led to additional differences between the APO-SUS and -UNSUS rat genomes. Interestingly, using a primer based on the topoisomerase II binding site consensus for PCR analysis of genomic DNAs digested with EcoRI or MboI revealed two differences between the APO-SUS (n\u00a0=\u00a03) and -UNSUS (n\u00a0=\u00a02) rat genomes of the original (F32) lines, designated products 4 and 5 (Fig.\u00a02c). Product 4 was present in APO-SUS but not in APO-UNSUS rat genomic DNA. In the replicate APO-SUS and -UNSUS lines (F18), the genomes of two APO-UNSUS rats did also not contain product 4, whereas it was present in two of the four APO-SUS rats tested. Product 5 was present in three of the four APO-UNSUS rats examined (in both the original and the replicate lines), whereas it was not observed in the seven APO-SUS rats tested (Fig.\u00a02c).\nEpigenetic variations between the APO-SUS and APO-UNSUS rat genomes\nWe then wondered whether, besides the five genetic variations, also epigenetic variations would be present between the APO-SUS and -UNSUS rat lines. AP-PCR analysis using primer AP-1 on RsaI- and HpaII-digested genomic DNAs from APO-SUS and -UNSUS (hypo)thalamus revealed one epigenetic variation, designated the E1-product (Fig.\u00a03a). The difference was observed in both the original APO-SUS and -UNSUS rats as well as the replicated lines. An \u223c1.4-fold reduced amount of the E1-product was observed in APO-SUS when compared with APO-UNSUS genomic DNAs (n\u00a0=\u00a012, P\u00a0<\u00a00.05). Since DNA methylation may be tissue specific, we decided to examine the E1-product in two other brain tissues. The level of the E1-product was \u223c2-fold reduced in genomic DNAs from the cerebellum of APO-SUS compared to APO-UNSUS rats (n\u00a0=\u00a04, P\u00a0<\u00a00.01), while no difference in the E1-levels was found in the hippocampus of the APO-SUS and -UNSUS rats (n\u00a0=\u00a04).\nFig.\u00a03Epigenetic variation in genomic DNAs from APO-SUS (S) and APO-UNSUS (U) (hypo)thalamus. Rats were from the original (F32) or the replicate (F18) rat lines. (A) Product E1 generated by AP-PCR using primer AP-1 on genomic DNAs digested with RsaI and HpaII. Arrow indicates a representative example of an AP-PCR product that did not show variable amounts using primer AP-1 (randomly chosen out of 38 products) and was used for normalization. (B) The AP-PCR E1-product generated from genomic DNAs from cerebellum, hippocampus and (hypo)thalamus. All rats were from different nests. The products indicated by the arrow were used for normalisation. Amounts for the epigenetic E1-product were significantly different between the APO-SUS and APO-UNSUS rats in the cerebellum (**P\u00a0<\u00a00.01; n\u00a0=\u00a04, plus s.e.m.) and (hypo)thalamus (*P\u00a0<\u00a00.05; n\u00a0=\u00a012, plus s.e.m.), but not in the hippocampus (n\u00a0=\u00a04, plus s.e.m.)\nChromosomal localizations of the genetic and epigenetic variations between the APO-SUS and -UNSUS rat genomes\nTo identify the locations of the genetic and epigenetic variations within the APO-SUS and -UNSUS rat genomes, AP-PCR fragments 1, 2, 3 and E1 were excised from the gel and each fragment was reamplified with the primer used for the original AP-PCR reaction. DNA sequence analysis of the amplified PCR fragments and database searches with the obtained nucleotide sequences revealed the chromosomal localisations of the four fragments; product 1 was located on chromosome 19q11, \u223c3.3\u00a0kb upstream of the first exon of the GAIP-interacting protein, C terminus (GIPC) gene; product 2 was part of a repeat sequence located on chromosome 2q34; product 3 was located in the first intron of the myosin 1b gene on chromosome 9q22; the epigenetic variation E1 was located on chromosome 6q31, downstream of the Jun dimerization protein 2 (NP_446346.1) and upstream of the ATF-like basic leucine zipper transcriptional factor B-ATF (SF-HT-activated gene 2; XP_216745.2). Since product 3 was localized within a gene, we decided to analyse this chromosomal region in more detail. Sequence analysis of the DNA region corresponding to AP-PCR fragment 3 and its surrounding region revealed a remarkably high number of genetic variations: 10 of the 1948 base pairs analysed were different between the APO-SUS and -UNSUS genomes (Fig.\u00a04). Comparison of the nucleotide sequences of the APO-SUS and -UNSUS DNA regions with the corresponding database sequence (geneID: 117057) revealed a 100% identity between the database and the APO-UNSUS sequences, indicating that the APO-SUS genome has diverged from the database sequence. Next, more APO-SUS and -UNSUS rats were genotyped for this region. All APO-UNSUS rats tested (n\u00a0=\u00a05) indeed showed the database sequence, whereas the APO-SUS rats tested (n\u00a0=\u00a05) all contained the relatively high number of variations in this chromosomal region.\nFig.\u00a04Nucleotide sequence of chromosomal region 9q22, corresponding to product 3, in APO-SUS and APO-UNSUS rats. APO-SUS\/-UNSUS genomic variations are indicated between brackets; the first nucleotide represents the APO-UNSUS sequence, the second nucleotide the APO-SUS sequence. The nucleotide sequences corresponding to the annealing sites of arbitrary primer AP-777 are underlined\nThe newly identified genetic and epigenetic variations, and apomorphine susceptibility in Wistar rats\nWe wondered whether the molecular-genetic difference between the APO-SUS and -UNSUS rats (the Aph-1b gene-dosage imbalance) would also be present in the Nijmegen outbred population of Wistar rats, or if the imbalance was generated during the breeding of the rat lines. PCR analysis of the Aph-1b locus in the 50 Wistar rats examined revealed in all cases the presence of three copies of the Aph-1b gene, suggesting that the reduction of Aph-1b copies in the APO-SUS rats had been induced during the breeding of the rats.\nWe next wondered whether the newly identified genetic and epigenetic variations in the APO-SUS and -UNSUS genomes were also induced during the breeding of the two rat lines, or if these variants are already present in Wistar rats. For this purpose, we performed AP-PCR analysis of (hypo)thalamic genomic DNAs from Wistar rats using primers AP-1, -7 and -777. The fingerprints revealed the presence of the APO-SUS as well as the -UNSUS variants of the genetic products 1, 2 and 3 (n\u00a0=\u00a08) and the epigenetic E1-product (n\u00a0=\u00a04) in Wistar rats (Fig.\u00a05), indicating that the newly identified variations were not induced during breeding of the APO-SUS and -UNSUS lines, but were already present in the original Wistar population.\nFig.\u00a05AP-PCR analysis (products 1, 2 and E1) and specific PCR (product 3) analysis of genomic DNAs from Wistar rats with low or high apomorphine susceptibility. (A) The presence or absence of products 1, 2 and 3 (genetic differences) was analysed in genomic DNAs from the (hypo)thalamus of Wistar rats with low (<10 gnaws in 45\u00a0min) or high (>500\u00a0gnaws in 45\u00a0min) apomorphine susceptibility. U: APO-UNSUS, S: APO-SUS. Lower panel: the amounts of the three products present in the Wistar rats were compared with the amounts found in the APO-SUS and -UNSUS rats. (\u25a0) the genotype of the genetic products 1, 2 or 3 in the Wistar rats with low apomorphine susceptibility is similar to the products 1, 2 and 3 genotype of the APO-UNSUS rats; (\u25a1) the genotype in the Wistar rats with low apomorphine susceptibility deviates from the APO-UNSUS genotype; \n() the genotype in the Wistar rats with high apomorphine susceptibility is comparable with the genotype in the APO-SUS rat; \n() the genotype in the Wistar rats with high apomorphine susceptibility deviates from the genotype in the APO-SUS rats. (B) AP-PCR analysis of the epigenetic E1-product (E1) on genomic DNAs from cerebellum and hippocampus of four Wistar rats. The product indicated by the arrow is an example of a product that did not show variable amounts and was used for normalisation of the E1-product. The amounts of the E1-products generated from the cerebellum or hippocampus were not different between the two Wistar rats with low gnawing scores (<10\u00a0gnaws in 45\u00a0min) and the two Wistar rats with high gnawing scores (>500\u00a0gnaws in 45\u00a0min)\nTo study whether the presence or absence of the genetic AP-PCR products observed in the APO-SUS and -UNSUS rats (products 1, 2 and 3) was linked to the apomorphine susceptibility of Wistar rats, we examined a group of five rats with low susceptibility to apomorphine (<10\u00a0gnaws per 45\u00a0min) and a highly susceptible group of three rats (>500\u00a0gnaws per 45\u00a0min). Product 1, which was less abundant in APO-SUS than -UNSUS rats, was not present in the three Wistar rats with high gnawing scores, but was also not found in two of the five Wistar rats with low gnawing scores. Of the eight rats examined, the APO-SUS-specific product 2 was found in two Wistar rats with a high and only one rat with a low degree of apomorphine susceptibility. The DNA region corresponding to product 3 was found twice as the APO-UNSUS sequence in the group consisting of the low-apomorphine-susceptible Wistar rats and once in the group of the high-apomorphine-susceptible rats (Fig.\u00a05a). These results indicate that none of the three products was directly linked to the apomorphine susceptibility of the Wistar rats. However, apomorphine susceptibility may not be the result of only a single genetic variation, but rather of multiple genetic and epigenetic alterations. Interestingly, 67% of the three genetic products present in the Wistar rats with low apomorphine susceptibility were also found in the APO-UNSUS genome, and 78% of the three products in Wistar rats with high apomorphine susceptibility were APO-SUS-specific variants (Fig.\u00a05a), indicating that a combination of the three genetic products may well be linked to apomorphine susceptibility. To study the link between the epigenetic E1-product and apomorphine susceptibility, two Wistar rats with low gnawing scores and two with high gnawing scores were examined for the amount of the E1-product in the cerebellum. Similar amounts of the E1-product as detected in APO-UNSUS rats were found in one Wistar rat with a low and in one Wistar rat with a high gnawing score. The amount of the E1-product detected in APO-SUS rats was found in one Wistar rat with low and in one Wistar rat with high apomorphine susceptibility, indicating that no direct link exists between the E1-product and apomorphine susceptibility. As observed in the APO-SUS and -UNSUS rats, no variation was detected in the level of the E1-product in hippocampal genomic DNAs from the four Wistar rats tested (Fig.\u00a05b).\nDiscussion\nIn this study, we investigated the genetic and epigenetic background of the phenotypically well-characterized APO-SUS and -UNSUS rats. Unravelling the molecular basis of this rat model may help in our understanding of complex human neurodevelopmental disorders, since many of the characteristics of the APO-SUS rat line are also observed in schizophrenic patients (Ellenbroek and Cools 2002). We recently identified a gene-dosage imbalance in the Aph-1b locus of the APO-SUS and -UNSUS rats, leading to a reduced expression of the Aph-1b gene in APO-SUS rats and a segregation with a number of behavioural parameters (Coolen et\u00a0al. 2005). Here, we used AP-PCR analysis as an approach to explore the presence of any other alterations in the two genomes and epigenomes. We indeed identified additional genetic and epigenetic variations, indicating that the Aph-1b locus might not be solely responsible for the observed phenotypes of the two rat lines and suggesting a multi-genetic and -epigenetic origin of the differences observed between the APO-SUS and -UNSUS rats. Importantly, we found similar AP-PCR patterns in both the original and replicate APO-SUS and -UNSUS lines. It is therefore highly unlikely that the observed variations between the APO-SUS and -UNSUS rat genomes were simply due to coincidence.\nDetailed analysis of one of the newly identified genetic alterations (in the DNA region corresponding to product 3) revealed in APO-SUS rats a cluster of variations in the myosin 1b gene. Hence, this cluster appears to be a hotspot for genetic instability. Product 1 was located upstream of the GIPC gene encoding a protein interacting with membrane-associated and transmembrane proteins, including the dopamine receptors D2 and D3 (Jeanneteau et\u00a0al. 2004). The genetic variation in this locus may therefore contribute to the differences in apomorphine susceptibility between the APO-SUS and -UNSUS rat lines. We also identified genetic alterations using a primer corresponding to a topoisomerase II binding site consensus. The rationale for this study was based on our present finding that a topoisomerase II binding site was located at the recombination site in the Aph-1b locus. The two newly identified, topoisomerase II-based genetic variations might point to a more general role for topoisomerase II binding sites in psychopathological mechanisms. We hypothesize that at these sites environmental factors, such as stress during early development, may cause an increase in the incidence of recombination and other mutagenic events, leading to brain dysfunction and affected behavior. Besides the genetic differences, one tissue-specific epigenetic variation was found between the APO-SUS and -UNSUS epigenomes. At present it is not clear what, if any, functional consequence should be attributed to the decrease in the methylation status of this CpG in the (hypo)thalamus and cerebellum, but not in the hippocampus, of APO-SUS relative to APO-UNSUS rats.\nEarlier microarray analysis of mRNA expression in the hippocampus of the APO-SUS and -UNSUS rats (\u223c7000 full-length sequences and \u223c1000 EST clusters) revealed that only Aph-1b was differentially expressed (Coolen et\u00a0al. 2005). The newly identified variations open the possibility that for an explanation of the background of the rat model one has to consider more genes that operate together in a multi-genetic and -epigenetic setting with several susceptibility loci. Thus, based on our present findings, more than one locus may be responsible for the complex phenotype of APO-SUS rats. Recently, it has been established that the contribution of genetic modifiers is also of importance for the outcome of a phenotype, since they can modulate the severity of the affected phenotype and the phenotypic characteristics without having a clear effect on the normal situation (Nadeau 2001; Nadeau and Topol 2006). Hence, as part of the genetic and epigenetic background of the APO-SUS and -UNSUS rat lines, our newly identified variants can be genetic and epigenetic modifiers influencing the phenotypic expression of the model. Insight into the genetic and epigenetic background may provide diagnostic tools, and clues for mechanisms and pathways to explain complex disorders.\nBesides the multiple variations in the APO-SUS and -UNSUS rat genomes and epigenomes, Aph-1b will presumably be a major player in the development of the complex APO-SUS phenotype, since it has a broad cellular effect via tissue-specific cleavage of many different substrates (Coolen et\u00a0al. 2006b) and is functional already during early development (Coolen et\u00a0al. 2006a). Remarkably, however, the number of Aph-1b gene copies was not linked to apomorphine susceptibility in Wistar rats. Whereas their apomorphine susceptibility varied, all unselected Wistar rats tested harboured three copies of the Aph-1b gene. Therefore, genetic and epigenetic factors other than Aph-1b will likely contribute to the susceptibility for apomorphine. We now indeed found a correlation between the newly identified genetic variations and the apomorphine susceptibility in the Wistar population, confirming our hypothesis that apomorphine susceptibility is caused by a number of genetic and epigenetic factors. A combination of the newly identified variations may have thus initially contributed to the degree of apomorphine susceptibility in the original Wistar rat population. Subsequent apomorphine injections to determine the susceptibility for the drug during the breeding of the APO-SUS and -UNSUS lines may have acted as an environmental stressor, triggering the Aph-1b recombination event at the topoisomerase II binding site only in rats with a high susceptibility for apomorphine. The induced gene-dosage imbalance of the Aph-1b gene, probably in combination with other genetic or epigenetic factors, could then have led to the complex phenotype observed in the APO-SUS rats.\nIn conclusion, the present findings suggest that psychopathological disturbances may be the result of multiple genetic as well as epigenetic factors. We infer that our newly identified variations are susceptibility loci for schizophrenia-like features in the rat and may give new insights into the genetic and epigenetic background of complex neurodevelopmental disorders.","keyphrases":["genetic and epigenetic variations","neurodevelopmental disorders","aph-1b","ap-pcr","apo-sus rat model"],"prmu":["P","P","P","P","R"]}
{"id":"Int_J_Biochem_Cell_Biol-1-5-1885942","title":"Keratinocyte serum-free medium maintains long-term liver gene expression and function in cultured rat hepatocytes by preventing the loss of liver-enriched transcription factors\n","text":"Freshly isolated hepatocytes rapidly lose their differentiated properties when placed in culture. Therefore, production of a simple culture system for maintaining the phenotype of hepatocytes in culture would greatly facilitate their study. Our aim was to identify conditions that could maintain the differentiated properties of hepatocytes for up to 28 days of culture. Adult rat hepatocytes were isolated and attached in Williams\u2019 medium E containing 10% serum. The medium was changed to either fresh Williams\u2019 medium E or keratinocyte serum-free medium supplemented with dexamethasone, epidermal growth factor and pituitary gland extract. The hepatic phenotype was then analysed using RT-PCR, immunohistochemistry, Western blotting and assays of liver function. Cells cultured in keratinocyte serum-free medium supplemented with dexamethasone, epidermal growth factor and pituitary gland extract maintained their phenotype for 3\u20134 weeks, based on expression of liver proteins, ureagensis and response to xenobiotics. In contrast, hepatocytes cultured in Williams\u2019 medium E rapidly lost the expression of liver proteins after 3 days. Cells cultured in keratinocyte serum-free medium supplemented with dexamethasone, epidermal growth factor and pituitary gland extract maintained their expression of liver-enriched transcription factors (C\/EBP\u03b1 and \u03b2, HNF4\u03b1 and RXR\u03b1) while expression was either lost or reduced in cells cultured in Williams\u2019 medium E. These results suggest that keratinocyte serum-free medium supplemented with dexamethasone, epidermal growth factor and pituitary gland extract can maintain the hepatic phenotype for a prolonged period and that this is probably related to the continued expression of the liver-enriched transcription factors.\n1\nIntroduction\nThe primary culture of hepatocytes from rodents constitutes an attractive model system for the study of liver function. However, a major limitation to such a model is the rapid and irreversible loss of differentiated hepatic functions in culture. De-differentiation is reflected not only in decreased liver-specific functions, but there is also an alteration of morphology: the cells flatten, depolarize, and lose many of the surface characteristics of normal hepatocytes in vivo. The mechanisms responsible for the loss of differentiated properties probably involve the downregulation of transcription factors involved in liver-specific gene expression (Padgham et al., 1993). The loss of differentiated functions has been attributed to the change in environmental conditions (extracellular matrix, hormonal conditions) following cell isolation (Padgham & Paine, 1993). The loss of the differentiated phenotype is most apparent in the rapid decline of total cytochrome P450s (CYPs) after isolation and in culture, particularly the CYP3A1 isoform (Padgham et al., 1993). Studies of liver function are therefore generally confined to the first few days of culture and this precludes longer-term studies. Conventional approaches to maintaining the differentiated properties of isolated hepatocytes in culture include supplementation of the medium with hormones such as dexamethasone (Dex) (Agius, Chowdhury, & Alberti, 1986; Enat et al., 1984), co-factors such as nicotinamide, pyruvate, DMSO and phenobarbital (LeCluyse, Bullock, Parkinson, & Hochman, 1996; Waxman, Morrissey, Naik, & Jauregui, 1990); the application of extracellular matrix components (Bissell, Arenson, Maher, & Roll, 1987; Gomez-Lechon et al., 1998) and co-culture with non-parenchymal epithelial cell-types (Rogiers & Vercruysse, 1993; Vallette et al., 1998).\nIn the present study we have introduced a new medium for the long-term culture of differentiated hepatocytes. This is keratinocyte serum-free medium (KSFM). It was tested both alone, and in combination with supplements (dexamethasone, EGF and pituitary gland extract), in comparison with the standard Williams\u2019 medium E, and was used to maintain rat hepatocytes in culture for up to 28 days. We performed immunohistochemical, Western blotting and RT-PCR analysis of the cells under the different culture conditions. We chose hepatic markers which are known to be either rapidly switched off after hepatocyte isolation or are important for detoxification. Expression of a particular gene or protein is normally taken as an indication of intact liver function. However, it is difficult to know simply from expression of a gene or protein that the associated function remains intact. For this reason we also assayed for hepatocyte ureagenesis, glycogen synthesis and response to xenobiotics. Lastly, to gain some mechanistic insight we determined the expression of several liver-enriched transcription factors. Our results suggest that KSFM, in combination with dexamethasone, EGF and pituitary gland extract can maintain the liver phenotype for between 21 and 28 days and it is likely that the ability to do this depends on the sustained expression of liver-enriched transcription factors.\n2\nMaterials and methods\n2.1\nMaterials\nCollagenase was obtained from Worthington Biochemical Corporation, dexamethasone, penicillin\/streptomycin antibiotics, l-glutamine, Williams\u2019 medium E and phenobarbital were purchased from Sigma Chemical Co. (St. Louis, MO). Fungizone (amphotericin B) and Keratinocyte serum-free medium (KSFM) and supplements, recombinant human epidermal growth factor and bovine pituitary extract at final concentrations of 5\u00a0ng\/ml and 50\u00a0\u03bcg\/ml, respectively, were obtained from GIBCO\u2122. Sources of other chemicals and media were as described previously (Shen, Slack, & Tosh, 2000; Tosh, Shen, & Slack, 2002).\n2.2\nRat hepatocyte isolation and culture\nMale Wistar rats (270\u2013330\u00a0g) were obtained from the Animal House of the University of Bath. Hepatocytes were isolated by the two-step collagenase perfusion technique as described previously (Tosh, Alberti, & Agius, 1988). Calcium-free EDTA perfusion medium was freshly prepared with the following composition: 0.05% (w\/v) KCl (Fisher Scientific) in 10\u00a0mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Sigma) buffer, 5\u00a0mM d-glucose (Sigma), 200\u00a0\u03bcM EDTA (Sigma) and 1\/1000\u00a0v\/v phenol red (Sigma) all in sterile phosphate buffered saline (PBS, pH7.4) The liver was perfused via the portal vein for approximately 10\u00a0min at a flow rate of 30\u201335\u00a0ml\/min and then replaced with a collagenase-containing perfusion medium [composition: 20\u00a0mM HEPES buffer, 5\u00a0mM d-glucose, 1\u00a0mM CaCl2 (Fisher Scientific), 0.001% (v\/v) phenol red (Sigma) all in sterile PBS (pH 7.4) and contained collagenase II (0.33\u00a0mg\/ml, w\/v)]. Approximately 150\u00a0ml was delivered to the liver for 10\u201315\u00a0min (with recirculation) at a flow rate of 30\u201335\u00a0ml\/min. Following collagenase perfusion, the liver was removed and hepatocytes dissociated using fine forceps. The cells were filtered through a 70\u00a0\u03bcm filter. Cells were washed three times (50\u00a0\u00d7\u00a0g for 2\u00a0min) in medium [20\u00a0mM HEPES buffer, 5\u00a0mM d-glucose, 1\u00a0mM CaCl2 (Fisher scientific), 0.001% (v\/v) phenol red (Sigma) all in sterile saline at pH 7.4]. Using trypan blue (Sigma), the cell viability was determined to be approximately 85%. Approximately 3\u20135\u00a0\u00d7\u00a0105 rat hepatocytes were initially placed in 35\u00a0mm culture dishes in hepatocyte attachment medium (Williams\u2019 medium E containing 10\u00a0U\/ml penicillin, 100\u00a0\u03bcg\/ml streptomycin, \u00a02mM l-glutamine, and 10% foetal bovine serum). After 6\u20138\u00a0h, the attachment medium was removed and replaced with either serum-free Williams\u2019 medium E or KSFM medium. Both media contained 10\u00a0U\/ml penicillin, 100\u00a0\u03bcg\/ml streptomycin, 2\u00a0mM l-glutamine, 50\u00a0\u03bcg\/ml gentamicin, 100\u00a0ng\/ml Fungizone with or without the indicated supplements. The composition with supplements was as follows: Williams\u2019 medium E only (W), Williams\u2019 medium E plus 1\u00a0\u03bcM Dex (WD), Williams\u2019 medium E plus 5\u00a0ng\/ml EGF (WE), Williams\u2019 medium E plus 1\u00a0\u03bcM Dex plus 50\u00a0\u03bcg\/ml pituitary gland extract (WDP), Williams\u2019 medium E plus 1\u00a0\u03bcM Dex plus 5\u00a0ng\/ml EGF (WDE), Williams\u2019 medium E plus supplements (both 5\u00a0ng\/ml EGF and 50\u00a0\u03bcg\/ml pituitary gland extract) (WS) and Williams\u2019 medium E plus supplements (5\u00a0ng\/ml EGF and 50\u00a0\u03bcg\/ml pituitary gland extract) plus 1\u00a0\u03bcM Dex (WDS); KSFM (K), KSFM plus 1\u00a0\u03bcM Dex (KD), KSFM plus 50\u00a0\u03bcg\/ml pituitary gland extract (KP), KSFM plus 5\u00a0ng\/ml EGF (KE), KSFM plus supplement (KS) and KSFM plus supplement plus 1\u00a0\u03bcM Dex (KDS).\n2.3\nImmunostaining and antisera\nFor immunofluorescence staining, cells cultured on glass coverslips were rinsed with PBS to remove any excess medium and then fixed with 4% PFA for 20\u201330\u00a0min. The cells were washed twice in PBS and kept at 4\u00a0\u00b0C prior to staining. Cells were immunostained as described previously (Shen et al., 2000). The coverslips were mounted with Gel\/Mount aqueous mounting medium (Biomeda, Foster City, CA). The primary and secondary antibodies used for immunofluorescence staining are listed in Table 1. For the detection of connexin 32 protein, the cells were fixed in acetone\/methanol (1:1, v\/v) then incubated in 1\u00d7 citrate buffer (Lab Vision Corporation, CA) at 37\u00a0\u00b0C for 30\u00a0min before blocking and staining.\n2.4\nWestern blotting\nProtein extracts from cultured rat hepatocytes were prepared by lysing pre-washed cells with lysis buffer [20\u00a0mM HEPES (pH 7.6), 150\u00a0mM NaCl, 1\u00a0mM EDTA, 2\u00a0mM dithiothreitol (DTT) and 1% (v\/v) Triton X-100] containing a 1\/100 dilution of protease inhibitor cocktail (Sigma) for 10\u00a0min on ice. The cell lysates were then centrifuged at 13,000\u00a0rpm at 4\u00a0\u00b0C and supernatants were stored at \u221280\u00a0\u00b0C until further use. The protein concentration of the supernatant was determined using the Bio-Rad protein assay reagent according to the manufacturer's instructions. Total protein (10\u201315\u00a0\u03bcg) was mixed with same volume of 2\u00d7 sample loading buffer [125\u00a0mM Tris\u2013HCl (pH 6.8), 4% (v\/v) SDS, 20% (v\/v) glycerol, 0.2\u00a0mM DTT and 0.02% (v\/v) bromophenol blue] and denatured by heating to 100\u00a0\u00b0C for 5\u00a0min. The proteins were then separated in 10% or 15% Criterion\u2122 pre-cast Tris\u2013HCl polyacrylamide gel (Bio-Rad) and transferred onto BioTraceNT\u00ae nitrocellulose membrane (Pall Corporation, Pensacola, FL). The membrane was blocked with 5% (v\/v) non-fat milk in 0.1% (v\/v) PBS\u2013Tween20 (Tween-20, was obtained from Sigma) at 4\u00a0\u00b0C overnight. The membrane was probed with primary and secondary antibodies (listed in Table 2) at room temperature for 2 and 1\u00a0h, respectively. Antibodies were diluted in 3% (v\/v) non-fat milk in 0.1% (v\/v) PBS\u2013Tween and are listed Table 2. The signals were detected with the ECL\u2122 Western blotting analysis system (Amersham) and developed on Hyperfilm\u2122 (Amersham).\n2.5\nQualitative and real-time RT-PCR\nTotal RNA was extracted from cultured rat hepatocytes using TRI reagent (Sigma, Poole, UK). The measurement of total extracted RNAs and reverse transcription were carried out as described previously (Li, Horb, Tosh, & Slack, 2005). Polymerase chain reactions containing the mixture of the same concentration of cDNA, 1.1X ReddyMix\u2122 PCR Master Mix (ABgene, Surrey, UK) and 50\u00a0ng sense\/antisense primers (listed in Table 3) were processed in a DNA thermal cycler using the following conditions: denatured at 94\u00a0\u00b0C for 1\u00a0min, amplification at 58\u00a0\u00b0C for 1\u00a0min and elongation at 72\u00a0\u00b0C for 1\u00a0min for indicated cycles. The samples were analysed in 1.2% agarose electrophoresis with 1\u00a0kb ladder marker (Invitrogen Life Technologies).\nTo obtain quantitative results, we performed real-time PCR using the Lightcycler system (Roche Diagnostics). The calibrators were the cDNA reverse transcribed from adult rat liver mRNA. The same amount of cDNA from calibrator and experimental samples were mixed with 1XSYBR@ Green Taq ReadyMix\u2122 (Sigma), 50\u00a0ng sense and antisense primers and transferred into Lightcycler capillaries (Roche Diagnostics). The following conditions were used for the amplification of PCR products: denaturation at 95\u00a0\u00b0C for 30\u00a0s; amplification at 58\u00a0\u00b0C for 5\u00a0s followed by 72\u00a0\u00b0C for 20\u00a0s for 40\u201350 cycles; cool-down at 40\u00a0\u00b0C for 2\u00a0min. The fluorescence signal was detected at the same time point of each cycle. Data were presented as the normalised ratio, which is the target\/reference ratio of the sample divided by the target\/reference ratio of the calibrator by Lightcycler Relative Quantification software.\n2.6\nPeriodic acid\u2013Schiff's (PAS) staining\nPAS staining was performed to detect glycogen. The cells were seeded onto coverslips and cultured for up to 4 weeks in KDS medium. The cells were then incubated with KDS plus 25\u00a0mM glucose for 24\u00a0h, fixed with 4% PFA and then permeabilised with 1% Triton X-100 at room temperature for 20\u201325\u00a0min. The cells were washed with tap water for 1\u20132\u00a0min and transferred to 1% periodic acid solution for 30\u00a0min. Next, the cells were washed with running tap water for 3\u00a0min and then incubated in the Schiff's reagent at room temperature for 30\u00a0min to develop. After washing in running tap water for 10\u00a0min, the slides were mounted in Gel\/Mount mounting medium.\n2.7\nUrea cycle assays\nTwo colorimetric assays, designed to detect secretion of urea and activity of arginase, were performed on the cultured rat hepatocytes. The urea assay was based on a previously published protocol (Meng, Zhang, & Wu, 2004). Briefly, 100\u00a0\u03bcl of urea standards (0\u201350\u00a0\u03bcg\/ml) and culture media collected over 24\u00a0h were incubated with 300\u00a0\u03bcl urease buffered solution (Sigma) at room temperature for 20\u00a0min. 600\u00a0\u03bcl of phenol nitroprusside, 600\u00a0\u03bcl of alkaline hypochlorite (both from Sigma) and 3\u00a0ml distilled water were then added, gently mixed and incubated at room temperature for 30\u00a0min. The absorbance of standards and samples were then measured at an optical density (OD) of 630\u00a0nm. Urea production was expressed as the amount accumulated in 24\u00a0h per culture dish (\u03bcg\/dish\/day).\nThe arginase assay was based on Corraliza, Campo, Soler, and Modolell (1994). Cultured rat hepatocytes were lysed in 0.1% (v\/v) Triton X-100 containing a 1\/100 dilution of protease inhibitor cocktail and shaken for 30\u00a0min. The lysate was then mixed with same volume of 25\u00a0mM Tris\u2013HCl (pH 7)\/5\u00a0mM MnCl2 and the enzyme was activated by incubation at 56\u00a0\u00b0C for 10\u00a0min. Activated lysate (25\u00a0\u03bcl) was then incubated with 25\u00a0\u03bcl of 0.5\u00a0M l-arginine at 37\u00a0\u00b0C for 1\u00a0h. The samples and the urea standards (0\u2013500\u00a0\u03bcg\/ml) were then incubated with 400\u00a0\u03bcl of an acid solution comprised of H2SO4:H3PO4:H2O at a ratio of 1:3:7 and 25\u00a0\u03bcl 9% (w\/v, dissolved in 100% ethanol) iso-nitro-propiophenone (Sigma) at 100\u00a0\u00b0C for 45\u00a0min. The urea production was detected at an OD of 540\u00a0nm. For urea assay, the secreted urea was presented as the amount of urea per dish per day. The urea production from the hydrolysis of arginine by arginase was taken as a proportional representation of endogenous arginase activity in the arginase assay experiments. The results were normalised with total cellular protein and shown as the amount of urea produced per mg protein per day (\u03bcg\u00a0urea production\/mg\u00a0total protein\/day).\n2.8\nImage collection\nFluorescent images were collected using a Zeiss LSM 510 confocal microscope and figures compiled using Adobe Photoshop 7.0. For cell counting and PAS staining experiments, numbers of random fields were selected using the 40\u00d7 objective lens of a Leica DMRB microscope. The cell numbers were visualised by 4,6-diamidino-2-phenylindole (DAPI) staining. Coverslips were incubated with DAPI (500\u00a0ng\/ml) in PBS for 30\u00a0min at room temperature before being mounted on to slides in Gel\/Mount mounting medium.\n2.9\nStatistical analysis\nThe data were expressed as mean\u00a0\u00b1\u00a0standard deviation. Comparison of individual treatments was conducted using Student's t-test or one-way ANOVA analysis with Fisher's LSD pairwise comparison.\n3\nResults\n3.1\nExpression of hepatic differentiation markers in rat hepatocytes cultured in Williams\u2019 medium E and KSFM\nDespite numerous attempts, it has been very difficult to maintain hepatocytes in a well-differentiated state for more than a few days in culture without significantly changing the culture conditions. We therefore sought to develop a protocol for long-term maintenance of hepatic functions in vitro by the use of a simple medium. Several groups previously used KSFM for culture of oesophageal keratinocytes, corneal epithelial cells and foreskin keratinocytes (Andl et al., 2003; Chen, Chang, Lee, Javier, & Azar, 2002; Vallette et al., 1998). Moreover, Katsura et al. (2002) previously used KSFM medium along with serum to culture adult human hepatocytes (but in the absence of the EGF and PGE supplements). We tested the KSFM medium for maintaining the liver phenotype in primary rat hepatocytes in the absence and presence of EGF and pituitary gland extract. Adult rat hepatocytes isolated by the collagenase perfusion technique were allowed to attach in Williams\u2019 medium E containing 10% serum and maintained for 6\u201310\u00a0h before changing to a serum-free defined Williams\u2019 medium E or KSFM supplemented with Dex and\/or EGF and pituitary gland extract. The initial measurements were taken for cultures at 72\u00a0h. Albumin expression was maintained in cells cultured in KSFM [either with Dex or EGF\/pituitary gland extract (referred to as KD and KS, respectively, see Section 2)] in comparison to cells cultured in WD or WS (Fig. 1A). We then compared expression of other liver-specific markers including serum proteins (\u03b11-antitrypsin, haptoglobin and transferrin), liver-enriched transcription factors (C\/EBP\u03b1, C\/EBP\u03b2, HNF-4\u03b1 and RXR\u03b1), enzymes associated with ammonia detoxification (GS and CPS) and Phase II metabolism (UDP-glucuronosyltransferase, UGT) in WS and KS. It was noted that KS maintained the hepatocyte heterogeneity in terms of populations of perivenous type GS and periportal type CPS-expressing cells (Fig. 1B). The results at 72\u00a0h showed that all the liver markers examined were maintained to a greater extent in KSFM-cultured conditions in comparison to Williams\u2019 medium E (Fig. 1B and C).\nTo test the active component in the supplements, we added EGF and pituitary gland extract separately to the Williams\u2019 medium E (Fig. 1C). Compared to EGF, pituitary gland extract had a greater effect on transferrin expression after 72\u00a0h of culture. However, we could not maintain the cells for more than 4\u20135 days, either with one or both the supplements. This result suggests that the KSFM medium itself must contribute to the maintenance of the hepatic phenotype, and it is not just an effect of supplements.\nVimentin is a cytoskeletal intermediate filament protein the expression of which has been associated with the de-differentiation of hepatocytes (Blaheta et al., 1998). We found that this was expressed in Williams\u2019 medium E cultured isolated rat hepatocytes but was absent in KSFM-cultured cells after a 72-h culture period (Fig. 1D). This suggests that KSFM is preventing de-differentiation as well as maintaining differentiated functions.\nConnexin 32 (Cx32) is the predominant gap junction protein expressed in hepatocytes (Kumar & Gilula, 1986; Nicholson et al., 1987). Gap junction intracellular communication plays an important role in regulating cell survival and apoptosis (Krysko, Leybaert, Vandenabeele, & D\u2019Herde, 2005), cell differentiation and proliferation (Cheng et al., 2004) and tumourigenesis (Luebeck, Buchmann, Schneider, Moolgavkar, & Schwarz, 2005). We examined the expression of Cx32 in rat hepatocytes cultured for 72\u00a0h in Williams\u2019 medium E or KSFM media. As shown in Fig. 2, gap junction-like arrays of Cx32 were only detected in hepatocytes cultured in KS and KDS (Fig. 2).\n3.2\nRat hepatocytes cultured in KDS medium retain their differentiated properties for up to 28 days\nAlthough the results for 3-day cultures with KSFM were encouraging, we wanted to know whether the medium could maintain the hepatic phenotype for longer culture periods. We began by examining the survival of hepatocytes under different culture conditions. Equal numbers of cells were seeded and cultured in six different KSFM culture conditions. The cell number at 24\u00a0h of culture was set as 100% and the percentages of hepatocytes at 1, 2 and 3 weeks were determined (Fig. 3A). In all cases there was a progressive loss of cells but the cells cultured in KDS exhibited the best survival rates, and we decided to use this combination for subsequent experiments. In addition, we also analysed the hepatic phenotype in the living cells by two different approaches. The first involved counting the number of DAPI-positive cells that also express liver proteins (Fig. 3B) or by immunostaining for the liver protein transferrin and collecting the corresponding transmitted light image to show the distribution of the cells (Fig. 3C). The results confirmed that more than 90% of the cells expressed liver proteins over 2 or 4 weeks of culture (Fig. 3B and C). Lastly, to determine whether KSFM prevents outgrowth of mesenchymal cells in long-term cultures, we co-stained for vimentin and transferrin after 2 and 4 weeks of KDS culture (Fig. 3C). At both time points we found only occasional vimentin positive cell (see insert in Fig. 3C) suggesting the KDS does indeed prevent outgrowth of mesenchymal cells.\nWe determined the expression of liver markers in KDS-cultured rat hepatocytes using RT-PCR (Fig. 4A), Western blotting (Fig. 4B) and immunostaining (Fig. 4C and D) and compared the results to fresh liver and freshly isolated hepatocytes (designated \u2018pre\u2019 and \u2018post\u2019). For RT-PCR, the primers were designed towards messages of detoxification enzymes and enzymes involved in amino acid metabolism. All the transcripts were highly expressed in KDS-cultured rat hepatocytes up to 3 weeks and most were still present after 4 weeks of culture (Fig. 4A). Using Western blotting, hepatic proteins including haptoglobin and the liver-enriched transcription factor hepatocyte nuclear factor 1\u03b1 (HNF1\u03b1) were maintained without diminution for at least 2 weeks (Fig. 4B). Furthermore, we also demonstrated that the expression of several liver proteins was maintained for at least 3 weeks. These include apolipoprotein B (ApoB), transferrin, albumin and Cyp2E1, C\/EBP\u03b1, RXR\u03b1 and HNF4\u03b1 (Fig. 4C and D). In general, the expression of both transcription factors and differentiation products was similar to those in fresh liver.\n3.3\nGlycogen storage and urea cycle activity is preserved in KSFM-cultured rat hepatocytes\nTo test whether hepatocytes cultured under different conditions maintained their functional capacity, we examined the potential to store glycogen and perform ureagenesis. We were able to detect glycogen in hepatocytes cultured with KDS for at least 4 weeks of culture (Fig. 5). We also investigated ureagenesis following culture of hepatocytes under KDS conditions. Assays were performed from 24\u00a0h up to 28 days of culture using either the detection of secreted urea (Fig. 6B), or by measurement of arginase activity (Fig. 6A). The results showed that both arginase activity and urea synthesis was generally maintained and are comparable to previously published values (Kang, Berthiaume, Nath, & Yarmush, 2004).\n3.4\nHepatic metabolising enzymes are induced by phenobarbital treatment in KDS-cultured hepatocytes\nPhenobarbital is widely used as an inducer of Phase I and II metabolising enzymes in liver. Following treatment by phenobarbital, the transcription of phase I (cytochrome P450s) and phase II (UGT) detoxification enzymes are activated through the binding of CAR-RXR (CAR, constitutive active receptor; RXR, 9-cis-retinoic acid receptor) heterodimers with the cis-acting element (DR4 motifs, such as NR1 and NR2 domain on the enhancer of Cyp2b10 gene) of target genes (Kakizaki et al., 2003). We initially examined the time course of expression of CAR and PXR (pregnane X receptor, an additional important nuclear receptor accounting for detoxification in hepatocytes) by RT-PCR in KDS-treated cells (Fig. 7A). The levels were well-maintained for 3 weeks and then started to drop off between 3 and 4 weeks of culture.\nTo examine the effect of phenobarbital on KDS-cultured rat hepatocytes, we analysed the mRNA expression of UGT, Cyp2B12, Cyp3A1 and Cyp7A1 by real-time RT-PCR. Rat hepatocytes were cultured for 7 or 14 days in KDS and then treated with and without phenobarbital for 3 days (total of 10 or 17 days culture). Fig. 7 shows that all the genes, including UGT and cytochrome P450s in the phenobarbital-treated cells, were upregulated 2\u201310 fold in comparison with control cells. The result demonstrates that hepatocytes cultured with KDS remain responsive to xenobiotics.\n4\nDiscussion\nMuch effort has been devoted in the past in attempting to develop conditions suitable for the in vitro culture of rodent hepatocytes. Some culture media have been described for sustaining the differentiated state of hepatocytes but they only maintain function in the short term, i.e., 7 days or less (Michalopoulos, Bowen, Mule, & Luo, 2003; Miyazaki et al., 1998). Alternative approaches to the resolving the problem for human liver includes isolating and maintaining embryonic liver cells in culture or adult human hepatocytes (Lazaro et al., 2003; Runge et al., 2000). Our work here shows that KSFM medium with dexamethasone, EGF and pituitary gland extract is able to maintain the differentiated properties of rat hepatocytes for between 21 and 28 days. This statement is based on the sustained expression of liver-specific proteins (including liver-enriched transcription factors), the ability to induce drug metabolising enzymes, store glycogen and produce urea. The main benefit of KSFM medium is that it is relatively simple in comparison with some of the culture conditions reported by other groups. In previous studies, various supplements have been utilised to maintain hepatocytes in culture. These include growth factors (EGF and hepatocyte growth factor) (Michalopoulos et al., 2003; Miyazaki et al., 1998), differentiation-promoting chemicals (nicotinamide and DMSO) (Mitaka, 1998), metal ions (ferrous iron, copper, manganese or zinc) (Kojima et al., 1996), and hormones (e.g. glucocorticoid) (Shelly, Tynan, Schmid, Schutz, & Yeoh, 1989). In contrast, the present medium is relatively simple to prepare it is commercially available and the only supplements required are dexamethasone, EGF and pituitary gland extract. It is also worth noting that extracellular matrices (Rana, Mischoulon, Xie, Bucher, & Farmer, 1994) are not required. We have cultured hepatocytes in KDS media on collagen, fibronectin, matrigel and glass for 2 weeks and then immunostained for albumin. There were no differences showing that the KDS medium alone is sufficient to maintain albumin expression.\nLiver-enriched transcription factors such as HNF1\u03b1, HNF4\u03b1 and C\/EBP\u03b1 are important in the control of hepatic differentiation (Costa, Kalinichenko, Holterman, & Wang, 2003). Indeed over-expression of HNF4\u03b1 by adenoviral infection prolongs the expression of liver-specific genes, cell viability and liver functions such as ammonia metabolism in cultured rat hepatocytes (Naiki, Nagaki, Asano, Kimata, & Moriwaki, 2005). In addition, C\/EBP\u03b2 has been shown to be a master regulator for the formation of transdifferentiated hepatocytes from pancreatic-derived cells (Kurash, Shen, & Tosh, 2004; Shen et al., 2000; Tosh et al., 2002). When we examined the expression of the liver-enriched transcription factors we found that C\/EBP\u03b1 and \u03b2, HNF4\u03b1 and RXR\u03b1 expression were reduced within 3 days of culture with Williams\u2019 medium E but were maintained when the cells were cultured in KDS. This observation suggests that loss of liver-enriched transcription factors is probably the cause of the loss of the differentiated hepatic phenotype.\nRecently, it was suggested that loss of connexin expression might be used as a prognostic marker for hepatocellular carcinoma (Sheen et al., 2004). The presence of connexin proteins in liver is associated with normal liver function. The induction of Cx32 and Cx26 proteins in DMSO-containing primary mouse hepatocytes cultures was also observed and confirmed the importance of connexin proteins in differentiating hepatocytes (Stoehr & Isom, 2003). In the present study, Cx32 was shown to be expressed in hepatocytes maintained in KDS culture medium for up to 3 weeks (Fig. 4C). The persistent expression of Cx32 may provide further evidence of the efficacy of KDS as a medium for primary hepatocyte culture.\nAlthough the KDS mixture does not constitute a defined medium, because of uncertainty about the precise composition of the pituitary extract, our results do show that KSFM, the EGF and the PGE all have a part to play in the maintenance of differentiated hepatocytes in culture. We note that PGE alone has previously been shown to maintain the differentiation status of mammary carcinoma cells (Kano-Sueoka et al., 1979; Platica et al., 1992).\nIn summary, we describe a simple, novel system for maintaining rat hepatocytes in culture and show that the differentiated phenotype can be maintained for long periods in culture. This method may provide a standard system for studies on drug metabolism and toxicity testing without the problem of overgrowth of mesenchymal cells. Primary hepatocytes are a better model for the liver in vitro than hepatoma cell lines, but they also provide a cheaper alternative and a more controllable system than the intact animal. The method may also have potential for reducing the number of animals used for this type of work. Finally, we believe that this long-term differentiated culture method could also pave the way for bioengineering applications such as the development of a bioartificial liver.","keyphrases":["liver-enriched transcription factors","hepatocyte culture","car, constitutive active receptor","w, williams\u2019 medium e","ksfm, keratinocyte serum-free medium","dex, dexamethasone","egf or e, human epidermal growth factor","pge or p, pituitary gland extract","c\/ebp, ccaat\/enhancer-binding protein","hnf, hepatocyte nuclear factor","rxr, retinoid x receptor","pxr, pregnane x receptor","cyps, cytochrome p450 proteins","gs, glutamine synthetase","cps, carbamoylphosphate synthetase","cx, connexin","rt-pcr, reverse transcription polymerase chain reaction","ugt, udp-glucuronosyltransferase","dmso, dimethyl sulfoxide","keratinocyte serum-free media"],"prmu":["P","P","P","R","R","R","R","M","M","M","M","R","R","M","M","M","R","R","M","R"]}
{"id":"Appl_Microbiol_Biotechnol-4-1-2243254","title":"Fumaric acid production by fermentation\n","text":"The potential of fumaric acid as a raw material in the polymer industry and the increment of cost of petroleum-based fumaric acid raises interest in fermentation processes for production of this compound from renewable resources. Although the chemical process yields 112% w\/w fumaric acid from maleic anhydride and the fermentation process yields only 85% w\/w from glucose, the latter raw material is three times cheaper. Besides, the fermentation fixes CO2. Production of fumaric acid by Rhizopus species and the involved metabolic pathways are reviewed. Submerged fermentation systems coupled with product recovery techniques seem to have achieved economically attractive yields and productivities. Future prospects for improvement of fumaric acid production include metabolic engineering approaches to achieve low pH fermentations.\nIntroduction\nFumaric acid (Fig.\u00a01) is a naturally occurring organic acid. It was first isolated from the plant Fumaria officinalis, from which it derives its name. Many microorganisms produce fumaric acid in small amounts, as it is a key intermediate in the citrate cycle. Fumaric acid is also known as (E)-2-butenedioic acid or trans-1,2-ethylenedicarboxylic acid. Sometimes the term \u201cfumarates\u201d is also used. In this review, this general term is not used to describe fumaric acid esters like dimethyl fumarate but only to describe the salts of fumaric acid (sodium fumarate, calcium fumarate).\nFig.\u00a01Current applications of fumaric acid production. Miscellaneous include: lubricating oil, inks, lacquers, carboxylating agent for styrenebutadiene rubber, personal care additives. a Anonymous 2007; b Willke and Vorlop 2004\nCurrently, fumaric acid is produced chemically from maleic anhydride, which in turn is produced from butane. However, as petroleum prices are rising rather quickly, maleic anhydride as a petroleum derivative has increased in price as well (Anonymous 2007). This situation has caused a renewed interest in the fumaric acid production by fermentation that was operational during the 1940s (Goldberg et al. 2006), but was discontinued and replaced by petrochemical processes. The fermentation process is also interesting because it involves carbon dioxide fixation, as will be discussed later. Fungi are known for their organic acid-producing capability and have been used in fermentation processes for fumaric acid production.\nProduction by filamentous fungi of organic acids, including fumaric acid, has recently been reviewed (Magnuson and Lasure 2004; Goldberg et al. 2006). These reviews focused on the microorganisms with their associated metabolic pathways. In contrast, the current review focuses on development of fumaric acid production processes. After summarizing fumaric acid properties, applications, and production, metabolic pathways for fumaric acid production will be discussed only briefly before turning to fermentation performance of the most prominent fumaric acid-producing strains. Furthermore, different methods that have been studied to optimize fermentation processes will be mentioned, and future prospects for process development will be discussed.\nProperties and applications of fumaric acid\nBecause of its structure (a carbon\u2013carbon double bond and two carboxylic acid groups), fumaric acid has many potential industrial applications (Fig.\u00a01). It can act as starting material for polymerization and esterification reactions (www.the-innovation-group.com\/ChemProfiles\/Fumaric%20Acid.htm; 10\/06\/07).\nAs raw material for polymerization, especially in the production of unsaturated polyester resins, maleic anhydride is currently preferred to fumaric acid because maleic anhydride [1.46\u20131.63 $\/kg; (Anonymous 2007)] is cheaper than fumaric acid, the latter historically being around 10% more expensive than maleic anhydride (http:\/\/www.chemweek.com\/inc\/articles\/t\/2007\/04\/04\/04\/022.html). However, fumaric acid could be a better option for the polymer industry among other carboxylic acids or their derivatives because of its nontoxic nature. In addition, special properties like greater hardness in the polymer structure can be achieved when fumaric acid is used (www.the-innovation-group.com\/ChemProfiles\/Fumaric%20Acid.htm; 10\/06\/07). In addition to polymerization, there are two potentially new applications for fumaric acid and both require a different grade of purity of it. The first is as a medicine to treat psoriasis, a skin condition (Altmeyer et al. 1994; Mrowietz et al. 1998). Psoriatic individuals are unable to produce fumaric acid in their body (which is not the case in normal individuals) due to a certain biochemical defect that interferes with adequate fumaric acid production in the skin. Therefore, psoriatic individuals need to take orally fumaric acid in the form of fumaric acid monoethyl or dimethyl ester to treat their disease. The second potential application of fumaric acid is as supplement in cattle feed. Recent studies indicate that a large reduction in the methane emissions of cattle can be achieved (up to 70%), if this cattle receives fumaric acid-based additive as a supplement in their diet (Mcginn et al. 2004). This could greatly reduce the total emission of methane, as farm animals are responsible for 14% of the methane emission caused by human activity.\nFumaric acid, together with the related succinic and malic acids, has been identified as one of the top ten building block chemicals that can be produced from sugars via biological or chemical conversion (Werpy and Petersen 2004). Compared to these other dicarboxylic acids, fumaric acid has a low aqueous solubility [7\u00a0g\/kg at 25\u00b0C; 89\u00a0g\/kg at 100\u00b0C (Stephen 1965)] and low pKa values [3.03 and 4.44 (Lohbeck et al. 1990)], which are properties that can be exploited for product recovery.\nFumaric acid production by petrochemical routes\nFumaric acid is currently produced by isomerization of maleic acid, which is produced from maleic anhydride. Maleic anhydride, in turn, is industrially produced by catalytic oxidation of suitable hydrocarbons in the gas phase. Benzene used to be the predominant starting material, but oxidation of n-butane or n-butane\u2013n-butene mixtures has become more important in recent years (Lohbeck et al. 1990). The butane oxidation reaction equation to maleic anhydride is: C4H10 + 3.5O2 \u2192 C4H2O3 + 4H2O.\nIn the most common maleic anhydride process, the catalyst is embedded in fixed-bed tubular reactors. The catalysts applied in this process are based on vanadium and phosphorus oxides. In this process, water is formed as a by-product, and a fairly small amount of it can be directly liquefied from the reaction gas by partial condensation. Organic solvents absorb maleic anhydride contained in the reaction gas. More than 98% of the anhydride can be absorbed in this way. The solvent\u2013anhydride mixture is then subjected to fractional distillation to separate maleic anhydride from the solvent, and the latter is returned to the absorption column (Lohbeck et al. 1990). The thus obtained pure maleic anhydride is then hydrolyzed into maleic acid according to the reaction equation C4H2O3+H2O \u2192 C4H4O4.\nSubsequently, the maleic acid is converted almost quantitatively by thermal or catalytic cis\u2013trans isomerization into fumaric acid (Lohbeck et al. 1990). The most common catalysts used are mineral acids, peroxy compounds, and thiourea. The crude fumaric acid obtained in this way is purified by crystallization from water (Fig.\u00a02). As will be shown later, a comparable purification may be used for fermentative fumaric acid production.\nFig.\u00a02Plant for fumaric acid production from malic acid. a Isomerization vessel; b Centrifuge; c Dissolving tank; d Filter; e Crystallizer; f Dryer (adapted from Felthouse et al. 2001)\nProduction capacity of maleic anhydride in 2007 was about 1,807,000\u00a0ton\/a (Anonymous 2007), with around 3% of this being used for fumaric acid production (http:\/\/chemicalmarketreporter.com\/home\/Default.asp?type = 17&liArticleID = 2015643...; 18\/04\/2007), corresponding to 90,000\u00a0ton\/a.\nEnzymatic conversion of maleic acid into fumaric acid\nIn the aforementioned chemical isomerization of maleic acid into fumaric acid, the conversion yield is restricted by the reaction equilibrium. This chemical conversion is occurring at high temperatures that causes formation of by-products from maleic and fumaric acids, and as a consequence, yields are below the equilibrium yields (Goto et al. 1998). This problem was the main motivation to look for a good enzyme that would facilitate isomerization, so that the equilibrium mixture would be obtained without formation of by-products.\nIt is known that the enzyme maleate isomerase (maleate cis-trans-isomerase) isomerizes maleic acid into fumaric acid. Microorganisms that produce maleate isomerase are Pseudomonas species (Otsuka 1961), Alcaligenes faecalis (Takamura et al. 1969), and Pseudomonas fluorescens (Scher and Lennarz 1969). However, maleate isomerase is unstable even at moderate temperatures (Takamura et al. 1969), and therefore, a heat-stable maleate isomerase is desired. Thermo-stable maleate isomerases derived from Bacillus stearothermophilus, Bacillus brevis, and Bacillus sp. MI-105 were reported by Goto et al. (1998), and according to the authors, the use of enzymes from these bacteria can improve the fumaric acid production process.\nFurthermore, in a more recent study using Pseudomonas alcaligenes XD-1, high rates of conversion of maleic acid into fumaric acid (6.98\u00a0g L\u22121 h\u22121) were reported (NakajimaKambe et al. 1997). This organism does not normally accumulate fumaric acid, but by heat treatment of the cells (70\u00b0C for 1\u00a0h), they lost their fumarase activity, which otherwise would be responsible for a conversion of fumaric acid into l-malic acid. This heat treatment did not affect the maleate isomerase activity. In addition, when calcium ions were added during the conversion step, the thermal stability of maleate isomerase was increased. With the use of P. alcaligenes XD-1, the highest reported conversion yield, 95%, was achieved in the conversion of maleic acid to fumaric acid (Ichikawa et al. 2003). Immobilization of the heat-treated cells is currently under investigation.\nMicrobial production of fumaric acid\nFumaric acid production by fermentation was operated in the United States during the 1940s, but later, this process was discontinued and replaced by chemical synthesis from petrochemical feedstocks, the latter being explained in a previous section. Nevertheless, the continuous increase of the petroleum prices has brought back the interest in fumaric acid production by submerged fermentation (Goldberg et al. 2006). Fumaric acid production by fermentation using Rhizopus species has been patented occasionally (Waksman 1943; Kane 1943; Lubowitz and La Roe 1958; La Roe 1959; Goldberg and Stieglitz 1986). In 1989, DuPont patented an improved fermentation process producing carboxylic acids (fumaric, succinic, malic, and mixtures thereof) by controlling dissolved oxygen levels between 30 and 80% (Ling and Ng 1989).\nIn the field of fumaric acid production by fermentation, there are many aspects determining the productivity of the fermentation process, such as the applied microbial strain and its morphology, the use of a neutralizing agent, and the applied feedstock. Those aspects are reviewed and analyzed in more detail in the following sections.\nFumaric acid-producing strains\nAfter the discovery of fumaric acid production in Rhizopus nigricans by Felix Ehrlich in 1911, Foster and Waksman (1938) screened 41 strains from eight different genera to identify high fumarate producing strains. The fumarate-producing genera identified were Rhizopus, Mucor, Cunninghamella, and Circinella species. Among these strains, Rhizopus species (nigricans, arrhizus, oryzae, and formosa) were the best-producing ones, yielding fumaric acid under aerobic and anaerobic conditions (Foster and Waksman 1938; Rhodes et al 1959; Kenealy et al. 1986; Cao et al. 1996; Carta et al. 1999). Table\u00a01 lists these fungi, and as is shown, the R. arrhizus NRRL 2582 and R. oryzae ATCC 20344 gave the highest volumetric productivity, product titer, and product yield values (Gangl et al. 1990; Cao et al. 1996).\nTable\u00a01Literature data on fumaric acid production by different Rhizopus generaStrainFermenterSubstrateProduct Titer (g l\u22121)Yield (g g\u22121)Vol. Prod. (g l\u22121 h\u22121)Time (h)Final pHReferenceR. nigricans 45Shake flaskGlucose14.70.50\u20131686.5Foster & Waksman 1938Shake flaskGlucose20.00.660.25806.5Romano et al. 1967R. arrhizus NRRL 2582Stirred tankGlucose90.00.701.22726.0Rhodes et al. 1962Stirred tankGlucose107.00.822.00536.0Ng et al. 1986Stirred tankGlucose73.00.720.501475.5Gangl et al. 1990R. arrhizus NRRL 1526Shake flaskGlucose97.70.811.02966.0Kenealy et al. 1986Fluidized bedMolasses17.50.360.36486.0Petruccioli et al. 1996Stirred tankGlucose38.00.330.46825.5Riscaldati et al. 2000R. oryzae ATCC 20344RBCa plus AdsorptionGlucose92.00.854.25244.5Cao et al. 1996Stirred tankGlucose65.00.650.90725.0Cao et al. 1996RBCaGlucose75.50.753.78245.0Cao et al. 199710-l air liftGlucose37.80.750.81465.0Du et al. 1997Stirred tankGlucose35.80.600.90405.5Zhou 1999Bubble columnGlucose37.20.531.03365.0Zhou et al. 2002R. formosa MUCL 28422Stirred tankCassava bagasse21.3\u2013\u2013\u20136.5Carta et al. 1999aRotatory biofilm contactor\nDespite the fact that the aforementioned experimental studies involve fungi, the use of bacteria has been considered as well. Donnelly et al. (2001) have suggested using a Lactobacillus host strain that lacks the malolactate enzyme, fumarase, and fumarate dehydrogenase. Introducing the maeA gene for the malic enzyme from Escherichia coli would result in a pathway from pyruvate to malate and hence in a malic acid-producing mutant. Additional genetic engineering would result in fumaric acid production.\nGenetic modification of microorganisms has hardly been explored for fumaric acid production but offers a potentially useful approach solution for improving yields and rates in fermentation.\nMetabolic pathways to fumaric acid\nFumaric acid is primarily an intermediate of the citrate cycle, but is also involved in other metabolic pathways. In 1939, it was suggested that fumarate biosynthesis involved a C-2 plus C-2 condensation in Rhizopus species (Foster et al. 1949). The reactions in this pathway seemed to be similar to those of the glyoxylate bypass (Foster et al. 1949). Years after, the glyoxylate bypass mechanism was ruled out because the theoretical molar yield for this pathway of 100% was not in agreement with the experimental yield of 140% (Rhodes et al. 1959; Romano et al. 1967). However, the main evidence for rejecting the glyoxylate bypass mechanism is that the key enzyme of the glyoxylate pathway, isocitrate-glyoxylate lyase, was repressed when high glucose concentrations were present like in the experiments used for fumaric acid production (Romano et al. 1967).\nIt was discovered that a C3 plus C1 mechanism involving CO2 fixation catalyzed by pyruvate carboxylase under aerobic conditions explained the high molar yields in fumarate production (Overman and Romano 1969). This CO2 fixation leads to oxaloacetic acid formation (Osmani and Scrutton 1985), so that C4 citrate cycle intermediates can be withdrawn for biosynthesis during the growth phase under aerobic conditions. When nitrogen becomes limiting and the growth phase stops, the metabolism of glucose and CO2 fixation could continue and lead to an accumulation of C4 acids (Romano et al. 1967). The maximal theoretical yield in a nongrowth situation is 2\u00a0mol of fumaric acid per mole of glucose consumed, upon fixation of 2\u00a0mol of CO2 via reductive pyruvate carboxylation. However, if the reductive pyruvate carboxylation would be the sole pathway, no ATP would be produced for maintenance or transport purposes. Therefore, the citrate cycle is also active during fumaric acid production (Rhodes et al. 1959; Kenealy et al. 1986).\nThe CO2 carboxylation enzyme, pyruvate carboxylase, is known to be localized exclusively in the cytosol, together with NAD-malate dehydrogenase and fumarase (that are present in the cytosol and in the mitochondria), leading to fumaric acid synthesis in this cell compartment (Osmani and Scrutton 1985). Studies performed by Peleg et al. (1989) indicated higher activities of these enzymes (especially the cytosolic isoenzymes) during fumaric acid production. Kenealy et al. (1986) used mitochondrial inhibitors to investigate their role in fumarate accumulation and found no direct involvement of such inhibitors of the citrate cycle in fumarate production. However, carbon-labeling studies have demonstrated the simultaneous utilization of both the citrate cycle and the reductive pyruvate carboxylation pathways under aerobic conditions (Fig.\u00a03). Besides the localization of fumarase isoenzymes, it was also found that addition of cycloheximide virtually eliminated the cytosolic isoenzyme and therefore caused a large decrease in the amount of fumaric acid produced by R. oryzae (Peleg et al. 1989).\nFig.\u00a03Citrate cycle pathway and reductive carboxylation pathway leading to fumaric acid accumulation (adapted from Kenealy et al. 1986). The ratio between the two pathways is not 1:1 as suggested by this figure\nThe carboxylation of pyruvate has been studied in more detail for the analogous microbial succinate production, but the understanding of the different metabolic fluxes involved is still incomplete (McKinlay et al. 2007).\nHigh-producing fumaric acid strains of Rhizopus not only produce fumaric acid but also other carboxylic acids like malic, lactic, acetic, succinic, and citric in smaller amounts than fumaric acid production (Rhodes et al. 1959; Carta et al. 1999). Sometimes, ethanol is also produced, like in the case of the high-producing strain R. oryzae ATCC 20344 (Cao et al. 1996). However, it was found that ethanol production can be reduced by a sufficient supply of oxygen to the culture (Cao et al. 1996).\nFumaric acid transport across the cell membrane\nThe transport mechanism of fumaric acid in fungi has not been studied yet. However, transport of l-malic acid and other dicarboxylic acids, including fumaric acid, has been studied in yeasts like Schizosaccharomyces pombe, Candida utilis, Candida sphaerica, and Hansenula anomala (Corte-Real et al. 1989; Corte-Real and Leao 1990; Saayman et al. 2000). These microorganisms may be comparable to fumaric-producing fungi. These studies showed that the initial uptake of malic acid was accompanied by disappearance of extracellular protons suggesting that the anionic form of the acid was transported by an accumulative dicarboxylate proton symporter. As fumaric acid seems to be a competitive inhibitor of l-malic acid uptake, it was suggested that fumaric acid uses the same import system.\nOn the other hand, these studies showed that undissociated dicarboxylic acid entered the cells slowly by simple diffusion. In addition, it was revealed that the rate of diffusion of the undissociated acid across the plasma membrane is subjected to opposite pH influences: an increase due to the relative increase of undissociated acid with decreasing pH and a decrease due to a decrease of the permeability of the cell membrane for the undissociated acid at decreasing pH (Corte-Real and Leao 1990). Increasing the number or activity of the dicarboxylic acid transporters could lower the intracellular fumarate concentration and could therefore have a positive effect on the production yield (van Maris et al. 2004).\nAlternative fermentation feedstocks\nGlucose has not been the only carbon source used for fumaric acid-producing strains. The use of xylose with immobilized R. arrhizus was studied, but the highest obtained volumetric productivity was only 0.087\u00a0g l\u22121 h\u22121 (Kautola and Linko 1989). Also, sucrose has been considered as a raw material in fumaric acid-production processes (Kautola and Linko 1989; Zhang et al. 2007), but sucrose has the disadvantage of being poorly metabolized by R. oryzae compared to glucose (Bulut et al. 2004).\nStarch-containing materials were also screened as feedstock for the fumaric acid production. Potato flour was used as feedstock for R. arrhizus, and although fumaric acid was the main metabolic product, a volumetric productivity of only 0.35\u00a0g l\u22121 h\u22121 was achieved (Moresi et al. 1991). On the other hand, it was found that the highest yields of fumaric acid were obtained from acid hydrolysates of starch-based materials (Moresi et al. 2002). The same feedstock was used by Federici et al. (1993) in studies on the effect of agitation speed and applied neutralizing agent. The tested neutralizing agent was a combination of CaCO3 and KOH\/KCO3, and it was concluded that comparable fumaric acid yields were obtained using these neutralizing agents and starch-based materials (Federici et al. 1993). Carta et al. (1999) optimized the fumaric acid production by R. formosa MUCL 28422 by using enzymatic hydrolysates of cassava bagasse, a high-starch-containing waste product. The productivity was not improved with respect to a fermentation where glucose was used as a feedstock, but the fact that these feedstocks are cheap and abundant makes these optimized fermentations economically attractive.\nNutrient requirements\nPhysical and nutritional requirements for fumaric acid fermentation have been studied in Rhizopus arrhizus (Rhodes et al. 1959; Rhodes et al. 1962). As Rhizopus species should enter a phase of limited growth during the fermentation, which can be achieved by nitrogen limitation, these studies showed that to achieve high yields during fumaric acid fermentation, the most critical parameter is the ratio of glucose to nitrogen. For example, a yield of fumaric acid on glucose of 85% (w\/w) was obtained using an initial C:N molar ratio of 200:1 for R. arrhizus 2582. When nitrogen limitation is not desired, phosphorus limitation can be used instead (Riscaldati et al. 2000). Trace metal concentrations of 500\u00a0ppm, 4\u00a0ppm, and 100\u00a0ppb for Mg++, Zn++, and Fe++, respectively, were found optimal for the formation of small (1\u00a0mm) spherical pellets that produced high concentrations of fumaric acid (36\u00a0g L\u22121; Zhou 1999).\nBecause CO2 is needed for the oxaloacetate formation from pyruvate by pyruvate carboxylase, the addition of calcium carbonate (CaCO3), which is used in many cases as a neutralizing agent, seems also important as a CO2 source during the production phase of the fermentation. However, in case that no CO2 or carbonate is added, the complete catabolism of a mole of glucose via the citrate cycle provides 6\u00a0mol of CO2 that may be used for pyruvate carboxylation (see Fig.\u00a03). In this case, the maximum theoretical yield would be 1.5\u00a0mol of fumaric acid per mole of glucose. This theoretical value is close to the value of 1.32\u00a0mol of fumaric acid per mole of glucose reported by Cao et al. (1996), who optimized a fermentation process without CO2 or carbonate feeding.\nNeutralizing agents\nContinuous neutralization of the pH has been necessary to obtain optimal yields in fumaric acid production by fermentation. Preferably, CaCO3 has been used as a neutralizing agent, but at the same time, this compound is causing viscosity problems due to the low aqueous solubility of calcium fumarate [21\u00a0g\/l at 30\u00b0C (Gangl et al. 1990)] that precipitates as a fermentation product. Furthermore, the cells can interact with the precipitated product, as has been found for R. oryzae, resulting in a highly viscous suspension. This has a detrimental effect on the rate of oxygen transfer which can be achieved, and hence, the fermentation might fail due to oxygen-limitation problems. Therefore, replacement of CaCO3 by other neutralizing agents like Na2CO3, NaHCO3, Ca(OH)2, and (NH4)2CO3 has been studied by different authors (Gangl et al. 1990; Riscaldati et al. 2000; Zhou et al. 2002). However, these studies showed that the fumarate production rates are the highest when CaCO3 is used as a neutralizing agent.\nBecause of the high solubility of sodium fumarate, fermentative production of fumarate using Na2CO3 as neutralizing agent leads to cheaper downstream processing than when CaCO3 is used. This is due to the fact that the fermentation product, sodium fumarate, has a higher solubility than CaCO3, and hence, there is no need of heating to recover the fermentation product (see Fig.\u00a04). In addition, cells can be reused (Gangl et al. 1990; Zhou et al. 2002). A similar situation was obtained when Rhizopus growth was limited by phosphorus so that (NH4)2CO3 could be used as neutralizing agent (Riscaldati et al. 2000). Nevertheless, it has been argued that a fermentation process without the use of neutralizing agents, and at the same time preventing product inhibition, will improve the economics of the general process (Gangl et al. 1990). However, when a high-yield process is developed without a carbonate as neutralizing agent, the required CO2 must be supplied by other sources. Fumaric acid fermentation systems without the use of neutralizing agents have been studied in the past and will be discussed in the following sections.\nFig.\u00a04Flow-sheet for fumaric acid production via fermentation. a Formulation tanks containing glucose and nutrients; b Seed fermentor; c Production fermentor; d Filter; e Acidification tank; f Filter; g Rotary dryer (adapted from Gangl et al. 1990)\nWith respect to fumarate salts inhibition, Rhodes et al. (1962) reported that production of soluble sodium or potassium fumarates was inhibited when the concentration of fumarate reached values of 34\u201340\u00a0g L\u22121 using R. arrhizus as a producer strain. Nonetheless, Gangl et al. (1990) found that addition of sodium fumarate (71.3\u00a0g L\u22121) was not inhibiting the same strain, although the cells needed 35\u00a0h to adapt to the high sodium fumarate concentrations. On the other hand, the free fumaric acid does inhibit its own production because the accumulated protons in the production medium decrease the pH, thus exerting a progressive inhibitory effect on fumaric acid production (Riscaldati et al. 2000). At low pH, excreted fumaric acid will passively diffuse back through the plasma membrane of the fungus decreasing its intracellular pH, and due to this phenomenon, the fermentation fails. Proposed methods to enable carboxylic acid fermentation at low pH are: genetic engineering of acid tolerant organisms such as yeasts and in situ product removal techniques (Cao et al. 1996; Sch\u00fcgerl 2000; van Maris et al. 2004).\nMorphology and oxygen-transfer problems\nOne of the difficulties of fermenting Rhizopus species is the morphology of these fungi. Rhizopus species tend to grow on the walls and on the stirrer of the reactor, and sometimes, clumps are formed. Therefore, the fermentation can suffer from oxygen limitation in particular when calcium fumarate is present. One way to solve this problem is to control the growth of the fungi and their morphology.\nA way to minimize oxygen mass transfer limitation to the cells is to stimulate formation of small spherical cell pellets (Zhou 1999). Small pellets can reduce clump formation during fermentation, and even if CaCO3 is present, the viscosity of the broth can be reduced. Moreover, pellets can facilitate the performance of a biomass retention system. For R. oryzae, low initial pH values for the cultivation media favored pellet formation, and good fumaric acid yields have been reached (Zhou et al. 2000).\nIn another morphology improvement study, Cao et al. (1997) used a rotary biofilm contactor (RBC) as fermentor with immobilized R. oryzae to produce fumaric acid. CaCO3 was used as neutralizing agent during this experiment. During the fermentation, the discs with immobilized cells were rotating, moving the cells from the gas phase of the fermentor to the liquid phase and back again (Cao et al. 1997). When the cells are exposed to the air, high oxygen transfer rates can be reached, while the cells can take up substrate and excrete the produced fumaric acid when they are submerged. In this system, additional agitation was not needed. The volumetric productivity was very high, compared to the volumetric productivity of an equivalent stirred vessel fermentor setup (see Table\u00a01). A disadvantage of this system could be the scalability potential of the RBC fermentor.\nImmobilization techniques for Rhizopus species have been investigated to open the possibility of a continuous operation mode for fumaric acid production and to reduce oxygen transfer problems as well. Buzzini et al. (1995) searched for the most suitable support for cell immobilization in fluidized-bed reactors during fumaric acid fermentation. Cork, expanded polystyrene, expanded clay, and wood shavings were investigated. With 6\u00a0mm pieces of cork, the highest titer, 24.1\u00a0g fumaric acid l\u22121 in 144\u00a0h, was achieved, which is comparable to the titer of 37.7\u00a0g l\u22121 after 166\u00a0h under conventional submerged conditions (Buzzini et al. 1995). The performance of a semi-continuous process was investigated with R. arrhizus immobilized on 5\u00a0mm cubic particles made of polyurethane sponge (Petruccioli et al. 1996). These particles were used in repeated batch fermentations in a fluidized-bed reactor (48\u00a0h, eight times). In the optimized process, the fumaric acid titer was 12.3\u00a0g l\u22121, and the volumetric productivity was 0.256\u00a0g l\u22121 h\u22121.\nFurthermore, as the oxygen mass transfer resistance through the boundary layer on the liquid side of the gas\u2013liquid interface can affect the interfacial oxygen transfer from the gas phase to the liquid phase, pressure pulsation was applied in a stirred tank fermentor using R. oryzae (Zhou 1999) to reduce this resistance. Mass yield and volumetric productivity of fumarate were 70.1% and 0.99\u00a0g L\u22121 h \u22121, respectively, which were higher than for traditional stirred tank fermentations. The same organism was studied in an airlift loop reactor as a fermentation system for fumaric acid production (Du et al. 1997). Here, the airlift loop reactor with porous sparger produced favorable conditions for mass transfer, and also, higher yields and productivities were reached than in stirred tank fermentations.\nIntegrated fermentation and recovery of fumaric acid\nFigure 4 presents a flow scheme proposed for fumaric acid production by batch fermentation (Gangl et al. 1990). Glucose and mineral salts are fed to the fermentor. The nitrogen source solution, (NH4)2SO4, is sterilized separately and fed to the seed fermentor. The harvested broth containing sodium fumarate (cells and trace amounts of Na2CO3) is filtered to remove the cells and then acidified by H2SO4 to pH\u00a01. After acidification, fumaric acid precipitates out of the solution and is sent to a rotary dryer to be completely recovered (Gangl et al. 1990). When CaCO3 is used as a neutralizing agent instead of Na2CO3, additional heating after the fermentation was supposed to be required to dissolve calcium fumarate and the excess of CaCO3 that usually stick to the cells. This problem leads to a tedious and expensive downstream processing.\nIn the downstream processing field, recovery techniques have not so much been studied for fumaric acid producers in submerged cultivations as it has been done for related fermentation products like succinic acid (Zeikus et al. 1999), citric acid (Heinzle et al. 2006), and lactic acid (Joglekar et al. 2006). Recovery systems like reactive extraction and membrane electrodialysis have not been studied yet. However, simultaneous fermentation and adsorption have been studied to remove fumaric acid during its formation to control fermentation pH at desired values and avoid product inhibition (Cao et al. 1996; Zhou 1999).\nCao et al. (1996) used a rotary biofilm contactor (RBC) setup as fermentor for R. oryzae, in combination with an adsorption column. The produced fumaric acid was removed from the broth by the adsorption column in a recycle loop, reducing product inhibition and thus increasing the production rate and sustaining cell viability. Polyvinyl pyridine (PVP) anion exchange resin in the hydroxide form was selected as adsorbent because it yielded the highest loading capacity for fumaric acid (0.31\u00a0g g\u22121 dry wt). The RBC, coupled with the adsorption column, increased the fumaric acid productivity significantly to 4.25\u00a0g l\u22121 h\u22121 because the total fermentation time was much less than in traditional stirred tank fermentations (see Table\u00a01). This volumetric productivity and the yield are the highest reported in literature. In this integrated system, the constant removal of the fumarate produced and the liberation of OH\u2212 from the adsorption column kept the fermentation pH at 4.5. The fumarate was desorbed from the adsorption resin by using 0.4\u00a0M NaOH so additional processing will be necessary to convert the sodium fumarate into neutral fumaric acid. Addition of extra CO2 was not necessary due to the fact that during the production stage, the biofilm was exposed to sterile air that was present in the headspace of the RBC enhancing the opportunity for CO2 fixation by the biofilm.\nA novel product recovery process by an ion exchanger resin was developed by Zhou (1999). Fumarate was recovered from the fermentation broth without the use of neutralizing agents, while keeping the pH at 5, by cycling the broth over a column of a resin (Amberlite IRA-900 with OH- as counterion). After eluting the loaded column with ammonium hydroxide, ammonium fumarate solution was obtained. This was passed through a Y-zeolite column that retained the ammonium and liberated the fumaric acid. The ammonium hydroxide solution can be recovered and recycled by thermal regeneration of the zeolite (Zhou 1999). However, this integrated process did not surpass yield values obtained in a stirred tank fermentor when fumaric acid fermentation was controlled by CaCO3 under pressure pulsation (see previous section), but a higher productivity value of 1.09\u00a0g L\u22121 h\u22121 was reached (Zhou et al. 2000).\nEconomics of fumaric acid production\nIn a theoretical study, Gangl et al. (1990) compared a fermentation and a benzene-based petrochemical process with respect to economy. The fermentation process taken for this has been described in the beginning of the previous section. Although Na2CO3 was assumed as a neutralizing agent in this study, the productivity (2\u00a0g l\u22121 h\u22121) and yield (0.82\u00a0g g\u22121) of the fumaric acid fermentation with CaCO3 obtained in a previous study were used (Ng et al. 1986).\nThis economic evaluation showed that the fermentation route was less favorable than the petrochemical route (economic evaluation included upstream and downstream sections). In particular, the raw material costs were higher for the fermentation process than for the petrochemical route. It was concluded that the fermentation route could become competitive with the petrochemical route if oil prices were around 61 $\/barrel. The latter price has been reached in the recent years (http:\/\/tonto.eia.doe.gov\/dnav\/pet\/xls\/PET_PRI_WCO_K_W.xls), while the productivity of the fermentation process has been improved significantly as is shown in Table\u00a01. Besides, one can roughly calculate from Gangl\u2019s study that the assumed sugar price was around 0.6\u00a0$\/kg of glucose, which is high compared to the current price (see Table\u00a02). Therefore, if the current glucose price is assumed in the calculations of Gangl\u2019s study, one can expect lower raw material costs for the fermentation route. On the other hand, the petrochemical route has also been improved significantly, now using butane instead of benzene. This will be reflected in the current maleic anhydride price, which is shown in Table\u00a02.\nTable\u00a02Comparison between petrochemical and fermentation route for fumaric acid productionParameterPetrochemical routeFermentation routeRaw materialMaleic anhydrideGlucoseReaction temperature (\u00b0C)90\u201310035Raw material price ($\/kg)1.46\u20131.63 (Anonymous 2007)0.46aProduct yield (% w\/w)112 (Lohbeck et al. 1990)85 (Cao et al. 1996)ahttp:\/\/www.ers.usda.gov\/briefing\/sugar\/data.htm; for 4th quarter of 2006\nThe simple comparison of petrochemical and fermentation routes for fumaric acid production given in Table\u00a02 suggests that the lower raw material price of the fermentative production might compensate the higher yields of the petrochemical production from maleic anhydride, and fermentation may become an economically viable alternative.\nConclusions and future prospects\nDue to increasing prices of fossil feedstock, fermentatively produced fumaric acid could become a cheaper alternative to the petrochemically based maleic acid as unsaturated dibasic acid in polyester resins in the nearby future. Based on the available literature, the microorganism with the highest productivity and yield of fumaric acid appears to be R. oryzae. This organism produces fumaric acid via a combination of the citrate cycle and reductive pyruvate carboxylation. In addition, the fumaric acid production by fermentation can be improved if the use of neutralizing agents is reduced or avoided and the morphology of the fungi is optimized. However, avoiding the use of a neutralizing agent will lead to product inhibition; therefore, it would be useful if metabolic engineering is applied to achieve fumaric acid production in suspended, acid-resistant microorganisms such as yeast. Another way to solve product inhibition problems is applying in situ removal of fumaric acid during the fermentation.","keyphrases":["fumaric acid","fermentation","rhizopus species","product recovery","maleate isomerase"],"prmu":["P","P","P","P","P"]}
{"id":"J_Headache_Pain-4-1-2386848","title":"Topiramate in the prevention of pediatric migraine: literature review\n","text":"Pediatric migraine is a disabling condition, which can cause a significant impact on quality of life. Currently, no drugs have been approved by the FDA for its preventive treatment. Our aim was to review the medical literature concerning the efficacy and tolerability of topiramate in the prophylactic treatment of migraine in children and adolescents. A total of five papers were reviewed: two randomized controlled trials (RCTs), a post-hoc subset analysis of adolescents who had been included in three RCTs carried out on adults and two open studies. Topiramate has been proven to reduce headache frequency and the accompanying disability. The frequency of side effects varied considerably among studies, the most frequent being weight loss, anorexia, abdominal pain, difficulties in concentrating, sedation and paresthesia. Since these adverse events, although often transitory, may be distressing for the child, we strongly recommend to assess the disability caused by the migraine episodes before deciding to initiate a prophylactic treatment. Nevertheless, dropout rates due to side effects in the studies were very low.\nIntroduction\nPediatric migraine is a disabling condition, which can cause a significant impact on quality of life. It can negatively influence the child\u2019s school performances, social and family life and often causes school absenteeism. Migraine prevalence in childhood ranges from 2.7 to 10% [1]. About 3\u20135% of school-aged children suffer from migraine and this proportion gradually increases to 20% through adolescence. Initially, there is a slight male predominance; however, through adolescence, there is a shift toward female predominance, which remains through adulthood [2, 3].\nManagement of pediatric migraine includes lifestyle changes (in order to avoid foods, habits or enviromental factors that may trigger a migraine attack), the use of abortive medications and preventive measures, which can be either nonpharmacological or pharmacological. Guidelines addressing the indications for the use of preventive measures in children are lacking, but to date, most authors recommend preventive measures in children when headache frequency exceeds three to four episodes per month and\/or the attacks are significantly disabling, as measured in scoring systems such as the Pediatric Migraine Disability Assessment Scale (PedMIDAS) [4, 5]. PedMIDAS is a validated six-item questionnaire based on the adult MIDAS tool [6] with developmentally appropriate changes and adjustments for childhood lifestyle. The questions deal with the impact of headache on school (school day absences, partial day absences, functioning at 50% or less ability in school), on household acitivities (ability to perform homework and chores) and on social functioning (including sports) [7, 8]. The assessment of migraine disability is particularly important in children to correctly evaluate and treat migraine.\nCurrently, no drugs have been approved by the Food and Drug Administration (FDA) for the preventive treatment of pediatric migraine. Topiramate is an antiepileptic drug, which has been approved by the FDA in the United States and in many other countries for the preventive treatment of migraine in adults and for the treatment of partial-onset seizures and primary generalized tonic\u2013clonic seizures as add-on therapy in children as young as 2\u00a0years. Our aim was to review the medical literature concerning the efficacy and tolerability of topiramate in the prophylactic treatment of migraine in children and adolescents.\nMaterials and methods\nWe searched Pubmed (1966\u2013July 2007) using the following keywords: \u201ctopiramate\u201d\u00a0+\u00a0\u201cheadache\u201d or \u201cmigraine\u201d\u00a0+\u00a0\u201cchildren\u201d or \u201cadolescents\u201d or \u201cpediatric.\u201d We included randomized controlled trials (RCT), open-label trials and retrospective studies. We excluded case-reports and studies conducted on less than 10 patients. We classified the studies in accordance with the AAN classification of evidence for therapeutic intervention (Table\u00a01) [9]. A total of five papers were reviewed.\nTable\u00a01AAN classification of evidence for therapeutic interventionClass I: Prospective, randomized, controlled clinical trial with masked outcome assessment, in a representative population.The following are required:\u00a0(a) Primary outcome(s) is\/are clearly defined\u00a0(b) Exclusion\/inclusion criteria are clearly defined\u00a0(c) Adequate accounting for drop-outs and crossovers with numbers sufficiently low to have minimal potential for bias\u00a0(d) Relevant baseline characteristics are presented and substantially equivalent among treatment groups or there is appropriate statistical adjustment for differencesClass II: Prospective matched group cohort study in a representative population with masked outcome assessment that meets (a)\u2013(d) above OR a RCT in a representative population that lacks one criteria (a)\u2013(d).Class III: All other controlled trials (including well-defined natural history controls or patients serving as own controls) in a representative population, where outcome is independently assessed, or independently derived by objective outcome measurement.Class IV: Evidence from uncontrolled studies, case series, case reports, or expert opinion.\nResults\nWe found two RCTs, a post-hoc subset analysis of 51 adolescents (12\u201317\u00a0years) who had been enrolled in three pivotal RCTs of topiramate for migraine prophylaxis in adults and two open studies.\nIn a recent Class I randomized, double-blind, placebo-controlled trial [10], 44 children were randomized (in a 1:1 ratio) to receive either placebo or topiramate (titrated to 100\u00a0mg a day) for 12\u00a0weeks. Participants had a diagnosis of migraine without aura (according to 2004 International Headache Society criteria) [11] and a frequency of two or more headaches per month for 3\u00a0months before entering the study. Children with comorbid medical associations or who were already on migraine prophylaxis were excluded. Primary outcome measures were the reduction in migraine frequency and severity; secondary outcome measures included number of analgesics taken and the functional disability. Migraine monthly frequency decreased from 16.14 (\u00b19.35) at baseline to 4.27 (\u00b11.95) at the end of the study in the treated group as compared with a decrease from 13.38 (\u00b17.78) to 7.48 (\u00b15.94) in the placebo group (P\u00a0=\u00a00.025). The percentage of topiramate-treated subjects showing >50% reduction in monthly migraine days during the double-blind phase was 95.2% as opposed to 52.4% in the placebo group (P\u00a0=\u00a00.002). The Pediatric Migraine Disability Assessment Score (PedMIDAS) also decreased significantly (P\u00a0=\u00a00.003) as did school absenteeism (P\u00a0=\u00a00.002). Side effects were rated mild to moderate, did not interfere with daily activities or cause any dropouts and included weight-loss (mean loss of 0.3\u00a0kg) in 81% of patients, paresthesia (23.8%), loss of appetite (23.8%), lack of concentration (19%), sedation (19%) and abdominal pain (14.3%). This seems a well-performed trial in which the mean monthly migraine frequency reduction is clearly evident in the topiramate-treated group. Patients treated with topiramate suffered at least five episodes a month before entering the study (range 5\u201330); a preventive treatment was, thus, clearly indicated. The reduction in the PedMIDAS score indicates that the drug side effects (difficulties in concentrating, sedation) interfered with school, household or social activities to a lesser extent than the migraine episodes.\nThe other Class I, randomized, double-blind, placebo-controlled trial [12] was carried out on 162 children (age 6\u201315\u00a0years) who were randomized in a 2:1 ratio to receive either topiramate (which was titrated over 8\u00a0weeks to 2\u20133\u00a0mg\/kg per day and maintained for 12\u00a0weeks) or placebo. Eligible criteria were the children with migraine with or without aura, weighing more than 20\u00a0kg, who experienced 3\u201310 migraine days\/month for the 3\u00a0months prior to the screening and during the 4-week prospective baseline phase. Exclusion criteria included chronic migraine, analgesic overuse and previous failure of \u22652 adequately dosed migraine preventive medications. One hundred and fifty-seven subjects were included in the intention to treat (ITT) population, defined as randomized subjects who had received at least one dose of study medication and had at least one postbaseline efficacy assessment, and a total of 131 children completed the study (per-protocol population); of these, 108 were treated with topiramate. Primary outcome measure was the reduction of migraine days per month in each treatment group of the ITT population. During the double-blind-phase, relative to the 4-week prospective baseline phase, there was a reduction of 2.6\u00a0\u00b1\u00a02.6 migraine days per month in the topiramate group compared with a mean reduction of 2.0\u00a0\u00b1\u00a03.1 migraine days per month in the placebo group (P\u00a0=\u00a00.061). In the per-protocol population, the reduction in monthly migraine days (2.8\u00a0\u00b1\u00a02.4 as opposed to 2.2\u00a0\u00b1\u00a02.1 in the placebo group) reached statistical significance (P\u00a0=\u00a00.033). During the last 28 days of treatment, the mean number of monthly migraine was reduced by 3.1\u00a0\u00b1\u00a02.6, as opposed to 2.4\u00a0\u00b1\u00a02.8 in the placebo group (P\u00a0=\u00a00.023). In the treated group, 32% of patients experienced a \u226575% reduction in mean monthly migraine days compared with a 14% reduction in the placebo group (P\u00a0=\u00a00.02). The dropout rate for adverse events was 6.3% for the topiramate group and 4% for the placebo group. In the treated group, the most common adverse events were anorexia (13%); weight decrease (10.2%), with a mean loss of 1.4\u00a0\u00b1\u00a02.6\u00a0kg (as opposed to 0.7\u00a0\u00b1\u00a03.9\u00a0kg in the placebo group); abdominal pain (10.2%); paresthesia (8.3%); and somnolence (8.3%). Serious adverse events occurred in four topiramate-treated patients and included infection (n\u00a0=\u00a02), severe migraine (n\u00a0=\u00a01) and suicidal ideation (n\u00a0=\u00a01).\nIn this study, the mean monthly migraine frequency reduction seems less evident when compared to the previous study [10], probably because migraine frequency at baseline was lower (range 2\u20139 in the ITT topiramate population) and children with chronic migraine were excluded. An important aspect is that the reduction is more evident in the last 28\u00a0days of treatment, indicating that it may take a few weeks before treatment with topiramate reaches its maximum efficacy.\nWinner et al. [13] performed a post-hoc subset analysis on 51 adolescents (12\u201317\u00a0years) who had been enrolled in three pivotal trials of topiramate for migraine prophylaxis in adults. Patients were to have had between 3 and 12 migraine attacks, and no more than 14 headache days per 28\u00a0days during the 3\u00a0months prior to the screening and during the 4-week prospective baseline phase. Also excluded were patients who overused analgesics or who had failed to respond to two or more adequate prophylactic treatments. Topiramate was administered at the dosages of 50, 100 or 200\u00a0mg a day for 26\u00a0weeks. When compared to baseline, there was a mean monthly migraine frequency reduction of 46, 63 and 65%, respectively, as opposed to a mean 16% reduction in the placebo group (P\u00a0=\u00a00.07, P\u00a0=\u00a00.02, P\u00a0=\u00a00.04, respectively). The most common adverse events (in the group treated with 100\u00a0mg a day) were as follows: paresthesia (38%), upper respiratory tract infections (23%), weight decrease (15%), abdominal pain (15%), anorexia (8%) and somnolence (8%). There were no dropouts in the group treated with topiramate. The incidence of adverse events was higher in the group treated with topiramate at the dosage of 200\u00a0mg, which did not appear to confer additional efficacy as opposed to the dosage of 100\u00a0mg a day.\nOne class IV study [14], assessing the efficacy of topiramate for pediatric migraine, included 97 children, 75 of which were reevaluated at a first follow-up visit (after 88.7\u00a0\u00b1\u00a035.7\u00a0days) and 41 at a second follow-up visit (after 203.1\u00a0\u00b1\u00a045.6\u00a0days). Included were children reporting more than three headaches per month. Topiramate was administered at the dose of 1.4\u00a0\u00b1\u00a00.74\u00a0mg\/kg\/day and headache frequency decreased from 16.5\u00a0\u00b1\u00a010 headaches\/month to 11.6\u00a0\u00b1\u00a010.2 headaches\/month (P\u00a0<\u00a00.001) at the first follow-up visit, with 43.1% of the patients experiencing a 50% or greater reduction in the number of headaches. By the second follow-up visit, headache frequency had decreased to 9.4\u00a0\u00b1\u00a08.4, with 56.1% of the patients experiencing a 50% or greater reduction in the number of headaches. Mean headache severity, duration and accompanying disability were also reduced. Disability was measured using the PedMIDAS score: a 50% reduction occurred in 48.6% of the patients at the first follow-up visit and in 62.5% of patients at the second follow-up visit. One quarter of patients complained of side effects, which declined in subsequent visits and included cognitive changes (12.5%), weight loss (5.6%) and sensory symptoms (2.8%). A possible bias of this study is that 50.7% of patients were on additional prophylactic medication (most often amitriptyline or divalproex).\nIn an open, prospective case series (class IV) study [15], topiramate was administered to 24 children with migraine, who had failed to respond to other prophylactics, at the mean dosage of 3.5\u00a0\u00b1\u00a01.7\u00a0mg\/kg for 4\u00a0months. Authors report a reduction in the duration and in the intensity of headaches; headache frequency reduction, however, did not reach statistical significance. Adverse events (emotional instability, paresthesia, anorexia, asthenia, weight loss) were experienced by one-third of patients. In our opinion, the baseline headache frequency (3.6\u00a0\u00b1\u00a02.7 a month with a range of 1\u201312) and duration (2\u20136\u00a0h) did not necessarily constitute an indication for prophylactic treatment in all treated cases.\nUnfortunately, the studies we reviewed were not homogeneous with regard to inclusion and exclusion criteria and with regard to outcome measures such as the \u201cresponder rate,\u201d which was not considered in all studies. Furthermore, the degree of disability caused by the migraine episodes, which is essential in the decision to initiate prophylactic treatment, was not assessed in all studies.\nDiscussion\nManagement of pediatric migraine can be tricky, seeing as no pharmacological treatment is formally approved or indicated in the prophylactic treatment of migraine in children and adolescents. A recent review of the pharmacological treatment options in pediatric migraine [16] found only flunarizine, which is not available in many countries, to be effective in rigorous controlled trials. Authors also concluded that there is conflicting evidence regarding propanolol and trazodone, while data from uncontrolled studies suggest cyproheptadine, amitriptyline, naproxen and antiepileptic drugs (topiramate, valproic acid and gabapentin) to be effective. Since this review was published, two randomized, placebo-controlled trials evaluating topiramate in the preventive treatment of pediatric migraine have been carried out [10, 12].\nTaken together, data from the papers we reviewed suggest that topiramate is effective in the preventive treatment of pediatric migraine. Furthermore, topiramate dosages, which seem to be effective in the treatment of migraine in children and adolescents (2\u20133\u00a0mg\/kg\/day) are much lower than those indicated for the adjunctive treatment of epilepsy (5\u20139\u00a0mg\/kg\/day) in children as young as 2\u00a0years.\nAs regards its safety and tolerability, serious adverse effects were very rare (one patient with suicidal ideation), though the risk of depression must be kept in mind. The percentage of patients experiencing side effects varied greatly among the studies, the most common ones being weight loss (which, in the RCTs [10, 12], was present in 81 and 10.2% of patients, respectively), anorexia (23.8 and 13%, respectively), abdominal pain (14.3 and 10,2%), difficulties in concentrating, somnolence\/sedation (19 and 8.3%) and paresthesia (23.8 and 8,3%). Side effects tended to decline over time and dropout rates due to side effects were very low (range 0\u20136.3%). Since these adverse events, although often transitory, may be clinically significant and distressing both for the child and its family, the decision to initiate treatment must be carefully weighed and should take the degree of disability caused by the migraine episodes into account.\nAn important aspect, which emerged from some of the studies, is a significant decrease in school absenteeism due to migraine [10] and a reduction in the disability caused by migraine (as assessed by PedMIDAS), leading to an improvement in the childrens\u2019 quality of life [10, 14].\nIn conclusion, topiramate seems to be a promising therapeutic option, though, clearly, further controlled trials are needed to confirm this data, as are studies comparing different drugs.","keyphrases":["topiramate","pediatric","migraine","children","headache"],"prmu":["P","P","P","P","P"]}
{"id":"Graefes_Arch_Clin_Exp_Ophthalmol-3-1-2082065","title":"Characteristics of dynamic processing in the visual field of patients with age-related maculopathy\n","text":"Purpose To investigate the characteristics of dynamic processing in the visual field of patients with age-related maculopathy (ARM) by measuring motion sensitivity, double-pulse resolution (DPR), and critical flicker fusion.\nIntroduction\nAge-related maculopathy (ARM) is the most common cause of loss of central vision in industrialized countries [17, 26, 30, 33, 34, 62]. Almost all clinical assessments of the consequences of ARM for basic visual functions and quality of life refer to foveal or macular vision. However, a few studies have shown that retinal defects can occur beyond 10\u00b0 eccentricity, which can be considered the maximum radial extent of the macula. Sunness et al., for instance, described histopathological anomalies outside the central retina, whereas sensitivity was unchanged in traditional static perimetry [55].\nCurcio et al. described that, beyond general age-related photoreceptor loss, five out of six ARM-affected donor eyes showed cone and rod loss in parafoveal regions [12]. The latter can be expected to reduce dark adaptation performance, which was confirmed up to 25\u00b0 eccentricity by Brown et al. [9]. In addition, ARM affects temporal aspects of visual information processing. Mayer et al. found reduced temporal resolution of ARM patients as measured by foveal flicker sensitivity with a stimulus of 2.8\u00b0 diameter [40, 41]. Brown and Lovie-Kitchin showed reduced flicker resolution in the fovea and at 10\u00b0 and 20\u00b0 eccentricity and concluded that \u201cthe functional effects of ARM are not confined to the central retina, but affect a large region of the visual field\u201d [11]. Falsini et al. found cone-mediated flicker sensitivity (CFS) losses in ARM by evaluating the focal electroretinogram (FERG) as a function of flicker modulation depth in the macula (9\u00b0 radius) [13].\nIn summary, the results of these studies show age-related as well as ARM-related functional loss of dynamic processing. However, whether the loss is confined to the macula has not been definitively answered. A further question is whether a loss outside the macula is specific to temporal resolution or whether other temporal aspects of retinal processing, such as motion sensitivity, can also be affected beyond the macula in patients suffering from ARM. Furthermore, we asked whether the impairment is confined to the magnocellular system or also involves the parvocellular pathway.\nMethods\nMotion contrast threshold\nExperimental setup\nThe experimental setup allowed measurements of peripheral motion perception up to 60\u00b0 eccentricity under photopic conditions (luminance\u2009=\u200975\u00a0cd\/m2). It consisted of a white, semicircular (180\u00b0) plastic screen (radius 90\u00a0cm, height 60\u00a0cm) with a rectangular opening in the center. A flat-screen LCD display was mounted behind the opening for the presentation of the test patterns. The subjects sat in front of the screen at a distance of 90\u00a0cm and monocularly fixated one of the fixation marks in primary view (fixation crosses with diameter\u2009=\u20094\u00a0cm and bar width\u2009=\u20090.5\u00a0cm). The marks were attached to the plastic background at 10\u00b0, 20\u00b0, 30\u00b0, 40\u00b0, and 60\u00b0 eccentricity (Fig.\u00a01). Fixation was monitored by a mirror mounted below the LCD display. Thus, the examiner could monitor the subject\u2019s eye movements during the presentation of a stimulus. Additionally, the mirror could be moved horizontally to allow a clear view of the patient\u2019s eye, even in extremely peripheral positions. We are confident that the examiner was able to detect saccadic eye movements of at least 5\u00b0 amplitude, which is still only half of the distance between two measuring locations.\nFig.\u00a01Experimental set up (top view). The subject\u2019s chair was rotated to maintain primary gaze for all tested fixation marks. a LCD monitor screen. b Semicircular plastic screen. c Fixation crosses (diameter\u2009=\u20094\u00a0cm, bar width\u2009=\u20090.5\u00a0cm). d Chin rest for stabilizing the head and keeping the viewing distance constant. e Subject\u2019s right or left eye\nMonitor calibration\nTo achieve specified stimulus luminance and contrast, the monitor\u2019s gamma curve (screen luminance vs. gray value) needs to be taken into account during stimulus presentation [4, 51, 52, 54]. We measured this for our monitor using Irtel\u2019s PXL software to set the screen uniformly at the 256 possible gray levels and measured center screen luminance with a digital luminance meter (Gossen Mavomonitor G04068, N\u00fcrnberg, Germany) [25]. A second order function was fit to the luminance data by non-linear regression (r2\u2009=\u200999%):\nwhere L is center screen luminance in cd\/m2 and g is the uniform gray value (range 0\u2013255). These function parameters are incorporated in our stimulus presentation software (see below). Target contrast is specified in the Michelson measure, .\nDesign of the motion stimulus\nThe test software for motion perception was custom-developed for these experiments and consists of two separate modules, one for stimulus generation and another for running the actual test program. Since threshold measurements are much faster and more reliable using four-alternative forced choice than two-alternative forced choice tasks, we employed four directions of motion (up, down, left, and right) to be discriminated by the subject. The stimuli were plaids composed of two orthogonal Gabor patterns of 45\u00b0 left and right tilt [15]:\nwhere L and L0 are the luminances of pattern and background, respectively,  is the square of radial diameter of the Gaussian envelope (the value where the amplitude has decreased to 1\/e), and \u03d5(t) is the spatial phase at time t (i.e. the shift within the envelope). For the plaids used here, we chose \u03c3\u2009=\u20090.8, which results in about 1.5 visible cycles (Fig.\u00a02).\nFig.\u00a02Gabor stimulus with double sinusoidal modulation in spatial quadrature. The entire patch was stationary, while the plaid pattern moved within the envelope in one of four possible directions. In addition, the stimulus was temporally modulated by a Gaussian envelope function, so that it slowly appeared out of the medium grey background and then merged back into it\nThe subject\u2019s task was to identify the direction of motion. Watson found that thresholds of motion detection and motion discrimination in healthy subjects are equal at sufficiently low spatial frequencies (e.g. 2 cpd) for both slow (1.5\u00a0Hz) and fast (12.4\u00a0Hz) movement of the test pattern [63]. Therefore, we used a low spatial frequency of 0.65 cpd and an intermediate speed of 5.71 \u00b0\/s (corresponding to 3.75\u00a0Hz local luminance change) for the motion stimulus. The size of the test pattern at the viewing distance of 90\u00a0cm was 3.8\u00b0. To minimize attracting transient attention by abrupt pattern onset, the test pattern gradually appeared and disappeared by using a Gaussian temporal envelope [27, 36, 37]. Due to the small changes between frames in these stimuli, \u201ctearing\u201d of the image was extremely unlikely and was indeed never observed [16].\nAdaptive algorithm\nMonocular contrast thresholds for motion perception were measured along the horizontal meridian in the nasal and temporal visual field at 10\u00b0, 20\u00b0, 30\u00b0 and 40\u00b0 eccentricity and in the temporal visual field additionally at 60\u00b0. The initial contrast value for all eccentricities was 30% (1.48 log %-contrast). For each eccentricity, the threshold was determined twice. Two blocks of nine measurements each were taken and the arithmetic mean calculated from the two results. The adaptive algorithm for threshold determination was based on Kesten and was modified according to Kaernbach to allow the additional response alternative \u201cI don\u2019t know\u201d [28, 29, 32, 58]. This paradigm, termed \u201cunforced choice task\u201d by Kaernbach, has been shown to be beneficial, especially for subjects who are unfamiliar with psychophysical testing procedures.\nKesten\u2019s algorithm is of the staircase kind, i.e. the intensity of the next presentation depends on the previous response only, not on the entire history or a longer sequence of preceding responses [32]. After a correct response, the log contrast of the stimulus was decreased by one incremental step, and after an incorrect response, it was increased by 1.67 incremental steps (5\/3). It thereby converged on the 62.5%-correct point (5\/8), i.e. the point of inflection \u03d5 on the psychometric function (of log contrast) in a four-alternative forced-choice task. The ratio V=5\/3=1:1.667 of up-down intensity changes results from Kesten\u2019s algorithm given the number n of response alternatives (four) by\nwhere \u03d5 is the probability at threshold or point of convergence,\nwith guessing rate \u03b3\u2009\u2009=\u20091\/n\u2009=\u20091\/4. (c.f. Treutwein\u2019s equation 16, the last term (Zn\u2013\u03d5) [58]). When the subject gave the indecisive response \u201cI don\u2019t know\u201d, the intensity of the next stimulus was increased by one incremental step, as specified by Kaernbach\u2019s extension which requires the specification of V as above [28]. The step width in Kesten\u2019s algorithm is reduced by a factor of m\/(m\u2009+\u20092) after a change in response category (correct-to-incorrect or vice versa) where m is the number of reversals (Treutwein\u2019s eq. 16). Since Kaernbach\u2019s extension requires the above-mentioned fixed ratio of up-down intensity changes, we used a step width reduction only every other reversal (i.e. m even). The Kesten rule is applied to log contrast, i.e. log Cn\u2009+\u20091=\u2009(logCn)\u00b1sn (where sn is the step width), which is equivalent to contrast being multiplied or divided by the antilog of step width.\nDouble-pulse resolution\nTreutwein and Rentschler developed a hardware and software combination for the measurement of double-pulse resolution (DPR) which allows simultaneous measurement at multiple visual field positions [60]. The technique has been used in experiments on patients with multiple sclerosis and glaucoma, and in an extensive study characterizing the temporal properties of the visual field across the life span [46, 48\u201350]. The experimental setup allowed (limited by screen size) measurement of DPR up to 20\u00b0 eccentricity, i.e. not as far into the periphery as for the motion contrast thresholds but well beyond the macular region.\nTest setup\nAn analogue 15\u2033 x-y-z display was used for stimulus presentation (Hewlett Packard model 1310, i.e. a CRT-display without a raster-scan generator), driven by a temporary buffer that stores the point coordinates and generates the control voltages for the display (\u201cpoint plot buffer\u201d; G. Finlay, Edmonton, Canada). Temporal control could be extended into the low microsecond range, so that the temporal resolution was better by a factor of 1,000 than in conventional setups [60]. A computer (PC) was used for running the experimental software that controlled stimulus generation, adaptive procedure, and data acquisition. The experimenter recorded the subject\u2019s responses via the computer keyboard.\nStimulus characteristics\nThe tests were performed monocularly at 30\u00a0cm viewing distance with a diagonal screen diameter of 40\u00a0cm. The stimuli in Treutwein\u2019s technique are eight squares of 1.15\u00b0\u2009\u00d7\u20091.15\u00b0 visual angle (5\u2009\u00d7\u20095 pixels) each, arranged in circles at 5\u00b0, 10\u00b0 , and 20\u00b0 eccentricity around a ninth square in the center [60]. Figure\u00a03 shows the stimulus time course. Eight of nine squares are continuously present for 80\u2009+\u2009\u03b3\u2009+\u2009280\u00a0ms. The target, one randomly selected stimulus, is switched off after 80\u00a0ms and then on again after a variable time gap \u03b3. The subject\u2019s task was to detect the gap and indicate the target position on the screen, which makes it a nine-alternative forced choice task. In supra-threshold trials, its location can be recognized as a short flicker of the target. A central fixation cross was presented continuously between stimulus presentations and was switched off 50\u00a0ms before the beginning of a new trial. The squares\u2019 luminance was 215\u00a0cd\/m2; subjects were light-adapted and the display background luminance was held constant in the low photopic range (luminance \u223c15\u00a0cd\/m2).\nFig.\u00a03Double-pulse stimulus characteristics (figure modified after Poggel and Strasburger, originally from Treutwein and Rentschler [46, 60]. a, b Time line diagram of stimulus display. c Stimulus positions (5\u00b0, 10\u00b0, and 20\u00b0 eccentricity)\nGap duration was varied by an adaptive thresholding algorithm (\u201cYAAP\u201d), based on maximum-likelihood psychometric function fitting [20, 59].\nFlicker frequency analyzer\nFor measurement of foveal flicker fusion frequencies we used the \u201cWiener Testsystem\u201d, a commercial computer-based diagnostic system with an emphasis on assessing driving fitness (Schuhfried, Moedling\/Vienna, Austria). The system was chosen as a reference, because it is an established standard system in Germany used for psychological testing; due to its construction it allows foveal measurement only. Measurements were performed by presenting a circular flickering red light stimulus (1.2\u00b0 diameter; luminance\u2009=\u2009270\u00a0cd\/m2; wave length 655\u00a0nm) on a white background in a tubular viewer. Thresholds were determined by the method of limits, i.e. the frequency of the flickering light is increased until permanent light is perceived and is then decreased until the light is once again perceived as flickering.\nThe subject indicated the perceived changes by a keystroke whereupon the critical frequency was recorded. The separate arithmetic means of the critical frequencies determined in the ascending and descending series are referred to as fusion frequency and flicker frequency, respectively. Each test cycle consisted of five training cycles, immediately followed by eight measurement cycles. For further analysis, the mean of the fusion frequency and the flicker frequency was calculated for each subject, which is referred to as the critical flicker fusion frequency (CFF).\nColor perception\nThe Lanthony D-15 color vision test (desaturated) was used for the examination of color perception. The test consists of 15 colored chips, which are used to determine the subject\u2019s capability to discriminate hues. At 50\u00a0cm viewing distance, every chip has a diameter of about 1.5\u00b0. The subject sorts the arbitrarily shuffled chips according to the arrangement in the color cycle. The chosen sequence was then documented in a test protocol and the color confusion score (CCS) and crossings over the color space were determined [1]. The measurement was done once under constant lighting conditions (color temperature \u223c4000\u00a0K).\nSubjects\nWe recruited three groups of subjects for this study: 14 subjects with ARM (19 eyes, mean visual acuity: 0.29, SE\u2009\u00b1\u20090.048; \u223c20\/63), a control group of age-matched subjects with healthy eyes (14 subjects; 18 eyes, mean acuity: 0.65, SE\u2009\u00b1\u20090.057; \u223c20\/32), and another control group of 7 young subjects (8 eyes, mean acuity: 1.25, SE\u2009\u00b1\u20090.047; 20\/16) (Table\u00a01). ARM in 16 of the 19 eyes of patients were classified as \u201cdry\u201d and three as \u201cwet\u201d. Six of 19 eyes in the ARM group had received intra-ocular lens implants during cataract surgery, which was also the case for 14 out of the 18 eyes of the age-matched control group. Since in most cases the implant causes mild myopia (\u22120.5 dpt), the patients-though presbyopic-could observe the fixation mark comfortably without near correction. Thus, optical artifacts from eyeglasses were prevented, such as aberrations or interference from the glasses\u2019 rims, which would otherwise occur at higher eccentricities.\nTable\u00a01Age and visual acuity of the subject groupsAgeVisual acuity\u00a0NMeanSEMinimumMaximumMeanSEARM group1973.792.360900.29 (20\/63)0.048Age-matched controls1871.51.560780.65 (20\/32)0.057Young controls827.11.721341.25 (20\/16)0.047Visual acuity is described in decimal notation and Snellen fractions\nVisual acuity (VA) was measured by a Landolt ring test (Binoptometer, Oculus, Germany) at 90\u00a0cm viewing distance (VA\u2009=\u20091\/\u03c9\u2032, where \u03c9\u2032 is the minimum angle of resolution in minutes). Based on these values, all subjects were capable of directing their gaze at the fixation mark without glasses. All subjects with ARM and the age-matched control group provided an up-to-date medical statement from an ophthalmologist, which appraised the condition of the retina and excluded other eye diseases (e.g. glaucoma, secondary cataract). All subjects were examined by their ophthalmologist with respect to clarity of the optical media, and none of them showed any evidence of changes in the visual pathways.\nThe complete examination (including the initial interview) took approximately 3 hours.\nThe ages of the subjects with ARM and the age-matched controls were sufficiently close; a t-test with a preceding test for normal distribution showed that the two groups did not differ significantly with respect to their age (p\u2009=\u20090.36). The study design had been approved by the ethics committee of the University of Munich and testing procedures were in accordance with the tenets of the Declaration of Helsinki. All subjects gave their informed consent for participation.\nResults\nMotion contrast threshold\nFigure\u00a04 shows the contrast thresholds for motion perception as a function of eccentricity in the visual field. Compared with the age-matched controls, the ARM group shows a distinct increase of contrast thresholds up to 20\u00b0 in the nasal and up to 40\u00b0 in the temporal field. Mann-Whitney U-tests of the contrast thresholds at 10\u00b0, 20\u00b0, 30\u00b0, and eccentricity on the horizontal meridian each showed significant differences between the two elderly groups (Table\u00a02).\nFig.\u00a04Mean contrast thresholds for motion perception of the subjects with ARM, age-matched controls, and young controls on the horizontal meridian. The broken lines show the increased contrast thresholds when stimuli were presented near the blind spot. The continuous lines show the regression (specified in the formula); omitted for the age-matched control group on the temporal side for clarityTable\u00a02Results of Mann-Whitney U-tests of contrast thresholds (ARM group vs. age-matched control group); U is the statistical value of nonparametric Mann-Whitney test for comparison of independent samples based on ordinal ranks; z\u2009 is the estimated statistical value of normalized distribution of U values; p\u2009 is the\u2009significance levelEccentricityNasal fieldTemporal fieldUzpUzp10\u00b016.00\u22124.71<0.00153.00\u22123.59<0.00120\u00b069.50\u22123.090.00272.50\u22122.830.00530\u00b0148.50\u22120.680.4996.00\u22122.280.02340\u00b0145.50\u22120.250.80102.00\u22122.100.03660\u00b0\u2212\u2212\u2212134.00\u22121.120.261\nDouble-pulse resolution\nWith respect to double-pulse resolution, the subjects with ARM showed strongly increased thresholds compared with the age-matched controls within the 20\u00b0 visual field covered by the test area (Fig.\u00a05). For better comparison with the motion sensitivity data, Fig.\u00a06 also shows the double-pulse results on the horizontal meridian. Both the comparison on the two horizontal meridians and on the entire circles at the investigated eccentricities (5\u00b0, 10\u00b0, and 20\u00b0) show significant group differences. We performed a Mann-Whitney U-test at every position on the horizontal visual field meridian (Table\u00a03).\nFig.\u00a05Mean double-pulse resolution (DPR) of the ARM group, age-matched controls and young controls at 5\u00b0, 10\u00b0, and 20\u00b0 eccentricity (stimuli positioned on concentric rings, Fig.\u00a03). The 0\u00b0-value represents the foveal DPR in the 5\u00b0-ring measurement. Note that the rather small SEM error bars indicate a limited range of variation of valuesFig.\u00a06Double-pulse resolution (DPR) of the ARM group, age-matched controls, and young controls along the horizontal meridianTable\u00a03Results of Mann-Whitney U-tests at each eccentricity for the ARM group and the age-matched control group; U is the statistical value of non-parametric Mann-Whitney test for comparison of independent samples based on ordinal ranks; z is the estimated statistical value of normalized distribution of U values; p is the significance levelEccentricityNasal fieldTemporal fieldUzpUzp5\u00b046.50\u22122.550.01146.50\u22122.560.01110\u00b045.00\u22123.83< 0.00179.50\u22122.780.00520\u00b076.00\u22122.890.00361.00\u22123.340.001\nCritical flicker fusion frequency (CFF)\nFigure\u00a07 shows critical flicker fusion frequency (CFF) as a function of age for the three subject groups. There is a slight but significant (p\u2009<\u20090.05) loss of CFF with age. It amounts to 0.059\u00a0Hz per year of age (i.e. around 5\u00a0Hz for the entire life span), which accounts for 30% of the inter-individual variance in the healthy subjects. Age-independent interindividual variance (70%) thus far exceeds the age-related loss. More importantly here, however, the graph also shows the marked loss of performance in the subjects with ARM. On average, the CFF in the ARM group is lower by 5.47\u00a0Hz, or approximately 14%, in comparison to the age-matched controls. Note that one 90-year-old subject reached the level of young normal subjects, while all other subjects with ARM showed lower performance than all young controls.\nFig.\u00a07Critical flicker fusion frequency as a function of age for the three groups. The regression line refers to the healthy controls\nThe analysis of variance (one-way ANOVA with group as factor and post-hoc test, Tamhane procedure) indicates a highly significant difference between the three groups (F\u2009=\u200925.16; df\u2009=\u20092; p\u2009<\u20090.001) which was caused predominantly by the highly significant difference between the age-matched control group and the ARM group (p<0.001).\nColor perception\nPerformance in color perception is a sensitive indicator of differential impairment of the parvo- vs. the magnocellular system [31]. The result from the Lanthony D-15 color vision test (unsaturated) allows the calculation of the color confusion score (CCS), which was used for the statistical analysis of the results. A one-way ANOVA with subject groups as factor and subsequent post-hoc test (Tamhane procedure) showed highly significant differences (F\u2009=\u200925.285; df\u2009=\u20092; p\u2009<\u20090.001) between the three test groups. The difference between the age-matched control group and the ARM group was also highly significant (p\u2009<\u20090.001). Thus, the subjects with ARM clearly showed impaired color discrimination. The comparison of crossings over the color space also shows significant differences between the subject groups (F\u2009=\u200920.394; df\u2009=\u20092; p\u2009<\u20090.001; mean crossings: ARM\u2009=\u20095.81, age-matched controls\u2009=\u20091.78, and young controls\u2009=\u20090).\nDiscussion\nMotion perception\nOur findings document deficits of dynamic visual field properties in ARM in a retinal region that extends far beyond the macula. In a sense, the well documented loss of visual function (e.g. foveal acuity) in the macula of subjects with ARM seems just like the proverbial tip of an iceberg. To our knowledge, this is the first study of dynamic characteristics of the peripheral visual field up to 60\u00b0 eccentricity in subjects with ARM. Functional studies on these patients in the past have either concentrated on foveal function only, or have used a large central stimulus that did not discriminate between foveal and nonfoveal function [5, 40\u201342, 57]. Only the study by Brown and Lovie-Kitchen has shown deficits in the temporal visual field up to 20\u00b0 eccentricity [13].\nThere are only a few studies on dynamic processing characteristics in ARM. Mayer et al. measured flicker sensitivity only foveally and suggest that it can be a predictor of exudative ARM [40, 41]. Normal aging of visual function \u2013 from which ARM losses need to be distinguished \u2013 has received only limited attention [19, 45, 46, 61].\nIn the present study, we determined the contrast thresholds for motion perception far into the periphery of the visual field on the horizontal meridian, up to 40\u00b0 on the nasal, and 60\u00b0 on the temporal side. We found pronounced impairment in the ARM group up to 20\u00b0 nasally and 30\u00b0 temporally, and smaller impairments still further out on the temporal side. The macula is commonly described as having a radius of 10\u00b0 [2, 22]. These elevated contrast thresholds for motion perception extend far beyond this eccentricity, in conflict with a widespread assumption that the effects of manifest ARM are restricted to the macula. The latter seems to be based on two facts. First, customary descriptions and classifications of the disease use morphological features that are visible by ophthalmoscopy or on fundus photographs [6\u20138]; and second, many experimental studies have tested vision in subjects with ARM only in central locations in the visual field as noted above, with a few notable exceptions [10, 11, 55].\nIn clinical practice, the assessment of the visual periphery is believed to play a minor role in ARM, for a lack of consequences in ophthalmic treatment and also because of the dramatic vision loss from damage to the central retina, immediately noticed by patients who report the loss of the ability to recognize fine spatial or contrast detail (e.g. small print). A loss of peripheral motion sensitivity, in contrast, is not easily recognized by the patient, possibly because the conscious (sustained) component of attention is normally directed towards central vision, whereas the transient (reflex-like) component of attention is not accessible by consciousness [44]. This phenomenon plays a well-known role in glaucoma which often goes unnoticed by the patient. Thus, the fact that patients do not complain about deficits in peripheral motion perception might lead the practitioner not to examine this retinal region in detail. Yet, practical consequences have to be considered. If diminished sensitivity to motion raises the possibility that a patient\u2019s transient attention might not be attracted to a moving stimulus, a behaviorally valuable eye movement may not be performed and a potentially hazardous situation may not be recognized.\nAdditionally, the tests available in routine clinical practice do not provide the appropriate stimuli to detect such defects. For instance, Holopigian et al. examined peripheral vision in patients with early ARM using standard electrophysiological and psychophysical tests (dark adaptation curves, electro-oculograms and electro-retinograms [23]. They found that only few subjects with early ARM developed disease-related effects in this region when examined with standard clinical tests.\nDouble-pulse resolution (DPR) and critical flicker fusion (CFF)\nThe results of DPR revealed distinct and statistically significant differences between the ARM group and age-matched controls up to 20\u00b0 eccentricity. Furthermore, the increased foveal thresholds of double-pulse resolution in subjects with ARM parallel the elevated foveal CFF thresholds, which also show a dependency on age and a significant increase in subjects with ARM.\nComparison of the age-matched with the young control group shows an age dependency which confirms earlier results [18, 46, 48, 61]. However, our results cannot be directly compared with the findings of Falsini et al. [13] who used a large homogeneous test field (18\u00b0 diameter) that covered visual field regions with pronounced differences in functional characteristics.\nThe fact that reduced dynamic performance in our patients was found for two quite different methods of measurement emphasizes the presence of impaired temporal vision performance far beyond the macula and suggests a common underlying pathogenesis.\nColor vision\nThe objective of the color vision test was to examine the relative involvement of the two neuronal subsystems of the primary visual pathway \u2013 magnocellular (M) and parvocellular (P) \u2013 in the previously obtained results. Both motion perception and temporal resolution are assumed to be predominantly conveyed by the M system, whereas color vision is predominantly mediated by the P system [31]. To simply find out whether color perception is intact, it is legitimate to use the color vision test foveally, or to allow the subjects to use the retinal area that serves them best. A (seemingly self-evident) conclusion that the deficits reported here may be caused by a specific damage to the M system can thus be rejected. Impaired color vision, as found here, shows that the P system is also affected.\nThe ARM group in our study showed a significantly lower foveal color vision performance than the age-matched control group. This would seem in conflict with the results of Frennesson et al. and Medina et al. who found no color vision deficits in subjects with ARM at an early stage [14, 42]. However, those studies used less sensitive tests with saturated colors, such as the Farnsworth D-15 test or the Farnsworth-Munsell 100 Hue test, whereas our findings are based on the more sensitive Lanthony D-15 test which uses unsaturated colors [14, 42]. Since (static) contrast sensitivity was not measured here, we cannot estimate how strongly the color vision findings were influenced by the expectedly reduced contrast sensitivity of these patients.\nConsequences\nEven though the perceptual limitations measured in this study are not immediately evident to subjects with ARM, their relevance for daily life should not be underestimated. Their importance is due especially to the fact that segments of the near periphery assist safe mobility in general and driving a car in particular [21, 39]. Visual perception of moving stimuli has two functions of great importance in daily life. First, we perceive a stable, stationary world in which we are moving, despite the fact that nearly all image components on the retina are moving [43]. Being able to distinguish between the effects of self-motion and those of moving objects requires the detection and analysis of relative motion [47]. Second, our attention is constantly redirected to interesting and potentially dangerous objects, which typically cause a reorientation of gaze. These functions were examined previously in connection with the distinction between sustained and transient attention [3, 38, 44].\nBecause of the impairment of the central visual field in ARM, motion perception with peripheral vision is of even greater importance for these patients. Measurements with dynamic test stimuli are especially important for the assessment of driving ability of visually impaired persons [24]. Marron and Bailey examined contrast sensitivity and visual acuity with regard to their importance for orientation and mobility [39]. Their results provide evidence that contrast sensitivity and the integrity of the visual field are of considerably greater importance for safe mobility than visual acuity. Studies from our own group show that dynamic peripheral sensitivity in a divided attention condition was the strongest predictor for safe driving in a group of healthy elderly drivers [53]. Recent studies have confirmed the importance of peripheral vision for driving by finding that contrast sensitivity and visual field limitations are the strongest predictors for mobility of visually impaired persons [10, 21, 35]. These studies demonstrate the importance of the assessment of visually guided performance regarding moving objects and contrast vision. In the tests at hand, these two tasks are linked. The growing possibilities for mobility of the elder generation with age-related visual impairment require examinations that take into account seniors\u2019 new lifestyles and can help ensure safe navigation.\nAnother relevant connection can be made with communication and the ability to read speech from the lips of a conversation partner, which is a difficulty faced by many elderly with additional hearing impairment. Although a general notion of the role of vision in speechreading has been established [56], the importance of motion sensitivity for speechreading has not been sufficiently investigated to date.","keyphrases":["visual field","arm","temporal resolution","motion perception","contrast sensitivity"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_Spine_J-2-2-1602206","title":"Dorsal root ganglionectomy for pseudotumor of the L3 dorsal root ganglion: a rare case and a rare treatment\n","text":"Dorsal root ganglia are oval enlargements on the dorsal nerve roots and contain the cell bodies of sensory neurons. Asymmetry of dorsal root ganglia may occur naturally, yet natural occurrence of gigantic dorsal root ganglion (DRG) is rare. The patient was 61-year-old woman who presented with atypical symptoms like neuropathic pain and urinary distention. Neuroimaging has shown left L3-4 far-lateral disc herniation and a gigantic L3 DRG. At surgery, the dural sheath of the ganglion had to be opened and a firm, yellow-colored abnormal tissue was exposed. The abnormal tissue considered to be a tumor of neural origin was gross totally excised and the patient\u2019s symptoms ceased immediately after surgery. Histopathological examination of the specimen revealed nothing more than normal DRG morphology. At 4 months postoperatively, the patient is well with mild L3 hyperesthesia and hyperalgesia. Dural sheath opening in neurosurgery is not a routine practice. The sheath may need to be opened when surgeon suspects of a tumor, a free disc fragment and any inflammation within the ganglion. Operative morphology of a severely edematous but non-tumoral (pseudotumor) ganglion has not previously been documented.\nIntroduction\nSmall neoplastic lesions of the vertebral column and the spinal canal are often misdiagnosed as intervertebral disc disease owing to the commoner occurrence of the latter [1]. Rarely, a neoplastic lesion may coincide with a herniated intervertebral disc usually without a topographic relationship in between [8]. Concomittant appearance of a disk herniation and a neoplastic lesion compromising the same nerve root represent a unique situation [1]. We herein report the unique case of a pseudotumor of the L3 dorsal root ganglion (DRG) adjacent to a far-lateral L3-4 disc herniation.\nCase history\nA 61-year-old physician presented with excruciating pain in her left leg. The mild leg pain that has been around the left knee and around the posterior hip for some weeks suddenly became unbearable one afternoon. The patient came to emergency room 8\u00a0h after the onset of the pain. Upon further questioning, the patient confessed that she had not emptied her bladder since the pain started. Past medical history was positive for a distant episode of trigeminal neuralgia, cluster headache, and an episode of significant back and right leg pain 7\u00a0years ago and a recent pulmonary embolus. Past surgical history included total hysterectomy and bilateral salpingo-oopherectomy after a serious pelvic inflammatory disease and abscess formation within the uterine tube.\nIn the emergency room, the patient was in extreme pain. Any movement or touch would exacerbate the pain. Right leg-raising test was positive at 60 and left leg-raising test was positive at 20\u00b0. Power was normal in both legs, including knee extension. Patellar and the ankle reflexes were normoactive bilaterally. Abdominal examination for a distended bladder was fruitless because of softened subcutaneous fat. Lumbar MR showed disc degeneration and mild bulging at L3-4, L4-5 and L5-S1 levels yet at L3-4 level there was foraminal and a round extraforaminal disc herniation (Fig.\u00a01a, b). Lumbar MR scan also verified a fully distended bladder (Fig.\u00a01c). Considering vaque radiological findings in contrast to serious pain and urinary retention, the MR examination was extended to cover the whole cervico-thoracic and thoracic levels. There was no cord compression.\nFig.\u00a01Axial T1-weighted (a) and T2-weighted (b) MR scans through L3-4 interspace show an extraforaminal lesion with questionable continuity with the disc space. The lesion is isointense on both T1- and T2-images. Please note that on T2-weighted axial image (b), the contralateral dorsal root ganglion (DRG) shows intermediate to high signal. c Parasagittal MR scan show L3-4 disk herniation obliterating the left neural foramen anteroinferiorly.Yet the left L3 nerve is of same width as the roots one above or below. d Parasagittal MR scan 5\u00a0mm lateral to section c shows that the enlarged DRG (arrows) appears to have been stuck in the distal neural foramen. Please note that the fat plane around the DRG is totally obliterated\nThe next morning, urinary retention re-occurred and an indwelling urinary catheter had to be placed. An ultrasound of the abdomen and pelvis was not contributory except identification of few benign adhesions secondary to previous pelvic surgery and a distended gall bladder. Meanwhile the left knee pain and the left sciatica got only a bit better but the patient described additional occasional electric shock like pain radiating medially from the left groin to the left knee. Lumbar computerized tomography (CT) to rule out a small bone chip originating from end-plate attached to a free disc fragment was done next. Axial CT scans showed the presence of a left-sided round hyperdense extraforaminal lesion at L3-4 level that was reported to be consistent with far-lateral disc herniation (Fig.\u00a02a). Coronal reformatted CT images better demonstrated the lateral extent of the disc herniation (Fig.\u00a02b). After an unsatisfactory 2-day narcotic treatment with codein 60\u00a0mg q6h, gabapentin 300\u00a0mg twice a day was started. On the third day of admission, a new pain emerged around the left inguinal area. By this time, the left patellar reflex diminished and left knee extension got 20% weaker. IV steroid (80\u00a0mg methyl prednisolon as the loading and 40\u00a0mg twice a day as the maintenance dose) was then started. Steroid alleviated a significant portion of the pain. Tapering steroid dose immediately caused return of intense pain, so surgery was considered as the last resort.\nFig.\u00a02Axial CT image through the lower end-plate of L3 (a) and coronal reformatted CT (b) image show a round hyperdense extraforaminal lesion consistent with far-lateral disc herniation\nThe patient was informed to consent for the removal of far-lateral disc herniation at L3-4 level. After medial facetectomy and ligamentous removal, the left L4 nerve and the L3-4 disc space were exposed. The bulging disc was compressing the L4 root from its shoulder. After standard discectomy, the L4 root became mobile. Next, lateral disc space was emptied by angling the rongeur laterally. Yet, disc compression alone was far from explaining the dramatic clinical picture. After total removal of left L3 inferior facet and L4 superior facet and undercutting the L3 pedicle, the left L3 root was identified and traced laterally. L3 nerve was more or less normal sized yet L3 DRG was extremely different than normal. The DRG was 3\u20134 times bigger than a normal DRG. The L3 root was totally immobile (Fig.\u00a03a). The DRG felt extremely gritty. The dural sheath of nerve root and the DRG was opened. A yellow-colored firm tissue came into view (Fig.\u00a03b, c). There was good cleavage from the dural sheath and the underlying septum. The healthy ventral rootlets were pushed anteriorly and inferiorly by the mass (Fig.\u00a03d). Overall, the mass lesion within the root sleeve was totally extirpated. Although no cerebrospinal fluid (CSF) was seen in the operative field, the dural sheath was sutured with interrupted prolene sutures and the suture was reinforced with tissue fibrin glue. On postoperative day 1, the urinary catheter was removed and the patient emptied her bladder spontaneously. The original pain was totally gone. The left knee function returned to normal. On third postoperative day, she complained of mild hyperesthesia and hyperalgesia over the left knee which did not affect her daily life. At 4\u00a0months postoperatively, she is leading a normal life with gabapentin 300\u00a0mg twice a day.\nFig.\u00a03Operative microphotographs show that: a the left L3 root and the DRG are extremely swollen, b after opening the dural nerve sheath, the upper end of a yellow colored tumor is being dissected, c the lower end of the tumor is being dissected with better cleavage, d after the removal of the tumor normal appearing ventral motor rootlets come into view, e root size return to normal after suture closure of the dural sheath (black arrows)\nThe histopathological specimen was extensively studied and did not reveal anything other than normal DRG tissue with mature ganglion cells, axons and Schwannian stroma supporting the axons (Fig.\u00a04).\nFig.\u00a04Photomicrographs a with hematoxylin and eosin and b with neurofilament (NF) protein immuno-stain demonstrate axons (left) and ganglion cells dispersed in a cellular stroma consisting of bundles of elongated spindle cells (right) (\u00d7200 original magnification). This represents normal DRG morphology\nDiscussion\nDorsal root ganglia are oval enlargements on the dorsal nerve roots and contain the cell bodies of sensory neurons. Although both the DRG and the ventral motor root are surrounded by a common dural sheath, a thin fibrous septum separates both. The DRG are surrounded by fat within the intervertebral foramen. According to the baseline anatomic information that Hasegawa et al. [5] obtained from male volenteers using MRI, the mean width, length and height is 5.7\u00d77.1\u00d77.3\u00a0mm3 for L3 DRG, 6.2\u00d78.4\u00d78.2\u00a0mm3 for L4 DRG, 5.9\u00d79.4\u00d78.3\u00a0mm3 for L5 DRG and 6.2\u00d711.2\u00a0mm2 for S1 DRG, respectively (data on the height of S1 DRG was not given in this study). The lower was the spinal level, the bigger was the ganglion size. In that study, the DRGs were located in lumbar intervertebral foramen in 92% of L1, 98% of L2, 100% of L3 and L4 and 95% of L5 levels. The S1 DRG was intraspinal in 79%. However analyzing patients presenting with low-back pain or sciatica, Hamanishi and Tanaka [4] found that 100% of L2 DRG, 48% of L3 DRG, 27% of L4 DRG and 12% of L5 DRG were located extraforaminally while 52% of L3 DRG, 72% of L4 DRG and 75% of L5 DRG were located intraforaminally.\nPreoperative radiological diagnosis of spinal nerve tumors producing radiculopathy or masquerading as spinal and\/or root pain has been facilitated by combined use of CT and MR. Intradural extramedullary tumors like schwannoma or extradural tumors like neurofibroma frequently affect the DRG and appear as dumbbell-shaped in 15% of cases [10, 14]. Diagnosis of tumors that had already enlarged the neural foramen is usually straightforward. However, a smaller pathology intrinsic to the root or the DRG will first be expected to cause an asymmetry as also depicted by MR scans in our patient. Yet asymmetry of DRGs may also occur naturally [4]. Hamanishi and Tanaka [4] found asymmetrical locations and sizes in 11% of the reviewed DRGs. Asymmetry at L3-4 level is rarer. Out of 104 patients that they reviewed, only one had DRG asymmetry at L3-4 level. Two cases who had asymmetrically gigantic DRGs similar to our case underwent surgery at different spinal levels [4]. In the first case, the lowered pedicle appeared to push down and kink the intraspinal portion of the L5 DRG. In the second case, the mass mimicking the gigantic DRG was histologically confirmed to be very old sequesterated nucleus pulposus at L5-S1 level. However, anteriorly compressed DRG could not be detected by MR scan at all [4].\nAs for MR signal intensity, schwannomas and neurofibromas will often appear iso- to hypointense on T1- and hyperintense on T2-weighted MR images [14]. The left L3 DRG in our case appeared isointense on both T1- and T2-weighted MR images. Intermediate signal on T2-weighted MR image usually suggests the presence of a tumor with numerous cellular and fibrous components and very little myxoid stroma. Unfortunately, paramagnetic contrast was not given in our case. One can argue that the preoperative radiological diagnosis of a tumor could have been possible if intravenous gadolinium was given. Although gadolinium enhanced MR imaging allows for better detection of intraneural edema with diffuse enhancement along the involved nerve roots, this characteristic probably would not have helped us in diagnosing a DRG tumor since DRG always enhance with contrast medium because of a less developed blood\u2013nerve barrier anyway. Recently, a French group studying enhancement pattern on 180 healthy DRG also concluded that 100% demonstrated significant and homogenous enhancement [3]. Therefore, even if we had used intravenous paramagnetic contrast preoperatively, it would probably be impossible to differentiate between an intraradicular tumor and a healthy DRG.\nIt was only at surgery that the left L3 DRG was found to be extremely swollen and had to be opened. A nerve root and a DRG may become swollen because of both biochemical irritation of nucleus pulposus and the mechanical compression of the disc materal [2, 6]. The mechanical compression of the DRG can increase the endoneural fluid pressure within the DRG almost threefolds [11]. Yet since the dural sheath is not an elastic tissue, a DRG can hardly ever reach the gigantic size that we have observed. Morever, the far-lateral disc protrusion at L3-4 level observed at surgery (in contrast to the extent of the herniation as seen on CT and MR images) could not have exerted the force necessary to cause a significant endoneural fluid pressure rise within the left L3 DRG. Therefore, the adjacent far-lateral disc herniation probably just coexisted with the gigantic left L3 DRG of unknown origin and served a better purpose by attracting our surgical attention to this part of the spine. Not only the size of the involved DRG, but also its firm feeling suggested the presence of something unusual inside it. Although intraradicular disc herniations occur more commonly at L5-S1 and L4-5 levels [12], the surgeon\u2019s impression before the microsurgical opening of the dural sheath was more inclined to a rare intradicular disc herniation rather than a tumor.\nDural opening in similar cases totally depend on surgeon\u2019s experience and\/or attitude or on the medicolegal restrictions of the preoperative consenting procedure. By opening the dural sheath of a nerve root or a DRG the surgeon not only will prolong the operative procedure but also will take the morbidity risk associated with CSF leak and difficulty with wound healing. Moreover, extra expense of tissue fibrin glue and\/or external lumbar drainage may also trigger an hesitation before dural opening in the currently managed health care systems. If a gross pathology like a tumor or a disc fragment is not readily encountered between the rootlets, chances are histopathology will be the only diagnostic tool. In addition to a real mass lesion like tumor or disc material, inflammatory reactions within a DRG\u2014although very rare\u2014may also cause a tumor-like lesion. A team from T\u00fcbingen, Germany recently has reported a very similar case presenting with low back pain and progressive hypesthesia of the thigh [10]. MR imaging with contrast enhancement revealed an intraspinal\u2013extradural mass at L1-2 level compressing the right L2 root. The surgical part of their case is almost identical to ours. The surgical team emptied the disc and the nerve was still compressed and appeared to be enlarged. In the end, they had to open the dural sheath and resect the indurated and swollen part of the ganglion. Histopathological diagnosis was ganglionitis characterized by loss of ganglion cells, increased number of nodules of Nagcotte and diffuse infiltration of T cells.\nGanglionectomy as performed for cancer pain or non-cancer pain (for failed back surgery syndrome or chronic lumbar radiculopathy) was originally described by Osgood et al. [9] and by definition is an extradural operation [13]. Wilkinson and Chan [15] in their recent publication advocated the use of microscope for the procedure. After exposure of the ganglion and surrounding plexus and compression of the venous plexus with cottonoids, the longitudinal incision path along the dural sheath is coagulated using low-current bipolar. Once the dural sheath is incised dorsally using a no. 11 knife, ganglion tissue will come into view. Young [16] described the DRG as a round, yellow, irregular structure. Wilkinson and Chan [15] described the ganglion as having an appearance similar to fat, with tiny \u201csparkles\u201d within. The latter description is for ganglia from patients with failed back surgery syndrome or chronic lumbar radiculopathy. In rare circumstances like we had, differentiation between an healthy and a tumoral ganglionic tissue may be extremely difficult even with the operating microscope. At this stage, the surgeon has to decide between a biopsy or total removal of the abnormal looking ganglionic tissue. Biopsy may be safer but then there is the possibility of sampling error. If total removal of the DRG is aimed (sensory ganglionectomy), then the afferents and efferents of the ganglion are elevated first and then the ganglion is separated from the underlying septum. To lessen the likelihood of neuroma formation, the proximal end of the ganglion is either hemoclipped or cut with CO2 laser [7]. If leakage of CSF is not encountered (which usually is the case) a watertight closure of the perineurium is not necessary. Some even advocated to leave an autogenous fat graft in the surgically created defect [15]. As for complication of the procedure, deafferentiation pain has been exaggerated because it is usually transient and never reaches to the point of anesthesia dolorosa [13, 15]. Taha et al. [13] states that there is no uniformly beneficial treatment for deaffarentiation pain after ganglionectomy but our patient definitely benefited from brief use of gabapentin.\nIn conclusion, we reported the unique case of a pseudotumor of L3 DRG. The patient presented with atypical symptoms like neuropathic pain and urinary distention. Preoperative MR scans showed a gigantic left L3 DRG yet this asymmetry was masked by the presence of an adjacent ipsilateral far-lateral disc herniation. Attention to foraminal and extraforaminal part of the spine is of utmost importance in the evaluation of lumbar MR scans. Surgery was both diagnostic and therapeutic in this case. Before opening the dural sheath of a nerve root or DRG, a surgeon should realize that if a tumor or a disc fragment does not come out at once, then it may only be through histopathology that a proper diagnosis can be reached.","keyphrases":["dorsal root ganglion","ganglionectomy","magnetic resonance imaging","microsurgery"],"prmu":["P","P","M","U"]}
{"id":"Ann_Surg_Oncol-3-1-1914247","title":"Long-Term Locoregional Vascular Morbidity After Isolated Limb Perfusion and External-Beam Radiotherapy for Soft Tissue Sarcoma of the Extremity\n","text":"Background Isolated limb perfusion (ILP) with tumor necrosis factor alpha (TNF-\u03b1) and melphalan, followed by delayed surgical resection and adjuvant external-beam radiotherapy is a limb salvage treatment strategy for locally advanced soft tissue sarcomas. The long-term vascular side effects of this combined procedure were evaluated.\nOptimum treatment strategies for locally advanced soft tissue sarcoma (STS) of the extremities have evolved considerably over the past 25 to 30 years. In 1982, Rosenberg et al.1 showed that amputations or exarticulations of the affected limb do not result in higher survival rates. This made preservation of the extremity and good limb function all the more important.2,3 An established limb salvage strategy is to perform a hyperthermic isolated limb perfusion (ILP) with tumor necrosis factor alpha (TNF-\u03b1) and melphalan, followed by delayed tumor resection and, in case of marginal or nonradical resection, by adjuvant external-beam radiotherapy (EBRT).4,5 Although ILP, in which high doses of chemotherapy are regionally delivered, is a technically demanding operative procedure, amputation necessitated by peri- or postoperative surgical complications occurs in <1% of cases.4 On the other hand, patients undergoing an intentionally limb-saving treatment with ILP still have a short- or long-term risk up to 40% of loss of the affected limb due to massive necrosis of the tumor and overlying skin resulting in a soft tissue deficit, late local recurrence, or critical leg ischemia.5\nIn ILP, cytotoxic agents are used, which cause a local toxic effect in the perfused extremity. After investigation of several perfusion agents for STS, the two-drug regimen of TNF-\u03b1 and melphalan turned out to be highly effective.4 The pathological tumor microvascularization is the primary target for TNF-\u03b1, resulting in coagulative and hemorrhagic necrosis of the tumors.6 Olieman et al.7 demonstrated by angiography findings of specific destruction of tumor vessels after perfusion with TNF-\u03b1, leaving the normal vasculature unchanged. The surgical procedure of ILP, however, exposes the cannulated blood vessels to intravascular manipulation, by means of arteriotomy, venotomy, and the insertion of catheters. Consequently, a 1% to 10% risk of developing acute vascular complications, such as thrombosis at the arteriotomy site and deep-vein thrombosis (DVT), has been described in the literature.8\u201310 Moreover, chemotherapy and atherosclerosis risk factors could have a synergistic role on the development of arterial stenosis.11 Adjuvant EBRT leads to a high incidence of early and late morbidity. Long-term vascular side effects, in particular arterial stenosis, can be induced by radiotherapy.12\u201314\nBecause little is known with respect to the late vascular morbidity of the combined procedure of ILP and EBRT, a retrospective study was conducted to evaluate the long-term locoregional vascular side effects of ILP, followed by delayed surgical resection and adjuvant high-dose EBRT.\nPATIENTS AND METHODS\nFrom 1991 to 2003, a total of 73 patients with a median age of 54 (range, 14\u201380) years and with locally advanced STS underwent 77 perfusions with a combination of TNF-\u03b1 and melphalan, with (n\u00a0=\u00a019) or without (n\u00a0=\u00a058) interferon gamma at the Division of Surgical Oncology of the University Medical Center Groningen, University of Groningen. The perfusion technique has been previously extensively described.15,16 Perfusion was followed by delayed local resection of the tumor remnant, and in cases of marginal or microscopically positive resection margins, high-dose postoperative EBRT was considered.\nIn 2005, 39 patients were still alive and in follow-up. Information about their past and current oncological and vascular status was obtained from the follow-up notes in the medical records. Seven patients were unable to participate in the study because of severe or advanced morbidity or comorbidity (n\u00a0=\u00a02) or because of nonmedical reasons (n\u00a0=\u00a05). Consequently, 32 patients, 14 men (44%) and 18 women (56%), with a median age of 47 (range, 14\u201371) years, were included (response rate, 82%). They completed a vascular checklist that asked questions about their medical vascular history, and they were screened for risk factors of vascular disease, such as smoking, hypertension, and diabetes. A blood sample was drawn to determine the cholesterol spectrum, which was used as an indicator of risk for atherosclerosis. All patients underwent a noninvasive arterial and venous vascular work-up that comprised duplex ultrasonography and vascular pressure measurements. The ankle-brachial index (ABI) and pulsatility index (PI), which are indications of vascular blood flow velocity, were measured to indicate the arterial status of the limb. For evaluation of veins, ultrasonography was performed to detect DVT and insufficiency. All investigations were performed by the same examiner (J.L.).\nFive patients had a STS of the upper extremity (16%) treated with axillary perfusion. The remaining 27 patients had a STS located in the lower limb (84%) and were treated with iliac (n\u00a0=\u00a013, 41%), femoral (n\u00a0=\u00a05, 16%), or popliteal (n\u00a0=\u00a09, 28%) perfusion. There were 27 primary STS and 5 recurrent STS. Twenty-four patients (75%) received adjuvant 60 to 70 Gy EBRT in fractions of 2 Gy. Adjuvant systemic chemotherapy was provided to seven patients. Definite histopathological classification of tumor (one embryonal rhabdomyosarcoma and one extraossal osteosarcoma) was the reason in two patients; three patients were treated with chemotherapy because they participated in an European Organization for the Research and Treatment of Cancer trial (EORTC 62931). Two patients received chemotherapy in a palliative setting for distant metastasis. All patients were treated after informed consent was obtained according to institutional guidelines. Twelve different histological types of STS were distinguished. The pathological grade of the tumor was scored following the criteria of Coindre et al.,17 and the stage of the tumor was assigned according to the American Joint Committee on Cancer.18 Patient and tumor characteristics are summarized in Table\u00a01.\nTABLE\u00a01.Characteristics of patientsAge (y)SexDiseaseLocalizationHistologyGradeAJCC stageLevelEBRTAmputationFollow-up (mo)18FPrimaryUpper legEmbryonal rhabdomyosarcoma33IliacNN15944FRecurrentPopliteal fossaMyxoid liposarcoma11IliacYN15243MPrimaryUpper legSynovial sarcoma33IliacYN15118MPrimaryLower legMyxoid chondrosarcoma23PoplitealYY14948FPrimaryUpper legWell differentiated liposarcoma11IliacYN14356FPrimaryProximal tibiaPNET33IliacYY13750FRecurrentFootPUS33PoplitealNN13525FPrimaryPopliteal fossaSynovial sarcoma23PoplitealYN13244MPrimaryUpper legMyxoid liposarcoma11IliacYN13124MRecurrentLower legSynovial sarcoma23PoplitealYN12937FPrimaryElbowMPNST23AxillaryYN12148MPrimaryPopliteal fossaMyxoid liposarcoma23IliacYN11163FRecurrentLower legPUS23PoplitealNY10737MPrimaryUpper legMyxoid liposarcoma11IliacYN10558MPrimaryUpper legPUS23IliacYN9871FPrimaryUpper legLeiomyosarcoma11FemoralNN9445FPrimaryUpper legPUS33IliacNN8256FPrimaryLower legPUS33PoplitealYN6163MPrimaryPopliteal fossaMyxoid chondrosarcoma3FemoralYN5037FPrimaryKneeLeiomyosarcoma11FemoralYN3628MPrimaryPopliteal fossaSynovial sarcoma23FemoralYN3657FPrimaryUpper legPUS33FemoralYN3542FPrimaryKneeSynovial sarcoma23IliacYY3247FPrimaryArmPUS33AxillaryYN3058FPrimaryLower legPUS33PoplitealYN2627MPrimaryArmEpithelioid sarcoma34AxillaryYN2671MRecurrentArmMyxoid fibrosarcoma11AxillaryNN2456FPrimaryArmExtraosseal osteosarcoma33AxillaryNN2214MPrimaryLower legPUS33PoplitealYN2065FPrimaryFootSynovial sarcoma23PoplitealNY1863MPrimaryUpper legSynovial sarcoma33IliacYN1771MPrimaryUpper legRhabdomyosarcoma33IliacYN17AJCC, American Joint Committee on Cancer; EBRT, external-beam radiotherapy; PNET, primitive neuroectodermal tumor; PUS, pleomorphic undifferentiated sarcoma; MPNST, malignant peripheral nerve sheath tumor.\nThe results were statistically analyzed by the Wilcoxon rank test and the Mann-Whitney U-test. Values of P\u00a0\u2264\u00a0.05 were considered significant. SPSS 12.0.1 for Windows statistical software was used (SPSS, Chicago, IL).\nRESULTS\nBefore ILP, none of the patients had a history of cerebrovascular insufficiency, coronary artery disease, peripheral arterial occlusive disease, or any other vascular disease. Risk factors for cardiovascular disease, hypertension, diabetes, smoking, obesity, hypercholesterolemia, and familiar arterial occlusive disease were analyzed (Table\u00a02).\nTABLE\u00a02.Risk factors for atherosclerosisRisk factorn%Dutch population (%)Hypertension10319.9a,bDiabetes mellitus4133.1bSmoking29.6b\u00a0\u00a0Smokers928\u00a0\u00a0Former smokers1031BMI\u00a0\u00a0BMI >25.0175346.5b\u00a0\u00a0BMI >30.041310.9bHypercholesterolemia (total cholesterol >6.5)61914cFamiliar cardiovascular disease1444NABMI, body mass index; NA, not available.a Range, 5% to 45%.b From http:\/\/www.cbs.nl\/.c From http:\/\/www.hartstichting.nl\/.\nTwo patients underwent vascular reconstruction after en-bloc tumor resection with the artery. One patient underwent a reconstruction of the femoral artery with an infragenual femoral-popliteal bypass, and the second patient underwent a popliteal artery reconstruction with an autologous vein graft. A DVT in the lower leg was diagnosed in the postoperative period in three patients (7.7%).\nNine (23%) of the 39 alive patients underwent a lower limb amputation at different levels after ILP with or without EBRT. No amputations of the upper extremity were performed. In two patients with microscopically involved resection margins, an amputation of the affected limb was indicated because adjuvant radiotherapy was contraindicated. One patient underwent amputation due to tumor recurrence 18 months after ILP and EBRT. Amputation was performed for critical leg ischemia due to atherosclerosis in two patients at 110 and 125 months after ILP and EBRT. Because of severe obstruction of the crural vessels, reconstructive therapy was not suitable.\nThe other four amputations were performed for extensive postperfusion necrosis of the involved tumor site (three patients at 0, 8, and 12 months after ILP with or without EBRT) and arterial occlusion without any vascular reconstructive possibilities (one patient 15 months after ILP and EBRT).5[A1] Figure\u00a01 illustrates treatment, outcome, and reasons for amputation. Five of the 32 patients included in this study underwent amputation and could therefore only partially undergo vascular follow-up.\nFIG.\u00a01.Flow chart illustrating treatment, outcome, and reasons for amputation. EBRT, external-beam radiotherapy; AMP, amputation; NI, not included in this study.\nArterial Vascular Disease\nWith a median follow-up of 88 (range, 17\u2013159) months, no patient experienced peripheral arterial occlusive disease. With duplex ultrasonography, however, two patients showed a complete arterial occlusion at the level of cannulation (6%). One of them underwent an iliac perfusion and EBRT because of a STS of the lower leg in 1993. After 10 years, she had signs of critical leg ischemia, which required a transfemoral amputation. She is now free of pain and can walk with a well-functioning prothesis. The other patient showed an axillary occlusion 10 years after an axillary perfusion, followed by resection and EBRT at the forearm. She did not have any symptoms of arterial occlusive disease, and her arm functions normally. Duplex ultrasonography showed good collateral flow. Two other patients (6%) showed an arterial stenosis at cannulation level at 22 and 30 months after axillary perfusions, followed by EBRT at the upper arm and axilla in one and systemic chemotherapy in both patients. They had normal arm function without any signs of ischemia.\nThe arteries at resection level were also evaluated. One patient with a fossa poplitea STS had complete occlusion of the superficial femoral artery 6 years after ILP and EBRT, with open arteries below knee level. A femoral-popliteal bypass was considered but was not performed because of the absence of any symptoms. At the time of evaluation, the ABI was .50; the patient did not have any complaints besides compression ulcers on his feet, which responded well to conservative therapy. The patient who needed an autologous vein graft of the popliteal artery has a stenosis at this level without clinical signs of arterial occlusive disease.\nTwenty-two of the total 32 patients included underwent ABI measurements; 22 had their PI determined at the femoral level and 20 at the popliteal level. Five patients were excluded because of amputation of the limb; another five were excluded because of a STS located in the upper limb. Of the remaining 22 patients, 18 received postoperative EBRT for the above-described indications.\nIn 21 of 22 patients, the ABI (normal,\u00a0\u22651.00) in the treated leg was .91 or more. As mentioned above, one patient had an ABI of .50 without any complaints. ABI measurements in the involved leg (median, 1.02; range, .50\u20131.20) showed a significant decrease compared with the contralateral leg (median, 1.09; range, .91\u20131.36; P\u00a0=\u00a0.001). Seven patients showed a decreased femoral PI (normal range, 5\u201310). Six of them underwent an iliac perfusion (86%). Popliteal PI (normal range, 6\u201312) was decreased in six patients. Perfusion at a more proximal level was performed in four of these patients (67%). When compared with the contralateral leg, PI was lower in the treated leg in 17 of 22 patients at femoral level (median, 6.30; range, 2.1\u201323.9 vs. median, 7.35; range, 4.8\u201321.9; P\u00a0=\u00a0.011) and in 19 of 20 patients at popliteal level (median, 8.35; range, 0\u201321.4, vs. median, 1.95; range, 8.0\u201332.6; P\u00a0<\u00a0.0005) (Table\u00a03). There were no differences in objective vascular side effects between men and women, between patients who did or did not undergo EBRT, or between patients <50 years old versus older patients (Table\u00a04). In patients with a follow-up period of >5 years, there was more often a decrease in ABI, compared with patients with shorter follow-up (13 of 14 patients vs. 4 of 8 patients, P\u00a0=\u00a0.024). With a longer follow-up period, there was also more often a decrease in PI at the femoral level (13 of 14 patients vs. 3 of 7 patients, P\u00a0=\u00a0.024), but not at the popliteal level (12 of 12 patients vs. 7 of 8 patients, P\u00a0=\u00a0.221) (Table\u00a05). No patient required vascular intervention during follow-up.\nTABLE\u00a03.Results of ankle-brachial index (ABI) and pulsatility index (PI) measurements in lower extremities of 22 patientsIndexTreated extremity, median (range)Contralateral extremity, median (range)P valueABI1.02 (.50\u20131.20)1.090 (.91\u20131.36).001PI femoral6.30 (2.1\u201323.9)7.35 (4.8\u201321.9).011PI popliteal8.35 (.0\u201321.4)10.95 (8.0\u201332.6)<.0005TABLE\u00a04. P values of differences between subgroups in decrease of ankle-brachial index (ABI) and pulsatility index (PI) for 22 patientsCharacteristicSexFollow-up after EBRTAgeFMYesNo>5 y<5 y<50 y>50 yNo. of patients1111184148139ABI.619.241.024.963\u2013\u2013\u2013\u2013PI femoral decrease.619.907.024.963\u2013\u2013\u2013\u2013PI popliteal decrease.269.937.221.414\u2013\u2013\u2013\u2013EBRT, external-beam radiotherapy.TABLE\u00a05.Differences in decreases in measurements according to length of follow-upDecrease in perfused legFollow-up, n (%)P value>5 y (n\u00a0=\u00a014)<5 y (n\u00a0=\u00a08)ABI13 (93%)4 (50%).024PI femoral13 (93%)4 (50%).024PI popliteal12 (100%)a7 (88%).221ABI, ankle-brachial index; PI, pulsatility index.a n\u00a0=\u00a012; data for two patients were not available.\nVenous Vascular Disease\nMany patients had some symptoms of venous insufficiency, such as lower limb pain, feelings of heaviness and discomfort, night palsies, paresthesia, and edema. One patient (3%) wore compression stockings because of superficial vena saphena magna insufficiency, and one patient (3%) had unilateral superficial varicosis in the treated leg. Another three patients (9%) showed uni- or bilateral deep insufficient venous vasculature by duplex ultrasonography, which in all cases was located at the level of resection. One DVT (3%) and one partial venous obstruction (3%) were found, both at the resection level. Venous vascular obstruction was not seen at the level of cannulation.\nDISCUSSION\nILP with TNF-\u03b1 and melphalan, with or without adjuvant EBRT, has become an approved treatment strategy for locally advanced STS of the extremities after the publication of the results of an European multicenter trial performed in the 1990s.4 Because our center was one of the first to use this treatment modality, we now have had more than 10 years\u2019 experience of ILP used to treat STS. This gives us the opportunity to evaluate the long-term treatment-related morbidity. Critical leg ischemia approximately 10 years after ILP, necessitating amputation in two patients, led to an interest of analyzing the vascular complications of ILP with or without EBRT.5\nDespite a complete destruction of tumor vasculature, ILP does not seem to have any effect on the macrovasculature of the perfused limb.7,19,20 However, because atherosclerosis risk factors and chemotherapy could play a synergistic role in the progression of arterial stenosis, patients who already have severe atherosclerotic disease are less suitable for ILP.11,19,20\nIn our study, many patients had one or more risk factors for atherosclerosis. In the study population, there seemed to be more patients with diabetes or hypercholesterolemia than in the standard Dutch population. The smoking rate is similar (28% vs. 30%); however, another 31% used to smoke (Table\u00a02).\nAcute vascular complications after ILP have been described in the literature. Deep-vein thrombosis (DVT) occurs in 1.7% to 10% of cases in the immediate postoperative period.10 In our studies, three patients (7.7%) had a successfully treated DVT after ILP. DVT seems to be extremely difficult to diagnose because the postoperative effects of ILP itself\u2014a warm, swollen edematous limb\u2014mask the classical symptoms. To prevent this undesired complication, anticoagulant therapy is recommended. Thrombosis at the arteriotomy site has also been mentioned in earlier studies. It was not encountered in this study but should be treated by prompt trombectomy.9,21 In case of repeated ILP in the same limb, perfusion through more proximal arterial and venous sites is recommended because fibrosis at the previous operative site does not allow insertion of the cannulas at the same level. All patients in this study underwent only one ILP.\nLong-term effects after ILP are mainly functional and consist of edema, stiffness, functional impairment, and muscle atrophy.21\u201323 Late vascular morbidity after ILP without EBRT has not been previously mentioned in other studies. In our study, one partial arterial occlusion (3%) was found. In contrast, long-term effects of EBRT have extensively been described in the literature. In a study of Butler et al.,12 three types of vascular damage have been described. At first, thrombosis occurs within 5 years after EBRT; a second fibrotic occlusion is seen within 10 years; and finally, a predisposition to the development of atheroma together with periarterial fibrosis is associated with a latent interval of 20 or more years. Rijbroek et al.24 mention claudication after a latent period. This is masked by the other effects such as pain, functional impairment, and edema. According to Kalman et al.,13 long-term vascular side effects of EBRT resemble the natural process of atherosclerosis, which is a combination of direct damage, periarterial fibrosis, and occlusion of vasa vasorum. The EBRT accelerates this process, sometimes 10 to 27 years after treatment. Little is known about the combined effects of ILP and EBRT. Vrouenraets et al.22 described one brachial arterial occlusion, but this was only 4 months after treatment. Olieman et al.16 described the occurrence of necrosis after ILP but did not find any increased tumor-related morbidity after ILP followed by EBRT. In another series, ILP was performed after EBRT, but it did not lead to more local toxicity or complications.25\nIn our study, amputation was performed in nine patients (23%). This is higher than the 9% to 21% described in the literature.4,26,27 However, amputations due to long-term complications like critical leg ischemia, which was performed in two patients (5.1%), have not been described in other studies. Furthermore, three complete (9%) and one partial (3%) arterial occlusion were found after the combined procedure.\nFortunately, these patients did not have invalidating symptoms of arterial occlusive disease. In most cases, vascular pressure measurements and duplex ultrasonographic evaluation showed statistically lower values compared with the contralateral extremity, but these were still within the normal range. This could explain the absence of symptoms.\nThe PI value normally increases progressively from aorta to ankle. A decrease in PI indicates stenosis in a more proximal vascular segment.28 In our study, six of seven patients with an abnormal femoral PI underwent iliac perfusion. This implies a vascular reaction at the level of cannulation. Furthermore, seven patients showed a decrease in popliteal PI compared with the femoral PI, which implies that vascular damage is present at a more proximal (i.e., cannulation or resection) level. However, with the present data, it is not possible to support this thesis because of the relatively small study population, and the absence of symptoms and other abnormal outcomes of the vascular work-up.\nIt is important to realize that blood vessels lose their elasticity with the years and are subject to the normal process of atherosclerosis. Furthermore, unilateral peripheral arterial occlusive disease is common. However, the fact that lower rates of ABI and PI were found in the treated leg in almost all patients and that no marked difference was found between younger and older patients makes the assumption of treatment-induced differences plausible.\nBecause only four patients with a lower leg STS were not treated with adjuvant EBRT, there are still no clinically relevant data about the late vascular damage of the single procedure of ILP. Thijssens et al.29 showed that adjuvant EBRT seems beneficial in all patients, so it might be impossible to evaluate the sole effects of ILP on the vascular system in the future.\nMajor late vascular complications after the combined procedure, like critical leg ischemia necessitating amputation, were found in two patients in this study. One patient had no risk factors for peripheral arterial occlusive disease besides smoking. He had had intermittent claudication 8 years after ILP and EBRT, and a normal ABI of 1.09. His condition did not improve after 2 years of conservative therapy and reconstruction was impossible, so he underwent an amputation 125 months after the initial limb salvage treatment strategy. The second patient, who had diabetes mellitus and familial cardiovascular disease, experienced no complications the first few years after ILP and EBRT. After 6 years, she experienced a pathological proximal tibia fracture due to radiation-induced osteonecrosis in the previous surgically treated area and needed to wear a brace. She lived for 3 years with a disabling function impairment of the knee and then developed signs of critical leg ischemia. An upper leg amputation was performed 110 months after the initial treatment. She now walks with a well-functioning prothesis.\nThe importance of leg preservation has been mentioned before. Even though most patients still are satisfied with the initial limb-saving treatment that they underwent, amputations due to major late complications with severe morbidity make the rationale of limb salvage worth reconsidering.\nRecently, one patient was successfully treated with thrombectomy for an acute popliteal artery occlusion. This patient, who underwent an ILP at femoral level followed by EBRT for a STS in the fossa poplitea 5 years before, had no complaints of peripheral arterial occlusive disease during his vascular work-up for this study 10 months earlier. ABI (1.03) and femoral PI (6.3) were normal; popliteal PI was low (5.3). With duplex ultrasonography, some atherosclerosis in the femoropopliteal section was seen. This patient has diabetes and hypertension, is obese (body mass index >30), and was a former smoker, so he was especially at risk for developing atherosclerosis.\nThis study shows that despite the importance of preventing major late vascular complications after the combined procedure of ILP and EBRT, a routine noninvasive vascular work-up does not seem to add value to normal follow-up. It is more important to realize that patients after the limb salvage treatment strategy of ILP, followed by delayed tumor resection and EBRT, are at risk for late vascular complications. This risk is substantially raised when personal risk factors such as smoking, diabetes, hypertension, obesity, and hypercholesterolemia are present. Because symptoms of vascular complications can be masked by the normal effects of the treatment itself, the possibility of vascular morbidity should be considered if the patient has any complaint. Furthermore, all patients must be convinced of the importance of reducing their additional personal risk factors for atherosclerosis.\nIn conclusion, objective measurements show a time-related decrease (>5 years\u2019 follow-up) of ABI and femoral PI in the treated leg, usually without subjective complaints. ILP followed by delayed resection and EBRT for a locally advanced STS can lead to major late vascular morbidity that requires amputation.","keyphrases":["vascular","perfusion","sarcoma","complications","radiation"],"prmu":["P","P","P","P","U"]}
{"id":"J_Occup_Rehabil-3-1-1915628","title":"Effects of Ambulant Myofeedback Training and Ergonomic Counselling in Female Computer Workers with Work-Related Neck-Shoulder Complaints: A Randomized Controlled Trial\n","text":"Objective: To investigate the effects of ambulant myofeedback training including ergonomic counselling (Mfb) and ergonomic counselling alone (EC), on work-related neck-shoulder pain and disability. Methods: Seventy-nine female computer workers reporting neck-shoulder complaints were randomly assigned to Mfb or EC and received four weeks of intervention. Pain intensity in neck, shoulders, and upper back, and pain disability, were measured at baseline, immediately after intervention, and at three and six months follow-up. Results: Pain intensity and disability had significantly decreased immediately after four weeks Mfb or EC, and the effects remained at follow up. No differences were observed between the Mfb and EC group for outcome and subjects in both intervention groups showed comparable chances for improvement in pain intensity and disability. Conclusions: Pain intensity and disability significantly reduced after both interventions and this effect remained at follow-up. No differences were observed between the two intervention groups.\nIntroduction\nWork-related musculoskeletal complaints in the upper extremity are common among workers in Western industrialised countries. In the Netherlands, about 15% of the working population report complaints in the neck, shoulders and arms [1] defined as pain, numbness, or tingling, resulting in loss of productivity, sick-leave or even disability. In 1998, about 8% of all Dutch employees were absent from work due to work-related musculoskeletal complaints, and 2% of the employee population was absent from work for more than four weeks [2]. These complaints impose a substantial economic burden in compensation costs, lost wages, and productivity. Apart from the individual suffering, the financial costs within member states of the European Union associated with musculoskeletal complaints are high [3].\nWork-related musculoskeletal complaints are multifactorial in origin and involve biomechanical, psychosocial, and individual components [4\u20136]. As a consequence different intervention approaches exist. Most often interventions address alterations of the physical work environment by adjustments of the work station and\/or education about working posture according to ergonomic principles [7\u201310]. There are several studies reporting on the effectiveness of ergonomic approaches [11, 12] and it is commonly accepted that properly designed work stations are prerequisites for healthy working. However, in spite of attention to ergonomics musculoskeletal complaints remain a considerable problem [13\u201315] and this is the rationale for the search for innovative interventions.\nA relatively new intervention approach addressing neck-shoulder complaints is myofeedback training based on the Cinderella-hypothesis [16]. The Cinderella-hypothesis is one of the most influential hypotheses explaining the process of development and persistence of pain in low intensity jobs [17] like computer work, and states that lack of sufficient muscle relaxation is a crucial factor in this process. Continuous muscle activity, even at low intensity levels, may result in homeostatic disturbances of the activated motor units due to affected blood flow and removal of metabolites [17]. Several studies have found an association between absence of moments of complete muscle rest and myalgia, especially for the commonly affected descending part of the trapezius muscle (e.g. [18\u201320]). Warning subjects when their muscle relaxation is insufficient could thus contribute to recovery, and this is the rationale for the Cinderella-based myofeedback training. This approach is different from traditional myofeedback training in which feedback is provided when muscle activity exceeds a certain level thereby aiming at decreasing muscle activation (e.g. [21, 22]). As the Cinderella-hypothesis suggests however that muscle relaxation is more relevant than muscle activation, a myofeedback-based intervention aiming at increasing muscle relaxation may be more beneficial.\nThe Cinderella-based myofeedback system [23] consists of a harness incorporating dry surface electrodes and a feedback unit. The system is ambulant and subjects can wear the harness under their clothes during working days. The harness is connected to a feedback unit worn at the waist which provides feedback by means of vibration and a soft sound when muscle relaxation is insufficient. A feasibility study using this equipment for four weeks in a group reporting work-related neck-shoulder complaints resulted in significantly decreased levels of pain intensity and also changed muscle activation patterns. These effects were still at hand four weeks after the myofeedback training ended [23]. However, as the study did not contain a control group and only investigated the lasting effects of the intervention for a short follow-up period (4 weeks), further research is needed.\nAs proper ergonomics are indispensable for healthy working, the myofeedback training is preferably applied in combination with an intervention approach aimed at improving ergonomics. Thus, the present study aimed at investigating the immediate and lasting effects of Cinderella-based myofeedback training including ergonomic counselling, compared to ergonomic counselling alone, on pain intensity and disability in females with work-related neck-shoulder complaints. As interventions focusing on multiple factors have shown to be related to decreased incidence of complaints [24] it was hypothesised that 4-weeks myofeedback training including ergonomic counselling would be more effective in reducing pain intensity and disability than the intervention based on ergonomic counselling alone.\nMethods and materials\nDesign and subjects\nA randomized controlled trial was performed to assess the effect of 4-weeks Cinderella-based myofeedback training combined with ergonomic counselling (together referred to as Mfb), compared to ergonomic counselling alone (EC), on pain intensity and disability. Measurements were performed prior to intervention but before randomisation (Baseline), immediately after four weeks of intervention (T0), and at three (T3) and six (T6) months after the intervention.\nParticipants were recruited in Sweden (area of G\u00f6teborg) and the Netherlands (area of Enschede) between March 2003 and June 2005. Computer workers like job counselors (Sweden) and (medical) secretaries (Sweden and the Netherlands) were approached by telephone and announcements, and volunteers were subsequently sent a screening questionnaire [25] which was developed within the EU-funded NEW project (Neuromuscular assessment in the Elderly Worker) [26]. Subjects eligible for participation were symptomatic female computer workers, predominantly over the age of 45 as the prevalence of complaints is especially high in this age category [27], working for at least 20 hours a week, and reporting perpetuating work-related musculoskeletal complaints in the neck and\/or shoulder region for at least 30 days during the last year.\nSubjects were excluded when they reported pain in more than 3 body regions, when they suffered from severe arthrosis or joint disorders, when they were using muscle relaxants, or when reporting other complaints in the upper extremity not related to (computer) work.\nPower calculation, based on the results of Hermens and Hutten [23], indicated that at least 35 subjects should be included in each intervention group (estimated proportion of subjects showing an effect on pain intensity was set at 0.6 in the Mfb group and 0.2 in the EC group; 1\u2212\u03b2\u00a0=\u00a00.90; \u03b1\u00a0=\u00a0.05). Block randomization was used to assign subjects to either Mfb or EC: When a new group of subjects started the intervention, half of them were assigned to Mfb and half of them were assigned to EC. The study was approved by the local Medical Ethics Committees and all participants gave their informed consent prior to participation.\nInterventions\nThe interventions were provided by three different therapists: One physiotherapist in Sweden and two health scientists in the Netherlands. They were thoroughly trained and practiced together prior to the start of the study to ensure that they would provide as identical interventions as possible. The character of the intervention made blinding of the therapists and the subjects to the intervention impractical. To prevent from information bias, subjects were informed that the aim of the current study was to compare the effects of two interventions and that there was no evidence favouring one of these interventions.\nErgonomic counselling (EC)\nSubjects received four weeks of intervention during which they kept a diary of activities and pain intensity scores. During the four weeks period they were visited weekly by their therapist. The first visit comprised an ergonomic workplace investigation by means of the risk inventory of Huppes et\u00a0al. [28]. This checklist contains questions to evaluate work tasks, working hours, work load, work station, and working methods. Based on the outcome, possible improvements were discussed with the subject. With regard to the work station, the focus was primarily on modifying the existing work station rather than providing new equipment. The remaining visits were used to further discuss the ergonomic aspects, the consequences of possible ergonomic adjustments etc., according to a manual to guarantee a uniform intervention.\nMyofeedback (Mfb)\nSubjects randomized to the Mfb group received Cinderella-based myofeedback training on top of EC. A two-channel ambulant myofeedback system combined with a harness incorporating dry surface Electromyography (sEMG) electrodes to enable a stable recording of upper trapezius muscle activity was used. The harness was connected to a sEMG processing and storage device (see Picture 1).\nPicture 1\nMyofeedback system: Harness and processing\/storage unit\nThe sEMG signal was amplified (15\u00d7), band pass filtered between 30 and 250\u00a0Hz, sampled at 512\u00a0Hz, digitized (22 bits ADC), and smooth rectified with removal of the low frequency components. Embedded software provided the detection and calculation of muscle rest, expressed as the sEMG parameter Relative Rest Time, which was defined as the percentage of time in which Root Mean Square was below a threshold (10\u00a0\u03bcV) for at least 0.12\u00a0s. This threshold was based on the noise level of the myofeedback system including mounted electrodes at the skin. Sensory feedback by means of vibration and a soft sound was provided after each 10\u00a0s interval when the relative duration of muscle relaxation in that particular interval was below 20%. The choice for a 10\u00a0s interval was based on the results of Voerman et\u00a0al. [29], and the 20% threshold was chosen based on the work of H\u00e4gg and \u00c5str\u00f6m [30]. Subjects were instructed that they should respond to the feedback by relaxation, which could be reached by slightly depressing the shoulders, or by sitting down quietly with the eyes closed, the hands in the lap while breathing deeply. Another relaxation strategy subjects were instructed was to maximally elevate the shoulders for three seconds to build up muscle tension and then to let loose this tension. This relaxation was anticipated to contribute to recovery of the Cinderella motor units. When there was no adequate response to the feedback, i.e. relaxation, the duration of the feedback signal progressively increased.\nImmediately after Baseline, subjects assigned to the Mfb group were given the myofeedback device and they were explained the working mechanism and background of the myofeedback training. Subjects wore the system for four weeks, for at least eight hours a week (distributed over two hours a day and two days a week as a minimum) while performing their regular work. During the weekly visits the sEMG-data from last week were scrutinized and discussed to give the subject insight in their relaxation patterns and to identify possible situations of concern. This procedure was facilitated by means of the diary.\nAssessment of pain intensity and disability\nPain intensity in the neck, shoulder (left and right), and upper back at time of the measurement was assessed by means of four Visual Analogue Scales [31]. Subjects were instructed to rate their subjectively experienced level of pain intensity at that moment for each body region. The Visual Analogue Scale consists of a 10\u00a0cm horizontal line with \u2018no discomfort at all' at the left and \u2018as much discomfort as possible' at the right endpoints of the line. Psychometric properties of the Visual Analogue Scale have been shown to be sufficient [32\u201334].\nThe level of subjectively experienced disability was assessed with the Pain Disability Index, a self-rating scale that measures the impact of pain on the abilities to participate in life activities [35]. The Pain Disability Index contains 7 items, one for each domain, i.e. (1) family and home responsibilities, (2) recreation (hobbies, sports, and leisure time activities), (3) social activity (participation with friends and acquaintances), (4) occupation (activities partly or directly related to working), (5) sexual behavior (frequency and quality of sex life), (6) self care (personal maintenance and independent daily living), and (7) life-support activity (basic life-supporting behaviors). Answers were provided on a categorical 11-points scale with \u2018not disabled\u2019 and \u2018fully disabled\u2019 at the extremes. In a chronic pain population, psychometric properties of the Pain Disability Index appeared to be sufficient [36].\nAnalysis\nData inspection showed non-normal distributions for all of the variables, and data were therefore log transformed, enabling parametric analyses. Analyses were performed both on the group as well as the individual level.\nFor each subject, VAS scores for the neck, left shoulder, right shoulder, and upper back were summed and averaged resulting in a combined neck-shoulder pain intensity score. Differences between the two interventions, i.e. Mfb and EC, for VAS and the Pain Disability Index were investigated using a linear mixed-model analysis technique for repeated measurements. The following variables were included in the model as fixed factors: Time of measurement (i.e. Baseline, T0, T3, and T6), intervention type (i.e. Mfb and EC), and study group (i.e. Sweden and the Netherlands), including two-way interactions. The factor study group comprises variance due to possible (socio)demographic differences as well as variance caused by the different therapists, organizations, and job characteristics in the two countries. The factor subject was included in the model as a random factor. Paired and independent samples t-tests were used for Post Hoc analysis.\nAt the individual level the percentages of subjects improving and the odds for improvement in both intervention groups were investigated. First, individual differences were calculated between Baseline and T0, Baseline and T3, and Baseline and T6 for pain intensity in the neck-shoulder region (i.e. the averaged score on the VAS for neck, left and right shoulders, and upper back) and Pain Disability Index. These differences were then dichotomized into \u2018clinically relevant improvement\u2019 or \u2018equal\/deteriorated.\u2019 For the Visual Analogue Scale the cut-off point for a clinically relevant improvement was defined at 13\u00a0mm which corresponds to the upper limit of the confidence interval of clinically relevant changes in Visual Analogue Scale scores in acute and non-acute pain patients as reported by Kelly in 2001 [37]. This cut-off point was also the result of the study of Todd [34]. Cut-off points for clinically significant changes in the Pain Disability Index scores have not been provided in literature. For a comparable measure, the Neck Disability Index which is a 10 items scale with 6 answering options resulting in scores varying from 0 to 50, the cut-off point was set at 5 which is 10% of the maximum score [38]. Using this as a starting point, a clinically relevant change was defined as a change of \u226510% of the maximum score of 70 of the Pain Disability Index, i.e. \u22657 units of the Pain Disability Index score.\nLogistic regression analyses providing Odds Ratios were performed to investigate whether the two intervention groups differed in terms of chances for clinically relevant improvements in pain intensity and disability. Three different models were investigated: Model I represents the crude Odds Ratio describing only the relationship between intervention type and odds for improvement without adjustment for confounding factors. The factors study group and baseline pain intensity\/baseline disability were assumed potentially confounding factors. To correct for these factors, two additional models were built as extensions of Model I: Model II incorporates study group as confounding factor, and Model III is an extension of Model II incorporating also the factor baseline pain intensity\/baseline disability level. For Models II and III interaction effects were additionally included in the analysis, to study effect modification, but these remained only included when significant. \u22122 Log Likelihood tests were used to select the best model. Odds Ratios including 95% Confidence Intervals were calculated and presented for each of the models.\nStatistical Package for Social Sciences 11.5 was used for statistical testing and alpha was set at .05 for statistical significance.\nResults\nSubjects\nSeventy-nine female computer workers with neck-shoulder complaints were included in this study: Forty-two subjects were assigned to the Mfb group and 37 to the EC group. Mean age was 52.0 (SD 5.8 years) in the Mfb group and 50.7 (SD 5.5) years in the EC group. Mean duration (days between Baseline and T0) of the interventions was 37 (SD 8) days for the Mfb group and 36 (SD 7) days for the EC group. Forty-one out of the 79 subjects were recruited in Sweden and the remaining 38 in the Netherlands. The number of subjects at Baseline, T0, T3, and T6 and the number of drop-outs are shown in Diagram 1.\nDiagram 1Flow chart subject recruitment and randomisation\nDrop-outs did not differ in age, weight, height, BMI, and pain intensity scores (Visual Analogue Scale), and disability (Pain Disability Index) from those fulfilling the intervention (p\u00a0>\u00a0.08).\nTable 1 provides an overview of sociodemographic characteristics of the subjects in the Mfb and EC groups. Thirty-eight subjects reported complaints in both the neck and shoulder, while 41 subjects reported complaints either in the neck or in the shoulder at the time of recruitment. Comparison of the characteristics between the Mfb and EC groups indicated that subjects assigned to the Mfb group reported more years within the same job compared to the EC group (p\u00a0<\u00a0.05).Table 1Characteristics of subject populationMfb (n = 42)EC (n = 37)Sociodemographics\u2003Body mass index25.2 (3.9)25.2 (3.7)\u2003Side dominance% Right-handed95%97%\u2003Living situation% Living alone16.710.8\u2003Working hours per week32.8 (7.8)32.8 (8.3)\u2003Working hours per week since (in years)17.0 (11.4)*12.0 (8.9)*Complaints\u2003Trouble in neck last yearYes92.991.9\u2003Trouble shoulders last yearYes, in both shoulders36.632.4Yes, in the right shoulder36.643.2Yes, in the left shoulder12.213.5\u2003Trouble in upper back last yearYes66.748.6\u2003Was work performance affectedYes31.718.9*Significant at the .05 level.\nAnalysis at group level\nPain intensity\nBaseline (geometric) mean VAS scores for each region separately were 27\u00a0mm (neck), 15\u00a0mm (left shoulder), 14\u00a0mm (right shoulder), and 13\u00a0mm (upper back) in the Mfb group and 24, 14, 19, and 18\u00a0mm in the EC group respectively.\nFigure 1 shows a box plot of the VAS score in the neck-shoulder region at Baseline, T0, T3, and T6 for the Mfb and the EC group. A clear decrease was observed at T0, T3, and T6 compared to Baseline, although at T6 pain intensity seemed to increase slightly in both groups.Fig. 1Box plot for averaged VAS score of pain intensity in the neck-shoulder region at Baseline, T0, T3, and T6 for both the Mfb and the EC group\nMixed linear modelling showed that pain intensity in the neck-shoulder region significantly changed over time (F\u00a0=\u00a012.08, p\u00a0\u2264\u00a0.01), without additional effects for the type of the intervention (F\u00a0=\u00a01.54, p\u00a0=\u00a00.22), study group (F\u00a0=\u00a0.48, p\u00a0=\u00a0.49), or interaction effects (F\u00a0\u2264\u00a0.87, p\u00a0\u2265\u00a0.35). Post Hoc comparisons revealed that the VAS score was significantly reduced at T0 (t\u00a0=\u00a04.37, p\u00a0<\u00a0.01), T3 (t\u00a0=\u00a05.10, p\u00a0<\u00a0.01), and T6 (t\u00a0=\u00a03.54, p\u00a0<\u00a0.01) compared to Baseline but also the reduction between T0 and T3 was significant (t\u00a0=\u00a02.85, p\u00a0=\u00a0.01).\nDisability\nFigure 2 shows a box plot of the Pain Disability Index score at Baseline, T0, T3, and T6 for the Mfb and the EC group. A comparable pattern to what was observed for pain intensity was found, with decreased disability levels at T0, T3, and T6 compared to Baseline.Fig. 2Box plot for the Pain Disability Index score at Baseline, T0, T3, and T6 for both the Mfb and the EC group\nDisability levels significantly changed over time (F\u00a0=\u00a017.68, p\u00a0<\u00a0.01) and were significantly different between the two study groups (i.e. Sweden and the Netherlands) (F\u00a0=\u00a05.30, p\u00a0=\u00a0.02). No additional effects were found for intervention type (F\u00a0=\u00a0.86, p\u00a0=\u00a0.35) nor the interaction terms (F\u00a0\u2264\u00a01.97, p\u00a0\u2265\u00a0.12). Post Hoc comparisons showed that subjects in the Swedish study group had lower Baseline values and reported reductions in disability only between Baseline and T0 (t\u00a0=\u00a02.20, p\u00a0=\u00a0.04) with a trend for reductions at T3 (t\u00a0=\u00a01.89, p\u00a0=\u00a0.07) and T6 (t\u00a0=\u00a01.81, p\u00a0=\u00a0.08). Subjects in the Dutch study group showed a significant decrease at T0 (t\u00a0=\u00a03.26, p\u00a0<\u00a0.01), T3 (t\u00a0=\u00a03.58, p\u00a0<\u00a0.01), and T6 (t\u00a0=\u00a03.51, p\u00a0<\u00a0.01) compared to Baseline.\nAnalysis at the individual level\nPain intensity\nFigure 3 gives an overview of the percentage of subjects in the Mfb and EC groups showing an improvement in pain intensity in the neck-shoulder region. About half of the subjects showed a clinically relevant improvement in pain intensity in the neck-shoulder region. Slightly more subjects in the Mfb group showed improvements compared to the subjects in the EC group immediately after the intervention period (T0) and at 6 months follow-up (T6).Fig. 3Percentage of subjects showing improvements in averaged VAS score of pain intensity in the neck-shoulder region at T0, T3, and T6 compared to Baseline for the Mfb and EC group [without correcting for confounding factors]\nCrude Odds Ratios (Model I) for improvement showed higher odds for clinically relevant improvement in the Mfb group at T0 and T6 compared to Baseline, while between T3 and Baseline subjects assigned to EC were more likely to show improvements (see Table 2). However, Odds Ratios were not significant (p\u00a0\u2265\u00a0.36) also not when corrected for potential confounding factors (p\u00a0>\u00a0.19). \u22122 Log Likelihood tests indicated that Model III, adjusting for the factors study group and baseline pain intensity\/disability level, superimposed the Models I and II (\u22122 LL; p\u00a0<\u00a0.05). As no significant interaction effects were found (p\u00a0>\u00a0.08) these were excluded from the final models as presented in Table 2.Table 2(Adjusted) odds ratios (95% confidence interval) for improvement in pain intensity in the neck-shoulder region for the Mfb group compared to the EC groupModel IModel IIModel IIIVAS1.561.593.42Baseline\u2013T0(.60\u20134.05)(.71\u20134.14)(.55\u201321.11)VAS.78.78.40Baseline\u2013T3(.29\u20132.08)(.29\u20132.09)(.08\u20131.99)VAS1.311.311.04Baseline\u2013T6(.47\u20133.60)(.47\u20133.64)(.29\u20133.77)Model I: Odds Ratio Crude.Model II: Odds Ratio Adjusted for factor study group.Model III: Odds Ratio Adjusted for factor study group and VAS at Baseline.\nDisability\nAn overview of the percentage of subjects in the Mfb and EC groups showing an improvement in disability is provided in Fig. 4.Fig. 4Percentage of subjects showing clinically relevant improvements in Pain Disability Index at T0, T3, and T6 compared to Baseline for the Mfb and EC group [without correcting for confounding factors]\nImmediately after the intervention period about twice as many of the subjects in the Mfb group showed clinically relevant improvements in disability compared to the EC group. This share of subjects increased somewhat in both groups after three months and then showed a small decline after six months where about one third of the subjects of the EC group and half of the Mfb group showed clinically relevant improvements in disability.\nSubjects assigned to the Mfb group had higher odds for improvement in disability at T0, T3, and T6: At T3, the odds for improvement in disability was 2.77 times higher in the Mfb group compared to the EC group which was significant (Model I; 95% CI 1.00\u20137.65; p\u00a0=\u00a0.05). When corrected for confounding variables, however, Odds Ratios for improvement were still higher for the Mfb group compared to the EC group, but this was not significant (p \u2265 .057) except for Model II for changes between Baseline and T3 (p\u00a0=\u00a0.04). Baseline disability levels significantly affected Odds Ratios: Model III was generally better than the models I and II (\u22122 LL; p\u00a0<\u00a0.05). As no significant interaction effects were found (p\u00a0>\u00a0.08) these were excluded from the final models as presented in Table 3.Table 3(Adjusted) odds ratios (95% confidence interval) for improvement in disability the Mfb group compared to the EC groupModel IModel IIModel IIIPain disability index2.702.721.48Baseline\u2013T0(.97\u20137.54)\u2020(.971\u20137.60)\u2020(.39\u20135.62)Pain disability index2.772.991.64Baseline\u2013T3(1.00\u20137.65)*(1.03\u20138.65)*(.34\u20137.97)Pain disability index2.542.611.48Baseline\u2013T6(.88\u20136.82)\u2020(.91\u20137.47)(.37\u20135.88)Model I: Odds Ratio Crude.Model II: Odds Ratio Adjusted for factor study group.Model III: Odds Ratio Adjusted for factor study group and Pain Disability Index at Baseline.*p\u00a0\u2264\u00a0.05.\u2020.05\u00a0\u2264\u00a0p\u00a0\u2264\u00a0.10.\nDiscussion\nThis randomised controlled trial investigated the effects of 4-weeks ambulant myofeedback training combined with ergonomic counselling in subjects with work-related neck-shoulder complaints relative to ergonomic counselling alone, on pain intensity and disability. The effects were evaluated immediately after the interventions, and at three and six months follow-up. Mean pain intensity and disability levels significantly reduced after both interventions (i.e. Mfb and EC). The effects were clinically relevant in a large part of the subjects: About 30\u201350% of the subjects showed clinically relevant improvements in pain intensity and\/or disability. No difference was found for the effect of the intervention on outcome and after correction for confounding factors subjects in both intervention groups did not differ with regard to chances for clinically relevant improvements in pain intensity and disability.\nSeveral studies have shown the beneficial effect of myofeedback training on pain reduction [21, 39\u201343] although Faucett and colleagues [22] reported changes in muscle activity rather than changes in pain intensity. The feedback approach used in these studies is different from the Cinderella-based myofeedback approach in that the traditional feedback method provides feedback when muscle activation is too high [21, 22, 42, 44\u201347], rather than when the time the muscle has relaxed is too short [23]. Two previous studies applying the Cinderella-based myofeedback training in subjects with work-related complaints [23] and subjects with a whiplash associated disorder [48] reported reduced pain intensity and disability levels. Compared to these studies, baseline pain intensity values were generally lower and the cut-off points for clinically relevant changes were set to higher levels in the current study, but the results indicate a comparable percentage of subjects reporting clinically significant reductions in pain intensity, i.e. between 35 and 50% of the subjects. This consistency in results adds to evidence for the effectiveness of (Cinderella-based) myofeedback training on pain intensity and disability in musculoskeletal neck-shoulder complaints.\nIn line with existing literature (e.g. [8, 10, 49, 50]) also subjects in the ergonomic counselling group reported reduced pain intensity and disability. It was however hypothesised that a treatment approach including both myofeedback training and ergonomic counselling (Mfb) would be more effective than treatment comprising only ergonomic counselling (EC) as interventions focusing on multiple factors have shown to be related to decreased incidence of complaints [24]. In the current study the two types of interventions did not differ in terms of outcome. This is in line with findings of other studies (e.g. [8, 10, 51\u201353]) that concluded that occupational interventions have generally comparable effects, although these studies did not include a myofeedback intervention. Newton-John and others [43] compared myofeedback with cognitive-behavioural therapy and a waiting list control group and found that both interventions showed favourable outcome in terms of pain intensity and disability compared to the waiting list control group without a difference between the two interventions.\nThere are possible explanations for the absence of differences between the two groups. One concerns the presence of subgroups in which the intervention is beneficial. Results showed that the effect is clinically relevant in about 30\u201350% of the subjects. Main question to be addressed here is whether, and how, these subjects can be characterised in terms of cognitive-behavioural characteristics and whether this characterisation can contribute to predict outcome of the intervention. This could substantially improve the efficiency and effectiveness of the interventions. Further, differences between the two groups may have been masked by using rather generic outcome measures. For instance, coping and patient-rated parameters may better represent the difference in outcome between different kinds of interventions [54]. Investigation into the specific working mechanisms of both interventions could clarify this. Finally, initial VAS and disability levels were low in both the Mfb and the EC group, especially in the study group from Sweden. This results in a smaller potential for improvement (floor effect) and as a result a smaller chance for finding differences between the Mfb and EC groups.\nMethodological considerations\nThis study did not include a non-intervention or placebo control group (for instance randomly administered feedback) which makes it hard to control for non-specific effects like regression to the mean or the Hawthorne effect [55]. This effect was already described in 1933 by Mayo, and is reported as a significant positive effect without causal basis in the theoretical motivation for the intervention, but is related to the effect on the participants knowing themselves to be studied in connection with the outcomes measured. An argument against such effect is that although it might occur in connection with the intervention (i.e. at T0) it is not likely that this effect would remain [56] after three to six months. In addition, the study population also contained subjects with chronic complaints (i.e. duration >6 months) who received a variety of treatments in the past, which were not successful. It is likely to expect that any non-specific effects of treatment would already have occurred during past treatments and that this effect in the current study would thus be small. Furthermore, results from previous studies learned that pain reduction in myofeedback-trained subjects were higher compared to a wait-list control group [43], a no intervention group [46], or subjects receiving a placebo [57]. Furthermore, an attention-only group showed no changed outcome in terms of disability and pain intensity [58].\nThe present study included a rather specific subject population: Participants were females, predominantly over the age of 45, still at work, and characterised by relatively low pain intensity and disability levels. Interpretation and extrapolation of results to other populations therefore requires caution, but as comparable effects of Cinderella-based myofeedback training have also been shown in mixed, younger subject populations [23, 48] and in a sample of patients who were on sick leave because of their neck-shoulder complaints [48], generalisation of findings might be legitimate. The subjects were selected based on self-reported complaints rather than a clinical evaluation. In a previous study [60] with female computer users above the age of 45, applying the same inclusion criteria as the present study, it was found that in subjects with neck-shoulder complaints the following clinical diagnoses were most prevalent: Trapezius myalgia (38%), tension neck syndrome (17%), and cervicalgia (17%). These diagnoses were found in 60% of the subjects reporting complaints. There were cases with supraspinatus tendinitis, frozen shoulder, and biceps tendinitis, but these were less common. This general pattern of clinical signs is likely to be at hand also in the present study. It could be hypothesised that myofeedback may be particularly relevant and helpful in cases with muscular pain syndromes.\nDespite extensive standardisations, the recruitment of subjects in two different study groups (Sweden and the Netherlands) resulted in heterogeneity of the subject population with regard to age, working hours, seniority, and working posture. To correct for this, the factor study group was considered a confounder needed to control for during analysis. This reduced the power of the study. An additional likely confounding factor is the compliance of the patient and therapist to the intervention. This is an often uncontrolled and thus potentially confounding factor in occupational intervention studies [59] and it is known that changes in knowledge and skills do not necessarily result in a behavioural change. In the Mfb group, the compliance was partly controlled as the number of hours the system was worn was recorded by the system itself, but the compliance to ergonomic knowledge and skills is hard to register objectively. This definitely is a challenge in future occupational intervention studies.\nThe drop-out rate in the Mfb group was higher than in the EC group (i.e. nine compared to five), which was probably related to the myofeedback system itself. Some subjects found the system inconvenient and disturbing in daily working activities. This may have suppressed the effect of the intervention. Improvement of the current device in terms of usability is therefore required to optimise the myofeedback training.\nConclusions\nFour-weeks of intervention significantly reduced pain intensity and disability, and this effect remained after three and six months follow-up. Myofeedback training combined with ergonomic counselling is thus beneficial for female computer workers over the age of 45, reporting pain and disability in the neck-shoulder region but no evidence was found favouring myofeedback training combined with ergonomic counselling over ergonomic counselling alone. Future research may aim at identifying possible subgroups of patients in which the interventions are especially beneficial. This may enhance the efficiency and the effectiveness of the interventions. Finally, as non-specific effects may have interfered with outcome, future studies could include a placebo control group for more insight in the specific effects of ambulant myofeedback training combined with ergonomic counselling.","keyphrases":["myofeedback training","work-related neck-shoulder complaints","neck-shoulder region"],"prmu":["P","P","P"]}
{"id":"Doc_Ophthalmol-4-1-2235911","title":"ISCEV guidelines for clinical multifocal electroretinography (2007 edition)\n","text":"The clinical multifocal electroretinogram (mfERG) is an electrophysiological test of local retinal function. With this technique, many local ERG responses, typically 61 or 103, are recorded from the cone-driven retina under light-adapted conditions. This document specifies guidelines for performance of the test. It also provides detailed guidance on technical and practical issues, as well as on reporting test results. The main objective of the guidelines is to promote consistent quality of mfERG testing and reporting within and among centers. These 2007 guidelines, from the International Society for Clinical Electrophysiology of Vision (ISCEV: http:\/\/www.iscev.org), replace the ISCEV guidelines for the mfERG published in 2003.\nIntroduction\nThe electroretinogram (ERG) is a mass potential, the result of the summed electrical activity of the cells of the retina. Full-field electroretinography is a well-established clinical technique for evaluating global retinal function [1]. The multifocal ERG (mfERG) technique was developed to provide a topographic measure of retinal electrophysiological activity. With this technique, many local ERG responses, typically 61 or 103, are recorded from the cone-driven retina under light-adapted conditions. In 2003, the International Society for Clinical Electrophysiology of Vision (ISCEV) published guidelines for recording the mfERG [2]. These were guidelines, not standards, to allow for further research before standards were set.\nAlthough the mfERG now has been used for more than 10\u00a0years to aid in the diagnosis of diseases of the retina, ISCEV decided that it is still premature to set specific standards. However, to take into consideration recent developments in technology and practice, this document provides revised guidelines for recording clinical mfERGs. These guidelines will be reviewed periodically, consistent with ISCEV\u2019s practice.\nDescription of multifocal electroretinography\nThe mfERG technique is a method of recording local electrophysiologic responses from different regions of the retina. Electrical responses from the eye are recorded with a corneal electrode as in conventional, full-field ERG recording. However, the nature of the stimulus and the form of the analysis differ. These differences allow a topographic map of local ERG activity to be measured. For the basic mfERG described here, the retina is stimulated with an array of hexagonal elements, each of which has a 50% chance of being illuminated every time the frame changes (Fig.\u00a01). Although the pattern appears to flicker randomly, each element follows the same pseudo-random sequence of illumination with the starting point displaced in time relative to other elements. By correlating the continuous ERG signal with the sequence of on- and off-phases of each element, the local ERG signal is calculated. Although we will refer to these local ERG signals as mfERG responses, it is important to keep in mind that they are not direct electrical potentials from local regions of retina, but rather they are a mathematical extraction of the signal. Further, because the stimulation rate is rapid, the waveform of the local mfERG response can be influenced both by preceding (\u2018adaptation effects\u2019) and subsequent stimuli (\u2018induced effects\u2019), as well as by the responses to light scattered on other retinal areas.\nFig.\u00a01(a) Representative hexagonal mfERG stimulus array with 61 elements scaled with eccentricity. Roughly half of the elements are illuminated at any one time. (b) Same as in panel A for an array with 103 elements\nThe typical waveform of the basic mfERG response (also called the first-order response or first-order kernel) is a biphasic wave with an initial negative deflection followed by a positive peak (Fig.\u00a02). There is usually a second negative deflection after the positive peak. These three peaks are called N1, P1 and N2, respectively. There is evidence that N1 includes contributions from the same cells that contribute to the a-wave of the full-field cone ERG, and that P1 includes contributions from the cells contributing to the cone b-wave and oscillatory potentials. Although there is some homology between the mfERG waveform and the conventional ERG, the stimulation rates are higher for the mfERG and, as noted above, the mfERG responses are mathematical extractions. Thus, the mfERG responses are not technically \u201clittle ERG responses\u201d. Therefore, the designations \u2018a-wave\u2019 and \u2018b-wave\u2019, used for the full-field ERG, are not appropriate to describe features of the mfERG waveform.\nFig.\u00a02Diagram of an mfERG response to show the designation of the major features of the waveform\nBasic technology\nElectrodes\nRecording electrodes\nPoor or unstable electrode contact is a major cause of poor quality records. It is important to follow the recommendations concerning fiber, foil, loop and contact lens electrodes in the full-field ERG Standard [1] and Pattern ERG (PERG) Standard [3]. In particular, electrodes that contact the cornea, or nearby bulbar conjunctiva, are required. In addition, good retinal image quality and proper refraction is desirable.\nReference and ground electrodes\nProper application of suitably conductive electrodes is essential for reliable mfERG recordings. Follow the recommendations made in other ISCEV Standards [1, 3].\nElectrode characteristics, stability and cleaning\nFollow the recommendations in the ISCEV full-field ERG and\/or PERG Standards [1, 3].\nStimulation\nStimulus source\nUntil recently, the mfERG stimuli were most commonly displayed on a cathode ray tube (CRT), i.e. a monitor. CRT monitors are being rapidly replaced with other devices such as liquid crystal display (LCD) projectors, arrays of light emitting diodes (LEDs) and organic LEDs (OLEDs). These alternative modes of stimulation can affect the amplitude and waveform of the mfERG making it essential to specify the type of display when reporting results.1\nFrame frequency\nA CRT frame frequency of 75\u00a0Hz has been used widely. (LCD displays use 60\u00a0Hz.) Use of different frequencies can substantially alter the amplitude and waveform of the mfERG response. Whatever frame frequency is used, normative values for normal healthy subjects need to be determined separately for that frequency. Further, it is essential to specify the frame frequency when reporting results.\nLuminance and contrast\nIn general, the luminance of the stimulus elements for CRT displays should be 100\u2013200\u00a0cd\/m2 in the lighted state and low enough in the dark state to achieve a contrast of \u226590%. This means that the mean screen luminance during testing will be 50\u2013100\u00a0cd\/m2. Although higher luminance levels can be used, the ability to detect local defects may be decreased due to the effects of stray light. In addition, the luminance requirements may differ if a non-CRT display is used. In any case, the surround region of the display (the area beyond the stimulus hexagons) should have a luminance equal to the mean luminance of the stimulus array.\nCalibration\nAs with other electrophysiologic signals, luminance and contrast affect the recorded signal and it is important to calibrate the stimulus following ISCEV guidelines [4]. The luminance of the dark and the light stimulus elements should be measured with an appropriate calibrator or spot meter. Many monitor screens are not of uniform brightness over the entire screen. While some variation is to be expected, a variation of greater than 15% is considered unacceptable. Some commercial systems are equipped to calibrate the display. If this ability is not present, we urge manufacturers to provide instructions for calibration of their devices.\nStimulus parameters\nStimulus pattern: The typical mfERG display is a hexagonal stimulus pattern scaled in size to produce mfERG responses of approximately equal amplitude across the retina. Thus, the central hexagons are smaller than the more peripheral ones. Different patterns may be useful in special cases (e.g. equal size hexagons for patients with eccentric fixation). These guidelines cover only the typical stimulus pattern, scaled to produce approximately equal size responses for healthy control subjects.\nFlicker sequence: Most commercial mfERG instruments use an m-sequence to control the temporal sequence (between light and dark) of the stimulus elements. An m-sequence, in which the elements can change with every frame, is recommended for routine testing. Different sequences, or the inclusion of global light or dark frames, have been suggested for specialized applications, but they are not a part of these guidelines, and should not be done in exclusion of a \u201cstandard\u201d mfERG for routine clinical purposes.\nStimulus size and number of elements: The overall stimulus pattern should subtend a visual angle of 20\u201330\u00b0 on either side of the fixation point. The stimulus region can be divided into different numbers of hexagons. The most frequently used patterns have 61 or 103 hexagons, with 241 hexagons occasionally used. The choice depends on balancing the need for good spatial resolution and a high signal-to-noise ratio, while minimizing the recording time. (See discussion below under clinical protocol).\nFixation targets: Stable fixation is essential for obtaining reliable mfERG recordings. Central fixation dots, crosses and circles are available with most commercial systems. The fixation targets should cover as little of the central stimulus element as possible to avoid diminishing the response. However, the examiner should always verify that the patient can see the fixation target. When the fixation targets are enlarged for low-vision patients, care should be taken not to obscure regions of interest. For example, a larger central cross will lead to a smaller central response simply due to occlusion of more of the stimulus.\nRecording, analyzing and presenting results\nAmplifiers and filters\nThe gain of the amplifier should produce recognizable signals without saturation. Appropriate band-pass filtering removes extraneous electrical noise, without distorting waveforms of interest. For a \u201cstandard\u201d mfERG, the high pass cutoff can range between 3 and 10\u00a0Hz and the low pass cutoff between 100 and 300\u00a0Hz. Filter settings, even within the ranges suggested, will influence the response waveform. Thus, the filter settings should be the same for all subjects tested by a given laboratory, as well as for the norms to which they are compared. Line-frequency or notch filters should be avoided.\nSignal analysis\nArtifact rejection: Because blinks and other movements can distort the recorded waveforms, commercial software usually includes \u2018artifact rejection\u2019 algorithms to eliminate some of these distortions. When applying an artifact rejection procedure after the recording, care should be exercised to assure that clinically important aspects of the waveform are not being modified. In any case, artifact rejection procedures should be specified when reporting mfERG results.\nSpatial averaging: In order to reduce noise and smooth the waveforms, some commercial programs allow the averaging of the response from each stimulus element with a percentage of the signal from the neighboring elements. Spatial averaging can help visualize the mfERG signal in noisy records. However, spatial averaging may obscure small, local changes or the borders of regions of dysfunction. Thus, it should be used with care and specified when reporting results. Further, the default conditions of commercial software should be examined, as spatial averaging may be a default condition.\nDisplaying results\nTrace arrays: All commercial programs can produce an array of the mfERG traces (Fig.\u00a03a, b). This display of the results is useful for visualizing areas of abnormality and for comparing the mfERG results to visual fields from perimetry. The trace array is the basic mfERG display and should always be included in the report of clinical results. Commercial software typically allows for these responses to be displayed with either a visual field view or retinal view as the frame of reference. The frame of reference should be indicated. Note that in Fig.\u00a03, each of the 61 (panel a) or 103 (panel b) responses are shown in approximately correct topographic representation. Adding contours to show the loci of different eccentricities, as in Fig.\u00a04, can help in determining whether the regions of low amplitude correspond to regions of the visual field that show a loss of sensitivity.\nFig.\u00a03Sample mfERG trace arrays (field view) with 61 elements (panel a) and 103 elements (panel b). (c, d) The 3-D response density plots (field view) associated with panels a and bFig.\u00a04The mfERG trace array (left panel, field view) and the probability plot from standard automated perimetry (right panel) for a patient with retinitis pigmentosa. The contours for a radius of 5 and 15\u00b0 are shown. The light gray, dark gray, and black squares indicate statistically significant field loss at the 5, 1 and 0.5 percent levels, respectively\nGroup averages: Commercial programs allow for the averaging of groups of responses from the trace arrays. This can be helpful for comparing quadrants, hemiretinal areas, normal and abnormal regions of two eyes, or successive rings from center to periphery. The latter is particularly useful for patients who have diseases that produce approximately radially symmetric dysfunction. In addition, responses from stimulus elements associated with a local area of interest can be averaged for comparison with a similar area in an unaffected eye or to data from control subjects. Figure\u00a05 shows the result of grouping the responses in Fig.\u00a03b by rings. The most commonly used display is response density (Fig.\u00a05a), in which the responses from the elements in each ring are summed and then divided by the area of these elements. Figure\u00a05b and c are alternative representations of the data in which summed responses from each ring are shown (panel c) or are normalized so that they have the same amplitude (panel b).\nFig.\u00a05The mfERG responses in Fig. 3b were grouped by concentric rings and summed to yield the \u2018Summed\u2019 responses in panel c. These summed responses are divided by the area of the elements of the ring for the \u2018Response Density\u2019 responses (panel a) and normalized so each has the same amplitude for the \u2018Normalized\u2019 responses (panel b)\nTopographic (3-D) response density plots: The 3-D plot (Fig.\u00a03c, d) shows the overall signal strength per unit area of retina. This display can sometimes be useful for illustrating certain types of pathology. In addition, the quality of fixation can be assessed by observing the location and depth of the blind spot. However, there are major dangers in using the 3-D plot in assessing retinal damage. First, information about the waveforms is lost. Thus large, but abnormal, or delayed responses can produce normal 3-D plots. Second, a central peak in the 3-D plot can be seen in some records without any retinal signal (see, Appendix: Artifact recognition examples for electrical noise and weak signals). Finally, the appearance of the 3-D plot from a given recording is dependent on how the local amplitude is measured. Whenever 3-D plots are presented, the method used to measure local amplitude should be identified, and the corresponding mfERG trace array also should be presented.\nSignal extraction: Kernels\u2014This document is aimed at the general mfERG user and only describes the basic response, the first-order kernel. Higher order kernels, particularly the second-order kernel, occasionally are reported, and used in special applications.\nClinical protocol\nPatient preparation\nPupils\nThe pupils should be fully dilated and pupil size noted.\nPatient positioning\nSubjects should sit comfortably in front of the screen. Relaxation of facial and neck muscles will reduce artifacts from muscles; a headrest may be helpful. The appropriate viewing distance will vary with screen size, in order to control the area (visual angle) of retina being stimulated.\nFixation monitoring\nGood fixation, both central and steady, is essential. Thus, fixation should be monitored, preferably by the use of monitoring instrumentation available on some units. When this option is not available, careful direct observation may be employed.\nRefraction\nAlthough there is some evidence that the mfERG is unaffected by moderate blurring of the retinal image in healthy individuals, we recommend refraction for optimal acuity. On some commercial machines, a manual adjustment of the viewing optics is possible. Alternatively, lenses can be placed in a holder positioned in front of the eye. In the latter case, the viewing distance must be adjusted to compensate for the relative magnification of the stimulus. Also care must be taken to avoid blocking the view of the stimulus screen by the rim of the lens or the lens holder and thus creating an apparent scotoma.\nMonocular versus binocular recording\nRecording is typically done with monocular stimulation. Those who record binocularly should be aware that signals can be altered by misalignment of the eyes.\nAdaptation\nPre-adaptation (before test): Subjects should be exposed to ordinary room lighting for at least 15\u00a0min prior to testing. Longer adaptation times may be needed after exposure to bright sun or bright lights such as those used for fundus photography or retinoscopy.\nRoom illumination: Moderate or dim room lights should be on and ideally should produce illumination close to that of the stimulus screen.\nStimulus and recording parameters\nStimulus Size\nThe stimulus should subtend 20\u201330\u00b0 of visual angle on either side of the fixation point.\nNumber of elements\nA display containing 61 or 103 elements should be used.\nDuration of recording\nA total recording time of at least 4\u00a0min for 61 element arrays, or 8\u00a0min for 103 element arrays, is recommended, although these times might be adjusted by experienced laboratories according to clinical needs. The overall recording time is divided into shorter segments (e.g. 15\u201330\u00a0s) so that subjects can rest between runs if necessary and also so that a poor record (from noise, movement or other artifacts) can be discarded and repeated without losing prior data.\nTrade-offs\nVarious manipulations will affect the signal-to-noise ratio (SNR) of the responses. In particular, decreasing the size (increasing the number for a fixed stimulus field size) of the stimulus elements and decreasing the duration of the recording will decrease the SNR of the responses. While decreasing the number (increasing the size) of elements will increase the SNR, it will decrease the spatial resolution of the test. In general, conditions with larger (i.e. 61) elements and a shorter recording time (e.g. 4\u00a0min) are easier for the patient and suitable for a general screening of macular function. On the other hand, conditions with 103 elements and a longer recording time (e.g. 8\u00a0min) are useful for assessing foveal function and mapping the outline of retinal defects. Very small elements (such as a 241 hexagon array) may sometimes be helpful for diseases with very small or irregular effects. Repeat recording is recommended to confirm small or subtle abnormalities.\nFurther, the choice of electrode type will also influence the SNR of the responses. For example, bipolar corneal contact electrodes yield recordings with the highest SNR. Thus, longer recording times, repeat measurements and\/or fewer stimulus elements are necessary to obtain comparable SNRs when a foil or fiber electrode is used.\nData reporting\nMode of display\nTrace arrays: It is essential to show the trace array when reporting mfERG results (see Fig.\u00a03a, b). These arrays not only show topographic variations, but also demonstrate the quality of the records, which is important in judging the validity of any suspected variations from normal. Trace lengths of 100\u00a0ms or more should be used for these displays. (It is hard to detect interference from line frequency and\/or kernel overlap in shorter trace lengths.).\nGroup averages: Arranging responses by groups can be a useful way to summarize the data. Concentric rings of traces, from the center outward, are most commonly used. Regions with fundus pathology can be averaged together if desired. Most laboratories report response density (Fig.\u00a05a).\nThree-dimensional plots: These should be used with caution and only when accompanied by trace arrays (see above). The 3-D plots (Fig.\u00a03c, d), without accompanying trace arrays, can be misleading (see Appendix). Note that if fixation is steady and central, a clear depression due to the blindspot should be present and located in the appropriate place.\nMeasurements calibration marks: Calibration marks must accompany all traces or graphs. This will enable comparisons among patients or within a patient on sequential visits.\nMeasuring mfERG amplitude and timing: The N1 response amplitude is measured from the starting baseline to the base of the N1 trough; the P1 response amplitude is measured from the N1 trough to the P1 peak (see Fig.\u00a02). The peak times (implicit times) of N1 and P1 are measured from the stimulus onset. Measurements of group averages should routinely include the N1 and P1 amplitudes and peak times.\nCommercial software provides measures of the overall amplitude and timing of the mfERG traces. There are various procedures for measuring amplitude (e.g. trough-to-peak amplitude), latency (e.g. response shifting, response stretching, time to peak), or overall response waveform (e.g. scalar product, root-mean-square (RMS)). A description of these techniques is beyond the scope of these guidelines. However, it should be noted that when a template is needed (e.g. for scalar product measures), the template should be formed from age-similar control data obtained from that laboratory.\nColor scales: The use of color scales is optional; care should be taken when reproducing color images on a gray scale as the luminance sequences may not be in the proper order.\nNormal values\nEach laboratory must develop its own normative data. Variations in recording equipment and parameters make the use of data from other sources inappropriate. Because electrophysiologic data are not necessarily described by a normal distribution, laboratories should report the median value rather than the mean, and determine boundaries of normality. The mfERG, like the full-field ERG, is somewhat smaller in amplitude in older individuals and in those with highly myopic eyes. Although these effects are generally not large, they can be important in the evaluation of some patients. In any case, age-adjusted normative data is recommended.\nReporting of artifacts and their resolution\nReports should indicate any problems with the recording such as movement, head tilt, poor refraction capability, poor fixation, etc. that might affect reliability and interpretation. Also, indicate explicitly any artifact reduction procedures or post-processing maneuvers used to prepare the data. This should include the type and number of artifact rejection steps, the spatial averaging with neighbors (noting the extent and number of iterations), and any other averaging or filtering procedures.","keyphrases":["multifocal electroretinogram","electroretinogram","clinical guidelines"],"prmu":["P","P","R"]}
{"id":"Purinergic_Signal-3-4-2072924","title":"Activation kinetics of single P2X receptors\n","text":"After the primary structure of P2X receptors had been identified, their function had to be characterized on the molecular level. Since these ligand-gated ion channels become activated very quickly after binding of ATP, methods with adequate time resolution have to be applied to investigate the early events induced by the agonist. Single-channel recordings were performed to describe conformational changes on P2X2, P2X4, and P2X7 receptors induced by ATP and also by allosteric receptor modifiers. The main results of these studies and the models of P2X receptor kinetics derived from these observations are reviewed here. The investigation of purinoceptors by means of the patch clamp technique following site-directed mutagenesis will probably reveal more details of P2X receptor function at the molecular level.\nPrimarily, P2X receptors are ligand-gated ion channels. This ion channel family, also known as ion channel-coupled receptors, mediates the fastest known signal transduction from extracellular messengers to the intracellular environment. This signal transduction mechanism is so simple (i.e., includes few steps) that it becomes fully activated in the low millisecond range. The best known biological structures using this approach are neuronal synapses in which receptors for the classic transmitters acetylcholine, glutamate, gamma-aminobutyric acid (GABA), glycine, or serotonin transmit excitation from one neuron to another in less than 1\u00a0ms [1]. This rapid activation kinetics of the ligand-gated ion channels is too fast to become analyzed by whole-cell current recordings or even slower fluorescent dye-based measurements of changes of the intracellular ion composition. The main reason for the low speed of these recording techniques compared to the receptor kinetics is the rate-limiting solution exchange at the extracellular side of the cell membrane where the ligand binding site is located. These obstacles in the investigation of ligand-gated channels can be overcome by the single-channel patch clamp technique where tiny parts (patches) are excised out of the cell membrane containing at best only one receptor. In such preparations, solution exchanges can be achieved within less than 1\u00a0ms [2]. Apart from the high time resolution, this technique has a resolution of ionic currents in the pA (picoampere) range sufficient to observe the ionic flow through and the gating (opening and closing) of single ion channels. With this technique, ion channels can be characterized and identified according to their conductance, macroscopic kinetics (time course of activation, i.e., opening of channels after ATP application, and of deactivation, i.e., closing of channels after ATP withdrawal), and microscopic kinetics (i.e., the characteristic mean times channels spend in closed or open configurations). The ultimate goal of the kinetic analysis is the development of a kinetic (normally Markovian) model of closed and open states which describes the agonist concentration dependency of the macroscopic and microscopic kinetics. Additionally, single-channel analysis may be able to discriminate whether various effects of agonists and antagonists are caused by changing agonist binding or efficacy or by altering the ion channel permeation behavior [3].\nAdditionally, patch clamping allows the separation of currents flowing through the ligand-gated channels from contaminating currents mediated by conductances activated downstream by secondary signals. In the case of P2X receptors, such signals may be changes in the intracellular concentration of Ca2+, Na+, K+, or H+ ions which can permeate the channel pore. The next steps in signal cascades may be changes of the cell volume with effects on the cytoskeleton, oxidative stress, and furthermore activation of kinases, phosphatases, or transcription. Therefore, investigating the time course of opening and closing of P2X receptors may help to understand signaling cascades they activate. It may also detect functional changes induced by receptor mutations more precisely than whole-cell current recordings or measurements of P2X receptor-induced changes of the intracellular Ca2+ concentration or even later effects.\nSoon after the introduction of the patch clamp technique, ATP-activated single channel events were discovered and analyzed. They were first described in rat sensory neurons [4] and cultured chicken myoblasts [5]. Later, different ATP-activated single ion channel currents were reported in smooth and cardiac muscle, glands, and other neurons where single-channel conductances between <1 and 60\u00a0pS were measured [6].\nMore detailed information about P2X receptor kinetics was obtained from measurements of the rise time of ATP-induced whole-cell currents which, as typical for ligand-gated receptors, decreased with increasing agonist concentration in the range of 5\u201325\u00a0ms [7, 8]. A more sophisticated kinetic analysis of P2X receptor function was performed in bullfrog dorsal root ganglion cells. The concentration dependence of the activation and deactivation time course as well as of the steady-state current during prolonged ATP application could well be described by a linear Markov model where the independent binding of three ATP molecules to equal binding sites leads to the opening of the channel which in the triliganded state is open 25% of the time:\nHere, the rate constants are given in s\u22121 and \u03b2\/\u03b1\u2009=\u20091\/4 [9]. Remarkably, the trimeric structure of cloned P2X receptor channels described much later [10\u201312] implies the necessity of binding of three ATP molecules and therefore coincides well with this model.\nInvestigations of the microscopic kinetics of native P2X receptors were used to characterize aspects of the gating behavior of the channels in distinct preparations but were not developed into kinetic models [5, 13\u201318].\nAfter cloning the P2X receptor subtypes, the kinetic analysis of heterologously expressed distinct P2X receptors became principally possible. However, the collection of enough single-channel data for establishing a kinetic model turned out to be too laborious or even seemed to be impossible on receptors which desensitize quickly (within milliseconds) and recover slowly (within minutes) [19, 20]. Therefore, models for P2X1 [21] and P2X3 [22] receptors have been developed which, like the model developed by Bean [9], are based on whole-cell recording and include additional desensitized receptor states.\nFor the slowly desensitizing P2X4 receptor, the effect of external Mg2+ ions was investigated on the single-channel level. The blocking effect of Mg2+ could be partially ascribed to the reduction of the mean open time [23]. A more detailed analysis of the single-channel kinetics of P2X4 receptors revealed three different mean open times and five mean closed times following activation of the receptor by 0.3\u00a0\u03bcM ATP, where desensitization is slow. Hence, it was concluded that a kinetic model should have at least three open and five closed states [24].\nA more comprehensive investigation of the single-channel kinetics under steady-state conditions (i.e., during prolonged application of constant extracellular ATP concentrations where the distribution of conformational states of receptors is not changing with time) was performed on the very slowly desensitizing P2X2 receptor [25]. It should, however, be mentioned that P2X2 receptors may exhibit substantial fast desensitization in excised patches [26]. The following model was developed:\nIn contrast to Bean\u2019s model [9] the three ATP binding sites are not equal in this case. Specifically, the second binding step possesses a higher association rate constant and a much higher dissociation rate constant compared to the first. Both together result in a decreased affinity. This could be interpreted in such a way that the binding of ATP at the first site induces a conformation change that leads to an easier accessibility of a second binding to ATP. But this second binding is weaker, possibly due to the repulsion of both anionic ATP4\u2212 molecules. Negative cooperativity due to ATP repulsion was also suggested for the human P2X7 receptor [27]. The addition of an open (O6) and a closed state (C7) to a simple C\u2013C\u2013C\u2013C\u2013O model was necessary to describe openings of the P2X2 receptor which occurred as single openings or as bursts. This model was however unable to explain P2X4 receptor kinetics of multichannel patches, and therefore a positive cooperativity toward opening of P2X4 receptor channels was assumed [28].\nThe P2X7 is another non-desensitizing P2X receptor which proved to be suitable for kinetic analysis [29]. A relatively simple model was used to describe both the ATP dependence of microscopic (open and shut times, open probability of single channels) and macroscopic kinetics (activation and deactivation time course) of the main gating mode measured by a combination of single-channel recording and an ultrafast solution exchange system:\nThe model could be simplified compared to that describing the kinetics of the P2X2 receptor, since the open time distribution could be described in most cases by one exponential component and the channel did not display a bursting behavior. The much lower association rate constants compared to the P2X2 model are owed to the low ATP affinity of the P2X7 receptor. The kinetics of a second gating mode of single P2X7 receptors were about four times slower but could principally also be described by this model.\nThe assumption of only two ATP binding sites was sufficient for the approximation of the measured P2X7 receptor kinetics. This is a further deviation from the models mentioned above. The finding that P2X7 receptors are putative trimers like other P2X receptors and therefore contain probably three ATP binding sites seems to be contradictory to the P2X7 receptor kinetics model. As yet, a comprehensive explanation cannot be given but there are indications for a third binding site that induces very small single ion channel currents (Riedel et al., unpublished).\nMean currents calculated by this model displayed an approximately monoexponential activation and deactivation time course with time constants in the range of 10\u201325\u00a0ms. It was much simpler than the kinetics of several measured P2X7 receptor-dependent whole-cell currents [11]. This indicates that under whole-cell conditions either other ion currents may become secondarily activated or the biophysical properties of single P2X7 receptors themselves become less complex due to washout of an essential receptor component. However, even in cell-attached recordings, in which the intracellular environment is preserved, the single-channel current measurements at P2X7 receptors gave indication neither of more complex activation kinetics nor of a dilatation of the channel pore which is assumed to occur during prolonged ATP application [11]. Moreover, these single-channel current characteristics are indistinguishable from those measured in human lymphocytes [16], corroborating the view that the native human P2Z receptor is a genuine P2X7 receptor. Detailed analysis of the permeation characteristics [30] revealed a stable single-channel conductance even in external Na+-free and low Ca2+ solutions during long-lasting application of large ATP concentrations where a permeability of the cell membrane to large organic cations was induced in whole-cell preparations [11, 31, 32]. Instead, the permeation behavior and the calculated pore size of about 8.5\u00a0\u00c5 was similar to P2X1 and P2X2 receptors [33, 34]. Accordingly, it can be concluded that the apparent pore dilatation of P2X7 receptors observed in macroscopic current recordings [32] has no equivalent at the single-channel level, suggesting that the P2X7 receptor-induced permeability increase is not due to pore dilatation but reflects the induction of additional conductances mediated by other proteins secondary to P2X7 receptor activation. As recently reported, such additional conductance may be established by pannexin [35, 36], which can be activated by intracellular Ca2+ ions [37] and membrane stretch [38]. Similarly, single-channel recordings from P2X2 [33] and P2X4 receptors [24] do not indicate any pore dilatation which is assumed to occur in whole-cell preparation [39\u201341].\nInstead of changing the permeation behavior, extracellular Na+ removal was found to drastically change the P2X7 ion channel kinetics [30]. The open probability, the mean open time as well as the activation and deactivation time constants considerably increased when Na+ was replaced by other monovalent cations. This effect was highly specific to Na+, suggesting that Na+ is not only a permeating ion but also a modulator of P2X7 receptor gating. The fact that the P2X7 receptor-induced increase of the cell membrane permeability to organic cations is promoted in Na+-free media [42, 43] can therefore be explained by an enhanced P2X7 receptor activation. This results in an increased Na+ and Ca2+ influx that may activate further permeation pathways.\nIt turned out that the kinetic model for P2X7 receptors [29] could easily explain the potentiating effect of Na+ removal, assuming that binding of Na+ to an extracellular site reduces the rate constant from O4 to C3 from 200\u00a0s\u22121 to 8\u00a0s\u22121. Although this is an allosteric effect not interfering with ATP binding, the concentration-response curve is shifted leftwards by Na+ removal, giving rise to a reduced apparent ATP binding constant. Such an apparent increase of a binding site affinity due to an increased efficacy has already been described by Colquhoun [44]. In this respect, single-channel recording has again proven to be the (possibly only) method to quantitatively measure and distinguish effects on single proteins of drug binding and the following conformational changes considered as efficacy. The model explains on the molecular level the known stimulating effect of Na+ substitution on effects mediated by P2X7 or P2X7-like native receptors [11].\nUnder conditions of hypoxia or tissue injury, ATP and K+ are secreted into the extracellular space [11]. Therefore, in metabolically compromised tissues, a replacement of extracellular Na+ by K+ may enhance the efficacy and potency of ATP on P2X7 receptors. Furthermore, ATP-mediated cell depolarization increases the driving force for K+ efflux and lowers the EC50 value of Na+ since the site seems to be located in the electrical field of the membrane [30]. In this way, K+ efflux may reinforce the ATP effect on P2X7 receptor-expressing cells, and even low concentrations of ATP may activate the P2X7 receptor to a substantial extent. However, whether such a mechanism is of relevance in vivo remains to be established.\nIn the future, the combination of single-channel recordings and molecular engineering methods will reveal more details concerning the function of P2X receptor-dependent ion channels. To this end, it may help to dissect and understand early and late steps of the signaling cascades which are started with the binding of ATP and are followed by biological processes known to be influenced by P2X receptors like contraction, secretion, perception, proliferation, and apoptosis [45]. Furthermore, patch clamp recording will continue to describe the effects of P2X receptor modulators on the single-channel level (for review of the investigations already performed, see North [11]).","keyphrases":["kinetics","p2x4","p2x7","model","single channel","purinergic receptor","voltage clamp"],"prmu":["P","P","P","P","P","M","M"]}
{"id":"Neurochem_Int-2-1-2194163","title":"Control of excitatory synaptic transmission by capsaicin is unaltered in TRPV1 vanilloid receptor knockout mice\n","text":"Several studies have shown that capsaicin could effectively regulate excitatory synaptic transmission in the central nervous system, but the assumption that this effect is mediated by TRPV1 vanilloid receptors (TRPV1Rs) has not been tested directly. To provide direct evidence, we compared the effect of capsaicin on excitatory synapses in wild type mice and TRPV1R knockouts. Using whole-cell patch-clamp techniques, excitatory postsynaptic currents (EPSCs) were recorded in granule cells of the dentate gyrus. First, we investigated the effect of capsaicin on EPSCs evoked by focal stimulation of fibers in the stratum moleculare. Bath application of 10 \u03bcM capsaicin reduced the amplitude of evoked EPSCs both in wild type and TRPV1R knockout animals to a similar extent. Treatment of the slices with the TRPV1R antagonist capsazepine (10 \u03bcM) alone, or together with the agonist capsaicin, also caused a decrease in the EPSC amplitude both in wild type and TRPV1R knockout animals. Both drugs appeared to affect the efficacy of excitatory synapses at presynaptic sites, since a significant increase was observed in paired-pulse ratio of EPSC amplitude after drug treatment. Next we examined the effect of capsaicin on spontaneously occurring EPSCs. This prototypic vanilloid ligand increased the frequency of events without changing their amplitude in wild type mice. Similar enhancement in the frequency without altering the amplitude of spontaneous EPSCs was observed in TRPV1R knockout mice.\n1\nIntroduction\nCapsaicin, the pungent ingredient of red peppers, severely affects pain sensation, inflammation or hyperalgesia. Systematic studies aiming to elucidate the effects of capsaicin revealed that this alkaloid primarily targets sensory fibers of the C type, where it activates a member of the transient receptor potential (TRP) channels, TRPV1 vanilloid receptors (TRPV1Rs) (Szallasi and Blumberg, 1999; Szolcsanyi, 2004). These receptors, cloned by Caterina et al. (1997), are non-selective cation channels gated by heat, low pH or endogenous ligands, such as anandamide (Tominaga et al., 1998; Zygmunt et al., 1999; Caterina et al., 1999; Smart et al., 2000). In addition, capsaicin could also affect the operation of both voltage-gated sodium and calcium channels (Balla et al., 2001; Lundbaek et al., 2005; Kofalvi et al., 2006), indicating that some of the capsaicin effects might not be linked to TRPV1Rs.\nIn contrast to the well-established function of TRPV1Rs in the periphery, its role is much less obvious in the central nervous system. Using autoradiography or immunohistochemistry, TRPV1Rs were shown to be present in several brain regions, including cortical structures (Acs et al., 1996; Mezey et al., 2000; Roberts et al., 2004; Toth et al., 2005; Cristino et al., 2006). Importantly, the specificity of signals in two of these reports has been confirmed in TRPV1R knockout mice (Roberts et al., 2004; Cristino et al., 2006), strongly arguing for the existence of TRPV1Rs in the CNS, yet their subcellular (synaptic or extrasynaptic) localization remains to be determined by high resolution electron microscopy. The functional role of TRPV1Rs in distinct brain regions was addressed by electrophysiological experiments. In the hippocampus, for example, an increase in paired-pulse depression of field potentials after application of capsaicin or anandamide has been noticed, and this effect was found to be sensitive to TRPV1R antagonists (Al-Hayani et al., 2001; Huang et al., 2002). In addition to the investigation of capsaicin effects on field potentials, other studies have examined the activation of putative TRPV1Rs on synaptic transmission more directly. These studies have found that in distinct parts of the brain, glutamatergic, but not GABAegic synaptic communication could be controlled by capsaicin, an effect that was also antagonized by TRPV1R antagonists (Sasamura et al., 1998; H\u00e1jos and Freund, 2002; Marinelli et al., 2002, 2003; Xing and Li, 2007). Interestingly, excitatory postsynaptic currents (EPSCs) evoked by electrical stimulation were found to be depressed after application of capsaicin, whereas the same treatment significantly increased the occurrence of spontaneous EPSCs without affecting their amplitude.\nTo reveal whether the effect of capsaicin on synaptic glutamate release is indeed mediated by TRPV1Rs, we investigated the properties of EPSCs in dentate granule cells after bath application of this prototypic vanilloid ligand both in wild type and TRPV1R knockout mice.\n2\nExperimental procedures\nExperiments were carried out according to the guidelines of the institutional ethical code and the Hungarian Act of Animal Care and Experimentation (1998. XXVIII. section 243\/1998). Wild type and TRPV1R knockout mice of both sexes (20\u201377 days old, C57BL\/6J strain) (Davis et al., 2000) were used. Mouse genotyping was performed on tail DNA. Neo PCR primer sequences were the following: NeoF 5\u2032-CCGGCCGCTTGGGTGGAGAGG and NeoR 5\u2032-products on 300\u00a0bp (targeted allele), and TRPV1Rs (VRF1 5\u2032-CATGGCCAGTGAGAACACCATGG and VRR2 5\u2032-AGCCTTTTGTTCTTGGCTTCTCCT) products on 150\u00a0bp (wild type allele). Amplification reactions were carried out in 25\u00a0\u03bcl total volume with presence of 1% dimethyl-formamide, 0.2\u00a0\u03bcM primer each, 0.2\u00a0\u03bcM dNTP, 1.5\u00a0mM MgCl2 (93\u00a0\u00b0C for 15\u00a0s, 58\u00a0\u00b0C for 15\u00a0s, 72\u00a0\u00b0C for 45\u00a0s, for 30 cycles). Amplification products were analyzed by agarose gel electrophoresis on 1.2% agarose gels. An example for the results of genotyping of a litter is shown in Fig. 1.\nThe animals were deeply anaesthetized with isoflurane followed by decapitation. After opening the skull, the brain was quickly removed and immersed into ice-cold cutting solution containing (in mM: NaCl 126, KCl 2.5, NaHCO3 26, CaCl2 0.5, MgCl2 5, NaH2PO4 1.25, glucose 10) bubbled with 95% O2\/5% CO2 (carbogen gas). Thick horizontal slices (300\u2013350\u00a0\u03bcm from mice) were prepared using a Leica VT1000S Vibratome. The slices were stored in an interface type chamber containing ACSF (in mM: 126 NaCl, 2.5 KCl, 26 NaHCO3, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, and 10 glucose) at room temperature for at least 1\u00a0h before recording.\nWhole-cell patch-clamp recordings were obtained at 34\u201336\u00a0\u00b0C from granule cells in the dentate gyrus visualized by infrared videomicroscopy (Versascope, Marton Electronics, Canoga Park, CA). Patch electrodes were pulled from borosilicate glass capillaries with an inner filament (1.5\u00a0mm o.d., 1.12\u00a0mm i.d.; Hilgenberg, Germany) using a Sutter P-87 puller. Electrodes (\u223c3\u20136\u00a0M\u03a9) were filled with a solution containing (in mM) 80 CsCl, 60 Cs-gluconate, 3 NaCl, 1 MgCl2, 10 HEPES, 2 Mg-ATP, and 5 QX-314 (pH 7.2\u20137.3 adjusted with CsOH; osmolarity 275\u2013290\u00a0mOsm). Excitatory postsynaptic currents (EPSCs) were recorded at a holding potential of \u221265\u00a0mV. Slices were perfused with ACSF containing 70\u2013100\u00a0\u03bcM picrotoxin to block inhibitory neurotransmission. The solution was bubbled with carbogen gas at room temperature and perfused at a flow rate of 2\u20133\u00a0ml\/min in a submerged type chamber. To evoke EPSCs, the stimulating electrode was placed in the stratum moleculare of the dentate gyrus. Pairs of electrical stimuli separated by 50\u00a0ms were delivered via a theta glass pipette (Sutter Instrument Company, Novato, CA) filled with ACSF at 0.1\u00a0Hz using a Supertech timer and isolator (Supertech Ltd., P\u00e9cs, Hungary, http:\/\/www.superte.ch). Access resistances (between 4 and 18\u00a0M\u03a9, compensated 65\u201370%) were frequently monitored and remained constant (\u00b120%) during the period of analysis. Signals were recorded with an Axopatch 200B (Molecular Devices, Sunnyvale, CA), filtered at 2\u00a0kHz, digitized at 6\u00a0kHz (National Instruments PCI-6024E A\/D board, Austin, TX), and analyzed off-line with the EVAN program (courtesy of Prof. I. Mody, UCLA, CA).\nThe drugs were perfused until the maximal effect was reached (usually 3\u20134\u00a0min). The effect of drugs on evoked EPSCs was calculated as follows: control EPSC amplitudes in a 2\u20133\u00a0min time window were compared to those measured after 5\u20136\u00a0min drug application for the same period of time. Only those experiments were included that had stable amplitudes at least for 10\u00a0min before drug application. The paired-pulse ratio was calculated from the mean amplitude of the second EPSCs divided by the mean amplitude of the first EPSCs. For spontaneously occurring EPSCs, the amplitude and the inter-event interval for individual events were calculated and medians of their distributions were compared before and after 5\u00a0min of capsaicin application. After each experiment, the tubing made of Teflon was washed with ethanol for 10\u00a0min and with ACSF for 15\u00a0min. For comparison of data, Wilcoxon matched pairs test or Mann\u2013Whitney U-test were used in STATISTICA 6.1 (Statsoft, Inc., Tulsa, OK). Data are presented as mean \u00b1S.E.M.\nPicrotoxin was purchased from Sigma\u2013Aldrich, while (E)-capsaicin and capsazepine were obtained from Tocris. Both drugs were dissolved in DMSO giving a 100\u00a0mM stock solution, which were stored at 4\u00a0\u00b0C.\n3\nResults\nThe effects of the prototypic TRPV1R agonist capsaicin on EPSCs evoked by focal stimulation of fibers in the stratum moleculare were measured in dentate granule cells of wild type mice and TRPV1R knockouts. Similar to what we found earlier (H\u00e1jos and Freund, 2002), bath application of 10\u00a0\u03bcM capsaicin significantly reduced the amplitude of EPSCs (by 36.6\u00a0\u00b1\u00a06.1% of control) in wild type mice (control: 143.8\u00a0\u00b1\u00a019.3\u00a0pA; capsaicin: 88.6\u00a0\u00b1\u00a012.1\u00a0pA; n\u00a0=\u00a06; p\u00a0<\u00a00.02; Fig. 2A and B). In TRPV1R knockout mice, a similar significant reduction was observed after capsaicin application, the amplitude of EPSCs was suppressed by 31.6\u00a0\u00b1\u00a04.1% of control (control: 146.9\u00a0\u00b1\u00a034.2\u00a0pA; capsaicin: 103.8\u00a0\u00b1\u00a026.9\u00a0pA; n\u00a0=\u00a06; p\u00a0<\u00a00.02; Fig. 2A and B). The inhibitory effect of capsaicin on the amplitude of EPSCs was indistinguishable in wild type mice and TRPV1R knockouts (p\u00a0>\u00a00.1).\nNext, we tested the effect of 10\u00a0\u03bcM capsazepine, a TRPV1R antagonist, on excitatory synapses. We found that bath application of this drug also significantly reduced the EPSC amplitude (by 34.9\u00a0\u00b1\u00a05.4% of control) in wild type animals (control: 165.1\u00a0\u00b1\u00a035.5\u00a0pA, capsazepine: 112.4\u00a0\u00b1\u00a028.2\u00a0pA; n\u00a0=\u00a05; p\u00a0<\u00a00.05; Fig. 3A and B). When we co-applied 10\u00a0\u03bcM capsaicin together with 10\u00a0\u03bcM capsazepine, the amplitude of evoked EPSCs was similarly decreased (by 33.3\u00a0\u00b1\u00a09.4% of control; control: 149.5\u00a0\u00b1\u00a021.2\u00a0pA; capsaicin\u00a0+\u00a0capsazepine: 99.6\u00a0\u00b1\u00a019.6\u00a0pA; n\u00a0=\u00a04; p\u00a0<\u00a00.05). In TRPV1R knockouts, suppression of the EPSC amplitude was comparable to that seen in wild type mice (i.e., by 30.2\u00a0\u00b1\u00a01.5% of control after capsazepine treatment; control: 205.2\u00a0\u00b1\u00a07.3\u00a0pA; capsazepine: 143.2\u00a0\u00b1\u00a06.4\u00a0pA; n\u00a0=\u00a04; p\u00a0<\u00a00.05; Fig. 3A and B). Similarly, the treatment of slices with a mixture of capsaicin and capsazepine reduced the EPSC amplitude by 38.1\u00a0\u00b1\u00a06.2% of control (control: 211.8\u00a0\u00b1\u00a04.4\u00a0pA, capsaicin\u00a0+\u00a0capsazepine: 131.3\u00a0\u00b1\u00a013.5\u00a0pA; n\u00a0=\u00a03; p\u00a0<\u00a00.05), just like in the wild types. These results suggest that capsazepine, as well as capsaicin alone can reduce the amplitude of EPSCs independent of TRPV1Rs, and their effects are not additive.\nBy a comparison of the paired-pulse ratios of evoked EPSCs, we next examined whether capsaicin and capsazepine affect excitatory synapses presynaptically or postsynaptically. If glutamate release is altered, then the paired-pulse ratio should change. If the reduction in EPSC amplitude is not accompanied by changes in the paired-pulse ratio, then the conductivity of glutamate receptors should be modified by the drug treatment. Therefore, we first investigated the effect of capsaicin on paired-pulse ratio in wild type mice and TRPV1R knockouts. After drug application, the ratio significantly increased to 121.3\u00a0\u00b1\u00a03.2% of control in wild types and to 132.5\u00a0\u00b1\u00a09.9% of control in knockouts (n\u00a0=\u00a06 each, p\u00a0<\u00a00.02). Comparable to these findings, capsazepine treatment also caused a significant increase in the paired-pulse ratio both in wild types (129.7\u00a0\u00b1\u00a010.1%; n\u00a0=\u00a05; p\u00a0<\u00a00.05) and knockouts (126.4\u00a0\u00b1\u00a08.8; n\u00a0=\u00a04; p\u00a0<\u00a00.05). Thus, the effects of both capsaicin and capsazepine appear to be presynaptic, reducing glutamate release from excitatory terminals both in wild type and TRPV1R knockout mice.\nIn further experiments, we investigated how capsaicin alters the properties of spontaneous EPSCs (sEPSCs). In wild type mice, bath application of capsaicin significantly increased the occurrence of spontaneous events (i.e., reduced the inter-event interval by 32.5\u00a0\u00b1\u00a011.8% of control, control: 0.43\u00a0\u00b1\u00a00.23\u00a0s; capsaicin: 0.2\u00a0\u00b1\u00a00.05\u00a0s; n\u00a0=\u00a05; p\u00a0<\u00a00.04, Fig. 4A and B) without changing their amplitude (control: 16.6\u00a0\u00b1\u00a02.1\u00a0pA; capsaicin: 16.1\u00a0\u00b1\u00a02.7\u00a0pA; n\u00a0=\u00a05; p\u00a0>\u00a00.1; Fig. 4A and B). Similarly, capsaicin also elevated the frequency of sEPSCs in TRPV1R knockouts, since the inter-event interval was reduced by 29.3\u00a0\u00b1\u00a07.4% of control (control: 0.16\u00a0\u00b1\u00a00.04\u00a0s; capsaicin: 0.12\u00a0\u00b1\u00a00.04\u00a0s; n\u00a0=\u00a06; p\u00a0<\u00a00.02; Fig. 4A and B). Similar to those observed in wild types, the amplitude of synaptic events did not change (control: 19.2\u00a0\u00b1\u00a03.6\u00a0pA; capsaicin: 17.7\u00a0\u00b1\u00a03.3\u00a0pA; n\u00a0=\u00a06; p\u00a0>\u00a00.1; Fig. 4A and B). The comparison of the decrease in the inter-event interval of sEPSCs between wild type mice and TRPV1R knockouts showed no difference (p\u00a0>\u00a00.1). These results provided further evidence that capsaicin affected synaptic glutamate release in wild type and TRPV1R knockout mice to a similar degree, in a similar manner.\n4\nDiscussion\nElectrophysiological data presented here strongly suggest that capsaicin actions on excitatory synaptic transmission are not mediated by TRPV1Rs, at least in the dentate gyrus. Our previous observations already raised this possibility (H\u00e1jos and Freund, 2002), as we have shown that the suppression of the amplitude of EPSCs after the second application of capsaicin was indistinguishable from that seen after the first application. This observation was not consistent with the known desensitization properties of TRPV1Rs upon repeated capsaicin application (Dray et al., 1989; Docherty et al., 1991; Caterina et al., 1997). Our results seem to contradict those pharmacological data, where the effect of capsaicin on synaptic transmission has been found to be fully blocked by antagonists specific for TRPV1Rs (e.g., capsazepine or iodo-resiniferatoxin) (Al-Hayani et al., 2001; Marinelli et al., 2003). Here we found that, in adult mice, capsazepine also effectively reduced the amplitude of EPSCs, similar to that seen after capsaicin application. In line with these data, a study by Kofalvi et al. (2003) has shown that glutamate release from synaptosomes prepared from the hippocampus of adult rats could be significantly suppressed by capsazepine. These findings are in contrast with our published results (H\u00e1jos and Freund, 2002), where capsazepine could antagonize the effect of capsaicin on EPSC amplitude recorded in slices from rats of P15-22. We repeated the experiments with capsazepine in young rats and found that this drug on its own could substantially enhance the amplitude of EPSCs (unpublished observations), which is in sharp contrast observed in adult animals (present study; Kofalvi et al., 2003). Thus, it seems likely that during development the molecular target of capsazepine changes its effect on synaptic transmission, or the binding site(s) of capsazepine might alter. We therefore propose that, in the hippocampus of young rats, the suppression of EPSC amplitude by capsaicin is counterbalanced by the enhancement caused by capsazepine, therefore no reduction in glutamate release can be observed.\nAs to the presynaptic mechanism of capsaicin actions, a recent study showed that iodo-resiniferatoxin as well as capsaicin (both applied in \u03bcM concentrations) could markedly reduce the high K+-induced Ca2+ entry (Kofalvi et al., 2006). Since transmitter release is highly sensitive to Ca2+ entry, one might assume that glutamate release from excitatory terminals in the dentate gyrus could be affected with a similar mechanism. In the present study we found that capsaicin, capsazepine, or co-application of the two, reduce the amplitude of EPSCs to a similar extent, suggesting that capsazepine might also decrease Ca2+ entry at the same site, where capsaicin acts.\nData from other laboratories, as well as our own results, showed that capsaicin could reduce the amplitude of evoked EPSCs, while it increased the frequency of spontaneous EPSCs without changing their amplitude (H\u00e1jos and Freund, 2002; Marinelli et al., 2002, 2003). These unconventional effects seem to suggest that the target molecule of capsaicin regulating glutamatergic transmission could be located at the presynaptic axon terminals, an assumption that is also supported by the observed increase in the paired-pulse ratio (present study). If capsaicin modulated postsynaptic glutamate receptors, the amplitude of sEPSCs should have also been altered, which was not the case (present study; Marinelli et al., 2003). The question arises how capsaicin could reduce the amplitude of evoked EPSCs, while in the same time increases the frequency of sEPSCs. One explanation might be that if capsaicin triggers release of retrograde messengers from postsynaptic neurons that directly promote fusion of vesicles irrespective of Ca2+ concentration within the terminals, this could enhance the action potential-independent glutamate release (i.e., the majority of sEPSCs under our circumstances). Although TRPV1Rs have been shown to be located in the dendrites and somata of hippocampal principal cells (Cristino et al., 2006), these receptors are unlikely to be involved in the enhancement of sEPSC frequency, since the same effect was observed in the TRPV1R knockout mice. Further studies should clarify the possible mechanisms underlying the capscaicin-induced changes in excitatory neurotransmission.\nIn summary, our data presented here suggest that TRPV1Rs may not be the target molecules for capsaicin regulating glutamatergic synapses in the hippocampal network, although they might have a function during pathological conditions like fever or osmotic changes in extracellular space (Caterina et al., 1997; Gavva et al., 2007; Liu et al., 2007).","keyphrases":["excitatory synapses","granule cell","dentate gyrus","glutamate","transmitter release","brain slices"],"prmu":["P","P","P","P","P","R"]}
{"id":"Anal_Bioanal_Chem-4-1-2262919","title":"Aptamers as molecular recognition elements for electrical nanobiosensors\n","text":"Recent advances in nanotechnology have enabled the development of nanoscale sensors that outperform conventional biosensors. This review summarizes the nanoscale biosensors that use aptamers as molecular recognition elements. The advantages of aptamers over antibodies as sensors are highlighted. These advantages are especially apparent with electrical sensors such as electrochemical sensors or those using field-effect transistors.\nIntroduction\nWith the completion of the Human Genome Project, we have a better understanding of disease-related biomaterials such as DNA, RNA, proteins and small biological molecules. Such knowledge can impact strongly on human health, because the identification of a disease-specific marker at an early stage of a disease can greatly improve the clinical success rate, and improve disease-related mortality. For early detection of disease, it is necessary to locate minute amounts of disease-related proteins, peptides or other small molecules in a patient\u2019s bloodstream or body fluid. Nanotechnology combined with biotechnology is expected to provide paradigm-breaking solutions in this area, as these technologies are extremely sensitive [1]. Already, nanoscale materials such as quantum dots [2] and metallic nanoparticles [3, 4], as well as nanodevices such as the nanocantilever [5], and nano field-effect transistors (FETs) [6\u20139] show potential for detecting tiny molecular signals.\nIn this review, we concentrate on nanoscale electrical biosensors with aptamers as molecular recognition elements. Biosensors are devices that can be used to detect the presence of a target analyte. As shown in Fig.\u00a01, biosensors are built up from a sample delivery component, a molecular recognition subsystem, a transducer changing the binding or reaction of target molecules into a measurable physical signal (mass, charge, heat or light) and a signal-processing unit. We are particularly interested here in electrical transducers, because they provide the most promising solutions for point-of-care disease detection. Biosensors using electrical transducers are rapid and convenient, and they do not require heavy optical instruments or expensive measuring devices.\nFig.\u00a01A biosensor. MIP molecularly imprinted polymer\nAptamers as molecular recognition elements\nFor the highly sensitive detection of a target analyte, the selectivity of the recognition element is vital in sensor performance. Until recently, immunosensors using antibody\u2013antigen recognition were employed for biodetection. However, highly sensitive and stable biosensors may use different recognition elements. In the 1980s, functional nucleic acids that can specifically bind and regulate viral or celluar proteins with high affinity were discovered [10]. The major breakthrough for functional nucleic acids occurred in 1990. First, the presence of small functional RNA molecules (TAR aptamers) that can inhibit viral replication [11] was revealed; they can effectively bind and inhibit the activity of viral protein, thereby raising the possibility that they may be used as therapeutic agents. In the second study in 1990, Tuerk and Gold [12] established the in vitro screening process termed \"systematic evolution of ligand by exponential enrichment\" (SELEX), to identify the RNA ligands with affinity for T4 DNA polymerase and various organic dyes [12]. Such nucleic acids were termed \"aptamers\" [from the Latin aptus (fit or appropriate)] by Ellington and Szostak [13].\nThe SELEX process is a technique for screening a very large library of oligonucleotides with random sequences by iterative cycles of selection and amplification. Figure\u00a02 shows a schematic diagram of the SELEX process [14]. The starting pool must be large enough to generate a high probability of producing a desired aptamer (normally around 1015 different sequences). The initial library of random-sequence oligonucleotides, which is obtained through combinatorial chemical synthesis, is incubated with a target of interest. Oligonucleotides showing affinity for the target are partitioned by affinity chromatography or filtration, and then amplified by PCR (for DNA libraries) or reverse transcription PCR (for RNA libraries) to create a new pool enriched in those oligonucleotides having a higher affinity for the target. As this cycle of selection and amplification is repeated, the abundance of the high-affinity oligonucleotides increases exponentially. Negative selection and counterselection are often employed in order to remove aptamers which bind to supports and molecules similar to the target, respectively. Typically after eight to 15 cycles, cloning and sequencing of the enriched library are carried out, revealing the sequence of oligonucleotides highly specific to the target. Once the sequence information has been obtained, the desired aptamer can be readily produced by chemical synthesis. For a given target molecule, DNA aptamers and RNA aptamers can be generated. However, they are quite different in sequence and three-dimensional structure since DNA lacks the 2\u2032 hydroxyl group of the RNA. For example, both the DNA version of the RNA aptamer for ATP and the RNA version of the DNA aptamer for ATP do not recognize ATP [15].\nFig.\u00a02The systematic evolution of ligand by exponential enrichment (SELEX) process [14]\nIn Table\u00a01, we summarize the advantages of aptamers over antibodies and the yellow-colored rows detail special advantages of aptamers as recognition elements [16]. First, aptamers possess high target affinity. It has been generally recognized that aptamer affinity is comparable to or even higher than that of antibodies, even though this is not always true. Unlike antibodies, aptamers can distinguish between targets even if the targets are in the same family. For example, anti-caffeine aptamers have lower affinity (by a factor of 104) for theophylline, despite the two compounds differing by only a single methyl group [17]. Because aptamers are selected using an in vitro selection process (SELEX), they are cost-effective, have uniform activity, and antitoxin aptamers can be readily synthesized. Antitoxin antibodies are more difficult to prepare, since they are cultured in animal cells. The immobilization of the recognition element can be crucial in biosensors, and this is easier with aptamers than with antibodies, because chemical modification of nucleic acids is simple and straightforward compared with that of antibodies. One of the biggest advantages of aptamers lies in their high stability. Being nucleic acids, aptamers are highly stable, and therefore have unlimited shelf lives. Also, because aptamers can undergo reversible changes of conformation with variations in temperature or salt concentration, aptamer-based sensors are potentially recyclable. The major limitation of aptamers (especially RNA aptamers) as molecular recognition elements is degradation by nucleases. However, it was shown that the problem can be overcome through chemical modifications of the sugar at the 2\u2032-position [18] or use of mirror-image analogs [19]. Recently chemically modified nucleic acid aptamers for in vitro selections have been reviewed [20].\nTable\u00a01Advantages of aptamers over antibodies [16]\nAs aptamers show high target affinity and high target specificity, they can be used as therapeutic agents, in affinity columns and as recognition elements in sensors detecting a wide range of targets. There are virtually no limits of targets for aptamers; small molecules [14], proteins [15] and antibiotics [21]. The first use of an aptamer as a molecular recognition element in sensors was reported in 1996, when fluorescent-tagged aptamers were employed for optical detection of human neutrophil elastase [22]. Since then, various transducers employing aptamers for recognition have been reported. These include the quartz crystal microbalance [23], the surface plasmon resonance technique [24], evanescent-wave-induced fluorescence [25], microcantilevers [26], electrochemical sensors [27\u201335] and FETs fabricated with single-walled carbon nanotubes (SWNTs) [36, 37]. In this review, we summarize recent advances in electrochemical and FET types of aptamer sensors, focusing on the high sensitivity and recyclability of the aptamer-containing devices.\nElectrical aptamer sensors\nElectrochemical aptamer sensors\nTypical electrochemical sensors operate by reacting with an analyte of interest and producing an electrical signal proportional to the analyte concentration. Many papers have reported the fabrication of electrochemical aptamer sensors where specific binding of an analyte to an aptamer on an electrode can produce an electrical signal [27\u201335]. Some authors use sandwich assays, employing a secondary aptamer for signal enhancement [28, 29]. However, a technique that is label-free and fast in response is favored especially for electrical types of biosensors. A difficulty with sandwich assays has recently been removed by Xiao et al. [31], who developed a \u201csignal-on\u201d architecture. Earlier electrochemical aptamer sensors used a \u201csignal-off\u201d architecture [30], in which the binding of the target analyte resulted in a decrease in the signal. In such cases, no more than 100% of the signal can be suppressed by target binding, and hence the dynamic range of the sensor is limited. Also, it is difficult to distinguish a real binding event from a false signal originating from contaminants or sensor degradation. To fabricate the signal-on electrochemical aptamer sensor, Xiao et al. introduced a short DNA sequence tagged with methylene blue (MB) that can hybridize with an aptamer. A single-stranded DNA aptamer, which binds to thrombin, was chosen as a model system. Thrombin is a proteolytic enzyme which facilitates blood clotting by converting fibrinogen into fibrin and is also regarded as a tumor marker in the diagnosis of pulmonary metastasis [38]. Figure\u00a03 shows a schematic diagram of the signal-on aptamer sensor. As shown, the sensor is constructed by immobilizing a thiolated thrombin aptamer to a gold electrode, and an MB-tagged partially complementary DNA is added to form a double-stranded complex. In this configuration, owing to the large persistence length of double-stranded DNA, the MB moiety is far away from the sensor surface, and only a small Faradaic current is observed. The persistence length, defined as the distance over which the direction of a polymer segment persists, provides a measure of elasticity in polymer chains. In the case of double-stranded DNA, the persistence length is about 50\u00a0nm, or 150\u00a0bp [39]. Since the thrombin aptamer is much shorter than 150\u00a0bp [40] (the recognition part is only 15\u00a0bp in length), the complex would be in a \u201cstanding\u201d position. When thrombin is introduced to such a complex, the thrombin aptamer binds to thrombin, and the MB-tagged single-stranded DNA approaches the electrode surface, improving the electron transfer with an increase of current. The persistence length of a single-stranded DNA is 1.5\u00a0nm in 2\u00a0M NaCl solution, and 3\u00a0nm in 25\u00a0mM NaCl solution [41].\nFig.\u00a03Thrombin binding with an engineered aptamer. MB methylene blue. (Reprinted with permission from [31], copyright 2005 American Chemical Society)\nIn the work of Xiao et al., a threefold signal gain was observed with 260\u00a0nM thrombin, while only a 40% signal decrease was observed using a signal-off sensor. The detection limit of the sensor thus rose to 3\u00a0nM as shown in Fig.\u00a04. Such a high sensitivity is comparable even with that given by sensors using amplification by labeling [28, 29] or optical sensors using evanescent-wave-induced fluorescence anisotropy [25]. Table\u00a02 summarizes the electrochemical aptamer sensors available for thrombin.\nFig.\u00a04Electrochemical signal from the signal-on aptamer sensor. (Reprinted with permission from [31], copyright 2005 American Chemical Society)Table\u00a02The detection limits of an electrochemical aptamer sensor for thrombin\u00a0Detection limitCharacteristicsReference13\u00a0nMUses an MB-tagged \"holding\" DNA sequence[31]280\u00a0nM\/3.5\u00a0nMPeroxidase-labeled thrombin gives an 80 nM detection limit. Biotin-labeled aptamer with horse radish peroxidase labeled streptavidin gives a detection limit of 3.5 nM[27]31\u00a0\u03bcMSandwich assay using two aptamers with different binding sites. Secondary aptamer labeled with glucose dehydrogenase[28]410\u00a0nMSame as for 3, but pyrroquinoline quinine glucose dehydrogenase is used for labeling[29]56.4\u00a0nMMB-tagged thrombin aptamer (signal-off sensor)[30]60.1\u20130.15\u00a0nMThrombin aptamer with ferrocene moiety (square-wave voltametry or chronopotetiometry used for the measurement)[32]711\u00a0nMMB-intercalated thrombin aptamer[25]MB methylene blue\nOwing to the unique three-dimensional structure of an aptamer, it is not even necessary to use \"holding\" DNA sequences. Baker et al. [34] reported the rapid, label-free detection of cocaine in both adulterated samples and biological fluid. The cocaine aptamer engineered by Stojanovic et al. [42] was used in this work. This aptamer is thought to form a partially unfolded structure, with only one of its three double-stranded stems intact, when target molecules are absent. In the presence of target, the aptamer folds into the cocaine-binding three-way junction structure of Fig.\u00a05a. With this unique approach, Baker et al. reported the detection of 500\u00a0\u03bcM cocaine in biological fluid, even when contaminants were present. The ability to detect cocaine in undiluted biological fluids (blood, for example) is crucial for simple forensic detection technology. The binding of small molecules to an aptamer is highly reversible, so it is possible to regenerate the sensor. Baker et al. [34] could regenerate the sensor to a degree of 99% by simply washing a used sensor with buffer.\nFig.\u00a05a Binding of coccaine with an MB-tagged aptamer. b Binding of platelet-derived growth factor (PDGF) with an MB-tagged aptamer. (a Reprinted with permission from [34], copyright 2006 American Chemical Society; b reprinted with permission from [35], copyright 2007 American Chemical Society)\nRegeneration of an aptamer sensor is also possible after protein binding. Lai et al. [35] reported the detection of picomolar levels of platelet-derived growth factor (PDGF) directly in blood serum. PDGF is a protein that regulates cell growth and division, and is very important for blood vessel formation (angiogenesis). While generally undetectable in normal cells, PDGF is overexpressed in a variety of disease such as atherosclerosis, and human tumors including sarcomas and glioblastomas [14, 43]. As shown in Fig.\u00a05b, specific binding of PDGF to the aptamer decreases the distance between MB and the electrode and consequently increases the electrical signal. The sensor could be also regenerated by 4-min incubation in 10% (w\/v) sodium dodecyl sulfate.\nIn this section, we have shown that engineered aptamers, employed as recognition elements in electrochemical transducers, can greatly improve sensor performance. It is an advantage of aptamers over antibodies that aptamers can be engineered to either enhance or activate signals. Recently, aptazymes (aptamers with catalytic activities) have been reported, which combine an enzymatic role with a recognition function [44]. Carefully designed aptazymes can be developed as sensors that do not require labeling. An electronic (electrochemical) sensor approach with carefully designed aptamers offer potential for highly sensitive, selective and reusable sensor platforms.\nAptamer sensors with carbon nanotube field-effect transistor transducers\nSince the first report on the biosensing ability of Si nanowire in 2001 [45] and the development of the chemical sensor operation of carbon nanotubes in 2000 [46], extensive research efforts have been initiated aimed at developing nanotube-based or nanowire-based sensors. Owing to the extremely large surface-to-volume ratio of one-dimensional nanostructures, it is possible to develop sensors with exquisite sensitivity. This is especially true in the case of carbon nanotubes, where all constituent atoms are at the surface. Any small disturbance of or adsorption to the surface could result in a large change in electrical conductance. Already, biosensors fabricated with semiconducting nanowires or nanotubes have been used as sensors that can detect a single virus [6], small molecules [47] or minute amounts of proteins in solution [7\u20139].\nFET sensors measure a change in the surface potential occurring during the binding between an analyte and a recognition element. Figure\u00a06 shows a schematic diagram of the working principle of carbon-nanotube-based FET sensors. FET sensors \u201cfeel\u201d the charges when target molecules bind with probe molecules immobilized on the sensor surfaces. Such an effect is often referred to as an \u201celectrostatic gating effect\u201d since the bound target molecules behave as an additional gate electrode. In the case of carbon nanotubes, SWNTs behave as p-type semiconductors in ambient atmosphere. In p-type FETs, positive gate voltage decreases the conductance (depletion) and negative gate voltages increase the conductance (accumulation) from the device. Therefore, if positively charged molecules bind with probe molecules on SWNT-FETs, this event appears as a decrease of conductance, while binding of negatively charged molecules appears as an increase of conductance in SWNT-FETs as denoted in Fig.\u00a06.\nFig.\u00a06The working principle of field-effect transistor (FET) sensors based on carbon nanotubes\nUsing FETs for immunosensing was first proposed by Shenk [48] in 1978. It was suggested that FETs could be used to detect surface polarization owing to the formation of an antibody and an antigen. This concept failed, however, because small ions in the solution caused a screening effect inhibiting detection of the changes [49]. Figure\u00a07 shows the charged semiconductor surface in solution. As shown in Fig.\u00a07, when a charged surface (this might be a metal electrode, a semiconductor or a charged dielectric) is brought into contact with an ionic solution, the surface charge potential is dissipated by redistribution of counter ions in the solution. The domains where the variation of electric potential occurs are called electrical double layers, and the depth of an electrical double layer can be determined by calculating the Debye screening length. The Debye screening length is the scale over which mobile charge carriers screen the electric field. In an electrolyte, the Debye screening length can be defined as follows:\nFig.\u00a07Electrical double layer at the sensor surface\nHere the Bjerrum length lB is defined as\nand is 0.7\u00a0nm at standard pressure and temperature. kB is the Boltzmann constant, k is defined as 1\/4\u03c0\u025b0, \u03c1i is the number density of ions and the zi values are valencies of various ions.\nThe Debye screening length, therefore, is proportional to the inverse square root of the ion number density, i.e., to ionic strength. For pure water, the Debye screening length is about 1\u00a0\u03bcm, while it is only 0.3\u00a0nm in 1\u00a0M NaCl solutions. In 1\u00a0M NaCl solution, therefore, two ions or charged species separated by 1\u00a0nm do not interact with each other. If we consider the sensor surface, any interactions occurring outside the Debye screening length cannot be detected using FET sensors. The biggest disadvantage of FET sensors is that they may not work with blood samples or body fluids, because the ionic strength of such physiological solutions is about 150\u00a0mM, which yields a Debye screening length of about 1\u00a0nm.\nUntil recently, FET-type sensors have employed an antibody as the recognition receptor. However, the typical size of an antibody is about 10\u201315\u00a0nm [50, 51]. In that case, the binding of an antigen with an antibody occurs far outside the Debye screening length in physiological solutions. Molecular recognition elements with high specificity, selectivity and small sizes are essential for applications of FET-type sensors. In this respect, aptamers are highly suitable. Figure\u00a08 shows the typical size ranges of antibodies and aptamers [52]. Because aptamers are mostly short nucleic acids ranging from 10 to 60\u00a0bp in length, the binding of target molecules with aptamers can occur inside the electrical double layer, even in 20\u201350\u00a0mM salt solutions.\nFig.\u00a08Size comparison of an aptamer with an antibody. IgG immunoglobulin G. (Reprinted from [52], copyright 2006 Elsevier)\nSuch a biggest merit of aptamers in FET sensors was first reported by So et al. [36] using a carbon nanotube biosensor with thrombin aptamers as molecular recognition elements. They also demonstrated the sensitivity and selectivity of aptamers in SWNT-FETs. Anti-thrombin aptamers (5\u2032-GGT TGG TGT GGT TGG-NH2-3\u2032) were immobilized onto the surface of the SWNT-FET, fabricated using a patterned growth technique, with carbonyl diimidazole\u2013Tween 20 as the linking molecules (Fig.\u00a09a). The authors observed an abrupt decrease in conductance upon reaction with thrombin, while almost no changes were observed with elastase (Fig.\u00a09b). Elastase is a member of the thrombin family and has almost the same molecular weight and isoelectric point as thrombin. Moreover, the sensor could be regenerated by simple washing with 6\u00a0M guanidine hydrochloride solution. The authors regenerated the sensor more than five times without significant loss of activity. As shown in Fig.\u00a09c, the authors measured the sensitivities of SWNT-FET sensors with aptamers as recognition elements. Before each measurement, the sensor was \"reset\" by washing it with guanidine hydrochloride solution. The lowest detection limit was around 10\u00a0nM thrombin, and the sensor signal was saturated at about 100\u00a0nM thrombin.\nFig.\u00a09a Single-walled carbon nanotube (SWNT) FET sensor with aptamers as molecular recognition elements. b Selectivity of an anti-thrombin aptamer-functionalized SWNT-FET. c Sensitivity of an anti-thrombin aptamer-functionalized SWNT-FET. The sensor was reset by washing it with 6\u00a0M guanidine hydrochloride solution before each measurement. CDI carbonyl diimidazole. (Reprinted with permission from [36], copyright 2005 American Chemical Society)\nMaehashi et al. [37] have recently addressed the size issue of recognition elements in FET-based sensors. In their work, immunoglobulin E (IgE) aptamer (5\u2032-NH2-GCG CGG GGC ACG TTT ATC CGT CCC TCC TAG TGG CGT GCC CCG CGC-3\u2032) and monoclonal anti-IgE antibody (IgE-mAb) were immobilized on carbon nanotubes, with the aid of the linking molecule 1-pyrenebutanoic acid succinimidyl ester. Figure\u00a010 a and b shows the responses of the aptamer-functionalized and antibody-functionalized sensors, respectively, when they were exposed to known concentrations of target IgE in 10\u00a0mM phosphate-buffered saline (PBS). As shown in Fig.\u00a010, the aptamer-functionalized sensor exhibited small changes with only 250\u00a0pM target protein molecules, while a negligible change in conductance was observed in the antibody-funtionalized sensor even with 140\u00a0nM IgE. In 10\u00a0mM PBS, the Debye screening length would be roughly 3\u00a0nm, and the binding of IgE with the aptamer would be safely inside the electrical double layer. With IgE-mAb, however, there is little hope that binding can be detected in the buffer solution used for the experiment, because the antibody molecule is about 10\u00a0nm long [53].\nFig.\u00a010The sensor signal measured from a an aptamer-functionalized SWNT-FET and b an antibody-functionalized SWNT-FET. IgE immunoglobulin E, PBS phosphate-buffred saline. (Reprinted with permission from [37], copyright 2007 American Chemical Society)\nAs discussed, aptamers may allow a wider range of analytes in FET sensors owing to their small size. SWNT-FET sensors with aptamers as recognition elements showed high sensitivity and selectivity, and could be readily regenerated. Highly sensitive carbon nanotube sensors combined with small, economic, highly selective and stable aptamers could provide cost-effective point-of-care testing devices.\nConcluding remarks\nThis review has focused on the advantages of electrochemical and FET sensor types with aptamers as recognition elements. Through use of aptamers for recognition, no labeling is required for electrochemical sensors, and signal-on architecture that is only possible with aptamers has made possible the sensitivity improvements. Also, as pointed out already, sensors can be recycled because of the reversibility of aptamer configurations. Alternatively, bound proteins may be simply washed off, without damaging the aptamers. In immunosensing, by contrast, it is practically impossible to remove bound antigens from antibodies without damaging the antibodies, because both antibodies and antigens are proteins in nature. Aptamers have proved their superiority over antibodies in nano-FET sensors. When no signal can be measured with an antibody\u2013antigen pair because of the large size of the antibody, the binding of small aptamers to various targets can occur inside the electrical double layer where the nanotube can \"feel\" the change.\nOwing to the rapid aging of society, there is great demand for paradigm-breaking biosensors to detect the onset of disease with speed, convenience and accuracy. In a few more years, electrical nanosensors, with engineered aptamers, will become prominent in the market. They will be small in size, high in sensitivity and competitive in price.","keyphrases":["aptamers","nanobiosensors","electrochemical aptamer sensors","field-effect transistor aptamer sensors"],"prmu":["P","P","P","R"]}
{"id":"Community_Ment_Health_J-2-2-1705498","title":"Mental Health and Substance Abuse Services Preferences among American Indian People of the Northern Midwest\n","text":"ABSTRACT\nINTRODUCTION\nA 2001 supplemental report on mental health to the Surgeon General describes the current United States public health perspective as a \u201cpopulation-based approach (that) is concerned with the health of the entire population, including its link to the physical, psychological, cultural, and social environments in which people live, work, and go to school (Dept of Health and Human Services (DHHS), 2001).\u201d This statement reflects a concept of holistic healing in a time of growing acceptance and utilization of alternative or complimentary health services beyond those available within professional medical systems. Rodenhauser (1994) notes that although psychiatry as a specific medical field shows increasing cultural awareness regarding clinical sensitivity within the academic literature, there has been little to no evidence of cultural factors found within mainstream journals for health administrators. In addition, very little research is available concerning the co-utilization of \u201calternative\u201d and western medical approaches, let\u00a0alone an examination of this overlap within specific cultures (Gurley et\u00a0al., 2001; Rhoades & Rhoades, 2000; but see Novins, Beals, Moore, Spicer, & Manson, 2004). An understanding of these services may be especially important within an American Indian cultural context where traditions often involve complex ceremonies and beliefs that require consideration far beyond that of mainstream religion (Rhoades & Rhoades, 2000).\nThe purpose of this study is to examine factors that influence the choice between traditional cultural and western mental health and substance use associated care among American Indians from reservations in the northern Midwest. Specifically, we examine preference in terms of perceived effectiveness and actual utilization of traditional vs. Western-based service outlets.\nBarriers to Health Services\nAmerican Indians and Alaskan Natives have been found to report highest rates (12.9%) of frequent mental distress among all ethnic groups, including Hispanics (10.3%), African Americans (9.7%), Whites (8.3%), and Asian\/Pacific Islanders (6.1%) (Centers for Disease Control (CDC), 1998). The 2000\u20132001 National Household Survey on Drug Abuse found American Indians\/Alaskan Natives to be second only to multi-ethnic individuals to have received mental health treatment in the past year. At the same time, American Indian\/Alaska Native respondents were second highest among all ethnic groups who had not received mental health care to report perceived unmet service needs (Barker et\u00a0al., 2004). A similar pattern is found for substance abuse where again, American Indians\/Alaskan Natives are second to multi-ethnic persons in terms of prevalence of illicit drug or alcohol dependence (Substance Abuse and Mental Health Services Administration (SAMHSA), 2002).\nA majority of Indian people within the United States receive physical and mental healthcare from Indian Health Services (IHS) (Gone, 2004). IHS\u2019s Indian Health Care System served around 1.6\u00a0million American Indians\/Alaska Natives residing on or near reservations in 2003 (IHS, 2003). Over one-third of service utilization within this system involves mental health and social service related concerns (IHS, 2002). Although Indian Health Services is meant \u201cto raise the physical, mental, social, and spiritual health of American Indiana and Alaska Natives to the highest level\u201d (IHS, 2002), a variety of barriers may impede American Indians\u2019 ability to receive adequate health care. Specific to mental health care delivery, and often overlapping with the characteristics of healthcare in general, Rodenhauser (1994) characterizes barriers into two major groups: (1) those within the current medical system, including under-funding and staffing issues, inconsistent services, insensitivities, fear of litigation and non-compliance with organizational requirements, and staff burnout; and (2) those inherent to American Indian\/Alaska Native cultures, such as fear, high rates of accidents\/violence, intrinsic conditions (i.e. denial, helplessness and hopelessness), and mistrust of government agencies.\nTraditional Help Seeking\nResearch and anecdotal evidence suggests that many American Indians report health related help-seeking via culturally traditional outlets. For example, Marbella, Harris, Diehr, Ignace, and Ignace (1998) surveyed 150 American Indian adult patients at the Milwaukee Indian Health Center and found that 38% had sought help from a traditional healer in addition to physician-administered care. Furthermore, 86% of those interviewed said that they would consider seeking a traditional healer in the future. In instances where traditional healer\u2019s and physician\u2019s advice differed, patients said they would rely on their healer\u2019s advice 61.4% of the time. In a separate study including respondents from 2 different American Indian tribes, Novins, Beals, Moore, Spicer, and Manson (2004) found traditional healing options to be prevalent and important to both tribes, whether used alone or in combination with biomedical services.\nAlthough traditional beliefs and practices can guide some American Indians through mental and physical illnesses and healing, there is disagreement concerning the degree of connection between traditional and professional medical services at an institutional level. A number of researchers discuss a gap wherein the processes of traditional and mainstream professional healing should be linked (Marbella et\u00a0al., 1998; Mohatt & Varvin, 1998; Novins, Beals, Sack, & Manson, 2000; Novins, Fleming, Beals, & Manson, 2000). Conversely, others feel that formal funding for traditional practices is difficult and perhaps inappropriate. Payment to a traditional healer by a patient may be compromised in a situation where billing must occur via a third party. Issues surrounding payment are complicated further considering the need to establish credentials for employees of government funded agencies like IHS (Rhoades & Rhoades, 2000). It is also important to consider the heterogeneity of the over 300 different tribal or language groups from which American Indian individuals belong (Beauvais, 1998). Tribal differences extend to various opinions regarding an integration of the two health care systems (Rhoades & Rhoades, 2000; see also Novins et\u00a0al., 2004).\nDespite some documentation on the preference of traditional vs. western medical treatment among American Indians who receive both, we found little empirical evidence that directly tests the factors that affect this choice (but see Gurley et\u00a0al., 2001; Novins et\u00a0al., 2004; Robin, Chester, Rasmussen, Jaranson, & Goldman, 1997). In addition, there is a lack of empirical research surrounding American Indian mental health\/substance use related services utilization (Manson, 2000), and what work has been done typically focuses on Indian children (i.e. Novins, Harman, Mitchell, & Manson, 1996; Costello, Farmer, Angold, Burns, & Erkanli, 1997; Novins et\u00a0al., 2000). This research begins to address such gaps in the literature by examining predictors of service preferences among a sample of American Indian adults.\nHypotheses\nIn a review of the literature, Manson (2000) includes differences related to culture (i.e. acculturation vs. enculturation) as important predictors of formal mental health service utilization. Elsewhere, higher levels of social support (here measured as community support) have been shown to be related to receiving a treatment recommendation for substance use problems (Novins et\u00a0al., 1996); in turn, being recommended for treatment may increase actual service utilization. In addition, rates of mental health treatment have been found to be higher for adults with a lower perceived health status (Barker et\u00a0al., 2004).\nBased on these findings and coupled with the barriers to care discussed earlier, we hypothesize that higher levels of enculturation, experiencing discrimination, and living on a reservation will be positively related to perceived effectiveness or preferences for informal services and negatively related to formal services. In addition, self-reported physical health is hypothesized to be negatively related to preferences of both service types (Barker et\u00a0al., 2004). We also predict that higher levels of social support will be positively related to use of both informal and formal service outlets. Lastly, we control for the effects of age, gender, education, and employment on service utilization preferences.\nMETHODS\nSample\nThese data were collected as part of the \u201cHealing Pathways Project,\u201d a 3-year lagged sequential study currently underway on four American Indian reservations in the Northern Midwest and five Canadian First Nation reserves. Because of national differences in health care systems, the data presented here includes only that from U.S. reservations. The data are from wave one of the study collected on two U.S. reservations from February through October 2002, and wave one on a second pair of U.S. reservations collected from February through October 2003. The reservations share a common cultural tradition and language with minor regional variations in dialects. The sample represents one the most populous Native cultures in the United States and Canada.\nThe project was designed in partnership with the participating reservations and reserves. Prior to the application funding, the research team was invited to work on these reservations, and tribal resolutions were obtained. As part of this agreement, the researchers promised that participating reservation names would be kept confidential in published reports. An advisory board was appointed by the tribal council at each participating reservation and is responsible for advising on difficult personnel problems, questionnaire development, reading reports for respectful writing, and assuring that published reports protected the identity of the respondents and the culture. Upon advisory board approval of the questionnaires, the study procedures and questionnaires were submitted for review and approved by an Institutional Review Board. All participating staff on the reservations were approved by the advisory board and were either tribal members themselves or non-members who are spouses of tribal members. To ensure quality of data collection, all interviewers underwent special training for conducting pencil-and-paper and computer-assisted personal interviewing for diagnostic measures, including feedback sessions. In addition, all of the interviewers completed a required human subject\u2019s protection training that emphasized the importance of confidentiality and taught procedures to maintain the confidentiality of data.\nEach tribe provided lists of families of enrolled children aged 10\u201312\u00a0years who lived on or proximate to (within 50\u00a0miles) the reservation or reserve. We attempted to contact all families with a target child within the specified age range. Families were recruited via personal visits from Native interviewers during which they were given an explanation of the project, a gift of wild rice, and an invitation to participate. After agreement to participate and later completion of interviews, each participating family member received $40 for their time. This recruitment procedure resulted in an overall response rate of 79.4%.\nSample Characteristics\nThe sample for this analysis is made up individuals from the U.S. reservations only and consisted of 865 parents\/caretakers (264 males and 601 females) of tribally enrolled children aged 10\u201312\u00a0years. Fathers\/male caretakers ranged in age from 21 to 70\u00a0years with an average age of 42\u00a0years; mothers\/female caretakers ranged in age from 17 to 78\u00a0years with an average of 39\u00a0years.\nMeasurement\nTo assess the perceived effectiveness of health service providers, respondents were given a series of questions asking them how effective various service outlets would be if ever there were to have (1) an emotional problem, or (2) a substance abuse problem. The response categories range from not at all effective to extremely effective. Traditional\/informal services include family, talking to an elder, sweat lodge, pipe ceremony, offering tobacco, traditional healer, traditional ceremony, and healing circle. Formal services include Indian Health Service (IHS), doctor, psychologist, social worker, counselor, psychiatrist, chemical dependency counselor, and nurse. Respondents were asked to evaluate the perceived effectiveness of formal services both on and off the reservation. For multivariate analysis a mean effectiveness score was computed for three categories of services: (1) traditional, (2) formal on reservation, and (3) formal off reservation, all within each realm of service need (emotional and substance abuse). Each mean score has a range from 1 to 5, with higher scores indicating greater perceived effectiveness.\nEnculturation is a latent construct assessed by three basic elements: (1) participation in traditional activities, (2) identification with American Indian culture, and (3) traditional spirituality (see Costello et\u00a0al., 1997; Whitbeck, McMorris, Hoyt, Stubben, & LaFromboise, 2002). The enculturation scale has high internal consistency (Cronbach\u2019s \u03b1\u00a0=\u00a0.79), and the standardized version of the scale is used in these analyses.\nPerceived discrimination is measured with an 11-item scale designed to tap a range of potential types and sources of discrimination. Respondents were asked to report how often they had experienced specific instances of discrimination. Those items included in the scale were; how often someone said something derogatory or insulting because of your race; how often a store owner, sales clerk or person working at a business has treated you in a disrespectful way because of your race; how often the police have hassled you because of your race; how often you have been ignored or exclude from an activity because of your race; how often someone has yelled a racial slur or racial insult at you; how often someone threatened physical harm because of your race; how often someone suspected you of doing something wrong because of your race; how often you have been treated unfairly because of your race; how often you have encountered whites who didn\u2019t expect you to do well because of your race; how often someone discouraged you from trying to achieve an important goal because of your race; and how often you have been treated unfairly in the courts because of your race. The four response categories range from never to always, with the mean of all 11 items used in these analyses. The scale has a high internal reliability with Cronbach\u2019s \u03b1 of .90.\nSocial support is measured with a 17-item scale in which respondents were read statements regarding community and neighborly cohesiveness and conflict. Statements include: this is a close knit community; people around here are willing to help their neighbors; there are adults in this community that children can look up to; there are long standing family grudges in the community; the community is split by politics; the community is split by alcohol or drugs; and so on. Respondents were asked to answer true or false to each statement. Items indicative of community conflict were reverse coded, and then all items were summed so that higher values represent higher community support. Cronbach\u2019s \u03b1 for this scale is .86.\nHealth status was assessed by response to the question \u201cIn general, how well would you say your health is?\u201d Response categories ranged from excellent (5) to poor (1). Values are coded so that higher scores indicate better health.\nSeveral control variables are included in the present analysis. Gender is a dummy variable coded as male\u00a0=\u00a00 and female\u00a0=\u00a01. Education is a five category item ranging from less than high school to an advanced degree. Employment is a dichotomous variable indicating if the respondents are employed full-time. Those with full-time employment are coded as 1. To assess the difference between those enrolled tribal members living on and off the reservation respondents were asked if they currently lived on the reservation at the time of their interview. Those who lived off of the reservation were given a value of 1.\nRESULTS\nDescriptive Characteristics\nThe adults who comprised this sample had an average educational level falling between a high school diploma and at least some secondary school experience (mean\u00a0=\u00a02.4; SD\u00a0=\u00a0.87), and more than half (57%) were employed full-time. Very few of the adults lived off of the reservation (12%) at the time of our interviews. The self-reported health status of the adults in our sample averaged between \u2018good\u2019 and \u2018very good\u2019 (mean\u00a0=\u00a03.3; SD\u00a0=\u00a01.0), and the overall level of reported social support in the communities was 8.4 (SD\u00a0=\u00a04.1). Enculturation is a standardized variable in these analyses (mean\u00a0=\u00a00; SD\u00a0=\u00a01), while the mean level of perceived discrimination was 1.5 (SD\u00a0=\u00a0.69), indicating that the average response across all of the discrimination questions fell between \u2018never\u2019 and \u2018a few times.\u2019\nAmong the dependent variables concerning service preferences among respondents, the highest mean scores were found for informal\/traditional services for both mental health (mean\u00a0=\u00a03.06; SD\u00a0=\u00a01.06) and substance use (mean\u00a0=\u00a03.09; SD\u00a0=\u00a01.15) related issues. Next highest were mean preference scores for on-reservation formal services (mental health\u00a0=\u00a02.51; SD\u00a0=\u00a01.01; substance use\u00a0=\u00a02.53; SD\u00a0=\u00a01.09), followed lastly by mean preference ratings for formal off-reservation care (mental health\u00a0=\u00a02.32; SD\u00a0=\u00a01.03; substance use\u00a0=\u00a02.34; SD\u00a0=\u00a01.09).\nPerceived Effectiveness of Services\nFigure\u00a01 illustrates the perceived effectiveness of services that are commonly used for mental health or substance abuse problems. The reported percentage indicates those respondents who felt the services would be very or extremely effective. There is a clear distinction of the perceived effectiveness between informal or traditional services and more formal services. Seventy-one percent of respondents felt that talking to a family member would be very or extremely effective. Nearly 60% felt talking to a tribal elder would be highly effective for dealing with mental health or substance problems. More traditional practices\/services such as offering tobacco and praying, seeing a traditional healer, traditional ceremonies, healing circle, sweat lodge, and pipe ceremony all were rated between 30% and 50%. Speaking to a counselor on the reservation was the highest rated professional service with 32.9% of respondents indicating it would be very or extremely effective. Most of the on reservation professional services rated higher than services off the reservation but lower than more traditional services. Those services that were seen as least effective were those professional services located off of the reservation.FIGURE\u00a01Perceived Effectiveness of Services\nUse of Informal and Formal Services\nTable\u00a01 reports the coefficients from ordinary least squares regression analyses for predictors of respondent perceptions of the effectiveness (i.e. preferences) of informal traditional and formal services for substance abuse and mental health concerns. Because this sample contains some cases where two adult reporters were interviewed within a household, reported coefficients are based on standard errors that have been adjusted to account for the potential bias of nested designs (analyses performed using STATA 7.0). The same sets of variables significantly predict informal service preference for both mental health (MH, Model 1A) and substance use (SU, Model 1B) related problems. For both models, females were more likely to prefer informal services (MH: \u03b2\u00a0=\u00a0.06, p\u00a0<\u00a0.05; SU: \u03b2\u00a0=\u00a0.05, p\u00a0<\u00a0.10), as were the employed (MH: \u03b2\u00a0=\u00a0.07, p\u00a0<\u00a0.05; SU: \u03b2\u00a0=\u00a0.07, p\u00a0<\u00a0.05). For both Model 1A and 1B, higher rates of social support (MH: \u03b2\u00a0=\u00a0.13, p\u00a0<\u00a0.001; SU: \u03b2\u00a0=\u00a0.13, p\u00a0<\u00a0.001), higher enculturation scores (MH: \u03b2\u00a0=\u00a0.49, p\u00a0<\u00a0.001; SU: \u03b2\u00a0=\u00a0.44, p\u00a0<\u00a0.001), and higher reports of perceived discrimination (MH: \u03b2\u00a0=\u00a0.09, p\u00a0<\u00a0.01; SU: \u03b2\u00a0=\u00a0.11, p\u00a0<\u00a0.01) were all positively associated with informal service preferences.TABLE\u00a01OLS Regression Models Predicting Perceived Effectiveness (Preferences) of Mental Health and Substance Use Related ServicesMental HealthSubstance Use1A2A3A1B2B3BInformalFormal (on res.)Formal (off res.)InformalFormal (on res.)Formal (off res.)B\u03b2B\u03b2B\u03b2B\u03b2B\u03b2B\u03b2Age\u00a0\u2212\u00a00.003\u00a0\u2212\u00a00.030.010.05\u00a0\u2212\u00a00.002\u00a0\u2212\u00a00.020.0010.010.010.07+0.000.003Gender (female\u00a0=\u00a01)0.150.06*0.130.06+0.040.020.130.05+0.050.002\u00a0\u2212\u00a00.03\u00a0\u2212\u00a00.01Education0.040.030.140.12**0.210.18***0.040.030.170.13***0.190.15***Employment0.140.07*0.080.04\u00a0\u2212\u00a00.01\u00a0\u2212\u00a00.010.150.07*0.020.01\u00a0\u2212\u00a00.06\u00a0\u2212\u00a00.03Live off reservation0.170.050.180.060.280.09*0.070.020.270.08*0.470.14***Physical health status0.010.010.030.030.020.02\u00a0\u2212\u00a00.02\u00a0\u2212\u00a0.010.020.010.010.01Social support0.030.13***0.030.13***0.020.08*0.040.13***0.050.17***0.020.09***Enculturation0.520.49***\u00a0\u2212\u00a00.05\u00a0\u2212\u00a00.05\u00a0\u2212\u00a00.05\u00a0\u2212\u00a00.050.510.44***\u00a0\u2212\u00a00.05\u00a0\u2212\u00a00.05\u00a0\u2212\u00a00.08-.07*Discrimination0.140.09**0.110.07+\u00a0\u2212\u00a00.07\u00a0\u2212\u00a00.050.190.11**0.170.1**\u00a0\u2212\u00a00.09\u00a0\u2212\u00a00.06Constant2.371.281.731.101.77R20.290.050.060.250.060.07Adjusted R20.280.040.050.240.050.06+p\u00a0<\u00a0.10; *p\u00a0<\u00a0.05; **p\u00a0<\u00a0.01; ***p\u00a0<\u00a0.001 (two-tail test).Sample size across models ranges from n\u00a0=\u00a0803 to n\u00a0=\u00a0822 after listwise deletion.\nMoving to predictors of formal service preferences on reservations (Models 2A & 2B), higher levels of social support were a significant predictor in both the MH (\u03b2\u00a0=\u00a0.13, p\u00a0<\u00a0.001) and SU (\u03b2\u00a0=\u00a0.17, p\u00a0<\u00a0.001) models, as were higher rates of perceived discrimination (MH: \u03b2\u00a0=\u00a0.07, p\u00a0<\u00a0.10; SU: \u03b2\u00a0=\u00a0.10, p\u00a0<\u00a0.01) and education (MH: \u03b2\u00a0=\u00a0.12, p\u00a0<\u00a0.01; SU: \u03b2\u00a0=\u00a0.13, p\u00a0<\u00a0.001). Age is significantly associated with formal on-reservation service preferences only for substance use problems (\u03b2\u00a0=\u00a0.06, p\u00a0<\u00a0.10), whereas females are more likely than males to prefer on-reservation formal care in the case of mental health problems (\u03b2\u00a0=\u00a0.06, p\u00a0<\u00a0.10).\nModels 3A and 3B illustrate the coefficients for predictors of formal off-reservation service preferences. Both models contain similar findings: higher education (MH: \u03b2\u00a0=\u00a0.18, p\u00a0<\u00a0.001; SU: \u03b2\u00a0=\u00a0.15, p\u00a0<\u00a0.001), living off of reservation lands (MH: \u03b2\u00a0=\u00a0.09, p\u00a0<\u00a0.05; SU: \u03b2\u00a0=\u00a0.14, p\u00a0<\u00a0.001), and higher levels of social support (MH: \u03b2\u00a0=\u00a0.08, p\u00a0<\u00a0.05; SU: \u03b2\u00a0=\u00a0.09, p\u00a0<\u00a0.05) were all positively associated with higher preferences for formal off-reservation services. In addition, Model 3B shows that those who scored higher on levels of traditional enculturation were less likely to perceive formal off-reservation care as effective in terms of substance use related problems (\u03b2\u00a0=\u00a0\u00a0\u2212\u00a0.07, p\u00a0<\u00a0.05).\nAcross all of the models in Table\u00a01, the independent variables explain proportionately more of the variance for the informal service models than those with formal services as the dependent variable. Adjusted R2 values for these models indicate that the predictors explain 28% and 24% of the variance in informal service preferences for MH and SU problems, respectively. These values for formal reservation-based care are 4% for MH and 5% for SU problems. The models for formal off-reservation care explain 5% of the variance in preferences for MH problems and 6% of the variance for SU problems.\nDISCUSSION\nThe adults in our sample felt that more culturally traditional services (such as family or a traditional healer) would be more effective than either professional services on the reservation, or professional services off the reservation. Off reservation services were perceived to be least effective. These findings support literature that suggests American Indians generally place considerable trust in traditional practices (i.e. Marbella et\u00a0al., 1998). The finding that mainstream professional services are perceived as less effective than traditional informal services and some of the on-reservation professional services underlies the previous discussion surrounding issues of trust and appropriateness of Western care in relation to American Indian cultures. It is also possible that a crisis-oriented care system may not be suited to adequately deal with mental health and substance abuse needs (Oetting & Beauvais, 1990\u20131991).\nThe multivariate analyses supported several of our hypotheses. Among the control variables, females were slightly more likely than males to report higher preference for both (MH & SA) types of informal care, as well as for on-reservation MH care. Though not explicitly hypothesized, these results correspond with previous help-seeking research that has found women more likely than men to view care as an appropriate remedy for psychological issues (Horwitz, 1987). Higher educational levels and employment were generally related to higher effectiveness ratings for services in this study, also corroborating previous research (see, for example, Horwitz, 1999, p. 66).\nAs expected, those who are more enculturated were more likely to utilize informal traditional services than those who are less enculturated. In addition, in the case of services for substance use disorders, higher levels of enculturation were negatively associated with preferences for formal off-reservation care (Model 3B). Those who lived off the reservation were more likely to use formal services, especially in terms of off-reservation care. Consistent with past research (Novins et\u00a0al., 1996) and as hypothesized, perceived social support was positively associated with higher perceptions of effective care across all types of services. Our hypothesis that higher rates of perceived discrimination would be associated with preferences for informal or traditional care is supported here. We found similar effects for discrimination and formal on-reservation care. These findings are congruent with Rodenhauser\u2019s (1994) description of the barriers to dominant-culture care in terms of mistrust of government agencies, all stemming from a history of colonization and continued institutional racism experienced by many American Indian peoples (see, for example, Duran & Duran, 1995). Overall, the results of this study highlight a preference for informal or culturally based care, especially among the enculturated.\nThe differences in the proportions of variance explained across each of the models in Table\u00a01 warrant discussion. These analyses include only one measure of cultural identity: enculturation. As discussed, this measure was significantly associated with ratings of informal service preference, but did little in terms of predicting formal service preferences. It may be that an important predictor of formal services is acculturation, or perhaps even biculturalism. Future work might include such measures to further our understanding of how cultural identity affects service preference and utilization (see Novins et\u00a0al., 2004).\nLimitations\nAlthough we interviewed a broad range of American Indian adults on multiple reservations that are dispersed geographically across two Midwestern states, these results pertain to a single culture and capture variations within this culture. We believe the findings represent the culture well, but they cannot be generalized to other Native cultures. A second caution regarding the sample is that it is made up of parents and caretakers of children aged 10\u201312\u00a0years. This could reflect a selection bias in that parents\/caretakers may be more likely to utilize services and to be currently mentally healthy and alcohol and drug free than individuals with no children. Finally, all of our measures are based on self-reports. We did not have access to service utilization rates from local clinics or reports of services use from traditional healers.\nPolicy Implications\nThese findings have important policy implications for those who provide health services to American Indian people. Cultural traditions are very much alive on U.S. reservations and are preferred methods of healing for mental health or substance abuse problems. This should be taken into account when designing health services systems either by providing alternative services onsite or through creatively engaging informal traditional services. Simply acknowledging informal service providers by asking the patient if they have seen a traditional healer and then contacting him or her for an opinion would be an important step. Health providers could include key community spiritual leaders and healers on health advisory boards or create health partnerships that would encourage mutual referrals. Perhaps the place to begin would be to respectfully seek the advice of elders on decreasing barriers between the two.\nActual integration of services is already taking place on many reservations, but implementation is not always straight forward (Marabella et\u00a0al., 1998; Mohatt & Varvin, 1998; Rhoades, & Rhoades, 2000). Issues about selection of traditional healers, and how to reimburse them such as whether they should charge fees for services and coverage for third party payments must be worked out culture by culture. Some view such integration as a potential acculturation risk.\nChanging the current short comings in mental health delivery first involves acknowledging that parallel systems exist in some cultures. The impetus for change may have to come from formal services providers who should demonstrate respect for traditional healers, invite their advice, and consult with them if patient\u2019s give permission. Clinicians may need to be proactive in offering their patients this option rather than waiting for the patient to request it. Creating this sensitivity may require specialized training for services providers on American Indian reservations. Grant funding groups may want to consider supporting model programs that bring together the two healing modalities.\nFuture Research\nThere is much to be done to increase American Indians\u2019 confidence in health services systems. One avenue for this would be to work more closely with informal traditional services that are trusted. We need control group trials of innovative services models that cross over between formal medical services and traditional approaches to healing to assess the efficacy of combining the approaches. Continued failure to acknowledge the strong preferences for traditional ways is to ignore a valuable health resource.","keyphrases":["mental health","substance abuse","american indians","service utilization"],"prmu":["P","P","P","P"]}
{"id":"Matern_Child_Health_J-2-2-1592144","title":"Family Planning Services: An Essential Component of Preconception Care\n","text":"Family planning services are necessary for the widespread adoption of preconception care for two reasons. First, preconception care is more likely if pregnancies are planned, and family planning services encourage pregnancy planning. Second, family planning services usually include counseling, and counseling provides an opportunity to discuss the advantages of preconception care. However, the potential of family planning services to promote preconception care is limited by underutilization of these services and inadequate attention to preconception care during family planning visits. This article suggests ways to reduce these problems.\n\u00a0\nIf all women of reproductive age, or at least those at elevated risk, are to benefit from preconception services, the use of family planning services must be increased and the content of such services expanded. Family planning services are essential for preconception care for at least two reasons. First, in the absence of such services, pregnancies will occur that have not benefited from preconception care. Preconception care during the reproductive years is dependent on women and men planning their pregnancies, not only in respect to their timing but also to health-related factors that would maximize the chances for a healthy pregnancy and a healthy infant. In the absence of such care, offered by family planning services, many pregnancies will not benefit from preconception advice. Second, family planning counseling provides an opportunity for promoting and providing preconception care. At the same time women and their partners are receiving advice about family planning, they can also receive instruction about the range of activities that lead to healthy pregnancies and healthy infants.\nUnfortunately, several factors prevent family planning from reaching its maximal potential for preconception care. These include underutilization of family planning services and inadequate attention to preconception counseling during family planning visits.\nUnderutilization of family planning services\nTwo types of data point to underutilization of family planning services with implications for decreasing opportunities for preconception care: the percentage of women actually seeking family planning services and the percentage of unintended pregnancies.\nVisits for family planning\nThe 2002 National Survey of Family Growth (NSFG) reported that 41.7% of women 15 to 44 years of age received at least one family planning service from a medical care provider in the 12 months prior to the interview [1]. This percentage is not as alarming as it might appear at first glance, because some of the women who did not seek family planning services already were pregnant, seeking to become pregnant, or infertile because of sterilization or other reasons. Nevertheless, this rather low percentage suggests that some women are not planning their pregnancies either deliberately or because they are experiencing problems obtaining family planning services.\nPregnancy planning\nIt is undoubtedly true that some women do not wish to plan the timing of their pregnancies. In many cases, these are married women who have religious objections to family planning. In Women of Crisis, one woman states \u201cyou don't think of life that way \u2013 of having children that way. You don't sit down and say you can afford to have a certain number of boys and girls; you have your children and try to do the best you can to be a good parent\u201d [2]. This attitude is probably not widely held, rather most women and their partners have some preferences in terms of the timing of births. This may be expressed in terms of age, marital status, or attainment of some educational, career, or economic goal. Despite this, a large percentage of pregnancies are unintended, including those that were experienced earlier than wanted (mistimed) or those that were not wanted at the time they occurred or at any future time (unwanted).\nAccording to the 2002 NSFG, 30.8% of all women 15 to 44 years of age had experienced an unintended birth at some time in their lives and, in the five years before the survey, 20.8% had had a mistimed birth and 14.1% an unwanted birth [3]. Using data from the 1982, 1988, and 1985 NSFGs, as well as abortion data, Henshaw estimated that in 1994, 49.2% of all pregnancies were unintended [4]. Employing a different set of questions to assess wantedness, the Pregnancy Risk Assessment Monitoring System (PRAMS) noted that in 1999 the percentage of unintended pregnancies resulting in live birth ranged from 33.7% to 52% across the 17 reporting states [5].\nAlthough the relationship between use of a family planning method and pregnancy planning is sometimes tenuous - an analysis of the 1995 NSFG found that 30.9% of the women who stated that their pregnancies had resulted from a contraceptive failure nevertheless classified the pregnancy as intended [6]. These data suggest that if preconception care were to be practiced widely, a larger percentage of women will need to seek family planning services to avoid unplanned pregnancies.\nReasons for underutilization\nMany reasons have been suggested for the underutilization of family planning services, including cost, availability, and limited contraceptive methods [7, 8].\nFinancial issues\nThe possible patient payment sources for family planning services include private health insurance, Medicaid, or out-of-pocket payments. The March 2005 Current Population Survey found 24.5% of women 18 to 20 years of age, 30.6% of those 21 to 24, 21.8% of those 25 to 34, and 17.1% of those 35 to 44 to be uninsured [9]. If these uninsured women want to receive family planning services, they must rely on their own funds or seek care from facilities that provide services free of charge or on a sliding fee scale. These include publicly supported facilities such as health department family planning clinics, community health centers and public hospitals, and facilities that combine public and private funding sources, such as Planned Parenthood centers, women's clinics, school-based health centers, and not-for-profit hospitals. Public funds for family planning services in such facilities come primarily from Medicaid and Title X (the family planning act). These funds are inadequate to meet the need. For women whose eligibility is due to a pregnancy, eligibility for all medical services, including family planning, ends 60 days postpartum, except in states with a waiver to extend the period. Title X tries to fill in the gaps in coverage but it is chronically underfunded. Even for those with employer-based health insurance, coverage for family planning services is not universal. In 2003, 93% of health plans offered an annual ob\/gyn visit, 88% covered oral contraceptives; 87%, sterilization; and 72%, all five reversible contraceptives. HMOs were more likely to offer contraceptives and sterilization than conventional plans, PPOs or POSs [10].\nAvailability\nFamily planning facilities need to be easily available both physically and psychologically. This means that they should be located where they can be reached easily by private or public transportation, be open days and hours that are convenient for potential users, offer the full range of contraceptive methods, provide educational materials in the languages of their patients, and employ personnel who can speak the language of the users and whose attitudes and behaviors are warm, friendly, and culturally sensitive. A recent four-state survey found that distance to a publicly financed family planning facility was not associated with teen or unintended pregnancies, suggesting that geographic availability is no longer a problem for family planning facilities [11], although it still is for abortion services [12]. The same study found major differences in terms of the other availability factors, such as limited hours, few contraceptive choices, and lack of translators [13]. Such problems may be partially responsible for inadequate contraceptive use.\nLimited contraceptive methods\nAlthough the number of family planning methods has increased in the past few decades, many women and men have difficulty finding one with which they are comfortable. The 2002 NSFG gave respondents a list of 19 family planning methods from which to choose. Among women 15\u201344 years of age, 61.9% currently were using a contraceptive method: the pill-18.9%; female sterilization-16.7% and the condom-11.1%. About 4% were using the least effective methods, including periodic abstinence and withdrawal. Among women who had intercourse in the three months before the interview and who were not sterile, pregnant, postpartum, or seeking pregnancy, 7.4% were not using a contraceptive - an increase from the 5.2% in the 1995 NSFG. (Of those at risk for an unintended pregnancy, 89.3% were currently using a method.) [1]. It is important to recognize that contraceptive use data are based on self-reports and provide no indication of how consistently or correctly the methods are being used. Failure rates are high possibly because of inconsistent or improper use. In one study of the 1995 NSFG, 9% of women experienced a contraceptive failure within one year of starting to use a reversible method of contraception: 7% of those on the pill, 9% of those relying on a male condom, and 19% of those practicing withdrawal [14].\nAfter female or male sterilization, hormonal methods are considered the most reliable form of contraception, but many women remain concerned about their safety. The side effects of many hormonal methods, especially Depo-Provera, discourage many women from their use [7, 8]. Further, the need to take a pill daily even in the absence of frequent intercourse creates problems for many women. Finally, the absence of a male contraceptive other than sterilization and the condom is a major barrier to effective and ongoing contraception.\nThe availability of one effective family planning method, emergency contraception (meant as back-up protection, not as a primary method), is currently limited by the refusal of the federal Food and Drug Administration to allow it to be sold over the counter and by the refusal of some pharmacists to fill prescriptions for this medication.\nInadequate attention to preconception counseling\nResearch is just beginning on the availability of preconception care overall although certain conditions, such as folic acid supplementation and counseling of diabetic women, have been studied extensively. The little existing evidence suggests that preconception care is not routinely integrated into family planning services, whether provided by obstetricians or family medicine specialists in private practice or HMOs, or by personnel in family planning facilities [15, 16]. Under such circumstances, the potential of this service delivery system for improving pregnancy and infant health through preconception care is not yet being realized.\nApproaches to increasing utilization\nSeveral evaluations indicate that the utilization of family planning services can be increased. California's Family Planning, Access, Care, and Treatment Program (Family Pact) provided contraceptive services to low income, medically indigent women increased use and reduced the numbers of unintended pregnancies [17]. State family planning waivers that expand Medicaid coverage for women beyond the 60 day postpartum limit have had a similar impact [18, 19]. Programs such as these should be expanded to more states, but they address only financial barriers. Attention should also be paid to other access-related problems, such as hours and days that facilities are open and language problems. Additional outreach also needs to be directed to low literacy and immigrant populations [20].\nApproaches to improving content\nMoos [15] reviewed many of the activities that will be essential if preconception care is to be integrated into health care generally. The American College of Obstetrics and Gynecology recently published a Committee Opinion, \u201cThe Important of Preconception Care in the Continuum of Women's Heath Care\u201d [21]. Here the focus is on adding preconception counseling to visits for family planning. The recent publication by the Centers for Disease Control and Prevention of ``Recommendations to Improve Preconception Health and Health Care - United States'' [22] should also lead to more and better preconception counseling.\nIn all likelihood, it may be most difficult to change patterns of family planning practice among physicians in private practice. In contrast, those in HMOs have the potential for exposure to more education about the need for such counseling. The establishment of standards for family planning services by the Health Plan Employer Data and Information Set (HEDIS) might also accelerate change. However, standard setting and financial and other incentives may be most effective when used with community health centers and health department and other publicly-funded sites of family planning services - as the women who use seek family planning at these sites may be those most in need of preconception care. Because such sites are funded by federal, state, and local governments, these units can insist that preconception care be integrated into their family planning services.\nConclusions\nProgress towards preconception care for all women will only be possible if a larger percentage of women and men plan their pregnancies. At present, although visits for contraceptive advice and methods provide an excellent opportunity for counseling about ways to achieve healthy pregnancies and healthy infants through preconception care, this potential is not being achieved. Increased use of family planning and increased attention to preconception care within family planning services may require that additional resources be devoted to public and professional education and to service delivery standards and financing.","keyphrases":["family planning","preconception care","pregnancy","access","intendedness"],"prmu":["P","P","P","P","U"]}
{"id":"Eur_Arch_Otorhinolaryngol-4-1-2217622","title":"Changes in CMDP and DPOAE during acute increased inner ear pressure in the guinea pig\n","text":"During and after an increase of inner ear pressure, induced by injection of artificial perilymph, the 2f1 \u2212 f2 and f2 \u2212 f1 distortion products (DPs) in cochlear microphonics (CM) and otoacoustic emissions (OAE) were recorded in the guinea pig. An inner pressure increase of \u223c600 Pa gave only small changes in CMDP and DPOAE. Along with a decrease in f1 amplitude, a small decrease in amplitude of the 2f1 \u2212 f2 and a small increase in the f2 \u2212 f1 were measured in CM. This matches a shift from a symmetrical position of the operating point for hair cell transduction, leading to an increase in even-order distortion and a decrease in odd-order distortion. Similar, a decrease in 2f1 \u2212 f2 DPOAE was expected. This might be the case at the generation sites but this effect was then more than compensated for by a better middle ear transfer, accounting for the increase of 0.4 dB of the 2f1 \u2212 f2 DPOAE amplitude. In conclusion, changes of overall inner ear fluid pressure have minor effects on cochlear function. This is a relevant finding for further understanding of diseases with changed inner ear fluid volumes, as M\u00e9ni\u00e8re\u2019s.\nIntroduction\nThe cochlea is a non-linear mechano-electrical transducer that transforms sound pressure waves into electrical information, the so-called mechanoelectric transduction (MET). The reverse electromechanical transduction is performed by the outer hair cell (OHC) which mechanically influences the vibration of the cochlear partition [7]. When the cochlea is stimulated by two tones, distortion products (DPs) can be measured in cochlear microphonic potentials (CMDP) and in the external auditory meatus as otoacoustic emissions (DPOAE). Both microphonic and acoustic DPs are considered to be reflections of OHC electromotility in the cochlea. Obviously, it is of interest to find a relation between these two functional modalities of the cochlea and the OHC in particular.\nIn this study the most prominent DPs, the 2f1\u00a0\u2212\u00a0f2 and f2\u00a0\u2212\u00a0f1, have been observed while increasing inner ear pressure. This was done by rapid injection of artificial perilymph into the scala tympani of the guinea pig\u2019s cochlea. Measurement of specific DPs may describe changes in gain and operating point of the non-linear cochlear amplifier. Furthermore, transfer characteristics of forward and backward transmission may be clarified by using CMDP and DPOAE [17]. These data could help gaining insight into pathophysiological mechanisms involved in pathologically increased inner ear pressures.\nWhen pressure variation is in the normal physiological range, this does not influence cochlear function as measured by compound action potentials in the guinea pig [2]. In humans, during activities like coughing or sneezing, perilymphatic pressure may undergo substantial fluctuations up to 10\u00a0mmHg (1.4\u00a0kPa).\nAn increased pathologic inner ear pressure may be the result of an increased intracranial pressure since the perilymphatic space is linked to the subarachnoid space via the cochlear aqueduct. Human OAE have been proven to be very sensitive to modifications of intracranial pressure [10]. Another clinical example of increased inner ear pressure or volume with an accompanying change of cochlear function is the endolymphatic hydrops in patients with M\u00e9ni\u00e8re\u2019s disease.\nIn a previous study [11], we measured solely the 2f1\u00a0\u2212\u00a0f2 DPOAE\u2019s in response to microinjection of artificial perilymph into the cochlea. Large changes of \u223c500\u00a0Pa in overall inner ear pressure gave only a small mean change of 0.7\u00a0dB in 2f1\u00a0\u2212\u00a0f2 DPOAE amplitude. The magnitude of change could (partly) be explained by a change in oval window stiffness. In this study, the same experimental setup was used, with the addition of the simultaneous recording of the 2f1\u00a0\u2212\u00a0f2 and f2\u00a0\u2212\u00a0f1 CMDP. We attempt to establish a better understanding of increased inner ear pressure effects on DPs in both CM potentials and OAE\u2019s.\nMaterials and methods\nExperiments were performed in eight guinea pigs (Harlan Laboratories, The Netherlands; body weight 350\u2013450\u00a0g) with a positive Preyer reflex. Animal care and use were in accordance with the principles of the declaration of Helsinki and approved by the animal experiment committee (protocol number 3047\/3103).\nGeneral anesthesia was induced by intramuscular administration of ketamine\/xylazine (60\/3.5\u00a0mg\/kg). Maintenance doses of the anesthetic were administered every hour. Muscle relaxation was obtained with succinylcholine (2.5\u00a0mg\/kg). The animals were artificially ventilated through a tracheostoma (Columbus Instruments, model 7950). Body temperature was maintained at 38\u00b0C with a heating blanket. Heart rate was monitored by skin electrodes placed on both sides of the thorax. The animal\u2019s head was kept in a stationary position by means of a steel bolt fixed to the skull with dental cement. Following a retroauricular incision, the bulla and external auditory canal were exposed. Subsequently, the bulla was opened equalizing middle ear pressure to normal air pressure [17]. By opening of the bulla, the round window was exposed. Through the round window membrane, the tip of a double-barreled micropipette was inserted into scala tympani. The double-barreled micropipettes were drawn from borosilicate glass (1.5\/0.84\u00a0mm diameter per barrel) and the tips were beveled (Narishige EG-40). The total tip diameter was around 60\u00a0\u03bcm, which is a compromise between a low enough flow resistance for fluid injection and tip smallness. One barrel of the pipette was used to measure inner ear pressure (WPI 900A micropressure system). Through the other barrel, artificial perilymph [9] was injected with a constant flow rate by applying a controllable pneumatic pressure to the barrel end. The injected volume was measured as the displacement of the fluid meniscus in the pipette, for which the inner diameter is precisely known (0.84\u00a0mm). The fluid injection rate was calculated as the total injected volume divided by the total injection time.\nDuring the experiment the DPOAE\u2019s were continuously measured using an Etymotic ER-10C DPOAE probe system. The two primary frequencies were set at 6\u00a0kHz (f1) and 7.4\u00a0kHz (f2), meaning a f2\/f1-ratio of 1.23. The low-level intensities were set at, respectively, 65\u00a0dB SPL (L1) and 55\u00a0dB SPL (L2). Full details of the measuring equipment are described elsewhere [11, 12].\nCochlear microphonic potentials were recorded with two differential Ni\u2013Cr electrodes with a diameter of 50\u00a0\u03bcm. One electrode was placed in scala tympani through the bony wall of the basal turn of the cochlea. The other was placed in scala vestibuli next to the oval window. The signals were routed via an amplifier to a spectrum analyzer and two lock-in amplifiers (SRS, model SR830 DSP). The lock-in amplifiers displayed the amplitude of the 2f1\u00a0\u2212\u00a0f2 and f2\u00a0\u2212\u00a0f1 CMDP which were referenced to an electronic 2f1\u00a0\u2212\u00a0f2 and f2\u00a0\u2212\u00a0f1-DP derived from the original primaries.\nA measurement contained a sequence of ten microinjections of artificial perilymph of 10\u00a0s each with pauses of 40\u00a0s. The rate of microinjection was 50\u00a0nl\/s. The repetitive microinjection was controlled with a precision electronic timer (Stanford DG535). During an experiment, National Instruments LabVIEW\u00ae was used for recording of the following output signals: amplitude and phase of the 2f1\u00a0\u2212\u00a0f2 DPOAE, 2f1\u00a0\u2212\u00a0f2 CMDP, f2\u00a0\u2212\u00a0f1 CMDP, f1 CM, and inner ear pressure. Calculations and fits to relevant portions of the obtained recordings were made off-line with an appropriate software package.\nResults\nSuccessful repeated sequences of injection with a rate of 50\u00a0nl\/s (n\u00a0=\u00a010) were performed in all guinea pigs (n\u00a0=\u00a08). The mean steady state inner ear pressure measured prior to injection of artificial perilymph was 205\u00a0Pa.\nTo determine the rise and fall times of the curves from the measured signals, proper fits were obtained with a simple exponential function: In some cases (Figs.\u00a01c, 2c, 4c) the curves needed an extra term in the formula for a proper fit: According to Wit et al. [13] time constants \u03c4 were calculated from the fits.\nFig.\u00a01a Averaged changes (n\u00a0=\u00a08) in inner ear pressure (Pa) during and after injection of 0.5\u00a0\u03bcl of artificial perilymph into scala tympani. The gray area depicts the injection period. Standard error bars are shown at t\u00a0=\u00a05, 15, and 40\u00a0s. b The shape of the mean inner ear pressure curve at the start of injection (time axis corresponds with Fig.\u00a01a). The recording (filled circles) is fitted (broken line) with an exponential function, yielding a time constant \u03c4. c The same is done for inner ear pressure recovery directly after injectionFig.\u00a02a Averaged changes (n\u00a0=\u00a08) in f1 CM amplitude (\u03bcV) during and after injection of 0.5\u00a0\u03bcl of artificial perilymph into scala tympani. The gray area depicts the injection period. Standard error bars are shown at t\u00a0=\u00a05, 15, and 40\u00a0s. b The shape of the mean f1 CM amplitude curve at the start of injection (time axis corresponds with Fig.\u00a02a). The recording (filled circles) is fitted (broken line) with an exponential function, yielding a time constant \u03c4. c The same is done for f1 CM amplitude recovery directly after injection\nIn Fig.\u00a01a, the averaged recorded inner ear pressure during and after injection of 0.5\u00a0\u03bcl of artificial perilymph is shown. At the start of injection the mean pressure increased to an \u223c600\u00a0Pa higher level within seconds. At the end of injection, the pressure immediately decreased, returning to its initial value within seconds. This pattern was consistently observed in all individual experiments. The time constant for increase and subsequent decrease of inner ear pressure were 1.25 and 1.16\u00a0s, respectively (Fig.\u00a01b, c).\nThe averaged f1 signal in the CM (Fig.\u00a02a) showed a mirrored behavior in respect to the inner ear pressure. Directly after the start of injection, the f1 CM decreased by 30\u00a0\u03bcV. The corresponding time constant was 1.17\u00a0s (Fig.\u00a02b), which is very close to the time constant for pressure increase. Conversely, at the end of injection the f1 amplitude returned to its initial value with \u03c4\u00a0=\u00a03.49\u00a0s (Fig.\u00a02c), which is slow with respect to the corresponding \u03c4 for pressure.\nThe averaged DPOAE 2f1\u00a0\u2212\u00a0f2-amplitude showed a similar typical course as the inner ear pressure (Fig.\u00a03a). Almost directly after the start of injection the 2f1\u00a0\u2212\u00a0f2-amplitude increased to reach a maximum of 0.4\u00a0dB above its original value at the end of the injection period. When the injection was instantaneously terminated, the amplitude dropped and returned to slightly above its onset level. There was a small observable time lag with respect to the pressure curve. The time constant for increase and subsequent decrease of the 2f1\u00a0\u2212\u00a0f2-amplitude were 1.52 and 3.43\u00a0s, respectively (Fig.\u00a03b,c).\nFig.\u00a03a Averaged changes (n\u00a0=\u00a08) in 2f1\u00a0\u2212\u00a0f2 DPOAE amplitude (dB SPL) during and after injection of 0.5\u00a0\u03bcl of artificial perilymph into scala tympani. The gray area depicts the injection period. Standard error bars are shown at t\u00a0=\u00a05, 15, and 40\u00a0s. b The shape of the mean 2f1\u00a0\u2212\u00a0f2 DPOAE amplitude (\u03bcPa) curve at the start of injection (time axis corresponds with Fig.\u00a03a). The recording (filled circles) is fitted (broken line) with an exponential function, yielding a time constant \u03c4. c The same is done for 2f1\u00a0\u2212\u00a0f2 DPOAE amplitude recovery directly after injection\nIn Fig.\u00a04a, the typical temporary decrease for the averaged 2f1\u00a0\u2212\u00a0f2 CMDP amplitude relative to the f1 CM is shown. The time constant for decrease in amplitude at the onset of injection was 1.12\u00a0s (Fig.\u00a04b), which corresponds with \u03c4 for pressure. The recovery is somewhat slower (2.28\u00a0s, Fig.\u00a04c), but still faster than the time constant for 2f1\u00a0\u2212\u00a0f2 DPOAE recovery.\nFig.\u00a04a Averaged changes (n\u00a0=\u00a08) in 2f1\u00a0\u2212\u00a0f2 CMDP relative to f1 CM amplitude during and after injection of 0.5\u00a0\u03bcl of artificial perilymph into scala tympani. The gray area depicts the injection period. Standard error bars are shown at t\u00a0=\u00a05, 15, and 40\u00a0s. b The shape of the mean 2f1\u00a0\u2212\u00a0f2 CMDP (relative to f1 CM) amplitude curve at the start of injection (time axis corresponds with Fig.\u00a04a). The recording (filled circles) is fitted (broken line) with an exponential function, yielding a time constant \u03c4. c The same is done for 2f1\u00a0\u2212\u00a0f2 CMDP (relative to f1 CM) amplitude recovery directly after injection\nFinally, in Fig.\u00a05, the averaged results of the f2\u00a0\u2212\u00a0f1 CMDP amplitude relative to f1 CM depict an increase during injection of artificial perilymph. When the injection was terminated, the f2\u00a0\u2212\u00a0f1 CMDP amplitude returned to the pre-injection level. Unfortunately, due to a low signal-to-noise ratio for the f2\u00a0\u2212\u00a0f1 CMDP, the obtained curve could not be fitted properly and no time constants could be derived.\nFig.\u00a05Averaged changes (n\u00a0=\u00a08) in f2\u00a0\u2212\u00a0f1 CMDP relative to f1 CM amplitude during and after injection of 0.5\u00a0\u03bcl of artificial perilymph into scala tympani. The gray area depicts the injection period\nDiscussion\nThe most striking result of this study is the minimal change in amplitude of the DPs in both OAE and CM during an acute increase of inner ear pressure. Besides, amplitudes changed to normal when inner ear pressure returned to its initial level. These results indicate a dynamically stable inner ear which is able to cope with a pressure increase of 600\u00a0Pa with only minimal and reversible cochlear function loss. According to B\u00f6hmer [2] this level of pressure increase would still be in the physiological range of the guinea pig ear varying between \u2212100 and +700\u00a0Pa.\nThe inner ear is an elastic structure completely encompassed by bone, in which pressure changes depend on the compliance of the membranous structures [14]. In normal ears, hydrostatic pressure in the perilymph equals pressure in the endolymph, and pressure changes applied to one compartment are immediately transmitted to the other through the elastic walls. The perilymph is in a dynamic equilibrium with the cerebrospinal fluid via the cochlear aqueduct. An excess of perilymph will escape through the cochlear aqueduct restoring inner ear fluid level.\nWhen increasing inner ear pressure, a cochlear disturbance with functional loss is expected. The observed decrease in CM f1 amplitude thus seems obvious. An increased cerebrospinal fluid pressure also suppresses the CM [16]. The increase in 2f1\u00a0\u2212\u00a0f2 DPOAE is counterintuitive, but in accordance with a previous publication in which we demonstrated the same change in cochlear function as measured by 2f1\u00a0\u2212\u00a0f2 DPOAE\u2019s [11]. According to B\u00fcki et al. [3] a change in stiffness of the oval window might slightly improve middle ear transfer and thus explain the small increase in DPOAE amplitude. In contrast to the 2f1\u00a0\u2212\u00a0f2 DPOAE, the 2f1\u00a0\u2212\u00a0f2 CMDP showed a decrease in amplitude during injection. Unfortunately, in literature very little can be found about the relation between DPOAE and CMDP. Still, it were Kemp and Brown [6] who found close parallels in the behavior of the 2f1\u00a0\u2212\u00a0f2 DPOAE and CMDP during and after anoxia in the guinea pig. Group latencies and third-tone suppression were found to be very similar, linking both signals to have a common origin.\nThe DPOAE and CMDP are generated in response to the same acoustic stimuli. The acoustic primaries are transferred through the external and middle ear to eventually reach the sensory epithelia in the cochlea. The cochlea as a non-linear mechano-electrical transducer transforms sound pressure waves into electrical information. This forward transduction process is called the MET. The CM potential is a direct result of this process and is thought to be mainly produced by OHCs. On the other hand, there is also a reverse electromechanical transduction in which the OHC mechanically influences the vibration of the cochlear partition [7]. Sound is transmitted back to the external ear where the acoustic DPOAE can be measured; the backward transmission.\nThe rise and fall times of the DPs in response to changes in inner ear pressure were derived from the fitted curves (Figs.\u00a02, 3, 4). As both CMDP and DPOAE were measured, this yielded information on forward and backward transmission. As anticipated, the time constants for the CMDP were smaller than the time constants for the DPOAE. Indeed, the latter would travel back and forth the middle ear which understandably costs more time. However, the difference has not the form of a simple longer delay. The explanation for this observation remains tentative. The averaged differences were 0.4 and 1.15\u00a0s for the 2f1\u00a0\u2212\u00a0f2 DP during increase and decrease of inner ear pressure, respectively.\nAt pressure increase all measured signals almost directly followed the change in inner ear pressure. Time constants varied from 1.17 to 1.52\u00a0s with respect to 1.25\u00a0s for the inner ear pressure curve. However, with pressure decrease there was a delay of 1\u20132\u00a0s for all measured signals. Measured differences cannot be accounted for by delay in measuring equipment, for this was <0.18\u00a0s. Assumed that the overall stiffness of the stapes-oval window-system governs the behavior of the DPOAE 2f1\u00a0\u2212\u00a0f2 amplitude, it is expected that it would follow stiffness changes directly and not 2\u00a0s later, as observed. It is conceivable that a delayed change in the endocochlear potential (EP) could be the cause of difference in \u03c4 values. However, in experiments by Salt and DeMott [9] the EP followed a temporary pressure change with a delay of <0.1\u00a0s. So, the mechanisms for the observed delays in inner ear pressure recovery remain to be elucidated.\nTwo-tone distortions are generated during non-linear mechanical amplification in the cochlea. Generation of the 2f1\u00a0\u2212\u00a0f2 depends on asymmetric component of a non-linear transfer function whereas the f2\u00a0\u2212\u00a0f1 depends on symmetric components. A change of the operating point is reflected in the level of the f2\u00a0\u2212\u00a0f1 distortion. In the gerbil low-frequency tones of 5\u00a0Hz were used to bias the position of the cochlear partition by Frank and Kossl [4, 5].\nBy low-frequency modulation of the 2f1\u00a0\u2212\u00a0f2 DPOAE, Bian et al. [1] described the forward MET by a sigmoid-shaped function, which related the displacement of the basilar membrane to the response of the hair cells.\nBy injecting artificial perilymph into scala tympani of the cochlea, the basilar membrane is temporarily displaced. Thus, by this displacement toward scala media, the operating state of the cochlear amplifier is also influenced. We measured a small decrease in the amplitude of the 2f1\u00a0\u2212\u00a0f2 distortion in CM (with respect to the amplitude of f1 in CM) and a small increase in the f2\u00a0\u2212\u00a0f1 in CM. Using the model by Bian et al. [1] this leads to the conclusion that the operating point for hair cell transduction is somewhat shifted from a symmetric position. This shift leads to an increase in even-order distortion and a decrease in odd-order distortion. Along these lines of reasoning we would also expect the 2f1\u00a0\u2212\u00a0f2 DPOAE to decrease. This might be the case at the generation sites [15], but this effect is then more than compensated for by an improved middle ear transfer.\nAnyhow, the observed changes are small. Changes of overall inner ear fluid pressure have minor effects on cochlear function. It may be that creating an artificial endolymphatic hydrops, by injecting artificial endolymph into scala media instead of scala tympani, has a larger effect. This is currently tested in further research, which will hopefully give insight into diseases with pathologically changed inner ear fluid volumes, as in M\u00e9ni\u00e8re\u2019s disease.","keyphrases":["cmdp","dpoae","inner ear pressure","guinea pig","injection","perilymph","2f1 \u2212 f2","f2 \u2212 f1"],"prmu":["P","P","P","P","P","P","P","P"]}
{"id":"Anal_Bioanal_Chem-3-1-1705481","title":"Analytical methods for PCBs and organochlorine pesticides in environmental monitoring and surveillance: a critical appraisal\n","text":"Analytical methods for the analysis of polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) are widely available and are the result of a vast amount of environmental analytical method development and research on persistent organic pollutants (POPs) over the past 30\u201340 years. This review summarizes procedures and examines new approaches for extraction, isolation, identification and quantification of individual congeners\/isomers of the PCBs and OCPs. Critical to the successful application of this methodology is the collection, preparation, and storage of samples, as well as specific quality control and reporting criteria, and therefore these are also discussed. With the signing of the Stockholm convention on POPs and the development of global monitoring programs, there is an increased need for laboratories in developing countries to determine PCBs and OCPs. Thus, while this review attempts to summarize the current best practices for analysis of PCBs and OCPs, a major focus is the need for low-cost methods that can be easily implemented in developing countries. A \u201cperformance based\u201d process is described whereby individual laboratories can adapt methods best suited to their situations. Access to modern capillary gas chromatography (GC) equipment with either electron capture or low-resolution mass spectrometry (MS) detection to separate and quantify OCP\/PCBs is essential. However, screening of samples, especially in areas of known use of OCPs or PCBs, could be accomplished with bioanalytical methods such as specific commercially available enzyme-linked immunoabsorbent assays and thus this topic is also reviewed. New analytical techniques such two-dimensional GC (2D-GC) and \u201cfast GC\u201d using GC\u2013ECD may be well-suited for broader use in routine PCB\/OCP analysis in the near future given their relatively low costs and ability to provide high-resolution separations of PCB\/OCPs. Procedures with low environmental impact (SPME, microscale, low solvent use, etc.) are increasingly being used and may be particularly suited to developing countries.\nIntroduction\nScope of the review\nPolychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs; aldrin, chlordane, DDT, dieldrin, endrin, heptachlor, mirex, toxaphene, hexachlorobenzene (HCB)) constitute ten of the twelve chemical substances\/groups currently defined under the Stockholm Convention on Persistent Organic Pollutants (POPs). While bans and phase-outs of these chemicals occurred during the 1970s and 1980s in most developed countries, they were not in place in many developing countries. Article 7 of the Stockholm Convention requires National Implementation Plans (NIPs) to be developed by signatory countries [1]. For countries where PCBs and OCPs are still in use or loosely regulated, the NIPs will eventually lead to the phase-out of OCPs for agricultural use and reduced use for disease vector control. However, many countries will be faced with the legacy of past use of these persistent chemicals. Residues in soils, sediments and biota will pose a risk for wildlife and human consumers. The problems of destroying existing stockpiles, especially of OCPs and PCBs in electrical equipment, as well as preventing migration of contaminants from storage areas, will need to be solved. There is a need to monitor levels and trends now and into the future in order to assess compliance with the POPs Convention within each country as well as on a regional and global basis.\nMethodology is available for PCBs and OCPs as a result of a vast amount of environmental analytical chemistry research and development over the past 30 to 40\u00a0years. However, the establishment of an analytical laboratory and the application of this methodology at currently acceptable international standards is a relatively expensive undertaking. Furthermore, the current trend to use isotope-labeled analytical standards and high-resolution mass spectrometry for routine POPs analysis is particularly expensive. These costs limit participation of scientists in developing countries and this is clear from the relative lack of publications and information on POPs from countries in Africa, south Asia and South\/Central America. Thus, this review will summarize the best practices in developed countries and new advances in PCB\/OCP analysis, while bearing in mind the need for low-cost methods easily implemented in developing countries.\nThis review will also mainly focus on methodology for the analysis of PCB\/OCPs in biological samples (fish, aquatic and terrestrial mammals and birds) as well as soils, sediment and passive samplers such as semipermeable membrane devices (SPMDs). These matrices and environmental compartments have higher concentrations of PCBs and most OCPs than water or air, making them more suitable for routine monitoring and more relevant in the context of exposure of humans and wildlife. Milk and blood are important matrices for POPs monitoring in humans and, in the case of blood, are increasingly used in programs involving live-captured marine mammals and birds. Thus analytical methods for milk and blood are briefly considered here. The advantages and disadvantages of various bioindicator species are discussed in detail by Tanabe and Subramanian [2].\nSubstances to be analyzed\nPCBs (except non-ortho-substituted congeners; no-PCB) and OCPs can be considered together because they are extracted and analyzed together in most cases, as discussed below. In practice, most laboratories determine about 30 or more individual PCB congeners, and 10\u201320 individual OCPs and their metabolites, regardless of the sample matrix. Ongoing POPs monitoring programs vary in their analyte lists. For example, the Integrated Atmospheric Deposition Network (IADN) in the Great Lakes region of North America includes over 100 PCB congeners [3], while the UNEP\/World Bank\/GEF project on Persistent Organic Pollutants, Food Security, and Indigenous Peoples in Arctic Russia included 15 PCB congeners [4]. The Arctic Monitoring and Assessment Program recommended 30 ortho-substituted PCBs [5].\nThe UNEP Global POPs monitoring workshop categorized individual PCB\/OCPs as \u201cessential\u201d and \u201crecommended,\u201d taking into consideration the need for various levels of analytical capacity building in developing countries in order to conduct the monitoring (Electronic Supplementary Material, Table\u00a01) [6]. For PCBs, a minimum of seven PCBs (28\/31, 52, 101\/90, 118, 138, 153 and 180) were regarded as essential for routine monitoring of fish and food products around the world. Using only seven congeners severely underestimates total PCB concentrations in some matrices\u2014particularly in abiotic samples such as air, soil, and sediment. On the other hand, these seven are robustly determined, having been the subject of numerous interlab comparisons (e.g., QUASIMEME [7]). UNEP [6] recommended a suite of 30 ortho-substituted congeners for more comprehensive monitoring.\nToxaphene and chlordane are multicomponent mixtures and analytical standards are available for some, but not all, major components, which makes them the most difficult to analyze among the eight OCPs on the POPs list. Current programs in Europe are determining three toxaphene congeners, Parlar (P) 26, 50 and 62, and interlaboratory comparisons have shown reasonable agreement for these congeners among laboratories [8]. However, P26 is interfered with by coeluting chlorobornanes in some matrices, and P62 can vary widely in its instrumental response. Furthermore, in North America, most datasets for toxaphene are still based on quantification with technical toxaphene, which yields a single value and no congener-specific information. Capacity for determining toxaphene outside of the USA, Canada and western Europe is very limited judging from the relatively large number of measurements from these countries and the dearth of measurements elsewhere.\nThe situation for chlordane is better because analytical standards are available for the major components (cis- and trans-chlordane; cis- and trans-nonachlor, heptachlor). Nevertheless, other octachloro- and nonachlorochlordane components are important [9] and are often found in top predators [10, 11], and analysts need to be aware of possible interferences from these compounds in seabird and marine mammal extracts.\nThe chlorinated insecticides endosulfan, lindane and methoxychlor are also candidates for inclusion in analytical methods where impacts of OCPs are being investigated. Global use of these insecticides, particularly endosulfan, is very significant [12, 13] These current-use pesticides can generally be isolated and quantified along with other OCPs.\nThe no-PCBs need to be considered separately because their analytical methodology is normally quite different from that used for ortho-substituted PCBs and OCPs (see Reiner et al. [14] in this issue).\nIn summary, the minimum number of PCB\/OCPs to be considered will vary with the objectives of a given study. A long list of analytes would be used for initial surveys where no information was available. Chemical structures and physical and chemical properties of selected PCBs and OCPs are listed in Electronic Supplementary Material Table\u00a02 [143, 144].\nAnalytical standards\nThe availability of accurate analytical standards is a fundamental requirement of an analytical program designed to quantify PCB\/OCPs. Standards are available from commercial chemical supply companies as well as from agencies involved in certification of reference materials, e.g., the National Institute of Standards and Technology (NIST) or from the Bureau Communautaire de R\u00e9f\u00e9rence (BCR) via the Sigma-Aldrich company. Interlaboratory studies have shown that incorrect analytical standards and calibration errors can be a major source of error in POPs analysis [15, 16]. These authors recommended that multilevel calibration at 4\u20136 levels should be used to define the response profile, that calibration solutions should be checked against independent solutions, either from another reliable laboratory or by preparing duplicate solutions, and that calibrants should be ampouled (in sealed glass vials) for long-term storage. They noted that refrigerator storage can loosen the caps on screw-top vials. Thus, storage of calibrant solutions in this manner should only be temporary.\nQuantification of PCBs can be carried out using two approaches; by Aroclor mixture or individual congener standard. The former method employs a standard that was available as a commercial product (e.g., Aroclor 1254 used in transformer oils) to quantitate the congeners contained in the Aroclor mixture against those matching in the sample. There are several mixtures available, with varying degrees of mean chlorination. Percent ratios of the congeners in various commercial mixtures have been reported [17]. The limitation of this method is the varying congener ratios within a given commercial mixture due to batch-to-batch production variability which, in turn, may lead to congener calculation error [18]. Also, a significant bias resulting in an overestimation of PCB measurements may occur if the PCB pattern in the sample does not match that of the standards. Sather et al. [19] suggested that accurate PCB determinations can be made if the chromatographic pattern of both the standards and the samples are similar. However, if the pattern differs, the study reported a bias doubling the values determined by the individual congener standard. The method of choice is congener determination using standard mixtures comprising composite individual congeners added at known quantities which were purchased individually. This provides an improved and known level of accuracy not possible with the commercial PCB mixture approach [19].\nCommonly used analytical methods for PCB\/OCP monitoring and surveillance\nOverview\nNumerous methods have been published over the past 30\u00a0years related to specific analytical techniques for the determination of PCBs and OCPs in food and environmental matrices. Laboratory standard operating procedures (SOPs) are available from the National Environmental Methods Index in the USA (which includes US EPA and US Geological Survey methods) (http:\/\/www.nemi.gov) and the Japan Environment Agency (http:\/\/www.env.go.jp\/en\/index.html), the National Oceanographic and Atmospheric Administration in the USA [20] and the US Food and Drug Administration [21]. Useful information may also be available from the International Committee for Exploration of the Sea [22], the Oslo\u2013Paris Commission [23] (http:\/\/www.ospar.org), the Helsinki Commission (HELCOM) (http:\/\/www.helcom.fi), the International Organization for Standardization (http:\/\/www.iso.org), the Association of Official Analytical Chemists International (http:\/\/www.aoac.org), and Gosstandart of the Russian Federation (http:\/\/www.krohne.ru\/russia_en\/downloads\/certificates\/cis\/russia\/gosstandard\/).\nNot all of these sources provide analytical SOPs for all environmental media. Alternatively, numerous books are available that summarize methods. For example, Keith [24] summarized US EPA methods for PCBs and OCP in sediment and biological materials. Wells and Hess [25, 26] have reviewed and recommended methods for the separation, isolation and recoveries of OCPs and PCBs from soils, sediment and biological matrices. De Boer and Law [27] provide a useful overview of current analytical methodology for OCP\/PCBs.\nThe UNEP POPs workshop on global monitoring (UNEP 2003) noted that, given the broad range of technical expertise for analysis of PCBs and OCPs, as evident from the extensive international participation in interlaboratory calibration projects for these compounds, no single, detailed, step-by-step analytical method can be recommended. Instead laboratories would use methods best-suited to their situation and take part in international interlaboratory comparisons to verify their work. This performance-based approach has also been adopted by the US EPA in an effort to introduce flexibility in conducting environmental monitoring. This approach also encourages analytical innovation by allowing the use of new and alternate methods provided that predetermined performance criteria are met [28].\nSampling\nAcquisition of suitably representative samples is fundamental to any environmental measurement program and should not be overlooked. Contamination during field collection can occur, particularly from PCBs in electrical equipment and building products (e.g., marine paints, joint sealants) [29, 30], or due to the ongoing use of OCPs. \u201cClean\u201d techniques need to be adopted, such as the use of special clothing and disposable gloves for sampling, sealed shipping containers, and field blanks. ICES [22], NOAA [20] and OSPAR [23] provide useful guidelines for sampling of fish\/crustaceans, mussels, and sediment, respectively. Azcue and Mudroch [31] provide a detailed overview of sampling methods for lakes and river sediments.\nSample containers should be supplied by the laboratory and checked for contamination. Appropriate sample containers for biological tissue that is to be analyzed for POPs are glass jars or glass vials with screw caps. Screw caps should be lined with solvent-rinsed aluminum foil or Teflon cap inserts. Clear polyethylene bags, and polypropylene jars, are also appropriate for temporary storage but may not be suitable for long-term storage because of the possibility of migration of plasticizers (such as phthalates) into the tissue, especially for samples with high lipid contents. Freezing and storage of multiple small samples suitable for analysis, rather than larger masses, is recommended to avoid multiple freezing and thawing of tissue [25] and to reduce sample handling, which in turn reduces the potential for contamination. Samples can be wrapped in aluminum foil and then inserted into plastic bags.\nBlood samples should be collected in ethylenediamine tetraacetic acid (EDTA) vials or vacutainers, centrifuged, and the plasma transferred to vials washed with hexane. Field blanks, consisting of sample containers taken to the lab and returned with other samples, should be included.\nAir sampling for POPs is normally conducted using polyurethane foam (PUF) or XAD resin [3]. From 300 to 10,000\u00a0m3 of air are passed through the sampling medium [3, 32]. Sampling media are then transferred to clean, wide-mouthed glass jars, sealed and stored at 4\u00a0\u00b0C prior to extraction. Some sampling cartridges can be sealed and transported and the sampling media transferred to Soxhlet or pressurized liquid extraction systems in a clean laboratory environment.\nWater sampling is particularly prone to contamination by PCBs due to ubiquitous contamination, especially on older ships and buildings, and to the very low sample concentrations likely to be encountered [33]. US EPA analytical methods for PCBs in water assume that drinking water is being analyzed [34, 35] and do not place much emphasis on sampling because the drinking water quality guideline is 0.5\u00a0ug\/L and only small <1\u00a0L samples need to be collected to assess compliance. Solid-phase extraction (SPE) cartridges have been widely used to extract relatively small volumes (1\u20135\u00a0L) for OCP analysis [36]. For sampling of natural waters where PCB concentrations are in the low pg\/L range, much larger samples are required. For example, Achman et al. [37] pumped lake water (\u223c100\u00a0L) directly into large-diameter filters (0.7\u00a0\u03bcm nominal pore size) and then through XAD-2 resin columns. Surrogate standards were added to the resin column prior to extraction. Similar solid-phase extraction approaches are used for sampling and extracting wet precipitation [38, 39]. Another large-volume application uses liquid:liquid extraction in a continuous flow approach [40]. This involves a one pass-through extraction unit where the sample is metered into a mixing chamber which contains a solvent (i.e., dichloromethane) and flows out to an overflow exit. The drawback to this method is the significant potential for contamination due to exposure to the surrounding air. Petrick et al. [41] describe an in situ sampler for the collection of very large volumes (\u223c500\u00a0L) of seawater on polyurethane foam for the determination of PCBs at low pg\/L concentrations. The studies by Petrick et al. [41] and Sobek and Gustafsson [42] illustrate the great care that is needed to properly determine PCBs at the low pg\/L level (femtograms\/L of individual congeners) that are found in ocean waters.\nSPMDs offer potential for relatively inexpensive sampling of water and air for POPs and potential for broad geographic coverage [43, 44]. For water sampling, lipid-based SPMDs (consisting of triolein inside polyethylene tubing) have generally been used to estimate bioavailable water concentrations. SPMD use in the aquatic environment has been validated in a series of studies [45]. The use of performance reference compounds (PRCs) added to the triolein prior to deployment has been shown to enhance the use of SPMDs by accounting for site-specific variables [45]. The PRCs are gradually lost from the sampler and thus permit an exposure adjustment factor or depuration rate to be calculated, which adjusts for changes in sampling rate.\nLipid-based passive air samplers have also been developed and deployed for air sampling of PCBs and OCPs [46]; however, more recently designs based on polyurethane foam (PUF) and XAD resin have become more widely applied [47, 48]. Care must be taken to prepare and transport the samplers under clean conditions. Following deployment, which is usually for several months for lipid-based and PUF samplers [49] for up to one year for XAD [44], samplers are placed in transport containers and returned to the lab. PRCs are also often added to the passive air samplers [47].\nSample storage and handling\nA basic requirement for analytical laboratories involved in the measurement of PCBs and OCPs is the availability of freezer and refrigerator capacity for sample storage and archiving. Wells and Hess [25] and ICES [22] provide recommendations for storage of biological and sediment samples. Kiriluk et al. [50] found no significant differences in concentrations of PCBs in whole fish homogenates stored at \u221220 or \u221280\u00a0\u00b0C over four years. Norstrom and Won [51] observed dehydrochlorination of DDT in long-term-archived egg homogenates, which they attributed to reactions occurring during freeze-drying. De Boer and Smedes [52] found no significant changes in the concentrations of PCBs and DDT components in fish tissue stored at \u221220 to \u221270\u00a0\u00b0C in the dark for up to 24\u00a0months. However, they did find changes in lipid extractability caused by oxidation of triglycerides to free fatty acids. The authors pointed out that this could affect calculations of lipid-normalized concentrations. To minimize any effects, they recommended long-term (>2\u00a0year) storage at \u221270\u00a0\u00b0C.\nSample preparation\nWells et al. [53] noted that sample preparation affected the magnitude of the variance in the analysis of PCB congeners in mussel tissue. They found that freeze-drying samples reduced recoveries of OCPs and PCBs, probably due to tighter binding and occlusion of residues in the dried material. Volatilization losses might also occur during freeze-drying of volatile compounds such as HCB, and there is a greater potential for contamination in the lab [54]. Although freeze-drying has been recommended by some authors for sediments and soils [25], lower recoveries of PCBs from freeze-dried sediments have also been reported [55]. Karl [56] noted that loss of water led to an increase in OCP concentrations in fish products during processing. Thus, in general, maintaining environmental sample tissues and sediments in their original wet state is regarded as the most appropriate approach for preparing samples for PCB\/OCP analysis. Furthermore, avoiding a drying step minimizes potential contamination from lab air, particularly in the case of PCBs that are ubiquitous contaminants of older (pre-1980s) buildings [30, 57]. Using wet material also avoids possible volatilization losses. Instead, homogenized samples should be mixed with a desiccant such as sodium sulfate, Celite or Hydromatrix to bind water. The desiccant must be certified to be free of analyates, e.g., by heating at high temperature in the case of sodium sulfate or pre-extraction (Celite; Hydromatrix).\nTables\u00a01 and 2 provide general guidance for various preparation\/extraction and isolation\/QA steps in the analysis of PCBs and OCPs and sources of information. Starting with sample preparation, the basic approach is to assure that the sample is prepared for extraction in a room that is free from significant contamination. Ideally this would involve a well-ventilated lab with air prefiltered through HEPA and carbon filters, but any clean chemical laboratory facility should be adequate for most work on PCBs and OCPs in most matrices except water or soils and sediments from remote locations. Laboratory buildings built after about 1990 are likely to be free of PCB-containing materials (electrical equipment\/paints\/sealants) or OCPs used as insecticides (or termiticides in foundations) because of the effects of bans on the use of these substances promulgated during the 1970s and 1980s. \nTable\u00a01Extraction techniques used for solid environmental samples (from Lopez-Avila [64]; Wells and Hess [25])TechniqueOverviewMethod referenceConventional SoxhletSample + desiccant mixture in glass or paper thimble is leached with warm (condensed) solvent for 4\u201312\u00a0hrs. Solvents are, e.g., diethyl ether, DCM, hexaneEPA 3540 [65]Automated Soxhlet (e.g., \u201cSoxtec\u201d)Extraction thimble is immersed in boiling solvent (30\u201360\u00a0min) then raised for Soxhlet extraction. Solvent can also be evaporated.EPA 3541 [65]Supercritical fluid extraction (SFE)Sample (usually +desiccant) placed in high-pressure cartridge and carbon dioxide at 150\u2013450\u00a0atm at temp of 40\u2013150\u00a0\u00b0C passed through. After depressurization, analytes are collected in solvent trapEPA 3560\u20133562High-speed blendingUseful for high water content samples such as plant material. Homogenizes sample with acetone and NaCl.[21, 66]Column extractionSample (+desiccant) placed in large column with filter and stopcock. Eluted with large volume of extraction solvent, e.g., hexane:DCM; hexane[67]Sonication-assisted extractionSample in open or closed vessel immersed in solvent and heated with ultrasonic radiation using ultrasonic bath or probe.Method 3550C [65]Microwave-assisted extraction (MAE)Sample in open or closed vessel immersed in solvent and heated with microwave energy.EPA 3546 [65]Pressurized liquid extraction (PLE)Sample (usually +desiccant) placed in extraction cartridge and solvent (heated, pressurized) passed through then dispensed in extraction vial.EPA 3545 [61, 65]Table\u00a02Guidance for various preparation, extraction and isolation steps in the analysis of PCBs and OCPsEnvironmental matrixAnalytical stepsGeneral proceduresEPA or other methodSoil and sedimentPreparationPrepare in a PCB- and pesticide-free room.[35, 86]Avoid air-drying. Wet sieve if necessary to remove large particles. Centrifuge sediment to remove excess water.Mix soils\/sediments with desiccant such as Na2SO4.Separate determination of dry mass by oven drying. For sediments total organic carbon should be determined.QAOne blank, soil CRM every ten samples; spike all samples with recovery surrogate standards. Bake glassware overnight at 200\u00a0\u00b0C or higher.ExtractionSoxhlet, PLE, sonication, or MAE with acetone: hexane or DCMSolvent evaporation, transfer to hexane.Sulfur removal with activated copper turnings required for sediment.Isolation\/cleanupAlumina, silica or Florisil elutions: non-polar (hexane) and polar (DCM:hexane or equivalent)VegetationPreparationHomogenize using food chopper or blender. Cryoblending using liquid nitrogen or dry ice is useful. Mix with dessicant. Separate determination of dry mass by oven-drying.[21, 35]QASame as soil. Use vegetation CRM if possibleExtractionSame as soil.Isolation\/cleanupSame as soil.Aquatic biotaPreparationSelect muscle or liver depending on species. For mussels and crustaceans use soft tissue. Select tissue that has not been in contact with the sample container. Homogenize using food chopper or blender. Cryoblending is useful.[21, 35]Mix with drying agent. Separate determination of lipid content.QASame as soil. Use fish or mussel SRMs.ExtractionSoxhlet, pressurized liquid extraction, or column extraction. Use acetone:hexane or DCM.Isolation\/cleanupRemove lipid using gel permeation chromatography if possible or by repeated washing of the extract with sulfuric acid. Follow with fractionation on silica or Florisil columns as described for soil.Marine mammal blubberPreparationSelect blubber that has not been in contact with the sample container. Blend or hand-mix with drying agent. Separate determination of lipid content.[10, 87]QASame as soil. Use fish oil or marine mammal SRMs.Isolation\/cleanupSame as for fish extracts.Air (high volume)Extraction,QA and cleanupAssuming that air is collected on polyurethane foams or XAD resin, these would be extracted in a Soxhlet or pressurized liquid extractor. Other steps as for soil or sediments[32]Semi-permeable membrane devices (SPMD)PreparationSPMDs would be removed from their transport cases and rinsed with precleaned water to remove accumulated dust (air-borne samplers) or periphyton (water samplers).[46]QAUse PRCsExtraction, and cleanupAssuming that the SPMD is lipid-based, extraction of POPs by \u201cdialysis\u201d into hexane would be achieved in a large glass cylinder.Water (including melted snow, ice and wet precipitation)ExtractionLiquid\u2013liquid, SPE (e.g., C18) extraction for small (<1\u00a0L) samples; XAD-2 or modified \u201cSpeedisk\u201d for >1\u00a0L.US EPA 508.1 [34] and 525.2 [36, 37, 78, 88]QA and cleanupPre-spike XAD columns with surrogates.Blood plasmaExtractionExtract blood plasma with ammonium sulfate\/ ethanol\/hexane (1:1:3) or C18 SPE extraction.[72, 73]Determine lipid content.QASame as fish. Use NIST 1589a SRM.Isolation and cleanupSulfuric acid partitioning to remove lipids.Acid\u2013base silica for additional lipid removal.MilkExtractionLiquid\u2013liquid partitioning with acetone:hexane or C18 SPE extraction. Determine lipid content.[75, 77]QABCR SRM 284 & 533 milk powder.Isolation and cleanupAs with plasma.\nRecommended extraction and isolation techniques for PCBs and OCP\nRecovery surrogates\/internal standards\nA standard QA step in the analysis of PCB\/OCPs is to include surrogate recovery standards in each sample. These are generally one or two PCB congeners (e.g., CB30 and CB204) and OCP-related, unlabeled compounds such as pentachloronitrobenzene, endrin ketone, 1,3,5-tribromobenzene, and other compounds that are readily separated from individual OCPs and not commonly found in the environment. If GC\u2013MS is being used as the detection system then 13C-labeled or deuterated surrogates should be used. These standards are carefully added by pipetting the appropriate volume of a standard solution onto the sample matrix prior to extraction. Surrogates do not define absolute recoveries because they are not incorporated into the matrix; however, they do provide information on analyte recovery throughout the isolation process and can also provide sample-to-sample extraction efficiency comparability. If bioanalytical techniques are being used to screen extracts, it may not be possible to include recovery standards, particularly 13C-surrogates, since they may respond similarly to the native compounds.\nExtraction techniques\nThe appropriately prepared sample can then be extracted by any one of a number of techniques (Table\u00a01). The main points to consider are to allow adequate exposure time for the solvent system in the sample matrix and to limit sample handing steps, i.e., avoid filtration steps by using Soxhlet (sample in a glass thimble), extraction columns (sample matrix eluted after soaking in solvent) or semi-automated systems (e.g., pressurized liquid extractors, PLEs). Cross-contamination from residues left behind by high levels in other samples is a concern at this stage and equipment must be thoroughly cleaned and checked from batch to batch. Purity of extraction solvents is a major consideration here. Only high-purity glass-distilled solvents should be used, because later evaporation steps will concentrate any contaminants. PLE is increasingly being used to replace Soxhlet and column extraction methods [58\u201360]. PLE uses much less solvent than Soxhlet or column elution. However, it suffers the disadvantage of initial high cost and the need for a stable power supply to avoid premature instrument shutdown. Also, operation of an automated PLE requires regular scheduled maintenance by trained service personnel. Preparation of a homogeneous dry sample from wet tissue or sediment for PLE can be a challenge due to the limited size of PLE extraction vials, currently set at 100\u00a0ml. A US EPA method (3545) using PLE for solid waste extraction is available [61]. This method recommends acetone\/hexane (1:1, v \/v) or acetone\/dichloromethane (DCM) for extraction of PCBs from solid waste, and these solvent systems appear to be the most commonly used [59]. There has been a recent trend toward not using chlorinated (potentially toxic) solvents such as chloroform and DCM, due to concerns over exposure to lab personnel. Binary mixtures such as hexane\/acetone (1:1) are regarded as equally effective for the extraction of POPs from solid samples [62]. In general, extraction with a polar:apolar binary mixture has been found to be more efficient for recovering OCPs and PCBs from fish tissues of low lipid content than an apolar solvent [63].\nThe time- and solvent-consuming nature of Soxhlet extraction (or related techniques involving percolation of a solvent through the sample) is generally thought to be related to the slow diffusion and desorption of the analytes from the sample matrix. Semivolatile compounds such as naphthalenes can also be lost from Soxhlet apparatus via volatilization [68]. The use of microwave, sonication, supercritical fluids, or elevated temperatures and pressure (as in PLE) increases the rates of diffusion and desorption and thus speeds up extraction [58, 64, 69]. Pressurized hot water extraction has even been used to extract PCBs from sediment and soil. Under pressure, the dielectric constant of the water can be manipulated to facilitate the extraction of nonpolar analytes [70]. Sonication, microwave-assisted extraction (MAE) and supercritical fluid (SFE) extraction have all been successfully employed to recover PCBs and OCPs from solid samples (Table\u00a01). Soxhlet is regarded as more reliable than sonication, SFE, or shake flask-type methods and equivalent to (but not as rapid as) MAE and PLE for a wide range of environmental matrices [25].\nBlood plasma lipids can be rapidly extracted using conventional solvent partitioning using a mixture of ammonium sulfate\/ethanol\/hexane (1:1:3) [71, 72]. Solid-phase extraction using C18 cartridges is also a proven technique [73]. Needham et al. [74] have reviewed methods for the analysis of POPs in human milk. PCB\/OCPs can be extracted from milk samples using conventional lipid extraction techniques involving liquid\u2013liquid extraction with hexane\/acetone [75]. Blending with acetonitrile and potassium oxalate [76] followed by C18-solid phase extraction has also been used in recent studies [77].\nWater sampling devices such as solid-phase extraction (SPE) cartridges, XAD or SPMDs are extracted by elution or dialysis (with hexane for lipid-based SPMDs). The elution of reverse-phase or XAD resin water sampler cartridges generally involves the use of a water miscible solvent first to remove water followed by a solvent of intermediate polarity such as DCM, methyl t-butyl ether or ethyl acetate. Combined extracts are then partitioned into hexane. Usenko et al. [78] compared six different SPE devices for the recovery of PCBs and OCPs from natural waters. These included XAD-2 resin, C18-filter disks and various C18 - and divinyl benzene-based adsorbents. They found that C18-based products, particularly \u201cSpeedisk,\u201d a modified divinylbenzene solid-phase extraction device, gave the best overall recoveries.\nAnother water sampling device, solid-phase microextraction (SPME), involves immersing a polydimethylsiloxane-coated syringe into 3\u20135\u00a0ml samples. Hydrophobic compounds are adsorbed onto the coated fiber and the syringe then placed into a hot injection liner which desorbs these compounds into the GC [79]. The benefit of this approach is that it requires no solvent or multistepped cleanup\/concentration procedures. A drawback is that the adsorption efficiency can be affected by complex water matrices and may not be suitable for all water sample types [80]. Particulate phases collected by filtration on glass fiber filters are treated like sediments and should be extracted by Soxhlet or pressurized liquid extraction.\nDetermination of lipid content\nLipid contents of biological samples should be determined during the analysis of POPs. Most studies have determined total extractable lipid gravimetrically by drying a fraction of the sample extract to constant weight [25]. However, results can vary widely among laboratories due to different extraction efficiencies of various combinations of solvent and extraction apparatus [81]. The benchmark method for total lipid is that of Bligh and Dyer [82]. A modified version of this method has improved sample handling and method precision [83]. Smedes [84] demonstrated that propanol:cyclohexane:water (8:10:11) was an effective substitute for the Bligh and Dyer chloroform:methanol:water. The Smedes method gave more consistent results for extractable lipids in fish tissue with low lipid content (\u223c1% lipid). Samples with very low fractions of lipid, such as blood plasma, are best analyzed using a colorimetric method [85].\nIsolation of analytes from coextractives\nThere are two aspects to this phase of POPs analysis. The analytes must be separated from nonvolatile materials which affect the performance of GC columns, such as pigments, inorganic sulfur, and triglycerides. Also, there is a need to separate the OCPs and PCBs from each other as much as possible prior to GC analysis in order to limit coelution problems. These isolation steps are also useful, and sometimes essential, for the application of bioanalytical methods to PCBs\/OCPs and dioxin-like activity (See \u201cBioanalytical methods for the quantification of OCPs and PCBs\u201d).\nAdsorption \u201ccleanup\u201d columns\nSeparation of PCB\/OCPs from coextractives can be relatively straightforward for low-lipid samples such as soils, sediments and vegetation (Table\u00a02). Generally, small silica gel or Florisil columns (either prepared in the lab or pre-purchased) should suffice. The purpose of this step is to remove coextractive pigments and to separate nonpolar PCBs (plus p,p\u2032-DDE) from more polar OCPs (HCH, most chlordanes, dieldrin\/endrin). This is achieved by applying the extract in a small volume of apolar solvent to fractionation by eluting with hexane followed by one or two other elutions of increasing polarity. Polar compounds are retained on the column. Basic alumina and silica gel columns have been effective for the separation of OCPs from fish lipids [89], although there is the possibility of minor losses due to dehydrochlorination of some OCPs, e.g., p,p\u2032-DDT, on the alumina. The effectiveness of these adsorption columns depends on the mass and the water content of the adsorbent together with the polarity of the solvent. In general 3\u20138\u00a0g of absorbent is used in a 0.5\u20131.0\u00a0cm-diameter column with silica gel or Florisil deactivated with a low percentage of water (0\u20135%) [51, 90]. Typically the sample is applied to the top of the column in hexane and then a first elution solely of hexane separates PCBs from many OCPs. Subsequent elutions with hexane\u2013diethyl ether or hexane\u2013DCM recover the OCPs (p,p\u2032-DDT, dieldrin, toxaphene) [21, 76]. Alumina and Florisil have the capacity to retain about 100\u00a0mg lipid per 10\u00a0g of adsorbent [26].\nSize-exclusion columns\nFor high-lipid samples (>10% lipid), such as some fish tissues (e.g., salmonid muscle, cod livers) and marine mammal blubber, a lipid removal step must be included. This can be achieved using size exclusion or gel permeation chromatography (GPC), either in automated systems, using HPLC columns or by gravity flow columns. A 60\u00a0g bed of Bio-Beads SX3, a neutral porous styrene divinylbenzene resin (BioRad Laboratories, Hercules, CA, USA) can accommodate up to 1\u00a0g of lipid and achieves the separation of fish lipids from most PCBs and OCPs with a size exclusion of \u223c400 Da [67]. Equivalent HPLC size-exclusion columns can be used [91]. The advantages of GPC are that it is nondestructive and that columns can be reused. A disadvantage is a requirement for large volumes of purified solvent (low-pressure or gravity systems) or expensive columns (HPLC). Automated GPC units are commercially available and allow for unsupervised elution of 12\u201324 samples. Due to required rinses of the unit sample lines, a portion of the sample is lost and must be accounted for when calculating final analyte concentrations. GPC eluates generally require an adsorption column fractionation step on silica or Florisil to remove remaining low molecular weight lipids, waxes and pigments that are not completely separated from the PCB\/OCPs.\nLipid destruction\nLipid removal using sulfuric acid washing or elution of the extract through sulfuric acid (50%) in silica or KOH-treated silica columns is also effective for most PCB\/OCPs, but does result in loss of some analytes such as dieldrin [92, 93]. Ethanolic\u2013KOH treatment of Soxhlet extracts is also suitable for lipid removal and works well for PCBs [87], but may degrade chlorinated aliphatics such as DDT, which is converted to DDE. The acid- or base-treated extracts containing the PCBs and OCPs are then subjected to an adsorption column fractionation step on silica or Florisil.\nSulfur removal\nSulfur is coextracted with PCBs and OCPs and presents a particular problem for GC\u2013ECD analysis of sediment extracts because of its strong response in this detector. It is also cytotoxic and needs to be eliminated prior to use with in vitro bioassays [94]. Sulfur can be removed by GPC but can also be removed using activated copper turnings (washed with concentrated HCl and held in an apolar solvent) or using mercury. The latter method removes sulfur more efficiently but is not recommended due to potential for contamination of the lab and lab effluent. Shaking with tetrabutylammonium sulfide has also been used to remove sulfur [95].\nEvaporation steps\nSolvent evaporation is generally used several times within all analytical methods for PCB\/OCPs and successful analyte recoveries critically depend on minimizing losses during this step. Surrogate standards may be added prior to evaporation to monitor compound losses [96]. The Pesticide Analytical Manual of the US FDA [21] includes a discussion of solvent evaporation and an overview of the most widely used equipment, rotary evaporators, Kuderna\u2013Danish apparatus and TurboVap equipment. The US FDA [21] overview favors the rotary evaporator because of greater range of use, e.g., for temperature-sensitive and semivolatile analytes, but notes that it is applicable to only one sample while both the TurboVap and K\u2013D apparatus can be set-up for multiple samples.\nPreparation for GC analysis\nFollowing fractionation on silica or Florisil, final extracts are prepared in GC vials for analysis. Addition of an internal standard to check solvent volume is recommended at this stage. Careful evaporation is required at this step, and only high-purity compressed gas (usually nitrogen) should be used. This can be done using a stream of regulated gas via a disposable glass pipet and heating block or via multineedle devices (e.g., \u201cN-Evap\u201d).\nQuantification methods\nOverview\nNumerous analytical approaches are available for quantifying PCBs and OCPs. As with extraction\/isolation steps, only general comments are provided here for GC analysis of ortho-substituted PCBs and OCPs. However, a major consideration is that the laboratory will have access to modern capillary GC equipment and either electron capture or mass spectrometry detection. Some general guidance on the application of gas chromatographic analysis of ortho-substituted PCBs and OCPs is provided in Table\u00a04. Cochran and Frame [97], in their review of the GC analysis of PCBs, noted that separation requires attention to (1) proper injection to minimize analyte band-broadening; (2) choice of carrier gas; (3) optimized carrier gas velocity; (4) GC oven programming; (5) column dimensions such as length, inside diameter, film thickness, number of plates; and (6) the type of column stationary phase.\nBioanalytical methods based on immunoassays, or in vitro bioassays for dioxin-like activity, have become widely available over the past ten\u00a0years for the screening of sample extracts for POPs. These methods and selected applications are briefly reviewed in this section.\nGC injection ports\nOptimization of injection conditions is critical to proper GC analysis [98, 99]. In the analysis of POPs and OCPs, problems often occur with nonvolatile coextractives such as triglycerides and pigments that, despite various isolation procedures, are still present in final extracts. Most GC applications for PCB and OCP analysis have employed split\/splitless injection systems, although on-column injection has also been used. Although on-column injection avoids artifacts associated with heated split\/splitless systems (i.e., degradation of labile compounds), it requires highly clean extracts to avoid matrix effects [99]. Retention gaps consisting of an empty capillary column have been used to reduce these effects and permit larger on-column injection volumes. Pressure programming allows improved control over carrier gas flow, resulting in improved separation of PCB and OCPs.\nGC columns\nFused silica open tubular capillary columns, generally coated with nonpolar or medium-polarity chemically bonded liquid phases are almost universally used for GC separation of PCBs and OCPs [99]. The use of capillary columns revolutionized PCB analysis, allowing identification of the individual congeners. This improved the comparability of the analytical data from different sources and helped to establish a basis for the understanding of geochemical cycles and toxicological implications. The basic technology for separation of PCB congeners described by Mullins et al. [100] has not changed greatly over the years. These authors used a 5% phenyl methyl silicone phase and a long temperature program (100\u00a0min) to achieve high-resolution separation of PCB congeners. Improved routine separations of PCBs have been achieved using 60\u00a0m\u00d70.25\u00a0mm i.d. columns with hydrogen carrier gas. PCBs within a homolog group elute according to their number of ortho chlorines: 4<3<2<1<0. Coelution of congeners remains a problem for routine analysis by GC\u2013ECD. However, several modified polydimethylsiloxane phases with n-octyl or n-octyldecyl substituents, e.g., DB-XLB (J&W Scientific, Folsom, CA, USA) and HT 8 (SGE Inc., Austin, TX, USA) can resolve all but four pairs of significant congeners and five pairs of minor congeners [18, 101] using mass spectrometry detection. The coelution of PCB congeners on the more commonly used 30\u00a0m DB-5 columns is outlined in Table\u00a03. Coelutions are thus important even for routinely monitored congeners such as CB 28, 31, 105 and 153. However, separation of these congeners can be routinely achieved on 60\u00a0m 5% phenyl methyl silicone phase capillary columns using H2 carrier gas and on n-octyl phases as well [18, 101]. Use of dual-column GC, in which the sample is injected simultaneously into nonpolar and medium-polar phase columns, is recommended if a suitable instrument is available. Alternatively, confirmation of peak identity in a subset of samples, using a second column of different polarity, can be done instead. Although other carrier gases such as He can be used, the best peak resolution and efficiency is achieved by using H2. \nTable\u00a03Significant PCB congener coelutions on 5% phenyl phases [97]ClassificationaCoeluting PCBsNumber of chlorinesA4, 101, 2A9, 72, 2A12, 132, 2A17, 152, 3 A27, 243, 3A32, 163, 3A28, 313, 3A33, 20, 533, 3, 4A43, 494, 4A47, 75, 484, 4, 4A44, 594, 4A37, 424, 4A71, 41, 644, 4, 4A66, 954, 5A56, 604, 4A84, 89, 101, 905, 5, 5, 5A117, 87, 1155, 5, 5A77, 1104, 5A135, 144, 1246, 6, 5A147, 1096, 5A123, 139, 149, 1185, 5, 6, 5A114, 1335, 6A131, 1226, 5A153, 132, 1056, 6, 5A176, 1307, 6A164, 163, 1386, 6, 6A158, 1296, 6A175, 1667, 6A173, 157, 2017, 6, 8A170, 1907, 7A198, 1998, 8A203, 1968, 8aCapital A indicates a major Aroclor congener\nCoelution of major OCPs with each other or with PCBs is also a problem in GC\u2013ECD analysis, despite preseparation by adsorption chromatography. For example, p,p\u2032-DDD can coelute with cis-nonachlor, CB99 with a trans-nonachlor isomer [10], p,p\u2032-DDE with dieldrin and CB85, and toxaphene congener Parlar 50 with CB128. Recently, the coelution of CB180 and brominated diphenyl ether congener 47 has also been noted [102].\nChiral GC separation of OCPs and PCBs\nMost OCPs on the POPs list have optically active or chiral isomers (e.g., \u03b1-HCH, o,p\u2032-DDT, the main constituents of technical chlordane, cis-\/trans-chlordane, heptachlor, as well as chlorobornanes in toxaphene) [103]. There are also 19 PCB atropisomers which are also chiral as a result of restricted rotation at the central biphenyl bond. While PCBs and OCPs are racemic mixtures when manufactured, microbial degradation in soils and water as well as biotransformation reactions in biota can result in nonracemic patterns in environmental samples. Crucial for chiral analysis is the availability of chiral capillary GC columns such as those with various cyclodextrins chemically bonded to a polysiloxane. These phases are relatively heat-stable and have low bleed [104]. Current methods range from the simple use of 30\u00a0m chiral columns to a two-dimensional \u201cheart-cutting\u201d technique. This involves a redirection of a targeted portion of the sample after elution from a methyl\/phenyl polysiloxane column into a chiral column via a computer-controlled pneumatic valve. This provides higher peak capacity and generally further separation of chiral compounds. While use of chiral GC separations is not part of routine OCP analysis, it is a well-developed technology that is relatively easy to implement in existing GC\u2013ECD and GC\u2013MS instruments. This technique has useful applications for tracking sources of OCPs and transformations of PCBs. For example, Jantunen et al. [105]) and Leone et al. [106] have shown that enantiomer fractions of chlordane isomers can be used to identify emissions of this pesticide from soils. Wong et al. [107] calculated minimum transformation rates for PCB atrophisomers CB95 and 136 in the Lake Superior food web using enantiomer ratios in predators and prey species.\nGC\u2013ECD\nSince the 1960s, POPs have been determined by GC with electron capture detection (ECD), initially using packed columns. Capillary GC\u2013ECD began to be routinely applied by the early 1980s. Use of GC\u2013ECD is recommended for routine analysis of OCPs, except for toxaphene, as well as for ortho-PCBs (Table\u00a04) but not for non-ortho-PCBs [14]. This instrumentation is widely available at relatively low cost from at least four instrument manufacturers. A substantial knowledge base exists on the use of this 40-year-old technology. GC\u2013ECD is capable of determining PCB\/OCPs at low ng\/g levels or higher in environmental matrices. Although at one time tritium-based ECDs were once available, the 63Ni detector is now universally used. This detector is operated at high temperatures, 300\u2013350\u00a0\u00b0C, which makes it relatively unaffected by column bleed. ECD suffers from the potential for false positives due to interferences such as those from sulfur, phthalate esters, and negative peaks generated by hydrocarbons. ECDs are normally operated with N2 or argon\/methane gas, which combines with the flow from the GC column (He or H2 carrier gas). Gases used for GC\u2013ECD must be ultrapure to protect both the GC column (which can be oxidized by trace oxygen or siloxanes hydrolyzed by trace water) and the ECD itself. Recent refinements in ECD technology include the use of microcells which have greater linear range than older detector cells [108] and can provide greater sensitivity. \nTable\u00a04General guidance on GC analysis and data reporting for PCBs and OCPsGC detectorAnalytesConfigurationAdvantages\/disadvantagesDetection limitsaCapillary GC - with electron capture detectionAll ortho-subsituted PCBs & all OCPs on the POPs list except toxaphene30 or 60\u00a0m\u00d70.25\u00a0mm id. Column with H2 carrier gas. Dual column nonpolar (DB-1) and intermediate polarity columns (DB-5).Relatively inexpensive and easy to operate. Similar response factors for most OCs.DDT\/DDE \u223c 1\u00a0pgGood sensitivity for all POPs. Adequate for routine tasks. High potential for misidentification of some POPs due to coeluting peaks.HCB \u223c0.5\u00a0pgQuadrupole mass spectrometry in electron ionization (EI) modeAll PCBs & all OCPs on the POPs list except toxaphene30\u00a0m\u00d70.25\u00a0mm i.d. low-bleed columns with He carrier gas. Selected ion mode for target POPs. Moderately expensive and more complex to operate and maintain. Newer instruments (post 1997) have adequate sensitivity for routine POPs monitoring at low pg\/\u03bcL concentrations. Much less potential for misidentification than with ECD.DDT\/DDE \u223c1\u201310\u00a0pgHCB \u223c1\u201310\u00a0pgDieldrin \u223c25\u00a0pgToxaphene \u223c500\u00a0pg (as tech mixture)Quadrupole mass spectrometry in electron capture negative ionization (ECNIMS) modeToxaphene and other highly chlorinated OCPs and PCB with >4 chlorines30\u00a0m\u00d70.25\u00a0mm i.d. low-bleed columns with He carrier gas. Selected ion mode for target POPs.Comparable sensitivity in ECNIMS mode to ECD in SIM mode for some POPs. Much less potential for misidentification than with ECD.DDT\/DDE \u223c0.1\u00a0pgHCB \u223c0.1\u00a0pgDieldrin \u223c1\u00a0pgToxaphene \u223c10\u00a0pg (as tech mixture)Ion trap mass spectrometry using MS\/MS modeAll PCBs, All OCPs on the POPs list30\u00a0m\u00d70.25\u00a0mm i.d. low-bleed columns with He carrier gas. Same columns as quadrupole MS.Comparable sensitivity to ECD in MS\/MS mode for some POPs. Much less potential for misidentification than with ECD.DDT\/DDE \u223c1\u00a0pgHCB \u223c1\u00a0pgDieldrin \u223c5\u00a0pgToxaphene \u223c100\u00a0pg (as tech mixture)High-resolution magnetic sector mass spectrometry in electron ionization (EI) modeAll PCBs, all OCPs on the POPs list except toxaphene30\u00a0m\u00d70.25\u00a0mm i.d. low-bleed columns with He carrier gas. Selected ion mode for target POPs at 10,000\u00d7 resolution.Comparable sensitivity to ECD in SIM mode. Highly reliable identification at low pg\/\u03bcL levels.DDT\/DDE \u223c0.05\u00a0pgHCB \u223c0.05\u00a0pgDieldrin \u223c0.1\u20130.5\u00a0pgToxaphene \u223c10\u00a0pg (as tech mixture)aInstrumental detection limits at S\/N of \u223c10\nGC\u2013MS\nCapillary GC coupled with mass spectrometry (GC\u2013MS) revolutionized environmental organic analysis in the 1980s [109], particularly with the advent of bench-top instruments. Current GC\u2013low-resolution (quadrupole) mass spectrometry (LRMS) instrumentation is capable of determining most PCB\/OCPs at low pg concentrations using electron ionization (EI) in selected ion mode. Electron capture negative ion ionization (ECNI) is capable of detection of low femtogram amounts of highly chlorinated OCPs such as chlordane and toxaphene congeners. In this mode, a reagent gas, usually methane, is introduced into the ion source and negative ions are monitored. The ECNI mode also has a more limited linear range than the EI mode, is more difficult to operate routinely due to greater sensitivity to temperature variations, and needs more frequent cleaning due to the reaction of the reagent gas with surfaces in the ion source.\nGC\u2013ion trap MS (ITMS) in MS\/MS mode offers an equivalent sensitivity to quadrupole ITMS in selected ion mode and improved specificity by examining product ions of major mass fragments. ITMS has been successfully applied to detect mono-ortho-PCBs at subpicogram levels in a range of environmental samples [110] and to determine pg levels of toxaphene congeners [90]. In general, both quadrupole and ion trap LRMS can suffer from false positives due to unit mass resolution. Thus experienced analysts are needed to interpret results using confirmatory information such as full-scan analyses, fragmentation patterns and ion ratios.\nGC coupled to HRMS provides much higher specificity for individual PCB\/OCPs due to its ability to provide millimass resolution and can also be used for the determination of all ortho-substituted PCBs (e.g., EPA Method 1668 [35]) and OCPs too, and provides a very high level of confidence in the results compared to GC\u2013ECD and LRMS.\nThe use of GC\u2013MS allows for the application of isotope-dilution techniques and these have increasingly come into routine use for PCB\/OCP analysis in environmental samples due to the sensitivities of the latest generation of quadrupole and ion-trap MS systems [111, 112]. A wide range of PCBs and OCPs are available as 13C-labeled compounds from several chemical supply companies, which, when added at the extraction step, increase precision and reproducibility for the native analytes.\nBioanalytical methods for the quantification of OCPs and PCBs\nEnzyme-linked immunoabsorbent assays (ELISA)\nELISAs have been used to quantify most DDT\/DDE, HCH isomers, toxaphene and cyclodiene OCPs as well as PCBs in environmental samples [113, 114]. ELISAs are based on competitive binding in which the binder molecule, an excess amount of labeled analyte or coating antigen, and the target analyte are allow to approach equilibrium. The sample antigen competes with the coated antigen for binding sites on the labeled antibody; after a wash step, detection is performed by adding substrate and chromophore. Quantification is generally performed via spectrophotometric measurements and the amount of analyte in the sample is interpolated from a calibration curve. Although widely used to screen for current-use herbicides and insecticides as well as their polar metabolites [115], the development of competitive immunoassays for neutral hydrophobic OCPs has lagged [114]. This is in part due to the need for low detection limits. Also, ELISA analysis for some OCPs such as HCH (and lindane) has been challenging due to the small size of the HCH molecule, its structural symmetry, and possibly most importantly, its lack of aromatic structures or other atomic groups capable of supporting delocalized electron networks [116]. The development of a sensitive ELISA for DDT required synthesis of a ligand with Cl replaced by b-alanine carboxamide and lindane after degradation to trichlorobenzenes [117]. Commercial ELISA kits for detection of PCBs and most OCPs are available from Millipore Corp. (Billerica, MA, USA) and Strategic Diagnostics (Newark, DE, USA). Detection limits for PCBs and OCPs are typically in the low \u03bcg\/L range in water or 0.1\u20131\u00a0\u03bcg\/g range in soil and plant extracts. These kits are meant to be used with relatively little sample preparation and, although semiquantitative, are ideal for screening samples and complement more elaborate techniques involving GC analysis (Fig.\u00a01).\nFig.\u00a01Illustration of the basic components of an ELISA for detection of OCPs and PCBs in environmental samples or extracts. Sample antigen (analyte) competes with antigen for binding sites on coating protein; after a wash step, detection is performed by adding substrate and chromophore\nShivaramaiah et al. [118] used an ELISA specific for DDE to survey DDT residues in surface soils in three river valleys of northern New South Wales, Australia. The technique allowed the analysis of large numbers of samples, thus permitting the areas of contamination to be mapped. Galloway et al. [119] used an ELISA to detect PCBs in extracts of mussels from New Bedford Harbor (USA). The ELISA and GC\u2013ECD results were highly correlated, although the latter were about 20% lower than quantitation by GC. Samples were extracted and lipid partially removed by chromatography on Florisil prior to exchanging the sample into a phosphate buffer\/methanol solution for the immunoassay. The use of the ELISA saved additional isolation steps and GC analysis. In general, fatty coextractive materials must be removed prior to application of ELISAs for OCPs. Skerritt et al. [120] examined the application of ELISAs for DDT and cyclodiene insecticides, heptachlor and endosulfan in plant-derived foods. They found that cleanup was necessary for foods that yielded highly colored extracts such as coffee and spinach and for oily products such as cottonseed.\nQuality assurance issues for PCBs and OCPs\nAncillary data\nA significant amount of ancillary data is required in order for the PCB\/OCPs measurements to be properly interpreted in terms of temporal and\/or spatial trends and exposure of wildlife and humans. Tanabe and Subramanian [2] have extensively reviewed the factors to be considered for the selection of bioindicator species, including human tissues. Analytical lab reports need to include quality assurance data so that results and lab performance can be assessed independently [6], as outlined in Table\u00a05. The objective here would be to have a record of the entire processing of the sample from preparation through to reporting concentrations that can be evaluated independently. Therefore, the individual labs should report concentrations for analytes, blanks and reference materials. Data reports should also include instrument calibration results and recoveries. This would enable method detection limits (MDLs) to be calculated independently of the lab if necessary. \nTable\u00a05Minimum reporting dataset for POPs analysisInformationDetailsSampling protocolsMethod, number, size and representativenessStorage temperature and locationSample tracking informationDate received, date analyzed, lab batch number or other unique identifiedPublished analytical methode.g., EPA methodLimit of detection\/quantificationSee \u201cQA procedures\u201dBlanksReagents and also field blanks if possibleRecoveriesDuplicatesCalibrationSource of standards; date stocks preparedSurrogate and internal standardsQA of cofactorsSuch as lipid, organic carbon and moisture contentConfirmatory testse.g., Use of second GC column or other detection systemData manipulationsBlank subtraction, recovery correction\nField and lab blanks\nDetermination of PCBs and OCPs requires the analysis of blank samples because of the ubiquitous nature of these contaminants, as discussed previously. For air and water, the sampling media (i.e., pre-extracted water, or the XAD resin, C18 cartridges, or SPMDs) can serve as blanks. Ideally these media should be transported to the field and exposed briefly to the sampling equipment, thus serving as a field blank. Additional samples of the media should be archived in the preparation lab for use as lab blanks. For soils and fatty samples, US EPA Method 1668 for PCBs recommends sand and corn oil, respectively, as lab reference blanks [35]. Clean facilities, ideally with HEPA and carbon-filtered air are recommended, but are not essential, for the determination of PCBs, except for ultralow levels in water. Contamination of lab air can be checked by use of SPMDs or by exposing C18-disks in the lab for a specified period.\nQA procedures\nKey elements in QA\/QC for POPs analysis are the use of reference materials, the use of quality charts, participation in interlaboratory studies, and the use of guidelines for sampling and analysis [121].\nRecovery studies in which individual analytes are spiked into the sample matrix prior to extraction are useful for determining the performance of the analytical method. However, they do not provide much information on the true extractability of analytes from naturally contaminated matrices. This can only be accomplished by extracting certified reference materials (CRMs) or, if none are available for a particular type of sample, some other well-characterized material that has been analyzed by an experienced reference lab [25, 74]. Recovery studies can be also useful for determining method detection limits if conducted at concentrations within 3\u20135 times the estimated limit of detection determined from the instrumental response or from blanks [122].\nAs a routine measure, spiking surrogate recovery standards into each sample provides useful information on losses of analyte from the extraction step onwards. However, no single PCB or OCP can be representative of all of the organochlorines being determined, and thus recovery correction should be performed with caution. Isotopically labeled surrogates are ideal for analyses of PCBs and OCPs that are being performed by LRMS and HRMS, and isotope dilution techniques correct for the recoveries of these surrogates.\nIf blanks are significant (for example, averaging greater than 10% of the average level of total PCBs), then blank correction should be carried out. This should only be done with a robust method blank based on at least seven samples or a blank known to be representative of a batch of samples. Ferrario et al. [29] subtracted an MDL value based on the blank+2\u00d7\u03c3b on the rationale that this ensured a low probability (5%) of false positives compared to subtraction of the average blank only.\nDetection limits\nDetection limits for PCB\/OCPs depend on the analytical method but also on the sample size and QA considerations, e.g., information available from blank or control samples and recovery studies. The selection of detection limits also depends on the goals of the program and how much emphasis is placed on the reliability of the results versus the need to achieve broad geographic coverage, to meet or exceed regulatory or risk assessment guidelines (Electronic Supplementary Material, Table\u00a03 [145\u2013152]). and to avoid reporting \u201cless thans\u201d for a high proportion of samples. A recent US EPA report has reviewed the approaches to calculating detection limits in the analysis of POPs and other environmental contaminants [122].\nDetection limits are usually expressed as a concentration, i.e., they are based on the average weight of the sample analyzed, as summarized in Table\u00a06. The recommended method for determining the MDL [122] is to analyze a series of samples, as free from the target analyte as possible (a minimum of seven replicates), that have a concentration of the analyte that is at least equal to, or in the same concentration range as (within a factor of five of), the estimated detection limit. All of the replicate aliquots are processed through the entire analytical method. The standard deviation (\u03c3) of the replicate measurements is determined and the MDL is then determined by multiplying the standard deviation by the Student\u2019s t-value at a 99% percentile for n\u22121 degrees of freedom. The MDL is thus an estimate of the measured concentration at which there is 99% confidence that a given analyte is present in a given sample matrix. The MDL is defined by the analytical laboratory and is likely to vary between labs because of different blanks and instrumental conditions. \nTable\u00a06Detection limits defined by various organizationsOrganizationTerminologyCalculationUS EPAMethod detection limit (MDL)Minimum level of quantitation (ML)American Chemical SocietyLimit of detection (LOD)Limit of quantitation (LOQ)International Organization for Standardization \/ International Union of Pure and Applied Chemistry (ISO\/IUPAC)Critical value (CRV) minimum detectable value (MDV)Limit of quantitation. (LOQ ISO\/IUPAC)\nThe MDL is analogous to the \u201cCRV\u201d described by Currie [123] and reported by ISO\/IUPAC, which is tn\u22121, 95\u00d7\u03c3b of a sample blank. Note that the t-value is based on the 95th percentile. Thus the MDL and CRV differ in the multiplier used. There may be practical difficulties with measuring some analytes if there no blank value, however the \u03c3 of a low-concentration standard can be used. The MDV is similar to the CRV but measured at the detection limit.\nLimit of detection (LOD) described by the American Chemical Society [124\u2013126] is equal to the field blank b plus three times the standard deviation of the blank (\u03c3b).  is numerically equivalent to the MDL as b approaches zero or when results are blank-subtracted. This definition has been widely used because of its publication in the peer-reviewed literature. It should be noted that the US EPA \u201cMDL\u201d and the ISO\/IUPAC \u201cCRV\u201d implicitly assume that results are adjusted for interferences or blank values.\nThe US EPA [122] and other organizations have also defined various lowest levels of quantitation which give a higher degree of confidence in the results. The \u201cminimum level of quantitation\u201d is the lowest level at which the entire analytical system must give a recognizable signal and an acceptable calibration point for the analyte. It is \u201cequivalent to the concentration of the lowest calibration standard, assuming that all method-specified sample weights, volumes, and clean up procedures have been employed. The ML is calculated by multiplying the MDL by 3.18 and rounding the results to the number nearest to (1, 2, or 5) \u00d710n, where n is an integer.\u201d [122]. The ACS LOQ is defined as being  b+10\u00d7\u03c3b above the gross blank. The ISO\/IUPAC LOQ is defined as \u201cthe signal or analyte (true) value that will produce estimates having a specified relative standard deviation (RSD), commonly 10%.\u201d [123]. It is calculated as:  where \u03c3Q is the standard deviation of the blank, and where KQ=10.\nTable\u00a07 presents some approximate values for MDLs that should be achievable assuming typical responses of ECD and MS systems, as well as three types of bioanalytical methods, to various OCPs\/PCBs and negligible blanks for the individual PCBs and OCPs in a 10 g sample. \nTable\u00a07Estimated MDLs for individual PCBs and OCPs (ng\/g wet wt), after Liem [127]AnalyteELISA (soil\/fly ash)ECDaMSb (low resolution; EI-SIM)MSc (high resolution; EI-SIM)PCB 28\u20130.050.10.01PCB 52\u20130.050.10.01PCB153\u20130.050.050.005PCB180\u20130.050.020.005p,p\u2032-DDE500.050.050.01Toxaphene5000.050.020.005Lindane4000.010.040.01HCB\u20130.010.020.005Dieldrin100\u20135000.010.020.01cis-Chlordane100\u20135000.030.050.01Total PCB 100\u20135000.1\u20131.00.1\u20131.00.01\u20130.1aAssumes MDL=3\u00d7SD of low-level spike (US EPA 2003), assuming 10\u00a0g sample and a sample volume of 0.5\u00a0mLbAssumes use of isotope dilution (13C) surrogates; electron ionization, selected ion monitoring mode with 10\u00a0g sample, and a sample volume of 0.5\u00a0mLcAssumes use of isotope dilution (13C) surrogates; electron ionization, selected ion monitoring mode with 10\u00a0g sample, and a sample final volume of 0.05\u00a0mL\nReference materials\nCertified reference materials (CRMs; also known as standard reference material or SRM) are essential for determining the accuracy of analytical methods for most PCBs and OCPs [128\u2013130]. The CRMs currently available are shown in Table\u00a08. While no single CRM is currently certified for all POPs, there are published uncertified values available for a broad array of individual PCBs, DDT isomers and toxaphene congeners, especially for NIST 1588a cod liver [131]. Sediment CRMs are freeze-dried and thus potentially not representative of sediment materials normally extracted. Some CRMs, notably SRM 1945a, cannot be shipped outside of the USA without CITES permits. \nTable\u00a08CRMs for PCBs\/OCPs in biota and sediment (adapted from de Boer and McGovern [128])CRMSourceTissue\/speciescis-CHLtrans-CHLDieldrinDDTHCBMirexPCB congenersSRM1974bNISTmusselXXXXSRM1976NISTlake troutXXXXSRM1588aNISTcod liverXXXXXSRM1945NISTwhale blXXXXXXSRM2974NISTmusselXXXSRM2977NISTmusselXXXXXSRM2978NISTmusselXXXX140\/OCIAEAplantXXXBCR598BCRcod liverXXXXXXCARP-1NRCCcarpXBCR349BCRcod liverXBCR350BCRmackerelXBCR682BCRmusselXBCR618BCRherringXEDF 2525CILlake troutXEDF 2514CILsoilXSRM1944NISTsedimentXXXXSRM1939aNISTsedimentXXXIAEA383IAEAsedimentXIAEA408IAEAsedimentXXXHS-1NRCCsedimentXHS-2NRCCsedimentXBCR536BCRsedimentXDX-1BCRsedimentDX-1BCRsediment\nCriteria for evaluating the desirability and efficacy of different analytical methods in environmental monitoring and surveillance\nOverview\nThe data generated by chemical analytical and bioanalytical methods for PCBs\/OCPs are often used to assess spatial and temporal trends of contamination in environmental media on a national, regional and international basis. The data will also be used by regulators and risk assessors to determine if concentrations exceed guidelines. Thus methods must give accurate results, which are comparable among labs, and at concentrations which are at or below action limits for food or guidelines on wildlife protection (see Electronic Supplementary Material Table\u00a03 [145\u2013152]).\nComparison of detection limits for widely used instrumentation for PCBs and OCPs in Table\u00a07 with guideline limits (Electronic Supplementary Material Table\u00a03) suggests that conventional chemical analytical methodology and bioanalytical methods can meet and exceed these limits in some cases by orders of magnitude. Sample size, lack of coextractive interferences, and final volume are important considerations.\nThe QA programs discussed above can assure laboratory personnel that accurate and reproducible results are being generated and can be validated by participating in regional, national or international interlaboratory comparisons using samples with assigned values [132]. In the context of implementation of the Stockholm POPs Convention, global comparability is the goal and thus successful participation in international programs should be a requirement of any participating lab. The challenge for future POPs monitoring, especially in developing countries, will be to assess the quality of data and to provide guidance for best laboratory practices.\nInterlaboratory comparisons\nInterlaboratory comparisons of POPs were conducted, along with the first environmental measurements, in the late 1960s, and became widely adopted in the 1990s [27]. The major international programs related to PCBs and OCPs are the QUASIMEME (Quality Assurance of Information for Marine Environmental Monitoring in Europe) program [53] and the determination of OCPs in food organized in 1993 and 1994 by the Global Environmental Monitoring Scheme (GEMS) of the World Health Organisation (WHO) [133]. Other important ongoing programs are the determination of OCPs\/PCBs in human milk under the auspices of AMAP [134] and the intercomparisons operated by the National Institute of Standards and Technology and the Marine Mammal Health and the National Oceanic Atmospheric Administration, National Marine Fishery Service. These programs have been a major stimulus for improved analysis of POPs globally in the past ten\u00a0years.\nThe results from interlab programs indicate that training and specific guidance on key aspects of the analytical methods, such as preparation of calibration solutions, are important [26].\nPCB\/OCP method accuracy\nHow accurate should analytical methods be? Obviously, they should be accurate enough to identify changes in temporal or spatial trends in contaminant concentrations [121]. Using the approach of Nicholson [135], accuracy is defined as being 95% certainty that the measured values will fall within upper and lower limits defined by: \nwhere \u03bc is the true concentration, \u03c3 is the precision, \u00b1b is the bias, where 1.645=tn\u22121, 0.95, and where the dataset is normally distributed. Both \u03c3 and \u2223b\u2223 can be calculated from multiple analyses of a CRM, where \u03c3 is the standard deviation of the analyte and \u2223b\u2223 is the relative percent deviation from the certified value (\u03bc). As a percentage of the true value, the accuracy is given as: \nBetween-laboratory accuracies of 15\u201320% have been achieved for PCB congeners among experienced labs participating in QUASIMEME [27].\nThe European Commission [136] has defined the acceptability of an analytical method based on repeated analysis of PCB\/OCPs in a CRM as follows: \n\u221250% to +20% for analytes in the range of <1\u00a0\u03bcg\/kg\u221230% to +10% for analytes >1\u00a0\u03bcg\/kg to 10\u00a0\u03bcg\/kg\u221220% to +10% for analytes >10\u00a0\u03bcg\/kg.\nThese percentages are also recommended acceptability guidelines for samples spiked with PCBs\/OCPs where no CRM is available.\nEmerging issues in analytical methods and future directions\nAnalytical methods for trace organic analysis are constantly evolving and improving, as can be seen from recent reviews of environmental analysis [58, 137, 138]. Although PCB\/OCP analysis is a mature area within environmental analytical chemistry, with basic methods that have remained unchanged for the past 20\u00a0years, there have been many new technologies that have been gradually adopted and undoubtedly more will emerge in the future. Some emerging procedures with low environmental impact (SPME, microscale glassware, low solvent use, etc.) may be particularly suited for developing countries where analytical budgets are small and product delivery times are lengthy. Thus, strategies must be considered that will allow improved techniques to be adopted by such labs.\nA major development in the analysis of POPs has been the introduction of semi-automated extraction instruments for PLE (Accelerated Solvent Extraction; Dionex Instruments, Sunnyvale, CA, USA). The PLE extraction can be combined with solid-phase adsorbents to extract and isolate analytes of interest, for example to remove lipids during the extraction of POPs from fatty samples [139]. In general, automated and semi-automated systems are available for cleanup and isolation of samples for PCB\/OCP using disposable solid-phase cartridges, as well as high-performance\/pressure LC columns.\nAdvances in GC that are potentially applicable to PCB\/OCP analysis in developing countries are the commercial availability of multidimensional GC (2D-GC) and \u201cfast GC.\u201d In GCxGC, or comprehensive two-dimensional GC, all of the analyte mass is transferred to a second column, and thus resolving power is increased by an order of magnitude [140]. When applied to PCB separations using GCxGC ECD, three- to four-fold lower LODs were obtained compared to one-dimensional GC, and dioxin-like no- and mo-PCBs could be separated from other congeners [141]. Rapid GC separation of PCB congeners by \u201cfast GC\u201d were obtained on narrow-bore (0.1\u00a0mm) columns which reduced peak widths and shortened total run times for the full PCB congener mix to minutes [142]. Both techniques can be run with a micro-ECD and are thus relatively inexpensive to implement.\nConclusions\nRegulators and managers require data on PCBs and OCPs concentrations in environmental media to assess water quality, food contamination, and to infer possible biological effects. Indeed, the global assessment of the success of the Stockholm POPs convention assumes a chemical measurement capability in all UNEP regions which will deliver data on concentrations in key media [6].\nThe analytical methodologies discussed here refer to an \u201canalytical system\u201d encompassing information on the collection and storage of samples, the procedures used to extract, isolate, concentrate, separate, identify, and quantify POPs residues in samples, as well as specific quality control and reporting criteria. All aspects of this system must be in operation for POPs to be analyzed and reported.\nThe chemical analytical methodology for the determination of PCBs and OCPs is a mature area within environmental analytical chemistry as a result of research and development over the past 30\u201340\u00a0years. Basic steps in the quantification of OCPs and PCB congeners have hardly changed in the past 20\u00a0years. Analytical methods for the determination of OCP\/PCBs in foods, soils, sediments, fish, birds, mammals (including human milk and blood) are available and could be implemented at relatively low cost in developing countries. However, access to modern capillary GC equipment with either electron capture or mass spectrometry (MS) detection to separate and quantify PCBs\/OCPs is required in order to conduct the analysis and to take part in regional and international intercomparsons.\nIn general, ELISAs are very useful tools for the rapid assessment of PCB\/OCPs contamination, especially in areas of former heavy use. They are particularly well-suited to laboratories in developing countries which may have access to spectrophotometric equipment but not to GC instrumentation.\nExisting analytical methods for PCB\/OCPs can determine over 100 individual components at low ng\/g concentrations in many environmental media using high-resolution capillary GC\u2013ECD. However, the number of certified values for OCP\/PCB congeners in certified reference materials is more limited (approximately 23 PCB congeners and 15 OCPs in NIST 1588a cod liver). At a minimum, the OCP\/PCBs for which there are certified values in readily available CRMs should be determined (approximately 38). With this number of analytes, the information would be useful for both regulatory actions as well as for source identification using multivariate analysis or other \u201cfingerprinting\u201d methods.\nInterlab comparisons of POPs analysis over the past ten\u00a0years have shown that availability of accurate analytical standards is a fundamental requirement of an analytical program designed to quantify trace organic contaminants such as POPs. Agencies such as GEF and UNEP Chemicals should give top priority to ensuring that certified analytical standards are available to all labs on a continuing basis.\nQuality assurance programs are critically important for demonstrating the performance of analytical methods for POPs within a lab and between labs. QA requirements for PCBs\/OCP analysis are well known and include the use of certified reference materials, field and laboratory blanks, the use of quality control charts to monitor long-term lab performance, participation in interlaboratory studies, and the use of guidelines for sampling and analysis.\nDetermination of PCBs\/OCPs requires the analysis of blank samples because of the ubiquitous nature of these contaminants. If blanks are significant (for example, averaging greater than 10% of the average level of total PCBs), then blank correction should be carried out.\nAs a routine measure, spiking surrogate recovery standards into each sample provides useful information on losses of analyte from the extraction step onwards. However, no single PCB or OCP can be representative of all the organochlorines being determined, and thus recovery correction should be performed with caution. Isotopically labeled surrogates are ideal for the quantification of PCBs and many OCPs via LRMS and HRMS; the application of isotope dilution techniques can correct for the recoveries of these surrogates.\nDetection limits depend not only on the analytical method used but also on the sample size and QA considerations, e.g., on information available from blank or control samples and recovery studies. Detection limits should be calculated as described by US EPA or by IUPAC\/ISO methodology. Comparison of detection limits for widely used instrumentation for POPs with action limits for POPs in food and tissue residue guidelines suggests that current GC\u2013ECD and GC-MS analytical methodology for PCB\/OCPs can meet and exceed these limits, in some cases by orders of magnitude.\nSome emerging new analytical techniques, such 2D-GC and \u201cfast GC\u201d using GC\u2013ECD, may be well-suited for use in developing countries in the near future given their relatively low cost and their ability to provide high-resolution separations of OCP\/PCBs. Procedures with low environmental impacts (microscale, low solvent use, etc.) may be particularly well-suited to developing countries where analytical budgets are small and product delivery times are lengthy. Thus, strategies must be considered that will allow improved techniques to be adopted by such labs.\nElectronic supplementary materials\nBelow is the link to the electronic supplementary material.\nTable\u00a01\n(DOC 4 kb)\nTable\u00a02\n(DOC 13 kb)\nTable\u00a03\n(DOC 6 kb)","keyphrases":["pcbs","organochlorines","pesticides","analysis","pops","review","extraction","gc\u2013ecd","gc\u2013ms","sample preparation and qa\/qc"],"prmu":["P","P","P","P","P","P","P","P","P","R"]}
{"id":"Diabetologia-3-1-2100429","title":"Vitamin B12 and folate concentrations during pregnancy and insulin resistance in the offspring: the Pune Maternal Nutrition Study\n","text":"Aims\/hypothesis Raised maternal plasma total homocysteine (tHcy) concentrations predict small size at birth, which is a risk factor for type 2 diabetes mellitus. We studied the association between maternal vitamin B12, folate and tHcy status during pregnancy, and offspring adiposity and insulin resistance at 6 years.\nIntroduction\nThe \u2018thrifty phenotype\u2019 hypothesis [1] introduced a new paradigm in the aetiology of chronic disease and stimulated research into the relationship between maternal nutrition, fetal growth and offspring risk of type 2 diabetes mellitus. India has the largest number of diabetic patients in the world [2] and this may be related to the fact that Indian babies are amongst the smallest in the world [3]. We were the first to demonstrate that low birthweight of Indian babies predicts insulin resistance and adiposity in childhood [4]. Subsequently we demonstrated that the apparently small Indian newborn babies are relatively adipose, hyperinsulinaemic and hyperleptinaemic compared with white babies [5, 6]. These babies grow into shorter, thinner (low BMI) but more adipose (higher body fat per cent and higher central fat) adults compared with whites, and have a many times higher risk of type 2 diabetes [7]. Thus, in addition to the role of putative genetic and modern day lifestyle factors, the intrauterine environment may also influence the risk of type 2 diabetes mellitus. The Pune Maternal Nutrition Study (PMNS) is the first study in India to investigate the relationship between maternal nutrition and offspring risk of type 2 diabetes and cardiovascular disease. Data were collected on the mother\u2019s size, diet, micronutrient status and physical workload during pregnancy [6, 8, 9], and the newborns were measured in detail at birth and every 6\u00a0months thereafter. At 6\u00a0years of age, the offspring were investigated for risk of type 2 diabetes and cardiovascular disease.\nWe have reported that a higher frequency of maternal intake of green leafy vegetables (GLVs), milk and fruit and higher erythrocyte folate concentrations are associated with larger newborn size [8]. In a small sub-sample we found that higher maternal circulating total homocysteine (tHcy) predicted low offspring birthweight [10]. Other studies in Pune have shown that vitamin B12 deficiency is common and contributes to hyperhomocysteinaemia, while folate deficiency is rare [11, 12].\nWe therefore hypothesised that vitamin B12 and folate deficiency in the mother during pregnancy would predict greater adiposity and insulin resistance in the offspring.\nMethods\nThe recruitment of mothers and follow-up of the children in the PMNS (Fig.\u00a01) has been described [6, 8, 9]. The study started in 1993. Non-pregnant married women living in six villages near Pune had detailed anthropometric measurements every 3\u00a0months and menstrual dates were recorded monthly. Women who became pregnant were assessed at 18\u2009\u00b1\u20092 and 28\u2009\u00b1\u20092\u00a0weeks of gestation for anthropometry, physical workload, dietary intake (24\u00a0h recall and food frequency questionnaire [FFQ]) and measurement of circulating nutrients. According to the National Nutritional Anemia Control Programme, women were given 100 tablets of iron (60\u00a0mg per tablet) and folic acid (500\u00a0\u03bcg per tablet) from 18\u00a0weeks of gestation. Newborn babies were measured within 72\u00a0h of birth (1994\u20131996), and the children were followed up post-natally with repeat anthropometry every 6\u00a0months.\nFig.\u00a01The PMNS: a flow diagram to describe data collection and exclusions from the study. LMP Last menstrual period\nAt 6\u00a0years of age (December 2000\u2013February 2003) the children were investigated for body size, body composition and risk factors for type 2 diabetes mellitus and cardiovascular disease. Families were instructed to continue their usual diet and activity prior to the study, and were transported to the Research Unit the previous evening. They were provided with a standard dinner and then water only until the next morning. A fasting blood sample was collected, followed by a 1.75\u00a0g\/kg body weight oral anhydrous glucose load and a further blood sample 120\u00a0min later.\nThe family\u2019s socioeconomic status (SES) was assessed using the Standard of Living Index devised by the National Family Health Survey [13]. Weight and height were measured using standardised protocols. Whole-body dual-energy X-ray absorptiometry (DEXA) scans (Lunar DPX-IQ 240; Lunar Corporation, Madison, WI, USA; paediatric software) were carried out to measure total, truncal and leg fat mass, percentage body fat and lean mass. Standard quality assurance tests were performed every day. The DEXA machine was installed 3\u00a0months after the study started, and therefore 59 children had DEXA measurements later than the other investigations.\nPlasma glucose was measured using an Hitachi 911 automated analyser (Hitachi, Tokyo, Japan) by the glucose oxidase peroxidase method. Plasma insulin was measured using a Delfia technique (Victor 2; Wallac, Turku, Finland) [14]. Insulin resistance was calculated using the fasting insulin and glucose concentrations (homeostatic model assessment of insulin resistance [HOMA-R]) [15]. As previously described, maternal erythrocyte folate concentrations were measured at the time of the original study (1993\u20131996), taking all necessary precautions in the collection, transport and storage of samples [8]. Based on the findings of a preliminary study [10], we measured plasma vitamin B12, tHcy and methylmalonic acid (MMA) concentrations in all the stored maternal fasting samples (\u221280\u00b0C) in February 2004, as described [11].\nEthical permission for the study was granted by the KEM Hospital Ethical Committee, and by the local village leaders. Parents gave informed written consent. None of these volunteers were paid for participating in the study.\nStatistics Skewed variables were transformed to normality using the following transformations: log to the base e (ln) (plasma vitamin B12 [18\u00a0weeks], tHcy and MMA and erythrocyte folate concentrations, maternal fat intake, and the child\u2019s trunk and leg fat mass), square root (maternal energy, protein, and carbohydrate intakes, and the child\u2019s total fat mass) and reciprocal of square root (maternal vitamin B12 concentrations at 28\u00a0weeks). Maternal pre-pregnant fat mass was calculated from the sum of four skinfold thickness measurements [16]. Maternal intakes of specific foods, based on the FFQ, were analysed as categorical variables. Low vitamin B12 and erythrocyte folate concentrations were defined as <150\u00a0pmol\/l and <283\u00a0nmol\/l, respectively. Elevated tHcy and MMA concentrations were defined as >10 and >0.26\u00a0\u03bcmol\/l [17]. Correlations between different nutritional measures in the mothers were tested using Pearson correlation coefficients. Relationships between maternal nutritional variables and outcomes in the children were analysed using multiple linear regression.\nAs a final stage of the analysis, we summarised the inter-relationships between maternal factors and outcomes in the children using principal components analysis (PCA) and conditional independence analysis. We first performed a PCA to condense the information contained in large groups of variables into a small number of \u2018component\u2019 variables. These components are independent of each other and therefore can be used in regression analysis without the problem of colinearity [18]. The groups of variables were: (1) maternal pre-pregnant size (height, skinfold thicknesses, head circumference, mid-upper-arm circumference [MUAC] and waist and hip circumferences); (2) maternal macronutrient intakes (energy, protein, carbohydrate and fat intakes at 18 and 28\u00a0weeks); (3) maternal micronutrient-rich foods in pregnancy (frequency of intake of dairy products, GLV, fruit and non-vegetarian items [meat, fish and eggs] at 18 and 28\u00a0weeks of gestation); (4) maternal micronutrient status (vitamin B12, folate, tHcy and MMA concentrations at 18 and 28\u00a0weeks); (5) newborn size (birthweight, length, skinfold thicknesses, MUAC, head and abdominal circumferences); and (6) 6\u00a0year body composition (height and DEXA measurements of lean mass, and total, truncal and limb fat). Other key variables (maternal SES and physical workload score [mean of 18 and 28\u00a0week scores], and HOMA-R in the child at 6\u00a0years) remained as single variables. These, and the relevant principal components were included simultaneously in a conditional independence analysis, which is a method of displaying \u2018pathways\u2019 of association between a pre-specified set of variables (in this case, the groups of variables described above). The partial correlation (that is, the correlation while holding the remaining variables constant in the set) of each pair of variables was calculated [19]. A \u2018path\u2019 diagram was then drawn, connecting pairs of variables that were significantly correlated (p\u2009<\u20090.01). Two-tailed significance was calculated at 5% level. Analyses were performed using SPSS 11.0 for windows (SPSS, Chicago, IL, USA) and STATA 7.0 (STATA, College Station, TX, USA).\nResults\nMany of the maternal size and nutritional measurements and their relationship to neonatal size have been described before [8]. The age (median [25th, 75th centiles]) of the mothers when they became pregnant was 21 (19, 23) years. They were short (152.0 [148.5, 155.4] cm) and had a low pre-pregnant BMI (17.8 [16.7, 19.1] kg\/m2) but a relatively high percentage body fat (20% [18, 24]). Their dietary intakes and micronutrient status during pregnancy are shown in Table\u00a01. The women\u2019s energy and protein intakes were low compared with the recommended dietary allowances of 10,565\u00a0kJ (2,525\u00a0kcal) and 65\u00a0g\/day, respectively [20]. At 18 and 28\u00a0weeks, their energy intakes were 1.57 and 1.43 times their calculated basal metabolic rate [21]. One-third of women were lacto-vegetarian, and only 15% of women ate non-vegetarian foods more frequently than once every alternate day. The portion sizes of non-vegetarian foods were small (<120\u00a0g\/day for chicken, fish and meat dishes and ~60\u00a0g\/day for eggs).\nTable\u00a01Maternal nutrition data during pregnancy (median and 25th and 75th centiles, unless otherwise stated)\u00a0Number18\u00a0weeks of gestationNumber28\u00a0weeks of gestationMacronutrient intakes\u00a0Energy (kJ)6927,293 (5,858, 8,774)6706,803 (5,523, 8,268)\u00a0Energy (kcal)6921,743 (1,400, 2,090)6701,626 (1,320, 1,976)\u00a0Carbohydrate (g)692313 (257, 376)670296 (236, 360)\u00a0Protein (g)69244.9 (35.4, 54.8)67041.7 (33.8, 51.3)\u00a0Fat (g)69232.7 (23.9, 42.9)67029.6 (22.4, 39.2)FFQ, n (%)\u00a0GLVs\u00a0Never69215 (2)67169 (10)\u00a0<1 per week692139 (20)671197 (29)\u00a0>1 per week692277 (40)671243 (36)\u00a0>Alternate day692261 (38)671162 (24)Dairy products, n (%)\u00a0Never692107 (15)67196 (14)\u00a0<1 per week692141 (20)671137 (20)\u00a0>1 per week692129 (19)671144 (21)\u00a0>Alternate day692315 (46)671294 (44)Non-vegetarian foods, n (%)\u00a0Never692228 (33)671254 (38)\u00a0<1 per week692182 (26)671181 (27)\u00a0>1 per week692182 (26)671149 (22)\u00a0>Alternate day692100 (14)67187 (13)Circulating micronutrients\u00a0Vitamin B12 (pmol\/l)638135 (103, 175)594122 (94, 160)\u00a0\u00a0<150\u00a0pmol\/l, n (%)380 (60)423 (71)\u00a0Erythrocyte folate (nmol\/l)618874 (687, 1,106)562961 (736, 1,269)\u00a0\u00a0<283\u00a0nmol\/l, n (%)1 (0.2)1 (0.2)\u00a0MMA (\u03bcmol\/l)6360.80 (0.50, 1.34)5940.73 (0.44, 1.18)\u00a0\u00a0>0.26\u00a0\u03bcmol\/l, n (%)586 (94)533 (90)\u00a0tHcy (\u03bcmol\/l)6398.1 (6.8, 10.3)5938.6 (6.7, 10.8)\u00a0\u00a0>10\u00a0\u03bcmol\/l, n (%)177 (28)193 (33)Dairy products refers to whole milk plus milk products (milk in tea and other beverages, yoghurt, buttermilk, ghee, ice cream and other milk-based preparations). Non-vegetarian foods are meat, fish and eggs\nMaternal vitamin B12 and folate status Women in whom vitamin B12, folate and related measurements were not available had similar pre-pregnant weight, SES and weight gain at 28\u00a0weeks of pregnancy compared with those who were studied. The majority of women (>60%) had low plasma vitamin B12 concentrations (Table\u00a01), over 90% had elevated MMA concentrations, and one-third were hyperhomocysteinaemic. On the other hand only one woman had a low erythrocyte folate concentration. Between 18 and 28\u00a0weeks of gestation, plasma vitamin B12, MMA and tHcy concentrations remained similar (p\u2009~\u20090.11, for all), while erythrocyte folate concentrations increased (p\u2009<\u20090.001). Plasma tHcy and MMA concentrations were inversely related to plasma vitamin B12 (p\u2009<\u20090.001 for both at 18 and 28\u00a0weeks of gestation). Low vitamin B12 concentration contributed 41 and 24% to the population attributable risk of hyperhomocysteinaemia, and 40 and 12% to high MMA at 18 and 28\u00a0weeks of gestation, respectively; the contribution of low folate concentration could not be calculated because of small numbers. Higher frequency of intake of dairy products and non-vegetarian foods was associated with higher plasma vitamin B12 concentrations (p\u2009=\u20090.005 and 0.04, respectively) and lower tHcy (p\u2009=\u20090.1 and p\u2009=\u20090.04) and MMA concentrations (p\u2009=\u20090.01 and p\u2009=\u20090.003). Plasma vitamin B12 concentrations were also related to protein intakes (28\u00a0weeks, p\u2009=\u20090.03) but not to energy intake. Higher frequency of GLV intake predicted higher erythrocyte folate concentrations (p\u2009=\u20090.001). All these associations were independent of the energy intake and SES.\nMaternal nutrition during pregnancy and newborn size Maternal vitamin B12 and MMA concentrations were unrelated to neonatal measurements. As previously described [8], lower maternal folate concentrations were associated with smaller newborn weight, MUAC and abdominal circumference (p\u2009=\u20090.003, 0.008, 0.008, respectively). Higher tHcy concentrations at 18\u00a0weeks were associated with smaller newborn size (MUAC, p\u2009=\u20090.02; abdominal circumference, p\u2009=\u20090.02; and subscapular and triceps skinfold thicknesses, p\u2009=\u20090.01 and p\u2009=\u20090.007).\nMaternal nutrition during pregnancy and offspring size, body composition and HOMA-R at 6\u00a0years At 6\u00a0years, the children were light, short and had a low BMI compared with an international (UK) reference [22] (Table\u00a02); none were overweight or obese as defined by International Obesity Task Force [23] criteria. However, skinfold thickness measurements showed that the children were relatively truncally adipose; the mean SD score for subscapular skinfold thickness was \u22120.42 compared with the UK growth standards [24], in contrast with \u22122.23 for weight and \u22121.86 for BMI. Higher fat mass and higher body fat per cent were associated with higher fasting insulin concentrations, higher HOMA-R and higher 120\u00a0min plasma glucose concentrations (p\u2009<\u20090.05 for all).\nTable\u00a02Characteristics of the children at 6\u00a0years (n\u2009=\u2009653)\u00a0Median (IQR)Mean SD scorea (SD)Age (years)6.1 (6.1, 6.2)Weight (kg)16.0 (14.8, 17.3)\u22122.23 (1.00)Height (cm)109.7 (106.7, 112.9)\u22121.39 (0.89)BMI (kg\/m2)13.3 (12.8, 14.0)\u22121.86 (0.90)Subscapular skinfold thickness (mm)5.0 (4.4, 5.6)\u22120.42 (0.68)Triceps skinfold thickness (mm)6.2 (5.4, 7.1)\u22121.41 (0.79)Fat mass (kg)3.1 (2.4, 3.7)Fat (%)18.8 (15.5, 22.2)Leg fat mass (kg)1.3 (1.0, 1.5)Trunk fat mass (kg)0.9 (0.7, 1.2)Lean mass (kg)12.7 (11.7, 13.7)Insulin resistance (HOMA-R)0.70 (0.37, 1.06)120\u00a0min glucose (mmol\/l)5.49 (4.82, 6.21)Vitamin B12 (pmol\/l)224 (167, 311)aSD scores at 6\u00a0years were derived from the UK growth standards [26, 28]IQR Interquartile range\nHigher frequency of maternal intake of GLVs and higher erythrocyte folate concentrations (28\u00a0weeks) were associated with higher fat mass and higher per cent body fat in the children (GLVs: p\u2009=\u20090.02, 0.08; and folate: p\u2009=\u20090.01, 0.002; Table\u00a03). None of the maternal nutritional variables were related to lean mass in the children.\nTable\u00a03Outcomes in the children at 6\u00a0years (body composition and insulin resistance) according to maternal vitamin B12 concentrations at 18\u00a0weeks, erythrocyte folate at 28\u00a0weeks and GLV and dairy product intake at 28\u00a0weeks\u00a0NumberHeight (cm)Fat mass (kg)Lean mass (kg)Insulin resistance (HOMA-R)Vitamin B12 at 18\u00a0weeks (pmol\/l)\u00a0<103146110.2 (4.3)3.3 (0.9)12.8 (1.6)0.78 (0.42, 1.34)\u00a0103\u2013134150109.3 (4.6)3.0 (1.0)12.7 (1.5)0.68 (0.35, 0.99)\u00a0135\u2013174145109.6 (4.9)3.1 (1.0)12.9 (1.7)0.69 (0.30, 0.95)\u00a0\u2265175151110.0 (4.9)3.2 (1.0)12.8 (1.7)0.61 (0.35, 1.00)\u00a0p valuea0.80.50.30.03\u00a0p valueb0.60.40.30.04Erythrocyte folate at 28\u00a0weeks (nmol\/l)\u00a0<734129109.2 (4.6)3.0 (1.0)12.8 (1.7)0.52 (0.28, 0.81)\u00a0734\u2013691136109.8 (4.5)3.1 (0.9)12.9 (1.6)0.65 (0.36, 0.92)\u00a0962\u20131,268131109.9 (4.7)3.2 (1.2)12.7 (1.8)0.71 (0.39, 1.12)\u00a0\u22651,269127110.7 (4.8)3.4 (1.1)13.0 (1.6)0.85 (0.51, 1.27)\u00a0p valuea0.020.0010.5<0.001\u00a0p valueb0.70.010.4<0.001GLVs at 28\u00a0weeks\u00a0Never63109.1 (5.5)3.0 (0.9)12.5 (1.7)0.83 (0.25, 1.23)\u00a0<Once per week183109.5 (4.7)3.0 (1.0)12.7 (1.7)0.73 (0.36, 1.05)\u00a0>Once per week231109.8 (4.6)3.2 (1.0)12.8 (1.7)0.69 (0.34, 0.99)\u00a0\u2265Alternate days146110.5 (4.1)3.3 (1.2)13.0 (1.6)0.66 (0.41, 1.00)\u00a0p valuea0.0020.0010.010.99\u00a0p valueb0.40.020.40.98Dairy products at 28\u00a0weeks\u00a0Never87108.4 (4.9)2.9 (0.9)12.6 (1.8)0.60 (0.32, 0.95)\u00a0<Once per week124110.2 (4.1)3.2 (1.0)12.8 (1.6)0.62 (0.37, 0.99)\u00a0>Once per week134109.4 (4.6)3.2 (0.9)12.7 (1.7)0.73 (0.40, 1.06)\u00a0\u2265Alternate days278110.3 (4.6)3.2 (1.2)12.9 (1.7)0.74 (0.37, 1.11)\u00a0p valuea0.0050.030.30.02\u00a0p valueb0.20.20.80.006Values are mean (SD) or median (interquartile range)ap value adjusted for the child\u2019s age and sexbp value adjusted for the above, plus: SES, the mother\u2019s pre-pregnant height and fat mass, the child\u2019s birthweight, skinfold thicknesses and gestation at delivery and the mother\u2019s protein intake at the time of measurement\nHigher frequency of maternal intake of dairy products at 28\u00a0weeks, and lower plasma vitamin B12 (18\u00a0weeks) and higher erythrocyte folate concentrations (28\u00a0weeks) were associated with higher HOMA-R in the children (Table\u00a03). The highest HOMA-R was in children whose mothers had the lowest vitamin B12 and highest folate concentrations (Fig.\u00a02). There was no statistically significant interaction, however, between vitamin B12 and folate concentrations in relation to HOMA-R (Table\u00a04). The associations remained significant after further adjustment for the child\u2019s own fat mass and plasma vitamin B12 concentrations. None of the other maternal dietary or macronutrient variables were related to HOMA-R in the children.\nFig.\u00a02Insulin resistance (HOMA-R) in the children at 6\u00a0years in relation to maternal vitamin B12 (18\u00a0weeks) and erythrocyte folate (28\u00a0weeks)Table\u00a04Multiple linear regression models for child\u2019s insulin resistance (HOMA-R) at 6\u00a0years as the dependent variableIndependent variableModel 1Model 2Model 3Model 4\u03b2p value\u03b2p value\u03b2p value\u03b2p valuePlasma vitamin B12 at 18\u00a0weeks (pmol\/l)\u22120.160.03\u22120.160.04Erythrocyte folate at 28\u00a0weeks (nmol\/l)0.38<0.0010.38<0.001Interaction term (vitamin B12\u2009\u00d7\u2009folate)\u22120.080.7The independent variable included maternal plasma vitamin B12 concentration at 18\u00a0weeks gestation, erythrocyte folate at 28\u00a0weeks gestation, child\u2019s age, sex, fat mass and standard of living index. The models progressively included vitamin B12, erythrocyte folate, both, and an interaction term (models 1 to 4, respectively)\nMaternal vitamin B12 concentrations were not related to the child\u2019s insulinogenic index (a measure of beta cell function) but 28\u00a0week erythrocyte folate concentration was directly related (p\u2009<\u20090.01).\nPCA and conditional independence analysis PCA and conditional independence analyses are summarised in Fig.\u00a03 and the Electronic supplementary material (ESM) Table 1. Mothers of higher SES had higher intakes of the micronutrient-rich foods, except non-vegetarian foods (correlation A). Larger maternal body size (correlation B) and larger size of the offspring at birth (correlation C) were associated with larger 6\u00a0year size. None of the maternal nutritional variables were related to 6\u00a0year body composition in the children, but the contrast between folate and vitamin B12 concentrations (micronutrient status, factor 3: higher maternal folate and MMA and lower vitamin B12) was associated with higher HOMA-R in the child at 6\u00a0years (correlation D).\nFig.\u00a03The groups of variables used in the PCAs are described in Statistical methods. The notation F1\u2013F3 indicates the first to third principal components, or factors (F), derived from each group of variables. Lines connecting boxes indicate significant positive correlations (p\u2009<\u20090.01), and bold lines denote correlations significant at p\u2009<\u20090.001. Correlations labelled A\u2013D are explained in the Results (PCA and conditional independence analysis). Veg Vegetables\nDiscussion\nWe have demonstrated for the first time in a purposeful, community-based prospective study an association between maternal nutritional measurements in pregnancy and two major risk factors for type 2 diabetes in the offspring. Among rural mothers in Pune, energy and protein intakes are lower than the recommended daily allowance, vitamin B12 status is poor but folate status is adequate. Maternal macronutrient intakes were unrelated to adiposity and insulin resistance in the offspring. However, higher maternal folate concentrations predicted greater adiposity (fat mass and body fat per cent) and higher insulin resistance, and lower vitamin B12 concentrations predicted higher insulin resistance. Children born to mothers with low vitamin B12 concentrations but high folate concentrations were the most insulin resistant. The 18\u00a0week vitamin B12 concentration was more strongly associated with insulin resistance than the 28\u00a0week value, while the reverse was true for erythrocyte folate. The lifespan of erythrocytes (4\u00a0months) means that 28\u00a0week erythrocyte folate reflects nutritional status earlier in pregnancy. Thus, early-mid pregnancy may be a critical period for the programming of adiposity and insulin resistance.\nThe PMNS children are short and thin but relatively adipose compared with white children, similar to the situation in Indian newborns [5, 6] and adults [25]. There were no excessively adipose or insulin-resistant children in this cohort, but adiposity measures were strongly related to metabolic risk factors (glycaemia and insulin resistance) in the normal range. Other studies have shown that higher levels of risk factors in the normal range in childhood predict an increased risk of disease in later life [26, 27].\nWe have demonstrated that the \u2018thin-fat\u2019 phenotype of Indians is associated with a higher risk of type 2 diabetes [7]. This phenotype reflects the simultaneous involvement of two major body compartments (less lean but more adipose), which contribute to the pathogenesis of type 2 diabetes [28]. Our study suggests that an intrauterine imbalance between two related micronutrients (vitamin B12 and folate) may be responsible. A related concept is that of sarcopenic obesity, which increases risk of metabolic and skeletal adverse outcomes [29].\nWe did not find an association between maternal vitamin B12 concentrations and size at birth, possibly because so many women had values in the deficient range. However, higher maternal tHcy concentrations (which were related to low vitamin B12 concentrations) predicted smaller newborn size. A study in Bangalore, South India, where mothers had higher vitamin B12 concentrations, found that low maternal vitamin B12 concentrations predicted fetal growth retardation [30]. These findings are consistent with the \u2018thrifty phenotype hypothesis,\u2019 which proposed that \u2018poor\u2019 maternal nutritional status increases the risk of type 2 diabetes in the offspring [1]. On the other hand, the positive associations of maternal folate and intakes of GLVs and dairy products with adiposity or insulin resistance in the children, are contrary to the hypothesis because they predicted larger offspring size at birth. Our results suggest a need for caution in designing nutritional strategies to improve fetal growth and future health based on relationships with birth size alone.\nPrevious studies linking maternal nutrition in pregnancy to cardiovascular risk factors in the offspring did not measure vitamin B12 or folate [31]. These two vitamins play a crucial role in nucleic acid synthesis and one-carbon metabolism. Low maternal folate status has been implicated in the aetiology of neural tube defects [32] and high tHcy levels have been associated with poor pregnancy outcomes [10, 33, 34]. This has led to a policy of supplementation with folic acid before and during pregnancy, and fortification of flour with folic acid in some countries. The Indian policy is to provide iron and folic acid (60\u00a0mg and 500\u00a0\u03bcg per day) to all pregnant mothers [35]. Despite the evidence of widespread deficiency [11] vitamin B12 supplementation is not a consideration in pregnant Indian women. Vegetarianism and low milk intakes contribute to low vitamin B12 status in Indians [36, 37]. Vegetarianism is multigenerational, and influenced by religious and socioeconomic factors. King Samrat Ashok (273 bc) first banned the killing of animals for food and this was institutionalised by three religions: Jainism, Hinduism and Buddhism. The role of socioeconomic factors is evident in the small portion sizes of non-vegetarian foods habitually eaten by our mothers. Adequate folate status in the PMNS indicates adequate dietary intake even before supplementation was started (18\u00a0weeks of gestation), which further increased the levels. In addition, it is our experience that many Indian obstetricians routinely prescribe high doses of folic acid (5\u00a0mg or more) in \u2018early\u2019 pregnancy with the intention of preventing neural tube defects, even though the majority of pregnant women approach the doctor after 12\u00a0weeks of gestation, when the neural tube is already closed. Thus religious and socioeconomic factors, and medical practices, contribute to create an imbalance in these two related vitamins. The association of higher maternal intake of dairy products with insulin resistance in the offspring requires further exploration. It is intriguing that higher milk consumption is associated with higher insulin resistance in European children [38].\nWe can only speculate about the possible mechanisms for our findings (Fig. 1). Vitamin B12 deficiency will trap folate as 5-methyltetrahydrofolate [39], prevent the generation of methionine from homocysteine and therefore reduce protein synthesis and lean tissue deposition. Elevated methylmalonyl-CoA could contribute to increased lipogenesis by inhibiting carnitine palmitoyltransferase [40] and thereby inhibit \u03b2-oxidation [41]. An analogous clinical situation is high-dose folic acid treatment of severely vitamin B12-deficient pernicious anaemia patients: anaemia improves but neurological damage worsens, possibly because of accumulation of odd-chain carbon fatty acids [42]. Epigenetic regulation, involving DNA methylation, may be another mechanism of nutritional programming, as demonstrated in animal models [43\u201345]. It would be interesting to study these aspects further.\nThe PMNS has many strengths including a population-based design and high participation and follow-up rates. The nutritional assessment methods were developed specifically for the population. The rural setting is representative of over 70% of the Indian population, in whom the prevalence of diabetes is rapidly rising [46]. The lacto-vegetarian food habits of our women are similar to ~40% of Indian households and ~10% of the world population [47]. Although vitamin B12 was measured in stored frozen samples, it is stable during long-term storage [48]. In the absence of accepted guidelines for interpreting vitamin B12 status in pregnancy [49], we cannot define the true extent of vitamin B12 deficiency, but high levels of MMA and tHcy suggested its presence in a majority of the women in our study [17]. Associations with folate and vitamin B12 concentrations but not with tHcy and MMA could be due to dependence of the latter two on non-nutritional factors that are altered in pregnancy (haemodilution and elevated glomerular filtration rate), although we did find an association between high maternal MMA and insulin resistance in the children in the conditional independence analysis. A limitation of the study is that it is observational, and therefore causality cannot be ascertained.\nIn conclusion, our data raise the important possibility that high folate intakes in vitamin B12-deficient mothers could increase the risk of type 2 diabetes in the offspring. This is the first report in humans to suggest that defects in one-carbon metabolism might be at the heart of intra-uterine programming of adult disease. There is a need for further studies to test our findings and to determine the correct management of low vitamin B12 concentrations in Indian mothers.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nFigure S1 Suggested metabolic mechanisms for adiposity, insulin resistance and altered gene expression in a situation of dietary vitamin B12 deficiency combined with adequate folate status (PDF 35.7\u00a0kb)\nTable S1\nUnrotated factor loadings from the PCA (PDF 75.3\u00a0kb)","keyphrases":["vitamin b12","folate","pregnancy","insulin resistance","offspring","maternal nutrition","adiposity"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Pediatr_Cardiol-4-1-2267487","title":"Polymorphisms of Human Leukocyte Antigen Genes in Korean Children with Kawasaki Disease\n","text":"Background Kawasaki disease is a leading cause of acquired heart disease in children. The prevalence rate varies in different ethnic groups. Recently, with the clinical application of molecular genetic technology, human leukocyte antigen (HLA) polymorphisms associated with several diseases have been identified by DNA analysis. This study aimed to assess the association of HLA alleles with susceptibility and complications of Kawasaki disease in Korean children.\nKawasaki disease is a systemic vasculitis that currently is a leading cause of acquired heart disease in children. The etiology of Kawasaki disease continues to be unknown, although it was described 4 decades ago in 1967 by Kawasaki [15]. Diagnosis continues to depend primarily on clinical manifestations.\nIt appears that the pathogenesis of Kawasaki disease is related to infection of a susceptible host coupled with epidemiologic and ethnic factors. In a study examining the epidemiologic characteristics of Kawasaki disease in San Diego, CA, USA, Asians\/Pacific Islanders were 2.7 times more likely and Hispanics one-third as likely to be hospitalized for Kawasaki disease compared with the children of other ethnic groups combined [4]. The sibling risk ratio is also known to be higher, than the risk of being affected with Kawasaki disease without sibling history, and cases of Kawasaki disease in parents and children have been reported [10, 21].\nFor these reasons, many reports have suggested involvement of the human leukocyte antigen (HLA) system. The results of such studies vary depending on the ethnic group studied. Several reports show the association of HLA genotypes with Kawasaki disease including HLA-Bw22 (now referred to as HLA-B54) in Japanese [14, 20], HLA-B51 in white populations [13, 16, 18, 19], and the major histocompatibility complex class I chain-related gene A (MICA) genes and others in southern Chinese population [6, 12].\nThe major histocompatibility complex (MHC) class II genes in Kawasaki disease show variable correlations [2, 8, 27]. To date, there have been no reports supporting a consistent relationship between Kawasaki disease and HLA genes in Korean patients. Therefore, the current study aimed to assess the association of HLA-A,-B,-C, and -DRB1 alleles with Kawasaki disease in Korean children.\nMaterials and Methods\nSubjects\nFrom March 2004 to December 2004, 74 patients with Kawasaki disease (44 boys and 30 girls) attending the pediatric outpatient clinic or admitted to St. Vincent\u2019s Hospital in Korea were recruited for this study. The mean age of the patients at diagnosis was 2.7 \u00b1 1.9 years. For comparison, genetically unrelated healthy Korean adults (n = 159) who had no history of Kawasaki disease were studied as a healthy control group. Blood samples were obtained after informed consent in compliance with the institutional review board of St. Vincent\u2019s hospital, College of Medicine, The Catholic University of Korea.\nThe diagnosis of Kawasaki disease was established by clinical manifestations meeting the defined criteria according to the report of the Research Committee on Kawasaki Disease of the Japanese Ministry of Health and Welfare in addition to exclusion of any other possible illnesses [25]. Coronary complications included the following lesions detected by echocardiography: an internal diameter exceeding 3 mm in patients younger than 5 years and exceeding 4 mm in patients age 5 years or older, dilation of a segmental luminal diameter to at least 1.5 times that of an adjacent segment, or a definitely irregular internal lumen of the coronary arteries. The clinical characteristics of the patients are shown in Table\u00a01.\nTable\u00a01Profiles of subjects with Kawasaki diseasen (%)Male\/female44\/30 (60\/40)Typical KD\/atypical KD65\/9 (88\/12)Family history of KD3 (4.1)Recurrence history of KD4 (5.4)Coronary complications21 (28)Other complications of KDGallbladder hydrops14 (19)Pyuria25 (34)Hepatopathy38 (51)Arthritis10 (14)KD, Kawasaki disease\nGenomic DNA was extracted by standard methods using the AccuPrep DNA extraction kit (Bioneer, Daejeon, Korea) from peripheral blood collected (4 ml) with ethylenediaminetetraacetic acid (EDTA) and kept at \u221220\u00b0C.\nHLA-A, -B, and -C Genotyping\nThe genotyping of HLA-A, -B, and -C was performed by the amplification refractory mutation system (ARMS)\u2013polymerase chain reaction (PCR) method. Each reaction contained a primer mix consisting of the allele- or group-specific primer pairs as well as internal control primers matching nonallelic sequences. Specific amplifications of the HLA-A, -B, and -C genes were performed using forward and reverse primers (44 for HLA-A, 47 for HLA-B, and 33 for HLA-C) designed according to the published nucleotide sequences [5, 17, 26].\nThe PCR procedure was carried out in a reaction (13\u00a0\u03bcl), containing 100 to 200 ng genomic DNA, 0.8 X buffer (40 mmol\/l KCl, 1.2 mmol\/l MgCl2, 8 mmol\/l Tris-HCl pH 8.8, 0.08% Triton X-100), 5% dimethylsulphoxide (DMSO), 200\u00a0\u03bcmol\/l of each dNTP, 0.25 U Taq DNA polymerase (Boehringer, Mannheim, Germany), 1\u00a0\u03bcmol\/l of each sequence-specific primer, and 0.2\u00a0\u03bcmol\/l of internal control primers.\nThe amplifications were performed in a My Cycler thermocycler (Bio-Rad, Hercules, USA). For amplification, 30 cycles were performed using the following steps: heating to 96\u00b0C for 1 min to denature the DNA; denaturation at 96\u00b0C for 25 s and at 70\u00b0C for 45 s; annealing and extension at 72\u00b0C for 30 s (for the first 5 cycles), 96\u00b0C for 25 s, 65\u00b0C 45 s, 72\u00b0C for 30 s (for the next 21 cycles); 96\u00b0C for 25 s, 55\u00b0C for 60 s, 72\u00b0C for 120 s (for the last 4 cycles); and a final 1 min extension at 72\u00b0C. The presence or absence of PCR products was determined after separation of the samples on a 1.5% agarose gel containing 0.5\u00a0\u03bcg\/ml of ethidium bromide.\nHLA-DRB1 Genotyping\nThe PCR sequence-specific primer (SSP) method was used for HLA-DRB1 genotyping. Each reaction contained a primer mix consisting of the allele- or group-specific primer pairs as well as the internal control primers that matched the nonallelic sequences. Specific amplification of the HLA-DRB1 gene was performed using 47 forward and reverse primers for HLA-DRB1 designed on the basis of the published nucleotide sequences [23].\nThe PCR procedure was carried out in a reaction (10\u00a0\u03bcl) containing 100 ng genomic DNA, 1 X buffer (60 mmol\/l Tris-HCl pH 9.0, 15 mmol\/l ammonium sulphate, 2.5 mmol\/l MgCl2), 200\u00a0\u03bcmol\/l of each dNTP, 0.25 U Taq DNA polymerase (Boehringer Mannheim, Germany), 1\u00a0\u03bcmol\/l of each sequence-specific primer, and 0.2\u00a0\u03bcmol\/l of the internal control primers.\nThe amplifications performed in a My Cycler thermocycler (Bio-Rad, Hercules, USA). For amplification, 27 cycles were performed using the following steps: heating to 96\u00b0C for 3 min to denature the DNA; denaturation at 96\u00b0C for 20 s; annealing and extension at 66\u00b0C for 60 s (for the first 10 cycles), 62\u00b0C for 80 s (for the next 10 cycles), and 61\u00b0C for 120 s (for the last 7 cycles); and a final 10 min extension at 72\u00b0C. The presence or absence of PCR products was determined after separation of samples on a 1.5% agarose gel containing 0.5\u00a0\u03bcg\/ml of ethidium bromide.\nStatistical Analysis\nStatistical differences between Kawasaki disease patients and healthy control subjects and between subgroups of Kawasaki disease patients were tested with chi-square and Fisher\u2019s exact test. The quantification of the relationship of the frequencies studied, in cases with Kawasaki disease, was performed by calculating the relative risks using the method of Woolf. Haldane\u2019s modification was applied for cases in which the variables included zero. A p value less than 0.05 was considered to be significant.\nIn this study, the data were not adjusted for multiple comparisons because the sample size was not large enough for multiple-comparison analysis correction.\nResults\nThe Genotype Distribution and Allele Frequencies of HLA-A for the Patients With Kawasaki Disease and the Healthy Control Group\nIn the analysis of the polymorphisms of HLA-A alleles, no statistical difference was found in the frequency of alleles between the patients with Kawasaki disease and the control group. However, the frequency of HLA-A26 alleles was significantly decreased in the Kawasaki disease patients without coronary complications (CC) compared with the healthy control group (p < 0.04; relative risk (RR) = 0.2). In a comparison between subgroups, with and without CC, no significant difference was identified (Table\u00a02).\nTable\u00a02The allele frequencies of human leukocyte antigen-A (HLA-A) in the patients with Kawasaki disease and the control subjectsHLA allelesHealthy controls (n =159) n (%)KD patients (n = 74) n (%)Coronary complicationsPresent (n = 21) n (%)Absent (n = 53 n (%)HLA-A015 (3.1)5 (6.8)2 (9.5)3 (5.7)0285 (53.5)43 (58.1)12 (57.1)31 (58.5)035 (3.1)3 (4.1)0 (0.0)3 (5.7)1131 (19.5)21 (28.4)6 (28.6)15 (28.3)2457 (35.8)33 (44.6)12 (57.1)21 (39.6)2625 (15.7)6 (8.1)4 (19.0)2 (3.8)a3020 (12.6)4 (5.4)1 (4.8)3 (5.7)3114 (8.8)6 (8.1)0 (0.0)6 (11.3)323 (1.9)0 (0.0)0 (0.0)0 (0.0)3338 (23.9)15 (20.3)2 (9.5)13 (24.5)681 (0.6)0 (0.0)0 (0.0)0 (0.0)KD, Kawasaki disease; RR, relative riskap < 0.04; RR = 0.2 (95% confidence interval [CI], 0.05\u20130.90) vs healthy control subjects\nThe Genotype Distribution and Allele Frequencies of HLA-B for the Patients With Kawasaki Disease and the Healthy Control Group\nIn analysis of the polymorphisms of HLA-B alleles, there was a significant increase in the frequency of HLA-B35 and -B75 alleles in the patients with Kawasaki disease compared with the control group (p < 0.006; RR = 3.1 vs p < 0.02; RR = 8.2). When the patients with Kawasaki disease were divided into two subgroups, with or without CC, the Kawasaki disease patients without CC showed a significantly increased frequency of HLA-B35 and -B75 alleles compared with the healthy control group (p < 0.02; RR = 3.1 vs p < 0.003; RR = 11) (Table\u00a03). Although the frequency of the HLA-B35 alleles were similar in the groups with and without CC (19% vs 18.9%), the HLA-B75 allele was found only in the Kawasaki disease patients without CC (0.0% vs. 13.2%). Therefore, the HLA-B35 allele was implicated in susceptibility to Kawasaki disease and the HLA-B75 allele in susceptibility to Kawasaki disease without CC. In a comparison between the subgroups, with and without CC, there was no overall significant difference identified between the two groups.\nTable\u00a03The allele frequencies of human leukocyte antigen-B (HLA-B) in the patients with Kawasaki disease and the control subjectsHLA allelesHealthy controls (n = 159) n (%)KD patients (n = 74) n (%)Coronary complicationsPresent (n = 21) n (%)Absent (n = 53) n (%)HLA-B0719 (11.9)9 (12.2)3 (14.3)6 (11.3)082 (1.3)0 (0.0)0 (0.0)0 (0.0)1320 (12.6)5 (6.8)2 (9.5)3 (5.7)146 (3.8)0 (0.0)0 (0.0)0 (0.0)2711 (6.9)3 (4.1)1 (4.8)2 (3.8)3511 (6.9)14 (18.9)a4 (19.0)10 (18.9)b375 (3.1)4 (5.4)2 (9.5)2 (3.8)387 (4.4)3 (4.1)0 (0.0)3 (5.7)392 (1.3)3 (4.1)2 (9.5)1 (1.9)4429 (18.2)10 (13.5)3 (14.3)7 (13.2)4618 (11.3)7 (9.5)2 (9.5)5 (9.4)470 (0.0)1 (1.4)0 (0.0)1 (1.9)4813 (8.2)2 (2.7)0 (0.0)2 (3.8)5129 (18.2)13 (17.6)5 (23.8)8 (15.1)529 (5.7)3 (4.1)2 (9.5)1 (1.9)5423 (14.5)10 (13.5)3 (14.3)7 (13.2)553 (1.9)4 (5.4)1 (4.8)3 (5.7)562 (1.3)0 (0.0)0 (0.0)0 (0.0)572 (1.3)1 (1.4)0 (0.0)1 (1.9)5815 (9.4)5 (6.8)0 (0.0)5 (9.4)594 (2.5)3 (4.1)0 (0.0)3 (5.7)6011 (6.9)7 (9.5)2 (9.5)5 (9.4)6123 (14.5)13 (17.6)6 (28.6)7 (13.2)6229 (18.2)14 (18.9)1 (4.8)13 (24.5)674 (2.5)2 (2.7)1 (4.8)1 (1.9)717 (4.4)1 (1.4)0 (0.0)1 (1.9)752 (1.3)7 (9.5)c0 (0.0)7 (13.2)dKD, Kawasaki disease; RR, relative riskap < 0.006; RR = 3.1 (95% confidence interval [CI], 1.29\u20137.62) vs healthy control subjectsbp < 0.02; RR = 3.1 (95% CI, 1.16\u20138.43) vs healthy control subjectscp < 0.02; RR = 8.2 (95% CI, 1.74\u201338.68) vs healthy control subjectsdp < 0.003; RR = 11 (95% CI, 2.79\u201351.13) vs healthy control subjects\nThe Genotype Distribution and Allele Frequency of HLA-C for the Patients With Kawasaki Disease and the Healthy Control Group\nIn the analysis of the polymorphisms of HLA-C alleles, there was a significant increase in the frequency of the HLA-Cw09 allele in the patients with Kawasaki disease compared with the healthy control group (p < 0.04; RR = 2.0). When the Kawasaki disease patients were divided into two subgroups, with or without CC, the Kawasaki disease patients without CC showed a significantly increased frequency of the HLA-Cw09 allele compared with the healthy control group (p < 0.05; RR = 2.1). However, no significant difference was found between the subgroups with and without CC (Table\u00a04).\nTable\u00a04The allele frequencies of human leukocyte antigen-C (HLA-C) in the patients with Kawasaki disease and the control subjectsHLA allelesHealthy controls (n = 159) n (%)KD patients (n = 74) n (%)Coronary complicationsPresent (n = 21) n (%)Absent (n = 53) n (%)HLA-Cw0159 (37.1)22 (29.7)6 (28.6)16 (30.2)020 (0.0)1 (1.4)0 (0.0)1 (1.9)0421 (13.2)11 (14.9)2 (9.5)9 (17.0)056 (3.8)1 (1.4)0 (0.0)1 (1.9)0625 (15.7)10 (13.5)5 (23.8)5 (9.4)0744 (27.7)20 (27.0)6 (28.6)14 (26.4)0829 (18.2)10 (13.5)3 (14.3)7 (13.2)0923 (14.5)19 (25.7)a5 (23.8)14 (26.4)b1039 (24.5)24 (32.4)5 (23.8)19 (35.8)1211 (6.9)4 (5.4)2 (9.5)2 (3.8)1434 (21.4)18 (24.3)7 (33.3)11 (20.8)158 (5.0)1 (1.4)1 (4.8)0 (0.0)KD, Kawasaki disease; RR, relative riskap < 0.04; RR = 2.0 (95% confidence interval [CI], 0.97\u20134.29) vs healthy control subjectsbp < 0.05; RR = 2.1 (95% CI, 0.92\u20134.87) vs healthy control subjects\nThe Genotype Distribution and the Allele Frequencies of HLA-DRB1 for the Patients With Kawasaki Disease and the Healthy Control Group\nAmong the HLA-DRB1 alleles, there was no increase in the frequency of alleles in the Kawasaki disease patients compared with the healthy control group. However, after subgrouping of the Kawasaki disease patients, with or without CC, the frequency of HLA-DRB1*11 was significantly increased in the Kawasaki disease patients with CC compared with the healthy control group (p < 0.04; RR = 4.7). In a comparison between the subgroups, with and without CC, the frequency of the HLA-DRB1*09 allele was increased in the Kawasaki disease patients with CC compared with the Kawasaki disease patients without CC (33.3% vs 9.4%; p < 0.04). However, there was no significant difference in the frequency of these alleles when each was compared with the healthy control group. The frequency of the HLA-DRB1*04 allele was slightly increased in the Kawasaki disease patients without CC compared with the healthy control group (p < 0.05; RR = 1.9) (Table\u00a05).\nTable\u00a05The allele frequencies of human leukocyte antigen-DRB1 (HLA-DRB1) in the patients with Kawasaki disease and the control subjectsHLA allelesHealthy controls (n = 159) n (%)KD patients (n = 74) n (%)Coronary complicationsPresent (n = 21) n (%)Absent (n = 53) n (%)HLA-DRB10129 (18.2)8 (10.8)1 (4.8)7 (13.2)037 (4.4)1 (1.4)0 (0.0)1 (1.9)0448 (30.2)30 (40.5)6 (28.6)24 (45.3)a0726 (16.4)8 (10.8)1 (4.8)7 (13.2)0832 (20.1)14 (18.9)3 (14.3)11 (20.8)0928 (17.6)12 (16.2)7 (33.3)b5 (9.4)104 (2.5)4 (5.4)2 (9.5)2 (3.8)1110 (6.3)8 (10.8)5 (23.8)c3 (5.7)1218 (11.3)11 (14.9)2 (9.5)9 (17.0)1332 (20.1)10 (13.5)3 (14.3)7 (13.2)1421 (13.2)10 (13.5)3 (14.3)7 (13.2)1535 (22.0)20 (27.0)5 (23.8)15(28.3)164 (2.5)2 (2.7)1 (4.8)1 (1.9)KD, Kawasaki disease; RR, relative riskap < 0.05; RR = 1.9 (95% confidence interval [CI], 0.96\u20133.82) vs healthy control subjectsbp < 0.04 between KD patients with and without coronary complicationcp < 0.04; RR = 4.7 (95% CI, 1.26\u201317.17) vs healthy control subjects\nDiscussion\nThe prevalence of CC among the patients with Kawasaki disease in this study was higher than previously reported (28.4% vs 5%) [22]. This is likely because the patients included had a previous diagnosis of Kawasaki disease and were attending our clinic for this reason. Patients with more severe Kawasaki disease, including those with CC, present for medical care more frequently than patients without CC, and this may explain the higher proportion of CC in our study. Three patients (4%) had a positive family history, and four patients (5.4%) had a history of Kawasaki disease recurrence. Prior studies reported the familial occurrence and recurrence rates for Kawasaki disease to be 1% and 3%, respectively [11, 24, 28].\nOur data were confined to Korean children and focused on patients who had Kawasaki disease with CC. Therefore, further research on the effect of the family history and recurrence of Kawasaki disease in association with HLA polymorphisms is needed.\nStudies have associated HLA alleles with many different human diseases, such as HLA-B27 with ankylosing spondylitis and Reiter syndrome, HLA-B35 with subacute thyroiditis, and HLA-DR8 with juvenile rheumatoid arthritis. Most of the early studies on the genetics of Kawasaki disease were conducted using serologic testing that measured HLA antigens. The association of the HLA-Bw22 allele with Kawasaki disease in Japanese studies was evaluated with the microcytotoxicity test used for tissue typing of 205 patients with Kawasaki disease and 500 control samples in the late 1970s [14]. On basis of these studies, a gene was thought to control susceptibility to Kawasaki disease and was linked to the Japanese-specific HLA antigen.\nSince the clinical application of genetic technology became available in the 1980s, polymorphisms at many HLA loci have been identified. In addition, there were changes in nomenclature, and the alleles were reclassified. For example, the previously labeled HLA-Bw22 allele was changed to the HLA B54 allele [1, 3].\nOur study on the polymorphisms of HLA genes was conducted using the PCR-ARMS method for the HLA class I genes and the PCR-SSP method for the HLA class II genes. There was a significant increase in the frequencies of HLA-B35, -B75, and -Cw09 alleles in the patients with Kawasaki disease compared with the healthy control group. As previously mentioned, this study showed that the HLA-B35 allele was related to susceptibility to Kawasaki disease and the HLA-B75 allele to Kawasaki disease without CC in Korean children. These results suggest that polymorphisms (in some of the B and C loci) in HLA class I genes are associated with Kawasaki disease in Korean children; consistent with prior reports on other ethnic groups [12, 14, 16, 19].\nReview of the medical literature on Kawasaki disease and HLA shows that there is a trend toward an association between the HLA-B loci and Kawasaki disease. However, to date, there is no confirmed relationship to a particular locus. It is likely that the HLA-B locus is not the only locus associated with Kawasaki disease. This can be the case because of its functional variation or the potential effects from other related genes around the HLA-B locus. The HLA-B locus and other associated genes might be useful genetic markers for Kawasaki disease.\nThe HLA-B35 gene is known to play a major role in a variety of infections. For example, in cases with HIV infection, the patients with an increased frequency of HLA-B35 show rapid progression of HIV [9]. Infection has been implicated in the etiology of Kawasaki disease. The finding in our study that the frequency of the HLA-B35 allele was increased in Korean children with Kawasaki disease suggests a role for infection in Kawasaki disease.\nThe HLA-DRB1 alleles showed no increased frequency in the Kawasaki disease patients compared with the healthy control group. However, when the patients were compared by subgroups, with and without CC, the frequency of HLA-DRB1*11 was significantly increased in the Kawasaki disease patients with CC (p < 0.04; RR = 4.7), and HLA-DRB1*04 was increased in the Kawasaki disease patients without CC (p < 0.05; RR =1.9) compared with the healthy control group. Comparison of the subgroups with and without CC showed that the frequency of the HLA-DRB1*09 allele was increased in Kawasaki disease patients with CC compared with the Kawasaki disease patients who had no CC (33.3% vs 9.4%; p < 0.04), and that there was no significant difference in comparison with the healthy control group. For the HLA-DRB1*11 allele, the finding of a higher allele frequency compared with the healthy control group was amplified by the subgrouping of Kawasaki disease patients with and without CC. Future studies with a larger sample size are needed to confirm these findings.\nSeveral reports describe the presence of HLA-DRB1 and the progression of infections [7]. However, most of the reports on the association of HLA-DRB1 with Kawasaki disease concluded that despite a regional association, their data failed to support a consistent role for HLA class II alleles in Kawasaki disease. If there was any role, it was predicted to be minor [2, 8].\nInterpopulation discrepancies of the frequencies of HLA alleles make generalization of results difficult. Matched population profiles are needed for disease association studies because ethnic differences can provide altered disease associations.\nIn this study, we compared HLA alleles of Kawasaki disease patients with known polymorphic loci of HLA genes in healthy Korean adults. The results we report on the polymorphisms of HLA genes in Kawasaki disease patients may be limited to the Korean population. However, this information may be helpful in future studies on HLA genetic polymorphisms in Kawasaki disease. Further studies with a larger sample size are needed for confirmation of our findings.","keyphrases":["polymorphism","gene","kawasaki disease","hla"],"prmu":["P","P","P","P"]}
{"id":"Immunogenetics-3-1-1914291","title":"MHC class I A region diversity and polymorphism in macaque species\n","text":"The HLA-A locus represents a single copy gene that displays abundant allelic polymorphism in the human population, whereas, in contrast, a nonhuman primate species such as the rhesus macaque (Macaca mulatta) possesses multiple HLA-A-like (Mamu-A) genes, which parade varying degrees of polymorphism. The number and combination of transcribed Mamu-A genes present per chromosome display diversity in a population of Indian animals. At present, it is not clearly understood whether these different A region configurations are evolutionarily stable entities. To shed light on this issue, rhesus macaques from a Chinese population and a panel of cynomolgus monkeys (Macaca fascicularis) were screened for various A region-linked variations. Comparisons demonstrated that most A region configurations are old entities predating macaque speciation, whereas most allelic variation (>95%) is of more recent origin. The latter situation contrasts the observations of the major histocompatibility complex class II genes in rhesus and cynomolgus macaques, which share a high number of identical alleles (>30%) as defined by exon 2 sequencing.\nIntroduction\nMajor histocompatibility complex (MHC) represents a multigene family that plays a crucial role in the generation of adaptive immune responses in vertebrate species. A key feature of the system is that some of its genes display abundant polymorphism at the population level. In addition, the number of Mhc class I or II genes may differ significantly between species, as well as between individuals of a species (Kelley et al. 2005). MHC polymorphisms may have a profound impact on several features such as disease susceptibility, organ transplantation, and reproductive success (Lagaaij et al. 1989; Goulder and Watkins 2004; Bontrop and Watkins 2005; Ziegler et al. 2005; Smith et al. 2006). The MHC systems of various primate species, including humans, have been studied extensively (Watkins 1995; Antunes et al. 1998; Bontrop et al. 1999; Adams and Parham 2001; de Groot et al. 2002; Lafont et al. 2004; Middleton et al. 2004; Marsh et al. 2005; Penedo et al. 2005; Abbott et al. 2006; Huchard et al. 2006). For example, the MHC of the rhesus macaque (MhcMamu), an Old World primate species, has been shown to share many similarities with the human leukocyte antigen (HLA) system in humans. The evolutionary orthologs of the HLA-DP, -DQ, and -DR genes are also present in rhesus monkeys, and as in humans, these loci are polymorphic (Bontrop et al. 1999). The number of Mamu-DRB region configurations appears to be expanded in comparison with humans, and some of these regions seem to harbor an extended number of genes (Doxiadis et al. 2000). Subsequent cDNA studies have illustrated, however, that in humans and rhesus macaques, comparable numbers of DRB genes are transcribed (de Groot et al. 2004).\nApart from Mamu-E (Knapp et al. 1998), -F (Otting and Bontrop 1993), -G (Boyson et al. 1996a; Castro et al. 1996), and -AG (Boyson et al. 1997), rhesus macaques also possess a B-like sequence, designated Mamu-I, which appears to be present in each haplotype (Urvater et al. 2000). As observed in humans, these nonclassical genes display low levels of polymorphism. An ortholog of the HLA-C gene appears to be absent in the rhesus macaque, whereas evolutionary equivalents of the classical HLA-A and -B genes have been described (Boyson et al. 1996b). The Mamu-A and -B genes have been subjected to several rounds of duplication as was shown by genomic sequencing (Daza-Vamenta et al. 2004; Kulski et al. 2004). Analysis of a panel of rhesus macaques, mainly originating from the Indian subcontinent, illustrated that the number and combination of Mamu-A and -B genes that are expressed per haplotype may differ at the population level (Otting et al. 2005). In the same study, marked differences in expression levels were shown. At this stage it is not known whether rhesus macaques originating from other geographic areas have unique Mhc class I alleles and\/or region configurations. Comparative studies have illustrated that many Mhc loci and lineages predate speciation events. The sharing of Mhc alleles between two primate species seems to be a rare event, and only a few cases have been documented (Cooper et al. 1998; Evans et al. 1998). An exception is provided by rhesus and cynomolgus macaques, which seem to share a high number of Mhc class II alleles as was defined by exon 2 sequencing (Blancher et al. 2006; Doxiadis et al. 2006). Whether this sharing is because of introgression or purifying selection remains to be elucidated. Thus far, about 50 Mafa-A sequences have been published for the cynomolgus macaque (Uda et al. 2004; Krebs et al. 2005). The absence of pedigreed material did not allow us to define Mafa-A loci or region configurations. For that reason, a panel of pedigreed cynomolgus macaques was incorporated in the present study. Comparison with rhesus macaque Mhc class I sequences obtained from different populations enabled us to draw conclusions on the evolutionary stability of A region variations.\nMaterials and methods\nAnimals and cell lines\nThe Biomedical Primate Research Centre houses a self-sustaining colony of approximately 1,000 rhesus macaques, mainly of Indian origin, that have been pedigreed based on the segregation of serologically defined MHC allotypes (Bontrop et al. 1999; Penedo et al. 2005; Doxiadis et al. 2006). Furthermore, a large collection of DNA samples as well as B-cell lines is available. In this study, B-cells derived from rhesus macaques of Chinese origin and from cynomolgus monkeys were used to isolate RNA. Most cynomolgus monkey samples originate from a pedigreed group housed at the campus of the University of Utrecht.\ncDNA cloning and sequencing\nRNA was isolated from B cells (Rneasy kit, Qiagen) and subjected to One-step reverse transcriptase polymerase chain reaction (RT-PCR), as recommended by the supplier (Qiagen). The primers (5\u2032MAS) AATTCATGGCGCCCCGAACCCTCCTCCTGG, and (3\u2032MAS) CTAGACCACACAAGGCGGCTGTCTCAC were used, which are specific for class I A transcripts in macaques. The final elongation step was extended to 30\u00a0min to generate a 3\u2032dA overhang. The RT-PCR products were cloned using the InsT\/Aclone kit (Fermentas). After transformation, 16 to 32 colonies were picked for plasmid isolations. The separate Mamu-A loci show differences in expression levels, as indicated by the numbers of clones, picked from each animal. The alleles with high expression levels (majors) may be associated with serotypes and thus involved in classical antigen presentation, whereas those with low expression levels (minors) are considered nonclassicals and may exhibit more specialized types of functions (Otting et al. 2005).\nSequencing reactions were performed using the BigDye terminator cycle sequencing kit, and samples were run on an automated capillary sequencing system (ABI Genetic Analyzer 3100) as has been described previously (Otting et al. 2005).\nLocus-specific PCR reactions\nTo test the presence of Mamu- and Mafa-A4*14 alleles, 1\u00a0\u03bcl of the RT-PCR samples was taken for a single-specific-primer PCR (SSP-PCR) reaction in advance of the cloning step. The relevant primers, (5\u2032A*14) GGGACCCGACGGGCGCCTCCAA and (3\u2032A*14) GGCCCTCCAGGTAGACTCTGTC have annealing sites in exon 3. Amplifications were carried out starting with 2\u00a0min at 94\u00b0C, followed by 25 cycles at 94\u00b0C, 65\u00b0C, and 72\u00b0C for 1\u00a0min each. The PCR products were subjected to direct sequencing, and the reactions were performed as described above.\nPhylogenetic analysis and nomenclature\nThe sequences were analyzed with the Sequence Navigator Software version 1.0.1 (Applied Biosystems), and alleles are based on at least three clones with identical full-length sequences. To define loci and lineages, alignments of the sequences were made using the MacVector\u2122 version 8.1.1 (Oxford Molecular Group), followed by manual adjustments. Phylogenetic analyses on the full-length (1,065\u00a0bp) sequences were also performed with the MacVector software. Neighbor-joining trees were constructed with the Kimura 2 parameter method. Bootstrap analyses were performed based on 1,000 replications.\nIn total, 130 unreported Mamu-A and Mafa-A sequences were submitted to the European Bioinformatics Institute and European Molecular Biology Laboratory (EBI-EMBL) database. Relevant information such as accession number and a reference cell-line are provided (Table\u00a01). Moreover, all novel Mamu-A and Mafa-A sequences are named in accordance with a generally accepted nomenclature proposal (Klein et al. 1990; Robinson et al. 2003; Ellis et al. 2006). For example, Mamu-A1*0101 defines a Mhc allele in the rhesus macaque, which is encoded by one of the class I loci: namely, A1. The first two digits after the asterisk define the lineage, whereas the third and fourth digits define the allele number. These allele numbers are arbitrary, as they reflect the order in which the alleles were discovered. A fifth and sixth digit are used to mark a synonymous basepair difference between two sequences. \nTable\u00a01Summary of Mamu-A and Mafa-A alleles detected in this studyAlleleAccessionAnimalsAlleleAccessionAnimalsMamu-A1Mafa-A1A1*0102AM295885EKKA1*0101AM295828YabaaA1*0103AM295886Ri056A1*0301AM295829HoebaA1*030102AM295887Ri185A1*0302AM29583077A1*0302AM295888r300A1*1002AM295831k71A1*0303AM2958898822A1*1003AM295832TrufoA1*0304AM295890Ri301A1*1702AM295833BufoA1*0305AM295891Ri037A1*1901AM29583477A1*040102AM295892Ri165A1*2202AM295835k94A1*0402AM295893Ri366A1*3101AM295836GayoA1*1001AM295894Ri002A1*3102AM295823BilboaA1*1002AM295895Ri142A1*3201AM29582480A1*1102AM295896Ri136A1*3801AM295825k66A1*1701AM295897Ri145A1*4002AM295826PedroA1*1801AM295898Ri011A1*4102AM295827k65A1*1802AM295899Ri018A1*4302AM295837k65A1*1803AM295900Ri260A1*5201AM29583888A1*1902AM295901Ri081A1*5301AM295839k390A1*1903AM295902BB10A1*5501AM295840k127A1*1904AM295903Ri197A1*5601AM295841k766A1*2201AM295904Ri009A1*5701AM295842k2,A1*2502AM295905Ri088A1*5801AM295843RastafaA1*2602AM2959068765A1*5901AM295844RozaA1*2802AM295907Ri082A1*6001AM295845VivaaA1*3201AM295908Ri094A1*6101AM295846k135A1*3301AM295909Ri253A1*6201AM29584781A1*4001AM295910Ri165A1*6301AM295848HippoA1*4101AM295911Ri284A1*6401AM295849BufoA1*4701AM295912Ri189A1*6402AM295850k95A1*4801AM2959138822A1*650101AM29585181A1*4901AM295914Ri088A1*650102AM295852k390A1*5001AM2959154053A1*6601AM295853k73A1*5101AM295916Ri281A1*6602AM295854MilvaA1*5201AM295917Ri136A1*6701AM29585583A1*5301AM295918Ri056A1*6801AM295856JuanitaA1*5401AM295919Ri260A1*6901AM295857ZazaaA1*5402AM295920Ri011A1*7001AM295858FrikoA1*5601AM295921Ri146A1*7101AM295859ZolaA1*5602AM295922Ri228A1*7201AM295860CanadaA1*5701AM295923Ri006A1*5801AM295924Ri026Mamu-A2Mafa-A2A2*050301AM295925Ri056A2*0501AM295861ClintA2*050302AM295930Ri226A2*0502AM29586284A2*050402AM295926Ri009A2*0503AM295863k95A2*0513AM295927Ri159A2*0504AM295864ClintA2*0514AM295928Ri095A2*0505AM295865BufoA2*0515AM295929Ri009A2*0506AM295866TrufoA2*0517AM295931Ri284A2*0507AM29586780,A2*0518AM295932Ri094A2*0508AM295868ZebraA2*0519AM295933Ri137A2*0509AM29586981A2*0520AM295934Ri197A2*0510AM295870MilvaA2*0521AM295935Ri126A2*0511AM295871HippoA2*0522AM295936Ri026A2*051201AM295872k73A2*0523AM295937Ri028A2*051202AM295876DojoA2*0524AM295938Ri301A2*0513AM29587381A2*0525AM295939Ri137A2*0514AM295874k95A2*0526AM295940Ri002A2*0515AM295875JuanitaA2*2402AM295941BB67A2*0516AM295878JawaA2*2403AM295942Ri366A2*0517AM295877LabaMamu-A3Mafa-A3A3*1307AM295943Ri136A3*1301AM295879k2,Mamu-A4Mafa-A4A4*1404AM295944Ri137A4*1401AM29588077A4*1402AM295881BufoMamu-A5Mafa-A5A5*300101AM295945BB67A5*3001AM295882GayoA5*300102AM295946Ri081A5*3002AM295883k766A5*3002AM295948Ri088A5*3003AM295947Ri366Mamu-A6Mafa-A6A6*0101AM295949Ri145A6*0101AM295884k73Mamu-A7A7*0101AM295950Ri082A7*0102AM295951Ri315A7*0103AM295952Ri009The Chinese rhesus macaques are indicated by Ri numbers. Nine alleles found in rhesus macaques of mixed breeding are also reported.\nResults and discussion\nPolymorphism and diversity of the Mamu-A region: comparison of Chinese and Indian rhesus macaques\nIn a previous communication, five different Mamu-A region configurations were defined in a population of Indian rhesus macaques (Otting et al. 2005). These configurations display diversity with regard to the number and combination of distinct Mamu-A genes present per chromosome. The loci have been designated Mamu-A1, -A2, -A3, and -A4, respectively (Fig.\u00a01). As can be seen, each region configuration comprises a Mamu-A1 gene characterized by high transcription levels (major) combined with one or two other Mamu-A genes characterized by lower transcription levels (minors). The Mamu-A1 gene is probably responsible for executing the classical antigen presentation function (Evans et al. 1999; Sidney et al. 2000; Sette et al. 2005), whereas the others can be considered as nonclassicals and may interact, for instance, with the KIR gene family present on NK cells. Two complete MHC haplotypes in the rhesus macaque are sequenced (Daza-Vamenta et al. 2004; Kulski et al. 2004), and they represent two-region configurations. One haplotype contains the -A1\/-A2 combination, whereas in the other, the -A1\/-A4 pair is observed. Both region configurations are present in the animals of Indian origin (Fig.\u00a01).\nFig.\u00a01Schematic representation of different Mamu-A region configurations observed in Indian rhesus macaques. The exact order and physical distances of the loci on the genome are still unknown. The relative levels of polymorphism and transcription of the loci are indicated. Two Mamu-A region configurations (1 and 5) are confirmed by the sequencing of the complete rhesus MHC region (Daza-Vamenta et al. 2004; Kulski et al. 2004)\nWithin the present panel of 42 Chinese animals, 59 different full-length Mamu-A cDNAs were detected that can be grouped into various loci and lineages (Fig.\u00a02). A complete listing of the alleles detected in the Chinese macaques is provided as electronic supplementary material (Table\u00a05). Because of codominant expression, rhesus macaques can be heterozygous for the Mamu-A1 gene and thus can express up to two allotypes. Nine animals were found to express three different Mamu-A1-like sequences. These animals shared one of these sequences in separated clades in phylogenetic tree (Fig.\u00a02; -A5, -A6, and -A7). The triplets of the three animals are indicated by asterisks. The clustering of the three alleles, obtained from one animal, in clades distinct from -A2, -A3, and -A4 indicates that one allele belongs to a separate locus. The newly detected loci are named Mamu-A5, -A6, and-A7, according to the generally accepted nomenclature system (Klein et al. 1990; Robinson et al. 2003; Ellis et al. 2006). Representatives of two loci, A5 and A6, are also observed in cynomolgus macaques that express more than two A1-like sequences.\nFig.\u00a02Phylogenetic tree of Mamu-A and Mafa-A gene\/alleles detected in this study. The -A2 locus is represented by four alleles only. The tree is based on full-length sequences, although analyses on only exon 2 and 3 showed no significant differences. The nomenclature of the new alleles is based on a more extended tree (not shown) containing all the Mamu-A and Mafa-A alleles now available. Asterisk, three alleles of animal Ri081, of which one represents the locus Mamu-A5. Double asterisks, three alleles of animal Ri145, of which one represents the locus Mamu-A6. Triple asterisks, three alleles of animal Ri078, of which one represents the locus Mamu-A7\nThirty-three new Mamu-A1 alleles were detected in the Chinese rhesus macaques. Although some of the alleles in this population group into lineages that are also present in Indian animals, sharing of alleles for the Mamu-A1 locus is rare and was observed only once. The Mamu-A1*26 allele is detected in both populations, whereas the Mamu-A1*03 and for -A1*04, alleles are highly similar and differ for one synonymous basepair substitution. Hence, it is concluded that most allelic polymorphism observed for the Mamu-A1 locus was probably generated after the rhesus macaque populations were separated. Sequence comparisons illustrated that most of the variations map at the contact residues of the peptide-binding site (data not shown). Thus, polymorphism at the highly divergent Mamu-A1 locus, characterized by high expression levels, must have resulted from positive Darwinian selection (Hughes and Nei 1988; Borghans et al. 2004).\nThe Mamu-A2 gene displays differential haplotype distribution in the Indian population of rhesus macaques (Fig.\u00a01). This also appears to be the case for Chinese animals, as Mamu-A2 cDNAs were detected in 39 out of 42 of them. In this panel, 16 alleles that differ from the ones earlier detected in Indian animals were defined. In the Indian population, low expression levels characterize the Mamu-A2 gene, which appears also to be the case in Chinese animals.\nFive Chinese macaques possess the Mamu-A3*1307 sequence, which is always observed in combination with Mamu-A1*1102. In concordance with the Indian animals, the number of Mamu-A3 clones detected also reflects low expression levels. A full-length Mamu-A4*1404 cDNA was detected in only three animals. In contrast to the Mamu-A4 allele in the Indian macaque, the Chinese allele has a stopcodon at the last triplet of exon 5 encoding the transmembrane part of the class I protein. This suggests that the corresponding gene product may settle in the membrane, but the signal transduction is impaired. As was observed earlier in Indian animals, this locus is characterized by extremely low expression levels (Fig.\u00a01). For that reason, the presence of Mamu-A4 cDNAs in the other animals was tested with SSP on the RT-PCR samples, and 17 out of 42 Chinese animals appeared to be positive for this locus. The Mamu-A5, -A6, and -A7 genes are also characterized by low expression levels and are considered to represent minors.\nIn contrast to the previously studied Indian population, the Chinese animals were selected randomly from a large population. Because of the lack of pedigree data and segregation profiles, it was not possible to firmly establish the segregation of different Mamu-A genes present per chromosome. Haplotypes can only be deduced based on sharing of sequences between animals, and such haplotypes are listed in Table\u00a02. At this stage, it is impossible to determine in which region configurations the Mamu-A5,-A6, and -A7 genes are present. \nTable\u00a02Chinese Mamu-A haplotypes deduced by sharing of alleles grouped into two-region configurations\u00a0Mamu-A1Mamu-A2Mamu-A3Number1*1801*15163*2201*05156*5201*051332*1102*13075\nMafa-A region polymorphism and diversity in cynomolgus monkeys\nSixty-two alleles were detected in the 96 cynomolgus macaques analyzed. As observed in the rhesus macaques, most animals had two polymorphic Mafa-A1 sequences, in combination with orthologs of the Mamu-A2, -A3, -A4, -A5, and\/or -A6 genes. In total, 38 Mafa-A1 alleles were detected, illustrating the high level of polymorphism of this locus. These alleles were compared with sequences reported earlier by two other research groups (Uda et al. 2004; Krebs et al. 2005) that have used different nomenclature systems. Only four alleles -A1*3101, -A1*3201, -A1*3801, and -A5*3001 detected in our panel are identical to the earlier described sequences A*310101, A*320101, A*380101, and A*300101.\nA total of 79 out of 96 animals have at least one Mafa-A2 sequence, and 18 alleles could be distinguished (Table\u00a01). The oligomorphic A2 locus is also present in the pigtailed macaque (Macaca nemestrina) as was recently described (Pratt et al. 2006; Lafont et al. 2007). In these studies, 90% of the animals possess the gene, and lower transcription levels were also observed in comparison to the other Mane-A alleles. The Mafa-A3 gene is observed in only two animals, whereas the Mafa-A4 locus is detected in 41 individuals. Two Mafa-A4 alleles were found that differ in only one basepair (nonsynonymous) substitution (Fig.\u00a02). As in the Chinese rhesus macaques, the Mafa-A4 alleles have a stopcodon at the end of exon 5. The Mafa-A5 and -A6 alleles were detected in, respectively, eight and four animals and display low levels of polymorphism. Phylogenetic analyses illustrate that apart from loci rhesus and cynomolgus monkeys also share lineages (Fig.\u00a02). As found in rhesus macaques, the Mafa-A1 alleles are characterized by high transcription levels, whereas the other loci display moderate or low expression levels.\nBecause most of the cynomolgus macaques are pedigreed, the combination of sequences that are inherited on one chromosome could be defined. As an example, the kinship tree of one breeding group is provided (Fig.\u00a03). The haplotypes are grouped based on the combination of loci (Table\u00a03). Seven combinations of loci or region configurations are recognized (Fig.\u00a04), and four of them (two to five) are also observed in the Indian rhesus macaques (Fig.\u00a01), which indicates that they predate speciation. Mafa-A5*3001 segregates in combination with Mafa-A1*6001 and -A1*7201 but not exclusively. The exact region configurations of Mafa-A6 containing haplotypes are not yet known.\nFig.\u00a03Pedigree of a cynomolgus macaque family showing segregation of Mafa-A alleles. The animals analyzed are indicated by shading. A question mark indicates that the sire has not been identifiedTable\u00a03Mafa-A haplotypes as defined by segregation grouped into seven-region configurations\u00a0Mafa-A1Mafa-A2Mafa-A3Mafa-A4Mafa-A5Number1A1*40026A1*58014A1*700132A1*0101A2*05144A1*1003A2*050610A1*1901A2*05133A1*3101A2*05044A1*5901A2*050812A1*6301A2*05017A1*6301A2*05113A1*6401A2*05059A1*6801A2*05153A1*6601A2*0512013A1*6602A2*051010A1*6801A2*051533A1*5701A3*130124A1*1702A4*140211A1*7101A4*1465A1*650102A2*05A4*1436A2*051202A4*1497A1*6001A5*30012A1*7201A5*30013One region configuration appears to lack the Mafa-A1 gene. At this stage, it is unknown whether the locus is really absent or was missed because of PCR failure, for example, because of a mutation in a primer site.Fig.\u00a04Schematic representation of different Mafa-A region configurations in cynomolgus macaques. Four configurations (2\u20135) are shared with the Indian rhesus macaques (Fig.\u00a01). One region configuration lacks the Mafa-A1 gene. It is not yet known whether the locus is really absent or missed because of PCR failure. Should a Mafa-A1 gene be found in extended studies than this region, configuration has to be deleted from the list\nSharing of -A1 and -A2 sequences between different macaque species\nA comparison of all available Mamu-A to Mafa-A sequences showed that six full-length cDNA transcripts are shared between both species of macaque. This is the case for five -A1 pairs and one -A2 pair (Table\u00a04). One of these cynomolgus monkey alleles is identical to an Indian rhesus allele, whereas the other five are shared with animals of Chinese origin. This observation was unexpected as the two rhesus macaque populations investigated share only one allele. The fact that cynomolgus macaques share more alleles with Chinese rhesus macaques than with Indian individuals may be explained by the fact that there is an overlap in the geographic areas inhabited by both species in Indochina (eastern Asia). It is known that rhesus macaques and cynomolgus monkeys can interbreed and produce offspring (Tosi et al. 2002). Sharing of alleles was not observed for the minors controlled by the A3\u2013A7 loci. \nTable\u00a04Identical Mhc class I sequences detected in the two species of macaqueMacaca mulattaMacaca fascicularisMamu-A1*0103Mafa-A*260101aMamu-A1*4001Mafa-A*400101aMamu-A1*1001Mafa-A1*1002Mamu-A*21bMafa-A1*0302Mamu-A1*5301Mafa-A1*5301Mamu-A2*0519Mafa-A2*051202aPublished by Krebs et al. 2005.bPublished by Otting et al. 2005.\nDifferent modes of selection operating on the Mhc class I and II genes in macaques\nAlthough Mamu-A and Mafa-A sequences are interspersed in the phylogenetic tree, the vast majority of the alleles are species unique, and for rhesus macaque, most of them appear to be population specific. This is in sharp contrast to the situation observed for the macaque class II region, where sharing for different exon 2 sequences at the Mhc-DR, but especially the -DQ and -DP genes, is far more common (Blancher et al. 2006; Doxiadis et al. 2006). About half of the Mafa-DPB1, -DQA1, and -DQB1 and one third of the Mafa-DRB sequences are identical to rhesus orthologs. As animals of the same populations have been used to study Mhc class I and II sequences, the present results exclude the possibility that this high sharing of Mhc class II sequences is because of introgression. A more likely explanation is that in macaques, the exon 2 sequences of the Mhc class II genes, which encode the peptide-binding site, have been subjected to purifying selection. As a consequence, many Mhc class II alleles in these two macaque species predate speciation processes. A databank search indicated that the phenomenon also extends to other macaque species (Robinson et al. 2003).\nThe Mhc class I alleles in macaques are largely unique, illustrating that selection has favored diversity. Mhc class I proteins are involved in the presentation of intracellular pathogens such as viruses and parasites that are known to evolve at high mutation rates. Recognition of Mhc class I molecules combined with a foreign peptide may result in the lysis of an infected cell. The Mhc class II molecules select peptides, originating from extracellular pathogens such as bacteria and fungi, for binding. Mhc class II-mediated activation may result in antibody production and\/or providing help to cytotoxic T cells. The present results illustrate that, probably because of coevolution with intracellular pathogens, macaques have generated a highly complex and divergent Mhc class I repertoire. The fact that Mhc class I alleles evolve quickly, even within a species, has been documented in humans for instance (Belich et al. 1992; Watkins et al. 1992). The high degree of sharing of Mhc class II sequences seems to be unique for macaques and has not been observed in any other group of vertebrate species.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material. \nTable\u00a05\nOverview of Mamu-A genes\/alleles in the panel of Chinese rhesus macaques. Sequences represented by two ciphers (for example A*05) are not confirmed and are not submitted to the to the EBI-EMBL database (PDF 127\u00a0KB).","keyphrases":["nonhuman primates","rhesus macaque","cynomolgus macaque","evolution"],"prmu":["P","P","P","U"]}
{"id":"Ann_Surg_Oncol-3-1-1914273","title":"Isolated Limb Perfusion with Tumor Necrosis Factor Alpha and Melphalan for Locally Advanced Soft Tissue Sarcoma: Three Time Periods at Risk for Amputation\n","text":"Background The aim of this study was to investigate the long-term limb salvage rate and overall survival after isolated limb perfusion (ILP) with tumor necrosis factor alpha and melphalan for locally advanced soft tissue sarcoma (STS).\nLimb salvage in patients with locally advanced extremity soft tissue sarcoma (STS) continues to be a challenge. Survival in these patients is determined by the development of distant metastases and is not improved with the amputation of the affected limb.1,2 Besides amputation, an extensive surgical procedure followed by radiotherapy is a treatment option.3 Rosenberg et al.1 showed the same disease-free and overall survival as amputation in the early 1980s with this treatment regimen. Preoperative therapies to improve limb salvage rates have been proposed. Suit et al.4 reported in 1981 on the use of preoperative radiotherapy. Eilber et al.2,5 combined preoperative (intra-arterial or systemic) chemotherapy and radiotherapy to improve resectability rates. In a randomized trial, O\u2019Sullivan et al.6 reported a greater risk of wound complications in the preoperative radiotherapy group compared with the postoperative radiotherapy group. The use of brachytherapy may also improve local control and avoid amputation.7 The current treatment strategy of high-grade limb sarcomas is wide local resection, with the goal of achieving a R0 resection with a 2-cm margin. If the margin is\u00a0<\u00a02 cm or if a R1 resection (microscopically involved margin) is performed, adjuvant radiotherapy with 50\u201370 Gy is indicated to reduce the risk of local failure.3 The question whether radiotherapy should be given before or after surgery is still unanswered.6\nAnother strategy for limb salvage in locally advanced extremity STS is to perform an isolated limb perfusion (ILP) with cytostatic agents. This procedure, which was originally developed for the treatment of melanoma of the limb in 1957, was also applied to the treatment of STS of the limb. In their first experience, Krementz et al.8 showed an early response rate of 83% with melphalan alone; however, complete regression of the tumor was rarely seen. Other perfusion agents in the treatment of limb STS were therefore investigated. Rossi et al.9 claimed that doxorubicin was efficacious; another study10 showed that doxorubicin alone was ineffective and that combined with melphalan, it was too toxic. Cisplatin also proved to be less effective than melphalan in the limb perfusion setting of sarcomas, and carboplatin was too neurotoxic.11\u201313\nWith the addition of tumor necrosis factor alpha (TNF-\u03b1) to the perfusion circuit, Lienard et al.14 made a step forward in the treatment of locally advanced extremity STS. A large European multicenter study proved the ILP concept in the limb salvage procedures for locally advanced STS with TNF-\u03b1 and melphalan. The objective response rate was 75%, and a limb salvage rate of 82% was achieved with minimal treatment-related morbidity.15 Since 1991, patients with locally advanced STS of the limbs have been treated at the University Medical Center Groningen by ILP with TNF-\u03b1 and melphalan with or without interferon gamma as perfusion agents, followed by delayed surgical excision and postoperative radiotherapy if a marginal resection or nonradical resection was performed. Recently we encountered long-term local morbidity, and therefore the aim of the present study was to analyze the limb salvage rate and survival in patients with locally advanced STS of the extremities that were treated in our center and to report the late effects of this treatment modality.\nPATIENTS AND METHODS\nPatient Characteristics\nDuring 1991\u20132003, a total of 73 patients with STS of the extremity underwent 77 perfusions with a combination of TNF-\u03b1 and melphalan, with (n\u00a0=\u00a019) or without (n\u00a0=\u00a058) interferon gamma. Thirty-six men and 37 women with a median age of 54 (range 14\u201380) years were treated. Tumors were considered unresectable because of size, their multicentricity in the limb, or fixation to the neurovascular bundle and\/or bone, and therefore amputation was the only treatment option. Perfusion was performed at the iliac level in 32 cases (42%), at the popliteal level in 23 cases (30%), and at the femoral and axillary level in 11 cases each (14%). There were 60 primary (82%) and 13 recurrent (18%) sarcomas. Sixty-two sarcomas were located in the leg (85%) and 11 were located in the arm (15%). All patients were treated after informed consent was obtained according to institutional guidelines. Nineteen different histological types of STS were distinguished. The pathological grade of the tumor was scored following the criteria of Coindre et al.,17 and the stage of the tumor was scored according to the American Joint Committee on Cancer (AJCC) criteria16 (Table\u00a01). Median tumor size was 16.2 (range 8.3\u201323) cm. In case of multifocal disease, the largest diameter was used.\nTABLE\u00a01.Histological grade and stage of tumorsaCharacteristicn%Grade\u00a0\u00a0I1014\u00a0\u00a0II2332\u00a0\u00a0III4054Stage\u00a0\u00a0I1014\u00a0\u00a0II11\u00a0\u00a0III5069\u00a0\u00a0IV1216a Grade according to Coindre et al.,17 and stage according to American Joint Committee on Cancer.16\nPerfusion Technique\nThe perfusion technique used at the University Medical Center Groningen is based on the technique developed by Creech et al.18 and described elsewhere.19 The major modifications during the last 30 years were the use of modern thermal blankets, improvement in leakage monitoring, and the introduction of a membrane oxygenator and heat exchanger to ensure optimal perfusion at 39\u201340\u00b0C. Because extensive washing with 6 L of saline is used, systemic inflammatory response syndrome (SIRS) is rarely seen.20 After surgery, patients can be monitored on the recovery ward instead of the intensive care unit.\nAssessment of Tumor Response, Tumor Remnant, and Follow-up\nResponses were assessed by standardized World Health Organization criteria and on the basis of physical examination and\/or imaging investigations (magnetic resonance imaging and computed tomographic scans).21 Complete response was defined as the disappearance of all measurable disease in the limb for\u00a0>\u00a04 weeks, partial response as regression of the tumor size by\u00a0>\u00a050% of the largest diameter for\u00a0>\u00a04 weeks, and no change as regression of\u00a0<\u00a050% of the tumor in the limb or progression of\u00a0<\u00a025% for longer than 4 weeks. Resection of the tumor remnants was performed 2\u201315 weeks (median, 8 weeks) after perfusion. After resection, response was also assessed by pathological examination. The tumor remnants were measured in three dimensions and the percentage of necrosis estimated in relation to the complete tumor volume. Representative tumor sections were taken, encompassing macroscopically different tumor areas, including necrosis. As a general rule, one section per centimeter largest diameter with a minimum of three was taken. On the basis of an integration of gross and microscopic findings, a final estimate of the percentages of viable and necrotic or regressive tumor was made.\nExcision margins were also evaluated on pathological examination and classified as radical when the resection margins were free of tumor cells (complete resection, R0), as R1 when resection margins were microscopically involved, or as R2 when resection margins were macroscopically positive involved. Postoperative radiotherapy (60\u201370 Gy) was considered indicated in case of\u00a0<\u00a095% necrosis on pathological examination of the tumor or with marginal or microscopically positive resection margins. All patients were followed after perfusion treatment in a standardized protocol. Median follow-up was 27 (range 2\u2013138) months.\nStatistical Analysis\nSurvival and limb salvage curves were calculated according to the Kaplan-Meier method and log rank test.22 Values of P\u00a0<\u00a00.05 were considered to be statistically significant. GraphPad Prism version 2.0 for Windows statistical software was used.\nRESULTS\nTumor Response\nA clinical complete response was observed after 19 ILPs (25%), a partial response after 53 ILPs (69%), and no change after 5 ILPs (6%); local progression was never observed. Resection of the remnant tumor was performed in 68 patients (93%). The pathological response is illustrated in Fig.\u00a01. After 17 ILPs (23%), no viable tumor cells were found on pathological examination. In 29 ILPs (37%),\u00a0\u2265\u00a090% necrosis was found on pathological examination. When both groups were added together, a good response to ILP was found in 60% of the patients. In 17 ILPs (22%), an intermediate response was found on pathological examination (necrosis 50%\u201380%). After eight ILPs (10%),\u00a0<\u00a020% of necrosis or no necrosis was found on pathological examination. In five patients (7%), tumor response was not assessed because of progression of distant metastases in four patients and a local recurrence in one patient that necessitated a second perfusion, resulting in 90% necrosis of the tumor. No correlation could be demonstrated between grade and percentage of tumor necrosis after perfusion (Pearson\u2019s correlation). Postoperative radiotherapy (total dose 60\u201370 Gy, 25\u00a0\u00d7\u00a02 Gy daily and 10\u201320 Gy boost) was given in 37 patients with microscopically involved or marginally free resection margins. Radiotherapy was initiated within 5\u20136 weeks after tumor resection. Radiation treatment was delivered through a multiple-field technique with computed tomographic treatment planning on a linear accelerator, 6\u201315 MV.\nFIG.\u00a01.Percentage of necrosis estimated at pathological examination of resected tumor remnant in relation to number of patients.\nAmputations and Limb Salvage\nA total of 21 amputations (28%) were performed. Table\u00a02 presents the time interval between ILP and amputation and the rationale for amputation. Overall 1, 5, and 10 years\u2019 limb salvage was 80.1%\u00a0\u00b1\u00a04.8%, 68.2%\u00a0\u00b1\u00a06.5%, and 60.6%\u00a0\u00b1\u00a09.2%, respectively (Fig.\u00a02). When we analyzed the limb salvage curve, we found that the risk for amputation could be divided into three time episodes. The first period occurs within the first year or year and a half after perfusion (n\u00a0=\u00a017 patients), with amputation performed to treat massive perfusion-induced necrosis of the tumor and overlying skin, which resulted in a soft tissue deficit (n\u00a0=\u00a06); tumor recurrence after perfusion (n\u00a0=\u00a05); wound complications after ILP followed by radiotherapy (n\u00a0=\u00a02); a microscopically involved resection margin with the rejection of the patient for adjuvant radiotherapy of the foot (n\u00a0=\u00a02); insufficient clinical response (one patient); and arterial thrombosis with no vascular reconstruction possibilities 2 months after resection of a local recurrence in the groin (n\u00a0=\u00a01).\nTABLE\u00a02.Amputations performed in 21 patients according to interval durationPatient no.DiagnosisAge (y)Interval (mo)ResectionRTRationale for amputationCurrent status1PUS600R0NoPostperfusion necrosis38NED 120 months2Angiosarcoma741R2NoLocal recurrenceDOD 11 months3Fibrosarcoma761R0NoPostperfusion necrosisNED 2 months4PUS672R0NoPostperfusion necrosisDOD 9 months5Epithelioid Sarcoma212R0NoPostperfusion necrosisDOD 54 months6Leiomyosarcoma172R0NoInsufficient clinical responseDOD 7 months7Liposarcoma602R1NoR1 resection, RT not possibleAWD 10 months8PNET623R0NoLocal recurrenceDOD 17 months9Synovial sarcoma393R0NoPostperfusion necrosisDOD 50 months10PUS633R1NoPostperfusion necrosisNED 72 months11Angiosarcoma804R1NoLocal recurrenceDOD 10 months12Synovial sarcoma654R1NoR1 resection, RT not possibleNED 6 months13Epithelioid Sarcoma226R2NoLocal recurrenceaDOD 39 months14Hemangioma pericytoma508R0YesWound complications after ILP with radiotherapyaAWD 65 months15PUS7112R0YesWound complications after ILP with radiotherapyaNED 14 months16PUS6115R0YesArterial occlusionAWD 17 months17Synoviosarcoma4218R1YesLocal recurrenceNED 20 months18Liposarcoma5337R0YesLocal recurrenceDOD 110 months19Liposarcoma3958R0NoLocal recurrenceDOD 120 months20PNET56110R0YesCritical leg ischemiaNED 118 months21Chondrosarcoma18125R0YesCritical leg ischemiaNED 134 monthsPUS, pleomorphic undifferentiated sarcoma; RT, radiotherapy; NED, no evidence of disease; AWD, alive with disease; DOD, death of disease; PNET, malignant peripheral nerve sheath tumor; R1 resection, microscopically involved resection margin; ILP, isolated limb perfusion.a Patients treated for second ILP with tumor necrosis factor alpha and melphalan.FIG.\u00a02.Limb salvage curve in patients with locally advanced soft tissue sarcoma treated with tumor necrosis factor alpha, melphalan, and isolated limb perfusion.\nThe second time period was within 5 years after ILP, with two amputations performed for late local recurrent disease (37 and 58 months after perfusion). The third episode occurred approximately 10 years after perfusion. Amputation was performed for critical leg ischemia with neuropathy due to treatment-induced atherosclerosis of the remaining tibial artery that was not suitable for arterial reconstruction (110 and 125 months after perfusion). An example of the clinical appearance of patient 21 is shown in Fig.\u00a03. In this patient, a popliteal ILP was performed at the age of 18 years for a chondrosarcoma. After marginal resection, this patient received 66 Gy adjuvant radiotherapy. Ten years after ILP, an amputation was performed because of critical leg ischemia. No recurrent disease was found on pathological examination of the amputated specimen. Another two patients developed a pathological fracture of the femur due to radiation-induced osteonecrosis (78 and 129 months after perfusion). These four patients with late post-ILP complications received high-dose postperfusion radiotherapy (60\u201370 Gy).FIG.\u00a03.Clinical appearance of lower leg of patient 21 (Table 3) before amputation for critical leg ischemia.\nSystemic Metastases and Survival\nTwelve patients presented with distant metastases at time of ILP (16% stage IV AJCC); half of these patients had lung metastases, and the other half had lymph node metastases. Eleven of these patients died of disease after a median period of 9 (range 2\u201354) months; one patient is alive with no evidence of disease after 11 months. During follow-up, 25 patients (36%) developed distant metastases at a median interval of 9 (range 2\u2013100) months. A statistically significant difference (P\u00a0<\u00a0.001) was observed between patients with no distant metastases at the time of ILP compared with patients with metastases at the time of ILP (Fig.\u00a04). Overall 1, 5, and 10 years\u2019 survival for all patients was 82.9%\u00a0\u00b1\u00a09.2%, 58.7%\u00a0\u00b1\u00a013.1%, and 42.5%\u00a0\u00b1\u00a018.2%, respectively (Fig.\u00a04).\nFIG.\u00a04.Overall survival in patients with locally advanced soft tissue sarcoma treated with isolated limb perfusion (ILP), tumor necrosis factor alpha, and melphalan. Statistically significant difference was observed between patients with no distant metastases at time of ILP (mets \u2212) compared with patients with metastases at time of ILP (mets +).\nDISCUSSION\nThe results of a European multicenter trial performed in the 1990s led to the approval of the use of TNF-\u03b1 for ILP in patients with locally advanced extremity sarcomas by the European Medicine Evaluation Agency.15 ILP with TNF-\u03b1 is currently available in more than 30 centers, and in 2002, a total of 350 so-called TNF-\u03b1 perfusions were performed. As one of the first centers that participated in the TNF-\u03b1 ILP experience, and with more than a decade\u2019s experience, we recently encountered long-term treatment-related morbidity necessitating amputation of the perfused limb 10 years after treatment. For this reason, we analyzed our results of ILP with TNF-\u03b1 and melphalan and describe our results in the present study.\nWe observed an overall response rate of 82%, which is in the range of the 63%\u201391% response rates reported in the literature.15,23,25 Although a suggestion has been made for a relation between the grade of sarcoma and the response to TNF-\u03b1 ILP, we could not demonstrate a correlation between grade and the percentage of necrosis after ILP with TNF-\u03b1. This is in concordance with the results of the Amsterdam group.23 Various reports have shown that a limb salvage rate of 81%\u201386% can be achieved in patients with locally advanced limb sarcoma.15,23,25 An independent review committee reconsidered the unresectability criteria of all patients enrolled onto the European study. Eighty percent of the patients in this study met the criteria for unresectability, and survival curves based on a match-control study with cases of the Scandinavian Soft Tissue Sarcoma Databank showed that TNF-\u03b1 ILP had no negative effect on survival.26\nWe used the Kaplan-Meier method to calculate limb salvage because this method adjusts for censored observations, i.e., patients who were alive and well at the time of last contact or patients who died of distant metastases but who retained preserved limb function. By use of this method, we calculated a 1-year limb salvage rate of 80%. Amputations were performed mostly because of postperfusion-related complications or early local recurrence in the first year after ILP. A second curve in limb salvage was observed within 5 years after TNF-\u03b1 ILP in two patients with late local recurrences. A third bend in the limb salvage curve was observed approximately 10 years after ILP. This was a new observation in two patients who had critical leg ischemia with ulceration and continuous pain. Besides ILP with TNF-\u03b1 and melphalan, both patients received adjuvant radiotherapy (66 and 70 Gy) after marginal tumor resection.\nWhat seems to be the cause of this late morbidity? Analysis of the functional and long-term morbidity in 97 patients with stage I melanoma treated with ILP with melphalan as the sole perfusion agent in our center showed after a median follow-up of 36 (range 12\u201376) months, no patients had critical leg ischemia.27 The Rotterdam and Amsterdam perfusion group reported long-term morbidity consisting of muscle atrophy or fibrosis in 11% of the patients after ILP with melphalan; however, cases of critical leg ischemia are not described.28 The fact that in our series no muscle atrophy or fibrosis was found might be explained by the fact that we always perform a lateral fasciotomy after ILP to prevent a compartment syndrome. When we performed a literature search for late morbidity after ILP with TNF-\u03b1 and melphalan, no studies could be retrieved.\nThe clinical importance of late morbidity after radiotherapy has evolved since Eifel et al.29 retrospectively reviewed the medical records of 1784 FIGO stage IB patients receiving primary radiotherapy at the M. D. Anderson Cancer Center between 1960 and 1989. She showed that after 5 years, there was a small but continuous risk of experiencing major complications of radiotherapy (i.e., urinary, rectal, and small bowel complications) at up to 20 years of follow-up. Johansson et al.30 described a high occurrence of severe neuropathy closely linked to the development of fibrosis around the nerve trunks after aggressive postoperative telecobalt therapy received in 1963\u20131965 in a group of 71 patients with breast cancer who were initially treated with modified radical mastectomy. Radiotherapy damage to the vascular system was demonstrated by Hopewell31 in an experimental setting; arteries of hamster cheek pouch showed localized constrictions after irradiation. These constrictions were caused by clones of endothelial cells and may be the predominant factor influencing the degeneration of the capillary bed after radiotherapy.32 Evidence of this occlusive effect of vessels by proliferating endothelial cells after radiation have also been reported by other investigators.33\nAnother argument to explain the observed late morbidity, at least in part, to radiotherapy is the fact that another two patients who received adjuvant radiotherapy after ILP with TNF-\u03b1 and melphalan developed a pathological fracture of the femur (78 and 129 months after ILP with TNF-\u03b1 and melphalan). Radiotherapy-induced osteonecrosis is a well-known phenomenon. Lin et al.34 described 12 fractures of the femur after surgery and irradiation for STS of the thigh. Treatment of these fractures was difficult and demanding, with only four bony unions after a mean follow-up of 37 months. When we add up the evidence of developing fibrosis after ILP with melphalan and the development of fibrosis after radiotherapy, the combination of the two regimens could explain the observed late morbidity rate in the present series.\nOverall survival for all patients showed a steadily decline, with a 10-year percentage of 42%. Even after 110 and 120 months, patients die of distant metastases. Sixteen percent of the patients had metastases at the time of ILP. A marked difference in survival was observed when comparing patients with pulmonary or lymph node metastases with patients lacking metastases at time of ILP. Five-year overall survival of 59% in this series is higher than the reported 5-year survival of 48% in the Amsterdam experience23 and the reported 32% of Lejeune et al.24 This is an unexpected observation because selection criteria for ILP with TNF-\u03b1 between the institutes are comparable. High-grade tumors and tumors\u00a0>\u00a05 cm in diameter have a great tendency to metastasize. Patients with such tumors could theoretically benefit from neoadjuvant chemotherapy. A quantitative meta-analysis of data from 14 trials of doxorubicin-based adjuvant chemotherapy showed a benefit from systemic adjuvant chemotherapy of 6% for local relapse-free interval, but there was no overall survival benefit at 10 years.35 Delaney et al.36 developed a regimen of preoperative chemotherapy consisting of mesna, Adriamycin, ifosfamide, and dacarbazine (MAID) interspersed with radiotherapy, followed by resection and postoperative chemotherapy with or without radiotherapy to improve outcome in patients with high-grade extremity STS. Compared with a historical group of control patients, outcome in the MAID group was better. In an update of 64 patients, 5 required amputation because of disease, 3 had unresectable disease, and 1 patient refused surgery. Estimated 3-year survival and local-regional control were 75.1% and 79.3%, respectively. These results are comparable with the results of the present study.37 However, systemic therapy is associated with systemic toxic effects, in contrast with the mild systemic side effects observed after ILP with TNF-\u03b1 and melphalan.\nSince 1992, we have not changed the indication for TNF-\u03b1 perfusion. Patients who were candidates for amputation of the involved limb, as assessed by preoperative magnetic resonance imaging, were offered an ILP with TNF-\u03b1 and melphalan, with the goal of preserving the limb with a locally advanced STS. After ILP patients received a delayed surgical resection, adjuvant radiotherapy was provided to patients with marginal or microscopically positive resection margins. This treatment resulted in a high limb salvage rate in patients with locally advanced STS, although late morbidity can occur, especially when adjuvant postoperative radiotherapy is applied. Therefore, continuous follow-up of these patients is warranted.","keyphrases":["isolated limb perfusion","tumor necrosis factor alpha","melphalan","sarcoma","amputation","radiotherapy","late morbidity"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Neuroradiology-2-2-1592467","title":"Cerebral blood volume, genotype and chemosensitivity in oligodendroglial tumours\n","text":"Introduction The biological factors responsible for differential chemoresponsiveness in oligodendroglial tumours with or without the \u22121p\/\u221219q genotype are unknown, but tumour vascularity may contribute. We aimed to determine whether dynamic susceptibility contrast (DSC) magnetic resonance imaging (MRI) could distinguish molecular subtypes of oligodendroglial tumour, and examined the relationship between relative cerebral blood volume (rCBV) and outcome following procarbazine, lomustine and vincristine (PCV) chemotherapy.\nIntroduction\nOligodendroglial tumours with combined loss of chromosomes 1p and 19q have a more indolent clinical evolution, respond more favourably to chemotherapy and show prolonged survival compared to those with intact 1p\/19q, but the basis of these clinical differences is not well understood [1\u20135]. In addition, up to 30% of oligodendroglial neoplasms with intact 1p\/19q may respond to chemotherapy [3, 6, 7], but the genetic and biological factors associated with response are unknown and not all patients with oligodendroglial tumours derive durable benefit from therapy. Assessment of the clinical utility of biological markers and non-invasive imaging techniques is essential to advance the clinical management of these patients.\nRecently dynamic susceptibility contrast magnetic resonance imaging (DSC-MRI) has been used to calculate perfusion parameters in gliomas [8, 9], guide biopsy [10], provide prognostic information [11] and demonstrate differences in the vascularity of low-grade astrocytomas compared with oligodendrogliomas [8]. It has been suggested that increased vascularity and concomitant improved drug delivery may contribute to the chemoresponsiveness of oligodendroglial tumours, especially as lipophilic drugs (procarbazine, lomustine and temozolomide), which easily cross the blood\u2013brain barrier, are used in the treatment of oligodendrogliomas [12\u201314]. Currently DSC-MRI is used in the radiological evaluation of gliomas, but its role in oligodendroglial neoplasms classified by genotype and the relationship with response and outcome following chemotherapy has not so far been investigated.\nThe aim of this study was to determine whether DSC-MRI obtained through routine pre-therapy MR diagnostic examinations may be used to distinguish molecular subtypes of oligodendroglial tumours, and to investigate the influence of tumour blood volume on outcome following procarbazine, lomustine and vincristine (PCV) chemotherapy compared to molecular genetics.\nMethods\nPatient selection\nPatients for investigation were selected from those participating in a prospective study of adult oligodendroglial tumours eligible for chemotherapy at the Walton Centre for Neurology and Neurosurgery\/Clatterbridge Centre for Oncology between May 2000 and June 2003 [7, 15\u201318]. The study had full ethical approval. For inclusion in this study, patients had to have (1) given research consent, (2) a histological diagnosis of oligodendroglioma or oligoastrocytoma based on the current World Health Organisation (WHO) classification [19], (3) a known 1p\/19q status, and (4) undergone DSC-MRI before or after biopsy (2 image-guided; 35 serial stereotactic), but prior to commencing therapy. All patients (with primary and recurrent disease) underwent biopsy prior to PCV. Patients with post-resection imaging were excluded.\nDCS-MRI\nImaging studies were obtained using a 1.5-T Signa MR scanner (General Electric Medical Systems). The MR imaging protocol consisted of T1-weighted spin-echo (SE) sagittal sections (400\/14 TR\/TE), axial fluid attenuated inversion recovery (FLAIR 8802\/142\/2200 TR\/TE\/TI), axial fast spin-echo (FSE) T2-weighted imaging (4360\/102 TR\/TE), 2D dynamic susceptibility-weighted, SE, echoplanar imaging (EPI 1900\/80\u00a0ms, bandwidth 62.5) and postcontrast T1-weighted SE (600\/14 TR\/TE) or FLAIR (2280\/11\/750 TR\/TE\/TI) axial imaging. The T2-weighted FLAIR, FSE and postcontrast T1-weighted images were used to define regions of T2 hyperintensity and T1 enhancement. For the dynamic EPI series, the tumour size and location, and the position of the margins were determined from the T2-weighted FLAIR or FSE images to equally define low-grade and high-grade tumours. Nine slices were selected to cover the majority of the tumour volume. Slice thickness was 7\u00a0mm, with no gap. A standard dose of 0.1\u00a0mmol\/kg body weight of dimeglumine gadopentetate (Gd DTPA) was injected intravenously manually at an approximate rate of 3\u00a0ml\/s. A series of T2*-weighted multislice image sets (field of view 30\u00d730, matrix 192\u00d7128) were acquired every 2\u00a0s during the first pass of the contrast agent. Image postprocessing was performed using commercial software (Functool2, version 6.0; Sun Microsystems). Negative enhancement integral (NEI) maps were generated for each patient relative to the signal intensity in the superior sagittal sinus (Fig.\u00a01). The axial slice with the greatest cerebral blood volume and tumour area was chosen for analysis.\nFig.\u00a01Calculation of rCBV using DSC-MR. CBV is derived from the drop in the T2* signal after injection of a bolus of paramagnetic compound [8, 9]. Using Functool2, the radiological tumour margin was delineated on the T2* sequence axial image and a single circular ROI placed over normal brain mirrored in the contralateral hemisphere (a, b). Negative enhancement integral (NEI) colour maps were generated (c, d) relative to the signal intensity in the superior sagittal sinus using image data between dynamic scans immediately before and after the contrast transient. CBV is proportional to the area under the contrast agent concentration-time curve and was calculated for each ROI by Functool2 using algorithms to integrate \u0394R2i values (where \u0394R2 is the change in the reciprocal of T2*). rCBV for each tumour was calculated from signal intensity time curves for individual ROIs placed within the delineated tumour region or in the large ROI in the contralateral hemisphere (as illustrated in e and f, arrows scans adjacent to the contrast transient) as described in the Materials and methods. a, c, e Low rCBV (0.73\u00b10.17, mean\u00b1SD) in a grade II oligoastrocytoma with intact chromosomes 1p and 19q and; b, d, f high rCBV (4.15\u00b10.87) in a grade II oligoastrocytoma with the \u22121p\/\u221219q genotype\nThe radiological tumour margin was delineated on the T2* sequence axial image using a nongeometric region of interest (ROI), and a single circular region was placed over normal brain mirrored in the contralateral hemisphere. Depending on tumour location, the mirrored area could include both white and grey matter. ROIs were placed according to published methods giving the lowest inter- and intraobserver variability [20] within the delineated tumour region. Briefly, five ROIs (area range 12\u201327\u00a0mm2) were placed over the tumour areas with the highest blood volume. Cystic and hemorrhagic regions were avoided and care was taken not to place ROIs over normal blood vessels or a biopsy tract where seen. Three independent analyses were performed by a neuroradiologist (T.S.S.) and a neurosurgeon (M.D.J.) blinded to histology and genotype, to assess inter- and intraobserver variability. For each observation, data were expressed relative to the internal reference (four ROIs placed within the delineated mirrored region of normal brain without deliberate bias towards either grey or white matter) to generate relative cerebral blood volume (rCBV) values (mean ROItumour\/mean ROInormal brain) and accommodate physiological differences between patients [21]. For comparison with clinical data, the mean rCBV from these three data sets was used.\nMolecular genetics\nFor each patient regions of tumour histology in pretherapy biopsy specimens (formalin-fixed, paraffin-embedded tissues or intraoperative diagnostic smear preparations) representative of the most aggressive tissue available and the overall pathology diagnosis were selected for laser capture microdissection to enrich the tumour component in the samples for analysis. Allelic imbalance was investigated using paired normal (microdissected uninvolved brain or DNA from blood) and tumour tissue samples and multiple simultaneous PCR amplification of a panel of microsatellite markers, capillary electrophoresis and data analysis as described previously [16\u201318]. The microsatellite panel included: chromosome 1 \u2013 D1S2667, D1S508, D1S214 (1p36); chromosome 19 \u2013 D19S412, D19S112, D19S596 (19q13); chromosome 17 \u2013 D17S796, D17S1176, D17S1353 (17p13); chromosome 10p \u2013 D10S89, D10S179 (10p12-10p15); chromosome 10q \u2013 D10S1687, D10s2491, D10S583, D10S587, D10S212 (10q22-10q26). Additional markers, D1S468, D1S2795 (1p36) and D19S217, D19S572 (19q13) were used in cases that lacked informativity for given loci. p53 mutations (exons 5\u20138) were detected by direct sequence analysis of PCR products.\nPCV chemotherapy, response and clinical outcome\nAll patients receiving oncology therapy had evidence of advancing disease (either radiographic or clinical progression) prior to therapy. Patients with mass effect or evidence of malignancy were treated at presentation; others were followed clinically until progression. Oncology therapy was based on individual patient\u2019s clinical status\/choice. Patients fit for PCV were offered PCV chemotherapy, or radiotherapy if fertility was an issue. PCV chemotherapy was administered according to standard clinical protocols [22]: lomustine (CCNU) (110\u00a0mg\/m2), procarbazine (60\u00a0mg\/m2) and vincristine (1.4\u00a0mg\/m2, maximum 2\u00a0mg) were given on days 1, 8\u201321, and 8 and 29. respectively. Cycles were repeated every 6\u00a0weeks for a maximum of six cycles. As described previously [18], a consultant neuroradiologist reviewed all available MR or CT images taken before, during and after chemotherapy, and at follow-up. The largest perpendicular diameters of the tumour were measured on axial sections of T2-weighted MR images or contrast-enhanced regions on T1-weighted MR or CT images. Response was assessed using the criteria of Macdonald et al. [23] and postcontrast T1-weighted MRI in 25enhancing tumours. In tumours showing no enhancement (n=6) or in those in which contrast enhancement was not assessable (n=1), response was based on T2-weighted images.\nInformation regarding current management, follow-up and outcome was collected prospectively. Progression-free survival (PFS) and overall survival (OS) were calculated from the start of PCV to progression, last seen, or death as appropriate.\nStatistical analysis\nInter- and intraobserver variability was determined using Bland and Altman plots [24]. Differences in rCBV between genetic and clinical factors were assessed using Student\u2019s t-test for numerical data and the Chi-squared or Fisher\u2019s exact test for categorical data. Receiver operator characteristics (ROC) analysis was performed to define the optimal rCBV cut-off for predicting 1p\/19q status. Kaplan-Meier survival curves were obtained and differences in OS or PFS were tested for statistical significance using the Log Rank test. Cox regression multivariate analysis of factors shown to be significant in univariate analysis was by forward stepwise entry of parameters at a significance of 0.05 for entry and 0.01 for removal. Probability (P) values less than 0.05 were considered significant.\nResults\nThe study included 30 patients with primary tumours without previous therapy and 7 with recurrent or persistent disease following radiotherapy. Median time between previous radiotherapy and MR imaging was 6.5\u00a0years (range 1.1\u201314.2\u00a0years). Median age was 44\u00a0years (range 28\u201371\u00a0years).\nInterobserver and intraobserver variability\nTumour rCBV was obtained from DSC-MRI (Fig.\u00a01) by two independent observers. Bland and Altman plots revealed low inter- and intraobserver variation (Fig.\u00a02). The closest observed limits of agreement were between the first observations of the neuroradiologist and neurosurgeon (mean \u22120.17, range \u22121.27 to 0.93; Fig.\u00a02a). The widest observed limits of agreement were between the repeat observations of the neurosurgeon (mean 0.08, range \u22121.59 to 1.95; Fig.\u00a02c). The greatest variation was observed in tumours with higher rCBV values.\nFig.\u00a02Bland and Altman plots illustrating interobserver (a, b) and intraobserver (c) variation of rCBV measurements. The greatest variation is seen at the right of the plot for tumours with the higher rCBV values\nHistopathology, genotype and rCBV\nOligodendroglial tumours of different histopathology subtypes and grade did not have significantly different rCBV measurements (Figure\u00a03a,b). Loss of both 1p36 and 19q13 was seen in 6\/9 grade II oligodendrogliomas, 6\/14 grade II oligoastrocytomas, 4\/4 grade III oligodendrogliomas, and 3\/10 grade III oligoastrocytomas. Tumours with the \u22121p\/\u221219q genotype had significantly higher rCBV than those with intact 1p and 19q (Fig.\u00a03c). Similar findings were observed when primary tumours only were analysed (data not shown). ROC analysis was used to determine the specificity and sensitivity of rCBV as a potential non-invasive tool to distinguish oligodendroglial neoplasms with or without the \u22121p\/\u221219q genotype. The area under the ROC curve was 0.853 (95% confidence interval: 0.778\u20130.927). A cut-off value of 1.59 for rCBV (sensitivity 92%, specificity 76%) best discriminated oligodendroglial tumours with and without the \u22121p\/\u221219q genotype (Fig.\u00a03c).\nFig.\u00a03Box plots of rCBV against (a) histopathology subtype (P=0.279*), (b) histopathology grade (P=0.442*), and (c) genotype (P=0.001*) in oligodendroglial tumours. The optimal rCBV cut-off that distinguishes tumour genotype is shown by the horizontal line. *Student\u2019s t-test\nTo facilitate comparison of rCBV with genotype and clinicopathological characteristics (Table\u00a01), tumours were considered to have high rCBV if this was greater than 1.59 as determined by the ROC analysis (Fig.\u00a04). High rCBV values were significantly more common in tumours with loss of 1p36 and 19q13 and less common in tumours with 17p13 loss and p53 mutation. Three tumours in the series had loss of chromosome 10q22\u201326, of which two also had loss of 10p11\u201315; all had high rCBV. One tumour with high rCBV had loss of 1p36 without loss of 19q13. As for numerical data, there was no significant association between high rCBV and histology subtype or grade. Contrast enhancement was weakly associated with high rCBV in the series but not in primary tumours. No associations were seen between tumour rCBV and patient age <50\u00a0years versus age \u226550\u00a0years, gender or temporal versus non-temporal location. Similar associations between rCBV, genotype and clinicopathological characteristics were obtained if only primary tumours were analysed (Table\u00a01). \nTable\u00a01rCBV, molecular genetics and clinical characteristics in the series and primary tumours\u00a0All tumoursPrimary tumoursnNumber of tumours with high rCBV P valueanNumber of tumours with high rCBV P valueaLoss of 1p36 and 19q13Yes1917 (89%)0.000*1614 (88%)0.000No184 (22%)142 (14%)Loss of 17p13Yes112 (18%)0.003*80 (0%)0.001No2619 (73%)2216 (73%)Loss of 10q 22\u201326 \u00b110p11\u201315Yes33 (100%)0.23811 (100%)1.0no3317 (52%)2915 (52%)p53 mutation(exon 5\u20138)Yes112 (18%)0.004*80 (0%)0.001No2518 (72%)2115 (71%)Patient age (years)<502513 (52%)0.491209 (45%)0.260>50128 (67%)107 (70%)Histology subtypeOligodendroglioma1310 (77%)0.091118 (73%)0.142Oligoastrocytoma2411 (46%)198 (42%)WHO histology gradeII2311 (48%)0.19199 (43%)0.118III1410 (71%)217 (78%)Contrast enhancementAbsent82 (25%)0.04682 (25%)0.092Present2819 (68%)2114 (68%)Tumour locationTemporal147 (50%)0.733136 (46%)0.713Nontemporal2314 (61%)1710 (59%)Tumour statusPrimary3016 (53%)0.674Recurrent75 (71%)Response to PCV\u00a0All tumoursResponders2114 (67%)0.1421711 (65%)0.097Nonresponders114 (36%)82 (25%)\u00a0Tumours with loss of 1p\/19qResponders1614 (88%)1311 (85%)Nonresponders00\u00a0Tumours with intact 1p\/19qResponders50 (0%)0.24540 (0%)0.515Nonresponders114 (36%)82 (25%)aProbability calculated by Fisher\u2019s Exact test. Responders showed complete, partial or minor response and non-responders had stable or progressive disease.Fig.\u00a04rCBV, molecular genetics and clinical characteristics. rCBV for each tumour is given as the mean\u00b1standard deviation of three independent observations. The arrow indicates tumours with rCBV above (high rCBV) and below the cut-off value of 1.59 (low rCBV). CR complete response (disappearance of all tumour, off steroids and neurologically stable or improved), PR partial response (50% or greater reduction in cross-sectional area, steroids stable or reduced, and neurologically stable or improved); MR minor response (>25 to <50% reduction in cross-sectional area, steroids stable or reduced, and neurologically stable or improved); PD progressive disease (25% or greater increase in cross-sectional area or any new tumour on CT\/MR images and\/or neurologically worse with steroids stable or increased); SD stable disease (all other situations); P primary tumour, R recurrent tumour\nrCBV and outcome following PCV chemotherapy\nOf the 37 patients, 33 had PCV chemotherapy following DSC-MRI (median 0.7\u00a0months, range 0.1\u201315.2\u00a0months), of whom 26 with primary tumours received PCV chemotherapy as the first oncology therapy, 6 with recurrent or persistent disease treated initially with radiotherapy received PCV as second oncology therapy, and 1 had PCV following radiotherapy and temozolomide. One patient with primary tumour had therapy switched to radiotherapy at the end of the first cycle because of reaction to procarbazine. The remainder received a median of four cycles of PCV (range one to six). Response to therapy was assessed in 32 patients (Fig.\u00a04). Measured radiological changes and clinical factors permitted response assessment using standard criteria of Macdonald et al. [23] in all patients showing enhancement. However, in tumours showing no enhancement assessed using T2-weighted MR images, three showed radiological change in cross-sectional area of >25% to <50%, accompanied by clinical benefit; these were included in the minor response (MR) category. None of those showing enhancement had measured radiological change in this range. Response (i.e. complete, partial or minor; see Fig.\u00a04 legend for definition) was seen in tumours with and without high rCBV. Similarly, non-responders (i.e. patients with stable or progressive disease) had either high or low tumour rCBV (Table\u00a01, Fig.\u00a04). Response was strongly associated with genotype; all 16 tumours with loss of 1p36 and 19q13 responded compared with 5 of 17 tumours with intact 1p36\/19q13 (Chi2 P<0.001). Of the 16 responders in the 1p36\/19q13 loss group, 14 had high rCBV. The five responders in the intact 1p36\/19q13 group all had low rCBV and the four tumours with high rCBV failed to respond.\nThe relationship between rCBV, genotype, histopathology and survival following PCV in the series and in those with primary tumour is given in Table\u00a02. The \u22121p\/\u221219q genotype and low-grade pathology were associated with longer PFS and OS. Histopathology subtype was not significantly associated with outcome. Increased rCBV was only associated with short PFS and OS when adjusted for the \u22121p\/\u221219q genotype. Tumours with intact 1p\/19q and high rCBV (two grade III primary oligoastrocytoma, one grade II recurrent oligodendroglioma, one grade III recurrent oligoastrocytoma) were associated with shorter PFS and OS than tumours with intact 1p\/19q and low rCBV or those with lp\/19q loss and high rCBV (Fig.\u00a05a,b). Similar findings were observed if primary tumour only were analysed (Fig.\u00a05c,d). Compared with significant clinicopathological parameters, high rCBV was an independent prognostic factor for PFS when adjusted for 1p\/19q status (Cox regression: PFS: hazard ratio 13.4, 95% CI 1.3\u2013133; P=0.008) and for PFS and OS in patients with intact 1p\/19q (Cox regression: PFS: hazard ratio 16.5, 95% CI 1.8\u2013152.2; P=0.004; OS: hazard ratio 7.4, 95% CI 1.6\u201333.3; P=0.013). \nTable\u00a02rCBV and survival in the series and in those with primary tumours. PFS and OS were calculated from start date of PCV (n number of cases, nc not calculated, nr median survival not reached). Contrast enhancement (present vs absent), age (<50 vs \u226550\u00a0years), ECOG performance status (0\u20131 vs \u22652) and gender (male vs female) were not significantly related to PFS or OS\u00a0nProgression-free survival (months)Overall survival (months)Median (95%CI)P valueaP valuebMedian (95%CI)P valueaP valuebAll tumoursLoss of 1p36 and 19q13Yes16>56 (nr)0.005>56 (nr)0.020No178 (0\u201321)>49 (nr)Histopathology subtypeOligodendroglioma11>56 (nr)0.0550.187>56 (nr)0.1130.288Oligoastrocytoma218 (9\u201341)50 (9\u201391)Histopathology gradeII19>56 (nr)0.0040.002>56 (nr)0.0070.012III144 (8\u201326)21 (8\u201334)rCBVLow14>46 (nr)0.7660.003>49 (nr)0.5850.002High1946 (nc)>56 (nr)Primary tumoursLoss of 1p36 and 19q13Yes14>56 (nr)0.037>56 (nr)0.056No12>39 (nr)49Histopathology subtypeOligodendroglioma10>56 (nr)0.0550.137All censored0.0920.209Oligoastrocytoma1646 (6\u201386)50 (6\u2013101)Histopathology gradeII17>56 (nr)0.0470.009>56 (nr)0.0480.041III96 (13\u201337)50 (nc)rCBVLow12>46 (nr)0.6810.007>49 (nr)0.6110.003High14>56 (nr)>56 (nr)aProbability calculated by the Log-Rank test.bProbability calculated by the Log-Rank test after adjustment for 1p\/19q genotype.Fig.\u00a05rCBV and outcome following PCV chemotherapy: a, b all patients; c, d patients with primary tumour only; a, c progression-free survival; b, d overall survival.\u2460 Patients with tumours with low rCBV (<1.59) and loss of 1p36 and 19q13 (all n=2, primary n=2).\u2461 Patients with tumours with high rCBV (>1.59) and loss of 1p36 and 19q13 (all n=15, primary n=12).\u2462 Patients with tumours with low rCBV (<1.59) with intact 1p36 and 19q13 (all n=12, primary n=10).\u2463 Patients with tumours with high rCBV (>1.59) and intact 1p36 and 19q13 (all n=4, primary n=2)\nDiscussion\nTo our knowledge this is the first study investigating the relationship between rCBV, genotype and outcome in oligodendroglial tumours treated with PCV chemotherapy. The cohort was drawn from a larger series in an ongoing prospective study of oligodendroglial neoplasms [15\u201318] and reflects the range of patients given PCV chemotherapy in routine clinical practice. Tumours with rCBV >1.59 were more likely to have 1p\/19q loss. PCV chemosensitivity was predicted by 1p\/19q loss, but high rCBV was not necessary for response. Following chemotherapy, rCBV was significantly associated with PFS and OS only after adjustment for genotype.\nDSC-MRI can be used to determine cerebral perfusion parameters such as rCBV, relative cerebral blood flow and permeability, but with the instrumentation available to us was restricted to rCBV. As in other studies [8, 20, 25, 26], placement of ROI in regions within tumour of greatest signal intensity yielded data with good inter- and intraobserver variability. The greatest variation was seen in tumours with high rCBV, reflecting heterogeneity of blood volume within these tumours. In this series, tumours with the \u22121p\/\u221219q genotype were more likely to have high rCBV than those with intact 1p\/19q. The ROC analysis determined an optimal cut-off (rCBV >1.59) for predicting the \u22121p\/\u221219q genotype with 92% sensitivity and 76% specificity. These data suggest that in conjunction with histopathology, rCBV may be used to distinguish tumours of the oligodendroglial lineage.\nMRI studies of tumour vasculature in gliomas have demonstrated that rCBV may be useful in the non-invasive determination of tumour grade and vascularity [21, 25, 27, 28]. Compared to their low-grade counterparts, high-grade tumours have elevated rCBV, associated with increased mitotic activity [29]. rCBV correlates with conventional angiographic assessment of tumour vascular density and histological measures of angiogenesis such as microvascular density and VEGF expression [26, 30\u201332], which reflects the histopathological finding of increasing neovascularization with grade [19]. However, most studies have investigated astrocytic gliomas or included a variety of glioma pathologies and large series of oligodendroglial neoplasms are uncommon. Recent reports suggest that rCBV values in oligodendrogliomas render DSC-MRI-based tumour grading inaccurate [28, 33], and rCBV may be similar in low- and high-grade oligodendrogliomas [33]. Indeed low-grade oligodendrogliomas have significantly elevated rCBV compared to low-grade astrocytomas [8]. In our series, rCBV did not distinguish oligodendrogliomas from oligoastrocytomas, nor was rCBV significantly different between grade II and III tumours. This lack of correlation between rCBV and grade is consistent with the increased microvessel density seen in both low- and high-grade oligodendroglial tumours [34, 35].\nIt has been postulated that the tumour vasculature in oligodendrogliomas contributes to their chemosensitivity [12\u201314]. In an anaplastic oligodendroglioma treated with PCV chemotherapy, post-therapy volume changes and normalization of rCBV were more evident in regions of the pretherapy tumour that showed high rCBV [13]. In the present study, tumours with and without the \u22121p\/\u221219q genotype, and high or low rCBV showed response to chemotherapy when assessed using conventional criteria [1, 23]. These data indicate that rCBV measured through DSC-MRI is not a key factor in determining PCV chemosensitivity. These findings may reflect the limitations of DSC-MRI associated with contrast recirculation and disruption of the blood\u2013brain barrier in tumours [8, 9]. Alternatively, chemosensitivity may be influenced more by blood flow than by volume. Further studies to measure additional parameters associated with vascular perfusion, including blood flow and permeability are essential to establish whether these factors contribute to chemosensitivity.\nThe \u22121p\/\u221219q genotype is known to be associated with longer PFS and OS [5] but the impact of tumour vasculature on prognosis in oligodendrogliomas is confined to a few histopathological [34, 36, 37] and imaging studies [12]. Although our cohort is small, we have presented preliminary data that the prognostic significance of rCBV may differ in tumours with or without the \u22121p\/\u221219q genotype. 1p\/19q loss and low-grade pathology were associated with favourable PFS and OS following PCV. In other studies of astrocytic gliomas [28] or mixed low-grade gliomas [11], high rCBV predicted poor prognosis. In our series, rCBV alone was an unreliable indicator of outcome, showing prognostic significance only after stratification for genotype, suggesting different effects for the two genetic subgroups at the cut-off used to classify tumours with high rCBV. With this cut-off, tumours with high rCBV and 1p\/19q loss were associated with longer survival than those with high rCBV and intact 1p\/19q. Unlike astrocytic gliomas, high rCBV values do not necessarily indicate aggressive biology associated with poor outcome in oligodendroglial neoplasms with 1p\/19q loss, suggestive of differences in the baseline biology of these two genetic subtypes. These preliminary data therefore indicate that further investigation of the prognostic significance of rCBV in molecular subtypes of glioma in a much larger series is warranted.\nConclusion\nIn conclusion, we have shown that high rCBV is more likely in oligodendroglial tumours with the \u22121p\/\u221219q genotype and may be a useful aid to diagnosis; however, rCBV does not predict chemosensitivity. Lack of correlation with histopathology grade and the data suggesting that the prognostic significance of rCBV may differ in patients with or without the \u22121p\/\u221219q genotype, indicates that different interpretation of rCBV data may be necessary for oligodendroglial compared with astrocytic gliomas. Further study is essential to fully evaluate the role of DSC-MRI in the non-invasive diagnosis and prediction of prognosis in gliomas with an oligodendroglial phenotype.","keyphrases":["chemosensitivity","oligodendroglial tumour","rcbv","outcome"],"prmu":["P","P","P","P"]}
{"id":"Pharm_World_Sci-3-1-1805038","title":"Determinants of potential drug\u2013drug interaction associated dispensing in community pharmacies in the Netherlands\n","text":"Objective: There are many drug\u2013drug interactions (D\u2013DI) of which some may cause severe adverse patient outcomes. Dispensing interacting drug combinations should be avoided when the risks are higher than the benefits. The objective of this study was to identify determinants of dispensing undesirable interacting drug combinations by community pharmacies in the Netherlands.\nStatements on the impact of the article on practice\nProcess and structure characteristics may influence the dispensing of undesirable interacting drug combinations in community pharmacies but probably to a minor degree.\nMedication surveillance in Dutch pharmacies seems to be effective.\nIntroduction\nDrug\u2013drug interactions (D\u2013DIs) are responsible for many adverse patient outcomes. Different studies suggest that D\u2013DIs may cause up to 3% of all hospital admissions [1\u20134]. A D\u2013DI is defined as a pharmacokinetic or pharmacodynamic influence of drugs on each other, which may result in desired effects, in reduced efficacy and effectiveness or in increased toxicity [5]. Although many D\u2013DIs exist, only a small part of these D\u2013DIs is clinically relevant [6\u20138]. The potential benefits of drug combinations should be weighed against the seriousness of the D\u2013DI, taking into account the availability of alternatives. Only in cases that the risks associated with the D\u2013DI are higher than the benefits or if a better alternative is available, the D\u2013DI should be avoided.\nIn the Netherlands, one of the tasks of the pharmacist is to intervene in case of D\u2013DIs, which involve a high risk for the patient. Hereto, the pharmacist uses patient characteristics and the medication history. All prescriptions, which are submitted to the pharmacy, are screened on potential interactions with the help of medication surveillance software. These D\u2013DIs are evaluated by the pharmacist who intervenes if necessary. This task is important but cumbersome, and requires great attention from the pharmacist. The organisational aspects, such as the tuning of the medication surveillance software and instructions of technicians, should be managed by the pharmacist in such a way that in case of D\u2013DIs with a high risk the pharmacist intervenes. This is important for the prevention of adverse patient outcomes [9].\nThe objective of this study was to assess process and structure characteristics associated with the dispensing of interacting drug combinations, which carry a high risk of adverse patient outcomes.\nMethods\nSetting\nThe data for this study were retrieved from the Drug Information Project, a division of the Health Care Insurance Board. This is a database containing the reimbursement data from eight health care insurance companies in the Netherlands. The reimbursement data from January 1st, 2001 until October 31st, 2002 were analysed. Eleven potential D\u2013DIs, that contained a high risk and could be substituted, because a good alternative was available, were selected and counted for each pharmacy in the database. These undesirable potential D\u2013DIs were mostly interactions between chronically used drugs which cannot be interrupted and short-term use of antibiotics or antimycotics, and were selected from the Dutch guidelines for the management of D\u2013DIs (Table\u00a01) [10, 11]. A D\u2013DI was counted as such, when the chronically used drug was dispensed both in the period 150\u00a0days preceding and in the period 150\u00a0days after the dispensing of antibiotics or antimycotics for short-term use in the same pharmacy. Pharmacies with less than 5,000 dispensings in the database were excluded.\nTable\u00a01Number of dispensings in the database of the individual drugs involved, the eleven potential D\u2013DIs and the calculated ratioDrug\u2013drug interactionNumber of dispensings drug A\u00a0\u00d7\u00a01,000 (range)Number of dispensings drug B\u00a0\u00d7\u00a0 1,000 (range)Number of D\u2013DIs counted (range)Average ratio (range)aDrug ADrug B1Erythromycin, clarithromycin, azithromycin, roxithromycinDigoxin440.8(0\u20132754)487.0(0\u20133064)3,993(0\u201341)1.39(0\u201318.52)2ItraconazoleDigoxin88.7(0\u2013349)487.0(0\u20133064)245(0\u20137)0.45(0\u201321.69)3CiprofloxacinTheophylline105.4(0\u2013769)100.9(0\u2013756)944(0\u201314)6.39(0\u2013534.38)4Miconazole oral gelAcenocoumarol, fenprocoumon44.6(0\u2013233)608.2(5\u20133156)154(0\u20133)0.38(0\u201321.30)5ErythromycinCarbamazepine49.7(0\u2013531)193.6(0\u2013871)35(0\u20134)0.24(0\u201340.92)6Erythromycin, clarithromycin, azithromycinDisopyramide426.6(0\u20132754)9.4(0\u2013151)61(0\u20134)\u20137Erythromycin, clarithromycinPimozide274.4(0\u20132004)57.4(0\u2013394)70(0\u201315)0.46(0\u201346.12)8Propranolol, oxprenolol, pindololbeta2-mimetics, inhalation corticosteroids250.6(1\u20131075)2,546.9(27\u201310504)5,127(0\u201394)0.54(0\u201312.98)9Erythromycin, clarithromycinCisapride274.4(0\u20132004)127.5(0\u2013821)586(0\u201311)1.16(0\u201340.45)10Itraconazole, fluconazole, ketoconazoleCisapride199.9(0\u2013727)127.5(0\u2013821)347(0\u201312)0.95(0\u201357.10)11Acenocoumarol, fenprocoumonAzapropazon608.2(5\u20133156)8.4(0\u2013164)32(0\u201319)\u2013aCalculated with Formula 1\nProcedure\nFor each pharmacy, we calculated the dispensing-ratios for the eleven potential D\u2013DIs with Formula 1. This formula was used because the risk of dispensing a D\u2013DI between drug A and drug B is dependent on the number of times each drug is dispensed. The more drug A or drug B are dispensed, the higher the risk that these drugs are combined on the basis of chance alone. In case the dispensing of drug A is independent from the dispensing of drug B and the D\u2013DI is never intervened, the ratio will on average be one. The number of times this ratio was above one was calculated, because a ratio above one might indicate that medication surveillance fails. In this calculation, there were 342 D\u2013DIs between norfloxacin and theophylline, which were excluded in the analysis because the guidelines concerning the management of this D\u2013DI were inconsistent. Two groups of pharmacies were selected, and the pharmacists were asked in August 2003 by the Inspectorate for Health Care (IHC) to fill in a questionnaire. The first group included pharmacies with a high risk of dispensing these D\u2013DIs, while the second group consisted of a random sample from the remaining pharmacies. These groups were equal in size. The selection criteria are described in Fig.\u00a01. The selection criterion for receiving a questionnaire (\u22654 times a ratio >1) was chosen on pragmatic reasons to have enough power for statistical analysis with manageable numbers. A concept questionnaire was composed on basis of a literature search and interviews with experts [9]. The questions concerned process and structure characteristics of several quality aspects and those questions were selected that could discriminate between high and low quality pharmacies. Mostly questions with objective answers were included, for example about written instructions for technicians, filing of data, tuning of the software (which signals were shown and which not) and personnel. The concept questionnaire was tested in three pharmacies and some questions were amended on the basis of their comments. The final questionnaire contained 183 questions, divided into 12 subjects (Table\u00a02). The questionnaire was accessible via the Internet. Pharmacies who had no access to the Internet received the questionnaire by post. Pharmacies who failed to fill in the questionnaire received reminders at regular intervals. A sample from both groups was visited by the IHC (Fig.\u00a01). Also here, the selection criterion (\u22655 times a ratio >1) was chosen to have enough power with manageable numbers. Thirty-seven questions from the questionnaire were selected and during the visits these questions were verified. The pharmacies were informed in advance that a selection of the pharmacies would be visited. The selected pharmacies were acquainted after completing the questionnaire. The visiting inspectors were blinded to the number of interacting drug combinations.\nFig.\u00a01The selection of the pharmacies receiving a questionnaire and IHC visitTable\u00a02The subjects and number of questions in the questionnaireChapterSubject (number of questions)General pharmacy dataOwnership of the pharmacy (1), cooperation with other pharmacies (1), cooperation with general practitioners (1), electronic submission of prescriptions (4)FacilitiesAlterations (2)Quality policySetting up and implementing a quality system (4), certification (2), attitude towards quality management (12)Quality measurementMeasurement of errors (2), complaints (1), patient satisfaction (2), interventions (3), and participation in mystery guest investigations (2)Receipt procedureNumber of personnel involved in dispensing a receipt (2), checks in dispensing a receipt (3)Medication surveillance\u2014tuning softwareMedication surveillance system used (1), tuning of the system e.g. which signals are showed and which are regarded as irrelevant (55a), surveillance of pharmacy preparations (2)Medication surveillance\u2014organisationThe way technicians are instructed to manage medication surveillance signals (5), the way this is supervised (2), number of interventions (1), use of resources (2), participation in courses (4), management of the D\u2013DI between carbamazepine and erythromycin (5) and between Sulfamethoxazole\/trimethoprim and Acenocoumarol (7)Medication surveillance\u2014recording managementThe way the management of signals is recorded (4)Pharmacy preparationsThe way instructions for pharmacy preparations are recorded (1), the way pharmacy preparations are supervised (3), the number of pharmacy preparations (2), the policy regarding analysing pharmacy preparations (3)Personnel and workloadSubjective workload (3), absence through illness (1), number of receipts dispensed per technician (2), personnel and experience of personnel (18)Patient careInformation given to patients (6), information exchange with hospitals (4), participation in health care projects (4)Farmacotherapeutic consultation groupsParticipation in farmacotherapeutic consultation groups (3), agreements made (3)aAs the questions for the four systems (Pharmacom, Aposys, Euroned, others) differed, pharmacists had to fill in only a quarter of these questions\nFormula 1:\nFormula 2:\nwhereki,ab: number of dispensings of interacting drug combination AB in pharmacy i;ki,a: number of dispensings of drug A in pharmacy i;ki,b: number of dispensings of drug B in pharmacy i;Ni: total number of dispensed drugs known in the database in pharmacy i;Ntot: total number of dispensed drugs known in the database in all pharmacies\nStatistical analysis\nFor each pharmacy, dispensing-ratios for the D\u2013DIs, comparable to the standardised mortality ratio, were calculated using Formula 2. With this formula, we standardise for the total number of dispensings per pharmacy in the database. In case all pharmacies dispense the D\u2013DIs in equal numbers, the ratio will be one for all pharmacies, and therefore the ratios have a better comparability. Pharmacies which have only a small number of dispensings in the database, will have extremely high numbers in case they dispense one or a small number of D\u2013DIs. Therefore, the results were equalized with Bayesian statistics to prevent extreme ups and downs due to low numbers of dispensings [12]. The pharmacies were divided into two sets. One set was used for the analyses and contained two-third of the pharmacies, the other set was used for the validation of the results obtained in the analyses. In the univariate analysis, correlations were searched between the answers in the questionnaire and these ratios. Correlations are only given if in both sets a correlation was found (p\u00a0<\u00a00.01). In the multivariate analysis, models were composed using the analysis set, predicting the dispensing of the interacting drug combinations. The models were validated using the validation set. The number of questions was too large for the multivariate analysis, and only a limited number of questions were selected. From every chapter, those questions were selected that correlated with the other questions and that could discriminate between pharmacies.\nResults\nThe database contained a total of 100,295,311 dispensings in the selected study period. One thousand one hundred and forty-two pharmacies were recorded in the database with 5,000 or more dispensings. The number of dispensings per pharmacy varied from 5,019 to 264,631. Because pharmacies receive reimbursements from several health care insurance companies and because not all health care insurance companies were included in the database, these numbers do not correspond with the total number of dispensings per pharmacy. The eleven potential D\u2013DIs were dispensed 11,594 times. In 5%, more than one pharmacy was involved. As these D\u2013DIs could not be assigned to a single pharmacy, they were excluded from further analyses. The number of dispensings and D\u2013DIs are shown in Table\u00a01. Disopyramide (D\u2013DI number 6) and azapropazon (D\u2013DI number 11) were not dispensed by 44% and 46% of the pharmacies, respectively. Therefore, a ratio could not be calculated for these pharmacies and these D\u2013DIs were excluded from the analyses.\nThe number of times a ratio above one was found was calculated (Table\u00a01) and pharmacies were selected as shown in Fig.\u00a01. Two hundred and sixty-eight pharmacies were selected to receive a questionnaire and 74 pharmacies were selected for a visit by the IHC. For several reasons, such as recent visitations and duplications in the database, 12 pharmacies were excluded. Eventually, 256 pharmacies received a questionnaire and 62 pharmacies were selected for a visit. Two hundred and forty-six questionnaires were filled in (response rate 96.1%) and 58 (93.5%) pharmacies were visited after the questionnaire was completed. The judgements during the visits by the IHC were compared with the answers by the pharmacists. In 33 of the 37 verified questions, the IHCs judgement matched in more than 90% the answer of the pharmacist. Except four questions, the judgement by the IHC was equally more positive and more negative than the answers by the pharmacist.\nIn the univariate analysis, all combinations between the questions and D\u2013DIs were searched for significant correlations. Two correlations were found with D\u2013DI number 1 between macrolide antibiotics and digoxin (Table\u00a03). Pharmacies, which are part of a health care centre dispensed this interacting drug combination more often than other pharmacies. A correlation with the type of medication surveillance system was also found. Pharmacies using the Euroned system dispensed this interacting drug combination more often, while pharmacies using the Pharmacom system dispensed this interacting drug combination less often.\nTable\u00a03Significant univariate correlations between the questionnaire and the number of dispensings of the D\u2013DIs between macrolide antibiotics and digoxin (number 1)QuestionCorrelationSignificanceIs the pharmacy part of a health care centre? (1 yes, 2 no) (yes n\u00a0=\u00a018, no n\u00a0=\u00a0228)\u22120.1650.009Which medication surveillance system is used in the pharmacy?Pharmacom (1 yes, 0 other) (n\u00a0=\u00a081)\u22120.2610.000Aposys (1 yes, 0 other) (n\u00a0=\u00a062)0.0880.170Euroned (1 yes, 0 other) (n\u00a0=\u00a089)0.1970.002\nFor the multivariate analysis, 32 variables were selected, representative of the whole range of questions. These variables were used in the analysis-set to compose models. The adjusted explained variance ranged from 2.6% to 28.9% (Table\u00a04). The model explaining the D\u2013DI between macrolide antibiotics and digoxin had by far the highest adjusted explained variance. The models were validated in the validation-set, calculating the unexplained variance (Table\u00a04). The six variables in this model explaining the D\u2013DI between macrolide antibiotics and digoxin are shown in Table\u00a05.\nTable\u00a04Predictability of the models composed in the multivariate analysisD\u2013DIAdjusted explained variance (R2) (analysis set)Unexplained variancea (validation set)128.90.61212.8\u22120.22317.331.547.0\u22120.18514.4\u22120.4176.56.4816.10.68914.0\u22120.43102.60.90aAn unexplained variance of zero means that the predictability found in the validation set equals the predictability in the analysis-set. The higher the unexplained variance, the worse the predictability in the validation-set compared to the analysis-setTable\u00a05The questions in the multivariate model predicting the dispensing of the D\u2013DI between macrolide antibiotics and digoxin (number 1)Variable:Answer (coding)Direction coefficientConstant3.3679Is the pharmacy part of a health care centre? (yes n\u00a0=\u00a018, no n\u00a0=\u00a0228)Yes (0) versus no (1)\u22122.2749Co-trimoxazole\u2014acenocoumarol: no appointments were made with the GPs. The drug will be dispensed.Option 1 \u2018with all GPs\u2019 (1) versus other option (0) (n\u00a0=\u00a011)ReferenceEight options of choice option 1 \u2018with all GPs\u2019 and optionOption 2 (1) versus other option (0) (n\u00a0=\u00a010)1.0308Eight \u2018with no GPs\u2019Option 3 (1) versus other option (0) (n\u00a0=\u00a04)0.3788Option 4 (1) versus other option (0) (n\u00a0=\u00a04)\u22120.4542Option 5 (1) versus other option (0) (n\u00a0=\u00a03)0.9026Option 6 (1) versus other option (0) (n\u00a0=\u00a02)\u22120.5100Option 7 (1) versus other option (0) (n\u00a0=\u00a04)\u22120.1912Option 8 \u2018with no GPs\u2019 (1) versus other option (1) (n\u00a0=\u00a0202)0.0886Are separate signal texts in the medication surveillance program adjusted to the situation in the pharmacy? (yes n\u00a0=\u00a072, no n\u00a0=\u00a0165)Yes (0) versus no (1)0.1793Is the management of signals traceably recorded on the receipt? (yes n\u00a0=\u00a0211, no n\u00a0=\u00a035)Yes (0) on the receipt, no not on the receipt (1)0.2691The supervision on management of signals takes place on the basis of signal lists (yes n\u00a0=\u00a0158, no n\u00a0=\u00a086)Yes (0) on the basis of signal lists, no (1) not on the basis of signal lists0.0723How many receipts are dispensed per year divided by the number of fte technicians<10\u20134\nDiscussion\nIn this study, we investigated determinants for the dispensing of 11 undesirable interacting drug combinations. In general, our results are in line with the expectation that the medication surveillance system plays an important role in medication surveillance. Although the 11 potential D\u2013DIs were counted 11,594 times which suggests that a considerable number of patients is exposed to potential and avoidable adverse patient outcomes, these results should be judged against a background of approximately 100 million dispensings. It is possible that in these cases due to particular circumstances any other option, such as substituting or not dispensing one of the drugs, is a less favourable choice than dispensing the D\u2013DI. In 5% of the total number of D\u2013DIs more than one pharmacy was involved, indicating the importance of communication. For the D\u2013DI between macrolide antibiotics and digoxin, two determinants were found. Although the type of medication surveillance system was a determinant, this does not mean that the differences are determined by the quality of the system itself because they may also correlate with the attitude of the pharmacists using the systems. The three medications surveillance systems differ in the extent to which communication with other healthcare providers is possible and developments were made in recent years. The Pharmacom system has the most advanced communication possibilities and compared to the other systems, new developments to the Euroned system were modest. Unexpectedly, pharmacies part of a health care centre dispensed this D\u2013DI more often than other pharmacies. In health care centres, the communication lines between pharmacists and general practitioners are much shorter, suggesting that intervening undesirable D\u2013DIs will be easier. Possibly, pharmacies which are part of a health care centre oppose the opinions from the general practitioners less often, to avoid harming the cooperation within the health care centre but, of course, there may be several other reasons.\nFor the other eight assessed D\u2013DIs no determinants were found in the univariate analysis, neither did the models in the multivariate analysis have a good predictability. A possible explanation is that the quality of medication surveillance in community pharmacies in the Netherlands is high. Therefore, the number of pharmacies dispensing high-risk D\u2013DIs seems to be small.\nOur study has some potential limitations. First, because we used strict inclusion criteria to prevent false-positive results, it is likely that the number of dispensings of undesirable interacting drug combinations in this study is an underestimation and it is possible that important determinants were not recognized or difficult to assess. In the univariate analyses, only those questions are given which had a significant (p\u00a0<\u00a00.01) correlation in two independent sets. Although we included 183 questions and nine D\u2013DIs in the univariate analysis, the possibility of including a significant correlation by chance was small (on average 0.16 question). Second, the reimbursement data from eight health care insurance companies were used. In the Netherlands, these companies work mostly regionally. It is nevertheless not to be expected that the determinants of dispensing interacting drugs differ per region or that pharmacies differ in their management of D\u2013DIs between patients of different health care insurance companies. Third, from all potential D\u2013DIs, only 11 (but highly clinically relevant ones) were selected for this study. According to the Dutch guidelines, for all 11 combinations the dispensing of an alternative was strongly advised as a good alternative was available. Nevertheless, it is possible that these dispensings were not an error because any other option was not possible. For example, when a patient is hypersensitive to the alternative drug recommended in the guidelines or when the alternative drug is not effective. In these cases, the benefit of both drug therapies should be weighed against the potential risks of the D\u2013DI. The potential risks can partly be avoided by taking appropriate measures such as monitoring of drug levels. In this study, we could not retrieve why the pharmacist had dispensed the interacting drug combination, and whether the dispensing was erroneous or not.\nFourth, the questionnaire was composed on the basis of a literature search and interviews with experts. It is possible that not all characteristics correlating with the dispensing of undesirable interacting drug combinations were disclosed, such as differences in population characteristics between pharmacies. For example, pharmacies with an elderly population using more drugs simultaneously have a higher risk of dispensing interacting drug combinations than pharmacies with a younger population. Also, it is possible that in areas with many general practitioners who use a medication surveillance system for prescribing, the background chance of a D\u2013DI is much smaller. Fifth, it is possible that the differences between pharmacies were too small compared with the power of this study to distinguish determinants.\nAll associations found in this study were directly related to the management of signals. In our questionnaire, we also included other topics, such as pharmacy preparations and patient care. Future research should focus on the management of a larger variety of signals than the ones in our study and on how D\u2013DI associated dispensing could be further reduced.\nConclusion\nIn conclusion, both medication surveillance systems and being part of a health care centre may play an important role in the management of D\u2013DIs and the avoidance of adverse patient outcomes. Pharmacies in a healthcare centre dispensed D\u2013DIs more often. For most D\u2013DIs, no determinants were found possibly indicating that the quality of medication surveillance in the Netherlands is high.","keyphrases":["drug\u2013drug interaction","pharmacy","netherlands","drug interaction","practice management"],"prmu":["P","P","P","R","R"]}
{"id":"Antonie_Van_Leeuwenhoek-4-1-2268727","title":"Control and possible applications of a novel carrot-spoilage basidiomycete, Fibulorhizoctonia psychrophila\n","text":"A novel cold-tolerant fungus, Fibulorhizoctonia psychrophila, was isolated from a refrigerated carrot storage facility and identified as an anamorph of Athelia, often classified in Rhizoctonia s.l. Growth of this fungus was observed between 0 and 20\u00b0C with an optimum at 9\u201312\u00b0C, while incubation of mycelium grown at 15\u201332\u00b0C resulted in absence of growth even after the fungus was transferred back to 15\u00b0C. Growth was inhibited in the presence of the antifungals sorbic acid or natamycin, in particular when the fungus was incubated at 18\u00b0C. F. psychrophila produces polysaccharide degrading enzymes that, when compared to enzymes from the ascomycete fungus Aspergillus niger, retain a larger proportion of their activity at lower temperatures. This indicates that F. psychrophila could be used as a source for novel industrial enzymes that are active at 4\u201315\u00b0C.\nIntroduction\nFungi occupy every natural environment on earth as well as man-made indoor environments. In fact, some fungi have ecological requirements and amplitudes that are ideally suited to some artificial (human made) environments. Some of these fungi create large problems with respect to human health (e.g. Aspergillus, Candida) or to food quality (e.g. Penicillium). Among standard methodologies to prevent food spoilage are the use of preservatives and refrigeration of food. The latter method, however, is not effective when cold-tolerant spoilage fungi are present. Cold-tolerant fungi are commonly found in nature and comprise species of different branches of the fungal kingdom, such as Geomyces, Leucosporidium (Panikov and Sivoza 2007), Cystofilobasidium and Mrakia (Nagakawa et\u00a0al. 2004).\nCell walls form the majority of the plant biomass and consist mainly of polysaccharides. As fungi are not able to import polymeric compounds into their cells, they rely on extracellular enzyme systems for the degradation of polysaccharides into monosaccharides and short oligosaccharides. Hydrolytic enzymes acting on polysaccharides are commonly produced by fungi. These enzymes have many applications in the food and feed, paper and pulp, textile, and pharmaceutical industries and have therefore been the subject of many studies (de Vries and Visser 2001; de Vries 2003). Most of these studies and applications involve enzymes of saprophytic ascomycete fungi (mainly species from Aspergillus and Trichoderma) that have optimal growth temperatures between 30 and 37\u00b0C. Consequently, most of these enzymes have optimum activities between 30 and 50\u00b0C and their activity rapidly decrease at lower temperatures (de Vries and Visser 2001). Efficient use of these enzymes in industrial applications is therefore often only possible by incubation above 30\u00b0C. This not only increases production costs due to the energy required to reach this temperature, but can also cause spoilage problems, especially with respect to food and feed applications.\nOne of the most commonly known cold-tolerant spoilage fungi is the basidiomycete Rhizoctonia\u00a0carotae (Adams and Kropp 1996; Jensen 1969; Jones and Aldwinckle 1990; Punja 1987), which is found in storage facilities of several vegetables such as carrot. Although the optimum growth-temperature of this fungus is between 15 and 20\u00b0C (Punja 1987), this fungus causes crater rot characterized by sunken lesions and abundant mycelial growth on carrots stored at temperatures between 1 and 4\u00b0C. The anamorph genus Fibulorhizoctonia (Adams and Kropp 1996) (as Fibularhizoctonia) was proposed to accommodate Rhizoctonia\u00a0carotae and R.\u00a0centrifuga L\u00e9v (Rader 1948). The teleomorphs of these species belong to Athelia Pers., while those of the Rhizoctonia\u00a0solani-complex belong to Thanatephorus Donk, currently placed in a different order (Hibbett et\u00a0al. 2007). The teleomorph of F.\u00a0carotae was identified as Athelia\u00a0arachnoidea (Berk.) J\u00fclich (Adams and Kropp 1996). However, there is some controversion considering the scope of this species, as the name has been used for litter decomposing specimens and lichen parasites. Arvidsson (1976) concluded that the use of the name A.\u00a0arachnoidea should be confined to the lichen parasite, while the material of Adams and Kropp (1996) contained only litter decomposers.\nOther basidiomycete species reported from cold storage are: Corticium\u00a0centrifugum (Bielenin 1986; Stalpers and Loerakker 1984; Weresub and Illman 1980), from stored apples and pears, causing fisheye rot. Although the name C.\u00a0centrifugum is connected with Athelia, the species concerned differs by having constant clamps at the septa, narrower hyphae and no sclerotia, and the production of a basidiome in culture, which is now known as Butlerelfia\u00a0eustacei (Weresub and Illman 1980). This species is known from Europe and North America.\nIn this paper we describe a new carrot-spoilage fungus, Fibulorhizoctonia\u00a0psychrophila, which is a predominant species detected in refrigerated storage facilities for carrots in The Netherlands. We have analysed the cold-tolerance of this fungus and its sensitivity to commonly used fungicides. In addition, we have analysed F.\u00a0psychrophila for production of plant polysaccharide degrading enzymes to assess its potential as a source of cold-active enzymes.\nMaterials and methods\nStrains and growth conditions\nF.\u00a0psychrophila CBS 109695 (IMI 395943) was isolated as a mycelial sample by H.A.B. W\u00f6sten from a wooden crate containing Daucus\u00a0carota in a refrigerated storage facility (4\u00b0C) in Bant, The Netherlands in 2002. Athelia\u00a0arachnoidea (Berkeley) J\u00fclich CBS 418.72 was isolated from fallen leaf-litter of Populus\u00a0sp. in The Netherlands. The type strain of Fibulorhizoctonia\u00a0carotae (Rader) G.C. Adams & Kropp CBS 464.48 and Aspergillus niger N402 were described previously (Adams and Kropp 1996; Bos et\u00a0al. 1988).\nF.\u00a0psychrophila CBS 109695 and A.\u00a0niger N402 were routinely propagated on malt extract agar and minimal medium (de Vries et\u00a0al. 2004), respectively. For growth of F.\u00a0psychrophila on carrot, potato and onion, these vegetables were ground using a coffee grinder. The vegetable pulp was used at a concentration of 10% in 1.5% agar plates in water. Liquid cultures of F.\u00a0psychrophila and A.\u00a0niger were performed in Schizophyllum\u00a0commune minimal medium (Dons et\u00a0al. 1979) and Aspergillus minimal medium (de Vries et\u00a0al. 2004), respectively, using 1% of a crude arabinoxylan preparation obtained from wheat after extraction of starch and proteins as the substrate. Plate cultures to determine the influence of several fungicides on growth of F.\u00a0psychrophila were performed on malt extract agar plates. Comparison of the optimal growth temperature for F.\u00a0psychrophila, Athelia\u00a0arachnoidea and F.\u00a0carotae was performed on malt extract agar and cherry decoction agar at temperatures of 0\u201327\u00b0C with intervals of 3\u00b0C.\nPowdered wood was obtained by grinding wood shavings of a storage crate in a coffee grinder until a fine powder was obtained. Of this powder, 1\u00a0g was added to 1.5\u00a0g agar and 100\u00a0ml water and autoclaved to prepare solid media. The crude arabinoxylan preparation was a gift from Latenstein (Nijmegen, The Netherlands) and is in fact a waste stream of a protein and starch extraction process from wheat.\nMolecular biology methods\nGenomic DNA of CBS 109695 was extracted using the FastDNA kit (Bio 101 Systems, Q-Biogene). The 5.8S gene and flanking ITS1 and ITS2 were amplified using the primers ITS1 and ITS4 (White et\u00a0al. 1990) and the sequence was deposited at genbank (Acc. Nr. EF492880). The sequence was compared with ITS sequences from F.\u00a0carotae (U85789) and A.\u00a0arachnoidea (U85791).\nEnzyme assays\nF.\u00a0psychrophila and Aspergillus\u00a0niger were grown at 15 and 30\u00b0C, respectively in liquid medium containing a crude arabinoxylan preparation. Culture filtrate was harvested over a nylon filter after 2\u00a0days of growth and analysed for enzyme activities at 4, 15 and 30\u00b0C. Enzyme activity was determined using p-nitrophenyl-\u03b2-d-galactopyranoside, p-nitrophenyl-\u03b2-d-glucopyranoside, p-nitrophenyl-\u03b1-d-galactopyranoside, and p-nitrophenyl-\u03b2-d-xylopyranoside (Sigma) as a substrate. A mixture was made consisting of 10\u00a0\u03bcl culture filtrate, 50\u00a0\u03bcl 50\u00a0mM sodium acetate (pH 5.0) and 30\u00a0\u03bcl sterile MiliQ water. The reactions were performed in triplicate and were started by the addition of 10\u00a0\u03bcl of 0.1% stock of the p-nitrophenyl-linked substrate and incubated for 2\u00a0h at 25\u00b0C, unless stated otherwise. The reactions were stopped by the addition of 100\u00a0\u03bcl 0.25\u00a0M Na2CO3 and measured at 405\u00a0nm in a micro plate reader (model 550, Bio-RAD). The amount of free p-nitrophenol was calculated using a calibration curve.\nResults and discussion\nTaxonomy\nFibulorhizoctonia\u00a0psychrophila Stalpers & de Vries spec. nov. (Fig.\u00a01).\nFig.\u00a01Colony morphology of Fibulorhizoctonia\u00a0carotae (464.84), Athelia\u00a0arachnoidea (418.72) and Fibulorhizoctonia\u00a0psychrophila (109659) after 2\u00a0weeks. (a) 0\u00b0C; (b) 6\u00b0C; (c) 18\u00b0C\nMycelium ad 9\u00b0C cotoneum, albidum, ad 18\u00b0C cremeum, avellaneum vel brunneum, velutinum vel crustosum. Liquor exsudatus brunneus. Sclerotia presentia vel absentia, irregularia, in statu maturitate subfusca a liquore exsiccata. Hyphae hyalinae vel subfuscae, (2.5\u2013)3\u20136\u00a0\u03bcm. Septa fibulatae vel afibulatae. Cellae sclerotiorum doliformes, tenui-tunicatae vel crasse-tunicatae, hyalinae vel fuscae. Fungus psychrophilus, ad temperaturas \u22123\u00b0 ad 21\u00b0C crescens.\nTypus: CBS 109695\nColonies at \u22123 to 9\u00b0C are cottony, rather high and reaching the lid of the Petri dish, white. Margin raised, rather dense. At increasing temperatures the mycelium grows less high and becomes nearly velvety at 18\u00b0C and nearly crustose at 21\u00b0C. At 12\u00b0 the centre of the colony is cream-coloured, at 15\u00b0C becoming Light Cinnamon Drab to Avellaneous (Munsell 7.5YR6\/2, 10YR6\/2, 10 YR6\/3). At these temperatures exudate droplets are not present. At 18\u00b0C the colony is low, nearly velvety, Ochraceous Tawny (7.5YR5\/8) caused by exudate drops and at 21\u00b0C there is hardly any growth; the mycelium is crustose and Cinnamon Brown to Prouts Brown (5YR4\/3, 5YR4\/4), and finally Mummy Brown. Sclerotia are generally not produced within 6\u00a0weeks, but have occurred in a tube; they start as white semiglobose pustules, generally up to 1\u00a0mm diameter, aggregated, often fusing, and producing a brown exudate, which dries in, leaving a dark brown sclerotium, up to 5\u00a0mm diam, finally Mummy Brown.\nMarginal hyphae at \u22123 to 3\u00b0C are irregular, rather long-celled, with swellings and granular contents, thin-walled, (2.5\u2013)3\u20136\u00a0\u03bcm wide, swellings up to 12\u00a0\u03bcm wide. Clamps present, not abundant. Branching at various angles, sometimes a constriction is present at the base of the side branch. At 6\u00b0C and above the hyphae are regular, thin- to firm-walled, 2.5\u20136(\u20137.5)\u00a0\u03bcm wide, contents hyaline to pale brownish, with abundant clamps, especially at the wider hyphae, but the narrower hyphae (2.5\u20133.4\u20134\u00a0\u03bcm) generally have clampless septa. Cell wall hyaline, but irregular brownish encrustations present at some hyphae, probably dried exudate.\nAerial mycelium regular, thin- to firm-walled, (2.5\u2013)3.2\u20136.5\u00a0\u03bcm wide, with hyaline to brownish contents. Most septa with clamps; clamps regular, but sometimes of the medallion type.\nSclerotia rarely produced, uniform, not forming a distinct cortex, consisting of thin- to thick-walled hyphae, usually consisting of slightly elongated swollen cells with granular contents, 4\u201310\u00a0\u03bcm wide, not unlike the (thin-walled) advancing hyphae at lower temperatures.Cardinal temperatures: minimum below \u22123\u00b0C, optimum range between 9 and 12\u00b0C, maximum 20\u00b0C.Holotype: CBS 109695 (IMI 395943), also preserved dried in herb. CBS.Mycobank number: MB501325.The sterile, sclerotium producing mycelia are traditionally classified in the artificial genus Rhizoctonia, which is currently restricted to basidiomycetous fungi and comprises anamorphs of various, not closely related genera such as Tulasnella, the Ceratobasidium\u2013Thanatephorus complex and Athelia (Stalpers and Anderson 1996). These groups have been elevated to the genus level as Epulorhiza, Rhizoctonia s.str. and Fibulorhizoctonia. Although teleomorphs have not been observed in F.\u00a0carotae and F.\u00a0psychrophila, morphological, physiological and molecular characters indicate that F.\u00a0psychrophila, F.\u00a0carotae and Athelia\u00a0arachnoidea are closely related and belong to Athelia, a corticioid genus, currently classified in the Atheliales (Rader 1948).\nF.\u00a0carotae has been described from cold stored carrots. It differs from F.\u00a0psychrophila in growing between 18 and 27\u00b0C (Fig.\u00a02), abundant production of sclerotia, and colour of the mat above 15\u00b0C. A.\u00a0arachnoidea has been described from both lichens and leaf litter. It differs from F.\u00a0psychrophila in its growth at higher temperatures (Fig.\u00a02), less abundant aerial mycelium, and pale colour at higher temperatures. ITS sequencing of F.\u00a0psychrophila revealed 95% identity to the ITS sequence of A.\u00a0arachnoidea and A.\u00a0carotae (data not shown), indicating that F.\u00a0psychrophila is very closely related to these two species.\nFig.\u00a02Growth comparison at different temperatures of Fibulorhizoctonia\u00a0psychrophila, Fibulorhizoctonia\u00a0carotae and Athelia\u00a0arachnoidea. The means of duplicate values are represented\nBoth morphological and molecular characters indicate without doubt, that F.\u00a0psychrophila, F.\u00a0carotae and Athelia\u00a0arachnoidea are closely related.\nTemperature-tolerance and growth of F.\u00a0psychrophila\nF.\u00a0psychrophila was inoculated on malt extract agar plates and incubated between 0 and 27\u00b0C. Growth was monitored by measuring the colony diameter. Optimal growth occurred at 9\u201312\u00b0C (Fig.\u00a02) and growth was absent above 20\u00b0C. When plates incubated for 3\u00a0days at 20 and 25\u00b0C were placed back at 15\u00b0C the fungus re-initiated growth, but incubation for 3\u00a0days at 32\u00b0C prevented growth of F.\u00a0psychrophila after the plates were transferred to 15\u00b0C (data not shown). Optimal growth of F.\u00a0carotae and A.\u00a0arachnoidea was at between 18 and 21\u00b0C (Fig.\u00a02). The strong decrease in growth of F.\u00a0psychrophila when the incubation temperature is raised above 15\u00b0C, suggests an even stronger adaptation of this fungus to cold biotopes.\nGrowth of F.\u00a0psychrophila was tested on three vegetable crops that are commonly stored under refrigerated conditions (carrot, potato and onion) in comparison to agar plates without carbon source. Water agar alone already permits growth of F.\u00a0psychrophila, but growth is significantly improved in the presence of carrot (data not shown). The presence of potato only resulted in a small increase in growth compared to water agar, but onion reduced growth of F.\u00a0psychrophila. These data demonstrate a preference of F.\u00a0psychrophila for carrot as a substrate. The reduced growth on onion is likely caused by the high levels of phenolic compounds in onions.\nInhibition of growth of F.\u00a0psychrophila using antifungals\nSorbic acid and natamycin are compounds commonly used for the inhibition of growth of spoilage fungi and the effectiveness of these compounds was tested on F.\u00a0psychrophila on malt- and water agar plates with powdered wood. F.\u00a0psychrophila is believed to survive on the crates used for the storage of carrots and therefore causes repeated spoilage when these crates are re-used. The presence of 250\u00a0mM sorbic acid resulted in a significant reduction in growth on malt extract agar, but not on powdered wood at 4\u00b0C (Fig.\u00a03a). However, higher levels of sorbic acid (625 and 1,250\u00a0mM) prevented growth of F.\u00a0psychrophila on both substrates. In light of the high concentrations of sorbic acid required to inhibit growth, the expected effectiveness of this compound in preventing spoilage by F.\u00a0psychrophila is limited.\nFig.\u00a03Influence of antifungals on the growth of Fibulorhizoctonia\u00a0psychrophila grown on malt extract agar (MA) or powdered wood from storages crates (HW). (a) sorbic acid, 18\u00b0C; (b) natamycin, 4\u00b0C; (c) natamycin, 18\u00b0C\nNatamycin was more effective in preventing growth of F.\u00a0psychrophila, resulting in a significant decrease in growth at 2\u00a0g\/l at 4\u00b0C. Increasing levels of natamycin reduced growth of the fungus at 4\u00b0C similarly on both malt extract agar and powdered wood, although a significant difference was observed at 50 and 100\u00a0g\/l (Fig.\u00a03b). However, at 100\u00a0g\/l natamycin, some residual growth could still be observed for powdered wood. A stronger inhibition with natamycin was observed when the incubations occurred at 18\u00b0C instead of 4\u00b0C, resulting in nearly complete inhibition of growth on both media at 0.5\u00a0g\/l natamycin (Fig.\u00a03c). The results described here indicate that spraying wooden crates with natamycin and incubating them at 18\u00b0C or higher likely reduces spoilage F.\u00a0psychrophila.\nF.\u00a0psychrophila produces cold-active hydrolytic enzymes during growth on polysaccharides\nBoth carrots and powdered wood consist largely of polysaccharides. As these are the main carbon sources for F.\u00a0psychrophila during growth in the storage facilities, it can be expected that the fungus produces polysaccharide degrading enzymes that are active at low temperatures. To study whether these enzymes have in fact a higher relative activity at low temperatures than those currently used in industrial applications, we compared enzyme activities from F.\u00a0psychrophila to those from Aspergillus\u00a0niger. A.\u00a0niger is one of the most commonly used fungi for the production of industrial enzyme preparations and produces a wide range of polysaccharide degrading enzymes (de Vries and Visser 2001). However, these enzymes are mainly active at higher temperatures.\nFor A.\u00a0niger, enzyme activities dropped to 30\u201350% when incubation at 15\u00b0C was compared to 30\u00b0C (Fig.\u00a04a). At 4\u00b0C, only 10\u201320% of the 30\u00b0C-activity was observed. For F.\u00a0psychrophila only a 5\u201335% drop was detected when comparing 30\u00b0C to 15\u00b0C and 30\u201360% of the activity at 30\u00b0C was still observed at 4\u00b0C (Fig.\u00a04b). The strongest difference was observed for \u03b1-galactosidase and \u03b2-glucosidase, where more than 90% of the activity was still detected at 15\u00b0C for F.\u00a0psychrophila, while for A.\u00a0niger only 35\u201345% of the activity was detected at this temperature.\nFig.\u00a04Relative hydrolase activities in culture filtrate of Fibulorhizoctonia\u00a0psychrophila and Aspergillus at different temperatures. (a) A.\u00a0niger culture filtrate; (b) F.\u00a0psychrophila culture filtrate. AGL\u00a0=\u00a0\u03b1-galactosidase, LAC\u00a0=\u00a0\u03b2-galactosidase, BGL\u00a0=\u00a0\u03b2-glucosidase, BXL\u00a0=\u00a0\u03b2-xylosidase. Activity at 30\u00b0 is set at 100%\nF.\u00a0psychrophila and other cold-tolerant fungi are potential sources for enzymes with high activity at low temperatures. This idea is supported by studies with cold-tolerant yeast and fungi, which were shown to produce cold-active pectinases (Nagakawa et\u00a0al. 2004, 2005a, b), although in these studies a direct comparison with industrially used enzymes was not made. Cold-active enzymes can be important for many applications, for example in detergents or for the removal of lactose from milk for lactose-intolerant people.","keyphrases":["carrot-spoilage","fibulorhizoctonia psychrophila","cold-tolerant fungus","cold-active enzymes"],"prmu":["P","P","P","P"]}
{"id":"Clin_Rheumatol-3-1-1820749","title":"Central sensitization: a biopsychosocial explanation for chronic widespread pain in patients with fibromyalgia and chronic fatigue syndrome\n","text":"In addition to the debilitating fatigue, the majority of patients with chronic fatigue syndrome (CFS) experience chronic widespread pain. These pain complaints show the greatest overlap between CFS and fibromyalgia (FM). Although the literature provides evidence for central sensitization as cause for the musculoskeletal pain in FM, in CFS this evidence is currently lacking, despite the observed similarities in both diseases. The knowledge concerning the physiological mechanism of central sensitization, the pathophysiology and the pain processing in FM, and the knowledge on the pathophysiology of CFS lead to the hypothesis that central sensitization is also responsible for the sustaining pain complaints in CFS. This hypothesis is based on the hyperalgesia and allodynia reported in CFS, on the elevated concentrations of nitric oxide presented in the blood of CFS patients, on the typical personality styles seen in CFS and on the brain abnormalities shown on brain images. To examine the present hypothesis more research is required. Further investigations could use similar protocols to those already used in studies on pain in FM like, for example, studies on temporal summation, spatial summation, the role of psychosocial aspects in chronic pain, etc.\nIntroduction\nChronic fatigue syndrome (CFS), as defined by the Centers for Disease Control and Prevention (CDCP), is a complex illness characterized by prolonged debilitating fatigue and multiple non-specific symptoms including headaches, recurrent sore throats, fever, muscle and joint pain, and neurocognitive complaints [1, 2]. In addition to the chronic fatigue, widespread and persistent pain is common in individuals with CFS [3\u20135]. A population-based study revealed that 94% of the persons diagnosed with CFS report muscle aches and pain and 84% report joint pain [6]. Nishikai et al. [7] reported muscle pain in 85 CFS patients of 114 patients (74.6%). Seventy-four patients (64.9%) complained of arthralgia. In another study, 24 of 44 patients suffered from chronic widespread pain [8]. Chronic fatigue accompanied by chronic musculoskeletal impairments such as myalgias and arthralgias could be considered an important subclass of CFS [9]. Evidence supportive of the clinical importance of widespread pain in CFS has been provided [10]: chronic pain accounts for up to 34% of the CFS patients\u2019 self-reported activity limitations and participation restrictions. Chronic pain is more disabling than chronic fatigue [10].\nGiven these facts, it may be surprising that the etiology of these pain complaints has not been studied extensively in patients with CFS. The systematic literature review by Meeus et al. [11] shows that only little progress has been made in understanding chronic widespread pain in patients with CFS. A few hypotheses have been proposed, but they have not been studied in depth or relatively little work has been performed to test these hypotheses. In contrast, a large body of scientific literature regarding the etiology of chronic pain complaints in fibromyalgia (FM) is currently available. The diagnosis of FM is based on the 1990 American College of Rheumatology criteria. Following these criteria, FM patients present with 11 of 18 positive tender points and with widespread pain [12]. Validity of both the definition for CFS and FM has been shown [12, 13].\nEspecially, investigations focusing on the phenomenon \u201ccentral sensitization\u201d are presented in force in FM. In CFS, the theory of central sensitization has only been suggested, to our knowledge [14]. Given the great overlap between CFS and FM [15] and given the dearth of studies focusing on the explanation for the chronic widespread pain in patients with CFS, it would be interesting to propose a theoretical model for the chronic pain in CFS based on the current knowledge of CFS and on the evidence for central sensitization in FM, giving rise to further research on that matter. Besides the knowledge on chronic pain in FM, it is necessary to gather knowledge on musculoskeletal pain in CFS.\nThe syndromes may overlap, but despite the similarities between the two syndromes, there are also differences. For example, immunological dysregulations such as the abnormal 2\u20135A synthetase\/RNase L pathway [16] have been revealed in CFS but have never been detected in FM patients. Furthermore, there is not yet any good evidence for similar pain mechanisms in CFS and FM. Some authors already found evidence suggesting differences in pain processing. For example, patterns of functional brain activity in patients with FM are quite different from those in patients with CFS.\nPatients with CFS, relative to controls, showed significantly lower blood perfusion in the brain stem [17, 18]. Patients with FM exhibited significantly lower rCBF levels, during rest, in the thalamus and the caudate nucleus [19]. Furthermore, Substance P has been found to be elevated in CSF of FM patients [20] and not in patients with CFS [21]. Therefore, the knowledge on pain in FM cannot be applied on CFS patients without further study. Based on the similarities and differences between the two syndromes, further research on pain in CFS is advised to get an image of pain processing in the two diseases.\nThe goal of this article is to provide a rational basis for future investigations. First, the concept of central sensitisation as a cause of chronic pain will be explained. This theoretical background will then be applied to FM and an overview of the evidence for central sensitization in FM will follow. Finally, based on the theoretical background and the findings in FM, the hypothesis concerning central sensitization in CFS will be unfolded, supported with the present knowledge on CFS.\nCentral sensitization\nIntroduction Pain is a complex perception that is influenced by prior experience and by the context within which the noxious stimulus occurs; \u201cnociception\u201d is the physiologic response to tissue damage or prior tissue damage [22]. The definition of pain is endorsed by the International Association for the Study of Pain: \u201cPain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage\u201d [23]. There are a host of physiologic mechanisms by which injuries lead to nociceptive responses and ultimately to pain [22]. However, not all nociceptive signals are perceived as pain and vice versa, not every pain sensation originates from nociception. Nevertheless, acute pain almost always originates from nociceptors in somatic or visceral tissue. Mainly two types of pain receptors are activated by nociceptive input. These include low-threshold nociceptors that are connected to fast conducting A-delta pain fibers, and high-threshold nociceptors that conduct impulses in slow (unmyelinated) C fibers. Within the dorsal horn of the spinal cord, these pain fibers synapse with spinal neurons via synaptic transmission. Many neurotransmitters (i.e., glutamate, substance P, etc.) are able to modulate the postsynaptic responses with further transmission to supraspinal sites (thalamus, anterior cingulated cortex, insular cortex, and somatosensory cortex) via the ascending pathways [22, 24, 25].The simplest form of plasticity in nervous systems is that repeated noxious stimulation may lead to habituation (decreased response) or sensitization (increased response) [26]. Prolonged or strong activity of dorsal horn neurons caused by repeated or sustained noxious stimulation may subsequently lead to increased neuronal responsiveness or central sensitization [25, 27]. Neuroplasticity and subsequent CNS sensitization include altered function of chemical, electrophysiological, and pharmacological systems [22, 28, 29]. These changes cause exaggerated perception of painful stimuli (hyperalgesia), a perception of innocuous stimuli as painful (allodynia) and may be involved in the generation of referred pain and hyperalgesia across multiple spinal segments [25, 30\u201333].While the exact mechanism by which the spinal cord becomes sensitized or in \u201chyperexcitable\u201d state currently remains somewhat unknown, some contributing factors have been proposed.\nTemporal summation or wind-up \u201cWind up\u201d refers to a central spinal mechanism in which repetitive noxious stimulation results in a slow temporal summation that is experienced in humans as increased pain [34]. In 1965, animal experiments showed for the first time that repetitive C-fiber stimulation could result in a progressive increase of electrical discharges from the second-order neuron in the spinal cord [35]. This mechanism of pain amplification in the spinal cord is related to temporal summation of second pain or wind-up. Second pain, which is more dull and strongly related to chronic pain states, is transmitted through unmyelinated C fibers to dorsal horn nociceptive neurons. During the C-fibres transmitted stimuli, N-methyl-d-aspartate (NMDA) receptors of second-order neurons become activated.It is well-known that NMDA activation induces calcium entry into the dorsal horn neurons [36]. Calcium entry into sensory neurons in the dorsal horn induces activation of nitric oxide (NO) synthase, leading to the synthesis of NO [37]. NO can affect the nociceptor terminals and enhance the release of sensory neuropeptides (in particular, substance P) from presynaptic neurons, therefore contributing to the development of hyperalgesia and maintenance of central sensitization [38]. Substance P (SP) is an important nociceptive neurotransmitter. It lowers the threshold of synaptic excitability, resulting in the unmasking of normally silent interspinal synapses and the sensitization of second-order spinal neurons [39].Furthermore, SP can extend for long distances in the spinal cord and sensitize dorsal horn neurons at a distance from the initial input locus. This results in an expansion of receptive fields and the activation of wide dynamic neurons by non-nociceptive afferent impulses [24].Wind-up can be elicited in human patients if identical nociceptive stimuli are applied to the skin or muscles more often than once every 3\u00a0s. The resulting progressive increase of pain sensations represents wind-up and has been demonstrated to result from a central rather than a peripheral nervous system mechanism, because the input from C nociceptors has been shown to decline or stay the same with stimulus repetition [40].\nEndogenous pain modulatory systems The presence of several pain inhibitory and facilitatory centers in the brainstem is well recognized. The dorsolateral funiculus appears to be a preferred pathway for descending pain inhibitory systems [41]. Experimental evidence for the existence of descending inhibitory pathways, and their connection with central sensitization, include the observations that bilateral lesions of the dorsolateral funiculus in the rat led to a significant decrease in latency for paw withdrawal to noxious stimulus [42]. Similarly, temporary spinal cord block (lidocaine) caused dorsal horn nociceptive neurons to expand their receptive fields and their responsiveness to afferent input [43]. In addition, selective chemical lesion of serotonergic inhibitory neurons in experimentally \u201cinflamed\u201d animals resulted in demonstrable behavioral \u201cpain\u201d hypersensitivity [42].The foregoing investigations suggest that disruption of one or more of the elements of the inhibitory system can result in, among other things, the equivalent of central sensitization [44]. One function of the descending inhibitory pathway is to \u201cfocus\u201d the excitation of the dorsal horn neurons. The effect is to generate a more urgent, localized, and rapid pain signal by suppressing surrounding neuronal activity [45]. This role is attributed to the \u201cdiffuse noxious inhibitory controls\u201d (DNIC) phenomenon [46]. According to this model, descending pathways effectively enhance the biologically valuable pain signal by reducing the level of irrelevant \u201cnoise\u201d in the system.Facilitatory pathways leading from the brainstem have also been identified. There is now behavioral evidence that forebrain centers are capable of exerting powerful clinically significant influences on various nuclei of the brainstem, including the nuclei identified as the origin of the descending facilitatory pathway [44]. The activity in descending pathways is not constant but can be modulated, for example, by the level of vigilance or attention and by stress [47]. Brosschot [48] refers to it as cognitive emotional sensitization. Forebrain products such as cognitions, emotions, attention, and motivation have influence on the clinical pain experience [44].Dubner and Ren [41] rewarded subjects for responding to a randomly delivered transient tissue threatening peripheral stimulus. It was found that sensitization of second-order pain pathway neurons was directly related to the strength of attention. The evidence suggests that selective attention to relevant stimuli activated descending pain modulatory systems, turning the balance in favor of facilitation. The dominance of descending facilitation then led to sensitization of second-order neurons [44]. Behavioral variables such as attention to a potentially threatening stimulus result in sensitization of dorsal horns spinal cord neurons. Moreover, behavioral modulation associated with selective attention to a perceived threat utilizes the same forebrain and brainstem structures and mechanisms as are involved in the development, amplification, and maintenance of persistent pain after actual tissue damage and inflammation [41].Certain cognitive styles and personality traits have been associated with amplification of pain and its extension in the absence of tissue damage. These include somatization, catastrophizing, and hypervigilance [49\u201352]. Thus, via descending pathways behavioral and cognitive therapies might also effect synaptic transmission in the spinal cord and thereby have the capacity to prevent or reverse long-term changes of synaptic strength in pain pathways [47].\nEvidence in FM\nExaggerated pain is common in whiplash and FM patients. Theoretically, peripheral mechanisms can account for the pain hypersensitivity. In FM, however, there is no evidence for peripheral sensitization as the cause of hyperalgesia, given the absence of real tissue damage. Peripheral sensitization is defined as a reduction in the threshold of nociceptive afferent receptors caused by a local change in the sensitivity of sensory fibers initiated by tissue damage [53]. Peripheral sensitization almost always depends on local inflammation, which may lead to decreased nociceptor thresholds. Despite extensive investigations, no tissue pathology, structural abnormalities, or evidence for a source of chronic stimulation of pain afferents have been detected in fibromyalgic patients [54].\nFurthermore, FM pain lacks a distinct spatial localization [55]. This suggests that pathophysiological central mechanisms contribute to or are responsible for FM pain [56]. This has led to the hypothesis that the central nervous system is hyperexcitable in these patients. Central hypersensitivity could explain exaggerated pain in the presence of minimal and undetectable tissue damage, in that the nociceptive signal is amplified by the hyperexcitable neurons [57].\nPain measurements Studies in patients with chronic pain after whiplash injury and with FM have demonstrated exaggerated pain responses after sensory stimulation of healthy tissues [55, 58\u201363]. For example, at same level of thermal stimulation, FM patients perceived pain as 49 and 52% more intense than healthy controls and patients with low-back pain [61]. After-sensations at 15\u00a0s after heat taps were regarded as painful on 83% of FM patients, compared to 37% pain reports of healthy controls. The late after-sensations (2\u00a0min after heat stimulation) were rated as painful in 55 and 5% of FM and control subjects, respectively [55]. It was not mentioned in the results if all FM patients were subject to increased sensitivity, but there were always significant differences between the FM patients and the healthy controls.Although most comparisons were made to pain-free subjects, some researchers, like Julien et al. [61], even compared the pain responses of FM patients to other pain patients, such as patients with chronic low-back pain. They could also report significant differences. Despite the lack of research into the contribution of psychological factors in wind-up, these results suggest that input to central nociceptive pathways is abnormally processed in patients with FM. Intramuscular electrical stimulation has been used to assess the efficacy of temporal summation of painful muscle stimuli. Temporal stimulation was found to be more pronounced and to cause stronger pain and larger referred areas in FM patients compared to controls [60].The increased efficacy of temporal summation in FM has even so been reproduced with cutaneous heat stimulation [55] and with cold and heat taps [59]. Facilitated temporal summation in patients with pain suggests that the efficacy of central processing is increased (central sensitization) in these patients [64]. In addition, after-sensation was greater in magnitude, lasted longer, and was more frequently painful in patients with FM [55]. The prolonged decay together with the augmentation provides evidence for the presence of central sensitization [24, 25]. Immersion of the arm in circulating noxious cold water resulted in a 49% more intense pain in FM patients compared to healthy controls [61]. Further evidence of central sensitization in FM is seen by enlarged referred pain areas. S\u00f6rensen et al. [60] found that fibromyalgic patients experienced stronger pain and larger referred areas after intramuscular injection of hypertonic saline.Moreover, spatial summation effect during increase of the stimulation area was found only in patients with FM and not in healthy controls or in patients with chronic low-back pain [61]. This indicates that pain inhibitory systems are not optimally recruited in patients with FM. FM patients perceived pain at the same intensities and unpleasantness during the ascending and the descending sessions (fingertip to shoulder and shoulder to fingertip immersion). In healthy controls and patients with low-back pain, the noxious stimulation of a large surface area results in an activation of a large population of nociceptive afferents that induce endogenous inhibitory responses, resulting in a decreased response in the dorsal horn neurons, and subsequently leads to lower pain intensities afterward.Given that FM patients experience similar pain intensities in the descending session after noxious stimulation of the whole arm, this study clearly demonstrated that FM patients present with a lack of activation of endogenous inhibitory systems [61]. Secondly, Lautenbacher and Rollman [65] showed that tonic painful and non-painful thermal stimulation of the foot increased the pain thresholds to electrical stimulation applied to a non-tender point (inner forearm) in healthy controls but had no effect on patients with FM. Kosek and Hansson [66] found that tourniquet ischemic pain in an arm increased the pressure pain threshold in healthy controls but not in FM patients, suggesting once more a deficiency in the latter of a pain-inhibitory phenomenon termed \u201cdiffuse noxious inhibitory control\u201d (DNIC) [66, 67]. Similarly, aerobic exercise has been shown to decrease wind-up pain in normal subjects but increased it in FM patients, suggesting the possibility of reduced endogenous analgesic mechanisms [68]. Also, isometric exercise (i.e., hand-grip exercise) resulted in increased thermal pain ratings and decreased pain thresholds, both ipsilateral and contralateral to the exercised extremity [69]. These investigations support a general hypothesis that FM reflects a disorder affecting modulation of pain sensitivity [66, 67]. In one such scenario, tonic DNIC is present in the normal situation, and its pathological absence results in the spontaneous pain and evoked pain sensitivity associated with FM [34].\nMeasurements of excitability The results of the above-mentioned studies, however, are based on pain reports of the patients and thus subjective in nature, and it was not clear whether this hypersensitivity was the result of central mechanisms or whether the hypersensitivity was the cause of hypervigilance. Banic et al. [57] could provide objective evidence by quantifying the minimal intensity of transcutaneous electrical stimulation of the sural nerve required to evoke flexion reflex in the biceps femoris. This study clearly demonstrates that spinal cord neurons are sensitized in chronic pain after whiplash and in patients with FM; because the stimuli were delivered at random time intervals and the latency of EMG response was measured, voluntary symptom amplification could be ruled out. Moreover, the electrical stimulation bypasses peripheral receptors.\nCognitive emotional sensitization Patients with FM or other pain disorders often receive the message that it is \u201call in their head\u201d. One construct that has been hypothesized to explain the pain amplification in FM is that of hypervigilance [34]. This hypothesis of hypervigilance has been argued by McDermid et al. [70]. FM showed an increased aversiveness to non-painful stimuli such as loud noise. Also, Crombez et al. [71] reported significant correlations between hypervigilance and pain intensity in FM patients. Furthermore, exposure to stressful situations, including noise, lights, and weather, is known to exacerbate symptoms of FM, including pain [72].In addition, FM patients with catastrophic thoughts report increased pain intensities [73\u201375]. Hassett et al. [74] found catastrophizing (27% of the variance) and depression (30% of the variance) to be significant predictors of pain. Finally, kinesiophobia and fear of pain are related to pain severity in patients with FM [76].The foregoing relations between emotions or cognitions and reported pain severity support the hypothesis of cognitive emotional sensitization in FM.\nCentral abnormalities in FM FM patients differ from healthy persons in regional cerebral blood flow (rCBF) distribution in several brain structures involved in pain processing and pain modulation both at rest and during experimental pain induction. Patients with FM exhibited significantly lower rCBF levels, during rest, in the thalamus and the caudate nucleus [17]. Dysregulation of thalamic activity and hypoperfusion of the caudate nucleus may contribute to the abnormal pain modulation, given the results of previous investigations [77\u201379]. In addition to nociceptive transmission, the thalamus also plays an important role in pain modulation. Animal studies proved that thalamic stimulation induces analgesia and lesions of the thalamus cause hyperalgesia [77, 78].Similarly, stimulating the caudate nucleus decreases pain behavior [79]. During pain induction in patients with FM, the absence of significant thalamic activation and a bilateral activation of the somatosensory cortices and the right anterior cingulated cortex was seen [80]. The patterns of cortical activation may be characteristic of patients with allodynia, and pain-induced activation of the right anterior cingulated cortex is associated with the use of maladaptive coping strategies [81]. Patients recruited by Bradley et al. [80] indeed reported significantly greater use of emotion-focused coping strategies (e.g., praying and hoping) during pressure stimulation. In addition, increased right frontal brain activity seemed related to increased pain sensitivity [82].Furthermore, FM patients are characterized by relatively high cerebrospinal fluid levels of substance P [20]. This finding indicates that abnormal brain activity in persons with FM is associated with abnormal CSF levels of a neuropeptide involved in pain transmission [80]. In addition, it is shown that a subgroup of FM patients present with mycoplasma infections [83]. Infection triggers the release of the pro-inflammatory cytokine interleukin-1\u03b2, which is known to play a major role in inducing cyclooxygenase-2 (COX-2) and prostaglandin E2 expression in the central nervous system [84].Peripheral nerve terminals can be sensitized by elevated COX-2 amounts and prostaglandin E2. Peripheral infections are even able to activate spinal cord glia, leading to the release of NO and proinflammatory cytokines, enhancing the pain response [85]. Physiological symptoms, such as pain, can be explained by these mechanisms (\u201csickness response\u201d).\nCentral sensitization in CFS?\nDirect evidence supporting the central sensitization hypothesis in CFS patients is currently lacking. But the present knowledge concerning CFS is suggestive of a central process similar to that seen in FM, given the great overlap between the two diseases and the observed similarities.\nFirst, lower pain thresholds at different sites (hyperalgesia) are reported in patients with CFS, compared to controls [86, 87]. Similar to FM, the lack of peripheral tissue damage and the lack of a distinct localization of the pain complaints are suggestive of a central abnormality responsible for the chronic widespread pain. To our knowledge, there are no investigations that focused on abnormal wind-up, temporal summation, or spatial summation in CFS, to collect evidence for central sensitization in CFS. Yet, evidence of a dysfunctional central anti-nociceptive mechanism in CFS has been proved by Whiteside et al. [88]. They reported a decrease of pain threshold in patients with CFS after graded exercise, while healthy controls present an increased pain threshold. These findings are similar to those of Vierck et al. [68] in FM patients.\nSecondly, the frequently reported opportunistic infections [89, 90] may lead to the \u201csickness response\u201d and complies with the central sensitization hypothesis, as explained above. Indeed, elevated NO levels have been documented in CFS patients [91]. As earlier mentioned, NO plays an important role in the history of central sensitization and, therefore, central sensitization caused by NO would be likely in patients with CFS. The release of excessive amounts of Substance P in the CSF, however, could not be documented in patients with CFS, in contrast to FM patients [21]. The CFS patients in this study, however, did not experience chronic widespread pain, and it is not clear if they fulfilled the 1994 CDC criteria [2]. Given that 70% of the CFS patients do fulfill the ACR criteria for widespread pain [12], it is striking that the patients included in the investigation of Evengard et al. [21] (focused on the source of pain) did not report these pain complaints. Further research on that matter should analyze the CSF of CFS patients (fulfilling the Fukuda criteria) suffering from chronic widespread pain.\nA third important argument in the central sensitization theory for CFS concerns the cognitive, psychological, and behavioral changes in patients diagnosed with CFS. CFS patients often present with depression [6, 92], catastrophizing [93, 94], somatization [95, 96], and kinesiophobia or fear avoidance [97\u201399]. In CFS, it is known that these psychosocial aspects are important factors in maintaining the complaints of CFS. These cognitions and emotions are able to influence pain perception via modulation of the descending pathways [44]. Furthermore, these \u201ccognitive styles\u201d and \u201cpersonality traits\u201d have earlier been mentioned to be associated with the amplification and the extension of pain [49\u201352].\nFinally, brain imaging already provided evidence for altered brain activity in CFS. Patterns of functional brain activity in patients with FM are quite different from those in patients with CFS. Patients with CFS, relative to controls, showed significantly lower blood perfusion in the brain stem [17, 18]. Patients with FM exhibited significantly lower rCBF levels, during rest, in the thalamus and the caudate nucleus [19]. However, the areas are different in FM and in CFS; both the affected areas could be related to central pain processing. Low brain stem rCBF levels may contribute to abnormal function of the locus ceruleus in patients with CFS. The locus ceruleus is involved in controlling descending anti-nociceptive pathways from the brain to the spinal dorsal horn [56]. In consequence, pain experiences of patients with CFS may be related to low resting state levels of functional activity in the brain stem [80].\nConclusion\nChronic widespread pain can be the consequence of central sensitization. Central sensitization is known as an increased central neuronal responsiveness and causes hyperalgesia, allodynia, and referred pain and hyperalgesia across multiple spinal segments, leading to chronic widespread pain. Possible triggers for sensitization of the spinal cord have extensively been discussed, such as wind-up or temporal summation, dysregulated descending inhibitory pathways, and upregulated facilitatory modulation. Wind-up or temporal summation is the result of repetitive noxious stimuli, leading to an increase in electrical discharges in the dorsal horn. Inhibitory modulation can be impaired by abnormalities in the central nervous system and the facilitatory pain pathways can be stimulated by certain behavioral and cognitive factors.\nThis theoretical background can be applied to FM. In FM, studies already provided evidence for central sensitization as the cause of chronic pain. Temporal summation was found to be more facilitated, and the inhibitory pain modulation seemed impaired in FM patients. These findings can explain the chronic spontaneous pain in FM. Furthermore, some central abnormalities could be examined\/objectified in FM: 1) hyperexcitability of the spinal cord, 2) decreased perfusion of pain-related brain structures, and 3) high levels of substance P in CSF. In addition, FM patients often present with pain hypervigilance, maladaptive coping strategies, and catastrophic thoughts, leading to cognitive central sensitization.\nBased on the knowledge on central sensitization, on FM and on CFS, it is suggested that chronic widespread pain in CFS is the consequence of central sensitization. There are arguments and probable mechanisms that could explain this phenomenon in CFS. Also, in other chronic pain populations, central sensitization may play a key role. In fact, there are many similarities between CFS patients and other chronic pain populations such as patients with chronic low-back pain, whiplash, FM, etc. The psychosocial factors, for example, have been proved to contribute to pain perception in these different pain populations. But the specific nature of CFS such as the immunological abnormalities, elevated NO amounts, preceding infections etc., invites further research, in particular, on the possible contributory role of these abnormalities to pain processing in CFS.\nIn FM, many researches have been conducted to prove the theory of central sensitization. In CFS, however, it sticks to \u201csupposing.\u201d To give a scientific basis to the theory, the protocols applied in FM investigations could be used for patients with CFS. It would, for example, be interesting to test the efficacy of temporal summation in CFS. The inhibitory control of pain could be another point of interest. The influence of exercise on pain tolerance has already been studied in CFS [88], however, on a relatively small sample. On the contrary, spatial summation has, to our knowledge, never been investigated in CFS. Furthermore, the role of depression, hypervigilance, kinesiophobia, catastrophising, etc. on chronic pain in CFS requires further research. To obtain more direct and objective information on central sensitization, the protocol described by Banic et al. [57] could be used to test the sensitivity of the central nervous system. Clearly, there are many possible research areas to test the hypothesis, but there is still a long way to go to elucidate the nature of the chronic pain in CFS.","keyphrases":["central sensitization","fibromyalgia","chronic fatigue syndrome","chronic pain"],"prmu":["P","P","P","P"]}
{"id":"Eur_J_Health_Econ-_-_-1388083","title":"Benefits and entitlements in the Hungarian health care system\n","text":"This contribution considers entitlements and benefits in the Hungarian health care system. After a brief introduction to the organizational structure of the system the decision-making processes are discussed in detail, including the most important actors, types and pieces of legislation, formal structures, decision-making criteria, and outputs in terms of benefit catalogues. Within the two main public financing systems (social insurance and tax-funded services) there are four types of regulatory regimes: (a) traditional political decision making, (b) price negotiations, (c) updating of classification systems for payment purposes, and (d) the procedure for the inclusion of registered medicines in the scope of the social health insurance system. As an example we discuss the benefit regulations and benefit catalogues in the category of services of curative care (HC.1) of the OECD classification of health services.\nWith the crisis and the collapse of the communist dictatorship Hungary witnessed the beginning of a thorough health sector reform. Replacing the tax-based financing of the state-socialist system, Hungary reverted to the earlier Bismarckian model of compulsory social insurance in 1990. New performance-based provider payment methods were introduced (capitation in primary care, fee-for-service for outpatient specialist services, and diagnosis-related groups (DRGs) in the acute inpatient care sector) together with cost-containment mechanisms to ensure that the preset budgets were not exceeded. Ownership of the majority of hospitals and other health care facilities was transferred to local governments. The vast majority of medical doctors and other health workers remained salaried public employees, with the only exception of primary care, where the bulk of family physicians work as contracted private entrepreneurs. The minimum salary for public employee physicians is determined by law, whereas the average salary in the health sector has remained among the lowest in the economy [1].\nEntitlement to health care is based mainly on the participation in the social insurance scheme (with compulsory membership, opting out is not permitted), and to a few services on citizenship. The national Health Insurance Fund (HIF) provides a virtually universal population coverage with an almost comprehensive benefit package, which applies to the whole country (i.e., there are no variations by region or by payer). Nevertheless, the HIF covers only the recurrent costs of services. The owners of health care facilities, mainly local governments, are obliged to cover the capital costs of services, which usually come from general and local taxation. Tax revenues are also used for covering the deficit of the HIF [1997 Act LXXX on Those Entitled for the Services of Social Insurance and Private Pensions and the Funding of these Services, Sect. 3(2)], certain special services (e.g., public health, catastrophe medicine, experimental medical technologies, family planning and maternal care), which are financed entirely from the central government budget [1997 Act LXXXIII on the Services of Compulsory Health Insurance, Sect. 18(5)a\u2013d, h; 1997 Act CLIV on Health, Sects. 141(2)b, 142(2)], and the copayment for certain medicines and therapeutic devices for socially disadvantaged (1993 Act III of on Social Services). In addition to informal payments to health workers, copayments for medicines and therapeutic devices constitute the most important private source of health care financing, which are almost exclusively out-of-pocket, as private health insurance is still insignificant in Hungary [1].\nDecision-making processes and actors\nEntitlement to publicly financed health services is regulated by various types of regulatory instruments (Table\u00a01). These represent the decisions of various decision-making actors, with varying decision-making rules\/processes and power. There is a hierarchy of regulations with the constitution on top, followed by governmental and then ministerial decrees. In the case of conflicting provisions the higher level regulation precedes the lower one, but the provisions of lower regulations are usually more detailed.Table\u00a01 Main types of regulations in Hungary [1949 Act XX on the Constitution of the Republic of Hungary; 1987 Act XI on Codification; Resolution No. 1088\/1994\u00a0(IX.21) on the Decision-Making Procedures of the Government; 1990 Act LXV on Local Governments]Type of regulationDecision makerMethod of decision makingConsultationLegal regulations, laws\u00a0\u00a0ConstitutionNational AssemblyTwo-thirds majority voteRequired\u00a0\u00a0Fundamental actsNational AssemblyTwo-thirds majority voteRequired\u00a0\u00a0ActsNational AssemblySimple majority voteRequired\u00a0\u00a0Governmental decreesCentral governmentSimple majority vote of the government or exceptionally the prime minister (one person)Required\u00a0\u00a0Ministerial decreesMinistersOne personRequired\u00a0\u00a0Local government decreesLocal government assembliesSimple majority voteRequiredRulings of the Constitutional CourtConstitutional CourtSimple majority vote\u2013Other means of state control\u00a0\u00a0ResolutionNational Assembly, central government, governmental committees, local governmentsAccording to decision maker\u2013\u00a0\u00a0OrderMinisters, heads of organizations with national scope of authorityOne person\u2013\u00a0\u00a0PolicyNational Assembly, central government, ministers, heads of national organizationsAccording to decision makerRequired\u00a0\u00a0Statement of interpretation of legislationsNational Assembly, central governmentAccording to decision maker?\u00a0\u00a0Announcement, communicationMinisters, heads of organizations with national scope of authorityOne person\u2013\nThe National Assembly (Parliament) and the central government (and Ministry of Health, MOH) are the key actors in national-level decision making. Parliament determines, for instance, the scope of publicly funded services, the benefit package, and the budget of the HIF. While most decisions of Parliament require a simple majority, the constitution and other fundamental acts (e.g., on local governments) can be changed only with a two-third majority vote. Passed bills are promulgated as acts, on the basis of which governmental and ministerial decrees are issued, which regulate the implementation of acts in detail [1].\nIn addition to acts, governmental, ministerial, and local government decrees, which represent generally valid and obligatory behavioral norms and are called \u201clegal regulations\u201d or \u201claws,\u201d there are lower level regulations, which are categorized as \u201cother means of state control,\u201d such as resolutions, orders, policies, statements, and announcements (1987 Act XI on Codification, Sects. 46\u201356) as well as organizational operational rules and procedures of various organizations and decision-making bodies. Resolutions, for instance, can be issued by the Parliament, the government, governmental committees, local governments, and their organs to regulate the tasks of organizations controlled by them, the rules of their own operation and plans within their scope of authority, while orders can be issued by ministers and the heads of organizations with national scope of authority to regulate the activities of the organizations under their control [1987 Act XI on Codification, Sect. 46(1)].\nThe process of decision making and the content of the benefit basket are usually regulated at least in ministerial decrees with only a few cases, when the decision on benefits has been decentralized to other organizations (Table\u00a02). This does not mean that the decision making has no input from a wide range of actors in the health care arena. The most important actors are as follows: (a) National Health Insurance Fund Administration (NHIFA), which administers the HIF, in particular its Department of Payment Informatics (formerly the Information Center for Health Care of MOH, (GY\u00d3GYINFOK)) which is responsible for the provider payment and performance measurement; (b) various advisory bodies and organizations of the MOH, including the national institutes of health and the professional colleges, which provide an expert input concerning a particular medical specialty; (c) professional organizations (e.g., Hungarian Medical Chamber, HMC; Hungarian Chamber of Pharmacists, HCP), unions, and provider and patient associations [1].Table\u00a02 Regulations of entitlements and benefits in HungaryRegulation (type according to Table 1)What is regulated?C\/PAct XX of 1949 (1)Right to healthCAct CLIV of 1997 (3)Right to health services; scope and broad content of health servicesCAct LXXX of 1997 (3)Participation in and contribution to the social insurance scheme (who is covered)\u2013Act LXXXIII of 1997 (3)Framework for benefits of social insuranceC, PGovernment Decree No. No. 217\/1997. (XII. 1 Korm (4)Executive order of Act LXXIII of 1997P, CDecree No. 46\/1997. (XII. 17) NM of the Minister of Welfare (5)Services excluded from public financingCGovernment Decree No. 284\/1997 (XII. 23) Korm (4)Copayments; exclusions (full fee)CAct XCIII of 1993 (3)Occupational health servicesCGovernment Decree No. 89\/1995. (VII. 14) Korm (4)Occupational health servicesCDecree No. 27\/1995. (VII. 25) NM of the Minister of Welfare (5)Occupational health servicesCDecree No. 5\/2004. (XI. 9) E\u00fcM of the Minister of Health (5)Benefit catalogue of balneotherapyCDecree No. 20\/1995. (VI. 17) NM of the Minister of Welfare (5)Benefit catalogue of treatment in sanatoriaCDecree No. 48\/1997. (XII. 17) NM of the Minister of Welfare (5)Benefit catalogue of dental careCDecree No. 49\/1997. (XII. 17) NM of the Minister of Welfare (5)Benefit catalogue of infertility treatmentsCDecree No. 47\/1997. (XII. 17) NM of the Minister of Welfare (5)Eligibility for free breast milkCDecree No. 50\/1997. (XII. 17) NM of the Minister of Welfare (5)Eligibility for patient transportationCDecree No. 51\/1997. (XII. 18) NM of Minister of Welfare (5)Benefit catalogue of screeningCDecree No. 56\/2003. (IX. 19) ESzCsM of the Minister of Health, Social and Family Affairs (5)Benefit catalogue of balneotherapyCDecree No. 19\/2003. (IV. 29) ESzCsM of the Minister of Health, Social and Family Affairs (5)Benefit catalogue of medical aids and prostheses (therapeutic devices)CGovernment Decree No. 43\/1999. (III. 3) Korm (Annex 8) (4)Benefit catalogue of chronic careCDecree No. 9\/1993. (IV. 2) NM of the Minister of Welfare (5)Benefit catalogue of outpatient specialist services; acute inpatient care; dental care; day cases of curative care; dialysis; chronic outpatient care;CDecree No. 1\/2003. (I. 21) ESzCsM of the Minister of Health, Social and Family Affairs (valid until 1 July 2005 then NHIFA announcement) (5, 12)Benefit catalogue of medicinesCDecree No. 20\/1996. (VII. 26) NM of the Minister of Welfare (5)Benefit catalogue of home careCDecree No. 49\/2004. (V. 21) ESzCsM of the Minister of Health, Social and Family Affairs (5)Tasks of MCH nursesCDecree No. 26\/1997. (IX. 3) NM of the Minister of Welfare (5)Tasks of school health services (physician, MCH, dentist, assistant)CGovernment decree No. 168\/2004. (V. 25) Korm (4)Regulatory regime (price negotiations for special medicines)PDecree No. 6\/1998. (III. 11) NM of the Minister of Welfare (5)Regulatory regime (payment)PGovernment Decree No. 112\/2000. (VI. 29) Korm (4)Regulatory regime (price negotiation)PDecree No. 32\/2003. (IV. 26) ESzCsM of the Minister of Health, Social and Family Affairs (5)Regulatory regime (medicines)POrder No. 6\/2005. (Eb. K. 3) OEP of the Chief Executive Officer of the National Health Insurance Fund Administration (9)Regulatory regime (medicines)PC content regulation,\nP Process regulation\nThe 1987 Act XI on Codification explicitly requires relevant nongovernmental and interest representation organizations to be consulted in the phase of preparation of laws (Sects. 27\u201332).\nGiven that the existing benefit catalogues are almost exclusively incorporated into acts, governmental decrees and ministerial decrees, the general features of the decision-making processes (regulatory regimes) on benefits are common, as described above. While in these cases it is straightforward who makes the decision on the basis of what method, the real issue is who is consulted in what form in the preparatory phase of the decision making process, for which the acts discussed so far only provide a very general and vague guidance.\nIn many cases the decision support mechanisms have not yet been formalized in lower level regulations, and therefore that the process of preparation and codification is based on tradition. For instance, the need for the creation of a ministerial decree can originate from the provision of an act or governmental decree\u2014in this case the MOH Legal Department initiates the codification process at the relevant professional department\u2014or it can be initiated internally or by an external actor\/stakeholder. Although each department must work on the basis of specific regulations on issue handling, in what cases, who is to be consulted, and how are usually not specified in these regulations but passed from one civil servant on the other (Zs. Kov\u00e1csy, personal communication, 2005).\nThis traditional regulatory regime is the most commonly used decision-making mechanism regarding entitlements and benefits (Fig.\u00a01). The two key acts in the center of the definition of the benefit package are 1997 Act CLIV on Health and 1997 Act LXXXIII on the Services of Compulsory Health Insurance. These acts define only a general framework in which both exclusions and inclusions are usually stipulated only at the level of broad functional categories. According to the 1997 Act CLIV, the right to health services is unconditional only for emergency life-saving services, services, which prevent serious or permanent health damage, and for the reduction in pain and suffering (Sect. 6). Patients have a right to other health services only within the limits set by another legislation (Sect. 7). The Act states that the state is responsible for the operation of the social insurance scheme to enable the individuals to exercise their right to health, and then lists the services, which must be financed from the central government\u2019s budget (Sects. 141, 142). The 1997 Act LXXXIII defines health services which are free of charge (Sects. 10\u201317), covered with copayment (Sects. 23\u201325), or excluded [Sect. 18(5\u20136)]. The starting point of the Act is that all health services are fully covered and exclusions are stipulated. In the frame of social insurance all professionally justified treatments can be used [Sect. 18(4)], but diagnostic and treatment protocols issued by the MOH can further specify the actual services for which the patients are entitled to [Sect. 19(1)]. Physicians are allowed not to adhere to the protocols if the deviation is justified by the status of the patient and by therapeutic considerations. It must be noted, however, that broad functional areas are also listed (i.e., there is a scope for implicit exclusions) in the Act in three main categories: (a) services for the prevention and early detection of diseases (Sect. 10), (b) curative services, including family physician services, dental care, outpatient specialist, and inpatient care (Sects. 11\u201314), and (c) other services including deliveries, medical rehabilitation, patient transport and emergency ambulance services (Sects. 15\u201317, 22).Fig.\u00a01 Traditional regulatory regime for entitlements and benefits in Hungary\nThese laws were passed by Parliament with a simple majority vote but are not updated or modified on a regular basis. Their modification can be initiated by the government (Minister of Health), the President of the Republic, Members of Parliament, Parliamentary Committees, or other stakeholders through these actors [Resolution No. 46\/1994 (IX. 30) OGY of the National Assembly].\nThe above acts contain a large number of clauses, which call the government, the Minister of Health, or both of them to further specify certain functional areas. There are two main types of ministerial decrees. The 1997 Act CLIV calls on the Minister of Health to regulate the professional requirements, including minimum standards and procedures of certain service categories, and these decrees may include provisions related to the benefit package. For instance, these types of ministerial decrees can specify the tasks which must be fulfilled in the framework of a particular service (e.g., school health services Decree No. 26\/1997 (IX. 3) NM of the Minister of Welfare on School Health Services) and what service can be ordered for what patients\u2019 conditions (e.g., rehabilitative treatments in sanatoria Decree No. 20\/1995 (VI. 17) NM of the Minister of Welfare on the Treatment in Sanatoria in the Frame of Medical Rehabilitation) and define who is allowed to provide specific services [e.g., home care; Decree No. 20\/1996 (VII. 26) NM of the Minister Welfare on Home Care)].\nOn the other hand, the 1997 Act LXXXIII authorizes ministerial (and governmental) decrees to specify entitlements and benefits within a particular service category [e.g., dental care; Decree No. 48\/1997 (XII. 17) NM of the Minister of Dental Services which Can Be Utilized in the Frame of the Compulsory Health Insurance]. In certain cases it is not the service concerned specified but the criteria of eligibility [e.g., the supply of breast milk; Decree No. 47\/1997 (XII. 17) NM of the Minister of Welfare on the Supply of Breast Milk in the Frame of the Compulsory Health Insurance]. The updating of these decrees is made on an ad hoc basis as the need arises. Benefits are uniform throughout the country and explicitly defined, although services are usually not detailed.\nNevertheless, there are certain areas, such as pharmaceuticals and therapeutic devices, where various factors such as the presence of powerful suppliers (manufacturers of medical goods) have led to more formalized decision making. These developments received a new impetus in Hungary as a result of the country\u2019s integration into the European Union, as the requirements of EU directives, for instance, in the case of pharmaceuticals, have had to be incorporated into Hungarian regulations. The decision-making process for the inclusion of a particular medicine in the benefit package of the social insurance scheme has been regulated down to the details of decision-making criteria.\nFurthermore, the introduction of new payment mechanisms created indirect means of defining benefits through the classification of cases and services for the purpose of provider reimbursement (Fig.\u00a02). In the case of outpatient care there is a list of services with the World Health Organization International Classification of Procedures in Medicine (ICPM) codes and point values, while acute inpatient cases are categorized into one of the 786 DRGs on the basis of the diagnosis (coded on the basis of the 10th edition of the International Classification of Diseases) and the procedures performed [Decree No. 9\/1993 (IV. 2) NM of the Minister of Welfare of the Social Insurance Financing of Specialist Services]. While the 1997 Act LXXXIII of declares that all professionally justified curative services are included in compulsory social health insurance financing, there is no incentive for the providers to provide services which are not reimbursed. Therefore these classification systems can be regarded as indirect benefit catalogues, with the updating procedure being their indirect modification. Since the introduction of the new payment mechanisms they have been regularly updated and the process of instituting changes has been formalized since 1998 [Decree No. 6\/1998 (III. 11) NM of the Minister of Welfare on the Regulation of Updating Professional Classification Systems and Financing Parameters Used in Health Care]. The updating process includes the modification and extension of the two fundamental classification systems as well as the various payment catalogues. The extension of the ICPM has special relevance for the benefit package, since those health care interventions, which are not listed in the Hungarian version of the ICPM have been excluded from public financing (1997 Act LXXXIII on the Services of Compulsory Health Insurance).Fig.\u00a02 Updated Classification Systems for Payment Purposes. [Decree No.\u00a06\/1998.\u00a0(III.\u00a011) NM of the Minister of Welfare; Procedure of the Payment Codes Updating Committee]\nThe key actors in the process are the GY\u00d3GYINFOK and the so-called Payment Codes Updating Committee (PCUC), which is an advisory body of the minister of health [Decree No. 6\/1998 (III. 11) NM of the Minister of Welfare on the Regulation of Updating Professional Classification Systems and Financing Parameters Used in Health Care]. The former is responsible for preparing decision support documents, including the collection and the analysis of the necessary financing data, while the PCUC makes the decisions formulated as proposals, guided by the criteria of public health impact and of the efficient allocation of resources [Procedure of the Payment Codes Updating Committee (Working Committee), December 2003, Sect. 2.5]. The ultimate decisions rest with the MOH. The list of outpatient specialist services, DRGs, day cases of curative care, chronic care services, and the various forms of dialysis (as well as their modifications) are always issued as ministerial decrees. Various announcements, communications, and guidelines are also published to promote the lawful use of payment catalogues [Decree No. 6\/1998 (III. 11) NM of the Minister of Welfare on the Regulation of Updating Professional Classification Systems and Financing Parameters Used in Health Care Sects. 2(2\u20134), 7(1)].\nThe PCUC has 15 permanent members, delegated by the MOH (2), CEO of the NHIFA (2), GY\u00d3GYINFOK (2), Hungarian Hospital Association (2), and HMC (1), while the Minister of Health appoints six medical experts and the head of the PCUC [Procedure of the Payment Codes Updating Committee (Working Committee), December 2003, Sect. 3]. The committee prepares its procedure, a yearly workplan and a methodological document as the basis of the updating process [Decree No. 6\/1998 (III. 11) NM of the Minister of Welfare on the Regulation of Updating Professional Classification Systems and Financing Parameters Used in Health Care Sect. 5(4)].\nThe updating process can be initiated in two ways. Regular updates are planned in the workplan of PCUC, while all the relevant stakeholders are allowed to ask for unscheduled updates which must then be evaluated and answered within 30\u00a0days [Decree No. 6\/1998 (III. 11) NM of the Minister of Welfare on the Regulation of Updating Professional Classification Systems and Financing Parameters Used in Health Care, Sect. 1(2)]; Procedure of the Payment Codes Updating Committee (Working Committee), December 2003, Sect. 2.4]. First, the latter proposals must be submitted to the head of the relevant national institute or directly to the Committee if there is no national institute concerned. The relevant professional college(s) then provides an expert opinion, and the GY\u00d3GYINFOK then prepares a cost estimation and budget impact analysis usually on the basis of data provided by a sample of health care institutions. The PCUC has at least one meeting per month, whose proceedings are to be submitted to the MOH in the form of a proposal for changes at least once in a year [Decree No. 6\/1998 (III. 11) NM of the Minister of Welfare on the Regulation of Updating Professional Classification Systems and Financing Parameters Used in Health Care, Sect. 5(6\u20137); Procedure of the Payment Codes Updating Committee (Working Committee), December 2003, Sect. 4.4].\nIn summary, there are four main regulatory regimes for the definition and modification of entitlements and benefits: (a) the traditional decision-making process, guided by the general rules of codification with less formalized preparatory phase, for services such as primary care and home care, (b) formalized as \u201cprice negotiations\u201d for therapeutic devices and balneotherapy, (c) formalized as the \u201cprocedure for the inclusion of registered medicines in the scope of the social health insurance system\u201d in line with the provisions of Council Directive 89\/105\/EEC (21 December 1988), and (d) formalized as the \u201cprocedure of updating professional classification systems and payment parameters\u201d for outpatient specialist and inpatient care services.\nAs a result of the various regulatory regimes, universal benefit catalogues exist for almost all service areas.\nEntitlements and benefits: services of curative care\nTable\u00a03 summarizes entitlements and benefits in the category of services of curative care (HC.1) of the International Classification for Health Accounts (ICHA) taxonomy [2].Table\u00a03 Entitlements and benefits: services of curative care (HC.1)HCFunctional categoryRegulatory regimeaBenefit catalogue (regulation no. from Table 2, method of classification, date passed, last updateda), taxonomy1.1Inpatient curative careUpdating of classification systems for payment purposes (4)Acute inpatient care\u2013somatic, intensive care, mental special area: transplantation21, Annex 3 (and Annex 8 for high cost high tech interventions; Annex 10 for course-type treatments); itemized by case (linked to diagnosis and therapy); 2 April 1993, 6 August 2004. 26 main groups (major diagnostic categories), altogether with 786 items (DRGs)1.2Day cases of curative careSpecialist outpatient care\u2014day surgery21, Annex 9; itemized by service; 2 April 1993, 6 August 2004.227 items in two lists: (a) 194 elective surgical procedures (Sect. I), (b) 33 other (\u201cclinical\u201d) interventions (Sect. III)Special area: hemodialysis21, Annex 11; itemized by service; 2 April 1993, 2 March 2001.6 items: (a) hemodialysis, hemofiltration, hemodiafiltration; (b) same for patients under 18 years old; (c) peritoneal dialysis, (d) hemoperfusion, (e) hemodialysis with reusable dialysator, (f) mobile treatment1.3Outpatient care1.3.1Basic medical and diagnostic services(Curative) primary careTraditional (2, 4)\u20131.3.2Outpatient dental careDental prophylaxis, care, surgeryTraditional (2, 4)13; itemized by service; 17 December 1997, 1 November 2001.3 main groups: (a) dental screening, with 7 items in three subgroups; (b) dental emergency services with 10 items; (c) dental primary and secondary care, defined as the payment list, detailed in a separate decree (no. 21)Updating of classification systems for payment purposes (4)21, Annex 12; itemized by service; 2 April 1993, 11 March 2003.125 items in 10 groups: (a) examinations, documentations, 9; (b) prevention, 5; (c) radiography, 5; (d) anesthesia and drug prescription, 5; (e) tooth preserving services and endodontia, 12; (f) oral diseases and parodontology, 11; (g) dental surgery, 24; (h) dental prosthetics, 26; (i) child, school, and youth dental services, 13; (j) orthodontia, 151.3.3All other specialized health careSpecialist outpatient care\u2013somaticUpdating of classification systems for payment purposes (4)21, Annex 2; itemized by service; 2 April 1993, 27 August 2004.List contains 3,166 (and together with the dispensary specific services 3,204) items (based on the 1978 ICPM of WHO)Specialist outpatient care\u2013mental; special area: psychoanalysis, -therapy21, Annex 15; itemized by service; 11 May 2004.Altogether 120 items (82 items overlap with Annex 2) in five main groups: (a) skin and STD, 29 items; (b) oncology, 26 items; (c) pulmonology, 25 items; (d) psychiatry, 19 items; (e) addictology, 21 items1.3.9All other outpatient curative careOutpatient care by other professionalsUpdating of payment classification systems (4)As with 1.3.3, which are included (e.g., logopedia, physiotherapy, dietetics, optometry, acupuncture); the rest are implicitly excluded (e.g., chiropody, bioenergy treatments, iris diagnostics, hydrotherapy)Alternative and complementary medicineSpecial area: balneotherapy and physiotherapyTraditional (4)11 (definition of entitlements, regulation of prescription, utilization, and professional requirements); itemized by service; indications and contraindications; 9 November 2004.Annex 1, balneotherapy services, 10 items; Annex 4, physiotherapy services, 13 items; Annexes 5 and 6, indications and contraindicationsPrice negotiations (4)18; itemized by service; 19 September 2003.3 groups with the same 10 items; copayment is different for spas, which have national, regional, and local qualification1.4Services of curative home careTraditional (4)23, Annex 1 (specifies professional requirements); itemized by service; 26 July 1996, 9 June 1999.13 service categories, 22 items (includes hospice at home)a Benefit catalogues with the traditional regulatory regime are updated on an ad hoc basis, while the updating of payment lists is regular. All benefit catalogues are issued as ministerial decrees.\nThe general framework for these services is set by the 1997 Acts CLIV and LXXXIII, as discussed above, but all health services in this category are considered within the frame of the social insurance scheme. Although the 1997 Act LXXXIII states that all professionally justified services are included, in most cases the detailed payment catalogues imply implicit exclusions, such as most services of alternative medicine. With the exception of family physician services (primary care), there are benefit catalogues for each service category, either as a list of cases\/services for payment purposes (1.1, 1.2, partly 1.3.2, 1.3.3, and partly 1.3.9) or for further specification of broad functional categories enumerated in the Act (partly 1.3.2, partly balneotherapy) or for the specification of professional requirements (1.4, partly balneotherapy).\nThese nationally valid benefit catalogues are the output of either the traditional decision-making process or the process of updating classification systems for payment purposes. They are issued as a ministerial decree in both cases, with the MOH being the final decision maker. Updating is ad hoc in the case of the former and regular in the case of the latter, where the preparatory phase is much more formalized and even a few decision-making criteria are defined (budget and public health impact), as discussed above.\nUndoubtedly the two most detailed benefit catalogues are the lists of DRGs for inpatient curative care (1.1), and the so-called WHO point, or German point system for outpatient specialist care (1.3.3, partly 1.3.9). The essence of the DRG based hospital payment is that it pays a standard fee for discharged acute hospital cases and not for any individual service items such as laboratory tests, hospital days, drugs, and procedures. The DRG system classifies cases into a manageable number of categories, which are more or less medically meaningful and in which resource use is the same or at least similar (homogeneous resource use) [3]. The DRG system was introduced countrywide in Hungary in 1993 after a pilot project that began in 1987. The DRG system in the United States was adapted to the local situation using the cost data collected [4]. The current version of the Hungarian adaptation of DRGs (homogeneous disease groups, HDGs) is 5.0, which contains 786 groups in 26 main diagnostic categories [Decree No. 9\/1993 (IV. 2) NM of the Minister of Welfare of the Social Insurance Financing of Specialist Services]. The first step of classifying patients is to determine the major diagnostic category, in which the main diagnosis and\/or the interventions can be a principal classification criterion, which is to be further modified by comorbidity and age. For instance there are three groups of classifying factors in the major diagnostic category 14 (pregnancy, birth and childbed): the type of pregnancy (without complications, pathological pregnancy), the type of delivery (cesarean, vaginal, vaginal with operation, vaginal with epidural anesthesia) and the conditions of delivery (high risk, other comorbidity; Fig.\u00a03). While in principle inpatient cases are sorted into HDGs primarily on the basis of diagnosis, which leaves hospital physicians to select treatment options freely, several treatment modalities are costly enough to be a principal classification criterion.Fig.\u00a03 Examples of the homogeneous disease groups in Hungary. [Decree No. 9\/1993. (IV.\u00a02) NM of the Minister of Welfare on the Social Insurance Financing of Specialist Services]\nThe WHO point system, however, is entirely itemized by service. It is based on the 1978 ICPM, but the original list has been modified frequently ever since it has been introduced as the basis of payment in the outpatient specialist setting. Along with the chronic outpatient care items, the current catalogue contains 3,204 service items and has been in effect since 27 August 2004. Each service has a five-digit identification code and the services are listed in numerical order, from item 110011 (first aid) to 97550 (ambulatory developmental-neurology follow-up care of infants with spina bifida) and the list closes with 12 items of \u201ccomplementary\u201d points for special transfusion (two items) for noncompliant patients who threaten or attack attending medical staff (one item), for supervision of patients after treatment (five items), for drug loading (one item), and for patients of young age (three items). The list in the ministerial decree is not structured in groups or subgroups, but it is generally service (e.g., ultrasound examinations) and organ-oriented [e.g., eye-examinations; Decree No. 9\/1993 (IV. 2) NM of the Minister of Welfare of the Social Insurance Financing of Specialist Services].\nDiscussion\nThe provision of universal and comprehensive coverage was the founding principle of the previous, state-socialist health care system. The tension between the changing and increasing needs and the available resources created shortages which have not been acknowledged and explicitly dealt with. Rationing effectively occurred through queuing, implicit waiting lists, the dilution of services, and informal payments [1].\nThe social insurance and payment reforms have brought about little change in this respect. Successive governments have faced the chronic deficit of the HIF, but measures to balance the budget have almost exclusively targeted the revenue side of the system, and only minor modifications have been implemented in the almost comprehensive benefit package [1]. Although entitlements are in principle linked to paying the contribution, the coverage is universal in practice since entitlement is not checked by the providers [5]. Despite payment catalogues the benefit package is defined rather negatively. While services financed by the HIF should be provided according to treatment protocols issued by the MOH, no such protocols have yet come into effect.\nThe general opinion in Hungary is that more explicit priority setting with more exclusions or more significant copayments would not be accepted by the majority of the population, and therefore politicians are reluctant to touch the issue of priority setting in a more systematic manner. On the other hand, the financial pressure on the system is high, and indirect, implicit rationing does occur, for instance, through informal payments. It is a question of how long this schism can be uphold, especially in the light of the challenges by joining the EU.\nThe plan of the current government is to revise entitlements to health care by expanding the scope of services to all emergency care for which all citizens are eligible [6]. The rest of the health services will be provided on the basis of participation in the social insurance scheme, but it will be checked whether the patient is in fact entitled to services. Furthermore, the government plans to revitalize the system of treatment protocols, which is obviously a means to exclude certain interventions. It is not yet known whether this is only a first step towards a more explicit priority setting, or whether the benefit package remains essentially unchanged.","keyphrases":["hungary","health services","health benefit plans","health priorities","national health programs"],"prmu":["P","P","R","R","M"]}
{"id":"Neuroimage-2-1-2225446","title":"Interpreting scan data acquired from multiple scanners: A study with Alzheimer's disease\n","text":"Large, multi-site studies utilizing MRI-derived measures from multiple scanners present an opportunity to advance research by pooling data. On the other hand, it remains unclear whether or not the potential confound introduced by different scanners and upgrades will devalue the integrity of any results. Although there are studies of scanner differences for the purpose of calibration and quality control, the current literature is devoid of studies that describe the analysis of multi-scanner data with regard to the interaction of scanner(s) with effects of interest. We investigated a data-set of 136 subjects, 62 patients with mild to moderate Alzheimer's disease and 74 cognitively normal elderly controls, with MRI scans from one center that were acquired over 10 years with 6 different scanners and multiple upgrades over time. We used a whole-brain voxel-wise analysis to evaluate the effect of scanner, effect of disease, and the interaction of scanner and disease for the 6 different scanners. The effect of disease in patients showed the expected significant reduction of grey matter in the medial temporal lobe. Scanner differences were substantially less than the group differences and only significant in the thalamus. There was no significant interaction of scanner with disease group. We describe the rationale for concluding that our results were not confounded by scanner differences. Similar analyses in other multi-scanner data-sets could be used to justify the pooling of data when needed, such as in studies of rare disorders or in multi-center designs.\nIntroduction\nTechniques utilizing in vivo MRI-derived measures of brain tissue morphometrics show increasing promise for aiding clinicians in assessment and diagnosis of disease. Studies that use longitudinal and\/or multi-site data-sets, such as the Alzheimer's Disease Neuroimaging Initiative (Mueller et al., 2005), have the potential to provide a wealth of information. The large numbers of subjects resulting from pooling multi-scanner data-sets has numerous advantages. It increases sensitivity thus allowing detection of subtle effects and enables the analysis of subgroups within a cohort. Additionally, pooling offers increased reliability and confidence about the size of effect by averaging out unforeseen confounds and hence a method for carrying out meta-analyses or analyses of rare subjects with orphan-diseases scanned at home rather than in distant centers. Any one study may have unforeseen bias which is lessened by pooling. However, one important confound of combining images gathered from different scanners is the potential for scanner effects to introduce systematic error, thus making the interpretation of results difficult. Partial volume effects and image intensity inhomogeneity can introduce error into automatic segmentation with any given scanner (Li et al., 2005). Noise of the electronics of the MRI system, subject-specific physiological noise and imaging gradient non-linearities also contribute to image intensity variability (Jovicich et al., 2006; Littmann et al., 2006). Furthermore, differences in subject positioning between sites add to the variability in distortion fields (Jovicich et al., 2006) and images can vary as a function of protocol differences between a baseline and a later scan or with drifts in instrument signal to noise over time (Preboske et al., 2006). The interaction of scanner differences with segmentation remains a particular concern and potential cause of varied measures of regional tissue volume in the brain (Ashburner and Friston, 2000). It is therefore vital to confirm that there is no important interaction between scanner and effect of interest and\/or account for the effects of different scanners in a principled manner before pooling from different scanners can be recommended as a routine.\nThe use of phantoms to calibrate different scanners and account for scanner drift or upgrades is well established (Tofts, 1998; Van Haren et al., 2003; Schnack et al., 2004; Jovicich et al., 2006) and a useful way to guarantee quality. Studies using a small number of healthy subjects repeatedly scanned on different scanners or after upgrades are also recommended to test reliability (Tofts, 1998; Han et al., 2006; Jovicich et al., 2006). However, it is not clear that the extra calibration efforts can actually correct for the majority of these sources of variability (Jovicich et al., 2006), or are even necessary. Also, it has yet to be established how confidently the results from multi-scanner data-sets can be interpreted irrespective of up-front calibration.\nMulti-scanner studies, though not common, are increasing. A longitudinal aging study by Raz and colleagues, one of the few which discussed the analysis of images derived from multiple scanners, concluded that using different scanners in 4 subjects did not affect measured intracranial volume with a manual tracing method (Raz et al., 2005). Likewise, manual hippocampal measurements performed on both 1.5\u00a0T and 3.0\u00a0T scanners in 8 healthy controls were not affected by field strength (Breillmann et al., 2001). We are not aware of any study that has described an automated analysis of a large, multi-scanner data-set with the aim of assessing whether scanner-associated biases are significant.\nWe set out to investigate scanner effects in an automatically preprocessed data-set of subjects with mild to moderate AD and cognitively normal elderly people whose MRI scans had been collected over a period of 10\u00a0years with multiple scanners and upgrades on a consistent platform and repeated calibrations. Even though the concern has been raised that segmentation errors are a particular problem in atrophic brains (Good et al., 2002), we did not find this to be an issue in our sample, possibly because the disease was early. We hypothesized that the changes due to AD pathology are large compared to scanner induced distortions and that there would be no interaction of scanner with case and\/or control groups. In this paper, we report the results in our data-set and outline a method by which multi-scanner data-sets might be pooled with confidence.\nMethods and results\nOur data-set included a total of 6 scanners and 136 subjects scanned over a 10-year period. None of the subjects were scanned more than once. All scans were done on the same platform, General Electric Signa 1.5\u00a0T scanners (slice thickness 1.6, matrix dimensions 256\u00a0\u00d7\u00a0192). There were minor variations in the TR, TE, and flip angle (see Table 1). Scanners also underwent upgrades over time. Importantly, all scanners were monitored with daily phantom quality checks which calibrated the gradients to within \u00b1\u00a01\u00a0mm over a 200-mm volume centered at iso-center, monitored signal to noise and radio frequency (RF) transmit gain. The major hardware elements (body resonance module gradient coil and birdcage head transmit\u2013receive volume coil) were unchanged throughout time and across scanners, except that for the oldest scanner (scanner 2), the wiring in the resistive shim set was not cooled to super conducting temperatures, whereas for the other 5 scanners, the shim coils were inside the dewar and were cooled to the superconducting range.\nWe used voxel-based morphometry (VBM) to evaluate the interaction of scanner and grey matter segmented modulated images for the 6 different scanners. VBM has the advantage of assessing the whole brain and not being biased to one particular region or structure. It entails a voxel-wise comparison of local volume of grey matter between groups after the images are spatially normalized into the same space, segmented, modulated, and smoothed. Voxel-wise statistical parametric maps result from statistically thresholded contrasts after corrections for multiple comparisons (Ashburner and Friston, 2000) using false discovery rate (FDR) (Benjamini and Hochberg, 1995).\nImages were visually inspected for artifacts or structural abnormalities unrelated to AD. They were firstly segmented into white (WM) and grey matter (GM) using SPM5 (Wellcome Trust Centre for Neuroimaging, Institute of Neurology, UCL, London UK \u2013 http:\/\/www.fil.ion.ucl.ac.uk\/spm). Then, WM and GM segments were further normalized to a population template generated from the complete image set using a diffeomorphic registration algorithm. This non-linear warping technique minimizes structural variation between subjects (Ashburner, 2007). For comparison, we also repeated the analysis using the more widely used standard SPM5 segmentation code (Ashburner and Friston, 2005) instead of the diffeomorphic registration algorithm. Resolution before normalization was \u2212\u00a0.9, .9, 1.6 and after normalization was \u2212\u00a01.5, 1.5, 1.5 for the diffeomorphic registration algorithm and \u2212\u00a02.0, 2.0, 2.0 with the standard SPM5 procedure. A separate \u2018modulation\u2019 step (Ashburner and Friston, 2000) was used to ensure that the overall amount of each tissue class was not altered by the spatial normalization procedure. Modulation was performed by multiplying the warped tissue probability maps by the Jacobian determinant of the warp on a voxel-by-voxel basis, which represents the relative volume ratio before and after warping, thus allowing voxel intensities in the segmented grey matter map, together with the size of the voxels, to reflect regional volume and preserve total grey matter volume from before the warp. Modulated grey matter scans were smoothed using a 6-mm full-width at half-maximum Gaussian kernel.\nThe smoothed grey matter images were analyzed in a factorial design, with the 6 different scanners as one factor (SCANNER) with 6 levels and the presence of AD (GROUP) as the second factor with two levels (present and absent). Age, gender, and total intracranial volume were entered as covariates (Fig. 1). We performed F-tests correcting for multiple comparisons across the brain (FDR correction). The degrees of freedom was 121 for all comparisons. The effect of group revealed strong effects (p\u00a0<\u00a00.001) in the left medial temporal lobe (\u2212\u00a029, \u2212\u00a024, \u2212\u00a09 [x, y, z]; F\u00a0=\u00a0100.34) and right medial temporal lobe (30, \u2212\u00a027, \u2212\u00a09 [x, y, z]; F\u00a0=\u00a091.44) (Fig. 2). The effect of scanner showed significant differences (p\u00a0<\u00a00.05) in the right (9, \u2212\u00a030, 0 [x, y, z]; F\u00a0=\u00a011.93) and left (\u2212\u00a09, \u2212\u00a011, 8 [x, y, z]; F\u00a0=\u00a09.69) thalami (Fig. 3), but this effect was less than the effect of group. T-tests contrasting each scanner against the others revealed the scanner effect in the thalamus was mostly due to the oldest scanner (scanner 2). One newer scanner (scanner 6) revealed an effect that did not survive FDR correction in the right thalamus (8, \u2212\u00a05, \u2212\u00a03 [x, y, z]; T\u00a0=\u00a04.03), whereas the oldest scanner contrasted with the others revealed a significant effect (p\u00a0<\u00a00.05) in the left thalamus (\u2212\u00a09, \u2212\u00a011, 6 [x, y, z]; T\u00a0=\u00a05.94). Despite the effect of scanner, there was no significant interaction of scanner with group, the highest Z-score being 3.82 with a corrected p value of 0.942. We performed F-tests for each possible combination of scanners and found no significant interaction with any of these groupings. When using the standard VBM procedure implemented in SPM5 for normalization and segmentation, the results of the various contrasts followed the same patterns as when using the diffeomorphic normalization\/segmentation procedure. We failed to find an interaction of scanner with group; the highest Z-score was 3.78 with an FDR corrected p value of 0.954. The effect of scanner showed significant differences (p\u00a0<\u00a00.05) in the left thalamus (\u2212\u00a010, \u2212\u00a010, 6 [x, y, z]; F\u00a0=\u00a09.49) and the right thalamus (8, \u2212\u00a028, \u2212\u00a04 [x, y, z]; F\u00a0=\u00a09.41). The effect of group also showed strong effects (p\u00a0<\u00a00.001) in the left medial temporal lobe (\u2212\u00a026, \u2212\u00a014, \u2212\u00a020 [x, y, z]; F\u00a0=\u00a0101.46) and right medial temporal lobe (26, \u2212\u00a08, \u2212\u00a018 [x, y, z]; F\u00a0=\u00a077.98).\nImportantly, we found no significant scanner effects in the medial temporal lobe cluster (\u2212\u00a029,\u2212\u00a024, \u2212\u00a09 [x, y, z]; F\u00a0=\u00a00.01) and the disease effect size in the thalamus was minimal compared to the effect size in the medial temporal lobe (9, \u2212\u00a030, 0 [x, y, z]; F\u00a0=\u00a01.06), suggesting minimal scanner effects in the areas that are most affected by AD and minimal disease effects in the areas showing scanner differences. Confidence intervals, which are reflective of the standard deviations, for the contrast estimates are shown in Fig. 4. At the voxel of greatest effect of group, the confidence interval is small relative to the effect size for the main effect of group. The opposite is true for the effect of scanner at the area of greatest disease, which is further evidence of lack of effect. For the main effect of scanner, confidence intervals are similar between the different scanner contrasts (see Fig. 4A), indirectly suggestive of relatively little variance across scanners.\nBecause there were 4 software upgrades, we also analyzed the interaction of software version and disease. Using the same basic design matrix as described for the interaction with scanners, this time the contrasts included cases and controls from each software version, covaried with age, gender and intracranial volume. As with the effect of scanner, there was no significant interaction of software version with group, the highest Z-score being 3.65 with a corrected p value of 0.880.\nDiscussion\nIn our data-set, we found the effect of disease to be substantially larger than the effect of scanner and failed to find a significant interaction of disease with scanner or software upgrades. In general, the effect of disease in AD is liable to be larger than the effect of scanner, which is supported by our result of no important interaction between scanner and effect of interest. Ideally, further studies with an even larger data set to better calculate the effect sizes and quantify the distance between the scanner effect cluster and group effect cluster could be done to validate our findings. However, comparison of the magnitude of the scanner effect versus disease effect in the medial temporal lobe cluster in our data-set demonstrates that scanner differences had minimal effects in the areas that are important in the study of AD. Furthermore, even though there are likely to be some differences among data from different scanners, our experiments were explicitly designed to detect scanner-related differences. By modeling appropriate confounds in the design matrix, it appears possible to remove these small effects. We were able to model this interaction easily because relatively homogeneous cases and controls were scanned in each machine. We are unable to say whether lesser or more subtle and distributed changes would be as resistant to scanner effects.\nThough we did not detect a significant interaction of scanner with disease, we cannot be absolutely certain it is indeed due to the absence of such effect. The lack of significance may be a reflection of the lack of statistical power, e.g., insufficient number of scans. Other causes such as a high average residual variance or residual variance inhomogeneities could also under-power the detection of the effects. However, the variance inhomogeneity was considered in our analysis by assuming unequal variance for the different levels of each of the two factors in our full factorial design (the two factors are the scanner and group). In a post-hoc manner, we explored the residual variance across scanners to assess whether that explained the lack of sensitivity in the results. In the area of greatest disease effect, the variance was low and did not reflect significant inhomogeneity, but in the area of greatest scanner effect there was lower average residual variance and more variability. Though our tests for variance inhomogeneity across the 6 scanners was not voxel-by-voxel over the whole brain volume, our findings seem to support that the variance inhomogeneity is location-dependent and should be accounted for when analyzing data acquired from different scanners as we attempted to do in our analysis.\nThe greatest effect of scanner was in the thalamus. The effect of the thalamus was largely driven by the scanner with the resistive shim set that was not cooled to superconducting temperatures, which suggests an impact of such hardware differences on thalamic segmentation. The composition of the thalamus is an issue of debate as it is not completely grey matter receiving numerous white matter tracts from other parts of the brain. Additionally, the grey matter intensity value of the thalamus is different from that of cortical grey matter. The intrinsically poor intensity contrast in the thalamus renders it susceptible to small differences in image contrast due to scanner differences. There is also less variability in this part of the brain, so tests will be more sensitive to such differences. These factors may contribute to the difficulty of accurate segmentation of the thalamus in addition to scanner effects.\nThe relatively small effect of scanner is potentially attributable to quality control measures during data acquisition and\/or the robustness of the segmentation method. The SPM5 algorithm, by using spatial information together with intensity information, should be more robust to such differences than a segmentation algorithm that is purely intensity-based. However, this study did not directly investigate which forms of preprocessing are most affected by scanner parameters. It is important to note, though, that the effect was not due to the diffeomorphic normalization procedure, since we obtained similar patterns to those with the standard SPM5 normalization. Presumably, the use of daily scanner calibrations allows for a relatively constant intensity contrast between grey and white matter voxels between scans. Routine procedures designed to minimize image intensity differences over time are useful, particularly since it has been demonstrated that several common scan functions can potentially introduce measurement errors as high as 100% (i.e., much greater than the disease effect of AD), without appropriate quality control measures in place (Preboske et al., 2006). Even though it is common experience that current generation scanners are remarkably stable over time, they do still drift in a way that is correctable with judicious calibration. Though we collected no data to determine the frequency and level of sophistication necessary, basic calibration akin to the current standard in most major centers is likely sufficient. Furthermore, while it can be useful to collect data on inter-subject variability by scanning the same subjects in different scanners, such efforts would not directly answer the question of whether data from different subjects on different scanners can be pooled.\nThe unified segmentation method of SPM5 (Ashburner and Friston, 2005) produces an estimate of the tissue class intensities from a fitting of spatial priors to the image, which allows for differences in image intensities between scans. Additionally, any image intensity differences due to subject to subject variation in coupling of the RF coil to the head that may be introduced by different head shapes and sizes are likely to be well-accounted for by bias corrections in the unified segmentation algorithm.\nAs long as provision for different scanners and\/or upgrades is made within an analysis, the effect of scanner regardless of magnitude is not likely to devalue the integrity of results. However, if there is a true physical interaction of the biological effect of interest with the method of measurement, perfect calibration or even using a single scanner would not prevent bias. On the other hand, any unusually large effect from one scanner would be attenuated by the totality of scans from different scanners that make up the template, which averages the different scanner effects for normalization.\nTo date, methodological differences between individual studies have prevented comprehensive pooling of data in meta-analyses of AD MRI studies (Zakzanis et al., 2003; Whitwell and Jack, 2005; Wahlund et al., 2005). To the extent that data pooling was possible, the meta-analysis compared regional volume between disease groups (Zakzanis et al., 2003). Although there may be concern about error introduced by the variability inherent in preprocessing many individuals across studies, automated VBM methods successfully overcome the problems to combine information from multiple scans and studies. VBM meta-analyses of pooled scans permit the usual range of analyses beyond simple categorical comparisons (e.g., regression, age interaction, subgroup, nonlinear, etc.).\nOur results suggest that for Alzheimer's disease, particularly if the imaging platform remains constant, variations attributable to individual scanners and upgrades may have negligible effects on segmented grey matter images. We expect, though do not yet have the data, that the approach is robust enough to accommodate using both 1.5\u00a0T and 3\u00a0T scans in the same meta-analysis. A principled approach to test the validity of a meta-analysis is to carry out an a priori interaction analysis of scanner by biological effect to be studied to exclude scanner-specific compromising effects. Additional comfort can be gained by finding that the biological effect is of greater magnitude than any effect of scanner. An alternative approach is to model each scanner's effects separately, thereby permitting a principled meta-analysis on pooled data. That data can be pooled from different scanners without corroding the integrity of results is reassuring for large multi-site studies.","keyphrases":["alzheimer's disease","multi-scanner","magnetic resonance imaging (mri)","voxel based morphometry"],"prmu":["P","P","M","M"]}
{"id":"J_Urban_Health-2-2-1705474","title":"An Analysis of Respondent Driven Sampling with Injection Drug Users (IDU) in Albania and the Russian Federation\n","text":"Injection drug users in Tirana, Albania and St. Petersburg, Russia were recruited into a study assessing HIV-related behaviors and HIV serostatus using Respondent Driven Sampling (RDS), a peer-driven recruitment sampling strategy that results in a probability sample. (Salganik M, Heckathorn DD. Sampling and estimation in hidden populations using respondent-driven sampling. Sociol Method. 2004;34:193\u2013239). This paper presents a comparison of RDS implementation, findings on network and recruitment characteristics, and lessons learned. Initiated with 13 to 15 seeds, approximately 200 IDUs were recruited within 8 weeks. Information resulting from RDS indicates that social network patterns from the two studies differ greatly. Female IDUs in Tirana had smaller network sizes than male IDUs, unlike in St. Petersburg where female IDUs had larger network sizes than male IDUs. Recruitment patterns in each country also differed by demographic categories. Recruitment analyses indicate that IDUs form socially distinct groups by sex in Tirana, whereas there was a greater degree of gender mixing patterns in St. Petersburg. RDS proved to be an effective means of surveying these hard-to-reach populations.\nIntroduction\nAccessing hard-to-reach populations for HIV prevention and research activities has historically been challenging. Certain high-risk populations are fundamental in the proliferation of HIV, particularly in the early stages of a country\u2019s epidemic. Owing to the stigmatized and often criminalized behaviors of injection drug users, for example, these populations avoid open contact with the rest of civil society. Respondent Driven Sampling (RDS) offers a solution to that dilemma, allowing researchers to access hard-to-reach populations through their social networks.2,3 A social network person includes other persons with whom he\/she has a social relationship and who tend to engage in the same behaviors as the index person. This paper presents approaches used to implement RDS in Tirana, Albania and St. Petersburg, Russia and lessons learned in these two cities.\nMaterials and Methods\nThe studies in St. Petersburg and Tirana utilized RDS to recruit IDUs into an HIV-related behavioral survey. While the methodology behind RDS has been described elsewhere,3 in brief it is a referral-based system whereby participants refer a limited number of friends or acquaintances (who are also members of the target group) to participate. Study participants receive an incentive both for participating and successfully recruiting others. Data are analyzed using the recruiting linkages and yield a probability sample of the population in the target area.\nFor inclusion in either study, participants had to be at least 15\u00a0years old and report having injected drugs recreationally in the past 6\u00a0months. Data collection for both surveys included face-to-face interviews and encompassed a 7\u20138\u00a0week period at three sites in each city. In Tirana, data collection took place in offices of two non-governmental organizations (NGOs), conducting outreach to IDU and in one treatment hospital. Data collection in St. Petersburg was conducted in three sites in various parts of the city. One was a fixed site in the center of the city. The other two sites were located just outside the city center and open on weekend afternoons, operating from mobile vans at stable locations. One site was selected due to its proximity to an illegal drug market.\nThe data collection team at each site and shift consisted of two interviewers and one person serving as coupon manager, supervised by a study coordinator. The coupon manager recorded data for each participant on a form that included each individual\u2019s self-reported network size, the coupon number brought to the interview, and the coupon numbers each was given for distribution. The Tirana sites also had a physician, laboratory technician and counselor as data collection included biological samples for HIV and other STIs.\nQuestionnaire topics included sociodemographic indicators, sexual and drug injection risk behaviors, knowledge of HIV and STIs, and previous HIV testing. Survey duration in Tirana was approximately 1.5\u00a0hour for both the behavioral and biological portions of the study, while in St. Petersburg data collection lasted approximately 45\u00a0minute. In accordance with RDS methodology, the initial participants of the study, known as \u201cseeds\u201d,4 were members of the target IDU population in their respective cities, and were selected to begin recruiting their peers into the study. In Tirana, 15 seeds launched the study, while in Russia recruitment was initiated with 11 seeds; 2 seeds were added later to generate more recruiting. The seeds selected in both cities were referred from local NGOs conducting outreach with the IDU population, as well as personal IDU contacts of study staff. Each participant in St. Petersburg received three coupons with an eight-week expiration date to recruit their peers while two coupons per participant were used in Tirana (no expiration dates). In St. Petersburg, three coupons continued to be distributed until approximately 200 participants were attained, after which participants were warned that the study would be ending within a few days. In Tirana, coupons were distributed until the desired sample size of 200 was reached; however, participants were warned from the beginning that once this was reached, no more coupons would be honored.\nIn Tirana, participants received cash incentives of 10\u00a0euros for participating and an additional 5\u00a0euros for each successful recruit. In St. Petersburg, use of monetary incentives was discouraged by all local stakeholders; therefore, participants were given a gift package consisting of a box of chocolates and personal hygiene products for completing the survey, and a box of chocolates or a can of coffee for each successful recruit. The gifts were equivalent to U.S. $10 for participating and U.S. $5 for recruiting.\nTwo hundred ten IDUs (not including seeds) were surveyed between July and August 2005 in Tirana, and 200 IDUs (not including seeds) were surveyed in St. Petersburg during the same time period.\nBiometric measures were taken in Tirana to discourage study members from participating in multiple sites. Measurements consisting of wrist circumference multiplied by forearm length were taken and recorded. Participants were told that this was a unique measurement that could be used to determine if they attempted to return or access other data collection sites. Project staff also rotated between sites to decrease the likelihood of participants visiting multiple sites. In St. Petersburg, biometric measurements were not taken since local researchers thought it would induce suspicion among IDUs and consequently discourage participation. Recognition of potential duplicate participants depended upon study staff, who were outreach workers familiar with the target population and operated out of the same site for the entire 8\u00a0weeks of data collection.\nThe data were analyzed using RDS Analysis Tool version 5.4 (RDSAT). RDS population estimates and confidence intervals as calculated by RDSAT are reported. The homophily index describes the extent of in-group ties. A homophily index of H\u2009=\u2009 1.0 reflects perfect homophily, indicating that ties are formed with other members of the same group. In contrast, H\u2009=\u2009 \u22121.0 reflects perfect heterophily, indicating that ties are formed completely outside of the group.3 Both studies were approved by the FHI Protection of Human Subjects Committee and the Tirana study was also approved by the Medical Ethical Committee of Albania.\nIn both cities, information about whether anyone refused to accept coupons was elicited from those who returned to collect their compensation for successful recruiting. Approximately 20% (Tirana) and 5% (St. Petersburg) of those approached to participate refused to accept the coupon from participants. Of a total of 450 coupons distributed in Tirana, 210 were returned. In St. Petersburg, 200 out of a total of 629 coupons distributed were returned. Furthermore, in Tirana, only two potential participants were found to be ineligible for participation and once consent had been given, no participants withdrew from the process. This information is not yet available for St. Petersburg.\nResults\nDemographic Characteristics\nTable\u00a01 describes the characteristics of the study samples in Tirana and St. Petersburg. The sample in Tirana consists predominately of male IDUs, with only 14 female IDUs recruited for the survey. This contrasts substantially from the sample in St. Petersburg, where more females were represented compared to Tirana, with 36% being female and 64% being male.\nTable\u00a01Demographic information\u00a0AlbaniaRussiaSex\u00a0Males91% (86, 96)64% (55, 73)\u00a0Females9% (4, 14)36% (27, 45)Education completed\u00a0Did not attend school9% (5, 16)0%\u00a0Primary17% (11, 26)0%\u00a0Secondary\/vocational67% (58, 76)84% (77, 92)\u00a0University7% (3, 8)16% (8, 23)Age\u00a015\u201322\u00a0years38% (33, 48)11% (7, 15)\u00a023\u201327\u00a0years36% (27, 41)38% (29, 47)\u00a028\u00a0years or more26% (19, 32)51% (42, 62)Current co-habitation situation\u00a0Married living with spouse12% (6, 18)9% (4, 15)\u00a0Married living with other sex partner0%4% (2, 9)\u00a0Married not living with any partner3% (1, 6)4% (1, 7)\u00a0Not married, living with sex partner20% (14, 26)33% (25, 42)\u00a0Not married, not living with sex partner65% (58, 72)49% (39, 60)The numbers in parentheses represent the 95% confidence intervals as calculated by RDS.\nIn Tirana, the majority had completed at least middle school (eight classes), with a third of the sample completing middle school and a third completing high school (12 classes). Only 7% in Tirana had completed university. In St. Petersburg, the majority (84%) had completed either secondary or vocational education, and 16% had completed university. In Tirana, the sample was comprised of young IDUs with 38% between 15 and 22\u00a0years of age and 74% below 27\u00a0years of age. The St. Petersburg study included an older IDU population, with over half of participants 28\u00a0years of age or older and only 11% between 15 and 22\u00a0years of age. In St. Petersburg, males were slightly older than females; a much higher proportion of males were 28\u00a0years of age or older (66 vs 26% of females; data not shown).\nThe majorities of the samples are unmarried (85% in Tirana and 82% in St. Petersburg). In Tirana, the majority also did not live with a sex partner (65%), while another 20% were unmarried but living with a sexual partner. A higher proportion of IDUs in Russia reported being unmarried but living with a sex partner (33%).\nNetwork Sizes\nAs a part of RDS methodology, respondents were asked about the size of their personal networks. Specifically, participants were asked how many people they know personally who used injecting drugs (i.e., \u201cyou know who they are, they know who you are, you have seen them in the last six months\u201d).\nIn Tirana, male IDUs reported a larger average personal network size than female IDUs. In contrast, in St. Petersburg, females had a larger average personal network size than males (20 vs 17, respectively).\nThe pattern of network size by age is again very different between IDUs in Tirana and St. Petersburg. In Tirana, network size increases with age (from 16 among the youngest age group to 21 in the oldest age group), whereas in St. Petersburg, network size decreases with age (from 25 in the youngest age group to 15 in the oldest age group). The same trend and differences between the two cities is observed with regard to education, which is likely due to the collinearity between age and education.\nSimilarly, in Tirana, there was a trend of increasing network size with increasing duration of injection drug use. However, in St. Petersburg, the network size was larger for recent initiates and long-term injectors compared to those who injected for 1\u20135\u00a0years (Table\u00a02).\nTable\u00a02History of injection drug useDuration of injection drug useAlbaniaRussia<1\u00a0year21% (11, 24)6% (3, 9)1\u20135\u00a0years72% (69, 83)43% (31, 51)>5\u00a0years6% (3, 11)52% (43, 63)\nRecruiting\nTable\u00a03 represents the recruitment patterns of IDUs in Tirana and St. Petersburg with respect to sex, age and length of injection. RDS provides the opportunity to learn about the degree to which people tend to affiliate with others sharing their same characteristics. In Tirana, the gender affiliation patterns demonstrate a strong heterophily in women, whereby women recruited only men (H\u2009=\u2009 \u22121.00), while men recruited women 7% of the time (H\u2009=\u2009 0.16; this is probably due to the small number of women in the Tirana sample, with only 14 women participating). From a possible 28 coupons given to these women, 16 coupons were returned (57%). In contrast, the gender affiliation patterns of IDUs in St. Petersburg reflects neither strong homophily nor heterophily. Both males and females recruited males about the same percentage of times (63 and 57%, respectively). The heterophily index in males was \u22120.02 and homophily index was 0.12 for females.\nTable\u00a03.Recruitment patterns by age, sex, and duration of drug injection in Tirana and St. PetersburgRecruitersRecruiteesTiranaSt. PetersburgYoungest (15\u201322\u00a0years)Middle (23\u201327\u00a0years)Oldest (\u2265 28\u00a0years)Youngest (15\u201322\u00a0years)Middle (23\u201327\u00a0years)Oldest (\u2265 28\u00a0years)Age\u00a0Youngest (15\u201322\u00a0years)42%35%23%10%54%36%\u00a0Middle (23\u201327\u00a0years)33%38%29%19%36%45%\u00a0Oldest (\u226528\u00a0years)29%39%32%12%44%44%SexMaleFemaleMaleFemale\u00a0Male93%7%63%37%\u00a0Female100%0%57%43%Duration of drug injectionNew (<1\u00a0year)Mid-term (1\u20135\u00a0years)Long-term (>5\u00a0years)New (<1\u00a0year)Mid-term (1\u20135\u00a0years)Long-term (>5\u00a0years)\u00a0New (<1\u00a0year)20%64%16%0%43%57%\u00a0Mid-term (1\u20135\u00a0years)8%57%35%7%40%54%\u00a0Long-term(>5\u00a0years)4%65%32%6%40%54%\nThe affiliation patterns of age recruiting relations in IDUs in Tirana reflects neither homophily nor heterophily. The homophily indices were 0.02 for the younger (15\u201322\u00a0years), 0.05 for the medium age (23\u201327\u00a0years) categories, and 0.09 for the older age group (\u226528\u00a0years). As can be seen, both youngest and the oldest age categories recruited the medium age group approximately one-third of the time. Further, both the youngest and the medium age groups equally recruited the oldest age group (approximately 23\u201329% of the time). Lastly, the medium and the oldest age groups recruited the youngest group with equal vigor at about one-third of the time.\nAmong IDUs in St. Petersburg, the affiliation patterns of age relations reflect neither homophily nor heterophily. The homophily indices were \u22120.03 for both the younger (15\u201322\u00a0years) and the medium age (23\u201327\u00a0years) categories, and \u22120.15 for the older age group (\u226528\u00a0years).\nIn Tirana, as with the relations by age, the affiliation patterns of relations based on duration of injection drug use reflects neither homophily nor heterophily. The homophily indices were 0.09 for new injectors (i.e., <1\u00a0year of injection experience), \u22120.10 for the mid-term users (1\u20135\u00a0years), and 0.09 for the longer-term injectors (>5\u00a0years). New users were recruited the least by the mid- and longer-term injectors (8 and 4%, respectively), and both the new and longer term injectors recruited mid-term injectors (64 and 65%, respectively).\nIn St. Petersburg, however, the affiliation patterns of relationships by duration of injection drug use differed between new injectors and longer-term injectors. The group of new injectors was completely heterophilous (H\u2009=\u2009 \u22121.0), reflecting a lack of in-group ties. However, both mid-term and longer-term injectors exhibited neither heterophily nor homophily (H\u2009=\u2009 \u22120.07 and H\u2009=\u2009 0.05, respectively).\nDiscussion\nThese two studies are among the first to report on the level of sexual and drug injection risk behaviors and knowledge about HIV prevention in a probability sample of IDUs in Tirana and St. Petersburg using RDS. The use of a systematic peer recruitment method afforded the possibility of capturing a wider range of IDUs than studies recruiting with outreach workers or from specific venues, such as STI clinics or needle exchange programs. Additionally, these two studies shed light on the social context by identifying social network relations among the IDUs.\nThe recruitment analysis suggests that IDUs in Tirana form socially distinct groups by sex. Female IDUs do not seem to interact with each other; no female IDU referred another woman. This suggests that intravenous drug use amongst females may be a highly stigmatized activity, or it may reflect their initiation into drug use by men.\nIn contrast, IDUs in St. Petersburg do not form socially distinct groups with regard to sex. The lack of strong ties within sex groups may be a reflection of injection behaviors in that they often inject in group settings which are likely to be of mixed sex and share needles with members of the opposite sex.\nIn Tirana, the lack of strong ties both within and between age groups most likely indicates that younger IDUs are probably not being initiated into injection drug use by injectors who are older in age. However, in St. Petersburg, there were no intra-group ties among younger females or among newer IDUs. All new IDUs appeared to be recruited only by long- and mid-term IDUs, and all younger female IDUs were primarily recruited by older male and female IDUs. This suggests that new IDUs, particularly female IDUs, have been initiated into injection drug use by older IDUs. The high prevalence of HIV and STIs5\u20137 in the IDU population in Russia places these new injectors at extremely high risk for HIV acquisition. The differences in average network sizes of male and female IDUs between Tirana and St. Petersburg may have implications for prevention programs. The larger network size of male IDUs compared to female IDUs in Tirana is not surprising given that the IDU population in Albania appears to be predominately male and clandestine for women. Therefore, it is apparently rare for female IDUs to know other female IDUs. In contrast, in St. Petersburg, female IDUs on average had a larger network size compared to male IDUs, which may be a reflection of the overlap of this population and the sex worker population. A sizable proportion are engaged in sex work so they likely know other sex workers from working in the same areas.\nThe younger age of IDUs in the Albanian sample indicates a potential for longer term drug use, allowing for greater transmission. Additionally, continued migration between borders with neighboring countries where prevalence amongst high-risk groups is higher (i.e., to Greece, Italy or Eastern European countries) may create a transmission\u2013migration effect.8\nThe male-to-female ratio of almost 2:1 in St. Petersburg is consistent with the decreasing trend in the male-to-female ratio of IDUs previously reported, from 7:1 in 1991 to 5:1 in 2001.9 However, it is also possible that previous studies may have underestimated the proportion of female IDUs. RDS may reach female IDUs better than other sampling methodologies. This could be evidence that RDS can capture a more diverse and possibly a more representative sample of IDUs in St. Petersburg.\nThe Tirana sample is proportionately younger than the St. Petersburg sample, perhaps describing an epidemic that is newer or reflecting the population structure in Albania where 40% is under 25\u00a0years of age.10 Records indicate an increase in substance abuse treatment since 2000 and the first HIV case was detected in Albania in 1993\u2014lending credence to the possibility of a younger epidemic.11\nThe co-habitation situation in Tirana differs from that in St. Petersburg, with a far greater proportion in St. Petersburg unmarried but living with a sex partner. It is possible that the more conservative nature of Albanian society and a holdover from the Albanian communist regime policies are reflected in this indicator.12 The finding in St. Petersburg that all new injectors were recruited only by long- and mid-term injectors is of concern. These social relations combined with the high prevalence of HIV in the IDU population in Russia place these new injectors at extremely high risk for HIV acquisition. This scenario is likely exacerbated by their large average network size (approximately 18).\nThe research conducted in Albania with IDUs is of vital importance. For the first time, policy makers and those involved with drug treatment and prevention of HIV and STIs have information to better plan and target interventions.\nThe pace of recruitment in both countries (approximately 8\u00a0weeks) seems to be fairly efficient. Differing numbers of coupons (two in Tirana and three in St. Petersburg) did not seem to affect overall recruitment with both studies reaching the desired sample size in approximately the same amount of time. In St. Petersburg, slow recruitment particularly at the fixed site at the city center was boosted by the addition of two seeds at week\u00a06; participation of only 50 IDUs at week\u00a06 increased to 200 by the eighth week. Over 200 IDUs were reached by the study in 7\u20138\u00a0weeks with 13\u201315 seeds in each city.\nThe body of literature regarding the use of RDS amongst hard-to-reach populations in developing countries is still in its nascence. Little is known about the effect of incentives on populations in developing countries or if random recruiting of peers (a fundamental assumption underlying the RDS methodology) is valid in countries where a large proportion of the population is poor. A greater body of research employing these methods in developing countries is needed.\nLessons learned from RDS methodology\nData collection during the summer was ideal in St. Petersburg given that some sites were mobile vans and participants had to wait outside. The season was also beneficial in Tirana, with data collection taking place before summer vacation. Given the large size of both cities, having three study sites in different parts of each city facilitated recruitment. Evening and Saturday hours were important to ensure that workers could participate. In St. Petersburg, upon recommendation from local NGOs, letters of support obtained from city police officials helped ensure that IDUs would not be harrassed at the study sites. In Tirana, biometrics were useful in creating a disincentive to participate multiple times. Involving NGO staff to recruit seeds and conduct data collection in the NGO offices enabled participants to feel comfortable about the process.\nLimitations\nPossible limitations to the study could have affected the results. In Tirana, the small numbers of women participating in the surveillance may indicate a strong desire to remain hidden, their limited numbers, or a reflection of poor recruiting. Because few women have been arrested or attended treatment facilities, there are no data regarding intravenous drug use amongst women in Tirana, or anywhere in Albania.11 In both cities, it is possible that RDS may have selectively attracted those IDUs desiring the incentives, which may have biased our sample. Formative research was conducted to assess if the amount would be coercive, so this is unlikely.\nThe major limitation of RDS is that it cannot control for non-response bias. Barriers to participation due to unknown characteristics of certain sub-groups or to factors beyond the control of the project could occur and are not measurable. Both studies attempted to determine the rate of non-response by asking participants returning to claim recruiting incentives about the number of attempts made before their coupons were accepted and the demographic characteristics of those who refused. They were also asked characteristics of those accepting the coupons. It is hoped that forthcoming analysis can assess if there is a difference between those who refuse, those who accept but do not show up, and those who accept and participate.\nThe coupon return rate in St. Petersburg was considerably lower than that of Tirana most likely due to the greater number of coupons given out by each participant in St. Petersburg (three coupons) than in Tirana (two coupons).\nConclusion\nBecause IDU participants were recruited through RDS, it is likely that the study sampled from a wide range of IDUs thereby increasing the sample representativeness in these two cities. Our study methodology likely drew a more diverse sample of IDUs compared to traditional recruitment by outreach workers or through needle exchange programs.\nThe studies in Tirana and St. Petersburg greatly enhance our understanding of IDUs and their network characteristics. They also provide evidence for the effectiveness of using RDS in recruiting hard-to-reach populations in environments where stigmatization and criminalization against their behaviors are commonplace.","keyphrases":["injection drug users","albania","respondent driven sampling."],"prmu":["P","P","R"]}
{"id":"Pediatr_Nephrol-3-1-1805049","title":"Transient severe metastatic calcification in acute renal failure\n","text":"Metastatic calcification, a known complication of prolonged end-stage renal disease, is herein described for the first time in a 10-month-old boy with acute renal failure, manifesting as a painful and swollen arm. Imaging revealed diffuse calcification and technetium-99 methylene diphosphonate (99Tc-MDP) uptake around the humerus and axilla. Calcium and vitamin D restriction, followed by intravenous administration of sodium thiosulfate caused a full symptomatic, radio- and scintigraphic improvement.\nIntroduction\nVascular (\u201cmetastatic\u201d) calcification (MC) (or calcific uremic arteriolopathy) is the most common type of extra-osseous calcification in end stage renal disease, manifesting as both medial and intimal calcification of large arteries [1]. It usually results from a deposition of calcified products in otherwise normal tissues as a result of hyperphosphatemia with or without hypercalcemia. It may affect the visceral organs, such as kidneys, lungs, and gastric mucosa, as well as joints, eyes, and skin [2]. The predisposing conditions of hyperphosphatemia and high (calcium\u00d7phosphorus) concentration products need to be persistent over weeks and months, since MC has been not been described in the setup of acute renal failure (even though a similar biochemical profile is seen there too).\nWe describe a case of MC in a 10-month-old child presenting with hemolytic uremic syndrome (HUS) and acute renal failure (ARF).\nCase report\nA 10-month-old male infant was recovering from his first episode of atypical HUS. He belonged to a Bedouin tribe previously diagnosed by us [3] with recessive factor H deficiency. His clinical course was remarkable for microangiopathic hemolytic anemia, oliguric renal failure and hypertension with a depressed myocardial function, associated with resistant hypocalcemia (total calcium as low as 5.4\u00a0mg\/dl) and hyperphosphatemia (as high as 13\u00a0mg\/dl). The latter was eventually controlled with phosphor binders (aluminum hydroxide at first for several days and, later, calcium gluconate), intravenous administration of calcium (mainly due to an episode of cardiac arrest without hyperkalemia, as previously described for HUS [4]), low phosphorus diet and alphacalcidol, 0.25\u00a0\u03bcg q.d. (the latter only after serum phosphorus had been normalized). Medications were given through a right femoral vein catheter and peripheral veins in the right arm. Peritoneal dialysis was started, using regular calcium (3.5\u00a0meq\/l) dialysate. Fresh frozen plasma (20\u00a0ml\/kg) was given daily until there was evidence of HUS remission, and was then switched to 20\u00a0ml\/kg twice a week, as previously done in homozygous factor H-deficient children [5].\nTen days after hospitalization, he was found to have a painful and mildly swollen left arm, mainly in the elbow area, with flexion limitation. There was no intravenous device in this arm. There were no high fever, local redness or swollen joints. The plain X-ray did not reveal any lesions or fractures. A week later the left shoulder and humeral area were more swollen and tender, with no specific nodule or bone deformity. On physical examination, the swelling was deeper than the dermis (mainly in the humeral area) and painful. The superficial skin did not show signs of ischemia, necrosis, ulceration or livedo reticularis. This time the plain X ray revealed diffuse calcification, involving mostly the arm muscles and the axilla (Fig.\u00a01a). Calcium and phosphorus levels were 9.9\u00a0mg\/dl and 6.6\u00a0mg\/dl, respectively. Renal function remained stable but impaired (serum creatinine and urea concentrations were as low as 1.05\u00a0mg\/dl and 90\u00a0mg\/dl, respectively). Parathyroid hormone (PTH) levels were normal (25.7\u00a0pg\/ml). Renal sonogram showed no evidence of nephrocalcinosis. A bone scan revealed significant technetium-99 methylene diphosphonate (99Tc-MDP) uptake along the areas that had seemed to be affected on the X-rays and physical examination (Fig.\u00a01b). Treatment with peritoneal dialysis, alphacalcidol and calcium salts was stopped. Ketoconazole 1\u00a0mg\/kg per day was given for 3\u00a0days, in addition to prednisolone 2\u00a0mg\/kg per day for a week. Renal function improved, but the (Pi\u00d7Ca) products remained above 60\u00a0mg2\/dl2. No improvement in the arm lesion was seen. On day 25 of hospitalization, intravenously administered sodium thiosulfate, 100\u00a0mg\/kg q.d., was started for 12\u00a0days. A gradual symptomatic improvement in arm range of movement and resolution of local pain was seen. At follow-up 8\u00a0weeks afterwards, X- ray and bone scan revealed significant resolution of MC (Fig.\u00a02). At long-term (>12\u00a0months) follow-up, no recurrence of MC was seen, in spite of recurrent events of HUS, eventually needing periods of peritoneal and hemodialysis.\nFig.\u00a01Chest X-ray on day 18 (a) black arrow calcification on the axilla, arrowheads calcification lines along the humerus. Tc-99m MDP scintigraphy (b) shows increased abnormal uptake in the soft tissue of the left shoulder, humerus and left scapula. Kidneys are enlarged, with increased diffuse abnormal radiotracer activityFig.\u00a02Repeated X-ray (a) and bone scan (b) after 1\u00a0month show no abnormal findings\nDiscussion\nThe unique MC features in our patient were its appearance early after the diagnosis of renal failure and its transient nature. Evidence for such early appearance of this complication comes only from an animal model of ARF (adenine-induced renal failure in rats), where microscopic metastatic calcification in aorta, coronary artery and other soft tissues were found after 4\u00a0weeks of uremia [6]. This was associated with hyperparathyroidism, hypocalcemia and hyperphosphatemia. In addition, a high index of suspicion may detect the MC early in the renal failure course, before it becomes ulcerated [7].\nCurrent sophisticated imaging techniques suggest an incidence of subclinical or asymptomatic MC in 20\u2013100% of patient with end-stage renal disease (ESRD) [8]. MC with soft tissue involvement in acute renal failure, to the best of our knowledge, has not been reported in children: using the PubMed and MESH search systems, and the terms \u201ccalcinosis\u201d and \u201ckidney failure, acute\u201d (the term \u201ccalciphylaxis\u201d is not an official indexing term), we found only three case reports of adults with such combination. There was a combination of risk factors for MC in our patient, including high dose parenteral calcium administration via both intravenous and peritoneal dialysis; high serum phosphorus levels due to renal failure and the hemolytic state; and the use of alphacalcidol [9]. These may have led to MC so early in the course of his disease. The possible correlation between renal failure due to HUS, a state where the basic damage is to the endothelium in small vessels, and MC is not supported by the literature search. Uremic calcification was thought to be a largely physico-chemical process facilitated by elevated Ca\u00d7P (i.e., \u201cmetastatic\u201d calcification). Evidence now shows that vascular smooth muscle cells actively take up phosphate to form bio-apatite. This process is associated with a phenotypic transformation of vascular smooth muscle cells during which they express osteoblast markers [10].\nSoft tissue accumulation of Tc99m MDP may be due to passive localization of tracer in slow fluid spaces (ascites, pleural effusion) when the blood concentration of tracer is high and these spaces do not clear as rapidly as the blood pool. Tracer may also bind in necrotic tissues (myositis, myonecrosis). Other causes of metastatic calcification include hypercalcemia from widespread destruction of bone (from metastatic cancer), parathyroid neoplasm and hypervitaminosis D. When the solubility product of calcium and phosphate is exceeded, there is precipitation of calcium in the extracellular space. Soft tissue calcification is, thus, another cause of soft tissue uptake of Tc99m MDP [11].\nReported therapeutic strategies for treating and preventing MC include: increasing dialysis dose, lowering serum calcium phosphorous and Ca\u00d7P solubility products, and avoiding calcium-based Pi binders and vitamin D analogs. Unfortunately, these methods have been ineffective in some patients with MC. Intravenously administered sodium thiosulfate (ST) increases the solubility of calcium deposits [12]. ST was shown to be successful in the treatment of both nephrolithiasis and tumoral calcinosis. ST has antioxidant effects on endothelial cells, but its exact mechanism of effect is unclear. It undergoes mainly renal clearance. Its half-life is increased from 15\u00a0min in the normal glomerular filtration rate (GFR) to 478\u00a0min in dialysis-dependent patients. The recommended adult dose is 12.5\u201325\u00a0g at the end of dialysis. The duration of this treatment is unclear. In our patient, a short ST course plus the withdrawal of enteral calcium and vitamin D were sufficient to resolve MC, which did not recur in the following events of HUS.","keyphrases":["sodium thiosulfate","calcific uremic arteriolopathy","hemolytic uremic syndrome","peritoneal dialysis","radionuclide imaging","alfacalcidol"],"prmu":["P","P","P","P","M","U"]}
{"id":"BMC_Fam_Pract-4-_-169167","title":"Measuring access to primary care appointments: a review of methods\n","text":"Background Patient access to primary care appointments is not routinely measured despite the increasing interest in this aspect of practice activity. The generation of standardised data (or benchmarks) for access could inform developments within primary care organisations and act as a quality marker for clinical governance. Logically the setting of targets should be based on a sound system of measurement. The practicalities of developing appropriate measures need debate. Therefore we aimed to search for and compare methods that have been published or are being developed to measure patient access to primary care appointments, with particular focus on finding methods using appointment system data.\nBackground\nPrimary care is under scrutiny along with other public services to improve access to its users. Access in primary care is typically conceptualised as the achievable access to appointments with clinical professionals, although it is not routinely measured in most practices. It is widely recognised that it represents an important dimension in determining the quality of care [1,2]. Nevertheless, it is also known that waiting times can vary widely in differing localities and countries, from patients being seen the same day to a wait of several weeks. Most primary care organisations have no more than a perception of variable demand and no method of comparing fluctuating levels of access to appointments within or between practices. Measuring patient access could generate useful information for patients, clinicians and practice managers. Demand management initiatives and ways to optimise access could then be audited bearing in mind the impact of such initiatives on opportunity costs [3-5].\nThere are potential difficulties, recognised by a recent discussion document [6], that have been accentuated by policy and political influences. The NHS Plan in the UK [7] suggested that patients should have access to primary care services within 48 hours, but the concept lacked sufficient definition. The proposed new GMS contract includes an optional 48 hour target for access to GP appointments. But what exactly should we measure? Access can be measured at many different interfaces, from the wait for traditional services such as appointments with a clinician to the alternative solutions of nurse triage, nurse led clinics, telephone advice (including NHS Direct) or electronic mail responses. Differing interpretation of terms can also cause confusion. How soon for example should a problem that is defined by a patient as urgent be seen in general practice? The perception of urgent differs between patients themselves [8] and between doctors and patients. The concept of routine appointments is easier to define and quantify. A proposed measure is the waiting time for the next available routine appointment but this provokes debate about whether this should be practice-based or specified for individual clinicians. If the latter, factors such as part-time working, practitioner popularity, the creation of multiple review appointments are likely to rapidly diminish a clinician's accessibility. How transparent would a practice want to be about such data and how useful or acceptable would it be to publish information at the clinician level? [9]\nRecognising the complex nature of this issue, we set out to review the literature. Our main aim was to search the international primary care literature for methods that had been, or were being developed to measure access to GP appointments, focusing on measures using appointment system data. Once identified, existing methods (tools, scales or other instruments) would be compared, with specific attention given to the type and levels of access they aimed to assess.\nMethod\nPreliminary searches indicated that this area did not have an extensive or long-standing research literature; most articles had been published after 1998. A broad but systematic search process was designed to allow for a poorly indexed publication pool. Medline, PubMed, Clin Psyc and ASSIA were searched for relevant publications between 1990 and 2001. The following MeSH terms were used: family practice, health service accessibility (organisation and administration, statistics and numerical data, standards, trends, methods, manpower), appointments and schedules (waiting lists), research design, health service needs and demand, weights and measures, quality of healthcare, management audit, patient satisfaction, health service needs and demand. In addition the following keywords were used: general practice, access, appointments (same day, urgent, routine) appointment systems, measurement, measures, tools, scales, demand, availability, audit and waiting times. Terms were used both singly and in combination. Title searches were used to increase the sensitivity. All citations and abstracts were appraised for relevance and full articles selected for examination by two researchers independently (GE and WJ). Key authors were contacted directly [1,12,16,18,21,25,27] and searches conducted on their previous work. Departments of General Practice in Universities in the United Kingdom were also asked to send details of any relevant research. Conference literature [21] and non-peer reviewed literature obtained from websites was also appraised. Relevant websites were identified using  and the search terms health service delivery, access to primary care and general practice. The following sites were reviewed: the Royal College of General Practitioners [6], the National Primary Care Research and Development Centre [1], The National Primary Care Development Team [10] and the Centre for Innovation in Primary Care [11].\nStudies or articles were included in the review if they described tools, scales, questionnaires or other methods of measuring actual patient access to appointments. We also included descriptions of methods that were currently being developed in this field, provided they had undertaken pilot studies and had completed one data collection exercise. Articles were excluded if they were purely editorial.\nResults\nA total of 1763 citations were initially identified and 38 articles retrieved for detailed assessment from the Pubmed and the Medline searches. Clin Psych and ASSIA searches provided some overlap but no new relevant material. The most helpful pointers to relevant publications were obtained from the website searches and personal communications rather than the traditional search engines. Two broad approaches to the measurement of patient access were identified. Firstly, appointment systems in organisations were analysed in differing ways to provide numerical data and, secondly, patient perceptions (reports) of access were evaluated using survey techniques.\nMethods using appointment system data\nTable 1 summarises the six identified methods that were based on appointment system data.\nTable 1\nComparison of methods based on appointments systems to measure access to primary care\nThird appointment [21]\nNEMAS [15]\nLedlow [14]\nAccess Response Index AROS [22]\nCampbell [12]\nKendrick [13]\nMeasurement\nThe 3rd available routine appointment at 12 midday, one day per week, for every clinician\nDate of patient call and appointment provided. GP requested and GP allocated.\nAppointments demanded but not available in US style primary care clinics compared to community clinics. Demand versus availability gap coded into 4 categories.\nNumber of days until next available routine appointment (with any clinician) at 4 pm, every working day\nNumber of appointments provided at the beginning of the day and the number still available. Total number of patients seen during the day, noting the number of 'extras'\nNumber of appointments available at the start of the day and the number of patients seen as 'extras' at the end of the day\nFrequency of data collection\nOnce a week\nContinuous\nDaily\nOnce a day\nTwice a day\nTwice a day\nWeighted for part time staff\nYes\nNo\nNo\nNo\nNo\nNo\nNamed clinician access measured\nYes\nYes\nNo\nNo\nNo\nNo\nData analysis\nWeekly median score and monthly average\nComputerised\nDemand versus availability gap\nComputer to work out 5 day moving average\nData related to practice list size, with rates given per 1000 patients\nDaily tally\nResults\nWeekly snapshot of patient access profile\nComplete computerised analysis of practice appointment system\nFeedback reports generated to clinic staff\nTrends across weekly schedules.\nBar charts represent number of appointments offered versus number of patients seen. Start of day appointment availability categorised as low, medium and high\nGraphical display of extras versus number of free appointments during the day\nExtent of and reason for use\nPrimary Care Collaborative in England. To inform implementation of advanced access\n145 teaching practices Audit\nUS Military Clinic Study\n10 practices To inform improvement\n19 practices Research Study\n1 practice Research Study\nCo-ordination\nNational Primary Care Development Team\nDepartment of General Practice, University of Glasgow.\nHealthcare Programs Central Michigan University.\nUniversity department of General Practice\nUniversity Department of General Practice, Edinburgh.\nDepartment of Primary Health Care, University of Southampton.\nThree methods determine appointment availability and\/or the satisfaction of demand on a daily basis but do not measure the days wait for appointments. Campbell measured clinician availability by recording the number of provided appointments at the beginning of each day plus the number of these un-booked at this time [12]. At the end of the day numbers seen and numbers of extras were counted, and adjusted for practice list size. Kendrick and Kerry recorded the number of available appointments at the beginning of the day and the number of extras seen at the end [13]. Ledlow also suggested a categoriations system for recording differing levels of unmet daily demand in a military medical service [14]\nThree methods measure access as days waited by representing appointment system data. A computerised package, NEMAS [15,16] enables practices to audit four areas: practice service provision (including 'appointment availability' and patient satisfaction), chronic disease management, drug monitoring and significant event analysis. The method calculates the mean time waited in days plus the minimum and maximum patient waits. Data can be presented for the whole practice or for individual GPs. Data entry involves using electronic forms to record the date of appointment request, the date the patient was seen, whether it was an elective, forced (i.e. next available clinician) or urgent appointment, the clinician requested and the clinician actually consulted. The costs of collecting data has varied considerably depending on which staff member is employed for the task [17]. Transfer of data from the practice system can be automatic with compatible systems (personal communication).\nThe National Primary Care Development Team is measuring access as part of the adoption of the system of 'Advanced Access', a system developed in the USA [18,19], as a response to patient waiting times of 4 \u2013 6 weeks for routine appointments in primary care [20]. This method collects data on one day per week (which can vary) at 12 noon. The number of days to the third available routine appointment for each clinician is recorded and a median figure calculated to represent an access score for the specified week. Over a month, the average of four median values is taken to represent a monthly access score. The third appointment is chosen, rather than the first, in order to negate the effect of sudden cancellations which otherwise could give false impressions of availability if at the moment of measuring there is one sudden cancellation in a schedule that is otherwise booked for several days ahead. The third appointment has been found by trial and improvement to best represent the actual waits involved (personal communication). Embargoed appointments are not included. A system of weighting has been developed [21] for part time workers to enable their scores to be incorporated.\nThe Access Response Index (AROS) was developed as a rapidly calculable measure of organisational access [22]. This index is derived by counting the number of days until the next available routine appointment, with any clinician, once during every normal working day. The data is recorded at four pm \u2013 a time chosen to avoid the influence of embargoed appointments that many organisations use to maintain urgent same day availability. The results are plotted on a graph, the daily fluctuation represented by the raw data is smoothed to a demonstrate trends by calculating a 5-day moving average (a data point which is the mean of every successive 5-day group). An example of the data produced by this measure is shown in Figure 1.\nFigure 1\nAROS scores for routine appointment availability (data from 11 practices)\nMethods using patient questionnaires\nFour patient experience questionnaires were found that contained access assessments and the relevant items are outlined in Table 2. The General Practice Assessment Survey (GPAS) [23] was developed by the National Primary Care Research and Development Centre, by adapting the Family Practice Assessment Survey (FPAS)[24]. A second survey named Europep [25,26] has been validated in 10 European countries. Thirdly Baker describes the use of a validated surgery satisfaction questionnaire (SSQ) that he developed [27,28] and finally Grogan's patient Satisfaction Questionnaire contains two sections that ask about access and appointment availability[29].\nTable 2\nPatient survey instruments: items used to determine access perceptions\nSurvey items\nResponse ratings\nGPAS [23]\n6.\nThinking of times when you want to see a particular doctor:\na) 5 point scale, 1 = same day, 5 = more than 5 days\na) How quickly do you get an appointment?\nb) How do you rate this?\nb) Range from 1 = very poor, 6 = excellent\n7.\nThinking of times when you are willing to see any doctor:\na) How quickly do you get an appointment?\n8. Yes \/ No \/ Not applicable \/ Don't know\nb). How do you rate this?\n8.\nIf you need an urgent appointment to see your GP can you normally get one on the same day?\nEUROPEP [25,26]\nWhat is your opinion of the general practitioner and\/or the practice over the past 12 months with respect to:\n5 point scale (poor to excellent)\n19)\nGetting an appointment to suit you?\n23)\nProviding quick services for urgent health problems?\nBaker [28]\n10)\nIt can sometimes be difficult to get an appointment with my doctor at this surgery.\n5 point agreement scale\n14)\nIt can be hard to get an appointment for medical care right away.\nGrogan [29]\n33)\nGetting an appointment at a convenient time is easy.\n5 point agreement scale\n34)\nAppointments are easy to make whenever I need them.\n35)\nIt is often difficult to get an appointment with a doctor.\n36)\nIt is easy to see a doctor of my choice.\nDiscussion\nPrincipal findings\nThis review of access measurement reveals the heterogeneous nature of the methods and the lack of any widely accepted conceptualisation of patient access. Identified measures are either practice centred using appointment data or patient orientated via surgery satisfaction questionnaires. It is clear that these two methods represent entirely different aims. It is not possible for episodic patient surveys to provide data that has enough currency or accuracy to inform organisational responses to patient demand.\nAlso demonstrated are inherent problems over definition of terms \u2013 defining what is to be measured and setting of targets. There appear to be three appointment categories over and above emergencies (which by definition cannot be given 'appointments'). Appointments for healthcare could be categorised into urgent, soon, and elective. Urgent appointments are typically seen as requests for same-day consultations. The soon category would fit problems that should be seen within two or three days to prevent escalation or symptom prolongation. Finally, routine or elective appointments suit individuals who value an agreed time window over other factors. The methods that represent patient waiting times for 'routine', i.e. elective appointments seem to hold promise as they avoid the definitional problems that surround 'urgent' appointments, and the different views that patients, clinicians and others have about 'urgency'. The task of deciding whether to represent access profiles for organisations or for individual clinicians also needs careful consideration. It may be more feasible (and less threatening) to routinely measure organisational access, especially if the data is to be used for benchmarking purposes.\nTo attempt to measure access means to obtain meaningful data from a dynamic system that is not always in equilibrium. The access experienced by an individual varies determined by demand, adequate appointment provision, sudden cancellations and block release of held appointments. We have distinguished two approaches to overcome this, either for a full statistical analysis using compatible systems or data retrieval software, or to use a simple snapshot method, deciding whether data-smoothing methods such as daily moving averages or aggregated weekly median scores are the best portrayal of overall access patterns.\nStrengths and weaknesses of the study\nMultiple search methods were used to ensure that the breadth of literature and online resources were examined as systematically as possible. The searches proved difficult and reflect the emergent status and the diversity of terms used in this area. We may have overlooked methods developed in other healthcare systems.\nImplications of the findings to healthcare services and research\nThe lack of a widely agreed measurement method to represent patient access to primary care services will make it impossible for practices to compare their response to patient demand with any degree of certainty. The 'third appointment' system is the most widely used method and is currently supported by the National Primary Care Development Team in England (but has no equivalent support in Scotland, Wales or Northern Ireland). It is however a relatively complex manipulation of appointment system data, and incorporates individual clinician availability. It seems from first principles that the important requirements of a tool designed to measure a dynamic concept such as patient access is simplicity and ease of regular data collection, so that longitudinal data patterns capable of indicating trends in organisations can be generated rather than data on individual clinician availability.\nA recent survey compared mechanisms used to manage requests for same day appointments[30]. Murray's proposal of doing today's work today (Advanced Access) eliminates appointment categories and the work involved in negotiating urgency by dealing with virtually all demand on the day it arises[31]. Access is an important determinant of healthcare quality but what are the implications of this approach to the balance of overall quality? Continuity of care, whilst not important for some problems is desirable for others [32,33]. Too drastic a shift in favour of access is likely to be at the cost of reduced continuity and a diminution of other services, such as screening and chronic disease management. Measures of organisational quality need to be aware of the dangers of focusing too much on one dimension, and should work towards the creation of measures that balance scores across internal and external requirements [34].\nAn effective consultation with a well trained clinician who knows the patient and who has access to a well structured longitudinal record will probably remain a gold standard and the issue of immediate 'access' should not be elevated above all the other components in this equation. Nevertheless, it seems prudent to develop measures that provide a better understanding of patient access to organisations with similar resources. Measuring the interval to the next available routine appointment, whilst measuring the measurable [35] will, if a consistent standard can be agreed, provide benchmark data, and mark an important step towards a compendium of methods to assess quality in primary care.\nConclusion\nThe two approaches of either using patient questionnaires or appointment system data to measure access are methods that represent entirely different aims. The latter method when used to represent patient waiting times for 'routine' elective appointments seems to hold promise as a useful tool and this avoids the definitional problems that surround 'urgent' appointments. The purpose for which the data is being collected needs to be borne in mind and will determine the chosen methods of data retrieval and representation\nCompeting Interests\nThe authors of this article have piloted the AROS Index.\nAuthors' Contributions\nPeter Edwards, Melody Emmerson and Glyn Elwyn were involved in the development of the Aros index. Wendy Jones and Glyn Elwyn conducted the Aros pilot study and the literature review with contributions from Adrian Edwards and Richard Hibbs.\nPre-publication history\nThe pre-publication history for this paper can be accessed here:","keyphrases":["measurement","access","primary care","appointments","waiting times."],"prmu":["P","P","P","P","P"]}
{"id":"Cell_Tissue_Res-3-1-2071963","title":"Localization of metabotropic glutamate receptors in the outer plexiform layer of the goldfish retina\n","text":"We studied the localization of metabotropic glutamate receptors (mGluRs) in the goldfish outer plexiform layer by light-and electron-microscopical immunohistochemistry. The mGluR1\u03b1 antibody labeled putative ON-type bipolar cell dendrites and horizontal cell processes in both rod spherules and cone triads. Immunolabeling for mGluR2\/3 was absent in the rod synaptic complex but was found at horizontal cell dendrites directly opposing the cone synaptic ribbon. The mGluR5 antibody labeled M\u00fcller cell processes wrapping rod terminals and horizontal cell somata. The mGluR7 antibody labeled mainly horizontal cell dendrites invaginating rods and cones and some putative bipolar cell dendrites in the cone synaptic complex. The finding of abundant expression of various mGluRs in bipolar and horizontal cell dendrites suggests multiple sites of glutamatergic modulation in the outer retina.\nIntroduction\nStimulation of vertebrate photoreceptors by light decreases the release of glutamate (Cervetto and MacNichol 1972; Murakami et al. 1972; Kaneko and Shimazaki 1976), which is sensed by glutamate receptors (GluRs) in the dendrites of second-order neurons. Glutamate receptors are roughly divided into ionotropic and metabotropic receptors (Eccles and McGeer 1979). Ionotropic receptors (iGluRs) form an integral ion channel, whereas metabotropic receptors (mGluRs) mediate responses through indirect mechanisms involving signal transduction cascades.\nSo far, eight different mGluRs have been cloned and classified into three groups based on their sequence similarities, second-messenger cascade, and pharmacology: group I comprises mGluR1 (Houamed et al. 1991; Masu et al. 1991) and mGluR5 (Abe et al. 1992) and their splice variants (1a\u2013d and 5a\u2013b); group II contains mGluR2 and mGluR3 (Tanabe et al. 1992; Tanabe et al. 1993); and group III includes mGluR4 (Tanabe et al. 1993), mGluR6 (Nakajima et al. 1993), mGluR7 (Okamoto et al. 1994; Saugstad et al. 1994), and mGluR8 (Duvoisin et al. 1995), and their splice variants (4a\u2013b, 7a\u2013b, 6a\u2013b, and 8a\u2013b). The lack of specific pharmacological tools to distinguish between mGluRs within a group makes it difficult to link a particular receptor type to a certain response property (for reviews, see Thoreson and Ulphani 1995; Schoepp et al. 1999).\nIn the retina, mGluRs play an important role in the transmission and modulation of visual signals. Light responses of ON-type bipolar cells (ON BCs) of a number of species, for instance, are sensitive to substances\/drugs that target group III mGluRs (Shiells et al. 1981; Slaughter and Miller 1981; Thoreson and Miller 1993). The receptor activated by these drugs is most likely mGluR6 (Nakajima et al. 1993; Akazawa et al. 1994; Masu et al. 1995; Ueda et al. 1997; Vardi and Morigiwa 1997; Laurie et al. 1997; Vardi et al. 2000). In fish, amphibians, and mammals, group III mGluRs modulate the glutamate release of cones (Koulen et al. 1999; Hirasawa et al. 2002; Hosoi et al. 2005). Furthermore, pharmacological activation of both group I and group III mGluRs modulates horizontal cell (HC) responses (Nawy et al. 1989; Takahashi and Copenhagen 1992; Linn and Gafka 1999), suggesting that multiple mGluRs must be present in the fish outer plexiform layer (OPL).\nAt the moment, however, data available on the identity and distribution of the various mGluRs in the teleost outer retina are scarce (Yazulla et al. 2001; Klooster et al. 2001). We have therefore performed a light and electron microscopy study of the mGluRs present in the goldfish OPL. An antibody against the ON BC marker, protein kinase C (PKC; Negishi et al. 1988; Suzuki and Kaneko 1990; Yazulla and Studholme 1992), has been used in order to identify possible localization in mixed-input ON BCs (ON MBCs).\nMaterials and methods\nSubjects\nGoldfish (Carassius auratus), 12\u201315\u00a0cm standard body length, were obtained from a commercial supplier and maintained at 16\u00b0C in aerated tanks filled with tap water circulating through a biological filter system. The fish were fed and kept on a 12\u00a0h\/12\u00a0h light-dark cycle. All animal experiments were approved by the ethical committee of the Royal Netherlands Academy of Arts and Sciences, acting in accordance with the European Communities Council Directive of 24 November 1986 (86\/609\/EEC).\nPreparation\nDissections took place early in the morning (between 7 a.m. and 9\u00a0a.m.) under ambient light. The fish used in this study were therefore all in the light phase of their circadian regime. After cervical transection, the cornea and lens of light-adapted animals were removed, and the remaining eyecups were cut in half through the equator. Half-eyecups were placed vitreous-side-down on a Millipore filter (13\u00a0mm diameter, 8\u00a0\u03bcm pore size; Millipore, Amsterdam, The Netherlands) placed on a filter holder. Suction was applied to remove the vitreous; the sclera and retinal pigment epithelium were peeled away.\nLight microscopy\nRetinas were fixed at room temperature for 10\u00a0min in 0.1\u00a0M phosphate-buffered 4% paraformaldehyde (pH 6.5) and for another 10\u00a0min in 0.1\u00a0M sodium carbonate-buffered 4% paraformaldehyde (pH 10.4). These short fixation times were chosen to prevent loss of antigenicity. After being rinsed in 0.1\u00a0M phosphate buffer (PB, pH 7.4), the tissue was cryoprotected at room temperature for 30\u00a0min in PB containing 12.5% sucrose and for 1\u20132\u00a0h in PB containing 25% sucrose.\nThe pieces of retina, still attached to the filter, were embedded in Tissue Tek (Sakura Finetek Europe, Zouterwoude, The Netherlands) in an aluminum boat and frozen in liquid nitrogen. Sections (8\u201310\u00a0\u03bcm thick) were cut in a cryostat, mounted on poly-L-lysine-coated slides (Menzel-Gl\u00e4ser, Braunschweig, Germany), dried and stored in a non-frost-free freezer at \u221220\u00b0C until use.\nRetinal sections were washed (2\u00d7, 10\u00a0min) in phosphate-buffered saline (PBS), blocked for 20\u00a0min in PBS with 2% normal goat serum (NGS; Jackson ImmunoResearch Lab, West Grove, USA), incubated for 18\u201348\u00a0h with the primary antibody (for dilutions, see Table\u00a01) in PBS containing 0.3% Triton X-100 and 5% NGS at 4\u00b0C. After several washes (15\u00a0min, 3\u00d7) in PBS, sections were incubated in the secondary antibodies diluted in PBS containing 0.3% Triton X-100 for 30\u00a0min at 37\u00b0C (Cy3-conjugated antibody: 1:500, Alexa-conjugated antibody: 1:600).\nTable\u00a01Primary antibodies (LM light microscopy, EM electron microscopy, aas amino acids, PKC protein kinase C, mGluR metabotropic glutamate receptor)AntibodySourceCatalog numberLotTypeHostPurificationAntigenDilution for LMDilution for EMBlot (kDa)aPKCbSigma-Aldrich, St. Louis, USAP57040641H4847MonoclonalMouseProtein ABovine, within aas 296\u2013317 of brain PKC1:5001:40080mGluR1\u03b1Chemicon, Temecula, USAAB1551196011PolyclonalRabbitAffinityRat, C-terminal, aas 1180\u20131199 (PNVTYASVILRDYKQSSSTL)1:1001:100140mGluR2\/3ChemiconAB1553196011PolyclonalRabbitAffinityRat, C-terminal, aas 860\u2013872 (NGREVVDSTTSSL)1:1001:100100 and 190mGluR5ChemiconAB159624010139PolyclonalRabbitAffinityRat, C-terminal, aas 1159\u20131171 (LIIRDYTQSSSSL)1:1001:100116mGluR7Upstate, Lake Placid, USA07\u201323925162PolyclonalRabbitProtein AHuman, C-terminal, aas 899\u2013912 (NSPAAKKKYVSYNN)1:5001:50097aWestern blots performed by external groups; relative mass values as reported in manufacturer\u2019s data sheetsbProduced after Parker et al. 1984 and Young et al. 1988\nSections were cover-slipped with Vectashield (Vector Labs, Burlingame, USA) and stored at \u221220\u00b0C. Slides were observed on a Leica DMRD (Leica Microsystems, Wetzlar, Germany) fluorescence microscope equipped with filter sets that were designed for fluorescein isothiocyanate and Cy3. Sections from double-label experiments were observed on an inverted Zeiss Axiovert 100\u00a0M microscope equipped with the LSM 510 META laser scanning confocal module (Carl Zeiss Jena, Jena, Germany).\nTwo types of controls were performed. To control for unspecific labeling attributable to secondary antibodies, experiments were performed by omitting the primary antibody. This eliminated all labeling. To control for unspecific labeling attributable to the primary antibodies, preadsorption experiments were performed by mixing, in a 20-fold molar excess, a synthetic peptide having the amino acid sequence against which each mGluR antibody had been raised (see below) with the corresponding primary antibody overnight at 4\u00b0C. Incubation proceeded as previously described, with the same concentrations for the primary antisera. Labeling was also completely eliminated in these preadsorption controls.\nElectron microscopy\nThe same fixation and cryoprotection procedures were used as described for light microscopy. The retinas were peeled off the filter and embedded in Tissue Tek (Sakura Finetek Europe, Zouterwoude, The Netherlands) in an aluminum boat. The boat was frozen in liquid nitrogen. Transverse frozen sections (30\u201340\u00a0\u03bcm thick) were obtained on a freezing microtome and collected in PB at room temperature (equivalent to freeze-thawing).\nThe retinal sections were incubated for 72\u00a0h with diluted antisera (for dilutions, see Table\u00a01). After being rinsed, the tissue was incubated in a poly-horseradish-peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit IgG (PowerVision, ImmunoVision Technologies, Daly City, USA). To visualize the peroxidase, the material was incubated in a TRIS-HCl diaminobenzidine (DAB, 0.05%) solution containing 0.03% H2O2. The DAB reaction product was subsequently intensified by the gold-substituted silver peroxidase method (Gorcs et al. 1986).\nSections were rinsed in sodium cacodylate buffer 0.1\u00a0M (pH 7.4) and post-fixed for 20\u00a0min in 1% OsO4 supplemented with 1.5 % potassium ferricyanide in sodium cacodylate buffer 0.1\u00a0M (pH 7.4). After being rinsed in sodium cacodylate buffer, the material was dehydrated and embedded in epoxy resin. Ultrathin sections were observed in a Philips EM201 electron microscope (Philips, Eindhoven, The Netherlands) and\/or Technai (CM) 12 electron microscope (FEI Company, Eindhoven, The Netherlands). Sections examined with the Philips electron microscope were counter-stained with uranyl acetate and lead citrate.\nSampling and production of photomicrographs\nAt least 20 retinal sections obtained from a minimum of five animals were used for a given light microscopy or electron-microscopy experiment (i.e., single-immunolabeling at the light-microscopy level for mGluR1\u03b1). About five photomicrographs were taken from the most representative sections in each experiment.\nLight micrographs of the single-label experiments were acquired as TIFF files directly from the Leica microscope by using a Leica 350\u00a0F digital B\/W camera (1284\u00d71028 pixels, 72 ppi). Light micrographs of the double-label experiments were acquired as TIFF files from the Zeiss LSM 510 META (1048\u00d71024 pixels, 72 ppi).\nFor electron-microscopy experiments, the same number of retinal sections\/animals as described above was examined at the light-microscopical level; labeled sections were chosen for ultra-thin sectioning. A minimum of 40 photoreceptor terminals were observed per ultra-thin section. Micrographs (n\u2009=\u200910\u201330) were taken from these sections, depending on the presence and amount of labeling.\nElectron micrographs obtained with the FEI electron microscope were directly acquired as TIFF files (1024\u00d71024\u00a0pixels, 72 ppi). Electron micrographs obtained from the Philips electron microscope were first printed from the negatives for analysis. The negatives of the prints that were selected for publication were scanned on a sprint scan 4000 scanner (Polaroid Nederland, Breda, The Netherlands) and acquired as TIFF files at 600 ppi.\nAll TIFF files were resampled at 400 ppi and subsequently re-sized and optimized for brightness and contrast by using Photoshop (Adobe Systems, San Jose, USA).\nImmunochemicals\nThe primary antibodies used in this study were purchased from Chemicon International (Temecula, USA), Sigma-Aldrich (St. Louis, USA), and Upstate (Lake Placid, USA). Details of these antibodies are listed in Table\u00a01. Goat anti-mouse Alexa was obtained from Molecular Probes (Breda, The Netherlands) and goat anti-rabbit Cy3 from Jackson ImmunoResearch Lab (West Grove, USA). For preadsorption controls, custom-made peptides were obtained from Eurogentec (Seraing, Belgium).\nWestern blotting\nGoldfish retinas and brains were homogenized with a Teflon pestle in ice-cold PBS (pH 7.4) containing a tablet of protease inhibitor cocktail (Boehringer Mannheim, Mannheim, Germany) per 50\u00a0ml PBS. After homogenization, a TRIS-buffered sample (pH 6.8) solution was added, with final concentrations of: 2% SDS, 5% \u03b2-mercaptoethanol, 10% glycerol, 0.005% bromophenol blue. The samples were boiled for 2\u00a0min and centrifuged for 10\u00a0min at 14,600\u00a0g; supernatants were stored at \u221220\u00b0C until use. The diluted samples were boiled for 2 min (3\u00d7), cooled, and then spun in a microfuge at 14,600g.\nFor sodium dodecyl-sulfate polyacrylamide gel electrophoresis, samples were run through a polyacrylamide stacking gel at 20\u00a0mA and through a 13% polyacrylamide gel at 30\u00a0mA. Protein standards (Bio-Rad Laboratories, Veenendaal, The Netherlands) were run in adjacent lanes. Gels were electroblotted on a poly-vinylidene-di-fluoride blot membrane (Millipore) overnight at a constant current of 80\u00a0mA. After being rinsed with a 0.5\u00a0M TRIS buffer containing 1.5\u00a0M NaCl and 5% Tween 200, the membrane was blocked in the same buffer containing 2% dry milk for 1 h.\nSubsequently, the membrane was incubated for 1 h in the primary antibodies (1:100), washed in the TRIS buffer, incubated in an HRP-conjugated goat anti-rabbit IgG (1:5000; Santa Cruz Biotechnology, Santa Cruz, USA), and washed in PBS. The immunoreaction was visualized by enhanced chemiluminescence (ECL, Amersham, Arlington Heights, USA) by using Kodak film. Exposures times were between 1 and 5\u00a0min.\nResults\nIn this study, we aimed at a qualitative description of the mGluR composition in the goldfish OPL. To have a parameter for comparisons with regard to ON BC labeling with the antibodies against the various mGluRs, we first investigated the immunoreactivity pattern yielded by the PKC antibody, a widely used ON BC marker. Therefore, the labeling for PKC will be described first, followed by each of the mGluRs studied.\nImmunolabeling for PKC\nThe PKC antibody was characterized by Western blot previously in a number of studies (Negishi et al. 1988; Suzuki and Kaneko 1990; Yazulla and Studholme 1992) and yielded similar results in various species (Table\u00a01).\nLight microscopy Immunolabeling for PKC in the goldfish retina at the light-microscopical level has been described in detail elsewhere (Suzuki and Kaneko 1990). Briefly, two populations of ON BCs were labeled: cells with small somata in the middle of the inner nuclear layer (INL) and small axonal terminations (cone-driven ON BCs), and cells with large flask-shaped somata and bulbous axon terminals (ON MBCs).\nElectron microscopy: rod synapses In the rod synaptic complex, the antibody against PKC labeled invaginating dendrites at the central position and processes at some distance from the ribbon (Fig.\u00a01a\u2013c, arrowheads). The labeled structures were elongated (Fig.\u00a01a), u-shaped (Fig.\u00a01b), or round (Fig.\u00a01c) and were found either directly opposite to the synaptic ribbon (Fig.\u00a01a,b) or a little distance away from it (Fig.\u00a01c). Dendrites with these characteristics and at these locations in the cyprinid retina were previously shown to belong to ON MBCs (Scholes 1975; Stell 1967, 1976, 1978; Saito et al. 1983, 1985; Klooster et al. 2001; Yazulla and Studholme 2001).\nFig.\u00a01Ultrastructural localization of protein kinase C (PKC) in the outer plexiform layer. In the rod spherules (a\u2013c), invaginating dendrites of putative mixed-input ON-type bipolar cells (ON MBCs) were positively labeled (arrowheads). Lateral elements of horizontal cells (HCs) were unlabeled (arrows), as were some invaginating processes that possibly belonged to OFF-type MBCs (asterisks). In the cone pedicles (d, e), PKC immunoreactivity was observed in small invaginating profiles at the base of the pedicles (arrowheads). These structures were never associated with the synaptic ribbons (SR). HC lateral elements were unlabeled (arrows). Bars\u20090.5\u00a0\u03bcmLateral processes immediately adjacent to the pre-synaptic ridge, previously classified as dendrites of rod-driven HCs (Stell 1967, 1976; Scholes 1975) were not labeled (Fig.\u00a01a\u2013c, arrows). Some invaginating processes were also not labeled (Fig.\u00a01a,c, asterisks); their position and fine structure, in combination with the lack of labeling, suggested that they were either dendrites of OFF-type MBCs or rod-driven HCs (Stell 1967, 1976; Saito et al. 1983, 1985).\nElectron microscopy: cone synapses PKC-immunoreactive structures were not abundant in the vicinity of the cone pedicles. When present, the labeled structures were never associated with the synaptic ribbon but were instead localized at the base of the cone pedicle (Fig.\u00a01d,e, arrowheads). These were probably ON MBC processes approaching the cone pedicle. In the closely related carp, dendrites of horseradish peroxidase-injected ON MBCs have been found at similar sites in the cone synaptic complex (Saito et al. 1983, 1985). Lateral elements were unlabeled (Fig.\u00a01d,e, arrows).\nImmunolabeling for mGluR1\u03b1\nWestern blotting The mGluR1\u03b1 antibody did not recognize any proteins in the goldfish retina, possibly because of the low concentration of the receptor in this tissue. The antibody produced a diffuse band at approximately 150\u00a0kDa in goldfish brain and rat samples (Fig.\u00a02c), close to the predicted molecular mass of mGluR1\u03b1 (Tables\u00a01, 2). The immunoreactive protein found in the rat retina was faint, probably reflecting the restricted expression of this receptor in retinal tissue.\nFig.\u00a02Immunolabeling for mGluR1\u03b1 (ONL outer nuclear layer, OPL outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer). a Immunoreactivity was concentrated at the OPL and outer half of the INL in which some BC somata were partially labeled (arrowheads). b Preadsorption with the control peptide (pread) abolished all labeling. c Western blot of total protein from goldfish retina (ret), goldfish brain (br), rat retina (ret), and rat cerebrum (cer). A protein at around 150\u00a0kDa, close to the predicted molecular mass of mGluR1\u03b1, was detected in goldfish brain, rat retina, and rat cerebrum. d, eRed and green channel confocal images of the same tissue section double-labeled with antibodies against mGluR1\u03b1 (Cy3, d) and PKC (Alexa, e). Some structures were positive for mGluR1\u03b1 but not for PKC (circles). Some small BCs were positive for the ON BC marker but not mGluR1\u03b1 (asterisks). f Merged image of the double-labeling experiment. A band of mGluR1\u03b1 immunoreactivity in the OPL partially overlapped with the dendritic processes of PKC-positive cells. Areas of overlap between the two proteins appear yellow. Bars\u200910\u00a0\u03bcmTable\u00a02BLAST (basic local alignment search tool) results and relative molecular mass (Mr) of fish mGluRs (aa amino acids)Source proteinZebrafish matchesSequence comparisonLength sourceLength zebrafishMr sourceMr zebrafishThis studyRat mGluR1(NP_058707)Similar to bifunctional mGluR forextracellular Ca and Glu (previouslysimilar to mGluR1-XP_690446a)SourcePNVTYASVILRDYKQSSSTL1199\u00a0aa1158\u00a0aa133\u00a0kDa130\u00a0kDa150\u00a0kDaConsensusP+VTYASVILRDYKQSSSTLZebrafishPSVTYASVILRDYKQSSSTLSimilar to bifunctional mGluR forextracellular Ca and Glu, partial(XP_001346460a)SourcePNVTYASVILRDYKQSSSTL271\u00a0aa30\u00a0kDaConsensusP+VTYASVILRDYKQSSSTLZebrafishPSVTYASVILRDYKQSSSTLRat mGluR2(EDL77281)Hypothetical protein (previousmGluR2 precursor-XP_692887)SourceNGREVVDSTTSSL872\u00a0aa874\u00a0aa96\u00a0kDa97\u00a0kDa100 and230\u00a0kDaConsensusNGREVVDSTTSSLZebrafishNGREVVDSTTSSLHypothetical protein (XP_001341692)SourceNGREVVDSTTSSL754\u00a0aa85\u00a0kDaConsensusNGREVVDSTTSSLZebrafishNGREVVDSTTSSLNovel protein similar to vertebratemGluR3 (CAM56446)SourceNGREVVDSTTSSL884\u00a0aa98\u00a0kDaConsensusNGRE+VDSTTSSLZebrafishNGREIVDSTTSSLHuman mGluR7(AAB51763)Similar to GluR (previously similar tomGluR7-XP_697214)SourceNSPAAKKKYVSYNN915\u00a0aa668\u00a0aa102\u00a0kDa75\u00a0kDa50\u00a0kDaConsensusNSPAAKKKYVSYN+ZebrafishNSPAAKKKYVSYNDRat mGluR5(NP_058708)Similar to bifunctional mGluR forextracellular Ca and Glu (previouslysimilar to mGluR1-XP_690446a)SourceLIIRDYTQSSSSL1171\u00a0aa1158\u00a0aa128\u00a0kDa130\u00a0kDa60 and100\u00a0kDaConsensus+I+RDY QSSS+LZebrafishVILRDYKQSSSTLSimilar to bifunctional mGluR forextracellular Ca and Glu, partial(XP_001346460a)SourceLIIRDYTQSSSSL271\u00a0aa30\u00a0kDaConsensus+I+RDY QSSS+LZebrafishVILRDYKQSSSTLSimilar to mGluR5 (XP_696823)SourceLIIRDYTQSSSSL1173\u00a0aa130\u00a0kDaConsensusL++R Y+QSSSSLZebrafishLVLRHYSQSSSSLAccession numbers listed below protein names. Codes in parentheses indicate accession numbers in the NCBI protein database. a Proteins whose DNA coding sequences are almost identical (only 2 nucleotide substitutions).\nLight microscopy mGluR1\u03b1 immunoreactivity in the goldfish retina was concentrated in the OPL (Fig.\u00a02a) and outer half of the INL in which the somata of some bipolar cells were labeled (Fig.\u00a02a, arrowheads). A confocal examination of the immunofluorescence pattern yielded by this antibody revealed diffuse labeling in the INL, inner plexiform layer (IPL), and ganglion cell layer (GCL; Fig.\u00a02d). Red and green images of a confocal view were taken from a retinal slice double-labeled for mGluR1\u03b1 (Fig.\u00a02d) and PKC (Fig.\u00a02e); the yellow areas in the merged image (Fig.\u00a02f) indicated the strong colocalization in ON BC processes in the OPL. However, mGluR1\u03b1 immunoreactivity in this layer was not restricted to PKC-positive processes. Furthermore, the small PKC-positive ON BCs were not positive for mGluR1\u03b1 (Fig.\u00a02d,e, asterisks). Conversely, the mGluR1\u03b1-antibody labeled structures (presumably amacrine cell somata) in the inner border of the INL that were not PKC-positive (Fig.\u00a02d,e, circles).\nElectron microscopy: rod synapses In the rod spherule, mGluR1\u03b1 immunoreactivity was present at invaginating dendrites in close apposition to the synaptic ribbons (Fig.\u00a03a\u2013d, arrowheads). These processes were elongated (Fig.\u00a03a), ellipsoidal (Fig.\u00a03b), goblet-shaped (Fig.\u00a03c), or round (Fig.\u00a03d), consistent with ON MBC dendrites. Roughly two invaginating dendrites were stained per rod spherule; the remaining invaginating dendrites (Fig.\u00a03a,b, asterisks) were not labeled. These were possibly OFF MBCs, rod-driven HCs, or a subtype of ON MBC negative for mGluR1\u03b1.\nFig.\u00a03Ultrastructural localization of mGluR1\u03b1 in the OPL. In the rod terminals (a\u2013d), ON MBC invaginating processes at the summit of the synaptic ribbon (SR) were positively labeled (arrowheads). Some invaginating processes were not labeled (asterisks); these might belong to OFF MBCs, to rod-driven HCs, or to a subtype of ON MBC negative for mGluR1\u03b1. Occasionally, a laterally positioned dendrite showed mGluR1\u03b1 immunoreactivity (thick arrow in d). In the cone pedicles (e, f), mGluR1\u03b1 immunoreactivity was found in small invaginating profiles that lay at the base of the pedicles or close to the ribbon (arrowheads) and that might have originated in BCs. In addition, a HC dendrite occupying the lateral position of the triads was positively labeled (thick arrows in e), whereas the other lateral element was unlabeled (arrows). The antibody also labeled HC spinules in the cone terminals (thick arrows in f). Bar 0.25\u00a0\u03bcmThe antibody also labeled single lateral elements facing the synaptic ribbon (Fig.\u00a03d, thick arrow). Whenever a lateral element was labeled, the other one was unlabeled (Fig.\u00a03d, arrow). Although processes in the lateral position are classically attributed as belonging to HCs (Stell 1967, 1976), we could not fully exclude the possibility that some of the labeled processes were aberrant ON MBC dendrites.\nElectron microscopy: cone synapses In the cone pedicles, various types of post-synaptic structures were labeled. In addition to small invaginating processes (Fig.\u00a03e,f, arrowheads), which could belong to BCs, mGluR1\u03b1 immunoreactivity was also present at one lateral dendrite of some cone triads (Fig.\u00a03e, thick arrows). The position and fine structure of these dendrites were consistent with those of cone-driven HCs (Stell 1967, 1976; Sakai and Naka 1983). When one lateral HC dendrite in a triad was positive for mGluR1\u03b1, the opposite HC dendrite was not (Fig.\u00a03e, thick and thin arrows, respectively). A few invaginations in the cone pedicle containing discontinuous patches of electron-dense material in the membrane and resembling HC spinules (Wagner 1980) exhibited mGluR1\u03b1 immunoreactivity (Fig.\u00a03f, thick arrows).\nImmunolabeling for mGluR2\/3\nWestern blotting The antibody against mGluR2\/3 reacted with proteins of approximately 100\u00a0kDa in goldfish brain, rat retina, and rat cerebrum (Fig.\u00a04c). This is the predicted molecular mass of mGluR2 and mGluR3 (Tables\u00a01, 2). In the goldfish retina, only a protein at about 200\u00a0kDa was detected, which also appeared in the other three tissue samples. This probably represents receptor dimers (Poli et al. 1999), which occur naturally in many G-protein-coupled receptors, including mGluRs (Bouvier 2001; Kubo and Tateyama 2005; Pin et al. 2005; Poli et al. 2003; Beraudi et al. 2007). In goldfish and rat retinas, a smaller band at around 40\u00a0kDa was detected.\nFig.\u00a04Immunolabeling for mGluR2\/3 (ONL outer nuclear layer, OPL outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer). a Immunoreactivity was seen as tiny bands in the OPL (thick arrows), irregular patches in the INL, and diffuse labeling in the IPL. b Preadsorption with the control peptide (pread) abolished all labeling. c Western blot of total protein from goldfish retina (ret), goldfish brain (br), rat retina (ret), and rat cerebrum (cer). A protein at around 100\u00a0kDa, close to the predicted molecular mass of mGluR2 and mGluR3, was detected in goldfish brain, rat retina, and rat cerebrum. The antibody also detected a protein at 200 kDa (probably receptor dimers) in all four tissues. d, eRed and green channel confocal images of the same tissue section double-labeled with antibodies against mGluR2\/3 (Cy3, d) and PKC (Alexa, e). f Merged image of the double-labeling experiment. No colocalization with ON BCs was found. Bars\u200910\u00a0\u03bcm. g, h In a few cone pedicles, mGluR2\/3 immunoreactivity was found in HC central and lateral elements of the cone triad (thick arrows). Note the unlabeled lateral processes (thin arrows) and synaptic ribbons (SR). Bars 0.5\u00a0\u03bcm\nLight microscopy Immunocytochemistry with the mGluR2\/3 antibody revealed signal in the OPL and INL and strong labeling in the IPL. Tiny discontinuous immunoreactive bands were visible in the OPL (Fig.\u00a04a, thick arrows). Confocal images showed a similar pattern, with a slightly stronger OPL labeling (Fig.\u00a04d, thick arrows). Clearly labeled somata could not be distinguished, although immunolabeling in the INL was concentrated in patches. Immunoreactivity for PKC (Fig.\u00a04e) did not colocalize with mGluR2\/3 (Fig.\u00a04d), as can be seen by the absence of yellow areas in the merged confocal image (Fig.\u00a04f).\nElectron microscopy: rod synapses No immunoreactive processes could be identified in the rod synaptic terminals.\nElectron microscopy: cone synapses HC dendrites occupying both the lateral and central position were positive for mGluR2\/3 in only a few cone pedicles (Fig.\u00a04g\u2013h, thick arrows). The fine structure of the central processes was consistent with that of HC dendrites because of their large dimensions compared with BC dendrites, cytoplasm devoid of organelles, some electron-dense amorphous material applied to the cytoplasmic surface, and concavity of the surface facing the synaptic ribbon (Stell 1967, 1976; Scholes 1975). No mGluR2\/3 immunoreactivity was seen in structures resembling BC dendrites, in agreement with our light-microscopical findings.\nImmunolabeling for mGluR5\nWestern blotting Immunoblots for mGluR5 revealed a protein at around 100\u00a0kDa in goldfish retina and brain and in rat cerebrum (Fig.\u00a05c). An additional band at 60\u00a0kDa was detected in the goldfish samples and in the rat retina. A heavier protein of around 145\u00a0kDa was detected in rat retina and cerebrum, roughly corresponding to the deduced molecular mass of rat mGluR5 (Koulen et al. 1997).\nFig.\u00a05Immunolabeling for mGluR5 (ONL outer nuclear layer, OPL outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer). a Immunoreactivity was concentrated in the ONL (open arrows), with some granular labeling in the OPL, INL, and IPL (thick arrow labeled HC body). b Preadsorption with the control peptide (pread) abolished all labeling. c Western blot of total protein from goldfish retina (ret), goldfish brain (br), rat retina (ret), and rat cerebrum (cer). A protein around 100 kDa was detected in the goldfish tissue and rat brain. The antibody also a smaller band (60\u00a0kDa) in the goldfish samples and in the rat retina, and a heavier band (145\u00a0kDa) in the rat retina and cerebrum. d, eRed and green channel confocal images of the same tissue section double-labeled with antibodies against mGluR5 (Cy3, d) and PKC (Alexa, e). f Merged image of the double-labeling experiment, showing no colocalization. Bars\u200910\u00a0\u03bcm. g Labeling is restricted to processes ensheathing photoreceptor terminals in the ONL (open arrows). h A similar labeling pattern (open arrows) is obtained with the monoclonal antibody against glial fibrilary acidic protein (GFAP; clone G-A-5, Sigma-Aldrich; 1:400), which labels exclusively M\u00fcller cells (SR synaptic ribbon). Bars 0.5\u00a0\u03bcm\nLight microscopy The mGluR5 antibody labeled vertical bands at the outer nuclear layer (ONL) in both conventional (Fig.\u00a05a, open arrows) and confocal microscopy (Fig.\u00a05d, open arrows) and yielded granular labeling in the OPL, INL, and IPL. Some HC somata were positively labeled at the outer margin of the INL (Fig.\u00a05a,d, thick arrows). There was no colocalization with PKC (Fig.\u00a05d\u2013f).\nElectron microscopy Ultrastructurally, mGluR5 immunoreactivity was seen in longitudinally oriented structures between photoreceptor terminals (Fig.\u00a05g, open arrows). This pattern was consistent with previous reports concerning the localization and ultrastructural appearance of M\u00fcller cell processes (Villegas 1960; Lasansky 1961; Pedler 1963; Miller and Dowling 1970). Furthermore, it resembled closely that of immunolabeling for glial fibrilary acidic protein (Fig.\u00a05h, open arrows), which is exclusively expressed by glial cells (Bignami and Dahl 1977; Liepe et al. 1994).\nImmunolabeling for mGluR7\nWestern blotting A protein of approximately 50\u00a0kDa was labeled in goldfish and rat samples, with an additional band of around 75\u00a0kDa in the goldfish brain. In the rat retina, a protein of 200\u00a0kDa was also recognized. A clear band could not be distinguished in rat cerebrum, although there was a larger precipitation around 50\u00a0kDa (Fig.\u00a06c), at some distance from the value reported by the producer (Table\u00a01).\nFig.\u00a06Immunolabeling for mGluR7 (ONL outer nuclear layer, OPL outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer). a Immunoreactivity was concentrated in the OPL (arrowheads), with some granular labeling in the INL, IPL, and GCL. b Preadsorption with the control peptide (pread) abolished all labeling. c Western blot of total protein from goldfish retina (ret), goldfish brain (br), rat retina (ret), and rat cerebrum (cer). A pronounced band at around 50\u00a0kDa was produced in all four samples. Additional immunoreactive proteins were found at 75\u00a0kDa in the goldfish tissue and at 200\u00a0kDa in the rat retina. d, eRed and green channel confocal images of the same tissue section double-labeled with antibodies against mGluR7 (Cy3, d) and PKC (Alexa, e). A BC soma (arrowheads) and an HC soma (thick arrows) were positive for mGluR7 but negative for PKC. f Merged image of the double-labeling experiment, showing no colocalization in ON BCs. g In rod spherules, lateral elements were labeled (thick arrows). h In the cone pedicles, small profiles not related to the synaptic ribbons (SR) were positive (arrowheads), as were lateral elements of the triads (thick arrow). Note the unlabeled lateral elements (thin arrows). Bars 0.25\u00a0\u03bcm\nLight microscopy The antibody against mGluR7 immunoreactivity yielded strong labeling in the OPL (Fig.\u00a06a, arrowheads) and granular labeling at the INL, IPL, and GCL. Occasionally, positive amacrine and ganglion cell somata were seen. Confocal images of double-labeling with PKC (Fig.\u00a06d\u2013f) did not show colocalization: mGluR7-positive areas were concentrated close to the outer border of the INL, immediately below the band of PKC-positive ON BC dendrites. Some mGluR7-positive somata that were not labeled for PKC could be identified in the outer half of the INL; their form and position was consistent with that of HCs (Fig.\u00a06d,e, thick arrow) and OFF BCs (Fig.\u00a06d,e, arrowhead).\nElectron microscopy: rod synapses Lateral elements of the rod triads were labeled, consistent with the labeling of putative HCs reported above (Fig.\u00a06g, thick arrows). We found no evidence of MBC labeling in rod spherules.\nElectron microscopy: cone synapses A few small processes away from the ribbons were labeled by the mGluR7 antibody (Fig.\u00a06h, arrowheads). Their small profiles resembled those of BCs. This observation, added to the finding that the antibody labeled PKC-negative BC somata (Fig.\u00a06d,e, arrow), suggested that these were OFF BC profiles. Lateral elements (Fig.\u00a06h, thick arrow) and spinules were also labeled, consistent with HC labeling at the light-microscopical level (Fig.\u00a06d,e, thick arrows). These labeled HC processes were however rare; most of the lateral elements were not positive (Fig.\u00a06h, thin arrows).\nDiscussion\nSummarizing scheme\nThis study shows that mGluRs of all three groups can be found in the goldfish OPL. The main findings are summarized in Fig.\u00a07. Cell types have been identified based on the results obtained at both light-microscopy and electron-microscopy levels.\nFig.\u00a07Representation of a rod spherule, a M\u00fcller cell process, and a cone pedicle, depicting the localization of PKC and the patterns yielded by the various mGluR antibodies (red group I mGluRs, green group II mGluRs, blue group III mGluRs, SR synaptic ribbon)\nIn the rod spherule, PKC labels invaginating dendrites of putative ON MBCs. The mGluR1\u03b1 antibody labels similar structures and some lateral elements. Lateral elements of rod triads are also positive for mGluR7. M\u00fcller cell processes between the rod spherules are positive for mGluR5. We have found no evidence for mGluR2\/3 in the rod synaptic complex.\nIn the cone pedicles, PKC labels fine dendrites at the base of the terminal or invaginating structures away from the ribbons. Elements at similar positions and with similar fine structure are positive for mGluR1\u03b1 and mGluR7. BC central processes in the cone triads are positive for mGluR1\u03b1. Spinules are positive for mGluR1\u03b1 and mGluR7, and lateral elements are labeled by the mGluR1\u03b1, mGluR2\/3, and mGluR7 antibodies. Finally, HC central elements are positive for mGluR2\/3.\nSpecificity of the antibodies\nNo data are available on the amino acid and\/or nucleotide sequences of goldfish mGluRs. The closest available information is from the zebrafish. This database is however not fully completed, and the various zebrafish mGluRs have not been unambiguously identified (Table\u00a02). The finding of candidates for zebrafish mGluR2 and mGluR6 has been reported, but the actual nucleotide and amino acid sequences have not been disclosed (Bjarnadottir et al. 2005).\nWe therefore used multiple data sources to infer the specificity of the antibodies used in this study: Western blots and BLAST searches in the zebrafish genome for the amino acid sequences in Table\u00a01 (at http:\/\/www.ncbi.nlm.nih.gov\/BLAST\/), the sequence alignments (with http:\/\/www.ch.embnet.org\/software\/LALIGN_form.html and http:\/\/bioinfo.genopole-toulouse.prd.fr\/multalin\/multalin.html), and the molecular mass calculations (at http:\/\/www.basic.northwestern.edu\/biotools\/proteincalc.html). We also \u201cBLASTed\u201d the proteins retrieved from the zebrafish database against all other species available, in order to check for protein similarities among species.\nTo choose good candidates for fish mGluRs among the BLAST hits, we relied on the scores and E-values; the cutoff was E<0.01. We adopted a stringent significance level, because the chance of obtaining random matches with short amino acid sequences was relatively large. All scores used for these comparisons were higher than 1,000. For the C-terminal mGluR5 peptide, the cutoff was lowered to E=0.35, since no candidates could be found with the same stringency as for the remainder of the peptide sequences.\nAs summarized in Table\u00a02, all of the anti-mGluR antibodies used in this study were raised against polypeptides that yielded mGluR-like hits when \u201cBLASTed\u201d against the zebrafish database. Two homologs were found for mGluR1, but one of these (XP_001346460) was an incomplete sequence that might have corresponded to a fragment of the other (XP_690446), since they only differed by one amino acid and their respective coding sequences by two nucleotides. For mGluR2\/3, three putative mGluRs were identified (Table\u00a02), but one of them had a slightly smaller molecular mass than the other two (85\u00a0kDa). We did not however detect multiple proteins around 80\u2013100\u00a0kDa in our immunoblots, which suggested that XP_001341692 was not present in the goldfish retina.\nAccording to the database search, the C-terminal peptides of mGluR5 and mGluR7 have one fish homolog each. The mGluR5 candidate shows lowest amino acid sequence similarity at the C-terminus, but the lack of overlapping expression patterns between mGluR1 and mGluR5 in the OPL of the goldfish retina excludes cross-reactivity of anti-mGluR5 and mGluR1.\nIn summary, we feel confident about the specificity of the commercially available anti-mGluR peptide antibodies, since the carboxytermini of mammalian and fish mGluR homologs are nearly identical, and the molecular masses of fish and mammalian homologs are similar (Table\u00a02).\nmGluRs of M\u00fcller cells\nThe finding of mGluR5 in M\u00fcller cells is in agreement with two previous studies of isolated M\u00fcller cells from the salamander; these studies have shown that activation of group I mGluRs has two major effects. First, it leads to the closure of the K+ conductance of these cells, which is by far the largest in M\u00fcller cells (Schwartz 1993). Since their glutamate uptake is both voltage-dependent (Brew and Attwell 1987; Schwartz and Tachibana 1990) and regulated by the internal K+ concentration (Barbour et al. 1988), one can imagine that mGluR5, by modulating this K+ conductance, indirectly modulates glutamate uptake by changing the membrane potential and\/or internal K+ concentration.\nThe second effect of group I mGluR activation in M\u00fcller cells is the generation of Ca2+ waves by triggering the release of Ca2+ from internal stores (Keirstead and Miller 1997). The physiological consequences of this effect are not completely clear, but they could contribute to lateral signaling among M\u00fcller cells, as in astrocytes (Kim et al. 1994).\nmGluRs of HCs\nHere, we show that rod and cone-driven HCs of the goldfish express mGluRs from all three groups, although the exact mGluR composition of these cells differs slightly from reports in other species (i.e., no mGluR1 in cat HCs: Cai and Pourcho 1999; no mGluR2\/3 in catfish HCs: Gafka et al. 1999).\nWe have found that lateral elements in the rod triads are positively labeled for mGluR1\u03b1 and mGluR7. Since the labeling for mGluR1\u03b1 is rare, we cannot at this point positively identify these processes as belonging to either rod-driven HCs or MBCs. In the cone pedicle, mGluRs seem to be expressed in a highly organized manner. mGluR1\u03b1 and mGluR7 tend to be localized to spinules and to dendrites occupying the lateral position in the cone triad, whereas mGluR2\/3 has been found at both lateral and central processes directly opposing the synaptic ribbon.\nThis extremely ordered pattern indicates that either a single cone-driven HC type expresses distinct mGluRs in different dendrites, or that the expression of a given mGluR is restricted to a certain subpopulation of HCs (i.e., the distribution is cell-type-specific). In cyprinid retinae, three physiological and morphological HC types have been described (MacNichol and Svaetichin 1958; Norton et al. 1968; Stell et al. 1982); one could easily imagine that each HC class expresses only one or two mGluRs at their dendritic tips.\nThe light responses of HCs are mediated by AMPA (2-amino-4-phosphonobutyric acid)\/kainic acid receptors (Lasater and Dowling 1982; Slaughter and Miller 1983; Zhou et al. 1993) and are not abolished by mGluR agonists or antagonists (Yang and Wu 1989; Luo and Liang 2003), showing that mGluRs are not implicated in the direct generation of light responses. They are most likely involved in the modulation of the light-driven conductance of these cells. In the fish retina, for instance, mGluR agonists have been shown to modify both light-modulated (Nawy et al. 1989; Yasui et al. 1990; Takahashi and Copenhagen 1992) and voltage-gated conductances of cone-driven HCs (Dixon and Copenhagen 1997; Linn and Gafka 1999; Gafka et al. 1999).\nmGluRs of ON MBCs\nThe labeling of ON MBC processes, in the rod spherule, with the antibody against mGluR1\u03b1 corroborates previous studies in fish (Klooster et al. 2001; Yazulla and Studholme 2001; Yazulla et al. 2001) and mammalian (Koulen et al. 1997) species, although it differs from the situation in the cat retina, in which mGluR1\u03b1 is pre-synaptic to rod-driven BCs (Cai and Pourcho 1999). Since no evidence has as yet been presented for the participation of group I mGluRs in the generation of the light responses of ON BCs, one can only speculate that this protein plays a modulatory role in the first synapse. In many parts of the central nervous system, group I mGluRs are involved in controlling the intracellular calcium concentration and membrane channel activity (for a review, see Fagni et al. 2000). In the spinal cord neurons of the lamprey, for instance, mGluR1 has been shown to regulate the resting membrane potential and thus the excitability of the cells by controlling a leak current (Kettunen et al. 2003).\nAlthough there is physiological evidence for the existence of a group III mGluR in fish ON MBCs (Shiells et al. 1981; Nawy and Copenhagen 1987; Kamermans et al. 2004), we have not been successful in obtaining specific immunoreactivity with the antibodies commercially available against (mammalian) mGluR4, mGluR6, and mGluR8. Furthermore, there is as yet no candidate for mGluR6 in the zebrafish database, which makes the production of custom-made antibodies against this protein difficult. One would however expect these cells to express an mGluR6-like receptor, given the many physiological similarities between the rod-driven conductance in fish and mammalian ON BCs.\nWhether and in what manner this mGluR6-like receptor and the mGluR1\u03b1-like protein interact in ON MBCs is still to be resolved. G-protein-coupled receptors can interact at many levels. For instance, gamma-aminobutyric acidB receptors form complexes with mGluR1\u03b1 dimers and thereby regulate the sensitivity of the latter to glutamate (for a review, see Kubo and Tateyama 2005). Interactions between mGluRs, however, have so far not been described. Another level of interaction lies within the second-messenger cascade. Until recently, only one G-protein (Go\u03b1) had been described in ON BCs (Vardi et al. 1993), which suggests that mGluR1\u03b1 and mGluR6 share (part of) the same intracellular machinery. Nonetheless, a second G-protein (Gi2\u03b1) has been shown to coexist with Go\u03b1 in rat ON BCs and not to couple to mGluR6 (Tian and Kammermeier 2006), providing an independent G-protein signaling pathway within these cells; this pathway might involve mGluR1\u03b1.","keyphrases":["metabotropic glutamate receptor","retina","immunohistochemistry","bipolar cell","horizontal cell","electron microscopy","goldfish, carassius auratus (teleostei)"],"prmu":["P","P","P","P","P","P","M"]}
{"id":"J_Mol_Biol-1-5-2098880","title":"Kinetic Analysis of the Slow Skeletal Myosin MHC-1 Isoform from Bovine Masseter Muscle\n","text":"Several heavy chain isoforms of class II myosins are found in muscle fibres and show a large variety of different mechanical activities. Fast myosins (myosin heavy chain (MHC)-II-2) contract at higher velocities than slow myosins (MHC-II-1, also known as \u03b2-myosin) and it has been well established that ADP binding to actomyosin is much tighter for MHC-II-1 than for MHC-II-2. Recently, we reported several other differences between MHC-II isoforms 1 and 2 of the rabbit. Isoform II-1 unlike II-2 gave biphasic dissociation of actomyosin by ATP, the ATP-cleavage step was significantly slower for MHC-II-1 and the slow isoforms showed the presence of multiple actomyosin\u2013ADP complexes. These results are in contrast to published data on MHC-II-1 from bovine left ventricle muscle, which was more similar to the fast skeletal isoform. Bovine MHC-II-1 is the predominant isoform expressed in both the ventricular myocardium and slow skeletal muscle fibres such as the masseter and is an important source of reference work for cardiac muscle physiology. This work examines and extends the kinetics of bovine MHC-II-1. We confirm the primary findings from the work on rabbit soleus MHC-II-1. Of significance is that we show that the affinity of ADP for bovine masseter myosin in the absence of actin (represented by the dissociation constant KD) is weaker than originally described for bovine cardiac myosin and thus the thermodynamic coupling between ADP and actin binding to myosin is much smaller (KAD\/KD \u223c 5 instead of KAD\/KD \u223c 50). This may indicate a distinct type of mechanochemical coupling for this group of myosin motors. We also find that the ATP-hydrolysis rate is much slower for bovine MHC-II-1 (19 s\u22121) than reported previously (138 s\u22121). We discuss how this work fits into a broader characterisation of myosin motors from across the myosin family.\nIntroduction\nThe myosin superfamily consists of at least 18 classes of ATP-dependent motor proteins that interact with actin filaments and are involved in a large variety of physiological processes, such as muscle contraction, phagocytosis, cell motility and vesicle transport.1,2 Class II myosins (found in muscle) consist of two heavy chains (MHC) and two pairs of light chains: the regulatory light chains (RLC) and essential light chains (ELC). The C termini of the myosin heavy chains dimerize and form a coiled-coil (\u201cmyosin tail\u201d) whereas the N-termini form the two myosin \u201cheads\u201d or \u201cmotor-domains\u201d. The most familiar activity of class II myosins is muscle contraction. All myosins appear to undergo the same ATP driven cycle of interaction of myosin with actin, known as the cross-bridge cycle, yet myosins show a wide variety of different mechanical activities. We are interested in the variety of different types of mechanochemical activities shown both within and between the members of the myosin family.\nMyosins from fast contracting, anaerobic, muscle fibres are amongst the best characterized myosins with events of the biochemical cross-bridge cycle having been correlated with mechanical and structural data from contracting muscle fibres. These fast myosins comprise a subgroup of the myosins family known as MHC-II-2 with three primary isoforms found in the mammalian adult skeletal muscle, 2a, 2b and 2x, each expressed from a different gene.3 The isoform MHC-II-1 is found in slow, aerobic skeletal muscle fibres and in ventricular cardiac muscle tissue where it is known as MHC-\u03b2. The myosin light chains (MLC) also differ between myosins with muscle-specific isoforms of both light chains.4 Whereas a single regulatory light chain isoform (MLC2s) is found in both the slow skeletal muscle and in the ventricle of mammals that have been examined, there are two isoforms of slow muscle essential light chains. One is found predominantly in the adult slow skeletal and the ventricle muscle (called MLC-1s\/v or MLC-1s\/b). The other (MLC-1sa) predominates in smooth and non-muscle tissue and in some skeletal muscle during development.5 The MLC-1s\/b is larger and includes and an N-terminal extension, rich in proline and lysine. Unlike molluscan and smooth muscle myosins, where light chain phosphorylation and calcium binding are the primary regulators of myosin's activity, the light chains of striated muscles seem to have relatively little effect on enzymatic activity of the isolated myosin head.6 However, the presence of specific light chain isoforms and their removal does have substantial effects on the generation of force and motility by striated myosin.6 The MLC stabilise the lever-arm or neck of the myosin head and they are in a position to modulate the translation of the isomerisation of the head into mechanical force or movement.7\nFast muscle fibres containing the MHC-II-2, as their name suggests, contract at higher velocities and are more susceptible to fatigue than slow muscle fibres containing MHC-II-1 and both properties have been related to the properties of the myosin heavy chain isoform expressed in the muscle. It is well established that ADP binding to actomyosin is much tighter for MHC-II-1 (slow isoform) than for MHC-II-2 (fast isoform).8 We recently examined the solution biochemical kinetic properties of the slow isoform MHC-II-1 from rabbit soleus muscle and compared it to the well characterised fast isoform MHC-II-2 from rabbit psoas muscle.9 This showed several significant differences between the two isoforms including:(1)Unlike MHC-II-2, the MHC-II-1 gave biphasic dissociation of actomyosin by ATP.(2)The tight binding of ADP to actomyosin was confirmed for MHC-II-I with at least three conformations of actomyosin\u2013ADP present in equilibrium.(3)The rate constant for ADP release from the predominant actomyosin\u2013ADP complex was the correct size to limit the shortening velocity of MHC-II-1 containing soleus muscle fibres.(4)MHC-II-1 has a significantly reduced rate constant for the ATP hydrolysis step.\nTogether these results indicate that the kinetic differences between the fast and slow MHC-II isoforms are greater than at first expected and that MHC-II-1 has much in common with some of the much slower smooth and non-muscle myosins (see Discussion).\nThree of the above novel observations (numbers (1), (2) and (4)) were in contrast to published data on the same isoform isolated from bovine ventricle muscle. Bovine MHC-II-1 has been widely studied from cardiac ventricle tissue and is an important source of reference work for cardiac muscle physiology.8,10 It is therefore essential to test if the differences observed between the rabbit and bovine isoforms are due to the experimental differences or species differences. The previous studies on bovine cardiac S1 (BCS1) reported that the ATP-hydrolysis rate is much higher (k+3\u00a0+\u00a0k-3\u00a0=\u00a0138 s\u22121)11,12 than the hydrolysis rate reported for rabbit soleus S1 under similar conditions (RSS1, k+3\u00a0+\u00a0k-3\u00a0=\u00a022 s\u22121).9 Also, unlike RSS1, the ATP-induced dissociation of AM, in the presence or absence of ADP, could not be fitted unambiguously to two components.10 In order to address these differences we used bovine MHC-II-1 isolated from the slow, bovine masseter muscle. Bovine masseter muscle is large, easily accessible and contains >95% MHC-II-1, which makes it convenient for large scale biochemical preparations.13 This tissue is readily available and the isolated myosin and S1 are stable when stored at \u221220\u00a0\u00b0C. In order to further examine potential species differences we also isolated myosin S1 from pig soleus fibres as another source of MHC-II-1 myosin. This tissue is not as pure in myosin isoform content14 but does provide a second reference material. Masseter muscle fibres have a maximum shortening velocity (V0\u00a0=\u00a00.27\u00a0\u03bcm s\u22121) of 40% of the value for rabbit soleus fibres (V0\u00a0=\u00a00.67\u00a0\u03bcm s\u22121) at 12\u00a0\u00b0C13 whereas the pig soleus fibres are even slower (V0\u00a0=\u00a00.17\u00a0\u03bcm s\u22121).13,15 Since V0 has been shown to be a property of the MHC isoform, the species differences in the isoforms used here are expected to be significant. The work presented here confirms the primary finding from the work of rabbit soleus, and extends these studies to slow bovine and porcine myosins. Of significance is that we show that the affinity of ADP for bovine masseter myosin is weaker than originally described for bovine cardiac myosin and thus the thermodynamic coupling between ADP and actin binding to myosin is much smaller10,11 (KAD\/KD \u223c5 instead of KAD\/KD\u00a0\u223c\u00a050). This may indicate a distinct type of mechanochemical coupling for this group of myosin motors. We also find that the ATP-hydrolysis rate is much slower for BMS1 (19 s\u22121) than reported previously for BCS1 (138 s\u22121).\nWe discuss how this work fits into a broader characterization of myosin motors from across the myosin family. Indeed the relevance of our observations on the significant differences between fast and slow muscle myosin isoforms in the release of ADP has been underlined by a recent study by Iwamoto et al.,16 published as we submitted this work. They have shown, using a novel X-ray scattering approach, that slow muscle acto\u2013\u2013S1 has a distinct movement of the lever arm on binding ADP, whereas little movement was detected with a fast muscle isoform. Larger movements were observed with the S1 from smooth and non-muscle myosin II isoforms. These observations are consistent with earlier electron micrograph images of ADP induced movements of the lever arm of smooth muscle17 and cytoplasmic myosin from the wider myosin family, such as myosin I18 and myosin VI.19 Thus, biochemical and structural studies are coming together to define how ADP release is a key event in defining the mechano-chemical coupling of different types of myosin.\nResults\nNucleotide binding to BMS1 in the absence of actin\nMHC-II-1 myosin was purified from bovine masseter muscle fibres (see Materials and Methods). Figure 1 shows the isolated myosin with two light chains and the purified BMS1 with only one light chain bound. The binding of ATP or ADP to myosin S1 is characterised by an increase in intrinsic protein fluorescence associated with nucleotide binding. Figure 2(a) shows the fluorescence change observed on rapidly mixing 25\u00a0\u03bcM or 400\u00a0\u03bcM ATP with 0.5\u00a0\u03bcM BMS1 at 20\u00a0\u00b0C (concentrations are post mixing). At 25\u00a0\u03bcM the transient increase in fluorescence (\u0394F) shown can be satisfactorily described by a two exponential function with kobs\u00a0=\u00a019.6 s\u22121 (\u0394F +11.8%) and 2.5 s\u22121 (\u0394F +4%). The fast phase is easily separated from the slower component and at low ATP concentrations (<50\u00a0\u03bcM) appears linearly dependent on ATP concentration with an apparent second-order rate constant of 0.97\u00a0\u00d7\u00a0106 M\u22121s\u22121. At higher ATP concentrations the kobs value saturates and a hyperbolic fit to the data gives a K0.5 value of 56\u00a0\u03bcM with a kmax of 117 s\u22121 (Figure 2(b)). The slow phase only appears as a single process at low ATP concentrations (\u226450\u00a0\u03bcM); at higher ATP there appear to be two components. Separating out these two phases across the full range of ATP concentrations is problematic but at ATP concentrations above 50\u00a0\u03bcM, the data can all be described by a small slow phase of amplitude 1\u20131.5% with a kobs of 2 s\u22121 and a medium rate phase with kobs of 12-15 s\u22121 and an amplitude of 4%. This is illustrated in Figure 2(a) for [ATP]\u00a0=\u00a0400\u00a0\u03bcM where the transient increase in fluorescence (\u0394F) shown can be satisfactorily described by a triple exponential function (with kobs\u00a0=\u00a0111 s\u22121 (\u0394F +10%), kobs\u00a0=\u00a013.5 s\u22121 (\u0394F +3.7%) and kobs\u00a0=\u00a02 s\u22121 (\u0394F +1%) for the fast, medium and slow phase, respectively). The analysis of the data shown in Figure 2(b) has been fitted assuming a constant slow phase of 2 s\u22121. The medium phase process at 12\u201315 s\u22121 is then only apparent when the fast component is faster than \u223c30 s\u22121 and then appears to be constant. In order to rule out contamination with ADP as the cause of the slower phases, BMS1 was extensively treated with apyrase but this had little effect on the observed transients.\nThe K0.5 and kmax of the fast phase can be assigned to K1 and k+2 of Scheme 1, respectively, the steps thatcontrol ATP binding as observed for many other myosin isoforms. One of the two slower phases could be due to the ATP hydrolysis step of ATP (k+3\u00a0+\u00a0k-3 in Scheme 1) that is known to generate changes in fluorescence for other myosins as described.20,21 In order to determine what is being observed here, quench flow measurements were done in order to establish the rate of ATP hydrolysis.\nBMS1 (5\u00a0\u03bcM) was mixed with excess ATP (50\u00a0\u03bcM) and incubated at 20\u00a0\u00b0C for desired time intervals (up to 350\u00a0ms). Figure 3 shows a rapid initial burst of ADP production, which can be described by a single exponential function with kobs of 19 s\u22121 and an amplitude of 0.61 ADP\/S1. The amount of irreversibly bound ATP to BMS1 at the end of the burst was determined by quenching the reaction into a hexokinase\/glucose buffer after 1\u00a0s and then adding 6.5% trichloroacetic acid (TCA) after a further 1\u00a0s. In controls this treatment hydrolysed any free ATP to ADP. After HPLC analysis of the reaction mixture 0.14 ATP\/S1 remained and therefore at the end of the burst phase 0.14 ATP\/S1 remains as tightly bound M*.ATP and 0.61 ADP\/S1 as M**.ADP.Pi (Scheme 1). Thus the value of K3\u00a0=\u00a0[M.ADP.Pi]\/ [M*.ATP]\u00a0=\u00a04.3. This is similar to the values reported for fast muscle S1.22,23 The value of the kobs can be assigned to k+3\u00a0+\u00a0k-3 and at 19 s\u22121 is similar to the medium phase of the stopped-flow fluorescence transient (kobs\u00a0=\u00a015 s\u22121). Thus, as for other myosins the hydrolysis step is accompanied by a fluorescence change. It is worth noting that this is quite a slow process, compared to the hydrolysis rate for fast skS1 from rabbit, which is tenfold faster, but similar to the hydrolysis rate for the slow myosin S1 from rabbit or pig soleus muscle9 (see Table 1). The origin of the slowest component of the fluorescence transient remains to be defined. This is small (1\u20131.5%) and has a constant kobs value of 2 s\u22121. This could arise from a small fraction of damaged myosin, from a contaminant myosin or from a fraction of the myosin that must isomerise before ATP can bind to it (see Discussion of ATP binding to actomyosin). The signals are very weak and represent a small fraction of the total making it very difficult to investigate and we have not pursued this further. Arrhenius plots of the maximal observed rates of the fast phase (k+2) and the medium phase (k+3\u00a0+\u00a0k-3) and van 't Hoff plot of K1 (not shown) show that between 8\u00a0\u00b0C\u201320\u00a0\u00b0C K1 does not depend on temperature. k+2 and k+3\u00a0+\u00a0k-3 have a marked temperature dependence with similar activation energies of 71\u00a0kJ\/mol and 75\u00a0kJ\/mol, respectively. The ratio of the amplitudes for the fast and medium phase remains fairly constant between 8\u00a0\u00b0C\u201320\u00a0\u00b0C (ratio\u00a0\u223c\u00a04:1).\nThe rate of ADP-association with BMS1 was also measured. In this case the fluorescence increase was smaller (\u0394F 3%) than seen for ATP. There was no evidence of a slow component of the reaction and the transient increase in fluorescence could be fitted to a single exponential. At high ADP concentrations kobs reaches a maximum value of 26 s\u22121 when fitted to a hyperbola (Figure 2(c)). The ADP-release rate k+6 (=k-D) can be determined from the intercept, k+6\u00a0=\u00a01.4(\u00b10.5) s\u22121. The maximal rate of ADP-binding represents k+6\u00a0+\u00a0k-6 (kmax,ADP)\u00a0=\u00a026 s\u22121 and K7\u00a0=\u00a0K0.5,ADP\u00a0=\u00a021\u00a0\u03bcM. The second-order constant of ADP binding k-6\/K7 (or k+D) can be defined by kmax,ADP\/K0.5,ADP\u00a0=\u00a01.24\u00a0\u00d7\u00a0106 M-1s\u22121 (see also Table 1) from which a value for the equilibrium dissociation constant can be calculated: KD (1\/K6K7)\u00a0=\u00a01.2\u00a0\u03bcM. Repeating the ADP-binding experiment using the slow myosin S1 isolated from pig soleus muscle, PSS1, showed that this myosin S1 had a slightly tighter affinity for ADP (KD\u00a0=\u00a00.9\u00a0\u03bcM; see Table 1).\nAn alternative method of determining k+6 (or k-D) is the displacement of ADP from BMS1 by addition of an excess ATP. Figure 4(a) and (b) shows the reaction observed on adding 100\u00a0\u03bcM ATP to 0.5\u00a0\u03bcM BMS1 in the presence of 0.2\u00a0\u03bcM ADP. The best fit of the fluorescence changes is the sum of three exponentials with kobs of 68 s\u22121, 9.6 s\u22121 and 1.1 s\u22121. Two of the observed three phases are due to the binding of ATP to ADP-free BMS1 (68 s\u22121) and the hydrolysis step (9.6 s\u22121), as described above, and the third phase, the binding of ATP, is limited by ADP release from BMS1-ADP (see Figure 4(a) and (b)), with an ADP-off rate of 1.1 s\u22121. Using a triphasic fit the observed rate constants are fairly independent of ADP concentration (average rate constants for the fast, medium and slow phase are 75 s\u22121, 8 s\u22121 and 0.9 s\u22121, respectively) but the measured amplitudes depend on the ADP concentrations. The amplitude of the fast phase (ATP-binding to BMS1) and medium phase (ATP-hydrolysis) decrease and of the slow phase (ADP dissociation) increases as the ADP concentration is increased (Figure 4(c)). The relative ratio of the amplitudes for the fast and medium phase remain constant up to 2\u00a0\u03bcM ADP (Afast: Amedium\u00a0\u223c\u00a04: 1). At higher ADP concentrations the amplitude of the ATP hydrolysis process, Amedium, becomes very small but the observed rate constant, kobs, is not affected by [ADP]. In order to be able to separate Afast and Amedium at high ADP concentrations, the relative ratio Afast\/Amedium was fixed at 4. The ADP dependence of the amplitudes could be described by hyperbolas with independent best-fit dissociation constants of 2.6\u00a0\u03bcM (fast phase), 3.0\u00a0\u03bcM (medium phase) and 3.0\u00a0\u03bcM (slow phase), resulting in an average KD\u00a0=\u00a02.8\u00a0\u03bcM. This is in reasonable agreement with the value found previously using the ADP binding experiment in Figure 2 (KD\u00a0=\u00a01.2\u00a0\u03bcM) and we will use the median value of 2.0. A tighter affinity of ADP for the S1 product of bovine cardiac MHC-1 myosin was reported (KD\u00a0=\u00a00.33\u00a0\u03bcM) when the interaction of ATP and ADP with bovine cardiac S1 was investigated.11\nNucleotide binding to MHC1 myosin S1 in the presence of actin\nThe ATP-induced dissociation of acto\u2013MHC-1 complexes can be followed by monitoring the fluorescence of a pyrene label covalently attached to Cys374 of actin. At ATP concentrations above 100\u00a0\u03bcM, the ATP-induced dissociation of bovine masseter myosin S1 (BMS1) is biphasic, as is illustrated in Figure 5(a) and (b), whereas below 100\u00a0\u03bcM ATP the fluorescence can be fitted to a single exponential. Figure 5(a) shows the resulting change in fluorescence upon mixing 0.5\u00a0\u03bcM pyrene-labelled acto\u2013BMS1 complex with 200\u00a0\u03bcM ATP. At this ATP concentration the reaction can be best described by a double exponential with kobs\u00a0=\u00a0278 s\u22121 (\u0394F 77%) and 58 s\u22121 (\u0394F 10%) for the fast and the slow phase, respectively. The fast phase shows a hyperbolic dependence on [ATP] and saturates at kobs\u00a0=\u00a0900\u20131100 s\u22121 whereas the slow phase remains constant at 56(\u00b113) s\u22121 at [ATP]\u00a0>\u00a0100\u00a0\u03bcM (Figure 5(b)). The rate constant of the slow phase (kslow\u00a0=\u00a056 s\u22121) is similar to the rate constant of ADP-release (94 s\u22121) and one could argue it is possible that the slow phase is due to contamination with ADP. However, prolonged incubation of BMS1 with apyrase did not remove the slow phase. The presence of another myosin isoform contamination might also cause the biphasic behavior. Bovine masseter fibres are not 100% MHC-1\u03b2 but contain a small amount (5%) of MHC-1\u03b1 isoform but this is a fast isoform and is therefore unlikely to cause an additional slow phase.13 The presence of non-muscle myosins (either class 2 or others) could produce a slow phase but the amplitude of the slow phase (15%) is far too large to come from such a source. For BMS1, according to scheme II, the kobs for the slow phase observed here can be assigned to k+\u03b1\u00a0=\u00a056 s\u22121. From the ratio of the amplitudes of the fast and slow phase of ATP-induced acto\u2013BMS1 dissociation (77:10) the value of K\u03b1\u00a0=\u00a07.7, can be assigned. Since K\u03b1\u00a0=\u00a0k+\u03b1\/k-\u03b1 and k+\u03b1\u00a0=\u00a056 s\u22121 then k-\u03b1\u00a0=\u00a07.3 s\u22121. The temperature-dependence of the ATP-induced dissociation of acto\u2013BMS1 was measured between 8\u00a0\u00b0C \u221232\u00a0\u00b0C at a low ATP-concentration (50\u00a0\u03bcM). From the dependence of kobs on temperature the activation energy can be calculated as Ea\u00a0=\u00a022\u00a0kJmol\u22121 (not shown).\nThe effect of ADP on the dissociation rate constant was measured at two different ATP concentrations: [ATP]\u00a0=\u00a050\u00a0\u03bcM and [ATP]\u00a0=\u00a0200\u00a0\u03bcM. At [ATP]\u00a0=\u00a050\u00a0\u03bcM, the ATP-induced dissociation of 0.5\u00a0\u03bcM pyrene-labelled acto\u2013BMS1, pre-incubated with 0\u2013250\u00a0\u03bcM ADP, yielded a fluorescence change of 55% that could be fitted to a single exponential. The resulting kobs was plotted against ADP concentration (see Figure 6(a)) and fitting against equation (4) resulted in a KAD of 9.6(\u00b11.4)\u00a0\u03bcM. Presuming the on-rate is diffusion- limited (\u223c107 M\u22121s\u22121) one can calculate the ADP-off-rate (k-AD\u00a0=\u00a096 s\u22121). Reducing the temperature to 12\u00a0\u00b0C reduced the measured KAD to 5\u00a0\u03bcM and thus also the calculated ADP off-rate (50 s\u22121). At higher ATP concentration (200\u00a0\u03bcM) the fluorescence change of the ATP-induced dissociation of BMS1-actin, in the absence of ADP, can be fitted to a double exponential and both kobs values decreased with increasing ADP concentrations. Plotting the kobs values versus the ADP-concentration (Figure 6(b)) yielded KAD values of 6.8(\u00b11.7)\u00a0\u03bcM (fast phase) and 6.6\u00a0\u03bcM(\u00b11.9)\u00a0\u03bcM (slow phase), indicating that the kobs of both phases are inhibited by ADP.\nThe temperature dependences of these two phases are measured at very high ATP concentrations ([MgATP]\u00a0=\u00a016\u00a0mM) in order to define the rate of ADP release. The ATP-induced dissociation of the acto\u2013BMS1 complex in the presence of saturating ADP concentrations (150\u00a0\u03bcM) is biphasic with a fast kobs\u00a0=\u00a094 s\u22121 (\u0394F 55%) and a slow kobs\u00a0=\u00a09.6 s\u22121 (\u0394F 10%) at 20\u00a0\u00b0C (see Figure 7(a)). According to the model depicted in Scheme 2, these two processes are due to the presence of two actomyosin complexes with ADP bound in solution, A\u00b7M'\u00b7D and A\u00b7M\u00b7D. The fast phase measured here (94 s\u22121) is due to the A\u00b7M'\u00b7D complex and corresponds well with the calculated ADP off-rate (96 s\u22121) and, according to Scheme 2 represents k-ADP. The other complex, A\u00b7M\u00b7D, is responsible for the slow phase observed here and, according to Scheme 2, represents the rate-limiting step of ADP-release (k+\u03b1D\u00a0=\u00a09.6 s\u22121). Between 12\u00a0\u00b0C\u201325\u00a0\u00b0C the temperature dependences of these observed fast and slow rates are almost identical (see Figure 6(b)) with similar activation energy values Ea\u00a0=\u00a0106\u00a0kJ (fast phase) and 98 kJ (slow phase). The ratio of amplitudes of the two phases, Afast:Aslow had a small dependence on the temperature with Af:As\u00a0=\u00a06.3 at 12\u00a0\u00b0C and 5.1 at 25\u00a0\u00b0C.\nThe small thermodynamic coupling (KAD\/KD) between actin and ADP binding to BMS1 (KAD\/KD\u00a0=\u00a04.8) can be validated by measuring the affinity of S1 for actin in the presence and absence of ADP (KDA\/KA). Phalloidin-stabilised pyr-actin (15\u00a0nM) incubated with various concentrations of BMS1 were mixed with ATP (10\u00a0\u03bcM), and the amplitude of the dissociation reaction was used to estimate the fraction of actin bound to BMS1. Without ADP present, kobs remained constant at all S1 concentrations (kobs\u00a0=\u00a019 s\u22121) and the amplitude increased hyperbolically with [BMS1]. In the presence of 250\u00a0\u03bcM ADP, the value for kobs was reduced (15\u00a0s\u22121) and the amplitudes increased hyperbolically with [S1] (see Figure 8). In the absence of ADP the best fit to the titration gave a KA\u00a0\u223c\u00a04\u20137\u00a0nM, whereas in the presence of ADP the best fit yielded a KDA of 37\u00a0nM. Thus, ADP reduced the affinity of S1 for actin by about fivefold (KDA\/KA\u00a0=\u00a037\/7\u00a0=\u00a05.2), which is similar to the reduction in ADP affinity for S1 caused by actin (KAD\/KD\u00a0=\u00a04.8).\nUsing MHC-II-1 myosin S1 from pig soleus muscle (PSS1) very similar results were obtained and these are summarised in Tables 1 and 2. The ATP-induced dissociation of acto\u2013PSS1 was biphasic at high ATP with a fast phase (kmax\u00a0=\u00a0800\u2013900 s\u22121) and a slow phase (kmax\u00a0=\u00a067 s\u22121), and a relative ratio Afast:Aslow\u00a0=\u00a04.8 (data not shown). This yields a value for K\u03b1\u00a0=\u00a04.8, and together with the measured value for k+\u03b1\u00a0=\u00a067 s\u22121 gives k-\u03b1\u00a0=\u00a014 s\u22121 (Table 2). In the presence of ADP (0\u2013200\u00a0\u03bcM) the ATP-induced dissociation of acto\u2013PSS1 also gave fluorescence transients that fitted a single exponential at low ATP concentrations (<100\u00a0\u03bcM) and a double exponential at high ATP concentrations (data not shown) and a KAD of 15\u00a0\u03bcM. At very high ATP concentrations (4\u00a0mM MgATP) the ATP-induced dissociation of the acto\u2013PSS1 complex in the presence of saturating ADP concentrations (50\u00a0\u03bcM) is also biphasic with a fast rate kobs,f\u00a0=\u00a048 s\u22121 and a slow phase kobs,s\u00a0=\u00a06.5 s\u22121 at 20\u00a0\u00b0C. The ratio of the two amplitudes Afast:Aslow is 6.5 (20\u00a0\u00b0C) and represents K\u03b1D (see Table 2).\nDiscussion\nThis paper set out to reexamine and extend the kinetic analysis of the bovine MHC-II-1 isoform of myosin found in slow skeletal muscle and the ventricle of cardiac muscle. Bovine masseter muscle fibres were successfully used to isolate the S1 construct (BMS1) of bovine MHC-II-1 myosin. Comparison of the kinetic parameters of BMS1 and the S1 construct isolated from the left ventricle of bovine cardiac tissue (BCS1),10,11 which is 100% MHC-II-1 and has the same light chain content, shows that theresults agree reasonably well with each other (Tables 1 and 2). The two parameters that deviate significantly between BMS1 and BCS1 are discussed below.\nThe ATP hydrolysis rate constant reported here is almost tenfold slower than that reported.12 The previously reported rate constant was based on the assumption that when ATP binds to S1, the maximum rate of the fluorescence change represents k+3\u00a0+\u00a0k-3 (Scheme 1), in analogy with measurements for fast myosin S1.20,21 However, the quench flow data reported here show that k+3\u00a0+\u00a0k-3 defines the medium phase observed in the stopped flow measurements and the maximum observed rate constant of the medium phase is k+3\u00a0+\u00a0k-3\u00a0=\u00a019 s\u22121, whereas the fast phase represents k+2. The ATP hydrolysis step of BMS1 is much slower than that of fast skeletal S1 from rabbit (k+3\u00a0+\u00a0k-3\u00a0=\u00a0131 s\u22121) but it is similar to the slow skeletal myosin II, MHC-II-1 from rabbit soleus (22 s\u22121)9 and pig MHC-II-1 (24.5\u00a0s\u22121).24 It may therefore be a general feature of slow myosin II isoforms. The role of this reduced ATP hydrolysis rate for MHC-II-1 could be related tothe slow ADP-release rates also observed for MHC-II-1. A slow ADP-release rate increases the time myosin is bound to actin (i.e. enhances the duty ratio). MHC-II-1 can compensate for this by a slow hydrolysis step thereby maintaining the low duty ratio typical of striated muscle myosins.\nThe affinity of ADP for BMS1 in the absence of actin (KD\u00a0=\u00a02\u00a0\u03bcM) is weaker than the value reported for BCS1 (KD\u00a0=\u00a00.33\u00a0\u03bcM) when the interaction of ATP and ADP with BCS1 was investigated.11 Due to aggregation problems in the BCS1 work, the interaction with ATP was reinvestigated a year later12 but as far as we are aware, the interaction with ADP has never been re-examined and this may therefore account for the discrepancy between the KD values. Table 1 shows that the k+6 values measured for BMS1 and BCS1 differ two- to threefold (k+6\u00a0=\u00a00.9\u20131.4 s\u22121 and 0.5 s\u22121, respectively) while the values for k-6\/K7 are fairly similar (1.24\u00a0\u00d7\u00a0106 M\u22121s\u22121 and 1.4\u00a0\u00d7\u00a0106 M\u22121s\u22121). The revised KD value for bovine MHC-II-1 S1 has implications for the thermodynamic coupling constant between the affinity of myosin for ADP and actin, KAD\/KD (see below).\nComparison of fast and slow muscle myosin II isoforms shows that in the absence of actin, most kinetic parameters are quite similar (Table 1). The major exception is the ATP-hydrolysis rate (k+3\u00a0+\u00a0k-3), which is about tenfold slower for the slow MHC-II-1 S1 isoforms compared to rabbit psoas S1. The ADP dissociation rate constant (k+6) is very similar for all slow isoforms (k+6 0.5\u20130.9 s\u22121) and about twofold slower than the ADP dissociation rate constant of fast skeletal S1 (k+6\u00a0=\u00a01.4 s\u22121). The ADP association rate constant (k-6\/K7) is similar for all slow isoforms. The different estimates of ADP binding and release result in a surprisingly wide range of values for the ADP-affinity for S1 isoforms listed from 0.4\u00a0\u03bcM (rabbit soleus) to 2\u00a0\u03bcM (bovine masseter). However, these KD values are well defined by both the ratio of rate constants (Figure 2) and the amplitude titration data (Figure 4), suggesting that this parameter does vary across the series of isoforms.\nThe data in the presence of actin (Table 2) indicate that the affinity of S1 for actin in the presence (KDA) and absence (KA) of ADP shows a sursprisingly large variation within the group of slow isoforms with a tenfold difference in KA value of bovine masseter andrabbit soleus and an even larger difference in KDA value. However, despite these differences in individual equilibrium constants, the slow isoforms show a similar range of values for their thermodynamical coupling constants (KAD\/KD and KDA\/KA). The slow isoforms show ratios of 3\u201320, all lower than the value for fast rabbit isoforms (30\u201360) and at the lower end similar to smooth muscle myosin (4\u20137)25,26 and non-muscle isoforms (\u223c1\u20135).\nTable 2 shows the parameters that control the ATP induced dissociation of acto\u2013S1 vary relatively little for the fast and slow muscle isoforms listed (K1k+2' and k+2' vary less than twofold). The major difference is that all of the slow isoforms show a two-phase ATP-induced dissociation of acto\u2013S1 whereas the fast muscle protein shows a single phase. Siemankowski & White10 stated that the ATP-induced dissociation of actoBCS1 (in the presence or absence of ADP) could not be fitted unambiguously to a single exponential, suggesting that it too has two components. Additional indications of two phases comes from data measured for other slow MHC-II-1 isoforms of the chicken, from slow skeletal, cardiac, and smooth muscle27 which all show the presence of an additional small slow phase. Thus, a biphasic reaction for ATP-induced dissociation of acto\u2013S1 appears to be common for slow muscle myosin isoforms. ATP-induced biphasic dissociation of acto\u2013S1 has also been reported for a some class I mammalian myosins and non-muscle myosin II.26 The biphasic transient has been interpreted in terms of scheme II in which a fraction of the acto\u2013S1 must isomerise before nucleotide can bind.\nThe work presented here also confirms the complex nature of the ADP release from acto\u2013S1 reported for soleus S1. It is not simply that ADP release is much slower for this group of myosins than for the fast muscle myosin but there is clear evidence for several A.M\u2013ADP complexes. We show here evidence for two complexes with ADP with an equilibrium constant between them (K\u03b1D) of 5.3 and rate constants of ADP release of 94 (k-ADP) and 9.6 (k+\u03b1D) s\u22121. Similar behavior is seen with the pig soleus (Table 2) and rabbit soleus (Table 3) but with variation across the three slow isoforms particularly in the value of K\u03b1D. The data with rabbit soleus provided evidence for a third AMD complex and by analogy we might expect a rapid collision complex between ADP and acto\u2013S1 not characterised here.\nThe value of the ADP release rate constant (k-ADP) has been assigned to the event during the actin-attached part of the ATPase cycle, which limits the maximum shortening velocity (V0) of a muscle fibre. The velocities of contraction are best characterised at 12\u00a0\u00b0C (0.17\u00a0M ionic strength) and the published values of V0 for the three slow muscle isoforms are listed in Table 3 along with the values of the ADP release constant at 12\u00a0\u00b0C. The maximum unloaded shortening velocity of muscle fibres (V0) of bovine masseter is 0.27\u00a0\u03bcm s\u22121 at 12\u00a0\u00b0C.13 From this, one can estimate the minimum value of the rate constant that limits the detachment of the cross-bridge (kmin) using the relationship:with V0\u00a0=\u00a0maximum sliding velocity (nm s\u22121) and d\u00a0=\u00a0working stroke (nm).8 Depending on the size of the working stroke, kmin is 54 s\u22121 (d\u00a0=\u00a05\u00a0nm) or 27 s\u22121 (d\u00a0=\u00a010\u00a0nm) for bovine masseter MHC-II-1. From Table 3 one can see that at 12\u00a0\u00b0C k-ADP is 27 s\u22121, which is close to the calculated kmin value. The data from the pig shows a similar correlation between kmin and k-ADP, as does the previously published data from rabbit.9 For all three MHC-II-1 isoforms the measured values of k+\u03b1D are much slower than the calculated kmin, indicating that this isomerisation step cannot be part of the main pathway of the unloaded cross-bridge cycle but represents a branched pathway that could become significant if the motor is contracting under loaded conditions. The velocity measurements are, by definition, made under zero load and the isomerisations controlling the ADP release steps are events which are potentially sensitive to the load on the actomyosin cross-bridge.\nThe evidence that ADP release is load-dependent has been discussed in general terms by Nyitrai & Geeves28 and specifically for the rabbit MHC-II-1 isoform by Iorga et al.9 The data for bovine and porcine isoforms are shown here to be similar to the rabbit isoform and a similar interpretation is possible. Briefly, there is substantial evidence from single-molecule mechanical measurements29\u201332 and electron micrographs of S1 decorated actin filaments17\u201319 that, for a range of non-muscle myosins and the smooth-muscle myosin there is a load-dependent structural change after the major force generating event. The force-generating event is associated with phosphate release and the second structural change is probably associated with ADP release. More recent studies with improved laser traps33 and X-ray scattering from S1-decorated, orientated actin filaments16 have extended these observations to striated muscle myosins. The laser trap observed a \u223c1\u00a0nm movement of the lever arm in the second step for the rat MHC-II-1 isoform and the rate constant we expect for the event limiting ADP release is compatible with the lifetimes measured in the laser trap. The X-ray data report a similar sized movement of the lever arm on binding\/release of ADP for the rabbit slow muscle S1. Together, the structural mechanical and biochemical kinetic studies support the hypothesis of a load dependent isomerisation of the actomyosin complex required to allow release of ADP.\nThe results presented here on the protein isomerisations that limit ADP release from the actomyosin of slow muscle may provide some insight into why, despite a tight affinity for ADP, slow muscles are more resistant to fatigue than fast muscles, or at least the part of fatigue associated with ADP build up. Fatigue is classically defined from studies of the effect of prolonged stimulation on the isometric tension of intact muscle fibres. The complete explanation of the loss of tension remains to be defined but any build up of ADP is likely to result in the enhancement of the lifetime of strongly bound A.M.\u2013ADP states as ADP competes with ATP induced detachment of the cross-bridge. The increased lifetime will increase steady-state tension as is seen on addition of ADP to skinned muscle fibres.34\u201336 Thus, ADP build up may compensate for other effects of fatigue which tend to reduce tension and the compensatory effect will be greater in slow than in fast muscle fibres.\nThe effect of ADP on velocity is different; the evidence is for a slowing of maximal shortening velocity in skinned muscle fibres.34,37 In a fast muscle the velocity is limited by the rate of ATP-induced dissociation of actin from A.M., and ADP competes with ATP for binding to the nucleotide pocket.38 The net rate constant for actin detachment is defined by:Thus, at mM ATP concentrations, ADP of the order of the KAD (\u223c100\u00a0\u03bcM) can produce a significant inhibition of the detachment rate constant. For slow muscle the detachment of myosin from actin is limited not by the ATP induced reaction but by the slower isomerisation preceding ADP release, controlled by k-ADP (Table 3 gives this as 27 s\u22121 for masseter at 12\u00a0\u00b0C). The following ATP-induced dissociation of AM remains much faster than this (\u223c1000 s\u22121 at mM ATP concentrations, in fact the K1, and k+2 values are similar for fast and slow myosin isoforms; see Table 2) and the velocity will only be slowed once the ADP competition effect reduces kdetach to <100 s\u22121 (equation (7)). This requires the ADP concentration to be many times the KAD value or for the ATP concentration to fall to \u223c100\u00a0\u03bcM.\nIn summary we have shown that the MHC-II-1 isoforms isolated from slow skeletal muscles from the cow and the pig are similar in general properties to MHC-II-1 isolated from bovine cardiac tissue and to the MHC-II-1 of the rabbit and these are quite distinct from the MHC-II-2 isoforms. Of significance is the slow ATP-hydrolysis rate, the low thermodynamic coupling between ADP and actin binding, the slow rates of ADP release (which are of the correct size to limit the maximum shortening velocity) and multiple actomyosin ADP complexes. Nucleotide access to and release from the binding site requires relatively slow isomerisations and these may be load-dependent. These properties are distinct from the fast muscle MHC-II-2 isoforms and similar to smooth and some non-muscle myosin isoforms, e.g. mammalian myosins 1b and 1c, and non-muscle myosin II. However, unlike the non-muscle isoforms, the MHC-II-1 isoforms have a low duty ratio and can produce relatively rapid movement in motility assays or in muscle fibres. These parameters suggest that slow muscle myosin isoforms may form a distinct functional group from the fast muscle isoforms and the slower group which we have called the strain-sensors; non-muscle myosins particularly a sub-set of class I myosins (myo1b and 1c) that appear to operate less as transport motors than as load-bearing and strain-sensing molecules. Slow muscle myosin, like fast muscle myosin, is required for movement but also has an additional role in efficient load bearing in postural muscles. Thus, the molecular properties of the MHC-II-1 motor are consistent with its physiological role.\nMaterials and Methods\nProteins\nMHC-II-1 myosin was purified from bovine masseter muscle fibres that had been frozen in liquid nitrogen prior to storage at \u221280\u00a0\u00b0C. Purification was based on the method described.39 Briefly, the tissue (40\u00a0g) was homogenized in ice-cold Guba-Straub buffer (pH 6.6) and the suspension was stirred for 30\u00a0min. After centrifugation, the supernatant was diluted tenfold with water. The precipitated protein was collected by centrifugation. The pellet was taken up in 3M KCl and ice-cold water was then added to precipitate the myosin and the protein was collected by centrifugation. This procedure was repeated three times and the pellet was then taken up in a small amount of 3M KCl and then adjusted to 125\u00a0mM KCl, 10\u00a0mM KPi, 2\u00a0mM EDTA, 4\u00a0mM dithiothreitol (DTT, final concentrations) for digestion. Subfragment 1 (BMS1) was prepared by digesting myosin using chymotrypsin using the method described.40 The soluble BMS1 protein was separated from the non-digested myosin and the insoluble myosin tails by centrifugation and purified over a Q-Sepharose column in 50\u00a0mM imidazole (pH 7) with a linear gradient from 0\u2013500\u00a0mM KCl. The fractions containing BMS1 were pooled and dialysed into 10\u00a0mM KPi buffer (pH 7.5) and typical yields were around 140\u00a0mg of protein. Figure 1 shows the isolated myosin with two light chains and the purified BMS1 with only one light chain bound. The gel suggests only one isoform of each light chain and the molecular mass of the essential light chain is consistent with this being the MLC-1b\/v isoform expected in slow skeletal and ventricle muscle. For myosin S1, isolated from pig soleus muscle, a similar procedure was followed to isolate the protein and from 58\u00a0g of tissue about 70\u00a0mg of pig soleus S1 (PSS1) was obtained. After dialysis, BMS1 and PSS1 were lyophilized in the presence of 1% (w\/v) sucrose and stored at \u221220\u00a0\u00b0C. Prior to use, BMS1 or PSS1 was dialyzed into 20\u00a0mM Mops, 30\u00a0mM KCl, 5\u00a0mM MgCl2, 1\u00a0mM sodium azide and remained stable in solution for several days. Rabbit skeletal muscle actin was prepared according to methods described41 and, when necessary, labelled with pyrene at Cys374.42\nATPase assay\nATP-ase activity was measured using an NADH-coupled assay at 20\u00a0\u00b0C in buffer containing 20\u00a0mM Mops (pH 7), 5\u00a0mM MgCl2, 30\u00a0mM KCl, 1\u00a0mM ATP, 40 units\/ml of lactate dehydrogenase, 200 units\/ml of pyruvate kinase, 1\u00a0mM phosphoenolpyruvate, and 200\u00a0\u03bcM NADH. Changes in A340 (\u03b5\u00a0=\u00a06220\u00a0M\u22121cm\u22121) were followed using a UV-VIS spectrophotometer. The basal Mg2+-ATPase activity of bovine masseter myosin S1 (0.012 s\u22121) agrees well with the value reported (0.017 s\u22121) for bovine cardiac myosin S1 isolated from bovine cardiac left ventricle.12 In the presence of 50\u00a0\u03bcM actin the Mg2+-ATPase activity of BMS1 increased more than tenfold (0.182 s\u22121) and the ATP-ase rate was linearly related to the actin concentration over the range of 0\u201350\u00a0\u03bcM. (Not shown).\nTransient kinetics\nAll kinetic experiments were performed at 20\u00a0\u00b0C in 20\u00a0mM Mops, 100\u00a0mM KCl, 5\u00a0mM MgCl2 and 1\u00a0mM azide (pH 7), unless indicated otherwise. All measurements were done with a High-Tech Scientific SF-61 DX2 stopped-flow system. Pyrene actin fluorescence was excited at 365\u00a0nm and emission was detected after passing through a KV389\u00a0nm cut-off filter (Schott, Mainz, Germany). Tryptophan fluorescence was excited at 295\u00a0nm and observed through a WG320 filter. The stated concentrations of reactants are those after mixing in the stopped-flow observation cell (\u201cpost\u201d) unless indicated otherwise. Stopped-flow data were analysed using the software provided by Hi-Tech (KinetAsyst) and the Origin software (Microcal).\nWithout actin present, the kinetics of S1 (either BMS1 or PSS1; M) with nucleotide (T,ATP; D,ADP) were interpreted based on the seven-step model described by Bagshaw etal.,43 where k+i and k-i are the forward and reverse rate constants and Ki (k+i\/k-i) represents the equilibrium constant of the reaction (Scheme 1).\nIn the presence of actin the kinetics of the interaction ofS1 with nucleotide were based on the model proposed for myo1b26,44 and is depicted in Scheme 2. In this model, AM exists in two conformations in the absence of nucleotide (A.M and A.M', equilibrium defined by K\u03b1\u00a0=\u00a0k+\u03b1\/k-\u03b1) and in the presence of ADP (A.M.D and A.M'.D, equilibrium defined by K\u03b1D\u00a0=\u00a0k+\u03b1D\/ k-\u03b1D). In the AM' state, the nucleotide can exchange with the solvent (\u201copen\u201d conformation) but not in the AM state (\u201cclosed\u201d conformation). Conversion of M to M' requires opening of the nucleotide binding pocket. The data presented here and summarised in Tables 1 and 2 allow assignment of all the rate and equilibrium constants of Scheme 2. The assignment of the constants to the data is summarized here.\nIn the absence of ADP, high ATP concentrations induce the dissociation of actin from S1 in two phases. The fast phase represents dissociation from A.M' with:\nThe slow phase is limited by the isomerisation from A.M to A.M' with the maximum value of kobs,slow\u00a0=\u00a0k+\u03b1. The ratio of the amplitudes of the fast and the slow phase of the ATP-induced dissociation defines the equilibrium constant between the two conformations in solution and since K\u03b1\u00a0=\u00a0k+\u03b1\/k-\u03b1, k-\u03b1 can be determined:\nFor fast myosin S1, such as skS1 and psoS1, K\u03b1\u00a0>>\u00a01 and only a single phase is observed.\nFor slow myosin S1, in the presence of ADP, the overall dissociation constant of ADP for the actin-S1 complex (KAD) is defined by equation (3):where KADP is the dissociation constant for ADP from A.M' and K\u03b1D the equilibrium constant between AM'D and AMD. If the binding of ADP is a rapid equilibrium event, controlled by KADP, ADP and ATP compete effectively for binding to AM and the observed rate constant kobs is defined by equation (4):where k0 is the observed rate constant of ATP-induced actin\u2013S1 dissociation in the absence of ADP and defined by equation (1). To titrate both A.M complexes to A.M.D, actin\u2013S1 was pre-equilibrated with a range of ADP concentrations before adding excess ATP. ATP-induced dissociation of actin from A.M.D occurs via the intermediate A.M'.D and A.M' and is limited by k+\u03b1D. If k+\u03b1 and k+\u03b1D differ by more than a factor of 3, then both fast and slow amplitudes seen at zero ADP are reduced as ADP is increased and replaced by a new phase with kobs\u00a0=\u00a0k+\u03b1D (this is the case for BMS1). The effect of ADP on the amplitude of the fast phase of ATP-induced dissociation of actin-S1 was analysed according to:where Ampfast represents the observed amplitude of the fast phase of the reaction, Amp0 is the amplitude at zero ADP concentration, [ADP] is the concentration of ADP before mixing, KAD is the dissociation equilibrium constant of ADP for actin\u2013S1, and C is the end point. The end point of the reaction defined by C was defined by the fluorescence at infinite high ADP concentrations. Amplitude is calculated as a percentage of the end point of the reaction, according to:\nWhere amplitude, expressed as %, equals the change in fluorescence observed during the reaction (\u0394F) divided by the fluorescence at the end point of the reaction (Ft\u221e), multiplied by 100.\nQuenched-flow experiments\nThe quenched-flow experiments were carried out with a Hi-Tech RQF-63 using a buffer containing 100\u00a0mM KCl, 5\u00a0mM MgCl2, 20\u00a0mM Mops (pH 7.0). BMS1 (5\u00a0\u03bcM) was mixed with excess ATP (50\u00a0\u03bcM) and incubated at 20\u00a0\u00b0C for desired time intervals (up to 350\u00a0ms). The reaction was quenched by the addition of an equal volume of 6.25% (w\/v) trichloroacetic acid (TCA). After neutralisation with NaOH and a clarification spin at 3000g for 5\u00a0min, the samples were diluted 1:10 with the fast protein liquid chromatography running buffer containing 125\u00a0mM KPi (pH 5.5). The separation of ADP and ATP was carried out on a fast protein liquid chromatography (FPLC) system (Amersham Biosciences) using a Hypersil ODS (3\u00a0\u03bcm) column and an isocratic flow. Integration of the peak areas provided the ratio of ADP and ATP at each time point. The ratio of the ADP to the total nucleotide concentration at each given time point was then used to calculate the hydrolysis rate of the myosin. The amount of irreversibly bound ATP to BMS1 at the end of the burst was determined by quenching the reaction into a 100\u00a0mM KCl, 20\u00a0mM Mops, 5\u00a0mM MgATP buffer containing hexokinase (2\u00a0\u03bcg\/\u03bcl) and glucose (1\u00a0mM) after 1\u00a0s and then adding 6.5% TCA after a further 1\u00a0s. In controls this treatment hydrolysed any free ATP to ADP. HPLC analysis of the reaction mixture gives information about the amount of ATP bound to S1 at the end of the burst phase.","keyphrases":["adp","actin","atpase","subfragment 1","mhc, myosin heavy chain","mlc, myosin light chain","rlc, regulatory light chain","elc, essential light chain","s1, myosin subfragment s1","bms1, bovine masseter s1","pss1, pig soleus s1","bcs1, bovine cardiac s1","rss1, rabbit soleus s1","tca, trichloroacetic acid","strain-sensor"],"prmu":["P","P","P","P","R","R","R","R","R","R","R","R","R","R","U"]}
{"id":"Matern_Child_Health_J-2-2-1592251","title":"Public Finance Policy Strategies to Increase Access to Preconception Care\n","text":"Policy and finance barriers reduce access to preconception care and, reportedly, limit professional practice changes that would improve the availability of needed services. Millions of women of childbearing age (15\u201344) lack adequate health coverage (i.e., uninsured or underinsured), and others live in medically underserved areas. Service delivery fragmentation and lack of professional guidelines are additional barriers. This paper reviews barriers and opportunities for financing preconception care, based on a review and analysis of state and federal policies. We describe states\u2019 experiences with and opportunities to improve health coverage, through public programs such as Medicaid, Medicaid waivers, and the State Children's Health Insurance Program (SCHIP). The potential role of Title V and of community health centers in providing primary and preventive care to women also is discussed. In these and other public health and health coverage programs, opportunities exist to finance preconception care for low-income women. Three major policy directions are discussed. To increase access to preconception care among women of childbearing age, the federal and state governments have opportunities to: (1) improve health care coverage, (2) increase the supply of publicly subsidized health clinics, and (3) direct delivery of preconception screening and interventions in the context of public health programs.\nIntroduction\nThe overarching goal of preconception care is to provide health promotion, screening, and interventions for the more than 62 million women of childbearing age [1] to reduce risk factors that might affect future pregnancies [2\u20134]. As elsewhere in this supplement, preconception care is defined as a set of interventions that aim to identify and modify biomedical, behavioral, and social risks to a woman's health or pregnancy outcome through prevention and management, emphasizing those factors which must be acted on before conception or early in pregnancy to have maximal impact. It includes care before a first pregnancy or between pregnancies (commonly known as interconception or internatal care) [5], and care that would typically be delivered in primary care settings [4].\nHealth professional leaders and professional organizations\u2019 guidelines have called for improvements in preconception care for more than 20 years [4, 6\u20139]. and several reviews of the literature have assessed the evidence for specific interventions and documented their effectiveness [10, 11]. Without changes in financing, however, it appears unlikely that the nation will improve its preconception health and, thereby, improve the health of women, their children, and their families. Since federal and state policies are important drivers of health care financing, this paper provides suggestions on how policymakers can improve financing for preconception care [12].\nAlthough many observers have suggested that providers would deliver more preconception care if they could bill payers for the service [13], a closer look at the situation suggests that more complex changes in women's health care financing may be necessary to support widespread use of preconception care. Affordability of care is a major concern for many women [14]. Many women lack basic health coverage, particularly non-pregnant, low-income women who do not meet of Medicaid's low income eligibility levels [15]. Securing private insurance coverage for additional preventive services may require more and better evidence about the effectiveness of interventions [4]. Other barriers to financing are more mechanical. Financing is required for an ongoing care process over a period of years and across the lifespan, not a single visit. Billing mechanisms and codes need refinement; in addition, cost-benefit analyses, and ongoing alignment with newly emerging approaches to care will also be required [16].\nImproving health coverage for women of childbearing age\nFinancial barriers to health care access are widespread in the United States, with many low-income individuals having no or inadequate health coverage. The Kaiser Family Foundation\u2019s, Women's Health Survey [17] does not focus on preconception care, but some of its results are relevant to understanding barriers to women's health care. Among women of childbearing age (18 to 44 years) 30 percent reported that they delayed or went without care because of cost. Moreover, 21% of women of all non-elderly women (18 to 64 years) in the survey reported that they did not fill a prescription for medication because of cost, including 40% of uninsured women [17].\nUninsured women\nMillions of Americans live without health coverage. Nearly 17 million American women are uninsured [17]. One-third of low income women (with income less than 200 percent of the federal poverty level), half of women with disabilities, and 18 percent of all non-elderly women were uninsured in 2003 [18]. Younger women were more likely than older women to be uninsured. Hispanic, Asian, and Black women were more likely than non-Hispanic white women to be uninsured, reflecting their income and employment status patterns (i.e., more likely to have incomes below 200 percent of poverty and less likely to be employed in jobs that offer health insurance) [17]. Moreover, recent data indicate that women are more likely to lose coverage than men. Between 2002 and 2003, nearly 900,000 women ages 18 to 64 became uninsured, compared to 600,000 men (53 and 47 percent, respectively). Such losses are attributed primarily to welfare reform policies and employer-based coverage trends [19].\nData for 2002\u20132003 reveal that the percentage of women ages 18\u201364 who were uninsured varies substantially, ranging from 30 percent in Texas to 8 percent in Minnesota. The proportion of uninsured low-income adult women shows similar variation, ranging from more than 50 percent in Texas to less than 25 percent in Massachusetts, Minnesota, Tennessee, and Wisconsin. Although to some extent these differences reflect Medicaid eligibility policy, the state-to-state variations also reflect employment patterns. For example, Minnesota had the highest percentage of adult women with private coverage and a fairly low proportion of women covered by Medicaid (83 percent and 8 percent, respectively). In contrast in Tennessee (under policies in effect at that time), 17 percent of adult women were Medicaid beneficiaries and only 70 percent had private coverage [20].\nWomen who are young, single, working part-time, or unemployed are most likely to uninsured. These factors are reflected in an age gradient, the percent of uninsured women ranging from 22 percent among 18 to 24 year olds, 21 percent among 25 to 34 year olds, 19 percent among 35 to 44 year olds, and 13 percent among 45 to 54 year olds (Fig. 1). Moreover, women ages 25\u201344 accounted for more than half (53 percent) of all uninsured women under age 65 [17].\nFig. 1Percent of Non-Elderly U.S. Women who are Unisured, By Age, 2004\nMedicaid as a source of coverage for women's health and PCC\nMedicaid is effective in improving access to health care for low income women. Medicaid coverage affords women access to essential preventive, primary, and reproductive health care services, thereby allowing them to reduce unintended pregnancy, choose the number and spacing of their pregnancies and, when desired, to plan a healthy birth [21]. Medicaid plays a particularly important role in financing ambulatory care for women of childbearing age [22]. While Medicaid coverage and enrollment for women declined during the mid-1990s as a result of changes in health and welfare policies, Medicaid demonstration waiver projects are now being used by some states expand to access to health care for women of childbearing age.\nUnder current Federal and State policies, Medicaid is the primary mechanism for extending health coverage for low-income, uninsured women. In 2003, 12 percent of all women of childbearing age (15\u201344 years) and 37 percent of poor women in that age group relied on Medicaid for health care coverage. Because nearly two-thirds (63 percent) of the women covered by Medicaid are of childbearing age (19\u201344 years), the program's performance is related to preconception care access and to the outcomes of pregnancy [17].\nFederal and state policies together define eligibility rules for Medicaid. Data from the Centers for Medicare and Medicaid Services (CMS) for female beneficiaries in the year 2000 indicate that 26 percent became eligible because they were pregnant and had low income, while 42 percent had welfare\/poverty-linked eligibility [23]. Federal law requires that states extend Medicaid eligibility to pregnant women with income below 133 percent of the federal poverty level (FPL) and many states exceed this level. No similar protections exist for non-pregnant women. As of July 2004, income eligibility thresholds, mainly parents with dependent children, ranged from 19 percent of FPL in Alabama to 275 percent FPL in Connecticut. Other women were enrolled because they are over age 65 and had below poverty income; this is a national policy that does not vary by state. Many other low income women do not, however, qualify for Medicaid because they do not have children under age 18, are not over age 65, or are undocumented. In addition, Medicaid managed care arrangements have had an impact on the source of services for women in their childbearing years [24].\nSome States have used waiver authority to expand Medicaid coverage to low income, uninsured adults \u2013 particularly the parents of children with publicly subsidized coverage. Parents in low-income families may be covered under so-called Section 1931 expansions, which do not require a waiver. More than 40 states and the District of Columbia provide Medicaid coverage to low-income parents through this option. The majority extend short-term coverage to unemployed parents, while other states extend coverage to parents with incomes to 50, 100, or 199 percent FPL, without regard to employment status. Washington State is one state that has used this option to large effect, extending eligibility to 200 percent FPL.\nIn nearly half of states, special waivers include a focus on adults and may have positive impact on coverage for low-income women of childbearing age. Three types of waivers are of particular importance here: (1) comprehensive waivers broadly designed to cover uninsured adults and make changes to benefits and cost sharing; (2) Health Insurance Flexibility and Accountability (HIFA) waivers, and (3) so-called \u201cfamily planning\u201d waivers [25]. All three types are authorized under Section 1115 of Medicaid and must be budget neutral. Each type of 1115 requires that states apply for and receive approval by the federal Centers for Medicare and Medicaid Services (CMS).\nComprehensive and HIFA waivers designed to increase health coverage among low-income adults\nNine states offer Medicaid coverage to low-income parents through Section 1115 waivers. All of these nine states extend coverage to families with incomes at or about 100 percent FPL. Again, some states extend coverage only to unemployed parents, whereas others extend coverage to parents with incomes to 50, 100, or 199 percent FPL, without regard to employment status. Minnesota's waiver extends coverage up to 275 percent FPL [26].\nAt the beginning of 2006, 11 states had active HIFA waivers, with the primary goal being to encourage new state approaches that increase the number of low-income individuals with health insurance coverage within current-level Medicaid and SCHIP resources [26]. Some of these (e.g., Maine and Michigan) extend coverage only to childless adults. More target the expansions to low-income parents with children enrolled in publicly subsidized coverage (e.g., Arizona, California, Illinois). Still other states sought a waiver for more general expansion of adult coverage.\nWith enactment of the Deficit Reduction Act of 2005 (DRA), states have increased flexibility to extend coverage, structure alternative benefit packages, and adopt premium and cost sharing requirements without a federal waiver. The DRA creates the potential for states to expand coverage to additional low-income adults, and some states (e.g., Kentucky, West Virginia) are considering expansions to parents of children with Medicaid or other publicly subsidized coverage under the State Children's Health Insurance Program (SCHIP). Such expansions may use limited benefit packages.\nFamily planning and interconception care waivers\nStates have received federal approval for Medicaid waivers programs that offer coverage to low-income women for family planning and reproductive health services. In 2001, nine state programs served 1.7 million women, and recent studies documented cost savings, reductions of unintended pregnancies, and improved use of family planning services in states with these programs [27]. By February 2005, 21 states had created such family planning waivers.\nIn recent years, 21 states have developed special programs to expand family planning services to women who do not otherwise qualify for Medicaid. Some states offer coverage to women who lose coverage after the birth of a baby or starting a job, while others offer family planning coverage based on income status to men and\/or women [28].\nOf review of 15 state family planning waivers prepared for the National Association of State Medicaid Directors (NASMD) found that core services covered were comprehensive health history and physical examination including appropriate laboratory testing, health education and counseling, approved birth control methods and supplies, and infertility services. Some states provide coverage for services that may be considered preconception care, such as screening for nutrition, folic acid intake, alcohol and tobacco use, genetic conditions, rubella serostatus, domestic violence, and other health risks [29].\nAn evaluation prepared for CMS of family planning waiver projects in six states found that they resulted in significant savings to both the Federal and State governments, as well as some evidence for expanded geographic availability of services and measurable reductions in unintended pregnancy [30]. This is true, despite the fact that the federal government matches state family planning expenditures at 90\/10 for each dollar spent. Greater potential savings and prevention, however, could result if States offered coverage for more comprehensive risk screening, health promotion, and interventions leading toward higher levels of preconception wellness. This would require an extension of states waiver authority aimed at coverage of preconception and interconception care.\nPublic financing to improve access to primary care\nPreconception care is part of a continuum of women's health services, typically provided in the context of well-woman visits or integral to chronic care management [31\u201333]. Most components of preconception care can be embedded in the process of primary and preventive care, rather than an isolated visit [34, 35]. Integration of preconception components into primary care can better serve women across the lifespan and at varying levels of risk [36, 37]. Primary care integrates a variety of health promotion, prevention, and acute care services to address a majority of personal health care needs and common health problems in a community setting [38]. It also may include screening for and ongoing management of chronic conditions in a primary care setting.\nNational survey data for 2004 indicate that 75 percent of U.S. women ages 18 to 44 had a health care visit in the past year, and most women of reproductive age obtain preventive health services in any given year [17], offering opportunities for clinicians to deliver preconception care. More than half (55 percent) of women had obstetrician\/gynecologist visits in the past year. Notably, however, one-third to one-half of women have more than one primary care providers (generally a family physician or internal medicine physician, and an obstetrician\/gynecologist) [14]. Some research indicates that having both a reproductive health provider and a primary care provider may increase utilization of clinical preventive services [39].\nHaving a usual or routine source of care (i.e., a place or provider they usually go when they need health care) is one indicator of health care access and a predictor of certain health behaviors [40]. Women who have a usual source of care are more likely to receive preventive care, to receive continuous care, and to have lower health care costs [14]. As with health coverage, the percentage of women reporting a usual source of care increases with age. Based on detailed survey data from 2002\u201303, the proportion of women with a usual source of care was 82 percent for ages 18\u201324, 86 percent for ages 25\u201334, and 91 percent for ages 35\u201344 [17]. Proportions also varied by race and ethnicity, with Hispanic women most likely to report no usual source of care. Non-Hispanic Black women were more likely to use a clinic and Non-Hispanic White women more likely to use a physician office as their routine source of care.\nThese data suggest that publicly subsidized clinics such as community health centers and other federally qualified health centers (FQHCs) might be used to increase access to preconception care among low-income and uninsured women. Community health centers\u2014local, non-profit, community-owned health clinics\u2014serve more than 15 million people in 3,600 low-income, medically underserved, urban and rural communities. Health centers are an important source of primary care for millions of low-income and uninsured women. Nearly 30 percent of all patients are women of childbearing age, and health centers provided prenatal care to over 330,000 women in 2003 [41]. Their patients account for one out of every 10 U.S. births [42]. Research on health centers shows that patients have less infant mortality and low birthweight [43], fewer health disparities [44], improved care for chronic conditions [42], and improved access to primary and preventive care [45]. A study of four states clinics suggests that FQHCs may be an important source of reproductive health services to reduce unintended pregnancies and teen births [46].\nAmong FQHCs, the Health Disparities Collaboratives Initiative is underway to improve the quality of primary care delivered, and over 600 FQHCs have participated [47]. This quality improvement model is tailored to fit with the staffing and patient patterns of FQHC and is supported by a partnership among community clinics, federal agencies, and national professional organizations. The Health Disparities Collaboratives started with a Chronic Diseases Care Model for quality improvement [48], but now are developing a Primary Health Care model and integrating with the Perinatal Care Collaboratives and other efforts [49].\nHealth centers receive about one-quarter of their total revenue from direct federal grants. Their single largest source of support is Medicaid, representing more than one-third of total revenues. Patients self-payments, other insurance, and large amounts of non-federal grants and contracts make up most of the remainder of health center funding [41]. Thus, federal and state fiscal decisions, in Medicaid and other health program appropriations, affect the fiscal well-being of FQHCs.\nFinancing public health programs and projects\nExisting public health programs serve millions of women each year. Preconception interventions can be incorporated into these existing programs targeted to reach women at highest risk [50]. Two program examples illustrate the potential, as well as the funding gaps.\nThe federal Family Planning program is authorized under Title X of the Public Health Service Act. Created in 1970, the Title X program is the only Federal program solely dedicated to family planning and reproductive health with a mandate to provide a broad range of voluntary, affordable, and effective family planning methods and services. The program is particularly designed to provide family planning services to low-income women, many of whom are uninsured and who would have no other source of care. The program provides funds to approximately 4,500 of the estimated 7,000 family planning clinics in the United States and provides reproductive health services to approximately 6.5 million persons each year [27]. Title X-funded clinics provide family planning education, contraceptives, and pregnancy tests; however, many do not offer more comprehensive risk screening and reproductive health promotion and reproductive life planning [51]. If Title X funding had been increased at the rate of inflation from its FY 1980 funding level, it would have been funded at over $590 million in FY 2002 [52, 53]. Federal funding was set at only $288 million for FY 2005 [54]. Without additional funding, it seems unlikely that Title X clinics will obtain the financial and staff resources to add components of preconception care [55].\nFederal and State public health programs funded by the Title V Maternal and Child Health Services Block Grant have the flexibility to give priority to preconception health and offer support for demonstration projects and evaluations of prevention programs. Authorized under Title V of the 1935 Social Security Act, this program provides block grants to states to improve the health of all women, children, youth and families. Title V funding is an important source of funding for infrastructure building, population-based services, enabling services, and direct health care services. Preconception care is a priority area for some state Title V programs and is being monitored with performance measures in additional states [51]. Many state Title V agencies have preconception or interconception projects underway or in the design phase, but most of these operate on a small scale or are targeted only to a special population such as teens. Opportunities exist to use Title V block grant and special project funds more broadly to fund related needs assessment, community-based prevention projects, health education, or preconception care for women. Without additional funding, however, it seems unlikely that new priorities will be adopted and new programs implemented by federal or state Title V-funded entities. As part of an overall funding cut for domestic discretionary programs, Congress reduced FY 2006 funding for the Title V program from $724 million to $700 million [56].\nAlthough the federally funded Healthy Start projects have interconception health activities, these projects located in communities with high infant mortality have opportunities to offer more systematic pre\/interconception screening, health promotion, and interventions. Healthy Start grantees are required to include interconception care activities as part of their overall project. Such activities might include linkage to local providers, tracking postpartum visits, providing case management services, and\/or directly provide interconception services [57]. Because Healthy Start funding is linked to Title V funding overall, resources for more extensive or systematic efforts are limited.\nPublic health programs funded through the Centers for Disease Control and Prevention (CDC), particularly those offering screening and related services for sexually transmitted diseases and HIV\/AIDS also could do more to provide preconception risk assessment and health promotion. Such programs targeted to high risk women are not, however, substitutes for access to primary care and health care coverage.\nConclusions\nPublic health policy can be correlated to women's health outcomes [58]. Three major policy and finance directions are proposed by this review paper. To increase access to care among women of childbearing age, the federal and state governments have opportunities to improve health care coverage, increase the supply of publicly subsidized health clinics, and encourage delivery of preconception screening and interventions in the context of public health programs. First and foremost, state and federal policymakers might increase health coverage for low-income women of childbearing age through Medicaid policy changes and waivers. Although health coverage in and of itself may not change health behaviors and health care utilization [59], having access\u2013financial and geographic\u2013to a provider can be seen as threshold requirements for improved health care utilization and delivery of preconception care. A second opportunity is to continue to increase support for community health centers and other federally qualified health centers in medically underserved communities. Last, but not least, policymakers might give greater attention to financing for health promotion and prevention programs, particularly programs such as Title X and Title V which focus on services to women of childbearing age [60\u201362].","keyphrases":["finance","policy","preconception","medicaid","title v","family planning","maternal health","infant health"],"prmu":["P","P","P","P","P","P","R","R"]}
{"id":"Matern_Child_Health_J-2-2-1592141","title":"Preconception and the Young Cancer Survivor\n","text":"Introduction\nWhile the diagnosis of cancer is not common in young men and women, cancer survivors younger than 40 are likely to be very concerned about the impact of their cancer on future fertility, their risk of cancer recurrence after treatment and risks to future offspring. It is estimated that 1 in 900 persons between 16 and 44 are cancer survivors [1], and that there are at least 250,000 women age 40 and younger who are breast cancer survivors in the United States. As cancer survival increases, the focus on quality of life issues increases. When questioned, at least 73% of young breast cancer survivors reported concerns about infertility due to cancer treatment, and 29% reported that this concern impacted their choice of cancer treatment [2].\nPremature ovarian failure\nIn women, the options for preserving fertility and risk of ovarian failure depends on the age at the time the cancer is treated, the type and location of cancer, and the specific treatment chosen. Some treatments, such as bone marrow transplantation (BMT) are accompanied by very high rates (>99%) of ovarian failure [3]. Even for those women who resume normal menstruation after chemotherapy, premature ovarian failure (POF) is not uncommon [4], and the risk of premature ovarian failure is 8-fold higher in cancer survivors [5]. The risk of POF increases with the woman's age, with less than 20% of women under 30 years experiencing POF, whereas most women older than 40 will become menopausal after chemotherapy [6].\nPOF and menopause are dependent on the type of therapy used. Rates of ovarian failure range from 15% for acute myelocytic leukemia (AML) to 50% for breast cancer, with the overall rate of ovarian failure of 34% [4]. The chemotherapy agents with the highest risk for ovarian failure were the alkylating agents. The higher the dose of these drugs, the higher the likelihood of ovarian failure.\nOvarian tissue damage is well documented with the use of radiation therapy. Radiotherapy is used for treatment of several cancer types affecting pre-menopausal women. The degree of damage to ovarian tissue is related to a woman's age, the total dose of radiation used, and the number of exposures. In one study, when the same total dose of radiation was delivered, exposure to multiple fractionated doses versus a single dose resulted in less damage to the ovaries [4]. When used in conjunction with chemotherapy, the risk of POF increases. Byrne et al. assessed the risk of premature ovarian failure in childhood cancer survivors and found the relative risk of early menopause was 9.2 for those treated with chemotherapy alone, and 27 for those treated with radiation plus chemotherapy [5].\nFertility preservation techniques\nAlthough an increasing number of techniques have become available for the preservation of fertility, many are experimental and have not been tested in randomized controlled trials. Table\u00a01 reviews the types of options available and their benefits and limitations.\nTable 1Fertility preservation options for women with malignanciesMethodBenefitsLimitationsIn vitro Fertilization (IVF)Most successful, widespreadDelay of therapy, use of fertility medicationsFrozen oocytesNo partner is neededDifficult to preserveOvarian tissue preservationLess tissue damage from freezing, can be used in pre-menarchal and young womenExperimentalTransposition of ovarian tissueLess radiation exposureLimited to women having radiation therapyPharmacological protectionLimit damage to ovarian tissueAdequate prospective studies needed\nIn vitro fertilization (IVF)\nThe best-studied and most successful option for preserving fertility in female cancer patients is ovarian stimulation followed by IVF and embryo cryopreservation. Ovarian stimulation needs to occur prior to cancer therapy because treatment can adversely affect ovarian reserve and response. The pregnancy rate with frozen embryos is lower compared to fresh IVF embryo transfer. A meta-analysis by the American Society for Reproductive Medicine reviewed over 88000 cycles of assisted reproductive technology (ART) treatment. When fresh oocytes were used, a delivery rate of 41.8% was found in more than 6500 cycles analyzed, compared to a delivery rate of 18.6% for frozen embryos [7].\nOne concern about ovarian stimulation with IVF is that it requires a delay of therapy, which could impact growth rate of tumors and survival rates. In addition, concerns have been voiced that the medications used to stimulate ovarian production could impact on estrogen-receptor positive tumors such as breast carcinoma. Anti-estrogens, such as tamoxifen, or aromatase inhibitors, (e.g. letrozole), have both been used in ovarian stimulation in breast cancer patients to maintain low estradiol levels during IVF cycles.\nFrozen oocytes\nFrozen oocytes are more difficult to preserve, resulting in a lower survival, fertilization and pregnancy rates. Damage is in part due to ice formation during the freezing process. Unfertilized frozen oocytes have a low success rate for live birth (2% per oocyte) [8], although the rates are now increasing. Rapid freezing of eggs by vitrification limits damage, which improves oocyte survival. Using intracytoplasmic sperm injection (ICSI) increases the fertilization rate even further.\nOvarian tissue cryopreservation\nFor pre-menarchal girls, young women with POF, women that cannot delay therapy or those in whom high doses of fertility medications are contraindicated, ovarian tissue cryopreservation is offered in some centers. Unlike frozen oocytes, primordial follicles in ovarian tissue are smaller and have less follicular fluid, and therefore may be at less risk from damage from ice crystals that form during freezing. It is hoped that transplanting thawed ovarian tissue will result in the recovery of fertility. Transplantation should be used with caution in women with ovarian or metastatic cancer, to prevent transferring malignant cells back after treatment. This treatment is still considered experimental. Although success in animals has been demonstrated, transplantation of cryopreserved ovarian tissue has resulted in only two livebirths [9, 10].\nOvarian transposition\nFor women having pelvic radiation, transposition of ovarian tissue outside of the field of radiation successfully preserves ovarian function. The radiation dose to the transposed ovary is approximately 5\u201310% the level of the non-transposed ovary [11].\nPharmacological protection\nTo limit or prevent chemotherapy-induced POF, some centers use gonadatropin releasing hormone (GnRH) agonists to reduce gonadatropins. This results in mimicking a pre-pubertal state, because the ovary appears to be less sensitive to gonadotoxic drugs prior to menarche. Because dividing cells are more susceptible to damage from cytotoxic agents, ovarian follicular cells are vulnerable. The mechanism of protection by GnRH agonists is unknown. One prospective clinical case series found that 70 of 75 women (93%) co-treated with GnRH agonists and chemotherapy resumed cyclic ovarian function verses 38 of 82 women (46%) treated with chemotherapy alone [12]. Adequately controlled prospective studies still are required, although data from animal studies are promising. Anti-apoptotic agents, such as sphingosine-1-phosphate (SIP), have prevented radiation-induced oocyte loss in mice [13]. However, since apoptosis is needed for the treatment of cancer, this method faces some obstacles for implementation.\nMale fertility\nIn men, sperm banking is routine and should be offered to all men prior to undergoing cancer treatment. When combined with intracytoplamic sperm injection (ICSI), sperm banking should preserve fertility for almost all males. There is no increased risk for birth defects when sperm is banked prior to therapy [14, 15]. Even in azoospermic males, testicular sperm extraction (TESE) with ICSI has resulted in successful pregnancies. By 2005, 11 of 29 men with azoospermia that had used TESE with ICSI had achieved a pregnancy which resulted in a livebirth [16]. For pre-pubertal males, sperm banking is not available because there is no spermatogenesis. The possibility of cryobanking of testicular tissue is being explored [16, 17]. An increase in sperm aneuploidy is seen up to 18 months post-chemotherapy, so it has been suggested that a delay of conception for 12\u201318 months after therapy is completed is reasonable [18].\nPregnancy after cancer\nOnce a woman has recovered from cancer treatment, no clear guidelines exist about the length of time to wait before attempting to become pregnant.\nEffect of cancer treatment on pregnancy\nOnce a cancer survivor achieves pregnancy, her health and the fetal health may be adversely affected by her prior treatment for cancer. Clinically significant heart disease occurs in 0.5\u20131% of women treated with anthracycline-based chemotherapy drugs, and may not develop until months or years after therapy is completed [19]. Also, women who have radiation to the left side of the abdomen or chest are at risk for heart damage, which could worsen during pregnancy or labor. Women with radiation to the abdomen or pelvic area or total body radiation are at increased risk for miscarriage, preterm birth and delivery of low birthweight infants [20]. A report from the Childhood Cancer Survivor Study (CCSS) did not show any increase in adverse pregnancy outcomes in women treated with most chemotherapy agents. However, an increased risk of stillbirth in women treated with abdominal radiation for Wilm's tumor has been noted [21].\nWomen who have completed treatment for breast cancer are often placed on selective estrogen receptor modulators (SERMs) such as tamoxifen to reduce the incidence of a second cancer. However, tamoxifen use during pregnancy is to be avoided because it is potentially teratogenic.\nEffect of pregnancy on cancer\nSome women are concerned that the hormonal changes from pregnancy may increase the risk of cancer recurrence, but pregnancy after breast cancer management does not increase the risk of cancer recurrence or cancer-related death [22, 23]. However, women who are carriers of BRCA1 or BRCA2 germline mutations and who have had a full-term pregnancy are at significantly higher risk of developing breast cancer by age 40 than mutation carriers who do not have children [24]. The risk for breast cancer is increased by 70% in the first 2 years after pregnancy for BRCA2 carriers, but not BRCA1 carriers [25].\nRisk to offspring\nSince cancer treatment by radiation and many chemotherapy agents are known to cause DNA mutations, cancer survivors are often concerned that the treatment may increase the risk for birth defects, genetic conditions or cancer in their offspring. Several studies have found no increased risk of congenital abnormalities in the offspring of cancer survivors. Epidemiologic data from Japanese survivors of the atomic bomb and their children did not show any evidence for new mutations. The CCSS also did not find any link between radiation therapy or chemotherapy for childhood cancer and genetic diseases or birth defects [26, 27].\nRisk of cancer in offspring\nNo increased risk for childhood cancer was identified in the offspring of childhood cancer survivors, but additional follow-up is needed [26]. In rare instances, the childhood cancer may be hereditary, with a risk of passing on the mutated gene to the offspring. Some cancers, such as breast, colon or ovarian cancer, are more likely to be hereditary when the cancer occurs at an earlier age. For women with breast cancer diagnosed prior to age 40 and no other family history of early breast cancer, the risk of a mutation in one of the hereditary breast cancer genes, BRCA1 or BRCA2 is estimated to be at least 13% [28].\nSummary\nAll women should be counseled about the impact of cancer treatment on fertility prior to undergoing chemotherapy, ovarian surgery or BMT, and offered fertility preservation options, of which ovarian stimulation with IVF is the most successful. Once women achieve pregnancy, the risk for birth defects or genetic conditions is not increased because of either radiation or chemotherapy. Because treatment options may increase the risk for adverse pregnancy outcome, review of therapy records is essential for determining potential risk to the pregnancy. For most young cancer survivors, fertility can be preserved and a successful pregnancy achieved, but discussing these issues prior to treatment is essential.","keyphrases":["preconception","ovarian failure","fertility preservation","pregnancy after cancer"],"prmu":["P","P","P","P"]}
{"id":"Arch_Dermatol_Res-3-1-1914293","title":"Lichen planus remission is associated with a decrease of human herpes virus type 7 protein expression in plasmacytoid dendritic cells\n","text":"The cause of lichen planus is still unknown. Previously we showed human herpes virus 7 (HHV-7) DNA and proteins in lesional lichen planus skin, and significantly less in non-lesional lichen planus, psoriasis or healthy skin. Remarkably, lesional lichen planus skin was infiltrated with plasmacytoid dendritic cells. If HHV-7 is associated with lichen planus, then HHV-7 replication would reduce upon lichen planus remission. HHV-7 DNA detection was performed by nested PCR and HHV-7 protein by immunohistochemistry on lesional skin biopsies from lichen planus patients before treatment and after remission. Biopsies were obtained from lichen planus lesions before treatment (n = 18 patients) and after remission (n = 13). Before treatment 61% biopsies contained HHV-7 DNA versus 8% after remission (P = 0.01). HHV-7-protein positive cell numbers diminished significantly after remission in both dermis and epidermis. Expression of HHV-7 was mainly detected in BDCA-2 positive plasmacytoid dendritic cells rather than CD-3 positive lymphocytes. HHV-7 replicates in plasmacytoid dendritic cells in lesional lichen planus skin and diminishes after remission. This study further supports our hypothesis that HHV-7 is associated with lichen planus pathogenesis.\nIntroduction\nLichen planus is clinically characterized by flat topped polygonal, itchy erythematous papules with Wickams\u2019 striae, nail deformation, and white mucosal patches [4]. The histopathological substrate comprises a dermal band like (lichenoid) lymphocytic infiltrate directed towards the basal epidermal layer with destruction of the basal membrane and apoptosis of epidermal cells (colloid bodies). A viral cause infecting skin cells has been proposed. Hepatitis C virus was epidemiologically linked to lichen planus [14], but this finding could not be confirmed in later studies [13].\nWe recently described an association of human herpes virus type 7 (HHV-7) DNA and large infiltrates of plasmacytoid dendritic cells (associated with virally induced inflammatory processes) in lesional skin of lichen planus patients but not in non-lesional skin of the same patients [5]. Moreover, skin of healthy volunteers and of patients with another inflammatory skin condition like psoriasis showed significantly less HHV-7 activity. Others have described plasmacytoid dendritic cell recruitment with interferon-alpha and antiviral protein MxA production, in oral and cutaneuous lichen planus lesions [16, 18]. The presence of HHV-7 in lesional skin could play a role in the pathogenesis of lichen planus. HHV-7 is an ubiquitous organism affecting more than 90% of the adult population worldwide, and primary infection occurs mainly during early childhood [1]. Like Epstein Barr virus reactivation is associated with Burkitt lymphoma, reactivation of HHV-7 during adulthood could be associated with lichen planus. To support this hypothesis, we quantified and compared the presence of HHV-7 DNA and protein in lesional lichen planus skin before, and after treatment-induced remission.\nMaterials and methods\nThe hospital medical ethics review board approved this study. Before entering the study, patients gave their written informed consent to participate. Skin biopsies were collected in a follow up study from the same patient group with lichen planus as described before [5]. In short, before treatment was initiated, two 4\u00a0mm full thickness skin biopsies were obtained from lesional skin. Patients were treated with 40\u00a0mg prednisone per os daily, diminished with 5\u00a0mg every other day until a daily dose of 20\u00a0mg was reached. From that point on, the prednisone dosage was diminished with 5\u00a0mg per week. If new lichen planus lesions recurred, the previous dose was administered and diminishment halted for 3\u00a0weeks. In case prednisone treatment was contra-indicated, other treatment options like topical corticosteroids, oral acitretin (0.5\u00a0mg\/kg body weight) and psoralen ultraviolet A combined photo therapy were considered, depending on the extent of lesions and underlying medical conditions.\nTreatment was terminated after complete clinical remission was achieved and 2\u00a0weeks later, provided no disease activity had recurred, another two biopsies were collected from recovered lesional skin adjacent to the pretreatment biopsy sites. Both time points biopsies were taken, one biopsy was processed for routine histopathology and examined by both a pathologist and a dermatologist separately to confirm the lichen planus diagnosis or signs of ongoing inflammation. The final diagnosis was made on mutual agreement and based on typical lichen planus associated dermatopathological signs like parakeratosis, wedge shaped hypergranulosis, saw-tooth configured rete ridges, vacuolar degeneration of the basal layer, colloid bodies, a band-like dermal inflammatory infiltration and pigment incontinence [17].\nThe second biopsy of each time point was snap frozen in liquid nitrogen, stored at minus 80\u00b0C, and thawed later for molecular biological analysis (HHV-7 DNA). DNA isolation and the nested PCR techniques have been previously described [5, 12]. The amplified fragment (296\u00a0bp) was identified by gel electrophoresis. The lower limit of detection of the PCR is 1\u20135 copies of HHV-7 DNA.\nWe performed immunohistochemistry as described before [5, 11]. The antibodies used in this study were directed against: HHV-7, clone 5E1 and clone KR4, both resulting in a similar specific staining pattern (data not shown; Advanced Biotechnologies Inc., Columbia, MD), CD3 (pan T cell marker; clone SK7, FITC-conjugated; BD Biosciences, San Jose, CA), and BDCA-2 (plasmacytoid dendritic cell marker; clone AC144, FITC conjugated; Miltenyi Biotec, Bergisch Gladbach, Germany). In the single-staining experiments we used clone 5E1 (directed against HHV-7 tegument protein pp85), but unfortunately this clone was no longer available at the time we performed the double staining. For this reason we used in the double staining experiments clone KR4 (directed against an unknown HHV-7 epitope). In brief the following sequence of steps was applied for single staining: blocking endogenous peroxidase activity; anti-HHV-7 (clone 5E1); biotin-conjugated goat anti-mouse antibody (Dako); horseradish peroxidase-conjugated streptavidin; and for clone KR4 a TSA amplification step (Perkin Elmer) was applied. In four patients specimens obtained before and after remission were double stained for HHV-7 and CD3 or for HHV-7 and BDCA-2. In this case the following incubation steps were performed: blocking endogenous peroxidase activity; anti-HHV-7 (clone KR4); biotin-conjugated goat anti-mouse antibody (Dako); horseradish peroxidase-conjugated streptavidin; TSA amplification according to the manufacturer\u2019s protocol (Perkin Elmer); an AEC kit to visualize the peroxidase activity (Vector); blocking potential available biotin, anti-mouse immunoglobulin, and peroxidase activity; FITC-BDCA-2 or FITC-CD3 antibody; rabbit anti-FITC (Dako); peroxidase-conjugated goat anti-rabbit (Dako) for BDCA-2 and phosphatase-conjugated goat anti-rabbit (Dako) for CD3; for BDCA-2 a TSA amplification step followed by phosphatase-conjugated streptavidin; and finally an AP kit (Vector) to visualize the phosphatase activity. All single-stained sections were counterstained with Mayer\u2019s hematoxylin (Fluka). The expression of single and double stained cells was counted in each entire section at 200\u00d7 magnification using a standard binocular light microscope (Olympus, Tokyo, Japan) equipped with a 0.5\u00a0\u00d7\u00a00.5\u00a0mm ocular grid. The number of cells in the epidermis was expressed per mm and the number in the dermis per mm2.\nDuring a follow up period of 1\u00a0year, visits were scheduled every 6\u00a0months. In case a patient experienced a relapse in between follow up visits, or after the initial 1\u00a0year follow up period, extra visits were performed at the patient\u2019s initiative for additional monitoring and treatment. Patients without a relapse on record were interviewed by telephone to exclude disease reoccurrence. The Wilcoxon rank sum test was used for statistical analysis of cell counts between samples before and after remission and the McNemar test for binominal data; P\u00a0\u2264\u00a00.05 was considered significant.\nResults\nEighteen patients entered this study with a mean age of 43.9\u00a0\u00b1\u00a011.5\u00a0years at the time of inclusion (mean age of 12 males was 41.7\u00a0\u00b1\u00a010.9 and for 6 females 48.4\u00a0\u00b1\u00a012.3\u00a0years). The mean disease duration was 4.7\u00a0\u00b1\u00a03.3\u00a0months (Table\u00a01). Lesional biopsies were taken from the lower leg in 14 cases, 3 times from the trunk and in 1 patient from the wrist. Fourteen patients were treated with oral prednisone, two patients received local corticosteroid treatment, one psoralen and ultraviolet A photo therapy and one acitretin. Three patients were lost to follow up, two patients refused biopsies after remission but their relapse data could be recorded. From the remaining 13 patients, biopsies could be collected after remission. The mean treatment duration until remission was 136\u00a0\u00b1\u00a060\u00a0days (in the prednisone treated subgroup 134\u00a0\u00b1\u00a060\u00a0days, in the subgroup who received other treatment modalities 148\u00a0\u00b1\u00a074\u00a0days). In 4 out of 13 biopsies a minimal remnant inflammatory infiltrate around the basal layer was still present, in spite of clinical remission.\nTable\u00a01Treatment characteristics and HHV-7 data before treatment and after remissionPatientDisease duration (months)Treatment modalityTreatment duration (days)Biopsy siteRelapse interval (months)Before treatmentAfter remissionHHV-7 positive epidermal cellsHHV-7 positive dermal cells BDCA2 positive cellsHHV-7\/BDCA2 double positive cellsHHV-7 PCRHHV-7 positive epidermal cellsHHV-7 positive dermal cellsBDCA2 positive cellsHHV-7\/BDCA2 double positive cellsHHV7 PCRPathology112Prednisone66Lower leg55,124,7Positive8,88,8NegativeNormal22Prednisone169Lower leg135,789,2Positive6,211,1NegativeNormal312Prednisone139Lower legNone4,711,7118,738,7Positive3,345,36511NegativeNormal45Prednisone72Lower leg16,753PositiveNANANANA56Prednisone64Foot62,429,4Negative4,720,5NegativeInflam61TopicalNAFootNA53,882,92813,3PositiveNANANANANANA76PUVA96Lower legNone22,339Negative2,35,9NegativeInflam81Prednisone75Foot4316,967Positive1,85,3NegativeNormal94Topical200Lower legNone9,62369,735,3Positive2,963812,3NegativeNormal102Prednisone205Wrist716115,9Negative3,226,9NegativeInflam112PrednisoneNALower legNA1234,9PositiveNANANANA122AcitretinNAFlankNone7,438,6PositiveNANANANA137Prednisone95Back513,466,118748Positive417,7426,7NegativeNormal147Prednisone247Lower leg44942,8Negative2,415,5NegativeNormal156Prednisone131Foot14,1979,5Negative24,7PositiveNormal162PrednisoneNABackNone7,435,6NegativeNANANANA173Prednisone154FootNone19,3119,3Positive0,82,5NegativeInflam184Prednisone187FootNone13,1106,832,75,7Negative0,83,2224,3NegativeNormalThe relapse interval was counted from discontinuation of treatment until the first relapse. HHV-7 immunohistochemical positive dermal and epidermal cells and BDCA2 positive cells were counted per mm2. Some biopsies after remission showed minimal basal layer inflammatory activity (inflam)NA not available\nDuring follow up (with a minimum period of 30\u00a0months) 9 out of 16 patients experienced a relapse of lichen planus symptoms, 6 patients within 1\u00a0year, 3 within 4\u00a0years. All relapses occurred in patients treated with oral prednisone. In 2 patients with relapses, remnant inflammation after remission had been noticed.\nIn 11\/18 (61%) patients lichen planus affected skin contained HHV-7 specific DNA before treatment compared to 1\/13 (8%) patients after remission (P\u00a0=\u00a00.01, McNemar test). In one patient (no. 15) HHV-7 DNA was detected after remission whereas before treatment no HHV-7 DNA was found.\nImmunohistochemical single staining for the presence of HHV-7 tegument protein revealed that before treatment the mean number of HHV-7 positive cells was 58.9\u00a0\u00b1\u00a033.4 per mm2 in the dermis and 12.7\u00a0\u00b1\u00a011.7 per mm in the epidermis (Fig.\u00a01). After remission the number of positive cells had dropped to 13.3\u00a0\u00b1\u00a012.2 and 3.3\u00a0\u00b1\u00a02.2, respectively (P\u00a0<\u00a00.05 with the paired samples t test).\nFig.\u00a01Mean cell count (in mm for the epidermis and mm2 for the dermis) of HHV-7 tegument pp85 positive cells with immunohistochemical staining in lichen planus affected epidermis and dermis, before treatment (white bars) and after remission (black bars). * and # P\u00a0<\u00a00.05 with the Wilcoxon rank sum test\nImmunohistochemical double staining was performed on skin specimens of four patients to investigate whether HHV-7 protein expression may be associated with T cells or plasmacytoid dendritic cells, which both represent major constituents of the inflammatory infiltrate. We found a considerable number of HHV-7\/BDCA-2 double positive cells in lesional lichen planus skin before treatment but not after treatment (Fig.\u00a02). The number of single stained BDCA2 positive cells decreased approximately twofold \u201cbefore treatment\u201d compared to \u201cafter remission\u201d (Table\u00a01). With HHV-7\/CD3 double staining, we predominantly observed single stained HHV-7 or CD3 cells whereas only an occasional double positive cell could be found (Fig.\u00a03).\nFig.\u00a02Double positive stained plasmacytoid dendritic cells reflects intracellular HHV-7 protein. BDCA-2 positive plasmacytoid dendritic cells (blue stain) containing HHV-7 protein (red stain) in lesional LP skin before (a) and after (b) treatment (overview 200\u00d7 and inlay 400\u00d7 magnification)Fig.\u00a03HHV-7 replication does not coincide with lymphocytes. HHV-7 positive cells (red stain) and CD3 positive lymphocytes (blue stain) cells in lesional lichen planus skin are in majority mutually exclusive (400\u00d7 magnification)\nDiscussion\nLichen planus is characterized by a band-like lymphocytic infiltrate, which seems to be involved in an attack of the basal epidermal layer. It is suggested that these T lymphocytes are directed towards infected skin cells expressing viral antigens. Molecular mimicry is a second conceivable inflammatory mechanism initiated by microbial antigens or microbial-reactive T lymphocytes that cross react with self antigens sharing homology (i.e., basal layer structures).\nIn our previous study we localized HHV-7 pp85 antigen containing cells in lesional lichen planus tissue around de dermo-epidermal junction, coinciding with the location of the inflammatory infiltrate [5]. In addition, we detected high numbers of plasmacytoid dendritic cells (cells that are associated with the clearance of virally induced diseases) within the infiltrate. The presence of HHV-7 DNA and HHV-7 antigen did not coincide with non-lesional lichen planus skin, psoriatic skin and normal skin from healthy donors. Since HHV-7 was not found in psoriatic skin (which is also characterized by a lymphocytic infiltrate) it is unlikely that HHV-7 in lichen planus is a reflection of an innocent bystander in a dense influx of lymphocytes. Moreover, in the current study we could localize HHV-7 protein, mainly in BDCA-2 positive plasmacytoid dendritic cells and sporadic in CD3 positive lymphocytes. This is remarkable since HHV-7 has been characterized as a T-lymphotropic virus [10]. Our results suggest that plasmacytoid dendritic cells can also host HHV-7. This is conceivable since plasmacytoid dendritic cells express CD4 [15], which is the main receptor used by HHV-7 to enter target cells [21].\nHere we investigated the effect of therapy-induced remission of lichen planus on HHV-7 replication. Both the number of HHV-7 DNA containing skin samples and the absolute number of HHV-7 antigen containing skin cells (in dermis and epidermis) diminished significantly after treatment-induced remission. Since we included a 2-week washout period between treatment termination and obtaining the post remission biopsies, a direct medication effect on viral replication is unlikely. In contrast to a fourfold diminution in HHV-7 positive cells after treatment, there was only a twofold reduction in the number of BDCA2 positive plasmacytoid dendritic cells. This makes it unlikely that the diminished HHV-7 expression only depends on a reduced number of plasmacytoid dendritic cells after treatment. Whether plasmacytoid dendritic cells are carriers of HHV-7 driving the disease activity or that these cells are infected as innocent bystanders, or actively induce the inflammatory process is not yet clear and needs further study.\nHHV-7, a member of the beta herpes virus subfamily was first discovered by Frenkel et al. [10] in CD4 positive T lymphocytes of healthy individuals. It is an ubiquitous virus with a worldwide prevalence exceeding 90%. Most of the primo infections occur during infancy [1]. Since the discovery of HHV-8 as the causative agent for Kaposi sarcoma [3], all three human herpes viruses (HHV-6, -7 and -8) discovered in the last few decades have been subjected to studies on their role in skin diseases of unknown origin like pitryasis rosea [7] and exanthema subitum [2, 19].\nA shared characteristic of all herpes viruses is their unique ability to persist in the host after primary infection, and become reactivated later in life (sometimes after many years of latency). Symptomatic reactivation of herpes viruses is a well-known phenomenon in the daily dermatological practice. For example, shingles afflicting patients later in life after acquisition of the causative varicella zoster virus during childhood, and the recurrence of labial and genital herpes simplex virus lesions after reactivation of viral DNA. Human herpes virus 6 reactivation is believed to cause the drug related exanthema and systemic side effect syndrome (DRESS) [6].\nIt is unlikely that HHV-7 primary infections cause lichen planus, since first transmission mostly occurs during childhood, whereas lichen planus affects mainly patients during midlife. Yet, it is conceivable that HHV-7 reactivation triggers lichen planus, in the same fashion as Epstein-Barr virus reactivation is associated with Burkitt lymphoma.\nThe apparent paradoxical efficacy of steroid therapy in lichen planus and a considered viral cause can be explained by a \u201chit and run\u201d model where the actual viral reactivation would be short lived but subsequently responsible for an independently propagated inflammatory skin reaction. This could also explain why not in all patients HHV-7 was detected at the time of inclusion. Moreover, in one patient (no. 15) HHV-7 DNA was detected after remission. It is this patient that experienced a relapse 1\u00a0month later and returned to the clinic for additional treatment. Possibly HHV-7 can trigger lichen planus episodes, including relapses, but resolve before clinical lichen planus symptoms appear. The other patient (no. 4) with a relapse within 1\u00a0month unfortunately refused us a post remission skin sample. On the other hand, the absence of HHV-7 replication in some of our patients could also be explained assuming a multi causal pathogenesis for lichen planus.\nFurther evidence for the causal association of HHV-7 and lichen planus could come from clinical studies on the efficacy of anti-herpetic medication. Which drug would be the best option is not yet clear. Guanoside analogons with little side effects like acyclovir show little activity against HHV-7 [8]. HHV-7 is more susceptible to cidofovir and foscarnet but these drugs are known for serious side effects [20].\nFredericks and Relman proposed a reconsideration of Koch\u2019s postulates for the identification of microbial antigens as causative agents in diseases [9]. With data presented here and in our previous study [5] we were able to meet with three out of their seven postulates for the establishment of a causal relationship between HHV-7 and lichen planus. (1) Microbial nucleic acid sequences were present in most cases in diseased sites and not in sites that lack pathology. (2) Fewer nucleic acid sequences were found in hosts without the disease. (3) Nucleic acid sequences correlates were present at a cellular level. The fourth postulate, a decrease of the number of nucleic acid sequences during resolution of the disease and increase with relapse, was met in this study as far as disease resolution is concerned. Unfortunately we did not collect lesional biopsies in those patients that suffered from relapses. HHV-7 reactivation during relapses will have to be investigated in future studies. (5) The number of nucleic acid sequences should correlate with disease severity and (6) reproducibility of the evidence will have to be met in future longitudinal studies. The seventh postulate, clinical features and pathology should be consistent with known biological characteristics of the microbe, will also have to be addressed in future studies since still little is known about the effects of HHV-7 in vivo yet. The hypothesis of HHV-7 as the causative agent for lichen planus deserves further research in larger studies.","keyphrases":["lichen planus","plasmacytoid dendritic cells","human herpes virus 7","skin disease","herpes virus replication"],"prmu":["P","P","P","P","R"]}
{"id":"Purinergic_Signal-4-1-2245997","title":"Pyrazolo-triazolo-pyrimidines as adenosine receptor antagonists: Effect of the N-5 bond type on the affinity and selectivity at the four adenosine receptor subtypes\n","text":"In the last few years, many efforts have been made to search for potent and selective human A3 adenosine antagonists. In particular, one of the most promising human A3 adenosine receptor antagonists is represented by the pyrazolo-triazolo-pyrimidine family. This class of compounds has been strongly investigated from the point of view of structure-activity relationships. In particular, it has been observed that fundamental requisites for having both potency and selectivity at the human A3 adenosine receptors are the presence of a small substituent at the N8 position and an unsubstitued phenyl carbamoyl moiety at the N5 position. In this study, we report the role of the N5-bond type on the affinity and selectivity at the four adenosine receptor subtypes. The observed structure-activity relationships of this class of antagonists are also exhaustively rationalized using the recently published ligand-based homology modeling approach.\nIntroduction\nAdenosine is an ubiquitous modulator, which exerts its functions through interaction with four G-protein-coupled receptors classified as A1, A2A, A2B and A3 [1]. In recent decades, intensive and successful efforts have been made by medicinal chemists to discover potent and selective ligands (both agonists and antagonists) for almost all adenosine receptor subtypes [2, 3]. Only the A2B subtype is still without a very potent and selective agonist ligand [4\u20136]. At the same time, great progress has been made in the field of human A3 adenosine receptor antagonists. This receptor seems to be associated with cerebroprotection [7, 8] and cardioprotection [9] and effects on the immune and inflammatory systems [10, 11]. The A3 adenosine receptor subtype, recently cloned from different species (e.g., rat, human, dog, sheep) [12, 13], is coupled to the modulation of at least two second-messenger systems: inhibition of adenylate cyclase and stimulation of phospholipase C and D [12, 13]. In humans, A3 receptors have been found in several organs, such as lung, liver, kidney, heart, and with a lower density, in the brain [9, 12, 13]. This receptor subtype is under examination in relation to its potential therapeutic applications. In particular, antagonists for A3 receptors seem to be useful for the treatment of inflammation or glaucoma [14, 15].\nIn the last 5\u00a0years, many efforts have been made in the context of searching for potent and selective human A3 adenosine antagonists. In this field, several different classes of compounds have been proposed, possessing good affinity (nM range) and with a broad range of selectivity [16].\nIn particular one of the most attractive human A3 adenosine receptor antagonists is represented by the pyrazolo-triazolo-pyrimidine family, reported by our group [17\u201323]. In Fig.\u00a01, the most interesting members of this class of compounds (1\u20133), which display high affinity and selectivity for this receptor subtype, are depicted [17\u201323].\nFig.\u00a01Structures and binding profiles of some representative pyrazolo-triazolo-pyrimidines as human A3 adenosine receptor antagonists\nThis class of compounds has been strongly investigated from the point of view of structure-activity relationships. In particular, it has been observed that fundamental requisites for having both potency and selectivity at the human A3 adenosine receptors are a small substituent at the N8 position and an unsubstituted phenyl carbamoyl moiety at the N5 position [17, 21]. In contrast, when the phenyl carbamoyl moiety (2) was replaced with a phenylacetyl group (3), a significant decrease in affinity at the hA3 adenosine receptors with a simultaneous retention or a slight increase in affinity at the hA2B adenosine receptor subtype was observed [24].\nThese data seem to suggest that bond type at the N5 position plays a fundamental role in the receptor recognition. For this reason, we decided to further investigate the effect of the bond nature at this position on affinity and selectivity, synthesizing two new derivatives bearing the benzoyl- (4) and benzensulfonamido (5) moieties at the N5 position.\nChemistry\nThe designed compounds (4, 5) were easily prepared by treating the well-known N8 methyl derivative (6) [18] with benzoyl (7) or benzensulfonyl (8) chlorides in dioxane in the presence of pyridine at reflux for 16\u00a0h (Scheme\u00a01).\nScheme\u00a01Preparation of designed compounds\nResults and discussion\nNewly synthesized (4, 5) and reference compounds (2, 3) were tested at the human A1, A2A and A3 receptors expressed in CHO cells; [3H]CCPA (A1) and [3H]NECA (A2A, A3) were used as radioligands in binding assays [25]. Radioligand binding at A2B adenosine receptors is problematic as no high-affinity ligand is readily available for this subtype. Therefore, inhibition of NECA-stimulated adenylyl cyclase activity was determined as a measurement of affinity of compounds (Table\u00a01).\nTable\u00a01Biological profile of synthesized (4, 5) and reference (2, 3) compoundsCompoundRhA1a (Ki nM)hA2Ab (Ki nM)hA2Bc (IC50 nM)hA3d (Ki nM)hA1\/hA3hA2A\/hA32CONHPh310 (295\u2013327)27.7 (13.3\u201357.8)3,440 (2,880\u20134,110)1.80 (0.88\u20133.68)172153COCH2Ph1,040 (864\u20131,260)282 (201\u2013375)12,620 (9,730\u201316,400)0.92 (0.80\u20131.06)1,1303604COPh2,030 (1,710\u20132,400)879 (643\u20131,200)>30,00015.7 (7.85\u201331.5)129565SO2Ph20,700 (16,700\u201325,700)6,060 (5,170\u20137,110)>30,000744 (534\u20131,040)288Data are expressed as geometric means, with 95% confidence limitsaDisplacement of specific [3H]-CCPA binding at human A1 receptors expressed in CHO cells, (n\u2009=\u20093\u20136)bDisplacement of specific [3H]-NECA binding at human A2A receptors expressed in CHO cellscIC50 values of the inhibition of NECA-stimulated adenylyl cyclase activity in CHO cells expressing hA2B receptorsdDisplacement of specific [3H]-NECA binding at human A3 receptors expressed in CHO cells\nA careful examination of the data clearly revealed significant differences in the binding profile of the reference compounds (2, 3) compared with the literature data (see Fig.\u00a01). In fact, in these binding studies, compound 2 was found to be ninefold less potent at the hA3 receptors with a consequent reduction in selectivity versus the other receptor subtypes, in particular versus hA2A (hA2A\/hA3\u2009=\u200915). In contrast, derivative 3 retained good affinity at the hA3 adenosine receptor and a quite good range of selectivity versus the other receptors in comparison with literature data. Also if these discrepancies in binding profile could be attributed to a different biological method utilized (e.g., radioligand), the data seem to indicate that the phenylacetyl moiety retains or enhances both potency and selectivity compared with the well-known phenylcarbamoyl chain. A significantly different binding profile was instead observed when the chain at the N5 position was alternatively modified. Introduction of a shorter amide (4) with respect phenylacetyl chain (3) led to a loss of potency at all four adenosine receptor subtypes, but a quite good retention of selectivity was observed. In contrast, the presence of a benzensulfonamido moiety at the N5 position induced a substantial loss of both potency and selectivity at the four adenosine receptor subtypes.\nMolecular modeling studies were performed on the pyrazolo-triazolo-pyrimidine derivatives 2\u20135 in order to identify the hypothetical binding motif of these N5 analogues and to rationalize their structure-activity relationship.\nFollowing our previously reported modeling studies [17\u201323], we built up a refined model of human A3 receptor by using a rhodopsin-based homology modeling (RBHM) approach [26\u201329]. Moreover, our recently described ligand-based homology modeling methodology (LBHM) has been used to simulate the conformational changes induced by ligand binding (methodological details are summarized in the Experimental Section) [29, 30].\nUsing this methodology, we found an \u201cexpanded\u201d conformational model of the human A3 receptor reverse agonist-like state, in which both shape and chemical complementarities have been specifically optimized around each ligand. Considering these new N5 analogues, the molecular volume of transmembrane (TM) binding cavity has been changed from 660 (A3 model obtained by the conventional rhodopsin-based homology modeling) to 840 \u00c53 (expanded A3 model obtained by ligand-based homology modeling) without altering the conventional rhodopsin-like receptor topology. The binding cavity reorganization induced by ligand binding is due to the conformational change in several amino acid side chains, such as Leu90 (3.32), Leu91 (3.33), Thr94 (3.36), His95 (3.37), Ile98 (3.40), Gln167 (EL2), Phe168 (EL2), Phe182 (5.43), Ile186 (5.47), Leu190 (5.51), Phe239 (6.44), Trp243 (6.48), Leu244 (6.49), Leu264 (7.35), and Ile268 (7.39).\nInterestingly, none of the new pyrazoloquinoline antagonists found an energetically stable docking pose in the conventional RBHM-driven A3 model. This is mainly due to the unfavorable topological complementarity among these antagonists and corresponding RBHM-driven TM binding cavity. In particular, highly destabilizing van der Waals interactions (steric conflicts) seem to be the reason for absent topological complementarities. These steric conflicts are drastically reduced or completely eliminated after the application of the LBHM approach.\nMolecular docking studies were carried out for the pyrazolo-triazolo-pyrimidine antagonists 2\u20134, using the \u201cexpanded\u201d conformational state of the receptor. As shown in Fig.\u00a02, we found a similar binding motif indicating that a common receptor-driven pharmacophore model can be depicted. This finding is in agreement with our previously reported studies [17\u201323].\nFig.\u00a02Hypothetical binding motif of the newly synthesized pyrazolo-triazolo-pyrimidine antagonists 2\u20134. The most energetically favorable docked conformation of each derivative is viewed from the membrane side facing TM helices 4 and 5. To clarify the TM cavity, the view of TM4 was omitted. Side chains of some amino acids important for ligand recognition are highlighted. Hydrogen atoms are not displayed\nIndeed, ligand recognition occurs in the upper region of the TM bundle, and the pyrazolo-triazolo-pyrimidine moiety is surrounded by TMs 3, 5, 6, 7 with the substituent in the N5 position oriented toward the intracellular environment. As shown in Fig.\u00a02, the furan ring at the 2-position is close to TMs 3 and 7. Interestingly, an important hydrogen bonding network can be observed in all energetically stable docked conformations of pyrazolo-triazolo-pyrimidine antagonists. In particular His95 (3.37) and Ser247 (6.52) are able to interact through hydrogen bonding with the N5-carbonyl oxygen of compounds 2\u20134 (2C=O\u00b7His95 ca. 3.0\u00a0\u00c5; 3C=O\u00b7Ser247 ca. 2.8\u00a0\u00c5; 4C=O\u00b7His95 ca. 2.9\u00a0\u00c5.)\nThese polar amino acids seem to be critical for the recognition of all antagonist structures and for receptor selectivity. In particular, Ser247 (6.52) of human A3 receptor subtype is not present in the corresponding position of A1 and A2 receptors, where the residue is replaced by a histidine (His251 in human A1, His250 in human A2A and His251 in human A2B). Histidine side chain is bulkier than serine, and possibly for this reason, large substituents at the N5 position of pyrazolo-triazolo-pyrimidine framework are not well tolerated by A1 and A2 receptor subtypes. In contrast, the hydroxyl group of Ser247 (6.52) of human A3 receptor is appropriately positioned to form a hydrogen-bonding interaction with the carbonyl oxygen of the N5-amide\/ureide group of compounds 2\u20134. These observations support the importance of an N5-acyl\/carbamoyl group in modulating receptor selectivity. The hydrophobic environment of the five nonpolar amino acids Ile98 (3.40), Ile186 (5.47), Leu190 (5.51), Phe239 (6.44), and Leu244 (6.49) can comfortably accommodate the phenyl ring of all N5-acyl\/carbamoyl derivatives.\nIn contrast, the introduction of the N5-sulfonamido moiety, as present in derivative 5, drastically reduces the affinity at the human A3 receptor. Interestingly, in this specific case molecular docking is not able to find an antagonist pose comparable to those described for the other N5-acyl\/carbamoyl derivatives. As shown in Fig.\u00a03, the rigid tetrahedral configuration associated with the N5-sulfonamido moiety avoids the sampling of energetically favorable antagonist poses in which the phenyl ring is linked to the N5 position in the hydrophobic pocket delimited by Ile98 (3.40), Ile186 (5.47), Leu190 (5.51), Phe239 (6.44), and Leu244 (6.49).\nFig.\u00a03Hypothetical binding motif of the newly synthesized N5-sulfonamido pyrazolo-triazolo-pyrimidine antagonist 5. The most energetically favorable docked conformation of each derivative is viewed from the membrane side facing TM helices 4 and 5. To clarify the TM cavity, the view of TM4 was omitted. Hydrogen atoms are not displayed\nThe most stable docking pose of 5 presents the N5-sulfonamido moiety close to TM3 and TM7, and the phenyl ring linked to N5 position is surrounded by a hydrophobic pocket delimited by Leu90 (3.32) and Ile268 (7.39). This antagonist pose is energetically less stable (ca. 15\u00a0kcal\/mol) with respect to those found for derivatives 2\u20134, due to the absence of the stabilizing interactions among the polar residues Thr94 (3.36), His95 (3.37), and Ser247 (6.52) and the N5-sulfonamido moiety. Structure superimposition of compounds 4 and 5 is shown in Fig.\u00a04.\nFig.\u00a04Structure superimposition of compounds 4 (in magenta) and 5 (in green) inside the receptor binding site\nThis severe steric constriction might explain the drastic reduction in affinity of derivative 5 at the human A3 receptor.\nConclusions\nThe present study has led to potent and selective human A3 adenosine receptor antagonists belonging to the class of the pyrazolo-triazolo-pyrimidine. In this study, we clarified the role of the N5-bond type on the affinity and selectivity at the four adenosine receptor subtypes. In particular, we confirmed the critical function played by the N5-acyl\/carbamoyl moiety in receptor recognition. The observed structure-activity relationship was also rationalized by the recently published ligand-based homology modeling approach, indicating that chemical and topological properties of the different ligands might induce important conformational changes in the antagonist-driven binding site of the human A3 adenosine receptor.\nExperimental section\nChemistry\nGeneral Reactions were routinely monitored by thin-layer chromatography (TLC) ion silica gel (precoated F254 Merck plates) and products visualized with iodine or potassium permanganate solution. Infrared spectra (IR) were measured on a Perkin Elmer 257 instrument. 1H NMR was determined in CDCl3 or DMSO-d6 solutions with a Bruker AC 200 spectrometer; peak positions are given in parts per million (6) downfield from tetramethylsilane as internal standard, and J values are given in Hz. Light petroleum ether refers to the fractions boiling at 40\u201360\u00b0C. Melting points were determined on a Buchi-Tottoli instrument and are uncorrected. Chromatographies were performed using Merck 60\u2013200 mesh silica gel. All products reported showed IR and 1H NMR spectra in agreement with the assigned structures. Organic solutions were dried over anhydrous magnesium sulphate. Elemental analyses were performed by the microanalytical laboratory of Dipartimento di Chimica, University of Trieste.\nGeneral procedures for the preparation of 5-(benzoyl)amino-8-methyl-2-(2-furyl)-pyrazolo[4,3 ]-1,2,4-triazolo[1,5-c]pyrimidine (4) and 5-benzensulfonamido-8-methyl-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (5)\nAmino compound (6) (10\u00a0mmol) was dissolved in freshly distilled dioxane (15\u00a0mL), and benzoylchloride or benzesulfonyl chloride (13\u00a0mmol) and pyridine (20\u00a0mmol) were added. The mixture was refluxed under argon for 16\u00a0h. Then the solvent was removed under reduced pressure and the residue was dissolved in EtOAc (30\u00a0ml) and washed twice with water (15\u00a0mL). The organic phase was dried on Na2SO4 and concentrated under reduced pressure. The residue was purified by flash chromatography (EtOAc-light petroleum 8:2) to afford the desired compounds (4, 5) as solids.\n5-(Benzoyl)amino-8-methyl-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (4)\nYield 78%, pale yellow solid, mp (EtOAc-light petroleum) 240\u00b0C; IR (KBr): 3,425-2,965, 1,675, 1,645, 1,620, 1,545, 1,450\u00a0cm\u22121; 1 H NMR (DMSO d6) \u03b4: 4.42 (s, 3H); 6.65 (dd, 1H, J\u2009=\u20092, J\u2009=\u20094); 7.24 (d, 1H, J\u2009=\u20094); 7.41\u20137.62 (m, 4H); 7.85\u20137.98 (m, 2H); 8.14 (s, 1H); 9.87 (bs, 1H). Anal. for C18H13N7O2 (MW 359.34). Calculated: C 50.16; H 3.69; N 27.29. Found: C 50.01; H 3.61; N 27.35.\n5-Benzensulfonamido-8-methyl-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (5)\nYield 62%, white solid, mp (EtOAc-light petroleum) > 300\u00b0C; IR (KBr): 3,375-2,985, 1,640, 1,600, 1,540, 1,355, 1,150\u00a0cm\u22121; 1H NMR (DMSO d6) \u03b4: 4.39 (s, 3H); 6.68 (dd, 1H, J\u2009=\u20092, J\u2009=\u20094); 7.22 (d, 1H, J\u2009=\u20094); 7.38\u20137.59 (m, 4H); 7.88-8.02 (m, 2H); 8.11 (s, 1H); 11.05 (bs, 1H). Anal. for C17H13N7O3S (MW 395.40). Calculated: C 51.64; H 3.31; N 24.80; S 8.11. Found: C 51.87; H 3.35; N 24.97; S 8.07.\nBiology\nAll pharmacological methods followed the procedures as described earlier [25]. In brief, membranes for radioligand binding were prepared from CHO cells stably transfected with human adenosine receptor subtypes in a two-step procedure. In a first low-speed step (1,000\u2009\u00d7\u2009g), cell fragments and nuclei were removed. The crude membrane fraction was sedimented from the supernatant at 100,000\u2009\u00d7\u2009g. The membrane pellet was resuspended in the buffer used for the respective binding experiments, frozen in liquid nitrogen and stored at \u221280\u00b0C. For the measurement of adenylyl cyclase activity only one high-speed centrifugation of the homogenate was used. The resulting crude membrane pellet was resuspended in 50\u00a0mM Tris\/HCl, pH 7.4 and immediately used for the cyclase assay.\nFor radioligand binding at A1 adenosine receptors, 1\u00a0nM [3H]CCPA was used, whereas 30 and 10\u00a0nM [3H]NECA were used for A2A and A3 receptors, respectively. Nonspecific binding of [3H]CCPA was determined in the presence of 1 mM theophylline, and in the case of [3H]NECA, 100\u00a0pM R-PIA was used. Ki values from competition experiments were calculated with the program SCTFIT [31].\nRadioligand binding at A2B adenosine receptors is problematic as no high-affinity ligand is available for this subtype. Therefore, inhibition of NECA-stimulated adenylyl cyclase activity was determined as a measurement of affinity of compounds. IC50 values from these experiments were converted to Ki values with the Cheng and Prusoff equation [32].\nComputational methodologies\nAll modeling studies were carried out on a 10 CPU (PIV-3.0GHZ and AMD64) Linux cluster running under openMosix architecture [33].\nHomology modeling, energy calculation, and docking studies were performed using the Molecular Operating Environment (MOE, version 2006.08) suite [34].\nAll docked structures were fully optimized without geometry constraints using RHF\/AM1 semiempirical calculations. Vibrational frequency analysis was used to characterize the minima stationary points (zero imaginary frequencies). The software package MOPAC (ver. 7) [35], implemented in MOE suite, was utilized for all quantum mechanical calculations.\nHomology model of the human A3 AR Based on the assumption that GPCRs share similar TM boundaries and overall topology, a homology model of the hA3 receptor was constructed. First, the amino acid sequences of TM helices of the A3 receptor were aligned with those of bovine rhodopsin, guided by the highly conserved amino acid residues, including the DRY motif (D3.49, R3.50, and Y3.51) and three proline residues (P4.60, P6.50, and P7.50) in the TM segments of GPCRs. The same boundaries were applied for the TM helices of the A3 receptor as they were identified from the X-ray crystal structure for the corresponding sequences of bovine rhodopsin [36], the CR coordinates of which were used to construct the seven TM helices for the hA3 receptor.The loop domains of the hA3 receptor were constructed by the loop search method implemented in MOE. In particular, loops are modeled first in random order. For each loop, a contact energy function analyzes the list of candidates collected in the segment searching stage, taking into account all atoms already modeled and any atoms specified by the user as belonging to the model environment. These energies are then used to make a Boltzmann-weighted choice from the candidates, the coordinates of which are then copied to the model. Any missing side-chain atoms are modeled using the same procedure. Side chains belonging to residues whose backbone coordinates were copied from a template are modeled first, followed by side chains of modeled loops. Outgaps and their side chains are modeled last.Special caution has to be given to the second extracellular (EL2) loop, which has been described in bovine rhodopsin as folding back over transmembrane helices [36] and, therefore, limiting the size of the active site. Hence, amino acids of this loop could be involved in direct interactions with the ligands. A driving force for this peculiar fold of the EL2 loop might be the presence of a disulfide bridge between cysteines in TM3 and EL2. Since this covalent link is conserved in all receptors modeled in the current study, the EL2 loop was modeled using a rhodopsin-like constrained geometry around the EL2-TM3 disulfide bridge. After the heavy atoms were modeled, all hydrogen atoms were added, and the protein coordinates were then minimized with MOE using the AMBER94 force field [37]. The minimizations were carried out by the 1,000 steps of steepest descent followed by conjugate gradient minimization until the rms gradient of the potential energy was less than 0.1\u00a0kcal mol\u22121 \u00c5\u22121. Protein stereochemistry evaluation was performed by several tools (Ramachandran and Chi plots measure phi\/psi and chi1\/chi2 angles, clash contacts reports) implemented in MOE suite [34].\nLigand-based homology modeling We have recently revisited the rhodopsin-based model of the human A3 receptor in its resting state (antagonist-like state), taking into account a novel strategy to simulate the possible receptor reorganization induced by the antagonist-binding [28]. We called this new strategy ligand-based homology modeling. Briefly, ligand-based homology modeling technique is an evolution of a conventional homology modeling algorithm based on a Boltzmann-weighted randomized modeling procedure adapted from Levitt [38] with specialized logic for the proper handling of insertions and deletions so that any selected atoms will be included in the energy tests and minimization stages of the modeling procedure. Ligand-based option is very useful when one wishes to build a homology model in the presence of a ligand docked to the primary template, or other proteins known to be complexed with the sequence to be modeled [34]. In this specific case, both model building and refinement take into account the presence of the ligand in terms of specific steric and chemical features. In order to generate an initial ensemble of ligand poses, a conventional docking procedure (see next section for details) with reduced van der Waals radii (equal to 75%) and an increased Coulomb-vdW cutoff (cutoff on 10\u00a0\u00c5; cutoff on 12\u00a0\u00c5) was performed. For each pose, an homology model is then generated to accommodate the ligand by reorienting nearby side chains. These residues and the ligand are then locally minimized. Finally, each ligand is re-docked into its corresponding low energy protein structures and the resulting complexes are ranked according to MOEScore [34].\nDifferent quantitative measures of molecular volume of the receptor binding cavities have been carried out by using MOE suite [34]. Prediction of antagonist-receptor complex stability (in terms of corresponding pKi value) and the quantitative analysis for non-bonded intermolecular interactions (H-bonds, transition metal, water bridges, hydrophobic) were calculated and visualized using several tools implemented into MOE suite [34].\nMolecular docking of the hA3 AR antagonists All antagonist structures were docked into the hypothetical TM binding site by using the MOE-dock tool, part of the MOE suite. Searching is conducted within a user-specified 3D docking box (the standard protocol selects all atoms inside 12\u00a0\u00c5 from the center of mass of the binding cavity), using the Tabu Search [39] protocol (standard parameters are 1,000 steps\/run, 10 attempts\/step, and 10 Tabu list length), and the MMFF94 force field [40]. MOE-dock performs a user-specified number of independent docking runs (50 in our specific case) and writes the resulting conformations and their energies in a molecular database file. The resulting docked complexes were subjected to MMFF94 energy minimization until the rms of conjugate gradient was <0.1\u00a0kcal mol\u22121 \u00c5\u22121. Charges for the ligands were imported from the MOPAC output files. To better refine all antagonist-receptor complexes, a rotamer exploration of all side chains involved in the antagonist-binding was carried out. Rotamer exploration methodology was implemented in MOE suite [34].","keyphrases":["adenosine receptors","ligand-based homology modeling","molecular modeling","antagonist binding"],"prmu":["P","P","P","R"]}
{"id":"Eur_J_Epidemiol-3-1-2071968","title":"Case-control study of environmental and social factors influencing cryptosporidiosis\n","text":"We report on the first case-control study to investigate the role of wider environmental and socioeconomic factors upon human cryptosporidiosis. Using GIS the detailed locations of 3368 laboratory-confirmed cases were compared to the locations of an equal number of controls. All cases were genotyped enabling Cryptosporidium hominis and Cryptosporidium parvum to be examined separately. When all cryptosporidiosis cases were analyzed, several location variables were strongly associated with illness: areas with many higher socioeconomic status individuals, many individuals aged less than 4 years, areas with a high estimate of Cryptosporidium applied to land from manure, and areas with poorer water treatment. For C. hominis cases, the strongly significant risk factors were areas with many higher socioeconomic status individuals, areas with many young children and urban areas. Socioeconomic status and areas with many individuals aged less then 4 years had a greater impact for infection with C. hominis than for C. parvum. Policy implications are discussed.\nIntroduction\nCryptosporidiosis is a common gastrointestinal disease, and is widespread in many developed and developing countries [1]. Our understandings of the causes of cryptosporidiosis are based upon a large number of outbreak studies and a smaller number of case-control studies. These have highlighted risks from drinking water from poorly treated public and private supplies, swimming in swimming pools, contact with farm animals and spread within institutions such as day care centres. Contact with young children is also known to be a risk factor [2]. There are 16 species of Cryptosporidium but C.\u00a0hominis and C.\u00a0parvum are the most important pathogens for humans [3]. In England and Wales there is a large environmental reservoir of C.\u00a0parvum in livestock and transmission is related to direct or indirect contact (e.g. through faecally contaminated drinking water) with these animals. The only major environmental reservoir for C.\u00a0hominis is humans, and so infection is acquired through direct or indirect contact with other infected or colonised humans particularly when associated with foreign travel [2, 4]. For both species once a primary infection has occurred, secondary infections may result from person-to-person transmission.\nEpidemiological studies into cryptosporidiosis have used questionnaires to identify proximal risk factors (patient activity) for cryptosporidiosis such as drinking water consumption or farm visits [5]. The objective of this study was to investigate the role of wider environmental and socioeconomic factors (e.g. water supply, socioeconomic status, land use, livestock densities and healthcare accessibility) upon human cryptosporidiosis.\nThis was achieved by linking small area data with postcoded cryptosporidiosis cases. All cases were genotyped to the species level enabling us to explore the aetiologies of the two main species C.\u00a0hominis and C.\u00a0parvum separately. We used a Geographical Information System (GIS) to derive detailed and aetiologically relevant indicators of environmental sources and points of contamination. We generated detailed measures of drinking water risks by tracing each individual\u2019s mains water supply back to the water treatment works and into the source catchment.\nBackground\nThe incidence of cryptosporidiosis varies geographically and studies have indicated that C.\u00a0hominis is more common in urban areas and C.\u00a0parvum in rural areas [6, 7]. However, few studies have examined the reasons behind this. The C.\u00a0parvum excess in rural areas may be related to higher probabilities of animal contact [6], a risk factor identified in a case-control study in England and Wales [2]. Higher population densities in urban areas may lead to greater person\u2013to-person transmission for C.\u00a0hominis. Socioeconomic status varies between geographical areas and this may lead to differences in reported incidence of disease. Higher social classes are more likely to undertake foreign travel [8] and use swimming pools [9]; known risk factors for cryptosporidiosis, especially C.\u00a0hominis. Conversely, individuals of higher social class are more likely to consume fresh fruit and vegetables [10], known to be negatively associated with cryptosporidiosis [2]. Agricultural workers are more likely to be exposed to Cryptosporidium [11] and their distribution varies spatially. Individuals with poor access to healthcare are less likely to seek treatment [12] and this may vary geographically or socially. Water supplies are a well-known risk factor for cryptosporidiosis [13] and the raw water quality and the efficiency and efficacy of treatment systems vary. Biofilms, organic coatings in water distribution systems, may play a role in prolonging the presence of Cryptosporidium in water distribution systems [14]. The probability of biofilm build-up is likely to be higher in long water distribution systems.\nMethods\nSpecies specific cryptosporidiosis incidence data were obtained from the UK Cryptosporidium reference unit for the 4\u00a0year period from January 2000, collected as part of the national collection of Cryptosporidium oocysts [15]. These data represent just under half of all the cases reported to national surveillance in England and Wales over that period (Chalmers et\u00a0al. in preparation). From the original dataset of 8075 cases, 3343 were analysed once a number of records were removed from the analysis. All cases where the patient had reported recent foreign travel (22%) were removed from the sample as the infection may have been acquired overseas. These data were based upon information collected by laboratories from the faecal sample request form that was filled in by the patient or physician. It is likely, based on information from previous work, that travel information is under-reported. Further cases were removed due to gaps in the geographical coverage of the independent variables (5%) and the inability to identify the species using PCR-based methods (2%).\nHowever, the main reason for the removal of cases were records which, had incomplete postcodes (59%) meaning that it was impossible to identify an accurate residential location. It is important to consider whether this omission may be correlated with any of our independent variables and hence introduce bias into the results. One source of missing postcodes is transcription omissions at individual laboratories but because these are likely to affect samples in a random manner they are unlikely to be correlated with any independent variables. The second major source is omissions by general practitioners due to them having inaccurate patient records or failing to include postcodes when submitting samples to laboratories for analysis. There is no evidence as to whether this varies systematically in a manner, which would bias the results of this research.\nFor each case one comparator control postcode was randomly selected from within the service area of the laboratory to which the case stool sample was sent. The process was weighted to account for differences in population between postcodes. Control postcodes were selected from within the same laboratory service area as the case postcode because different laboratories have different selection criteria and screening protocols for cryptosporidiosis [16] and so differences in incidence between laboratory service areas may be artefactual. Service areas were generated by assuming that each sample was sent to its nearest laboratory (based on shortest road distance). This resulted in 135 service areas across England and Wales and the method was validated successfully by comparing the postcodes of the cases, to the laboratory to which their sample was sent. This indicated that 87% of cases were assigned to the correct laboratory. These service areas varied in size from 106\u00a0km2 to 3,770\u00a0km2 with an average size of 1,142\u00a0km2. They were consequently large enough to contain a variety of different drinking water sources and encompass a range of agricultural landscapes and socioeconomic characteristics.\nOur control postcodes represent areas where no cryptosporidiosis was reported. However, due to issues of under reporting [17] and asymptomatic infection [18, 19] some of the control postcodes may have contained affected individuals. However, the reported rate of cryptosporidiosis in England and Wales is only 9 per 100,000 per year and so even taking into account under reporting and asymptomatic infection, the probability of a control postcode containing cases remains extremely low. Furthermore, such a bias would only tend our results to the null hypothesis.\nFor each case postcode and control postcode explanatory variables were derived using GIS (ArcGIS9.1). A measure of the degree of rurality was obtained using the Rural and Urban Area Classification [20]. This groups census areas into one of eight categories ranging from \u201curban areas\u201d to \u201chamlets and isolated dwellings in sparse surroundings\u201d. A buffer zone of 2.5\u00a0km was created around the centre of each postcode and within these areas the GIS was used to extract estimates of the total amount of Cryptosporidium applied to land through animal manures. These were based upon a 1\u00a0km2 map of manure applications developed by the Agricultural Land Advisory Service [21]. This was created by combining information from the agricultural census with land use data, information from animal excreta and manure management surveys and estimates of oocyst concentrations in manure [22]. The estimates will consist of C.\u00a0parvum and other species\/genotypes common in animals, which are unlikely to include C.\u00a0hominis.\nA range of socioeconomic variables were obtained by identifying the 2001 Output Area within which each postcode was located. Output areas are the smallest area for which census data are released and contain approximately 125 individuals. Within each Output Area the percentage of people in each of the eight socio-economic status bands were identified. These ranged from higher managerial and professional occupations to those in routine occupations. Additionally, the proportion of the population aged 0\u20134\u00a0years and the percentage employed in agriculture was obtained. Finally a measure of health care accessibility was derived by calculating the travel time from each postcode to the nearest GP.\nIndividuals receive their water from one or more water treatment works, which abstract their water from one or more sources and are subject to different forms of water treatment. Consequently, a number of volume-weighted measures were derived to describe the water supply of each case and control postcode. Information on the proportion of water supplied from different sources (surface vs. groundwater) and subject to different treatments (e.g. membrane filtration, simple disinfection) was obtained from the England and Wales Drinking Water Inspectorate. These data included any changes in treatment that occurred during the 2000\u20132003 period. Information could only be obtained for public water supplies. For each surface or groundwater abstraction, catchments were calculated using the GIS and the density of Cryptosporidium applications to land, sewage discharges and sewage overflows in each were calculated. The probability of biofilm build-up was simulated by calculating the volume weighted, average straight line distance between each postcode and the water treatment works supplying their water.\nIn total over 50 explanatory variables were produced. Multivariable models were then constructed using conditional logistic regression for all Cryptosporidium infections followed by separate models for C.\u00a0hominis and C.\u00a0parvum. A forward regression technique was performed by adding the most significant variable in turn. Collinearity was avoided by ensuring that the addition of each variable did not lead to significant changes in the coefficients or significance of any other variables in the model. Standardised forms of the independent variables were entered into the models. Results are presented in terms of Odds Ratio (OR) estimates and the 95% Confidence Interval (CI). The statistical analysis was undertaken using STATA\/SE 8.2.\nMuch of our understanding of the aetiology of cryptosporidiosis is obtained from an analysis of reported outbreaks. However, these only represent about 8% of all cases reported to national surveillance. In order to examine whether outbreak cases were significantly influencing the results presented in this paper all our models were fitted with and without outbreak cases. Cases associated with a particular outbreak are identified within the national collection of Cryptosporidium oocysts used for this study from records held by local Health Protection Teams or Units. The omission of outbreak cases had a minimal impact upon the results implying that outbreak cases are not significantly influencing the results presented. Consequently, the models presented in this paper are inclusive of all cases.\nResults\nThe results for all Cryptosporidium infections are shown in Table\u00a01. These indicate that living in an area with higher amounts of Cryptosporidium applied to land in a 2.5\u00a0km buffer around each postcode (OR 1.084 P\u00a0=\u00a00.022), larger proportions of individuals in the 0\u20134\u00a0years age group (OR 1.145 P\u00a0<\u00a00.001) and more individuals in the highest socioeconomic status groups (OR 1.203 P\u00a0<\u00a00.001) were all positively associated with risk. Drinking water subject to superior water treatment (OR 0.770 P\u00a0<\u00a00.001) and groundwater sourced drinking water (OR 0.821 P\u00a0=\u00a00.001) were negatively associated with risk. Once these two drinking water variables were included in the model the proportion of the water supply which was from non superiorly treated surface water sources was negatively associated with risk (OR 0.869 P\u00a0=\u00a00.019).\nTable\u00a01Multivariate model for all Cryptosporidium infectionVariableOR95% CIP valueStandardised quantity of Cryptosporidium applied to land in a 2.5\u00a0km buffer1.0841.012\u20131.1630.022Standardised proportion of population in ages 0\u201341.1451.090\u20131.203<0.001Standardised proportion of population in highest socioeconomic status groups (1\u20134)a1.2031.140\u20131.270<0.001Standardised proportion of water supply that is subject to superior treatmentb0.7700.679\u20130.874<0.001 Standardised proportion of water supply that is groundwater0.8210.729\u20130.9250.001Standardised proportion of water supply from non superiorly treated surface water0.8690.772\u20130.9770.019 R2\u00a0=\u00a02.23%, estimated from 3368 case and 3368 control postcodesaWorking in managerial and professional occupations, intermediate occupations and small employers and own account workersbDefined as water treated with coagulation and flocculation, slow sand filtration, cartridge or membrane filtration\nThe dataset was then split into C.\u00a0parvum and C.\u00a0hominis. The results for C.\u00a0parvum are shown in Table\u00a02 and indicate that living in an urban area was negatively associated with risk (OR 0.852 P\u00a0<\u00a00.001). Living in an area with higher amounts of Cryptosporidium applied to land in a 2.5\u00a0km buffer around each postcode (OR 1.167 P\u00a0<\u00a00.005), larger proportions of individuals in the 0\u20134 age group (OR 1.094 P\u00a0=\u00a00.018) and more individuals in the highest socioeconomic status groups (OR 1.109 P\u00a0=\u00a00.010) were all positively associated with risk. Drinking water subject to superior water treatment (OR 0.738 P\u00a0<\u00a00.001) and groundwater sourced drinking water (OR 0.679 P\u00a0<\u00a00.001) were negatively associated with risk. Once these two drinking water variables were controlled for two further variables became significant. An interaction between the proportion of groundwater supply and the amount of Cryptosporidium applied to land in the catchment (OR 1.289 P\u00a0<\u00a00.001) was positively associated with risk. The proportion of the water supply which was not superiorly treated, from a surface water source with a high amount of Cryptosporidium applied to land in the catchment was negatively associated with risk (OR 0.846 P\u00a0=\u00a00.009).\nTable\u00a02Multivariate model for Cryptosporidium parvum infectionVariableOR95% CIP valueStandardised urban area0.8520.779\u20130.932<0.001Standardised quantity of Cryptosporidium applied to land in a 2.5\u00a0km buffer1.1671.047\u20131.2300.005Standardised proportion of population in ages 0\u201341.094 1.015\u20131.1790.018Standardised proportion of population in highest socioeconomic status groups (1\u20134)a1.109 1.025\u20131.2000.010 Standardised proportion of water supply that is subject to superior treatmentb0.738 0.646\u20130.842<0.001 Standardised proportion of water supply that is groundwater0.679 0.554\u20130.833<0.001Standardised interaction between the groundwater proportion and the C. parvum density in catchment1.289 1.088\u20131.527<0.001Standardised proportion of water supply from non superiorly treated surface water with high C. parvum density in catchment0.846 0.746\u20130.9590.009 R2\u00a0=\u00a04.24%, estimated from 1,623 case and 1,623 control postcodesaWorking in managerial and professional occupations, intermediate occupations and small employers and own account workersbDefined as water treated with coagulation and flocculation, slow sand filtration, cartridge or membrane filtration\nThe results for C.\u00a0hominis are presented in Table\u00a03 and indicate positive associations with living in an urban area (OR 1.261 P\u00a0<\u00a00.001), an area with a high proportion of individuals from social classes 1\u20134 (OR 1.297 P\u00a0<\u00a00.001) and many individuals in the 0\u20134 age group (OR 1.189 P\u00a0<\u00a00.001).\nTable\u00a03Multivariate model for Cryptosporidiumhominis infectionVariablesOR95% CIP valueStandardised urban area1.2611.154\u20131.378<0.001Standardised proportion of population in highest socioeconomic status groups (1\u20134)a1.2971.201\u20131.401<0.001Standardised proportion of population in ages 0\u201341.1901.112\u20131.274<0.001R2\u00a0=\u00a03.57, estimated from 1,720 case and 1,720 control postcodesaWorking in managerial and professional occupations, intermediate occupations and small employers and own account workers\nDiscussion\nIn the full model all Cryptosporidium illness was positively associated with the quantity of Cryptosporidium applied to land in a 2.5\u00a0km buffer around each postcode. This may be due to the increased probability of direct contact with the environmental reservoir of Cryptosporidium in animal manures. When the analysis was split into C.\u00a0hominis and C.\u00a0parvum, surrounding Cryptosporidium applications to land was only a significant risk factor for C.\u00a0parvum. This is unsurprising as animal manures are unlikely to include C.\u00a0hominis.\nThere were positive associations between cryptosporidiosis and higher social classes for all Cryptosporidium illness but when the individual species were examined the social class gradient was significantly stronger for C.\u00a0hominis in comparison to C.\u00a0parvum (OR 1.297 vs. 1.109). There are a number of explanations for this. If we consider C.\u00a0parvum, then there is a large environmental reservoir and individuals of higher social classes are more likely to undertake recreational activities such as walking in the countryside [9] which may increase the chance of animal-to-human transmission. Additionally individuals of higher social classes are more likely to undertake foreign travel [8] a known risk factor for cryptosporidiosis but especially for C.\u00a0hominis [4]. Foreign travel cases were removed from our analysis, though it is likely that some travel related cases remained within the dataset. Furthermore, the association with social class may indicate community transmission from travel cases to other individuals in the same social group with whom they are most likely to interact. This may be direct person-to-person transmission [2] or indirect transmission such as through swimming pools. It is known that recreational swimming is positively associated with higher social groups [9]. It is also worth highlighting that these positive associations with higher social classes are overriding other factors. Individuals of a higher social class are more likely to consume fresh fruit and vegetables [10], known to be negatively associated with cryptosporidiosis [2].\nLiving in an area with a higher proportion of individuals aged 0\u20134\u00a0years is a risk factor for all illness with Cryptosporidium and for C.\u00a0parvum and C.\u00a0hominis individually. It is known that cryptosporidiosis incidence is highest in this age group. However, the observation that the magnitude of effect is greater for C.\u00a0hominis than for C.\u00a0parvum (OR 1.190 vs. 1.094) is surprising as previous studies have shown a greater proportion of C.\u00a0parvum (33%) occur in children in the 0\u20134 age group compared to C.\u00a0hominis (20%) [2]. Toileting contact with a child under 5\u00a0years, even in the absence of symptoms, has been shown to be a risk factor for C.\u00a0hominis but not C.\u00a0parvum (2). The authors of this study suggested that asymptomatic carriage in young children may be one of the main reservoirs of C.\u00a0hominis.\nThe association with children in the 0\u20134\u00a0year age group was examined further by subdividing the C.\u00a0parvum and C.\u00a0hominis models into those cases where the affected individual was 4\u00a0years or younger and those where the individual was 5\u00a0years and older. To ensure comparability, identical explanatory variables were fitted to each sub model as in the original models. The resulting models were then analysed for major shifts in the significance of the explanatory variables. The only major difference for C.\u00a0parvum was that the percentage of individuals aged 0\u20134 in the area was not significant (P\u00a0=\u00a00.9) in the model for those 5\u00a0years and older. This suggests that young children are not a risk factor for C.\u00a0parvum illness in older individuals. In contrast the percentage aged 0\u20134 was a significant risk factor in both young and old for C.\u00a0hominis, highlighting the importance of person-to-person transmission between young and older individuals. In the 4\u00a0years and younger model, living in an urban area was an insignificant risk factor (P\u00a0=\u00a00.7) for C. hominis.\nAll illness with Cryptosporidium showed negative associations with areas having superior water treatment, and negative associations with areas supplied by large amounts of groundwater. When the models were split by species, this effect was observed for only C.\u00a0parvum. Although there have been outbreaks of C.\u00a0hominis associated with sewage contamination of drinking water, C.\u00a0parvum dominates in the environment and so the lack of association between C.\u00a0hominis and drinking water is unsurprising.\nOnce water source and treatment were included in the model, further variables became significant in the overall model and in the C.\u00a0parvum model. These need to be interpreted with caution as they will be greatly impacted by any measurement error in our source and treatment drinking water variables. In the overall model the proportion of poorly treated water supplied from surface water sources was negatively associated with risk. In the C.\u00a0parvum model a similar variable became significant namely the proportion of poorly treated water supplied from surface water sources from catchments with high amounts of Cryptosporidium applications to land. Both these associations indicate reductions in risk from the drinking water supplies most at risk of Cryptosporidium contamination. This may highlight immunity in a population periodically exposed to Cryptosporidium [19]. Alternatively new Cryptosporidium regulations to improve water supplies have led to improved water treatment at sites most at risk of Cryptosporidium contamination [23] and these improvements have been incorporated into our water supply database. Consequently the negative association with the most at risk sites may be evidence of the new regulations leading to the improved monitoring and attention being paid to these most at risk plants [23].\nIn the C.\u00a0parvum model a positive association was observed in the interaction variable between the proportion of groundwater sourced drinking water and the quantity of Cryptosporidium applied to land in the groundwater catchment. This highlights that, although generally of lower risk, if there is a high density of Cryptosporidium in the groundwater catchment then these can be a risk factor for illness with C.\u00a0parvum.\nThe links between cryptosporidiosis and drinking water are noteworthy as this period covers the new drinking water regulations to control Cryptosporidium implemented in 2000 [23]. Since this time a reduction in cryptosporidiosis has been reported, especially the reduction in the size of the spring peak nationally [24]. Consequently, although the regulations appear to have had success in reducing illness, drinking water remains a significant risk factor for cryptosporidiosis in England and Wales.\nThe urban\u2013rural gradient was not a significant variable in the full model. However, in the C.\u00a0parvum model negative associations were found with urban areas. It is important to note that these associations were independent of any impacts relating to water supply and Cryptosporidium applications to land, which we would expect to be correlated with urban areas. It may be that other factors associated with living in urban areas are important. It may indicate that these individuals are less likely to interact with their surroundings reducing the probability of C.\u00a0parvum infection. An alternative explanation is that our water treatment variables reflect the supply pattern in 2003. In earlier years it may be that the association with land use is accounting for the greater uncertainty in the water treatment variables. This is plausible as rural water supplies are likely to be at greater risk of C.\u00a0parvum contamination from agricultural and possibly wildlife sources. This argument is strengthened by the observation that when the data were stratified into 2000\u20132001 and 2002\u20132003 the land use variable was only significant in the earlier period.\nFor C.\u00a0hominis a positive association with urban areas was observed. This may be due to increased opportunities for person-to-person transmission in these areas due to factors such as more nursing homes, day care centres and enhanced availability of swimming pools. The association between increased risk of a person-to person-transmitted disease and increased population density is in line with that predicted by epidemiological theory [25]. In the model of all illness with Cryptosporidium the urban\u2013rural density is not a significant variable implying that the different relationships observed for C.\u00a0parvum and C.\u00a0hominis are cancelling each other out in the full model. The observation of different associations with urban areas for C.\u00a0parvum and C.\u00a0hominis is also noteworthy because of our earlier concern about omissions of postcodes by general practitioner and whether this could bias the study results. It could be suggested that postcode completeness varies between general practitioners in urban and non-urban areas. However, the models for C.\u00a0parvum and C.\u00a0hominis show different signs for the urban area variable. This implies that any bias due to differential general practitioner completeness of postcode information between urban and non-urban areas is likely to be minimal.\nIn this study no associations were found between cryptosporidiosis and the proxy variable for biofilm build-up, accessibility to healthcare or the proportion of the population employed in agriculture. This suggests that these variables are not of major importance to cryptosporidiosis aetiology or that our methodology was unable to create effective measures of them.\nIn conclusion, this is the first study to examine the influence of the wider environment upon illness with Cryptosporidium and of the two species C.\u00a0hominis and C.\u00a0parvum separately. It has shown separate aetiologies for each species highlighting the importance of separating C.\u00a0hominis and C.\u00a0parvum in epidemiological studies. For the first time it has also demonstrated all illness with Cryptosporidium to be higher in areas populated by higher social classes and this effect is greater for illness with C.\u00a0hominis. It also supports studies indicating the importance of young children to illness with both Cryptosporidium species. This is the first study to highlight the importance of the agriculture surrounding place of residence as a risk factor for C.\u00a0parvum illness. Elevated incidence of C.\u00a0parvum illness was found in rural areas and elevated levels of C.\u00a0hominis illness in urban areas, effects that were independent of water supply and agricultural factors. The analysis highlighted that, in spite of new regulations to reduce Cryptosporidium concentrations in drinking water, water supply remained a risk factor for illness with C.\u00a0parvum. Risk was lowered in areas with superior water treatment and in areas supplied by groundwater. There was also evidence that, in certain circumstances, groundwater could be a risk factor for C.\u00a0parvum illness. Finally there were indications of a lowering of risk of C.\u00a0parvum illness in the most at risk water supplies, an impact that was independent of superior treatment. This may be evidence of population immunity or effective regulation governing water treatment and monitoring.","keyphrases":["cryptosporidiosis","cryptosporidium","water","social class","agriculture","environment and public health"],"prmu":["P","P","P","P","P","R"]}
{"id":"Eur_J_Appl_Physiol-4-1-2225999","title":"Periprandial changes of the sympathetic\u2013parasympathetic balance related to perceived satiety in humans\n","text":"Food intake regulation involves various central and peripheral mechanisms. In this study the relevance of physiological responses reflecting the autonomic nervous system were evaluated in relation to perceived satiety. Subjects were exposed to a lunch-induced hunger-satiety shift, while profiling diverse sensory, physiological, and biochemical characteristics at 15 min intervals. Sensory ratings comprised questionnaires with visual analogues scales about their feeling of satiety, desire to eat, fullness, and hunger. Physiological characteristics included heart rate, heart rate variability, and blood pressure, while biochemical markers such as cortisol levels and \u03b1-amylase activity were monitored in saliva. The four sensory ratings correlated with heart rate and salivary \u03b1-amylase suggesting a higher sympathetic tone during satiety. Furthermore, heart rate variability was associated with age and waist-to-hip ratio and cortisol levels negatively correlated with body mass index. Finally, neither chewing nor swallowing contributed to a heart rate increase at food consumption, but orosensory stimulation, as tested with modified sham feeding, caused a partial increase of heart rate. In conclusion, after meal ingestion critical physiological alterations reveal a elevated sympathetic tone, which is a potential measure of satiety.\nIntroduction\nRegulation of food intake comprises both homeostatic and hedonic components controlled by hunger and satiety signals in both peripheral and central pathways. Afferent and most peripheral signals provide information about the body\u2019s nutritional state and the central nervous system mediates and integrates episodic hunger and satiety signalling towards a functional output that controls metabolism with accompanying behaviour (Schwartz et al. 2000). While communication between periphery and brain is crucial in coordinating integrative processes for metabolism, such signals can also be anticipatory as they can rise even before food is ingested through visual, auditory, and olfactory stimuli (Mattes 1997), or even appear, like insulin secretion, when no food was presented to na\u00efve subjects (Bellisle et al. 1985).\nIn hunger and satiety signalling, endocrine systems and the autonomic nervous system (ANS), play crucial roles. The sympathetic (SNS) and parasympathetic nervous system (PNS), support distinct and opposite functions in alertness and physiology. They respond to changes in both the external and internal environments and are also important in regulation of metabolism and energy homeostasis (Tempel and Leibowitz 1994; Bray 2000). In relation to a hunger-satiety shift after food intake in humans, changes of the SNS and PNS activity can occur before those in nutritional hormone levels, as demonstrated by instant changes in the states of alertness (Craig and Richardson 1989; Geisler and Polich 1992; Holt et al. 1999), muscle sympathetic nerve activity (Fagius and Berne 1994), and cardiac output (Waaler et al. 1991; Waaler and Eriksen 1992), which may be part of cephalic phase responses (Mattes 1997; Nederkoorn et al. 2000). An inverse relationship of SNS activity and body fat emphasize the importance of the SNS in food intake, suggesting a role of \u03b2-adrenergic receptors in peripheral and central control of eating (Bray 2000). Tracking of ANS characteristics and how they are changing in relation to appetite and satiety helps to understand the role of the ANS in the context of food intake regulation, overweight and obesity.\nThe present study assessed whether time course and patterns of physiological responses in humans may serve as consistent characteristics of the SNS\u2013PNS balance and indicate perceived satiety. We hypothesised that ANS characteristics are affected by satiety. Subjectively rated satiety, desire to eat, fullness, and hunger were evaluated over a session of 4.75\u00a0h, including a lunch-induced hunger-satiety shift, while heart rate (HR), heart rate variability (HRV) and blood pressure were monitored. Cortisol levels and \u03b1-amylase activity were also measured from the subject\u2019s saliva as two other determinants reflecting the SNS\u2013PNS balance. Related to food intake, cortisol, as the end product of the hypothalamic\u2013pituitary\u2013adrenal-axis, has a primary function in controlling nutrient ingestion and metabolism over the circadian cycle (Tempel and Leibowitz 1994), the hypothalamus is a key target for cortisol (Stricker-Krongrad and Beck 2002; Harthoorn et al. 2005), and adrenalectomy causes a reduction of food intake (Drazen et al. 2003, 2004). Salivary \u03b1-amylase is a biomarker for SNS activity because it serves as a correlate of adrenergic activity under stress, and is predictive of plasma catecholamine levels (Chatterton et al. 1996; Rohleder et al. 2004). Finally, chewing, swallowing, and orosensory stimulation by food in a modified sham-feeding model were examined for a possible contribution to HR alterations.\nMethods\nHealthy subjects, 9 males and 9 females, aged between 23 and 63\u00a0years (average 42.9\u00a0years) participated in this study. Smokers and subjects with prescribed medication, except for contraceptives, and excessive weight loss during the last 3\u00a0months were excluded from participating in this study. The subjects were paid for their involvement and informed consent was obtained according to the guidelines of the Ethical Committee of Wageningen UR. Weight, height, and waist and hip circumferences, were measured while subjects wore light indoor clothing with emptied pockets and no shoes. To estimate body weight, 1\u00a0kg was subtracted from clothed body weight. Body mass index (BMI) was the weight divided by height squared. Waist circumference was measured at the level midway between the lower rib margin and the iliac crest, with participants in a standing position and breathing out gently. Hip circumference was measured at the level of the widest circumference over the greater trochanters. The waist-to-hip ratio (WHR) was calculated as waist circumference divided by hip circumference.\nDesign and procedure\nEach subject was tested alone in a quiet furnished room with appropriate ventilation and lighting from 11:45 a.m. to 16:30 p.m. All subjects had their breakfast as usual at least 3\u00a0h before starting the test session. Subjects chose their lunch from a standard menu, which was served at exactly 12:30 p.m., and was finished within 20\u00a0min. The lunch was ad libitum, and the left-over of each meal was weighed and calculated into energy content to calculate total intake. The meals eaten were between 2.5 and 3.3 MJ, which is about 30% of the recommended regular daily intake, and contained 15\u201320%(energy) protein, 35\u201340%(energy) fat, and 40\u201345%(energy) carbohydrates. The meals did not contain caffeine or capsaicin. During the session a Polar S810 device (Polar Electro Oy, Kempele, Finland) recorded beat-by-beat periods (R\u2013R intervals) continuously. Every 15\u00a0min systolic and diastolic blood pressure were measured by an OMRON M4-I fully automatic monitor (OMRON Healthcare Europe, Hoofddorp, The Netherlands), saliva was donated, and subjects were asked to rate \u201chow satiated are you\u201d, \u201chow is your desire to eat\u201d, \u201chow full are you\u201d, and \u201chow hungry are you\u201d, using 100-mm non-structured visual analogue line scales (VAS) all on one page and anchored at their ends by the descriptors \u201cvery little\u201d and \u201cvery much\u201d (Flint et al. 2000).\nHR and HRV\nFor determination of HR and HRV, individual datasets were obtained from the Polar S810 device. Data processing and analysis were performed by Polar Precision Performance software (version 4.00.020), based on the consecutive numbers of successive R\u2013R intervals (ms) during the test session. HR was derived and expressed as beats\/min of each successive 15\u00a0min. Determining R\u2013R interval over 15\u00a0min allowed HR and HRV measurement with high (<3\u00a0ms) timing accuracy (Ruha et al. 1997). Power spectral analysis of HRV was computed from the R\u2013R formats over data segments of 15\u00a0min each, with cubic interpolation for the R\u2013R series at 4\u00a0Hz and spectrum estimation based on fast Fourier transformation. The two main rhythmic oscillations in the R\u2013R intervals were a high frequency (HF) component between 0.15 and 0.40\u00a0Hz synchronous to respiratory activity, and a low frequency (LF) component between 0.04 and 0.15\u00a0Hz related to vasomotor waves. For both LF and HF components, the centre frequency and associated power, i.e. area under the curves of individual components (ms2), were calculated. The power in the HF band represents PNS activity, while the power in the LF band characterises SNS activity and part of the PNS activity (Berger et al. 1986; Pagani et al. 1986; Malliani et al. 1994; Perini and Veicsteinas, 2003). A reciprocal relation exists between the HF and LF rhythms from which an index characterising the SNS\u2013PNS balance can be derived. The LF-to-HF ratio (%) was determined for every 15\u00a0min.\nSaliva biochemistry\nEvery 15\u00a0min subjects chewed on a rolled 25-cm2 sheet of parafilm (Parafilm\u00ae M) for up to 1\u00a0min and spat into a 12\u00a0ml polystyrene tube (Greiner Bio-One, Kremsm\u00fcnster, Austria). The saliva was immediately placed into a freezer at \u221240\u00b0C. For further biochemical analyses, saliva samples were centrifuged at 1,000\u00a0g for 2\u00a0min. The salivary cortisol level was determined on 25\u00a0\u03bcl of thawed saliva using a Cortisol Enzyme Immunoassay (EIA) Kit (DSL-10-67100) (Diagnostic Systems Laboratories DSL, Sinsheim, Germany) with an intra-assay variation between 1.9 and 4.8% and an inter-assay variation between 3.8 and 7.2%. Determination of in vitro salivary \u03b1-amylase activity was performed on 10\u00a0\u03bcl of saliva by means of an \u03b1-Amylase Kinetic Reaction Kit (Salimetrics LLC, State College, PA, USA) using 2-chloro-p-nitrophenol linked with maltotriose as chromagenic substrate and measured at 405\u00a0nm. The intra-assay variation was between 2.5 and 7.2%, and the inter-assay variation between 3.6 and 5.8%.\nOral activities and orosensory stimulation\nOf the 18 subjects, 6 men and 6 women (BMI between 21.5 and 28.4), were exposed to distinct sessions of 15\u00a0min in which different oral activities and processing were examined for their influence on HR. Baseline of HR was measured for 15\u00a0min. Sessions consisted of either chewing on parafilm for 15\u00a0min, drinking 200\u00a0ml of water during the first 5\u00a0min of the 15\u00a0min session, consuming 200\u00a0ml of yoghurt-drink (Friesland Foods, The Netherlands, 1.0 MJ, containing: 3.0%(w\/w) protein, 17%(w\/w) carbohydrates, 16%(w\/w) sugars, and 3.0%(w\/w) fat) during the first 5\u00a0min of a 15\u00a0min session, or modified sham feeding of this yoghurt-drink, a \u201cchew and spit\u201d technique in which foods are smelled, chewed, and tasted, but not swallowed (Robertson et al. 2001), during 5\u00a0min of the 15\u00a0min session. HR was monitored over data segments for resting and chewing periods and of 5\u00a0min periods for drinking and sham feeding.\nData analyses\nData are expressed as the mean \u00b1 SEM, unless otherwise specified. Of each measure and per time point, values were averaged over 18 subjects. Correlations were calculated between each of the four VAS ratings and HR, LF-to-HF ratio, blood pressure, cortisol, and \u03b1-amylase across the 20 time points of the session. Across subject correlations were done between age, BMI, WHR and both preprandial and postprandial levels of HR, LF-to-HF ratio, blood pressure, cortisol, and \u03b1-amylase per individual. For statistical assessment, postprandial values were compared to preprandial level and differences were established with repeated measures analysis of variance (ANOVA). Differences with P values of 0.05 or less were considered to be statistically significant. All data were analysed by using Statistica (version 6.0; StatSoft, Tulsa, OK, USA).\nResults\nMean BMI was 25.0\u00a0\u00b1\u00a03.5\u00a0kg\/m2 and mean WHR was 0.89\u00a0\u00b1\u00a00.07 (Table\u00a01). As shown in Fig.\u00a01, prior to lunch, desire to eat and hunger ratings increased from about 35 to 70\u00a0mm. Satiety ratings decreased from 50 to 35\u00a0mm, whilst fullness scores did not change markedly during the preprandial period. Lunch induced changes in all four sensory ratings followed by a steady reduction of these shifts over time. Average ratings of satiety and fullness were increased over 30\u00a0min by about 40\u00a0mm, while desire to eat and hunger ratings decreased to about 10\u00a0mm. After these shifts, ratings gradually declined to about preprandial scores over the next 4\u00a0h.\nTable\u00a01Anthropometric values for 18 subjects and session means of physiological characteristicsAverage\u00a0\u00b1\u00a0SDRangeAge (years)42.9\u00a0\u00b1\u00a013.023\u201363Body mass index (kg\/m2)25.0\u00a0\u00b1\u00a03.521.5\u201336.1Waist-to-hip ratio0.89\u00a0\u00b1\u00a00.070.77\u20131.00Heart rate (beats\/min)75.3\u00a0\u00b1\u00a07.863.3\u201389.3LF-to-HF ratio (%)409\u00a0\u00b1\u00a0282103\u20131,182Blood pressure (mmHg)\u00a0Systolic126\u00a0\u00b1\u00a013107\u2013150\u00a0Diastolic78\u00a0\u00b1\u00a01056\u201395Salivary cortisol (nmol\/l)14.2\u00a0\u00b1\u00a06.16.0\u201330.3Salivary \u03b1-amylase (U\/ml)139\u00a0\u00b1\u00a06914\u2013263Fig.\u00a01Satiety (a), desire to eat (b), fullness (c), and hunger (d) rated on VAS for 18 subjects. Bars are SEM. Grey-shaded bar represents lunch\nHR, HRV, and blood pressure\nShortly after starting the lunch, HR increased by 10\u00a0\u00b1\u00a02.5% (P\u00a0<\u00a00.02) for about 20\u00a0min, and then decreased for a following 30\u00a0min (Fig.\u00a02a). Forty-five to 50\u00a0min after starting the lunch, a second and more prolonged increase of HR was found (P\u00a0<\u00a00.02) that lasted at least 1\u00a0h.\nFig.\u00a02HR (a), LF-to-HF ratio (b), and blood pressure (c) for 18 subjects. Bars are SEM. Grey-shaded bar represents lunch. * Different from preprandial level (P\u00a0<\u00a00.02)\nIn the HRV a bi-phasic trend of postprandial fluctuations for LF-to-HF ratio was found, but these were not significantly different from the preprandial level (Fig.\u00a02b). Systolic pressure demonstrated minor fluctuations during the session, which were not significantly different from before lunch, however, diastolic pressure showed a postprandial decrease (P\u00a0<\u00a00.02) (Fig.\u00a02c).\nSaliva biochemistry\nSalivary cortisol levels were high at the beginning of the session, followed by a gradual decrease during the day (Fig.\u00a03a) without any significant influences of the lunch. Similar to the bi-phasic increase of HR, in vitro salivary \u03b1-amylase activity displayed a similar pattern over the session (Fig.\u00a03b). An increase of 60\u00a0\u00b1\u00a015% as compared to preprandial level in \u03b1-amylase activity was observed (P\u00a0<\u00a00.01) early after starting the lunch meal. A second and more lengthened increase of \u03b1-amylase activity was demonstrated between 1 and 3\u00a0h after starting the lunch (P\u00a0<\u00a00.01).\nFig.\u00a03Salivary cortisol levels (a) and in vitro \u03b1-amylase activity (b) for 18 subjects. Bars are SEM. Grey-shaded bar represents lunch. * Different from preprandial level (P\u00a0<\u00a00.01)\nSensory ratings and ANS characteristics\nCorrelations were found between VAS satiety, desire to eat, fullness, and hunger. Variations in satiety rating across time correlated with fullness (r\u00a0=\u00a00.99, P\u00a0<\u00a00.001), desire to eat (r\u00a0=\u00a0\u22120.97, P\u00a0<\u00a00.001), and hunger (r\u00a0=\u00a00.96, P\u00a0<\u00a00.001). Desire to eat was correlated to fullness (r\u00a0=\u00a0\u22120.96, P\u00a0<\u00a00.001) and hunger rating (r\u00a0=\u00a00.99, P\u00a0<\u00a00.001), and fullness rating was correlated with hunger (r\u00a0=\u00a0\u22120.95, P\u00a0<\u00a00.001).\nHR showed correlations to satiety (r\u00a0=\u00a00.74, P\u00a0<\u00a00.001), desire to eat (r\u00a0=\u00a0\u22120.74, P\u00a0<\u00a00.001), fullness (r\u00a0=\u00a00.74, P\u00a0<\u00a00.001), and hunger (r\u00a0=\u00a0\u22120.66, P\u00a0<\u00a00.01). LF-to-HF ratio was not significantly correlated to the four sensory ratings. Although systolic pressure was not associated to any of the sensory ratings, diastolic pressure showed correlations to satiety (r\u00a0=\u00a0\u22120.48, P\u00a0<\u00a00.05), desire to eat (r\u00a0=\u00a00.53, P\u00a0<\u00a00.05), fullness (r\u00a0=\u00a0\u22120.50, P\u00a0<\u00a00.05), and hunger (r\u00a0=\u00a00.58, P\u00a0<\u00a00.01). Salivary cortisol levels were not significantly correlated with any of the sensory ratings, whereas salivary \u03b1-amylase was correlated to satiety (r\u00a0=\u00a00.78, P\u00a0<\u00a00.001), desire to eat (r\u00a0=\u00a0\u22120.83, P\u00a0<\u00a00.001), fullness (r\u00a0=\u00a00.80, P\u00a0<\u00a00.001), and hunger (r\u00a0=\u00a0\u22120.83, P\u00a0<\u00a00.001).\nAnthropometrics and physiological responses\nA correlation was found between age and both preprandial and postprandial LF-to-HF ratio (r\u00a0=\u00a00.55, P\u00a0<\u00a00.02 and r\u00a0=\u00a00.52, P\u00a0<\u00a00.05, respectively) and age and postprandial HR (r\u00a0=\u00a00.49, P\u00a0<\u00a00.05). WHR and postprandial LF-to-HF ratio appeared to be correlated (r\u00a0=\u00a00.55, P\u00a0<\u00a00.02), but this relation was also found to be mediated by age. Preprandial cortisol levels were associated to BMI (r\u00a0=\u00a0\u22120.47, P\u00a0<\u00a00.05).\nOral activities and orosensory stimulation\nChewing, swallowing and orosensory stimulation by food in a modified sham-feeding model were examined for their possible effect on instant increases of HR. Neither chewing on parafilm nor drinking of water showed any increase of HR in comparison to baseline (Fig.\u00a04). Drinking 200\u00a0ml of yoghurt, which was without chewing, increased HR about 10% (P\u00a0<\u00a00.01). This was a similar increase as under the lunch fed conditions. Finally, orosensory stimulation by the same type of yoghurt in sham feeding way increased HR about 5% (P\u00a0<\u00a00.01).\nFig.\u00a04HR during chewing on parafilm, drinking water or yoghurt, and modified sham feeding with the same yoghurt-drink, expressed as stimulus-to-control ratio in relation to baseline for HR. Both consumption of yoghurt-drink and modified sham feeding of the yoghurt caused an increase in HR (P\u00a0<\u00a00.01). Bars represent SEM for 12 subjects\nDiscussion\nSensory ratings scales for satiety, desire to eat, fullness, and hunger were used in combination with measures to assess the relevance and temporal pattern of responses reflecting the sympathetic\u2013parasympathetic (SNS\u2013PNS) balance in relation to perceived satiety in humans.\nSatiety and hunger are often interpreted as being reciprocal, although they are mediated and signalled by different mechanisms, in which leptin, insulin, and cholecystokinin are important (Schwartz et al. 2000). For this reason several rating scales were used about feelings of satiety and hunger. Over a 4.75-h test session, including a lunch-induced hunger-satiety shift, all four VAS ratings showed consistent changes. The temporal profile of satiety rating was paralleled by fullness and inversely related to desire to eat and hunger ratings. Preprandially, increases of desire to eat and hunger ratings were larger than decreases of satiety and fullness ratings. Ingestion of a standard meal of 2.5\u20133.3 MJ changed all four VAS ratings 40\u201360\u00a0mm, but with regard to the lunch-induced hunger-satiety shift, changes in desire to eat and hunger ratings were found to be larger than the changes of ratings of satiety and fullness. This suggests that ratings of \u201cdesire to eat\u201d and \u201chunger\u201d are more sensitive to the evaluation of appetite periprandially than ratings of \u201csatiety\u201d and \u201cfullness\u201d. Sensory ratings tracked over time and combined with food intake demonstrated a gradual recovery to preprandial rating scores over about 4\u00a0h after meal consumption. This might reflect the gastrointestinal changes and processes over the same time that affect appetite. Although, differences may result from different types of food intake or different types of satiety measurements, recovery time also depends on sensory attributes (De Graaf 1993). The finding of a similar 4-h recovery time for all four sensory descriptors used, appears to be consistent following different types of meals (Green et al. 1997; Erdmann et al. 2004), and different rating techniques (Stubbs et al. 2001).\nPhysiological responses reflecting the SNS\u2013PNS balance were simultaneous to altered patterns of perceived satiety. In this food-related context, these ANS characteristics not only provide additional insights into the integration of central and peripheral regulatory processes for metabolism, but also render a potential measure of satiety in humans.\nBoth the SNS and PNS are segmented multifunctional systems, which can be evaluated in several ways (Morrison 2001). Changes in HR, HRV, and blood pressure are some of the variables that can be used to assess the SNS\u2013PNS balance, in which a higher HR and cardiovascular load and output is typified by a higher SNS\u2013PNS ratio. High salivary \u03b1-amylase levels are indicating a higher sympathetic tone, are predictive of high plasma catecholamine levels, and are reduced by adrenergic beta-blockers (Speirs et al. 1974; Chatterton et al. 1996). The SNS and PNS regulate processes and metabolic pathways important for recovery and energy homeostasis (Bray 2000; Szekely 2000). The changes of the SNS\u2013PNS balance, therefore, are relevant to satiety and may occur before those in circulating hormones that stimulate or inhibit food intake. With regard to the effects of food intake on appetite, two distinct phases have been considered within the postulated \u201csatiety cascade\u201d, called \u201csatiation\u201d and \u201csatiety\u201d, the first defined as the process which develops during eating and which brings eating to end, and the second defined as the state of inhibition over further eating (Green et al. 1997). This satiety cascade takes into account properties of food, physiological processing of nutrients that contribute to overall control of food intake, and also conceptualises differences between immediate postingestive effects and those produced later. The digestive period is divided into three to four phases depending on the site at which food acts to stimulate neural and endocrine organs, i.e. the cephalic and orosensory, gastric, and intestinal phases, which are sequential and follow on the progression of a meal.\nBoth short- and long-term autonomic alterations occur towards a high sympathetic tone after a meal based on increases of HR and salivary \u03b1-amylase. The short-term increases may belong to cephalic and orosensory phase responses, whereas the long-term increases cohere to the gastrointestinal mechanisms. While the importance of \u03b1-amylase is on starch digestion, the association of both HR and \u03b1-amylase with the VAS ratings show the possibility of measures for perceived satiety. Moreover, HR and \u03b1-amylase have a recovery over about 4\u00a0h after a meal. Further, a diastolic blood pressure decline might characterize satiety, however, with weaker correlations. The haemodynamic changes illustrate that eating places demand on the cardiovascular system, most presumably specifically on the visceral compartment of the body. A postprandial increase in HR points to enhanced cardiac output, while a lower blood pressure during a high feeling of satiety may indicate a lower systemic resistance especially to visceral parts of the body, which results in a higher mesenteric blood flow (Jager et al. 1986), and may influence postprandial behaviour. The LF-to-HF ratio showed a similar bi-phasic trend as HR and salivary \u03b1-amylase, however, it was a less consistent measure of SNS\u2013PNS balance. In addition, a postprandial bi-phasic SNS predominance indicates immediate triggering of cephalic or orosensory responses and the more gradual activation of metabolic mechanisms within time frames coherent to the putative distinction between \u201csatiety\u201d and \u201csatiation\u201d (Green et al. 1997). The instant increase of HR with food intake can be part of a cephalic phase response triggered by oral physical activity, non-metabolic food cues, or sensory perception. Neither chewing, swallowing, nor water intake were responsible for a changed sympathetic tone as indicated by HR, however, orosensory stimulation with modified sham feeding enhanced HR to about half of the increase that was reached after intake of a meal. An instant increase in HR is, thus, induced in the oral cavity most presumably initiated by perception of taste, flavour, or energy-rich substrates. An alternative explanation for the bi-phasic effect on a higher SNS\u2013PNS balance is that the decrease in HR for about 30\u00a0min after finishing food intake is caused by inhibition of autonomic functioning. The satiety hormones cholecystokinin, glucagon-like peptide 1, and gastric inhibitory peptide could be involved as their release from the gastrointestinal tract in response to eating (Matzinger et al. 2000; Burcelin 2005) inhibit autonomic functioning at the central level as demonstrated in rodents, where cholecystokinin inhibites brainstem presympathetic neurons and reduces HR (Sartor and Verberne 2002; Fan et al. 2004).\nAside from the close correlation between SNS and PNS balance and perceived satiety in a periprandial period, there is an inverse relationship between SNS activity and obesity, consistent with that most types of obesity are associated with low sympathetic activity (Bray 1991). Further personal characteristics like age, BMI, and WHR, were associated with the ANS characteristics. In addition to BMI as a marker of overweight, WHR serves as indicator of fat storage in abdominal depots. A negative correlation between salivary cortisol and BMI was found. The role of cortisol in obesity is complex with both high and low cortisol secretion patterns (Bj\u00f6rntorp and Rosmond 2000). Cortisol is higher in subjects with the night-eating syndrome (Birketvedt et al. 1999) and binge eating disorder and bulimia nervosa (Gluck et al. 2004). In overweight subjects, a hypoactive hypothalamic\u2013pituitary\u2013adrenal-axis, not knowing whether it is cause or consequence of a dysregulated SNS, may contribute to greater hunger and over-consumption. A mechanism of hypothalamic\u2013pituitary\u2013adrenal-axis inhibition is described in rats, showing a negative feedback of visceral fat on the hypothalamus that lowers cortisol levels (Dallman et al. 2003, 2004; La Fleur et al. 2005).\nIn conclusion, within a periprandial context a high sympathetic tone is a potential measure of satiety in humans, and activation of the SNS might be a satiety system, which may also help regulation of food intake and energy expenditure from body fuel stores. HR and salivary \u03b1-amylase may be implemented to measure satiety in eating disorders and overweight.","keyphrases":["human","eating","heart rate","saliva","satiation"],"prmu":["P","P","P","P","P"]}
{"id":"Environ_Manage-3-1-1914239","title":"Community Capacity for Implementing Clean Development Mechanism Projects Within Community Forests in Cameroon\n","text":"There is a growing assumption that payments for environmental services including carbon sequestration and greenhouse gas emission reduction provide an opportunity for poverty reduction and the enhancement of sustainable development within integrated natural resource management approaches. Yet in experiential terms, community-based natural resource management implementation falls short of expectations in many cases. In this paper, we investigate the asymmetry between community capacity and the Land Use Land Use Change Forestry (LULUCF) provisions of the Clean Development Mechanism within community forests in Cameroon. We use relevant aspects of the Clean Development Mechanism criteria and notions of \u201ccommunity capacity\u201d to elucidate determinants of community capacity needed for CDM implementation within community forests. The main requirements are for community capacity to handle issues of additionality, acceptability, externalities, certification, and community organisation. These community capacity requirements are further used to interpret empirically derived insights on two community forestry cases in Cameroon. While local variations were observed for capacity requirements in each case, community capacity was generally found to be insufficient for meaningful uptake and implementation of Clean Development Mechanism projects. Implications for understanding factors that could inhibit or enhance community capacity for project development are discussed. We also include recommendations for the wider Clean Development Mechanism\/Kyoto capacity building framework.\nIntroduction\nThere is a growing assumption that payments for environmental services including carbon sequestration and greenhouse gas emission reduction could provide an opportunity for poverty reduction and the enhancement of sustainable development within integrated natural resource management approaches (Asquith and others 2002; Pagiola and others 2005). Studies have identified community forest management as a model that could meet the triple objectives of providing mechanisms and incentives for community management of carbon, forest conservation and local development needs (Klooster and Masera 2000; Poffenberger and others 2002; Smith and Scherr 2003). Yet community-based natural resource management performance remains mixed (Agrawal 2001; Armitage 2005; Barrett and others 2005). This study seeks to find out whether or not and in what ways communities currently managing forests in Cameroon meet the capacity requirements to handle the Clean Development Mechanism (CDM) projects.\nThe CDM is one of three \u201cflexible mechanisms\u201d in the Kyoto Protocol designed to accomplish the objectives of the UNFCCC. It makes provision for investment by industrialised countries and industry in projects related to carbon emissions reduction and carbon sequestration in developing countries. These projects should contribute to sustainable development in developing countries (i.e., Non-Annex 1 countries) while enabling developed countries (i.e., Annex 1 countries with quantified emission reduction targets) to meet the Kyoto emission reduction and quantified emission limitation targets (Art. 12.2 of the Kyoto Protocol).\nLand Use Land Use Change and Forestry (LULUCF) and energy projects are required to meet certain conditions in order to acquire Certified Emission Reductions from the Executive Board of the CDM. Main conditions include additionality (mitigation effects \u201cwith project\u201d must be additional to what would have happened \u201cwithout project\u201d); leakage (project mitigation effects must not be offset by project impacts outside the accounting boundary); and contribution to sustainable development (to be demonstrated according to host country rules). Certified Emission Reductions represent the emission reduction or sequestration output of a project, and constitute the basis on which payments are made.\nBrown and others (2000) state that current modalities and information requirements for CDM are beyond the scope of community capabilities and skills. However, few studies have attempted to test the dimensions of this alleged asymmetry between CDM modalities and procedures and local community capabilities and skills. In this paper, we seek to review this gap by examining local community capacity for the development and implementation of CDM projects within Community Forests in Cameroon.\nWe use conceptual notions of CDM criteria and community capacity to empirically analyse two community forestry cases in Tinto and Bimbia Bonadikombo, Cameroon. The intention is to identify, document, and interpret local strategies and conditions affecting past community forestry successes and failures, in order to recognise and understand those factors that might enhance or limit community capacity for CDM implementation.\nThe paper is structured as follows. Section 2 highlights the criteria and conditions for CDM projects and the main features of community forestry in Cameroon. Appropriate community capacity requirements for CDM projects are derived in section 3. Section 4 presents the methods used and the study context. In section 5, we evaluate community capacity and draw implications for CDM and the wider CDM\/Kyoto policy framework.\nBy \u201ccommunity\u201d is meant certain characteristics referred to in community-based natural resource management including, having reasonably defined decision-making processes; being a homogenous unit (sometimes ethnic) with shared goals and values; having traditional resource use systems and livelihood strategies; and also having a clear spatial or conceptual boundary (Armitage 2005; Li 2002). While these are good conceptual characteristics to work with, Li (2002) sees these as very \u201cstrategic simplifications.\u201d In many instances, there is no crisp boundary between the state and the community, communities are not homogenous, having many individuals or groups that do not share community resource management goals. Property rights and decision-making systems may not also be as defined. We thus attempt to reflect these in our discussions.\nWe, therefore, see community capacity as the collective ability of individuals and groups acting in concert toward sustainable development in a given locality. In operational terms, community forests in Cameroon are managed by legal entities or community-based organisations constituted by a given \u201ccommunity\u201d for the purpose. But first, we present the requirements for CDM forestry projects and community forestry modalities in Cameroon.\nCDM Requirements and Community Forestry\nCDM Requirements\nCDM projects are expected to meet a set of requirements prior to the issuance of certified emission reductions by the CDM Executive board. These requirements are articulated in the Kyoto Protocol and in subsequent decisions taken during the Conference and Meetings of Parties (mainly in Decisions 19\/CP.9 and 14\/CP.10 and the Marrakech Accords). These requirements can be summarised under the following categories: additionality, acceptability, externalities, and certification.\nIt suffices to mention that these rules apply to afforestation and reforestation. These two are the only land use land use change and forestry activities accepted under the CDM.\nAdditionality\nSequestration or emission reductions due to the project activities must be \u201cadditional\u201d to any that would occur in the absence of the project (Paragraphs 18\u201322 of Decision 19\/CP.9). In other words, additionality implies that projects must result in a net storage of carbon and, therefore, a net removal of carbon from the atmosphere. Other forms of additionality include programme, financial, and investment additionality. Programme additionality refers to project demonstration that its emission reductions are additional to emissions required by law or government policy. Financial additionality refers to the fact that funding for the implementation of projects must not come from overseas development or environment assistance funds. Investment additionality refers to the demonstration that the creation of carbon offsets will involve costs that would not be incurred in the \u201cbusiness as usual\u201d scenario. Though not a requirement per se, it is a way of demonstrating \u201cintent\u201d and effort through financial analysis.\nAcceptability\nThe Kyoto Protocol states that all carbon offset projects in developing countries are required to contribute to sustainable development (Article 2.1 and 12.2). Host countries have to have criteria for sustainable development by which projects will be judged. In addition, projects must be consistent with other international agreements and guidelines such as the Convention on Biodiversity, Agenda 21, Ramsar, and others.\nExternalities (Environmental Impact and Leakage)\nProjects must demonstrate a clear strategy to deal with all impacts\/effects that may arise from project implementation. These impacts could include positive or negative social, cultural, economic, or environmental impacts. Projects have to show how the negative impacts would be mitigated or countered.\nA prominent aspect in externalities is the question of leakage. Leakage can be defined as unplanned emissions that could occur outside project boundaries as a result of project activities. Leakage should not disqualify a project except in instances where projections of emissions are substantial enough to negate projected carbon offsets. However, project analysis must show how leakage has been estimated and what measures will be put in place to minimize it.\nCertification\nThe concreteness, measurability, and long-term characteristics of the project will have to be checked independently by a third-party (i.e., a Designated Operational Entity) accredited by the CDM executive board. This takes place in three stages during the CDM project cycle namely validation, verification, and certification.\nValidation is the process of independent evaluation of project activity based on the Project Design Document against the CDM requirements. The outcome is the registration of the project.\nVerification is the independent review process of monitored reductions or sequestration that occurred as a result of a registered project activity for a given period. This is an ex-post check to confirm whether or not and to what extent carbon offsets have actually been attained.\nCertification is the process by which the designated operational entity gives written assurance of the emission reductions or sequestrations achieved by the project during a specified time period as verified. The result is the issuance of Certified Emissions Reductions (CERs).\nSmall-Scale Afforestation and Reforestation Projects\nFollowing discussions on the complications and costs involved in responding to the rules or requirements outlined above, baseline, monitoring, and certification modalities were simplified for \u201csmall-scale projects.\u201d Decision 14\/CP.10 defines small-scale projects as those that will result in net anthropogenic greenhouse gas removals by sinks of less than 8 kilo tonnes of carbon dioxide per year during the crediting period. However, the host country has to approve that the project developers are a low-income community or individuals.\nCommunity Forests\nMany authors have argued that community forest management has the potential of fulfilling the triple objectives of biodiversity conservation, supporting local development, and providing forest services such as carbon sequestration (GEF 2000; Klooster and Masera 2000; Smith and Scherr 2003). Hence, if well managed, it could contribute substantially to the achievement of CDM objectives.\nFurthermore, the area of forests under various forms of community management has been increasing in the world. White and Martin, (2002) note that 14% of forests in the most forested countries are owned by communities, whilst some 8% more are controlled by communities. More forests in Cameroon are coming under community management following new forest legislation in 1994, introducing community forestry. By January 2006, there were 334 applications by communities in the Ministry of Forests and Fauna (MINFOF). Of the 334 applications, 90 community forests were under full community management, indicating that the figure more than quadrupled from 17 in December 2001. At this rate, total area under community forestry could attain 1 million hectares in five years (i.e., 200 community forests at a maximum of 5000ha).\nCommunity forestry in Cameroon was chosen for this study because the policy provisions for community forestry provides a good institutional and regulatory framework (though not sufficient) for project appraisal, approval and verification by the Sub-Directorate of community forestry in the Ministry of Forests and Fauna (Minang and others 2007). Rules and regulations are elaborated in the Manual of Procedures and Norms for the Management of Community Forests (MINEF 1998). No other forest management type in Cameroon has such a regulatory framework. We briefly present the concept of community forestry in Cameroon in the following paragraphs.\nCommunity Forest is defined as \u201cthat part of non-permanent forest estate (not more than 5000ha) that is the object of an agreement between government and a community in which communities undertake sustainable forest management for a period of 25 years renewable\u201d (MINEF 1998).\nGovernment approves a community forest application and signs a management agreement upon community fulfilment of the following requirements, The community has constituted a legal entity and appointed a community forest manager who shall represent them in negotiations with government in matters of community forestry;The community has delineated and mapped the intended community forest area;The community has completed an 8\u201310% inventory of the timber, non-timber forest products, and wildlife of the forest;The community has provided a description of previous activities carried out in the intended forest area;The community presents a simple management plan for the intended forest; andThe community shows proof of stakeholder agreement on the intentions of forest management.\nOnce the management agreement is signed, policy requirements are as follows:That 100 % inventories are carried out in the compartments prior to the commencement of activities;The management of community forests provide annual activity plans for approval;The management of community forests provide annual reports to government; andThe community forest management plans are reviewed every five years.\nMany community forests in Cameroon are a mix of natural and secondary forests. Some cocoa agroforests are also found within community forests as well. Therefore, a broad spectrum of activities including regeneration, afforestation, logging, and non-timber forest product collection is implemented within community forests.\nHowever, current CDM rules only accept afforestation and reforestation type forestry; therefore, natural forest management by communities is not eligible. But the possibility exists that forest management could be taken up by the Kyoto protocol in the future (post 2012) under different rules (Santili and others, 2005).\nPrivate individuals, companies, or government own most current CDM projects institutions where decision-making and management are likely to have more structure and simplicity. Community ownership and management is complex and problematic in terms of resource tenure, project responsibilities, benefit allocation, and governance aspects. CDM rules have not been tested in those complex communities that harbour tremendous biosphere carbon management potential.\nCommunity Capacity Assessment Framework\nProject developers are required to put forth arguments and supporting evidence for each CDM requirement in a Project Design Document. Special knowledge, skills, technology, and infrastructure are also needed for collecting and analysing the required evidence. Furthermore, planning, coordination, and management skills will be required in the project development process.\nIn the ensuing paragraphs, we review specific tasks of each requirement in order to elucidate dimensions of community capacity requirements. Table\u00a01 presents a summary of the resulting assessment framework of community capacity for CDM forestry projects.\nTable\u00a01CDM community capacity assessment frameworkCDM requirementCommunity Capacity requirementsAdditionalityDoes the community have access to adequate financial resources for baseline and other analysis?Does community have access to required technology for data collection and analysis?Does community have access to necessary human resources (knowledge and skills)?AcceptabilityAre the necessary national sustainable development policy analysis knowledge and skills available within the community?ExternalitiesAre the necessary impact assessment and leakage analysis knowledge and skills available within the community?CertificationHow adequate is the community forest monitoring system?How adequate is the community information infrastructure?Is the relevant CDM information (forest inventory, socio-economic) available?Does community have required financial resources to engage Designated Operational Entities?Management CapabilityAre actors effectively participating in decision-making and implementation?How effectively are resource rules being implemented?How good are actor relationships in forest management?Are communities receiving adequate government and NGO support?\nAdditionality: The draft tool for the demonstration of additionality proposes a five-step screening and analysis procedure for CDM projects including,Preliminary screening based on the starting date of the project activity and the specific features of the afforestation and reforestation activity (mapping and map analysis, land use analysis);Identification of alternatives to the project activity consistent with current laws and regulations (land use analysis, estimates\/measurements of carbon stocks, projections, baseline development);Investment\/financial analysis (Internal Rates of Return, Net Present Value, cost benefit ratio, sensitivity analysis, etc.)Barrier analysis (investment, institutional, technological, cultural, social, ecological, and other kinds of barriers); andImpact of CDM registration (Expected).The above-mentioned steps are indicative of the knowledge and skills required to provide valuable arguments and evidence on additionality.\nThe draft tool for additionality also specifies that evidence regarding land use can be provided from land use and land cover maps and satellite images of around 1990. This means access to mapping technology as well as other technology for measurements of soil carbon or biomass estimation will be required. Lee (2004) concludes that most of the information is technical, requiring good knowledge and skills and technology to collect and manage.\nIt can be argued that communities may hire these services, but the costs can be very high. Recent studies in Tanzania revealed that costs for carbon inventories done by communities (with minimal supervision) would be 10 times, or more, cheaper than when experts are contracted (Zahabu 2006). Community inventory costs ranged between 2.5 and 21 $\/ha\/yr. Transaction costs for afforestation and reforestation have been estimated at between $8\u201331\/tC (Poffenberger and others 2002) and $0\u201370 \/tC (de Jong and others 2000).\nEligibility: Demonstrating compliance with national sustainable development rules involves engagement with the national authorities and providing evidence. For community forestry in Cameroon, \u201csustainability\u201d implies creating a legal entity, ensuring participation, developing a simple management plan, and developing a benefit sharing mechanism. Technical expertise and resources (financial and material) are thus required to provide the evidence.\nExternalities: Environmental impact assessments and social impact assessments are required of each project. The project is also required to show how they will mitigate or solve any negative impacts identified by studies. The same holds for leakage. Relevant knowledge and skills as well as resources are thus required to carry out these studies and design mitigation measures.\nCertification: A monitoring plan must be provided for all the variables estimated in the project design document. This requires a demonstration of how information would be collected and archived to enable validation and verification by the designated operational entity. The data collection, processing, storage, retrieval, and sharing with the operational entities demand a certain level of data and information infrastructure. The term data or information infrastructure means the relevant base collection of technologies, policies, and institutional arrangements that facilitate the availability of and access to data relevant for the implementation of carbon forestry.\nNegotiations, contracting services, and communication with the operational entities involve costs and specific skills. Ecosecurities (2002) estimates the costs of validation at between $18,900 and $37800 and verification costs at about $9400 per audit. Prototype Carbon Fund cost estimates are slightly higher (Lee 2004).\nManagement Capabilities and Conditions: Though not a direct CDM requirement, management remains a critical success factor for CDM projects, hence it is being incorporated by operational entities in validation processes. Nelson and de Jong (2003) demonstrate the importance of institutional arrangements in rule setting, enforcement, and monitoring for carbon forestry projects in Chiapas, Mexico. Poffenberger and others (2002) cite interalia, effective institutions, democratic leadership, transparency in decision-making and public expenditures, and minimizing social conflicts as important success factors for community carbon forestry. Subak (2000) also underscores the role of governments and NGOs in providing technical and institutional support for carbon mitigation projects in Costa Rica. FERN (2000) reported conflicts resulting from resource tenure perceptions that created serious problems for a carbon project in East Africa.\nBased on the preceding paragraphs, we raise key questions for community capacity assessment under the four CDM requirements. We also include a category on Management capabilities and conditions\u2014see Table\u00a01 (Ivey and others 2004). The various issues discussed in this framework are interrelated; hence, we try to show these interactions as much as possible based on our empirical evidence.\nMethods and Context\nMethods\nThis study aims at evaluating community capacity to meet CDM conditions. To do so, we create a framework (Table\u00a01) and seek empirical evidence by way of case studies. We identified two communities in Cameroon for the study: Tinto and Bimbia Bonadikombo (hereinafter called Bimbia). The choice of these communities was based on the willingness to provide data and the relative homogeneity\/heterogeneity and accessibility of the communities. All three villages in Tinto and four villages in Bimbia are typical small rural livelihood-based settings, while two settlements in Bimbia are relatively larger and peri-urban. The fact that these cases were not initially conceived for CDM purposes constitutes a limitation in the study.\nData collection tools included semi-structured interviews (19), structured-interviews\/questionnaires (84), focus group discussions (6), secondary data review, and forest transect walks. Given the substantive nature of the dimensions of community capacity, more discursive data collection tools were selected (Frankfort-Nachmias and Nachmias 1996; Yin 1994). Questionnaires were used for selected community resource persons to understand the relevant community knowledge and skills pool. Data sources included community forest legal entities, Ministry staff, traditional authorities, NGO staff, community\/user groups, and municipal authorities. Documents analysed included various planning, monitoring, seizure, and study reports. Interviews were used to obtain insight and check the information from secondary sources and other tools. Thematic and issue based content analysis was used to analyse the transcripts and secondary information for answers to questions in the community capacity assessment framework.\nThe Empirical Setting\nThe Tinto community consists of three neighbouring villages of the same clan namely Bessinghe, Kerieh, and Mbu. The total population of between 1700\u20132000 is very homogenous with less than 1% \u201coutsiders.\u201d It is typically rural, but is an administrative (District) headquarters with a forestry office. Most farmers grow cocoa or coffee as cash crops, alongside cassava, maize, and other subsistence crops. Forest activities include hunting, collecting non-timber forest products, and timber. Tinto began a community forest planning process in November 1999 and signed a management agreement with government for an evergreen lowland forest area of 1295 ha in December 2002. But little has happened by way of management to date.\nThe Bimbia Bonadikombo community is a complex of many villages namely, Mbonjo, Chopfarm, Banangombe, Bonabile, Dikolo, Mabeta, Ombe Native (Bamukong), Bonadikombo, and several plantation worker camps. Two of these settlements (Bonabile and Dikolo) are larger and peri-urban in character and located on the fringes of the Limbe (Victoria) town (see Fig.\u00a01). Limbe and the surrounding areas have a population of about 123,900 inhabitants (RCDC 2002). It is highly heterogeneous with few local people (of the Bakweri tribe). Forest extraction activities in order of importance include collection of non-timber forest products, fuel wood and timber, charcoal burning and hunting. The community has been managing a 3700\u2013ha forest since mid 2002. Vegetation is evergreen with different types: littoral vegetation, mangrove, freshwater swamp forest, stream and riverside vegetation, and lowland rainforest.\nFig.\u00a01Location of study areas in Cameroon\nCommunity Forest Actors\nA nested institutional structure can be observed in both the Tinto and Bimbia communities. There are user groups, community-based organisations created for community forest management purposes, and traditional authorities, all of which are moulded and developed within the locality. These organisations work closely with the forest administration and NGOs. Table\u00a02 presents a summary of actors, their interests and responsibilities within community forests in these communities.\nTable\u00a02Summary description of community forestry actorsActorInterests and Responsibility1. Bimbia Bonadikombo Natural Resource Management Council (BBNRMC)Manages Bimbia forest; Has an elected Board and a Forest Management Officer overseeing day-to-day operations2. Tinto Clan Community Forest\u2013Common Initiative Group (TCCF\u2013CIG)Manages Tinto forest; Has an elected Management Committee and a Forest Management Officer in charge of day-to-day operations.3. ChiefsVillage heads; custodians of forests; authorise access to all resources and land; in both cases are members of the BBNRMC board and TCCF-CIG committee, respectively4. Forest User GroupsIncludes all user groups; interested in access rights; participate in general assemblies of organisations; In the case of Bimbia, each user group has a representative on the Board5. Women in communitiesInterested in access rights for non-timber forest products and farmland6. Elites\u201cSuccessful\u201d sons and daughters living outside the community (as defined by these communities): interested in broad village development;7. Ministry of Forests and Fauna (MINFOF)Mandated to ensure sustainable forest management; provide technical support; conflict resolution8. Municipal AuthoritiesInterested in contributions of community forest to development of municipality9. Non-Governmental Organisations (NGOs)Interested in sustainable forest management; provides technical, institutional, and financial support;Mount Cameroon Project supported Bimbia, while Living Earth Foundation supported Tinto.\nThe makeup of institutional structures differs slightly between the two communities and some actors such as charcoal burners are only found in the Bimbia community. It is worth noting that forest use and livelihood activities often involve combinations of activities. But we present the actors in terms of forest use activities in order to capture specific issues that could otherwise be diluted in the analysis of various activity combinations.\nAssessing Community Capacity for CDM project development\nIn this section, we evaluate community capacity in the light of CDM requirements and the corresponding community capacity requirements (Table\u00a01), including mainly additionality, acceptability, externalities, certification and management capabilities and conditions. (Table\u00a05) provides a summary of the findings.\nAdditionality\nThe key additionality capacity question to address is whether or not communities have access to the financial, technological and human resources required to fulfil additionality requirements.\nIn terms of financial resources, total annual income in 2005 for Bimbia stood at $31, 200 (see Table\u00a03). At the end of that year, Bimbia was running at a deficit of about $3000. Total income in Tinto stood at $10,150. One hundred percent of the reported income for Tinto during this period was an advance payment for timber exploitation. Prior to this deposit, all income for the Tinto community forest over three years was a grant from Living Earth amounting to $800.\nTable\u00a03Estimated income and expenditure of community forests (January\u2013December 2005)DescriptionBimbia BonadikomboTintoIncome\u00a0\u00a0Total (XAF\/USD)14,867,000\/302005,000,000a\/10150\u00a0\u00a0From forest operations- wood (%)28.5100\u00a0\u00a0From grants\/donations (%)10.40\u00a0\u00a0From service delivery (ecotourism and tree care services to Urban Council (%)230\u00a0\u00a0Fines and auction sales (%)19.10\u00a0\u00a0Loans (%)18.80Expenditure\u00a0\u00a0Total (XAF\/USD)15,910,000\/32300940,000\/1900\u00a0\u00a0Operational costs, Office (%)11100\u00a0\u00a0Operational costs, Field (%)23.40\u00a0\u00a0Salaries (%)62.80\u00a0\u00a0Investments (%)00aThis amount represents a deposit made by a potential timber exploiter in November 2005, as proof of liquidity\nConsidering mitigation potential and transaction costs for various averted deforestation, reduced impact logging and regeneration carbon scenarios as in Table\u00a04, and the financial resources of both communities, the investment requirements will be extremely difficult if not impossible for these communities without external support. Table\u00a04 was calculated by using a chronosequence of mean carbon content and rates of carbon saved under different land use options in Cameroon. We estimated that the Bimbia forest could mitigate between 7\u201312.4 Kt C y\u22121 and the Tinto forest between 1.7\u20135.3 (see Table\u00a04). With production potentials of less than 8 Kt Co2 y\u20131 (2182 tC y\u22121), both projects could qualify for small-scale CDM. This means they can reduce transaction costs by up to 50%. Yet with the current financial situation, they are unlikely to meet the investment requirements.\nTable\u00a04Projected carbon mitigation potential for community ForestsWithout ProjectWith Project Scenario 1cWith Project Scenario 2cTinto Community\u00a0\u00a0Total area (ha)1295\u00a0\u00a0Vegetation typeNatural forest\u00a0\u00a0Scenario descriptionConversionAverted DEForestationReduced Impact Logging(ADEF)- Conservation(RIL)\u00a0\u00a0Potential area (ha)\/yr7.7752\u00a0\u00a0Carbon gain -tC ha \u22121a220104\u00a0\u00a0Total carbon saving (Kt C y \u22121)b\u20141.75.3Bimbia Community\u00a0\u00a0Total area (ha)3714\u00a0\u00a0Vegetation typeNatural forest (50%) and mixed cocoa farms and secondary forests (50%)\u00a0\u00a0Scenario descriptionConversionADEF \u2014 Conservation of natural forest\/Conservation\u00a0+regeneration Conservation\u00a0+\u00a0regeneration(RIL)\/conservation + regeneration\u00a0\u00a0Potential area (ha)\/Yr\u201411 (ADEF)\/ 928 (Conservation)74 (RIL)\/928 (Conservation)\u00a0\u00a0Carbon gain -tC ha\u22121a\u2014220 (tC ha\u22121)\/5 tC ha\u22121y\u22121104 \/ 5 tC ha\u22121 y\u22121Total carbon saving (Kt C y\u22121)\u20142.4\/4.647.7\/4.647.0412.34aMean Annual Carbon gain values for various project scenarios are assumed from reported studies in Cameroon as follows: ADEF (Kotto-Same and others 1997), RIL (Justice and others 2001), and Regeneration (Palm and others 2000).bTotal carbon savings are given as \u2211(Carbon gain \u00d7 potential area)cGeneral Scenario assumptions are:\u2022 Secondary forests are made available for conservation and regeneration\u2022 There will be no fires, droughts or disasters during the project lifetime\u2022 Illegal logging will be minimal and not sufficient to significantly affect project\u2022 Forest areas do not include roads, water bodies and minor human settlements\u2022 Exercise of usufruct rights for subsistence purposes including fuel wood, and non timber forest product harvesting is unlikely to significantly affect carbon flows\nCommunity forestry as practiced in both communities is multi-activity and can entail prohibitive negotiation costs (Smith and Scherr 2003). De Jong and others (2000) reported costs of participation, negotiation, and conflict prevention in the Scolel Te project in southern Mexico ranging from $52\u2013325\/ha. Such costs and those to be incurred on impact prediction, validation, and verification are not part of the concessions on small-scale CDM projects, yet these costs in themselves could be prohibitive to CDM project uptake and development.\nSecondly, an analysis of pre-2005 financial records in both communities revealed they received overseas development assistance for community forestry implementation (11.70% of income for Bimbia and 100% for Tinto; Table\u00a03). Because communities received overseas development assistance for completely different project purposes, they may become ineligible under the financial additionality criterion.\nTechnical resources such as satellite images, GPSs, and tree height measurement instruments would be helpful in providing information required for baseline estimation. None of these communities have direct access to satellite images or facilities to process them. Bimbia can access images through the GIS unit of nearby Mount Cameroon Botanic Gardens and Conservation Centre. But they have to pay for it. Access to free satellite data from the Internet would be difficult given the very weak connectivity services in the region. Bimbia has one GPS that can allow them to map current land use. Tinto has a compass that can be useful for inventories.\nThe human resources required for related additionality analysis is currently limited in both communities. Fourteen Bimbia and 11 Tinto community members received training on timber inventories during the process of developing the simple management plans. The main skills acquired included doing physiological measurements (tree heights, dbh), using the compass and or the GPS, tree identification, and laying out sample plots and transects. But the skills required for carbon estimation are more complex, including biomass estimation, using allometric equations, root biomass estimation, measuring trees of all diameters, destructive sampling, and so on. These technical carbon estimation skills are absent in both communities.\nSkills for financial and investment analysis are absent in these communities. One of the staff in Bimbia has basic undergraduate course knowledge in cost-benefit analysis but this is not enough. The community would thus have to hire such services at high costs. In Tinto, no one had such skills and knowledge.\nAcceptability\nThe main capacity question in the acceptability criterion is do communities have the knowledge and skills to provide evidence of project contribution to sustainable development? According to the current community forestry regulations, \u201csustainability\u201d is demonstrated by the development of a simple management plan, a viable legal entity, a benefit-sharing mechanism, and planned community projects (MINEF 1998). Both communities fulfilled these conditions, thanks to previous financial and technical assistance from NGOs and projects (McCall and Minang 2005). Mount Cameroon Project helped Bimbia while Living Earth Foundation assisted Tinto. Hence, we can say that both communities adhere to the sustainable development criteria that exist.\nHowever, problems may emerge if national sustainability criteria for CDM eventually include international environmental conventions.\nExternalities\nThe key capacity question addressed below is do communities have the knowledge and skills for the required impact assessment and leakage analyses.\nFive persons (three employees and two board members) in Bimbia have at least undergraduate knowledge and some experience of environmental and social impact assessments. This means they have a good chance of providing the evidence required by this criterion of the CDM. On the other hand, none of the Tinto members had any knowledge of environmental or social impact assessments. No respondent in both communities had any understanding of leakage.\nCertification\nCommunities would have to collect, analyse, archive, and eventually share information with designated operational entities responsible for validation and verification. Hence, the key capacity questions for certification include, how functional are community forestry monitoring systems? How adequate are community information infrastructures? And do communities have enough financial resources to engage Designated Operational Entities for validation?\nMonitoring and reporting mechanisms in both communities suffer serious inadequacies. Monitoring and reporting in Bimbia are characterised by monthly management council board meetings at the managerial level, and by forest patrols at an operational level, 186 patrols in 2005. These meetings and patrols result in reports. However, the patrols are for the most part erratic, triggered by tip-offs on illegal activity. In 2003, control posts or check points that could be manned for 24 hours were made at strategic outlets from the forest, but were later abandoned by the operation committee members. It is alleged that the control posts system did not give the room for corrupt practices desired by some operation committee members, hence, the abandonment. Some of them negotiate and collect fines from defaulters without the knowledge of the management officer. In extreme cases, they cooperate with illegal timber exploiters. In the case of Tinto, no monitoring or reporting of any sort has been done since December 2002 when the management agreement was signed. The community explains that with \u201czero\u201d activities in the community forest, there is no reason to report. Given that these monitoring systems were not meant for CDM projects, they would need to be overhauled if these projects are to be registered within the CDM. However, the corruption and institutional planning problems currently experienced would still pose serious difficulties for CDM monitoring.\nNeither community has conducted post-management agreement forest surveys or inventories of designated exploitation compartments as required by law. They have equally failed to convene general assemblies of stakeholders to discuss and review progress as their statutes demand since the commencement of community forest activities.\nCommunity information systems were found in both cases to have relevant biophysical, socio-economic, and market data that could be used for further analysis on impact, baselines, leakage, and other CDM requirements. However, inventory data available to these community forests are inadequate for carbon estimation as they were done for timber exploitation as required by extant community forestry rules. Most of these studies were done with the help of NGOs prior to the management agreement. As demonstrated in the preceding text, further collection of complementary data for CDM without such assistance may be less reliable because communities do not have adequate skills.\nMaterial and technological resources (infrastructure) can tremendously influence project information management. Bimbia currently has limited office space within the premises of the Divisional Office for Limbe. They have four operational computers and a digital filling system for all reports. Hard copies of reports are stored on shelves totalling about four metres in length. With a motorbike, 12 staff and little funds to hire cars regularly, transportation is a serious hindrance to monitoring efforts. In Tinto, activities are run from the forest manager\u2019s house. All information for the Tinto community forest is found in two cardboard folders.\nManagement Capability and Conditions\nIn this section, we examine the extent to which community decision-making processes, rules compliance, actor relationships, and relationships with government and NGOs are adequate for carbon project development.\nParticipation and Decision-making\nInvolving actors in building consensus and decision-making for CDM project implementation is important for local communities (de Jong and others 2000; Smith and Scherr 2003). Participation in decision-making is largely by actor representation in the decision-making bodies within the legal entities managing the forests on behalf of the communities. The management board in Bimbia is made up of chiefs, elected user group representatives, and some employees such as the forest manager. It meets on average 10 times a year. Attendance sheets show charcoal burners as the only consistent user group participating at these meetings. Timber exploiters and the fuel wood harvesters have been persistently absent. Latent power struggles within the leadership have weakened decision-making processes. The six chiefs in Bimbia sent a letter to the acting forest manager complaining that they were not being sufficiently consulted on day-to-day forest management issues such as issuance of permits (Letter of January 2005).\nIn Tinto, the management committee is made of all three chiefs, village representatives, and the forest manager. It has met about five times since December 2002. Interviewees reported about four ad hoc meetings between the manager, two chiefs, and one board member for consultation on proposals for sale of standing volume within the community forest between 2003 and 2005. Power struggles over money have also weakened decision-making in Tinto. All three chiefs in Tinto complain of not being consulted by the manager especially on financial issues. Given observed actor reactions and comments during informal discussions, it is our reasoned judgement that this is an important issue, which contributed to the inertia in the take off of community forestry activities in Tinto. These systems still harbour many weaknesses that may inhibit consensus-building processes for carbon forestry.\nAnnual general assemblies in which popular community participation is expected have not been convened in either case since mid-2002, implying that actors have not had the opportunity to participate in more strategic decision-making in community forestry.\nImplementation of Community Forestry Rules\nAn assessment of community forestry rules compliance produced mixed results. In Bimbia, annual legal timber exploitation has been between 500 to 700m3. About 5000 trees have been planted in two of three compartments envisaged in the management plan. There has also been good cooperation with charcoal burners in the implementation of management rules.\nHowever, in Bimbia the rules have also been seriously flouted. Deforestation and degradation has been accelerated by farming and illegal timber exploitation. Illegal fuel wood harvesting is rife in the accessible southeast and western compartments of the forest. For example, in 2005, 186 forest monitoring patrols were conducted. During these patrols, the following were confiscated, 49 chain saws, 2000 kg of charcoal (100 bags of about 100 kg), 301 small fuel wood chunks, and 1254 sawn timber boards of various sizes (4\u00a0\u00d7\u00a08 cm; 4 \u00d7 12 cm. etc.). Income from fines and sales of confiscated products amounted to about $5700 (2, 840, 000 XAF), representing about 67% of total income from about 655m3 of legal logging from the Bimbia forest. These numbers are explained by easy accessibility to the Bimbia forest, which is located a few kilometres from Limbe town.\nThe Tinto community had agreed to exploit 2000 m3 of timber annually from the forest. But in three years nothing has happened. They advance the absence of an access road and lack of start-up resources as reasons for the inertia. Negotiations for timber exploitation are ongoing. The above evidence suggests many potential inadequacies regarding rules compliance for carbon forestry.\nActor Relations\nGood actor relationships are necessary for success in carbon forestry. Figure\u00a02 presents the state of relationships between the actors in the Tinto and Bimbia communities. The figure was developed mainly from interviews and secondary data, and discussed and validated with other actors especially those not interviewed.\nFig.\u00a02Sociogram showing community forest actor relationships in both communities\nVaried land tenure perceptions have affected the actor relationships in community forestry implementation in Bimbia. In Bimbia, it was agreed with the representatives of all actors during the land use plan and the simple management plan phases that each farmer within the forest would pay a registration fee of 2000FCFA ($3.5), after which his or her farm would be assessed and annual rents determined. Less than 100 of the estimated 1000 farmers have registered. Farmers think registration is only a pretext and that rents might eventually be prohibitive, thereby kicking them out. They emphasise that the spirit of pre-community forest indigenous organisations such as the Victoria Lands and Forest Conservation and the Victoria Area Rainforest Common Initiative Group that aimed at ejecting \u201cnon-native usurpers\u201d from their forest still prevails. Such land tenure perceptions and issues of trust explain the poor relationships between the management council and farmers or fuel wood collectors (Fig.\u00a02) especially because many users of the forest are non-native.\nRelations between communities and government can influence community project development and risk management in terms of enabling training and improving access to resources and technology. It could also stifle progress when conflicts arise in their relationship.\nCommunity forestry policy stipulates that the Ministry of Forests and Fauna (MINFOF) is supposed to provide technical support to communities in forestry activities, but working relationships have been poor in both cases. Poor working relationships arise partly from lack of clarity in roles and responsibilities. The Bimbia community accuses MINFOF of illegally issuing about 14 logging permits within their forest, and lack of transparency with auction sale dues from joint seizures. MINFOF says Bimbia has no right to sanction defaulters while Bimbia insists the law allows them to deal with minor offences. The line between major and minor is not clear even for the neutral interpreter of policy. These examples of disagreements have created conflicts between the community and forest administration staff (see Fig.\u00a02).\nIt is clear that communities will need external assistance with complex carbon measurement and monitoring tasks such as the use of allometric equations, soil laboratory measurements, access to remote sensing technology, and information management. This would be expected from MINFOF, but the question is whether this government body is in a position to provide such assistance. To start with, technological knowhow and resources are in short supply in this government department. Existing allometric equations were developed for known marketable timber species only. Available and useful remote sensing and GIS data are limited to 1950 and 1991 aerial photographs covering some forest areas in the country. These could be used as evidence for land use and land cover for critical date requirements for CDM, i.e., December 31, 1989, for example. Staff and resources in the ministry are inadequate for supporting communities (Ekoko 2000). The ratio of staff to forest area in the southwest province, in which both Tinto and Bimbia are located, is about 1:15000ha. A senior forestry official acknowledged that they are so badly equipped that they sometimes ask for transportation or material support from communities in the fulfilment of their tasks. Staffs in most cases have little knowledge of carbon issues.\nRelations between communities and NGOs have been good. NGOs or bilateral projects have provided the support communities\u2019 need for forest management. For example, in Bimbia, Mount Cameroon Project (GTZ-DFID funded) facilitated workshops on community forestry regulations and helped with the establishment of the legal entity. It also financed hired expertise for the training of community members and subsequent implementation of mapping, forest inventories, and the development of simple management plans. Living Earth Foundation used a similar approach in the case of Tinto. This support by NGOs continued through the entire community forestry-planning process and the early stages of implementation (late 2003 to early 2004).\nBut very often these NGOs have been using overseas development assistance funds. Such funds may not be acceptable under the CDM given the financial additionality criterion. This makes a compelling argument for the creation of a national fund to support CDM project development.\nA summary of the findings is presented in Table\u00a05. While the synthesis shows that communities have benefited in terms of income, knowledge, and skills acquisition and employment, it also reveals serious inadequacies in human, financial, and technological resources required for successful community forestry implementation. In comparative terms, Bimbia fared better with regards to resources. Perhaps Tinto\u2019s remote location and relatively smaller size explains the difference in resource availability.\nTable\u00a05Summary of findingsRequirementBimbia BonadikomboTintoAdditionality\u00a0\u00a0Access to financial resourcesInsufficient financial resources. Deficit of $3000 in 2005 accounts; Little experience with high interest loans; Eligibility unlikely due to use of ODA funds in forestry implementationInsufficient financial resources. Functioning for past three years with $784 in total; No experience with loans; Eligibility unlikely due to use of ODA funds in forestry development.\u00a0\u00a0Knowledge and skillsLimited knowledge and skills. One person with undergraduate knowledge of cost benefit analysis and none in investment or financial analysis methods. Fourteen employees have knowledge and skills in timber inventories only.Knowledge and skills are extremely limited. No knowledge or experience of financial or investment analysis in community. Eleven people have knowledge and skills in timber inventories only.\u00a0\u00a0Access to technologyLittle or no access to satellite data and other technologies required.Same as in BimbiaAcceptability\u00a0\u00a0Knowledge and SkillsCommunity fulfilled \u201csustainability\u201d criteria in the development of management plans with NGO support. Hence have some relevant experience.Same as in BimbiaExternalities\u00a0\u00a0Environmental impact and leakageThree members in community have knowledge skills and experience in environmental and social impact assessment, therefore good potentialNo knowledge or skills in environmental or social impact assessment observedNo knowledge or understanding of leakage observedSame as in BimbiaCertification\u00a0\u00a0Monitoring systemsMonitoring systems are functional, but inadequate for CDM because they do not involve ongoing inventories; Short of manpower (ratio of staff to forest area is 1:285 ha)No documented evidence of monitoring. The lone staff cannot ensure any proper monitoring for an area of 1 295ha.\u00a0\u00a0Information systemBoth physical and digital Information systems (computers) are operational. This can allow for \u201csharability\u201d; hence, the system is potentially adaptable to CDM archiving requirements.Information system consists of two cardboard folders. Hence, inadequate in form, content, and quality for CDM purposes.\u00a0\u00a0CDM Information availabilitySome relevant geographical, socio-economic, ecological, and general information is available for CDM use within current systems. But much more is requiredSame as in Bimbia\u00a0\u00a0Financial resourcesInadequate financial resources to pay for validation servicesSame as in BimbiaManagement Capability\u00a0\u00a0Actor relationshipsConflicts exist, (i) between 6 chiefs and management board of community forest; (ii) between community\u2014MINFOF over 14 permits and proceeds from auctioning of seizures; and (iii) between farmers and forest management.Conflicts between 3 chiefs and management officer.\u00a0\u00a0Forestry rules enforcementIllegal activity income accounts for about 67% of income from legal forest activities and 19% of total revenue in 2005. It is significant and poses threats to the success of potential carbon project.No illegal activity observed. Rules are being respected.\u00a0\u00a0Government community relationshipGovernment short of forestry personal (staff to forest area ratio in the province is about 1:1500ha); Government staff do not understand the CDM and have no relevant skills to support communitiesSame as in Bimbia\u00a0\u00a0NGO\u2013community relationshipThe Cameroon Mountain Conservation Foundation (CAMCOF) is interested in providing support for carbon forestry in the area, but lack the knowledge, skills and resources.Living Earth Foundation is interested in providing support for carbon forestry in the community, but lacks the knowledge, skills and resources.\nRegarding management capability and conditions, a mix of similarities and differences can be noted. In similarities, both communities have received NGO support and have also had conflicts with forest administration on various issues. In differences, Bimbia has experienced substantial internal conflicts, high levels of illegal activity, forest degradation, and staff corruption. In Tinto, the forest has remained intact due to the absence of activities, owing mainly to the remoteness of the area, and lack of startup resources and entrepreneurship. These differences illustrate that local community capacity is a result of the unique manner in which these attributes coalesce in particular places and, therefore, should also be seen to a very large extent on a case-by-case basis (Agrawal 2001; Armitage 2005).\nConclusions and Practical Implications\nSeveral studies have supported payment for environmental services within the commons as an emerging model of sustainable development for poor communities. Yet community capacity to implement such models often falls short of expectations. This paper set out to assess the capacity of communities to implement CDM projects in Cameroon. From experiences of success and failure in community forest implementation, we draw conclusions on the implications for CDM implementation within community forestry setups, i.e., in the case where communities currently managing forests decide to add on carbon sequestration as another land use.\nAnalysing the dimensions and determinants of community capacity for CDM in both the Bimbia and Tinto cases points to substantial inadequacies. It indicates that taking up CDM carbon management procedures complicates the challenges of local communities already grappling with huge community forest management difficulties within host country modalities and procedures. The inadequacies revealed serve as good arguments for varying dimensions of additionality in the CDM certification process. But more importantly however, evidence from the Bimbia and Tinto cases, though limited, also points to a number of generalizable observations on community capacity to manage terrestrial CDM projects.\nFirstly, proactive capacity-building measures are needed to increase project uptake in poor countries. Our case studies highlight evidence that local communities lack the knowledge, skills, and technical and financial resources to accommodate current CDM rules. Management capabilities and conditions are also deficient in many ways. Tasks for baseline estimation, investment, financial, environmental and social impact, and leakage analysis cannot be met under poor community conditions in Cameroon and many parts of sub-Saharan Africa. This evidence supports previous explanations for why Africa is lagging behind in CDM project development, i.e., currently accounting only for 1.7% of all projects in the CDM pipeline (Desanker 2005). This raises serious questions about the potential of CDM contributing to sustainable development in its current form.\nDue attention should, therefore, be given to multiple partnership arrangements and most especially to NGO capacity for supporting CDM implementation processes (Nelson and de Jong 2003). The Bimbia and Tinto cases point to the potential role NGOs can play in enabling project uptake and development. However, any community capacity approach for CDM needs to carefully consider tasks to be undertaken by communities and those to be undertaken by external agencies or consultants. This is because NGOs or consultants are developing many energy projects within the CDM and in some cases they get paid a proportion of the anticipated credits. Replicating such a scenario in CDM forestry could mean ignoring local competencies, thereby diverting vital community benefits in the form of credits. Sharing such roles and responsibilities especially vis-\u00e0-vis project developers and other partners could help reduce conflicts and facilitate institutional relations and hence resource governance. This could be beneficial for the development of other environmental services systems for water catchments or biodiversity.\nSecondly, managing actor relationships as influenced by their motivations, perceptions, and resources, within multiple use forestry projects, is a great challenge regarding risk of project failure, leakage, and costs of CDM projects. Involving actors in building consensus and compliance is difficult and costly for local communities (de Jong and others 2000; Smith and Scherr 2003). The knowledge and facilitation skills required are enormous, therefore taking on carbon as another community forestland use will compound the knowledge and skills demands. Such evidence should support the prioritization of institutional capacity building for developing countries called for in Decision FCCC\/CP\/2004\/L.11, and other documents within the Subsidiary Body for Implementation capacity building framework of the Kyoto Protocol.\nThirdly, this research brings a number of cross-scale CDM issues to the fore. It shows that community capacity depends on and is part of a forest\/land use policy framework. The CDM framework assumes that the necessary macro-institutional and regulatory support for micro-level implementation would be available. This study found out that neither sustainable development criteria nor supportive institutions and personnel exist in Cameroon.\nDeveloping national CDM guidelines for both forestry and energy projects might be a necessary (but not sufficient) condition for CDM project development at a local level. It can be beneficial in providing institutional structure and for specifying critical standards on impact assessment, monitoring, measures preventing risk of project failure, and information management for CDM forestry projects. This supports earlier contentions that some regulation may be required to reduce livelihood risks and increase social benefits (Smith and others 2003; Minang and others 2007)\nSupporting Designated National Authorities and allied ministerial services to provide proactive capacity building to poor communities is imperative for Kyoto Protocol processes if CDM is to succeed. This could help provide badly needed support from government institutions in the areas of resource\/incentive provision, training, information management, monitoring, and marketing. Costa Rica is an example of a non-annex 1 country that has instituted proactive measures of this kind (FAO 2004; Subak 2000). An opportunity exists for the creation of a national fund to support CDM projects to help provide startup funds for communities in Cameroon. Such a fund could tap from the coffers of the Special Forestry Development Fund, a mechanism that enables a proportion of forestry tax revenues to be reinvested into forestry development. Putting such funds into capacity building and not project implementation could be justifiable under the CDM.\nFinally, there is a need for a rethinking of current CDM forestry modalities. Current rules are complex, unfeasible, and unfairly beyond the capacity of poor communities such as those assessed in this study, thus confirming previous conclusions in Brown and others (2000) and Poffenberger and others (2002). It may also explain (at least in part) why India, China, Brazil, and Mexico combined hosted 83% of all CDM projects, while Africa hosted only 2% by June 2006. Provisions for small-scale CDM forestry projects are not far-reaching enough. They do not currently consider basic environmental and social impact and community negotiations costs, but our research demonstrates that they pose equally strong challenges to CDM project development even in instances where less than the small-scale threshold mitigation value of 8 kt C y\u22121 applies. If the sustainable development objectives of the CDM and the Kyoto Protocol must be attained in the poorest countries, further consideration should be given to CDM modalities in the ongoing post-2012 forestry negotiations. This study and others provide a growing body of evidence on community capacity for carbon forestry that could help in the development of more realistic and equitable CDM rules.","keyphrases":["community capacity","clean development mechanism","community forests","cameroon"],"prmu":["P","P","P","P"]}
{"id":"Eur_Spine_J-3-1-2071961","title":"Low back pain in general practice: cost-effectiveness of a minimal psychosocial intervention versus usual care\n","text":"An intervention that can prevent low back pain (LBP) becoming chronic, may not only prevent great discomfort for patients, but also save substantial costs for the society. Psychosocial factors appear to be of importance in the transition of acute to chronic LBP. The aim of this study was to compare the cost-effectiveness of an intervention aimed at psychosocial factors to usual care in patients with (sub)acute LBP. The study design was an economic evaluation alongside a cluster-randomized controlled trial, conducted from a societal perspective with a follow-up of 1 year. Sixty general practitioners in 41 general practices recruited 314 patients with non-specific LBP of less than 12 weeks\u2019 duration. General practitioners in the minimal intervention strategy (MIS) group explored and discussed psychosocial prognostic factors. Usual care (UC) was not protocolized. Clinical outcomes were functional disability (Roland\u2013Morris Disability Questionnaire), perceived recovery and health-related quality of life (EuroQol). Cost data consisted of direct and indirect costs and were measured by patient cost diaries and general practitioner registration forms. Complete cost data were available for 80% of the patients. Differences in clinical outcomes between both the groups were small and not statistically significant. Differences in cost data were in favor of MIS. However, the complete case analysis and the sensitivity analyses with imputed cost data were inconsistent with regard to the statistical significance of this difference in cost data. This study presents conflicting points of view regarding the cost-effectiveness of MIS. We conclude that (Dutch) general practitioners, as yet, should not replace their usual care by this new intervention.\nIntroduction\nLow back pain (LBP) affects a large number of people each year. Lifetime prevalence rates range from 49 to 70% [45]. LBP causes not only great discomfort, but also great economic loss due to work absenteeism [28, 45]. In the UK, LBP is one of the most expensive conditions for which an economic analysis has been carried out [28]. Economic evaluations in which both the costs and clinical outcomes of two or more interventions are compared, are becoming increasingly important, as health care expenditures rise while budgets remain limited [44]. The importance of economic evaluations is illustrated by the fact that some authors suggest that an intervention might be implemented when it is less effective but saves substantial costs [8].\nAs especially chronic LBP is associated with substantial costs to society [15], a large amount of costs can be saved by interventions that prevent acute LBP becoming chronic. The rationale is that it will be more cost-effective to address a wider target population early with simple low-cost interventions than to expend considerable time and resources on rehabilitating the smaller group of back pain patients who have become incapacitated by chronic pain. As psychosocial factors have been shown to play an important role in the transition from acute to chronic back pain [25, 33], one may assume that early interventions focusing on these factors prevent chronicity.\nWe tested this assumption by conducting a cluster-randomized clinical trial in general practice, comparing a minimal intervention strategy (MIS) aimed at psychosocial factors for patients with (sub)acute LBP to usual care (UC) by the GP, which was not standardized. Our theory on the working mechanisms of MIS was that identification and discussion of psychosocial factors would lead to modification of these factors, eventually leading to better functioning. Unfortunately, MIS appeared to be no more effective than UC in improving the following clinical outcomes: the degree of functional disability, the recovery rate and the number of patients on sick-leave due to LBP [20]. These findings are in line with Linton and Andersson [26], who showed that their cognitive-behavioral intervention was not more effective than usual care in reducing the degree of back pain and generic function status. However, their intervention was effective in reducing the number of visits to a physician for spinal pain and number of days of sick-leave, implicating time savings for physicians and thus substantial cost savings for society. These results may indicate an increase in coping or self-care with the pain; patients who received the psychosocial intervention, less often visited a physician and had less days of work while they had the same degree of functional disability as the patients who received usual care. These promising results, in combination with the fact that self-care with the pain was also a goal in our MIS, the recent emphasis on health care budgets, and the call for more high quality economic evaluations on the cost-effectiveness of treatments for LBP [43] stimulated us to conduct an economic evaluation from a societal perspective with a follow-up of 1\u00a0year. We hypothesized that MIS would be cost-effective compared to UC.\nMaterials and methods\nStudy design\nThe study is designed as a full economic evaluation alongside a cluster-randomized controlled trial and was approved by the Medical Ethics Committee of the VU University Medical Center in Amsterdam, the Netherlands.\nRandomization and training sessions\nRandomization took place at the level of the general practice in blocks of four practices, according to a random numbers table prepared before recruitment of general practitioners. General practitioners were informed about their allocation after they had given final consent to participation. Twenty practices (28 GPs) were randomized to the MIS group and 21 practices (32 GPs) to the UC group. The GPs randomized to the MIS group received two training sessions of 2.5\u00a0h each which were given by a GP (HvdH) with extensive expertise in development and training of psychosocial interventions. The training consisted of theory, role-playing and feedback on the practiced skills. In addition, a treatment manual was provided. The contents of the training sessions and its evaluation by GPs have been described in more detail elsewhere [21].\nPatients and interventions\nParticipating GPs were asked to select ten consecutive patients who consulted them for LBP. Inclusion criteria were age 18\u201365, non-specific LBP of less than 12\u00a0weeks\u2019 duration (i.e. (sub)acute LBP) or an exacerbation of mild symptoms, and sufficient knowledge of the Dutch language. Exclusion criteria were specific LBP (i.e. LBP caused by specific pathological conditions), LBP currently treated by another healthcare professional, and pregnancy. Patients, but not their GPs, were kept unaware that two different interventions were studied.\nPatients received a minimal intervention strategy (MIS) or usual care (UC). The MIS was aimed at identification and discussion of psychosocial prognostic factors. The MIS consultation lasted about 20\u00a0min and consisted of three phases: exploration, information and self care. During the exploration phase, the GP explored the presence of psychosocial prognostic factors by asking standardized questions that could be rephrased to fit the style of communication of the doctor and the patient. The following psychosocial prognostic factors were explored: the patient\u2019s own ideas on the cause of their LBP, fear avoidance beliefs, worries\/distress, pain catastrophising, pain behaviors and reactions from the social environment (family, friends, work). In the information phase the GP provided general information on the cause, course and (im)possibilities of treatment of LBP, thereby giving specific attention to psychosocial factors identified in the exploration phase. Finally, in the self care phase, the GP and patient set specific goals on resuming activities or work. Follow-up consultations were not protocolized, but we advised GPs to make an appointment for a follow-up visit in case they identified obstacles to recovery and suspected an increased risk of chronic LBP.\nGPs in the UC group provided care as usual. We did not protocolize the content and number of UC consultations, and assumed that GPs would generally follow the guideline for LBP of the Dutch College of General Practitioners [13]. For acute LBP (<6\u00a0weeks\u2019 duration) this guideline advises a wait and see policy. For subacute LBP (6\u201312\u00a0weeks\u2019 duration) the guideline advises referral for physical therapy in the case of persistent functional disability. Explicit guidance on psychosocial factors is lacking. The contents of both interventions have been described in more detail elsewhere [20].\nData collection\nClinical outcomes\nBaseline data were collected during a home visit by a research assistant, while follow-up data after 12\u00a0months were collected using postal questionnaires. Primary clinical outcome measures were functional disability, perceived recovery, and health related quality of life. Functional disability was measured at baseline and after 12\u00a0months by the Roland\u2013Morris disability questionnaire (0\u201324) [35]. Perceived recovery was scored by the patient on a 7-point Likert scale (very much\/much\/slightly improved, no change, slightly\/much\/very much worse) after 12\u00a0months [42]. As a score of at least \u201cmuch improved\u201d has been denoted a minimal clinically important change [32], patients were a priori defined as recovered if they reported at least \u201cmuch improvement\u201d. Health related quality of life was measured at baseline and after 3, 6 and 12\u00a0months by the EuroQol (0\u20131) [12], covering five domains: mobility, self-care, usual activities, pain\/discomfort, and anxiety\/depression. The EuroQol scores were transformed into utilities using a representative British sample and time trade-off methods. The utilities were then multiplied by the amount of time a patient spent in this particular health state, with transitions between health states linearly interpolated [7]. This results in quality adjusted life years (QALYs).\nIn order to describe the study population characteristics in more detail and to compare baseline similarity of both intervention groups, five other outcome measures were assessed at baseline: (1) pain severity during the day (0\u201310) [27]; (2) perceived general health (1\u20135), using the first question of the subscale \u201cgeneral health perceptions\u201d of the short form health survey (SF-36) [47]; (3) fear avoidance beliefs, using the 4-item physical activity subscale of the fear avoidance beliefs questionnaire (FABQ, 0\u201324) [46]; (4) catastrophising thoughts, using the 6-item subscale of the coping strategies questionnaire (CSQ, 0\u201336) [36] and (5) distress, measured by the 16-item subscale of the four-dimensional symptom questionnaire (4DSQ, 0\u201332) [37].\nCost data\nThe economic evaluation was conducted from a societal perspective, indicating that all costs and consequences of the competing interventions are taken into account regardless of who pays for or benefits from them [9]. A societal perspective incorporates direct health care costs, direct non-health care costs and indirect costs due to LBP. All cost data were collected by prospective cost diaries [16] that patients completed for the periods baseline to 3\u00a0months, 3\u20136\u00a0months, 6\u20139\u00a0months, and 9\u201312\u00a0months. In addition, GPs were contacted after 12\u00a0months follow-up to provide information on follow-up consultations due to LBP in the last year (date, referrals, medication prescribed by physician). GPs used their medical records to complete these registration forms.\nTable\u00a01 summarizes the cost categories, and the prices and sources used for valuing. \u201cMedication\u201d in Table\u00a01 includes both the over-the-counter medication and medication prescribed by physicians, while self-prescribed alternative interventions are included in \u201ccomplementary care\u201d. Costs of absenteeism from paid labor due to LBP were calculated according to the friction cost approach [23]. This approach is based on a mean income of the Dutch population according to age and gender, and defines a friction period as 154\u00a0days [31]. As most cost data were collected during the year 2002, prices were adjusted using consumer price index figures.\nTable\u00a01Prices used for valuing resources (year 2002)ResourcesEuroDirect health care\u00a0Primary care costs\u00a0\u00a0General practitioner (consultation)a19.8\u00a0\u00a0MIS-consultation, including intervention costs (consultation)b40.9\u00a0\u00a0Physical therapist (treatment session)a22.3\u00a0\u00a0Manual therapist (treatment session)c30.8\u00a0\u00a0Exercise therapist (treatment session)a22.5\u00a0\u00a0Back school (treatment session) [18]66.3\u00a0\u00a0Chiropractor (treatment session)d40.0\u00a0\u00a0Physiofitness (treatment program, 10 sessions)b120.0\u00a0\u00a0Professional homecare (per hour)a21.3\u00a0\u00a0Psychologist (treatment session)d75.0\u00a0Secondary care costs\u00a0\u00a0Outpatient appointmenta54.8\u00a0\u00a0Hospitalization general hospital (per day)a330.0\u00a0\u00a0Surgery (per hour)b884.7\u00a0\u00a0Radiographb48.2\u00a0\u00a0MRI-scanb212.9\u00a0MedicationeDirect non-health care\u00a0\u00a0Complementary care d\u00a0\u00a0Informal care (per hour)a8.1\u00a0\u00a0Equipment aidsfIndirect costsAbsenteeism from paid workgAbsenteeism from unpaid worka8.1MIS Minimal intervention strategy, MRI Magnetic resonance imagingaPrice according to Dutch guidelines [30]bCalculated unit costscPrice according Dutch Central Organisation for Health Care Charges [10]dPrice according to professional organisation or health care providerePrice of medication prescribed by the GP according to Royal Dutch Society for Pharmacy [48]; price of over-the-counter medication according to cost diariesfPrice according to cost diariesgCosts based on mean income of Dutch population according to age and sex [23, 31]\nStatistical analysis\nFirstly, baseline similarity was studied. Secondly, we compared baseline characteristics of patients with complete cost data to those with incomplete cost data by using logistic regression analysis. Thirdly, clinical outcomes and total costs were compared. As cost data are characterized by large variation and irregular distributions and as a complete cost dataset was available for 80% of our participants (250\/314), we decided that our primary analysis would be a complete case analysis. Differences between groups (MIS minus UC) were calculated for the clinical outcomes: (1) functional disability, by calculating change scores between baseline and 12\u00a0months follow-up; (2) perceived recovery at 12\u00a0months follow-up; and (3) health-related quality of life gained over 12\u00a0months (i.e. QALYs). Students t tests were used to analyze the change scores between the treatment groups for functional disability and quality of life, and a Chi-square test for perceived recovery. To compare costs between the two groups, confidence intervals (CIs) for the mean differences in costs were calculated using bias-corrected and accelerated bootstrapping (2000 replications) [11]. Bootstrapping incorporates drawing samples with replacement and is a preferred method for the analysis of cost data, as it uses the observed distributions of the data without making assumptions about the shape of the distribution [38]. Fourthly, cost-effectiveness analyses were performed. Incremental cost-effectiveness ratios were calculated in which the mean difference in total costs (MIS minus UC) was divided by the mean difference in improvement on the clinical outcomes (MIS minus UC). Uncertainty around the ratios was calculated using the bias-corrected percentile bootstrapping method (5,000 replications) [5] and plotted on a cost-effectiveness plane.\nSensitivity analyses\nA complete case analysis has some disadvantages. Due to the missing data the power of the analysis is reduced and bias may be introduced due to selective drop-out. Imputation can be used to replace missing data by statistical estimates of the missing values. To explore the robustness of our primary analysis, we performed two sensitivity analyses in which we imputed missing data: in one analysis we imputed all missing cost data, and in the other we imputed only missing days of absenteeism. Imputation was done using the Expectation Maximization algorithm (SPSS 10.1).\nResults\nBetween September 2001 and April 2003, 314 patients were enrolled in our study: 143 in the MIS group and 171 in the UC group. Table\u00a02 shows that baseline characteristics of GPs and patients were largely similar for the two groups. Less than 9% of all patients withdrew from the study during follow-up. Reasons were \u2018no time and no complaints anymore\u2019 (MIS n\u00a0=\u00a01, UC n\u00a0=\u00a04), \u201cburden too high due to psychological problems\u201d (UC n\u00a0=\u00a03) or \u201cunknown\u201d (MIS n\u00a0=\u00a010; UC n\u00a0=\u00a08). The flow chart of this study, including information on refusals, exclusions and drop-outs has been published in our previous paper [20].\nTable\u00a02Baseline characteristics of general practitioners and patientsMIS UC GPsn\u00a0=\u00a028n\u00a0=\u00a032Demographic characteristics\u00a0Age (years)a, mean (SD)43.0 (7.2)45.7 (7.4)\u00a0Gender, female6\/2812\/32Number of included patients per GP\u00a00 1\/285\/32\u00a01\u2013517\/2814\/32\u00a0>5 10\/28 13\/32Patientsn\u00a0=\u00a0143n\u00a0=\u00a0171Demographic characteristics\u00a0Age (years), mean (SD)43.4 (11.1)42.0 (12.0)\u00a0Gender, % female47.647.4\u00a0Nationality, % Dutch97.297.7\u00a0Health insurance, % public70.667.8Educational level and work status\u00a0Education levela, %\u00a0\u00a0Primary35.0 33.1\u00a0\u00a0Secondary46.252.7\u00a0\u00a0College, university 18.814.2\u00a0Paid job, % yes81.881.3\u00a0Sick leave because of LBPa (among the working population), % yes34.841.0Characteristics of LBP\u00a0Duration current episode (days), median (IQR)11 (5\u201321)14 (7\u201321)\u00a0Frequency of LBP episodes last year, %\u00a0\u00a01 or 2 episodes58.060.8\u00a0\u00a03 or more episodes 19.618.7\u00a0\u00a0Exacerbation of persisting LBP22.420.5\u00a0Pain radiating below kneea, %12.814.6Clinical outcomes, primary measures\u00a0Functional disability (RDQ, 0\u201324), mean (SD)11.7 (5.4)12.2 (5.0)\u00a0Health related quality of life (EuroQol), mean (SD)0.61 (0.25)0.61 (0.24)Clinical outcomes, secondary measures \u00a0Pain severity during the day (0\u201310)b, mean (SD)4.9 (2.0)4.8 (2.0)\u00a0Severity of the main complaint (0\u201310)a, mean (SD)7.0 (1.9)6.8 (2.0)\u00a0Perceived general health (SF-36, 1\u20135)a, mean (SD)2.7 (0.8)2.8 (0.8)Clinical outcomes, psychosocial measures \u00a0Fear-avoidance beliefs (FABQ, 0\u201324), mean (SD)14.3 (5.6)15.3 (5.2)\u00a0Catastrophising thoughts (CSQ, 0\u201336), mean (SD)10.3 (6.6)11.2 (6.9)\u00a0Distress (4DSQ, 0\u201332)b, mean (SD)8.3 (7.0) 9.5 (7.3)GP General practitioner, MIS Minimal intervention strategy, UC Usual care, n number, SD Standard deviation, IQR Inter quartile range (25th\u201375th percentile), LBP Low back pain, RDQ Roland\u2013Morris disability Questionnaire, SF-36 Short-form 36, FABQ Fear avoidance and beliefs questionnaire, CSQ Coping strategies questionnaire, 4DSQ 4D symptom questionnaire (higher scores means more functional disability, worse health, more fear-avoidance, more catastrophising or more distress)aTwo missing valuesbOne missing value\nFor 116 patients (81%) in the MIS group and 134 patients (78%) in the UC group complete cost data were available. Logistic regression analysis comparing baseline characteristics of patients with complete cost data to those with incomplete cost data showed that patients with incomplete cost data on average were younger and scored higher on distress at baseline.\nClinical outcomes\nAfter 1\u00a0year follow-up both the groups showed similar improvements in clinical outcomes. Sixty-nine percent of the patients were defined as recovered after 1\u00a0year. The difference between both the groups in mean improvement on functional disability was \u22120.74 points on the RDQ (95% CI, \u22122.31 to 0.83), and \u22122% (95% CI, \u221214 to 10%) on recovery rate. Over the follow-up period of 1\u00a0year the mean difference in quality of life was 0.004 QALYs (95% CI, \u22120.04 to 0.03). All differences favored UC, but were neither clinically relevant nor statistically significant (Table\u00a05).\nCost data\nTable\u00a03 lists per patient the mean utilization of resources (i.e. health care, help, absenteeism). In both the groups, resource utilization was low and largely similar. Only two statistically significant differences were found. In the year following randomization patients in the MIS group had more consultations with a GP (MIS 2.7 vs. UC 0.9), excluding the consultation leading to recruitment but including the 20\u00a0min consultation aimed at psychosocial measures as specified by the study protocol. Patients in the UC group reported more consultations with a manual therapist (MIS 0.1 vs. UC 0.4) but the proportions of patients who received such a treatment (MIS 2.6% vs. UC 9%) were very low.\nTable\u00a03Mean resource use (SD) per patient (n\u00a0=\u00a0250) for MIS and UC during 12 months follow-up, and the percentage of patients who made use of that specific resourceResourcesMIS (n\u00a0=\u00a0116)UC (n\u00a0=\u00a0134)Direct health care \u00a0Primary care\u00a0\u00a0General practitioner (consultations)*2.7 (1.0)99.1%0.9 (1.2)44%\u00a0\u00a0Physical therapy (treatment sessions)2.3 (5.8)17.2%3.2 (5.8)35.1%\u00a0\u00a0Manual therapy (treatment sessions)*0.1 (0.7)2.6%0.4 (1.7)9%\u00a0\u00a0Exercise therapy (treatment sessions)0.3 (1.8)2.6%0.9 (3.5)9%\u00a0\u00a0Back school (treatment sessions)0.5 (0.6)0.9%0 (0)0%\u00a0\u00a0Chiropractor (treatment sessions)0 (0)0%0.1 (0.9)2.2%\u00a0\u00a0Physiofitness (treatment programs)0.1 (0.2)6%0.1 (0.2)4.5%\u00a0\u00a0Professional home care (hours)0 (0)0%0.6 (6.5)0.7%\u00a0\u00a0Psychologist (treatment sessions)0.1 (0.8)0.9%0 (0)0%\u00a0Secondary care\u00a0\u00a0Outpatient visit (number)0.1 (0.4)4.3%0.2 (0.7)7.5%\u00a0\u00a0Hospitalization (days)0.02 (0.2)0.9%0.01 (0.1)0.7%\u00a0\u00a0X-ray (number)0.1 (0.3)5.2%0.1 (0.4)9.7%\u00a0\u00a0MRI-scan (number)0.01 (0.1)0.9%0.03 (0.2)2.2%\u00a0\u00a0Medication (% yes)\u201374.1%\u201373.9%Direct non-health care\u00a0Complementary care\u00a0\u00a0Homeopathy (treatment sessions)0.04 (0.3)1.7%0 (0)0%\u00a0\u00a0Osteopathy (treatment sessions)0.1 (0.5)3.4%0.2 (0.3)0.7%\u00a0\u00a0Massage (treatment sessions)0.1 (0.8)1.7%0.4 (3.0)3.7%\u00a0\u00a0Acupuncture (treatment sessions)0 (0)0%0.04 (0.4)1.5%\u00a0\u00a0Magnetizer (treatment sessions)0.3 (2.3)1.7%0.1 (1.0)0.7%\u00a0Orthomanual therapy (treatment sessions)0.03 (0.3)1.7%0.03 (0.4)0.7%\u00a0\u00a0Podiatrist 0.03 (0.2)1.7%0.03 (0.3)1.5%\u00a0\u00a0Informal care (hours)0 (0)0%0.7 (6.3)3%\u00a0\u00a0Equipment aids (% yes)\u201310.3%\u20136.0%Indirect costs\u00a0\u00a0Absenteeism paid work (days)4.2 (12.1)28.4%8.6 (23.1)38.8%\u00a0\u00a0Absenteeism unpaid work (hours)2.2 (10.9)12.1%3.3 (10.4)17.2%*Significant difference (P\u00a0<\u00a00.05)SD Standard deviation, n number, MIS Minimal intervention strategy, UC Usual care\nTable\u00a04 shows the mean total costs in both the treatment groups and the difference in costs with 95% CI. Total indirect costs, especially absenteeism from paid work, were the largest contributor to the total costs. The difference in total costs amounted to 490 \u20ac (95% CI \u2212987 to 92 \u20ac) in favor of the MIS group (MIS 799 \u20ac; UC 1288 \u20ac), but this difference was not statistically significant.\nTable\u00a04Mean costs (SD) in Euros per patient in the MIS and UC group and differences between both the groups during follow-up of 52\u00a0weeksMIS (n\u00a0=\u00a0116)UC (n\u00a0=\u00a0134)Mean difference (95% CI)Direct health care costs181 (287)175 (275)6 (\u221265; 73)\u00a0Primary care costs159 (181)152 (242)7 (\u221252; 54)\u00a0Secondary care costs22 (179)23 (101)\u22121 (\u221244; 35)\u00a0Medication costs9 (18)9 (14)0 (\u22124; 4)Direct non-health care costs30 (75)31 (129)\u22121 (\u221225 ; 27)\u00a0Complementary care costs18 (65)21 (115)\u22122 (\u221224; 22)\u00a0Informal care6 (34)8 (58)\u22122 (\u221212; 11)\u00a0Equipment aids5 (22)2 (14)3 (\u22122; 6)Indirect costs587 (1,636)1,081 (2,463)\u2212495 (\u2212921; 158)\u00a0Absenteeism paid work569 (1,631)1,055 (2,448)\u2212486 (\u2212932; 50)\u00a0Absenteeism unpaid work18 (89)27 (84)\u22129 (\u221231; 18)Total costs798 (1,820)1,288 (2,594)\u2212490 (\u2212987; 92)None of the mean differences between MIS and UC are statistically significant (P\u00a0>\u00a00.05)SD Standard deviation, MIS Minimal intervention strategy, UC Usual care, n Number, 95% CI 95% confidence interval\nCost-effectiveness\nTable\u00a05 shows the incremental cost-effectiveness ratios (ICERs) for the three outcome measures. MIS resulted in less improvement than UC, but saved money. The ICER for functioning was 690 \u20ac, indicating that per point less improvement on the RDQ MIS saved 690 \u20ac, while per percent less improvement in recovery rate MIS saved 239 \u20ac. The difference in QALY\u2019s gained during 1\u00a0year between both the groups was very small, resulting in a large ICER of 47,348 \u20ac. The large majority of the bootstrapped ICERs presented on the cost-effectiveness planes are located in the southern quadrants (Fig.\u00a01), indicating that the costs of MIS were lower than the costs of UC.\nTable\u00a05Incremental cost-effectiveness ratios for functional disability, perceived recovery and health-related quality of lifeOutcome measureMISUCMIS versus UCCostsEffectsaCostsEffectsaICERFunctional disability (RDQ; 0\u201324) Change score between baseline and 1\u00a0year follow-up798 \u20ac (n\u00a0=\u00a0116)8.378 (n\u00a0=\u00a0116)1,307 \u20ac (n\u00a0=\u00a0132)9.116 (n\u00a0=\u00a0132)MIS results in less improvement, but saves 690 \u20ac per RDQ pointRecovery rate Score at 1\u00a0year follow-up 798 \u20ac (n\u00a0=\u00a0116)68.1% (n\u00a0=\u00a0116)1,288 \u20ac (n\u00a0=\u00a0134)70.1% (n\u00a0=\u00a0134)MIS results in less improvement, but saves 239 \u20ac per 1% Quality of life (EuroQol; QALY) Score over 1\u00a0year follow-up819 \u20ac (n\u00a0=\u00a0102)0.833 (n\u00a0=\u00a0102)1030 \u20ac (n\u00a0=\u00a0111)0.837 (n\u00a0=\u00a0111)MIS results in less improvement, but saves 47,348 \u20ac per QALY MIS Minimal intervention strategy, UC Usual care, ICER Incremental cost-effectiveness ratio, n Number, QALY Quality adjusted life yearaDifference in clinical outcomes between baseline and 12\u00a0months follow-upFig.\u00a01Cost-effectiveness plane for functional disability (RDQ) in which MIS is compared to UC\nSensitivity analyses\nImputation of missing cost data led to a mean difference of \u2212628 \u20ac (95% CI \u22121123 to \u221281 \u20ac) in total costs. Imputation of missing data on days of absenteeism led to a mean difference of \u2212545 \u20ac (95% CI \u22121031 to \u221240 \u20ac) in total costs. Both differences are statistically significant and in favor of MIS.\nDiscussion\nThe results of our primary analysis showed no statistically significant differences in total costs or clinical outcomes between our psychosocial intervention and UC in patients with (sub)acute LBP in general practice. However, the results of our sensitivity analyses are inconsistent with those of the primary analysis. In this discussion section we will focus on the interpretation of our cost data and the methodological issues involved when interpreting cost data. In previous papers we have discussed several methodological issues involved when interpreting the clinical outcomes (e.g. the quality of the training sessions and interventions) [20, 21].\nComparison to the literature\nA recent review on the cost-effectiveness of treatments for patients with LBP reported that 6 of the 17 studies concluded that the intervention of interest was more cost-effective than the control intervention [43]. Unfortunately, no definite conclusions for the cost-effectiveness of a specific intervention could be given as the number of economic evaluations per type of intervention was limited. More recently the cost-effectiveness analyses of two trials on psychosocial primary care interventions for LBP have been published. The UK BEAM Trial compared spinal manipulation, exercise classes incorporating cognitive behavioral principles, spinal manipulation plus exercise classes, and best care [40, 41]. Although the authors conclude that spinal manipulation is a cost-effective addition to \u201cbest care\u201d, as it generates 0.04 more QALYs for an extra 279 \u20ac [37], others question this conclusion [6, 39]. Another study in primary care compared advice directed towards promoting self-management and modifying beliefs and behavior, and routine physiotherapy treatment with advice only [14, 34]. The authors conclude that advice should be considered as the first-line treatment, as out-of-pocket expenses of patients receiving routine physiotherapy were significantly higher (41\u00a3, 59 \u20ac), while differences in either QALYs (0.02) or National Health Service costs (20\u00a3, 29 \u20ac) were not significantly different [34]. Both the above mentioned studies found lower QALYs and higher health care costs over 12 months than we found in our study. The shorter duration of the LBP episode at baseline of our patients and, thus, their more favorable prognosis may (partly) explain these results. In contrast to our study, both the trials did not include indirect costs in their cost-effectiveness analyses, even though it is generally agreed upon that indirect costs associated with work absence due to LBP account for high economic costs in western societies [28, 45].\nMethodological issues and secondary analyses\nIn our study, we used the British sample as reference population for the utilities as, until recently, these were the only reference data available in the Netherlands. Recently, a EuroQol transformation has been introduced based on a Dutch sample [24]. We decided not to use this Dutch transformation as (1) it has only been internally validated through bootstrapping methods but not yet externally validated; (2) the representativeness of the, rather small (n\u00a0=\u00a0298), Dutch sample can be debated [4] and, finally (3) use of the Dutch transformation would complicate comparison across international studies. A comparison between the use of a Dutch or UK sample showed that, although QALYs based on the Dutch sample were slightly higher compared to the British sample, the mean differences between the groups, in this case two primary care interventions for treatment of depression, were largely the same [2].\nWe based our sample size calculations on demonstrating a clinically relevant difference of 2.5 points on the RDQ instead of demonstrating the cost-effectiveness [20]. Although the usefulness of sample size calculations in economic evaluations has been debated [1], the required sample size in an appropriately powered cost-effectiveness study is expected to be much larger than in a clinical effectiveness trial [3].\nWork absence due to LBP was the main contributor to the total costs associated with LBP. The number of days of absenteeism was twice as high in the UC group as in the MIS group. Although not statistically significant, this finding induced us to explore whether MIS was cost-effective among patients having a paid job (n\u00a0=\u00a093 MIS; n\u00a0=\u00a0110 UC). This post-hoc subgroup analysis showed that the difference in total costs amounted to \u2212548 \u20ac (95% CI \u22121137 to 200 \u20ac) in favor of MIS, which was not statistically significant. As this subgroup analysis was clearly insufficiently powered, and as other early interventions have been shown to reduce sick-leave in workers [17, 19, 22] one may hypothesize that MIS may be cost-effective in an occupational setting. The sensitivity analyses with imputed data were less underpowered and, interestingly, showed statistically significant differences in total costs between groups. The question now arises which analysis we should value most.\nInterpretation of the conflicting results\nA complete case analysis has the advantage over an imputed analysis that no assumptions have to be made regarding imputation of data that are characterized by large variation and irregular distributions. However, a complete case analysis does not include all participating patients, which reduces the statistical power and which may introduce bias. Advocates of the (conclusions of our) complete case analysis may, among others, be (1) researchers who do not favor imputation techniques when data are available for 80% of the cases; (2) GPs, as application of MIS will increase their workload, with no evidence of improvement of the patients\u2019 functional disability, recovery rates, or pain intensity. Advocates of the (conclusions of our) sensitivity analysis may among others be (1) researchers who favor imputation techniques; (2) policy makers, who are interested in all relevant costs and effects regardless of who will pay the costs and who may benefit; or (3) managers in workplace settings as they may be especially interested in the reduction of costs due to sick-leave.\nThe cost-effectiveness planes do not provide a solution for the impasse. The plane for functional disability showed that 96% of the bootstrapped cost-effect pairs, although very close to the origin of the plane, were located in the southern quadrants. Does this indicate cost-effectiveness of MIS? The answer to this question will depend on the way one defines cost-effectiveness. Obviously, when a new intervention is clinically more effective and less costly than the old intervention (south\u2013east quadrant), there will be no debate about cost-effectiveness and implementation of the new intervention. When a new intervention is clinically more effective and more costly (north-east quadrant), a new intervention will be considered cost-effective when costs stay below a certain threshold. Dowie (2004) [8], however, questions why an intervention should not also be implemented when it is less effective but does save substantial costs, pleading for defining a threshold that extends into the south-west quadrant. This threshold can be a straight line from the north-east to the south-west quadrant, or kinked at the origin of the cost-effectiveness plane, indicating that the selling price for an unit of effect is greater than the buying price of an additional unit of effect [29].\nAlthough there are various points of view, we conclude that (Dutch) GPs should not replace their UC by MIS in patients with (sub)acute LBP. This conclusion is based on the results of the complete case analysis, because this was our pre-planned analysis. The second reason is that the difference in costs, if any, was mainly caused by a small proportion of the population who reported sick leave due to back pain. We cannot rule out the possibility that this was a chance finding. The third reason is that MIS is a new approach in general practice. Implementation of MIS will meet many difficulties, given the lack of evidence for clinical effects, the need for training of GPs and the increase of GP workload. The final, and maybe most important reason is that our study is the first and, as far as we know, the only study that investigated the cost-effectiveness of a psychosocial intervention in patients with (sub)acute LBP in general practice.\nConclusions\nResults of only one study are insufficient to establish firm evidence on the cost-effectiveness of an intervention. More studies on the cost-effectiveness of psychosocial interventions in general practice are needed, thereby taking into account some of the factors that may explain why our intervention was not more successful than usual GP care as described in previous papers [20, 21]. As yet, we conclude that (Dutch) general practitioners should not replace their usual care by our new intervention.","keyphrases":["low back pain","general practice","cost-effectiveness","psychosocial factors","minimal intervention strategy"],"prmu":["P","P","P","P","P"]}
{"id":"BMC_Bioinformatics-4-_-317283","title":"PubMatrix: a tool for multiplex literature mining\n","text":"Background Molecular experiments using multiplex strategies such as cDNA microarrays or proteomic approaches generate large datasets requiring biological interpretation. Text based data mining tools have recently been developed to query large biological datasets of this type of data. PubMatrix is a web-based tool that allows simple text based mining of the NCBI literature search service PubMed using any two lists of keywords terms, resulting in a frequency matrix of term co-occurrence.\nBackground\nWith the advent of high throughput genomic and proteomic approaches, the ability to generate data has outstripped the ability to assign biological relevance. Searching the MEDLINE literature database of greater than 14 million entries one-by-one makes establishing biological significance a daunting task. The basic PubMed search window contains a typical single search box for the input of simple keyword combinations. PubMed does allows complex searches using advanced search options, but this requires some knowledge of string search assembly, and an understanding of the PubMed Entrez programming utilities. These are still relatively obscure for many molecular biologists.\nLiterature mining approaches have been developed to place multiplex biological datasets into context relative to published medical literature. Computational tools such as PubGene [1], VxInsight [2], MedMiner [3], EASE [4], MeshMap[5], XPLORMED [11], AbXtract [12], and HAPI [13] are available which allow the user to query more complex gene name or keyword combinations, including multiplex proteomic or cDNA microarray results, versus literature citations in PubMed with defined types of output. These computational tools use different strategies in literature mining and in some cases produce statistical significance or graphical displays.\nSimilarly, literature based annotation projects such as the GO project [9] have devised a complex hierarchical annotation system using expert annotators based on a defined controlled vocabulary. This is important due to significant problems with nomenclature in molecular biology. Although quite useful, the GO approach does not allow for flexibility, variable interpretation, or individual investigator input in the hierarchies assigned to individual genes. In this report, we describe a simple, freely accessible, web based application for basic literature mining of PubMed that performs automatic multiplex Boolean queries. This allows the naive user to assign biological relevance and annotate gene lists through PubMed.\nImplementation\nPubMatrix is a CGI front-end application, which submits queries consisting of search and modifier terms against NCBI's PubMed database and presents the results as a matrix of document hits. Results are stored in a database for retrieval and are presented as hyperlinks to the user for rerunning individual queries of interest. The application runs on an Apache http server using the PERL programming language and a MySQL database for storing terms and results.\nResults\nPubMatrix is a simple intuitive multiplex comparison tool requiring no user understanding of algorithm design, perl, or scripting, and minimal instruction time. It allows automatic systematic searching of PubMed followed by a quick intuitive survey of results, and as such, dramatically reduces the investigator time needed to query PubMed as compared to a gene-by-gene approach. Moreover, it is systematic and objective. Unlike typical PubMed searches performed on an ad hoc basis, the 1st and the 1000th query are searched in an unbiased manner. This helps avoid becoming distracted with seemingly interesting comparisons early on in a traditional literature search session. It is flexible in the sense that any list of terms in almost any combination may be used. With PubMatrix, large lists of keywords can be compared and, when used with lists of gene names and function, may be used to analyze and annotate microarray gene lists and datasets. GO keywords may be used to annotate gene lists in a semi-automated fashion. Additionally, the ability to save searches in an archive allows sharing among collaborators and public accessibility of curated lists of useful search results and search terms.\nFigure 1 shows the web interface for PubMatrix. Data entry begins with creating two lists of keyword terms, (figure 1) SEARCH TERMS and MODIFIER TERMS. These can be any simple words or strings of words. Typically official gene symbols (TP53, CTLA4, etc.) can be used for microarray or proteomic data. These lists can be pasted into the appropriate data entry boxes on the page or loaded as a text file through the browser. Essentially any search combination that can be loaded into the basic PubMed search window can be run in PubMatrix in matrix file format. This gives a broad search capability as compared to PubGene [1], which is limited to gene identifiers only. Terms in either list can contain PubMed field limits (i.e. author, title, abstract, journal, publication date, etc.) subject to PubMed\/Entrez formatting specifications [10]. Compound terms in either list can be used as well. Like PubMed, success in using PubMatrix is dependent upon the quality of the input terms. If terms are general, you may end up with general results, if they are too specific, you may limit the search inappropriately. As in PubMed, PubMatrix makes no provision for nomenclature curation and provides no measure of statistical significance. General terms may be appropriate for some searches while specific terms may be useful for others. If the terms used are inappropriate or non-specific, the search will return inappropriate and low quality results; quisquiliarum ineo quisquiliarum egredior.\nFigure 1\nThe PubMatrix Data Input Page \nThere is currently a 100 SEARCH TERM and 10 MODIFIER TERM limit on the search capability due to NLM automatic script searching restrictions. The size and speed of PubMatrix is largely determined by NLM restrictions. In unrestricted tests, PubMatrix rapidly returned results using term lists of over 10,000 terms. The size limit of 100 \u00d7 10 will generally accommodate a gene list from a published summary table of microarray results or genes from a cluster produced from a typical clustering program. After data entry, the PubMatrix program then performs pairwise comparisons of each SEARCH term against each MODIFIER term using the Boolean operator \"AND\", capturing only the pairwise frequency counts found in PubMed. It does not capture the actual PubMed record. Relative frequencies are then presented in an html matrix table (Fig 2) of control terms versus search terms. This table can then be surveyed or sorted for positive hits. Clicking on a single frequency number in the matrix table then performs a de-novo individual query for that single pairwise combination in PubMed. In this way the user can drill down or focus on specific interesting combinations.\nFigure 2\nThe report page from a simple search of gene names versus neural modifier terms. All reported numbers are hyperlinked and will initiate a de-novo search for that specific term combination.\nProductive searches using broadly useful control term lists have been assembled into an associated PubMatrix public archive. These previously used search term lists may be commonly useful and may aid other investigators in subsequent searches. Examples include, official gene symbols, functional descriptors, cytogenetic bands, or polymorphic markers. Likewise, non-molecular term lists have been used and archived which may be broadly useful, such as commonly prescribed drugs, common diseases, and meeting speakers, among others. For example, prior to a meeting, an attendee can rapidly search all the meeting speakers names against important keywords found in the meeting program to provide a one-page summary of speakers scientific interests. Archived term lists can be copied to initiate new searches with new modifier term combinations. Table 1 gives examples of categorical lists of terms used in PubMatrix. Searches using these term lists can be found in the PubMatrix: Public Results page.\nTable 1\nExamples of categorical search lists\nCategory\nExamples\nOfficial Gene Symbols\nAPOB, ACE, BDNF, CD45, ...\nPolymorphic markers\nD1S478, D6S470, D13S193, ...\nDNA sites\nAAATTT, CAGCAG, TTTTTT, ...\nChromosomal bands\n1ter*, 1p36*, 1p35*....Xq27*, Xter*\nCountries\nsweden, canad*, mexic*, finland, ...\nCommon Prescription drugs\nacetaminophen, acyclovir, albuterol, alprazolam, ...\nCommon diseases\natopic dermatitis, asthma, crohn's, Celiac, Graves',...\nDate of Publication\n1973 [dp], 1974 [dp]......2000 [dp], 2001 [dp], 2002 [dp]\nMeeting Speakers\nWeiss A, Pierce SK, Kupfer A,...\nPubMatrix tables can be combined or processed further to provide visual displays or summaries of complete experiments or large datasets in the context of medical literature. Figure 3 shows a display of cDNA microarray results of an ovarian cancer cell line treated with the drug cisplatin. The literature results are displayed in the context of gene expression ratios (Z-ratios). Similarly, Figure 4 shows a comprehensive term list of human cytogeneic bands covering the human genome (1p36*, 1p35*, 1p34*...Xter*) versus the single term \"Autoimmune\". When displayed by chromosome, major autoimmune loci are identified. These include the major histocompatibility complex, chromosome 2q (CTLA4), as well as APECED on 21q. Figures 3 and 4 were displayed in MS EXCEL using the 3-D chart view option. Moreover, PubMatrix tables can be pasted into spreadsheet programs and imported into other types of data visualization programs such as CLUSTER\/TREEVIEW [11] or SPOTFIRE [12] for graphic display and manipulation.\nFigure 3\nGene expression results (Z-ratio) of cisplatin treatment of an ovarian tumor cell line versus keywords relevant to cisplatin resistance. This graph was constructed in MS EXCEL directly from a PubMatrix search result table using the 3D chart view option after adding gene expression values (Z-ratios).\nFigure 4\nVisual display of chromosomal-band term list versus the term. \"autoimmune\". Search terms were 313 sequential human chromosomal bands (1pter, 1p36, 1p35, 1p34, etc....Xq26, Xq27, Xq28, Xqter) versus the single modifier term \"autoimmune\". This graph was constructed in MS EXCEL using 3D chart view option after separating individual chromosome results into individual columns.\nConclusions\nPubMatrix allows a simple systematic approach to query the medical literature in PubMed with comparative keyword lists. It performs simple automatic queries and greatly reduces analysis time. In this way, increasingly large datasets generated by high-throughput multiplex assays such as proteomic or microarray assays can be mined, archived, displayed, and annotated for biological and disease relevance.\nAvailability and requirements\nPubMatrix is available for free use at this URL:\nAuthors' contributions\nKGB and TJB conceived the approach and participated in early design and testing. DAH, GD, RAL, and CC participated in software design and testing. JE participated in web design, database development, and algorithm modification. All authors read and approved the final manuscript","keyphrases":["pubmed","literature mining","microarray","proteomic"],"prmu":["P","P","P","P"]}
{"id":"Matern_Child_Health_J-2-2-1592142","title":"Trends in Folic Acid Awareness and Behavior in the United States: The Gallup Organization for the March of Dimes Foundation Surveys, 1995\u20132005\n","text":"Objective: To summarize changes in folic acid awareness, knowledge, and behavior among women of childbearing age in the United States since the U.S. Public Health Service (USPHS) 1992 folic acid recommendation and later fortification. Methods: Random-digit dialed telephone surveys were conducted of approximately 2000 women (per survey year) aged 18\u201345 years from 1995\u20132005 in the United States. Results: The percentage of women reporting having heard or read about folic acid steadily increased from 52% in 1995 to 84% in 2005. Of all women surveyed in 2005, 19% knew folic acid prevented birth defects, an increase from 4% in 1995. The proportion of women who reported learning about folic acid from health care providers increased from 13% in 1995 to 26% in 2005. The proportion of all women who reported taking a vitamin supplement containing folic acid increased slightly from 28% in 1995 to 33% in 2005. Among women who were not pregnant at the time of the survey in 2005, 31% reported taking a vitamin containing folic acid daily compared with 25% in 1995. Conclusions: The percentage of women taking folic acid daily has increased modestly since 1995. Despite this increase, the data show that the majority of women of childbearing age still do not take a vitamin containing folic acid daily. Health care providers and maternal child health professionals must continue to promote preconceptional health among all women of childbearing age, and encourage them to take a vitamin containing folic acid daily.\nIntroduction\nSpina bifida and anencephaly \u2013 the most common neural tube defects (NTDs) \u2013 are serious birth defects that occur early in pregnancy, often before a woman realizes she is pregnant. Important studies show that intake of the B vitamin folic acid reduces the incidence of NTDs by 50\u201370% when taken before conception and during the first trimester of pregnancy [1, 2]. This compelling research prompted the 1992 U.S. Public Health Service (USPHS) recommendation that all women of childbearing age who are capable of becoming pregnant consume 400 micrograms (mcg) of folic acid daily [3]. For women who previously had an infant with an NTD and were planning a pregnancy, it was recommended that they consume a much larger dose, 4,000 mcg of folic acid per day at least one month before becoming pregnant and during the first three months of pregnancy [3].\nIn 1998, the 1992 USPHS recommendation was reaffirmed by the Institute of Medicine, which stated that women capable of becoming pregnant should take 400 micrograms of \u201csynthetic folic acid\u201d daily from fortified foods or supplements or a combination of the two, in addition to consuming natural folate from a varied diet [4]. Additionally, in 1998 the Food and Drug Administration (FDA) began requiring folic acid fortification of enriched breads, cereals, flours, pastas, rice, and other grain products [5], to aid in increasing folic acid consumption by an estimated average of 100 mcg daily in women of childbearing age. Other organizations, such as the American Academy of Pediatrics and the American College of Obstetricians and Gynecologists, have supported the USPHS recommendation [6, 7]. National and state organizations implemented educational and media campaigns during the mid-1990s to promote the awareness and consumption of folic acid among women of childbearing age [8, 9]. Since the implementation of national policies, the number of pregnancies affected by NTDs in the U.S has decreased approximately 26% \u2013 from 4,000 affected pregnancies prior to fortification to 3,000 affected pregnancies after fortification [10]. Despite these efforts, the 50 percent reduction anticipated by the USPHS in 1992 has not been forthcoming. This report summarizes results from nine surveys conducted between 1995\u20132005 after the USPHS 1992 recommendation and folic acid fortification. It documents the steady increase in awareness and knowledge of folic acid \u2013 and the as yet modest change in consumption of a vitamin containing folic acid \u2013 among U.S. women of childbearing age.[11].Table 1Awareness, knowledge, behavior, and source of knowledge regarding folic acid among women of childbearing age \u2013 United States, 1995\u20132005bCharacteristic199519971998200020012002200320042005Awareness\u2003Heard or read about folic acid526668757980797784Knowledgea\u2003Knew folic acid can help prevent birth defects41013141920212419\u2003Knew folic acid should be taken before pregnancy2671071010127Behavior\u2003Take folic acid daily (all women)283232342933324033\u2003Take folic acid daily (non-pregnant women)253029322731303731Source of knowledgea\u2003Health-care provider131519202225302926\u2003Magazine\/Newspaper353631302727252826\u2003Radio\/Television102223242525211918Good sources of folic acida\u2003Green leafy vegetables\u2013\u2013\u2013242327282526\u2003Fortified cereals\u2013\u2013\u2013244335Source: March of Dimes Birth Defects Foundation. From 1995\u20132004 the margin of error for estimates is \u00b13%. In 2005, the margin of error for estimates was \u00b12%.Note:\u2212=Not available.aOf women who report being aware of folic acidbStatistical estimates were weighted to reflect the total population of women aged 18\u201345 years in the contiguous United States who resided in households with telephones\nMethods\nThe data for this study include a national sample of approximately 2000 women (per survey year) aged 18\u201345 years from 1995\u20132005. The March of Dimes Birth Defects Foundation contracted the Gallup Organization to conduct random-digit-dialed telephone surveys. Since 1995, response rates have ranged from 24% to 52%. The response rate for the 2005 survey was 32% (2,647 women). Statistical estimates were weighted based on Current Population Survey (CPS) data to reflect the total population of women aged 18\u201345 years in the contiguous United States who resided in households with telephones. In 2005, the sampling design included an oversampling of women ages 18\u201325. In 1995\u20132004, the margin of error for estimates based on the total sample size was \u00b13% and in 2005 it was \u00b12% . The questionnaire and methods used were described previously and were the same in all survey years [12].\nResults\nTable\u00a01 displays the percentage of women reporting awareness of and knowledge about folic acid, and their daily use of vitamins containing folic acid. Between 1995 and 2001, the percentage of women reporting having heard or read about folic acid increased from 52% in 1995 to 79% in 2001. Since 2001, awareness of folic acid has remained relatively constant at about 80%. Of all women surveyed in 2005, 19% knew that folic acid prevented birth defects, compared to 4% in 1995. Also in 2005, 7% of women knew that folic acid should be taken before pregnancy, compared to 2% in 1995. The proportion of all surveyed women who reported taking a vitamin supplement containing folic acid daily has remained almost unchanged but increased gradually from 28% in 1995 to 40% in 2004, but decreased to 33% in 2005. Of women surveyed in 2005 who were aware of folic acid, 26% identified green leafy vegetables as good sources of folic acid. However, in 2000, fortified cereals were added to the list of possible sources, and only 2\u20135% of women identified fortified cereals as a good source of folic acid.\nTable\u00a01 also displays women's reported sources of information about folic acid. From 1995 to 2005, the proportion of women who reported learning about folic acid from health care providers showed a two-fold increase from 13% in 1995 to 26% in 2005. Of women surveyed in 2005, 31% reported that their health care provider discussed the benefits of folic acid with them, compared to 24% in 2001. Of the same group of women in 2005, 30% reported being told by their health care provider that folic acid prevented birth defects. However, only 7% of women in 2005 reported that their health care provider said folic acid needs to be taken before pregnancy. This statistic has ranged from 3% in 2004 to 8% in 2003 since its inclusion in the survey in 2002.Table 2Percentage of women of childbearing age 18\u201345 who reported awareness of folic acid by selected sociodemographic characteristics \u2013 United States, 2000\u20132005aCharacteristic200020012002200320042005Race\/ethnicity\u2003White788282838187\u2003Non-White626971686871\u2003Hispanic626969727173\u2003Non-Hispanic768081807984Age group (yrs)\u200318\u201324687370737072\u200325\u201334768284828088\u200335\u201345778081818087Education\u2003Less than high school546059565658\u2003High school\/Trade\/Technical\/ Vocational Training667273767078\u2003College (any)838687868690Annual household income\u2003< $25,000617168696672\u2003$25,000\u2013$39,999767776797984\u2003$40,000\u2013$49,999807784878287\u2003>=$50,000848888868790Source: March of Dimes Birth Defects Foundation. From 1995\u20132004 the margin of error for estimates is \u00b13%. In 2005, the margin of error for estimates was \u00b12%.aStatistical estimates were weighted to reflect the total population of women aged 18\u201345 years in the contiguous United States who resided in households with telephones.\nFor the last ten years, the percentage of women learning about folic acid from media sources such as radio and television increased from 10% in 1995 to 18% in 2005, whereas information about folic acid obtained from magazines or newspapers decreased from 35% in 1995 to 26% in 2005. These results are shown in Table\u00a01.\nTable\u00a02 displays the percentage of women of childbearing age 18\u201345 who reported awareness of folic acid by selected sociodemographic characteristics. In 2005 the women least likely to be aware of folic acid were non-white women (71%), women aged 18\u201324 years (72%), those women with less than a high school education (58%), and those with a household income of less than $25,000 (72%). These results are consistent with years prior to 2005.\nTable\u00a03 displays the percentage of women of childbearing age 18\u201345 who reported taking folic acid daily, by selected sociodemographic characteristics. Of all women surveyed in 2005, those least likely to consume a vitamin containing folic acid daily include non-white women (23%), those women aged 18\u201324 (24%), those with less than a high school education (20%), and those with household incomes of $25,000 or less (27%). Among women who were not pregnant at the time of the survey in 2005, 31% reported taking a vitamin containing folic acid daily compared with 25% in 1995. Among women who were surveyed in 2005 who were currently pregnant, 90% reported taking a vitamin containing folic acid daily compared to 79% in 1997.Table 3Percentage of women of childbearing age 18\u201345 who reported taking folic acid daily, by selected sociodemographic characteristics \u2013 United States, 1995\u20132005aCharacteristic199519971998200020012002200320042005Race\/ethnicity\u2003White\u2013\u201333363036344336\u2003Non-White\u2013\u201326262325283123\u2003Hispanic\u2013\u201329362326293827\u2003Non-Hispanic\u2013\u201332342934334034Age group (yrs)\u200318\u201324212323292222253124\u200325\u201334303634343237343936\u200335\u201345303534363036354637Education\u2003Less than high school222020182621211920\u2003High school\/Trade\/Technical \/Vocational Training263027312429283231\u2003College (any)303736383238374836Annual household income\u2003< $25,000202228262223243027\u2003$25,000\u2013$39,999313229342634314028\u2003$40,000\u2013$49,999323431372833394837\u2003>=$50,000334338403540384638Pregnancy status\u2003Currently pregnant\u20137979848074828190\u2003Not currently pregnant253029322731303731Source: March of Dimes Birth Defects Foundation. From 1995\u20132004 the margin of error for estimates is \u00b13%. In 2005, the margin of error for estimates was \u00b12%.Note: -=Not availableaStatistical estimates were weighted to reflect the total population of women aged 18\u201345 years in the contiguous United States who resided in households with telephones\nIn 2005, 67% of childbearing age women reported they did not take daily supplements. The most common reasons for not taking any daily vitamin or mineral supplements are that they \u201cforget to take them\u201d, stated by 28% of women, or \u201cdon't feel they need them\u201d as reported by 16% of women. However, in 2005, 86% of women who were not currently taking daily vitamins or mineral supplements reported they would be likely to start taking a multivitamin daily if advised by their health care provider or physician.\nDiscussion\nThe findings in this study show that whereas a large percentage of women have heard of folic acid, only a small proportion are aware that folic acid prevents birth defects and should be taken before pregnancy. Furthermore, despite the USPHS recommendation in 1992 [3] the percentage of women taking a vitamin supplement containing folic acid daily has only modestly increased between 1995 and 2005. There was a substantive increase in the percentage of women taking folic acid daily in 2004, but this increase was not sustained in 2005. Clearly, the observed increases in awareness and knowledge about folic acid are not translating into increased multivitamin use.\nIncreased awareness of the importance of folic acid is due in part to educational outreach activities undertaken over the years by CDC, the March of Dimes Birth Defects Foundation and its chapters, the Spina Bifida Association of America, and members of the National Council on Folic Acid. These activities culminated in a national meeting in January 1999 to coordinate a folic acid promotional campaign (May through September 1999) with continuing independent educational outreach efforts [8]. Although there were no substantial changes in folic acid use, recent dietary habits might provide insight into the sudden increase in folic acid use in 2004. A recent study found that women on low-carbohydrate diets were 50% more likely to take folic acid daily than women on other diets; however, in 2005, folic acid use was similar across dieting types [13]. The results from the 2004 survey may be an anomalous finding, given that many of the findings returned to pre-2004 levels (e.g., multivitamin use). Our findings may suggest the need for more continued and increased media messages to encourage women to consume adequate amounts of folic acid every day through fortified foods, dietary supplements, and a folate-rich diet. Furthermore, communities should identify food products that provide recommended levels of folic acid available in local grocery stores, and encourage women to select diets with sufficient folic acid.\nRecent educational efforts alone do not appear to be adequate to demonstrate an impact on changing behavior with regard to folic acid consumption. The findings from this report can be used to develop multi-pronged targeted public health programs to increase the number of women of childbearing age consuming a vitamin containing folic acid daily. Demographic, sociocultural, and environmental factors impact behavior and more attention must be given to these factors. Results from these surveys indicate that variations exist in folic acid awareness, knowledge, and use among women of different race\/ethnicity, age groups, education levels, and socioeconomic status. This suggests that the linkage between folic acid awareness, knowledge, and use is more complicated than simply increasing awareness and knowledge. Non-white women, those between the ages of 18\u201324, those less educated, and those of a lower socioeconomic status are associated with both lower folic acid knowledge and lower use of a vitamin containing folic acid. Targeted messages and teachable opportunities of communication for younger women, women of racial and ethnic groups, and women of low socioeconomic status need to be identified and mobilized to increase the number of childbearing age women taking folic acid daily, regardless of pregnancy plans.\nOur results indicated that despite the fact that some women learn about folic acid from their health care provider, most women are not currently following their health care provider's advice even though they might feel the recommendation is important. This is apparent when looking at the proportion of women taking a multivitamin daily. In addition, women report they do not take a vitamin containing folic acid because they \u201cfeel they don't need to\u201d, or are not receiving the folic acid message from their health provider. More research is needed to understand what the motivators and barriers are for consuming a multivitamin containing folic acid daily to design appropriate interventions (whether service delivery, policy, media messages, or educational in nature) for women of childbearing age. It is important to continue to target educational efforts at health care providers with emphasis on the importance of women of childbearing age taking folic acid daily to prevent NTDs. But, education of health care providers may not be enough by itself. Changing health care provider practices is not merely a matter of knowledge acquisition. Public health programs need to forge stronger alliances with the health care delivery systems at the local and national level to develop and incorporate meaningful folic acid messages into routine counseling and existing health programs and services, since preconceptional counseling is not available to all women of childbearing age. Strategies are needed to understand the context of patient and provider interaction to promote consumption of a daily vitamin containing folic acid among all women of childbearing age, regardless of whether or not they are planning a pregnancy.\nThe findings from this survey are subject to several important limitations. First, because women under the age of 18 were not included in the survey, the full extent of folic acid knowledge and consumption among the entire population of women of childbearing age is unknown. Second, women without telephones were excluded; therefore, data may underestimate the number of women of childbearing age from low socioeconomic groups. Third, folic acid knowledge and consumption of nonparticipants might have been different from those of participants: participating women were more highly educated than the total U.S. population. The prevalence of consuming vitamin supplements might have been higher among these women than among U.S. women in general because vitamin consumption is positively correlated with education [11, 12]. Fourthly, folic acid use may have been under ascertained since we did not have information on women's dietary folate consumption or their folate levels. Further, self-report of dietary intake, such as taking multivitamins, could be biased by social desirability \u2013 the tendency of respondents to respond in a way so as to avoid disapproval, rather than reporting actual beliefs or actions. Finally, surveys of a more representative sample of women of childbearing age in the United States are needed to obtain more precise estimates of folic acid use among this population.\nPolicy and practice implications\nFortification efforts are an effective method of reducing the number of NTD-affected pregnancies, and have led to a 26% decline in the number of NTD-affected pregnancies since the 1998 mandate that enriched cereal grain flours be fortified with 140 mcg per 100 gram (g) [10]. Although this is a substantial decline, it does not reach the 50% reduction estimated by the USPHS in 1992 [1]. While the U.S. is seeing a decline in the number of NTDs, other countries have seen similar or greater declines. In Chile for example, wheat flour was fortified in 2000 with 220 mcg folic acid per gram of the pre-mix already used in wheat flour. Nine public hospitals in Santiago, Chile, which account for approximately 25% of the births in that country, reported a 40% decline in the number of NTDs [14]. Canada's flour was fortified with folic acid in 1998 with 150 mcg of folic acid per 100 g of grain and certain provinces reported almost a 50% decline of NTDs[15].\nSome countries have chosen to recommend the use of folic acid supplementation for women of childbearing age instead of fortification. One study examined data from thirteen birth defect registries from areas in Europe and Israel with over 8,000 cases of spina bifida and anencephaly to determine the impact of recommendations on the rate of NTDs [16]. Although some countries saw a slight decline in rates, the majority of countries saw no detectable decrease in the rates of NTDs using recommendations for supplementation alone [16]. This small decline implies that although recommendations might be an important aspect of reducing the rate of NTDs, programs such as fortification could have a broader and more effective impact on the overall rates of NTDs. Countries who try to use a combination of methods \u2013 such as fortification and supplementation, along with recommendations and education programs \u2013 might see a greater decline in the rates of NTDs.\nConclusions\nThe Healthy People 2010 objectives call for an increase to 80% in the number of nonpregnant women aged 15\u201344 years consuming daily amounts of 400 mcg of folic acid through fortified foods or dietary supplements (Objective 16\u201316a) and to increase the median red blood cell (RBC) folate level to 220 ng\/ml among nonpregnant women aged 15 to 44 years by 2010 (Objective 16\u201316b) [17]. Although substantial increases in RBC folate levels among women of childbearing age has been achieved [18], more aggressive efforts are needed to increase the number of women consuming 400 mcg of folic acid daily through fortified food or dietary supplement to reduce the prevalence of NTDs.\nTo succeed in eliminating all folic acid preventable birth defects beyond what fortification has already achieved, more must be done besides educating women and their health care providers. Pregnancies and births affected by spina bifida or anencephaly have profound physical, emotional, and financial effects on families and communities. This year there will be an estimated 3,000 NTD-affected pregnancies. This study reinforces the need for public health practitioners to mobilize multiple sectors, including health care professionals, government organizations, state and local organizations, advocacy groups, and concerned citizens to coordinate efforts and activities to help alleviate a devastating public health issue.","keyphrases":["folic acid consumption","childbearing age women","multivitamin use"],"prmu":["P","P","P"]}
{"id":"Exp_Brain_Res-4-1-2214825","title":"The effect of the \u201crod-and-frame\u201d illusion on grip planning in a sequential object manipulation task\n","text":"We investigated the effect of visual context (i.e., a visual illusion) on the planning of a sequential object manipulation task. Participants (n = 13) had to grasp a rod embedded in a \u201crod-and-frame\u201d illusion and insert the rod-end into a tight hole in a pre-defined way. The grip type (defined by start posture, either pronated or supinated; and end posture, either comfortable or uncomfortable) used to grasp the rod was registered as a macroscopic variable of motor planning. Different rod orientations forced the participants to switch between grip types. As expected, most participants switched between pronated and supinated start postures, such that they ended the movement with a comfortable end posture. As it has been argued that planning is dependent on visual context information, we hypothesized that the visual illusion would affect the specific rod orientation at which participants would switch into a different grip type. This hypothesis was confirmed. More specifically, the illusion affected the critical spatial information that is used for action planning. Collectively, these findings are the first to show an effect of an illusion on motor planning in a sequential object manipulation task.\nIntroduction\nAt present, there is a lively debate about the effects of visual illusions on the planning and control of discrete grasping actions (for reviews, see Glover 2004; Carey 2001). An influential model in this respect is the perception-action model of Milner and Goodale (1995; Goodale and Milner 1992, 2004). The perception-action model posits a dissociation between two functionally and structurally different visual pathways in the brain: a ventral stream processing \u201cvision for perception\u201d (the \u201cwhat-system\u201d) and a dorsal stream processing \u201cvision for action\u201d (the \u201chow-system\u201d). The ventral pathway processes information used for the conscious recognition and identification of objects. The dorsal pathway, however, has the purpose to guide goal directed actions, and is therefore dependent on information about the spatial properties and coordinates of an object with respect to the actor. Since these different pathways depend on different sources of visual information (i.e., context dependent information for the ventral stream and context independent information for the dorsal stream), the perception-action model predicts that a visual illusion will affect perception, but not action, a prediction for which ample evidence exists (e.g., Aglioti et al. 1995; Haffenden and Goodale 1998; Dyde and Milner 2002).\nRecently, Glover proposed the planning-control model (Glover 2002; Glover and Dixon 2001a, 2001b, 2002), in which a dissociation between visual representations that subserve planning and those that are used for on-line control of action is postulated. This model posits that representations responsible for planning entail a broad range of current visual and cognitive information about (1) spatial (e.g., size, shape, orientation) and non-spatial (e.g., function, weight, fragility) properties of the target object, (2) the overarching goal of the action, and (3) the visual context surrounding the target. This information is integrated with knowledge from past experience (Glover 2004, p. 4). Representations responsible for on-line control, however, are solely aimed at minimizing the spatial error of the movement and are focused on the spatial characteristics of the target object. Support for the planning-control model has been found in experiments investigating the effects of a visual illusion on action. As an example, Glover and Dixon (2001b) had participants grasp a bar placed in front of a grated background in such a way that an orientation illusion was induced. The bar could be grasped with an overhand grip or an underhand grip. The results showed that grip choice was affected by the visual background. Hence, these findings indicate that the selection, or planning, of a particular grip type is subject to the visual context surrounding the target. In a second experiment, using the same experimental set-up, Glover and Dixon investigated planning and control by measuring kinematic parameters during transport of the hand to the bar. Contrary to the first experiment, participants were not free in their grip choice, and had to use the same, predefined grip during the whole session. In line with the predictions of the planning-control model, hand orientation was affected by the illusion in the initial part of the movement, but this effect decreased when the hand approached the bar. Thus, initial planning, as evidenced by grip type and initial kinematic parameters of the reach, was affected by the illusion, but during transport of the arm the kinematic parameterization was corrected.\nAlthough studies that investigated the impact of the visual context surrounding the target object on the planning and control of action are abundant (e.g, Aglioti et al. 1995; Jackson and Shaw 2000; Danckert et al. 2002; Franz 2001; Franz et al. 2005; Mendoza et al. 2006; Van Doorn et al. 2007), the evidence is restricted to simple prehension tasks that did not demand any further action with the grasped object: the goal of the action was to merely to grasp and lift the object. No studies have yet investigated the effect of visual context in situations where the overarching goal of the action does not coincide with simply grasping and lifting the object. This is surprising, since tasks such as a sequential object manipulation task, in which a target is grasped for a specific purpose, provide a method \u2018par excellence\u2019 to evaluate planning processes. By using a sequential object manipulation task, it can be assessed whether planning processes also take the visual context surrounding of an object into account when the overarching goal of the action requires further manipulation of the grasped object.\nA distinctive feature of sequential object manipulation tasks is that they require anticipatory planning, i.e., the forthcoming perceptual-motor demands associated with the goal of the action sequence need to be taken into account when initially grasping an object (cf., Johnson-Frey et al. 2004). In other words, the type of grasp is not only determined by the characteristics of the target object and its visual surrounding, but must also accommodate the ensuing task requirements. Ample evidence for such anticipatory planning can be found in the studies of Rosenbaum and co-workers. They showed that the selection of a particular grip type is indicative for motor planning. Participants preferred to grasp an object with a grip type that enabled them to end the task in a comfortable posture, the so-called \u2018end-state comfort\u2019 effect (Rosenbaum and Jorgensen 1992; Rosenbaum et al. 1992, 1993, 1996; Cohen and Rosenbaum 2004; Short and Cauraugh 1999). Stated differently, participants sacrifice initial comfort for the sake of final comfort, implying anticipatory motor planning. For example, in the Rosenbaum and Jorgensen study (1992) participants had to grasp and rotate a bar that was mounted on a clock-face. Positions on the clock-face were separated by 45\u00b0, and participants were instructed to take hold of the bar and rotate it to a pre-defined target position. The results showed that participants adapted their initial posture, such that it enabled them to end the task in a comfortable posture. Obviously, to attain this comfortable end posture, they switched between overhand and underhand grip types when grasping the bar (for similar findings from a different theoretical background, see Kelso et al. 1994). Summing up, the studies of Rosenbaum et al. showed that initial grip type is indicative for motor planning in a sequential object manipulation task, and second, they showed that initial comfort is sacrificed to attain posture comfort at the end of the task. Additionally, previous studies have shown similar effects for kinematic parameterization. For example, Marteniuk et al. (1987, see also Gentilucci et al. 1997) showed that the goal of the second movement was reflected in movement kinematics of the first movement. Likewise, Steenbergen et al. (1995) showed that the strength of joint couplings of the first movement was critically dependent on the task constraints of the second movement.\nAt present, no study has scrutinized the effects of visual context on movement planning in a sequential task that requires anticipatory planning. Earlier studies on the effects of visual context on the planning of action used simple grasping movements. For instance, in the experiments of Glover and co-workers the action goal was to grasp the bar, no further manipulation was required. Hence, the posture with which the bar was grasped was identical to the posture at the end of the task. Consequently, no conflict occurred between comfort of the initial posture and comfort of the end posture. In a sequential task however, the initial posture with which an object is grasped and the posture at the end of the task are not necessarily the same because additional movements are made after grasping the object. Therefore, when planning the initial posture in a sequential object manipulation task, participants have to take the constraints arising from the end posture into account as well, i.e., they are engaged in anticipatory planning (Johnson-Frey et al. 2004).\nIn the present study, the effect of visual context on anticipatory planning of a sequential object manipulation task was investigated. Specifically, participants had to grasp a rod embedded in a \u201crod-and-frame\u201d illusion (i.e., a rod surrounded by a tilted frame) and subsequently place the rod-end vertically in a tight hole. Both, rod and frame could be independently rotated. Following Rosenbaum et al. (1992) we expected adaptations in the initial posture such that a comfortable end posture is reached. Specifically, participants are expected to switch between pronated and supinated initial postures at a specific rod orientation. Based on the planning-control model (Glover 2002, 2004), in which context effects are not distinguished with respect to the different components of planning, it is hypothesized that the exact rod orientation at which this switch occurs is affected by the rotation of the frame.\nMethod\nParticipants\nThirteen right-handed college students (three males, ten females), aged 18\u201327\u00a0years (mean age 22.6\u00a0year\/month, SD 2.10\u00a0year\/month) participated in the experiment for money or course credit (see Table\u00a01 for participant information). All participants had normal or corrected-to-normal vision, were na\u00efve to the purpose of the experiment, and had no known neurological deficits. This study was approved by the local ethics committee and performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.\nTable\u00a01Participant informationPartM\/FAge (year)PercepPro: supicomf:uncomfStrategy1.F230.90100:125225:0Comfortable ender2.F220.9224:201225:0Comfortable ender3.F250.97101:124223:2Comfortable ender4.F200.82108:117225:0Comfortable ender5.F21\u00d752:173224:1Comfortable ender6.M250.9082:143222:3Comfortable ender7.F270.7987:138225:0Comfortable ender8.F200.90123:102225:0Comfortable ender9.M260.80146:79225:0Comfortable ender10.M27\u00d7138:87225:0Comfortable ender11.F180.90225:038:187Pronation starter12.F190.74225:0144:81Pronation starter13.F190.70224:156:169Pronation starterPart: participant number; M\/F male\/female; age: age in years; Percep: Perception task\u2014percentage correct answers; Pro:supi: Start posture\u2014number of pronated start postures: number of supinated start postures; Comf:uncomf: End posture\u2014number of comfortable end postures: number of uncomfortable end postures; Strategy: strategy used in the action task (see text for description)\nExperimental set-up and apparatus\nThe participants were comfortably seated in a chair positioned in front of a table upon which the experimental set-up was placed (see Fig.\u00a01). The stimulus consisted of a white 3D \u201crod-and-frame\u201d illusion that was placed in front of a black curtain (220\u00a0\u00d7\u00a0105\u00a0cm). This curtain was used to prevent any visual cues of veridical frames of reference, such as the ceiling or the floor. Both the rod (length: 15\u00a0cm, diameter: 3.5\u00a0cm) and the surrounding frame (30\u00a0\u00d7\u00a030\u00a0\u00d7\u00a02.3\u00a0cm) could be rotated independently such that the rod-and-frame illusion was created. The rod had a grey marker on one side signifying the end that had to be placed upwards in the hole (diameter: 5\u00a0cm) of a box. After the participant had placed the rod in the hole, an experimenter sitting next to the participant replaced the rod to the set-up and scored the used grip type. Rotation of rod and frame was performed manually by a second experimenter who sat behind the curtain. Participants wore liquid crystal occlusion goggles to prevent them seeing the rotation of the frame and rod in-between trials. The goggles could be switched from opaque to transparent in less than 30\u00a0ms.\nFig.\u00a01Schematic drawing of the experimental setup, viewed from above (a) and a photograph of the first author grasping the rod (b)\nProcedure\nThe study consisted of two experimental sessions that were conducted in succession. First, an action task was performed, second we performed a perception task to assess participants\u2019 perceptual sensitivity for the illusion [these tasks are denoted as (1) Action task and (2) Perception task in what follows]. Standard rest breaks were present between sessions, and on participants\u2019 demands.\nAction task\nThe action task consisted of a pre-measurement and the main experiment. The procedure for both was as follows. A trial started when the participant pressed the button on the button-box with the index finger of the preferred (right) hand. Subsequently, the goggles were closed and the second experimenter manually changed the rod and frame orientation. When ready (i.e., within 2\u00a0s) the goggles opened, which was the start-signal for participants to grasp the rod as quickly as possible and place it vertically with the marker facing upwards in a hole of a tight fitting box that was located in front of them, slightly to the right of the body midline. Participants were asked to grasp the rod with a power grip, i.e., with the thumb on one side of the rod and the fingers on the other side. Once the rod was grasped, participants were not allowed to change the grip type during rotation of the rod. This was necessary, because it urged participants to plan the task prior to grasping the rod. If this had not been the case and participants were allowed to manipulate the rod in-hand, then it would not have been strictly necessary for participants to plan the movement prior to grasping the rod.\nAs dependent variable, the grip type that participants used to grasp the rod was measured. The grip types were evaluated on two criteria: the start posture of the hand and the end posture of the hand. The start posture was scored as either a \u201cpronated\u201d (overhand) or a \u201csupinated\u201d (underhand) posture. The end postures were scored as \u201ccomfortable\u201d when a grip with the thumb towards the marker was used and as \u201cuncomfortable\u201d when a grip with the thumb away from the marker was used (see also, Steenbergen et al. 2000; Rosenbaum and Jorgensen 1992). Since the start and end posture are not fully independent of each other, the combination of start and end posture was labeled as the grip type. Three different grip types were distinguished: grip type 1, a pronated initial posture resulting in a comfortable end posture, grip type 2, a supinated initial posture resulting in a comfortable end posture and grip type 3, a pronated initial posture resulting in an uncomfortable end posture (see Fig.\u00a02). The combination of a supinated initial posture resulting in an uncomfortable end posture was theoretically possible. However, this combination was never used, and will therefore not be mentioned in what follows.\nFig.\u00a02The grip type scoring system used to establish the grip type that participants used. Grip types were defined by the combination of the initial posture (pronated or supinated) and the end posture (comfortable or uncomfortable). Explanation, see text\nWith respect to the grip type, our primary interest was the rod orientation at which a switch into another grip type occurred. The rod orientation at which there was an equal chance to observe both grip types was denoted the \u201cswitch point\u201d.\nPre-measurement\nAs the location of the switch point differed between individuals, we performed a pre-measurement prior to the main experiment. In this pre-measurement the individual switch point of each participant was established. In general, switches in grip types occur in the lower half of the \u201cclock face\u201d (e.g., Rosenbaum et al. 1992; Steenbergen et al. 2000), but individual differences are present as to the exact orientation of the rod where the switch occurs. During the pre-measurement the frame was not rotated. Rod orientations were presented in a range of 180\u00b0, from the horizontal rod orientation with the marker on the left side (denoted as \u221290\u00b0), via the vertical rod orientation with the marker facing downwards (denoted as 0\u00b0) to the horizontal rod orientation with the marker on the right side (denoted as 90\u00b0). Thirteen rod orientations were tested, separated by equal angles of 15\u00b0 (see Fig.\u00a03). Every rod orientation was presented three times in a completely randomized order, resulting in a total of 39 trials. The switch point was determined by the rod orientation where participants switched between two different grip types, thus, at this rod orientation there was an equal chance to observe both grip types. For most participants the switch point was restricted to one rod orientation. When the grasping pattern consisted of a range of rod orientations, the mathematical middle of that range was taken to be the switch point for that participant. The pre-measurement took approximately 15\u00a0min.\nFig.\u00a03Schematic drawing of the 13 rod positions used in the pre-measurement of the action task. In this figure the rod is oriented at 45\u00b0. The black side of the rod represents the marker. Note that the color coding is inconsistent with the experiment where we used a black background, whereas the rod and the surrounding frame were colored white (see Figs.\u00a01b, 2 )\nThe main experiment\nThe rod orientations during the main experiment were normalized to the individual switch points, which allowed us to study the individual switch region into detail without overloading participants with too many trials. Measurements were performed in a range of 80\u00b0 surrounding the individual switch point, separated by angles of 10\u00b0. This resulted in a total of nine rod orientations that were tested in the main experiment (\u221240\u00b0, \u221230\u00b0, \u221220\u00b0, \u221210\u00b0, 0\u00b0, 10\u00b0, 20\u00b0, 30\u00b0, 40\u00b0 relative to individual switch point). Negative orientations are clockwise rod orientations compared with the individual switch point, whereas positive orientations are directed counterclockwise to the switch point. During the experiment we also manipulated the orientation angle of the frame, such that the \u201crod-and-frame\u201d illusion was created. The frame was rotated in either a clockwise (CW) or a counterclockwise (CCW) direction. A total of five frame orientations were used (20\u00b0 CCW, 10\u00b0 CCW, 0\u00b0, 10\u00b0 CW, 20\u00b0 CW) yielding a total of 45 unique conditions. In each condition, five trials were performed in a completely randomized order. The main experiment, involving 225 trials, took about 45\u00a0min for each participant.\nPerception task\nWe performed a perception task to assess participants\u2019 perceptual sensitivity for the illusion. It was examined whether different rotations of the surrounding frame affected the perceived orientation of the rod. To that end, two rod-and-frame combinations were sequentially shown to the participant. First, a rod surrounded by a tilted frame was shown, followed by either the same or a different oriented rod surrounded by Frame 0\u00b0. In between presentations, the goggles were closed for less than 2\u00a0s. Participants had to report if the orientation of the rod was the same or different in the two displays. In the majority of the trials the rod orientation did not change between presentations (for example, when the first display was a combination of Rod \u221230\u00b0 and Frame 20\u00b0 CCW, the second display combined Rod \u221230\u00b0 with Frame 0\u00b0). In this perception task, 4 frame rotations\u00a0\u00d7\u00a09 rod orientations\u00a0\u00d7\u00a03 repetitions were tested, yielding 108 trials. In addition, we also added 72 \u201ccatch trials\u201d (4 Frame rotations\u00a0\u00d7\u00a09 rod orientations\u00a0\u00d7\u00a02 directions of rod changes), where the rod orientation actually did change between the two presentations, either 10\u00b0 CW or 10\u00b0 CCW. The main reason to add catch trials was to prevent that participants could anticipate that the two rods were the same in all trials. However, catch trials were not used in the analyses. The total of 180 trials was presented in a completely randomized order. The perception-task took about 45\u00a0min to be carried out.\nData analysis\nAction task\nAnalysis of pilot recordings revealed that participants used two strategies to perform the action task. Although all participants used grip type 1 in some of the trials, at the individual switch point differences in grip type choice appeared. While most of the participants switched to an underhand initial posture resulting in a comfortable end posture (grip type 2), some participants switched to an overhand initial posture resulting in an uncomfortable end posture (grip type 3). Consequently, two movement strategies could be delineated. One group of participants switched between grip type 1 and grip type 2 and always ended with a comfortable end posture (this strategy is denoted as \u201ccomfortable enders\u201d), whereas the other group of participants switched between grip type 1 and grip type 3 and always started with a pronated initial posture (this strategy is denoted as \u201cpronation starters\u201d).\nAt the individual switch point every participant used grip type 1 in approximately 50% of the trials, irrespective of the strategy employed, because participants either switched between grip type 1 and grip type 2 (comfortable enders) or between grip type 1 and grip type 3 (pronation starters). This allowed us to collapse the data and to use the same scoring method for both strategies, that is, the frequency of grip type 1. For every participant individually, logistic (S-shaped) functions were fitted through the mean frequency of grip type 1, separately for the five different frame orientations and on the basis of a least squares fitting method (see Van Doorn et al. 2007 for a similar method). The function was of the form where y is the assigned score, i.e., the location of the switch point, x is the rod orientation, c is the rod orientation of the switch point and k is a measure of the slope at that point Using this method, for every participant the location of the switch point (i.e., the rod orientation where a participant switched between grip types) was determined for the five frame orientations. In order to calculate the illusion effect, the value of the switch point in the control condition (0\u00b0 frame rotation) was subtracted from the value of the switch point in the experimental conditions (where the frame was rotated). As our prime interest was the effect of visual context on planning, rather than the direction of the illusion effect, we used absolute difference scores. Moreover, the direction of the illusion effect was not similar among participants, a finding that is not uncommon in the \u201crod-and-frame\u201d illusion literature (e.g., Beh and Wenderoth 1971; DiLorenz and Rock 1982). The absolute difference scores were analyzed using a repeated measures Analysis of Variance (ANOVA) with frame as within subjects factor.\nPerception task\nThe number of errors per condition were analyzed using a 4 (frame: 20\u00b0 CCW, 10\u00b0 CCW, 10\u00b0 CW, 20\u00b0 CW)\u00a0\u00d7\u00a05 (rod: \u221220\u00b0, \u221210\u00b0, 0\u00b0, 10\u00b0, 20\u00b0) repeated measures ANOVA. Frame as a factor in the ANOVA denoted the first frame that is presented to the participant. The second frame was always the same, i.e., 0\u00b0.\nResults\nAction task\nDuring the experiment, participants showed the same grasping behavior as in the pre-measurement phase, that is, they switched between different grips at a particular rod orientation. The average switch point over all conditions was at rod orientation \u22123\u00b0 (for the \u201ccomfortable enders\u201d at rod orientation \u22126\u00b0 and for the \u201cpronation starters\u201d at rod orientation 6\u00b0), all were in the lower half of the clock face. For the negative rod orientations (i.e., rod orientations that are rotated clockwise compared with the individual switch point) participants used grip type 1, which is an overhand initial posture resulting in a comfortable end posture. Conversely, at the positive rod orientations (i.e., rod orientations that are rotated counterclockwise compared with the individual switch point) the grip patterns were less consistent. Most participants (n\u00a0=\u00a010) switched to an underhand initial posture leading to a comfortable end posture, while some (n\u00a0=\u00a03) switched to an overhand initial posture leading to an uncomfortable end posture (\u201ccomfortable enders\u201d and \u201cpronation starters\u201d, respectively, see also Table\u00a01).\nTo answer our main research question (does visual context affect anticipatory planning?), we analyzed the effect of frame orientation on the location of the switch point. For each participant individually, we calculated at which rod orientation they switched between grips for all frame orientations, using a logistic function. This way, we could calculate the magnitude (in degrees) by which the switch point had shifted in the experimental conditions compared with the control condition. In Fig.\u00a04 the data of four participants are shown (participants 9, 10, 12 and 13). In the figure, the different frame orientations are depicted on the x-axis (with Frame 0\u00b0 as the control condition), whereas the y-axis represents the location (i.e., rod orientation) of the switch point. It can be derived that the location of the switch point is different in the control condition and the experimental conditions. However, the effect of frame was not in the same direction for all participants. Therefore, absolute different scores between the switch point of the control condition (i.e., Frame 0\u00b0) and the switch point in the four experimental conditions (i.e., Frame 10\u00b0 CW, Frame 20\u00b0, Frame 10\u00b0 CCW and Frame 20\u00b0 CCW) were calculated as a measure of the illusion effect. The mean absolute illusion effect (i.e., the amount of degrees that the switch point had shifted compared with the control condition) was 5.3\u00b0 for Frame 20\u00b0 CCW, 9.3\u00b0 for Frame 10\u00b0 CCW, 7.4\u00b0 for Frame 10\u00b0 CW and 6.0\u00b0 for Frame 20\u00b0 CW (see Fig.\u00a05). A repeated measures ANOVA revealed a significant effect of Frame [F(4,48)\u00a0=\u00a03.29, P\u00a0<\u00a00.05 with Greenhouse Geisser correction for sphericity]. Pairwise comparisons showed that the illusion effect of Frame 20\u00b0 CCW, Frame 10\u00b0 CCW and Frame 20\u00b0 CW were significantly different from the control condition (all P\u00a0<\u00a00.05).\nFig.\u00a04Location of the switch point (i.e., rod orientation) in the five frame rotation conditions in four participants (9, 10, 12, 13). On the x-axis the five frame orientations are depicted, whereas the y-axis represents the rod orientation of the switch pointFig.\u00a05The absolute effect of frame rotation on the switch point for the four frame orientations (averaged over all participants). Error bars indicate 2 SE of the mean. The absolute effects are calculated by subtracting the value of the switch point in the control condition from the experimental conditions, hence, the control condition is not depicted here. On the x-axis the different frame orientations are plotted, whereas the y-axis represents the magnitude (in degrees) by which the switch point was shifted compared with the control condition\nPerception task\nData of two participants (participants 5 and 10, see Table\u00a01) were not used for analyses due to technical problems. The mean score of all participants was 0.85 (SD 0.084), indicating that in 85% of the trials participants correctly reported that the perceived rod orientation in the two presentations was not different. The percentages of correct answers varied between 70% (participant 13) and 97% (participant 3). The mean score in the \u2018comfortable end posture\u2019 group was 87% compared with 78% in the \u201cpronation start posture\u201d group. However, this between subjects effect of strategy just failed to reach significance [F(1,9)\u00a0=\u00a03.74, P\u00a0=\u00a00.085]. A repeated measures ANOVA on the total number of errors revealed a significant effect of Frame [F(3,27)\u00a0=\u00a04.14, P\u00a0<\u00a00.05]. The percentages of correct answers were 86% for Frame 20\u00b0 CCW, 90% for Frame 10\u00b0 CCW, 87% for Frame 10\u00b0 CW and 77% for Frame 20\u00b0 CW. Post hoc comparisons showed significant differences between Frame 20\u00b0 CW and Frame 10\u00b0 CW (P\u00a0=\u00a00.051) and between Frame 20\u00b0 CW and Frame 10\u00b0 CCW (P\u00a0<\u00a00.05).\nDiscussion\nThe purpose of the work reported here was to evaluate the influence of visual context on the planning of a sequential object manipulation task. Earlier research on the effects of visual illusions on action was limited to simply grasping a target object without any further purpose. By contrast, in the present study we asked participants to grasp a target object to subsequently place it in a hole. This task requires anticipatory planning, in which constraints arising from the end posture prevail in initial grip choice. That is, the initial grip must accommodate the upcoming movements. As far as we know, no other study has scrutinized visual context effects in such a sequential, object manipulation task. In our study, a rod was embedded in a typical \u201crod-and-frame\u201d illusion configuration. We used a wide range of rod orientations that would force participants to switch between different grip types if they were to reach a comfortable posture at the end of the task. The effect of visual context on anticipatory planning processes was investigated by measuring if the location of the switch point shifted when the surrounding frame was tilted.\nThe main finding of our study was that the frame manipulations affected the location of the switch point (i.e., the rod orientation where participants switched between grip types), and thus the motor planning of the initial grip type towards the target object. Although earlier findings have already shown that the kinematics and joint couplings in the first movement towards a target object are affected by the upcoming second movement (Gentilucci et al. 1997; Marteniuk et al. 1987; Steenbergen et al. 1995), our results extend these finding by showing that initial grip planning is also affected by the visual context. However, in line with earlier findings on the illusion effects of the \u201crod-and-frame illusion\u201d (e.g., Beh and Wenderoth 1971; DiLorenz and Rock 1982) the results did not show a consistent direction of the illusion effect among participants. This phenomenon is due to the complex interaction between the specific location of the individual switch point with the frame orientation and the individual sensitivity for the illusion.\nOur results are in line with at least three contemporary models that make specific predictions about the effect of illusions on action (planning). First, following the predictions of Glovers\u2019 model (2004) we hypothesized that the visual context would affect the specific rod orientation at which participants switch to a different grip. This hypothesis was confirmed as the location of the switch point was affected by the surrounding frame. These findings extend observations of Glover and Dixon (2001b; Glover et al. 2005; see also Van Doorn et al. 2007), in which an orientation illusion was shown to affect grip choice in a simple grasping task. Second, our findings are in line with the predictions stemming from the perception-action model (Milner and Goodale 1995). Goodale and Milner propose that the ventral stream is responsible for \u201cthe perceptual representation of the perceptual world that is used in the planning of actions\u201d (Goodale and Milner 2004, p. 38), thus assuming action planning to be subject to a visual illusion. Support for the hypothesis that the ventral stream plays an important role in action planning has also been reported in a patient study by Dijkerman et al. (2003), in which two patients with ventral stream lesions did not show appropriate switching when grasping bars in different orientations. Finally, our results can also be accommodated by the common-representation model of Franz (2001), in which it is proposed that a visual illusion affects both perception and action. It is important to note here that our study was not aimed at providing a critical test for one of these models. Rather, we aimed to examine what components of planning are affected by visual context.\nThe \u201crod-and-frame\u201d illusion has been investigated by Dyde and Milner (2002), who found that the illusion influenced perception but not action. At first glance, these results may appear contradictory to our findings, but we argue that the difference in task constraints may have contributed to the different findings. As Smeets et al. (2002) argued, different tasks necessitate different types of spatial information to be used for action. For example in the Dyde and Milner study (2002) participants grasped the ends of the rod between their thumb and forefinger and participants were therefore dependent on the visual information regarding the position of the ends of the rod. In contrast, the orientation of the rod constituted the relevant action-related information source for participants in our study. Smeets and Brenner (1995) and Smeets et al. (2002) have proposed that an illusion only effects on action when the critical spatial characteristics of the target in the relation to the to-be-performed action are affected by the illusion. In our study this critical spatial characteristic was the orientation of the rod, whereas in the Dyde and Milner study it was the position of the ends of the rod.\nFinally, two issues should be mentioned here, namely, the comparison between the perception task and the action task, and second, the unexpected finding of two strategy groups. The first issue concerns the comparison of illusion effects on perception and action. As Franz (2001) pointed out, an inherent problem in visual illusion studies is the comparison between the perception and the action task, as these tasks are predominantly measured by different methods, as was also the case in the present experiment. However, although our study does not allow us to compare perception and action in a quantitative way, the perception task did provide information about how participants perceived the rod orientation when surrounded by a tilted frame. Specifically, participants\u2019 perception of the rod orientation was affected by the surrounding frame.\nThe second issue concerns the finding of two strategies. Our results showed that the means (i.e., grip type) by which the end goal was reached was affected by the visual illusion, however, participants reached the end goal differently, viz used different strategies. Most participants switched between pronated and supinated start postures, such that they ended the movement with a comfortable end posture. Still, three participants in our study did not obey this \u201cend-posture comfort\u201d rule. They used a pronated start posture that resulted in both uncomfortable and comfortable end postures. Importantly, however, irrespective of the strategy used, the effect of the visual context on grip planning was consistent. That is, tilting the frame affected the rod orientation where participants switched their grip, but depending on the strategy most participants switched between grip type 1 and 2 and some participants switched between grip type 1 and 3. This unexpected finding begs the question as to why some participants used a strategy that did not enable them to end the task in a comfortable end posture? The \u201cposture based motion planning\u201d-model of Rosenbaum et al. (2001) assumes that prior to movement execution an end posture is chosen from the stored posture base. The model further assumes a time constraint for this search process. If enough time is allowed, the search will most likely result in a posture that satisfies the end comfort criterium. If, however, insufficient time is allowed for the search, end postures may be selected that are not optimal. More specifically, these postures may be uncomfortable or even unfit for the task (see Meulenbroek et al. 2001 for model simulation and validation). In our study we instructed participants to perform the task \u201cas fast as possible\u201d. Therefore, it may be speculated that the \u201cpronation starters\u201d have put more emphasis on the speed of responding, thereby not completely searching their stored posture base. As the group of \u201cpronation-starters\u201d was small (n\u00a0=\u00a03), we cannot draw any definite conclusions on this matter but further examination is warranted.","keyphrases":["visual illusion","motor planning","perceptual judgment","grip selection"],"prmu":["P","P","M","R"]}
{"id":"J_Headache_Pain-4-1-2386850","title":"Impact of headache in Europe: a review for the Eurolight project\n","text":"A recent health economic survey in Europe has suggested that migraine is the costliest among the neurological disorders. According to many studies, migraine and other disorders lead to widespread suffering, reduction of quality of life, and marked impairment of participation, both in work and social activities. The present literature survey was made in order to summarize what is known on the subject, as a preparation for a EU-supported study to assess the impact in several EU countries with similar methodology and the same research instrument. Previous studies have yielded relatively reliable data only for migraine, whereas the impact of tension-type headache is virtually unknown or only very incompletely known for most dimensions of headache impact. Some data do suggest, however, that this headache may be as important from a health economic and a public health perspective as migraine. In future studies it is important to get population-based data from various countries relevant for estimation of indirect (mostly absenteeism from work and reduced working efficiency when having headache) and direct costs (related to medication, consultations, investigations and hospitalisations). Also, the impact on ability to get education and participate in the workforce is very relevant, as is the impact on love life and family planning. The quality of life of headache patients should be measured by validated instruments. To get a complete picture, one should also ask about the effect on the life of partners and children, and on the possible impact even when headache-free (e.g. fear of the next attack).\nIntroduction\nFor a just and rational distribution of means to health-care services and health-related research, reliable data on the individual and societal impact of different disorders are crucial. In recent years several initiatives have been launched to raise the awareness that headache is not only a nuisance for some individuals, but also that it entails widespread suffering and loss of opportunities for patients and their families, and large cost for the society. The recently published report on the prevalence and burden of headache [1] is a premise for the campaign \u201cLifting the burden: The Global Campaign to reduce the burden of headache\u201d[2]. In Europe, much data on both the economic costs of migraine have been collected and presented in connection with the \u201cCost of Brain Disorders in Europe\u201d project [3], in which migraine is treated along with many of the other (neurological and psychiatric) \u201cbrain disorders\u201d. The Eurolight project (http:\/\/www.eurolight-online.eu) is an initiative supported by the EC Public Health Excecutive Agency launched in May 2007. Its objectives are to bring together the relevant medical, scientific and lay organizations, and to gather updated reliable comparable information regarding migraine, tension-type and chronic headache. It will be the first data collection on headaches at EU level focusing on a holistic, patient-driven and scientifically validated approach, aiming to fill in the main holes in our knowledge by performing comparable studies on headache prevalence and its impact in selected European countries (Austria, France, Germany, Italy, Lithuania, the Netherlands, Spain, UK, Ireland, Luxembourg). A pilot study has already been performed in Luxembourg. The present review of the existing literature on both economic and non-economic impact was performed as a part of the Eurolight project, to assess the current state of knowledge, and to build up a questionnaire to measure all the most relevant aspects of headache impact. This study presents the results of this review and a suggestion of the main dimensions that ought to be covered in the headache impact tool to be used in the Eurolight study.\nEconomic impact of headache\nHeadache may have considerable economic consequences, both for the patient and for the society as a whole. There are more studies about the societal costs than about the individual economic losses of the patients.\nRelation to socioeconomic status, education and employment\nIn a large Norwegian study (the HUNT study), both migraine and headache in general was associated with low socioeconomic status [4], which has also been found in North America [5, 6] but not in some smaller European studies [7\u201311]. The question whether this is a consequence or a cause of headache is not satisfactorily answered, but in one Swedish study, half of the patients reported a negative influence of migraine on their ability to pursue studies and one third a negative influence on their finances [9]. In a US study it was found that headache patients have somewhat reduced labour force participation [12], but employment status has not been found to be related to headache in some European studies [7, 13].\nAbsenteeism from work\nIn two relatively old studies, one from Finland in 1979 [14] and one from San Marino in 1986 [15], 7% of working individuals had been absent from work in the previous year due headache. In a Danish study of 1992 [16], it was found that 43% of migraineurs (5% of the population) and 12% of TTH patients (9% of the population) had been absent from work during the previous year due to headache, i.e. a total of 14% of the population. In a Swedish study of 2004 [9], it was found that 65% of migraineurs reported some degree of absence from either school or work during the previous year. These data are, however, of relatively limited interest from an economical viewpoint as they do not indicate the number of days that the headache sufferers are away from work.\nThe number of days with work absence due to headache is relatively consistent across studies from different countries. In some previous studies it has varied between 2 and 6\u00a0days per year among headache patients in general [17], and between 1.5 and 4.2\u00a0days per year in migraineurs [9]. A study among migraineurs in Sweden revealed that 35% were never absent from work due to migraine, and 54% were absent 1\u20132\u00a0days per year [9]. Compared to headache-free individuals, migraine patients in the HUNT study from Norway lost on average 4.4 workdays per year, and persons with non-migrainous headache lost 2.5 workdays per year [18]. In the Danish study from Copenhagen [16], the TTH patients who had been absent seem to have been as much or more absent from work than the migraine patients, and the number of workdays lost due to migraine was 270 and to TTH 820 per 1,000 persons per year, i.e. a total of 1,090\u00a0days. In a study from England in 2003 [13], 15% had been absent from work or had reduced ability to work due to headaches in the previous 3 months. Per year, headache accounted for 1,327 missed and 5,213 reduced ability days per 1,000 workers per year, representing 0.5 and 2.0% of all working days in the adult population, irrespective of headache status. This study did not relate absenteeism to different headache diagnoses. In an English study of 2003, an estimated 5.7 workdays per year was missed by migraineurs working or attending school [8]. This seems to be higher than in France where a diary-based registration of absenteeism published in 1999 showed that migraineurs were away from work 2.18\u00a0days per year due to headache [19].\nEffectiveness when working with headache\nWorking with migraine results in a 35% productivity loss on average according to some European studies [20]. This figure is, however, largely based on migraineurs\u2019 self-report, which may give a too high estimate according to a recent US study from a workplace[21]. In this study, it was found that the working ability assessed by self-report was much lower than the objectively measured working efficiency (20 vs. 8%). The relatively small decline in working ability led the authors to conclude that workers with even relatively severe headache find creative ways to cope with the pain and maintain standards.\nHealth economic studies\nFor the headache part of the \u201cCost of Brain Disorders in Europe\u201d project, a literature search for studies containing cost data for migraine and other headaches identified eight European studies evaluating the direct or indirect costs of migraine from a societal perspective [20], from France [22, 23], Germany [24], The Netherlands [25], Spain [26], Sweden [27] and the UK [28, 29]. No studies analysing the cost of TTH or other non-migraineous headaches were found. There were large variations in costs across the six European countries where data were available, ranging from around \u20ac100 per patient per year in Sweden to nearly \u20ac900 in Germany. These variations are probably mostly due to different methodologies and differences in the year when the studies were conducted. An important finding was that the vast majority of total costs, between 72 and 98%, was indirect costs, due to lost productivity, either in the form of work absence or reduced efficiency levels when working with migraine. Women tended to lose more workdays than men, but indirect costs were similar due to lower salaries and labour force participation amongst women. The direct costs, related to consultation, diagnostic investigations, treatments, and hospital admissions accounted for less than 30% of total costs in most studies.\nThe cost estimate for migraine in the European report was based on an average of the most representative cost estimates, from the UK, Germany and France. An average annual cost of \u20ac585 per migraine patient was estimated for these Western European countries. The 1-year prevalence of migraine was 14% among adults in Europe according to the review of epidemiological studies [1], i.e. 41 million adult Europeans with active migraine. Per patient migraine was the least costly disorder among the brain disorders. However, due to the high prevalence, the total cost of migraine was estimated to be \u20ac27 billion for whole Europe in 2004, which was the highest cost among the purely neurological disorders. Many of the psychiatric afflictions were even more costly according to this review. It is, however, likely that the available cost data in Europe would tend to underestimate the actual costs of headache, mainly because no cost data existed on the most common headache type (TTH), but also because children and adolescents were not considered, and because cost connected with more expensive medication (triptans) were not included, since most cost studies were performed before this class of drugs was introduced.\nIn a separate paper summarizing the prevalence and cost data for headache in Europe [30] a more speculative estimate for the cost of headache, rather than migraine alone, was derived by using the results of the Danish [31] and British [32] population-based studies which demonstrated that around 1,100\u20131,300\u00a0days per 1,000 workers were missed due to headache each year. The British study also suggested that the number of days with reduced efficacy was around four times higher than the number of days missed. Assuming a reduced efficiency of 35% when working with headache, and that the direct costs of headache constitute the same proportion of the total costs as for migraine, the average total cost per headache patient was estimated to be roughly \u20ac420 per year (of which \u20ac390 would be due to indirect costs and \u20ac30 due to direct medical costs). Since headache in general was found to affect nearly 50 % of Europeans, this estimate, if true, would make headache a much more costly disorder than migraine alone.\nIt is of interest to compare the European cost study [3] with more recent cost studies in some individual European countries. In one study from Spain [33] the annual costs of migraine was only about 50% of the sum given in the European Cost study for the same country. The difference may partly be explained by somewhat lower prevalence figures (12 vs. 14%) for migraine used in the Spanish study, but the main difference may be that this study did not employ a bottom-up design, but used published statistics and data to estimate resource use and productivity losses, which may have led to an underestimation of some costs. A recent study from France [34], restricted to the direct costs in 1999, found that these costs were at least twice as high (\u20ac128) as in the European migraine cost study (<\u20ac60). This study included both \u201cstrict\u201d migraine (IHS 1.1 and 1.2) and \u201cmigraineous disorder\u201d (IHS 1.7, corresponding to 1.6 in ICHD-2), which together affected 17% of the population. For the whole country the direct costs amounted to more than 1 billion \u20ac, which was 0.068% of the gross national product. Non-migraineous episodic headache, affecting 9.2% of the population, entailed a considerably lower cost of \u20ac28.\nIt may also be of interest to compare the European studies with one US study using a quite different methodology to assess direct costs. In this study, all types of medical care costs (not only those related to headache) were derived from the claims records of a large health plan, whereas diagnostic status (migraine or not) and comorbid and demographic status was ascertained using a telephone interview among members of the health plan [35]. Migraineurs incurred on average $700 more per year in total medical care costs than the controls. Interestingly, this statistically significant difference disappeared when psychiatric comorbidity variables (anxiety and depression) were entered into the model. The much higher costs per patient reflected in this study than in the French study [34] and Spanish study [33] may therefore at least partly be due to the differences in cost assessment methodology, indicating that the direct costs specifically related to migraine and not to comorbid disorders are most reliably assessed by a direct method, questioning patients about use of health-care resources.\nMedication for headache constitutes an important part of the direct costs. In France in year 2000, the most frequently used acute medications for migraine were paracetamol, salicylates and NSAIDs. Triptans were used by 8% of migraineurs, and prophylactic treatment was used by 6% [36]. In Denmark 26% of migraineurs had used triptans in 2001, but less than 5% of those with pure migraine had used prophylactic medication [37].\nNon-economic impact\nFrom a purely humanitarian perspective, but also from a public health perspective, the pain, suffering and disability caused by headaches are as important as the economic consequences. In a study performed in young women in nine Western European countries, 86% of migraineurs stated that their life would have been better if they did not suffer form migraine [38]. A German study showed that, on average, patients with migraine or TTH had around 1\u00a0month every year affected by headaches [10]. The main burden of headache is carried by a minority of sufferers, and a Swedish study has shown that 27% of migraine patients had 68% of all attacks [9]. Three to 4% of the European population have headache half of the days or more per month [1].\nDisability\nIt has been calculated that in the US, 300,000 persons stay in bed each day (24\u00a0h) due to headaches [39]. A Swedish study has shown that the disability is not only related to the attacks since many migraine patients report an impairment also between attacks [40]. Nine % of patients report that they have some residual disability since they do not recover completely between attacks, and in addition, many patients live in a constant worry about the next attack [9, 38].\nIn some studies, the level of disability due to migraine has been evaluated with the Migraine Disability Assessment Scale (MIDAS). With this instrument, days with work absence (job or household chores), days with \u226550% reduction in productivity, and days with inability to participate in social activities are counted during a 3-month period. In France, among those with active migraine, 22% (1.5% of the whole population) had grades III or IV disability (moderate or severe disability, indicating 11\u00a0days or more during the last 3\u00a0months\u2019 period when headache affected work\/household chores 50% or more, or leisure activities) [36]. MIDAS III or IV were about twice as common among migraineurs in one US study (54%) [41], as it was in the multinational Latin American study (50%) [42]. Among patients with headache in general (both migraine and non-migraineous headache, comprising 70% of the study population), 10.3% (7.2% of the population) had MIDAS grade III or IV disability [13]. Comparing the percentage of the general population in France with MIDAS disability grade III\u2013IV due to migraine (1.5%) [36], with the percentage of population in England with same disability due to headache in general (7.2%) [13], it seems that non-migraineous headache causes more disability on a population than does migraine. The headache-attributed lost time (HALT) index is a close derivative of MIDAS (http:\/\/www.liftingtheburden.org\/ \u2192 Resources \u2192 Burden measure) to be used for headache burden studies, which will be conducted by the Lifting The Burden Campaign [43].\nAccording to the World Health Organisation (WHO), the preferred measure of disease burden is \u201cDisability Adjusted Life Years\u201d (DALYs), which is a sum of the years of life lost (YLL) and the years lived with disability (YLDs). The YLDs are determined by the incidence and duration of the disorder, and by a disability weight ranging between 0 and 1 [44]. Although migraine entails no increased mortality (i.e. YLL\u00a0=\u00a00), it ranked the 19th among the leading causes of DALYs among women aged between 15\u201344 years, and with regard to YLDs, it was 19th for both sexes, and the 12th for women, irrespective of age. Using the WHO data for a calculation of the burden of \u201cbrain disorders\u201d (i.e. the psychiatric and neurological disorders) in Europe, the weight of migraine was lower than that of the major psychiatric disorders, dementias, stroke and injuries, but higher than that of epilepsy, multiple sclerosis and Parkinson\u2019s disease[45].\nIn a recent report on the global prevalence and burden of headache disorders, the burden of migraine and TTH were measured in a similar way as the DALYs by combining data on prevalence, mean number and duration of headache attacks, and headache intensity, from studies containing such information. For the world as a whole, it was demonstrated that TTH resulted in a higher population burden (approximately 55% of total burden) than migraine (45%). The data for Europe indicated an even higher burden due to TTH compared to migraine [1]. If one uses the European data from this study it can be calculated that the hours with migraine headache would add up to between 34 and 100\u00a0h per year, if distributed on each adult individual in the population. The data on TTH are too scarce to use for similar calculations.\nStudies using validated QoL-instruments\nThe SF-36 is a validated instrument to measure quality of life (QoL), containing eight dimensions. One US study [46], recruiting migraine patients from a medication trial, demonstrated that migraineurs had lower QoL than the general US population, most marked for bodily pain, physical role limitations and social functioning.\nA Dutch population-based study found that migraine had a negative influence on all a dimensions compared to controls. The negative influence on QoL was larger than that of e.g. asthma, and it increased with increasing headache frequency [47]. Two population-based studies from Spain among chronic daily headache sufferers showed a marked negative influence, most marked for those with medication overuse, but similar for those with a headache of a migraine or a tension type [48]. One of these showed that the headache frequency may have a greater impact than headache intensity on QoL [48], and the other that chronic headache with medication overuse was associated with a decrease in all QoL aspects studied with SF-36, most marked for physical role and bodily pain [49]. A study from UK showed that migraineurs with high or moderate disability had a marked reduction on all dimensions on the SF-36 [50]. One Swedish study compared SF-36 results in the two sexes and in participants with different pain conditions. There was a gender difference for headache, which in men influenced physical function, physical role and bodily pain most, and in women vitality, social functioning, emotional functioning and mental health [51]. In a French study, migraineurs had significantly lower scores than headache-free controls on all SF-36 dimensions, and lower scores on the pain dimension than those with other headaches or with TTH [52].\nOne study comparing migraineurs in the US and the UK used a shorter QoL instrument, the SF-12, which contains a physical and a mental component [53]. In both countries, migraineurs had lower scores than controls on both components also after adjusting for socioeconomic status and for depression. However, in those with both migraine and depression, the QoL was significantly reduced in comparison to those who were not depressed.\nIn another French study, using a disease-specific QoL instrument called QVM, the QoL was found to be lowest among those with chronic headache, intermediate among migraineurs and highest among subjects with other forms of episodic headache [54].\nThe total burden of headache patients may not only be related to the headache per se, but also to comorbid conditions. European population-based studies have demonstrated that depression and\/or anxiety occur two to three times more often among migraineurs than in the general population [55, 56]. Depression adds to the reduction in QoL in migraine [53]. This comorbidity may be as important for non-migrainous headache [57], but it is not known how this comorbidity influences the QoL in other headaches. In addition, it has been found that headache is also comorbid with other bodily pain, both in Finnish children [58] and Norwegian adults [59].\nFamily impact of migraine\nMigraine also affects the patients\u2019 spouses and children. In a population-based Swedish study [9], the percentage of migraine sufferers who reported a negative impact of migraine was 76% for attendance to work, 67% for family situation, 59% for leisure time, 48% for pursuing studies, 46% for sexual life, 37 % for their social position, 31% for love, 30% for their financial situation, 27% for making a career, and 11% for making friends.\nOne study has measured the impact on the family in two population samples of similar size in US and UK [60]. The impact was very similar in both countries. More than 60% of patients reported a marked impact on the ability to do household chores because of their migraine during the past 3\u00a0months, and it was markedly reduced also in 20% of the patients\u2019 partners. Almost 46% of patients, and 24% of partners had missed days of family or social activities due to the proband\u2019s migraine, and 16% of patients and 12% of partners had avoided making plans for family or social activities due to the proband\u2019s migraine. As to the impact on the children of patients, more than 60% stated that it had a moderate to marked influence on the relation with their children, 40% stated that they would have been a better guardian or parent without migraine, more than 10% stated that their children had missed school, and 10% that their children had been late to school because of their headache. Forty-six percent of patients stated that they would have been better partners without headaches, and 5% stated that they had had fewer children because of headache, 0.4% that they had avoided having children, and 15% that they had avoided oral contraception. Compared with a control group, the partners of migraine patients were significantly more dissatisfied with the demands, responsibility and duties placed upon them, and with their ability to perform.\nConclusions\nHealth economic studies have documented that the costs of headache disorders are huge; the costs only for migraine amounting to \u20ac27 billion in the EU countries, and the cost for other headaches are probably as large. However, better population-based cost studies are needed to assess the cost involved with TTH. Headache sufferers tend to have lower income and education, and more of them may be unemployed, but it is still uncertain whether this is true for most European countries, and also whether it may be a cause of or an effect of headache. It is also amply documented that migraine confers a high degree of disability with more forced absence from work and leisure activities, and migraineurs also have a measurably reduced quality of life. In addition, there is a marked impact on family life, and headaches also put considerable strains on partners and children. A minority of headache sufferers chose to have fewer children than they would have had if they had not had headaches.\nBased on the present review we have identified some main domains, summarized in the Table\u00a01, that should be covered in order to capture as much as possible of the headache burden. The investigations should be performed in population-based samples, and to assess the whole burden, it is particularly important that not only migraine is included but also TTH and the chronic headaches. Different studies show marked variations between countries, both with regard to prevalence and burden of headache. At present it is not possible to determine with certainty whether these differences are real or due to variations in methodology. This highlights the need to study several countries with the same methodology and instrument, which will be done in the ongoing studies of the Eurolight. If further research shows that there are real and important differences between various countries and regions, investigations to determine the causes of such differences may elucidate ways to lower the burden of headache in a population. In any case, we believe that the Eurolight studies will provide the evidence needed to let headache disorders get the resources for treatment and research that they deserve according to the burden they place on people in Europe.\nTable\u00a01The main domains that should be covered in headache burden studiesEconomic burden\u00a0Direct costMedication, consultation, investigations, hospitalisations\u00a0Indirect costsWorkdays lostDecreased effectiveness when working with headacheLost career and education opportunitiesNon-economic burden\u00a0DisabilityMIDAS or HALTTime with disability: Headache frequency x duration x intensity\/disability\u00a0Impact outside attacksResidual disability and fear of next attack\u00a0Quality of LifeSF 36, SF 12, WHOQual etc\u00a0Family impactImpact on the life of partner and\/or childrenImpact on marriage and love lifeImpact on family planning and\/or contraception\u00a0Psychiatric complaintsVarious scales to measure anxiety and\/or depression as consequent or comorbid disorders (e.g. HADS)","keyphrases":["impact","headache","eurolight","migraine","burden","disability"],"prmu":["P","P","P","P","P","P"]}
{"id":"Anal_Bioanal_Chem-4-1-2324128","title":"Hydrophilic interaction liquid chromatography (HILIC) in proteomics\n","text":"In proteomics, nanoflow multidimensional chromatography is now the gold standard for the separation of complex mixtures of peptides as generated by in-solution digestion of whole-cell lysates. Ideally, the different stationary phases used in multidimensional chromatography should provide orthogonal separation characteristics. For this reason, the combination of strong cation exchange chromatography (SCX) and reversed-phase (RP) chromatography is the most widely used combination for the separation of peptides. Here, we review the potential of hydrophilic interaction liquid chromatography (HILIC) as a separation tool in the multidimensional separation of peptides in proteomics applications. Recent work has revealed that HILIC may provide an excellent alternative to SCX, possessing several advantages in the area of separation power and targeted analysis of protein post-translational modifications.\nIntroduction\nRecent years have seen a significant increase of interest in hydrophilic interaction liquid chromatography (HILIC). Although HILIC was first introduced in the 1970s [1], it took until the start of this century for a considerable number of HILIC applications to appear, highlighting its versatile nature [2\u20137]. A major cause of this increase is the growing need for the analysis of polar compounds that do not bind to reversed-phase (RP) materials and the constantly increasing complexity of samples [8]. Additionally, the exceptional growth of liquid chromatography\u2013electrospray ionisation\u2013mass spectrometry (LC\u2013ESI\u2013MS) for analytical analyses has led to a widening search for acceptable chromatographic materials. In this search it has been noted that the buffer conditions that are used for HILIC are highly compatible with MS, and that the high organic contents of these buffers can potentially increase sensitivity in ESI\u2013MS[9, 10]. Moreover, an HILIC separation is \u201corthogonal\u201d to RP separation, which makes it viable for the multidimensional separation of complex samples [11\u201313]. Finally, HILIC has been found to allow enrichment and the targeted analysis of post-translational modifications (PTMs) such as glycosylation [14], N-acetylation [12] and phosphorylation [15] in proteomics applications.\nAlthough HILIC applications cover a large range of bioactive compounds, in this review we will focus on the use of HILIC in proteomics. We will describe how HILIC can be used for the separation of peptides in multidimensional chromatography and provide insights into its sensitivity, selectivity, separation power and \u201corthogonality.\u201d We will also discuss how HILIC has recently been used for the enrichment of post-translational modifications at both peptide and protein levels.\nHILIC\nHILIC is characterized by the use of a hydrophilic stationary phase and a hydrophobic organic mobile phase [16], which was originally described by Linden et al. in 1975 [1]. The order of elution is reversed relative to reversed-phase chromatography (RP), with hydrophilic compounds being retained longer than hydrophobic compounds. Therefore, HILIC can simply be seen as a form of normal-phase (NP) chromatography. However, the acronym HILIC was suggested to distinguish it from NP, as NP is typically performed with nonaqueous, non-water-miscible solvent buffers, while HILIC is performed with water-miscible solvents and elution is achieved by a water gradient [2, 9, 17, 18].\nIn recent years, several stationary phases have emerged that are specifically made for HILIC approaches. Popular phases include underivatized silica stationary phases that contain functional groups such as siloxanes, silanols with (or without) a small quantity of metals [2, 9, 19, 20], derivatized silica, such as the cation exchanger polysulfoethyl A [17, 18], the weak cation exchanger Polycat A [21], the weak anion exchanger PolyWAX [22, 23], TSKgel amide 80 [24, 25], zwitterionic (ZIC)-HILIC [14] and \u201cclick\u201d saccharides [26]; see Fig.\u00a01 for structures of typical HILIC phases. Each of these materials display different retention characteristics and separation selectivities and require distinct buffer constitutions for optimal results [10].\nFig.\u00a01Chemical structures of the functional groups in common HILIC stationary phases\nA HILIC buffer typically contains more than 70% acetonitrile [17]. Other eluents have been tested, for instance methanol or isopropanol, but they resulted in poor chromatography or no analyte retention [2]. It is believed that the hydrophilic stationary phase enriches water from the buffer and thus generates an aqueous layer [17]. This allows for hydrophilic analytes to partition between the hydrophilic layer and the hydrophobic elution buffer. Elution is obtained through increasing the hydrophilicity of the mobile phase by increasing the water content. The final separation mechanism of elution, however, is most probably a superpositioning of partitioning and electrostatic interactions or hydrogen bonding to the stationary phase [2]. The extent to which each mechanism dominates is dependent on the actual type of stationary phase used and the buffer conditions, including the level and type of organic content, the type and concentration of salts and the pH [10]. Charged stationary phases, such as the abovementioned anion or cation exchangers and deprotonated silanol groups, are most likely to display some degree of electrostatic interaction [2]. Buffering salts in the mobile phase can decrease these electrostatic interactions through disruption. The choice of salts is however limited, due to the highly organic conditions of HILIC buffers, which makes it difficult to dissolve some salts in them [10]. Typically, ammonium acetate and formate are chosen because of their compatibility with MS, but ammonium bicarbonate, triethylamine phosphate (TEAP), sodium perchlorate and sodium methylphosphonate (Na-MePO4) have also been found to be applicable [2, 23]. Additionally, the same salt-based disruption can decrease the retention of analytes and can be useful during elution [17]. However, in some instances an increase in retention was seen upon increasing the salt concentration [10]. This was rationalized by the suggestion of a mechanism in which salt is enriched in the aqueous layer, which in turn increases the hydrophilicity of this liquid layer around the stationary phase [10].\nUnsurprisingly, the type of salt that is used can also influence the retention behavior. It was observed that basic peptides were retained longer with TEAP in the buffer, while with Na-MePO4 these were the first peptides to elute with the weak anion exchanger PolyWAX as stationary phase [23]. It was reasoned that TEAP, as a counterion, will generate a sublayer where one negative charge of the salt is attracted to the positively charged stationary phase, while the second negative charge is free to attract basic peptide residues. In the case of Na-MePO4, there is only one negative charge that interacts with either the stationary phase or basic residues, leaving enough stationary phase available for interaction with acidic peptides [23].\nA special type of HILIC called electrostatic repulsion\u2013hydrophilic interaction chromatography (ERLIC) actually specifically utilizes the electrostatic interactions in HILIC [23]. In ERLIC, a stationary phase is chosen that has a similar charge to the analytes to be separated. Analytes are on the one hand repelled by the stationary phase but on the other they are retained in the aqueous layer around the stationary phase. These opposing interactions allow isocratic resolution [23]. ERLIC can also be exploited in the selective isolation of phosphopeptides from a tryptic digest [23, 27].\nAnother factor that influences the retention characteristics in HILIC is the pH of the buffer. Whether the buffer pH is above or below the pKa of the analyte determines its charge state, which in turn affects the hydrophilicity of the analyte and likewise the interaction with the stationary phase [10, 12]. For example, large differences were found in the retention profiles of a highly complex tryptic digest run over the same column at pH 3 and pH 8 [12]. At pH 3, acidic peptide residues are protonated and so the overall hydrophilicity of peptides containing these residues is decreased, leading to poorer retention of these peptides, with the effect being magnified with higher numbers of acidic residues [12].\nFor a more detailed overview of different HILIC stationary phases and their applications outside the field of proteomics, see H\u00ebmstrom et al. [2].\nHILIC in multidimensional peptide separation\nShotgun proteomics involves the analysis of entire proteomes in single experiments. Analysis of such samples raises a number of issues, including enormous complexity, large dynamic range and low levels of available analyzable material. Often, SDS PAGE-based [28] or LC separation techniques are used to reduce the sample complexity, followed by RP LC-MS as the final readout. Major advances have been made in the miniaturization of LC\u2013MS, since reducing the flow rate improves mass spectrometric sensitivity [29], reduces nonspecific adsorption onto separation devices, and improves chromatographic resolving power [30], but improvements are still required. The complexity of a sample can be further reduced prior to mass spectrometric analysis by the addition of extra dimensions of separation. At present, the method of choice is the combination of strong cation exchange (SCX) with RP [31\u201334]. Over time, a number of alternative configurations have been developed, including size exclusion chromatography (SEC)-RP [35, 36] and RP at pH\u00a010 followed by RP at pH\u00a02.6 [37]. However, the low separation power of SEC [36] and the correlative separation of RP at different pH levels has not yielded the optimal two-dimensional (2D) LC system yet.\nRecently, HILIC was introduced as a dimension for 2D-LC\u2013MS and was shown to remove a few of the disadvantages of existing techniques [11, 12]. Evidently, RP and HILIC buffers are not directly compatible and so online hyphenation of these stationary phases will be difficult. Online hyphenation possesses certain advantages, such as minimal loss of sample, no vial contamination, and no sample dilution [38, 39]. However, performing an experiment in an offline fashion is not necessarily inferior since overloading of the second dimension is no longer an issue with offline fractionation as it is for online approaches. Moreover, in an offline mode, columns and\/or buffers that are normally not compatible with the two consecutive separations can be used [38, 40]. Finally, a conventional gradient for peptide separation can be used in offline setups, and this has been shown to be generally superior to step elution [40].\nGilar et al. separated and analyzed a mixture of approximately 200 peptides using different stationary phases, including SEC, HILIC, RP at pH 2.6 and pH 10 and SCX. HILIC was shown to have a separation power superior to both SCX and SEC [11]. The orthogonality\u2014i.e., how different the separation mechanisms are\u2014of the stationary phases was determined by plotting the peptide retention times of two dimensions against each other (see Fig.\u00a02). Although this study did not actually experimentally connect the different stationary phases, since all LC\u2013MS runs were one dimensional, it revealed that HILIC is slightly more orthogonal to RP than SCX [11].\nFig.\u00a02Two-dimensional plots of normalized peptide retention times in HILIC, SCX and RP separation. Both HILIC and SCX have separation mechanisms that are orthogonal to RP, but the clustering of similarly charged peptides in SCX makes this a less optimal first dimension. From the work of Gilar et al. [11] and reproduced with permission from the American Chemical Society\nA genuine two-dimensional HILIC\u2013RP system was presented recently [12]. A microliter flow-scale ZIC\u2013HILIC system was connected offline with a nanoliter flow-scale RPLC\u2013MS. For semi-automation, a microliter-scale fraction collector was connected to the HILIC output to mix the eluent with RP- and MS-compatible aqueous buffer and to allow direct consecutive analysis of the HILIC fractions by MS. In this study, a level of orthogonality to RP was found that matched the theoretical picture presented by Gilar et al. [11]. SCX separation can be limited by the fact that similarly charged peptides cluster in narrow elution windows (see also Fig.\u00a02), and it was shown that HILIC did not exhibit this behavior [11, 12].\nAnalysis of data acquired using this two-dimensional system shed further light on how HILIC can be orthogonal to RP. Indeed, a separation mechanism was observed that is based on both retention by hydrophilicity due to partitioning with the aqueous sublayer and retention by charge due to electrostatic interaction with the stationary phase [11, 12]. These electrostatic interactions ensure that HILIC separation is more than merely the reverse of RP, while the presence of hydrophilic interactions made similarly charged peptides elute over a wider window [12].\nIt was further noted that the orthogonality of ZIC\u2013HILIC with RP is dependent on the buffer pH. At pH 3, a higher orthogonality was seen than at pH 6.8 and pH 8 [12].\nThe studies discussed so far were all performed with HILIC columns packed with particles. A further improvement in separation could be obtained by using monolithic structures instead of porous particles. The higher porosity of monolithic materials enables the use of longer columns and\/or higher flow rates, increasing the separation efficiency without increasing the backpressure [41]. Recently, attempts to develop HILIC columns that possess monolithic characteristics have been reported [41\u201345]. Horie et al. demonstrated the use of HILIC monoliths for peptide separations [42]. The authors applied increased flow rates and confirmed that short gradients of 3\u201310\u00a0min were possible with 20-cm-long 200\u00a0\u03bcm ID columns, while the peak capacities were comparable to a similar monolithic RP setup. The poor peptide sequencing speed of a mass spectrometer can be a potential drawback in the direct coupling of such columns to MS. Currently, mass spectrometers can sequence 2\u20133 peptides per second, which is significantly lower than the number of peptides that will be delivered by the monolith column. However, such columns are highly applicable as a first dimension, where time is not a parameter.\nHILIC is proving to be an attractive choice among the range of separation methods available for the proteomics researcher. The combinations chosen so far for 2D-LC\u2013MS, as reported in the literature, have been limited to HILIC\u2013RP, but the versatility of the chromatographic material will also allow combinations such as SCX\u2013HILIC and three-dimensional approaches can be envisaged. The high organic levels utilized in HILIC buffers compared to RP buffers have the additional benefit that they improve electrospray ionization, which increases the sensitivity of ESI\u2013MS [9], thus suggesting that it may be an interesting final dimension [11].\nHILIC for the analysis of PTMs\nThe analysis of protein PTMs is a very important albeit challenging task in proteomics. PTMs are generally present at much lower levels than their unmodified counterparts, which makes it very easy to \u201cmiss them\u201d in untargeted analyses, such as those performed using shotgun proteomics techniques [46]. Protein modifications such as phosphorylation and glycosylation have proven to be difficult to investigate in a routine fashion. This is mainly because these moieties are prone to elimination and adsorption if care is not taken with sample preparation and MS analysis protocols [14, 47]. Recently, some of these MS problems have been overcome by using alternative MS peptide fragmentation techniques, such as electron capture dissociation (ECD) [48] and electron transfer dissociation (ETD) [49]. Several approaches have been developed to counter some of the problems presented by the low abundance and highly labile nature of PTM peptides. The use of chelation [50\u201352] or specific chromatographic materials [47] have proven to be successful strategies, and in the latter category HILIC has proven to be a worthy addition.\nHILIC in the targeted analysis of phosphorylated peptides\nPhosphorylation is probably the most studied protein post-translational modification in proteomics; it provides an important function in signal transduction, metabolic maintenance and cell division [53]. The development of techniques to enrich for phosphopeptides has involved major efforts in phosphoproteomics. One of the more successful approaches targets the enrichment of phosphopeptides by SCX at low pH [47]. At pH\u00a03, acidic residues such as glutamic and aspartic acid are neutral while phospho-serine\/threonine\/tyrosine will still be negative. Such a distinction allows proteolytic tryptic phosphopeptides to be separated from \u201cnormal\u201d tryptic peptides due to an earlier elution. However, other peptide subgroups with reduced net charge, such as N-acetylated tryptic peptides, will also exhibit a shorter retention time, and thus a second level of enrichment is required to separate coeluting nonphosphorylated peptides from phosphorylated peptides [54]. Furthermore, the trypsinization of proteins is less efficient for regions where phosphomoieties are present [55]. Thus a large fraction of proteolytic phosphopeptides will contain miscleavages (i.e., additional basic residues), and will not be enriched. Therefore, other techniques for the targeted enrichment of phosphopeptides are required and are generally performed after SCX, such as those based on immobilized metal affinity chromatography (IMAC) [50, 54] or the metal oxide TiO2 [51, 52, 56]. One other limitation of SCX for phosphopeptide enrichment is that it is based on poor retention. This means that multiply phosphorylated peptides, another large subgroup, are difficult to retain or are even lost. The use of strong anion exchange (SAX) stationary phases immediately following SCX retention can circumvent some of these problems [57]. The generally lower pI values of phosphopeptides will allow stronger retentions on SAX than \u201cnormal\u201d peptides. However, the extra LC separation increases the analysis time and the likelihood of sample loss.\nFinally, a mixed-bed ion exchange method with both cation and anion exchange was presented recently and was shown to recover more normal tryptic peptides than SCX and to exhibit orthogonal separation towards RP [58]. Phosphopeptides could be eluted by applying a single final salt step. However, in this step only a subset of phosphopeptides\u2014the acidic ones\u2014could be resolved.\nHILIC is presented as an alternative enrichment technique, as it exploits the increase in hydrophilicity of peptides through the attachment of a phosphogroup [15, 22, 23, 27]. McNulty et al. applied a TSKgel Amide-80 based HILIC system to the phosphopeptide analysis of a Calyculin A-treated HeLa cell lysate digest. The authors demonstrated that the system, under optimal conditions, allowed phosphorylated peptides to be separated from nonphosphorylated peptides and acidic peptides that might interfere with subsequent IMAC enrichment. Interestingly, the phosphopeptides eluted in the middle of the HILIC separation and would also allow a considerable level of fractionation. This rudimentary analysis permitted more than 1000 unique phosphopeptides to be identified after further IMAC enrichment and LC\u2013MS analysis [15].\nYtterberg et al. evaluated the use of HILIC in a preparative separation set-up using HILIC material packed in micro tips [22]. Different combinations of SCX, RP and two types of HILIC [polyhydroxyethyl aspartamide (PHEA) and PolyWAX] were tested for the enrichment of phosphopeptides from a saliva digest. As expected, most of the phosphorylated peptides were retained strongly under typical HILIC analysis conditions. Furthermore, the separation power of the stepwise elution was sufficient to separate peptides with differing numbers of phosphorylated residues. The conditions used with PolyWAX are ERLIC conditions and seem to be very useful for phosphopeptide enrichment [23]. Normal tryptic peptides are simultaneously repelled by the weak anion exchange material and attracted by hydrophilic interactions, while phosphopeptides are attracted by both. This mode of separation is very responsive to salt contents and the pH of the buffer and can thus be tweaked to enrich for highly phosphorylated peptides containing up to six phosphorylated residues [22].\nIn contrast to these results obtained with TSKgel Amide-80, PHEA and PolyWAX, the enrichment of phosphopeptides was not observed with ZIC\u2013HILIC [12]. This might be explained by the zwitterionic nature of this chromatographic material: the negative charge could potentially repel the phosphate group, resulting in phosphopeptides eluting with nonphosphorylated peptides.\nThe microcolumn approach is a simple and undemanding preparative method. However, HPLC-mode HILIC has the potential for automation and fractionation, as the HILIC buffers are directly compatible with IMAC and TiO2. It might be a valuable alternative to SCX, with improved fractionation and better retention of multiply phosphorylated peptides, and so it potentially could allow a more comprehensive analysis of the phosphoproteome. While it should be noted that the application of HILIC to phosphoproteomics is still in its infancy, it is likely that the actual power of HILIC in this field will become clear in the next few years.\nHILIC in the targeted analysis of N-terminally acetylated peptides\nThe positive N-terminal charge of a protein can be neutralized by different post-translational modifications, each potentially influencing protein function, stability and interaction with other biomolecules [59]. Amongst the biologically relevant N-terminal modifications are acetylation, methylation and myristoylation [60]. As mentioned above, tryptic N-acetylated peptides can be enriched by SCX [33, 54, 61]. The acetylation neutralizes the N-terminal charge, lowering the net charge compared to the unmodified counterpart. Therefore, N-acetylated tryptic peptides will, like phosphopeptides, elute in the first few SCX fractions.\nZIC\u2013HILIC has been proposed as another means of enriching N-acetylated tryptic peptides [12]. When it contains a neutralized N-terminus, the hydrophilicity of an N-acetylated peptide is decreased. This reduction in polarity is even more pronounced at pH 3, where only basic peptide residues are charged and acidic residues are protonated. Through the use of a ZIC\u2013HILIC separation and fractionation, Boersema et al. showed that this subgroup of trypsinized peptides that are N-terminally blocked could be enriched due to their shorter retentions than \u201cnormal\u201d trypsinized peptides (see Fig.\u00a03) [12]. Similarly, other subgroups of N-terminally modified peptides, such as those that contain an N-terminal pyroglutamic acid, were also found primarily in the first ZIC\u2013HILIC fractions [12].\nFig.\u00a03Distribution of phosphorylated and N-acetylated peptides over ZIC\u2013HILIC fractions. First dimension: ZIC-HILIC, 200\u00a0\u03bcm\u2009\u00d7\u2009160\u00a0mm, 3.5\u00a0\u03bcm, 200 \u00c5. Flow rate 1.5\u00a0\u03bcL\/min, 1\u00a0min.fractions. Number of peptides: bare line, total; squares, N-acetylated; triangles, phosphorylated. ZIC\u2013HILIC provides clear enrichment of N-acetylated peptides in the initial fractions. From the work of Boersema et al. [12], reproduced with permission from the American Chemical Society\nUnfortunately, as trypsin cannot cleave acetylated lysine residues, peptides containing this modification could not be enriched. The miscleavage-containing K-acetylated tryptic peptide will possess a net charge that is the same as \u201cnormal\u201d tryptic peptides [12].\nInterestingly, and in contrast with SCX [33], no significant enrichment of C-terminal peptides was observed in this ZIC\u2013HILIC set-up, although the hydrophilicity of these peptides is also different from \u201cnormal\u201d tryptic peptides, due to the absence of a terminal arginine or lysine residue. It was hypothesized that this might be explained by the difference in charge distribution after losing an N-terminal positive charge compared to lacking a C-terminal positive charge. A C-terminal peptide would as a result be more polar\u2014and thus more hydrophilic\u2014compared to an N-terminally blocked peptide [12]. As suggested above, the ZIC\u2013HILIC method will enrich for any peptide containing a neutralized N-terminus and thus would be applicable as a first step in the enrichment of formylated, carboxylated, hydroxylated, palmitoylated and myrisotylated peptides.\nHILIC in the targeted analysis of glycosylated peptides\nGlycosylation, the attachment of a carbohydrate moiety to a protein, is a frequent but very heterogeneous PTM. It modulates the physicochemical and biological properties of proteins and serves as a recognition determinant between molecules, molecules and cells, or between cells [62]. Glycosylation of outer cell wall proteins are, for instance, involved in host\u2013pathogen interactions. It has been estimated that more than half of all proteins in a biological system are glycoproteins [63]. The heterogeneity of glycan structures makes a global proteomics characterization of glycosylation extremely difficult, as the signal intensity of glycosylated peptides with the same amino acid backbone is dispersed over peptides with different oligosaccharide chains [64]. The proteomics analysis of glycopeptides is made even more tedious by the fact that MS\/MS data rarely allow the confident identification of peptide sequences due to the relatively large molecular weights of glycosylated peptides [65] and the complex fragmentation that originates from both peptide and glycan cleavages [14]. To overcome some of these issues in proteomics analysis, the labile sugar groups are often removed by deglycosylation prior to MS analysis [66]. The problem of low glycopeptide abundance can be tackled by enrichment techniques, mostly those based on lectin-mediated affinity capture [66]. Recently, HILIC was introduced as a promising additional enrichment step for glycopeptides [14].\nThe hydrophilicity of glycopeptides makes them ideal candidates for separation by HILIC. A first glycopeptide application for HILIC was found in the analysis of differentially sialylated glycopeptides from interferon-\u03b3 [67]. Fractions of glycopeptides separated by RP were further separated by HILIC. Their orthogonal retention on the HILIC column was shown to correlate well with the number of sialyl groups. Further studies by ESI\u2013MS revealed that the interaction of N-glycans with ZIC\u2013HILIC is based on a partitioning mechanism, while the separation of differently sialylated N-glycans could be explained by an electrostatic repulsion interaction mechanism [68]. Recently, more global proteomics approaches were reported that targeted protein glycosites using HILIC to enrich glycopeptides [14, 66, 69\u201372]. Generally, in these approaches glycoproteins were first selectively captured by lectin-mediated affinity chromatography, followed by SDS-PAGE and in-gel digestion. The peptides obtained were then further enriched for glycosylation using ZIC\u2013HILIC microcolumns. The glycopeptides that were primarily found in the later fractions were then enzymatically degycosylated prior to MS analysis. Figure\u00a04 shows the relative intensities after solid-phase extraction (SPE) of a tryptic digest of TIMP-1 with RP (R2) and HILIC microcolumns. The relative abundance of glycopeptides was significantly increased following enrichment by HILIC. SPE by RP caused the matrix-assisted laser desorption\/ionization (MALDI) spectrum to be dominated by nonglycosylated peptides, while, in comparison, SPE with HILIC resulted in a spectrum crowded with glycosylated peptides [70].\nFig.\u00a04MALDI-TOF spectra after SPE of a tryptic digest of TIMP-1 by RP (R2, upper spectrum) and HILIC (lower spectrum) microcolumns. HILIC clearly enriches for glycopeptides. This figure was kindly provided by Dr. P. H\u00f8jrup (similar to [70])\nSmall diversions from this general protocol have been reported and include lectin-mediated affinity capture at the peptide level [69] and the further separation of glycopeptides by SCX [66]. It was shown that the unambiguous localization of glycosylation sites is facilitated by leaving a single GlcNac residue on the site [14] or by tagging the site using 18O isotope labeling [66, 69].\nUsing the HILIC method, 62 glycosylation sites could be identified for 37 glycoproteins in human plasma [14], while a further study of the Cohn IV fraction of human plasma revealed 103 N-glycosylation sites and 23 fucosylated N-glycosylation sites [66]. 1465 N-glycosylated sites were found on 829 proteins in C. elegans [72]. However, this study was performed using three 50\u2013200\u00a0mg samples for affinity chromatography on three different lectin columns, and each LC\u2013MS analysis was performed in triplicate [72], unlike the Cohn IV work, where a 1\u00a0mg fraction was used [66].\nThe glycosylation sites were determined in these studies, but not the actual glycan structure. A few studies have used HILIC for the desalting and enrichment [65, 70] or the separation [73] of intact glycopeptides. However, these studies all used relatively simple protein mixtures or even single proteins, indicating that the unambiguous characterization of peptide glycan structures at the proteome level is still rather challenging.\nThese early experiments show that HILIC can be used as an effective and relatively simple tool for the targeted analysis of protein glycosylation. Future studies are likely to follow, wherein HILIC might also have an important role to play in not only glycosite elucidation but also in the compositional analysis of glycan structures, as HILIC was already found to be convenient for the desalting and enrichment [64] or even the separation [74] of glycans themselves.\nHILIC in the targeted analysis of histone modifications\nThe regulation of the chromatin structure, and therefore DNA transcription and replication, is driven by post-translational modifications of the core histones. These modifications, including methylation, acetylation, phosphorylation, sumoylation and ubiquitination, are primarily found at the histone N-terminal tails [75]. Different modifications can occur at the same histone, and it is emerging that not just single site modification but rather the interdependence of these modifications is very important to regulation [76]. The analysis of histone modifications on intact histone proteins by MS is very demanding, due to the enormous heterogeneity of the modifications [77]. The separation of differentially modified proteins prior to MS is therefore essential.\nHILIC has been used in the last decade for the analysis of acetylated and methylated histones [5, 7, 21, 78\u201380]. In these studies, RP and HILIC phases are generally combined to separate different histones. In analyses where LC\u2013MS analysis is used for the characterization of histone modifications, this is performed on the peptide level. However, information on the interdependence of modifications is lost due to the hydrolysis step, because different modifications will end up at different peptides [5, 6, 79\u201381]. To preserve this information, a HILIC separation of intact histones was recently performed using a top-down MS approach, i.e., at the level of intact proteins [77, 82]. Histone H4 was purified by RP while HILIC was used for the further separation of differentially modified forms. Subsequently, these H4 HILIC fractions were analyzed by ESI\u2013Q\u2013FTMS\/MS with ECD fragmentation [82]. A similar but semi-top-down approach was performed for the analysis of H3.2. As H3.2 is not very soluble in the highly organic HILIC buffers, GluC digestion was employed to generate a 50 amino acid-long N-terminal peptide that contains most of the modification sites [77]. Figure\u00a05 clearly shows the separation power of HILIC in the analysis of histones. It simultaneously separates acetylated and methylated histones, greatly reducing the complexity of the sample. Acetylation is the major determinant of separation. The separation of proteins for which acetylation was induced by butyrate resulted in more peaks at lower retention times. The smaller peaks within these larger peaks represent proteins with different levels of methylation.\nFig.\u00a05HILIC separation of GluC-generated long N-terminal peptides from histone H3.2. HILIC primary separation is controlled by the number of acetylations (the three bigger peaks for the +butyrate sample), and within these peaks a secondary separation is observed, relating to the number of methylations (the narrower peaks). Adapted by permission from Macmillan Publishers Ltd: Nature Methods [77]\nIn these histone analyses, HILIC delivered the extra separation step that was needed to sufficiently reduce the complexity of the samples and to separate the most important histone modifications.\nConcluding remarks\nRecent years have witnessed an increased interest in HILIC. More versatile and diverse stationary phases have become available, leading to reports of an exciting and broad range of applications. The unique separation and orthogonality of HILIC towards RP (the most commonly used peptide separation method) make it an ideal method for multidimensional chromatography that can extend separation power. As far as selectivity is concerned, HILIC can compete well with RP and SCX, which are the two main chromatographic techniques applied today. Focusing solely on proteomics applications, HILIC has been shown to be very versatile in analyses of protein modifications. The retention of hydrophilic compounds can be effectively exploited in the enrichment of phosphorylated, N-terminally blocked and glycosylated peptides. Moreover, in combination with RP it allows the separation of complex differentially modified histones, an incredibly challenging area. It is therefore expected that the number of applications of HILIC in the proteomics field will increase significantly in the years to come, and that the development of novel HILIC stationary phases and\/or monolithic columns will continue. Much progress is expected.","keyphrases":["hilic","proteomics","post-translational modification","two-dimensional liquid chromatography"],"prmu":["P","P","P","R"]}
{"id":"Neuropharmacology-2-1-2194164","title":"CB1 receptor-dependent and -independent inhibition of excitatory postsynaptic currents in the hippocampus by WIN 55,212-2\n","text":"We investigated the effect of a synthetic cannabinoid, WIN 55,212-2 on excitatory postsynaptic currents (EPSCs) evoked by stimulation of Schaffer collaterals in CA1 pyramidal cells. Bath application of WIN 55,212-2 reduced the amplitude of EPSCs in dose-dependent manner tested between 0.01 nM and 30 \u03bcM. In rats and mice, this cannabinoid ligand inhibited excitatory synapses in two steps at the nM and \u03bcM concentrations. When the function of CB1 cannabinoid receptors (CB1R) was impaired, either by the application of a CB1R antagonist AM251, or by using CB1R knockout mice, WIN 55,212-2 in \u03bcM concentrations could still significantly reduced the amplitude of EPSCs. WIN 55,212-2 likely affected the efficacy of excitatory transmission only at presynaptic sites, since both at low and high doses the paired pulse ratio of EPSC amplitude was significantly increased. The inactive enantiomer, WIN 55,212-3, mimicked the effect of WIN 55,212-2 applied in high doses. In further experiments we found that the CB1R-independent effect of 10 \u03bcM WIN 55,212-2 at glutamatergic synapses was fully abolished, when slices were pre-treated with \u03c9-conotoxin GVIA, but not with \u03c9-agatoxin IVA.\n1\nIntroduction\nThe type 1 cannabinoid receptors (CB1Rs) have been shown to control the release of different neurotransmitters, but the mechanisms underlying the regulation of synaptic communication could substantially vary between brain regions (Freund et\u00a0al., 2003). Pharmacological results, suggesting a presynaptic locus of action of cannabinoid receptor ligands, have been fully supported by immunohistochemical data. Several studies demonstrated at the electron microscopic level that CB1Rs decorated both inhibitory and excitatory axon terminals (Katona et\u00a0al., 1999, 2006; Kawamura et\u00a0al., 2006). In addition, recent high-resolution quantitative studies established that CB1Rs were found all around the axon membrane, but were enriched in the perisynaptic annulus and on preterminal segments, whereas immunolabelling was weaker in the synaptic active zone (Nyiri et\u00a0al., 2005; Kawamura et\u00a0al., 2006). This subcellular distribution of CB1Rs might imply an action on several regulatory mechanisms of transmitter release, including the control of Ca2+ entry via voltage-dependent Ca2+ channels (primarily by receptors located in the perisynaptic annulus), the reduction of axonal conduction (by receptors present on the preterminal segments), or a direct action on exocytosis (Wilson et\u00a0al., 2001; Diana and Marty, 2003).\nIn spite of the direct anatomical evidence, several pharmacological observations suggest that some synthetic cannabinoid agonists (mainly WIN 55,212-2) could also have a CB1R-independent action on synaptic glutamate release. This possibility has been fuelled primarily by experiments using CB1R knockout mice. Our laboratory was the first to show that, in the absence of CB1Rs, WIN 55,212-2 was still able to reduce excitatory, but not inhibitory postsynaptic currents in CA1 pyramidal neurons (H\u00e1jos et\u00a0al., 2001). Moreover, WIN 55,212-2 was more potent in suppressing GABAergic than glutamatergic transmission (Hoffman and Lupica, 2000; Ohno-Shosaku et\u00a0al., 2002; H\u00e1jos and Freund, 2002), providing further support for the possible presence of CB1R-independent binding site at excitatory synapses. Importantly, AM251, a CB1R antagonist prevented the reduction of synaptic inhibition after application of WIN 55,212-2, whereas glutamatergic transmission could still be suppressed by about 50% in the presence of AM251 (H\u00e1jos and Freund, 2002). In contrast to the above findings showing that hippocampal glutamatergic synapses were effectively regulated independent of CB1Rs, electrophysiological data from other groups suggested that CB1Rs were solely responsible for the cannabinoid modulation of excitatory synaptic transmission in the hippocampus (Ohno-Shosaku et\u00a0al., 2002; Domenici et\u00a0al., 2006; Takahashi and Castillo, 2006).\nTo shed light on the reasons behind the contradictory findings regarding the involvement of CB1R-dependent vs. -independent mechanisms in the regulation of hippocampal excitatory synapses, we re-examined the effect of WIN 55,212-2 on\u00a0monosynaptically evoked excitatory postsynaptic currents (EPSCs) in CA1 pyramidal cells. All these experiments were performed in a modified submerged recording conditions (H\u00e1jos et\u00a0al., 2005).\n2\nMethods\nExperiments were carried out according to the guidelines of the institutional ethical code and the Hungarian Act of Animal Care and Experimentation (1998. XXVIII. section 243\/1998.). Male Wistar rats (14\u201318\u00a0days old), as well as wild type and CB1R knockout mice (15\u201325\u00a0days old, CD1 strain) were used. The animals were deeply anaesthetized with isoflurane followed by decapitation. After opening the skull, the brain was quickly removed and immersed into ice-cold cutting solution containing (in mM: sucrose 252; KCl 2.5; NaHCO3 26; CaCl2 0.5; MgCl2 5; NaH2PO4 1.25; glucose 10). The solution had been bubbled with 95% O2\/5% CO2 (carbogen gas) for at least 30\u00a0min before use. Thick horizontal slices (350\u00a0\u03bcm from mice and 400\u00a0\u03bcm from rats) were prepared using a Leica VT1000S Vibratome. The CA3 region was removed to prevent epileptic burst firings. The slices were stored in an interface type chamber containing ACSF (in mM: 126 NaCl, 2.5 KCl, 26 NaHCO3, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, and 10 glucose) at room temperature for at least 1\u00a0h before recording. After the initial incubation period, slices were transferred individually into a submerged type recording chamber.\nWhole-cell patch-clamp recordings were obtained at 30\u201332\u00a0\u00b0C from CA1 pyramidal cells visualized by infrared DIC videomicroscopy (Zeiss Axioscope, Germany). Patch electrodes were pulled from borosilicate glass capillaries with an inner filament (1.5\u00a0mm O.D.; 1.12\u00a0mm I.D., Hilgenberg, Germany) using a Sutter P-87 puller. Electrodes (\u223c3\u20136\u00a0M\u03a9) were filled with a solution containing (in mM) 80 CsCl, 60 Cs-gluconate, 3 NaCl, 1 MgCl2, 10 HEPES, 2 Mg-ATP, and 5 QX-314 (pH 7.2\u20137.3 adjusted with CsOH; osmolarity 275\u2013290\u00a0mOsm). Excitatory postsynaptic currents (EPSCs) were recorded at a holding potential of \u221265\u00a0mV. Slices were perfused with ACSF containing 70\u2013100\u00a0\u03bcM picrotoxin to block inhibitory neurotransmission. The solution was bubbled with carbogen gas at room temperature and perfused at a flow rate of 3\u20134.5\u00a0ml\/min in a slice chamber optimized for laminar flow to ensure the stability of the amplitude of evoked currents and a better oxygenation of submerged slices (H\u00e1jos et\u00a0al., 2005). To evoke EPSCs, the stimulating electrode was placed in the stratum radiatum of CA1. Pairs of electrical stimuli separated by 50\u00a0ms were delivered via a theta glass pipette (Sutter Instrument Company, Novato, CA) filled with ACSF at 0.1\u00a0Hz using a Supertech timer and isolator (Supertech LTD, P\u00e9cs, Hungary, http:\/\/www.superte.ch). Access resistances (between 4 and 18\u00a0M\u03a9, compensated 65\u201370%) were frequently monitored and remained constant (\u00b120%) during the period of analysis. Signals were recorded with a Multiclamp 700A (Molecular Devices, Sunnyvale, CA), filtered at 2\u00a0kHz, digitized at 6\u00a0kHz (National Instruments PCI-6024E A\/D board, Austin, TX), and analyzed off-line with the EVAN program (courtesy of Prof. I. Mody, UCLA, CA).\nThe drug was perfused in a given concentration until the maximal effect was reached. The time needed for maximal inhibition was usually 6\u20138\u00a0min. To avoid the possible effect of a changing pH, we added the same amount of HCl to the control solution. The concentration response relationship for WIN 55,212-2 was obtained as follows: control EPSC amplitudes in a 2\u20133\u00a0min time window were compared to those measured after 10\u00a0min drug application for the same period of time. Only those experiments were included that had stable amplitudes at least for 10\u00a0min before drug application. After each experiment, the tubing made of Teflon was washed with ethanol for 10\u00a0min and with ACSF for 15\u00a0min. Each data point represents the mean\u00a0\u00b1\u00a0SEM of the maximal inhibition of the evoked EPSCs (n\u00a0=\u00a03\u20137). EC50 values\u00a0were estimated by fitting a curve to the points of the dose response plots\u00a0obtained in rats or wild type mice using the equation of f(x)\u00a0=\u00a0a\/(1\u00a0+\u00a0exp(\u2212(x\u00a0\u2212\u00a0c)\/b))\u00a0+\u00a0(100\u00a0\u2212\u00a0a)\/(1\u00a0+\u00a0exp(\u2212(x\u00a0\u2212\u00a0e)\/d)), where \u2018c\u2019 and \u2018e\u2019 give the values for high and low affinity binding sites, respectively. The data points obtained in the presence of AM251 or in CB1 knockout mice were fitted by the equation of f(x)\u00a0=\u00a0a\/(1\u00a0+\u00a0exp(\u2212(x\u00a0\u2212\u00a0c)\/b)), where \u2018c\u2019 gives the value of EC50. The curve fitting was done using Origin 7.5 (OriginLab Corporation, MA). The paired pulse ratio was calculated from the mean amplitude of the second EPSCs divided by the mean amplitude of the first EPSCs. The paired pulse ratio after drug treatment was compared with the control using Wilcoxon matched pairs test in Statistica 6.1 (Statsoft, Inc., Tulsa, OK). Data are presented as mean\u00a0\u00b1\u00a0SEM.\nPicrotoxin, WIN 55,212-2 and WIN 55,212-3 were purchased from Sigma-Aldrich, AM251 was obtained from Tocris, while \u03c9-conotoxin GVIA and \u03c9-agatonix IVA from Alomone Labs. For all experiments, WIN-55,212-2 was dissolved in 0.1N HCl giving a 20\u00a0mM stock solution stored at 4\u00a0\u00b0C. AM251 was dissolved in DMSO (100\u00a0mM) and stored at \u221220\u00a0\u00b0C. WIN 55,212-3 dissolved in DMSO (100\u00a0mM) was stored at 4\u00a0\u00b0C. From these stock solutions, the final dilution of drugs was done in ACSF containing picrotoxin under constant stirring and the prepared solution was bath applied. In control solutions, the vehicle was diluted in the same concentration as in the solutions containing drugs. Bovine serum albumin (BSA) was added in a concentration of 0.1\u00a0mg\/ml to the solutions used for experiments with WIN 55,212-3.\n3\nResults\nThe effects of the cannabinoid agonist WIN 55,212-2 on EPSCs evoked by focal stimulation of Schaffer collaterals were measured in hippocampal CA1 pyramidal cells. First we performed concentration response analyses for the inhibitory effects of WIN 55,212-2 on evoked EPSC in rat slices (Fig.\u00a01a). WIN 55,212-2 bath applied between the concentrations of 0.1\u00a0nM and 30\u00a0\u03bcM suppressed the amplitude of EPSCs in two steps. The apparent EC50 values from the fitted curve were 2.91\u00a0nM and 3.77\u00a0\u03bcM (Fig.\u00a01c). Then we investigated the WIN 55,212-2-sensitivity of EPSCs, when AM251, a CB1R specific antagonist was added to the bath solution in the concentration of 2\u00a0\u03bcM. In spite of the presence of AM251, the cannabinoid agonist could still reduce the amplitude of evoked currents, but only in the \u03bcM range (Fig.\u00a01a). The estimated EC50 value for this effect was 1.69\u00a0\u03bcM (Fig.\u00a01c).\nIn the next set of experiments, we examined the concentration response relationship for the WIN 55,212-2-induced reduction of evoked EPSCs in mouse slices (Fig.\u00a01b). The sensitivity of synaptic currents for WIN 55,212-2 was tested between the concentrations of 0.01\u00a0nM and 30\u00a0\u03bcM. Similar to that observed in rat slices, the cannabinoid agonist also decreased the amplitude of EPSCs in two steps. The EC50 values estimated by fitting a curve to the points of the dose response plot were 1.91\u00a0nM and 12.1\u00a0\u03bcM (Fig.\u00a01c). To reveal whether WIN 55,212-2 could still suppress excitatory transmission in CB1R-independent manner in mice, we examined the effect of the cannabinoid agonist in CB1R knockout animals. As shown in Fig.\u00a01b, WIN 55,212-2 effectively reduced the amplitude of EPSCs, but only in the \u03bcM range. The apparent EC50 value estimated from the curve fitting was 8.32\u00a0\u03bcM (Fig.\u00a01c).\nThese results obtained both in rats and mice suggest that WIN 55,212-2 in nM concentrations inhibits excitatory synaptic transmission exclusively via CB1Rs, whereas in \u03bcM concentrations it has a mixed CB1R-dependent and -independent effect on glutamatergic transmission at Schaffer collateral synapses.\nBy a comparison of the paired-pulse ratios of evoked EPSCs, we next investigated whether the CB1R-independent action of WIN 55,212-2 is presynaptic, i.e. whether it is inhibiting glutamate release similar to that seen earlier for CB1Rs. We first examined the effect of 10\u00a0nM WIN 55,212-2 on the paired-pulse ratio in rats and wild type mice. After drug application, the ratio significantly increased to 132.5\u00a0\u00b1\u00a09.4% of control in rats and to 129.5\u00a0\u00b1\u00a014.2% of control and mice (Fig.\u00a02; n\u00a0=\u00a05 each, p\u00a0<\u00a00.05, Wilcoxon test). These data are in line with both electrophysiological and anatomical results, suggesting a presynaptic locus of CB1R-dependent action. To check that the changes in the paired-pulse ratio were due to the activation of CB1Rs, we contrasted these values before and after the application of 10\u00a0nM WIN 55,212-2 in the presence of AM251. As expected, the paired-pulse ratio was not altered (98.4\u00a0\u00b1\u00a06.1%, n\u00a0=\u00a06; p\u00a0>\u00a00.1, Wilcoxon test; Figs. 2a,c). Next we compared the paired-pulse ratio before and after the application of 30\u00a0\u03bcM WIN 55,212-2. The ratio of evoked currents was significantly increased to 145.1\u00a0\u00b1\u00a05.3% of control in rat slices (n\u00a0=\u00a05; p\u00a0<\u00a00.05, Wilcoxon test) and to 141.3\u00a0\u00b1\u00a011.8% in slices from wild type mice (n\u00a0=\u00a07; p\u00a0<\u00a00.05, Wilcoxon test). To reveal that the CB1R-independent action of WIN 55,212-2 also modifies transmitter release, we investigated the paired-pulse ratio after application of 30\u00a0\u03bcM WIN 55,212-2, while 2\u00a0\u03bcM AM251 was included in the bath. The ratio of the amplitude of evoked EPSCs still significantly increased, to 136.4\u00a0\u00b1\u00a08.4% of control (Figs. 2a,c; n\u00a0=\u00a07, p\u00a0<\u00a00.05, Wilcoxon test). Similar to these results, 30\u00a0\u03bcM WIN 55,212-2 also raised the paired pulse ratio to 121.1\u00a0\u00b1\u00a02.9% of control in CB1R knockout mice (Figs. 2b,c; n\u00a0=\u00a06, p\u00a0<\u00a00.05, Wilcoxon test). Thus, the CB1R-independent effect of WIN 55,212-2 also appears to be presynaptic, reducing glutamate release from Schaffer collateral terminals.\nAs reported earlier (Shen and Thayer, 1998), WIN 55,212-2 in \u03bcM concentrations could directly alter Ca2+ currents independent of CB1Rs, an effect that could be mimicked by its inactive enantiomer WIN 55,212-3. To test whether at glutamatergic axon terminals a similar mechanism would be responsible for the reduction of EPSC amplitude, WIN 55,212-3 was bath applied in two different concentrations to rat slices. This inactive enantiomer significantly suppressed the amplitude of evoked EPSCs by 42.8\u00a0\u00b1\u00a010.7% (n\u00a0=\u00a05) and 54.7\u00a0\u00b1\u00a012.1% (n\u00a0=\u00a03) in 10\u00a0\u03bcM and 30\u00a0\u03bcM concentrations, respectively (Fig.\u00a01c). These effects were indistinguishable from those values, which were obtained in the presence of AM251 after application of 10\u00a0\u03bcM (46.9\u00a0\u00b1\u00a07.8%; n\u00a0=\u00a05) or 30\u00a0\u03bcM (59.6\u00a0\u00b1\u00a03.2%; n\u00a0=\u00a04) WIN 55,212-2 (p\u00a0>\u00a00.1, Mann\u2013Whitney U-test). These results suggest that the CB1R-independent action of WIN 55,212-2 on glutamatergic transmission might be due to the direct inhibition of Ca2+ entry into the presynaptic boutons.\nTo get deeper insight into the mechanisms underlying the CB1R-independent effects of WIN 55,212-2, we specifically examined the involvement of voltage-gated Ca2+ channels in this process. Rat slices were pre-incubated either in 250\u00a0nM \u03c9-agatoxin IVA (a specific blocker of P\/Q-type Ca2+ channels) or in 250\u00a0nM \u03c9-conotoxin GVIA (a specific inhibitor of N-type Ca2+ channels) at least for an hour. After placing the pre-treated slices in the recording chamber, we bath applied 10\u00a0\u03bcM WIN 55,212-2 in the presence of 2\u00a0\u03bcM AM251. In slices pre-treated with \u03c9-agatoxin IVA, the amplitude of EPSCs was reduced by 39.7\u00a0\u00b1\u00a08.3% (n\u00a0=\u00a04, p\u00a0<\u00a00.05, Wilcoxon test; Fig.\u00a03a), whereas there was no change in the synaptic currents after application of WIN 55,212-2 in slices pre-incubated with \u03c9-conotoxin GVIA (95.8\u00a0\u00b1\u00a03.2% of control, n\u00a0=\u00a06, p\u00a0>\u00a00.1, Wilcoxon test; Fig.\u00a03b). The results of these experiments suggest that the CB1R-independent action of WIN 55,212-2 at glutamatergic synapses is mediated via inhibition of N-type Ca2+ channels.\n4\nDiscussion\nOur data presented here demonstrate that excitatory synapses of Schaffer collaterals in CA1 pyramidal cells are inhibited by WIN 55,212-2 both via CB1R-dependent and independent mechanisms. In low nM concentrations, this cannabinoid ligand solely acts as a CB1R agonist reducing glutamate release. In contrast, WIN 55,212-2 in the \u03bcM range suppresses glutamatergic synaptic transmission via activation of CB1Rs as well as inhibiting N-type Ca2+ channels independent of CB1Rs.\nShen et\u00a0al., (1996) were the first to show that excitatory transmission in the hippocampus could be reduced by WIN 55,212-2, a finding that has been strengthened later by several other laboratories (Misner and Sullivan, 1999; Al-Hayani and Davies, 2000; H\u00e1jos and Freund, 2002; Ohno-Shosaku et\u00a0al., 2002; Hoffman et\u00a0al., 2003). Similarly to that observed in the hippocampus, WIN 55,212-2 was also shown to suppress excitatory synapses in other brain regions, including the cerebellum (Levenes et\u00a0al., 1998; Takahashi and Linden, 2000), neocortex (Domenici et\u00a0al., 2006), basolateral amygdala (Azad et\u00a0al., 2003; Domenici et\u00a0al., 2006), or striatum (Gerdeman and Lovinger, 2001; Huang et\u00a0al., 2001). In earlier studies, the lack of immunostaining for CB1Rs at excitatory terminals (Katona et\u00a0al., 1999; H\u00e1jos et\u00a0al., 2000), taken together with experiments showing that WIN 55,212-2 could significantly reduce glutamate release in CB1R knockouts (H\u00e1jos et\u00a0al., 2001; Kofalvi et\u00a0al., 2003), fuelled the concept that distinct cannabinoid receptors control synaptic excitation and inhibition. This interpretation was supported by the unequivocal demonstration of high densities of CB1Rs on GABAergic axons, while adjacent glutamatergic terminals remained negative (Katona et\u00a0al., 1999; H\u00e1jos et\u00a0al., 2000; Nyiri et\u00a0al., 2005), as well as by the complete disappearance of cannabinoid sensitivity of IPSCs in CB1R knockouts (H\u00e1jos et\u00a0al., 2000, 2001; Varma et\u00a0al., 2001; Wilson et\u00a0al., 2001). Moreover, H\u00e1jos and Freund (2002) showed that AM251 could fully antagonise the effect of WIN 55,212-2 at GABAergic, but not at glutamatergic synapses, further strengthening the existence of a novel cannabinoid-sensitive binding site at hippocampal excitatory synapses. Recently this concept was substantially challenged both by anatomical and electrophysiological experiments, including studies from our own laboratory. First, using a different type of antibody, CB1Rs were convincingly shown to be present on glutamatergic terminals, although in much smaller quantities than on GABAergic axons (Katona et\u00a0al., 2006; Kawamura et\u00a0al., 2006). Specificity of the staining has been confirmed in CB1R knockout tissue. Second, excitatory transmission was found to be insensitive to the application of WIN 55,212-2 in distinct strains of transgenic mice lacking CB1Rs (Ohno-Shosaku et\u00a0al., 2002; Domenici et\u00a0al., 2006; Takahashi and Castillo, 2006). The discrepancy between earlier and recent data might be resolved by the present findings suggesting that at low nM concentrations WIN 55,212-2 specifically activates CB1Rs, whereas in the \u03bcM range the agonist could further reduce glutamate release via direct inhibition of presynaptic Ca2+ entry independent of CB1Rs (present study; Shen and Thayer, 1998; Kofalvi et\u00a0al., 2007). This difference in the specificity of WIN 55,212-2 as a function of its concentration can be noticed already in studies reported by the Kano laboratory. WIN 55,212-2 in 100\u00a0nM caused a large reduction in the amplitude of EPSCs in wild type mice, but in CB1R knockouts less then 5% suppression was found (Ohno-Shosaku et\u00a0al., 2002). However, in 2\u00a0\u03bcM concentration, WIN 55,212-2 inhibited excitatory transmission by about 20% in CB1R knockout mice, an effect that was unaltered in the presence of CB1R antagonists (Kawamura et\u00a0al., 2006). Further support to the hypothesis that WIN 55,212-2 in \u03bcM concentrations can reduce excitatory transmission via CB1R-independent mechanisms come from the work of Hoffman et\u00a0al. (2005), where the authors showed that in the presence of AM251, 3\u00a0\u03bcM WIN 55,212-2 significantly (appr. by 40%) reduced excitation. This view, however, is not supported by two recent studies using CB1R knockout animals, in which 5\u00a0\u03bcM WIN 55,212-2 was found to be completely ineffective at excitatory synapses (Domenici et\u00a0al., 2006; Takahashi and Castillo, 2006). The explanation for these negative findings remains to be investigated.\nAnother finding of the present study that deserves discussion is that the effective concentration of WIN 55,212-2 that significantly inhibited the amplitude of synaptic events in slice preparations was 100 fold lower than it was earlier reported by several laboratories using similar recording circumstances (Takahashi and Linden, 2000; Robbe et\u00a0al., 2001; H\u00e1jos and Freund, 2002; Hoffman et\u00a0al., 2005). Compared to earlier studies, we changed some conditions that could account for the distinct efficacy of WIN 55,212-2, which allowed clearly separating CB1R-dependent and independent effects. The preparation and storage of slices, as well as the flow rate of the solution during recordings was modified: slices were cut in a sucrose containing solution and stored in an interface-type chamber before recordings, and a higher flow rate was used in the recording chamber, ensuring a better oxygenation of the tissue (H\u00e1jos et\u00a0al., 2005). Under these circumstances, the amplitude of evoked synaptic currents became more stable, and, more importantly, the efficacy of WIN 55,212-2 to suppress excitatory synapses was comparable with those measured in cell cultures (Ohno-Shosaku et\u00a0al., 2002) or binding assays (Felder et\u00a0al., 1995).\nSeveral studies in different brain regions suggested that the CB1R-independent effect of WIN 55,212-2 might significantly alter synaptic communication among neurons (H\u00e1jos et\u00a0al., 2001; Pistis et\u00a0al., 2004; Kofalvi et\u00a0al., 2005; Matyas et\u00a0al., 2006), presumably through a direct blockade of Ca2+ entry at the presynaptic terminals (Shen and Thayer, 1998; Kofalvi et\u00a0al., 2007). In the present study, we provided evidence that the CB1R-independent effect of WIN 55,212-2 at glutamatergic synapses was mediated by inhibiting N-type Ca2+ channels.\nThe question arises whether WIN 55,212-2 in high doses could also alter GABAergic transmission independent of CB1Rs, since GABA release from CB1R-expressing axon terminals is known to depend on N-type Ca2+ channel activation (Wilson et\u00a0al., 2001). Indeed, we found that in CB1R knockout mice 10\u00a0\u03bcM WIN 55,212-2 substantially reduced the amplitude of IPSCs to 62.8\u00a0\u00b1\u00a020.5% of control (n\u00a0=\u00a04). In contrast, when CB1R function was intact, both low and high doses of WIN 55,212-2 led to a comparable reduction of IPSC amplitudes (in 3\u00a0nM: 47.9\u00a0\u00b1\u00a015.4% of control, n\u00a0=\u00a04; in 10\u00a0\u03bcM: 45.2\u00a0\u00b1\u00a019.7% of control, n\u00a0=\u00a04), similar to results obtained earlier (H\u00e1jos and Freund, 2002). The reason why WIN 55,212-2 in 10\u00a0\u03bcM concentration did not result in an additional suppression of inhibitory events in the presence of functional CB1Rs may be explained by the fact that CB1R-dependent inhibition of GABAergic currents is entirely due to blocking N-type Ca2+ channels, which occludes the CB1R -independent action of WIN 55,212-2 directly on the same Ca2+ channels. Testing this hypothesis, and other novel aspects of cannabinoid modulation of GABAergic transmission is the subject of another line of investigations in our laboratory.\nThe question arises whether at glutamatergic synapses, under some experimental conditions, endocannabinoids can reduce the efficacy of neurotransmission via CB1R-dependent and -independent mechanisms, similar to high concentrations of WIN 55,212-2, which can modify Ca2+ entry directly. Some results indeed imply that endocannabinoids could directly inhibit different types of voltage-gated Ca2+ channels independent of CB1Rs (Chemin et\u00a0al., 2001; Fisyunov et\u00a0al., 2006), yet other data suggest that endocannabinoids released upon depolarization of a postsynaptic neuron (or exogenously applied) are unable to suppress excitatory transmission in CB1R knockout mice (Ohno-Shosaku et\u00a0al., 2002; Straiker and Mackie, 2005, but see Rouach and Nicoll, 2003). Nevertheless, the importance of identifying a CB1R-independent binding site for WIN 55,212-2 as N-type Ca2+ channels at excitatory terminals lies in resolving some contradictions in pharmacological and behavioural studies that emerged partly due to the use of WIN 55,212-2 in widely varying concentrations (see a thorough discussion of this issue in Haller et\u00a0al., 2007).","keyphrases":["hippocampus","cannabinoids","pyramidal cell","glutamate","transmitter release","brain slices"],"prmu":["P","P","P","P","P","R"]}
{"id":"Eur_J_Health_Econ-_-_-1388086","title":"Definition of the \u201cHealth Benefit Basket\u201d in Poland\n","text":"The subject of \u201chealth benefit basket\u201d has been hotly debated for years among the Polish public, but until recently the debate has tended to be largely theoretical and abstract and therefore has lacked an effect on public policy. The situation changed in 2004, for two reasons: first the verdict of the Constitutional Tribunal invalidating the existing health insurance law and, second, Poland\u2019s accession to the European Union. The first problem was solved in part by defining a list of specific exclusions in the law and a promise to establish an institution for health technology assessment. The second issue remains open, although to some extend it is being dealt with legally by regulations issued from the Ministry of Health on acceptable waiting times for health services.\nFundamentals of the health care system\nHealth care in Poland remains the responsibility of various sectors, but it is carried out principally by the social security system. Most aspects of the general health care framework were founded in 1997 by Article 68 of the Constitution which states that: (a) Everyone has a right to health care. (b) Citizens, regardless of their economic status, shall be provided with equal access to health care services financed from the public funds by the public authorities; the conditions and the scope of services are to be detailed in the appropriate laws. (c) Public authorities are obliged to provide special health care services to children, pregnant women, disabled persons, and the elderly. (d) Public authorities are obliged to counter epidemic diseases and prevent potentially health-threatening outcomes of environmental degradation.\nPrior to 1989 the Polish health care system fell under a public integrated model, sometimes called the \u201cSemashko\u201d model, named for its Soviet ideologist, and changed gradually over time. In this system the government was both the principle insurer and the major provider of services, via health facilities owned and administered by regional representatives of the government (Voivods, \u201cGovernors\u201d). The benefit basket in a given area was determined by the physical presence or absence of specialists and equipment. The country had begun to reform the health care system as early as the 1980s, albeit rather slowly. Until the end of the 1990s the reform process focused basically on redefining the role of the state and introducing quasimarket mechanisms, greater individual freedom, and decentralized responsibility and management. On 1 January 1999 the institutions of universal health insurance commenced their activity by virtue of the Law of Universal Health Insurance of 1997, with amendments from 1998. This established 16 Regional Health Insurance (Sickness) Funds along with the Health Insurance Fund for so-called \u201cuniformed workers\u201d (army workers, policemen, railway workers) and their families, which operated at interregional level. According to the Law of Universal Health Insurance, Health Funds sign contracts with health care institutions. Regional Health Funds covered particular voivodships (administrative regions) populated by 1\u20136 million inhabitants. After 2000 there were no legal barriers to the Health Funds extending their activities to other regions. Citizens were free to choose the Health Fund irrespective of their place of living. Health Funds had become autonomous organizational and property institutions, but they still remained public and managed funds collected through premiums under the supervision of a public \u201cboards,\u201d with the principle aim of providing insofar as their budget allowed the best health care to all insured persons in their region.\nIn reality, however, there was little competition between Health Funds for patients, except in some neighboring regions when certain Health Funds began absorbing persons in adjacent areas. Still however, they were focused rather on preserving quality and \u201cproper relations\u201d with providers in their own regions. In 2001 the Social-Democratic Government initiated a centralization of the system by merging all Health Funds into a unified universal National Health Fund (NHF) with 16 regional branches. The process of recentralization of the system lasted 2\u00a0years and was burdened with substantial technical problems and shortcomings. During the period from 2001 until 2005 there were six Ministers of Health, and the Health Insurance Act was completely re-written twice, one after the intervention of the Constitutional Tribunal. One of the reasons given by the Tribunal in declaring the law unconstitutional was the lack of definition concerning \u201cconditions and scope of services\u201d to which an insured person is entitled under the health insurance law.\nRegulation of the benefit basket\nThere are various legal acts and documents defining the scope of services and conditions required for entitlement. In the hierarchy of regulations the most important and most general are those based upon the Constitution. More specific are the laws ratified by Parliament and the President, which regulate certain areas of social life and activities. The laws listed below include obligations of public organs and institutions, to provide and ensure health benefits to persons in need:Health insurance law which regulates the universal and obligatory insurance scheme that covers some 99% of Polish citizens and a large proportion of noncitizens, residents, both short- and long-term (Ustawa, 27 August 2004, r. o \u015bwiadczeniach opieki zdrowotnej finansowanych ze \u015brodk\u00f3w publicznych, Dz.U. Nr 210 z 2004 r. poz. 2135).Labor code and occupational medicine law which regulates relations between employers and employees, including the health protection measures that employers are obliged to pay (Ustawa, 26 June 1974, r. Kodeks pracy-tekst jednolity, Dz.U. nr 21 z 1998 r. poz.94 z p\u00f3\u017an.zm; (Ustawa, 27 June 1997, o s\u0142u\u017cbie medycyny pracy r., Dz.U. Nr 96 poz.593).Laws which regulate the obligations of public authorities and individuals regarding protection against and coping with specific problematic health areas, such as infectious diseases (Ustawa z, 6 September 2001, r. o chorobach zaka\u017anych i zaka\u017ceniach, Dz.U. Nr 126 z 2001 r. poz.1384 z p\u00f3\u017an.zm), mental health (Ustawa, 19 August 1994, r. o ochronie zdrowia psychicznego, Dz. U. Nr 111, poz. 535, z p\u00f3\u017an. zm.), drug (Ustawa, 24 April 1997, r. o przeciwdzia\u0142aniu narkomanii, Dz. U. z 2003 r. Nr 24, poz. 198 i Nr 122, poz. 1143), and alcohol abuse (Ustawa, 26 October 1982, r. o wychowaniu w trze\u017awo\u015bci i przeciwdzia\u0142aniu alkoholizmowi, Dz. U. z 2002 r. Nr 147, poz. 1231, z p\u00f3\u017an. zm.10) problems-which.Laws on the rescue system (Ustawa, 25 July 2001, r. o Pa\u0144stwowym Ratownictwie Medycznym, Dz. U. Nr 113, poz. 1207, z p\u00f3\u017an. zm.13) and services (Ustawa, 6 December 2002, r. o \u015bwiadczeniu us\u0142ug ratownictwa medycznego, Dz. U. Nr 241, poz. 2073 oraz z 2003 r. Nr 99, poz. 920) which regulate the functioning and financing of the national rescue system and care provided to persons in need.Social security law which establishes rules for social security coverage for various population groups, both occupationally active and nonactive (Ustawa, 13 October 1998, o systemie ubezpiecze\u0144 spo\u0142ecznych, Dz. U. Nr 137, poz. 887 z p\u00f3\u017an. zm.).Penal code which regulates the issues of crime and treatment of persons convicted of committing crimes (Ustawa, 6 January 1997, r.-Kodeks karny wykonawczy, Dz. U. Nr 90, poz. 557, z p\u00f3\u017an. zm.9).Law on foreigners which regulates the rights and obligations of non-Polish residents who are under care of Polish authorities (Ustawa, 13 January 2003, r. o cudzoziemcach, Dz. U. Nr 128, poz. 1175 oraz z 2004 r. Nr 96, poz. 959 i Nr 179, poz. 1842).Road traffic code which regulates the rights and obligations of public roads users (Ustawa, 20 January 1997, r. o ruchu drogowym, Dz.U. Nr 58 z 2003 r. poz.515 z p\u00f3\u017an.zm.).\nMany of these laws contain so-called delegations to regulations\/decrees regulating on a technical level both the scope and conditions of services available to individuals in Poland. The underlying logic of the legislative process in Poland provides that laws establish the fundamental principles and mechanisms while regulations\/decrees govern their practical application. Both are by nature obligatory, and there are legal and administrative sanctions for noncompliance. In addition to legal acts, there are additional documents referring to the benefit basket which are not of legal nature. The most important of these and the only one discussed here is a catalogue of services (so-called \u201ccontracted products\u201d) which are purchased by the NHF. The catalogues are appendices to the procurement documentation in the procedures of purchasing health services and goods within the universal health insurance system. The \u201cproducts\u201d included in the catalogues are then the subject of contracts between the NHF and the service providers, to be provided to the \u201cbeneficiaries.\u201d\nTable\u00a01 presents a list of laws and decrees which determine the benefit basket in each of the existing subsystems. The level of detail in defining the scope of the benefits to which persons are entitled, differs in the Acts. It is worth noting that in each case the Act states who is entitled (covered) to a certain kind of protection and what protection it is, sometimes also defining the circumstances and conditions under which it is available. For present purposes we can differentiate three levels of explicitness of definitions in the benefit basket:Table\u00a01 List of documents determining the benefit basket and its level of explicitnessExplicitSemiexplicitImplicit1. Health insurance law+++\u00a0\u00a0Decree on dental care+\u2013\u2013\u00a0\u00a0Decree on basic and supplementary pharmaceuticals+\u2013\u2013\u00a0\u00a0Decree on pharmaceuticals for chronically ill+\u2013\u2013\u00a0\u00a0Decree on prevention services+\u2013\u2013\u00a0\u00a0Decree on prevention services, school medicine+\u2013\u2013\u00a0\u00a0Decree on medical good and materials+\u2013\u2013\u00a0\u00a0Decree on medical transportation benefits+\u2013\u2013\u00a0\u00a0Decree on spa therapy services\u2013+\u2013\u00a0\u00a0Decree on highly specialized procedures+\u2013\u2013\u00a0\u00a0Services procurement documentation+\u2013\u20132. Mental health protection law\u2013\u2013+3. Social insurance law\u2013\u2013+\u00a0\u00a0Decree on rehabilitation measures\u2013+4. Farmers social insurance law\u2013\u2013+\u00a0\u00a0Missing decree\u2013\u2013\u20135. Penal code\u2013+\u20136. Alcohol abuse law\u2013+\u20137. Drugs abuse law\u2013+\u20138. Law on foreigners\u2013\u2013+9. National rescue system law\u2013\u2013+10. Rescue services law\u2013\u2013+11. Labor code\u2013\u2013+\u00a0\u00a0Decree on prevention measures for employees\u2013+\u2013\u00a0\u00a0Decree on prevention measures for self-employed and others\u2013+\u201312. Occupational medicine law\u2013+\u2013\u00a0\u00a0Decree on occupational medicine in defense services+\u2013\u2013\u00a0\u00a0Decree on occupational medicine in internal affairs services+\u2013\u2013\u00a0\u00a0Decree on occupational medicine in penitentiary services+\u2013\u2013\u00a0\u00a0Decree on occupational medicine in National Railways+\u2013\u201313. Infectious diseases law\u2013+\u2013\u00a0\u00a0Decree on immunization of employees+\u2013\u2013\u00a0\u00a0Decree on obligatory immunizations+\u2013\u201314. Road traffic law\u2013+\u2013\u00a0\u00a0Decree on obligatory drivers health tests+\u2013\u2013Explicit: That which is understood as providing specific medical procedures and actions (e.g., immunization for hepatitis B), namely those mentioned in the law or regulationSemiexplicit: That which outlines a certain area of health care to which beneficiaries have rights (e.g., primary care)Implicit: That which mentions generally that health care is provided, sometimes with additional formulation as all necessary, efficient, etc.In some of the Acts included in Table\u00a01 some benefits are determined in more detail and some others in less detail, which means that the level of explicitness of defining the benefit basket ranges from implicit to explicit. As a rule, regulations (decrees) are more detailed in the benefits definition than laws, and the laws are more specific than the Constitution. However, in the case of the most prominent Act, the Universal Insurance Law, the Act encloses the benefits definition on all three levels of detail.\nObligations of the public authorities derived from the legal regulations are financed from public sources, which amount to 70% of the total health expenditure in Poland. Private expenditure (estimated as 30% of the total health expenditure) is mainly out-of-pocket payment with minor supplemental role of private insurance and quasi-insurance. As regards private expenditure, no benefit basket can be discussed; the only exception is occupational medicine, discussed below.\nRoles of partners in defining the benefit basket\nParliament is the supreme legislative body in Poland. The central government, respecting (in theory) a subsidiary principle, regulates the activities of both individuals and institutions and, through its administration and agencies supervises their compliance with legal regulations. Local (regional) governments are responsible for public activities on their territories. Laws establish principles and mechanisms, while regulations\/decrees establish their practical applications. Regulations\/decrees cannot exceed the area determined by the specific law, and they are issued by each of the government ministries. With regards to the benefit basket, the National Assembly, i.e., a joint session of Sejm and Senate (lower and the upper chambers of Parliament), adopts the Constitution, among others, with its provisions dedicated to health protection. The Sejm drafts laws that must be approved by the Senate and signed by the President. The Government ministries define various parts of the \u201cbenefit basket\u201d issuing regulations to existing legal provisions.\nThe NHF defines catalogues of services (so-called \u201cproducts\u201d) which are purchased by the health insurance institution that implements universal mandatory health insurance. The process of defining the list of products was not formalized until recently, although in 2005 a formalized process of setting general conditions of contracting was established, which take places under the NHF protectorate and with participation of providers associations representing more the 10,000 professionals. The catalogues are published as appendices to the procurement documentation in the process of purchasing health services and goods within the universal health insurance system. The \u201cproducts\u201d from catalogues are later placed in contracts between the NHF and the providers of the services.\nDefinitions of the benefit basket in certain areas of health care\nHealth care is a wide area, and sometimes it is difficult to define which actions belong to it and which, for example, to social security. For the present study, the OECD functional classification (International Classification for Health Accounts\u2013Health Functions) was used to define boundaries of the health care system. Table\u00a02 presents areas\/subsystems of health care in Poland with their respective regulations regarding the benefit basket and their characteristics; the individual Acts presented in Table\u00a01 are aggregated here into following areas:Table\u00a02 Benefit basket and an explicitness of its description in distinguished areas (L law, R regulation,\nA nonlegal document,1 least precise, 3 most precise)Social health insurance systemSocial insurance systemOccupational medicineRescue systemMental health and substance abuseInfectious diseasesPrisoners, foreigners under state careLRALRALRALRALRALRALRAHC.1 Services of curative care\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u20131\u2013\u2013HC.1.1 Inpatient curative care233\u2013\u2013\u2013\u2013\u2013\u20132\u201331\u2013\u20131\u20133\u2013\u2013\u2013HC.1.2 Day cases of curative care1\u20133\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u20131\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013HC.1.3 Outpatient care2\u20133\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u20131\u2013\u20131\u20133\u2013\u2013\u2013HC.1.3.1 Basic medical and diagnostic services2\u20133\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u20131\u2013\u20131\u2013\u2013\u2013\u2013\u2013HC.1.3.2 Outpatient dental care233\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u20131\u2013\u20131\u20133\u2013\u2013\u2013HC.1.3.3 All other specialized health care2\u20133\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u20131\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013HC.1.3.9 All other outpatient curative care2\u20133\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u20131\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013HC.1.4 Services of curative home care2\u20133\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013HC.2 Services of rehabilitative care\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u20131\u2013\u2013HC.2.1 Inpatient rehabilitative care1\u20132123\u2013\u2013\u2013\u2013\u2013\u20131\u20132\u2013\u2013\u2013\u2013\u2013\u2013HC.2.2 Day cases of rehabilitative care1\u20133\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u20131\u20132\u2013\u2013\u2013\u2013\u2013\u2013HC.2.3 Outpatient rehabilitative care1\u201331\u20133\u2013\u2013\u2013\u2013\u2013\u20131\u20132\u2013\u2013\u2013\u2013\u2013\u2013HC.2.4 Services of rehabilitative home care1\u20133\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u20131\u20132\u2013\u2013\u2013\u2013\u2013\u2013HC.3 Services of long-term nursing care\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013HC.3.1 Inpatient long-term nursing care2\u20133\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u20131\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013HC.3.2 Day cases of long-term nursing care1\u20133\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u20131\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013HC.3.3 Long-term nursing care: home care2\u20133\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u20131\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013HC.4 Ancillary services to health care\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u20131\u2013\u2013HC.4.1 Clinical laboratory2\u20133\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u20131\u2013\u20132\u20133\u2013\u2013\u2013HC.4.2 Diagnostic imaging2\u20133\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u20131\u2013\u20132\u20133\u2013\u2013\u2013HC.4.3 Patient transport and emergency rescue222\u2013\u20133\u2013\u2013\u20131\u201321\u2013\u2013\u2013\u2013\u20131\u2013\u2013HC.4.9 All other miscellaneous services\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013HC.5 Medical goods dispensed to outpatients\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u20131\u2013\u2013HC.5.1 Pharmaceuticals and medical nondurables\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u20131\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013HC.5.1.1 Prescribed medicines23\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u20131\u2013\u201323\u2013\u20132\u2013HC.5.2 Therapeutic devices and medical durables\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013HC.5.2.1 Glasses and vision products23\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013HC.5.2.2 Orthopedic devices and other prosthetics23\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013HC.5.2.3 Hearing aids23\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013HC.5.2.4 Medicotechnical devices, incl. wheelchairs23\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013HC.5.2.9 All other miscellaneous medical durables23\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013HC.6 Prevention and public health services\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u20131\u2013\u2013HC.6.1 Maternal and child health223\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u20131\u2013\u20132\u20133\u2013\u2013\u2013HC.6.2 School health services223\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u20132\u20133\u2013\u2013\u2013HC.6.3 Prevention of communicable diseases123\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013233\u2013\u2013\u2013HC.6.4 Prevention of noncommunicable diseases123\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u20131\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013HC.6.5 Occupational health care\u2013\u2013\u2013\u2013\u2013\u201312\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013HC.6.9 All other miscellaneous public health services\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u20131\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013Social health insurance system, regulated by health insurance law and related decrees. Procurement documentation belongs to this system, which is not a legal act but is discussed here because of its role in the system.Social insurance system, which is a wider concept than social health insurance, but here it contains functions regulated by social insurance law and farmers\u2019 social insurance law (Ustawa z dnia 20 grudnia 1990 o ubezpieczeniu spo\u0142ecznym rolnik\u00f3w i ich rodzin, Dz.U. z 1998r. Nr 7 poz.25 z p\u00f3\u017an.zm.) and related decrees.Occupational medicine, which is a privately funded system, according to OECD definitions, often in the form of prepaid arrangements of employers, covering prevention services and often some forms of outpatient health care.Rescue system, comprising benefits resulting from regulations on national rescue system and rescue services.Mental health and substance abuse, comprising benefits for persons suffering from mental disorders and substance abuse problems.Infectious diseases, covering prevention and treatment services, partly provided on a mandatory basis.Prisoners and foreigners residing in closed centers, covering all necessary medical interventions for persons with limited freedom.Table\u00a02 describes the kind of document that regulates the benefit basket in each subsystem and the level of precision in determining the basket, which refers to the distinctions mentioned above (1 the least precise, 3 the most precise).\nSocial health insurance system\nMost health services and goods are delivered within the social health insurance system, which we therefore discuss in more detail. After lengthy debate and two major changes in the law in 1997 and 2003 and the Law of 27\/08\/2004 on health care services funded from public resources was adopted in 2004 to replace existing health insurance laws (Letter of Issue no. 210, 2004, pos. 2135). Under the new law the beneficiaries, i.e., EU citizens with insurance rights, gained the right to access the entire range of available health care services. Publicly funded services are divided into 22 different categories, for example, diagnostic tests including medical laboratory diagnostic, services aimed at health protection, prevention of diseases and injuries, early detection of diseases, including compulsory vaccinations, primary health care, outpatient specialist services, and inpatient care. The Minister of Health has several legal obligations for issuing the regulations partially forming the benefit basket. These are specified in Table\u00a01.\nAccording to Article 16 of the Act on health care services financed from public funds, the benefit basket does not cover some services, such as reimbursement of the cost of certificates required for a driving license and other medical statements or forensic expertise obtained at the request of the court or prosecutor, which are covered by separate public funds. Patients residing at nursing homes must cover the cost of food and accommodation themselves. There is a negative list of services excluded from the guaranteed public health service basket, such as vaccinations not included in the compulsory vaccination package, plastic and cosmetic surgery when it is not necessary treatment for malformation, injury, illness, and gender change operations but also treatment of epilepsy with stimulation of nervus vagus, diagnostics and treatment of the taste disorders, hyperbaric chamber in SM treatment. A full list of these services was appended to the law, which means that it must be ratified by Parliament. Moreover, according to Article 25 of the Act, the NHF does not reimburse the costs of treatment or diagnostics of an insured person that are incurred abroad unless these services were provided in accordance with Regulation 1408\/71\/EC. On the basis of these regulations and previously presented legal provisions the NHF defines the so-called \u201ccontracted products\u201d that are purchased in the scheme. The catalogue of health care products includes a number of parts\/chapters, as presented in Table\u00a03.Table\u00a03 Categories of \u201ccontracted products\u201d purchased by National Health Fund with classification by SHA categories and short description, valid for 2005SHA categoryCharacteristicsPrimary careHC.1.3.1, HC.6.1, HC.6.2, HC.6.3Provided by general practitioners, pediatricians, and internal medicine specialists. The care is supported by general-practitioner nurses and midwives. Contains also school nurse activityOutpatient care, surgicalHC.1.3.3Contains approx. 50 invasive (surgical) procedures provided by outpatient care physiciansOutpatient care, medical and diagnosticsHC.1.3.3Contains consultation services of outpatient care physicians, together with a set of diagnostic tests that must be provided by them at their own costOutpatient care, diagnosticsHC.4.2Contains 22 imaging procedures financed separatelyHospital care, generalHC.1.1Contains approx. 1400 different hospital care cases; both surgical (with invasive procedure) and medical (conservative therapy)Hospital care-radiology, nuclear medicineHC.1.1, HC.1.2Contains nuclear medicine therapies, provided with or without hospitalizationHospital care, oncological therapyHC.1.1, HC.1.2Contains chemotherapy of neoplasm, both in hospital care and day careHospital care, nononcological therapyHC.1.1, HC.1.2Contains chemotherapy for nononcological cases (e.g., hepatitis B), provided both in hospital and day careDentistry, childrenHC.1.3.2Contains approx. 40 procedures available to children (up to 18\u00a0years) onlyDentistry, generalHC.1.3.2Contains approx. 70 procedures available to all insurersDentistry, pregnant womenHC.1.3.2Contains services that are available for free to pregnant womenSpasHC.2.1, HC.2.3Contains rehabilitative and treatment services available in spas (a category of settings), both in inpatient and ambulatory mannerEmergencyHC.4.3Contains rescue and emergency transportation services, both air and groundSeparately contracted servicesHC.1.2Contains approx. 20 not otherwise classified services, mainly provided in day care (incl. hemodialysis, oxygen therapy, etc.)Psychiatric and substance abuse therapyHC.1.1, HC.1.3.3, HC.1.2Contains services provided by psychiatrists, in all kind of settingsRehabilitationHC.2.1, HC.2.2, HC.2.3, HC.2.4Contains rehabilitation services provided by physicians and physiotherapists, in all kinds of settingsLong-term careHC.3.1, HC.3.2, HC.3.3Contains long- term nursing care, but also palliative care and hospices, in all kinds of settingsHealth promotion and preventionHC.6.4Contains prevention and early detection programs for noncommunicable diseases\nThe catalogue of services is not published in a uniformed way but rather as a set of appendices to various models of contracts with various groups of providers. It is published in electronic form usually in October of the year preceding the year of contracting. Table\u00a03 was developed on a basis of an official publication of the NHF and presents an aggregate of a product list from a variety of NHF internal documents. The logic of products nomenclature reflects the way in which it is used for payment and monitoring purposes. Some parts of the catalogue are published in the form of legal acts and appendices and are rewritten into contractual conditions, for example, dentistry, spas, and medical products (including orthopedic) while others are developed fully by the NHF experts.\nOther schemes\nA number of laws regulate the other schemes providing services and goods to Polish inhabitants (see Table\u00a01). General characteristics of the remaining schemes are that they usually rely on public funds and implement social and public policy in delivering services and goods to various population groups. Some of the schemes obligatorily engage private funds, for example, occupational medicine which obliges employers to provide certain kind of services to employees. The special regulations often focus on underprivileged populations, such as the mentally ill and alcohol and drugs addicts, to make available necessary services to persons who would otherwise not receive them. It is interesting to note that many of the functions specified in these special regulations oblige the NHF to finance it, as in the case of infectious diseases.\nDiscussion\nThis study presents the legal and administrative regulations regarding health benefits to which various groups in Poland are entitled. The range of available benefits is clearly quite wide, and the justifications for respective entitlements are also varied. However, in both public and professional opinion there is no such a thing as the \u201cbenefit basket\u201d in Poland [1]. This is also frequently the reason adduced to explain why the health system works so poorly. There is no systematic research in this area, but the different parties in the system appeal for the provision of a benefit basket clearly for different reasons: patients who feel lost in the system see in the envisioned benefit basket a way to secure their rights, and professionals have claimed that the lack of explicit listing of benefits make patients demand more then necessary, which places physicians under strong pressure to prescribe and perform many services that are not eventually covered by the payer. On the other hand, explicit formulation of \u201cproducts\u201d purchased in the insurance system creates substantial controversies and criticism because some medical procedures are omitted.\nIn 1999\u20132000 the Ministry of Health and Social Welfare publicly expressed its commitment to the development of a defined basic benefit basket. This proposal was hotly disputed at the time, with a number of groups opposing the concept because it was often seen as a way of limiting available health care and thus saving money. The debate prompted the Ministry to seek outside assistance in the form of consultants\u2019 advice. The consultation project was aimed at \u201cverifying and arranging the range of services purchased by the Health Funds\u201d regarding determination of the range of guaranteed services, the so-called basic benefit basket, with specification and terms of their provision [2]. The formulated range and description of services were expected to serve as the basis for the Health Funds in drafting contracts with service providers. It was also intended to serve as a tool for performance assessment of particular Sickness Funds, made by the Health Insurance Supervisory Office and the Ministry of Health.\nIn January 2004 the Polish Constitutional Tribunal announced its finding (Trybuna\u0142 Konstytucyjny orzeczenie, 7 January 2004, r. (Sygn. Akt K 14\/03) that the existing health insurance law was unconstitutional because it failed to define with sufficient precision the scope of services that beneficiaries may expect from the national health system as mentioned in the Constitution (\u201cConditions and scope of services will be detailed in the appropriate law,\u201d Paragraph 62). The Government was given approx. 1\u00a0year to prepare a new law to fulfill this requirement.\nIn formulating the new law many different possibilities were considered. One suggestion was that the law should contain a list of medical procedures which are performed in the system. This idea was considered unrealistic, however, especially because of the short period available for drafting the law. There were strong voices to the effect that some one must distinguish clearly between those services that are provided fully free of charge within the system and those that must be paid for either partially or in full by the patients. This differentiation was made by a group of experts assembled by the Minister of Health [3]. The concept also considered the establishment of a special institution, which would make technology assessments of medical interventions and participate in the decision-making process on inclusion and exclusion of the interventions in the benefit basket for universal health insurance. Finally, because there was no prevailing opinion as to the final role of the technology assessment institution, it was not established. However, the Minister of Health was required by stipulations in the last paragraph of the insurance law to \u201cundertake efforts to establish an organizational structure, whose task would be to make health technology assessment of medical procedures, with special regard to procedures, which were subject to purchasing by the Fund.\u201d Certain changes in insurance law, namely attachment of a list of excluded procedures, together with a promise to establish an institution of health technology assessment, solved the legal controversy between Parliament and Constitutional Tribunal.","keyphrases":["poland","health services","health benefit plans","health priorities","national health programs"],"prmu":["P","P","M","M","R"]}
{"id":"Qual_Life_Res-3-1-1832149","title":"Feasibility, reliability, and validity of adolescent health status measurement by the Child Health Questionnaire Child Form (CHQ-CF): internet administration compared with the standard paper version\n","text":"Aims In this study we evaluated indicators of the feasibility, reliability, and validity of the Child Health Questionnaire-Child Form (CHQ-CF). We compared the results in a subgroup of adolescents who completed the standard paper version of the CHQ-CF with the results in another subgroup of adolescents who completed an internet version, i.e., an online, web-based CHQ-CF questionnaire.\nIntroduction\nDuring the past two decades, several measures have become available to describe generic health-related quality of life in pediatrics, but adolescent self-report questionnaires received relatively little attention until now [1, 2]. The Child Health Questionnaire (CHQ) is one of the most widely used pediatric health-related quality of life measures and has been translated into 21  languages (32  countries). There is a form for parents and also a self-report form for adolescents, the Child Health Questionnaire-Child Form (CHQ-CF) [2\u20137]. The CHQ covers physical and psychosocial aspects of health, and includes the impact of child health problems or handicaps on family life [3]. This study focuses on the evaluation of missing answers at the item level, distribution of the scale scores, reliability, and validity of the CHQ-CF in an adolescent population.\nWe expect the commonly used paper format of the CHQ and other health questionnaires to be increasingly replaced by internet versions, especially in adolescent populations that are accustomed to the use of computers and the internet [8]. From the perspective of clinicians and researchers, the advantages of using the internet include avoiding paper work, on-line data-entry, and procedures designed to reduce the number of missing answers and the length of questionnaires [9, 10].\nIn general, the mode of questionnaire administration (e.g., written questionnaire, face to face interview, telephone interview, computer questionnaire) may affect the participation rate, number of missing answers, psychometric properties, and actual scores [11\u201314]. With regard to health questionnaires, several studies demonstrated some differences between the commonly used paper versions and computer versions of the same questionnaires [15\u201317]. Especially in studies comparing paper and computer questionnaires on sensitive topics, administration via computer was found to increase reporting of e.g., drug use or unsafe sexual behaviors, as this medium is apparently perceived as providing more privacy than a paper form [18\u201320].\nWith regard to online, i.e., internet or web-based administration of health questionnaires, several studies have demonstrated that online health questionnaires are feasible in various settings, especially among adolescents [21, 22]. However, very few randomized studies have evaluated whether psychometric properties and scores differ between the paper and the internet mode of administration of the same health questionnaire [23\u201326].\nIn this study, we compared indicators of the feasibility, reliability, and validity of the CHQ-CF in a subgroup of adolescents who completed the standard paper version of the CHQ-CF with the same indicators in another subgroup of adolescents who completed a newly developed internet version of the questionnaire. Additionally, we compared the mean CHQ-CF scores and distributions of the scale scores between both subgroups. A randomized parallel group design was applied in a large adolescent population (13\u20137  years old), ensuring that both subgroups were comparable.\nThe study assessed and compared the paper and internet mode of CHQ-CF administration with regard to the following indicators:\nthe number of missing answers (indicator of feasibility),the distribution of the scale scores including mean scale scores in the whole sample and in gender and age specific subgroups,the internal consistency reliability of multi-item scales (indicator of reliability),the ability of the CHQ-CF to discriminate between subgroups with and without self-reported chronic conditions (indicator of construct validity).\nMethods\nStudy population\nIn 2003, 1,071  students in 55  classes of various educational levels in the 3rd year of seven secondary schools (13\u20137 years old) in the area of Vlaardingen (metropolitan area) and Harderwijk (rural area), The Netherlands, were invited to complete the Child Health Questionnaire Child Form (CHQ-CF). The parents and students each received written information about the study several weeks before data collection; parents could refuse their child\u2019s participation, and participation by the students was voluntary.\nData collection\nThe CHQ-CF consists of 87  items with 4, 5, or 6 response options divided over 10 multi-item scales and two single-item scales (Table 1) [3]. To reduce respondent burden, the item \u201cchange-in-health\u2013was not fielded in this study, and the CHQ-CF scales \u201crole functioning-emotional\u2013and \u201crole functioning-behavioral\u2013were combined into a single scale. The combination of the two role functioning scales is a departure from the CHQ-CF instructions that makes the test analogous to the parent form of the CHQ in this regard [3]. For each scale, items were summed up (some recoded\/recalibrated) and transformed into a 0 (worst possible score) to 100 (best possible score) scale [3]. Items on standard socio-demographic variables and the prevalence of seven chronic conditions were included in the questionnaire. From the conventional paper format, using the same wording of the items and instructions, an internet version of the questionnaire was developed through a generic internet tool using PHP (4.0.1), MySQL (3.22), and JavaScript (1.3) [27]. The internet version of the questionnaire listed the items of each CHQ-CF scale on a separate web-page. The internet version did not allow the respondent to select more than one answer to each item of the CHQ-CF and it checked the questionnaire for missing answers before the respondent could \u201clogout\u2013 If one or more of the items were not answered, the internet version prompted the respondent to go back to complete those items; but, if the user failed to \u201clogout\u2013properly, missing answers would remain.Table 1CHQ-CF scales, items per scale, and interpretation of low and high scoresaCHQ-CF ScalesNumber of itemsDescription low scoreDescription high scorePhysical functioning (PF)9Child is greatly limited in performing all physical activities, including self-care, due to healthChild performs all types of physical activities, including the most vigorous without limitations due to healthRole functioning: Emotional (RE)b3Child is greatly limited in school work or activities with friends as a result of emotional problemsChild has no limitations in schoolwork or activities with friends as a result of emotional problemRole functioning: Behavioral (RB)b3Child is greatly limited in school work or activities with friends as a result of behavior problemsChild has no limitations in schoolwork or activities with friends as a result of behavior problemsRole functioning: Physical (RP)3Child is greatly limited in school work or activities with friends as a result of physical healthChild has no limitations in schoolwork or activities with friends as a result of physical healthBodily pain (BP)2Child has extremely severe, frequent and limiting bodily painChild has no pain or limitations due to painGeneral behavior (BE)17Child very often exhibits aggressive, immature, delinquent behaviorChild never exhibits aggressive, immature, delinquent behaviorMental health (MH)16Child has feelings of anxiety and depression all of the timeChild feels peaceful, happy, and calm all of the timeSelf esteem (SE)14Child is very dissatisfied with abilities, looks, family\/peer relationships and life overallChild is very satisfied with looks, family\/peer relationships and life abilities, overallGeneral health perceptions (GH)12Child believes his\/her health is poor and likely to get worseChild believes his\/her health is excellent and will continue to be soChange in health (CH)c1Child\u2019s health is much worse now than 1  year agoChild\u2019s health is much better now than 1  year agoFamily activities (FA)6The child\u2019s health very often limits and interrupts family activities or is a source of family tension.The child\u2019s health never limits or interrupts family activities nor is family a source of tensionFamily cohesion (FC)1Family\u2019s ability to get along is rated \u201cpoor\u2013Family\u2019s ability to get along is rated \u201cexcellent\u2013aReproduced with permission [3]bThe CHQ-CF scales \u201cRole functioning-emotional\u2013and \u201cRole functioning-behavioral\u2013were merged into a single scale \u201cRole functioning-emotional\/behavioral\u2013(REB) in this studyc This single-item scale was not fielded in this study\nRandomization\nWithin each school class, students were randomly assigned to either the paper or the internet mode of administration using SPSS-generated random numbers. Students completed the questionnaires, either on paper or online in a classroom with computers linked to the internet, under the supervision of a research assistant; the students were allowed adequate privacy.\nAnalysis\nPreparatory secondary vocational education was labelled as \u201clower secondary education\u201d; secondary schools that prepare students for higher professional training as \"intermediate secondary education\", and university preparatory secondary education as \"higher secondary education\". Differences between the characteristics of the participants allocated to the paper versus the internet versions of the questionnaires were tested by Student\u2019s t-test and the \u03c72 test. We assessed the frequency of missing answers to CHQ-CF items; the difference in the number of missing answers between the two formats was assessed by the Mann-Whitney U test. We assessed the distributions of the CHQ-CF scale scores to evaluate floor and ceiling effects (\u226525% of the respondents having the lowest\/highest score) for both modes of administration. Differences between CHQ-CF scale scores by format in the total sample were assessed by Mann-Whitney U tests. Additionally, after transforming the scale scores into ranks, ANOVA was applied to test whether the mode of questionnaire administration interacted with the variables gender (male n = 432; female n = 501) and age (13\u20134  year olds, n = 399; 15\u20137  year olds, n = 534). Cohen\u2019s effect sizes, defined as d = [Mean(a) Mean(b)]\/SD, where the denominator was the square root of [(na-1)SDa2 + (nb-1)SDb2] \/ [(na-1) + (nb-1)], were applied to indicate the relative magnitude of score differences between modes of administration. Here, the letters \"a\" and \"b\" refer to the subgroups administered the paper and internet forms of the test, respectively [28]. Following Cohen\u2019s suggested guidelines, 0.20 \u2264d <  0.50 indicated a \u201csmall effect\u2013 0.50 \u2264d <  0.80 a \u201cmedium effect\u2013 and d \u22650.80 a \u201clarge effect\u2013[28]; Norman et al. have suggested that, in general, d = 0.50 can be considered as threshold for a \u201cminimally important difference\u2013(MID) [29]. Cronbach\u2019s \u03b1 was applied to evaluate the internal consistency reliability of CHQ-CF multi-item scales by format; \u03b1 of 0.70 or higher was considered to indicate sufficient internal consistency reliability [30]. We applied statistical tests of the hypothesis that the Cronbach\u2019s \u03b1 reliability coefficients of CHQ multi-item scales in the sample administered the test on paper were equal to those administered the test online [31]. We applied item-level discriminant tests to evaluate whether the CHQ-CF items represent separate domains. For each mode of questionnaire administration, we evaluated whether (on average) correlation coefficients (Pearson-r correlation coefficients) between the items and their own scale score (without the item under consideration) were higher than the correlation coefficients between these items and any other scale. The average Pearson-r correlation coefficients were calculated by applying Fisher\u2019s z transformations [32]; we tested whether the differences between the average Pearson-r correlation coefficients in the subgroup administered the paper form and in the subgroup administered the test online were statistically significant [33]. We assessed the CHQ-CF\u2019s ability to discriminate between subgroups with 0, 1 or 2, and 3 or more chronic conditions, after having transformed the CHQ-CF scale scores into ranks, by ANOVA with the independent variables \u201cnumber of chronic conditions\u2013 \"mode of questionnaire administration\u2013 and the interaction term \u201cnumber of chronic conditions\u2013\u201cmode of questionnaire administration\u2013 Cohen\u2019s effect sizes d = [Mean(a) Mean(b)]\/SD in the condition subgroup were calculated for 1 or 2 versus 0 conditions, and for \u22653 versus 0 conditions. The designations \"a\" and \"b\" refer to the subgroups without chronic conditions and those with chronic conditions, respectively [28].\nAll analyses were done using SPSS, Version 11.0.1. The medical ethical committee of the Erasmus MC-University Medical Center Rotterdam, approved the study.\nResults\nParticipants and randomization\nThe participation rate was 87%. The age range of the participants was 13\u20137  years (mean age 14.7  years; SD 0.68), 54% were female, 93% were born in the Netherlands, and the majority attended lower secondary education (Table 2). The prevalence of self-reported chronic conditions was as follows: asthma, 8%; allergies, 25%; hearing problems, 7%; visual problems, 8%; headaches or migraine, 17%; chronic lower back pain, 17%; and depression or anxiety attacks, 8% (Table 2). These characteristics were equally distributed in the groups assigned to the paper and internet versions of the questionnaires (P \u22650.05; Table 2). The demographic characteristics of the participants (age, gender, country of birth, and educational level) reflected those of the general population of Dutch adolescents [34].Table 2Characteristics of study participants (total sample: n = 933; participants assigned to the paper questionnaire:n = 475; participants assigned to the internet questionnaire: n = 458)Total study group (n = 933)Group with paper mode of administration (n = 458)Group with Internet administration (n = 475)P-valueMean (SD) or Rangen% of ParticipantsMean (SD) or Rangen% of ParticipantsMean (SD) or Rangen% of ParticipantsInternet versus paper modeDemographic characteristicsAge (years)Mean (SD)14.7 (0.68)14.7 (0.68)14.7 (0.68)0.61aRange13\u2013713\u2013713\u20137GenderWomen50154%24451%25756%0.17bBorn in the NetherlandsYes86693%44193%42593%0.90bEducational level of the schoolLower secondary education54558%27458%27159%Intermediate secondary education17919%9420%8519%0.88cHigher secondary education20922%10723%10222%Chronic conditions:AsthmaYes768%408%368%0.81bAllergiesYes22925%11825%11124%0.88bProblems with hearingYes627%306%327%0.70bProblems with seeingYes788%398%399%0.91bHeadaches or migraineYes15917%8117%7817%1.00bChronic low back painYes15917%8819%7116%0.22bDepression and\/or anxiety attacksYes748%368%388%0.72ba Student\u2019s t-testb Chi square test df = 1c Chi square test df = 2\nDifference in the number of missing answers between different modes of CHQ-CF administration\nAt the item level, use of the paper version of the CHQ-CF resulted in more missing answers (0\u2013.89% per item; mean 0.54%) compared with the internet version (0\u2013.22% per item; mean 0.04%; P <  0.01).\nCHQ-CF scores by mode of administration\nA ceiling effect was observed for four CHQ-CF scales in the subgroup that completed the paper questionnaire, and 3 CHQ-CF scales in the subgroup that completed the internet questionnaire (Table 3). Four CHQ-CF scales, i.e., \u201cgeneral behavior\u2013 \u201crole functioning-physical\u2013 \u201cmental health\u2013 and \u201cfamily activities\u2013 resulted in statistically significant, higher scores for paper versus internet administration (P <  0.05), but the effect sizes (d) were \u22640.21 (Table 3). The mode of questionnaire administration did not interact significantly with gender (P \u22650.05 regarding all scales), nor with age (P \u22650.05 regarding six scales), except for the CHQ-CF scales \u201crole functioning-emotional\/behavioral\u2013(P <  0.05), \u201cmental health\u2013(P <  0.05), \u201cself esteem\u2013(P <  0.05), and \u201cgeneral health\u2013(P <  0.01). Regarding these 4 CHQ-CF scales, administration of the paper version resulted in lower scores than online administration (or nearly equal scores in the case of \u201cmental health\u2013 in the subgroup of 13\u20134  year olds, while in the subgroup of 15\u20137  year olds, paper administration resulted in higher scores compared with internet administration; the Cohen\u2019s effect sizes (d) for these differences, regardless of sign, were \u22640.21 (data not shown).Table 3Comparison of mean scores, distributions of the scale scores, and other psychometric properties of CHQ-CF scales in subgroups with paper (n= 475) and internet modes (n = 458) of questionnaire administrationCHQ-CF scalesa (range 0\u201300)Mode of admini strationMean (SD)Paper versus internet mode of administrationRange of scores% maxd% mine25th %tile50th%tile75th%tileCronbach\u2019s alphaAverage item-own scale correlation fAverage item-other scale correlationP-value (MWU)bEffect size dcPhysical FunctioningPaper96.0 (6.9)0.370.0144\u201300560961001000.690.400.19Internet95.8 (7.2)37\u201300530931001000.720.440.18Role funct.-Emo\/behPaper89.4 (17.2)0.140.000\u201300600781001000.810.650.35Internet89.5 (15.1)0\u201300540861001000.70 g0.51 g0.28Role funct.-PhysicalPaper95.0 (12.9)0.020.0522\u2013008101001001000.860.740.30Internet94.4 (11.7)22\u201300750891001000.76 g0.60 g0.25Bodily PainPaper73.5 (22.7)0.56\u20130.050\u20130025060801000.880.800.35Internet74.7 (21.4)0\u2013002416080900.890.810.30General BehaviorPaper80.9 (10.6)0.000.2125\u201300107582880.830.440.26Internet78.6 (11.4)35\u201300007279870.850.460.26Mental HealthPaper76.5 (15.4)0.010.1214\u201300206980890.920.630.35Internet74.6 (14.8)14\u201300006777840.90 h0.600.31Self EsteemPaper74.7 (12.2)0.990.0220\u201300106875820.880.560.30Internet74.4 (11.6)25\u201300007076820.870.550.28General HealthPaper73.5 (16.5)0.590.0519\u201300306376860.820.500.28Internet72.6 (16.7)20\u201300106375860.810.580.28Family ActivitiesPaper80.0 (17.7)0.030.118\u2013001706783960.810.580.24Internet78.1 (16.9)25\u2013001306779920.780.540.24Family CohesionPaper70.6 (23.5)0.910.000\u201300182608585nana0.36Internet70.7 (23.0)0\u201300162608585nana0.33aThe CHQ-CF scales \u2018Role functioning-emotional\u2013and \u2018Role functioning-behavioral\u2013were merged into a single scale in this studybTwo-sided Mann-Whitney U test of scale scores of the group using paper, and the group using the internet version of the questionnairecDifference of mean scores divided by weighted average of SDs in groups given the paper and internet version of the questionnaires [28, 29]d\/e% of respondents with best\/worst possible score (ceiling\/floor)fEach item was correlated with the applicable ad-hoc scale without the item under considerationg\/hStatistically significant differences of Cronbach\u2019s alpha\/average Pearson-r correlation coefficients in subgroups given paper or internet questionnaires (gP \u22640.01; hP \u22640.05) [31, 33]. na Not applicable; Role funct.-Emo\/beh - Role functioning-emotional behavioral; Role funct.-Physical - Role functioning-physical\nInternal consistency reliability of scales by mode of administration\nCronbach\u2019s \u03b1s for the two formats were adequate for all CHQ-CF scales, except \u201cphysical functioning\u2013in the subgroup administered the paper version of the questionnaire (\u03b1 =  0.69). The two \u201crole functioning\u2013scales and \u201cmental health\u2013showed statistically significant, higher Cronbach\u2019s \u03b1s in the subgroup administered the paper version of the questionnaire compared with the alphas in the subgroup administered the internet version (P <  0.01, respectively P <  0.05) (Table 3). All multi-item scales, regarding both modes of administration, showed higher average (corrected) item-own scale correlation coefficients than average item-other-scale correlation coefficients. The two \u201crole functioning\u2013scales showed statistically significant, higher average item-own scale correlation coefficients in the subgroup administered the paper version of the questionnaire compared with the item-own scale correlation coefficients in the subgroup administered the internet version (P <  0.01) (Table 3).\nConstruct validity by mode of administration\nAll mean CHQ-CF scale scores were lower in the subgroup with one or two reported conditions and in the subgroup with three or more reported conditions when either was compared with the subgroup with no reported conditions. For both modes of questionnaire administration, and for all CHQ-CF scales, the more chronic conditions that were reported, the higher the effect sizes compared with the subgroup with no chronic conditions. ANOVA showed statistically significant CHQ-CF score differences by \u201cnumber of chronic conditions\u2013for all scales (P <  0.01) (Table 4). The mode of questionnaire administration did not interact significantly with the variable \u201cnumber of chronic conditions\u2013(P \u22650.05 for all scales) (Table 4).Table 4Ability of the CHQ-CF scales to discriminate between subgroups differing in the participants\u2013number of chronic conditions, for the group that was assigned to complete the paper version (n = 475) and for the group that was assigned to complete the internet version (n = 458)Number of chronic conditions per participantCHQ-CF scalesa (range 0\u201300): Mode of admini- stration0 conditions1 or 2 conditions\u2265 conditions1 or 2 versus 0 conditions\u2265 versus 0 conditionsANOVA P-value Number of chronic conditionsANOVA P-value Internet versus paper modeANOVA P-value Interaction term Number of chronic conditions by mode of questionnaire administrationn = 224 (Paper)n = 206 (Internet) Mean (SD)n = 210 (Paper)n = 217 (Internet) Mean (SD)n = 41 (Paper)n = 35 (Internet) Mean (SD)Effect size dbEffect size dbPhysical functioningPaper97 (5)96 (7)90 (10)0.240.80*Internet97 (5)95 (7)89 (12)0.280.64*0.000.950.52Role funct.-emo\/behavPaper93 (13)88 (18)77 (24)0.250.64*Internet91 (14)89 (15)79 (18)0.150.66*0.000.200.72Role funct.-physicalPaper97 (11)94 (13)91 (17)0.190.34Internet96 (10)94 (13)91 (13)0.140.340.000.070.69Bodily painPaper82 (18)70 (22)47 (24)0.52*1.45*Internet80 (18)72 (21)55 (24)0.391.03*0.000.200.32General behaviorPaper84 (9)80 (10)71 (13)0.400.98*Internet81 (10)78 (11)69 (14)0.260.90*0.000.030.65Mental healthPaper83 (10)74 (16)57 (16)0.56*1.58*Internet80 (11)72 (15)59 (18)0.501.20*0.000.260.29Self esteemPaper79 (10)73 (12)61 (12)0.52*1.44*Internet77 (9)73 (12)66 (14)0.330.80*0.000.260.06General healthPaper80 (14)71 (15)54 (14)0.57*1.82*Internet79 (13)70 (17)55 (18)0.54*1.29*0.000.740.58Family activitiesPaper83 (16)79 (18)70 (20)0.260.67*Internet81 (16)78 (17)66 (15)0.170.97*0.000.030.51Family cohesionPaper77 (21)68 (23)53 (26)0.380.89*Internet74 (20)68 (24)64 (28)0.240.380.000.180.05a The CHQ-CF scales \u2018Role functioning-emotional\u2013and \u2018Role functioning-behavioral\u2013were merged into a single scale in this studybDifference of the means divided by SD in the subgroup with chronic condition(s), where * indicates at least a \u201cminimally important difference\u2013(d \u22650.50) [28, 29]Role funct.-Emo\/beh - Role functioning-emotional behavioral; Role funct.-Physical - Role functioning-physical\nDiscussion and conclusions\nIn this study we applied a randomized design to compare the results of the Child Health Questionnaire-Child Form (CHQ-CF) administered by a paper questionnaire and by an online questionnaire. The results provided support for the feasibility, internal consistency reliability, and construct validity of the CHQ-CF scales. Both modes of questionnaire administration yielded comparable scale scores and showed comparable psychometric properties. Additionally, the study provided reference\/norm scores for clinical studies (general population of 13\u20137 year olds).\nStrengths of the current study\nThe participation rate was high. Study group characteristics (age, gender, country of birth, and educational levels) were representative of those of the general population of Dutch adolescents [34]. Randomization to either the paper or internet mode of administration of the CHQ-CF was successful with respect to the evaluated characteristics.\nLimitations\nWe applied a randomized parallel group design that allows for the comparison of indicators of feasibility, reliability, and validity at the group level between a subgroup that completed a paper version and a subgroup that completed an internet version of the CHQ-CF. However, this did not allow an evaluation of whether the same person would provide equivalent or different answers to the same CHQ-CF questionnaire administered by the alternative mode, which would require a randomized crossover design [25, 35]. Such an evaluation at the individual level requires the respondent to forget all previously provided answers at the second assessment, e.g., by waiting 1 or 2 weeks between the two measurements. It also requires that there is no effect from having previously completed a CHQ-CF questionnaire by any mode, paper or internet, at the second assessment, and that scores by the same mode of administration after a relatively short interval, in the absence of changes in health status, are exactly the same. However, in an evaluation of retesting with the same paper version of the CHQ-CF after 2 weeks, 5 out of 10 CHQ-CF scales showed statistically significant, higher scores at the second measurement with Cohen\u2019s effect sizes ranging from 0.25 to 0.40, while intraclass correlation coefficients between the first and second measurement ranged from 0.06 thru 0.84 [7]. Furthermore, in a randomized crossover design, \u201ccarry-over\u2013effects may be present, i.e., completing an internet version before a paper version may have a different effect on the second assessment, than does completing a paper version before an internet version [35]. Despite the logistical and the above-mentioned methodological challenges, we recommend future studies comparing the paper and internet versions of the CHQ-CF applying a randomized crossover design to evaluate congruency of answers to CHQ-CF items at the individual level.\nIn this study, internet and paper questionnaires were completed in a controlled environment with adequate privacy and supervision. This may not be the case during future applications. We are unaware of the impact less privacy during completion of the questionnaires may have, but this would apply to both the paper and the internet versions of the questionnaire.\nFor both modes of questionnaire administration, we did not evaluate correlations between CHQ-CF scores and a relevant parent-rated questionnaire such as the CHQ-PF50 [2, 3]. Test-retest reliability of the CHQ-CF and its responsiveness and sensitivity to changes in health were not evaluated in the current study. The CHQ-CF has a relatively large number of items; therefore, we recommend developing a shorter version in the future.\nPsychometric properties\nThe psychometric properties, with only a few exceptions, were equal between the two modes of questionnaire administration. The Cronbach\u2019s \u03b1 of the scale \u201cphysical functioning\u2013in the subgroup administered the paper version of the questionnaire was just under 0.70, and the difference with the alpha in the subgroup administered the internet version was not statistically significant.\nMissing values\nCompared with the paper version, the internet version was successful in reducing the quantity of missing data. Theoretically, differences in selective partial non-response between formats might have contributed to differences in scores that were reported in this study. In our study, in the subgroup (n = 86) that had at least one missing answer to a paper CHQ-CF item, all scale means were somewhat lower than in the subgroup (n = 389) with no missing answers, but these differences were not significant (P \u22650.05). Thus, missing answers are not a source of the observed score differences.\nScore differences between modes of questionnaire administration\nRecently, Ritter et al. found no statistically significant score differences between internet and paper modes of administration for 16 health-related measures, but the study was conducted in an opportunity sample retrieved from the internet, which limits its generalizability [23]. In a randomized internet-paper comparison among adolescents concerning various health measures other than the CHQ-CF, only one statistically significant score difference was reported among 21 topics [24]. In another randomized adolescent study, a medical consumption index and 11 indicators of fruit consumption and determinants of fruit consumption did not show statistically significant score differences between internet and paper administration of the questionnaire, except for one measure that showed small score differences between modes of administration [25]. The International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire did not show statistically significant score differences between internet and paper administration in two randomized adolescent studies [25, 26].\nIn our study, in the whole sample, the paper version resulted in slightly, yet statistically significant, higher scores for 4 of 10 CHQ-CF scales compared with the internet version. One plausible explanation is chance, since it should be considered that given multiple comparisons, there is a 1-in-20 chance of a false association for each comparison (Type I error at \u03b1 = 0.05) [36]. A commonly used Bonferroni correction for 10 comparisons would imply an adjusted \u03b1 =  0.05\/10 =  0.005 [36]; at \u03b1 =  0.005, only one score difference (regarding the scale \u201cgeneral behavior\u2013 was significant. Furthermore, given Cohen\u2019s suggested guidelines for the interpretation of effect sizes, three of the four statistically significant differences between modes of administration can be considered as negligible (d \u22640.12), and one difference regarding the CHQ-scale \u201cgeneral behavior\u2013(d = 0.21) can be considered as small [28]; all effect sizes were far below d = 0.50 that was suggested as an approximate threshold for \u201cminimally important differences\u2013by Norman et al. [29]. This study provides no explanations for the established small score differences between paper and internet administration, or for the established statistically significant, but small interaction effects of administration mode with age in the case of four CHQ-CF scales.\nConclusions\nWith increasing application of online health questionnaires rather than questionnaires on paper, especially in adolescent populations, it should be noted that comparison of results requires that the scores between these modes of administration do not show meaningful statistically significant differences. This study showed that, overall, paper and internet versions of the CHQ-CF yielded only a few, negligible or small, differences. Paper and internet modes of CHQ-CF administration may be combined in a single study, although researchers should consider the possibility of minor score differences depending on the mode of administration for some scales. We recommend repeated studies in other populations, including clinical populations, to confirm or reject our results.","keyphrases":["feasibility","reliability","validity","adolescents","health status measurement","health-related quality of life","internet questionnaire","online questionnaire","reference \/ norm scores","web-based questionnaire","child health questionnaire child form 87 items (chq-cf87)"],"prmu":["P","P","P","P","P","P","P","P","P","R","M"]}
{"id":"J_Chem_Ecol-4-1-2373414","title":"Defense-Inducing Volatiles: In Search of the Active Motif\n","text":"Herbivore-induced volatile organic compounds (VOCs) are widely appreciated as an indirect defense mechanism since carnivorous arthropods use VOCs as cues for host localization and then attack herbivores. Another function of VOCs is plant\u2013plant signaling. That VOCs elicit defensive responses in neighboring plants has been reported from various species, and different compounds have been found to be active. In order to search for a structural motif that characterizes active VOCs, we used lima bean (Phaseolus lunatus), which responds to VOCs released from damaged plants with an increased secretion of extrafloral nectar (EFN). We exposed lima bean to (Z)-3-hexenyl acetate, a substance naturally released from damaged lima bean and known to induce EFN secretion, and to several structurally related compounds. (E)-3-hexenyl acetate, (E)-2-hexenyl acetate, 5-hexenyl acetate, (Z)-3-hexenylisovalerate, and (Z)-3-hexenylbutyrate all elicited significant increases in EFN secretion, demonstrating that neither the (Z)-configuration nor the position of the double-bond nor the size of the acid moiety are critical for the EFN-inducing effect. Our result is not consistent with previous concepts that postulate reactive electrophile species (Michael-acceptor-systems) for defense-induction in Arabidopsis. Instead, we postulate that physicochemical processes, including interactions with odorant binding proteins and resulting in changes in transmembrane potentials, can underlie VOCs-mediated signaling processes.\nIntroduction\nPlants respond to herbivore damage with the release of volatile organic compounds (VOCs) that signal the presence of herbivore prey to predators and parasites and thereby serve as an indirect defense mechanism (e.g., Heil 2008). Research has demonstrated that these VOCs can also be perceived by neighboring plants or intact, systemic parts of the damaged plant (Baldwin et al. 2006; Heil 2008). Particularly, green-leaf volatiles (GLVs) have been associated with induced resistance in intact plants (Arimura et al. 2000; Engelberth et al. 2004; Farag et al. 2005; Ruther and Kleier 2005; Kost and Heil 2006;). However, little is known about the identity of VOCs that are active in this context or about a structural motif that active VOCs might have in common. In an attempt to explain the inducing activity of such VOCs on gene expression, it has been suggested that GLVs with an \u03b1,\u03b2-unsaturated carbonyl group such as (E)-2-hexenal can trigger defense responses in Arabidopsis through their activity as reactive electrophile species (Almeras et al. 2003).\nIn the present study, we used lima bean (Phaseolus lunatus L.) to search for traits that characterize defense-inducing VOCs. Lima bean responds to herbivore damage with the jasmonate-mediated production of VOCs and extrafloral nectar (EFN), an aqueous, sugar-containing secretion on nonreproductive plant organs that attracts predatory arthropods (mainly ants). The natural blend of VOCs that is released from a herbivore-damaged lima bean induces EFN secretion in intact neighboring plants (Kost and Heil 2006) and serves as a within-plant signal (Heil and Silva Bueno 2007). Among the quantitatively dominant VOCs that are released from induced lima bean, however, only (Z)-3-hexenyl acetate significantly induced EFN secretion when used as pure compound (Kost and Heil 2006). In the present study, we applied different structurally related esters to lima bean plants and monitored their EFN secretion.\nMaterials and Methods\nPlants were cultivated from seeds collected in the coastal area of Puerto Escondido, Oaxaca, M\u00e9xico (15\u00b055.596 N and 097\u00b009.118 W, elevation 15\u00a0m). Seedlings were cultivated under ambient conditions in 250\u00a0ml pots filled with soil from the original growing site. The plants were watered daily and fertilized 6\u00a0weeks after germination with commercial fertilizer: 10\u00a0ml per plant of a solution of 3\u00a0mg l\u22121 of \u201cFertilisante foliar de alta concentraci\u00f3n\u201d (Grupo Bioqu\u00edmico Mexicano, Aaltillo, Coah., Mexico). Experiments were conducted with plants of an age of 8\u201310\u00a0weeks. The following compounds were dissolved in Lanolin paste (all at 0.1\u00a0\u03bcg \u03bcl\u22121): (A) (Z)-3-hexenyl acetate, (B) (E)-3-hexenyl acetate, (C) (E)-2-hexenyl acetate, (D) 5-hexenyl acetate, (E) (Z)-3-hexenyl isovalerate, (F) (Z)-3-hexenyl butyrate, (G) 2-ethylhexanol, (H) (E)-2-hexanal, (I) decanal, and (K) nonanal (Lanolin paste and all compounds were purchased from Sigma Aldrich). Each 0.25\u00a0g paste per plant was applied on green plastic stripes (MAX Bindeband) attached to the plant in order to avoid direct contact of the paste with the plant. The plants were then packed in perforated PET foil bags (Bratenschlauch, Toppits, Minden, Germany) and in nets to protect them from EFN consumers. Amounts of VOCs released from the lanolin paste into the plants\u2019 headspace under these conditions were monitored in a parallel experiment that used a closed-loop stripping system as described previously (Kost and Heil 2006). EFN secretion was quantified from the five youngest leaves 24\u00a0h later. In short, EFN concentration was measured with a portable refractometer, and nectar volume was measured with glass capillaries (Kost and Heil 2006) to calculate the total amount of soluble solids secreted, which in the case of lima bean EFN are mainly glucose, fructose, and sucrose. Leaves were then collected and dried to calculate EFN secretion as soluble solids secreted per gram leaf dry mass and per 24\u00a0h. Plants to which Lanolin paste without any compound added had been applied served as controls (Lanolin control, LC). Per day, two groups each comprising all 11 treatments were investigated, and in total eight plants per treatment were used.\nResults and Discussion\nAmounts of VOCs released from the lanolin paste into the plant\u2019s headspace resembled those released from an induced plant, i.e., all individual VOCs were present at 80\u2013130% of the amount of (Z)-3-hexenyl acetate that is released under comparable conditions from five induced leaves of lima bean (data not shown, for amounts of VOCs released from an induced plant see Kost and Heil 2006). The different VOCs to which lima bean plants had been exposed affected their EFN secretion significantly (General linear model: F77,10\u2009=\u200910.05, P\u2009<\u20090.001). Compounds A\u2013F consisted of acyl hexenols in which the configuration and position of the double bond was systematically shifted from the polar head to the aliphatic terminus of the molecule. Moreover, the importance of the size of the acyl moiety was evaluated by using acetates, butyrate, and isovalerate of (Z)-3-hexenol. Although only one of the tested VOCs is naturally released from lima bean, exposure to the six compounds (A\u2013F) elicited EFN secretions that were significantly higher than those of the controls (LSD posthoc analysis: P\u2009<\u20090.05, see Fig.\u00a01). These compounds were (Z)-3-hexenyl acetate, (E)-3-hexenyl acetate, (E)-2-hexenyl acetate, 5-hexenyl acetate, (Z)-3-hexenyl isovalerate, and (Z)-3-hexenyl butyrate. Hence, EFN secretion by lima bean can be induced by VOCs that are not released naturally from this species, while several VOCs that are released from induced plants did not significantly change EFN secretion in previous experiments (Kost and Heil 2006).\nFig.\u00a01EFN secretion in \u03bcg soluble solids secreted per gram leaf dry mass and per 24\u00a0h is depicted. Plants were exposed for 24\u00a0h to (A) (Z)-3-hexenyl acetate, (B) (E)-3-hexenyl acetate, (C) (E)-2-hexenyl acetate, (D) 5-hexenyl acetate, (E) (Z)-3-hexenyl isovalerate, (F) (Z)-3-hexenyl butyrate, (G) 2-ethylhexanol, (H) (E)-2-hexanal, (I) decanal, and (K) nonanal dissolved in Lanolin paste. Plants to which pure Lanolin paste had been applied served as controls (LC). Lower and upper whiskers represent the 5% and the 95% percentile, lower and upper margins of boxes the 25% and the 75% percentile, the lines within boxes indicate medians. Different letters appearing above boxes mark treatment effects that differ significantly from each other (P\u2009<\u20090.05 according to LSD posthoc analysis), and structures of compounds leading to a significant induction of EFN secretion as compared to the control are graphically presented. LogP values of all compounds were calculated with ChemDraw Ultra 6.0 and are given under the structures (compounds A\u2013F) and in the insert (G\u2013K)\nHow are VOCs perceived by plants, and via which mechanisms do they affect defense expression patterns? It has been suggested that molecules with an \u03b1,\u03b2-unsaturated carbonyl group can trigger defenses in Arabidopsis through their activity as reactive electrophile species (Almeras et al. 2003). In principle, VOCs could also be perceived by binding to specific receptor-proteins or to odorant binding proteins with a preference for a class of compounds, similar to animal olfactory systems.\nApparently, neither the configuration nor the position of the double bound is a critical factor. Compounds reported to prime or induce gene activity or phenotypic defenses in intact corn plants comprise (Z)-3-hexen-1-ol, (Z)-3-hexenal, and (Z)-3-hexenyl acetate (Engelberth et al. 2004; Farag et al. 2005; Ruther and Kleier 2005). We found that (Z)-3-hexenyl acetate, (E)-3-hexenyl acetate, (E)-2-hexenyl acetate, and 5-hexenyl acetate all elicited particularly high EFN secretion rates in lima bean. The majority of these substances lack an \u03b1,\u03b2-unsaturated carbonyl group, which thus cannot be a generally required motif. In addition, a compound such as 5-hexenyl acetate cannot yield an electrophile such as (E)-2-hexenal via the sequence of ester hydrolysis, oxidation, and isomerization, as it is principally possible for (E or Z)-3-hexenyl acetate or (E or Z)-2-hexenyl acetate. Moreover, unlike (Z)-3-hexenyl acetate the potential end product of the transformation sequence, (E)-2-hexenal, had only a weak effect on the nectar flow. Similarly, the observation that (Z)-3-hexenyl acetate and (E)-3-hexenyl acetate elicited almost identical EFN secretion rates contradicts the involvement of classical receptor proteins, since these usually require a specific stereochemistry of the interacting molecule. We further calculated LogP values to check for putative importance of the compound\u2019s lipophilicity but did not find any clear relation between the EFN-inducing activity of a compound and its octanol-water partition coefficient (Fig.\u00a01).\nMore studies are required to understand which VOCs induce plant defenses via which mechanism. However, our results point to a new mechanism. Changes in transmembrane potentials\u2014occurring through modulations of ion fluxes\u2014are involved in early signaling events in the cellular response to stress (Maffei et al. 2007), and exposition to VOCs indeed changes membrane potentials in intact lima bean leaves (M. Maffei, personal communication). It is thus tempting to speculate that the dissolving of VOCs in the membranes coupled to interactions with membrane proteins, similar to the odorant binding proteins of insects (Campanacci et al. 2001), leads to changes in transmembrane potentials and thereby induces gene activity. This hypothesis gains support from the observation that the induction of EFN secretion by VOCs appears to be a gradual one rather than a clear \u2018yes-or-know\u2019 response. Several of the compounds that are released naturally from lima bean showed a trend towards an increase in EFN secretion, although the difference was not significant: EFN secretion in response to Linalool was on average higher by 40% than in control plants, and DMNT ((3E)-4,8-dimethylnona-1,3,7-triene) increased EFN secretion by 50% (see Table 1 in Kost and Heil 2006). Similarly, plants exposed to ethylhexanol or (E)-2-hexanal in the present study showed a trend towards higher EFN secretions than controls (Fig.\u00a01). EFN responds to a comparably wide variety of structures, and slight changes in the molecular structure gradually alter the induction effect. This observation is best explained by a comparably simple physicochemical process, whose detailed nature remains to be elucidated.\nHow can signals evolve that apparently lack chemical specificity? The answer might be simply that plants seldom are exposed to GLVs or chemically related compounds that are not released from attacked plants. In evolutionary terms, the probability of this situation was probably too low to cause any selection towards a more specific signal perception.","keyphrases":["herbivore-induced volatiles","indirect defense","signal","hexenyl acetate","induced defense","plant-plant communication"],"prmu":["P","P","P","M","R","M"]}
{"id":"Anal_Bioanal_Chem-4-1-2324126","title":"Application of at-line two-dimensional liquid chromatography\u2013mass spectrometry for identification of small hydrophilic angiotensin I-inhibiting peptides in milk hydrolysates\n","text":"A two-dimensional chromatographic method with mass spectrometric detection has been developed for identification of small, hydrophilic angiotensin I-inhibiting peptides in enzymatically hydrolysed milk proteins. The method involves the further separation of the poorly retained hydrophilic fraction from a standard C18 reversed-phase column on a hydrophilic interaction liquid chromatography (HILIC) column. The latter column is specifically designed for the separation of hydrophilic compounds. Narrow fractions collected from the HILIC column were analysed for their angiotensin I-converting enzyme (ACE) inhibiting potential in an at-line assay. Fractions showing significant inhibition of ACE were analysed by LC\u2013MS for structure elucidation. With this method the main peptides responsible for ACE-inhibition in the hydrophilic part of a milk hydrolysate could be determined. The ACE-inhibiting peptides RP, AP, VK, EK, and EW explained more than 85% of ACE-inhibition by the hydrophilic fraction.\nIntroduction\nHypertension is one of the major health problems in the western world. A variety of food ingredients that help to reduce blood pressure have been described. Particularly attractive are hydrolysed proteins from fermented milk containing specific peptides that can inhibit the angiotensin I-converting enzyme (ACE), an enzyme involved in blood-pressure regulation [1\u20133]. For the development of optimized anti-hypertensive food products, it is crucial to know which specific peptide(s) in the complex protein hydrolysate is (are) responsible for the ACE-inhibition.\nIdentification of active ingredients in complex mixtures is generally done using assay-guided repeated fractionation. For the specific case of ACE-inhibiting peptides in milk-protein hydrolysates often only part of the total activity of the product can be assigned to specific peptides [4]. A significant part of the remaining activity is present in fractions that are poorly retained by the standard reversed-phase columns used in the fractionation procedure. Nakamura et al. [1] for example could only explain part of the activity of a casein hydrolysate by the tri-peptides VPP and IPP eluting in one of the later fractions. The remaining activity was present in the unretained fraction. In our laboratory similar samples were seen to have over 30% of the total activity in the unretained fraction. Identification of the oligopeptides in this fraction was not further pursued but would have been extremely difficult because the fraction contains far too many compounds for unambiguous activity assignment. Moreover, there are also too many interfering compounds present in the first-eluting fractions for reliable MS identification. Similar situations are also described by other authors [5\u20137]. In a previous investigation we presented a new at-line method for the identification of ACE-inhibiting (oligo)peptides in hydrolysed caseinate [4]. Approximately 70% of the activity of these samples was caused by peptides nicely retained by the two reversed-phase columns used in the fractionation. Twenty percent of the activity was present in the hydrophilic fraction. The remaining 10% was caused by a large number of peptides with low ACE-inhibiting activity. Identification of the peptides in the hydrophilic fraction was impossible due to the large number of peptides present and the presence of co-eluting compounds, such as salts and sugars, that resulted in severe suppression of ionisation. Two reversed-phase columns were used in an at-line 2D set-up to provide improved resolution of the very complex mixture. Unfortunately, the highly hydrophilic peptides escaped identification since they were unretained on both columns. To identify the peptides responsible for the ACE inhibition of the unretained hydrophilic fraction improved chromatographic retention and separation is required. Hydrophilic interaction chromatography (HILIC) could be an attractive way of achieving this.\nThe use of HILIC for separation of hydrophilic substances such as proteins, peptides, and nucleic acids was first described by Alpert in 1990 [8]. Since then HILIC has been used for the separation of many different substances, for example denaturants [9], polar oligomers [10], and pharmaceutical ingredients [11]. Many publications can be found describing the increased retention of hydrophilic peptides on HILIC columns in comparison with reversed-phase columns. A detailed review is published by Yoshida [12]. Literature describing the application of reversed-phase separation in combination with HILIC chromatography is scarce. A notable exception is the compositional analysis of an enzymatic protein hydrolysate obtained from acid-deaminated wheat gluten by Schlichtherie-Cerny [13]. The hydrolysate was first fractionated by GPC. The fraction corresponding to a molecular weight range of 100\u2013750Da was then further fractionated by RPLC. Finally, the peptides in the unretained peak from the RPLC column were separated and characterised by HILIC\u2013API-MS. Using this method ten amino acids, eight di-peptides and one tri-peptide, were identified in this fraction.\nIn this paper we describe the development of a method for the identification of ACE inhibiting peptides in the poorly retained fractions of a reversed-phase fractionation of an enzymatically hydrolysed milk protein. The possibility of improving chromatographic resolution by using a HILIC separation as a second-dimension separation after the first reversed-phase isolation is studied. The aim of the new method is to extend the applicability of a previously developed system for the identification of ACE-inhibiting peptides in complex peptide mixtures such as protein hydrolysates [4]. The applicability of the method is demonstrated by the identification of hydrophilic ACE-inhibiting peptides in an enzymatically hydrolysed milk protein.\nExperimental\nChemicals\nThe model peptides VPP, IPP, and LPP (purity >98%) were purchased from Bachum (D\u00fcbendorf, Switzerland). All other peptides were synthesised by JPT Peptide Technologies (Berlin, Germany). For HPLC analysis, acetonitrile, formic acid, and trifluoroacetic acid were purchased from Merck (Amsterdam, The Netherlands). Aqueous ammonia (Merck) was used for neutralization of the HPLC fractions. For instrument tuning PEG 300, PEG 600, and PEG 1000 were purchased from Sigma Chemicals (St Louis, MO, USA). Ammonium acetate was obtained from Merck. For the at-line assay angiotensin I-converting-enzyme (ACE) and hippuryl\u2013histidyl\u2013leucine (HHL) were purchased from Sigma\u2013Aldrich Chemie (Zwijndrecht, The Netherlands). PBS buffer was purchased from Gibco (Paisley, UK). The hydrolysed milk powder was an enzymatically hydrolysed milk protein obtained from Calpis (Tokyo, Japan).\nInstrumentation\nAll peptide separations and identifications were performed with a Waters Alliance 2795 HT HPLC coupled to a Micromass QTOF-Ultima hybrid time-of-flight mass spectrometer equipped with a lock spray option for accurate mass determination (Waters, Almere, The Netherlands). Fractions were collected in polypropylene 300-\u03bcL 96-well plates (Nunc, Roskilde, Denmark) using a Mark IV fraction collector also from Waters. Solvent evaporation was performed using an Ultravap 96-well evaporation device (Porvair, Shepperton, UK). Quantification of HHL and HL in the at-line assay was performed on the QTOF instrument described above.\nTwo-dimensional analytical separation\nThe first-dimension reversed-phase separation was performed on two serially connected 150 \u00d7 2.1mm Inertsil 5 ODS3 columns, particle size 5\u03bcm, operating in the gradient mode (Varian, Middelburg, The Netherlands). The guard column was a 20 \u00d7 3.9mm SymmetryShield RP8 column packed with 5-\u03bcm particles (Waters, Etten-Leur, The Netherlands). Mobile phase A consisted of 0.1% TFA in Milli-Q water. Mobile phase B consisted of 0.1% TFA in acetonitrile. The initial eluent composition was 100% A. After a 5min hold a linear gradient was started to 5% B in 15min, followed by a linear gradient to 70% B in 75min. Finally, mobile phase composition was programmed to 99% B in 1min and kept at 99% B for another 4min. At the end of the gradient the eluent composition was reduced to 100% A in 6min and the system was allowed to re-equilibrate for 14min. The total run time was 120min. The eluent flow rate was 0.2mL min\u22121 and the column temperature was 60\u00b0C. In-house experiments revealed that at this temperature isomeric peptides such as IPP and LPP are separated. UV detection was performed at 215nm and 254nm. Fractions of 200\u03bcL were collected in 96-well plates. After neutralization and solvent evaporation the residues were reconstituted in 50\u03bcL of an 80:20 mixture of solvents A and B for the second-dimension HILIC analysis.\nThe second dimension HILIC analysis was performed on a 150 \u00d7 2.1mm HILIC Atlantis column with a particle size of 3\u03bcm (Waters, Etten-Leur, The Netherlands). Here solvent A consisted of 0.1% formic acid in acetonitrile and solvent B of 10mmol L\u22121 aqueous ammonium acetate solution+0.1% formic acid in Milli-Q water. The separation was performed at 40\u00b0C, the maximum temperature specified by the manufacturer, and a flow rate of 0.2mL min\u22121. The initial eluent composition was 95% A. A linear gradient was used to 70% B in 30min, followed by a linear gradient to 90% B in 5min where it was kept for another 5min. At the end of the run the eluent was reduced to 95% C in 2min. The eluent flow was then increased to 0.3mL min\u22121 in 1min. This flow rate was maintained for another 7min. Finally the flow was reduced to 0.2mL min\u22121 in 1min. and the system was allowed to equilibrate for 3min. The total run time was 54min. 45\u03bcL of each fraction collected from the ODS3 column was injected. At the outlet of the second dimension HILIC column fractions of 200\u03bcL were collected in 96-well plates. The fractions were neutralized, evaporated, and transferred to the activity assay (next section).\nAt-line Matsui assay\nACE inhibition by the collected fractions was measured in a 96-well plate assay according to the at-line method described in an earlier publication [1]. The method is based on the conversion of hippuryl\u2013histidyl\u2013leucine (HHL) into hippurate (H) and histidyl\u2013leucine (HL) by the angiotensin I-converting enzyme (ACE). Briefly, after evaporation of the eluent the fractions were reconstituted in 50\u03bcL PBS buffer and vortex mixed for 1min. This solution (40\u03bcL) was used for the at-line assay. Percentage inhibition was calculated from the peak areas of HHL and H at 280nm. For the active fractions the remaining 10\u03bcL was used for identification of the peptides.\nStructural identification of ACEI peptides\nPeptide identification was performed on the QTOF MS instrument using HPLC\u2013MS and MS\u2013MS in the accurate mass mode. Fractions collected after the second dimension that showed activity were separated on the HILIC column now coupled to the MS using the gradient described above. The source and desolvation temperatures were 100\u00b0C and 300\u00b0C, respectively. The cone and desolvation gas flows were 100L h\u22121 and 800L h\u22121, respectively. The capillary voltage was 4kV and the cone voltage 35V. The collision energy was 10eV in full-scan mode and 25eV in MS\u2013MS mode. The collision gas was argon and the analyser pressure was 4 \u00d7 10\u22125 mbar. In the lock spray probe a mixture of PEG 300, PEG 600, and PEG 1000 dissolved in 10mmol L\u22121 ammonium acetate in methanol\u2013water 1:1 (v\/v) at concentrations of 0.01, 0.02, and 0.04\u03bcg mL\u22121, respectively, was used. The reference flow was 5\u03bcL min\u22121. Spectra were recorded at a resolution of 10,000. Masslynx software (Waters) was used for data acquisition and evaluation.\nResults and discussion\nIn a reversed-phase fractionation of a protein hydrolysate a significant percentage of the activity can be present in the unretained hydrophilic fraction. To be able to calculate the contribution of this fraction of our milk hydrolysate to the total activity, an ACE-inhibition profile of the entire product was produced. To this end 20\u03bcL of a 20mg mL\u22121 solution of the milk hydrolysate in solvent A was injected on to the 300mm ODS3 column. Fractions of 200\u03bcL each were collected from 0 to 65min, with an interval time of 1min, in a 96 well plate. The ACE inhibition of each fraction was determined. Figure 1a shows the MS TIC of the fermented milk and Fig.\u00a01b shows the activity profile of the same analysis. From the data in Fig.\u00a01b it was calculated that the hydrophilic compounds in fractions 6 to 15 represent approximately 25% of the total ACE inhibition. MS\u2013MS showed that the high activity in fractions 27 to 31 resulted from the known ACE inhibiting peptides IPP and LPP. The activity in fractions 21 to 23 resulted from the peptide VPP. These peptides have been identified to be the main contributors to the ACE-inhibition of a Lactobacillus helveticus-fermented milk [2] and represent 40% of the total ACE inhibition. The remaining activity between fractions 16 and 40 is most likely caused by a mixture of ACE-inhibiting peptides of different chain lengths. This is still under investigation. In the region from fraction 6 to fraction 15 a large number of peptides co-elute and the spectra of the fractions are highly complex. Figure 2, for example, shows the mass spectrum of fraction 7 collected from the first dimension ODS 3 columns and the ion traces of three of the ACE-inhibiting peptides discussed later in this manuscript. At least 16 ions can be observed with intensities above 10% of the base peak, all representing different peptides. In order to be able to assign the measured activity to one or more of these peptides a further separation in an additional chromatographic dimension is required. To achieve this, fractions collected from the first-dimension ODS column were injected on the second dimension HILIC column. Again 200-\u03bcL fractions were collected in 96-well plates. After neutralization and evaporation the residues were reconstituted in 50\u03bcL Milli-Q water. Of this, 40\u03bcL was used for ACE inhibition testing, the remaining 10\u03bcL for MS identification. As an example of the results of these experiments, Fig.\u00a03 shows the TIC chromatogram obtained from full-scanning analysis of fraction 7 of the ODS3 columns, now separated on the second dimension HILIC column. The TIC chromatogram clearly shows that the composition of this fraction is indeed very complex. To identify the peptide(s) responsible for the ACE-inhibition, the fractions collected in the 96-well plate were subjected to the ACE-inhibition assay. Figure 4 displays the activity distribution of ODS fraction 7. The data in Fig.\u00a04 are corrected for the background recorded using a blank gradient, i.e. a gradient without injection. Blank analysis of the HILIC column showed slightly negative values compared with a reference of pure PBS. The reason for these negative responses is still unknown. The results in Fig.\u00a04 clearly show the strong activity in HILIC fractions 17 to 19 and somewhat lower activity in fractions 12 to 16 from the HILIC column. LC\u2013MS separations were performed on these fractions and the MS data were searched for peptides in the two active regions. In Fig.\u00a05 the mass spectrum of fraction 18 is given. In the spectrum three abundant ions can be observed. A library search and MS\u2013MS measurements revealed that m\/z 272.1721 represents the molecular ion of the di-peptide RP (\u22120.7ppm), while m\/z 253.1190 represents the molecular ion of HP (\u22122.1ppm) and m\/z 235.1305 the loss of water from m\/z 253.1190. Identification of the peptides in the remaining fractions of the HILIC column showed that fraction 7 of the first-dimension ODS3 column consisted of three free amino acids, 19 di-peptides and 2 tri-peptides while six compounds remained unidentified. The positively identified amino acids were E, Q, and K and the di-peptides were QD, KY, ER, RE, KP, HP, RP, AP, VK, EK, EW, and PH. The di-peptides ET, TP, TQ, PQ, KV, KE, and HK and the tri-peptides APK and VRG were tentatively identified. Structure confirmation was based on elution time, measured exact mass (error <5ppm) and MS\u2013MS fragmentation pattern compared with those of the model compounds. Using this method the remaining hydrophilic fractions 5 to 15 collected from the ODS3 column were also analysed in the 2D mode. In Fig.\u00a06 a three-dimensional plot of the activity distribution over the fractions of the two columns is given. In total, five amino acids, 35 di-peptides, 13 tri-peptides, one penta-peptide and 18 not yet identified compounds were found. The identities of all amino acids and the sequence of 27 di-peptides were again confirmed by use of model compounds. Table\u00a01 lists the sequences of the identified peptides together with their reported IC50 values and ACE inhibition data at 20\u03bcmol L\u22121 established in house (Foltz et al. manuscript in preparation). For peptides for which no model compounds were available, identification was based solely on the fragmentation pattern in MS\u2013MS.\nFig.\u00a01LC separation of a 20\u00a0mg mL\u22121 solution of the milk hydrolysate powder on the ODS-3 reversed-phase column. a MS-TIC chromatogram. b Activity profile. Error bars indicate the standard deviation (n\u2009=\u20093). Fraction volume 200\u00a0\u03bcLFig.\u00a02Mass spectrum of fraction 7 collected from the first-dimension ODS3 columnFig.\u00a03MS-TIC of fraction 7 from the ODS3 column analysed on the HILIC column. The inset shows the selected ion traces of three of the most active peptidesFig.\u00a04ACEI profile of fraction 7 from the ODS3 column analysed on the HILIC columnFig.\u00a05Mass spectrum of fraction 18 collected from the HILIC columnFig.\u00a06Three-dimensional display of the ACEI distribution of the fractions collected from the ODS3 column and the HILIC columnTable\u00a01Peptides identified in fractions 6 to 15 of the ODS3 column, analysed on the HILIC columnelution (min) timepeptide sequenceIC50 value (\u03bcM) literatureLiterature referenceInhibition at 20\u00a0\u03bcM (%)peak area (counts)elution time (min)peptide sequencevalue (\u03bcM) literatureLiterature referenceInhibition 20\u00a0\u03bcM (%)peak area (counts)11.64Fn.r.\u00a0\u00a08316.34FP1215.713n.m.63111.90Yn.r.\u00a0\u00a0227416.41HSMn.r.\u00a0\u00a065612.02EIn.r.\u00a038.2222116.43EPFn.r.\u00a0\u00a018612.36IEn.r.\u00a03.7459116.43DKIn.r.\u00a0\u00a018712.40YP72013n.m.5816.52KY1.63,7.8,1316,13,17n.m.1312.59EVn.r.\u00a025.462216.66VYP28813\u00a012512.59VEn.r.\u00a041.02217.00EWn.r.\u00a065.1157812.59IP1291317.710017.22VK12.91357.216712.84VP5751345.980117.23ERn.r.\u00a0n.m.23613.21En.r.\u00a0n.m.28817.30SGYn.r.\u00a0\u00a062413.26TEn.r.\u00a026.034417.35MEn.r.\u00a024.64213.44LQn.r.\u00a011.274817.55Kn.r.\u00a0n.m.49713.74KP16,22,3013,1533.714417.61EKn.r.\u00a041.833713.81VNEn.r.\u00a0\u00a0137817.84REn.r.\u00a0n.m.16614.08LNn.r.\u00a014.727117.86KEn.r.\u00a013.48314.10Qn.r.\u00a0n.m.51117.89KV33\u00a0n.m.23514.10QDn.r.\u00a0n.m.79118.04VVRn.r.\u00a0\u00a011414.28TDVENn.r.\u00a0\u00a0201918.08HPn.r.\u00a011.1142714.50PTn.r.\u00a03.1100118.18KVPn.r.\u00a0\u00a04514.56NVPn.r.\u00a0\u00a0274418.24VPQn.r.\u00a0\u00a050514.61YQn.r.\u00a0n.m.3618.63RP21,91,1821336.4134014.64SPPn.r.\u00a0\u00a033518.97PHn.r.\u00a00.030614.72TQn.r.\u00a00.017519.03QPn.r.\u00a0n.m.40214.86AHn.r.\u00a07.310119.11KP16,22,3013,1533.614414.91PP2284.7180.0175919.22APKn.r.\u00a0n.m.26615.01AP29,26913,1011.481120.18VRGn.r.\u00a0n.m.31315.18PQn.r.\u00a00.0104120.47HKn.r.\u00a00.0398The sequences of the bold printed peptides were confirmed by MS\u2013MS analyses of reference peptides, n.r., not reported in the literature; n.m., not measured\nTo verify whether the identified peptides indeed fully explain the measured activity of the hydrophilic fraction the concentration of all peptides should be determined. Next a mixture should be prepared which is then analysed using the two-dimensional method described in this article. The ACE-inhibition profile of this mixture should be identical with that of the sample. Such an experiment will hardly be feasible because it requires the availability of all the components. To estimate of the completeness of identification, the contribution of each peptide was estimated from the measured peak area and the IC50 value obtained from literature or from unpublished in-house data. To estimate the contribution of an individual peptide the following relationship was used: CA \u2248 Ap\/IC50p. In this equation CA represents the contribution of the peptide to the measured ACE-inhibition, Ap the peak area, and IC50p the IC50 value of the peptide. Clearly this proportionality equation is only approximate as it assumes that the MS response for all peptides is identical. When both the peak area and the IC50 value are taken into consideration it can be concluded that the di-peptides AP, RP, VK, and EK are the main peptides responsible for the measured ACE-inhibition of the hydrophilic fractions 6 to 10 collected from the ODS3 column (Fig.\u00a07). In Fig.\u00a08 the activities of all peptides in each fraction are summarized and the profile is compared with that of the measured profile. The two profiles are similar. The differences between the graphs can be explained from the fact that no correction was applied for differences in MS responses of the various peptides.\nFig.\u00a07Distribution of the ACE-inhibiting activity, calculated from Table 1, in hydrophilic fractions 5 to 10 collected from the ODS3 columnFig.\u00a08Calculated (bottom) and measured (top) ACE inhibition profiles\nThe remaining fractions 11 to 15 of the ODS3 column were analysed in the same way as fractions 6 to 10. These analyses showed that the peptide EW is responsible for the major part of the ACE-inhibition measured in these fractions. In total, the five peptides identified in the fractions 6 to 15 are responsible for approximately 85% of the activity measured in the hydrophilic fractions. The most important contribution is that of the di-peptide RP, which is responsible for 34%. Together with the peptides VPP, IPP, and LPP now approximately 65% of the total activity of the product has been explained. The remaining 35% is distributed over a large number of peptides with relatively low overall activities.\nWhen compiling the list of published IC50 values for the peptides identified in this study in some cases large differences were seen for the IC50 values of proline-containing peptides in different publications. The reported IC50 values for AP, for example, vary between 29 and 269\u00a0\u03bcmol L\u22121 [14]. This large difference might be caused by variations in the cis-to-trans ratio of the proline present in synthetic model compounds as a result of differences in the synthetic routes (Fmoc or Boc), as was shown for the peptide DKIHP by G\u00f3mez-Ruiz [15]. In our calculations the trans-Pro value of 29\u00a0\u03bcmol L\u22121 for AP was used, because trans-Pro is known to be dominant in natural products [15]. Many of the identified di-peptides were found in milk hydrolysates for the first time. For most of the newly identified ACE active peptides it is actually the first time they are reported at all. The long list of newly identified peptides found here clearly demonstrates the potential of the two dimensional separation approach of HILIC and reversed-phase HPLC described here.\nConclusions\nTwo-dimensional liquid chromatography in combination with mass spectrometry was successfully used for identification of poorly retained peptides present in enzymatically hydrolysed milk protein. A standard C18 reversed-phase column was used for the first separation followed by a second dimensional separation on a HILIC column. This two-dimensional procedure significantly improves the separation of hydrophilic peptides that elute almost unretained on a reversed-phase column and co-elute with numerous other compounds such as carbohydrates and salts. These compounds suppress the MS ionization and complicate identification. The method enabled the identification of hydrophilic peptides in complex mixtures. In the hydrophilic fraction of the milk hydrolysate investigated 71 compounds were found, including five free amino acids, 35 di-peptides, 12 tri-peptides, one penta-peptide, and 18 not yet identified compounds. Five peptides, RP, AP, VK, EK, and EW were responsible for approximately 85% of the measured activity of the hydrophilic fraction; of these RP made the highest contribution of 34%. None of the five peptides has, to our knowledge, been reported earlier in milk hydrolysates or related products such as yogurt or cheese. Most of the peptides involved remained undetected in single-dimensional chromatography.","keyphrases":["reversed-phase","hilic","ms","hplc","angiotensin-converting enzyme","blood pressure-lowering peptides"],"prmu":["P","P","P","P","M","M"]}
{"id":"Pflugers_Arch-4-1-2226063","title":"Release of cardiac troponin I from viable cardiomyocytes is mediated by integrin stimulation\n","text":"Elevated cardiac troponin-I (cTnI) levels have been demonstrated in serum of patients without acute coronary syndromes, potentially via a stretch-related process. We hypothesize that this cTnI release from viable cardiomyocytes is mediated by stimulation of stretch-responsive integrins. Cultured cardiomyocytes were treated with (1) Gly\u2013Arg\u2013Gly\u2013Asp\u2013Ser (GRGDS, n = 22) to stimulate integrins, (2) Ser\u2013Asp\u2013Gly\u2013Arg\u2013Gly (SDGRG, n = 8) that does not stimulate integrins, or (3) phosphate-buffered saline (control, n = 38). Cells and media were analyzed for intact cTnI, cTnI degradation products, and matrix metalloproteinase (MMP)-2. Cell viability was examined by assay of lactate dehydrogenase (LDH) activity and by nuclear staining with propidium iodide. GRGDS-induced integrin stimulation caused increased release of intact cTnI (9.6 \u00b1 3.0%) as compared to SDGRG-treated cardiomyocytes (4.5 \u00b1 0.8%, p < 0.001) and control (3.0 \u00b1 3.4%, p < 0.001). LDH release from GRGDS-treated cardiomyocytes (15.9 \u00b1 3.8%) equalled that from controls (15.2 \u00b1 2.3%, p = n.s.), indicating that the GRGDS-induced release of cTnI is not due to cell necrosis. This result was confirmed by nuclear staining with propidium iodide. Integrin stimulation increased the intracellular and extracellular MMP2 activity as compared to controls (both p < 0.05). However, despite the ability of active MMP2 to degrade cTnI in vitro, integrin stimulation in cardiomyocytes was not associated with cTnI degradation. The present study demonstrates that intact cTnI can be released from viable cardiomyocytes by stimulation of stretch-responsive integrins.\nIntroduction\nTroponins are myofibrillar proteins involved in the regulation of actin\u2013myosin interaction, thereby controlling contraction and relaxation. Cardiac troponins (cTnT, cTnT, and cTnC) are predominantly bound, via tropomyosin, to actin filaments of sarcomeres, with only a small proportion of cTnT (6\u20138%) and cTnI (3\u20138%) found in the soluble cytoplasmic pool [1]. Due to high cardiac specificity, serum concentrations of cTnI and cTnT are well-established diagnostic and prognostic markers of irreversible myocardial damage in acute coronary syndromes [9]. However, elevated serum levels of cardiac troponins have also been observed in patients without acute coronary syndromes in whom irreversible myocardial cell injury was not a prominent aspect [8, 11, 20]. Several studies reported elevated serum levels of troponins in patients with cardiomyopathy [29], heart failure [18], unstable angina pectoris [14], pulmonary embolism [17], renal insufficiency [5], and in ultra-endurance athletes [19]. The exact mechanism underlying the release of troponins in the absence of necrosis, without lethal disruption of the sarcolemma, remains to be elucidated and most likely differs from the release of troponins due to necrotic cell death.\nA potential explanation for the cTnI release without lethal sarcolemmal disruption is the cellular release of proteolytic cTnI degradation products. Indeed, in isolated rat hearts, Feng et al. [6] showed degradation of cTnI upon increasing preload, which was independent of ischemia. They proposed that the cTnI degradation might be caused by increased myocardial stretch per se.\nMechanical stretch of cardiomyocytes, as occurs during pressure or volume overload, initiates a cascade of intracellular signals, including increased intracellular calcium concentration, increased intracellular NO formation, and the activation of intracellular proteases such as MMP2 and MMP14 [28, 36]. MMP2 is able to degrade cTnI intracellularly [39] and may be involved in the stretch-induced release of cTnI and its degradation products. In contrast, other studies suggest that cTnI leaks from reversibly damaged cardiomyocytes as an intact non-degraded molecule [8, 20].\nMechanical stretch of cardiomyocytes is sensed by stimulation of integrins, which are transmembrane glycoprotein receptors that link the extracellular matrix (ECM) to the intracellular cytoskeleton [26]. Integrins act as mechanotransducers and bidirectional signaling molecules and participate in overload-induced hypertrophic and post-infarct remodeling [26]. The Arg\u2013Gly\u2013Asp (RGD) sequence that is present in fibronectin and other ECM proteins is an agonist for integrins [27], which can be used to simulate myocardial mechanical stretch in vitro.\nWe hypothesize that the stretch-related process through which cTnI can be released from viable cardiomyocytes is mediated by integrin stimulation. The purpose of the present study is therefore to investigate whether (1) RGD-induced integrin stimulation causes release of intact and\/or fragmented cTnI from cardiomyocytes in the absence of necrotic cell death, (2) integrin stimulation is associated with MMP2 activation and MMP2-related cTnI degradation, and (3) cTnI release in the absence of necrosis differs from the release of cTnI in the presence of necrosis.\nMaterials and methods\nPrimary cultures of neonatal cardiomyocytes\nPrimary cultures of cardiomyocytes were prepared from the ventricles of 2-day-old Wistar rats as described previously [24]. Briefly, ventricles were minced, and cells were isolated enzymatically using collagenase type I (CLS, Lakewood, NJ, USA) at 37\u00b0C. The cell suspension was centrifuged, and the cell pellet was resuspended in growth medium containing Ham\u2019s F10 (Flow Laboratories, Irvine, UK), 10% fetal bovine serum, 10% horse serum (HS), 100\u00a0U\/ml penicillin, and 100\u00a0\u03bcg\/ml streptomycin (all from Invitrogen, Breda, The Netherlands). The cells were seeded in 6-cm diameter Primaria\u00ae-coated plastic culture dishes (Falcon, Becton Dickinson, Etten-Leur, The Netherlands), and after 45\u00a0min, non-adherent cells representing the cardiomyocytes were collected and plated in six-wells plates (\u2205 35\u00a0mm per well, Costar, Corning, NY, USA) at a density of \u22481.5\u2009\u00d7\u2009106 cells per well. Cardiomyocytes were incubated in a humidified incubator at 37\u00b0C and 5% CO2 and culture medium [1:1 (v\/v) mixture of DMEM (Invitrogen) and Ham\u2019s F10 supplemented with 5% HS, penicillin (100\u00a0U\/ml) and streptomycin (100\u00a0\u03bcg\/ml)] was refreshed after 20\u00a0h and 48\u00a0h. After 3\u00a0days of culturing, a monolayer of spontaneously beating cardiomyocytes had formed. The investigations had the approval of the Animal Experiments Committee of the LUMC according to Dutch law.\nExperimental protocol for integrin stimulation\nThree days after cell isolation, cardiomyocyte cultures were washed twice in HEPES-buffered salt solution (HBSS), containing (in millimole per liter) NaCl (125), KCl (5), MgSO4 (1), KH2PO4 (1), CaCl2 (2.5), NaHCO3 (10), d-glucose (5), HEPES (20), pH\u00a07.4 at 37\u00b0C. Cardiomyocytes were incubated for 24\u00a0h in 1\u00a0ml of HBSS supplemented with (1) the pentapeptide Gly\u2013Arg\u2013Gly\u2013Asp\u2013Ser containing the GRGDS sequence (Sigma, St. Louis, MO, USA) at various concentrations (100, 200, or 300\u00a0\u03bcg\/ml) to stimulate integrins, with (2) Ser\u2013Asp\u2013Gly\u2013Arg\u2013Gly (SDGRG, the reverse sequence of GRGDS, Sigma), which does not affect integrin function, at a concentration of 300\u00a0\u03bcg\/ml, or with (3) phosphate-buffered saline (PBS) as a control. After 24\u00a0h, medium was separated from the cells, cells were taken up in 1\u00a0ml of ice-cold lysis buffer (100\u00a0mmol\/l Tris\u2013HCl, 0.1% Tween, pH\u00a07.5), both cell and medium samples were kept on ice awaiting assay of lactate dehydrogenase (LDH) activity on the same day, and subsequently stored at \u221220\u00b0C for later cTnI and MMP2 assays.\nTroponin-I ELISA\nPurified human heart cTnI (Calbiochem, LaJolla, CA, USA) was dissolved in urea\/Tris buffer according to the manufacturer\u2019s instructions. Monoclonal mouse anti-cTnI clone 19C7 (directed against amino acid sequence 40\u201350) and monoclonal mouse anti-cTnI clone 6F9 (directed against amino acid sequence 189-195) were purchased from HyTest (Turku, Finland). The enzyme-linked immunosorbent assay (ELISA) was based on the sandwich principle as described previously [13]. The percentage cTnI released from each culture equalled . Detection limit of the assay was 6\u00a0ng\/ml. Inter-assay variability was 6 and 10% at a TnI concentration of 200 and 25\u00a0ng\/ml, respectively. The intra-assay variability was 6% at TnI concentrations of 100\u00a0ng\/ml.\nWestern blot analysis\nProtein extracts were size-fractionated on NuPage Novex 12% Bis\u2013Tris gels (Invitrogen) and transferred to Hybond PVDF membranes (Amersham Biosciences, Roosendaal, The Netherlands). Non-specific binding sites were blocked using blocking solution consisting of 20\u00a0g\/l ECL advance blocking agent (Amersham Biosciences) in TBS-Tween (10\u00a0mmol\/l Tris\u2013HCl, 150\u00a0mmol\/l NaCl, 0.05% Tween pH\u00a08.0). Membranes were incubated for 1\u00a0h with the anti-TnI antibody (K83085G, Biodesign, Saco, ME, USA), which detects various cTnI degradation products. After four washes in TBS-Tween, membranes were incubated with horseradish peroxidase-labeled secondary antibody (rabbit anti-goat IgG, Santa Cruz Biotechnology, Heidelberg, Germany). Chemiluminescence was induced by ECL advance detection reagent (Amersham Biosciences) and detected by exposure to Hyperfilm ECL (Amersham Biosciences).\nLDH activity assay\nNecrotic cell death was quantified by assay of LDH activity released from cells into medium according to Wroblewski and Ladue [41]. LDH activities of medium and cell samples were determined using a spectrophotometer (Ultrospec 3000, Pharmacia Biotech, Roosendaal, The Netherlands) at 25\u00b0C. The total LDH activity in each culture, i.e., cell+medium, was determined, and the LDH activity in cell and medium samples was expressed as a percentage of total LDH activity. The detection limit of the LDH assay was 5\u00a0U\/l, the intra-assay variability was 3% (110\u00a0U\/l) and 6% (48\u00a0U\/l), and inter-assay variability was 12% (110\u00a0U\/l).\nNuclear staining with propidium iodide\nAn additional assessment of cell death was performed by quantifying the nuclei of necrotic cells using propidium iodide and fluorescence microscopy. For this purpose, cardiomyocyte cultures were treated with PBS, GRGDS, and SDGRG for 24\u00a0h, then incubated with propidium iodide (5\u00a0mg\/l, Molecular Probes, Eugene, OR, USA) for 15\u00a0min in the dark, washed in PBS, and viewed under a fluorescence microscope (Zeiss, Hamburg, Germany) at 535\/617-nm excitation\/emission. Subsequently, cultures were incubated with digitonine (10\u00a0\u03bcmol\/l) for 5\u00a0min to induce necrotic cell death in all cardiomyocytes. The extent of necrosis equalled the number of necrotic nuclei before digitonine incubation as a fraction of the number of necrotic nuclei after digitonine incubation (100%).\nIn vitro degradation of purified cTnI by active MMP2\nTo study whether active MMP2 is able to degrade cTnI in vitro, purified human cardiac TnI (500\u00a0pg\/\u03bcl, Calbiochem) was incubated with human recombinant active MMP2 (1.5\u00a0ng\/\u03bcl, Oncogene, San Diego, CA, USA) in enzyme buffer at 37\u00b0C for 0, 60, and 120\u00a0min. In a separate series of experiments, MMP2 was preincubated with an inhibitor of MMP2, o-phenanthroline (100\u00a0\u03bcmol\/l) for 15\u00a0min. As a control, cTnI was incubated without active MMP2 at 37\u00b0C. Intact cTnI and cTnI degradation products were visualized by Western blotting.\nZymography\nGelatinolytic activities of MMP2 in cell and medium samples of control, GRGDS- and SDGRG-treated cardiomyocytes were analyzed by zymography as described previously [12, 33]. Briefly, cell samples (tenfold concentrated by freeze-drying for 18\u00a0h) and medium samples (unconcentrated) were prepared in sample buffer and were loaded on gels. After electrophoresis, the gels were washed twice in 2.5% Triton X-100, were washed in enzyme buffer, and were incubated in enzyme buffer at 37\u00b0C overnight. Gels were stained with amino black and subsequently destained. Gelatinase activity was quantified by measuring the extent of gelatin digestion using a scanning densitometer (2202 Ultrascan, LKB, Paramus, NJ, USA).\nExperimental protocol for necrotic cell death\nThree days after cell isolation, cardiomyocyte cultures were washed twice in HBSS, pH\u00a07.4, at 37\u00b0C, and subsequently incubated in 1\u00a0ml of HBSS, supplemented with sodium azide (1\u00a0mmol\/l, n\u2009=\u20097) at 37\u00b0C. After 24\u00a0h of incubation, medium was separated from the cells, cells were taken up in 1\u00a0ml of ice-cold lysis buffer, both cell and medium samples were kept on ice awaiting assay of lactate dehydrogenase (LDH) activity on the same day, and subsequently stored at \u221220\u00b0C for later cTnI assay.\nStatistics\nResults are expressed as mean\u2009\u00b1\u2009SD. Statistical analysis was performed by Student\u2019s t test and by one-way analysis of variance followed by Bonferroni\u2019s post hoc test. Differences were regarded as statistically significant if p\u2009<\u20090.05. SPSS14 for Windows (SPSS, Chicago, IL, USA) was used for statistical analysis.\nResults\nEffect of integrin stimulation on release of cTnI\nCell and medium samples were assayed for intact cTnI by ELISA (Fig.\u00a01a) and for cTnI degradation products using Western blotting (Fig.\u00a02a\u2013d). Cellular cTnI content in ten cultures was 0.27\u2009\u00b1\u20090.10\u00a0\u03bcg per culture (100%). Control cardiomyocytes had released \u22483% of total cTnI, probably as a result of cell death of a few cardiomyocytes during 24\u00a0h of incubation. GRGDS treatment at a concentration of 100 or 200\u00a0\u03bcg\/ml did not show considerable release of cTnI (\u22482%). However, a concentration of 300\u00a0\u03bcg\/ml GRGDS resulted in a significant extra release of intact cTnI (to \u224810%), compared with control cardiomyocytes (p\u2009<\u20090.001) and cardiomyocytes treated with 100 or 200\u00a0\u03bcg\/ml GRGDS (both p\u2009<\u20090.001; Fig.\u00a01a). Incubation of cardiomyocytes with 300\u00a0\u03bcg\/ml SDGRG, the reverse sequence of GRGDS that has no effect on integrin function, did not induce a significant extra release of intact cTnI (Fig.\u00a01a). In addition, Fig.\u00a02b and d shows that cTnI was released as an intact protein of 29\u00a0kDa and that integrin stimulation is not associated with the release of cTnI degradation products.\nFig.\u00a01a Release of cardiac troponin-I (cTnI) from cardiomyocytes incubated with PBS (control, n\u2009=\u200938), GRGDS (100\u00a0\u03bcg\/ml, n\u2009=\u20095; 200\u00a0\u03bcg\/ml, n\u2009=\u20094; 300\u00a0\u03bcg\/ml, n\u2009=\u200922), or SDGRG (300\u00a0\u03bcg\/ml, n\u2009=\u20098), detected by ELISA. b Release of LDH from cardiomyocytes incubated with PBS (control, n\u2009=\u200938), GRGDS (300\u00a0\u03bcg\/ml, n\u2009=\u200922), or SDGRG (300\u00a0\u03bcg\/ml, n\u2009=\u20098). (*p\u2009<\u20090.001 vs control, #p\u2009<\u20090.001 vs GRGDS 100, $p\u2009<\u20090.001 vs GRGDS 200, \u2021p\u2009<\u20090.001 vs GRGDS 300, and \u00a7p\u2009<\u20090.001 vs SDGRG 300)Fig.\u00a02Western blots of intact cTnI (29\u00a0kDa) and cTnI degradation products present in cell samples (left, 2.5\u00d7 diluted) and medium samples (right, 10\u00d7 concentrated) of cardiomyocytes incubated with PBS (control), GRGDS (300\u00a0\u03bcg\/ml, a and b), SDGRG (300\u00a0\u03bcg\/ml, c and d), or sodium azide (1\u00a0mmol\/l, e and f) for 24\u00a0h\nEffect of integrin stimulation on cell viability\nNecrotic cell death was quantified by the release of LDH activity from cardiomyocytes into the medium (Fig.\u00a01b). Cellular LDH activity of ten cultures was 0.47\u2009\u00b1\u20090.24\u00a0U per culture (100%). In control cardiomyocytes, \u224815% of total LDH was released in 24\u00a0h, which confirms previous findings of cellular necrosis in cardiomyocytes after 24\u00a0h in serum-free medium [25]. LDH release from integrin-stimulated cardiomyocytes (GRGDS, 300\u00a0\u03bcg\/ml) equalled LDH release from control cardiomyocytes (Fig.\u00a01b). Surprisingly, cardiomyocytes treated with SDGRG (300\u00a0\u03bcg\/ml) for 24\u00a0h showed significantly less LDH release (9.5%) than control and GRGDS-treated cardiomyocytes (both p\u2009<\u20090.001).\nAssessment of cell death using nuclear staining with propidium iodide showed a similar pattern. The percentage of necrotic cells in controls (7.2\u2009\u00b1\u20092.1%, n\u2009=\u20098) equalled that of GRGDS-treated cardiomyocytes (8.5\u2009\u00b1\u20092.2%, n\u2009=\u20098, p\u2009=\u2009N.S.). Again, the percentage of necrotic cells in SDGRG-treated cardiomyocytes was significantly lower (6.2\u2009\u00b1\u20091.9%, n\u2009=\u20098) compared with GRGDS-treated cardiomyocytes (p\u2009<\u20090.05), confirming the results of the LDH assay.\nThese results demonstrate that integrin stimulation by GRGDS participates in the release of cTnI from cardiomyocytes in the absence of necrosis.\nEffect of integrin stimulation on MMP2 activity\nIn control experiments, we verified that recombinant active MMP2 is able to degrade purified cTnI in vitro in a number of fragments of 26, 14 and 8\u00a0kDa (Fig.\u00a03). However, as we observed no cTnI degradation upon integrin stimulation (Fig.\u00a02), we studied whether this was due to the lack of MMP2 activation during integrin stimulation, measuring the zymographic gelatinolytic activities of MMP2. In control cardiomyocytes, gelatinolytic MMP2 activities were detected in both cell and medium samples, indicating a baseline level of MMP2 in cultured cardiomyocytes (Fig.\u00a04). Total MMP2 levels were higher in medium than in cells (\u224890 vs \u224810% of total MMP2, respectively).\nFig.\u00a03Western blot of intact cTnI (29\u00a0kDa) and cTnI degradation products after in vitro incubation of purified intact cTnI without and with active MMP2 and with active MMP2 in combination with a MMP2 inhibitor (o-phenanthroline) for 0, 60, and 120\u00a0minFig.\u00a04Gelatinolytic activities of MMP2 isoforms in cell samples (left, 10\u00d7 concentrated) and medium samples (right, unconcentrated) of cardiomyocytes incubated with PBS (control, n\u2009=\u200912), GRGDS (300\u00a0\u03bcg\/ml, n\u2009\u2265\u20099), or SDGRG (300\u00a0\u03bcg\/ml, n\u2009\u2265\u20093) for 24\u00a0h\nIn cells samples, several gelatinolytic activities of MMP2 were detected corresponding to a rodent-specific glycosylated MMP2 (75\u00a0kDa), proMMP2 (72\u00a0kDa, the major gelatinase activity), an active intermediate MMP2 (64\u00a0kDa), and active MMP2 (62\u00a0kDa; Fig.\u00a04, left). GRGDS-treated cardiomyocytes demonstrated significantly higher levels of cellular proMMP2 (72\u00a0kDa) and active MMP2 (64\u2009+\u200962\u00a0kDa) than control and SDGRG-treated cardiomyocytes (Fig.\u00a04, left), whereas levels of glycosylated MMP2 (75\u00a0kDa) were unaffected by GRGDS. In medium samples (Fig.\u00a04, right), several gelatinolytic activities of MMP2 were detected, corresponding to a rodent-specific glycosylated MMP2 (75\u00a0kDa), proMMP2 (72\u00a0kDa), and active MMP2 (62\u00a0kDa), respectively. GRGDS-induced integrin stimulation resulted in significantly higher medium levels of glycosylated MMP2 (75\u00a0kDa) and active MMP2 (62\u00a0kDa) compared to medium samples of control and SDGRG-treated cardiomyocytes (both p\u2009<\u20090.05; Fig.\u00a04, right). Medium levels of proMMP2 (72\u00a0kDa) were unaffected by integrin stimulation.\nRelease of cTnI from necrotic cardiomyocytes\nTo compare the release of cTnI from viable cardiomyocytes during GRGDS-induced integrin stimulation with the release of cTnI upon necrotic cell death, cardiomyocyte cultures were treated with sodium azide (1\u00a0mmol\/l) to induce necrotic cell death. After 24\u00a0h of azide-treatment, LDH release from cardiomyocytes was 73.4\u2009\u00b1\u200910.5% (n\u2009=\u20097), indicating extensive necrotic cell death. Release of intact cTnI from these necrotic cardiomyocytes was 32.7\u2009\u00b1\u200913.8% (n\u2009=\u20097). Notably, in contrast to the release of intact cTnI from integrin-stimulated cardiomyocytes (Fig.\u00a02b), cardiomyocyte necrosis leads to the release of at least four cTnI degradation products with molecular weights of 26, 20, 17, and 12\u00a0kDa (Fig.\u00a02f).\nDiscussion\nThe major findings of this study are that integrin stimulation in cardiomyocytes by GRGDS leads to (1) release of intact cTnI from viable cardiomyocytes in the absence of necrosis and (2) increased levels of intracellular and extracellular MMP2 activity that, however, does not result in degradation of cTnI into its fragments. This study therefore indicates that the release mechanism of cTnI from viable cardiomyocytes upon integrin stimulation differs from cTnI release from necrotic cardiomyocytes, which is associated with extensive cTnI degradation.\nElevated plasma cTnI levels are frequently found in pathological conditions in which irreversible myocardial cell injury is not a prominent aspect. In patients with heart failure, elevated plasma cTnI levels have been reported with normal plasma levels of CK-MB [2, 18]. Some studies have suggested that elevated serum levels of cTnI may be the result of myocardial strain [15, 20], as occurs during pressure or volume overload. Logeart et al. [15] studied 71 patients with heart failure and found in 19 patients elevated cTnI concentrations associated with LV remodeling and increased plasma brain natriuretic peptide levels. They postulated that increased LV wall strain may have led to the increased cTnI release, but necrotic cell death was not excluded in that study. In acute pulmonary embolism, several reports showed elevated cTnI levels [17, 21] possibly related to the presence of myocardial strain. In addition, Feng et al. [6] demonstrated that increased preload in isolated rat hearts was sufficient to cause release of cTnI as the result of myocardial stretch independent of ischemia.\nOverload-induced stretch at the cardiomyocyte level is sensed by integrins, mechanotransducers molecules that link the extracellular matrix to the intracellular cytoskeleton [32]. In the present study, we showed that GRGDS-induced integrin stimulation of cardiomyocytes resulted in a threefold increased release of cTnI that occurred in the absence of necrosis, as cell viability remained unchanged by GRGDS treatment compared with control cardiomyocytes. Control cardiomyocytes demonstrated baseline levels of necrosis after 24\u00a0h, consistent with previous findings of cell death in cardiomyocytes after 24\u00a0h in serum-free medium [25]. Surprisingly, we found significantly less necrosis in SDGRG-treated cardiomyocytes than in control cardiomyocytes with both LDH assay and nuclear staining with propidium iodide. The mechanism responsible for this protective effect is unlikely to be related to integrin function but may be related to protection against necrosis exerted by certain proteins such as serum components.\nSeveral studies have suggested that release of cTnI from viable cardiomyocytes, without lethal disruptions of the cardiomyocyte sarcolemma, does not involve intact cTnI (29\u00a0kDa) but rather proteolytic degradation products of cTnI formed by intracellular proteases as MMP2 and calpain-I [6, 7]. In the present study, we demonstrated that purified intact cTnI can indeed be degraded by active MMP2 in vitro. MMPs are a family of zinc-containing enzymes involved in degradation of the extracellular matrix during tissue remodeling [34]. However, MMP2 also acts intracellularly and may, therefore, be responsible for degradation of myofilament proteins including cTnI and myosin light chain-1 (MLC-1) [30, 39]. MMPs are synthesized by a variety of cells, including cardiomyocytes [4], in a latent form (proMMP) that is activated by either proteolytic cleavage or by conformational changes induced by cytokines, reactive oxygen species, and peroxynitrite [22, 35, 38]. Previously, Wang et al. [37] have demonstrated that mechanical stretch of neonatal cardiomyocytes induced expression and activation of MMP2. In addition, integrin stimulation by GRGDS has been reported to upregulate MMP2 expression in fibroblasts and certain tumor cell lines [31, 40], suggesting an important role for integrins in regulating MMP2 activity. In the present study, we showed that GRGDS-induced integrin stimulation of cultured cardiomyocytes was indeed associated with increased activity of intracellular proMMP2 and active MMP2 and increased release of active MMP2 into the medium. In cardiomyocytes incubated with SDGRG, neither cellular levels nor medium levels of proMMP2 or active MMP2 were altered compared to control cardiomyocytes, indicating that increased MMP2 activity is specific for GRGDS-induced integrin stimulation.\nHowever, despite the increase of MMP2 activity during integrin stimulation and the ability of active MMP2 to degrade purified cTnI in vitro, integrin stimulation was not associated with cTnI degradation. If cTnI degradation had occurred, we would have been able to detect that in this system (Figs.\u00a03 and 2f). The reason for the lack of cTnI degradation in the presence of GRGDS-induced MMP2 activation is unclear. The discrepancy between the in vitro degradation of purified cTnI by active MMP2 and the lack of cTnI degradation in GRGDS-treated cardiomyocytes may be explained by a difference in specific MMP2 activity. The MMP2 activity per nanogram cTnI in the study with purified cTnI was fourfold higher than that in cardiomyocytes treated with RGD. In addition, purified cTnI may be more susceptible to MMP2 cleavage than cTnI that is complexed to cTnT, cTnC, and tropomyosin. In cardiomyocytes, 93\u201397% of cTnI is complexed to cTnT, cTnC, and tropomyosin which may protect the MMP2 cleavage sites on the cTnI molecule.\nThe mechanism responsible for the release of intact cTnI from viable integrin stimulated cardiomyocytes may be leakage of free intact cTnI from the cytosolic pool. Several studies have demonstrated that mechanically induced transient disruptions (wounding) of the sarcolemma are a constitutive event in vivo [3, 10, 16, 23]. This mechanism is responsible for the release of proteins, such as myocyte-derived growth factors (fibroblast growth factors 1 and 2), which are released despite the lack of a classic signal peptide sequence normally associated with exocytotic secretion. These mechanically induced alterations in cardiomyocyte\u2019s sarcolemmal permeability may similarly be involved in the release of cTnI from the cytosolic pool of cardiomyocytes in the absence of necrotic cell death. Further study is needed to address this issue.\nConclusions\nThe present study demonstrates that viable cardiomyocytes release cTnI as an intact protein by a stretch-related mechanism mediated by integrins. This finding may possibly explain why, in several pathological conditions, plasma cTnI levels are elevated in the absence of myocardial necrosis.","keyphrases":["cardiomyocyte","stretch","cell death","mechanoreceptor","cell culture"],"prmu":["P","P","P","U","R"]}
{"id":"Eur_J_Pediatr-3-1-1876255","title":"What is new in otitis media?\n","text":"The \u201cwait and see\u201d approach in acute otitis media (AOM), consisting of postponing the antibiotic administration for a few days, has been advocated mainly to counteract the increased bacterial resistance in respiratory infections. This approach is not justified in children less than 2 years of age and this for several reasons. First, AOM is an acute inflammation of the middle ear caused in about 70% of cases by bacteria. Redness and bulging of the tympanic membrane are characteristic findings in bacterial AOM. Second, AOM is associated with long-term dysfunction of the inflamed eustachian tube (ET), particularly in children less than 2 years of age. In this age group, the small calibre of the ET together with its horizontal direction result in impaired clearance, ventilation and protection of the middle ear. Third, recent prospective studies have shown poor long-term prognosis of AOM in children below 2 years with at least 50% of recurrences and persisting otitis media with effusion (OME) in about 35% 6 months after AOM. Viruses elicit AOM in about 30% of children. A prolonged course of AOM has been observed when bacterial and viral infections are combined because viral infection is also associated with ET dysfunction in young children. Bacterial and viral testing of the nasopharyngeal aspirate is an excellent tool both for initial treatment and recurrence of AOM. Antibiotic treatment of AOM is mandatory in children less than 2 years of age to decrease inflammation in the middle ear but also of the ET particularly during the first episode. The best choice is amoxicillin because of its superior penetration in the middle ear. Streptococci pneumoniae with intermediary bacterial resistance to penicillin are particularly associated with recurrent AOM. Therefore the dosage of amoxicillin should be 90 mg\/kg per day in three doses. In recurrent AOM with \u03b2-lactamase-producing bacilli, amoxicillin should be associated with clavulanic acid at a dose of 6.4 mg\/kg per day. The duration of the treatment is not established yet but 10 days is reasonable for a first episode of AOM. OME may be a precursor initiating AOM but also a complication thereof. OME needs a watchful waiting approach. When associated with deafness for 2\u20133 months in children over 2 years of age, an antibiotic should be given according to the results of the bacterial resistance in the nasopharyngeal aspirate. The high rate of complications of tympanostomy tube insertion outweighs the beneficial effect on hearing loss. The poor results of this procedure are due to the absence of effects on ET dysfunction. Pneumococcal vaccination has little beneficial effects on recurrent AOM and its use in infants needs further studies. Treatment with amoxicillin is indicated in all children younger than 2 years with a first episode of AOM presenting with redness and bulging of the tympanic membrane. Combined amoxicillin and clavulanic acid should be given in patients with \u03b2-lactamase-producing bacteria. The duration of treatment is estimated to be at least 10 days depending on the findings by pneumo-otoscopy and tympanometry. Bacterial and viral testing of the nasopharyngeal aspirate is highly recommended particularly in children in day care centres as well as for regular follow-up. The high recurrence rate is due to the long-lasting dysfunction of the eustachian tube and the immune immaturity of children less than 2 years of age.\nIntroduction\nThe number of publications relating to acute otitis media (AOM) and otitis media with effusion (OME) has been increasing markedly within the last 2\u00a0years [40\u201349]. It points to the difficulties in giving generally accepted guidelines for treatment. The confusion can be partially explained by the absence of uniform definitions and inhomogeneous groups of patients being included in clinical studies.\nAOM is an acute inflammation of the middle ear, frequently elicited by viral infections and characterized by redness and bulging of the tympanum. Infections with bacteria can be identified in at least 70% of the cases by culture of the middle ear purulent fluid containing mainly polymorphonuclear cells [35].\nOME is an effusion of the middle ear without symptoms of acute inflammations such as pain and fever, with frequently an amber or opaque aspect of the tympanum, the effusion containing mainly macrophages. Retraction of the tympanum with the light reflex still present may render the diagnosis difficult. Cultures for bacteria are positive in about 50% of cases whereas viruses have been identified in about 30% [35]. OME may be a precursor event initiating suppurate AOM but it can be the continuum of AOM as well. OME is the direct consequence of impaired middle ear ventilation.\nTympanometry (Fig.\u00a01) shows a flat curve type b in both AOM and OME. In contrast, in OME associated with incomplete obstruction of the eustachian tube (ET) a curve c with a peak in the negative pressure zone is observed. Moreover, lower peak height and greater width in the negative pressure zone are frequent findings in OME [54].\nFig.\u00a01The three main types of tympanograms showing: a a peak near atmospheric pressure expressed in daPa with stapedius reflex for different Hz frequencies, b a flat curve without stapedius reflex for 500, 2000, 4000\u00a0Hz pointing to presence of middle ear exudate: AOM or OME, c a lower peak in the negative pressure zone (\u2212235\u00a0daPa) with an enlarged width pointing to OME with partial permeability of ET to air entrance and still a stapedius reflex\nThis overview summarizes the main new findings about AOM and OME which are in accordance with some basic well-known but sometimes forgotten data and facts, with particular emphasis on the vulnerability of young children below the age of 2\u00a0years.\nAetiopathogenesis\nRole of the eustachian tube\nThe great importance of the first attack of AOM in young children lies in the subsequent long-lasting dysfunction of the ET. Indeed, the main functions of the ET are ventilation, protection and clearance of the middle ears (Fig.\u00a02) [5, 35] and play a determining role in the recurrence of AOM. Ventilation of the middle ear occurs by each deglutition by action of the tensor velum palatinum muscle leading to air equilibration with the atmospheric pressure. By obstruction of the ET, a negative pressure develops inside the middle ear resulting in effusion and aspiration of nasopharyngeal secretions. Poor ventilation leads to decreased PO2 which results in decreased bactericide power of polymorphonuclear cells. Impaired clearance results in proliferation not only of aerobic but also anaerobic bacteria in the middle ear. Conversely, reflux otitis occurs with decreased compliance of the ET due to abnormal flaccidity.\nFig.\u00a02Three physiologic functions of the eustachian tube in relation to the middle ear. NP nasopharynx, ET eustachian tube, TVP tensor velum palatinum muscle, ME middle ear, MAST mastoid, TM tympanic membrane, EC external canal\nET dysfunction is a major problem in the young child: the small calibre of the ET as well as its horizontal direction [5] (Figs.\u00a03 and 4) are responsible for both the high incidence of AOM and the frequent relapses by each viral infection. This explains the poor long-term prognosis evidenced in a prospective study comprising 210 AOM patients of less than 2\u00a0years of age by Damoiseaux et al. [16]. At least 50% of the patients had recurrences of AOM and persistence of OME was observed in 47% of them after 3\u00a0months and in 35% after 6\u00a0months. Yet, only 50% of these patients were treated with amoxicillin. Unfortunately, the authors do not mention either the delay of treatment or the dosage of the antibiotic.\nFig.\u00a03The difference in the angle of the eustachian tube between infants and adults [5]Fig.\u00a04The tympanic membrane forms the lateral wall of the box-shaped middle ear. The function of the eustachian tube is to equilibrate middle ear pressure with that in the nasopharynx. Bacteria and viruses resident in the nasopharynx may reach the middle ear during pressure equilibration. One-third of the middle ear mucosa and the entire eustachian tube are lined with mucociliary epithelium to transport bacteria from the middle ear back to the nasopharynx. Air from the middle ear enters the mastoid air cells by way of the aditus\nIn other words, the first episode of AOM determines the evolution and the recurrences because of the associated acute inflammation involving also the ET.\nRole of viruses and bacteria\nViruses\nMost children are infected with respiratory syncytial virus (RSV) in their first year of life. A prospective study [2] in 42 children aged 2\u201324\u00a0months with bronchiolitis showed that 26 of them had AOM at entry or within 10\u00a0days and an additional 10 developed OME; only 6 patients remained free of both AOM and OME during a 3-week observation period. These findings were confirmed in a recent study showing that in patients with persistent RSV antigen in the middle ear effusion, 31% relapsed despite a favourable outcome of the first AOM episode [50].\nThe association of AOM with several viral infections has been largely documented. The prevalence of respiratory viruses in the middle ear fluid of 456 children, aged 7\u00a0months to 7\u00a0years, with AOM was 41% [21]. RSV was the most frequent virus isolated followed by parainfluenza, influenza, enteroviruses and adenoviruses. These findings have been confirmed by others [18, 32, 33, 41, 50] and rhinovirus, coronavirus and metapneumovirus should be added to the above-mentioned list [53]. Even the measles virus has recently been isolated from the middle ear fluid in two patients [64].\nBacteria\nIn 70% of patients with AOM, bacteria can be found by culturing the middle ear fluid [35]. The most frequent species isolated are Haemophilus influenzae and Streptococcus pneumoniae [27]. Nasopharyngeal aspirate culture may give valuable information on the bacteria involved in AOM [28, 51, 55, 58]. The presence of conjunctivitis points to Haemophilus influenzae infection [4].\nHeikkinen et al. found Streptococcus pneumoniae in 25% of their patients, Haemophilus influenzae in 23% and Moraxella catarrhalis in 15% [21]. It has been demonstrated that recurrences of AOM are associated with positive bacterial culture in the nasopharynx [23], even at the end of the antibiotic treatment [28] and in most cases the pathogen was Streptococcus pneumoniae [58]. However, recurrence more than 14\u00a0days after an initial AOM episode is mostly due to a new infection, i.e. not a true relapse [27]. In a most recent prospective study on purulent meningitis [11], the association of AOM was found in half of the patients [14].\nBacteria and viruses\nBulut et al. performed both bacterial and viral testing in 120 children with AOM [8]. These patients, aged between 6\u00a0months and 12\u00a0years, did not receive an antibiotic for 2\u00a0weeks prior to the study. A positive bacterial culture was obtained in 54% of the children and respiratory viruses were identified in 32% of them; combined viral and bacterial infection was demonstrated in 12% of the samples.\nPersistent otitis was documented in about 50% of the children with combined viral and bacterial infection in a prospective study of 271 children because viral infections contribute to the ET dysfunction [12].\nOther risk factors\nThe immaturity of the immune system of young children makes them incapable of killing encapsulated bacteria. This, along with the ET dysfunction, explains the long-lasting course of AOM as well as the high recurrence rate.\nThis is particularly important for infants attending day care centres [37, 38]. It is sometimes necessary to withdraw these infants from these institutions during the winter to avoid recurrences. Additional risk factors are passive smoking, young siblings going to school and siblings with previous AOM episodes.\nThese factors were identified in a prospective study on 2,253 children aged 2\u00a0months to 2\u00a0years published in 1997 [37]. The proportion of patients developing at least one episode of OME was 79% at 12\u00a0months and 91% at 24\u00a0months. Breast feeding and exposure to tobacco smoke contributed little to the outcome, but a lower socioeconomic status and repeated exposure to other children at home or in day care centres were the most important risk factors.\nChildren with cleft palate are particularly prone to otitis and should be treated by early tympanostomy tube insertion [60].\nComplications: mastoiditis\nMastoiditis should be treated by antibiotics according to the bacterial resistance and by temporarily inserting tympanostomy tubes to improve drainage [65] because of the narrow calibre of the aditus ad antrum. [22] (Fig.\u00a04).\nTreatment\nGeneral considerations\nA \u201cwait and see\u201d approach has been advocated in the treatment of AOM in children mainly because of the increased antimicrobial resistance of bacteria causing respiratory infections [13]. Indeed, a meta-analysis of 5,400 children, aged 6\u00a0months to 18\u00a0years, and collected from 33 randomized trials concluded that there was a significant but modest impact of antibiotics on the primary control of AOM [45]. Similar results emerged from a subsequent study published by Damoiseaux et al. in 2000 [15]. This randomized double-blind prospective trial carried out by general practitioners in 240 children, aged 6\u00a0months to 2\u00a0years, showed that only 7 and maybe 8 of them needed antibiotics. Both studies, however, suffer from major flaws; the absence of long-term data, in particular the recurrence rate of AOM, and OME not being considered an end point. Moreover, the former study included patients with a very large age range and therefore blunted the effect of the critical age group of the patients below the age of 2\u00a0years. Indeed, over 70% of AOM patients present their first attack during the first year of life and over 90% within 2\u00a0years of age [35]. The visualisation of the tympanum in this age group is not always easy especially when cerumen obstructs the narrow external ear channel. Yet, as already mentioned above, bulging and redness of the tympanic membrane are important diagnostic signs of bacterial AOM [27, 39].\nTreatment of AOM\nThe crucial role of the first attack of AOM makes the immediate administration of antibiotics mandatory in all patients with AOM with redness and a bulging tympanum (Table\u00a01) [26]. A \u201cwait and see\u201d approach can only increase the inflammation and hence the ET dysfunction. The choice of the antibiotic must take into account the penetration in the middle ear and the type of bacteria involved. The penetration for amoxicillin\u2014about 40% of the blood concentration\u2014is much higher than for cephalosporins (about 20%) and macrolides (about 10%) but lower than for cotrimoxazole (60%) [31]. The penetration of amoxicillin is not changed by the addition of clavulanate to amoxicillin [10, 52].\nTable\u00a01Guidelines for treatment of AOMEpisodeSigns or symptomsTreatment or approachChildren <2\u00a0years of ageFirst episodeAmoxicillin 90\u00a0mg\/kg per day for at least 10\u00a0days according to the bacterial resistanceWeekly follow-up by tympanometry and pneumo-otoscopyRecurrent episodeWith redness and bulging tympanumAmoxicillin or amoxicillin 90\u00a0mg\/kg per day + clavulanic acid 6.4\u00a0mg\/kg per day according to bacterial resistanceWithout bulging\u201cWait and see\u201d approachChildren >2\u00a0years of ageWith redness and bulging tympanum or otorrhoeaSame treatment as \u201crecurrent episode\u201dWithout evident bulging tympanum\u201cWait and see\u201d approach\nBecause of the increased incidence of Streptococcus pneumoniae with intermediary resistance [57, 61], the dosage of amoxicillin should be doubled, i.e. 90\u00a0mg\/kg per day given in three administrations as recommended by the Centers for Disease Control and Prevention [3].\nHaemophilus influenzae is more likely to become \u03b2-lactamase positive after previous antibiotic administration [9]. Therefore, the addition of clavulanate\u2014at a dose of 6.4\u00a0mg\/kg per day\u2014to amoxicillin makes sense. Most Moraxella catarrhalis strains are nowadays also \u03b2-lactamase positive [42].\nThe duration of the antibiotic treatment is a matter of discussion. For children less than 2\u00a0years of age, a 10-day course is reasonable. Pneumo-otoscopy and tympanometry can help to appreciate the permeability of the ET. Careful follow-up is necessary.\nNasopharyngeal aspirate cultures may give valuable results and are highly recommended in children remaining in day care centres or with a history of previous AOM episodes [28, 51].\nFor children more than 2\u00a0years of age the previously watchful guidelines can be maintained but some flexibility is recommended [22, 36].\nTreatment of OME: tympanostomy tubes?\nThe approach to OME must take into account the natural history of spontaneous healing and the main complication which is temporary hearing impairment [1]. The absence of spontaneous healing after 6\u00a0weeks to 2\u00a0months in children over 2\u00a0years of age is an indication for a 10-day antibiotic course according to the results of the nasopharyngeal aspirate cultures. It is our experience that this approach has several advantages. First, the effect even temporarily on the hearing defect is frequently successful. Second, it decreases the recurrence of AOM by improving the ET functions and third, it allows postponement or avoidance of tympanostomy tube insertion, because improved hearing points to improved permeability of the ET.\nThe delay of 6\u00a0weeks or 2\u00a0months before treatment of OME (Table\u00a02) is based on the duration of OME. Indeed, in a prospective study by Renko et al. [43] of 90 children with AOM, 0.6\u20137\u00a0years of age, treated with amoxicillin or cefuroxime axetil for 10\u00a0days, the mean duration of OME was 10.2\u00a0days (range: 1\u201358\u00a0days); only 10 of 90 still had OME after 29\u201343\u00a0days.\nTable\u00a02Guidelines for treatment of OME. SMZ sulfamethoxazole, TMP trimethoprimIndicationApproachTreatmentWithout deafness\u201cWatchful waiting\u201d approachWith deafnessAfter 6\u00a0weeks-2\u00a0months in children \u22652\u00a0yearsAmoxicillin 90\u00a0mg\/kg per day or amoxicillin 90\u00a0mg\/kg per day + clavulanic acid 6.4\u00a0mg\/kg per day for 10\u00a0days according to the results of the bacterial resistanceNo swimmingRecurrent OME or OME of long durationAzithromycin 10\u00a0mg\/kg per day, 3\u00a0days\/week for 4\u00a0weeksCotrimoxazole 30\u00a0mg SMZ + 6\u00a0mg TMP\/kg per day according to the results of the bacterial resistanceAdenoidectomy in children \u22653\u00a0years of age with nasopharyngeal obstruction and recurrent adenoiditisNo swimming\nIn a long-term prospective study in 6,350 healthy infants treated according to specified guidelines, Paradise et al. [38] observed bilateral OME in only 5.6% of the 161 children who had not received tympanostomy tubes at the age of 4.\nThe indication to insert tubes is equivocal. The large prospective studies on 3,686 infants and young children by the Paradise group have shown the absence of or negligible effects of tympanostomies on developmental outcomes at 3\u20134\u00a0years [34, 37]. On the other hand, insertion of tympanostomy tubes during the first 3\u00a0years of life is associated with complications in at least 80% of the patients [63]. Tympanostomy can be a source of bacterial infections [24] and of tympanosclerosis.\nExtensive reviews by Kubba et al. [25], Rosenfeld et al. [46], Rovers et al. [48] and the recent Cochrane Database by Lous et al. [30] concluded that there was only a small benefit of the conventional ventilation tube. The main reason for the poor results of tympanostomy tubes is the absence of effects on the ET dysfunction. It has never been demonstrated that tympanostomy tubes improve the functions of the ET.\nThe recommendations by the American Academy of Pediatrics [1] are to insert tubes in conditions with persisting hearing loss of more than 40\u00a0dB particularly with posterosuperior retraction pockets, ossicular erosions and adhesive atelectasis because of the increase of structural damage with effusion duration.\nWatchful waiting for 3\u20136\u00a0months in the winter period seems advisable in children with mild hearing deficit.\nAdenoidectomy should be considered in children \u22653\u00a0years of age with nasopharyngeal obstruction and recurrent adenoiditis [45].\nVaccination\nThe effects of pneumococcal vaccination on recurrent AOM have been studied extensively. In a randomized controlled trial by Brouwer et al. [6] in 383 children, aged 1\u20137\u00a0years, with recurrent AOM, the effects in 190 children treated with heptavalent pneumococcal vaccine were compared with 193 children receiving hepatitis A or B vaccines. The study concluded that there was no beneficial effect. A similar conclusion was drawn from a study by Van Kempen et al. [62] in 74 children aged 1\u20137\u00a0years and in the Cochrane Database by Straetemans et al. [56].\nA shift of the causative pathogen has been observed from Streptococcus pneumoniae to Haemophilus influenzae or to other strains of pneumococci than the ones included in the vaccine [20, 59]. A slight reduction of the incidence of AOM of 6\u20138% was observed in the USA [7].\nInfants and prematures\nA high incidence of OME has been observed in neonatal intensive care units due to the nasally placed tubes for ventilatory assistance. It has been suggested that immune immaturity with impaired neuromotor function predisposes to this complication [17]. Vaccination with the heptavalent pneumococcal vaccine has been advocated in premature babies and neonates [19], but this study does not provide information on the actual AOM prevalence in a control group receiving only hepatitis B vaccine. It should be remembered that meconium aspiration into the entry of the middle ear is associated with increased risk of AOM and therefore early nasopharyngeal suction is mandatory [29].","keyphrases":["acute otitis media","aom","otitis media with effusion","ome","tympanostomy tube insertion","vaccination","guidelines for treatment","mastoiditis","eustachian tube dysfunction","otitis media in infants"],"prmu":["P","P","P","P","P","P","P","P","R","R"]}
{"id":"World_J_Urol-4-1-2413091","title":"Maximum tumor diameter is not an independent prognostic factor in high-risk localized prostate cancer\n","text":"Objectives Previous studies suggest that maximum tumor diameter (MTD) is a predictor of recurrence in prostate cancer (PC). This study investigates the prognostic value of MTD for biochemical recurrence (BCR) in patients with PC, after radical prostatectomy (RP), with emphasis on high-risk localized prostate cancer.\nIntroduction\nThe characteristics mostly used to predict progression of prostate cancer (PC), after radical prostatectomy (RP), are Gleason score, pre-operative PSA level, pathological staging, positive surgical margins, and tumor volume (TV). [1] The first three are also used to define high-risk localized PC [2]. TV in the RP specimen is also clearly associated with prognosis. However, whether it is an independent prognostic factor, both for the total group and for the high-risk group, still remains controversial [3].\nEpstein et al. [4] stated that, although TV is a predictor of progression, it did not provide additional information over Gleason score and pathological stage. This was also found in a study by Salomon et al. [5] where only Gleason score and pathological stage were independent factors to predict progression after RP. If these parameters were known, TV did not provide any additional information. This is in contrast with Stamey et al. [6] who found that TV is an independent prognostic parameter for PC progression. These findings were also observed in other studies [7\u20139]. None of these studies, however, specifically addressed the high-risk subgroup. An explanation of these conflicting results might be that TV estimations can be done with the help of several techniques, as Humphrey and Volmer [10] described earlier. Moreover, these techniques are rather complex and time-consuming. Therefore, maximum tumor diameter (MTD) has been suggested as a fast and easy proxy for TV. Renshaw et al. [11] noted that simple measurements of the largest diameter of the largest tumor from glass slides is a predictor of PSA failure. This was supported by Eichelberger et al. [12] who showed that MTD correlates with TV and other potential prognostic factors for clinical outcome. However, these studies had either a small number of patients with a relatively short follow-up (57 patients, median follow-up 27.2\u00a0months) or did not investigate the correlation between MTD and biochemical recurrence (BCR).\nTherefore, in this present study, with a large cohort of 542 RP specimens, and a median follow-up of 39.5\u00a0months, we studied the prognostic value of MTD, as a surrogate for tumor volume, for BCR in patients with PC, with special attention to the high-risk group.\nMaterials and methods\nBetween 1992 and 2005, 617 patients were treated with RP for clinically localized PC at our institute. Of these patients, 75 were excluded from analyses because of HIFU treatment, hormonal pre-treatment or irradiation before RP. Of the remaining 542 patients, five had incomplete follow-up data. Charts were examined retrospectively for clinical follow-up data and risk group classification. These risk groups were classified according to D\u2019Amico et al. [2]. High risk was defined as stage \u2265T2c or PSA level >20\u00a0ng\/ml or Gleason score \u22658, and we formed one group of the low and intermediate risk group (stage\u00a0<\u00a0T2c, PSA level\u00a0\u2264\u00a020\u00a0ng\/ml and Gleason score\u00a0\u2264\u00a07).\nAll RP specimens were fixed overnight, inked, and cut into serial, transverse, 4\u00a0mm thick slices according to a standard protocol by Ruijter et al. [13]. In brief, the apical and basal margins were amputated to a thickness of 4\u00a0mm and sectioned parasagittally at 4\u00a0mm intervals. The seminal vesicles were sectioned parallel and entirely submitted. The prostate was then sectioned perpendicularly to the long axis (apical to basal) of the gland along the posterior rectal surface at 4-mm intervals. Tumor was outlined on the microscopic glass slides and schematically drawn on the macroscopically photographed transverse sections in order to be able to reconstruct tumor extension and tumor multifocallity. Detailed pathological analysis, including number of tumors, total volume of all tumors, MTD, pathological staging, Gleason score, extracapsular extension and positive margins, was performed by a single pathologist (CAHK). Tumors were staged using the American Joint Committee on Cancer (AJCC) TNM staging criteria of 2002 [14]. MTD was defined as the largest diameter of the largest tumor. MTD was determined by marking both ends of the tumor and measuring the distance at the glass slide directly. If the tumor was present on consecutive glass slides, thickness of the slices of the concerning sections was summed. This reconstructed diameter was considered as MTD if it was larger than the diameter, and was visualized directly on the glass slides.\nPSA values were obtained before surgery and at every follow-up point. BCR after RP was defined as two subsequent PSA levels above 0.10\u00a0ng\/ml among patients who reached non-measurable levels after RP.\nStatistical analysis\nBaseline characteristics are summarized with median and interquartile ranges (IQR). Associations between MTD and clinical or pathological characteristics were examined by univariate regression models. Kaplan\u2013Meier curves were used to assess the risk of BCR. Proportional hazards multivariable regression models were composed to determine prognostic factors for BCR. SPSS version 12.0.1 for Windows was used for all statistical analysis.\nResults\nPatient and tumor characteristics are summarized in Table\u00a01. The age of the patients at time of RP was 62.7\u00a0years (IQR 58.4\u201366.5\u00a0years) and they had a median Gleason score of 6. In the specimens, a median MTD of 24.0\u00a0mm (IQR 16.0\u201332.0\u00a0mm) was found. Patients were followed for a median period of 39.5\u00a0months (IQR 17.1\u201367.3\u00a0months). Overall, 114 (21%) patients developed BCR after RP. The median time to PSA recurrence was 20.3\u00a0months (IQR 10.3\u201346.9). The overall 5-year Kaplan\u2013Meier risk of BCR was 25% (95% CI\u00a0=\u00a020.4\u201329.6) for the whole group. For the high-risk group and low\/intermediate risk, these were 27.4 (95% CI\u00a0=\u00a022.2\u201332.6) and 10% (95% CI\u00a0=\u00a02.2\u201317.8), respectively. Totally, 432 of the 542 patients fulfilled the criteria for high-risk localized PC, 72 patients were low\/intermediate risk, and 38 patients never reached non-measurable PSA levels.\nTable\u00a01Patient and pathological characteristics (n\u00a0=\u00a0542)MedianIQRAge (years)62.758.4\u201366.5Follow-up period (months)39.517.1\u201367.3Pre-operative PSA (ng\/ml)8.05.5\u201313.0Gleason score65\u20137Number of tumors (n)21\u20133Index tumor volume (cm3)1.60.6\u20133.4Total tumor volume (cm3)2.00.8\u20133.9Maximal tumor diameter (mm)24.016.0\u201332.0Data are presented as median and interquartile ranges (IQR)\nAssociations between MTD and pathological characteristics are summarized in Table\u00a02. Overall we found 359 organ-confined (T2) tumors, 116 extracapsular extended tumors (T3a), and 52 tumors invaded into seminal vesicles (T3b). Half of all the patients (n\u00a0=\u00a0271) had positive surgical margins and eight patients had lymph node involvement. Pre-operative PSA levels ranged from 0.1\u201387.2\u00a0ng\/ml (median 8.0\u00a0ng\/ml). Kruskall\u2013Wallis tests or Mann\u2013Whitney U test revealed that MTD was significantly associated with pathological stage (P\u00a0<\u00a00.001), Gleason score (P\u00a0<\u00a00.001), margin status (P\u00a0<\u00a00.001), number of tumors (P\u00a0<\u00a00.001) and pre-operative PSA levels (P\u00a0<\u00a00.001). Positive surgical margins were associated with a larger MTD compared to negative surgical margins. Single tumors were also found to have a larger MTD than multiple tumors. Extracapsular extension (T3a) and invasion to seminal vesicles (T3b) were both associated with larger maximal tumor diameters compared to those of organ-confined (T2) tumors. MTD in the high-risk group was significantly higher then in the low\/intermediate risk group (median 24.5 vs.14.0\u00a0mm, P\u00a0<\u00a00.001). We also calculated the correlation coefficient for MTD and all other factors. We found that MTD was significantly correlated to index TV (=largest tumor) (r\u00a0=\u00a00.60, P\u00a0<\u00a00.001) and total TV (r\u00a0=\u00a00.61, P\u00a0<\u00a00.001) in the total group. In the high-risk group, these correlations were weaker. In addition, significant correlations with MTD were found for Gleason score (r\u00a0=\u00a00.17, P\u00a0<\u00a00.001), the number of tumors (r\u00a0=\u00a0\u22120.21, P\u00a0<\u00a00.001) and pre-operative PSA levels (r\u00a0=\u00a00.38, P\u00a0<\u00a00.001).\nTable\u00a02Associations between MTD and pathological characteristicsNo. of patientsMaximum tumor diameterMeanMedianRangeP-value*Pathological stagea (2002)\u00a0T235921.020.01\u201360<0.001\u00a0T3a11630.828.012\u201360\u00a0T3b5231.131.03\u201355Gleason scorea\u00a0\u2264630122.521.01\u201365<0.001\u00a03\u00a0+\u00a0413626.225.08\u201360\u00a04\u00a0+\u00a032225.024.03\u201349\u00a0\u226588028.828.04\u201360Margin statusb\u00a0Negative27020.520.01\u201352<0.001\u00a0Positive27128.528.03\u201365Number of tumorsb\u00a0124127.326.01\u201365<0.001\u00a0\u2265130122.322.01\u201350Pre-operative PSAa\u00a0<45617.415.03\u201346<0.001\u00a04\u20131028923.624.01\u201355\u00a010\u20132012324.524.02\u201355\u00a0>207034.435.510\u201365Risk groupsb\u00a0High 43225.824.51\u201365<0.001\u00a0Low\/intermediate7214.314.01\u201336*\u00a0P-values were assessed by aKruskal\u2013Wallis test or bMann\u2013Whitney U test\nUnivariable analyses of MTD as a continuous variable revealed that it is weakly associated with risk of BCR (HR\u00a0=\u00a01.02 per mm increase, 95% CI\u00a0=\u00a01.00\u20131.04, P\u00a0=\u00a00.024). In the high-risk group, this association was lost (HR\u00a0=\u00a01.01 per mm increase, 95%CI\u00a0=\u00a00.99\u20131.03, P\u00a0=\u00a00.18).\nIn the total group TV, pre-operative PSA levels, pathological stage, and Gleason score were associated with risk of BCR, as presented in Table\u00a03. Proportional hazards multivariable regression models were composed to determine significant independent prognostic factors (Table\u00a03). Positive surgical margins, higher Gleason score, advanced pathological stage, and multiple tumors were the main prognostic factors for BCR. Neither MTD nor tumor volume turned out to be significant in either group.\nTable\u00a03Univariable and multivariable analysis of clinical and pathological characteristics associated with time to BCRCovariatesUnivariableMultivariableHR95% CIAHR95% CIPre-operative PSA1.021.01\u20131.04*Pathological stage\u00a0pT21.01.0\u00a0pT3a2.121.39\u20133.23*1.260.81\u20131.97#\u00a0pT3b3.902.33\u20136.53*1.791.02\u20133.13*Gleason score\u00a0\u226461.01.0\u00a03\u00a0+\u00a042.641.64\u20134.25*1.971.20\u20133.23*\u00a04\u00a0+\u00a037.273.18\u201316.63*5.712.47\u201313.20*\u00a0\u226585.683.60\u20138.97*3.572.17\u20135.89*Extracapsular extension2.711.87\u20133.94*Invasion seminal vesicle3.071.94\u20134.87*Margin status4.643.02\u20137.12*3.752.36\u20135.96*Number of tumors1.020.88\u20131.17#1.181.02\u20131.36*Maximal tumor diameter1.020.99\u20131.04#Index tumor volume1.041.02\u20131.07*Total tumor volume1.041.02\u20131.07*HR hazard ratio, CI confidence interval, AHR adjusted hazard ratio*\u00a0P\u00a0<\u00a00.05, #\u00a0P\u00a0>\u00a00.05\nDiscussion\nPrediction of outcome after RP in patients with localized PC is important to tailor follow up, and for example, to consider adjuvant therapy in patients at high risk for recurrence. Several studies have assessed the value of morphological and clinical variables in this respect. Tumor stage, Gleason score, and pre-operative PSA levels are obvious parameters, and also used to define patients at high-risk for recurrence [2]. MTD, as easy to determine surrogate for tumor volume [12], is another potential variable considered as a potential predictor of BCR. In our study of 542 patients, we found that MTD correlates with index TV, total TV, pathological stage, Gleason score, number of tumors, and pre-operative PSA levels irrespective of risk classification. However, MTD was not an independent predictor of BCR in the total or in the high-risk subgroup. For the whole group, these results are in contrast with previously reported findings.\nOne of the first studies addressing the association of MTD and BCR was by Renshaw et al. [3]. In this study, with 57 patients undergoing RP, it was found that MTD was strongly correlated with total TV. In this small series, a high MTD was associated with a slight increased risk for BCR (HR 1.12) that was marginally significant. Their subsequent study was a larger cohort of 434 patients. The median MTD was 13\u00a0mm. The median follow-up was 12.9\u00a0months and 27.2% of patients had PSA failure. This study confirmed that MTD is an independent predictor of BCR, even after adjustment for other risk factors [11].\nTheir next study by Dvorak et al. [15] had an even larger cohort (781 patients) and longer median follow-up (64.8\u00a0months). MTD remained the same with a median of 13\u00a0mm, and BCR in 31% of the patients. Again they found that MTD was significantly associated with time to BCR (HR\u00a0=\u00a01.04, P\u00a0=\u00a00.004) adjusted for pre-op PSA, Gleason score, and pathological stage. But when surgical margin status was added, MTD lost statistical significance (P\u00a0=\u00a00.07). One important feature of these studies is that the RP specimens were not uniformly processed. Therefore, we have to be taking into account the variation in evaluation of the prognostic factors. Another difference with our results is that the median MTD in these three studies (13\u00a0mm) are less than in our study (24\u00a0mm). This might be explained by the way we determine our MTD. Renshaw et al. limited their measurements to the single focus of tumor that could be demonstrated on one slide (slice-MTD). We, on the other hand, take the largest diameter of the largest tumor after reconstruction (real-MTD). To see if we could explain this difference, we also measured the slice-MTD in a part of our cohort. In 250 consecutive cases from 2000 to 2005, we found a median slice-MTD of 19\u00a0mm and a median real-MTD of 24\u00a0mm. This strongly suggests that part of the difference in MTD is caused by the different way MTD is measured. The difference that still exists could be explained by the fact that Renshaw et al. used partially submitted prostates, thus missing part of the prostates for evaluation. The only study where they did use uniformly processed specimens is by Eichelberger et al., but this study has a very short follow-up. They analyzed the prognostic value of MTD in a cohort of 364 patients treated with RP, and found a mean MTD of 17.3\u00a0mm and biochemical recurrence in 12% of the patients [16]. In this study with a median follow-up of only 12\u00a0months, they found that MTD was a significant predictor of BCR, adjusted for Gleason score and surgical margin status (HR\u00a0=\u00a01.70, P\u00a0=\u00a00.011). In our study we found a median time to BCR of 20.3\u00a0months, hence a follow-up of 12\u00a0months seems not long enough to investigate BCR.\nAnother striking difference is the percentage of positive surgical margins. In our study this is relatively high (50%) compared to other studies (Renshaw et al. 31 and 26%, Eichelberger et al. 23%, Dvorak et al. 27%). Our median BCR rate (21% after 39.5\u00a0months), however, was comparable to the median of these studies [Renshaw 27% (22.5\u00a0months), Eichelberger 12% (12\u00a0months), Dvorak 31% (64.8\u00a0months)]. An explanation for the high number of positive surgical margins, next to surgical techniques, is that by evaluating only partially included prostates you miss positive surgical margins.\nThe difference between our study and studies like Eichelberger et al, in which the entire prostate was histologically examined using whole mount sections, are less easily explained, but here the interval at which the prostates were sliced is higher (mean 7.8\u00a0mm according to the used protocol described by Qian et al. [17], personal communication). From this, we can conclude that MTD is not an independent prognostic marker if surgical resection margins are accurately documented.\nFinally, none of the mentioned studies separately looked at the group where prediction might be of most importance, the high-risk group. As clearly indicated, in this group, neither MTD nor TV is of any significant value.\nIn all, we do not advice to use MTD as a prognostic factor for BCR, since after proper follow-up and with adequate pathological techniques it does not add anything to other readily available prognostic factors. Moreover, in the high-risk group, TV is not an independent prognostic factor at all.\nConclusions\nThe results of our study suggest that MTD is positively correlated to total TV, as can be expected. MTD was weakly associated with risk of BCR and this association was lost in the high-risk group. If adjusted for positive surgical margins, higher Gleason score, advanced pathological stage, and multiple tumors, MTD did not provide additional information.","keyphrases":["localized","prostate","prostatectomy","prognosis","high risk"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_J_Nucl_Med_Mol_Imaging-_-Immediate_Access-2292795","title":"The diagnostic value of 124I-PET in patients with differentiated thyroid cancer\n","text":"Background The purpose of this prospective study was to evaluate the clinical diagnostic value of iodine-124 (124I)-positron emission tomography (PET) in patients with advanced differentiated thyroid carcinoma (DTC) and to compare the 124I-PET imaging results with the 131I whole-body scan (WBS).\nIntroduction\nPapillary and follicular thyroid cancer are the most frequent histological types of thyroid cancer (85-90%) [1, 2]. Total thyroidectomy with or without lymph node dissection is the initial therapy, followed by radioactive iodine therapy.\nIn the routine follow-up of low-risk patients, diagnostic 131I whole-body scanning (WBS) is not recommended in the recently published guidelines [3] (http:\/\/www.british-thyroid-association.org\/draft_thyca_23.12.06.pdf; http:\/\/www.oncoline.nl\/uploaded\/docs\/Schildkliercarcinoom\/schildklier%20ebro\/ijn%20schildklier.pdf), but is still considered to be valuable in the follow-up of the high-risk patients. Measurement of the serum level of thyroglobulin (Tg), under recombinant human thyroid-stimulating hormone (rhTSH) stimulation or suppression, and ultrasonography have gained a more central role in monitoring for recurrent thyroid cancer [3, 4-7] (http:\/\/www.british-thyroid-association.org\/draft_thyca_23.12.06.pdf). In patients with increasing or elevated Tg, a blind treatment with high-dose 131I can be applied, followed by a post-treatment 131I scan which also serves as a diagnostic tool [21]. However, with this strategy, unnecessary high radiation exposure and high TSH level must be taken into account especially in patients who subsequently have no 131I uptake on their post-treatment scan. Improvement of diagnostic imaging for the detection of recurrent or metastatic disease and a better (anatomical) localisation, e.g. using advanced imaging technique such as 124I-positron emission tomography (PET)\/CT would allow more selective application of 131I therapy and might avoid unnecessary high-dose treatments.\nSeveral iodine isotopes such as 123I, 125I, 131I play an important role in nuclear medicine, both for diagnostic purposes and for therapy. Iodine-124 is a positron emitting isotope and therefore suitable PET imaging. Its half-life is 4.2\u00a0days with a very complex decay scheme leading to extra non- or partially annihilation radiation coincidence detection [8, 9]. Approximately 23% of the desintegrations results in positron emissions.\nWhile the radioisotopes 123I and especially 131I are used on a wide scale in diagnosis and treatment of all thyroid disorders, 124I has received little attention. This isotope would allow thyroid cancer imaging using the high-resolution PET technique [8, 10]. Iodine-124 has so far mainly been used for dosimetry or thyroid volume measurements [10-16]. However, the recent development of combined PET\/CT scanners may increase clinical application in thyroid cancer patients, as detailed anatomical information is combined with the location of iodine positive tissue [17]. Iodine-124 has recently been applied for staging of differentiated thyroid cancer, as reported in a few case reports and small series, with promising results [17-19]. However, the diagnostic value of 124I-PET imaging as compared to (low- and high-dose) 131I scintigraphy has to be further investigated.\nIodine-124 PET may be able to detect recurrent or residual disease in differentiated thyroid carcinoma (DTC) with a higher sensitivity than the conventional (diagnostic) 131I scans because of the higher spatial resolution. In this way, 124I-PET imaging could possibly be a useful diagnostic tool during the follow-up of DTC patients. Therefore, we have performed a pilot study in the early treatment phase of thyroid cancer to get an impression of the diagnostic potential of 124I-PET for detection and staging of advanced DTC. We compared the 124I-PET imaging results with the diagnostic and post-treatment 131I-WBS.\nMaterials and methods\nTwenty patients with histologically proven advanced DTC, including extrathyroidal tumour growth (T4), extra-nodal tumour growth or distant metastasis (M1), were examined in this study. These patients had undergone (near) total thyroidectomy. Four to 6\u00a0weeks after surgery, diagnostic 131I-WBS after 37\u00a0MBq of 131I was obtained, followed by an ablative dose of 5,550\u00a0MBq 131I. Post-treatment WBS was obtained after 10\u00a0days. At the time of ablation, serum Tg and Tg antibodies (TgAb) levels were also determined.\nAll 20 studied patients underwent diagnostic 131I-WBS, 124I-PET scan and post-treatment 131I-WBS 4\u00a0months after ablation therapy (Fig.\u00a01). The Tgoff levels (under TSH stimulation after thyroid hormone withdrawal or after rhTSH injection) were also measured just before the administration of the diagnostic low-dose 131I. The Medical Ethics Committee of University Medical Center Groningen approved the study protocol, and all patients gave written informed consent.\nFig.\u00a01The diagram showed a schematic course of the investigations performed 4\u00a0months after ablation\nTg was measured by immunoradiometric assay (Brahms, Henningsdorf, Germany), with a functional sensitivity (i.e. the Tg concentration with a vital capacity of 20%) of 0.3\u00a0ng\/ml as determined by the laboratory. Thyroglobulin antibodies (TgAb) were detected by both immunoradiometric assay (Brahms, Henningsdorf, Germany) with a cutoff of 60\u00a0U\/ml and by immunoluminometric assay (Abbott, Hoofddorp, Netherlands) on the Architect i 2000 platform (Abbott) with a cutoff of 4.1\u00a0U\/ml. Both cutoff concentrations were determined by Brahms and Abbott, respectively. In 19 patients, the Tg level was determined after thyroid hormone withdrawal and in one patient after rhTSH due to poor physical condition (Table\u00a01).\nTable\u00a01Patient characteristics and imaging resultsPtAge, sexTNMTgoff (ng\/ml)TSH (mU\/l)\n124I-PETPt 131I-WBSDx 131I-WBSValidation159MpT1N0M14850CV, pelvis +++CV, pelvis +++CV, pelvis +MR cv: p, MRI pelvis:p267FfT4N1M120148Neck, thyroid bed +++, lung ++Neck, thyroid bed +++ lung ++Neck ++CT lung: no abnormal lesions, MRI neck: p385MfT4N0M12.6a\n0.01a\nSkull, CV, pelvis +++CV+++CV +Skull: p, MR cv: pb\n448FpT4N1M04977Neck ++Neck ++-MRI:neck: p574FpT4N1M0<0.3044Neck ++Neck ++-MRI:neck: p674MfT2N0M1691>200Neck\/SC +++Neck\/SC, pelvis +++-MRI pelvis: pb\n773FfT4N1M010556Neck\/thyroid bed +Neck\/thyroid bed ++-US neck\/FNAC: p818FpT4N1M1<0.3051Pelvis ++Pelvis +-MRI pelvis:p939MpT4N1M08575Neck +++Neck +++-MRI neck: p1073FpT4N1M0343Neck +--US neck and FNAC: inconclusive, FDG PET: n1159MfT3N0M1168073Femur prosthesis ++--MRI pelvis\/femur : reactive tissue around the femur prosthesis; FDG PET: lesions in costae and pelvis1273MpT3N0M1<0.3033 -skull ++-MRI skull: p, FDG PET: n1367MpT4N0M0<0.30142-pelvis +-X-pelvis: n, bone scan: n1442FpT4N1M01581---US neck and FNAC: n, FDG PET: n1549FpT4N0M01866---US neck and FNAC: n, FDG PET: n1659MpT4NxM02.876---MRI neck, US neck and FNAC: n, FDG PET: n1768MpT2N0M11444---US neck\/FNAC: n, FDG PET and CT: lung lesions1879FfT4NxM01.653---MRI neck: p, US neck: lymph node not accessible for FNA, FDG PET: n1971FfT4N0M00.5436----2037MpT1N1bM0<0.3095----\np Papillar, f follicular, Pt \n131\nI-WBS post-treatment 131I-WBS, Dx \n131\nI-WBS diagnostic 131I-WBS, CV cervical vertebrae, SC sternal clavicular, \u2212 not visible, + just visible, ++ visible, +++ clearly visible, n negative, p positive, FNAC fine needle aspiration cytology, FDG-PET fluorodeoxyglucose positron emission tomography\narhTSH stimulated\nbIn these two patients, no additional radiologic imaging of the neck (no. 3) and pelvis (no. 6) was performed to confirm the findings on the PET and 131I-WBS due to poor physical condition and due to the lack curative therapeutic options\nTracer\n124I-sodium iodide solution was obtained from Ritverc Isotope Products, St. Petersburg, Russia and was imported by I.D.B. Holland BV (Baarle-Nassau, The Netherlands). Radiochemical purity using instant thin layer chromatography and radionuclide purity using germanium (HP-Ge) detector were tested before release according to standard radiopharmaceutical procedures. Before use, the solution was filtered using a Millipore bacterial filter (0.22\u00a0\u03bcm) and diluted with sterile saline. A sterility test was performed on each batch, but data were only available after administration due to the length of the test procedure (7\u00a0days).\nPET scanning\nDiagnostic 124I-PET imaging was performed 24\u00a0h after intravenous administration of 74\u00a0MBq of 124I [17]. A Siemens PET camera (Exact HR+, Knoxville, TN, USA) was used for imaging. The patient was positioned in the scanner, and a standard clinical whole-body PET study was performed in 2D ETTE mode over seven to eight positions (from the upper thigh up until the top of the skull) of 5-min emission and 3-min transmission, standard energy window setting of 350-650\u00a0keV. Images were reconstructed with attenuation-weighted OSEM, two iterations and eight subsets. The total time needed for the scan was approximately 60\u00a0min.\nIn the initial four patients, the 124I-PET images were repeated 96\u00a0h after 124I administration (thus, 72\u00a0h after therapeutic dose of 131I) using the same acquisition settings as after 24\u00a0h. Narrowing of the energy window (425-650 or 460-562\u00a0keV, 3D mode) for 124I-PET imaging during high-dose 131I therapy to reduce the effects of \u03b3-rays (364, 637\u00a0keV) of 131I and 124I (602\u00a0keV) and to improve image quality, as described in the phantom study (and clinical application in one thyroid cancer patient) by Lubberink et al. [20], was not possible in our institution due to technical reasons, e.g. camera characteristics. The 96-h 124I-PET images were very noisy and blurred and were excluded from the study. This poor image quality could partially be explained by the poor statistics due to the very low radioactivity left in the body after 96\u00a0h and partially by technical reasons as described above.\nData analysis\nThe 131I-WBS and 124I-PET scans were visually interpreted by two independent, experienced nuclear medicine physicians (HTP, PLJ). The findings on the 124I-PET scans were compared with the findings on the diagnostic and post-therapeutic 131I-WBS. Correlation with radiologic imaging (US, CT, MRI) and\/or cytological (fine needle aspiration cytology, FNAC) investigation was done to confirm the findings or in case of discordant findings on the 124I-PET scan and 131I-WBS. If no abnormal uptake was seen on the PET scan and 131I-WBS, additional radiologic imaging (MRI, US), with or without FNAC, and fluorodeoxyglucose (FDG) PET were also performed to detect local or metastatic disease and\/or used as a follow-up diagnostic tool.\nResults\nFrom December 2005 until April 2007, 20 consecutive patients with advanced DTC were included in this prospective study. The group consisted of ten women and ten men, median age of 67\u00a0years (range 18-85\u00a0years). Individual patient characteristics and the findings on the 124I PET scan and 131I-WBS are summarised in Table\u00a01.\nPhysiological uptake of 124I was observed in the salivary glands, esophagus, gastrointestinal tract and bladder as it is also normally seen on the 131I scans.\n124I-PET vs diagnostic 131I-WBS\nEleven patients (no. 1-11) showed uptake on the 124I-PET scan. In only three of them (no. 1-3) was the uptake also observed on the diagnostic 131I scan, but the uptake was clearer and the abnormalities were more extensive on the 124I-PET scan (Fig.\u00a02). In nine patients (no. 12-20), no uptake was observed on both the scans. Results of additional radiologic imaging (MRI and\/or US-FNAC) are also listed in Table\u00a01.\nFig.\u00a02This patient (no. 1) showed a clearly visible lesion in the cervical vertebrae on the 124I-PET (a, arrow), comparable with the lesion visible on the post-treatment 131I-WBS (c, arrow). This lesion was vaguely visible on the diagnostic 131I-WBS (b, arrow). Physiologic uptake in the esophagus, gastrointestinal tract and bladder is observed on the 124I-PET\nIt has also been noticed that the Tgoff level (after endogenous TSH stimulation) was not detectable (<0.30\u00a0ng\/ml, TSH >30\u00a0mU\/L) without the presence of Tg antibodies in five (Table\u00a01, no. 5, 8, 12, 13, 20) of the 20 patients. However, two of these five patients showed lesions on the 124I-PET (and post-treatment 131I scan) confirmed by MRI. Diagnostic 131I-WBS was negative in all five.\n124I-PET vs post-treatment 131I-WBS\nNine (no. 1-9) out of the 11 patients with uptake on the 124I-PET scan had lesions which were also visible on the post-treatment scan (Fig.\u00a03). In two patients (no. 10, 11), no uptake was observed on the post-treatment 131I scan and no anatomical localisation could be confirmed. FDG PET showed, however, lesions in the costae and pelvis in patient no. 11 (Fig.\u00a04).\nFig.\u00a03This patient (no. 6) showed a clearly visible lesion in the left lower neck region or on the 124I-PET (a, arrow), comparable with the lesion visible on the post-treatment 131I-WBS (c, arrow). No uptake was observed on the diagnostic 131I-WBS (b)\nFig.\u00a04Clearly, uptake in the right costae (arrow) and left hip is seen on the FDG-PET (c) in patient no. 11, whereas the post-treatment 131I-WBS (a) was negative. The 124I-PET (b) showed only uptake around the femur prosthesis on the right side, which is also observed on the FDG-PET, which could be explained by reactive tissue on the MRI. The complementary uptake of radioiodine and FDG is also known as the flip-flop phenomenon, which was first described by Joensuu and Ahonen [27]\nTwo patients (no. 12, 13) showed uptake on the post-treatment 131I scan, which was not visible on the 124I-PET scan: In one, MRI confirmed the 131I uptake in the skull, and in the other, no anatomical localisation could be found for the 131I uptake in the pelvis region. In seven patients (no. 14-20), no uptake was observed on both the scans. In four of seven patients (no. 14-17), no lesions could be found with MRI and\/or US-FNAC; in one of these four (no. 17), FDG-PET showed uptake in the lungs which was confirmed by CT. One patient (no. 18) showed slightly enlarged lymph node in the superior mediastinum on the MRI which was not accessible for US-guided FNAC. In two patients (no. 19, 20), MRI\/US of the neck will be obtained (due to patient delay). Results of additional radiologic imaging are listed in Table\u00a01.\nDiscussion\nThis pilot study showed that 124I-PET detected more abnormalities in comparison to the diagnostic 131I-WBS, but showed comparable findings with the post-treatment 131I-WBS. Eleven patients had positive 124I-PET scanning, and only three had visible abnormalities with the low-dose 131I scan. Moreover, 124I-PET also showed abnormalities in two of the five patients with undetectable Tg, while the low-dose diagnostic 131I scan was negative in all five. These findings suggest that 124I-PET better predicts the outcome of high-dose 131I treatment and would be better suited as a diagnostic tool to base clinical decisions on such as additional surgery or application of high dose 131I. Therefore, 124I-PET should be performed before considering high-dose 131I treatment. A negative 124I-PET could mean omitting 131I treatment, and further additional imaging should be performed to detect (non-iodine avid) metastatic disease.\nTwo patients (no. 12, 13) showed uptake in the skull and pelvis region, respectively, on the post-treatment 131I-WBS which was not visible on the 124I-PET. Possible explanations for these findings might be the low dose of 124I in which (small) lesions in the skull might be missed and false-positive uptake of 131I in the bowel which could be mistaken for abnormality.\nOur results are in agreement with the study by Freudenberg et al. [17]. In their study, 12 patients with advanced DTC underwent high-dose 131I-WBS, 124I-PET, CT, combined 124I-PET\/CT, FDG-PET and US post-thyroidectomy during routine clinical staging. The overall lesion detectability for high-dose 131I-WBS was comparable with the 124I-PET, 83 vs 87%, respectively. Moreover, combined 124I-PET\/CT modality, which showed an overall lesion detectability of 100%, resulted in a change of staging in two patients and a change in management in one.\nIt has been questioned why 124I-PET would be a suitable diagnostic tool when blind treatment is given anyway in the clinical practice. Blind treatment with high-dose 131I in patients known with negative low-dose diagnostic 131I scanning but with elevated Tg has been used as a diagnostic, therapeutic and prognostic tool. Patients without iodine accumulation on the post-treatment WBS, which could be an indication for tumour dedifferentiation, had a worse prognosis compared to those with a positive post-treatment WBS [21]. Repeating high doses of 131I may bear the risks in terms of long-term risk of secondary malignancies. In addition, repeated long-lasting TSH stimulation might have adverse effects on tumour growth. This aspect of long-lasting TSH stimulation may be prevented when performing the 124I-PET after rhTSH stimulation before the decision of giving additional high-dose 131I. However, comparative studies are needed to evaluate the yield of the 124I-PET under endogenous TSH stimulation and after rhTSH stimulation.\nAdditional advantages of 124I-PET would include the better resolution of this tomographic method and the ease of combining this with CT data to increase the diagnostic value, as shown by Freudenberg et al. [17]. Moreover, 124I-PET would allow more precise dosimetric calculations [25, 26, 28, 29], although that was not a focus of this study.\nAnother aspect which favours performing of 124I-PET before blind high-dose 131I is the radiation dose. The effective dose of 124I is in the same order of magnitude as 131I [10, 22-24]. The effective dose of 124I-iodide is 0.095\u00a0mSv\/MBq with a thyroid uptake of 0% and increases to1.5\u00a0mSv\/MBq with a thyroid uptake of 35% [23]. The total radiation exposure has been calculated at 7.0\u00a0mSv for a dose of 74\u00a0MBq 124I. The administered dose is comparable to the dose administered for routine nuclear medicine scans. However, the total radiation exposure of 124I is just a fraction compared to the total radiation exposure of the therapeutic dose of 131I (340\u00a0mSv for 5,550\u00a0MBq [23], which was also mentioned in the study by Freudenberg et al. [17].\nThe issue whether stunning is a real phenomenon and its clinical relevance\/consequence is debatable. Stunning effect after (higher) diagnostic dose of 131I has been described and discussed in the literature. The applied diagnostic 131I dose could impair the ability of the residual thyroid carcinoma tissue to accumulate the subsequently applied high-dose 131I dose. The degree of stunning probably depends on the absorbed radiation dose and the time between the diagnostic and therapeutic 131I dose.\nStunning was not seen with diagnostic 131I doses of 185\u00a0MBq (5\u00a0mCi) or lower [30, 31], whereas stunning was frequently observed after a diagnostic dose of 370\u00a0MBq (10\u00a0mCi) 131I [32]. There is also evidence that a short time interval between the administration of the diagnostic and therapeutic 131I dose may diminish the effect of stunning [31]. Most authors who have described stunning administered the therapeutic 131I dose several days after the completion of the diagnostic 131I scan [32-35]. Stunning is not seen when the therapeutic dose is administered within several hours. Furthermore, the time interval between the administration of a high dose of 131I and the performance of a post-therapy WBS may influence the observation of stunning [30, 36]. A longer time interval allows more time for soft tissue clearance of 131I, which results in a higher sensitivity of the post-therapy WBS. No stunning was seen at the post-therapy 131I-WBS, performed 5-10\u00a0days after doses of 1,110-3,700\u00a0MBq (30-100\u00a0mCi) 131I after 74\u00a0MBq (2\u00a0mCi)) and 370\u00a0MBq (mCi) diagnostic scan [37].\nIn our study, we used a rather a low dose of 124I (74\u00a0MBq) and a short interval (1\u00a0day) between the administration of the diagnostic dose of 124I and the administration of the therapeutic 131I dose and a long interval (10\u00a0days) between the therapeutic 131I dose and the post-therapy 131I-WBS. If stunning does exist after 124I, it is therefore unlikely to have reduced the efficacy of 131I treatment in our study.\nIodine-124 is, however, poorly available with high costs, but the advantages of 124I-PET could outweigh these disadvantages and can lead to more clinical application in the follow-up of DTC patients when 124I will become more available.\nConclusions\nIn this study, 124I-PET proved to be a superior diagnostic tool as compared to low-dose diagnostic 131I scans and adequately predicted findings on subsequent high-dose post-treatment 131I scans. In combination with the high resolution and the possibilities to combine with CT, this could lead to improved clinical decision making.","keyphrases":["diagnostic value","differentiated thyroid cancer","iodine-124","positron emission tomography"],"prmu":["P","P","P","P"]}
{"id":"Histochem_Cell_Biol-4-1-2386528","title":"The mammalian centrosome and its functional significance\n","text":"Primarily known for its role as major microtubule organizing center, the centrosome is increasingly being recognized for its functional significance in key cell cycle regulating events. We are now at the beginning of understanding the centrosome\u2019s functional complexities and its major impact on directing complex interactions and signal transduction cascades important for cell cycle regulation. The centrosome orchestrates entry into mitosis, anaphase onset, cytokinesis, G1\/S transition, and monitors DNA damage. Recently, the centrosome has also been recognized as major docking station where regulatory complexes accumulate including kinases and phosphatases as well as numerous other cell cycle regulators that utilize the centrosome as platform to coordinate multiple cell cycle-specific functions. Vesicles that are translocated along microtubules to and away from centrosomes may also carry enzymes or substrates that use centrosomes as main docking station. The centrosome\u2019s role in various diseases has been recognized and a wealth of data has been accumulated linking dysfunctional centrosomes to cancer, Alstrom syndrome, various neurological disorders, and others. Centrosome abnormalities and dysfunctions have been associated with several types of infertility. The present review highlights the centrosome\u2019s significant roles in cell cycle events in somatic and reproductive cells and discusses centrosome abnormalities and implications in disease.\nIntroduction\nOver 120\u00a0years have passed since the discovery of centrosomes (Flemming 1875; Van Beneden 1876) and yet, we are only at the beginning of understanding the multiple and central roles of this intriguing cellular organelle. Boveri (1901) who coined the name centrosomes had produced a remarkable wealth of new information characterizing and analyzing centrosomes during fertilization and cell division (Boveri 1901) by using iron hematoxylin as a major cytological staining technique, and he brilliantly recognized the very important role of centrosomes in cancer (1914). Flemming had called the discovery of centrosomes as important as the discovery of the nucleus (Flemming 1891) yet, research on centrosomes stagnated for decades for a number of reasons including war events in Europe that slowed all research and because studies using the electron microscope did not reveal a structure that could be easily defined. Centrosome structure was described as amorphous osmiophilic material surrounding a pair of well-structured centrioles, which stimulated and generated new research on centrioles while research on centrosomes became less appreciated. With the development of immunological probes and molecular techniques coupled with immunofluorescence microscopy centrosomes reclaimed their status and Boveri\u2019s earlier work was vigorously pursuit by reproductive biologists, and by cellular and cancer biologists who were able to confirm and build on Boveri\u2019s remarkably detailed and accurate data and visionary research. Our understanding of centrosomes has advanced enormously and very rapidly in recent years and its central role in cellular biology has clearly been recognized. It can be predicted that a number of diseases other than cancer are linked directly or indirectly to centrosome dysfunctions. Centrosome isolations, forward and reverse genetics, RNA-mediated interference, mass-spectrometry-based proteomics, live cell imaging particularly with GFP-tagged proteins, and laser microsurgery are some of the newer more recently employed techniques that have been applied to centrosome research and rapidly moved the field into new frontiers. The present paper will review the state of our knowledge of centrosomes and analyze its impact on cellular functions.\nDefinition of the centrosome and its key functions\nThe centrosome is a subcellular non-membrane bound semi-conservative organelle, approximately 1\u00a0\u03bcm in size, that serves as the cell\u2019s primary microtubule organizing center (MTOC) and plays a major role in numerous cellular functions. Through its microtubule organizing functions the centrosome facilitates many cellular activities including cell motility, polarity, maintenance of cell shape, cell division, transport of vesicles, and targeting of numerous signaling molecules. A typical mammalian centrosome consists of a proteinaceous scaffold containing a large number of centrosome proteins including \u03b3-tubulin and the gamma-tubulin complex (\u03b3-TuRC) that typically surround a pair of perpendicularly oriented cylindrical centrioles, therefore referred to as pericentriolar material (PCM). The PCM scaffold undergoes shape changes throughout the cell cycle to produce cell-cycle-specific microtubule organizations for specific cellular functions. Lacking a defined membrane boundary, the centrosome\u2019s three-dimensional architecture is maintained through specific protein\u2013protein interactions. Microtubules are anchored at the centrosome with their minus ends (Bornens 2002) and microtubule growth is regulated by distal plus-end addition of tubulin subunits (McIntosh and Euteneuer 1984). In interphase, the centrosome is juxtapositioned and closely associated with the nucleus. At the G2\/M transition it undergoes significant cell cycle-specific reorganizations and matures into the division-competent center of the mitotic poles. Because the centrosome nucleates microtubules and controls microtubule numbers and lengths it directs most microtubule-related functions including transport of macromolecular complexes, positioning of cell organelles, cell motility, cell shape, polarity, and segregation of chromosomes during cell division. The mitotic centrosomes are critically important to mediate the strictly balanced bipolar separation of chromosomes. In addition to the regular function as MTOC the centrosome orchestrates various major important cell cycle events including entry into mitosis, anaphase onset, cytokinesis, G1\/S transition, and monitoring of DNA damage (Kramer et al. 2004).\nWhile a typical centrosome consists of a pair of centrioles surrounded by PCM, centrioles are not always present within the centrosome structure as will be detailed later in this paper. In general, centrioles are important for centrosome integrity and to aid in the recruitment of centrosomal proteins into the PCM. The PCM is a fibrous scaffolding lattice that consists of a large amount of coiled-coil centrosome proteins and anchors signaling molecules and components of the \u03b3-TuRC (Fig.\u00a01). Some centrosome proteins are permanently associated with the centrosome core structure such as \u03b3-tubulin, the \u03b3-TuRC, and centrin (addressed below in more detail) while several other centrosomal proteins are temporarily associated with the centrosome core structure and include centrosome proteins such as the Nuclear Mitotic Apparatus protein (NuMA). Such centrosomal proteins serve cell-cycle-specific functions and are needed for specific cell cycle activities. The cell cycle-specific centrosome proteins are different from those proteins or enzymes (kinases, phosphatases and others) that may be colocalized with centrosomes and may utilize centrosomes as a docking station but are not centrosome proteins that are directly involved in performing centrosome-specific functions. So far, about 500 different proteins have been associated with the centrosome by mass spectroscopy (Andersen et al. 2003) although it is not clear whether all of those proteins are centrosome proteins or proteins or enzymes that use centrosomes as docking station. Regulators of various cell cycle events have been co-localized with centrosomes and include the mitogen activated protein kinase (MAPK) (Sun et al. 2002) which may use centrosomes as a docking station to perform and coordinate multiple cell cycle-specific functions. Vesicles that are translocated along microtubules to and away from centrosomes may also carry enzymes or substrates that use centrosomes as docking platform (Fig.\u00a02a, b).Fig.\u00a01A typical mammalian centrosome is composed of two centrioles surrounded by a meshwork of a proteins embedded in matrix called the pericentriolar material (PCM). Gamma-tubulin and the gamma-tubulin ring complex that nucleate microtubules along with associated proteins are embedded in the PCM. Highlighted in this diagram are two centrosomal complexes, the microtubule nucleating complex and the microtubule anchoring complexFig.\u00a02a The centrosome serves as central station to mediate substrate and enzyme activities along microtubules towards the minus ends of microtubules driven by dynein (white circles) and toward the plus ends driven by kinesin (black circles). b Through its microtubule-organizing capabilities the centrosome mediates distribution of cell organelles such as mitochondria, Golgi, and vesicles of various sizes, many containing regulatory enzymes for cell cycle regulation\nFunctional homologues of centrosomes are spindle pole bodies of yeast (Knop et al. 1999), the nucleus-associated body of the cellular slime mold (Daunderer et al. 1999), and nucleating sites in plants (Chan et al. 2003). In addition, multiple plasma membrane-associated sites in Drosophila epidermal cells (Mogensen 1999) and in Xenopus oocytes (Pfeiffer and Gard 1999) have also been identified to serve as MTOCs. New research has established that virus, bacteria, and parasites exploit the host cell\u2019s centrosomal capabilities and recruit centrosomal material for their own survival within host cells (reviewed in Scaplehorn and Way 2004; Coppens et al. 2006).\nTaken together, the centrosome is a truly central and main cellular station that directs, coordinates, and regulates most cellular functions either directly or indirectly through its microtubule organizing capabilities.\nCentrosome proteins and their specific functions\nNumerous centrosome proteins have been described and their functions have been determined but the list of newly discovered centrosome proteins continues to grow and includes centrosome proteins that have multifunctional capabilities. Furthermore, the list of diseases in which centrosome protein dysfunctions play a role also continues to grow as research on centrosomes has progressed rapidly and new research teams have formed around the growing centrosome field. The initial discovery of centrosomal proteins in an autoimmune antibody has provided a most powerful tool to stain centrosomes with immunological probes (Calarco-Gillam et al. 1983) and since then, a large number of centrosomal proteins have been identified based on purification of autoimmune sera and subsequent production of specific centrosome antibodies.\nThe reported number of centrosome proteins varies depending on the methods applied. Because the centrosome is a non-membrane-bound organelle it does not have defined borders and isolation methods can be flawed by removing critical centrosome proteins or including contaminants that are not centrosome proteins. In addition, because the centrosome is a flexible structure that undergoes cell cycle-specific changes the number of centrosome proteins varies in different cell cycle stages. Functional screens have been used but because of the nature of centrosomes may not detect all centrosome proteins. Mass spectroscopy has identified as many as 500 centrosomal proteins (Andersen et al. 2003) while a more conservative number of 100 have been identified with different methods at various cell cycle stages with about 60 being present in the interphase centrosome (reviewed by Wilkinson et al. 2004) and may reflect the average centrosome protein quantities that are characteristic for interphase centrosomes in typical mammalian somatic cells. Various definitions have been used to describe the centrosome. It is generally agreed that the centrosome core structure consists of permanently associated centrosome proteins that remain after treatment of the centrosome complex with microtubule depolymerizing agents such as cold, nocodazole, colchicine derivatives, and others. The well-studied \u03b3-tubulin is a protein of the centrosome core structure. Centrosome core proteins are permanently associated with the centrosome structure throughout cell cycle-dependent structural changes in most systems. Such cell cycle-specific changes are particularly apparent in reproductive cells after fertilization in, which rapid changes of centrosomes and microtubule formations are required for rapid cell cycle functions. Other centrosome proteins associate with the core structure during different cell cycle stages to perform cell cycle-specific functions (discussed below). Such proteins include NuMA (reviewed in Sun and Schatten 2006) that performs essential functions in mitosis. The analysis of determining the number of centrosome proteins is further complicated by the great number of proteins that use centrosomes as a central docking station or platform where regulatory complexes accumulate and communicate by signaling through the microtubule network. Such complexes may co-localize with centrosomes but should not be included in the definition of the centrosome. Known and newly identified centrosome proteins that localize to human centrosomes are reviewed in Wilkinson et al. (2004). Centrosome proteins that are known to be present in purified centrosomes and identified by mass spectrometric analysis include the structural proteins alpha tubulin, beta tubulin, gamma-tubulin, gamma-tubulin complex components 1\u20136, centrin 2 and 3, AKAP450, pericentrin\/kendrin, ninein, pericentriolar material 1 (PCM1), ch-TOG protein, C-Nap1, Cep250, Cep2, centriole-associated protein CEP110, Cep1, centriolin, centrosomal P4.1 associated protein (CPAP), CLIP-associating proteins CLASP1 and CLASP 2, ODF2, cenexin, Lis1, Nudel, EB1, centractin, myomegalin; the regulatory molecules cell division protein 2 (Cdc2), Cdk1, cAMP-dependent protein kinase type II\u2014alpha regulatory chain, cAMP-dependent protein kinase\u2014alpha catalytic subunit, serine\/threonine protein kinase Plk1, serine\/threonine-protein kinase Nek2, serine\/threonine-protein kinase Sak, Casein kinase I, delta and epsilon isoforms, protein phosphatase 2A, protein phosphatase 1 alpha isoform, 14-3-3 proteins, epsilon and gamma isoforms; the motor and related proteins dynein heavy chain, dynein intermediate chain, dynein light chain, dynactin 1, p150 Glued, dynactin 2, p50, dynactin 3; and the heat shock proteins heat shock protein Hsp90, TCP subunits, and heat shock protein Hsp73.\nCentrosomes can be depleted of many PCM components by treatment with high salt which also extracts \u03b3-tubulin (Moritz et al. 1998) leaving behind centrosome scaffolds. A centrosome matrix has been described in several studies and has been visualized in the classic sea urchin invertebrate model system with low voltage field emission scanning electron microscopy (LVFESEM) on cold treated and isolated centrosomes (Thompson-Coffe et al. 1996; reviewed in Schatten et al. 2000a; Schatten and Chakrabarti 2004), which revealed a particle structure of 1\u20132\u00a0\u03bcm and repeating subunits.\nCentrosome proteins that have been studied more fully in various cell systems include \u03b3-tubulin, pericentrin, centrin, and NuMA.\nGamma-tubulin is an essential centrosome protein which had been discovered through fungal genetics (Oakley and Oakley 1989) and since then described in all eukaryotic cell groups. Gamma tubulin is not only found in centrosomes but it also can serve as nucleating sites in animal cells that do not display typical centrosomes (Tulu et al. 2003). Gamma-tubulin is localized at the oocyte cortex in amphibians which has been shown by Pfeiffer and Gard (1999). In plants that do not have typical centrosomes, \u03b3-tubulin is thought to nucleate unfocused microtubules within the cell and it is found along the length of microtubules (Canaday et al. 2000). Gamma-tubulin is a highly conserved protein in eukaryotes. It is vital for centrosome functions and elimination of \u03b3-tubulin is lethal to cells and to the organism (Oakley et al. 1990; Joshi et al. 1992) while elimination of components that bind to \u03b3-tubulin has little effect on cell viability. The function of \u03b3-tubulin was clearly determined by purification of the \u03b3-tubulin ring complex (\u03b3-TuRC) that revealed a ring shape and a substructure that were able to nucleate microtubule polymerization in vitro (Zheng et al. 1995). The ca. 2.2-MDa \u03b3-TuRC consists of 12 or 14 \u03b3-tubulin molecules and is the nucleating complex for microtubules in all cells studied so far (Hannak et al. 2002). Gamma tubulins may laterally interact with each other. Several accessory proteins are associated with this complex (Tassin and Bornens 1999; Gunawardane et al. 2000; Murphy et al. 2001) providing new challenges for further investigations. The \u03b3-TuRC has been well characterized by electron microscopy including immunoelectron tomography showing an open ring structure of ca. 25\u00a0nm diameter (Moritz et al. 1995; Zheng et al. 1995; Oegema et al. 1999; Moritz et al. 2000). The \u03b3-TuRC is capable of functionally capping the minus ends of microtubules facilitating growth of microtubule protofilaments. Six components of the \u03b3-TuRC in addition to \u03b3-tubulin have been identified so far (Gunawardane et al. 2003; Murphy et al. 2001; Tassin et al. 1998). Gamma-tubulin complexes are required for microtubule assembly which has been shown by using salt-extracted centrosomes that lack \u03b3-TuRCs and are not able to recruit microtubules while addition of \u03b3-tubulin complexes from cytoplasmic extracts restored nucleation competency although various other components are needed to anchor the \u03b3-TuRC to the centrosome structure including the large coiled-coil A-kinase anchoring proteins (Tassin et al. 1998; Murphy et al. 1998; Dictenberg et al. 1998; Doxsey et al. 1994; Flory and Davis 2003; Gillingham and Munro 2000; Kawaguchi and Zheng 2004; Keryer et al. 2003; Steadman et al. 2002; Takahashi et al. 2002) and Cep135 (Ohta et al. 2002). The microtubule-minus-end-binding proteins including \u03b3-TuRC are concentrated at the proximal ends of centrioles while tubulin polyglutamylation of the centriole walls modulates interaction between tubulin and microtubule associated proteins. The centriole-interacting proteins can bind and aid in the recruitment of matrix proteins.\nA cell cycle-dependent regulation of \u03b3-TuRC is thought to account for differences between interphase and mitotic microtubule structure formations and activities. In interphase \u03b3-TuRC nucleates fewer but longer microtubules while in mitosis, more \u03b3-TuRC is associated with the centrosome, which is part of the centrosome maturation process that takes place from interphase to mitosis (discussed below). Mitotic microtubules are shorter, larger in number, and highly dynamic. They are regulated by a number of cell cycle-specific proteins that participate in centrosome regulation such as the small GTPase Ran, Aurora A kinase, polo-like kinases and others.\nPericentrin is a centrosome protein that plays a role in centrosome and spindle organization (Doxsey et al. 1994; Dictenberg et al. 1998; Young et al. 2000). It forms a ca. 3 MDa-complex with \u03b3-tubulin and depends on dynein for assembly onto centrosomes (Young et al. 2000). Pericentrin plays a role in recruiting \u03b3-tubulin to centrosomes in vertebrate cells (Dictenberg et al. 1998) along with several other proteins; it is part of the pericentrin\/AKAP450 centrosomal targeting (PACT) domain (Gillingham and Munro 2000). Mutation of the pericentrin gene results in loss of recruitment of several other centrosomal proteins aside from \u03b3-tubulin, which argues for its essential role in the centrosome complex; it may provide a link between centrioles and centrosomes (Martinez-Campos et al. 2004).\nCentrins are small proteins and members of a highly conserved subgroup of the EF-hand superfamily of Ca2+-binding proteins that is associated with centrioles (Bornens 2002; Jurczyk et al. 2004). Centrin is also an intrinsic component of centrosomes that has an essential role in the duplication of centrosomes (Levy et al. 1996; Salisbury 1995; Lutz et al. 2001; reviewed in Manandhar et al. 2005) which has been clearly determined in mutation experiments (Salisbury et al. 2002).\nNuMA (Nuclear Mitotic Apparatus protein) is an essential cell cycle-dependent centrosome-associated protein that plays a significant role in the organization of the mitotic apparatus during mitosis. It is a multifunctional protein (reviewed in Sun and Schatten 2006, 2007) that performs nuclear functions as nuclear matrix protein during interphase and translocates from the nucleus to spindle poles at the onset of mitosis to form a crescent-shaped complex around centrosomes tethering microtubules into the accurate bipolar organization (addressed below in more detail).\nMany of the other centrosomal proteins also play significant roles in centrosome functions but are not discussed in detail in this section. Several centrosomal proteins have been implied in microtubule anchoring to the centrosome including ninein that has strongly been implied to serve as microtubule minus-end anchoring protein (Mogensen et al. 2000) and dynactin that has a major role in microtubule anchorage at centrosomes as well as at non-centrosomal anchorage sites. It is preferentially localized to the mother centriole and plays a role in microtubule organization (Quintyne et al. 1999; Schroer 2001; Quintyne and Schroer 2002).\nCentrosome cycle within the cell cycle\nLive cell imaging and immunofluorescence microscopy have clearly shown the remarkable shape changes of the centrosome which is best visualized in reproductive cells after fertilization requiring rapid centrosome and microtubule dynamics to position the pronuclei and prepare the zygote cell for mitosis. Figure\u00a03 shows stages of the centrosome cycle in a sea urchin egg which is the model system used by Boveri (1901) for many of his centrosome studies. Centrosomal material disperses around the zygote nucleus (Fig.\u00a03a) to become bipolar in late prophase (Fig.\u00a04c). It becomes maximally condensed during metaphase (Fig.\u00a03e) and expands within the anaphase spindle poles (Fig.\u00a03g) before it becomes compacted again when telophase nuclei form. Figure\u00a03b, d, f, and h are images of microtubule labeling for either the same cell or cells of equivalent cell cycle stages.Fig.\u00a03The structural cell cycle-dependent changes of centrosomes are shown here in sea urchin eggs during the first cell cycle after fertilization. This system had been used by Theodor Boveri for the majority of his classic studies on centrosomes. Centrosome material disperses around the zygote nucleus (aarrows) and separates to the poles during prophase (carrows). Centrosomes become densely compacted in metaphase (earrows) and disperse during anaphase (garrows). The correlated images for microtubules either from the same cell or from corresponding cell cycle stages are shown in b, d, f, and h. Immunofluorescence microscopy of centrosomes, microtubules, and DNA (blue). Centrosomes are displayed in green; microtubules are displayed in green (b, d) or red (f, h). Reprinted with permission from Schatten et al. 2000aFig.\u00a04In interphase, a single centrosome is juxta-positioned to the nucleus. a, c, and d show small GFP-centrin-labeled centrosomes in mouse 3T3 cells. Microtubules are detected with \u03b1-tubulin and shown in red; b is of an LNCaP prostate cancer cell labeled with human autoimmune antibody SPJ displaying multiple centrosomal foci perhaps indicating centrosome abnormalities; e shows a porcine fibroblast cell labeled with \u03b3-tubulin to detect the centrosome and Mitotracker Rosamine to detect mitochondria. Microtubules are shown in green; b reprinted with permission from Schatten et al. 2000b\nIn interphase, a single centrosome containing a pair of centrioles is juxta-positioned and closely connected to the nucleus as shown in Fig.\u00a04 in an LNCaP prostate cancer cell (Fig.\u00a04b), mouse 3T3 cells containing GFP-centrin label to detect centrosomes and labeled with \u03b1-tubulin to detect microtubules (Fig.\u00a04a, c, d), and a pig fibroblast cell labeled with \u03b3-tubulin to detect the centrosome and Mitotracker Rosamine to detect mitochondria (Fig.\u00a04e). Centrosomes duplicate shortly before the G2 stage of the cell cycle in a precisely orchestrated program to ensure precise duplication into two and not more centrosomes. This process begins with disorientation of the pair of centrioles, centriole duplication, centrosome disjunction, and results in sister centrosome separation as reviewed in Mack et al. (2000) and Ou et al. (2004). While a wealth of data has been accumulated on the duplication and separation of centrioles data on the molecular events underlying centrosome duplication, disjunction and separation are only slowly accumulating. Centrosome separation refers to the spatial separation of centrosome material around the nucleus which is driven by plus- and minus-end directed microtubule motor proteins. The Nek2 kinase is involved in centrosome disjunction (Meraldi and Nigg 2001; reviewed by Fry 2002). Phosphorylation of the centrosomal protein centrin plays a role in centrosome disjunction at the G2\/prophase transition.\nPolo like kinases are required for multiple stages of mitotic progression and have clearly been implicated in centrosome separation. Specific kinases have been linked to centrosome and microtubule dynamics and Plk1 plays a critical role in centrosome and microtubule organization (Sun et al. 2001a, b, c, d, 2002; Tong et al. 2002, 2003; Fan et al. 2003). Plk1 and Plk3 both have been implicated in microtubule and centrosome functions in interphase and in mitosis (Fenton and Glover 1993; Donaldson et al. 2001; Wang et al. 2002). Loss of Plk3 function has also been associated with loss of cell shape which indicates loss of microtubule functions underneath the plasma membrane (Wang et al. 2002).\nWhile the role of centrosomes in microtubule nucleation and chromosome separation has been studied in great detail, centrosomes have only more recently been implicated in cytokinesis. Khodjakov and Rieder (2001) showed that 30\u201350% of cells with laser-ablated centrosomes failed to complete cytokinesis perhaps because spindles formed without orientation and did not have attachment to the cell cortex resulting in incomplete chromosome separation. The molecular mechanisms by which centrosomes exert an effect on cytokinesis are not clear but their role as docking station for cytokinesis-required regulatory proteins is implied and may include Plk and microtubule motor proteins.\nThe role of centrosomes as docking station where regulatory complexes accumulate for cell cycle activities has only been studied in recent years. The centrosome serves as a platform for a wide variety of regulatory molecules that play a role in cell cycle-specific functions but are not themselves centrosome proteins. These complexes regulate peripheral events through microtubule-dependent transport which indicates a role of the centrosome as command center that integrates signals using microtubules as signaling routes. As such, centrosomes play critical roles in cell cycle regulation in both mitosis and interphase by providing a central signaling station and controls to trigger the next phases of the cell cycle by integrating, regulating, and amplifying signaling pathways.\nPhosphorylation and posttranslational modifications are other important aspects in centrosome regulation required for cell cycle-specific changes which has been reviewed in Wilkinson et al. (2004).\nCentrosome duplication in a regular cell cyle is typically well synchronized with the DNA cycle. Studies on duplication of centrosomes have been performed in recent years to reveal that there is a block to reduplication which assures that centrosomes are duplicated accurately only once within a normal cell cycle. Elegant studies to determine the block to reduplication have been performed by Wong and Stearns (2003) who fused G1 phase cells containing one centrosome with G2 phase cells containing two centrosomes to determine if an already duplicated centrosome would re-duplicate under these conditions. Interestingly, the G1 centrosome duplicated while the G2 centrosome did not which led to the conclusion that there is an intrinsic block to reduplication within the centrosome. Initiation of centrosome duplication is under cytoplasmic control and driven by cyclin-dependent kinase 2 (Cdk2) complexed with cyclin E or cyclin A that rises during the late G1 stage (reviewed in Sluder 2004). These results were compiled by several laboratories in the late 1990s using Xenopus eggs (Hinchcliffe et al. 1999; Lacey et al. 1999) and mammalian somatic cells (Mantel et al. 1999; Matsumoto et al. 1999). It was later shown that initiation of centrosome duplication also requires calcium\/calmodulin-dependent kinase II (CaMKII) (Matsumoto and Maller 2002). CaMKII phosphorylates centrosome proteins in vitro (Pietromonaco et al. 1995) and is localized to spindle poles (Ohta et al. 1990). Ubiquitin-mediated proteolysis of centrosomal proteins may be involved in the block to reduplication as proposed by several investigators (Tugendreich et al. 1995; Freed et al. 1999; Gstaiger et al. 1999) who showed localization of a variety of components of the SFC proteolysis pathway as well as the 26-S proteasome to centrosomes in human cells.\nCentrosome duplication and DNA replication both require hyperphosphorylation of the retinoblastoma (RB) protein and activation of Cdk2. Although the centrosome cycle and DNA cycle are normally coupled through cell cycle-dependent checkpoints it has been possible to dissociate the centrosome cycle from other cell cycle events. Application of cycloheximide to Xenopus oocytes has resulted in centrosome duplication while other cell cycle events were inhibited (Gard et al. 1990). Hydroxyurea or aphidicolin also resulted in continuation of the centrosome cycle in CHO cells producing supernumerary centrosomes while DNA replication was inhibited (Balczon et al. 1995).\nNormally, the DNA cycle and the centrosome cycle are intimately coupled and synchronized in a regular cell cycle. When the nucleus or DNA is damaged, centrosomes become inactivated in a well-orchestrated process. The \u03b3-TuRC dissociates from centrosomes, the spindle fails to assemble resulting in failure of chromosome segregation. Figure\u00a05 is a schematic representation of a typical centrosome cycle within a typical cell cycle. Please see figure legend for description.Fig.\u00a05A typical mammalian centrosome cycle within the cell cycle. The single interphase centrosome (a) is closely associated with the nucleus and nucleates an array of interphase microtubules. Centrosome duplication occurs during the S phase in synchrony with DNA dupliction (b). Centrosome separation of the duplicated centrosome toward opposite poles takes place in the early prophase stage (c). The bipolar mitotic apparatus becomes established when centrosomes have reached the opposite poles and the nuclear envelope has broken down (d). During this stage interphase centrosomes mature into mitotic centrosomes acquiring mitosis-associated centrosomal proteins including NuMA that moves out of the nucleus to become a mitotic centrosome-associated protein. The metaphase centrosome (e) becomes highly compacted to organize the metaphase spindle with microtubules attached to the kinetochores. Anaphase is the stage when centrosomal material becomes decompacted again (f) before reorganizing into interphase centrosomes that associate with the nuclei of the separating daughter cells (g). Centrosomes shaded yellow with centrioles and PCM displayed in black; nuclei shaded orange; microtubules displayed as black rods; chromosomes displayed in black. Modified from Sun and Schatten 2007\nCentrosome maturation into mitotic centrosomes\nOne of the most important functions of the centrosome is its role in establishing the bipolar mitotic spindles that orchestrate the precisely balanced segregation of chromosomes. To accomplish this important function a major reorganization of centrosomal material occurs at the G2\/M transition in vertebrate cells which is referred to as centrosome maturation. During this time some centrosomal proteins become diminished while others such as the \u03b3-TuRC become enriched (Khodjakov and Rieder 1999) and \u03b3-tubulin increases three to five-fold (Khodjakov and Rieder 1999).\nCyclin-dependent kinases play a significant role in centrosome cycle progression. The G2\/M transition requires Cdk1\/cyclin B as well as Cdk1\/cyclin A, which has been reviewed in detail by Fry and Hames (2004). Cdk1 is localized to centrosomes at the onset of mitosis (Bailly et al. 1989; Pockwinse et al. 1997) and it has been shown that Cdk1\/cyclin B activation is detected in centrosomes during prophase (Jackman et al. 2003).\nThe centrosome cycle is tied to the centriole cycle and both are closely coupled with the DNA cycle. At the transition from the G1 to S phase centriole duplication begins and continues through G2 when the replicating pro-centriole elongates. Two duplicated juxta-positioned centrioles are clearly apparent at the G2 stage. The two centrosome complexes are held to each other throughout interphase and undergo disjunction prior to entry into mitosis (Mayor et al. 1999). Centrosome maturation takes place by acquiring mitotic centrosome proteins including Plk1 (Golsteyn et al. 1995), NuMA (Merdes et al. 1996) and others while interphase centrosome proteins such as C-Nap1 (Fry et al. 1998) or Nlp (Casenghi et al. 2003) are removed. Increased recruitment of \u03b3-TuRC to the centrosome assures increased nucleation of microtubules for spindle formation. While centriole separation has been studied in great detail in recent years, our knowledge about centrosome separation is still incomplete and we do not yet fully understand how centrosomes are separated into two. Separation is complete when cells re-enter G1 with a single centrosome surrounding a pair of centrioles.\nOne of the well-studied essential mitotic centrosome-associated proteins is NuMA. After nuclear envelope breakdown (NEB), NuMA becomes a most significant centrosome-associated protein (reviewed in Sun and Schatten 2006, 2007). NuMA does not associate with interphase centrosomes. It serves as nuclear matrix protein in the interphase nucleus and becomes dispersed into the cytoplasm during NEB, a process in which cyclin B plays a critical role. Cdk1\/cyclin B dependent phosphorylation is important for translocation of NuMA from the nucleus into the cytoplasm (Saredi et al. 1997; and references therewithin) where it associates with microtubules using a dynein\u2013dynactin-mediated mechanism to be translocated along microtubules to the centrosome area. Next, to become a functional mitotic protein NuMA needs to be translocated from the cytoplasm to the mitotic centrosomes. In this highly regulated process microtubules and dynein\u2013dynactin play crucial roles in transporting NuMA to the mitotic poles where it forms an insoluble crescent around centrosomes that tethers microtubules precisely into the bipolar configuration that forms the mitotic apparatus (Merdes and Cleveland 1998). NuMA is distributed to the separating centrosomes during early mitosis. It ensures minus-end binding and stabilization of microtubules on the centrosome area that faces chromosomes. NuMA facilitates cross-linking of spindle microtubules, aiding in the organization and stabilization of spindle poles from early mitosis to anaphase. To exit its mitotic functions and to relocate to the nucleus NuMA needs to be dissociated from the mitotic spindle poles through a process that requires cdc1\/cyclin B (Gehmlich et al. 2004). Destruction of cyclin B promotes exit from mitosis, which starts at the mitotic poles and is linked to disassembling the mitotic centrosome complex. Failure of NuMA to relocate to the nucleus will result in cytoplasmic NuMA spots that organize abnormal microtubule formations (Gehmlich et al. 2004).\nAs nuclear matrix protein NuMA is involved in DNA replication and transcription. It displays distinct staining patterns in the nucleus (Merdes and Cleveland 1998; Zeng 2000; Gobert et al. 2001) and stains heavily in mitosis when using anti-NuMA antibodies (Saredi et al. 1997; Merdes et al. 2000; Gobert et al. 2001; Zhong et al. 2005; Liu et al. 2006). Inaccurate NuMA regulation and atypical NuMA distribution will result in abnormal mitosis as has been reported for cancer cells (Saunders et al. 2000).\nSome of the important mitotic cell cycle regulators are concentrated at the centrosome and include Polo and Aurora A kinases (Barr and Gergely 2007), and cdc2\/cyclin B kinase (Jackman et al. 2003). Some of these proteins bind to the anaphase promotion complex\/cyclosome (APC\/C). The activated APC\/CCdc20 degrades cyclin B and securin to allow cell cycle exit from mitosis (Kramer et al. 2000; Huang and Raff 1999; Wakefield et al. 2000).\nMicrotubule motor proteins are essential for the composition of a functional mitotic centrosome as many centrosome proteins are shuttled along microtubules to the centrosome core structure including pericentrin and NuMA. Pericentriolar satellites (or PCM-1 granules) may function as cargo vehicle for various proteins including ninein, centrin, and pericentrin (Dammermann and Merdes 2002). These PCM-1 granules move along microtubules in a dynein-dependent process (Kubo et al. 1999; Kubo and Tsukita 2003).\nThe microtubule motor proteins dynein and kinesin are crucial for transport of cargo along microtubules and balanced transport is important to maintain cellular homeostasis. In most cells, transport of cellular organelles such as mitochondria and vesicles along microtubules is mediated by the kinesin family of motor proteins to the plus end of microtubules and by cytoplasmic dyneins and its co-factor dynactin to the minus ends (reviewed by Welte 2004). Bidirectional transport of cell organelles and vesicles is also possible (Haggeness et al. 1978). Imbalanced transport can lead to pathologies related to centrosome and microtubule functions as well as to failures in organelle and vesicle distribution. Centrosome and microtubule pathologies are the result of disrupted transport. Disruption of transport is the cause or effect in a chain of signal transduction events; disruption depletes the resources of proteins and generates signals regulating transcription. Following disruption is a cascade of declines resulting in secondary pathologies. Microtubule-based transport processes are vital for cell functions and dysfunctional transport along microtubules has been implicated in cellular deterioration and disease such as Alzheimer\u2019s.\nAs is the case for transport toward the minus end transport of microtubules toward centrosomes dynein is involved in the mediation of transloction along microtubules to the plus ends towards the cell periphery. Dynein motors may be linked to actin or intermediate filaments as has been shown for neurons in which dynein and dynactin are responsible for translocation of microtubules from the centrosome in the cell body into the axon (Baas 1998; Ahmad et al. 1999). Myosin may play a role in connecting microtubules to actin filaments (Cao et al. 2003).\nSupernumerary centrosomes: role in genomic instability and cancer\nBoveri\u2019s visionary book on centrosome abnormalities in cancer had originally been translated from German into English as its significance and powerful implications had been recognized from the onset (Boveri 1914). This book has recently been re-edited, again because of its significance that continues to fascinate investigators in the centrosome field as well as others. The visionary research and intellectually remarkable interpretations implied one cell with abnormal centrosomes as the primordial tumor cell.\nBoveri\u2019s observations of dispermic eggs developing multipolar mitoses and his brilliant thought process to imply supernumerary centrosomes in tumor has found enormous recognition during the past decade when several groups followed up on Boveri\u2019s initial findings and analyzed archived tumor tissues of various types. Since then multipolar mitoses have been identified in a large number of cancers as hallmarks of tumor tissue with direct implications in the causes for aneuploidy (Lingle and Salisbury 1999, 2000; Lingle et al. 1998; Pihan et al. 2003; Pihan and Doxsey 1999; Schatten et al. 1998a, b, 1999a, b, 2000a, b, c; Brinkley and Goepfert 1998; and others; reviewed in Goepfert and Brinkley 2004). Moreover, overexpression of specific centrosome proteins resulted in abnormal centrosome configurations and aneuploidy (Lingle et al. 2002; Katayama et al. 2003) confirming the important role of centrosomes in cancer development and progression. Increased centrosome number and volume, supernumerary centrioles, accumulation of increased PCM, and abnormal phosphorylation of centrosomes have all been associated with cancer cell centrosomes followed by loss of cell polarity (Lingle et al. 1998; Schatten et al. 2000c).\nDeregulation of centrosome duplication and genes implicated in centrosome amplification are processes that lead to cascades of cell cycle-related abnormalities. The loss of p53 is associated with multiple cycles of centrosome duplication in one S phase resulting in multiple centrosome numbers (Pihan and Doxsey 1999). P53 might play a role in the block to reduplication in synchrony with the DNA cycle. Viral oncoproteins that inactivate p53 also result in cells with supernumerary centrosomes as has been shown for the papilloma virus (reviewed in M\u00fcnger and Duensing 2004). However, different investigators have challenged that loss of p53 directly affects centrosome duplication (Meraldi et al. 2002) and attribute centrosome abnormalities to missing checkpoint functions after loss of p53.\nThe breast cancer suppressor gene BRCA1 may also play a role in deregulation of centrosome duplication as targeted deletion of BRCA1 exon11 leads to centrosome amplification (Xu et al. 1999). However, the mechanism by which BRCA1 affects centrosome duplication remains to be fully investigated. BRCA1 plays a role in G2\/M checkpoint functions which may result in loss of the block to centrosome reduplication.\nAurora kinases play central roles in the mitotic process and cell division and Aurora A has been implicated in centrosome amplification in breast cancer. Aurora A localizes to centrosomes and overexpression of Aurora A causes multipolar mitotic spindles and is implicated in early development of mammary tumors.\nOncogenic insults may result in a high number of mutation rates in cells predispositioned to tumor and the mutation rate increases in cells that have reached replicative senescence. In contrast, cervical carcinogenesis has strongly been associated with infections by high-risk human papillomavirus (HPVs). It has been suggested that the HPV E7 oncoprotein may induce primary centrosome duplication errors and act as mitotic mutator (reviewed in M\u00fcnger and Duensing 2004). As with other cancers, abnormal multipolar mitoses resulting from supernumerary centrosomes have clearly been associated with HPV-associated lesions and centrosome abnormalities are already detected in early stages of tumor development. Studies have shown that uncoupling of the cell division cycle from the centrosome cycle subverts centrosome homeostasis (Duensing et al. 2000).\nAn emerging field in centrosome research is their role in aging. Supernumerary centrosomes are thought to be associated with aging and have been shown in senescing cells (Schatten et al. 1999a). Many of the cell cycle regulators that play a role at the transition from G2 to M phases are downregulated in aging cells (Ly et al. 2000), which is the most significant phase for reorganizations of microtubules and centrosomes. As mentioned above, centrosomes undergo major changes in phosphorylation at the G2\/M transition and it has been shown that centrosomes in aging cells have lower activity in centrosome-associated protein kinases (Cande 1990; Huang 1990). Plk has been identified as one of the gene products that are down-regulated in aging cells (Ly et al. 2000), which will have significant consequences for centrosome and microtubule organization. This is important considering that many diseases of aging involve inaccurate microtubule organization coupled with transport dysfunctions.\nCentrosome-independent pathways and non-centrosomal microtubule arrays\nTypical centrosomes are absent in most plant cells and centrosome material without centrioles compose the meiotic spindle in animal oocyte cells. Somatic cells also can assemble bipolar spindles in the absence of typical centrosomes (Meraldi and Nigg 2002; Manandhar et al. 2005). In animal cells, microsurgery to remove the centrosome from transformed mouse fibroblast cells (L929 cells) resulted in reorganization of microtubules without a centrosome but absence of a mitotic spindle and cell cycle progression arrest implying that the centrosome is important for cell cycle progression. Although the centrosome is vital for animal cells and there are no viable mutants in animal cells that lack centrosomes there are centrosome-independent pathways that have recently been explored by destroying centrosomes in situ with focused pulses of high-energy (laser) light, termed the \u201cablative photo-decomposition\u201d approach (reviewed in Khodjakov and Rieder 2004). These studies used Green Fluorescent Protein (GFP) fusion for detection of centrosomes for precise centrosome targeting and to follow the extent of centrosome destruction in live cells. These studies showed that some acentrosomal spindle formation is possible when centrosomes are destroyed.\nIn parthenogenetic human, rhesus, and bovine embryos a bipolar spindle is formed at the time of first mitosis without apparent \u03b3-tubulin and pericentrin foci at the spindle poles which argues that mitotic spindles can self-assemble without functional MTOCs. Acentrosomal meiotic and mitotic spindles have been described (Lee et al. 2000; Megraw et al. 2001; Bonaccorsi et al. 2000; Khodjakov et al. 2000; Khodjakov and Rieder 2001; Raff 2001). It is not entirely clear how the spindle poles are formed but microtubule motor activities have been implicated in this process (Walczak et al. 1998).\nRelease of entire microtubules from the centrosome complex has been well documented as the main mechanism to produce non-centrosomal, free microtubules in neurons (Ahmad et al. 1999). The microtubule-severing protein katanin has been implicated in this process (Ahmad et al. 1999; Baas 1999; McNally and Vale 1993; McNally et al. 2000). Katanin may also play a role in M-phase severing processes at the spindle poles in mammalian cells when spindle pole microtubules become depolymerized (McNally et al. 1996; McNally and Thomas 1998). Katanin also plays a role in severing of axonemal microtubules (Lohret et al. 1998) and katanin-like proteins have been identified in plants (Burk et al. 2001).\nNon-centrosomal microtubule arrays have been described for polarized epithelial cells (reviewed by Morgensen 2004) to serve various specialized functions of differentiated cells. In these cells centrosomes are present but organize fewer microtubules while most of the cellular microtubules are non-centrosomal and the minus ends are localized toward the plasma membrane without \u03b3-tubulin anchorage. Such organization is seen in rat Sertoli cells (Vogl et al. 1995). Other examples of non-centrosomal microtubules with significant cellular functions are found in the organ of Corti in the inner ear, and during vertebrate myogenesis. The microtubule minus end-anchoring protein ninein is localized to the non-centrosomal microtubule minus-end sites in MDCK polarized epithelial cells while \u03b3-tubulin or pericentrin are absent.\nMicrotubule-associated proteins (MAPs) at the minus ends may play a role in minus end-capping of microtubules preventing their depolymerization. Plus-end capping involves Rho GTPases and the downstream effector mDia (mouse diaphanous-related formins) (Gundersen 2002; Palazzo et al. 2001). Other plus-end capping candidates are CLIP-170, EB1, dynein\/dynactin, and APC (adenomatis polyposis coli) protein (reviewed in Mogensen 2004). It is thought that these non-centrosomal microtubules originate from centrosomes. Dynactin also localizes to the non-centrosomal microtubule minus-end sites in MDCK polarized epithelial cells in further support for anchorage sites at the cell apex. A microtubule release and capture mechanism has been implicated in the generation of non-centrosomal apico-basal microtubule formations in polarized epithelial cells. The non-centrosomal microtubule organization provides support for the idea that two functionally distinct microtubule minus-end-associated complexes are independent from each other and comprise the nucleating complex and the anchoring complex. The role of ninein in microtubule anchorage at the centrosomal PCM has been reported by Mogensen et al. (2000). The minus end microtubule anchoring capabilities by ninein have been demonstrated by overexpression of ninein causing an increase of microtubule anchorage and decrease in microtubule release. The loss of microtubule radial organization has been demonstrated by using either anti-ninein antibodies or RNAi depletion of ninein (Dammermann and Merdes 2002).\nCentrosome abnormalities and mitotic cell death\nAlterations in centrosome organization and function are associated with cellular stress and may be part of the mitotic catastrophe response characteristic for programmed mitotic cell death. Centrosome structure and function are directly affected by chaotropic agents such as formamide (Schatten et al. 2000d) and in response to heat shock and DNA damage. While mild heatshock treatment triggers repair mechanisms and centrosome thermotolerance severe heat shock leads to cell death by a mitotic catastrophe mechanism. Formamide induces the formation of multipolar spindles perhaps as a result of centrosomal protein denaturation (Schatten et al. 2000d). Tripolar and fractionated centrosomes after treatment of cells with formamide are shown in Fig.\u00a06a and b, respectively. The effects of heat shock and other environmental stresses have been studied in various cell systems that all displayed centrosome alterations. Hsp70 was localized to centrosomes in HeLa cells (Rattner 1991); Nakahata et al. (2002) reported centrosomal dysfunction and non-apoptotic mitotic catastrophe in human tumor cells which has also been reported for cells exposed to radiation (reviewed in Sato et al. 2004). Some of the causes for mitotic catastrophe are related to checkpoint or DNA repair failures which is thought to be a genetically programmed response to genotoxic stress leading to centrosome inactivation (Sibon et al. 2000). Loss of \u03b3-tubulin, loss of pericentrin and multipolar spindles as well as additional PCM foci and structural alterations are some of the characteristics associated with heat shock (Vidair et al. 1995, 1996, 1993; Barrau et al. 1978; Knox et al. 1991; Debec et al. 1990; Marcaillou et al. 1993; Debec and Marcaillou 1997). Cells with mitotic catastrophe and multipolar spindles are eliminated from a cell population by a process resulting in cell death which has been reported for mammalian somatic cells as well as for Drosophila cells in culture (Schatten et al. 1999a). This process is used in addition to apoptosis to eliminate cells with dysfunctional mitotic cells from a normal cell population. It has also been shown to account for causes of embryo abnormalities after somatic cell nuclear transfer (SCNT) (reviewed in Schatten et al. 2005; Zhong et al. 2007). Such cells undergoing mitotic cell death are negative for TUNEL staining and isolated DNA is not characterized by laddering that is typical for apoptosis (Roninson et al. 2001; Nabha et al. 2002). Apoptotic inhibitors are not effective in these cells and apoptotic bodies are not formed while fragmentation of centrosomes appears characteristic for mitotic cell death.Fig.\u00a06The chaotropic agent formamide causes centrosomal damage and results in tripolar (a) or multiple (b) centrosomal foci as shown here in an example using sea urchin eggs as model system. Centrosomes labeled red (a) or green (b). Microtubules in b labeled red a= modified from Schatten et al. 2000d\nIonizing radiation is well known to induce aberrations in centrosome number in tumor cell lines but the underlying causes are not well understood (Sato et al. 1983, 2000a, b). As discussed above for heat shock damage the DNA checkpoint pathways may play a role in radiation-induced centrosome aberrations or it may directly affect centrosome structure. Mitotic cell death is seen in most cell types after ionizing radiation while apoptosis is primarily seen in lymphoma and leukemia cells. Exposure to gamma-irradiation at a single dose of 10\u00a0Gy resulted in increased numbers of centrosomes with varied abnormalities and cell fractionation (reviewed in Sato et al. 2004). Mitotic cell death after mitotic catastrophe is also caused by other chemotherapeutic agents such as doxorubicin, cisplatin, bleomycin, and taxol (Roninson et al. 2001; Schatten et al. 2000a, b). The causes for centrosome number increases after irradiation are not clear but loss of cell cycle regulators such as p53 and its downstream targets p21 and Gadd45 have been implicated in supernumerary centrosomes (Fukasawa et al. 1996; Hollander et al. 1999; Mantel et al. 1999; Carroll et al. 1999). Dissociation of centrosome cycles from DNA cycles may be among the causes for centrosome aberrations and multipolar spindle formations after irradiation. Aberrant hypermethylation has recently been implicated in inactivation of checkpoint genes that may influence cell cycle-dependent centrosome abnormalities as reported for pancreatic cancer (Ohki et al. 2000; Sato et al. 2003).\nEnvironmental stress can result in the formation of aggresomes which are oftentimes localized close to centrosomes and are thought to be the result of misfolded proteins (Ellgaard et al. 1999; Johnston et al. 1998; Wojcik and DeMartino 2003; Kopito 2000; Roth et al. 2008). Some of the aggresomes contain \u03b3-tubulin and are associated with disease or neurodegenerative disorders such as Parkinson\u2019s disease and dementia (McNaught et al. 2002).\nCentrosomes in reproduction including fertilization and somatic cell nuclear transfer (SCNT)\nBoveri\u2019s brilliant discoveries on centrosomes primarily came from his studies on centrosomes during fertilization and cell division in sea urchin eggs (Boveri 1901), which resulted in a wealth of profound information on the important contribution of sperm centrosomes for successful fertilization. His work on reproduction also resulted in the remarkable insights that supernumerary centrosomes are at the core of malignant tumors (Boveri 1914) which was based on his observations that dispermic eggs (eggs fertilized with two sperm) developed multipolar mitotic poles. An example of a dispermic sea urchin egg is seen in Fig.\u00a07. Boveri had recognized that sperm contribute the dominant centrosomal material that was detected by staining with iron hematoxylin and could be traced throughout cell division and development. In more recent years, staining of centrosomes with immunological probes confirmed that in most animal species except for the mouse (Schatten et al. 1986, 1987) dominant centrosomal material is contributed by sperm (reviewed in Manandhar et al. 2005; Sun and Schatten 2006, 2007) and biparental centrosome contributions to the zygote are typical for most species. More detailed studies using specific immunofluorescent probes to specific centrosome proteins provided additional insights into centrosomal contributions during gametogenesis and fertilization (reviewed by Manandhar et al. 2005). These studies showed that the sperm retains its proximal centriole while losing most of the PCM. The oocyte, on the other hand degenerates centrioles while retaining centrosomal proteins. The sperm\u2019s proximal centriole recruits egg proteins shortly after sperm incorporation including \u03b3-tubulin, centrin, pericentrin, and NuMA to the sperm centriolar complex. The recruitment of maternal \u03b3-tubulin to the sperm\u2019s centrosomal \u03b3-tubulin results in a significant accumulation of \u03b3-tubulin. The blended centrosome after fertilization also contains centrin from both sperm and egg. The zygotic centrosome that is closely associated with the decondensing male pronucleus then organizes a radial aster that grows toward the female pronucleus. The centriole-centrosome complex duplicates during the pronuclear stage. A zygote aster forms around the appositioned (or fused) male and female pronuclei (zygote nucleus) and centrosomes separate to form the bipolar mitotic apparatus in preparation for cell division (reviewed in Sun and Schatten 2006, 2007). Dysfunctional sperm centrosomes, dysfunctional zygote centrosomes, and polyspermy have been implicated in causes for male-derived infertility (reviewed in Schatten 1994) while aging of oocyte centrosomes is among the major causes for infertility in older women (Battaglia et al. 1996) as dysfunctional centrosomes are implied in the causes for aneuploidy. It is estimated that the rates of aneuploidy in preimplantation human embryos is as high as 52-61% (Munne and Cohen 1998).Fig.\u00a07A tetrapolar spindle displaying microtubules (green) and chromosomes (red) as a result of dispermy in a sea urchin egg. Such results on dispermic eggs had stimulated Boveri to propose that centrosome abnormalities are at the core of malignant tumors. Image produced in collaboration at the Integrated Microscopy Resource at the University of Wisconsin\nCentrosome remodeling after nuclear transfer: communication between embryonic and somatic cells\nCloning of animals by somatic cell nuclear transfer (SCNT) involves cellular communication of embryonic cells with somatic cell nuclei and somatic cell centrosomes from donor cells. In recent years, cloning of animals by SCNT has opened up new avenues to produce genetically modified species with higher nutritional value or improved traits that are useful in agriculture. Genetically modified pigs have been produced as models for human disease and for various other biomedical applications (reviewed in Prather 2000, 2007; Schatten et al. 2005). However, one of the major difficulties to overcome in cloning is the low cloning efficiency that ranges from 1 to 5% in different animals. Centrosomal dysfunctions have been identified as possible causes for low cloning efficiencies in several species including the pig (Zhong et al. 2007) and rhesus monkeys (Simerly et al. 2003) and centrosomal dysfunctions may also play a role in developmental abnormalities that are frequently associated with cloning.\nSCNT involves transferring a somatic cell nucleus with known genetic value into an enucleated oocyte from a different animal followed by electrical activation. Subsequently, SCNT requires communication of enucleated oocytes with the donor cell nucleus and its associated centrosome. The complex molecular regulation and functions of centrosomes in reproductive cells are complicated by the fact that somatic cell centrosomes need to be remodeled by the enucleated oocyte (reviewed in Sun and Schatten 2007), which is crucially important as the donor cell\u2019s centrosome is required to perform all functions that are normally fulfilled by the blended sperm and egg centrosomal material. Under these conditions, the enucleated oocyte\u2019s cytoplasmic regulatory machinery is required to regulate the donor cell centrosome for embryo-specific functions. Centrosome dysfunctions may occur as the oocyte\u2019s cell cycle regulation systems are different from those used by somatic cells. Indeed, multipolar mitoses resulting from supernumerary centrosomes have been reported in reconstructed pig oocytes (Zhong et al. 2007) which may be among the underlying causes for decreased cell numbers as a result of increased mitotic cell death.\nWhile blending of sperm and egg centrosomal material into a functional centrosome after fertilization is necessary for embryo-specific functions, it is not known whether the donor cell centrosome also attracts centrosomal components from the oocyte or whether the somatic cell centrosome is able to nucleate and organize microtubules for oocyte-specific functions without oocyte centrosomal components. Aberrant composition of centrosome proteins will result in abnormal microtubule organizations as has been discussed for cancer cells. As the donor cell centrosome contains \u03b3-tubulin and \u03b3-TuRC it is not clear whether it attracts additional \u03b3-tubulin from the oocyte as is the case for sperm centrosomes. Analysis of cancer cell centrosomes revealed increased \u03b3-tubulin associated with centrosomes resulting in more than the normal number of microtubules leading to aneuploidy (Lingle et al. 1998; Schatten et al. 2000a, b). Increased accumulation of microtubules in the reconstructed egg may also play a role in centrosome dysfunctions resulting in abnormal microtubule organization and aneuploidy (Zhong et al. 2007; Martin et al. 2007). The composition of the centrosome in reconstructed oocytes is not known and needs further investigation to determine whether oocyte-stored centrosome proteins are recruited to the donor cell centrosome.\nSCNT also challenges nucleo-cytoplasmic interactions as several centrosome proteins such as NuMA are localized in the nucleus, adding further complexities to centrosome remodeling. The oocyte\u2019s regulatory systems have to communicate with the donor cell\u2019s nuclear and centrosomal material as many of the cell-cycle-dependent centrosome proteins are of nuclear origin and depend on nucleo-cytoplasmic interactions for cell cycle-specific and developmentally regulated functions. Such functions include governing microtubule-mediated translocation of mitochondria for spatio-temporal requirements for ATP, translocation of macromolecular complexes, vesicles that may contain enzymes, and translocation of transient centrosome-associated proteins that are needed for molecular centrosome restructuring to perform cell cycle-specific centrosome functions. We do not yet have information on the role of cyclin B in centrosome remodeling in reconstructed embryos which will be important information to obtain as cyclin B is one of the major regulators of centrosomes and is crucial for centrosome maturation and for cell cycle-dependent molecular centrosome dynamics. Cyclin B is a critical regular of NuMA association with centrosomes and its release from spindle poles during exit from mitosis (Saredi et al. 1997; Merdes and Cleveland 1998; Gehmlich et al. 2004). In contrast to mammalian somatic cells in which cyclin B synthesis increases during G2 and M mainly as the result of cyclin B gene transcription increase, in embryonic cells cyclin B synthesis is constant throughout the cell cycle and cyclin B accumulation is the result of decrease in its degradation rate.\nTaken together, studies of centrosomes in reconstructed embryos will provide new avenues to obtain information on centrosomal regulation by cytoplasmic factors.\nConcluding remarks and future directions\nRenewed interest in centrosome research started about 25\u00a0years ago when it was shown that certain autoimmune sera from CREST patients would reliably label centrosome material in animal cells. Since then, rapid progress has been made to identify specific centrosome proteins and determine their functions, which led to renewed appreciation of the enormous significance that centrosomes carry as cellular organelles and as major platform for cellular regulation. Centrosome dysfunctions have been linked to various human genetic diseases (reviewed in Badano et al. 2005) and centrosomes can directly be affected by environmental and genotoxic stresses. Adverse effects on centrosomes will result in multiple cascades of cellular dysfunctions as centrosomes regulate distribution of cellular organelles including mitochondria which are the major source for ATP production and depend on microtubules for distribution to their cell cycle-specific functional destinations. Based on these new insights it is easy to understand that centrosomes are linked to many diseases in which transport along microtubules is impaired which includes Alzheimer\u2019s and several neurological diseases in which extensive trafficking along microtubules takes place. This area of research with a new focus on centrosomes is just at the beginning.\nThe entire field of aging research is open to investigations of centrosomes in senescing cells. Our previous studies on Drosophila cells in culture have linked multipolar mitoses to cellular senescence (Schatten et al. 1999a) and further studies are needed to explore centrosome regulation in aging cells. Other new areas of research include the effects of environmental stresses on centrosomes and manipulation and exploitation of centrosomes and the associated microtubule cytoskeleton by pathogens which has been explored for Toxoplasma parasites (Coppens et al. 2006) and has been reviewed by Scaplehorn and Way (2004) in a recent book dedicated to various aspects of centrosome research (Nigg 2004). Taken together, we are in the middle but also at the beginning of new appreciation for the centrosome as key organelle and as main station for directing and regulating cellular processes through its microtubule highway system and further research will undoubtedly result in uncovering and perhaps also repairing pathologies related to centrosome dysfunctions.","keyphrases":["centrosomes","microtubules","cell cycle","mitosis","mtoc","centrosome proteins","centrosome functions"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Eur_Radiol-2-2-1705542","title":"MR imaging in sports-related glenohumeral instability\n","text":"Sports-related shoulder pain and injuries represent a common problem. In this context, glenohumeral instability is currently believed to play a central role either as a recognized or as an unrecognized condition. Shoulder instabilities can roughly be divided into traumatic, atraumatic, and microtraumatic glenohumeral instabilities. In athletes, atraumatic and microtraumatic instabilities can lead to secondary impingement syndromes and chronic damage to intraarticular structures. Magnetic resonance (MR) arthrography is superior to conventional MR imaging in the diagnosis of labro-ligamentous injuries, intrinsic impingement, and SLAP (superior labral anteroposterior) lesions, and thus represents the most informative imaging modality in the overall assessment of glenohumeral instability. This article reviews the imaging criteria for the detection and classification of instability-related injuries in athletes with special emphasis on the influence of MR findings on therapeutic decisions.\nIntroduction\nIn the last decade, understanding of the biomechanics and pathophysiology of the athletic shoulder has significantly improved. Especially in overhead athletes and throwers several pathologic mechanisms have been identified which could not be explained by the traditional concepts of instability and impingement. With an expanded definition, glenohumeral instability is now believed to play the crucial role in the etiology of the painful athletic shoulder. Instability related to sports is not infrequently unrecognized and initiates a vicious circle of secondary impingement, muscular dysfunction, and damage to intraarticular structures that can be devastating and may end the athlete\u2019s career [1\u20135].\nThe term \u201cinstability\u201d constitutes a spectrum of disorders which includes hyperlaxity, subluxation, and dislocation. Principally, glenohumeral instability can be classified according to its etiology, degree, frequency, and direction. The classic categorization of affected individuals into two groups with traumatic and atraumatic instability represented by the mnemonics TUBS and AMBRII has been supplemented by a further grouping that is mainly comprised of overhead athletes with so-called \u201cmicroinstability\u201d (microtraumatic instability) and that has been labelled with the acronym AIOS (Table\u00a01). However, it should be emphasized that congenital or acquired hyperlaxity, microinstability, and traumatic instability can overlap particularly in athletes engaged in overhead sports [2, 6]. \nTable\u00a01Mnemonics for classification of glenohumeral instabilityTUBSAMBRIIAIOSTraumaticAtraumaticAcquiredUnidirectionalMultidirectionalInstabilityBankart lesionBilateralOverstressSurgeryRehabilitationSurgeryInferior capsular shift(Rotator) Interval closure\nWith reference to the complex relationships beween instability and impingement in overhead athletes, Jobe and co-workers developed a classification system (Table\u00a02) with four groups of pathologic conditions [1]. Whereas groups 1 and 4 represent clearly defined entities with either impingement or instability, groups 2 and 3 include pathologies in which both mechanisms coexist. Group 1 corresponds to classic subacromial impingement, which appears to be relatively rare in athletes, especially in younger individuals. Group 2 refers to microinstability caused by repetitive microtraumatization of the capsulo-labral structures as the main pathologic process, leading to secondary impingement syndromes such as posterosuperior impingement. Group 3 equals the AMBRII group, with athletes that have congenital hyperlaxity of both shoulders as well as of other joints. Group 4 finally includes pure traumatic instabilities without secondary impingement as seen in the TUBS group. In the majority of athletes, traumatic instability occurs in an anterior direction; sports-related posterior \u201cmacroinstability\u201d is very rare [1, 2, 4, 7, 8]. \nTable\u00a02Modified classification of instability and impingement in the athletic shoulder [1, 2, 4]GroupPathologyEtiologyPattern of Lesions1Primary impingementSubacromial stenosisRotator cuff lesionsNo instability2Primary instabilityRepetitive microtraumaLabro-ligamentous lesionsSecondary impingementRotator cuff lesionsSLAP lesionsPosterosuperior impingement3Primary instabilityGeneralized hyperlaxityLarge capsular volumeSecondary impingementRotator cuff lesionsLabral abnormalities4Primary instabilityAcute trauma (dislocation)Labro-ligamentous injuriesNo impingementSLAP lesions\nMagnetic resonance (MR) imaging plays an important role in the diagnostic work-up of the painful athletic shoulder and with advanced techniques has in many instances already replaced diagnostic arthroscopy. This article discusses the role of MR imaging and MR arthrography in the context of the most common forms of sports-related glenohumeral instability and presents the typical MR arthrographic appearance of their corresponding intraarticular lesions and patterns of injury.\nTechnical considerations\nParticularly in athletes, MR arthrography has been advocated for accurate depiction of labro-ligamentous injuries as well as rotator cuff pathology [5, 7\u201311]. With the exception of examinations following acute shoulder dislocation, where joint effusion or hemorrhage serve as \u201cnatural\u201d contrast media, at our institution MR arthrography is performed on a routine basis in individuals with clinical evidence of shoulder instability and in athletes with unclear shoulder pain. For intraarticular injection of contrast media, several different techniques have been described that can all successfully be employed in MR arthrography [9]. We inject 12\u201320\u00a0ml of a 2.5\u00a0mmol\/l solution of a gadolinium chelate following fluoroscopically guided puncture of the glenohumeral joint via a classic anterior approach and verification of the intraarticular position of the needle tip by application of a small amount of iodinated contrast media. With the use of a dedicated shoulder coil transverse, coronal oblique, and sagittal oblique T1-weighted spin-echo (SE) sequences are then obtained. Fat-suppression is routinely applied in the coronal oblique plane, but not necessarily in the transverse and sagittal oblique planes. The examination is supplemented by acquisition of a coronal oblique intermediate weighted (echo time=33\u201345\u00a0ms) turbo spin-echo (TSE) sequence with spectral fat-saturation. In patients with anterior instability or suspected posterosuperior glenoid impingement we generally add a T1-weighted SE and\/or an intermediate weighted TSE sequence in the ABER (abduction and external rotation) position, achieved by transverse oblique orientation of the images along the long axis of the humerus after flexing the elbow and placing the patient\u2019s hand behind his head or neck (Fig.\u00a01) [5, 9, 12]. Geometric parameters are identical for all pulse sequences with a section thickness of 3 mm and an in plane resolution of 0.4\u00d70.3\u20130.5 mm.\nFig.\u00a01a, bABER position: normal findings. a Coronal localizer image of the shoulder obtained in abduction and external rotation shows correct orientation of transverse oblique sections aligned with the humeral shaft. b Resulting transverse oblique T1-weighted MR arthrogram demonstrates normal appearance of anteroinferior labrum (white arrow) with taut IGHL (arrowheads), posterosuperior labrum (black arrow), and supraspinatus tendon (**) (T greater tuberosity of humeral head)\nThe non-enhanced MR imaging protocol performed after acute shoulder dislocation includes a coronal oblique T1-weighted SE sequence, coronal oblique and transverse intermediate weighted TSE sequences with fat-saturation, and a sagittal oblique T2-weighted TSE sequence with the same geometric parameters as mentioned above.\nTraumatic glenohumeral instability\nTraumatic glenohumeral instability is typically initiated by a specific traumatic event, followed by other episodes of dislocation or subluxation with a unidirectional pattern [2, 7]. In the vast majority of athletes traumatic instability occurs in an anteroinferior direction when a sudden force overwhelms the anterior capsular structures while the athlete\u2019s arm is in an abducted, externally rotated, and extended position [13]. The resulting combination of injuries represents the source of chronic instability, particularly those involving the inferior glenohumeral ligament (IGHL), which, according to the current opinion, is the most important passive stabilizer of the shoulder joint. The IGHL is formed by an anterior and a posterior band, which represent condensations of the capsule running from the inferior labrum to the humeral neck. The anteroinferior labrum and the anterior band of the IGHL together form the anteroinferior labro-ligamentous complex. The labrum is thought to serve more as an insertion site for the IGHL than to provide stability to the glenohumeral joint by deepening the glenoid fossa [13\u201315]. Although generally common in sports, this type of instability is rarely observed in throwers or overhead athletes, but, if present, can cause secondary damage to the rotator cuff and the superior and posterior labrum [2].\nLabro-ligamentous injuries\nThe high diagnostic accuracy of MR arthrography in the detection of labro-ligamentous lesions has been demonstrated in several studies. MR arthrography identifies labral tears with a sensitivity of 88\u201396% and a specificity of 91\u201398% [14, 16\u201319]. For the detection of lesions of the superior, middle, and inferior glenohumeral ligaments, Chandnani and co-workers reported sensitivities and specificities of 88\u2013100% [20]. The sensitivities and specificities of unenhanced MR imaging for the diagnosis of labro-ligamentous injuries vary widely in the literature. Although a direct comparison with MR arthrography has not yet been performed in a larger series, we regard the role of standard MR imaging in the diagnostic work-up of shoulder instability as questionable, particularly with a view to chronic cases and the depiction of associated pathology [7, 9, 14, 21, 22]. The advantages of MR arthrography are obvious and result from capsular distension with separation of anatomic structures and improved delineation of tears following entrance of contrast media [8, 9]. MR arthrography thereby allows more confident distinction of pathologic conditions from the common anatomic variations of labral morphology as well as from congenital variants of the glenohumeral ligaments and the labro-ligamentous unit, such as the Buford complex [7, 9, 14, 23\u201326]. Furthermore, the imaging appearance of Bankart variant lesions has been described only with MR arthrography [7, 14, 27, 28].\nFew studies have directly compared MR arthrography and computed tomography (CT) arthrography in the diagnosis of labro-ligamentous injuries. The results suggest that, with the exception of bony changes, CT arthrography is less sensitive than MR arthrography [5, 16]. However, CT arthrography with the use of new multidetector techniques has not yet been fully evaluated in the shoulder. First results indicate that due to its capability of isotropic data acquisition it will probably perform equivalent to MR arthrography [8, 26].\nSince modern surgical stabilization procedures aim at restoration of normal anatomy, a more differentiated view at labro-ligamentous injuries has developed in the recent years, and several variants of traumatic lesions of the anteroinferior labrum and\/or capsular structures have been described. The following types of lesions should be distinguished (Fig.\u00a02).\nFig.\u00a02a\u2013fClassification of Bankart and Bankart variant lesions. a Bankart lesion, b bony Bankart lesion, c Perthes lesion, d ALPSA (anterior labro-ligamentous periosteal sleeve avulsion) lesion, e GLAD (glenolabral articular disruption) lesion, f HAGL (humeral avulsion of glenohumeral ligaments) lesion. (LLC anteroinferior labro-ligamentous complex, P scapular periosteum, HH humeral head, AC articular cartilage of glenoid, IGHL inferior glenohumeral ligament)\nBankart lesion\nThe classic labral injury described by Bankart (Fig.\u00a02a) is a complete detachment of the anteroinferior labro-ligamentous complex from the glenoid associated with a rupture of the scapular periosteum [29, 30]. The Bankart lesion represents the most common form of labro-ligamentous injury in patients with first-time traumatic dislocations of the shoulder [31, 32]. Due to the lost contact with the periosteum the lesion shows no tendency to heal. Surgical treatment is by reattachment of the labro-ligamentous complex to the glenoid either arthroscopically or during an open procedure (Bankart repair) [31, 32]. MR arthrography (Fig.\u00a03) typically shows a deformed anteroinferior labrum which is completely separated from the glenoid and therefore is \u201cfloating\u201d in the anterior capsular recess adherent to the anterior band of the IGHL [7]. In many cases the torn periosteum can be visualized by transverse MR arthrograms.\nFig.\u00a03a\u2013cBankart lesion. a Transverse and b coronal oblique T1-weighted MR arthrograms show complete detachment of the anteroinferior labrum (arrows) which \u201cfloats\u201d within the anterior capsular recess but remains attached to the IGHL (arrowheads). c Fat-suppressed T1-weighted MR arthrogram obtained in ABER position reveals separation of the anteroinferior labrum from the glenoid edge as well as disruption of the periosteum (arrow). Note taut IGHL adherent to the labrum (arrowhead)\nBony Bankart lesion\nIn a bony Bankart lesion (Fig.\u00a02b) an osseous fragment of variable size is avulsed from the anteroinferior glenoid together with the labro-ligamentous complex. The size of the fragment influences the therapeutic concept, since larger glenoidal defects can give rise to recurrent instability after stabilizing soft tissue procedures such as the Bankart procedure, particularly in contact athletes [33, 34]. In a study with cadaveric specimens and experimentally created defects of the glenoid rim the critical fragment size was >7 mm in average width [34]. Burkhardt and De Beer coined the term \u201cinverted peer\u201d to describe the visual impression of a significant bone defect of the glenoid at arthroscopy. The \u201cinverted peer\u201d geometry is the reverse of the normal peer-shaped glenoid, which means that its anteroposterior diameter below the midglenoid notch is smaller than that above it [33]. This configuration usually precludes arthroscopic repair and represents an indication for fragment refixation or bone grafting. MR arthrography is less accurate in depiction of bony Bankart lesions compared with CT or CT arthrography [35]. Smaller osseous fragments can easily be overlooked, particularly if fat suppression is used, whereas glenoid fractures with larger fragments can usually be diagnosed with sufficient confidence. Sagittal oblique images are useful to estimate the geometry of the glenoid (Fig.\u00a04).\nFig.\u00a04a\u2013dBony Bankart lesion. a Coronal oblique and b transverse T1-weighted MR arthrograms show avulsion of a large osseous fragment (arrowheads) from the glenoid together with the attaching anteroinferior labrum and IGHL (arrow). c Sagittal oblique T1-weighted MR arthrogram demonstrates \u201cinverted peer\u201d geometry of glenoid in presence of a \u201crelevant\u201d bony Bankart lesion (arrowhead). Note smaller anteroposterior diameter of glenoid below (**) midglenoid notch than above (++) it. For comparison, normal peer-shaped configuration of glenoid is shown on d sagittal oblique T1-weighted MR arthrogram from individual with stable shoulder (C coracoid process)\nPerthes lesion\nThe Perthes lesion (Fig.\u00a02c) is a variant of the Bankart lesion, which also occurs in patients with acute anterior instability. In the Perthes lesion the anteroinferior labro-ligamentous complex is detached from the glenoid, but unlike in the injury described by Bankart, the periosteum remains intact and is stripped anteromedially [27, 31, 36]. Therefore, unless only loosely attached, the labrum may remain in its normal anatomic position. The integrity of the periosteum allows partial \u201chealing\u201d of the labrum, which might also become resynovialized and thus, although incompetent, can look normal on arthroscopic inspection. Because scar tissue may prevent contrast media from entering the labral tear, nondisplaced Perthes lesions can be difficult to detect on MR arthrography as well [14, 27]. MR arthrograms obtained in the ABER position have been reported to be more sensitive in the diagnosis of Perthes lesions compared with conventional transverse MR arthrograms, although these results are based on a relatively small series of patients [12, 27]. In our experience, the ABER position can at times help to visualize nondisplaced Perthes lesions by separating the base of the anteroinferior labrum from the glenoid and consecutively allowing the entrance of contrast media. On the other hand, displaced or slightly displaced lesions can be realigned with the glenoid and therefore be less conspicuous on ABER images than on transverse images obtained in neutral position (Fig.\u00a05).\nFig.\u00a05a, bPerthes lesion. a Transverse fat-suppressed T1-weighted MR arthrogram shows detachment and slight displacement of the anteroinferior labro-ligamentous complex (arrow) and stripping of the intact scapular periosteum (arrowhead). On b corresponding T1-weighted MR arthrogram obtained in ABER position the anteroinferior labrum is realigned with the glenoid edge by the taut IGHL (arrowhead). Although the tear is still demarcated by contrast media (arrow), it is less conspicuous than on the conventional transverse section in this case. (M middle glenohumeral ligament\nALPSA lesion\nThe ALPSA (anterior labro-ligamentous periosteal sleeve avulsion) lesion (Fig.\u00a02d) has also been termed \u201cmedialized Bankart lesion\u201d and is more common in patients with recurrent than with first-time traumatic dislocations of the shoulder [14, 30]. It represents an anteroinferior labral avulsion that leads to medial displacement and inferior rotation of the labro-ligamentous complex together with the intact periosteum in a sleeve-like fashion, thereby causing incompetence of the IGHL and consecutively anterior instability. The labrum may eventually heal and be resynovialized in this abnormal position [7, 30]. Transformation of the ALPSA lesion into a Bankart lesion by dissection of the complex from the glenoid followed by anatomic refixation (Bankart repair) has been recommended as the treatment of choice [30]. MR arthrography (Fig.\u00a06) shows the typical medial and inferior displacement of the deformed labro-ligamentous complex which is best seen on transverse and coronal oblique images. Contrast medium often outlines a crease or cleft between the glenoid and the nodular shaped fibrous tissue on the glenoid neck, whereas the glenoid edge typically lacks a normal labral structure [7, 14, 30]. Familiarity with these signs can prevent misinterpretation of the ALPSA lesion as an anatomic variant in cases with only slight displacement of the labrum.\nFig.\u00a06a, bALPSA lesion. a Coronal oblique and b transverse T1-weighted MR arthrograms demonstrate a nodular shaped anteroinferior labroligamentous complex (arrows) that is displaced medially and inferiorly on the scapular neck. Note cleft (arrowheads) between fibrous tissue and glenoid\nGLAD lesion\nThe GLAD (glenolabral articular disruption) lesion (Fig.\u00a02e), as described by Neviaser is a superficial tear of the anteroinferior labrum in combination with an articular cartilage lesion of the anterior inferior quadrant of the glenoid. The injury is thought to result from glenohumeral impaction while the arm is abducted and externally rotated. Persistent anterior shoulder pain after a fall onto an outstretched arm is the most common clinical presentation, and arthroscopic debridement has been proposed as the treatment of choice [37].\nThe labral component of the abnormality typically represents an inferior based flap tear without evidence of capsulo-periosteal stripping. Since the anterior fibers of the IGHL remain strongly attached to the labrum and glenoid, the GLAD lesion is usually not associated with anterior instability. The degree of articular cartilage damage is variable and ranges from softening and fibrillation to deep surface defects with loose fragments of cartilage which might remain attached to the anterior labrum and can be undermined by contrast media on MR arthrography (Fig.\u00a07) [7, 14, 28, 37]. Particularly if the chondral lesion is small and therefore hardly visible on MR imaging, the GLAD lesion can look similar to instability-related labral injuries. It is therefore important to note that the labrum is nondisplaced and both the IGHL as well as the periosteum are intact.\nFig.\u00a07GLAD lesion. Transverse T1-weighted MR arthrogram shows a nondisplaced tear of the anterior labrum (arrow) associated with a full-thickness chondral defect of the anterior glenoid (arrowhead). A small fragment of hyaline cartilage adheres to the torn labrum\nHAGL lesion\nIn contrast to the Bankart lesion and its variants, the HAGL (humeral avulsion of glenohumeral ligaments) lesion (Fig.\u00a02f) does not involve the labro-ligamentous complex at the glenoid, but represents an isolated tear of the IGHL at its humeral insertion following vigorous shoulder dislocation [38, 39]. The injury is associated with anterior instability and represents a pitfall at arthroscopy as well as open shoulder surgery, since it can be overlooked if the area of the humeral neck is not specifically searched for this finding [39, 40]. The majority of patients in whom a HAGL lesion is detected are male and are involved in contact sports, such as rugby, (American) football, or ice hockey [33, 40]. The injury therefore is thought to be relatively rare in countries where these kinds of sport are unpopular. Treatment is by surgical reattachment of the IGHL to its humeral insertion [38, 40]. Particularly in subacute cases with absence of joint effusion, the HAGL lesion can only be diagnosed with confidence if MR arthrography is used. As a sequel of humeral avulsion the IGHL is seen as a J-shaped rather than a U-shaped structure on coronal oblique arthrograms, and contrast extravasation can occur at its insertion site (Fig.\u00a08). Acute injuries are usually associated with edematous changes of the soft tissues anterior to the humeral neck [39]. Approximately 20% of HAGL lesions occur with avulsion of a bony fragment from the humerus (bony HAGL=BHAGL) [41]. On rare occasions the HAGL lesion can be associated with a classic Bankart lesion. Due to the complete isolation of the anterior band of the IGHL which results from this combination of injuries, the lesion has been described as the \u201cfloating AIGHL\u201d [42]. Other associated injuries include subscapularis tears and Hill-Sachs defects as well as other osteochondral lesions [38, 40].\nFig.\u00a08a, bHAGL lesion. a Coronal oblique fat-suppressed T1-weighted MR arthrogram and b corresponding intermediate weighted TSE image demonstrate avulsion of the IGHL at its humeral insertion causing a J-shaped configuration of the axillary recess (arrowheads). Contrast extravasation can be seen at the site of the tear (arrows). Bone marrow edema within humeral head (*) was caused by acute Hill-Sachs defect (not shown)\nParticularly in athletes, the decision whether an arthroscopic or an open procedure should be performed to achieve the most favourable outcome, can be very difficult and is subject to a controverse discussion. Criteria in favour of open rather than arthroscopic surgery include engagement in contact sports, presence of a relevant glenoid defect and\/or Hill-Sachs defect, a HAGL lesion, and poor quality of the labro-ligamentous complex [14, 33, 38, 43]. A recent study could demonstrate the potential of MR arthrography in classification of anteroinferior labro-ligamentous injuries with a view to therapeutic decisions [14]. With arthroscopy as the \u201cgold standard,\u201d Bankart and Bankart variant lesions could be correctly classified with a sensitivity of 77%, a specificity of 91%, and an accuracy of 84%. Furthermore, MR arthrography was capable of identification of injuries with poor quality of the anterior labrum and IGHL, which in high concordance with arthroscopy could not be assigned to one of the specific types of lesions mentioned above. All of these \u201cnonclassifiable\u201d lesions with degenerative changes and scar tissue formation were observed in patients with chronic anterior instability. On MR arthrography (Fig.\u00a09) these injuries are characterized by a swollen inferior glenohumeral ligament complex without distinct depiction of the labrum, IGHL, and scapular periosteum [14].\nFig.\u00a09Nonclassifiable anteroinferior labro-ligamentous injury. Transverse T1-weighted MR arthrogram shows markedly deformed and enlarged labro-ligamentous complex without differentiability of labrum, IGHL, and scapular periosteum. The lesion was associated with chronic anteroinferior instability\nHill-Sachs defect\nThe Hill-Sachs defect represents a compression injury of the posterolateral humeral head which develops during anteroinferior dislocation as the relatively large and soft humeral head impacts against the comparatively small and hard bony glenoid [13]. The incidence of the lesion at arthroscopy varies considerably from 47 to 100% among different series of patients with first-time traumatic dislocations [31, 32, 44]. The depth and size of a Hill-Sachs lesion, ranging from shallow chondral defects to deep osteochondral impaction fractures, is thought to reflect the tightness of the joint capsule [44]. In athletes with hyperlaxity at the time of the first shoulder dislocation, the humeral injury therefore typically is small or absent. Osteochondral Hill-Sachs defects that involve less than one third of the circumference of the humeral head are usually regarded as prognostically irrelevant. Larger lesions, especially if oriented with their long axis parallel to the glenoid, can engage the anterior corner of the glenoid in abduction and external rotation and thus lead to repeated subluxations or redislocations (\u201cengaging\u201d Hill-Sachs defect) [33].\nOn transverse MR images, the Hill-Sachs defect is typically seen at or slightly above the level of the coracoid process (Fig.\u00a010). False positive diagnoses can occur due to misinterpretation of a normal anatomic groove at the posterolateral humerus, which can often be found caudad to that position [45]. In acute or subacute cases with edema of adjacent bone marrow, the impaction injury can easily be detected on fat-suppressed intermediate\/T2-weighted or STIR images. Chronic lesions, particularly if small, are often better visualized by CT [35]. However, clinically significant osteochondral defects are sufficiently demonstrable by MR and rarely necessitate further imaging.\nFig.\u00a010a\u2013cHill-Sachs defect: MR appearance. a Acute lesion: transverse fat-suppressed intermediate weighted TSE image shows impaction of the posterolateral humeral head (arrowhead) with adjacent bone marrow edema following acute anteroinferior dislocation. b, c Chronic lesions: b transverse fat-suppressed T1-weighted and c transverse T1-weighted MR arthrograms obtained in two different patients several months after anteroinferior dislocation depict circumscribed (b) and extensive (c) osteochondral defects (arrowheads) of the humeral head (C tip of coracoid process)\nAtraumatic glenohumeral instability\nAtraumatic glenohumeral instability is typically multidirectional and usually evident in individuals with congenital hypermobility syndrome. The diagnosis is predominantly based on the clinical examination, which uncovers bilateral affection of the shoulders in association with generalized hyperlaxity of ligaments and joints [1, 2, 35, 46]. The increased baseline laxity of the shoulder seen in athletes with generalized hypermobility is advantageous for several types of sports, but at the same time harbors a high risk of long-term injury with damage of intra- and periarticular structures [1, 2]. If conservative management fails, multidirectional instability is usually treated surgically by inferior capsular shift and closure of the rotator interval [6].\nThere are no specific findings to diagnose atraumatic glenohumeral instability by MR imaging or MR arthrography [46]. In most non-athletic AMBRII patients, MR arthrography shows an increased capsular volume but no or little substantial alterations of the intraarticular structures. In athletes, laxity of the capsule is frequently associated with secondary damage to the labrum and the rotator cuff [2]. The labrum may be hypoplastic or torn, but can as well appear swollen and increased in signal intensity due to more or less extensive degenerative change (Fig.\u00a011). Further morphologic alterations can involve the biceps anchor and the articular surface of the rotator cuff [2]. MR arthrography can help with therapeutic decisions by identification or exclusion of significant intraarticular pathology that might represent an indication for surgical repair in addition to capsular reduction.\nFig.\u00a011a, bAtraumatic glenohumeral instability: labral degeneration. a Transverse fat-suppressed T1-weighted and b corresponding sagittal oblique T1-weighted MR arthrograms reveal markedly swollen and enhanced but nondisplaced anterior glenoid labrum in an athlete with multidirectional shoulder instability. Extensive labral degeneration was verified by arthroscopy (not shown)\nMicrotraumatic glenohumeral instability\nMicrotraumatic glenohumeral instability is thought to result from chronic traumatization of the capsular structures in throwers and overhead athletes, such as tennis players, swimmers, and handball players. In contrast to atraumatic glenohumeral instability, microinstability is typically seen unilaterally only in the dominant shoulder of the athlete. Whereas repetitive abduction and external rotation cause microtrauma of the anterior capsule and thus can provoke anterior instability, repetitive overhead activity with abduction, flexion, and internal rotation might give rise to posterior microinstability as a result of injury to the posterior labro-ligamentous elements [4, 7, 9, 35, 46].\nStructural abnormalities consecutive to microtraumatic glenohumeral instability can be depicted by MR arthrography and include laxity of the anterior or posterior capsule, labral injuries ranging from degeneration and fraying to tearing and detachment, SLAP (superior labral anteroposterior) lesions, and tears of the rotator cuff caused by secondary impingement [2].\nPosterosuperior glenoid impingement (PSI)\nPSI is a form of internal impingement which represents a common problem in throwers and overhead athletes, presenting with acute or chronic posterior shoulder pain [4, 5, 47, 48]. The basic observation was reported by Walch, who described impingement between the articular side of the supraspinatus tendon and the posterosuperior edge of the glenoid evident in abduction and external rotation [49]. In throwers and overhead athletes, PSI can lead to a typical pattern of injuries, the so-called \u201ckissing lesions\u201d, which includes corresponding lesions of the undersurface of the rotator cuff, the posterosuperior labrum, the greater tuberosity, and the superior bony glenoid [47, 48, 50\u201352]. The development of PSI has been attributed to chronic injury (repetitive stretching) of the anterior capsular structures, particularly the IGHL, with consecutive anterior microinstability, which causes anterior subluxation of the humeral head in abduction and external rotation during overhead movements, and thus allows excessive contact between the rotator cuff and the posterosuperior glenoid [47, 48]. However, this theory is not generally accepted. Other authors described a contracture of the posteroinferior capsule and a posterior SLAP lesion as the essential lesions for the development of PSI in throwers [3]. Although the basic mechanism is still subject to discussion, the high coincidence of PSI and SLAP lesions is undoubted. Furthermore, there is apparently a huge overlap of clinical symptoms in athletes with SLAP lesions, PSI, or both. It is important to stress that contact between the undersurface of the rotator cuff and the posterosuperior glenoid, as seen on arthroscopy, is not pathologic per se. Posterosuperior glenoid impingement should only be diagnosed if this contact is associated with clinical symptoms and corresponding lesions of the involved anatomic structures [2, 13, 51, 52]. Conservative treatment is usually suited in athletes with minor structural abnormalities, whereas surgical debridement and repair (eventually in combination with capsular plication) are indicated in the presence of relevant rotator cuff and labral lesions.\nMR imaging can be used to confirm the diagnosis of PSI and to help with therapeutic decisions by demonstrating the extent of articular damage. Since MR arthrography has been shown to be more accurate than conventional MR imaging in the detection of partial rotator cuff tears, labral tears, and SLAP lesions, it is also regarded as the method of choice in athletes with suspected PSI, which is accompanied by a combination of these abnormalities [50, 53\u201355]. MR arthrograms (Fig.\u00a012) typically show articular-sided partial tearing of the supraspinatus and\/or infraspinatus tendons. The supraspinatus lesion, unlike in patients with subacromial impingement, usually involves the posterior part of the tendon. Damage to the posterosuperior labrum ranges from degenerative change and fraying to tears and detachment and might be associated with ganglia and SLAP lesions. Alterations of bone at the greater tuberosity and the superior glenoid are best demonstrated by fat-suppressed intermediate or T2-weighted images and include erosions, bone marrow edema, cyst formation, and sclerosis [50\u201352]. MR arthrograms obtained in the ABER position (Fig.\u00a012d,e) usually show contact between the lesions of the rotator cuff and the superior labrum\/glenoid and might even reveal interposition of the supraspinatus tendon pinched between the glenoid and the greater tuberosity. Furthermore, the ABER position is useful in demonstration of subluxation of the humeral head and depiction of abnormalities of the anterior capsule. The anterior band of the IGHL can appear attenuated and elongated or might even be torn [50].\nFig.\u00a012a\u2013ePSI: spectrum of findings on MR arthrography. a Transverse fat-suppressed T1-weighted MR arthrogram and b, c corresponding coronal oblique images obtained in a tennis player with posterior shoulder pain show a posterosuperior labral tear (arrow in a) associated with an articular-sided partial tear of the posterior aspect of the supraspinatus tendon (arrowhead in b) and a SLAP type 2 lesion (arrow in c). Note large volume of anterior capsule (* in a). d, e T1-weighted MR arthrograms obtained in ABER position from two athletes with PSI: d Arthrogram demonstrates the \u201ckissing lesion\u201d pattern with corresponding tears of the posterosuperior labrum (white arrow) and the undersurface of the supraspinatus tendon (black arrowhead) accompanied by sclerosis of the greater tuberosity of the humeral head at the contact zone with the glenoid (black arrow). Note elongation of the IGHL (white arrowhead). e Arthrogram shows interposition of the supraspinatus tendon (*) between the greater tuberosity and the superior glenoid as well as a tear of the IGHL (arrowhead) with consecutive contrast extravasation\nSLAP lesions\nSLAP lesions of the shoulder are common injuries in athletes which represent tears of the superior glenoid labrum that extend in an anterior to posterior direction [55, 56]. Four different types of SLAP lesions have been described in the original classification by Snyder: \ndegenerative fraying of the superior labrum;avulsion of the superior labrum and biceps anchor from the glenoid;bucket-handle tear of the superior labrum with preserved biceps anchor;bucket-handle tear of the superior labrum involving the long head of biceps tendon [56].\nThis classification has been expanded with several further types of lesions, which mainly represent combinations of the most common form, the SLAP type 2 lesion, with other injuries of the labrum, medial glenohumeral ligament, or rotator cuff. However, only the Snyder classifications have found wide acceptance, and most institutions categorize these injuries as type 1\u20134 with or without asscociated lesions. Whereas type 1 and 2 lesions are typically caused by repetitive torsion of the biceps anchor (the so-called \u201cpeelback mechanism\u201d) in throwers and overhead athletes, types 3 and 4 lesions are thought to occurr more often after a fall onto an outstretched arm or onto a flexed elbow [3, 55, 56]. Furthermore, type 2 lesions are frequently observed in athletes with traumatic anterior shoulder instability, where they develop in association with lesions of the anteroinferior labro-ligamentous complex (most often a classic Bankart lesion). In these cases, the labro-ligamentous avulsion can be considered as the main lesion, with the SLAP lesion being an associated injury [55]. Surgical treatment is indicated in all types of SLAP lesions except type 1 lesions which are usually of no clinical relevance. Since type 2 and 4 lesions impair the stabilizing function of the biceps insertion and therefore can provoke glenohumeral instability, secondary impingement, and rotator cuff lesions, they are commonly treated by refixation. Type 3 bucket-handle tears are usually not surgically reattached but treated by simple debridement [55, 56].\nMR arthrography has proved to be superior to conventional MR imaging with regard to the identification of SLAP lesions and has been shown to be reliable in assessment of the stability of the biceps anchor and the detection of associated injuries [55, 57\u201359]. However, its ability to correctly classify the different types of lesions is limited [55].\nMost SLAP lesions are best depicted on MR arthrograms oriented in the coronal oblique plane (Fig.\u00a013). Increased signal intensity and\/or surface irregularity of the superior labrum can be seen in type 1 lesions which, however, can only infrequently be diagnosed by MR imaging. Type 2 lesions are characterized by linear extension of contrast media into the superior labrum and the biceps anchor. This most common type of SLAP lesion also represents the most problematic in terms of differential diagnosis. False positive and false negative diagnoses of a SLAP type 2 lesion can be caused by misinterpretation of a sublabral recess as a tear and vice versa [55]. The sublabral recess (Fig.\u00a014) is a very common anatomic variant with an incidence of >70% in cadaveric studies [26, 60]. It represents a variably sized synovialized cleft between the superior labrum and the bony glenoid which is typically oriented medially and points at the supraglenoidal tubercule [26, 55, 60]. The most important criterion for the presence of a SLAP type 2 lesion rather than a sublabral recess therefore is lateral or superior extension of the tear. If the tear extends medially (like a sublabral recess) it often shows irregular margins and\/or a relatively wide separation between the labrum and the glenoid. In individuals with traumatic anterior instability the type 2 lesion often originates from an anteroinferior tear of the labro-ligamentous complex that extends cranially up into the superior labrum and the biceps anchor (Fig.\u00a015) [55]. Type 3 lesions are characterized by a vertical and an additional horizontal contrast interface which separate the avulsed superior labrum as a triangular-shaped fragment from the intact biceps tendon. In Type 4 lesions the horizontal component of the tear additionally extends into the long head of biceps tendon. The bucket-handle fragment therefore is composed of the superior labrum as well as a portion of the tendon and might be more or less displaced from the superior glenoid [55, 59].\nFig.\u00a013a\u2013dSLAP lesions: classification according to Snyder [56] on coronal oblique MR arthrograms (from [35]; modified). a SLAP type 1 lesion, b SLAP type 2 lesion, c SLAP type 3 lesion, d SLAP type 4 lesion (LBC labral-bicipital complex, HH humeral head, G glenoid)Fig.\u00a014Sublabral recess. Coronal oblique T1-weighted MR arthrogram demonstrates medially oriented cleft (arrowhead) between the superior labrum and the glenoid outlined by contrast media which was also diagnosed as a sublabral recess with a stable biceps insertion on arthroscopy (not shown). Note meniscoid shape of superior labrum (*) and integrity of the biceps anchor (arrow)Fig.\u00a015a\u2013dSLAP type 2 lesion in association with a Bankart lesion in traumatic anterior glenohumeral instability. a Coronal oblique fat-suppressed T1-weighted MR arthrogram shows superior extension of contrast media into the superior labrum and biceps anchor (arrowhead). b Corresponding sagittal oblique MR arthrogram reveals tearing of the entire anterior labrum (arrowheads) extending from inferior to superior. c, d Corresponding transverse MR arthrograms demonstrate detachment of the anterior labrum that continues as a classic Bankart lesion anteroinferiorly (arrows)\nConclusion\nSports-related glenohumeral instability is a complex and in many aspects still controversial subject. MR imaging can play an important role in evaluation of the athlete\u2019s shoulder if it aims at providing essential information for therapeutic decisions at eye level with diagnostic arthroscopy. Therefore, the radiologist should be familiar with the mechanisms and classification of injuries as well as the advantages and limitations of his examination techniques. At present, MR arthrography represents the best evaluated and most informative imaging technique for the assessment of shoulder pathology in many aspects and thus, appears to be most valuable for the diagnostic work-up of glenohumeral instability and unclear shoulder pain in athletes.","keyphrases":["magnetic resonance","mr arthrography","shoulder dislocation","shoulder mr","shoulder injuries"],"prmu":["P","P","P","R","R"]}
{"id":"Pediatr_Nephrol-4-1-2413095","title":"Genetic polymorphisms of the RAS-cytokine pathway and chronic kidney disease\n","text":"Chronic kidney disease (CKD) in children is irreversible. It is associated with renal failure progression and atherosclerotic cardiovascular (CV) abnormalities. Nearly 60% of children with CKD are affected since birth with congenital or inherited kidney disorders. Preliminary evidence primarily from adult CKD studies indicates common genetic risk factors for CKD and atherosclerotic CV disease. Although multiple physiologic pathways share common genes for CKD and CV disease, substantial evidence supports our attention to the renin angiotensin system (RAS) and the interlinked inflammatory cascade because they modulate the progressions of renal and CV disease. Gene polymorphisms in the RAS-cytokine pathway, through altered gene expression of inflammatory cytokines, are potential factors that modulate the rate of CKD progression and CV abnormalities in patients with CKD. For studying such hypotheses, the cooperative efforts among scientific groups and the availability of robust and affordable technologies to genotype thousands of single nucleotide polymorphisms (SNPs) across the genome make genome-wide association studies an attractive paradigm for studying polygenic diseases such as CKD. Although attractive, such studies should be interpreted carefully, with a fundamental understanding of their potential weaknesses. Nevertheless, whole-genome association studies for diabetic nephropathy and future studies pertaining to other types of CKD will offer further insight for the development of targeted interventions to treat CKD and associated atherosclerotic CV abnormalities in the pediatric CKD population.\nIntroduction\nChronic kidney disease (CKD) is irreversible and progressive [1]. In children, CKD is underappreciated, understudied, and an important cause of morbidity and mortality [1, 2]. Furthermore, adjusted mortality rates since 1991 among the pediatric end-stage renal disease (ESRD) population increased by 5% to 26.6 per million general population in 2005; and cardiovascular (CV) mortality among pediatric ESRD patients has increased from 17.7 deaths per 1,000 patient years at risk in 1991 to 23.4 in 2005 [3]. Children with CKD live with the consequences of abnormal renal function for their entire lives, with nearly 60% affected since birth with congenital or inherited kidney disorders [2]. Diabetic nephropathy and hypertension, which are the dominant causes of CKD in adults, are rare causes of CKD in childhood. CKD in children is the result of heterogeneous diseases of the kidney and urinary tract that range from common congenital malformations of the urinary tract to rare inborn errors of metabolism. Although some patients have stable kidney function for years, others have a rapid decline in function. The factors associated with an accelerated decline in kidney function include: the cause of CKD, proteinuria, hypertension, anemia, hyperphosphatemia, and metabolic acidosis [4\u20139]. In common with children and adults with CKD, progression to kidney failure occurs via a final common pathway characterized by progressive interstitial fibrosis, peritubular capillary loss with hypoxia, and destruction of functioning nephrons because of tubular atrophy [10]. Despite the diverse initiating and secondary factors noted above, CKD progression is strongly influenced by common inflammatory mechanisms [11].\nCKD is a well-known risk factor for atherosclerotic CV disease [12\u201316]. Children with kidney failure receiving chronic dialysis have a cardiac death rate 1,000-fold higher compared with children in the general population [15]. Children with mild to moderate CKD have a high prevalence of traditional risk factors for atherosclerotic CV disease, including hypertension, hyperlipidemia, and elevated homocysteine levels [17\u201319]. Left ventricular hypertrophy (LVH), a pathophysiologic adaptation of the myocardium, is viewed as a marker for early CV disease in pediatric patients with CKD [20, 21]. The long-standing and progressive atherosclerotic CV abnormalities that begin in childhood CKD contribute to the increased CV morbidity in adulthood [22]. CKD promotes maladaptive interactions between the heart and kidneys, which in turn amplifies the progressive failure of these organs [23]. The emerging evidence suggests that cytokines may play a vital regulatory role in initiation and progression of both renal and CV disease in patients with CKD [24].\nPreliminary evidence primarily from adult CKD studies [24] indicates common genetic risk factors for CKD and atherosclerotic CV disease. Although multiple physiologic pathways share common genes for CKD and CV disease, substantial evidence [10, 25\u201328] supports our attention to the renin angiotensin system (RAS) and the interlinked inflammatory cascade because they modulate the progression of renal and CV disease. Current research suggests that the natural variations of the genes involving the RAS-cytokine pathway influence the rate of progressions for renal and CV disease in CKD patients [24]. Insights gained by understanding how variations in this pathway influence the progressions of renal and CV disease will lead to hypotheses for targeted interventions to treat CKD and associated atherosclerotic CV abnormalities in the pediatric CKD population. This article reviews clinically relevant candidate genes of the RAS-cytokine pathway and the fundamentals of genotype\u2013phenotype association studies.\nCKD\u2013CV disease link: RAS-cytokine pathway\nIntervention trials in adults with CKD have demonstrated that blockade of the RAS slow progression of renal disease via antihypertensive and anti-inflammatory mechanisms [26\u201328]. The RAS generates circulating angiotensin II (AT2), which regulates blood pressure and intravascular volume. In contrast to its endocrine function, tissue RAS produces AT2 that is involved in autocrine and paracrine signaling within all bodily organs, including the heart, blood vessels, and kidneys [29]. Tissue RAS exerts a pivotal role in the regulation of cytokine signaling, potentially modulating the inflammatory response associated with renal disease progression and susceptibility for CV dysfunction.\nTissue RAS via AT2 regulates the cytokine pathway responsible for progressive injury in the kidney and heart [25, 30\u201332]. As depicted in Fig.\u00a01, activation of tissue RAS increases the local production of AT2. After AT2 stimulates the AT2 receptor, a number of signaling systems are triggered, including that of nuclear factor kappa B (NF-\u03baB), which is responsible for upregulation of proinflammatory cytokines [33]. The cytokine signaling modulates endothelial dysfunction, adhesion and migration of circulating immune cells (monocyte, leukocytes, or neutrophils) into the interstitium, and activation of resident fibroblasts [10, 11]. Cytokines are soluble polypeptides that act as important humoral modulators in immunoregulation, hematopoiesis, and inflammation. Cytokines act in a highly complex coordinated network with considerable overlap and redundancy between the function of individual cytokines. Being pleiotropic in their actions, these molecules can induce or repress their own synthesis as well as that of other cytokines and cytokine receptors [24, 34].\nFig.\u00a01Activation of the renin angiotensin system (RAS) and an increase in the local production of angiotensin II (AT2) triggers the inflammatory host response\nIn the kidney, the inflammatory host response leads to renal interstitial fibrosis and progression [10]. These actions within the kidney are mediated by proinflammatory [tumor necrosis factor (TNF)-\u03b1, interleukin (IL)-1, IL-6] and profibrotic cytokines [TGF-\u03b2 and plasminogen activator inhibitor (PAI)-1] [10, 35]. Proteinuria stimulates interstitial inflammation and fibrosis in the kidney; it also is a risk factor for future decline in kidney function [36]. NF-\u03baB activity is stimulated by albumin [37] and is the pathway that links proteinuria and tubulointerstitial inflammation and fibrosis in the kidney [38, 39]. In the heart, the AT2-stimulated inflammatory response leads to LVH [40, 41]. The myocardial hypertrophy is caused by an increase in cell size and accompanied by changes in gene expression in response to AT2 [42, 43]. In addition to AT2, proinflammatory cytokines IL-1, IL-6, TNF-\u03b1, and TGF-\u03b2 are responsible for myocyte hypertrophy and interstitial fibrosis [44, 45]. In the blood vessel, the AT2 pathway is the molecular mechanism leading to atherosclerosis [25, 32]. There is preliminary evidence indicating impaired flow-mediated dilation among hypertensive patients with mutations in the promoter region of the NF-\u03baB gene [46]. Hence the upregulation of RAS-cytokine pathway activity is associated with renal progression, LVH, and atherosclerosis. The magnitude of this response may depend on genetic polymorphisms, which may either increase or decrease expression of these genes [24, 47].\nGenetic polymorphisms\nDifferent versions of a gene at a specific chromosomal location, or loci, that encode a trait are called alleles (see Table\u00a01 for a glossary of common terms). Complementary alleles are inherited from each parent. A change in one nucleotide (base pair) within a gene is called a single nucleotide polymorphism (SNP). About 11 million SNPs with minor allele frequencies (MAF) of at least 1% are estimated to exist in the human genome. SNPs that affect native protein function, i.e. functional SNPs, can occur in gene promoter regions, coding regions, splice junctions, and 3\u2019-untranslated regions (UTR) and may be causally involved in the etiology of human disease. Other types of polymorphisms include insertion\/deletion (indel) polymorphisms and mini- and microsatellites (di-, tri-, and tetranucleotide repeats) [48]. Variation in phenotypic expression of a gene may be affected by epigenetic factors, where gene expression is affected by mechanisms other than alterations in the nucleotide sequence, but this is beyond the scope of this review.\nTable\u00a01Glossary of common genetic termsAlleles: Alternate sequences of the same gene, one inherited from each parent.Biological pathway: A set of proteins that interact to produce normal and abnormal physiology.Candidate gene: A gene in which variants could plausibly explain a given phenotype, such as severity of disease or variable response to drug. Methods to identify candidate genes include basic science studies, identifying DNA sequences conserved across species, human genetics, epidemiologic association studies, or genome-wide analyses.Epigenetics: Heritable change in the pattern of gene expression mediated by mechanism other than alterations in the primary nucleotide sequence of gene.Genome: The collection of all DNA in an organism. Only a small proportion (probably <3%) of human genomes encodes proteins.Genotype: The genetic makeup of an individual, which may refer to the whole genome or to specific genes or regions of genes.Haplotype: A set of genetic variants that are inherited together. Polymorphisms that are coinherited more often than by chance alone are in linkage disequilibrium (LD). Haplotype blocks may include many individual polymorphisms in high LD; as a result, establishing genotype at any single polymorphic site with such a block may establish genotypes at linked sites within the block. Individual single-nucleotide polymorphisms (SNPs) that can be used to establish genotype within a haplotype block are termed tag SNPs.Heterozygous: Having different alleles in a specific region of DNA.Homozygous: Having the same alleles in a specific region of DNAPhenotype: Measurable characteristics of an organism. These may derive from genotype, environment, or the combination. Organisms with the same phenotype can have different genotypes.Polymorphisms: DNA variants that are common, often defined as >1% in a given population. Polymorphisms can be in coding regions (where they may be synonymous or nonsynonymous) or, more commonly, in noncoding regions, and often vary by ethnicity. The most common type of polymorphism is a change in one nucleotide (base pair) in a DNA sequence, referred to as an SNP. Other polymorphisms are insertion and deletion of multiple sequential nucleotides (indels); variable numbers of repeats, such as doublets or triplets; or large-scale duplications or deletions. Although some genetic variants are known to alter protein abundance or function, the functional consequences of most polymorphisms are unknown.Tag SNPs: These are maximally informative SNPs that characterize common haplotypes.\nThe study of individual SNPs has yielded exciting insight into the factors involved in CKD progression. Although candidate-gene-based approaches are a logical first step, they are unlikely to provide a complete answer. The progressions to renal and CV disease are complex traits involving multiple genes. As discussed later, the cooperative efforts among scientific groups and the availability of robust and affordable technologies that can identify thousands of SNPs across the genome make genome-wide association studies an attractive paradigm for studying polygenic diseases such as CKD. Whole-genome association studies are being used to identify the genetic basis for CKD, with large consortiums investigating the genetic predisposition to diabetic nephropathy in Europe and North America [49, 50].\nAlthough a complete overview of all known genetic polymorphisms of the RAS-cytokine pathway is beyond the scope of this review, a complete listing can be found at the National Center for Biotechnology Information (NCBI) SNP database (dbSNP) (www.ncbi.nlm.nih.gove\/projects\/SNP\/). There are polymorphisms of the RAS-cytokine genes that have been reported to be associated with renal progression and\/or CV morbidity and are summarized below (Table\u00a02). Despite some of the potential weaknesses of the studies included, these candidate-gene association studies offer some preliminary information worthy of further investigation.Table\u00a02Candidate-gene polymorphisms and associations with renal and cardiovascular (CV) diseases among subjects with chronic kidney disease (CKD)AuthorGene and genotypeaStudy population\/study typebSample size (n)Clinical significanceRenin Angiotensin System (RAS)Boright A [53]ACE haplotypesUS diabetic nephropathy CKD (DCCT-EDIC)\/cohort studyRestricted to Caucasian cohort = 1,365The genetic variation of the ACE gene is associated with microalbuminuria and diabetic nephropathy.Hadjadj S [49]ACE insertion\/deletion polymorphismEuropean diabetic nephropathy CKD\/case-control studyAdult diabetic nephropathy cases = 1,057, controls = 1,127The haplotype including the ACE deletion allele was associated with diabetic nephropathy.Haszon I [57]ACE insertion\/deletion polymorphismEuropean Vesicoureteral reflux CKD\/case-control studyPediatric VUR cases = 77, controls = 80A deletion at both alleles is linked to renal scarring in VUR.Hohenfellner K [55]ACE insertion\/deletion polymorphismEuropean CKD (Nutritional Treatment of Chronic Renal Failure in Childhood Study)\/cohort studyPediatric cohort = 95A deletion at both alleles is linked to a higher risk of renal progression among children with congenital renal malformation.Lovati E [58]ACE insertion\/deletion polymorphismEuropean ESRD\/case-control studyAdult ESRD cases = 260, controls = 327A deletion at both alleles is linked to a higher risk of renal progression among adults.Ng D [54]ACE insertion\/deletion haplotypeUS diabetic nephropathy CKD\/case-control studyAdult Caucasians type 2 with diabetic nephropathy cases = 291, controls = 167The deletion allele haplotype is associated with diabetic nephropathy.Papp F [56]ACE insertion\/deletion polymorphismEuropean ESRD\/case-control studyPediatric ESRD cases = 20, controls = 150A deletion at both alleles is linked to ESRD.Hsu C [64]AGT-6 G>AUS CKD (ARIC Study)\/ cohort studyRestricted to Adult African American CKD cohort = 3,381 cGenotype A\/A is linked to a higher risk of renal progression among African American CKD patients.Reis K [111]AGT M235TEuropean kidney transplant\/case-control studyAdult kidney transplant cases = 82, controls = 100Genotype Thr\/Thr linked to chronic allograft dysfunction.Buraczynska M [66]AT1R A>CEuropean ESRD\/case-control studyAdult ESRD cases = 745, controls = 520Genotype C\/C or A\/C is linked to higher risk of renal progression in adults.Interleukin (IL)-1Wetmore JB [73]IL-1\u03b1 g.-889 C>TUS ESRD\/case-control studyAdult ESRD cases = 239, controls = 252Genotype T\/T is linked with risk for ESRD.Amoli M [82]IL-1\u03b2 g.-511 C>TEuropean Henoch-Schonlein purpura disease (HSP)\/case-control studyAdult and pediatric HSP cases = 49, controls = 146Carriage of the T allele linked to severity of renal involvement with Henoch-Schonlein purpura.Interleukin-1 Receptor AntagonistBuraczynska M [83]IL-2RN*2European ESRD\/case-control studyAdult ESRD cases = 602, controls = 433Homozygous for the IL2RN*2 allele linked with more rapid progression in patients with glomerulonephritis and diabetic nephropathy and risk for ESRD.Wetmore JB [73]IL-1RN*2US ESRD\/case-control studyAdult ESRD cases = 239, controls = 252Homozygous for the IL2RN*2 allele is linked to risk for ESRD.Watanabe M [81]IL-12N*2Japanese IgA nephropathy CKD \/case-control studyAdult IgA Nephropathy cases = 106, controls = 74Carriage of the IL2RN*2 allele is linked to severe proteinuria and increased creatinine in IgA nephropathy.Interleukin-6Balakrishnan V [72]g.-174G>CUS ESRD (HEMO Study)\/cross-sectional studyAdult ESRD cohort = 187Genotype G\/G or G\/C is linked to increased comorbid conditions and decreased functional status among dialysis patients.Losito A [86]g.-174G>CEuropean ESRD\/case-control studyAdult ESRD cases = 161, controls = 169Carriage of the C allele linked to LVH in hemodialysis patients, especially those with diabetes.Muller-Steinhardt M [87]g.-174G>CEuropean Kidney transplant\/cohort studyAdult kidney transplant cohort = 158Carriage of the C allele linked to decreased kidney allograft survival.Interleukin-10Girndt M [88]g.-1082 G>AEuropean ESRD\/cohort studyAdult ESRD cohort = 300The A\/A genotype is linked to a lower production of IL-10 and increased CV morbidityTumor Necrosis Factor (TNF)-\u03b1Balakrishnan V [72]g.-308 G>AUS ESRD (HEMO Study)\/cross-sectional studyAdult ESRD cohort = 187Genotype A\/A or A\/G is linked to low serum albumin, increased comorbid conditions, and decreased functional status among dialysis patients.Transforming Growth Factor-\u03b2Sato F [106]g.-509C>T and g.+869T>CJapanese IgA Nephropathy CKD\/cross-sectional studyAdult IgA nephropathy cases = 329, controls = 297The -509C\/C and 869C\/C genotypes are linked with heavy proteinuria and mesangial cell proliferation in patients with IgA nephropathy.Rao M [107]g.+915G>CUS ESRD (HEMO Study)\/cross-sectional, and cohort studiesAdult ESRD cohort = 187Genotype G\/C vs G\/G was linked with risk for prevalent vascular disease, new onset cardiac morbidity and cardiac mortality in HD patients.Plasminogen Activator Inhibitor (PAI)-1Aucella F [113]4G\/5GEuropean ESRD\/cohort studyAdult ESRD cohort = 417Genotype 4G\/4G is linked to increased risk for fatal MI among HD patients.Reis K [111]4G\/5GEuropean Kidney transplant\/case-control studyAdult kidney transplant cases = 82, controls = 100Carriage of the 4G allele linked to chronic allograft dysfunction.Wong A [112]4G\/5GChinese Systemic lupus erythematosus CKD\/case-control studyAdult diabetic nephropathy cases = 95, controls = 46Genotype of the 4G\/4G linked to increased severity lupus nephritis among SLE patients.ACE angiotensin converting enzyme, AGT angiotensin, AT1R angiotensin II type 1 receptor, DCCT Diabetes Control and Complications Trial, EDIC Epidemiology of Diabetes Interventions and Complications, ESRD end-stage renal disease, ARIC Arthrosclerosis Risk in Communities, HEMO hemodialysis, VUR vesicoureteral reflux, HD hemodyalysis,  SLE systemic lupus erythematosus, MI myocardial infarctiona The gene and gene polymorphism of interestb Study population is given to indicate potential for population stratification and type of CKD population (name of cohort study)\/study type by: cross-sectional, cohort, or case controlc In the study by Hsu et al., 3,449 subjects had AGT genotyping, whereas 3,381 subjects had both AGT and AT1R genotyping\nThe renin-angiotensin system\nPolymorphisms in the RAS system are associated with clinically significant renal and CV disease morbidity and thought to occur through a proinflammatory mechanism. The inflammatory response is activated by the RAS through the recruitment of proinflammatory cells to the site of injury [51, 52] and the upregulation of adhesion molecules on vascular endothelial cells and smooth muscle cells [30].\nA naturally occurring variant in the angiotensin-converting enzyme (ACE) gene, located on 17q23, is a 250-base pair deletion in intron 16. The D\/D genotype is associated with a high ACE level, whereas the I\/I genotype is associated with a low ACE level. In subjects with type 1 and type 2 diabetes, the haplotype insertion allele of the ACE gene has been associated with lower risk of diabetic nephropathy compared with the haplotypes including the deletion allele [49, 53, 54]. The D\/D genotype has been associated with renal progression in children and adults with CKD [55\u201358]. The D\/D polymorphism has been associated with LVH and QTc interval prolongation in patients with ESRD [59, 60].\nPolymorphisms in angiotensinogen (AGT), located on 1q42-q43, are associated with increase in risk for renal progression and CV disease. Reported frequently in the literature, the M235T SNP is a methionine (Met) to threonine (Thr) amino acid substitution at codon 235. The Thr\/Thr genotype is associated with an increased risk for hypertension in the general population [61, 62] and in kidney transplant patients with chronic allograft dysfunction. Investigators have recently recommended a name change of the SNP M268T for the substitution at amino acid 268, to be consistent with accepted human gene mutation nomenclature [63]. Another SNP is the AGT-6 G\/A promoter variant, which is associated with a higher risk for renal progression in the African American CKD population [64].\nA polymorphism in the AT2 type 1 receptor (AT1R) gene polymorphism, located on chromosome 3 (3q21-q25) [65], has also been associated with risk of renal progression and CV disease. The polymorphism of interest is a nucleotide change from an adenine (A) to cytosine (C) in the 3\u2032-UTR at nucleotide 1166. The C\/C genotype has been associated with a more rapid onset of renal failure compared with those with the A\/A genotype [66]. Furthermore, the C\/C genotype also is associated with the development of hypertension and coronary artery disease [67, 68].\nInterleukin-1 and IL-1 receptor antagonist (IL-1Ra)\nThe IL-1 family consists of two proinflammatory cytokines, IL-1\u03b1 and IL-1\u03b2, and a naturally occurring anti-inflammatory agent, the IL-1Ra. The balance between IL-1 and IL-1Ra in local tissues plays an important role in the susceptibility to and severity of many diseases [69, 70]. Plasma IL-1 and IL-1Ra have been shown to predict cardiovascular outcome [71] and mortality in ESRD [72]. The genes of the IL-1 complex map to the 430-kb region on the long arm of chromosome 2. The IL-1\u03b1 gene has an SNP g.-889C>T, which is a base pair change from a cytosine to a thymine (C\u2192T) [73]. The IL-1\u03b2 gene has an SNP g.-511C>T, which has a base pair change exchange (C\u2192T) [73] and g.+3953 [74]. The IL-1Ra gene contains a variable number of tandem repeat (VNTR) polymorphisms in intron 2 (IL-1RN) [75]. The IL-1RN allele 2 is related to increased production of IL-1\u03b2 [76]. Polymorphisms in IL-1\u03b1 have been associated with ESRD [73]. Gene polymorphisms of IL-1\u03b2 and IL-1RN have been associated with hypertension [77], atherosclerosis [78\u201380], CAD [78\u201380], and progression of renal disease [81\u201383].\nInterleukin-6 (IL-6)\nIL-6 is a proinflammatory cytokine that stimulates the production of C-reactive protein (CRP) and fibrinogen. There is a promoter polymorphism at position \u2013174 of the IL-6 gene (g.-174G>C). Carriage of the C allele is associated with higher levels of IL-6 production in response to pathologic stimuli [84]. The polymorphism is associated with a higher risk of CV disease in the general population [84, 85]. In dialysis patients, the carriage of the C allele is associated with high blood pressure, LVH, and decrease in functional status [47, 86]; in kidney transplant recipients, it is associated with decreased graft survival [87].\nInterleukin-10 (IL-10)\nIL-10 attenuates the inflammatory response [47]. Decreased production of IL-10 is associated with increased CRP and higher cardiovascular mortality [88]. The IL-10 gene is located on 1q31-32 and is composed of five exons and has SNPs at positions g.-592 C>A, g.-819 C>T, and g.-1082 G>A [89, 90]. The low producer genotype A\/A of the g.-1082G>A SNP is associated with increased CV mortality in ESRD patients [88].\nTumor necrosis factor-\u03b1 (TNF-\u03b1)\nTNF-\u03b1 production is stimulated by AT2 and associated with tubulointerstitial fibrosis [91]. Furthermore, elevated TNF-\u03b1 levels are associated with CV disease comorbidities: coronary artery disease [92], LVH [93], and congestive heart failure [94]. The TNF-\u03b1 gene is located on chromosome 6 and is highly polymorphic. Numerous promoter-region SNPs exist and are located at the upstream positions -1031, -863, -857, -851, -419, -376, -308, -238, -163, and -49 relative to the transcription start site; another SNP is at +488 in the intron [95, 96]. In hemodialysis patients, the polymorphism g.-308 G>A in the promoter region of the TNF-\u03b1 gene has been associated with significant comorbidity; the carriage of the A allele is associated with a low serum albumin, higher burden of comorbid conditions, and a low Karnofsky score [47].\nTransforming growth factor-\u03b2 (TGF-\u03b2)\nTGF-\u03b2 is a cytokine that regulates cell growth, differentiation, and extracellular matrix production [97]. TGF-\u03b2 transmits the profibrotic signaling of AT2 that promotes interstitial fibrosis in the kidney [98]. Blockade of AT2 by ACE inhibitors and AT2 receptor blocker (ARB) drugs reduces intrarenal TGF-\u03b2 [99]. Furthermore, TGF-\u03b2 is responsible for the production of additional fibrosis-promoting molecules such as connective tissue growth factor (CTGF) and PAI-1 [10]. Overproduction of TGF-\u03b21 is associated with renal progression [10, 100], hypertension [101], and LVH [102]. Several polymorphisms have been identified in the TGF-\u03b2 gene [103]. There are two polymorphisms in the signal peptide sequence Leu10->Pro (g.+869T>C), Arg25\u2192Pro (g.+915G>C) associated with higher production of TGF-\u03b21 [104, 105]. Gene polymorphisms in TGF-\u03b21 have been associated with an increase in proteinuria and mesangial cell hypertrophy in patient with IgA nephropathy [106]. Furthermore, the +915G\/C genotype at codon 25 (Arg\/Pro) may be a genetic susceptibility factor for the development of atherosclerosis due to the genotype\u2019s association with an increased risk for cardiac morbidity and cardiac-specific mortality in hemodialysis (HD) patients [107].\nPlasminogen activator inhibitor-1 (PAI-1)\nUpregulation of PAI-1 favors extracellular matrix accumulation and fibrosis by inhibiting fibrinolysis [108]. AT2 signaling via the type 1 receptor increases the production of PAI-1 [108]. Furthermore, regression of sclerosis is associated with blockade AT2 and a reduction in PAI-1 [109]. The gene polymorphism of interest is a 4G\/5G insertion\/deletion 675 base pairs from the start of the promoter. The polymorphism affects the binding of nuclear proteins involved in the regulation of PAI-1 gene transcription, leading to higher rate of synthesis with the 4G\/4G genotype [110]. The genotype is associated with chronic kidney allograft nephropathy [111] and increased activity of lupus nephritis [112], The PAI-1 4G\/5G polymorphism is associated with fatal and nonfatal myocardial infarction in dialysis patients [113]. In summary, much of the data supporting the link between RAS-cytokine gene polymorphisms and the progression of renal and CV disease stem from adult studies that involve a significantly larger prevalent population. However, there are preliminary data supporting efforts for genotype\u2013phenotype association studies in children [55\u201357]. Although there is heterogeneity for the causes of CKD between the adult and pediatric populations, progression for renal and CV abnormalities shares the same final common pathway, as discussed above.\nFinding the link between genotype and phenotype\nAs stated previously, the natural variations of the genes involving the RAS\u2013cytokine pathway potentially influence the rate of progressions for renal and CV disease in CKD patients. For example, a study by Balakrishnan et al. demonstrates the relationship between cytokine gene polymorphism and cytokine secretion from peripheral blood monocytes (PBMC) in hemodialysis patients [47]. Genotyping was performed for SNPs in the promoter region of IL-6 (-174 G>C), TNF-\u03b1 (-308G>A), and IL-10 (-1082 G>A) in 183 ESRD patients. Plasma cytokine levels by endotoxin-stimulated PBMCs were measured by enzyme-linked immunosorbent assay (ELISA). Plasma IL-6 levels were higher in the circulating blood from patients having -174G\/C or C\/C genotype; and IL-10 secretion was increased in -1082 G\/G genotype (Table\u00a03). The inflammatory response to the uremic milieu is variable and associated with cytokine gene polymorphisms in CKD patients.Table\u00a03Relationship between genotype and plasma cytokine levels [72]CytokineGenotypeTranscription\/secretion level (expected)Plasma level in pg\/ml mean \u00b1 SDInterleukin (IL)-6-174 C\/CLow12.2\u2009\u00b1\u20095.1-174 G\/G, G\/CHigh15.01\u2009\u00b1\u200917.4aTumor necrosis factor (TNF)-\u03b1-308 G\/GLow998.8\u2009\u00b1\u20091156.2-308 G\/A, A\/AHigh1131\u2009\u00b1\u20091616.2IL-10-1082 A\/ALow344.8\u2009\u00b1\u2009356.3-1082 G\/AIntermediate391.0\u2009\u00b1\u2009440.5-1082 G\/GHigh627.4\u2009\u00b1\u2009506.2baLevene\u2019s test for unequal variance, p=0.05, bKruskall\u2013Wallis test, p\u2009=\u20090.01\nBy analyzing differences in DNA sequences in large cohort studies, the disease phenotype can be mapped according to known genetic markers (i.e. known locations of SNPs) by linkage analysis. Unlike monogenic disorders, polygenic disorders as in CKD and CV abnormalities are the result of complex interactions of intra- and intercellular systems that are governed by multiple gene loci (polygenic), often modified by gene\u2013environment and gene\u2013gene interactions (epistasis) [24]. The genetic determinants of renal and CV disease progression are cumulative variations of gene transcription and function in these interdependent pathways.\nA consistent picture of genotype\u2013phenotype relationships in CKD and CV disease are lacking, probably because: (1) other unknown functional loci may be present; (2) polymorphisms that are known but which have not yet been recognized to be functional may exist; (3) inconsistent definitions of CKD; (4) heterogeneity of diseases that cause CKD; (5) limitations due to sample size, especially in the pediatric CKD population. Polymorphisms do not exist in isolation, and it may be the combination of base changes at several proximal sites along the allele, i.e. the haplotype that influences the function. Haplotype methods may capture a large proportion of the genetic variation across sizable regions using a minimal number of tag SNPs [114, 115], as discussed further below.\nHaplotype and HapMap\nAlleles that are in close proximity along a DNA strand tend to cross over together during recombination and comprise a haplotype. In a population, common haplotypes can be inherited among many individuals from a common ancestor, with complementary haplotypes being given by each parent. The characterization haplotype variants among human populations (http:\/\/www.hapmap.org) offers new opportunities in the genetic analysis of CKD through whole-genome association studies.\nAt this time, the International Haplotype Map (HapMap) Consortium is characterizing where and how frequently sequence variants occur in the human genome in four different ethnic groups worldwide [116]. Here, SNPs along a chromosome that tend to be inherited together can define a haplotype. Although a chromosomal region may contain many SNPs, there only a few tag SNPs that offer the most information about genetic variation in that region. Within candidate genes, the number of common polymorphisms is finite [117], but direct assay of all existing common polymorphism is inefficient, because genotypes at many of these sites are strongly correlated. Selection of the maximally informative set of a common tag SNP set can comprehensively interrogate for main effects of the haplotype [118, 119].\nThe HapMap project has opened the way for whole-genome association studies. Association refers to the statistical dependence between two variables, which is a measure of the degree to which the frequency of a risk factor, in this case a genetic marker, is different between persons with disease compared with those without disease. Hence, studies to determine the genes that influence CKD progression will compare CKD patients with a more rapid decline in glomerular filtration rate (GFR) to those with slower progression. Regions where the two groups differ in their haplotype frequencies might contain genes associated with renal progression. However, the dawning of new technology for rapid sequencing of DNA has implications of advancing this field of research beyond the HapMap [120].\nPotential limitations of gene association studies\nThe presence of an association does not imply that the observed relationship is one of cause and effect. A judgment of causation from epidemiologic data relies on assessing the validity of the observed statistical association and the cumulative evidence from a variety of sources in order to support a causal inference regarding the genetic marker of interest. An association between a gene and disease may be indicative of a true relationship between the gene and the disease; however, the association may not be valid due to chance (type I error), limited sample size (type II error), bias, or confounding. Ideally, a gene association study will have sufficient cases and controls (on the order of thousands) to identify a gene locus or region that is truly associated with the disease given a threshold of significance on the order of P value <\u200910\u22126 [121]. However, an investigation of this nature is not likely to be achieved immediately in the pediatric CKD population without preliminary evidence from studies of modest sizes varying in degrees of quality. Furthermore, no single center alone will have a sufficient number of pediatric CKD subjects to power such an analysis and would likely need a cooperative effort among a number of pediatric CKD centers.\nType I and type II error\nTypically, investigators may submit their data sets for genotyping with a standard, very large, set of SNPs, which will include markers located in the candidate gene along with a few thousand which are not. Spurious associations where the genetic marker of interest is correlated with the disease by chance may occur, especially in the presence of multiple comparisons [122]. To illustrate the multiple comparisons problem, we use a hypothetical case-control study of 10,000 unrelated SNPs to be tested for association with the disease of interest, with the threshold for statistical significance set to a P value of 0.01. If 10,000 statistical tests are performed to assess the association between each of the SNPs and the disease, by chance alone, we would expect 100 SNPs at random to be statistically associated with the disease, even though there is no true relationship; this is a type I error. With increasing number of genetic markers being typed and multiple intermediate phenotypes being tested, strict guidelines for publication of gene association studies have been proposed [123\u2013125].\nIn studies of modest size, SNP markers in the candidate gene may produce relatively modest evidence in favor of association with a level of P\u2009<\u20090.01, even with hundreds of subjects [126]. Thus, a marker having a real but modest effect is not expected to produce an odds ratio (OR) with a smaller P value than markers producing apparently significant results by chance [126]. In addressing this limitation, setting a stringent P value (P\u2009<\u200910\u22126) is a generally accepted convention for large studies with markers of low prior probability of true association [121]. Applicable to studies of smaller magnitude, alternative methods relying on Bayesian strategies have been proposed [126, 127]. Under these approaches, the prior probability of association based on preliminary evidence is used to weight the P value obtained. This supports the general recommendation for having biologic plausibility of the observed association such that the genetic variant of interest is involved the pathogenesis of disease [122, 124, 128]. It is worth considering whether the known function of the gene is linked to the observed phenotype. Furthermore, the association between gene and disease is less likely to be spurious if the relationship is observed in other independent studies. Although mildly controversial [129], there is general agreement that the initial findings of a gene\u2013disease association study be replicated by other studies [130, 131].\nMany gene association studies are not replicated for a number of reasons. A small sample size may limit a study\u2019s ability to detect an association if one truly exists. Concluding that there is no association when one truly exists defines a type II error. The magnitude of the contribution of single gene variants to polygenic disorders is small, with a typical effect sizes corresponding to ORs of 1.2\u20131.6 [132]. Compared with the smaller sample sizes needed to detect a larger effect (e.g. OR \u2265 2.0), detecting associations of smaller magnitude requires a much larger sample size, which may dramatically increase the recruitment costs and make some studies unfeasible [132].\nFurthermore, the lack of consistency in proposed gene\u2013disease association across studies may also be reflective in the inherent complexity and heterogeneity of common diseases, including CKD [131], which are beyond the scope of this review. A proposed solution for studies with insufficient power are to: (1) emphasize replication and obtain data to determine biologic plausibility; (2) synthesize results of individual studies for meta-analysis; or (3) obtain data on individual subjects from several studies to perform a pooled analysis [125].\nLimitations in study design\nBiased estimates for an observed association between genotype and phenotype may originate from a flaw in the design or conduct of the study that has introduced systematic error or bias into the result. There are numerous considerations to this point, but a few topics deserve some mention in this general overview. A more complete discussion of these considerations can be found elsewhere [122, 125, 133, 134]. The adequacy of any epidemiologic study design depends on the scientific question [135]. A study that is designed to detect an association may \u201coverselect\u201d cases in order to detect an association. However, the measures of the association from studies designed to detect genotype\u2013phenotype associations are not valid or generalizable measures of association for the population [125].\nThe case\u2013control study is a common study paradigm for genome association studies because it is economically efficient, allows for the evaluation of diseases with long latent periods, and can examine multiple etiologic factors for one disease; however compared with other study paradigms, case\u2013control studies are particularly prone to bias if controls are not properly selected [136]. Genetic association studies may be biased or confounded by population stratification [133] and genotyping errors [134]. Population stratification can create the appearance of a SNP\u2013disease association and arises when race and\/or ethnicity is related both to the SNP under investigation (e.g. differences in allele frequencies within distinct ethnic groups) and to the disease of interest. Similar to epidemiologic studies needing to address race and ethnicity as potential confounders, gene polymorphism studies should assess the potential for bias and confounding due to population stratification [137].\nOf additional concern, genotyping errors can be significant, leading to null results or erroneous conclusions [134]. Genotyping errors can stem from a number of causes, including: (1) inadequate sample quality; (2) artifacts due to biochemical or equipment problems; (3) errors from the DNA amplification process; (4) human factors. Genotyping errors in phenotype\u2013genotype association studies will tend to bias estimates of association toward the null assuming that errors occur at equal frequency across case status [134]. However, if cases and controls are genotyped using different assays or run separately in distinct batches, differential errors may occur resulting in either over- or underestimation of the true association [134].\nConclusions\nGenetic association studies have the potential to provide new insights into the factors responsible for CKD renal and CV progression. These investigations provide hope for new drug targets to treat or modify individual disease risk. In the case of CKD, genetic polymorphisms in the RAS\u2013cytokine pathway may be responsible for the intraindividual variation in renal and cardiac progression in patients with CKD and may offer new targets for drug therapy.\nThe Human Genome and HapMap projects have made it possible to evaluate a multitude of candidate genes that might be linked to CKD progression. The enthusiasm for these investigations must be tempered by acknowledging the limitations of gene association studies. Attention to biologic plausibility and appropriate study design will help the interpretability of published results. Independent investigations replicating initial findings are needed to support an inference of a causal association between the gene polymorphism of interest and the disease phenotype.\nWhole-genome association studies are becoming widely available and are being performed to investigate the genetic predisposition to diabetic nephropathy [49, 50]. In an effort to understand the risk factors for progression of CKD and CV disease, the ongoing CKD cohort studies in adult and pediatric patients [138, 139] are evaluating known risk factors for CKD progression, including etiology of CKD, proteinuria, and hypertension. These cohort studies are collecting biologic and genetic samples for future studies of cytokines or their genetic polymorphisms, which may yield scientific insight into the pathophysiologic mechanisms of CKD progression in both adults and children.","keyphrases":["genetic","polymorphism","cytokine","chronic kidney disease","progression","pediatric","cardiovascular disease"],"prmu":["P","P","P","P","P","P","R"]}
{"id":"Biol_Psychol-1-5-2075532","title":"Anxiety and orienting of gaze to angry and fearful faces\n","text":"Neuroscience research indicates that individual differences in anxiety may be attributable to a neural system for threat-processing, involving the amygdala, which modulates attentional vigilance, and which is more sensitive to fearful than angry faces. Complementary cognitive studies indicate that high-anxious individuals show enhanced visuospatial orienting towards angry faces, but it is unclear whether fearful faces elicit a similar attentional bias. This study compared biases in initial orienting of gaze to fearful and angry faces, which varied in emotional intensity, in high- and low-anxious individuals. Gaze was monitored whilst participants viewed a series of face-pairs. Results showed that fearful and angry faces elicited similar attentional biases. High-anxious individuals were more likely to direct gaze at intense negative facial expressions, than low-anxious individuals, whereas the groups did not differ in orienting to mild negative expressions. Implications of the findings for research into the neural and cognitive bases of emotion processing are discussed.\n1\nIntroduction\nResearch into the cognitive and neural mechanisms underlying emotion processing indicates neural circuitry, involving subcortical and cortical structures (including the amygdala and prefrontal cortex), which is responsible for detecting threat-related cues in the environment and triggering a variety of cognitive, behavioural and physiological responses, in particular, attentional vigilance (LeDoux, 1996; Davis and Whalen, 2001). Neuroimaging studies show that the amygdala, which plays a central role in this circuitry, is reactive to threat-related cues, such as fearful faces (e.g., Whalen et al., 1998). Individual differences in the operation of this threat-processing system may underlie individual differences in vulnerability to anxiety and anxiety disorders (Davis and Whalen, 2001).\nFrom a clinical perspective, cognitive models of anxiety also assume the existence of a threat-processing system and propose that anxiety is characterized by cognitive biases (in particular, in stimulus evaluation and selective attention) which favour the processing of threat cues. These biases are proposed to be responsible for individual differences in anxiety vulnerability (e.g., Beck and Emery, 1985; Williams et al., 1997; Mogg and Bradley, 1998). Cognitive models, such as Beck and Emery's (1985), are important because they provide the basis for cognitive-behaviour therapy (CBT), which aims to remove these threat-processing biases and is an effective treatment for many anxiety disorders (e.g., Ballenger, 1999). Research into cognitive models of anxiety indicates that anxious individuals have an enhanced attentional bias for threat cues, compared with non-anxious individuals, and that this bias operates in early aspects of processing (e.g., review by Mogg and Bradley, 1998).\nIn order to understand the functional properties of this postulated threat-processing system, it is necessary to clarify the type of stimuli to which it is sensitive. Neuroimaging research typically indicates that the amygdala is more reactive to fearful than angry faces (e.g., Whalen et al., 2001; Blair et al., 1999; Davis and Whalen, 2001; but see Yang et al., 2002). Davis and Whalen (2001) suggested that fearful faces elicit more amygdala activity because they are ambiguous (i.e. they signify the presence of danger, but do not provide information about its source) and that the threat-processing system is more reactive to ambiguous (or indirect) threat cues because it is designed to promote attention to stimuli which require more detailed processing in order to determine appropriate responding (e.g., escape or approach). Neuroimaging evidence has been reported supporting the hypothesis of enhanced processing of ambiguous threat cues, as indexed by amygdala responses to angry and fearful faces (Adams et al., 2003). Thus, according to Davis and Whalen's (2001) theoretical view, fearful faces should be particularly effective in capturing attention and in eliciting vigilance.\nBiases in visuospatial orienting to threat faces have been investigated using visual-search, visual-probe and eye-movement paradigms. Visual-search studies require participants to search arrays of faces for discrepant emotional expressions and results have indicated faster detection of schematic angry faces, relative to positive faces (e.g., \u00d6hman et al., 2001; Fox et al., 2000). In the visual-probe task, pairs of faces (e.g., angry face and neutral face of the same individual) are briefly presented, followed by a probe stimulus (e.g., small dot) to which the participant responds. Response times (RTs) to probes reflect the allocation of attention to the faces, as RTs are typically faster to probes which appear in attended, rather than unattended, locations. Visual-probe studies provide evidence of enhanced attentional biases for angry faces in individuals with high non-clinical anxiety, and in anxious patients (e.g., Bradley et al., 1998, 1999; Mogg et al., 2004). They also indicate that the attentional bias increases as the intensity of the angry expression increases (Wilson and MacLeod, 2003). Eye-movement methodology has extended this research, as it provides a direct and ecologically-valid measure of visual orienting, and further indicates that anxious individuals have a greater bias to direct their gaze initially towards angry faces (relative to neutral or happy faces), compared with non-anxious individuals (Bradley et al., 2000; Mogg et al., 2000), which suggests that the attentional bias for threat operates at a relatively early stage of visual processing and guides eye-movements. However, there has been little research using these paradigms to assess biases in visual orienting towards fearful faces. For example, one study using a visual-search task suggested that angry, but not fearful, faces preferentially attract attention (Williams et al., 2005), whereas another study using a visual-probe task indicated an attentional bias for fearful faces that had been perceptually degraded (i.e. low spatial frequency; Holmes et al., 2005). Neither study examined the influence of anxiety.\nConsequently, the main aim of the present study was to compare biases in initial orienting of gaze towards fearful versus angry faces in high- and low-anxious individuals. A second aim was to examine the effect of manipulating the emotional intensity of the faces (cf. Blair et al., 1999; Wilson and MacLeod, 2003) on attentional responses. Neuroimaging research (Davis and Whalen, 2001) predicts that fearful faces should elicit stronger vigilance responses than angry faces. In addition, cognitive models of anxiety predict that high-anxious individuals should show greater biases in initial orienting of gaze towards both angry and fearful faces, than low-anxious individuals. Furthermore, if biases in initial orienting to threat-related cues are primarily a function of the affective salience of the stimuli (Mogg and Bradley, 1998), then these biases in orienting should increase as the intensity of the fearful and angry expressions increases.\n2\nMethod\n2.1\nParticipants\nParticipants were undergraduates who were screened on the trait version of the State-Trait Anxiety Inventory (STAI; Spielberger, 1983); those with trait anxiety scores of 40 or less were allocated to the low anxiety group, and those scoring 50 or more to the high anxiety group. Additional selection criteria were fluency in spoken English and visual acuity within normal limits. Nine volunteers did not complete the study due to equipment calibration problems and six participants had excessive missing eye-movement data (described later). The final sample comprised 28 participants (3 M, 25 F) in the low-anxious group, and 21 (3 M, 18 F) in the high-anxious group.\n2.2\nMaterials and apparatus\nTwenty-four face stimuli were selected from the NimStim Set of Facial Expressions (http:\/\/www.macbrain.org\/faces\/).1 They consisted of angry, fearful and neutral prototypical expressions posed by eight models (four female and four male numbered: 01f, 03f, 07f, 08f, 21m, 23m, 27m, 34m). For each model and type of facial expression (fearful or angry), the emotional face was blended with the neutral face to produce a continuum comprising five exemplars of increasing emotional intensity: i.e. neutral prototype (0%), three morphed expressions (25%, 50% and 75%) and the emotional prototype (100%); see Fig. 1. The preparation of the morphed faces used Gryphon Morph v2.5 software (Gryphon Software Corporation, 1994). Each emotional face (i.e. 25%, 50%, 75% or 100% exemplar of a fearful or angry face) was paired with the neutral expression (0%) of the same model to create 64 face pairs for use in the attentional task.\nThe task was administered using MEL2 software (Schneider, 1995), Pentium 450\u00a0MHz PC, 15\u00a0in. VGA monitor and MEL2 response box. Eye-movements were monitored with 120\u00a0Hz infrared pan\/tilt eye tracking system (Model 504, Applied Scientific Laboratories, Bedford, Massachusetts) and E5000 software (Applied Science Group, 2000) which was run on a Pentium 333\u00a0MHz PC. Testing was conducted in a dimly lit room.\n2.3\nProcedure\nParticipants completed a visual acuity check and were seated 1\u00a0m from the monitor, with the eye tracking camera positioned 50\u00a0cm in front of them, below the right eye. The equipment was calibrated by displaying the numbers 1 to 9 on the screen in a 3\u00a0\u00d7\u00a03 array and recording the direction of gaze whilst participants looked at each number in turn.\nEach trial of the attentional task started with a central fixation-cross shown for 1000\u00a0ms, followed by a pair of pictures presented side by side for 500\u00a0ms. The pictures measured 90\u00a0mm\u00a0\u00d7\u00a0110\u00a0mm, with their inner edges 45\u00a0mm apart. Immediately after the offset of the picture-pair, a probe (pair of dots : or ..) was presented in the position of one of the preceding pictures (visual angle of 7.7\u00b0 between two probe positions) until a manual response. Participants were asked to press one of two keys as quickly as possible to indicate the type of probe. Inter-trial interval varied randomly between 750 and 1250\u00a0ms. Participants were instructed to keep their head still throughout the task and to look at the fixation-cross at the start of each trial. Eye-movement data were recorded from the onset of the fixation-cross until the manual response. There were eight practice trials followed by two blocks, each consisting of two buffer trials and 256 experimental trials, with a short rest-break between the blocks. Across the whole task, the 64 face pairs were presented eight times, balanced for emotional face location and probe location (left vs. right). Trials were presented in a new random order for each participant. If eye calibration quality deteriorated during the task, the task was briefly interrupted to repeat the calibration procedure.\nAfter the attentional task, participants completed three face rating tasks: forced-choice discrimination, anger and fear ratings, which were included to check the effectiveness of the manipulation of emotional intensity. Each task presented the faces from the attentional task, one at a time, in random order. Each trial consisted of a central fixation-cross (500\u00a0ms), followed by a face and rating scale, displayed until a keyboard response. In the forced-choice discrimination task, participants indicated whether each face was angry or fearful. In the anger and fear rating tasks, they rated each face for how angry or fearful it appeared on a scale ranging from 0 (not at all) to 8 (extremely). Finally, they completed questionnaires including the state and trait versions of the STAI, and short-form Social Desirability Scale (SDS; Strahan and Gerbasi, 1972; the latter was included because defensiveness can have a confounding effect on measures of anxiety and attentional bias, e.g., Eysenck, 1997).\n2.4\nData preparation\n2.4.1\nEye-movement data\nThese were prepared using the Eyenal Data Analysis Program (Applied Science Group, 2000). Direction of gaze was measured in degrees approximately once every 8\u00a0ms. If eye-movements were stable within 1\u00b0 of visual angle for 100\u00a0ms or more, this was classified as a fixation to that position. Initial fixations to the pictures were calculated if the following criteria were met: (i) gaze was fixated in the central region before picture onset, (ii) fixations occurred at least 100\u00a0ms after picture onset, and (iii) fixations were directed at either the left or right picture (>1.3\u00b0 wide of central fixation position on the horizontal plane). Six participants had excessive missing data (85% or more of trials lacked identifiable fixations), so were excluded from all analyses. The amount of missing data did not vary as a function of anxiety group, F(1, 47)\u00a0<\u00a01.7, emotional face type, emotional face intensity, or their interaction, F(3, 141) <1.5, all p's\u00a0>\u00a0.2. Gaze-direction bias scores were obtained for each participant and each level of intensity of fearful and angry faces by calculating the number of trials in which the first shift of gaze was towards the emotional face, as a proportion of the number of trials in which the initial shift in gaze was made to either the emotional or neutral face (bias scores\u00a0>\u00a0.5 reflect an orienting bias towards the emotional face; .5\u00a0=\u00a0no bias).\n2.4.2\nRT data\nData from error trials (2%) and RTs <200\u00a0ms or >1250\u00a0ms (1%) were excluded. The groups did not differ in missing data or overall mean RT, ts\u00a0<\u00a01. An inverse transformation was applied to the RT data to reduce the influence of skewness and outliers (Ratcliff, 1993). Attentional bias scores were calculated using a standard formula (e.g., Bradley et al., 1998), for each participant and each level of intensity of fearful and angry faces, by subtracting the mean RT when the emotional face and probe were in the same location from the mean RT when the emotional face and probe were in different locations. RT bias scores were corrected (reverse sign and multiply by 106) so that they would be easier to comprehend, i.e. positive values of bias scores reflect an attentional bias for emotional faces, relative to neutral faces (0\u00a0=\u00a0no bias).\nKolmogorov\u2013Smirnov tests showed that the distributions of RT bias scores and gaze-direction scores were within normal limits.\n3\nResults\n3.1\nGroup characteristics\nThe high- and low-anxious groups differed significantly in trait and state anxiety (see Table 1), but not in social desirability scores, age or gender ratio.\n3.2\nEye-movement data\nGaze-direction bias scores were entered into a 2\u00a0\u00d7\u00a02\u00a0\u00d7\u00a04 mixed design ANOVA with anxiety group (high, low) as the between-subjects independent variable (IV), and emotional face type (angry, fearful) and intensity of emotional face (25%, 50%, 75%, 100%) as within-subject IVs (see Fig. 2 for means). There were significant main effects of anxiety group, F(1, 47)\u00a0=\u00a04.68, p\u00a0<\u00a0.01, , and emotion intensity, F(3, 141)\u00a0=\u00a010.49, p\u00a0<\u00a0.01, , which were qualified by a significant anxiety group\u00a0\u00d7\u00a0emotion intensity interaction, F(3, 141)\u00a0=\u00a03.02, p\u00a0<\u00a0.05, . There were no other significant results, e.g., anxiety group\u00a0\u00d7\u00a0emotion intensity\u00a0\u00d7\u00a0type of emotional face: F\u00a0<\u00a01. Separate ANOVAs of bias scores from each group showed a significant main effect of emotional intensity on gaze direction in the high-anxious group, F(3, 60)\u00a0=\u00a09.54, p\u00a0<\u00a0.001, , but not in the low-anxious group, F(3, 81)\u00a0=\u00a02.04, p\u00a0=\u00a0.12, . To clarify further the anxiety group\u00a0\u00d7\u00a0emotion intensity interaction, the groups were compared on their bias scores at each level of intensity (averaged across angry and fearful faces).\nThere was no significant difference between the high- and low-anxious groups in bias scores for 25% negative faces, (Ms\u00a0=\u00a0.508 vs. .500, t\u00a0<\u00a01, NS, d\u00a0=\u00a0.11), 50% negative faces (.533 vs. .533, t\u00a0<\u00a01, NS, d\u00a0=\u00a0.003), or 75% negative faces (.561 vs. .528, t(47)\u00a0=\u00a01.34, NS, d\u00a0=\u00a0.39). A one-sample t-test was used to contrast the RT bias scores against a value of .5 (which indicates no bias); results showed that, irrespective of group, participants showed no attentional bias for 25% negative faces (.504, t\u00a0<\u00a01, NS, d\u00a0=\u00a0.05), but a significant bias for 50% negative faces (.533, t(48)\u00a0=\u00a02.99, p\u00a0<\u00a0.01, d\u00a0=\u00a0.43) and also for 75% negative faces (.542, t(48)\u00a0=\u00a03.39, p\u00a0<\u00a0.01, d\u00a0=\u00a0.48).\nFor 100% negative faces, the high-anxious group showed a significantly greater bias in orienting towards them, compared with the low-anxious group (.629 vs. .547, t(47)\u00a0=\u00a03.21, p\u00a0<\u00a0.01, d\u00a0=\u00a0.91). One-sample t-tests showed a significant bias in gaze towards 100% negative faces, relative to neutral faces, in both the high-anxious (.629, t(20)\u00a0=\u00a05.66, p\u00a0<\u00a0.001, d\u00a0=\u00a01.24) and low-anxious (.547, t(27)\u00a0=\u00a03.26, p\u00a0<\u00a0.01, d\u00a0=\u00a0.62) groups. Thus, the bias for participants to direct their gaze initially at prototypical (100%) negative faces, was significantly stronger in the high-anxious group.\n3.3\nManual RT data\nRT bias scores were entered into a 2\u00a0\u00d7\u00a02\u00a0\u00d7\u00a04 mixed design ANOVA with anxiety group, type of emotional face and intensity of emotional face as IVs. There was a significant group\u00a0\u00d7\u00a0emotion intensity interaction, F(3, 141)\u00a0=\u00a02.83, p\u00a0<\u00a0.05, , and no other significant results (e.g., group\u00a0\u00d7\u00a0intensity\u00a0\u00d7\u00a0face type: F\u00a0<\u00a01). The pattern of significant findings corresponded to that in the eye movement data. The high- and low-anxious groups did not significantly differ in RT bias scores for 25% negative faces (\u22127.4 vs. 16.4, t(47)\u00a0=\u00a01.40, NS, d\u00a0=\u00a0.40), 50% negative faces (13.1 vs. 19.0, t\u00a0<\u00a01, NS, d\u00a0=\u00a0.11), or 75% negative faces (4.5 vs. 24.9, t(47)\u00a0=\u00a01.17, NS, d\u00a0=\u00a0.33). Contrasts of RT bias scores against zero showed that, irrespective of group, participants showed no attentional bias for 25% negative faces (6.2 vs. zero, t\u00a0<\u00a01, d\u00a0=\u00a0.10), a significant bias for 50% negative faces (16.4, t(48)\u00a0=\u00a02.12, p\u00a0<\u00a0.05, d\u00a0=\u00a0.30), and a near-significant bias for 75% negative faces (16.2, t(48)\u00a0=\u00a01.87, p\u00a0<\u00a0.07, d\u00a0=\u00a0.27).\nFor 100% negative faces, there was a significant group difference in attentional bias (t(47)\u00a0=\u00a02.04, p\u00a0<\u00a0.05, d\u00a0=\u00a0.60). Contrasts of the bias scores against zero showed a significant attentional bias for 100% negative faces in the high-anxious group (37.4, t(48)\u00a0=\u00a03.48, p\u00a0<\u00a0.01, d\u00a0=\u00a0.76), but not in the low-anxious group (4.2, t\u00a0<\u00a01, d\u00a0=\u00a0.07).\nThe RT bias and gaze-direction bias scores for 100% negative faces significantly correlated with each other (r\u00a0=\u00a0.35, p\u00a0<\u00a0.05). There were no significant associations between these two measures of attentional bias for negative faces at lower levels of emotion intensity (r\u00a0=\u00a0.11, .10 and .13 for 25%, 50% and 75% negative faces, respectively, all NS).2\n3.4\nRating tasks\n3.4.1\nForced choice discrimination task\nA 2 (anxiety group)\u00a0\u00d7\u00a02 (emotional face type)\u00a0\u00d7\u00a04 (intensity of emotional face3) ANOVA of the proportion of faces correctly classified as angry or fearful showed a significant main effect of intensity, F(3, 141)\u00a0=\u00a0173, p\u00a0<\u00a0.001, , as, unsurprisingly, more intense emotional expressions were classified more accurately. Mean proportion of correctly classified negative faces with 25%, 50%, 75% and 100% intensity was .70, .94, .97 and .96, respectively. There were no other significant results (e.g., group\u00a0\u00d7\u00a0intensity, F(4, 188)\u00a0=\u00a01.26, p\u00a0>\u00a0.25; group\u00a0\u00d7\u00a0intensity\u00a0\u00d7\u00a0face type: F\u00a0<\u00a01).\n3.4.2\nAnger ratings\nA 2 (anxiety group)\u00a0\u00d7\u00a05 (emotion intensity) ANOVA of ratings of faces from the anger continuum showed only a significant main effect of intensity, F(4, 188)\u00a0=\u00a0594, p\u00a0<\u00a0.001, , with no effects involving group, Fs\u00a0<\u00a01. Mean anger ratings of 0%, 25%, 50%, 75% and 100% angry faces were 1.6, 2.8, 4.8, 6.2 and 7.0, respectively. Bonferroni-corrected contrasts showed that each mean differed significantly from each other, p\u00a0<\u00a0.005.\nA 2\u00a0\u00d7\u00a05 ANOVA of anger ratings of faces from the fear continuum showed only a significant effect of intensity, F(4, 188)\u00a0=\u00a011.63, p\u00a0<\u00a0.001, , with no effects involving group, Fs\u00a0<\u00a01. Mean anger ratings of 0%, 25%, 50%, 75% and 100% fearful faces were 1.6, 1.1, 0.8, 0.8 and 0.7, respectively. Bonferroni-corrected contrasts showed that faces with fearful content (i.e. 25\u2013100%) were rated as less angry than neutral faces, p\u00a0<\u00a0.005.\n3.4.3\nFear ratings\nA 2\u00a0\u00d7\u00a05 ANOVA of fear ratings of faces from the fear continuum showed a significant main effect of intensity, F(4, 188)\u00a0=\u00a0515, p\u00a0<\u00a0.001, , with no effects involving group (group: F\u00a0<\u00a01; group\u00a0\u00d7\u00a0intensity: F(4, 188)\u00a0=\u00a01.20, NS). Mean fear ratings of 0%, 25%, 50%, 75% and 100% fearful faces were 1.3, 2.3, 4.4, 5.9, and 6.6, respectively. Bonferroni-corrected contrasts showed that each mean differed significantly from each other, p\u00a0<\u00a0.005.\nA 2\u00a0\u00d7\u00a05 ANOVA of fear ratings of faces from the anger continuum showed a significant main effect of emotion intensity, F(4, 188)\u00a0=\u00a02.72, p\u00a0<\u00a0.05, , and no effects involving group, Fs\u00a0<\u00a01. Mean fear ratings of the 0%, 25%, 50%, 75% and 100% angry faces were 1.3, 1.5, 1.2, 1.0 and 0.9, respectively. Bonferroni-corrected contrasts did not reveal significant differences between these ratings.\n4\nDiscussion\nThe present findings indicate that fearful and angry faces elicited similar biases in visuospatial orienting. There was a greater tendency for participants to direct gaze initially towards faces with moderate or intense threat-related facial expressions (50\u2013100% intensity), relative to neutral faces, whereas mild threat-related facial expressions (25% intensity) did not elicit a bias in initial orienting. Moreover, visuospatial orienting to both fearful and angry faces was significantly influenced by individual differences in anxiety: high-anxious individuals showed a greater tendency to direct gaze at prototypical (100%) threat-related faces, irrespective of whether the faces depicted fear or anger. The secondary measure of attentional bias, which was obtained from manual RTs, showed a similar pattern of results to that obtained in the eye-movement data.\nThe enhanced attentional bias in high-anxious individuals, which was found for prototypical angry faces, is compatible with previous findings, e.g., from visual-probe or eye-movement studies (e.g., Mogg and Bradley, 1998; Mogg et al., 2000). The present results further indicate that high- and low-anxious individuals did not differ significantly in visual orienting to threat-related faces which had weaker emotional expressions. This may explain why some studies might fail to find evidence of an effect of individual differences in anxiety on attentional bias, if they use less salient exemplars of emotional facial expressions than the prototypical faces used here.\nThe finding that fearful and angry faces elicited a similar pattern of attentional bias is consistent with cognitive and neural models which posit the existence of a threat-processing system which modulates vigilance for potential sources of danger. Previous research using prototypical expressions suggests that this bias in visual orienting of gaze is specific to threat-related faces, as, for example, anxious patients have a greater bias to shift their gaze initially towards angry faces, but not happy or sad faces, compared with non-anxious controls (Mogg et al., 2000). Given this previous finding, the present study did not include these additional face types; moreover, these would have made the task too long and fatiguing for participants.\nThe finding of an equivalent pattern of attentional bias for fearful and angry faces is not consistent with expectation from some recent neuroimaging research findings (discussed earlier) which indicated that the amygdala, which is proposed to modulate attention to threat-related cues, is more sensitive to fearful than angry faces (Davis and Whalen, 2001). However, this might be resolved by considering the different component processes of attention, namely, shifting versus maintenance (LaBerge, 1995; Serences et al., 2005). Thus, the amygdala may indeed modulate attention to threat, but its level of activation may be a function of both initial orienting and maintained attention. Fearful and angry faces may have a similar capacity to attract attention initially (as indicated by the present findings), but may differ in the extent to which they hold attention. After initial orienting to angry faces, attention may be subsequently directed away from them, e.g., due to emotion-regulation processes (Mogg and Bradley, 1998; Rohner, 2002; Garner et al., 2006); whereas attention may be maintained longer on fearful faces if they require more detailed processing to determine appropriate responding, as suggested by Davis and Whalen (2001). This hypothesis could be addressed by both cognitive and neuroimaging studies: for example, eye-movement studies, which employ longer display times to assess the time-course of attentional responses (e.g., >1\u00a0s), could compare biases in both initial orienting and gaze dwell time for fearful versus angry faces.4 Neuroimaging studies could examine whether amygdala response to angry faces is positively associated with initial orienting to threat, but inversely associated with subsequent attentional avoidance; as suggested by research indicating that amygdala activity is suppressed by inhibitory influences of the prefrontal cortex, which mediates emotion-regulatory processes including attention control (Nomura et al., 2004; Ochsner and Gross, 2005). Thus, although many neuroimaging studies have focused largely on the role of the amygdala in emotion processing, it is important to consider it within the context of a broader network involving other structures (e.g., prefrontal cortex, thalamus, hippocampus, periaqueductal gray, locus coeruleus), with which it has reciprocal connections and which subserve a variety of functions, including attention, startle, escape and avoidance (Davis and Whalen, 2001; McNaughton and Corr, 2004).\nThe results from the face rating tasks confirmed the content validity of the morphed face stimuli, which were well-differentiated in emotion intensity. The groups did not differ in their explicit ratings of the faces, which is compatible with previous research that has also failed to reveal anxiety-related differences in ratings of emotional faces, and which might be due to demand or social desirability effects obscuring anxiety-related effects on self-report data (e.g., Wilson and MacLeod, 2003). These null results contrast with those from the attentional task which indicated that individual differences in anxiety played an important role in determining the processing of emotional stimuli, namely, in visual orienting. Thus, to clarify the neurocognitive mechanisms underlying emotion processing, it would seem helpful for neuroimaging studies to make greater use of tasks that are sensitive to individual differences in anxiety, such as attentional paradigms (e.g., Monk et al., 2006), and also to routinely assess individual differences in anxiety, given that this is an important determinant of attentional responses to threat.","keyphrases":["anxiety","fearful faces","angry faces","attentional bias","eye movements"],"prmu":["P","P","P","P","P"]}
{"id":"Support_Care_Cancer-4-1-2413096","title":"Specialized palliative care services are associated with improved short- and long-term caregiver outcomes\n","text":"Goals of work The goal of this study was to evaluate, at a population level, the association between specialized palliative care services (SPCS) and short- and long-term caregiver outcomes.\nIntroduction\nIn the more than 7,300 specialized palliative-care services (SPCS) internationally [6], a variety of models of service delivery are in place [42]. A common model of SPCS relies on trained specialist providers, whose work is largely in palliative care; in this model, coordination of care occurs wherever the patient is located.\nTo date, evaluation of SPCS has been difficult [1, 28]. A recent meta-analysis demonstrated that SPCS had a positive impact on patients\u2019 pain, other symptoms, and caregiver satisfaction. Evidence is lacking for other caregiver outcomes, or for economic benefit to justify the community\u2019s healthcare investment in these services [31].\nPalliative care is person-centered care that focuses on optimizing function and comfort in the setting of life-limiting illness. The patient, family member(s), and informal caregiver(s) comprise the \u201cunit of care.\u201d\nSPCS must be judged by their net health impact not only on the patient but also on caregivers including short-term (while the individual performs the functions of caregiver) and long-term (once the role has ceased) outcomes [14]. There are extensive demands on informal caregivers serving patients with life-limiting illness; the caregiver burden encompasses physical, emotional, financial, existential, and social responsibilities [2, 20, 53]. Many caregivers perform a variety of roles, and those with a larger number of roles exhibit greater caregiver strain [33]. Caregiving may compromise health and is associated with premature mortality long after the death of the patient [9, 47, 50, 56, 58]. Depression and restriction of activities are common among caregivers [5, 22, 66]. Caregiver outcomes depend upon a host of variables including gender, age, socioeconomic status, living situation, and the type and quality of relationship between the caregiver and the recipient of care [43].\nIn the short term, SPCS coordinate support and services in an attempt to minimize caregiver distress and respond to unmet needs [40, 59]. \u201cUnmet needs\u201d, defined as needs not being addressed plus needs receiving insufficient attention, can be psychological, social, informational, physical (activities of daily living and household management), existential, legal, or financial [40]. The experience of caregiving has been described in five domains, each of which carries support needs and outcome repercussions; these domains are (1) disrupted schedule, (2) financial problems, (3) lack of family support, (4) loss of physical strength, and (5) caregiver self-esteem [24, 43]. Inadequate support of caregivers may predict financial burden and poorer caregiver health [59]. A summary measure for differences in caregiver outcomes can be the total number of unmet needs [40, 59].\nIn the intermediate to long term, SPCS programs are designed to facilitate families\u2019 and caregivers\u2019 adjustment to the loss that they have experienced [34]. High-level long-term caregiver outcomes, with which to evaluate these programs, are poorly developed [27, 28, 68]. One such potential caregiver outcome is \u201cmoving on.\u201d Inability to \u201cmove on\u201d has been identified as a marker of a complicated course for caregivers after the death [50, 51]. Conversely, caregivers often reflect that they are starting to move on with their life at some point after death. Psychiatric and social-work literature defines moving on as establishing a new caregiver-defined equilibrium after having experienced a period of disequilibrium and integrating the potentially life-changing impact of caregiving into one\u2019s new self-perception and life roles in areas including relationships, intimacy, work, and finances [7, 13, 17, 19, 55]. Moving on is not simply \u201ctaking up where one left off\u201d [7]; rather, it implies some degree of re-adjustment and integration of the caregiving and bereavement experiences. It may be characterized as returning to a sense of well-being, \u201creframing,\u201d or \u201cadapting\u201d [53, 54, 65]. Despite the widespread use of the concept, a validated instrument for measuring moving on has not yet been developed. Nonetheless, the concept of enabling a caregiver to move on can be a first approximation of the long-term impact of SPCS on caregivers.\nHistorically, efforts to assess SPCS\u2019 influence on short- and long-term caregiver outcomes have been limited because of difficulties in identifying a population of caregivers for people with life-limiting illnesses who do not interact with SPCS [2, 41, 63]. For a population-based assessment of SPCS outcomes, the appropriate denominator must include all caregivers for people with life-limiting illnesses, not only those who are referred to SPCS [15, 48]. We have previously described a population-based survey methodology, the South Australian Health Omnibus Survey, for identifying caregivers of people with life-limiting illnesses where SPCS were and were not involved; we used this methodology again in the current study [16].\nFigure 1 depicts the conceptual model being tested in this study. Its aims are to determine whether:\nSPCS meet the short-term needs of caregivers at the time they are providing support for someone with a life-limiting illness; andAn association exists between SPCS use and differences in caregiver-defined long-term outcomes in the years after they have completed their role.Fig.\u00a01Conceptual model of the relationship between caregiving, \u2018moving on\u2019, specialized palliative care services (SPCS), and unmet needs\nThe null hypothesis was that SPCS were associated neither with differences in unmet needs nor with caregivers\u2019 ability to move on after bereavement.\nMethods\nSetting and subjects South Australia has a population of 1.47 million people [4]. The South Australian government provides funding for SPCS to support general practitioners and community nurses. The programs offered by SPCS in South Australia reflect service structures in the United Kingdom (UK), United States (US), and elsewhere [26, 28, 31, 44]. Services delivered include care for inpatients, hospital consultation, outpatient clinics, and community visits. Patients access SPCS through referral from any source. The primary eligibility criterion is a life-limiting illness; approximately 85% of people referred to SPCS have cancer.\nSurvey methodology The data for this survey were collected in the South Australian Health Omnibus Survey, a state government-associated face-to-face health survey conducted annually since 1991 with approximately 3,000 randomly selected respondents each year. The full survey methodology has been detailed elsewhere [16, 67]. Previously, the content and construct validity in palliative care service planning has been validated [16]. The survey has Ethics Committee (aka, Internal Review Board; IRB) approval.\nThe survey is revised and piloted annually with 50 people using the planned methods for the main survey (Fig.\u00a02). Anonymous face-to-face interviews were conducted by trained interviewers in the respondents\u2019 homes annually during September to November in 2001, 2002, and 2003. A total of 13,200 households across these 3\u00a0years were selected for the survey. Metropolitan households were selected in a skip pattern from a randomly selected starting point within 340 Australian Bureau of Statistics collectors\u2019 districts. In nonmetropolitan areas, households were selected using 100 starting points; all towns with a population greater than 10,000 were included, and towns with a population above 1,000 were randomly selected with probability proportional to size. One interview was conducted per household with the person aged 15 or older who most recently had a birthday. Data were double-punched; missing responses were followed up by telephone. Supervisors recontacted 5% of the study population to verify accuracy of the data.\nFig.\u00a02All palliative care survey questions embedded in the South Australian Health Omnibus Survey, 2001\u20132003\nCaregiving was defined for the respondent as follows: \u201c\u2018Care\u2019 includes attention to any of the needs of the person, including hands-on care, overnight care, respite, shopping, collection of medications, taking to appointments, emotional support, bathing, etc.\u201d To incorporate differing levels of caregiver burden into the analysis, respondents were asked if they provided: \u201cday-to-day hands-on care\u201d (care 5\u20137\u00a0days per week); \u201cintermittent hands-on care\u201d (care 2\u20134\u00a0days per week); or, \u201crare hands-on care\u201d (care 1 or less days per week). While this method does not encompass information on caregivers\u2019 multiple roles (e.g., parent, employee, spouse, and caregiver), it does stratify respondents along one axis of caregiver burden\u2014the intensity of care provided.\nIn the 2003 Health Omnibus Survey, a list of response categories was provided to patients to elicit their perceptions of specific unmet needs (Fig.\u00a02). \u201cUnmet needs\u201d were categorized into 14 types of extra support which the respondent felt would have helped him\/her as a caregiver; these were selected based on our clinical experience with patients and caregivers in palliative care, the published literature, and pilot testing of the Health Omnibus Survey [40]. These categories map to the five domains of caregiver experience described by Given et al. [24] in the following way: disrupted schedule (better out-of-hours care, respite care); financial problems (financial support\/financial planning, legal planning); lack of family support (emotional support for me, emotional support for the person who died, other emotional support, spiritual support, bereavement support); loss of physical strength (support with the physical care of the person who died); and caregiver self-esteem (emotional support for me). Additionally, the study response categories included informational and medical management needs (information about what would happen as the illness progressed, information about services available as the illness progressed, assistance with medications, assistance with physical care and control of symptoms).\nAmong the 50 pilot respondents in the 2003 survey year, those who answered the respondent-defined moving on question were subsequently asked, \u201cWhat does the concept of \u2018moving on\u2019 mean to you?\u201d Responses were incorporated into the categories for the survey (Fig.\u00a03).\nFig.\u00a03Bereaved caregivers\u2019 definitions of \u2018moving on\u2019 (n\u2009=\u200924)\nData analysis The survey respondents were standardized against the population of all South Australia for gender, 10-year age group, socioeconomic status, and region of residence per the 2001 Australian Census [4] using direct standardization [18] and macros combining multiple survey years obtained from the South Australian Department of Human Services [16, 67]. Each respondent was assigned a standardized weight and only weighted data were analyzed. Annual datasets were compared; there were no statistically significant differences, before combining the datasets, between the years in response rate, demographics, recency of death, deaths due to cancer, family\/friend relationship between the respondent and the deceased, or care provided.Descriptive statistics were used to summarize respondent characteristics and responses. Relationships between categorical variables were tested using the chi-square test or chi-square test for trend, as appropriate. Relationships between continuous variables were tested using the Student\u2019s t test or one-way analysis of variance (ANOVA), as appropriate. Two-tailed p values were reported; statistical significance was assumed if p\u2009<\u20090.05. After testing for multi-collinearity (all tolerances >0.9266), variables were entered into a multivariable logistic regression model (PROC LOGISTIC) to determine the factors accounting for categorical caregiver outcomes, or a linear regression model (PROC GLM) with Tukey corrections [10] to determine the factors accounting for continuous outcomes. The Statistical Analysis Software (SAS) statistical package was used for analysis (The SAS System, release 8.02, Cary, North Carolina, USA).\nSample size calculation A sample size requirement was calculated for the comparison of the mean number of unmet needs (\u201cextra supports needed\u201d) among caregivers where SPCS were used vs. SPCS not used. Assuming that a difference in one unmet need was clinically meaningful and a standard deviation of 2.1, then a sample size of 94 caregiver respondents was needed for an alpha 0.05, beta 0.80, and ratio of cases to controls of 0.6 [21]. A sample size requirement was also calculated for the dichotomous comparison of the proportion moving on by use of SPCS. If the baseline rate of moving on was 70% among those who did not use SPCS and an improvement of 10% with SPCS was deemed clinically meaningful (i.e., increased rate of moving on to 80% when SPCS was used), then a sample size of 385 caregiver respondents was needed for an alpha 0.05, beta 0.80, and ratio of cases to controls of 0.60 [21]. Both sample-size expectations were met.\nResults\nResponse rate Of the 13,200 households approached, the weighted number of respondents was 9,088. The response rate was 70.7% after exclusion of 401 uninhabited houses. Of those approached, reasons given for not responding included refusal (too busy or not interested; 2,006, 15.2%), unable to contact (1,059, 8.0%), respondent unable to speak English (195, 1.5%), selected respondent away for duration of survey (176, 1.3%), illness or mental incapacity precluded participation (173, 1.3%), and terminated interview (6, 0.05%).\nPopulation affected by a death About 37% of the population (3,340) indicated that someone close to them had \u201cdied of a terminal illness like cancer, motor neuron disease, or emphysema\u201d in the preceding 5\u00a0years (Table\u00a01). Respondents reporting a death of someone close to them were significantly more likely to be older, married, or widowed, in rural settings, working, and finished with school than those who did not report a death (all p\u2009\u2264\u20090.01).\nTable\u00a01Characteristics of respondents who reported a death of a loved one due to a terminal illness and bereaved respondents who identified themselves as caregiversCharacteristicAll respondents who reported death of a loved one due to terminal illnessRespondents who identified themselves as caregivers of the deceased individualsRespondents who were not caregivers of the deceased individualsP value between caregivers and noncaregivers of the deceased individualsaN (% of all respondents)3,340 (37)949 (10)2,391 (26)Age group (years)15\u201329692 (21)184 (19)507 (21)0.508730\u201344946 (28)274 (29)673 (28)45\u201359912 (27)274 (29)638 (27)60\u201374518 (15)147 (15)371 (15)75 and over273 (8)70 (7)203 (8)GenderMale1,558 (47)362 (38)1,196 (50)<0.0001Female1,782 (53)587 (62)1,196 (50)Marital statusMarried or de facto2,215 (66)603 (63)1,612 (67)<0.0001Separated or divorced273 (8)62 (7)210 (9)Widowed210 (6)111 (12)99 (4)Never married643 (19.173 (18)470 (20)Residential locationMetropolitan2,270 (68)645 (68)1,625 (68)0.9786Rural1,071 (32)304 (32)767 (32)Country of birthEnglish-speaking background2,992 (90)859 (90)2,133 (89)0.4447Non-English-speaking background348 (10)90 (9)258 (11)Missing1 (0)1 (0)Highest level of educational attainmentDid not complete school, high school or still studying1,660 (50)479 (50)1,182 (49)0.6010Trade qualification, certificate, diploma or higher1,680 (50)471 (50)1,210 (51)Work statusWork full-time or part-time1,852 (55)526 (55)1,326 (55)0.9910Not working including home duties, student, retired, unemployed and other1,488 (45)423 (44)1,066 (45)Values are numbers (percentages)aChi-square test or chi-square test for trend, as appropriate.\nCaregivers affected by a death Approximately 10% (949) of respondents (28.4% of people bereaved) identified themselves as caregivers (Table\u00a01) for someone who had died from a life-limiting illness in the past 5\u00a0years. When compared to all noncaregivers who reported death of a loved one, caregivers were significantly more likely to be female and widowed (Table\u00a01). About 905 (95.4%) caregivers knew if a SPCS was involved in the care of the deceased individual. Caregivers who knew about SPCS usage were more likely to be close family members (p\u2009<\u20090.0001; chi-square test for trend) but otherwise reported similar patient and caregiver profiles to that of those who \u201cdid not know\u201d if SPCS were involved. Subsequent analyses describe the 905 caregivers who knew about SPCS usage.\nAbout 321 of 905 (35.5%) individuals provided day-to-day hands-on care for 5 to 7\u00a0days per week; 344 (38.0%) intermittent hands-on care for 2 to 5\u00a0days per week; and 240 (26.5%) rare hands-on care for one or less days per week (Table\u00a02). On average, all caregivers provided care for 22.3 (SD 37.2) months, with day-to-day hands-on caregivers providing care for 24.3 (SD 39.9) months, intermittent caregivers for 23.2 (SD 38.9) months, and rare caregivers for 18.2 (SD 30.4) months (p\u2009=\u20090.518; one-way ANOVA).\nTable\u00a02Relationship between the caregiver and the decreased individual and characteristics of the care providedRelationship between the caregiver and the deceased individualCaregivers (n)a905Family relationshipSpouse\/partner92 (10)Child (of a parent who died)209 (23)Parent (of a child who died)61 (7)Sibling37 (4)Other relative303 (33)Friend175 (19)Other27 (3)Close relation (spouse\/parent\/child)362 (40)Distant relation or friend543 (60)Level of care providedDay-to-day hands-on care321 (35)Intermittent hands-on care344 (38)Rare hands-on care240 (26)Duration of care providedbMonths [mean (SD)]22.3 (37)Care for <1\u00a0year134 (45)Care for \u22651\u00a0year163 (55)Values are numbers (percentages) except as indicatedaReports are from caregivers who knew if a palliative care service was used (n\u2009=\u2009905, 95% of caregivers).bReports are from caregivers responding to the 2003 survey only (n\u2009=\u2009314).\nUse of SPCS Caregivers reported that SPCS provided support in at least 60% of deaths of terminally-ill people in South Australia (Table\u00a03) and more frequently in people who died of cancer than of noncancer illnesses (84.3 vs. 64.7%, p\u2009<\u20090.0001; chi-square test).\nTable\u00a03Characteristics of the deceased individuals and use of specialized palliative care services (SPCS)Characteristics of the deceased individualSPCS involved in the care of the deceased individualSPCS not involved in the care of the deceased individualAll deceased individuals cared for by caregiver respondents in this surveyP valueaDeceased individualsb539 (60)366 (40)905 (100)Recency of deathMonths [mean (SD)]25.3 (17)26.0 (18)25.6 (17.6)0.5578Cause of deathCancer454 (84)235 (64)689 (76)<0.0001Noncancer85 (16)128 (35)213 (24)Values are numbers (percentages) except as indicatedaFrom student t test or chi-square test, as appropriate.bReports are from caregivers who knew if a palliative care service was used (n\u2009=\u2009905, 95% of caregivers).\nUnmet needs of caregivers while delivering care and the use of SPCS The relationship between SPCS involvement and additional caregiver supports needed during illness through death was evaluated using the 2003 survey dataset. Approximately 154 (48.1%) caregivers identified that some type of extra support was needed\u201492 (28.8%) identified physical support needs, 52 (16.3%) information needs, 78 (24.4%) emotional needs, 29 (9.1) financial needs, and 26 (8.1%) other needs\u2014with a mean 1.3 (SD 2.1) extra supports identified per deceased individual. In a linear regression model evaluating the relationship between number of supports needed, level of caregiving, and involvement of a SPCS, involvement of the SPCS helped to predict unmet needs for hands-on caregivers (Fig.\u00a04). For those without SPCS involvement, the number of extra supports needed increased with the level of caregiving (t tests for means; noncaregivers vs rare hands-on caregivers, 0.52 vs. 0.81, p\u2009=\u20090.2433; noncaregivers vs intermittent caregivers, 0.52 vs. 1.56, p\u2009<\u20090.0001; noncaregivers vs day-to-day caregivers, 0.52 vs. 2.35, p\u2009<\u20090.0001); this trend was not noted when SPCS were involved, indicating that caregiver gain, in which unmet needs were addressed, occurred in the context of SPCS. Day-to-day caregivers indicated fewer extra support needs when SPCS were involved in the care of the deceased individual (t tests for means; no SPCS vs. SPCS, 0.95 vs. 2.3, p\u2009=\u20090.0031). The frequency of the top five extra support needs of day-to-day caregivers, by SPCS use or not, is presented in Table\u00a04.\nFig.\u00a04Relationship between the number of categories of additional supports needed during the period of the illness through death, level of care provided, and specialized palliative care service (SPCS) involvement (p\u2009=\u20090.0014 for the interaction of level of care and SPCS). Data from 2003 survey (total of 994 respondents and 314 caregiver respondents). Linear regression model with Tukey corrections (PROC GLM); numbers are least squares means of the number of extra support needs identified by respondents. Vertical bars represent 95% confidence intervalsTable\u00a04Five most frequently identified unmet needs of day-to-day hands-on caregivers (by percentage of respondents)Need forPercentage of respondents identifying this unmet needSPCS involved in the care of the deceased individual (n\u2009=\u2009120)More help with the physical care of the person22%More information about what to expect as the illness progressed13%Better emotional support for the caregiver13%Better emotional support for the person who had the life-limiting illness13%More information about services that could be accessed12%SPCS not involved in the care of the deceased individual (n\u2009=\u200988)Better emotional support for the caregiver30%Better emotional support for the person who had the life-limiting illness23%More information about services that could be accessed22%More help with the physical care of the person21%Better symptom control19%\nImpact of SPCS on long-term caregiver outcomes When SPCS were involved in care, caregivers were significantly more likely to be able to move on with their lives after the death (Table\u00a05; 86.4 vs 77.2%, p for trend\u2009=\u20090.0016). More than 80% of people who used SPCS were able to move on by 1\u00a0year, whereas it took 2\u00a0years for 80% of people who did not use SPCS to move on (Fig.\u00a05). About 3\u00a0years after the death, 10\u201314% of all caregivers still had not been able to move on.\nFig.\u00a05Relationship between time and the ability to \u201cmove on\u201d with life for those with and without involvement from a specialized palliative-care service (SPCS). Chi-square tests were used at each timepoint (likelihood of moving on by use of SPCS)Table\u00a05Impact of specialized palliative care services (SPCS) on caregiver outcomesOutcomeSPCS involved in the care of the deceased individualSPCS not involved in the care of the deceased individualP value for trendaN537364Ability to \u201cmove on\u201d with life\u00a0\u201cI have been able to move on with my life\u201d464 (86)281 (77)0.0016\u00a0\u201cI am starting to move on with my life\u201d60 (11)67 (18)\u00a0\u201cI have not been able to move on with my life\u201d13 (2)16 (4)Experience vs. expectations\u00a0Better than expected97 (18)55 (15)0.6310\u00a0As expected172 (32)121 (33)\u00a0Worse than expected240 (45)163 (45)\u00a0I didn\u2019t know what to expect27 (5)22 (6)Values are numbers (percentages)aChi-square test for trend.\nPredictors of caregivers\u2019 ability to move on In a multivariable logistic regression model, use of SPCS was positively associated with an ability for caregivers to move on [odds ratio (OR) 0.54, CI 0.36\u20130.80; Table\u00a06]. This translates to a 46% improvement on the ability to move on when a SPCS is involved. No other factors in the model positively influenced ability to move on.\nTable\u00a06Multivariable logistic regression model (PROC LOGISTIC) predicting caregivers unlikely to \u201cmove on\u201d with lifeVariablesOdds-ratios (95% confidence interval)Age (years)0.99 (0.98, 1.01)Gender (reference: female)Male0.87 (0.58, 1.32)Marital status (reference: married)Separated or divorced1.25 (0.58, 2.79)Widowed1.75 (0.93, 3.29)Never married1.25 (0.67, 2.32)Residential location (reference: metropolitan)Rural0.87 (0.57, 1.33)Country of birth (reference English-speaking background)Non-English-speaking background1.23 (0.65, 2.31)Highest level of educational attainment (reference: trade qualification, certificate, diploma, or higher)Didn\u2019t complete school or still studying0.96 (0.63, 1.45)Work status (reference: Work full or part time)Not working, home duties or other1.43 (0.90, 2.25)Recency of death (reference: death >2\u00a0years ago)Death in past 2\u00a0years2.58 (1.68, 3.97)Cause of death (reference: cancer)Noncancer0.77 (0.47, 1.25)Family relationship (reference: distant relation or friend)Close relation (spouse\/parent\/child)3.34 (2.13, 5.23)Level of care provided (reference: rare hands-on care)Day to day hands-on care3.72 (1.93, 7.16)Intermittent hands-on care2.42 (1.25, 4.69)Expectations vs experience (reference: Better than expected)As expected1.33 (0.63, 2.78)Worse than expected\/I didn\u2019t know what to expect3.43 (1.76, 6.68)Specialized palliative care service (SPCS) involvement (reference: No SPCS involvement)SPCS involved in the care of the deceased individual0.54 (0.36, 0.80)Unlikely to \u201cmove on\u201d with life is defined as \u201cI am starting to move on or I have not moved on with my life\u201d since the death of the individual\nPredictors of caregivers inability to move on In a multivariable logistic regression model, caregivers who were unlikely to have \u201cmoved on\u201d were significantly more likely to have: had a worse-than-expected experience between diagnosis and death (OR 3.43, CI 1.76\u20136.68; Table\u00a05); provided day-to-day or intermittent hands-on care (OR 3.72, CI 1.93\u20137.16, and OR 2.42, CI 1.25\u20134.69); been in a close family relationship with the deceased (OR 3.34, CI 2.13\u20135.23); and been bereaved in the preceding 2\u00a0years (OR 2.58, CI 1.68\u20133.97).\nDiscussion\nSPCS are a significant health-system investment. For 30\u00a0years, researchers have been working to define the improved outcomes associated with SPCS use [2, 12, 25, 30, 31, 45, 46, 48, 49, 61, 64]. The Health Omnibus data support that SPCS are associated with better meeting of needs for day-to-day hands-on caregivers while in the caregiving role and that SPCS have a subsequent long-term impact in improving a caregiver-defined outcome, moving on.\nFinding the whole caregiver population (rather than only those referred to services) has been the dominant challenge in establishing caregiver benefits from SPCS involvement [41]. By using a population-level method [16], this study avoided the bias introduced in studies which only access caregivers through clinicians, case note audits, or registries (clinical or death).\nImpact of SPCS on caregivers while in the role Our study builds on evidence that caregivers providing constant care who accessed SPCS had fewer unmet needs than did an otherwise identical population who did not access SPCS [31]. Considering the five domains of the caregiving experience described by Given et al. [24], family support was the main domain identified as a source of unmet need; the proportion of respondents who identified unmet needs in this domain decreased when SPCS were involved. An additional category of unmet needs important to the caregivers in this study pertained to information needs and help with physical and medical aspects of caring. The largest proportion of identified unmet needs reported were in this category; fewer needs in this category were reported by caregivers of deceased individuals who had SPCS involvement.\nImpact of SPCS on caregiver long-term outcomes SPCS need proactively to minimize the health risks associated with caregiving [23]. Other population approaches have shown associations of benefit with SPCS use in caregiver morbidity and mortality [63]. Christakis and Iwashyna [15] analyzed 31,000 spousal survivors of someone who died from 1 of 13 frequent causes of expected death vs. propensity-matched to controls from the same US Medicare data set. Mortality rates for the surviving spouses were compared 18\u00a0months after the death. There was decreased mortality in the group who used SPCS for 24,721 female caregivers (5.4 vs 4.9%, OR 0.92, CI 0.84\u20130.99) and 6,117 male caregivers (13.7 vs 13.2%, OR 0.95, CI 0.84\u20131.06). The Omnibus study adds to the work of Christakis by suggesting not only that caregivers who interact with SPCS might have an association with less post-role mortality, but that they may adjust more rapidly to their new life after having been a caregiver.\nLike Christakis\u2019 work, this current study cannot attribute a cause-and-effect relationship, although it can demonstrate strength of association. Those willing to access services may have had a better outcome because of problem-focused coping strategies [53].\nMcCorkle et al. [37] conducted a randomized controlled trial (RCT) evaluating a home nursing intervention to support palliative caregivers. Despite initial improvement in psychological morbidity of bereaved caregivers in the intervention group, differences that were clinically and statistically significant at 6 and 13\u00a0months were no longer apparent by 25\u00a0months. Zisook and Shuchter [69] demonstrated that caregivers\u2019 self-rated \u201cadjustment\u201d on a categorical scale showed progressive reductions in the first year. Such patterns are mirrored by our data.\nIn the McCorkle, Zisook, and Omnibus studies, there is a sizeable group of caregivers who still could not move on after several years. The fact that caregiver distress can last for so long after the death of the person for whom they have cared means that the measurement of service impact demands longitudinal approaches [32, 48, 60].\nAn RCT from Norway randomized access to SPCS by whole populations in a setting comparable to the current study [54]. The primary outcome for \u201cclose family members\u201d (whose level of caring was not clear) was the intensity of grief reactions as measured in the second part of the Texas Revised Inventory of Grief. There was no difference in grief reactions at 13\u00a0months in the 183 family members of the 434 patients who were originally enrolled in the study. By contrast, the higher level question, \u201cHave you been able to \u2018move on\u2019?\u201d in our study elicited a substantial difference over time between people who did and did not access SPCS. Although moving on constitutes only part of the definition for complicated grief [51] (or prolonged grief reaction), the measured rates of complicated grief in similar populations in the literature appear to be of the same order of magnitude, and at the same time after the death of the care recipient, as the findings from the Omnibus study [11, 39, 52].\nOther predictors of caregiver outcomes Our study supports other broad population-based observations that the intensity of the level of care provided directly correlates with longer-term caregiver outcomes including burden, health, and mortality [57, 59]. The level of caregiving helped to predict both unmet needs and the ability to move on.\nOne of the strongest predictors of an inability to move on was if the experience from diagnosis to death was \u201cworse than expected;\u201d this finding is consistent with other work in the area [8, 29]. The discrepancy between expectations and what actually happens can amplify feelings of lack of control over a situation. Conversely, a perceived sense of control is known to be related to overall wellbeing [36], and may contribute to an ability to move on.\nLimitations to the study Analysis was limited to the information collected by the South Australian Health Omnibus Survey, a retrospective population-based approach which is dependent upon recollections of bereaved respondents. Methodologically, a prospective study would have been preferable in order to establish potential causal relationships between SPCS and short- and long-term caregiver outcomes; however, it would not be practically feasible to conduct such a prospective controlled research study among caregivers at a population level.\nBecause the area of caregiving is a relative newcomer to the realm of clinical research, a paucity of robust assessment measures is, as yet, a major limitation of studies in this population. In assessment of short-term caregiver outcomes, we developed a variable to indicate level of intensity of caregiving, which we could link to caregiver strain. We could not encompass in this variable other factors not provided by the 2001\u20132003 Omnibus dataset, such as roles that caregivers might have been simultaneously performing.Thus, within our measure of caregiver strain defined by intensity of caregiving, we were unable to differentiate those caregivers who performed multiple roles from those who were solely caregivers.\nWith respect to long-term caregiver outcomes, as noted above, a validated instrument to measure moving on has not yet been developed; in the absence of such a measure, we acknowledge the respondent-defined nature of the concept as utilized in this study. As a first step toward defining this construct, we asked the 50 respondents for the 2003 annual Health Omnibus Survey pilot for input regarding what the concept of moving on meant to them. Results appear in Fig.\u00a03 and provide some initial parameters for development of a measure of this construct.\nPeople who live in remote South Australia and those without caregivers were not represented, and people from some cultural backgrounds may not be seen in these data. All results were based upon the recall of the respondent, which is a validated approach [35, 38, 62]. Caregiver-derived information about the uptake of SPCS is directly derived from first-hand knowledge. Other limitations of the approach have been outlined previously [16].\nThe programs offered through SPCS and their funding varies widely across the world, making global assessments of the impact of SPCS difficult [3, 15, 42]. This study reflects a variety of palliative care service types ranging from single-nurse-led rural services to large regional metropolitan interdisciplinary programs. These results can be generalized to similar health settings internationally.\nFuture directions\nThe relationship between fewer unmet needs and SPCS suggests that SPCS do provide substantial support in helping to plan care and identify contingencies in future care. An understanding of the specific attributes of SPCS that make the most difference in meeting caregivers\u2019 needs will be pursued through future work using similar methods.\nMoving on was defined by respondents of a single year of the Health Omnibus Survey; broader validation is planned. Subsequent information will enable evaluation of whether moving on, as a measurable outcome, can be improved. An interesting line of inquiry, which would be made possible by inclusion of a baseline question in a future Health Omnibus Survey, is whether respondents who accessed SPCS were further along in the trajectory of psychological acceptance than were respondents who had not accessed SPCS. A positive correlation might indicate a self-selection bias in which respondents who had used SPCS and who had succeeded in moving on had a predisposition to move on, in comparison to respondents who had not chosen to access SPCS. While of academic interest, this information would not impact the utility of the finding that SPCS facilitated moving on among those individuals who accessed services.\nThe methodological strength of the approach used in this study, in which we probed data collected via the Health Omnibus Survey to answer a health services question on the population level, could be improved by favorable comparison of data from the Omnibus on caregiver assessment to data collected using a validated caregiver assessment instrument. This step represents a possible future avenue of study but is not yet possible due to the lack of such an instrument.\nThe relationship between caregivers\u2019 expectations (diagnosis through death) and outcomes will be explored with two subsequent years of data. All results need to be confirmed in other health-delivery systems.","keyphrases":["palliative care","caregivers","outcome assessment (health care)","health services needs and demands"],"prmu":["P","P","R","R"]}
{"id":"Calcif_Tissue_Int-4-1-2268729","title":"Bone Degeneration and Recovery after Early and Late Bisphosphonate Treatment of Ovariectomized Wistar Rats Assessed by In Vivo Micro-Computed Tomography\n","text":"Bisphosphonates are antiresorptive drugs commonly used to treat osteoporosis. It is not clear, however, what the influence of the time point of treatment is. Recently developed in vivo micro-computed tomographic (CT) scanners offer the possibility to study such effects on bone microstructure in rats. The aim of this study was to determine the influence of early and late zoledronic acid treatment on bone in ovariectomized rats, using in vivo micro-CT. Twenty-nine female Wistar rats were divided into the following groups: ovariectomy (OVX, n = 5), OVX and zoledronic acid (ZOL) at week 0 (n = 8), OVX and ZOL at week 8 (n = 7), and sham (n = 9). CT scans were made of the proximal tibia at weeks 0, 2, 4, 8, 12, and 16; and bone structural parameters were determined in the metaphysis. Two fluorescent labels were administered to calculate dynamic histomorphometric parameters. At week 16, all groups were significantly different from each other in bone volume fraction (BV\/TV), connectivity density, and trabecular number (Tb.N), except for the early ZOL and control groups which were not significantly different for any structural parameter. After ZOL treatment at week 8, BV\/TV, structure model index, Tb.N, and trabecular thickness significantly improved in the late ZOL group. The OVX and ZOL groups showed, respectively, higher and lower bone formation rates than the control group. Early ZOL treatment inhibited all bone microstructural changes seen after OVX. Late ZOL treatment significantly improved bone microstructure, although the structure did not recover to original levels. Early ZOL treatment resulted in a significantly better microstructure than late treatment. However, late treatment was still significantly better than no treatment.\nPostmenopausal osteoporosis affects millions of women worldwide and results in loss of bone mass and bone microstructural changes, which lead to reduced bone strength. It remains unknown how exactly the microstructure is affected over the time course of osteoporosis development and to what extent this structure can be recovered by drug treatment. Since the assessment of bone structure in humans is still limited to a few peripheral sites, the ovariectomized rat is used in many studies as an animal model for osteoporosis in cross-sectional evaluations. Numerous cross-sectional studies have determined the loss of trabecular bone in the proximal tibia in rats following ovariectomy (OVX) at different time points by measuring bone structural parameters [1\u20133]. OVX has been shown to decrease bone volume fraction, connectivity, and trabecular number and to increase trabecular separation and structure model index, which indicates that trabecular bone changes from plate-like to more rod-like in the proximal tibial metaphysis of the female rat. However, conflicting results were found for the effects of OVX on trabecular thickness, showing increases, decreases, or no response at all [4\u20136]. These cross-sectional studies are limited to comparing averages per group only, which provides a reduced sensitivity for detection of differences. In addition, no information is obtained regarding the specific locations of bone changes.\nRecently, however, in\u00a0vivo micro-computed tomographic (CT) scanners became available, which can monitor the process of bone loss and microstructural changes in small, living animals. So far, two studies have been reported in which this equipment was used to monitor changes in rat bones after OVX. Interestingly, these studies reported, respectively, significant increases [5] and decreases [7] in trabecular thickness after OVX, indicating that the response may vary. While the process of bone degeneration after OVX is not fully understood, even less information is available for the process of recovery after drug treatment. The most widely used drugs for treatment of diseases associated with increased bone resorption, such as postmenopausal osteoporosis, are now bisphoshonates.\nMany cross-sectional studies have determined the effects of bisphosphonates in rats either as a preventive treatment, before development of OVX-induced bone loss, or as a recovering treatment after development of bone loss. Although the results are difficult to compare directly due to differences in experimental design, preventive treatment in animals may lead to the same amount of final bone mass as recovering treatment. The associated microstructure in terms of number and thickness of trabeculae, however, can be different. While the influence of the time point of treatment is highly relevant clinically, no studies were found that directly compared the effects of a preventive and a recovering treatment on final bone mass and associated microstructure. In addition, the fact that only results of cross-sectional studies have been reported complicates comparison of the final and original bone structure and provides little or no information on the changes in structure over time. Here also we expect that longitudinal in\u00a0vivo micro-CT imaging will potentially provide more information about the specific changes in the bone, both spatially and temporally.\nFor the present study, we had two goals: (1) to determine the process of bone loss and bone microstructural changes in ovariectomized rats and (2) to determine the effects of early and late treatment of osteoporotic rats with a bisphosphonate. We used an in\u00a0vivo high-resolution micro-CT scanner to image the proximal tibia of female adult Wistar rats. For the treatment, zoledronic acid (ZOL) was used, since this is a third-generation bisphosphonate that proved a potent osteoclast inhibitor. It has shown to be effective in cancer patients with bone metastases and is currently being tested in clinical trials as a promising therapeutic agent for osteoporotic patients [8, 9].\nMethods\nAnimals\nThirty-six female, 30-week-old, retired breeding Wistar rats were obtained from Harlan Laboratories (Horst, The Netherlands) and allowed to acclimate for 7 days before the start of the experiment. The rats were maintained with a cycle of 12\u00a0hours light and 12\u00a0hours darkness and allowed to eat and drink ad libitum. The experiment was approved by the Animals Ethics Committee of the University of Maastricht, The Netherlands. The rats were divided into four groups based on weight: control (n\u00a0=\u00a09), OVX (n\u00a0=\u00a09), OVX and early ZOL (n\u00a0=\u00a09), OVX and late ZOL (n\u00a0=\u00a09). All rats were ovariectomized at week 0, and the control group underwent sham OVX. Success of OVX was confirmed at necropsy by determining atrophy of the uterine horns. ZOL (kindly donated by Novartis Pharmaceutical, Basel, Switzerland) was dissolved in a saline vehicle. The early ZOL group was administered ZOL at a single dose of 20\u00a0\u03bcg\/kg body weight s.c. at OVX (comparable to the phase III clinical trial dose used in osteoporotic patients). The late ZOL group was administered the same single dose 8\u00a0weeks after OVX. Eleven and 4\u00a0days before necropsy, the fluorescent labels calcein (Fluka, Buchs, Switzerland) and alizarin (Merck, Dietikon, Switzerland) were administered s.c. in a saline vehicle to all rats at doses of 30\u00a0mg\/kg and 20\u00a0mg\/kg body weight, respectively, to evaluate bone formation dynamics in the proximal tibial metaphysis. Rats were killed at 16\u00a0weeks by exsanguination.\nMicro-CT Scanning\nDirectly after the operation, a 6-mm micro-CT scan (70\u00a0kV, 85\u00a0\u03bcA, 1,000 projections per 180 degrees, 350\u00a0ms integration time) with an isotropic resolution of 15\u00a0\u03bcm was made of the proximal tibia using an in\u00a0vivo micro-CT scanner (vivaCT 40; Scanco Medical, Bruettisellen, Switzerland). The CT scanner was calibrated, and a beam-hardening correction algorithm was applied to all scans. Follow-up in\u00a0vivo CT scans were made after 2, 4, 8, 12, and 16\u00a0weeks to monitor bone structure. One CT scan took 35\u00a0minutes, during which the animal was anesthetized with isoflurane and the scanned leg was placed in a custom-made leg-fixating device. The design of the rat holder was such that the left leg was not exposed to radiation while scanning the right leg. Radiation damage to the scanned bone was not expected to occur, based on a previous study in which 8-weekly CT scans with the same radiation dose caused no detected bone damage [10]. In that study, we also showed that the reproducibility of all structural parameters was high, with a coefficient of variation of about 1%.\nImage processing included gaussian filtering and segmentation and was described elsewhere in detail [10]. In brief, the same filtering and segmentation values were used for every measurement of each animal (sigma\u00a0=\u00a00.7, support\u00a0=\u00a01, threshold density\u00a0=\u00a00.504\u00a0g hydroxyapatite\/cc, equivalent to 22% of maximal gray-scale value). From every baseline and follow-up CT scan, the metaphyseal trabecular bone was manually selected and bone structural parameters (bone volume fraction [BV\/TV], connectivity density [Conn.D], structure model index [SMI], trabecular number [Tb.N], trabecular thickness [Tb.Th], and trabecular separation [Tb.Sp]) were automatically determined. The average attenuation coefficient of the trabecular bone tissue was determined for all measurements using a protocol provided by the manufacturer of the micro-CT scanner. With this protocol, the gray levels of voxels near the trabecular surfaces are not included, to ensure that the measurements are not affected by partial volume effects. Also, cortical thickness of the metaphysis was analyzed. The cortical bone starting from the tibia\u2013fibula junction was manually selected for each measurement, resulting in a region approximately 1.8\u00a0mm long. The same filtering and segmentation values were used as for the trabecular analysis. Cortical thickness was automatically determined from the selected region.\nWhen two CT scans of the same animal are made at different time points, the position of the animal in the scanner will not be exactly the same and, therefore, the CT scans will also differ. In order to detect bone structural changes on a micro level, one CT scan needs to be translated and rotated to match the other. We developed image registration software that registers two scans based on minimizing the correlation coefficient [11]. Every follow-up scan is registered with the first scan, thereby revealing any possible changes in the microstructure and minimizing errors in the region of interest for the structural parameters.\nMechanical Testing\nAfter sacrifice, all left tibiae were dissected and used for two different mechanical tests. First, a standard three-point bending test was applied. The tibiae were placed on the lateral surface on two rounded supporting bars with a distance of 2.4\u00a0mm. A preload of 1\u00a0N was applied (Z020; Zwick, Ulm, Germany) at the medial surface of the diaphysis by lowering a third rounded bar. A constant displacement rate of 6\u00a0mm\/minute was applied until failure. Displacement was measured from the actuator displacement transducer of the testing machine. Stiffness, ultimate force, and ultimate displacement were calculated for each sample.\nTo assess the mechanical properties of the proximal tibia, a proximal tibia compression test was performed similar to the method of Hogan et al. [12]. A 4-mm slice of the proximal metaphysis including both cortical and trabecular bone was sawed below the growth plate using a diamond saw (Accutom-5; Struers, Ballerup, Denmark). This slice was axially compressed (Z020) during five preconditioning cycles between 1 and 70\u00a0N. After the last preconditioning cycle, axial compressive load was applied at 0.5\u00a0mm\/minute until failure, while axial displacement was measured using an extensometer. Stiffness, ultimate force, and ultimate displacement were determined for all samples.\nHistomorphometry\nAt necropsy, all right tibiae were fixed in 70% ethanol and embedded in methylmethacrylate (Technovit 9100; Heraeus Kulzer, Wehrheim, Germany). Thin sections (5\u00a0\u03bcm) were cut to determine fluorochrome-based dynamic histomorphometric parameters of bone formation using an Axiophot photomicroscope (Zeiss, Oberkochen, Germany) linked to a camera (CF 15\/4 MC; Kappa, Gleichen, Germany), and a QUANTIMET 600 image analysis system was used to calculate the amount of mineralized surface per bone surface (MS\/BS, percentage), corrected mineral apposition rate (MAR) (\u03bcm\/day), and bone formation rate (BFR) per bone surface (\u03bcm\/day). Calculations of the dynamic parameters were performed as recommended previously [13].\nStatistics\nSince this experiment concerned repeated measures in multiple groups, several statistical tests were performed to reveal changes in parameter values over time and between groups. For all structural parameters, the percentage change at different time intervals was calculated for each animal individually. A one-way analyses of variance (ANOVA) with repeated measures was performed on the percentage change for all structural parameters and all groups. Furthermore, a one-way ANOVA with a Bonferroni post-hoc test was used to determine differences between the groups at all time points for each structural parameter. Also, a one-way ANOVA with a Bonferroni post-hoc test was used to determine differences between the groups in cortical thickness, histomorphometric results, and mechanical results. For each group, a paired Student\u2019s t-test was performed on the absolute values of the structural parameters of each time point compared to baseline values, to determine the earliest point at which a detectable change was found. A paired Student\u2019s t-test was used to determine late ZOL treatment effects within this group. Finally, a paired Student\u2019s t-test was performed on the absolute values between each measurement for each group and structural parameter. P\u00a0<\u00a00.05 was considered significant.\nResults\nOvariectomy\nBased on the uterine weight, we determined seven unsuccessful OVXs. The rats concerned were removed from further analyses, which left nine rats in the control group, five in the OVX group, seven in the OVX and late ZOL group, and eight in the OVX and early ZOL group. For each animal, we determined the percentage change in structural parameters compared to the values at week 0. Figure\u00a01 shows the average percentage change and upper standard deviation for all groups for BV\/TV, Conn.D, SMI, Tb.N, Tb.Th, and Tb.Sp. The OVX group without treatment showed large changes in structural parameters, indicating the development of OVX-induced bone loss (Figs.\u00a01 and 2). Paired Student\u2019s t-test indicated significant changes within 2\u00a0weeks after OVX for all structural parameters. BV\/TV, Conn.D, and Tb.N decreased during the experiment and SMI and Tb.Sp increased. Both Tb.Th and Tb.N decreased within 2\u00a0weeks, indicating that both thinning and complete resorption of trabeculae started directly after OVX. The initial rapid loss of bone and connectivity was largely accompanied by trabecular thinning, while secondary, slower loss of bone was concomitant with a decrease in Tb.N. While BV\/TV, Conn.D, SMI, Tb.N, and Tb.Sp showed continuous changes in the same direction throughout the experiment, Tb.Th initially decreased significantly until 4\u00a0weeks after OVX and then increased again until after 16\u00a0weeks Tb.Th was significantly higher than in the control group.\nFig.\u00a01Average percentage change in structural parameters in the metaphyseal proximal tibia and upper standard deviation for all groups at all time points. Brackets indicate P\u00a0<\u00a00.05 at week 16Fig.\u00a02Same slice of an unprocessed CT scan of the same rat in the OVX group taken at weeks 0 (a), 2 (b), 4 (c), 8 (d), 12 (e), and 16 (f). Images show typical trabecular bone loss due to OVX in the metaphysis. Green line shows the analyzed metaphyseal bone\nThe absolute average values and standard deviations for all structural parameters of all measurements are shown in Table\u00a01. At the starting point, some differences in the values between the groups were present. This was due to the fact that some animals, which mostly had a relatively low BV\/TV, were removed from the study because they did not respond to the OVX. However, since we compared the relative changes in each animal, the results were most likely not affected by the difference in absolute values.\nTable\u00a01Mean values and standard deviation of all structural parameters of all groups at the start of the experiment GroupBV\/TV (1) Conn.D (1\/mm)SMI (1)Tb.N (1\/mm) Tb.Th (\u03bcm) Tb.Sp (\u03bcm) Control0.17 (0.056)41 (47)1.52 (0.53)1.89 (1.38)105 (49)613 (368)OVX0.26 (0.055)141 (76)1.22 (0.39)3.77 (1.66)84 (10)335 (174)OVX\u00a0+\u00a0late ZOL0.23 (0.062)86 (66)1.23 (0.34)2.77 (1.55)89 (13)482 (227)OVX\u00a0+\u00a0early ZOL0.17 (0.056)38 (21)1.70 (0.39)1.86 (0.80)95 (7)647 (207)\nZOL Treatment\nThe early ZOL treatment and control groups were not significantly different at all time points for all structural parameters.\nUp until 8\u00a0weeks after OVX, when ZOL was administered to rats in the late treatment group, the OVX group and the late treatment group were not significantly different for all structural parameters. BV\/TV, SMI, Tb.Th, and Tb.N were significantly improved in the late ZOL group 4\u00a0weeks after the single ZOL injection (Table\u00a02). In addition, Tb.Sp significantly improved within 8\u00a0weeks after injection, while Conn.D showed no significant response. Between weeks 12 and 16, all structural parameters stayed constant.\nTable\u00a02P values of paired Student\u2019s t-test in the late ZOL group after ZOL treatment Structural parameterP for weeks 8\u201312P for weeks 8\u201316BV\/TV0.001*0.004*Conn.D0.2490.181SMI0.003*0.004*Tb.N0.041*0.005*Tb.Th0.000*0.000*Tb.Sp0.3440.008**Significant (P\u00a0<\u00a00.05)\nAt week 16, BV\/TV, Conn.D, and Tb.N were significantly different between all groups except for the early treatment and control groups (Table\u00a03). Tb.Th was only significantly different between the control and OVX groups. Although substantial differences in Tb.Sp and SMI were seen between all groups, except for the early treatment and control groups, significant differences were only found between the OVX and control groups and between the OVX and early treatment groups.\nTable\u00a03P values of ANOVA plus Bonferroni post-hoc test comparing all groups at all time points for all structural parametersGroup \u0394BV\/TV\u0394Conn.D\u0394SMI2481216248121624812161 vs. 20.010*0.000*0.000*0.000*0.000*0.025*0.001*0.000*0.000*0.000*0.027*0.008*0.004*0.001*0.003*1 vs. 30.005*0.000*0.000*0.000*0.000*0.3410.021*0.001*0.000*0.005*0.9290.4730.1410.3050.241 vs. 40.9990.9990.9990.9990.3220.9990.9990.9990.9990.9990.9990.9990.9990.9990.9992 vs. 30.9990.9990.5790.000*0.000*0.9990.9060.8110.042*0.027*0.5980.4430.6670.1480.372 vs. 40.000*0.000*0.000*0.000*0.000*0.0790.001*0.000*0.000*0.000*0.011*0.005*0.003*0.001*0.003*3 vs. 40.000*0.000*0.000*0.000*0.000*0.90.023*0.001*0.000*0.000*0.4210.290.1040.3310.206Group \u0394Tb.N\u0394Tb.Th\u0394Tb.Sp2481216248121624812161 vs. 20.0880.001*0.000*0.000*0.000*0.003*0.002*0.6730.9990.013*0.210.006*0.001*0.000*0.000*1 vs. 30.1080.019*0.005*0.007*0.018*0.008*0.002*0.3250.9990.9990.0990.0720.20.8020.9991 vs. 40.9990.9990.9990.9990.9990.9990.9990.9990.9990.9990.9990.9990.9990.9990.9992 vs. 30.9990.9990.3530.001*0.000*0.9990.9990.9990.9990.1520.9990.9990.1280.001*0.000*2 vs. 40.450.001*0.000*0.000*0.000*0.001*0.001*0.2650.9990.06*0.4920.007*0.001*0.000*0.000*3 vs. 40.6230.027*0.004*0.003*0.002*0.003*0.001*0.1090.9990.9990.2790.0840.1870.6670.999Groups: 1, control; 2, OVX; 3, late ZOL; 4, early ZOL*Significant (P\u00a0<\u00a00.05) \nCT-derived mineralization values in all groups did not change over time and did not differ between groups at all time points.\nAging\nAn ANOVA with repeated measures on all structural parameters of the control group was performed to determine if there was a significant effect of aging. BV\/TV and Tb.N were found to significantly decrease and Tb.Sp was found to significantly increase due to aging. All other structural parameters did not change significantly in 16\u00a0weeks.\nAn ANOVA with repeated measures was performed on the percentage change in structural parameters comparing the control and early treatment groups to determine any significant differences. BV\/TV and Tb.N showed a significant effect of age, treatment, and age * treatment and Tb.Sp showed a significant effect of age and age * treatment. While BV\/TV decreased significantly over time in both the early treatment and control groups, it was found to decrease at a higher rate in the latter. Tb.N significantly decreased over time in the control group; however, it did not significantly change in the early treatment group as a net result of OVX, aging, and ZOL treatment. Tb.Sp significantly increased in the control group due to aging, while it did not in the early treatment group.\nCortical Thickness\nOVX initially led to a decrease in metaphyseal cortical thickness, and at 4\u00a0weeks both the OVX and the late ZOL groups were significantly lower than the early ZOL group (Fig.\u00a03). However, after 4\u00a0weeks it increased again in the OVX group, and at 16\u00a0weeks cortical thickness was significantly higher than in the control group. Early ZOL treatment inhibited the changes in cortical thickness. The late ZOL group, just like the OVX group, showed an increase in cortical thickness between 4\u00a0and 16\u00a0weeks, although this was less pronounced.\nFig.\u00a03Average percentage change in cortical thickness in the metaphyseal proximal tibia and upper standard deviation for all groups at all time points. At week 4 the OVX and the late ZOL groups were significantly lower than the early ZOL group, and at week 16 the OVX group was significantly higher than the control group\nMechanical Testing\nExperimental test results were obtained for seven (control), five (OVX), six (OVX and late ZOL), and seven (OVX and early ZOL) tibiae. No significant differences were found between all parameters determined in the three-point bending test (Fig.\u00a04a).\nFig.\u00a04Stiffness, ultimate force, and ultimate displacement determined from three-point bending tests on diaphyseal tibia (a) and axial compression tests (b) on metaphyseal bone. Groups: 1, control; 2, OVX; 3, late ZOL; 4, early ZOL. *Significant difference between groups based on ANOVA and Bonferroni post-hoc test (P\u00a0<\u00a00.05)\nFor the results from the proximal tibia compression test, a significant difference in ultimate displacement was found between the OVX group and the early ZOL group and between the OVX and the control groups (Fig.\u00a04b). Although not significantly determined, a trend was observed in which OVX resulted in a lower stiffness and ultimate force, while late ZOL treatment partially recovered these values and early ZOL treatment did to a larger extent.\nHistomorphometry\nHistomorphometric results were obtained for five (control), five (OVX), six (OVX and late ZOL), and five (OVX and early ZOL) rats. The OVX group showed a significantly higher mineralizing surface (MS\/BS) and BFR and a substantially higher mineral apposition rate (MAR) than the control group (Fig.\u00a05). Both ZOL groups showed a significantly lower MS\/BS and BFR than the OVX group and control group, while MAR was significantly lower than only the OVX group. Additionally, the late ZOL group had a significantly lower MS\/BS and a nonsignificantly lower BFR than the early ZOL group.\nFig.\u00a05Dynamic bone histomorphometric parameters for all groups in the metaphyseal proximal tibia. Groups: 1, control; 2, OVX; 3, late ZOL; 4, early ZOL. Based on ANOVA and a Bonferroni test, asignificantly different from group 1, bsignificantly different from group 2, csignificantly different from group 3, and dsignificantly different from group 4 (P\u00a0<\u00a00.05)\nDiscussion\nIn this study we assessed the effects of OVX and of early and late ZOL treatments after OVX on the microstructure of metaphyseal bone in the proximal tibia of aged, female Wistar rats over a 16-week time course, using in\u00a0vivo high-resolution micro-CT.\nOVX induced significant changes in all structural parameters within 2\u00a0weeks, indicating the rapid development of OVX-induced bone loss. An initial phase of rapid decreases in BV\/TV and Conn.D was seen, followed by a second phase in which changes took place at lower rates. The initial rapid loss of bone and connectivity was largely caused by trabecular thinning, while the secondary, slower loss of bone was accompanied by a decrease in Tb.N. OVX induced large decreases in BV\/TV, Conn.D, and Tb.N and large increases in Tb.Sp and SMI, indicating the change of more plate- to rod-like bone, which agrees with the literature on the effects of OVX in rat tibiae [2, 14\u201316]. A different response was, however, seen in Tb.Th, which initially decreased until 4\u00a0weeks after OVX and then increased until at the end point of the experiment it was higher than in the control group. A possible explanation for this response could be that, due to increased osteoclast activity, initially most trabeculae will become thinner, while only few are completely resorbed. After a certain period of resorption, the thinner trabeculae will be perforated and completely resorbed. Since the thicker trabeculae remain present, the average Tb.Th will increase. Another possible explanation would be that mechanical adaptation takes place. Since much bone is lost after OVX and the number and thickness of trabeculae decrease, all load bearing goes through the remaining trabeculae. Therefore, the stress increases in these trabeculae, which stimulates bone formation and would lead to increased Tb.Th. It is also possible that a combination of these mechanisms actually takes place. In the two studies published to date regarding in\u00a0vivo micro-CT monitoring of the effects of OVX on the metaphyseal proximal tibia in rats, the loss of trabecular bone was similar to what was seen here [5, 7]. However, Tb.Th significantly increased in one study and decreased in the other directly after OVX. Although the same rat strain was used and the same anatomical location was studied, it could be that differences, such as the use of retired breeders vs. virgin rats, age, or type of data analysis, led to variable results.\nEarly ZOL injection at the time of OVX completely prevented all changes in structural parameters and, thus, the development of OVX-induced bone loss. Aging caused a significant decrease in BV\/TV, Conn.D, and Tb.N and an increase in Tb.Sp in the control group, which agrees with the literature [7]. However, a significant decrease was merely seen in BV\/TV in the early ZOL group and at a significantly lower rate than in the control group. This indicates that a single injection of ZOL was powerful enough to inhibit even natural deterioration of bone volume and microstructure due to aging. One year of weekly ZOL injections of a concentration as low as 0.3\u00a0\u03bcg\/kg completely suppressed the effects of OVX in vertebrae of Sprague-Dawley rats, and 1.5\u00a0\u03bcg\/kg was needed to retain all structural parameters and mechanical properties in the femurs [8, 17]. Similarly, 69\u00a0weeks of weekly ZOL injections in OVX rhesus monkeys reduced bone loss in a dose-dependent manner [18]. In a phase II study, both a yearly and a quarterly ZOL dose increased spinal and femoral bone mineral density in postmenopausal women [19]. In the current study, we have shown the long-term, potent inhibiting effects of ZOL on both BV\/TV and the microstructure in rats.\nWhile the preventive effects of ZOL have previously been studied in rats [8, 17], the effect of ZOL treatment after the development of OVX-induced bone loss on the microstructure, which more closely resembles the clinical situation, is new. Late ZOL injection after 8\u00a0weeks of bone loss development inhibited further bone loss. Possibly as a result of the filling of resorption cavities [20], a temporary but significant increase in BV\/TV and thickness and a significant decrease in SMI were seen directly after ZOL administration. Four weeks later, however, all structural parameters stayed constant. This is in contrast with the OVX group, which still showed deterioration of bone structure after 8\u00a0weeks. Overall, bisphosphonate treatment after the onset of osteoporosis is known to stabilize or increase BV\/TV and Tb.Th in both humans and other animals, which agrees with the data presented here [21\u201323]. It is generally thought that the underlying mechanism for an increase in BV\/TV and Tb.Th is the inhibiting effect of bisphosphonates on bone resorption preventing further thinning of trabeculae and the reduction of remodeling space by filling of resorption cavities, leading to increased bone mass [20, 24].\nCortical thickness first decreased as a result of OVX, later increased again, and finally was higher than that of the control group. Both increases and decreases in metaphyseal tibial cortical thickness have been reported in cross-sectional studies, which may be due to different analytic methods or different time points of sacrifice [25, 26]. Early ZOL treatment resulted in a similar cortical thickness as seen in the control groups, which was fairly constant. Late ZOL treatment resulted in a similar pattern as in the OVX group, although less pronounced, which may be explained by the inhibiting effect of ZOL on bone remodeling rate. Cortical thickness profiles in all groups were similar to those for Tb.Th, which may be interpreted as an indication that mechanical adaptation takes place in both cortical and trabecular bone after sustained bone loss.\nNo significant differences were found in mechanical properties determined in the three-point bending test between the OVX and control groups, which agrees with the literature [27, 28]. Also, no significant influence of ZOL treatment was found on the mechanical properties determined in the three-point bending test, which may be expected as ZOL inhibits bone resorption and does not increase formation. In the proximal tibia compression test, ultimate displacement was found to be higher in the OVX group than in the other groups. ZOL treatment partially recovered this increase in ultimate displacement. Although not significantly, ultimate force and stiffness tended to be lower in the OVX group, while ZOL treatment inhibited this. The low number of animals per group and variability in the data unfortunately limit the interpretation of these results. It is known that while endosteal bone resorption takes place during aging and is increased as a result of OVX, periosteal apposition increases as well [29\u201334], which was also seen in our CT scans (data not shown), thereby compensating for the loss of endocortical and trabecular bone [35]. Therefore, loss of trabecular and endocortical bone may not necessarily mean loss of strength. While first-generation bisphosphonates, such as etidronate, have been shown to directly decrease mineralization of bone [9, 36], it is thought that ZOL may increase mineralization by inhibiting resorption and thereby allowing longer mineralization [37, 38]. Furthermore, microdamage could accumulate, and both of these effects could alter the mechanical properties of the bone tissue. Nonetheless, the trends in the mechanical properties resembled the trends in BV\/TV and showed that ZOL inhibited the changes resulting from OVX.\nBone-forming activity was slightly higher in the OVX compared to the control group, reflecting an increased turnover rate. Both ZOL groups displayed lower bone-forming parameters compared to the control and OVX groups, probably due to suppression of bone formation and resorption. Additionally, the late ZOL group tended to show slightly less bone-forming activity than the early ZOL group, possibly due to the later time point of treatment. These results agree with previous reports of decreased osteoblast and osteoclast numbers after ZOL injection and decreased number of resorption lacunae [20, 39]. CT-derived estimates of mineralization of metaphyseal trabecular bone were, however, not significantly increased in ZOL-treated rats. This would indicate that ZOL treatment did not influence mineralization in this time frame.\nAs previously mentioned, at times, substantial differences were found between groups; however, due to a relatively high standard deviation, no significant differences unfortunately were detected. This high standard deviation was mostly seen in the OVX and late treatment group. This was most likely caused by the fact that retired breeders were used, which are known to show a variable response to OVX. Due to an unsuccessful OVX, seven animals were removed from the study. This decreased the number of animals used in the data analysis and, since these were mostly animals with a relatively low BV\/TV, the average BV\/TV at the start of the study was higher in the OVX and late treatment groups. Therefore, we performed statistical tests on percentage changes in structural parameters rather than on the absolute values. The trend for the percentage and absolute changes looked similar and the P values were similar for most cases. However, when ANOVA was performed on the absolute values comparing all groups at week 16, less significant differences in structural parameters were found, indicating the more sensitive approach of the in\u00a0vivo experimental design.\nAnother limitation to this study was the fact that on average, at 8\u00a0weeks in the study, the OVX group was in a more deteriorated condition than the late treatment group, while there was no demonstrable explanation for this. Although the ANOVA showed no significant differences between the groups, the treatment group still had a more favorable starting point when ZOL was administered. However, because after ZOL injection significant improvement took place while the OVX group still deteriorated, it is shown that this will not have affected the final conclusions.\nThe method of in\u00a0vivo CT scans combined with image registration software presented here has enabled us to study our goals in a more efficient way in terms of number of animals needed in the experiment. Follow-up micro-CT scans were made of the same animals, enabling us to use paired statistics and relative percentage changes in structural parameters. By registering all CT scans, we were able to select the same part of the metaphyseal trabecular bone for every measurement, thereby decreasing the variation in structural parameters due to a varying selected region of interest.\nIn conclusion, OVX induced rapid changes in trabecular bone mass and structure. A single injection of ZOL at OVX completely inhibited these changes in bone mass and structure seen in osteoporotic rats. A single ZOL injection after OVX-induced bone loss improved bone mass and structure, per group and compared to the control group. BV\/TV and the associated bone microstructure at the end point of this study were, however, significantly less favorable than in the early treatment group.","keyphrases":["bisphosphonate","rat","in vivo micro-computed tomography","osteoporosis"],"prmu":["P","P","P","P"]}
{"id":"Dev_Genes_Evol-4-1-2292478","title":"Do teashirt family genes specify trunk identity? Insights from the single tiptop\/teashirt homolog of Tribolium castaneum\n","text":"The Drosophila teashirt gene acts in concert with the homeotic selector (Hox) genes to specify trunk (thorax and abdomen) identity. There has been speculation that this trunk-specifying function might be very ancient, dating back to the common ancestor of insects and vertebrates. However, other evidence suggests that the role of teashirt in trunk identity is not well conserved even within the Insecta. To address this issue, we have analyzed the function of Tc-tiotsh, the lone teashirt family member in the red flour beetle, Tribolium castaneum. Although Tc-tiotsh is important for aspects of both embryonic and imaginal development including some trunk features, we find no evidence that it acts as a trunk identity gene. We discuss this finding in the context of recent insights into the evolution and function of the Drosophila teashirt family genes.\nIntroduction\nHomeotic genes are responsible for defining regional identity, and mutations in these genes often transform one body part into another. The best characterized homeotic genes are the Hox genes, which have been identified in representatives of most animal phyla (reviewed by Martindale 2005) and have been extensively studied in several organisms. However, some non-Hox genes have also been classified as homeotic genes based on their mutant phenotypes. One such gene encodes the zinc finger transcription factor Teashirt (Fasano et al. 1991). Loss-of-function (LOF) teashirt (tsh) mutations are reported to cause partial transformation of the larval prothorax to a labial identity due to ectopic expression of the Hox gene Sex combs reduced (Fasano et al. 1991; Roder et al. 1992). The trunk (thoracic and abdominal) segments of tsh\u2212 larvae are shorter than normal and the denticle belts (a trunk-specific feature) are severely disorganized. Moreover, patches of sclerotic cuticle, which have been interpreted as head structures, form between the remaining denticle belts (Fasano et al. 1991). Conversely, ectopic expression of tsh results in the development of denticle belts in the head (de Zulueta et al. 1994). On the basis of these phenotypes, tsh has been suggested to function as a specifier of trunk vs head identity (Roder et al. 1992).\ntsh is believed to act in parallel with the trunk Hox genes (Scr, Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B)) to determine segmental identity (de Zulueta et al. 1994; Roder et al. 1992). When all of these Hox genes are inactivated, trunk segments develop with what has been interpreted as a mixed head\/trunk identity. That is, sclerotized tissue develops in the posterior compartment of each trunk segment, whereas denticle belts (a trunk-specific feature) still develop in the anterior compartment (Sato et al. 1985; Struhl 1983). When tsh function is also eliminated, the trunk segments are more severely affected in that denticle belts disappear and additional head-like cuticle is observed (Roder et al. 1992).\nRecent data suggests that tsh may play an even greater role in trunk development than was previously recognized. In the absence of tsh function, its paralog tiptop (tio) is ectopically expressed in the trunk region (Laugier et al. 2005). When tio and tsh functions are both absent, the trunk denticles are even more severely affected than in mutants that lack only tsh, suggesting that tio can partially substitute for tsh and reduce the effects of tsh mutations.\nRobertson and coworkers (2004) proposed the existence of a system of zinc-finger proteins that divides the Drosophila embryo into sections and limits the functional domains of Hox genes. They suggest that within this system tsh specifies the trunk region, while disconnected (disco) specifies the gnathal region. In the absence of tsh, disco (as well as its paralog disco-related) is ectopically expressed in the trunk, and there is some evidence that ectopic disco expression is responsible for at least some features of tsh mutants (e.g., loss of denticles and abnormal sensory neurons) that are thought to represent a switch to head\/gnathal identity (Robertson et al. 2004).\nManfroid et al. (2004) speculated that the trunk-specifying function of tsh family members might be conserved in vertebrates, as tsh genes from mice are expressed in the trunk region and can also rescue the phenotype of Drosophila tsh mutants when ectopically expressed. However, such conjecture requires a rather large phylogenetic leap, as it is not clear that the role of tsh in trunk specification is conserved even in other insects. tsh family genes of the firebrat, Thermobia domestica, and the milkweed bug, Oncopeltus fasciatus, are expressed predominantly in the gnathos and thorax, arguing against a function in trunk specification (Herke et al. 2005; Peterson et al. 1999). Furthermore, when Herke et al. (2005) knocked down the function of the Oncopeltus tsh family gene by RNA interference (RNAi), they observed transformations of distal leg to antenna, but did not report any changes in trunk identity. Finally, our previous studies suggest that genetic specification of trunk identity in the red flour beetle, Tribolium castaneum, differs significantly from that in Drosophila. Removal of trunk Hox genes alone is sufficient to produce dramatic transformation of trunk to head identity (Brown et al. 2002). Antennae develop on all gnathal and trunk segments of embryos homozygous for a deficiency that removes the Tribolium orthologs of Deformed (Dfd), Scr, Antp, Ubx, and abd-A (Stuart et al. 1991). Thus, there is apparently no need to invoke the function of another gene for trunk identity. Peterson et al. (1999) predicted that either the Tribolium tsh gene has no function in trunk specification or that the expression of tsh is controlled by Hox genes. Taken together, these observations suggest that the trunk-specifying role of tsh observed in Drosophila might not be conserved in other insects.\nTo better understand the evolution of tsh family gene function, we cloned and characterized a tsh homolog from Tribolium. This gene, Tc-tiotsh, is the sole member of this family in the Tribolium genome. It has a broad embryonic expression domain, encompassing not only the trunk region but much of the head as well. RNAi experiments revealed that Tc-tiotsh functions in several embryonic and imaginal tissues, but they did not suggest a conserved function in trunk specification. We discuss these results and reexamine the classification of tsh as a trunk-specifying gene in Drosophila.\nMaterials and methods\ncDNA cloning\nA genomic fragment of the Tc-tiotsh gene was amplified using degenerate primers based on the conserved amino acid motifs VWLGKG and CKVCDK (first round) and ILKCMW and CKVCDK (second round). Additional, overlapping fragments were identified by RACE, using the 5\u2032 and 3\u2032 RACE Systems (GibcoBRL), by polymerase chain reaction (PCR) screening a Tribolium castaneum cDNA library as described previously (Nie et al. 2001), or by inverse PCR (Ochman et al. 1988). To confirm that these overlapping fragments represent a contiguous sequence, primers were designed near the ends of the putative contig, and reverse transcriptase (RT)-PCR was performed using total embryonic RNA (0\u201330\u00a0h of development) as template. The resulting PCR product was cloned and sequenced (GenBank accession number AF356647).\nSequence analysis\nThe nucleotide contig for Tc-tiotsh was assembled using the Seqman module of Lasergene (DNASTAR). To identify putative tsh family members in other genomes, we performed tBLASTn searches using Drosophila melanogaster Tsh and Tio as query sequences. Multiple alignments were created with the AlignX module of Vector NTI 8 (InforMax). Minor manual adjustments were performed to maximize the alignments. The final output of the multiple alignment was created using BOXSHADE (available at http:\/\/www.ch.embnet.org\/software\/BOX_form.html). Phylogenetic analyses were performed using MEGA version 3.1 (Kumar et al. 2004) or TreePuzzle (http:\/\/bioweb.pasteur.fr\/seqanal\/interfaces\/Puzzle.html), and TreeView (http:\/\/taxonomy.zoology.gla.ac.uk\/rod\/treeview.html) was used to visualize the output.\nIn situ hybridization and immunostaining of embryos\nA digoxigenin-labeled fragment of Tc-tiotsh was hybridized to whole-mount wild-type (Georgia-1) embryos as previously described (Brown et al. 1994b). Embryos were secondarily immunostained with either \u03b1-Invected antibody (4D9) (Patel et al. 1989) or with \u03b1-Even-skipped antibody (2B8) (Patel et al. 1994), which cross-react with Tc-Engrailed and Tc-Even-skipped, respectively. Tc-spalt (Tc-sal) expression was assayed using the probe described by Tomoyasu et al. (2005).\nRNA interference (RNAi)\ndsRNA of two different, non-overlapping fragments of Tc-tiotsh was synthesized using the PCR template method described by Kennerdell and Carthew (2000). Briefly, primers were chosen to amplify a fragment of Tc-tiotsh (bases 1358\u20131786 or 1848\u20132581 of GenBank sequence AF356647) and the T7 promoter sequence (TAATACGACTCACTATAGG) was added to the 5\u2032 end of each primer. The resulting primers were used to amplify template for RNA synthesis. The resulting product was purified and 1 \u03bcg used as template for RNA synthesis using the Megascript T7 High Yield Transcription kit (Ambion).\nFor embryonic RNAi, 0\u20132\u00a0h eggs were collected from wild-type beetles (GA-1) and injected with dsRNA (2\u20134\u00a0\u03bcg\/\u03bcl) as previously described by Brown et al. (1999). After several days, hatched larvae and unhatched eggs were treated with lactic acid\/ethanol. Additional larvae were documented by SEM as described by Curtis et al. (2001).\nInjection into non-virgin adult females was performed by manually opening the elytra and wings of ether-anesthetized beetles and injecting 2\u00a0\u03bcg\/\u03bcl dsRNA into the dorsal abdomen. Larval RNAi was performed as described by Tomoyasu and Denell (2004).\nResults\nTribolium has a single teashirt family gene\nWe cloned a member of the tsh gene family from the red flour beetle, Tribolium castaneum (see Materials and methods for details). Comparison of the cDNA sequence to the recently completed Tribolium genome sequence revealed three exons distributed over about 90\u00a0kb (Fig.\u00a01). Most of this distance can be attributed to an approximately 86-kb intron between the first and second exons.\nFig.\u00a01Gene structure of the Tc-tiotsh gene and its Drosophila homologs. Exons are denoted by boxes and introns by thin lines. The coding region is shaded gray and zinc finger-encoding motifs are shown in black\nOnly three full-length Tsh family proteins have been described from insects: the Drosophila melanogaster paralogs Tsh and Tio, and the single member of the Tsh family in Anopheles gambiae, Ag-TioTsh (Fasano et al. 1991; Laugier et al. 2005). In addition, small fragments of tsh family genes have been amplified from several other insects (Herke et al. 2005; Peterson et al. 1999). Two conclusions have been drawn from these limited data. First, the two Drosophila genes seem to be the result of a recent duplication in the Drosophila lineage. Second, all of the Tsh family members from other insects appear to be more similar in sequence to Tio than to Tsh. In this paper, we present data that support and greatly strengthen these conclusions.\nWe performed a tBLASTn search of several recently sequenced insect genomes for tsh family members (Table\u00a01). The Tribolium castaneum gene that we cloned is the only member of the tsh family present in the genome sequence of this insect. Likewise, the Apis mellifera (honeybee) genome contains only a single member of this gene family. In contrast, all of the additional Drosophila species we searched (D. pseudoobscura, D. simulans, D. yakuba and D. virilis) have orthologs of both tio and tsh. Consistent with the situation in D. melanogaster (Laugier et al 2005), tio and tsh are closely linked (less than 250\u00a0kb apart) in all of the Drosophila species examined, suggesting that these genes likely arose from a relatively recent tandem duplication event.\nTable\u00a01Teashirt family genes in sequenced insect genomesOrganismOrdertsh family genesGenome ReferenceTribolium castaneumColeoptera1The Tribolium Genome Sequencing Consortium 2008Apis melliferaHymenoptera1The Honeybee Genome Sequencing Consortium 2006Anopheles gambiaeDiptera1Holt et al. 2002Drosophila simulansDiptera2 (tio and tsh)Drosophila 12 Genomes Consortium 2007Drosophila yakubaDiptera2 (tio and tsh)Drosophila 12 Genomes Consortium 2007Drosophila pseudoobscuraDiptera2 (tio and tsh)Richards et al. 2005Drosophila virilisDiptera2 (tio and tsh)Drosophila 12 Genomes Consortium 2007tBLASTn searches were used to determine the number of teashirt (tsh) family genes in several assembled insect genomes. All Drosophila species examined had orthologs of both tsh and tiptop (tio), while the non-drosophilid species each contained only one tsh family member.\nAs the Tribolium gene is related to both tio and tsh, we have named it Tc-tiotsh, after the convention established by Laugier et al. (2005) for the single Anopheles gene. We compared the predicted Tc-TioTsh protein to the other insect Tsh family members for which full-length predicted proteins are available. (Although, we were able to identify a tsh family gene in Apis, the 5\u2032 portion of the transcription unit was apparently missing from the genome sequence.) Like tio and Ag-tiotsh (Laugier et al. 2005), Tc-tiotsh encodes four zinc fingers (Fig.\u00a01), three of which are clustered near the middle of the protein (Fig.\u00a02a), and one that is located near the C terminus (Fig.\u00a02b). Besides the zinc finger regions, several additional short regions of sequence identity (shaded in Fig.\u00a02 and data not shown) are present in all of the full-length predicted proteins. The three dipteran proteins are highly similar at their N termini, while Tio, Ag-TioTsh, and Tc-TioTsh share a C-terminal motif (in addition to the fourth zinc finger) that is absent in Tsh. Regions shared between the beetle and dipteran proteins have been conserved over a long phylogenetic distance, suggesting that they likely have important functions.\nFig.\u00a02Multiple alignments of Teashirt family members. Amino acids identical or similar in at least two of the proteins are shaded black or gray, respectively. Tc Tribolium castaneum; Ag Anopheles gambiae, Dm Drosophila melanogaster; Mm Mus musculus; Dr Danio rerio. a Multiple alignment of the three clustered zinc fingers. Zinc fingers (ZF) 1\u20133 are indicated above the sequence. Regions underscored with asterisks were concatenated and subjected to phylogenetic analysis. b Multiple alignment of the C-terminal zinc finger region. Zinc finger 4 is delimited above the alignment. Tsh lacks this zinc finger. c Distinct N-terminal motifs in dipteran Tsh family members (upper) and in Tribolium and vertebrates (lower)\nTo better understand the evolutionary history of the tsh gene family, we have performed phylogenetic analyses using conserved portions of the zinc-finger region (underlined in Fig.\u00a02) from the full-length insect Tsh family proteins that are currently available (Drosophila, Tribolium, and Anopheles). Both maximum likelihood (Fig.\u00a03) and neighbor-joining (data not shown) methods produce trees in which the two Drosophila proteins cluster together, suggesting these genes were produced by duplication after the divergence of the mosquito and fly lineages. The branch lengths of the Drosophila proteins are much longer than those of Anopheles and Tribolium, suggesting rapid divergence of both genes after duplication.\nFig.\u00a03Phylogenetic analysis of insect Teashirt family members. The two Drosophila paralogs cluster together in an unrooted tree produced by maximum likelihood analysis, suggesting that these genes resulted from a recent duplication in the Drosophila lineage. The bootstrap value of 100 indicates that the tree is very robust\nThe mouse and human genomes each have three tsh family members (Caubit et al. 2000; Manfroid et al. 2004), which are much more similar to one another than they are to any of the insect proteins. Interestingly, vertebrate Tsh family proteins share an N-terminal motif (MPRRKQ) with the Tribolium protein (Fig.\u00a02c), which is distinct from the N-terminal motif shared by the dipteran family members (Laugier et al. 2005). The conservation of this sequence in an insect and vertebrates suggests it has an important function and that it was probably present in the common ancestor of protostomes and deuterostomes.\nTaken together, these comparisons of tsh family members indicate that the ancestor of protostomes and deuterostomes had a single tsh family gene. We were unable to identify any tsh family members in the genomes of two non-bilaterian metazoans: the placozoan Trichoplax adhaerens (http:\/\/genome.jgi-psf.org\/Triad1\/Triad1.home.html) and the cnidarian Nematostella vectensis (Putnam et al. 2007), suggesting this gene family may have arisen after the bilaterian\/non-bilaterian split. Within the vertebrate lineage, duplication events (perhaps in association with whole genome duplications) produced at least three paralogs. A duplication in the Drosophila lineage (after divergence from the mosquito lineage) resulted in the tsh and tio paralogs.\nTc-tiotsh embryonic expression is not limited to the trunk\nWe examined the embryonic expression pattern of Tc-tiotsh (Fig.\u00a04) by in situ hybridization, using the posterior compartment marker Engrailed (Brown et al. 1994a; Patel et al. 1989) to determine the stage of embryonic development (segments are added sequentially) and provide a reference for the register of Tc-tiotsh expression. Tc-tiotsh expression develops in a dynamic, pair rule-like manner (Fig. S1 and S2), but eventually encompasses not only the trunk segments (thorax and abdomen), but also the gnathal segments and the labrum and intercalary segment in the procephalon (Fig.\u00a04a). Tc-tiotsh expression in the thorax and the first abdominal segment appears somewhat stronger than in other regions (Fig.\u00a04b). As appendages develop, Tc-tiotsh is expressed in the proximal portion of each leg (Fig.\u00a04d).\nFig.\u00a04Tc-tiotsh expression in Tribolium embryos. Engrailed expression is shown in brown, while Tc-tiotsh expression is in purple. Embryos are oriented with anterior to the left (except in panel d in which anterior is to the top). a During germband elongation, Tc-tiotsh is expressed in the gnathal and trunk segments as well as two procephalic regions (the intercalary segment [Ic] and the labrum [Lr]). b When germband elongation is completed, Tc-tiotsh expression is strongest in the thoracic (T1\u2013T3) and first abdominal (A1) segments. c Slightly later, strong Tc-tiotsh expression appears in the posterior terminal region of the embryo (arrowhead). dTc-tiotsh is expressed in only the proximal portion (arrow) of the developing legs. e In embryos undergoing germ-band retraction, Tc-tiotsh expression is present in the six Malpighian tubule primordia that evert from the hindgut. f In a Df(HOMC) homozygote lacking most Hox genes, Tc-tiotsh expression in the segmented portion of the germband is greatly reduced. In contrast, expression in the Malpighian tubule primordia remains strong\nAfter germ-band elongation, Tc-tiotsh is expressed in the posterior region of the embryo. Initially, expression is seen in a patch on either side of the ventral midline (Fig.\u00a04c), and later in six buds that extend from the invaginating hindgut (Fig.\u00a04e). These structures are the primordia of the Malpighian tubules; they also express Tc-dachsund (Prpic et al. 2001) and Tc-Cut (data not shown).\nspalt does not appear to repress Tc-tiotsh in Tribolium embryos\nIn Drosophila, the limits of tsh expression are controlled either directly or indirectly by spalt-major (salm). In a salm mutant, the tsh expression domain expands both anteriorly and posteriorly (Roder et al. 1992). To assess whether the broader expression of Tc-tiotsh (compared to tsh) might reflect a difference in Tribolium spalt (Tc-sal) expression, we examined the embryonic expression pattern of the single Tribolium sal gene (Tomoyasu et al. 2005). We find that the Tc-sal expression domain (Fig. S3) significantly overlaps that of Tc-tiotsh, suggesting that Tc-sal does not repress Tc-tiotsh expression. It is possible that Tribolium has lost the regulatory interaction between these genes. Alternatively, regulation of tsh by sal may have evolved more recently, after the divergence of these lineages.\nTc-tiotsh expression is reduced in embryos lacking Hox genes\nIn Drosophila, some aspects of tsh expression are regulated by the Hox genes (Roder et al. 1992). We have examined expression of Tc-tiotsh in embryos homozygous for Df(HOMC), a deletion that removes most of the Tribolium Hox cluster. Tc-tiotsh expression is severely reduced in the ventral region of these embryos (Fig.\u00a04f). These results suggest that reduction of Tc-tiotsh expression might contribute to the transformations seen in Df(HOMC) embryos. However, our functional studies of Tc-tiotsh (presented below) reveal no evidence for this.\nTc-tiotsh functions in the embryonic labial and thoracic segments\nWe used RNAi to ascertain the probable LOF phenotype of Tc-tiotsh. Pupae injected with Tc-tiotsh dsRNA produced very few eggs upon reaching adulthood, and none of these progeny showed an abnormal phenotype. To circumvent the apparent effect of Tc-tiotsh on oogenesis, we injected Tc-tiotsh dsRNA either directly into eggs or into the abdomens of non-virgin adult females (egg production from these females quickly tapers off, further indicating an effect on oogenesis). Injection of Tc-tiotsh dsRNA by either technique produced embryos with defects in the labial and thoracic segments (Fig.\u00a05, Table\u00a02, and data not shown). During normal Tribolium embryonic development, the two labial appendages fuse at the ventral midline and migrate anteriorly to a position between the maxillary appendages (Fig.\u00a05a). In moderately affected Tc-tiotsh RNAi larvae (Fig.\u00a05b), the labial appendages are smaller than normal, and in the most severely affected embryos (Fig.\u00a05c) these appendages fail to fuse. In all of these cases, the abnormal labial appendages fail to migrate to their normal position between the maxillary palps. These abnormalities produce secondary effects on the orientation of the other head appendages.\nFig.\u00a05Scanning electron micrographs of Tc-tiotsh RNAi larvae. For all panels, the scale bar represents 50um. a\u2013c Ventral views of larval heads. Maxillary (pink) and labial (green) appendages have been shaded. d\u2013e Lateral views with leg segments labeled as follows: coxa (C), trochanter (Tr), femur (F), tibiotarsus (Tt), and pretarsus (Pt). a Wild-type larval head showing the proper position of the maxillary (Mx) and labial (L) appendages. b Moderately affected Tc-tiotsh RNAi larva in which the labial appendages (especially the proximal region) are highly reduced. c Severely affected Tc-tiotsh RNAi larva in which the labial appendages have failed to fuse. Note that in both b and c, the ventral, posterior region of the head (presumed to derive from the labial segment) is almost completely absent. d Wild-type larva. eTc-tiotsh RNAi larva with short legs. On each leg, the trochanter is presumably fused with the reduced coxaTable\u00a02Tc-tiotsh RNA interference cuticular phenotypesdsRNA fragmentSeverity of EffectNoneMildModerateSevere1358\u2013178612\/74 (16.2%)4\/74 (5.4%)14\/74 (18.9%)44\/74 (59.5%)1358\u201317862\/58 (3.4%)14\/58 (24.1%)39\/58 (67.2%)3\/58 (5.2%)1848\u201325810\/24 (0%)4\/24 (16.6%)20\/24 (83.3%)0\/24 (0%)All results were obtained by injection of dsRNA into eggs. dsRNA fragment names refer to the nucleotides of GenBank sequence AF356647 included in the fragment. Severity of effect was classified as follows. Larvae with mild effects had short legs resulting from reduction and fusion of proximal segments. Moderately affected larvae had reductions in the ventral head and labial appendages in addition to short legs. Severely affected larvae were distinguished from those with moderate effects by the failure of the labial appendages to fuse at the ventral midline.\nShort legs were the most common feature seen in Tc-tiotsh RNAi larvae, being present in all larvae with labial defects, as well as in some larvae with apparently normal labial appendages (Table\u00a02). The reduction in leg length stems from effects on the proximal leg segments. The coxa and trochanter are reduced and apparently fused, and the femur is slightly shortened (Fig.\u00a05e). This phenotype is similar to that observed in the adult legs of hypomorphic Drosophila tsh mutants (Erkner et al. 1999) and thus may indicate a conserved role for tsh family genes in proximal limb development.\nWe also noted that Tc-tiotsh RNAi larvae show a pronounced curvature of the head and thoracic segments, such that the head bends forward (Fig.\u00a05e). This phenotype apparently results from reduction in length (anterior to posterior) of the ventral portions of the thoracic segments and posterior head (Fig.\u00a05b,c,f).\nTc-tiotsh is important for proper development of adult trunk segments\nWe examined the role of Tc-tiotsh in adult development using larval RNAi (Tomoyasu and Denell 2004). The resulting adults display a number of defects in the thoracic and abdominal segments (Fig.\u00a06). Most noticeably, these individuals have abnormalities of the T2 notum that prevent the elytra from closing properly (data not shown). We also observed mild defects in the proximal legs. In addition, adults resulting from Tc-tiotsh larval RNAi show abnormalities in many of the ventral sclerites (sternites) (see El-Kifl 1953 for a description of wild-type structures). In the ventral prothorax (T1) of RNAi-treated individuals, the epimera, which are normally located posterior to the T1 coxae, are missing and the basisternum is reduced in size resulting in the exposure of membranous areas (Fig.\u00a06b,d). In addition, the shape of the sternellum (the process extending between the T1 legs) is abnormal. Ventral T2 is not noticeably affected, but the T3 sternite is missing its anterior process (meso-spinasternum) (Fig.\u00a06d). The anterior abdominal segments are also affected by Tc-tiotsh larval RNAi. In wild-type adults, the first and second abdominal segments (A1-A2) form an internal portion of the T3 leg sockets and are only lightly sclerotized, while A3 is distinguished by a sharply pointed process extending between the T3 legs (Fig.\u00a06e). In Tc-tiotsh RNAi individuals, an additional anterior region (A1 and\/or A2) is heavily sclerotized and A3 lacks its anterior process, thus resembling the more posterior abdominal segments (A4\u2013A6) (Fig.\u00a06f).\nFig.\u00a06Adult phenotypes resulting from Tc-tiotsh larval RNAi. All images are ventral views with anterior to the top. a Wild-type adult head and thorax. b Head and thorax of Tc-tiotsh RNAi individual. The T1 basisternum (Bs) is smaller than normal. c Enlarged view of region outlined in A. d Enlarged view of region outlined in B. The T1 epimera (Epm) and the T3 meso-spinasternum (Ss) are reduced or missing. e Wild-type adult abdomen. f Abdomen of Tc-tiotsh RNAi adult. The third abdominal segment (A3) lacks its anterior process, and an additional heavily sclerotized segment is present anterior to A3\nDiscussion\nWe have identified and cloned the lone member of the tsh gene family from Tribolium castaneum. Our analysis of this gene provides important insights into the evolution of the tsh gene family at the levels of both sequence and function.\nEvolution of tsh family genes\nSearches of several non-drosophilid insect genome sequences identified only a single tsh family member in each genome. These results corroborate previous suggestions (based on more limited data) that most insects have only a single tsh-related gene and that this is the ancestral state (Herke et al. 2005; Laugier et al. 2005). A key implication of this model is that the Drosophila genes tio and tsh are the result of a recent duplication event that occurred sometime after the divergence of mosquitoes and flies. Since that duplication, tsh has apparently diverged from the ancestral sequence faster than has tio (although both have diverged more than the Tribolium and Anopheles homologs). More rapid divergence by one member of a recently duplicated gene pair is not unprecedented. For example, the Hox3 paralogs zerkn\u00fcllt (zen) and bicoid (bcd) are believed to have arisen by duplication late in the dipteran lineage (Brown et al. 2001; Stauber et al. 1999, 2002), but the sequence of Zen is more similar to that of the Hox 3 proteins of other insects than it is to Bcd.\nMuch of the sequence conservation between Tsh family members is found in the zinc finger regions. However, a few additional conserved motifs are present, particularly at the protein termini. The C-terminal region is well conserved between Tc-TioTsh, Ag-TioTsh, and Tio, but this conserved region is not found in Tsh. The conservation of the N-terminal region is particularly interesting. The Tsh family members for which N-terminal sequence is available fall into two distinct classes (Fig.\u00a02c). Ag-TioTsh and both Drosophila proteins share an extended region of identity that is completely different than the short motif (MPRRKQ) shared by the Tribolium and vertebrate proteins. This pattern of conservation suggests that an ancestral Tsh family member had the MPRRKQ motif, which was replaced with a different motif sometime before the separation of Anopheles and Drosophila within the dipteran lineage. It is not clear what the function of these disparate motifs might be, as the vertebrate Tsh proteins function very similarly to Dm-Tsh when expressed in Drosophila (Manfroid et al. 2004). Perhaps these motifs are important in a particular tissue or at a different stage in development than those assayed. As additional insect Tsh family protein sequences become available, it will be possible to more accurately determine the point at which these N-terminal sequences began to diverge.\nEvolution of tsh family gene expression\nAlthough the sequences of other insect tsh family members are most similar to tio, their expression patterns (where known) seem to be more similar to tsh. The embryonic expression patterns of tsh and tio in Drosophila are strikingly different (Laugier et al. 2005). tsh is expressed throughout the ectoderm of trunk segments, while tio is expressed in only a few cells of the trunk ectoderm (as well as in the Malpighian tubules and parts of the CNS and gut). In tsh mutants, however, tio is more broadly expressed in the trunk, indicating that tsh represses tio expression in this region. Likewise, tsh is ectopically expressed in the stomodeal region when tio expression is knocked down by RNAi.\nThe Tc-tiotsh expression pattern includes similiarities to both the tsh and tio expression patterns. The ectodermal expression in the segmented region of the germband is reminiscent of, although more extensive than, that of tsh. On the other hand, Tc-tiotsh is expressed in the Malpighian tubule primordia, similar to tio. The expression of tio near the stomodeum might also correspond to the expression of Tc-tiotsh in the labrum and intercalary segment, although it is difficult to compare expression patterns in the cephalic regions of these insects due to the derived structure of the Drosophila larval head.\nIt is interesting that the Tc-tiotsh expression pattern approximates the sum of the tsh and tio patterns. This may indicate that the expression pattern of the ancestral gene that gave rise to tsh and tio had an expression pattern similar to Tc-tiotsh and that parts of its expression pattern (and perhaps its function) have been split between its daughters. Such division of labor resulting from complementary loss of particular expression domains or functions is termed subfunctionalization and has been suggested to play a key role in the maintenance of duplicated genes (Force et al. 1999).\nAmong other insects, expression patterns of tsh family members have been reported only for the firebrat, Thermobia domestica, and the milkweed bug, Oncopeltus fasciatus (Herke et al. 2005; Peterson et al. 1999). In both cases, these genes are expressed in the gnathal, thoracic, and first abdominal segments, with very weak (if any) expression in the remainder of the abdomen. Both insects also show expression in the posterior tip of the embryo (see below). Oncopeltus has additional expression in the procephalon. The leg expression pattern in firebrats has not been described, but Herke et al. (2005) reported that milkweed bugs show expression throughout the legs, which fades from the distal tips later in development.\nInterestingly, all of the insect tsh family genes whose expression has been described are expressed in the post-abdominal region. However, these domains have been interpreted differently. In firebrats, expression in this region is reported to represent the anlagen of the cerci and the median caudal filament (appendages of the 11th abdominal segment) (Peterson et al. 1999). The posterior expression of tsh has been interpreted as the future anal opening (Fasano et al. 1991), and that of Oncopeltus tiptop is described only as the caudal region (Herke et al. 2005). Our results show that Tc-tiotsh is expressed in the Malpighian tubule primordia. Likewise, tio is expressed in the developing Malpighian tubules (Laugier et al. 2005). Thus, Malpighian tubule expression may have been part of the ancestral expression pattern of this gene family. It is possible that the posterior expression domains in other insects will, upon closer examination, be found to represent the developing Malpighian tubules as well.\nEvolution of tsh family function\nTc-tiotsh is required for the proper development of the proximal portion of developing larval legs. Drosophila larvae lack external appendages, but tsh is expressed in the proximal regions of leg imaginal discs and hypomorphic tsh alleles cause proximal leg defects (Erkner et al. 1999). These results suggest that proximal leg expression and function were probably a feature of a tsh-like gene in the ancestor of beetles and flies. In contrast, Herke et al. (2005) reported that knockdown of Oncopeltus tiptop caused transformations of the distal legs to antennae. Based on this observation, they concluded that Oncopeltus tiptop acts as a distal leg selector gene, and suggested that tio or tsh might play a similar role in Drosophila. tsh expression is known to be restricted to the portion of the imaginal disc that will form the body wall and proximal leg segments (Gonzalez-Crespo and Morata 1996). Similarly, Tc-tiotsh is expressed in the proximal region of embryonic legs and leg imaginal discs (Fig.\u00a04d and data not shown). We have also examined the expression of tio in Drosophila leg imaginal discs and found that expression is restricted to the body wall and proximal portion of the developing leg. Thus, it is highly unlikely that the \u201ctiptop-like\u201d distal leg-specifying function suggested by Herke et al. (2005) is performed by Tio, either alone or in combination with Tsh.\nWe cannot rule out additional, internal functions for Tc-TioTsh. In particular, the expression of Tc-tiotsh in the Malpighian tubules is intriguing, as Drosophila tio (and possibly other homologs) show similar expression.\nThe striking effects of larval Tc-tiotsh RNAi would not have been predicted based on Drosophila experiments, as relatively few adult effects of LOF tsh mutations have been reported. Other than the proximal leg defects that have been discussed above, the only other adult phenotypes reported are defects in wing posture (Soanes et al. 2001) and in structures that develop from the eye-antennal disc, namely, the eyes (Singh et al. 2002) and the maxillary palps (Bhojwani et al. 1997). This dearth of adult phenotypes could be a consequence of the pre-adult lethality of many tsh allele combinations. However, it might also be due to functional redundancy with tio. We examined tio expression in Drosophila imaginal discs and found that it is indeed expressed in a pattern very similar to that of tsh (data not shown). Thus, loss of both tio and tsh during Drosophila imaginal development might produce a more severe phenotype.\nThe principal question prompting our studies of the Tribolium Tc-tiotsh gene was whether the trunk-specifying function reported for tsh in Drosophila was conserved in other insects. However, insights from our work and other recent reports compel us to first reexamine whether tsh itself is really a trunk-specifying gene. The key observations, which originally led to this conclusion, were the presence of head-like sclerites in embryos lacking tsh, and the positive correlation between expression of tsh and the presence of denticle belts (a trunk-specific feature) (de Zulueta et al. 1994; Fasano et al. 1991; Roder et al. 1992). The later discovery that tsh and disco form mutually exclusive domains in the trunk and head, respectively, seemed to support this view (Robertson et al. 2004). Below we synthesize recent studies that may lead to new interpretations of these observations.\nA number of mutant phenotypes in Drosophila, including those of tsh and some Hox genes, feature small patches of sclerotic tissue. The previous interpretation of such patches as ectopic head tissue has been called into question. Mace et al. (2005) showed that the sclerites that form in a segment lacking Hox gene expression are correlated with breaches in epidermal integrity and the resulting activation of a wound response pathway. If in fact, the sclerotized cuticle seen in tsh mutants is scar tissue rather than head cuticle, the interpretation of tsh as a trunk-specifying gene would be less convincing, but still possible.\nThe case for tsh acting as a trunk specifier was apparently strengthened when Robertson et al. (2004) proposed the attractive hypothesis that a system of zinc finger proteins acted in conjunction with the Hox genes to specify body regions. Within this system, disco specified gnathal segments and tsh specified trunk segments. However, the same laboratory recently reported that the conserved role of disco is to specify the medial portion of appendages, and suggested that the boundary between disco and tsh expression is actually an artifact of maggot morphology (Patel et al. 2007). That is, the reduced Drosophila larval head might lack proximal, tsh-expressing tissue (body wall) while retaining medial appendage tissue, whereas the trunk retains tsh-expressing tissue but has lost most appendage tissue. Taking this scenario a step further, ectopic expression of tsh might \u201crestore\u201d the body wall tissue of the head, creating larger, more complete segments. As the genes controlling segment formation also control denticle belt patterning, these enlarged head segments would have denticle belts as well. This interpretation is consistent with the observation that the additional denticle belts produced by ectopic tsh expression are interspersed with head-specific structures (de Zulueta et al. 1994). When considered in this light, perhaps tsh is no more a trunk identity gene than are other genes involved in segment formation and denticle patterning such as wingless, rhomboid, or shavenbaby. On the other hand, tsh does seem to provide the spatial information that limits full segment formation to the trunk, and thus in Drosophila could be considered a trunk-specifying gene.\nIn contrast, Tc-tiotsh does not appear to act in any way as a trunk identity gene. This is not to say that Tc-tiotsh has no role in the development of trunk segments, but rather that we find no evidence that it specifies trunk vs head fates. In embryos depleted of Tc-tiotsh the ventral portions of the labial and thoracic segments are reduced, but not visibly transformed. Interestingly, some of the labial abnormalities observed in Tc-tiotsh RNAi larvae are reminiscent of those produced by loss of function alleles of prothoraxless (Tc-Antp) (Shippy, unpublished data). Knockdown of Tc-tiotsh during imaginal development produces adults with patterning defects in the thoracic and abdominal sclerites, including possible transformations of anterior abdominal sclerites to a more posterior identity. These phenotypes, together with the reduction in Tc-tiotsh expression seen in Df(HOMC) embryos, suggest that Tc-tiotsh is regulated to some extent by Hox genes and may carry out a subset of the functions of these genes in both the head and the trunk.\nOur studies of Tc-tiotsh indicate that trunk specification is not a universal function of tsh family members. It is possible that this function has been lost in Tribolium. However, expression pattern data from other insects and functional analysis in Oncopeltus argues against this conclusion. A more likely scenario is that tsh family members acquired a role in defining trunk at some point after the divergence of the coleopteran and dipteran lineages. Analysis of tsh function in additional holometabolous insects should help pinpoint when this event occurred.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nFig.\u00a0S1\nDynamic expression of Tc-tiotsh during germband elongation. Tc-tiotsh expression always appears in an odd-numbered parasegment and the anterior compartment of the adjacent posterior even-numbered parasegment before it is detected in the posterior compartment of the adjacent anterior even-numbered parasegment. An arrowhead marks the mandibular Engrailed stripe in each embryo, while numbers indicate parasegment identity. aTc-tiotsh expression is first observed spanning the mandibular Engrailed stripe. The mesoderm, which has not yet invaginated at the ventral midline, does not show Tc-tiotsh expression. bTc-tiotsh is expressed in PS3 and the anterior compartment of PS4, but not yet in the posterior compartment of PS2. c An embryo with a gap in Tc-tiotsh expression in the posterior compartment of PS10. Tc-tiotsh expression is also visible in the intercalary segment and the developing labrum. d An embryo in which the gap in Tc-tiotsh expression corresponds to the posterior compartment of PS12 (GIF 0 kb)\nFig.\u00a0S2\nTc-tiotsh and Tc-Even-skipped (Tc-Eve) expression. Tc-Eve expression is shown in brown, while Tc-tiotsh expression is in purple. Embryos are shown in ventral view with anterior to the left. Numbers refer to Tc-Eve stripes. a The first Tc-Eve primary stripe has resolved into two secondary stripes, 1a and 1b. The Tc-tiotsh expression domain at this time covers not only the Tc-Eve first primary stripe region, but also the region anterior to it. The second Tc-Eve primary stripe has formed too, but no Tc-tiotsh expression is present in this region yet. Tc-tiotsh is also expressed in the amnion. b The second Tc-Eve primary stripe is resolving into 2a and 2b, but there is still no Tc-tiotsh expression in this region. The expression of Tc-tiotsh in the amnion is still visible at this stage. c The third Tc-Eve primary stripe has formed and is just beginning to split into secondary stripes. A new stripe of Tc-tiotsh expression covers the region delimited by Tc-Eve stripes 2a and 2b (PS3 and the anterior compartment of PS4). No Tc-tiotsh expression is seen in the posterior compartment of PS2 yet. d A much older embryo than in A, B, and C shows the same relationship between Tc-Eve and Tc-tiotsh expression in the posterior region of the embryo (GIF 0 kb)\nFig.\u00a0S3\nTc-sal expression. Anterior is to the top. In a wild-type embryo in which the abdominal segments are just beginning to form, Tc-sal is expressed in most of the gnathal and thoracic segments (GIF 0 kb)","keyphrases":["teashirt","trunk","tiptop","tribolium","homeotic"],"prmu":["P","P","P","P","P"]}
{"id":"Crit_Care-9-1-1065088","title":"Clinical review: Treatment of heat stroke: should dantrolene be considered?\n","text":"Rapid and efficient cooling is the most important therapeutic objective in patients with heat stroke (HS). This article reviews the mechanism of action and rationale for the use of dantrolene as a potential supportive cooling method in the treatment of HS. Relevant studies were included to support discussion of the role of dantrolene for the treatment of HS. In some studies dantrolene was shown to accelerate cooling rate when administered after the development of exertional HS. Dantrolene was also found to be effective in reducing the extent of HS signs when given as pretreatment in an animal model. Accumulated data do not support the routine use of dantrolene as an adjuvant cooling technique in HS, but administration of this drug in severe cases, or in which no improvement is observed, appears rational. Further trials are needed in order to assess the true effectiveness of dantrolene in HS.\nIntroduction\nHyperthermia is defined as any core temperature rise to above the hypothalamic set-point at which heat-dissipating mechanisms are impaired. Normal core temperature values are in the 36.5\u201337.5\u00b0C range at rest and can rise to 40\u00b0C during strenous exercise. Hyperthermia is usually caused by an imbalance between total heat (metabolic and environmental heat) accumulated and total heat lost from the body. Hyperthermia per se may be physiological and can be compensated for (as in the moderate elevation in core temperature that occurs in an exercising and otherwise asymptomatic individual), or it may be pathophysiological but generally well tolerated. It may also be associated with adverse pathophysiological consequences such as inability to continue physical exertion (heat exhaustion). At the extreme, hyperthermia represents a state in which the elevation in core temperature is either already accompanied by organ injury or is sufficient to produce such injury if left untreated.\nSeveral syndromes have been reported to be associated with extreme hyperthermia, including malignant hyperthermia (MH), neuroleptic malignant syndrome (NMS) and heat stroke (HS). The mainstay of treatment of these syndromes includes the administration of basic resuscitative measures together with simultaneous cooling aimed at reducing body temperature [1,2]. Cooling may be achieved by several methods, such as immersion in cold water and evaporation of water over the skin [3-7]. Because heat is primarily produced by hyperactivity of muscles in the various syndromes, dantrolene sodium, a muscle relaxant, has also been suggested to accelerate cooling [8-10]. In MH, dantrolene administration resulted in a rapid reduction in mortality rate, and therefore it is now considered as an essential part of treatment in this syndrome [11-13]. Dantrolene has also been recommended for the treatment of NMS [14]. To date, no drug has shown significant efficacy in improving the outcome of patients with HS.\nExperience with dantrolene use in HS is in its initial phases (Tables 1,2,3). In some studies a possible benefit from dantrolene treatment was reported, whereas in others it was not found to be effective [15-22]. We reviewed relevant studies concerning dantrolene use in HS in order to establish its role in the treatment of this condition.\nStudies included in this review were identified by means of a comprehensive Medline search, limited to the English language literature, using the following keywords: 'dantrolene' and 'hyperthermia' or 'heat'. Each article identified was further examined for relevancy.\nDantrolene\nDantrolene sodium is a hydantoin derivative. It acts directly on muscle contractile elements, attenuating the amount of calcium released from the sarcoplasmic reticulum of skeletal muscle to the cytosol [23-25]. As a result, calcium dependent excitation\u2013contraction coupling and consequent muscle contraction are inhibited.\nThe mean biological half-life of dantrolene in healthy volunteers or patients was reported to range from 6 to 9 hours, with extremes of 3 and 22 hours [25-30]. It is primarily metabolized in the liver, with 15\u201325% of the dose administered being excreted by the kidneys [27,29,30]. Dantrolene is marketed as Dantrium (Norwich Eaton Pharmaceuticals, Norwich, NY, USA) and can be administered intravenously (in solution with mannitol and sodium hydroxide) or orally.\nWhen dantrolene is used as an emergency therapy, such as in acute hyperthermic syndromes, it is unlikely to cause major adverse effects [25]. However, prolonged use may be associated with undesirable side effects that include weakness, drowsiness, diarrhea and malaise [25]. Hepatotoxicity, which can be irreversible, is also a major concern. The risk for hepatic injury appears to be about twofold greater in females, in patients receiving doses over 300 mg\/day and in those treated for over 60 days [31]. In addition, dantrolene sodium solution is highly alkaline (pH 9.6), and intravenous injection may cause extravasation and tissue necrosis [25].\nHeat stroke and 'thermic stress syndrome'\nHS is a life-threatening illness characterized by an elevation in core body temperature to above 40\u00b0C with central nervous system (CNS) dysfunction that results in delirium, convulsions, or coma [32,33] in the absence of any other cause of CNS dysfunction, and skin dryness. The affected individual may suffer from a characteristic multiple organ clinical and pathological syndrome caused by temperature-induced tissue damage [1,34]. HS results from exposure to a high environmental temperature, in which case it is called 'classic' heat stroke (CHS; or 'nonexertional' HS), or from strenuous exercise, in which case it is called 'exertional' heat stroke (EHS) [34-36]. CHS is usually seen in the very young or elderly and in poor socioeconomic settings with limited access to air conditioning. EHS is seen more commonly in exercising individuals, for instance soldiers in army training. Further reviews on HS are available [32-36].\nEpidemiological incidence data for HS is imprecise because of varying definitions of heat-related death and under-diagnosis [35]. One study conducted in urban USA found a variation from 17.6 to 26.5 cases per 100,000 capita. Data from Saudi Arabia show a seasonal incidence pattern, ranging from 22 to 250 cases per 100,000 capita. The crude mortality rate in Saudi Arabia has been estimated at 50%. Treatment consists of immediate cooling and support of organ system function.\nSome authors believe that HS, as well as MH and other hyperthermic syndromes, are different manifestations of a broader underlying condition called 'thermic stress syndrome' [9,10,15,17,37]. This common malady, which is triggered by different mechanisms, is characterized by an extreme elevation in body temperature and CNS dysfunction, which may be complicated by bleeding diathesis, elevated serum enzymes levels and renal failure. Because dantrolene was found to be effective in the treatment of MH and NMS, its usage has also been recommended for the treatment of HS [8-10,15].\nDantrolene use in heat stroke\nTwo reports from the early 1980s demonstrated a beneficial effect from using dantrolene in HS. Lydiatt and Hill [15] reported on a patient with EHS who responded favourably to dantrolene (4 mg\/kg intravenously) after sympthomatic therapy had failed. In a case report, Denborough [8] reported that intravenous dantrolene (20 mg bolus) therapy produced rapid recovery in a young soldier suffering from EHS after a march.\nIn a controlled study, the adjuvant effects of dantrolene in comparison with passive cooling were investigated in a canine model of CHS [16]. Intravenous administration of dantrolene (5 mg\/kg) did not significantly improve cooling rates, haemodynamic parameters, pathological changes, or clinical outcome when compared with passive cooling.\nChanna and coworkers [17] conducted a study in which 20 EHS patients were randomly assigned to two groups with only one group receiving dantrolene (2.45 mg\/kg intravenously). Patients in both groups were cooled either using the Makkah body cooling unit or by 'conventional methods', namely spraying of tap water over the patients while fanning. Cooling rates were found to be significantly higher in the dantrolene group. All patients survived and there was no difference in the incidence of neurological sequelae between the groups.\nThe effects of dantrolene were also evaluated in 53 patients suffering from CHS [21]. All patients were treated by using a body cooling unit in which atomized water was sprayed over the victim while they were fanned with hot air. In addition, treatment with either dantrolene (2 mg\/kg) or placebo was given intravenously after blind, randomized selection.\nDantrolene administration did not result in any enhancement of the cooling rate, and there was no significant difference in the number of hospital stays between the two groups. It should be noted that in this study both treatment groups achieved satisfactory cooling in under 1 hour. Therefore, a possible positive effect of dantrolene in extreme cases of HS, in which cooling times might be longer, cannot be ruled out. It should further be noted that in this study Bouchama and coworkers [21] treated their patients with dantrolene and mannitol simultaneously. Thus, it could not be concluded whether the reported positive effects were attributed to dantrolene or to mannitol.\nMannitol may be beneficial in the setting of HS because of its effect on expanding the intravascular volume and reducing tissue injury after ischemia by scavenging oxygen free radicals [38,39]. In order to address this question, Zuckerman and coworkers [19] designed a randomized controlled study in which heat stroked piglets were treated by one of the following four methods: passive cooling, 'conventional cooling' (intravenous fluid resuscitation, sponging with tap water, mechanical fanning and ice gastric lavage), conventional cooling along with dantrolene, and conventional cooling along with mannitol. Although dantrolene significantly shortened the cooling time when compared with conventional cooling, it did not shorten the cooling time when compared with mannitol. Moreover, it was shown that dantrolene did not improve cardiovascular parameters when administered in addition to conventional methods.\nTayeb and Marzouki [20] investigated whether dantrolene might be advantageous in prophylaxis against HS in exercising sheep. They found that pretreatment with dantrolene (3 mg\/kg and 1.5 mg\/kg intravenously) caused a significant reduction in body temperature, and decreased the extent of HS signs as well as some of the induced changes in enzymes and hormones. These effects were more significant in the group receiving dantrolene 3 mg\/kg than in the group receiving 1.5 mg\/kg. The dantrolene group also exhibited a trend toward reduced mortality from HS (0\/12) in comparison with the group that did not receive dantrolene treatment (2\/6).\nUsing a rat model of EHS and CHS, Moran and coworkers [18] studied the efficacy of dantrolene both as a prophylactic agent and as a therapeutic drug. Administration of high doses of dantrolene (140 mg\/kg intravenously) before heat stress delayed the development of HS in the sedentary animals. Estimated temperatures were lower in the exercising rats that received Dantrolene pretreatment, but this was most reasonably attributed to the limited physical capacity induced by dantrolene. When dantrolene, at the same dose, was administered after the development of HS, it significantly improved the cooling rate in the exercising rats \u2013 an effect that was not seen in the sedentary group.\nDiscussion\nThe rationale for the use of dantrolene in HS emerged from the overlap in the systemic manifestations of HS and other hyperthermic syndromes, particularly MH. However, some researchers claimed that, although hyperthermia was characteristic of both MH and HS, these syndromes represent two clinical entities with different heat generating cellular mechanisms. Although MH is characterized by muscular rigidity, which causes active heat production, HS usually manifests in the form of flaccidity, and heat production does not continue uncontrolled [17,40]. Therefore, dantrolene, which uncouples the excitation\u2013contraction sequence heat generating mechanism, may not be beneficial in the treatment of HS. Moreover, MH is a genetically transmitted disease, which is triggered by several depolarizing muscle relaxants or volatile anaesthetic agents [41,42]. In comparison, to the best of our knowledge, no genetic predisposition has been definitely incriminated in the typical HS patient.\nHowever, several reports suggest a possible link between HS and MH. Tobin and coworkers [43] reported a case of fatal HS in a patient who experienced MH 8 years earlier and speculated whether other HS victims are MH susceptible. An abnormal caffeine\u2013halothene contracture test, which is considered indicative for MH susceptibility, was found in more than 40% of individuals who survived EHS, whereas less than 1% of the general population exhibited this response [44]. It is important to note that adequate recovery time must be given before performing such tests after a bout of rhabdomyolysis. Muscle function takes considerable time to recover and, inferentially at least, so does muscle metabolism. Therefore, care must be taken before relying on post-HS muscle contracture tests. A possible underlying myopathy in EHS patients was demonstrated by Bendahan and coworkers [45], who found abnormal muscle energetics in such patients by using a 31P magnetic resonance spectroscopy. Therefore, there is some evidence to suggest that a subset of individuals with HS may also be predisposed to MH. Theoretically, this subset of individuals might continue to have a relatively high level of heat production even after collapse. They may also be at increased risk for EHS because an attenuated muscular efficiency can increase muscular caloric production at the same absolute work intensity. The fact that some individuals with HS might have a contribution of MH-type pathophysiology might also explain why there has been anecdotal evidence suggesting a possible benefit from dantrolene in selected cases.\nThe above studies indicate that the cooling rate was the only parameter among those evaluated that improved when dantrolene was administered after the development of HS [17-19]. However, this effect was not demonstrated in all studies. The diversity of results may be accounted for by the models investigated. When dantrolene was administered in an exercising model, it was found to be more effective than in a CHS model. This is not surprising because dantrolene inhibits muscle contraction, which does not play a role in the pathophysiology of CHS.\nCollectively, most of the studies suggest that dantrolene is not beneficial in reducing mortality rate when it is given for the treatment of HS. However, survival rates in EHS currently approach 95%, and the studies were not large enough to exhibit a reduced mortality [1,46]. Moreover, some reports suggest that dantrolene may even prevent death in cases of severe HS [15,17]. For example, one patient in the study conducted by Channa and coworkers [17] who received dantrolene made a complete recovery in spite of an initial core temperature of 44\u00b0C. An EHS patient described by Lydiatt and Hill [15] had a rectal temperature of 42\u00b0C for more than 30 min and responded only to dantrolene. Noteworthy, the authors did not state the upper recording limit of their thermometer. If a clinical thermometer has an upper limit of 42\u00b0C, then it will record 42\u00b0C when the actual temperature is 46\u00b0C. Despite successful cooling for 30 min, the thermometer still shows 42\u00b0C even though the temperature has been reduced by 4\u00b0C. Taking dantrolene at this instant will lead to an impressive, albeit misinterpreted, result.\nSome researchers indicated that the relative ineffectiveness of dantrolene in some of the studies may be related to an inadequate dose [21,47]. Dantrolene was administered in doses of 2\u20135 mg\/kg, which were selected according to recommended protocols with regard to the use of this agent in MH. However, the optimal dose of dantrolene in MH is still questionable. Although Britt [48] reported a complete recovery from MH when dantrolene was used at a dose of 6 mg\/kg, others [31] postulated that an increased dose of dantrolene may be associated with hepatotoxicity. The risk for hepatotoxicity is even more concrete in HS patients because the liver is commonly injured in HS and increasing the dose of dantrolene may worsen this hepatic damage. Nevertheless, hepatotoxicity has never been reported in HS victims who received dantrolene.\nDespite the absence of outcome-based data, it might nevertheless be pathophysiologically rational to try dantrolene in selected cases. Dantrolene may be beneficial where there is evidence of ongoing excessive heat production, such as in cases of HS that are accompanied by muscular rigidity. In these cases, the apparent HS may be misdiagnosed, and the patient may actually be suffering from MH or NMS. Finally, dantrolene might also have a role to play in limiting muscle injury and its consequences in rhabdomyolysis by impairing calcium release from the sarcoplasmic reticulum [49,50]. Loss of calcium homeostasis in muscle cells may be associated with muscle injury resulting from impairment in mitochondrial respiration and ATP production, activation of phospholipase A2 with production of leukotrienes and prostaglandins, increased production of free radicals, and activation of calcium-activated proteases [49]. Although there is no evidence that dantrolene reduces the extent of muscle injury in EHS, it was found to protect against muscle injury associated with exercise in several studies [51-54].\nDantrolene pretreatment reduced the severity of HS signs in an exercising animal model. This effect may be attributed to decreased muscular production of metabolic heat or to an unknown modulation of neurotransmitter release [20]. However, the effect of dantrolene on muscle contracture may result in limited physical capacity. Furthermore, the administration of dantrolene before HS is more theoretical than real because the incidence of EHS is negligible in comparison with the number of individuals who are engaged in physical activity and\/or are exposed to heat strain.\nConclusion\nTherapy with the accepted MH prophylactic dose of 2\u20135 mg\/kg resulted in a possible benefit from dantrolene treatment in HS in some studies, whereas in others it was not found to be effective. The limited literature does not support the routine use of dantrolene as an adjuvant cooling technique in the setting of HS. Nevertheless, because dantrolene does appear to increase cooling rate, furthur studies are advised for dantrolene use in severe cases or cases in which no improvement is observed with other cooling methods. Wider trials are still needed to evaluate the efficacy of dantrolene in reducing mortality, thereby justifying its use in HS. Noteworthy, dantrolene should not be used as a single cooling method, and its administration should only be in addition to the well established 'conventional methods'.\nCompeting interests\nNone declared.\nAbbreviations\nCHS = classic heat stroke; CNS = central nervous system; EHS = exertional heat stroke; HS = heat stroke; MH = malignant hyperthermia; NMS = neuroleptic malignant syndrome.","keyphrases":["heat stroke","dantrolene","cooling","hyperthermia","temperature","exertional heat stroke"],"prmu":["P","P","P","P","P","P"]}
{"id":"Ann_Biomed_Eng-2-2-1764593","title":"NFAT and NF\u03baB Activation in T Lymphocytes: A Model of Differential Activation of Gene Expression\n","text":"Mathematical models for the regulation of the Ca2+-dependent transcription factors NFAT and NF\u03baB that are involved in the activation of the immune and inflammatory responses in T lymphocytes have been developed. These pathways are important targets for drugs, which act as powerful immunosuppressants by suppressing activation of NFAT and NF\u03baB in T cells. The models simulate activation and deactivation over physiological concentrations of Ca2+, diacyl glycerol (DAG), and PKC\u03b8 using single and periodic step increases. The model suggests the following: (1) the activation NFAT does not occur at low frequencies as NFAT requires calcineurin activated by Ca2+ to remain dephosphorylated and in the nucleus; (2) NF\u03baB is activated at lower Ca2+ oscillation frequencies than NFAT as I\u03baB is degraded in response to elevations in Ca2+ allowing free NF\u03baB to translocate into the nucleus; and (3) the degradation of I\u03baB is essential for efficient translocation of NF\u03baB to the nucleus. Through sensitivity analysis, the model also suggests that the largest controlling factor for NFAT activation is the dissociation\/reassociation rate of the NFAT:calcineurin complex and the translocation rate of the complex into the nucleus and for NF\u03baB is the degradation\/resynthesis rate of I\u03baB and the import rate of I\u03baB into the nucleus.\nIntroduction\nNuclear factor of activated T cells (NFAT) and nuclear factor \u03baB (NF\u03baB) are Ca2+-dependent and PKC\u03b8-dependent transcription factors that mediate the immune response in T lymphocytes (T cells).8,16,25,44,49 In resting cells both NFAT and NF\u03baB reside primarily in the cytoplasm, NFAT in its phosphorylated form and NF\u03baB bound to the inhibitor I\u03baB. T cell receptor stimulation initiates a cascade of intracellular reactions causing an increase in the cytosolic calcium concentration and a resulting translocation of both NFAT and NF\u03baB to the nucleus.19,48 Both NFAT and NF\u03baB may bind to promoter regions of DNA and, in concert with other transcription factors, allow expression of genes responsible for the production of the interleukins IL-2 by NFAT50 and IL-8 by NF\u03baB.58\nIn the NFAT pathway (Fig.\u00a01a), Ca2+ activates the phosphatase calcineurin, which binds to and dephosphorylates NFAT.34,45,59 Dephosphorylation exposes nuclear localization signals,10 resulting in rapid translocation of the NFAT-calcineurin complex to the nucleus where it can participate in promoting gene transcription.51 The cytoplasm-to-nucleus translocation is opposed by kinases such as GSK-3 and possibly protein kinase A that block the Ca2+-calcineurin dephosphorylation of NFAT in the cytoplasm, or that rephosphorylate free NFAT in the nucleus.9,43 Association of NFAT with activated calcineurin inhibits the kinase activity; in the absence of activated calcineurin, dephosphorylated NFAT in the nucleus is rephosphorylated and exported. This futile shuttling of NFAT into and out of the nucleus results in little gene transcription, thus gene expression activated by NFAT requires continuously active calcineurin.54,62\nFigure\u00a01.\n(a) Schematic diagram of the mechanism of action of NFAT. At rest, NFAT is in its phosphorylated form (NFAT:Pi) and located mainly in the cytosol. Upon activation, a rise in [Ca]i activates calcineurin (C) which binds NFAT:Pi and dephosphorylates it, allowing its import into the nucleus where it can act on the DNA. (b) Schematic diagram of the mechanism of action of NF\u03baB. At rest, NF\u03baB is bound to its inhibitor I\u03baB mainly in the cytosol (NF\u03baB:I\u03baB). Activation of calcineurin (C) synergizes with PKC\u03b1 activating an early phase of the kinase IKK and activation of PKC\u03b8 activates a later phase of IKK which phosphorylates I\u03baB. It then dissociates from NF\u03baB allowing NF\u03baB to be imported into the nucleus where it can act on the DNA.\nIn contrast to NFAT, NF\u03baB is retained in the cytoplasm (Fig.\u00a01b) not by phosphorylation but by association with the inhibitory I\u03baB proteins.7 In resting cells, NF\u03baB and I\u03baB can dissociate in the cytoplasm and translocate to the nucleus.14 Reassociation of NF\u03baB and I\u03baB masks nuclear localization signals on both species resulting in nuclear export. NF\u03baB has little opportunity to bind to DNA during this shuttling and so little transcription occurs. Stimulation of the T cell receptor, however, initiates a cascade of reactions proceeding through Ca2+48 and PKC\u03b833,56 that results in activation of the I\u03baB kinase IKK. IKK phosphorylates I\u03baB associated with NF\u03baB, allowing the I\u03baB to dissociate from NF\u03baB and marking the I\u03baB for subsequent polyubiquitination and degradation by the proteasome.20,36 NF\u03baB is freed by this degradation, nuclear localization signals on NF\u03baB are exposed, and it is translocated to the nucleus where it can bind to DNA and promote gene transcription. I\u03baB is resynthesized when the calcium and PKC\u03b8 concentration decrease. Newly formed I\u03baB translocates to the nucleus where it binds to NF\u03baB and masks its nuclear localization signals. The NF\u03baB:I\u03baB complex is then exported to the cytoplasm thus completing the activation-deactivation cycle. Recently, Hoffman and co-workers showed that oscillations in the I\u03baB concentration are observed experimentally and modeled its mechanism.26\nIn many cells, interplay of the sources and sinks of Ca2+ results in oscillations in the intracellular calcium concentration when surface receptors are stimulated by agonist.11 In particular, T cells display calcium oscillations in response to T cell receptor activation.21 The transcription factors NFAT and NF\u03baB respond in different ways to the frequency of these oscillations, thus differentiating the activation of NFAT and NF\u03baB. In this way differential transcription factor activation and consequent gene expression is conferred through a ubiquitous second messenger, namely Ca2+.22 Calcium oscillations have also been shown to increase the level of NFAT activation at low levels of stimulation over a steady rise in Ca2+, increasing the efficiency of low-level signal detection.22 Furthermore, overexpression of calcineurin partially replaces the calcium requirement for NFAT activation, as does the expression of constitutively active calcineurin.39,15\nDefects in the activation of NFAT and NF\u03baB and expression of IL-2 and IL-8, respectively, have been implicated in a number of human diseases and conditions. In several patients, failure to activate NFAT was shown to cause severe combined immunodeficiency disease, SCID.23 Overexpression of IL-8 can cause psoriasis and rheumatoid arthritis.58 Other conditions related to defects in NFAT and NF\u03baB activation include asthma, allergy, inflammation, and septic shock.\nThese pathways also contain an important target for drugs such as cyclosporin A and FK506, which act as powerful immunosuppressants by blocking the activation of calcineurin\u2019s phosphatase activity,15,18,39 thereby suppressing activation of NFAT and NF\u03baB in T cells. Furthermore, the PKC\u03b8 inhibitor rottlerin can block PKC\u03b8 translocation to the immunological synapse (IS),57 which suppresses the activation of NF\u03baB in T cells. These properties make these drugs useful not only in combating immune system diseases but also in preventing the rejection of transplanted tissues. But the general immune system suppression that results with cyclosporin A and FK506 leads to severe side effects, such as secondary tumors and opportunistic infections,30 and progressive loss of renal function and neurotoxicity.5 Additionally, inhibition of transcription factor activation in other types of cells can have unintended and undesirable consequences.1\nTo understand better how signal transduction pathways function, to help find therapies and cures for immune system diseases, and to find better ways to control the immune system in transplant patients it would be useful to understand the NFAT and NF\u03baB pathways in a quantitative fashion.\nModels and Mechanisms\nWe have developed mathematical models for the action of NFAT and NF\u03baB that simulate the activation and deactivation of each factor by changes in intracellular calcium concentration. Two separate models have been developed, one for NFAT and one for NF\u03baB. We required both models to incorporate experimentally determined reaction pathways and to simulate activation and deactivation of the transcription factors through experimentally determined intermediates and over physiological calcium concentrations. Both models use parameters derived, as much as possible, from experimental observations (Tables\u00a01\u20134). We required the models to be capable of simulating the system response to calcium concentration oscillations and, in line with experimental observations,22 to show a different response in the activation of NFAT and NF\u03baB to changes in the frequency of the oscillations. We expect the NFAT model to show that oscillations in the concentration of calcium enhance the activity of NFAT at low calcium concentrations and that overexpression of calcineurin replaces the requirement for calcium in the activation of NFAT. For both the NFAT and NF\u03baB models we expect the models to require sustained calcium signaling for gene expression.TABLE\u00a01.NFAT rate constants.Rate constantValueSourcek10.0000256\u00a0s\u22121Estimatek20.00256\u00a0s\u22121Loh et\u00a0al.34k30.005\u00a0s\u22121Estimatek40.5\u00a0s\u22121Estimatek50.0019\u00a0s\u22121Shibasaki et\u00a0al.52k60.00092\u00a0s\u22121Shibasaki et\u00a0al.52k70.005\u00a0s\u22121Estimatek80.5\u00a0s\u22121Estimatek90.5\u00a0s\u22121Estimatek100.005\u00a0s\u22121Estimatek116.63\u00a0\u03bcM\u22121\u00a0s\u22121Adjusted to control rate of activation observed by Shaw et\u00a0al.51k120.00168\u00a0s\u22121Adjusted to control rate of deactivation observed by Beals et\u00a0al. 10k130.5\u00a0s\u22121to agree with similar reactions given by Klee et\u00a0al.32k140.00256\u00a0s\u22121Loh et\u00a0al.34k150.00168\u00a0s\u22121Adjusted to control rate of deactivation observed by Beals et\u00a0al.10k166.63\u03bcM\u22121s\u22121Adjusted to control rate of activation observed by Shaw et\u00a0al.51k170.0015\u00a0s\u22121Zhu and McKeon62k180.00096\u00a0s\u22121Zhu and McKeon62k191.0\u03bcM\u22123s\u22121Klee et\u00a0al.32; Kakalis et\u00a0al.29k201.0\u00a0s\u22121Klee et\u00a0al.32, Kakalis et\u00a0al.29k210.21\u00a0s\u22121Estimate and adjusted relative to k22 to give equal calcium ion concentrations in nucleus and cytoplasmk220.5\u00a0s\u22121EstimateTABLE\u00a02.Concentration and volume parameters.ParameterValueSourceCell diameter9000\u00a0nMEstimate (Alberts et\u00a0al.2)Nucleus diameter6000\u00a0nMEstimate (Alberts et\u00a0al.2)Nucleus volume (vn)113\u00a0\u03bcm3CalculatedCytosol volume (vc)269\u00a0\u03bcm3CalculatedResting cell [Ca2+]100\u00a0nMAlberts et\u00a0al.2Activated cell [Ca2+]1000\u00a0nMAlberts et\u00a0al.2Total calcineurin concentration60.0\u00a0nMCrabtree17Total NF\u03baB concentration52.9\u00a0nMCarlotti et\u00a0al.13Total NFAT concentration7.23\u00a0nMEstimateTotal I\u03baB concentration55.0\u00a0nMCarlotti et\u00a0al.13Number calcium ions required to activate each calcineurin molecule3Kakalis et\u00a0al.29Total PKC\u03b8 concentration2000\u00a0nMEstimateTotal PKC\u03b1 concentration1000\u00a0nMEstimateTABLE\u00a03.PKC\u03b1 membrane-binding parameters.ParameterDefinitionValueSourceKCaDissociation constant22.0\u00a0\u03bcMNalefski et\u00a0al.38hCalcium binding cooperativity1.5Nalefski et\u00a0al.38nStoichiometric coefficient2\u00a0mol\/molNalefski et\u00a0al.38KDAGDissociation constant10.2\u00a0nMAnanthanarayanan et\u00a0al.3[DAG]Diacyl glycerol concentration2000.0\u00a0nMMogami et\u00a0al.37tr2aI\u03baB constitutive mRNA synthesis1.54\u00a0\u00d7 10\u22126\u00a0\u03bcM\u00a0s\u22121Hoffmann et\u00a0al.25tr2I\u03baB inducible mRNA synthesis1.65\u00a0\u00d7 10\u22122\u03bc M\u22121\u00a0s\u22121Hoffmann et\u00a0al.25tr3I\u03baB mRNA degradation2.8\u00a0\u00d7 10\u22124\u00a0s\u22121Hoffmann et\u00a0al.26TABLE\u00a04.NF\u03baB rate constants.Rate constantValueSourcek50.0019\u00a0s\u22121Shibasaki et\u00a0al.52k60.00092\u00a0s\u22121Shibasaki et\u00a0al.52k191.0\u03bcM\u22123s\u22121Klee et\u00a0al.32; Kakalis et\u00a0al.29k201.0\u00a0s\u22121Klee et\u00a0al.32, Kakalis et\u00a0al.29k210.21\u00a0s\u22121Estimate and adjusted relative to k22\u00a0to give equal calcium ion concentrations in nucleus and cytoplasmk220.5\u00a0s\u22121Estimatek230.614\u00a0\u03bcM\u22121\u00a0s\u22121Carlotti et\u00a0al.14, Huang et\u00a0al.27k240.00184\u00a0s\u22121Carlotti et\u00a0al.14k250.002\u00a0s\u22121Adjusted to control rate of activation\/deactivation observed by Dolmetsch et\u00a0al.22k261.0\u00a0\u03bcM\u22121\u00a0s\u22121Adjusted to control rate of activation\/deactivation observed by Dolmetsch et\u00a0al.22k270.00026\u00a0s\u22121k28\/k27\u00a0=\u00a050; Carlotti et\u00a0al.14k280. 0134\u00a0s\u22121Estimated from Birbach et\u00a0al.12k290.010\u00a0s\u22121k30\/k29\u00a0=\u00a02, Carlotti et\u00a0al.14k300.02\u00a0s\u22121Estimatek310.000034\u00a0s\u22121Estimatek320.000034\u00a0s\u22121Estimatek330.02\u00a0s\u22121Estimatek340.000034\u00a0s\u22121Estimatek350.000000036\u00a0s\u22121Adjusted to agree with Trushin et\u00a0al.56k360.00008\u00a0s\u22121Adjusted to agree with Trushin et\u00a0al.56k370.0000016\u00a0s\u22121Adjusted to agree with Trushin et\u00a0al.56k380.0008\u00a0s\u22121Adjusted to agree with Trushin et\u00a0al.56k390.0016\u00a0s\u22121Adjusted to agree with Trushin et\u00a0al.56k400.0006\u00a0s\u22121Adjusted to agree with Trushin et\u00a0al.56k410.02\u00a0s\u22121Adjusted to agree with Yang et\u00a0al.60k420.000177\u00a0s\u22121Adjusted to agree with Yang et\u00a0al.60k430.02Estimated from Trushin et\u00a0al.56k440.009Estimated from Trushin et\u00a0al.56\nNFAT Model\nA schematic diagram of the mechanism for the action of NFAT is shown in Fig.\u00a01b. A rise in the cytosolic calcium concentration leads to an increased calcium\u2013calmodulin concentration which activates calcineurin (denoted by C*). Calcineurin binds to and dephosphorylates the cytosolic species NFAT:Pi, the phosphorylated form of NFAT.40,46 Without calmodulin, the phosphatase activity of calcineurin is much reduced.31 The dephosphorylated NFAT-calcineurin complex (NFAT:C*) translocates to the nucleus, where it may bind to promoter regions of DNA and assist in initiation of gene transcription. A lowering of the calcium concentration results in deactivation of calcineurin and its dissociation from the NFAT-calcineurin complex. Free nuclear NFAT may then be phosphorylated by ubiquitous kinases, masking nuclear localization signals and resulting in translocation from the nucleus to the cytoplasm. Thus rising and falling calcium ion concentrations result in activation and deactivation of the transcription factor NFAT.\nThere are several simplifying assumptions made in the model for NFAT activation: (1) the total cellular concentration of NFAT remains constant, that is, the degradation and synthesis rates are balanced; (2) dephosphorylated NFAT bound to calcineurin in the nucleus is the transcriptionally active form of NFAT; (3) calcineurin dephosphorylates NFAT in one composite dephosphorylation site (i.e. all sites are treated as one); (4) The cytosolic and nuclear free calcium concentrations are equal and are controlled by a calcium clamp protocol simulating the procedure used by Dolmetsch et\u00a0al.;22 (5) calcineurin binds three calcium ions to become active; and (6) NFAT and calcineurin clearly bind to other molecules in the cell, however, these interactions are ignored for simplicity.\nA reaction scheme consistent with this mechanism is given in Fig.\u00a02a. Here nuclear species are shown in the lower half of the figure and given the subscript n. Cytosolic species are shown in the upper half of the figure and given the subscript c. At the low calcium concentrations present in the resting cell the dominant NFAT-containing species is phosphorylated NFAT in the cytoplasm, designated (NFAT:Pi)c (Fig.\u00a02a \u2013 dark gray box at left). At the high calcium concentrations present in the activated cell the dominant species is NFAT associated with activated calcineurin in the nucleus, (NFAT:C*)n (Fig. 2a \u2013 light gray box at right). The model treats cytosolic and nuclear entities as distinct, and thus includes 12 separate chemical species, and cytosolic and nuclear Ca.2+. The arrows between species represent forward and reverse chemical reactions, or nuclear import and export. The rates of reaction, and of import and export, are represented by the 22 rate constants, designated k1 to k22.\nFigure\u00a02.\n(a) Reaction scheme for the NFAT mechanism. The activation path for NFAT is shown by the light gray arrows. An increasing calcium concentration activates calcineurin (C), which binds to and dephosphorylates NFAT:Pi (dark gray box). The NFAT-activated calcineurin complex NFAT:C* is imported to the nucleus where it may be transcriptionally active (light gray box). The deactivation path for NFAT is shown by the dark gray arrows. A decreasing calcium concentration allows dissociation of NFAT and activated calcineurin (NFAT:C*) in the nucleus (light gray box). Free NFAT is phosphorylated and exported to the cytoplasm (dark gray box). (b) Reaction scheme for the NF\u03baB mechanism. The activation path for NF\u03baB is shown by light gray arrows. An increase in the calcium ion concentration activates calcineurin in synergy with PKC\u03b1 which, through a sequence of reactions, activates an early phase of the kinase IKK. Activation of PKC\u03b8 leads to activation of a later phase of IKK. IKK phosphorylates the NF\u03baB:I\u03baB complex (dark gray box) which then dissociates. The phosphorylated I\u03baB is degraded and the free NF\u03baB imported into the nucleus to be the transcriptionally active form (light gray). The deactivation path for NF\u03baB is shown by dark gray arrows. A decrease in the calcium ion concentration allows resynthesis of I\u03baB:Pi, which enters the nucleus and associates with NF\u03baB. The resulting complex dephosphorylates and is exported from the nucleus.\nUsing the law of mass action, this reaction scheme yields a system of 12 coupled first order differential equations describing the change in concentration with time for each of the 12 distinct chemical species (Appendix A). The parameters in the equations include the 22 rate constants and the number of calcium ions required to activate the phosphatase activity of each calcineurin molecule, designated by m in the figure. The time course for the concentrations of each of the chemical species from a given initial condition is determined by numerical solution of the system using a standard fourth order Runge\u2013Kutta technique.\nTable\u00a01 lists the rate constants used in the NFAT model. They may be loosely divided into three groups: (1) calculated from published data, (2) estimated from published data, and (3) adjusted to fall within expected physiological ranges in order to make the model conform to published experimental data.\nNFkB Model\nA schematic diagram of the mechanism of action of NF\u03baB is shown in Fig.\u00a01b. As previous studies have shown, PKC\u03b8 and calcineurin act as co-activators of the transcription factor NF\u03baB.55,56 A high calcium concentration activates calcineurin and PKC\u03b1, which through a sequence of reactions, co-activates the IKK complex with PKC\u03b8. TCR\/CD3 activation leads to the translocation of PKC\u03b8 to the immunological synapse (IS), and CD28 activation leads to PKC\u03b8 migration to the central supramolecular activation cluster (cSMAC) in the IS.28 IKK phosphorylates I\u03baB in the complex NF\u03baB:I\u03baB, leading to immediate recognition of I\u03baB by the F-box\/WD-domain protein E3RS,I\u03baB61 polyubiquitination of the I\u03baB20 and subsequent degradation of the I\u03baB by the proteasome. Our model does not explicitly include the polyubiquitination but includes it in the degradation rate of I\u03baB which is essential for NF\u03baB translocation and hence for gene transcription. Once freed of I\u03baB, NF\u03baB translocates to the nucleus where it may bind to DNA and promote gene transcription. When the calcium concentration falls to resting levels, newly synthesized I\u03baB rapidly translocates to the nucleus where it combines with NF\u03baB, masking nuclear localization signals on both species.6 The NF\u03baB:I\u03baB complex is then exported from the nucleus to the cytoplasm.\nThere are several simplifying assumptions used to render the model computationally tractable: (1) the total cellular NF\u03baB concentration is constant due to a balance between its synthesis and degradation processes; (2) I\u03baB production is enhanced by activated NF\u03baB as described by Hoffman et\u00a0al.;26 (3) the cytosolic and nuclear free calcium concentrations are equal and are controlled by a calcium clamp protocol simulating the procedure used by Dolmetsch et\u00a0al.;22 (4) the IKK kinases are lumped into one reaction; (5) in the model I\u03baB represents I\u03baB\u03b1; (6) the amount of free NF\u03baB in the nucleus is used as a measure of the activation of the transcription factor; (7) calcineurin binds 3 calcium ions to become active; and (8) the binding of calcineurin, I\u03baB, and NF\u03baB to other molecules in the cell clearly occurs, but is not included in the model for simplicity.\nA reaction scheme consistent with this schematic diagram is shown in Fig.\u00a02b. As in the reaction scheme for NFAT, nuclear species are shown in the lower half of the figure and given the subscript n. Cytosolic species are shown in the upper half of the figure and given the subscript c. In the resting cell, the dominant NF\u03baB containing species is NF\u03baB:I\u03baB in the cytoplasm (Fig.\u00a02b \u2013 dark gray box at right). In the active cell, the dominant species is NF\u03baBn (Fig. 2b \u2013 light gray box at left). The NF\u03baB model has 17 different chemical species with the nuclear and cytosolic concentrations represented separately. As in the NFAT model, the arrows between species represent forward and reverse chemical reactions, or nuclear import and export. The rates of reaction, and of import and export, are represented by the 26 rate constants, designated k5 and k6 and k19 to k42. Note that k41 represents the rate of degradation of I\u03baB and k42 the rate of its resynthesis.26 From the NF\u03baB reaction scheme a system of 17 coupled first order differential equations may be written, one for the change in concentration with time of each of the 17 distinct chemical species (Appendix B). Given the initial concentration of each species, numerical integration of the system yields the time course of the concentration for each species.\nResults and Discussion\nA number of simulations were performed to duplicate experimental results to validate the model. In all simulations, the parameters listed in Tables\u00a01\u20134 were used unless stated in the figure legends. The only difference in the simulated results was the calcium clamp protocol used.\nNFAT Model Results\nUsing the parameter values in Tables\u00a01 and 2, the model was allowed to come to steady state at rest ([Ca]i\u00a0=\u00a00.1\u03bcM). The resting state concentrations are shown in Table\u00a05. These concentrations were used as initial values when simulating the evolution of the concentration of each species during a transition from the resting state ([Ca]i\u00a0=\u00a00.1\u00a0\u03bcM) to the active state ([Ca]i\u00a0=\u00a01.0\u00a0\u03bcM). The results are shown in Fig.\u00a03a. The high calcium concentration activates calcineurin, which associates with and dephosphorylates NFAT:Pi in the cytoplasm; the concentration of NFAT:Pi in the cytoplasm falls rapidly. Only a small amount of NFAT:Pi:C* is formed in the cytoplasm when activated calcineurin associates with NFAT:Pi because it is rapidly dephosphorylated to NFAT:C*, which in turn is rapidly translocated to the nucleus where it is transcriptionally active. Note in this figure that the change in concentration of the nuclear species appears large relative to the concentrations of the cytosolic species because the volume of the nucleus is smaller than the volume of the cytoplasm by a factor of about 2.4. The light gray arrows in Fig.\u00a02a show the NFAT activation pathway on the reaction scheme for NFAT. Calcineurin associates with calcium ion, activating calcineurin. The activated calcineurin associates with phosphorylated NFAT and dephosphorylates it. The NFAT:C* resulting from this dephosphorylation is rapidly imported to the nucleus where it is transcriptionally active.\nFigure\u00a03.\n(a) Activation of NFAT from the resting state. (b) Deactivation of NFAT from the active state. The reaction paths (Fig.\u00a02a) for the activation and deactivation of NFAT are determined by observing the sequence of the formation of the various species in the reaction scheme.\nTABLE\u00a05.\nNFAT resting steady-state concentrations.\nSpecies\nConcentration (nM)\nNFATn\n0.5219\nNFATc\n0.1101\nC*n\n0.0505\nC*c\n0.0091\n(NFAT:Pi)n\n0.2272\n(NFAT:Pi)c\n9.4397\n(NFAT:Pi:C*)n\n0.0025\n(NFAT:Pi:C*)c\n0.0022\n(NFAT:C*)n\n0.9477\n(NFAT:C*)c\n0.0061\nCn\n49.198\nCc\n9.7108\nCan2+\n100\nCac2+\n100\nA simulation of the deactivation of the activated state is shown in Fig.\u00a03b. Here the initial concentrations were those of the activated steady state, and the calcium ion concentration was set to 0.1\u00a0\u03bcM. Figure\u00a03b shows that the concentration of the transcriptionally active species (NFAT:C*)n falls rapidly as NFAT and calcineurin dissociate. Consequently the concentration of NFATn rises as NFAT is freed by the dissociation, and then falls as it is phosphorylated. The concentration of the phosphorylated species (NFAT:Pi)n never rises very high because this species is rapidly exported from the nucleus. The dark gray arrows in Fig.\u00a02a show the NFAT deactivation pathway on the reaction scheme for NFAT. After (NFAT:C*)n dissociates, free NFAT is phosphorylated and exported from the nucleus.\nDolmetsch et\u00a0al.22 suggest that non-linearity in the response of NFAT to calcium ion concentration is important in determining how the activity of NFAT depends on oscillations in the calcium ion concentration. Their experiments show that oscillation frequency is the critical factor in differentiating the response of NFAT and NF\u03baB. Our models for both NFAT and NF\u03baB simulate the effect of calcium ion concentration oscillations on the activation of the transcription factors. One such simulation for NFAT is shown in Fig.\u00a04a. The initial concentrations of the species were those of the resting state and the oscillation period of the calcium ion concentration was 100\u00a0s. The calcium ion concentration was fixed at 1.0\u00a0\u03bcM during the initial 10\u00a0s and 0.1\u00a0\u03bcM during the final 90\u00a0s of each oscillation. Figure\u00a04a shows that the concentration of the transcriptionally active (NFAT:C*)n rises rapidly during the high calcium concentration pulse and then decays more slowly during the low calcium concentration portion of the period. The concentration of (NFAT:Pi)c follows the reverse trend, decreasing rapidly when the calcium ion concentration is high and increasing slowly when the concentration is low. A fraction of the NFAT exists as free NFAT in the nucleus, oscillating between the form NFATn and (NFAT:C*)n as calcineurin associates with and dissociates from NFAT. We can easily see that in the absence of constant signaling the concentration of transcriptionally active (NFAT:C*)n would soon fall to resting state levels. This is shown in Fig.\u00a04a where at 1500\u00a0s the calcium ion concentration is set to 0.1\u00a0\u03bcM. The concentration of the transcriptionally active (NFAT:C*)n falls and that of (NFAT:Pi)c rises, both approaching their resting levels as time increases.\nFigure\u00a04.\n(a) Activation of NFAT by oscillations in the calcium ion concentration. During each period the calcium concentration was high (1.0\u00a0\u03bcM) for 10\u00a0s and low (0.1\u03bcM) for 90\u00a0s. At 1500\u00a0s the calcium concentration was set to the resting state value (0.1\u00a0\u03bcM) and the system returned to the resting state. (b) The percent of NFAT in the active state is plotted as a function of calcium ion oscillation period. (c) The percent of NFAT in the active state is plotted as a function of average calcium ion concentration for steady state and oscillating calcium ion concentrations. The percent of NFAT in the active steady state decreases below 0.4\u00a0\u03bcM when the calcium concentration is constant. The percent of NFAT in the active state persists at a high level to less than 0.2\u00a0\u03bcM average concentration when the calcium concentration oscillates.\nTo study how the degree of NFAT activation varies with oscillation frequency, we simulated calcium concentration oscillations using 10\u00a0s intervals of high concentration and varying intervals of low concentration, with the total period ranging from 100 to 1800\u00a0s (Fig.\u00a04b). Because the concentration of the transcriptionally active (NFAT:C*)n varies throughout the period, a sufficient number of periods was simulated to reach a stable state, then the lowest value of (NFAT:C*)n during the last period was chosen to calculate the percent of NFAT in the transcriptionally active form. We reasoned here that NFAT involved in shuttling in and out of the active form was not likely to bind to DNA and assist in initiation of transcription, thus the lowest value of the concentration of the active form was justified. In this way the fraction of NFAT in the transcriptionally active form was found as a function of the oscillation period. Figure\u00a04b shows that the percent of NFAT that is transcriptionally active is almost 60% with a 100\u00a0s period, and that this fraction falls to less than 10% with a 900\u00a0s period. These results agree qualitatively with the results presented by Dolmetsch et\u00a0al.,22 in their Fig.\u00a03b for the percent of gene expression as a function of oscillation period. Their data shows 50% gene expression with a 100\u00a0s oscillation, with the percent expression falling to zero for periods of 400\u00a0s or more. The quantitative differences between our simulated results and the experimental observations by Dolmetsch et\u00a0al.22 possibly occur because they have determined percent gene expression while we calculate percent of NFAT that is transcriptionally active. There might be additional non-linearity or cooperativity introduced in transcription factor binding to DNA and transcription. For example, as mentioned earlier, Fiering et\u00a0al.24 observed that the binding of 3 NFAT molecules to a constructed promoter on a DNA strand produced cooperativity of transcription. They also suggested a similar requirement for NF\u03baB activation of gene expression. While inclusion of these factors would result in a more quantitatively accurate reproduction of the reporter gene expression data, we chose to simplify the model to emphasize the mechanisms for activation of the transcription factors.\nDolmetsch et\u00a0al.22 show that the percent of cells expressing a lacZ+ reporter gene, sensitive to transcriptionally active NFAT, decreased when the steady-state calcium concentration fell below about 0.35\u00a0\u03bcM. But if the calcium concentration exhibited oscillations, the gene expression persisted to an average calcium concentration at least as low as 0.2\u00a0\u03bcM. The model displays similar behavior (Fig.\u00a04c). To simulate oscillations, 10\u00a0s periods of high calcium concentration and 90\u00a0s periods of low calcium concentration were used. The low calcium concentration was fixed at 0.1\u00a0\u03bcM, and the concentration during the high calcium intervals was fixed in each of several tests to produce a set of experiments each having a different average calcium concentration. Model data for the steady state was obtained following the protocol described previously. Figure\u00a04c shows that simulating oscillations does indeed enhance the activity of NFAT at low calcium concentrations relative to the activity at a constant calcium concentration. The shape of the curves representing steady state and oscillating calcium concentration behavior are quite similar to those shown by Dolmetsch et\u00a0al.,22 in their Fig.\u00a02c. The two calculated curves meet at a calcium concentration of 0.39\u00a0\u03bcM, very close to the 0.35\u00a0\u03bcM shown in the experimental work. This corresponds to a period of approximately 30\u00a0s. If the period is longer than 30\u00a0s (the average calcium is lower than 0.39\u00a0\u03bcM), then the oscillations are more potent than steady-state calcium with the same average at activating NFAT. If higher, the opposite is true. The model data shows enhanced NFAT activity down to about 0.2\u00a0\u03bcM, below which the activity decreases rapidly.\nFinally, the NFAT model was tested to determine if it correctly simulates the observation that overexpression of calcineurin partially replaces the requirement for calcium, as reported by Luo et\u00a0al.35 These simulations were carried out by holding the calcium ion concentration fixed at the resting state value, 0.1\u00a0\u03bcM, and varying the calcineurin concentration, followed by calculating the degree of activation of the NFAT. Figure\u00a05 shows the results obtained when the calcineurin concentration was increased as much as a factor of 25 times above the baseline concentration. The percent of NFAT in the transcriptionally active form does indeed increase when the calcineurin concentration increases, and high levels of activation are achieved with high calcineurin concentrations.\nFigure\u00a05.\nPercent active NFAT as a function of calcineurin concentration at fixed low calcium ion concentration. The calcium concentration was fixed at a level characteristic of the resting state (0.1\u00a0\u03bcM) and the calcineurin concentration increased to simulate overexpression of calcineurin. Each data point shows the steady state concentration of transcriptionally active NFAT at a fixed calcineurin concentration. Overexpression of calcineurin replaces the requirement for calcium at fixed low calcium concentration.\nNF\u03baB Model Results\nThe NF\u03baB model was characterized in a manner similar to that for the NFAT model. Using the parameters from Tables\u00a02\u20134, the calcium concentration was fixed at 0.1\u00a0\u03bcM and the time evolution of the concentration of each species calculated until steady state values were observed. These concentrations were taken to be those of the steady state at rest (Table\u00a06).TABLE\u00a06.NF\u03baB resting steady-state concentrations.SpeciesConcentration (nM)NF\u03baBn7.1779NF\u03baBc0.0731(I\u03baB:Pi)n0.2035(I\u03baB:Pi)c0.0525(NF\u03baB:I\u03baB:Pi)n0.2343(NF\u03baB:I\u03baB:Pi)c0.1076(NF\u03baB:I\u03baB)n0.2376(NF\u03baB:I\u03baB)c52.961C*n0.0505C*c0.0091Cn49.198Cc9.7108100100\nFigure\u00a06a shows the concentrations of species in the NF\u03baB model during a transition from the resting to active states. Starting with the resting steady state concentrations, the calcium ion concentration was fixed at 1.0\u00a0\u03bcM and the time evolution of the concentration of each species calculated. The high calcium concentration activates calcineurin, which in synergy with PKC\u03b1 and PKC\u03b8, co-activates the kinase IKK. The activated kinase phosphorylates I\u03baB associated with NF\u03baB in the cytoplasm. Subsequent polyubiquitination and I\u03baB degradation result in a decrease in the cytoplasmic NF\u03baB:I\u03baB:Pi concentration, a dissociation of NF\u03baB and I\u03baB, and rapid translocation of NF\u03baB into the nucleus. The concentration of cytosolic NF\u03baB never rises very high because of its rapid nuclear import, therefore the concentration of this species is not shown in Fig.\u00a06a. The light gray arrows in Fig.2b show the NF\u03baB activation pathway on the reaction scheme for NF\u03baB. A rise in the calcium concentration activates calcineurin, which through a sequence of reactions, and in synergy with PKC\u03b1, activates an early phase of the kinase IKK. CD3\/CD28 activate PKC\u03b8, leading to activation of a later phase of IKK.56 Activated IKK phosphorylates the NF\u03baB:I\u03baB complex in the cytoplasm. After dissociation, the phosphorylated I\u03baB is polyubiquitinated and degraded, and free NF\u03baB is imported to the nucleus where it may be transcriptionally active.\nFigure\u00a06.\n(a) Activation of NF\u03baB from the resting state. (b) Deactivation of NF\u03baB from the active state. The sequence of events depicted in Fig.\u00a02b can be determined by observing the time evolution of the concentration of the different reaction species.\nDeactivation of the activated state of NF\u03baB is shown in Fig.\u00a06b. The initial concentrations were those of the activated state at equilibrium, and the calcium ion concentration was fixed at 0.1\u00a0\u03bcM. The most important step in deactivation is synthesis of new I\u03baB, which is rapidly translocated to the nucleus forming the phosphorylated complex with NF\u03baB. This complex is dephosphorylated and rapidly translocated into the cytoplasm. The result is an overall increase in concentration of NF\u03baB:I\u03baB in the cytoplasm as the concentration of NF\u03baB in the nucleus decreases. The dark gray arrows in Fig.\u00a02b show the pathway for deactivation on the reaction scheme for NF\u03baB. Newly synthesized I\u03baB is imported to the nucleus where it associates with NF\u03baB. The resulting complex dephosphorylates, and the NF\u03baB:I\u03baB complex is exported to the cytoplasm. As for NFAT, Dolmetsch et\u00a0al.,22 have studied the degree of gene expression caused by NF\u03baB as a function of steady-state calcium ion concentration. In their paper, Fig.\u00a03a shows that NFAT and NF\u03baB behave in a similar manner; they exhibit a highly non-linear sigmoidal dependence, with very little gene expression at calcium ion concentrations of 0.1\u00a0\u03bcM or less, a rapid rise in gene expression at calcium concentrations between 0.1\u00a0\u03bcM and 0.5\u00a0\u03bcM, and near 100% gene expression at calcium concentrations over 0.5\u00a0\u03bcM. Since we follow the calcium clamp protocols used by Dolmetsch and co-workers, which does not involve CD3 or CD28 activation and hence does not involve activation of PKC\u03b8, we do not activate PKC\u03b8 in these simulations. Figure\u00a07 shows our simulated results for NF\u03baB without PKC\u03b8 activation, with the percent of NF\u03baB that is transcriptionally active plotted as a function of the calcium ion concentration. These results were obtained by setting the calcium ion concentration to the desired value, then calculating the concentration of transcriptionally active NF\u03baB as a function of time until a steady state was reached. There is good agreement between our simulated behavior and the experimental results of Dolmetsch et\u00a0al.22 Both show activation of NF\u03baB over the physiological range of calcium ion concentrations, with little activity at low concentrations and full activity at high concentrations. The sigmoidal shape of the calculated curve agrees with the experimental results.\nFigure\u00a07.\nDependence of the percent active NF\u03baB on the steady-state calcium concentration. For each data point the calcium ion concentration was fixed at the indicated value.\nAs was done for the NFAT model, the NF\u03baB model will simulate the system response to calcium oscillations. An example of NF\u03baB activation by calcium concentration oscillations is shown in Fig.\u00a08a. The initial concentrations were those of the resting steady state and the oscillation period was 100\u00a0s. The calcium ion concentration was fixed at 1.0\u00a0\u03bcM during the initial 10\u00a0s of each period and at 0.1\u00a0\u03bcM for the remaining 90\u00a0s of each period. When the calcium concentration is high, the concentration of NF\u03baB:I\u03baB in the cytoplasm falls rapidly, and when the calcium concentration is low the concentration of this species increases slowly. The concentration of NF\u03baB in the nucleus follows the reverse trend, increasing rapidly during the periods of high calcium concentration and falling more slowly when calcium concentration is low.\nFigure\u00a08.\n(a) Activation of NF\u03baB by oscillations in the calcium ion concentration. During each period the calcium concentration was high (1.0\u00a0\u03bcM) for 10\u00a0s and low (0.1\u00a0\u03bcM) for 90\u00a0s. The concentration of the NF\u03baB:I\u03baB complex in the cytoplasm falls rapidly during periods of high calcium concentration. The concentration of the phosphorylated NF\u03baB:I\u03baB complex first rises, then falls as I\u03baB is degraded. Free NF\u03baB is imported to the nucleus, steadily increasing the concentration of the transcriptionally active nuclear NF\u03baB. (b) Differentiation of the activity of NFAT and NF\u03baB by calcium ion oscillation period. The activity of NF\u03baB decreases as the oscillation period increases (upper curve) but not as much as for NFAT (lower curve); the percent active NF\u03baB is greater than that of NFAT for a given calcium concentration oscillation period. This dissimilarity in behavior differentiates the response of NFAT and NF\u03baB to calcium concentration oscillations.\nDuring the calcium oscillations, the concentration of the transcriptionally active nuclear NF\u03baB rises, showing little oscillation because of the relatively slow rate of the polyubiquitination and I\u03baB degradation steps. In the absence of constant signaling the concentrations of the species return to their resting levels. In Fig.\u00a08a the concentration of Ca2+ is set to 0.1\u03bcM at 2000\u00a0s, after which the concentration of nuclear NF\u03baB falls, and that of (NF\u03baB:I\u03baB)c rises, to levels characteristic of the resting state.\nWe studied how the degree of NF\u03baB activation varied with calcium concentration oscillation period as we did for the NFAT model. Figure\u00a08b shows the results of simulations with periods varying from 100 to 2000\u00a0s. For the shortest period, the percent of active NF\u03baB was 70%, and at the longest period the percent active NF\u03baB was 20%. These results agree qualitatively with those shown by Dolmetsch et\u00a0al.,22 in their Fig.\u00a03b. Their figure shows over 90% gene expression with a 100\u00a0s period, falling to less than 20% gene expression for an 1800\u00a0s period. Note that they have determined percent gene expression while we calculate percent of transcriptionally active NF\u03baB, a difference that may explain the quantitative difference in the results.\nFigure\u00a08b combines the model results for NFAT and NF\u03baB to compare the effect of oscillation frequency on activation of NF\u03baB to the activation of NFAT by including the curve from Fig.\u00a04b Figure\u00a08b shows that a higher fraction of the NF\u03baB is transcriptionally active than that of NFAT for long oscillation periods. These results agree qualitatively with those shown in Dolmetsch et\u00a0al.,22 Fig.\u00a03b, where gene expression by NFAT declined rapidly with oscillation frequency but persisted to long periods for NF\u03baB.\nFigure\u00a09a simulates the results of Yang et\u00a0al.,60 who studied the degradation of I\u03baB\u03b1 with time in fibroblasts. To simulate this, we have used Ca2+ oscillations with a period of 100\u00a0s and assumed that degradation is the same for all members of the I\u03baB family and then plotted the percent of I\u03baB remaining with time. We have also assumed that the active PKC\u03b8 and PKC\u03b1 concentrations drop after 30\u00a0min based on the experimental observations of Szamel et\u00a0al.53 who report that PKC\u03b8 and PKC\u03b1 translocated rapidly to the membrane within 1\u201310\u00a0min and start to decline after 30\u00a0min. This was simulated with the model by reducing the amounts of active PKC\u03b8 and PKC\u03b1 concentrations to resting values at 40\u00a0min (Fig.\u00a09a). The simulation shows that there is near complete degradation of I\u03baB in about 30\u00a0min, which agrees with the experimental results shown in Yang et\u00a0al.60 in their Fig.\u00a02. They observed an increase in I\u03baB concentration after about 40\u00a0min.\nFigure\u00a09.\n(a) Degradation of I\u03baB with time during Ca2+ oscillations with period 100\u00a0s. Activation of PKC\u03b8 and PKC\u03b1 is terminated at 40\u00a0s. (b) Percent active NF\u03baB as a function of the rate of degradation of NF\u03baB. At steady state and at high calcium concentration the percent of active NF\u03baB is sensitive to the rate of I\u03baB degradation assumed in the model. As the degradation rate decreases the percent of active NF\u03baB decreases. Thus the degradation rate must be sufficiently high to obtain significant activation of NF\u03baB. The NF\u03baB model uses a degradation rate of 0.02\u00a0s\u22121.\nFigure\u00a09b shows that the rate of degradation of I\u03baB is an essential factor in the activation of NF\u03baB. Here the dependence of the percent active NF\u03baB is shown as a function of the I\u03baB constitutive degradation rate, k41 in the NF\u03baB model. Our NF\u03baB model uses a degradation rate of 0.02\u00a0s\u22121. As the degradation rate decreases below this value the percent of active NF\u03baB decreases, thus a sufficiently high I\u03baB degradation rate is required for efficient NF\u03baB activation.\nSensitivity Analysis\nTo further characterize the system, a sensitivity analysis was performed to determine which steps in the reaction schemes for NFAT and NF\u03baB exerted the most influence on the activation of the transcription factors. To this end, each rate constant was increased and decreased by 10%. The percent change of steady-state active transcription factor at 1.0\u00a0\u03bcM calcium over the control was calculated (Fig.\u00a010). The sensitivity was calculated by\nFigure\u00a010.\n(a) Sensitivity analysis for NFAT activation. (b) Sensitivity analysis for NF\u03baB activation. In both cases, the rate constants were increased and decreased by 10%. The sensitivity of the steady state concentration of active transcription factor at 1.0\u00a0\u03bcM calcium to changes in the rate constants is shown.\nFor NFAT (Fig.\u00a010a), the rate constants that seem to exert the greatest effect on activation are k9, k10, k13, k14, k15, k16,k19, and k20. The rate constants k9 and k10 control the rate of nuclear import and export of NFAT and the rate constants k13 and k14 control the phosphorylation and dephosphorylation of NFAT by calcineurin. The sensitivities here suggest that increasing the binding affinity of NFAT and calcineurin (k15 and k16) will increase activation of NFAT. This is consistent with mutational studies that demonstrate that alteration of the calcineurin binding site on NFAT decreases activation of NFAT.4,41 It is interesting to note that even though the sensitivities of NFAT activation to changes in k11 and k12 are in the expected directions, the values of these sensitivities are relatively small. Also, increasing the rate of activation of calcineurin through calcium binding (k19 and k20) will also increase activation of NFAT. Other rate constants that exert a small but still significant effect are k5 and k6 suggesting that an increase in the amount of free cytosolic calcineurin will increase activation of NFAT. This is consistent with Fig.\u00a05 and the experiments that demonstrate that increasing calcineurin increases the activation of NFAT.\nFor NF\u03baB (Fig.\u00a010b), the rate constants k30, k41, k42 and tr3 exert the greatest control on the activation of NF\u03baB. The rate constants k23, k24, k25, k26, k27, k28, k43k44, tr2 and tr2a also exert a significant influence on activation of NF\u03baB. The rate constants k41 and k42 control the degradation and synthesis of I\u03baB. The rate constants tr2, tr2a and tr3 control the rate of I\u03baB mRNA synthesis and degradation. The importance of these rate constants re-emphasizes the importance of the removal of free I\u03baB from the cystosol for activation of NF\u03baB as indicated by Fig.\u00a09. The rate constants k43 and k44 control the shuttling of IKK between the cytoplasm and the nucleus. The rate constants k30 and k29 control the import of I\u03baB into the nucleus. Preventing the import of I\u03baB into the nucleus increases the activation of NF\u03baB as it lowers the amount of I\u03baB available to bind NF\u03baB which leads to its export from the nucleus. The rate constants k23 and k24 control the dissociation of NF\u03baB and I\u03baB. Increasing the dissociation rate increases activation of NF\u03baB. The effect of the rate constants k25 and k26 demonstrate that increasing the rate of phosphorylation of I\u03baB by IKK increases the activation of NF\u03baB. Finally, decreasing k27, the rate constant for export of NF\u03baB out of the nucleus, or increasing k28, the rate of import NF\u03baB to the nucleus, increases the amount of activation of NF\u03baB. This effectively increases the amount of NF\u03baB in the nucleus. It is interesting that although reducing the rate of phosphorylation of IKK by calcineurin does inhibit NFkB translocation to the nucleus, this is not one of the most sensitive parameters. The sensitivity analysis suggests that there are several other targets that will block NFAT and NF\u03baB translocation. For several of the targets on NFAT, there is experimental data to support the model predictions about the sensitivities. On the other hand, the prediction that blocking of NFAT translocation decreases transcription has yet to be verified experimentally. This might be accomplished by developing a compound that either binds the nuclear import site of NFAT or that blocks the import mechanism. For NF\u03baB the prediction of this analysis is that the most effective target would be to block I\u03baB degradation or increase its re-synthesis. Another potent target would be to block NF\u03baB translocation. Other possible interventions to block NF\u03baB translocation would be to introduce compounds that increase the affinity of NF\u03baB for I\u03baB or that increase the activity of IKK. These are not the only possible sites as many accessory pathways regulate this system. Future studies that build on this model and explore these pathways will give further insight.\nConclusions\nWe have developed models for the calcium and PKC\u03b8 mediated activation and deactivation of the transcription factors NFAT and NF\u03baB that incorporate experimentally determined reaction pathways and that simulate this action over physiological calcium concentrations. The model reproduces experimentally observed behaviors of both the NFAT and NF\u03baB systems under a variety of conditions.\nSeveral parameters values for T cells were not available and therefore were derived from experimental observations in cells other than the T cell. For the most part these choices are reasonable estimates, but should be examined further as new data on signaling during T cell activation becomes available. Variations in some estimates will have a greater influence on model outcomes than the others. For example, the total concentration of NF\u03baB was taken from fibroblast data,13 but this mostly affects the amount activated rather than the percent activated yielding a small control coefficient (0.0127). On the other hand, sensitivity analysis indicates that the degradation rate of I\u03baB that was estimated using data from fibroblasts60 would exert a greater influence on model results and thus its choice is more critical.\nWhile the model is qualitatively accurate, simulation of the reporter gene levels observed by Dolmetsch and colleagues might be more closely approximated by the inclusion of additional features to the model. Several of these enhancements are obvious and consist of including elements both upstream and downstream of the system modeled. For example, binding of the transcription factors to the DNA might be added along with steps to describe the expression of the reporter genes observed in the experiment. Inclusion of these steps would require adding significant complexity to the model that might obstruct demonstration of the mechanisms governing activation of the calcium dependent transcription factors NFAT and NF\u03baB. Thus, addition of these steps has been left for future work.\nAlthough this model is able to simulate the calcium clamp in the in vitro experiments used by Dolmetsch and co-workers, in vivo the T cell has more complex calcium dynamics. One aspect of these dynamics is the calcium activation of PKC and its feedback on phospholipase C (PLC). Many studies have indicated that PKC acts as a co-activator of the transcription factors NFAT and NF\u03baB through its effect on calcineurin.18,55 According to Pfeifhofer et\u00a0al.,42 PKC\u03b8 is predominately expressed in T-lymphocytes. They found that PKC\u03b8 increases NFAT and NF-\u03baB activation by reducing the intracellular calcium elevation through a reduction in IP3 production. They also found that PKC\u03b1 does this to a lesser degree. The current model includes the activation of PKC\u03b8 and its effect on IKK complex. Future work might include any feedback of PKC on PLC and possibly the actions of other PKCs.47\nTwo computational models have been developed by other authors to model the dynamics of NF\u03baB in T-lymphocytes. The model developed by Carlotti et\u00a0al.,13 describes the association and dissociation of NF\u03baB and I\u03baB and their translocation into the nucleus both in the associated and dissociated forms. Their model demonstrates that NF\u03baB is localized in the cytoplasm at rest due to its association with I\u03baB and the export of NF\u03baB from the nucleus. A second model, developed by Hoffman and co-workers, demonstrated that the temporal control was due to coordinated degradation and resynthesis of I\u03baB and that I\u03baB provides a strong negative feedback that can turn off the NF\u03baB response.","keyphrases":["t cell","immune response","calcium","computational model"],"prmu":["P","P","P","P"]}
{"id":"J_Med_Internet_Res-9-2-1913941","title":"How Complementary and Alternative Medicine Practitioners Use PubMed\n","text":"Background PubMed is the largest bibliographic index in the life sciences. It is freely available online and is used by professionals and the public to learn more about medical research. While primarily intended to serve researchers, PubMed provides an array of tools and services that can help a wider readership in the location, comprehension, evaluation, and utilization of medical research.\nIntroduction\nThis study considers the use of PubMed (www.pubmed.gov) by practitioners of complementary and alternative medicine (CAM). In 2001, Americans invested US $50 billion in this form of health care, according to John Weeks, editor of The Integrator, a newsletter tracking \u201cthe business of alternative medicine,\u201d with 40% of American adults turning at some point to alternative medicine [1]. While some in the medical research community have argued that \u201cthere is no alternative medicine\u201d as \u201cthere is only scientifically proven, evidence-based medicine supported by solid data, or unproven medicine for which there is no scientific evidence\u201d [2], the US National Center for Complementary and Alternative Medicine (one of the National Institutes of Health) sponsors research in this field and serves as a clearinghouse for clinical trials and research summaries.\nWithin the scope of its standing as the largest bibliographic index on the life sciences, PubMed (containing Medline, formerly known as Index Medicus, as a subset) indexes medical research articles and includes material useful for physicians and pharmacists, as well as traditional Chinese medicine practitioners [3,4]. A study of nurses, health care assistants, midwives, and health visitors (registered nurses or midwives who promote mental, physical, and social well-being in the community) has shown that, among those who had sufficient access to online sources of health information, the access to journal literature was felt to have led to improved patient care [5].\nA prerequisite for the successful pursuit of health information is what Norman and Skinner identify as eHealth literacy, or the skills that enable people \u201cto seek, find, understand, and appraise health information\u201d [6]. This study focuses on how, among a group of CAM practitioners, those abilities are supported by an array of PubMed tools and services. Up to this point, investigators have recognized that users of online health services are not a monolithic group. Studies have been conducted on the reading levels demanded of cancer patients [7], the overwhelming quantity of information available for first-time mothers [8], the limited search skills of college students studying health [9], the growing use of electronic resources by nurses with limited search skills [10,11], and the growing confidence among doctors in their search strategies, although few use this skill on a real-time basis with patients [12].\nThe variety of approaches that these studies apply testifies to the idea that reading health literature online is situation specific and calls for context, and that investigations into eHealth literacy are fruitful when specific populations are delineated. Information retrieval studies have been done on citation retrieval [13] and literature searching [14] of PubMed, as well as on the use of the medical subject heading (MeSH) and Limits services [15]. Work has also been done in advising users how to best conduct CAM searches [16,17] with at least one study finding that PubMed indexing is not yet adequate for this field of medicine, especially with CAM clinical trials [17].\nThis study is intended to complement the more common focus among information science researchers on users\u2019 information retrieval strategies with indexing services such as PubMed [18,19]. It addresses the value of PubMed\u2019s specific tools and services for CAM practitioners, as these tools are introduced to them in the context of their own interests in the life sciences and their interest in learning about the use of PubMed. In focusing on the value and role of the tools and services, we are drawing on literacy and learning research in the subject domains, or \u201cdomain knowledge\u201d as Alexander and others refer to it [20]. Alexander has established the degree to which a combination of personal interest and background knowledge contribute to the degree of learning in domain areas such as biology and physics, and she holds, in particular, that \u201cone\u2019s knowledge base is a scaffold that supports the construction of all future learning\u201d [21].\nIn this sense, PubMed\u2019s provision of tools and services (such as Related Articles and Bookshelf) can be seen to help users easily and immediately augment, in a limited way, shortcomings in their knowledge base. The tools and services compensate for shortcomings not only in searching for articles but also when comprehending, evaluating, and utilizing abstracts and full-text articles. The tools and services (such as Full-Text Access, Email Alert, Send To) also appear to be poised to support the user\u2019s interest in and engagement with the indexed material (which is the other critical factor Alexander has identified for domain learning). By introducing CAM practitioners to the tools and services, one by one, and gaining from them a sense of the perceived value and contribution of each, this study seeks to determine the potential contribution of these tools and services to the knowledge-base scaffolding and the personal interest of health care workers who lie outside of PubMed\u2019s originally intended audience of researchers and physicians. This work has obvious implications for the design of the tools, in PubMed\u2019s effort to serve as broad an audience of health care practitioners as possible, and as a way, in turn, of extending the impact and benefits of life science research.\nMethods\nSample\nThe participants in this study were drawn from among those practising CAM. They were recruited from three sources: (1) participants 1-5 were drawn from among students taking an online professional development course at a community college on research literacy for the health professions, (2) participants 6-9 were recruited from the Massage Therapy Association of British Columbia, and (3) participants 10-18 were drawn from the Canadian Memorial Chiropractic College. The eight women and 10 men who agreed to participate in the study ranged in age from their 20s to their 50s. Of the 18 participants, 10 were pursuing or possessed a chiropractic degree, seven were registered massage therapists, and one practised homeopathy. A total of 13 participants had a university degree, with one holding a master\u2019s degree and another, a PhD. In terms of background with online research, participants 1-5 were taking a course on research literacy, while participants 13-18 had been introduced to the topic in a session with a librarian, with some one-on-one follow-up.\nDesign\nParticipants were offered an opportunity to learn how to use PubMed in one-on-one sessions with the researcher (MQ-R) in exchange for helping the researchers understand the value of PubMed\u2019s various features to users such as themselves. The 10 PubMed tools and services considered in this study (Table 1) can be divided into three functions: information retrieval (Boolean Search, Limits, Related Articles, Author Links, MeSH), information access (Publisher Link, LinkOut, Bookshelf), and information management (History, Send To, Email Alert). The sessions with the participants took place between April 2004 and December 2006 and averaged 2 hours in length. Sessions held with participants 1-9 were conducted over the phone, with both participant and researcher accessing PubMed through the Internet, while sessions with participants 10-18 were conducted face-to-face with a single computer connected to the Internet, which the participant operated. Participants were introduced to between six and 10 PubMed tools. The participants in the phone sessions covered 7.1 items on average compared to 7.7 items covered by participants in the face-to-face sessions.\nPrior to their PubMed training session, the researcher asked participants by email to identify a health science topic of interest to them. The researcher then found a suitable article based on the interest of the 14 participants who responded and sent it to them in advance of the PubMed session (Multimedia Appendix 1). The participants were asked to read the paper prior to the session. The starting point for each session, after introductions and preliminary questions about the participant\u2019s background, was an initial search of PubMed on a topic of interest. Participants were then guided through a range of six to 10 PubMed tools and services depending on the pace of the 2-hour session and where the interests of the participants led (Multimedia Appendix 2). For each tool and service, they were introduced to how the feature worked, using concepts and papers of interest to them, and were asked to provide their thoughts on its potential value for their use of PubMed.\nTable 1\nPubMed tools and services introduced to participants\nTool\/Service\nFunction\nBoolean Search\nSearch terms can be entered using Boolean operators AND, OR, NOT.\nSearch Limits\nAllows the user to limit the search by language; type of journal; gender; human or animal subjects; age; type of article; date; inclusion into PubMed.\nRelated Articles\nThis appears as a button\/link with each citation and brings up a list of matching articles based on the original article.\nFull-Text Access\nThis is provided through the publisher\u2019s link on the abstract page and on the LinkOut page, with publisher, aggregator, PubMed Central, and\/or subscribing libraries providing open access for roughly 15% of articles.\nSend To\nSend To allows users to email citations, convert citations to a text file, or save citations for editing and further action.\nSearch History\nThis tracks terms, time, and number of results in searches, while allowing Boolean searches combining different searches from the Search History.\nBookshelf\nThis provides links to relevant passages in a set of full-text life science books that range from basic science to clinical practices.\nMeSH Vocab\nMeSH is a controlled vocabulary database for ascertaining the most commonly used terms in PubMed.\nEmail Alert\nThe National Center for Biological Information (NCBI) of the National Library of Medicine (NLM) offers email notification of new work in specified topics.\nAuthor Links\nAuthors\u2019 names have links leading to a listing of all of their work in PubMed.\nAnalysis\nThe sessions were transcribed (Multimedia Appendix 3), and the two researchers created a summary table of the results by rating the responses for each tool as (+) positive (\u201cthat\u2019s valuable\u201d), (++) positive with emphasis (\u201cI\u2019d really use that\u201d), or (\u2212) negative or indifferent (\u201cthat makes sense\u201d). Because of the session\u2019s 2-hour time constraint and the semistructured handling of the sessions, not every tool was covered with every participant. As a follow-up to the PubMed session, the researcher emailed the participants 2 weeks later, asking them to describe their current use, if any, of PubMed and other Internet resources.\nResults\nWhat follows is a detailed qualitative analysis of the participants\u2019 responses to each of the 10 PubMed tools and services covered in this study, with the aim of better understanding how such aspects of PubMed might be seen as contributing to its use by the participants. Differences in the responses among the participants (on the basis of training or university degree) was not the focus of the study, although, as might be expected, those with prior training and with university degrees tended to value PubMed tools and services more highly. Participants with prior training in the use of online research literature thought that, on average, 6.0 out of the 7.5 tools and services that were introduced to them would be valuable in their use of PubMed, while those without such training responded positively to 4.6 of the 7.1 items introduced to them (Table 2). Similarly, those with a university degree responded positively to 5.7 out of 7.4 items, compared to those without a university degree, who gave a positive evaluation to 4.8 out of 7.4 items.\nTable 2\nValue of PubMed tools and services for participants (N = 18)\nParticipants\nPubMed Tools and Services\u2021\nFurther Use of PubMed\nCode\u2020\nUniversityDegree\nPrior Online ResearchTraining\nBooleanSearch\nSearchLimits\nRelatedArticles\nFull-TextAccess\nSendTo\nSearch History\nBookshelf\nMeSHVocab\nEmailAlert\nAuthorLinks\nMF1\nyes\nyes\n+*\n+*\n+\n++\n+\n++*\n\u2212\n\u2212*\n+*\nyes\nCM2\nyes\nyes\n++\n+\n++\n+\n+*\n\u2212\nyes\nHF3\nno\nyes\n++*\n+*\n++*\n+\n+\n++\n\u2212\n+\nno\nMF4\nyes\nyes\n+*\n+*\n+*\n+*\n++\n++\nMF5\nyes\nyes\n+*\n+\n+\n++\n++\n+\nMM6\nno\nno\n+\n++\n++\n++*\n\u2212\n+\n\u2212\n\u2212\nMF7\nno\nno\n++\n\u2212\n++\n+\n+\n++\nyes\nMF8\nno\nno\n\u2212\n\u2212\n+\n++\n\u2212\n\u2212\n+\n\u2212\nyes\nMM9\nyes\nno\n+\n\u2212\n\u2212\n+\n\u2212\n++\n\u2212\nno\nCF10\nno\nno\n++\n++\n\u2212\n+\n\u2212\n+\n\u2212\nyes\nCM11\nyes\nno\n++\n\u2212\n\u2212\n++\n\u2212\n++\n+\n++\n+\nyes\nCF12\nyes\nno\n+\n+\n\u2212\n++\n++\n++\nyes\nCM13\nyes\nyes\n+\n\u2212\n+\n+\n\u2212\n+\n\u2212\n+\n+\nno\nCM14\nyes\nyes\n+\n\u2212\n+*\n+\n+*\n++*\n\u2212\n+\nCM15\nyes\nyes\n\u2212\n++\n++\n++\n++\n+*\n+\n\u2212\nyes\nCM16\nyes\nyes\n+*\n+\n++\n+*\n++\n+\n\u2212\nCM17\nyes\nyes\n+*\n+*\n+\n++*\n++\n+\n\u2212\n\u2212*\n+\nCM18\nyes\nyes\n\u2212\n++\n\u2212*\n+*\n+\n\u2212\n+\n*Prior familiarity with tool or service.\n\u2020First letter of code: M=registered massage therapist; C=chiropractor; H=homeopath. Second letter of code: M=male; F=female.\n\u2021 +: positive evaluation of tool or service; ++: additional positive emphasis; \u2212: negative or indifferent response to tool or service\nBlank cells indicate that tool or service was not covered in the participant\u2019s session or participant did not respond to email follow-up.\nBoolean Search\nThe basic starting point with PubMed, as with any online index or database, is the search, which typically allows the combining of search terms with AND, OR, or NOT for more accurate searching. These Boolean operators were familiar to a number of participants, but were a little vague for others: \u201cI might have heard of it\u201d (CF12). Learning about them made a considerable impression on a number of participants:\nCM2\nI will use the Boolean search methods even when using the different features of the database. This is incredibly important to know, use, and understand. It\u2019s like the building blocks of searches. I\u2019ve got to practise that one afterwards.\nAt least one participant recognized that the search results were profoundly influenced by the terms of the search: \nCM11\nThe difficulties is [sic] the prejudice you take to your search question. Maybe you missed something because you describe[d] it badly.\nSearch Limits\nA further search strategy that the participants found useful was the ability to apply Limits to the searches, including dates, author, links to free full-text articles, language, gender, human subjects, type of article, and so on (Figure 1): \u201cI have used Limits with dates mainly...and if I am searching for a particular author\u201d (MF1). Limits seemed useful for participants for finding specific information, such as when they needed to answer a particular question: \u201cI think I would use this one [Limits] to look for something very specific, but if I wanted to browse around I would use Related Articles\u201d (MF5), and \u201cI think I would use the Limits if I have a problem patient who comes at me with a Google paper, like if she was a 45-year-old female who found something and said, \u2018Why don\u2019t I try this?\u2019\u201d (CF10). The participants found Limits useful because it allowed them to be selective about the information they would find, such as limiting their searches specifically to include only core clinical journals. This is because \u201cthey [core clinical journals] are well recognized in the medical community\u201d (MF7), and, as another participant explains, \u201cBecause sometimes I don\u2019t want to know...what a really alternative journal says, I want to read the larger stuff\u201d (CF12). \nFigure 1Limits tab on PubMed\nRelated Articles\nThe ability to identify one very promising article and then find all of the related articles by clicking on a hyperlink (Figure 2) was highly valued by five participants: \u201cI really like Related Articles; I saw it in the other database, and it is useful because it brings me to things that I would probably not have found otherwise, outside my own searching\u201d (CM2). However, participants also intuited how Related Articles could be used in relation to Boolean searches to achieve a very efficient focus on the desired research: \u201cThese look really great.\u2026 I could spend hours just seeing what comes up from this feature after starting from one search\u201d (HF3), and \u201cThis list [based on Related Articles] makes more sense to me than the original list that came up\u201d (MF4). It did seem to a number of the participants that \u201cRelated Articles worked perfectly\u201d (MF7). Yet, Related Articles did not work for everyone, as one chiropractor commented, \u201cWhat [PubMed] thinks is similar is not what I think is similar\u201d (CM18).\nFull-Text Access\nPubMed provides access to the complete articles that it indexes through a publisher\u2019s link on the article\u2019s abstract page (see Figure 2), as well as a link to PubMed Central if the journal\u2019s contents have been placed in this open-access repository. In addition, LinkOut provides links to sites where the article can be accessed, including sites of the publisher, journal aggregators such as EBSCO and ProQuest, and university libraries that hold print and\/or electronic editions of the article (which are accessible to members of those libraries). Many of the publishers and the aggregators provide nonsubscribers the ability to purchase access. For example, Elsevier\u2019s Science Direct changes US $30 to purchase an article in any of its 2200 journals. In addition, PubMed clearly identifies that a small proportion (roughly 15%) of the literature has been made \u201copen access\u201d by its publisher, either immediately upon publication or after a certain period (\u201cmoving wall\u201d model), typically from 6 to 24 months following publication.\nFigure 2Partial results of a PubMed search on \u201ccomplementary medicine,\u201d showing icons (from top to bottom) for \u201cno abstract,\u201d \u201cabstract only,\u201d and \u201copen access to full-text article\u201d (although no abstract is available) and hyperlinks to \u201cRelated Articles\u201d and \u201cLinks\u201d (to the full text on publisher and\/or library websites)\nWhile the article\u2019s abstract was typically included in PubMed\u2019s indexing of an article, it was judged insufficient by participants as a way of both understanding and evaluating the research, whether they had had very little experience with the research or were well oriented to their field\u2019s literature. As one first-time PubMed user stated as she looked at an abstract, \u201c[I] definitely need to read the article. It [abstract] does not tell us enough\u201d (MF8). This response is consistent with previous studies on the shortcomings of abstracts [22-24].\nFor most participants, the abstract was necessary as a starting point but was insufficient as source of information on its own. It served as a screen, as it would for a researcher: \nCM2\nSo an abstract is really useful. If I felt the abstract was interesting, I would definitely need the full text to actually find the article useful to my work. An abstract only provides enough information to pass the first screening test.\nThis chiropractor initially felt, on first looking through the list of PubMed search results, that \u201cThis information is not what I am looking for; it is too technical, too specialized.\u201d However, by the time he had had the chance to download and look at an open-access article, he was expressing regrets: \u201cThis is incredible; it is so easy; I wish I could just finish reading the article right now\u201d (CM2). Frustration over not being able to view full-text articles was a common theme: \u201cThere was an article that I was interested in looking at, and it said that it would not be available for about a year or 6 months\u201d (CM14). \u201cThe only problem,\u201d as a registered massage therapist put it, \u201cis that the ones that are free are not the ones I was wanting\u201d (MF8).\nFor some, visiting a research library was not an option, and the cost of purchasing a single article posed a serious obstacle: \u201cIt deters me, big time, having to pay for articles\u201d (MF8), and \u201cOh, you can order papers; I don\u2019t have money to order papers\u201d (CM14). For others, using a credit card online was an issue: \u201cI don\u2019t think that that\u2019s a safe measure, with my credit card online\u201d (MF5). Yet the interest in seeing an article was sufficient that a number of participants were prepared to give serious consideration to paying to download it: \u201cIt is incredibly important to have access to the full text of the article. I would even pay to access a full-text article\u201d (CM2); \u201cI think I\u2019ll look for mostly free articles so I could actually read them. If I was desperate to find information and couldn\u2019t find anything, then I might actually buy it\u201d (HF3), and \u201cdepending on how valuable the article is I might be willing to do that\u201d (MF7).\nFor one chiropractor, who administered a professional chiropractic organization, having access to the full-text article was a must. He felt that the full text offered a more accurate picture of the research and provided the language he needed to use for his own work, which was to lobby the Canadian government to further integrate chiropractic into standard public health policies. As he explained,\nCM11\nNo one who has ever done research has ever done an abstract that exactly covers the detail of what\u2019s done inside. So when you actually read the research paper you see more\u2026. The abstract\u2026never provides that robust meaty quote that I need.\nHe went on to give an example from the New England Journal of Medicine in which the results reported in the body of the article placed the chiropractor\u2019s treatment of childhood asthma in a far better light than the abstract did.\nAnother chiropractor conveyed the frustration of coming across a very good source of research, only to find access to the resource restricted. In this case, it was a systematic review conducted by the Cochrane Collaboration, which are not freely available:\nCM14\nIt says [reading from the abstract] \u201cevidence which is based on the Cochrane review of the subject.\u201d This review is like topnotch.... It\u2019s called a Cochrane collaboration, and we don\u2019t have free access to that.... It would be phenomenal if we had access to that.... It\u2019s all based on a strict protocol, like how many people were in the study.... If you want to know the vast information about a subject, you should go to Cochrane reviews.\nEven in the case of a chiropractor who felt that \u201coften times I get most of the information I need from the abstract\u201d (CM17) and thus did not need the full text, there was a recanting after a few moments:\nCM17\nWell, that\u2019s not true. I do [need access to the full text]. Often times you are stuck because they tell you \u201cin the article we describe\u201d such and such. And then you do everything to get it. And we have different ways to get articles.\nA registered massage therapist noted how much she appreciated it when the researcher sent, prior to her session, the full text of an article of interest: \nMF7\nI especially valued that it was not just a summary.... It is very relevant to my work as a therapist, and as an instructor. [I] also found the references very helpful.\nAnd finally, a registered massage therapist who had recently obtained her MSc from a Canadian university, and thus had lost her ready access to the scholarly literature upon graduation, had learned about the issue of access in terms of how libraries were signing a new type of agreement with the journal publishers, which carefully controlled access:\nMF1\nThe contracts that the academic institutions sign with the publishers, limiting access to students and faculty only, mean less access for the public. When hard copies were available, anyone could read, digest, copy relative sections, etc. Now, depending on the institutional policy, it may be difficult even to get access to view a full text. Needless to say, the cost of individual articles is exorbitant.\nThe library would allow the public access to its online journals at a small number of \u201cpublic\u201d terminals (for those designated as \u201cwalk-ins\u201d in the contract), but as this participant noted, \nMF1\n[The university libraries] control [public access] very, very, very tightly. I went in and asked if I could use the computer.... I had 6 articles I wanted to see, and they let me do that.\nShe concluded that this represented \u201cthe publisher\u2019s stranglehold on the universities\u201d (MF1). Another participant had caught wind of a hopeful agreement that would greatly increase access to medical research, not only for him, but the public at large:\nMM6\nI was talking to a librarian here at the public library and she said in [British Columbia, Canada] they were...trying to link the public library with the university library so that anybody that has a library card [could view the journals].\nIt was PubMed\u2019s clear identification of, as well as links to, the open-access articles that was the one aspect which participants consistently valued. The participants felt strongly about the importance of being able to read the full text, and while a few had access to research libraries (McGill and University of British Columbia cited), others were willing to consider paying for that access. But in all cases, the value of open access to this literature was judged as a critical aspect in becoming better informed about what the research had to offer their professional practice.\nSend-To Tools\nUsers are able to select, organize, and save the citations they find by sending the selected citations to a file, text, printer, clipboard, email, or RSS feed. Five of the participants were very impressed by how this tool allowed one to build a personal set of research resources: \nCM2\nI\u2019ve done this before [email search results to self] when I have found research on the Web. I think it\u2019s great; it is very easy, and I like to keep things in my inbox. This is a great tool that I would use.\nAnother chiropractor commented on how it enabled him to integrate PubMed into his work: \nCM11\nOver the course of the day, I can sort it and then I have my list in order. Now I can take that display list and send it to text.\nSearch History\nPubMed keeps a record of each search that a user conducts, under History, allowing users to not only review their previous searches but to combine them using Boolean operators. \u201cWow. That\u2019s amazing. If I ever want to go back to what I did, this is how. It\u2019s like you don\u2019t have to write anything down because it is all recorded automatically. This is really, really great\u201d (HF3). While this tool only made it into the sessions of 10 participants, only one of those participants proved relatively indifferent to its powers: \u201cThat makes sense\u201d (CM18).\nBookshelf\nPubMed has integrated a number of full-text medical and life science books that can be searched separately or in conjunction with an article abstract to gain background information about key terms in the abstract (which are highlighted with links to the relevant passages in the books). Only one participant saw how access to the books might be used for providing background: \u201cI really like it; if I am writing a thesis or a paper, I would see this as very useful\u201d (MF5). More frequently, the participants in the study made a number of observations about the currency and focus of the research articles as the reasons they preferred them over books: \u201cWell that\u2019s interesting [referring to Bookshelf], but I like articles because they are more current\u201d (MF1), and \u201cI like journal articles because they are more specific\u201d (HF3).\nA chiropractor reinforced this idea, to a degree that suggested how the background role of PubMed\u2019s Bookshelf program was being missed by some:\nCM14\nThat\u2019s interesting [referring to Bookshelf]. But...once you get into reading research articles, they are much better than reading books. Books are always behind a few years.... There is a lot of bias behind the author of the book\u2014reading an article, you get the actual truth.\nSuch responses suggest that the participants were picking up ideas about the importance of currency and the leading edge role of journals in the life sciences, ideas that need to be refined, certainly, but that are not far removed from common thinking within the research culture.\nMeSH Vocabulary Database\nThe MeSH Database enables readers to look up the controlled vocabulary used to index the research literature. Among the 10 participants introduced to MeSH, there were challenges initially in comprehending it, to a degree not experienced with the other tools and services: \u201cI don\u2019t understand; what is this [MeSH Database]? Is this everything with the keywords?\u201d (CF12), and \u201cWell, I have taken the tutorial [on the MeSH Database], and I\u2019ve tried to use it, and I know it provides you with headings\u201d (MF1). However, with further explanation and a chance to work with it, at least one participant found it \u201ctotally awesome and very useful\u201d (MF8), and another appreciated that \u201cit helps to distinguish things, and so you can go in one direction or another depending on what you want\u201d (MM9). However, that same participant pointed out that what he felt was an element of bias:\nMM9\nThe word \u201csubluxation\u201d...means something quite different to a medical doctor than it does to a chiropractor when it comes to small tiny restriction in motion or small tiny misalignment. But the medical profession still doesn\u2019t believe such things can occur, so I see here that there is no such thing here as subluxation in the sense that the chiropractor means it\u2014very interesting.\nPubMed\u2019s sharing of its vocabulary used for indexing also spoke to the value of this service as a source of engagement and reflection.\nEmail Alert and Notification Services\nThe National Center for Biotechnology Information (NCBI) of the National Library of Medicine (NLM) is responsible for PubMed and offers users a personalized service that includes email alerts when materials of interest to them are indexed. While this tool came up in only five of the sessions, there was recognition of its value by all the participants: \u201cI have an automatic referral for [from] NCBI every so often about updates of new research in this area\u201d (MF1). A chiropractor had signed up with a similar service, PubCrawler, developed by a genetics research lab at Trinity College, Dublin, for alerting readers to new articles in areas of interest: \u201cI\u2019ve signed up to PubCrawler, which alerts me to new research in my area, and so [I] quite often scan these [almost daily]\u201d (CM11).\nAuthor Links\nThe ability to find other works by the same author, simply by clicking on the author\u2019s name, was not generally valued by the participants with the exception of the homeopath and one of the chiropractors: \u201cSo I have heard of him before. I would think it would be interesting; it would be interesting to know what his perspective was on other things\u201d (CF12). With the six participants who encountered this feature divided between interest in and indifference toward it, it may seem that this way of focusing on the work of an individual researcher is not as valued as the other means of tracking work.\nComprehending Research\nIn the course of reflecting on PubMed tools and services, some of the participants reflected on their understanding, as well as their critical reading and use, of the research they were searching. For example, a number of participants commented on the value of reading the abstract as a necessary first step in reading research: \u201cI wouldn\u2019t read a paper without an abstract\u201d (HF3). By the same token, the abstract was not enough: \u201cDefinitely need to read the article. [Abstract] does not tell us enough here\u201d (MF8). A chiropractor noted that the figures and illustrations available in the full text were needed to make sense of the orthopedic tests used in the study:\nCM15\n[The abstract is] a good summary, but...if the article had pictures and stuff, because maybe these are new tests that have come out that the school hasn\u2019t taught us yet, and they might be better than the ones we\u2019ve learned...[then access] would be important.\nAnother chiropractor spoke of the importance of seeing the research methodology described in detail:\nCM14\nThe abstracts usually don\u2019t show enough about the methods of an article.... It depends on the method, if the paper is actually good.\nSimilarly, a third chiropractor, who was satisfied with the abstract alone when it came to \u201cadvice or recommendations,\u201d felt that when the \u201ctreatment\u201d of a client was at issue, then more than the abstract was needed: \nCM18\nI can\u2019t break down the methods they used [with the abstract alone], and if there\u2019s any flaws in it...I won\u2019t get it from the abstract. So I would like to see the whole thing.\nThe participants in this study were aware of their limits in understanding the research, which concerned specific domains of knowledge, as this chiropractor made clear: \u201cI am trying to find one that is not as \u2018biochemistry\u2019 to read; those ones are terrible to read.\u201d Yet, within a given search, the same participant was able to find research studies which, in dealing with \u201cexercise for treating fibromyalgia,\u201d were extremely helpful and comprehensible to the point where he \u201cwould want to prescribe\u201d those exercises to his clients (CM16). More than one registered massage therapist was prepared to challenge the limits on her own understanding: \u201cThere\u2019s a lot of qualitative research out there on complementary therapies, but not a lot of quantitative [work], so I am looking for quantitative-oriented stuff\u201d (MF5). However, she realized that she was not clear on what \u201cthe p factor\u201d (sic) was and stated that \u201cI\u2019m not really up with statistics.... I can understand some but not all\u201d (MF5). A second registered massage therapist was willing to push her learning beyond the concerns of clinical practice: \u201cWell, it\u2019s not useful for me, for what I do, but it does tell me some information that I didn\u2019t know before: the cell type that they are talking about, the engagement of it\u201d (MF7).\nIf participants felt somewhat overwhelmed by the research at times\u2014\u201cIt\u2019s a little above my head\u201d (CM13)\u2014they also knew how to direct their reading in relation to their strengths. As a registered massage therapist put it: \u201cThis is a little too theoretical and general...[while I\u2019m] looking for protocols for treating low back pain.... I\u2019d be more on \u2018protocol,\u2019 \u2018orthopedic,\u2019 \u2018massage therapy\u2019\u201d (MM9). Or, as a chiropractor explained: \u201cIt\u2019s a bit too heavy; it is going to go beyond what I need clinically\u201d (CF10). What was perhaps most notable about the participants\u2019 responses to the research was their perception of the value of this public resource to their professional practice: \u201cIt\u2019s pretty encouraging to find this [material in PubMed]; it\u2019s not intimidating\u201d (MF7), and \u201cI use the database and feel quite comfortable with it\u201d (CF10).\nA Growing Research Culture\nThis research on CAM practitioners\u2019 use of a life sciences research index reflects a growing influence of a research culture among the health professions and the public. Two participants made reference to their clients bringing in online health information:\nMF5\nMy problem is telling [my clients] that what they are finding in Google is not that sound scientific information because any practitioner out there, anybody can put something out onto Google or their office website, but it\u2019s not necessarily valid. I always have clients who are asking me about information that has come out on certain treatments, so that is primarily what I would tend to use this [PubMed] for.\nThat same increased interest in recent research had been expressed within the CAM community as well, as the administrator of a chiropractic organization stated: \nCM11\nOur membership expects us to know things instantly if they are in the public domain. They say, \"Why don\u2019t you know? Everyone else knows\u201d.\nThe increasing prominence of research in the health services sector was also reflected in the training received by graduates of the Canadian Memorial Chiropractic College:\nCM17\nThis school is very evidence-based. So a lot of the assignments are pretty much looking up research: sensitivity, specificity, tests, prognosis.... If you are efficient with any of these search engines, you can find the information quite quickly. I have used it in university as well, but not to a great extent. When I got here I had to use it much more. It depends on the clinician that you get; some are really, really, really, evidence-based, meaning they follow everything the research says.\nAnother participant noted:\nCM16\nThey [at the Canadian Memorial Chiropractic College] talk so much about evidence-based care in medicine and chiropractic, and you have to have research articles in order to attempt to that. You need a way to do it and PubMed is the only way I know.\nHe went on to add that \u201cit is free.\u201d This was further confirmed by a classmate: \u201cThey [the school] thrust that down our throats: \u2018Make sure you are up to date on that\u2019\u201d (CM15).\nIn turn, CAM practitioners are participating in the debates on the growing prominence of certain forms of medical research but in the following example also demonstrate limited understanding of the methodology:\nCM11\nI wrote an article about [how]...it is more or less impossible to do a [randomized clinical trial] of chiropractic. You can\u2019t blind a practitioner; it\u2019s more or less impossible to blind the patient, so any effect of randomization is nonsense.\nOverall, there is a growing global awareness of the important role played by research in the life sciences, and these CAM practitioners are learning that this body of knowledge has much to offer, with PubMed providing one means of obtaining access to it: \u201cThere\u2019s a lot going on in Europe and Asia that I know nothing about unless they publish in Lymphology or unless they present at the ISL [International Society of Lymphology]\u201d (MF7).\nContinuing Use of PubMed\nIn response to follow-up emails that were sent out some weeks after the session with the researcher, seven participants affirmed that they were using or were planning to use PubMed:\nCM2\nYes, I have used PubMed since our last conversation. I use it on a weekly basis. I use it to find information for patients and\u00a0for myself. I am in the process of developing an acupuncture-related website and writing a book on health-related topics.\nIssues of access to research articles persisted in the use of PubMed for this chiropractor: \nCM2\nI have not purchased any articles, although I have been tempted. I tend to look at the full-text articles, and I have used the links LinkOut and Bookshelf with success.\nAs well, he had begun to see how he could best use PubMed in his practice:\nCM2\nI think I\u2019ll use it to get research, to get on-the-fly research to answer questions. But I wouldn\u2019t send my patients to use the database or conduct searches on this database after a consult with them because I think it would be too complicated. I would not refer them to PubMed to find their own research.\nThere were also indications from the participants of PubMed raising the quality of knowledge that informs their practice: \u201cI\u2019m using [PubMed] now to look something up instead of Google\u201d (CF12).\nOther participants spoke in the follow-up emails of their plans for continuing use of PubMed: \nCM15\nNow that I know more about it, I think I\u2019ll use it more.... Now that we have had this session, I\u2019ll be able to understand it a little bit better, especially this \"Related Articles\".\nThis same participant had explained in his session with the researcher how the Limits tool would serve his practice: \u201c[This] would be great if I had a patient come in with a problem, and I had to really specify my search according to his needs\u201d (CM15).\nAnother chiropractic student spoke of PubMed use in terms of its public access as a source of professional development:\nCM17\nI am not going to be in academia much longer. It would be great to know as much as I could before I leave. I plan on using this when I leave as well.\nOf course, not everyone who participated in this study went on to use PubMed afterward:\nHF3\nI often learn about research studies from [online] medical news sources [eg, Medical Post] and access them through links provided in the article versus searching PubMed. There is so much information out there, and I find it hard to find time to access it all!.\nA similar theme was sounded by a massage therapist: \nMM9\nI have no urgent reason to [use PubMed], and my time is too occupied to browse.... Currently, I do not [need PubMed].\nAnd a chiropractor wrote to say that while he is sometimes \u201cdirected to PubMed as a resource\u201d at his school, the problem is that he \u201ccould never really get the entire PDF files\u201d that he wanted (CM13).\nDiscussion\nThis study confirmed our hypothesis that CAM practitioners would find that certain PubMed tools and services, on being introduced to them, had the potential to contribute to their engagement with and understanding of the research that interested them. This study did not seek to measure the actual contribution of these tools and services to the users\u2019 learning. It was designed to establish whether these users perceived the tools and services as having value, as a first step to subsequent studies that will assess the differences that individual tools might make to the participants\u2019 learning.\nAmong the PubMed tools and services to which the participants were introduced, only the service of full-text access proved a positive asset for engagement, comprehension, evaluation, and utilization. As well, the Boolean Search and Related Articles were also strong contributors to the participants\u2019 work with PubMed, and all tools and services impressed more than a few participants as to their value and contribution.\nIn terms of the specific design of PubMed, it is clear that the NLM is continuing to improve its design and functionality [25]. For example, the participants found MeSH to be a particularly difficult tool to grasp; however, the NLM has since provided three animated tutorials (eg, \u201dSearching with MeSH\u201d) with voice-over, which make its operation much clearer, with at least one recent study attesting to MeSH as the preferred search strategy [26]. Still, one of this paper\u2019s reviewers wisely advised that training in MeSH would greatly improve user searches, and the ways in which a user\u2019s health vocabulary contributes to recognition and comprehension has been the subject of a recent study [27].\nThere have been design improvements, as well, with Related Articles, Limits, and other tools. With Bookshelf, the only feature that left over half of the participants who tried it seeing little value in it, PubMed has withdrawn the book links from an article\u2019s abstract, while still offering users the ability to search its book holdings. Yet, these medical and basic science books could well serve as background resources when a reader is stymied by concepts in biochemistry and statistics, to name two areas that this study\u2019s participants identified as weak for them [28]. There is reason, then, for the NLM to continue exploring ways of integrating the books into the literature searches in a way that encourages professionals in the health services to seek background clarification and context, as a way of improving their engagement with the research.\nThe study also has bearing on what has developed, with the rise of the Internet, into the \u201caccess issue.\u201d This goes back to at least 1999, when Harold Varmus, then director of the National Institutes of Health, proposed that all medical research journals make their work freely available in PubMed Central 6 months after publication, an initiative that met with the successful resistance of commercial and society publishers [29]. More recent measures include the National Institutes of Health Public Access Policy, which asks funded researchers to voluntarily deposit copies of their published work in PubMed Central, but which has managed to achieve a less than 4% compliance rate to date [30]. As well, the proposed Federal Research Public Access Act of 2006 is currently before the Senate, which would mandate open access within 6 months of publication for a large proportion of US government\u2013funded research [31]. The results of this study would seem to support efforts to increase access to research by making it clear how greater access could contribute to the practice of CAM, as well as to more traditional forms of clinical practice.\nThe participants\u2019 consistent interest in having access to the full text of the articles indexed in PubMed leads to a strong and immediate recommendation to improve that access: The NLM should make every effort to capture an otherwise missing and substantial source of open access to research and scholarship, namely, the published health sciences research that has been posted by authors in institutional repositories and on websites. While PubMed has an excellent system for identifying and connecting to open-access articles made available by publishers, it needs to develop similarly effective systems for tapping the published work that authors have posted, with the publisher\u2019s permission, in archives and on websites. With numerous archiving mandates, both in place and pending, for this form of open access to research that has been funded by governments and foundations, PubMed needs to ensure that it is able to take advantage of this movement to greater openness. For example, PubMed could include a means of searching the more than 800 repositories worldwide.\nFor those who study the use of health information, we see a need to push beyond the obvious limitations of this study. There are sufficient grounds for further studies carefully designed to assess the contribution that particular aspects of these tools provide for different types of users, with a focus on how the design of the tools and services augments the users\u2019 comprehension, evaluation, and utilization of the materials they encounter.\nThe prospect of the ongoing development of resources such as PubMed, along with an increasing degree of public and professional expectation of access to research and scholarship, holds much promise for the continuing educational quality of the Internet and society at large. While only a fraction of CAM practitioners and a proportion of their clients will pursue these forms of knowledge, there is a larger point to this. Through the combined efforts and commitment of the NLM, a good number of life science journals, and the researchers themselves (who post open-access copies of their work), the quality of knowledge that is publicly and universally available is increasing, adding to people\u2019s understanding as well as to the health care practices that affect their lives. This study is one small demonstration of how the benefits of this greater access extend to a larger community than has been commonly considered when it comes to public resources such as PubMed.","keyphrases":["complementary and alternative medicine","pubmed","literacy","information retrieval","professional development","information management","open access","research dissemination"],"prmu":["P","P","P","P","P","P","P","M"]}
{"id":"Glycoconj_J-4-1-2413117","title":"The structural plasticity of heparan sulfate NA-domains and hence their role in mediating multivalent interactions is confirmed by high-accuracy 15N-NMR relaxation studies\n","text":"Considering the biological importance of heparan sulfate (HS) and the significant activity of its highly-sulfated regions (S-domains), the paucity of known functions for the non-sulfated NA-domains is somewhat puzzling. It has been suggested that chain dynamics within the NA-domains are the key to their functional role in HS. In this study, we investigate this hypothesis using state-of-the-art nuclear magnetic resonance (NMR) experiments at multiple frequencies. To resolve the problem of severe overlap in 1H-NMR spectra of repetitive polysaccharides from proteoglycans, we have prepared oligosaccharides with the chemical structure of HS NA-domains containing the 15N nucleus, which has enough chemical shift dispersion to probe the central residues of octasaccharides at atomic resolution using 600 MHz NMR. By performing NMR relaxation experiments at three magnetic-field strengths, high quality data on internal dynamics and rotational diffusion was obtained. Furthermore, translational diffusion could also be measured by NMR using pulse field gradients. These experimental data were used, in concert with molecular dynamics simulations, to provide information on local molecular shape, greatly aiding our relaxation analyses. Our results, which are more accurate than those presented previously, confirm the higher flexibility of the NA-domains as compared with reported data on S-domains. It is proposed that this flexibility has two functional roles. First, it confers a greater area of interaction from the anchoring point on the core protein for the bioactive S-domains. Secondly, it allows multiple interactions along the same HS chain that are dynamically independent of each other.\nIntroduction\nHeparan sulfate (HS), a member of the glycosaminoglycan (GAG) family, is thought to have appeared early in metazoan evolution [1, 2]. It is ubiquitous in vertebrate extracellular matrices as a constituent of proteoglycans and its extensive repertoire of biological roles, via interactions with hundreds of proteins, make it the most intensely studied of the GAGs. In particular, HS has been implicated in several important biological functions that involve binding to anti-thrombin (AT), fibroblast growth factors (FGFs), cytokines and chemokines [3]. Chemically, the HS biopolymer consists of repeating disaccharide units of variably-sulfated uronic acid (d-glucuronic or l-iduronic acid) and glucosamine (N-acetylated or N-sulfated) monosaccharides. It is this variability in chain epimerization and sulfation (see Fig.\u00a01) that appears to be responsible for the specificity of interactions between HS and proteins [4], rather than the basic linkage pattern of the polymer, which is conserved in all forms.\nFig.\u00a01The basic disaccharide repeating unit of heparan sulfate. In the compounds studied here, the number of repeats (n) is 2, 3 and 4, for compounds 1, 2 and 3 respectively. In these compounds, R1\u2009=\u2009COCH3 and R2\u2009=\u2009R3\u2009=\u2009R4\u2009=\u2009OH, the uronic acid residue is glucuronic acid and the terminal uronic acid residue is 4\u20135 unsaturated\nIn some cases, such as the interaction between anti-thrombin (AT) and a HS pentasaccharide sequence (which includes the rare 3-O-sulfated glucosamine) [5], the local spatial arrangement of sulfo groups is postulated to be the major driver of specificity. Furthermore, the internal and global conformation and dynamics of the chains also play important roles in HS bioactivity [6\u20138]. These conformational contributions to function are not easy to separate from the others, since the density and arrangement of the opposing sulfo groups may profoundly influence the local dynamics of HS-chains, creating an ambiguity in relating HS structure to function. However, solution-state nuclear magnetic resonance (NMR) studies can be used to resolve this ambiguity by directly measuring molecular conformation and dynamics on the nanosecond time scale with atomic resolution [9, 10].\nNumerous NMR studies have been conducted to gain insight into the 3D-structure of HS chains. Much of the early literature was concerned with the conformation and dynamics of the l-iduronate residues [7, 11\u201313], and not without controversy [14]. From these studies, which used NMR coupling-constants and basic relaxation data, a picture emerged of iduronic acid residues that have high internal flexibility, but their dynamic segmental motions are decoupled from neighboring residues [6]. More recent studies of HS chain dynamics using model-free analysis [15] of NMR relaxation data have attempted to describe the internal and overall motion of the various residues of HS [16\u201318]. Interpretation of these data has not been without controversy either. Hricovini et al. [18] compared the dynamics of the non-sulfated region of HS (NA-domain) with those from a highly sulfated HS fragment (S-domain) using the model-free analysis and found that the NA-domain was more rigid. This was ascribed to the lack of flexible iduronic acid residues in the NA-domains. However, a more recent publication by Angulo et al. [16] (also using the model-free approach) showed very restricted motion of the internal iduronic acid residues, in contrast to earlier investigations. At the heart of these conflicting results lies: (a) the difficulty in separation of internal motion due to inter-converting ring conformations of the flexible iduronic acid residue and motion due to flexibility of the glycosidic linkages, (b) problems associated with gathering of high quality data for these nearly linear biopolymers, and (c) interpretation of NMR data from polydisperse samples [11]. These difficulties can be overcome by: (a) restricting the analysis to rigid glucosamine rings (which are not epimerized), (b) increasing the NMR chemical shift dispersion using isotopic enrichment, and (c) improved purification protocols.\nIn protein NMR, isotopic labeling in conjunction with modern heteronuclear filtering experiments (which remove all proton resonances not correlated to a labeled nucleus) are used to overcome issues of signal overlap. In particular, 15N-enrichment is frequently used because the nitrogen atom is highly sensitive to changes in its local environment and consequently has a large range of NMR chemical shifts. The fact that there is often only one nitrogen atom per residue, as opposed to several protons, further adds to spectral simplicity. For dynamic molecular studies, the 15N-nucleus is also desirable since the 1H-15N system within an amide moiety can be considered as an isolated spin-pair for relaxation purposes, simplifying the analysis. Furthermore, 15N has a small negative gyromagnetic ratio, making it more sensitive to dynamics compared to the 13C nucleus (which has a larger and positive gyromagnetic ratio). For example, recent use of 15N-labeling in the GAG hyaluronan has permitted full residue-specific NMR signal assignment and dynamical studies of oligosaccharides of up to eight residues, with unprecedented accuracy [19].\nIn this study, isotopic 15N-labeling of the glucosamine residues was used to overcome the difficulties associated with previous NMR relaxation analyses of HS dynamics. The polymer used here was the capsular polysaccharide from E. coli K5 bacteria, which is chemically identical to the NA-domains from HS. By growing the bacteria in 15N-enriched media, quantitative isotopic labeling of the polysaccharide can be achieved on the milligram scale, as described previously [20]. The polysaccharide was degraded using K5-lyase enzymatic digestion and separated into pure oligosaccharides using large-scale ion-exchange chromatography to facilitate the study of dynamics at the atomic level. Three 15N-labeled oligosaccharides (see Fig.\u00a01) were purified to homogeneity and used for NMR studies: a tetrasaccharide (1), a hexasaccharide (2) and an octasaccharide (3). 1H-15N NMR relaxation experiments (T1 and heteronuclear nOe experiments) were fitted to both isotropic and anisotropic models of molecular diffusion to assess the validity of assuming isotropic overall motion in these non-sulfated molecules. The libration of the amide group (which contributes to the measured dynamics) was taken into consideration using experimental J-coupling data. Furthermore, diffusion ordered spectroscopy (DOSY) [21, 22] and molecular dynamics (MD) simulations were used to assist in evaluating the models of molecular shape.\nThe approach presented here can easily be extended to the various modified forms of HS, as these can be produced by chemical and enzymatic modification of the K5 capsular polysaccharide [23]. Our novel findings shed light upon the role of dynamics in HS, and it is intended that these approaches will be adopted more generally, leading to higher quality dynamic data with atomic precision for these most interesting and important biomolecules.\nTheory\n15N NMR relaxation and rotational diffusion\nThe dynamic behavior of biomolecules on the sub-nanosecond timescale can be probed by NMR relaxation, by measuring T1- and T2- relaxation rates and the nOe enhancement (\u03b7) at multiple magnetic fields [9] and interpreting the results using the Lipari\u2013Szabo model-free approach [15]. For small molecules (as described here) the T1-and T2-relaxation rates are similar for the 1H-15N system, thus in this case only T1 and \u03b7 are considered [19]. Several (often confusing) treatises of these equations are present in the literature, thus the basic theory is briefly reconsidered here.\nRelaxation rates for an isolated N\u2013H spin-pair are related to the spectral density function, J(\u03c9), through Eqs.\u00a01 and 2, where \u03b3 is the gyromagnetic ratio, \u0127 is Planck\u2019s constant over 2\u03c0, \u03c9 is the relevant nuclear Larmor frequency, \u0394\u03c3N is the difference between the axial symmetric 15N chemical shift tensor (estimated to be \u2212135\u00a0ppm for N-acetylglucosamine see also methods section) and rNH is the N\u2013H inter-nuclear distance (a value of 1.01\u00a0\u00c5 is used here, see also methods section).\nFor an isotropic (spherical) model of molecular diffusion the spectral density function is given by Eq. 3, where \u03c4 is the average rotational correlation time (radians s\u22121) and D is the rotational diffusion coefficient. In the case of a symmetric top (cylindrical) model of molecular diffusion the anisotropic spectral density function is given by the Eqs. 4 and 5, where  and  are the correlation time and rotational diffusion coefficient perpendicular to the symmetry axis,  and  are the corresponding variables parallel to the symmetry axis (by this definition ), and \u03b1 is the angle between the N\u2013H vector and the cylindrical axis of symmetry.\nWhen the internal motion (\u03c4e) is much faster than the global rotational correlation time (\u03c4M), these two phenomena can be separated using the model free approach of Lipari and Szabo [15]. In their treatment, if it is assumed that molecular tumbling is isotropic, the spectral density function is represented by Eqs.\u00a06 and 7, where S2 is the generalized order parameter. The corresponding anisotropic relationship is shown in Eq.\u00a08, where Janiso(\u03c9) is described by Eq.\u00a04 and the  are given in Eq.\u00a09.\nIt should be noted that this derivation (resulting in Eq. 8) is consistent with the original proposal of Lipari and Szabo [15] and slightly different to a recent and broadly-used implementation of that theory [24].\nDiffusion measurements\nThe average translational diffusion rate can be measured by pulsed-field-gradient (PFG) NMR [25], often referred to as diffusion ordered spectroscopy (DOSY) [21, 22]. In this experiment, the diffusion coefficients can be estimated by varying the gradient amplitude (g) and fitting the experimental data to the theoretical Stejskal\u2013Tanner Eq.\u00a010 [26].\nIn Eq.\u00a010, S is the signal amplitude, S0 is the echo amplitude that would have resulted had there been no diffusion, Dt is the translational diffusion coefficient (not to be confused with the rotational diffusion rates described above), \u03b4 is the gradient pulse width and \u0394\u2032 is the diffusion time corrected for the effects of a finite gradient pulse width.\nOften the relative diffusion coefficient is sought, but when the absolute value is of interest, a more correct value of Dt can be obtained by taking the systematic errors (due to non-uniform field gradients) into account by fitting to a different expression, which compensates for this, Eqs.\u00a011 and 12 [27], where the coefficients cn are probe and pulse-sequence dependent.\nThe hydrodynamic radius of a molecule can be shown to be related to the translational diffusion coefficient, Dt. This radius, and thus Dt for an isolated polymer in solution, scales (via an exponent, v) with molecular mass (M), Eq. 13. The value of v can be shown to be 3\/5 and 1\/3 for fully solvated and collapsed polymer chains, respectively [28]. A polymer is fully solvated in a solvent where its interactions with the solvent are favored over self-association\u2014the converse is true for a collapsed polymer. In a so-called Flory or \u03b8 solvent, the polymer behaves as a random coil, where v\u2009=\u20091\/2 [28].\nResults\n15N-labeled polymer with a chemical structure identical to the non-sulfated domains of heparan sulfate (HS) was prepared from E. coli. K5 capsular polysaccharide and enzymatically digested. The resulting oligomers were purified and three samples were prepared that had 4, 6 and 8 sugar units (see experimental section for details). 1D 1H-NMR spectra and mass spectrometry (not shown) determined that the oligosaccharides were uniformly 15N-labeled and 1H-15N-HSQC spectra (see Fig.\u00a02) confirmed that the samples contained no major contaminants.\nFig.\u00a0215N-1H HSQC spectrum of the tetra-, hexa- and octa-saccharides (from top to bottom) of the NA-domain of heparan sulfate at a proton-resonant frequency of 900\u00a0MHz. The abscissa shows 1H chemical shift [ppm]. Labels on the spectrum correspond to amide groups, labeled according to their position within the oligosaccharide, using Greek nomenclature. Variability in the line shapes are due to acquisition and processing of the NMR spectra\nRelaxation experiments (1H-15N T1 and heteronuclear nOe) were measured for each of the oligosaccharides (1, 2 and 3) at three magnetic field strengths (14.1, 18.1 and 21.1 T). In each case the 1H-15N HSQC-based T1-relaxation experiments were repeated 12 times with a variable relaxation delay and the resultant peak heights were fitted to exponential curves (relaxation curves for the octasaccharide at 14.1 T are shown in Fig.\u00a03). The heteronuclear nOes were calculated from the ratio of peak heights in 1H-15N spectra, recorded with and without 1H-presaturation.\nFig.\u00a03T1-relaxation curves for an octasaccharide unit from the NA-domain of heparan sulfate at the lowest magnetic field strength used in this study (14.1\u00a0T). The resonances from the amide groups of the central residues (\u03c8 and \u03b3) are resolved and have slightly different relaxation times. For the specific assignments see Fig.\u00a02\nTable\u00a01 shows the calculated exponential decay constants (T1) and the corresponding nOe ratios (\u03b7). The errors in the tabulated T1 values were taken to be twice the exponential curve fitting errors, while the errors in the nOe ratios were estimated by calculating the ratio of the sum of the amplitudes (A) of the two HN signals from the anomeric amide group (\u03b1\/\u03b2) and the same signal in the \u03c9-amide group (clearly resolved in all spectra, see Fig.\u00a02), according to Eq. 14.\nTable\u00a01Fitted 1H-15N T1-relaxation data and measured nOe (\u03b7) ratios for three lengths (4-, 6- and 8-saccharide units denoted 1, 2 and 3 respectively) of the NA-domain from heparan sulfate at three magnetic-field strengths (14.1, 18.1 and 21.1 T)\u00a014.1T18.1T21.1T\u03b7T1[ms]\u03b7T1[ms]\u03b7T1[ms]1\u03b1\u22121.351,790\u22121.231,625\u22120.421,526\u03b2\u22121.631,614\u22121.221,524\u22120.561,445\u03c9\u22121.891,255\u22120.951,183\u22120.391,1162\u03b1\u22120.961,613\u22120.851,473\u22120.281,400\u03b2\u22121.121,456\u22120.791,368\u22120.341,310\u03b3\u22121.07995\u22120.339420.14889\u03c9\u22121.171,050\u22120.38978\u22120.019393\u03b1\u22120.811,437\u22120.841,362\u22120.141,342\u03b2\u22120.991,318\u22120.751,278\u22120.331,252\u03b3\u22120.587550.047970.50803a\u03c8\u22120.77831\u22120.178240.40803a\u03c9\u22120.92946\u22120.189100.25908aHigh resolution spectrum not collected\u2014value estimated from a low-resolution spectrum (see experimental section).\nThe heteronuclear experiments for the octasaccharide (3) were collected using two different sweep-widths. In the first experiment a larger sweep width was used, where all nuclei were present and unfolded, but in which the \u03b3- and \u03c8-amide groups could not be resolved. These resonances were resolved using a second, narrower spectral width (not shown). The spectral width of the second experiment was chosen so that aliased peaks did not interfere with the resonances of interest.\nConversion of the resultant relaxation measurements into 3D-molecular information was performed via the model-free method for calculating the spectral density function (as needed by relaxation Eqs.\u00a01 and 2). Due to the potential shape anisotropy of the oligosaccharides, two methods were used to estimate the spectral density functions, one that assumed spherical symmetry and one that incorporated cylindrical symmetry, see Eqs.\u00a06 and 8. Fitting the relaxation data to these equations resulted in order parameters, overall and internal tumbling times, and in the case of anisotropy, a set of angles. It was found that the relaxation data was not sensitive to the internal correlation time (\u03c4e) and thus it varied significantly when allowed to change during the fitting process. However, this parameter is of secondary interest, and by fixing the internal correlation time at 30\u00a0ps (consistent with molecular dynamics simulations), higher quality and more robust fits to the experimental data could be produced, as noted previously [29]. The uncertainty in this variable is reflected in previous literature data for similar compounds, where \u03c4e ranges from a few picoseconds to several hundred picoseconds in similar compounds (15\u201350\u00a0ps for a HS pentasaccharide and 110\u2013290\u00a0ps for a HS hexasaccharide) and with large errors (\u00b125\u201385\u00a0ps) [16\u201319, 30]. The results, given in Table\u00a02, therefore assume a constant internal correlation time of 30 ps and clearly show the expected trend in generalized order parameter (S2), where the amide groups at the central residues are more ordered (higher S2 values) than those at the terminal residues. Also, the overall correlation times increase as the oligosaccharides get longer, as expected a priori.\nTable\u00a02Calculated model-free parameters for the tetra-, hexa- and octa-saccharides (1, 2 and 3 respectively) of the NA-domain of heparan sulfate, using a constant internal correlation time of 30\u00a0ps, fitted to isotropic and anisotropic models of rotational diffusionModels of rotational diffusionIsotropicS2S2calc1\u03c4M [ns]0.57\u03b10.320.46\u03c4e [ns]0.03\u03b20.350.59\u03c90.480.622\u03c4M [ns]0.71\u03b10.350.49\u03c4e [ns]0.03\u03b20.36\u00a0\u03b30.600.70\u03c90.550.613\u03c4M [ns]0.74\u03b10.32\u00a0\u03c4e [ns]0.03\u03b20.34\u00a0\u03b30.87\u00a0\u03a80.82\u00a0\u03c90.67AnisotropicS2\u03b1[\u00b0]S2calc\u03b1calc[\u00b0]1 [ns]0.35\u03b10.3315.40.4664.2 [ns]0.65\u03b20.3640.10.5962.11.88\u03c90.5658.60.6263.1\u03c4e [ns]0.03\u00a02 [ns]0.57\u03b10.313.80.4962.8 [ns]0.79\u03b20.3649.5\u00a01.39\u03b30.6258.60.7064.4\u03c4e [ns]0.03\u03c90.6184.70.6163.33 [ns]0.39\u03b10.3975.0\u00a0 [ns]1.00\u03b20.4166.7\u00a02.59\u03b30.652.2\u00a0\u03c4e [ns]0.03\u03a80.6623.2\u00a0\u03c90.5431.1\u00a0These values were calculated from experimental data collected at three magnetic-field strengths (14.1, 18.1 and 21.1 T). The S2calc and \u03b1calc values were calculated by averaging data extracted from 50\u00a0ns molecular dynamics simulations\nMolecular shape was probed using the DOSY experiment, applied to the three samples. The diffusion coefficients were estimated from their respective methyl peaks at 2.03\u00a0ppm (Table\u00a03) using the Stejskal\u2013Tanner Eq. 10. The resulting diffusion values were used to calculate the scaling-factor exponent (\u03bd) in Eq. 13, which provides information about the overall molecular shape. Figure 4 shows a plot of log(Dt) against log(M), showing the slope of the fitted line (\u03bd) to be 0.48 .\nFig.\u00a04Plot of log(Dt) vs. log(M) for three different lengths of the NA-domain of heparan sulfate, consisting of 4, 6 and 8 sugar units (Dt is the translational diffusion time and M the weight-averaged molecular mass). The dotted line is the best-fit to a straight line (as described in the theory section, Eq. 13)Table\u00a03Calculated translational diffusion times using the methyl resonance of the various oligomers (tetra-, hexa- and octasaccharides labeled in table as 1, 2 and 3 respectively) of the NA-domain from heparan sulfate at a proton frequency of 400\u00a0MHzCompoundD [10\u22126 cm2\/s]13.08\u2009\u00b1\u20090.000422.55\u2009\u00b1\u20090.000732.22\u2009\u00b1\u20090.0007\nMolecular dynamics (MD) simulations were performed on the tetra- and hexa-saccharides in water boxes. Figure 5 shows 40 snapshots from the simulation of the hexasaccharide (stripped of ions and water molecules), superimposed on the atoms from the central linkage. The simulation data were analyzed to give theoretical values of both S2 and the angle (\u03b1), defined as the angle between the molecular axis and the N\u2013H vector (see Table\u00a02). The calculated S2 values show similar trends to the experimental data, but are generally higher. The \u03b1 values for both the tetra- and hexa-saccharide show nearly random behavior, consistent with an isotropic model.\nFig.\u00a05Overlay of 40 snapshots from 50\u00a0ns of molecular dynamics simulations of the hexasaccharide of unsulfated heparan sulfate. The molecules are superimposed using the atoms in the adjacent linkages. The position of the nitrogen atom (blue sphere) in each structure is highlighted\nThe coupling constants of 1 and 2 were measured from 1D 1H spectra except for the central residues of 2 where there was overlap in the 1D spectrum. In this case, cross-peaks in the 1H-15N HSQC were fitted to 2D Lorentzian line-shapes using a non-linear fitting scheme (these data are given in Table\u00a04).\nTable\u00a043J(H2\u2212HN)-coupling constant (in Hz) of the tetra- and hexa-saccharides (1 and 2 respectively) from the NA-domains of heparan sulfate\u00a03J(\u03b1\u2212 [H2\u2212HN]) [Hz]3J(\u03b2\u2212 [H2\u2212HN]) [Hz]3J(\u03b3\u2212 [H2\u2212HN]) [Hz]3J(\u03c9\u2212 [H2\u2212HN]) [Hz]18.438.559.1428.308.659.149.24All values have an estimated error of \u00b10.05 Hz.\nDiscussion\nAnalysis of the NMR relaxation data\nThe results clearly show the advantages of 15N-labeling, where even the amide groups in the central residues of an octasaccharide are well resolved (Fig.\u00a02). In addition to the gain resolution, an excellent spread of relaxation data is found due to the use of the 15N-nucleus (Table\u00a01). The average change in nOe enhancement (\u03b7) over the different fields is 1 unit, with some of the \u03b7-values passing through zero. In addition, much more precise measurements can be made of the longer T1-relaxation times in the 1H-15N system (compared to 1H-13C). The data follows a clear trend and even without further analysis the difference in relaxation properties of the central and terminal residues of the octasaccharide can be appreciated, as well as the difference between the various lengths of the polymer.\nThe experimentally measured relaxation datasets were interpreted using the model-free approach [15], which relates the measured relaxation values at multiple fields to dynamic conformational properties. In the model-free approximation it is assumed that internal motion (libration) and global motion (diffusion) are uncoupled and on significantly different timescales. Furthermore, it is assumed that molecular dynamics occurs with respect to a rigid diffusing molecular frame. While neither of these assumptions are entirely true for biomolecules, they yield good results if an appropriate model for molecular diffusion is chosen. The two simplest models for diffusion are based on the isotropic sphere and the anisotropic symmetric top (rod or cylindrical shape) [15]. In proteins, for example, the simpler isotropic spherical shape usually suffices, but this model may not be accurate for rod-shaped carbohydrate polymers [18], which are expected to become more anisotropic (rod shaped) with increasing length of the polymer chain. Previous studies on HS analogues have modeled diffusion using an anisotropic model, improving the agreement with experimental data [6, 11, 16\u201318, 30, 31]. In such a symmetric-top model, the correlation time about the symmetry axis of the rod shape  is separated from that perpendicular to the symmetry axis . Values of the parallel correlation time  tend to be similar in linear carbohydrates (~0.5\u00a0ns) and (by definition) are always smaller than the perpendicular correlation time ; the ratio of the two correlation times can be used as a measure of anisotropy. Other parameters in the model-free approach are the internal correlation time (\u03c4e), the generalized order parameter (S2) and, in the anisotropic model, an angle (\u03b1).\nIsotropic or anisotropic model of rotational diffusion?\nFrom the diffusion ordered spectroscopy (DOSY) data in Table\u00a03 and Fig.\u00a04 it is apparent that the overall polymeric shape of the NA-domain of HS is not consistent with a rigid rod. In fact, the slope of the line in Fig.\u00a04 is almost equal to the theoretical value for a random coil (\u03bd\u2009=\u20091\/2), which indicates that even the oligosaccharides have significant segmental motion. This is supported by the model-free analysis of the tetra- and hexa-saccharides (using an isotropic model of diffusion), where relatively low values of S2 are found. However, when similar analysis was used to interpret relaxation measurement from the octasaccharide, relatively higher S2-values were found at the central amide groups. In contrast, the corresponding parameters, calculated using the anisotropic model of diffusion, are consistent with a random coil. These data therefore suggest that the tetra- and hexa-saccharides undergo nearly isotropic diffusion, whereas the octasaccharide diffuses anisotropically (all with high internal and segmental motion). It is likely that it is the \u03b1-linkages within the oligosaccharides that are responsible for a compact dynamic shape at shorter lengths, which only starts to behave anisotropically as an octasaccharide.\nThe lower order parameters calculated using an anisotropic diffusional model are due to the introduction of an additional parameter (\u03b1) that is the angle between the inter-nuclear N\u2013H vector and the molecular cylindrical-symmetry axis. This parameter adds another dimension of complexity, providing a more accurate interpretation of the experimental data than using isotropic diffusion. For example, in the isotropic diffusional model, if there is a large amplitude motion along an N\u2013H vector that is coincident with the long axis of the molecule, the T1 and \u03b7 values will be interpreted as low amplitude motion, which will be compensated for by an increase in the overall rotational diffusion. However, in the anisotropic model, the angular dependence of the internal motion with respect to the molecular frame is taken into account, and the data fitted so that the angle (\u03b1) is also optimized. Inspection of Table\u00a02 reveals that the amide groups at the central residues in the octasaccharide are indeed coincident with long axis of the molecule (small \u03b1), explaining the decrease in order parameter compared to the isotropic model. Further inspection shows that whenever the N\u2013H vector is along the long axis, the anisotropic model predicts more motion and, when the vector is pointing along the short axis (large \u03b1), the converse is true.\nMore importantly, the anisotropic interpretation indicates that the two amide groups in the central residues of the octasaccharide have similar dynamics, as inferred from their S2 values (Table\u00a01), in contrast to the results from the isotropic analysis, where the \u03b3-amide group appears to be more ordered (higher S2) than the more central \u03c8-amide group. Furthermore, na\u00efve interpretation of the raw experimental relaxation values (see Fig.\u00a03, for example) would agree with the conclusion of the analysis via the isotropic model. However, this initial inspection is likely to be erroneous, because one would logically expect that the amide group in the most central residue (i.e., \u03c8 in Fig.\u00a02) would be more ordered (or at least similarly ordered) than that in a neighboring residue, which is in agreement with the initially counterintuitive results from the anisotropic diffusional model. Therefore, these findings (in addition to the DOSY data) substantiate the use of an anisotropic model in the analysis of octasaccharide dynamics.\nConsidering now analysis of the shorter oligosaccharides using an anisotropic model of diffusion, it is found that both the parallel  and perpendicular  diffusional rates increase, whereas one would expect only  to increase. This suggests that there is no well-defined anisotropic interpretation and the simpler isotropic model is preferable. Such an observation is further substantiated by molecular dynamics simulations performed for the tetra- and hexa-saccharide (see Fig.\u00a05 and Table\u00a02). In these simulations it was found that the angle \u03b1 is nearly random for all amides, consistent with an isotropic model.\nMicroscopic interpretation of S2\nNMR scalar coupling constants (J-couplings) [32] can also be used to measure dynamics due to bond librations (via a suitable empirical equation) and the 3J(H2\u2212HN) coupling constant is a direct reporter of amide dynamics in these oligosaccharides. Theoretical calculations have shown that, given the same chemical environment, a decrease in the coupling constant is associated with increased amide librations. From Table\u00a04, it can be seen that in 2 the central \u03b3-amide group has a higher coupling constant compared to that of the terminal \u03c9-amide group. This is contrary to expectation, as the central amide group is regarded to be more rigid. In this case, it appears that the change in chemical environment causes the unexpected observation. This is not expected to be the case when comparing coupling constants measured in 1 and 2 from amides in similar chemical environments, i.e., those at the \u03b1-, \u03b2- and \u03c9-positions, where a variability of 0.1\u00a0Hz is observed in the coupling constants. This can be attributed to slight changes in amide libration of a few degrees (using a previously derived relationship between coupling constants and torsional libration [32]). A similar relationship between dihedral angle and S2 indicated that these angular variabilities would correspond to deviations in S2 of less than 0.01 [15]. Furthermore, the expected differences in amide group librations across individual molecules are less than 5\u00b0 (using the above mentioned relationship), which would result in small changes in S2 of less than 0.05. Therefore, differences in amide libration are not responsible for the more dramatic change in S2 observed at specific amide positions (Table\u00a02). The major remaining site-specific contributor to S2 in the NA-domains from HS (which contain relatively rigid 4C1-puckered sugar rings) must thus be due to segmental motions induced by linkage librations. The S2-values reported here can therefore be seen as direct reporters of the chain dynamics in these polymers.\nRole of dynamics in HS\nThe S-domains of HS have previously been investigated using relaxation data from C\u2013H vectors and an interpretation based on an anisotropic diffusional model (see Angulo [16] and references therein). The values reported for  of such domains are 1.6, 1.7 and 2.6 ns for penta-, hexa- and hepta-saccharide fragments, respectively. In addition, these fragments show a much higher degree of anisotropy compared to the NA-domain analyzed here. This would suggest that the S-domains are comparatively more rigid, as reflected in the higher S2 values reported: 0.91, 0.91 and 0.93 for central residues of the penta-, hexa- and hepta-saccharide fragments, respectively (cf. ~0.65 for the NA-domains found here).\nFrom the findings presented here it thus appears that the NA-domains of HS are much more dynamic than the S-domains. At the molecular level, the only chemical difference between the NA- and S-domains of HS is sulfation and epimerization, and it has been suggested previously that both share a similar local 3D-chain conformation [33]. Therefore, it is more likely that the major contributor to chain rigidity of S-domains is the repulsion of multiple negative charges, rather than specific cross-residue intramolecular interactions, but this remains to be validated by further experimentation.\nFrom a biological viewpoint this confirms the hypothesis that the primary function of the NA-domains is to give the HS chains flexibility, since the S-domains are significantly stiffer. This would give proteoglycan HS chains the ability to rapidly and effectively explore space around their tethering points on the core protein (Fig.\u00a06). It would also allow them to dynamically adapt their shapes and present themselves effectively to a range of protein targets. In addition, this would permit HS chains to interact in a multivalent fashion, without the dynamics of one interaction greatly affecting others. This may explain the omnipresence of functional S-domains in HS, which are separated by completely unsulfated NA-domains [34]. Supporting evidence is provided by Stringer and Gallagher [8], who found an HS fragment with specifically high affinity for the platelet factor 4 (PF4) tetramer. This HS fragment consisted of two S-domains separated by an NA-domain long enough to allow for interaction of the two S-domains with each side of the PF4 tetramer. This argument is also consistent with the diversity of molecules that HS interacts with and the picture that its primary role is in regulation and control [2, 4, 35].\nFig.\u00a06Schematic diagram illustrating the proposed increase in flexibility due to the NA-domains of heparan sulfate and the concomitant increase in spatial range that can be covered together with the potential for mediating multivalent interactions\nConversely, the current study would suggest that the S-domain rich heparin proteoglycans do not have the local plasticity of HS proteoglycans. Therefore, in order to provide the same effective local concentration of sulfo-groups, heparin may be required at higher concentrations than HS. This is supported by the fact that when mast cells are activated their cytoplasmic granules (where heparin is thought to act as a depository for cationic molecules i.e. histamines, serotonin, etc.) are released into the extracellular matrix resulting in a high concentration of highly active (S-domain rich) heparin molecules [36]. Such action would allow heparin to quickly interact with proteins at the site of release, a response which is important in inflammatory processes, where heparin acts as an anticoagulant [36]. However, as its concentration falls due to diffusion from the point of release its highly potent effects would drop-off rapidly.\nConclusions\nHeparan sulfate (HS) NA-domains have been investigated using NMR and computational methods, to determine whether their dynamic properties are consistent with previous hypotheses, suggesting that NA-domains add flexibility to HS. For these nearly-linear biomolecules conventional 1H-NMR methods yield poor spectra with overlapping resonances that are difficult to interpret. We have demonstrated that these difficulties can be overcome by use of 15N-labeling and efficient purification protocols. This approach, which could easily be extended to sulfated domains, allows the dynamical properties of NA-domains of HS to be studied with unprecedented accuracy.\nIn this study, three lengths of the enzymatically-digested polymer (consisting of 4, 6 and 8 sugar units, respectively) were isolated, purified and high-quality 15N-NMR relaxation data was collected for each at three magnetic-field strengths. The resultant data was analyzed using the model-free approach using both isotropic and anisotropic models of rotational diffusion. To complement these studies, and to ease their interpretation, molecular shape was further probed by measuring translational diffusion (using NMR pulse field gradients) and by performing solvated molecular dynamics simulations. The resultant order parameters (S2) from the model-free analysis reveal a high degree of disorder in the oligosaccharide chains, with S2-values that are similar in the hexa- and octa-saccharide, suggesting that these values represent polymer behavior. Following these analyses, it was determined that the NA-domains of HS behave as random coils and that for shorter oligosaccharides their rotational diffusion can be approximated to be isotropic, whereas for the octasaccharide the results are more consistent with anisotropic diffusion.\nThe flexibility exhibited by the NA-domains is contrary to literature data on the sulfated S-domains of HS, which appear to be much more rigid [16]. It is proposed that the NA-domains primarily serve a motional role in HS, consistent with reports of the domain structure of HS. It is therefore argued that the motional properties of the NA-domains increase the efficiency of HS in control and regulation. These findings also suggest that heparin does not have this flexibility, a view consistent with the known roles of heparin, which include storage of ionic molecules and involvement in processes requiring a fast, local response.\nMethods and materials\nBiochemical preparation of oligosaccharides\nK5 capsular polysaccharide was prepared from cultures of Escherichia coli Bi8337\/41 [O10:K5:H4], originally supplied by Professor Ian S. Roberts. Cells were grown in a starter culture of 50\u00a0ml of LB broth, 1\u00a0ml of that media was pelleted and resuspended in 500\u00a0ml of M9-minimal media containing 1g\/l 15N-ammonium chloride (15N>99%) as the only nitrogen source. The polysaccharide purification procedure was similar to that described previously and is summarized here [37]. After 16\u00a0h of growth the cells were harvested (5,000\u00a0g, 10\u00a0min) and washed once with 50\u00a0ml of phosphate-buffered saline (pH\u00a07.2). Cell pellets were resuspended and incubated (30\u00a0min, 37\u00b0C) in 50\u00a0ml extraction buffer (50\u00a0mM Tris\u2013Cl, 5\u00a0mM EDTA, pH\u00a07.3) four times, retaining the supernatants. Polysaccharide was precipitated from the pooled supernatants by addition of sodium cetyl-3-ethyl ammonium bromide to 0.1% (w\/v) followed by a 16\u00a0h incubation at room temperature. The precipitate was recovered by centrifugation (10,000\u00a0g, 20\u00a0min), re-suspended in 1\u00a0M NaCl, precipitated by addition of ethanol to 80% (v\/v) and again recovered by centrifugation. Following dissolution in distilled water (5\u00a0ml), the preparation was dialysed against distilled water, centrifuged (100,000\u00a0g, 1\u00a0h) and the supernatant containing purified polydisperse K5 polysaccharide (20\u00a0mg\/l) was lyophilized.\nLyophilized 15N-enriched K5 polysaccharide (~30\u00a0mg) was dissolved in 5\u00a0ml of digest buffer (100\u00a0mM Tris\u2013HCl, 100\u00a0mM Na-acetate, pH\u00a08.5) and incubated at 37\u00b0C for 16\u00a0h with 180\u00a0U of K5-lyase enzyme (kindly supplied by Professor Ian S. Roberts). The reaction was stopped by incubation in a boiling water bath for 5\u00a0min and the precipitated enzyme was removed by centrifugation (13,000\u00a0g, 2\u00a0min).\nDiffering lengths of oligosaccharides from the K5-polysaccharide digest were separated using anion exchange FPLC on an \u00c4KTA system (GE Healthcare). Briefly, digest supernatant was loaded onto a Sepharose-Q HR column (GE Healthcare), equilibrated in Milli-Q H2O at flow rate of 10\u00a0ml\/min, and eluted with a gradient of 0\u2013100\u00a0mM NaCl over 90\u00a0min. The eluent was monitored at both 214 and 232\u00a0nm. Following lyophilization, each oligosaccharide was desalted using a Bio-Gel P2 column (BioRad) running in 50\u00a0mM HN4Ac, and then repeatedly lyophilized to remove residual NH4Ac by sublimation. Purity was confirmed by analytical ion-exchange chromatography using a Mono-Q HR 5\/5 (GE Healthcare), NMR and mass spectrometry.\nSample preparation\nSamples of oligosaccharides for NMR spectroscopy were prepared from lyophilized material reconstituted in 5% (v\/v) D2O, 0.02% (w\/v) NaN3, 0.3\u00a0mM DSS, pH\u00a06.0; pH\u00a06.0 was chosen to allow amide HN protons to be observed. The final concentration of all samples was calculated to be approximately 5\u00a0mM.\nNMR spectroscopy\nThe T1-relaxation and nOe experiments were performed at 24.5\u00b0C, using standard pulse sequences [40]. All experiments were repeated at three different magnetic-field strengths: 14.1, 18.1 and 21.1T. The T1-relaxation delays were: 4, 12, 16, 32, 48, 80, 120, 160, 240, 300, 360 and 480\u00a0ms, performed in a random order to avoid systematic errors. 1024 complex data points were acquired in the direct (1H) dimension and 18 complex points in the indirect (15N) dimension, using a spectral window of 10\u00a0ppm in the direct dimension and 2.2\u00a0ppm in the indirect dimension. In addition, a second spectrum was collected for three (for all experiments), which had a narrower spectral width of 0.35\u00a0ppm in the indirect dimension.\nThe data was processed using the NMRPipe processing software [41]. The data was typically zero-filled in the acquisition dimension to 16,384 complex points and 1,024 complex points in the indirect dimension. The acquisition dimension was apodized using an exponential window function (typically with 15\u00a0Hz line-broadening) and the indirect dimension was apodized using a Lorentzian to Gaussian apodization function, in which the line broadening and narrowing parameters were adjusted as appropriate for the spectrum. The various peaks were picked using the peak-picking algorithm within the NMRPipe software and the peaks heights were used in all cases. The nonlinLS line shape fitting algorithm within the NMRPipe software was used on a subset of the peaks to validate this approach, and the agreement between the two approaches was found to be excellent (with the zero-filling used here). The J-couplings were measured by finding the difference between peak frequencies, determined also using the nonlinLS algorithm.\nThe assignment of the NH resonances follow the strategy employed by Blundell et al. [38, 39], applied to the oligosaccharides of hyaluronan. The full assignment of these oligosaccharides will be published elsewhere, the assignment of the resonances of interest, however, is given here. The assignment of the tetrasaccharide is straightforward as there are only three cross-peaks, two of which are weaker than the third. The weaker peaks have intensities equal to 60 and 40% of the intensity of the third peak. Based on their intensities, the weaker peaks were assigned to the anomeric protons (\u03b1 and \u03b2), which are further assigned based on their intensities, since the \u03b1-anomer is dominant with preserved ratio in both free GlcNAc [32] and in the terminal GlcNAc of hyaluronan [39] (this assignment is further verified by a NOESY cross-peak between the NH proton and the previously assigned [37] anomeric proton at position 1 of \u03b1\/\u03b2-GlcNAc). The remaining cross-peaks can thus be assigned to the remaining NH atoms in the \u03c9-residue of the tetrasaccharide. In the hexasaccharide all of the cross-peaks present in the tetrasaccharide are observed together with an additional cross-peak. This additional peak is assigned as the more central \u03b3-residue, as it does not experience the same \u201cend effects\u201d (see also references to end effects in Blundell et al. [38, 39]). Finally, the octasaccharide was assigned based on the known assignment of the shorter fragments and the trend towards polymer properties. The cross-peaks for the NHs at \u03b1-, \u03b2- and \u03c9-positions were nearly identical to those in the hexasaccharide, as these experience the same end-effects and could thus be readily assigned. The position of the NH cross-peaks of the \u03b3- and \u03c8-residues in the octasaccharide are close to each other. However, one of these resonances has 1H and 15N chemical shifts that are almost identical to \u03b3-residue of the hexasaccharide and is thus assigned as the \u03b3-amide in the octasaccharide. The remaining cross-peak is therefore due to the \u03c8-amide. These assignments were further verified using the HSQC spectrum of a decasaccharide. In this spectrum, the intensity of the cross-peak corresponding to the \u03c8-amide increases (not shown), due to convergence to polymer properties and overlap, whereas the intensity of the remaining resonances are unchanged. This tendency towards polymer behaviour was additionally used to verify the assignment of the NH cross-peak in the \u03c8-residue of the octasaccharide, which experiences the least amount of end-effects.\nModel-free NMR relaxation analysis\nComputer programs were written in the C-programming language to calculate the relaxation times for an isolated N\u2013H vector, according to the equations presented in the theory section. The internal correlation time at each vector was fixed at 30\u00a0ps (as described in the text), with an associated variable S2 at each position and for each molecule a single variable for the isotopic model. For the anisotropic model, the rotational correlation time  and angles between the long axis and each N\u2013H vector (\u03b1) had to be additionally considered. Predictions were compared with experimental data using the \u03c72 function and optimized using a Monte Carlo algorithm [19]. This process was carried out ten times for each oligosaccharide and the mean and standard deviation values for each fitted parameter were reported.\nThe N\u2013H bond length and the chemical shift anisotropy of the amide nitrogen were calculated by ab initio DFT calculations on N-acetyl-d-glucosamine [32] (while the calculations were performed previously these values have not been reported before). Briefly, the computation of the chemical shift anisotropy was performed using the GIAO method in the Gaussian software at the B3LYP\/IGLOO-III level of theory, which resulted in a value of \u2212135\u00a0ppm, in close agreement with previously-reported values that used similar methods [42, 43]. Similarly, the N\u2013H bond length was taken from quantum mechanical geometry optimization at B3LYP\/631G(d,p) level of theory. The N\u2013H distance was found to range from 1.008 to 1.020\u00a0\u00c5, depending on solvation model used (1.008, 1.020 and 1.015\u00a0\u00c5 for calculations performed in vacuo, with implicit solvent and with explicit solvent, respectively), which is in agreement with similar calculations on proteins [42, 44]. Due to this small variability of the N\u2013H distance, the relaxation calculations were performed using a bond length of both 1.01 and 1.02\u00a0\u00c5 (the results of the latter calculation are included in supplementary Table\u00a02).\nMolecular dynamics simulations\nMolecular dynamics (MD) simulations were performed using CHARMm and a force-field suitable for glycosaminoglycans, as described previously [45] using an integration time-step of 2 fs. Briefly, oligosaccharides were built in a low-energy conformation consistent with preliminary NOESY measurements (unpublished results) and placed into rhomboidal dodecahedral water-boxes. The boxes had dimensions of 4.0 and 4.5\u00a0nm, containing 1504 and 2141 TIP3P water molecules for the tetrasaccharide and hexsaccharide, respectively. Long-range electrostatic interactions were treated using the particle-mesh Ewald approach and truncated to zero between 0.8 and 1.2\u00a0nm. Simulations (using the CPT ensemble) were carried out at 298\u00a0K on both \u03b1 and \u03b2-anomers of the tetrasaccharide and on the \u03b2-anomer of the hexasaccharide, for a total of 50\u00a0ns in each case. Coordinates were stored every 5\u00a0ps for later analysis.\nThe S2 values were calculated as the autocorrelation of N\u2013H vectors extracted from the MD simulation. The N\u2013H vectors were extracted by overlaying all frames from the MD simulation on the same set of atoms. These atoms were the linkage atoms adjacent to each amide (O4 of the same residue GlcNAci to C1 of previous residue GlcAi\u22121 (d-glucuronic acid) and O4 of next residue GlcNAci+1 to C4 of that residue GlcNAci+1). For the GlcNAc residue at the reducing terminus the corresponding anomeric atoms (O1 and its attached proton) were used instead (as there is no i\u00a0+\u00a01 residue).\nValues for cos(\u03b1) were calculated from the simulation by alignment (rotation and translation) of each coordinate frame such that the radius of gyration tensor was diagonal. Each N\u2013H vector was normalized and then projected along the unit vector coincident with the long axis of the aligned molecule, which resulted in estimates for cos(\u03b1). These values were arithmetically averaged over the whole simulation.\nDiffusion ordered spectroscopy (DOSY) experiments\nDiffusion measurements were carried out without spinning on a 400\u00a0MHz Varian Inova instrument, using a 5\u00a0mm diameter indirect detection probe equipped with a z-gradient coil, allowing gradient pulses up to 30\u00a0G cm\u22121. No sample temperature control was used; experiments were carried out at the probe quiescent temperature in a room air-conditioned to about 20\u00b0C. Nominal gradient strengths were calibrated according to manufacturer\u2019s recommendations. The Oneshot pulse sequence [46] was used with a diffusion delay \u0394 of 0.2\u00a0s and a net diffusion-encoding pulse width \u03b4 of 2\u00a0ms (i.e., each bipolar pulse-pair consisted of two 1\u00a0ms gradient pulses) with 10 gradient strengths, ranging from 3.0 to 27.3\u00a0G cm\u22121, chosen to give equal steps in gradient squared. A spectral width of 4,000\u00a0Hz and 16,384 complex data points were acquired for each gradient strength. Correction for instrumental imperfections by reference deconvolution [25], using the TSP peak as reference, were performed on the DOSY data using the standard instrument software (Vnmr6.1C). The diffusion coefficients were fitted to Eq.\u00a010 using four coefficients in the power series, where the coefficients for correction of the non-uniform field gradients (0.928, \u22129.78\u2009\u00d7\u200910\u22123, \u22123.83\u2009\u00d7\u200910\u22124 and 2.51\u2009\u00d7\u200910\u22125) were obtained as previously described [27].\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nSupporting Table\u00a01\nExperimental and calculated nOe ratios (\u03b7), and T1 relaxation data for isotopic models of diffusion. (PDF 24.5\u00a0kb)\nSupporting Table\u00a02\nCalculated model-free parameters for the tetra-, hexa-, and octa sacchrides (1, 2, and 3 respectively) of the NA-domain of heparan sulfate, using a constant internal correlation time of 30 ps, fitted to isotopic and anisotropic models of rotational diffusion (PDF 23.5\u00a0kb)","keyphrases":["dynamics","nuclear magnetic resonance (nmr)","polysaccharide","proteoglycan","glycosaminoglycan","carbohydrate","heparin","n-acetyl heparosan"],"prmu":["P","P","P","P","P","P","P","M"]}
{"id":"J_Abnorm_Child_Psychol-4-1-2277450","title":"Societal Burden of Clinically Anxious Youth Referred for Treatment: A Cost-of-illness Study\n","text":"A prevalence-based cost-of-illness study using a societal perspective was conducted to investigate the cost-of-illness in clinically anxious youth aged 8\u201318 in The Netherlands. Discriminant validity of the cost diary used was obtained by comparing costs of families with an anxious child (n = 118) to costs of families from the general population (n = 41). To examine the convergent validity, bottom-up acquired costs derived from cost diaries were compared to top-down acquired costs obtained from national registrations. Bottom-up acquired costs measured by means of cost diaries amounted to \u20ac2,748 per family of a clinically referred anxious child per annum. Societal costs of families with clinically anxious children were almost 21 times as high compared to families from the general population. With respect to convergent validity, total health care costs using the bottom-up approach from clinically anxious children were quite comparable to those of top-down data of anxious children, although costs within the subcategories differed considerably. Clinical anxiety disorders in childhood cost the Dutch society more than 20 million euros a year. Based on results of discriminate and convergent validity, the cost diary seems a valid method in establishing cost-of-illness in childhood anxiety disorders.\nAnxiety disorders are among the most common childhood mental disorders. Prevalence rates for children with anxiety disorders vary considerably in the international literature from 1.8 (Anderson et al. 1987) to 25% (Kessler et al. 1994). In The Netherlands, a 6-month prevalence rate of 23.5% for anxiety disorders was found in 13- to 18-year-old adolescents. When impairment in daily functioning is taken into consideration, Klein and Pine (2002) estimated the prevalence rate to be between 5 and 10%. A Dutch study revealed that the prevalence rate of severely interfering child anxiety disorders is 9.7% (Verhulst et al. 1997). The yearly prevalence rate of children with a mental disorder seeking treatment in mental health care ranges from 1.6 (Sytema and Koopmans 1998) to 8.1% (Leaf et al. 1996). In The Netherlands, merely 3.5% of 4- to 18-year-old children with a mental disorder had been referred yearly to mental health care for treatment (Verhulst and van der Ende 1997). This latter percentage resembles the 4% of Dutch people with anxiety disorders who are referred for further treatment by their general practitioner (van Wieren et al. 2007).\nWith respect to costs, the total costs for anxiety disorders in adults were highest of all mental illnesses, namely 46.6\u00a0billion dollars, accounting for 31.5% of total expenditures for mental health in 1990 in the USA (Dupont et al. 1996). Greenberg and colleagues (1999) estimated the annual costs of anxiety disorders in adults to be 63.1\u00a0billion dollars in the USA in 1998 by extrapolating the costs of 1990. Besides direct costs of treating anxiety disorders, indirect costs due to impaired social functioning, such as financial dependence and unemployment also are found in adults with anxiety disorders (Leon et al. 1995). In The Netherlands, health care costs of anxiety disorders across all ages were 180\u00a0million euro in 1999, accounting for 0.5% of the health care budget. More specifically, health care costs due to anxiety disorders in 1- to 24-year-old children and young adults amounted to 21.6\u00a0million euros (Polder et al. 2002).\nAn analysis to measure the magnitude of costs related to illness is called a cost-of-illness study and involves an estimation of the total economic burden which an illness imposes on society. The main objective of a cost-of-illness study is to translate the burden on society into monetary costs. Typically in a cost-of-illness study all costs associated with a particular illness are identified and measured, including health care costs, patient and family costs and costs occurring in other sectors. The results of a cost-of-illness study can be used to gain insight in how much society is spending on a disease, and how much potentially can be saved if effective treatment is offered. Also, it identifies the different cost categories and the size of the contribution of each sector in society. The information coming from cost-of-illness studies can be helpful in setting priorities for health care efficiency research (Ament and Evers 1993; Polder et al. 2002; Rice and Miller 1995).\nEssentially there are two approaches to establish the cost-of-illness, namely the bottom-up and the top-down approach. The bottom-up approach acquires data on the patient level by means of registrations or self-report measures. Self-report measures contain retrospective cost questionnaires, retrospective cost interviews or prospective cost diaries (Bruijnzeels et al. 1998a; Sleed et al. 2005). The bottom-up approach enables one to capture resource use that is likely to vary from patient to patient and may not be easily extracted from existing data sources. Multiplying the costs per patient by the national prevalence rate results in the total cost-of-illness for a particular patient group. An advantage of the bottom-up approach is that detailed data can be obtained regarding costs outside the health care sector, such as costs due to productivity losses, out-of-pocket costs, costs of informal care, and costs due to absence from school. A disadvantage of this method may be that the sample of patients used for the cost-of-illness calculations is not representative of the entire patient group, for example due to strict inclusion and exclusion criteria (Oostenbrink et al. 2004; Polder et al. 2002). The top-down approach involves gathering data from existing (national) registrations on total costs of a service category such as hospital care, and dividing these costs by an appropriate unit, such as a patient group using the service category, mostly per year if appropriate. While there are benefits using this approach such as being less resource intensive, this calculation may also prove to be less accurate (Byford et al. 2003). Furthermore, when using a top-down approach it is difficult, if not impossible, to obtain costs outside the health care sector (Oostenbrink et al. 2004; Polder et al. 2002).\nAnother important distinction in cost-of-illness studies is that costs can be determined from a prevalence-based or incidence-based perspective. In a prevalence-based approach, the costs of a disorder are determined for a fixed period of time, as a result of the prevalence of the disorder (number of cases of a disorder at a specific time). This period is called the base period and most often studies investigate the annual economic burden, thus based on a year (Ament and Evers 1993; Rice and Miller 1995). A disadvantage of the prevalence-based approach is that it may be difficult to isolate potential differences in costs by stage or duration of the disorder (Greenberg et al. 1999). The incidence based approach estimates the lifetime costs of an illness from its onset, thus costs are assigned to the year in which the illness begins (Ament and Evers 1993; Rice and Miller 1995). Incidence based studies can isolate the differences in costs by stage or duration of the disease. However, a disadvantage of using this approach is that follow up research is required.\nIn this article, a prevalence-based cost-of-illness analysis was performed in 8- to 18-year-old children and adolescents with anxiety disorders, who were referred to treatment. The primary aim of this study was to establish the societal costs of illness in families with a clinically anxious child, using the bottom-up approach by means of a prospective cost diary. A secondary aim of this study was to investigate the discriminative validity of the prospective cost diary by comparing the costs related to anxiety, psychological, physical and other problems in families with a clinically anxious child to the same costs in families from the general population. The third aim of this study was to establish convergent validity by comparing bottom-up acquired health care costs obtained with the cost diaries of the clinically anxious children with top-down acquired health care costs of children with a primary diagnosis of anxiety disorder.\nMaterials and Methods\nParticipants\nFor the cost-of-illness study and the discriminative validity study, participants were clinically anxious children and adolescents from the general population, respectively. The clinically anxious group consisted of 118 families with a child referred for community mental health treatment because of anxiety, and participated in a multi-centre randomized clinical trial comparing family Cognitive Behavioural Therapy (CBT) with individual CBT (Bodden et al. submitted for publication). This study was conducted from March 2002 to November 2005, inclusion ended in March 2004. Inclusion criteria were (1) age 8\u201318, (2) a primary anxiety disorder (except for obsessive-compulsive and post-traumatic stress disorder), (3) IQ\u2009\u2265\u200980, and (4) at least one parent willing to participate. Children were excluded if suffering from (1) substance abuse, (2) current suicide attempts, (2) psychoses, (3) autism-spectrum disorders, or (4) untreated Attention Deficit Hyperactivity Disorder (ADHD). Children using medication for their anxiety were only included if dosage was kept constant during treatment and follow-up. There were 74 girls (63%) and 44 boys (37%), range 8\u201317\u00a0years (M\u2009=\u200912.4, SD\u2009=\u20092.6). Most were Caucasian (n\u2009=\u2009117, 99%) and 53 (45%) attended primary education. The remaining 65 (55%) attended secondary education.\nAccording to the compound diagnosis on the Anxiety Disorders Interview Schedule (ADIS; Silverman and Albano 1996), the mean number of diagnosis per child was 2.9 (SD\u2009=\u20091.7), whereas the mean severity level of the primary diagnosis was 7.1 (SD\u2009=\u20091.0). Furthermore, primary anxiety diagnoses consisted of social phobia (n\u2009=\u200937, 31%), separation anxiety (n\u2009=\u200932, 27%), generalized anxiety disorder (n\u2009=\u200921, 18%), specific phobia (n\u2009=\u200920, 17%), and panic disorder with or without agoraphobia (n\u2009=\u20098, 7%). Of the 118 children, 94 (80%) had one or more comorbid anxiety disorder(s); separation anxiety disorder (n\u2009=\u200920, 17%), social phobia (n\u2009=\u200943, 36%), specific phobia (n\u2009=\u200951, 43%), panic disorder (n\u2009=\u200920, 17%), generalized anxiety disorder (n\u2009=\u200942, 36%), obsessive-compulsive disorder (n\u2009=\u20095, 4%), and post-traumatic stress disorder (n\u2009=\u20097, 6%). Moreover, 29 (25%) had comorbid dysthymic disorder or depression, 9 (8%) ADHD, 2 (2%) conduct disorder, and 1 (1%) oppositional defiant disorder.\nThe sample from the general population consisted of a group of 41 families who were recruited via advertisements. Children of these families were comparable to children from the clinically anxious group based on gender, \u03c72 (1)\u2009=\u200919, p\u2009>\u20090.1, age, F (1, 154)\u2009=\u20090.0, p\u2009>\u20090.1, and educational level, F (1, 154)\u2009=\u20092.6, p\u2009>\u20090.1. There were 27 girls (66%) and 14 boys (34%), range 8\u201317\u00a0years (M\u2009=\u200912.5, SD\u2009=\u20092.5). Most were Caucasian (n\u2009=\u200940, 98%) and 16 (39%) attended primary school. The remaining 25 (61%) attended secondary education. According to the compound diagnosis on the ADIS, the prevalence of anxiety disorders in this sample was 12% (n\u2009=\u20095) and 2.4% (n\u2009=\u20091) for ADHD, closely resembling Dutch prevalence of anxiety disorders and ADHD, respectively 9.7% and 2.3% (Verhulst et al. 1997). The primary anxiety diagnoses consisted of social phobia (n\u2009=\u20091, 20%) and specific phobia (n\u2009=\u20094, 80%) with a mean severity level of 4.8 (SD\u2009=\u20090.8). The overrepresentation of simple phobia and the relatively low severity rates of the anxiety disorders in the control group also support the \u201cnormality\u201d of the control children.\nAll children and parents received and signed a written informed consent after receiving oral and written information on the study. Clinically anxious children, their siblings and parents were assessed before treatment and families from the general population were only measured once. Furthermore, all family members had to fill in a battery of questionnaires, including a prospective cost diary.\nAnxiety Disorders Interview Schedule\nChildren\u2019s diagnostic status was assessed using the Dutch ADIS-C\/P (Siebelink and Treffers 2001), which is a semi-structured diagnostic interview measuring anxiety disorders and other DSM-IV childhood psychopathology. The severity score consists of impairment in daily functioning per diagnosis and ranges from 0 to 8, 4 indicating a clinical diagnosis. Based on criteria, child and parents ratings are combined in a compound summary. The ADIS-C\/P possesses good psychometric properties (Silverman et al. 2001). In our study, the total inter-rater agreement for all ADIS diagnoses (kappa) was averaged and was 0.89 for the ADIS-C and 0.83 for the ADIS-P. The mean inter-rater agreement for the severity score was calculated with correlations and was 0.98 for the ADIS-C and 0.83 for the ADIS-P.\nProspective Cost Diary\nA cost diary was used to determine the resources used (Freer 1980; Goossens et al. 2000) in both the cost-of-illness study as well as in the discriminative validity study. Cost dairies were completed prospectively by one of the parents and covered a period of 2\u00a0weeks. Families of clinically anxious children received the cost diary 2\u00a0weeks before treatment and the sample of families from the general population received the same cost diary during their one time assessment. Each cost diary was presented in a booklet form with instructions and an example diary. The cost diary was designed such that all costs which are potentially relevant to society were captured, including health care costs, patient and family costs and costs in other sectors. Parents recorded the following resource use: visits to psychologist or psychotherapist, visits to the general practitioner, outpatient consultations at the hospital, medication, day treatment, costs of paid house help, children\u2019s day care, after school care, informal care, productivity losses due to absence of work by the parents, loss of household activities or voluntary work, loss of leisure time, absence from school of the child, and out-of-pocket costs. Informal care means that persons near the family, such as relatives, friends, or neighbors, take care of the family and possibly take over some domestic tasks. Out-of-pocket costs are actual expenses made by the family (and are therefore paid \u2018out of the pocket\u2019), and include for example transportation costs, parking costs, over-the-counter medication and own monetary contributions for health care services which are not fully reimbursed by health insurance companies. Also, parents recorded the name of the family member for whom the resource use was applicable (child, sibling, mother or father), as well as the reason for the resource use by that family member. For analysis, the reported reasons were grouped into (1) due to anxiety, (2) due to psychological problems, (3) due to physical problems, and (4) due to other reasons.\nUnit Prices\nCosts were calculated by multiplying the resources used, obtained with the prospective cost diaries, by the unit price of each resource. Costs were calculated at 2003 euros (1\u20ac\u2009=\u20090.087$, July 30th 2003). It should be noted that, whenever possible, unit prices and not transfer payments such as charges, reimbursements or patient bills were used to value health care resource use. Money transfers may bear little relation to actual resource consumption as a result of (local) budgeting agreements and political negotiations (Finkler 1982). For example, in The Netherlands hospitals receive an annual budget. As a consequence, the reimbursement of a day of hospitalization (which is the amount a hospital charges to a health care insurance company) may vary from year to year as it is used as a balancing item for a budget overdrawing. The key point is that the amount that exchanges hands between providers, third-party payers and patients are not necessarily a good measure of resources used. Therefore, for the valuation of resource consumption in health care, mostly actual unit prices are used, which are obtained by performing unit price calculations. Unit prices consist of personnel, material and capacity costs, as well as costs of housing and overhead.\nAlmost all unit prices were obtained by using published Dutch guideline prices (Oostenbrink et al. 2004). However, medication prices were obtained from the Medication and Aid Information Project database on the Internet (Board of Health Insurances 2005) and were based on the Daily Defined Dosage (DDD), which indicates the mean medication usage per person a day with claw back and value added tax (VAT) (20%). An average unit price was calculated for medication for anxiety problems and medication for psychological problems. As actual unit prices for day treatment and institutionalization were not available, the prices were derived from the average of actual reimbursements (Board Tariff Rates Health Care and Care Authority 2005). It should be noted that the unit price of informal care, loss of leisure time, voluntary work and domestic activities was based on a \u2018shadow price\u2019 as stated in the Dutch guideline (Oostenbrink et al. 2004). A key characteristic of informal care, leisure time, voluntary work and domestic activities is that it is unpaid. So, these activities by definition do not have an official unit price, since no actual payments take place. If an official unit price is not available, resource use should be valued against a \u2018shadow price\u2019. This means that informal care, loss of leisure time, voluntary work and domestic activities were valued against the wage rate per hour of a housekeeper. To determine the costs associated with absence from school, actual annual tuition, which consists of a contribution by parents and a state-subsidy per child (National Institute of Budget information 2005), was divided by the total annual hours at school according to the standard, resulting in a price per hour of school attendance. As the larger part of school costs is fixed, such as the costs of housing, (teaching) personnel and school materials, a child missing school does not result in actual savings. Therefore, the actual costs per hour of school attendance were used to value an hour of school absence. Productivity costs of the parents due to absence from paid work were calculated by means of the friction cost method, based on a mean added value of the Dutch working population (Oostenbrink et al. 2004). This method only takes into account production losses confined to the period needed (usually 90\u00a0days) to replace a sick employee.\nCost-of-illness\nThe primary aim of this study was to calculate the societal burden of illness of families of clinically anxious youth in The Netherlands. The cost-of-illness study was performed from a societal perspective and included direct health care costs, direct non-health care costs, indirect costs and out-of-pocket costs.\nThe costs per family over a period of 2\u00a0weeks were extrapolated to a period of 1\u00a0year (i.e. multiplied by 26) under the assumption that data obtained with the cost diaries were representative for that period (Goossens et al. 2000). Subsequently, the annual costs per family were multiplied by the Dutch prevalence figure for anxiety disorders, which is 9.7% (Verhulst et al. 1997) and the percentage of referred children of 3.5% (Verhulst and van der Ende 1997), which results in a multiplication factor of 0.34%, to calculate the total annual societal cost-of-illness of families of clinically anxious children referred for treatment. It was assumed that the percentage of referred children with a mental disorder (3.5%) could be attributed to anxiety disorders as well. Furthermore, a low and high estimation of the cost-of-illness was calculated. The low multiplication factor of 0.08% was derived by multiplying the low prevalence rate of children with a mental disorder seeking treatment in mental health care of 1.6% with the low prevalence rate of impairment in functioning of 5% (i.e. 1.6\u2009\u00d7\u20095%). The high multiplication factor of 0.81% was derived by taking the high prevalence rate of children with a mental disorder seeking treatment in mental health care of 8.1% and multiply this with the high prevalence rate with impairment in functioning of 10% (i.e. 8.1\u2009\u00d7\u200910%).\nDiscriminative Validity\nTo determine the discriminative validity of the prospective cost diary, the societal costs of the 118 families of anxious children were compared to the societal costs of 41 families from the general population. For this purpose, all costs related to the child, irrespective of reason, were taken into account (i.e. psychological, physical or other). For both groups, the costs per family over a period of 2\u00a0weeks were extrapolated to a period of 1\u00a0year to obtain the annual costs per child for both groups. It was hypothesized that costs for anxiety problems were higher in the clinically anxious group compared to the general population but comparable between the two groups for psychological, physical and other problems.\nConvergent Validity\nTo establish convergent validity, health care costs per child per year obtained with the prospective cost diaries of 118 clinically anxious children were compared with the health care costs of children with a primary diagnosis of anxiety disorder, according to top-down registrations. For this comparison, only health-care costs due to anxiety of the child were considered, because the costs obtained with the top-down registration applied to children with a primary diagnosis of anxiety disorder and only consisted of costs related to the health care sector.\nThe top-down data were obtained from a generic cost study based on national registrations (Polder et al. 2002). In this study, the total costs of health care in 1999 were assigned to diagnosis groups based on actual delivered care, according to distribution codes which are chosen in such a way that they represent equal units of care. These distribution codes are adjusted to the best available data about the care usage (for more details see Polder et al. 2002). This was done for each of the 21 health care sectors including the mental health care sector. Diagnosis groups were based on the International Statistical Classification of Diseases, injuries and causes of death (ICD-9; World Health Organization 1977). The ICD-9 is divided into 17 chapters, including chapter V, mental and behavioural disorders. Furthermore, a more intricate classification is used, by itemizing the chapters into 96 specific diagnosis groups, including anxiety disorders. All anxiety disorders are taken into account (ICD-9 300.0-3 and 300.5-9) (World Health Organization 1977).\nThe top-down data could not be compared directly to the group of 8\u201318\u00a0year old children included in the bottom-up cost-of-illness analysis, because costs were presented for the age groups 5- to 9-, 10- to 14- and 15- to 19-year-olds. Therefore, the assumption was made that costs were equally distributed within the age groups, so the corresponding ages (8\u201318) could be filtered out by averaging the costs and multiplying them by each age year (Polder et al. 2002). Since top-down data were based on costs in 1999, for comparison with the bottom-up cost-of-illness approach, costs were actualized to 2003 euros by using the Consumer Price Index (CPI; Statistics Netherlands 2005).\nStatistical Analysis\nStatistical analysis could only be performed on the cost-of-illness study and on the discriminative validity study, since top-down data were not available on child level. Missing items in the cost diaries were handled with the Missing Value Analysis of SPSS based on the regression models using available data of the group mean, the available data of the individual and the pattern of change of the group. To investigate whether data were normally distributed, a Kolmogorov\u2013Smirnov test was performed. Due to highly skewed cost distributions, bootstrap simulations were conducted in order to get insight in significant differences on subtotal and total costs between families of clinically anxious children and families from the general population and the uncertainty surrounding these costs. The bootstrap method estimates the sampling distribution of a statistic through 1,000 simulations, based on sampling with replacement from the original data (Briggs, Wonderling and Mooney 1997). Bootstrap methods are increasingly being used for analysis of cost data, as traditional parametric and nonparametric statistical methods to analyse the difference in mean costs between groups may be inappropriate when data are skewed (Barber and Thompson 2000; Desgagn\u00e9 et al. 1998).\nResults\nCost-of-illness\nAll resources used and costs made related to the anxiety of the child by families with a clinically anxious child were summed and averaged for 2\u00a0weeks and 1\u00a0year (Table\u00a01). The mean costs per family over 2\u00a0weeks were extrapolated to a year and amounted to \u20ac2748 per family with a clinically anxious child. Costs were not normally distributed, Kolmogorov\u2013Smirnov Z\u2009=\u20094.5, p\u2009<\u20090.01, and skewed to the left, indicating that a minority of families (n\u2009=\u200925, 21%) reported costs related to the anxiety of the child. A logistic regression was performed using families with and without costs as the dependent variable and age, gender, severity and number of diagnoses as covariates within an enter method. When comparing families who reported costs to families who reported no costs, binary logistic regression revealed only one main effect for age, corrected for gender, severity and number of diagnoses, Wald (1)\u2009=\u200911.4, p\u2009<\u20090.01, O.R.\u2009=\u20091.4. No main effect was found for gender, severity or number of diagnoses, Wald (1)s\u2009<\u20090.2, ps\u2009>\u20090.1. This indicates that in families who reported costs related to the child\u2019s anxiety, the child was older compared to families who reported no costs, irrespective of gender, severity and number of diagnoses. The main contributor to costs were costs related to institutionalized treatment (26% of total costs), day treatment (23% of total costs), productivity loss of the parents (23% of total costs) and school absence (17% of total costs).\nTable\u00a01Mean total resource use, subtotal and total societal costs due to anxiety reasons using a bottom-up approach (n\u2009=\u2009118)\u00a0Costs per unitTotal resource use (2\u00a0weeks)Total costs (2\u00a0weeks)Total costs per child (2\u00a0weeks)Total costs per child a year\u00a0\u00a0SumSumM (SD)M (SD)Direct health care costsPsychologist124\/contact10.000.000.000.00Psychiatrist76.00\/contact10.000.000.000.00G.P practice20.20\/contact13.0060.600.51 (4.14)13.35 (108)G.P. telephone10.10\/contact11.0010.100.09 (0.93)2.23 (57.98)Anxiety medication0.72\/ddd214.2510.260.09 (0.50)2.26 (13.00)Pharmacists fee6.45\/med.114.2591.910.78 (4.50)20.28 (117)Institutionalized treatment164\/day520.003,28627.85 (213)724 (5,537)Day treatment95.87\/day530.002,87624.37 (152)634 (3,940)\u2003Subtotal\u00a0\u00a06,33553.69 (261)1,396 (6,773)\u2003Bootstrapped subtotal\u00a0\u00a0\u00a052.78 (24.05)1,371 (583)Direct non-health care costsPaid house keeper12.70\/h12.0025.440.22 (2.34)5.61 (60.84)Informal care8.30\/h132.002692.28 (19.04)59.23 (495)\u2003Subtotal\u00a0\u00a02942.50 (19.16)64.84 (498)\u2003Bootstrapped subtotal\u00a0\u00a0\u00a02.43 (1.76)68.15 (47.59)Indirect costsPaid work34.98\/h183.292,88824.48 (154)636 (4003)Unpaid work8.30\/h14.0033.600.28 (3.09)7.40 (80.34)Loss of leisure time8.30\/h143.003613.06 (16.47)79.59 (428)Household work8.30\/h16.5054.600.46 (4.03)12.03 (105)School absence4.37\/h34742,07017.55 (57.89)456.20 (1,505)\u2003Subtotal\u00a0\u00a05,40845.83 (198)1,191.64 (5,135)\u2003Bootstrapped subtotal\u00a0\u00a0\u00a046.12 (18.22)1,193.64 (473)Out-of-pocket costsTransportation0.16\/km112620.200.17 (1.41)4.42 (36.66)Own contribution alternative treatment\u00a0\u00a03502.97 (32.22)77.12 (838)Own contribution medication not prescribed\u00a0\u00a063.300.54 (5.83)13.95 (152)\u2003Subtotal\u00a0\u00a04343.67 (38.22)95.52 (994)\u2003Bootstrapped subtotal\u00a0\u00a0\u00a03.86 (36.48)97.52 (94.08)Total costs\u00a0\u00a012,471106 (340)2,748 (8,841)Bootstrapped total costs\u00a0\u00a0\u00a0104 (32.45)2,749 (815)1Source of volume price: Oostenbrink et al. 2004, 2Gip databank, 3Nibud, 4Cost diary, 5Actual costs\nThe total number of Dutch children aged 8\u201318 in 2003 was computed using data of the Statistics Netherlands (2005), and resulted in 2,175,382 children. Multiplying this figure by 0.34% resulted in 7,385 Dutch children with an anxiety disorder that interferes with daily functioning and who are referred to treatment. Hence, the annual societal cost-of-illness accumulated to \u20ac2,748\u2009\u00d7\u20097,385\u2009=\u2009\u20ac20,293,958 for families of clinically anxious children referred for treatment in the Netherlands. The same calculation was performed using the low and high multiplication factors. This resulted in a low estimate of the cost-of-illness of \u20ac4,783,839 and a high estimate of \u20ac48,436,370.\nDiscriminative Validity\nThe mean total annual societal costs related to anxiety, psychological, physical and other reasons for families with a clinically anxious child and families from the general population are presented in Table\u00a02. In the clinically anxious group, costs due to anxiety reasons were highest (89% of total costs), followed by costs due to physical reasons (6% of total costs), costs due to other reasons (3% of total costs) and psychological reasons (3% of total costs). The total annual costs of families with clinically anxious children due to anxiety problems, psychological problems, physical problems and other amounted to a mean of \u20ac3,084 (SD\u2009=\u20098,945) per child.\nTable\u00a02Subtotal and total costs for clinically anxious children and children from the general population per year using a bottom-up approach (n\u2009=\u2009118)\u00a0Anxiety reasonPsychological reasonPhysical reasonOther reasonTotal a year\u00a0Cl. anx.Gen.Cl. anx.Gen.Cl. anx.Gen.Cl. anx.Gen.Cl. anx.Gen.Direct health care costsPsychologist\/psychiatrist0.000.000.000.000.000.000.000.000.000.00G.P practice13.350.004.450.0013.350.000.000.0028.860.00G.P. telephone2.230.002.230.004.450.000.000.008.840.00Hospital visit0.000.000.000.0034.3798.8017.190.0051.4898.80Emergence visit hospital0.000.000.000.0030.630.000.000.0030.680.00Medication2.260.002.290.006.804.680.220.0011.704.68Pharmacist fee20.250.0018.480.0029.8420.541.420.0069.9420.54Day treatment6340.000.000.000.000.000.000.006340.00Institutionalized treatment7240.000.000.000.000.000.000.007240.00\u2003Subtotal1,3960.0027.440.0011912418.830.001,559124\u2003Bootstrapped subtotal\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a01,521124Direct non-health care costsHouse keeper5.610.000.000.000.000.008.410.0014.040.00Informal care59.230.000.000.000.000.0014.810.0074.100.00\u2003Subtotal64.830.000.000.000.000.0023.210.0088.050.00\u2003Bootstrapped subtotal\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a087.210.00Indirect costsPaid work6360.000.000.000.000.007.670.006440.00Unpaid work7.400.000.000.000.000.000.000.007.280.00Leisure time loss79.590.000.000.000.000.000.000.0079.560.00Household work12.030.000.000.000.000.000.000.0011.960.00School absence4560.0051.010.0048.600.0015.880.005720.00\u2003Subtotal1,1920.0051.010.0048.600.0023.550.001,3150.00\u2003Bootstrapped subtotal\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a01,3050.00Out-of-pocket costsTransportation4.450.000.760.003.680.000.850.009.620.00Alcohol0.000.000.000.000.000.0011.130.2911.180.29Caffeine0.000.000.000.000.500.000.780.001.300.00Tobacco0.000.000.000.000.000.007.400.007.280.00Own expenses91.070.000.007.291.3816.490.000.0092.5623.66\u2003Subtotal95.520.000.767.295.5516.4920.160.2912223.92\u2003Bootstrapped subtotal\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a012523.89Total costs2,7480.0079.217.2917414185.750.293,084148Bootstrapped total2,7900.0078.447.2317314286.990.323,070147Incremental costs (95% CI)\u22122,790 (\u22124,530 to \u22121,336)\u221271 (\u2212205 to 0)\u221231 (\u2212199 to 131)\u221287 (\u2212152 to \u221233)\u22122,923 (\u22124,505 to \u22121,470)\nCosts of families from the general population were highest for physical reasons (95% of total costs). A small proportion of costs was assigned to psychological reasons (5% of total costs) and other reasons (0.2% of total costs). Despite the fact that five children in the control group met criteria for an anxiety disorder, families reported no costs for anxiety reasons. The total annual costs obtained from families of the general population amounted to a mean of \u20ac148 (SD\u2009=\u2009523) per family.\nCosts in both groups were not normally distributed (Kolmogorov\u2013Smirnov Z\u2009>\u20091.6, p\u2009<\u20090.05). The bootstrapped results indicated that total societal costs were significantly higher in families with clinically anxious children compared to families from the general population. Subdividing the costs into reason showed significant higher costs related to anxiety and other reasons in families with a clinically anxious child compared to families from the general population. Costs related to psychological reasons were borderline significantly higher for the families with a clinically anxious child (see Table\u00a02).\nConvergent Validity\nThe total health care costs of 8- to 18-year-old children with a primary diagnosis of anxiety disorder amounted to \u20ac8,018,000 in 1999 using the top-down approach, which augmented to \u20ac9,075,613 in 2003 when corrected with CPI. The total number of anxious 8- to 18-year-old children referred for treatment in 2003 was 7,385 (i.e. 2,175,382\u2009\u00d7\u20090.34%). Dividing the total health care costs due to anxiety in children by this number resulted in \u20ac1,229 per anxious child that was referred (see Table\u00a03).\nTable\u00a03Direct health care costs, anxiety based per child a year based on the Bottom-up and top-down approach\u00a0Bottom-up costsTop-down costsA day of hospitalization0.00203Day treatment0.001.84Polyclinic visit0.0042.51Categorical hospital0.0032.71Remaining0.007.60\u2003Subtotal hospital care0.00287General practitioner15.5858.27Paramedical care0.001.23\u2003Subtotal Primary care92.7059.50Pharmaceutical help36.4967.50Mental health care1,358785Management and care insurances0.0029.92Total costs1,4101,229\nDirect health care costs derived from the bottom-up method were used to compare with the top-down approach and amounted to \u20ac1,410 per referred anxious child (see Table\u00a03). Total health care costs for clinically anxious children resulted in \u20ac10,412,187 with a low estimate of \u20ac2,453,541 and a high estimate of \u20ac24,842,036. Although the overall difference in total health care costs between the bottom-up and top-down approach per anxious child per year were about \u20ac180 (top-down 13% lower than bottom-up), the differences in costs per health care sector were considerably higher. For instance, costs of medical hospital care were \u20ac287 higher using the top-down approach (23% of total costs) compared to costs obtained with the bottom-up approach (0% of total costs). On the other hand, mental health care costs were \u20ac573 higher (top-down 42% lower than bottom-up) using the bottom-up approach (96% of total costs) compared to the costs obtained with the top-down approach (64% of total costs).\nDiscussion\nTo the authors\u2019 knowledge, this is the first cost-of-illness study in clinically anxious children referred for treatment. The aim of this study was threefold. The first aim was to measure the societal costs of illness in clinically anxious children and their families, using a bottom-up approach by means of a prospective cost diary. The second aim was to investigate the discriminative validity of the prospective cost diary by comparing the costs of families with a clinically anxious child to the costs of families from the general population. The third aim of the study was to establish convergent validity by comparing bottom-up acquired health care costs of the clinically anxious children with top-down acquired health care costs of children with a primary diagnosis of anxiety.\nWith respect to results regarding cost-of-illness, bottom-up acquired total societal costs for families of clinically anxious children referred for treatment amounted to more than \u20ac20\u00a0million per year in the Netherlands with a low estimation of 4.8 and a high estimation of 48\u00a0million euros, which is a quite broad range. Costs amounted to \u20ac2,748 per family with a clinically anxious child per year. These costs seem relatively low compared to costs of other childhood psychopathology. Knapp et al. (1999) conducted a pilot study on ten children aged 4\u201310 with conduct disorders. Cost amounted to \u20ac22,272 per family per year, which is about eight times as high as the costs per family with a clinically anxious child in our study. However, a more elaborate study (n\u2009=\u200980) on 3- to 8-year-old children with anti-social behaviour (Romeo et al. 2006), reported lower mean annual total costs, namely \u20ac8781 per child, still 3.2 times higher than those of anxious children. In another study on 11 children with autism spectrum disorders, societal costs were \u20ac51,844 per child per year (J\u00e4rbrink et al. 2003), which is 19 times higher than the costs per anxious child. It should be noted that the prevalence rate for anxiety is almost four times as high as conduct disorder (2.6%, Verhulst et al. 1997) and 39 to 44 times higher than the prevalence rate for autism spectrum disorders (0.22\u20130.25%, van der Gaag et al. 1996). Consequently, the relative costs for society of childhood anxiety disorders, combining absolute costs with prevalence, are similar to conduct disorders and higher than those of autism spectrum disorders.\nIn line with Knapp et al. (1999), Romeo et al. (2006) and J\u00e4rbrink et al. (2003) a large part of the costs in our study can be attributed to the parents\u2019 productivity loss due to anxiety disorders of the child (23% of total costs). Knapp et al. (1999) found that per family with a child with conduct disorder, the annual costs due to lost employment were \u20ac5,683 (30% of the total costs). Costs to the family related to extra time spend on household tasks, the need for repairs and time off work due to the anti-social behaviour of the child were \u20ac4,637, which is 78% of the total costs. J\u00e4rbrink et al. (2003) showed that more than 50% of the total costs of children with an autism spectrum disorder were attributable to parents and costs related to income loss were \u20ac17,405 (34% of total costs). Our study also found high costs due to school absenteeism (17% of total costs). School absence can have long-term economic consequences such as decreased job opportunities or even unemployment. Taken together, both productivity costs of parents and school absence costs of children seem very important and should be considered in future costing studies. Furthermore, logistic regression analysis revealed that in families with costs related to the child\u2019s anxiety, the child was older compared to families with no costs, irrespective of gender, severity and number of diagnoses. This is in line with the finding that top-down health care costs related to anxiety disorders and health care costs in general increase with age (Polder et al. 2002). Perhaps older children are more prone to react against their parents and are more independent, thereby reducing the influence of parents (Hudson et al. 2002). It can be speculated that less influence from the parents may lead to more school absenteeism among older children. Indeed, 39% of the children aged 18\u201324 reported school absenteeism in the last 2\u00a0months compared to 13% of the children aged 12\u201317 (Statistics Netherlands 2005). Also, the older child may be more aware of the anxiety complaints, possibly increasing the costs of mental health care. Future research should investigate this issue more profoundly.\nAn additional finding of this study concerns the difference in measuring costs in children with anxiety disorders compared to cost-of-illness studies in adults with anxiety disorders. The main difference of measuring costs in children with anxiety disorders, as other mental disorders in children, is that anxiety in the child does not only affect the child, but also the child\u2019s family. Therefore, the costs concerning the child\u2019s anxiety should also be measured at the family level. Although many would argue that the costs of illness for adults spill over to the family as well, studies on costs of anxiety disorders in adults usually do not include family members, especially not their children. So, in future research it may be interesting to assess the costs of adult anxiety disorders on a family level as well. For example, they might be more protective of their children and therefore increase their children\u2019s health care consumption or more often let their child miss a day from school. Moreover, in adult cost-of-illness studies, productivity losses due to absence of paid work are measured. In children, these kind of productivity losses do not occur. However, alternatives such as costs due to absence from school should be taken into account, since school participation can be regarded as a productive activity, and (regular) absence from school may serve as a proxy for future productivity losses.\nWith respect to the results regarding discriminative validity, total costs of families with clinically anxious children using a bottom-up approach were almost 21 times higher than costs of families from the general population. In line with the hypothesis, the difference in costs between families of clinically anxious children and families from the general population can largely be subscribed to the significant difference in anxiety-related costs, showing good discriminative validity of the cost diary on anxiety related costs. However, significant differences were also found on costs due to other reasons. In the prospective cost diary, the reasons for resource use were recorded based on subjective judgments by the parents. However, since anxiety is an internalizing disorder, parents may not always have been able to interpret children\u2019s symptoms correctly. Therefore, they may have attributed costs mistakenly to non-anxiety reasons. For example, parents regularly reported a baby-sitter for a child old enough to stay on its own, if the child would not have been anxious. Another item that was reported as not being related to anxiety was extra travel expenses for the separation anxious child, who otherwise could have stayed at home.\nWith respect to convergent validity, total direct health care costs related to anxiety were 13% lower using a top-down approach than using a bottom-up approach, which seems quite comparable. However, subdividing the costs into several cost categories showed that costs were distributed differently among the cost categories. For example, bottom-up acquired mental health care costs were 58% higher than top-down acquired mental health care costs. Furthermore, 23% of the total costs using a top-down approach were attributed to hospital care, whereas no hospital care costs were found using the bottom-up approach. Hence, convergent validity on total direct health care costs were quite comparable, while the specific cost categories differed between bottom-up and the top-down approach. These findings suggest that children, who are not yet referred for mental health care, might consume more medical hospital care compared to children who are referred for mental health care. Therefore, from a cost-of-illness perspective, it would also be interesting to investigate costs of families with children with anxiety disorders who are not referred for mental health treatment, using the cost dairy developed for this study. It might well be that these families have more medical health care costs, as the comparison with top-down acquired data suggests.\nThis study has some limitations, which should be addressed. The first limitation is that the low and high multiplication factor used in this study caused a broad range of the societal costs of illness. This can be explained by the broad range of internationally reported prevalence rates and percentages of children seeking treatment due to differences in measurements, populations, and criteria that need to be fulfilled. As a Dutch population was investigated, the most appropriate prevalence rate is 9.7% (Verhulst et al. 1997), which was obtained through the use of a reliable structured diagnostic interview. The same is true for the 3.5% of children with mental disorders seeking treatment (Verhulst and van der Ende 1997). Besides, 4% of the Dutch people with anxiety disorders are referred for further treatment by their general practitioner (van Wieren et al. 2007). Therefore, the prevalence and referral rates used in this study seem reliable.\nThe second limitation of the current study is that it can be argued that 2-week cost diaries filled in by parents are not representative enough to reliably assess 1-year bottom-up costs due to the limited time period. However, Goossens et al. (2000) have found that keeping a diary in a limited period namely 1\u00a0week every month, 2\u00a0weeks every 2\u00a0months or an entire year resulted in similar outcomes in fibromyalgia and pain patients. Another study of Bruijnzeels et al. (1998b) found that there was a substantial agreement between a 3-week cost diary, filled in by parents, and medical records concerning general practitioner visits of children. Moreover, the inclusion period of this study lasted 2\u00a0years in which the cost diaries were filled out, therefore possible seasonal influences are ruled out. However, it is likely that service use patterns may vary over the year and do not follow a stable trajectory. So, for some families the 2-weeks period for collecting costs may have led to an underestimation of the total annual costs, whereas for other families this may have led to an overestimation of the total annual costs. Nevertheless, as the mean annual costs are based on cost data from each of these families, it is expected that possible over- and underestimations of annual costs are balanced.\nThe third, and most serious, limitation of this study is that although total health care costs obtained with the top-down and bottom-up method seemed quite comparable, the sample of children with a primary diagnosis of anxiety disorder used in the top-down study by Polder et al. (2002) might not be fully comparable to the clinically anxious children from our bottom-up approach in at least three respects. First, the children in the bottom-up sample were clinically anxious children with severe anxiety disorders. The mean number of anxiety disorders was 3 and the mean severity level 7, indicating severe interference with daily functioning. The children with a primary diagnosis of anxiety disorder in the top-down sample might suffer from different levels of anxiety disorders; some may have minor anxiety problems, while others may have severe anxiety problems. This kind of information was not available using the top-down data. Second, while the clinically anxious children in the bottom-up sample were awaiting CBT in a community mental health care setting, the anxious children in the top-down sample might be in different types and\/or phases of treatment. Again, this kind of information was not available using the top-down data. Third, the clinically anxious children in the bottom-up sample were diagnosed using a universally used semi-structured interview, the ADIS which is based on DSM-IV criteria (American Psychiatric Association 1994). In the top-down sample, children were diagnosed by the doctor in attendance, who attributed the anxiety diagnosis to the patient based on the ICD-9 code, without a structured interview. Due to the classification method, the top-down estimation is possibly less reliable than the bottom-up cost estimation. Therefore, results of the comparison between top-down and bottom-up costs should be interpreted with caution.\nIn conclusion, this cost-of-illness study shows that the societal costs of families with a clinically anxious child who seek treatment amount to more than 20\u00a0million euros a year in the Dutch population, and were 21 times higher than in families of the general population. An effective treatment could decrease these costs on the long term. A recent cost-effectiveness study on family CBT versus individual CBT showed that societal costs in families with a clinically anxious child increased during treatment (due to total costs of 12 sessions CBT a \u20ac1,612) but decreased directly after treatment to baseline level. Even more, during the 1-year follow-up societal costs dropped below the costs at baseline (Bodden et al. submitted for publication). Since anxiety disorders tend to last into adulthood (Newman et al. 1996), societal costs are likely to be higher at the long term. This results in anxiety disorders accounting for the highest costs of all mental illnesses in adulthood in the USA (Dupont et al. 1996). Hence, societal costs in adults with anxiety disorders may be saved by providing effective treatments to children with clinical anxiety disorders.","keyphrases":["cost-of-illness","cost diary","children","anxiety disorders"],"prmu":["P","P","P","P"]}
{"id":"BMC_Bioinformatics-4-_-166134","title":"Information extraction from full text scientific articles: Where are the keywords?\n","text":"Background To date, many of the methods for information extraction of biological information from scientific articles are restricted to the abstract of the article. However, full text articles in electronic version, which offer larger sources of data, are currently available. Several questions arise as to whether the effort of scanning full text articles is worthy, or whether the information that can be extracted from the different sections of an article can be relevant.\nBackground\nMost applications of information extraction from the scientific medical bibliography use the Abstract of the publication (for review see for example [1-3]). In the context of information extraction in molecular biology it is usually understood that the information to be extracted from an article are words regarding biological concepts that could synthesize the main points of the article (keywords). Therefore the Abstract of a paper is a good target for information extraction because by definition an abstract synthesizes the content of the article. Moreover, abstracts are available in public databases. However, nowadays most journals are also available in electronic version, and thus full text articles can be used for information extraction.\nIt is obvious that the full text of an article contains more information than its Abstract. However, in approaching full text analysis several problems must be tackled. On the one hand, the storage of full text articles requires more disk space and the analysis needs more computational capacity. On the other hand, an Abstract, as a summary, contains a high frequency of relevant terms (keywords), but this may not be the case of the rest of the article.\nOther questions regard the quality of the information carried by different sections of an article. First of all, is the information in full text organized enough so that keywords can be extracted? Secondly, different biological concepts (for example, gene and protein names, tissue names, organisms, experimental conditions, etc.) may be located in different parts of the article. Or it could be that a word has a different meaning depending on the section where it is located (the word has a context dependent meaning). For example, regarding gene names, those found in the Methods section may refer mostly to analytical tools rather than being relevant to the biological phenomenology described in the whole article. In summary, it would be good to quantify and qualify the information in a full text article before embarking in large scale extraction of particular items of information.\nWith this goal in mind, we analyzed in this work the kind of information that is attached to different parts of an article and we tried to quantify how much information can be found in each section of an article. This should help to state some guidelines for researchers attempting to extract particular keywords (words synthesizing the content of the article) from full text articles.\nResults\nText Corpus\nAs previously stated, the major objective of this work was to compare the information defined as keyword content carried by different sections of a paper, especially the differences between the Abstract and the rest. Therefore, as source for our analysis we used a set of full text articles with a regular section structure, in our study having a defined Abstract, Introduction, Methods, Results, and Discussion (A, I, M, R, D). Another requirement was certain homogeneity of style across the articles (for example, a similar length of the Methods section) and, since there is great interest in the field of data mining on the detection of gene names, the subject should be related to Genetics. Thus, we chose the 104 articles published in Nature Genetics from June 1998 (volume 19, issue 2) to June 2001 (volume 28, issue 2), which comply with the AIMRD structure. Note that other journals, or even the Letters of the very same Nature Genetics, might have a different structure (for example, lacking separated I, M, R, D sections).\nSelection of Keywords\nTo simplify matters, and following our previous work [4], we focused on the extraction of relevant words (keywords) regarding objects, detected as nouns from natural text by a standard grammatical tagger (TreeTagger, Helmut Schmid, IMS, Stuttgart University, ). In order to derive keywords from the section of an article, we first compute the associations between the words in the section. Here, we take the sentence as the unit of text to look for associations, that is, two words are associated in the context of a section if they co-occur repeatedly in sentences within that section (see METHODS).\nSince words associated strongly to many other words are relevant to the matter that is dealt in the article [5] we use a score (K) that is higher for words with many and strong relations to other words (see METHODS). This measure is used to select words as keywords, in this case, related to objects such as proteins, genes, organisms, etc.\nIn order to evaluate the performance of the keyword detection, we observed how the selected keywords matched the MeSH (Medical Subject Headings, ) terms attached by indexers at the National Library of Medicine to these 104 articles (18.6 on average). Since MeSH terms can be composed of several words (for example, \"Learning Disorders\"), we selected those composed of a single word (6.80 terms on average). We noted that the most unspecific (for example, animal) were often not present in the text and thus could not be matched by a keyword as opposed to species names (mouse, mycobacterium, human), or anatomical terms (hippocampus, cerebellum, breast). Of those single-word MeSH terms, 4.91 were found on average in the article (as nouns), and 2.22 were among the set of selected keywords (above K >= 0.3). Obviously, a more accurate comparison to MeSH terms would require the detection of bigrams, and trigrams (keywords composed of multiple words), but this is out the scope of our work. The recall when matching the original MeSH terms (6.80 on average) went down from 4.91 \/ 6.80 = 0.72 in the dictionary of 470.6 different nouns present in an article to 2.22 \/ 6.80 = 0.33 in the 66.6 keywords selected. However, since the size of the list of all nouns found in an article (470.6) is much larger than the number of keywords (66.6), the precision in matching the MeSH terms of an article increased from 4.91 \/ 470.6 = 0.010 to 2.22 \/ 66.6 = 0.033.\nKeyword Selection by Section\nThe number of words selected upon a threshold in the K value varies for different sections (see Figure 1). The first observation is that there are a small number of words that have much better K scores than the rest. This means that the organization of words makes it possible to extract keywords for all the five considered sections.\nFigure 1\nAverage number of keywords versus K for A, I, M, R, and D sections. The average number of nouns per section is, A = 52, I = 171, M = 404, R = 600, D = 331.\nThe number of selected words is very similar for all sections for very high values of K (above 0.8). Above a threshold on K (K >= 0.5; see Table 1) the resulting number of keywords is quite similar for Introduction and Methods (around 15 for each) with the other three sections producing around nine keywords. However, if one accounts for the size of the sections it is obvious that the frequency of keywords (selected with K >= 0.5) per noun is the best in the Abstract (0.18), followed by the Introduction (0.08), with Methods, Results, and Discussion lagging behind. This justifies data mining strategies that focus in the analysis of Abstracts in order to minimize computational resources. However, this result already indicates that not all keywords are in the Abstract, and that therefore mining the rest of the article may be worthy.\nTable 1\nKeyword selection per section.\nall\nK >= 0.3\nK >= 0.4\nK >= 0.5\nA\n52.17\n19.44\n14.42\n9.77\nI\n171.32\n31.03\n20.47\n14.00\nM\n404.19\n54.24\n28.50\n15.80\nR\n599.98\n24.74\n12.74\n7.85\nD\n331.04\n26.16\n14.25\n8.75\nAverage number of nouns per section (all), or number of those selected as keywords for three different thresholds on the K score.\nSections Display Heterogeneous Information\nAs a way to show that the keyword content in different sections is heterogeneous, we examined which keywords (if any) were selected in all the sections of an article. Our results indicate that, as it could be expected, not many keywords are present in every section and those are not very relevant. Even for a low threshold of K >= 0.3, there is on average only one of such general keywords per article. Those are often non-informative words such as \"gene\", or \"protein\". This indicates that the information is unevenly distributed across the sections of the article, that is, different sections contain different kind of information.\nWe illustrate the heterogeneity of the information by section with the keywords selected (for K >= 0.5) for a particular article [6] (Figure 2). This work deals with a mutation of the Nf1 gene of mouse (an exon loss) that produces learning deficits. The only keyword present in every section is the organism under study, the mouse. If the Methods section is excluded, only one single more keyword (mutation) is selected. Other three-section overlaps give more interesting keywords such as the name of the gene under study (Nf1, neurofibromin), a domain contained in the resulting protein (GAP), the method for testing learning performance of mice (maze), or the resulting phenotype (impairment, lethality). Keywords unique to different sections tend to correspond to the different information contained in each section. For example, the keywords unique to the Methods section deal with reagents and techniques (antibody, amersham, tris, primer).\nFigure 2\nThe keywords selected for an article [6] with a K >= 0.5 are represented as they appear in the different sections of the article.\nIn order to quantify the differences and similarities of content across the article we have used the number of keywords that are shared between different sections (Table 2). The values indicate that the Methods section is the most different of all. In Methods, the content is usually focused on the techniques and protocols used, and not so much on the biological phenomena that is the main subject of the article. This alone explains why those keywords present in every section (for example protein, gene) are scarce and uninteresting.\nTable 2\nAverage number of keywords (K >= 0.5) shared by two sections for the corpus of 104 articles.\nA\nI\nM\nR\nD\nA\n2.01\n0.92\n1.77\n2.20\nI\n2.01\n0.81\n1.34\n2.02\nM\n0.92\n0.81\n1.55\n1.02\nR\n1.77\n1.34\n1.55\n1.99\nD\n2.20\n2.02\n1.02\n1.99\nRegarding similarities between sections, A, I, and D are evenly similar among them, and R is the closest to M, as it is shown when plotting the distance matrix of Table 2 as a dendogram (see Figure 3). This is probably due to the fact that the Results section deals with the protocols used, although not as explicitly as the Methods section. The Discussion focuses again on the biological results (stressing their relation to the current knowledge) without detailing the techniques that have already been explained in Methods and justified in Results.\nFigure 3\nIn order to display graphically the similarity between sections regarding keyword content, we took the inverse of the average number of shared keywords (Table 2) as a measure of dissimilarity between sections, and we plotted it as a dendogram (using [19]).\nThis result indicates that each section contains certain keywords that are unique to the section. In the following we try to characterize what are the differences in content between sections.\nQualitative Analysis of Subjects per Section\nTo make a deeper analysis of the kind of information present in each of the sections, we classified in seven categories a set of words present in our corpus of 104 articles (among the most frequent nouns). In order to do so as unambiguously as possible, we selected words that matched MeSH descriptors also consisting on that single word and belonging to only one major MeSH category (see METHODS). We added another category not present in MeSH, that of \"Units, Dimensions, & Parts\" in order to account for many terms that are currently not MeSH terms but are of interest to us.\nThe results (See Figure 4a) indicate that the large sections are a good source of keywords, obviously Methods gathering many terms related to techniques. Introduction, Results and Discussion contain a good deal of information regarding diseases. However, again, the Abstract section is shown as the best source for most subjects regarding frequency of keywords (Figure 4b) except for those typical of the Methods section (Techniques & Equipment; Chemicals & Drugs).\nFigure 4\nWord categories present in the five sections under analysis. Classes according to MeSH are A (Anatomy), B (Organisms), C (Diseases), D (Chemicals & Drugs), E (Techniques & Equipment), G (Biological Sciences). An additional class X was defined in this work (Units, Dimensions, & Parts). The number of words used for the analysis was 36 (class A), 14 (B), 11 (C), 47 (D), 33 (E), 41 (G), 49 (X). (a) Average number of occurrence of words of each subset per section. (b) Frequency of words of each subset per total number of words for each section.\nDistribution of Gene Names\nSince the detection of gene and protein names is a very important subject, broadly used for the detection of macromolecular interactions (see for example [7]), and because, as stated in the introduction, we are concerned about the relevance of matching gene names in different sections of an article, we examined the distribution of gene names across sections.\nFrom a long list of genes names derived from the SWISSPROT database [8], we selected a very restricted set of 539 genes whose names are composed of three letters followed by one single digit, thus very difficult to be mistaken to other words not being genes. For example, there are gene names called Not or That. Shorter names (e.g. A6) can also be a problem. A total of 224 gene names out of the 539 was matched in 76 of the 104 articles. The Results section was the one with the greatest number of unique gene names (Figure 5a). Again, the Abstract, and then the Introduction, are the sections with the highest frequency of these names (Figure 5b).\nFigure 5\nDistribution of matches to a set of 224 gene names across sections. (a) Average number of unique gene names per section. (b) Frequency of different gene names per total of nouns for each section.\nIn order to illustrate the problems that affect gene-name identification if context is ignored (even using gene names apparently easy to recognize) (discussed for example in [9]) we checked manually the context of gene names that were exclusively mentioned in the Methods section. Of the 224 genes, just 24 were mentioned in the Methods section of the corresponding 14 articles and not elsewhere (see Table 3). In five of the 14 articles, the name was referring to a non-gene object (three restriction endonucleases, a vector name, and a fibroblast cell strain). In five articles, the gene was mentioned in a technical context (usually, the gene mRNA level was used for analysis of cell state) and no biological process involving the gene was described. In only five articles we found the mention of the gene name relevant (See Table 3). Additionally, we noted that of these 24 gene names, at least two (Pbp2, Pom1) could refer to two non-homologous (unrelated) genes, and another one (Sac1) to four; such polysemous gene names complicate gene identification from text. Biologists are aware of such problems (see for example [10]). In summary, extreme caution should be applied with gene names appearing uniquely in the Methods section because the context of gene names there is very different to that seen in the rest of the article. If automated methods to extract gene names from text are applied to the Methods section, those that explore the context of gene names using part-of-speech tagging (for example, [11]) or Hidden Markov Models (for example, [7]) should then perform better than those that just take co-occurrences of gene names [12,13].\nTable 3\nDetection of gene names appearing only in the Methods section.\nRef\nRestriction endonucleases\nMsp1\nv27.n3.277\nIncorrect:\nPst1\nv19.n4.340\nSac1\nv27.n4.375\nVector name\nPsg5\nv23.n3.287\nCell strain\nTig3\nv26.n3.291\nDefinition of a Yeast strain\nCan1, Leu2, Lys2, Trp1\nv26.n4.415\nIn array\nFaf1\nv20.n3.266\nCorrect (technical context):\nGrowth detection\nMcm5, Mcm6\nv25.n3.263\nPlatelet mRNA analysis\nPbp2\nv23.n2.166\nPrimers used to determine embryo sex\nZfy1, Zfy2\nv27.n1.31\nAnalysis of mutant phenotypes\nPmd1\nv24.n4.355\ncDNA probe\nRab2\nv19.n2.134\nCorrect:\nSNP found in cDNA\nAdd3, Npr2\nv22.n3.239\nIdentifier given\nPom1\nv28.n3.223\nDetection of meiosis specific genes\nMei4, Mek1, Sps4, Zip1\nv26.n4.415\nRef: reference of the article in Nature Genetics by volume, issue number and first page of the article.\nDiscussion\nThere is a clear need for doing information extraction of biological data from full text scientific articles and the means for doing it are there with computers better suited for faster computation every day and new methodologies for Natural Language Processing that can be used for biomedical literature (see for example [14]). Regarding the source of data, the full text electronic versions of journals are now more the rule than the exception, with initiatives in the way towards the construction of large public repositories of such information (although hotly debated; see about PubMed Central [15,16]).\nIn this work we have shown that the distribution of information (as keywords) in full text articles is heterogeneous and that there is certain correspondence of article sections with different kind and density of relevant data. The Abstracts are shown as the best repository from the point of view of having many keywords in a short space, justifying previous information extraction approaches. The lack of large repositories of full text articles in contrast to the current eleven million of references (many of them with their abstract) in the MEDLINE database, are another advantage of the Abstract approach.\nHowever, we have shown that there is much more relevant information (at least in a ratio of 1:4 regarding gene names, anatomical terms, organism names, etc.) in the rest of the article. We have demonstrated that the information is structured enough to get important numbers of relevant keywords, but that for certain words (such as gene names) caution has to be taken regarding the context of the word.\nWe propose that the text mining of full text articles should be approached with different strategies for different sections. Beyond the Abstract, the Introduction looks like the best place to look for protein and gene names (and interactions) since it is probably describing current knowledge. The Discussion section, that interprets the results and put them in context with the current knowledge, looks like the third best place for mining such information, with Methods probably as the worst place. The Results section could be problematic given its mixed nature between Methods and the rest.\nRegarding other subjects, such as keywords about biological concepts (species, tissues, diseases, etc.), again the Abstract and then the Introduction section look like the best sections to mine regarding frequency of such keywords, but Results and especially Discussion seem better from a quantitative point of view. The Methods section is clearly appropriated for looking for technical data, measurements, and chemicals. Respect to chemicals, again, their context can be completely different in this section compared to the rest.\nConclusions\nExtraction of biological information from full text looks promising, but context must be regarded. Part of this context is given by the situation of the text under analysis within the article. Therefore, tuning the extraction of information to the section is probably a good strategy, and for particular tasks some sections should be avoided.\nWe have shown that the kind of simplistic annotation that constitutes tagging a fragment of an article as belonging to a characteristic section is already useful for text mining. But further tagging using markup codes in XML style [17] identifying biological objects and concepts (under development; see for example [18] or the GENIA project ) could ultimately make text mining a children's game. We hope for future interfaces for writers of Molecular Biology articles that should do the job upon validation by the authors (for example, marking every occurrence of a gene name with a unique and stable link to any of the existing gene sequence databases). For this to happen, the collaboration between both scientists and publishers will be very important.\nMethods\nDerivation of Associations between the words of a section\nGiven a section from an article, we split the text in sentences using a standard part of speech tagger (TreeTagger). We only computed associations between the words identified from the tagging as nouns. Following [4], the association between two words (wi,wj) (for example, \"cell\" and \"cycle\") can be modeled as the degree of inclusion of one word into the other () which can defined as the fuzzy binary relation given by: , that is, the ratio of the number sentences where both words wi and wj co-occur to the number of sentences the word wi occurs. This is an asymmetric relation very appropriate to model hierarchical relations between words as they happen in natural text. For example, in some Cell Biology context, the word \"cycle\" could appear always related to the word \"cell\" (as in \"cell cycle\"), but the word \"cell\" can be related to many other words such as in \"cell growth\", \"cell membrane\", or \"cell nucleus\". Accordingly, the inclusion value of the word \"cycle\" into \"cell\" will be close to one and the inclusion value of the word \"cell\" into the word \"cycle\" will be close to zero.\nSelection of Keywords\nWe identify a word as relevant for the text analyzed if it establishes many and strong relations to other words (following [4]). Therefore, in a given section, we define a score for a word wi that is equal to , normalized to the maximum value found for K of any word in that section. Then, the keywords of the section are defined as those words that have a K score above a certain value.\nClassification of Words in Subjects\nIn order to classify words into categories we used the following procedure. We chose the MeSH (Medical Subjects Headings) classification from the National Library of Medicine. All MeSH terms (including official synonyms) composed of one single word were selected and then the stem of the word was computed using TreeTagger. The words present in our corpus of 104 articles were ordered by frequency and all words occurring more than 200 times were selected. Those matching the selected single-word MeSH headers from six categories (A, B, C, D, E, and G; See the caption of Figure 4 for descriptions) were selected as belonging to those classes. In order to avoid possible miss-annotations, words matching more than one category were discarded. Manual analysis of the resulting table of associations was carried out in order to check the associations and to make new ones. A new class not present in MeSH (the X class of \"Units, Dimensions, & Parts\") was generated in order to include a large number of terms mainly present in the Methods section.\nAuthors' Contributions\nPS carried out the analysis of the keyword distribution from a database of full text articles. CP developed and applied the method to compute keywords. MA prepared the figures (except fig 2 by PS) and conceptualised the structure of the paper. PB and MA co-directed the project and contributed to the final manuscript. All authors collaborated during the whole length of the project. All authors read and approved the final manuscript.","keyphrases":["information extraction","keyword","full text article","gene name","data mining","text mining"],"prmu":["P","P","P","P","P","P"]}
{"id":"Clin_Rheumatol-3-1-2092406","title":"Sick leave and work disability in patients with early arthritis\n","text":"We studied the occurrence of sick leave and work disability, the presence of workplace adaptations and the usage of professional guidance related to working problems in patients with early arthritis. Inclusion criteria were arthritis symptoms of less than 2 years duration and a paid job at the time of diagnosis. Assessments were done in connection with an early arthritis clinic (EAC) at entry into the cohort and 12 months thereafter by means of a questionnaire comprising questions on sick leave (absenteeism from work reported to the employer), work disability (receiving a full or partial work disability pension), unemployment, work adaptations and professional guidance related to working problems. Fifty-seven of the 69 participants (83%) had an arthritis symptom duration of <6 months. The number of patients with sick leave due to arthritis in the past 12 months decreased from 28 (41%) at study entry to 18 (26%) after 12 months of follow-up. The number of patients receiving a work disability pension increased from 5 (7%) at study entry to 13 (19%) after 12 months of follow-up (10 partial and 3 full). Sick leave in the 12 months before study entry appeared to be the most important predictor of the institution or increase in a work disability pension (odds ratio, 16.1; 95%CI, 1.8\u2013142.8). Between study entry and follow-up, the number of patients with workplace adaptations increased from 20 (29%) to 28 (42%), whereas the number of patients receiving vocational guidance decreased from 48 (70%) to 36 (52%). In patients with early arthritis and a paid job, arthritis-related sick leave was common and occurred in part before patients entered the EAC and a diagnosis was made. About 20% of the patients became permanently work disabled, with partial work disability being more common than full work disability. Considerable proportions of patients received workplace adaptations and professional guidance with working problems.\nIntroduction\nRheumatoid arthritis (RA) and other chronic inflammatory rheumatic diseases are disabling conditions that affect the lives of individual patients in many ways. The prevalence of work disability is high among patients with RA [1], as well as other forms of chronic arthritis, such as ankylosing spondylitis [2], systemic lupus erythematosus [3] and to a lesser extent psoriatic arthritis [4, 5]. In RA patients, it was found that most work disability develops rapidly after onset [6], with recent studies showing work disability rates among RA patients 1\u00a0year after diagnosis vary between 15 and 30% [7\u20139].\nSo far, research has focussed mainly on permanent work disability and its determinants [10]. Over the recent years, the interest in sick leave, which is considered to precede permanent work disability in chronic arthritis, is increasing. In cross-sectional studies in early RA (disease duration <2\u00a0years), the proportions of patients on sick leave varied from 5\u201355% [7, 11\u201313], whereas in follow-up studies, 50\u201360% of the patients reported sick leave over a period of 1\u00a0year [7, 14, 15].\nUntil now, the number of studies on work disability and sick leave in early arthritis is confined to the situation after diagnosis, with no data being available on the occurrence of sick leave or permanent work disability in the preceding period. In addition, little is known about the extent of adaptations to the work place or guidance by health professionals in these early stages of the disease.\nThe aim of this study was therefore to describe the occurrence of sick leave and permanent work disability and its predictors in the very early phases of the disease. Moreover, this study aimed to describe the provision of adaptations to the workplace and professional guidance in patients with chronic arthritis.\nMaterials and methods\nPatients\nThis work disability study was originally designed as a multicenter study, including the Leiden University Medical Center (LUMC) Leiden, the Haga-Leyenburg Teaching Hospital and Bronovo Hospital, The Hague and the Reinier de Graaf Hospital, Delft, The Netherlands. We aimed to recruit patients in connection with two cohorts, the early arthritis clinic [16] (EAC; LUMC patients only) and the BeSt study [17] (LUMC, Haga-Leyenburg Teaching Hospital, Bronovo Hospital and Reinier de Graaf Gasthuis). BeSt is an abbreviation of treatment strategies (in Dutch: BehandelStrategie\u00ebn) for RA, with four drug treatment strategies all aimed at achieving low disease activity being compared in patients with early, active RA [17]. The Medical Ethics Committees of all three participating hospitals approved the protocol of this additional work disability study.\nDue to logistical problems, the actual recruitment of patients in the period 2000\u20132003 has only been carried out in connection with the EAC in the LUMC. The EAC is a facility aimed at early diagnosis and treatment of arthritis, including all patients suffering from arthritis in one or more joints, an arthritis symptom duration of less than 2\u00a0years, and not being diagnosed with gout, chondrocalcinosis or traumatic arthritis or a serious disease other than a rheumatic condition. General practitioners are requested to refer patients with suspected arthritis as soon as possible; all patients can be seen within 2\u00a0weeks after referral [16]. Only patients who had a paid job 12\u00a0months before the first visit in connection with the EAC were included.\nOn average, a definite diagnosis was made within 2\u00a0weeks after the first visit in connection with the EAC. At the time the present study was conducted, patients who were diagnosed with RA were promptly treated with either methotrexate or salazopyrine. RA patients fulfilling the inclusion criteria for the BeSt study [RA according to the American College of Rheumatology, formerly American Rheumatism Association, criteria [18], a disease duration of less than 2\u00a0years, aged 18\u00a0years or older and active disease indicated by \u22656 of 66 swollen joints, \u22656 of 68 tender joints and at least one of the following two criteria: an erythrocyte sedimentation rate (ESR) of \u226528\u00a0mm\/h or a global health score of \u226520\u00a0mm on a 0\u2013100\u00a0mm visual analogue scale (VAS), where 0\u2009=\u2009best and 100\u2009=\u2009worst] were randomized to four treatment groups. These groups included sequential monotherapy (group 1), step-up combination therapy (group 2), initial combination therapy with tapered high-dose prednisone (group 3), or initial combination therapy with infliximab (group 4). For patients failing on their medication, the study protocol prescribed a number of subsequent treatment steps [17].\nAll patients included in the work disability study gave written informed consent.\nAssessment methods\nAssessments were done at study entry (at study inclusion of the EAC cohort) and after 12\u00a0months of follow-up. Basic sociodemographic and clinical data were obtained from the baseline assessments of the EAC, whereas data on the working situation and mental health were gathered by means of additional questionnaires.\nSociodemographic and clinical characteristics\nSociodemographic variables included age, sex, status of living (living with a partner yes\/no) and education level, divided into three categories: primary education (0\u20138\u00a0years), secondary education (9\u201316\u00a0years) and higher vocational education \/university (17\u00a0years or more).\nDisease activity was measured by means of (a) the ESR (mm\/h); (b) the Ritchie score, concerning pain in 26 selected joints on a scale of 0\u20133 (0\u2009=\u2009no pain and 3\u2009=\u2009worst pain), with the final score ranging from 0\u201369 [19] and (c) the patient\u2019s global assessment of disease activity, pain and fatigue on three separate VAS. All VAS were 10\u00a0cm horizontal lines, with the anchors on the left being no disease activity, no pain and no fatigue, whereas the anchors on the right were the worst imaginable disease activity, severe pain and severe fatigue.\nPhysical functioning was measured by the Health Assessment Questionnaire (HAQ). The HAQ is a 20-item questionnaire comprising eight domains of activities of daily living, with the final score ranging from 0 (no disability) to 3 (severe disability) [20].\nAnxiety and depression were measured by means of a Dutch version of the Hospital Anxiety and Depression Scale (HADS) [21]. The higher the scores on the anxiety or depression scales the more anxious or depressed the subject is (range, 0\u201321 per scale).\nJob characteristics, sick leave, work disability, work adaptations and guidance with working problems\nCurrent occupation was grouped into four categories: (1) predominantly mental demands and absence of physical demands, (2) a combination of physical and mental effort, (3) light physical demands and (4) heavy physical demands [22].\nSick leave was defined as absenteeism with a duration of 2\u00a0weeks or more, related to the arthritic complaints and reported to the employer. At the time the study was conducted, in the Dutch social security regulations, the maximum duration of sick leave was 1\u00a0year, and sick leave could be either complete (reported sick for all hours that one is engaged) or partial (reported sick for part of the hours that one is engaged). The duration of sick leave in the 12\u00a0months before study entry and 1\u00a0year thereafter was divided into four categories: (1) 2\u20134\u00a0weeks, (2) 1\u20133\u00a0months, (3) 3\u20136\u00a0months, (4) 6\u20139\u00a0months and (5) 9\u201312\u00a0months.\nPermanent work disability was defined as receiving a complete or partial work disability pension related to the rheumatic disease. In the Dutch social security regulations, a partial work disability pension in combination with a part-time job is common. In this situation, sick leave may occur during the hours active at work.\nThe presence of material or immaterial adaptations at the workplace (including adapted tools, adapted furniture, reduction in tasks or duties, help of colleagues, flexible working hours, opportunities for more breaks, less working hours or reduced work pace) was recorded. Reduced working hours were only considered as an adaptation at work when this adaptation did not pertain to official, incomplete sick leave.\nGuidance with working problems was recorded by means of a question concerning the receipt of help in maintaining the job. Health professionals listed for giving help were: the rheumatologist, occupational physician, general practitioner, social worker, physical therapist, occupational therapist or nurse. In addition, it was registered whether patients received support from their employer and\/or colleagues. Satisfaction with the received guidance was recorded. In addition, patients were asked by whom they would have liked to be helped more.\nBackground information on sick leave and work disability\nBackground information on sick leave and work disability in the Dutch population in the period the study was conducted was derived from the Internet site of the Dutch Central Bureau of Statistics, http:\/\/www.cbs.nl.\nStatistical analysis\nData entry was performed using Microsoft Office Access 2000. Statistical analyses were executed using SPSS 10.0 for Windows.\nContinuous variables were presented as medians with ranges and categorical variables as number and percentages. Comparisons of the median age and the sex distribution of patients included in the study and all other patients of 65\u00a0years or younger who entered the EAC cohort in the study period were done by means of the Mann\u2013Whitney U and chi-square test, respectively.\nProportions of patients being on sick leave or receiving a work disability pension at study entry and after 12\u00a0months of follow-up were compared using McNemar tests for paired comparisons of categorical data, whereas the average numbers of hours per week patients were working were compared by means of the Wilcoxon signed rank test. These analyses were repeated excluding all patients with a symptom duration >6\u00a0months at study entry.\nSociodemographic, disease and job characteristics in the group of patients in whom the working situation deteriorated after 12\u00a0months of follow-up (defined as being entitled a partial or full work disability pension for the first time or changing from a partial to a full work disability pension) and in the group of patients in whom the working situation remained stable or improved (defined as receiving no disability pension or the same partial work disability pension at either time point of changing from a partial work disability pension to no disability pension) were compared by Mann\u2013Whitney U and chi-square tests.\nThen, a multivariate backward logistic regression analysis on predictors of deterioration of the working status was performed. Only variables that showed a significant difference in the above-mentioned analysis (p values\u2009\u2264\u20090.1) were entered as independent variables in logistic regression analysis.\nThe numbers of patients receiving adaptations in the workplace or vocational guidance in the past year at study entry and after 12\u00a0months of follow up were compared by the McNemar test, whereas the median total numbers of workplace adaptations per patient at both time points were compared by means of the Wilcoxon signed rank test.\nResults\nIn the study period, in total, 418 patients entered the EAC cohort. One hundred and five of these 418 patients (25.1%) were older than 65\u00a0years. From the remaining 313 patients, 81 patients (25.9%) were found eligible and willing to participate. The reasons for not being included were not recorded on the individual level. A group of patients was not eligible for the study because they were not having a paid job 12\u00a0months before the first visit in connection with the EAC cohort. In addition, a number of patients were eligible but declined participation, and some patients were not invited because of logistic reasons.\nTwelve of the initially included patients were excluded within 2\u00a0weeks after enrollment because a diagnosis of gout, chondrocalcinosis, sarcoidosis or a non-rheumatic disease was confirmed after additional diagnostic procedures so that they did not meet the inclusion criteria for the EAC.\nThe 69 patients included in the study were younger (median age, 42.0\u00a0years; range 18\u201364) than the 244 patients in the EAC cohort aged 65\u00a0years or less who were not included (median age, 48.0\u00a0years; range, 18\u201365; p\u2009=\u20090.08). Moreover, the proportion of female patients was lower in the 69 included patients (38\/69, 55%) than in the 244 patients who were not included (158\/244, 65%; p\u2009=\u20090.09).\nSociodemographic and disease characteristics, including physical and mental function, of the 69 included patients are shown in Table\u00a01. In total, 69 patients were recruited from the EAC cohort. Thirty-nine patients (57%) had definite RA, 8 patients (12%) had probable RA, 10 (15%) had psoriatic arthritis, 3 (3%) had spondylarthropathy and 9 (13%) had unclassified arthritis, with the arthritis still being unclassified after 12\u00a0months of follow-up. Twelve of the 39 patients with definite RA also participated in the BeSt study; two were randomized to treatment group 1, three to treatment group 2, five to treatment group 3 and two to treatment group 4.\nTable\u00a01Sociodemographic and disease characteristics of 69 patients with early arthritisCharacteristicsn (%)Sociodemographic characteristics\u00a0Age, in years; median (range)42 (18\u201364)\u00a0Female patients, n (%)38 (55)\u00a0Living with partner\/family, n (%)57 (83)\u00a0Educational level, n (%)\u00a0\u00a0High18 (26)\u00a0\u00a0Medium32 (46)\u00a0\u00a0Low19 (28)Disease characteristics\u00a0Diagnosis, n (%)\u00a0\u00a0RA (definite)39 (57)\u00a0\u00a0RA (probable)8 (12)\u00a0\u00a0Psoriatic arthritis10 (15)\u00a0\u00a0Arthritis unspecified9 (13)\u00a0\u00a0Spondylarthropathy3 (4)\u00a0Duration of arthritis symptoms, in years; median (range)0.3 (0\u20132)\u00a0VAS, 0\u201310\u00a0cm; median (range)\u00a0\u00a0Disease activity patient5.3 (0\u201310.0)\u00a0\u00a0Pain patient4.7 (1.0\u20139.8)\u00a0\u00a0Fatigue patient4.8 (0\u201310.0)ESR, in mm\/hr; median (range)20 (2\u2013105)Ritchie index (0\u201368); median (range)6 (1\u201332)HAQ (0\u20133); median (range)0.63 (0\u20131.88)HADS anxiety; median (range)5 (0\u201316)HADS depression; median (range)4 (0\u201313)\nIn total, 6 patients (9%) had a symptom duration of >1\u00a0year, 6 patients (9%) had a symptom duration between 6\u00a0months and 1\u00a0year and 57 patients (83%) had a symptom duration of <6\u00a0months.\nOverall, disease activity was moderate, with the median VAS disease activity score being 5.3 (range, 0\u201310.0)\u00a0cm, the median ESR 20 (range, 2\u2013105)\u00a0mm\/h and the median Ritchie score being 6 (0\u201332).\nData on the working status are presented in Table\u00a02. Patients were distributed fairly evenly over the four job categories.\nTable\u00a02Working status in 69 patients with early arthritis\u00a0Study entryFollow-up (12\u00a0months)p valueaJob characteristics\u00a0Occupational category, n (%)\u00a0\u00a0Mental demands18 (26)\u00a0\u00a0Mixed mental\/physical demands14 (20)\u00a0\u00a0Light physical demands20 (29)\u00a0Heavy physical demands17 (25)\u00a0Hours of work per week; median (range)36 (5\u201370)34 (4\u201370)b0.455\u00a0Full-time employment, n (%)34 (50)29 (42)0.375Sick leave\u00a0Current sick leave, n (%)22 (32)\u00a0Sick leave for the past 12\u00a0months, n (%)28 (41)18 (26)0.031\u00a0Extent of sick leave, n (%)\u00a0\u00a0Complete sick leave15 (54)12 (67)\u00a0\u00a0Partial sick leave7 (25)3 (17)\u00a0\u00a0Unknown6 (21)3 (17)\u00a0Duration of sick leave for the past 12\u00a0months, n (%)\u00a0\u00a02\u20134\u00a0weeks5 (18)7 (41)0.754\u00a0\u00a01\u20133\u00a0months14 (50)2 (12)0.002\u00a0\u00a03\u201312\u00a0months8 (29)8 (44)1.0\u00a0\u00a0Unknown1 (4)1 (4)1.0Work disability\u00a0Work disability pension\u00a0\u00a0Complete or partial, n (%)5 (7)13 (19)0.008\u00a0\u00a0Complete, n (%)03 (4)0.250\u00a0\u00a0Partial, n (%)5 (7)10 (15)0.180ap value of Wilcoxon signed rank test or McNemar where appropriate.bOnly patients who did not start to receive a work disability pension or an extension of a work disability pension were considered.\nAt study entry, 22 patients (32%) were currently on sick leave. In The Netherlands, in the period 2000\u20132003, the average proportion of persons on sick leave in the working population ranged between 4.8 and 8.4%, depending on the branch (public service or industry) [http:\/\/www.cbs.nl]. Twenty-eight patients reported sick leave over the past 12\u00a0months, whereas after 12\u00a0months follow-up, 18 patients reported sick leave in the preceding period. This difference was mainly accounted for by a decrease in the number of patients reporting sick leave for periods of 1\u20133\u00a0months. In total, 34 patients (49%) reported sick leave in either 12\u00a0months before study entry or 12\u00a0months thereafter. Of the 27 patients who reported sick leave due to arthritis in the past 12\u00a0months with a specific duration at study entry, 5 were partially or fully work disabled after 12\u00a0months of follow up, 10 reported long-term sick leave (1\u20133\u00a0months), 4 reported short-term sick leave (2\u20134\u00a0weeks) and 8 reported no sick leave due to arthritis.\nFive patients (7%) received a partial disability pension at study entry. In two of these patients, this entitlement was extended to a full work disability pension after 12\u00a0months, whereas in the other three patients, the disability pension remained unchanged. In addition, eight patients (12%) started receiving a work disability pension in the 12-month follow-up period (one patient a full disability pension and seven a partial disability pension).\nIn The Netherlands, the average proportion of employees in the working age range (15\u201364\u00a0years) becoming work disabled over a period of 1\u00a0year ranged between 1 and 1.1% in the period the study was conducted (http:\/\/www.cbs.nl). None of the patients became unemployed during follow-up.\nA repetition of the analyses excluding the six patients with a symptom duration of >1\u00a0year showed similar results (data no shown).\nTable\u00a03 shows sociodemographic, disease and job characteristics of patients in whom the work status deteriorated (institution or extension of work disability pension) and of patients in whom the working situation improved or remained stable. In the patients in whom the work status deteriorated, the proportions of patients with a diagnosis of definite RA, the occurrence of sick leave in the previous 12\u00a0months and a duration of sick leave >4\u00a0weeks were significantly higher than in the group of patients in whom the working status remained stable. Moreover, patients in whom the working status deteriorated had a significantly higher VAS disease activity compared to patients in whom the working status remained stable or improved.\nTable\u00a03Deterioration of working statusa in 12\u00a0months follow up in 69 patients with early arthritis\u00a0More work disability, n\u2009=\u200910Stable work (dis)ability n\u2009=\u200959p valuebSociodemographic characteristics\u00a0Age, years; median (range)42 (28\u201358)44 (18\u201364)0.484\u00a0Female patients, n (%)5 (50)33 (56)0.996\u00a0Educational level, n (%)\u00a0\u00a0High5 (50)13 (22)0.141\u00a0\u00a0Medium3 (30)29 (49)0.435\u00a0\u00a0Low2 (20)17 (29)0.846Disease characteristics\u00a0Diagnosis, n (%)\u00a0\u00a0RA (definite)9 (90)30 (51)0.049\u00a0\u00a0RA (probable)08 (14)0.481\u00a0\u00a0Arthritis unspecified1 (10)8 (14)1.0\u00a0\u00a0Psoriatic arthritis010 (17)0.356\u00a0\u00a0Spondylarthropathy03 (5)1.0\u00a0Duration of symptoms, years; median (range)0.4 (0\u20130.9)0.3 (0\u20132)0.955\u00a0VAS, 0\u201310\u00a0cm; median (range)\u00a0\u00a0Disease activity patient7.5 (4.1\u20139.8)5.1 (0\u201310.0)0.038\u00a0\u00a0Pain patient5.7 (2.1\u20139.8)4.6 (1.0\u20139.6)0.096\u00a0\u00a0Fatigue patient5.9 (5.0\u20139.8)4.0 (0\u201310.0)0.131\u00a0Disease activity\u00a0\u00a0ESR, mm\/h; median (range)26 (6\u2013105)17 (2\u201393)0.264\u00a0\u00a0Ritchie index (0\u201368); median (range)14 (2\u201317)6 (1\u201332)0.068\u00a0Physical and mental functioning\u00a0\u00a0HAQ (0\u20133); median (range)0.88 (0\u20131.75)0.63 (0\u20131.88)0.174\u00a0\u00a0HADS anxiety; median (range)6.5 (0\u201313)5 (0\u201316)0.650\u00a0\u00a0HADS depression; median (range)5 (1\u201313)3 (0\u201311)0.195Job characteristics\u00a0Current occupational category, n (%)\u00a0\u00a0Mental demands3 (30)15 (25)1.0\u00a0\u00a0Mixed mental\/physical demands3 (30)11 (19)0.689\u00a0\u00a0Light physical demands1 (10)19 (32)0.292\u00a0\u00a0Heavy physical demands3 (30)14 (24)0.977\u00a0Sick leave related to arthritis 12\u00a0months before study entry, n (%)9 (90)19 (32)0.002\u00a0Duration of sick leave related to arthritis in 12\u00a0months before study entry, n (%)\u00a0\u00a02\u20134\u00a0weeks05 (26)0.767\u00a0\u00a01\u20133\u00a0months5 (56)9 (47)0.036\u00a0\u00a03\u201312\u00a0months4 (44)5 (26)0.026aDeterioration of work status defined as starting to receive a work disability pension or altering from a partial to a full work disability pension after 12\u00a0months of follow-up.bp value of Mann\u2013Whitney U test or chi-square where appropriate\nIn the multivariate logistic regression analysis, the occurrence of sick leave in 12\u00a0months before study entry was found to be the only significant predictor of deterioration of the working situation (odds ratio, 16.1; 95%CI, 1.8\u2013142.8).\nThe presence of material and immaterial adaptations at the workplace is shown in Table\u00a04. The number of patients in whom the workplace was adapted increased from 20 (29%) at baseline to 28 patients (42%) after 1\u00a0year follow-up. The numbers of patients using adapted tools or furniture, flexible time-management or performing different tasks increased significantly with time. Moreover, the average number of workplace adaptations per patient increased significantly.\nTable\u00a04Presence of material and immaterial adaptations at the workplace in 69 patients with early arthritis\u00a0Study entry, n\u2009=\u200969Follow-up (12\u00a0months), n\u2009=\u200966p valueaNo. of patients with adaptations, n (%)20 (29)28 (42)0.134No. of adaptations per personb; median (range)2.5 (1\u20136)3.0 (1\u20139)<0.001Types of adaptations\u00a0Adapted tools\/furniture, n (%)1 (3)11 (17)0.002\u00a0Different tasks (extra education), n (%)2 (3)12 (18)0.013\u00a0Reduction of tasks\/duties, n (%)9 (13)18 (27)0.064\u00a0Help of colleagues, n (%)9 (13)15 (23)0.210Flexible working hours, n (%)10 (15)7 (11)0.581Flexible time-management, n (%)7 (10)17 (26)0.021Less working hours, n (%)8 (12)12 (18)0.424Reduced work pace, n (%)5 (7)7 (11)0.774ap value of McNemar test or Wilcoxon signed rank test where appropriate.bOnly considering patients with one or more adaptations.\nTable\u00a05 shows the guidance of other persons with the aim of the maintenance of the paid job. At study entry, 48 patients (70%) and, in the follow-up period, 36 patients (52%) received professional guidance, with the rheumatologist, general practitioner and physical therapist being the most frequently mentioned health care professionals. The proportions of patients being satisfied with the guidance were the lowest for the occupational physician. Employers and\/or colleagues provided help in one third of patients, for the large majority of patients to their satisfaction.\nTable\u00a05Usage of and satisfaction with professional guidance and help of employers\/colleagues with respect to arthritis-related problems at work in 69 patients with early arthritis\u00a0Study entryFollow-up (12\u00a0months)UsageSatisfactionUsageSatisfactionRheumatologist, n (%)37 (54)35 (95)21 (30)21 (100)General practitioner, n (%)34 (49)29 (85)12 (17) 12 (100)Physiotherapist, n (%)24 (35)20 (83)21 (30)21 (100)Occupational physician, n (%)16 (23)12 (75)19 (28)13 (68)Social worker, n (%)6 (9)6 (100)4 (6)4 (100)Clinical nurse specialist, n (%)6 (9)6 (100)9 (13)8 (89)Occupational therapist, n (%)5 (7)5 (100)9 (13)8 (89)Total48 (70)41 (59)Employer\/colleagues, n (%)23 (33)21 (91)21 (30)19 (91)All values expressed as numbers (%)\nThirteen patients (19%) would have liked to have more guidance with problems at work, especially from their rheumatologist, a nurse specialized in rheumatology or from their employer.\nDiscussion\nThis study in patients with early chronic arthritis shows that about half of the patients reported arthritis-related sick leave, and 19% became permanently work disabled over a period 1\u00a0year before and 1\u00a0year after entering an EAC cohort. Partial work disability was more common than full work disability, and sick leave appeared to be its most important predictor. In addition, work place adaptations, professional guidance and help from employer and\/or colleagues were common in this population.\nWith respect to the occurrence of sick leave, our results are in accordance with those of previous follow-up studies in patients with early arthritis, where sick leave rates of 50\u201360% over a period of 1\u00a0year were reported [7, 14, 15]. As in our study, 83% of the patients had a disease duration of less than 6\u00a0months; our data on the occurrence of sick leave in the 12\u00a0months before study entry suggest that in a considerable proportion of patients, sick leave may occur before they enter an EAC, and a diagnosis of arthritis has been made. In our study, the occurrence of sick leave decreased with time, a finding that cannot be completely interpreted as a positive observation, as it was in part explained by patients on sick leave becoming permanently work disabled (Table\u00a06).\nTable\u00a06Working situation after 12\u00a0months of follow-up in 27 patients with early arthritis who reported sick leave at study entry (in one patient the duration of sick leave was unknown)Study entryFollow-upDuration of sick leavePartial work disabilityFull work disabilitySick leave 2\u20134 weeksSick leave 1\u20133 monthsSick leave 3\u201312 monthsNo sick leave2\u20134\u00a0weeks (n\u2009=\u20095)0012021\u20133\u00a0months (n\u2009=\u200914)2131343\u201312\u00a0months (n\u2009=\u20098)110042\nIn our study, 19% of the patients were permanently work disabled after 2\u00a0years of follow-up. This figure is in the same range or somewhat more favourable than work disability rates found in previous studies in patients with early RA [7\u20139]. In fact, our results may even be more favourable, as work disability was partial in the majority of patients, whereas in other studies, only complete job loss was considered. It should be noted, however, that our study concerned various forms of chronic arthritis, and a deterioration of the working situation appeared to be more frequent among patients with a diagnosis of definite RA than in patients with other forms of arthritis. Our results are in concordance with the observation that the incidence of permanent work disability among RA and AS patients appears to be declining over the last decades, most probably related to new developments in pharmacological therapy [23\u201325]. Indeed, the patients included in our study were promptly treated, with the medical treatment for the patients with RA consisting of either monotherapy with methotrexate or salazopyrine or treatment according to the BeSt protocol [17].\nIn this study, the duration of symptoms varied among patients, so that not all patients were at equal risk of developing sick leave or work disability. The impact of the symptom duration may however be limited, as one of our analyses showed that symptom duration had no effect on the development of more work disability in the follow-up period of 12\u00a0months.\nIn this study, no control group was used for comparison. Data from the Dutch Central Bureau of Statistics (http:\/\/www.cbs.nl) indicate that there is a considerable \u201cbackground risk\u201d of sick leave and work disability in the general population. However, as in this study, patients were explicitly asked to report sick leave and work disability due to arthritis; it is doubtful whether this background risk needs to be taken into account. It appeared that sick leave and work disability rates among patients with early arthritis were considerably higher than those in the general population; however, for valid comparisons, a separate study with similar registration methods among patients with early arthritis and the general population is needed.\nWith respect to the determinants of work disability, similar to studies in RA [26], disease severity was, in general higher, and physical and psychological functioning worse among patients in whom the working status deteriorated than in those in whom the working status remained stable. The fact that the association of some measures of disease severity or physical or psychological functioning with changes in the working status did not reach statistical significance may have been due to the relatively small sample size of this study. Moreover, our finding that the occurrence of sick leave in the 12\u00a0months before study entry was associated with a deterioration of the working situation is in concordance with the results of another study by our group among another group of patients with various form of chronic arthritis [27]. The results of both studies underscore the importance of the recognition of sick leave as a risk factor for work disability in patients with chronic arthritis. Previously, it has been questioned whether rheumatologists do sufficiently recognize patients\u2019 working problems [28] of which sick leave could be considered as one of its manifestations. Apart from paying attention to the presence of sick leave, the use of instruments that have been specifically developed for the identification of those individuals with chronic arthritis having difficulties at work could be helpful because by using these tools, working problems could be recognized in earlier stages. Examples of those instruments are the RA-Work Instability Scale [29] and the Work Limitations Questionnaire [30]. In addition, Detaille et al. [31] showed which issues are important from the patients\u2019 perspective and recommended a topic list for various health professionals to use during consultations. So far, these instruments are only partly validated, have been mainly used in studies, and are not developed to be applied at the individual level. At present, valid and brief questionnaires specifically designed for clinical practice are not yet available. It is however conceivable that simply asking patients about working problems or sick leave is another sensitive method to identify patients at risk of work disability.\nThe results of this study show that over a period of 2\u00a0years, in 30\u201340% of the patients, the work situation was adapted, about 30% received help from the employer or colleagues and 60\u201370% of patients received professional guidance to maintain their job. With respect to the implementation of work place adaptations and help from the employer or colleagues, these figures are in line with those of a recent study in early RA patients [32]. In that study, it was demonstrated that support and help of co-workers, employer help, shortened work days, new skills and a modified work station or equipment were among the work-related factors that significantly ameliorated work ability. With respect to professional guidance, a recent study in Dutch employing RA patients with a short disease duration found that according to the patients, the mobilization of health care professionals would help them tackle their threats to work ability [33].\nIn our study, patients were generally very appreciative of help concerning their working problems, with the lowest proportions of patients being satisfied obtained for the occupational physician. In The Netherlands, the importance of the role of occupational physician in the maintenance of a paid job in patients with chronic diseases is generally recognized. However, in a previous study, the communication between occupational physicians and rheumatologists was found to be hampered [34], factor that could have contributed to the relatively low satisfaction scores.\nA limitation of the present study may be that the total number of patients was relatively low, and various types of chronic arthritis were included, hampering conclusions about specific diagnosis groups. Moreover, in this study, about 25% of all patients in the working age range (<65\u00a0years) who entered the EAC participated in the work disability study. This proportion is significantly lower than the percentages of patients with a paid job at symptom onset in similar age groups in early RA cohorts, with frequencies ranging between 50 and 75% [11, 35\u201337]. As the sociodemographic and clinical characteristics of the non-participants were not systematically gathered, it remains unclear to what extent the population included in this study differs from all patients with early arthritis who have a paid job at symptom onset. Given the probability of selection bias, our results have to be interpreted with care.\nIn conclusion, this study in patients with early chronic arthritis and a paid job showed that the occurrence of arthritis-related sick leave, before and directly after a diagnosis is made, is substantial. In contrast, full permanent work disability developed in a relatively small minority. Work place adaptations, professional guidance and help from employers or colleagues to maintain a paid job were fairly common. Future research should aim at the detection of patients at risk for the development of sick leave and permanent work disability, as well as the optimization of the process of vocational guidance.","keyphrases":["sick leave","work disability","arthritis","occupational health","vocational rehabilitation"],"prmu":["P","P","P","R","M"]}
{"id":"Qual_Life_Res-4-1-2248606","title":"Measurement of overall quality of life in nursing homes through self-report: the role of cognitive impairment\n","text":"Measuring quality of life is a necessity for adequate interventions. This paper concerns the usefulness of six self-report measures for overall quality of life for nursing home residents with various levels of cognitive impairment. It was investigated which proportion of residents from four cognition groups could complete a scale, and internal consistency and construct validity of the scales were studied. Data collection took place in ten Dutch nursing homes (N = 227). The proportion of residents that could complete each scale varied. The Depression List could be administered most often to the cognitively most impaired group (43%; Mini Mental State Examination-scores 0\u20134). In the three cognition groups with MMSE-score >5, internal consistency of the Depression List, Geriatric Depression Scale and Negative Affect Scale was adequate in all three groups (alpha \u2265.68). Intercorrelation was highest for the Philadelphia Geriatric Center Morale Scale, the Depression List, and the Geriatric Depression Scale (rho \u2265.65). Nonetheless, self-report scales were not strongly correlated with two observational scales for depression, especially in cognitively severely impaired residents (rho \u2264.30). In conclusion, it may not be possible to measure overall quality of life through self-report, and possibly also through observation, in many nursing home residents.\nIntroduction\nOver the past decades, quality of life has become a focal point in scientific research and clinical practice. Although researchers disagree on the domains that make up quality of life, the general consensus is that quality of life measurement should focus on the subjective experience of the individual. This implies that the individual in question is the most valid source of information [1, 2]. However, nursing home residents may not be able to respond to self-report measures or lose this ability during their stay, f.i. due to dementia, which complicates the assessment and monitoring of a resident\u2019s quality of life across time [3\u20135]. Although self-report is a complex process of introspection and evaluation [6], research has asserted that moderately demented patients still can report on their quality of life, even when they have poor insight into and awareness of their dementia [4, 7\u20139]. According to Kane et\u00a0al. [8], 60% of the nursing home population would be able to reliably report on their quality of life. It would, therefore, be helpful to know which scales can be applied to nursing home residents with varying degrees of cognitive impairment.\nMost scales measure separate dimensions of quality of life [10]. This has the advantage of a higher responsiveness to change than a measure for overall quality of life (OQOL). However, OQOL is an attractive outcome that can be measured as a single subjective result of weighing unspecified dimensions that are considered to be relevant by the patient. The administration is also less burdening, which is an important factor in a very frail elderly population. Therefore, several researchers (also) use a single overall measure [e.g. 3, 7].\nIn our approach to quality of life [11, 12], OQOL is represented by subjective (i.e. psychological) well-being. Given this, scales for subjective (psychological) well-being can be used as OQOL scales. Although not always considered as the central outcome, psychological well-being is an important dimension of quality of life in many other approaches to quality of life in the elderly [13\u201319]. It encompasses both positive and negative affect, and life satisfaction (i.e. morale and contentment) [14, 20, 21], but most often a selection of these concepts is used for measurement. Instruments for psychological well-being that are used in the elderly are, for instance, the Philadelphia Geriatric Center Morale Scale (PGCMS) [22] and the Bradburn Affect Balance Scale [23]. For measuring affect in the elderly, the Positive and Negative Affect Scales [24, 25] and the observational Philadelphia Geriatric Center Positive and Negative Affect Scales [26] have been recommended [21]. Both positive affect and negative affect are important dimensions in quality of life scales [7, 17\u201319, 27]. Sometimes, however, only negative scales, such as scales for depression, are used in the measurement of quality of life [28]. As the absence of depression does not automatically imply that a resident is happy or content, this poses the question of whether positive and negative scales do, indeed, measure separate constructs and thus, whether or not a negative scale can be used as a single scale for OQOL.\nIn this paper the aim is to investigate the usefulness of six self-report measurement scales for OQOL, by studying whether they can be administered reliably and validly in a large group of nursing home residents, and whether this is related to cognitive impairment. We hypothesized that if all scales measure OQOL in nursing home patients validly and reliably, they would correlate highly, within all cognition groups. Moreover, the scales should be related to observational scales that measure OQOL.\nMethods\nData were collected in ten nursing homes in the Netherlands. The Medical Ethics Committee of the VU University Medical Center had approved the research proposal, and written informed consent was obtained from the participants or their legal representative. Data were collected on a maximum sample of 30 residents over a period of 3\u00a0months per nursing home, with an equal distribution of residents with mainly physical handicaps (in so-called \u2018somatic\u2019 units) and mainly dementia syndromes (in so-called \u2018psychogeriatric\u2019 units).\nThe principal investigator (DLG, a trained psychologist) administered the self-report OQOL scales and the cognitive test (see later), while the nursing staff carried out the observational assessments. The completeness of the interview data depended on the resident\u2019s cognitive and physical abilities and willingness to answer questions. The scales were administered in random order. The administration of a scale was terminated when a resident proved to be unwilling or unable to respond to the questions that were asked. To assure the validity of cross-sectional comparisons, the self-report and observational scales for each resident were both assessed within the same 4-week period.\nMeasurement instruments\nThe scales that were selected to measure OQOL had been used before in published research among nursing home residents or frail elderly populations. A distinction was made between scales that ask about OQOL literally, scales that focus on positive affect, negative affect or life-satisfaction, and scales that indicate clinical depression.\nSelf-report OQOL scales\nA general question on OQOL (GEN-QOLQ) was asked: \u2018Overall, how would you rate the quality of your life at the moment?\u2019. This is a modification of the general question on quality of life that is part of Brod and co-workers\u2019 scale for quality of life in people with dementia [7]. To our question \u2018at the moment\u2019 was added, because a pilot study showed that, without this explicit time-limit, the residents tended to evaluate the whole of their past life. The response scale, which is presented in the form of a card, consists of the following response categories: 1\u00a0=\u00a0bad, 2\u00a0=\u00a0moderate, 3\u00a0=\u00a0good, 4\u00a0=\u00a0very good, and 5\u00a0=\u00a0excellent.\nThe Philadelphia Geriatric Center Morale Scale (PGCMS) [22] is a self-report scale that has been developed to assess elderly people in institutions, and has regularly been used as an outcome measure in research on quality of life and well-being in the elderly [e.g. 29, 30]. It consists of 17 dichotomous items measuring life satisfaction, and the scores are summed, with a high score indicating high quality of life. The scale has been found to be reliable, valid and sensitive [30], and internally consistent (KR-20 of .79) [31].\nThe Positive And Negative Affect Scales (PANAS) [24], were also used. The Positive Affect Scale (PAS) consists of 10\u00a0items concerning positive feelings, such as enthusiasm, interest and determination. The Negative Affect Scale (NAS) consists of 10\u00a0items concerning \u2018negative\u2019 feelings, such as fear, sadness, anxiety and hostility. For this study, the time frame \u2018today\u2019 was chosen, and instead of the original 5-category scale, a 2-category response scale was used, because a pilot study showed that very few residents were able to answer the 5-category scale. The administration was visually mediated, following the procedure proposed for the Depression List (see further). Summing the item-scores yielded two separate total scores, ranging from 0 (no positive\/negative affects confirmed) to 10 (all positive\/negative affects confirmed). The PAS and the NAS were found to be suitable for use in the elderly [21, 25]. Earlier reported internal consistency with the time-frame of \u2018today\u2019 yielded a Cronbach\u2019s alpha of .90 for the PAS and .87 for the NAS in the general population [24].\nThe Depression List (DL) is a Dutch self-report screening instrument for depression, especially suitable for the assessment of (elderly) people with cognitive impairment [32]. It consists of 15 keywords that are presented on cards, one by one, accompanied by a simple question. For instance, a card with \u2018down\u2019 printed on it is accompanied by the question \u2018do you feel down?\u2019. Sum-scores range from 0 (no depressive complaints) to 30 (many depressive complaints). In psychometric research, the reported internal consistency of the DL was .82 in a group of visitors to a psychogeriatric day-care clinic [32].\nThe Geriatric Depression Scale (GDS) is a self-report screening instrument for depression in the elderly that is of known reliability and validity, also in long-term care [33, 34]. It consists of 30 dichotomous questions, which are summed into total scores, ranging from 0 (no depressive complaints) to 30 (many depressive complaints).\nOther scales\nThe Mini Mental State Examination (MMSE) is a test for cognition, and has scores ranging from 0 (very severe cognitive impairment) to 30 (no cognitive impairment). It is widely used and has been validated, also in long- term care populations [35, 36].\nThe GIP-sad behavior [37, 38] is a sub-scale of the Behavior Observation Scale for Geriatric Inpatients (GIP), which is widely used in nursing homes in the Netherlands. The 6-item GIP-sad behavior (GIP-S) measures the behavior of elderly people in intramural care settings that expresses sadness, unhappiness, and anxiety, and is used in the present paper as a scale for OQOL. Sum-scores range from 0 (no sad behavior) to 18 (frequent sad behavior). When first published, the internal consistency (Cronbach\u2019s alpha) of the scale was .84, and the inter-rater reliability (Pearson\u2019s r) was .74 [37]. In a validation study, internal consistency was found to be .86, and the average inter-rater reliability of the items (Cohen\u2019s weighted kappa) was .43 [39].\nThe MDS Depression Rating Scale (DRS) is an observational scale, based on items from the Minimal Data Set of the Resident Assessment Instrument [40], which can be used to screen for depression [41]. The DRS consists of seven MDS items that are summed. The scores range from 0 (no depressive behavior) to 14 (frequent depressive behavior). The internal consistency (Cronbach\u2019s alpha) when it was developed was .75 in the derivation sample and .71 in the validation sample. Its sensitivity against a psychiatric diagnosis of depression was 91% [41]. In a validity study the internal consistency of the DRS was .68, and its correlations with the Geriatric Depression Scale (GDS) and the Hamilton Depression Rating Scale were .19 and .24 respectively [42].\nAnalyses\nIn order to determine whether cognitive status relates to the psychometric properties of the scales, the total group of residents was divided into four MMSE score-groups. An attempt was made to find a division based on known cut-off points that also resulted in equally large groups. The traditional MMSE cut-off point indicating cognitive impairment is 22\/23 [43]. Among the reported cut-off points for severe cognitive impairment are 16\/17 and 17\/18 [36], and known cut-off points for reliable self-report assessment are 9\/10 [e.g. 7] and 14\/15 [e.g. 44]. The division into cognition groups was carried out as follows: a MMSE score below 5 (very low cognition group); scores from 5 to 12 (low cognition group); scores from 13 to 21 (moderate cognition group); and scores of 22 or higher (\u2018high\u2019 cognition group).\nFor each MMSE score-group, the number of residents who could complete each scale was calculated, and compared with the number of residents to whom it was offered. Cronbach\u2019s alphas were calculated to determine the internal consistency of the scales, and were compared across the different cognition groups. Cronbach\u2019s alpha is considered to be fairly good if higher than .70, but should not be higher than .90 [45]. For construct validity, Spearman coefficients for the interrelationships of the self-report OQOL scales and for the relationships of the OQOL scales with the observational scales for OQOL were calculated and compared across the different cognition groups. Significant correlation coefficients are described in the results and interpreted as follows: .21\u2013.40\u00a0=\u00a0fair correlation, .41\u2013.60\u00a0=\u00a0substantial correlation, and .61\u2013.90\u00a0=\u00a0strong correlation.\nResults\nSample description\nThe overall sample consisted of 227 residents. Their average age was 80.5 (SD 9.26; range 52\u2013100), and 78% were female. The average score on the MMSE (N\u00a0=\u00a0200) was 11.8 (SD 9.26); 26.5% had a score below 5, and 18.5% had a score of 22 or higher. The internal consistency of the MMSE in this sample was .89. The curves of the NAS and the DL were slightly positively skewed. The scores on the NAS scales were found to be quite low (Table\u00a01, descriptives total group).\nTable\u00a01Descriptives and practical utility of self-report overall quality of life-scalesScales (max. range)Descriptives total groupAvailable MMSE scores% residents who could complete a scale, per MMSE score groupaMean (range)SDNN22\u20133013\u2013215\u2013120\u20134PositiveGEN-QOLQ (1\u20135)2.7 (1\u20135)1.011911797 (35\/36)81 (35\/43)80 (43\/54)11 (4\/36)PGCMS (0\u201317)10.3 (0\u201317)4.511211094 (34\/36)84 (36\/43)72 (39\/54)3 (1\/36)PAS (0\u201310)4.4 (0\u201310)2.610298100 (30\/30)97 (31\/32)94 (34\/36)21 (3\/14)NegativeNAS (0\u201310)1.6 (0\u20137)1.99894100 (30\/30)97 (30\/31)94 (31\/33)21 (3\/14)DL (0\u201330)8.0 (0\u201326)5.4143135100 (23\/23) 96 (26\/27)100 (65\/65)43 (21\/49)GDS (0\u201330)10.8 (0\u201326)7.0106102100 (37\/37)91 (39\/43)72 (26\/36)0 (0\/14)aEach fraction presents the number of residents who completed the scale in relation to the total number of residents who were offered the scale. Because the scales were not offered to all 227 participants, the numbers in the denominators do not add up to 227, and differ for each scale\nDue to practical considerations and the frailty of the residents, not all scales were administered to all residents. Therefore, the number of completed scales varied.\nProportion of completed scales\nTable\u00a01 reports that in the high cognition group (MMSE score of 22 or higher), all six scales could be completed by 94\u2013100% of the residents. In the moderate cognition group (MMSE score between 13 and 21), all scales, except the GEN-QOLQ and the PGCMS, could be completed by 91\u201397% of the residents. The GEN-QOLQ and the PGCMS could be completed by 84% and 81% in this group, respectively. In the low cognitiongroup (MMSE score 5\u201312) the DL still could be completed by all residents (100%), the PAS and the NAS by 94%, the GEN-QOLQ by 80%, and the PGCMS and the GDS by 72%. In the very low cognition group (MMSE score below 5) the DL could still be completed by 43% of the residents, but only 21% or less could complete the other scales. The GDS was not completed by any of these residents, and the PGCMS only by 3%.\nOnly 3 MMSE-groups were used for all further analyses, because in the very low cognition group (N\u00a0=\u00a053) too few residents could complete the scales.\nInternal consistency\nThe GEN-QOLQ consisted of one question, so internal consistency analysis was not applicable. Table\u00a02 shows that internal consistency of the DL and GDS was satisfactory in all cognition groups; that of the NAS was also acceptable, although this scale was somewhat less consistent in the high cognition group (.68). The PGCMS shows good consistency, except in the moderate cognition group (.53). The PAS is the least reliable scale, as it only reaches an acceptable alpha in the moderate cognition group. Further, the scales showed no linear trends of decreasing internal consistency with increasing cognitive impairment, but there were some variations between cognitive groups.\nTable\u00a02Internal consistency of self-report overall quality of life-scales for different cognition groupsTotal groupHigh cognition MMSE \u226522Moderate cognition MMSE <22 & \u226513Low cognition MMSE <13 & \u22655Alphaa (miic) bNcAlpha (miic)NAlpha (miic)NAlpha (miic)NPositivePGCMSd.72 (.17)108.81 (.26)33.53 (.14)35.83 (.22)38PAS.55 (.18)98.47 (.20)29.77 (.25)30.68 (.18)32NegativeNAS.72 (.18)97.68 (.16)29.81 (.31)30.71 (.17)31DL.81 (.22)139.87 (.30)22.72 (.15)26.79 (.21)64GDS.91 (.24)98.90 (.24)34.92 (.29)37.89 (.22)23aAlpha\u00a0=\u00a0Cronbach\u2019s alpha; bMiic\u00a0=\u00a0mean inter-item correlationcThe Ns of the MMSE-groups do not add up to the N of the total group, because some participants (N\u00a0=\u00a027 of N\u00a0=\u00a0227) did not have an MMSE-scoredAs it consists of one question, no alpha coefficient could be calculated for GEN-QOLQ\nConstruct validity\nConstruct validity, inter-relationship\nTable\u00a03 presents the Spearman correlation coefficients for the self-report OQOL scales in the three cognition groups. Each correlation coefficient for two scales pertains to all residents who completed both scales. Therefore, the group-sizes differ for the correlation coefficients. Also, the group sizes may be larger than in Table\u00a02, because scale-scores were also calculated when one of the scale\u2019s items was missing, using mean substitution.\nTable\u00a03Spearman correlation coefficients between self-report overall quality of life scales, for the separate MMSE score-groupsGEN-QOLQPGCMSPASNASDLHCaMCLCHCMCLCHCMCLCHCMCLCHCMCLCPositivePGCMS .38*.61**.22N343537PAS.01.30.47**.19.21.34N303031293132NegativeNAS\u2212.06\u2212.32.13\u2212.51**\u2212.69**\u2212.47**\u2212.17\u2212.10.16N302929293030303031DL\u2212.43*\u2212.28\u2212.35*\u2212.75**\u2212.79**\u2212.76**\u2212.36\u2212.27\u2212.25.45.62**.44*N232143232139192134192131GDS\u2212.47**\u2212.60**\u2212.36\u2212.80**\u2212.66**\u2212.65**\u2212.20\u2212.43*\u2212.25.64**.65**.49*.88**.77**.69**N353424343525303023302922232026aHC\u00a0=\u00a0High cognition group (MMSE score \u226522); MC\u00a0=\u00a0moderate cognition group (MMSE score <22 \u226513); LC\u00a0=\u00a0Low cognition group (MMSE score <13 \u22655)*\u00a0P\u00a0<\u00a0.05; **\u00a0P\u00a0<\u00a0.01 \nTable\u00a03 shows that the PGCMS, DL and GDS had the strongest intercorrelation. These three scales correlated significantly (P\u00a0<\u00a0.01) in all cognition groups. Also the NAS was substantially correlated with the GDS and the DL. The other relationships between the scales varied. As far as the different cognitive groups are concerned, although the correlations between the scales were mostly lowest in the low cognition group, no clear linear trend across cognition groups was visible. The strength of the relationships between the positive PGCMS and the negative NAS, DL and GDS further indicates that these constructs are far from independent, as often has been found.\nConstruct validity, relationship with GIP-S and DRS\nAs Table\u00a04 shows, the GIP-S correlated significantly with the GEN-QOLQ, PGCMS, NAS, DL and GDS, but only in the moderate cognition groups. The DRS correlated only significantly to the GEN-QOLQ (only for the high cognition group), PGCMS (for both high and moderate cognition), DL (for moderate cognition) and GDS (for both high and moderate cognition). None of the self-report scales correlated significantly with the observational scales in the low cognition group. Remarkably, there was no correlation between the NAS and the DRS. The correlation coefficients for the GIP-S and the DRS (not shown in Table\u00a04) were .57 in the high cognition group (N\u00a0=\u00a034); .48 in the moderate cognition group (N\u00a0=\u00a039); and .42 in the low cognition group (N\u00a0=\u00a062). In the very low cognition group, in which almost no self-report scales could be administered, the correlation between these two observational scales was .28 (N\u00a0=\u00a070).\nTable\u00a04Relationships of self-report overall quality of life-scales with observational measures for overall quality of life, for the separate MMSE score-groupsPositiveNegativeGEN-QOLQ N\u00a0=\u00a0119PGCMS N\u00a0=\u00a0112PAS N\u00a0=\u00a0102NAS N\u00a0=\u00a098DL N\u00a0=\u00a0143GDS N\u00a0=\u00a0106HCaMCLCHCMCLCHCMCLCHCMCLCHCMCLCHCMCLCGIP-Srho\u2212.26\u2212.41*\u2212.28\u2212.24\u2212.40*\u2212.06.01.01\u2212.08.36.50**.02.37.42*.14.39.43**.01N\u00a0=\u00a0213N353142343239302833302730232364373525DRSrho\u2212.43*\u2212.25\u2212.09\u2212.36*\u2212.36*\u2212.06.03\u2212.24.11.23.30\u2212.12.12.51*.17.44**.52**.30N\u00a0=\u00a0220N323341323437272932272829222463343725aHC\u00a0=\u00a0High cognition group (MMSE score \u226522); MC\u00a0=\u00a0Moderate cognition group (MMSE score <22 \u226513); LC\u00a0=\u00a0Low cognition group (MMSE score <13 \u22655)*\u00a0P\u00a0<\u00a00.05, ** P\u00a0<\u00a00.01\nDiscussion\nThe aim of this paper was to investigate the usefulness of six self-report scales for measuring OQOL in nursing home residents. Hence, it was examined what percentage of residents could complete each scale, how high internal consistency and construct validity of the scales were, and whether these results were associated with level of cognitive impairment. It appeared that, of all the scales considered here, the DL could be administered to the most residents, even to almost half (43%) of the residents with very severe cognitive impairment. The other scales could only be administered to a small minority of this group. However, in the higher cognition groups, all scales (except for the PGCMS in the 5\u201312 MMSE group) could be administered to 80% to 90% of the residents. This percentage is higher than the 60% that was reported by Kane et\u00a0al. [8], but refers to shorter scales that only measure OQOL instead of various dimensions of quality of life.\nIn addition to residents being able to complete a scale, the resulting psychometric properties of the scales are of importance. It appeared that all scales, with the exception of the PAS and, to a lesser extent, the PGCMS, had an acceptable internal consistency. Although the alphas varied across the cognition groups, there was no linear trend of decreasing consistency with increasing cognitive impairment. With regard to validity, the PGCMS, DL and GDS (and the NAS to a lesser extent), were strongly interrelated in all cognition groups, although the correlations in the low cognition group overall were somewhat lower. These four scales also had the strongest relationships with the observational scales for OQOL. However, these relationships were not very strong. Moreover, in the low cognition group the scales were not related to the observational scales. This suggests that level of cognitive impairment has a substantial influence on the validity of self-report OQOL-scales.\nThe PAS performed worst on all aspects. The low internal consistency in the high cognition group could be explained by one item (\u2018determined\u2019). When it was omitted, Cronbach\u2019s alpha increased from .52 to .70. Furthermore, its disappointing characteristics may partly be explained by the fact that the PAS measures a somewhat different construct than the other scales. Its low correlation with the NAS, the other PANAS scale, is not unexpected. Positive and negative affect are considered to be largely independent. Considering the item content of the five scales, six affects (items) of the PAS were not included in the PGCMS, the DL or the GDS (i.e. interested, exited, strong, proud, inspired, and determined), whereas only three affects of the NAS were not included (i.e. guilty, hostile, and shameful). The PAS therefore must be considered to measure a different construct than the other scales, which also explains the absence of a correlation with the GIP-sad behavior and the Depression Rating Scale. Although conceptually it may be a good addition to the measurement of OQOL,- as psychological well-being is made up of positive affect, negative affect and life satisfaction -, its poor psychometric performance make it unsuitable in its present form. In contrast, the other PANAS scale, the NAS, had quite good properties, although the mean score on the NAS was low. The rating of affective states ideally involves a consideration of intensity, duration and frequency [21]. However, in cognitively impaired residents it is important to use a self-report scale that is as simple as possible, which may lead to loss of information, and therefore a loss of psychometric quality. For instance, the dichotomized response scale and the time-frame of \u2018today\u2019 that was used for the PAS and the NAS may have resulted in less discriminatory and lower scores, but increasing the response categories and the time-frame would threaten its reliability. We therefore suggest further research into the optimal time frame and response categories of the NAS.\nAlthough the DL and the GDS have been developed as screening instruments for depression, they correlated very strongly with the PGCMS, which is a scale for life satisfaction. Studying the item content of the scales, it appeared that the items of these three scales have many similarities. The items of the DL and the GDS contain positive as well as negative affects and also contentment (e.g. \u2018satisfied\u2019, \u2018happy\u2019 and \u2018hopeful\u2019). Remarkably, although the PGCMS is considered to be a positive scale, it has more items that contain negative affects or cognitions (12 out of 17; 71%) than the GDS (20 out of 30; 67%), and especially the DL (7 out of 15; 47%). Therefore, despite the fact that the names of the GDS and the DL suggest a negative scale, they both contain ample positive items. Likewise, the PGCMS cannot be considered to be a solely positive scale. So, with adapted scoring methods, each of these scales could be used as a single measure for OQOL, covering both positive and negative aspects.\nAs in most proxy studies [2, 46\u201349], in the present study low correlation was found between self-report and proxy assessment in low cognition groups (in this study: observational scales, rated by the nursing staff). This may suggest that the validity of self-report scales decreases with the level of cognition, but also that the validity of observational scales is lower in the low cognition group. Indeed, the correlation between the two observational scales in this study was lower in the low cognition group. Nevertheless, the relationships between the self-report and observational scales were also not strong in the high cognition group. This suggests that, although they are certainly related, self-report and observational scales may measure a different construct, irrespective of the cognitive functioning of the resident. The fact that both observational scales were negative may have complicated matters even further. Additional research into the relationship of self-report and observational scales for OQOL as well as proxy-report measures (following Edelman et\u00a0al. and Sloane et\u00a0al. [46, 48]) in relation to cognitive performance is therefore necessary. This research should use positive observational scales for OQOL, for instance the Philadelphia Geriatric Center Positive and Negative Affect Scales [26]. It should also apply a shorter maximum time-interval for collecting observational data than the four week-period used in this study, since this may have resulted in an underestimation of the association between the self-report scales on the one hand and the observational scales on the other.\nFor now, the decision as to whether or not a self-report scale can still be administered reliably and validly in a cognitively impaired resident could best be made for individual assessments when administering a quality of life-scale. The research on quality of life measurement in dementia has shown that one can use screening questions, incorporated in the scale, and thus tailored to the specific cognitive demands of the scale, in order to determine whether a resident is cognitively able to answer the questions [7, 50]. Nevertheless, even if a resident appears to be able to understand the questions, it is not certain that her answers are a true reflection of her inner state. Therefore, further research should focus on developing guidelines on when the administration of a scale can be considered as reliable and valid. This research could, for instance, study the possibility of examining test-retest reliability by repeating questions of a scale throughout the assessment. Such test-retest reliability in the assessment of cognitively impaired residents is an important indication that the resident has understood the questions and that she really communicates her true subjective state. In addition, repeating the assessment on another day and calculating a mean score for the two assessments can result in a more stable self-report OQOL score.\nIn conclusion, measuring overall quality of life reliably and validly through self-report may not be possible in nursing home residents with at least moderate cognitive impairment. The quality of observational assessment of OQOL may also be lower in cognitively impaired residents. Before drawing definite conclusions about the usefulness of self-report scales, it is necessary to study their reproducibility. Nevertheless, in clinical practice, using self-report scales will provide interesting information on the experience of the residents, and is therefore in itself a valuable addition to observational data. The Depression List is a useful scale in this respect, especially for the assessment of nursing home residents with mild to moderate cognitive impairment.","keyphrases":["overall quality of life","self-report outcome measures","long term care","age-related memory disorders"],"prmu":["P","R","M","U"]}
{"id":"Pediatr_Nephrol-4-1-2259257","title":"Cyclosporine-A-induced nephrotoxicity in children with minimal-change nephrotic syndrome: long-term treatment up to 10 years\n","text":"The impact of cyclosporine A (CsA) therapy in patients with steroid-dependent nephrotic-syndrome (SDNS) on long-term renal function is controversial. Data beyond 5 years are rare. Long-term renal function was evaluated in children with SDNS with and without CsA therapy, especially beyond 5 years. Twenty children were treated with CsA (study group) for a mean of 5.4 \u00b1 2.2 years (ten patients for 5\u201311 years). Glomerular filtration rate (GFR) was calculated before and after 3 and 12 months and at latest follow-up of therapy. Fifteen children with cyclophosphamide-treated SDNS without CsA served as controls. In the study group, GFR decreased within 12 months from 136 \u00b1 19 to 120 \u00b1 31, to 114 \u00b1 14 ml\/min per 1.73 m2 at latest follow-up (p < 0.0001). Patients with CsA > 5 years had a GFR of 111 \u00b1 14 ml\/min per 1.73 m2 at latest follow-up without a GFR below 90 ml\/min per 1.73 m2. No CsA toxicity was found in biopsies. In the control group, GFR dropped within 3 months, from 137 \u00b1 27 to 130 \u00b1 24, to 126 \u00b1 19 ml\/min per 1.73 m2 at latest follow-up (p = 0.1). Patients with and without nephrotoxic CsA therapy showed a drop in GFR. In CsA-treated patients, GFR was about 12% lower at latest follow-up compared with patients without nephrotoxic therapy but always remained within normal range. CsA seems to be safe, even in long-term treatment for more than 5 years.\nIntroduction\nMinimal-change nephrotic syndrome (MCNS) in children is characterised by steroid responsiveness, subsequent relapses and a benign prognosis concerning renal function [1]. Patients with steroid-dependent nephrotic syndrome (SDNS) and frequent relapsing nephrotic syndrome (FRNS) with steroid-toxic side effects are recommended for treatment with cyclophosphamide (CP). The overall rate of cumulative sustained remission after therapy with CP, however, was only 24% after 10\u00a0years [2]. The efficacy of cyclosporine A (CsA) in the treatment of steroid-sensitive nephrotic syndrome (SSNS) has been well demonstrated [3\u20138]. Early withdraw of CsA leads to relapses of the SDNS. So patients remain dependent on CsA for years. CsA was identified as being nephrotoxic, inducing tubulointerstitial fibrosis [9], vasoconstriction leading to reduced renal plasma flow and glomerular filtration rate (GFR) [10]. The potential risk of long-term CsA therapy to induce chronic renal failure in patients with MCNS has been discussed controversially. Seikaly et al. [11] found no drop in GFR but a histological progression of tubulointerstitial lesions in patients treated with CsA in comparison with those without CsA. Hulton et al. [5, 12] demonstrated a significant reduction of GFR within the first 3\u00a0months after introduction of CsA. Thereafter, the GFR remained stable. In contrast, Inoue et al. [6] could not detect a significant impairment of renal function in patients with CsA therapy. The interpretation of several studies focussing on the impact of CsA therapy on long-term renal function is limited and controversial.\nMost authors investigate the influence of CsA on renal function in nephrotic children with different histologic entities, not considering their different risk to progress to chronic renal failure. Thus Niaudet et al., Habib and Niaudet, and Gregory et al. [7, 13, 14] did not differentiate between MCNS and focal segmental glomerulosclerosis (FSGS) in their studies evaluating long-term renal function in nephrotic children with CsA therapy.The physiological drop in GFR after temporary hyperfiltration during a relapse often has been neglected. Relapse-associated hypoproteinemia and hypoalbuminaemia lead to oedema and increased GFR [15]. Hulton et al. [12] considered that concomitant therapy with steroids could contribute to an increase of GFR due to the mineralocorticoid effect. Consequently, steroid cessation could lead to a physiological drop in GFR.Several studies claim to address the long-term effect of CsA therapy on renal function, but actually, many of them do not extend more than 2\u20133\u00a0years [5, 6, 7, 12, 16], depicting the short-term effect of CsA on renal function.No study evaluated an additional control group without nephrotoxic therapy to demonstrate the physiological development of GFR in patients with SDNS.\nIn this study, we focus on the long-term renal function in 20 children with biopsy-proven MCNS treated with CsA for a mean of 5.36\u2009\u00b1\u20092.2\u00a0(range 2\u201311)\u00a0years. The development of GFR in these patients is compared with a control group with SDNS without CsA therapy.\nPatients and methods\nBetween 1993 and 2004, 20 children (eight girls and 12 boys) with biopsy-proven MCNS were treated with CsA for a mean of 5.4\u2009\u00b1\u20092.2\u00a0(median 5.0, range 2.1\u201311.0) years. Patients only qualified for this retrospective analysis after a minimum CsA therapy duration of 2\u00a0years. The definitions and criteria for NS, remission, relapse and steroid dependency were those used by the International Study of Kidney Disease in Children (ISKDC) and the Arbeitsgemeinschaft P\u00e4diatrische Nephrolgie (APN) [17, 18].\nAll patients had SDNS and were treated with cyclophosphamide (CP) prior to CsA without achieving sustained remission. CsA treatment was started with a dosage of 100\u2013150\u00a0mg\/m2 per body surface area (BSA) in two divided doses. CsA dose was adjusted to a target blood level of 80\u2013120\u00a0ng\/ml. Side effects such as hypertension, hypertrichosis and gingival hyperplasia were not consistently documented and could not be considered in this study. Renal function (GFR) was calculated by the Schwartz formula ([19], \u03ba-factor 0.55) before and after 3 and 12\u00a0months and at the latest follow-up of CsA therapy. Renal biopsy under CsA therapy was performed in five patients after 4.9\u20137.0\u00a0years. All patients starting CsA therapy were still on a standard relapse treatment with prednisone (40\u00a0mg\/m2 every 48 h) that was discontinued after 4 weeks. At the reviews after 3 and 12\u00a0months and latest follow-up, all patients were in remission and had no steroid therapy.\nIn the control group, 15 children (seven girls, eight boys) with SDNS treated with CP were evaluated for long-term renal function. GFR was calculated before and after therapy with CP and at the latest follow-up (mean 4.9\u2009\u00b1\u20093.4\u00a0years). According to APN recommendations, these patients were treated with CP with 2\u00a0mg\/kg body weight for 12\u00a0weeks [18, 20]. As concomitant medication, all patients in the control group received prednisone 60\u00a0mg\/m2 every 48\u00a0h tapered to 10\u00a0mg\/m2 every 48\u00a0h and discontinued when CP was stopped. At latest follow-up, all patients were in remission without further steroid therapy.\nStatistical analysis\nStatistical analysis was performed employing SPSS 12.0 for windows. Data were expressed as mean \u00b1 standard deviation. Student\u2019s t test was performed, and a p value <0.05 was regarded as significant.\nResults\nTwenty paediatric patients with SDNS were treated with CsA due to failing CP therapy for a mean of 5.4\u2009\u00b1\u20092.2\u00a0 (median 5.0, range 2.1\u201311.0) years. The mean starting dosage was 130\u2009\u00b1\u200932\u00a0mg\/m2 BSA per day. The mean CsA dosage after 12\u00a0months was 126\u2009\u00b1\u200926\u00a0mg\/m2 BSA per day, with a trough level of 99\u2009\u00b1\u200927\u00a0ng\/ml. The mean age at the beginning of CsA therapy was 8.4\u2009\u00b1\u20093.0\u00a0 (median 8.6, range 3.1\u201314.2) years. GFR calculated at the beginning, at months 3 and 12 and at the latest follow-up of CsA treatment was 136\u2009\u00b1\u200919 (median 136), 130\u2009\u00b1\u200920 (median 135), 120\u2009\u00b1\u200931 (median 113) and 114\u2009\u00b1\u200914\u00a0(median 114) ml\/min per 1.73\u00a0m2, respectively (Table\u00a01 and Fig.\u00a01). Unfortunately, data of only 12 patients were available for the calculation of GFR at month 3. The decline in GFR from the beginning to month 12 and to the latest follow-up was significant, with p\u2009=\u20090.025 and p\u2009<\u20090.0001, respectively (Fig.\u00a02). The drop in GFR within the first 12\u00a0months was 11.8% and during the whole follow-up 14.9%. Three patients reestablished their GFR after cessation of CsA. No patient had a GFR below 90\u00a0ml\/min per 1.73\u00a0m2, and 17 patients had a GFR above 105\u00a0ml\/min per 1.73\u00a0m2 at latest follow-up. CsA trough level at latest follow-up was 85\u2009\u00b1\u200962\u00a0ng\/ml;  mean CsA dosage was 105\u2009\u00b1\u200934\u00a0mg\/m2 BSA per day.\nTable\u00a01Patient characteristics [mean \u00b1 standard deviation (SD)] and renal function calculated by glomerular filtration rate (GFR in ml\/min per 1.73\u00a0m2; mean \u00b1 SD) in patients with cyclosporine A (CsA, study group) and with cyclophosphamide (CP, control group)\u00a0Study group n\u2009=\u200920Control group n\u2009=\u200915Age at start of MCNS (years)4.4\u2009\u00b1\u20092.2 4.0\u2009\u00b1\u20092.9Age at start of CP (years)5.7\u2009\u00b1\u20092.26.0\u2009\u00b1\u20093.2Age at start of CsA (years)8.4\u2009\u00b1\u20093.0\u2013CsA trough level at  month 12 (ng\/ml)98.8\u2009\u00b1\u200926.9\u2013Follow-up time (years)5.4\u2009\u00b1\u20092.24.9\u2009\u00b1\u20093.4GFR at start of CP138.7\u2009\u00b1\u200924.6p\u2009=\u20090.4137.7\u2009\u00b1\u200927.6p\u2009=\u20090.3Median 132Median 131.6Range 119\u2013181Range 95\u2013196GFR at stop of CP130\u2009\u00b1\u200930.9130.3\u2009\u00b1\u200924.7Median 135.9Median 131.1Range 82\u2013164Range 93\u2013178GFR at start of CsA136.3\u2009\u00b1\u200919.0p<0.0001Median 136.5Range 97\u2013186GFR at latest follow-up114.5\u2009\u00b1\u200914.5126.4\u2009\u00b1\u200919.8p\u2009=\u20090.1Median 114.2Median 124.6Range 89\u2013135Range 92\u2013156MCNS minimal-change nephrotic syndrome, GFR glomerular filtration rateFig.\u00a01Development of the glomerular filtration rate (GFR) of all patients on cyclosporin A (CsA) therapy (n\u2009=\u200920)Fig.\u00a02Boxplot demonstrating the development of glomerular filtration rate (GFR) in patients on cyclosporin A (CsA) therapy (n\u2009=\u200920)\nCsA was effective in 19 of 20 patients, leading to long-term remission or reduction of relapses to an infrequent relapsing NS (IRNS). Chlorambucil was introduced in one patient due to frequent relapses after CsA cessation. In three patients, arterial hypertension was treated; in one patient, antihypertensive therapy could be stopped after CsA cessation. Ten patients were treated with CsA for more than 5\u00a0(median 6.5, mean 7.0\u2009\u00b1\u20091.8,\u00a0 range 5.0\u201311.0)\u00a0years. GFR at latest follow-up was 111\u2009\u00b1\u200914\u00a0  (median 109, range 90\u2013132) ml\/min per 1.73\u00a0m2. Five patients underwent renal biopsy after 5.1\u20137.3\u00a0years after CsA therapy had been started before they were transferred to adult nephrologists. CsA-associated nephrotoxicity was not reported by an experienced renal pathologist (Prof. U. Helmchen, Hamburg, Germany). CsA was stopped in nine patients (mean age 13.1\u2009\u00b1\u20092.5\u00a0years) after a mean of 4.9\u2009\u00b1\u20091.9\u00a0(median 4.4, range 2.3\u20139.0) years due to long-term sustained remission in all but one patient. GFR remained normal after CsA cessation, with 120\u2009\u00b1\u200925 (median 116) and 123\u2009\u00b1\u200910\u00a0(median 125, range 105.18\u2013134.98) ml\/min per 1.73\u00a0m2 at time of therapy cessation and at latest follow-up, respectively.\nThe control group consisted of 15 patients with SDNS who were treated with CP at a mean age of 6.0\u2009\u00b1\u20093.2\u00a0(median 4.6) years. At the start of CP, GFR was 138\u2009\u00b1\u200928\u00a0(median 132) ml\/min per 1.73\u00a0m2. After 3\u00a0months, GFR dropped to 130\u2009\u00b1\u200925\u00a0ml (median 131) ml\/min per 1.73 m2 (p\u2009=\u20090.3).   GFR at latest follow-up (mean follow-up 4.9\u2009\u00b1\u20093.4\u00a0years) was 126\u2009\u00b1\u200920\u00a0(median 125) ml\/min per 1.73\u00a0m2  (p\u2009=\u20090.1, Figs.\u00a03 and 4). This drop was in the order of 8% but not statistically significant.\nFig.\u00a03Development of glomerular filtration rate (GFR) of patients without cyclosporin A (CsA) therapy (n\u2009=\u200915)Fig.\u00a04Boxplot demonstrating the development of glomerular filtration rate (GFR) in patients without cyclosporin A (CsA) therapy (n\u2009=\u200915)\nTable\u00a01 summarises the development of GFR in both patient groups, starting with the GFR before CP therapy. Patients of both groups had an initial decline in GFR during CP therapy.\nDiscussion\nHistologically, the nephrotoxic effect of CsA has been defined by Mihatch et al. [9]: most frequent lesions attributable to CsA are tubular atrophy, interstitial fibrosis and arteriolar hyalinosis. However, a review of the literature demonstrates that it is difficult to predict the influence of CsA on long-term renal function in patients with MCNS. Some authors did not find a significant reduction in renal function under CsA therapy in nephrotic children [4, 8] and only minor changes in renal histology without progression of CsA tubulointerstitial lesions [14, 21]. Others emphasised reduced GFR [5, 12], reduced renal plasma flow [22] or progression of tubulointerstitial lesions histologically [11, 23] in patients with NS and CsA therapy. Histology progression could not be associated with decrease in renal function [11, 23] or duration of CsA therapy [7]. Myers et al. [24] postulated that in case of striped interstitial fibrosis, some nephrons undergo atrophy while their function could be compensated for by unaffected nephrons. This may mask nephrotoxicity by measurements of normal GFR. In summary, a clear proven association between impaired renal function and long-term CsA therapy in patients with MCNS could not be found. But hints permit the question regarding the safety of long-term CsA therapy in children with a benign renal disease.\nComparison of results of this study with established opinions (e.g. [8, 12, 23]) is difficult:\nMost studies mix different aetiological entities with different potential to progress to chronic renal failure (MCNS vs. SRNS and FSGS; [4, 7, 13, 16, 21, 22]). The impairment of renal function in patients with FSGS and other types of SRNS cannot reliably be distinguished from CsA nephrotoxicity, as those entities have a high risk for chronic renal failure on their own.In most studies, the follow-up period is restricted to 2\u20133\u00a0years; in single studies up to 5\u00a0years. In this study, at least ten patients underwent long-term CsA therapy > 5\u00a0years. No other study investigated patients with such a long treatment period [5\u20138, 11\u201314, 23].\nNeuhaus et al. and Ganesan et al.  [8, 23] evaluated patients with MCNS only and an extended follow-up time of 5\u00a0years. They evaluated nine patients, with one being identified with FSGS as the underlying disease in the follow-up biopsy. All patients received CsA with higher trough levels and higher CsA dosage (level: mean of 220\u00a0ng\/ml, range 141\u2013270\u00a0ng\/ml, CsA dosage 126\u2009\u00b1\u200929\u00a0mg\/m2 per day) than accepted for our study group. The method for CsA level measurements was a polyclonal assay resulting in higher levels compared with monoclonal assays. Nevertheless, the CsA levels and dosage were still higher than in our study group. CsA toxicity was proven in 3\/9 patients histologically. Ganesan et al. [23] found histological signs for CsA toxicity in 79% and renal insufficiency with a GFR < 80 in 4\/19 patients without correlation to histological changes. Again, that study mixed patients with steroid-resistant and steroid-responsive NS, not distinguishing between the different clinical entities.\nIn our study, only patients with histologically proven MCNS were evaluated for long-term renal function under CsA therapy. Patients qualified for the study only after a minimum treatment duration of 2\u00a0years. The follow-up period extended over a mean of 5.4\u00a0years, with ten patients who had been treated between 5 and 11\u00a0years. At least in half of the patients with long-term CsA therapy > 5\u00a0years, CsA toxicity was excluded by biopsy. Also in our study group, the GFR of patients with CsA therapy remained within the normal range, with a drop within the first 12\u00a0months (mean drop 11.8%) and remaining stable thereafter, with a mean GFR of 115\u2009\u00b1\u200916\u00a0ml\/min per 1.73\u00a0m2. No patient progressed to chronic renal failure, even after therapy duration of more than 10\u00a0years. Hulton et al. [5, 12] showed comparable results for patients with CsA therapy, with an initial drop in GFR and a constant renal function afterwards. But the study is restricted to a short follow-up of 2\u00a0years in four patients only. In our study, a significant reincrease in GFR after therapy cessation was not found. In general, these patients showed excellent renal function in the long-term follow-up (mean 123\u00a0ml\/min per 1.73\u00a0m2). In contrast, patients in Hulton et al.\u2019s studies showed a worse GFR of 93 that reincreased back to a GFR > 100\u00a0ml\/min per 1.73\u00a0m2 after CsA had been stopped.\nInterestingly, in our control group of patients without CsA therapy, an initial drop in GFR was also found. Those patients showed a mean drop in GFR of about 5% within the 12\u00a0weeks of CP therapy, and GFR even dropped about 8% until the latest follow-up. Probably, the observed decrease in GFR in patients with CsA therapy cannot only be attributed to the hitherto described nephrotoxic effect of CsA.\nHulton et al. [12] mentioned the potential increase of GFR caused by the mineralocorticoid effect of concomitant steroid medication. One could speculate that the cessation of steroids might lead to a physiological drop in GFR. Additionally, a possible but not proven interpretation should be discussed: Part of the GFR reduction may be attributable to normalisation of hyperfiltration in nephrotic children during the first months after initiation of immunosuppressive therapy. In patients with relapsing nephrotic syndrome, long-term immunosuppressive drugs (e.g. cyclophosphamide, CsA, chlorambucil) are initiated after urine remission has been achieved; complete remission of serum albumin normally is not awaited.\nRelapse-associated hypoproteinemia and hypoalbuminemia lead to oedema and an increase in GFR. This is explained by the decrease of the colloid osmotic pressure in the glomerular capillary. Reduced total serum protein leads to decreased colloid osmotic pressure and consequently to increased GFR [15]. Normalisation of the increased filtration extends over weeks and leads to a decrease in GFR physiologically. This phenomenon may explain the drop in GFR, even in patients without nephrotoxic treatment.\nIn summary, this study offers long-term follow-up of 20 patients with proven SSNS treated with CsA for a median of 5\u00a0years. Initially, the patients showed a drop in GFR but remained stable afterwards. None developed chronic renal failure. Nevertheless, the generalisation of these results are limited, as the study was retrospective, and only 5\/20 patients underwent renal biopsy under long-term nephrotoxic treatment. It is questionable whether a prospective study to evaluate the outcome of long-term CsA therapy in children with MCNS is reasonable in the future. Newer, nonnephrotoxic drugs, e.g. mycophenolate mofetil (MMF) are of concern and need to be evaluated for efficacy and safety in the treatment of SDNS and FRNS. However, our analysis demonstrates that even long-term therapy with CsA for more than 5\u00a0years in children with MCNS is safe and does not impair renal function.","keyphrases":["nephrotoxicity","minimal-change nephrotic syndrome","cyclosporine a","glomerular filtration rate","long-term outcome"],"prmu":["P","P","P","P","R"]}
{"id":"Purinergic_Signal-3-4-2072927","title":"The role of ATP and adenosine in the brain under normoxic and ischemic conditions\n","text":"By taking advantage of some recently synthesized compounds that are able to block ecto-ATPase activity, we demonstrated that adenosine triphosphate (ATP) in the hippocampus exerts an inhibitory action independent of its degradation to adenosine. In addition, tonic activation of P2 receptors contributes to the normally recorded excitatory neurotransmission. The role of P2 receptors becomes critical during ischemia when extracellular ATP concentrations increase. Under such conditions, P2 antagonism is protective. Although ATP exerts a detrimental role under ischemia, it also exerts a trophic role in terms of cell division and differentiation. We recently reported that ATP is spontaneously released from human mesenchymal stem cells (hMSCs) in culture. Moreover, it decreases hMSC proliferation rate at early stages of culture. Increased hMSC differentiation could account for an ATP-induced decrease in cell proliferation. ATP as a homeostatic regulator might exert a different effect on cell trophism according to the rate of its efflux and receptor expression during the cell life cycle. During ischemia, adenosine formed by intracellular ATP escapes from cells through the equilibrative transporter. The protective role of adenosine A1 receptors during ischemia is well accepted. However, the use of selective A1 agonists is hampered by unwanted peripheral effects, thus attention has been focused on A2A and A3 receptors. The protective effects of A2A antagonists in brain ischemia may be largely due to reduced glutamate outflow from neurones and glial cells. Reduced activation of p38 mitogen-activated protein kinases that are involved in neuronal death through transcriptional mechanisms may also contribute to protection by A2A antagonism. Evidence that A3 receptor antagonism may be protective after ischemia is also reported.\nHistorical overview\nThe physiological roles of adenosine triphosphate (ATP) and its metabolite adenosine have been historically linked to cell metabolism since ATP is an ubiquitous intracellular energy source in a number of enzymatic processes. However, over the years both metabolites have emerged as very versatile molecules of biological systems, being implicated in a variety of cell processes, from platelet aggregation to neurotransmission. The term \u2018purinergic signalling\u2019 was first introduced in the scientific literature by Burnstock [1]. An implicit concept for sustaining the hypothesis of purinergic neurotransmission proposed in the 1970s was the existence of purinergic receptors. The first evidence in this direction suggested the existence of two different subfamilies of such \u2018purinoceptors\u2019, identified as P1 and P2 receptors, selective for adenosine and ATP respectively [2]. Four different subtypes of P1-G-protein-coupled receptors: A1, A2A, A2B and A3 are known at present [3], whereas P2 purinoceptors belong to two major families: P2X ligand-gated ion channel receptors and P2Y G-protein-coupled receptors [4]. Cloning experiments supported this classification and helped to subdivide P2 receptors into seven P2X and eight P2Y subtypes [5] (plus the recently deorphanized GPR17 receptor [6]).\nRoles of ATP in neurotransmission under normoxic conditions\nThe first studies on the role of extracellular ATP in hippocampal neurotransmission indicated prominent inhibitory action of this purinergic nucleotide on synaptic activity [7, 8]. However, several lines of evidence led to the hypothesis that ATP-mediated inhibition of hippocampal neurotransmission was probably mediated by adenosine acting on A1 receptors. In fact, this response was theophylline-sensitive [7\u20139] and absent in A1 knockout mice [10]. This assumption was supported by the fact that, in the hippocampus, extracellular ATP is rapidly converted into adenosine by ecto-ATPases and ecto-nucleotidases [11] and that adenosine acting on A1 receptors exerts a well-described inhibitory role on CA1 excitatory neurotransmission [3]. In recent years, a more detailed observation of ATP-evoked effects, supported by the synthesis of new pharmacological tools, has helped to clarify the effective role of purinergic nucleotides in the hippocampus. In particular, it emerged that ATP mediates inhibitory effects by P2 receptor activation [12\u201314].\nIn a recent paper, we contributed to elucidating the role of ATP on CA1 hippocampal neurotransmission, and we reinforced the concept that ATP-mediated effects are not necessarily linked to adenosine formation in this brain region [15]. ATP is hydrolysed by ecto-NTPDase, enzymes located on the cell surface in the CNS that limit ATP, ADP and AMP spatio-temporal activity [11, 16]. There are three different known NTPDases: NTPDase1 hydrolyzes ATP and ADP equally well, NTPDase2 has a high preference for ATP, NTPDase3 is a functional intermediate, preferably hydrolyzing ATP [17]. In our study, we took advantage of some recently synthesized compounds: ARL 67156, which, at micromolar concentrations, inhibits rat NTPDase1 and 3 transiently transfected in Chinese hamster ovary cells, showing negligible activity on NTPDase2 [18]; BGO 136, a new inhibitor described as a selective NTPDase1 and 2 blocker with Ki values in the high micromolar range [19]; and the recently synthesized PV4, which strongly inhibits rat NTPDase1, 2 and 3 with Ki values in the nanomolar range [20]. By using these inhibitors that are able to block NTPDase activity without interfering with P2 receptor activation, we demonstrated that ATP exerts an inhibitory action, independent of its degradation to adenosine. As shown in Fig.\u00a01, the application of ATP during a NTPDase activity blockade still elicits a decrease in evoked synaptic responses which is even more pronounced than that evoked by ATP alone.\nFig.\u00a01a, bThe inhibitory effect induced by ATP on fEPSP amplitude is potentiated in the presence of different NTPDase inhibitors. a Time-course of fEPSP amplitude before, during and after the application of ATP in the absence or in the presence of the NTPDase1,2,3 inhibitor PV4. Each point in the graph represents the mean \u00b1 SE of fEPSP value measured as percent of baseline, pre-drug level. b Columns in the graph summarize the average amplitude (mean \u00b1 SE) of evoked fEPSP recorded from CA1 hippocampal region in control conditions, 5 min after superfusion of ATP alone and 5 min after ATP superfusion in the presence of different ecto-ATPases inhibitors. Note that the inhibitory effect of ATP on fEPSP amplitude is potentiated by BGO 136, PV4 and ARL 67156. *P\u2009<\u20090.05 one-way ANOVA, Newman-Keuls multiple comparison post-hoc test versus pre-drug value. \u00a7P\u2009<\u20090.05, one-way ANOVA, Newman-Keuls multiple comparison post-hoc test versus 10\u00a0\u03bcM ATP treated slices. (Modified from [15])\nAn excitatory effect of exogenous ATP on hippocampal neurotransmission has also been reported by different authors. This effect, which was observed after drug removal, was described to persist in in vitro preparations for a relatively prolonged period, up to 1\u00a0h [21\u201325]. This \u2018long-lasting\u2019 potentiation of synaptic responses was compared to electrically evoked LTP, firstly described in the hippocampus by Abrams and Kandel [26] and called \u2018ATP-induced LTP\u2019. In agreement, we observed a potentiation of the synaptic responses after drug removal when slices were superfused in the presence of the metabolically stable ATP-analogue ATP\u03b3S (Fig.\u00a02) [15]. In addition to this \u2018long-lasting\u2019 excitatory effect evoked by the exogenous application of P2 agonists, we also demonstrated an excitatory tone exerted by endogenous ATP. Slices superfused with P2 antagonists show a small but significant reduction in synaptic transmission (Fig.\u00a03). These data demonstrate that tonic activation of P2 receptors contributes to glutamatergic excitatory neurotransmission in the hippocampus, an observation that is in line with previous work [27].\nFig.\u00a02Inhibitory and excitatory effects of the stable ATP analogue ATP\u03b3S. Averaged time-course (n\u2009=\u20094) of PS amplitude before, during and after the application of different concentrations of ATP\u03b3S. PS amplitude (mean \u00b1 SE) is measured as percent of baseline level. Upper panels represent single traces recorded in a typical experiment before, during and after ATP\u03b3S application at different concentrationsFig.\u00a03Excitatory effects of endogenous ATP. Bars in the graphs represent the average of fEPSP amplitude in the presence of P2 antagonists: PPADS (30 \u03bcM) and MRS 2179 (10\u00a0\u03bcM). *P\u2009<\u20090.05, paired Student\u2019s t-test. (Modified from [15])\nRoles of ATP in ischemic conditions\nThe role of ATP may become critical during pathological conditions such as ischemia, when extracellular ATP concentrations increase. An enhanced outflow of radioactive ATP from hippocampal slices during in vitro ischemic-like insults was first reported by Juranyi and co-workers [28], and the first demonstration that ATP outflow increases in vivo during the induction of focal ischemia in the rat was reported by Melani and colleagues [29]. Thus, at the ischemic site, levels of extracellular nucleotides may remain elevated for long periods of time after injury. Evidence supports the idea that, under such pathological conditions, released ATP may exert an excitotoxic role by acting on its receptors, thus enhancing Ca2+ inward currents and altering synaptic activity and cellular plasticity. Non-selective antagonists of P2 receptors, suramin and PPADS, and the selective antagonists, BBG and MRS 2179 of P2X7 and P2Y1 receptors respectively, prevent the irreversible failure of neurotransmission induced by a prolonged period of OGD in hippocampal slices [15]. Moreover, they protect from development of anoxic depolarization (AD), which is a rapid and regenerative wave of depolarization that propagates in tissue and represents an unequivocal sign of sufferance [15]. Antagonists of P2 receptors have been proved protective against cell death induced by either ATP itself, hypoglycemia or glutamate exposure in primary cultures of brain-derived neurones [30\u201333]. Intrastriatal ATP injection in rats induced, 24\u00a0h later, a clearly lesioned area [34]. The ATP-induced damage was concentration-dependent, mimicked by ATP\u03b3S and \u03b1,\u03b2 meATP (but not by ADP or adenosine) and blocked by RB2, a non-specific P2 antagonist [34]. Suramin, another non-specific P2 receptor antagonist, administered 30\u00a0min before occlusion of the middle cerebral artery, resulted in a significant decrease in infarct and oedema volume 6\u00a0h after brain injury [35]. In agreement, it was demonstrated that RB2 [36] and PPADS [37] improve neurological deficit and reduce the damage induced in rats in a model of focal ischemia in vivo. Moreover, RB2 induces the expression of P2X7 receptors on reactive microglia in the remote ipsi and contralateral cingulate and medial frontal cortex and striatum. Although a pro-apoptotic role has been attributed to the P2X7 receptor, results suggest that microglial cells expressing the P2X7 receptor can be implicated in tissue damage as well as in the defence and repairing processes in the remote ipsi and contralateral undamaged areas [36].\nIn considering the effect of ATP during ischemia, it must be taken into account that ATP itself is involved in control of cerebrovascular regulation [38] and that the metabolite of ATP, ADP, that interacts with P2Y1\/P2Y12 receptors, is a potent platelet aggregator. However, clinical studies that checked the antiplatelet therapy of the selective P2Y12 receptor antagonist clopidogrel reported a response variability [39, 40].\nAlthough evidence suggests that ATP exerts a detrimental role under ischemia, there are reports that it may exert a trophic role in terms of cell division and differentiation in both differentiated and undifferentiated cells [41\u201343], including adult neural stem cells [44]. Interesting data correlate these trophic effects with the occurrence of spontaneous Ca2+ waves that propagate among adjacent cells in a self-renewing manner. Kawano et al. [45] reported that ATP-induced initiation and propagation of intracellular Ca2+ waves in human mesenchymal stem cells (hMSCs) promote activation of transcription factors (e.g. NFAT) that are involved in cell differentiation. In the same study, they demonstrated that ATP-induced Ca2+ waves disappear in the fully differentiated adipogenic phenotype. We recently reported that ATP is spontaneously released from hMSCs during the early stages of culture (P0-P5). Moreover, we reported that ATP decreases proliferation rate (Fig.\u00a04) and modulates specific ionic current hMSCs [46]. A high extracellular ATP concentration at earlier cell culture passages suggests an important role of ATP in regulating cell differentiation. Increased hMSC differentiation may account for an ATP-induced decrease in cell proliferation.\nFig.\u00a04a, bHuman mesenchymal stem cells in culture spontaneously release ATP that modulates cell proliferation. a Extracellular concentrations of ATP were measured in the medium containing hMSCs and in control medium not containing cells. Data are expressed as mean \u00b1 SE, n\u2009=\u200911, unpaired Student\u2019s t-test: *P\u2009<\u20090.0001 vs medium alone. b Effect on hMSC proliferation after daily application of ATP (10\u00a0\u03bcM) and P2 antagonists, PPADS (30\u00a0\u03bcM) and MRS 2179 (10\u00a0\u03bcM). Data are expressed as percentage of proliferation. Proliferation of untreated cells was assumed as 100%. The cell number was determined after 5\u00a0days of culture by a culture counter. Each column bar represents the mean \u00b1 SE of n\u2009=\u20094 for each experimental condition. Paired Student\u2019s t-test: *P\u2009<\u20090.05 vs respective control; one-way ANOVA, Newman-Keuls post-test: \u00a7P\u2009<\u20090.05 vs 10\u00a0\u03bcM ATP-treated cells. (Modified from [46])\nTherefore, it can be envisaged that ATP, as an autocrine\/paracrine homeostatic regulator, exerts different effects on cell trophism according to its extracellular concentrations, distinct cell populations involved, differential expression and recruitment of P2 receptors and of more or less sustained stimulation of the same receptor. Balancing these effects may be relevant in the post-ischemic brain, when a neuroregenerative process could promote tissue repair [47]. On this basis, determination of ATP extracellular concentrations at various times after ischemia induction may help to identify which types of receptors may be stimulated under ischemia and explain the role of detrimental versus trophic ATP during ischemia. ATP present in the extracellular space is rapidly metabolised by membrane-bound NTPDase. Extracellular ATP concentrations, evaluated in the brain to date, have been underestimated, since it has not been possible to selectively inhibit ecto-NTPDase. In fact, the selective inhibitor ARL 67156, tentatively used thus far to inhibit ecto-NTPDase, interferes with the ATP assay method [29].\nRole of adenosine in cerebral transmission under normoxic conditions\nAdenosine exerts an important tonic modulation of synaptic transmission in the brain. This tonic inhibition of synaptic transmission is evoked by stimulation of A1 receptors, as demonstrated in several brain regions, such as the hippocampus, striatum and olfactory cortex [48, 49]. The inhibitory effect of adenosine A1 receptor stimulation has a pre- and postsynaptic component. Activation of the presynaptic A1 receptors reduces Ca2+ influx through the preferential inhibition of N-type and, probably, Q-type channels [50, 51]. Inhibition of presynaptic calcium currents decreases transmitter release [52], and adenosine, by stimulation of A1 receptors, has been found to inhibit the release of virtually all classical neurotransmitters: glutamate, acetylcholine, dopamine, noradrenaline and serotonin (see in [53]). In particular, powerful suppression of glutamate release from presynaptic terminals has been described in the hippocampus [54, 55], where adenosine A1 receptor activation reduces the number of quanta released (but not the size of individual quanta nor postsynaptic glutamate receptor sensitivity) in the Schaffer collateral-commissural pathway [56]. The postsynaptic effect of A1 receptors consists of direct hyperpolarisation of neurones via activation of GIRK channels (Kir 3.2 and 3.4 channels: potassium inward rectifiers) [57, 58]. Endogenous adenosine exerts tonic inhibition of excitatory neurotransmission. The selective A1 antagonist, DPCPX, causes a 15% increase in synaptic potential amplitude in in vitro brain slices [59]. This is an expected result in a brain region where the adenosine concentration at a receptor level was calculated around 200\u00a0nM [59, 60] and A1 receptors, whose affinity for adenosine is in the low nanomolar range, are highly expressed. These data are confirmed by the fact that, in slices taken from homozygous A1 receptor knockout mice, no evidence was found for an endogenous inhibitory action by adenosine in the Schaffer collateral pathway in the CA1 region of the hippocampus or at the mossy fibre synapses in the CA3 region [61].\nOpposite effects from A1-mediated synaptic inhibition are elicited by A2A receptor activation, which has been shown to mediate excitatory actions in synaptic function [62\u201364]. In the hippocampus in vitro, A2A receptor stimulation results in a Ca2+-dependent release of acetylcholine [65, 66]. Furthermore, the application of CGS 21680, a selective A2A receptor agonist, decreases the ability of A1 receptor agonists to inhibit excitatory neurotransmission [9, 67]. This effect suggests that A2A receptor stimulation increases synaptic transmission through A1 receptor desensitisation [68, 69]. However, there is also evidence that A2A receptors increase excitatory amino acid release. In fact, the selective stimulation of adenosine A2A receptors augments the amount of glutamate released in the hippocampus and striatum of young rats [70\u201372].\nIn spite of the excitatory role in neurotransmission brought about by A2A receptors, the net effect of adenosine is an inhibitory tonus on neurotransmission, in accordance with observations suggesting that activation of A2A receptors requires protracted stimulation to induce evident effects on synaptic transmission [59]. It is worth noticing that the role of A2A receptors in the striatum is recently gaining interest in light of their heterodimerisation with D2 dopamine receptors. The association between A2A and D2 receptors results in an antagonistic interaction that provides a rationale for evaluating A2A-selective antagonists in Parkinson\u2019s disease, supported by epidemiological evidence indicating an inverse relationship between caffeine consumption and the risk of developing this pathology [73, 74]. It was suggested that A2A antagonists not only provide symptomatic relief but also decelerate dopaminergic neurone degeneration in patients.\nDiscrepancies about the role of adenosine A3 receptors in the brain are present in the literature. Activation of this receptor subtype has been associated with both excitatory and inhibitory effects, even in the same brain region. An excitatory role of A3 receptors has been supported by evidence indicating that, in the rat hippocampus, their activation attenuates LTD and allows induction of LTP elicited by a subliminal weak-burst protocol [75]. In addition, in the same brain area, A3 receptor activation through a selective adenosine A3 agonist has been shown to antagonize the adenosine A1 receptor-mediated inhibition of excitatory neurotransmission [8]. Moreover, A3 receptor stimulation always attenuates the inhibition of hippocampal slice neurotransmission, in a PKC-dependent manner, caused by presynaptic metabotropic glutamate receptors [76]. Whole-cell patch clamp recordings in CA3 hippocampal pyramidal neurones demonstrate that A3 receptor activation results in a significant potentiation of high threshold hippocampal Ca2+ currents by a PKA-dependent mechanism [77]. Finally, facilitation of the onset of epileptiform discharge has been observed in the presence of the selective A3 receptor agonist Cl-IB-MECA [78], and a reduction in such epileptic activity was observed when A3 receptors, activated by endogenously released adenosine during seizures, were blocked by the selective antagonist MRS 1191 [79].\nContrary to previous results, an inhibitory action has been attributed to A3 receptors by Brand and colleagues [80], who demonstrated that, in rat cortical neurones, the selective activation of this adenosine receptor subtype is involved in inhibition of excitatory neurotransmission, suggesting a synergistic action with the inhibitory effect of adenosine brought about by A1 receptor activation. Despite results obtained by A3 receptor stimulation, evidence that a selective block of A3 receptors does not affect neurotransmission in the CA1 region of the hippocampus under normoxic conditions indicates that endogenous adenosine at physiological concentrations does not exert tonic activation of A3 receptors [8, 81]. This is in line with evidence that A3 receptor activation requires micromolar levels of extracellular adenosine, which can be reached only during pathological conditions of impairment in energy supply (for example during hypoxia or ischemia [82\u201385]).\nRole of adenosine in ischemic conditions\nExtracellular adenosine concentrations increase dramatically during ischemia [82, 86, 87]. During ischemia, following the imbalance between ATP degradation and resynthesis, the intracellular concentration of adenosine increases. Therefore, increased adenosine concentrations in the extracellular space during ischemia are likely due to the equilibrative transporter that carries adenosine out of cells. Adenosine formation can also take place at the extracellular level, through the hydrolysis of extracellular ATP operated by NTPDases and ecto-5\u2032-nucleotidase (e5\u2032-NTs) (see Fig.\u00a05). Hence, these enzymes have a dual function in modulating purinergic neurotransmission: (1) they rapidly interrupt ATP-mediated signalling by degrading extracellular nucleotides and (2) they give rise to extracellular adenosine, which activates P1 receptors. However, recent evidence in vitro suggests that released ATP does not substantially contribute to the adenosine concentration in the extracellular milieu during ischemia [88].\nFig.\u00a05Schematic drawing of intracellular and extracellular adenosine formation. In the extracellular space, adenosine and ATP act on own purinergic receptor subtypes: P1 and P2 receptors, respectively. ADP adenosine diphosphate, AMP adenosine monophosphate, ATP adenosine triphosphate, e5\u2032-NT ecto-5\u2032-nucleotidase, 5\u2032-NT 5\u2032-nucleotidase, NTDPase ecto-nucleoside triphosphate diphosphohydrolases, P1 adenosine receptor, P2 ATP receptor, SAH S-adenosylhomocysteine, T bidirectional nucleoside transporter. (Modified from [142])\nAdenosine-potentiating agents, which elevate endogenous adenosine levels by either inhibiting its metabolism by adenosine deaminase or kinase [89, 90] or preventing its transport [91, 92], offer protection against ischemic neuronal damage in different in vivo ischemia models. Moreover, adenosine infusion into the ischemic striatum during transient focal ischemia proves to significantly ameliorate the neurological outcome and reduce infarct volume [93].\nA temporal correlation exists between adenosine outflow and synaptic potential inhibition in rat hippocampal slices during ischemia-like conditions [84, 88]. Synaptic inhibition during ischemia is greatly dependent on adenosine, which, by stimulating A1 receptors, exerts a protective role by reducing the Ca2+ influx, thus counteracting the presynaptic release of excitatory neurotransmitters [55, 94] and, in particular, glutamate, which exerts an excitotoxic role during ischemia mainly by overstimulation of NMDA receptors [95]. By directly increasing the K+ and Cl\u2212 ion conductances, adenosine stabilises the neuronal membrane potentials, thus reducing neuronal excitability [96]. Consequent reductions in cellular metabolism and energy consumption [97] and moderate lowering of the body\/brain temperature [98] are protective in ischemia.\nA1 receptor agonists are shown to attenuate ischemic or excitotoxic neuronal damage in both in vitro and in vivo models of cerebral ischemia (for review, see [99, 100]). In accordance, adenosine A1 antagonists given acutely exacerbate the damage induced by ischemia in different animal models of ischemia. An unselective A1 receptor antagonist, theophylline, increased mortality [101, 102]. Unlike acute treatment, chronic administration of A1 agonists worsened survival and increased neuronal loss [103], a phenomenon thought to depend on A1 receptor desensitization.\nAlthough data converge in demonstrating a neuroprotective effect of adenosine through A1 receptors during ischemia, the use of selective A1 agonists is hampered by unwanted peripheral effects, e.g. sedation, bradycardia, hypotension [104]. Von Lubitz and co-workers [105] have reported that post-ischemic administration of the A1 receptor agonist adenosine amine congener (ADAC), which induces fewer undesirable effects, increases survival in gerbils. Moreover, we may consider that administration of agents that elevate the local concentration of adenosine at the injury site, by inhibiting its metabolism to inosine or rephosphorylation to AMP or reuptake, may have the advantage of restricting the effect of such inhibitors to areas of injury-induced adenosine release [106].\nMore recently, the role of A2A receptors in ischemic neuroprotection has been studied. Gao and Phillis [107] demonstrated for the first time that the non-selective A2A receptor antagonist CGS 15943 reduces cerebral ischemic injury in the gerbil following global forebrain ischemia. Subsequently, many reports have confirmed the neuroprotective role of A2A receptor antagonists in different models of ischemia. The selective A2A receptor antagonist SCH 58261 reduced ischemic brain damage in neonatal [108] and adult [83, 109] rat models of focal cerebral ischaemia. The same antagonist, subchronically administered, was protective against both brain damage and neurological deficit in the adult rat model of focal cerebral ischemia [110, 111]. Studies in A2A receptor knockout mice supported the neuroprotective role of A2A receptor antagonists on ischemic brain damage [112]. The beneficial effects of A2A antagonists in stroke were mainly attributed to reduced glutamate outflow [83, 110, 113]. Prolonged application of the A2A selective agonist CGS 21680 significantly reduced synaptic depression brought about by OGD in the hippocampus [59] and the same agonist promotes glutamate release under normoxic and ischemic conditions [70, 71, 114, 115]. In addition to neurones, adenosine A2A receptors are located on microglia [116, 117] and astrocytes [118]. On glial cells, A2A receptors mediate inhibition of the glutamate uptake transporter, GLT-1, and stimulate glutamate outflow [119]. A2A antagonists prevent the increase in glutamate levels induced by glutamate uptake inhibitors [120]. Therefore, the protective effects of A2A antagonists in brain ischemia may be largely due to reduced glutamate outflow from neurones and glial cells.\nFurther support of a major role for glia in the neuroprotective effect of A2A antagonism in ischemia comes from the observation that subchronic administration of the A2A receptor antagonist SCH 58261 reduces p38 mitogen-activated protein kinase (MAPK) activation in striatal and cortical microglia 24\u00a0h after permanent focal ischemia [111]. Evidence indicates that p38 MAPK is activated in glia up to 24\u00a0h after ischemia [121, 122] and is involved, through transcriptional mechanisms, in neuronal death [123, 124]. Since SCH 58261 reduces glutamate outflow in the first hours after ischemia [83], reduced p38 MAPK activation may be due to a direct effect on glial A2A receptors or may be secondary to a reduction in the excitotoxic cascade that primes p38 activation [125]. The importance of A2A receptors under ischemia is highlighted by the observation that A2A receptor expression increases on neurones and microglia after ischemia [126]. Selective inactivation of A2A receptors on bone-marrow-derived cells (wild-type mice transplanted with A2A receptor knockout bone marrow cells) attenuates infarct volumes and ischemia-induced expression of several proinflammatory cytokines in the brain [127]. Therefore, protective effects of A2A antagonists may be attributed to inhibition of inflammation product production.\nIn several studies, A2A receptor agonists have been found to be protective in the global ischemia model in the gerbil [128, 129]. Jones and co-workers [130] show that peripheral administration of the A2A receptor agonist CGS 21680 protects the hippocampus against kainate-induced excitotoxicity. However, the direct injection of CGS 21680 into the hippocampus failed to afford protection, while the A2A antagonist ZM 241385, when injected directly into the hippocampus, reduced kainate-induced neuronal damage [131]. These data suggest that the neuroprotective properties of A2A agonists are mainly due to peripherally mediated effects. Major mechanisms that may account for A2A-mediated protection include inhibition of platelet aggregation and vasodilation [89] and anti-inflammatory actions. A2A receptors on neutrophils may account for inhibition of adhesion to endothelial cells and ensuing production of free radicals [132, 133].\nOn this basis, adenosine and its receptors are considered targets for therapeutic implementation in the treatment of stroke. At the moment, a possible adenosynergic therapeutic strategy after ischaemia that is worth consideration is that of increasing adenosine concentrations at the ischemic sites by inhibitors of adenosine metabolism or reuptake in association with adenosine A2A antagonists. When considering the possible use of adenosine kinase inhibitors, it should be taken into account that adenosine represents only a small percentage of nucleotide content [134], therefore inhibition of its rephosphorylation to ATP by adenosine kinase inhibitors does not weigh upon the ATP content.\nThe few studies present in the literature concerning the role of A3 receptors in the pathophysiology of cerebral ischemia are rather contradictory. We have demonstrated that selective antagonism of A3 receptors facilitates the recovery of synaptic activity induced by ischemic preconditioning in rat hippocampal slices [81]. A harmful role of A3 receptors during in vitro OGD was confirmed by our observation that blocking the A3 adenosine receptor consistently abolishes or delays the occurrence of anoxic depolarization (AD) and significantly protects from the irreversible disruption of excitatory neurotransmission caused by a severe ischemic episode [135]. These results are in agreement with the observation that acute administration of a selective adenosine A3 agonist exacerbates the damage elicited by global ischemia in the gerbil [136]. On the contrary, it was demonstrated that chronic pre-ischemic administration of an A3 agonist protects against ischemic neuronal damage [136]. This effect may be attributed to desensitisation of A3 receptors. In fact, both human and rat A3 receptors are desensitised within a few minutes after agonist exposure [137, 138].\nContrary to the above information, Hentschel and colleagues [139] demonstrated that under hypoxic conditions, selective activation of A3 adenosine receptors brings about an inhibition of excitatory neurotransmission on cortical neurones, indicating that A3 receptors may sustain the neuroprotective action of adenosine induced by A1 receptors. Consistent with these reports, mice lacking A3 adenosine receptors show increased neurodegeneration in response to repeated episodes of moderate hypoxia [140] or an increase in cerebral infarction after transient ligation of the middle cerebral artery [141]. These opposite results regarding an excitatory or inhibitory role of A3 receptors on synaptic activity under hypoxia\/ischemia may be reconciled by our recent data [135], suggesting that in a first phase of ischemia, A3 receptors play a protective synergistic role with A1 receptors. Severe ischemia would transform the A3 receptor-mediated effects from protective to injurious.\nTaken together, these data suggest that the outcome of A3 receptor stimulation on synaptic transmission during hypoxic\/ischemic phenomena depends on the intensity and duration of stimulation.\nConcluding remarks\nPurinergic signalling, e.g. adenosine and ATP, by activating specific membrane receptors (P1 and P2 respectively), is strictly correlated in orchestrating brain cell functions either under physiological normoxic or ischemic circumstances. However, the respective contribution of each single element is hard to discern from the total outcome, due to the rapid and ubiquitous enzymatic interconversion of these molecules.\nIn the brain, both compounds are likely to play a role in neuronal activity, and the inhibitory action of endogenous adenosine in excitatory neurotransmission (mainly due to A1 receptor activation) is well documented. Conversely our data, in line with previous observations in the literature, suggest that tonic activation of P2 receptors contributes to excitatory transmission in the hippocampus. Such an effect is modest, but it is unmasked by the application of P2 purinergic antagonists that reduce evoked synaptic responses.\nAn inhibitory effect elicited by exogenous ATP application has been frequently described and is mostly due to enzymatic degradation to adenosine and the subsequent activation of A1 receptors. However, our evidence demonstrates that ATP itself may inhibit hippocampal synaptic transmission since an even more pronounced effect is observed when enzymatic ATP degradation is blocked. Moreover, in our studies in the hippocampus, a potentiation of neurotransmission by the ATP analogue, ATP\u03b3S, develops a few minutes after the inhibitory effect has been washed out, and it persists for 45\u00a0min to 1\u00a0h. Within the brain, ATP is involved in the synaptic plasticity phenomenon and may be an important mediator of long-lasting effects in synaptic plasticity.\nEither adenosine or ATP seems to become a particularly important signalling molecule under pathological conditions, such as ischemia, when the extracellular concentration of either compound drastically rises. It is well known that adenosine exerts important neuroprotective effects during brain ischemic insults by activating adenosine A1 receptors, which profoundly inhibit synaptic transmission and in particular the release of glutamate, known to contribute to ischemic damage. Under severe ischemia, a prolonged stimulation of both A2A and A3 receptors may be deleterious. The role of endogenously released ATP during cerebral ischemia is mainly deleterious, as found in our work and in the literature, since the application of P2 antagonists always results in a reduction in ischemic damage. The mechanisms underlying these effects are still unknown, but they can be correlated to the tonic excitatory action of endogenous ATP found in many brain regions (for example in the hippocampus and prefrontal cortex).\nPurinergic signalling has an ancient phylogenetic origin. Expression of P2 receptors on cell membrane and responses to extracellular ATP are found in primitive prokaryotic species up to evolved animals and plants, suggesting an important and highly conserved role of extracellular purine nucleotides during evolution. Similarly, ontogenetic development in several species seems to involve purinergic signalling, especially during its first stages. Our observation that hMSCs, an undifferentiated line of cells able to originate a number of different cell lineages (adipocytes, chondrocytes, osteoblasts, neurones), spontaneously release ATP into the extracellular space and express functional purinergic P2 receptors, which modulate different kinds of membrane currents, and that ATP, during the early stages of culture, inhibits proliferation indicates an autocrine\/paracrine mechanism of action for extracellular ATP in modulating cell functions of undifferentiated stem cells at early developmental stages. Such effects may be important in brain neurogenesis during development and in responses to neurodegenerative stimuli.","keyphrases":["atp","adenosine","ischemia","ecto-atpase inhibitors","adenosine a2a receptors","p2 purinergic receptors"],"prmu":["P","P","P","P","P","R"]}
{"id":"Int_J_Biochem_Cell_Biol-2-1-2279807","title":"A2 isoform of mammalian translation factor eEF1A displays increased tyrosine phosphorylation and ability to interact with different signalling molecules\n","text":"The eEF1A1 and eEF1A2 isoforms of translation elongation factor 1A have 98% similarity and perform the same protein synthesis function catalyzing codon-dependent binding of aminoacyl-tRNA to 80S ribosome. However, the isoforms apparently play different non-canonical roles in apoptosis and cancer development which are awaiting further investigations. We hypothesize that the difference in non-translational functions could be caused, in particular, by differential ability of the isoforms to be involved in phosphotyrosine-mediated signalling.\n1\nIntroduction\nTranslation elongation factor 1A (eEF1A) is one of the core members of the elongation machinery providing high efficiency and processivity of the protein synthesis process in the eukaryotic cell (Negrutskii & El'skaya, 1998). eEF1A forms a ternary complex with aminoacyl-tRNA and GTP and delivers the correct aminoacyl-tRNA to the A site of mRNA programmed ribosome in the GTP hydrolysis-dependent mode. The GDP-bound form of eEF1A has been shown to interact with deacylated tRNA (Petrushenko et al., 1997) and can transport it to aminoacyl-tRNA synthetase as the tRNA recycling arm of a tRNA channelling cycle (Petrushenko, Shalak, Budkevich, Negruskii, & El'skaya, 2002).\nIt is believed that the translation function of eEF1A might be combined with its involvement in cytoskeletal network maintenance and various signalling pathways (Lamberti et al., 2004). eEF1A has been shown to interact with phospholipase C-gamma1 (PLC-gamma1) (Chang et al., 2002; Kim et al., 1999) and non-receptor tyrosine kinase Txk of the Tec family (Maruyama, Nara, Yoshikawa, & Suzuki, 2007). Importantly, Txk can form a complex with poly(ADP-ribose) polymerase 1 and eEF1A to influence interferon-gamma gene transcription in Th1 cells. Recently Lau, Castelli, Lin, and Macaulay (2006) identified eEF1A as a novel binding partner for Akt2 rho-associated kinase. In most cases it is not known whether interactions of eEF1A with protein kinase might lead to phosphorylation of eEF1A. Neither has the role of eEF1A in signal transduction pathways been identified.\nIn mammalian cells, two tissue- and development-specific isoforms, eEF1A1 and eEF1A2, are present. The expression of the 98% similar isoforms is mutually exclusive (Kahns et al., 1998). Interestingly, eEF1A2, which is present normally only in muscles and neurons, was recently associated with tumor development in tissues that normally express only eEF1A1 (Amiri et al., 2007; Anand et al., 2002). How eEF1A2 isoform is involved in malignant transformation is not as yet understood. Since tyrosine kinases are prominent players in cancer development, direct comparison of the ability of eEF1A1 and eEF1A2 to be involved in phosphoTyr-specific signalling processes could help to interpret cancer-related properties of eEF1A2.\nIn this study, we compared for the first time the interactions of eEF1A1 and eEF1A2 with SH2 and SH3 domains of various signalling molecules. Contrary to eEF1A1, eEF1A2 was able to interact with SH2 domains of Grb2, RasGAP, Shc and Shp2 as well as with SH3 domains of Crk, Fgr, Fyn and PLC-gamma1. Both eEF1A1 and eEF1A2 formed complexes with SH2 domain of PLC-gamma1. In vivo interaction between endogenous eEF1A and tyrosine phosphatase Shp2 was confirmed in HEK293 cell line and tyrosine phosphorylation of eEF1A1 and eEF1A2 was demonstrated by Western blots with anti-phosphotyrosine-specific antibodies. Importantly, the level of phosphorylation of eEF1A2 was higher than eEF1A1 when isolated from tissues, and also when overexpressed in cells. Furthermore, the level of phosphorylation has been shown to be crucial for in vitro complex formation of eEF1A2 and SH2 domain of Shp2.\n2\nMaterials and methods\n2.1\nCell lines and plasmid constructs\nHEK293 cells were purchased from the American Type Culture Collection (Manassas, VA) and grown according to their instructions. To produce eEF1A1 and eEF1A2 stable cell lines pcDNA 3.1 eEF1A1 and eEF1A2 with the C-terminal His-tags were linearized by MunI and transfected in HEK293 cells using ExGene500 (Fermentas) according to manufacturer recommendations. G418 was used to select stable cell lines.\n2.2\nBioinformatics\nScanSite Program was used for bioinformatic identification of possible SH2 and SH3 domains binding sites within eEF1A1 and eEF1A2. ScanSite employs the matrix of selectivity values for amino acids at each position relative to an orienting residue as determined by the oriented peptide library technique (Obenauer, Cantley, & Yaffe, 2003).\n2.3\nPurification of the eEF1A1 and eEF1A2 isoforms\neEF1A1 was purified from rabbit liver using a combination of the gel filtration, anion exchange, cation exchange and hydroxyapatite chromatographies as described previously (Budkevich et al., 2002). eEF1A2 was isolated from rabbit muscles using the same procedure except the gel-filtration step was omitted in some cases. To confirm the biological activity of the isolated isoforms, the GDP\/[3H]GDP exchange was carried out as in (Carvalho, Carvalho, & Merrick, 1984).\n2.4\nPull down assay\nPurified GST-SH2 and GST-SH3 domains of various signalling molecules were kindly provided by Prof. Ivan Gout (London, UK) (Gout et al., 1993). In a pull down assay, 1.5\u00a0\u03bcg of GST, GST-SH2 or GST-SH3 domains fusion protein was incubated with glutathione-Sepharose 4B beads (Amersham Biosciences) for 2\u00a0h at 4\u00a0\u00b0C. The beads were then washed to remove unbound proteins and incubated at 4\u00a0\u00b0C for 3\u00a0h with 1.5\u00a0\u03bcg of purified eEF1A1 or eEF1A2. Non-specific interactions were removed by extensive washing and bound proteins eluted by boiling in Laemmli sample buffer. Eluted proteins were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and detected by immunoblotting with specific antibodies.\n2.5\nPhosphatase treatment\nCalf intestine alkaline phosphatase (CIAP) (Fermentas) has been used to dephosphorylate eEF1A1 and eEF1A2. The proteins were precipitated from the lysates of stable cell lines on NiNTA and extensively washed with 1\u00d7 reaction buffer for CIAP (10\u00a0mM Tris\u2013HCl (pH 7.5), 10\u00a0mM MgCl2), then 10\u00a0U of CIAP was added and reaction mix was incubated at 37\u00a0\u00b0C for 1\u00a0h. Thereafter the beads were washed two times with 1\u00d7 reaction buffer and the proteins bound were eluted by boiling in Laemmli sample buffer.\nFor dephosphorylation of total protein fraction HEK293 cells overexpressing eEF1A1 or eEF1A2 were extracted with 1\u00d7 CIAP reaction buffer. To facilitate lysis the cells were passed through a 20-gauge needle 20 times and whole cell extracts were centrifuged at 10,000\u00a0\u00d7\u00a0g for 15\u00a0min at 4\u00a0\u00b0C. The supernatant was incubated with 10\u00a0U of CIAP at 37\u00a0\u00b0C for 1\u00a0h, in control samples sodium orthovanadate was added to final concentration 1\u00a0mM.\n2.6\nImmunoprecipitation\nHEK293 cells were treated under indicated conditions, then washed with ice-cold phosphate-buffered saline (PBS) and extracted with the lysis buffer containing 10\u00a0mM Tris\u2013HCl (pH 7.5), 150\u00a0mM NaCl, 5\u00a0mM EDTA, 50\u00a0mM NaF, 1% Triton X-100, 1\u00a0mM sodium orthovanadate and a mixture of protease inhibitors (Roche Applied Science). Whole cell extracts were centrifuged at 10,000\u00a0\u00d7\u00a0g for 15\u00a0min at 4\u00a0\u00b0C. Endogenous protein complexes were immunoprecipitated from 0.5\u00a0mg of total cell lysates with corresponding antibodies (1\u00a0\u03bcg\/probe) and immobilized on protein A-Sepharose beads (Amersham Biosciences). Immune complexes were washed three times with the lysis buffer and used for immunoblotting. As a loading control Western blotting (WB) of total cell lysate with anti-\u03b2-actin has been used. The effective serum starvation (24\u00a0h in medium without serum) and stimulation (10% FBS, 30\u00a0min) has been confirmed in WB of total cell lysates with antibodies against phosphorylated residues S240\/S244 in ribosomal protein S6, which is phosphorylated by S6K1\/2 (effector kinases in PI3K\/mTOR pathway) in response to growth factors stimulation.\n2.7\nImmunoblot analysis\nProteins were separated by SDS-PAGE, transferred onto polyvinylidene difluoride membrane, and incubated for 1\u00a0h with blocking solution (2% BSA, Tris-buffered saline, 0.1% Tween-40). After blocking the membranes were probed 2\u00a0h at 4\u00a0\u00b0C with anti-GST, anti-eEF1A (Upstate Biotechnology), anti-His tag (Cell Signalling), anti-Shp2 (Cell Signalling) or anti-pTyr 4G10 (Upstate Biotechnology) antibodies. After extensive washing with the solution of Tris-buffered saline and 0.1% Tween-40, the membranes were incubated with secondary horseradish peroxidase-conjugated antibody for 1\u00a0h at room temperature. The antigen\u2013antibody complexes were detected using the ECL system (Millipore). When immunoblots had to be re-probed, the membranes were initially stripped and re-blocked prior to incubation with another type of primary antibody.\n3\nResults\n3.1\nExistence of SH2\/SH3 domain binding sites in the eEF1A1 and eEF1A2 molecules are predicted by bioinformatic analysis\nFirst we examined the possibility if the 98% similar eEF1A1 and eEF1A2 might have distinct binding motifs for signalling molecules. A low stringency Scansite (Obenauer et al., 2003) search revealed potential SH2\/SH3 domain containing protein partners and predicted the existence of several phosphorylation sites for tyrosine kinases in both isoforms (Fig. 1). However, the SH3 recognition motif specific for Src kinase and adaptor protein Crk was found only in the eEF1A2 molecule. This is an important indication of the possibility of differential involvement of eEF1A2 in the formation of signalling complexes.\n3.2\neEF1A1 and eEF1A2 interact in vitro with SH2\/SH3 domains of different proteins\nThe ability of eEF1A1 and eEF1A2 to form complexes with SH2 and SH3 domains of different signalling molecules was directly analysed in pull down experiments. The preparative amounts of native eEF1A1 and eEF1A2 were purified from rabbit tissues as described in Section 2. Importantly, the rabbit and human proteins are 100% identical. SH2 domains of signalling proteins Grb2, RasGAP, PLC-gamma1, Shc, Shp2 and SH3 domains of Crk, Csk, Fgr, Fyn, Src were expressed as recombinant proteins fused with GST, purified from bacteria, immobilized on glutathione sepharose and mixed with eEF1A1 or eEF1A2. Complexes of eEF1A1 and eEF1A2 with SH2 and SH3 domains of different proteins were detected (Fig. 2A and B). Both eEF1A1 and eEF1A2 interacted with SH2 domain of PLC-gamma1, while eEF1A2 exclusively formed complexes with SH2 domains of Grb2, RasGAP, Shc, Shp2 and SH3 domains of Crk, Fgr, Fyn, PLC-gamma1.\nIt should be noted that principal possibility of the interaction of PLC-gamma1 and eEF1A was shown earlier. The interactions of SH2 and SH3 domains (Kim et al., 1999) or PH domain (Chang et al., 2002) of PLC-gamma1 with eEF1A were reported. Our results confirmed the interaction of eEF1A with SH2 and SH3 domains of PLC-gamma1 and were used as internal positive control of interaction with known eEF1A binding partner.\nContrary to eEF1A1, eEF1A2 was found to interact with SH2\/SH3 domains of several adaptor proteins. Grb2 and Shc provide a critical link between cell surface growth factor receptors and the Ras and Myc signalling pathways via interactions with cytoplasmic Tyr kinases, G-proteins and other cellular Tyr-phosphorylated proteins. Interestingly, Shc is constitutively activated (tyrosine-phosphorylated) in most breast cancer cells where non-typical expression of eEF1A2 was reported (Frackelton et al., 2006; Tomlinson et al., 2005).\nIdentification of RasGAP protein as a direct partner of G-protein eEF1A2 may be interesting in two ways. On the one hand, RasGAP may play GTPase activating role for eEF1A2, because the initial rate of GTP hydrolysis by the intrinsic eEF1A2 GTPase is slightly lower when compared with eEF1A1 (Kahns et al., 1998 and our unpublished observation). On the other hand, eEF1A2 may influence RasGAP activity and consequently modulate Ras.\neEF1A2 interacts also with SH3 domain of Crk which is involved in MAPK signalling, actin cytoskeleton reorganization and insulin receptor signalling (Feller, 2001). It should be noted that eEF1A is involved in actin cytoskeleton regulation as well (Gross & Kinzy, 2005; Shiina, Gotoh, Kubomura, Iwamatsu, & Nishida, 1994).\nFgr and Fyn are Tyr kinases that participate, in particular, in the MAPK\/ERK and insulin receptor signalling pathways involved in the control of initiation of translation (Alper & Bowden, 2005). The interaction of eEF1A2 with SH3 domains of Fgr and Fyn suggests eEF1A2 as a prospective substrate for these enzymes.\nThus, eEF1A2 is capable of forming complexes with SH2 and SH3 domains of several signalling molecules. The potentially increased role of eEF1A2, as compared to eEF1A1, in phosphoTyr-mediated signalling pathways lends support for the putative oncogenic role assigned to eEF1A2 earlier (Amiri et al., 2007; Lamberti et al., 2004; Lee, 2003).\n3.3\neEF1A1 and eEF1A2 are in complex with Shp2 in vivo\nVerification of the in vitro formed complexes was done in vivo taking as an example interaction of eEF1A with Shp2.\nFirst, HEK 293 stable cell lines overexpressing eEF1A1-His, eEF1A2-His or GFP protein were used to confirm that endogenous Shp2 protein interacts with eEF1A2. Interestingly, specific binding of endogenous Shp2 to both isoforms of eEF1A was detected (Fig. 3A). Since only the C-terminal SH2 domain of Shp2 was used as a bait in the experiments in vitro, we suggest eEF1A1 and eEF1A2 bind to different domains of Shp2.\nAlso, a complex of endogenous eEF1A and Shp2 was immunoprecipitated from HEK293 cells using anti-eEF1A antibodies (Fig. 3B). The complex eEF1A\/Shp2 was detected in growing cells. Interestingly, complex formation was more effective in cells synchronized by growth factors starvation and less efficient in cells stimulated with serum, indicating the regulatory nature of this interaction (Fig. 3B). Importantly, eEF1A2 was shown earlier to accumulate in G0 rather than in G1 and S phase cells (Ann et al., 1991). However, since anti-eEF1A monoclonal antibodies are specific both for eEF1A1 and eEF1A2, and the presence of both isoforms in embryonic cells is possible, it is difficult to determine precisely which isoform of eEF1A interacts with Shp2 in vivo.\n3.4\neEF1A2 and Shp2 interaction is sensitive to dephosphorylation\nTo test if the interaction of eEF1A2 with C-terminal SH2 domain of phosphatase Shp2 is dependent on phosphorylation status of eEF1A, equal amounts of total cell lysates of the stable cell lines expressing eEF1A1 or eEF1A2 were treated with calf intestine alkaline phosphatase, or, in control samples, incubated with phosphatase inhibitor (sodium orthovanadate). Specific interaction of SH2-Shp2 and eEF1A2 was detected only in the samples not treated with CIAP indicating that phosphorylation of eEF1A2 is a necessary condition for eEF1A2\/Shp2 complex formation (Fig. 3C).\n3.5\neEF1A1 and eEF1A2 are phosphorylated at Tyr in vivo\nThe interaction of eEF1A with SH2 domains of different proteins in vitro as well as the eEF1A\u2013Shp2 complexes formation in vivo indicate the possibility of tyrosine phosphorylation in the eEF1A molecules. To test if native eEF1A1 and eEF1A2 contain such modifications we probed the isolated eEF1A proteins with monoclonal antibodies specific to phosphorylated tyrosines (4G10). As a result, tyrosine phosphorylation of both isoforms was detected (Fig. 4A). Moreover, the phosphorylation of eEF1A1 and eEF1A2 at tyrosine residues was confirmed using the His-tagged eEF1A isoforms precipitated on Ni-NTA sepharose (Fig. 4B). The eEF1A1 and eEF1A2 samples dephosphorylated with calf intestine alcaline phosphatase were used to control specificity of 4G10 antibodies. Notably, eEF1A2 was characterized by greater phosphorylation level as compared to eEF1A1 in both experiments.\n4\nDiscussion\nBesides its function in translation, eEF1A is thought to be involved in several cellular processes, including embryogenesis, senescence, oncogenic transformation, cell proliferation and organization of cytoskeleton (reviewed in Gross & Kinzy, 2005; Lamberti et al., 2004; Lee, 2003). There are two tissue-specific isoforms of eEF1A. eEF1A1 functions in all mammalian tissues except neurons, cardiomyocytes and myotubes, where eEF1A2 is taking role and is expressed. Recently eEF1A2 was also found to express in human pancreatic islets (Ahmed, Forsberg, & Bergsten, 2005). Few attempts to differentiate the specific functions of the isoforms have been carried out so far. In vitro, eEF1A2 has slightly decreased translation functions [our unpublished observation]. Discovery of putative oncogenic role of eEF1A2 in ovarian cancer (Lee, 2003) as well as opposite apoptosis-related functions of the isoforms (Chang & Wang, 2007) indicated a possibility of distinct participation of eEF1A1 and eEF1A2 in cellular signalling processes. The mechanisms of cancer-related functions of eEF1A2 are not known. We suggested that difference of the isoforms could be related to their distinct ability to be involved into phosphoTyr-mediated signalling pathways.\nTo examine such a possibility we used the panel of cloned SH2 and SH3 domains of various signalling molecules. SH2 domains are known to associate with specific phosphoTyr-containing sites (Schlessinger & Lemmon, 2003). SH3 domains of adaptor proteins can support the association with a number of signalling proteins and upon cell stimulation can engage adaptor proteins with Tyr-phosphorylated cytoplasmic or membrane-attached partners (Li, 2005). As a result, we detected the interaction of eEF1A1 and eEF1A2 with SH2 domain of PLC-gamma1 indicating that both isoforms could contain phosphoTyr residues. In addition, eEF1A2 was found associated with SH2 domains of Grb2, RasGAP, Shc and Shp2 (Fig. 2A). This suggests eEF1A2 plays a more universal role in signal pathways. The finding that eEF1A2 rather than eEF1A1 can interact with SH3 domains of Crk, Fgr and Fyn supports this notion (Fig. 2B).\nThe association of eEF1A and Shp2 is very appealing in terms of the role Shp2 is playing in the cell-activation of Ras, with subsequent activation of PI3K and MAPK signalling pathways (Mohi & Neel, 2007). In addition, Shp2 regulates actin cytoskeleton reorganization by modulating the activity of small GTP-binding protein Rho (Burks & Agazie, 2006; Harada, Katoh, & Negishi, 2005). It is interesting that eEF1A is an actin-binding protein which can regulate actin bundling as well, but its target substrates have not been identified so far (Murray, Edmonds, Liu, & Condeelis, 1996). Since direct binding of eEF1A to Shp1 was recently found to have some regulatory consequence, namely eEF1A2-dependent increase of Shp1 phosphatase activity (Nandan, Yi, Lopez, Lai, & Reiner, 2002) the examination of possible effect of eEF1A2 on the Shp2 activity is a tempting task for future investigations.\nWe have observed direct interaction of eEF1A2 with SH2 domain of RasGAP protein. Here we can only speculate about possible involvement of eEF1A2 in the activation of PI3K and MAPK pathways by Ras through RasGAP inactivation (Yarwood, Bouyoucef-Cherchalli, Cullen, & Kupzig, 2006). Otherwise, RasGAP may play a role of GAP for eEF1A2. The last notion could be supported by the observation that the subunits of eEF1B, regular exchange factor for eEF1A1, were not identified as interacting partners for eEF1A2 (Mansilla et al., 2002).\nThe phosphorylation of native and overexpressed eEF1A1 and eEF1A2 at tyrosine residues was confirmed by WB with phosphotyrosine-specific antibodies (Fig. 4A and B). Several preps of eEF1A1 and eEF1A2 were analysed and eEF1A2 always demonstrated higher phosphorylation level than eEF1A1 (data not shown).\nRecently, phosphorylation of Tyr 29 and Tyr 141 of eEF1A was found during an extensive search for all proteins containing phosphotyrosine in human cancer cells (Rush et al., 2005). However, since both identified tryptic peptides contained phosphoTyr29 or phosphoTyr141 (which are identical in eEF1A1 and eEF1A2) and the overall search was limited by the peptides containing phosphotyrosine, the unambiguous identification of the phosphorylated isoform in that case was hardly possible.\nThus, we have identified several binding partners of eEF1A2 previously unknown. Novel interaction of eEF1A with tyrosine phosphatase Shp2 has been confirmed also in vivo. Potentially augmented ability of eEF1A2 as compared to eEF1A1 to be involved in key signal transduction complexes could be linked to the putative oncogenic role of the former found recently in ovarian cancer (Anand et al., 2002). The definite role of eEF1A2 in activation of cell proliferation awaits further investigations.","keyphrases":["eef1a1","eef1a2","sh3 domain","sh2 domain","shp2"],"prmu":["P","P","P","P","P"]}
{"id":"Invest_New_Drugs-3-1-2039853","title":"Intravenous ATP infusions can be safely administered in the home setting: a study in pre-terminal cancer patients\n","text":"Summary\nIntroduction\nIn recent years favorable effects of adenosine 5\u2032-triphosphate (ATP) on nutritional and functional status and survival in cancer patients were reported [1\u20135]. However, ATP [6\u201310] and its breakdown product adenosine [11\u201318], given either as a continuous intravenous infusion or by intravenous bolus administration, were also reported to have side effects, which might hamper administration especially in an outpatient setting.\nIn cancer patients, ATP was administered as continuous intravenous infusions over several hours, in doses varying from 25 to 100\u00a0\u03bcg kg\u22121 min\u22121. In a Phase I study [4], 14 patients with advanced cancer received 96-h infusions of ATP once per month for a total of at least three infusions, as tolerated by the patient. In the first three patients, ATP was given at a dose of 50\u00a0\u03bcg kg\u22121 min\u22121. If well tolerated, subsequent dose escalations were planned in groups of three patients in increments of 25\u00a0\u03bcg kg\u22121 min\u22121 until the maximally tolerated dose (MTD) was reached. Patients who completed therapy without serious toxicity and later progressed were eligible for retreatment at a higher dose of ATP. Fourteen patients received a total of 43 ATP infusions: 11 patients received 29 infusions at a dose of 50\u00a0\u03bcg kg\u22121 min\u22121, six patients received 11 ATP infusions at a dose of 75\u00a0\u03bcg kg\u22121 min\u22121, and three patients who had tolerated 50\u00a0\u03bcg kg\u22121 min\u22121 very well were reconsented and received one cycle each at a dose of 100\u00a0\u03bcg kg\u22121 min\u22121. In total, 179 adverse events were reported, whether or not these could be ascribed to ATP. The majority of side effects (dyspnea, chest pain, hypotension, gastrointestinal side effects, dizziness, headache, fever and local injection site reaction) were minor (104 adverse events grade 1 (58%) and 54 adverse events grade 2 (30%) based on the US National Cancer Institute Toxicology Criteria. Dose-limiting toxicities of ATP were reported in seven patients, i.e. asymptomatic electrocardiographic evidence of cardiac ischemia (n\u2009=\u20092), grade 3 cardiac toxicity (angina without evidence of infarction), and grade 4 pulmonary toxicity (dyspnea at rest), usually occurring as a characteristic cardiopulmonary syndrome including an initial feeling of chest \u2018tightness\u2019 and a sensation of \u2018needing to take a deep breath\u2019 (n\u2009=\u20095). Dose-limiting cardiopulmonary toxicity appeared in all three 100\u00a0\u03bcg kg\u22121 min\u22121 ATP infusions, in four (36%) 75\u00a0\u03bcg kg\u22121 min\u22121 infusions, and in five (17%) 50\u00a0\u03bcg kg\u22121 min\u22121 infusions.\nIn a subsequent uncontrolled phase II study [5], 15 previously untreated patients with stage IIIB\/IV non-small-cell lung cancer (NSCLC) received infusions of ATP at doses of 50 (first course) and 65\u00a0\u03bcg kg\u22121 min\u22121 (subsequent courses) for 96\u00a0h every 4\u00a0weeks. Reported side effects were, in decreasing frequency, chest pain, dyspnea, coughing, anxiety, injection site pain, chest tightness, headache, insomnia, and hot flushes. According to the authors, six patients reported severe adverse events, of which CTC 4 dyspnea occurred in two patients; this led to the cessation of treatment in five patients [5].\nIn the first randomized clinical trial with ATP [1], 28 patients with stage IIIB\/IV NSCLC received a total of 176 intravenous ATP courses, at doses of maximally 75\u00a0\u03bcg kg\u22121 min\u22121 (tailored to the maximum tolerance of each patient) for 30\u00a0h every 2\u20134\u00a0weeks. Twenty-nine percent of the infusions were given at rates of 25\u201340\u00a0\u03bcg kg\u22121 min\u22121, 27% at 45\u201360\u00a0\u03bcg kg\u22121 min\u22121 and 44% at 65\u201375\u00a0\u03bcg kg\u22121 min\u22121 [19]. The most frequent side effects were chest discomfort (15%) and the urge to take a deep breath (10%), both of which resolved within minutes after lowering the ATP dose. Electrocardiography (ECG) was performed in patients with chest pain\/discomfort during the ATP infusions, but no ECG changes suggestive of myocardial ischemia were detected [19]. In all of the above studies, ATP was administered on an inpatient basis under medical supervision.\nBased on the previously reported favorable effects of ATP on quality of life, nutritional status and survival [1, 3], we initiated a new randomized clinical trial to investigate whether these effects of ATP could be confirmed in patients with different types of cancer in the pre-terminal stage of disease (life expectancy 1\u20136\u00a0months). In view of the short life expectancy of these patients, and based on the favorable safety profile of ATP, we decided to administer ATP in the home setting.\nThe aim of the present study was to investigate the safety of ATP administration at home in pre-terminal cancer patients. We hypothesized that side effects of ATP infusions would occur mainly during the first ATP infusion, especially during assessment of the MTD of ATP. We also hypothesized that the presence of cardiac disorders and\/or lung cancer would lead to a lower MTD, and a higher frequency of side effects.\nPatients and methods\nStudy population and design\nPatients were recruited through the Departments of Medical Oncology and Pulmonology of five hospitals in different regions in The Netherlands (Maastricht, Eindhoven, Utrecht and Heerlen) and through general practitioners in the region of Maastricht. Eligible were patients with cytologically or histologically confirmed cancer, for whom medical treatment options were restricted to supportive care, who had a life expectancy <6\u00a0months, had a World Health Organization (WHO) performance status 1 or 2, and who at the time of inclusion suffered from at least one of the following complaints: fatigue, anorexia, or weight loss >5% in the previous 6\u00a0months. Exclusion criteria were: symptomatic angina pectoris, symptomatic heart failure, atrio-ventricular block (checked by standard ECG in all patients before inclusion), life expectancy of less than 4\u00a0weeks, concurrent palliative chemotherapy at the time of inclusion, cognitive dysfunction, and the presence of other diseases hampering adequate follow up. The eligibility of patients was evaluated by a medical oncologist or pulmonologist. After baseline measurements, patients were randomly allocated to ATP or control treatment. The present report on the safety of ATP administration is restricted to patients randomized to ATP treatment. The study was approved by the Ethical Committee of University Hospital Maastricht and Maastricht University, and all patients signed written informed consent prior to the study. Details of the trial design have been published elsewhere [20].\nIntervention\nAdenosine 5\u2032-triphosphate (ATP-Na2) of >98% purity was purchased from Pharma Waldhof GmbH and co. KG (D\u00fcsseldorf, Germany). For each infusion, 2.5\u00a0g of ATP were dissolved in 500\u00a0ml NaCl 0.9%, sterilized by ultrafiltration (Minisart 0.22\u00a0\u03bcM), and supplied in sterile containers. Patients received weekly intravenous ATP infusions of 8\u201312\u00a0h, over a period of 8\u00a0weeks. All ATP infusions were started at a dose of 20\u00a0\u03bcg kg\u22121 min\u22121 and then increased in steps of 10\u00a0\u03bcg kg\u22121 min\u22121 every 10\u00a0min, until a maximum dose of 50\u00a0\u03bcg kg\u22121 min\u22121, or the MTD, if this was lower, had been reached. Thereafter, ATP was infused at a continuous rate. If any side effects occurred, the dose was reduced until side effects disappeared. Blood pressure and pulse were registered before starting the infusion, at 30\u00a0min after determining the MTD, and just before the infusion was stopped.\nSince initiation of ATP infusions under medical supervision in a clinical setting would facilitate the treatment of possible side effects, the first two ATP infusions were given at the day care center of the participating hospitals. Based on evaluation of safety data in the first 22 patients, in view of the mild character of the noted side effects during the first two infusions in these patients, the Ethical Committee granted permission to administer only the first ATP infusion at the day care center and all subsequent infusions at home. The description below applies to the procedures in case of only one hospital infusion.\nAt the end of the first infusion, the safety and tolerated dose of ATP was evaluated for each individual patient. Subsequent infusions were given at the patients\u2019 home by an experienced and trained nurse of a specialized infusion team, usually embedded in the regional Community Care Organization. The MTD as determined during the first infusion at the day care center was also the maximum dose for this patient for the next infusions at home. The same rule was applied for subsequent home infusions, so that the infusion rate in any subsequent ATP infusion course was never higher than the MTD during the previous ATP infusion. Patients and their partners were instructed extensively regarding the infusion procedures; also, they were instructed to immediately call the involved infusion team in case of any side effects.\nDocumentation of side effects and adverse events\nSide effects of ATP infusions\nSide effects of all ATP infusions, adjustments of dosage schedules, and any other events occurring during the infusions were registered by the infusion nurse, either a. during the infusions, or b. on completion of each infusion. In addition, the researcher called all patients after each infusion to ask whether any side effects had occurred. Side effects were scored using the NCI Common Toxicity Criteria [21] on a four-point scale, according to seriousness. In this system, dyspnea is graded as follows: 0, no change; 1, not defined; 2, dyspnea on significant exertion; 3, dyspnea at normal activity; 4, dyspnea at rest. Since most terminal cancer patients are at rest, dyspnea would in these cases automatically be classified as CTC grade 4, which would misrepresent the actual severity of the symptoms and disregard the intention of the NCI Common Toxicity Criteria (i.e. CTC grade 4\u2009=\u2009life-threatening). For this reason, we decided to apply the general toxicity grading of the CTC also for dyspnea, i.e. 1, mild; 2, moderate; 3, severe; and 4, life-threatening.\nBased on Agteresch et al. [19], we pre-defined 13 side effects: chest discomfort, urge to take a deep breath, nausea, flushing, light-headedness, dyspnea, headache, sweating, mood alteration-anxiety, palpitations, epistaxis, symptoms of cardiac ischemia, injection side reaction or other (to be specified). In case of persistent or severe chest pain, suspect for potential cardiac ischemia, an ECG was made in order to exclude this.\nStatistical analysis\nData were entered in a database using Microsoft Access 2000 and checked for errors by means of double data entry by two independent persons. Data were analyzed using SPSS 13.0 for Windows. Changes in blood pressure and heart rate over time were tested for statistical significance by Student\u2019s paired t test. To test our hypothesis that side effects would especially occur during the first infusion, we calculated the average number of side effects per infusion over all infusions. To investigate whether the MTD and frequency of side effects were dependent on the presence of cardiac disorders and\/or lung cancer, linear regression models were fitted. As dependent variables, we used mean MTD per patient over all courses, and frequency of side effects per infusion. As independent variables, we included dummy variables for the presence of cardiac disorders (yes\/no), and lung cancer (yes\/no).To investigate the relation between the seriousness of side effects (CTC grades 2\u20134 vs. CTC grade 1) and the presence of cardiac disorders and\/or lung cancer, Fisher\u2019s exact test was used. P values of less than 0.05 indicated significance.\nResults\nStudy population\nFifty-one patients with different types of pre-terminal cancer were included in the present study. Baseline characteristics of the patients are listed in Table\u00a01. Mean age was 68.1\u00a0years (range: 45\u201387\u00a0years) and the majority of patients were male (69%). The most frequent diagnosis was lung cancer (41%), followed by colon cancer (16%). The majority of patients (65%) had WHO performance status 1, and 35% WHO status 2. Twelve patients (24%) suffered from cardiac disorders.\nTable\u00a01Baseline characteristics of ATP-treated patients (n\u2009=\u200951)\u00a0NumberPercent (%)Age (years)68.1 (45\u201387)aGender\u00a0Male3569\u00a0Female1631WHO performance scoreb\u00a013365\u00a021835Tumor type\u00a0Lung2141\u00a0Colon816\u00a0Gastro-intestinal other612\u00a0Prostate510\u00a0Other1121Presence of cardiac disorders1224aMean (range)bWHO 1: restricted in physically strenuous activity but ambulatory and able to do light work; WHO 2: ambulatory (not more than 50% in bed) and capable of self-care but unable to carry out any work\nOut of 51 patients, six patients died during the intervention period because of disease progression (Fig.\u00a01). Three patients did not start because of rapid deterioration of their condition and 18 patients stopped with the ATP infusions. Reasons to stop were deterioration in medical condition (n\u2009=\u200911), fear of side effects (n\u2009=\u20094), or being unsatisfied with the effect of ATP (n\u2009=\u20093).\nFig.\u00a01Flow diagram of the study\nOver the study period of 8\u00a0weeks, a total of 266 ATP infusions were administered in 48 patients. Fifteen patients (31%) received one to three ATP courses, nine patients (19%) received four to seven courses, and 24 patients (50%) completed all eight ATP infusions. Eleven infusions (4%) of 266 infusions were given at the lowest dose of 20\u00a0\u03bcg kg\u22121 min\u22121, 85 infusions (32%) at a dose of 25\u201340\u00a0\u03bcg kg\u22121 min\u22121, and 170 (64%) at the highest dose of 45\u201350\u00a0\u03bcg kg\u22121 min\u22121.\nHeart rate and blood pressure\nHeart rate decreased from 86\u2009\u00b1\u20091 (mean\u2009\u00b1\u2009SEM) at baseline to 83\u2009\u00b1\u20091 at 30\u00a0min after reaching the MTD (p\u2009<\u20090.001), and thereafter remained constant at 83\u2009\u00b1\u20091 beats\/min, as measured at 8\u201310\u00a0h of ATP administration (comparison with baseline: p\u2009<\u20090.001). Systolic blood pressure decreased from 126\u2009\u00b1\u20091 at baseline to 124\u2009\u00b1\u20092 at 30\u00a0min (p\u2009<\u20090.001), but then increased to 133\u2009\u00b1\u20092\u00a0mm Hg at 8\u201310\u00a0h of ATP infusion (comparison with baseline: p\u2009<\u20090.001). Diastolic blood pressure also initially decreased from 75\u2009\u00b1\u20091\u00a0mmHg to 72\u2009\u00b1\u20091\u00a0mmHg (p\u2009<\u20090.001), but then increased to 77\u2009\u00b1\u20091\u00a0mm Hg at 8\u201310\u00a0h of ATP infusion (comparison with baseline: p\u2009=\u20090.02).\nSide effects\nTen patients out of 48 had no side effects during any of the infusions. Out of 266 infusions, 167 infusions (63%) were without any side effects. In 48 infusions (18%) 1 side effect occurred, in 29 infusions (11%) 2 side effects, and in 22 infusions (8%) >2 side effects. In total, 192 side effects were reported (Fig.\u00a02): 138 side effects (72%) were reported to the nurse, of which 96 during the infusions and 42 afterwards; and 54 (28%) to the researcher when specifically asked for. The average number of side effects per infusion was 0.72 (i.e. 192 side effects in 266 infusions).\nFig.\u00a02Reported side effects of ATP according to location (day care centre vs. home), time of reporting side effects (during vs. after the infusion), and person to whom the side effects were reported (nurse vs. researcher)\nAt the day care center, 63 side effects were reported in 95 infusions (0.66 side effect per infusion). Of these 63 side effects, 56 (89%) were reported to the nurse, of which 55 during the infusions and 1 afterwards; and 7 (11%) to the researcher when specifically asked for. At home, 129 side effects were reported in 171 infusions (0.75 side effect per infusion); of these, 82 (64%) were reported to the nurse, of which 41 during the infusions and 41 afterwards; and 47 (36%) to the researcher when specifically asked for. All side effects were transient and resolved within minutes after lowering the ATP infusion rate.\nIn a total of 99 infusions, one or more side effects were reported by patients. In the 51 infusions with one or more side effects reported during ATP administration, the infusion was stopped in 13 infusions (25%), lowered in 30 infusions (59%) and not changed in 8 infusions (16%). Of the latter eight infusions, the side effects had already disappeared before lowering the infusion rate in four infusions, whereas in three infusions the side effects had already been present before the start of the ATP infusions; in one infusion, extravasation occurred, and the ATP infusion was temporarily interrupted. In 48 out of the total of 99 infusions with one or more side effects, the side effects were reported by patients to the nurse or researcher only after completion of the infusion, and the infusion rate was therefore not adapted.\nThe frequency of specific side effects observed during the ATP infusions is shown in Table\u00a02. Dyspnea was the most frequent side effect, appearing in 36 out of 266 infusions (14%), followed by chest discomfort (12%) and the urge to take a deep breath (11%). No symptoms of cardiac ischemia occurred in any of the infusions.\nTable\u00a02Side effects during a total of 266 intravenous ATP cycles in 51 patients; CTC-gradingNo side effectsSide effects according to CTC gradeTotal side effects1234CTC grade unspecifiedNumberPercent of inf. (%)Cardiac ischemia2660000000Chest discomfort2342614013212Dyspnea2303510003614Epistaxis2660000000Flushing2588000083Headache252112001145Injection side reaction2615000052Lightheadedness25492010125Mood alteration-anxiety2624000042Nausea251141000156Othera2352820013112Palpitations2651000010Sweating2602300162Urge to take a deep breath23818100002811Total16122414192In some courses more than one side effect was observedaThe side effects classified as \u2018Other\u2019 were cold shivering, feelings of coldness, pain in infusion arm, dry eyes, sleepiness, burning sensation of the throat, vomiting, diarrhea, pain neck, general discomfort and unspecified\nFrom the 192 side effects, one side effect (lightheadedness) occurred at the day care center and was scored by the nurse as CTC 4 because of prolonged stay (one night) at the hospital after the first infusion; there were no abnormalities on ECG. Four side effects were scored as CTC 3 (in all cases chest discomfort), and 22 side effects as CTC 2. Two CTC 3 side effects occurred at the day care center in two patients, with no abnormalities on ECG. Of these, one patient decided to stop with the ATP infusions. The other patient wanted to continue; for safety reasons, all subsequent infusions in this patient were given at the day care center. The remaining two CTC 3 side effects occurred at home: one in a patient during the normal procedure of stepwise increasing the infusion rate by the nurse according to protocol, in the other patient during constant ATP infusion after the nurse had left; in the latter case, the nurse was called by telephone by the patient or informal caregiver and immediately arrived. In both cases, the infusions were stopped immediately for that very day. The first patient decided to continue the ATP infusions; in this patient, all four subsequent infusions until the end of the study were given at the day care center at the highest ATP dose (50\u00a0\u03bcg kg\u22121 min\u22121), and no further side effects occurred. The second patient decided to stop the ATP infusions.\nThe following CTC 2 side effects were reported by patients:\nTo the nurse during the infusions, by seven patients in seven infusions:\nUrge to breathe deeply, headache, sweating and cold shivering in one patient at the day care center (the same patient with the CTC 3 chest discomfort and CTC 4 lightheadedness)Sweating in one patient at the day care center at the end of the infusion, due to flushing the infusion line (violation of the administration protocol)Pain in the infusion arm (one patient) and dyspnea (one patient), both at the day care centerUrge to breathe deeply, headache and sweating in one patient at home, during the period of stepwise increasing the infusion rate by the nurseChest discomfort (once) and lightheadedness (twice) at home in two patients, who called the home nurseOut of these seven patients with CTC 2 side effects, two patients decided to stop the ATP infusions for the rest of the study.To the nurse after completion of the infusion, by three patients (four infusions)To the researcher when specifically asked for, by one patient (five infusions)These nine CTC 2 side effects (eight times urge to breathe deeply, once nausea), were probably not sufficiently serious to call the home nurse. All of these patients continued the ATP infusions.\nTiming of side effects\nSide effects were not only present during the first infusion but also during subsequent infusions, even though the number of side effects per infusion appeared to be lower during the last three infusions (Fig.\u00a03). At the day care center, of the 55 side effects reported during the infusions, 28 (51%) were reported during MTD assessment and 27 side effects (49%) after MTD assessment had been completed (Fig.\u00a02). At home, 41 side effects were reported during the infusion, 22 side effects (54%) during MTD assessment, and 19 side effects (46%) after MTD assessment had been completed. In the latter 19 cases of side effects, the nurse (and once the general practitioner) was called by the patient and visited the patient to lower or stop the infusion rate.\nFig.\u00a03Average number of side effects per ATP infusion in subsequent infusions\nEffect of cardiac or lung disorders on MTD and frequency of side effects\nMean MTD was 48\u2009\u00b1\u20092 (mean\u2009\u00b1\u2009SEM)\u00a0\u03bcg kg\u22121 min\u22121 in patients without cardiac disorders and\/or lung cancer (Table\u00a03). In the presence of cardiac disorders mean MTD was 8\u00a0\u03bcg kg\u22121 min\u22121 lower (p\u2009<\u20090.001) and 4\u00a0\u03bcg kg\u22121 min\u22121 lower in the presence of lung cancer (p\u2009=\u20090.02). The mean number of side effects per infusion was 0.71\u2009\u00b1\u20090.28 in patients without cardiac disorders and\/or lung cancer. In the presence of cardiac disorders, the number of side effects was significantly higher: 2.25\u2009\u00b1\u20090.49 (p\u2009=\u20090.02). The presence of lung cancer had no significant impact on the frequency of side effects (0.66\u2009\u00b1\u20090.34). Neither the presence of cardiac disorders, nor the presence of lung cancer had a significant impact on the frequency of specific complaints (chest discomfort, urge to breathe deeply, nausea, lightheadedness, or dyspnea). However, in the presence of cardiac disorders, the number of other side effects was significantly higher (p\u2009=\u20090.006). The proportion of CTC grade 2\u20134 side effects in the presence of cardiac disorders and\/or lung cancer (21 CTC grades 2\u20134 out of 133 side effects, i.e. 16%) was similar to patients without such disorders (seven CTC grades 2\u20134 out of 59 side effects, i.e. 12%).\nTable\u00a03The independent effect of the presence of cardiac disorders and\/or lung cancer on MTD and frequency of side effectsMeanNo cardiac disorders and\/or lung cancerCardiac disordersLung cancerMTD48\u2009\u00b1\u2009240\u2009\u00b1\u20093***44\u2009\u00b1\u20092*All side effects0.71\u2009\u00b1\u20090.282.25\u2009\u00b1\u20090.49*0.66\u2009\u00b1\u20090.34Chest discomfort0.14\u2009\u00b1\u20090.050.32\u2009\u00b1\u20090.090.12\u2009\u00b1\u20090.06Urge to breathe deeply0.08\u2009\u00b1\u20090.050.25\u2009\u00b1\u20090.080.09\u2009\u00b1\u20090.05Nausea0.06\u2009\u00b1\u20090.040.18\u2009\u00b1\u20090.070.03\u2009\u00b1\u20090.05Lightheadedness0.06\u2009\u00b1\u20090.040.14\u2009\u00b1\u20090.080.06\u2009\u00b1\u20090.05Dyspnea0.12\u2009\u00b1\u20090.050.35\u2009\u00b1\u20090.090.17\u2009\u00b1\u20090.06Other0.09\u2009\u00b1\u20090.060.42\u2009\u00b1\u20090.11**0.05\u2009\u00b1\u20090.07Reference group: patients with no cardiac disorders and\/or lung cancer*p\u2009<\u20090.05**p\u2009<\u20090.01***p\u2009<\u20090.001\nContinuation of infusions after completion of the study\nOf the 24 patients who completed all eight ATP infusions, 12 decided to continue ATP administration (nine patients with lung cancer, one with prostate cancer, one with a malignant melanoma, one with liver cancer). Until the censoring date, these patients had received a total of 116 ATP infusions after completion of the regular 8-week study period (range 1\u201334 additional infusions). Of these 116 infusions, 11 infusions (10%) reached a dose of \u226420\u00a0\u03bcg kg\u22121 min\u22121, 72 infusions (62%) were given at a dose of 25\u201340\u00a0\u03bcg kg\u22121 min\u22121, and 33 (28%) reached a dose of 45\u201350\u00a0\u03bcg kg\u22121 min\u22121 ATP. A total of 38 side effects were reported over 116 infusions (0.33 side effects per infusion), a number significantly lower (p\u2009<\u20090.001) than in the first 8 infusions in the same 12 patients.\nDiscussion\nThe aim of this study was to investigate whether ATP can be safely administered to patients with pre-terminal cancer in the home setting. A total of 266 infusions were administered for 8\u201312\u00a0h. Although statistically significant changes in blood pressure and heart rate were observed, these were minor and clinically irrelevant, corroborating earlier results [19]. In total, 192 side effects were reported during the 266 ATP infusions, i.e. an average of 0.72 side effects per infusion. Most side effects were mild and transient, presumably due to the short plasma half-life of ATP and adenosine (0.6\u20131.5\u00a0s) [22].\nWe saw more side effects than in a previous study in 28 lung cancer patients by Agteresch et al. [19]. These authors observed 91 side effects during a total of 176 ATP cycles of 30\u00a0h (0.52 side effects per infusion), even though these infusions were administered for a longer period of time (30\u00a0h vs 8\u201310\u00a0h in our study). One possible explanation of this discrepancy may be the different procedures of data collection in these two studies. For safety reasons, the safety procedure adopted in the present study was that the researcher contacted the patients after each home infusion to ask for side effects, specifically naming a number of the most common potential side effects (based on side effects listed by Agteresch et al. [19]. Our results clearly demonstrate that this procedure lead to a higher frequency of reported side effects in our study: out of a total number of 192 side effects, 54 side effects (28%) were reported only afterwards to the researcher when specifically asked for. If these side effects (i.e. side effects which had not spontaneously been reported to the nurse during or after the infusion) are excluded, the frequency of the remaining side effects per infusion in the present study (138\/266; 0.52) is remarkably similar to the report by Agteresch et al. [19] (91\/176; 0.52), in which trained research nurses in a dedicated clinical research unit registered the side effects.\nIn both our study and the studies by Haskell et al. [4, 5] and Agteresch et al. [19], chest discomfort\/pain, dyspnea and the urge to take a deep breath were frequent side effects.\nResults showed that in 43 out of 51 infusions (84%) with side effects reported during ATP administration, the infusion rate was lowered or stopped according to protocol. Reasons for not lowering the infusion rate in eight infusions were the fact that in four infusions complaints disappeared automatically; in three infusions the reported complaints were already present before the start of the infusion; and in one infusion extravasation occurred with a temporary interruption of the ATP infusion.\nIn 48 infusions with side effects, side effects had only been reported after completion of the concerned ATP infusion and the infusion rate was therefore not adapted. One potential explanation for this finding may be that patients were eager to receive ATP at the highest possible dose, as some patients expressed their worry that the potential effectiveness of ATP could fade in case of lowering the dose (similar to chemotherapy), despite our explicit explanation to patients prior to the study that there is currently no evidence that the efficacy of ATP would be better at higher ATP doses. Another possible explanation is that patients in our study population were used to experiencing far more serious complaints caused by progression of the disease and\/or previous antitumor treatment.\nOne CTC 4 side effect, four CTC 3 side effects, and 22 CTC score 2 were observed. The CTC 4 side effect (see \u201cResults\u201d), and two CTC 3 side effects (chest discomfort) occurred at the day care center, with no abnormalities on ECG. The other two CTC 3 side effects occurred at home, one with the nurse still being present; and in the other case, the nurse immediately arrived.\nSide effects were not limited to the period of MTD assessment, nor were they limited to the first ATP infusion. The implication of this finding is that alertness with regard to potential side effects of ATP administration is necessary during all subsequent ATP infusions, and not just during the first infusion or MTD assessment.\nThe lower MTD found in the presence of cardiac disorders or lung cancer indicates that patients with these disorders tolerate lower doses of ATP without side effects than patients without these disorders. Moreover, even at the lower MTD, side effects tend to be more frequent in the presence of cardiac disorders. Because all patients with presence of COPD also had cardiac disorders or lung cancer, this subgroup was not analyzed separately.\nBased on our findings, we conclude that ATP administration at home is safe. First, the majority of infusions (63%) are without side effects, and second, our results confirm a previous study in lung cancer patients [19] showing that side effects that do appear are mild and transient, resolving within minutes after lowering the ATP infusion rate. Nevertheless, it remains possible that some side effects could be prevented by encouraging patients to report any side effects without delay. This emphasizes the need for careful instruction and facilitating patients reporting and monitoring of side effects when ATP infusions are administered at home. As patients with cardiac disorders and lung cancer appear to have a lower MTD and more side effects, these patients should be monitored with special care.","keyphrases":["atp","pre-terminal cancer","side effects","home-infusion"],"prmu":["P","P","P","U"]}
{"id":"Ann_Surg_Oncol-3-1-2077910","title":"Prophylactic Mastectomy in BRCA1\/2 Mutation Carriers and Women at Risk of Hereditary Breast Cancer: Long-Term Experiences at the Rotterdam Family Cancer Clinic\n","text":"Background BRCA1\/2 mutation carriers and women from a hereditary breast(\/ovarian) cancer family have a highly increased risk of developing breast cancer (BC). Prophylactic mastectomy (PM) results in the greatest BC risk reduction. Long-term data on the efficacy and sequels of PM are scarce.\nWomen with a germ-line BRCA1 or BRCA2 mutation as well as 50% risk carriers from a hereditary breast(\/ovarian) cancer (HB(O)C) family are at increased risk of developing breast and\/or ovarian cancer compared with the general population.1\u20134 Options to reduce this risk are regular surveillance, chemoprevention, or prophylactic surgery. Prophylactic surgery includes prophylactic mastectomy (PM) and\/or prophylactic bilateral salpingo-oophorectomy (PBSO). PM implies either a bilateral prophylactic mastectomy (BPM) in high-risk unaffected women as well as in high-risk women with a history of breast cancer (BC) previously treated with breast conserving therapy (BCT), or a contralateral prophylactic mastectomy (CPM) after a unilateral therapeutic mastectomy. Several studies have shown that PM strongly reduces the risk of developing (contralateral) breast cancer, while PBSO reduces the risk of ovarian as well as primary breast cancer.5\u201310 These strategies therefore have commonly been accepted at this moment as risk-reducing strategies for women being at increased risk of HB(O)C.\nPM, however, is a drastic and irreversible intervention, and in case of breast reconstruction (BR), is accompanied by a substantial complication rate.11 Further issues of concern with respect to PM include changes in a woman\u2019s body image and self-esteem, changes of sexual function, and in psychological distress.\nAt the Rotterdam Family Cancer Clinic, 35\u201351% of women carrying a BRCA1 or BRCA2 mutation opt for either bilateral or contralateral PM.12,13 For women who are contemplating this intervention, it is imperative to have reliable data on the outcomes of PM in a well-defined cohort to make a good informed decision and to minimize postoperative feelings of deception. At the Rotterdam Family Cancer Clinic there is ample and long-term experience with sufficient numbers of women undergoing PM. We previously reported data concerning the occurrence of breast cancer after BPM in unaffected women with a proven BRCA1\/2 mutation,5,14 complications of PM with breast reconstruction,11,15 and psychological aspects of PM in combination with BR.16\nIn the current analysis, we report on an extended series with longer follow-up of women having undergone a PM at the Rotterdam Family Cancer Clinic because of either a proven BRCA1\/2 mutation or a genetic susceptibility (50% risk carriers from a HB(O)C family). Our study sample was large enough to discriminate between unaffected women and women with a history of BC (affected). Special attention is paid to the prevalence of (pre)malignant lesions in prophylactically removed mastectomy specimens. Further, we report on the postoperative complications of PM in combination with breast reconstruction.\nPATIENTS AND METHODS\nAs of the start of the Rotterdam Family Cancer Clinic in 1991, PM and\/or PBSO are being discussed as risk-reducing strategies with women at increased risk of hereditary BC and\/or ovarian cancer. In early years, PM was discussed with BRCA1\/2 mutation carriers as well as with women from a HB(O)C family without a proven mutation (so-called 50% risk carriers), and applied for unaffected as well as affected (with a history of breast cancer) women. Due to the development of more advanced mutation-detection methods enabling the performance of a complete gene mutation screen, there has been a shift in more recent years to discuss the option of PM only with identified mutation carriers. Before 1996, the decision to undergo a PM and\/or PBSO was discussed individually by the doctor and the woman in question. As of 1996, women opting for either PM and\/or PBSO are additionally discussed in the multidisciplinary Committee on Hereditary Tumors. For this purpose, institutional guidelines concerning the surveillance schedule and indications regarding PM\/PBSO have been further elaborated and implemented as of 2000, which were updated as knowledge progressed and more evidence-based data became available.\nBefore 2000, no additional examinations were performed before PM, irrespective of the individual situation (unaffected\/affected; mutation\/50% risk carrier). Women were seen biannually for physical examination, while a mammography was performed annually. As of 2000, institutional guidelines from the working party on hereditary tumors recommended to perform clinical breast examination (CBE) and imaging examination within 3 months prior to PM, to minimize the risk of finding unexpected malignant changes at PM. At first, imaging examination consisted of either mammography or magnetic resonance imaging (MRI) scan, while more recently MRI has been preferred. Breast ultrasound (US) and, if necessary, fine-needle aspiration cytology (FNAC) are additionally performed in case lesions are found at CBE or one of the imaging examinations. Further, the guidelines recommend the discussion of the case in the multidisciplinary Committee on Hereditary Tumors and a standard visit with a psychologist. For affected women, the guidelines are extended with dissemination investigations to rule out recurrent or distant breast cancer activity (chest x-ray, liver ultrasound, bone scan, liver functions and determination of Ca15.3\/Ca125). Where women with a history of ovarian cancer were previously eligible for PM, at the moment this is not discussed anymore in this setting, because the prognosis is mainly dictated by the ovarian cancer. In the sample, these women were classified as \u201cunaffected,\u201d unless they also had a history of BC.\nTo evaluate the short-term and long-term medical effects of prophylactic surgery in high-risk women, a combined retrospective and prospective, longitudinal study was activated at our institution, including all genetically susceptible women who had opted for prophylactic surgery (either PM and\/or PBSO). Women were informed by oral and written information and were asked for written consent. The protocol was approved by the institutional review board (project EMC-DDHK 98-15).\nSurgical Technique\nAt our institute, the oncological and plastic surgeon perform the PM and BR as a team. During the operation, the patient is under general anesthesia in a half-supine position. A skin-sparing mastectomy is performed through a vertical, peri-areolar incision, which extends from just above the nipple down the submammary fold. The breast tissue, including the superficial fascia (creating thin skin flaps), the axillary tail, the inframammary fold, the nipple-areolar complex, and the fascia of the pectoral muscle are removed. In case of immediate breast reconstruction, either a subpectoral silicone implant is inserted in a pocket created below the pectoral muscles in a one-stage procedure, or autologeous tissue is used. Autologeous reconstruction encompasses a broad range of procedures incorporating the patient\u2019s own tissues to recreate the breast. The transverse rectus abdomis myocutaneous (TRAM) flap and latissimus dorsi flap are two standard myocutaneous flaps used for breast reconstruction. More recent modifications to the traditional techniques led to the use of the deep inferior epigastric perforator (DIEP) flap. Nipple reconstruction is offered after 6 months and consists of three small transposition flaps; the areola is mimicked by tattooing the desired skin color. Breast reconstruction is not always performed in the same operation as the mastectomy; the techniques for these delayed reconstructions, however, are as described previously.\nMicroscopic Examination of Mastectomy Specimens\nAs of 1995, a standard procedure has been followed for meticulous microscopic examination of prophylactically removed mastectomy specimens to rule out the presence of \u201coccult\u201d (microscopic) malignant alterations. The protocol prescribes that mastectomy specimens are cut into slices of 0.5\u20131 cm thickness, whereby each slice is carefully inspected and palpated for abnormalities. Standard, three randomly selected parenchymal tissue samples from each quadrant and a transverse section through the nipple are submitted for histology, in addition to samples of all visible or palpable abnormalities. Further, three samples from each quadrant of the mastectomy specimens are snap frozen for the tissue bank. Radiographic examination of breast tissue specimens is not performed on a routine basis.\nStudy Design\nThe current study included all women at increased risk of hereditary BC, according to previously described criteria,17 who underwent prophylactic bilateral or contralateral mastectomy between January 1, 1994 until December 31, 2004. Of our study cohort 310 women (86.6%) underwent PM at our clinic, while 48 women (13.4%) were treated elsewhere, e.g., due to a waiting list at our clinic, or the fact that previous surgery was performed elsewhere. The latter women were only eligible for this analysis if the follow-up after PM took place at our clinic, and a copy of the pathology report was available. In general, DNA testing was performed before the prophylactic surgery, although some women choose for prophylactic surgery without or irrespective of DNA testing. DNA analysis was performed according to standard procedures, as has been previously described.18,19 DNA testing was not an inclusion criterion for participation in the study. Proven noncarriers from a family BRCA mutation were excluded from the study.\nRelevant data were extracted from the hospital records. For each woman, including deceased women, the following information was obtained: date of birth, death, and PM, performance (yes\/no) and type of breast reconstruction, PBSO, diagnosis of breast and\/or ovarian cancer, mutation status, duration of follow-up after PM (end date being either the date of death or the date of last clinic visit in case of loss to follow-up, or the end date of this study, i.e. December 31, 2004), and type and number of complications after breast reconstruction. Regarding the latter, we distinguished between early (within 6 weeks) and late postoperative complications (after 6 weeks). Early complications consisted of infection, necrosis, bleeding, and luxation of the prosthesis. Late complications were divided in surgical complications (such as capsular formation, infection, necrosis, and luxation of the prosthesis), and complaints related to cosmetic outcome (such as poor symmetry and dog ears). Nipple reconstruction is regarded as part of the breast reconstruction and therefore has not been registered as a cosmetic complication. A computerized database (MS-Access) was used to process the data. Data were entered retrospectively as well as prospectively after each clinic visit.\nStatistical Analysis\nDescriptive statistics (median, range, and frequency) were computed. When appropriate, statistical significance testing between relevant subgroups was performed using the chi-square test for categorical variables and a t-test for continuous variables. A P value of less then .05 was considered statistically significant.\nRESULTS\nStudy Population\nIn Table\u00a01 the characteristics of the women who underwent a PM are shown. A total of 358 women, with a median follow-up after PM of 4.5 years, fulfilled the study eligibility criteria, consisting of 181 (50.6%) women with a history of breast cancer (affected women), and 177 (49.4%) women without a history of BC (unaffected women). A total of 236 (65.9%) women were BRCA1\/2 mutation carriers, while the other 122 women (34.1%) were 50% risk carriers from a HB(O)C family. The unaffected group mainly consisted of BRCA1\/2 mutation carriers as compared with 50% risk carriers (82% vs 18%, P < .001), whereas the affected group consisted of an equal number of mutation carriers and 50% risk carriers (91 mutation carriers versus 90 50% risk carriers, P = .94). This difference in distribution was highly significant (P < .001).\nTABLE\u00a01.Characteristics of the study populationHISTORY OF BCNo (Unaffected, n = 177)Yes (Affected, n = 181)P valuedBRCAHB(O)CBRCAHB(O)Cn(%)an(%)an(%)an(%)aNo. of women (N)145(82)b32(18)b91(50)c90(50)c<.001e\u00a0\u00a0Death due to cancer0(0)1(3)9(10)7(8)Age at PM (years)\u00a0\u00a0Median36.038.542.047.0<.001\u00a0\u00a0Range22\u20136528\u20135525\u20136526\u201368\u00a0\u00a0<30 years18(12)3(9)6(7)2(2)<.001\u00a0\u00a030\u201339 years74(51)14(44)26(29)22(24)\u00a0\u00a040\u201349 years36(25)10(31)38(42)31(34) \u00a0\u00a0\u226550 years17(12)5(16)21(23)35(39)Duration of follow-up (years)4.44.73.94.5Mutation status\u00a0\u00a0BRCA1115(79)\u201376(84)\u2013\u00a0\u00a0BRCA230(21)\u201315(16)\u2013PBSO\u00a0\u00a0Yes83(57)3(9)61(67)12(13).13\u00a0\u00a0No62(43)29(91)30(33)78(87)Age at PBSO (years)\u00a0\u00a0Median40.045.043.049.0<.01\u00a0\u00a0Range29\u20135735\u20134532\u20136537\u201358Timing of PBSO\u00a0\u00a0Before PM18(22)2(67)14(23)6(50).11\u00a0\u00a0At PM35(42)1(33)16(26)5(42)\u00a0\u00a0After PM30(36)0(0)31(51)1(8)Ovarian cancer before PM4(3)0(0)2(2)1(1)Unexpected (p)MF at PM3(2)2(6)4(4)1(1)Cancer after PM\u00a0\u00a0Breast cancer1(1)0(0)0(0)0(0)\u00a0\u00a0Ovarian cancer2(1)0(0)1(1)0(0)BC, breast cancer; HB(O)C, hereditary breast(\/ovarian) cancer; PM, prophylactic mastectomy; PBSO, prophylactic bilateral salpingo-oophorectomy; (p)MF, (pre)malignant findings.a Percentage of the number of women in column in question, unless stated otherwise.b Percentage of unaffected women.c Percentage of affected women.d Difference between unaffected and affected BRCA1\/2 mutation carriers, unless stated otherwise.e Difference in distribution between unaffected and affected women.\nThe median age at PM in the unaffected and affected group was significantly different, being 37 and 44 years, respectively (Table\u00a02, P < .001). In the affected group, mutation carriers were significantly younger at PM than 50% risk women (42 vs 47 years, respectively; P = .045). In the unaffected group, we found no significant difference in this respect (36 vs 38.5 years, respectively; P = .102). A history of ovarian cancer was present in four mutation carriers from the unaffected group (2.3%), and in three women (1.7%) in the affected group.\nTABLE\u00a02.Comparison between unaffected and affected womenUnaffectedAffectedP valueNo. of women (N)177 (%)181 (%)Age at PM (years)\u00a0\u00a0Median3744<.001\u00a0\u00a0Range22\u20136525\u201368\u00a0\u00a0<30 years21 (12) 8 (4)<.001\u00a0\u00a030\u201339 years88 (50)48 (27)\u00a0\u00a040\u201349 years46 (26)69 (38)\u00a0\u00a0\u226550 years22 (12)56 (31)PBSO\u00a0\u00a0Yes86 (49)73 (40).12\u00a0\u00a0No91 (51)108 (60)Age at PBSO (years)\u00a0\u00a0Median4044<.001\u00a0\u00a0Range29\u20135732\u201365PM, prophylactic mastectomy; PBSO, prophylactic bilateral salpingo-oophorectomy.\nIn Fig.\u00a01, the number of PMs per year, separately for unaffected\/affected BRCA mutation carriers, as well as for unaffected\/affected 50% risk carriers is shown. In the unaffected group, the annual number of women undergoing PM was always larger in mutation carriers compared with 50% risk carriers, except for the first 2 years (1994\/1995). Further, the number of unaffected BRCA mutation carriers undergoing PM widely differed over the years, with two peaks in 1998 and 2001, respectively. In contrast, the number of unaffected 50% risk carriers undergoing PM was quite stable over the years.\nFIG.\u00a01.Annual number of PM in unaffected\/affected BRCAa\/2 mutation carriers and 0% risk carriers from HB(O)C families.\nIn the affected group, however, the pattern was different, whereby a shift has taken place through the years. In the early years of the study period (1994\u20131997), mainly women without a proven mutation underwent PM in this group. Between 1997 and 2001, approximately as many mutation carriers as 50% risk carriers underwent a PM. As of 2002, more mutation carriers have undergone a PM, although the number of PMs in 2004 again was not different between mutation and 50% risk carriers.\nProphylactic Bilateral Salpingo-oophorectomy\nA considerable part of the BRCA mutation carriers undergoing PM also opted for PBSO, being 57% in the unaffected versus 67% in the affected group, respectively (Table\u00a01, P = .13). In the HB(O)C group, however, only a minority of the women underwent a PBSO, being 15 of 122 50% risk carriers (12%). The median age at PBSO was younger in BRCA mutation carriers compared with the 50% risk carriers, both in the affected and the unaffected group. This difference, however, was not significant (P = .13 and P = .40, respectively). Further, the median age at PBSO was lower in the unaffected compared with the affected group, being 40 versus 44 years, respectively (Table\u00a02, P < .001). In addition, unaffected BRCA mutation carriers underwent PBSO at a younger age compared with mutation carriers with a history of BC (40 vs 43 years, respectively, P < .01, Table\u00a01).\n(Pre)malignant Findings at PM\nIn 10 of the 358 women (2.8%), abnormal findings were unexpectedly found in the mastectomy specimens (table\u00a01). Prior to intended PM there was no suspicion to justify an axillary nodal dissection in combination with the PM procedure. This occurred in five \u201cunaffected\u201d (2.8 %) as well as in five previously affected women (2.8%), and in both mutation carriers (3%) and 50% risk carriers (2.5%).\nThe characteristics of the unexpected (pre)malignant findings as well as the preoperative screening results are chronologically described in detail in Table\u00a03. In 1995, in one woman both preoperative clinical breast examination (CBE) and mammography were suspicious for a malignancy. However, additional investigation, consisting of ultrasound and cytology, did not reveal a malignancy. Nevertheless, histological examination of the mastectomy specimens revealed an invasive ductal carcinoma (IDC), eventually staged as a pTxN1Mo. The patient died of metastatic breast cancer 4 years after the PM. In another two women, undergoing PM in 1996 and 1997, a lesion was found preoperatively (CBE and mammography, respectively) and classified as probably benign. No malignant abnormalities were seen at subsequent ultrasound examination, which is the reason a FNAC was not performed. However, histological examination of the mastectomy specimens revealed a small ductal carcinoma in situ (DCIS) in both women. In 2002, in one woman preoperative MRI revealed a lesion classified as probably benign. Indeed, no malignant abnormalities were found at additional ultrasound examination. Histological examination of the mastectomy specimens, however, revealed an IDC in the right, and an invasive medullar carcinoma (IMC) in the left breast. Preoperative screening in the remaining six women, performed 1\u20136 months preceding PM, did not show suspicious abnormalities. Still, another invasive carcinoma, three cases of DCIS, and two cases of lobular carcinoma in situ (LCIS) were found in the mastectomy specimens. All unexpected (pre)malignant findings in the affected women were found in the contralateral breast.\nTABLE\u00a03.Characteristics of unexpected (pre)malignant findings in the PM specimensPreoperativeYear of PMGenetic risk groupHistory of BC before PMHistologyGradeTumor size (mm)ER\/PR statusCBEMxMRI1995HB(O)CNoIDCNANANASCSCa, b1996HB(O)CNoDCISII<2NAPBnla1996BRCA1NoLCISNANANAnlnl1997HB(O)CYesDCISINANAnlc1997BRCA1YesDCISII<2NAnlPBa1997BRCA1YesIDCII3Negativenlc1998BRCA1YesDCISNA<2NAnlnl2000BRCA1YesLCISNANANAnlnl2002BRCA1NoIDCIII5NegativenlnlPBaIMCIII6Positive2003BRCA2NoDCISII<2NAnlnlPM, prophylactic mastectomy; BC, breast cancer; DCIS, ductal carcinoma in situ; LCIS, lobular carcinoma in situ; IDC, invasive ductal carcinoma; IMC, invasive medullar carcinoma; CBE, clinical breast examination; Mx, mammography; MRI, magnetic resonance imaging; SC, suspicion of cancer; PB, probably benign; nl, normal; NA, not applicable.a Additional investigation normal.b Macroscopic palpable tumor; microscopic no clear border, therefore tumor size and grade not determinable.c Treated previously for BC at another hospital. No information about preoperative imaging available.\nCancer During Follow-up After PM\nAfter PM, no incident breast cancer cases were observed in 50% risk carriers.\nIn BRCA mutation carriers, ovarian cancer was detected in two women in the unaffected group, and in one woman in the affected group (Table\u00a01).\nOne BRCA1 mutation carrier from the unaffected group presented in 2001, 3.5 years after PM (no malignant findings at histological examination), with metastatic adenocarcinoma in an axillary lymph node, morphologically and immunohistochemically consistent with breast cancer. Additional dissemination examinations also revealed metastases in bone and liver. Review of the preoperative data (physical examination and mammography) at the presentation of metastatic disease did not show malignant alterations, while meticulous reexamination of all pathology slides and additional investigation of frozen tissue material did not detect a primary breast cancer.\nReconstructive Breast Surgery\nIn Table\u00a04, the numbers and types of reconstructive breast surgery, as well as the numbers and types of complications after breast reconstruction (BR) are shown. Of the total population (n = 358), 276 women underwent BR in combination with PM, being 60% unaffected and 40% affected women. The presence or absence of a BRCA1\/2 mutation did not influence the BR rate (P = .23 for unaffected, and P = .10 for affected women), or the type of BR (P = .25 for unaffected, and P = .68 for affected women). Unaffected women mainly opted for BR (94%), consisting of 163 immediate and three delayed reconstructions, while a considerable part of the affected women did not opt for BR (37%) (P < .001). Further, unaffected women mainly opted for BR by means of (silicon) prosthesis (96%). In contrast, affected women opting for BR (102 immediate, and 8 delayed) more often had a reconstruction by means of autologeous tissue compared with unaffected women, being 13% versus 3%, respectively (P = .004).\nTABLE\u00a04.Breast reconstruction (BR) in women undergoing PMHistory of BCNo (Unaffected)Yes (Affected)P valueNumber of women177(%)181(%)Breast reconstruction (BR)\u00a0\u00a0No9(5)68(37)<.001\u00a0\u00a0Yes166(94)110(61)\u00a0\u00a0Unknown a2(1)3(2)Type of BR\u00a0\u00a0Silicon prosthesis159(96)95(86).004\u00a0\u00a0Autologeous tissue6(3)14(13)\u00a0\u00a0Unknown a1(1)1(1)Therapy first BC b\u00a0\u00a0BCT\u2013\u20137(6)\u00a0\u00a0BCT\/RT\u2013\u201333(30)\u00a0\u00a0MAST\u2013\u201367(67)\u00a0\u00a0MAST\/RT\u2013\u20133(3)Women with complications after BR\u00a0\u00a0No84(51)55(50).92\u00a0\u00a0Yes82(49)55(50)Total number of complications after BR c12788\u00a0\u00a0Early (<6 weeks after BR)42(33)31(35).74\u00a0\u00a0Late (>6 weeks after BR)85(67)57(65)Surgery due to late complication\u00a0\u00a0No38(30)24(27).67\u00a0\u00a0Yes89(70)64(73)Early complicationsSurgery due to early complication\u00a0\u00a0No27(64)17(55).42\u00a0\u00a0Yes15(36)14(45)Type of early complication Infection8(19)14(45).09 Necrosis11(26)4(13) Bleeding20(48)12(39) Prosthesis luxation2(5)0(0) Poor arterial inflow0(0)1(3) Pneumothorax1(2)0(0)Late complicationsSurgery due to late complication\u00a0\u00a0No11(13)7(12).91\u00a0\u00a0Yes74(87)50(88)Type of late complication\u00a0\u00a0Infection4(4)0(0).33\u00a0\u00a0Necrosis1(1)3(5)\u00a0\u00a0Capsular formation31(37)20(35)\u00a0\u00a0Prosthesis luxation2(2)3(5)\u00a0\u00a0Poor cosmetic appearance d31(37)19(34)\u00a0\u00a0Dog ear16(19)12(21)PM, prophylactic mastectomy; BC, breast cancer; BR, breast reconstruction; BCT, breast conserving therapy; BCT\/RT, BCT in combination with radiotherapy; MAST, therapeutic mastectomy; MAST\/RT, MAST in combination with radiotherapy.a Surgery performed at another hospital, not included in P value calculation.b Data shown for women with a BR after PM.c One woman can have \u22651 complication.dIncluding asymmetry.\nIn 137 of 276 women opting for BR (49.6%) one or more complications were registered, totaling 215 complications. Surgical reinterventions were performed in 153 cases (124 for late complications).\nConcerning the number of complications, this was not different between unaffected and affected women, neither for the moment of the complication (i.e., early or late) (P = .74), the necessity of reintervention due to the complication (P = .67), nor for the type of complication (P = .09 for early complications and P = .33 for late complications).\nDISCUSSION\nIn this study we updated and extended the long-term experiences at the Rotterdam Family Cancer Clinic with prophylactic mastectomy (PM) in proven BRCA1\/2 mutation carriers and in 50% risk carriers from a HB(O)C family. We compared the data of PM and breast reconstruction (if performed) in women with (affected) and without a personal history of BC (unaffected women) and further distinguished between women with a BRCA 1 or 2 mutation, and women without a proven mutation. While within the unaffected group, especially BRCA1\/2 mutation carriers opted for PM, we observed that within the affected group an equal number of mutation carriers and 50% risk carriers from a HB(O)C family opted for PM. Women carrying a BRCA 1 or 2 mutation are known to have an increased risk of developing contralateral primary BC,20,21 which is even more apparent among women who are younger when diagnosed with a primary breast carcinoma (age <50 years).22,23 For high-risk women without a proven mutation inconsistent results on the risk of developing a contralateral breast cancer (CBC) were reported. Shahedi et al.24 as well as Kirova et al.25 reported an increased risk of developing CBC in non-BRCA1\/2 women, while Tilanus et al.26 concluded that the rate of CBC was only slightly and insignificantly increased in non-BRCA1\/2 compared with sporadic breast cancer patients. In view of these results, one expects that after a first diagnosis of breast cancer, especially mutation carriers will opt for prophylactic removal of the remaining breast tissue. In our study sample, however, also a considerable part of the women without a proven BRCA1\/2 mutation opts for prophylactic mastectomy, especially after a history of breast cancer. It has to be mentioned that, since information on the BRCA1\/2 mutation status is not always known in the latter group and genetic testing is missing a number of mutations, it is likely that some of these patients are in fact mutation carriers. Further, the group of women with a history of breast cancer, with and without a gene mutation, may partly consist of women who initially chose for surveillance, and eventually opted for PM after the diagnosis of breast cancer. This is in accordance with an earlier report indicating that women may be more likely to undergo PM after a previous diagnosis of BC.27 This might also partly explain the higher age at the time of PM in the group with a history of BC.\nWe found that the age at PM was younger in unaffected women, both for mutation carriers as for 50% risk carriers. Further, we found a significant difference between unaffected and affected mutation carriers in the distribution of the numbers of PM over the various age categories, with the highest numbers of PM in the age group of 30\u201340 for unaffected and 40\u201350 for affected carriers, respectively. Moreover, the distribution of PM over the various age categories remained completely identical to the age distribution reported in a previous study on PM from our institute,5 indicating consistency over time.\nOur data show that, despite preoperative (imaging) examination, the presence of unexpected microscopic (pre)malignant findings in this group of high-risk women is real (3%). Other studies reporting on high-risk and\/or pathologic findings in prophylactically removed breast tissues, described percentages varying from 0.1\u201357%.8,27\u201329 However, the comparison of frequencies of unexpected (pre)malignant findings between studies is hampered by differences in population selection, preoperative screening methods, pathological examination of the specimens, and definition of what is considered (pre)malignant [e.g., lobular carcinoma in situ (LCIS)]. The percentage of unexpected invasive carcinomas in these studies ranged from 0.1\u20137.7% (0.8% in our study). Most of these studies, however, did not provide information about the outcome of preoperative physical breast or imaging examination, which at the moment is a standard procedure at our institution. It might be that since the implementation of institutional guidelines concerning preoperative breast examination in 2000, and the introduction of magnetic resonance imaging (MRI), being more sensitive in detecting carcinomas in high-risk women,30,31 as detection tool, the number of unexpected malignant findings in the PM specimens is decreasing.\nWith a 3% incidence of unexpected microscopic (pre)malignant findings, the potential role of sentinel node biopsy (SN) for all patients undergoing PM has been discussed. However, the majority of the (pre)malignant findings we found in this series, represents DCIS\/LCIS; settings for which a sentinel node biopsy is not standardly indicated. Invasive cancer was found in only 0.8% of the patients in this series. Therefore, in our opinion, routine use of SN in all patients undergoing PM is not warranted, which is also supported in the paper by Boughey et al.32\nA previous study from our institution, investigating the efficacy of PM in unaffected women with a proven BRCA1\/2 mutation, observed no cases of breast cancer after PM.5 The mean follow-up in that study was 3 years. In the current cohort, one BRCA1 mutation carrier presented with metastatic disease 3.5 years after PM (no primary BC found), suggesting the presence of an occult primary tumor that was never found, despite a thorough reexamination of the specimen at the presentation of the metastatic disease. This finding emphasizes the fact that despite thorough examination of the mastectomy specimens, the presence of an occult breast cancer cannot be ruled out completely and indicates that a form of surveillance after PM might be relevant.\nThe number of reconstructions after risk-reducing mastectomy was lower in the affected group. This may be due to the fact that BR after previous radiotherapy and\/or therapeutic mastectomy not always leads to satisfactory cosmetic results.15,33 Some patients abandon, in consultation with and\/or at the advice of their (oncological\/plastic) surgeon, from BR for this reason. Other women have accepted the mutilation\/alteration of body image caused by mastectomy, are reluctant to undergo renewed surgery, and prefer the use of external prosthesis.\nDuring the follow-up period of this study, 49.6% of the women with immediate or delayed BR after PM showed complications. In total 215 complications were registered, leading to surgical reintervention in 153 cases. These findings are consistent with several other reports,34,35 though there are also studies reporting lower,11,15,33 or even higher36 complication rates after (immediate) BR. However, the literature in this area is difficult to compare, in part because not all previous series compare bilateral prophylactic mastectomy in unaffected women with risk-reducing mastectomies in women after a previous therapy for breast cancer. Furthermore, data may not be comparable because of different definitions of complications. Moreover, some studies describe the complication rate as a percentage of the total number of reconstructions,11,15,33 while others, like our study, present the percentage of women with complications.34\u201336\nWe found no differences in the numbers of complications after (immediate) breast reconstruction in unaffected women compared with previously affected women in this study. This finding appears to be in contrast with earlier reports (also from our institution) describing the occurrence of more complications after mastectomy followed by (immediate) breast reconstruction in affected women. These studies report negative effects of preoperative radiotherapy on the cosmetic outcome of the reconstruction, in particular the risk of capsular formation would be increased, having negative consequences on the symmetry of the breasts. Further, asymmetry can be expected to occur more often after previous therapeutic mastectomy. Although we have no explanation for our findings, it is possible that the experience of the surgeons at our institution is important. Indeed, where previously BR by means of silicon prosthesis after breast conserving therapy was performed, this is not done anymore.\nIn summary, we confirmed our previous findings that prophylactic mastectomy strongly reduces the risk of developing breast cancer in both BRCA1\/2 mutation carriers and 50% risk carriers. As the frequency of unexpected cancers in this high-risk group remains real, preoperative imaging and careful histological examination is warranted. Further, we found a substantial complication rate after breast reconstruction, which mainly concerned late cosmetic issues, almost always leading to additional surgery. In this respect, patients should be informed preoperatively that an optimal cosmetic effect cannot unconditionally be achieved in just one single operation. Concerning the complication rate after BR, we did not find a significant difference between affected and unaffected women. In our opinion, our data are providing additional data on this issue and may help to inform women considering prophylactic mastectomy and their physicians, in the complex process of decision-making.","keyphrases":["mastectomy","brca1\/2","breast cancer","complications","prevention","unexpected carcinomas"],"prmu":["P","P","P","P","U","R"]}
{"id":"Purinergic_Signal-4-2-2377326","title":"Oxygen-glucose deprivation induces ATP release via maxi-anion channels in astrocytes\n","text":"ATP represents a major gliotransmitter that serves as a signaling molecule for the cross talk between glial and neuronal cells. ATP has been shown to be released by astrocytes in response to a number of stimuli under nonischemic conditions. In this study, using a luciferin-luciferase assay, we found that mouse astrocytes in primary culture also exhibit massive release of ATP in response to ischemic stress mimicked by oxygen-glucose deprivation (OGD). Using a biosensor technique, the local ATP concentration at the surface of single astrocytes was found to increase to around 4 \u03bcM. The OGD-induced ATP release was inhibited by Gd3+ and arachidonic acid but not by blockers of volume-sensitive outwardly rectifying Cl\u2212 channels, cystic fibrosis transmembrane conductance regulator (CFTR), multidrug resistance-related protein (MRP), connexin or pannexin hemichannels, P2X7 receptors, and exocytotic vesicular transport. In cell-attached patches on single astrocytes, OGD caused activation of maxi-anion channels that were sensitive to Gd3+ and arachidonic acid. The channel was found to be permeable to ATP4\u2212 with a permeability ratio of PATP\/PCl = 0.11. Thus, it is concluded that ischemic stress induces ATP release from astrocytes and that the maxi-anion channel may serve as a major ATP-releasing pathway under ischemic conditions.\nIntroduction\nAstrocytes do not merely support, but actively regulate, neuronal functions by forming highly elaborate networks with other astrocytes and neurons [1\u20134]. Astrocytes communicate with neurons by releasing extracellular signaling molecules such as ATP and glutamate, while they communicate with each other via gap junctions, in addition to these gliotransmitters. Astrocytic ATP release has been shown to be induced in response to receptor stimulation by UTP [5], glutamate [6], and noradrenaline [7], to mechanical stimulation [8\u201310], to osmotic swelling [11], and to exposure to low Ca2+ solution [8, 9, 12]. Although ischemic stress is known to induce ATP release from rat hippocampal slices [13], it is not known whether astrocytes respond to ischemia with ATP release. The first purpose of the present study was to address this question.\nAstrocytes express maxi-anion channels with a large single-channel conductance of 200\u2013400\u00a0pS [14\u201316]. Recently, we have demonstrated that astrocytic maxi-anion channels are activated by chemical ischemia and serve as one of the major pathways for glutamate release [17]. The maxi-anion channel was found to possess a pore with a size large enough to permeate ATP [18] and was found to actually conduct anionic forms of ATP in mammary C127 cells under hypotonic conditions [19], in kidney macula densa cells in response to increased luminal NaCl concentrations [20], and in cardiac myocytes under hypotonic or ischemic conditions [21]. Thus, there is a possibility that maxi-anion channels serve as one of the pathways for ATP release from astrocytes under ischemic conditions. The second purpose of this study was to test this possibility.\nMaterials and methods\nCells\nAstrocytes were obtained from neonatal mouse brain cortex and cultured, as previously described [17, 22], in Eagle\u2019s MEM supplemented with L-glutamine, 100\u00a0U\/ml penicillin, 0.1\u00a0mg\/ml streptomycin, 10% FBS, and 2.2\u00a0mg\/ml NaHCO3.\nHEK293 cells stably transfected with recombinant P2X2 purinergic receptors were prepared and cultured, as described elsewhere [23]. The HEK-P2X2 cells were used after dissociation into single cells and culturing in suspension for over 10\u00a0min.\nSolutions and chemicals\nThe standard Ringer solution contained (mM): 135 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 5 Na-HEPES, 6 HEPES, and 5 glucose (pH 7.4, 290\u00a0mosmol\/kg H2O). For inside-out experiments, we used standard Ringer solution both in the bath and in the pipette. The pipette solution for biosensor whole-cell experiments contained (mM): 125 CsCl, 2 CaCl2, 1 MgCl2, 10 EGTA, and 5 HEPES (pCa 7.6, 275\u00a0mosmol\/kg H2O, pH 7.4 adjusted with CsOH). For ATP conductance measurements in the inside-out configuration, the bath solution was substituted with a solution of 100\u00a0mM Na4ATP.\nOxygen-glucose deprivation (OGD) stress was applied by switching the perfusate from the standard Ringer solution to the OGD solution. The OGD solution was prepared by replacing D-glucose in Ringer solution with 2-deoxyglucose and was continuously bubbled with 100% argon gas for more than 1\u00a0h before and during experiments. The oxygen concentration (pO2) measured using an oxygen sensor (LICOX A3R, GMS, Kiel-Mielkendorf, Germany) was 118.4\u2009\u00b1\u20091.1\u00a0mmHg (n\u2009=\u20098) for standard Ringer solution exposed to air in the experimental perfusion chamber. Under OGD conditions, the oxygen concentration in the perfusate solution decreased to a stable value of 7.5\u2009\u00b1\u20090.6\u00a0mmHg (n\u2009=\u20099) at the inlet and 40.1\u2009\u00b1\u20091.8\u00a0mmHg (n\u2009=\u20098) at the outlet of the experimental patch-clamp chamber.\nGdCl3 was stored as a 50-mM stock solution in water and added directly to solutions immediately before each experiment. Arachidonic acid, phloretin, brefeldin A (BFA), 1-octanol, probenecid, glibenclamide, and apyrase were purchased from Sigma-Aldrich (St. Louis, MO, USA) and BAPTA-AM from DOJINDO (Kumamoto, Japan); they were added to solutions immediately before use from stock solutions in DMSO. DMSO did not have any effect when added alone at concentrations employed in the present study (less than 0.1%).\nOsmolality of all solutions was measured using a freezing-point depression osmometer (OM802, Vogel, Kevelaer, Germany).\nLuciferin-luciferase ATP assay\nThe bulk extracellular ATP concentration was measured by a luciferin-luciferase assay (ATP Luminescence Kit; AF-2L1, DKK-TOA, Tokyo, Japan) with an ATP analyzer (AF-100, DKK-TOA, Tokyo, Japan), as previously described [19, 21, 24], using astrocytes cultured in 12- or 24-well plates. After the culture medium was totally replaced with isotonic Ringer solution (1,000 and 425\u00a0\u03bcl for 12- and 24-well plates, respectively), cells were incubated for 60\u00a0min at 37\u00b0C. An aliquot (100\u00a0\u03bcl) of the extracellular solution was collected as a control sample for background ATP release. An OGD challenge was then applied by gently removing most of the remaining extracellular solution (875 and 300\u00a0\u03bcl for 12- and 24-well plates, respectively) and adding the OGD solution (1,000 and 400\u00a0\u03bcl for 12- and 24-well plates, respectively), and then placing the plates in an airtight incubator where normal air was replaced with 100% argon. At the specified time points, the plate was carefully rocked again to ensure homogeneity of the extracellular solution, and samples (20 and 50\u00a0\u03bcl for 12- and 24-well plates, respectively) were collected from each well for the luminometric ATP assay. The ATP concentration was measured by mixing 20 or 50\u00a0\u03bcl of sample solution with 530 or 500\u00a0\u03bcl normal Ringer solution and 50\u00a0\u03bcl of luciferin-luciferase reagent. At this ratio, the ionic salt sensitivity of the luciferase reaction was negligible. When required, drugs were added to the OGD solution to give the final concentrations as indicated. Since Gd3+ is known to interfere with the luciferin-luciferase reaction [25], we supplemented the luciferin-luciferase assay mixture with 600\u00a0\u03bcM EDTA when the samples contained Gd3+. Other drugs employed in the present study had no significant effect on the luciferin-luciferase reaction.\nBiosensor ATP measurements\nTo measure the local concentration of ATP released from single astrocytes, the biosensor technique was employed, as described previously [23]. ATP-dependent changes in whole-cell P2X2 receptor currents were recorded from a biosensor HEK-P2X2 cell at a holding potential of \u221250\u00a0mV. Before the actual measurements of ATP release, a calibration curve was constructed by recording the biosensor responses to known concentrations of ATP. The biosensor cell in the whole-cell configuration was lifted by means of a micromanipulator and positioned near a handmade local microperfusion device consisting of several inlet tubes. Each of the inlet tubes was filled with a different concentration of ATP solution ranging from 1 to 20\u00a0\u03bcM. To minimize the effect of desensitization, the sensor cell was exposed to ATP puffed every 3\u00a0min for 1\u00a0s to a maximum of five applications. During the measurements of ATP from an individual astrocyte under OGD stress, the biosensor cell was gently touched to the surface of the target cell. From the recorded currents, the concentration of ATP was calculated according to the calibration curve.\nPatch-clamp experiments and data analysis\nPatch-clamp experiments were performed, as described previously [17, 19], using an EPC-9 patch-clamp system (HEKA Electronics, Lambrecht\/Pfalz, Germany). The membrane potential was controlled by shifting the pipette potential (Vp). Currents were sampled at 3\u00a0kHz and filtered at 1\u00a0kHz. Data acquisition and analysis were done using Pulse+PulseFit (HEKA Electronics, Lambrecht\/Pfalz, Germany). Whenever the bath Cl\u2212 concentration was altered, a salt bridge containing 3 M KCl in 2% agarose was used to minimize variations of the bath electrode potential. Liquid junction potentials were corrected online. All experiments were performed at room temperature (23\u201325\u00b0C).\nSingle-channel amplitudes were measured by manually placing a cursor at the open and closed channel levels. Background currents were subtracted and the mean patch currents were measured at the beginning (first 25\u201330\u00a0ms) of current traces in order to minimize the contribution of voltage-dependent current inactivation. The reversal potentials were calculated by fitting I\u2013V curves to a second-order polynomial [18] or measured directly from ramp currents. Permeability ratios were calculated, as described previously [19, 21]. Data were analyzed in Origin 6 or 7 (OriginLab Corporation, Northampton, MA, USA). Pooled data are given as means \u00b1 SEM of n observations.\nStatistical differences of the data were evaluated by analysis of variance (ANOVA) and the paired or unpaired Student\u2019s t-test where appropriate and were considered significant at P\u2009<\u20090.05.\nResults\nOGD induces massive release of ATP from astrocytes\nUnder control conditions, when astrocytes were incubated in standard Ringer solution, the basal release of ATP was very low and did not exceed 0.25\u00a0nM over 40\u00a0min incubation time (Fig.\u00a01a: open circles). In contrast, when cells were transferred to OGD conditions, the bulk ATP in the incubation medium rapidly increased from the basal level to a peak level of approximately 2\u00a0nM within 15\u201320\u00a0min. After this period, the extracellular ATP concentration remained high up to 30\u00a0min of incubation and then slightly decreased at 40\u00a0min of incubation under OGD stress (Fig.\u00a01a: filled circles).\nFig.\u00a01OGD-induced ATP release from astrocytes detected by the luciferin-luciferase assay. a Time course of ATP release during incubation with control standard Ringer solution (open circles) and with the OGD solution (filled circles). b Pharmacological profiles of ATP release measured 20\u00a0min after application of OGD solution. Each data point or column represents the mean \u00b1 SEM (vertical bar). *Significantly different from the control OGD value in the absence of drugs at P\u2009<\u20090.05\nIn the following pharmacological experiments, we used an incubation time point of 20\u00a0min, when the net ATP release induced by OGD reached a steady-state level. Application of extracellular 50\u00a0\u03bcM Gd3+ nearly completely inhibited OGD-induced ATP release from cultured mouse astrocytes. Arachidonic acid (20\u00a0\u03bcM) also significantly inhibited OGD-induced ATP release. In contrast, OGD-induced ATP release was not significantly affected by blockers of the volume-sensitive outwardly rectifying (VSOR) Cl\u2212 channel, phloretin (100\u00a0\u03bcM) and glibenclamide (200\u00a0\u03bcM), of the CFTR Cl\u2212 channel, glibenclamide (200\u00a0\u03bcM), and of the transporter MRP, probenecid (1\u00a0mM). Also, the ATP release was not sensitive to inhibitors of connexin or pannexin hemichannels, 1-octanol (2\u00a0mM) and carbenoxolone (100\u00a0\u03bcM), and a blocker of the P2X7 receptor, brilliant blue G (1\u00a0\u03bcM). Preincubation of the cells with an inhibitor of vesicular transport, brefeldin A (BFA, 5\u00a0\u03bcM), for 120\u00a0min and together with an intracellular Ca2+ chelator, BAPTA-AM (5\u00a0\u03bcM), for 30\u00a0min had no significant effect on the OGD-induced release of ATP (Fig.\u00a01b).\nIn order to detect ATP release at the surface of single astrocytes, we employed a biosensor ATP assay technique [23]. We first established the calibration curve by recording the responses of biosensor HEK-P2X2 cells to different known concentrations of ATP (Fig.\u00a02a). We then gently put the biosensor cell close to the surface of a target astrocyte and recorded the whole-cell current responses from the biosensor cell. As shown in Fig.\u00a02b (top panel), virtually no ATP-evoked currents were observed during perfusion of standard Ringer solution. However, inward current spikes were consistently recorded upon perfusion with OGD solution after a lag period of 7.2\u2009\u00b1\u20091.0\u00a0min (n\u2009=\u20095) (Fig.\u00a02b: middle panel). The OGD-induced inward current responses were eliminated by application of an ATP-hydrolyzing enzyme, apyrase (Fig.\u00a02b: bottom panel). OGD-induced responses were never observed in biosensor cells alone which were positioned remote from target astrocytes. Using the calibration curve, the local ATP concentration was estimated to be 3.7\u2009\u00b1\u20090.1\u00a0\u03bcM at the surface of single astrocytes subjected to OGD stress.\nFig.\u00a02Cell surface ATP release induced by OGD from single astrocytes. a Calibration of ATP measurements by the biosensor technique. Top panel: representative current responses evoked by puff applications of 1\u201320\u00a0\u03bcM ATP in a biosensor HEK-P2X2 cell under whole-cell clamp. Bottom panel: concentration-response curve of the ATP-induced currents recorded from biosensor cells. Each data point represents the mean \u00b1 SEM (vertical bar). b Representative current traces recorded from biosensor cells in close proximity to target astrocytes after perfusion with standard Ringer solution (top panel) or with the OGD solution in the absence (middle panel) or presence (bottompanel) of 0.1\u00a0mg\/ml apyrase, which was applied after current responses appeared. When apyrase was applied from the beginning of OGD application, no current response was observed in biosensor cells positioned next to astrocytes (data not shown)\nFrom these data, it is concluded that in response to OGD stress, Gd3+- and arachidonic acid-sensitive ATP release from astrocytes is induced, increasing the extracellular ATP concentration to a level high enough to stimulate most types of purinergic receptors.\nOGD activates ATP-conductive maxi-anion channels in astrocytes\nThe fact that both Gd3+ and arachidonic acid are potent blockers of maxi-anion channels in mouse astrocytes [17] suggests that OGD-induced ATP release involves the activity of maxi-anion channels. In cell-attached experiments, OGD stress actually induced activation of anion channels with a large conductance (Fig.\u00a03a), as seen to occur in a similar manner upon stimulation by hypotonicity or chemical ischemia in mouse astrocytes [17]. The unitary I\u2013V relationship for these events exhibited slight outward rectification, and the mean slope conductance was 427\u2009\u00b1\u200923\u00a0pS at +60\u00a0mV and 262\u2009\u00b1\u200921\u00a0pS at \u221250\u00a0mV (Fig.\u00a03b). In cell-attached patches, OGD-induced activation of the maxi-anion channel started with a lag time of several minutes and reached its peak within 10\u00a0min (8.8\u2009\u00b1\u20091.2\u00a0min, n\u2009=\u20096), as shown in Fig.\u00a03c.\nFig.\u00a03OGD-induced single-channel activity of the maxi-anion channel in cell-attached patches on astrocytes. a Representative current trace recorded during application of alternating pulses (the protocol shown in inset above). Note that voltage-dependent inactivation of channel activity took place shortly after application of step pulses to \u00b1 50\u00a0mV. b Current-voltage (I\u2013V) relationship for unitary events of the maxi-anion channel. The reversal potential was \u22124.6\u2009\u00b1\u20091.9\u00a0mV. Each data point represents the mean \u00b1 SEM (vertical bar). c Time course of OGD-induced activation of the maxi-anion channel. The mean patch currents recorded at \u00b150\u00a0mV are plotted as a function of time\nThe single-channel activity induced by OGD persisted after excision of the patch (Fig.\u00a04: control). In the inside-out mode, however, the channel activity was blocked by extracellular Gd3+ (50\u00a0\u03bcM) in backfilled pipettes and by intracellular (bath) application of 20\u00a0\u03bcM arachidonic acid (Fig.\u00a04: Gd3+ and arachidonate). In contrast, phloretin (100\u00a0\u03bcM) failed to affect the maxi-anion channel activity (Fig.\u00a04: phloretin).\nFig.\u00a04Pharmacological profile of OGD-activated maxi-anion channel events in inside-out patches excised from astrocytes. a Representative current traces recorded during application of alternating pulses (the protocol shown in inset above) in the absence (control) and presence of Gd3+ (50\u00a0\u03bcM in the pipette), arachidonic acid (20\u00a0\u03bcM in the bath), and phloretin (100\u00a0\u03bcM in the bath). b Effects of Gd3+, arachidonic acid, and phloretin on the OGD-induced single-channel activity recorded at \u00b1\u200925\u00a0mV. Data are normalized to the mean currents before application of drugs. Each column represents the mean \u00b1 SEM (vertical bar). *Significantly different from the control value at P\u2009<\u20090.05\nIn inside-out patches, when the bath solution was changed from standard Ringer solution to a 100\u00a0mM Na4ATP solution, the reversal potential shifted from 0\u00a0mV to \u221220.6\u2009\u00b1\u20090.9\u00a0mV (n\u2009=\u20096); from this result, a PATP\/PCl value was estimated to be 0.11\u2009\u00b1\u20090.01 (Fig.\u00a05).\nFig.\u00a05Representative ramp I\u2013V records of single-channel events of the maxi-anion channel preactivated by OGD in an inside-out patch exposed to standard Ringer solution (chloride) and one exposed to 100\u00a0mM Na4ATP solution (ATP). The inward currents recorded in the ATP-exposed patch represent ATP4\u2212 currents. The amplitude of outward currents became smaller when ATP4\u2212 was present in the bath, suggesting a blocking of currents by ATP4\u2212, as observed in mammary C127 cells [19]\nThese results indicate that in astrocytes OGD stress activates a maxi-anion channel which can conduct anionic forms of ATP and is sensitive to Gd3+ and arachidonic acid.\nDiscussion\nIn the brain, bidirectional communication takes place between glial cells and neurons. Astrocytes clear synaptically released glutamate from the synaptic cleft, receive signals from neurons at certain receptors, and release molecules, called gliotransmitters, that modulate synaptic transmission, protect neurons from impairment, or repair neuronal tissues after damage. ATP and glutamate represent major gliotransmitters. It has been reported that astrocytes release ATP in response to a number of stimuli, including receptor activation [5\u20137], mechanical or osmotic perturbation [8\u201311], and deprivation of extracellular Ca2+ [8, 9, 12]. The present study demonstrated that astrocytes also respond to ischemic stress mimicked by OGD with ATP release (Figs.\u00a01a and 2b). ATP released from astrocytes has been shown to regulate glutamatergic synaptic transmission [6, 7, 10], inhibit excitability of retinal neurons [26], activate microglia [27, 28], modulate some functions of astrocytes [11], and protect astrocytes themselves from H2O2-induced cell death [29]. Pathophysiological functions of ischemia-induced ATP release from astrocytes remain to be elucidated.\nThere has been much controversy about the pathway of nonlytic release of ATP from a variety of cell types [30, 31]. The candidate pathways proposed for astrocytic ATP release include connexin hemichannels [8], MRP [11], P2X7 receptors [12], and exocytotic vesicular transport [9]. In the present study, however, OGD-induced ATP release was shown to be insensitive to blocking agents for connexin hemichannels (1-octanol, carbenoxolone), MRP (probenecid), P2X7 receptors (brilliant blue G), and exocytosis (BFA plus BAPTA-AM) (Fig.\u00a01b). Recently, it was reported that pannexins form ATP-conductive channels when expressed in Xenopus oocytes [32]. However, a possible involvement of the pannexin hemichannel in OGD-induced ATP release from astrocytes can be ruled out by the present observation (Fig.\u00a01b) of insensitivity to carbenoxolone, which is a potent blocker of not only connexin but also pannexin hemichannels as well [33]. Although VSOR anion channels were shown to be permeable to ATP at large positive potentials in endothelial cells [34], potent VSOR blockers (phloretin, glibenclamide) failed to affect OGD-induced ATP release from astrocytes in the present study (Fig.\u00a01b). In contrast, Gd3+ and arachidonic acid were found to prominently suppress ATP release from mouse astrocytes subjected to OGD (Fig.\u00a01b). Our previous study [17] showed that in mouse astrocytes, maxi-anion channels are activated by chemical ischemia and are sensitive to both Gd3+ and arachidonic acid. The present study demonstrated that maxi-anion channels are activated by OGD (Fig.\u00a03) and that OGD-activated maxi-anion channels are sensitive to Gd3+ and arachidonic acid (Fig.\u00a04) and can conduct anionic forms of ATP (Fig.\u00a05). On balance, it appears that the maxi-anion channel serves as a major pathway for OGD-induced ATP release from mouse astrocytes in primary culture. Our preliminary study has shown that in mouse hippocampal slices, astrocytes also respond to OGD with the activation of maxi-anion channels (H.-T. Liu, R.Z. Sabirov, Y. Okada, unpublished observations). Since ischemia-like stress was reported to induce ATP release from hippocampal slices [13], it is highly possible that the maxi-anion channel also provides a pathway for ischemia-induced ATP release from astrocytes in slice preparations.","keyphrases":["oxygen-glucose deprivation","atp release","maxi-anion channel","astrocytes"],"prmu":["P","P","P","P"]}
{"id":"Protein_Expr_Purif-1-5-1885977","title":"Vectors for expression of proteins with single or combinatorial fluorescent protein and tandem affinity purification tags in Dictyostelium\n","text":"We constructed a series of expression vectors for purification of native proteins and protein complexes in Dictyostelium. Protein purification is achieved by either a C-terminal or N-terminal fusion of the protein of choice to the tandem affinity purification (TAP) tag. The TAP tag consists of a protein A tag and a calmodulin binding peptide (CBP) and has been successfully used for purification of native protein complexes from yeast and animal cells. Protein expression is driven by the constitutive actin 15 promoter and the vectors optionally carry additional green- or yellow fluorescent protein (GFP or YFP) tags for fusion at either a C- or N-terminal location. Tandem affinity purification of native Dictyostelium protein complexes was tested by using pArc-34, one of the members of the well characterized Dictyostelium Arp2\/3 complex, as bait. After denaturation and SDS\u2013PAGE separation of the pArc-34 associated proteins all members of the Arp2\/3 complex could be identified.\nDictyostelium discoideum is a genetically tractable model system for studying remodeling of the actin cytoskeleton during cell locomotion, cell division, phagocytosis and vesicle trafficking and for elucidation of signaling pathways that control chemotaxis and development. Several metazoan-type cytoskeletal components were identified for the first time in Dictyostelium\n[1\u20133], and the process of pleckstrin homology domain mediated recruitment that plays such crucial roles in leukocyte chemotaxis and phagocytosis was also first demonstrated in the Dictyostelids [4]. The Dictyostelium genome is now completely sequenced and each gene is therefore available for functional analysis by gene disruption, overexpression and mutagenesis [5]. Genetic approaches to create tagged mutations, such as REMI (restriction enzyme mediated integration), have identified numerous genes with crucial roles in developmental or cellular processes [6]. However in only a few cases has it been possible to use genetic approaches, such as screens for suppressor mutations, to determine epistatic relationships between genes [7]. Or, in other words, to determine in what order their cognate proteins act on each other in a pathway. In other genetically tractable systems, such as the fruit fly and the nematode, epistatic relationships are usually determined by crossing in loss- and gain-of-function mutants of genes in the pathway. The poor accessibility of the Dictyostelid sexual stage, the macrocyst, has prohibited this approach.\nOther genetic screens, such as the yeast two-hybrid system or the split-ubiquitin method are being successfully used to identify interacting proteins in a pathway [8,9]. However, these methods depend on successful heterologous expression of the proteins in yeast, which is not possible for many proteins. The TAP tag was developed about seven years ago to purify native protein complexes from yeast [10,11] and was used for systematic identification of protein complexes in yeast [12\u201315] and metazoans [16,17]. For C-terminal tagging, the TAP tag contains in tandem, a calmodulin binding peptide tag, a TEV protease cleavage site and a protein A tag. For N-terminal tagging the order of these units is reversed. We have constructed a series of vectors for C- and N-terminal tagging of proteins expressed in Dictyostelium. Protein expression is driven by the strong constitutive actin15 promoter. Additional cloning sites were introduced to allow replacement of this promoter by an inducible promoter. A third green- or yellow fluorescent protein tag was introduced for C- or N-terminal expression in some of the vectors for rapid assessment of the levels of protein expression and the cellular location of the expressed proteins.\nMaterials and methods\nConstruction of plasmid vectors\nGene fragments were amplified by polymerase chain reaction (PCR)2 with a 4:1 mixture of the Taq and Pfu DNA polymerases (Promega, Madison, WI). All oligonucleotide primers used in this study are listed in Table 1. The integrating Dictyostelium vector EXP4(+) [18] was used as starting material. An XbaI and a HindIII site upstream of the actin15 promoter were first successively destroyed by filling in the overhanging sites after digestion with the Klenow fragment of polymerase I, followed by religation, resulting in vector EXP4(-XH). This also generated a unique Nhe1 site replacing the HindIII site.\nA long multiple cloning site (MCS) was generated by deleting the existing polylinker by BglII\/XhoI digestion of the vector and by ligating a duplex of primers MCS1 and MCS2 that carries compatible sticky ends into the BglII and XhoI sites. The resulting vector, EXP5(+) was used as the backbone for all subsequent constructs containing TAP and\/or EGFP\/EYFP fragments (Table 2).\nThe N-terminal TAP fragment (NTAP) was amplified from the plasmid pBS1761 [11] using primers NTAP1 and NTAP2 and ligated into the BamHI and HindIII sites of EXP5(+), yielding pDV-NTAP. The C-terminal TAP fragment (CTAP) was amplified from plasmid pBS1479 [10] using primers CTAP1 and CTAP2 and ligated into the ClaI and XhoI sites of EXP5(+), yielding vector pDV-CTAP. Enhanced yellow- and green fluorescent protein genes (YFP) and (GFP) were amplified from the vectors pEYFP-N1 and pEGFP-C3 (Clontech, Mountain View, CA), respectively. For N-terminal location of YFP, primers tapYFP1 and tapYFP2 were used and insertion occurred in the HindIII and SpeI sites of EXP5(+), yielding pDV-NYFP. For C-terminal location of YFP or GFP, primers tapYFP3 and tapYFP4 were used and insertion occurred into the EcoRI and ClaI sites of EXP5(+), yielding pDV-CYFP or pDV-CGFP. All constructs were validated by sequencing across the entire tag and MCS and are listed in Table 2.\nGeneration and transformation of construct p34-ArcCTAP\nThe Dictyostelium p34-Arc protein is encoded by the ArpE gene [5,19]. The 1251\u00a0bp ArpE coding region was amplified by PCR from genomic DNA isolated from wild-type AX2 cells, using primers p34Arc1 and p34Arc2 (Table 1). The BamHI\/XbaI digested PCR product was ligated into the BamHI and XbaI sites of vector pDV-CTAP, yielding vector p34-ArcCTAP. The p34-ArcCTAP, pDV-CGFP-CTAP and pDV-CTAP constructs were transformed by electroporation into D. discoideum strain AX2 and transformants were selected by growth in the presence of 20\u00a0\u03bcg\/ml G418.\nPurification of TAP-tagged protein complexes\nTAP purification was performed as described previously [10,11] with some modifications. Briefly, 2\u00a0\u00d7\u00a0109 exponentially growing AX2 cells expressing TAP constructs were harvested, washed twice in 10\u00a0mM Na\/K-phosphate buffer, pH 6.5 and resuspended in 10\u00a0ml IPP (150\u00a0mM NaCl in 10\u00a0mM Tris, pH 8.0), containing one tablet of Complete Protease inhibitor cocktail (Roche, Lewes, UK) per 50\u00a0ml. Cells were lysed by addition of Triton X-100 to 1% (v\/v) and incubated for 30\u00a0min on ice. Lysates were cleared by centrifugation for 20\u00a0min at 20000g and 4\u00a0\u00b0C. The supernatant was rotated for 1\u00a0h at 4\u00a0\u00b0C with 0.1% (v\/v) Nonidet P-40 (NP-40) and 200\u00a0\u03bcl bed volume of IgG-agarose beads (Sigma, St Louis, MO) in Polyprep columns (Bio-rad, Hercules, CA). The beads were washed three times with IPPN (0.1% NP-40 in IPP), and once with 10\u00a0ml TEV cleavage buffer (1\u00a0mM DTT and 0.5\u00a0mM EDTA in IPPN). Bound complexes were liberated by rotating the column for 2\u00a0h at 16\u00a0\u00b0C with 100 units of TEV protease (Invitrogen, Carlsbad, CA) in 1\u00a0ml TEV cleavage buffer and recovered by elution.\nSubsequently 3\u00a0\u03bcl of 1\u00a0M CaCl2, 3\u00a0ml CaM binding buffer (1\u00a0mM imidazole, 1\u00a0mM Mg-acetate, 10\u00a0mM \u03b2-mercaptoethanol and 2\u00a0mM CaCl2 in IPPN) and 200\u00a0\u03bcl of bed volume Calmodulin Affinity Resin (Stratagene, La Jolla, CA) were added per milliliter of eluate. The mixture was rotated for 1\u00a0h at 4\u00a0\u00b0C in a second Polyprep column. Beads were washed 3 times with 10\u00a0ml CaM binding buffer and bound material was eluted in 5 fractions of 200\u00a0\u03bcl each with CaM elution buffer (1\u00a0mM imidazole, 1\u00a0mM Mg-acetate, 10\u00a0mM \u03b2-mercaptoethanol and 2\u00a0mM EGTA in IPPN). Proteins were separated by SDS\u2013PAGE on 4\u201312% Bis\u2013Tris gels (Invitrogen, Carlsbad, CA) and visualized with a Colloidal Blue Staining kit (Invitrogen, Carlsbad, CA).\nProtein mass fingerprinting\nProtein mass fingerprint data was obtained by MALDI-TOF-TOF (MS\/MS) analysis performed at the University of Dundee \u2018Fingerprints\u2019 Proteomics Facility using an Applied Biosystems 4700 Proteomic Analyser. Protein bands were excised, in-gel reductively alkylated and digested in 20\u00a0mM NH4HCO3 containing 0.1% N-octylglucoside and 12.5\u00a0\u03bcg\/ml trypsin [20]. One-tenth of each digest was applied to a 192 well MALDI sample plate (Applied Biosystems (AB)), allowed to air dry and mixed with 0.5\u00a0\u03bcl of a solution of 5\u00a0mg\/ml \u03b1-cyano-4-hydroxy-trans-cinnamic acid matrix (Sigma, StLouis, USA), 50% (v\/v) acetonitrile, 0.1% (v\/v) trifluoroacetic acid in 10\u00a0mM NH4H2PO4, and then allowed to air dry prior to analysis.\nThe mass spectrometer was internally calibrated using the AB 4700 Proteomic Analyser Calibration Mix. Using the 4000 series Explorer Software (AB), MS spectral data were acquired from the samples and an MS\/MS list was automatically generated for further analysis based on the top 5 most intense ions present (trypsin and major keratin ions were excluded). The MS and MS\/MS spectral data obtained were exported from the 4700 using the Global Proteome Server (GPS) Explorer Software (AB). The data were then submitted to a local MASCOT search engine (Matrixscience, London, UK) for protein database searching against the Uniprot database (http:\/\/www.ebi.ac.uk\/swissprot\/) for identification.\nResults and discussion\nCloning strategies\nThe integrating Dictyostelium vector EXP4(+) was chosen as the backbone for all constructs. This vector is derived from pATSP [21], itself a derivative of the standard cloning vector pAT153. EXP4(+) carries the Act6NeoR cassette for G418 selection in Dictyostelium, and a cassette for protein expression consisting of the constitutive actin 15 (A15) promoter, the 2H3 terminator and a small polylinker with 6 restriction sites [18]. Because some of the cloning steps used to make this vector could not be retraced, we sequenced about 900\u00a0bp each around the points of fusion of the 2H3 terminator with the actin6 promoter, and the NeoR terminal region with the original pAT153 vector backbone. This revealed that the 2H3 terminator was preceded by 257\u00a0bp of the 2H3 coding region. However, since this coding sequence is downstream of the stopcodon in EXP4(+) and all derived vectors, it is not incorporated in the expressed tagged proteins. The complete sequence of EXP(4+) was reconstructed and a vector map is presented in Fig. 1.\nTo obtain a larger multiple cloning region, we first successively destroyed the XbaI and HindIII sites upstream of the A15 promoter, creating a novel NheI site. In addition to the existing SalI site, this site can be used to exchange promoters or to insert genes with their own promoter. The existing polylinker was removed by BglII and XhoI digestion and replaced by an adaptor duplex of oligonucleotides MCS1 and MCS2 (Table 1), yielding vector EXP5(+) (Fig. 1). The new multiple cloning site (MCS) contains an ATG start codon, preceded by 5 adenine residues to generate the Dictyostelium Kozak sequence and followed by 7 unique restriction sites:\nThe MCS was subsequently used to clone in the TAP tags and enhanced YFP or GFP tags that were amplified by PCR. This is possible in any configuration i.e. TAP alone, YFP(GFP) alone or combinations of TAP and YFP(GFP) at the same site or at opposing sites. When cloned at the same site, the YFP(GFP) is in between the gene of interest and the TAP sequence as indicated above. Here, NTAP and CTAP stand for N- and C-terminal TAP, respectively, while NYFP and CYFP(GFP) stand for N- and C-terminal GFPs and YFPs. Vectors with single NTAP, CTAP, NYFP or CYFP(GFP) tags were created first and validated by DNA sequencing across the entire tag and MCS. The CTAP fragment contained a stopcodon before the XhoI restriction site. Vectors with TAP\u00a0+\u00a0GFP(YFP) tags were then generated by removing a tag from one of these vectors and cloning it in the appropriate position in the other vectors. Some restriction sites cannot be used anymore: BglII will digest both the NTAP and the CTAP tags, while EcoRI will digest the CTAP tag. NheI will digest the CTAP tag, which then leaves only SalI to introduce a different promoter. All vectors prepared in the course of this work are listed in Table 2 and their sequences are deposited in GenBank.\nPurification of the Dictyostelium Arp2\/3 complex with TAP-tagged p34-Arc\nTo validate that the TAP tag can be used to purify native protein complexes in Dictyostelium we used a TAP tagged D. discoideum p34-Arc protein as bait for purification of members of the well-characterized Arp2\/3 complex. The Arp2\/3 complex is a complex of 7 proteins, including the p34-Arc protein, involved in the nucleation of actin polymerization [19]. We prepared a C-terminal fusion of the TAP tag with D. discoideum p34-Arc, as was previously done for yeast and human p34-Arc (named Arc35 and ARP2C, respectively) [14]. The p34-Arc gene was amplified from D. discoideum genomic DNA using the primers p34Arc1 and p34Arc2 (Table 1), which harbour BamH1 and XbaI sites, respectively, and cloned into the BamHI and XbaI digested pCTAP vector. This yielded the vector p34-Arc-CTAP, which was transformed into D. discoideum AX2 cells. AX2 cells transformed with the pDV-CTAP vector and with a pDV-CGFP-CTAP vector served as controls. For each transformed cell line, lysates were prepared by detergent treatment from 2\u00a0\u00d7\u00a0109 cells and adsorbed to IgG agarose. After extensive washing, the 4.8\u00a0kD CBP moiety of the TAP tag with fused \u201cbait\u201d and complexed \u201cprey\u201d were released from IgG agarose by TEV protease treatment. The proteins or protein complexes were further purified over calmodulin resin, eluted with EGTA, denatured and size-fractionated on SDS\u2013PAGE gels. Fig. 2 shows that a few faint bands were visible in preparations from cells that contained the empty pDV-CTAP vector. Extracts from pDV-CGFP-CTAP transformed cells showed a strong band at 32\u00a0kD, the expected size of the CGFP\u2013CBP fusion (26.8\u00a0+\u00a04.8\u00a0kD) after successful cleavage. Extracts of cells transformed with p34-Arc-CTAP displayed a pattern of 8 bands, similar to that observed after purification of the Dictyostelium Arp2\/3 complex by traditional chromatography [19]. Bands 1\u20138 were cut out from the gel, proteins were digested in gel with trypsin and sequences of the extracted peptides were determined by MALDI-TOF\/TOF mass spectrometry. This yielded 12\u201318 different peptide sequences for each of the 4 larger bands and 7\u201311 different peptide sequences for each of the four smaller bands. Query of the Uniprot protein database with the peptide sequences yielded all members of the D. discoideum Arp2\/3 complex for bands 1\u20134 and 6\u20138 (Fig. 2). Band 5, which is also present in the pDV-CTAP and pDV-CGFP-CTAP extracts was identified as discoidin B, a highly abundant Dictyostelium lectin, which is most likely a non-specific contaminant.\nConclusions\nWe have constructed eleven integrating plasmid vectors for protein expression in Dictyostelium that carry the TAP tag for tandem affinity purification of native proteins or protein complexes. In addition to the TAP tag for N-terminal or C-terminal fusion, a subset of vectors carry an additional enhanced GFP or enhanced YFP tag at either the same or opposite location as the TAP tag. The vectors are constructed in such a manner that the existing constitutive actin15 promoter can easily be swapped for an inducible promoter. Alternatively the pDV-CTAP vectors also allow cloning of a gene under its own promoter. In addition, the neomycin selection cassette can be exchanged for a different selection marker e.g. blasticidin resistance, which requires only a single copy of the bsr gene.\nPreferentially, the \u201cbait\u201d gene of choice should be expressed under its own promoter from a single copy vector in a \u201cbait\u201d null mutant. This will avoid artefacts due to ectopic expression and competition of the endogenous \u201cbait\u201d with the tagged \u201cbait\u201d for binding to cellular proteins. Moreover, rescue of the null mutant phenotype by the tagged \u201cbait\u201d will ensure that the tags are not interfering with protein function. However, it may often be desirable to overexpress the \u201cbait\u201d to obtain a sufficient yield of protein complexes, although this also increases the probability of pulling down unrelated proteins.\nWe show that for the pDV-CGFP-CTAP vector, the highly fluorescent GFP protein is efficiently released and purified by the two affinity steps and the TEV protease treatment. We also show that by using a p34-Arc-CTAP fusion protein as bait, the 6 associated members of the Arp2\/3 complex could be isolated with negligible contamination. We therefore expect that these vectors will prove to be useful tools for identification of native protein complexes in Dictyostelium.","keyphrases":["tandem affinity purification","expression vector","native protein complexes","yellow fluorescent protein","dictyostelium discoideum"],"prmu":["P","P","P","P","P"]}
{"id":"Ann_Surg_Oncol-4-1-2373867","title":"Laparoscopic Pelvic Autonomic Nerve-Preserving Surgery for Sigmoid Colon Cancer\n","text":"Background To test the feasibility of laparoscopic approach in performing the simultaneous pelvic autonomic nerve preservation during standard anterior resection of sigmoid colon cancer.\nPreservation of pelvic autonomic nerves to retain genitourinary function without compromise of oncologic clearance of tumor has been extensively studied in rectal cancer surgery.1\u201312 However, there is still not much information on sexual and bladder function after the curative resection of sigmoid colon cancer. Theoretically, the pelvic autonomic nerve-preserving surgery is technically much easier during a sigmoid colon resection, as apposed to rectal surgery, because the pelvic deep-seated neural structures, such as pelvic plexus (inferior hypogastric plexus), lateral ligament, neurovascular bundles of Walsh, and periprostate plexus, were not within the boundary of dissection.13\u201317 It is therefore our opinion that during the anterior resection of sigmoid colon cancer, preservation of autonomic nerves is a priority\u2014that is, autonomic nerve preservation is appropriate unless there are oncologic reasons for the resection of nerves, e.g., the nerves were directly involved by cancer, in terms of better postoperative genitourinary function for the patients. Recently, more and more sigmoid colon cancers were resected laparoscopically.18\u201320 Therefore, we conducted the present study to test the feasibility of laparoscopic approach in performing the total pelvic autonomic nerve preservation during the standard oncologic resection of sigmoid colon cancer. We hypothesized that given a well-illuminated magnified view by laparoscopy, the autonomic nerves can be well protected from inadvertent surgical damage.\nPATIENTS AND METHODS\nPatients meeting the following eligibility criteria were recruited for the present study. The inclusion criteria were: (1) curative and elective surgery; (2) sigmoid adenocarcinoma with tumor location generally 15 to 40\u00a0cm above anal verge; (3) American Society of Anesthesiology function class I to class III patients; (4) age between 18 to 65\u00a0years. The exclusion criteria were: (1) emergency or urgent surgery; (2) evidence of tumor invasion to adjacent organs or distant metastasis; (3) previous major abdominal or pelvic surgery; (4) abnormal preoperative baseline genitourinary function on the basis of validated questionnaire-based interview.10,21,22 Before entering the present study, all patients were well explained regarding the surgical procedures, and informed consents were obtained. The present study was approved by the Institutional Review board of National Taiwan University Hospital and was designated as NTUH 93S-024.\nSurgical Strategy\nThe related neuroanatomy for the nerve-preserving surgery of sigmoid colon cancer is shown in Fig.\u00a01A. The preservation of autonomic nerves was focused on the following three anatomic sites: (1) inferior mesenteric plexus (Fig.\u00a01B); (2) superior hypogastric plexus (Fig.\u00a01C); and (3) the paired hypogastric nerves (Fig.\u00a01D). The details of surgical procedures were well recorded and are shown in the associated video.\nFig.\u00a01(A) Surgical anatomy of pelvic anatomic nerves related to the nerve-preserving surgery of sigmoid colon cancer. (B) The inferior mesenteric plexus (yellow arrow) encircles the root of inferior mesenteric artery (IMA). (C) The superior hypogastric plexus overlying the interiliac trigone area. (D) The right hypogastric nerve travels across the presacral areolar tissue and then adheres to the proper rectal fascia.\nTo ensure the visualization and preservation of the nerves at all three critical points, we used a specific point-by-point checklist during the operations. The dissection commences at the medial side of sigmoid mesentery at the level of pelvic promontory. By gentle tenting of the sigmoid mesocolon ventrally, the superior hypogastric plexus was identified and preserved at the interiliac trigone just below the peritoneum. Subsequently, the dissection was continued upward along the ventral side of abdominal aorta. The preaortic plexus were then preserved by sparing the preaortic connective tissue. Thereafter, the medial-to-lateral laparoscopic dissection along the areolar tissue between sigmoid mesocolon and Gerota fascia was performed. The medial approach is well adapted for laparoscopy because it provides the working space and demands the least handling of sigmoid colon.23\nDuring the mobilization of sigmoid mesocolon, we did not identify the ureter because the loose connective tissue between Gerota fascia and sigmoid mesocolon is a natural dissection plane. Normally, the ureter and gonadal vessels are covered by the Gerota fascia, and therefore, if the ureter is clearly or intentionally identified, it means that the Gerota fascia is opened and the dissection plane may be imprecise. By simultaneous upward and medial-to-lateral dissection, we subsequently reach the junction of inferior mesenteric artery (IMA) and abdominal aorta. The IMA was divided 1 to 2\u00a0cm distal to its origin, or just distal to the left colic branch. This is because the sympathetic fibers encircle the IMA at the level of IMA\u2019s origin from abdominal aorta. If the nerves are not adequately cleared from \u201cbehind\u201d the IMA, they can be easily damaged at this point (Fig.\u00a01B). Subsequently, the \u201choly plane\u201d at the transition of the mesosigmoid to the mesorectum was meticulously dissected to progressively displace the hypogastric nerves dorsally and laterally (Fig.\u00a01D), and thus preserve them.\nAssessment of Urinary Function\nUrinary function was evaluated on the basis of questionnaire-based interview of patients preoperatively and 3\u00a0months after surgery. In patients with major surgical complications, for example, anastomotic leakage, the urinary function was evaluated 3\u00a0months after recovery from surgical morbidity, i.e., 3\u00a0months after the closure of defunctioning ileostoma. The preoperative functional questionnaire was based on the recollection of function before the development of symptoms from sigmoid colon cancer. Routinely, the urine Foley catheter was inserted for the patients immediately before the operation. In the postoperative period, the date for urine removal of the Foley catheter and the nature of any voiding problems, such as those requiring recatheterization, were recorded. The duration of indwelling urine Foley catheter was counted from the time of insertion to the time of its removal. The questionnaire used was based on International Prostate Symptom Score (IPSS), the parameters of which included incomplete bladder emptying, frequency, intermittency, urgency, weak stream, straining, and nocturia. Further subdivisions were added to the seven items.10 The scoring system for voiding function described above was based on a 0 to 5 scale, as follows: 0, not at all; 1, less than one time in five; 2, less than half the time; 3, approximately half the time; 4, more than half the time; 5, almost always. IPSS was calculated by adding the item scores. We ranked the urinary function as good (IPSS, 0\u20137); fair (IPSS, 8\u201314), and poor (IPSS, 15\u201335). Any voiding problems recovered within 3\u00a0months after surgery was considered to be transient bladder voiding dysfunction; otherwise, the voiding problems were deemed persistent.\nAssessment of Sexual Function\nSexual function was evaluated preoperatively and 6\u00a0months after operation or recovery from surgical complications, if present.\nIn male patients, the sexual function was evaluated on the basis of potency and ejaculation. The ejaculatory function was graded as good, fair (decrease in ejaculatory amounts up to 50%), and poor (retrograde ejaculation, failure to ejaculate). The grade of potency disorder was measured on the basis of the International Index of Erectile Function (IIEF), which contains five index domains (erectile function, orgasm function, sexual desire, intercourse satisfaction, and overall satisfaction) and 15 items.21 Each item of the IIEF is scored on a 5- or 6-point Likert score. Score for each domain are variable, and the total score range is 5 to 75. We classified the male potency function as good (IIEF, 60\u201375), fair (IIEF, 44\u201359), and poor (IIEF, 5\u201343).\nIn female patients, sexual function was evaluated on the basis of the Female Sexual Function Index (FSFI), which is a 19-item validated questionnaire that was designed to evaluate female sexual dysfunction, especially female sexual arousal disorder.22 The scale is divided into six domains: desire, arousal, lubrication, orgasm, satisfaction, and pain. Each item is scored with a 5- or 6- point Likert scale. The scales for each domain are variable, and the total score range is 4 to 95. For the present study, we classified the female sexual function as good (FSFI, 76\u201395), fair (FSFI, 58\u201375), and poor (FSFI, 4\u201357). Furthermore, the specific sexual problems in women such as libido, arousal, lubrication, orgasm, and dyspareunia were investigated.\nData Management\nData on patients undergoing laparoscopic autonomic nerve-preserving surgery for sigmoid colon cancer between January 2004 and December 2006 were entered into a prospective database of the present study. Until June 2007, the mean time of follow-up for the patients was 18 months, ranging from 6 to 36\u00a0months. The genitourinary function of the patients was analyzed by the intention-to-treat principle. Patients were first contacted by telephone by a research assistant who described the nature of the project. Those who consented to the present study were given a questionnaire-based interview by the research assistant. The data were managed by a statistician. To prevent bias of evaluation, the research assistant was not told which surgical procedure was performed for each individual patient.\nBecause genitourinary function is influenced by preoperative function and numerous other variables, including the emotional and physiological status of the patients, as well as other influences such as chemotherapy, each patient was used as his or her own control in evaluating the change of function before and after surgery. The two sets of measurements on the same patient were compared, and the P value was calculated from paired t-tests. A statistically significant level of difference was set at a P value of\u00a0\u2264.05.\nRESULTS\nA total of 112 patients were operated on with the intent of total preservation of pelvic autonomic nerves and curative resection of sigmoid colon cancer. The demographics and clinicopathologic data are shown in Table\u00a01. The nerve-preserving surgery was considered as failed in four patients when the videos of the surgical procedures were rescrutinized by coauthor colorectal surgeons and one general surgeon. The nerve-preserving anterior resection of sigmoid colon was performed with acceptable operation time, little blood loss, and low morbidity. The number of dissected lymph node was adequate, and the short-term oncologic result was good.\nTable\u00a01Demographics, clinicopathologic data, and surgical outcomes of patients undergoing laparoscopic autonomic nerve-preserving surgery for sigmoid colon cancer (n\u00a0=\u00a0112)ParameterValueaAge (years)55.8\u00a0\u00b1\u00a06.4Sex (M\/F)58\/54Body mass index (weight\/height2, kg\/m2), mean (range)\u00a0\u00a0\u00a0\u00a0Male23.4 (20.3\u223c31.6)\u00a0\u00a0\u00a0\u00a0Female24.8 (22.0\u223c32.5)ASA class\u00a0\u00a0\u00a0\u00a0I62\u00a0\u00a0\u00a0\u00a0II46\u00a0\u00a0\u00a0\u00a0III4TME stage (pathologic) \u00a0\u00a0\u00a0\u00a0I8\u00a0\u00a0\u00a0\u00a0II54\u00a0\u00a0\u00a0\u00a0III50Operative time (minutes)184.4\u00a0\u00b1\u00a044.8Conversion rate .9% (n\u00a0=\u00a01)Blood loss (mL)84.5\u00a0\u00b1\u00a024.0Postoperative ileus (hours)48.0\u00a0\u00b1\u00a08.4Hospitalization (days)8.0\u00a0\u00b1\u00a02.0Postoperative pain (visual analog scale)3.5\u00a0\u00b1\u00a0.4Postoperative complication\u00a0\u00a0\u00a0\u00a0Total7.1% (n\u00a0=\u00a08)\u00a0\u00a0\u00a0\u00a0Wound infection4\u00a0\u00a0\u00a0\u00a0Deep vein thrombosis1\u00a0\u00a0\u00a0\u00a0Pneumonia 1\u00a0\u00a0\u00a0\u00a0Anastomotic leakage2No. of lymph nodes collected14.4\u00a0\u00b1\u00a04.0Tumor recurrence rate8.0% (n\u00a0=\u00a09)ASA, American Society of Anesthesiology; TNM, tumor, node, metastasis system.a Continuous data are presented as mean\u00a0\u00b1\u00a0SD.\nIn patients with successful nerve-preserving surgery (96.4%, n\u00a0=\u00a0108), 104 patients completed the evaluation of urinary function (Table\u00a02). The median duration of the indwelling urine Foley catheter was 3.0\u00a0days, ranging from 1.0 to 7.0\u00a0days. Preoperatively, the baseline urinary function of all patients was ranked as good (IPSS, 0\u20137). However, after postoperative removal of the urine Foley catheter, the ranks of IPSS were changed to good in 98 patients (94.2%), fair (IPSS, 8\u201314) in 5 (4.8%), and poor (IPSS, 15\u201335) in 1 (1.0%). There was no significant difference of preoperative and postoperative IPSS score (P\u00a0=\u00a0.075, paired t-test) in patients with successful nerve-preserving surgery. The six patients with fair or poor bladder function experienced only transient dysfunction and recovered thereafter. Forty-four male and 42 female patients who were sexually active before the operation, i.e., whose ejaculatory function, IIEF, and FSFI score were all ranked as good, responded to the assessment of sexual function. Postoperatively, in male patients (Table\u00a03), the ranks of ejaculatory function were changed to good in 40 (90.9%), fair (decrease in ejaculatory amounts) in 3 (6.8%), and poor (retrograde ejaculation, failure of ejaculation) in 1 (2.3%). The grades of the potency were changed to good (IIEF, 60\u201375) in 41 (93.2%), fair (IIEF, 44\u201359) in 2 (4.5%), and poor (IIEF, 5\u201343) in 1 (2.3%). There was no significant difference of preoperative and postoperative IIEF score (P\u00a0=\u00a0.082, paired t-test) in male patients whose pelvic autonomic nerves were successfully preserved during surgery. In female patients (Table\u00a03), the ranks of sexual function were changed to good (FSFI score, 76\u201395) in 36 (85.7%), fair (FSFI score, 58\u201375) in 4 (9.5%), and poor (FSFI score, 4\u201357) in 2 (4.8%). However, there was no significant change of FSFI score (P\u00a0=\u00a0.122, paired t-test) for female patients before and after successful nerve-preserving surgery. Remarkably, specific sexual problems in women with autonomic nerve preservation included vaginal lubrication in 14.3% (n\u00a0=\u00a06), dyspareunia in 9.5% (n\u00a0=\u00a04), sexual arousal in 4.7% (n\u00a0=\u00a02), and orgasm in 4.7% (n\u00a0=\u00a02).\nTable\u00a02Changes in urinary function before and after surgery for patients with successful nerve-preserving surgery (n\u00a0=\u00a0104)Urinary function (range of IPSS)Before operationAfter operationP valuea\u00a0\u00a0\u00a0\u00a0Good (0\u20137)10498\u00a0\u00a0\u00a0\u00a0Fair (8\u201314)05\u00a0\u00a0\u00a0\u00a0Poor (15\u201335)01IPSS score (mean\u00a0\u00b1\u00a0SD)3.20\u00a0\u00b1\u00a01.723.68\u00a0\u00b1\u00a02.82.075IPSS, International Prostate Symptom Score.aP value was calculated on the basis of statistics of paired t-test.Table\u00a03Changes in sexual function for male and female patients with successful nerve-preserving surgeryFunctionBefore OperationAfter OperationP valueaMale patients (n\u00a0=\u00a044)Ejaculatory function\u00a0\u00a0\u00a0\u00a0Good4440\u00a0\u00a0\u00a0\u00a0Fair03\u00a0\u00a0\u00a0\u00a0Poor01Erectile function (IIEF Score) \u00a0\u00a0\u00a0\u00a0Good (60\u201375)4441\u00a0\u00a0\u00a0\u00a0Fair (44\u201359)02\u00a0\u00a0\u00a0\u00a0Poor (5\u201343)01IIEF score (mean\u00a0\u00b1\u00a0SD)72.4\u00a0\u00b1\u00a04.670.3\u00a0\u00b1\u00a08.4.082Female patients (n\u00a0=\u00a042) Sexual function (FSFI Score) \u00a0\u00a0\u00a0\u00a0Good (76\u201395)4236\u00a0\u00a0\u00a0\u00a0Fair (58\u201375)04\u00a0\u00a0\u00a0\u00a0Poor (4\u201357)02FSFI score (mean\u00a0\u00b1\u00a0SD)89.0\u00a0\u00b1\u00a09.285.4\u00a0\u00b1\u00a016.4.122IIEF, International Index of Erectile Function; FSFI, Female Sexual Function Index.aP value was calculated on the basis of statistics of paired t-test.\nOf the four patients (3.6%) who failed the procedures of the nerve-preserving surgery, three were female and one was male (Table\u00a04). In them, the mean duration of indwelling urine Foley catheter was 6\u00a0days (range, 4\u20138\u00a0days). Although the genitourinary function for the four patients was normal before surgery, the single male patient\u2019s IPSS and IIEF rank declined to the level of \u201cfair\u201d and he experienced retrograde ejaculation after the operation. The IPSS rank of the three female patients changed to the \u201cfair\u201d level in two, but the other patient remained at a \u201cgood\u201d level after operation. The sexual function was adversely affected by the failure of nerve-preserving surgery, with a \u201cfair\u201d rank of FSFI in one patient and a \u201cpoor\u201d rank in the remaining two patients. All three female patients experienced specific sexual problems, including vaginal lubrication, dyspareunia, sexual arousal, and orgasm.\nTable\u00a04Changes of values in specific domains of the scoring system for evaluation of genitourinary function in patients who failed the procedures of nerve-preserving surgery (n\u00a0=\u00a04)DomainPatient 1 (male)Patient 2 (female)Patient 3 (female)Patient 4 (female)Before OPAfter OPBefore OPAfter OPBefore OPAfter OPBefore OPAfter OPIPSS (score range)\u00a0\u00a0\u00a0\u00a0Total0100209012\u00a0\u00a0\u00a0\u00a01. Incomplete emptying (0\u20135)02010404\u00a0\u00a0\u00a0\u00a02. Frequency (0\u20135)02010102\u00a0\u00a0\u00a0\u00a03. Intermittency (0\u20135)01000202\u00a0\u00a0\u00a0\u00a04. Urgency (0\u20135)01000001\u00a0\u00a0\u00a0\u00a05. Week stream (0\u20135)01000000\u00a0\u00a0\u00a0\u00a06. Straining (0\u20135)02000203\u00a0\u00a0\u00a0\u00a07. Nocturia (0\u20135)01000000IIEF (score range) \u00a0\u00a0\u00a0\u00a0Total7544\u2013\u2013\u2013\u2013\u2013\u2013\u00a0\u00a0\u00a0\u00a01. Erection function (1\u201330)3018\u2013\u2013\u2013\u2013\u2013\u2013\u00a0\u00a0\u00a0\u00a02. Orgasmic function (0\u201310)106\u2013\u2013\u2013\u2013\u2013\u2013\u00a0\u00a0\u00a0\u00a03. Sexual desire (2\u201310)106\u2013\u2013\u2013\u2013\u2013\u2013\u00a0\u00a0\u00a0\u00a04. Intercourse satisfaction (0\u201315)157\u2013\u2013\u2013\u2013\u2013\u2013\u00a0\u00a0\u00a0\u00a05. Overall satisfaction (2\u201310)107\u2013\u2013\u2013\u2013\u2013\u2013FSFI\u00a0\u00a0\u00a0\u00a0Total\u2013\u2013955895279532\u00a0\u00a0\u00a0\u00a01. Desire (2\u201310)\u2013\u2013108104105\u00a0\u00a0\u00a0\u00a02. Arousal (0\u201320)\u2013\u20132012205206\u00a0\u00a0\u00a0\u00a03. Lubrication (0\u201320)\u2013\u20132010206206\u00a0\u00a0\u00a0\u00a04. Orgasm (0\u201315)\u2013\u2013159153154\u00a0\u00a0\u00a0\u00a05. Satisfaction (2\u201315)\u2013\u2013159153155\u00a0\u00a0\u00a0\u00a06. Pain (0\u201315)\u2013\u20131510156156IIEF, International Index of Erectile Function; FSFI, Female Sexual Function Index.\nDISCUSSION\nThe present study confirmed our hypothesis that by taking advantage of the optic properties of laparoscopy, we can precisely preserve the pelvic autonomic nerve at the following three anatomic sites: inferior mesenteric plexus, superior hypogastric plexus, and paired hypogastric nerves. However, we failed to preserve the autonomic nerve in four (one male and three female) patients of this case series\u2014a finding that needs to be addressed. After the completion of nerve-preserving surgery, we routinely tented the superior hypogastric plexus to check the total preservation of associated nerves. In our scrutiny of the surgical videos, we found that the mechanisms of nerve injury for the three female patients were the same: the nerve injuries were all located at the level of the hypogastric nerves and were bilateral. In our experience, the fibers of pelvic autonomic nerve were generally thinner in female that than in male patients. Furthermore, anatomically, the paired hypogastric nerves run downward, close to or tethering the proper rectal fascia along the pelvic wall (Fig.\u00a01D). At this point, the right-sided hypogastric nerves of the three female patients were subjected to avulsion injury. On the other hand, the left-side hypogastric nerves of these three female patients were mistaken as pelvic fibrotic bands and were cauterized. We thought that some female patients might be predisposed to chronic pelvic inflammation, thus blurring the pelvic dissection plane, and therefore the hypogastric nerves and retroperitoneal fibrotic bands could not be clearly differentiated, even under the well-illuminated and magnified view of laparoscopy. In the only male case of autonomic nerve injury, we found that the patient\u2019s body mass index (BMI) was 32.0\u00a0kg\/m2. During the surgery, the hypogastric nerve was coated by fatty tissues and was inadvertently severed bilaterally. Because obese patients (BMI\u00a0>\u00a030\u00a0kg\/m2) represented only 6 (10.3%) of the 58\u00a0male and 4 (7.4%) of 54\u00a0female patients in this case series and nerve injury related to obesity occurred in only 1\u00a0male patient, we therefore were not sure whether high BMI was a risk factor for nerve injury. Further recruitment of more obese patients for study will be necessary to solve this interesting question.\nRemarkably, CaverMap Surgical Aid (CM; Blue Torch Medical Technologies, Inc., Ashland, MA) has been recommended to assist surgeons in intraoperatively localizing the autonomic nerves, ultimately leading to improved preservation of autonomic nerves and sexual function.24 We thought that CaveMap might be useful in case the nerve-preserving procedures were for the treatment of rectal cancer, in which case the neural structures to be preserved were deep-seated in the pelvis. However, for the oncologic resection of sigmoid colon cancer, the autonomic nerves at the above-mentioned three critical anatomic points were usually be clearly identified. In the rare cases when exact identification of the autonomic nerves was difficult, we recommended that the surgeons find the precise dissection plane between sigmoid mesentery and retroperitoneal tissue, and thus a plate of nerve-containing tissue overlying the abdominal aorta and interiliac trigone area can be left intact. On the other hand, the present study showed that laparoscopic nerve-preserving surgery for sigmoid colon cancer was technically feasible and could achieve function-preserving outcomes as good as those of traditional open surgery.10,11 However, the functional results of most reported series to date were based on the patients with rectal cancer undergoing open laparotomy. Therefore, further randomized studies in this respect that focus on sigmoid colon cancer are necessary.\nNeurophysiologically, the internal urethral orifice is innervated by sympathetic signals and has dual functions: one to ensure urinary continence, and the other to prevent backflow of semen during ejaculation. The present study provided the insights into the functional significance of the internal urethral orifice by the clinical observation that the female patients manifested some degree of urinary incontinence and the male patients experienced retrograde ejaculation as a result of the failed preservation of bilateral hypogastric nerves. However, the influence of damage of pelvic sympathetic nerves on female sexual function has been ambiguous but generally considered because it would most likely result in impairment of vaginal lubrication. The manifestation of specific sexual problems for the three female patients with inadvertent sympathectomy supported this hypothesis. Remarkably, our previous study on the canine modal showed that when one side of hypogastric nerve was severed, the other side of hypogastric nerve could compensate for the genitourinary functional loss to a normal level.15 To clarify the role of pelvic sympathetics in the human genitourinary function, further recruitment of more patients with iatrogenic sympathetic nerve injury for study is required. Because the intentional severance of sympathetic nerve during the surgical practice is not ethical, we think that further study subjects will be focused on patients with an advanced rectosigmoid cancer in which unilateral or bilateral sympathectomy was a necessity for the radical resection of cancer.\nIn the present study, 6 patients (5.8%) had transient voiding disorder, 4 (9.1%) of 44\u00a0male patients had ejaculatory disorder, and up to 14.3% (6 of 42) female patients had sexual problems after the operation, even though their sympathetic nerves were successfully preserved during surgery. Our explanations for this phenomenon were as follows: first, anatomically, the inferior mesenteric plexus, thoracolumbar sympathetic splanchnic nerves, and superior hypogastric plexus were grossly manifested as a continuous plate of weblike connective tissues underlying the root of IMA and overlying the lower abdominal aortic and the interiliac trigone area. Therefore, some minor partial injury of nerve fibers may not be detected during surgery. Second, the retroperitoneal dissection by electrocauterization sometimes causes side injury and\/or ischemia change of nerve fibers and thus compromised the neurophysiologic function. Third, the sexual function is influenced by various psychosocial factors. For example, the alteration of body image and\/or bowel function after the operation may also have negative effect on sexual function.\nGenitourinary functional disorders are generally subjective symptoms, and their measurement, either by questionnaire-based interviews or laboratory manometric and volumetric studies, are vulnerable to evaluation bias. Therefore, before the translation of technical feasibility of nerve-preserving surgery into the retaining of normal genitourinary function, the methodology used for the evaluation of functional outcomes in the present study needs to be further addressed. First, we used IPSS, IIEF, and FSFI to measure the genitourinary function of patients. These three kinds of validated questionnaires have been generally considered to be highly sensitive, and they have been widely adopted internationally for the study of genitourinary function.21,22\u00a0Second, in this study, both the patients and the assessors were informed of the intent to preserve the sympathetic nerves during sigmoidectomy but actually had no idea whether the nerve-preserving procedure was successful. The blinding of both assessors and patients to the surgical procedure can lessen the placebo effect. Third, the genitourinary function varies at different time points of evaluation. In this study, the evaluation of postoperative genitourinary function was conducted at the standardized time points before and after the operation. However, the preoperative baseline data was based on patient recall of genitourinary function before the appearance of bowel symptoms. The evaluation of baseline genitourinary function was therefore inevitably subject to methodology limitations, i.e., the potential recall bias. However, to validate the preoperative baseline data of patients, the spouses or sexual partners of some selected patients were also interviewed. We emphasize that patients were included in this study only when their normal baseline genitourinary function was ensured.\nIn conclusion, the genitourinary function of patients can be successfully preserved with good technical efficiency during laparoscopic sigmoidectomy. The present study should facilitate further study regarding the impact of preservation of genitourinary function on the postoperative quality of life for patients with sigmoid colon cancer.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\n(WMV 24856 kb)","keyphrases":["pelvic autonomic nerve","sigmoid colon cancer","laparoscopic surgery"],"prmu":["P","P","R"]}
{"id":"Intensive_Care_Med-3-1-1915647","title":"Meta-regression analysis of high-frequency ventilation vs conventional ventilation in infant respiratory distress syndrome\n","text":"Objective There is considerable heterogeneity among randomized trials comparing high-frequency ventilation (HFV) with conventional mechanical ventilation (CMV) in premature neonates with respiratory distress syndrome. We investigated what factors explained differences in outcome among these trials.\nIntroduction\nHigh-frequency ventilation (HFV) has been compared with conventional mechanical ventilation (CMV) since the 1980s. In HFV, patients are ventilated with small tidal volumes, even smaller than the dead space of their airways, at high frequencies, normally between 5\u202fand 10\u202fHz. Because HFV combines high mean airway pressures with small tidal volumes, this technique of ventilation has been regarded by some to be the most optimal form in patients with infant respiratory distress syndrome (IRDS), adult respiratory distress syndrome (ARDS), and other forms of severe lung disease\u00a0[1].\nThe HFV has been extensively investigated in premature neonates with IRDS, a\u00a0population specifically at risk for chronic lung disease (CLD). Unfortunately, the results of these studies were equivocal\u00a0[2, 3]; thus, the question remains whether or not HFV better prevents CLD than conventional mechanical ventilation (CMV) in patients with severe lung disease. A\u00a0significant number of meta-analyses have been performed to answer this question\u00a0[4, 5, 6, 7, 8]. Pooled estimates of pulmonary outcomes failed to show clinically relevant differences among HFV and CMV\u00a0[7]; however, significant heterogeneity existed between studies included in these meta-analyses. In a\u00a0recent cumulative meta-analysis we identified improvements of the conventional treatment of IRDS and ventilation strategies applied in both HFV and CMV as important sources of heterogeneity\u00a0[4]. These associations could be confounded by other explanatory variables. Although a\u00a0meta-analysis may pool results from randomized trials, differences among trials will not be randomly or independently distributed. A\u00a0meta-analysis constitutes an observational study of trials, subjected to bias inherent to observational research. In a\u00a0meta-regression analysis it is possible to adjust for confounding covariates. A\u00a0number of alternative hypotheses have been formulated to explain heterogeneity between trials:\nThe observed regression of the cumulative relative risks to the level of unity was due to publication bias.Use of the Sensormedics ventilator resulted in better results in HFV treated patients.A\u00a0prolonged ventilation on CMV before initiating HFV treatment could reduce the benefits of HFV.In subgroups of more premature neonates with lower birth weight with a\u00a0higher susceptibility for CLD, HFV could result in better pulmonary outcome.With outcome rates increasingly representing more severe disease, HFV could have an increasing advantage over CMV\u00a0[9, 10]; therefore, we used meta-regression analysis to better estimate relative treatment effects through adjustments for factors that could explain trial heterogeneity.\nMethods\nTrials were included based on a\u00a0previous meta-analysis that we had conducted\u00a0[4]. The same search strategy, as well as the same inclusion and exclusion criteria as in our previous meta-analysis, were used for an update, yielding two more studies that could be included for this meta-regression analysis. Validity of studies was assessed by criteria published by Jadad et al.\u00a0[11]. The validity was generally deemed as high with adequate allocation concealment in all trials. Blinding of treatment was not possible due to the nature of the interventions.\nData extraction was performed as has been reported in our previous meta-analysis. The following outcome measures were used: mortality, chronic lung disease (CLD) as defined by supplemental oxygen need or ventilator dependency at the age of 30\u201336\u202fweeks post-menstrual. A\u00a0number of explanatory variables were extracted as well: year of publication; type of ventilator used for HFV (Sensormedics 3100A ventilator versus other); ventilation strategies applied in the HFV and CMV treatment groups were obtained as previously described\u00a0[4]; time on CMV before study initiation; gestational age and birth weight; and outcome rates in the control population were taken as proxy for baseline disease severity in the source population. The Sensormedics ventilator was singled out because previous research suggested better performance compared with other oscillator ventilators\u00a0[2, 4].\nStatistical analysis\nAll data were extracted according to the intention-to-treat principle. The number of patients surviving without chronic lung disease was subtracted from the total number of randomized patients in each treatment arm to calculate the composite outcome of death or CLD. To calculate the risk of CLD, the number of surviving patients was put in the denominator. Publication bias was assessed by visual appraisal of symmetry of funnel plots and performing rank tests. Smaller studies could show different results than larger studies which could suggest publication bias, but in fact was caused by systematic differences among studies; therefore, an analysis of publication bias stratified for ventilation strategies was performed to determine whether the observed association between the inverse of the standard error with the risk ratio was confounded by ventilation strategies. Meta-regression analysis was used to evaluate other hypotheses. The dependent variables, RR of CLD and RR of CLD or death, were natural log transformed to linearize the regression models. Individual studies were weighted by inverse variances of relative risks of outcomes of interest so that the more precise studies had more influence in the analysis. Firstly, linear regression analyses were applied to explanatory variables. Secondly, linear regression analyses with continuous covariates were conducted stratified by HLVS, LPVS, and use of surfactant. Finally, multivariable linear regression analyses were performed to calculate adjusted contributions of different explanatory variables of rivalling hypotheses to changes in RR. The relative effects of covariates were evaluated by relative risk ratios (RRR). A\u00a0relative risk ratio quantifies the relative change in RR that is associated with a\u00a0specified change of a\u00a0covariate. For continuous variables the RRR was calculated for the ranges of minimum and maximum values of covariates that were reported in trials. For example, the RRR for year of publication was calculated by using the range between the publication year of the first year and the publication year of the last included trial. The RRR for year of publication thus estimates the relative change in RR due to the difference in years of publication between the first and last trials. All analyses were conducted using SPSS 12.0.1 for Windows software (SPSS, Chicago, Ill.) and Excel (Microsoft, Redmond, Wash.).\nResults\nFor the analyses 15 studies were available that specified either CLD in survivors or death or CLD as outcome measures\u00a0[2, 3, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24]. In 11 trials a\u00a0high frequency oscillatory ventilator was used\u00a0[2, 3, 12, 13, 16, 17, 19, 20, 22, 23, 24], in 7 of these trials this was the SensorMedics ventilator\u00a0[2, 12, 13, 17, 22, 23]. Two studies used a\u00a0high-frequency jet ventilator\u00a0[14, 15] and in two studies a\u00a0high-frequency flow interrupter ventilator was used\u00a0[18, 21]. In the HFV group a\u00a0total of 1141 patients were included for the outcome of CLD with 373 events and a\u00a0total of 1457 patients with 671 events for the outcome death or CLD. In the CMV group a\u00a0total 1159 patients were reported for the outcome of CLD with 428 events and a\u00a0total of 1473 patients with 730 events for the outcome death or CLD. A\u00a0forest plot of these trials can be found in the Electronic Supplement.\nTable\u202f1 summarizes the main characteristics of the studies. The outcome of CLD was available in all studies but one\u00a0[22]. Time on CMV to start of the study was not reported by Plavka et al.\u00a0[17] and Craft et al.\u00a0[21]. In only one study was surfactant not used as concomitant treatment\u00a0[12]. A\u00a0high lung volume strategy (HLVS) was used in all but two studies\u00a0[14, 16]. A\u00a0ventilation strategy in the CMV-treated patients that could qualify as lung protective (LPVS) was reported in the most recent 9 studies\u00a0[2, 3, 18, 19, 20, 21, 22, 23, 24]. Studies were published over a\u00a0range of 13\u202fyears. Other reported ranges of covariates were 8.7\u202fh average time on CMV before start of study, 5\u202fweeks average gestational age, and 0.65\u202fkg average birth weight. These ranges were used to calculate RRRs. Two studies dominated the analyses by virtue of the weight they received in the analyses: Johnson et al.\u00a0[3] and Courtney et al.\u00a0[2] (together 69% for CLD and 73% for death or CLD as outcome).\nTable\u00a01Study characteristicsReferenceYearTime on CMVAgeBirth weightSensorMHLVSLPVSSurfCLD lnRRWeightDeath or CLD lnRRWeight\u00a0[12]1992 9.0281.100YYNN-1.290.01-0.580.01\u00a0[13]1996 3.0311.500YYNY-0.670.04-0.550.02\u00a0[14]1996 7.2270.950NNNY 0.020.01-0.230.10\u00a0[15]1997 8.0271.020NYNY-0.700.03 0.480.03\u00a0[16]1998 1.0281.100NNNY 0.000.00 0.310.00\u00a0[17]1999260.850YYNY-1.030.01-0.740.01\u00a0[18]1999 0.5270.870NYYY 0.090.06 0.010.04\u00a0[19]2001 2.6260.840YYYY-0.980.02-0.590.02\u00a0[20]2001 0.3280.990NYYY-0.200.05-0.060.05\u00a0[2]2002 2.7260.850YYYY-0.060.16-0.220.13\u00a0[3]2002 1.0260.850NYYY-0.010.54-0.020.60\u00a0[23]2003 1.0291.200YYYY 0.320.03 0.270.04\u00a0[22]200314.0270.980YYYY-0.040.05\u00a0[21]2003260.726NYYY 0.100.05 0.090.03\u00a0[24]2005 0.3270.880NYYY-1.440.01-1.200.00Year: year of publication. Time CMV: Mean time on CMV before start of the study in hours. Age: Mean gestational age (weeks). Birth weight: mean birth weight (kg). HLVS: high lung volume strategy in the HFV group, LPVS: lung protective ventilation strategy in the CMV group, Surf: use of surfactant in the study. CLD: chronic lung disease, defined as on oxygen at 30\u201336 weeks postmenstrual age, LnRR: natural log of the relative risk\nA\u00a0funnel plot of the inverse of the standard error against the natural logarithm of the RR for CLD was indicative of publication bias because of asymmetry round the line of the pooled effect (Fig.\u202f1). A\u00a0rank test showed a\u00a0p-value of 0.112. A\u00a0stratified analysis of publication bias is indicated by different colors in Fig.\u202f1. To visually evaluate publication bias within subgroups of ventilation strategy, the distribution of trials round the corresponding colored lines (mean effect size within subgroup) was assessed. Stratification by ventilation strategy (HLVS and LPVS vs either no HLVS and\/or no LPVS) showed p-values of 0.456 and 0.851, respectively, indicating less evidence of publication bias. The distribution of stratified studies round the lines of pooled estimates showed less asymmetry (Fig.\u202f1). Publication bias for the composite outcome of death or CLD was less likely with a\u00a0p-value of 0.329. Stratified analysis showed p-values of 0.677 and 1.000.\nFig.\u00a01Funnel plot. Selection bias in reporting RR of chronic lung disease (CLD) as suggested by asymmetry of the distribution of studies. x-axis: inverse of the standard error of the RR; y-axis: natural logarithm of the RR. Blue diamonds: studies with either no high lung volume strategy (HLVS) or no lung protective volume strategy (LPVS); pink diamonds: studies with both HLVS and LPVS; dotted line: estimated pooled RR including all studies; dashed colored lines: pooled RR of subgroups of studies. Publication bias was visually appraised by assessing symmetry of distribution of studies around the lines of pooled estimates. CMV conventional mechanical ventilation\nFigures\u202f2, 3, 4 show the results of the linear meta-regression analyses for continuous explanatory variables with relative risk of CLD as dependent variable. Two studies dominate these figures, designated by the weight they received in the analyses\u00a0[2, 3]. Over the years the reported benefit of HFV over CMV seemed to diminish (Fig.\u202f2). A\u00a0longer time on CMV prior to study initiation and a\u00a0higher gestational age and increase of birth weight (data not shown) seemed to be positively associated with a\u00a0relatively better outcome in HFV (Figs.\u202f3,\u00a04). Table\u202f2 shows the results of linear meta-regression analyses, showing significant associations with year of publication (3.1 times higher RR with change of publication year from 1992 to 2005) and whether or not a\u00a0protective ventilation strategy was applied (1.9 times higher RR with change of protective ventilation from no to yes; Table\u202f2). In the linear regression analyses with death or CLD as composite outcome no significant associations were detected. Whether or not a\u00a0Sensormedics high-frequency oscillatory ventilator was used and baseline incidence in CMV (0.75 vs 0.08) treated patients displayed the smallest effects on trial outcome (RRR\u202f=\u202f0.84 and 0.90 for CLD and RRR\u202f=\u202f0.85 and 0.99 for death or CLD, respectively).\nFig.\u00a02Linear regression analyses. Crude and subgroup linear regression analyses of the effect of year of publication, prior time on CMV and gestational age with natural logarithm of RR of CLD as dependent variable. y-axis: natural logarithm of the RR; x-axis: explanatory variables. Blue diamonds: studies with either no HLVS or no LPVS; pink diamonds: studies with both HLVS and LPVS. The size of the diamonds reflects the weights the individual trials contribute to the analyses. Thin blue line: regression line including all studies; thick pink line: regression line including only studies with both HLVS and LPVS. CMV conventional mechanical ventilationFig.\u00a03Same as Fig.\u00a02Fig.\u00a04Same as Fig.\u00a02Table\u00a02Univariable linear regression analysis95% confidence interval95% confidence intervalCrude BSig.LowerUpperRRRLowerUpperboundaryboundaryboundaryboundaryAll studiesCLD Year 0.090.025 0.010.163.131.18 8.27 SensorM-0.170.351-0.550.210.840.58 1.24 (no to yes) TimeCMV-0.090.055-0.190.000.440.19 1.02 Age-0.080.237-0.230.060.660.32 1.36 Weight-0.760.163-1.870.350.540.22 1.33 HLVS-0.110.883-1.741.520.890.17 4.57 LPVS 0.640.009 0.191.101.911.21 3.00 Surf 1.210.168-0.593.003.340.5620.03 CMV-0.180.774-1.531.170.900.42 1.92Death or CLD Year 0.050.096-0.010.122.010.86 4.65 SensorM-0.170.132-0.390.060.850.67 1.06 TimeCMV-0.010.590-0.050.030.920.65 1.29 Age-0.020.733-0.130.100.910.52 1.61 Weight-0.220.611-1.160.710.840.40 1.77 HLVS-0.370.698-2.441.690.690.09 5.45 LPVS 0.190.275-0.180.561.210.84 1.76 Surf 0.520.289-0.511.561.690.60 4.75 CMV-0.020.963-0.910.870.990.60 1.63Studies with surfactant, HLVS, and LPVSCLD Year 0.000.971-0.230.220.960.0517.34 TimeCMV-0.050.698-0.340.250.660.05 8.75 Age 0.040.727-0.220.301.220.33 4.49 Weight 0.410.693-1.992.811.380.20 9.44Death or CLD Year 0.010.846-0.150.171.200.15 9.72 TimeCMV 0.000.819-0.050.040.960.65 1.43 Age 0.060.406-0.100.211.340.61 2.92 Weight 0.550.396-0.891.991.550.49 4.90Simple linear regression analyses were calculated for chronic lung disease (CLD), defined as on oxygen at 30\u201336\u202fweeks postgestational age, and\ndeath or CLD. The following co-variates were evaluated: Year: Number of years after the first included study; SensorM:\nWhether or not a Sensormedics type of HFV was used; TimeCMV: Mean time on CMV before start of the study in hours; Age: Mean gestational age (weeks); Weight: mean birth weight (kg); HLVS:\nhigh lung volume strategy in the HFV group; LPVS: lung protective ventilation strategy in the CMV group; Surf:\nuse of surfactant in the study; B was the estimated crude coefficient; RRR: relative risk ratio\u202f=\u202fRRcovariate=1\/RRcovariate=0,\nfor binary variables (SensorM, HLVS and LPVS), for continues\nvariables the extreme values reported in the studies were used to calculate the ranges, 13 for years, 8.7 for Time on CMV,\n5 for Age, 0.8 for Weight and 0.65 for CMV (RRRyears\u202f=\u202fRRyear=2005\/RRyear=1992,\nRRRtime on CMV\u202f=\u202fRRtime=9\u202fh\/RRtime=0.3\u202fh, RRRage\u202f=\u202fRRage=31\u202fweeks\/RRyear=26\u202fweeks,\nRRRweight\u202f=\u202fRRweight=1.5\u202fkg\/RRyear=0.7\u202fkg, RRRincidence\nof CLD in CMV=0.75\/RRincidence=0.08)\nFigure\u202f5 shows how the incidence of CLD in the CMV treated patients was related to the incidence in HFV treated patients for each of the studies. The diagonal line represents the line of no effect in this figure. A\u00a0trend line was fitted by weighted linear regression, showing a\u00a0small effect of change in incidence in CMV on incidence in HFV-treated patients.\nFig.\u00a05Linear regression analysis of incidence of CLD in CMV on incidence of CLD in HFV. y-axis: incidence of CLD in HFV; x-axis: incidence of CLD in CMV. Thin pink line: regression line including all studies\nYear of publication was not related to change in relative risk of CLD in the subgroup of studies with HLVS, LPVS and concomitant use of surfactant (RRR\u202f=\u202f0.96). There was only a\u00a0small increase in relative risk for death or CLD (RRR\u202f=\u202f1.20; Fig.\u202f2; Table\u202f2). Opposite effects of gestational age (RRR\u202f=\u202f1.22 for CLD and 1.38 for death or CLD vs RRR\u202f=\u202f0.66 for CLD and 0.91 for death or CLD, respectively) and birth weight were detected in the subgroup analysis (Fig.\u202f4; Table\u202f2). Prior time on CMV exerted less effect on outcome compared with the crude analysis, RRR\u202f=\u202f0.66 for CLD and 0.96 for death or CLD and RRR\u202f=\u202f0.44 for CLD and 0.92 for death or CLD in the adjusted and crude analyses, respectively (Fig.\u202f3; Table\u202f2).\nMultivariable linear regression analyses were conducted to assess the independent contributions to change in RR by explanatory variables (Table\u202f3). The RRRs in Table\u202f3 have the same meaning as in Table\u202f2, only they represented adjusted RRRs. Year of publication was not considered as an independent explanatory variable but rather as proxy for changes in treatment and patient population. Gestational age and birth weight were collinearly related by nature; only gestational age was fitted in the model. One study contributed to the fact that surfactant was not used; therefore, surfactant was not used in the multiple linear regression analyses. Two models were fitted. Model\u202fA used Sensormedics, time on CMV, gestational age, HLVS, and LPVS as covariates. The largest estimated effects were caused by ventilation strategies, HLVS, and LPVS, adjusted for use of Sensormedics ventilator, prior time on CMV, and gestational age. These estimations were consistent for the outcomes CLD (RRR\u202f=\u202f0.42 and RRR\u202f=\u202f2.02 for HLVS and LPVS, respectively) and death or CLD (RRR\u202f=\u202f0.42 and RRR\u202f=\u202f1.98 fro HLVS and LPVS, respectively). Use of a\u00a0Sensormedics ventilator seemed to have a\u00a0much smaller effect on RR for outcome. The RRR of gestational age, comparing 26\u202fweeks with 31\u202fweeks, for CLD and death or CLD were larger (RRR\u202f=\u202f1.17 and RRR\u202f=\u202f1.47). The effect of a\u00a0difference in prior time on CMV of 8.7\u202fh on CLD vs death or CLD was not consistent (RRR\u202f=\u202f0.85 and RRR\u202f=\u202f1.07, respectively).\nTable\u00a03Multivariable linear regression analysisAdjusted95% confidence interval95% confidence intervalBSig.Lower boundaryUpper boundaryRRRLower boundaryUpper boundaryModel ACLD (Constant)-0.660.900-13.0311.70 SensorM-0.040.884 -0.75 0.660.960.47 1.94 TimeCMV-0.020.903 -0.38 0.340.850.0419.22 Age 0.030.850 -0.36 0.421.170.16 8.32 HLVS-0.880.306 -2.80 1.040.420.06 2.84 LPVS 0.700.506 -1.73 3.142.020.1823.12Death or CLD (Constant)-1.860.412 -7.22 3.49 SensorM-0.170.309 -0.55 0.210.850.58 1.24 TimeCMV 0.010.722 -0.05 0.061.070.68 1.69 Age 0.080.299 -0.09 0.251.470.62 3.47 HLVS-0.880.407 -3.38 1.620.420.03 5.06 LPVS 0.680.127 -0.28 1.651.980.76 5.19Model BCLD (Constant) 0.070.904 -1.21 1.35 SensorM-0.060.698 -0.38 0.260.940.69 1.30 HLVS-0.810.203 -2.14 0.520.440.12 1.68 LPVS 0.720.011 0.21 1.232.061.23 3.43Death or CLD (Constant) SensorM-0.110.318 -0.33 0.120.900.72 1.13 HLVS-0.790.363 -2.66 1.080.450.07 2.93 LPVS 0.460.089 -0.09 1.011.590.92 2.74Multiple linear regression analyses were calculated for chronic lung disease (CLD),\ndefined as on oxygen at 30-36 weeks postgestational age, and death or CLD. The following co-variates were evaluated:\nSensorM whether or not a Sensormedics type of HFV was used; TimeCMV\nmean time on CMV before start of the study (in hours). HLVS high lung volume\nstrategy in the HFV group, LPVS lung protective ventilation strategy in the\nCMV group. B was the adjusted estimated coefficient. RRR relative risk\nratio=RRcovariate=1\/RRcovariate=0, for binary variables (SensorM,\nHLVS and LPVS), for continues variables the extreme values reported in the studies were used, 8.7 for Time on\nCMV (RRRage\u202f=\u202fRRage=31\u202fweeks\/RRyear=26\u202fweeks)\nA\u00a0sensitivity analysis was conducted by fitting a\u00a0second model (model\u202fB) with the most important variables, HLVS and LPVS, combined with whether or not a\u00a0Sensormedics ventilator was used. The reported RRRs were comparable to those in the first model. Type of ventilator did not have a\u00a0large effect compared with ventilation strategies (RRR\u202f=\u202f0.94 and RRR\u202f=\u202f0.90). The HLVS was associated with a\u00a0decrease of the RRs comparing HFV with CMV (RRR\u202f=\u202f0.44 and RRR\u202f=\u202f0.45), while LPVS increased the RRs to the line of no effect (RRR\u202f=\u202f2.06 and RRR\u202f=\u202f1.59).\nDiscussion\nOur meta-regression analysis showed a\u00a0clear trend of decreasing differences in pulmonary outcome between HFV and CMV in randomized trials conducted in premature neonates with IRDS over the years. The most likely hypothesis for this trend was the application of a\u00a0LPVS in the most recent studies. Use of surfactant could also have a\u00a0significant contribution, but only one study did not use surfactant\u00a0[12]. In previous meta-analyses, subgroup analyses or cumulative methods were used to explore heterogeneity\u00a0[4, 5, 7]. Subgroup analysis is equivalent to meta-regression with a\u00a0categorical trial-level covariate. Considering subgroup analysis formally as a\u00a0meta-regression has advantages, since it focuses on differences between subgroups as is appropriate, rather than the effects in each subgroup separately. Furthermore, it is appropriate to use meta-regression to explore sources of heterogeneity, even if an initial overall test for heterogeneity is non-significant. This test often has low power and therefore a\u00a0non-significant result does not reliably identify lack of heterogeneity\u00a0[25].\nIn this meta-regression analysis we evaluated in a\u00a0quantitative way a\u00a0number of hypotheses that were raised to account for different results between randomized trials. A\u00a0relatively large proportion of well-conducted trials were available for the analyses. For most explanatory variables there were important differences among trials. The effects of the two most important covariates, HLVS and LPVS, were consistent in the different models and were even increased in effect size by adjusting for other covariates. None of the competing hypotheses were more likely to influence results as shown by calculating the RRRs. Common pitfalls in meta-regression analysis can occur, such as multiple or post-hoc analyses, and lead to data dredging and a\u00a0high probability of false-positive conclusions\u00a0[25]. We, therefore, restricted our analyses to a\u00a0limited number of pre-specified explanatory covariates.\nPublication bias was considered unlikely as an explanation of the apparent diminishing relative effect of HFV. Publication bias is selection bias. If trials are selectively published either because of their size or because of significant results, this would result in an association between trial size and\/or precision and the trial outcome. Strictly speaking, funnel plots probe whether studies with little precision (small studies) give different results from studies with greater precision (larger studies). Asymmetry in the funnel plot may therefore result not from a\u00a0systematic under-reporting of negative trials but from an essential difference between smaller and larger studies that arises from inherent between-study heterogeneity\u00a0[26]; thus, if larger studies were also associated with changes in ventilation strategies and these strategies resulted in changes in reported RRs, the assumed publication bias would be, in fact, a\u00a0real association between ventilation strategy and study outcome; therefore, we conditioned the association between precision and effect size, presumably caused by publication bias, on ventilation strategies. This resulted in a\u00a0lower p-value for publication bias and more symmetrical distribution of studies in subgroups in the funnel plots; therefore, what appeared to be publication bias could also be explained by differences in ventilation strategies related to both study size and observed relative risks. However, it should be pointed out that the strength of this evidence is difficult to assess because fewer studies in the subgroups automatically resulted in less power to detect publication bias.\nOther alternative hypotheses that have been formulated to explain differences among studies were also less compatible with the evidence\u00a0[9]. The type of ventilator, Sensormedics vs other types of high-frequency ventilators, displayed RRR close to one. In the crude analyses, prior time on CMV before study initiation showed contradictory effects to what was hypothesized\u00a0[10]. The adjusted analyses showed conflicting results depending on the outcome. Gestational age and birth weight could also influence the magnitude of the effect of HFV compared with CMV. In the adjusted analysis gestational age did not change the RR for CLD but showed an increase of the RR for less premature neonates. Finally, an increased risk of CLD was not accompanied by a\u00a0greater relative benefit of HFV as compared with CMV.\nThe observed effects of continuous variables, such as time on CMV or gestational age, could be exaggerated by small studies with outlying results. For the covariate, time on CMV, the two largest studies showed results that were compatible with the hypothesis that this had no important impact on the results of these trials\u00a0[2, 3]. The same fact applied to the effect of baseline incidence of CLD or death or CLD. Gestational age and weight were comparable between the two largest trials, which made it more difficult to ascertain the relevance of the hypothesis that in smaller and more premature infants HFV performed better than CMV treatment. The observed direction of the effect of gestational age and birth weight, however, was opposite to what the hypothesis predicted. If gestational age was to be interpreted as a\u00a0higher risk of acquiring CLD, one would expect that an increase in the incidence of CLD was associated with a\u00a0relatively lower incidence of CLD in HFV treated patients; however, linear regression analysis showed perfectly equal increase in both treatment groups. Still, the possibility remains that the relationship with patient averages, such as gestational age and birth weight, across trials was not the same as the relationship for patients within trials, and therefore an effect of these patient characteristics cannot be excluded but only considered in relation to other covariates\u00a0[25].\nSimilar findings of the effects of ventilation strategies have been reported by us and other authors as well\u00a0[4, 5]; however, meta-analyses are subject to bias when differences among trials are used to explain differences in reported RRs. In this meta-regression analysis we were able to estimate adjusted association measures, thereby diminishing the effects of possible confounders\/effect-modifiers. By calculating less biased estimates of the effects of ventilation strategies and the effect of using a\u00a0Sensormedics ventilator instead of other ventilators on the outcome in the different HFV trials we were able to reinforce the hypothesis that ventilation strategies are more important than type of ventilator to prevent CLD.\nThe results of this meta-analysis stresses the importance of using appropriate ventilation strategies to prevent ventilator-induced lung damage in a\u00a0highly vulnerable group of patients; therefore, in clinical practice the question of how to use the ventilator is more important than the question of which ventilator should be used. The major theoretical advantage of HFV to CMV is delivery of smaller tidal volumes to an optimally recruited lung. As this meta-regression analysis did not confirm that subgroups of more premature neonates, avoidance of CMV prior to initiating HFV, or neonates with higher risk of CLD were more likely to benefit form elective HFV in IRDS, future research should be directed at identifying patients in whom HFV does have a\u00a0benefit over CMV. To improve the robustness of these conclusions and to avoid the limitations of meta-analysis of trials, an individual-patient-data-based meta-regression analysis should be conducted.\nConclusion\nIn conclusion, confining randomized trails to smaller or more premature children with IRDS did not seem to result in better pulmonary outcomes of HFV compared with CMV. A\u00a0generally held opinion that a\u00a0prolonged ventilation time on CMV prior to initiating HFV diminished the benefits of HFV was not in agreement with the current evidence. The most important effects resulting in differences among trials were probably caused by ventilation strategies applied in HFV- and CMV-treated patients.\nElectronic supplementary material\nElectronic Supplementary Material (DOC 514K)","keyphrases":["meta-regression analysis","high-frequency ventilation","infant","respiratory distress syndrome","conventional mechanical ventilation"],"prmu":["P","P","P","P","P"]}
{"id":"Virchows_Arch-4-1-2234444","title":"PEComas: the past, the present and the future\n","text":"The perivascular epithelioid cell (PEC) is a cell type constantly present in a group of tumors called PEComas. PEC expresses myogenic and melanocytic markers, such as HMB45 and actin. Recently, recurrent chromosomal alterations have been demonstrated in PEC. At present, PEComa is a widely accepted entity. In the past 10 years, the use of this term has allowed to report and describe numerous cases permitting to start highlighting the biology of this group of lesions. PEComas are related to the genetic alterations of tuberous sclerosis complex (TSC), an autosomal dominant genetic disease due to losses of TSC1 (9q34) or TSC2 (16p13.3) genes which seem to have a role in the regulation of the Rheb\/mTOR\/p70S6K pathway. There are some open questions about PEComas regarding its histogenesis, the definition of epithelioid angiomyolipoma and the identification of the histological criteria of malignancy. An innovative therapeutic trial using rapamycin is under way for tumors occurring in TSC such as renal angiomyolipoma and lymphangioleiomyomatosis. Its success could provide the rationale for the use of the same drug in other lesions composed of PECs, especially in the malignant ones.\nWhat is the perivascular epithelioid cell?\nThe perivascular epithelioid cell (PEC) (Fig.\u00a01) is a cell type constantly present in a group of tumors including angiomyolipoma (AML), clear-cell \u201csugar\u201d tumor (CCST) of the lung and extrapulmonary sites, lymphangioleiomyomatosis, clear-cell myomelanocytic tumor of the falciform ligament\/ligamentum teres and rare clear-cell tumors of other anatomical sites.\nFig.\u00a01Renal angiomyolipoma: perivascular epithelioid cells arranged around a blood vessel; H&E \u00d720\nIt has morphologic, immunohistochemical, ultrastructural and genetic distinctive features such as an epithelioid appearance with a clear to granular cytoplasm, a round to oval, centrally located nucleus and an inconspicuous nucleolus. PEC has mild to any atypia and a typical perivascular location [13]. At present, PEC has not a known normal counterpart.\nImmunohistochemically, PEC expresses myogenic and melanocytic markers, such as HMB45, HMSA-1, MelanA\/Mart1, microophtalmia transcription factor (Mitf), actin and, less commonly, desmin [13, 55, 126]. Its immunoreactivity for vimentin is usually incospicuous.\nAt ultrastructural analysis, PEC contains microfilament bundles with electron-dense condensation, numerous mithocondria and membrane-bound dense granules [11, 118].\nIt is thought that PEC can modulate its morphology and immunophenotype: given all the characteristics described above (Fig.\u00a02), PEC can show muscular features with a spindle shape and a stronger positivity for actin than for HMB45 or it can have an epithelioid feature with a strong positivity for HMB45 and a mild, if any, reaction for actin (Fig.\u00a03). PEC can also become vacuolized acquiring the feature of an adipocyte. Progesterone receptor positivity has been described in PEC with spindle morphology; this suggests a possible role of progesterone in this morphologic modulation [13, 107].\nFig.\u00a02Diagram demonstrating the modulation of morphology and immunophenotye of PECsFig.\u00a03Hepatic angiomyolipoma: strong granular HMB45 immunoreactivity in perivascular epithelioid cells; HMB45 \u00d720\nRecently, recurrent chromosomal alterations have been demonstrated in PEC [87].\nWhat is a PEComa?\nThe World Health Organization defines PEComa as \u201ca mesenchymal tumor composed of histologically and immunohistochemically distinctive perivascular epithelioid cells\u201d [37].\nAt present, this neoplasm is a widely accepted entity. However, some authors cast doubts on the existence of PEComa as a distinctive tumor, particularly as regards the uterine ones that they view as leiomyosarcoma with aberrant expression of HMB45 [102\u2013104]. Although we understand their perplexity, we would like to underline some points. First, it has been demonstrated that there are different gene expression profiles among uterine smooth muscle tumors, demonstrating their heterogeneity [105, 106, 119]. Second, we think that it is difficult to explain, in uterine smooth muscle neoplasms, both HMB45 and Mitf positivity as an aberrant expression or antibody crossreactivity involving both antigens [81]. Third, we think that the absence of a recognized normal counterpart for PEC is not a sufficient reason to reject the concept of PEComa; in fact, there are other well-accepted soft-tissue neoplasms, such as alveolar soft-part sarcoma and epithelioid sarcoma, that still have not a known normal counterpart.\nSome authors have questioned about the use of the term \u201cPEComa\u201d. They consider it ambiguous because it is not clear whether this term should be restricted to purely epithelioid tumors as CCST or whether the term should be more broadly applied to include AML and lymphangioleiomyomatosis [80, 111].\nWe believe that AML, CCST and pulmonary lymphangioleiomyomatosis are composed of PECs in different stages of modulation, and these lesions, together with clear-cell myomelanocytic tumor of the falciform ligament\/ligamentum teres, belong to the same family of tumors, the PEComas.\nNevertheless, we think that it is better to continue to name some of them (i.e. AML and pulmonary lymphangioleiomyomatosis) also with their established names which identify their clinical and morphological aspects and are well known by clinicians and pathologists [15].\nMoreover, in the past 10 years, the use of the term PEComa has permitted to report and describe numerous cases with the morphological and immunohistochemical features of this tumor permitting to start to understand the biology of this group of lesions.\nPEC and tuberous sclerosis\nPEComas are related to the tuberous sclerosis complex (TSC) or, better, to the genetic alterations of TSC, an autosomal dominant genetic disease due to losses of TSC1 (9q34) or TSC2 (16p13.3) genes [112, 116] and characterized by mental retardation, seizures and cellular proliferations (AMLs, subependymal giant cell tumors, cutaneous angiofibromas, cardiac rabdomyomas, lymphangioleiomyomatosis, pulmonary multifocal micronodular hyperplasia). Similar alterations of the TSC genes have been demonstrated in a significant number of PEComas, both occurring within the TSC and in sporadic cases.\nIn recent years, great advances have been made in our knowledge of TSC and related lesions. In particular, TSC genes seem to have an important role in the regulation of the Rheb\/mTOR\/p70S6K pathway [62].\nKenerson et al. [59] have recently demonstrated increased levels of phospho-p70S6K, a marker of mTOR activity, in sporadic AMLs. The associated reduced phospho-AKT expression is consistent with the disruption of TSC1\/2 function.\nSimilar findings were obtained analysing extrarenal PEComas.\nPEComa: the past\nPEC was first described in 1943 by Apitz [5] as an \u201cabnormal myoblast\u201d in renal AML, as reported by Masson [77] in his famous book.\nThe idea of a possible link between AML and other two lesions, CCST and lymphangioleiomyomatosis, came from the fact that the same epithelioid cell was observed in them. Moreover, this cell was HMB45-positive in all three lesions [9, 93, 94].\nLater, ultrastructural examination revealed the presence of premelanosomes both in CCSTs of the lung and in epithelioid clear-cell component of renal and hepatic AMLs [9, 42, 43, 93, 94, 118].\nIn addition, the possibility of a relationship between AML and lymphangioleiomyomatosis was suggested by the fact that both occur in TSC [10].\nWe proposed that CCST should be also included in this syndrome [10], and the first description of this tumor in a patient with TSC confirmed this idea [35].\nIn 1996, we described the first case of pancreatic CCST and suggested the name PEComa for neoplasms composed of a pure proliferation of PECs [125].\nMoreover, the fact that the same clear epithelioid cell composing pulmonary \u201csugar\u201d tumor was present in AML and was the only component of some small renal microhamartoma (Fig.\u00a04) suggested to us the idea of the existence of a monotypic epithelioid AML of the kidney. We looked for them, and we found them [96].\nFig.\u00a04Renal microhamartoma completely composed of perivascular epithelioid cells; H&E \u00d720\nPEComa: the present\nPEComas have been described in different organs and are considered ubiquitous tumors.\nKidney\nPEComas of the kidney include classic AML, microscopic AML (so-called microhamartoma), intraglomerular lesions, cystic AML, epithelioid AML, oncocytoma-like AML and lymphangiomyomatosis of the renal sinus.\nClassic angiomyolipoma is the most common mesenchymal tumor of the kidney. Being composed of a variable mixture of adipose tissue and spindle and epithelioid smooth muscle cells mixed together with abnormal thick-walled blood vessels, AML is the prototype of the capacity of PEC to modulate its morphology [29, 76].\nFor a long time, AML has been considered a hamartoma rather than a true neoplasm, but, at present, its clonal nature has been demonstrated [19, 57, 101]. In patients with TSC, renal AMLs are found in both sexes, in the third and fourth decades of life, with a female predominance; they are usually asymptomatic, bilateral, small and multifocal. Sporadic AMLs occur in older patients, in the fourth to sixth decades of life, with a female predominance; they are single, unilateral and larger than those associated with TSC [76]. Classic AML contains more than one cell type; if a particular cell type predominates, AML is consequently named (lipoma-like AML or leiomyoma-like AML) [6].\nClassic AML has a benign outcome. Multifocality and regional lymph node involvement can occur, and this is considered to represent a multifocal growth pattern rather than a metastasis [1, 110]. Three cases of sarcoma developing in sporadic AML have been reported. Similar cases have not been described in TSC patients [22, 32, 73].\nBoth inherited and sporadic AML frequently demonstrates loss of heterozigosity of chromosome 16p (containing the TSC2 locus). The TSC1 gene occasionally shows loss of heterozigosity [17, 50].\nMicroscopic angiomyolipomas (so-called microhamartomas) They are small nodules often present in a kidney with an AML. They are not homogeneous in appearance and display all the heterogeneous morphologic aspects of AML; microscopic AMLs do not contain thick-walled blood vessels [13, 20].\nIntraglomerular lesions with similar features of AML have been reported in patients with and without tuberous sclerosis and in the TSC2\/PKD1 contiguous gene syndrome, a disease with a deletion disrupting both TSC2 and PKD1 (autosomal dominant polycystic disease gene) [74].\nCystic angiomyolipoma is a recently described variant of AML [25, 33]. It is a solid-cystic lesion that, microscopically, is composed of epithelial cysts lined by cuboidal to hobnail cells, positive for cytokeratin; a compact subepithelial \u201ccambium-like\u201d layer of stromal cells positive for HMB45, Melan-A, CD10, estrogen and progesterone receptors and a solid extracystic component with the morphology of a muscle-predominant (Fig.\u00a05) AML, which is positive for HMB45, estrogen and progesterone receptors, smooth muscle actin and desmin and which is associated with irregular blood vessels. Some authors think that the subepithelial \u201ccambium-like\u201d layer is a manifestation of a mullerian differentiation of PEC. If the epithelial cysts represent entrapped renal tubular epithelium [33] or are also related to PEC is unknown.\nFig.\u00a05Cystic angiomyolipoma: strong positivity for actin in the solid extracystic component; SM ACT \u00d710Only 1 of the 16 cases described to date had a history of TSC [33].\nEpithelioid angiomyolipoma is another recently described variant of AML. It is composed of purely epithelioid cells with melanogenesis markers immunoreactivity arranged in sheets. Both adipocytes and abnormal blood vessels are not present. The cytoplasm of the neoplastic cells varies from faintly eosinophilic to clear. Tumor cells can display considerable nuclear atypia, and necrosis can be present. This tumor can recur locally and metastasise causing death. On the basis of histology alone, it is not possible to predict malignant behaviour in these neoplasms, and further data are needed to better define it. However, at the present time, all epithelioid AMLs should be closely followed clinically. Epithelioid AMLs has been described in patient with or without evidence of TSC and in the TSC2\/PKD1 contiguous gene syndrome.Loss of heterozigosity of TSC2 have been reported in some cases of sporadic epithelioid AML [28, 72, 74, 84, 96].\nOncocytoma-like angiomyolipomas are tumors composed of a homogeneous population of HMB45-positive polygonal cells with strongly eosinophilic cytoplasm. They have been described in patients with and without TSC. Recognition of this variant is important because oncocytomas in the same kidney with AMLs have been reported repeatedly and in patients with TSC oncocytomas seem to occur more frequently than in general population [75].\nLymphangiomyomatosis of the renal sinus is a plaque-like mass in the wall of the renal pelvis. All three cases reported to date also had renal AMLs, but in only one of them careful postmortem examination of the lungs revealed pulmonary lymphangiomyomatosis [74, 78].\nBladder and prostate\nIn 2003, Pan et al. [85, 86] have reported two PEComas of the genitourinary tract in patients without TSC. Both tumors were composed of a variable percentage of epithelioid and spindle cells with clear to granular cytoplasm arranged in nests separated by a vascular stroma. Neoplastic cells were positive for HMB45 but not for epithelial markers, vimentin and S100 protein. The prostatic tumor (Fig.\u00a06) showed a low mitotic activity, coagulative necrosis and a malignant behaviour, whereas the neoplasm of the bladder (Fig.\u00a07), lacking these histologic findings, behaved in a benign fashion. Another case of primary PEComa of the bladder has recently been described; also in this case, the patient is well and alive at 48 months after surgery [90].\nFig.\u00a06Prostatic PEComa composed of epithelioid cells with clear cytoplasm arranged in nests; H&E \u00d74Fig.\u00a07Bladder PEComa composed of epithelioid cells with clear cytoplasm arranged in nests, H&E \u00d74\nFinally, Weinreb et al. [120] have recently described a case of PEComa of the urachal cyst composed of pleomorphic cells and containing necrosis and a high mitotic activity. In this case, they also found a peculiar aspect that they named \u201cpecosis\u201d: remote from the tumor, there were isolated capillaries surrounded by a single layer of clear cells.\nUterus\nThe first case of PEC tumor of the uterus was reported by Pea et al. [95]: it was a polypoid neoplasm involving the endometrium, which showed morphological features overlapping those of the CCST of the lung (Fig.\u00a08).\nFig.\u00a08Uterine PEComa composed of epithelioid cells with a clear cytoplasm and well-defined cell borders; H&E \u00d710\nVang and Kempson [115] described eight cases of uterine PEC tumor (\u201cPEComa\u201d). They distinguished a morphologic spectrum of neoplasms varying from tumors with a tongue-like growth pattern composed of sheets of HMB45-positive clear epithelioid cells, which they called group A, to circumscribed tumors composed of hyalinized stroma and neoplastic cells focally positive for HMB45 and extensively immunoreactive for actin and desmin, which they refer to as group B. In two of these eight cases, pelvic lymph nodes contained lymphangiomyomatosis.\nLesions considered to be uterine involvement of lymphangioleiomyomatosis are usually asymptomatic, and some of them correspond to an incidental finding in patients with TSC.\nPEComas of the uterus have usually shown benign behaviour, but 13 tumors, 2 of them associated with TSC, were aggressive [16]. In two of six uterine PEComas described by Folpe et al. [39], patients have metastases and one of six patients died of PEComa.\nFadare et al. [31] described a case of uterine PEComa in a patient with TSC in which there was intra-abdominal \u201cPEComatosis\u201d: surgeon found small foci of epithelioid cells in the lamina propria of the small intestine, myometrium and ovarian hilum.\nOvary, vulva and vagina\nA tumor with a strong and diffuse HMB45 expression morphologically corresponding to an epithelioid angiomyolipoma has been reported in the ovary [4].\nTazelaar et al. [111] reported a case described as primary extrapulmonary clear-cell \u201csugar\u201d tumor of the vulva.\nFinally, cases of vaginal PEComa have also been described [39, 83].\nLung\nPEComas of the lung include lymphangioleiomyomatosis and clear-cell \u201csugar\u201d tumor.\nLymphangioleiomyomatosis (LAM) (Fig.\u00a09) is a rare and progressive disease that affects the lungs of women, usually in premenopausal age; occasionally, it can be extrapulmonary (as described above). It consists of a nodular, often widespread and bilateral interstitial proliferation of HMB45, actin and desmin-positive smooth muscle cells which can vary from small spindle-shaped cells to large epithelioid cells, usually arranged around thin-walled, branching vascular channels; this proliferation is associated with dilated lymphatics and cystic changes [9, 11, 18, 113].\nFig.\u00a09Pulmonary lymphangioleiomyomatosis composed of epithelioid cells arranged around a vascular channel; H&E \u00d720\nLAM is usually sporadic; patients with TSC are frequently afflicted.\nIn many cases of LAM, there is a slow progression to pulmonary failure, and the only therapy is lung transplantation. In no-transplanted patient, there is a median survival of 8\u201310 years [113].\nCCST (Fig.\u00a010) was originally described in the lung [68]. It is a rare and benign neoplasm composed of a uniform population of round-to-polygonal epithelioid cells, with a clear or eosinophilic cytoplasm and well-defined cell borders. Tumor cells are surrounded by prominent and thin-walled vascular channels. CCST has a nested or alveolar appearance. We have also observed adipocytic cells in a few cases of CCST [12]. Tumor cells are positive for HMB45 (Fig.\u00a011) [12, 41, 93].\nFig.\u00a010Pulmonary clear-cell \u201csugar\u201d tumor composed of epithelioid cells with a clear cytoplasm and well-defined cell borders. A prominent and vascular channel is present; H&E\u2009\u00d710Fig.\u00a011Pulmonary clear-cell \u201csugar\u201d tumor: HMB45 immunoreactivity in tumor cells; HMB45 \u00d720\nPulmonary CCST is rarely associated with TSC [35]; in the vast majority of cases, it is a sporadic lesion.\nPancreas\nPEComa of the pancreas was reported for the first time in 1996 by Zamboni et al. [125]. It was actually the first time that the very term PEC was introduced (Fig.\u00a012).\nFig.\u00a012Pancreatic clear-cell \u201csugar\u201d tumor: this tumor has overlapping features of the clear-cell \u201csugar\u201d tumor of the lung: epithelioid cells, with a clear cytoplasm and with a nested or appearance; H&E \u00d74\nThe paper reported a tumor with overlapping features of the \u201cbenign clear-cell sugar tumor of the lung\u201d. This finding was in our opinion consistent with the hypothesis that similar tumors could possibly arise in many if not all locations.\nThus the term PEComa was introduced to include all similar lesions arising outside the lung. At the time, we believed this to be a unique case. However, in the following years we have observed other two cases of PEComa of the pancreas with overlapping morphological, phenotypical and clinical features.\nThus, while still a very rare disease of the pancreas, PEComa is not an extraordinary finding in this location.\nThe clinical aspects of PEComa of the pancreas are interesting because they are discovered incidentally during echography of the abdomen and then investigated usually with cyto-aspiration. The observation of clear epithelioid cells can understandably lead to the wrong diagnosis of clear-cell carcinoma of the pancreas and thus lead to duodenocephalopancreasectomy.\nA primary angiomyolipoma of the pancreas has also been described. It was a cephalopancreatic nodule in a non-TSC woman [51].\nLiver\nApart from kidney, liver is the most likely organ involved by angiomyolipoma (Fig.\u00a013), either classic or epithelioid [44, 114, 118]. Frequently, hepatic AML shows a prominent component of large epithelioid cells [114]. There has also been described a case of CCST of the common bile duct [98].\nFig.\u00a013Hepatic angiomyolipoma: solid component made of large epithelioid cells; H&E \u00d710\nOther sites\nLymphangioleiomyomatosis has also been reported in extrapulmonary sites including mediastinal and retroperitoneal lymph nodes, soft tissue of the mesentery and the renal sinus, as previously described. Usually, extrapulmonary LAM presents as a localized well-circumscribed mass called \u201clymphangiomyoma\u201d [78].\nApart from kidney and liver, classic or even epithelioid angiomyolipoma can occur in different visceral and somatic sites as gastrointestinal tract, pelvis [14, 34, 47], nasal cavity [7], soft tissues [49] retroperitoneum [63], bone [53] and orbit [54].\nClear-cell \u201csugar\u201d tumor has been described in a variety of extrapulmonary sites, apart from uterus and pancreas: skin [26], soft tissues [40], breast [45], skull base [66], gastrointestinal tract [8, 111], inter-atrial cardiac septum [111] and oral mucosa [61].\nClear-cell myomelanocytic tumour of the falciform ligament\/ligamentum teres (CCMMT) is a PEComa showing predominantly spindled cell morphology.\nIt has been first described in 2000 by Folpe et al. [36] who presented seven cases of PEComa arising in the abdomen. All patients but one were women ranging from 3 to 21years of age. These neoplasms were composed of spindle cells arranged in fascicles and nests, positive for HMB45 and, in three cases, also for Melan-A, microophtalmia transcription factor (Mitf) and actin. Also in these cases, premelanosomes were ultrastructurally detected. At follow-up, in one patient presumed lung metastasis developed.\nCCMMT has also been described in thigh [38] and in the skin [79].\nPEComa: the future\nPEComas are a group of ubiquitous neoplasms sharing morphological, immunohistochemical, ultrastructural and genetic distinctive features.\nThere are some open questions about PEComas: the histogenesis and the normal\/physiological counterpart of PEC, the definition of epithelioid AML and the identification of the histological criteria of malignancy.\nThe histogenesis and the normal\/physiological counterpart of PEC are unknown, but some hypotheses have been proposed. One hypothesis is that PEC derives from undifferentiated cells of the neural crest that can express dual smooth muscle and melanocytic phenotype; a second hypothesis is that PEC has a myoblastic, smooth muscle origin with a molecular alteration that brings to expression of melanogenesis and melanocytic markers [107]; a third hypothesis is that PEC has a pericytic origin. As regards PEComa\u2019s histogenesis, the involvement of TSC pathway in these neoplasms can suggest some possibilities: it has been previously proposed that B-raf activity in cells lacking TSC2 may play a role in cell differentiation [56]. Moreover, TSC pathway regulates negatively Wnt\/beta-catenin pathway [71] and beta-catenin regulates transcription of genes involved in cell proliferation and differentiation. Nevertheless, more cases should be analysed to better understand PEC origin and PEComa\u2019s histogenesis.\nThe second issue regarding PEComas is which cases should be classified as epithelioid AML particularly in the kidney and liver, where AML occurs with higher frequency. We defined epithelioid AML as a neoplasm composed of purely epithelioid cells with melanogenesis markers immunoreactivity arranged in sheets, without adipocytes and abnormal blood vessels.\nHowever, in otherwise classic AML, areas of epithelioid cells can be observed, raising the question how much they should be represented to call a tumor \u201cepithelioid angiomyolipoma\u201d. The collection of such cases and a consensus meeting could be useful tools to answer to this problem.\nMalignant PEComa can be a very aggressive disease leading to multiple metastases and death as expected with a high-grade sarcoma [3, 14, 16, 32, 39, 52, 66, 67, 70, 85, 96, 99, 109, 121, 124]. A few malignant PEComas metastasised after several years (7\u20139 years) [27, 73, 89, 100] (Table\u00a01).\nTable\u00a01Reported cases of malignant PEComas in literatureReferenceDiagnosisSiteSex\/ageOutcomeCommentsSale and Kulander [100]CCSTLungn.a.\/n.a.AWD at 10\u00a0yearsHepatic metastases at 10\u00a0yearsFerry et al. [32]Epithelioid AMLKidneyF\/49DOD at 5.5\u00a0months\u00a0Al-Saleem et al. [3]Epithelioid AMLKidneyF\/21DOD at 3\u00a0months\u00a0Pea et al. [96]Epithelioid AMLKidneyF\/24DOD at 1\u00a0yearPelvic and hepatic metastasesPea et al. [96]Epithelioid AMLKidneyM\/29DOD at 18\u00a0monthsPulmonary and hepatic metastasesChristiano et al. [21]Epithelioid AMLKidneyM\/42AWD at 15\u00a0months\u00a0L\u2019Hostis et al. [67]Epithelioid AMLKidneyF\/71DOD at 2\u00a0years\u00a0Folpe et al. [36]PEComaLigamentum teres falciform ligamentM\/29DOC at 1\u00a0yearRadiographically suspected lung metastasis at 3\u00a0monthsMartignoni et al. [73]Epithelioid AMLKidneyF\/50AWD at 10\u00a0yearsLocal recurrence at 7\u00a0years; pulmonary, pelvic and abdominal metastases at 10\u00a0yearsBonetti et al. [14]PEComaUterusF\/41ANED at 6\u00a0monthsOvarian mass at presentationBonetti et al. [14]PEComaUterusF\/19AWD at 18\u00a0monthsAggressive local recurrence at 1\u00a0month; lung and bone metastases at 11\u00a0monthsBonetti et al. [14]PEComaTerminal ileum and cecumF\/28DOD at 28\u00a0monthsHepatic metastasisCibas et al. [22]Epithelioid AMLKidneyF\/49AWD at 6\u00a0months\u00a0Saito et al. [99]CCSTKidneyF\/23DOD at 1\u00a0yearRetroperitoneal recurrence at 3\u00a0monthsYamamoto et al. [122]Epithelioid AMLKidneyM\/47DODDOD with spinal, lung and lymph node metastases (autopsy case)Dimmler et al. [27]PEComaUterusF\/61AWD at 7\u00a0yearsLung metastasesGreene et al. [46]PEComaUterus and pelvic side wallF\/79DOD at several monthsPelvic and mesenteric recurrence at 2\u00a0yearsLau et al. [63]Epithelioid AMLRetroperitoneumM\/29ANED at 18\u00a0yearsLiver metastases at 9\u00a0years; thymus and lung metastases at 17\u00a0yearsLeclerc et al. [64]Epithelioid AMLKidneyF\/36ANEDLymph node metastasesLin et al. [69]Epithelioid AMLKidneyn.a.\/58AWDLiver and lymph node metastasesOng et al. [82]Epithelioid AMLKidneyF\/74AWDAdrenal metastasesPan et al. [85]PEComaProstateM\/46DOD at 4\u00a0yearsSubmitted to adjuvant chemotherapy; lung metastases at 3\u00a0yearsTakahashi et al. [109]Epithelioid AMLKidneyF\/40DOD at 18\u00a0monthsSubmitted to adjuvant chemotherapy; pulmonary metastasesTakahashi et al. [109]Epithelioid AMLKidneyM\/44DOD at 60\u00a0monthsSplenic and retroperitoneal metastases at 3\u00a0years; hepatic metastases at 60\u00a0monthsYanai et al. [123]PEComaJejunumF\/32AWD at 25\u00a0monthsPelvic wall recurrence\/local metastases at 13\u00a0months; ovarian metastases at 25\u00a0monthsDarai et al. [24]Epithelioid AMLUrachF\/n.a.AWDLymph node metastasesHarris et al. [49]PEComaSoft tissue near kneeM\/87AWD at 40\u00a0monthsInguinal lymph node and lung metastases at 13\u00a0monthsLehman [66]PEComaSkull baseF\/49DOD at 3\u00a0monthsParaspinal and lung metastasis at 6\u00a0weeksBosincu et al. [16]PEComaUterusF\/59DOD at 1\u00a0yearPelvic recurrence at 6\u00a0monthsEvert et al. [30]PEComaRectovaginal spaceF\/56AWDPulmonary metastases at presentationFolpe et al. [39]PEComaNeckF\/77ANED at 6\u00a0monthsRe-excision and submitted to adjuvant radiotherapyFolpe et al. [39]PEComaForearmM\/71ANED at 10\u00a0monthsRe-excision and submitted to adjuvant radiotherapyFolpe et al. [39]PEComaBroad ligamentF\/16ANED at 18\u00a0monthsRe-excisedFolpe et al. [39]PEComaFalciform ligamentF\/15ANED at 35\u00a0monthsRe-excisedFolpe et al. [39]PEComaUterusF\/56AWD at 11\u00a0yearsSubmitted to adjuvant radio and chemotherapy; lung and bone metastasesFolpe et al. [39]PEComaPelvic soft tissueF\/72AWD at 15\u00a0monthsLocal recurrenceFolpe et al. [39]PEComaOmentumM\/40AWD at 24\u00a0monthsExtensive intra-abdominal recurrence\/local metastasesFolpe et al. [39]PEComaUterusF\/59AWD at 30\u00a0monthsSubmitted to adjuvant chemotherapy; liver and lung metastases at 30\u00a0monthsFolpe et al. [39]PEComaMesenteryF\/46DOD at 27\u00a0monthsSubmitted to adjuvant chemotherapy; extensive intra-abdominal recurrence and liver metastases at 22\u00a0monthsFolpe et al. [39]PEComaUterusF\/36DOD at 39\u00a0monthsSubmitted to adjuvant chemotherapy; lung metastases at 12\u00a0months and liver metastases at 36\u00a0monthsSvec and Velenska [108]Epithelioid AMLKidneyF\/47ANED at 5\u00a0monthsRecurrence at 3\u00a0monthsYu et al. [124]Epithelioid AMLKidneyF\/12DOD at 9\u00a0monthsLymph node metastases; submitted to adjuvant chemotherapyAgaimy and W\u00fcnsch 2006 [2]PEComaIleumF\/63AWD at 14\u00a0monthsAbdomino-pelvic recurrenceKim et al. [60]CCMTBroad ligamentF\/12AWD at 1\u00a0yearIliac fossa recurrenceMai and Belanger [70]PEComaThighM\/56DOD at 1\u00a0yearPulmonary and brain metastasesMai and Belanger [70]PEComaThighF\/60DOD at 1\u00a0yearPulmonary metastasesMai and Belanger [70]PEComaGroinM\/46DOD at 2\u00a0yearsLymph node metastasesParfitt et al. [89]PEComaLiverF\/60AWD at 10\u00a0yearsHepatic recurrence and pulmonary, pancreatic and muscular metastases at 9\u00a0years; bladder metastases at 10\u00a0yearsParfitt et al. [91]PEComaLung\/Adrenal glandF\/53AWDBrain metastases at several mothsYamamoto et al. [121]PEComaColonF\/43DOD at 38\u00a0monthsPeritoneal dissemination at 20\u00a0monthsGupta et al. [48]Epithelioid AMLRetroperitoneumF\/80AWD at 1\u00a0yearHepatic and rib metastasesHuang et al. [52]Epithelioid AMLKidneyF\/78DOD at 5\u00a0months\u00a0Park et al. [92]Epithelioid AMLKidneyM\/69AWD at 8\u00a0monthsHepatic and peritoneal metastasesPark et al. [92]Epithelioid AMLKidneyF\/46AWD at 12\u00a0monthsHepatic and lymph node metastasesWeinreb et al. [120]PEComaRetroperitoneumF\/49AWDBrain and lung metastasesANED Alive, not evidence of disease; AWD alive with disease; DOC dead of other causes; DOD dead of disease; n.a. not available\nRecently, Folpe et al. [39] reported 26 cases of PEComas of soft tissue and gynaecologic origin proposing criteria for the classification of these tumors as \u201cbenign\u201d, \u201cof uncertain malignant potential\u201d and \u201cmalignant\u201d. In this study, they observed a significant association between tumor size >5cm, infiltrative growth pattern, high nuclear grade, necrosis and mitotic activity >1\/50 HPF and subsequent aggressive clinical behaviour of PEComas.\nWe think that this approach is the best available at the moment. While more cases with long follow-up are needed to verify the effectiveness of this prognostic classification, we believe that all cases of PEComa should be classified according to the criteria proposed by Folpe et al.\nAnother future challenge regarding PEComas is their management.\nSurgery seems to be the only approach for aggressive cases, as chemio- and radiotherapy has not shown significant results. However, this derives from anecdotical cases as no therapeutic trial has so far been implemented. There are obvious difficulties to perform a therapeutic trial mainly due to the rarity of the disease. An international cooperative study is needed to address this problem.\nA very different problem is posed by lymphangioleyomyomatosis. In fact, lymphangioleyomyomatosis is composed of a population of cells with no atypia. Mitotic activity is virtually absent or extremely low. In spite of this, the disease usually progresses inexorably towards lung-function impairment.\nRecently, Kenerson et al. [59] demonstrated TSC1\/2 inactivation and m-TOR hyperactivation in non-TSC AMLs and in extrarenal PEComas using immunohistochemistry and Western blot analysis. In particular, m-TOR hyperactivation can be studied in such lesions using immunohistochemical detection of p70S6K. As we have previously mentioned, p70S6K is a protein kinase activated by m-TOR. Immunohistochemical reaction for p70S6K can stain both p70S6K, which gives a cytoplasmic signal (Fig.\u00a014), and a second isoform of kinase, p85S6K, which is also activated by m-TOR and which gives a nuclear signal.\nFig.\u00a014Hepatic angiomyolipoma: cytoplasmic signal in immunohistochemical reaction for p70S6K; p70S6K \u00d710\nRapamycin is a specific inhibitor of m-TOR [23] which is approved by the US Food and Drug Administration for immunosuppression therapy after renal transplant [117], and it has recently been approved also for therapy of renal cell carcinomas and acute myeloid leukemia [88, 97]. Preclinical studies in animal models of TSC have shown significant in vivo response to rapamycin [58, 65].\nAs regards LAM, an innovative therapeutic trial is under way. Rapamycin seems to block signaling through the Akt cell growth and survival pathway (for up-to-date information, see http:\/\/www.thelamfoundation.org).\nA similar trial has already shown a positive effect on renal AML, and we can hope to see the same effect on LAM.\nIf this will be the case, this could provide the rationale for the use of the same drug in other lesions composed of PECs.","keyphrases":["pecoma","pec","tsc","mtor","angiomyolipoma","rapamycin","lymphangioleiomyomatosis","sugar tumor"],"prmu":["P","P","P","P","P","P","P","P"]}
{"id":"Mol_Genet_Genomics-3-1-2129107","title":"Identification of InuR, a new Zn(II)2Cys6 transcriptional activator involved in the regulation of inulinolytic genes in Aspergillus niger\n","text":"The expression of inulinolytic genes in Aspergillus niger is co-regulated and induced by inulin and sucrose. We have identified a positive acting transcription factor InuR, which is required for the induced expression of inulinolytic genes. InuR is a member of the fungal specific class of transcription factors of the Zn(II)2Cys6 type. Involvement of InuR in inulin and sucrose metabolism was suspected because of the clustering of inuR gene with sucB, which encodes an intracellular invertase with transfructosylation activity and a putative sugar transporter encoding gene (An15g00310). Deletion of the inuR gene resulted in a strain displaying a severe reduction in growth on inulin and sucrose medium. Northern analysis revealed that expression of inulinolytic and sucrolytic genes, e.g., inuE, inuA, sucA, as well as the putative sugar transporter gene (An15g00310) is dependent on InuR. Genome-wide expression analysis revealed, three additional putative sugar transporters encoding genes (An15g04060, An15g03940 and An17g01710), which were strongly induced by sucrose in an InuR dependent way. In silico analysis of the promoter sequences of strongly InuR regulated genes suggests that InuR might bind as dimer to two CGG triplets, which are separated by eight nucleotides.\nIntroduction\nSome plants store part of their carbon as sucrose and as polymers of fructose (fructans). Plant fructans, often referred to as inulins, have predominantly \u03b2-2,1 linkages between the fructose moieties and terminate with a sucrosyl residue. Inulin is present in a range of plant species. In some species such as chicory, garlic, Jerusalem artichoke and Dahlia tubers about 15\u201320% of the plant fresh weight consists of inulin (Gupta and Kaur 1997; Kaur and Gupta 2002). Apart from the use of inulin for the production of fructose rich syrups, inulin and fructo-oligosaccharides (FOS) have attracted considerable research attention because of the large number of health benefits obtained from inulin and FOS in the diet of humans (see for a review Kaur and Gupta 2002).\nAspergillus niger is a saprophytic fungus, mainly present in the soil, feeding preferably on organic matter such as plant cell wall polysaccharides (cellulose, hemi-cellulose and pectin), and on plant storage polysaccharides (starch and inulin). A. niger is able to produce various inulin degrading enzymes including an exo-inulinase, an endo-inulinase and an invertase (Moriyama et al. 2003; Ohta et al. 1998; Akimoto et al. 1999; Berges et al. 1993; Boddy et al. 1993; L\u2019Hocine et al. 2000). The inulinolytic enzymes are simultaneously induced in the presence of inulin or sucrose, indicating that the expression of the genes encoding these enzymes is co-regulated and controlled by a common transcription factor (Yuan et al. 2006).\nTranscriptional activation of catabolic enzyme networks is often accomplished through transcription factors of the Zn(II)2Cys6 type. This type of transcription factor is unique to fungi. Gal4 is the transcriptional activator for the expression of genes involved in galactose utilization, and is the best characterized Zn(II)2Cys6 transcription factor. In general, Zn(II)2Cys6 transcription factors possess a well conserved N-terminal localized DNA binding motif (CX2CX6CX5\u201316CX2CX6\u20138), followed by a less well defined conserved domain known as a fungal specific transcription factor domain (Pfam04082). The C-terminal part of the Zn(II)2Cys6 transcription factor normally contains the activation domain. In the genome sequence of Saccharomyces cerevisiae, 54 putative Zn(II)2Cys6 transcription factors have been identified (Akache et al. 2001). They are involved in the transcriptional control of a wide variety of cellular processes (Todd and Andrianopoulos 1997; Akache et al. 2001) including genes involved in amino acid metabolism (ArgRp, Crabeel et al. 1995; Leu3p, Kohlhaw 2003; Lys14p, El Alami et al. 2000), sugar metabolism (Mal63p, Ni and Needleman 1990), pyrimidine biosynthesis (Ppr1p, Flynn and Reece 1999). In the genome of filamentous fungi even more Zn(II)2Cys6 transcription factors are present. In the recently released genome sequence of A. niger CBS 513.88, 296 ORFs were identified that contain the Zn(II)2Cys6 motif and similar numbers are predicted from the genomes of A. fumigatus and A. nidulans (see for comparison Pel et al. 2007). The role of only a few of the Zn(II)2Cys6 transcription factors in filamentous fungi have been studied in detail. Filamentous fungal Zn(II)2Cys6 proteins are involved in the regulation of various processes which include secondary metabolite production (e.g. AflR, Woloshuk et al. 1994), pigmentation (e.g. Cmr1, Tsuji et al. 2000), nitrogen metabolism (NirA, Burger et al. 1991), proline utilization (PrnA, Cazelle et al. 1998; Leucine biosynthesis (Leu3p, Kirkpatrick and Schimmel 1995), alcohol utilization (AlcR, Kulmburg et al. 1992) and sexual development (RosA, Vienken et al. 2005). Well studied examples of fungal Zn(II)2Cys6 transcription factors required for polysaccharide catabolism include AmyR, a transcriptional activator of starch degrading enzymes (Petersen et al. 1999; Gomi et al. 2000; Tani et al. 2001a), and XlnR, a transcriptional activator for xylanolytic enzymes (van Peij et al. 1998a, 1998b; Gielkens et al. 1999; Hasper et al 2004).\nIt is well established that the genes encoding many proteins required for secondary metabolite production are clustered in the genome (Woloshuk et al. 1994; Walton 2000). In addition to several biosynthetic enzymes that synthesize the secondary metabolites, such clusters also contain a Zn(II)2Cys6 transcription factor which is required for the transcriptional activation of the genes within the cluster. Gene clustering is not limited to secondary metabolite pathways, but also found for the proline utilization genes (PrnA, Gomez et al. 2002) and for some of the amylolytic genes under the control of the regulator AmyR. Adjacent to the gene for the AmyR transcription factor are genes encoding alpha-glucosidase (AgdA) and an alpha-amylase (AmyA) (Gomi et al. 2000). Also in A. parasiticus, a sugar utilization gene cluster has been identified (Yu et al. 2000). However, it is clear that clustering of genes with related functions is not a general rule, since the genes adjacent to the XlnR transcription factor do not seem to be involved in xylan degradation.\nIn a previous study, we showed that the expression of the genes encoding inulin degrading enzymes is co-regulated and specifically induced in the presence of inulin and sucrose (Yuan et al. 2006). The availability of the genome sequence of A. niger allowed the rapid identification of Zn(II)2Cys6 transcription factors adjacent to or in close proximity to the genes encoding (putative) enzymes involved in inulin catabolism. We have identified a Zn(II)-cluster transcription factor (InuR) which is adjacent to a gene encoding a sugar transporter and a recently identified intracellular invertase (SucB) with transfructosylation activity (Goosen et al. 2007).\nIn this paper, we show that deletion of the inuR gene results in strongly impaired growth on inulin and sucrose, and also abolishes the induction of the genes encoding the extracellular enzymes involved in inulin and sucrose degradation. The gene encoding the putative sugar transporter located next to inuR is co-regulated with the genes encoding the inulinolytic enzymes. These results show that InuR acts as a positive acting transcriptional activator for the induced expression of genes involved in the breakdown of inulin and sucrose and the uptake of inulin breakdown products.\nMaterials and methods\nStrains, culture conditions and transformation\nAspergillus niger strain N402 used in this study was derived from the wild-type strain A. niger van Tieghem (CBS 120.49, ATCC 9029) (Bos et al. 1988). The A. niger strain used by DSM to sequence the genome is NRRL 3122. Strain AB4.1 is a pyrG negative derivative of N402 (van Hartingsveldt et al. 1987) and was used to construct disruption strains. A. niger strains were grown in Aspergillus minimal medium (MM) (Bennet and Lasure 1991), or Aspergillus complete medium (CM) consisting of MM medium with the addition of 0.5% (w\/v) yeast extract and 0.1% (w\/v) casamino acids. Growth medium was supplemented with 10\u00a0mM uridine (Serva) when required. For shake flask cultures, A. niger strains were grown in MM supplemented with 1% (w\/v) carbon source and 0.1% (w\/v) casamino acids. The conidiospores were inoculated at a concentration of 2\u00a0\u00d7\u00a0106 spores per ml. Glucose and sucrose (BDH chemicals), maltodextrin (Avebe), starch (Windmill Starch, Avebe), xylose, fructose and maltose (Sigma-Aldrich), raffinose (Sigma chemicals) and inulin (Sensus Frutafit, Cosun) were used as carbon sources.\nFor transfer experiments, A. niger strains were pre-grown in MM supplemented with 2% (w\/v) xylose and 0.1% (w\/v) casamino acids for 18\u00a0h at 30\u00b0C on a rotary shaker at 300\u00a0rpm. Then mycelium was harvested by suction over a nylon membrane and washed with MM without carbon source. Aliquots of 1.6\u00a0g wet weight of mycelium were transferred to 300-ml Erlenmeyer flasks containing MM supplemented with 1% (w\/v) carbon source and incubated at 30\u00b0C for the time indicated in the text. The mycelium was harvested over Myracloth (Calbiochem) and frozen in liquid nitrogen, followed by storage at \u221280\u00b0C prior to the isolation of total RNA. Conidiospores were obtained by harvesting spores from CM plates after 4\u20136\u00a0days of growth at 30\u00b0C, using a 0.9% NaCl solution. Transformation of A. niger AB4.1 was done as described by Punt and van den Hondel (1992) using lysing enzymes (L1412, Sigma) for protoplastion. The bacterial strain used for transformation and amplification of recombinant DNA was Escherichia coli XL1-Blue (Stratagene). Transformation of XL1-Blue was performed according to the heat shock protocol as described by Inoue et al. (1990).\nConstruction of the inuR::pyrG deletion strain\nThe A. nigerinuR gene was deleted by the replacement of the complete open reading frame of the inuR gene with the Aspergillus oryzaepyrG gene. The plasmid used to disrupt inuR was constructed as follows. The DNA fragments flanking the inuR ORF were amplified by PCR using N402 genomic DNA as template. Fragments with a length of 0.8\u00a0kb of 5\u2032 flanking DNA and 1.0\u00a0kb of 3\u2032 flanking DNA were amplified using primers inuRP1 and inuRP2, inuRP3 and inuRP4 (Supplementary Table\u00a01), respectively. Each primer was adapted with restriction sites as indicated for further cloning. The amplified PCR fragments were digested with NotI and XhoI or XhoI and KpnI, respectively, and cloned into pBlue-Script II to obtain plasmid pInuRF5 and pInuRF3. Subsequently, the 0.8\u00a0kb NotI-XhoI fragment containing 5\u2032 flanking of inuR from pInuRF5, was ligated into NotI\/XhoI digested pInuRF3 to give pInuRF53. The A. oryzaepyrG gene was isolated as a 2.8\u00a0kb SalI\u2013BamHI fragment, obtained from plasmid pAO4-13 (de Ruiter-Jacobs et al. 1989) and ligated into XhoI\/BamHI digested pInuRF53 to obtain p\u0394inuR (pXY3.1). Before transformation, the plasmid p\u0394inuR was linearized with NotI and transformed into A. nigerpyrG- strain AB4.1. Uridine prototrophic transformants were selected by incubating protoplasts on agar plates containing MM without uridine. Transformants were screened by PCR using primers PAO9 and inuRP5 or PAO10 and inuRP6 (Supplementary Table\u00a01). Only transformants with a targeted deletion of the inuR gene should result in the amplification of a 1.2 or 1.4\u00a0kb PCR fragment, respectively. PCR positive transformants were verified by Southern blot analysis as described by Sambrook et al. (1989).\nChromosomal DNA from positive PCR transformants was isolated as described by Kolar et al. (1988). Ten \u03bcl of genomic DNA was digested for 3\u00a0h with 10\u00a0U of XhoI or EcoRI, respectively. The 1.0\u00a0kb of the 3\u2032-flanking DNA fragment of inuR was used as probe for the detection of inuR disruptants. A probe was generated by digestion of the pInuRF3 plasmid containing the inuR 3\u2032-flanking fragment with XhoI and BamHI. Fragments were purified from gel and [\u03b1-32P]dCTP labeled probes were synthesized using Rediprime II DNA labeling System (Amersham Pharmacia Biotech) according to the instructions of the manufacturer.\nComplementation of inuR mutant\nThe A. niger inuR gene was amplified by PCR using primers inuRP7 and inuRP8 (Supplementary Table\u00a01) and N402 genomic DNA as a template. The primers corresponded to 1\u00a0kb upstream and 0.5\u00a0kb downstream of the A. niger inuR gene, respectively. PCR was performed using Phusion\u2122 High-Fidelity DNA Polymerase (Finnzymes) according to the manufacturer\u2019s instruction. The amplified 4.1\u00a0kb PCR fragment was cloned into pGEMT-easy vector (Promega) to obtain the inuR complementation plasmid pXY5.1. pXY5.1 was then co-transformed with pAN7.1 harboring hygromycin gene as selection marker (Punt et al. 1987) into A. niger \u0394inuR strain XY3.1 (see in the text). Hygromycin resistant transformants were selected by incubating protoplasts on agar plates containing MM with 200\u00a0mg\/ml of hygromycin. Southern blot analysis was performed on selected transformants as described by Sambrook et al. (1989).\nConstruction of the \u0394inuR \u0394amyR double deletion strain\nTo make a pyrG-strain of the \u0394inuR strain (XY3.1) this strain was incubated on MM plates containing 1\u00a0mg\/ml of 5\u2032FOA and 10\u00a0mM uracil. 5\u2032FOA resistant, uracil requiring mutants were transformed with pAB4.1 harboring the A. nigerpyrG or with pPyrE harboring the A. niger pyrE gene on a 4.3\u00a0kb SstII subclone, respectively. One of the selected 5\u2032FOA resistant mutants, XY4.1, was complemented with the pyrG gene, indicating that the uracil auxotrophy was caused by a mutation in the pyrG gene. The strain XY4.1 was then used to construct a \u0394inuR \u0394amyR double disruption strain by transforming plasmid p\u0394amyR which also contains the pyrG selection marker (Yuan et al., submitted). Uridine prototrophic transformants were selected by incubating protoplasts on agar plates containing MM without uridine. Transformants that showed normal growth on glucose but not on starch were selected. One possible \u0394inuR\u0394amyR double mutant (XY5.1) was verified by Southern analysis and used for further analysis.\nNorthern blot analysis\nTotal RNA isolation, Northern analysis and synthesis of DNA probes was performed as described in Yuan et al. (2006). The primers used to generate probe An15g00310 are shown in Supplementary Table\u00a01.\nMicrotiter plate growth assay\nGrowth of A. niger strains were determined using a HTS7000 BioAssay Reader (Perkin Elmer Life Sciences). Spores (1\u00a0\u00d7\u00a0104) were inoculated in each well of a 96-well microtiter plate (Nalge Nunc International, USA) and incubated at 32\u00b0C for 56\u00a0h. Each well contained 200\u00a0\u03bcl of MM containing 1% (w\/v) of one of the various carbon sources each supplemented with 0.01% (w\/v) casamino acids to stimulate spore germination. Six replicates of each condition were made. Growth was monitored by measuring turbidity (OD595) at 2-h intervals.\nMicroarray experiments and data analysis\nRNA extracted from the A. niger \u0394inuR strain and wild-type strain (N402) grown on different carbon sources were used for microarray experiments using custom made \u201cdsmM_ANIGERa_coll\u201d Affymetrix GeneChip\u00ae microarrays kindly provided by DSM Food Specialties (Delft, The Netherlands). For microarray experiments, mycelia were isolated from grown cultures by transfer experiment (see above) and each growth condition was performed in duplicate as independent biological experiments.\nTotal RNA was isolated from mycelia using TRIzol reagent (Invitrogen) and RNA quality was verified by analyzing aliquots with glyoxal\/DMSO gel electrophoresis and Agilent Bioanalyzer \u201cLab-on-chip\u201d system (Agilent Technologies, USA). Processing, labeling and hybridization of cRNA to A. niger Affymetrix GeneChips were performed according to the corresponding Affymetrix protocols for \u201cEukaryotic Target Preparation\u201d and \u201cEukaryotic Target Hybridization\u201d. For probe array washing and staining, the protocol \u201cAntibody Amplification for Eukaryotic Targets\u201d was followed. Hybridized probe array slides were scanned with Agilent technologies G2500A Gene Array Scanner at 3\u00a0\u03bcm resolution and a wavelength of 570\u00a0nm. Affymetrix Microarray Suite software MAS5.0 was used to calculate the signal and P values and to set the algorithm\u2019s absolute call flag, which indicates the reliability of the data points according to P (present), M (marginal) and A (absent). The data on each chip were globally scaled to an arbitrary target gene intensity of 500.\nThe pre-scaled chip data from each hybridization experiment were normalized using Genespring 7.0 software (Silicon Genetics). Normalization with default parameters in Genespring software (Per Chip: Normalize to 50th percentile, Per Gene: Normalize to median) was used. For this study, we focused on sucrose induced genes. Therefore, pre-filtering of data was performed to select for genes whose detection calls are present in both sucrose duplicate samples of wild-type strain N402. The selected data set was further performed for one-way ANOVA analysis. Fold changes in expression levels between two different conditions were then computed for genes with P\u00a0<\u00a00.05 based on One-way ANOVA analysis and changes by more than twofold were considered significant and are reported here. Microarray data were deposited into ArrayExpress with an accession number 19095 at http:\/\/www.ebi.ac.uk\/at-miamexpress.\nIn silico analysis of regulatory elements of co-regulated genes\nThe 1.0\u00a0kb promoter sequences of the seven sucrose induced InuR dependant genes: An12g08280 (InuE), An11g03200 (InuA), An08g11070 (SucA), An15g04060, An15g00310, An15g03940, and An17g01710 were obtained from the CBS513.88 strain. The 1.0\u00a0kb promoter regions were analyzed on both strands using DNA Analyzer module of Phylosopher 6.5.1 (Genedata A.G., Basel, Switzerland) using the Gibbs Sampling Strategy for Multiple Alignment (Lawrence et al. 1993).\nResults\nIdentification of potential transcriptional regulators involved in inulin catabolism\nA. niger is able to produce various inulinolytic enzymes involved in the modification or degradation of inulin and sucrose. The transcriptional regulation of the genes encoding the inulinolytic enzymes of A. niger, including an exo-inulinase (InuE; An12g08280), an endo-inulinase (InuA; An11g03200), and an invertase (SucA; An08g11070), has been studied previously (Moriyama et al. 2003; Yuan et al. 2006). In addition to the extracellur enzymes, two additional potential intracellular inulinolytic enzymes (SucB; An15g00320 and SucC; An06g02420) were predicted from the A. niger genome sequence (Yuan et al. 2006). These proteins contain all the conserved domains of the GH32 family to which inulinolytic enzymes belong. Finally, a potential ORF was found in the genome which showed homology to the group of exo-inulinases. Because the predicted ORF lacked several of the conserved domains and contained several frame shift mutations, this gene (inuQ, An11g03210) was considered to be a pseudogene (Yuan et al. 2006).\nThe expression of the genes encoding the extracellular inulinolytic enzymes is co-regulated and induced on inulin and sucrose, suggesting that these genes are under control of a single transcription factor (Yuan et al. 2006). Some transcriptional activators involved in sugar catabolism are clustered in the genome with their target genes (Gomi et al. 2000; Yu et al. 2000). To identify possible candidate transcription factors involved in the regulation of inulinolytic genes, we searched the A. niger genome sequence for transcription factors located close to the genes encoding the inulinolytic enzymes. We found that inuA (An11g03200) and inuQ (An11g03210) are located next to each other and that also a Zn(II)2Cys6 transcription factor encoding gene (An11g03220) was located adjacent to inuQ. The orientation of the three ORFs is in the same direction indicating that they do not share a common promoter region (Fig.\u00a01).\nFig.\u00a01Schematic representation of the clustering of putative transcription factors with inulinolytic genes in the genome of A. niger. An11g03220 and An08g11040 and inuR encode putative transcription factors. The genes related to inulin degradation include: inuA (endo-inulinase), inuQ (pseudogene), sucA (invertase), sucB (homologous to sucA) and An15g00310 (putative sugar transporter). An08g00160 encodes a hypothetical protein without known function. The arrows indicate the transcriptional orientations of the genes\nIn the proximity of sucA (An08g11070), a second transcription factor encoding gene was found. This transcription factor contains a Cys2His2 zinc finger DNA binding motif. Between this gene, An08g11040 and sucA an additional ORF (An08g11060) is present. This 273 amino-acid protein encodes a hypothetical protein without any conserved motifs. A BlastP search revealed that this protein has no close orthologs in other fungal genomes. The transcription factor, encoded by An08g11040, shows strong sequence similarity to the S. cerevisiae Zpr1p (e-value 1e-100), an essential transcription factor that contributes to normal cell proliferation (Gangwani et al. 1998).\nClustered with sucB (An15g00320) a third putative transcription factor encoding gene (An15g00300) was found which also belonged to the Zn(II)2Cys6 transcription factor family. Between sucB and An15g00300, an additional ORF (An15g00310) is located (Fig.\u00a01). The protein encoded by this gene has all the characteristics of a sugar transporter protein. In the direct proximity of the two remaining genes encoding inulinolytic enzymes (InuE and SucC) no potential transcription factors were found. The three transcription factors identified via clustering were considered to be good candidates to be involved in the transcriptional regulation of the inulinolytic system of A. niger. In order to study their possible role in inulin utilization, deletion strains of these transcription factors were constructed. Deletion of An11g03220 did not have any measurable effect on growth on inulin or sucrose in comparison with the wild-type strain, which indicates that this gene is not required for inulin utilization. Growth of the deletion strain on xylose, glucose, fructose, starch and maltose was also identical to the growth of the wild-type strain (data not shown). A deletion strain of An08g11040 was never obtained although over 500 transformants were screened. A possible explanation might be that An08g11040 is an essential gene. As indicated above, An08g11040 is most homologous to the essential Zpr1p transcription factor of S. cerevisiae. Since the homology with Zpr1p does not indicate involvement of this transcription factor in inulin utilization, no further attempts were made to obtain the deletion strain. As shown in detail below, deletion of An15g00300 resulted in a strain which showed a severe growth defect on inulin and on sucrose. For the remainder of the paper, we will refer to An15g00300 as inuR.\nInuR homologs were also identified in the genomes of other Aspergilli. The alignment of the InuR proteins is given in Fig.\u00a02. The strong conservation of the transcription factor among Aspergilli, suggest that the regulation of inulinolytic gene expression is mediated in the various Aspergilli by the InuR proteins present in the different fungi.\nFig.\u00a02Alignment of InuRp and its orthologs in Aspergilli. The amino acid sequences of the Zn2Cys6 binuclear DNA-binding motif (Pfam00172) and the Fungal specific transcription factor domain (Pfam04082) are underlined. The conserved six cystein residues are indicated by stars. AoInuRp, AgInuRp, AfInuRp and AdInuRp are InuR protein orthologs from A. oryzae, A. niger, A. fumigatus and A. nidulans, respectively\nInuR encodes a Zn(II)2Cys6 transcription factor that is required for growth on inulin\nBased on the analysis of the CBS513.88 genomic sequence, the predicted open reading frame of the inuR encoding gene is 2,466\u00a0bp long and interrupted by 4 introns with sizes of 153, 80, 58 and 48\u00a0bp, respectively. The predicted InuR protein sequence is comprised of 709 amino acid residues which would result in a protein with a calculated molecular mass of 78.3\u00a0kDa. Analysis of the predicted InuR protein indicated that InuR contains two conserved domains. One is the Zn(II)2Cys6 (CX2CX6CX5CX2CX6C) binuclear cluster which represents the DNA-binding domain at the NH2-terminal end (residues 35\u201361). The domain is very well conserved (Pfam00172) and binds two Zn atoms which coordinate folding of the domain. A BlastP search revealed orthologous transcription factors in the genome of A. nidulans, A. oryzae and A. fumigatus (see above, Fig.\u00a02). The most similar transcription factors to InuR which have been functionally characterized, apart from the proposed orthologs in A. oryzae, A. fumigatus and A. nidulans, are the AmyR transcription factors from the different Aspergillus species. Subsequent phylogenetic analysis indicated that the subgroup of InuR transcription factors is most closely related to the group of AmyR transcription factors (data not shown). Besides the Zn(II)2Cys6 motif, a second conserved domain, known as the middle homology region (MHR) or Pfam04082 domain, is present in Zn(II)2Cys6 transcription factors. The region is thought to assist the Zn(II)2Cys6 cluster in DNA target discrimination (Schjerling and Holmberg 1996). In the A. niger InuR protein, the MHR\/Pfam04082 domain compromises aa 353\u2013425, and the domain is also present in the InuR proteins of the other Aspergilli.\nConstruction and growth properties of the inuR disruptant in A. niger N402\nAn inuR disruptant mutant was constructed as described in the \u201cMaterials and methods\u201d. Putative disruption strains were identified by PCR screening, and further verified by Southern blot analysis. Strain XY3.1 was selected to further characterize the InuR function and we will refer to this strain as the \u0394inuR strain.\nThe effect of the disruption of the inuR gene on the utilization of various carbon sources was analyzed on agar plates. Growth and conidiation of the \u0394inuR strain was strongly reduced on plates containing inulin in comparison to the wild-type strain (Fig.\u00a03). As shown, growth of the \u0394inuR strain on inulin was not completely abolished. After 7\u00a0days of growth, a thin colony with a reduced degree of conidiation was formed. Some effect on growth and conidiation was also observed for the \u0394inuR strain growing on plates containing sucrose and to a lesser extent on raffinose. The differences in growth on sucrose and raffinose were most clear after 3\u00a0days of growth (Fig.\u00a03). Both the colony size and the degree of conidiation of the \u0394inuR strain and the wild-type strain after 7\u00a0days of growth were very similar (data not shown). The \u0394inuR strain showed a similar growth phenotype to the wild-type on MM plates containing fructose, glucose, maltose, starch, xylose, sorbitol or glycerol as sole carbon source (Fig.\u00a03 and data not shown).\nFig.\u00a03Effects of A. niger strains on the utilization of different carbon sources. The wild-type (N402) and inuR disruptant (\u0394inuR), amyR disruptant (\u0394amyR) and double mutant (\u0394amyR\u0394inuR) strains were grown on MM plates containing 1% different carbon sources at 30\u00b0C for 3\u00a0days\nGrowth of the \u0394inuR strain on different carbon sources was also determined by analyzing growth in a microtiter plate well containing MM with various carbon sources. Equal numbers of spores of the wild-type strain (N402) and \u0394inuR strain were inoculated and plates were incubated at 32\u00b0C. For each condition at least four replicates were made and the average value of the replicates and its standard deviation value were determined as shown in Fig.\u00a04. The \u0394inuR showed a clear growth defect on inulin and sucrose. A small, but reproducible growth difference in growth on raffinose on agar plates was also observed in the microtiter plate analysis. Initially, the growth curves of the wild-type and the \u0394inuR strain on raffinose were identical, but after 24\u201332\u00a0h growth of the \u0394inuR strain seem to be retarded (Fig.\u00a04). The \u0394inuR strain grew equally well as the wild-type strain on MM with maltodextrin, maltose, glucose, xylose or fructose as sole carbon source (Fig.\u00a04 and data not shown). In conclusion, the growth analysis of the \u0394inuR strain indicates that the Zn(II)2Cys6 transcription factor is required for the efficient utilization of inulin and and to a lesser extent raffinose, and it may therefore act as a positive transcriptional activator of the genes encoding the inulinolytic enzymes. To further substantiate that the defective growth of the \u0394inuR strain on inulin was caused by the disruption of the inuR gene, a plasmid containing the inuR gene with 1\u00a0kb upstream and 0.5\u00a0kb downstream region was constructed and co-transformed into the \u0394inuR strain with pAN7.1 (Punt et al. 1987). Hygromycin resistant transformants were analyzed for growth on inulin and subjected to Southern blot analysis. Transformants that showed restored growth on inulin all harbor the inuR gene, while transformants showing defective growth on inulin did not contain the inuR gene (data not shown). The restoration of the wild-type growth on inulin upon retransformation of the \u0394inuR strain with a functional copy of the inuR gene indicated that the inability to grow on inulin was indeed a consequence of inuR disruption.\nFig.\u00a04Microtiter plate assay of growth of inuR disruptant on different carbon sources. A. niger wild-type (N402; open circles) strain and inuR disruptant (\u0394inuR; solid circles) were inoculated in microtiter plate wells and each well was inoculated with 1\u00a0\u00d7\u00a0104 spores in 200\u00a0\u03bcl of MM containing 1% carbon source and 0.1% casamino acids. The growth was monitored by measuring culture turbidity (OD595) and read every 2\u00a0h. Each condition was performed in six replicates. The standard deviation value is indicated\nInuR is required for the induction of inulinolytic genes\nTo further establish the role of InuR as a positive acting transcriptional regulator for inulinolytic gene expression, Northern blot analysis was performed to determine the mRNA level of the various inulinolytic enzyme encoding genes both in the \u0394inuR strain and the WT strain. We previously showed that the expression of the genes encoding the exo-inulinase (inuE), endo-inulinase (inuA), invertase (sucA), was detected only in the presence of inulin and sucrose and not on other carbon sources tested such as xylose, glucose, maltose or starch. Expression of the sucB gene was low on all the carbon sources tested and slightly induced in the presence of inulin and sucrose (Yuan et al. 2006).\nIn order to clarify whether the expression of these inulinolytic genes was controlled by InuR, both the wild-type strain and the \u0394inuR strain were pre-grown for 18\u00a0h in MM containing xylose as a carbon source. After the pre-growth, equal amounts of mycelium was transferred to fresh MM containing 1% (w\/v) inulin or 1% (w\/v) sucrose as a carbon source. At different time points after the transfer RNA was extracted from mycelia and subjected to Northern blot analysis (Fig.\u00a05). The transfer to inulin and sucrose resulted in an induction of the inulinolytic enzymes in the wild-type strain. The induction of genes is much faster on sucrose than on inulin and is explained by a much more rapid degradation and uptake of the carbon source as observed previously (Yuan et al. 2006). In the \u0394inuR strain, no induction of expression was found for the inuE and inuA genes after transfer to inulin and a very low level of sucA mRNA was detected after the transfer to inulin. The transfer to sucrose did not result in a detectable induction of any of the three inulinolytic genes in the \u0394inuR strain. A slight induction of expression of sucB in the presence of inulin and sucrose seems also dependent on InuR (Fig.\u00a05). The expression of the gene encoding a putative sugar transporter, An15g00310 in relation to inulin and sucrose was also examined. As discussed above, An15g00310 is clustered and located between the inuR and the sucB genes (Fig.\u00a01). Expression of An15g00310 is strongly induced in response to the presence of inulin and sucrose and dependant on the InuR transcription factor (Fig.\u00a05). No or very low expression levels of An15g00310 were detected when grown on maltose, glucose, fructose or xylose or after a transfer to maltose indicating that An15g00310 expression is specific for growth on inulin and sucrose (data not shown). The inuR gene was constantly expressed at a relatively low level on the various carbon sources tested (inulin, sucrose, maltose, fructose or glucose, data not shown), suggesting that the activation of the InuR transcription factor is regulated post-transcriptionally.\nFig.\u00a05Northern blot analysis of inulinolytic genes in wild-type (N402) and the \u0394inuR strain from mycelia transferred from xylose (preculture) to inulin or sucrose. Total RNA was isolated from mycelia growing in inulin or sucrose at different time points (2, 4, 8 and 24\u00a0h) as indicated after the transfer\nGenome wide transcriptional profiling of the InuR regulon in A. niger\nThe experiments described above suggest that InuR is a transcriptional activator of inulinolytic genes and required for the efficient utilization of inulin and sucrose. In addition to regulating the inulinolytic genes, it is possible that InuR also regulates other genes in the genome, through direct or indirect processes in the pathway of inulin or sucrose utilization. To identify additional genes with a possible role in inulin or sucrose metabolism, we performed genome wide expression analysis using A. niger Affymetrix GeneChip arrays. The A. niger wild-type strain N402 and the \u0394inuR strain were pre-grown for 18\u00a0h in 2% (w\/v) xylose and transferred to fresh medium containing 1% (w\/v) sucrose as carbon source and grown for 2\u00a0h on sucrose. As a control, the mycelium from the N402 strain was also transferred to fresh 1% (w\/v) xylose medium. For each culture condition a duplicate experiment was carried out. RNA was isolated from each of the samples and subjected to microarray analysis (see \u201cMaterials and methods\u201d).\nTo identify genes that were induced by sucrose, we first compared the expression profile of A. niger wild-type strain N402 grown in sucrose to that of A. niger wild-type strain N402 grown in xylose 2\u00a0h after transfer. The expression of 97 genes was more than twofold increased by sucrose (One-way ANOVA analysis P\u00a0<\u00a00.05) (Supplementary Table\u00a02). To identify genes that were induced on sucrose in an InuR dependant way, the expression profile of the A. niger wild-type strain N402 grown in sucrose was compared to that of the \u0394inuR strain after the 2\u00a0h transfer to sucrose. A total of 40 genes were identified where expression was significantly increased on sucrose in the N402 strain, but not in the \u0394inuR strain (One-way ANOVA analysis P\u00a0<\u00a00.05 and >2-fold change) (Supplementary Table\u00a03.) Finally, by combining these 2 data sets, 26 genes were identified for which expression was induced by sucrose in an InuR dependent manner (Table\u00a01).\nTable\u00a01Description of sucrose induced and InuR dependent genes in A. niger at 2\u00a0h after transfer from precultureORF No.N402 sucrose N402 xylose amyR sucrose Fold inductionP valueFunctionSucrose versus xyloseN402 vs \u0394inuRCarbohydrate transport and metabolism\u00a0An08g11070103.40\u00a0\u00b1\u00a021.54aP 0.11\u00a0\u00b1\u00a00.01A 0.26\u00a0\u00b1\u00a00.45A930.3391.10.008Invertase \u00a0An15g0406036.82\u00a0\u00b1\u00a01.36P 0.18\u00a0\u00b1\u00a00.03A 0.29\u00a0\u00b1\u00a00.05A 204.2126.60.009Putative fructose facilitator\u00a0An12g08280145.30\u00a0\u00b1\u00a027.90P 1.81\u00a0\u00b1\u00a00.28P, M 1.97\u00a0\u00b1\u00a00.21P, M 80.373.80.008Exo-inulinase\u00a0An11g0320029.27\u00a0\u00b1\u00a00.80P 0.63\u00a0\u00b1\u00a00.23A 0.74\u00a0\u00b1\u00a00.06A46.239.60.007Endo-inulinase\u00a0An15g0031020.64\u00a0\u00b1\u00a02.95P 1.34\u00a0\u00b1\u00a00.17A 1.76\u00a0\u00b1\u00a00.97P 15.411.70.018Putative monosaccharide transporter \u00a0An15g03940167.30\u00a0\u00b1\u00a01.20P 65.74\u00a0\u00b1\u00a03.04P 61.68\u00a0\u00b1\u00a047.66P 2.52.70.027Putative monosaccharide transporter Amino acid transport and metabolism\u00a0An04g0134044.31\u00a0\u00b1\u00a03.39P 16.25\u00a0\u00b1\u00a01.27P 10.16\u00a0\u00b1\u00a014.17P 2.74.40.041Putative asparagine synthase \u00a0An14g027207.75\u00a0\u00b1\u00a00.04P 2.37\u00a0\u00b1\u00a00.17p 2.88\u00a0\u00b1\u00a01.17P 3.32.70.037Putative neutral amino acid permease Lipid transport and metabolism\u00a0An08g0367010.76\u00a0\u00b1\u00a00.09P 4.45\u00a0\u00b1\u00a00.14P 3.91\u00a0\u00b1\u00a02.36P, A 2.42.80.015Putative ethanolamine kinase \u00a0An16g061902.42\u00a0\u00b1\u00a00.14P 1.16\u00a0\u00b1\u00a00.07P, A 0.97\u00a0\u00b1\u00a00.19A 2.12.50.039Putative glycerophosphoinositol transporter Phosphate transport and metabolism\u00a0An02g001808.04\u00a0\u00b1\u00a00.06P 2.85\u00a0\u00b1\u00a00.03P 0.50\u00a0\u00b1\u00a00.22A 2.816.20.001Putative phosphate-repressible phosphate permease Nucleotide transport and metabolism\u00a0An02g0996041.96\u00a0\u00b1\u00a04.97P 8.61\u00a0\u00b1\u00a00.73P 16.54\u00a0\u00b1\u00a07.61P 4.92.50.033Putative phosphoribosylformylglycinamidine synthase Other transport mechanism\u00a0An17g0171054.97\u00a0\u00b1\u00a015.19P 2.83\u00a0\u00b1\u00a00.65P 5.71\u00a0\u00b1\u00a010.10P, A 19.49.60.049Putative multidrug transporter\u00a0An08g0233026.99\u00a0\u00b1\u00a04.40P 5.73\u00a0\u00b1\u00a00.31P 12.12\u00a0\u00b1\u00a00.63P 4.72.20.024Putative ABC multidrug transporter\u00a0An17g0024040.24\u00a0\u00b1\u00a03.78P 12.14\u00a0\u00b1\u00a00.61P 19.54\u00a0\u00b1\u00a08.12P 3.32.10.038Putative ABC transporterTranscription\u00a0An02g054609.25\u00a0\u00b1\u00a01.05P 2.60\u00a0\u00b1\u00a00.23A 4.26\u00a0\u00b1\u00a00.83M, A 3.62.20.040Putative DNA-directed RNA polymerase\u00a0An02g126103.81\u00a0\u00b1\u00a00.25P 1.55\u00a0\u00b1\u00a00.02A 1.21\u00a0\u00b1\u00a00.89P, A 2.53.20.043Putative Ran-GTPase-activating proteinProtein synthesis\u00a0An02g1264048.21\u00a0\u00b1\u00a06.42P 9.16\u00a0\u00b1\u00a01.17P 18.71\u00a0\u00b1\u00a01.32P 5.32.60.041Putative RNA-binding protein \u00a0An17g0181555.05\u00a0\u00b1\u00a01.42P 15.21\u00a0\u00b1\u00a00.37P 24.59\u00a0\u00b1\u00a06.90P 3.62.20.004Putative translation initiation factor\u00a0An01g0604010.17\u00a0\u00b1\u00a02.79P 3.21\u00a0\u00b1\u00a00.04P 3.88\u00a0\u00b1\u00a00.04P 3.22.60.025Putative 60S ribosome biogenesis protein\u00a0An02g1408023.20\u00a0\u00b1\u00a00.69P 9.94\u00a0\u00b1\u00a00.25P 10.87\u00a0\u00b1\u00a07.87P 2.32.10.010Putative asparaginyl-tRNA synthetaseProtein fate\u00a0An11g0498036.47\u00a0\u00b1\u00a01.45P 9.59\u00a0\u00b1\u00a01.06P 12.82\u00a0\u00b1\u00a00.53P 3.82.80.010Putative importin unit beta-4Unclassified\u00a0An01g003409.56\u00a0\u00b1\u00a00.97P 2.26\u00a0\u00b1\u00a00.42P 3.13\u00a0\u00b1\u00a00.32P, A 4.23.10.036Unknown protein\u00a0An15g0315013.42\u00a0\u00b1\u00a00.68P 4.01\u00a0\u00b1\u00a00.16P 4.02\u00a0\u00b1\u00a00.26P 3.43.30.008Unknown protein\u00a0An07g026905.27\u00a0\u00b1\u00a00.03P 1.69\u00a0\u00b1\u00a00.02A 2.22\u00a0\u00b1\u00a00.27P 3.12.40.001Unknown protein\u00a0An01g055003.77\u00a0\u00b1\u00a00.06P 1.25\u00a0\u00b1\u00a00.03P 1.46\u00a0\u00b1\u00a00.35P 3.02.60.005Unknown proteinaThe expression level was based on the geometric mean value of the duplicate samples and the deviation values between the duplicate samples are indicated. P, M or A, representing detection calls for present, marginal or absent respectively. The P-value was based on One-way ANOVA analysis. Genes were divided over different functional groups according to FunCat (Ruepp et al. 2004)\nThe predicted protein sequences of these 26 genes were assigned to various functional categories (Ruepp et al. 2004; Mewes et al. 1997) and shown in Table\u00a01. Interestingly, genes with the highest fold changes (>10-fold change) almost all fall into the category of carbohydrate transport and metabolism. Among them are the genes encoding the extracellular inulinolytic enzymes, sucA, inuE and inuA, as well as the above mentioned putative sugar transporter gene (An15g00310), supporting the previous Northern analysis. An additional gene that showed a very high fold-change and belongs to the category of carbohydrate transport and metabolism is An15g04060. An15g04060 encodes a putative sugar transporter which is over 200-fold induced by sucrose in a wild-type strain N402 when compared to xylose and the induction was greatly reduced by 127-fold when inuR gene was deleted. BlastP search revealed that the transporter displayed the highest identity to a fructose transporter protein (Frz1) from Zygosaccharomyces bailii (Pina et al. 2004). An additional putative sugar transporter gene (An15g03940) was induced on sucrose in an InuR dependant way. In contrast to the other two transporters (An15g00310 and An15g04060) this transporter is relatively highly expressed on both xylose and in the \u0394inuR mutant, resulting in relative small fold-changes. An additional gene (An17g01710) was strongly induced on sucrose (about 19-fold), in an InuR dependant way. The protein encoded by An17g01710 is annotated as a multidrug resistance (MDR) protein because of its highest identity to other MDR proteins of the Major Facilitator Superfamily of transporters. Since the substrates of MDR proteins are hard to predict on the basis of the amino acid sequence, the annotation should be interpreted with care and does not exclude the possibility that this protein has a role in sugar transport. Apart from the An15g04060 transporter which might function as a fructose transporter based on the strong homology towards Frz1p (Pina et al. 2004), a useful prediction of substrates of the other putative transporters can not be made.\nIn the list of induced genes (Table\u00a01), a considerable number are related to transcription, translation and amino acid metabolism. The increase in expression of those genes might indicate an overall increase of metabolic activity on sucrose compared to xylose and a subsequent higher expression of the protein transcription\/translation machinery.\nThe genome wide analysis indicated that three extracellular inulinolytic genes and four putative sugar transporter genes were strongly induced on sucrose in an InuR dependant way. To identify possible elements in the promoter regions of these genes that could mediate InuR binding an in silico analysis of 1-kb upstream promoter sequences of these seven genes was performed. As shown in Fig.\u00a06, optimal alignment of the promoter sequences revealed a consensus sequence (CGGN8CGG) which is present at least once in the promoter region of all genes. At least one of the triplets consistently is preceded by a T, resulting in a conserved TCGG motif in every proposed element. Within the second triplet, the conservation of the GG is consistent, but conservation of the C is not. This putative binding site of the InuR protein (CGGN8CGG) is identical to the binding site of the AmyR transcription factor which binds and induces the expression of amylolytic genes (Petersen et al. 1999). To examine whether AmyR is also involved in the regulation of inulinolytic genes, the \u0394amyR mutant was constructed and analysed for growth on various carbon sources. As shown in Fig.\u00a03, deletion of the amyR transcription factor did not result in a growth defect on inulin, sucrose or other carbon sources. The \u0394amyR strain did show a strong growth reduction on maltose (data not shown). To examine a possible overlapping role of the InuR and AmyR transcription factors for inulin and\/or sucrose utilization, the \u0394amyR\/\u0394inuR double mutant was constructed. Growth analysis of the double mutant on inulin showed a minor further reduction in growth and sporulation on inulin and sucrose (Fig.\u00a03) indicating AmyR and InuR have independent functions. Fig.\u00a06In silico prediction of putative InuR binding sites in the promoter of sucrose induced genes. Conserved nucleotides are coloured in gray. Numbers beside the sequences indicate the position relative to the ATG of the downstream located gene\nDiscussion\nWe previously showed that the genes encoding extracellular inulinolytic enzymes in A. niger are co-regulated at the transcription level and identified sucrose, but not fructose or glucose as an efficient inducer of inulinolytic genes. It was proposed that there might be a common transcription factor to activate the expression of inulinolytic genes in response to the presence of inducing carbon sources (Yuan et al. 2006), similar to that which has been found in other carbohydrate degrading enzymes networks such as the amylolytic genes (AmyR) or the xylanolytic genes (XlnR). In this study we reported the successful identification of an inulinolytic transcriptional activator, InuR. Previous studies to isolate the AmyR and XlnR transcription factors were based on genetic screens to identify mutants with impaired expression of amylolytic and xylanolytic enzymes (Petersen et al. 1999; Gomi et al. 2000; van Peij et al. 1998a). Here, we used a different approach which made use of the full genome sequence of A. niger. The approach is based on the observation that in some cases, e.g., in the case of the AmyR transcription factor, the target genes of the transcription factor are adjacent to the transcription factor itself. In the case of the inulinolytic genes, three possible inulinolytic gene clusters were identified, each of which contains a gene encoding a putative transcription factor (Fig.\u00a01). Disruption mutants were successfully obtained for two of the three transcription factors and subsequent phenotypic analysis indicated that only InuR (An15g00300) was involved in the inulin utilization pathway. The \u0394inuR mutant showed severe reduced growth on agar plates and in submerged cultures containing sucrose or inulin as sole carbon source. Furthermore, we showed that the induction of the inulinolytic genes by inulin and sucrose is dependant on the presence of InuR. In addition to the transcription factor itself the inulinolytic gene cluster identified in A. niger consists of a sugar transporter and sucB which encodes an intracellular invertase with transfructosylating activity (Goosen et al. 2007).\nTo examine whether the position and clustering of the InuR orthologs was conserved among aspergilli, we compared the genes adjacent to the InuR homologous transcription factors in A. nidulans, A. oryzae and A. fumigatus. The clustering of the three genes, inuR (An15g00300), An15g00310 (putative sugar transporter) and sucB (An15g00320) in A. niger was conserved in A. nidulans (AN3835; e-value 5e-144, AN3836; e-value 7e-158 and AN3837; e-value 5e-142), and A. oryzae (AO090701000036; e-value 0.0, AO090701000037; e-value 0.0 and AO090701000038; e-value 0.0, respectively). In A. fumigatus, the InuR orthologue (Afu6g05010; e-value 0.0) is located next to the SucB orthologue (Afu6g05000; e-value 0.0), but no homolog to the putative sugar transporter (An15g00310) was present in the gene cluster in A. fumigatus. The initial gene model of the inuR gene in the A. niger annotation was lacking the ZnII(2)Cys6 binuclear cluster domain. Repositioning the intron\/exon positions in N-terminal region resulted in the prediction of a 709 amino acid long protein containing a complete Zn(2)Cys6 binuclear cluster domain. BlastP searches with the A. niger InuR protein revealed orthologous genes in the three Aspergillus species examined (A. nidulans, A. oryzae and A. fumigatus). The closest homologs in A. oryzae and A. nidulans did not contain a Zn(II)2Cys6 domain and the A. fumigatus homolog only a partial Zn(II)2Cys6 cluster. The gene models of the other Aspergilli were manually inspected and by improving the gene model a Zn(II)2Cys6 domain was found to be present in the proteins. Interestingly, it turned out that the gene model of A. niger was completely conserved in the other Aspergili. Thus, the gene model of the A. nigerinuR gene could be used as a template for the prediction of the inuR gene models in the other Aspergilli. Both the numbers of introns as well as the intron\/exon positions were conserved among the four Aspergilli. This result implies that gene model prediction in Aspergilli can be improved by comparison of the gene models of orthologous genes.\nThe inuR gene is situated in the genome next to a putative sugar transporter gene (An15g00310) and sucB. We recently showed that the sucB gene encodes an intracellular invertase with transglycosylating activity (Goosen et al. 2007). As depicted in Fig.\u00a01, the putative sugar transporter (An15g00310) in the cluster shares the same promoter region with the sucB gene. The Northern analysis and microarray analysis showed that the two genes are not coregulated indicating that the common promoter region does not act bidirectionally as has been observed, e.g., in the case of the niiA and niaD genes (Punt et al. 1995). Expression of the gene An15g00310 was highly induced by sucrose or inulin and the induction was dependent on InuR (Fig.\u00a05), while the sucB gene was lowly expressed on various carbon sources and hardly induced on sucrose and inulin. The function of An15g00310 remains to be elucidated but it is tempting to suggest that it involved in the transport of sucrose, or its hydrolysis products fructose or glucose.\nThe function of SucB in relation to inulin or sucrose metabolism is also currently unknown. We showed that deletion of the sucB did not result in a phenotype related to growth on inulin or sucrose (Goosen et al. 2007). The constitutive low level of expression of the sucB gene might suggest that SucB acts as a scouting enzyme to \u201csense\u201d the presence of a useful carbon source by generating an inducer molecule which can further activate transcription of additional inulinolytic genes. A possible explanation for the lack of phenotype of the sucB deletion mutant is the presence of an addition intracellular invertase, encoded by the sucC gene. Both the enzymatic activity of the SucC protein as well as the effect of deletion of sucC and simultaneously deletion of sucB and sucC will be the subject of future research.\nThe genome wide transcription analysis using Affymetrix microarrays identified a limited number of genes that were strongly induced by sucrose in an InuR dependent way. These genes encode the extracellular inulinolytic enzymes and four genes encoding putative transporters. The specificity of the sugar transporters should be determined biochemically as has been done previously for A. niger sugar transporters (vanKuyk et al. 2004) since phylogenetic comparison to suggest possible substrates is not reliable (Patricia vanKuyk, personal communication). The limited number of genes that are induced by sucrose in an InuR dependant way suggests a relatively simple regulatory mechanism regarding sucrose catabolism in A. niger. Unpublished additional microarray data showed that the same three extracellular enzymes were induced on inulin in an inuR dependant way. The expression of the putative sugar transporters on inulin differed from the pattern observed on sucrose. Two of the four transporters (An17g01710 and An15g04060) were not induced on inulin after 2 and 8\u00a0h of growth on inulin. The transporter encoded by the An15g03940 gene was four to fivefold induced on inulin compared to xylose after 8\u00a0h of the transfer. Expression of this sugar transporter is also high on xylose (Table\u00a01) and maltose, Yuan et al., unpublished results), indicating that the expression of this sugar transporter is not very tightly regulated and suggest a broad specificity of the sugar transporter. The fourth transporter (An15g00310) was more highly expressed on inulin 8\u00a0h after the transfer than on maltose and xylose (four to fivefold induction), and the induction was InuR dependant. The difference between the observed expression patterns of the extracellular enzymes and the putative sugar transporters suggest that under the different growth conditions the fungus expresses the same set of extracellular enzymes, but that the fungus fine-tunes the expression of the sugar transporters in order to allow efficient uptake. Such a fine-tuning mechanism could explain why such a large number of sugar transporters is present in the genome of A. niger as 461 proteins have been identified that belong to the Major Facilititator Superfamily of transporters (Pel et al. 2007). Such a mechanism allows the fungus to express the sugar transporters with the optimal properties in relation to substrate diversity, substrate concentration, environmental pH, and other environmental factors and nutrient conditions.\nThe InuR transcription factor is a member of the Zn(II)2-Cys6 DNA transcription factor family which is a fungal specific type of transcription factor. In Aspergilli, this family of transcription factors has expanded significantly when genome data is compared to that of the yeast S. cerevisiae or Neuropora crassa. In S. cerevisiae and N. crassa 54 and 89 genes, respectively (Akache et al. 2001; Borkovich et al. 2004), have been identified that contain the Zn(II)2-Cys6 DNA binding domain, the genomes of Aspergilli contain 216 (A. nidulans), 188 (A. fumigatus) or 296 (A. niger) Zn(II)2-Cys6 DNA transcription factors. The actual number of proteins containing a Zn(II)2-Cys6 DNA binding domain might still increase in Aspergilli and other fungi. We showed that the predicted gene model for the inuR gene in several Aspergilli was incorrect, resulting in a protein lacking the Zn(II)2-Cys6 DNA binding domain. Closer examination of the A. nidulans and A. niger genome annotations revealed that the difficulty of the automatic annotation in predicting the correct gene model for this class of transcription factors is not limited to inuR gene, but is a more general problem. Manual improvement of the gene models identified 326 Zn(II)2-Cys6 DNA binding domain containing proteins in A. nidulans and 480 Zn(II)2-Cys6 DNA binding domain containing proteins in A. niger (Ram and Punt, unpublished data). Thus, it is likely that other fungal genomes contain more as yet unidentified Zn(II)2-Cys6 DNA binding domain containing proteins, which could be revealed with improved gene models or manual annotation.\nTranscription factors containing the Zn(II)2-Cys6 DNA binding domain usually bind the promoter region of their target genes via a conserved DNA binding site. Often, but not always, the binding site consists of conserved terminal trinucleotides, usually in symmetrical configuration (direct repeat, everted repeat or an inverted repeat) and spaced by an internal variable sequence of a defined length. Examples are the Gal4p and Lac9p transcription factors that bind to CGGN11CCG, Ppr1p and UaY that bind to CGGN6CGG, and Put3 that binds to CGGN10CCG (Todd and Andrianopoulos 1997). The binding sites for the AmyR transcription factor have been studied in detail. Several promoter regions of amylolytic genes contain a CGGN8(A\/C)GG site in their promoter region to which AmyR can bind and activate transcription (Tani et al. 2001b; Ito et al. 2004).\nComparison of the 1-kb upstream promoters of the strongly InuR dependent sucrose induced genes revealed that a common consensus sequence (CGGN8CGG) is present in the promoters of all genes. Strikingly, in every putative element at least one repeat consists of a TCGG sequence (Fig.\u00a06). This observation might be explained by a speculative, but testable, model of InuR binding and activation. We propose that InuR will bind as a dimer to the CGGN8CGG motif, and that the first InuR molecule will bind with high affinity to the TCGG DNA sequence. This binding may recruit a second InuR molecule to the site that will bind to the second motif, possibly with a weaker affinity for the DNA because of the sequence variation. Such a model for cooperative binding of a dimeric transcription factor has been experimentally substantiated for the AmyR transcription factor (Ito et al. 2004). The proposed InuR binding site (CGGN8CGG) might be conserved among filamentous fungi, as the shared promoter of the inuC and inuD genes in Penicillium sp. strain TN-88 contains a similar site (Moriyama et al. 2006).\nThe proposed InuR binding site is very similar to the AmyR binding site which is present in the promoter of many amylolytic genes in A. niger (Yuan et al., unpublished results) and raises immediately the question of how the specificity of the InuR and AmyR transcription factors is accomplished. Comparison of the Zn(II)2Cys6 domain of several Zn(II)2Cys6 transcription factors revealed that the Zn(II)2Cys6 domain of InuR and AmyR is not more related to each other than they are related to unrelated Zn(II)2Cys6 transcription factors (XlnR and FacB). Interestingly, the MHD of InuR and AmyR showed a high degree of identity and suggest that the MHD domain may assist in binding to the DNA target sequence. After establishing that the conserved CGGN8CGG motif in the promoter of inulinolytic genes is involved in the binding and activation of expression of the target genes, the AmyR and the InuR transcription factors are interesting subjects for further research aimed to understand how Zn(II)2-Cys6 transcription factors recognize their target genes and what determines the specificity of the binding. Analysis of the \u0394amyR\u0394inuR double mutant indicated that both transcription factors have independent functions. The slightly decreased growth and sporulation of the \u0394amyR\u0394inuR double mutant on sucrose compared to growth of the \u0394inuR mutant on sucrose might be explained by the activity of \u03b1-glucosidases that are under control of the AmyR transcription factor. It is well established that sucrose can be hydrolyzed either by \u03b2-fructosidase, that splits sucrose by attacking the terminal, non-reducing \u03b11, \u03b22 linked fructose moiety, but also by an \u03b1-glucosidase catalyzing the hydrolysis of sucrose at the terminal non-reducing \u03b11\u20134 linked glucose residue (Geber et al. 1992; Kelly and Kwon-Chung 1992; Alberto et al. 2004). A. niger contains several \u03b1-glucosidase encoding gene of which two of them are under control of the AmyR transcription factor. Disruption of the amyR transcription factor further decreases the level of agdA and agdB transcripts (Yuan et al., unpublished results), which might result in lower \u03b1-glucosidase activity and a subsequent decrease in the efficiency by which sucrose can be hydrolyzed.\nIn conclusion, we have identified InuR as the transcriptional activator required for utilization of inulin and sucrose. The results in this paper suggest that A. niger metabolizes inulin and sucrose by inducing the expression of a limited number of extracellular enzymes and sugar transporters that are under transcriptional control of a single transcription factor, InuR. The mechanism by which the InuR transcription factor is activated in response to the presence of inulin and sucrose and the mechanism that determines the specificity of the binding to the InuR target genes are the subjects of future research.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nSupplementary Tables (DOC 224 kb)","keyphrases":["sucrose","transcription factor","invertase","affymetrix genechip arrays","inulinase","carbohydrate metabolism"],"prmu":["P","P","P","P","U","R"]}
{"id":"Eur_Spine_J-2-2-1602196","title":"Pathogenesis, diagnosis and management of pneumorrhachis\n","text":"Pneumorrhachis (PR), the presence of intraspinal air, is an exceptional but eminent radiographic finding, accompanied by different aetiologies and possible pathways of air entry into the spinal canal. By reviewing the literature and analysing a personal case of traumatic cervical PR after head injury, we present current data regarding the pathoanatomy, clinical and radiological presentation, diagnosis and differential diagnosis and treatment modalities of patients with PR and associated pathologies to highlight this uncommon phenomenon and outline aetiology-based guidelines for the practical management of PR. Air within the spinal canal can be divided into primary and secondary PR, descriptively classified into extra- or intradural PR and aetiologically subsumed into iatrogenic, traumatic and nontraumatic PR. Intraspinal air is usually found isolated not only in the cervical, thoracic and, less frequently, the lumbosacral regions but can also be located in the entire spinal canal. PR is almost exceptional associated with further air distributions in the body. The pathogenesis and aetiologies of PR are multifold and can be a diagnostic challenge. The diagnostic procedure should include spinal CT, the imaging tool of choice. PR has to be differentiated from free intraspinal gas collections and the coexistence of air and gas within the spinal canal has to be considered differential diagnostically. PR usually represents an asymptomatic epiphenomenon but can also be symptomatic by itself as well as by its underlying pathology. The latter, although often severe, might be concealed and has to be examined carefully to enable adequate patient treatment. The management of PR has to be individualized and frequently requires a multidisciplinary regime.\nIntroduction\nPneumorrhachis (PR), the phenomenon of intraspinal air, is an exceptional imaging finding, caused by various, mainly traumatic and iatrogenic aetiologies. It is associated with different pathologies and possible pathways of air entry into the spinal canal. Although PR has been previously described, mainly in the radiological literature, spine specialists are less acquainted with this unusual pathological entity.\nTherefore, based on a complete review of the medical literature and the analysis of a personal case description, we offer contemporary theories of the development of PR and current management strategies for this uncommon epiphenomenon of coincident underlying injuries and diseases to elucidate this rare phenomenon and to raise awareness to PR and possible coexisting pathologies.\nMaterials and methods\nA comprehensive literature search of the U.S. National Library of Medicine\u2019s Medline bibliographic database was conducted and the international medical literature up to December 2005 has been reviewed for the term \u201cpneumo(r)rhachis\u201d and related appropriate medical subject headings such as intraspinal air, intraspinal pneumoc(o)ele, spinal epidural and subarachnoid pneumatosis, spinal and epidural emphysema, aerorachia, pneumosaccus, air myelogram, pneumomyelogram or pneumomyelography.\nEach bibliography and retrieved article were then obtained in complete form, analysed and searched for further citations. Pertinent references cited in the identified articles and other relevant literature were also scanned. Further references of each text were identified, and the text was obtained in complete form and analysed. A new case of cervical PR due to severe head injury was added.\nResults\nAfter application of the methods and criteria described above, only isolated case reports and no series of more than three cases of this condition could be found. In total, 71 articles were identified that reported on 86 cases. In addition, we describe a previously unreported case of traumatic cervical PR, which was diagnosed in our department.\nIllustrative case\nA 51-year-old woman was admitted comatose after an automobile accident and attempted unsuccessful endotracheal intubation with prolonged insufficient mask ventilation. Multiple skin lacerations were present as were blunt thoracic trauma with several rib fractures and severe head injury. The patient was decerebrated with bilateral fixed and dilated pupils. Cranial computerized tomography (CT) examination (Fig.\u00a01) revealed a dislocated left occipital skull fracture radiating into the foramen magnum and petrosal bone and a fracture of the sphenoid sinus accompanied by a large acute right hemispheric subdural haematoma causing midline brain shift, traumatic subarachnoid haemorrhage, cerebral oedema and signs of hypoxemia. Internal pneumocephalus with diffuse air distribution in the basal, prepontine and perimesencephal cisterns and fourth ventricle could be detected. Additional spinal CT (Figs.\u00a02, 3) demonstrated air lucency in the cervical vertebral column indicating extradural PR. Intraspinal air was caused by the transphenoid sinus and petrosal bone fractures thus allowing direct communication of pneumatized air containing cavities with the intracranial space. The penetrated air was then forced caudally due to the elevated intracranial pressure resulting from severe brain injury with diminution of capacity of the intracranial space. Horizontal and head-down position of the patient finally allowed the entrapped air to pass through the foramen magnum into the spinal canal.\nFig.\u00a01Axial cranial CT scans (corresponding soft-tissue and bone window setting) revealing severe head injury accompanied by traumatic internal pneumocephalus with air distributed prepontine, perimesencephally and intraventricullary accompanied by skull fractures of the sphenoid, left occipital and petrosal bone leading to PRFig.\u00a02Sagittal multiplanar reformatted spinal CT images (corresponding soft-tissue and bone window setting) demonstrating traumatic cervical PRFig.\u00a03Axial CT scans of the cervical spine (corresponding soft-tissue and bone window setting) showing extradural air collection within the ventral spinal canal\nThe patient remained decerebrated, was transferred to the intensive care unit, considered brain death after adequate examinations and devoted to organ donation.\nDiscussion\nPathogenesis\nDefinition and classification\nFree air surrounding the dura mater spinalis is an uncommon phenomenon that was primarily reported by Gordon et al. [30] in 1977 and described under various terms such as intraspinal pneumocele [28, 38, 50] or pneumocoele [78], spinal epidural and subarachnoid pneumatosis [4\u20136, 9, 18, 19, 32\u201334, 41, 47, 61, 74], spinal and epidural emphysema [4, 11, 23, 27, 28, 33, 59, 65, 69, 70, 72, 77], aerorachia [18, 22, 28, 34], pneumosaccus [9, 28, 62], air myelogram [2, 30, 76], pneumomyelogram [2, 3, 12, 17, 20, 28, 30, 76] or pneumomyelography [26]. The term PR itself was first coined 10\u00a0years later by Newbold and co-workers [54]. PR per se usually represents an asymptomatic, probably underdiagnosed epiphenomenon of coincident underlying injuries and diseases. As imaging techniques improve, this pathologic entity has become somewhat more often diagnosed.\nPR can be classified descriptively into internal, intradural (intraspinal air within the subdural or subarachnoid space) and external, extradural (intraspinal, epidural air) PR. External PR by itself is usually innocuous, whereas internal traumatic PR frequently is associated with major trauma and believed to be a marker of severe injury [28].\nPathomechanism and aetiology\nApart from artificial, usually traumatic spinal leaks and penetrating spine injuries as a possible explanation for a direct route of intraspinal entry, air may entrap due to a one-way air valve mechanism and dissect indirectly between the paraspinal soft tissues into the epidural space of the spinal canal via the neural foramina and along the vascular and nerve root sheaths, and vice versa, thereby producing PR.\nVarious conditions may directly or indirectly produce PR including trauma [1\u20133, 9, 10, 12, 13, 17, 20\u201322, 26\u201328, 30, 32, 37\u201342, 45, 50, 51, 54, 60, 62, 64, 66\u201368, 73, 74, 76, 78], respiratory complications and conditions that produce high intrathoracic pressure and barotraumas [5, 6, 11, 14, 19, 22\u201324, 33, 40, 47, 55, 56, 58, 59, 61, 69, 70, 72, 77], recent iatrogenic manipulations during surgical, anaesthesiological and diagnostic interventions [7, 9, 15, 18, 31, 33, 35, 36, 44, 48, 52, 53, 57, 65, 71], malignancy and its associated therapy [29, 49, 63, 75] or it may occur spontaneously [4, 24]. In some cases, PR is an accidental discovery [2] and the conditions and lesions causing PR remain unclear or undetected [12]. The pathologies leading to PR can be generally subsumed and further classified into iatrogenic, traumatic and nontraumatic causes [28].\nThirteen cases are found in the literature describing PR caused by violent coughing due to bronchial asthma or acute bronchitis [11, 14, 23, 24, 40, 55, 56, 58, 59, 70, 72], single cases of PR after cardiopulmonary resuscitation [58], airway obstruction due to foreign body aspiration [69] and two cases each of PR after physical exertion [19, 77] and inhalational drug abuse of 3,4-methylenedioxymethamphetamine (\u201cEcstasy\u201d) [6] or marijuana [34]. Furthermore, three cases of PR because of prolonged and forceful emesis with diabetic ketoacidosis were documented [5, 47, 61]. Other authors describe PR secondary to traumatic causes including isolated head [3, 10, 17, 30, 38, 39, 62, 68, 50, 76, 78], cervical [2], thoracic [21, 22, 40, 60, 64, 66], abdominal [42] and pelvic injuries [13] or combinations of different injury patterns including spinal trauma [1, 9, 12, 20, 26, 27, 32, 37, 41, 45, 51, 54, 67, 73, 74].\nInvasive tumour progression and postradiation changes [29, 49, 63, 75], surgical interventions [33, 36, 65, 71], nasotracheal intubation [18] and peri- or epidural anaesthesia involving a lumbar puncture [7, 9, 15, 31, 35, 44, 48, 52, 53, 57] have also been reported as a cause of PR. Introduction of air as a negative contrast medium into the lumbar subarachnoid space for diagnostic pneumoencephalography or pneumomyelogram, first performed and described by Dandy in 1919, is now only of historical interest [16].\nLocalization and distribution\nTwo documented cases with air in the entire spinal canal [12, 55, 56], single cases of air bubbles distributed in the cervical, thoracic and lumbar region [35], and in the cervical and lumbar spine [10], six cases of air in the thoracic and lumbar region [7, 9, 15, 26, 57] and eight cases with both cervical and thoracic PR were previously described [1, 11, 26, 32, 47, 52, 63, 77]. Most cases demonstrate isolated PR of the cervical [2, 3, 5, 10, 17, 20, 30, 38, 39, 45, 50, 51, 54, 60, 66, 68, 69, 74, 76, 78], thoracic [4, 6, 14, 18, 19, 21\u201324, 27, 29, 33, 34, 37, 40\u201342, 49, 58, 59, 61, 64, 65, 67, 70\u201375] or lumbosacral region [9, 10, 13, 31, 36, 44, 48, 53, 57, 62, 70, 72, 74]. Nevertheless, this might be due to the fact that in some cases of PR not a complete examination of the whole spine was carried out. Furthermore, location and distribution of air within the spinal canal is probably depending on the site of air dissection, rate and volume of intraspinal air with large volumes spreading widely, capacity of intraspinal space and positioning of the patient. In cases of external PR, because of the lower resistance from the loose connective tissue, as compared with the rich vascular network that is present anteriorly, the epidural air usually collects in the posterior epidural space [6].\nPR may be primary or secondary depending on whether intraspinal air is the cause and origin or effect of associated further occurrence of air in the body.\nAlmost exceptionally, PR is found in combination with associated air distribution in other compartments and cavities of the body: particularly, in conjunction with pneumocephalus, pneumothorax, pneumomediastinum, peumopericardium or subcutaneous emphysema [3\u20136, 9\u201311, 13, 14, 17\u201324, 26, 27, 29, 30, 32\u201334, 36\u201339, 42, 47, 49\u201351, 54\u201356, 58\u201366, 68\u201370, 72\u201374, 76\u201378]. Thus, diagnosis of PR implicates the possibility of the coincidence of associated and hidden further air distributions in the body. Especially, patients with traumatic cervical and intradural PR will almost certainly have injuries of the anterior cranial fossa usually causing subdural PR and skull base or middle and posterior cranial fossae fractures with intracranial subarachnoid air and pneumocephalus, but also penetrating spinal injuries may cause isolated subarachnoid PR without accompanying head injury [2, 17, 76].\nDiagnosis\nDiagnostic procedures\nSince intraspinal air is usually asymptomatic and clinically unspecific, PR is primarily a radiographic and not a clinical diagnosis. The diagnostic work-up of patients with PR should include plain roentgenograms and CT scanning of the spine.\nX-ray may be helpful as an initial examination for the early detection of possible associated injuries and to detect larger amounts of intraspinal air [28, 56]. A linear lucency along the spinal canal on a lateral chest radiograph was described as a useful sign in the detection of PR [6]. Moreover, PR itself to some extent acts as a negative contrast agent to delineate canal morphology [78].\nThe diagnostic tool of choice for a reliable and prompt detection of PR is CT [9, 11, 49, 56]. Nevertheless, on CT, intra- and extradural PR may be difficult to differentiate [28, 60, 67]. Since PR is often combined with air distribution in other parts of the body and traumatic PR is a marker of severe injury, these observations suggest a wider indication for the application of CT, and if necessary, even a systematic total body CT scan or magnetic resonance imaging (MRI). After the initial CT examination follow-up of patients with PR should primarily rely on clinical observations.\nOther, not only more sensitive but also more extensive diagnostic techniques such as MRI or myelography are indicated for determining coexisting aetiologies of PR and differential diagnostically examinations [56].\nDifferential diagnosis\nIntraspinal air has to be clearly differentiated from free intraspinal gas collections due to degenerative [25], malignant [43], inflammatory [8] and infectious diseases by gas-forming organisms [46]. It has been demonstrated gas-chromatographically that in cases associated with the so-called vacuum disc phenomenon, the gas collection contains up to 92% nitrogen combined with oxygen, carbon dioxide and other trace gases, in contrast to atmospheric air with a lower nitrogen (78%) and a higher oxygen composition (21%) [25].\nIntraspinal gas, which usually has the same low density as air collections on CT, cannot be clearly differentiated by CT examination. It can simulate PR and therefore has to be included in the differential diagnosis of PR. Furthermore, the coincidence of PR and intraspinal gas has to be considered.\nManagement\nSymptomatology\nPR in itself usually is asymptomatic, does not tend to migrate and reabsorbs spontaneously and completely with the air being passed directly into the blood in several days without recurrence [3, 4, 6, 10\u201314, 16, 19, 22\u201324, 27, 34, 36, 69, 70, 74]. Therefore, patients with PR usually are managed conservatively. As the entrapped air occupies parts of the cerebrospinal compartment thus may cause syndromes of both intracranial and intraspinal hypertension as well as hypotension secondary to either an increase or decrease of intracranial and intraspinal pressure.\nRarely, PR per se is symptomatic and associated with discomfort and pain or even neurological deficits [29, 31, 35, 41, 44, 48, 52, 53, 57, 71, 78]. One case of traumatic PR leading to sensory symptoms has been reported. It was associated with pneumocephalus resulting from closed head injury and treated conservatively by the administration of supplemental oxygen [78]. A single case of a patient with progressive motor deficit of the lower limbs as a result of entrapped intraspinal air, which compressed the spinal cord due to presumptive introduction of air into the intraspinal arachnoid space after repeated lumbar puncture, was demonstrated by Uemura et al. [71]. Altered intrathecal pressure caused by the adhesion of the arachnoid following spinal surgery for lipoma of the thoracic spine may have caused PR in this case. The patient was reoperated with removal of the spinal compression and closure of the dural defect. Gonzales et al. [29] reported a case of reversible spinal cord and lower cervical root dysfunction caused by air dissection through a bronchopleural-epidural-cutaneous fistula likely due to tumour erosion and postradiation changes. The patient underwent surgical exploration of the chest with improvement of symptoms postoperatively. Identification of the epidural space through loss of resistance to air is a widely applied anaesthetic procedure during placement of an epidural catheter. In contrast, intraspinal air may cause undesirable inadequate analgesia during epidural anaesthesia [7, 15]. But injected air also can act as a space-occupying lesion and exert pressure on nervous structures within the spinal canal [7, 15, 31, 48, 52, 53, 57]. Eight cases with postanaesthetic neurological symptoms and pain thought to be complications associated with the application of intraspinal air were reported in the literature [31, 35, 44, 48, 52, 53, 57] including two case descriptions of accidentally injected air into the epidural space via a permanent epidural catheter for continuous epidural anaesthesia [35, 44]. The treatment consisted primarily of removal of the spinal catheter, bed rest and medication for pain relief, without permanent sequelae in most cases [31, 35, 48, 57]. Other treatment modalities included intravenous dexamethasone [53], decompression of the epidural space by percutaneous insertion of a Tuohy needle with air aspiration [44], administration of high concentrations of inspired oxygen with the idea of promoting reabsorption of air from the subdural space or even trials of hyperbaric oxygen therapy [34, 57].\nTreatment\nBecause of the rareness and the different pathogenesis and aetiologies, no empiric guidelines for the treatment of PR and standards of care exist. PR is thought to be associated with an increased morbidity and mortality [14]. Therefore, the whole extent of the conditions causing PR has to be evaluated and the contributing causes leading to PR have to be appropriately treated. PR associated with decreased intraspinal pressure secondary to cerebrospinal fluid leakage usually has a more benign character, whereas entrapped intraspinal air under pressure entering the cranio-spinal compartment usually in combination with a one-way air valve mechanism might cause tension PR and pneumocephalus with nervous tissue compression requiring intervention. Therefore, the authors suggest recognizing the importance of differential diagnosis of the phenomenon of altered intraspinal pressure with either hypo- or hypertension of the cerebrospinal compartment to allow adequate management.\nBecause of the higher risk of possible meningitis, demonstrable cerebrospinal fluid leaks, if significant or persistent, may have to be repaired neurosurgically [50, 55] or treated by a temporary lumbar spinal catheter [38]. Furthermore, in some cases the underlying pathologies causing PR such as fistulous tracts between intrathoracic structures and the subarachnoid space (thoracoarachnoid fistulas) [29, 63], traumatic lung injury [66, 73] or lung herniation into the spinal canal [37] may require surgical intervention. Although operative repair usually may be uncomplicated, diagnosis and identification of the fistula remains challenging. Other treatment modalities include bronchoscopy for extraction of aspirated foreign bodies causing airway obstruction [69] and transient high concentration oxygen therapy to achieve nitrogen washout [17, 34, 78]. The use of application of prophylactic antibiotic treatment to prevent meningitis and its potential benefit is not instituted and discussed controversial [47, 78]. In principle, apart from antibiotic treatment of underlying infectious diseases such as acute bronchitis [11], even in traumatic PR, we do not recommend prophylactic management with antibiotics in cases of extradural PR and in patients with intradural PR without signs and symptoms of meningitis. The management of patients with PR has to be decided on an individual basis and frequently requires an inter- and multidisciplinary regime.\nIf intraspinal air is present, especially if caused by trauma, the spine physician has to consider PR as an initial sign of potentially associated, hidden and severe diseases or injuries [55, 56].\nIf general anaesthesia is required in a patient with PR, because of the presumptive diagnosis of a perforation of the dura mater and additional pneumocephalus, the involved anaesthetist should not use inhalational nitrous oxide, because it may cause expansion of intracavitary air and result in an increase in CSF pressure, as nitrous oxide diffuses into the air-filled space. In addition, pressurisation of the oro- and nasopharynx should be avoided, and alternative anaesthetic techniques such as intermitted positive pressure ventilation with transient high-concentration oxygen should be used, thus preventing an increase in the volume of any intraspinal and intracranial air and promote faster reabsorption of air [17, 34, 57].\nThe radiologist necessarily has to search for other pathological conditions during the evaluation of a patient with PR and should further investigate the underlying disease or whole extent of injuries. For further treatment of underlying pathologies, other disciplines such as internal medicine, thoracic surgery or ORL may have to be consulted.\nThe subjects of patients with severe and life-threatening causes leading to PR should be carefully monitored, followed-up and considered for admission to an intensive care unit.\nConclusions\nPneumorrhachis can be caused by a multitude of sources and the evaluation of aetiologies of PR could be a diagnostic challenge. Although PR per se usually is self-limiting and without further therapeutic consequences, prompt recognition of the underlying cause is essential. The attending spine specialist has to carefully evaluate the associated pathologies leading to PR to enable adequate therapy.","keyphrases":["pneumorrhachis","intraspinal air","spinal canal"],"prmu":["P","P","P"]}
{"id":"Neurochem_Res-3-1-1832150","title":"Human Coenzyme Q10 Deficiency\n","text":"Ubiquinone (coenzyme Q10 or CoQ10) is a lipid-soluble component of virtually all cell membranes and has multiple metabolic functions. Deficiency of CoQ10 (MIM 607426) has been associated with five different clinical presentations that suggest genetic heterogeneity, which may be related to the multiple steps in CoQ10 biosynthesis. Patients with all forms of CoQ10 deficiency have shown clinical improvements after initiating oral CoQ10 supplementation. Thus, early diagnosis is of critical importance in the management of these patients. This year, the first molecular defect causing the infantile form of primary human CoQ10 deficiency has been reported. The availability of genetic testing will allow for a better understanding of the pathogenesis of this disease and early initiation of therapy (even presymptomatically in siblings of patients) in this otherwise life-threatening infantile encephalomyopathy.\nIntroduction\nCoenzyme Q10 (CoQ10) is the predominant human form of endogenous ubiquinone. Synthesized in the mitochondrial inner membrane, CoQ10 is comprised of a ubiquinone head group attached to a tail of 10 five-carbon isoprenoid units, that anchors the molecule to the membranes [1]. In additions to its central role in the mitochondrial respiratory chain as electrons carrier from complex I and II to complex III [2], CoQ10 is now thought to be involved in a number of cellular functions (Table\u00a01) [1]. Intracellular synthesis is the major source of CoQ10, although a small proportion is acquired through diet. The complexity of the biosynthesis (Fig.\u00a01) suggests that defects in different biosynthetic enzymes or regulatory proteins may cause different clinical syndromes.\nTable\u00a01Functions of coenzyme Q (modified from [1])Electron carrier in mitochondrial respiratory chain [3]Extra-mitochondrial electron transport [4]Antioxidant [5]Regulation of mitochondrial permeability transition pore [6]Regulation of physiochemical properties of membranes [7]Modulation of the amount of \u03b2-integrins on the surface of blood monocytes [8]Modulator of endothelial function [9]Oxidation of sulfide in yeast [10]Introduction of disulfide bonds in bacteria [11]Fig.\u00a01CoQ10 biosynthetic pathway with eight known biosynthetic enzymes denoted as COQ1-8. CoQ10 is composed of a benzoquinone and a decaprenyl side chain. While the quinone ring is derived from amino acids tyrosine or phenylalanine, the isoprenoid side chain is produced by addition of isopentenyl pyrophosphate molecules to geranylgeranyl pyrophosphate (derived from mevalonate pathway) by decaprenyl diphosphate synthase. After para-hydroxybenzoate and decaprenyl pyrophosphate are produced, at least seven enzymes (encoded by COQ2-8) catalyze condensation, methylation, decarboxylation, and hydroxylation reactions to synthesize CoQ10\nPrimary CoQ10 deficiency is an autosomal recessive condition with a clinical spectrum that encompasses at least five major phenotypes: (1) encephalomyopathy characterized by the triad of recurrent myoglobinuria, brain involvement and ragged red fibers; (2) severe infantile multisystemic disease; (3) cerebellar ataxia; (4) Leigh syndrome with growth retardation, ataxia and deafness; and (5) isolated myopathy [12\u201326].\nGeneralized weakness, exercise intolerance, and recurrent myoglobinuria are the distinctive features in the first four patients reported with the encephalomyopathic form, [12\u201315]; however, myoglobinuria was absent in another patient [16]. All the patients described so far have proximal muscle weakness [12\u201316] The brain involvement is variable; seizures are a common finding [12, 13], in association with cognitive impairment [12] and cerebellar symptoms [12, 15, 16].\nThe second variant, described in three families, manifests as an infantile encephalopathy with renal involvement. R\u00f6tig and colleagues initially described two siblings with defects of multiple quinone-dependent enzymes ascribed to a deficiency of CoQ10 in various tissues [17]. The patients presented with retinitis pigmentosa, optic nerve atrophy, bilateral sensorineuronal deafness, nephrotic syndrome, progressive ataxia, and cardiomyopathy. In Rahman\u2019s patient, hypothermia, lactic acidosis, cerebral and cerebellar atrophy, and developmental delay were associated with renal tubulopathy and ventricular hypertrophy [18]. Last year, we described two siblings who presented with nephrotic syndrome due to glomerulosclerosis, and the older sibling developed hypotonia, psychomotor delay, seizures, stroke-like episodes, and cerebellar and cerebral atrophy [19]. Interestingly, in these infantile-onset patients, deficiency of CoQ10 is present not only in skeletal muscle, but also in other tissues and cells including skin fibroblasts. In addition, two adult sisters with encephalopathy, growth retardation, infantilism, ataxia, deafness and lactic acidosis have been described by Van Maldergam [23].\nCerebellar ataxia is the most common phenotype associated with CoQ10 deficiency with 21 reported patients [20\u201322, 26]. Cerebellar ataxia and cerebellar atrophy are present in all the patients. Epilepsy is the most common associated feature, but pyramidal signs, mental retardation, myopathic weakness, and delayed motor milestones are other variably associated symptoms and signs. Except for two adult brothers with cerebellar ataxia and hypogonadism [22], the condition begins in childhood or adolescence. Muscle morphology does not show ragged-red fibers and lipid storage myopathy and CoQ10 is moderately reduced in skin fibroblasts.\nThe clinical presentation of the variant isolated myopathy, recently described in four patients, is subacute onset of exercise intolerance and proximal limb weakness at variable ages. All patients have lipid storage and ragged-red fibers in muscle, as well as increased serum lactate and creatine kinase (CK) [24, 25]. In all the patients with different phenotypes, respiratory chain enzymes analyses show reduced activities of complex I\u00a0+\u00a0III and II\u00a0+\u00a0III with normal activities of isolated complex I and III.\nIn most of the reported patients, the exact site and nature of the defects in the biosynthesis of CoQ10 have not yet been identified. Because ubiquinone biosynthesis is complex and not fully defined (Fig.\u00a01), identification of the molecular genetic defect is not straightforward.\nUndetectable CoQ10 and low levels of decaprenylpyrophosphate, an intermediate compound in the synthesis of the lateral chain of CoQ10 were found in the fibroblasts of the patient reported by R\u00f6tig and colleagues suggesting a defect in the synthesis of the decaprenyl side-chain, but they did not find any mutation in the cDNA of COQ1, the gene encoding transprenyltransferase, the enzyme that synthesizes decaprenylpyrophosphate [17].\nThis year, in two siblings with the infantile form of CoQ10 deficiency, we reported a homozygous missense mutation (Y297C) in the COQ2 gene, which encodes para-hydroxybenzoate (PHB) polyprenyl transferase. PHB-polyprenyl transferase mediates the conjugation of the benzoquinone ring with the decaprenyl side chain. Biochemical assays measuring incorporation of radiolabeled PHB or decaprenyl-PP into CoQ10 in skin fibroblasts from the proband showed 23\u201325% of control fibroblasts activity [27] confirming the defect of CoQ10 biosynthesis.\nThe finding of a homozygous stop codon mutation in the APTX gene which is known to cause ataxia-oculomotor-aprataxia 1 (AOA1), in three siblings with cerebellar ataxia and CoQ10 deficiency, supports the hypothesis that the ataxic form is a genetically heterogeneous disease in which CoQ10 deficiency can be secondary [28]. Aprataxin is a member of the histidine triad superfamily and may be involved in nuclear DNA single strand break repair [29\u201331]. Despite the lack of an obvious link between aprataxin and regulation of CoQ10 synthesis or catabolism, our findings, coupled with the clinical improvement of patients after CoQ10 supplementation, suggest that CoQ10 deficiency plays a pathogenic role in AOA1. Intriguingly, both CoQ10 and cholesterol share a common biosynthetic pathway, therefore, in AOA1, altered levels of these molecules could be due to aberrant biosynthesis.\nPatients with all forms of CoQ10 deficiency have shown clinical improvement with oral CoQ10 supplementation. The beneficial effects of exogenous CoQ10 require high doses and long-term administration [32, 33]; its oral bioavailability is poor due to its extreme hydrophobicity; therefore, only a small fraction of orally administered CoQ10 reaches the circulatory system, and augmentation of mitochondrial CoQ10 content is even less effective. Beneficial effects of CoQ10 supplementation are supported by in vitro correction of biochemical and histological abnormalities [12\u201314, 17]. However, while muscle abnormalities improve clinically and biochemically, in general, cerebral symptoms are only partially ameliorated [16]. This difference could be explained by the presence of irreversible structural brain damage before treatment or the poor penetration of CoQ10 across the blood-brain barrier. Increases in plasma concentrations of CoQ10 after oral supplementation in both humans and animals has been well-documented, but early work questioned whether CoQ10 accumulates in tissues [34] and studies of 45-day-old rodents showed little or no change in rodent brain CoQ10 concentrations after oral administration perhaps because levels of CoQ10 are tightly regulated in young animals or may be saturated in membranes [34, 35]. In contrast, administration of very high doses of CoQ10 (200\u00a0mg\/kg daily) to 12\u201324\u00a0month-old rats produced significant increases in brain CoQ10 levels [36], but a more recent study of the regional distribution of CoQ in 16\u00a0week-old rat brain before and after 4\u00a0weeks of moderately high exogenous dietary CoQ10 (20\u00a0mg\/kg daily) supplementation showed that the concentration of CoQ was essentially unchanged in the cortex and in the striatum [37]. That study also demonstrated that the cerebellum, of the 7 brain regions dissected, contains the lowest levels of CoQ. If human brain has the same regional distribution of CoQ10 as rat brain, the cerebellum may have the narrowest safety margin and therefore would be the first tissue to suffer from a pathological shortage of CoQ10. Moreover, to function as an antioxidant, CoQ10 must be in the reduced form but normally only 20% of the lipid is reduced in the brain. The high proportion of oxidized CoQ10 in the brain could be a reflection of the high oxygen consumption in this tissue, causing an increased demand of anti-oxidants [38]. Therefore, CoQ10 deficiency could be another form of inherited ataxia due to oxidative damage, as vitamin E deficiency.\nOxidative damage and mitochondrial dysfunction have also been implicated in neurodegenerative diseases such Parkinson disease (PD), Alzheimer disease (AD), and Friedreich\u2019s ataxia (FRDA) [39\u201341]. Preclinical studies have indicated that CoQ10 can protect the nigrostriatal dopaminergic system and high doses (1200\u00a0mg daily) of CoQ10 for 16\u00a0months appeared to slow progression PD [40, 42]. In 10 FRDA patients treated for 47\u00a0months high doses of CoQ10 (400\u00a0mg daily) with vitamin E (2100\u00a0IU daily), improved bioenergetics in heart and skeletal muscle, improved cardiac function by echocardiography, and, possibly less clinical decline compared to historical controls [43]. Larger studies are necessary to better define the short- and long-term effects of CoQ10 as primary or adjunctive therapy in neurodegenerative diseases.\nDeficiency of CoQ10 has been associated with a variety of neurological disorders that respond to oral supplementation. We have demonstrated that CoQ10 deficiency can be primary due to a defect of biosynthesis or secondary as in patients with APTX mutations. The response of both groups of patients to oral supplementation suggests that exogenous CoQ10 is taken up by affected tissues and can correct biochemical derangements. Further studies to define CoQ10 deficiency syndrome will reveal new causes, enhance our understanding of its pathogenesis, and may provide novel insights that may be relevant to other neurodegenerative diseases.","keyphrases":["coq10","myopathy","cerebellar ataxia","encephalopathy","mitochondria"],"prmu":["P","P","P","P","U"]}
{"id":"Ann_Surg_Oncol-4-1-2190340","title":"Diagnostic Accuracy of Fine Needle Biopsy for Metastatic Melanoma and Its Implications for Patient Management\n","text":"Background The use of fine needle biopsy (FNB) for the diagnosis of metastatic melanoma can lead to the early removal and treatment of metastases, reduce the frequency of unnecessary surgery, and facilitate the staging of patients enrolled in clinical trials of adjuvant therapies. In this study, the accuracy of FNB for the diagnosis of metastatic melanoma was investigated.\nFine needle biopsy (FNB) is frequently used in the diagnostic workup of clinically or radiologically detected mass lesions that are suspicious for metastatic melanoma. By determining whether they represent metastatic melanoma, the use of FNB in melanoma patients can expedite detection of metastases, leading to earlier removal and treatment; facilitate the staging of patients enrolled in clinical trials of adjuvant therapies (particularly in deep-seated lesions); reduce the frequency of unnecessary surgery; and assist in the planning of the most appropriate surgery.\nFNB is a swift, minimally invasive and cost-effective technique employed in the diagnostic workup of mass lesions occurring in a wide variety of organs.1,2 The technique has been extensively evaluated in assessing the nature of lesions located in the tissues of the breast, thyroid, lung, liver, pancreas, lymph nodes, salivary glands, and kidneys, among other locations.3\u20136\nThere have been several previous case studies assessing the use of FNB in patients with melanoma. The majority of these studies lacked sufficient case numbers to precisely determine the diagnostic accuracy of FNB.7\u201310 However, in 1986, Perry and colleagues11 analyzed almost 300 FNBs from melanoma patients and found the procedure to be accurate, with a sensitivity of 86.5% and a specificity of 96.1%. Since this study, there have been important changes in the procedure, including improvements in immunochemical characterization and radiological guidance. In 2000, Voit and colleagues12 published a study of 739 FNBs from melanoma patients with palpable suspicious lymph nodes or small lesions that were only detectable by ultrasound B-scan examination. Although the authors reported a sensitivity of 97.8% and a specificity of 100.0%, the sensitivity depended on lesion size.\nThe objective of this retrospective cohort study was to evaluate the diagnostic accuracy of the FNB procedure in the detection of metastatic melanoma. To accomplish this, a very large consecutive sample was collected of FNBs performed on melanoma patients who attended the Sydney Melanoma Unit (SMU), Sydney, Australia. This large sample also allowed evaluation of the effect of several clinicopathologic features and factors related to the procedure on the diagnostic accuracy of FNB.\nMATERIALS AND METHODS\nPatients\nFor all patients with melanoma who attended the SMU and gave informed consent, clinical and histologic details of their disease were recorded on the SMU database, and follow-up information was entered prospectively. Patients with suspicious clinically palpable or radiologically identified mass lesions, detected by a variety of imaging modalities, were further investigated by FNB. All SMU patients who underwent an FNB that had been reported by cytopathologists from the Department of Anatomical Pathology at the Royal Prince Alfred Hospital in Sydney, Australia, between January 1992 and December 2002 were identified from the SMU database, and their clinical records and FNB reports were reviewed. Details of the patients are provided in Table\u00a01.\nTABLE\u00a01.Patient characteristicsCharacteristicn%Sex\u00a0\u00a0Male88862.7\u00a0\u00a0Female52837.3Age at melanoma diagnosis (y)\u00a0\u00a010\u2013301107.8\u00a0\u00a031\u2013401359.5\u00a0\u00a041\u20135025618.1\u00a0\u00a051\u20136030321.4\u00a0\u00a061\u20137030221.3\u00a0\u00a070\u20138021915.5\u00a0\u00a081+624.4\u00a0\u00a0Unknown292.0Cancer diagnosis\u00a0\u00a0Other cancer diagnoses15911.2\u00a0\u00a0More than one other cancer diagnoses151.1No. of primary melanoma lesions\u00a0\u00a01123687.3\u00a0\u00a0214810.4\u00a0\u00a0\u22653322.3No. of FNBs\u00a0\u00a0195367.3\u00a0\u00a0228720.3\u00a0\u00a03986.9\u00a0\u00a0>3785.5Total no. of patients1416100.0FNB, fine needle aspiration biopsy.\nFNB Procedure\nFor palpable lesions, the FNBs were performed by the reporting cytopathologist or a trainee pathologist under their supervision. Following localization and stabilization of the lesion with one hand, a hollow bore needle (22G, 23G, or 25G) was inserted directly into the mass and the needle was moved swiftly in and out for approximately 10 seconds. Aspiration with a syringe was not used in the vast majority of cases, hence our preference for the term \u2018fine needle biopsy\u2019 and \u2018fine needle aspiration biopsy\u2019. In our experience, the use of a needle without an attached syringe allows better control of the movement of the needle. Furthermore, aspiration often yields blood, promotes clotting and hampers optimal interpretation of cytologic detail. The procured material was ejected from the needle onto glass slides by pushing air from a syringe through the needle. The material was spread evenly across the slide using another glass slide. One slide was air-dried and stained immediately with Diff-Quik (Lab Aids, Narrabeen, NSW, Australia) and another fixed in alcohol and later stained by the Papanicolaou method.2 Residual material was washed into Hanks balanced salt solution for later preparation of cell blocks using the serum-prothrombin method2 or by cytocentrifuge preparations, to be used for immunochemistry. The air-dried slides were examined by the cytopathologist at the time of the procedure. Further passes were performed if necessary, depending on the amount and type of cellular material obtained.\nReview of Clinical Material and Follow-up\nThe accuracy of the FNB procedure in diagnosing metastatic melanoma was evaluated by two reference standards: a) histopathologic evaluation of the excised lesion (1120 cases) or b) follow-up in those cases for which histologic material was not available (462 cases). The duration of follow-up was 6 months or greater (mean 50.2 months, median 45.7 months, range 6.1\u2013144.4 months) in 456 cases. In six cases, the length of follow-up was less than 6 months (mean 4.2 months, median 4.0 months, range 3.0\u20135.7 months). The mass was considered benign if it was stable in size or resolved after clinical follow-up.\nCytodiagnosis\nCytodiagnoses were categorized as positive, suspicious, or negative for metastatic melanoma (Table\u00a02). Cases were considered positive for metastatic melanoma if the specimen included sufficient numbers of well-preserved malignant cells with typical cytological features, pigment, and\/or confirmatory immunochemistry for a confident diagnosis of melanoma to be made. On verification, these samples were classified as truly positive (n = 800, 50.5%) or falsely positive (n = 5, 0.3%).\nTABLE\u00a02.Categories of cytodiagnosesCharacteristicTotal n%ConfirmedNot confirmedPositive for Metastatic Melanoma\u00a0\u00a0Malignant cells\u2014melanoma108949.4805284Suspicious for Metastatic Melanoma\u00a0\u00a0Malignant cells\u2014suspicious for melanoma442.0395\u00a0\u00a0Malignant cells\u2014unknown cancer331.52211\u00a0\u00a0Suspicious for malignancy401.8355Negative for Metastatic Melanoma\u00a0\u00a0Malignant cells\u2014other cancer924.24745\u00a0\u00a0No malignant cells\u2014other cells present41618.9308108\u00a0\u00a0No malignant cells\u2014scant other cells29613.420492\u00a0\u00a0No malignant cells\u2014no other cells1757.912253\u00a0\u00a0No malignant cells\u2014procedure not performed190.9019Total22041001582622\nSamples that contained cells from unclassified\/unspecified malignancies or cases categorized as suspicious for melanoma (those with small numbers of atypical cells, poorly preserved cells, and cells that lacked specific features of melanoma, such as cytoplasmic pigment, and where insufficient material was present for immunochemistry) were classified as suspicious for metastatic melanoma. These were determined to be true suspicious (n = 78, 4.9%) or falsely suspicious (n = 18, 1.1%) results after verification.\nFNBs classified as negative for metastatic melanoma contained no material that could be diagnosed as metastatic melanoma. They included cases containing malignant cells diagnosed as another neoplasm or b) various amounts of cellular material from the tissue of the site that was sampled. After verification, these procedures were found to be either true negative (n = 612, 38.7%) or false negative (n = 69, 4.4%).\nStatistical Analysis\nAll analyses were performed using the S-PLUS software package (Insightful Corporation, Seattle, WA) and Microsoft Excel, version 2000 (Microsoft, Redmond, WA). Diagnostic accuracy of FNB for metastatic melanoma was measured by the sensitivity and specificity of the test.\nThe effect of the type of tissue involved by melanoma, anatomic location, and 15 clinicopathologic and procedural factors were analyzed. These factors included features of the primary melanoma such as tumor thickness, dermal mitotic rate, presence of ulceration, predominant cell type and histopathologic subtype of melanoma; patient attributes such as sex, American Joint Committee on Cancer (AJCC)\/International Union Against Cancer (UICC) stage,13 age at FNB and location of the FNB (local or distant to the primary lesion); aspects of the FNB procedure such as number of needle passes, needle size, the experience (based on caseload) of the reporting cytopathologist; year of procedure, use of immunostains; and presence of necrosis. Statistical significance was determined by the 95% confidence intervals (95% CIs) of these parameters. When comparing different samples, a two-sample test for binomial proportions was used. All equations used have been described elsewhere.14,15\nRESULTS\nIn the 11-year study period, 2204 consecutive FNBs were performed in 1416 patients. Nineteen FNBs (0.9% of all FNB cases) were excluded from the analysis because the procedure could not be performed, i.e., no aspirate was obtained (Table\u00a03). In half of these cases, the lesion could not be located (inappropriate referrals), but patient pain tolerance and difficulties with the procedure also contributed.\nTABLE\u00a03.Fine needle biopsy procedures that could not be performedLocationExplanation for failureConfirmationHistologyLeft neckUnable to locate lesionNegative\u2014follow-up\u2013Left face (subcutis)PainNot confirmed\u2013Right faceUnable to locate lesionNot confirmed\u2013Left neck (LN)Unable to locate lesionNegative\u2014surgeryNo evidence of malignancyRight breastAdjacent to prothesisNegative\u2014follow-up\u2013Right neckPainNot confirmed\u2013Right axilla (LN)Unable to locate lesionNegative\u2014follow-up\u2013Right neckPainNegative\u2014surgeryNo evidence of malignancyLeft neck (LN)PainNegative\u2014follow-up\u2013Left neck (LN)Unable to locate lesionNegative\u2014follow-up\u2013Right groinPainNegative\u2014surgeryNo evidence of malignancyLeft axilla (LN)Unable to locate lesionPositive\u2014surgeryMelanomaRight axilla (LN)Unable to locate lesionNegative\u2014follow-up\u2013Right neck (LN)PainNot confirmed\u2013Left axilla (LN)Unable to locate lesionNot confirmed\u2013Left neckUnable to locate lesionNegative\u2014follow-up\u2013ThyroidUnable to locate lesionNegative\u2014follow-up\u2013Right axilla (LN)Unable to locate lesionNegative\u2014follow-up\u2013Right sternum (LN)Unable to locate lesionNot confirmed\u2013LN, lymph node.\nVerification of the remaining 2185 FNBs was partial; in 1582 (71.8%), the true disease status was confirmable by either histopathologic evaluation of the excised lesion or by clinical follow-up (Fig.\u00a01). A total of 1120 FNBs were verified by histopathologic evaluation of the excised lesion; all confirmed positive FNBs and FNBs with false-positive or negative results were verified histologically. In those cases (mostly FNBs with negative results) where histopathologic material was not available (462 cases), verification was made by clinical follow-up. The mass was considered benign if it was stable in size or if it resolved after clinical follow-up. The duration of follow-up was >6 months in 456 cases and 3 to 6 months in 6 cases. Of the verified FNBs, 1435 (90.7%) were palpation-guided FNBs performed by trainee cytopathologists or cytopathologists, while 147 (9.3%) were image-guided FNBs performed by radiologists or other physicians.\nFIG.\u00a01.Flow diagram showing fine needle biopsy (FNB) result distribution.\nA total of 603 FNBs (27.4%) could not be verified; this was due to loss to follow-up, particularly by death in patients with advanced metastatic melanoma, uncertainty regarding sites (where FNBs from more than one site were obtained at the same visit to the clinic), and where there were multiple metastases, which led to uncertainty in lesion location and the correlation of FNB and histopathologic results. Unconfirmed FNBs differed from confirmed procedures in several ways. They were more likely to be from lesions located in visceral organs or to be in patients with AJCC\/UICC stage IV disease. FNBs with inconclusive cytodiagnoses were more likely to be followed up with clinical observation or further biopsy.\nConfirmation of FNBs was unaffected by the year of procedure, the number of needle passes during sampling, the use of immunostains, the reporting cytopathologist, or the age or sex of the patient.\nApproximately 12% of the patients were diagnosed with additional cancers (most commonly breast cancer, colorectal cancer, and chronic lymphocytic leukaemia), with some suffering from multiple types of other cancers. Multiple primary melanomas occurred in an eighth of this patient population. Almost a third of the patients underwent multiple FNBs. These procedures were performed both concurrently and sequentially.\nThere were 1582 FNB procedures for metastatic melanoma with histologic verification or clinical follow-up. The overall sensitivity was 92.1% (95% CI, 93.7\u201390.0) and the specificity was 99.2% (95% CI, 99.7\u201398.1). Five cases were determined to be false positive, resulting in a false-positive rate of 0.6% (Table\u00a04). The false-negative rate was 10.2%, with no metastatic melanoma identified in 69 FNB cases in which metastatic melanoma was identified by later histologic evaluation (Fig.\u00a02).\nTABLE\u00a04.False-positive fine needle biopsy findings for metastatic melanomaCytodiagnosisHistologyLocationCommentMelanomaMetastatic adenocarcinomaRight axillary (LN)\u2013MelanomaMetastatic papillary carcinomaLeft supraclavicular fossa (LN)\u2013MelanomaHematomaLeft axilla\u2013MelanomaChronic osteomyelitisLeft skull (bone)S100 positiveaMelanomaMetastatic adenocarcinomaRight axilla\u2013LN, lymph node.aS100-positive histiocytes were identified in the excision specimen, which probably caused the misdiagnosis.FIG.\u00a02.Distribution of false-negative fine needle biopsy (FNB) cytodiagnoses.\nThe large numbers of confirmed cases permitted detailed analysis of this procedure for metastatic melanoma. The effect of FNB site was studied (Table\u00a05). Lymph node tissue was the most common site for FNB evaluation, with 926 procedures, 753 of them confirmed. The sensitivity and specificity of FNB for metastatic melanoma in lymph nodes were not significantly different compared with that for all sites. However, the sensitivity for FNBs performed on lymph nodes located in the axilla was approximately 9% less compared with FNBs performed on lymph nodes in other locations (z = \u22123.9, P = 0.0001). Increased FNB sensitivity (by 4%) was found when the procedure was performed on lesions located in the skin and subcutis compared with other sites. However, this was only of borderline significance (z = 1.9, P = 0.05). Too few confirmed FNBs were conducted on visceral organs (n = 79) to allow conclusions to be drawn regarding the success of the procedure in these locations.\nTABLE\u00a05.Diagnostic accuracy of FNB: Effect of tissue type and anatomic locationLocationnConfirmed%TPFNTSFSFPTNSn(95% CI)Sp(95% CI)All FNB2204158271.880069781856120.92(0.90\u20130.94)0.99(0.98\u20131.00)Lymph nodes92675381.34134341732460.91(0.88\u20130.93)0.99(0.97\u20131.00)\u00a0\u00a0Neck23518578.710431541580.97(0.92\u20130.99)0.98(0.91\u20131.00)\u00a0\u00a0Axilla38331381.71552912221130.84a(0.78\u20130.89)0.98(0.94\u20131.00)\u00a0\u00a0Groin27423385.0144111100670.93(0.88\u20130.96)1.00\u2013\u00a0\u00a0Other342264.710031081.00\u20131.00\u2013Skin and subcutis71150470.92701726401870.94b(0.91\u20130.96)1.00\u2013\u00a0\u00a0Head and neck1319874.8545700320.92(0.82\u20130.96)1.00\u2013\u00a0\u00a0Trunk28819668.11014620830.96(0.91\u20130.99)1.00\u2013\u00a0\u00a0Limbs29221071.911581320720.93(0.88\u20130.97)1.00\u2013Visceral organs1767944.9303520390.91(0.76\u20130.97)1.00\u2013\u00a0\u00a0Liver562239.371100130.88(0.53\u20130.98)1.00\u2013\u00a0\u00a0Lung944345.7182410180.90(0.70\u20130.97)1.00\u2013Other39124662.98766521400.94(0.87\u20130.97)0.99(0.95\u20131.00) TP, true positive; FN, false negative; TS, true suspicious; FN, false suspicious; FP, false positive; TN, true negative; SN, sensitivity; SP, specificity; 95% CI, 95% confidence interval.aAxilla lymph nodes had significantly reduced sensitivity compared with other sites (z = -3.9, P = .0001).bSkin and subcutis FNBs had significantly increased sensitivity compared with other sites (z = 1.9, P = .05).\nFifteen clinicopathologic and procedural factors were analyzed to assess their effect on the diagnostic accuracy of FNB for metastatic melanoma (Table\u00a06). The FNB confirmation rate was similar for all clinicopathologic factors, with the exception of AJCC\/UICC stage and location (distant or regional to the primary lesion), as described above.\nTABLE\u00a06.Diagnostic accuracy of fine needle biopsy effect of clinicopathologic factorsFactorNConfirmed%TPFNTSFSFPTNSN(95% CI)SP(95% CI)All FNB2204158271.880069781856120.92(0.90\u20130.94)0.99(0.98\u20131.00)AJCC\/UICC stage at FNB\u00a0\u00a0Stage I40032380.8128922631550.93(0.88\u20130.97)0.98(0.95\u20130.99)\u00a0\u00a0Stage II70556980.72833124612240.90(0.86\u20130.93)1.00(0.98\u20131.00)\u00a0\u00a0Stage III83656167.13102427511940.93(0.90\u20130.95)0.99(0.97\u20131.00)\u00a0\u00a0Stage IV26312949.0795510390.94(0.87\u20130.97)1.00(0.91\u20131.00)Location\u00a0\u00a0Regional1340107780.46175858643340.91(0.89\u20130.93)0.99(0.97\u20131.00)\u00a0\u00a0Distant86450558.418311201212780.94(0.90\u20130.97)1.00(0.98\u20131.00)Use of immunochemistry\u00a0\u00a0Yes58340669.631662681490.98a(0.96\u20130.99)0.98(0.90\u20131.00)\u00a0\u00a0No1621117672.548463521045630.88(0.86\u20130.91)0.99(0.98\u20131.00)Year\u00a0\u00a01992\u2013199439228572.71441515511050.91(0.85\u20130.94)0.99(0.95\u20131.00)\u00a0\u00a01995\u2013199755541474.62161513611630.94(0.90\u20130.96)0.99(0.97\u20131.00)\u00a0\u00a01998\u2013200069350072.22412126322070.92(0.88\u20130.95)0.99(0.97\u20131.00)\u00a0\u00a02001\u2013200256438367.91991824411370.92(0.87\u20130.95)0.99(0.96\u20131.00)Sex\u00a0\u00a0Male137099472.650048451013900.91(0.89\u20130.93)1.00(0.99\u20131.00)\u00a0\u00a0Female83458870.53002133842220.93(0.90\u20130.96)0.98(0.96\u20130.99)Age at FNB\u00a0\u00a0\u226450 y57241472.41981222121790.94(0.90\u20130.97)0.99(0.96\u20131.00)\u00a0\u00a0>50 y1632116871.660257561734330.91(0.89\u20130.93)0.99(0.98\u20131.00)No. of FNB attempts\u00a0\u00a0142630972.52324320680.98b(0.96\u20130.99)1.00(0.95\u20131.00)\u00a0\u00a0251837171.61851321301490.93(0.89\u20130.96)1.00(0.97\u20131.00)\u00a0\u00a0331521969.5791312601090.86(0.77\u20130.92)1.00(0.97\u20131.00)\u00a0\u00a0\u2265419213670.833172111630.66(0.52\u20130.78)0.98(0.92\u20131.00)\u00a0\u00a0Unknown75354772.62712221642230.92(0.89\u20130.95)0.98(0.96\u20130.99)Needle sizec\u00a0\u00a022G1336045.1222410310.92(0.74\u20130.98)1.00(0.89\u20131.00)\u00a0\u00a023G17313879.8667820550.90(0.81\u20130.95)1.00(0.93\u20131.00)\u00a0\u00a025G91066473.03512439712420.94(0.91\u20130.96)1.00(0.98\u20131.00)Necrosis present\u00a0\u00a0Yes674161.224490040.86(0.69\u20130.94)1.00(0.51\u20131.00)\u00a0\u00a0No2137154172.177665691856080.92(0.90\u20130.94)0.99(0.98\u20131.00)Pathologist caseload\u00a0\u00a0<100 cases16410463.4517540370.88(0.77\u20130.94)1.00(0.91\u20131.00)\u00a0\u00a0100\u2013500 cases65147472.82272927321860.89(0.84\u20130.92)0.99(0.96\u20131.00)\u00a0\u00a0>500 cases1389100472.352233461133890.94d(0.92\u20130.96)0.99(0.08\u20131.00)First primary Breslow thickness \u00a0\u00a0\u22642 mm95070574.23322334843040.94(0.90\u20130.96)0.99(0.97\u20130.99)\u00a0\u00a0>2 mm91966272.03673833602180.91(0.87\u20130.93)1.00(0.98\u20131.00)\u00a0\u00a0Unknown33521564.210181141900.93(0.86\u20130.96)0.99(0.94\u20131.00)First primary ulceration\u00a0\u00a0Yes57140771.32471618101250.94e(0.90\u20130.96)1.00(0.97\u20131.00)\u00a0\u00a0No100975775.036040351043080.90(0.87\u20130.93)0.99(0.97\u20131.00)\u00a0\u00a0Unknown62441867.01931325711790.94(0.90\u20130.96)0.99(0.97\u20131.00)First primary lesion mitotic rate (\/mm2)\u00a0\u00a0\u2264141430072.51361012421360.93(0.88\u20130.96)0.99(0.95\u20131.00)\u00a0\u00a01 to <448135273.21701920211400.90(0.85\u20130.93)0.99(0.96\u20131.00)\u00a0\u00a04 to <833223771.41417810800.95(0.91\u20130.98)1.00(0.95\u20131.00)\u00a0\u00a0\u2265839229675.5175221440810.89(0.84\u20130.93)1.00(0.95\u20131.00)\u00a0\u00a0Unknown58539767.91781124721750.94(0.90\u20130.97)0.99(0.96\u20131.00)First primary lesion histologic subtype\u00a0\u00a0Desmoplastic1309774.6426630400.88(0.75\u20130.94)1.00(0.91\u20131.00)\u00a0\u00a0SSM51237072.31681022411650.94(0.90\u20130.97)0.99(0.97\u20131.00)\u00a0\u00a0NM46134374.41982014411060.91(0.86\u20130.94)0.99(0.95\u20131.00)\u00a0\u00a0Other75454972.82772622412190.91(0.88\u20130.94)1.00(0.97\u20131.00)\u00a0\u00a0Unknown34722364.311571432820.94(0.89\u20130.97)0.98(0.92\u20130.99)First primary lesion predominant cell type\u00a0\u00a0Epithelioid44332974.31851713401100.92(0.87\u20130.95)1.00(0.97\u20131.00)\u00a0\u00a0Spindle1378965.0476900270.89(0.77\u20130.95)1.00(0.88\u20131.00)\u00a0\u00a0Mixed17311164.2586830360.91(0.81\u20130.96)1.00(0.90\u20131.00)\u00a0\u00a0Unknown1451105372.651040481154390.93(0.90\u20130.95)0.99(0.97\u20131.00)TP, true positive; FN, false negative; TS, true suspicious; FN, false negative; FP, false positive; TN, true negative; SN, sensitivity; SP, specificity; 95% CI, 95% confidence interval; SSM, superficial spreading melanoma; NM, nodular melanoma.aFNBs that used immunostains had significantly increased sensitivity compared with those that did not (z = 4.8, P < .001).bFNBs obtained in one pass had significantly increased sensitivity compared with FNBs which required more than one pass (z = 4.8, P < .001).cThe size of the needle used for the FNB procedure was not known in 988 cases.dFNBs reviewed by a cytopathologist who reported >500 cases had significantly increased sensitivity compared with FNBs reported by cytopathologists who had reported <500 cases (z = 2.4, P = .02).eFNBs obtained from patients with an ulcerated first primary lesion had increased sensitivity compared with FNBs from patients without ulcerated first primary lesions (z = 1.9, P = .05).\nPatients with an ulcerated primary lesion experienced slightly better FNB test sensitivity compared to those with no ulceration (z = 1.9, P = 0.05). AJCC\/UICC stage, anatomic location, year of procedure, needle size, sex, age at FNB, primary lesion thickness, dermal mitotic rate, predominant cell type, histologic subtype of melanoma, and presence of necrosis did not affect the diagnostic accuracy of the test (Table\u00a06).\nThree factors related to the conduct of the procedure affected FNB test accuracy. These were the number of needle passes needed to collect the sample, the number of FNBs for metastatic melanoma the cytopathologist had reported during the study period (caseload), and the use of immunostains. Samples that required only one attempt at FNB had a sensitivity increase by >10% compared to samples with more than one attempt (z = 4.8, P < 0.001). Sensitivity fell in a linear manner with each subsequent attempt (Fig.\u00a03). Cytopathologists who had caseloads of >500 FNBs (2 cytopathologists) had greater FNB test sensitivity by about 5% compared to those who had reported <500 FNBs (16 cytopathologists) (z = 2.4, P = 0.02). Finally, FNB tests in which immunostains were used had better sensitivity (z = 4.8, P <0.001) (Table\u00a06).\nFIG.\u00a03.Sensitivity of fine needle biopsy (FNB) in the diagnosis of metastatic melanoma. Sensitivity is reduced in a linear manner as the number of FNB attempts needed to obtain the sample increases. Bars, 95% confidence intervals.\nDISCUSSION\nMetastatic melanoma is an aggressive tumor with a high mortality rate. Patients with primary melanomas that are at high risk of metastasizing who attend the SMU are followed closely for evidence of metastatic disease with radiological investigations and regular clinical follow-up. Early surgery for metastatic disease may lead to longer disease-free periods and may ultimately improve survival in some of these patients. This underscores the importance of early detection of metastatic disease.\nIn some melanoma treatment centers, FNB has been used for many years to verify clinically suspicious lesions and radiological abnormalities in patients with melanoma, prior to traumatic or costly surgical or adjuvant treatment. FNB is a cost-effective, rapid procedure that is well tolerated by patients when performed by proficient operators.2 However, the ability of the test to accurately diagnose suspicious lesions as metastatic melanoma has been infrequently studied.\nSensitivity and specificity are measures that are used extensively in the FNB literature. They were chosen as the best measures of diagnostic accuracy of FNBs in patients with melanoma because of the binary quality of the data.16 In this study, a large number of consecutive FNB procedures (2204) for metastatic melanoma were analyzed, with 1582 procedures confirmed by either histopathology after surgical resection or by clinical follow-up. This cohort represents more FNB procedures in melanoma patients than all previous reported series combined.7\u201312 The overall sensitivity of FNB for metastatic melanoma was 92.1%, and the specificity was 99.2%. These results are superior to those obtained by Perry and colleagues11 in their study of 298 (261 confirmed) cases of metastatic melanoma FNB more than 20 years ago (sensitivity of 86.5% and specificity of 96.1%). Other similar case series did not have the power to determine the true diagnostic accuracy of FNB because of their low numbers of FNBs (between 56 and 108 confirmed cases).7\u201310 Voit and colleagues12 examined 739 FNBs for metastatic melanoma and found the technique to be highly sensitive (97.9%) at their institution. However, regional node basins in their patient population were routinely evaluated by ultrasound B-scan, and a third of FNB cases were performed under ultrasound guidance.\nStudies of FNB are frequently retrospective investigations and thus verification bias may be a hazard in these studies. Not only are there different reference tests, which are determined by the results of the FNB procedure, namely histopathology after surgical resection for all positive FNB tests and clinical follow-up for most negative procedures, but verification is also partial. Not every consecutive FNB can be verified. Differential verification can lead to overestimation of the measures of diagnostic accuracy.17,18 This bias is related to the quality of the reference tests. Confirmation of melanoma metastases by histopathologic examination of tissue removed after surgery is better at identifying true disease status than clinical follow-up of patients with negative FNB results.19 Melanoma metastases may regress spontaneously, resulting in failure to identify all false-negative results by clinical follow-up, or they may cause truly positive findings to be incorrectly classified as false positives if the lesion resolves before it is excised or during follow-up.20,21\nIn this study, there were differences in those FNBs that were verified by the reference tests compared with those that were not. Unconfirmed FNBs were more often performed on nonpalpable lesions in visceral sites, and in patients with advanced disease. Verification rates of negative, positive, and suspicious FNBs were 68.2%, 73.9%, and 82.1%, respectively.\nDespite the high specificity rate, as a result of the large number of confirmed biopsy samples, five false-positive findings were detected (Table\u00a04). The cytologic features of these cases have been reviewed in more detail elsewhere.22 In all cases, misinterpretation of the cellular material had occurred, three of which were found to be adenocarcinoma. Two of these represented metastatic breast adenocarcinoma in axillary nodes, and one was a metastatic papillary adenocarcinoma of renal origin. Two other false-positive cases were caused by the misinterpretation of large histiocytes or reactive fibroblasts as metastatic melanoma cells.\nIn 69 FNBs, no metastatic melanoma cells were identified cytologically; however, the presence of metastatic melanoma was subsequently identified by histopathologic examination (false negatives) (Fig.\u00a02). Astute clinical and\/or radiologic surveillance results in the detection of small suspicious lymph nodes. Difficulties in performing FNBs on such small suspicious lymph nodes contribute to some of the false-negative diagnoses (particulary small, mobile axillary nodes). In fact, approximately a third (n = 21) of these biopsy samples (which were reported as \u201cno malignant cells identified\u201d) did not contain any cellular material, suggesting that an absence of sampling of the malignant cells was the reason for the false-negative result. Further investigation is usually necessary to identify the cause of the mass lesion. Surgical examination is usually the next step when the index of clinical suspicion is high.\nThere were several sources of difficulty in performing the FNB procedure for metastatic melanoma. Often it occurred when there was a failure to locate the suspicious lesion because of its small size or its location was not communicated with adequate precision. Failure to identify metastatic melanoma occurred when cellular material showed too few typical morphologic characteristics of metastatic melanoma, or these characteristics were destroyed or masked by necrosis.\nThe large number of confirmed FNB in this study permitted the analysis of subgroups. Most FNB for metastatic melanoma were performed in lymph nodes as well as skin and subcutaneous tissues. No difference in sensitivity was noted among these tissue groups. However, FNBs of lymph nodes of the axilla were significantly less sensitive compared with those performed at other sites and with FNBs performed in lymph nodes of the groin and neck. A number of factors are likely to contribute to the low sensitivity of FNBs performed in the axilla, including the greater difficulty in gaining access to and locating lymph nodes, particularly those high in the axilla, and the presence of large amounts of fatty tissue in axillary lymph nodes (\u201chorseshoe\u201d nodes).\nFifteen clinicopathologic factors were examined for their effect on the accuracy of FNB for metastatic melanoma, most of which had no effect on the diagnostic accuracy of the test. However, the following four variables did influence the sensitivity of the FNB procedure. (1) Ulceration of the primary lesion led to a small increase in the sensitivity of the test (P = 0.05). (2) Additional needle passes were performed if the initial sampling failed to obtain sufficient cellular material; thus, the number of needle passes is an indicator of the difficulty in obtaining a sample from a specific lesion. FNBs that were performed in only one needle pass were found to have superior sensitivity than those which required additional passes to obtain an adequate sample. Furthermore, the sensitivity of the test seemed to decrease in a linear manner with each subsequent pass. (3) Training and experience have been shown to significantly influence the interpretation of FNB of the breast,23 and in this study, we found that the caseload (i.e., level of experience) of the cytopathologist who reviewed the slides influenced the sensitivity of the test. Those cytopathologists who reviewed >500 metastatic melanoma FNB samples in the study period performed better. Those cytopathologists who performed <100 reviews seemed to do as well as those who had a caseload of 100 to 500 cases. (4) The use of immunostains was associated with improved sensitivity of the test. However, this may reflect the fact that immunochemistry was only performed in those cases in which a sufficiently high cellular yield was obtained, in which case it may have been the high cellular yield that lead to an improvement in sensitivity, rather than the use of immunostains per se. The diverse cytological presentation of metastatic melanoma may also be a factor in the association of the use of immunochemistry with increased sensitivity.24,25 Nasiell et al.26 found that immunochemical characterization was necessary to conclusively diagnose >50% of metastatic melanomas that presented with an equivocal cytological picture. However, immunological characterization cannot be considered definitive when the FNB lacks typical cytologic features expected of metastatic melanoma. For example, one of the false-positive cases exhibited S100 positivity, whereas two of the false-negative FNBs contained melanoma cells that seemed to be S100 negative. This suggests that cytopathologists should be cautious when reporting the results of immunostains on limited samples.\nOur study shows that FNB for metastatic melanoma is a procedure with very high specificity and good sensitivity. Several clinicopathologic factors were found to influence the diagnostic accuracy of the test for metastatic melanoma. These included factors relating to the original primary melanoma lesion, location of the sampled lesion, and factors relating to the performance of the test. The SMU employs an on-demand FNB service with assessment of the cytologic material and delivery of a provisional result to the clinician at the time of the patient\u2019s visit. This helps guide subsequent diagnostic and therapeutic measures, and it reduces costs (e.g., by decreasing the need for additional patient visits to the clinic). A multidisciplinary approach involving clinicians, pathologists and radiologists enables an efficient and cost-effective management strategy in melanoma patients.","keyphrases":["diagnostic accuracy","fine needle biopsy","melanoma","diagnosis","pathology","cytology"],"prmu":["P","P","P","P","P","P"]}
{"id":"Cell_Calcium-1-5-1885971","title":"Dynamic and static calcium gradients inside large snail (Helix aspersa) neurones detected with calcium-sensitive microelectrodes\n","text":"We have used quartz Ca2+-sensitive microelectrodes (CASMs) in large voltage-clamped snail neurones to investigate the inward spread of Ca2+ after a brief depolarisation. Both steady state and [Ca2+]i transients changed with depth of penetration. When the CASM tip was within 20 \u03bcm of the far side of the cell the [Ca2+]i transient time to peak was 4.4 \u00b1 0.5 s, rising to 14.7 \u00b1 0.7 s at a distance of 80 \u03bcm. We estimate that the Ca2+ transients travelled centripetally at an average speed of 6 \u03bcm2 s\u22121 and decreased in size by half over a distance of about 45 \u03bcm. Cyclopiazonic acid had little effect on the size and time to peak of Ca2+ transients but slowed their recovery significantly. This suggests that the endoplasmic reticulum curtails rather than reinforces the transients. Injecting the calcium buffer BAPTA made the Ca2+ transients more uniform in size and increased their times to peak and rates of recovery near the membrane. We have developed a computational model for the transients, which includes diffusion, uptake and Ca2+ extrusion. Good fits were obtained with a rather large apparent diffusion coefficient of about 90 \u00b1 20 \u03bcm2 s\u22121.This may assist fast recovery by extrusion.\n1\nIntroduction\nChanges in free intracellular calcium ([Ca2+]i) play a vital role in intracellular signalling, notably in coupling electrical excitation to secretion, contraction and other activation processes [1,2]. Many of the processes activated by a rise in [Ca2+]i are believed to occur in the nucleus [3,4], which in large neurones may be far from the cell membrane. It is well-established that a brief depolarization of most nerve cells causes an influx of Ca2+ ions across the plasma membrane and thus a local rise in [Ca2+]i. The spatiotemporal characteristics of this rise in calcium are defined by the interactions between Ca2+ ions and the cytoplasmic milieu. Calcium-induced calcium release (CICR) processes via either IP3 or ryanodine receptors can facilitate the regenerative spread of the calcium signal throughout the cell, whilst immobile calcium buffers will slow and attenuate the signal. If the increase in sub-membrane [Ca2+]i is to act as a signal at a distance, the speed at which the Ca2+ ions travel from the cell membrane to their target depends critically on the rate of diffusion of Ca2+ ions in the cytoplasm. Most studies of calcium homeostasis and signalling in nerve and muscle cells have been made using optical methods to measure [Ca2+]i via the binding of Ca2+ ions by fluorescent or other indicators [5]. Such indicators tend to increase intracellular Ca2+ buffering and mobility [6]. Many authors (for example [7\u201311]) have modelled the effect of mobile indicators on Ca2+ transients, but very few have produced data on intracellular Ca2+ movement from the periphery towards the centre inside real neurones in the absence of such alien additives. We now present such data, obtained with Ca2+-sensitive microelectrodes (CASMs), for the first time.\nCASMs offer an alternative but rarely-used method for measuring [Ca2+]i. They have the advantage of measuring [Ca2+]i in a small volume without adding any extraneous compounds which might interact with Ca2+ throughout the cell. But they are rather slow to respond, and only a realistic choice for large cells since they tend to create a leak at the site of penetration [12\u201315].\nIn previous work, with CASMs made from aluminosilicate glass, the CASMs were rarely moved once a stable potential had been obtained from deep inside the neurone [e.g. 13,14,16]. Recently a modified electrode construction technique has allowed us to use CASMs with very slender tips and longer lives made from thin-walled quartz glass. We have also found it possible, with a motorized micromanipulator, to move the CASMs in repeatable steps up and down, usually without apparently causing damage to the cell.\nWe have sought to record Ca2+ transients at different distances from the inside of the membrane at the far side of the penetrated cell. Our recordings at different depths were quite repeatable, and enabled us to reconstruct the radial inward movement of the entering Ca2+ towards the centre of the cell. We have done this in large neurones with initially no added Ca2+ indicator or buffer. Although our method does tend to induce a leak of Ca2+ ions at the penetration site, we believe that the leak simply increases the steady state level without apparently influencing the kinetics of the Ca2+ transients. We investigated the role of the endoplasmic reticulum by blocking its Ca2+ uptake pump with cyclopiazonic acid. We also injected the mobile Ca2+ buffer BAPTA, which has very similar Ca-buffering properties to many fluorescent Ca2+ indicators, to measure its effects on Ca2+ diffusion. We have modelled the Ca2+ increases mathematically and have been able to model the transients closely by assuming that calcium signalling in these cells can be described by Ca2+ influx, diffusion, extrusion, sequestration and buffering. Good fits were obtained with a diffusion coefficient of about 90\u00a0\u00b1\u00a020\u00a0\u03bcm2\u00a0s\u22121, which is higher than reported in other cell types.\n2\nMaterials and methods\n2.1\nGeneral\nAll experiments were done on large (150\u2013250\u00a0\u03bcm diameter) snail neurones in isolated sub-oesophageal ganglia [14,16]. Cells were voltage-clamped using two microelectrodes to \u221250\u00a0mV, and depolarized at intervals by 40 or 50\u00a0mV to generate an influx of Ca2+ ions. The resulting changes in [Ca2+]i were measured using CASMs. The main differences from previous methods were that we made the CASMs with quartz rather than aluminosilicate glass and used a motorized micromanipulator (Luigs & Neumann, Ratingen, Germany) to move the CASM up and down in reproducible steps. Rotating the Z-control wheel by 360\u00b0 moved the CASM up or down by 40\u00a0\u03bcm, thus for a 20\u00a0\u03bcm step we rotated it manually by 180\u00b0.\n2.2\nPreparation\nAn aestivating snail, Helix aspersa, was killed humanely by rapid removal of the circumoesophageal ring of ganglia. These ganglia were mounted dorsal side uppermost on a plastic bath insert. The thick connective tissue covering the back of the visceral and right and left pallial ganglia was then removed and the bath insert with ganglia was mounted in the experimental chamber at an angle of 50\u00b0 to the vertical. Once covered with snail Ringer (flow rate 1.0\u20131.2\u00a0ml\u00a0min\u22121, bath volume 0.1\u00a0ml) the inner connective tissue was torn with a fine tungsten hook to expose some of the neurones. All experiments were carried out at room temperature, 18\u201322\u00a0\u00b0C, starting at least an hour after the dissection.\n2.3\nSolutions\nThe normal snail Ringer contained (mM): 80 NaCl, 4 KCl, 7 CaCl2, 5 MgCl2, 20 HEPES, titrated with NaOH to pH 7.5. Nominally Ca-free Ringers had the same composition but with no CaCl2 and 12\u00a0mM MgCl2. The EGTA Ca-free solution had an additional 1\u00a0mM EGTA. The CO2 Ringer was the same as normal Ringer except that it had 20\u00a0mM NaHCO3 instead of HEPES, was bubbled with 2.5% CO2 in air and contained 0.1\u00a0mM NaH2PO4. The high K low Ca solution for BAPTA iontophoresis calibration contained 100 KCl, 5 NaCl, 0.6 MgCl2, and 10\u00a0mM HEPES. The pH was adjusted to 7.2 with KOH. Cyclopiazonic acid (CPA) was kept frozen as a 50\u00a0mM stock solution in dimethyl sulphoxide and diluted in snail Ringer on the day of use. All these chemicals were from Sigma.\n2.4\nMicroelectrodes\nConventional micropipettes were pulled from 1.2\u00a0mm filamented borosilicate glass tubing and backfilled with either 2\u00a0M CsCl (for passing current), or 2\u00a0M KCl (for recording membrane potential). Tips were broken if necessary by touching a pin in the bath to give resistances between 5 and 15\u00a0M\u03a9. BAPTA was iontophoresed from similar micropipettes filled with 100\u00a0mM BAPTA (K salt). CASMs were made by a method refined from those described previously [16,17]. Every few days we pulled 10\u201312 long, sharp micropipettes from unfilamented quartz tubing (1.0\u00a0mm\u00a0o.d., 0.7\u00a0mm\u00a0i.d.) using a Sutter P-2000 puller (Sutter Instrument Co, Novato, Cal.). We then broke the tips on a glass rod to o.d. 1.0\u20131.5\u00a0\u03bcm. We then left the micropipettes in air for 2\u20134\u00a0h before silanizing them with bis(dimethylamino)dimethylsilane (Fluka 14755) as described before [16].\nIndividual CASMs were prepared as follows on the day of the experiment. First we backfilled 2 or 3 silanized micropipettes with 1\u00a0mM CaCl2 (instead of the 100\u00a0mM KCl, 0.1\u00a0mM CaCl2 used before, which we realised shortened the CASM's useful life) using a 10\u00a0ml syringe connected to the back of each micropipette via a silicone rubber tube to force air out of the tips. We then sucked a column (150\u2013250\u00a0\u03bcm) of sensor cocktail into the tip of each micropipette, taking care to avoid any air gap between cocktail and backfill. The cocktail composition was (mg\/g): 12 Ca ionophore ETH 129 (Fluka 21193), 6 Na tetrakis[3,5-bis(trifluoromethyl)phenyl]borate, 200 2-nitrophenyloctylether, 17 high molecular weight PVC, and 765 tetrahydrofuran (all from Fluka). Filled microelectrodes were left in air to allow the tetrahydrofuran to evaporate for at least 2\u00a0h before use, and had useful lives of 2\u20133 days. During the experiment we applied 80\u2013120\u00a0kPa of air pressure to the back of the CASM, since this maximizes its response [15]. Before use each microelectrode was tested in the experimental bath, initially perfused with normal snail Ringer. Once the potential had stabilised the perfusate was switched to Ca-free (EGTA) Ringer's solution. Any electrode showing a potential change of less than 150\u00a0mV was discarded. The potentials recorded from the CASMs during the experiments were displayed as VCa (Vm being automatically subtracted when both electrodes were in the same cell) and we have not converted them either to pCa or nM free [Ca2+]i except for modelling purposes. Calibration of CASMs is difficult because, in our hands, their responses tend to change when they are withdrawn from a cell at the end of an experiment. Previous work [14] suggests that their responses are essentially linear with pCa between 2 and 7(10\u00a0mM\u2013100\u00a0nM), with a potential change of 28\u00a0mV per decade. Half-response times ranged from 0.2 to 0.5\u00a0s, but were not routinely measured. Results were discarded if on withdrawal the electrode potentials had changed by more than 7\u00a0mV.\n2.5\nEstimating the BAPTA intophoresis transport number\nWe first used CASMs to measure how much BAPTA was required to change the VCa of 1\u00a0ml of the high K, low Ca solution from \u221250 to \u2212110\u00a0mV (CASM zeroed in normal snail Ringer). The BAPTA was injected into the 1\u00a0ml aliquots using an oocyte microinjector. The average quantity of BAPTA needed was 75\u00a0nmol. We then made droplets (volumes 15\u201325\u00a0nl) of the same solution under oil, measured VCa as with a snail neurone, and injected BAPTA iontophoretically. We found that to achieve the equivalent VCa change as in the 1\u00a0ml samples we had to inject an average 0.6\u00a0\u03bcC\u00a0nl\u22121, giving a transport number of 0.01.\n2.6\nData collection and analysis\nPotentials from the microelectrodes were led via Ag\/AgCl wires to preamplifiers in the cage. Outputs from these and the clamp amplifier were filtered (8-pole Bessel) and recorded at 20\u00a0Hz on a PC via a CED micro 1401 interface and Spike 2 data collection program (Cambridge Electronic Design, UK). The CASM potential was differentially recorded with the Vm microelectrode potential as reference. Figures were prepared from the CED data after loading into Microsoft EXCEL. Large spikes in the VCa records generated by electronic pickup were erased, and the clamp current records were restricted in range. For more details of most of the apparatus see [14,16]. Data are presented as means\u00a0\u00b1\u00a0S.E.M. of n observations. The statistical significance of observed shifts in means was determined by a one-tailed Wilcoxon rank sum test. The difference between means was considered significant when the p-value was higher than 0.95. For baseline adjustment, selecting transients, time to peak and exponential fitting, a home made Matlab script with a graphical user interface was used.\n2.7\nComputational modelling\nThere is an extensive body of literature on the modelling of Ca2+-signalling in a wide variety of cell types, for example [18\u201321]. In this study we set out to develop a minimal computational model of Ca2+-signalling in snail neurones. We used the model to validate and explain the results obtained from the experiments. Furthermore, we used the model to estimate the mobility of Ca2+ in these large neuronal cell bodies, which have not been exposed to mobile Ca2+-indicators.\nThe model includes the following dynamic processes: Ca2+ diffusion, influx, extrusion, sequestration and buffering. We assume that influx and extrusion mechanisms at the plasma membrane are uniformly distributed over the cell's surface. The CASM and the axon attached to the soma only represent a small fraction of the cell's volume (<1%) and surface area (<5%), furthermore transients were measured near the far side of the cell, opposite to the site of penetration. Since we could not detect any major deformation of the cell looking through the microscope we assume that the Ca2+ transients measured close to the membrane can be modelled with spherical symmetry. Using these assumptions we can reduce the three-dimensional diffusion problem to a one-dimensional radial diffusion equation in a sphere. Similar computational models for Ca2+ diffusion in a spherical cell can be found elsewhere [9,22].\nThe removal of Ca2+ after a brief influx is relatively slow, with time constants ranging from 5 to 15\u00a0s, while Ca2+ buffering occurs on the micro- and millisecond time scale. We therefore can separate these different processes kinetically and remove the explicit buffering terms from the transport equation [20]. In so doing we have to replace the Ca2+ diffusion coefficient DCa (\u03bcm2\u00a0s\u22121) with an apparent diffusion coefficient Dapp:\nHere we used the notation from [7]; \u03bae and\u03bab denote the endogenous and exogenous Ca2+ binding ratio, respectively; the associated diffusion coefficients are De and Db. The diffusion coefficient of free cytoplasmic Ca2+ is denoted by DCa and was assumed to be 220\u00a0\u03bcm2\u00a0s\u22121\n[23]. Furthermore, all other transport processes have to be scaled down by the total Ca2+ binding ratio \u03batot\u00a0=\u00a01\u00a0+\u00a0\u03bae\u00a0+\u00a0\u03bab.\nThe total Ca2+ binding ratio \u03batot depends on the Ca2+ concentration; however, when the concentration is much lower than the dissociation constant it may be approximated to be constant. Moreover, although some data are available on buffering in snail neurones [24], precise measurements that separate the buffering and sequestration processes are not. We have therefore worked with apparent fluxes and apparent parameters in most cases. Multiplication of these apparent parameters with the total buffering power should yield the real parameters.\nThe general transport equation that describes the calcium dynamics in the cell, taking into account the different aforementioned considerations then becomes:\nThe first term on the right hand side represents the radial diffusional process that is driven by the concentration gradient in the cell. The transmembrane fluxes jin,app and jout,app describe the apparent influx of calcium through channels and apparent efflux of calcium by the pump, respectively, Jseq,app denotes the apparent calcium flux associated with sequestration to stores and mitochondria in the cell's cytoplasm and finally jbasal,app represents the basal influx of Ca2+ at rest. The surface-to-volume ratio rsv (m\u22121) is necessary to convert the flux density j (mol\u00a0m\u22122\u00a0s\u22121) into a concentration flux J (mM\u00a0s\u22121). The concentration [Ca2+]i and all fluxes are functions of position r (m) and time t (s); however, for readability we do not write this explicitly.\nThere are few precise quantitative data available about the different transport processes involved. To prevent the introduction of many unknown parameters we therefore kept the mechanistic complexity to a bare minimum. Consequently we modelled the influx as a square pulse with the same duration as the depolarisation:where the apparent Ca2+-current denoted by ICa,app (A) relates to the real current as follows: ICa,app\u00a0=\u00a0ICa\/\u03batot. S denotes the surface area (m2) of the cell and F is Faraday's constant. The extrusion of Ca2+ by the pump is modelled by the following linear equation (for example see [25]):where Pout,app denotes the apparent extrusion permeability (m\u00a0s\u22121) and relates to the real permeability as follows: Pout,app\u00a0=\u00a0Pout\/\u03batot. The flux associated with sequestration was also assumed to be linear:where kseq,app denotes the apparent sequestration rate constant (s\u22121) and relates to the real rate constant as follows: kseq,out\u00a0=\u00a0kseq\/\u03batot\u00b7[Ca2+]s is the steady state Ca2+ concentration. This approximate model is also known as a pool model [26]. Finally the basal influx jbasal,app was directly calculated from the steady state Ca2+ concentration and the extrusion parameter: jbasal,app\u00a0=\u00a0Pout,app [Ca2+]s.\n2.8\nMicroelectrode model\nIn this study we used CASMs to measure the Ca2+ concentration after short depolarisations. If we assume equal activity coefficients in the Ringer and cytoplasm, the steady state cytoplasmic Ca2+ concentration can be directly estimated from the Nernst equation applied to the CASM potential:where the steady state Nernst-potential is denoted by ECa (V), R is the gas constant and the temperature T was 293\u00a0K. The backfill Ca2+ concentration [Ca2+]CASM in the microelectrode was 1.0\u00a0mM. The reference potential E0 was manually adjusted prior to the experiment such that the measured potential was 0\u00a0mV in Ringer solution; the Ringer solution contained 7\u00a0mM Ca2+.\nWere the resistance and capacitance of the CASMs to be infinitely small and VCa to follow the Nernst equation over the whole range it could be used directly to estimate the cytoplasmic Ca2+ concentration. VCa is indeed close to the Nernstian value at [Ca2+]i over 100\u00a0nM [14]. Unfortunately the resistance of these microelectrodes is very high, which will introduce a delayed microelectrode response. To allow for this delay we used a RC filter to calculate the model microelectrode potential VCa:The response time of the microelectrode is denoted by \u03c4CASM (s). We assume that it is constant and depends only weakly on the microelectrode potential and [Ca2+]i.\n2.9\nIntegration and parameter fitting\nAssuming a linear transport equation a rather complex analytical solution consisting of an infinite series of exponentials with different time constants can be obtained using standard methods [27]:\nThe amplitudes An and the time constants \u03c4n are:\nThe constants \u03b1n are the positive roots of the following equation:where the diffusion time constant \u03c4D\u00a0=\u00a0d2\/(4D) and the extrusion time constant \u03c4p\u00a0=\u00a0d\/(2P).\nThe slowest time constant determines the overall recovery time. When there is no sequestration this time constant depends on the cell diameter d, the extrusion rate constant and the diffusion coefficient in a complex way. However, when diffusion is much faster than extrusion (extrusion limited) the time constant is simply \u03c40\u00a0=\u00a0d\/(6Pout) and vica versa when diffusion is much slower than extrusion (diffusion limited) the time constant is simply \u03c40\u00a0=\u00a0d2\/(4\u03c0Dapp).\nTo compare the model with the results obtained from the experiments, we solved the transport equation simultaneously with the differential equation for the microelectrode potential. Although the analytical solution is very useful for theoretical analysis and testing, it requires many terms to obtain an accurate solution and does not allow for non-linearity. Therefore, we used a numerical approach for solving both equations, allowing more flexibility. Time integration was done using the forward Euler scheme and diffusion was calculated using a finite volume scheme (see for example [22]). The value of surface-to-volume ratio rsv in Eq. (2) using this finite volume scheme is simply the ratio of the cell surface S and the volume of the first compartment at the membrane. Both solutions were calculated using a Matlab 6.5 mex-routine written in C++.\nIn a typical experiment several Ca2+ transients were measured in succession at different depths. The relative distance was reconstructed from the micromanipulator movements that were all either 10 or 20\u00a0\u03bcm. The diameter of the cell was estimated from all of these successive steps. Unfortunately the absolute position (offset) of the transient measured closest to the membrane was not known exactly. We therefore allowed the depth as an adjustable parameter within the limits of \u00b110\u00a0\u03bcm. The transients measured closer to the middle were therefore more reliable. Furthermore, the response time of the electrode was estimated to be about 1.5\u20132.5\u00a0s and was allowed to vary within these limits. The remaining four unknown parameters in the transport equation, together with the response time, were obtained from an optimal fit. A simplex search algorithm was used to find this least square fit. To increase reliability of the parameter estimates, several transients measured at different depths were fitted simultaneously. Also, any slow baseline drift was removed and the first two seconds of the transients, that show an electrical artefact, were not used during the fitting process.\n3\nResults\n3.1\nChanges in measured calcium with depth of penetration\nAt the start of all experiments the tip of a CASM was placed centrally on the top of the chosen neurone and moved down in 20\u00a0\u03bcm steps until there was a sudden fall in the potential, as shown in Fig. 1A. Inside the cell the CASM registers both the membrane potential (Vm) and the change in VCa (the voltage, set to zero outside the cell, proportional to pCa). In this representative example the CASM was moved down in four steps before its potential changed from zero to about \u221250\u00a0mV. During a further six steps downward the CASM potential gradually became more negative, eventually more negative than \u2212150\u00a0mV. By this time the electrode tip was 200\u00a0\u03bcm from the top of the cell, which had a diameter of about 240\u00a0\u03bcm. We then inserted the membrane potential microelectrode (the output of which was electrically subtracted from the CASM potential to give VCa) and clamp current microelectrode, using manually-operated micromanipulators. To test that the CASM and Vm electrodes were recording the same change in Vm, we then hyperpolarised the cell by 30\u00a0mV for 20\u00a0s. There were only small transient changes in VCa, generated by electrical pickup and imperfect subtraction of rapid changes. We then (not shown in Fig. 1A) started to depolarize the cell by 50\u00a0mV for 1\u00a0s, repeated every 2\u00a0min, to allow an influx of Ca2+ ions. The recorded increase in [Ca2+]i was very small; VCa increased from \u2212107.5 to \u2212106.4\u00a0mV, and then recovered. After the first depolarisation we raised the CASM by 60\u00a0\u03bcm and left it unmoved for 15\u00a0min to allow recovery from the initial penetrations.\nAs shown in detail in Fig. 1B, we then moved the CASM down every 2\u00a0min, between the 9th and 16th depolarisations, in six steps of 20\u00a0\u03bcm and two steps of 10\u00a0\u03bcm. (The third of these steps made the clamp current increase from \u22123 to \u22127\u00a0nA, but this was not correlated with any change in [Ca2+]i.) As the CASM tip was moved down the transient increases in VCa after each depolarisation became larger, and the steady-state VCa after each recovery became more negative. After the sixth step VCa had fallen to \u2212127\u00a0mV, although after the next two it had risen slightly to \u2212126\u00a0mV. After the last step down VCa rose to \u2212118\u00a0mV before any depolarisation, suggesting the CASM had gone too far; although there was no change in the clamp current. We therefore then withdrew the CASM in several steps\u2014two each of 10 and 20\u00a0\u03bcm. After the first 20\u00a0\u03bcm step up, the VCa paradoxically decreased, as if the upward movement somehow allowed a better seal. Such paradoxical improvements on withdrawal were often seen in the early part of an experiment.\nFig. 2 shows the VCa responses to the first nine depolarisations of Fig. 1B in more detail. As the CASM tip was moved closer to the far side of the cell the responses to the depolarisations became larger and faster. Presumably this reflects the fact that during the depolarisations Ca2+ entered the cytoplasm only at the cell membrane. Once Vm is returned to \u221250\u00a0mV, entry ceased and the Ca2+ both diffused into the cell interior and was pumped out by the plasma membrane Ca ATPase, or PMCA. In some experiments we were able to make several different sequences of downward and upward movements in the same cell to a depth at which, as shown in Fig. 3A, the apparent [Ca2+]i rose rapidly. This experiment was done on a cell of diameter about 140\u00a0\u03bcm, and the depths at which the apparent [Ca2+]i began to increase were, respectively, 140, 150 and 140\u00a0\u03bcm. In other words, it appears that when the CASM tip reached the far side of the cell it suffered a loss of sensitivity or caused a local leak. We could see no change in the clamp current associated with the sudden increases in VCa. In many other cells, once the CASM created a sudden rise in [Ca2+]i it took many minutes to recover, if at all.\nThe simplest explanation of the decreases in apparent [Ca2+]i as the CASM was advanced deeper and deeper into a cell is that the electrode insertion induced a leak at the site of penetration. As the CASM was pushed in, its tip moved further away from the leak, which itself may be reduced by better sealing. During long experiments, as shown below, CASM movement had less dramatic effects on steady-state levels, suggesting that the leak declined with time.\nIn Fig. 3B we show mean steady-state VCa values against the estimated distance of the CASM tip from the bottom of the cell, from the early part of a total of six experiments in which the CASM was moved down in steps, like those of Figs. 1 and 3A. The bottom of the cell was taken as the point at which VCa rose rapidly without a change in membrane potential. Transients recorded within 20\u00a0\u03bcm of this were plotted as \u201cclose\u201d to the membrane. The mean VCa close to the cell membrane was \u2212126\u00a0mV, which corresponds to a pCa of 6.7; similar to the 6.76 (equivalent to a [Ca2+]i of 170\u00a0nM) measured in snail neurones which had not been injected with buffers [13].\nThe VCa transients became faster as the CASM tips were moved closer to the far side of the cell. Fig. 3C shows the mean times to peak of the VCa increases from the same six experiments. From a best-fit line through the points, we can calculate that the mean rate at which the VCa peak moved towards the centre of the cell was 6\u00a0\u03bcm\u00a0s\u22121. We also show in Fig. 3C the average size of the same transients, in mV from base to peak. The distance over which the transients decreased to half their (extrapolated) initial size of about 16\u00a0mV was about 45\u00a0\u03bcm, assuming that close to the membrane is equivalent to about 10\u00a0\u03bcm.\n3.2\nThe effect of removing external calcium\nIf calcium ions, driven by the large electrochemical gradient, normally leak into the top of the cell around the tips of the microelectrodes, reducing external calcium should reduce the apparent [Ca2+]i. We have tested the effect of a possible leak by exposing cells to either nominally Ca-free solutions or the same with added EGTA. Measured with a CASM, the [Ca2+] of the nominal solution was about 20\u00a0\u03bcM, that of the EGTA Ca-free Ringer less than 10\u00a0nM. Fig. 4 shows a representative experiment in which the cell was superfused with the Ca-free solutions with the CASM at three different depths.\nFor the first two exposures to Ca-free solution the CASM was 80\u00a0\u03bcm deep in a cell of diameter about 170\u00a0mm. The Ca-free solutions caused VCa to decrease by 11\u00a0mV from, respectively, \u2212115 and \u2212116\u00a0mV. There was little difference between the effect of the two Ca-free solutions except that the EGTA solution almost abolished the second depolarisation-induced rise in Ca. As the CASM was moved deeper into the cell the steady-state VCa fell to \u2212129\u00a0mV, and the reduction seen with Ca-free Ringer decreased to 4 and 3\u00a0mV, respectively. While some of the decreases seen with the removal of external Ca may reflect increased pump activity, it seems most likely that the elevated VCa levels seen near the middle of the cell are due to leakage round the microelectrodes where they cross the cell membrane.\n3.3\nThe effect of CO2-bicarbonate buffering\nWe have done most of our experiments in HEPES-buffered Ringer, although it is known that CO2-bicarbonate-buffered solutions greatly increase intracellular pH buffering while reducing pH buffering power extracellularly [28,29]. It is possible that such a change in pH buffering might influence the diffusion of Ca2+ ions. We have therefore recorded the effects of changing the extracellular buffer from HEPES to CO2-bicarbonate on Ca2+ transients recorded at different depths. While it is well established [16,30] that the intracellular pH (pHi) decrease caused by CO2 entry leads to a decrease in steady-state [Ca2+]i, the Ca2+ transients were little changed (not shown). By 20\u00a0min after changing the superfusate to CO2 the Na-dependent chloride\/bicarbonate exchanger [28] should have returned pHi to normal, so we then again moved the CASM down in steps. This time the VCa baseline did not change, and the transient times to peak were very similar to those recorded in HEPES. In a total of four similar experiments, the average time to peak at distances of 0\u201320\u00a0\u03bcm was 4.20\u00a0\u00b1\u00a00.45\u00a0s in HEPES, and 4.35\u00a0\u00b1\u00a00.68\u00a0s in CO2-bicarbonate.\n3.4\nThe role of calcium stores.\nIt seems possible that the VCa gradients described above, and the higher steady-state apparent [Ca2+]i than previously reported in cells which had added buffer, were partly due to mechanically-induced leakage from the endoplasmic reticulum calcium stores. Having penetrated the cell membrane the CASM must have been moved down through the endoplasmic reticulum and might have caused significant injury. We have therefore tested the effects of the SERCA pump inhibitor cyclopiazonic acid (CPA). It is well-established that CPA empties the Ca2+stores over a period of several minutes by blocking their uptake mechanism [31], and emptying the stores will presumably stop them leaking. Fig. 5 shows one of four similar experiments with CPA, in all of which (once the stores were empty) it had little effect on either the baseline VCa or the near-membrane VCa changes with CASM movement. The times-to-peak are trending to be later in CPA treated cells, although this was not very pronounced close to the membrane and statistically not significant (p-value assuming time-to-peak is later after CPA: 0.635 (n\u00a0=\u00a04)). For distances of 20, 40 and 60\u00a0\u03bcm from the membrane the times to peak were significantly longer (p-values assuming times-to-peak are later after CPA 0.952(5), 0.952(5) and 0.952(5)). The recovery time after the Ca2+ transients was clearly much longer in CPA treated cells (p-values assuming recovery times are longer after CPA: 0.984(4), 0.984(5), 0.972(5) and 0.996(5)), as shown in Fig. 5C. Interestingly the effect of CPA on recovery times was more pronounced for transients measured close to the membrane. Furthermore, the transient size showed a downward trend and decreased on average about 15% (nominal [Ca2+]i) in CPA treated cells. Although this percentage was statistically not significant (p-values assuming transients are smaller after CPA: 0.794(4), 0.845(5), 0.79(5) and 0.726(5)), it may suggest that a small fraction of the increase in [Ca2+]i is caused by release of Ca2+.\n3.5\nThe effect of BAPTA injection\nExogenous calcium buffers have been widely shown to influence Ca2+ signalling in a variety of cells; for example in nerve terminals [32] and Xenopus oocytes [33]. The fast buffer BAPTA is particularly interesting since it has similar properties to many of the indicators used to study Ca2+ signalling. We have therefore examined the effects of BAPTA, injected iontophoretically, on both the steady-state VCa and the Ca2+ transients induced by depolarisations. Fig. 6A shows a representative part of one of four similar experiments. Before the BAPTA injection we recorded VCa transients at several different CASM depths. At the closest approach to the membrane the time to peak was 2.9\u00a0s and VCa reached a peak of \u2212107\u00a0mV. The first two BAPTA injections loaded the cell to give an estimated BAPTA concentration of 9\u00a0\u03bcM. At the closest approach to the membrane the VCa transient then had a time to peak of 6.6\u00a0s. After two more BAPTA injections giving an estimated final concentration of 135\u00a0\u03bcm, the transient time to peak at the closest point was again slower, at 9.1\u00a0s. In three of the experiments we were able to measure times to peak and the size of the VCa transients at three different depths before and after injecting BAPTA to a final concentration of 100\u2013150\u00a0\u03bcm. The collected data are plotted in Fig. 6B and C. This shows that BAPTA tended to make the Ca2+ transients more uniform throughout the cell, both in times to peak and overall sizes.\nAlthough the first two BAPTA injections had little effect on the steady-state VCa, the second two lowered VCa from \u2212128 to \u2212131\u00a0mV. In a total of four similar experiments large BAPTA injections (raising the BAPTA concentration by over 100\u00a0\u03bcM) also lowered the steady state VCa by an average 4\u00a0\u00b1\u00a01.1\u00a0mV. This effect of BAPTA is similar to that reported before in snail neurones [14] in which intracellular CASM responses were calibrated by pressure-injecting BAPTA calibration solutions.\n3.6\nComputational modelling of Ca2+ transients in large snail neurones\nIn this section we present a minimal computational model that agrees fairly well with our experimental results. In the model we assume that during a 1\u00a0s current pulse (ICa,app) Ca2+ enters the cell at the plasma membrane whence it diffuses into the interior with an apparent diffusion coefficient Dapp. Furthermore, Ca2+ is removed at the plasma membrane by a Ca2+ pump with extrusion permeability Pout,app; we also allow for sequestration everywhere in the cell with rate kseq,app. Further mathematical details can be found in Section 2.\nIn modelling Ca2+ transients at different depths we overcame our imprecise knowledge of the different parameters by estimating them using a least square fit. Fig. 7A shows eight simultaneously fitted transients that were measured in the middle of a long experiment at various depths ranging form close to the membrane at about 20\u00a0\u03bcm to about 70\u00a0\u03bcm away from the membrane. The depths estimated from micrometer movements are the lower values shown above the peaks. To fit the data these positions were allowed to vary within the limits of half a CASM step. The depths obtained from the fit (upper values) are reasonably close to the micrometer positions. Interestingly, modelled positions near the membrane are slightly shifted towards the centre of the cell, which might indicate lower mobility close to the membrane.\nThe optimal fit yielded an apparent diffusion coefficient of Dapp\u00a0=\u00a080\u00a0\u03bcm2\u00a0s\u22121. Furthermore, the fit required an apparent sequestration rate constant of kseq,app\u00a0=\u00a00.047\u00a0s\u22121, which corresponds to a time constant of \u223c20\u00a0s. Attempts to fit the transients without sequestration failed.\nThe extrusion permeability was Pout,app\u00a0=\u00a011\u00a0\u03bcm\u00a0s\u22121, which corresponds to a time constant of about 3.6\u00a0s, much shorter than the diffusional time constant of 18\u00a0s (see Section 2). This suggests that clearance is limited by diffusion rather than by the pump rate. In most cells we observed that after influx a rather steep gradient develops in the direction of the membrane, which fits the diffusion-limited case.\nThe response time was estimated to be \u03c4CASM\u00a0=\u00a02.1\u00a0s, which was slower than the 1\u00a0s that was measured in 10\u00a0\u03bcM Ca2+ Ringer. In most experiments the times-to-peak very close to the membrane ranged from 2.5 to 3.5\u00a0s. Because the time-to-peak near the membrane should equal the duration of the influx, the response time of the electrode in cytoplasm is about 1.5\u20132.5\u00a0s.\nThe estimated apparent diffusion coefficient for the recordings shown in Fig. 7 of Dapp\u00a0=\u00a080\u00a0\u03bcm2\u00a0s\u22121 is higher than reported in other studies: \u223c14\u00a0\u03bcm2\u00a0s\u22121 in frog muscle [34], \u223c13\u00a0\u03bcm2\u00a0s\u22121 in cytoplasmic extracts of Xenopus oocytes [24], \u223c20\u00a0\u03bcm2\u00a0s\u22121 in the axoplasm of metacerebral cells of Aplysia californica\n[23] and \u223c10\u00a0\u03bcm2\u00a0s\u22121 in rod photoreceptor outer segments [35]. A diffusion coefficient of \u223c10\u00a0\u03bcm2\u00a0s\u22121 has been reported in intact Myxicola axoplasm [36], but much higher values were seen in ATP-depleted axoplasm, and with high [Ca2+]i.\nIn most of these studies the diffusion coefficient was estimated using a simple model with only a few parameters. Our model contained more parameters that we varied to obtain a good fit. The parameters for influx, extrusion and sequestration and to some extent the positions and the response time may compromise the estimate of Dapp. We therefore calculated the mean error variance as a function of the apparent diffusion coefficient of four fits obtained from four different cells. While fixing the apparent diffusion coefficient, all other parameters were allowed to vary in order to obtain an optimal fit. In each cell we fitted at least three transients measured at distances \u223c40, \u223c60 and \u223c80\u00a0\u03bcm and calculated the mean error variance as a function of Dapp. All cells gave a reasonable fit with a minimum error at 90\u00a0\u00b1\u00a020\u00a0\u03bcm2\u00a0s\u22121 (Fig. 7B). Since we allow all other parameters to compensate for an incorrect diffusion coefficient this value should be a conservative estimate. Attempts to fit the transients with Dapp values smaller than 50\u00a0\u03bcm2\u00a0s\u22121 failed. Some cells fitted better than others, since the expected transient size did not always match perfectly when multiple transients were used. However, perfect fits could be obtained with any individual transient. Close inspection of the traces revealed that baseline variations could affect the transient size. Possibly the Ca2+ influx is reduced or removal mechanisms are enhanced at higher basal [Ca2+]i levels. We still conclude that the diffusion coefficient is considerably higher (five to seven times) than reported for other cell types.\nA large fraction of the cytoplasmic Ca2+ is bound to buffers. The properties and distribution of these Ca2+ buffers are therefore critical for Ca2+ mobility in the cytoplasm. Analysis of Ca2+ transients under differing free Ca2+ concentrations and exogenous Ca2+ buffering in snail neurones indeed showed that Ca2+ is quite well buffered in snail neurones [24]. To explain the high diffusion coefficient a large fraction of the Ca2+ buffers must be mobile. When we injected the cells with BAPTA, it clearly changed the VCa transients considerably, promoting spatially uniform global signals as described in Xenopus oocytes [33]. These were loaded with 48\u00a0\u03bcm Oregon Green to record [Ca2+]i, which may itself have influenced Ca2+ movement. Our Fig. 7C shows three transients measured at 50\u201360\u00a0\u03bcm from the membrane with different BAPTA concentrations; the nominal concentrations were 0, 50 and 150\u00a0\u03bcM. BAPTA slightly increased the times to peak (10.2, 10.6 and 12.6\u00a0s), reduced the size of the transients and slowed the recovery time significantly (13.6, 18.1 and 30.0\u00a0s). These values are consistent with a reduced clearance rate. Typically, if the Ca2+ mobility is not changed, reduction of the clearance rate slows the recovery time but also increases the time-to-peak. After normalisation of the times to peak we could only detect a slight reduction in the latency (<0.5\u00a0s) between 0 and 150\u00a0\u03bcM BAPTA, suggesting that the mobility of BAPTA is comparable to that of the endogenous Ca2+ buffers.\nWe fitted the three transients simultaneously to the model allowing a different exogenous Ca2+ binding ratio for each transient. The model gives a reasonable fit with an endogenous buffer diffusion coefficient of De\u00a0=\u00a0100\u00a0\u03bcm2\u00a0s\u22121 and Ca2+ binding ratio of \u03bae\u00a0=\u00a065. This suggests that the endogenous Ca2+-buffers have a comparable Ca2+ binding ratio to that in Aplysia axons [25] but appear to be much more mobile. The apparent diffusion coefficients for Ca2+ at the different BAPTA concentrations are: Dapp\u00a0=\u00a0102\u00a0\u03bcm2\u00a0s\u22121 (0\u00a0\u03bcM BAPTA), Dapp\u00a0=\u00a012\u00a0\u03bcm2\u00a0s\u22121 (50\u00a0\u03bcM BAPTA) and Dapp\u00a0=\u00a0151\u00a0\u03bcm2\u00a0s\u22121 (150\u00a0\u03bcM BAPTA). The estimated diffusion coefficient of BAPTA was Db\u00a0=\u00a0183\u00a0\u03bcm2\u00a0s\u22121, which slightly increases the mobility of Ca2+ at higher BAPTA concentrations. Similar values for the BAPTA diffusion coefficient were reported by others [37\u201339].\n4\nDiscussion\nWe have shown that with CASMs it is possible to record steady-state calcium levels and depolarisation-induced Ca2+ transients at different depths in large snail neurones. The decrease in calcium levels as the CASM was pushed deeper was probably due to increasing the distance from a site of leakage around the CASM and other microelectrode insertions at the top of the cell. We found that the sizes and kinetics of the Ca2+transients measured after brief (1\u00a0s) depolarisations to zero or \u221210\u00a0mV varied systematically and repeatably with distance from the plasma membrane at the bottom of the cell. Blocking the endoplasmic reticulum SERCA pump with CPA had little effect on the transient sizes and times to peak, but significantly slowed subsequent recovery times. Injecting BAPTA both reduced and slowed the transients close to the membrane while magnifying them deep inside the cell. By fitting our results to a mathematical model we calculate that the apparent Ca2+ diffusion coefficient is about 90\u00a0\u03bcm2\u00a0s\u22121.\n4.1\nThe problem of leakage\nIon-sensitive and other non-patch microelectrodes inevitably make a hole in the cell membrane, which allows leakage in or out of the cell. Given the gradients, the ions whose leakage is most likely to distort the measured intracellular level are Na+ and Ca2+. Thus, Vaughan-Jones [40] recorded [Na+]i in crab muscle with a Na+-sensitive microelectrode (NASM) inserted radially (perpendicular to the long axis) and found that low external Na caused a surprisingly rapid fall in [Na+]i. He considered that this might have been caused by the low external Na reducing a leak. This was later confirmed by experiments in which it was shown that NASMs inserted along the long axis into a cannulated fibre recorded only small changes in [Na+]i when external Na was reduced, but when inserted radially the recorded [Na+]i was at first high but fell as the NASM was pushed deeper [12].\nPerhaps because the gradient is small and pHi very well buffered no one has considered that pH-sensitive microelectrode measurements have been contaminated by leakage. On the other hand, [Ca2+]i is very low and only moderately buffered. In our hands CASMs unfortunately do not perform well unless their tips are at least 1\u00a0\u03bcm in diameter [15,17]. Furthermore, only after much manipulation did CASMs record a low [Ca2+]i which increased on depolarization. The initially high levels were recorded even in cells loaded with Fura-2 whose fluorescence indicated a normal low [Ca2+]i. Experiments in which CASMs were calibrated while inside neurones by injecting buffers led to the conclusion that the true [Ca2+]i in quiescent voltage clamped snail neurones with injected buffer was 40\u00a0nM (equivalent to a CASM VCa of about \u2212140\u00a0mV) and that CASMs usually cause a leak at the point of insertion [14].\nThe early experiments with CASMs were all done with a manually-operated micromanipulator, so that systematic movements were not attempted once a stable VCa was obtained. The current results were obtained with a motor-driven and remote-controlled micromanipulator. Typically, the CASM was moved down in several 20\u00a0\u03bcm steps before its potential changed at all. This shows that the cell membrane is very extensible, since the cell as a whole did not visibly move as the CASM was inserted. For example in Fig. 1A, the CASM was moved down by 80\u00a0\u03bcm before the potential first fell. As the CASM tip was then moved deeper the potential became more and more negative. This was probably due to a combination of movement away from the site of leakage, and a reduction in the size of the leak. It is hard to distinguish between these two possibilities, but the reproducible effects of movement as shown for example in Fig. 3A suggest that the leak was relatively constant, although tending to reduce with time. In some experiments the steady-state VCa appeared to stabilize some distance from the apparent far side of the cell, as shown in the control part of Fig 6A, and was not reduced further as the tip approached the far side of the cell. Presumably the leakage was by then too small to have a significant effect.\n4.2\nCa2+ influx rather than Ca2+ release determines the increase in [Ca2+]i\nOur finding that the peak of the VCa transient diffuses away from the membrane at a speed of about 6\u00a0\u03bcm\u00a0s\u22121 confirms the long-held view that Ca2+ signalling which relies purely on diffusion will inevitably have a slow response time. The lack of effect of CPA, and the decline of the signal size with distance, rather unexpectedly both rule out significant regeneration of the signal as it spreads inwards. This is surprising, since snail neurones respond well to caffeine and certainly have the capacity for Ca-induced Ca2+ release (CICR) [41,16]. For example, the addition of a low concentration of caffeine slightly enhanced Ca2+ transients [41], while the removal of caffeine temporarily reduced the peak amplitudes suggesting that the stores were at least partly emptied [16].\nEither the signal is large enough without any store contribution, or something about our experimental conditions has inhibited CICR. We depolarised the cell for 1\u00a0s to ensure a large Ca2+ load every 2\u00a0min, but this may have in some way have inhibited store release. On the other hand in other experiments we have avoided depolarising the cell for long periods, and not found that the first subsequent response was different. The distance over which the VCa transients fell to half their extrapolated initial size was about 45\u00a0\u03bcm, similar to the distance between the cell membrane and the nucleus. In normal-sized neurones of course the nucleus would be much closer.\nWe conclude that the main mechanism in our experiments that leads to an increase in [Ca2+]i is transmembrane Ca2+ influx through voltage-gated Ca2+ channels opened by depolarisation and not massive release triggered by a small Ca2+ influx as in vertebrate cardiac muscle. The only previous direct measurements of Ca2+ movement in nerve cells without added buffers were described by Gorman et al. [42]. They were able to detect a rise in [Ca2+]i only within about 40\u00a0\u03bcm of the membrane in Aplysia neurones after a 5\u00a0s depolarisation. Their CASMs were made with a different sensor (ETH 1001) to that which we used, and appear to have been relatively insensitive at low values of [Ca2+]i.\n4.3\nThe transmembrane pump and sequestration by cytoplasmic organelles determine Ca2+ clearance\nThe depolarisation-induced Ca2+ influx is presumably cleared by extrusion across the cell membrane and sequestration to cytoplasmic organelles. The PMCA or Ca2+:H+ ATPase in snail neurones appears to exchange two extracellular protons for one intracellular Ca2+ at the expense of ATP [43]. When it is blocked by ortho-vanadate, the [Ca2+]i does not fully recover from an influx but ends in a plateau. This suggests that the PMCA is the essential removal mechanism, since the endoplasmic-reticulum and mitochondrial uptake mechanisms are not inhibited by cytoplasmic o-vanadate [16].\nNevertheless, for many different cell types it has been shown that mitochondria participate in Ca2+ clearance when Ca2+ concentrations are high [44,45]. It is very difficult to measure the action of mitochondria in snail neurones directly. However, an increased muffling power at high Ca2+ concentrations [24] suggests that mitochondria may play a role close to the membrane where high transients occur.\nInstead of reinforcing the Ca2+ signals, and thereby generating a fast travelling Ca2+ wave into the cell, the stores clearly play a role in the recovery of the Ca2+ transients. CPA significantly increased the Ca2+ clearance time from \u223c13 to \u223c18\u00a0s; these were measured in the centre of the cell and were corrected for the CASM response time. From this we estimate that the stores contribute about 25% to the clearance. This is lower but comparable to the result found in Purkinje cell somata from rat cerebellar slices, where blocking of the endoplasmic Ca2+ pump also significantly slowed the recovery time after influx [46].\n4.4\nCalcium transients change systematically with depth\nAlthough the steady-state VCa levels were probably elevated by continuous Ca2+ entry, the recorded transients and the way they changed with depth were, we believe, physiologically significant. It seems unlikely that the kinetics of the diffusion of Ca2+ from the far side of the cell towards the centre would be changed by a constant or slowly-declining leak. Our average value for the speed at which a depolarisation-induced Ca2+ transient flows from the periphery to the centre of a nerve cell with no added mobile buffer was \u223c6\u00a0\u03bcm\u00a0s\u22121. We can find no previous measurements of this parameter in excitable cells, although several groups have modelled the process; (for example [8,12]). The only real measurement we have found was done with 100\u00a0\u03bcm Fluo-3 in an isolated atrial myocyte with inhibited sarcoplasmic reticulum Ca2+ pumps [10]. Our estimate from their Fig. 1 is that the calcium peak moved at least 10 times faster than in our observations, but the authors\u2019 model showed that this was probably due to the Fluo-3. Their modelled data as shown in their Fig. 4 suggest that with no added mobile buffer their Ca2+ transients might travel to the centre of their model myocyte at a similar speed to that which we measure, although their time axis does not extend far enough. Furthermore, when the speed at which Ca2+ transients travel depends on diffusion, the velocity is not constant but varies with distance; typically velocity first increases and then decreases when the centre of the cell is approached. In contrast, true waves are constantly regenerated and tend to move with a constant speed [47].\n4.5\nCalcium diffusion in large snail neurones without added buffer\nIf recovery from a brief Ca2+ influx relies on extrusion at the plasma membrane, diffusion will become important for large cells because Ca2+ has to diffuse to the plasma membrane to be extruded. Moreover, the fastest possible clearance without sequestration is the diffusion limit. When the extrusion rate is much faster than diffusion, the recovery time constant is proportional to the square of the cell's diameter and inversely proportional to the diffusion coefficient (see Section 2). Because large snail neurones mainly rely on transmembrane extrusion mechanisms and do not seem to reinforce Ca2+-signals, the diffusion coefficient becomes a critical parameter for Ca2+ clearance and spatial signalling. Fig. 8 shows the recovery times, assuming that diffusion is the rate-limiting process, for various different diffusion coefficients as function of the cell's diameter. The recovery time of large snail neurones is about 10\u201320\u00a0s (dashed line), which requires a diffusion coefficient between 60 and 120\u00a0\u03bcm2\u00a0s\u22121. However, most snail and other animal nerve cell bodies are much smaller, with diameters between 5 and 50\u00a0\u03bcm. The recovery times of these cells is typically in the order of seconds (dotted line). Hence, with diffusion coefficients <20\u00a0\u03bcm2\u00a0s\u22121 the cell can still be cleared readily by the pump.\n4.6\nWhy is Ca2+ more mobile in large snail neurones?\nIt is possible that large cells, that do not use Ca2+release mechanisms for signalling and rely on extrusion at the membrane for reasonably fast recovery, require a high Ca2+ mobility to function. A high mobility will also ensure fast signal transmission to the nucleus. This is quite different from large cells that use release mechanisms to trigger rapid on\/off responses such as muscle, oocytes and chromaffin cells. These cells typically have low Ca2+ mobilities and do not rely as much on extrusion, but recycle internally released Ca2+. Small neurones and axonal regions with high surface to volume ratios represent another class of geometries, which do not require high Ca2+ mobility for fast recovery.\nBecause Ca2+ is well buffered with an estimated binding ratio of 65, the mobility of the endogenous mobile buffers must be high to explain the high apparent mobility of Ca2+. Possibly the ratio of mobile buffers to immobile buffers is higher in large snail neurones, or the diffusion coefficient of the mobile buffer fraction is much higher than in other cells. This may be related to less cytoplasmic crowding, for example by reduced cytoskeleton, causing the movement of proteins to be less constricted [48]. Axonal regions and muscle cells contain high concentrations of cytoskeletal components that may restrict or slow considerably the movement of larger calcium binding proteins compared to large snail neurones.\nThe great majority of studies on intracellular Ca2+ signalling have been done with indicators such as Fura 2, which can be used on all sizes and types of cell. But the interaction of the Ca2+ signals with the indicator may have subtle effects, which are hard to predict. It is to be regretted that CASMs so far have such large tips and slow responses that they can be used only on exceptionally large cells.","keyphrases":["neurone","diffusion","intracellular calcium"],"prmu":["P","P","P"]}
{"id":"Methods-1-5-1895922","title":"Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals\n","text":"The resazurin assay utilising microtitre-plate, described by Drummond and Waigh in 2000, has been modified to achieve more accuracy in the determination of the minimum inhibitory concentration (MIC) values of natural products, including crude extracts, chromatographic fractions or purified compounds against various bacterial strains. This modified resazurin method is simple, sensitive, rapid, robust and reliable, and could be used successfully to assess antibacterial properties of natural products.\n1\nIntroduction\nAntibiotics have revolutionised mankind\u2019s health status, allowing treatment of life threatening infections. However with the increasing occurrence of bacterial resistance against available antibiotics, it has now become essential to look for newer antibiotics. Most of the antibiotics available today come from natural origin, especially from various microbial or marine sources. Plants also produce compounds to protect themselves from microbial attacks. For screening natural products, e.g. crude extracts, chromatographic fractions or purified compounds for antibacterial activities, it is essential to employ an in vitro antibacterial assay that is simple, rapid, efficient, reliable, sensitive, safe and cost-effective. Moreover, most often the small quantities of natural products, especially purified compounds, that are available for antibacterial screening, can be a limiting factor in any viable screening programme. The conventional methods, e.g. disc diffusion method, may be time consuming and require significant quantities of the test materials, and there are also a few other problems associated with this method as discussed by Drummond and Waigh [3].\nAt the beginning of our research project, aimed at screening Scottish plants for antibacterial activities [1,2], the resazurin assay utilising a microtitre-plate as described by Drummond and Waigh in 2000 [3] seemed useful. This was because this method uses an indicator, resazurin, which allows the detection of microbial growth in extremely small volumes of solution in microtitre plates without the use of a spectrophotometer. However, it was soon realised that this published method incorporated changes in the concentration of both the test material as well as the bacterial suspension. Thus, although there is a serial dilution of the test materials, this method results in a decrease in the bacterial concentration serially, and as a consequence cannot be a \u2018true\u2019 indicator of the minimum inhibitory concentration (MIC) which is supposed to be assessed in this assay. Moreover, in a number of other antibacterial assays, the bacterial concentration is only approximate as they are compared to the Macfarland standard. This approach is subjective and results are often not reproducible in two different laboratories. To overcome these drawbacks, we have modified the resazurin assay, especially the dilution protocols, and utilised a standard concentration of bacterial suspension so that a \u2018true\u2019 MIC value can be obtained. By challenging the test materials with a standardised inoculum, reproducible and meaningful results can be obtained. We now present a detailed description of the modified resazurin method, which is simple, sensitive, rapid, robust and reliable, and could be used successfully to assess antibacterial properties of natural products.\n2\nMethod\n2.1\nGeneral\nIncubator at 35 and 37\u00a0\u00b0C; pipettes of various sizes (Gilson); sterile tips, 100, 200, 500, and 1000\u00a0\u03bcL; 5\u00a0mL multichannel pipette (Fischer Supplies); centrifuge tubes (Fischer Supplies); vortex mixer (Fischer Supplies); centrifuge (Fisons); petridishes (Fischer Supplies); sterile universal bottles (Fischer Supplies); UV spectrophotometer (Shimadzu); sterile resazurin tablets (BDH Laboratory Supplies); sterile normal saline; sterile isosensitest broth (Oxoid); sterile isosensitest agar (Southern Group Laboratory, SGL); antibiotic solutions (Sigma\u2013Aldrich); sterile solution of 10% (v\/v) DMSO in water (Sigma\u2013Aldrich).\n2.2\nMedium\nIsosensitest medium was used throughout this assay, as it is pH buffered. Although NCCLS recommends the use of Mueller Hinton medium for susceptibility testing [4], the isosensitest medium had comparable results for most of the tested bacterial strains [5].\n2.3\nUse of standardised bacterial colony numbers\nThe method wherein turbidity is compared to Macfarland standards usually 0.5 is not able to give a standardised number of CFU for all strains, because this is operator driven and is thus subjective. It also makes it difficult to compare different bacterial species as they have differing optical densities. Thus to ensure that a uniform number of bacteria were always used, a set of graphs of killing\/viability curves for each strain of bacterial species was prepared. A final concentration of 5\u00a0\u00d7\u00a0105\u00a0cfu\/mL was adopted for this assay. Thus different strains and different bacterial species could be compared.\n2.4\nPreparation of bacterial culture\nUsing aseptic techniques a single colony was transferred into a 100\u00a0mL bottle of isosensitest broth, capped and placed in incubator overnight at 35\u00a0\u00b0C. After 12\u201318\u00a0h of incubation, using aseptic preparation and the aid of a centrifuge, a clean sample of bacteria was prepared. The broth was spun down using a centrifuge set at 4000\u00a0rpm for 5\u00a0min with appropriate aseptic precautions. The supernatant was discarded into an appropriately labelled contaminated waste beaker. The pellet was resuspended using 20\u00a0mL of sterile normal saline and centrifuged again at 4000\u00a0rpm for 5\u00a0min. This step was repeated until the supernatant was clear. The pellet was then suspended in 20\u00a0mL of sterile normal saline, and was labelled as Bs. The optical density of the Bs was recorded at 500\u00a0nm, and serial dilutions were carried out with appropriate aseptic techniques until the optical density was in the range of 0.5\u20131.0. The actual number of colony-forming units was calculated from the viability graph. The dilution factor needed was calculated and the dilution was carried out to obtain a concentration of 5\u00a0\u00d7\u00a0106\u00a0cfu\/mL.\n2.5\nPreparation of resazurin solution\nThe resazurin solution was prepared by dissolving a 270\u00a0mg tablet in 40\u00a0mL of sterile distilled water. A vortex mixer was used to ensure that it was a well-dissolved and homogenous solution.\n2.6\nPreparation of the plates\nPlates were prepared under aseptic conditions. A sterile 96 well plate was labelled (Fig. 1). A volume of 100\u00a0\u03bcL of test material in 10% (v\/v) DMSO or sterile water (usually a stock concentration of 1\u00a0mg\/mL for purified compounds, and 10\u00a0mg\/mL for crude extracts) was pipetted into the first row of the plate. To all other wells 50\u00a0\u03bcL of nutrient broth or normal saline was added. Serial dilutions were performed using a multichannel pipette. Tips were discarded after use such that each well had 50\u00a0\u03bcL of the test material in serially descending concentrations. To each well 10\u00a0\u03bcL of resazurin indicator solution was added. Using a pipette 30\u00a0\u03bcL of 3.3\u00d7 strength isosensitised broth was added to each well to ensure that the final volume was single strength of the nutrient broth. Finally, 10\u00a0\u03bcL of bacterial suspension (5\u00a0\u00d7\u00a0106\u00a0cfu\/mL) was added to each well to achieve a concentration of 5\u00a0\u00d7\u00a0105\u00a0cfu\/mL. Each plate was wrapped loosely with cling film to ensure that bacteria did not become dehydrated. Each plate had a set of controls: a column with a broad-spectrum antibiotic as positive control (usually ciprofloxacin in serial dilution), a column with all solutions with the exception of the test compound, and a column with all solutions with the exception of the bacterial solution adding 10\u00a0\u03bcL of nutrient broth instead (see Fig. 2).\nThe plates were prepared in triplicate, and placed in an incubator set at 37\u00a0\u00b0C for 18\u201324\u00a0h. The colour change was then assessed visually. Any colour changes from purple to pink or colourless were recorded as positive (Fig. 1). The lowest concentration at which colour change occurred was taken as the MIC value. The average of three values was calculated and that was the MIC for the test material and bacterial strain.\n3\nResults\nResazurin is an oxidation\u2013reduction indicator used for the evaluation of cell growth, particularly in various cytotoxicity assays [6]. It is a blue non-fluorescent and non-toxic dye that becomes pink and fluorescent when reduced to resorufin by oxidoreductases within viable cells. Resorufin is further reduced to hydroresorufin (uncoloured and nonfluorescent). A resazurin reduction test has also been used for decades to demonstrate bacterial and yeast contamination of milk [6,7].\nThe effectiveness of this modified resazurin assay has been demonstrated with methanol extracts of selected Scottish plants, purified compounds (Fig. 1), and the positive control ciprofloxacin (Table 1), and a direct comparison of the MIC determination of the antibiotics using the old and modified resazurin methods (Table 2) using the antibiotics norfloxacin, cefotaxime, and amoxicillin.\n4\nConcluding remarks\nChoosing the correct assay to assess the antimicrobial potential of extracts and compounds is important for generating high-quality data with the greatest accuracy, speed and efficiency, enabling the addition of potential new antimicrobial compounds and extracts to our armamentarium. This modified resazurin assay corrected the dilution inaccuracies, especially in relation to the MIC determinations, as described by Drummond and Waigh [3], and enabled results to be comparable for the test material for different bacterial strains. The method described here is easy to follow and accurate. The generation of an accurate MIC value, which can be compared to existing antibiotics, empowers the scientist with the knowledge to decide whether the extracts and compounds are worth pursuing further in terms of their antimicrobial potential.","keyphrases":["antibacterial assay","resazurin","serial dilution","plant extract"],"prmu":["P","P","P","R"]}
{"id":"Gene-2-1-2270349","title":"Experimental assessment of bioenergetic differences caused by the common European mitochondrial DNA haplogroups H and T\n","text":"Studies of both survival after sepsis and sperm motility in human populations have shown significant associations with common European mitochondrial DNA haplogroups, and have led to proposals that mitochondria bearing haplogroup H have different bioenergetic capacities than those bearing haplogroup T. However, the validity of such associations assumes that there are no non-random influences of nuclear genes or other factors. Here, we removed the effect of any differences in nuclear genes by constructing transmitochondrial cybrids harbouring mitochondria with either haplogroup H or haplogroup T in cultured A549 human lung carcinoma cells with identical nuclear backgrounds. We compared the bioenergetic capacities and coupling efficiencies of mitochondria isolated from these cells, and of mitochondria retained within the cells, as a critical experimental test of the hypothesis that these haplogroups affect mitochondrial bioenergetics. We found that there were no functionally-important bioenergetic differences between mitochondria bearing these haplogroups, using either isolated mitochondria or mitochondria within cells.\n1\nIntroduction\nAnalysis of the variation in normal human mitochondrial DNA (mtDNA) has identified many haplogroups\u2013specific patterns of polymorphisms that have arisen over the last 150,000\u2013200,000\u00a0years\u2013that have been used to study the origin, radiations and evolution of human populations (Cann et al., 1987; Wallace et al., 1999; Wallace, 2005). These mtDNA haplogroups have been less studied bioenergetically than mtDNA mutations that lead to disease (Dimauro and Davidzon, 2005; Taylor and Turnbull, 2005), but normal variation in mtDNA may affect disease susceptibility (Herrnstadt and Howell, 2004) and longevity (Santoro et al., 2006). These effects could be explained by differences in mitochondrial coupling efficiency (the percentage of oxygen consumption used for ATP synthesis rather than heat generation) or mitochondrial production of ROS (reactive oxygen species). It has been proposed that particular haplogroups cause inefficient oxidative phosphorylation and greater heat production and were therefore selected during the radiations of humans into Arctic environments (Mishmar et al., 2003; Ruiz-Pesini et al., 2004; Wallace, 2005; Montiel-Sosa et al., 2006), but this proposal is controversial (Elson et al., 2004; Kivisild et al., 2006; Ruiz-Pesini and Wallace, 2006) and is not supported by direct measurement of mitochondrial coupling efficiencies in mitochondria carrying representative \u201carctic\u201d and \u201ctropical\u201d mtDNAs (Amo and Brand, 2007; Elson et al., 2007).\nTwo lines of evidence suggest that common European haplogroups may affect bioenergetic function: there are associations of both survival after sepsis and sperm motility with haplogroup H. In a prospective study of intensive care patients in Newcastle-upon-Tyne, U.K., individuals with haplogroup H (about 40% of the population studied) were more than twice as likely to survive 180\u00a0days after sepsis than those with non-H haplogroups (primarily the closely-related haplogroups J and T; about 30% of the population) (Baudouin et al., 2005). Mitochondrial dysfunction may be linked to sepsis-induced multiple organ failure (Protti and Singer, 2007), so altered mitochondrial bioenergetics might be the causal link between haplogroup and survival. Two possibilities have been proposed: haplogroup H might protect through greater heat generation (because of higher electron transport rates or looser coupling) (Baudouin et al., 2005), or through greater ROS production (because of tighter coupling and raised protonmotive force), which could reduce bacterial infection (Wallace, 2005). There have been no direct experimental tests of these possibilities.\nThe second line of evidence comes from studies of sperm motility, which is correlated with mitochondrial enzymatic activities and depends on the activity of the mitochondrial electron transport chain (Ruiz-Pesini et al., 1998; Ruiz-Pesini et al., 2000). Haplogroup T was over-represented in men with asthenozoospermia (reduced sperm motility), whereas haplogroup H was over-represented in men with other fertility problems. Sperm with haplogroup H performed better in a test of motility and had higher cytochrome oxidase activity than those with haplogroup T (Ruiz-Pesini et al., 2000), suggesting that mitochondria carrying haplogroup H make more ATP than those with haplogroup T. This conclusion has been extended to sublineages of haplogroup U (Montiel-Sosa et al., 2006), but associations between haplogroups and reduced male fertility and sperm motility have been disputed by others (Pereira et al., 2005; Pereira et al., 2007). Less well-coupled mitochondria make less ROS (Korshunov et al., 1997; Liu, 1997; Lambert and Brand, 2004), so people having such mitochondria might suffer less from neurodegenerative diseases caused by ROS (Wallace, 2005). Some epidemiological studies support this prediction; e.g. haplogroup T is under-represented in Alzheimer's disease patients (Chagnon et al., 1999). However, other studies have not replicated this finding (Elson et al., 2006), and correlations between mitochondrial haplogroups and neurodegenerative diseases are controversial (Raule et al., 2007). Again, there have been no direct experimental tests of possible differences in the bioenergetic properties of mitochondria that might underlie the reported associations of haplogroup and phenotype.\nIn the present study, we analyse bioenergetic capacity and coupling efficiency in mitochondria isolated from cytoplasmic hybrids (cybrids) carrying haplogroups H and T with identical nuclear DNA. Furthermore, to investigate the effects of haplogroups H and T on the bioenergetic status of mitochondria at the cellular level, we analyse the respiratory capacities and mitochondrial coupling efficiencies of intact cybrids.\n2\nMaterials and methods\n2.1\nSubjects\nHealthy volunteers were recruited by advertisement and written informed consent was obtained. Ethical approval was obtained from the Cambridge Research Ethics Committee. DNA was extracted from buccal swab samples using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Mitochondrial haplogroups were determined by PCR-RFLP analysis according to published criteria (Wallace et al., 1999). Entire mtDNAs were amplified in several overlapping fragments by PCR (Torroni et al., 1997). Each fragment was digested by restriction enzymes and resolved on agarose gels. 15\u00a0ml of blood were taken from each of three volunteers who had haplogroup H, and three who had haplogroup T, for platelet preparation and cybrid construction. Haplogroup H (Achilli et al., 2004) is subdivided into at least 15 sub-haplogroups (H1-H15). In the Newcastle sepsis study, there were no significant differences in survival of sub-haplogroups H1 (\u2212\u00a03008 TaqI), H2 (\u2212\u00a04769 AluI), H3 (+\u00a06773 NlaIII) and the remaining H sub-haplogroups combined (Baudouin et al., 2005). Our haplogroup H volunteers were H1, H3 and (H, not H1, H2 or H3). Haplogroup T (Macaulay et al., 1999) is subdivided into at least 5 sub-haplogroups (T1\u2013T5) (Pike 2006). Our haplogroup T volunteers were one T (sub-haplogroup not checked), one T1 (\u2212\u00a012629 AvaII) and one (T, not T1). No major differences in cybrid phenotypes between sub-haplogroups were observed.\n2.2\nGeneration of cybrid cell lines\nCybrid cell lines are constructed by repopulation of mtDNA-less (\u03c10) cells with exogenous mitochondria (King and Attardi, 1989). A549.B2 \u03c10 (mtDNA-less) cells derived from human lung carcinoma A549 (originally carrying mitochondrial DNA of haplogroup H) were cultivated in Dulbecco's modified Eagle's medium (DMEM) containing 4.5\u00a0g\/l glucose, 110\u00a0\u00b5g\/ml sodium pyruvate, 10% (v\/v) fetal bovine serum (FBS) and 50\u00a0\u00b5g\/ml uridine. Platelets (which have no nuclei) were isolated from the volunteers' blood samples and fused with A549.B2 \u03c10 cells as described elsewhere (Chomyn, 1996). The resultant cybrids had mtDNA from the different donors, but their nuclear DNA was identical. After many generations of cybrid growth (diluting platelet-derived nuclear-encoded subunits), all nuclear-encoded mitochondrial protein subunits will be specified by the host cell, but all mitochondrial-encoded subunits will be specified by the donor mtDNA. The cybrid cell lines were constructed and routinely maintained in DMEM with 10% dialysed FBS. Stocks of cell lines were frozen and kept at \u2212\u00a080\u00a0\u00b0C until required. DNA was extracted from cultured cybrid cells as described previously (Laird et al., 1991) to confirm mitochondrial haplogroups by PCR-RFLP analysis.\n2.3\nMitochondrial respiration and membrane potential\nHuman A549 cell mitochondria were prepared from cultured cybrids as previously described (Amo and Brand, 2007). Mitochondrial oxygen consumption was measured at 37\u00a0\u00b0C using a Clark electrode (Rank Brothers, Cambridge, UK) calibrated with air-saturated medium comprising 0.115\u00a0M KCl, 10\u00a0mM KH2PO4, 3\u00a0mM Hepes (pH 7.2), 2\u00a0mM MgCl2, 1\u00a0mM EGTA and 0.3% (w\/v) defatted BSA, assumed to contain 406\u00a0nmol atomic oxygen\u00b7ml\u2212\u00a01 (Reynafarje et al., 1985). No correction was made for consumption of oxygen by the electrode, so oxygen consumption at low rates will be slightly overestimated. Mitochondrial membrane potential (\u0394\u03c8) was measured simultaneously with respiratory activity using an electrode sensitive to the lipophilic cation TPMP+ (triphenylmethylphosphonium) (Brand, 1995). Mitochondria were incubated at 1.0\u00a0mg\u00b7ml\u2212\u00a01 (for succinate respiration) or 1.5\u00a0mg\u00b7ml\u2212\u00a01 (for 2-oxoglutarate\u00a0+\u00a0malate respiration) in the presence of 80\u00a0ng\u00b7ml\u2212\u00a01 nigericin (to collapse the pH gradient so that the protonmotive force was expressed exclusively as \u0394\u03c8) and 4\u00a0\u00b5M rotenone (to inhibit complex I). The TPMP+-sensitive electrode was calibrated with sequential additions of TPMP+ up to 2\u00a0\u00b5M, then 4\u00a0mM succinate or 3.2\u00a0mM 2-oxoglutarate\u00a0+\u00a00.8\u00a0mM malate (with rotenone omitted) was added to initiate respiration. Experiments were terminated with 1.6\u00a0\u00b5M FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), allowing correction for any small baseline drift. \u0394\u03c8 was calculated from the distribution of TPMP+ across the mitochondrial inner membrane using a binding correction factor of 0.35\u00a0mg protein\u00b7\u00b5l\u2212\u00a01. Respiratory rates and respiratory control ratios (the state 3 respiration rate with 0.8\u00a0mM ADP divided by the state 4 rate with oligomycin) with 2-oxoglutarate\u00a0+\u00a0malate as substrate were determined in the absence of nigericin.\n2.4\nModular kinetics\nTo investigate differences in oxidative phosphorylation caused by mitochondrial DNA variants, we applied a systems approach: modular kinetic analysis (Amo and Brand, 2007). This analyses the kinetics of the whole of oxidative phosphorylation divided into three modules connected by their common substrate or product, \u0394\u03c8. The modules are (i) the reactions that produce \u0394\u03c8, consisting of the substrate translocases, dehydrogenases and other enzymes and the components of the respiratory chain, called \u2018substrate oxidation\u2019, (ii) the reactions that consume \u0394\u03c8 and synthesize, export and dephosphorylate ATP, consisting of the ATP synthase, the phosphate and adenine nucleotide translocases and any ATPases that may be present, called the \u2018phosphorylating system\u2019, and (iii) the reactions that consume \u0394\u03c8 without ATP synthesis, called the \u2018proton leak\u2019 (Brand, 1990). The analysis reports changes anywhere within oxidative phosphorylation that are functionally-important, but is unresponsive to changes that have no functional consequences. Comparison of the kinetic responses of each of the three modules to \u0394\u03c8 obtained using mitochondria isolated from different cybrids reveals any effects of mitochondrial haplogroup on the kinetics of oxidative phosphorylation. Oxygen consumption and \u0394\u03c8 were measured simultaneously using mitochondria incubated with 80\u00a0ng\u00b7ml\u2212\u00a01 nigericin and 4\u00a0\u00b5M rotenone. Respiration was initiated by 4\u00a0mM succinate or 3.2\u00a0mM 2-oxoglutarate\u00a0+\u00a00.8\u00a0mM malate (with rotenone omitted). The kinetic behaviour of a \u2018\u0394\u03c8-producer\u2019 can be established by specific modulation of a \u0394\u03c8-consumer and the kinetics of a consumer can be established by specific modulation of a \u0394\u03c8-producer (Brand, 1998). To measure the kinetic response of proton leak to \u0394\u03c8, the state 4 (non-phosphorylating) respiration of mitochondria in the presence of oligomycin (0.8\u00a0\u00b5g\u00b7ml\u2212\u00a01; to prevent any residual ATP synthesis), which was used solely to drive the proton leak, was titrated with malonate (up to 0.5\u00a0mM). In a similar way, state 4 respiration was titrated by FCCP (up to 0.8\u00a0\u00b5M) for measurement of the kinetic response of substrate oxidation to \u0394\u03c8. State 3 (maximal rate of ATP synthesis) was obtained by addition of excess ADP (0.8\u00a0mM). Titration of state 3 respiration with malonate (up to 1.15\u00a0mM) allowed measurement of the kinetics of the \u0394\u03c8-consumers (the sum of the phosphorylating system and proton leak). The coupling efficiencies of oxidative phosphorylation were calculated from the kinetic curves as the percentage of mitochondrial respiration rate at a given \u0394\u03c8 that was used for ATP synthesis and was therefore inhibited by oligomycin. Note that any slip reactions will appear as proton leak in this analysis (Brand et al., 1994).\n2.5\nCell respiration\nCell respiration was measured at 37\u00a0\u00b0C using a Seahorse XF24 Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, MA, USA), which is fully described elsewhere (Wu et al., 2007). Cybrids were thawed and subcultured in Petri dishes in DMEM to which had been added 4.5\u00a0g\/l glucose, 110\u00a0\u00b5g\/ml sodium pyruvate, 0.02\u00a0vol of penicillin\/streptomycin (5000\u00a0units\/5000\u00a0\u00b5g\/ml), 0.02\u00a0vol of 200\u00a0mM l-glutamine and 0.1\u00a0vol Fetal Bovine Serum (heat inactivated). Cybrids were seeded at 20,000\u201350,000\u00a0cells per well in Seahorse XF24 24-well plates and grown for 3\u20135\u00a0d in a 37\u00a0\u00b0C incubator under 95% air\/5% CO2, then washed and incubated for about 1\u00a0h in a 37\u00a0\u00b0C incubator under air in 700\u00a0\u00b5l of assay medium (comprising 120\u00a0mM NaCl, 3.5\u00a0mM KCl, 2\u00a0mM MgCl2, 1.3\u00a0mM CaCl2, 1.2\u00a0mM Na2SO4, 0.4\u00a0mM KH2PO4, 15\u00a0mM d-glucose and 0.4% (w\/v) BSA, pH adjusted to 7.4 with NaOH). Experiments were carried out to a paired design: each plate was seeded half with one H-haplotype cybrid line and half with one T-haplotype cybrid line. Appropriate amounts of concentrated stocks of oligomycin, FCCP, rotenone and myxothiazol dissolved in 70% ethanol were diluted into 75\u00a0\u00b5l of assay medium and loaded into the appropriate addition ports in a measuring cartridge. The plate and measuring cartridge (containing waveguides and fluorescent oxygen sensors together with reagents for automated pneumatic additions of inhibitors and uncouplers) was loaded into the machine and after the calibration procedure (30\u00a0min calibration, 10\u00a0min rest), measurement cycles of 1\u00a0min sample mixing, 2\u00a0min waiting and 3\u00a0min measurement of oxygen consumption rate were initiated. All respiration rates were calculated as a percentage of the rate in the same well for the fourth measurement point \u2014 the basal rate just preceding addition of oligomycin (see Fig. 4). This corrected for differences in cell density between wells. In the range measured, absolute rates of oxygen consumption were linearly related to cell numbers seeded. Absolute basal oxygen consumption rates for the parental A549 cell line are reported to be about 1.7\u00a0nmol\/min\/106 cells (Wu et al., 2007). Respiration rates at each time-point from three or four replicate wells were averaged.\n2.6\nStatistics\nValues are given as mean\u00a0\u00b1\u00a0SEM for n cell clones (n\u00a0=\u00a015 cell clones for Figs. 1\u20133, five different clones for each donor and three donors for each haplogroup, and n\u00a0=\u00a06 cell clones for Fig. 4, two different clones for each donor and three donors for each haplogroup). To test for significant differences in modular kinetics between haplogroups, we interpolated the mean respiration rates of mitochondria from the individual clones at a range of different potentials between the highest and lowest common membrane potential. The significance of differences between means was assessed by unpaired Student's t-test using Microsoft Excel X; P values\u00a0<\u00a00.05 were taken to be significant.\n3\nResults\n3.1\nModular kinetic analysis of oxidative phosphorylation\nHaplogroup H is a subgroup of haplogroup HV, and haplogroup T is a subgroup of haplogroup JT; HV and JT diverged about 40,000\u00a0years ago (Kivisild et al., 2006). Haplogroup H is found in about 41% of Europeans and haplogroup T is found in about 15% (Wallace et al., 1999). Haplogroup H differs from haplogroup T by four non-synonymous mutations in proteins (one each in the complex I subunits, ND1 and ND2, and two in the complex III subunit, cytochrome b), two mutations in tRNAs (tRNAarg and tRNAthr) and three mutations in rRNA (two in 16S rRNA and one in 12S rRNA) (Kivisild et al., 2006), so differences in oxidative phosphorylation between these haplogroups are plausible.\nTo compare mitochondrial functions between haplogroup H and T, we measured the kinetics and coupling efficiency of oxidative phosphorylation using mitochondria isolated from cybrid cells. Fig. 1A, B and C shows the kinetics of the three modules of oxidative phosphorylation using succinate as respiratory substrate. Fig. 1A shows the kinetic response of substrate oxidation to its product, \u0394\u03c8; Fig. 1B shows the kinetic response of proton leak to its driving force, \u0394\u03c8, and Fig. 1C shows the kinetic response of the ATP phosphorylating pathway to its driving force, \u0394\u03c8. The three sets of kinetics curves using succinate were indistinguishable between mtDNA haplogroup H (closed symbols) and T (open symbols). We also measured the kinetics of substrate oxidation using 2-oxoglutarate\u00a0+\u00a0malate as substrate instead of succinate to check for any effects of differences in the mitochondrial complex I genes. Once again, the kinetic response of the substrate oxidation system to \u0394\u03c8 using 2-oxoglutarate\u00a0+\u00a0malate as substrate was not significantly different between haplogroups H and T (Fig. 1D).\nFrom the kinetic curves shown in Fig. 1, we can analyse the coupling efficiency of oxidative phosphorylation. The classic measure is the respiratory control ratio: respiration at maximal rate of ATP synthesis (state 3) divided by respiration with no ATP synthesis (state 4) (Fig. 2A and B). A more precise measure of coupling efficiency is given by the percentage of respiration that is coupled to ATP synthesis. This is calculated by subtracting the rate of proton leak (Fig. 1B) from the total rate of respiration (Fig. 1A) at any chosen membrane potential, and expressing the difference as a percentage of the total rate. If this percentage is lower, then a greater proportion of the redox energy is diverted to heat production. Mitochondria in cells normally operate at less than maximal rates and values of \u0394\u03c8. Fig. 2C reports the coupling efficiency across the whole range of rates of ATP synthesis and values of \u0394\u03c8 from state 3 (maximal rate of ATP synthesis) to close to state 4 (where ATP synthesis is minimal). As expected (Brand et al., 1993), coupling efficiency decreased as mitochondria moved from state 3 towards state 4. There were no significance differences in rates, respiratory control ratio or coupling efficiencies between haplogroups H and T.\nWe also independently measured the respiratory control ratio using 2-oxoglutarate\u00a0+\u00a0malate as substrate and calculated coupling efficiency from the data in Fig. 1 (Fig. 3). Once again, there was no clear difference between haplogroup H and T, either in respiratory rates (Fig. 3A), respiratory control ratio (Fig. 3B) or coupling efficiencies across a range of rates and potentials (Fig. 3C).\nIn summary, at the mitochondrial level we observed no significant functional differences between mitochondria isolated from cybrids of haplogroup H and haplogroup T.\n3.2\nCoupling efficiency of respiration in intact cybrid cells\nTo check whether differences in mitochondrial function between haplogroups were lost when mitochondria were isolated from the cells and measured at a standardised protein concentration in an artificial incubation medium, we measured the respiration rates and coupling efficiencies of mitochondria in intact cybrids. Cybrids were grown on 24-well plates and oxygen consumption was measured using a Seahorse XF24 Extracellular Flux Analyzer. Fig. 4A shows that oxygen consumption was inhibited when ATP synthesis was prevented by addition of oligomycin, stimulated when oxidation was uncoupled from phosphorylation by addition of the uncoupler FCCP, and inhibited again when mitochondrial electron transport was abolished by addition of rotenone (to inhibit complex I of the electron transport chain) and myxothiazol (to inhibit complex III).\nThe residual rate in the presence of rotenone and myxothiazol represents non-mitochondrial oxygen consumption. This comprised 16.7\u00a0+\u00a02% of basal respiration rate in H-haplotype cybrids and 18.2\u00a0+\u00a02.7% in T-haplotype cybrids. These values were not significantly different. Subtraction of this non-mitochondrial respiration allowed assessment of the rates and coupling efficiency of the remaining mitochondrial respiration.\nOligomycin inhibits mitochondrial ATP synthesis, so mitochondrial respiration that is insensitive to oligomycin is due to uncoupled respiration, i.e. to proton leak pathways through the mitochondrial inner membrane, and respiration that is sensitive to oligomycin is due to ATP synthesis occurring in the cells. Because oligomycin prevents the energy-utilizing pathway of ATP synthesis, the mitochondrial proton motive force will rise after oligomycin addition, and the proton leak rate will increase, leading to an overestimate of the proton leak rate and an underestimate of the ATP synthesis rate that occurred before oligomycin was added (Brand, 1990). With this proviso, Fig. 4B shows the estimates of the percentages of basal respiration used for ATP synthesis and proton leak within the cells. There was no difference between the haplogroups. The percentage of respiration used for ATP synthesis is the coupling efficiency, and, with a value of 85% of mitochondrial respiration, it was comparable to the coupling efficiency at intermediate respiration rates found in mitochondria isolated from the cells (Figs. 2 and 3), similar to the value of 80% measured previously using the parental cell line (Wu et al., 2007), and similar to values found in a range of other cell types (Brand, 2005).\nRespiration rates in the presence of FCCP are not directly limited by ATP synthesis, and represent the maximal capacity of substrate oxidation by the cells under the conditions of measurement. In some cells this rate is equivalent to the maximum rate of the electron transport chain, but in others there is strong rate-limitation imposed by the supply of substrates to the mitochondria, particularly when ATP levels drop as a secondary result of uncoupling and substrates like glucose and fatty acids cannot be activated to glucose-6-phosphate and fatty acyl CoA. Fig. 4A shows that the FCCP-uncoupled respiration rates of A549 cybrids were initially high and rather variable, but then diminished with time, consistent with increasing limitation by substrate availability. There was no significant difference in uncoupled rates between haplogroups (Fig. 4B).\nIn summary, at the intact cell level we observed no significant functional differences between mitochondria within cybrids of haplogroup H and haplogroup T.\n4\nDiscussion\nThere are published associations between mitochondrial haplogroups H and T and phenotypes that might be expected to have a strong bioenergetic component. However, our results show no significant bioenergetic differences in mitochondria with H or T-haplogroup mtDNAs in a constant nuclear background, at either the mitochondrial or the cellular level. We have previously shown that our methodology can pick up 10% differences in respiratory chain activity (Amo and Brand, 2007), so if haplogroup does affect mitochondrial bioenergetics, any effects must be very small. The lack of effect of haplogroup on mitochondrial coupling efficiency in intact cybrids shows that even if haplogroup affects mitochondrial proliferation or retrograde signalling, such changes do not affect coupling efficiency in the cells under basal conditions.\nThe strengths of our approach are (i) we used a direct empirical measurement, rather than inference from genetic and physiological traits, (ii) we separated the effects of mitochondrial haplogroup from any confounding effects of nuclear DNA background and (iii) we used a simple in vitro system with the powerful modular kinetic approach where the relevant variables could be tightly controlled and manipulated. However, our approach also has some weaknesses. (i) A small effect of mitochondrial haplogroup operating over hours or days might still be too small to be detected by biochemical experiments. (ii) We measured the bioenergetic capacity using isolated mitochondria and cybrid cells in simple defined media, but effects of mitochondrial haplogroups might only emerge under special conditions (for example, they might require triggers such as sepsis or energetic stress or might require particular nuclear genes to be expressed, and so be tissue-specific or suppressed in the lung carcinoma cell line we employed).\nOur results fail to provide support for the hypothesis that the common European mitochondrial haplogroups H and T have significant effects on bioenergetics that cause the associations with survival after sepsis and sperm motility that have been reported. If such associations stand up to further scrutiny, they presumably operate through more subtle mechanisms than respiratory rates, mitochondrial coupling efficiencies or ATP supply.","keyphrases":["sepsis","sperm motility","cybrid","coupling efficiency","oxidative phosphorylation","\u03c10, mtdna-less","\u03b4\u03c8, mitochondrial membrane potential","tpmp, triphenylmethylphosphonium","fccp, carbonyl cyanide p-trifluoromethoxyphenylhydrazone"],"prmu":["P","P","P","P","P","R","R","M","M"]}
{"id":"Eur_Spine_J-2-2-1602188","title":"Renal cement embolism during percutaneous vertebroplasty\n","text":"Percutaneous vertebroplasty (PVP) is an effective treatment for lesions of the vertebral body that involves a percutaneous injection of polymethylmethacrylate (PMMA). Although PVP is considered to be minimally invasive, complications can occur during the procedure. We encountered a renal embolism of PMMA in a 57-year-old man that occurred during PVP. This rare case of PMMA leakage occurred outside of the anterior cortical fracture site of the L1 vertebral body, and multiple tubular bone cements migrated to the course of the renal vessels via the valveless collateral venous network surrounding the L1 body. Although the authors could not explain the exact cause of the renal cement embolism, we believe that physicians should be aware of the fracture pattern, anatomy of the vertebral venous system, and careful fluoroscopic monitoring to minimize the risks during the PVP.\nIntroduction\nPercutaneous vertebroplasty (PVP) is an effective, minimally invasive procedure in which polymethylmethacrylate (PMMA) cement is injected into a diseased vertebral body. This technique provides pain relief and strengthens weakened vertebral bodies. Vertebroplasty has gained widespread popularity for the treatment of benign or malignant compression fractures since its initial description 21\u00a0years ago [9]. However, complications can still occur during the procedure even though PVP is considered to be minimally invasive. The reported complication rates range from 1 to 10%, but are generally minor [3]. This paper reports our experience of a renal cement embolism during PVP, with a review of the literature.\nCase report\nA 57-year-old man, who had fallen from a 2.4\u00a0m ladder 4\u00a0months earlier, complained of severe back pain. He had been a healthy farmer prior to the accident. However, since his fall, he had suffered from debilitating back pain refractory to conservative treatment for 2\u00a0months. In particular, he complained of activity-related pain corresponding to the level of a compression fracture. The outside computed tomography (CT) scans demonstrated a compression fracture of the L1 body without destruction of the posterior wall and significant vertebral collapse. The magnetic resonance imaging (MRI) showed a slight step-off of the anterior cortical margin of the compressed L1 vertebral body, with lower marrow signal intensity on T1-weighted image (Fig.\u00a01).\nFig.\u00a01The T1 weighted sagittal magnetic resonance image of the lumbar spine demonstrates a slight step-off of the anterior cortical margin of the compressed L1 vertebral body, with decreased marrow signal intensity\nIt was decided to proceed with PVP of the L1. The patient was placed in the prone position. An 11-gauge needle was advanced into the vertebral body via the left unilateral transpedicular approach using sterile technique and fluoroscopic guidance under local anesthesia. The PMMA (Zimmer Inc., IN, USA) cement has two components: an ampule of liquid and a fine powder of PMMA containing barium sulfate, e.g., 10\u00a0ml ampoule of liquid to 20\u00a0g of powder. Under careful fluoroscopic visualization, the mixture of PMMA cement and barium sulfate powder having a consistency similar to that of toothpaste was slowly injected into the vertebral body, diffusing throughout the intertrabecular marrow space. Before its injection, the mixture was loaded into several 1\u00a0ml syringes. However, when approximately 8\u00a0cc of PMMA had been injected unilaterally, multiple bone cement was detected at the bilateral renal fossae. The procedure was immediately stopped. After the procedure, the back pain had improved by more than 50%, but he still complained of severe right flank pain and fever. His renal function was impaired and the BUN to Cr ratio was high (27.2\/1.3). On the same day, both kidneys were swollen (13\u00a0cm), and were found to contain a large amount of hyperechoic materials on the abdominal ultrasound (Fig.\u00a02). The DMSA (99mTc dimercaptosuccinic acid) scan has been found to be quite useful for examining the bilateral kidney damage in detail, but the patient refused this. The CT scans showed the origin of the leak arising at the level of the vertebroplasty, and showed cement in the left anterior external venous plexus draining into both renal veins. Multiple tubular opacities, which corresponded to the course of the renal vessels, were detected, with some of the cement scattered through the left posterior external venous plexus on the CT scans (Fig.\u00a03). The patient was diagnosed with a renal embolism and was treated conservatively. On the sixth day after the procedure, the BUN to Cr ratio had returned to normal, and the patient was released from hospital without pain. A follow-up ultrasound 8\u00a0months later revealed prominent atrophy of the right upper renal cortex with compensatory hypertrophy of the left kidney. However, the patient is currently doing well and has no subjective symptoms.\nFig.\u00a02Both kidneys are swollen (13\u00a0cm), and the abdominal ultrasound shows large amounts of hyperechoic materials (a, right kidney; b, left kidney)Fig.\u00a03Computed tomography scans show the origin of the leak arising at the level of the vertebroplasty (a), revealing cements in the left anterior external venous plexus draining into both renal veins (b)\nDiscussion\nImage-guided percutaneous vertebral augmentation or PVP, was first performed in France in 1984, when Deramond and Galibert et al. [9] injected PMMA into the C2 vertebra, which had been partially destroyed by an aggressive hemangioma. Over the next 15\u00a0years, many groups advocated expanding the indications for PVP to include osteoporotic compression fractures, traumatic compression fractures, and painful vertebral metastasis [2, 6, 7, 11, 18, 19]. Our case had the anterosuperior fracture of the L1 body without destruction of the posterior wall and significant vertebral collapse so that it might not lead to a technically difficult vertebroplasty procedure; or might not constitute relative contraindications [6].\nEven though PVP has many advantages including its simplicity, easy approach, and minimally invasiveness, many authors have reported several complications. The reported complication rates range from 1 to 10%, with a higher incidence of complications in cases with metastatic lesions [3]. However, complications are rare (1~2%) with osteoporosis, and are generally asymptomatic and transient [2, 11, 19]. The reported complications associated with these procedures include hypotension, rib fractures [18], dural tear [1], pulmonary cement embolism [1, 4, 14], adult respiratory distress syndrome [18], cerebral cement embolism [15], root compression due to intraforaminal cement leakage [6], paraplegia due to spinal cord [6] and cauda equina compression [16], intravascular extension of cement [1], infection, and cement toxicity [12].\nThe vascular leakage of PMMA, although rare, might have disastrous consequences. The viscosity of the PMMA cement is a crucial aspect during the procedure, and progressively increases as a result of methyl methacrylate polymerization. The rate of polymerization depends on several difficult-to-evaluate factors, including the ambient temperature and the quantity of the solvent. A paste is preferred to a liquid consistency because the latter may cause leakage of the PMMA into the venous system, particularly when the lesion is a highly vascular or extensive osteolytic tumor [5, 6]. However, it is not always possible to maintain the appropriate viscosity of PMMA at all times prior to the injection because the viscosity of the PMMA changes over time. The possibility that the renal cement embolism was caused by insufficient polymerization of the PMMA at the time of injection could not be excluded.An adequate amount of the infusing PMMA is also recommended. Cotten et al. [6] reported that pain relief does not appear to be proportional to the degree of lesion filling by the PMMA. In addition, Martin et al. [13] reported that complications were mainly related to an excessive PMMA injection.Good-quality fluoroscopy is essential for the early detection of a minimal cement leakage into the perivertebral vein. Jensen et al. [11] recommended a barium\/tungsten combination in order to allow for adequate visualization of venous flow as well as early detection of venous PMMA migration during fluoroscopy. In our case, renal cement embolism caused by perivertebral venous migration was not early recognized. Injection was performed under lateral fluoroscopy, paying particular attention to the epidural space, the spinal canal, and the perivertebral veins. But real-time detection of laterovertebral leakage remained difficult owing to overlap of the cement filling vertebral body, even though intermittent anteroposterior fluoroscopy could not overcome this problem. Gangi et al. [8] recommended that CT guidance increases precision, improves the results, and reduces complications. But this is also the lack of real-time visualization of cement leak. The vertebral venography should be carried out before the PMMA injection. However, its value is also contentious. Jensen et al. [11] advocated the use of antecedent venography to decrease the incidence of complications associated with the needle placement within the basivertebral venous plexus and to delineate the route of cement egress. However, Deramond et al. [7] reported that the lesion is stained by the injection of contrast media in the case of spinal tumors, which interrupts the early venous leakage, and that the different flow characteristics of the contrast material and PMMA hamper the predictive value of venography [14]. Venography is not usually performed at our center. In our case, the PMMA leaked outside of the left anterior cortical fracture site of the L1 vertebral body and multiple tubular bone cement migrated to the course of the renal vessels via the anterior external vertebral venous plexus. Anatomically, the vertebral venous system is a large valveless collateral venous network within and around the vertebral column, extending from the sacral hiatus along the entire length of the vertebral column up to the foramen magnum [10]. There are also a large number of connections to the subcutaneous and vertebral veins as well as the sacral venous plexus. We do not know whether the cause of the renal cement embolism was via the vertebral venous network or the inappropriate PMMA viscosity. The patient\u2019s position is also clinically important. Vogelsang reported that the intraosseous pressure recorded in the lumbar spinous process increased by compressing the inferior vena cava, which he called \u201cretrograde congestion\u201d [17]. Under this condition, the vertebral pressure is increased, which might reduce the risk of fat, bone marrow, air and bone cement extrusion into the IVVP and EVVP. Our procedure used a hand injection method for the PMMA in order to prevent an increase in the injection velocity.We could not determine the precise cause after a lengthy review of the whole procedure, but it is possible that there might have been some vascular anomaly, such as congenital communication, between the vertebral venous system and renal vessels.\nConclusion\nThe authors encountered an unexpected renal embolism during PVP. We could not explain the exact cause of the renal cement embolism, but we believe that physicians should be aware of the fracture pattern, anatomy of the vertebral venous system, and careful fluoroscopic monitoring to minimize the risk during the PVP.","keyphrases":["embolism","percutaneous vertebroplasty","bone cement","vertebral venous system"],"prmu":["P","P","P","P"]}
{"id":"Histochem_Cell_Biol-4-1-2323029","title":"The art of cellular communication: tunneling nanotubes bridge the divide\n","text":"The ability of cells to receive, process, and respond to information is essential for a variety of biological processes. This is true for the simplest single cell entity as it is for the highly specialized cells of multicellular organisms. In the latter, most cells do not exist as independent units, but are organized into specialized tissues. Within these functional assemblies, cells communicate with each other in different ways to coordinate physiological processes. Recently, a new type of cell-to-cell communication was discovered, based on de novo formation of membranous nanotubes between cells. These F-actin-rich structures, referred to as tunneling nanotubes (TNT), were shown to mediate membrane continuity between connected cells and facilitate the intercellular transport of various cellular components. The subsequent identification of TNT-like structures in numerous cell types revealed some structural diversity. At the same time it emerged that the direct transfer of cargo between cells is a common functional property, suggesting a general role of TNT-like structures in selective, long-range cell-to-cell communication. Due to the growing number of documented thin and long cell protrusions in tissue implicated in cell-to-cell signaling, it is intriguing to speculate that TNT-like structures also exist in vivo and participate in important physiological processes.\nIntroduction\nCommunication with the environment is a basic principle of any biological system. With the increasing complexity of higher organisms, cells had to evolve diverse mechanisms to exchange spatial and temporal information crucial for tissue organization and the maintenance of the organism as a whole. Certainly, tissues are not just a loose accumulation of individual cells, but a highly organized population of interacting cells. Thus, in the various types of animal and plant tissues, cell-to-cell communication is important for maintaining a supracellular organization. Probably the best-characterized example of such an organization is given by the animal central nervous system, which is composed of a complex network of interconnected neuron and glial cells. It is widely accepted that the complexity of the brain reflects its enormous number of intercellular links provided by synaptic connectivity (including gap junctions) between axons and dendrites. Gap junctions, through which molecules <1\u00a0kDa can penetrate, are also important players in establishing supracellular organization in other tissues containing electrically excitable cells as heart and smooth muscle cells. Furthermore, these proteinaceous channels interconnect cells of the multicellular layer forming epithelial tissues. Finally, cell-to-cell coupling via gap junctions is a general phenomenon during early embryogenesis, where most cells are electrically coupled. Perhaps due to the presence of a thick cell wall, plant cells have evolved thin membrane channels referred to as plasmodesmata (PD) to allow direct connections over longer distances (Balu\u0161ka et al. 2004a; Cilia and Jackson 2004; Gallagher and Benfey 2005). Because these bridges provide both membrane and cytoplasmic connectivity between cells, plants are regarded as a supracellular assembly (Balu\u0161ka et al. 2004b, c). PD, in addition to the functions known for gap junctions in animals, facilitate the transfer of ribonucleoparticles, transcription factors and viruses (Ruiz-Medrano et\u00a0al. 2004).\nIn 2004, a new principle of cell-to-cell communication between animal cells, based on the formation of thin membrane channels, was reported (Rustom et al. 2004). These channels, referred to as tunneling nanotubes (TNT), were initially found in cultures of rat pheochromocytoma (PC12) cells. As PD, they were shown to mediate membrane continuity between connected cells. TNT permit the direct intercellular transfer of organelles, cytoplasmic molecules, and membrane components (Gerdes et\u00a0al. 2007). Subsequent studies on other cell types revealed morphologically similar structures directing intercellular transfer of cargo, including pathogens. It thus becomes apparent that nanotubular bridges provide an important and general mechanism of cell-to-cell communication between animal cells. In the following, we summarize the current knowledge and development in the field of nanotubular communication. As awareness of the potential physiological implications of these structures, we will give a short overview on published data describing long and thin cellular protrusions in tissue implicated in cellular communication.\nDiversity of TNT-like structures\nTNT connecting PC12 cells are typically 50\u2013200\u00a0nm in diameter and can reach lengths up to several cell diameters. They are stretched, interconnecting cells at their nearest distance (Fig.\u00a01) (Hodneland et\u00a0al. 2006; Rustom et\u00a0al. 2004). Their structural integrity is sensitive to mechanical stress, chemical fixation, and even to prolonged light exposure. The feature that most strikingly distinguishes TNT from other cellular protrusions is that they hover in the medium and have no contact to the substratum (Fig.\u00a01a, a1). A structural characterization of TNT between PC12 cells revealed that they contain F-actin as a prominent cytoskeleton element but no microtubules (Table\u00a01). Further characterization at the ultra-structural level showed a seamless transition of the surface membrane of the TNT with the plasma membranes of the connected cells (Fig.\u00a01b, b1, b2). In addition, a continuous membrane with both connected cells was evident in transmission electron micrographs (Fig.\u00a02a, a1, a2) (Table\u00a01). These observations, together with the documentation of a limited lateral diffusion of fluorescent membrane proteins occurring between the plasma membranes of TNT-connected cells, led to the proposal that TNT mediate intercellular membrane continuity (Fig.\u00a03a) (Gerdes et\u00a0al. 2007; Rustom et\u00a0al. 2004).\nFig.\u00a01Architecture of TNT between cultured PC12 cells. (a) 3D fluorescence image ((x\u2212y)-maximum projection of 40 consecutive 400\u00a0nm sections) of a wheat germ agglutinin-stained TNT connecting two live PC12 cells. (a1) (x\u2212z)-projection in the plane of the TNT indicated in (a). (b) Scanning electron micrograph (SEM) showing the ultra-structure of a TNT between two PC12 cells. The boxed areas are shown as higher magnification images (b1, b2). Modified from Rustom et al. (2004) Science 303:1007\u20131010. Scale bars, a, a1, b, 5\u00a0\u03bcm; b1, b2, 500\u00a0nmTable\u00a01TNT-like structures in vitroaCell type TNT-like structures Cytoskeletal components Membrane continuity\/ \u201copen-ended\u201d Cargo PC12 cells (Rustom et al. 2004) F-actin, myosin Va (+)b Endosome-related organelles, lipid-anchored proteins (EGFP-f), EGFP-actin NRK cells (Rustom et al. 2004)c (F-actin, myosin Va)c ND Endosome-related organelles EBV-transformed human B cell line (721.221) (\u00d6nfelt et al. 2004) ND ND GPI-GFP, (HLA-Cw6-GFP, in coculture with human peripheral blood Natural Killer cells)d Between neonatal rat CM and adult human EPC (Koyanagi et al. 2005) ND ND Mitochondria, soluble proteins (GFP) Primary cultures of rat astrocytes (Zhu et al. 2005) F-actin, myosin Va ND ND DC (Watkins and Salter 2005) ND (+)e Calcium fluxes, surface receptors (HLA-A,B,C class I MHC)d THP-1 monocytes (Watkins and Salter 2005) F-actin (+)e Calcium fluxes, the fluid phase marker lucifer yellow, surface receptors (HLA-A,B,C class I MHC)d Between DC and THP-1 monocytes (Watkins and Salter 2005) ND (+)e Calcium fluxes Human monocyte-derived macrophages (bridges with diameter \u22650.7\u00a0\u03bcm) (\u00d6nfelt et al. 2006) F-actin, microtubules (+)f Mitochondria, endosome-related organelles, lysosomes Human monocyte-derived macrophages (nanotubes with diameter <0.7\u00a0\u03bcm) (\u00d6nfelt et al. 2006) F-actin ND Surfing Mycobacterium bovis bacillus Calmette-Gu\u00e9rin Cos-1, XC and HEK 293 cells (Sherer et al. 2007) F-actin (\u2212)b MLV Jurkat T cells (Sowinski et al. 2008) F-actin (\u2212)b,e HIV-1 protein Gag aOnly those publications that fulfill at least one of the listed criteria are shownbAccessed by electron microscopycGurke, S., Barroso, J., Bukoreshtliev, N., Gerdes, H.-H., unpublished datadThese molecules were shown to localize in TNT-like structures, but their intercellular transfer was not proveneAccessed by the measurement of calcium fluxesfProposed from the observation of a seamless transition between microtubules of the bridge with microtubular networks of both connected cellsND: not determinedFig.\u00a02Transmission electron micrographs (TEM) showing the ultra-structure of distinct TNT-like bridges in different cell types. (a) Open-ended TNT connecting two PC12 cells reconstructed from images of two consecutive 80\u00a0nm sections. The boxed areas are shown as higher magnification images (a1, a2). A continuous membrane is observed between the nanotube and the plasma membrane of the two connected cells. Modified from Rustom et al. (2004) Science 303:1007\u20131010. (b) Close-ended TNT-like bridge connecting two T cells and displaying a junctional border, reconstructed from images of 13 consecutive 60\u00a0nm sections. The boxed areas are shown as higher magnification images (b1, b2). The nanotube formed by one cell (b2) protrudes into an invagination (arrowhead) of the connected cell (b1). Modified from Sowinski et al. (2008) Nat Cell Biol 10:211\u2013219. Scale bars, a, b, 1\u00a0\u03bcm; a1, a2, b1, b2, 500\u00a0nmFig.\u00a03Schematic representations of three distinct nanoscaled cellular protrusions and proposed modes of cell-to-cell communication. (a) A TNT-mediating membrane continuity between cells. (a1) Organelles like endocytic vesicles and mitochondria are transported uni-directionally between cells by an actin-dependent mechanism. (b) Nanotubular bridge between cells displaying a junctional border. (b1) Distinct viral particles are transported either at the surface of the nanotube by a receptor-dependent mechanism using actin retrograde flow or inside the cellular nanotube by an actin-dependent mechanism. (c) Cellular nanotube (cytoneme) extending toward a target cell by chemotaxis. (c1) Signaling molecules secreted by the target cell are proposed to be endocytosed by a receptor-mediated mechanism at the tip of the cytoneme and transported in a retrograde manner toward the cell body of the receiving cell. The arrows (a\u2013c) indicate the direction of transfer\nBased on the morphological criteria defined for PC12 cells, similar TNT-like connections were subsequently identified for several permanent cell lines and primary cultures (for a comprehensive overview see (Gerdes et al. 2007)) (Table\u00a01). TNT-like bridges between dendritic cells (DC) (Watkins and Salter 2005) and perhaps also those connecting neonatal rat cardiomyocytes (CM) and adult human endothelial progenitor cells (EPC) (Koyanagi et\u00a0al. 2005), appear to be most closely related to the TNT described for PC12 cells. The flow of cytoplasmic molecules suggests membrane continuity between connected cells (Table\u00a01). This contrasts the TNT-like bridges between T cells, which have been characterized lately in considerable detail (Sowinski et al. 2008). The observed T-cell nanotubes have an average length of \u223c20\u00a0\u03bcm, a diameter of 180\u2013380\u00a0nm, and are not tethered to the substratum. Moreover, as in PC12 cells, only F-actin but not microtubules were detected in T-cell nanotubes (Fig.\u00a03). However, despite the overall similarity in architecture and capacity to facilitate intercellular transfer of cargo between nanotubes of PC12 cells, DC, CM\/EPC and T-cells, the latter do not mediate membrane continuity between connected cells (Fig.\u00a02b, b1, b2) (Table\u00a01). This was concluded from the observation that nanotubes between T-cells (i) do not facilitate the transfer of cytoplasmic molecules, (ii) do not permit free diffusion of fluorescent plasma membrane components and (iii) display a junctional border between the nanotube and the connected T-cell at the ultra-structural level (Fig.\u00a02b1). Accordingly, they were classified as \u201cnot open-ended\u201d cellular nanotubes (Fig.\u00a03b). Other identified close-ended nanotubular bridges that share some morphological features with TNT are murine leukemia virus-induced \u201cfilopodial bridges\u201d (Sherer et\u00a0al. 2007) (Table\u00a01).\nInterestingly, it has been shown that different classes of nanotubes exist even within a single cell type. In the case of macrophages, not only thin, F-actin-containing bridges similar to other TNT-like structures were detected, but also a thicker type of cellular connection (\u22650.7\u00a0\u03bcm diameter), which contained both F-actin and microtubules (\u00d6nfelt et\u00a0al. 2006). This thicker type of connection was suggested to be open ended due to a seamless transition of the microtubules inside the bridge with the microtubular networks of both connected cells (\u00d6nfelt et\u00a0al. 2006) (Table\u00a01). Thin, and thicker microtubule-containing connections were also found between prostate cancer cells (Vidulescu et\u00a0al. 2004).\nFinally, another type of long and thin F-actin-containing cellular protrusions that share striking features with TNT-like intercellular bridges, are cytonemes. These membrane nanotubes were discovered in Drosophila imaginal wing discs, emanating from the periphery of the columnar cell sheet toward the signaling center associated with the anterior\/posterior border (Ram\u00edrez-Weber and Kornberg 1999). They are thought to extend toward the target cells by chemotaxis (Ram\u00edrez-Weber and Kornberg 1999) (Fig.\u00a03c) to accomplish the receptor-mediated uptake of the secreted morphogen Decapentaphlegic (Dpp) during spatial patterning (Hsiung et al. 2005). Once endocytosed, the morphogen is thought to be delivered to the cell body by retrograde transport along the cytoneme (Fig.\u00a03c1). Thus, these structures are proposed to fulfill an important task in long-range cell-to-cell signaling during embryonic development. In contrast to TNT-like structures, cytonemes have not been shown to physically bridge cells. Nevertheless, functionally similar structures, emanating from tracheal cells in response to Branchless (Bnl)-Fibroblast Growth Factor (FGF) signaling, appear to do so (Sato and Kornberg 2002).\nThe identification of TNT-like structures in cell cultures has been mainly based on the morphological criteria defined for PC12 cells (Rustom et al. 2004). In some cases, a proper comparison was hindered by their limited characterization. Nonetheless, heterogeneity regarding formation, structure, and functional properties across cell types and even within a given cell type has emerged (Table\u00a01). Certainly, more detailed information is necessary for a proper classification of all these structures and thus, as things are now, we shall refer to them as TNT-like structures, irrespective of whether membrane continuity was observed.\nFormation of TNT-like structures\nTNT-like structures form de novo between cells on a time-scale of several minutes by apparently two distinct mechanisms. With respect to the first mechanism, initial studies on PC12 cells showed that TNT could be established by a seemingly directed outgrowth of swaying filopodia-like protrusion(s) toward a neighboring cell. Once contact is made, a single dilated and bended bridge is often observed along with the degeneration of remainder protrusions. This bended structure is then remodeled into a straight and thin bridge showing the characteristic morphology of typical TNT (Rustom et\u00a0al. 2004). One can speculate that the seemingly directed filopodia outgrowth preceding TNT formation is under control of chemotactic guidance. Evidence for this is provided for related processes like the formation of cytonemes toward a Bnl-FGF gradient in in\u00a0vitro cultures of Drosophila cells (Ram\u00edrez-Weber and Kornberg 1999) (Fig.\u00a03c) or murine leukemia virus (MLV)-induced nanotubular bridges emanating from non-infected fibroblast cells toward infected cells (Sherer et\u00a0al. 2007). The observation that TNT-like bridges also emerge when attached PC12 (Rustom et\u00a0al. 2004) or immune cells (\u00d6nfelt et al. 2004, 2006; Sowinski et al. 2008) depart from each other, led to the proposal of a second mechanism of TNT formation (Gerdes et\u00a0al. 2007; \u00d6nfelt et\u00a0al. 2004), which a\u00a0priori excludes any dependence on chemotactic guidance of filopodia. Subsequent detailed studies on T cells revealed that cell-to-cell interaction for at least a few minutes before dislodging is required for successful nanotubular bridge formation (Sowinski et al. 2008). It is of note that, in contrast to PC12 cells, a filopodium-dependent mechanism of nanotubular bridge formation between immune cells was not reported to date (\u00d6nfelt et al. 2004, 2006; Sowinski et al. 2008).\nActin polymerization is thought to be a key event for both mechanisms of TNT formation (Gerdes et\u00a0al. 2007). It drives filopodia outgrowth important in the first mechanism and is most probably important for the stabilization of TNT-like structures emerging by the second mechanism. In support of this, TNT formation is not observed in the presence of actin-depolymerizing drugs (Rustom et al. 2004), and TNT-like structures between T cells only form if the cells diverge with a speed below that of processes driven by actin polymerization (Cameron et al. 1999; Sowinski et al. 2008). Zhu et al. (2005) obtained further evidence that actin polymerization and TNT formation are linked by demonstrating their concurrent induction by H2O2 in primary cultures of rat astrocytes.\nTNT-like structures have a dynamic nature leading to only transient bridges as observed in in\u00a0vitro cultures. The nanotubular bridges between T cells (Sowinski et al. 2008), PC12 cells or normal rat kidney (NRK) cells (Bukoreshtliev, N., Gerdes, H.-H., unpublished data) have variable lifetimes, ranging from a few minutes to less than 60\u00a0min for the former and even up to several hours for the latter two. These considerable differences in lifetime, even at the cellular level, may reflect the existence of different subclasses of TNT-like structures. It is interesting to speculate that the fusion of the nanotube with the plasma membrane of the connected cell is part of a time-dependent maturation step leading to such heterogeneity.\nStudies on model artificial membrane tubes such as those created by pulling tethers from synthetic lipid vesicles or cellular plasma membrane provide a complementary approach to obtain mechanistic insights into the formation of TNT-like structures. The morphology of such nanotubes resembles that of TNT-like structures and it seems plausible that the same physical laws govern the formation and architecture of both structures. The exploitation of such in\u00a0vitro models revealed that the elongation of plasma membrane nanotubes requires a membrane flow from the cell plasma membrane into the growing tube. It is suggested that cells maintain a membrane reservoir (e.g. ruffles, invaginations), controlled by the cytoskeleton, to provide a buffer against membrane tension over several micrometers of tube elongation (Raucher and Sheetz 1999; Sun et\u00a0al. 2005). Thus, the available membrane reservoir may restrict the number, total length, and lifetime of TNT-like connections for a given cell.\nTNT-like structures are conduits for the delivery of cargo\nAn obvious advantage of establishing direct bridges between cells is an improvement in selective communication even over long distances. In agreement with this assumption, the common feature of all TNT-like structures characterized in more detail, is the transfer of cargo between connected cells (Table\u00a01).\nCellular components\nDirect evidence for the intercellular exchange of cargo via TNT-like structures was obtained by video-microscopic studies. By employing fluorescent dyes, organelles belonging to the endosomal\/lysosomal system (Rustom et al. 2004) (Gurke, S., Gerdes, H.-H., unpublished data) as well as mitochondria (Koyanagi et\u00a0al. 2005) were shown to traffic uni-directionally along TNT-like structures between cells over long distances (Fig.\u00a03a1) (Table\u00a01). The occurrence of an intercellular transfer of organelles raises the question as to what kind of information is transferred and how this information is integrated in the target cell. In the case of the endosomal system, early endocytic vesicles are particularly interesting candidates for TNT-mediated delivery. These organelles are one of the major reloading points for a variety of signaling complexes resulting from the endocytosis of activated cell surface receptors (Miaczynska et\u00a0al. 2004). Once delivered to the target cells, the transferred endocytic vesicles are able to fuse with their counterparts (Rustom et\u00a0al. 2004), and provide a potential way to integrate information. In essence, the transfer of endocytic organelles propagates signaling information from the single cell to a larger community, which may lead to coordinated cell behavior. Mitochondria were shown to transfer via TNT from neonatal rat CM to adult human EPC and it was suggested that this event could contribute to the acquisition of a cardiomyogenic phenotype by the progenitor cells (Koyanagi et\u00a0al. 2005) (Fig.\u00a04). Moreover, the active transfer of this organelle from adult stem cells and somatic cells to mammalian cells with non-functional mitochondria was shown to rescue aerobic respiration in the latter (Csord\u00e1s 2006; Spees et\u00a0al. 2006). The obtained data are consistent with a TNT-related delivery, although alternative mechanisms were not excluded.\nFig.\u00a04Emerging physiological implications of TNT-like structures\nIn addition to the transfer of organelles, plasma membrane components such as lipid-anchored proteins can laterally transfer along the TNT-like bridge into the plasma membrane of connected cells. This was demonstrated for both the inner and outer leaflets of the plasma membrane by using glycosylphosphatidylinositol conjugated to GFP (GPI)-GFP (\u00d6nfelt and Davis 2004; \u00d6nfelt et\u00a0al. 2004) and the farnesylation signal of c-Ha-Ras fused to the C-terminus of EGFP (EGFP-f) (Rustom et\u00a0al. 2004), respectively (Table\u00a01). Such a transport is consistent with membrane continuity between connected cells. Surface receptors could also be transferred directly by lateral diffusion in the plane of the cell surface, in addition to an endosome-dependent delivery. Interestingly, the transfer of membrane proteins between myeloid cells was found to involve scavenger receptors (Dr. R. D. Salter, Univ. of Pittsburgh, personal communication). It is of note that major histocompatibility complex (MHC) class I receptors were detected at the entire length of TNT-like bridges connecting immune cells (\u00d6nfelt et al. 2004; Watkins and Salter 2005). This points to a key role of TNT in a faster and more efficient presentation of antigens, in particular at the immunological synapse (Groothuis et\u00a0al. 2005; Williams et\u00a0al. 2007) (Fig.\u00a04), defined as a highly specialized interface between immune cells (Norcross 1984; Paul and Seder 1994). In this case, the lateral transfer of only a few molecules can fulfill important roles in a coordinated immune response. Interestingly, in analogy to TNT, membrane continuity was found at the immunological synapse (Stinchcombe et\u00a0al. 2001). The characterized \u201cmembrane bridges\u201d had a diameter of 50\u201395\u00a0nm as observed by electron microscopy (Stinchcombe et\u00a0al. 2001).\nFinally, the proposed model of membrane continuity suggests a cytoplasmic bridge in analogy to PD, which permits the free flow of soluble molecules up to a certain size limit defined by the free space along the interior of the nanotube. In support of this view, EGFP-actin was transferred between TNT-connected PC12 cells (Rustom et al. 2004), GFP between CM and EPC (Koyanagi et al. 2005), and the fluid phase marker Lucifer yellow between THP-1 monocytes (Watkins and Salter 2005) (Table\u00a01). Notably, both GFP and the small dye molecule calcein, with a molecular weight of 400\u00a0Da, were apparently impeded to diffuse passively between TNT-connected PC12 cells (Rustom et al. 2004). Thus, TNT-like structures, like PD, may have a variable size exclusion limit depending on the cell type (Gerdes et al. 2007). With regard to soluble markers, certainly the most physiologically relevant finding was the TNT-dependent propagation of calcium signals between THP-1 monocytes as well as from DC to THP-1 monocytes (Watkins and Salter 2005) (Table\u00a01). Stimulation by contact or exogenous soluble stimulators of both DC and THP-1 monocytes led to a calcium flux that propagated through a network of TNT-connected cells up to 500\u00a0\u03bcm away from the point of stimulation. The elicited calcium wave propagated uni-directional with an initial speed of 35\u00a0\u03bcm\/s that slowed down rapidly to 10\u201315\u00a0\u03bcm\/s (Watkins and Salter 2005). This rather low and rapidly decreasing speed supports a TNT-mediated signal spread along the cellular network, which is driven by a chemical gradient rather than an action potential. Such a view also corroborates a TNT-mediated membrane continuity providing a cytoplasmic bridge between connected cells (Table\u00a01). Most remarkably, the transfer of calcium ions through TNT led to the flattening and membrane extension of the receiving cells, as occurs during physiological responses in preparation for phagocytosis (Watkins and Salter 2005). Thus, this study provided the first evidence for functional connectivity accomplished by TNT and suggests a crucial role of these structures in immune defense (Fig.\u00a04).\nPathogens\nAn emerging topic is the efficient spread of pathogens, like bacteria and mammalian viruses, along cell membrane protrusions (Figs.\u00a03, 4). By using established or induced nanotubular connections between cells, pathogens circumvent the rate-limiting step of diffusion and efficiently accelerate their spreading (Sherer et\u00a0al. 2007; Sowinski et al. 2008). Among the viruses shown to exploit cellular protrusions to transfer from cell-to-cell are the vaccinia virus (Cudmore et\u00a0al. 1995,. 1996), pseudorabies virus (Favoreel et\u00a0al. 2005, 2006), the herpes simplex virus (La Boissi\u00e8re et\u00a0al. 2004), MLV (Sherer et\u00a0al. 2007) and human immunodeficiency virus type 1 (HIV-1) (Sowinski et\u00a0al. 2008). In the following, we will focus on the latter two, which were characterized in much detail and gave rise to two different mechanisms of virus transmission.\nSowinski et al. (2008) proposed membrane nanotubes between T cells as a new route for HIV-1 transmission (Fig.\u00a03b1), in addition to cellular contact points known as virological synapses (Jolly et\u00a0al. 2004; Jolly and Sattentau 2004; Sol-Foulon et\u00a0al. 2007). Using a recombinant HIV-1 expressing Gag-GFP, they demonstrated its uni-directional move through innate nanotubular bridges and subsequent transmission into connected T cells (Sowinski et\u00a0al. 2008) (Table\u00a01). Furthermore, the requirement of cell surface receptors for viral transmission implied, in agreement with ultra-structural data, a junctional border between the nanotube and the connected T cell. It should be emphasized that the transmission of HIV-1 through nanotubular bridges can minimize the exposure of the virus to extracellular antibodies or complement. However, a nanotube-dependent spread may open new avenues in the development of antiviral drugs.\nA different, even though nanotube-dependent, mechanism of transmission was reported for MLV (Sherer et\u00a0al. 2007). The cell-to-cell transmission of MLV was directly visualized by infecting cultured cells with a recombinant virus expressing the viral capsid protein Gag and the envelope protein Env as a fusion construct with fluorescent proteins. MLV induces the outgrowth of thin and long filopodia from uninfected cells toward infected cells (Sherer et\u00a0al. 2007). These filopodia are proposed to stably anchor to the cell body of an infected cell by viral envelope glycoprotein (Env)\/receptor interactions. Subsequently, MLV moves along the outer surface of these nanotubular bridges to reach the cell body of target cells, where it finally enters (Sherer et\u00a0al. 2007) (Fig.\u00a03b1). In addition to viruses, bacteria were reported to surf along TNT-like structures connecting macrophages (\u00d6nfelt et\u00a0al. 2006) (Table\u00a01).\nMechanism of transfer\nIt is possible that more than one transfer mechanism for shipping cargo via TNT-like bridges has evolved considering their structural diversity and the multitude of transferred cargo. For organelles and viruses, an F-actin-dependent, uni-directional transport prevails (Fig.\u00a03), with the exception of a microtubule-dependent, bi-directional movement of organelles in thick bridges connecting macrophages (\u00d6nfelt et\u00a0al. 2006). In the case of endocytic organelles, an acto-myosin-dependent transport system is presumed (Fig.\u00a03a1) due to the presence of the barbed-end directed actin-specific motor myosin Va in TNT (Rustom et\u00a0al. 2004; Zhu et\u00a0al. 2005) (Bukoreshtliev, N., Gerdes, H.-H., unpublished data) (Table\u00a01) partially co-localizing with the respective organelles (Rustom et\u00a0al. 2004). This model is supported by the low transport velocity in the range of actin-dependent transport (Rustom et\u00a0al. 2004) and implies that the F-actin fibers inside the bridge have the same polarity. Also HIV-1 particles were suggested to move in an actin-dependent manner through nanotubular bridges (Sowinski et al. 2008) (Fig.\u00a03b1). Interestingly, even in the case of MLV surfing along filopodia bridges with a speed \u223c7\u00a0times slower as compared to that of HIV-1 within the bridges, an acto-myosin transfer mechanism appears to be in place (Sherer et\u00a0al. 2007). This results in the uni-directional movement of all viral particles from infected to target cells, presumably using myosin II controlled retrograde flow of actin and cognate Env-receptor interactions (Fig.\u00a03b1). In contrast to organelles and viruses, the uni-directional propagation of calcium waves is likely to be driven by a chemical gradient rather than an active transport mechanism. Passive diffusion is likely to control the transfer of other small cytoplasmic molecules and also some plasma membrane components, if membrane continuity is present.\nRegarding the modality by which shipped cargo enters the target cell, two different scenarios are conceivable. In the case of membrane continuity as observed in PC12 cells, an open-ended transport along cytoskeleton elements without border crossing is envisaged, consistent with the observed uniform movement of transferred organelles (Rustom et\u00a0al. 2004). In analogy to PD, gating mechanisms may exist to control this open-ended transport (Gerdes et\u00a0al. 2007). In the case of close-ended cell-to-cell bridges containing junctional borders, as are the innate nanotubes connecting T cells and the MLV-induced cellular bridges, the cargo has to traverse the plasma membrane boundary. This is consistent with the finding that a receptor-dependent transmission of HIV-1 Gag could be blocked by the HIV-1 fusion inhibitor T20 (Sowinski et\u00a0al. 2008).\nUltra-fine cellular extensions in tissue\nDoes intercellular communication mediated by TNT-like structures play a physiological role in tissue? This is probably the most interesting and important question that needs to be addressed in the near future. The absence of a TNT-specific marker does not allow an explicit answer to this question yet. However, studies employing cell cultures already point to important physiological implications of TNT-like structures in intercellular communication (reviewed in Gerdes et\u00a0al. (2007)) (Fig.\u00a04). It could be argued that cell culture models are rough representations of physiological systems and that TNT-like structures are the sole result of stress conditions in this in vitro situation. However, the widely documented richness of cellular protrusions in tissue implicated in intercellular communication (R\u00f8rth 2003) (Table\u00a02) disagrees with such a possibility and instead supports an in\u00a0vivo function. Cellular extensions have been documented in diverse tissues and during various physiological processes such as embryogenesis, stem cell differentiation, cell migration, and wound healing.\nTable\u00a02Selected examples of cell protrusions in tissueProtrusions Tissue Cytoskeletal components Cargo Length [\u03bcm] Diameter [nm] Growth speed [\u03bcm\/min] Thin, elongated, active filopodia and lamellipodia Primary mesenchyme sea urchin embryos (Gustafson and Wolpert 1967; Miller et al. 1995) F-actinaND 80a200\u2013400b25a Cytonemes Drosophila wing imaginal discs (Ram\u00edrez-Weber and Kornberg 1999) F-actin ND <700 200 15 Cytoneme-like Mouse limb bud cells (Ram\u00edrez-Weber and Kornberg 1999); Drosophila wing imaginal discs (Jacinto et al. 2000) F-actinbND 10bND 1bApical peripodial extensions Drosophila wing\/eye imaginal discs (Gibson and Schubiger 2000) Microtubules Mitochondriac 5\u201330 ND ND Myopodia Drosophila embryonic muscle cells (Ritzenthaler et al. 2000; Ritzenthaler and Chiba 2003) F-actin ND 40 ND ND Myopodia-like Mouse transverse muscle of thorax (Misgeld et al. 2002); Xenopus (Kullberg et al. 1977) ND ND 10dND ND Cellular extensions Drosophila eye imaginal discs (Chou and Chien 2002) F-actin Scabrous vesicles 40 ND ND Apical and lateral cell protrusion Drosophila wing imaginal discs (Demontis and Dahmann 2007) F-actin GFP-Rab5c, GFP-Tkvc1\u201310 200 ND aData from Miller et al. (1995)bData from Jacinto et al. (2000)cThese molecules were shown to localize in cell protrusions, but their intercellular transfer was not provendData from Misgeld et al. (2002)ND: not determined\nThe formation of cellular extensions (\u201cpseudopods\u201d) during embryogenesis became first evident by studying sea urchin morphogenesis (Gustafson and Wolpert 1967; Miller et\u00a0al. 1995), suggesting a function of these structures in force generation or cell recognition. During subsequent studies over the last decades, a variety of similar cellular extensions were found to be associated with developmental processes. Interestingly, most of them appear to be actin-rich as TNT-like structures. The Drosophila wing imaginal disc provides an attractive system for studying cell protrusions within a developing tissue. The first described example of such protrusions were the F-actin-containing cytonemes extending from the periphery of the columnar cell sheet toward the signaling center within this layer (Ram\u00edrez-Weber and Kornberg 1999). This discovery inspired further studies describing other cellular protrusions in the wing disc extending not only within (Chou and Chien 2002; Demontis and Dahmann 2007) but also between cell layers (Demontis and Dahmann 2007; Gibson and Schubiger 2000). In a very recent study on live wing discs, Demontis and Dahmann (2007) observed two new types of cellular extensions, i.e. lateral protrusions interconnecting distant cells of the columnar epithelium, and protrusions of the apical surface connecting to the squamous epithelium. Furthermore, ectopically expressed GFP-actin and the early endosome marker GFP-Rab 5 were detected inside these protrusions, resembling the finding made for TNT-like structures between cultured cells. These different protrusions may be involved in the intercellular signaling necessary for growth and patterning of wing imaginal discs (Demontis and Dahmann 2007).\nThin, actin-based membrane extensions were also found to be crucial for embryonic dorsal closure in Drosophila (Jacinto et\u00a0al. 2000). Data obtained by confocal imaging of living fly embryos suggest that these long protrusions participate in both the mechanics of epithelial adhesion and in the search for a correct partner within the opposing epithelium. Likewise, related fundamental processes such as gastrulation, neural crest closure (Bard 1992), and wound healing (Wood et\u00a0al. 2002) seem to involve similar actin-rich membrane extensions.\nOther examples of actin-based cellular extensions during development are the so-called myopodia in Drosophila embryonic muscle cells that can be observed in whole mount. These dynamic postsynaptic microprocesses, with lengths up to 40\u00a0\u03bcm and highly sensitive to photoillumination, cluster at the site of motoneuron innervation while interacting with presynaptic filopodia. It was proposed that they play a role in the process of synaptic target recognition by contributing to direct long-distance cellular communication (Ritzenthaler et\u00a0al. 2000; Ritzenthaler and Chiba 2003). Importantly, similar structures have been described in Xenopus (Kullberg et\u00a0al. 1977) and mouse (Misgeld et\u00a0al. 2002).\nFinally, long, thin cellular extensions are also associated with guided cellular migration during development (Ribeiro et\u00a0al. 2002; Sato and Kornberg 2002), immune defense such as the migration of macrophages through the endothelial cell layer (Blue and Weiss 1981) and tumor cell invasion (Vignjevic and Montagnac 2008). Certainly, there are many more examples of long cellular nanotubes in tissue involved in cellular communication besides the ones listed here (Table\u00a02). This suggests that extensions in tissue are a rather common feature and that cells in culture preserve the ability to form similar, physiologically relevant structures. But, an important question remains: Do TNT-like structures, observed in cell culture models, have mechanistically and\/or functionally related counterparts in live tissue? Furthermore, with respect to TNT, which have been found to mediate membrane continuity between cultured cells, it will be of foremost interest to find out, if this type of connection also exists in\u00a0vivo.","keyphrases":["tunneling nanotube","tnt","intercellular transfer","cytoneme","viral transmission"],"prmu":["P","P","P","P","P"]}
{"id":"Diabetologia-3-1-1781097","title":"Pten (phosphatase and tensin homologue gene) haploinsufficiency promotes insulin hypersensitivity\n","text":"Aims\/hypothesis Insulin controls glucose metabolism via multiple signalling pathways, including the phosphatidylinositol 3-kinase (PI3K) pathway in muscle and adipose tissue. The protein\/lipid phosphatase Pten (phosphatase and tensin homologue deleted on chromosome 10) attenuates PI3K signalling by dephosphorylating the phosphatidylinositol 3,4,5-trisphosphate generated by PI3K. The current study was aimed at investigating the effect of haploinsufficiency for Pten on insulin-stimulated glucose uptake.\nIntroduction\nType 2 diabetes mellitus is a multifactorial disease with a complex pathophysiology that includes defects in insulin production and a failure in peripheral tissues to take up glucose in response to insulin. Signalling via phosphatidylinositol 3-kinase (PI3K) is a key pathway in the regulation of glucose uptake by insulin [1]. Activation of PI3K via insulin receptor substrate-1 causes PI3K to phosphorylate inositol-containing phospholipids at the 3\u2032-position of the inositol ring, increasing the levels of the lipid messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3), among others. PIP3 activate a phosphoinositide kinases, which in turn phosphorylate and activate protein kinase B (PKB), also known as Akt, a key effector kinase for many of the downstream metabolic effects initiated by insulin via PI3K. The phosphatase and tensin homologue (PTEN) is a phosphatase which recognises both protein and lipid substrates. As a lipid phosphatase, PTEN dephosphorylates PIP3 at the 3\u2032-position of the inositol ring, thereby acting as an antagonist to PI3K signalling [2, 3]. PTEN was originally identified as a tumour suppressor gene, and is one of the most commonly mutated genes in human cancer [4\u20136]. Mutation or deletion of PTEN has been identified in many cancers including prostate, brain, breast, endometrium and skin [4\u20138].\nAs many of the metabolic outcomes of insulin are achieved through recruitment of PI3K and the subsequent rise in PIP3 levels, PTEN may have a critical role in modulating sensitivity to insulin-stimulated glucose uptake. Recent studies in which Pten was ablated specifically in liver, adipose tissue and muscle in mice by Cre recombinase-based strategies have shown a role for Pten in the regulation of insulin sensitivity in those organs, and highlighted the contributions of those organs to glucose homeostasis of the whole animal [9\u201311]. However, the effect of global reduction of Pten levels on glucose uptake and metabolism has not yet been shown in an in vivo mouse model. Unfortunately, experimental genetic inactivation of both Pten alleles (Pten\u2212\/\u2212) results in early embryonic lethality [6, 12]. Thus mice harbouring inactivation of one Pten allele (haploinsufficiency, Pten+\/\u2212) are an important experimental model for studying the role of Pten in vivo. Haploinsufficiency for Pten has important consequences for cell proliferation and tumorigenesis in mice [6, 12\u201314]. Pten+\/\u2212 mice exhibit defective apoptosis in T cells, B cells and macrophages and spontaneous neoplasms in various tissues including prostate, liver, endometrium and others [12\u201314]. Here we show that Pten haploinsufficiency results in insulin hypersensitivity and enhanced insulin-mediated glucose uptake.\nMaterials and methods\nExperimental animals All laboratory animals were cared for and used according to guidelines of the Canadian Council on Animal Care. Pten+\/\u2212 mice were generated by R. Parsons [12]. These mice were backcrossed with C57BL6 mice for more than ten generations. The genotypes of the mice were determined as described [12].\nBlood glucose determination For determination of blood glucose levels, glucose levels in blood samples taken from tail vein were determined using a One Touch Ultra blood glucose monitor (LifeScan Inc., Milpitas, CA, USA). For the glucose tolerance test, mice were fasted overnight (15\u00a0h) prior to i.p. injection of glucose (2\u00a0mg\/g body weight). Blood glucose levels were then determined at the indicated time-points following glucose injection. For the insulin challenge test, mice were fasted for 15\u00a0h prior to i.p. injection of 0.6\u00a0mU insulin\/g body weight. Blood glucose levels were then determined at the indicated time-points following insulin injection.\nDetermination of insulin in plasma Mice were fasted for 15\u00a0h, then approximately 100\u00a0\u03bcl of blood was collected from the tail vein using pipette tips precoated with heparin. The mice were then injected i.p. with 2\u00a0mg glucose\/g body weight, and a second blood sample was obtained 15\u00a0min following injection. The blood samples were centrifuged at 805\u00a0g in a table-top centrifuge, and insulin in the plasma fraction was determined using an insulin ELISA system (ALPCO Diagnostics, Salem, NH, USA) according to the manufacturer\u2019s instructions.\nIsolation of pancreatic islets and glucose-stimulated insulin release Islets were isolated from pancreata of mice as described [15]. Pancreata from three mice were pooled. After 24\u00a0h in culture, islets were preincubated in Krebs\u2013Ringer bicarbonate buffer plus 0.1% BSA (KRBB-BSA) and 1.67\u00a0mmol\/l glucose. The islet cells were incubated for 1\u00a0h in 1\u00a0ml KRBB-BSA containing the desired concentration of glucose (1.67 or 16.7\u00a0mmol\/l). The incubation medium was then collected, and centrifuged and the supernatant fractions were used for assay of insulin content by ELISA. Total extractable insulin in islets was determined by adding 0.5\u00a0ml lysis buffer (1\u00a0mol\/l acetic acid with 0.1% BSA) to the islet pellet. Cell debris was pelleted by centrifugation, and the supernatant fraction was used for insulin assay.\nFluorescent immunohistochemistry of pancreatic islets Immunocytochemistry has been described [16]. Pancreata from wild-type and Pten+\/\u2212 mice were embedded in paraffin. Five-micron sections were incubated for 1\u00a0h at room temperature with guinea-pig anti-insulin antibody (1:100 dilution; Dako, Mississagua, ON, Canada) and rabbit anti-glucagon antibody (Dako; 1:75 dilution). Sections were visualised by incubation with Alexa\u2013Fluor 488 goat anti-guinea-pig (Invitrogen, Molecular Probes, Burlington, ON, Canada; 1:100 dilution) and Texas Red donkey anti-rabbit antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA; 1:100 dilution). Sections were examined by fluorescence microscopy.\nBeta cell mass calculation Following insulin immunostaining, pancreatic sections were captured under an FITC filter using a Zeiss Axioplan 2 microscope equipped for epifluorescence and Pathvysion imaging software. The insulin immunopositive area (as a proportion of pancreatic area) was determined in at least six fields per section using Zeiss AxioVision software, performed on eight pancreatic sections per mouse. Beta cell mass was calculated as pancreatic mass times the percent beta cell area for each mouse.\n2-[18F]fluoro-2-deoxyglucose uptake and microPET scanning Mice were fasted for 15\u00a0h prior to i.p. injection with 0.6\u00a0mU insulin\/g body weight. Twenty minutes after insulin injection, mice were injected with 2-[18F]fluoro-2-deoxyglucose (18FDG) (4.1\u00a0MBq) via the tail vein, and whole-body distribution of 18FDG was monitored by positron emission tomography (PET) using a microPET R4 tomograph (Concorde Microsystems, Knoxville, TN, USA). The image data were attenuation-corrected using a 68Ge rod source. Wild-type and Pten+\/\u2212 mice were positioned side-by-side and scanned simultaneously for 60\u00a0min after 18FDG injection. Data were analysed using the manufacturer\u2019s software, ASIPro VM 5.0.1.0. For data analysis, regions of interest (ROIs) were specified in the coronal section of a hindlimb for each mouse. The 18FDG activities in each ROI were summed across all coronal planes, and expressed as a proportion of 18FDG in the whole mouse (total 18FDG activity was also taken as the sum of activity across coronal planes).\nPrimary myocyte culture Primary myocytes were cultured from fore- and hindlimbs from wild-type and Pten+\/\u2212 mice according to a protocol adapted from Springer et al. [17]. Skeletal muscle was excised and minced and then incubated at 37\u00b0C for 2\u00a0h with 2.4\u00a0units\/ml dispase II. Dispase digestion was halted by addition of 10\u00a0ml F-10-based myocyte medium (400\u00a0ml F-10 nutrient mixture, 100\u00a0ml fetal bovine serum, 25\u00a0\u03bcg basic fibroblast growth factor, penicillin\/streptomycin). The tissue slurry was then strained through a fine metal mesh and centrifuged at 500\u00a0g for 5\u00a0min. The supernatant fraction was removed to a fresh centrifuge tube and the pellet was resuspended and plated to a 5-cm collagen-coated culture dish in a total of 4\u00a0ml F-10-based medium. The supernatant fraction was re-centrifuged and the pellet was pooled with the first. The cells were passaged when they reached 70\u201380% confluence. To enrich for myocytes, the old medium was removed by aspiration, the cells washed with PBS, then incubated in a film of this buffer for 15\u00a0min. Myocytes were loosened from the dish by striking the dish several times sharply on the bench top. Cells were washed from the dish with PBS. The PBS incubation was repeated and the cells were pooled. Cells kept in culture for more than 5\u20136\u00a0days began to exhibit the elongated morphology typical of myotubes [18]. Cells in these older cultures also exhibited spontaneous twitching. All experiments were performed with cells on days 2\u20133 of culture.\n2-Deoxy[3H]glucose uptake Experiments were performed on wild-type or Pten+\/\u2212 cells from passage two to eight, grown on 24-well (1-cm diameter wells) plates. Cells were preincubated in serum-free medium for 12\u00a0h prior to experiments. The cells were washed twice with HEPES-buffered saline (140\u00a0mmol\/l NaCl, 20\u00a0mmol\/l HEPES, 2.5\u00a0mmol\/l MgSO4, 1\u00a0mmol\/l CaCl2, 5\u00a0mmol\/l KCl, pH 7.4), then incubated with 10\u00a0\u03bcmol\/l 2-deoxy[3H]glucose (0.037\u00a0MBq\/ml) for 5, 10, 20 or 30\u00a0min, in the absence or presence of 0.1\u00a0\u03bcmol\/l insulin. At the end of the incubation period, the radioactive substrate was removed, and the cells were washed three times with ice-cold 20\u00a0mmol\/l glucose. Cells were lysed with 0.2\u00a0ml 2% SDS, and the lysates were used for scintillation counting. Non-specific diffusion of 2-deoxy[3H]glucose was determined by incubating cells in the presence of 2-deoxy[3H]glucose and 10\u00a0\u03bcmol\/l cytochalasin B, a specific inhibitor of glucose uptake [19].\nDetermination of cell-surface GLUT1 and GLUT4 levels Primary myocytes were grown in 10-cm culture dishes. The cells were rinsed with PBS, and incubated with 0.5\u00a0mg\/ml sulpho-NHS-LC-biotin (Pierce Biotechnology, Rockford, IL, USA) in PBS for 20\u00a0min. Cells are impermeable to this reagent and it labels the amine groups of cell-surface proteins. Following the incubation, the cells were washed three times with PBS, then lysed with 0.5\u00a0ml lysis buffer (1% NP-40, 150\u00a0mmol\/l NaCl, 50\u00a0mmol\/l Tris pH 7.5, including protease and phosphatase inhibitors). Protein was quantified with a BCA protein assay kit (Pierce). Streptavidin-agarose beads (Pierce) were pre-blocked by incubation with 2% BSA in lysis buffer. Each sample (1.2\u00a0mg protein) was incubated with the streptavidin beads (100\u00a0\u03bcl, 50% bead slurry) for 1\u00a0h at room temperature. The beads were washed five times, for 30\u201360\u00a0min each wash, using lysis buffer containing 0.1% SDS. After the final wash, 50\u00a0\u03bcl SDS sample-loading buffer were added, and heated at 95\u00b0C for 10\u00a0min, then vortexed to elute the biotinylated proteins. The proteins in the supernatant fractions were analysed by SDS-PAGE, then transferred to nitrocellulose. Proteins were probed using polyclonal antibodies against GLUT1 and GLUT4 (also known as SCLA2A1 and SCLA2A4, respectively).\nWestern immunoblotting for PKB, phospho-PKB and phospho-glycogen synthase kinase 3 (GSK3)-\u03b1\/\u03b2 Total protein extracts of primary myocytes were prepared by lysing cells in NP-40-based lysis buffer. Thirty to fifty micrograms of protein per sample were analysed by SDS-PAGE, then transferred to nitrocellulose. Rabbit polyclonal anti-PKB, anti-phospho-PKB (Ser473) and anti-phospho-GSK3-\u03b1\/\u03b2 (Ser21\/9) antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Mouse monoclonal anti-vinculin antibody was obtained from Sigma (St Louis, MO, USA). Horseradish peroxidase-conjugated secondary antibodies were obtained from Dako. Protein bands were visualised using an enhanced chemiluminescence system and autoradiography by exposure to X-ray film.\nStatistical analysis Statistical analysis was performed using Student\u2019s t test. Significance was assessed at the 95% confidence level.\nResults\nAs glucose metabolism may be altered due to a change in insulin sensitivity in Pten+\/\u2212 mice, blood glucose levels in wild-type and Pten+\/\u2212 mice were determined. Slight but statistically significant hypoglycaemia was observed in Pten+\/\u2212 mice during ad libitum feeding (11.3% lower blood glucose in Pten+\/\u2212 mice) (Table\u00a01). Fasting caused a drop in glucose levels in both groups, with slight but significant hypoglycaemia in the Pten+\/\u2212 group (10.8% lower blood glucose) relative to wild-type mice. An insulin challenge test revealed hypersensitivity to insulin in Pten+\/\u2212 mice (Fig.\u00a01a). Following i.p. insulin injection (0.6\u00a0mU\/g body weight), blood glucose levels in Pten+\/\u2212 mice remained depressed at all time-points measured (up to 120\u00a0min). In contrast, wild-type mice started to recover after 30\u00a0min, and glucose levels returned to fasting levels 120\u00a0min after injection. A glucose tolerance test showed that Pten+\/\u2212 mice had greater glucose tolerance than wild-type mice (Fig.\u00a01b). After i.p. glucose injection, blood glucose levels in Pten+\/\u2212 mice returned to fasting levels in less than 60\u00a0min, approximately twice as rapidly as in wild-type mice. \nTable\u00a01Blood glucose levels in wild-type and Pten+\/\u2212 mice during ad libitum feeding and after a 15-h fast\u00a0Mean blood glucose\u00b1SD (mmol\/l)p value (independent Student\u2019s t test)Wild-typePten+\/\u2212Ad libitum feeding7.1\u2009\u00b1\u20090.4 (n\u2009=\u20095)6.3\u2009\u00b1\u20090.2 (n\u2009=\u20094)0.006Fasting6.5\u2009\u00b1\u20090.3 (n\u2009=\u20095)5.8\u2009\u00b1\u20090.3 (n\u2009=\u20095)0.008Fig.\u00a01Insulin hypersensitivity in Pten+\/\u2212mice. a Insulin challenge test. Mice were fasted for 15\u00a0h prior to i.p. injection of insulin (0.6\u00a0mU\/g body weight). Blood glucose levels were determined at the indicated times. Means\u00b1SEM for six wild-type mice (white circles) and six Pten+\/\u2212 mice (black circles). b Glucose tolerance test. Mice were fasted for 15\u00a0h prior to i.p. injection of glucose at 2\u00a0mg\/g body weight. Means\u00b1SEM for six wild-type mice (white circles) and seven Pten+\/\u2212 mice (black circles)\nThe observed difference in ability to normalise blood glucose levels could be due to differences in insulin sensitivity or islet function and insulin production. We therefore investigated insulin levels in the mice, as well as properties of pancreatic cell morphology and function. Fasting plasma insulin levels were slightly but significantly lower in Pten+\/\u2212 mice (Table\u00a02), which is consistent with increased peripheral insulin sensitivity. Fifteen minutes after i.p. glucose administration (2\u00a0mg glucose\/g body weight), similar increases in plasma insulin were observed in the two groups (Table\u00a02), suggesting that there is no marked difference in insulin production in the Pten+\/\u2212 mice. Insulin released from islets isolated from Pten+\/+ and Pten+\/\u2212 mice was also examined. Islets were cultured as described in Materials and methods, and incubated in the presence of low (1.67\u00a0mmol\/l) or high (16.7\u00a0mmol\/l) glucose. Insulin released into the medium was determined by ELISA and calculated as percentage of total insulin content in the islets. Per cent of total insulin release (presented as mean value\u00b1SD) was similar under conditions of low glucose (Pten+\/+, 3.3\u2009\u00b1\u20090.4% vs Pten+\/\u2212, 2.8\u2009\u00b1\u20090.3%; p\u2009>\u20090.05 no significant difference, n\u2009=\u20093 per group) and high glucose (Pten+\/+, 4.2\u2009\u00b1\u20090.7% vs Pten+\/\u2212, 3.7\u2009\u00b1\u20090.2%; p\u2009>\u20090.05 no significant difference, n\u2009=\u20093 per group). \nTable\u00a02Plasma insulin levels in wild-type and Pten+\/\u2212 mice after fasting, and 15\u00a0min after i.p. glucose administration (2\u00a0mg glucose\/g body weight)\u00a0Mean plasma insulin\u00b1SD (ng\/ml)p value (independent Student\u2019s t test)Wild-type (n\u2009=\u20097)Pten+\/\u2212 (n\u2009=\u20096)Fasting0.50\u2009\u00b1\u20090.040.36\u2009\u00b1\u20090.100.05Post-glucose challenge1.8\u2009\u00b1\u20090.52.4\u2009\u00b1\u20090.4>0.05 (not significant)\nImmunofluorescence of pancreatic sections revealed similar morphology of islets of wild-type and Pten+\/\u2212 mice (Fig.\u00a02), suggesting Pten haploinsufficiency has no marked effect on islet morphology. Accordingly, beta cell mass was not found to be significantly different between wild-type and Pten+\/\u2212 mice (Table\u00a03).\nFig.\u00a02Pancreatic islet morphology and immunohistochemistry for insulin (green) and glucagon (red) in wild-type (a) and Pten+\/\u2212 (b) mice. Pancreatic sections were immunostained as described in Materials and methods and visualised by fluorescence microscopy. Original magnification \u00d740Table\u00a03Pancreatic beta cell mass of Pten+\/+ and Pten+\/\u2212 littermates\u00a0Mean beta cell mass\u00b1SE (mg)Pten+\/+1.24\u2009\u00b1\u20090.30 (n\u2009=\u20093)Pten+\/\u22121.29\u2009\u00b1\u20090.15 (n\u2009=\u20093)\nSkeletal muscle is an important tissue involved in insulin-stimulated glucose uptake [10, 20]. In vivo glucose uptake was assessed by monitoring distribution of 18FDG into muscles in real-time by PET. 18FDG is transported into cells by glucose transporters and is phosphorylated by hexokinase, but is not a substrate for further metabolism in the glycolytic pathway [21]. To compare insulin-stimulated glucose uptake in wild-type and Pten+\/\u2212 mice, the mice were fasted for 15\u00a0h, then injected i.p. with insulin (0.6\u00a0mU\/g body weight) 20\u00a0min prior to injection with 18FDG via the tail vein. Mice were imaged for 18FDG uptake into the hindlimb for 60\u00a0min (Fig.\u00a03a). 18FDG activity in hindlimbs was analysed as a proportion of total body activity, and was found to be higher in Pten+\/\u2212 than in wild-type mice at all time-points (Fig.\u00a03b).\nFig.\u00a03Insulin-stimulated uptake of 18FDG in wild-type and Pten+\/\u2212 mice. Mice were fasted for 15\u00a0h prior to i.p. injection of 0.6\u00a0mU insulin\/g body weight. Twenty\u00a0minutes after insulin injection, mice were injected with 18FDG via a tail vein, and whole-body distribution of 18FDG was monitored by microPET for 60\u00a0min. The experiment was repeated three times with similar results. Typical results are shown. a\u2013f Imaging of 18FDG distribution in coronal section; wild-type mouse is on the left and Pten+\/\u2212 mouse is on the right (a 5\u00a0min; b 15\u00a0min; c 25\u00a0min; d 35\u00a0min; e 45\u00a0min; f 55\u00a0min). Hindlimb muscles were highlighted as regions of interest, and 18FDG activity in each region of interest quantified across coronal planes at each time-point, as described in Materials and methods. g Graphical representation of relative 18FDG activity. Data are shown as proportion of 18FDG activity in hindlimbs relative to activity in whole body for wild-type mice (circles) and Pten+\/\u2212 mice (triangles) (mean\u00b1SD; n\u2009=\u20093 per point)\nTissues from Pten+\/\u2212 mice exhibited slightly reduced levels of Pten protein relative to wild-type mice [14, 22\u201324]. In order to examine cellular glucose uptake and intracellular signalling events, primary myocytes were cultured from hindlimb muscle of wild-type and Pten+\/\u2212 mice. In the absence of insulin, cells from Pten+\/\u2212 mice showed increased 2-deoxy[3H]glucose uptake at all time-points measured, to a maximum increase of twofold relative to wild-type cells at the 3\u00a0h time-point (Fig.\u00a04a). This result, together with the lower fasting glucose level of Pten+\/\u2212 mice, suggest that the PI3K\/PKB pathway was chronically activated in Pten+\/\u2212 animals. In the presence of insulin, Pten+\/\u2212 cells exhibited a further increase in glucose uptake, up to threefold greater than wild-type cells. This observation is consistent with the above results of the insulin and glucose challenge tests and 18FDG uptake experiment in showing a hypersensitivity to insulin due to Pten haploinsufficiency.\nFig.\u00a04a 2-Deoxy[3H]glucose uptake in wild-type and Pten+\/\u2212 myocytes. Myocytes were serum-starved for 12\u00a0h prior to incubation with 10\u00a0\u03bcmol\/l 2-deoxy[3H]glucose (0.037\u00a0MBq\/\u03bcmol\/l) for the indicated times with 0 or 1\u00a0\u03bcmol\/l insulin. Following incubation cells were lysed and aliquots of lysates were taken for determination of radioactivity by scintillation counting. Means\u00b1SD for three separate determinations. White circles, wild-type cells 0\u00a0\u03bcmol\/l insulin; black circles, wild-type cells 1\u00a0\u03bcmol\/l insulin; white triangles, Pten+\/\u2212 cells 0\u00a0\u03bcmol\/l insulin; black triangles, Pten+\/\u2212 cells 1\u00a0\u03bcmol\/l insulin. b Cell-surface GLUT1 and GLUT4 levels. Levels of cell-surface GLUT1 and GLUT4 proteins in wild-type (WT) and Pten+\/\u2212 cells were determined as described in Materials and methods\nGlucose transport into skeletal muscle occurs by a facilitated diffusion process mediated primarily by the glucose transporter proteins, GLUT1 and GLUT4. The level of GLUT4 at the plasma membrane is tightly regulated by insulin, whereas GLUT1 is found constitutively on the cell surface [25]. Since 2-deoxy[3H]glucose uptake was elevated in Pten+\/\u2212 cells even in the absence of insulin (Fig.\u00a04a), we analysed cell-surface GLUT4 and GLUT1 by western immunoblot as described in Materials and methods (Fig.\u00a04b). This analysis showed increased GLUT4 at the cell surface in Pten+\/\u2212 cells, while similar GLUT1 levels were observed in the two cell types.\nAs PKB is a key effector kinase that mediates many of the downstream cellular events induced by the PI3K signalling pathway, including insulin-stimulated glucose uptake [26, 27], we determined the phosphorylation status of PKB in wild-type and Pten+\/\u2212 myocytes. Little to no phosphorylation was observed in the absence of insulin, and phosphorylation at Ser473 was observed upon addition of insulin (Fig.\u00a05). Whereas PKB phosphorylation started to decrease after 4\u00a0h in wild-type cells in the presence of insulin, phosphorylation was sustained in Pten+\/\u2212 cells during the course of the experiment (up to 6\u00a0h). Immunoblotting for total PKB protein showed that PKB levels were consistent in Pten+\/\u2212 and Pten+\/+ cells (Fig.\u00a05). Immunoblotting for vinculin was included as a protein-loading control. An immediate downstream substrate of PKB is GSK3\u03b2. This enzyme becomes inactivated upon phosphorylation by PKB, in turn allowing its substrate, glycogen synthase, to mediate glycogen synthesis and thus promote glucose storage. Consistent with the observed enhancement of PKB phosphorylation in Pten+\/\u2212 cells, these cells also exhibited a higher degree of GSK3\u03b2 phosphorylation in comparison with Pten+\/+ cells (Fig.\u00a05).\nFig.\u00a05Phosphorylation of PKB and GSK3\u03b2. Myocytes were serum-starved for 12\u00a0h prior to incubation with 1\u00a0\u03bcmol\/l insulin for the indicated times. Cell lysates were prepared, and proteins were separated by PAGE followed by western immunoblot analysis for vinculin, total PKB, phospho-PKB (p-PKB S473) and phospho-GSK3\u03b2 (p-GSK3\u03b2) as indicated. This experiment was repeated three times with typical results shown. WT, wild-type\nDiscussion\nRecent studies utilising tissue-specific deletion of Pten in liver, adipose and muscle have shown that these organs make important contributions to the glucose homeostasis of the animal and clearly establish Pten as a negative regulator of insulin signalling in vivo [9\u201311]. In a separate report, in vivo antisense treatment resulted in 90% reduction of Pten levels in the liver of diabetic mice, normalising blood glucose levels and improving performance in an insulin tolerance test [28]. While the above studies focused on the role of Pten in specific tissues, the current work defines the effects of partial systemic reduction of Pten on the regulation of insulin sensitivity in the context of the whole animal.\nPTEN expression\/activity is tightly regulated by a complex mechanism that is currently poorly understood. PTEN is transcriptionally regulated by transcription factors such as p53, Egr-1, NF\u03baB and SMADs, while protein levels and activity are modulated by phosphorylation, oxidation, subcellular localisation, phospholipid binding and protein stability [29]. Previous studies showed that Pten haploinsufficiency results in only a 15\u201350% reduction in Pten protein, leaving a substantial amount of Pten protein remaining in the tissues examined [14, 22\u201324, 30]. In our hands, western blot analysis indicated similar decreases in tissue Pten levels (data not shown). The present study provides the first evidence that Pten haploinsufficiency leads to insulin hypersensitivity in vivo, suggesting that even small changes in Pten levels or activity can lead to dramatic effects on insulin responses. Thus partial systemic inhibition of PTEN activity may represent a novel strategy to ameliorate the pathology of type 2 diabetes.\nThe PI3K\/PKB signalling axis is central to maintenance of normal glucose homeostasis [1]. Pten-deficient cells typically have elevated levels of intracellular PIP3 [31, 32]. The sustained phosphorylation of PKB\/Akt and a downstream effector, GSK3\u03b2, observed in the current study is consistent with previous reports of elevated PKB\/Akt activation as a result of impaired desensitisation of PI3K-dependent signals under conditions of Pten inactivation or inhibition [9\u201311, 28]. A subtle reduction of Pten in the Pten+\/\u2212 mice appears sufficient to achieve a prolonged and robust insulin signal. The roles of PIP3 and PKB\/Akt in insulin-mediated glucose uptake have been well-established; therefore, our data collectively suggest that sustained PI3K\/PKB\/Akt\/GSK3 signalling may, in part, contribute to insulin hypersensitivity in Pten+\/\u2212 mice. However, other PKB\/Akt-independent signalling pathways, as well as additional signals delivered by the protein phosphatase and\/or adaptor functions of Pten may also contribute to the insulin hypersensitivity phenotype manifested by Pten haploinsufficiency.\nGLUT1 glucose transporters are constitutively present at the cell surface, while GLUT4 cycles between intracellular vesicles and the cell surface in a manner controlled by insulin-mediated signalling mechanisms [25]. In unstimulated fat and muscle cells, the majority of GLUT4 exists in intracellular compartments, while the insulin signal stimulates the exocytosis of GLUT4 to the cell surface. Studies involving PKB\/Akt-null animals and silencing of PKB\/Akt by RNA interference have demonstrated that PKB\/Akt is of primary importance in GLUT4 translocation [33\u201336]. In the current study, we observed an enhancement of 2-deoxyglucose uptake in Pten+\/\u2212 cells even in the absence of insulin stimulation, which was consistent with our observation of increased biotinylation of GLUT4 at the cell surface, while GLUT1 levels were unaffected. However, we detected no increase in phosphorylation of PKB\/Akt in the absence of insulin in Pten+\/\u2212 cells. Consistent with these observations, a recent study in which Pten was specifically knocked down by RNA interference reported slight but significant enhancement of deoxyglucose uptake in adipocytes, but no apparent increase in phosphorylation of PKB\/Akt [37]. These results could reflect a mechanism(s) affecting glucose transport involving Pten\/PIP3 but independent of PKB\/Akt. Such a mechanism could involve the atypical protein kinases C-\u03bb and -\u03b6 (PKC-\u03bb and PKC-\u03b6), which, like PKB\/Akt, are subject to control by PI3K via PIP3 and 3-phosphoinositide-dependent protein kinase-1 (PDK-1) [38\u201340]. A number of studies have implicated PKC-\u03bb\/\u03b6 in insulin-stimulated glucose transport and GLUT4 translocation [38, 41\u201343]. These studies indicate a role for PKC-\u03bb\/\u03b6 which appears to be complementary or parallel to that of PKB\/Akt.\nThe importance of the PI3K pathway in insulin signalling has raised interest in developing pharmaceutical antagonists targeting PIP3 phosphatases such as PTEN or Sh2-containing inositol 5\u2032-phosphatase 2 (SHIP2; also known as Inppl1) in an effort to enhance insulin responsiveness in type 2 diabetes [44]. While a study on Ship2-null mice initially implicated Ship2 as a key regulator of glucose uptake [45], it was subsequently recognised that these mice also had deleted a neighbouring gene, Phox2a, which may have contributed to the observed phenotype (corrigendum appears in Nature vol. 431, p. 878). A new gene-targeted mouse has since been generated in which only the gene encoding Ship2 is deleted [46]. Interestingly, this mouse does not exhibit insulin hypersensitivity but is protected against diet-induced obesity,  [46, 47]. In the light of these new findings, our observations of dramatic enhancement of insulin sensitivity and glucose uptake resulting from Pten haploinsufficiency suggests that PTEN plays a role in the desensitisation of insulin signalling, and strengthens the hypothesis that PTEN is a key regulator of insulin-stimulated glucose uptake.\nType 2 diabetes is associated with insulin deficiency and resistance to insulin signalling [48, 49]. The current study provides further support for PTEN as a candidate for drug intervention in treating type 2 diabetes. However, enthusiasm for systemic PTEN inhibitors as a therapeutic approach for treating type 2 diabetes is tempered by the potential side-effects of such agents. Most notably, a modest reduction of Pten in Pten+\/\u2212 mice promotes tumour formation in a wide variety of tissue, suggesting that long-term chronic administration of systemic PTEN inhibitors may lead to enhanced cancer development and progression. Furthermore, the PI3K pathway is an important regulator of numerous normal cellular processes and systemic PTEN inhibition may cause dysfunction of many cells and tissues, leading to alterations in liver, kidney, brain function, etc. For example, hepatocyte-specific Pten-deficient mice exhibit hepatomegaly and liver steatosis [9]. Systemic inhibition of PTEN may therefore be unacceptable clinically because of these inherent potential toxicities [44]. However, targeted delivery of PTEN antagonists to muscle cells or adipocytes may overcome these limitations and they hold promise as clinically viable agents in treatment of type 2 diabetes [44]. This study shows that partial inhibition of Pten is sufficient to enhance insulin sensitivity. This observation is important from a therapeutic standpoint, as most drugs are unlikely to completely inhibit enzyme activity in vivo, nor would complete inhibition be desirable given the potential for cancer development resulting from chronic or overactivation of the PI3K\/PKB pathway [12, 14, 23].\nIn conclusion, Pten haploinsufficiency results in insulin hypersensitivity, which underscores the importance of PTEN in attenuating insulin signals, and suggests that small-molecule drugs that antagonise PTEN activity may represent a novel class of insulin-sensitising agents and targeted delivery of these agents to muscle cells or adipocytes may have potential clinical utility in treating insulin resistance observed in type 2 diabetes.","keyphrases":["insulin hypersensitivity","glucose uptake","insulin sensitivity","pten haploinsufficiency"],"prmu":["P","P","P","P"]}
{"id":"Infect_Genet_Evol-1-5-2080692","title":"Chitotriosidase deficiency is not associated with human hookworm infection in a Papua New Guinean population\n","text":"Human chitotriosidase (CHIT1) is a chitinolytic enzyme with suggested anti-fungal properties. Previous studies have suggested that chitotriosidase may also protect individuals against filarial nematode infections and malaria. A mutant allele, which renders chitotriosidase unstable and enzymatically inactive, is found at a frequency of >20% in Caucasians and other populations. This allele is found at much lower frequency in parts of West Africa where malarial and intestinal helminth infections are endemic. Here, we investigate whether there is a significant association between chitotriosidase genotype and the intensity of hookworm infection in 693 individuals from five villages in Papua New Guinea. Individuals were genotyped for chitotriosidase using a PCR-based assay. There was no association between CHIT1 genotype and the intensity of hookworm infection as determined by faecal egg counts. The frequency of the mutant allele was 0.251, very similar to that found in non-endemic countries. The extent of geographical variation in allele frequencies across worldwide populations was not high (Fst = 0.11), and does not provide evidence for directional selection at this locus between different geographical areas. We conclude that the CHIT1 genotype does not play a crucial role in protection against hookworm infection. This does not correlate with a previous study that linked the mutant CHIT1 genotype to filariasis susceptibility. The possible reasons for this discrepancy are discussed.\n1\nIntroduction\nChitotriosidase is a chitinolytic enzyme which was recently identified in man and is synthesised in large quantities by activated macrophages (Hollak et al., 1994). As humans lack endogenous chitin, a role for chitotriosidase is not fully understood. However the anti-fungal action of homologous plant chitinases has led to the suggestion that chitotriosidase may degrade chitin containing pathogens (Boot et al., 1998). The chitotriosidase gene (CHIT1) consists of 12 exons located on chromosome 1q31\u201332 (Boot et al., 1998). A 24 base pair duplication in exon 10 leads to the activation of a cryptic 3\u2032 splice site that results in an abnormally spliced mRNA with an inframe 87 nucleotide deletion (Boot et al., 1998). The mutant protein lacks amino acids 344\u2013372 that are required for the formation of the TIM-barrel catalytic core (Boot et al., 1998; Fusetti et al., 2002). Macrophages from chitotriosidase deficient individuals express only small amounts of mRNA and secrete virtually no chitotriosidase protein (Boot et al., 1998).\nIn Dutch and Ashkenazi Jewish populations 6% of individuals are homozygous for the mutant allele, whereas 35% and 34%, respectively, are heterozygous carriers (Boot et al., 1998) with a mutant allele frequency of 0.23. A similar allelic frequency is present in Portugal (0.22), Sicily (0.27) and Sardinia (0.21) (Malaguarnera et al., 2003). However in two West African countries, Benin and Burkina Faso, that are mesoendemic for Plasmodium falciparum malaria and endemic for gastrointestinal helminths, a total absence of the homozygous mutation and significant reduction in heterozygous individuals has been reported, with mutant allele frequency 0.00 and 0.02, respectively (Malaguarnera et al., 2003). This observation led to the hypothesis that chitotriosidase may be involved in resistance to protozoan or helminth infections common in tropical countries (Malaguarnera et al., 2003).\nParasitic nematodes are known to contain chitin, and are thus a potential target of human chitotriosidase. Chitin is found in the egg-shell of both free-living and parasitic nematodes, and in the microfilarial sheath surrounding the first-stage larvae of filarial nematodes (Fuhrman and Piessens, 1985). More recently, chitin has been demonstrated in the pharynx of both the free-living Caenorhabditis elegans (Zhang et al., 2005) and the gastrointestinal parasite Oesophagostomum dentatum (Neuhaus et al., 1997). There are two chitin synthase genes in C. elegans, chs-1 and chs-2, encoding eggshell and pharynx chitin, respectively, and chs-2 knockdown leads to lack of pharyngeal function and starvation (Zhang et al., 2005). This suggests that host chitotriosidase could potentially interfere with parasitic nematode feeding. Two studies to date have investigated associations between CHIT1 genotype and filarial nematode infection. Choi et al. (2001) found significant association between susceptibility to lymphatic filariasis (Wuchereria bancrofti) and homozygosity for the mutant allele in an Indian population, but in a similar study in Papua New Guinea, no association between CHIT1 genotype and lymphatic filariasis was observed (Hise et al., 2003).\nThere have been no previous studies investigating CHIT1 genotype and gastrointestinal nematode infection. Human hookworm infection is an abundant chronic gastrointestinal nematode infection in sub-tropical and tropical countries, causing significant morbidity, principally due to iron-deficiency anaemia (Brooker et al., 2004). Human hookworm burden (as assessed by faecal egg count) is known to be under host genetic control (Williams-Blangero et al., 1997), but the genes responsible have not been identified. The aims of the current study were (1) to investigate associations between CHIT1 genotype and human hookworm infection in an endemic population from Madang Province, Papua New Guinea and (2) to investigate evidence for directional selection at the CHIT1 locus, by comparing worldwide allele frequency variation at the CHIT1 locus to that observed at other loci across the genome.\n2\nMaterials and methods\n2.1\nGeographical location and study population\nThe study population consisted of five villages in lowland Madang Province, Papua New Guinea, where Necator americanus is the only hookworm species (Pritchard et al., 1990). Villages were censused and pedigree information collected in 1998, and sample containers for faecal collection offered to all individuals aged 4 years and above. Faecal egg counts were performed using a modified McMaster salt flotation method and results were expressed as eggs per gram (epg) of faeces (Quinnell et al., 2004). Venous blood was then collected, and all individuals were offered treatment with albendazole or pyrantel pamoate. DNA was extracted from buffy coats using phenol\/chloroform. In September 2001, a second faecal sample was taken from three of the five villages and reinfection hookworm burden determined; some previously untreated individuals provided samples in 2001. Hookworm epg has been shown to be a heritable phenotype in the study population (Breitling and Quinnell, unpublished results). The study was approved by the Medical Research Advisory Committee of Papua New Guinea and informed consent was received from all subjects or their parents.\n2.2\nChitotriosidase genotyping by PCR\nThermal cycling reactions consisted of Reddy Mix PCR master mix (ABgene), 4.5\u00a0pmol forward and 4.5\u00a0pmol reverse chitotriosidase primers (forward: AGCTATCTGAAGCAGAAG; reverse: GGAGAAGCCGGAAAGTC) (Sigma) and 2\u00a0\u03bcl genomic DNA in a final volume of 15\u00a0\u03bcl. Samples were run in 96 well PCR plates (ABgene) on a Peltier PTC-200 Thermal Cycler (MJ Research). The thermal cycling protocol was: 94\u00a0\u00b0C for 5\u00a0min; 35 cycles of 94\u00a0\u00b0C for 30\u00a0s, 54\u00a0\u00b0C for 30\u00a0s and 72\u00a0\u00b0C for 30\u00a0s; and 72\u00a0\u00b0C for 5\u00a0min. Thermal cycling products with 10 base pair markers (Promega) were separated on 4% high resolution agarose (Sigma) gels in 1\u00d7 TBE buffer and ethidium bromide. Gels were viewed using the Gene Genius Bio-imaging System and Gene Snap software (Syngene). The size of wild type product is 75 base pairs whereas the mutant product is 99 base pairs due to the mutant allele containing a 24 base pair duplication in exon 10 (Boot et al., 1998).\n2.3\nStatistical analysis\nVariation in faecal egg counts by CHIT1 genotype was analysed under both a codominant and recessive model. Faecal egg counts were highly overdispersed, so analysis was performed using a generalised linear model with a negative binomial error structure in Stata 9.1. This method has been shown to be more accurate than analysis of log-transformed parasite burden data (Wilson et al., 1996). Significant covariates included in the analysis were age, village, faecal consistency, anthelmintic treatment and the age\u00a0\u00d7\u00a0treatment and village\u00a0\u00d7\u00a0treatment interaction terms. Egg counts from both years were analysed together; since some individuals were sampled both pre- and post-treatment, standard errors were adjusted for the non-independence of samples from the same individual using the \u2018cluster\u2019 option. To control for non-independence of individuals due to genetic relatedness, further analysis was performed with the total test of association in a variance components framework using the programme QTDT (Abecasis et al., 2000). Finally, to control for potential population stratification, transmission disequilibrium testing was carried out using the orthogonal association model in QTDT. The phenotypic variable used for these analyses was the residuals from a negative binomial regression of egg counts against significant covariates; where two samples were taken from the same individual, the mean residual for that individual was calculated.\nTo quantify geographic variation in allele frequencies, Fst values were calculated according to Cockerham and Weir (1984) using the programme FSTAT 2.9.3 (Goudet, 1995). Allele frequencies were compared across five broad geographical areas, using the weighted mean allele frequency for each geographical area (Europe\/Mediterranean, Africa, South Asia, East Asia and Papua New Guinea). To compare observed values of Fst to those expected for neutral markers, Fst was also calculated across comparable populations selected from the Human Genome Diversity Panel using published data for 210 genome-wide short insertion\/deletion polymorphisms genotyped by the Mammalian Genotyping Service (data available from http:\/\/research.marshfieldclinic.org\/genetics\/) (Soranzo et al., 2005). The populations selected were European (French\/Italian), African (Yoruba), South Asia (Sindhi), East Asia (Chinese Han) and Papuan.\n3\nResults\nIn total 693 individuals from Papua New Guinea were genotyped for the chitotriosidase mutation. The allelic frequency of the 24\u00a0bp duplication in 279 unrelated individuals, determined by PCR genotyping, was 0.251. The percentage of individuals who were homozygous mutant was 6% (16\/279), heterozygous 39% (108\/279) and homozygous wild type 56% (155\/279). Genotype frequencies did not differ from those expected under Hardy\u2013Weinberg equilibrium (\u03c72\u00a0=\u00a00.25, d.f.\u00a0=\u00a01, P\u00a0=\u00a00.62).\nBoth egg counts and CHIT1 genotypes were available for a total of 602 individuals (574 sampled pre-treatment and 210 sampled post-treatment). The prevalence and intensity of infection (mean egg count) in these individuals were 81% (2574\u00a0epg) pre-treatment, and 92% (1727\u00a0epg) post-treatment. Egg counts were very similar across CHIT1 genotypes both pre-treatment and after reinfection (Table 2). Combined analysis of both years\u2019 data revealed no significant relationship between egg count and genotype under either a codominant (\u03c72\u00a0=\u00a00.03, d.f.\u00a0=\u00a01, p\u00a0=\u00a00.87) or recessive model (\u03c72\u00a0=\u00a00.02, d.f.\u00a0=\u00a01, p\u00a0=\u00a00.90). Further analysis using the total test of association in QTDT, controlling for non-independence of individuals due to genetic relatedness, again showed no significant association between CHIT1 genotype and egg count (\u03c72\u00a0=\u00a00.39, d.f.\u00a0=\u00a01, p\u00a0=\u00a00.53). Similarly, transmission disequilibrium testing, which controls for population stratification, was not significant (\u03c72\u00a0=\u00a00.58, d.f.\u00a0=\u00a01, p\u00a0=\u00a00.45).\nPublished allele frequencies for CHIT1 in different geographical areas are shown in Table 1. Using these data and data from the current study, Fst values were calculated to measure variation in allele frequency across five broad geographical areas (Europe, Africa, South Asia, East Asia, and Papua New Guinea). The Fst across all populations for CHIT1 was 0.11. This was comparable to the median Fst of 0.12 for 210 indels typed in populations from similar geographical areas. Pairwise comparisons showed higher Fst values between Africa and East Asia (Fst\u00a0=\u00a00.69) and Africa and South Asia (Fst\u00a0=\u00a00.54), than those between other areas (Fst\u00a0<\u00a00.40). In each case the observed Fst was greater than that for all but 7 of 210 indels, giving an empirical P\u00a0=\u00a00.038 (8\/210).\n4\nDiscussion\nThe results from the present study in Papua New Guinea strongly suggest that chitotriosidase does not have a critical role in protection against N. americanus. The relative number of people who are wild type homozygous for chitotriosidase, heterozygous and homozygous mutant is similar to that seen in countries where this infection is not endemic, suggesting no advantage for the wild type allele in endemic areas. Moreover, there was no evidence for an association between CHIT1 genotype and hookworm burden in our study population.\nThe mutant chitotriosidase genotype has been associated with an increased susceptibility to W. bancrofti infection in a study of 216 individuals from South India (Choi et al., 2001). Previously it has been demonstrated that the microfilariae, oocytes and zygotes of the filarial nematode Brugia malayi contain chitin (Fuhrman and Piessens, 1985; Schraermeyer et al., 1987). In the rodent filarial nematode Acanthocheilonema vitae, there are distinctive chito-oligomeric N-glycans that contain projections of up to six GlcNAc residues that may be involved in parasite-host interactions (Haslam et al., 1999). Additionally chitin synthase is required for sheath development in microfilarial progeny. Chitin has also been identified in the cuticle of mature W. bancrofti (Araujo et al., 1993). The presence of chitin in the sheath and cuticle of filarial nematodes and the requirement for chitin synthase in reproduction show that chitin has an important role in the lifecycle of these species. If host-derived chitotriosidase can access this chitin, thus interfering with the parasite's growth and development, then it may explain why susceptibility to W. bancrofti is increased in individuals with the mutant genotype. However, a second study by Hise and coauthors (Hise et al., 2003) in 906 individuals in a region endemic for bancroftian filariasis in Papua New Guinea did not find any significant correlation with infection status or disease phenotype. Thus the issue is in further need of clarification.\nIn contrast to filarial worms, gastrointestinal nematodes such as hookworm do not have a microfilarial stage, and the chitin-containing eggs are not accessible to host chitotriosidase. However, chitin has recently been demonstrated in the pharynx of the free-living nematode C. elegans (Zhang et al., 2005). It is not known whether chitin occurs in the pharynx of N. americanus, but it has been demonstrated in the related strongyloid parasite O. dentatum (Neuhaus et al., 1997). Pharyngeal chitin provides a potential target for host chitotriosidase, both in tissue-dwelling larval stages, which may be accessible for macrophage or neutrophil-derived chitotriosidase, and blood-feeding adults. Chitotriosidase levels can be elevated in serum, e.g. in acute falciparum malaria (Barone et al., 2003), and as adult hookworms are thought to ingest 0.03\u20130.2\u00a0ml of blood daily, they may be exposed to high levels of chitotriosidase. However, the lack of an association between CHIT1 genotype and hookworm burden seen in the present study suggests that chitotriosidase is not an important effector mechanism against hookworm infection. Further studies would be useful to examine the bioavailability of nematode chitin, its accessibility for exogenous chitinases, or their effect on parasite viability.\nThe mutant allele frequency observed in the current study was higher than that in another Papuan population from East Sepik Province (Hise et al., 2003). Such variation within Papua New Guinea is not surprising, and differences between these populations have been observed at other loci (Main et al., 2001). Analysis of worldwide variation in allele frequencies showed that the degree of population differentiation at the CHIT1 locus (Fst\u00a0=\u00a00.11) was very similar to the median value for 210 indels across the genome. Though the populations sampled were not identical, this suggests that there has not been directional selection at this locus across different geographical areas, and that the worldwide variation in allele frequencies is consistent with genetic drift (Akey et al., 2002). There was some evidence for higher than average population differentiation at the CHIT1 locus between African and Asian populations, which may suggest some local adaptation, though this would not be significant after correction for multiple testing.\nMalaguarnera et al. (2003) suggest that an intact CHIT1 genotype may be advantageous in protection against malaria, based on the very low frequency of the mutant genotype in Burkina Faso and Benin, where malaria is meso-endemic. However, our results are not consistent with the hypothesis that chitotriosidase is involved in protection against malaria, or any tropical disease. There is no evidence for directional selection worldwide, and the greatest difference in allele frequencies is observed between Africa and other tropical countries, also endemic for malaria (Choi et al., 2001; Chien et al., 2005). Although Barone et al. (2003) found that chitotriosidase levels are elevated in the serum in acute P. falciparum malaria, there is no evidence that elevated serum chitotriosidase levels have an impact on the severity and outcome of malaria, and no study has compared genotypes with disease status. Indeed, increased serum chitotriosidase may actually increase malaria transmission, by inhibiting the formation of the peritrophic membrane in the anopheline gut (Di Luca et al., 2007). As the study area in Papua New Guinea is endemic for malaria, we have performed an association analysis which showed no significant difference between chitotriosidase genotype and the presence of P. falciparum and P. vivax malaria infection (data not shown). The caveat of this analysis is that it was based on a single time point assessment of parasitaemia, rather than repeated measurements. As parasitaemia is very variable, we cannot make a reliable statement regarding the effect of the CHIT1 genotype on malarial infection, and further studies are needed.\nTaken together, our data suggest that the chitotriosidase genotype does not play a major role in protection against hookworm infection, but this may be different in the case of lymphatic or cutaneous filariasis. However, there is no evidence for selection at this locus mediated by malaria or another tropical disease.","keyphrases":["chitotriosidase","papua new guinea","malaria","necator americanus","chit1 mutation"],"prmu":["P","P","P","P","R"]}
{"id":"J_Med_Internet_Res-4-3-1761945","title":"eEurope 2002: Quality Criteria for Health related Websites\n","text":"Background A number of organisations have begun to provide specific tools for searching, rating, and grading this information, while others have set up codes of conduct by which site providers can attest to their high quality services. The aim of such tools is to assist individuals to sift through the mountains of information available so as to be better able to discern valid and reliable messages from those which are misleading or inaccurate.\nIntroduction\nHealth related web sites are now amongst the most frequently accessed sites on the internet with current estimates indicating that there are now over 100,000 sites offering health related information [1]. As a result of the wealth of information available and its apparent popularity, a number of organisations have begun to provide specific tools for searching, rating, and grading this information, while others have set up codes of conduct by which site providers can attest to their high quality services. The aim of such tools is to assist individuals to sift through the mountains of information available so as to be better able to discern valid and reliable messages from those which are misleading or inaccurate.\nRecognising that European citizens are avid consumers of health related information on the internet and recognising that they are already using the types of rating system described above, the European Council at Feira on June 19-20 2000 supported an initiative within eEurope 2002 to develop a core set of Quality Criteria for Health Related Websites.\nAccordingly a series of meetings was held during 2001 which drew together key players from Government departments, International Organisations, non-governmental organisations and industry, to explore current practices and experiments in this field. Some sixty invited participants from all the Member States, Norway, Switzerland, and the United States of America took part in the kick-off meeting of June 7-8, 2001: they included delegates from industrial, medical, and patient interest groups, delegates from Member States' governments, and key invited speakers from the field of health information ethics. These individuals, and many others, also took part in the web-based consultation which was open from august to November 2001 [2].\nThe focus of the discussions was primarily on the reliability of health related websites as a potential vehicle for health related messages, rather than on the substance and content of the health messages themselves. The specific aim was to draw up a commonly agreed set of simple quality criteria on which Member States, as well as public and private bodies, may draw in the development of quality initiatives for health related websites. These criteria should be applied in addition to relevant Community law [3].\nAs a result of the meetings, as well as a web-based public consultation, a core set of quality criteria was established. The criteria may be used as a basis in the development of user guides, voluntary codes of conduct, trustmarks, accreditation systems, or any other initiative adopted by relevant parties, at European, national, regional or organisational level. By using a common set of criteria as a starting point, such initiatives can develop in a focused manner across the European Union.\nThe objectives for the criteria were defined as follows:\nThe quality criteria should address issues of both supplier and user education: one document that simultaneously tells suppliers how to comply with key quality criteria and educates users as to what they ought to expect from a good health website;\nThe quality criteria should address both passive information-giving sites as well as sites that allow for transactions between service or information providers and users (i.e. information, products and services).\nThe quality criteria should facilitate compliance with EU Directives, other current guidelines, and technical standards relevant to this area.\nIt should be noted that the objective was not to develop a method for the implementation of such criteria at a European level. Although some actors in the field have called for an EU trustmark for health related websites which would operate in a way similar to the CE marking of certain goods [4,5], such initiatives are not within the ambit of the eEurope2002 action. They may, however, be considered within future eEurope action plans and other European programmes.\nQuality Criteria for Health Related Websites\nDeveloped in widespread consultation with representatives of private and public eHealth websites and information providers, other industrial representatives, public officials, and representatives of government departments, international organisations, and non-governmental organisations.\nThese criteria should be applied in addition to relevant Community law\nTransparency and Honesty\nTransparency of provider of site - including name, physical address and electronic address of the person or organisation responsible for the site (see Article 5 and 6 Directive 2000\/31\/EC on Electronic Commerce).\nTransparency of purpose and objective of the site\nTarget audience clearly defined (further detail on purpose, multiple audience could be defined at different levels).\nTransparency of all sources of funding for site (grants, sponsors, advertisers, non-profit, voluntary assistance).\nAuthority\nClear statement of sources for all information provided and date of publication of source.\nName and credentials of all human\/institutional providers of information put up on the site, including dates at which credentials were received.\nPrivacy and data protection\nPrivacy and data protection policy and system for the processing of personal data, including processing invisible to users, to be clearly defined in accordance with community Data Protection legislation (Directives 95\/46\/EC and 2002\/58\/EC).\nUpdating of information\nClear and regular updating of the site, with date of up-date clearly displayed for each page and\/or item as relevant. Regular checking of relevance of information.\nAccountability\nAccountability- user feedback, and appropriate oversight responsibility (such as a named quality compliance officer for each site).\nResponsible partnering - all efforts should be made to ensure that partnering or linking to other websites is undertaken only with trustworthy individuals and organisations who themselves comply with relevant codes of good practice.\nEditorial policy - clear statement describing what procedure was used for selection of content.\nAccessibility\nAccessibility- attention to guidelines on physical accessibility as well as general findability, searchability, readability, usability, etc.\nRelevant Community Law is listed in reference 3. Terms in italics are further discussed in the Glossary of Terms.\nIt should also be noted that while this Communication is addressed to the Member States of the European Union and private or public bodies operating in those States, due consideration should be given to the global nature of information disseminated through websites. Accordingly bodies adopting measures to implement the criteria should be aware of the fact that their information will be accessed by many individuals of different nations and cultures. In particular, attention should be paid to the fact that the developing world is a keen consumer of health information and that culturally specific content should be clearly identifiable as such.\nTextbox 1 sets out the resulting quality criteria; the ensuing text then explores some of the ways in which they may be implemented. This illustrates what could be done at national or regional level to promote high quality, accessible health related information to the European citizen. The table may easily be detached from the present text to form a simple reminder of the key Quality Criteria for Health related Websites.\nTailoring the Criteria For Different Types of Health Related Content\nThe criteria set out above are designed to be applicable to the development and maintenance of a health related site irrespective of the type of information or audience to whom the information is targeted. However, one essential quality criterion is that a health-related web site should state clearly what is its target audience and that care should be taken to ensure that both the style and nature of the information, and its presentation, are appropriate for the chosen audience. A number of the respondents to the consultation on the draft criteria, which was conducted between August and October 2001 via the eEurope website, identified the need to address not only site development and maintenance, but also the specific quality issues particular to health related content [6].\nWhen tailoring the content to a chosen audience, a number of factors should be borne in mind in addition to those set out above which should govern the construction of a site. These factors may be considered under the same broad headings as the general site development criteria:\nTransparency of Health Related Content\nTransparency of the health related objectives of the provider of the information, including the purpose and objective of content provision, should be clearly defined and stated.\nWhere advice or information on particular conditions, lifestyles or medications is given, funding from producers of products thereby implicitly or explicitly endorsed should be transparent to the site user.\nExisting Community legislation already contains information and transparency requirements. For example Article 5 of Directive 2000\/31\/EC on electronic commerce concerns the general information to be provided by an Information Society Services provider; Article 6 of Directive 2000\/31 which concerns additional information to be provided in the case of commercial communications which are part of or constitute an information society service and Article 10 of Directive 95\/46\/EC on the protection of individuals with regard to the processing of personal data and on the free movement of such data also applies.\nAuthority of Health Related Content Providers\nWhere a policy of using only accredited medical professionals to generate content is adopted, this should be clearly stated and adhered to.\nWhere a mixed group of content providers is used, (medical professionals, journalists, personal testimony, etc) the category of content provider of each item should be clearly identifiable.\nWhere scientific evidence is cited, the sources of such evidence should be easily identifiable to the user.\nWhere a medicinal product is recommended, EU legislation on Medicinal Product advertising should be adhered to, and any documents authorised by a regulatory authority should be made available to the site user.\nWhere advice is offered, the site provider should always include a reminder that internet based advice, whether personalised or not, cannot replace a face to face consultation with a healthcare practitioner.\nPrivacy and data protection of Health Data\nWhere any personal information is collected and further processed by the site user, including data processing invisible to the users, the requirements of Directive 95\/46\/EC on the protection of individuals with regard to the processing of personal data and on the free movement of such data, in particular article 8 on sensitive and health data, should be carefully assessed and full compliance assured.\nUpdating of Health Related Information\nWhere specific health related data are provided, the relevance of such content should be regularly verified.\nAccountability for Health Related Content\nWhere specific health related user feedback is provided by the site, particularly where personalised medical advice is offered, every effort should be made to ensure that such advice is bona fide and that advisors are suitably qualified to offer advice.\nAccessibility in Health Related Content\nWhere a particular type of audience is targeted (eg children), the presentation and content of information should be appropriate to the chosen target audience.\nThe use of a metadata labelling system may be used to make health data more findable. Such a system may also be used in conjunction with quality criteria to give higher ranking by search engines to those sites or pages labelled as complying with defined quality criteria.\nApply International or European standards, wherever possible, in order to facilitate notably the interoperability between different services and the cross-border provision of web based health services.\nImplementation of the Quality Criteria For Health Related Websites\nIssues for the European Community\nThe purpose of the eEurope 2002 action on Quality Criteria for health-related Websites was to encourage the adoption of a common set of basic quality criteria for such sites. The issue of whether and how these criteria might be implemented at European level was not within the terms of the action. The implicit assumption was that this was a matter to be addressed in Member States at national or regional level, making use of the wide range of private and not-for-profit organisations which are already operating systems for implementing quality criteria for health-related websites.\nIn view of the rapid increase in health-related websites in the European Union and the increase in the number of European Union citizens consulting such sites, it could be argued that there would be merit in the Community establishing its own system for implementing agreed quality criteria. Such a Community-sponsored system would however require considerable resources to set up and operate, and it is unclear that it would offer clear value added to the Member States. The Commission therefore considers that at the moment the difficulties inherent in a Community system would outweigh any possible advantages. Nevertheless, the issue of how and how effectively quality criteria are being implemented is of considerable significance at the European level. To ensure that European citizens have access to reliable health information on the Internet implies not only that there is a consensus on the necessary quality standards, but that those standards are satisfactorily implemented right across the European Union.\nThis does not mean that the same method of implementation should be used everywhere - indeed it must be doubtful that any particular mechanism would be appropriate in all circumstances and in all countries For example, in pharmaceuticals the Commission is considering ways of meeting the growing demand by patients to be able to access information directly about their medicines. The Commission has included proposals within the current review of EU pharmaceutical legislation, Review 2001, to take account of this growing demand. This has also been recently reinforced by the work of the High Level Group on Innovation and Provision of Medicines - G10 Medicines - which has covered this area in their reports.\nHowever, whatever system adopted, there should be clarity about the mechanisms being used in the different Member States and the extent of the involvement of the national and regional health authorities. With the forthcoming enlargement of the European Union, this requirement for transparency becomes even greater.\nSome Examples of Methods of implementing Quality Criteria\nSimple Codes of Conduct\nA number of organisations have adopted an approach similar to that described in this Communication, of setting up meetings and consultations between experts in order to establish by consensus a set of quality criteria. The eHealth Code of Ethics adopted in May 2000 by the Internet Health Coalition [7] is perhaps the best known of such `codes of conduct'.\nThe object of this and other similar codes is to offer a process of self-assessment by health site providers. However, a basic code of good conduct, or set of quality criteria will form the basis of all the approaches described below.\nThe way in which such codes are implemented varies. Where the code is adopted by an umbrella organisation, such as the Pharmaceutical Group of the European Union, then the organisation itself seeks to ensure that all members comply with the code. In other cases a code has been adopted for the purposes of in-house application only, as in the case of the American Medical Association. Although other organisations may cite the code, and claim to use it, the organisation developing the code makes no attempt to ensure that other parties are in fact implementing it.\nThe costs of the code of conduct approach are generally rather low, requiring only an initial outlay on meetings to draw up the code. However, the benefit of such codes can also be rather limited given the absence of effective enforcement mechanisms.\nA code of conduct which addresses issues concerning the protection of personal could in itself form a Code of Conduct relevant to a specific area of practice as foreseen in Article 27 of Directive 95\/46\/EC on Data Protection. Any such draft community code, and amendments or extensions to existing Community codes, may be submitted to the Working Party established by Article 29 of Directive 95\/46\/EC on data protection. Similarly, a Code of Conduct which addresses the particular issues of electronic commerce in the health domain may be drafted in accordance with the framework foreseen in Article 16.1 of the Directive 2000\/31\/EC on Electronic Commerce.\nSelf Applied Code of Conduct or Quality Label\nA next step in the implementation of a code of conduct can be characterised as the self applied quality label. In such a case a third party organisation develops a code of conduct and allows those who undertake to abide by the terms of the code to display a label, seal or logo which certifies compliance with the code.\nThe oldest, and perhaps best known, of such labels is the Health on the Net Foundation (HON) [8] label whose eight point set of quality criteria is currently used by more than 3000 internet sites worldwide. A site provider wishing to use the HON label has to make a formal application and a commitment to strictly observe all the HON code principles. Compliant sites identify themselves by the HON code hyperlink (or \"active\") seal displayed at a prominent location. The seal is termed `active' because clicking on it links the user to the HON site. Conformity with the HON code principles is verified by the team of checkers at HON. HON cannot prevent dishonest operators from simply cutting and pasting the HON code seal onto their Web sites in a bid to enhance their credibility. It does, however, conduct random checks on subscribers to ensure they remain compliant with the HON code. By way of additional policing, the Internet community is invited to report misuse of the label.\nThe costs of this system of applied labelling are not very high, requiring a relatively small team to process applications for use, maintain random checks of sites displaying the label and respond to any reports of misuse. The benefits may be significant in drawing to the attention of users the importance of the criteria inherent in the label. However, the benefits must be weighed against the requirement of the users of the sites to understand the nature of the label, and perhaps more importantly, to care about its aims and objectives.\nUser Guidance Tools\nA further application of the code of good conduct takes the form of a user guidance tool. In this case compliance with a code is demonstrated not by a label, but by a link to a guidance tool which invites the user to check for him or herself if a site and its contents comply with pre-set criteria.\nA typical such tool is displayed by the site as a logo on which the user may click to reveal a series of questions with which to interrogate the site so as to assess whether the information offered is trustworthy. Such tools may be specific to a particular type of information, such as DISCERN [9] which provides a brief questionnaire through which users gain a valid and reliable way of assessing the quality of written information on treatment choices for a health problem. Other tools seek to give guidance on the trustworthiness of any health-related information. An example of this is NETSCORING [10], which uses a questionnaire of 49 criteria falling into eight categories: credibility, content, links, design, interactivity, quantitative aspects, ethics, and accessibility. Yet other tools are targeted at particular categories of internet users. For example, the QUICK [11] tool seeks to provide children with a step-by-step guide to assessing health related information on the internet.\nWhile such tools are frequently adopted for the guidance of users by national health portals (such as National Health Service DIRECT in England and Wales), they may also be used as site development tools by authors and publishers of information since they define the standards which users are entitled to expect.\nThe financial costs of the user guide are low, often not extending beyond the initial development costs. However, the burden of the use of this kind of tool falls on the internet user, mostly because of the time it takes to apply, which reduces the incentive to use it.\nFiltering tools\nWhere a guidance tool is provided by a third party to a user to apply for him or herself, a filtering tool is applied to provide a searchable database of filtered and accredited information. Such filtering tools are often based on the gateway approach to organising access to Internet. The fundamentals of this approach are that Internet resources are selected for their quality and relevance to a particular target audience. They are then reviewed and resource descriptions created, which are stored, generally with the associated metadata, and generally in a structured database. The consequence of this effort is to improve the recall, and especially the precision, of Internet searches for a particular group of users.\nAn example of this type of tool is found in the OMNI site (Organising Medical Networked Information) [12] which provides a gateway to evaluated, quality Internet resources in health and medicine, aimed at students, researchers, academics and practitioners in the health and medical sciences.\nThe costs of such a filtering tool are relatively high in that a team of trained experts must be employed to search for, abstract and classify information on the internet in order that it may be entered into the database. The benefits of such a tool, for the initiated user, are also high since it provides a valuable shortcut to individual searches of the internet using non-specific search engines.\nThird Party Quality and Accreditation Labels\nThe most advanced, and also most costly, of the mechanisms available for implementing quality criteria for health related websites, is the third party accreditation system. A third party issues a label to certify the compliance of the site with the criteria of evaluation.\nA range of implementations fall into this category, from lower cost intra-organisation bodies for quality certification, acting in a similar to the notified bodies used in CE marking, to high cost external independent assessors who perform audits and grant accreditation.\nAt present no third party accreditation bodies are fully operational in Europe, although two noteworthy pilots are running in MEDCERTAIN (a demonstration project of the European Union \"Safer Internet Action Plan\") and TNO QMIC, a pilot study of the Netherlands Organisation for Applied Scientific Research.\nIn the case of MEDCERTAIN [13] a series of levels of accreditation are envisaged, starting with a self-certification label in which the provider of the site uses the MEDCERTAIN metalabelling system which incorporates a machine read language to describe and evaluate health information on the Internet. These labels are then in turn used to place a given item of site correctly within a gateway system, such as the OMNI system described above. The next level envisaged by MEDCERTAIN is one in which non-medical experts personally check the site for compliance with the level 1 tagging and also against the agreed set of quality criteria. The highest level involves medical assessment of the content and a rating of the content by relevant healthcare professionals.\nThe QMIC [14] system, on the other hand, envisages a system similar to the ISO 9000:2000 standard. The QMIC system is based on a complex set of standards drawn up by the third party (TNO in this case) but implemented by the site provider through an internal `quality certification body' who is in turn regularly assessed by the third party organisation to ensure that it is performing its function of internal quality assurance properly. The site, once duly assessed by the internal notified body is then admitted to a portal maintained by the third party who undertakes to ensure that the sites linked into the portal are applying the internal quality assurance system with due care.\nPurposes of Implementation of Quality Criteria For Health Related Websites\nThe general purpose of any quality initiative, whatever method of implementation is chosen, must be the protection of the consumer. However, in some cases that general purpose may be best achieved through educating the user of the service while in other cases the provider of the service will be the target of the quality initiative. In order to assist in the selection of an appropriate implementation method, the targeted purposes of the various methods are examined in more detail below\nEducating Users\nIn their daily lives as consumers of information delivered via the traditional media, most people learn to use a wide range of assessment tools: judging the nature of the outlet providing the information (a general or specialist bookshop or a work exclusively available from the author), the look and feel of the publication as a whole (a magazine with several contributions or a one page pamphlet). In addition, most people know whom to contact for further information (librarian, bookshop assistant, publisher).\nIn the world of internet content, however, it is less evident what are the relevant indicators of quality. It is for this reason that quality marks and user guides have proliferated, namely to educate the consumer and to provide a recognisable \"quality\" label which site creators may use to promote their sites. Accordingly, for such codes to be effective it is highly important that the public are informed about the existence of the Codes through public education campaigns.\nAssisting searchers\nThe purpose of quality marks is not, however, simply to provide access to qualified information but also to assist the citizen in coping with the torrent of information which a search on a health related subject might produce: it has been said that \"trying to get information form the internet is like drinking from a fire hose. You don't even know what the source of the water is\" [15]. In order to try to manage the flow from the fire hydrant into a steady stream from a tap, some organisations have developed and applied tools for rating web sites in order that they may offer pre-selected and more easily searchable sources to their consumers (see for example OMNI or MEDCERTAIN).\nEducating Site Providers\nThe problem is not only with the torrent of information, but also with the behaviour of its purveyors. Whilst it may take considerable effort to find an outlet for unusual or extreme ideas in the traditional media, virtually anyone with a modicum of computer skills and very little money can create their own website. The objective of many of the code of conduct initiatives is therefore to educate both the providers and consumers of information about the processes and good practices that a website should be able to demonstrate.\nIn order to educate not only the provider, but also the consumer of information a further set of actors have developed a wide range of user assessment tools. Such tools are usually in the form of on-line check lists which ask the consumer to check off types of information as they find them: statement of aim, explicit statement of source of information, explicit date of information, etc. Some may be rather short (HON), some quite detailed (NETSCORING); some are aimed at specific markets (DISCERN - for treatment choices) and some aimed at children (QUICK) to mention but a few.\nAssuring Quality\nMost of the organisations publishing and administering such codes operate on a simple selflabelling processes in which the site provider undertakes to follow the code and in return displays its \"trustmark\" relying on spot check and vigilant users to identify those who are not complying with the given code of conduct. While this may not be as effective as a fully policed trustmark system of the type we are used to seeing as regards, for example, electronic products, it nonetheless addresses a need in a reasonably effective manner.\nConclusions\nThe eEurope initiative was launched by the European Commission on 8th December 1999, with the adoption of the Communication `eEurope - An Information Society for all (COM (1999), 687 final, of 8.12.1999)'.\nThe \"eEurope 2002 Action Plan - An Information Society For All\", was adopted by the Commission on 14th June 2000, and politically endorsed by the European Council in Feira (Portugal) on 19-20 June 2000. It detailed the policy actions which are required to meet these objectives by 2002.\nThe eEurope 2005 Action Plan (COM (2002) 263 final, of 28.5.2002), was adopted by the Commission on 28 May 2002 and politically supported by the European Council in Sevilla (Spain) on 21 - 22nd June 2002. It, notably, set the objective for Europe to have, by 2005, \" Modern online public services\". To achieve this objective, one of the proposed actions is to promote e-health services. It also commits the Commission to monitor \" actions taken by Member States to make health information as accessible as possible to citizens as well as initiatives to implement quality criteria for web sites\".\nIn this respect, the e Europe 2005 Action Plan affirms that \" it is critical that e-health content and services are developed efficiently, are available for all and health related web sites comply with established quality criteria\".\nWith respect to the enlargement of the European Union it should also be noted that the eEurope+ Action Plan, which was adopted by the accession States to mirror the eEurope 2002 Action Plan, includes similar action on quality criteria for health related websites.\nAccordingly it will also be important to monitor the activities undertaken pursuant to that Action Plan.\nThis Communication sets the scene for the implementation of a core set of quality criteria in Member States for health related web sites, within the context of the relevant existing Community legislation (as listed in footnote 3) and in accordance with the requirements of that legislation. The set of quality criteria is based upon a broad consensus among specialists in this field, health authorities, and prospective users. It is now to be expected that national and regional health authorities, relevant professional associations, and private medical website owners will:\nimplement the Quality Criteria for Health Related Websites in a manner appropriate to their website and consumers.\ndevelop information campaigns to educate site developers and citizens about minimum quality standards for health related websites.\ndraw on the wide range of health information offered across the European Union and localise such information for the benefit of citizens (translation and cultural adaptation).\nexchange information and experience at European level about how quality standards are being implemented.\nFinally, within the context of the Information Society activities and as part of the implementation of the European Union public health programme, consideration will be given to the possibilities of developing and operating a joint action, with the plans drawn up under eEurope, to improve availability to the general public on the Internet of information on health matters, and considering the possibilities for establishing a system of recognizable Community seals of approval for Internet sites.","keyphrases":["guidelines","privacy","quality assurance","internet\/standards","ethics, professional","social control, formal","health care quality","health care\/standards","commerce\/standards","information management\/standards","medical informatics\/standards","quality control","informed consent"],"prmu":["P","P","P","R","R","M","R","R","R","R","M","M","M"]}
{"id":"J_Mol_Biol-1-5-1892151","title":"Restriction Endonucleases that Bridge and Excise Two Recognition Sites from DNA\n","text":"Most restriction endonucleases bridge two target sites before cleaving DNA: examples include all of the translocating Type I and Type III systems, and many Type II nucleases acting at their sites. A subset of Type II enzymes, the IIB systems, recognise bipartite sequences, like Type I sites, but cut specified phosphodiester bonds near their sites, like Type IIS enzymes. However, they make two double-strand breaks, one either side of the site, to release the recognition sequence on a short DNA fragment; 34 bp long in the case of the archetype, BcgI. It has been suggested that BcgI needs to interact with two recognition sites to cleave DNA but whether this is a general requirement for Type IIB enzymes had yet to be established. Ten Type IIB nucleases were tested against DNA substrates with one or two copies of the requisite sequences. With one exception, they all bridged two sites before cutting the DNA, usually in concerted reactions at both sites. The sites were ideally positioned in cis rather than in trans and were bridged through 3-D space, like Type II enzymes, rather than along the 1-D contour of the DNA, as seen with Type I enzymes. The standard mode of action for the restriction enzymes that excise their recognition sites from DNA thus involves concurrent action at two DNA sites.\nIntroduction\nRestriction\u2013modification (R-M) systems can be classified into four main groups on the basis of their genetic and polypeptide organisations, their modes of action and their co-factor requirements: Types I, II, III and IV.1,2 Most bacterial genomes encode multiple R-M systems: about 35% of the total are Type I systems, 40% Type II, 10% Type III and 15% Type IV.3 The restriction enzymes from the Type I and the Type III systems are ATP-dependent proteins that initiate DNA translocation at specific recognition sites and then cleave DNA some distance from the sites, usually upon collision of two complexes translocating from separate sites.4,5 Type I restriction enzymes normally require two recognition sites in cis to cleave DNA, though they can cleave circular DNA with one site, while the Type III nucleases almost always need two sites.6,7 The Type IV systems are GTP-dependent enzymes that restrict DNA that has been methylated at two or more sites.5,8 The endonucleases from the Type I, III and IV families thus constitute \u223c60% of the restriction enzymes found in nature, and they all interact with two target sites before cleaving DNA.9\nWhile the Type I and Type III systems feature multi-subunit proteins with both endonuclease and methyltransferase activities,4,5 Type II R-M systems generally comprise two separate proteins, one for each activity,10 though in some cases both functions are fused into a single polypeptide.11,12 Unlike the NTP-dependent systems, the Type II endonucleases cleave DNA at fixed positions in or near their recognition sites.3 Most (but not all)13 need Mg2+ as a cofactor,10,14 though some of the fused proteins additionally need the methyl donor S-adenosyl methionine (AdoMet) not only for methyltransferase but also for nuclease activity.11,12 The standard Type II restriction enzymes, such as EcoRV and BamHI, are homodimers that recognise palindromic sequences, typically 4\u20138\u00a0bp long, and cut both strands at specified positions within the sequence.14,15 They cleave DNA with multiple sites by means of separate reactions at each site.16\u201318\nOf the \u223c4000 Type II restriction enzymes identified to date3 many differ from the standard and have been classified into various sub-types.2 One tenet of this classification is their mode of action at multiple recognition sites. Numerous Type II enzymes, maybe 50% of the total,9,18 cannot cleave DNA without first interacting with two copies of their sites, either looping out the DNA between two sites in cis on the same chain of DNA, or bridging sites in trans on separate molecules of DNA.19\u201323 The enzymes that need two sites can be identified by comparing their activities on DNA substrates with one or two copies of the requisite sequence.24,25 Under most circumstances, an enzyme that binds two DNA loci has a higher affinity for sites in the same chain over sites in separate molecules,9 so will usually cleave two-site DNA more rapidly than one-site DNA.24 However, an enzyme that needs two sites but which has a high affinity for its site may have sufficient affinity for sites in trans to give its Vmax on a DNA with one site.25 Nevertheless, DNA\u2013protein interactions are weakened at elevated ionic strengths so even if an enzyme gives equal rates on the one and two-site substrates at low ionic strength, it may cleave the two-site DNA faster than the one-site DNA at raised ionic strength.16,18,26 Alternatively, these enzymes can be identified from whether they can be activated to cleave plasmids with one target site by adding oligonucleotide duplexes that also carry the target sequence, as a result of interactions in trans between plasmid and oligoduplex.20,25,27\nThe Type II restriction enzymes that need two sites have been categorised into different sub-types on the basis of how they cleave DNA with two or more copies of their site.2 The Type IIE enzymes, such as EcoRII, NaeI and Sau3AI,27\u201329 have two (or more)30 dissimilar DNA-binding clefts: one has the catalytic functions for cleaving the recognition sequence but is inactive unless a second copy of the sequence is bound to an activator cleft. The Type IIF enzymes, such as SfiI, NgoMIV and SgrAI, function as tetramers with two identical DNA-binding clefts, each made from two subunits22,31 but they are virtually inactive unless both clefts are filled with cognate DNA.24,32 Consequently, while both Type IIE and IIF enzymes cleave two-site substrates more rapidly than one-site substrates, Type IIE enzymes cut two-site DNA initially at just one site and then, in a much slower reaction, the second site.33 In contrast, Type IIF enzymes act concertedly, to give as their initial product DNA cut at both sites.22,24,34,35\nType II restriction enzymes have also been categorised on the basis of the positions at which they cleave the DNA relative to their recognition sequences.2 While many Type II enzymes cleave within palindromic sites, the Type IIS nucleases recognise asymmetric sequences and cleave both strands at specified positions on one side of the site.3 For example, FokI cleaves DNA downstream of the sequence GGATG, 9 and 13 bases away in \u201ctop\u201d and \u201cbottom\u201d strands, respectively. Its recognition site is thus usually noted as GGATG(9\/13). Most Type IIS nucleases, including FokI, have very low activities against DNA with one cognate site, and become active only after interacting with two sites.36 However, they vary in their modes of action at two DNA sites: some cut just one strand at one site,13 others both strands at one site, like Type IIE enzymes,36\u201338 while further examples cut both strands at both sites, like Type IIF enzymes.39,40\nCategorisation of R-M systems from their positions of DNA cleavage yields a further subset,2 the Type IIB enzymes (Table 1) exemplified by BcgI.41\u201345 The Type IIB systems have bipartite recognition sequences that are usually asymmetric though some, such as BplI and FalI, have palindromic sites (Table 1).3 Their recognition sites look like sites for Type I rather than Type II systems but the Type IIB systems belong unambiguously to the II family because their endonucleases cut DNA at specified rather than random positions. Even so, the similarities between the Type I and the IIB systems extend to their functional organisations.42,44,46\u201348 Like Type I enzymes,4 each IIB system features a multi-functional protein with both methyltransferase and endonuclease activities. BcgI contains two polypeptides, A and B, in a 2:1 ratio.42 The A subunit has the amino acid sequences for both methylation and cleavage activities but by itself has neither activity. The B subunit resembles a DNA specificity subunit from a Type I enzyme and is thus likely to direct the protein to its recognition site.44 Several other Type IIB enzymes contain two subunits akin to the A and B subunits of BcgI,49 though in BplI these are at a 1:1 rather than a 2:1 ratio.50 In contrast, some IIB enzymes carry the endonuclease, the methyltransferase and the DNA recognition functions in three separate blocks of amino acid sequence within a single polypeptide.46\u201348 The IIB systems all require AdoMet for DNA methylation and Mg2+ for DNA cleavage but, as is common among combined restriction\u2013modification proteins, many also need the methylation cofactor for their nuclease activity (Table 1).43\nThe positions of DNA cleavage by Type IIB nucleases are, like Type IIS enzymes, fixed distances from their recognition sites, 7\u201315 bases away depending on the system in question (Table 1).3 While the Type IIS enzymes make a double-strand break on one side of the site, the Type IIB nucleases make double-strand breaks on both sides of their sites,41,46\u201350 so cutting a total of four phosphodiester bonds at each site. They thus release their intact recognition sites from the remainder of the DNA on a short fragment, 30\u201335\u00a0bp long depending on the enzyme (Table 1). Perhaps as a result of remaining bound to its 34\u00a0bp product, the BcgI nuclease functions stoichiometrically, completing just one turnover, rather than catalysing multiple turnovers.42\u201345 However, the complete cleavage of a plasmid with two BcgI sites required less of the BcgI protein than a plasmid with one site, so it was suggested that BcgI interacts with two copies of its target sequence before cutting DNA.45 Many restriction enzymes have to bridge two sites before they can cleave DNA but it has yet to be determined whether this is a general characteristic of the Type IIB endonucleases, or unique to BcgI. This question was examined here by analysing the reactions of several Type IIB restriction proteins on DNA molecules that have either one or two copies of the relevant recognition sequence. The enzymes were from commercial suppliers, at concentrations given in terms of units (U) of activity rather than molarity, and were initially studied under the conditions advised by the supplier (Table 2).\nResults\nBcgI\nPrevious studies had shown that the amount of BcgI protein needed to cleave a plasmid with two BcgI sites (pBR322) was smaller than that required to cleave a plasmid with one site (pUC19), so it was suggested that BcgI needs to interact with two copies of its recognition site to cut DNA.45 However, the activities of restriction enzymes can be affected by non-specific sequences flanking the specific site and, for enzymes with bipartite recognition sequences as is the case with BcgI (Table 1), by the sequence of the spacer within the site.17,51\u201353 While one of the two BcgI sites in pBR322 is the same as that in pUC19, the other differs in both flanking and spacer sequences. The enhanced activity of BcgI on pBR322 could thus be due to the context of the novel site. Furthermore, the different amounts of enzyme needed to cleave these two plasmids may reflect different affinities for each DNA rather than different reaction rates. A derivative of pUC19 was therefore constructed that had two BcgI sites, both in the same sequence context as the pUC19 site: pDG5 (Supplementary Data, Figure S1).18\nWhen tested at a concentration of BcgI that resulted in the cleavage of almost all of both one-site and two-site plasmids, the initial rate for the reaction on pUC19 was found to be about sixfold lower than that on pDG5 (Table 3). The difference in reaction rates cannot be due to either sequence differences surrounding the specified bp of the recognition sequence, nor to insufficient enzyme for complete cleavage of either DNA. Instead, the data support the previous proposal45 that BcgI interacts with two copies of its recognition site, preferentially in cis, before cutting the DNA.\nProteins can mediate long-range communications between distant DNA sites by either tracking along the 1-D contour of the DNA from one site to the other without losing contact with the DNA; or by binding concurrently to both sites upon the juxtaposition of the sites in 3-D space, so looping out the intervening DNA.9,18 These routes can be distinguished by comparing a DNA catenane containing two interlinked circles with one site in each ring to a single circle of DNA with two target sites.33\u201335,54 The catenane cannot support 1-D tracking from one site to the other54 but, since the 3-D distance between two sites in the separate rings of a supercoiled catenane will be similar to that between two sites in a supercoiled plasmid,55 the catenane should be as effective as the two-site plasmid for 3-D looping.33 This strategy was applied previously to several other restriction enzymes with the plasmid pMLE2,34,35 a DNA with two target sites for numerous Type II enzymes, including BcgI, interspersed with two directly repeated res sites from the transposon Tn21. It can be converted by Tn21 resolvase into a catenane containing two interlinked rings of DNA with one BcgI site in each ring.\nInitial rates for the reactions of BcgI on the parental plasmid with two BcgI sites (Figure 1(a)) and on the catenane with one BcgI site in each ring (Figure 1(b)) were measured from the decline in the concentration of the supercoiled substrates with time: the initial rates on the plasmid and on the catenane, 0.40\u00a0nM\/min and 0.37\u00a0nM\/min, respectively, were almost identical and in both cases considerably faster than that of 0.07\u00a0nM\/min on a plasmid with one BcgI site under comparable conditions (data not shown). Moreover, almost all of the catenane was cleaved by BcgI in a highly concerted manner to give directly the final products with double-strand break(s) in both rings: neither the nicked forms, that would have arisen from single-strand breaks in one or both rings, nor the individual circles, from double-strand break(s) in the other ring, accumulated appreciably during the course of the reaction (Figure 1(b)). Hence, BcgI must bridge two copies of its recognition sequence through 3-D space and then introduce at least one double-strand break at each site before releasing the DNA.\nAloI, BaeI and BplI\nTo examine whether other Type IIB endonucleases need to interact with two sites, a series of plasmids were constructed with either one or two copies of their recognition sequences (Supplementary Data, Figures S1\u2013S3). Most of the constructs involved cloning a duplex of synthetic oligonucleotides at one location in the vector, to give a plasmid with one target site for each enzyme: then inserting into that plasmid at a separate location a second copy of the duplex, to give a plasmid with two copies of each target. In these cases, the recognition sites for the test enzyme on the one-site substrate, and both of its sites on the two-site substrate, were all within the same sequence context. On the two-site substrates made here, the distances between the pairs of sites varied from 673\u00a0bp to 1288\u00a0bp (Table 3; on pDG5, the BcgI sites are 1810\u00a0bp apart). As the reactions were carried out on supercoiled plasmids, this variation is not significant. In a supercoiled plasmid, the mean separation of sites in 3-D space is only marginally affected by their 1-D contour separation along the DNA.56 Furthermore, even the shortest separations span several persistence lengths, thus nullifying any effect due to twisting and bending of the intervening DNA.9 A further consideration for the test plasmid is that proteins capable of bridging two asymmetric DNA sequences are often affected by the orientation of the sequences: some prefer directly repeated sites, others sites in inverted orientation.40 This variation was removed by using as the new two-site substrates only plasmids with inverted sites.\nInitial tests were carried out on three Type IIB enzymes, AloI, BaeI and BplI, as these embody several different aspects of the IIB systems (Table 1). Both BaeI and BplI contain two polypeptide chains, A and B, but these are present in BplI in a 1:1 rather than the 2:1 ratio seen with BcgI.49,50 Conversely, AloI is composed of a single polypeptide that carries endonuclease, methyltransferase and DNA recognition functions.47 BplI also differs from most Type IIB enzymes in having a palindromic recognition site while both BaeI and AloI have asymmetric targets, as is usual amongst IIB enzymes. Indeed, the recognition sequence for BaeI is the most asymmetric of the IIB sites; one segment of its bipartite sequence is 5\u00a0bp long, the other only 2\u00a0bp (Table 1). A plasmid with solitary recognition sites for AloI, BaeI and BplI, pJM1, was constructed and this used in turn to make a plasmid, pJM2, with two sites for each of these enzymes. Both AloI and BaeI cleaved the plasmid with two copies of their respective sites more rapidly than the plasmid with one copy (Figure 2(a) and (b)): fourfold faster in the case of AloI, similar to BcgI; by a larger factor, 13-fold, in the case of BaeI (Table 3). In contrast, BplI cleaved the supercoiled plasmids with one and two BplI sites at identical rates (Figure 2(c)).\nAnother strategy to determine whether a restriction enzyme needs two sites is to see if it can be activated to cleave a plasmid with one cognate site by an oligoduplex that has the recognition sequence.20,27 Interactions spanning separate molecules of supercoiled DNA are generally disfavoured so the interaction in trans needed to cleave the one-site plasmid can, at an appropriate concentration of duplex, be achieved more readily between plasmid and duplex than between two molecules of plasmid.24,25 However, higher concentrations of the duplex will inhibit plasmid cleavage as the enzyme becomes fully occupied with the duplex.24,39 When this test was applied to BaeI (Figure 3(a)), a specific oligoduplex with the cognate sequence for BaeI enhanced the cleavage of the plasmid with one BaeI site when present at relatively low concentrations, but abolished plasmid cleavage at higher concentrations. A control non-specific duplex lacking the sequence for BaeI failed to activate cleavage of the one-site plasmid, though high concentrations of this duplex led to a small reduction in plasmid cleavage, presumably due to the non-specific sequence acting as a weak inhibitor. Similar results were obtained when specific and non-specific duplexes were added to AloI reactions (data not shown). With BplI (Figure 3(b)), the non-specific duplex again had no effect on the cleavage of the one-site plasmid but in this case the specific duplex with the BplI site acted solely as an inhibitor of plasmid cleavage: no activation was observed at any duplex concentration tested.\nTaken together, the above data show that both AloI and BaeI need to interact with two copies of their recognition sites in order to cut DNA. The faster reaction rates of these enzymes on the two-site plasmid, relative to the one-site DNA, are likely due to the intrinsic preference for interactions in cis, spanning sites in the same molecule of DNA, over interactions in trans, across separate molecules. Nevertheless the reactions of both AloI and BaeI on the one-site plasmid could be activated by adding a duplex with the requisite recognition sequence. These two enzymes are therefore not limited to sites in cis, but can also become active by binding two sites in trans. In contrast, the reactions of BplI on one and two-site plasmids, and the effect of a specific duplex on its one-site reaction, all match the expectations of a restriction enzyme that acts at individual sites, like EcoRV or BglI.17\u201319\nOther Type IIB enzymes\nThe above experiments raise two possibilities. One is that the requirement of two recognition sites, as seen with BcgI, BaeI and AloI, is a general property of the Type IIB enzymes and that BplI is a solitary exception. The other is that the requirement for two sites is shared by only a fraction of the Type IIB enzymes and that BplI represents a major portion of these enzymes. Further plasmids with one or two copies of the requisite recognition sites were constructed to test most of the other Type IIB enzymes currently available (Table 1): AlfI, BsaXI, CspCI, FalI, PpiI and PsrI. When tested under the recommended reaction conditions for the enzyme in question (Table 2), four out of the six additional enzymes, AlfI, CspI, FalI and PpiI, cleaved their two-site substrate at significantly faster rates than their one-site substrate: by factors that varied from sixfold with AlfI and FalI to 43-fold with CspCI (Table 3).\nAlfI and FalI recognise palindromic sequences, a feature of the BplI system but not of many other Type IIB systems (Table 1). Hence, Type IIB enzymes with palindromic sites do not necessarily act like BplI. The reactions of FalI on the plasmid with one FalI site were also examined in the presence of varied concentrations of oligoduplexes with and without its recognition sequence (data not shown). The specific oligoduplex activated FalI-cleavage of its one-site substrate, exactly like BaeI (Figure 3(a)), while the non-specific DNA had no effect apart from a slight inhibition at high concentrations, again like BaeI. FalI is therefore a Type IIB enzyme with a palindromic recognition sequence but which has to interact with two copies of its sequence to cut DNA, as is also AlfI (see below; Figure 5(a)).\nUnder their standard reaction conditions, the remaining two enzymes, BsaXI and PsrI, cleaved their two-site plasmids at rates that were \u22642-fold faster than those on their one-site substrates (Table 3; Figure 4(c)). A restriction enzyme that acts at individual sites will cleave a two-site DNA twice as fast as a one-site DNA if the DNA concentration is above that of the enzyme but below the Km value, simply due to the twofold difference in substrate concentration (i.e. recognition sites). Alternatively, if present at a >2-fold excess over the DNA, an enzyme that acts at individual sites will again carry out its initial reaction on a two-site DNA twice as fast as on a one-site DNA, as two molecules of the enzyme can then act simultaneously on the two-site DNA, one at each site. However, Type II restriction enzymes that need two sites often cleave their one and two-site substrates at similar rates in reactions at low ionic strength, but cleave the two-site DNA faster than the one-site DNA at high ionic strength.16,18,26,39 Consequently, to find out why BsaXI and PsrI cleave their one and two-site substrates at similar rates, and why BplI cleaves its equivalent substrates at identical rates, the reactions of these three enzymes were studied in buffers of varied ionic strength (Figure 4; Table 3).\nThe recommended reaction buffer for BplI contains 66\u00a0mM KOAc (Table 2) and, under these conditions, BplI cleaved to completion its one and its two-site substrates at identical rates (Figure 2(c)). An increase in the concentration of KOAc to 200\u00a0mM again allowed complete cleavage of both substrates, even more rapidly than at 66\u00a0mM, but a further increase to 800\u00a0mM led to incomplete cleavage of both substrates (Figure 4(a) and (b)). However, at all KOAc concentrations tested, the progress curves for the BplI reactions on its one and two-site substrates were superimposable. Similar results were obtained when the ionic strength was varied by adding NaCl instead of KOAc: the addition of NaCl affected both the rates and amplitudes of the BplI reactions, but NaCl had the same effect on the one-site and the two-site reactions (data not shown). In contrast, while PsrI had cleaved its two-site DNA at a marginally higher rate than its one-site DNA in a reaction buffer with 66\u00a0mM KOAc (Figure 4(c)), it cleaved the two-site DNA at a considerably faster rate than the one-site DNA in the presence of 200\u00a0mM KOAc (Figure 4(d)): 12-fold faster instead of the twofold difference under standard conditions (Table 3). For BsaXI, the ratio of its reaction rates on its two and one-site substrates likewise increased as the KOAc concentration was raised: from 1.6-fold at 50\u00a0mM to fourfold at 200\u00a0mM (Table 3).\nModes of action\nApart from BplI, all of the Type IIB enzymes tested here are capable of cleaving plasmids with two cognate sites more rapidly than plasmids with a single site, so they must act on the two-site DNA by bridging sites in cis. Among the restriction enzymes that bridge DNA sites in cis, the Type IIE systems first cleave one site and then in a separate reaction the other, often at a much slower rate.20,21 In contrast, the Type IIF enzymes act concertedly on two-site substrates and liberate as their initial product the DNA cut in both sites, bypassing intermediates cut at one site.19,22,24 These mechanisms can be distinguished by examining the transient intermediates formed during their reactions on a supercoiled (SC) DNA with two recognition sites.9,25,33 Such a DNA can be cleaved to give, successively: the open-circle (OC) form cut in one strand at one or both sites; the full-length linear (FLL) form cut in both strands at one site; and, as the end products, the two linear fragments after cutting both sites. If a restriction enzyme cleaves the DNA by means of separate but kinetically equal reactions at each site, the amount of the FLL form should rise to a maximum of 40% of the total DNA before declining, upon cleavage of the second site.17,57 If it cleaves one site more rapidly than the other, as is the case with Type IIE enzymes,30,33 the amount of the FLL form will reach a maximum of >40% of the total. Conversely, if the enzyme first cuts one site at a slow rate and then the second site at a faster rate, as is the case with the Type IIF enzymes,16,24 the maximal amount of FLL DNA will be <40% of the total. Three examples of this strategy are shown: AlfI (Figure 5(a)), CspCI (Figure 5(b)) and BcgI (Figure 1(a)).\nIn all three examples, and also with all of the other Type IIB enzymes examined here apart from AloI (data not shown), their reactions yielded none of the OC form of the DNA that would have arisen if products cut in one strand, at one or both sites, had accumulated during the reaction. A SC DNA with two recognition sites for a Type IIB enzyme has eight scissile phosphodiester bonds, in top and bottom strands on either side of both sites. Cleavage of any one of these eight bonds will convert the SC to the OC form but if the next phosphodiester bond to be cleaved is chosen at random from the seven remaining targets, six out of the seven leave the DNA in its OC state and only one, that opposite the initial nick, will result in the OC form being converted to the linear product with one double-strand break. Hence, Type IIB enzymes might be expected to generate large amounts of the OC intermediate before creating any linear product. The observation that no OC DNA accumulated shows instead that, after cutting one strand at a single locus, Type IIB enzymes must cut the second strand at that locus very shortly thereafter, within the lifetime of a single DNA\u2013enzyme complex.\nThe reaction of the AlfI endonuclease on its two-site substrate yielded not only, as noted above, none of the OC form but also very little of the FLL form (Figure 5(a)). (No distinction is made here between the products from bilateral as opposed to unilateral cleavages at individual sites: see Materials and Methods.) The amount of FLL DNA reached a maximum at about 15% of the total, far below the value of 40% expected for kinetically equal reactions at each site. The DNA cut at one AlfI site is therefore almost always cleaved at the other site before being liberated. Hence, after bridging two copies of its recognition sequence, AlfI acts concurrently at both sites in a highly concerted manner. The reaction profile of AlfI is thus reminiscent of SfiI and the other Type IIF restriction enzymes.16,24,33 Several other Type IIB enzymes also converted major fractions of their two-site substrates directly to the final products with double-strand breaks at both sites, liberating none of the OC and only small amounts of the FLL intermediate: they included FalI, PpiI and PsrI (data not shown). AlfI and the other Type IIB enzymes that act in this manner therefore fall into the Type IIF category.\nOn the other hand, the CspCI endonuclease converted the SC substrate with two CspCI sites first to the FLL form and only later, after a lag phase, to the two linear products cut at both sites: as before, intermediates cut in one strand were not detected (Figure 5(b)). Hence, CspCI first cuts both DNA strands at one recognition site, and then in a separate reaction, creates one or more double-strand breaks at the other site. The amount of FLL DNA formed during the CspCI reaction reached a maximum of \u223c40% of the total DNA. This level matches the expectation for a restriction enzyme that cleaves a two-site substrate in separate but equal reactions at each site.17,57 AloI, BaeI and BsaXI also cleaved their two-site substrates to give first the FLL linear form, again to a maximal yield of \u223c40% of the total, and only later the linear fragments with double-strand breaks at both sites. Though these enzymes all need to bind two copies of their recognition sites to become fully active, they cleave each site in separate reactions.\nIn the reaction of BcgI on pMLE2, a plasmid with two BcgI sites (Figure 1(a)), the FLL form of the DNA was observed as a transient intermediate but even at its peak, the FLL species constituted <30% of the DNA, below the level indicative of two separate but equal reactions. This shows that BcgI sometimes dissociates from this plasmid after cutting one site but sometimes cuts both sites before dissociating. Other plasmids that have two BcgI sites, but with different flanking\/spacer sequences from those on pMLE2, are cleaved without releasing any of the FLL DNA (S. Ganguly, J.J.T.M. & D.M.G., unpublished results). Moreover, BcgI cleaved the catenane derived from pMLE2 in a highly concerted manner: it released virtually none of the individual circles that would have been liberated by making a double-strand break in one ring (Figure 1(b)). Hence, BcgI rarely dissociates from the catenane after cutting just one site and instead remains bound until it has opened both rings. Other restriction enzymes were observed previously to act more concertedly on catenanes with one target site in each ring than on plasmids with two sites in cis.33 A synaptic complex between two sites in the separate rings of a catenane is likely to be more stable than one spanning sites in cis, as the juxtaposition of the sites in the catenane will not impose any deformation of the intervening DNA. The enzyme may therefore be able to cut both sites within the lifetime of the catenane synapse but not within the lifetime of the plasmid synapse.\nAmongst the Type IIB enzymes that cleave plasmids with two cognate sites more rapidly than plasmids with one target, the largest differences observed under standard reaction conditions were with BaeI and CspCI, which cleaved their two-site substrates 13 and 43-fold faster, respectively, than their one-site substrates (Table 3). Yet these two enzymes cleaved two-site plasmids by means of separate but kinetically equal reactions at each site (Figure 5(a)). To determine how these two enzymes communicate between distant DNA sites, their reactions were studied on catenane substrates that had in each ring either one BaeI site or one CspCI site (data not shown). When tested with the relevant enzyme, the catenanes were cleaved at similar rates to the parental two-site plasmid for that enzyme, in both cases more rapidly than the corresponding one-site plasmid. Moreover, like BcgI (Figure 1(b)), both BaeI and CspCI cleaved the catenanes much more concertedly than the plasmids: they released almost none of the individual circles from cutting the catenane in a single ring. Hence, both BaeI and CspCI act rapidly on DNA with two copies of their recognition sites by first spanning the sites through 3-D space, as opposed to tracking along the DNA. They can then act concertedly at both sites. BaeI and CspCI thus also belong in the Type IIF category of restriction endonucleases.\nDiscussion\nThe general case\nThe Type IIB endonucleases constitute a discrete subset of the Type II R\/M systems,2 delineated by their bipartite recognition sequences and by their bilateral sites of DNA cleavage either side of their recognition sites (Table 1). To determine whether Type IIB enzymes need to interact with two target sites in order to cleave DNA, the reactions of ten IIB nucleases were examined on plasmids that carry either one or two copies of the requisite sequence. With one exception, BplI, they all cleaved their two-site substrates more rapidly than the one-site substrates (Table 3) though two, BsaXI and PpiI, gave only twofold (or smaller) differences under their standard reaction conditions. These two both exhibited larger differences at elevated ionic strengths (Figure 4, Table 3). Moreover, all of the Type IIB enzymes tested against catenanes cleaved the catenane with one site in each ring as readily as the parental plasmid with two sites in a single ring (Figure 1). In addition, in all cases tested apart from BplI, the Type IIB enzymes cleaved plasmids with a single cognate site at enhanced rates in the presence of cognate oligoduplexes (Figure 3).\nThese experiments confirm the original proposal of Kong & Smith,45 that BcgI interacts with two copies of its recognition sequence before cutting DNA, and show further that the vast majority of the Type IIB enzymes act in the same way. Proteins that interact with distant DNA sites intrinsically prefer sites in cis over sites in trans,9,24,25 which accounts for why these enzymes cleave the two-site plasmids more rapidly than their one-site substrates. Nevertheless, the catenane experiments show that they capture their two sites in cis through 3-D space and not by tracking along the DNA, like the Type I enzymes.34,54 They can, however, also act in trans, by forming a complex bridging one recognition site on a plasmid and another on an oligoduplex.\nThe Type IIB endonucleases that cleave DNA after binding two cognate sites include some composed of two polypeptide chains, such as BcgI and BaeI,42,49 and some composed of a single polypeptide, such as AloI and PpiI.46\u201348 They also include some with asymmetric recognition sequences, like BsaXI, CspCI and PsrI, and some with palindromic sites, like AlfI and FalI. Furthermore, they include some that require AdoMet for their nuclease activities, viz. BcgI and CspCI, and others with no requirement for AdoMet, viz. BsaXI and PsrI (Table 1). The need to interact with two sites before cutting the DNA is thus a general property of the Type IIB enzymes, common to a wide range of these enzymes even though they differ from each other in many other respects. Two main mechanisms exist for the Type II restriction enzymes that use two sites:9,19\u201321,33 some, the Type IIE systems, employ one site as an activator to enhance cleavage of another site;27\u201330 others, the Type IIF systems, act concertedly at both sites.22,24,32\u201335 The Type IIB nucleases fall into the latter category.\nThis study extends the range of restriction endonucleases that are currently known to cleave DNA only after interacting with two target sites. In addition to the Type I, III and IV systems,1,4,5 which constitute \u223c60% of the R-M systems found in nature,3 the set includes a substantial fraction of the Type II enzymes: a portion of those that act within palindromic sites;18 the majority of the Type IIS systems that act on one side of an asymmetric site;36\u201340 and now, from this study, essentially all of the Type IIB enzymes that act bilaterally either side of a bipartite sequence. The enzymes that need two sites may therefore constitute 80% of the total and those that act at individual sites only 20% of the restriction enzymes present in vivo, even though the latter include the so-called archetypes like EcoRI, EcoRV and BamHI, the enzymes most widely used as tools for DNA manipulations in vitro.58,59 Though enzymes acting at individual sites might seem to be more efficient at restricting DNA than those requiring two sites, the widespread requirement for two sites indicates an advantage over the one-site systems.19 The need to interact with two copies of the recognition sequence before cleaving the DNA, rather than a single copy, may function as a double-check to ensure these enzymes only cleave DNA in response to the cognate sequence, and thus avoid untoward reactions at non-cognate sites.9,38,60 However, other restriction enzymes can utilise a cognate site to initiate cleavage of a non-cognate site.22,35,61,62 In these cases, the bridging interaction between cognate and non-cognate sites serves to broaden the range of sequences that the enzyme cleaves.61\nThe special case\nOn the other hand, one of the ten Type IIB enzymes tested, BplI, showed none of the characteristics of a restriction enzyme that interacts with two sites. Instead, BplI catalyses separate and independent reactions at each copy of its recognition site, in the same manner as the standard restriction enzymes like EcoRV.19 Under all reaction conditions employed with BplI, the progress curves for the utilisation of its one-site and two-site substrates were superimposable, even though its overall activity varied considerably over the range of conditions tested (Figures 2(c), 4(a) and (b)). Unlike Psr I for example, which cleaved its one and two-site substrates at similar rates at low ionic strength but at dissimilar rates at higher strengths (Figure 4(c) and (d)), no difference was observed with BplI even when the salt concentration was too high to allow complete DNA cleavage. Moreover, BplI cleaved the plasmid with two BplI sites by means of separate reactions at each site, albeit at the same rate (data not shown). In addition, an oligoduplex with the recognition sequence for BplI inhibited rather than enhanced its cleavage of a plasmid with one BplI site (Figure 3(b)), which again indicates that BplI can bind only one copy of its recognition sequence at a time.\nBplI clearly acts in an atypical manner for a Type IIB endonuclease, but its unusual properties cannot be due to its palindromic recognition sequence. Though most Type IIB enzymes recognise asymmetric sites (Table 1), some such as AlfI and FalI have palindromic sites yet still need two sites to cleave DNA. However, the unique properties of BplI might be a consequence of its subunit composition. Like BcgI, BplI is made up of two polypeptide chains but seemingly in a 1:1 ratio instead of the 2:1 ratio reported for BcgI.42,50 A related possibility is that BplI is not really a Type IIB enzyme, and is instead a novel Type IIS system with a palindromic recognition site. The restriction enzymes currently considered as Type IIS systems all have asymmetric sites and cut DNA at specified positions on one side of the site.2,3 A protein must bind to an asymmetric sequence in one particular orientation, which fixes the position of its catalytic residues on the DNA,57 but it can bind to a palindromic site in either of two orientations. BplI cleaves DNA either side of its site 8 and 13 bases away (Table 1)50 so on 50% of the occasions that it binds to its site, its catalytic functions may be located 8\/13 bases to the \u201cleft\u201d of the site and, on the 50% of the time, 13\/8 bases to the \u201cright\u201d of the site. A Type IIS enzyme with a palindromic site would thus make bilateral cleavages either side of its site in the characteristic manner of a Type IIB enzyme. Though most of the Type IIS enzymes need to interact with two sites for full activity,13,37\u201340 some need only one site.36\nMaterials and Methods\nEnzymes\nTn21 resolvase was purified as before.33,34,54 All other enzymes were obtained from commercial suppliers, stored at \u201320\u00a0\u00b0C and used essentially as advised by the supplier. The suppliers of the Type IIB restriction enzymes studied here, and the primary reaction buffers for each enzyme, are listed in Table 2. The concentrations of these enzymes were given in terms of units (U) of enzyme activity per ml, as specified by the supplier. While all of the commercial enzymes were free from non-specific nucleases, the purities of the preparations were not known and could not be assessed here. Most of the supplied samples contained insufficient amounts of protein for further analysis: the number of units in each purchase varied from 50\u00a0U to 1250\u00a0U and Type II restriction enzymes typically have specific activities of 106\u2013107\u00a0U\/mg of protein.10,19\nDNA\nThe plasmid pUC1963 and its derivatives pMDS2(a),34 pMLE1 and pMLE2,33 and pDG5,18 have been described. Specifically, pDG5 had been constructed from pMLE1 by inserting a 56\u00a0bp duplex that contained the same sequence as that around the BcgI site in pUC19: pDG5 is thus a 3806\u00a0bp plasmid with two BcgI sites 1810\u00a0bp apart that are identical to each other and to that in pUC19.\nFurther plasmids, with one or two copies of Type IIB recognition sites were constructed from pDG5 and pMDS2a (Supplementary Data, Figures S1\u2013S3). Typically, the vector was first linearised with a restriction enzyme and then ligated to a DNA duplex with appropriate single-strand extensions: the duplexes carried recognition sequences for several Type IIB enzymes and were made by annealing pairs of synthetic oligodeoxyribonucleotides (MWG Biotech). The ligation mixtures were used to transform Escherichia coli HB101 and the plasmids from several transformants sequenced across the site of the insertion (University of Dundee Sequencing Service). From each ligation, some transformants were found to contain a plasmid with a single copy of the insert at the requisite site. These plasmids were then used as vectors to clone a second duplex at a separate site, to give plasmids with two inserts in either inverted or directly repeated orientation. The constructs with inverted (head-to-head) inserts were used as the two-site substrates.\nThe transformants were cultured in M9 minimal media containing 37 MBq\/l [methyl-3H]thymidine (GE Healthcare) and the plasmids purified by CsCl density-gradient centrifugations.31\u201334 The preparations generally contained 80\u201395% supercoiled monomeric plasmid, with 5\u201320% open-circle and dimeric forms. The supercoiled forms of pMLE2, pJM2 and pIS2 were converted into catenanes by reactions with Tn21 resolvase at a 24-fold molar excess over the plasmid, as noted before.18,33,54 DNA concentrations were evaluated from A260 measurements.\nReactions\nReactions were usually carried out by adding 0.5\u20134\u00a0\u03bcl of the restriction enzyme (diluted, if necessary, in the buffer advised by the supplier) to 200\u00a0\u03bcl of 3H-labelled DNA (5\u00a0nM) in the appropriate buffer at the requisite temperature (Table 2). One 10\u00a0\u03bcl aliquot (the zero-time point control) was removed before adding the enzyme and further aliquots at timed intervals thereafter. The aliquots were mixed immediately with 5\u00a0\u03bcl of an EDTA Stop-Mix.24,32,38 In some instances, reactions were carried out in 20\u00a0\u03bcl volumes containing constant amounts of enzyme and plasmid but with varied concentrations of an oligoduplex: some duplexes contained, others lacked, the recognition sequence for the test enzyme. After a fixed time interval, these reactions were quenched with Stop-Mix as described above.\nThe quenched samples were analysed by electrophoresis through agarose.26,40 From reactions on plasmid substrates, the following forms of the DNA were separated: the intact supercoiled (SC) plasmid; the nicked open-circle (OC) form; the full-length linear (FLL) DNA cut in both strands at one site; and, for substrates with two sites, the two linear products (L1 and L2) from making double-strand breaks at both sites.16,25 With catenane substrates, the intact catenane was separated from the catenanes with nicks in one or both rings; and from both the separate rings and the linear products due to double-strand breaks in one or both sites.7,33\u201335 Type IIB enzymes cleave DNA on both sides of their recognition sites, but the linear product from cutting on one side of a site is only 27\u2013 33\u00a0bp longer than that cleaved on both sides of the same site. As the plasmids used here were \u223c4\u00a0kb in size, with \u223c1\u00a0kb between the sites in the two-site substrates, the products from bilateral cleavages were not separated from those with unilateral cleavages. Moreover, the 27\u201333\u00a0bp fragments excised from the remainder of the DNA were too small to detect on the agarose gels used here. It was assumed that DNA products cut in both strands at one or both recognition sites had been cleaved bilaterally at those sites.\nSegments of the gels were analysed by scintillation counting,25,40,57 to determine the concentration of each form at each time point. The concentrations shown in Figures 1\u20135 are the means from three independent experiments: for clarity, the error bars for the standard deviations (typically <10% of the mean values) have been omitted. Initial rates were evaluated from the mean values for the concentration of supercoiled DNA, by using GRAFIT (Erithacus Software, Horley, UK) to fit to a linear slope the decrease in these concentrations with time, starting at time zero and extending over the zero-order stage of the reaction: the errors in the reaction velocities cited in Table 3 are the standard errors from the fitting procedure.17,57 The errors on the ratios of the rates on the one and two-site plasmids were calculated from the error limits on each of the individual rates.\nFor the majority of the Type IIB enzymes tested here, the concentrations of their substrate ([S]) declined linearly during their reactions, for \u226550% of the total reaction: for example, BaeI (Figure 2(b)). However, for some such as AloI (Figure 2(a)), [S] declined in an exponential rather than a linear fashion. For substrate utilization to follow an exponential curve, one of the following must pertain: the reaction contains [E0]\u00a0>\u00a0[S] and thus has the characteristic first-order kinetics for a single-turnover reaction; or it is under multiple-turnover (steady-state) conditions with [E0]\u00a0<\u00a0[S], but with an initial [S] below the Km value so that the reaction velocity then declines in parallel with [S].52 The former may apply to BcgI, as this enzyme seems to act stoichiometrically rather than catalytically.42,45 Further experiments using BcgI that had been purified in this laboratory (S. Ganguly, S. Milsom, J.J.T.M & S.E.H., unpublished results) show that all of the BcgI reactions described here contained enzyme in excess of the substrate. It has yet to be determined which of these two possibilities apply to the other Type IIB enzymes that give exponential curves, as their concentrations were known only in terms of enzyme units, not molarities. On the other hand, a linear decline in [S] can only arise from a zero-order steady-state reaction involving multiple turnovers of the enzyme. Consequently, to permit comparisons of different enzymes, all of the reaction rates were evaluated as zero-order velocities from the decline in [S] with time over the initial portion of the reaction.","keyphrases":["restriction enzyme","dna\u2013protein interaction","adomet, s-adenosyl methionine","koac, mg(oac)2 and tris-oac, potassium, magnesium and tris acetate, respectively","r-m, restriction\u2013modification","u, units of restriction enzyme activity","fll, full-length linear","oc, open-circle","sc, supercoiled","enzyme mechanism","dna excision","dna looping"],"prmu":["P","P","R","M","M","R","R","R","R","R","R","R"]}
{"id":"Purinergic_Signal-3-4-2072913","title":"Ectonucleotidases in M\u00fcller glial cells of the rodent retina: Involvement in inhibition of osmotic cell swelling\n","text":"Extracellular nucleotides mediate glia-to-neuron signalling in the retina and are implicated in the volume regulation of retinal glial (M\u00fcller) cells under osmotic stress conditions. We investigated the expression and functional role of ectonucleotidases in M\u00fcller cells of the rodent retina by cell-swelling experiments, calcium imaging, and immuno- and enzyme histochemistry. The swelling of M\u00fcller cells under hypoosmotic stress was inhibited by activation of an autocrine purinergic signalling cascade. This cascade is initiated by exogenous glutamate and involves the consecutive activation of P2Y1 and adenosine A1 receptors, the action of ectoadenosine 5\u2032-triphosphate (ATP)ases, and a nucleoside-transporter-mediated release of adenosine. Inhibition of ectoapyrases increased the ATP-evoked calcium responses in M\u00fcller cell endfeet. M\u00fcller cells were immunoreactive for nucleoside triphosphate diphosphohydrolases (NTPDase)2 (but not NTPDase1), ecto-5\u2032-nucleotidase, P2Y1, and A1 receptors. Enzyme histochemistry revealed that ATP but not adenosine 5\u2032-diphosphate (ADP) is extracellularly metabolised in retinal slices of NTPDase1 knockout mice. NTPDase1 activity and protein is restricted to blood vessels, whereas activity of alkaline phosphatase is essentially absent at physiological pH. The data suggest that NTPDase2 is the major ATP-degrading ectonucleotidase of the retinal parenchyma. NTPDase2 expressed by M\u00fcller cells can be implicated in the regulation of purinergic calcium responses and cellular volume.\nIntroduction\nGlial cells play an active role in the regulation of neuronal activity. By release of so-called gliotransmitters, in particular glutamate and adenosine 5\u2032-triphosphate (ATP), activated glial cells provide both excitatory and inhibitory effects on neighbouring neurons. Excitation is mediated predominantly by glutamate, whereas ATP, after extracellular conversion to adenosine, causes neuronal suppression [1\u20133]. In the neural retina, stimulation of M\u00fcller glial cells by receptor agonists such as ATP, dopamine and thrombin, or electrical and mechanical stimuli, triggers intracellular calcium responses associated with a release of ATP from the cells [4, 5]. It has been suggested that ATP released from M\u00fcller cells is extracellularly converted to adenosine, which in turn activates A1 receptors in a subset of retinal ganglion cells, resulting in cellular hyperpolarisation [5]. In addition to the involvement in glia-to-neuron signalling, glia-derived nucleotides in the retina have autocrine effects. ATP released from retinal glial cells and subsequent activation of purinergic receptors evokes long-range calcium waves in the glial cell network [4] and inhibits the swelling of M\u00fcller cells under conditions of osmotic stress [6].\nExtracellular metabolism of ATP has been implicated in the glia-to-neuron signalling in the retina [5]. It is known that M\u00fcller cells express ecto-5\u2032-nucleotidase (CD73) [7\u20139], at least during the period of postnatal vasculogenesis [10]. CD73 hydrolyses nucleoside monophosphates such as AMP to the respective nucleosides [11]. However, it is unclear whether M\u00fcller cells also express ectoenzymes that degrade ATP, such as nucleoside triphosphate diphosphohydrolases (NTPDases) or ectonucleotide pyrophosphatase\/phosphodiesterases (ENPPs). NTPDase1 (CD39, ectoapyrase) and NTPDase2 (CD39L1, ecto-ATPase), as well as NTPDases 3 and 8, are capable of hydrolysing nucleoside 5\u2032-tri- and diphosphates such as ATP and ADP, albeit with considerably different substrate preference. Whereas NTPDase1 hydrolyses ATP and ADP about equally well, NTPDase2 preferentially degrades ATP, and NTPDases 3 and 8 reveal intermediate substrate preferences [12, 13]. In the neural retina, ADPase enzyme histochemistry and CD39 immunohistochemistry allocated NTPDase1 exclusively to the vasculature [14, 15]. In the brain, NTPDase1 is associated with microvessels and microglial cells but not with neurons or astrocytes [16]. NTPDase2 is expressed in the germinal zones of the developing and adult brain and by subpial astrocytes [17, 18]. In the peripheral nervous system, NTPDase1 is expressed by blood vessels and NTPDase2 by various types of glial cells [19]. ENPP1 is an ectoenzyme with both 5\u2032-nucleotide phosphodiesterase and nucleotide pyrophosphatase activity [20]. It is expressed in cell lines derived from brain glial cells and has also been allocated to brain capillaries and synaptosomal plasma membranes [12, 21\u201323]. The aim of our study was to investigate whether extracellular nucleotide metabolism is implicated in the inhibitory effect of ATP on the osmotic swelling of M\u00fcller cells, to determine the types of ectonucleotidases expressed by M\u00fcller cells and whether the retinal expression of ectonucleotidases is altered after transient retinal ischaemia reperfusion.\nMaterials and methods\nMaterials\nMitotracker Orange (chloromethyltetramethylrosamine) and Fluo-4\/AM were purchased from Molecular Probes (Eugene, OR, USA). Adenosine-5\u2032-O-(\u03b1,\u00df-methylene)-diphosphate (AOPCP), 6-N,N-diethyl-D-\u00df,\u03b3-dibromomethylene ATP (ARL-67156), 8-(3-chlorostyryl) caffeine (CSC), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), N6-methyl-2\u2032-deoxyadenosine-3\u2032,5\u2032-bisphosphate (MRS2179), N-nitrobenzylthioinosine (NBTI), p-nitrophenyl phosphate (pNPP), pyridoxal-phosphate-6-azophenyl-2\u2032,4\u2032-disulfonic acid (PPADS), and all other substances used were obtained from Sigma-Aldrich (Taufkirchen, Germany) unless stated otherwise. The following antibodies were used: rabbit anti-NTPDase1 (1:200) [16], rabbit anti-NTPDase2 (1:500) [17], goat anti-ENPP1 (1:200; Santa Cruz), goat anti-ecto-5\u2032-nucleotidase (1:200; Santa Cruz), rabbit anti-P2Y1 (1:100; Alomone), rabbit anti-A1 receptor (1:200; Santa Cruz), mouse anti-vimentin (1:200; V9 clone, Santa Cruz), mouse anti-glutamine synthetase (1:250, Chemicon) and mouse anti-cellular retinaldehyde-binding protein (CRALBP; 1:1000; Acris). The secondary antibodies were Cy3-conjugated goat anti-rabbit immunoglobulin (Ig)G (1:400; Dianova), Cy2-coupled goat anti-mouse IgG (1:200; Dianova), Cy3-coupled donkey anti-goat IgG (1:200; Dianova) and Cy2-coupled donkey anti-mouse IgG (1:200; Dianova).\nAnimals\nAll experiments were performed in accordance with the European Communities Council Directive 86\/609\/EEC and were approved by the local authorities. Adult Long-Evans rats (250\u2013350\u00a0g), NTPDase1 (cd39)-deficient mice, and wild-type mice were used. The generation of NTPDase1-deficient mice from wild-type animals has been described previously [24]. Animals had free access to water and food in an air-conditioned room on a 12-h light\u2013dark cycle. Transient retinal ischaemia was induced in one eye of the rats, whereas the other eye remained untreated and served as control (three animals). Anaesthesia was induced by intramuscular ketamine (100\u00a0mg\/kg) and xylazine(5\u00a0mg\/kg). The anterior chamber of the treated eye was cannulated from the pars plana with a 27-gauge infusion needle connected to a bag containing normal saline. The intraocular pressure was increased to 160\u00a0mmHg for 60\u00a0min by elevating the saline bag. The animals were killed by carbon dioxide 3\u00a0days after reperfusion, and the eyes were removed.\nM\u00fcller cell swelling\nTo determine volume changes of M\u00fcller glial cells evoked by hypotonic stress, the somata of the cells in the inner nuclear layer of rat retinal slices were recorded. Acutely isolated slices (thickness, 1\u00a0mm) were placed in a perfusion chamber and loaded with the vital dye Mitotracker Orange (10\u00a0\u03bcM). This dye is selectively taken up by M\u00fcller cells, whereas retinal neurons, astrocytes and microglial cells remain unstained [25]. The stock solution of the dye was prepared in dimethylsulfoxide and resolved in saline. The slices were examined with a confocal laser scanning microscope (LSM) LSM 510 Meta (Zeiss, Oberkochen, Germany). Mitotracker Orange was excited at 543\u00a0nm, and emission was recorded with a 560\u00a0nm long-pass filter. In the course of the experiments, the somata of M\u00fcller cells were recorded at the plane of their maximal extension.\nA gravity-fed system with multiple reservoirs was used to perfuse the recording chamber continuously with extracellular solution; the hypotonic solution and test substances were added by rapidly changing the perfusate. The extracellular solution consisted of (mM) 136 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 10 hydroxyethylpiperazine ethanesulfonic acid (HEPES) and 11 glucose, adjusted to pH 7.4 with Tris. The hypotonic solution (60% of control osmolarity) was made up by adding distilled water. Barium chloride was added to the extracellular solution and was preincubated for 10\u00a0min. Blocking substances were preincubated for 15\u201345\u00a0min, and agonists were administered simultaneously with the hypotonic solution.\nCalcium imaging\nWholemounts of the rat retina (9\u00a0mm2) were placed in a perfusion chamber and incubated for 1\u00a0h at room temperature in extracellular solution containing the calcium-sensitive fluorescence dye Fluo-4\/AM (11\u00a0\u03bcM). After 10\u00a0min of washing by continuous perfusion of extracellular solution, ATP was added by a fast change of the perfusate. ARL-67156 was preincubated for 25\u00a0min. Fluo-4 was excited at 488\u00a0nm; emission was recorded with a band-pass filter between 505 and 550\u00a0nm.\nImmunohistochemistry\nIsolated rat retinas were fixed in 4% paraformaldehyde for 2\u00a0h. After several washing steps in buffered saline, the tissues were embedded in saline containing 3% agarose (w\/v), and 60-\u03bcm-thick slices were cut by using a vibratome. For double labelling, the slices were incubated in 5% normal goat or donkey serum plus 0.3% Triton X-100 in saline for 2\u00a0h at room temperature and, subsequently in a mixture of primary antibodies overnight at 4\u00b0C. After washing in 1% bovine serum albumin, secondary antibodies were administered for 2\u00a0h at room temperature. Control slices were stained without primary antibodies; no unspecific labelling was observed following incubation with secondary antibodies (not shown). Images were taken with the LSM.\nImmunocytochemistry\nTo prepare suspensions of dissociated cells, isolated rat retinas were stored for 30\u00a0min in saline containing 0.4\u00a0mg\/ml papain (Boehringer, Mannheim, Germany) at 37\u00b0C. After fixation with 4% paraformaldehyde for 10\u00a0min and washing with saline, the tissues were triturated with a pipette until single cells were dissociated. The cells were blocked and permeabilised for 30\u00a0min with 5% normal goat serum and 0.3% Triton-X 100, followed by incubation with primary antibodies for 6\u00a0h at 4\u00b0C. After washing in 1% bovine serum albumin in saline, the secondary antibodies were applied for 1\u00a0h at room temperature.\nEnzyme histochemistry\nMice were deeply anaesthetised with pentobarbital (800\u00a0mg\/kg) and perfused intracardially with phosphate-buffered saline containing heparin (0.5\u00a0mg\/ml) followed by 0.05\u00a0M cacodylate-buffered 4% paraformaldehyde, pH 7.4. Subsequently, eyes were removed and immersed at 4\u00b0C in the same fixative for 4\u00a0h. The eyes were cryoprotected overnight with 30% sucrose and frozen in isopentane at \u221280\u00b0C. Frozen tissues were stored at \u221280\u00b0C until sectioning. Frozen sections (14\u00a0\u03bcm) were deposited on 3-aminopropyl-triethoxysilane-coated slides and allowed to dry for 1\u00a0h. The dry sections were stored at \u221280\u00b0C until further processing. For localisation of ectonucleotidase activity, a lead phosphate method was applied, as previously described [16]. In brief, frozen sections were warmed to room temperature. The enzyme reaction was performed for 1\u00a0h at room temperature in a TMS-buffered substrate solution (in mM: 2 Pb(NO3)2, 5 MnCl2, 2 CaCl2, 50 Tris-maleate, pH 7.4, plus 0.25\u00a0M sucrose) stabilised with 3% dextran T 250 (Roth, Karlsruhe, Germany) containing the substrates ATP, ADP or pNPP (sodium salts; 1\u00a0mM each). In control experiments, the substrate was omitted from the incubation solution. After washing sections with demineralised water, the lead orthophosphate precipitated as a result of nucleotidase activity was visualised as a brown deposit by incubating sections in an aqueous solution of (NH4)2S (1% v\/v).\nData analysis\nTo determine the extent of swelling of M\u00fcller cell somata, the cross-sectional area of Mitotracker-Orange-stained cell bodies in the inner nuclear layer of retinal slices was measured off-line using the analysis software of the LSM. Bar diagrams display the mean [\u00b1 standard error of the mean (SEM)] cross-sectional areas of cell somata that were measured after 4-min perfusion with hypotonic solution relative to the somatic area measured before hypotonic challenge (100%). To evaluate the calcium responses, the fluorescence of Fluo-4 was normalised to the prestimulus value by calculation of the ratio F\/F0, whereby F0 represents the baseline fluorescence before agonist application. Statistical analysis was performed using the Prism program (Graphpad Software, San Diego, CA, USA); significance was determined by Student\u2019s t test or Kruskal-Wallis test followed by Dunn\u2019s comparison for multiple groups.\nResults\nPurinergic signalling inhibits osmotic swelling of M\u00fcller glial cells\nTo determine whether extracellular nucleotide metabolism is implicated in the inhibitory effect of ATP on the osmotic swelling of retinal glial cells, we investigated the alterations in size of M\u00fcller cell somata in acutely isolated rat retina slices in response to hypotonic stress. As described previously [26, 27], administration of a hypotonic solution (containing 60% of control osmolarity) did not alter the size of M\u00fcller cell somata in slices of control retinas, whereas M\u00fcller cell somata swelled in the presence of barium ions (Fig.\u00a01a,b). Similarly, M\u00fcller cell somata in postischaemic retinas displayed hypotonic swelling. We recently showed that various purinergic receptor agonists, such as ATP, ADP and adenosine, inhibit osmotic swelling of M\u00fcller cell somata and that glutamate also inhibits the swelling via activation of a purinergic signalling cascade [6]. This signalling cascade is apparently mediated by autocrine mechanisms in M\u00fcller cells, as indicated by the lack of effect of the blocker of voltage-gated sodium channels tetrodotoxin on the action of glutamate, ATP and adenosine (Fig.\u00a01c). The swelling-inhibitory action of glutamate and ATP were blocked by suramin and the inhibitor of P2Y and distinct P2X receptors, PPADS (Fig.\u00a01d). The selective P2Y1 receptor antagonist, MRS2179, prevented the effects of glutamate and ATP but not of adenosine (Fig.\u00a01e), whereas the antagonist of adenosine A1 receptors, DPCPX, prevented the effects of all three agonists investigated (Fig.\u00a01f). The selective A2a receptor antagonist, CSC, had no effect (Fig.\u00a01f). The data suggest that the swelling-inhibitory effects of glutamate and ATP are mediated by activation of P2Y1 and A1 receptors, and that the activation of A1 receptors occurs downstream from P2Y1 receptor activation. Apparently, glutamate evokes a consecutive release (or formation) of ATP and adenosine, which successively activates P2Y1 and A1 receptors in M\u00fcller cells.\nFig.\u00a01A purinergic signalling cascade inhibits the osmotic swelling of M\u00fcller glial cells. Rat retina slices were perfused with a hypotonic solution (60% of control ionic strength), and the cross-sectional area of M\u00fcller cell somata was recorded. In (c\u2013h), retinal slices were perfused with a hypotonic solution in the presence of barium chloride (1\u00a0mM). a Time-dependent alterations in the soma area of M\u00fcller cells in response to a hypotonic solution. M\u00fcller cell somata were recorded in a control retina in the absence and presence of barium (1\u00a0mM) and in a postischemic retina. Insets: soma of a M\u00fcller cell before (left) and during (right) hypotonic exposure. Bar 5\u00a0\u03bcm. b Soma area of M\u00fcller cells under hypotonic conditions in control and postischemic retinas. c Glutamate, adenosine 5\u2032-triphosphate (ATP) and adenosine, respectively, inhibited the hypotonic swelling of M\u00fcller cells somata. This effect remained unaltered in the presence of tetrodotoxin (TTX; 1\u00a0\u03bcM). d Suramin (200\u00a0\u03bcM) and pyridoxal-phosphate-6-azophenyl-2\u2032,4\u2032-disulfonic acid (PPADS) (10\u00a0\u03bcM) prevented the swelling-inhibitory effects of glutamate and ATP. e The effects of glutamate and ATP (but not of adenosine) were abolished by the selective blocker of P2Y1 receptors, MRS2179 (30\u00a0\u03bcM). f The antagonist of adenosine A1 receptors, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (100\u00a0nM), prevented the effects of glutamate, ATP and adenosine. The antagonist of adenosine A2a receptors, 8-(3-chlorostyryl) caffeine (CSC) (200\u00a0nM), did not inhibit the effect of adenosine. g The effect of glutamate involves extracellular metabolism of ATP. The glutamate effect was prevented by the ectoapyrase inhibitor 6-N,N-diethyl-D-\u00df,\u03b3-dibromomethylene ATP (ARL-67156) (50\u00a0\u03bcM) and remained largely unaltered in the presence of the ecto-5\u2032-nucleotidase inhibitor, adenosine-5\u2032-O- (\u03b1,\u00df-methylene)-diphosphate (AOPCP) (250\u00a0\u03bcM). hThe swelling-inhibitory effect of glutamate was abolished in the presence of the antagonist of nucleoside transporters, N-nitrobenzylthioinosine (NBTI) (10\u00a0\u03bcM). Agonists were coadministered with the hypotonic solution and tested at the following concentrations: glutamate, 1\u00a0mM; ATP, 10\u00a0\u03bcM; adenosine, 10\u00a0\u03bcM. Bar diagrams display mean \u00b1 standard error of the mean soma areas, which were measured after a 4-min perfusion of the slices with the hypotonic solution (n\u2009=\u20094\u201316 cells per bar). **P\u2009<\u20090.01, ***P\u2009<\u20090.001 compared with data obtained before osmotic challenge (100%). \u2022\u2022P\u2009<\u20090.01, \u2022\u2022\u2022P\u2009<\u20090.001 compared with data obtained from swollen cells. \u25cb\u25cbP\u2009<\u20090.05, \u25cb\u25cb\u25cbP\u2009<\u20090.001 compared with the agonist effects\nThough P2Y1 receptors are suggested to be preferentially activated by ADP [28], there are also studies that show an activation by ATP [29, 30]. To determine whether extracellular nucleotide metabolism is involved in the swelling-inhibitory effect of glutamate, we tested an inhibitor of ectoapyrases (ARL-67156) and an inhibitor of the ecto-5\u2032-nucleotidase (AOPCP). The effect of glutamate was prevented in the presence of ARL-67156 (Fig.\u00a01g), suggesting that glutamate evoked a release of ATP from M\u00fcller cells, which is extracellularly degraded to ADP\/adenosine monophosphate (AMP). On the other hand, AOPCP was largely ineffective in preventing the effect of glutamate (Fig.\u00a01G), indicating that the majority of adenosine, which is released after stimulation with glutamate, is not formed by extracellular degradation of AMP. Most of the adenosine, which mediates the glutamate effect, is released by facilitated transport, as indicated by the blocking effect of the antagonist of nucleoside transporters, NBTI (Fig.\u00a01h). The data suggest that the swelling-inhibitory effects of glutamate and ATP involve extracellular degradation of ATP to ADP but not of AMP into adenosine.\nATP-evoked calcium responses\nBy rapid degradation, ectonucleotidases are suggested to limit the actions of purinergic receptor agonists. In rat retina M\u00fcller cells, intracellular calcium responses can be evoked by various purinergic agonists, such as ATP, ADP and uridine triphosphate (UTP) [31]. The predominant P2 receptor subtype that evokes calcium responses in rat M\u00fcller cells is the P2Y1 receptor [31]. ATP-evoked calcium responses in rat M\u00fcller cells are restricted to the inner half of the cells, i.e. to the processes that traverse the inner plexiform layer, and the cell endfeet at the inner surface of the retina [6, 32]. To determine whether extracellular nucleotide metabolism modulates purinergic calcium responses, we recorded the calcium responses in the endfeet of M\u00fcller cells in acutely isolated retinal wholemounts upon administration of ATP. ATP-evoked calcium responses were observed in M\u00fcller cell endfeet that ensheath the ganglion cell bodies, as well as in glial processes that surround retinal vessels (Fig.\u00a02a). As purinergic receptor agonists do not evoke P2X receptor-mediated cation currents in rat M\u00fcller cells [33, 34], the calcium responses are suggested to be evoked by activation of P2Y receptors. Inhibition of ectoapyrases by ARL-67156 caused a significant increase in the ATP-evoked calcium responses in the endfeet of M\u00fcller cells (Fig.\u00a02b,c). It is concluded that the activity of ectonucleotidases regulates the purinergic receptor-mediated calcium responses in M\u00fcller glial cells.\nFig.\u00a02Extracellular nucleotide metabolism modulates the adenosine 5\u2032-triphosphate (ATP)-evoked calcium responses in rat retina M\u00fcller cell endfeet. a Views onto the nerve fibre\/ganglion cell layers of a retinal wholemount. The images show the fluorescence of Fluo-4 and were taken before (control) and during the peak calcium response upon bath appplication of ATP (50\u00a0\u03bcM). Asterisk, retinal vein. Arrow, capillary. Arrowhead, nerve fibre bundles. Bar 20\u00a0\u03bcm. b Time-dependent changes of the relative fluorescence ratio in M\u00fcller cell endfeet. The ATP (10\u00a0\u03bcM)-evoked responses were recorded in the absence (control) and presence of 6-N,N-diethyl-D-\u00df,\u03b3-dibromomethylene ATP (ARL-67156) (50\u00a0\u03bcM). A fluorescence ratio of one means no change in the cytosolic free calcium level. c Mean (\u00b1 standard error of the mean) amplitude of the ATP (10\u00a0\u03bcM)-evoked calcium responses (n\u2009=\u200938 and 50 endfeet per bar). *P\u2009<\u20090.05 compared with the ATP control\nImmunolocalisation of ectonucleotidases\nWe stained retinal slices and isolated cells to determine the type of ectonucleotidases expressed by rat M\u00fcller cells. In retinal slices, immunoreactivity for NTPDase1 was predominantly localised in large blood vessels within the nerve fibre\/ganglion cell layers. In addition, faint staining was observed in the microvessels of the inner nuclear layer (arrows in Fig.\u00a03a). Retinal neurons and glial cells, as well as isolated M\u00fcller cells (Fig.\u00a03b), were apparently devoid of immunoreactivity for NTPDase1. M\u00fcller cells express NTPDase2, as indicated by the glutamine-synthetase-expressing M\u00fcller cell fibres that traverse the inner plexiform layer in retinal slices (filled arrowheads in Fig.\u00a03a), and the immunolabeling of isolated M\u00fcller cells (Fig.\u00a03b). The NTPDase2 protein is localised at the whole plasma membrane of M\u00fcller cells, including the membranes of the cell endfeet and somata. In addition to M\u00fcller cell fibres, NTPDase2 is expressed in both plexiform (synaptic) layers. Immunoreactivity for ENPP1 was largely absent in the inner retina, whereas a slight staining of M\u00fcller cell fibres in the outer retina (unfilled arrowheads in Fig.\u00a03a) was apparent in some (but not all) retinal slices investigated. M\u00fcller cells expressed immunoreactivity for the ecto-5\u2032-nucleotidase, in particular, at the inner stem processes (filled arrowheads in Fig.\u00a03a) and endfeet, but also at the outer stem processes (Fig.\u00a03b). In addition, neuronal cell bodies in the ganglion cell and inner nuclear layers may express immunoreactivity for ecto-5\u2032-nucleotidase (Fig.\u00a03a). The data suggest that M\u00fcller cells express enzymes for the extracellular degradation of ATP and AMP (NTPDase2 and ecto-5\u2032-nucleotidase).\nFig.\u00a03Immunohistochemical localisation of ectonucleotidases and purinergic receptors in rat retina M\u00fcller glial cells. a Retinal slices. b Isolated M\u00fcller cells. c Slices of postischemic retinas that were obtained from animals 3\u00a0days after transient retinal ischemia. The tissues and cells were coimmunostained against the glial-cell-specific proteins vimentin, glutamine synthetase or mouse anti-cellular retinaldehyde-binding protein (CRALBP). Filled arrows, blood vessels. Filled arrowheads, M\u00fcller cell fibres in the inner nuclear layer (INL). Unfilled arrows, M\u00fcller cell endfeet. Unfilled arrowheads, a, c M\u00fcller cell fibres in the outer nuclear layer (ONL) and b M\u00fcller cell somata, respectively. GCL ganglion cell layer, IPL inner plexiform layer, PRS photoreceptor segments. Bars 20\u00a0\u03bcm\nWe found that the inhibition of osmotic M\u00fcller cell swelling is mediated by the consecutive activation of P2Y1 and adenosine A1 receptors (see above). M\u00fcller cells express the immunoreactivities for both receptor proteins, which is indicated by the staining of vimentin-positive M\u00fcller cell fibres in the inner plexiform layer (filled arrowheads in Fig.\u00a03a). In addition, neuronal cell bodies in the inner nuclear and ganglion cell layers apparently express immunoreactivities for P2Y1 and A1 receptors (Fig.\u00a03a). Induction of osmotic swelling is a characteristic feature of M\u00fcller cells after transient retinal ischaemia (Fig.\u00a01a,b) [26], which can be inhibited by activation of the purinergic signalling cascade described above [6]. We stained retinal slices that were obtained 3\u00a0days after transient retinal ischaemia and found no alteration in the overall localisation of the various enzyme and receptor proteins investigated (Fig.\u00a03c and not shown). An apparent upregulation of the immunoreactivities in the inner retina [ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL)] may be caused by the strong decrease in thickness of the inner retinal layers (in particular, of the IPL), which is a typical degeneration pattern of rodent retinas after transient ischemia.\nEctonucleotidase histochemistry\nTo further identify the nature of retinal ectonucleotidases, we analysed retinal tissues of NTPDase1-deficient and wild-type mice using enzyme histochemistry. When ATP was used as substrate, histochemical staining was observed throughout the retinal tissue of both wild-type and knockout mice, with preferential reaction deposit in the outer retina (Fig.\u00a04a). This suggests that deletion of the NTPDase1 gene has no significant effect on ATPase activity in the retina. Even though the resolution of the enzyme histochemical images is limited due to dispersion of the reaction product, they correspond closely to the immunostaining for NTPDase2 in the rat retina, except that blood vessels are stained in addition (Fig.\u00a03a). In contrast, when ADP was applied as substrate in wild-type mouse retinas for the same length of time as ATP, staining of the retinal parenchyma was largely abolished. However, strong staining of the vessels was maintained (Fig.\u00a04b). This pattern of enzyme histochemical staining was very similar to the immunohistochemical staining for NTPDase1 (Fig.\u00a03a). The enzyme histochemical staining at blood vessels for ADP was abolished in retinas of NTPDase1-deficient mice (Fig.\u00a04b). No staining of the neural retina was observed when pNPP was applied as a substrate of alkaline phosphatase (Fig.\u00a04c). The pigment epithelium of both wild-type and knockout mice was strongly stained with all substrates used, suggesting that it is rich in nonspecific phosphatases (alkaline phosphatase). No reaction product was obtained in the absence of substrates (not shown). The data suggest that NTPDase2 (that has a very high preference for ATP as a substrate, ATP to ADP hydrolysis ratio of \u223c10:1 [35]), is the major ATP-degrading ectonucleotidase of the neural retina, where it is associated with M\u00fcller cells. NTPDase1 (that equally hydrolyses ATP and ADP) is restricted to blood vessels, and activity of alkaline phosphatase at physiological pH is essentially absent. The lack of enzyme histochemical staining for ecto-ADPase in the NTPDase1 knockout mice (Fig.\u00a04b) also suggests the absence of NTPDase3 and NTPDase8.\nFig.\u00a04Enzyme histochemical localisation of ectonucleotidase activity in retinal slices of nucleoside triphosphate diphosphohydrolases (NTPDase1) wild-type (cd39+\/+) and NTPDase1-deficient (cd39\u2212\/\u2212) mice. a When adenosine 5\u2032-triphosphate (ATP) is used as substrate, the reaction product is distributed throughout the retinal tissue, with most intense staining in the outer plexiform layer. b The use of adenosine 5\u2032-diphosphate (ADP) as substrate results in a staining of retinal blood vessels (arrows). This staining is lost in NTPDase1-deficient mice. cp-nitrophenyl phosphate (pNPP), a substrate of alkaline phosphatase, reveals no retinal staining except of the pigment epithelium. The pigment epithelium has been lost in some sections. GCL ganglion cell layer, INL inner nuclear layer, IPL inner plexiform layer, ONL outer nuclear layer, PRS photoreceptor segments. Bars 20\u00a0\u03bcm\nDiscussion\nThe data presented here suggest that extracellular ATP metabolism is involved in the autocrine regulation of the M\u00fcller cell volume in response to osmotic stress. Obviously, there is a multilevel signalling cascade resulting in a prevention of cellular swelling under hypoosmotic stress. Whereas we focused on the role of ectonucleotidases in this study, other aspects of this cascade were described in a previous study [6]. One of the first steps is the release of glutamate by retinal neurons, which activates metabotropic glutamate receptors on M\u00fcller glial cells [6]. Because a blockade of neuronal activity by tetrodotoxin did not abolish the effect of glutamate, we concluded that neurons are not involved in the cascade downstream from activation of metabotropic glutamate receptors. The pharmacological data presented in Fig.\u00a01 suggest the further course of the cascade. Activation of glial metabotropic glutamate receptors evokes a release of ATP, which is assumed to be metabolised to ADP. This assumption is supported by the finding that the inhibitor of ectoapyrases, ARL-67156, prevented the swelling-inhibitory effect of glutamate (Fig.\u00a01g). In addition to the unspecific blockers of P2 receptors, suramin and PPADS, the P2Y1-specific inhibitor, MRS2179, efficiently suppressed the effects of glutamate and ATP, suggesting that ADP resulting from the action of ectoapyrases activates P2Y1 receptors on M\u00fcller cells. Expression of this receptor subtype on rat M\u00fcller cells was demonstrated previously [31, 36]. The further degradation of ADP to AMP and adenosine does not play a decisive role in the cascade because the blocker of ecto-5\u2032-nucleotidases, AOPCP, reduced the glutamate effect nonsignificantly. However, adenosine is obviously involved in the cascade. Whereas inhibition of P2Y1 prevented the effects of glutamate and ATP, it did not alter the action of adenosine. Thus, the effect of adenosine is an event downstream from the activation of glutamate and P2Y1 receptors. Using two different subtype-specific adenosine receptor blockers, we concluded that the adenosine effect is mediated by activation of A1 receptors (Fig.\u00a01f). Because extracellular formation of adenosine is likely to be only a minor source of this nucleoside, the blocker of nucleoside transporters, NBTI, was tested and prevented the effect of glutamate. This suggests that the main part of adenosine is liberated via a transporter. We did not investigate the final steps of the cascade downstream from the activation of A1 receptors. It was concluded from previous data [6, 37] that activation of A1 receptors causes the opening of potassium and chloride channels in the M\u00fcller cell membrane; thus, the cells extrude potassium and chloride ions, which is associated with a water efflux that prevents swelling under hypoosmotic conditions. Considering the results presented in this study, the extracellular enzymatic degradation of ATP to ADP might play a crucial role in the purinergic cascade involved in the autocrine regulation of the M\u00fcller cell volume.\nThe activity of ectonucleotidases controls the level of purinergic receptor agonists in the extracellular space. We found that inhibition of ectoapyrases increased the ATP-evoked calcium responses in M\u00fcller cell endfeet, suggesting that ectonucleotidases control the availability of extracellular ATP that evokes calcium responses predominantly by activation of P2Y1 receptors [31]. The relatively small effect of the ectoapyrase inhibitor may be explained by the possibility that ATP-evoked ATP release from M\u00fcller cells may overwhelm the extracellular degradation of ATP. By rapid inactivation of purinergic signalling, ectonucleotidases may regulate the light-evoked purinergic neuron-to-glia signalling in the retina [32] and the long-range purinergic calcium signalling in the network of retinal glial cells [4].\nIn a third step of the study, we investigated the expression of enzymes involved in extracellular nucleotide degradation in M\u00fcller cells. We found that M\u00fcller cells of the rat express immunoreactivities for NTPDase2 and ecto-5\u2032-nucleotidase but not for NTPDase1 and only faintly for ENPP1. The expression of ecto-5\u2032-nucleotidase is in agreement with previous studies [7\u20139]. The expression of  NTPDase2 by M\u00fcller cells is a novel finding and is in agreement with data that showed an expression of NTPDase2 (but not NTPDase1) in distinct glial cells of the brain and peripheral nervous system [16, 17, 19], as well as with a recent study that described a localisation of ecto-ATPase activity at the electron microscopic level in M\u00fcller cell membranes (in addition to synaptic membranes) [38]. The enzyme histochemical experiments revealed that NTPDase2 is the major ATP-degrading ectonucleotidase of the neural mouse retina, whereas NTPDase1 activity is restricted to blood vessels and alkaline phosphatase is essentially absent at physiological pH. The data also rule out the possibility of a significant expression of NTPDases 3 and 8 in the retinal parenchyma. Our data confirm previous studies describing an exclusive localisation of NTPDase1-like activity and protein to the vasculature of the neural retina [14, 15]. According to our data, ATP can be rapidly hydrolysed within the retinal parenchyma, whereas (due to the very low ADPase activity of NTPDase2) ADP will be degraded with a considerable delay. The retinal pigment epithelium apparently expresses the activity of nonspecific phosphatases (alkaline phosphatase) and may also express various other extracellular nucleotide-degradative enzymes [39].\nThe enzyme histochemical data are in agreement with the results of the cell-swelling experiments indicating that ecto-5\u2032-nucleotidase activity is not involved in the regulation of M\u00fcller cell volume, likely due to the absence of the substrate AMP. The small, nonsignificant effect of the 5\u2032-nucleotidase inhibitor (Fig.\u00a01g) may reflect the very slow and delayed degradation of ADP to AMP by NTPDase2 [35]. However, further investigations are necessary to confirm the involvement of extracellular nucleotide metabolism in the regulation of M\u00fcller cell volume. The nucleotide analogue ARL-67156 has been suggested to inhibit the activities of ectoapyrases in several tissues [40\u201342]. A recent study using a capillary electrophoresis enzyme assay has shown that ARL-67156 blocks predominantly the activity of recombinant NTPDases1 and 3 and has only a minor effect on the activity of recombinant NTPDase2 [43]. Our experimental data imply that NTPDase2 was inhibited by ARL-67156 in retinal slices. It remains to be determined whether the functional state of NTPDase2 (such as its oligomeric state [35]) can govern its sensitivity to certain inhibitors or whether ARL-67156 may exert additional effects in intact tissues in situ, such as a blockade of the release of ATP from agonist-stimulated cells [44].\nBoth extracellular degradation of ATP and the formation of adenosine have been implicated in the suppression of neuronal activity in the retina [5]. It has been suggested that ATP released from M\u00fcller cells is converted extracellularly to adenosine, which in turn activates A1 receptors in a subset of retinal ganglion cells, resulting in cellular hyperpolarisation [5]. The present results indicate that both neuronal cells and M\u00fcller cells express ecto-5\u2032-nucleotidase, that forms adenosine from AMP. On the other hand, we found that only the activity of ecto-ATPases (and not of the ecto-5\u2032-nucleotidase) is implicated in the regulation of M\u00fcller cell volume and that comparable ecto-ADPase activity (which may deliver AMP as the substrate of ecto-5\u2032-nucleotidase) is absent from the retinal parenchyma. The reason for the discrepancy with previously published data [5] is unclear and remains to be solved in future investigations. It cannot be ruled out that pharmacological modulation of the glial purinergic signalling may also indirectly alter neuronal activity via alterations of the glial cell volume and, therefore, of the extracellular space volume. Moreover, differences in the local microenvironment and the spatial relationship of ectonucleotidases, purinergic receptors and transporter molecules may explain some of the different findings.\nIn summary, we found that NTPDase2 is the major ATP-degradative ectonucleotidase in the parenchyma of rodent retinas, whereas NTPDase1 is expressed solely in retinal vessels. The activity of NTPDase2 is suggested to be implicated in the regulation of the M\u00fcller cell volume under osmotic stress conditions. This regulation is likely mediated by extracellular degradation of released ATP and subsequent transporter-mediated release of adenosine. The activity of NTPDase2 is also involved in the regulation of ATP-evoked calcium responses in M\u00fcller cells; this may have functional importance for the purinergic neuron-to-glia signalling observed in response to light stimulation [32]. Additional investigations are required to determine the functional roles of NTPDase2 and ecto-5\u2032-nucleotidase in the reciprocal signalling between neurons and glia in the retina.","keyphrases":["ectonucleotidase","m\u00fcller glial cell","retina","a1 receptor","p2 receptor","cellular swelling"],"prmu":["P","P","P","P","P","P"]}
{"id":"Exp_Brain_Res-3-1-2080346","title":"Visual and musculoskeletal underpinnings of anchoring in rhythmic visuo-motor tracking\n","text":"Anchoring, that is, a local reduction in kinematic (i.e., spatio-temporal) variability, is commonly observed in cyclical movements, often at or around reversal points. Two kinds of underpinnings of anchoring have been identified\u2014visual and musculoskeletal\u2014yet their relative contributions and interrelations are largely unknown. We conducted an experiment to delineate the effects of visual and musculoskeletal factors on anchoring behavior in visuo-motor tracking. Thirteen participants (reduced to 12 in the analyses) tracked a sinusoidally moving visual target signal by making flexion\u2013extension movements about the wrist, while both visual (i.e., gaze direction) and musculoskeletal (i.e., wrist posture) factors were manipulated in a fully crossed (3 \u00d7 3) design. Anchoring was affected by both factors in the absence of any significant interactions, implying that their contributions were independent. When gaze was directed to one of the target turning points, spatial endpoint variability at this point was reduced, but not temporal endpoint variability. With the wrist in a flexed posture, spatial and temporal endpoint variability were both smaller for the flexion endpoint than for the extension endpoint, while the converse was true for tracking with the wrist extended. Differential anchoring effects were absent for a neutral wrist posture and when gaze was fixated in between the two target turning points. Detailed analyses of the tracking trajectories in terms of velocity profiles and Hooke\u2019s portraits showed that the tracking dynamics were affected more by wrist posture than by gaze direction. The discussion focuses on the processes underlying the observed independent effects of gaze direction and wrist posture on anchoring as well as their implications for the notion of anchoring as a generic feature of sensorimotor coordination.\nIntroduction\nAlthough cyclical movements have often been understood and modeled as self-sustained oscillators or limit cycles (Beek et al. 1996; Haken et al. 1985; Kay et al. 1987), their trajectories in phase space (velocity against position) are typically wrinkled and asymmetric, rather than perfectly harmonic and symmetric. A better approximation can be achieved by adding Gaussian white noise to the limit cycle description (Kay 1988), but this is insufficient to account for the observation that cyclical movements are characterized by specific regions of reduced kinematic (i.e., spatio-temporal) variability, which are often, but not solely, located at or around the maximal angular excursions or movement endpoints. Such regions have been dubbed \u201canchor points\u201d, implying that they serve as \u201c\u201cintentional attractors\u201d or \u201corganizing centers\u201d within, and for\u201d the entire cycle production (Beek 1989; pp 183\u2013184; cf. Beek et al. 1992). Beek (1989) conjectured that at, or around, anchor points critical task-specific information is available for organizing a cyclical act (in his case ball juggling). In line with this conjecture, Kelso and Jeka (1992) concluded from the representation of 4-limb patterns as a single trajectory on a 3-D torus that the essential information for coordination is confined or localized to discrete regions in phase space. In several subsequent studies on rhythmic movement conducted from a dynamical systems perspective, anchor points or anchoring phenomena have been observed, discussed and even modeled. In order to motivate the purpose and design of the present study, it is necessary to highlight the various aspects of anchoring that have been identified so far in the literature.\nFurther evidence for anchoring was obtained in a study by Byblow et al. (1994) in which participants performed bimanual cyclical wrist movements in both in-phase and antiphase coordination, either at a self-paced tempo or to the beat of a metronome that increased in frequency. In both cases, a local reduction of spatial variability was found at maximal angular excursions, which was more pronounced for the dominant than for the non-dominant hand, and more so for pronation than for supination (see also Carson et al. 1994). In a subsequent study, Byblow et al. (1995) reported the same spatial anchoring phenomenon for unimanual rhythmic movements that were synchronized to either a discrete or a continuous visual pacing signal, which led the authors to conclude that anchoring is not dependent on discrete information pulses, but may also occur if the external pacing signal is continuous. Importantly, in this series of studies, anchoring was defined in terms of reduced endpoint variability. The authors assumed that the movement reversal points were also the \u201cregions of the kinematics where information is specified\u201d (p. 124). However, for paced movements, this need not be the case because reduced endpoint variability does not necessarily imply that participants also timed their maximal excursions to the (discrete or continuous) external signal. For this reason, it is important to distinguish between anchoring as reduced spatial variability and as reduced variability in the timing of the movement excursions relative to the beats or the reversal points of the metronome. This difference is illustrated in Fig.\u00a01, which shows the phase plane of a unimanual rhythmic wrist movement that is synchronized to a continuous visual signal with a stationary frequency. As can be readily appreciated from the figure, the reduced variability at one of the endpoints (as indicated by the arrows) does not imply that the temporal locations of the visual signal (as indicated by the circles) coincided with the movement reversal points. Therefore, both aspects should be addressed in a complete analysis of anchoring behavior.\nFig.\u00a01Exemplary phase portrait of rhythmic isofrequency wrist oscillations during an in-phase visuo-motor tracking task. Spatial anchoring, that is points or regions of reduced movement variability, are typically observed at or around movement reversal points (viz. at peak wrist flexion (left) in this example). White and gray circles represent the time indices of left and right target turning points (i.e., the movement reversals of the visual metronome), which were used to determine temporal anchoring\nThe finding of Byblow et al. (1994) that anchoring depended on whether a pronation or a supination movement was performed was replicated by Byblow et al. (1995) and led to the notion that anchor points are differentiated in terms of their stability due to differences in musculoskeletal properties. Further support for this notion was found by Carson (1996) and Carson and Riek (1998) in an experimental set-up in which participants were instructed to either flex or extend their index finger to an auditory metronome. Performance was found to be more stable in the flex-on-the-beat pattern than in the extend-on-the-beat pattern, especially when the forearm was in a supine position as opposed to a prone or neutral position. With this series of studies, the notion of anchoring became confined to the situation in which a particular point in the movement cycle is synchronized with an auditory metronome, in spite of its more generic original definition. As a result of this narrowing, anchoring was reduced to a purely local effect, which gave Fink et al. (2000) the opportunity to emphasize that anchoring, so defined, also carries global consequences for the overall coordinative pattern, even though this aspect was already inherent to the original formulation of anchoring by Beek (1989). The point was driven home by showing that the bimanual coordination was more stable when both movement reversal points of each finger movement (i.e., peak flexion and peak extension) were paced or \u2018anchored\u2019 (so-called double-metronome condition) as opposed to when only one reversal point was \u2018anchored\u2019 (single-metronome condition). These effects were explicitly modeled by Jirsa et al. (2000) using a parametric stabilization term which preserves the stability properties of bimanual coordination as captured by the well-known model of Haken et al. (1985) for phase transitions in rhythmic arm and hand movements, while also explaining the varying stability of movement under the two metronome conditions.\nAnother form of anchoring in the literature on eye\u2013hand coordination is known as \u201cgaze anchoring\u201d, which refers to the phenomenon of an enforced ocular target fixation for the duration of the entire pointing movement (Neggers and Bekkering 2000, 2001, 2002). When pointing to a target the pointing movement is preceded by a saccadic eye movement to the target (Prablanc et al. 1979). This saccadic eye movement is not only correlated to the start of the arm movement, but the coupling between gaze and aiming movements is also observable after pointing initiation, and it appears that the central nervous system demands ocular fixation of the pointing target until pointing is completed (Neggers and Bekkering 2000, 2001, 2002). When participants do not foveate on the homing-in phase of movements, pointing accuracy deteriorates (Neggers and Bekkering 1999; Prablanc et al. 1979; Vercher et al. 1994), which suggests that gaze anchoring serves to facilitate the planning and execution of pointing movements.\nThe significance of gaze fixations for the dynamical characteristics of cyclical movements became apparent in a recent study on the (de)stabilizing effects of transformed feedback on visuo-motor tracking by Roerdink et al. (2005). When feedback was absent (i.e., during normal tracking), right endpoint variability was smaller than left endpoint variability when participants fixated at the right, whereas the converse was true when participants fixated at the left. Left and right endpoint variability was similar during gaze fixations at the center and when smoothly pursuing the target signal. Those findings may be interpreted to imply that participants actively created visuo-motor anchors by fixating their gaze at one of both endpoints, without being instructed to do so (i.e., gaze direction was not manipulated in the experiment). However, the observation that gaze fixations may promote motor anchoring does not exclude the possibility that musculoskeletal properties played into the observed anchoring phenomena as well. Indeed, Roerdink et al. (2005) also found clear indications that anchoring was affected by musculoskeletal properties. In the conditions with visual feedback gaze fixation at the right resulted in reduced right endpoint variability, as was the case without feedback, but now gaze fixation at the center also resulted in reduced endpoint variability at the right, while no anchoring was observed for gaze fixation at the left. Detailed analyses of the data revealed that, compared to the condition without visual feedback, participants had shifted the center of their hand excursions to the right in the conditions with visual feedback in an apparent effort to align the feedback signal with the target signal. This rightward shift could well have affected the relative contributions of antagonistic muscle groups (i.e., by increasing the active contribution of the wrist extensors and decreasing that of the wrist flexors to the hand excursions that were performed with the right hand), as well as the possibilities for storage and recovery of elastic strain energy in muscles and tendons. Although the viscosity of the wrist joint is negligible, and passive elastic torques are generally small, the latter become sizeable at larger wrist excursions (Lehman and Calhoun 1990), and are known to affect the muscular production of wrist movement (Schieber and Thach 1985). In sum, Roerdink et al. (2005) found firm support for visual underpinnings of anchoring in the form of anchoring promoting effects of gaze fixations, as well as clear indications of musculoskeletal contributions, in line with the existing evidence in the literature that anchoring phenomena are governed by both visual and musculoskeletal factors.\nHowever, as the preceding summary of the pertinent literature illustrates, the effects of visual and musculoskeletal factors of anchoring have not yet been investigated in any systematic fashion, and several theoretically important issues still need to be addressed. For one, the relative contributions of visual and musculoskeletal factors to anchoring are unknown, with researchers placing theoretical emphasis on either type of factor. Furthermore, it is unknown whether the effects of visual and musculoskeletal factors on anchoring are independent (i.e., additive) or dependent (i.e., interactive). To resolve those issues, we conducted an experiment that was specifically designed to systematically delineate the effects of visual and musculoskeletal factors on anchoring behavior in visuo-motor tracking. Our choice for this particular experimental task and set-up was motivated from our previous study on the effects of visual feedback on visuo-motor tracking, which revealed clear instances of visuo-motor anchoring with marked effects of gaze direction and strong hints of wrist posture effects, even though neither of these factors was varied explicitly (Roerdink et al. 2005). Based on those previous results, we expected that systematic manipulations of both factors\u2014with other variables such as tracking frequency and tracking mode being kept constant\u2014would have clearly discernible effects on anchoring, both in terms of its spatial and temporal characteristics. Due to the absence of relevant data, however, we had no specific expectations with respect to their relative contributions to anchoring or the possibility of interaction effects in that regard. In line with the original definition of anchoring, as well as the insights of Fink et al. (2000), we expected both gaze direction and wrist posture to not only influence local features of the dynamics of visuo-motor tracking but also its global organization.\nMethods\nParticipants\nThirteen persons (2 men and 11 women, aged 21\u201350\u00a0years) volunteered to participate in the study. All participants were right-handed, according to their scores on a shortened version of the Edinburgh handedness inventory (Oldfield 1971), and had normal or corrected to normal vision. Participants gave their written informed consent prior to the experiment, which was approved by the ethics committee of the Faculty of Human Movement Sciences at VU University Amsterdam.\nApparatus\nParticipants were seated on a height-adjustable chair behind a rack on which a vertically oriented manipulandum was mounted (Fig.\u00a02, upper panel). The manipulandum was attached to a rotatable horizontal lever whose axis was aligned with the wrist\u2019s flexion\u2013extension axis. The right hand was strapped to the flat manipulandum (fingers extended and thumbs up), allowing flexion and extension movements about the wrist. Angular position of the wrist was registered by means of a potentiometer (Labmaster, sampling frequency 1,000\u00a0Hz), which was positioned underneath the manipulandum. An armrest with two adjustable supports, located on both sides of the forearm just proximal of the wrist joint, was used to prevent forearm movement.\nFig.\u00a02Schematic representation of the experimental set-up. Upper panel Schematic side view of the experimental set-up. Lower panel LED bow from participant\u2019s perspective, with the calibration field superimposed by means of the nine white circles\nA semicircular projection bow was positioned in front of the participant at a distance of 113\u00a0cm from the rotation axis of the potentiometer, matching the radius of the bow (see Fig.\u00a02, lower panel). The projection bow consisted of a continuous array of 448 light-emitting diodes (LEDs). A harmonically oscillating target signal was projected on the LED bow. Concurrent visual feedback of the angular position of the wrist was provided by means of a laser pointer attached to the manipulandum pointing 22\u00a0mm below the trace of the target signal.\nThe participant\u2019s chin was placed on a height-adjustable chinrest and the head was secured with straps between two vertical supports to prevent head movements. A horizontal cover was attached to the chinrest, which prevented vision of the moving hand (Fig.\u00a02). Left eye orientation was monitored via reflection of pupil\u2013cornea boundaries in near-infrared light (Applied Systems Laboratories, series 5000 Eye Tracker, sampling frequency 50\u00a0Hz, spatial error <0.6\u00b0). To determine the participant\u2019s point-of-gaze, eye orientation was calibrated using a nine-point calibration field attached to the LED bow (Fig.\u00a02, lower panel). The experimenter received online feedback of the point-of-gaze on a video screen. Eye orientation was recalibrated when necessary (i.e., in case of an apparent mismatch between the actual point-of-gaze and its appearance on the video screen).\nThe rack with armrest and manipulandum could be positioned in different orientations with respect to the LED bow, allowing manipulation of wrist posture (see below). Each rack orientation was calibrated to the LED bow to facilitate direct comparison of target and feedback signals as well as for offline comparison of target signal and potentiometer data (both expressed in \u00b0). Point-of-gaze was expressed in \u00b0, allowing for a comparison of point-of-gaze and the target signal. Eye-tracker data were synchronized with potentiometer and LED bow data.\nProcedure\nIn the experimental trials, participants were instructed to follow the oscillating target signal. The amplitude of the target signal was selected to correspond to 20\u00b0 of hand motion, because Peper and Beek (1998) had established that this was, on average, the preferred amplitude for this kind of experimental task and set-up. The target signal oscillated at 1.8\u00a0Hz, that is, with a frequency at which frequency-locked point-of-gaze oscillations were found to be absent for this experimental task and set-up (cf. Roerdink et al. 2005).\nParticipants tracked the target signal under experimental manipulation of gaze direction and wrist posture. Participants were instructed to direct their gaze to one of three fixation points defined in relation to the oscillating target signal, that is, the left target turning point, the right target turning point, or the center in between the two target turning points (Fig.\u00a03). Wrist posture was manipulated by bringing the wrist in a flexed, neutral, or extended posture (Fig.\u00a03). In the flexed posture the rack with armrest and manipulandum was positioned such that the participant\u2019s forearm was rotated 40\u00b0 clockwise with respect to the neutral position, whereas in the extended posture the forearm was rotated 40\u00b0 counterclockwise with respect to the neutral posture. Prior to the experiment the participant\u2019s range of motion was determined: the hand was secured to the manipulandum and the voluntarily achieved maximal wrist flexion and extension positions were measured. The center of this range was considered the neutral wrist posture for the participant in question. In the neutral posture the wrist was on average flexed 12.7\u00b0 (\u00b13.5\u00b0) relative to the position in which the forearm was aligned with the palm of the hand. For all participants, the range of experimental wrist postures fell well within the individually determined maximal range of motion (154.6\u00b0\u00a0\u00b118.5\u00b0), covering on average 77.4% when the additional excursions corresponding to the amplitude of the target signal were taken into account.\nFig.\u00a03Schematic representation of the experimental design, consisting of all nine combinations of wrist posture (flexed, neutral, extended), induced by altering the orientation of the forearm with respect to the oscillating hand, and gaze direction (left, center, right), schematically represented by transparent rectangles\nThe three wrist postures (i.e., flexed, neutral, and extended) were crossed with the three gaze directions (i.e., left, center, and right region of the target signal), resulting in nine experimental conditions (see Fig.\u00a03 for a schematic representation). Participants performed all conditions five times, amounting to a total of 45 trials per participant. Trials were presented in blocks with the three wrist postures providing the first level of blocking (3\u00a0\u00d7\u00a015 trials) and gaze direction (3\u00a0\u00d7\u00a05 trials) the next. The order of the wrist posture blocks was counterbalanced over participants (with each order being performed by two participants; full counterbalancing was possible because the data of one participant were discarded, see below). The gaze direction blocks within the wrist posture blocks were presented in random order. Prior to each wrist posture block, participants were invited to familiarize themselves with each new wrist posture through making self-paced wrist movements. The laser pointer provided direct feedback of the hand movement during both familiarization and experimental trials. Participants were instructed to move their hand in phase with the oscillating target signal as accurately as possible while directing their gaze to the instructed fixation point (left, center, or right). To facilitate trial initiation, the target signal always started at the attended area. All trials lasted 20\u00a0s (i.e., 36 cycles).\nData analysis\nThe data of one participant were excluded from the analysis because the point-of-gaze data were disturbed (i.e., pupil\u2013cornea boundaries were often undetectable due to \u2018squeezing\u2019 of the eyes, i.e., partial eye closures). For the remaining 12 participants point-of-gaze was assessed (in \u00b0) for each trial to ensure that they had adhered to the task instructions regarding gaze direction. Specifically, the horizontal point-of-gaze data were classified according to three groups, viz. left, center and right, defined by regions of \u00b110\u00b0 centered around \u221220\u00b0 (left target turning point), around 0\u00b0 (center in between the two target turning points) and around +20\u00b0 (right target turning point), respectively. As is common in eye-tracking, missing or disturbed values occasionally occurred due to blinking or brief eye closures. For a trial to be included in the analyses, at least 80% of the horizontal point-of-gaze samples had to fall within the range from \u221230\u00b0 to 30\u00b0, while in turn at least 80% of those samples had to fall within the instructed region. Based on this nested criterion, 63 out of the total of 540 trials were discarded. For the 477 included trials gaze was directed to the instructed region (i.e., left, center or right) on average 98.8% of the time. All participants successfully performed the tracking task in that the mean tracking frequency, defined as the inverse of the period between maximal extensions of the wrist, of the included trials was 1.80\u00a0Hz with a very small overall standard deviation (\u00b14.0\u00a0\u00d7\u00a010\u22125\u00a0Hz). One trial was removed because of phase wrapping, which occurred to catch up after a late start.\nPre-processing\nPotentiometer data (hand movement) and LED coordinates (target) of the included trials were transformed into \u00b0 and low-pass filtered using a bi-directional second-order Butterworth filter (cut-off frequency: 15\u00a0Hz). The first five cycles of each trial were excluded from analysis to eliminate possible transient effects. From the remaining 31 cycles several dependent variables were calculated. Those variables related to tracking performance, anchoring, and global kinematics, respectively.\nTracking performance\nTracking accuracy was determined by calculating the root mean square (RMS) of the continuous error between target and hand movement, which was obtained by subtracting the actual position from the required position. In addition, the continuous relative phase (in \u00b0) between the target signal and the hand movement was calculated by subtracting the phase of the hand oscillations from that of the target oscillations. Mean relative phase (\u03d5) and its transformed circular variance (TCV) were quantified using circular statistics (cf. Burgess-Limerick et al. 1991; Mardia 1972). \nAnchoring\nIn both discretely and continuously paced cyclical movements, as well as in \u2018self-paced\u2019 rhythmic movements, anchoring typically occurs at, or around, the movement reversal points (see also Fig.\u00a01), where it may become manifest as a reduction of spatial (e.g., Byblow et al. 1994, 1995; Fink et al. 2000; Roerdink et al. 2005) or temporal (e.g., Roerdink et al. 2007) variability, or both. We therefore determined (a) the spatial variability of maximal wrist flexion and extension excursions by calculating the respective standard deviations (SD in \u00b0), and (b) the temporal variability between the time instances of the left and right target turning points and the corresponding time instances of maximal wrist flexion and extension excursions (SD in ms). To quantify the presence or absence of anchor points in the tracking trajectories, variants of the following general anchoring index (AI, dimensionless) were calculated in which SDl and SDr represent the spatial or temporal variability corresponding to the left and right target turning point, respectively. If SDl and SDr are equal, AI\u00a0=\u00a00.5. AI\u00a0<\u00a00.5 corresponds to smaller spatial or temporal variability at the left than at the right target turning point (i.e., anchoring at peak flexion), whereas the opposite is true for AI\u00a0>\u00a00.5 (i.e., anchoring at peak extension). AIspatial and AItemporal denote the anchoring index for spatial and temporal variability, respectively.\nThe anchoring index captures local effects of wrist posture and gaze direction in predefined regions of the tracking trajectories. In theory, anchor points or regions may be found elsewhere in the movement cycle, but a first inspection of the data indicated that anchoring occurred at or near the endpoints (see also Fig.\u00a01), as indeed was found in the previous studies on anchoring cited in the Introduction. However, to avoid that we would miss important kinematic aspects of the tracking trajectories, we not only focused on the effects of wrist posture and gaze direction around movement reversal points, but also on more global properties of the tracking trajectories.\nGlobal kinematics\nThe global properties of the tracking trajectories were assessed by means of the velocity profiles (i.e., wrist angular velocity as a function of cycle time) and Hooke\u2019s portraits (i.e., wrist angular acceleration as a function of wrist angular position). To assess those properties, wrist angular position time series were normalized to the amplitude of the target signal (i.e., \u22121 implies target turning point on the flexion side, +1 implies target turning point on the extension side), after which the normalized position time series were mean centered. Next, velocity and acceleration time series were computed from the position time series by means of a conventional 3-point difference algorithm and normalized to the angular velocity of the target signal (i.e., divided by 3.6\u03c0). Based on the minima of the position time series, corresponding to maximal wrist flexion, each cycle was cut from the velocity time series (i.e., from maximal flexion via maximal extension to maximal flexion). The individual velocity profiles were time-normalized to 200 points using a spline interpolation procedure, i.e., a normalization to percentage cycle time, and the average velocity profile was calculated for each participant for each included trial. From this average velocity profile the duration of flexion and extension movements were taken and summarized in the movement duration index (MDI, dimensionless), expressed as: in which MDflexion (MDextension) is the duration from maximal wrist extension (flexion) to maximal wrist flexion (extension). If MDflexion and MDextension are equal, MDI\u00a0=\u00a00.5. MDI\u00a0<\u00a00.5 corresponds to shorter duration of the flexion than the extension half cycle, whereas the opposite is true for MDI\u00a0>\u00a00.5. Furthermore, peak velocities for the flexion and extension movements were taken (i.e., PVflexion and PVextension, respectively) and represented in the peak velocity index (PVI, dimensionless), expressed as: \nIf PVflexion and PVextension are equal, PVI\u00a0=\u00a00.5. PVI\u00a0<\u00a00.5 indicates that flexion peak velocity is smaller than extension peak velocity, whereas the opposite is true for PVI\u00a0>\u00a00.5. Note that for harmonic wrist oscillations, PVI and MDI both approach 0.5.\nThe Hooke\u2019s portraits were constructed as follows. Based on the extrema in the normalized wrist angular position time series, corresponding to peak flexion and extension, each half cycle was cut from the position and acceleration time series (i.e., from peak flexion to peak extension and from peak extension to peak flexion). The position and acceleration half cycles were time-normalized to 100 points using a spline interpolation procedure. Next, the average position and acceleration time series were computed for every half cycle for each trial of each participant. A linear (harmonic) oscillator implies a straight line in the Hooke\u2019s portrait, whereas a deviation from a straight line represents the contribution of nonlinear components. The amount of variance that can be attributed to a harmonic oscillation can be readily quantified by the r2 of the linear regression of position onto acceleration (i.e., r2\u00a0=\u00a01 for a purely harmonic oscillation such as the target signal). The explained variance of the summed contribution of nonlinear terms was expressed as NL\u00a0=\u00a01 \u2212 r2 (Mottet and Bootsma 1999; for a similar approach see Beek and Beek 1988).\nStatistical analysis\nTo determine the effects of wrist posture and gaze direction on tracking behavior, each dependent variable was submitted to a repeated measures analysis of variance (ANOVA) with within-subject factors wrist posture (3 levels: flexed, neutral, extended) and gaze direction (3 levels: left, center, right). Individual scores per condition were obtained by averaging the values of the included trials for the condition in question. Significant effects (P\u00a0<\u00a00.05) are reported, and effect sizes are represented as partial eta squared values \nPost-hoc analysis was performed using two-tailed paired-samples t tests (with Bonferroni correction). One-sample t tests were conducted to see whether AIspatial and AItemporal differed significantly from 0.5 (the value indicating that spatial or temporal variability corresponding to the left and right target turning point are similar).\nResults\nEffects of wrist posture\nWrist posture had significant effects on the variability of the relative phase between tracking and the target signal (TCV), the spatial and temporal anchoring indices (AIspatial, AItemporal), the velocity profile characteristics (MDI, PVI) and the harmonicity (NL) of tracking trajectories (see Table\u00a01). Tracking accuracy (RMS; mean\u00a0\u00b1\u00a0standard error\u00a0=\u00a06.0\u00b0\u00a0\u00b10.3\u00b0) and mean relative phase (\u03d5; \u22125.2\u00b0\u00a0\u00b12.1\u00b0, indicating that wrist oscillations were slightly leading the target signal) did not differ significantly over wrist postures. Post-hoc analysis indicated that TCV was significantly larger for the extended wrist posture (21.7\u00b0\u00a0\u00b11.2\u00b0) than for the flexed posture (19.4\u00b0\u00a0\u00b10.9\u00b0), while TCV for the neutral posture (19.9\u00b0\u00a0\u00b11.4\u00b0) was not significantly different from the flexed and extended posture.Table\u00a01Results of the repeated measures ANOVA on dependent variablesDependent variableWrist postureGaze directionPosture\u00a0\u00d7\u00a0gazeF(2, 22)PF(2, 22)PF(4, 44)PRMS1.67NS0.130.60NS0.051.85NS0.14\u03d50.49NS 0.049.46<0.0050.460.84NS0.07TCV4.38<0.050.281.39NS 0.111.20NS0.10AIspatial36.10<0.0010.7781.60<0.0010.880.39NS0.03AItemporal32.55<0.0010.750.26NS 0.021.39NS0.11PVI48.25<0.0010.8113.11<0.0010.541.45NS0.12MDI41.73<0.0010.7914.79<0.0010.571.55NS0.12NL7.09<0.0050.393.13NS 0.221.73NS0.14Main effects of wrist posture and gaze direction and wrist posture\u00a0\u00d7\u00a0gaze direction interaction effects are presentedRMS root mean square of the continuous error between target and hand movement, \u03d5 mean relative phase between target and hand movement, TCV transformed circular variance of \u03d5, AIspatial spatial anchoring index, AItemporal temporal anchoring index, PVI peak velocity index, MDI movement duration index, NL contribution of nonlinear terms to tracking trajectories \nAIspatial for the extended wrist posture differed significantly from that for the neutral and flexed posture, while AItemporal differed significantly between all wrist postures. Importantly, both anchoring indices differed from 0.5 for the flexed and extended postures, but not for the neutral posture (see Fig.\u00a04, left panels). One-sample t tests revealed that AIspatial and AItemporal were significantly smaller (0.450 and 0.487, respectively) than 0.5 in the flexed posture (t(11)\u00a0=\u00a0\u22124.72, P\u00a0<\u00a00.001 and t(11)\u00a0=\u00a0\u22123.57, P\u00a0<\u00a00.005, respectively) and significantly greater (0.601 and 0.525, respectively) than 0.5 in the extended posture (t(11)\u00a0=\u00a010.01, P\u00a0<\u00a00.001 and t(11)\u00a0=\u00a06.33, P\u00a0<\u00a00.001, respectively), indicating that anchoring occurred at maximal flexion and extension, respectively.Fig.\u00a04Spatial and temporal anchoring indices as a function of wrist posture (left panels) and gaze direction (right panels). Asterisks indicate significant differences with AI\u00a0=\u00a00.5. Error bars represent standard error\nThese indications of anchoring were accompanied by significant changes in the tracking trajectories, as can be appreciated from the averaged velocity profiles depicted for each wrist posture in Fig.\u00a05 (left column). The cycle duration of the flexion (extension) phase was shorter than 50% for tracking with the wrist in a flexed (extended) posture. Furthermore, flexion peak velocity was higher than extension peak velocity for tracking with a flexed wrist posture, while the opposite was the case for tracking with an extended wrist posture. In a neutral wrist posture, the velocity profile was nearly harmonic, with similar flexion and extension half cycle durations and peak velocities. These observations were all confirmed statistically. Specifically, a significant main effect of wrist posture was observed for PVI and MDI (see Table\u00a01): post-hoc analysis indicated that PVI and MDI differed significantly for all three wrist postures. One-sample t tests revealed that, in the flexed wrist posture, the duration of the flexion phase was significantly shorter than that of the extension phase (i.e., MDI\u00a0=\u00a00.473; t(11)\u00a0=\u00a0\u22123.47, P\u00a0<\u00a00.01), while flexion peak velocity was significantly higher than extension peak velocity (i.e., PVI\u00a0=\u00a00.539; t(11)\u00a0=\u00a03.92, P\u00a0<\u00a00.005). In the extended wrist posture, the converse was true (MDI\u00a0=\u00a00.538; t(11)\u00a0=\u00a07.52, P\u00a0<\u00a00.001; PVI\u00a0=\u00a00.444; t(11)\u00a0=\u00a0\u22126.53, P\u00a0<\u00a00.001), indicating shorter extension phase duration and higher extension peak velocity. In the neutral wrist posture condition, both PVI (0.510) and MDI (0.494) did not differ significantly from 0.5.Fig.\u00a05Mean normalized velocity profiles as a function of wrist posture (left panel) and gaze direction (right panel). For a harmonic oscillator extension and flexion peak velocities equal 1 and \u22121, respectively, while the zero crossing of the velocity profile occurs at the vertical dashed line, indicating that the extension and flexion phase have the same duration\nThe most striking aspect of the Hooke\u2019s portraits was the general tendency to deviate from a straight line (representing harmonic oscillation) with the wrist flexed or extended. Fig.\u00a06 shows averaged Hooke\u2019s portraits of the flexion and extension half cycles for each combination of wrist posture and gaze direction. The panels in the rows show the Hooke\u2019s portraits for each wrist posture. As is evident from the figure, the harmonicity was reduced in the flexed and extended wrist posture compared to the neutral wrist posture (larger deviations from the line acceleration\u00a0=\u00a0\u2212position), especially for tracking with the wrist extended. This was also reflected in the variance that was attributed to nonlinear components as quantified by NL (reflecting the residual of the linear regression of position onto acceleration). In particular, a significant main effect of wrist posture was observed for NL (see Table\u00a01). Post-hoc analysis revealed that the contribution of nonlinear terms was significantly greater in the extended wrist posture compared to the neutral wrist posture (NL\u00a0=\u00a00.154\u00a0\u00b1\u00a00.02 and 0.073\u00a0\u00b1\u00a00.01, respectively). NL for tracking with the wrist in a flexed posture (NL\u00a0=\u00a00.110\u00a0\u00b1\u00a00.02) did not differ significantly from the other two wrist postures. Note that the reduced harmonicity of tracking in the extended wrist posture may well have caused the significant increase in TCV in the extended wrist posture relative to that of the flexed posture.Fig.\u00a06Hooke\u2019s portraits as a function of gaze direction (columns: left, center, right) and wrist posture (rows: flexed, neutral, extended) with flexion and extension half cycles indicated in gray and black, respectively. The dotted black line represents the Hooke\u2019s portrait for the harmonically moving target signal\nEffects of gaze direction\nGaze direction had significant effects on mean continuous tracking relative phase (\u03d5), the spatial anchoring index (AIspatial) and velocity profile properties (MDI, PVI), but not on tracking accuracy (RMS), relative phase variability (TCV), the temporal anchoring index (AItemporal) and the harmonicity (NL) of tracking trajectories (see Table\u00a01). Post-hoc analysis revealed that \u03d5 was significantly less negative when gaze was directed to the right (\u22121.6\u00b0\u00a0\u00b12.6\u00b0) than when gaze was directed to the left (\u22126.5\u00b0\u00a0\u00b11.4\u00b0) or the center (\u22127.5\u00b0\u00a0\u00b11.9\u00b0) region.\nPost-hoc analysis further revealed that AIspatial differed significantly between all three gaze directions. Moreover, one-sample t tests revealed that AIspatial was significantly smaller than 0.5 when gaze was directed to the left target turning point (0.433; t(11)\u00a0=\u00a0\u22126.73, P\u00a0<\u00a00.001), indicating smaller left (corresponding to maximal flexion excursions) than right (corresponding to maximal extension excursions) endpoint variability (see Fig. 4, upper right panel). Similarly, AIspatial was significantly larger than 0.5 when gaze was directed to the right target turning point (0.593; t(11)\u00a0=\u00a07.77, P\u00a0<\u00a00.001), indicating anchoring at the right (extension) endpoint. AIspatial was not significantly different from 0.5 when gaze was directed at the center region between the two target turning points (AIspatial\u00a0=\u00a00.515). Whereas gaze direction had a strong effect on the spatial anchoring index  the effect of gaze direction on AItemporal was not significant, as can be appreciated from Fig.\u00a04 (lower right panel; see also Table\u00a01).\nThe right panel in Fig.\u00a05 depicts the averaged velocity profiles as a function of gaze direction. The effect of gaze direction on the velocity profile characteristics PVI and MDI was much smaller than that of wrist posture (i.e., smaller deviations from 0.5; compare also the corresponding effect sizes in Table\u00a01). Nevertheless, the significant main effect of gaze direction entailed that when gaze was directed to the right target turning point PVI and MDI were significantly different from the other two gaze directions (for left, center, and right, respectively: PVI\u00a0=\u00a00.508, 0.499, and 0.486; MDI\u00a0=\u00a00.494, 0.500, and 0.511). One-sample t tests indicated that when gaze was directed to the target turning points (left or right), the hand movements in that direction were shorter in duration with a larger peak velocity. As is apparent from a comparison of the Hooke\u2019s portraits depicted in the columns of Fig.\u00a06, gaze direction only had a limited influence on the nonlinear terms in the hand oscillations (particularly if compared to the effect of wrist posture, as presented in the three rows of Fig.\u00a06). The effect of gaze direction on NL was not significant (left 0.115\u00a0\u00b1\u00a00.01; center 0.105\u00a0\u00b1\u00a00.01; right 0.118\u00a0\u00b1\u00a00.01).\nWrist posture\u00a0\u00d7\u00a0gaze direction interaction effects\nNone of the dependent variables showed a significant wrist posture\u00a0\u00d7\u00a0gaze direction interaction effect (see Table\u00a01), implying that the respective effects of the two factors were independent and additive in case both yielded significant main effects. A case in point is the spatial anchoring index AIspatial (see Table\u00a01). Both wrist posture and gaze direction had a significant effect on AIspatial in the absence of a significant wrist posture\u00a0\u00d7\u00a0gaze direction interaction effect. Fig.\u00a07, presenting AIspatial for all combinations of wrist posture and gaze direction, illustrates the additive manner in which the two factors affected spatial anchoring (cf. the \u2018staircase\u2019 pattern in each wrist posture condition).\nFig.\u00a07Spatial anchoring index for all combinations of wrist posture (flexed, neutral, extended) and gaze direction (left [L], center [C], right [R]). Asterisks indicate significant differences with AIspatial\u00a0=\u00a00.5. Error bars represent the standard error\nDiscussion\nIn the present experiment we sought to systematically delineate the effects of visual and musculoskeletal factors on anchoring phenomena, that is, local reductions in kinematic variability, which may reflect \u201corganizing centers\u201d for perceptual-motor control. In particular, we investigated the relative contributions of gaze direction and wrist posture on both spatial and temporal anchoring in the performance of a rhythmic, unimanual visuo-motor tracking task. In addition, we examined the velocity profiles and Hooke\u2019s portraits of the full tracking trajectories to gain insight into the relationship between the local anchoring phenomena and the global organization of the tracking movements. In the following, we first outline the main findings of both types of analysis before discussing their broader implications for the theoretical understanding of anchoring.\nEffects on spatial and temporal anchoring \nAs stated in the introduction, previous research revealed that the degree of anchoring is a function of musculoskeletal properties, such as the type of movement performed (i.e., flexion versus extension, pronation vs. supination) and the relative length of flexor and extensor muscles. Besides musculoskeletal influences, Roerdink et al. (2005) also reported evidence for visual underpinnings in that anchoring was found to depend on gaze direction. Based on those findings we expected that systematic manipulations of gaze direction and wrist posture would have clearly discernable effects on anchoring, which was indeed the case.\nBoth gaze direction and wrist posture significantly affected endpoint variability in the expected direction (see Fig.\u00a04, upper panels). Specifically, when gaze was directed to the left target turning point, left (i.e., flexion) spatial endpoint variability was reduced while the converse was true when gaze was directed to the right target turning point. In contrast, differential anchoring effects were absent when gaze was fixated in between the two target turning points. Furthermore, endpoint variability was smaller for the extension endpoint than for the flexion endpoint when the wrist was in an extended posture, whereas differential anchoring effects were absent when tracking was performed with the wrist in a neutral posture. All of these findings are fully in line with those of Roerdink et al. (2005), even though gaze direction and wrist posture were systematically manipulated only in the present study. Moreover, the current systematic variations of wrist posture also revealed that for the flexed wrist posture flexion endpoint variability was lower than extension endpoint variability.\nIn addition to spatial anchoring we examined temporal anchoring, that is, local reductions in the variability of the timing of the maximal movement excursions relative to the reversal points of the visual metronome, an aspect that was not addressed in previous studies. This complementary analysis revealed that temporal anchoring was affected by wrist posture but not by gaze direction, suggesting that wrist posture had a different, more pronounced effect on anchoring than gaze direction (see Fig.\u00a04, lower panels). Specifically, with the wrist in a flexed posture the variability in the timing of flexion excursions relative to the target was lower than that of extension excursions while the converse was true with the wrist extended. No differential effect in timing variability was observed for tracking with the wrist in a neutral posture nor for the three different gaze directions. These findings suggest that movements were actively timed or anchored on maximal flexion with the wrist in a flexed posture and on maximal extension with the wrist in an extended posture. In contrast, the absence of an effect of gaze direction on temporal anchoring may imply that gaze direction only affected the spatial accuracy of the tracking movements but not their timing.\nFor reasons explained in the introduction, we had no specific expectations with respect to the issue whether the effects of visual and musculoskeletal factors on anchoring were independent (i.e., additive) or dependent (i.e., interactive) in nature. The results indicated that gaze direction and wrist posture affected anchoring in an additive rather than interactive fashion (see Fig.\u00a07), because no significant interactions between gaze direction and wrist posture were found for any of the dependent variables (see Table\u00a01). The fact that gaze direction affected only spatial anchoring and not temporal anchoring provided further support for the conclusion that the contributions of gaze direction and wrist posture to anchoring were independent.\nEffects on global tracking behavior \nThe analyses of the velocity profiles and Hooke\u2019s portraits of the full tracking trajectories revealed that wrist posture had a much larger effect than gaze direction on the global tracking behavior. As can be observed in Fig.\u00a05, velocity profiles were asymmetric when tracking was performed with the wrist flexed or extended as opposed to a neutral wrist configuration, which resulted in symmetric, bell-shaped velocity profiles. The three different gaze directions only affected the velocity profiles to a limited yet significant degree. Congruently, wrist posture affected the harmonicity of the wrist movements much more than gaze direction, as can be appreciated from Fig.\u00a06 (viz. greater deviation from the straight line representing harmonic oscillation).\nThose qualitative observations were reflected in significant differences in the duration, peak velocity and harmonicity of the flexion and extension phases of the movement as determined from the velocity profiles and Hooke\u2019s portraits. Wrist posture strongly affected the global tracking behavior and led to marked differences between flexion and extension for wrist postures other than neutral. For the flexed (extended) wrist posture, the flexion (extension) phase lasted shorter, had higher peak velocity and was less harmonic than the extension (flexion) phase. Gaze direction also significantly affected movement duration and peak velocity of the flexion and extension half cycles, albeit to a much smaller degree. If gaze was directed to the left (right) target turning point, the duration of the flexion (extension) phase was shorter than that of the extension (flexion) phase, accompanied by higher flexion (extension) peak velocity. When gaze was fixated in between the two target turning points velocity profiles were symmetric. Tracking harmonicity was not significantly affected by gaze direction.\nFurthermore, Hooke\u2019s portraits for tracking with the wrist flexed and extended should be rich in information about specific conservative and dissipative nonlinear components giving rise to the observed anharmonicity (Beek and Beek 1988; see also Beek et al. 1992; Buchanan et al. 2003, 2006; Mottet and Bootsma 1999, 2001). As can be appreciated from Fig.\u00a06, local stiffness tended to increase towards the anchored endpoint, indicating a so-called hardening spring corresponding to an additional Duffing term in the equation of motion (Beek and Beek 1988; Mottet and Bootsma 2001). Besides this nonlinear conservative term, also nonlinear dissipative terms appeared to be operative towards the anchored endpoint given the asymmetry in the acceleration and deceleration parts of the half cycle in question. Considering that zero acceleration (i.e., peak velocity) occurred in the first part of the flexion (extension) phase for a flexed (extended) posture, a self-sustaining Rayleigh oscillator is a likely candidate for the dissipative terms contributing to the anharmonic tracking behavior (Beek and Beek 1988; Mottet and Bootsma 2001). Finally, visual inspection of the nine panels of Fig.\u00a06 suggests a graded modulation of those nonlinear conservative and dissipative components as a function of wrist posture and gaze direction, underscoring again that both factors had an additive rather than an interactive effect on the organization of tracking.\nMusculoskeletal underpinnings of anchoring\nThe marked effects of wrist posture on anchoring and the global organization of tracking may be related to a variety of musculoskeletal factors including the possibility of saving mechanical energy from half cycle to half cycle owing to the ability of muscles to store mechanical energy in a potential, elastic form towards the end of each half cycle to facilitate the production of the next half cycle (Guiard 1993). Although this storage and release of elastic strain energy in muscles and tendons is considered to play a limited role in rhythmic wrist movements, passive elastic torques become sizeable at larger wrist excursions (Lehman and Calhoun 1990; Schieber and Thach 1985). Hence, this energy-saving mechanism may well be relevant for tracking with the wrist flexed or extended and underlie the conservative and dissipative terms identified in the Hooke\u2019s portraits, as will be discussed further below.\nThe temporal anchoring analysis revealed that movements were actively timed or anchored on maximal flexion with the wrist in a flexed posture and on maximal extension with the wrist in an extended posture. In either case, movement durations were shorter (and peak velocities larger) towards the anchored endpoint. Following a similar line of reasoning, the observed deviation from harmonicity in the Hooke\u2019s portraits for a flexed (extended) wrist posture suggests that flexion (extension) movements are actively steered to a specific point in the perceptual-motor workspace (i.e., the movement cycle is anchored on maximal flexion (extension)), while the extension (flexion) half cycle simply serves to bring the hand out again for the next flexion (extension), perhaps in part passively through the release of potential energy stored in the elastic tissues around the wrist. The observed local and global features of tracking are surely consistent with such an interpretation.\nIn any case, in a flexed (extended) wrist posture, the flexion (extension) reversal point or phase indeed appears to act as an \u201corganizing center\u201d within and for the entire cycle production (Beek 1989; Beek et al. 1992). Thus, by actively timing or controlling the flexion (extension) phase, the movement cycle as a whole is produced to satisfy the imposed synchronization demand of the tracking task. Beek (1989) submitted that anchor points contain critical task-specific information for organizing a cyclical act. The results of the present study suggested that the anchor points may also possess functional task-specific mechanical properties that can be utilized in the organization of the cyclical act (in our case, the possibility to store and release energy). Tracking was controlled in a (partly) discrete manner, with the actively controlled flexion (extension) phase apparently acting as a kind of biological escapement for fueling the potential energy capacity of the extensors (flexors).\nVisual underpinnings of anchoring\nThe absence of interaction effects between gaze direction and wrist posture, in combination with the observations that gaze direction did not affect temporal anchoring and that the global tracking dynamics was more strongly influenced by wrist posture than by gaze direction implied that both factors had different and independent effects on the tracking dynamics. The results strongly suggested that gaze direction predominantly affected the spatial accuracy of the tracking movements and (by and large) not its temporal organization, and is thus associated only with spatial control. Surely, this conclusion is consistent with the results of ample pointing, aiming and reaching studies. For example, pointing accuracy suffers when participants do not foveate at a target during the homing-in phase of aiming movements (e.g., Bekkering et al. 1995; Neggers and Bekkering 1999; Prablanc et al. 1979; Vercher et al. 1994). The tight coupling between gaze and aiming movements is underscored further by the finding that the eyes cannot saccade away from the target until the aiming movement is completed, which has led to the insight that this so-called \u201cgaze anchoring\u201d serves the planning and execution of pointing movements (Neggers and Bekkering 2000, 2001, 2002). Although online concurrent visual feedback of the tracking movements was provided in the present study, the positive effect of gaze direction (or \u201cgaze anchoring\u201d in general) on spatial endpoint accuracy appears independent of the availability of visual information from the pointing or tracking movement itself (either directly or indirectly through extrinsic visual feedback). For example, Neggers and Bekkering (2001) also observed \u201cgaze anchoring\u201d phenomena without vision of the moving arm, while Roerdink et al. (2005) observed reduced endpoint variability in the direction of gaze during tracking in the absence of visual tracking feedback and vision of the moving hand. These findings indicate that the spatial anchoring results mediated by gaze direction observed in the present experiment do not necessarily imply increased accuracy in the foveated direction due to an alignment of visual target and feedback signals, but that also more generic \u201cgaze anchoring\u201d mechanisms (see Neggers and Bekkering 2000, 2001, 2002) are implicated in the observed anchoring phenomena. Future studies should be conducted to reveal the precise contribution of \u201cgaze anchoring\u201d on endpoint variability during rhythmic tracking, for instance by comparing unimanual in-phase and antiphase tracking (without direct or extrinsic visual feedback of the moving hand) under fixed left, center, and right gaze directions.\nTheoretical implications with regard to anchoring\nOver the years the study, and with it the notion, of anchoring has become confined to synchronization of a particular point in the movement cycle with a discrete or continuous metronome. Against this development we would like to revive the more generic definition of anchoring as originally given by Beek (1989), who described anchor points as \u201corganizing centers within, and for\u201d cyclical movements. Inherent to this description are at least four theoretically and methodologically relevant features. First, anchoring can also occur without an external pacer, as has been reported for juggling (Beek 1989; see also Beek et al. 1992) and self-paced wrist cycling (Byblow et al. 1994). In a similar vein, we expect that the present results regarding wrist posture will be preserved with self-paced rhythmic wrist movements. Second, in the case of paced movements, performing a task at the prescribed frequency does not necessarily imply anchoring (e.g., as a case in point, in the present study AI\u2019s did not differ from 0.5 for a neutral wrist posture when gaze was fixated in between the two target turning points, see Fig.\u00a07). Conversely, it is only useful and possible to examine anchoring to a pacing signal if the task is performed at, or at least very close to, the prescribed frequency. Third, anchoring is not a purely local effect but has implications for the overall coordination (Beek 1989; Fink et al. 2000; Jirsa et al. 2000; Maslovat et al. 2006), as is captured in the statement that anchor points serve as organizing centers within and for the entire cycle production (Beek 1989). By timing the movement to a particular point in the cycle, the movement cycle as a whole is timed and stabilized. Pattern stability may be improved, however, by employing more than one anchor point (Fink et al. 2000; Jirsa et al. 2000; Maslovat et al. 2006). Finally, although in cyclical tasks anchoring is typically observed in the form of a local reduction of kinematic variability at or near the movement endpoints, anchor points may in principle be observed anywhere throughout the cycle (e.g., the point of ball release in juggling; Beek 1989). Moreover, anchor points are not fixed in the perceptual-motor workspace but can be actively created or assembled, for example, by fixating gaze at one of both target turning points (Roerdink et al. 2005).\nCoda\nIn the present study we delineated the effects of gaze direction and wrist posture on anchoring phenomena. A novel methodological feature of the study was that the analysis of spatial endpoint variability was complemented with a temporal counterpart, by quantifying the timing variability between movement reversals and target turning points in an analogous manner. We found that both visual and musculoskeletal factors affected spatial and temporal anchoring phenomena in different ways: the former by making use of task-specific visual information available at the gaze anchored point, the latter by exploiting task-specific mechanical properties. The results are in line with the original definition of anchoring (Beek 1989; see above), which was broadened by suggesting that, besides perceptual information, task-specific mechanical properties may also be available and actively utilized at the anchor point in favor of task performance or task economy.\nFinally, we would like to emphasize that anchoring is a generic aspect of coordination dynamics that may have considerable potential for practical applications, such as in learning complex coordination patterns (e.g., Maslovat et al. 2006) or in restoration of pathological gait (e.g., Roerdink et al. 2007). For example, the latter study on the effect of acoustically paced treadmill walking on gait coordination in stroke patients found that the footfalls of the nonparetic limb were anchored to the tones of the metronome (Roerdink et al. 2007). The analysis of temporal anchoring provided insight into the manner in which asymmetric gait patterns are coordinated to symmetric acoustic pacing rhythms. More importantly, the notion of anchoring may also provide an entry point for improving the organization of paced walking in stroke patients in physical therapy (Roerdink et al. 2007), for example, by instructing stroke patients to coordinate or anchor footfalls of the paretic limb to the beat of the metronome.","keyphrases":["anchoring","eye\u2013hand coordination","visual information","coordination dynamics","manual tracking","wrist position","visuo-motor control","movement trajectories"],"prmu":["P","P","P","P","M","R","R","R"]}
{"id":"Virchows_Arch-4-1-2329727","title":"Presence of HIF-1 and related genes in normal mucosa, adenomas and carcinomas of the colorectum\n","text":"Expression of the transcription factor hypoxia-inducible factor 1 (HIF-1), which plays a key role in cellular adaptation to hypoxia, was investigated in normal colorectal mucosa (ten), adenomas (61), and carcinomas (23). Tissue samples were analyzed for HIF-1\u03b1, its upstream regulators, von Hippel\u2013Lindau factor, AKT, and mammalian target of rapamycin (mTOR) and its downstream targets glucose transporter 1 (GLUT1), carbonic anhydrase IX, stromal-cell-derived factor 1 (SDF-1) by immunohistochemistry. In normal colorectal mucosa, HIF-1\u03b1 was observed in almost all nuclei of surface epithelial cells, probably secondary to a gradient of oxygenation, as indicated by pimonidazole staining. The same staining pattern was present in 87% of adenomas. In carcinomas, HIF-1\u03b1 was present predominantly around areas of necrosis (78%). Active AKT and mTOR, were present in all adenomas, carcinomas, and in normal colorectal mucosa. GLUT1 and SDF-1 were present in the normal surface epithelium of all adenoma cases, whereas in the carcinoma GLUT1 was located around necrotic regions and SDF-1 was present in all epithelial cells. In conclusion, HIF-1\u03b1 appears to be physiologically expressed in the upper part of the colorectal mucosa. The present observations support that upregulation of HIF-1\u03b1 and its downstream targets GLUT1 and SDF-1 in colorectal adenomas and carcinomas may be due to hypoxia, in close interaction with an active phosphatidylinositol 3-kinases\u2013AKT\u2013mTOR pathway.\nIntroduction\nColorectal carcinomas arise from normal epithelial cells of the colorectal mucosa through an adenoma stage. The transition from benign adenomas to malignant carcinomas, occurring in about 5% of all adenomas, is only beginning to be explored in appreciable detail. Biological changes involved in this process include important signaling pathways like the mammalian target of rapamycin (mTOR)\u2013AKT pathway while recently also a role for hypoxia-driven signaling was shown [6, 14]. Both pathways are mediated by the transcription factor hypoxia-inducible factor 1 (HIF-1) as depicted in Fig.\u00a01. HIF-1 is involved in cellular adaptation to hypoxia in solid tumors, including stimulation of angiogenesis and glycolysis [33]. HIF-1 consists of two subunits of which the \u03b2-subunit is constitutively expressed, whereas HIF-1\u03b1 is regulated in particular by the oxygen level. Under normoxia, the HIF-1\u03b1 protein is hydroxylated by prolyl hydroxylases [15, 16], which enables the von Hippel\u2013Lindau factor (VHL) to bind, resulting in ubiquination and rapid degradation by the proteosome [27]. HIF-1\u03b1 is overexpressed in many solid tumors [38] and its presence has been correlated with, i.e., poor prognosis in breast and colorectal cancer patients [4, 36]. Stromal expression of hypoxia-regulated proteins is an adverse prognostic factor in colorectal carcinomas.\nFig.\u00a01Schematic representation of the interaction of the analyzed proteins. The PI3K\u2013AKT\u2013mTOR pathway is inducing HIF-1\u03b1. In addition, the absence of hypoxia prevents HIF-1\u03b1 from degradation mediated by VHL. When HIF-1\u03b1 is present, it binds to HIF-1 \u03b2 in the nucleus. The formed complex activates target genes, like GLUT1, SDF1, and CA IX\nHigh protein levels of HIF-1\u03b1 are not only caused by hypoxia but can also be induced by the absence of wild-type protein 53, mutated VHL and other tumor suppressor genes, as well as by activation of oncogenes like Her2neu (c-erbB-2) [24]. In addition, growth factors play a role. However, both hypoxia and signaling-mediated HIF-1 stimulation are mediated by the phosphatidylinositol 3-kinase (PI3K)\u2013AKT, and the mTOR pathway [8]. Interestingly, both the serine\u2013threonine protein kinase AKT as well as mTOR have been suggested to play a role in an early stage of colorectal carcinogenesis [2, 30]. Furthermore, the presence of a non-hypoxic HIF-1 induction has also been suggested, in colorectal adenomas [9].\nUpon HIF-1 activation, multiple target genes become transcriptionally active, including genes involved in anaerobic glycolysis, metabolism, and angiogenesis. This includes glucose transporter 1 (GLUT1), which facilitates cellular glucose uptake [11, 26] and the angiogenesis-associated genes vascular endothelial growth factor and stromal-cell-derived factor 1 (SDF-1) also known as CXCL12 [5]. Furthermore, carbonic anhydrase IX (CA IX) which is prominently induced by hypoxia and is therefore considered to be an intrinsic hypoxia marker, which correlates with a chemical marker of hypoxia, pimonidazole [1, 17, 23, 28]. An additional function of CA IX is in the regulation of the maintenance of the physiological equilibrium in normal gastrointestinal tissue [31].\nThe present study aims to analyze the role HIF-1 in normal colorectal mucosa, adenomas, and carcinomas. To this end, the presence of HIF-1\u03b1 as well as of its downstream targets GLUT1, SDF-1, and CA IX were analyzed in normal colon tissue, colorectal adenomas, and carcinomas. To explore the possible cause of HIF-1 overexpression, i.e., hypoxia or oncogenic stimulation, both the presence of the chemical hypoxia marker pimonidazole and of the HIF-1\u03b1 upstream regulators, VHL, AKT, and mTOR, were investigated.\nMaterials and methods\nTumor material\nA total of 84 tissue samples were studied, consisting of 61 colorectal adenomas, 20 of which contained already a focus of carcinoma and are therefore referred to as progressed adenomas, in contrast to the 41 adenomas with dysplasia only, which are referred to as nonprogressed adenomas. In addition, 23 colorectal carcinomas were analyzed. These samples were obtained from 75 patients, 37 of which were female and 38 male. Mean age was 67.8\u00a0years (range 40\u201389). Normal mucosa samples were obtained from the resection margins of colectomy specimens from ten patients with colorectal cancer. The study was approved by the Institutional Review Board and was in accordance with Dutch medical ethical guidelines.\nDetermination of hypoxic regions\nHypoxic regions in mouse tissues were visualized using the Hydroxyprobe-1 kit (Chemicon International, Temecula, CA, USA). These experiments were approved by the animal experimentation ethics committee, according to local and governmental regulations.\nBriefly, mice were injected intravenously with pimonidazole (60-mg\/kg body weight), which forms adducts from reductively activated pimonidazole in hypoxic cells. Mice were killed 90\u00a0min later by asphyxiation in CO2. The Hydroxyprobe-1 monoclonal antibody was biotinylated using D-biotinyl-\u025b-aminocaproic acid N-hydroxysuccinimide ester (Boehringer Mannheim) and was used to stain pimonidazole adducts in formalin-fixed paraffin-embedded tissues according to recommendations by the manufacturer.\nImmunohistochemistry\nImmunohistochemistry was performed on 4-\u03bcm-thick full-block consecutive sections. Slides were deparaffinized, followed by rehydration, and subsequently staining in batches with the antibodies described in Tables\u00a01 and 2. HIF-1\u03b1 staining was performed as described previously [3]. In brief, antigen retrieval was performed in antigen retrieval solution (DAKO, Glostrup, Denmark) for 45\u00a0min at 96\u00b0C. The primary antibody against HIF-1\u03b1 was incubated at a dilution of 1:500 for 30\u00a0min at room temperature and the catalyzed signal amplification system (DAKO) was used for detecting HIF-1\u03b1 staining.\nTable\u00a01Details of the immunohistochemical stainingsAntibodySpeciesCompany and institutesDilutionIncubationAntigen retrievalDetectionHIF-1\u03b1MouseBD Pharmingen1\/50030\u2032 RTWB TRS 97\u00b0C 45\u2032CSAAktRabbitCell Signaling1\/50o\/n 4\u00b0CWB Citr 97\u00b0C 10\u2032ABCmTORRabbitCell Signaling1\/50o\/n 4\u00b0CWB Citr 97\u00b0C 10\u2032ABCVHLMouseBD Pharmingen1\/10060\u2032 RTWB Citr 97\u00b0C 20\u2032PowervisionCA IXMouseLab Harris1\/5030\u2032 RTnoneEnvision+Glut-1RabbitDAKO1\/40060\u2032 RTWB Citr 97\u00b0C 20\u2032ABCSDF-1MouseR&D systems1\/400o\/n 4\u00b0CWB Citr 97\u00b0C 10\u2032ABCHydroxyprobe-1MouseChemicon International1\/20040\u2032RT0.01% pronase in PBS 40\u00b0C for 40\u2032ABCBD Transduction laboratories, Mab monoclonal antibody, PAb polyclonal antibody, RT room temperature, WB water bath, TRS target retrieval solution (DAKO), Citr citrate buffer 10\u00a0mM pH 6.0, CSA catalyzed signal amplification system (DAKO), ABC avidin\u2013biotinylated peroxidase complex, Powervision from immunologic R&D systemsTable\u00a02Number (and percentages) of HIF-1\u03b1, P-AKT, P-mTOR, VHL, CA IX, GLUT1, and SDF-1 expression in nonprogressed and progressed colorectal adenomas and adenocarcinomas in the respective categories of staining intensity\u00a0Nonprogressed Adenoma, n\u2009=\u200941Progressed adenoma, n\u2009=\u200920Adenocarcinoma, n\u2009=\u200923Kruskal\u2013Wallis test, P-valueHIF-1Neg6 (15)2 (10)5 (22)0.8+0 (0)2 (10)1 (4)++7 (17)3 (15)3 (13)+++28 (68)13 (65)14 (61)AKTNeg0 (0)0 (0)0 (0)0.1+12 (29)7 (35)0 (0)++11 (27)5 (25)10 (43)+++18 (78)8 (40)13 (57)mTORNeg2 (5)0 (0)a0 (0)0.4+2 (5)3 (16)0 (0)++12 (29)6 (32)7 (30)+++25 (61)10 (53)16 (70)VHLNeg0 (0)a1 (5)0 (0)0.9+7 (18)0 (0)1 (4)++12 (30)11 (55)11 (48)+++21 (53)8 (40)11 (48)CA IXNeg10 (24)4 (20)1 (4)0.3+0 (0)0 (0)0 (0)++1 (2)2 (10)2 (9)+++30 (73)14 (70)20 (87)GLUT1Neg0 (0)1 (5)0 (0)0.02+8 (20)6 (30)0 (0)++15 (37)4 (20)6 (26)+++18 (44)9 (45)17 (74)SDF1Neg8 (20)a7 (37)a0 (0)<0.005+4 (10)4 (21)0 (0)++13 (33)2 (11)5 (22)+++15 (38)6 (32)18 (78)aOne case not tested\nFor CA IX staining, no antigen retrieval step was used. Slides were incubated with a mouse primary antibody to CA IX in a 1:50 dilution for 30\u00a0min at room temperature. Detection was performed with the Envision+ system\u2013horseradish peroxidase system for mouse primary antibodies (DAKO).\nGLUT-1 staining was performed with a rabbit polyclonal anti-GLUT1 (antibody clone A 3536, DAKO, dilution 1:400) without antigen retrieval and subsequently developed with a standard avidin\u2013biotinylated peroxidase complex (DAKO).\nStaining procedures for AKT and mTOR were identical. After antigen retrieval, endogenous peroxidase activity was blocked for 10\u00a0min in methanol containing 0.3% hydrogen peroxide. The AKT antibody (Phospho-Akt (Ser473)) and mTOR antibody (Phospho-mTOR (Ser2448)) both obtained from Cell Signaling (Danvers, MA, USA) were incubated overnight at 4\u00b0C in a 1:50 dilution and subsequently detected with a standard avidin\u2013biotinylated peroxidase complex.\nSDF-1 staining was performed similar to the AKT and mTOR staining, except for the secondary antibody, which was a goat anti-rabbit antibody in this case.\nFor VHL staining, antigen retrieval was performed and endogenous peroxidase was blocked as the AKT and mTOR staining. Primary antibody against VHL was incubated for 60\u00a0min by room temperature, followed by Powervision (Immunologic, Duiven, the Netherlands) incubation for 30\u00a0min and subsequently the staining was detected with diaminobenzidine (SIGMA FAST\u2122 3,3\u2032-diaminobenzidine tablets, Sigma Aldrich, St. Louis, MO, USA).\nBefore the slides were mounted with cover slips, all sections were counterstained for 30\u00a0s with Mayer\u2019s hematoxylin and dehydrated in 70%, 96%, and subsequently 100% ethanol and finally in xylene.\nEvaluation and statistics\nThe intensity of the staining was compared between the normal mucosa, nonprogressed, progressed adenomas, and carcinomas and scored by two investigators. Staining intensity was scored in four classes (0 to 3) taking the negative control as a reference for score 0 and the positive control (clear cell renal adenocarcinomas (Grawitz tumor) as a reference for score 3). For HIF-1\u03b1, only nuclear staining was scored, for CA IX, membrane staining, and AKT and SDF-1 cytoplasmic staining, for mTOR predominantly cytoplasmic but also membrane staining was scored. Significance of differences between categories was analyzed by means of the Kruskal Wallis test. P-values\u2009<\u20090.05 were considered to be significant.\nResults\nHIF-1\u03b1 expression and its regulators hypoxia and VHL\nIn normal colorectal mucosa nine out of ten samples showed HIF-1\u03b1 present in nuclei of the surface epithelium (Fig.\u00a02). To evaluate whether hypoxia played a physiologic role in the induction of HIF-1\u03b1 in normal colorectal mucosa, we used mice that were injected with pimonidazole, a chemical marker of hypoxia that can be visualized by immunohistochemistry. The presence of tissue hypoxia was demonstrated in the surface epithelium of normal colon mucosa of the treated mice (Fig.\u00a03). The pattern of hypoxic cells in the normal colorectal mucosa in mice matched with the distribution of HIF-1\u03b1 immunohistochemical staining in human colorectal mucosa.\nFig.\u00a02Serial sections of normal colon mucosa, colorectal adenoma, and adenocarcinoma analyzed for HIF-1\u03b1 and VHL expression. HIF-1\u03b1 staining was observed in the nuclei of surface epithelium of normal mucosa and colorectal adenoma, whereas in adenocarcinomas HIF-1\u03b1 was present near hypoxic regions with necrosis. VHL expression was observed in the cytoplasma of all epithelial cells of normal mucosa, adenomas, and adenocarcinomasFig.\u00a03Detection of hypoxia in murine colorectal mucosa by pimonidazole. a Colorectal mucosa of a non-injected mouse, b colorectal mucosa of a mouse that was injected intravenously with pimonidazole. After 90\u00a0min, the mouse was killed and colon tissue harvested and formalin fixed. Immunohistochemical staining against pimonidazole revealed hypoxia in the surface epithelium of the normal colon mucosa\nThe presence of HIF-1\u03b1 in normal colorectal mucosa was compared with that in adenomas and carcinomas. Adenomas displayed HIF-1\u03b1 expression in 53 of the 61 cases, predominantly at the surface, gradually decreasing towards the center of the adenomas. No differences were seen between nonprogressed and progressed adenomas (85% and 90%, respectively), as shown in Table\u00a02. Of the carcinomas, 78% (n\u2009=\u200918) were positive, showing nuclear staining of HIF-1\u03b1 in epithelial tumor cells. In carcinomas, a decreasing gradient of HIF-1\u03b1 staining was observed from necrotic areas towards the well-vascularized stroma. Besides the epithelial tumor cells, in carcinomas, inflammatory cells in the tumor-associated stroma, and in five cases B-cells in lymph follicles, were positive for HIF-1\u03b1. Statistical analysis showed no significant differences in intensity of HIF-1\u03b1 staining between normal mucosa, adenomas, and carcinomas.\nVHL, the regulator of HIF-1\u03b1 degradation, was also analyzed by means of immunohistochemistry (Fig.\u00a02). VHL expression was observed in all epithelial cells in normal colorectal mucosa as well as in colorectal adenomas and carcinomas.\nAKT and mTOR, oncogenic pathways regulating HIF-1\u03b1\nTo investigate whether, in addition to hypoxia, oncogenes and\/or growth factors can contribute to the elevated HIF-1\u03b1, we studied the presence of phosphorylated forms of AKT and mTOR, which reflect an active state of the oncogene-driven pathway of HIF-1\u03b1 activation. Low-intensity cytoplasmic staining for the active form of AKT (P-AKT) was seen in epithelial cells of normal mucosa in all tissues (Fig.\u00a04). Positive staining was also seen in the smooth muscle cells of the muscularis propria and muscularis mucosae and in the walls of blood vessels. In adenomas, staining in epithelial cells was more intense, present throughout the neoplastic tissue, and observed in all cases. Staining patterns in adenocarcinomas were similar and also observed in all samples analyzed.\nFig.\u00a04Typical immunohistochemical staining patterns of the active forms of AKT (P-AKT) and mTOR (P-mTOR) in normal colon mucosa, colorectal adenoma, and adenocarcinomas. Presence of active AKT is seen in the cytoplasm of all epithelial cells with increasing intensities from the normal mucosa towards adenocarcinoma. Presence of active mTOR is restricted to the surface epithelium in normal mucosa and adenomas. In adenocarcinomas, cells surrounding necrotic areas show active mTOR as well. In addition, in adenocarcinomas, intracellular localization of mTOR changes from cytoplasm towards cellular membrane staining\nActive mTOR was only present as a cytoplasmatic staining; nuclear staining was not observed in any of the cases (Fig.\u00a04). In normal mucosa, mTOR was detected in the surface epithelium in all tissues. Likewise, in adenoma samples, mTOR was expressed in the surface epithelium but also more diffusely throughout the adenomas. In adenocarcinomas, staining patterns were diffused across the whole tumor, but more intense expression was detected in areas surrounding necrosis. Higher levels of active mTOR were observed at the invasive front of the tumor. In the adenocarcinomas, besides the cytoplasmic staining in some cells, a clear cellular membrane staining was observed. In all normal mucosa samples, adenomas, and carcinomas, mTOR was present, except for 5% of the nonprogressed adenomas, which were negative for mTOR (Table\u00a02).\nExpression of the HIF1 downstream targets Glut1 and SDF-1\nTo investigate to what extent downstream pathways were affected by HIF-1 upregulation, several HIF-1 target genes, including GLUT1 and SDF-1, were analyzed in normal mucosa, adenomas, and adenocarcinomas. Expression of GLUT1, which is involved in glucose metabolism, was closely correlated with HIF-1\u03b1 in all colon tissues analyzed. In normal colon mucosa, GLUT1 expression showed a gradient from the surface epithelium towards the basis of the crypts (Fig.\u00a05). All normal tissues showed a positive GLUT1 staining. In nonprogressed adenomas, all cases showed GLUT1 expression compared to 95% of progressed adenomas. All adenocarcinomas (n\u2009=\u200923) were positive for GLUT1 as well. Staining was localized at the cell membrane of epithelial cells in normal mucosa, adenomas, and adenocarcinomas (Fig.\u00a05). Most intense staining was seen at the surface epithelium of adenomas and in the vicinity of necrosis in carcinomas. As to staining intensity, a significant difference was detected between the adenomas and the carcinomas (Table\u00a02).\nFig.\u00a05Typical immunohistochemical staining patterns of HIF-1\u03b1, GLUT1, SDF-1, and CA IX in normal colon mucosa, colorectal adenomas, and adenocarcinomas. The downstream targets of HIF-1, i.e., SDF-1, GLUT1, and CA IX, have predominantly overlapping staining with the presence of HIF-1. However, in normal mucosa, CA IX is not expressed in the HIF-1\u03b1 positive surface epithelium but is expressed in the basal of the crypts. In adenocarcinomas, SDF-1 is not only present in HIF-1\u03b1-positive cells but in all epithelial cells\nIn normal colon tissue, SDF-1 expression was comparable to that of HIF-1\u03b1, i.e., it was detected in a gradient from the surface epithelium towards the basis of the crypts. However, in all colorectal adenomas and adenocarcinomas, intense SDF-1 expression was observed in the epithelial cells, which was located in the cell membrane and cytoplasm (Fig.\u00a05). Because this epithelial SDF-1 expression was seen in all epithelial cells, it co-localized only partly with HIF-1\u03b1 presence. In contrast to the comparable intensity of the HIF-1\u03b1 staining in adenomas and adenocarcinomas, the SDF-1 staining intensity was much higher in adenocarcinomas than in the adenomas (Table\u00a02). In addition to the epithelial expression of SDF-1, endothelial cells of blood vessels stained positive for SDF-1 (Fig.\u00a06).\nFig.\u00a06SDF-1 expression in epithelial and endothelial cells of normal detected by immunohistochemistry\nExpression of CA IX\nCA IX was originally identified in gastrointestinal tissues and later recognized as a HIF-1 target gene in many tissues. In normal mucosa, the presence of CA IX actually did not co-localize with that of HIF-1\u03b1. CA IX was observed in the bottom parts of the crypts, i.e., in the proliferative compartment, in contrast to HIF-1\u03b1, which was present in the surface epithelium. Seventy percent of the normal tissues showed CA IX expression. In adenomas and particular adenocarcinomas, CA IX and HIF-1 expressions had more resemblance. In adenomas, CA IX was present in a more diffuse pattern throughout the whole adenoma, including the surface epithelium (Fig.\u00a05). In the adenocarcinomas, CA IX showed a peri-necrotic staining, mainly co-localizing with the HIF-1\u03b1 expression.\nDiscussion\nThe present study demonstrates the presence of nuclear HIF-1\u03b1 in the surface epithelium of normal colon and adenomas and in areas associated with peri-necrotic areas of colon adenocarcinoma. The data support a hypoxia-induced HIF-1 cascade, both in normal and neoplastic colorectal mucosa, as suggested by pimonidazole and HIF-1\u03b1 patterns in association with GLUT1 and SDF-1 in normal colorectal mucosa and adenomas, as well as peri-necrotic HIF-1\u03b1 and CA IX patterns in colorectal carcinomas. The occurrence of hypoxia around the surface epithelium was confirmed by pimonidazole staining of unaffected murine colon. Furthermore, hypoxia and the oncogenic activation of the AKT\u2013mTOR pathway both contributed to HIF-1 expression in adenomas and adenocarcinomas. Moreover, the expression patterns of CA IX in normal mucosa and SDF-1 in neoplastic mucosa pointed to additional regulations independent from HIF-1.\nThe hypoxia\u2013HIF-1 cascade\nThe presence of HIF-1\u03b1 under physiological circumstances in the surface epithelium of normal colorectal mucosa observed in the present study may be due to the anaerobic environment in the lumen of the colon, which contributes to a relatively hypoxic state of especially this surface epithelium. Indeed, we observed accumulation of the chemical hypoxia marker pimonidazole particularly in the surface epithelium, indicating low oxygen levels [19]. The presence of pimonidazole showed a gradient towards the basis of the crypts, suggesting that the cause of hypoxia was indeed related to the anaerobic lumen. Apparently, the capillaries in the surface regions of the colon epithelium were unable to cope with the high oxygen demand of the tissue. This strongly suggests that these hypoxic areas may induce HIF-1\u03b1, which contributes to a sustained pressure on the stimulation of angiogenesis and glycolysis to adjust the energy levels of these cells.\nAlthough immunohistochemical analysis of patient samples does not allow a formal analysis of functional relationships, it is highly likely that the HIF-1 detected in normal colorectal mucosa was transcriptionally active because HIF-1\u03b1 was detected in the nuclei and the HIF-1 downstream targets GLUT1 and SDF-1 were present in the same regions where HIF-1\u03b1 was found.\nIn 87% of colorectal adenomas, HIF-1\u03b1 was present, showing staining predominantly in the nuclei of the surface epithelium. In colorectal adenocarcinomas, the presence of HIF-1\u03b1 changes from the surface epithelium to epithelium cells surrounding regions of necrosis, indicating severe hypoxia. Presence of HIF-1\u03b1 in the nuclei of epithelial cells went along with expression of the downstream targets GLUT1 and SDF1 in all different stages of the colon carcinogenesis.\nIn contrast to SDF-1 and GLUT1, the downstream target CA IX, known as a stringent hypoxia marker [35], did not co-localize with HIF-1\u03b1 staining in normal colorectal mucosa. It should be noted that CA IX was originally described to have a role in maintaining the physiological equilibrium in the gastrointestinal tract. In fact, CA IX was more abundant in the basal half of the crypts, containing the proliferating compartment, rather than in the upper half of the mucosa where HIF-1\u03b1 expression was observed. Indeed, co-localization of CA IX with Ki-67, a proliferation marker, has been found in colorectal cancer [31]. The lack of HIF-1\u03b1 in the CA IX-positive regions indicates an HIF-1\u03b1-independent regulation of CA IX in normal colon epithelium. CA IX expression was reported to be also regulated by the transcription factor SP1 [18, 31].\nOncogenic upregulation of HIF-1\nOncogene-dependent regulation of HIF-1 can be mediated by the AKT\u2013mTOR pathway [24, 39]. In the present study, in epithelium of all normal colorectal mucosa, a weak staining of P-AKT was observed, confirming data of Itoh et al. [14]. In all adenomas and adenocarcinomas analyzed, the active (phosphorylated) forms of AKT and mTOR were detected as well. Especially active mTOR showed co-localization with HIF-1\u03b1 in normal tissue as well as in adenomas and adenocarcinomas. Co-localization of P-AKT with HIF-1\u03b1 was less evident. Recently, in breast cancer, it has been shown that in the absence of P-AKT, only low levels of HIF-1\u03b1 could be detected while in the presence of P-AKT high HIF-1\u03b1 protein levels were observed [10]. In colorectal tumorigenesis, this regulation mechanism may be less relevant because staining was already observed in normal tissue. In addition, the presence of active AKT was not HIF-1 related because in adenocarcinomas P-AKT was present in a higher percentage of cells present than HIF-1\u03b1. The downstream target of AKT, mTOR, displayed comparable cellular expression patterns to HIF-1\u03b1 in normal, adenomas, and carcinomas. This suggests that the AKT\u2013mTOR\u2013HIF-1\u03b1 axis may be important in tumor growth and development as was seen in prostate epithelial cells [25].\nExpression of VHL\nAn alternative mechanism that can lead to HIF-1\u03b1 overexpression is loss of VHL expression. In normal colon mucosa, VHL is expressed, while in colorectal tumors VHL has been found to be lost or mutated in 11% of cases [7, 21]. Mutations in the VHL gene have been reported to result in cytoplasmic staining of HIF-1\u03b1 in colorectal cancer in one study [22], while this could not be confirmed in another study of 80 patients [40]. Also in the present study, no cytoplasmic staining of HIF-1 \u03b1 was seen at all, and VHL expression was seen in all epithelial cells of normal mucosa, colorectal adenomas, and carcinoma.\nExpression of SDF1\nSDF-1 was investigated in the present study as a downstream target of HIF-1 [13, 32]. Surprisingly, epithelial cells showed much stronger SDF-1 expression than endothelial cells lining blood vessels. In addition, expression was not merely restricted to areas showing HIF-1\u03b1 staining but was observed in all epithelial adenomas and adenocarcinomas. SDF-1 staining in tumors cells has also been reported in breast cancer patients [29] and in clear cell\u2013renal cell carcinomas, in which HIF-1\u03b1 is constitutively expressed [37]. The intense staining of the colon tumor cells suggests that SDF-1 not only has a function in attracting circulating progenitor cells towards the angiogenic areas [12]. Indeed, SDF-1 stimulates intestinal epithelial cell migration and enhances the integrity of the mucosal barrier [34].\nThe level of expression of SDF-1 might also play a role in tumorigenesis because it is related with prognosis in colorectal cancer. The presence of its coding mRNA and the mRNA of its receptor, CXCR4, have been shown to be a poor prognostic factor [20]. In tumor formation, SDF-1 may bear impact on the recruitment of various types of progenitor cells needed for enhanced growth of the tumor tissue [29]. Our study supports a role of SDF-1 in carcinogenesis because SDF-1 staining becomes more intense with the progression of normal colon tissue toward carcinomas. However, future studies are needed to substantiate such role.\nConclusion\nThe common expression of HIF-1\u03b1 in the surface epithelium of the colorectal mucosa suggests that HIF-1 may play a role in the physiology of normal colon tissue. HIF-1\u03b1 presence does not appear to be related to progression of colorectal carcinogenesis in contrast to GLUT1 and SDF-1, which are expressed more intensely at later stages of this process.","keyphrases":["hif-1","mtor","sdf-1","ca ix","colon"],"prmu":["P","P","P","P","P"]}
{"id":"Plant_Foods_Hum_Nutr-4-1-2413119","title":"Liver Protective Effects of Morinda citrifolia (Noni)\n","text":"This study evaluated the protective effects of Noni fruit juice on acute liver injury induced by carbon tetrachloride (CCl4) in female Sprague-Dawley (SD) rats. Liver damage (micro-centrilobular necrosis) was observed in animals pretreated with 20% placebo (drinking water) + CCl4. However, pretreatment with 20% Noni juice in drinking water + CCl4 resulted in markedly decreased hepatotoxic lesions. Furthermore, serum alanine aminotransferase and aspartate aminotransferase levels were significantly lower in the Noni group than the placebo group. In a correlative time-dependent study, one dose of CCl4 (0.25 mL\/kg in corn oil, p.o.) in female SD rats, pretreated with 10% placebo for 12 days, caused sequential progressive hepatotoxic lesions over a 24 h period, while a protective effect from 10% Noni juice pretreatment was observed. These results suggest that Noni juice is effective in protecting the liver from extrinsic toxin exposure.\nIntroduction\nCarbon tetrachloride (CCl4) is a common environmental pollutant and liver carcinogen. Due to its detoxifying function in protecting the body, the liver is a target for CCl4 toxicity [1]. Antioxidants and anti-inflammatory agents play a critical role in liver protection by scavenging superoxide anion radicals (SAR) and neutralizing lipid peroxides (LPO). They also suppress the inflammatory response [2\u20134]. Therefore, there is need for a natural product that protects the liver, but is also cost-effective and safe, without long-term side effects. This study evaluated the liver protective potential of the fruit juice of Morinda citrifolia (Noni), from Tahiti, a strong antioxidant and anti-inflammatory nutritional supplement.\nWe have previously reported that Noni juice is an effective antioxidant, demonstrating a dose-dependent effect against SAR and LPO in vitro [5]. Based on the data from our laboratory and others, we suggested that Noni juice may be effective in protecting the liver against acute CCl4 toxicity. To test our hypothesis, a classic CCl4-induced liver injury model was chosen to study the liver protective effect in female SD rats. Morphological change is the most direct sign of liver injury. Histopathological changes were evaluated for morphological evidence of the liver protective properties of Noni juice. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) assays that measure liver function were performed on the Noni group (Noni + CCl4) and compared to those of the positive control group (placebo + CCl4).\nMaterials and Methods\nMaterials\nCarbon tetrachloride was purchased from Sigma Chemical Co., St. Louis, MO, USA. Noni juice was made by Morinda Holdings Inc. (Provo, UT, USA) from Noni fruit originating in Tahiti. The placebo was prepared using the same procedures and ingredients for making Tahitian Noni\u00ae Juice (TNJ), but without the Noni component.\nAnimals\nSix-week-old female SD rats were purchased from Charles River Inc. (Wilmington, MA, USA). The animals were housed in a room maintained at 25\u00a0\u00b0C with a 12\u00a0h photoperiod. They were fed a laboratory chow diet and water ad libitum. The experimental design for these tests was approved by the Institutional Ethics Review Committee for animal experiments at the National Cancer Center.\nExperiment 1\nAnimal Treatment\nSixteen female SD rats were divided into two groups of eight each. Both groups were provided drinking water for 7\u00a0days. Twenty percent placebo was added to the drinking water in one group and 20% Noni juice in the other. On day\u00a08, four animals from each group were fed 0.5\u00a0mL\/kg CCl4 in corn oil. All animals were sacrificed at 6\u00a0h post-CCl4 administration. The liver was removed and fixed in 10% neutral formalin for histological examination. Blood was collected from each animal for serum AST and ALT activity to examine liver function.\nLM Examination\nLiver samples were preserved in 10% neutral buffered formalin and dehydrated in a graded alcohol series. Following xylene treatment, the specimens were embedded in paraffin blocks and cut into 5\u00a0\u03bcm thick sections. Consecutive sections were stained with hematoxylin and eosin (H & E) and examined under an Olympus BH2 light microscope. The number of foci\/mm2 was counted to assess the degree of necrosis [6]. Digital images were recorded with the Pixera Visual Communication Suite.\nALT and AST Assay\nSerum was separated from clotted blood by centrifugation at 1,600\u00a0rpm for 10\u00a0min at 4\u00a0\u00b0C. Serum ALT and AST activities were measured using an automatic biochemical analyzer calibrated with quality control standards (E.I. du Pont de Nemours) in a clinical laboratory (Family Medicine Laboratory, UIC College of Medicine at Rockford).\nExperiment 2\nAnimal Treatments\nThirty-six female SD rats were divided into two groups of 18 each. While dose range-finding experiments revealed that maximum protection occurred with 20% Noni juice in drinking water, optimum protection (per amount ingested) occurred with 10%. Therefore, each group was supplied 10% placebo or 10% Noni juice in drinking water for 12\u00a0days. On day\u00a013, 15 animals from each group were fed 0.25\u00a0mL\/kg CCl4 in corn oil. Three animals from each group were sacrificed at 1, 3, 6, 16, and 24\u00a0h post-CCl4 administration. The remaining three animals in each group were fed 1\u00a0mL\/kg corn oil and sacrificed 1\u00a0h later, to serve as vehicle placebo and Noni controls. Multiple liver-tissue samples were removed from each rat to be fixed for LM examination.\nLM Examination\nLiver samples for experiment 2 were prepared and observed for light microscopy as described in experiment 1.\nStatistical Analysis\nStatistical analysis was done by a Student two-tail T test [7]. Differences were considered as significant at P\u2009<\u20090.05 and highly significant at P\u2009<\u20090.01. Results were expressed as mean \u00b1 SD.\nResults and Discussion\nIn experiment 1, the liver lobular structure among rats treated 6\u00a0h earlier with 20% placebo or 20% Noni juice did not reveal any noticeable histological changes (Fig.\u00a01a,b), which indicates that high-dose pretreatment with Noni juice or placebo does not produce hepatotoxicity. Hepatic sections, from rats pretreated with 20% placebo followed by CCl4 treatment, showed typical CCl4-induced liver injury (Fig.\u00a01c). Sections revealed extremely vacuolized hepatocytes (balloon liver cells and apoptotic necrotic cells) adjacent to the central vein (arrows and arrowhead, Fig.\u00a01c), indicating acute CCl4-induced hepatotoxicity. Hepatocytes were significantly protected from CCl4-induced toxicity by 20% Noni juice pretreatment (Fig.\u00a01d), and demonstrated a normal appearance, except for slight steatosis (fatty deposition) in hepatocytes pericentral to the vein. In the CCl4 + 20% placebo group, 23.8% of cross-sectional areas contained swollen balloon cells, whereas in the CCl4 + 20% Noni group, only 1.9% contained vacuolized cells (P\u2009<\u20090.001). Furthermore, the apoptotic cells were dramatically decreased by 50% in the Noni pretreated group when compared with the CCl4 + placebo group (P\u2009<\u20090.001).\nFig.\u00a01H & E liver sections (250X), from female SD rats showing protective effect of 20% Noni pretreatment d on acute hepatotoxicity at 6\u00a0h post-CCl4 exposure (0.5\u00a0mL\/kg CCl4 in corn oil). The rats in a (control) and b (noni treated) received no CCl4 exposure, and show no signs of liver-cell damage. With CCl4 exposure, there are significant hepatotoxicity effects: balloon liver cells and apoptotic necrotic cells adjacent to the central vein (arrow and arrowhead (c)). d In contrast, with Noni pretreatment + CCl4 exposure, shows no signs of hepatotoxicity. b and d (noni treated) show cell patterns that are more ordered, and show no signs of hepatotoxicity\nIn experiment 2, 10% Noni + CCl4 pretreatment resulted in a delayed appearance of damaged hepatocytes with a significantly decreased number of necrotic foci (80%) and apoptosis (50%), compared to the placebo group at 3, 6, and 16\u00a0h (arrows and arrowhead, Fig.\u00a02b,d,f). These results indicate that Noni juice protects the liver from acute CCl4 exposure. Cellular damage consisted of vacuolated hepatocytes and inflammatory cells surrounding the central vein in the placebo + CCl4 group (Fig.\u00a02b,d,f). Conversely, liver lobules in the Noni juice + CCl4 group maintained a normal appearance in each of the post-treatment time intervals (Fig.\u00a02a,c,e).\nFig.\u00a02Rat liver sections (100\u00d7), showing representative sequential hepatopathology at 3, 6, or 16\u00a0h following pretreatments with 10% Noni juice, or placebo, plus 0.25\u00a0mL\/kg CCl4. Panels: a 3\u00a0h post-CCl4 + Noni juice (normal hepatic structure); b 3\u00a0h post-CCl4 + placebo, (first stages of hepatocyte swelling pericentral to vein; arrow denotes swollen hepatocytes); c 6\u00a0h post-CCl4 + Noni (normal hepatic structure); d 6\u00a0h post-CCl4 + placebo, (increased number and degree of swollen hepatocytes); e 16\u00a0h post-CCl4 + Noni (balloon cells adjacent to pericentral vein, and delayed response of hepatotoxicity); and f 16\u00a0h post-CCl4 + placebo (increase in number of damaged foci, and an extension of damaged area midzonal to vein; arrow indicate swollen hepatocytes, arrowhead denotes inflammatory foci)\nComparative histopathological changes of the Noni juice and placebo-treated groups, at 3\u00a0h post-0.25\u00a0mL\/kg CCl4 treatment, are shown in Fig.\u00a03. The effects of 10% Noni juice on reducing inflammation surrounding the central vein area (Fig.\u00a03a), and the absence of swollen hepatocytes are clearly shown in the H & E sections. In Fig.\u00a03b, a similar liver section from the CCl4 + 10% placebo treatment reveals swollen hepatocytes and a massive influx of inflammatory cells (arrow and arrowheads, Fig.\u00a03a,b).\nFig.\u00a03H & E cross sections (150\u00d7), of pericentral vein areas showing inhibitory effect of 10% Noni juice on inflammation at 3\u00a0h post-0.25\u00a0mL\/kg CCl4-induced acute hepatotoxicity (a), versus placebo + CCl4 (b). Arrows denote increased foci of inflammatory cells, arrowheads indicate swollen hepatocytes\nNoni Juice Suppresses ALT and AST Activities\nSerum ALT and AST activities were determined for enzymatic evidence of liver injury (Table\u00a01). The protective effect of Noni juice against CCl4-induced hepatotoxicity was demonstrated by a significant 50% suppression (P\u2009<\u20090.05) of ALT activity compared to the placebo. AST activity in the Noni juice pretreated group was significantly suppressed by 68% (P\u2009<\u20090.01), compared to the placebo. As noted in the table, the 20% placebo or 20% Noni juice in drinking water for 7\u00a0days did not influence liver function compared with the control. In contrast, the 20% placebo + CCl4 treatment produced acute liver damage as measured by elevated serum ALT and AST activities.\nTable\u00a01Effect of Noni juice on acute CC14-induced hepatitis in SD ratsTreatmentALT (IU\/L)AST (IU\/L)Control67\u2009\u00b1\u200931**222\u2009\u00b1\u2009121**Placebo (20%)67\u2009\u00b1\u200926**222\u2009\u00b1\u200999**Noni juice (20%)65\u2009\u00b1\u200959**184\u2009\u00b1\u200931**Placebo (20%) + CCl4 0.5\u00a0mL\/kga136\u2009\u00b1\u2009411135\u2009\u00b1\u2009466Noni juice (20%) + CCl4 0.5\u00a0mL\/kg68\u2009\u00b1\u200912**358\u2009\u00b1\u200969*aData from each group were compared to that of the positive control group (20% placebo + 0.5\u00a0mL\/kg CC14). Data are expressed as means \u00b1 SD (n\u2009=\u20094). Student two-tail T test*P\u2009<\u20090.05, **P\u2009<\u20090.01\nThe present study supports the protective effects of Noni juice on acute CCl4-induced hepatotoxicity. Necrosis of hepatocytes was minimized in livers of animals pretreated with Noni juice in both experiments. Noni juice prevented free-radical-induced oxidative-pathological events.\nDamaged hepatocytes release ALT and AST enzymes following acute exogenous toxin exposure. Therefore these elevated serum enzyme levels are good chemical indicators of liver damage. Noni juice is able to normalize liver function after acute exposure to CCl4. Serum ALT and AST activity, a measure of defense enzyme levels, was significantly suppressed after CCl4 exposure in the Noni juice pretreated animals, compared with the elevated enzyme levels in the placebo pretreated animals. Therefore, pretreatment with Noni juice indeed protected hepatocytes from acute CCl4 exposure.\nA few reports have suggested the involvement of Noni juice in the development of chemically induced hepatitis in a limited number of cases in Europe. However, no causal role has been established in any of these instances. Further, an official European investigation of these cases determined that no relationship between Noni juice and hepatitis was evident and that consumption of Noni juice is unlikely to induce adverse human liver effects [8]. Animal and human studies have revealed that Noni juice is not hepatotoxic, even at very high doses [9, 10].\nConclusions\nOur investigation suggests that Noni juice exerts effective protection against acute extrinsic chemical induced hepatic injury by inhibiting inflammatory response and suppressing elevated liver enzyme activities; thus preventing consequent cell membrane damage. Histopathological observations revealed morphological evidence, and bioassays revealed functional evidence, that Noni confers resistance to acute CCl4-induced hepatotoxicity. High dosages of Noni and placebo pretreatments did not induce liver damage. Noni may thus be an efficacious natural hepatoprotective nutritional supplement against chemically induced hepatotoxicity. Future studies should focus on how Noni juice influences the inflammatory and cytotoxic cytokines involved in liver injury.","keyphrases":["liver protection","morinda citrifolia","noni","carbon tetrachloride"],"prmu":["P","P","P","P"]}
{"id":"Pediatr_Radiol-3-1-1950215","title":"Congenital anomalies of the male urethra\n","text":"The spectrum of congenital anomalies of the male urethra is presented. The embryologic basis of each anomaly, when known, is discussed. Clinical and imaging features of each entity are presented.\nIntroduction\nThe development of the male genitourinary system is complex. Numerous anomalies of the male urethra exist, either as isolated anomalies or in combination with other disorders. The current understanding of the embryology and anatomy of normal male urethral development might help explain the causes of the various anomalies as well as their relationships.\nEmbryology of the male urethra\nBetween weeks four and five of fetal life, the cloaca is separated from the amniotic cavity by the cloacal membrane [1]. The cloaca is divided into the urogenital sinus and the hindgut by the urorectal septum, which descends in a rostral-to-caudal fashion (Fig.\u00a01). The urogenital sinus is the precursor of the bladder and posterior urethra. The urorectal septum is formed by proliferation, migration and fusion of mesodermal folds that arise from the lateral walls of the cloaca. When the urorectal septum reaches the caudal aspect of the cloaca, the cloacal folds, which lie on the dorsal portion of the cloacal membrane, fuse to form the rudimentary perineum. The cloacal membrane subsequently undergoes disruption such that the hindgut and phallic cloaca open separately to the exterior, divided by the urorectal septum (Fig.\u00a02).\nFig.\u00a01Embryologic development of the male genitourinary tract. The cloaca is divided into the urogenital sinus (precursor of the bladder and posterior urethra) and the hindgut by the urorectal septum (adapted from reference 2)Fig.\u00a02Embryologic development of the male genitourinary tract. The urorectal septum continues to descend and the cloacal membrane undergoes disruption. The hindgut and phallic cloaca now open separately to the exterior (adapted from reference 4)\nThe most caudal aspect of the cloaca (phallic cloaca) extends distally through the developing genital tubercle. Proliferation of rostral mesoderm of the genital tubercle displaces the cloaca so that it lies on the caudal aspect of the developing glans (Fig.\u00a03). Failure of this step results in epispadias. The endodermal lining of the phallic cloaca appose to form the urethral plate. Portions of the cloacal membrane also contribute to the urethral plate (Fig.\u00a04). The urethral plate undergoes median cleavage. Following mesodermal proliferation and fold fusion extending proximally to distally, the shaft urethra and proximal two-thirds of the glandular urethra are formed. This is lined by endoderm. The distal urethra at the glans penis is formed from fusion of distal folds that arise from the apical part of the glans distal to the cloaca and subsequent urethral plate (Fig.\u00a04). It is lined by ectoderm [2\u20134]. Discontinuity in the urethral plate or abnormality of fusion of the urethral folds can lead to hypospadias.\nFig.\u00a03Embryologic development of the male genitourinary tract. The phallic cloaca extends distally through the developing genital tubercle. Proliferation of rostral mesoderm of the genital tubercle displaces the cloaca so that it lies on the caudal aspect of the developing glans (adapted from reference 2)Fig.\u00a04Embryologic development of the male genitourinary tract. Median cleavage of the urethral plate occurs. The urethral plate forms the deep and lateral walls of the proximal urethra. The phallic cloaca opens to the exterior at the base of the glans (adapted from reference 4)\nNormal anatomy of the male urethra\nThe male urethra is divided into the prostatic, membranous and penile (cavernous) urethra (Figs.\u00a05 and 6). The prostatic urethra courses through the prostate and is the widest part of the urethra. On the posterior wall of the prostatic urethra lies the urethral crest, an elevated longitudinal ridge of tissue. The verumontanum (seminal colliculus) is located at the midportion of the urethral crest and is the site of the ejaculatory duct orifices. Additionally, the orifice of the prostatic utricle, a glandular grouping, is located at the summit of the verumontanum. The utricle, a derivative of the Mullerian duct and urogenital sinus, and the male homologue of the uterus and vagina, is a blind indentation that extends dorsally into the prostate and can measure up to 6\u00a0mm. The prostatic ducts drain into the prostatic sinus, the fossa on either side of the elevated urethral crest. The intermuscular incisura produces an indentation on the anterior aspect of the prostatic urethra, at the level of the verumontanum, and results from prominent collagenous tissue.\nFig.\u00a05Normal male urethral anatomyFig.\u00a06Voiding cystogram in a 3-year-old with prior urinary tract infection. Normal urethral anatomy is shown (P prostatic urethra, short arrow incisura, long arrow verumontanum)\nThe membranous urethra is the shortest, least distensible portion of the urethra. It is surrounded by the sphincter urethrae membranaceae (external sphincter), which in addition to vessels, nerves and deep transverse perineal muscle represents the urogenital diaphragm. The internal sphincter is located at the bladder neck.\nThe penile urethra is the longest portion of the urethra. The proximal portion of the penile urethra, the bulbar urethra, is dilated and is the site of drainage of Cowper\u2019s ducts. The penile urethra is also dilated distally at the glans penis, where it forms the fossa navicularis. The midportion of the penile urethra is tethered superiorly by the suspensory ligament [5].\nCongenital anomalies of the urethra\nPosterior urethral valves\nPosterior urethral valves (PUV), first described in 1717 by Morgagni, occur in one of every 5,000\u20138,000 male infants [6]. PUV can lead to bilateral renal obstruction, bladder dysfunction, vesicoureteral reflux, hydronephrosis and renal damage. In 1919, Young proposed a classification system consisting of three types of valves. Type I valves (the most common) arise from the verumontanum and extend distally to attach to the lateral walls of the urethra as two leaflets (type Ia) or as a unicuspid leaflet (type Ib). Type II valves extend proximally from the verumontanum to the bladder neck, and type III valves represent a diaphragm attached to the circumference of the urethra with a central hole, distal (type IIIa) or proximal (type IIIb) to the verumontanum [1, 8]. The existence of Young\u2019s type II valves is debated [7]. Type III valves are considered by many to be the same entity as Cobb\u2019s collar and are discussed in the section on congenital urethral strictures [1, 7].\nThe valves are composed of connective tissue interspersed with smooth muscle, similar to the tissue encasing the ejaculatory ducts, and are lined by stratified squamous epithelium rather than the transitional epithelium that lines the posterior urethra [1].\nThe embryologic defect leading to the development of PUV is not known. Many authors believe the anomaly is caused by abnormal integration of the Wolffian ducts into the urethra, while others consider it a result of persistence of the cloacal membrane [1, 7].\nAutopsy studies have shown that the valves represent a single obliquely oriented diaphragm rather than a bicuspid structure as originally believed [7, 9]. The term congenital obstructive posterior urethral membrane (COPUM) has, therefore, been introduced [10].\nMany cases of PUV are detected by prenatal sonography. Normal fetal renal parenchyma and amniotic volume do not necessarily predict good postnatal renal function [11]. Intrauterine intervention, while improving hydronephrosis, does not prevent the development of renal dysplasia [12]. Newborns might present with pulmonary hypoplasia secondary to oligohydramnios, or an abdominal mass caused by hydronephrosis or bladder distension. In older patients clinical signs include failure to thrive, poor urinary stream, sepsis, poor renal function or salt-losing nephropathy [13]. Treatment is by fulguration of the valve, although in infants vesicostomy or pyeloureterostomy might be performed prior to valve ablation. Posterior urethral dilatation persists after fulguration in 20% of patients. Despite therapy, 40% of patients have poor renal growth and another 40% develop end-stage renal disease [12, 14]. A normal or near normal serum creatinine level or GFR at 1\u00a0year of life, following decompression of the urinary tract, are useful positive prognostic indicators [12, 14]. In contrast, the development of proteinuria is associated with a poor prognosis [14].\nUltrasonography is usually the initial imaging modality used to evaluate patients with suspected PUVs. US reveals a thick bladder wall and bilateral but asymmetric hydronephrosis. Transperineal sonography can demonstrate dilatation of the posterior urethra (the \u201ckeyhole\u201d sign) (Fig.\u00a07) [15]. A perirenal urinoma from forniceal rupture or urinary ascites can also be seen. PUV is the most common cause of urinary ascites in the newborn [16]. Echogenic kidneys might also be seen and are associated with a poor prognosis [14].\nFig.\u00a07Posterior urethral valves. Sonography demonstrates a thick-walled bladder and dilatation of the prostatic urethra (arrow), the \u201ckeyhole sign\u201d\nTypical findings of PUV on voiding cystourethrography (VCUG) include a trabeculated bladder with diverticula, dilatation of the prostatic urethra and a discrepancy between the caliber of the prostatic urethra and the bulbar and penile portions of the urethra (Fig.\u00a08). During voiding, the contrast stream between the prostatic urethra and bulbous portion of the urethra is more dorsal in position than normal. Vesicoureteral reflux is present in 50% of cases and is more often unilateral than bilateral. Reflux into the prostate might also be seen.\nFig.\u00a08Posterior urethral valves. An image from a VCUG demonstrates a thick-walled trabeculated bladder and dilatation of the posterior urethra (long arrow), and the location of the valve (small arrow)\nPrune-belly syndrome\nPrune-belly syndrome (PBS) was first described in 1950 by Eagle and Barret as a triad of deficient abdominal wall muscles, urinary tract anomalies and cryptorchidism. Associated abnormalities include cardiac, limb and intestinal abnormalities (30% of patients have malrotation). Although PBS is primarily seen in males, it has been described in females; female patients have genital abnormalities including bicornuate uterus and vaginal atresia [17].\nUrinary tract abnormalities, which can be evaluated by sonography and VCUG, include both upper tract disease and lower tract disease. Hydroureter, more pronounced than the coexisting hydronephrosis, is caused by poor ureteral peristalsis and poor bladder emptying rather than true obstruction. Varying degrees of renal dysplasia with or without cystic change might be present. Characteristic findings on VCUG include a large, smooth-walled bladder without trabeculations and marked bilateral vesicoureteral reflux into tortuous and laterally positioned ureters. A urachal diverticulum might be present, resulting in anterior and midline tethering of the bladder dome (Fig.\u00a09). Calcification is rarely identified within the bladder and urachal diverticulum and is secondary to urinary stasis [18].\nFig.\u00a09Prune-belly syndrome. a Images from a VCUG show the characteristic smooth-walled bladder that is tethered anteriorly by a urachal remnant (long arrow). Note the utricle (short arrow) arising from the dilated prostatic urethra, which is positioned dorsal to the wide bladder neck. b Voiding film shows dilatation of the proximal urethra consistent with megalourethra. Megalourethra can coexist in these patients\nThe prostatic urethra is dilated secondary to or in association with prostatic aplasia or hypoplasia. (Pathologically, the verumontanum is small and the ejaculatory ducts are normal.) Unlike PUVs, the prostatic urethra in patients with PBS is high in position and its posterior wall is elongated relative to its anterior wall. A normal utricle might be present. The penile urethra might be normal, or additional urethral anomalies such as megalourethra, urethral atresia or coexisting posterior urethral valves can be present. Patients with urethral obstruction do not survive [17].\nCongenital urethral stricture\nAlthough most urethral strictures in males are post-traumatic, there are rare reports of congenital urethral strictures of the bulbous urethra in neonates and older children [19]. These might be secondary to a failure of canalization of the cloacal membrane during fetal development [1, 20] and have also been referred to as Cobb\u2019s collar, Moormann\u2019s ring and Young\u2019s type III valve [7, 19]. Patients present with urinary tract infection. Older children might have diurnal enuresis. Diagnosis is by VCUG or retrograde urethrography. VCUG will demonstrate focal narrowing of the bulbous urethra, while retrograde urethrography will confirm a normal penile urethra. The site of urethral narrowing in congenital urethral stricture is distal to the external urethral sphincter, which differentiates this entity from PUV (Fig.\u00a010). Vesicoureteral reflux can be seen in up to 53% of patients [20]. Treatment is by transurethral incision. Other causes of urethral stricture, including trauma, must be excluded.\nFig.\u00a010Congenital urethral stricture. A markedly dilated urethra is seen proximal to a congenital stricture in the bulbous urethra. Retrograde urethrography in this patient (not shown) demonstrated a focal narrowing at the bulbous urethra with a normal penile urethra. The site of obstruction is more distal than that seen with PUV\nCongenital urethral polyps\nCongenital urethral polyps are benign and arise from the prostatic urethra near the verumontanum. They are composed of vascular connective tissue, although glandular and nerve tissue have been described. Because they have a stalk, these polyps are mobile and can move proximally into the bladder or distally into the bulbous urethra. They can be a cause of urethral obstruction or bleeding. VCUG is diagnostic and demonstrates a mobile filling defect in the bladder neck or below the verumontanum (Fig.\u00a011). Endoscopic resection is the treatment of choice [21, 22].\nFig.\u00a011Urethral polyp. a Image from a VCUG demonstrates a polypoid filling defect arising from the prostatic urethra (arrow). b Note change of position of the filling defect (arrow) during the examination (images courtesy of J. Michael Zerin, MD)\nMullerian duct remnants: enlarged prostatic utricle and Mullerian duct cyst\nThe normal prostatic utricle (Latin for \u201cpouch of the prostate\u201d) is a minute, blind opening located at the verumontanum [23, 24]. It is a glandular grouping lined with epithelial cells with differentiation similar to those lining the main prostatic ducts and acini [23]. It has no function and is the male homologue of the female uterus and vagina (Fig.\u00a012).\nFig.\u00a012Utricle. Voiding cystourethrogram in a child with urinary tract infection demonstrates a normal utricle that was incidentally detected (arrow). The ventral urethra is not filled with contrast material\nMidline cystic structures arising at the dorsal aspect of the prostatic urethra represent two distinct categories of Mullerian duct remnants. They are described as enlarged prostatic utricles when they communicate with the urethra and as Mullerian duct cysts when they do not. These terms have been used interchangeably, contributing to confusion in nomenclature.\nAn enlarged prostatic utricle is a congenital abnormality. Although its embryogenesis is not certain, theories include its derivation from the Mullerian duct or urogenital sinus [23] or secondary to an error in the production or sensitivity to Mullerian regression factor [25]. Unlike normal prostatic utricles, enlarged prostatic utricles are lined by squamous epithelium. Patients present with urinary tract infection, epididymitis, and postvoid dribbling [23\u201326]. Diagnosis is made by VCUG. During voiding, filling of a structure arising from the dorsal aspect of the prostatic urethra is seen (Fig.\u00a013). Retrograde urethrography is also diagnostic. In some cases, urethroscopy demonstrates absence of the verumontanum [23].\nFig.\u00a013Enlarged prostatic utricle. There is filling of a large utricle (arrow) that arises from and communicates with the posterior urethra in this patient with intersex abnormality. The patient has had penile urethral reconstruction (asterisk bladder)\nEnlarged prostatic utricles are classified according to a grading system. In grade 0, the opening is located in the prostatic urethra but the utricle does not extend over the verumontanum. In grade I the utricle is larger but does not extend to the bladder neck. In grade II, it extends over the bladder neck, and in grade III, the opening of the utricle opens into the bulbous urethra rather than the prostatic urethra [23]. Enlarged prostatic utricles are often seen in patients with intersex, hypospadias (11\u201314%), and cryptorchidism. There is a direct relationship between the degree of hypospadias and utricular size [25].\nIn contrast to enlarged prostatic utricle, Mullerian duct cysts are acquired abnormalities. They present later in life, are not associated with abnormal genitalia and do not communicate with the urethra. Histologically, they are lined by columnar or cuboidal epithelium identical to that of a normal prostatic utricle. Kato et al. [23, 24] have proposed that Mullerian duct cysts develop at a later stage than enlarged prostatic utricles, secondary to narrowing or obstruction of the communication between the normal utricle and the urethra. Patients present with an incidental rectal mass, constipation, urinary retention, hematuria or obstructive azospermia [23\u201326]. On US, CT or MRI a cystic mass is identified dorsal to the prostatic urethra (Fig.\u00a014). This may contain debris. VCUG is not helpful, since these cysts do not communicate with the urethra. Surgical excision is curative. Of note, there is a 3% incidence of malignancy in Mullerian duct cysts, most commonly occurring in the fourth decade [25].\nFig.\u00a014Mullerian duct cyst. Transverse sonographic image of the bladder in this 15-year-old boy with recurrent epididymitis demonstrates a cystic mass with internal debris (arrow) posterior to the bladder (asterisk)\nCowper\u2019s syringocele\nCowper\u2019s glands are small paired glands located dorsal to and on either side of the membranous urethra. They secrete a mucous substance during ejaculation that acts as a lubricant. The main duct draining Cowper\u2019s glands drain below the urogenital diaphragm into the ventral aspect of the bulbous urethra [27, 28]. During VCUG, the main duct and Cowper\u2019s glands can fill with contrast material and appear as a tubular channel paralleling the ventral aspect of the undersurface of the bulbous membranous urethra and ending at the urogenital diaphragm (Fig.\u00a015) [29]. This finding is usually of no clinical significance.\nFig.\u00a015Cowper\u2019s gland. During voiding there is filling of a structure at the ventral aspect of the bulbourethra (arrow)\nCowper\u2019s syringocele, a rare anomaly, occurs when there is dilatation of the main draining duct. Maizels et al. [30] described four types of Cowper\u2019s syringocele: simple syringocele, in which there is reflux into a minimally dilated duct; imperforate syringocele, in which the orifice draining the dilated duct is closed and there is cystic dilatation of the distal duct at the level of the bulbourethra; perforate syringocele, in which the orifice draining the duct is patulous and there is free reflux into the duct resembling a diverticulum; and ruptured syringocele, in which the distal portion of the duct is dilated but is not in communication with the more proximal portion of the main duct [30]. Cowper\u2019s syringocele can also be classified as open or closed, open if it communicates with the urethra and closed if it does not [31].\nClinically, patients present with frequency, urgency, dysuria, post-void incontinence, hematuria or urinary tract infection. Diagnosis is made by VCUG, retrograde urethrography or urethrocystoscopy. Treatment requires marsupialization of the syringocele.\nAnterior urethral valves and diverticula\nAnterior urethral valves and diverticula are rare. Most anterior valves (40%) are located in the bulbous urethra, but they can also be located at the penoscrotal junction (30%) and penile urethra (30%) [32]. They are composed of folds located on the ventral aspect of the urethra that rise during voiding, resulting in urethral obstruction [33]. Anterior urethral diverticula communicate with the urethra and are found on the ventral aspect of the urethra between the bulbous and mid-penile urethra [32].\nAlthough many authors distinguish between anterior urethral valves and diverticula, others consider them the same entity. It has been proposed that valves cause proximal urethral dilatation with the formation of a saccular diverticulum [32]. Conversely, progressive enlargement of a diverticulum can result in a distal valve-like flap. Embryologic theories of anterior urethral diverticula formation include a developmental defect in the corpus spongiosum leading to formation of a diverticulum, cystic dilatation of urethral glands, and sequestration of an epithelial rest [33]. McLellan et al. [28] have demonstrated connections between an anterior urethral diverticula and cowperian ducts, proposing that anterior urethral diverticula and valves arise from the anterior lip of a ruptured cowperian duct syringocele.\nClinical presentation depends on the patient\u2019s age and the degree of urethral obstruction and includes difficulty voiding, incontinence and recurrent urinary tract infections. VCUG is diagnostic (Fig.\u00a016). Some authors describe a difference in appearance between anterior diverticula and valves; in valves, the urethral dilatation forms an obtuse angle with the ventral floor of the urethra, while in a urethral diverticulum, the angle is acute [31]. Vesicoureteral reflux can be found in up to one-third of patients with anterior urethral valves and 20% of patients with diverticula [32]. Sonography has also been used for diagnosis [34]. Stone formation within a diverticulum has been reported [35]. Treatment consists of transurethral valve ablation or, in the case of diverticula, transurethral or open diverticulectomy and urethroplasty [32].\nFig.\u00a016Anterior urethral diverticulum. Arrow points to a diverticulum arising from and communicating with the ventral aspect of the urethra\nAnterior urethral valves occurring in the fossa navicularis, the most distal aspect of the urethra, are referred to as valves of Guerin. These are described as a septum partially separating the lacuna magna (a widening at the dorsal aspect of the distal urethra at the glans penis) from the urethra. Valves of Guerin might have a different embryologic basis than other types of anterior urethral valves. Some propose a failure of alignment between the glans urethra and penile urethra while others believe they represent an anatomic variant, as many patients are asymptomatic [36]. Valves of Guerin have been associated with urethral bleeding. VCUG is diagnostic and will demonstrate a small collection of contrast material at the dorsal aspect of the distal urethra (Fig.\u00a017). Marsupialization of the valve into the urethral lumen is the treatment of choice [37].\nFig.\u00a017Valves of Guerin. A small contrast-filled diverticulum (arrow) is present, arising from the dorsal aspect of the glans urethra. There is no urethral obstruction\nMegalourethra\nMegalourethra is caused by defective formation of the corpus spongiosum and corpora cavernosa secondary to a mesodermal defect. There are two types of megalourethra: (1) scaphoid, in which there is ventral urethral dilatation and hypoplasia of the corpus spongiosum, and (2) fusiform, in which there is circumferential urethral dilatation and hypoplasia of the corpus spongiosa and corpora cavernosa. Megalourethra is often associated with other congenital abnormalities including cryptorchidism, renal agenesis, hypospadias, primary megaureter, PBS, VACTERL complex [38] and severe gastrointestinal anomalies [39]. These patients have a functional rather than anatomic urethral obstruction, causing stasis and back pressure into the upper urinary tracts. Diagnosis is by VCUG (Fig.\u00a018). Reconstructive surgery is required.\nFig.\u00a018Megalourethra. Single view from a VCUG in a boy with partial sacral agenesis demonstrates focal dilatation of the urethra (arrow). Note the wide bladder neck\nUrethral duplication\nUrethral duplication is a rare anomaly frequently seen in association with other anomalies including hypospadias, epispadias, cleft lip and palate, congenital heart disease, tracheoesophageal fistula, imperforate anus and musculoskeletal anomalies [40].\nDuplication commonly occurs along the sagittal plane. The ventral urethra is the more functional urethra and contains the verumontanum and sphincters [40]. When urethral duplication is present along the coronal plane, bladder duplication is always present [41].\nUrethral duplication can be classified into three types using Effmann\u2019s classification [42]. In type I, there is partial duplication of the urethra. In type II, there is complete duplication of the urethra. Type II urethral duplication can be subclassified as IIA1 if both urethras arise from separate bladder necks, type IIA2 if one channel arises from the other, and type IIB if there is duplication with one meatus. IIA2 Y-duplication occurs when one urethra arising from the bladder neck or posterior urethra opens to the perineum. This type of urethral duplication often coexists with stenosis of the anterior portion of the normally positioned urethra [43] and other severe congenital anomalies [44]. Type III urethral duplication consists of complete duplication of the urethra and bladder.\nEmbryologically, urethral duplication can be caused by abnormal Mullerian duct termination and growth arrest of the urogenital sinus [40] or misalignment of the termination of the cloacal membrane with the genital tubercle [41].\nDepending on the type of duplication, patients might be asymptomatic. Symptoms include urinary tract infection, epididymitis, and incontinence [43]. Diagnosis can be made by VCUG or retrograde urethrography (Figs.\u00a019 and 20). Urodynamic studies are helpful to confirm the position of the functional urethra in order to distinguish it from congenital urethroperineal fistula (see below) [44]. One-third of patients have associated vesicoureteral reflux [41].\nFig.\u00a019Complete urethral duplication (type II). Image from a voiding cystourethrogram demonstrates contrast agent filling a dorsal and ventral urethra (arrows). Vesicoureteral reflux is presentFig.\u00a020Partial urethral duplication (type I). There is partial duplication of the urethra in this patient with a history of hypospadias repair. The two channels (arrows) join to a single channel, which is demarcated by the catheter\nCongenital urethroperineal fistula\nAlthough congenital urethroperineal fistula (CUF) resembles Y-type urethral duplication, it should be considered a separate entity. In Y-type urethral duplication the ventral urethra opens to the perineum and, as in all urethral duplications, is the functional urethra. In contrast, in CUF, the dorsal urethra is the functional urethra and the ventral urethra (fistula) is hypoplastic [41, 45]. The differentiation between Y-type duplication and CUF is particularly important in the surgical management of these patients. In Y-duplication the functional ventral channel should not be resected, while in the CUF resection of the ventral channel is curative [45].\nThe embryology of CUF is not clear. Diagnosis is made by retrograde urethrography or VCUG to determine the dominant urethra. In the case of congenital urethroperineal fistula, voiding will be predominantly through the dorsal urethra. Urodynamics is important to determine the position of the external urethral sphincter [39].\nAnorectal malformations\nCongenital anorectal malformation represents a complicated spectrum of anomalies that range from anal stenosis to imperforate anus with communication with the urogenital system [46]. In males, the anomaly is characterized by gastrointestinal anomalies, imperforate anus and frequently other congenital anomalies including tracheoesophageal fistula, heart disease, limb anomalies and radial and renal abnormalities (VACTERL complex) [47]. The malformation can be classified as low or high\/intermediate. Fistulous communications between the blind-ending rectum and the urinary tract are more common in males with high\/intermediate anomalies. Rectobulbocutaneous fistulous tracts have also been described in which a fistula arises from the rectum, communicates with the bulbous urethra at its mid-portion and continues ventrally to terminate at the scrotum wall [48, 49]. A concurrent rectourethral fistula to the posterior urethra or, less commonly, the bladder, has been described in 28% of patients of imperforate anus [50] and there is a high incidence of other urethral anomalies as well [39, 40].\nEmbryologically the normal sequence of cloacal division by the urorectal septum into the urogenital sinus and rectum is disrupted. Preexisting fusion of mesoderm at the dorsal aspect of the cloacal folds might be present, which prevents normal caudal descent of the urogenital septum and results in a urorectal septal defect. Recent reports describe a possible genetic link between abnormalities of the Sonic Hedgehog gene (Shh) and midline developmental abnormalities such as those seen with the VATERL association [40, 47, 51].\nDiagnosis is made by VCUG or distal loop study following colostomy (Figs.\u00a021 and 22).\nFig.\u00a021Anorectal malformation. Image from a VCUG demonstrates an anourethral fistula (arrow) in a newborn. This is also referred to as an H-type fistula because of its configurationFig.\u00a022Anorectal malformation. Distal loop study in an infant with a high imperforate anus demonstrates a fistula extending from the rectum to the posterior urethra\nConclusion\nThe development of the male urethra is complex. Although understanding its embryology can help explain certain anomalies, there are others in which the embryologic defect has not been elucidated. Imaging, including VCUG and retrograde urethrography, is helpful in defining the anatomy and identifying associated findings.","keyphrases":["congenital","anomalies","male urethra","embryology"],"prmu":["P","P","P","P"]}
{"id":"Eur_J_Epidemiol-4-1-2190786","title":"The Generation R Study Biobank: a resource for epidemiological studies in children and their parents\n","text":"The Generation R Study is a population-based prospective cohort study from fetal life until young adulthood. The study is designed to identify early environmental and genetic causes of normal and abnormal growth, development and health from fetal life until young adulthood. In total, 9,778 mothers were enrolled in the study. Prenatal and postnatal data collection is conducted by physical examinations, questionnaires, interviews, ultrasound examinations and biological samples. Major efforts have been conducted for collecting biological specimens including DNA, blood for phenotypes and urine samples. In this paper, the collection, processing and storage of these biological specimens are described. Together with detailed phenotype measurements, these biological specimens form a unique resource for epidemiological studies focused on environmental exposures, genetic determinants and their interactions in relation to growth, health and development from fetal life onwards.\nIntroduction\nThe Generation R Study is a population-based prospective cohort study from fetal life until young adulthood. The background and specific research projects of the study have been described in detail previously [1, 2]. Briefly, the Generation R Study is designed to identify early environmental and genetic causes of normal and abnormal growth, development and health from fetal life until young adulthood. The study focuses on four primary areas of research: (1) growth and physical development; (2) behavioural and cognitive development; (3) diseases in childhood; and (4) health and healthcare for pregnant women and children. The main outcomes and determinants are presented in Tables\u00a01 and 2. The general aims of the study are:To describe normal and abnormal growth, development and health from fetal life until young adulthood;To identify biological, environmental and social determinants of normal and abnormal growth, development and health from fetal life until young adulthood;To examine the effectiveness of current strategies for prevention and early identification of groups at risk.Table\u00a01Main outcomes per research areaGrowth and physical developmentFetal growth patterns and organ developmentPregnancy complicationsPostnatal growth patternsObesityRisk factors for development of cardiovascular diseaseRisk factors for type 2 diabetesBehavioural and cognitive developmentMaternal and paternal psychopathologyFetal and postnatal brain developmentBehaviour, psychopathology and cognitionNeuromotor developmentChronic painAttachmentStress reactivityDiseases in childhoodInfectious diseases in childhoodDevelopment of the immune systemAsthma and asthma related symptomsParoxysmal neurological disordersHealth and healthcareQuality of lifeHealth care utilizationEffectiveness of screening programmesTable\u00a02Main determinantsBiological determinantsParental anthropometrics and blood pressureFetal and postnatal growth characteristicsEndocrine and immunological factorsGenetic variantsEnvironmental determinantsMaternal and childhood dietParental life style habits (including smoking, alcohol consumption)Housing conditionsSocial determinantsParental education, employment status and household incomeParental marital statusEthnicity\nFor these aims, a detailed and extensive data collection has been conducted in the prenatal phase of the study and is currently conducted in the early postnatal period [2]. This data collection comprises also biological specimens including DNA, blood for phenotypes and urine samples. These biological specimens form the Generation R Study Biobank, which enables future epidemiological studies focused on environmental exposures, genetic determinants and their interactions in relation to growth, health and development from fetal life onwards. In this paper, the design, collection, processing and storage of these biological specimens in the prenatal phase of the study are described.\nDesign and biological samples collection\nThe Generation R Study is a population-based prospective cohort study from fetal life until young adulthood [2]. Mothers with a delivery date between April 2002 and January 2006 were eligible. Enrolment was aimed in early pregnancy but was allowed until birth of the child. Extensive assessments were carried out in mothers and their partners in pregnancy and are currently performed in their children. Assessments in pregnancy were planned in early pregnancy (gestational age <18\u00a0weeks), mid-pregnancy (gestational age 18\u201325\u00a0weeks) and late pregnancy (gestational age \u226525\u00a0weeks) and at birth. The individual time scheme of these assessments depended on the specific gestational age at enrolment [2]. The partners were assessed once in pregnancy. All children form a prenatally recruited birth-cohort that is currently being followed until young adulthood.\nAdditionally, more detailed assessments of fetal and postnatal growth and development are conducted in a randomly selected subgroup of Dutch children and their parents, referred to as the Generation R Focus Cohort. Studies conducted in this subgroup examine etiological associations using more time-consuming methods that cannot be applied in the whole cohort.\nBiological materials have been collected in early, mid- and late pregnancy and at birth. Figure\u00a01 shows the design of the collection of biological materials in this phase. In early, mid- and late pregnancy, biological materials were collected at the visits to one of our dedicated research centres. The planned amounts of blood taken by ante-cubital venous puncture were 35 ml in early pregnancy and 20\u00a0ml in mid-pregnancy from the mother and 10\u00a0ml from her partner. When mothers were enrolled in mid- and late pregnancy, 35\u00a0ml was taken at the first visit. Urine samples of mothers have been collected in early, mid- and late pregnancy from February 2004 until November 2005. Directly after delivery, midwives or obstetricians collected 30\u00a0ml cord blood from the umbilical vein.\nFig.\u00a01Design data collection biological specimens\nThe Generation R Study is approved by the Medical Ethical Committee of the Erasmus Medical Center, Rotterdam. Mothers and their partners received written and oral information about the study. Participants were asked for their written informed.\nStudy cohort\nIn total, 9,778 mothers were enrolled in the study. Of these mothers, 91% (n\u00a0=\u00a08,880) was enrolled in pregnancy. Only partners from mothers enrolled during pregnancy were invited to participate. In total, 71% (n\u00a0=\u00a06,347) of all partners were enrolled. The general characteristics of the mothers and their partners are presented in Table\u00a03. Of all participating mothers, enrolment was in early pregnancy in 69% (n\u00a0=\u00a06,748), in mid-pregnancy in 19% (n\u00a0=\u00a01,857), in late pregnancy in 3% (n\u00a0=\u00a0275) and at birth of their child in 9% (n\u00a0=\u00a0898). The cohort comprises various ethnic groups. The largest ethnic groups are the Dutch, Surinamese, Turkish, Moroccan, Dutch-Antilles and Cape Verdian mothers.\nTable\u00a03Characteristics of mothers and their partners MothersPartners(n\u00a0=\u00a09,778)(n\u00a0=\u00a06,347)Gestational age at enrolment (%)\u00a0\u00a0\u00a0\u00a0Early pregnancy69\u2013\u00a0\u00a0\u00a0\u00a0Mid-pregnancy19\u2013\u00a0\u00a0\u00a0\u00a0Late pregnancy3\u2013\u00a0\u00a0\u00a0\u00a0Birth9\u2013Pregnancy number in study (%)\u00a0\u00a0\u00a0\u00a01st pregnancy 94\u2013\u00a0\u00a0\u00a0\u00a02nd pregnancy 6\u2013\u00a0\u00a0\u00a0\u00a03rd pregnancy 0.1\u2013Age at enrolment (years)a29.7 (5.3)32.7 (5.8)Parity (%)\u00a0\u00a0\u00a0\u00a0055\u2013\u00a0\u00a0\u00a0\u00a0131\u2013\u00a0\u00a0\u00a0\u00a0\u2265214\u2013Ethnicity (%)\u00a0\u00a0\u00a0\u00a0Dutch, other-European 5870\u00a0\u00a0\u00a0\u00a0Surinamese96\u00a0\u00a0\u00a0\u00a0Moroccan 74\u00a0\u00a0\u00a0\u00a0Turkish 96\u00a0\u00a0\u00a0\u00a0Dutch Antilles 43\u00a0\u00a0\u00a0\u00a0Cape Verdian 42\u00a0\u00a0\u00a0\u00a0Others 99Education (vocational training) (%)\u00a0\u00a0\u00a0\u00a0Lower 138\u00a0\u00a0\u00a0\u00a0Intermediate4541\u00a0\u00a0\u00a0\u00a0Higher 4251Values are percentages aMean (standard deviation)\nLogistics\nAll biological samples were bar coded with a unique laboratory number. Blood or urine samples collected at one visit have the same bar code with an additional specific tube number. This combination forms a unique registration number. All following steps in processing, storing and data management of the samples are linked to this unique number.\nAll blood samples from the mother and partner were taken by research nurses and temporally stored at our research centre or one of the obstetric departments at room temperature for a maximum of 3\u00a0h. At least every 3\u00a0h, these blood samples were transported to a dedicated laboratory facility of the regional laboratory in Rotterdam, the Netherlands (STAR-MDC) for further processing and storage. Participants delivered their child either at home or at one of the hospitals in Rotterdam. The midwife or obstetrician collected cord blood samples. Subsequently, courier services with a 7-days and 24-h availability were responsible for transportation of these cord blood samples to our laboratory within 2\u00a0h.\nAfter collection and transportation, all blood and urine samples have been centrally processed and stored at the STAR-MDC laboratory. Samples for DNA extraction have initially been stored as EDTA whole blood samples and subsequently transported to the Erasmus Medical Center for further processing.\nAll blood and urine samples are registered in Labosys (Philips) [3]. Both the anonymous person unique study numbers and all sample numbers are registered which enables matching of each sample to a study subject. Also, picking and defrosting of samples in registered in Labosys. A back-up of this registration is available at the Erasmus Medical Center.\nResponse rates\nResponse rates for collection of biological specimens are presented in Table\u00a04. Overall response rates for blood collection in mothers are high. To get these high response rates, we have used the venous punctures that were already planned in routine care for additional blood sample collection when this was possible. Blood for DNA extraction is available in 91, 83 and 67% of the eligible mothers, partners and children. The larger number of missings in partners and children were mainly due to lack of consent and logistical constraints during delivery, respectively. As Table\u00a04 shows, response rates for the various blood samples at one occasion were similar. Absolute numbers of urine samples are lower than the absolute number of blood samples because the urine sample collection was performed during a limited period in the prenatal phase of the study. Tables\u00a05 and 6 show characteristics of children and partners with and without available DNA. In children, the number of missing blood samples in children with perinatal complications including low birth weight and preterm birth is higher because of the limited possibilities and priorities during these deliveries. At this moment, efforts are carried out for completing DNA collection in the children by buccal samples.\nTable\u00a04Response rates biological specimensEligibleAvailable % (n)Mother\u00a0\u00a0\u00a0\u00a0DNA8,88091 (8,055)\u00a0\u00a0\u00a0\u00a0Early pregnancy\u00a0\u00a0\u00a0\u00a0Plasma (EDTA)6,74895 (6,398)\u00a0\u00a0\u00a0\u00a0Serum6,74894 (6,337)\u00a0\u00a0\u00a0\u00a0Urine2,79785 (2,375)\u00a0\u00a0\u00a0\u00a0Mid-pregnancy\u00a0\u00a0\u00a0\u00a0Plasma (EDTA)8,24193 (7,682)\u00a0\u00a0\u00a0\u00a0Serum8,24193 (7,631)\u00a0\u00a0\u00a0\u00a0Urine3,38097 (3,279)\u00a0\u00a0\u00a0\u00a0Late-pregnancy\u00a0\u00a0\u00a0\u00a0Urine3,91896 (3,762)Partner\u00a0\u00a0\u00a0\u00a0DNA6,34783 (5,289)Child\u00a0\u00a0\u00a0\u00a0DNA8,82167 (5,908)\u00a0\u00a0\u00a0\u00a0Plasma (EDTA)8,82166 (5,857)\u00a0\u00a0\u00a0\u00a0Serum8,82166 (5,821)Number of eligible subjects reflects those participating in the study during pregnancy (DNA mother and partner) and visit our research centre in early, mid- or late pregnancy (plasma, serum and urine). The number of eligible subjects for urine sample collection is lower since this data-collection was added after starting the study. Number of eligible children reflects the number of live born children of mothers who were enrolled in the prenatal phase of the study with a known date of birthTable\u00a05Characteristics of partners with and without DNA availablePartners (n\u00a0=\u00a06,347)DNA available (n\u00a0=\u00a05,289)No DNA available (n\u00a0=\u00a01,058)Age (years)32.8 (5.7)32.2 (5.7)Ethnicity (%)\u00a0\u00a0\u00a0\u00a0Dutch, other-European 68.266.1\u00a0\u00a0\u00a0\u00a0Surinamese6.86.6\u00a0\u00a0\u00a0\u00a0Moroccan 3.86.2\u00a0\u00a0\u00a0\u00a0Turkish 6.87.2\u00a0\u00a0\u00a0\u00a0Dutch Antilles 2.93.4\u00a0\u00a0\u00a0\u00a0Cape Verdian 2.71.9\u00a0\u00a0\u00a0\u00a0Others 8.88.6Values are percentages or means (standard deviation)Table\u00a06Characteristics of children with and without DNA availableChildren (n\u00a0=\u00a08,821)DNA available (n\u00a0=\u00a05,908)No DNA available (n\u00a0=\u00a02,913)Male (%)50.650.1Birth weight (grams)3,444 (520)3,280 (671)Birth weight\u00a0<2,500\u00a0g (%)3.310.8Gestational age (weeks)40.1 (37.1\u201342.0)39.9 (34.7\u201342.1)Gestational age\u00a0<37\u00a0weeks (%)3.911.2Ethnicity (%)\u00a0\u00a0\u00a0\u00a0Dutch, other-European 64.754.6\u00a0\u00a0\u00a0\u00a0Surinamese7.49.0\u00a0\u00a0\u00a0\u00a0Moroccan 6.57.9\u00a0\u00a0\u00a0\u00a0Turkish 7.59.5\u00a0\u00a0\u00a0\u00a0Dutch Antilles 3.84.8\u00a0\u00a0\u00a0\u00a0Cape Verdian 2.43.5\u00a0\u00a0\u00a0\u00a0Others 7.710.7Values are percentages, means (standard deviation) or median (95% range)\nDNA\nFor DNA extraction, one 10\u00a0ml EDTA tube from mothers, partners and children at birth have been collected and stored directly as whole blood sample at \u221280\u00b0C. After completion of the blood sample collection, DNA extraction was started from white blood cells. First, DNA extraction from all children has been conducted manually using the Qiagen FlexiGene Kit (Qiagen Hilden, Germany). Currently, DNA extraction from 5\u00a0ml whole blood samples from the mothers and partners is performed by a Hamilton STAR multi-channel robot using AGOWA magnetic bead technology (at 72 samples per run) and is expected to be finished in spring 2008.\nExtracted DNA is automatically collected in stock tubes (2D Matrix, Micronic) that are organized in 96-wells format, but that can be individually addressed. These stock samples are split into two tubes that are stored at different locations. One of the stocks will then be used for normalising the DNA concentrations in 96 deep well (DW) plates. This protocol, as well as other manipulations of the DNA samples are performed on a Caliper ALH3000 pipetting robot (8\/96\/384 channels) with a Twister module and a 96\/384 wells Tecan GENios plus UV reader. In total, 90 DNA samples are normalised per 96 DW plate with 6 water blancs per plate. Additionally, a random selection of 5% of the total number of samples are put into separate 96 DW plates, again with 6 blancs per plate, for control purposes. For normalisation, sample and diluent volumes are automatically calculated and produced to obtain equal DNA concentrations for all samples (25\u201350\u00a0ng\/\u03bcl). From these stock 96-DW plates, 384 DW plates are created, which comprise 24 blancs each. Subsequently, replica PCR plates (384 wells) with a concentration of 1\u00a0ng\/\u03bcl are created for genotype studies.\nStock 96-well plates (DW and 2D Matrix, Micronic) are stored at \u221220\u00b0C at two different locations within the Erasmus Medical Center (Genetic Epidemiology Laboratory, Genetic Laboratory Internal Medicine). The 384 DW stock and PCR plates are stored at the Genetic Laboratory Internal Medicine, Erasmus Medical Center. All genotyping studies with Generation R DNA material are performed in-house at the high throughput genotyping facility in this Laboratory.\nGenome Wide Association database\nA new approach to identify most important common genetic variants that contribute to explain variance in phenotypes by a systematic analysis of our genome has only recently become available as the Genome Wide Association (GWA) analysis [4]. The GWA analysis is now generally considered a critical tool to identify genes for complex diseases and traits. The GWA analysis is based on genotyping large numbers (>500,000\u20132,000,000) of Single Nucleotide Polymorphisms (SNPs), and subsequently performing association analyses of the SNP genotypes (or haplotypes thereof) with the phenotype of interest. The power of GWA analyses has been recently demonstrated with the identification of susceptibility genes involved in a wide range of disorders varying from common and rare diseases including coronary heart disease, diabetes and multiple sclerosis [5\u20137]. These developments have led to a stream of novel disease genes, highlighting new etiological pathways and are expected to improve the understanding of the molecular basis of these diseases. Currently, we are preparing a GWA analysis in all children participating in the Generation R Study of whom DNA is available. For this, the infrastructure, used for the GWA analysis in Rotterdam Study, a population-based cohort study in about 15,000 adults in Rotterdam, the Netherlands is used [8].\nBlood for phenotypes\nResults of analyses performed in blood samples (5\u00a0ml EDTA plasma) for routine care for pregnant women (Hb, Ht, HIV, HBsAg, Lues, Rhesus factor and irregular antibodies) are obtained from midwife and obstetric registries.\nAll other EDTA plasma and serum samples from mothers and children have been processed and stored for future studies. Processing was aimed to be finished within a maximum of 3\u00a0h after venous puncture. Total processing time took 15\u00a0min. The samples were spun and the plasma and serum volume was distributed into 250\u00a0\u03bcl aliquots and transferred to 0.65\u00a0ml polypropylene tubes (Micronic) by a Tecan automatic liquid handler. From the mother, a set of on average 16 aliquots tubes (with a total volume of 4\u00a0ml) each from one plasma and serum sample were divided over four different microtiter trays. From the plasma and serum samples from the children, on average 11 tubes were filled with 250\u00a0\u03bcl aliquots (in total 2.75\u00a0ml) divided over three microtiter trays. The trays were immediately stored at \u221280\u00b0C. Each Micronic tube is uniquely coded on the bottom with the Traxis\u00ae 2D code.\nThe aliquots were divided over 4 trays of 96 tubes for storage in the freezers. Figure\u00a02 shows the distribution of the samples in the 96 trays. All samples are stored at \u221280\u00b0C at one location (STAR-MDC laboratory) where they are distributed over four different freezers with different power supplies.\nFig.\u00a02Distribution of blood samples in the 96 trays (see text)\nUrine\nUrine samples (65\u00a0ml) were added to the data collection between February 2004 and November 2005. The urine samples were kept at room temperature for a maximum of 3\u00a0h and transported to the STAR-MDC laboratory for further processing. At the laboratory, the urine was filled out manually in one 5\u00a0ml and three 20\u00a0ml tubes. Before the urine was filled out, a gravity measurement was performed. The 5\u00a0ml urine tubes were send to the Department of Medical Microbiology of the Erasmus Medical Centre for Chlamydia analysis [9]. The remaining urine tubes (20\u00a0ml) were each stored at \u221220\u00b0C at the STAR-MDC laboratory.\nOther population-based birth cohort studies\nThe Generation R Study is not the first birth-cohort study with an extensive collection of biological specimens. Compared to other birth-cohort studies, the size of the Generation R Study cohort is not larger but the measurements are more detailed and the study group has a large ethnic variety [10\u201314]. Studies having a similar design and biological specimens collection include the Avon Longitudinal Study of Parents and Children (ALSPAC) in Bristol, UK, the Danish National Birth Cohort Study, the Norwegian mother and child Cohort Study, the Southampton Women\u2019s Survey (SWS) in Southampton, UK and The National Children\u2019s Study in the USA.\nCollaboration with these studies for funding, standardization, replication and quality management is necessary. Currently, we are exploring possibilities for formal collaboration.\nRequests for biological samples use\nThe study has an open policy with regard to collaboration with other research groups. Request for collaboration and use of biological specimens should primarily be pointed to Albert Hofman (a.hofman@erasmusmc.nl). These requests are discussed in the Generation R Study Management Team regarding their scientific merits, study aims, overlap with ongoing studies, logistic consequences and financial contributions.\nGeneral policy is that collaborating researchers and groups are responsible themselves for the finances and that all laboratory tests are conducted in the Eramus Medical Center, Rotterdam. After approval of the project by the Generation R Study Management Team and the Medical Ethical Committee of the Erasmus Medical Center, the collaborative research project is embedded in one of the four research areas supervised by the specific principal investigator.","keyphrases":["biobank","child","parents","cohort study"],"prmu":["P","P","P","P"]}
{"id":"Photosynth_Res-4-1-2173912","title":"Structure and organization of phycobilisomes on membranes of the red alga Porphyridium cruentum\n","text":"In the present work, electron microscopy and single particle averaging was performed to investigate the supramolecular architecture of hemiellipsoidal phycobilisomes from the unicellular red alga Porphyridium cruentum. The dimensions were measured as 60 \u00d7 41 \u00d7 34 nm (length \u00d7 width \u00d7 height) for randomly ordered phycobilisomes, seen under high-light conditions. The hemiellipsoidal phycobilisomes were found to have a relatively flexible conformation. In closely packed semi-crystalline arrays, observed under low-light conditions, the width is reduced to 31 or 35 nm, about twice the width of the phycobilisome of the cyanobacterium Synechocystis sp. PCC 6803. Since the latter size matches the width of dimeric PSII, we suggest that one PBS lines up with one PSII dimer in cyanobacteria. In red algae, a similar 1:1 ratio under low-light conditions may indicate that the red algal phycobilisome is enlarged by a membrane-bound peripheral antenna which is absent in cyanobacteria.\nIntroduction\nIn cyanobacteria and red algae, the main accessory light-harvesting complexes are the phycobilisomes (PBSs), large and highly structured assemblies of phycobiliproteins associated to the cytoplasmic surface of the thylakoid membrane (Gantt 1980; Grossman et\u00a0al. 1993; MacColl 1998; Adir 2005). They capture and transfer energy primarily to chlorophyll (Chl) a in Photosystem (PS) II (Gantt 1986; Grossman et\u00a0al. 1993, 1995; Sidler 1994; MacColl 1998; Samsonoff and MacColl 2001; Adir 2005). The PBSs are composed of \u03b1- and \u03b2- polypeptides bearing covalently attached open-chain tetrapyrroles (Betz 1997). They assemble in disc-shaped aggregates that are stacked in the peripheral rods of the PBS. Colourless linker polypeptides between adjacent discs stabilize the PBS structure and regulate energy transfer (Liu et\u00a0al. 2005).\nPBSs in the unicellular red alga Porphyridium cruentum were first observed about 40\u00a0years ago (Gantt and Conti 1966) and described as hemiellipsoidal. Based on electron microscopic results, a preliminary model was proposed (Gantt et\u00a0al. 1976) involving a tricylindrical core subassembly in the semi-spherical centre and several peripheral rods radiating out from the centre to form the rounded surface of the semi-sphere. The core contains allophycocyanin (APC), while the peripheral rods are mainly composed of phycocyanins (PC) and phycoerythrins (PE). In other systems, hemidiscoidal PBSs have been identified of which the 3-D structure was recently characterized in some detail by single-particle electron microscopy (Barber et\u00a0al. 2003; Yi et\u00a0al. 2005). However, such detailed information of hemiellipsoidal PBSs is still lacking.\nDespite the fact that energy transfer and mutational studies have indicated that phycobilisomes can couple directly to PSI as well as to PSII, little is known about the structural aspects of the interaction. The precise association of phycobilisomes to PSII and PSI is not known, but there are some hints which PSII subunits could be involved in interaction. A structural comparison between the core parts of green plant and cyanobacterial PSII shows differences in the position of the cyt b559 subcomplex and of the CP47 core antenna with respect to the D1\/D2 reaction centre. Within the D1\/D2 proteins, there are clear differences between plants and cyanobacteria at the stromal ends of membrane-spanning \u03b1-helices, even though these proteins are highly homologous. The differences in subunit arrangements in these two complexes may reflect an adaptation to the peripheral antenna systems, membrane-extrinsic phycobilisomes in the case of cyanobacteria and membrane-bound peripheral LHCII subunits in the case of green plants (B\u00fcchel and K\u00fchlbrandt 2005).\nState transitions are a physiological adaptation mechanism that changes the interaction of the peripheral antenna proteins with the PSI and PSII core complexes. In green plants and green algae, membrane-bound peripheral antenna proteins play a specific mobile role to (re)direct part of the excitation energy either towards PSI or PSII ; whereas in cyanobacteria the phycobilisomes are considered to play this role. It was found with FRAP measurements that the phycobilisomes diffuse quite rapidly, but that PSII is immobile on the timescale of the measurement, indicating that the linkage between PBSs and the photosystems is unstable (Sarcina et al. 2001, 2006; Joshua et al. 2005). Based on these data it was proposed that the lateral diffusion of PBSs is involved in regulation of photosynthetic light-harvesting (state 1-state 2 transitions) (Mullineaux et\u00a0al. 1997). It was found that phycobilisomes diffuse, on average, nearly three times faster in a PsaL(-) mutant than in the wildtype (Aspinwall et\u00a0al. 2004). It is currently believed that light-induced state transition depends completely on a movement of PBSs towards PSI or PSII (Li et\u00a0al. 2006).\nDespite the fact that high-resolution data on the interaction of PBSs and photosystems are lacking, models for their organization have been presented (Bald et\u00a0al. 1996). For hemidiscoidal phycobilisomes the key features are the PSII dimer as prerequisite for interaction and a row-like organization of PSII dimers as a template for phycobilisome attachment. Two PSII monomers form the \u2018contact area\u2019 within the PSII-PBS supercomplex under so-called \u2018state 1\u2019 conditions. In this model attachment of PBS with Photosystem I (PSI) under so-called \u2018state 2\u2019 conditions was proposed (Bald et\u00a0al. 1996). This model stresses that the multimeric state of the PSI and PS II complexes will be of relevance. In cyanobacteria, PSI can be present as monomers or trimers and PSII as monomers or dimers. In red algae, both PSI and PSII are different because they bind several copies of membrane-associated antenna copies, which are lacking in cyanobacteria (S. Kere\u00efche, R. Kou\u0159il, G.T. Oostergetel, A. B. Doust, E.J. Boekema, J.P. Dekker, unpublished data). Moreover, PSI only exists as a monomer (Gardian et\u00a0al. 2007).\nWe have studied the supramolecular conformation of hemiellipsoidal PBSs in Porphyridium cruentum, as well as\u2014for comparison\u2014hemidiscoidal PBS in a Synechocystis PCC 6803 mutant by single particle electron microscopy. PBS-thylakoid vesicles provide relatively uniform PBS structure in contrast to isolated PBSs, and allow us to acquire a spatial view of hemiellipsoidal structure. It is suggested that one PBS is likely to associate and line-up with one PSII dimer, in both cyanobacteria and red algea despite the fact that the red algal PBSs are two times larger in width. The larger PBS width in red algae can, however, be compensated by a membrane-bound peripheral antenna, absent in cyanobacteria.\nMaterials and methods\nIsolation of isolated PBSs and PBS-thylakoid membranes\nPorphyridium cruentum was grown in an artificial sea water medium. Flasks were supplied with 3% CO2 in air through a plug of sterile cotton at a constant temperature of 18\u00b0C. Cultures were illuminated continuously with light provided by daylight fluorescent lamps at different light intensities, 6\u00a0Wm\u22122 (low-light) and 15\u00a0Wm\u22122 (high-light). The separation of isolated PBSs followed a published protocol (Glazer 1988).\nThe PBS-membrane vesicles were prepared as described by Mustardy et\u00a0al. (1992) with slight modifications. Cells were harvested by centrifugation (5000\u00a0g; 10\u00a0min), rinsed with 0.5\u00a0M potassium phosphate buffer (pH 7.0), and then suspended in SPC medium (0.5\u00a0M sucrose, citrate, 0.5\u00a0M potassium phosphate, 0.3\u00a0M potassium citrate, pH 7.0). After homogenization, the cells were broken in a French press at 4,000 psi in order to achieve large membrane sheets with minimal disturbance. The broken cell mixture was layered on a two-step sucrose gradient (1.0\u00a0M and 1.3\u00a0M) with the same buffer conditions as the cell suspension. After centrifugation for 30\u00a0min in a micro-centrifuge, the PBS-containing thylakoids were collected from the 1.0\u20131.3\u00a0M sucrose interface.\nSamples for imaging were prepared by placing glow-discharged, collodion-carbon-coated copper grids (200\u00a0mesh\/inch) onto a droplet of sample solution for 10\u00a0min, excess liquid was then blotted off and the samples were fixed with 2% glutaraldehyde, followed by successive washes on three droplets of double-distilled water. Staining was achieved by placing the grid on a droplet of 2% uranyl acetate. Finally, the staining solution was blotted off.\nElectron microscopy\nElectron microscopy was performed on a CM120 electron microscope (Philips, Eindhoven, The Netherlands) operated at 120\u00a0kV. Images were recorded under low dose conditions (a total dose \u223c25\u00a0e\u2212\/\u00c52) with a 4,000 SP 4\u00a0K slow-scan CCD camera (Gatan, Pleasanton, CA). Images of 2-D arrays of membrane-bound phycobilisomes were recorded at 45,000 magnification, at a defocus value of \u2212600\u00a0nm with a pixel size of 6.5\u00a0\u00c5 at the level of the specimen, (after binning the images). From the sample of non-crystalline phycobilisomes, 600 images were recorded at 80,000 magnification, at a defocus value of \u2212380\u00a0nm with a pixel size of 3.75\u00a0\u00c5 at the level of the specimen after a binning of 2. In total, about 12,000 single particle projections were selected for single particle image analysis, which was performed with the Groningen Image Processing (GRIP) software package on a PC cluster. Selected single-particle projections were aligned by multi-reference and reference-free alignment procedures (Penczek et\u00a0al. 1992; van Heel et\u00a0al. 2000). Particles were then subjected to multivariate statistical analysis followed by hierarchical classification (van Heel et\u00a0al. 2000).\nResults\nMembranes with attached PBSs were studied with electron microscopy. By cross-linking with gluteraldehyde, the integrity of the PBS organization on the membrane was preserved. Under low-light growth conditions (6\u00a0W\/m\u22122), the PBSs on the membrane were mostly arranged in ordered domains with a high packing density of about 560\u00a0\u00b1\u00a020 PBSs per \u03bcm2 (Fig.\u00a01, left frame). In contrast, at higher light intensities the distribution of PBSs is largely disordered, and the density is significantly lower, 384\u00a0\u00b1\u00a045 PBSs per \u03bcm2 (Fig.\u00a01, right frame). This is in agreement with earlier reports of the dependence of pigment density in P.\u00a0cruentum on growth conditions (Jahn et\u00a0al. 1984; Gantt et\u00a0al. 2003), but this is the first time that it is shown that the PBS arrangement is also affected.\nFig.\u00a01Typical examples of negatively stained cyanobacterial membranes and associated phycobilisomes showing multiple ordered 2-D arrays under low-light conditions (left frame) and a random distributiom under high-light conditions (right frame) and the space bar equals 1\u00a0\u03bcm for the left frame and 400\u00a0nm for the right frame\nSingle particle analysis was performed on large sets of electron microscopy projections, selected from EM specimens with negatively stained membranes, on which many PBSs were still membrane-bound and smaller numbers lying around the membranes. The best preserved details were found in the top view of the membrane-bound PBS, which has maximal dimensions of 60\u00a0\u00d7\u00a041\u00a0nm in projection (Fig.\u00a02A). Free PBSs have about the same features but lack sharp details (Fig.\u00a02B). A trapeziform projection shows the PBS in a side view position, but it appears rather fuzzy, despite the fact that over 4,000 projections were processed (Fig.\u00a02C). Smaller projections are almost featureless (Fig.\u00a02D, E). A small, almost circular-shaped projection with 3-fold symmetry and a diameter of about 11.5\u00a0nm is a common impurity and is very likely to be a PBS fragment released during storage or EM specimen preparation (Fig.\u00a02F). Presumably this is a single disc of phycobiliproteins with a linker polypeptide incorporated as can be discerned from the electron density in the centre.\nFig.\u00a02Single particle electron microscopy of phycobilisome projections from Porphyridium cruentum (A) View in the membrane plane of membrane-bound phycobilisomes (B) View in the membrane plane of free phycobilisomes (C\u2013E) side views of free phycobilisomes. (F) Most common phycobilisome fragment. (G) image of A with 2-fold rotational symmetry imposed after analysis (H) Image of C, with mirror symmetry imposed after analysis. The number of summed projections for A-F is 1520, 484, 4096, 380, 499 and 746, respectively. The space bar is 25\u00a0nm\nIn an attempt to study the types of packing of PBSs within 2D arrays we compared several hundred areas. In parts with a low amount of ordering the PBSs have a tendency to associate into specific linear structures with their long edges. This is also true for those arrays with the highest amount of ordering. These membrane parts do not show a uniform type of packing, as is exemplified by two average sums of arrays showing a different repeating motif and size, which is about 55\u00a0\u00d7\u00a035\u00a0nm in Fig.\u00a03C and 45\u00a0\u00d7\u00a031\u00a0nm in Fig.\u00a03D, respectively.\nFig.\u00a03Comparison of semi-crystalline arrays of phycobilisomes. (A) the membrane-attached phycobilisome (B) APC core structure of Synechocystis PCC 6803 ck mutant, sum of 5019 projections for comparison (A.A. Arteni, R. Ajlani, E.J. Boekema, unpublished results). (C) 2D array with semi-crystalline phycobilisomes, an average of 11 fragments (D) 2D array with semi-crystalline phycobilisomes, an average of 107 fragments (E) PSII dimer (from Thermosynechococcus elongatus) (F) PSII double dimer, found after solubilization of membranes with dodecyl maltoside, a sum of 59 projections (G) PSII double dimer, found after solubilization of membranes with digitonin, a sum of 46 projections. The space bar is 50\u00a0nm\nSince there may be specific interactions of the PBSs and the underlying membrane arrangement of PSII, we also compared these arrays with the dimensions of PSII and with those of a hemi-discoidal type PBSs of cyanobacteria. The width of cyanobacterial PBSs can be derived accurately from the central PBS component, which is the APC core structure. In the top view position, the APC core structure has a length and width of 22.5 and 15\u00a0nm, respectively (Fig.\u00a03B). The width is about the same of a PSII core dimer (Fig.\u00a03E). During previous structural studies on PSII from Thermosynechococcus elongatus we have observed small numbers of a higher type of association of PSII, which were double dimers (Fig.\u00a03F). These dimers are also present in low numbers after membrane solubilization with digitonin, without further purification steps (Fig.\u00a03G; Folea and Boekema, unpublished data).\nDiscussion\nSingle particle electron microscopy revealed the overall features of the hemispherical PBS of Porphyridium cruentum. The PBSs attached to the membranes have the best preserved features, whereas the side views of free PBSs appear fuzzy, indicating some amount of structural flexibility. Some of the flexibility could have been caused by the air drying of the negatively stained EM specimens. On the other hand, the packing of two types of 2D arrays also differs, because the total surface in one type of packing is substantially smaller than in the other one (Fig.\u00a03), suggesting intrinsic flexibility.\nThe supramolecular organization of the red algal and cyanobacterial membranes is not understood in detail, because we do not know how the PBSs are precisely associated to PSII and PSI. There is for instance no information on the subunits involved in this interaction. Some low-resolution interaction concepts are based on freeze-fracture electron microscopy, which has revealed that cyanobacterial PSII can be arranged in a row-like organization in the membrane with a repeat of 10\u201312\u00a0nm (M\u00f6rchel and Schatz 1987) or 14\u00a0nm (Nilsson et\u00a0al. 1992). The double dimers presented in Fig.\u00a03 and first discussed in Kuhl et\u00a0al. 1999, are considered to be remnants of the row-like PSII organization. Based on these data, a model for the arrangement of PBSs was proposed as described in Bald et\u00a0al. (1996). In this model the PBSs associate along the rows with their short axis as a repeat. This model makes sense for photosynthetic organisms with hemi-discoidal PBSs because there is a 1:1 match between the 15\u00a0nm width of the APC core, as presented in Fig.\u00a02, and the PSII dimmer, which has about the same width.\nThe situation in the red alga P.\u00a0cruentum is, however, obviously different because of the much larger size of its PBSs. It could have a different or no special arrangement of its PBSs in relation to row-like PSII. In particular it was shown that the tricylindrical core of the PBSs in P.\u00a0cruentum is two times wider than that of the hemidiscoidal forms in cyanobacteria (Redecker et\u00a0al. 1993), which we confirm in the current study. This is commensurate with the suggestion that the PBSs in red algae bind to a tetrameric PSII complex, on the basis of freeze-fracture EM imaging (Redecker et\u00a0al. 1993; Teckos et\u00a0al. 2004). Further corroboration is derived from the stoichiometry of PBSs and PSII complexes which is about 1:3\u20134 (Cunningham et\u00a0al. 1989). These authors show that the stoichiometry of the photosystems and PBSs in P.\u00a0cruentum is largely unaffected by light intensity (Cunningham et\u00a0al. 1989).\nHowever, we have to take into account that red algal PSI and PSII have a peripheral membrane-embedded antenna system that makes the total PSI and PSII supercomplexes substantially larger in surface area. The precise arrangement of the peripheral antanna in red algal PSII is unknown, but it was recently found that in the related unicellular cryptophyte Rhodomonas SC 24 the peripheral antenna extends the width of PSII by 50% on one of its sides (S. Kereiche, R. Kou\u0159il, G.T. Oostergetel, A. B. Doust, E.J. Boekema, J.P. Dekker, unpublished data, see Appendix I). If such an antenna arrangent would also exist on both sides of P.\u00a0cruentum PSII, the width would have been doubled to about 30\u00a0nm, and become similar to the PBS width in one of the 2-D arrays (Fig.\u00a03D). This would mean that a 1:1 ratio between rows of PSII and PBSs is also possible in red algea, despite the giant dimensions of the PBSs. This would be a meaningful ratio for light-harvesting under low-light conditions, where ordered arrays have been observed. But whether a 1:1 or a lower 1:2\u20134 ratio is true or not needs to be established by further work on the supramolecular organization of PSII within the membrane and by analyzing the size and shape of supercomplexes composed of PSII and its peripheral antenna by single particle electron microscopy.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\n(DOC 156 kb)","keyphrases":["phycobilisome","red alga","porphyridium cruentum","electron microscopy","single particle analysis"],"prmu":["P","P","P","P","P"]}
{"id":"J_Gastrointest_Surg-3-1-1852376","title":"The Role and Limitations of 18-Fluoro-2-deoxy-d-glucose Positron Emission Tomography (FDG-PET) Scan and Computerized Tomography (CT) in Restaging Patients with Hepatic Colorectal Metastases Following Neoadjuvant Chemotherapy: Comparison with Operative and Pathological Findings\n","text":"Background Recent data confirmed the importance of 18-fluoro-2-deoxy-d-glucose positron emission tomography (FDG-PET) in the selection of patients with colorectal hepatic metastases for surgery. Neoadjuvant chemotherapy before hepatic resection in selected cases may improve outcome. The influence of chemotherapy on the sensitivity of FDG-PET and CT in detecting liver metastases is not known.\nIntroduction\nThe liver is the most common, and often the only, site of distant metastases from colorectal cancer (CRC).1 Hepatic resection is the only effective therapy for a subset of patients with CRC metastatic to the liver, and is associated with 5-year survival rates ranging from 25 to 40%.2\u20135 From 60 to 65% of patients will, however, develop recurrent tumors after hepatic resection, indicating that they had harbored unrecognized intra- or extrahepatic tumor foci at the time of liver resection.6 Moreover, several studies report unresectable disease in 40\u201370% of patients that undergo laparotomy for liver resection.2,7,8 These data indicate that better patient selection is needed to avoid unnecessary operations. There are several potential ways of improving patient selection, one of which is the administration of neoadjuvant therapy followed by reevaluation and better preoperative staging.\nPositron emission tomography with the glucose analog 18-fluoro-2-deoxy-d-glucose (FDG-PET) is a sensitive diagnostic tool for the detection of colorectal metastases. Approximately 25% of patients are discovered to have new intra- or extrahepatic tumors on FDG-PET performed after standard imaging.9\u201313 Screening with FDG-PET before hepatic resection for CRC significantly improves the survival rates of resected patients, probably by improving patient selection.14\nThe role of neoadjuvant chemotherapy to down-stage nonresectable liver metastases and to improve outcome following hepatic resection of resectable liver metastases is an evolving concept, but one that is not yet established. With the recent application of new chemotherapeutic agents, such as irinotecan, oxaliplatin, and bevacizumab, improved response rates can be achieved and the use of these agents in the neoadjuvant setting would appear to be especially relevant for patients with nonresectable disease or patients with high risk of recurrence.15\u201318\nThe aim of our study was to examine the effect of neoadjuvant chemotherapy for hepatic colorectal metastases on CT and FDG-PET\/CT findings and to define the role of these imaging techniques in this setting. To do so, we compared CT and FDG-PET\/CT findings with histopathological reports.\nPatients and Methods\nPatients\nPatients with colorectal liver metastases were assigned to receive either an immediate liver resection (group 1) or neoadjuvant chemotherapy (group 2). The criteria for neoadjuvant treatment were: \nNonresectable tumors due to size, location, and number and assessment of the surgical team that complete (R0) resection was not technically possible.High risk of recurrence according to the Memorial Sloan\u2013Kettering Cancer Center (MSKCC) clinical risk score to assess risk of recurrence.19 Specifically, patients with two or more risk factors [number of metastases >1, disease-free survival <12\u00a0months, carcinoembryonic antigen (CEA) levels >200\u00a0ng\/ml, metastases from the colonic tumor to regional lymph nodes, size of the largest metastases >5\u00a0cm] were assigned to neoadjuvant treatment.Presence of extrahepatic disease.Oncologist\u2019s preference\u2014this applied to patients with MSKCC >2 that were referred from other hospitals for immediate surgery. The decision not to administer neoadjuvant therapy was not necessarily in agreement with our policy.Patient\u2019s preference\u2014patients who refused neoadjuvant therapy were assigned to immediate surgery when feasible.\nNeoadjuvant Chemotherapy\nTreatment consisted of a neoadjuvant chemotherapeutic combination of 5-fluorouracil, leucovorin, and either oxaliplatin (FOLFOX) or irinotecan (FOLFIRI). Seventeen patients (35%) were also given bevacizumab. Most of the group 2 patients were given neoadjuvant irinotecan unless they were enrolled on a multicenter study whose protocol consisted of the administration of neoadjuvant oxaliplatin.\nStaging\nBefore undergoing neoadjuvant chemotherapy, all group 2 patients underwent a triphasic contrast-enhanced CT scan, and a FDG-PET\/CT was performed in 30 (62.5%) of them. All 75 patients in group 1 and group 2 underwent FDG-PET\/CT and abdominal CT before liver surgery. The time interval between the last course of chemotherapy and the FDG-PET\/CT scan was at least 2\u00a0weeks, and surgical exploration took place within 1\u00a0month following the FDG-PET\/CT scan in most of the cases. Because we used an integrated PET\/CT technique, precise anatomical localization could be achieved and confirmed with the standard triphasic abdominal CT findings.\nPET\/CT\nThe patients were asked to fast for at least 4\u00a0h before undergoing PET\/CT. Earlier lab tests had shown that they all had glucose levels <150\u00a0mg%. The patients received an intravenous injection of 370\u2013666\u00a0MBq (10\u201318\u00a0mCi) of 18F-FDG. Data acquisitions by an integrated PET\/CT system (Discovery LS; GE Medical Systems, Milwaukee, WI, USA) were performed within 60\u2013120\u00a0min after injection. Iodinated oral contrast material was given to opacify loops of the bowel on the CT image. Data acquisition was as follows: CT scanning was performed first, from the head to the pelvic floor, with 140\u00a0kV, 80\u00a0mA, a tube rotation time of 0.5\u00a0s, a pitch of 6, and a 5-mm section thickness, which was matched to the PET section thickness. Immediately after CT scanning, a PET emission scan that covered the identical transverse field of view was obtained. Acquisition time was 5\u00a0min per table position. PET image data sets were reconstructed iteratively by applying the CT data for attenuation correction, and coregistered images were displayed on a workstation (Xeleris, Elgems, Haifa, Israel).\nStudies of all patients were retrieved and read in consensus by two experts (U.M. and E.E.-S.). All suspected sites of metastatic disease showing an increased FDG uptake were recorded. The location of hepatic lesions was recorded according to the Couinaud segmental classification.\nHepatectomy\nAll patients without extensive extrahepatic disease underwent surgical exploration and intraoperative ultrasound (IOUS). Resections of all metastatic sites were performed by either anatomic or R0 nonanatomic resection, with a tendency toward maximal parenchymal preservation with nonanatomic resections.\nComplete radiological response to neoadjuvant chemotherapy was defined as the complete resolution of all metastatic sites according to the CT and PET-CT. In these cases, careful palpation and IOUS were performed in search of remaining tumor or scarring. When there was no evidence of either, the tumor sites were resected according to the findings on the original imaging (i.e., before any response to neoadjuvant treatment).\nDetection of Hepatic Metastases\nTo define the sensitivity of CT and FDG-PET\/CT for liver metastases, imaging results were compared with the presence and size of liver lesions as demonstrated and measured by histopathological reports.\nResults\nPatients\nBetween June 2002 and September 2005, 75 patients with 155 suspected metastatic lesions from a primary CRC underwent hepatic resection in our department. Group 1 included 27 patients with 33 lesions who underwent immediate liver resection and group 2 included 48 patients with 122 lesions who first received neoadjuvant chemotherapy before subsequently undergoing liver resection. The patient\u2019s profiles are outlined in Table\u00a01. Table\u00a02 lists the operative procedures that were performed in the two groups. \nTable\u00a01Study Patients\u2019 Profiles\u00a0Group 1 (n\u2009=\u200927)Group 2 (n\u2009=\u200948)P valueSex ratio (F\/M)0.500.920.22Mean age, years (std deviation)66 (9.8)61.25 (10.9)0.06Site\u00a0Colon9 (71%)32 (66%)0.74\u00a0Rectum8 (29%)16 (33%)LN metastases (Duke\u2019s >B in colonic specimen)81.5%82%0.73No. of liver tumors (mean) (std deviation)1.19 (0.4)2.52 (1.9)0.0001Max tumor diameter (largest) (std deviation)3.53\u00a0cm (2.84)3.9\u00a0cm (1.84)0.49Extrahepatic disease (no. of patients)790.56Prior liver resection461Mean MSKCC risk score (range)1.82 (0\u20134)2.48 (2\u20135)0.003Group 1, immediate hepatic resection; Group 2, hepatic resection following neoadjuvant chemotherapyLN\u2009=\u2009lymph nodeTable\u00a02Operative ProceduresOperative procedure (no. of patients; lesions)Group 1(n\u2009=\u200927)Group 2 (n\u2009=\u200948)Right hepatic lobectomy58Left hepatic lobectomy44Central hepatectomy03Right trisegmentectomy10Nonanatomic resections1529Left lat segmentectomy22Explorative laparotomy (no resection)02Group 1, immediate hepatic resection; Group 2, hepatic resection following neoadjuvant chemotherapy\nDetection of Hepatic Metastases\nThe overall findings, the sensitivity, specificity, and accuracy of triphasic contrast-enhanced CT and FDG-PET\/CT in the detection of viable liver metastases compared to the pathological results are presented in Table\u00a03. FDG-PET and CT had a statistically significant higher sensitivity in detecting liver metastases in patients who did not receive chemotherapy compared to patients who received chemotherapy (Table\u00a03). Statistical analysis also revealed that triphasic contrast-enhanced CT had a higher sensitivity than PET\/CT in detecting colorectal metastasis following neoadjuvant treatment (65.3 vs 49%, respectively, P\u2009<\u20090.0001), but not in patients who did not receive neoadjuvant therapy (87.5 vs 93.3%, P\u2009=\u20090.625). \nTable\u00a03FDG-PET and CT\u2014Comparison With Pathological Results\u00a0Group 1 n\u2009=\u200933Group 2 n\u2009=\u2009122P valuePET\u00a0TP2948\u00a0True negative (complete response)\u201320\u00a0FP24\u00a0FN250\u00a0Sensitivity93.3%49%<0.0001\u00a0Specificity\u201383.3%CT\u00a0TP2864\u00a0True negative (complete response)\u201318\u00a0FP16\u00a0FN434\u00a0Sensitivity87.5%65.3%0.038\u00a0Specificity\u201375%Group 1, immediate hepatic resection; group 2, hepatic resection following neoadjuvant chemotherapy\nFour of the six false-positive (FP) results on FDG-PET involved patients who had previously undergone hepatic resection. These lesions were discovered on follow-up FDG-PET\/CT. Uptake was observed along the resection site, and these patients underwent nonanatomic liver resections for suspected locally recurrent lesions. Pathologic evaluation failed to reveal any tumor cells. The positive predictive value of FDG-PET\/CT for metastasis recurrence in the resection site was only 33%, and specificity was 60%.\nSensitivity of FDG-PET in the detection of colorectal metastasis correlated with the size of the metastasis (Table\u00a04). Average size of the metastases in the two groups was 33.9\u00a0mm (standard deviation 19) in group 1 and 18.9\u00a0mm (standard deviation 19) in group 2, P\u2009<\u20090.0001. \nTable\u00a04Sensitivity of FDG-PET: Correlation With Tumor SizeTumor size<1\u00a0cm1\u20133\u00a0cm>3\u00a0cmGroup 1 sensitivity (total no. of lesions)33% (n\u2009=\u20093)100% (n\u2009=\u200915)92% (n\u2009=\u200913)Group 2 sensitivity (total no. of lesions)17% (n\u2009=\u200935)78% (n\u2009=\u200941)100% (n\u2009=\u200922)Group 1, immediate hepatic resection; group 2, hepatic resection following neoadjuvant chemotherapy\nWe also compared the sensitivity of CT and FDG-PET for patients who received FOLFIRI or FOLFOX (n\u2009=\u200931) with patients who received the same regimen plus bevacizumab (n\u2009=\u200917). The results are outlined in Table\u00a05. We found that the sensitivity of FDG-PET, but not of CT, was lower in patients who received bevacizumab, although the difference did not reach statistical significance. \nTable\u00a05FDG-PET and CT in Patients who Received Chemotherapy With or Without Bevacizumab: Comparison With Pathological Results\u00a0Bevacizumab \u2212Bevacizumab +P valuePET\u00a0TP2919\u00a0True negative (complete response)173\u00a0FP22\u00a0FN2030\u00a0Sensitivity59%39%0.068CT\u00a0TP3331\u00a0True negative (complete response)135\u00a0FP60\u00a0FN1618\u00a0Sensitivity67%63%0.9\nDetection of Extrahepatic Metastases\nIn group 1, there were one FP result for extrahepatic disease (suspected recurrence in colonic anastomosis, abdominal wall), one true-positive (TP) result (recurrence in mesocolic lymph nodes), and one false negative (FN) result (in a patient with peritoneal metastases). In group 2, there was one FP result (for suspected peritoneal metastasis), three TP results (recurrence in paraaortic lymph nodes and solitary lung metastasis), and two FN results (for peritoneal metastases).\nDiscussion\nThe role of neoadjuvant chemotherapy followed by hepatectomy for colorectal liver metastases has not yet been clearly established. New chemotherapeutic agents, including irinotecan, oxaliplatin, and the biologic agent bevacizumab, have yielded improved response rates in the treatment of advanced CRC. These agents may have a potential role in the neoadjuvant setting for down-staging both nonresectable disease to resectability15,16 and resectable disease, probably mostly for patients with high risk of recurrence.17 Our policy is to administer neoadjuvant treatment to patients with nonresectable disease, those with extrahepatic disease, and those with resectable disease who have two or more risk factors according to the MSKCC clinical risk score.19 One of the theoretical benefits of neoadjuvant treatment is that patients who develop additional extrahepatic or intrahepatic metastases during this time period are spared a futile major operative procedure. Accurate staging before the beginning of neoadjuvant treatment and restaging following the treatment are crucial for optimal patient selection.\nThe standard preoperative staging of patients with colorectal liver metastases includes combined abdominal CT and chest x-ray or chest CT. It was recently demonstrated that FDG-PET as a complementary staging method improves the therapeutic management of patients with colorectal liver metastases.20 Preoperative screening with FDG-PET results in an increased survival rate of patients who undergo liver resection.14 This can be explained by the detection of occult intra- and extrahepatic metastatic disease, thus obviating futile explorations. In the current study, the sensitivity of FDG-PET\/CT following neoadjuvant therapy was only 49% compared to a sensitivity of 93.3% in patients who did not receive neoadjuvant treatment (P\u2009<\u20090.0001). The influence of the chemotherapeutic drugs on the sensitivity of FDG-PET in detecting extrahepatic metastases is not known, but we could assume that it is influenced in a similar way. This may result in a higher-than-expected rate of nonresectable disease discovered at the time of laparotomy and more extrahepatic recurrences following resection. In our series, only three of the 48 patients (6.25%) who received neoadjuvant chemotherapy were found to have nonresectable disease (one had diffuse liver metastases and two had peritoneal spread) that was not discovered preoperatively by either abdominal CT or FDG-PET\/CT. We believe that one of the reasons for the high operability rate is the fact that a significant number of patients underwent a baseline FDG-PET\/CT before the administration of neoadjuvant chemotherapy. We therefore recommend performing a baseline FDG-PET scan for all candidates for liver resection before the administration of neoadjuvant treatment. A longer follow-up is needed to assess the results of our application of this protocol.\nThe decreased sensitivity of FDG-PET\/CT in detecting liver metastases should also be a consideration when planning the extent of liver resection. We believe that the extent of resection should be guided by additional imaging modalities, including abdominal CT and IOUS, in patients who received neoadjuvant treatment. In our series, triphasic contrast-enhanced abdominal CT had a higher sensitivity than FDG-PET\/CT in detecting colorectal metastasis in patients who received neoadjuvant chemotherapy (65.3 vs 49%, P\u2009<\u20090.0001). The higher sensitivity of CT alone compared to FDG-PET\/CT in detecting small colorectal metastasis has been reported by Ruers et al.,20 and this may be even greater in patients who received chemotherapy. An attractive solution is the integrated PET\/CT scanner on which a diagnostic triphasic abdominal CT scan can be performed at the same setting as the PET scan.\nThere are several possible explanations for the decreased sensitivity of FDG-PET\/CT in the detection of colorectal metastases following neoadjuvant therapy: \nSize of the lesion. The sensitivity of FDG-PET in detecting colorectal metastasis was reported as being directly related to the size of the lesions.20 We found similar results in our series (Table\u00a04). The average size of the metastases following neoadjuvant treatment was significantly smaller than that in patients who did not receive chemotherapy (33.9\u00a0mm in group 1 and 18.9\u00a0mm in group 2, P\u2009<\u20090.0001). Two FN results in group 1 and 32 in group 2 involved tumors smaller than 1\u00a0cm. We can assume that one of the main reasons for the decreased sensitivity of FDG-PET following chemotherapy is the decrease in size of the metastases.Chemotherapy and \u201cmetabolic shutdown.\u201d It has been demonstrated that the sensitivity of FDG-PET is diminished in cancer patients who undergo the examination less than 2\u00a0weeks following the administration of chemotherapy,21 presumably due to a temporary metabolic \u201cshutdown.\u201d Although the scans in our study were done with a minimal interval of 2\u00a0weeks from the last course of chemotherapy, partial response to therapy may have caused decreased FDG uptake in metastatic lesions, making them undetectable in comparison to the physiological background uptake of FDG in the liver. This may have been a contributing factor to the FN results in our series. We found a lower sensitivity of FDG-PET (but not of CT) in detecting liver metastases following regimens including bevacizumab compared to regimens that did not include bevacizumab, although the difference did not reach statistical significance. This result may have significant clinical implications; however, it needs to be verified in larger series.Time interval between the FDG-PET and surgery. In our series, two patients with FN results (two hepatic lesions) underwent surgery more than 2\u00a0months after FDG-PET was performed. Viable tumors were discovered at the site of the original metastases which had disappeared on FDG-PET following neoadjuvant treatment. Although it is conceivable that the relatively long interval between FDG-PET and surgery may have contributed to the FN results, we believe that these tumors may have been FN due the small size of the lesions following partial pathological response to chemotherapy.Nonavid tumors. PET avidity of the tumors can be assessed only in patients who undergo a baseline FDG-PET before neoadjuvant treatment. It has been reported that FDG-PET is less sensitive for mucinous adenocarcinoma.22 In our series, ten FN results (lesions) were in patients with nonavid mucinous adenocarcinoma (sensitivity 37.5%).\nThere were two FP results in group 1 and four FP results in group 2. Four of the six FP results were in patients who had undergone a previous hepatic resection, for which follow-up FDG-PET detected uptake in the same location of the resected metastasis. These patients underwent nonanatomic resections of the \u201clesions.\u201d The pathological examination revealed only foreign body reaction without any tumor cells. In our current study, FDG uptake in the tumor bed following previous resection had a positive predictive value of 33% (2\/6). The specificity of FDG uptake in the tumor bed for recurrence was 60%. We believe that FDG uptake in the tumor bed following a previous liver resection is not specific for tumor recurrence, especially if the CEA levels are normal. Nguyen et al. demonstrated that FDG uptake may be high in various granulomatous lesions,23 possibly explaining the FP results along resection margins. Therefore, biopsy or follow-up should be considered in these cases.\nConclusions\nThe sensitivity of FDG-PET in detecting colorectal hepatic metastases decreases significantly following neoadjuvant chemotherapy. This may result in a higher-than-expected rate of nonresectable disease discovered at the time of laparotomy and in more extrahepatic recurrences following resection. We recommend staging patients with a \u201cbaseline\u201d contrast-enhanced FDG-PET\/CT both before and after the administration of neoadjuvant therapy. The extent of hepatic resection should be guided by systematic integration of data from all additional imaging modalities (abdominal CT, IOUS), as well as by the original imaging findings (before the neoadjuvant treatment). We recommend resection of all metastases that achieved complete radiological response, whenever technically possible. Longer follow-up and further studies are required to justify neoadjuvant treatment and screening with FDG-PET\/CT in patients with colorectal metastases to the liver who are at high risk of recurrence.","keyphrases":["fdg-pet","neoadjuvant chemotherapy","colorectal liver metastases"],"prmu":["P","P","P"]}
{"id":"Eur_J_Nucl_Med_Mol_Imaging-4-1-2275776","title":"Variations in 123I-metaiodobenzylguanidine (MIBG) late heart mediastinal ratios in chronic heart failure: a need for standardisation and validation\n","text":"Background There is lack of validation and standardisation of acquisition parameters for myocardial 123I-metaiodobenzylguanidine (MIBG). This lack of standardisation hampers large scale implementation of 123I-MIBG parameters in the evaluation of patients with chronic heart failure (CHF).\nIntroduction\nRadiolabelled metaiodobenzylguanidine (123I-MIBG), an analog of the false neurotransmitter guanethidine, localises in adrenergic nerve terminals primarily via the norepinephrine (NE) transporter (uptake-1) system [1, 2]. In the past two decades, a large number of investigators have demonstrated decreased myocardial 123I-MIBG uptake in heart failure patients and have shown that those with the lowest uptake tend to have the poorest prognosis [3\u201311]. There have also been findings suggesting that abnormalities of myocardial 123I-MIBG uptake may be predictive of increased risk for ventricular arrhythmia and sudden cardiac death (SCD) [12, 13].\nOne factor that has constrained acceptance of cardiac 123I-MIBG imaging as a clinical patient management tool in heart failure has been the variability of technical aspects of the procedure. Although most publications have included the heart-to-mediastinum ratio (H\/M) as the measure of myocardial uptake, the methods used to obtain this parameter show considerable variation. In addition, the influence of procedural and acquisition parameters on the reproducibility of this measurement technique have only occasionally been considered. While the effect of collimator selection on H\/M ratios has been studied, similar attention has not been paid to the impact of administered activity, acquisition time and duration [14]. Given the absence of published guidelines for cardiac 123I-MIBG imaging, standardisation of procedures among individual users of this agent becomes even more important.\nThe objective of the present study was to examine the influence of procedural variables associated with 123I-MIBG imaging when a standardised technique was used to determine the H\/M ratio.\nMaterials and methods\nStudy design\nMyocardial 123I-MIBG scintigrams on 290 heart failure patients (male and female subjects \u226518\u00a0years of age with New York Heart Association [NYHA] Classes II\u2013IV) from six sites in five European countries were retrieved and analysed. These imaging studies were performed between 1993 and 2002 and were collected for a previously described trial [15]. This trial was designed to show that standardised methodology could be applied to the analysis and processing of 123I-MIBG myocardial images to obtain reproducible quantitative results. As the large majority of patients had only late planar 123I-MIBG images, the analysis of these images is the sole focus of this paper.\nInvestigational sites sent copies of all digital image files to an Imaging Core Laboratory (ICL) for evaluation and analysis. Data on amount of 123I-MIBG activity injected and image acquisition parameters (i.e. delay after injection of 123I-MIBG and start of acquisition, acquisition duration and type of collimator used) were collected.\nThe primary efficacy objective of the original trial was to determine the prognostic significance of the late H\/M ratio in relation to occurrence of major cardiac events (MCE), defined as either: 1) cardiac death due to all causes, including myocardial infarction (MI), progressive heart failure and sudden cardiac death (SCD); 2) cardiac transplant; 3) potentially fatal arrhythmic event, including resuscitated cardiac arrest or appropriate internal cardiac defibrillator (ICD) discharge. Results of that analysis have been published. For the purposes of this paper, H\/M thresholds associated with low-, intermediate- and high-risk for MCEs were defined to provide the basis for the multivariate analysis described below.\nImage analysis\nAn experienced nuclear medicine technologist not affiliated with the ICL processed all the planar images to determine the heart\/mediastinum (H\/M) ratio. The heart region of interest (ROI) was drawn manually to include both ventricles and any atrial activity that was clearly visible. A square mediastinal ROI was drawn in the upper mediastium, using the apices of the lungs as anatomic landmarks (Fig.\u00a01). The H\/M ratio was calculated as the ratio of the counts\/pixel in the two ROIs.\nFig.\u00a01Example of processing procedure for late planar 123I-MIBG images. The positioning of the mediastinal ROI was standardized in relation to the lung apex, the lower boundary of the upper mediastinum and the midline between the lungs\nStatistical analysis\nData are presented as mean \u00b1 standard deviation, unless indicated otherwise. Kaplan\u2013Meier survival analysis and the log-rank test were used to analyse differences in event rate for different subject groups defined by the late H\/M thresholds. Differences between these groups for continuous data were compared using analysis of variance (ANOVA) with a post hoc Bonferroni. Categorical data were compared using the Fisher\u2019s exact test. A forward stepwise multivariate regression analysis was performed to determine independent predictors of late H\/M. Explanatory variables were related to patient characteristics (i.e. gender, age, NYHA class, left ventricular ejection fraction [LVEF], and underlying etiology of heart failure) and to acquisition parameters (i.e. amount of injected 123I-MIBG activity, collimator type, delay after injection and start of acquisition, and duration of acquisition). Multivariate forward stepwise regression analysis was performed for all study subjects. The overall goodness-of-fit for each model was expressed as the adjusted R2. The F test was used to assess whether a model explained a significant proportion of the variability. A p\u2009<\u20090.05 was considered to indicate a statistically significant difference. All statistical analyses were performed with SPSS (SPSS for Windows, version 11.01, SPSS Inc, Chicago, IL, USA).\nResults\nAs previously described, there were 67 major cardiac events. Figure\u00a02 shows a Kaplan\u2013Meier survival analysis for three different groups of late H\/M. Quantitative estimates for low risk (late H\/M >2.0, 2-year survival of 95%), intermediate risk (late H\/M 1.4\u20132.0, 2-year survival of 76%) and high risk (late H\/M <1.4, 2-year survival of 45%) could be determined. The differences in event rate were statistically significant between the three groups (all p\u2009<\u20090.001).\nFig.\u00a02Kaplan\u2013Meier curve showing the relation between survival and time to first major cardiac event for three groups with different H\/M values: late H\/M <1.4 vs late H\/M 1.4\u20132.0, p\u2009<\u20090.0001; late H\/M<1.4 vs late H\/M >2.0, p\u2009<\u20090.0001; late H\/M 1.4\u20132.0 vs late H\/M>2.0, p\u2009<\u20090.0001\nTable\u00a01 shows the baseline characteristics and the parameters related to the 123I-MIBG acquisition of all subjects and for the three different groups of late H\/M. Overall, the majority of subjects were male and were in functional class II according the NYHA classification and had a LVEF<50% (n\u2009=\u2009262). Between the three late H\/M groups, there were differences in age (subjects with a late H\/M >2.0 were younger), NYHA classification, LVEF (increasing from subjects with a late H\/M <1.4 to subjects with a late H\/M >2.0) and HF etiology. In addition, there were no differences in parameters of heart failure (LVEF and NYHA classification) between the different centres.\nTable\u00a01Patient and scintigraphic parameters for all subjects and subdivided by late H\/M 123I-MIBG\u00a0All (N\u2009=\u2009290)Late H\/M<1.4 (N\u2009=\u200951)1.4\u20132.0 (N\u2009=\u2009143)>2.0 (N\u2009=\u200996)Patient characteristics\u00a0Male\/Female237\/5339\/12*117\/26*81\/15*\u00a0Age (years)53\u2009\u00b1\u200911 (21\u201383)55\u2009\u00b1\u200910\u202154\u2009\u00b1\u200910\u202149\u2009\u00b1\u200911\u00a0NYHA (II\/III\/IV)165\/116\/911\/34\/6\u202085\/55\/3\u202069\/27\/0\u2020\u00a0LVEF (%)32\u2009\u00b1\u200914 (7\u201385)23\u2009\u00b1\u20099\u202031\u2009\u00b1\u200913\u202139\u2009\u00b1\u200914*\u00a0Etiology of heart failure (isch\/non-isch)121\/16920\/31\u202170\/73\u202131\/65\u2021Scintigraphic parameters\u00a0Collimator type (LEGP\/LEHR\/ME)50\/216\/24\u202024\/25\/2\u202024\/104\/15\u20202\/87\/7\u2020\u00a0Activity (MBq)164\u2009\u00b1\u200953 (72\u2013370)145\u2009\u00b1\u200931\u2020173\u2009\u00b1\u200964160\u2009\u00b1\u200941\u00a0Delay after injection (h)4.3\u2009\u00b1\u20090.6 (1.9\u20136.9)4.1\u2009\u00b1\u20090.5\u20214.3\u2009\u00b1\u20090.5\u20214.5\u2009\u00b1\u20090.6\u00a0Duration of acquisition (min)9.6\u2009\u00b1\u20093.3 (1\u201315)11.3\u2009\u00b1\u20094.3\u20209.2\u2009\u00b1\u20093.49.4\u2009\u00b1\u20092.0*Data are presented as mean \u00b1 standard deviation (range).NYHA New York Heart Association, LVEF left ventricular ejection fraction, Isch ischemic, Non-isch non-ischemic, LEGP low-energy all purpose, LEHR low-energy high resolution, ME medium energy, MBq mega-Becquerel, h hours, min minutes.Categorical data (Fisher\u2019s exact test, for each group): *p\u2009=\u20090.53; \u2020p\u2009<\u20090.001; \u2021p\u2009<\u20090.035.Continuous data (ANOVA with posthoc Bonferoni): *p\u2009<\u20090.01 compared to late H\/M <1.4; \u2020p\u2009<\u20090.01 compared to late H\/M 1.4\u20132.0; \u2021p\u2009<\u20090.01 compared to late H\/M >2.0.\nOverall, most acquisitions were performed with low-energy high-resolution (LEHR) collimators (74%), followed by low-energy general purpose (LEGP) collimators (17%). Only 9% of acquisitions were made with medium-energy (ME) collimators, all from one centre. The amount of activity injected ranged from 72 to 370\u00a0MBq (mean 164\u2009\u00b1\u200953\u00a0MBq). There was a large variation in the time-span after between injection of 123I-MIBG and imaging, ranging from 1.9 up to 6.9\u00a0h (mean delay 4.3\u2009\u00b1\u20090.6\u00a0h). The duration of acquisition could be classified in seven categories: 1, 2, 3, 4, 5, 10 and 15\u00a0min. The majority of acquisitions was performed with a 10-min duration (n\u2009=\u2009203). There were differences between the three late H\/M groups in collimator type, amount of injected 123I-MIBG, delay after injection, and duration of acquisition (Table\u00a01).\nTable\u00a02 shows the results of the stepwise multivariate regression analysis for all subjects and all parameters. LVEF, collimator type, duration of acquisition, NYHA classification and age were independent predictors of late H\/M. This model predicted approximately 30% of the variation in late H\/M results (p\u2009<\u20090.0001). Even after exclusion of the very low acquisition durations (<5\u00a0min), the duration of acquisition remained an independent predictor of late H\/M.\nTable\u00a02Multivariate analysis of clinical and acquisition related parameters as predictors of late H\/M for all subjects (N\u2009=\u2009290)VariablesCoefficient bStandard error bP value\u00a0Constant0.5490.411\u00a0LVEF0.0150.02<0.001\u00a0Collimator type0.5310.087<0.001\u00a0Duration of acquisition0.0450.0140.001\u00a0NYHA\u22120.1210.0550.028\u00a0Age\u22120.0060.0030.034Goodness-of-fit of the model: adjusted R2\u2009=\u20090.312; P value\u2009=\u2009<0.0001\nIn subjects with a late H\/M <1.4, stepwise multivariate regression analysis showed that NYHA classification and delay after injection were the only independent predictors of late H\/M (Table\u00a03). This model predicted approximately 10% of late H\/M (p\u2009=\u20090.03). In subjects with a late H\/M 1.4\u20132.0, stepwise multivariate regression analysis showed that LVEF and collimator type were the only independent predictors of late H\/M (Table\u00a04). This model predicted approximately 10% of late H\/M (p\u2009=\u20090.001). In subjects with a late H\/M >2.0, duration of acquisition was the only independent predictor of late H\/M (Table\u00a05). However, duration of acquisition predicted only 1% of late H\/M and explained a statistically non-significant proportion of variability (p\u2009=\u20090.347).\nTable\u00a03Multivariate analysis of clinical and acquisition related parameters as predictors of late H\/M for subjects with a late H\/M<1.4 (N\u2009=\u200951)VariablesCoefficient bStandard error bP value\u00a0Constant1.6800.161\u00a0NYHA\u22120.0550.0270.049\u00a0Delay after injection\u22120.0680.0300.031Goodness-of-fit of the model: adjusted R2\u2009=\u20090.104; P value\u2009=\u20090.030Table\u00a04Multivariate analysis of clinical and acquisition related parameters as predictors of late H\/M for subjects with a late H\/M 1.4\u20132.0 (N\u2009=\u2009143)VariablesCoefficient bStandard error bP value\u00a0Constant1.3700.078\u00a0LVEF0.0040.0010.001\u00a0Collimator type0.0860.0300.005Goodness-of-fit of the model: adjusted R2\u2009=\u20090.100; P value\u2009=\u20090.001Table\u00a05Multivariate analysis of clinical and acquisition related parameters as predictors of late H\/M for subjects with a late H\/M >2.0 (N\u2009=\u200996)VariablesCoefficient bStandard error bP value\u00a0Constant0.0221.376\u00a0Duration of acquisition0.1190.0570.040Goodness-of-fit of the model: adjusted R2\u2009=\u20090.013; P value\u2009=\u20090.347\nDiscussion\nIn a heterogeneous population of heart failure patients, the most important determinant of cardiac uptake of 123I-MIBG is the degree of impairment of the NE transporter function in presynaptic adrenergic neurons [3\u201311]. As such, considerable variation in late H\/M would be expected between patients with otherwise similar clinical presentations (NYHA class, LVEF, etc). Nevertheless, the findings of the present study indicate that a proportion of the variation in the late H\/M 123I-MIBG can also be explained by differences in acquisition parameters. Of particular interest, the factors contributing to this variation differed for different ranges of late H\/M.\nAbnormalities in myocardial 123I-MIBG imaging have been demonstrated in patients with both common cardiac pathologies such as CAD, CHF, and non-ischemic cardiomyopathy [3\u201312, 16\u201318] and more uncommon entities such as Syndrome X [19], Brugada Syndrome [20] and idiopathic ventricular fibrillation [13]. However, there has been limited standardisation of the acquisition and quantitative analysis techniques used by various investigators, and most studies have involved small numbers of subjects and locally defined reference standards and measures of outcome. Thus, despite the large number of studies on cardiac 123I-MIBG imaging, these methodological and analytical limitations have hampered the implementation of this technique for the evaluation and management of individual patients.\nAcquisition duration was one of the independent predictors of late H\/M. The positive correlation suggests that a longer acquisition time is associated with a higher late H\/M. However, it is more likely that this association reflects that a longer acquisition time results in a higher number of counts and the allied improvement of count statistics. In particular, in patients with reduced myocardial uptake (i.e. heart failure patients), the higher signal-to-noise ratio for a longer acquisition (\u226510\u00a0min in the present study) plays a pivotal role in the accurate assessment of late H\/M. In addition, delay after injection was in high-risk subjects (late H\/M <1.4), an independent predictor of late H\/M. The negative correlation suggests that a longer acquisition time is associated with a lower late H\/M.\nHowever, in analogy to acquisition duration, it is more likely that this association reflects that a longer delay results in a lower number of counts and the allied deterioration of count statistics. In particular, in patients with reduced myocardial uptake (i.e. heart failure patients), the higher signal-to-noise ratio for a shorter delay after injection plays a pivotal role in the accurate assessment of late H\/M.\nThe most well-validated influence on the measured late H\/M is the collimator type. Because of the high-energy photons emitted by 123I, septal penetration affects estimation of the H\/M ratio in 123I-MIBG imaging with a low-energy (LE) collimator [21]. Medium-energy (ME) collimators have been shown to improve quantitative accuracy in 123I studies [14, 21, 22]. While these results would suggest that semi-quantitative cardiac 123I-MIBG imaging might best be performed using ME collimators, there are practical limitations to such a recommendation. Almost all nuclear cardiology procedures are now performed on a multi-head gamma camera, and many dedicated dual-head cardiac cameras are not supplied with ME collimators (at least for SPECT).\nThe precise impact of collimator choice on the prognostic value has not been evaluated yet. However, it seems feasible that the improved accuracy of the ME collimators will decrease the number of patients with an intermediate risk likelihood and thereby improving the prognostic accuracy of myocardial uptake parameters of 123I-MIBG. It is therefore important to establish the degree to which the collimator used affects prognostic utility of the H\/M measurement.\nRigorous and uniform analysis of cardiac 123I-MIBG parameters minimises inter-and intra-individual variation [15, 23]. In the analysis of the entire population, numerous variables (LVEF, collimator type, duration of acquisition, NYHA classification and age) contributed to the prediction of H\/M, but only two of these variables are under the control of the imager. Furthermore, the present results suggest that the severity of cardiac sympathetic neuronal dysfunction also modulates the impact of acquisition parameters on the H\/M determinations. For example, in the low-risk group (H\/M >2.0), none of the parameters had a meaningful impact on the numerical result, indicating that preserved myocardial uptake of 123I-MIBG can be reliably determined under widely different acquisition conditions. By comparison, considering user-controlled variables, assessment of high-risk subjects (H\/M <1.4) was affected by delay after injection, while the assessment of H\/M in the intermediate risk subjects was affected by the collimator employed. Standardisation therefore is most important in assessing patients with more severe disease, although in the present study the procedural variables only contributed a relatively small fraction of the overall variability in H\/M determinations.\nAging is associated with changes in the cardiovascular autonomic regulation, both under resting and stimulated conditions. At rest, elderly subjects show increased sympathetic and decreased parasympathetic activity as well as impaired arterial baroreflex function [24\u201334]. The negative correlation, although weak, between age and late H\/M is in line with the finding that sympathetic activity increases with age typically reflected by increased circulating catecholamine levels, and that it is associated with decreased myocardial uptake of 123I-MIBG, an observation previously reported by Somsen et al. [35] and Estorch et al [36].\n123I-MIBG has been shown by individual centres to have both good reproducibility and acceptable variability, and to have prognostic value in CHF patients [3\u201311, 23, 37, 38]. These single-centre experiences, however, do not necessarily allow for extrapolation of the obtained results to other centres. In spite of this, quantitative thresholds are often implemented without inter-institutional validation. Because there are differences between centres in hardware, acquisition parameters and post-acquisition processing, the extrapolation to other centres, or generalisation of these single-centre findings, is not per se justified. Especially due to the lack of well-defined data sets, validation in each separate centre and between centres has not been possible.\nRecently, a realistic 3D-gated cardiac phantom, the Amsterdam gated cardiac phantom (AGATE phantom), was developed and validated [39]. Such a phantom could be used as a tool for the validation in each separate centre and for standardisation between centres. However, a phantom can only partially simulate patient characteristics. Therefore, the phantom results obtained will give only an impression of repeatability and variability, but they do not necessarily reflect the accuracy of measurements in patients. Therefore, in addition to the phantom measurements, a set of reference patient studies should also be included in a validation of the technique [40]. Moreover, the results should be validated in each separate centre and monitored for reproducibility and consistency.\nSeveral medications used to treat patients with heart failure may influence myocardial 123I-MIBG uptake. However, in our study the data on medication use were relatively complete, available for about 80% of the study subjects, so any further analysis could only be based on this subset. The problem with pursuing a medication analysis is that any effect on 123I-MIBG uptake is related to individual physiology and efficacy of treatment rather than on the technical attributes of the imaging, which was the main focus of this manuscript. In addition, the main manuscript already included results examining these effects, specifically that subjects that had events were less likely to be on \u03b2-blockers than those who did not have events [15]. A specific analysis of the effect of medication was not deemed appropriate to this manuscript. In addition, the use of medication that interferes with myocardial uptake of 123I-MIBG (such as tricyclic antidepressants), was not significant and did not warrant a separate analysis.\nThe primary limitation of the present study has already been noted, namely, that the largest influence on the H\/M is cardiac disease status rather than image acquisition parameters. Although collimation and acquisition duration were independent predictors of late H\/M, their individual contributions to measurement variance were <10% each. Because of the retrospective nature of this multi-centre study, it was not possible to control for the effects of numerous variables that might have had undetected impact on late H\/M. Studies using phantoms have already established the effect of collimator selection on H\/M, and similar studies could be performed to examine the influence of count rate and total counts. Additional patient studies that included multiple acquisitions of different durations and times post-injection could establish the effect of these parameters on H\/M with greater clarity.\nIn conclusion, the limited standardisation in acquisition parameters reported in literature was also reflected in the results of this multi-centre study. A wide variety of collimators, delay after injection of 123I-MIBG to the start of the acquisition and acquisition durations were reported. In general, validation and standardisation require commitment and perseverance. Moreover, without proper validation and standardisation, the role myocardial 123I-MIBG in the clinical management of CHF will continue to be constrained. Without these requirements, the use of myocardial 123I-MIBG will be limited to a single centre research tool for understanding the physiology of myocardial sympathetic neuronal activity under different conditions and after interventions. With application of appropriate standardisation, myocardial 123I-MIBG imaging could become a routine procedure for evaluation of heart failure and other cardiac disease populations.","keyphrases":["mibg","heart failure","prognosis","instrumentation"],"prmu":["P","P","P","U"]}
{"id":"Eur_J_Pediatr-3-1-1829443","title":"Insulinoma misdiagnosed as juvenile myoclonic epilepsy\n","text":"We report a girl presenting with drug-refractory myoclonia and generalized tonic-clonic seizure (GTCS) on awakening who, initially, was misdiagnosed as having juvenile myoclonus epilepsy (JME) before insulinoma was detected.\nMany conditions may mimic or cause epileptic seizures, including psychiatric and movement disorders, cardiac arrhythmias and metabolic abnormalities. Toxic and metabolic causes are potentially curable, are refractory to antiepileptic drugs, and may be fatal if untreated. Insulinoma is a rare and treatable cause of hypoglycemic seizures which might be misdiagnosed as intractable epilepsy.\nCase report\nIn May 2001 a 13-year-old girl was referred to the hospital because of GTCS on awakening. The family history was unremarkable and her personal history revealed one simple febrile seizure at age 3\u00a0years. One month before admission two episodes of confusional states were reported which lasted 5 and 30\u00a0min and were characterized by unresponsiveness and psychomotor slowing.\nPhysical and neurological examinations were normal, weight was 62\u00a0kg (97th centile), height 162\u00a0cm (90th centile), and menarche was at age 13\u00a0years (March 2001, 1\u00a0month before the first seizure). Awake EEG the day after GTCS revealed normal background activity, bilateral synchronous and asynchronous high-amplitude delta waves over the parietotemporal regions, and right frontal sharp-slow waves. Blood count, blood chemistry (glucose 4.5\u00a0mmol\/l, calcium 2.4\u00a0mmol\/l, potassium 4.0\u00a0mmol\/l), and magnetic resonance imaging (MRI) of the brain were normal. Confusional states were interpreted as complex partial seizures and GTCS as a secondary generalization. Low-dose carbamazepine (6.3\u00a0mg\/kg per day) was introduced. Two weeks later, a second GTCS on awakening occurred, followed by two short episodes of unresponsiveness before breakfast in front of the television. Another few days later, perioral and eyelid myoclonus and jerks in the arms and legs were noticed early in the morning. After sleep deprivation a series of myoclonic jerks at awakening in the morning and after several naps were registered coinciding with generalized low-amplitude spikes or polyspikes. JME was diagnosed and carbamazepine replaced by valproate (20\u00a0mg\/kg per day) which was not effective. Blood glucose was normal (4.2\u00a0mmol\/l 4\u00a0h after food intake). Two months later (August 2001) a second overnight long-term video-EEG revealed generalized discharges of spikes or polyspikes and slow waves in the second part of the night. Again myoclonic jerks in the hands and legs with a single generalized low-amplitude spike and slow waves were registered on awakening (Fig.\u00a01).\nFig.\u00a01Ictal EEG on awakening: generalized low-amplitude spike with simultaneous registration of myoclonic jerks of the hands and legs. Although the EEG pattern is not so typical for JME, together with the history and the clinical symptoms this EEG seemed compatible with this diagnosis\nIn September 2001, 6\u00a0months after the manifestation of the first seizures, low preprandial blood glucose (1.8\u00a0mmol\/l, normal 3.8\u20136.1) and high insulin (31\u00a0\u03bcU\/ml, normal 1.9\u20132.3) were detected because the patient was sluggish and dizzy (glucose and insulin were measured by routine methods). Inappropriate insulin secretion was suspected and confirmed by pre- and postprandial glucose and insulin measurements (preprandial: glucose 2.8\u00a0mmol\/l, insulin 33\u00a0\u03bcU\/ml and 50\u00a0min after food intake: glucose 3.1\u00a0mmol\/l, insulin 153\u00a0\u03bcU\/ml); therefore, a 72-h fasting test was not done. Abdominal ultrasound (US) and MRI revealed a round and circumscribed hypoechoic lesion (2.1\u2009\u00d7\u20091.8\u2009\u00d7\u20091.9\u00a0cm) with sharp but uneven borders in the pancreatic body without evidence of metastases. Insulinoma was suspected. Because multiple endocrine neoplasia type I syndrome (MEN I) is associated in 5\u201310% of the cases with insulinoma [5, 9], prolactin, thyroid-stimulating hormone, parathyroid hormone, and thyroxin (T3, T4) were determined, which were all normal; therefore, mutation analysis of exons 2\u201310 of the MENIN gene was not done. Valproate was stopped and the patient was advised to have food intake every 2\u00a0h and three times during the night. Since then she has been seizure free, but myoclonic jerks recurred occasionally when meals were delayed by >5\u00a0h during the night. Her weight had increased from 62 to 84\u00a0kg within the last 10\u00a0months before surgery.\nSurgery was performed 5\u00a0months later (March 2002) in Moscow and two insulinomas were revealed by intraoperative US: one located on the superior-posterior surface between the head and body (diameter 2.5\u00a0cm) and the other on the anterior surface of the pancreas (diameter 0.7\u20130.9\u00a0cm). Before the surgery, US and MRI disclosed only one lesion. Both insulinomas were removed in toto and the endocrine pancreas was suppressed by Sandostatin (octreotide) for 4\u00a0days. Histology confirmed the diagnosis of two benign beta-cell adenomas. The patient fully recovered, remained seizure free during the next 4\u00a0years, has a current weight of 62\u00a0kg (75th centile), and is mentally normal.\nDiscussion\nMyoclonus and GTCS after awakening are hallmarks of JME, especially when adolescents are affected [2]. Hence, in our patient the interpretation of the clinical symptoms together with spike waves was indicative of JME. Confusional states and drug-refractory seizures on the other side are less compatible with the diagnosis of JME, but fit well with hypoglycemic events. The latter, however, were erroneously thought to be excluded by two normal blood glucose measurements. Furthermore, hypoglycemia in nondiabetic adolescents is much rarer than JME. Insulinoma, responsible for the profound hypoglycemia in our patient, has its onset usually in middle [10] or older age [4] and is very rare in children [9]. Symptoms are prominent following prolonged fasting states, especially in the morning as in our patient, but also in the late afternoon [4]. Hypoglycemia can cause loss of consciousness, sluggishness, confusion, asthenia, deep coma, dizziness, disturbances in vision, and epilepsy. An international review of 1,067 cases [10] showed such neuropsychiatric symptoms in 92% of the patients. Irreversible damage of the central nervous system occurred in 6.8% of the cases [10]. Forty-one percent of the patients have amnesia during the hypoglycemic event [4]. Therefore, the history has to be obtained from family members or friends. Clinical details about the onset, duration, and type of symptoms as well as temporal association with fasting and the relief of symptoms after food intake are helpful information for making the diagnosis. Considerable weight gain as in our patient was reported in 39% of the patients [4]. Taking into account that an actually normal glucose level does not exclude episodes of hypoglycemia, a prolonged fasting test up to 72\u00a0h under close monitoring is recommended to assess inappropriate insulin secretion [3].\nThe median interval from the onset of symptoms to the diagnoses of insulinoma is 2\u00a0years, with a wide range of 1\u00a0month to 30\u00a0years [4]. Initial misdiagnosis is frequent. In a series of 59 patients with insulinoma, prior diagnosis included neurological disorders in 39 (64%) and especially epilepsy in 23 (39%) patients. Seven patients (12%) were treated with antiepileptic drugs [4]. The seizure symptoms are variable. During severe hypoglycemia generalized and focal seizures occurred in children [6]. In patients with insulinoma, shaking of all four limbs at night [8], jerking and twitching [1], and symptoms suspicious for partial and complex partial seizures with and without secondarily generalized seizures are reported [3, 5]. Varying degrees of EEG changes and sometimes massive spiking were recorded during hypoglycemia [7]. In patients with insulinoma, focal and widespread EEG slowing [1, 3] combined with epileptic activity [5] are mentioned.\nConclusions\nThe combination of seizures and episodic confusional states should prompt the search for hyperinsulinism, especially when the episodes occur in fasting states and when they are refractory to antiepileptic treatment. Inappropriately high insulin with low blood glucose is diagnostic; this constellation has to be investigated resolutely (e.g., by the classic 72-h fasting test) but with caution under strict clinical and chemical monitoring. Timely diagnosis of an insulinoma is of paramount importance to prevent sequelae for such patients.","keyphrases":["insulinoma","juvenile myoclonic epilepsy","myoclonus","confusional state","hypoglycemia"],"prmu":["P","P","P","P","P"]}
{"id":"Clin_Auton_Res-2-2-1705534","title":"The effect of stimulation technique on sympathetic skin responses in healthy subjects\n","text":"The aim of this study was to collect normative data for sympathetic skin responses (SSR) elicited by electrical stimulus of the ipsilateral and contralateral peripheral nerves, and by magnetic stimulus of cervical cord. SSRs were measured at the mid-palm of both hands following electrical stimulation of the left median nerve at the wrist and magnetic stimulation at the neck in 40 healthy adult volunteers (mean age 52.2 \u00b1 12.2 years, 19 males). The onset latency, peak latency, amplitude and area were estimated in \u201cP\u201d type responses (i.e., waveforms with a larger positive, compared to negative, component). SSR onset and peak latency were prolonged when the electrical stimulus was applied at the contralateral side (i.e., the SSR recorded in the right palm P < 0.001). The onset latency was similar on both sides during cervical magnetic stimulation. However, peak latency was faster on the left side (P < 0.03). Comparison of electrical and magnetic stimulation revealed that both the onset and peak latency were shorter with magnetic stimulation (P < 0.001). The latency of a SSR varies depending on what type of stimulation is used and where the stimulus is applied. Electrically generated SSRs have a longer delay and the delay is prolonged at the contralateral side. These factors should be taken into account when interpreting SSR data.\nIntroduction\nSympathetic skin response (SSR) testing is commonly used to assess sympathetic function [18]. Abnormal SSR occur in both central [7, 8] and peripheral autonomic dysfunction [1, 16, 21].\nThe SSR is a complex polysynaptic reflex, which courses through a common centrifugal path via sympathetic post-ganglionic unmyelinated fibers [2, 24]. However, its exact pathway through the central nervous system is not fully understood. A variety of stimuli can generate a SSR, which illustrates the complexity of the afferent inputs to the reflex.\nAlthough measuring SSR is relatively simple, interpreting these measurements is not so straightforward. Different afferent stimuli may well lead to a wide range of normal SSR values. It is difficult to attribute abnormalities of SSR to sympathetic dysfunction alone, particularly in cases of polyneuropathy, due to the potential influences by somatic fibers sensory inputs [24].\nThe aim of this study was to collect normative SSR data. SSRs were measured in both mid-palms following electrical stimulation of the left medial nerve and magnetic stimulation of the neck in healthy controls. SSR onset and peak latency, amplitude and area were analyzed. Electrically generated SSR in the ipsilateral (left) and contralateral (right) palms were compared to the SSR evoked by magnetic stimulation.\nMethods\nSubjects\nForty healthy adult volunteers [9 males\/21 females, mean age 52.21\u00a0\u00b1\u00a012.23 (range 28\u201375) years, height 172\u00a0\u00b1\u00a011.3\u00a0cm] were studied. All subjects were healthy, with no history of cardiovascular or neurological disease. All subjects denied previous alcohol or drug abuse. None of the subjects had contraindications for magnetic stimulation, such as metal prosthesis or pacemaker. All participants gave written informed consent prior to the start of the study.\nProcedure\nThe study was performed using a 2-channel Counterpoint, Medtronic-Dantec (Medtronic-Dantec Electronics, Skovlunde, Denmark) electromyographic unit. SSR recordings were based on the technique described by Shahani et alal. [20]. Briefly, subjects lay supine in an air-conditioned, quiet room with temperature maintained at 24\u00b0C and skin temperature at 33\u00a0\u00b1\u00a00.5\u00b0C. The same investigator performed all experiments, and care was taken to avoid all external stimulation. Active standard disk ECG Ag\/AG\/Cl electrodes were attached to the mid-palm of both hands and referenced to the corresponding dorsal surfaces. Skin temperature was verified as being above 32\u00b0C by measuring close to the recording electrodes. The recording sites were the same in all experiments.\nElectrical stimulation\nSingle square-wave electrical pulses of 0.1\u00a0milliseconds duration were applied to the left median nerve at the wrist at irregular intervals (30\u201360\u00a0seconds) to produce a SSR. The sweep speed was 1,000\u00a0milliseconds\/D, the amplifiers sensitivity was 100\u00a0\u03bcV\/div and the amplifier filters were set at 0.5\u00a0Hz and 2\u00a0kHz for the low and high frequency filter, respectively. Five trials were recorded simultaneously from both palms and on each side the response with the largest amplitude and shortest latency were taken. A fixed stimulus intensity of 60\u00a0mA was used throughout.\nMagnetic stimulation\nA Magstim 200 stimulator, equipped with a circular coil of 9\u00a0cm inner diameter and a 2.0-T maximum magnetic field strength, was used to apply magnetic stimulation at the cervical area. The center of the circular coil was placed in the midline over the 7th cervical spinous process. The current was set to anti-clockwise flow in the coil (side A visible) and the stimulus intensity fixed at 60% of maximum output. Five stimuli were delivered randomly every 20\u201330\u00a0seconds.\nAnalysis of waveforms\nTo avoid habituation 5\u00a0minutes rest was given between electrical and magnetic stimulation. To maintain uniformity of the results only \u201cP\u201d type responses (waveforms with a larger positive, compared to negative, component) were analyzed.\nFigure\u00a01 shows how the components of the waveform were analyzed. The onset latency was taken from the time of the stimulus artefact to the start of the positive deflection. The peak latency was taken from the time of the stimulus artefact to the peak of the positive deflection. The peak-to-peak amplitude and the area under the SSR curve were also measured.\nFigure\u00a01A typical sympathetic skin response recording. Typical SSR recordings taken from the right palm (upper trace) and left palm (lower trace) (a) following electrical stimulus of left median nerve and (b) magnetic stimulation of cervical cord\nStatistical analysis\nDifferences between stimulus procedures in the same subjects were determined by using paired t-tests. The tests were two tailed. Statistical significance was set at P\u00a0<\u00a00.05. All analyses were performed using SPSS for Windows (10.0; SPSS Inc., Chicago, IL).\nResults\nClear SSR recordings with a main positive component (i.e., P waveforms) were obtained in all participants.\nLatency\nMean SSR onset and peak latency on the right side, which was contralateral to the electrical stimulus, was significantly prolonged compared to the left side, which was ipsilateral to the electrical stimuli (P\u00a0<\u00a00.001, Tables\u00a01 and 2). No differences in the onset latency were found between right and left sides following cervical magnetic stimulation. However, a weak interside difference in peak latency (P\u00a0<\u00a00.03) was observed (faster on the left side). Comparison between electrical and magnetic stimulus revealed that both onset and peak latency on either side were significantly shorter for magnetic stimulation (P\u00a0<\u00a00.001, Tables\u00a01 and 2).\nTable\u00a01Normative sympathetic skin response data in healthy subjectsElectrical ipsilateral (L)Electrical contralateral (R)Magnetic (L)Magnetic (R)Latency-onset (seconds)1.79\u00a0\u00b1\u00a00.221.90\u00a0\u00b1\u00a00.24a1.63\u00a0\u00b1\u00a00.25c1.66\u00a0\u00b1\u00a00.28dLatency-peak (seconds)2.54\u00a0\u00b1\u00a00.332.68\u00a0\u00b1\u00a00.38a2.40\u00a0\u00b1\u00a00.27c2.43\u00a0\u00b1\u00a00.28b,dAmplitude (mV)2.67\u00a0\u00b1\u00a01.492.35\u00a0\u00b1\u00a01.32a2.74\u00a0\u00b1\u00a01.612.70\u00a0\u00b1\u00a01.69Area (mV\u00a0seconds)2.85\u00a0\u00b1\u00a01.532.63\u00a0\u00b1\u00a01.413.24\u00a0\u00b1\u00a01.993.22\u00a0\u00b1\u00a01.96dAll data are mean\u00a0\u00b1\u00a0SD. Electrical stimulation was applied at the left medial nerve of the wrist. SSR were recorded in both the left (ipsilateral) and right (contralateral) palms. Statistically significant difference (P\u00a0<\u00a00.05)a Electrical stimulus\u2014between sidesb Magnetic stimulus\u2014between sidesc Left side\u2014between electrical and magnetic stimulusd Right side\u2014between electrical and magnetic stimulusTable\u00a02The difference in sympathetic skin response onset latencies evoked by electrical and magnetic stimulationLatency difference (milliseconds)Electrical stimulation. R\u2013L105\u00a0\u00b1\u00a085 (0\u2013320)*Magnetic stimulation. R\u2013L27\u00a0\u00b1\u00a086 (\u2212110\u2013330)Left electrical\u2013magnetic158\u00a0\u00b1\u00a0183 (\u221250\u2013640)*Right electrical\u2013magnetic237\u00a0\u00b1\u00a0201 (0\u2013780)*Differences in SSR onset latency measurements during 3 types of stimulation (left and right electrical stimulation and magnetic stimulation). All data are mean\u00a0\u00b1\u00a0SD (and range). L, left; R, right. * Significance of P\u00a0<\u00a00.05 (comparison by one sample t-test)\nAmplitude and area\nDuring electrical stimulation amplitude was higher of the stimulated (left) side compared to the non-stimulated side. Amplitude was similar during magnetic and contralateral electrical stimulation. The area of the SSR measured at the right side was greater for magnetic stimulation compared to electrical stimulation.\nDiscussion\nThe main finding of this study was that the latency of a SSR depends on whether an electrical or magnetic evoking stimulus is used. We also found a mean latency difference between contralateral and ipsilateral evoked electrical responses of 105\u00a0milliseconds.\nSSR are generated through a complex somato-sympathetic reflex with spinal, bulbar and suprabulbar components [13]. Until now, they have not been precisely defined in humans [26]. SSR can be evoked by various types of stimuli including auditory, deep breathing, painful sensation of the limbs or face etc., and are therefore mediated by different afferent pathways [2]. Moreover, SSR are evoked by impulses that originate in a different area to which the response is produced. For example, electrical pulses over the sternal skin can be used to elicit SSR in the palms [19], which highlight the complexity of the internal neuronal network involving polysynaptic relays and connections between different vertical levels along the neuraxis and in both sides of the human body [11]. It is, therefore, not surprising that measurement of a SSR depends on the site and the type of stimulus used to initiate the reflex.\nThe longer latency in the contralateral side of 105\u00a0milliseconds may be explained by the crossing over of the signal from one side of the body to the other. The cross over time of around 100\u00a0milliseconds suggests that the reflex circuits are supraspinal [13]. Data from animal models indicate that the crossing occurs at multiple levels, including the brainstem and spinal cord [26]. However, patients with cervical or high thoracic lesions resulting in complete tetraplegia or paraplegia have no SSR from the hands and feet [6], suggesting that supraspinal centers are essential in the generation of SSR.\nA constant latency to alternatively electrical stimulation of median nerves was suggested in a previous study [3]. However, latency was measured with 20 consecutive electrical stimulations, and thus the effects of adaptation (or habituation) cannot be excluded.\nMagnetic stimulation may be most reproducible method to evoke SSR [14]. In theory, strong stimulation generated by the magnetic field close to the spine avoids any possible interference by affected somatic sensory fibers, providing a measure of efferent sympathetic function alone. In the case of the lower motor neurons, it has been showed that cervical magnetic stimulation excites the motor roots at or very near the exit foramen or even further distally [5]. In respect of SSRs, magnetic stimulation could, in analogy to motor neurons, excite the sensory roots at their entry [14] or directly activate the sympathetic trunks within the spinal canal [25].\nIn our study, the latency of the SSR evoked by magnetic stimulation was shorter than the corresponding values of the electrically elicited responses. The shorter latency of the magnetically generated SSR could be caused by impulses bypassing the peripheral afferent part of the reflex. Given that the difference between electrical stimulation at the wrist and magnetic stimulation at the neck was 158\u00a0milliseconds and assuming that the average distance from wrist to C7 is 70\u00a0cm, the conduction velocity of afferent somatic fibers is calculated to be approximately 5\u00a0m\/second.\nPrevious studies suggested that the efferent path is served by sympathetic B and C fibers conducting at approximately 2\u00a0m\/second [12, 17], whereas the afferent path is served by somatic myelinated sensory groups II and III conducting at rates of 50\u20135\u00a0m\/second [12]. In good agreement with our results, other investigators have reported a latency difference between electrically and magnetically elicited SSR of 80\u00a0milliseconds [14] and 130\u00a0milliseconds [4], with the former being always longer.\nBased on our results, it can be assumed that the SSR onset latency to spinal cord magnetic stimulation was the shortest, whereas that to contralateral peripheral nerve electrical stimulation was the longest. Furthermore, the difference in latency between SSR to magnetic and ipsilateral electrical stimuli was close to that between SSR to ipsilateral and contralateral electrical stimuli. A satisfactory explanation for the observed differences would not be offered without the risk of being highly speculative.\nThe morphology is another element of SSRs instability. Two main types of SSR waveform were previously recognized; P type which has a larger positive component and N type with a larger negative component [15, 22, 23]. It has been shown that strength of stimulation was positively correlated with P type amplitude and inconsistently with N type and that strong stimuli probably produced P type responses [22]. To ensure similarity of measurements and facilitate comparison of responses to different kinds of stimuli, we arbitrarily choose to study only subjects with predominant P type responses. In none of our participants the change of stimulation from electrical (ipsilateral and contralateral) to magnetic resulted in switching of P to N type of SSR. This can be the consequence of using relatively high intensity in electrical and magnetic stimulus.\nThe latency values in our subjects were slightly higher than those in previous reports. In those reports, the mean SSR latency from the palm to electrical stimulation of the median nerve ranged between 1.27\u00a0seconds and 1.51\u00a0seconds [4, 10, 14, 24]. This discrepancy should be attributed to a methodological difference. The negative onset of the response is generated by the electrical activation of the sweat gland. The peak of the negative phase is probably variable and generated by the relationship between sweat production and the surround tissue [24]. Under the particular recording conditions applied, the majority of the examined individuals had potentials with an initial positive peak. Therefore, instead of measuring the onset latency to the first negative peak, which is most often used, we measured the onset latency from stimulus artifact to the onset of the main positive wave irrespective of the existence or not of a proceeding small negative component. As regards to the peak latency values, they showed similar to onset latency differences in this study. Thus, peak latency is not suggested as an additional parameter, which could be useful in clinical practice.\nAmplitude is the SSR parameter that has been reported to be highly variable. It can be influenced by several factors such as temperature, time of the day, emotion and for this reason several authors have not used it as measure of disease [2, 9]. The relative stability of amplitude and area measurements among the SSRs evoked by different stimuli in our subjects is noticeable. These findings could be attributed to the stable conditions of recordings (single session for all SSR recordings in each subject and long interstimulus interval to avoid habituation).\nThere are certain potential limitations in the study design. First, the fact that SSRs were not assessed for reproducibility in individual subjects. However, the phenomenon of habituation [3] did not allow us to compare measurements (particularly for the amplitude) of consecutive responses in a single individual.\nSecond, since it is known that a variety of stimuli are capable of producing SSR, startle could intermix with the electrical stimulus and, mostly, auditory could interfere with the magnetic stimulus, leading to shorter SSR latencies. A study comparing parameters of SSR elicited by pure auditory as opposed to other types of stimulation should clarify this point. However, in a previous study, where ear-plugs were used to exclude auditory stimuli, the SSR latency measurements to neck magnetic stimulation were even shorter than in our subjects [14], suggesting that the intermixed auditory stimulation is not a major limitation. Finally, habituation, in a broad sense including the central processing and familiarity, cannot be completely ruled out by a 5-minute interval between techniques, although intervals of 40\u201360\u00a0seconds between trials were reported elsewhere [10, 14, 22]. Application of electrical followed by magnetic stimulation in some subjects and the inverse procedure in others might have been used, although this would have increased the complexity of the study design.\nIn brief, SSRs\u2019 have been introduced as an easy to perform and painless mean for the evaluation of post-ganglionic sympathetic function [20]. In the filed of autonomic nervous system where the diagnostic options are limited, and despite the expression of certain doubts, mainly related to the large diversion of individual values, SSR remains at present the most widely applied neurophysiological test for the sympathetic nervous system. According to the results of the present study, latency which is the most widely quoted parameter, varied depending on the stimulation type, being shorter at ipsilateral than contralateral electrical stimulus and even less short when magnetic stimulation at the neck was applied. Further studies on the challenging issue of SSR waveform and its possible clinical relevance are necessary to explore it in detail.","keyphrases":["sympathetic skin response","methods","autonomic nervous system","galvanic skin response","sudomotor function"],"prmu":["P","P","P","M","M"]}
{"id":"Eur_J_Clin_Pharmacol-4-1-2235908","title":"Teaching clinical pharmacology and therapeutics with an emphasis on the therapeutic reasoning of undergraduate medical students\n","text":"Background The rational prescribing of drugs is an essential skill of medical doctors. Clinical pharmacologists play an important role in the development of these skills by teaching clinical pharmacology and therapeutics (CP&T) to undergraduate medical students. Although the approaches to teaching CP&T have undergone many changes over the last decennia, it is essential that the actual teaching of CP&T continues to be a major part of the undergraduate medical curriculum.\nIntroduction\nThe field of modern clinical pharmacology began to develop in the second half of the 19th century when physicians began to realise that agents such as heavy metals and plant extracts, then in use, more often made diseases worse instead of better [1]. In 1969, a study group on clinical pharmacology was convened by the World Health Organisation (WHO) in order to demarcate the scope of this relatively new discipline [2]. They concluded that the tasks of the clinical pharmacologist include: (1) research into the action of drugs in humans, (2) services such as providing information on drugs and (3) teaching clinical pharmacology and therapeutics (CP&T) to medical students, hospital staff and physicians [2]. While both research and services are important areas of clinical pharmacology, it is in their roles as teachers of CP&T to medical students that clinical pharmacologists have an extremely important effect on the development of rational prescribing by medical doctors. However, despite this importance, the undergraduate teaching of CP&T has not, in contrast to the two other functions of the clinical pharmacologist, achieved an international level of recognition [3, 4]. The lesser attention given to teaching CP&T is also reflected in the small number of scientific publications. For example, only about 20 articles on teaching CP&T were published in the European Journal of Clinical Pharmacology between 1980 and 2000.\nIn many medical schools, undergraduate students learn little about the therapeutic use of drugs and even after graduation, some doctors read little on the subject and rely too much on the promotional efforts and information from the pharmaceutical industry. As indicated by a survey conducted under the auspices of the WHO in 1989, in European medical schools, an average of only 28\u00a0h was devoted to teaching clinical pharmacology, even though over 100\u00a0h were devoted to pharmacology [5, 6]. However, the number of hours spent teaching CP&T is probably underestimated since clinical pharmacology and particularly therapeutics are taught implicitly during the clinical clerkships.\nThe European Association of Clinical Pharmacology and Therapeutics (EACPT) was founded in 1993 to promote the field of CP&T. One of the important objectives of the EACPT was to develop CP&T in Europe by improving and harmonising the teaching of the rational use of drugs at the undergraduate and postgraduate levels. An Education Subcommittee of the EACPT was subsequently established in 1997 to increase the amount of attention given to as well as the quality of teaching CP&T. The first task of this subcommittee was to identify the educational requirements of medical students in terms of CP&T. A positive consequence of the expanded attention for the teaching of CP&T has been the substantial increase of scientific publications on teaching CP&T during the past decade: there have been about 32 articles published between 2000 and 2007 in the European Journal of Clinical Pharmacology, which is twice as many as during the preceding two decades.\nUnfortunately, despite the increasing amount of attention being given to the teaching of CP&T, CP&T educational programmes are still not optimal in many medical schools [7]. Nowadays, many graduates still feel insufficiently prepared to assume prescription responsibilities after graduation [7]. Therefore, it is important to improve the undergraduate teaching in CP&T.\nIn this review, we shall endeavour to indicate the essential aspects of teaching CP&T to undergraduate medical students. To this end, we first describe what is known about the general learning objectives in CP&T that medical students should master before graduation. Subsequently, we focus on an essential (in our opinion) core learning objective: \u2018therapeutic decision-making\u2019. We go on to describe how medical students and physicians arrive at a therapeutic decision and discuss the possibilities of teaching medical students rational pharmacotherapy. Finally, we recommend approaches for organising, presenting and assessing CP&T in the undergraduate medical curriculum.\nFrom core learning objectives to competencies\nIn many curricula, the teaching in clinical disciplines is centred on symptoms and diagnosis, and little or no time is given to the principles of drug treatment. In the past, CP&T was only taught as short courses in medical schools in which information was presented in the form of dogmatic indications of which drug to use and when. In some medical schools, however, clinical pharmacologists participated in basic pharmacology courses, usually given early in the clinical part of the curriculum [4]. Notwithstanding, much of the postgraduate and continuing education of the practicing physician has been left to the persuasive methods of the pharmaceutical industry [1].\nIt was only in 1989 that the Council for Medical Student Education in Clinical Pharmacology and Therapeutics stressed the importance of defining a core curriculum for clinical pharmacology in the medical curriculum. One year later, Nierenberg developed a core curriculum for medical students in CP&T [9]. Based on consensus between 40 clinical pharmacology teachers, Nierenberg formulated a list of core knowledge, skills and attitudes that every medical student should master before graduation in order to be able to prescribe effectively and safely [8, 9].\nCore knowledge in clinical pharmacology deals with the facts that are necessary to make rational and optimal therapeutic plans; it includes the principles needed to organise these facts into useful information and to recognise when essential facts are missing [5, 8]. Core skills are in many ways similar to core knowledge because knowledge must be learned and used as the medical student develops skill in practicing rational therapeutics. Core attitudes include attitudes about what constitutes valid information, what kind of information is likely to be in error and what new information must be continuously acquired from reliable sources.\nBased on the learning objectives as formulated by Nierenberg, a questionnaire on core learning objectives was subsequently developed in the United Kingdom by Walley [10]. Senior academic clinical pharmacologists who were active in teaching CP&T in the UK or Ireland assessed the relative importance of each learning objective as an element of a core curriculum. This resulted in a list of core knowledge, skills and attitudes [11] that largely matched the learning objectives as formulated by Nierenberg [8].\nIn addition to the approach of Nierenberg and Walley to defining the core curriculum in CP&T, Orme described two other approaches to developing a core curriculum for CP&T [12]. First, based on the WHO concept of an \u2018essential drug list\u2018, he developed two lists of drugs; one list consisted of approximately 120 essential drugs that students would be expected to know in detail, and a shorter list consisted of drugs that students would be expected to be familiar with but were not required to know in any detail. This list of essential drugs was then examined by the Education Subcommittee of the EACPT. The second approach to defining a core curriculum for CP&T was the disease-based approach. Three types of disease process were defined: (1) 67 diseases that were common and that the student must know how to manage; (2) 158 diseases that were less common but that students must be able to diagnose, after which the appropriate therapy could be found in the literature; (3) 36 diseases that were rare but that students should be aware of.\nDefining a core curriculum for CP&T as presented by Nierenberg, Walley and Orme is important to defining the shape and boundary of the discipline and may be helpful as part of a medical student's examination. However, in addition to knowledge, physicians must also have prescribing skills in order to prescribe rationally.\nTherefore, the WHO Action Programme on Essential Drugs has developed, in collaboration with the University of Groningen, a manual for undergraduate medical students on the principles of rational prescribing, the so-called Guide to Good Prescribing (GGP). This manual provides a normative model for therapeutic reasoning and prescribing and provides a six-step guide to the process of rational prescribing that will be necessary throughout the clinical career of the medical student (Table\u00a01). All six steps are based on the core learning objectives, knowledge, skills and attitudes as formulated by Nierenberg and Walley and on positive experience in the Netherlands [13, 14].\nTable\u00a01World Health Organisation six-step model of rational prescribing [18]StepsDescriptionStep 1Define the patient\u2019s problemStep 2Specify the therapeutic objectiveStep 3aChoose your standard treatment (P-drug)Step 3bVerify the suitability of your treatment (P-drug)Step 4Start the treatmentStep 5Give information, instructions and warningsStep 6Monitor (and stop?) treatment\nIn addition, the content is based on an observational study among general practitioners and clinicians in the Netherlands and on 10\u00a0years of experience with pharmacotherapy courses for medical students in Dutch medical faculties and abroad. The GGP has also been reviewed and examined by a large body of international experts in the teaching of pharmacotherapy. As reported by De Vries et al., a short interactive training course in pharmacotherapy, using the GGP, was evaluated in a controlled study among 219 undergraduate international medical students [15]. This study indicated that undergraduate students who used the GGP performed significantly better than students who did not used the GGP. In addition, students not only remembered how to solve old problems, but they could also apply their skills to new problems, a so-called transfer effect.\nA new framework for the innovation of medical curricula has been recently introduced, called the CanMEDS. The CanMEDS framework describes aspects of competence related to seven roles of a clinical specialist: the role of Medical Expert, Communicator, Health Advocate, Collaborator, Manager, Scholar and Professional [16]. Despite the fact that many doctors agree with the importance of these aspects of competence and that many countries have already adopted the CanMEDS roles in their curricula, there is limited information on how these roles can be applied in an international context and in different specialities, such as CP&T. Therefore, before clinical pharmacology as a discipline can adopt the CanMEDS competencies, the core learning objectives as determined in the last decades will have to be translated into the CanMEDS competencies.\nIn conclusion, in order to improve the competency of future doctors to prescribe effectively and safely, several clinical pharmacologists have attempted to determine what every medical student should master before graduation. A common feature of all of the competencies formulated is that all students must acquire a knowledge of the clinical pharmacology of essential drugs and diseases and must master prescribing skills in order to become competent in the rational prescription of drugs.\nTherapeutic decision-making\nAlthough, as described previously, there are many essential therapeutic skills, the final choice of a (drug) treatment for a patient can be looked upon as a core skill in therapeutics. In order to find effective ways to teach our future doctors, it is important to explore how expert doctors arrive at this final therapeutic decision.\nTherapeutic decision-making or therapeutic reasoning is, together with diagnostic reasoning, an important part of the process of clinical reasoning. Therapeutic reasoning can be defined as the step in clinical reasoning that pertains to the choice of therapy [17]. However, in contrast to the process of diagnostic reasoning, which has been investigated extensively, little is known about the process of therapeutic reasoning. To our knowledge, the first attempt to investigate therapeutic reasoning in real practice was a small observational study carried out in 1984 in the Netherlands. The results of this unpublished study constituted the basis for the development of the WHO six-step model [18]. Over 500 patient consultations by 25 general practitioners and 25 clinical specialists were observed and recorded. In addition, all doctors were interviewed about their therapeutic reasoning. Based on this study, it became evident that doctors generally based their choice of (drug) treatment on two steps: (1) doctors initially used treatment guidelines or drug formularies as a starting point; (2) this was followed by a verification of the suitability of this treatment for the individual patient and a modification of the choice of treatment if necessary (for example, in case of co-morbidity or co-medication). Moreover, it was also observed that the more experienced physicians knew more standard treatments by heart compared to less experienced physicians.\nThe first study that described the process of how therapeutic decisions are made in practice in greater detail was performed by Denig [19]. By interviewing 169 different general practitioners and 72 hospital physicians, she studied how doctors arrive at a choice of drug and what factors influence this drug choice [19, 20]. This study indicated that when a doctor is confronted with a diagnosis \u2013 for example, a patient with both essential hypertension and a renal disorder \u2013 he or she immediately thinks of a number of pharmacotherapeutic possibilities, referred to as the \u2018evoked set\u2019. Depending on the diagnosis, the \u2018evoked set\u2019 consists of 1.7\u20135 different pharmacotherapeutic options that are influenced by many factors, such as refresher courses, the literature, experience and advertisements from the pharmaceutical industry. The ultimate choice out of the \u2018evoked set\u2019 for an individual patient may be either \u2018unreasoned\u2019 (routine) or \u2018reasoned\u2019 (evaluation of the different options). However, how the final choice of treatment is made is still unknown.\nIn contrast to the scarcity of information available on therapeutic decision-making, much research has been done on diagnostic reasoning. When an experienced doctor is confronted with a patient with certain symptoms and signs, so-called illness scripts are called up from memory. Illness scripts contain clinically relevant information on diseases, their consequences, the context in which diseases develop, including the personal circumstances, and the experience of the doctor with previous patients. These scripts are generated by the frequent solving of diagnostic clinical problems [21, 22]. Based on recognition, experienced doctors are able to choose the right script for solving a specific diagnostic problem efficiently, particularly in routine cases [23].\nIn order to verify whether this is the right script for the individual patient, two types of diagnostic reasoning may be used \u2013 analytical and non-analytical. Analytical reasoning is characterised as a slow and relatively time-consuming process that is carried out consciously and systematically and, if possible, evidence-based. Less or inexperienced doctors, such as medical students, mostly use this type of reasoning, mainly because they do not possess the ability to call up so-called \u2018illness scripts\u2019. In contrast to analytical reasoning, non-analytical reasoning is carried out rapidly and subconsciously and is based on experience and pattern recognition. This type of reasoning is used especially by experienced doctors. However, when an experienced doctor is confronted with a complex patient case, he or she will also use analytical reasoning.\nBased on the similarities between therapeutic and diagnostic reasoning, it is possible to construct a hypothetical model of therapeutic reasoning (Fig.\u00a01) [24]. When the diagnosis has been determined, one or more treatment scripts will be called up from the memory. In order to determine the right treatment, an analytical or non-analytical process, or a combination of both, will start. This process is similar to the process of diagnostic reasoning. The chosen treatment and its effect will contribute to the modification of the existing treatment scripts or may result in a new treatment script.\nFig.\u00a01Hypothetical model of therapeutic reasoning [24]\nHowever, in order to validate this hypothetical model, further research should be performed, particularly in terms of how experienced doctors arrive at their choice of treatment. In addition, we need to find out how these experienced doctors differ from less-experienced doctors, such as medical students and interns. The answers to these questions will provide more insight into how we should teach therapeutics to students although it can already be stated that early clinical practice will support the development of treatment scripts by undergraduate medical students.\nHow to teach and learn therapeutics\nGaining knowledge and at the same time applying this knowledge in practice is essential for learning in general and, presumably, also for the development of treatment scripts by medical students. This so-called context-learning seems to be more effective in many ways than sequential learning, in which learning and applying knowledge is separated [22, 25, 26]. The positive effect can be explained by theories from cognitive psychology and medical problem-solving [22, 26\u201328]. These theories suggest that the way in which knowledge is stored in the brain is essential for its recall and application. Therefore, storing pharmacotherapeutic knowledge in combination with the situation in which this knowledge will be applied benefits the speed and quality with which the information is recalled [21, 22, 29].\nContext-learning is defined as learning in a setting that is similar to the setting of the future profession [30]. Context-learning is based on four basic principles: setting, repetition, feedback and responsibility [30]. The setting in which therapeutics is taught or learned should be the same as or as similar as possible to the setting of the future profession; for medical students, this is the clinical setting, such as in primary health care, hospitals or nursing homes. This clinical setting gives students the opportunity to gain experience the same way doctors do, allowing them to generate networks of organised knowledge in their memory and to develop illness and treatment scripts [31]. Subsequently, students should be given the opportunity to repeat the therapeutic problem-solving process as much as possible. Repetition allows students to generate networks of organised knowledge in the brain. Frequent exposure to patients and pharmacotherapeutic problems gradually condense these networks into readily accessible therapeutic scripts. Furthermore, students should also receive feedback immediately after their performance to assure the condensation of correct therapeutic scripts. Argumentation and motivational feedback may be a rapid way to reveal the process of therapeutic thinking and its possible errors. Finally, students should be responsible for their own learning. It is the student's own responsibility to repair any lack of knowledge or skills discovered during their clinical work and feedback sessions.\nApart from the most extreme form of context-learning, which is the clinical setting, different variations with lower levels of concreteness of the context are possible, varying from role-playing sessions with standardised patients in a simulated practice setting to the solution of written patient problems in small working groups and patient demonstrations during lectures. However, there are many ways to improve the setting, such as by using real case histories instead of written patient problems or through the use of video materials, laboratory test results or roentgenograms.\nOne possible approach to organising a context-learning programme in therapeutics is described by Vollebregt et al. [32]. These authors described a context-learning programme consisting of weekly-organized, role-playing sessions in the form of consulting hours. The role-playing sessions consist of three phases: consultation, argumentation and feedback. First, a \u2018student\u2019 doctor must carry out three therapeutic consultations of 10\u00a0min each. Before the start of a consultation, the doctors are given a written patient case. Subsequently, the \u2018student\u2019 patient and \u2018student\u2019 assessor enter the consultation room, and the doctor has to choose and prescribe the (drug) treatment interactively with the patient. The second phase (argumentation) starts immediately after the consultations. In this phase, the doctor has to substantiate the chosen therapy. Finally, during the third phase, all students sit together and discuss the various (drug) treatments and the performance of the doctors, guided by a clinical pharmacologist.\nIn addition to teaching therapeutics, assessing students\u2019 therapeutic knowledge and skills is an essential component of the medical curriculum. In 1990, Miller suggested a framework for clinical assessment (Fig.\u00a02) [33]. According to this framework, students, residents or physicians must have knowledge in order to carry out the required professional functions effectively. Students must also know how to use the knowledge that they have accumulated. For example, they must develop the skill that is needed to acquire information from a variety of human and laboratory sources, to analyse and interpret data and, finally, to translate such findings into a rational diagnostic or therapeutic plan. When this quality is functionally adequate, it is defined as a competence. Furthermore, students must not only be able to demonstrate that they know and know how, but also to show how they do it when confronted with a patient, which is called performance. Finally, it is important what a graduate actually does when functioning independently in clinical practice. According to Miller, this action component of professional behaviour is the most difficult to measure.\nFig.\u00a02\u2018Pyramid\u2019 by Miller as an illustrative framework for discussing the assessment of clinical skills, competence and performance [33]\nThe Objective Structured Clinical Examination (OSCE) and structured clinical examinations in general are well-known and approved methods for assessing competence and performance [34]. As far as the OSCE is concerned, this has also been shown to be useful for assessing therapeutic competence. Nevertheless, it is important that the manner in which the students are assessed is as similar as possible to the approach used in teaching them.\nIn conclusion, based on theories as to how physicians arrive at their therapeutic decisions, context-learning seems to be an effective way to teach CP&T to medical students. It is obvious that the ideal situation, i.e. real practice with real patients, is not always attainable, but various suboptimal forms of context-learning and assessment can be applied.\nConclusions and recommendations\nIn this document, the learning objectives of teaching CP&T to undergraduate medical students have been described, with an emphasis on therapeutic decision-making. Based on current theories of cognitive psychology and medical education, we have also discussed context-learning as an effective approach to teaching medical students how to prescribe rationally.\nUntil recently, there were only two groups of people who were permitted to prescribe drugs \u2013 registered medical practitioners and registered dental practitioners. Since 2006, some nurses, pharmacists and physician assistants have also been able to prescribe drugs for medical conditions within their area of competence [35]. Consequently, given this increasing number of registered prescribers with different qualifications, it has become increasingly important to train prescribers sufficiently in how to choose and prescribe drugs rationally. In addition, most curricula are changing from discipline- and subject-based teaching to competence and integrated or problem-based learning, resulting in less visibility of CP&T. The importance of teaching CP&T is further supported by the fact that many graduates still feel insufficiently prepared to assume prescribing responsibilities after graduation [7]. Furthermore, many hospital admissions and even deaths are caused by possibly avoidable medication errors [36].\nClinical pharmacologists should play an important role in the development of prescribing skills by teaching CP&T to undergraduate medical students. It is recommended that they formulate a CP&T context-learning curriculum within the medical curriculum \u2013 of course, in collaboration with physicians. This curriculum must be based on the final learning objectives of the CP&T education programme in which the required level of therapeutic competence of medical graduates has been determined. In addition, the curriculum must fulfil the criteria of context-learning, i.e. gaining knowledge and skills simultaneously in a (simulated) clinical practice setting. Furthermore, for the sake of clarity, it is stressed that the CP&T curriculum should be a visible part of the medical curriculum and that students must know, from the beginning, the required level of competence when they graduate. Current medical students often do not recognise the various CP&T teaching activities since they are scattered through the medical curriculum.\nA draft of a CP&T context-learning curriculum is presented in Fig.\u00a03. At the start of the curriculum, the emphasis lies on gaining CP&T knowledge and simultaneously learning to apply this knowledge by training skills in therapeutics. During this phase, little attention is given to the prescription of drugs in clinical practice. In the following study years, as CP&T knowledge and therapeutic skills increase, increased emphasis is given to prescribing in clinical practice, while the acquisition of knowledge and skills diminish. An example of a more specific CP&T curriculum is presented in Box 1.\nFig.\u00a03A design of a clinical pharmacology and therapeutics (CP&T) context-learning curriculum (adapted from HJM van Rossum)\nIn conclusion, effective undergraduate teaching of CP&T is essential to improve rational prescribing and will immunize students against factors that may induce irrational prescribing after graduation. Therefore, based on current knowledge about learning, cognitive psychology and research in therapeutic teaching, a CP&T curriculum should be a prominent part of the medical curriculum. The CP&T curriculum should also be linked to postgraduate and continuing education in order to maintain an optimal competence in rational prescribing after graduating. Finally, to achieve a CP&T curriculum, allies should be found because clinical pharmacologists can and should not work on this alone. Medical students are already allies; they are interested in clinical pharmacology and really want to learn how to prescribe drugs rationally. Physicians should also become allies, since they can provide the clinical context for teaching and can prevent students from copying the bad prescribing habits of some of the physicians that train medical students. Associate clinical pharmacologists must collectively determine the current state and perspectives of the undergraduate education of CP&T in Europe. To this end, a European research project, which is organised jointly by the EACPT and British Pharmacological Society, has recently been started.\nUltimately, clinical pharmacologists, students and physicians collectively may be able to convince the policy-makers, such as the faculty boards, of the need for a CP&T curriculum. Such a joint effort is truly necessary because, unfortunately, the following words, spoken by Miller in 1990, are, to a great extent, still valid: \u201cIt will not be easy to convince conservative medical faculties, reasonably comfortable with the current conventions that allow clinical impressions to substitute for systematic accumulation of behavioural evidence, that change (in teaching, ed.) is in order\u201d.\nBox 1. Illustration of a CP&T curriculum\nFigure\u00a04 shows the different learning activities and their mutual relations. The horizontal lines represent the three learning components: knowledge [upper line: basic pharmacology, clinical pharmacology, therapeutics (1) and therapeutics (2)], clinical practice (middle line: video patients, patient demonstrations, observation of consultations and prescribing drugs) and skills [lower line: prescription, drug formulary, prescribing (6-steps) and CP&T literature]. The vertical lines describe the simultaneous use of these three components in order to realise a context-learning curriculum.\nFig.\u00a04Outline of a CP&T curriculum\nLet us use the example of a clinical pharmacology department that provides a course of lectures in basic and clinical pharmacology during the first three years of a classical medical curriculum. The aim of the department is to expand its teaching activities and use the context-learning methodology as much as possible. Therefore, the lectures in basic and clinical pharmacology must be transformed according to the criteria of context-learning, i.e. by adding patient cases (clinical practice) and prescription writing (skills). For example, the lecture about \u2018P-450-dependent oxidation\u2019 is placed into the clinical context by presenting a video of a patient case illustrating a drug interaction as the result of a wrong drug choice (WHO step 3b). The lecture concludes by discussing the patient case according to the six-step approach, including how to write a new prescription [18]. The clinical pharmacology lectures in the second and third year can be transformed in a similar way by presenting real patients, followed by lectures on different classes of drugs. Simultaneously with the clinical pharmacology lectures, students must develop a personal drug formulary by, for example, using an E-learning programme [3]. In the fourth year, students start on their clinical clerkships. In collaboration with their clinical colleagues, the clinical pharmacology department introduces a therapeutic assignment during these clerkships. Students must observe several consultations by clinicians in a structured way according to the WHO six-step approach and discuss these in small groups during therapeutic sessions with a clinical pharmacologist and\/or physician. Concurrently, students must follow a skills training programme in order to learn how to prescribe rationally (six steps). During the clinical clerkships in the fifth and sixth years, prescriptions written by the students are evaluated by a clinical pharmacologist in collaboration with a physician and pharmacist during therapeutic sessions. Finally, students must attend a course on how to evaluate CP&T literature and how they must apply this knowledge in practice.\nIt is evident that in an integrated or problem-based medical curriculum it will be difficult to organise lectures. However, an advantage of this type of curriculum is that teaching is already centred on patient cases. As a result, clinical pharmacologists can introduce learning tasks with respect to basic and clinical pharmacology and therapeutics. In such a curriculum, it will probably be easier to train students in the six-step approach in small group-teaching sessions and practice prescribing in a clinical setting.\nObviously, there are various possibilities for context-learning teaching activities, and the above-mentioned are just a few examples of these. More detailed information on how to determine learning objectives for therapeutics and on methods for teaching and assessing therapeutics can be found in the WHO Teacher\u2019s Guide to Good Prescribing [37]. In addition, this guide also provides information on how to mobilise support for changing and implementing a CP&T curriculum and how to perform research in this field.","keyphrases":["teaching","undergraduate","education","pharmacotherapy","clinical pharmacology & therapeutics"],"prmu":["P","P","P","P","M"]}
{"id":"Neurogenetics-4-1-2238789","title":"Towards a pathway definition of Parkinson\u2019s disease: a complex disorder with links to cancer, diabetes and inflammation\n","text":"We have previously established a first whole genome transcriptomic profile of sporadic Parkinson\u2019s disease (PD). After extensive brain tissue-based validation combined with cycles of iterative data analysis and by focusing on the most comparable cases of the cohort, we have refined our analysis and established a list of 892 highly dysregulated priority genes that are considered to form the core of the diseased Parkinsonian metabolic network. The substantia nigra pathways, now under scrutiny, contain more than 100 genes whose association with PD is known from the literature. Of those, more than 40 genes belong to the highly significantly dysregulated group identified in our dataset. Apart from the complete list of 892 priority genes, we present pathways revealing PD \u2018hub\u2019 as well as \u2018peripheral\u2019 network genes. The latter include Lewy body components or interact with known PD genes. Biological associations of PD with cancer, diabetes and inflammation are discussed and interactions of the priority genes with several drugs are provided. Our study illustrates the value of rigorous clinico-pathological correlation when analysing high-throughput data to make optimal use of the histopathological phenome, or morphonome which currently serves as the key diagnostic reference for most human diseases. The need for systematic human tissue banking, following the highest possible professional and ethical standard to enable sustainability, becomes evident.\nIntroduction\nOur understanding of Parkinson\u2019s disease (PD) is largely incomplete. However, the pace of discovery in this field is rapidly accelerating. It took more than 100\u00a0years for the key region of neuronal damage, the substantia nigra to be identified [1], and it took almost 80\u00a0years for the first disease-causing mutation to be discovered [2]. Not even 10\u00a0years later, the first whole genome transcriptome analysis had been performed [3], and a number of other microarray studies focusing on known sequences were carried out (e.g. [4\u20136]). We now provide the complete list of 892 highly dysregulated PD nigral genes derived from a brain tissue-validated whole genome expression microarray data set. In addition, predicted interactions of a number of these genes are reported as potential drug targets. We would like to emphasise that the neurohistological validation that is so crucial for our work and which has already led to the identification of two novel Lewy body components predicted on the basis of this dataset [7, 8] could not have been performed without generous brain donations. In addition, the iterative analysis performed combining histological phenome (morphonome) data and clinical criteria within silico data mining would not have been possible without significant advances in computing, notably virtual machine technology. It is readily apparent that a publication of this format requires the Internet as it would not have been possible to publish its almost 3,200 hyperlinked files on paper, which are provided as electronic supplemental material.\nPD is a severely disabling neurodegenerative disorder second in frequency only to Alzheimer\u2019s disease and has a significant socio-economic impact. Unlike in Alzheimer\u2019s disease, however, the brain region taking the brunt of the disease process is rather well circumscribed. In addition, there is widespread consensus on diagnostic criteria both clinically and neuropathologically (http:\/\/www.ICDNS.org). This is at least in part due to the fact that the leading motor symptoms are less complex and easier to recognize and define than the clinical signs in disorders mainly affecting higher brain functions such as cognition. Furthermore, there is symptomatic treatment for PD pointing to key pathways involved. All these are important prerequisites when working with high throughput technologies such as microarrays which require precise tissue sampling because the procedures employed are both laborious and expensive.\nMajor known pathways involved in PD include the ubiquitin-proteasome system dysfunction of which may lead to abnormal protein deposition, mitochondrial failure and decreased expression of synaptic proteins [6, 9\u201311]. Oxidative stress has been traditionally implicated in the aetiology of the disease but the changes observed could be secondary. The concept of \u2018neuroinflammation\u2019 has become very popular recently [12\u201314], but our own work in this field does not currently support a role for microgliosis as a driver of the disease process [15]. The effective failure of recent studies employing non-steroidal anti-inflammatory drugs supports this notion [16, 17]. Thus, there are leads and popular ideas but the big picture of PD pathogenesis is still missing. A true understanding of PD and its subtypes will require integrated knowledge from several system biological levels, ranging from genomics to proteomics and metabonomics as well as clinical data and neuroimaging. Through this study, we aim to contribute a validated transcriptomic data layer.\nMaterials and methods\nData set used\nThe 94.CEL files used for this study have been deposited at the National Center for Biotechnology Information, Gene Expression Omnibus (http:\/\/www.ncbi.nlm.nih.gov\/projects\/geo) with GEO Series accession number GSE8397 (scheduled release date: 1 January 2008). The patient samples employed have been described previously [3]. This data set is based on Affymetrix HU_133A and HU_133B gene chips set and has been extensively validated over a period of 2\u00a0years using qRT-PCR, immunocytochemistry and in situ hybridisation to cellularly \u2018back-map\u2019 sequences of interest [7, 8, 10, 18].\nData analysis procedures employed\nIn silico analyses were performed with the help of programme packages from different suppliers. Our initial microarray data analysis was performed using ArrayAssist 3.0 (Stratagene) but there were intermittent problems with some versions of the software (3.2\u20133.4). We have repeated our analysis using newer (4.0, 4.1; Linux and\/or Windows) and the latest version of this program (ArrayAssist 5.5 for Windows, Stratagene). In addition, we have reproduced our results using an independent software package for microarray analysis, PathwayStudio 5.0. The (GC-)RMA algorithm which has become a gold standard for Affymetrix microarray normalisation was applied in all cases. In addition, PathwayAssist (Ariadne) and Pathway Architect (Stratagene) were employed during phases of our work. For ease of use, most software installations were performed in virtual machines (VMware Workstation and Fusion, respectively; http:\/\/www.vmware.com) and two virtual machines were frequently run in parallel on Windows, Linux, or Macintosh platforms. The ability to create virtual machine \u2018snap shots\u2019 of critical stages of the analysis proved invaluable for the backtracking of results and to allow comparability over time. It proved essential in the case of the most complex network analyses where software stability proved to be a factor.\nWe have used the microarray data in a hypothesis generating rather than hypothesis testing way (cf. [4, 10]). Since our original predictions based on the ArrayAssist 3.0 dataset turned out to be very reliable with respect to subsequent in situ tissue validation results, we have performed our refined analysis by means of this programme following extensive comparison with readings generated by the latest versions of ArrayAssist 5.5 and PathwayStudio 5.0. Anyone intending to reproduce our results is referred to the original CEL files (GEO ID GSE8397).\nThe following original cases were excluded from our refined analysis to obtain more homogeneous cohorts taking newly obtained histological and expression data into account: Con4, 8 and MS155 as well as the sample of medial substantia nigra from PD22. Thus, nine control nigra and 23 PD nigra samples remained in the study. To find differentially expressed genes, the p value cut off was kept at 0.001 (differential expression\u2009=\u20091 log2). Multiple testing corrections (FDR, Bonferroni) were carried out for comparative purposes. The 892 top genes identified on the basis of 1,145 probes are referred to as the \u2018priority genes\u2019.\nHierarchical clustering was executed on both rows and columns using ArrayAssist 5.5 (Pearson centred distance metric, centroid linkage rule). Similarity images were produced to visualise the quality of clustering results (SI_Figure_1). In addition, self-organising map clustering was performed on both rows and columns using a Euclidean distance metric in ArrayAssist 5.5 (maximum number of iterations 50, number of grid rows 3, number of grid columns 4, initial learning rate 0.03, initial neighbourhood radius 5, grid topology hexagonal, neighbourhood type bubble).\nFor finding cell processes regulated by the differentially expressed genes, PathwayStudio software (version 5.0) was used. The 164 top up-regulated priority genes showing a differential expression >1 log2 were selected and the find common targets algorithm was employed in the build pathway tool setting the cell process filter option. The procedure was repeated for diseases entity type. Cell processes and disease conditions showing the highest number of biological associations, i.e. the strongest probabilistic relationship based on literature evidence to the group of 164 top up-regulated priority genes were selected. Furthermore, known interactions between all 892 dysregulated genes were identified using the ResNet 5.0 database of molecular interactions which has been derived from the published literature by means of a natural language processing technology called Medscan [19]. A filter for the parameters, promoter binding, expression and regulation was applied in the latter case.\nFor additional validation, the commercial PathArt database (PathwayAssist plug-in, Jubilant Biosys) containing a large number of manually curated pathways was queried. A number of GEO datasets (e.g. astrocytes and microglia in culture) were downloaded to evaluate cell-type specificity of expression of individual priority genes. A search for novel secreted biomarkers was also performed of which NPTX2 [8] may be one.\nTo retrieve known drug interactions of the 892 priority genes, more than 9,000,000 database objects were checked in ResNet 5.0 (PathwayStudio 5.0). Subsequently, all known interactions of individual relevant drugs contained in ResNet 5.0 were retrieved and the results overlaid with the set of priority genes. Drugs scoring high in terms of the number of their known interactions with the 892 priority genes were identified and the relationship to PD analysed employing the PubMed database (http:\/\/www.ncbi.nlm.nih.gov\/sites\/entrez).\nResults\nThe extended set of 892 PD priority genes\nScreening of histological and clinical parameters combined with iterative cycles of data mining enabled us to identify the most comparable cases in the cohorts which are detailed in SI_Figure_1. The number of highly significantly dysregulated genes (p\u2009<\u20090.001) increased from 429 (570 probes) to 892 (1,145 probes) while 97% of the originally identified genes [3] were confirmed. The complete list of all 892 PD priority genes is provided in SI_Table_1a. It should be mentioned that there are still some sequences that show highly significant dysregulation in PD on which there is no information available from ResNet 5.0, the IDconverter database [20] or NetAffx. However, all 1,145 probes (SI_Tables_1b&c) were used for the clustering (Fig.\u00a01 and SI_Figure_1). Information on the 1,145 Affymetrix probes available from the public IDconverter database is provided in SI_Table_1d. SI_Table_1e shows the probe information not found in IDConverter or the ResNet database, respectively, that was obtained via NetAffx (http:\/\/www.affymetrix.com\/analysis\/index.affx). Thirty-one priority genes have been previously designated PD candidate genes (http:\/\/www.pdgene.org\/) (SI_Table_1f), and it is worth noting that 33 are considered AD candidate genes as well (SI_Table_1g) with HSPA1A, HSPA1B, HTR2A, KCNJ6, SLC6A3, SNCA, SNCG, TF and UCHL1 being present in both lists. A PubMed search revealed that another 10 priority genes have known functional links to PD (SI_Table_1h). Finally, \u2018whole genome\/whole human body\u2019 clustering using an independent data set derived from 64 different organ samples which is based on Affymetrix Human Genome U133 Plus 2.0 arrays allowed categorical separation of all nervous tissue samples including three different ganglia (SI_Figure_2).\nFig.\u00a01Dendrogram showing the results of unsupervised hierarchical clustering of the 1,145 probes encompassing the 892 priority genes (corresponding Affymetrix probe IDs and expression data are provided online in SI_Figure_1). The algorithm separates the control (left) from the Parkinson\u2019s disease group (right) (rows: red, expression above average; black, average expression; green, below average). An explanation of sample designations, patient data and a quantitative colour code are also provided in SI_Figure_1\nGene ontology and pathway analysis of the differentially expressed genes\nThe statistically most significant overlap (Fisher Exact test) between the 892 priority genes and GO groups was found for GO ID 0016020 (membrane) and GO IDs 0005515\/0045308 (protein binding or protein degradation tagging activity, respectively). Further details are provided in the supplemental material (SI_Table_2). The main cell processes predicted to be influenced by the top up-regulated priority genes (164 genes showing differential expression >1 log2) are illustrated in Fig.\u00a02a. A hyperlinked version of this figure providing details on all genes and their interactions is available online (SI_Figure_3a). A search of 192 canonical pathways and 555 signalling pathways in PathwayStudio 5.0 yielded the RET_HSF1 signalling pathway which shares six priority genes as the highest ranking result (SI_Table_3, SI_Pathway_1).\nFig.\u00a02a Cell processes predicted to be influenced by the top up-regulated priority genes (164 genes showing a differential expression >1 log2) based on in silico analysis employing the ResNet database (PathwayStudio 5.0, Ariadne). P and expression values of all 1,145 probes can be found in SI_Table_1c. An online version of this figure with hyperlinks is provided as SI_Figure_3a. b The three disease conditions showing the strongest biological association with the group of top up-regulated priority genes which may serve as drivers of the disease process underlying PD. In silico analysis was performed employing the ResNet database of molecular interactions (Pathway-Studio 5.0, Ariadne). An online version of this figure with hyperlinks is provided as SI_Figure_3b\nPredicted interactions of a subset of the 892 priority genes are shown in Fig.\u00a03. A total of 417 known connections were retrieved from ResNet. This figure is provided for orientation purposes. Hyperlinked permutations of this figure showing details of all genes and their interactions are available online (SI_Figure_4).\nFig.\u00a03Hypothetical \u2018super pathway\u2019 not stratified for cell type illustrating known direct interactions between the 892 PD priority genes (regulation, expression and promoter binding only). A total of 417 interactions (relations) are shown and any unlinked entities were removed. Display style: by effect; cellular layout; colour codes: promoter binding, violet; green, positive regulation; red, negative regulation; grey and\/or broken lines, unknown (Resnet 5.0 database, unedited). Blue shading around selected genes indicates their involvement in the cellular process and disease conditions depicted in Fig. 2a and b, respectively. Permutations of this figure with hyperlinks are provided online as SI_Figures_4a-c\nKnown components of Lewy bodies and how they relate to the group of priority genes are presented in Fig.\u00a04. The probes for most of these components were found to cluster together in the self-organising map shown in SI_Figure_5. A hyperlinked version of Fig.\u00a04 with expression values overlaid is available online (SI_Figure_6). Interactions of known PD genes with the priority genes of this study are demonstrated in Fig.\u00a05 (SI_Figure_7).\nFig.\u00a04Neuronal pathway containing proteins found in Lewy bodies [11]. Priority genes of this study are marked by the blue shading. An online version of this figure with hyperlinks and an overlay of expression values is provided as SI_Figure_6. Symmetrical layout, display by effect (PathwayStudio)Fig.\u00a05Interactions of known PD genes (marked yellow) with the priority genes of this study. Expression value overlay: red indicates up- regulation and blue indicates down-regulation in PD nigra. An online version of this figure with hyperlinks and an overlay of significance values is provided as SI_Figure_7. Cellular layout, display by effect (ResNet)\nWe did not observe an effect for gender (cf. [21]). Hierarchical clustering of the male and female PD patients on the basis of the expression values of the 892 priority genes did not separate the groups nor did a whole genome clustering omitting sex chromosomal sequences.\nRelationship to diseases and drug interactions\nA search of the ResNet database identified three disease conditions that showed the strongest probabilistic relationship to the group of top up-regulated priority genes, cancer, diabetes and inflammation as illustrated in Fig.\u00a02b. A hyperlinked version of this figure providing details on all genes and their interactions is available online (SI_Figure_3b). Known drug interactions of some of the priority genes were retrieved from the ResNet 5.0 database through checking of more than 9,000,000 database objects. It is noteworthy that drugs such as clozapine, cocaine and haloperidol, which are used in the treatment of PD or which cause Parkinsonian side effects, appear to interact with a large number of PD priority genes (SI_Figure_8). A search of the ResNet database also yielded information on the interactions of two cytostatic drugs, paclitaxel and vincristine with the priority genes identified in this study (SI_Pathways_2&3). Both paclitaxel and vincristine have been reported to induce parkinsonian side effects [22, 23].\n\u2018Hub\u2019 vs \u2018peripheral\u2019 genes\nA number of genes known to have numerous interactions with other genes were found amongst the priority genes. These represent so-called network \u2018hubs\u2019 and include HSPA1A, NFKBIA, CDC42, GSK3B, ACHE, AGTR1, IGF1R and TH as well as about 200 others (50 to >1,600 connectivities). Figure 2a and b contain a number of hubs. However, almost 30% of the priority genes have no known interactions according to the ResNet 5.0 database although the cellular localisation is known in some instances. Such genes may be called \u2018peripheral\u2019 genes of the human interactome [24, 25]. Apparently, peripheral priority genes with still unknown pathway connections and cellular localisation are ACTR10, ANKRD29, ANKRD34, ANKRD50, ARMCX4, ARRDC2, ASMTL, ATAD1, BLOC1S2, CAP2, CCDC4, CCDC85A, CKMT1A, CNIH3, DBNDD1, DCUN1D4, DIRAS2, DOPEY1, EHBP1, ELMOD1, FARSLB, FBXO9, FHOD3, GABARAPL3, GARNL4, GPRASP2, GPRIN3, GUSBP1, HISPPD1, HNRPUL2, IPW, KLHL1, KRT222P, LRRC49, LRRC55, LYNX1, MANEAL, MAP1LC3A, MAP9, MGC22265, MGC39606, MGC4677, MIA3, MRC1L1, NCDN, NGFRAP1L1, NRIP3, NUDT11, OCIAD2, OGDHL, OSBPL10, PCYOX1L, PFAAP5, PGM2L1, PLCXD3, PNMA6A, PRKY, PRMT8, PRPS1, RFPL1S, RUTBC2, SLC35F1, SNX10, SNX25, TAGLN3, TBC1D24, TBC1D9, TMEM130, TMEM132B, TMEM35, TRIM4, TRIM9, TSGA14, TTC7B, TUBB2B, TUBB3, UBPH, USP34, WDR47, XKR4, ZNF204.\nUp-regulated peripheral genes are shown in Table\u00a01. Following the above criteria, a number of Lewy body components and genes known to interact with the established PD genes seem peripheral to main brain metabolic pathways but their exact status remains to be determined.\nTable\u00a01Up-regulated \u2018peripheral\u2019 priority genes\nDiscussion\nOur study reveals a significant up-regulation of substantia nigra genes in PD which have known biological associations with cancer, diabetes and inflammation. This includes major \u2018hub\u2019 genes [24\u201326] such as p53, somatic mutations of which can cause cancer. This is of note as p53 forms part of a molecular network that integrates tumour suppression and ageing [27]. DJ-1 is another cancer- and Parkinson\u2019s disease-associated protein [28], and it is of special interest in the present context that the ubiquitin-proteasomal pathway has an established role in neoplastic processes [29]. Furthermore, both parkin and PINK1 might be tumour suppressor genes, and it has been suggested that although cancer is rare in PD, unraveling the link between PD and cancer [30\u201331] may open a therapeutic window for both diseases [32]. The finding of a molecular biological association between diabetes and PD is not truly surprising either [33\u201337]. Thirdly, the link of PD with inflammation which emerges from our unsupervised analysis seems almost expected considering the very lively debate of this topic in the literature. The whole genome transcriptome data presented here certainly justify additional scrutiny of the underlying mechanisms in relation to PD pathogenesis.\nThe problem of defining what causes PD at a system level has become more complex with the recent finding that disease-relevant genes may reside at the periphery of disease networks. It is interesting to note that the neighbour of a disease node appears more likely to be another disease protein, which also preferentially interacts with other disease nodes [38]. Proteins that are associated with the same disease show a 10-fold increased tendency to interact with each other than those not associated with the same disease [26]. This should direct our attention also to genes that do not form major network hubs but which are either likely to be involved in PD on cell biological grounds (e.g. Lewy body components) or which interact with PD causing genes. A significant fraction of the genes identified in this study still represent functionally ill-characterised entities.\nThe on-line material of this manuscript illustrates some of the pathways and biological association networks that emerge from our analysis. Networks are now recognized to pervade all aspects of human biology and the question where function lies within a cell is shifting from a simple focus on genes to the understanding that behind each cellular function there is a discernible network module consisting of genes, transcription factors, RNAs, enzymes and metabolites [39]. However, \u2018network medicine\u2019 is still in its infancy and the present study may be the first where an iterative multidimensional tissue analysis approach, http:\/\/www.neurogenetics.net\/Multidimensional.html, has been applied to a human neurological disorder. The ultimate goal of such analyses is the precise cellular localisation of all expressed human disease genes in their affected tissues. The present PD dataset has so far yielded two novel components of Lewy bodies [7, 8] but much more back-mapping work will need to be performed.\nFor instance, the exact mechanism of cell death in PD is still unknown [40]. Recent evidence has suggested that one mechanism linked to the death of terminally differentiated neurons is aberrant re-entry into the cell cycle, and possible connections between oxidative stress and unscheduled cell cycle re-entry in PD have been proposed [41]. However, as neuroscientists, we may have to move beyond the description of the cell cycle that has been propagated by those in the cancer field because the regulation of the cell cycle in the neuron is much more nuanced (K Herrup, http:\/\/www.alzforum.org\/new\/detailprint.asp?id=1688). This raises the possibility that some of the data supporting cell processes such as mutagenesis in this study may have to be re-read and interpreted in a modified way. It is worth noting in this context that absence of RET signalling in mice causes progressive and late degeneration of the nigrostriatal system [42]. We would also like to point out that the present study provides additional evidence for the importance of changes in the neuronal cytoskeleton in PD [43\u201344] because neurofilament subunit as well as microtubuli-associated protein genes were found to be highly dysregulated. Dysregulation of signal transduction, heat shock and synaptic proteins also featured very prominently.\nThe view that the 892 nigral genes presented here are relevant for sporadic PD is supported by the finding that their pattern of expression is characteristic of nervous tissue (SI_Figure_2). It is further clear from our data that the human substantia nigra in PD does not represent dead tissue but that there is an active ongoing disease process understanding of which may hold the key to halting PD. The dysregulated priority genes may reside at the core of the disease process and could serve as novel targets for therapeutic intervention. This idea is supported by the observation that a number of priority genes interact with drugs whose actions are associated with a Parkinsonian clinical phenotype.\nThe uncertainty whether inflammatory processes truly represent a causative factor in the aetiology of PD [45] requires an answer. Our own work and that of others suggests a direct role of primary glial degeneration in the pathogenetic process underlying PD [10, 46]. This means that PD extends beyond the neuron. The disease is also not confined to the substantia nigra anatomically. Detailed cellular back-mapping of all priority genes to brain tissue will help to settle these questions. New algorithms are required to explain the links of PD as defined in the living and in the microscope with the underlying high-throughput datasets.\nCollection of links to Electronic Supplemental Material (ESM)\nSI_Table_1a List of all 892 highly dysregulated genes (\u2018priority genes\u2019) contained in the ResNet 5.0 database (Ariadne) http:\/\/www.morphonom.net\/ng\/ESM\/t\/SI_Table_1a.xls\nSI_Table_1b List of the 1,145 Affymetrix probes identifying highly dysregulated sequences including all 892 \u2018priority genes\u2019 http:\/\/www.morphonom.net\/ng\/ESM\/t\/SI_Table_1b.txt\nSI_Table_1c P and differential expression values for the 1,145 Affymetrix probes http:\/\/www.morphonom.net\/ng\/ESM\/t\/SI_Table_1c.xls\nSI_Table_1d Publicly available information on the 1,145 Affymetrix probes from IDconverter (http:\/\/idconverter.bioinfo.cnio.es\/) http:\/\/www.morphonom.net\/ng\/ESM\/t\/SI_Table_1d.xls\nSI_Table_1e Information on gene sequences not found in the ResNet database obtained via NetAffx (http:\/\/www.affymetrix.com\/analysis\/index.affx) http:\/\/www.morphonom.net\/ng\/ESM\/t\/SI_Table_1e.xls\nSI_Table_1f List of priority genes that are published PD candidate genes (http:\/\/www.pdgene.org\/) http:\/\/www.morphonom.net\/ng\/ESM\/t\/SI_Table_1f.xls\nSI_Table_1g List of priority genes that are published AD candidate genes http:\/\/www.morphonom.net\/ng\/ESM\/t\/SI_Table_1g.rtf\nSI_Table_1h List of priority genes with known functional links to PD (PubMed) http:\/\/www.morphonom.net\/ng\/ESM\/t\/SI_Table_1h.xls\nThe literature references for this table can be found in http:\/\/www.morphonom.net\/ng\/ESM\/r\/SI_References_1.rtf\nSI_Table_2 Gene Ontology analysis of the 892 genes http:\/\/www.morphonom.net\/ng\/ESM\/t\/SI_Table_2.xls\nSI_Table_3 Signalling pathway analysis of the 892 genes (PathwayStudio 5.0) http:\/\/www.morphonom.net\/ng\/ESM\/t\/SI_Table_3.xls\nSI_Figures_1a Hierarchical clustering of the 1,145 probes (dendrogram description: clustering on rows and columns, Pearson centred distance metric, centroid linkage rule); the clustering separates PD cases (green, right) from controls (red, left); designation of samples as indicated in the dataset submitted to GEO (see \u2018Materials and methods\u2019 section). http:\/\/www.morphonom.net\/ng\/ESM\/f\/SI_Figure_1a.png\nAbbreviations used: F, female; M, male; LN, lateral substantia nigra; MN, medial substantia nigra; CON, PDC, controls; PD, Parkinson\u2019s disease; followed by the number indicating the age of each subject (cf. Moran et al. [3]); LN, MNCON10, M, 71; LN, MNCON2, M, 77; MNCON3, M, 81; LN, MNCON9, M, 57; LN, MNPD01, F, 87; LN, MNPD02, M, 83; LN, MNPD04, M, 68; LN, MNPD07, M, 78; LN, MNPD09, F, 86; LN, MNPD10, F, 81; LN, MNPD16, F, 85; MNPD20, M, 75; MNPD21, M, 76; LN, MNPD28, M, 82; LN, MNPD29, M, 76; MNPD32, M, 89; MNPD34, F, 84; MNPD36, M, 76; LN, MNPDC1, M, 76.\nAn explanation for the colour coding is provided in http:\/\/www.morphonom.net\/ng\/ESM\/f\/SI_Figures_1a&c-Color_range.jpg\nThe labels used for the clustering and the corresponding Affymetrix probe set IDs are explained in http:\/\/www.morphonom.net\/ng\/ESM\/f\/SI_Figures_1a&c-Labels_used_for_clustering.xls\nThe four probes at the very bottom of the figure (XIST, X (inactive)-specific transcript) in PD01, 9, 10, 16 and 34 identify the female patients in our refined cohort. They served as an internal control.\nSI_Figures_1b Column similarity image for SI_Figure_1a.png http:\/\/www.morphonom.net\/ng\/ESM\/f\/SI_Figure_1b.png\nSI_Figures_1c Self-organising map corresponding to SI_Figure_1a (dendrogram description: clustering on rows and columns, Euclidean distance metric, maximum number of iterations 50, number of grid rows 3, number of grid columns 4, initial learning rate 0.03, initial neighbourhood radius 5, grid topology hexagonal, neighbourhood type bubble) http:\/\/www.morphonom.net\/ng\/ESM\/f\/SI_Figure_1c.png\nSI_Figure_2 Hierarchical clustering of the 1,145 probes using 64 whole genome array datasets (Affymetrix Human Genome U133 Plus 2.0 arrays) representing individual organ samples including 20 brain regions and three ganglia [47]. GSM numbers refer to the respective file names in the complete dataset which comprises 353 whole genome arrays (GEO database, GSE ID GSE3526). There is a complete separation of nervous tissue (left) from other organs on the basis of the 1,145 probes. http:\/\/www.morphonom.net\/ng\/ESM\/f\/SI_Figure_2.png\nThe designations of all samples and their code numbers are provided in http:\/\/www.morphonom.net\/ng\/ESM\/r\/SI_References_2.rtf\nSI_Figure_3a Online version (with links) of Fig.\u00a02a http:\/\/www.morphonom.net\/ng\/ESM\/f\/Cell_Processes.html\nSI_Figure_3b Online version (with links) of Fig.\u00a02b. (XIST was not included in this analysis). http:\/\/www.morphonom.net\/ng\/ESM\/f\/Disease.html\nSI_Figures_4a-c Online permutations of Fig.\u00a03\nLayout by cellular localisation with links http:\/\/www.morphonom.net\/ng\/ESM\/f\/892.html\nSymmetrical layout with links http:\/\/www.morphonom.net\/ng\/ESM\/f\/892s.html\nHierarchical layout with links http:\/\/www.morphonom.net\/ng\/ESM\/f\/892h.html\nSI_Figure_5 Known components of Lewy bodies are indicated by the blue highlighting (this figure is identical to SI_Figure_1c except that it has a lower resolution) http:\/\/www.morphonom.net\/ng\/ESM\/f\/SI_Figure_5.png\nSI_Figure_6 Online version of Fig.\u00a04 with expression values overlaid (red indicates up- and blue indicates down-regulation in PD nigra, respectively). The blue shading of the priority genes has been replaced with yellow. http:\/\/www.morphonom.net\/ng\/ESM\/f\/Lewy_Body.html\nSI_Figure_7 Hyperlinked online version of Fig.\u00a05. The gene colours range in this figure indicates significance: high, white; low, red. The blue shading indicates that the respective gene is a priority gene. http:\/\/www.morphonom.net\/ng\/ESM\/f\/PD_genes_interactions_with_892_direct_no_DorCP.html\nSI_Figures_8a-c Known drug interactions of some of the priority genes as derived from the ResNet 5.0 database (more than 9,000,000 database objects were checked). It is noteworthy that drugs such as clozapine, cocaine and haloperidol which are used in the treatment of PD or which cause Parkinsonian side effects appear to interact with an especially large number of PD priority genes.\nClozapine http:\/\/www.morphonom.net\/ng\/ESM\/f\/SI_Figure_8a.png\nCocaine http:\/\/www.morphonom.net\/ng\/ESM\/f\/SI_Figure_8b.png\nHaloperidol http:\/\/www.morphonom.net\/ng\/ESM\/f\/SI_Figure_8c.png\nSI_Pathway_1 This signalling pathway was identified based on a search of 192 canonical pathways and 555 signalling pathways in PathwayStudio 5.0 and ranked highest (also see SI_Table_3). Six priority genes are represented in this pathway and are marked by the blue shading. http:\/\/www.morphonom.net\/ng\/ESM\/p\/RET_HSF1_signaling_pathway.html\nSI_Pathway_2 Interactions of the cytostatic drug, paclitaxel with a total of 13 priority genes (blue shading) are shown. Display by effect. Hierarchical layout. http:\/\/www.morphonom.net\/ng\/ESM\/p\/paclitaxel_interactions.html\nSI_Pathway_3 Interactions of the cytostatic drug, vincristine with 3 priority genes (violet shading, bottom of figure) are illustrated. Red shaded genes also showed dysregulation in the PD nigra (p\u2009<\u20090.001). Display by references count (darker blue indicates a larger number of references supporting the respective connection). Hierarchical layout. http:\/\/www.morphonom.net\/ng\/ESM\/p\/vincristine_interactions.html\nSI_References_1 References for SI_Table_1h http:\/\/www.morphonom.net\/ng\/ESM\/r\/SI_References_1.rtf\nSI_References_2 Designations and code numbers for SI_Figure_2 http:\/\/www.morphonom.net\/ng\/ESM\/r\/SI_References_2.rtf","keyphrases":["lewy bodies","microarrays","network medicine","aposklesis","expression analysis","neurodegeneration","synaptic dysfunction"],"prmu":["P","P","P","U","R","U","R"]}
{"id":"Eur_J_Clin_Pharmacol-3-1-1779627","title":"Medication administered to children from 0 to 7.5 years in the Avon Longitudinal Study of Parents and Children (ALSPAC)\n","text":"Objective To present data on the parentally-reported use of all types of medicinal products in children from 0 to 7.5 years, in a large cohort in south-west England.\nIntroduction\nIt is now well recognised that there is a need for more studies to obtain paediatric information on medicines used in children with the aim of reducing the amount of unlicenced and off-label use of medicines in this population. In 2002, 15% of children under 5 and 20% of children aged 5\u201315 were reported as having a long-standing illness [13]. In addition, young children may experience a range of relatively minor ailments requiring medical treatment. A previous report using data from this cohort showed that cold, cough, high fever, colic\/stomach ache, diarrhoea and vomiting were very common symptoms in the pre-school years, and that cough, cold, earache and high temperature are the most common symptoms to present to medical services [6]. In 2004, 35.7 million prescription items were dispensed in the community for children under 16 at a cost of \u00a3293.8 million [7]. This represents an average of 4.5 items per head in the age range 0\u201315\u00a0years [7]. The categories of drugs most frequently prescribed for children are antibacterials, analgesics and bronchodilators [12]. Prescribing outside the terms of the product licence occurs in drugs commonly prescribed in general practice such as systemic antibiotics [11].\nAlthough children are entitled to free medicines on a National Health Service prescription in the UK, parents may also choose to purchase products over-the-counter (OTC) for various reasons, such as convenience and confusion over their entitlements [2]. In a US survey involving 8,145 3-year-olds, 54% had been given an OTC medication in the last 30\u00a0days, most commonly paracetamol and cough\/cold medicines [8], and a Norwegian study concluded that paracetamol was an important tool for parents in managing their children\u2019s illnesses [9]. A significant proportion of OTC medicines used in children may be purchased from non-pharmacy outlets, where advice on appropriate use is unavailable [3].\nThis study describes the patterns of parentally-reported use of all types of medicinal products in a large community sample of young children, at four data collection times between the ages of 4\u00a0weeks and 7.5\u00a0years.\nMethods\nThe data for this study were collected as part of the Avon Longitudinal Study of Parents and Children (ALSPAC): a longitudinal study designed to investigate the health and development of children [5]. The study was approved by the ALSPAC law and ethics committee, and the three local research ethics committees.\nPregnant women resident in the three Bristol-based health districts in the former county of Avon in southwest England, and with an expected delivery date falling between 1 April 1991 and 31 December 1992, were eligible to be enrolled. The core ALSPAC sample consists of 14,212 women who had a total of 14,541 study pregnancies, involving 14,676 fetuses, resulting in 14,062 live births. An additional 548 children from 542 eligible pregnancies not included in the core sample were included in the study from age 7. Cohort retention has been good, and we are currently in regular contact with approximately 11,000 families. Further details can be obtained from the ALSPAC website, http:\/\/www.alspac.bris.ac.uk.\nFrom various studies performed at the planning stage, the Avon population was considered to be broadly similar to that of the whole of Great Britain. After delivery, the representative nature of the sample was assessed. As in similar studies, the ALSPAC cohort has a slight shortfall in the less affluent families and a shortfall in ethnic minority mothers. Birth weights and lengths and growth measures in ALSPAC are very similar to national standards [5].\nThe primary source of data collection is via postal questionnaires about the health, development and home environment of the study children completed by each child\u2019s main carer, usually the mother. This paper presents data obtained from four such questionnaires administered when the study children were 4\u00a0weeks, 54, 78 and 91\u00a0months old. Each of these asked questions, designed by the ALSPAC study team, about the use of medicinal products.\nThe 4-week questionnaire (sent out between 1991 and 1993), asked the baby\u2019s mother to: \u201cPlease list all the ointments, pills and medicines that have been given to your baby while he\/she has been at home. Check: have you included ointments to prevent nappy rash, eyedrops, herbal remedies, etc.\u201d\nThe questionnaire sent out between 1995 and 1997, when the child was 54\u00a0months of age (4.5\u00a0years) asked the following: \nChildren often have accidents or illnesses that need treatment. Please indicate which of the following have been given to your child since he\/she was 3\u00a0years old \nCough medicineAntibiotics\/penicillinThroat medicineVitaminsParacetamol\/calpolOintment for skinEye ointmentDiarrhoea mixture\/pillsDimotapp\/decongestantEar dropsEye dropsIronLaxativesHomeopathic medicineHerbal medicineOtherFor each category, the carer was asked to tick whether the response was \u2018Never\u2019, \u2018Yes, for one episode only\u2019 or \u2018Yes, for 2 or more episodes\u2019; and \u2018If yes, please give full names of substances if you can\u2019.Are there any pills, ointments or medicines that he\/she has taken every day or nearly every day for the last 3\u00a0months? (Include vitamins, skin cream, inhaler, laxatives as well as antibiotics, homeopathic and herbal remedies, etc.) If yes, please describe.\nAt 78\u00a0months (6.5\u00a0years, data collection 1997\u20131999) and 91\u00a0months (7.5\u00a0years, data collection 1998\u20132000) the first question was expanded slightly as follows: \nChildren often have accidents or illnesses that need treatment. Please indicate which of the following have been given to your child in the last 12\u00a0months. \nCough medicineAntibiotics\/penicillinThroat medicineVitaminsParacetamol\/calpolOintment for skinEye ointmentDiarrhoea mixture\/pillsDimotapp*\/decongestantEar dropsEye dropsIronLaxativesHomeopathic medicineHerbal medicineAsthma medicationVaporiserOther\n(*active ingredients: brompheniramine, phenylephrine and phenylpropanolamine)\nThe tick box response options, the request for full name of the product, and the second question were the same as at 54\u00a0months.\nAfter a questionnaire was sent out, if a response was not received within 3\u00a0weeks, a reminder letter was sent. If the questionnaire had still not been received after a further 2\u00a0weeks, a second reminder letter was sent. Finally, if no response had been received after 3\u00a0months, a member of the study team either rang the mother or visited her home, and encouraged or helped her to complete the questionnaire. The date of completion is included on each questionnaire, so whilst all returned questionnaires are added to the database and included in the overall response rate, those that were completed after a defined cut-off date can be excluded from individual analyses.\nMost of the self-completion responses on the questionnaires are self-coding\u2014the ticked reply box contains a printed number that can be directly keyed. However, the actual names of medicinal products used (including vitamins and supplements, herbal products, homeopathic remedies, over-the-counter and prescription products) were collected as free text responses.\nAn ALSPAC drug dictionary has been developed based on the World Health Organisation (WHO) Drug Dictionary (first quarter 2000 version, provided free of charge by the Uppsala Monitoring Centre). The ALSPAC dictionary assigns a Drug Name and Anatomical\u2013Therapeutic\u2013Chemical (ATC) classification as described in the WHO Drug Dictionary to an individual product. The latter classification groups medications according to the organ or body system on which they are designed to act and their chemical, pharmacological and therapeutic properties. Some drugs have several ATC codes as they can be used in different preparations on different body systems. The ALSPAC dictionary codes preparations, and thus a single ATC, has been assigned to each product. In the few cases where more than one ATC code would have been possible, a decision was made as to the most likely use in this population. Additional details about the WHO Drug Dictionary can be found on the WHO website: http:\/\/www.whocc.no\/atcddd\/.\nIn cases where a category of medicinal product was ticked as having been used but no further details were provided, a non-specific code for that category has been added to the database of text responses.\nFor the purposes of this report, all responses indicating that a product in the defined category had been used have been included, thus indicating \u2018ever use\u2019 of these categories of medicinal products rather than taking into account frequency of use.\nResults\nThe response rate was good: Table\u00a01 gives the number of questionnaires mailed and returned at each age. The majority of questionnaires were completed at or close to the intended age. For example, 90% of the returned month 54 questionnaires had been completed by 55\u00a0months and 99% by 58\u00a0months, so all data from the 54\u201391\u00a0month questionnaires have been included. However, as the purpose of the question at 4\u00a0weeks was to ascertain what medicinal products were used on the study children as very young babies, a cut-off was applied so that only questionnaires completed before the child was 8\u00a0weeks old were included. Using this criterion, at least one questionnaire was completed for 12,525 study children and all 4 were completed for 6,635 children. \nTable\u00a01Total number of medicinal products recorded per child at each age\u00a04 weeks (<8 weeks)54 months78 months91 monthsNo. of questionnaires sent out13,65912,37410,91411,251No. of completed questionnaires10,897a9,7258,5788,269Total no. products recorded per child02,57312933941013,0295571,0101,13922,5941,4001,7461,91131,5022,0201,7151,75946871,8341,3701,31753201,3821,0017716130939590451739578346227813352179133910205139771010351431174333212492412133013714256715161061619191710132181251900220212110220231aCompleted within 8\u00a0weeks of birth\nFor most of the defined categories, the number of tick box responses not supported with a text response giving the name of the product increased between the ages of 54 and 91\u00a0months (at 4\u00a0weeks, responses to the medication question were all collected as free text).\nThe week 4 questionnaire was completed before the child was 8\u00a0weeks of age in 10,897 cases (80% of those sent, 88% of those returned). Around three-quarters of study children were exposed to some form of medicinal product before 8\u00a0weeks of age. At this age, dermatological products were most commonly reported, mainly those for treatment and protection against nappy rash, such as \u2018Sudocrem\u2019, a proprietary product containing zinc oxide used in 29% of babies, and zinc and castor oil cream used in 22%. Simeticone (activated dimeticone) in products for treatment of colic and flatulence, such as \u2018Infacol\u2019, was given to 16% of babies. Gripe water (terpeneless dill seed oil and sodium bicarbonate) was used in 13% of the cohort. Other commonly reported products included oral and topical antifungals and ophthalmic antibiotics (mostly chloramphenicol). A few babies (2.3%) were given vitamin drops. Some were given herbal drinks (2.2%), homeopathic products (1.2%) and a variety of topical complementary and alternative medicine products, such as calendula and camomile creams (3.3%).\nSeveral OTC products not licensed for use in this age group were reported. For example, inhalant decongestant products not licensed for children under 3\u00a0months of age\u2014Snuffle-babe Vapour Rub (eucalyptus oil, menthol and thyme oil), Karvol Decongestant Capsules for inhalation (levomenthol with chlorobutanol, pine oils, terpineol and thymol) and Vicks Vaporub (levomenthol, camphor, eucalyptus oil and turpentine oil)\u2014 were used in over 6% of the cohort, and xylometazoline nasal spray was used on 0.6%. Calpol (paracetamol), which is licensed from 2\u00a0months, was given to over 1% when they were under 8\u00a0weeks old.\nAt the older ages, 54, 78 and 91\u00a0months, 9,728 (78% of those mailed), 8,578 (78%) and 8,269 (73%) questionnaires were completed and returned, respectively. In these latter questionnaires, relatively few children were reported not to have used any medicinal products in the preceding 12\u201318\u00a0months. At 54\u00a0months (4.5\u00a0years), only 129 children (1.3% of those returning the questionnaire) were not reported as having used a medicinal product since the age of 3. At 78 and 91\u00a0months, the figures were 339 (4.0%) and 329 (4.0%), respectively. Table\u00a01 shows the number of products reported per child at each age.\nTable\u00a02 shows the frequency of use of medicinal products, by ATC code, at each data collection time. The data are presented as the total number of drugs reported in an ATC class per number of children in which any products within this class were used (i.e. more than one product in a class may have been reported per child). The pattern of use of different categories of medicinal product varied with age. Usage of several product categories was higher at 54\u00a0months than at 78 or 91\u00a0months. For example, at 54\u00a0months, 1,052 children (10.2%) were reported to have used topical steroids (mostly hydrocortisone) compared with 7.4% at 78\u00a0months and 4.9% at 91\u00a0months. Paracetamol (as a single ingredient) was recorded for 9,211 children (94.7%) at 54\u00a0months, 7,595 (88.5%) at 78\u00a0months and 7,247 (87.6%) at 91\u00a0months. In addition, systemic antibiotics (mainly amoxicillin), laxatives (mostly lactulose), treatments for diarrhoea (mainly rehydration salts), various cough and cold products, emollient and protective skin preparations (mostly paraffin-based proprietary products), topical antipruritics, antihistamines and local anaesthetic products were all greater at 54\u00a0months than at the older ages. Conversely, use of other categories, such as throat preparations (sprays and lozenges), and anti-inflammatory products, increased with increasing age. Use of \u2018asthma\u2019 medication (mainly salbutamol and beclomethasone) also increased with increasing age; at 54\u00a0months, 6.8% of the cohort reported use of anti-asthmatics, by 78\u00a0months this had increased to 12.0% and to 14.2% at 91\u00a0months. Of the 1,174 children reported as having used asthma medication at 91\u00a0months, 1,038 (88%) had previously had a diagnosis of \u2018asthma\u2019 from a doctor and 907 (77%) reported having asthma in the past 12\u00a0months. \nTable\u00a02Frequency of use of medicinal products, by ATC code, at each age. Total number of drugs reported in a class\/number of children in which this class of product was used (individual children may have used more than one product in a class). ATC level 1 (in bold) includes all reported cases. ATC level 2 is given where 10 or more children reported a product in this category in at least one questionnaireATC code\u00a0<8 weeks54 months 3\u20134.5 years78 months 5.5\u20136.5 years91 months 6.5\u20137.5 yearsNo. of questionnaires returned with data10,8979,7258,5788,269\u00a0ATC text\u00a0\u00a0\u00a0\u00a0AGastrointestinal preparations4,470\/3,4166,733\/4,1884,747\/3,1304,262\/2,973A01Stomatological preparations691\/649114\/9457\/4635\/30A02Antacids, drugs for peptic ulcer and flatulence3,422\/2,69031\/2725\/2315\/13A03Antispasmodics, anticholinergics and propulsives29\/2913\/1213\/119\/9A06Laxatives41\/41528\/421277\/200328\/262A07Antidiarrhoeals, intestinal anti-inflammatories and anti-infectives39\/38693\/682196\/191227\/224A11Vitamins247\/2475,169\/3,5673,143\/2,1213,748\/2,692A12Mineral supplements1\/1170\/15540\/27158\/149BBlood and blood forming organs61\/4757\/5028\/2132\/25B03Antianaemic preparations46\/3248\/4128\/2131\/24CCardiovascular system13\/116\/58\/74\/3DDermatologicals9,618\/6,3975,105\/3,2693,538\/2,6532,988\/2,441DDermatologicals unspecified25\/241,015\/1,010915\/9071,376\/1,373D01Antifungals for dermatological use584\/557173\/163185\/173125\/118D02Emollients and protectives7,908\/5,6551,954\/1,45312,18\/928690\/519D04Antipruritics, including antihistamines, anaesthetics 6\/6158\/15191\/8959\/57D06Antibiotics and chemotherapeutics, topical98\/95169\/161154\/142107\/99D07Corticosteroids, topical125\/1211,358\/1,052790\/631507\/408D08Antiseptics and disinfectants843\/785246\/223161\/154114\/110D11Other dermatological preparations25\/2516\/1411\/113\/3GGenito-urinary system and sex hormones1\/117\/1518\/169\/8HSystemic hormones, excl sex hormones3\/369\/5963\/5649\/40H01Pituitary, hypothalamic hormones and analogues02\/212\/814\/11H02Corticosteroids for systemic use1\/164\/5548\/4634\/28JGeneral anti-infectives for systemic use354\/3416,505\/5,9763,322\/3,1512,434\/2,358J01Antibacterials for systemic use346\/3356,498\/5,9733,309\/3,1462,419\/2,349LAntineoplastic and immunomodulating agents001\/12\/1MMusculo-skeletal system063\/62111\/107119\/118M01Anti-inflammatory and antirheumatic products062\/61105\/103117\/116NNervous system163\/1599,561\/9,1847,857\/7,6207,400\/7,261N02Analgesics157\/1549,510\/9,1817,802\/7,6167,344\/7,256N03Anti-epileptics1\/139\/2838\/2125\/15N06Psychoanaleptics (antidepressants\/stimulants)1\/14\/25\/524\/17PAntiparasitics, insecticides and repellents087\/8090\/8458\/54P02Anthelmintics066\/6562\/5937\/36P03Ectoparasiticides, scabicides, insecticides, repellents014\/1322\/2217\/16RRespiratory system1,168\/1,0329,609\/6,9958,615\/5,4927,796\/5,131R01Nasal preparations468\/456705\/655437\/391403\/382R02Throat preparations0471\/471514\/511649\/646R03Anti-asthmatics01,178\/6582,422\/1,0291,920\/1,174 R05Cough and cold preparations697\/6437,839\/6,4345,058\/4,6784,684\/4,391 R06Antihistamines for systemic use3\/3216\/186184\/163140\/127SSensory organs1496\/14112954\/22131051\/8841090\/861 S01Ophthalmologicals1,316\/1,2571,852\/1,444536\/438633\/499 S02Otologicals3\/3737\/728426\/416405\/403 S03Ophthalmological and otological preparations177\/177365\/31489\/8452\/49VVarious855\/7051,082\/745955\/7051,139\/796 V03All other therapeutic products (including CAM)587\/5041,079\/744954\/7041,138\/795 V07All other non-therapeutic products (eg baby toiletries)295\/21401\/10\nTreatments for less common conditions were also reported by this community sample. By 91\u00a0months, 6 children were using insulin, 17 reported methylphenidate (for attention deficit hyperactivity disorder) and 2 reported thyroid therapy. At 54\u00a0months, 28 children reported antiepileptic medication but this had reduced to 15 children by 91\u00a0months.\nBetween 54 and 91\u00a0months, a very small number of children were reported to have been given an OTC medicine, whilst they were younger than the age specified on the Patient Information Leaflet or Summary Product Characteristics, for example, aspirin, \u2018Dequadin\u2019 lozenges (dequalinium chloride), \u2018Night Nurse Liquid\u2019 (paracetamol, pomethazine hydrochloride, dextromethorphan hydrobromide) and \u2018Diocalm\u2019 (morphine hydrochloride and attapulgite).\nDiscussion\nThe advantage of collecting data on children\u2019s use of medicinal products from their parents\/carers, as opposed to from medical records and prescription databases, is that the former are in a position to know what was actually given to their children, rather than what may have been prescribed but not collected or taken by the child. In addition, the data potentially includes medication obtained from all sources\u2014GP\u2019s, pharmacies, supermarkets, CAM practitioners, friends, relatives and neighbours. However, potential issues include the accuracy of recall (especially over long periods of 12\u201318\u00a0months, as used at the older ages in this study) and parental knowledge and understanding about the medicines given to their child.\nThese data were collected over the period 1991\u20132000. Between 1994 and 1998, the annual number of children aged 5\u201315\u00a0years prescribed antibacterial drugs ranged from 322 to 402 per 1,000 [12]. This compares favourably with the current study where the number reporting use of antibiotics over the previous 12\u00a0months was 367 per 1,000 at 6.5\u00a0years of age. At 7.5\u00a0years, the rate was lower at 284 per 1,000, but these latter data reflect use of antibiotics between 1997 and 2000, when a substantial decline occurred in the prescribing of antibiotics by general practitioners, in an effort to curb the spread of antibiotic resistance [14]. Children under 5 are known to be the greatest users of antibiotics [10, 12], and the data from this study are consistent with this in that antibiotic use reported at 4.5\u00a0years was considerably greater than at 6.5 and 7.5\u00a0years.\nThe number of children aged 5\u201315\u00a0years prescribed bronchodilators over the period 1994\u20131998 ranged between 135 and 141 per 1,000 boys and 113\u2013118 per 1,000 girls [12]. The prevalence of treated asthma in the same age group was 128\u2013132 per 1,000 boys and 100\u2013106 per 1,000 girls, and around 94% of these are prescribed bronchodilators [12]. In this study, the reported use of all \u2018asthma\u2019 medication for boys and girls combined was 120 per 1,000 at 6.5\u00a0years and 142 per 1,000 at 7.5\u00a0years. Thus, the figures from this study do not suggest a lack of recall. Indeed, at 7.5\u00a0years, the reported use of asthma medication is slightly higher than the national figures. However, as the latter combine data over a 10-year age range (5\u201315\u00a0years) any differences at individual ages may be masked.\nTherefore, as the questionnaire data on use of prescribed antibiotics and anti-asthmatics in this study is in the range expected from prescription data, parentally-reported use of non-prescription medication in this cohort is also likely to be reasonably reliable.\nThe findings suggest that most children use some form of medication from a very young age. Medicines available over-the-counter were widely used, although it was not possible to determine from these data whether they were actually purchased over-the-counter or prescribed. The reason for use was not collected, so it was also not possible to assess to what extent products were appropriately used. For example, paracetamol has been found to be used by parents to calm and settle their children and aid sleep [1, 9]. Some products were used in children younger than the age for which they were licensed. It was not possible to assess general adherence to information given in the patient information leaflets or whether advice was sought prior to using a product outside the specifications, but parents of young children should be encouraged to follow the labelling advice or seek advice from a health professional.\nThe ALSPAC database provides a resource to investigate the use of commonly used medications with later health and development; for example paracetamol, which is widely used and may be associated with development of asthma [4]. Future work may also examine differences in usage by sex of the child and socio-economic status of the parents.","keyphrases":["medication","children","alspac","paediatric drug use"],"prmu":["P","P","P","R"]}
{"id":"J_Autism_Dev_Disord-4-1-2335292","title":"Play Behavior and Attachment in Toddlers with Autism\n","text":"Play helps to develop social skills. Children with autism show deviances in their play behavior that may be associated with delays in their social development. In this study, we investigated manipulative, functional and symbolic play behavior of toddlers with and without autism (mean age: 26.45, SD 5.63). The results showed that the quality of interaction between the child and the caregiver was related to the development of play behavior. In particular, security of attachment was related to better play behavior. When the developmental level of the child is taken into account, the attachment relationship of the child with the caregiver at this young age is a better predictor of the level of play behavior than the child's disorder.\nIntroduction\nPlay is important in the development of a child because it allows children to learn and practice new skills in safe and supportive conditions (Boucher 1999). In play children have the opportunity to develop not only motor skills but also cognitive and social skills (e.g. Pellegrini and Smith 1998).\nPlay shows developmental steps; cognitive development is reflected in, in order, manipulative, functional and symbolic or representational play. First children handle toys in oral and manipulative ways by feeling, licking, sniffing, turning them around, throwing them away, etc. This manipulation creates opportunities to learn about different objects, relations, and about ways to interact and influence the direct environment (Gibson 1988; Piaget 1962; Ruff 1984; Williams 2003). Functional play develops at approximately 14\u00a0months of age (Bretherton 1984), and is defined by Ungerer and Sigman (1981) as \u2018the appropriate use of an object or the conventional association of two or more objects, such as a spoon to feed the doll, or placing a teacup on a saucer\u2019. The child assigns a function to an object that it contains in daily life, even when an object is miniaturized. Around 24\u00a0months of age, symbolic play emerges, although it is difficult to define the point at which play becomes truly \u2018symbolic\u2019 (Jarrold et\u00a0al. 1993). Symbolic play is considered a higher level of play, because it involves pretence, whereas pretence is not necessarily present in functional play.\nThe social part in play development starts with the step from the child\u2019s playing by itself to noticing the play of others. This social aspect develops further by participating in the play of others, which creates the opportunity to deal with \u2018interference\u2019 of others and to develop cooperation skills. Play forms also the context for learning about trust, negotiation and compromise, and with these skills the child has the opportunity to form and maintain friendships (Jordan 2003).\nThe quality of the relationship with the parent may have an impact on motivational aspects of play behavior as well as on the quality of play. A secure relationship with a trusted attachment figure optimizes the opportunity for the child to explore the environment under safe and supportive conditions (Ainsworth 1978; Bowlby 1982). Indeed it has been found that children with secure attachment relationships display more sophisticated, complex and diverse play during interaction with their mother and during solitary play (e.g., Bornstein et\u00a0al. 1996; Fiese 1990; Haight and Miller 1992; O\u2019Connell and Bretherton 1984; Slade 1987; Tamis-LeMonda et\u00a0al. 2002).\nPlay Behavior and Autism\nOne of the core deficits in autism is a severe deficit in social behavior. In children with Pervasive Developmental Disorders (PDD) this is demonstrated in play behavior at various levels. Deviations in play behavior can be detected in the first year of life (Ungerer and Sigman 1981; Van Berckelaer-Onnes 2003) and continue through all phases of play development.\nThe first phase of play development, which involves exploratory\/manipulative behavior of objects, is in children with autism characterized by a number of unusual features. They tend to restrict their play to a limited selection of objects (Van Berckelaer-Onnes 2003), or even an isolated part of an object (Freeman et\u00a0al. 1979). They prefer proximal senses of touch and taste above visual exploration (Williams 2003) and can become intensely preoccupied for long periods of time with non-variable visual examination of just one object (Freeman et\u00a0al. 1979), or non-play (Ruff 1984), which impairs further development of play (Van Berckelaer-Onnes 2003).\nAlthough several studies reported children with autism to produce the same number of functional acts under spontaneous as well as structured conditions (e.g. Baron-Cohen 1987; Van Berckelaer-Onnes 1994; Charman 1997; Lewis and Boucher 1988; Libby et\u00a0al. 1998; Williams et\u00a0al. 2001), it has also been found that children with autism spend significantly less time playing functionally than controls (Lewis and Boucher 1988; Jarrold et\u00a0al. 1996; Sigman and Ungerer 1984), show lower levels of appropriate object use (Freeman et\u00a0al. 1984), less variety in their functional play (Sigman and Ungerer 1984), more repetition (Atlas 1990; Williams et\u00a0al. 2001) and fewer functional acts (e.g. Mundy et\u00a0al. 1990; Sigman and Ungerer 1984; Ungerer and Sigman 1981).\nChildren with autism spectrum disorders (ASD) may experience particularly difficulties in symbolic play. When symbolic play is performed, their play behavior may be more like \u2018learned routine\u2019 rather than spontaneous play (Williams et\u00a0al. 2001). The lack of this particular type of play in the behavioral repertoire of children with autism does however not necessarily imply a specific impairment in their symbolic abilities. It might reflect a more general cognitive or social deficit associated with autism impinging on the whole range of play development (Jarrold et\u00a0al. 1993).\nThis Study\nPlay behavior in children with autism has been studied before, under various circumstances and on different levels. However, most studies involved subjects older than 42\u00a0months of age. The control groups in these studies were mainly subjects matched on mental age (MA), which created substantial differences in chronological age. In this study we investigated play behavior of children with and without ASD, but also of atypically and typically developing controls under the age of 36\u00a0months. Several domains of play behavior were analyzed to investigate differences in play behavior between clinical and non-clinical children, and between clinical children with and without ASD. Observing play behavior at this young age provides the opportunity to detect whether the basic play skills of children with ASD are disturbed, or whether the differences appear at a later age when higher levels of play are expected to be shown. We expected the ASD children to lag behind in their level of play behavior already from their first years of life.\nPlay behavior in children with ASD was also examined in relation to attachment quality. We expected that ASD children with secure attachment relationships would be more playfully engaged and socially involved compared to insecurely attached children with the same disorder. Furthermore, as disorganized attachment is the most insecure type of attachment, it was expected that disorganized children would show more delay in \u2018social\u2019 play behavior compared to children without disorganized attachment.\nMethod\nDiagnostic Assessments\nThis study was part of a study on early screening for autism. For further details regarding recruiting see Dietz et\u00a0al. (2006), and Swinkels et\u00a0al. (2006). The children were recruited between the age of 14 and 36\u00a0months, based on social developmental delay, but final psychiatric diagnosis was obtained at 42\u00a0months. Psychiatric examinations included a series of six visits that were scheduled within a period of 5\u00a0weeks. At each weekly visit, the social and communicative behavior of the child was observed in a small group of young children and their parents. The assessments included a standardized parental interview, developmental history, and the Vineland Social-Emotional Early Childhood Scales (Sparrow et\u00a0al. 1997); the Autism Diagnostic Interview\u2014Revised (ADI\u2014R; Lord et\u00a0al. 1994); standardized behavior observation (Autism Diagnostic Observation Schedule ADOS-G; DiLavore et\u00a0al. 2000), and pediatric examination and medical work-up. On the basis of all available information, and on the basis of clinical judgment, a diagnosis was given by an experienced child psychiatrist. The inter-rater reliability for two diagnostic categories; ASD or other than ASD was calculated. Agreement among three child psychiatrists (HvE, JB, ED) was reached in 92% of 38 cases. Agreement corrected for chance was 0.74 (Cohen\u2019s Kappa). Agreement for all diagnostic categories was reached in 79% of 38 cases. Agreement corrected for chance was 0.67 (Cohen\u2019s Kappa). Diagnostic discrepancies were resolved at a consensus meeting. If appropriate, children and their parents were offered \u201ccare as usual\u201d.\nThe cognitive level of the child was measured with the Mullen Scales of Early Learning (Mullen 1995).\nDiagnostic Groups\nForty-one clinical children participated in this study. At the age of 42\u00a0months they were classified with AD (n\u00a0=\u00a012; mean age 30.25\u00a0months (SD 4.81) and developmental level 51.17 (SD 4.06)), PDD-NOS (n\u00a0=\u00a011; mean age 27.73\u00a0months (SD 7.42) and developmental level 71.36 (SD 15.98)), MR (n\u00a0=\u00a010; mean age 26.50\u00a0months (SD 5.38) and developmental level 55.10 (SD 4.09)) or LD (n\u00a0=\u00a08; mean age 27.75\u00a0months (SD 5.68) and developmental level 83.63 (SD 8.48)). No difference in age was detected between the clinical groups, but developmental level was significantly different, F(3, 40)\u00a0=\u00a023.44, p\u00a0<\u00a0.01.\nBesides the clinical diagnosis, the groups of children were also divided into the group ASD, including children with AD and PDD-NOS (n\u00a0=\u00a023, mean age 29.04\u00a0months (SD\u00a0=\u00a06.18) and a mean developmental level 60.83 (SD\u00a0=\u00a015.19)). The other group (non-ASD) contained children with the developmental disorders MR and LD (n\u00a0=\u00a018, mean age 27.06\u00a0months (SD\u00a0=\u00a05.38), developmental level 67.78 (SD\u00a0=\u00a015.85)). No differences in age and developmental level were detected between clinical children with and without ASD.\nWe included two control groups. One control group contained children who were referred to the hospital due to doubt about the development (AC (Atypical Controls), n\u00a0=\u00a016). Clinical investigation, however, showed that these children were free from clinical diagnoses. The other control group recruited through well-baby clinics contained typically developing children (NC (normal controls), n\u00a0=\u00a016). Based on parental reports and observations of the psychologists, these children were free from any child psychiatric disorder.\nThe group of children with atypical development was younger (AC; M\u00a0=\u00a020.50, SD\u00a0=\u00a03.03) compared to the typical developing control group (NC; M\u00a0=\u00a028.00, SD\u00a0=\u00a01.75), t\u00a0=\u00a0\u22128.57, p\u00a0<\u00a0.01. Although the children in the atypical control group were not showing severe developmental delays, their overall developmental level was lower (AC; M\u00a0=\u00a085.00, SD\u00a0=\u00a010.46) compared to the typically developing control group (NC; M\u00a0=\u00a098.44, SD\u00a0=\u00a012.18) t\u00a0=\u00a0\u22123.35, p\u00a0<\u00a0.01. However, no significant differences were detected in the play behavior of the both control groups.\nDescriptive characteristics of the children are presented in Table\u00a01.\nTable\u00a01Characteristics of the various diagnostic groupsADPDDNOSMRLDACNCM (SD)M (SD)M (SD)M (SD)M (SD)M (SD)Total12111081616Boy\/Girl8\/48\/38\/28\/013\/36\/10Age30.25 (4.81)27.73 (7.42)26.50 (5.38)27.75 (5.68)20.50 (3.03)28.00 (1.75)Dev. level51.17 (4.06)71.36 (15.98)55.10 (4.09)83.63 (8.48)85.00 (10.46)98.44 (12.18)Aut. Symp.36.25 (5.99)21.55 (14.19)23.30 (9.86)7.38 (2.97)11.13 (6.85)Security of Attachment\u22121.96 (3.03)1.06 (3.34)\u22121.28 (3.57)2.19 (2.52)1.54 (2.32)2.17 (1.86)Disorg. of Attachment5.17 (2.95)2.82 (2.64)4.00 (3.04)2.00 (2.33)2.40 (2.41)1.50 (1.10)Attachment classification\u00a0\u00a0\u00a0\u00a0Insecure-avoidant212000\u00a0\u00a0\u00a0\u00a0Secure3627613\u00a0\u00a0\u00a0\u00a0Insecure-ambivalent022023\u00a0\u00a0\u00a0\u00a0Disorganized723120\nMeasures\nStrange Situation Procedure\nAinsworth (Ainsworth et\u00a0al. 1978) developed the SSP to observe the attachment behavior of the child towards the mother in a standardized and stressful laboratory setting. The SSP was coded by two trained observers (SS & MBK), who were blind for the diagnoses of the children. Agreement for the four attachment classifications (n\u00a0=\u00a028) corrected for chance was .74 (Cohen\u2019s Kappa). Besides the attachment classifications, we also used the simplified Richters et\u00a0al. (1988) algorithm to compute continuous scores for attachment security (Van IJzendoorn and Kroonenberg 1990) on the basis of the interactive SSP scale scores for proximity seeking, contact maintaining, resistance and avoidance. Disorganization was coded using the Main and Solomon (1990) 9-point rating scale for disorganized\/disoriented attachment.\nPlay Behavior\nPlay behavior was observed according to protocol. The child and the mother were left in the room for ten minutes. The mother was instructed not to stimulate the child to play but to join when the child asked for cooperation in this free play situation. All children received the same set of toys.\nVideotapes were coded by means of \u2018The Observer\u2019 (Noldus 1991) using an ethogram developed to analyze the play behavior of 2-year-old children, who cannot speak or speak very little. The behaviors mouthing, manipulative play, exploration, functional play, representational play 1 and 2, container play, grouping and stacking 1 and 2 were defined by Largo and Howard (1979). Doll directed play and relational play were derived from Williams et\u00a0al. (2001). Fenson et\u00a0al. (1976) provided descriptions of the behaviors symbolic acts and banging. From Leslie\u2019s (1987) definitions of symbolic play, only subject substitution was used.\nThe various play behaviors were categorized in manipulative, functional or symbolic play, and these variables were used in the analyses. The amount of time that a child spent actually playing was calculated as the percentage of time the child played of the total time of the play session. The amounts of time the child performed manipulative, functional and\/or symbolic play, were calculated as percentages of the time spent playing. When the child did not play, it would typically show other behaviors such as sitting passively. Reliability among the three coders for play behavior was based on 50% of the videotapes. Agreement was reached in 92% of 38 cases. Mean agreement corrected for chance was 0.74 (Cohen\u2019s Kappa).\nThe variable \u2018level of play\u2019 was calculated based on the three different levels of play behavior; manipulative, functional and symbolic play. Durations of the three kinds of play behavior were included in the calculation with the following formula: ((1 x duration of manipulative play)\u00a0+\u00a0(2 x duration of functional play)\u00a0+\u00a0(3 x duration of symbolic play)) \/ total duration of play. Differential weights were thus assigned to the social and cognitive levels of play. The variable \u2018change toys\u2019 was based on the frequency per minute the child initiated play with another toy. The preference of toys was measured by calculating which toys were preferred most during play.\nCorrelations between play behavior, child characteristics and attachment related variables are presented in Table\u00a02.\nTable\u00a02Correlations between play, characteristics of children and attachment related variablesAge Developmental levelAttachment security Attachment disorganizationManipulative playFunctional playSymbolic playLevel of playDuration of playNumber of autistic characteristics.23\u2212.66**\u2212.47**\u2212.41**.28*\u2212.20\u2212.19\u2212.23\u2212.08Age in months\u2013\u2212.21.01\u2212.00\u2212.02.06.03.08.07Developmental level\u2013.43**\u2212.44**\u2212.33**.30*.21.35**.19Attachment security\u2013\u2212.49**\u2212.00.08.09.14.14Attachment disorganization\u2013.05\u2212.23\u2212.09\u2212.30*\u2212.30*Manipulative play\u2013\u2212.59*\u2212.15\u2212.38**.08Functional play\u2013\u2212.20.78**.66**Symbolic play\u2013.37**.10Level of play\u2013.87***Correlation is significant at the .05 level**Correlation is significant at the .01 level\nAnalyses\nPlay Behavior and Clinical Diagnoses\nNo gender differences were found for the variables \u2018duration of play\u2019, \u2018manipulative\u2019, \u2018functional\u2019 and \u2018symbolic\u2019 play, the \u2018overall level of play\u2019 and \u2018change toys\u2019. Analyses started with an overall analysis (ANOVA) for all groups, taking differences in developmental level and age into account. Next, differences between the clinical groups with and without ASD were analysed. Preference for toys was investigated using ANOVA for the whole sample, taking differences in developmental level and age into account, and for clinical children with and without ASD.\nContribution of Attachment\nTo examine whether security of attachment and attachment disorganization contributed to differences in play behavior and preference for toys, analyses with attachment were performed overall, in clinical children and in the group of children with ASD. Play behaviors and preference of toys of children with and without secure attachment, and children with and without disorganized attachment were analyzed.\nResults\nDuration of Play Behavior\nNo differences were detected for duration of play time after controlling for difference in age and developmental level (F(5, 72)\u00a0=\u00a01.87, p\u00a0=\u00a0.11). Also, no differences were detected between clinical children with and without ASD (t\u00a0=\u00a0\u22121.09, p\u00a0=\u00a0.29). Mean values for the play variables are presented in Table\u00a03.\nTable\u00a03Duration, level and type of play, and change of toys of the various diagnostic groupsDuration of play (%time)Level of playChanging toys (freq\/min)nM (SD)M (SD)M (SD)AD 1257.32 (22.24)15.66 (7.29).04 (.01)PDDNOS 1168.19 (12.61)20.00 (5.05).05 (.02)MR 1070.20 (10.73)18.47 (5.03).05 (.02)LD 866.38 (22.45)20.96 (8.74).04 (.02)AC 1669.50 (15.03)21.47 (5.89).05 (.02)NC 1677.19 (15.67)24.00 (5.34).05 (.02)Manipulative playFunctional playSymbolic playnM (SD)M (SD)M (SD)AD 1222.86 (20.68)32.28 (20.62)2.19 (7.35)PDDNOS 1124.90 (18.32)34.74 (19.55)8.54 (12.43)MR 1032.38 (18.65)35.01 (21.32)2.81 (5.08)LD 817.91 (10.05)37.53 (26.57)10.93 (10.99)AC 1614.66 (9.33)50.36 (20.48)4.49 (7.45)NC 1615.62 (14.95)56.33 (21.61)5.25 (7.55)\nManipulative, Functional and Symbolic Play\nOverall analysis showed no differences for percentage of time actually spent on manipulative (F(5, 72)\u00a0=\u00a01.00, p\u00a0=\u00a0.43), functional (F(5, 72)\u00a0=\u00a02.08, p\u00a0=\u00a0.08), or symbolic play (F(5, 72)\u00a0=\u00a01.32, p\u00a0=\u00a0.27) when differences in age and developmental level were taken into account. Mean values of the percentage of time for the three different forms of play are presented in Table\u00a03. Neither were differences detected for manipulative play between clinical children with and without ASD (t\u00a0=\u00a0\u2212.38, p\u00a0=\u00a0.71), for functional play between clinical children with and without ASD (t\u00a0=\u00a0\u2212.39, p\u00a0=\u00a0.70), and for symbolic play between clinical children with and without ASD (t\u00a0=\u00a0\u2212.39, p\u00a0=\u00a0.70), when differences in age and developmental level were taken into account. Mean values of the percentage of time for the three different forms of play for clinical children with and without ASD are presented in Table\u00a04.\nTable\u00a04Play behaviors of children with and without ASDDuration of play (%time)Level of playChanging toys (freq\/min)nM (SD)M (SD)M (SD)ASD 2362.52 (18.72)17.74 (6.56).04 (.02)Non-ASD 1868.50 (16.50)19.58 (6.82).04 (.02)Manipulative play (%time)Functional play (%time)Symbolic play (%time)nM (SD)M (SD)M (SD)ASD 2323.84 (19.17)33.46 (19.96)5.23 (10.38)Non-ASD 1825.95 (16.75)36.13 (23.09)6.42 (8.98)\nLevel of Play\nLevel of play, taking differences in developmental level and age into account, did not show any differences between the different groups (F(5, 72)\u00a0=\u00a01.20, p\u00a0=\u00a0.32). Mean values of the level of play are presented in Table\u00a03. Moreover, no differences were detected between clinical children with and without ASD either (t\u00a0=\u00a0\u2212.87, p\u00a0=\u00a0.39), see Table\u00a04.\nPreference for Toys and Change of toys\nAnalyses were performed for the duration of play that the children were involved with the toys \u2018car\u2019, \u2018doll\u2019, \u2018puzzle\u2019, \u2018daily utensils\u2019, bricks\u2019, \u2018book\u2019 and \u2018ball\u2019, to analyze whether there were any differences in preference for toys. Again, differences in age and developmental level were taken into account. An overall difference was found for playing with \u2018daily utensils\u2019 F(5, 72)\u00a0=\u00a02.53, p\u00a0=\u00a0.04. Children with AD spent significant less time playing with daily utensils. Overall differences were also found for reading a book, F(5, 72)\u00a0=\u00a02.61, p\u00a0=\u00a0.03 and playing with a puzzle F(5, 72)\u00a0=\u00a03.11, p\u00a0=\u00a0.01. No differences were detected between children with and without ASD for the time spent playing with daily utensils and playing with a puzzle. However, children with ASD spent significantly less time (M\u00a0=\u00a02.12, SD\u00a0=\u00a04.60) reading a book compared to clinical children without ASD (M\u00a0=\u00a013.11, SD\u00a0=\u00a017.62), t\u00a0=\u00a0\u22122.88, p\u00a0<\u00a0.01.\nNo differences were detected for changing toys in the overall group, taking differences in age and developmental level into account F(5, 72)\u00a0=\u00a0.47, p\u00a0=\u00a0.80. Mean values of the frequency of changing toys are presented in Table\u00a03. There were also no differences between the clinical children with and without ASD (t\u00a0=\u00a0.37, p\u00a0=\u00a0.72), see Table\u00a04.\nQuality of Attachment and Play\nChildren with a secure attachment showed higher levels of play (M\u00a0=\u00a021.74, SD\u00a0=\u00a06.32) compared to children without secure attachment (M\u00a0=\u00a018.24, SD\u00a0=\u00a06.61), t\u00a0=\u00a0\u22122.18, p\u00a0=\u00a0.03. However, because of the higher percentages of secure attachment in children without a clinical disorder, the difference was also tested in the group of children with clinical diagnoses. In the clinical group children with secure attachment (M\u00a0=\u00a020.88, SD\u00a0=\u00a07.02) showed significantly higher levels of play than children without a secure attachment relationship (M\u00a0=\u00a016.35, SD\u00a0=\u00a05.73) as well, t\u00a0=\u00a0\u22122.20, p\u00a0=\u00a0.04.\nChildren with ASD who were securely attached spent more time playing symbolic play compared to children with ASD without a secure attachment (t\u00a0=\u00a0\u22122.37, p\u00a0=\u00a0.03). Level of play was also higher in children with ASD with a secure attachment relationship compared to children with ASD without a secure attachment relationship (t\u00a0=\u00a0\u22123.27, p\u00a0<\u00a0.01) (Fig.\u00a01). Moreover, children with ASD with a secure attachment relationship spent more time actually playing compared to children with ASD without a secure attachment relationship (t\u00a0=\u00a0\u22122.74, p\u00a0=\u00a0.01). The differences within the group of children with ASD with and without secure attachment relationships remained significant after taking differences in age and developmental level into account; for symbolic play F (1, 23)\u00a0=\u00a04.47, p\u00a0=\u00a0.05, for level of play F (1, 23)\u00a0=\u00a08.88, p\u00a0<\u00a0.01, and for duration of play F (1, 23)\u00a0=\u00a07.19, p\u00a0=\u00a0.01, see Table\u00a05.\nFig.\u00a01Secure and disorganized attachment and mean level of play in children with ASDTable\u00a05Play behaviors of children with ASD with and without secure and disorganized attachmentDuration of play (%time)Level of playChanging toys (freq\/min)nM (SD)M (SD)M (SD)ASD-nonB 1454.99 (20.16)a*14.78 (5.65)a*.04 (.02)ASD-B 974.22 (7.09)a*22.33 (5.25)a*.05 (.02)ASD-nonD 1470.74 (12.46)b*20.24 (5.38)b* .05 (.02)ASD-D 949.73 (20.22)b*13.84 (6.58)b*.04 (.01)Manipulative playFunctional playSymbolic playnM (SD)M (SD)M (SD)ASD-nonB 1422.77 (18.59)30.74 (19.82)1.48 (4.40)a**ASD-B 925.50 (21.08)37.67 (19.87)11.05 (14.22)a**ASD-nonD 1427.40 (21.07)35.98 (16.78)7.36 (12.31)ASD-D 918.30 (15.25)29.53 (24.09)1.90 (5.44)aASD-B versus ASD non-BbASD-D versus ASD non-D*p\u00a0<\u00a0.05, **p\u00a0<\u00a0.01\nDisorganized Attachment Relationship and Play\nChildren with a disorganized attachment classification showed lower levels of play (M\u00a0=\u00a016.39, SD\u00a0=\u00a06.83) than children without a disorganized attachment classification (M\u00a0=\u00a021.32, SD\u00a0=\u00a06.20), t\u00a0=\u00a02.51, p\u00a0=\u00a0.02. Within the clinical group no difference was shown for level of play of children with and without disorganized attachment. However, children without a disorganized attachment relationship spent more time playing (M\u00a0=\u00a069.29, SD\u00a0=\u00a015.67) compared to children with a disorganized attachment classification (M\u00a0=\u00a055.50, SD\u00a0=\u00a019.28) t\u00a0=\u00a02.25, p\u00a0=\u00a0.04.\nChildren with ASD with disorganized attachment showed lower levels of playing than children with ASD without disorganized attachment (t\u00a0=\u00a02.44, p\u00a0=\u00a0.03), see Fig.\u00a01. This difference remained significant after taking differences in age and developmental level into account, F (1, 23)\u00a0=\u00a05.29, p\u00a0=\u00a0.03. Moreover, children with ASD without disorganized attachment spent more time playing compared to children with ASD with disorganized attachment (t\u00a0=\u00a011.94, p\u00a0=\u00a0.02). Again, the difference remained significant after taking differences in age and developmental level into account, F (1, 23)\u00a0=\u00a09.40, p\u00a0<\u00a0.01 (see Table\u00a05).\nChildren with and without secure or disorganized attachment relationships did not differ on preference for toys.\nDiscussion\nOur findings highlight the importance of attachment in the development of play of children with autism and other developmental disorders. Attachment quality explained play behavior regardless of the clinical status of the children. Taking developmental level of the child into account, we found that children with a secure attachment relationship spent more time playing. They also showed a higher level of play and more symbolic play behavior. Children with a disorganized attachment relationship spent less time playing, and within the group of children with ASD disorganized attachment was related to lower levels of play.\nOur earliest understanding about the world and our own actions may have a social rather that a cognitive origin (Hobson 2002; Jordan 2003; Vygotsky 1978). Social deficits belong to the core deficits of children with autism. Nevertheless, children with autism are able to develop a secure attachment relationship with the primary caregiver (Naber et\u00a0al. 2007; Rutgers et\u00a0al. 2004), which contributes to better play outcomes in children with autism. We indeed found that children with secure attachment relationships showed more exploration and higher levels of play, whereas children with disorganized attachment showed less exploration, even after controlling for developmental level. Especially in children with autism the quality of attachment relationship was associated with the development of \u2018social\u2019 play.\nUnexpectedly, for duration of play, level of play or changing toys no differences were detected between children with and without ASD, or even between children with and without a developmental disorder, after controlling for developmental level. Nevertheless, similar to Williams et\u00a0al. (2001), we found that children with autism preferred toys that were based on \u2018simpler\u2019 play behavior. Children with autism did not prefer daily utensils or books to play with.\nSeveral explanations may account for the absence of a difference in most play variables. First, we used a free play situation with the mother. Although the parents were instructed only to follow the child leads and not to structure the setting, the presence of the parent may have motivated the child to continue playing. Second, the lower levels of play behavior typical for this young age period may still be within the reach of children with autism. At a later age, when the children are expected to show symbolic play behavior at higher levels, differences in the amount or quality of symbolic play may emerge. Third, play behavior in children with autism may show delays compared to other children (Beyer and Gammeltoft 2000; Howlin 1986; Lord 1984; Lord and Magill 1989; Wolfberg 1999), but as Jarrold (2003) pointed out, the absence of pretend\/symbolic play in children with autism may result from assessing individuals who are (mentally) too young to be expected to show pretend\/symbolic play. In the current young age group few children showed much symbolic play, and no difference emerged. Fourth, we used ethological measures to observe play behavior in an objective manner, but the context and coherence or patterning of the behavior was not taken into account. A more holistic approach with global ratings of play behavior may uncover differences between children with and without autism.\nLimitations of the Study\nAlthough studies of play behavior are required at an early age to get more insight into its development, the young age of the children is also a limitation because symbolic play was not yet in reach of many subjects in the current study. However, retrospective parental reports and screening studies mention differences in play behavior already at this young age. Longitudinal studies are needed to follow the development of these children\u2019s play across time to see whether differences in play behavior arise at a later stage, and to examine whether the effects of a positive attachment relationship are lasting.\nConclusions and Future Research\nAs pointed out in a review of Jarrold et\u00a0al. (1993), studies that indicate a lack of symbolic play behavior in children with autism cannot be seen as convincing proof of the inability of the child to produce this type of play, because MA matched controls would be needed. In our study, we matched the clinical groups both on chronological and on MA. Due to this matching we were able to compare play behavior of children with and without autism. We found no differences in play between children with and without ASD. This may be due to the mental and chronological age of the toddlers included in our study; the lower levels of play behavior typical for this age period may still be within the reach of children with autism. At a later age, children with autism may start to lag behind which might be shown in delayed and infrequent occurrence of symbolic play. We hope to follow-up the current sample to test this interpretation.\nWhat we did find were striking differences between children with and without secure or disorganized attachment relationships. The quality of the parent-child relationship appears to contribute substantially to the development of play in young children regardless of their autistic symptoms. Intervention studies based on play behavior have shown to positively contribute to the development of play in children with ASD during the intervention period. However, no long-term effects have been documented. Our findings show the importance of attachment and suggest that interventions focusing on the improvement of play behavior of children with autism should also focus on enhancing the quality of the attachment relationship. The early intervention study with PDD children by Mahoney and Perales (2005) explored this approach in stimulating cognitive, communicative and socio-emotional functioning. Attachment-based video-feedback intervention has been proven to be effective in typically developing children (Bakermans-Kranenburg et\u00a0al. 2003; Juffer et\u00a0al. 2007) and this approach might not only in the short run but also long-term lead to improvement of quality of attachment as well as level of play behavior in children with autism.","keyphrases":["play behavior","attachment","autism","developmental disorder"],"prmu":["P","P","P","P"]}
{"id":"J_Assoc_Res_Otolaryngol-4-1-2413083","title":"Higher Sensitivity of Human Auditory Nerve Fibers to Positive Electrical Currents\n","text":"Most contemporary cochlear implants (CIs) stimulate the auditory nerve with trains of amplitude-modulated, symmetric biphasic pulses. Although both polarities of a pulse can depolarize the nerve fibers and generate action potentials, it remains unknown which of the two (positive or negative) phases has the stronger effect. Understanding the effects of pulse polarity will help to optimize the stimulation protocols and to deliver the most relevant information to the implant listeners. Animal experiments have shown that cathodic (negative) current flows are more effective than anodic (positive) ones in eliciting neural responses, and this finding has motivated the development of novel speech-processing algorithms. In this study, we show electrophysiologically and psychophysically that the human auditory system exhibits the opposite pattern, being more sensitive to anodic stimulation. We measured electrically evoked compound action potentials in CI listeners for phase-separated pulses, allowing us to tease out the responses to each of the two opposite-polarity phases. At an equal stimulus level, the anodic phase yielded the larger response. Furthermore, a measure of psychophysical masking patterns revealed that this polarity difference was still present at higher levels of the auditory system and was therefore not solely due to antidromic propagation of the neural response. This finding may relate to a particular orientation of the nerve fibers relative to the electrode or to a substantial degeneration and demyelination of the peripheral processes. Potential applications to improve CI speech-processing strategies are discussed.\nIntroduction\nPolarity sensitivity of auditory nerve fibers has been extensively studied in animal and computational models using both monophasic and biphasic stimuli. Responses to monophasic cathodic pulses show, in general, lower thresholds and longer latencies than anodic responses (Miller et al. 1998, 1999a, 2004). Similarly, the most effective phase of sinusoidal stimuli was shown to be the cathodic phase in single-fiber recordings from the cat (Hartmann et al. 1984). However, polarity sensitivity may also depend on the electrode position relative to the fibers and on the nerve morphology (Ranck 1975; McIntyre and Grill 1999; Rubinstein 1993; Rattay 1999; Rattay et al. 2001a). If so, then, the findings obtained in the vast majority of animal experiments may not apply to human cochlear implant (CI) listeners (Miller et al. 1998).\nIn human CI stimulation, charge-balanced stimuli are required to prevent damaging electrochemical reactions (Brummer and Turner 1977). Consequently, the effect of absolute polarity, which is usually studied using monophasic pulses, remains largely unknown in humans. Asymmetric pulses, where the second phase is lower in amplitude and longer in duration than the first, have provided a way to study polarity sensitivity in CI users. In such pulses, the contribution of the short, high-amplitude phase is increased at the expense of the long, low-amplitude one while maintaining the charge-balancing requirement. We recently showed that inverting the polarity of an asymmetric pulse stimulus yielded differences in most comfortable levels (MCLs) of CI users, giving lower MCLs when the short, high-amplitude phase was anodic than when it was cathodic (Macherey et al. 2006). Because short pulses require less charge to evoke the same loudness as longer ones (at least for phase durations less than approximately 500\u00a0\u03bcs; Moon et al. 1993; Macherey et al. 2006), it is possible that loudness was determined primarily by the higher-amplitude phase of our asymmetric pulses. This result, then, suggests that the human auditory nerve might be more sensitive to anodic stimulation\u2014the opposite to what one expects from animal measures of auditory-nerve activity (Hartmann et al. 1984; Miller et al. 1998, 1999a, 2004). The following series of experiments directly test this hypothesis in eight CI users, using both electrophysiological and psychophysical measures. The experimental design relies on the characteristics of the auditory nerve fibers which, after a stimulation pulse, undergo a period of absolute refractoriness, lasting approximately 400\u00a0\u03bcs, followed by a progressive recovery (Stypulkowski and van den Honert 1984; Miller et al. 2001; Morsnowski et al. 2006). Experiment 1 provides an insight into the morphology of anodic and cathodic phase responses. Experiments 2 and 3 measure the masking effectiveness of each individual polarity phase using electrophysiological and psychophysical masking, respectively.\nGeneral methods\nSubjects\nEight users of the CII or HiRes 90k CIs, manufactured by Advanced Bionics, participated in this series of electrophysiological (experiment 1 and 2) and psychophysical (experiment 3) experiments. The subjects\u2019 biographical data are shown in Table\u00a01. Testing was approved by the Cambridge and Leuven Local Research Ethics Committees and informed consent was obtained from all subjects.\nTABLE\u00a01Biographical data of the subjects, including age, duration of deafness (DD) and duration of CI use (CI), in years and etiologySubjectAge (years)DD (years)CI (years)EtiologyS15544GeneticS25113MeningitisS370<23Unknown suddenS43127Unknown progressiveS566>61Unknown progressiveS66032Unknown progressiveS761183UnknownS85214Meningitis\nElectrically evoked compound action potential recordings and psychophysical testing\nMost contemporary CI systems allow the direct recording of the auditory nerve response, known as the electrically evoked compound action potential (ECAP). This is a noninvasive technique that consists of recording the intracochlear potential evolution after a stimulation pulse. To minimize electrical artifacts, we used the \u201cmasker\u2013probe paradigm\u201d (Brown et al. 1990), which relies on the fact that the neural response to a \u201cprobe\u201d pulse is reduced by an immediately preceding \u201cmasker.\u201d By presenting the probe and masker either alone or in combination, it is possible to derive the neural response to the probe with the artifact \u201csubtracted out\u201d (cf. Fig.\u00a01). In experiments 1 and 2, ECAPs were measured using several combinations of masker and probe. In all cases, masker and probe were presented in monopolar mode, on an electrode located in the middle of the array (electrode 9) with reference to the case (extracochlear) electrode of the implant. The responses were recorded using electrode 7 (approximately 2\u00a0mm apical to electrode 9) with reference to the ring ground electrode for S5 and the case electrode for all other subjects and were averaged over 128 sweeps. In experiment 3, psychophysical masking patterns were measured using the APEX experimental platform and the BEDCS software (cf. Macherey et al. 2006; Laneau et al. 2005). Masker and probe were also presented in monopolar mode, the masker on electrode 9 and the probe on an adjacent electrode (8 or 10).\nFIG.\u00a01.Description of the masker\u2013probe paradigm for artifact cancellation (Brown et al. 1990). Four traces are recorded: masker alone (1), masker and probe (2), probe alone (3), and zero stimulation (4) to correct for any DC offset. This method is based on the assumption that the nerve fibers are in their absolute refractory state when the probe is presented after the masker (2). The subtraction (1) + (3)\u00a0\u2212(2)\u00a0\u2212\u2009(4) therefore yields the probe neural response with the artifact removed.\nExperiments\nExperiment 1: ECAP responses to anodic and cathodic phases\nRationale and methods\nIn experiment 1, we recorded ECAPs from seven subjects (S1\u2013S7). The masker was a cathodic-first, symmetric biphasic pulse with a 32-\u03bcs phase duration presented at a comfortable level, determined subjectively for each subject before testing. The probes were either anodic-first (Fig.\u00a02A) or cathodic-first (Fig.\u00a02B) asymmetric waveforms with a phase separation of 2.9\u00a0ms. The first (short) phase of the pulse was 32-\u03bcs long, and the second (long) phase was eight times longer and lower in amplitude. The 2.9-ms phase separation present in the probe allowed us to measure the neural response to the first phase independently of the second. The two probes had the same level (between most comfortable and comfortably loud) and were each presented 500\u00a0\u03bcs after the end of the masker. Additionally, to differentiate neural responses from imperfectly cancelled stimulation artifacts, we recorded, in four of the subjects (S3\u2013S6), the responses to several probe levels, spanning the entire electrical dynamic range.\nFIG.\u00a02.ECAP responses to anodic and cathodic phases. A Masker (thick line) and probe (thin line) used to measure the anodic phase response. B Masker and probe used to measure the cathodic-phase response. C and D ECAPs evoked by the first phases of anodic-first (C) and cathodic-first (D) phase-separated asymmetric probes. The time reference corresponds to the onset of the probe. The second phase is not displayed as it occurs 2.9\u00a0ms after the first one. The top panels show an example of a level series obtained with subject S5. The different curves correspond to probe levels ranging from a soft (320\u00a0\u03bcA) to a comfortably loud level (576\u00a0\u03bcA) by steps of 64\u00a0\u03bcA, with the size of the fluctuations increasing monotonically with increasing current level. The other panels show the responses to the highest probe levels for all the other subjects. The N1 peak of the anodic response occurs, on average, 290\u00a0\u03bcs after the onset of the probe.\nResults\nAn example of a level series is shown in the top panels of Figure 2C (anodic phase) and D (cathodic phase) for S5. The first narrow, short-latency peak observed for both anodic-first and cathodic-first probes almost certainly reflects a residual artifact because, for most subjects (with the exception of S2 and S6), its polarity is dependent on the probe polarity (positive and negative for anodic-first and cathodic-first probes, respectively), and its latency is similar for the two probes. For all subjects, the response to the anodic phase shows a biphasic shape with a first-negative peak N1 followed by a less robust positive peak P2. Only one (S4) of the seven subjects showed a measurable response to the cathodic phase, and only at the highest level of the dynamic range. For this subject, the latency of the response was longer for the cathodic-first (420\u00a0\u03bcs) than for the anodic-first (270\u00a0\u03bcs) probe, consistent with reports of animal auditory excitation (Miller et al. 1998, 1999a, 2004). Most of these animal data were, however, obtained using a recording electrode located at the nerve trunk, whereas our data reflect intracochlear recordings. The position of the recording electrode may have an important influence on the response latencies, and we will further consider this parameter in \u201cDiscussion.\u201d For now, it is worth noting that the major discrepancy with common animal data was that the amplitude of the response was, in our case, always greater for the anodic phase.\nExperiment 2: masking effectiveness of anodic and cathodic phases\u2014ECAP measures\nRationale and methods\nThe reliability of the masker\u2013probe paradigm strongly depends on the effectiveness of the masker, as the neural response obtained represents the subtraction of the masked response from the unmasked one. Instead of reflecting different neural responses, the anodic\/cathodic amplitude difference obtained in experiment 1 may relate to the fact that the biphasic masker did not effectively mask the cathodic phase. This would be expected if, for example, the excitation sites for the biphasic masker and the cathodic probe were the central axon and the peripheral process, respectively. The peripheral process may, in such a case, not be driven in its refractory state by the masker, and the masker\u2013probe paradigm would consequently subtract out the neural response. We performed, in experiment 2, another set of ECAP measures in six subjects (S1\u2013S6) to verify that the anodic phase was also a more effective masker than the cathodic phase. Unlike the previous experiment, we introduced a 2.9-ms phase separation in the symmetric biphasic masker and assumed that the probe would be mainly masked by the masker\u2019s second phase (denoted as the masking phase). We recorded the responses to anodic-first and cathodic-first symmetric biphasic probes with no phase separation in the two conditions, \u201canodic masking\u201d (Fig.\u00a03A) and \u201ccathodic masking\u201d (Fig.\u00a03B) phase, and also in a third \u201cbiphasic masking\u201d (Fig.\u00a03C) condition. This last condition, where the masker was a cathodic-first, symmetric biphasic pulse with no phase separation, was performed for two reasons. First, because this protocol is routinely used in clinics, the morphology of the resulting ECAP response is well documented. It usually consists of an N1\u2013P2 complex (Brown et al. 1990), similar to the response obtained in experiment 1 for the anodic phase. Second, it allowed us to compare the latencies of the probes\u2019 responses. If the anodic phase is the most excitatory phase of a biphasic pulse, we would expect the response to show a longer latency when the anodic phase is the second phase of the probe than when it is the first.\nFIG.\u00a03.Combinations of maskers (thick lines) and probes (thin lines) used in experiment 2. A Anodic masking condition. B Cathodic masking condition. C Biphasic masking condition. For each masking condition, two different symmetric biphasic probes were used: an anodic-first and a cathodic-first.\nResults\nFor all masking conditions, a large uncancelled artifact was present at the probe onsets. Figure 4 shows examples of these artifacts obtained with S5 using biphasic cathodic-first (A) and anodic-first (B) probes in the anodic masking condition. The size of this artifact did not depend on the probe level and was still present at subthreshold levels (thick lines of Fig.\u00a04). For means of clarity and because this uncancelled artifact was clearly distinct from the neural response, subsequent figures will only show the neural response part of the recordings. Note that an advantage of the present approach is that it compares the effects of different maskers, whereas the artifact occurred near the onset of the probe.\nFIG.\u00a04.Examples of uncancelled artifacts obtained in experiment 2 for S5. ECAP responses to cathodic-first (A) and anodic-first (B) biphasic probes in the anodic-masking condition at subthreshold (thick line) and suprathreshold (thin line) levels.\nFigure 5A shows the anodic-first (thick line) and cathodic-first (thin line) probe responses in the biphasic masking condition for S1. The response latency of the N1 peak was shorter for the anodic-first probe, consistent with the neural activation being elicited primarily by the anodic phase. All subjects demonstrated this trend with a latency difference ranging from 0 to 80\u00a0\u03bcs (Fig.\u00a05B), opposite to results obtained in the guinea pig (Klop et al. 2004). A t test performed on the data from the six subjects revealed that this difference was significant (df\u2009=\u20095, p\u2009=\u20090.011). Apart from this latency difference, the responses to anodic-first and cathodic-first probes had similar amplitudes. To further improve artifact cancellation, the two responses were averaged for data analysis.\nFIG.\u00a05.Masking effectiveness of anodic and cathodic phases. A Example of ECAP recordings obtained with subject S1 for an anodic-first (thick line) and a cathodic-first (thin line) biphasic probe in the biphasic masking condition. The response latency is shorter for the anodic-first probe. B Latency of the cathodic-first probe response vs. latency of the anodic-first probe response. For each subject, the two probes were presented at an identical level (comfortably loud) and also at the same level as the biphasic masker. C, D, and E Example of ECAPs evoked by a symmetric biphasic probe with no phase separation in the three masker conditions: anodic masking (C), cathodic masking (D), and biphasic masking (E) for subject S1. Each trace corresponds to the average of the anodic-first and cathodic-first biphasic probe responses. In the cathodic masking condition, the neural response was sometimes not clearly measurable. In D, for example, there is no excursion of the trace in the negative portion of the graph. Nevertheless, the recording still shows a voltage increase in the same region where the probe neural response occurs. The amplitude of the probe response was, in this case, assumed to be the magnitude of this increase. F Summary of ECAP amplitudes obtained in the anodic-masking (black bars), cathodic-masking (white bars), and biphasic-masking (gray bars) conditions.\nAn example of the averaged responses of S1 is illustrated in Figure 5 for anodic masking (C), cathodic masking (D), and biphasic masking (E). For the six subjects tested, the amplitude of the response (difference between N1 and P2) was greater when the masking phase was anodic than when it was cathodic (Fig.\u00a05F). This confirmed the previous finding that the anodic phase is more effective in driving auditory nerve fibers in their refractory state. Furthermore, the probe responses in the anodic masking and biphasic masking conditions were strikingly similar, suggesting that the anodic phase is the effective phase of a symmetric biphasic pulse. This last observation also argues strongly against the responses being influenced by an uncancelled masker artifact. Note that, although the anodic and cathodic maskers had the same level, and differed only in their leading polarity, the level of the biphasic masker was presented, on average, 2.4\u00a0dB higher to have a loudness similar to that of the other two maskers. This increase in level reflects the influence of phase separation on loudness reported in previous publications (McKay and Henshall 2003; Carlyon et al. 2005; van Wieringen et al. 2005; Macherey et al. 2006). For S2 and S6, the amplitude is negative in the cathodic masking case because the subjects exhibited an inverted response (with a positive peak). This may mean that the cathodic phase acts as a facilitator and not as a masker for these specific subjects, possibly because of residual potential summation (Miller et al. 1997; De Balthasar et al. 2003).\nExperiment 3: masking effectiveness of anodic and cathodic phases\u2014psychophysical measures\nRationale and methods\nBecause ECAPs in CIs can only be measured intracochlearly and not directly on the nerve trunk, the recordings potentially contain the neural response traveling not only toward the central auditory system (orthodromically) but also in the opposite direction, toward the fibers\u2019 periphery (antidromically; Miller et al. 2004). It is possible that this phenomenon occurred and that the larger ECAP amplitude of the anodic response obtained in experiment 1 did not reflect a larger response propagating toward the central auditory system (Cartee et al. 2005). Although the results of experiment 2 argue against this hypothesis, a perhaps more direct way to tease out this effect is to perform psychophysical measures, which require perceptual judgments from the subjects, thereby reflecting neural responses propagating centrally. The aim of experiment 3 was to compare the masking effectiveness of each polarity phase psychophysically and also to measure the most effective phase of an asymmetric pulse: Is it the short, high-amplitude or the long, low-amplitude one? We used two asymmetric pulse\u2013train stimuli (anodic- and cathodic-first) with a 6.4-ms phase separation as maskers of a biphasic pulse\u2013train probe presented 1\u00a0ms either after the first or after the second phase of the masker (Fig.\u00a06A). Contrary to experiments 1 and 2 where we were constrained by the limited duration of the recording window, we used a longer phase separation to minimize refractory effects of one phase of the masker over the other. The masker was a 76-pulse per second pulse train, either anodic-first (conditions 1a and b) or cathodic-first (conditions 2a and b) with a short phase of 22\u00a0\u03bcs and a long phase eight times longer and lower in amplitude. Masker and probe had a total duration of 400\u00a0ms and were temporally interleaved, the masker being presented on electrode 9 and the probe on electrode 8, or, in an additional set of measures, on electrode 10. Four subjects (S1\u2013S3 and S8) were asked to detect the anodic-first biphasic probe in the presence of one of the two maskers, in a three-interval, two-alternative forced-choice task where only one interval contained the probe. Feedback was provided and the level of the probe was adaptively varied in a two-down, one-up adaptive procedure that stopped after eight reversals. For subjects S1\u2013S3, the step size was 32\u00a0\u03bcA for the first two and 4\u00a0\u03bcA for the last six reversals. For subject S8, the phase duration of the biphasic probe had to be increased to 43\u00a0\u03bcs to be able to reach a comfortably loud level, and the step sizes were 64 and 8\u00a0\u03bcA, respectively, for the first two and the last six reversals. Each estimate was the average value of the last six reversals. We also performed the same four measures with the polarity of the probe reversed (cathodic-first instead of anodic-first). We did this because, in a previous study (Macherey et al. 2006), we reported the presence of a low-amplitude current flow between the two phases of stimuli with relatively long phase separations (similar to the separation used here). The polarity of this current flow is opposite to the polarity of the preceding stimulus phase and is due to the passive discharge of the implant\u2019s DC-blocking capacitors. This current may influence the detection of the probe by predepolarizing or prehyperpolarizing the neural membrane after each phase. The results obtained in this condition were similar to the ones obtained with the initial stimulus polarity and will therefore not be shown here.\nFIG.\u00a06.Psychophysical masking patterns. A Description of the four experimental conditions. Anodic-first asymmetric masker (thick line) with a phase separation of 6.4\u00a0ms followed by a biphasic probe presented 1\u00a0ms after the end of the short, high-amplitude phase (1a) or 1\u00a0ms after the end of the long, low-amplitude phase (1b) of the masker. Conditions 2a and 2b are identical to 1a and 1b except that the masker is reversed in polarity. B Difference between masked and unmasked threshold for the four conditions (1a, 1b, 2a, and 2b) and the four subjects (S1\u2013S3 and S8) for a probe on electrode 8. The probe dynamic ranges were 6.5, 4.2, 3.9, and 11.1\u00a0dB for S1, S2, S3, and S11, respectively. C Same as B with the probe on electrode 10.\nResults\nThe four CI subjects (S1\u2013S3, S8) were first asked to balance in loudness the two opposite-polarity maskers at their MCL following a procedure described in Macherey et al. (2006). On average, the level of the cathodic masker was adjusted 1.6-dB higher than the anodic one, consistent with the anodic-first pulse being more effective than the cathodic-first one. These levels were used in the following part of the experiment where the subjects had to detect the presence of the biphasic probe in each of the four conditions and also in the unmasked case. Figure 6B shows the threshold shifts (difference between masked and unmasked thresholds) obtained with the probe on electrode 8. For an anodic masker, the short, high-amplitude (anodic) phase produced more masking than the long, low-amplitude (cathodic) phase for all subjects (conditions 1a vs. 1b). No clear pattern was, however, obtained in the case of a cathodic masker (2a vs. 2b), showing across-subject variability. More interestingly, although presented at a lower level, the short, high-amplitude anodic phase (1a) was a more effective masker than the short, high-amplitude cathodic phase (2a). Similar results were obtained with the probe on electrode 10 (Fig.\u00a06C). These observations were corroborated by a two-way repeated measures ANOVA performed on the mean data of all subjects. The treatment factors were the probe channel (8 or 10) and the probe condition. First, there was a significant effect of the probe condition (F(3,9)\u2009=\u20095.01, p\u2009=\u20090.026) but no effect of the probe channel (F(1,3)\u2009=\u20090.52, p\u2009=\u20090.52) or of the interaction factor (F(2.91,8.73)\u2009=\u20090.22, p\u2009=\u20090.88). Second, pairwise comparisons revealed that the short, high-amplitude anodic phase was a better masker than the long, low-amplitude cathodic phase (1a\u2009>\u20091b, p\u2009=\u20090.037) and also than the short, high-amplitude cathodic phase (1a\u2009>\u20092a, p\u2009=\u20090.008). No significant difference was, however, found between the short cathodic and the long anodic phases (p\u2009>\u20090.4). These results demonstrate that the larger ECAP response previously measured for anodic stimulation was not solely caused by antidromic propagation and strengthens the finding that auditory nerve fibers are more sensitive to anodic stimuli.\nDiscussion\nMain findings\nWe have reported the results of three electrophysiological and psychophysical masking experiments designed to compare the responses of the human auditory nerve to anodic and cathodic current flows. In experiment 1, the anodic phase produced the usual N1-P2 ECAP morphology for all subjects, whereas the cathodic phase showed no measurable response except for one subject. This observation led us to conclude that the anodic phase was more effective than the cathodic phase. There are, however, at least three alternative explanations for this trend.\nFirst, the cathodic phase response of experiment 1 may have had a shorter latency than the anodic phase response and may have consequently been mixed within the uncancelled stimulus artifact. Although this hypothesis is not supported by animal data, usually showing longer latencies for cathodic stimuli (Miller et al. 1998, 1999a, 2004), a potentially important confounding variable is the location of the recording electrode. Most animal ECAP data are recorded using an electrode positioned on the nerve trunk. Consequently, the shorter the response latency, the more central the excitation site is. This may, however, not be the case for intracochlear recordings where the recording and the stimulating electrodes are close to each other (Miller et al. 2004). Here, we used a recording electrode 2\u00a0mm apical to the stimulating one, similar to what is used clinically, and this choice may have affected the morphology and the latency of the ECAP responses. We note that our second experiment controlled for any possible effects of response latency to the probe by studying the effects of masker polarity for a fixed probe and by showing that, although the leading polarity of a biphasic probe did affect the response latency, it was always the case that responses were larger with an anodic than with a cathodic masker.\nSecond, as previously stated, the response to the anodic phase obtained in experiment 1 may not only represent the neural response traveling toward the central auditory system. As the recording electrode is located at the periphery of the nerve fibers, the recorded response possibly contains the antidromic response of the fibers. Although this is possible, this would necessarily imply that the nerve fibers still have remaining peripheral processes to allow the propagation of antidromic spikes. In such a case, this phenomenon may not occur in subjects with a long duration of deafness and presumably a poor peripheral processes\u2019 survival. Furthermore, the psychophysical report of experiment 3 demonstrates that the orthodromic response (propagating centrally) is also larger for the anodic than for the cathodic phase. Antidromic propagation cannot therefore be the only account for our finding.\nThird, the cathodic phase may excite a different subpopulation of nerve fibers than a biphasic pulse with no phase separation. In such a case, the masker-probe paradigm that we used in Experiments 1 and 2 would not be effective in the measure of the cathodic phase response, i.e., if the masker does not effectively mask the probe, the subtraction method would not allow the neural response to appear. Although the present data cannot rule out this possibility, it is worth noting that the masker-probe paradigm was successfully used in the measure of the anodic phase response. This would mean that a biphasic pulse (whether anodic- or cathodic-first) with no phase separation excites the same subpopulation as a single anodic phase but a different subpopulation than a single cathodic phase. Consequently, this would not change the conclusion that the anodic phase is the most \u201ceffective\u201d phase of a biphasic pulse and that the ECAPs recorded clinically most likely represent the response to the anodic phase of biphasic pulses.\nAlthough the observation of a higher sensitivity to anodic current flows is surprising, some animal data have already shown such a result. Miller et al. (1998) recorded ECAPs to monophasic anodic and cathodic pulses from the cat and the guinea pig. They observed lower cathodic thresholds for cats but lower anodic thresholds for guinea pigs. The reason for this species\u2019 discrepancy was, however, unclear, and the authors proposed that it may relate to differences in anatomy or in electrode size and position. Other stimulus parameters have shown to affect human and animal subjects in different ways (Shepherd and Javel 1999; Carlyon et al. 2005), and several factors may underlie those species\u2019 differences, including differences in anatomy, neural properties, neural survival, and electrode geometry (reviewed by Miller et al. 1999b).\nFactors affecting polarity sensitivity\nThe simplest model of extracellular stimulation that can account for a lower cathodic threshold is the activating function of Rattay (1989). This model describes the spatial pattern of polarization of an infinite, homogeneous fiber subjected to an extracellular electrical current. A cathodic stimulus produces a large depolarization of the fiber proximal to the electrode and a smaller hyperpolarization at both sides of the depolarized region, whereas an anodic stimulus produces the exact opposite pattern of polarization. This model is, however, too restrictive in the case of auditory nerve fibers, which are made of several excitable elements (peripheral process, unmyelinated cell body, and central axon) that presumably have different biophysical properties and, hence, different activation thresholds. Moreover, auditory nerve fibers are curved, and the spatial pattern of polarization produced by an electrical pulse may show multiple regions of depolarization and hyperpolarization (Rattay 1999; Rattay et al. 2001a). There are at least three factors that may influence polarity sensitivity: the electrode position, the nerve parameters, and the stimulus current level. They are discussed in the following paragraphs.\nRanck (1975) reviewed the different mechanisms of electrical excitation of the central nervous system and showed that the electrode position relative to the fiber had a crucial effect on its sensitivity to polarity. He reported that myelinated fibers were more easily excited by cathodic stimuli when the electrode was placed over the central axon. However, for an electrode located near the cell body, on the opposite side from the central axon, the lowest threshold was often obtained with an anodic stimulus. Rattay (1999) and McIntyre and Grill (1999) found similar place-dependent polarity effects on their modeled central neurons. In another modeling study, Rubinstein (1993) focused on the excitation process near the axon termination. He also demonstrated the extreme dependence of polarity sensitivity on the exact electrode position. As a CI electrode presumably excites a large number of nerve fibers with different orientations and locations, it is possible that each fiber is excited differently by anodic and cathodic phases (Rattay et al. 2001b). The present study was restricted to electrodes mid-way along the electrode array. Subsequent studies should explore other electrode positions and other electrode designs.\nComputational models have provided a simple way to investigate the influence of neural parameters. In two different modeling studies, Rattay et al. (1999, 2001a) suggested that intact human auditory nerve fibers would be more sensitive (showing lower excitation thresholds) to cathodic stimuli, whereas fibers with degenerated or demyelinated peripheral processes would be more sensitive to anodic stimuli. First, they predicted that, for intact fibers, the depolarization magnitude would be maximal at the level of the peripheral process and would be larger for a cathodic than for an anodic pulse. Second, for fibers without peripheral processes, their model showed that the only site that could be depolarized by a cathodic pulse was the cell body, whereas an anodic pulse could depolarize the central axon. Given that the cell body was modeled using a channel density ten times smaller than for the central axon, it had a much higher threshold than the central axon, thereby leading to a lower anodic threshold. A similar effect occurred in the case of demyelination of the peripheral process because unmyelinated fibers need much stronger currents to be excited than myelinated ones. Given that peripheral processes in deafened ears are believed to be somewhat degenerated (Fayad and Linthicum 2006) and that this degeneration is often accompanied by demyelination, the predictions of Rattay et al. (1999, 2001a) give a plausible explanation for the higher sensitivity to anodic stimulus currents observed in implanted subjects. This would therefore imply that the excitation site is at the level of the central axon, which is consistent with a report of van den Honert and Stypulkowski (1984) on the responses of cats\u2019 fibers with and without peripheral processes and cell bodies. They found the responses of the degenerated fibers to closely resemble those of the normal fibers stimulated at high current levels. Although the exact location of the excitation site is still a matter of debate and probably depends on the electrode configuration and on the stimulus current level, Miller et al. (1999a) demonstrated that cats\u2019 fibers were almost always excited at the level of their central axon with monopolar stimulation. The response latencies obtained in experiment 1 for anodic stimulation (290\u00a0\u03bcs on average) also argue for a stimulation at the level of the central axon, given that they are similar to the latencies commonly observed in human CI subjects for symmetric biphasic waveforms but also comparable to those measured intracochlearly in cats for anodic pulses (Brown et al. 1990; Miller et al. 2004). Given that the main morphological discrepancies between humans and cats are the length of the peripheral processes (longer in humans) and the size and degree of myelination of the spiral ganglion cells (larger and unmyelinated in humans; Liberman and Oliver 1984; Spoendlin and Schrott 1989; Rattay et al. 2001a), we would expect significant ECAP latency differences if the auditory nerve fibers were excited at their peripheral process or cell body sites but not if they were excited at the level of their central axon. To test whether neural degeneration and demyelination are the causes of our finding, additional psychophysical measures may be performed. Specifically, in ears with substantial degeneration, the dominance of the anodic phase should be enhanced, the effect of phase separation on threshold should be reduced (Prado-Guitierrez et al. 2006), and the slopes of the strength-duration functions should be shallower (Miller et al. 1995).\nFinally, we reported in a previous study (Macherey et al. 2006) a comparison of thresholds and MCLs for anodic- and cathodic-first asymmetric pulses. Although we did find lower MCLs for anodic-first pulses\u2014consistent with the present results\u2014we did not observe any difference at threshold. It is possible that the higher sensitivity to anodic stimuli is specific to suprathreshold levels. One hypothesis proposed by Macherey et al. (2006) was that cathodic-first asymmetric pulses may produce a cathodal block at high levels. This seems, however, unlikely given that cathodal blocking should occur at very high levels above threshold (approximately eight times higher; Ranck 1975). This would correspond to a current level 18-dB higher than threshold, therefore exceeding the common dynamic range values of CI users (for example, the probe dynamic ranges of experiment 3 ranged from about 4 to 11\u00a0dB). Another possibility is that the cluster of fibers recruited at threshold is equally sensitive to anodic and cathodic phases or that, alternatively, approximately the same number of fibers is excited by an anodic and by a cathodic phase. At higher levels, more spatially remote fibers may be recruited, and it is possible that a majority of these fibers are more sensitive to anodic stimulation. Interestingly, in their spatial modeling study of the human cochlea, Rattay et al. (2001b) predicted that the nerve fibers close to the stimulating electrode would demonstrate lower cathodic than anodic thresholds but that fibers more spatially remote would in some cases exhibit the opposite trend. However, we note that in experiment 3, the greater masking effectiveness of the anodic phase was observed for probes presented to electrodes adjacent to that used for the masker.\nApplications to CIs\nOur results have implications not only for theories of electrical stimulation of the human auditory nerve but also for the development of future CIs. Asymmetric pulses may be used in a CI speech processing strategy for two reasons: to reduce power consumption or to increase spatial selectivity.\nFirst, we previously suggested that asymmetric pulses presented in monopolar mode would achieve some worthwhile power savings (Macherey et al. 2006). If such pulses were implemented in a speech processing strategy, then, the results of experiment 3 suggest that the short, high-amplitude phase of the pulses should be anodic relative to the active electrode. In this case, the effect of the long, low-amplitude phase on neural excitation should be kept minimal.\nSecond, for so-called longitudinal bipolar stimulation, where each intracochlear electrode is stimulated with reference to another nearby intracochlear electrode, the pattern of excitation along the cochlea can sometimes show a bimodal shape with peaks of excitation near the two stimulating electrodes (Kral et al. 1998; Chatterjee et al. 2006). Frijns et al. (1996) suggested that this bimodality may be reduced by the use of asymmetric pulses. Their computational model showed that the nerve fibers proximal to the electrode for which the short, high-amplitude phase is cathodic would be more effectively excited than the fibers proximal to the other electrode. Our results suggest that a similar reduced bimodality and enhanced spatial selectivity may be obtained in CIs, but, crucially, with more excitation near the electrode presenting the short, anodic phase. Note that, in the present work, we used asymmetric pulses with relatively long phase separations, which are unlikely to be useful in multichannel CIs because they would require an extremely low repetition rate. However, we would expect this enhanced spatial selectivity to also hold for bipolar asymmetric stimuli with a shorter or no phase separation, which may be more appropriate candidates for implementation in a CI speech processing strategy.","keyphrases":["auditory nerve","cochlear implants","psychophysical masking","asymmetric pulses","stimulus polarity","electrical stimulation"],"prmu":["P","P","P","P","P","P"]}
{"id":"Neuroimage-1-5-1971136","title":"Correlation of quantitative MRI and neuropathology in epilepsy surgical resection specimens\u2014T2 correlates with neuronal tissue in gray matter\n","text":"Newer MRI methods can detect cerebral abnormalities not identified on routine imaging in patients with focal epilepsy. Correlation of MRI with histopathology is necessary to understand the basis of MRI abnormalities and subsequently predict histopathology from in vivo MRI. The aim of this study was to determine if particular quantitative MR parameters were associated with particular histological features.\nIntroduction\nThe structural basis of focal epilepsy has a major effect on the natural history of the condition in an individual patient (Semah et al., 1998). In 30% of patients, epilepsy is refractory to current drug treatments. In patients with drug-resistant focal epilepsy, surgical resection may be curative, and the nature of the underlying lesion influences post-operative outcome (McIntosh et al., 2001; Luders and Schuele, 2006).\nMRI has greatly facilitated assessment for epilepsy surgery by revealing underlying structural cerebral abnormalities. When optimal MRI and post-operative neuropathological examination of the resection specimen do not identify an underlying structural abnormality, seizure outcome from surgery is less likely to be good (Blume et al., 2004; Jeha et al., 2006), reflecting the absence of a discrete, removed abnormality or one that is currently undetectable by imaging or pathology. This is to some extent a self-fulfilling strategy, such that patients in whom no structural abnormality is identified may not be considered for surgery (Spencer et al., 2005). Even optimal MRI, however, may not reveal known underlying pathological substrates such as focal cortical dysplasia and mild malformations of cortical development (Palmini et al., 2004), which are evident on histopathological examination of the resected tissue (Lee et al., 2005; Widdess-Walsh et al., 2006).\nThus, there is a pressing need for further developments in MRI, such as higher field strengths to improve signal-to-noise ratio, and newer sequences that may better pick out culpable neuropathological lesions in the neocortex.\nThe correlations between hippocampal volumes and T2 measures with glial and neuronal densities were established more than a decade ago (Cascino et al., 1991; Van Paesschen et al., 1995). For the more recently developed MR contrasts, the correlation between MR parameters and the underlying neocortical structure is not well understood and it is important for this to be clarified if these contrasts are to contribute to presurgical evaluation.\nQuantitative histopathology has developed in parallel with MRI and led to more accurate and sophisticated interpretation of microscopic tissue structure (Thom et al., 2005). We have previously shown that temporal lobe resection specimens and their resultant histological slides can be aligned with pre-operative volumetric MRI data, allowing comparison of advanced MRI data sets in regions of interest to the equivalent areas on histopathological slides (Eriksson et al., 2005). Thus, quantitative MRI data and quantitative histopathological data can be directly compared. In this study we compared the data obtained using two quantitative MRI contrasts, T2 relaxation time maps (T2); and magnetization transfer ratio (MTR), with quantitative neuropathological data from the resected material. The aim was to explore the clinical utility of T2 and MTR and determine if particular quantitative MR parameters were associated with particular histological features.\nMethods\nSubjects\nWe studied nine consecutive patients for temporal lobe epilepsy surgery at the National Hospital for Neurology and Neurosurgery who had had pre-operative advanced MRI and in whom resection specimens were processed according to our protocol (Eriksson et al., 2005). The study was approved by the Joint Research Ethics Committee of the National Hospital for Neurology and Neurosurgery and Institute of Neurology, UCL. All patient and control subjects gave written informed consent to participate in the study.\nThe age range of subjects was 31\u201346\u00a0years (median 36\u00a0years) and 4 were male. Seven had a right anterior temporal lobe resection, and two had surgery on the left. The surgical procedure was selected after our standard presurgical work-up, including EEG-video telemetry, conventional MRI (T1- and T2-weighted, proton density and FLAIR), psychological and psychiatric assessments and multi-disciplinary case conference.\nControl groups for the advanced MRI consisted of healthy volunteers with no history of neurological disease and normal conventional MRI and were the same subjects as reported previously (Salmenpera et al., 2007). Twenty-nine volunteers served as a control population for the analysis of T2 maps and 26 volunteers for MTR.\nMR acquisition\nAll scans were acquired on the same 1.5\u00a0T GE Signa MR scanner (GE Medical Systems, Milwaukee, WI). The T1-weighted volume sequence from the conventional scan protocol for all subjects was required for the MR:pathology correlation. The sequence parameters were: TE\/TR\/TI\/NEX, 4.2\/15\/450\/1; flip angle 20\u00b0; acquisition matrix 256\u00a0\u00d7\u00a0192; field of view 24\u00a0\u00d7\u00a018\u00a0cm; 3\/4 phase FOV, 124 contiguous 1.5\u00a0mm slices, giving a voxel size of 0.94\u00a0\u00d7\u00a01.25\u00a0\u00d7\u00a01.5\u00a0mm.\nSubjects had two additional scans to generate quantitative MR data:\nT2 mapping\nDual-echo CSF suppressed, FFT2 (fast FLAIR T2) data sets were acquired using TR\/TI\/TE1,2\u00a0=\u00a05000\/1638\/15,120\u00a0ms. Twenty-eight contiguous axial slices were obtained with an acquisition matrix of 256\u00a0\u00d7\u00a0256 and a 24\u00a0cm in-plane field of view, giving a voxel size of 0.94\u00a0\u00d7\u00a00.94\u00a0\u00d7\u00a05\u00a0mm. T2 signal intensity was calculated pixel-by-pixel using the dual-echo data sets, one set from the early echo (TE1) and one from the late echo (TE2), according to the equation: T2 signal\u00a0=\u00a0(TE2\u00a0\u2212\u00a0TE1)\u00a0\/\u00a0ln (S1\/S2) where S1 and S2 represent the signal intensities for the early and late echoes respectively (Rugg-Gunn et al., 2005). Total acquisition time was 13\u00a0min and 50\u00a0s.\nMagnetization transfer imaging\nMagnetization transfer (MT)-weighted and non-MT-weighted three-dimensional data sets were acquired using TR\/TE\u00a0=\u00a022.6\/5.4\u00a0ms. Volume matrix was 256\u00a0\u00d7\u00a0256 (in-plane)\u00a0\u00d7\u00a0124 over a 240\u00a0\u00d7\u00a0240\u00a0\u00d7\u00a0180\u00a0mm field of view, giving a voxel size of 0.94\u00a0\u00d7\u00a00.94\u00a0\u00d7\u00a01.5\u00a0mm. MT-weighted images were collected after the application of a pre-pulse to saturate the broad resonance of immobile macromolecule protons. The MT radiofrequency pulse used was a three-lobe Hamming apodized sinc pulse with a duration of 6.4\u00a0ms and of 5.1\u00a0\u03bcT, applied 2\u00a0kHz off-resonance (Rugg-Gunn et al., 2003). Total acquisition time was 13\u00a0min and 54\u00a0s.\nMRI quantitation\nVoxel-by-voxel statistical comparisons were performed on each of the two image contrasts. This process used SPM99 (Wellcome Dept. of Imaging Neuroscience, Institute of Neurology, London, UK) to produce intensity probability maps that were normalized to a standard space defined by a template of all the controls (Salmenpera et al., 2007). The normalized T2 maps were smoothed with a 4\u00a0mm and MTR maps an 8\u00a0mm isotropic Gaussian kernel to improve signal to noise ratio, to allow the images to conform more closely to a Gaussian field model and to increase the validity of statistical inference. A standard univariate t-test was applied using SPM99 to every voxel in the image in order to create a map from which statistical inference was drawn. Significant increases or decreases in MR contrast signal intensity were detected at an individual voxel threshold of p\u00a0<\u00a00.001. The theory of Gaussian fields was used to calculate a corrected multiple comparison p value of 0.05. We tested for regional differences in intensity between each patient and the control group. Further details of the image processing are given in a previous paper (Salmenpera et al., 2007).\nQuantitative MR values were obtained by overlaying a region of interest on the quantitative MR data. The mean intensity value of all voxels within the region of interest was calculated.\nMR:pathology visual correlation\nAll subjects had a standard anterior temporal lobe resection performed by the same surgeon. During and after resection, specimens were processed according to standard histopathological and co-registration protocols and digital images of the tissue blocks were correlated with the pre-operative volumetric MRI data (Eriksson et al., 2005). Briefly, after fixation the resected temporal lobe was sliced in 5\u00a0mm blocks perpendicular to the maximum linear extent of the superior temporal sulcus. The MR volume data were rotated and reformatted into an oblique coronal plane that matched the orientation of these blocks. These oblique coronal images were visually inspected in comparison to the pathological blocks. Using standardized criteria, the best fit MRI slice for any one pathological block was identified and matches for adjacent blocks of tissue ensued. Two raters (SF and SE) performed this task separately and reached a consensus in any case with discrepancy.\nAll subjects had post-operative MRI scans to indicate the extent of the resection.\nHistopathology\nStandard laboratory protocols were used to prepare resected tissue in sections stained for Nissl (cresyl violet\/LFB), glial fibrillary acidic protein (GFAP; Dako, Cambridge, UK; polyclonal 1:1500) and neuronal nuclear antigen (NeuN; Chemicon, Temecula, CA, USA, monoclonal 1:2000). An experienced epilepsy neuropathologist (MT) made a qualitative tissue assessment for clinical purposes. For quantitative histopathology the following sections were used: the standard 7\u00a0\u03bcm GFAP sections; additional 20\u00a0\u03bcm sections stained with NeuN; 20\u00a0\u03bcm sections stained for microtubule associated protein (MAP2; SIGMA, Saint Louis, MO, USA; 1:2000); 7\u00a0\u03bcm sections stained with anti-synaptophysin antibody (Dako, Cambridge, UK; 1:100). All sections were processed in the same batch to ensure uniform immunostaining, which is important when assessing the field fraction, which relies on the intensity of staining. A commercial image analysis system (Histometrix, Kinetic Imaging, Liverpool, UK), attached to a Zeiss Axioskop microscope, was used to generate quantitative histopathology measures. Two regions of tissue were assessed in each subject: ROI1 in the deep white matter of the middle temporal gyrus (a region identified from MRI-directed correlation, see below); and ROI2, in the cortex in the gyral crown of the middle temporal gyrus. Quantitative measures in ROI1 were: stereological counts of neurons; field fraction estimates of immunostaining for GFAP, MAP2 and synaptophysin. Quantitative measures in ROI2 were stereological counts of neurons and field fraction estimates of staining for NeuN and GFAP. The stereological neuronal counts were performed as previously described (Eriksson et al., 2006). For field fraction estimates the ROI was outlined on the computer screen of the image analysis system at \u00d7\u00a02.5 magnification. The number of fields within the ROI to be analyzed was chosen (70\u201390 in our study) and the computer software randomly chose a starting point and then a field spacing to cover the whole ROI at the relevant sampling rate. All cortical layers were included in the analysis. Magnification was then increased to \u00d7\u00a040 and each field displayed on the computer screen. In the first field for analysis, light intensity parameters were optimized to visualize the immunopositive pixels and the RGB (red-green-blue) parameters representing immunopositive pixels were set. Light intensity and RGB parameters were kept constant for all subsequent fields. The software program then automatically estimated the number of immunopositive pixels as a proportion of the entire field. An average of field fraction for all fields was calculated, thus reporting a single field fraction value for each ROI in a given patient. RGB parameters were defined uniquely for each case before analyses as signal intensity can vary slightly between cases. An example of images used for field fraction analyses in one patient is shown in Fig. 1. Measures in ROI1 were made by an experienced epilepsy neuropathologist (MT) and measures in ROI2 by a neurologist (SE) with extensive experience of quantitative neuropathology.\nHistopathology and MRI correlation\nMRI-directed\nAbnormal regions in the SPM results for the MRI data were observed for several subjects (Table 1). To correlate MRI data with histopathological analyses in the same area in all cases, region of interest analyses were performed. A region that had abnormal T2 signal on voxel based analysis in one subject that was within the deep white matter of the middle temporal gyrus was selected and designated ROI1. This region was of sufficient size to provide robust histopathological data (Eriksson et al., 2006) and was present in tissue which had been resected in all 9 subjects. Since ROI1 and all quantitative MRI data used for the SPM analyses were normalized to SPM space, quantitative MTR and T2 data could be achieved by superimposing ROI1 on each case's normalized MTR and T2 data.\nTo achieve quantitative histopathological data, the ROI was aligned with the volume MRI of each patient by a sequence of two normalizations as follows (Fig. 2).\nEach subject's volume MRI (cropped and tilted as used for the initial MR:pathology correlation) was normalized to the T1 image template in SPM99 using the default normalization protocol in SPM99 (Fig. 2). The T1 image template was in the same image space as ROI1, thus ROI1 could be associated with each subject's normalized MRI. The normalized volume was then back normalized to the volume MRI in each subject's native image space. The parameters used for the back-normalization were also used to transform ROI1 from T1 space back to the native space (Fig. 2). This ROI could then be transferred to the equivalent pathological space by visual inspection of the previously determined MRI:pathology correlation (Fig. 2). Thus ROI1 was located on the pathology slices of each subject. This region, directly drawn onto the pathology slides of interest, was used for subsequent quantitative histopathology. An example of the images of ROI1 for one subject is shown in Fig. 3.\nWe also defined a region in the pathological data and determined its MR characteristics. To achieve this, the MRI volume data were segmented in subject space using intensity thresholding and manual drawing in Analyze AVW 5.0 (BIR, Mayo Clinic, Minnesota). The entire resection volume and the middle temporal gyrus within the resection volume were extracted by manual drawing guided by visual inspection of the previously determined MR:pathology correlation. Intensity thresholding and visual inspection were used to separate gray and white matter. A final manual segmentation step was used to separate the gray matter of the gyral crown (ROI2), from the gray matter in the rest of the middle temporal gyrus. This step used similar criteria to characterize the limits of the gyral crown as were used by the neuropathologist in the quantitative neuropathology. The data from the initial MR:pathology correlation were used to determine which MR slices within the middle temporal gyrus in each subject corresponded to ROI2. Each subject's MR volume data were registered to the advanced MR image data in the native subject space using SPM99 and the default registration option. Because of the different qualities of the T2 maps and MTR image data, different registration was performed and different templates used that more closely resembled the two data sets.\nFor the MTR images, the segmented gray and white matter (summed) were used, while the MR volume data were registered to the proton density image which was acquired simultaneously with the T2 data, thus effectively registering the MR volume to the T2 data. Once optimal registration parameters were identified, these were used to register ROI2 in the MR volume data to the T2 maps and MTR data sets (Fig. 4). Thus, for example, gray matter in the middle temporal gyrus could be identified on the T2 image and T2 values determined for the gray matter in that region. An example of the images of ROI2 for one subject is shown in Fig. 5.\nQuantitative MR and histopathology measures were compared using SPSS (version 11.0.0).\nResults\nCareful correlation of abnormal regions in the SPM data and the resected tissue indicated that regions that were abnormal on SPM analysis were physically present in the resected tissue in four patients (Table 1). In one patient (patient 3) qualitative neuropathology identified a hamartia (a subtle malformation of cortical development) in the white matter approximately 3\u00a0mm in diameter (Table 2). This was not detected using the SPM analysis and was not a part of ROI1 or ROI2. The abnormal region in one patient (T2 region in patient 5) was in the white matter of the middle temporal gyrus and was labeled as the region of interest, ROI1. The quantitative histopathology and quantitative MR data for this region for the nine patients are given in Table 3. Spearman Rho correlation did not reveal any significant correlations between any of these pathological and MR measures. This region had an abnormal average T2 value in one other patient (patient 1) but MT values were within the normal range for all subjects. Clustering analysis of the histopathological data could not separate the two patients with abnormal T2 values from those subjects with normal T2 values in ROI1.\nQuantitative histopathology was determined for the gray matter in the gyral crown of the middle temporal gyrus. The registration process enabled us to determine quantitative MR data for the same region (ROI2) (Table 4). There was a significant Spearman Rho negative correlation between the field fraction measure of NeuN and the T2 value in ROI2 (correlation coefficient \u2212\u00a00.72, p\u00a0=\u00a00.028).\nDiscussion\nWe have previously shown that in vivo MRI and pathological blocks may be aligned using visual inspection and a standardized protocol (Eriksson et al., 2005). We have now shown that, with additional registration protocols, quantitative parameters from MRI and histopathology can be directly compared. There was a negative correlation between the histopathological field fraction measure of NeuN immunopositivity and the MR measure of T2 relaxation time in the gray matter of the middle temporal gyrus. This is the first correlation between a quantitative MR parameter and histopathological data from neocortex in patients with epilepsy.\nWe wished to compare the same area in all patients because it is not known how much histopathological measures and MR parameters differ as a function of location within cerebral tissue. Although some of the histopathological features of interest were readily quantified with a semi-automated protocol, stereological measures require significant expert time input. In consequence, we restricted this study to two regions of interest. The first region investigated corresponded to a region of abnormal T2 signal on SPM analysis in one patient and was chosen since it was of sufficient size to provide robust histopathological data and was available in all resections. Our second region was identified in each subject's histopathological sections, the gray matter of the crown of the middle temporal gyrus overlying ROI1, giving us data in adjacent gray matter.\nThe limitation of the initial MR and pathology matching is that the matching was visual and was guided by anatomy and was to some extent subjective. Our previous study, however, showed that the protocol gave consistent, reliable and accurate registrations within the structural limitations of the histopathological data (Eriksson et al., 2005). The subsequent matching of specific MR or pathology regions with their respective pathology or MR regions was carried out using computational normalization and registration, both of which were entirely repeatable. This may seem a complex procedure when, for example, the middle temporal gyrus is generally readily identified on anatomical MRI. However, the advanced quantitative MR sequences are optimized for the appropriate MR parameter and not for visualization of anatomy and it can therefore be difficult to confidently identify anatomical features using these contrasts. Identifying the structure of interest in a high quality anatomical scan and then using registration and\/or normalization protocols reduced the number of subjective steps (only one definition of the tissue structure was required rather than one in each MR scan) and therefore reduced computation time and increased repeatability.\nThe T2 signal in MRI is dependent upon the relationship between bound and free water in a tissue, which is itself dependent upon the macromolecular environment. Disruption of tissue integrity can result in an increase in the free water in tissue and an increase in T2 relaxation time. Changes in T2 in the cerebrum have been related to, among other processes, gliosis, demyelination, edema, neuronal loss and infarction. In an earlier study from our group, significant increases in T2 signal were observed in 51% of patients with cryptogenic focal epilepsy (Rugg-Gunn et al., 2005). The MTR signal is dependent upon the exchange of magnetization between free and bound protons in tissue and this depends upon the integrity of the macromolecular environment. Changes in MTR have been related to histopathologic changes of axonal loss, gliosis, demyelination and edema (Lexa et al., 1994; Brochet and Dousset, 1999). Our earlier study found reductions of MTR in acquired lesions, malformations of cortical development and in regions that were normal on conventional MRI (Rugg-Gunn et al., 2003). In the present study ROI1 was defined by an abnormal T2 value in one subject. Additional analysis revealed that this region also had an abnormal T2 signal in one other subject. MTR values were normal in this region in all subjects. There were no significant linear correlations between the MR values and the histopathological values in ROI1 in our group. The two subjects with abnormal T2 values did not differ from the other seven subjects in relation to their standard or quantitative histopathological data. The T2 and MTR values were all normal in ROI2.\nThere was a significant negative correlation between the T2 relaxation time and the field fraction estimate of NeuN staining in the cortex of the middle temporal gyrus. A decrease in NeuN staining could suggest a reduction in the number of mature neurons in the tissue, altered packing density or alterations in cell size or NeuN immunoreactivity. An increase in T2 signal has been correlated with neuronal loss and increased gliosis (Van Paesschen et al., 1997; Rugg-Gunn et al., 2005). Increased gliosis may further reduce the relative concentration of neurons in a region, although sometimes, tissue sclerosis or gliosis causes reduction in tissue volume which could paradoxically increase packing density. We did not observe any relationship between T2 value and GFAP field fraction. Thus, the correlation detected may reflect a consistent biological relationship focused on neuronal loss. However, one subject had neocortical neuronal loss observed during routine qualitative neuropathology (Case 4). This subject had the lowest value of neuronal count in the gray matter of ROI2 (the crown of the middle temporal gyrus) but also the lowest value of T2 in ROI2 which appears contrary to the correlation observed in the whole group. The reason for this is not clear. The cortical NeuN field fraction in this case was 0.11 (range 0.08\u20130.13).\nGray matter neuronal counts did not correlate with the field fraction estimate of NeuN. The gray matter neuronal counts are a count for each neuron observed in the region of interest expressed as a proportion of the total area assessed. The field fraction estimate of NeuN is an estimate of the area occupied by tissue immunopositive for NeuN as a proportion of the total area assessed. This tissue is predominantly neuronal cell bodies with some axons and dendrites. Thus 5 large neurons will occupy a greater area of immunopositivity than 5 small neurons. The neuronal count would be the same in each case but the field fraction would be different. This may explain the lack of correlation between the field fraction and the neuronal count. The relation of these two measures with the T2 relaxation value suggests that the T2 relaxation is dependent on more than just the number of neurons, also on their size or complexity, which may reflect their function. Further staining protocols and analyses of neuronal size are necessary to elucidate further this difference.\nThe lack of any other correlations between the MR and the histopathology measures could be due to a number of factors. A real correlation may exist and not have been detected, possibly because of the limited number of subjects in the study. A change of MR scanner curtailed data collection for this series and we are now recruiting more subjects imaged using a new 3\u00a0T scanner. We could also have missed a true correlation because the registration between the MR region and the pathological region was inadequate as discussed previously.\nAlternatively, there may not be any other true correlations between the MRI sequences and pathological measures studied. The pathological measures characterize real physical features \u2014 for example, the actual number of neurons and the proportion of tissue affected by gliosis. However, the underlying physical significance of the MR data is not yet well characterized. We may have chosen pathological measures which do not relate to the features characterized by the MR signals. Myelin content, for example, might influence both T2 and MTR. We did not think it likely that myelin studies would be of relevance in epilepsy in the context of this paper, but additional histopathological analyses, such as LFB for myelin or myelin basic protein, may lead to further useful data in future studies.\nThere was also limited tissue specificity for absolute T2 and MTR values between white and gray matter (see Tables 3 and 4 for details). Thus the MR measures and the histopathology measures may be measuring different features of the tissue and would not be expected to correlate. A hamartia, seen as a cluster of neurons in the white matter microscopically, was not detected as an abnormal MR region with either MTR or T2 using SPM. There are new histopathological techniques in development and some of these may prove a better match to the MR signal changes. MR acquisition on a 3\u00a0T scanner will produce data with better signal-to-noise qualities and improved spatial resolution and may increase the sensitivity of quantitative MR measures. Further correlations of quantitative histopathology and quantitative MRI measures may lead to derivation of MRI measures that are most sensitive to pathological changes.\nOur protocol for matching MR and pathology has demonstrated how we can investigate both the histopathology directed by an MR finding and the MR data directed by a histopathological finding. We identified a biologically plausible correlation between an increased T2 relaxation time and a reduction in neuronal labeling in the cortex of the middle temporal gyrus. If newer, quantitative MRI sequences are to be adopted more widely in routine clinical practice, further research exploring their underlying biological and pathological substrates will be necessary and may ultimately lead to a reduction in the proportion of patients with drug-resistant focal epilepsy whose optimal MRI is said to be \u201cnormal\u201d.","keyphrases":["mri","epilepsy","histology","mtr","fast flair t2"],"prmu":["P","P","P","P","P"]}
{"id":"J_Neurol-3-1-1915650","title":"Tremor in multiple sclerosis\n","text":"Tremor is estimated to occur in about 25 to 60 percent of patients with multiple sclerosis (MS). This symptom, which can be severely disabling and embarrassing for patients, is difficult to manage. Isoniazid in high doses, carbamazepine, propranolol and gluthetimide have been reported to provide some relief, but published evidence of effectiveness is very limited. Most trials were of small size and of short duration. Cannabinoids appear ineffective. Tremor reduction can be obtained with stereotactic thalamotomy or thalamic stimulation. However, the studies were small and information on long-term functional outcome is scarce. Physiotherapy, tremor reducing orthoses, and limb cooling can achieve some functional improvement. Tremor in MS remains a significant challenge and unmet need, requiring further basic and clinical research.\nSearch strategy and selection criteria\nReferences for this review were identified from searches of Pubmed (from 1966 to 2005) and the Cochrane Library (2005, Issue 2) with the search terms \u201cmultiple sclerosis\u201d, \u201ctremor\u201d, \u201cataxia\u201d, \u201cdisability\u201d, \u201cprevalence\u201d, \u201csurgery\u201d, \u201cthalamotomy\u201d, \u201cdeep brain stimulation\u201d and \u201ctreatment\u201d. Articles were also identified through searches of the reference tables of identified papers. Furthermore we searched the ISI Science Citation Index for relevant articles citing identified papers. Only articles in English and German were included in the review; publications in abstract form were not considered.\nIntroduction\nTremor is such a common problem in multiple sclerosis (MS) that the famous French neurologist Charcot (1825\u20131893) described it as a part of his triad of characteristic symptoms in MS, together with nystagmus, and scanning speech.\nTremor of the upper limbs can be very disabling and seriously impair many activities of daily living and quality of life. In practice the treatment of tremor in MS is often frustrating. In this review we discuss medical, surgical and other treatment options.\nEpidemiology\nAlthough the epidemiology of symptoms in MS has been studied extensively, it remains difficult to ascertain the prevalence of tremor in historic study populations, mostly because Kurtzke\u2019s functional systems scale for cerebellar function, which is most widely used in clinical studies in MS, does not record tremor [55]. In a large long-term follow-up study, Weinshenker and colleagues found functionally relevant cerebellar deficits in one third of 259 patients [94]. In another study, disability and dependency of patients with MS were shown to correlate with the severity of ataxia [95].\nTwo newer studies assessed the prevalence of tremor in MS. One study by Alusi and coworkers examined 100 patients randomly selected from a London MS-clinic and found tremor in 58% of patients. The tremor was minimal in 27%, mild in 16% and moderate to severe in 15% of all patients [6]. As patients were selected from a specialist clinic, the relatively high prevalence and severity of tremor might be due to selection bias.\nIn a prevalence cohort study by Pittock and colleagues, 200 MS patients living in Olmsted County, Minnesota, USA were assessed for tremor and measures of disability. Tremor was found in 25.5% and severe tremor in 3% of the study population [74]. Probably the community based population in the latter study gives a more realistic estimate of tremor prevalence in MS. In both studies, however, tremor was associated with greater disability as measured on the expanded disability status scale (EDSS). In the Olmsted County population, patients with tremor of any severity were more likely to be unemployed or retired early because of disability.\nTremor subtypes\nTremor in MS can involve the head, neck, vocal cords, trunk and limbs, whereas involvement of the tongue, jaw or palate has not been reported [5].\nThe different types of tremor are currently classified according to a working consensus of the Movement Disorder Society [29]. In MS, the two most prevalent tremor forms are postural tremor (tremor present whilst voluntarily maintaining a position against gravity) and intention tremor (tremor occurring during target directed movement where tremor amplitude increases during visually guided movements towards the target). True rest tremor (tremor present in a body part that is not voluntarily activated and is completely supported against gravity) is unusual in patients with MS, and Holmes (or \u201crubral\u201d) tremor is also very uncommon.\nIn the two main prevalence studies, [6, 74] rest tremor was observed only in the Olmsted County survey (1% of patients), whereas Holmes tremor was observed in neither study. An overview of the affected body parts observed in both studies is given in Table\u00a01. In both studies tremor was most commonly found in the arms. In the London study population, 36% of patients suffered from bilateral arm tremor, making this the most common pattern of limb involvement.\nTable\u00a01Details of the two main prevalence studies on tremor in MSLondon [6] (n\u00a0=\u00a0100)Olmsted County [74] (n\u00a0=\u00a0200)Total patients with tremor58 (58%)51 (25.5%)Patients with severe tremor15 (15%)6 (3%)Arm tremor56 (56%)47 (23.5%)Bilateral arm tremor36 (36%)not reportedLeg tremor10 (10%)12 (6%)Head tremor9 (9%)7 (3.5%)trunk tremor7 (7%)not reported\nPathophysiology of tremor\nThe pathophysiology of tremor in MS is a difficult area of investigation, partly because MS is by definition a multifocal disease, so that tremor occurrence cannot easily be linked to a single neuroanatomical site. Systematic postmortem studies on the link between lesion site and the clinical phenomenon of tremor have never been undertaken.\nThe predominance of action tremors (postural and intention) in patients with MS point to the cerebellum and its connections as the most likely source of tremor production, whereas the rarity of rest tremor argues against an involvement of the basal ganglia. The common occurrence of bilateral tremor might indicate that damage to the cerebellum and its connections is often multifocal.\nAnother link to the cerebellum in the pathophysiology of tremor in MS is the effect of peripheral cooling on intention tremor. Intention tremor is thought to be modulated through increased long latency stretch reflexes [30]. Cooling has been shown to decrease the sensitivity of muscle spindles [64] and the velocity of peripheral nerve conduction [56]. In two experimental studies, cooling of the arms markedly reduced intention tremor severity in patients with MS [77, 35]. The authors argue that this effect might have been partly due to decreased muscle spindle function and decreased nerve conduction velocity, which in turn resulted in decreased input into tremor producing cerebellar circuits.\nAlusi and coworkers drew attention to the placement site of deep brain stimulation (DBS) electrodes to help understand the neuronal circuits involved in tremor production. They stated that neurosurgeons increasingly chose the nucleus ventralis oralis posterior (VOP) of the thalamus rather than the classic target, the nucleus ventralis intermedius (VIM). This is interesting because the VOP is the basal ganglia output nucleus of the thalamus [6], suggesting that the cerebellar tremors seen in MS might actually be generated by the basal ganglia. The standard electrode placement site reported in the literature, however, is the VIM and no studies comparing placement sites have been published. Whittle and coworkers comment on the difficulty of electrode placement in MS patients: in most patients there are major brain distortions due to demyelination, plaque formation and e vacuo hydrocephalus, and it is therefore uncertain whether the anatomy of these patients conforms to standard stereotactic atlasses [100]. Keeping these comments in mind, it seems unwise to base pathophysiological theories purely on DBS electrode placement site.\nA range of animal experiments have been undertaken to indentify the anatomical structures involved in tremor production (for review [101]). It has been shown that damage or removal of the cerebellar cortex does not induce intention tremor in monkeys, [31, 76] whereas partial or complete cerebellectomy leads to tremor during movement and posture [75, 42, 40]. A slow, 3\u20135 Hz tremor during target directed movements can also be induced by reversible cooling of the dentate nucleus (the origin of most cerebellar efferents) in monkeys [22].\nMost cerebellar efferents project from the dentate nucleus via the superior cerebellar peduncle (brachium conjunctivum) to the red nucleus and the thalamus. According to some studies, transection of the superior cerebellar peduncle causes intention tremor in monkeys [34, 93], whereas other authors do not mention this effect.\nInterestingly, Carpenter and colleagues report that tremor induced by transection of the superior cerebellar peduncle can be alleviated by a second lesion in the lateroventral or centromedian thalamus. [19, 20] The results of these animal studies suggest that damage to cerebellar efferents (through lesions of the dentate nucleus or superior cerebellar peduncle) may cause disinhibition of thalamic nuclei which are the main producers of intention tremor.\nAlthough this is an interesting pathophysiologic model of intention tremor production, it remains uncertain whether the results of animal studies can be generalized to patients with MS.\nIn summary, clinical observation, animal studies and some experimental evidence in humans favour the cerebellum and the thalamic nuclei connected to it as the major locus of intention tremor production, but more research is needed to evaluate the role of the basal ganglia and other systems in tremor production in MS.\nAssessment of tremor\nDepending on the subtype of tremor, several methods for the assessment of tremor severity and its impact on the lives of patients have been developed. Rest tremor is often assessed with the tremor subscale of the Unified Parkinson\u2019s Disease Rating Scale (UPDRS). Fahn and colleagues devised the most comprehensive tremor scale for non-parkinsonian tremor in 1984 [33]. This instrument measures tremor in nine body parts at rest, while maintaining a posture and during goal directed movements. It also includes an assessment of arm tremor while writing and pouring water as well as a subscale for functional disability (interference of tremor with dressing, writing, eating etc.). Some studies included in this review use a modified and heavily abbreviated version of this scale, but most studies assess tremor by clinical examination only (e.g. by finger-to-nose testing, drinking from a cup, nine-hole-peg-test or writing and drawing tasks) or use a simple ordinal severity scale, often classifying tremor as absent, mild, moderate or severe. A simple 0\u201310 tremor severity scale devised by Bain and coworkers [11] has been shown to be a valid and reliable measurement tool in patients with MS [7], but has so far only been used in few clinical studies [6, 4, 15].\nAccelerometry and polarized light goniometry are neurophysiologic methods of tremor assessment. While these methods offer an objective measurement of tremor severity, they can only measure one aspect of an often complex movement problem at a time, and e.g. cannot measure the ataxia which often complicates tremor in MS.\nMatsumoto and colleagues devised a more complex \u201cquantitative movement analysis technique\u201d, which records the patients\u2019 goal directed movements in three dimensions using an electromagnetic tracking device, but the complexity of this method [62] as well as the computer aided tracking tasks employed by Aisen and coworkers [2] prohibit their widespread use.\nOne of the most important aspects of tremor in MS is its impact on the daily life of the patients. Functional status in MS-patients is often measured by asking the patients to complete questionnaires assessing activities of daily living such as writing, eating or dressing. Often these scales are self-devised and not validated. Validated scales used in the studies reviewed in this article include the Frenchay Activities Index [85] and the quality of life subscale of the Functional Assessment of Multiple Sclerosis Scale (FAMS) [62].\nMedical treatment\nA summary of the published studies on medical treatment of tremor in MS is given in Table\u00a02. Most of the published literature on medical treatment consists of case reports and uncontrolled open label studies. The few randomized controlled trials comprised small numbers of patients and very likely lacked the power to reveal small treatment effects.\nTable\u00a02Studies on medical treatment of tremor in MSStudyn*study designintervention(s)tremor assessmentpatients with tremor reduction (%)patients with improved functional status (%)adverse effects (n)Koller, 1984 [52]3double-blind placebo-controlled crossoverpropranolol (not specified) isoniazid (po.) 1200mg\/d ethanol (iv.) 50ml of 10% solutionwriting tasks, patient self assessment, clinical examination, accelerometryno effecthepatitis while on isoniazid treatment (1)Sabra et al. 1982 [80]4case reportsisoniazid (po.) 400 to 1200mg\/dclinical examination4 (100%)4 (100%)abnormal liver function tests (1)Duquette et al. 1985 [32]12open labelisoniazid (po.) 500\u20131000mg\/dpatient self assessment, clinical examination, blinded evaluation of video tapes10 (83%)0drowsiness, dysphagia, increased bronchial secretions (6) abnormal liver function tests (2) fatigue (2)Morrow et al. 1985 [67]5open labelisoniazid (po.) 700\u20131200 mg\/dclinical examination4 (80%)4 (80%)increased weakness (2) drowsiness (1)Francis et al. 1986 [38]5open labelisoniazid (po.) 1200 mg\/dclinical examination, polarised light goniometry4 (80%)0anorexia and nausea (3)Hallet et al. 1985 [44]6double-blind placebo-controlled crossoverisoniazid (po.) 1200 mg\/dself-rating scales, accelerometry, blinded evaluation of video tapes6 (100%)0NoneBozek et al. 1987 [16]8double-blind placebo-controlled crossoverisoniazid (po.) 12 or 20 mg\/kgclinical examination, accelerometry, blinded evaluation of video tapes6 (75%)4 (50%)somnolence (3) **Sechi et al. 1989 [86]7single-blind placebo controlledcarbamazepine (po.) 400 or 600 mg\/dclinical tremor rating scale, accelerometry7 (100%)not reportednone reportedRice et al. 1997 [78]16double-blind placebo-controlled crossoverondansetron (iv.) 8 mg (single dose)nine-hole peg-test, writing tasks, patient self assessment12 (75%)not reportedshort-lasting foot dystonia (1)Gbadamosi et al. 2001 [39]14open labelondansetron (iv.) 8 mg (single dose)nine-hole peg-test, writing tasks, patient self assessmentno effectnone reportedAisen et al. 1991 [2]6open labelGluthetimide 750 to 1250 mgblinded functional assessment by occupational therapist, computer-aided tracking tasks5 (83%)5 (83%)sedation (4)Clifford, 1983 [21]8open label\u03949-THC (po.) 5 to 15 mgclinical examination5 (63%)2 (25%)noneZajicek et al. 2003 [103]365\u2020double-blind randomized placebo controlledcannabis extract (po.) \u03949-THC (po.)patient self assessmentno effectcannabis extract: adverse events in 12 \u03949-THC: adverse events in18 placebo: adverse events in 20Wade et al. 2004 [92]13\u2021double-blind randomized placebo controlledcannabis extract (po.)visual analogue scale symptom reductionno effectcannabis extract:$ 112 adverse events in 80 patients placebo:$ 53 adverse events in 80 patientsFox et al. 2004 [37]14double-blind randomized placebo controlled crossovercannabis extract (po.)tremor rating scaleno effectcannabis extract: adverse events in 10 placebo: adverse events in 2THC\u00a0=\u00a0tetrahydrocannabinol* MS patients completing the study** in this study, one additional patient was withdrawn because of a severe adverse event (dyspnoea, fever, rash, obtundation)\u2020 the main topic of the study is the effect of cannabinoids on spasticity in MS, the patients were asked to assess treatment effect on other symptoms\u2021 in this study, patients were asked to name their most troublesome symptom; of the 160 included patients, 13 named tremor$ No details are reported on the subgroup of the 13 tremulous patients, the total number of adverse events is reported for the whole groups on active treatment and on placebo (80 patients in each group)\nBeneficial effects have been reported for a variety of drugs: case reports and small uncontrolled open label studies claim a positive effect of primidone [47], gluthetimide [2], intrathecal baclofen [96] and isoniazid. [80, 32, 67, 38] Controlled clinical trials have been published on the use of propranolol [52], ethanol [52], isoniazid, [52, 44, 16] carbamazepine [86], ondansetron [78] and dolasetron [65].\nKoller evaluated the effect of propranolol, isoniazid and ethanol on tremor in three tremulous MS patients in a double blind crossover trial and found no treatment effect for any of the treatments [52]. To the best of our knowledge, no further trials with propranolol have been published, but it is interesting to note that Alusi and coworkers excluded two tremulous MS patients from a thalamotomy trial because they had achieved functional improvement after propranolol use [4].\nIn an open study the hypnotic-sedative drug glutethimide exhibited visible functional benefit in six of eight MS patients with tremor [2]. Apparently, a controlled trial to confirm these findings was never undertaken.\nTwo double-blind placebo-controlled trials using isoniazid were published. In the first study reduction of tremor occurred in six of eight patients [16], with functional improvement in four patients. In the second study all six patients had measurable tremor reduction after treatment with isoniazid but this did not lead to functional improvement [44]. Doses of isoniazid used to treat MS-related tremor were very high (up to 1200 mg a day), and treatment was in some patients associated with anorexia and nausea [38] or with a combination of drowsiness, dysphagia and increased bronchial secretion. [32, 67] Other reported side effects were abnormal liver function tests, [80, 32] fatigue [32] and increased weakness [67]. Isoniazid inhibits GABA aminotransferase activity and increases GABA in the central nervous system, but no correlation was found between the degree of GABA elevation in the cerebrospinal fluid and clinical response [16].\nSechi and coworkers published a small single-blind placebo-controlled trial with carbamazepine [86]. They reported improvement of tremor as assessed by clinical examination and accelerometry in all seven included patients, but failed to report whether this translated into functional improvement.\nA placebo controlled, double-blind, crossover study, suggested that a single intravenous dose of the 5-HT3 receptor antagonist ondansetron led to tremor reduction in twelve, and to functional improvement in nine of the sixteen included tremulous MS patients [78]. A subsequent open label study by Gbadamosi and colleagues which used the same intervention found no significant treatment effect [39]. Likewise, a small clinical trial with dolasetron, another 5-HT3 receptor antagonist, showed no significant treatment effect on cerebellar ataxia [65].\nWeiss and coworkers reported a positive effect of intrathecal baclofen in a single patient with bilateral arm tremor [96]. The tremor amplitude decreased almost linearly as the dosage increased and tremor was abolished at a dosage of 250\u00a0\u00b5g per day. This possible treatment option has, however, not been observed or evaluated in other studies.\nIn recent years, there has been growing interest in cannabis as a possible therapeutic in MS. In the 1980\u2019s a case report [63] and a small uncontrolled study [21] on the beneficial effects of cannabis on tremor and spasticity in MS were published and a survey taken among MS patients revealed that many patients experienced positive effects of smoked cannabis on MS-related symptoms [23].\nBaker and colleagues reported a decrease of tremor and spasticity in an animal model of MS after treatment with \u03949-tetrahydrocannabinol, the active ingredient of cannabis [12]. All this furthered hopes of cannabis as a possible new treatment option for tremor in MS, but much to the disappointment of tremulous MS patients, several well conducted randomized controlled trials did not show a significant effect of orally administered cannabis extracts [103, 37, 92] or oral \u03949-tetrahydrocannabinol [103] on tremor.\nSurgical treatment\nThe surgical treatment options for tremor in MS are stereotactic thalamotomy and DBS. An overview of the published studies on surgical treatment is given in Tables\u00a03 and 4. Most of the studies are small observational retrospective studies. When reviewing the literature on surgical treatment, it is surprising as well as disappointing that the majority of studies are remarkably imprecise in providing basic information on the length of follow-up, on adverse effects and-most importantly-on the effect on functional status and tremor associated disability.\nTable\u00a03Studies on stereotactic surgery for the treatment of tremor in MSStudyn*patient characteristics**lesion sitefollow-uptremor and disability assessmentpatients with tremor reduction (%)\u00a7patients with improved functional status (%)\u00a7patients with permanent adverse effects (n)Cooper, 1960a [25]2disabling intention tremorVL3 to 12 moclinical examination2 (100%)not reportednoneCooper, 1960b [24]6disabling intention tremorunilateral (n\u00a0=\u00a05) or bilateral (n\u00a0=\u00a01) VLNot specifically reportedclinical examination, assessment of filmed tremor5 (83%)not reportedincrease of contralateral hemiparesis (1)Krayenb\u00fchl et al. 1962 [54]4disabling bilateral intention tremorunilateral (n\u00a0=\u00a03) or bilateral (n\u00a0=\u00a01) VL3 weeks to 6 moclinical examination4 (100%)4 (100%)noneBroager and Fog, 1962 [18]4severe intention tremorunilateral VL1 to 6 moclinical examination4 (100%)2 (50%)generalized seizure (1) mental change (1)Cooper et al. 1967 [26]32disabling bilateral intention tremorunilateral or bilateral VL12 to 96 moclinical examination27 (85%)not reportedincrease of contralateral hemiparesis (2)Samra et al. 1970 [81]25disabling bilateral arm intention tremorunilateral or bilateral VLnot specifically reportedclinical examination, assessment of filmed tremor22 (88%)not reportedincrease of contralateral hemiparesis (1)Riechert and Richter, 1972 [79]29disabling intention (n\u00a0=\u00a029) and postural (n\u00a0=\u00a018) tremorunilateral (n\u00a0=\u00a028) or bilateral (n\u00a0=\u00a01) VLnot specifically reportedclinical examination, patient self assessment questionnaires29 (100%)\u201ctwo thirds\u201d of patientsincrease of leg paresis (2)Arsalo et al. 1973 [10]26disabling bilateral intention tremorunilateral VL and subthalamus3 to 97 moclinical examination21 (80%)not reportedsubdural haematoma (1)Andrew et al. 1974 [8]4disabling postural (n\u00a0=\u00a02) and intention (n\u00a0=\u00a04) tremorunilateral VIM6 to 36 moclinical examination4 (100%)not reportedhemiplegia (1)Van Manen, 1974 [91]4severe intention tremorunilateral VL3 to 86 mo\u2020clinical examination2 (50%)not reportednot reported separately for MS subgroupHauptvogel et al. 1975 [45]11severe intention tremorunilateral (n\u00a0=\u00a010) or bilateral (n\u00a0=\u00a01) VL15 to 86 moclinical examination7 (63%)4 (36%)not reportedMundinger and Kuhn 1982 [68]84severe action tremorZI, VOP36 to 120 moclinical examination filmed tremor patient self assessment questionnaire70 (83%)not reportednot specifically reportedSpeelman and van Manen, 1984 [89]11severe intention tremorunilateral VL3 weeks to 132 moclinical tremor and functional rating scales8 (73%)0 (0%)hemiparesis (4) micturition disturbance (2) speech disorder (1)Kandel and Hondcarian, 1985 [51]20severe intention tremorunilateral (n\u00a0=\u00a015) or bilateral (n\u00a0=\u00a05) VL (ZI, FF)12 to 120 monot reported14 (70%)not reportednoneHitchcock et al. 1987 [48]30tremorThalamus24 moclinical examination functional rating scales50%25%not reportedWester et al. 1990 [97]9severe intention tremorunilateral VOA and VOP3 to 89 mo mean: 24 mo\u2020questionnaire sent to treating neurologist6 (66%)6 (66%)hemiparesis (5) mental changes (3) dysphasia (3) dysarthria (2) subdural haematoma (1)Goldman et al. 1992 [43]2severe intention tremor unresponsive to medicationunilateral VL3 mo 34 moclinical tremor rating scale2 (100%)0 (0%)dysarthria (1)Whittle and Haddow, 1995 [98]9severe rest, kinetic, postural or intention tremorunilateral VL12 moclinical examination and evauation of video tapes9 (100%)2 (22%)depression (2)Shahzadi et al. 1995 [87]33severe tremorunilateral VIM3 to 120 moclinical examination ability to drink from a waterfilled cup22 (67%)17 (51%)not reportedHooper and Whittle, 1998 [50]6severe postural (n\u00a0=\u00a04) or intention tremor (n\u00a0=\u00a02)unilateral VL14 to 73 mo mean: 51 moBarthel Index1 (16%)1 (16%)not reportedCritchley and Richardson, 1998 [27]24disabling intention tremorunilateral (n\u00a0=\u00a022) or bilateral (n\u00a0=\u00a02) VIMmean: 26 moclinical tremor and functional rating scales18 (75%)2 (8%)hemiparesis (1) seizure (2) MS relapse (3) dysarthria (1)Schuurman et al. 2000 [85]5severe arm tremorunilateral VIM\u20216 moclinical tremor and functional rating scales5 (100%)0 (0%)severe gait or balance disturbance (2)Niranjan et al. 2000 [72]***3severe action tremorunilateral VIM2 to 11 mo median 6 mo\u2020clinical tremor rating scale, patient self assessment of functional improvement3 (100%)3 (100%)noneAlusi et al. 2001 [4]11severe postural and intention tremorunilateral VOP (n\u00a0=\u00a07), ZI (n\u00a0=\u00a03), STN (n\u00a0=\u00a01)12 moclinical tremor and functional rating scales11 (100%)7 (64%)depression (3) seizures (2) hemiparesis (1) dysphasia (1)Matsumoto et al. 2001 [62]6severe tremorunilateral VIM3 to 12 moclinical tremor and functional rating scales, novel movement analysis tool6 (100%)0 (0%)MS relapse (2)Bittar et al. 2005 [15]10disabling postural and intention arm tremorunilateral VOP (distal tremor), unilateral ZI (proximal tremor) or unilateral VOP and ZI (mixed tremor)12 to 50 mo: mean 16 moclinical tremor rating scalenot individually reported, overall improvement of mean tremor scores: postural: 78% intention: 72%not reportedhemiparesis (3) seizures (1)VOP\u00a0=\u00a0nucleus ventralis oralis posterior; VOA\u00a0=\u00a0nucleus ventralis oralis anterior; VIM\u00a0=\u00a0nucleus ventralis intermedius; ZI\u00a0=\u00a0zona incerta; VL\u00a0=\u00a0nucleus ventralis lateralis; STN\u00a0=\u00a0nucleus subthalamicus; FF\u00a0=\u00a0Forel\u2019s Field* MS patients with completed surgical intervention and remaining in the study until end of follow up** an effort is made to distinguish between predominance of intention or postural tremor although many terms to describe tremor subtypes are used in the studies*** In this study, gamma-knife radiosurgery is used\u00a7 improvement as described in case reports or measured at the end of follow-up on any scale used in the study\u2020 in this study, MS patients were grouped together with patients with other movement disorders, no details are given for the MS-subgroup\u2021 thalamotomy was followed six months later by contralateral DBS electrode implantation in patients with bilateral tremorTable\u00a04Studies on DBS for the treatment of tremor in MSStudyN*patient characteristicsstimulation sitefollow-uptremor and disability assessmentpatients with tremor reduction (%)\u00a7patients with improved functional status (%)\u00a7patients with permanent adverse effects (n)Brice and McLellan, 1980 [17]2severe bilateral arm intention tremorbilateral subthalamic5 mo 6 moclinical examination2 (100%)2 (100%)**Nguyen and Degos, 1993 [71]1severe unilateral distal postural arm tremorunilateral VIM17 moclinical tremor and functional rating scales1 (100%)1 (100%)not reportedSiegfried and Lippitz, 1994 [88]9severe intention tremorunilateral (n\u00a0=\u00a08) or bilateral (n\u00a0=\u00a01) VIMnot reportednot reported9 (100%)not reportednot reportedBenabid et al. 1996 [13]4severe arm tremor\u2020VIM\u2020\u2265 6 moclinical tremor rating scale0 (0%)\u2021no detailed report\u2020intracerebral haemorrhage (1)Geny et al. 1996 [41]13severe postural tremor (n\u00a0=\u00a012), moderate intention tremor (n\u00a0=\u00a01)unilateral VIM8 to 26 mo mean: 13 moclinical tremor and functional rating scales9 (69%)12 (92%)MS relapse (3)Whittle et al. 1998 [99]***5severe arm tremorVLnot reportednot reportednot reportednot reportednot reportedHay, 1999 [46]1head and limb tremorunilateral thalamus2 monot reported1 (100%)not reportednot reportedMontgomery et al, 1999 [66]14disabling arm tremorunilateral VIMvariableclinical tremor rating scale15 (100%)not reportedMS relapse (1)#Schulder et al. 1999 [83]5severe bilateral postural and intention arm tremorunilateral VIM>6 moclinical tremor rating scale, patient self assessment of functional improvement5 (100%)3 (60%)Ms relapse (2)Taha et al. 1999 [90]2bilateral limb, head or voice tremor\u2020bilateral VIM (bilateral DBS or unilateral DBS plus contralateral thalamotomy)mean: 10 moclinical tremor rating scale2 (100%)not reportednot reported separately for MS subgroup\u2020Schuurman et al. 2000 [85]5severe arm tremorunilateral or bilateral VIM6 moclinical tremor and functional rating scales3\u20135 (60\u2013100%)$0 (0%)dysarthria (2) severe gait or balance disturbance (1) arm ataxia (1)Krauss et al. 2001 [53] Loher et al. 2003 [61]$$2severe tremorunilateral or bilateral VIM\u20203 to 24 mo, mean: 12 mo\u2020clinical tremor rating scales, assessment of video tapes2 (100%)not reportednot reported separately for MS subgroup\u2020Matsumoto et al. 2001 [62]3severe tremorunilateral VIM3 to 12 moclinical tremor and functional rating scales, novel movement analysis tool3 (100%)0 (0%)noneHooper et al. 2002 [49]10disabling arm tremorunilateral thalamus12 moclinical tremor and functional rating scales10 (100%)0 (0%)intracerebral haemorrhage (2) generalized seizure (2)Nandi et al. 2002 [70]1severe bilateral postural and intention tremorunilateral ZI12 moclinical examination1 (100%)1 (100%)increased dystonic posturing of left foot impairing ambulation (1)Berk et al. 2002 [14]12disabling arm tremorunilateral VIM12 moclinical tremor and functional rating scales, patient self assessment questionnairesignificant tremor reduction, not individually reportedno significant improvementwound infection (2)Wishart et al. 2003 [102]4bilateral arm tremorbilateral VL15 to 31 moclinical tremor rating scale4 (100%)4 (100%)MS relapse (1) dysarthria (1)Schulder et al. 2003 [84]9disabling arm tremorunilateral thalamus9 to 48 mo,clinical tremor and functional rating scales, patient self assessment8 (88%)3 (33%)MS relapse (3)Nandi et al. 2004 [69]10disabling arm tremorunilateral (n\u00a0=\u00a06) or bilateral (n\u00a0=\u00a04) VOP and ZI3 to 23 mocomputer-aided tracking taskssignificant tremor reduction, not individually reportednot reportedseizure (1) dysarthria (1) wound infection (1)Bittar et al. 2005 [15]y10disabling postural and intention arm tremorunilateral VOP (distal tremor), unilateral ZI (proximal tremor) or unilateral VOP and ZI (mixed tremor)3 to 23 mo:clinical tremor rating scalenot individually reported, overall improvement of mean tremor scores: postural: 64% intention: 36%not reportedmonoparesis (1)VIM\u00a0=\u00a0nucleus ventralis intermedius; ZI\u00a0=\u00a0zona incerta; VL\u00a0=\u00a0nucleus ventralis lateralis* MS patients with completed surgical intervention and remaining in the study until the end of follow up\u00a7 improvement as described in case reports or measured at the end of follow-up on any scale used in the study** in this study two patients had complete surgery and in three, surgery was aborted. A worsening of dysarthria is reported in one patient, it is unclear whether this patients had completed surgery*** This study mostly discusses difficulties in target localisation and patient selection# in this study, one patient did not complete surgery due to an intraoperative VIM haemorrhage\u2020 in this study, MS patients were grouped together with patients with other movement disorders, no details are given for the MS-subgroup\u2021 For the whole group, tremor was \u201cinconsistently, less significantly [than tremor of Parkinson\u2019s Disease and essential tremor] or not improved. If improvement was achieved it lasted only a few months\u201d$ The exact number of patients with improved tremor cannot be ascertained the way the data is presented in this study$$ Both studies are on one patient cohort, the relevant data on MS patients are derived from both studies\nThe first study on thalamotomy for tremor in MS was published by Cooper in 1960 [25]. Brice and colleagues were the first to report improvement of tremor through continuous thalamic DBS in 1980 [17].\nStrategies for patient and treatment site selection\nThe earlier studies on thalamotomy used the thalamic nucleus ventralis lateralis (VL) as the target, whereas the nucleus VIM was chosen in most DBS studies and most later thalamotomy studies. This preference for the VIM is probably due to the experience with this thalamic nucleus in the treatment of tremor in Parkinson\u2019s disease (PD) and essential tremor (ET). Research groups in Oxford and London used the nucleus VOP and the zona incerta (ZI) as targets for both lesional surgery and DBS, but the results yielded with these targets [4, 69, 15] were not different from those reported for VIM or VL thalamotomy or stimulation.\nThe same researchers advocate the use of tremor frequency analysis during movement tasks as a method to identify patients likely to benefit from surgery [60]. This may be a valuable tool for patient selection, although it has only been validated in a small number of patients and studies in which this technique is used [4, 69, 15] do not report better results than studies without this selection method.\nThe site for lesional surgery or electrode placement is classically chosen relative to the site of the anterior and posterior commissures using a standardized atlas. More recently, surgeons tried to refine this placement strategy with microelectrode recording within the target area. Neurons discharging synchronous to peripheral tremor are identified and the treatment site is placed in an area where the most tremor related neuronal activity is found. [73, 59, 57, 58]\nThis method of treatment site selection is often used in studies on tremulous patients with PD and ET, but only a small number of MS-patients were included in these studies. [87, 13, 66, 62, 84] Since this method offers a theoretical advantage over the classical methods of treatment site selection, it should be further evaluated.\nOutcome after surgical treatment\nBecause of the many shortcomings of the published studies, the results need to be interpreted with great caution. It does, however, seem as if almost all patients experience tremor reduction immediately after thalamotomy, and roughly 70% of patients continue to benefit from thalamotomy beyond a follow-up period of one year (Table\u00a03). In the three studies on thalamic DBS with a follow-up period longer than one year, 69% to 100% of the patients experienced reduced tremor [41, 84, 102]. Functional improvement after both thalamotomy and DBS is much more variable and unfortunately not reported in many studies (Tables\u00a03 and 4).\nNiranjan published an interesting study on gamma-knife thalamotomy as a possible alternative to neurosurgery. In this study all three patients with MS-related tremor experienced marked improvement of tremor after radiosurgery. Unfortunately, no further studies have been published to evaluate this interesting non-invasive treatment option [72].\nThere are two trials in which thalamotomy and DBS were compared in patients with MS. In a randomized controlled trial conducted by Schuurman and colleagues [85] patients with ET, tremor due to PD and tremulous MS patients were randomized to undergo either VIM thalamotomy or DBS. Five tremulous MS patients were randomized to each group. After a short follow-up period of six months, there were no significant differences between thalamotomy and DBS in functional outcome for the MS subgroup. In the same study, fewer adverse effects were observed in the DBS group, except for the subgroup of MS patients, where adverse effects were about equal for both interventions (Tables\u00a03 and 4). Bittar and colleagues compared cumulative tremor scores for intention and postural tremor between patients undergoing VOP\/ZI thalamotomy or DBS in a non-randomized study [15]. Ten patients were in each group. Interestingly, after a mean follow-up period of 15 to 16 months, patients in the thalamotomy group had a better outcome (tremor reduction: 78% for postural tremor and 72% for intention tremor) than patients in the DBS group (tremor reduction: 64% for postural tremor and 36% for intention tremor). However, as there were more adverse effects in the thalamotomy group, no clear recommendations could be given.\nAdverse events reported for the neurosurgical interventions comprised increase of hemiparesis, dysarthria, dysphasia, mental changes, depression, seizures, intracerebral haemorrhage, subdural haematoma, wound infection and MS relapse. Thalamotomy was associated with a higher risk of adverse events than DBS. Bilateral thalamotomy carries such a high risk of adverse effects, that it is no longer recommended. If bilateral treatment is necessary, either bilateral DBS or unilateral thalamotomy followed by contralateral DBS are possible treatment options.\nOther treatment options\nElectromagnetic fields, limb cooling, physiotherapy, weight bracelets, orthoses and specialized software have been advocated as additional treatment options.\nSandyk and Dann reported a reduction of intention as well as postural tremor in three tremulous MS patients treated with pulsed electromagnetic fields [82], but these interesting findings have not been substantiated in a larger trial.\nAlbrecht and coworkers published a small clinical trial on the effect of arm cooling on intention tremor [3]. In their study, patients achieved significantly better results on a clinical testing battery after immersion of the tremulous arm in ice water. As this effect lasted for about 45 minutes the authors recommend limb cooling to achieve transient tremor control for activities such as working with a PC, signing a document or self-catherisation. In a similar study, Feys and colleagues report tremor reduction lasting for about 30 minutes after limb cooling with a special cooling device [35].\nWeighted wrist cuffs are a mechanical tool to reduce tremor amplitude, and one article reported their beneficial effects in three MS patients [28]. Although wearing bracelets decrease tremor amplitude and therefore may offer some benefit to tremulous patients, their effect on intention tremor is small and their use is therefore unlikely to yield important functional improvement. A more advanced computer aided tremor reducing orthosis provided functional benefit in a small case series including patients with MS [1].\nPhysiotherapy aimed at improving ataxia in MS was evaluated by Armutlu and coworkers. In their small pilot study they found that rehabilitative physiotherapy using Johnstone pressure splints was superior to physiotherapy alone [9]. Unfortunately no larger trials followed this pilot study, so that it remains uncertain which patients may benefit from which form of physiotherapy.\nThe use of a mouse driven computer system is a special challenge for tremulous MS patients. Feys and colleagues published a study on the use of specialized software developed to aid computer use in 36 tremulous MS patients and found significant improvement in the time needed to complete some basic mouse driven computer operations [36].\nConclusion\nThe treatment of tremor remains a great challenge for everyone caring for patients with MS. Drug treatment with currently available medication is unsuccessful in most cases and much more research on the pathophysiology and biochemistry of tremor production in MS will be necessary before an efficient medical treatment can be developed. Stereotactic surgery can be an effective means to treat severe tremor, but it is currently uncertain whether lesional surgery or DBS is the treatment of choice. Larger clinical trials comparing both interventions are needed. Other treatment options, including physiotherapy, tremor reducing orthoses, and limb cooling can lead to valuable improvements in activities of daily living.","keyphrases":["tremor","multiple sclerosis","ataxia","treatment"],"prmu":["P","P","P","P"]}
{"id":"Neuroradiology-3-1-1786189","title":"Reperfusion injury following cerebral ischemia: pathophysiology, MR imaging, and potential therapies\n","text":"Introduction Restoration of blood flow following ischemic stroke can be achieved by means of thrombolysis or mechanical recanalization. However, for some patients, reperfusion may exacerbate the injury initially caused by ischemia, producing a so-called \u201ccerebral reperfusion injury\u201d. Multiple pathological processes are involved in this injury, including leukocyte infiltration, platelet and complement activation, postischemic hyperperfusion, and breakdown of the blood\u2013brain barrier.\nIntroduction\nIn the treatment of acute stroke, restoration of the blood supply can reduce more extensive brain tissue injured by salvaging a reversibly damage penumbra of tissue [1]. This mechanism provides a rationale for clinical trials which have demonstrated that reperfusion after thrombolysis improves clinical outcome in selected patients with acute stroke [2].\nReperfusion, however, carries certain risks. Some patients experience disastrous outcomes in the form of fatal edema or intracranial hemorrhage following thrombolysis [3]. In some animal stroke models [4, 5], reperfusion after a long ischemic period can cause a larger infarct than that associated with permanent vessel occlusion. Thus, while reperfusion may reduce infarct size and improve clinical outcome in some patients, in others it may exacerbate the brain injury and produce a so-called \u201ccerebral reperfusion injury\u201d [4, 6, 7]. Cerebral reperfusion injury can be defined as a deterioration of ischemic but salvageable brain tissue after reperfusion.\nThrombolysis [2] and embolectomy [8, 9] usually result in reperfusion of the infarcted brain tissue and therefore carry the risk of causing reperfusion injury. Thus reperfusion injury deserves the attention of those interested in the diagnosis and treatment of acute stroke. Strategies to reduce or minimize cerebral reperfusion injury require the understanding of the pathophysiology of cerebral reperfusion injury, and the way the reperfusion injury is visualized by magnetic resonance imaging (MRI). Therapeutic options for preventing or attenuating cerebral reperfusion need to be considered.\nCerebral reperfusion injury: mechanisms and correlation with MRI\nLeukocyte infiltration and secondary ischemia as depicted with DW MRI\nLeukocytes play an important role in the development of cerebral reperfusion injury. During reperfusion, activated leukocytes interact with endothelial cells and plug capillaries, disrupt the blood-brain barrier (BBB) through the release of neutrophil-derived oxidants and proteolytic enzymes, extravasate from capillaries and infiltrate brain tissue, and release cytokines which mediate inflammation. These processes produce an inflammatory cascade, resulting in the deterioration of the salvageable penumbra [7].\nEvidence of the deleterious effects caused by leukocytes is provided by animal studies. For example, Zhang et al. [10], using a rat model of 2\u00a0hours transient focal cerebral ischemia induced by advancing a nylon monofilament to occlude the middle cerebral artery (MCA), showed that neutrophils accumulate at the site of neuronal injury 6\u00a0hours after restoration of cerebral circulation. The neutrophil accumulation occurred earlier and to a greater extent in reperfusion tissue than in tissue permanently deprived of blood supply [10]. Furthermore, this study showed that the infarct volume increased dramatically between 6 and 24\u00a0hours following the start of reperfusion, and that the period of maximal infarct expansion correlated closely with the time course of neutrophil infiltration.\nThe contribution of leukocytes to cerebral reperfusion injury is also supported by the beneficial effects of neutrophil depletion. Bednar et al. [11] investigated the effect of administering antineutrophil antiserum treatment on brain infarct size in a rabbit model of transient ischemia. The regional cerebral blood flow (CBF) of neutropenic rabbits recovered from less than 5\u00a0ml\/100\u00a0g per minute to 20\u201330\u00a0ml\/100\u00a0g per minute following reperfusion, while in the non-neutropenic rabbits it remained at less than 10\u00a0ml\/100\u00a0g per minute. Correspondingly, the infarct size was significantly smaller in the neutropenic animals. Similar results have been reported in rats treated with antineutrophil monoclonal antibodies [12].\nThe evidence from animal experiments points to the role of neutrophils in restricting CBF and increasing infarct size during reperfusion. High-quality evidence of reperfusion-induced secondary ischemic injury has been obtained using diffusion-weighted imaging (DWI) in animals. Furthermore, the evolution of reperfusion injury can be depicted by observing dynamic changes with DWI (Fig.\u00a01) [13].\nFig.\u00a01DWI images of a rat model with 1\u00a0hour of MCA occlusion (MCAO) followed by 10\u00a0hours of reperfusion. These images show the evolution of ADC at different times: before ischemia (control), at the end of MCAO, and at different time points of reperfusion. Note the transient recovery of ADC during the early phase of reperfusion, followed by secondary deterioration (reproduced with permission from Olah et al. [13])\nOlah et al. [13] investigated the dynamics of the apparent diffusion coefficient (ADC) in a rat model with 1\u00a0hour of MCA occlusion (MCAO) followed by 10\u00a0hours of reperfusion. They found that the ADC was decreased at the end of ischemia, then significantly improved during the first 2\u00a0hours of reperfusion (Fig.\u00a02), but decreased again at later time points with a secondary increase of hemisphere lesion volume. The ADC change most likely reflects a secondary ischemic injury. Although a potential role for apoptosis cannot be excluded, the observed secondary deterioration of ADC did not support apoptosis as a major mechanism, because the ADC decline indicated cell swelling and not loss of cell volume, which is a fundamental and universal characteristic of programmed cell death [14]. Similarly, Neumann-Haefelin et al., also using a rat MCAO model, showed that DWI abnormalities reversed transiently during the early reperfusion period but recurred after several hours of restored blood flow [15]. Likewise, Li et al., using a rat model with 10\u201330\u00a0minutes of temporary MCAO, observed that the DWI hyperintensity seen during the initial ischemic insult reverted to normal approximately 60\u201390\u00a0minutes after the start of reperfusion, with secondary hyperintensities emerging 12\u00a0hours after reperfusion [16]. The secondary increase of DWI signal intensity observed in these studies is likely a marker of cerebral reperfusion injury [15].\nFig.\u00a02Temporal evolution of mean relative ADC in the end-ischemic lesion volume in a rat model with 1\u00a0hour of MCA occlusion (MCAO) followed by 10\u00a0hours of reperfusion. Although a significant improvement in ADC attends reperfusion, there is a secondary deterioration likely as a consequence of the reperfusion-related injury (reproduced with permission from Olah et al. [13])\nSeveral interesting characteristics of the ADC changes during reperfusion have been reported in animal studies. First, the time of onset of the secondary reduction of ADC varies amongst different animal studies, ranging from 2\u201372\u00a0hours after reperfusion [12, 16\u201318]. The reason is probably due to the different durations of the initial ischemic insult and differences in the animal model used. Second, the secondary deterioration of ADC can be predicted by early changes of T2 during reperfusion. Olah et al. [13], in their rat model study, observed that an increase in T2 value early in the ischemic period predicts a secondary deterioration in ADC after reperfusion. This suggests the possibility of using T2 value clinically to predict the extent of reperfusion injury likely to follow recanalization, and may be useful in assessing patients\u2019 likelihood of benefiting from thrombolysis.\nHemorrhagic transformation (HT) follows hyperperfusion and disruption of the BBB (see below), but some of the imaging studies are reviewed in this section because there is a close relationship between the ADC changes and the subsequent HT after cerebral ischemia. In a rabbit model of thromboembolic stroke, Adami et al. [19] compared the percentage of pixels with very low ADC at various time points after the start of ischemia with the subsequent occurrence of HT (as determined on histopathological sections). The authors found that, at each time point, regions populated by pixels with an ADC value below the threshold of 550\u2009\u00d7\u200910\u22126 mm2\/s were likely to undergo HT, and that the percentage of pixels below this threshold correlated with the severity of HT. This result has also been confirmed in several human studies. In a group of acute stroke patients treated with intravenous recombinant tissue plasminogen activator (rtPA) within 3\u00a0hours from symptom onset, Selim et al. [20] demonstrated that the absolute number of voxels with ADC values \u2264550\u2009\u00d7\u200910\u22126 mm2\/s was much higher in patients with HT than in those without. The authors suggested that, although patients with both small and large DWI lesions have the tendency to develop HT, the volume of ischemic tissue with ADC \u2264550\u2009\u00d7\u200910\u22126 mm2\/s may best predict the risk of intracerebral HT after thrombolysis.\nADC values have also been reported to correlate with recanalization and clinical outcome in ischemic stroke patients undergoing local intra-arterial thrombolysis. Taleb et al. [21] identified two patient groups on angiographic findings: one group with more than 50% recanalization and the other with less than 50%. Relative ADC values were found to be higher in the first group of patients. Moreover, all patients in the recanalization group had a Barthel index of 80 or more, whereas eight of ten patients in the nonrecanalization group had a Barthel index of 50 or less.\nMolecular mechanisms of leukocyte infiltration\nThe mechanisms of leukocyte infiltration are characterized by leukocyte activation, leukocyte\u2013endothelial interaction resulting in accumulation in the vascular bed, followed by leukocyte extravasation into the interstitial space. Each of these mechanisms is briefly reviewed.\nThe accumulation of leukocytes, along with red blood cells and platelets during reperfusion, may plug capillaries, preventing the restoration of blood flow and resulting in a \u201cno-reflow\u201d phenomenon and secondary cerebral ischemia [22\u201327]. Del Zoppo et al. [27] used a microscopic imaging system in a baboon model of transient ischemia. There was significant capillary obstruction caused by leukocytes reperfusing the microvascular bed within 60\u00a0minutes after the restoration of cerebral circulation. The \u201cno-reflow\u201d phenomenon explains well the better recovery of regional CBF in neutropenic rabbits [11].\nThe molecular mechanisms by which leukocytes infiltrate the parenchyma during reperfusion have been elucidated. This multistage process can be conceptually divided as follows: (1) leukocytes \u201crolling\u201d on the endothelium, (2) firm adherence of leukocytes to the endothelium, and (3) transmigration into the tissue [1, 28\u201331]. Superoxide free radicals produced during ischemia and reperfusion cause the upregulation of endothelial P-selectin. Once leukocytes reach the ischemic territory the endothelial P-selectin interacts with its leukocyte counter-receptor P-selectin glycoprotein 1 [31]. This initial interaction facilitates the low affinity \u201crolling\u201d of leukocytes on the endothelium. Firm adherence is developed with subsequent interaction of the leukocyte \u03b22 integrins CD11a\/CD18 and CD11b\/CD18 with endothelial intercellular adhesion molecule 1 (ICAM-1).\nThe transmigration of leukocytes results from the expression of platelet-endothelial cell adhesion molecule-1 (PECAM-1) along the endothelial cell junction. Once the leukocytes infiltrate the parenchyma, they then release various chemical mediators including neutrophil elastase, reactive oxygen species (ROS), leukotrienes, and prostaglandins, resulting in increased microvascular permeability, edema, thrombosis and parenchymal cell death [1, 27, 32]. This process compounds the initial injury that occurred in these tissues during the hypoxic insult.\nPlatelet-mediated reperfusion injury\nPlatelets play a synergistic role with leukocytes in reperfusion injury. Several studies suggest that platelets are activated after cerebral ischemia and reperfusion [33\u201335]. Following activation, platelets may adhere to both leukocytes and cerebral microvascular endothelial cells, contributing to the \u201cno-reflow\u201d phenomenon and furthering tissue injury. Additionally, activated platelets release a variety of biochemical mediators of tissue injury.\nSeveral studies have confirmed that platelet activation occurs in patients with stroke. Zeller et al. [36] used flow cytometric detection of platelet adhesion molecules as a marker of platelet activation, and reported a substantially greater activation in acute stroke patients than in normal controls. Similarly, van Kooten et al. [37], using urinary excretion of 11-dehydro-thromboxane-B2 as a marker of platelet activation, demonstrated a 347% increase in the marker excretion in ischemic stroke patients compared with matched controls.\nChong et al. [38] investigated changes in platelet\u2013leukocyte adhesion using a rat model of transient MCA ischemia. The authors reported that platelet\u2013neutrophil adhesion was significantly increased from 16.6% in the control group to 39% in the reperfusion group (P\u2009<\u20090.01). Platelets in the stroke group expressed high levels of P-selectin. It was hypothesized that enhancement of platelet\u2013leukocyte adhesion may be mediated by increased P-selectin expression on the platelet membrane.\nStudies on the role of platelets in coronary reperfusion of the heart suggest that, in addition to causing mechanical obstruction, platelets can cause temporary vasospasm by releasing serotonin, thromboxane A2, and free radicals. This can cause diminished reperfusion of the ischemic territory [33]. Further, activated platelets can release mediators that may cause chemotaxis and migration of leukocytes, thus exacerbating the inflammatory cascade [33]. These mechanisms may also prove to be important in cerebral reperfusion injury after ischemic stroke.\nComplement-mediated reperfusion injury\nExperimental studies have shown that complement activation is an important component of reperfusion injury in several organ systems. Complement may be activated during reperfusion through the antibody-dependent classical pathway, the alternative pathway, or the MBL\/MASP (mannan-binding lectin\/mannan-binding lectin-associated serine proteases) pathway [39, 40]. Complement activation results in the formation of several inflammatory mediators, including anaphylatoxins C3a, C5a, and the distal complement component C5b-9 (membrane attack complex, MAC) [39]. C5a and the MAC are the best-characterized effectors of tissue damage within the complement system [41, 42]. C5a may stimulate leukocyte infiltration into the ischemia and reperfusion injury-affected tissue by its strong chemotaxis action. C5a may also further amplify the inflammatory response by inducing the release of several proinflammatory cytokines, including IL-1, IL-6, monocyte chemoattractant protein 1 (MCP-1) and tumor necrosis factor \u03b1 (TNF-\u03b1). MAC can cause cellular membrane injury directly and can increase cell membrane permeability. Furthermore, MAC plays an essential role in mediating the recruitment of leukocytes to the reperfused tissue via local induction of IL-8. Several agents that inhibit all or part of the complement components such as cobra venom factor (CVF) [43], C5a monoclonal antibodies [44], and Cl esterase inhibitor [45], have been shown to significantly attenuate the degree of reperfusion injury in animal models, providing further evidence that complement plays an important role in reperfusion injury.\nPostischemic hyperperfusion and PWI\nPostischemic hyperperfusion is recognized as a frequent occurrence in stroke. Several animal studies suggest that the restoration of cerebral circulation consistently results in a hyperperfusion phase. Hyperperfusion may contribute to the development of reperfusion injury by causing brain edema or hemorrhage. Further, following postischemic hyperperfusion, a phase of secondary hypoperfusion can occur, which results in harmful effects on the reperfused tissue [1, 46, 47].\nHeiss et al. [46] observed the effects of hyperperfusion in cats using a model of temporary MCA occlusion of varying durations (30, 60, and 120\u00a0minutes). In the 30-minute group, only a transient reactive hyperperfusion (compared with preocclusion values) was found. All animals in this group survived during the observation period, and no cerebral injury was found on histological study. In the 60- and 120-minute groups, the degree of hyperperfusion was significant, reaching up to 300% of preocclusion values. Most importantly, a clear difference was found between surviving and dying cats. In dying cats, postischemic hyperperfusion was long and severe, large infarcts developed and intracranial pressure increased fatally. In surviving cats postischemic hyperperfusion was transient and less severe, infarcts were smaller and intracranial pressure was significantly lower. A report of a similar study design suggested that the degree of hyperperfusion correlates significantly with occurrence of intracerebral petechial hemorrhages in the damaged cortex [47].\nPerfusion-weighted imaging (PWI) provides quantitative and qualitative maps of CBF, cerebral blood volume (CBV), and mean transit time (MTT), allowing identification of regions of relative hypo- and hyperperfusion [48]. Kidwell et al. [48] used PW MRI to characterize hyperperfusion in 12 patients following intra-arterial thrombolysis. Hyperperfusion was visualized in 5 of 12 patients. On day 7, 79% of voxels with hyperperfusion demonstrated infarction, whereas only 36% not showing hyperperfusion demonstrated infarction. Despite the voxel-by-voxel association of hyperperfusion with infarction, there were no significant differences in the degree of clinical improvement in patients with regions of hyperperfusion versus those without. It has been reported that reduction in volume of hypoperfused brain on PWI is a marker of response to treatment and improved function in ischemic stroke patients [49]. Larger future studies are necessary to assess the extent to which both hyperperfusion and hypoperfusion may result in unfavorable clinical outcome.\nBreakdown of the BBB and contrast-enhanced MRI\nBreakdown of the BBB during cerebral reperfusion may lead to the development of vasogenic edema, HT and infarction, all contributing to cerebral reperfusion injury. This hypothesis has been supported by several clinical and animal studies [5, 50\u201352].\nSage and Duffy [50] investigated the increase in BBB permeability after transient global cerebral ischemia in a rat model. Permeability of the BBB was increased at 15\u00a0minutes after reperfusion of the brain, and cerebral edema occurred 15\u201330\u00a0minutes after reperfusion. Another study reported a disruption of the BBB after 3\u00a0hours of reperfusion, following a 3-hour temporary occlusion [5]. In contrast, the BBB remained intact after 6\u00a0hours of occlusion in a permanent occlusion group. Moreover, the temporary occlusion group had increased ipsilateral hemispheric water content, suggesting that cerebral edema had occurred. In addition to these reports, numerous histological studies with various tracers have established that reperfusion may exacerbate vasogenic edema due to BBB disruption [6, 53, 54].\nT2-weighted imaging is useful for detecting vasogenic edema. Neumann-Haefelin et al. [15] used several MRI techniques to monitor the dynamics of secondary ischemic damage, BBB disturbances, and development of vasogenic edema during the reperfusion phase after focal cerebral ischemia in rats. After 2.5\u00a0hours of ischemia, signal intensity on both DWI and T2-weighted images increased rapidly in the previously ischemic region due to BBB disruption (enhancement on postcontrast T1-weighted images) and edema formation. More importantly, early BBB damage after reperfusion appeared to be predictive of relatively pronounced increases in T2 lesion size due to vasogenic edema at subacute time points (1 to 2\u00a0days).\nUsing a novel MRI marker that can image BBB disruption, Latour et al. [55] studied a total of 144 acute stroke patients, to test the association between reperfusion, HT and clinical outcome. Nearly two-thirds (63%) of the patients had evidence of reperfusion within 1\u00a0week. BBB disruption was more common in patients who reperfused (45%) than in patients who did not reperfuse (18%). Both HT and BBB disruption were more common in patients undergoing intra-arterial rtPA therapy (31% and 55%), than those not treated (14% and 25%). In the reperfused group, patients with BBB disruption were more likely to have a poor clinical outcome (63%) than those without disruption (25%). Moreover, early BBB disruption was an independent predictor of HT. This was a key study because it associated early BBB disruption with HT and poor clinical outcome in humans.\nContrast-enhanced T1-weighted MRI can detect disruption of the BBB during reperfusion. Under normal physiological conditions, contrast agent does not cross the intact BBB. Mechanisms that cause BBB disturbances, such as reperfusion injury, result in leakage of the contrast agent into the brain. In a recent study, rats were subjected either to permanent MCA occlusion, reperfusion after 1\u00a0hour (early reperfusion), or reperfusion after 3\u00a0hours of MCAO (late reperfusion) [56]. This protocol allowed monitoring the evolution of BBB disruption during permanent or temporary MCAO. Contrast agent extravasation was found in all animals with permanent MCAO and late-reperfusion animals, but in only 40% of early-reperfusion animals. Postischemic hyperperfusion was demonstrated in late-reperfusion animals, but not in early-reperfusion animals. Thus, using contrast-enhanced MRI, the disruption of the BBB was correlated with the hemodynamic and biophysical consequences of reperfusion.\nFenstermacher et al. [57] employed the Patlak plot processing of gadolinium-shifted T1 relaxation-time images to obtain quantitative estimations of BBB opening in rats. Following 2.5\u00a0hours of reperfusion, the \u201cblood-tissue distribution\u201d of gadolinium-T1-shifted protons was serially imaged every 3\u20134\u00a0minutes for 20\u201330\u00a0minutes. After that, the value of blood-to-brain transfer constants (K1) for gadolinium was calculated from the serial changes of the relaxation time via Patlak plots [58] of MRI-T1 datasets. The K1 for gadolinium in ischemia and reperfusion areas was around 200 times larger than that of normal areas, indicating a huge impairment of BBB function during ischemia and reperfusion. This approach may be utilized to monitor the time-course of BBB function in transient cerebral ischemia.\nThe disruption of the BBB has also been observed in ischemic stroke patients undergoing rtPA therapy. A retrospective study of 213 patients used the postcontrast enhancement of the cerebrospinal fluid (CSF) in FLAIR images (termed hyperintense acute reperfusion marker, HARM) to characterize early BBB disruption [59]. BBB disruption was more common in patients with reperfusion (45%) than in patients without reperfusion (18%, P\u2009=\u20090.006). In multiple logistic regression, reperfusion was the strongest independent predictor of early BBB disruption (OR 4.09, 95% CI 1.28 to 13.1, P\u2009=\u20090.018). HARM was associated with HT and worse clinical outcome. This is a key study for two reasons: first, the association between reperfusion, BBB disruption and HT was established in humans, and second, it may be possible to reduce the complications of rtPA by targeting adjunctive therapies against BBB disruption. In another recent study, three of the six patients receiving rtPA showed early parenchymal enhancement, and all three of them went on to develop HT, whereas none of the patients without early parenchymal enhancement developed hemorrhage [60]. The results suggest that early parenchymal enhancement may be significantly correlated with subsequent symptomatic HT and may be helpful in identifying patients at risk after thrombolytic therapy.\nPotential therapeutic strategies\nWith the progress made in the understanding of the mechanisms in cerebral ischemia and reperfusion injury, an increasing number of strategies have been developed for limiting or preventing further brain damage during reperfusion [61\u201364]. Many of these strategies interfere with leukocyte infiltration. Two previously described animal studies indicated that leukocyte depletion with either antineutrophil antiserum treatment or with antineutrophil monoclonal antibody leads to better recovery of regional CBF and significantly reduces infarct size after cerebral reperfusion [11, 12]. Similarly, anti-ICAM-1 monoclonal antibodies reduce neutrophil infiltration and lesion size after reperfusion in rats, while they are ineffective in permanent ischemia [65], suggesting that ICAM-1 may be an attractive therapeutic target that specifically addresses the problem of reperfusion injury.\nHowever, the translation of these promising antileukocyte strategies into effective therapies in humans has been disappointing. A randomized controlled trial (RCT) evaluated the use of enlimomab, a murine ICAM-1 antibody, in 625 ischemic stroke patients [66]. Patients were randomized to receive either enlimomab for 5\u00a0days or placebo within 6\u00a0hours after stroke onset. At 90\u00a0days, the modified Rankin scale score was worse in patients treated with enlimomab. Moreover, the hazard of death was 43% higher on enlimomab than on placebo. There are several possible explanations for the failure of enlimomab to produce the desired effects. It is possible that the positive results seen with anti-ICAM therapy in animal studies do not reflect the immunological response in human stroke. Alternatively, ICAM-1 therapy may be effective only after reperfusion [65], and it is believed that only a minority of patients experience complete reperfusion after ischemic stroke [67]. Thus, it may have been more appropriate to try enlimomab in patients who received thrombolytic therapy.\nAnother RCT randomized 966 ischemic stroke patients presenting within 6\u00a0hours of acute stroke to either placebo or UK-279,276, a CD11b\/CD18 inhibitor. Of these patients, 204 also received tPA when indicated [68]. The trial was prematurely terminated when an interim analysis showed that the trial would be unable to demonstrate the benefit of this therapy in terms of improvement in Scandinavian Stroke Scale score at 90\u00a0days.\nSome conventional drugs have been shown in animal experiments to provide effective neuroprotection. For example, studies in a rat model have demonstrated that both dextran sulfate and unfractionated heparin can effectively prevent leukocyte infiltration and reduce infarct volume after reperfusion [69, 70]. Although the actual mechanism is still unclear, these findings suggest a possible role for these agents alongside other therapeutic modalities that cause recanalization (i.e. intravenous\/intra-arterial rtPA, mechanical thrombolectomy) [69, 70]. Trials investigating this approach in humans have yet to be conducted. Besides these drug therapies, there are two management strategies, brain cooling and conditioned blood perfusion, which are reviewed in some detail because they can modulate several pathways\/mechanisms that contribute to reperfusion injury. Both strategies are promising, and interventional neuroradiologists may have a role in administering these therapies in the future.\nBrain cooling\nReports on the role of hypothermia in humans are encouraging. Two large RCTs established conclusively the neuroprotective effects and safety of systemic hypothermia after cardiac arrest [71, 72]. A meta-analysis of the results [73] showed that patients in the hypothermia group were more likely to be discharged with no or only minimal neurological damage (risk ratio 1.68; 95% CI 1.29\u20132.07). This translated to six as the a number needed to treat in order to prevent one death or serious neurological deficit.\nThere are no hypothermia efficacy studies in ischemic stroke patients. Several animal studies of temporary MCAO have shown that systemic hypothermia applied up to 3\u00a0hours after reperfusion has started is effective in reducing infarct volume (by 50\u201390%) and reducing animal mortality [74, 75]. Moreover, hypothermia exerts its neuroprotective effects partly by suppressing the BBB opening [76, 77] and by attenuating neutrophil infiltration in the ischemic penumbra [78, 79]. Hence, hypothermia exerts at least some of its neuroprotective effects by modulating mechanisms responsible for reperfusion injury.\nIn most clinical studies, hypothermia is induced by surface cooling with the use of cooling blankets, alcohol applied to exposed skin, or ice bags to groin, axilla and neck. Although whole-body surface cooling is the simplest and most cost-effective option for inducing hypothermia [80], it has two major drawbacks. First, it takes several hours to reach the target body core temperature. All studies report a 3- to 7-hour time period for cooling down to 32\u201334\u00b0C [81\u201384]. Target temperature should be reached as soon as possible to prevent reperfusion injury; the 3\u20137\u00a0hour time period to reach target temperature is too long and the therapeutic window will be missed for many patients. Theoretical models predict that intracerebral hypothermia can be achieved up to 30 times faster than whole-body hypothermia [85]. The second drawback of whole-body cooling is the high incidence of complications such as pneumonia, arrhythmias\/bradycardias, hypotension and coagulopathies [81\u201383, 86, 87]. Selective brain cooling may dramatically improve the safety profile of therapeutic hypothermia. Selective brain cooling can be achieved by means of an endovascular catheter that infuses the cerebral vasculature with cold saline. A recent study examined whether cold saline (20\u00b0C) infused intra-arterially into the ischemic territory of rats that had undergone MCA occlusion could selectively induce cerebral hypothermia and reduce brain injury from stroke [88]. Following cold saline infusion, cerebral temperature fell within 1\u00a0min to 33.4\u00b0C, while rectal temperature was maintained above 36\u00b0C at all times. A significantly reduced infarct volume was found in ischemic rats that received a local cooling infusion compared with control animals (Fig.\u00a03). Further clinical research is needed to explore the therapeutic potential of selective brain cooling and the yet-unknown adverse effects, as well as to establish the extent to which selective brain cooling specifically modulates reperfusion injury.\nFig.\u00a03Graph showing percentages of infarct volume in the four ischemic rat groups (stroke, stroke with local infusion of saline at 20\u00b0C and 37\u00b0C, and stroke with systemic infusion at 20\u00b0C). A significantly (P\u2009<\u20090.001) reduced infarct volume was found in ischemic rats that received a local cooling infusion compared with the rats in the other three groups (reproduced with permission from Ding et al. [88])\nConditioned blood reperfusion\nConditioned blood reperfusion is a therapeutic strategy in which the ischemic organ is perfused with autologous conditioned blood at the onset of reperfusion. Blood conditioned by filtration is depleted of leukocytes and platelets, both at levels far greater than 99.99%, and the complement cascade is modulated in such a way as to prevent the deposition of MAC onto ischemic tissue [89]. Filtering the reperfused blood in this manner may reduce further tissue damage after restoration of circulation [89, 90]. Blood can be filtered using the conditioned blood reperfusion application (CoBRA) filter (Pall B-1328 filter; Pall Corporation, East Hill, N.Y.) at the onset of reperfusion [89]. Withdrawn arterial blood can be filtered through the CoBRA filter and returned back to the cerebral circulation via an endovascular catheter. A small feasibility and safety clinical trial of blood filtering during primary percutaneous transluminal coronary angioplasty for acute myocardial infarction demonstrated that integration of the CoBRA filter into an active hemoperfusion system did not delay the procedure or cause any complications [89]. Furthermore, conditioned blood reperfusion resulted in an acute improvement in ventricular function. Animal models of hypothermic circulatory arrest have demonstrated improvement of neurological outcomes following conditioned blood reperfusion [91\u201393]. In one of these studies, pigs received blood filtered using the CoBRA filter [92]. Each pig received a common carotid artery filtered perfusion for 10\u00a0minutes. Pigs receiving conditioned perfusion had significantly better neurological scores than control pigs (P\u2009<\u20090.005). These findings suggest that multifocal intervention aimed at leukocytes, platelets and complement attenuation can dramatically improve neurological recovery from ischemic insult. Future studies will establish the efficacy of this strategy and the incidence of possible adverse effects, such as coagulapathies and infections.\nFurther studies are needed to evaluate the role of local hypothermia and conditioned blood perfusion in reducing postischemic reperfusion injury. It is possible, yet still speculative, that in the future interventional neuroradiologists may use these approaches following recanalization with thrombolytic agents or mechanical embolectomy to lessen reperfusion injury.","keyphrases":["reperfusion injury","cerebral ischemia","magnetic resonance imaging","hypothermia","conditioned blood reperfusion"],"prmu":["P","P","P","P","P"]}
{"id":"Ann_Biomed_Eng-2-2-1705503","title":"Optimisation of Biochemical Condition and Substrates In Vitro for Tissue Engineering of Ligament\n","text":"In this work, we analysed the effect of growth factors on in vitro cell proliferation and collagens synthesis by fibroblasts cultured for 72 h on different substrates (silicon sheet with or without 1% gelatin, and glass as control surface) for ligament tissue engineering. A human fibroblast cell line (CRL-2703) was used. The synthesis of type I and type III collagens were evaluated qualitatively and quantitatively by RT-PCR and confocal microscopy, respectively. Cell proliferation was evaluated by two methods: (1) MTT assay (2) cell cycle analysis. It was found that PDGF-AB stimulate the proliferation of fibroblast cultured on gelatin coated silicon sheet in dose dependant manner with a maximum effect at 10 ng ml\u22121. The exogenous TGF-\u03b21 induced the expression of type I and type III collagens in a dose and substrate-dependant manner. We deduce from this work that biochemical conditions and substrates have an important impact for optimisation of the tissue neo synthesis.\nINTRODUCTION\nTissue engineering is a multidisciplinary field at the crossroads of biochemistry, mechanics, engineering and materials science to develop substitutes for replacing injured or diseased tissues.14,15,25,48 For all applications, it is necessary to take into account of several points: cell source, growth factors, mechanical forces, substrates, and the behaviour of the cells on the substrate.\nSubstrates or scaffolds are defined as the matrix that provides a suitable environment for cell proliferation and differentiation. Some scaffolds have been prepared from natural and synthetic materials, such as collagen, poly (glycolic acid) (PGA), and copolymers of l-lactic acid and \u0454-caprolactone. It is necessary to improve the physical and chemical nature of scaffold for the superior induction of tissue regeneration. However, the chemical composition of the scaffold can have great influence on its biocompatibility.5,38 The source, solubility, degree of cross linking, physical form of the protein coating substrates, and the substrates itself can influence the cell functions like proliferation and metabolic activity.5\nCells and materials are two essential components in ligament tissue engineering. Materials could interfere with cells adhesion, proliferation, and differentiation. Currently, there are two common approaches to modify the materials to improve their biocompatibility, coating with biocompatible materials, or surface modification. As nearly all interaction between cells and artificial surfaces are mediated by a layer of absorbed protein, many proteins have been coated on the surface of target materials to promote adhesion, (i.e. fibrinogen, collagen, hyaluronan,...).13 Protein coated substrates are provided as a two dimensional substrates. Moreover, it has been shown that fibroblast attachment, morphology, proliferation, and collagen synthesis varied from different culture substrates and the type of the fibroblast obtained from different organs\/tissue.9\nAn appropriate scaffold for ligament regeneration must be able to provide mechanical strength to initially withstand the in vivo force and degrade safely at an appropriate rate in vivo.39In vitro studies, a planar artificial substrate (i.e. silicon membrane) is usually used for growing cells and subjected them to mechanical loading.\nFurnishing scaffolds with biochemical factors are also promising. Many biochemical factors including growth factors could promote tissue regeneration and\/or healing processes.13\nGrowth factors are known to play an important role in the healing response of connective tissues.27 Several reports indicate the significance of growth factors in the intrinsic healing process of injured ligaments.24,32 Many growth factors affect the tendon and ligament healing process after injury by stimulating extra-cellular matrix (ECM) synthesis and cell proliferation.3,17,21,47 It has been shown that exogenous Transforming Growth Factors \u03b21 (TGF-\u03b21) increased the collagen synthesis of human anterior cruciate ligament cells and human ligamentum flavum.22,35 Platelet Derived Growth Factor-AB (PDGF-AB), one of the major serum growth factor, is known to enhance cell proliferation and healing responses,26,46 as well as to increase rupture force, stiffness and breaking energy of the tissue.31\nThe definition of the optimal condition of culture, such as coating of the substrate, synergistic effect of the substrate and growth factors are required for ligament tissue engineering.\nIn order to define the optimal concentration of growth factors and the evolution of cells culture on silicon membrane, which will be subjected to cyclic stretch for later mechanical study, the objective of the present work was to investigate the influences of individual growth factors and substrates on the cell proliferation and ECM synthesis. In this work, we studied the cell viability, proliferation, collagen production of fibroblast seeded on the different substrates and the effect of PDGF-AB on the cell proliferation and TGF-\u03b2 on the ECM synthesis to allow the optimisation of culture conditions, which could be used for in vitro reconstruction of ligament\/tendon tissues.\nMATERIALS AND METHODS\nCells and Culture Conditions\nHuman skin fibroblast cell line (CRL-2703) was purchased from American Type Culture Collection (ATCC, No. 2584882, Manassas, VA). The cells were placed in 75\u00a0cm2 plastic culture flask with Dulbecco\u2019s modified Eagle\u2019s medium\/Nutrient F-12 (GibcoBRL, Life Technologie, France) containing 10% decomplemented foetal bovine serum (FBS) supplemented with 100\u00a0U\/ml penicillin, 100\u00a0\u03bcg\/ml streptomycin, 4\u00a0mM l-glutamine, 2,5\u00a0\u03bcg\/ml fungizone, 1% non-essential amino acids (GibcoBRL, Life Technologie, France), 150\u00a0\u03bcg\/ml l-ascorbic acid, 1.5\u00a0g\/l NaHCO3 (Sigma, France) and were incubated at 37\u00b0C [air (95%) and CO2 (5%)]. The medium was changed every 2\u00a0days. At confluence, cells were detached from the culture flask with 0.125% trypsin\u2013EDTA mixture (Sigma, France) for 10\u00a0min at 37\u00b0C, and then resuspended in complete medium until confluence.\nSubstrate Surface Treatment\nTransparent, elastic silicon sheet (thickness: 0.0254\u00a0cm) (Specialty manufacturing INC, USA) was punched out into round sheets (diameter: 1.4\u00a0cm). Glass slips (diameter: 1.4\u00a0cm) (Polylabo, France) and silicon sheets were sterilised by overnight immersion in 75% ethanol aqueous solution, air- dried, then placed in a 24-well multiwell culture plate, and sterilised by 10\u00a0min UV irradiation. Gelatin solutions (1% w\/v in distilled water) (Sigma, France) were sterilised by filtration before use. Cells were seeded into 24 wells culture plates at a density of 3\u00a0\u00d7\u00a0104\u00a0cells\/cm2 on different surface glass slips (control surface), silicon sheet, coated or not with gelatin for 24\u00a0h at 37\u00b0C to allow cell attachment.\nBiochemical Stimuli\nThe concentrations of growth factors used in this study included those noted to be effective in previous studies.37,42 The concentration range for TGF-\u03b2 was 0.12\u201325\u00a0ng\u00a0ml\u22121 and from 1 to 100\u00a0ng\u00a0ml\u22121 for PDGF-AB. In our study, five concentrations of TGF-\u03b21 (0.1, 0.5, 1, 5, 10\u00a0ng\u00a0ml\u22121) and four concentrations for PDGF-AB (1, 5, 10 and 50\u00a0ng\u00a0ml\u22121) were used. These concentrations were added to the cultures in order to determine their effects on the cell proliferation for PDGF-AB and collagen production for TGF-\u03b21. Cell proliferation and collagen synthesis were determined 72\u00a0h after the addition of the growth factors. Culture medium containing 10%FBS without growth factors was used as control.\nFibroblast Proliferation Experiments\nMTT Assay\nAfter 72\u00a0h PDGF-AB treatment, MTT 3-[4,5-Dimethylthiazol-2,5-diphenyltetrazolium bromide, blue Thiazolyl] (Sigma, France) was prepared in PBS at a final concentration of 2\u00a0mg\/ml. Then 125\u00a0\u03bcl of the solution were added to each well containing 500\u00a0\u03bcl of DMEM without red phenol. The cells were incubated for 4\u00a0h at 37\u00b0C to allow the yellow dye to be transformed into blue formazan crystals by the mitochondrial dehydrogenases. The insoluble product was dissolved by addition of 800\u00a0\u03bcl of acidified isopropanol (0.04\u00a0N HCl in isopropanol), and thoroughly mixed to dissolve the dark blue crystals. The supernatants were removed, centrifuged, and read within 30\u00a0min by using a Beckman DU-600 spectrophotometer (USA) with a wavelength of 570 and 700\u00a0nm was used as reference wavelength. The optical density difference OD\u00a0=\u00a0OD570\u00a0nm\u2212OD700\u00a0nm was estimated.\nCell Cycle Analysis\nAfter 72\u00a0h PDGF-AB treatment, cells were trypsinisation then collected by centrifugation (500g) for 10\u00a0min. Cells pellets were washed twice with 2\u00a0ml of PBS. 50\u00a0\u03bcl of the washed cell suspension (15\u00a0\u00d7\u00a0104 cells) were stained according the kit instruction (Coulter DNA Prep Reagents kit; Beckman-Coulter, Miami, USA) in order to analyse the cell cycle by flow cytometry (EPICS XL Coulter; Beckman-Coulter). Primary histograms were generated by gating the cells according to the surface and the peak of the fluorescence signal in order to avoid taking doublets into account. These histograms were then submitted to computerised calculations of the cell cycle by using polynomial analysis derived from the Dean and Jett model Multi Cycle AV (Phoenix Flow System, San Diego, USA). For each testing condition, the percentage of cells in each phase was determined. DNA content was analysed in at least 105 nuclei in each sample.\nRNA Isolation and RT-PCR\nTotal cellular RNA were isolated from fibroblasts after dissolution in TRIzol reagent (Invitrogen, France) according to the manufacturer\u2019s instructions. RNA were extracted using chloroform, then samples were centrifuged (9500g) for 15\u00a0min at 4\u00b0C. Isopropanol was added to precipitate the RNA and the samples were centrifuged for 10\u00a0min under the same conditions. The RNA pellet was washed by addition of 1\u00a0ml of 75% ethanol and centrifuged at 5600g for 5\u00a0min. The pellet was resuspended in 50\u00a0\u03bcl DEPC-treated water. The concentrations of RNA were determined spectrophotometrically at 260\u00a0nm and the purity of RNA were controlled by the ratio DO260\u00a0nm\/DO280\u00a0nm. The first strand cDNA was prepared from the RNA with reverse transcriptase (kit ThermoScript RT-PCR System) (BioRad, USA). The cDNA was amplified by polymerase chain reaction (Platinum Taq DNA Polymerase) (Invitrogen, France) using the primers shown in Table\u00a01. Gel electrophoresis of RT-PCR products was performed in a 2% agarose (Sigma, France) run in 0.5\u00d7 Tris\/-boric acid EDTA (TBE) buffer (Bio-Rad, USA). The gel was stained in a 0.5\u00a0\u03bcg\/ml ethidium bromide solution for 20\u00a0min and the images were obtained using a Bio-Rad GS-2000 Molecular Imaging System. Bands intensities were evaluated against background quantified by Quantity One Image software (Bio-Rad, USA) and normalized to \u03b2-actin.\nTABLE\u00a01.Prime sequence for RT-PCR.MRNAPrimer sequenceProduct size (bp)Type I collagen5\u2032-TCC CCA GCC ACA AAG AGT CTA CA-3\u20321555\u2032-GTG ATT GGT GGG ATG TCT TCG TC-3\u2032Type III collagen5\u2032-CTG CCA TCC TGA ACT CAA GAG TGG-3\u20324475\u2032-CCA TCC TCC AGA ACT GTG TAG G-3\u2032\u0392-actin5\u2032-ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG CG-3\u20328385\u2032-CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC-3\u2032\nImmuonofluorescence Labelling\nAfter incubation with TGF-\u03b21, the cells were rinsed with PBS. The cells were incubated for 45\u00a0min with anti-collagen I (1\/50 dilution) or anti-collagen III (1\/100 dilution) rabbit IgG polyclonal (Calbiochem, France) in DMEM without red phenol (GibcoBRL, Life Technologie, France) containing 0.5% (w\/v) BSA. The cells were then rinsed thoroughly in DMEM without red phenol and incubated with the second antibody, Alexa\u2122-488 conjugated goat anti-rabbit IgG (\u03bbex\u00a0=\u00a0488\u00a0nm, \u03bbem\u00a0=\u00a0515\u00a0nm) (1\/200 dilution) for 30\u00a0min in the dark. After washing with DMEM without red phenol, the cells were fixed with 1% para-formaldehyde (Sigma, France) in PBS for 10\u00a0min, and rinsed in PBS. The cells were observed under a fluorescence confocal microscope (SP2-Leica micro systems, Germany).\nStatistical Analysis\nDifferences in cell proliferation and collagen synthesis were analysed by one-way ANOVA analysis to evaluate differences between groups with PLSD Fisher correction (Statview IV\u00ae, Abacus Concepts Inc, Berkley,CA, USA). A p value less than 0.05 was considered statistically significant. All data were expressed as the mean\u00a0\u00b1\u00a0standard deviation (SD).\nRESULTS\nAll the works were realized on three types of substrates (silicon sheet, silicon sheet with 1% gelatin coating as testing surfaces and glass surface used as a control).\nEffect of PDGF-AB on Cell Proliferation\nWe studied the effect of PDGF-AB on fibroblast proliferation according to substrates and PDGF concentrations by two different and complementary methods.\nMTT Assays\nThe results of the MTT assay are summarized in Fig.\u00a01. These results were obtained from fibroblast cultured on different substrates at day 3 in the presence or absence of four different concentrations of PDGF-AB. On the glass and gelatin coated silicon sheet cultures, PDGF-AB induced a dose-dependent increase in the value of A570. On glass culture, the significant increase was observed in the range of 5\u201350\u00a0ng\u00a0ml\u22121 PDGF-AB (p\u00a0<\u00a00.01). On the gelatin coated silicon sheet culture, the significant increase was observed in the range of 1\u201350\u00a0ng\u00a0ml\u22121 PDGF-AB (p\u00a0<\u00a00.01). On the silicon sheet culture, the values of A570 decrease when the concentration of PDGF-AB was above 5\u00a0ng\u00a0ml\u22121.\nFIGURE\u00a01.Dose dependent effect of PDGF-AB on cell proliferation by MTT assays. Cells were treated with different concentrations of PDGF-AB for 72\u00a0h. The metabolic activity of fibroblast seeded on glass, silicon sheet with or without 1% gelatin was measured by MTT assays. # p\u00a0<\u00a00.05 versus control glass, *p\u00a0<\u00a00.05 versus control silicon sheet, \u00a4 p\u00a0<\u00a00.05 versus control gelatin coated silicon sheet, n\u00a0=\u00a012.\nNevertheless, we observed a loss of cells on silicon sheet at 50\u00a0ng\u00a0ml\u22121 PDGF-AB.\nCell Cycle Analysis\nTo confirm the MTT results, we also studied the effect of PDGF-AB on cell cycle (Figs.\u00a02 and 3). Cell cycle analysis was performed on day 3 of the culture. It was observed in the three types of substrates that PDGF-AB induced a concentration-dependant decrease in the percentage of cells in G0\/G1 phase and an increase in S and G2\/M phase.\nFIGURE\u00a02.Cell cycle analysis by flow cytometry. Representative cell cycle histogram of the cells cultured on glass (a), gelatin coated silicon sheet (b). After 3\u00a0days of treatment with PDGF-AB (1, 5, 10, 50\u00a0ng\u00a0ml\u22121), cells were recovered and DNA staining with propidium iodide (IP) was performed. Ten thousand cells were analysed for each sample, n\u00a0=\u00a09.FIGURE\u00a03.Effect of different concentrations of PDGF-AB on fibroblast cell cycle regulation. Cells cultured on glass (a), silicon sheet (b), gelatin coated silicon sheet (c). Cells were subjected to different concentrations of PDGF-AB (1, 5, 10, 50\u00a0ng\u00a0ml\u22121). The percentage of cells in each phase of the cell cycle was analysed by flow cytometry. Results are represented as mean\u00a0\u00b1\u00a0SD of three independent experiments for each substrate. p\u00a0<\u00a00.05 versus control, n\u00a0=\u00a09.\nOn gelatin coated silicon sheet, the percentage of cells in S-phase was higher than that without gelatin or glass (Fig.\u00a03C). Moreover, cells exposed to 10\u00a0ng\u00a0ml\u22121 of PDGF-AB had the highest percentage of cells in G2\/M phase (p\u00a0<\u00a00.01).\nEffect of TGF-\u03b21 on the Expression of Collagens\nEffect of TGF-\u03b21 on Type I and Type III Collagens mRNA Expressions\nThe gene expressions of collagens in cells cultured on different substrates were measured by RT-PCR. RT-PCR analysis of type I and type III collagen were performed at day 3 in the presence or absence of five different concentrations of TGF-\u03b21. The RT-PCR results on human fibroblasts seeded on the three substrates are shown in Fig.\u00a04. The quantitative results concerning type I and type III collagen are summarised in Fig.\u00a05.\nFIGURE\u00a04.Effect of different concentrations of TGF-\u03b21 on the mRNA expression of type I and type III collagen. Representative RT-PCR results of human fibroblasts seeded on glass (a), silicon sheet (b), gelatin coated silicon sheet (c). The OD of each band was normalised to the values of housekeeping gene, \u03b2-actin. The cells were cultured on different substrates in the presence of different concentrations of TGF-\u03b21 (0.1, 0.5, 1, 5, 10\u00a0ng\u00a0ml\u22121) for 3\u00a0days.FIGURE\u00a05.Quantitative measurement of the RT-PCR results. a: type I collagen, b: type III collagen. Results are expressed as mean\u00a0\u00b1\u00a0SD of a representative of three different experiments carried out in duplicate. Black bars represent cell culture on glass; bright grey bars represent cell culture on silicon sheet; dark grey bars represent cell culture on gelatin coated silicon sheet. # p\u00a0<\u00a00.05 versus control glass, *p\u00a0<\u00a00.05 versus control silicon sheet, \u00a4 p\u00a0<\u00a00.05 versus control gelatin coated silicon sheet, n\u00a0=\u00a06.\nThe mRNA expression of collagen type I is shown in Fig.\u00a05a. On the glass culture and gelatin coated silicon sheet cultures, there was no significant difference in the mRNA expression of collagen type I between the TGF-\u03b21 treated group and the control group. Otherwise, on the silicon sheet culture, the mRNA expression of collagen type I increased significantly in the presence of 0.1\u00a0ng\u00a0ml\u22121 TGF-\u03b21.\nThe mRNA expression of collagen type III is shown in Fig.\u00a05b. On the glass culture, we can see a cyclic effect of TGF-\u03b21 on the expression of collagen type III and it reached a maximum response at 10\u00a0ng\u00a0ml\u22121 TGF-\u03b2. Also, compared to control group, the mRNA expression of collagen type III was decreased significantly at a concentration of 0.5 and 1\u00a0ng\u00a0ml\u22121 TGF-\u03b21 (p\u00a0<\u00a00.05).\nOn the gelatin coated silicon sheet cultures, the significant increase in the mRNA expression of collagen type III was observed by the addition of 0.1 and 10\u00a0ng\u00a0ml\u22121 TGF-\u03b21 (p\u00a0<\u00a00.05).\nOn the silicon sheet culture, the mRNA expression of collagen type III decrease when the concentration of TGF-\u03b21was above 0.5\u00a0ng\u00a0ml\u22121.\nQualitative Expression of Type I and Type III Collagens\nIn parallel of the RT-PCR experiments, we carried out some experiments by confocal microscopy to visualise the expression of collagen. The protein expression of collagen type I and type III of the cells cultured on glass were revealed by indirect immunofluorescence staining. As shown in Fig.\u00a06, collagen type I occurred as spots dispersed around the cells, and collagen type III was organised as network-like fibre. There is no difference in the expression of type I collagen with the different concentration of TGF-\u03b21. In contrast, the expression of type III collagen was increased after the addition of TGF-\u03b21. The same expression model of collagens was observed on the other substrates (data not shown).\nFIGURE\u00a06.Immunofluorescence staining of collagen in monolayer cultures of fibroblast on glass visualised by confocal microscopy. (A) Type I collagen is identified on the 3rd day of culture by use of anti collagen type I antibodies. Only spots of this type of collagen were secreted by the cells (arrowhead). (B) Type III collagen is identified by use of anti collagen type III antibodies. It appears like network in the cell (arrow). (objectif 40\u00d7\/NA 0.80, Leica, Germany). Bar 40\u00a0\u03bcm. (a): Control cell (DMEM\u00a0+\u00a010%FBS); (b): Cell treated with 0.5\u00a0ng\u00a0ml\u22121 TGF-\u03b21; (c): Cell treated with 10\u00a0ng\u00a0ml\u22121 TGF-\u03b21.\nDISCUSSION\nAs cell proliferation and matrix synthesis are important parameters in tissue engineering, the aim of this study was to optimise the in vitro culture conditions on ligament\/tendon tissues for future experiments in bioreactors. The effect of PDGF-AB and TGF-\u03b21 on the cell proliferation and matrix synthesis of fibroblast cultured on three different types of substrates (silicon sheet with or without 1% gelatin, glass) for 3\u00a0days were investigated in this study. For the tissue regeneration, it is better to use three-dimensional scaffolds to investigate the effects of the properties of the material on cell behaviour. However, there are many parameters of the scaffolds to be considered, such as porosity, pore size, pore interconnectivity, surface property, topography and chemical composition. Therefore, in this study, two-dimensional substrates were used to investigate the influence of substrate surface property on the proliferation and ECM synthesis of fibroblast.\nFirstly, we used PDGF-AB, which was widely used as a mitogenic growth factor and as a chemotactic agent for cells such as fibroblasts.45 Our experiments demonstrated that cell proliferation was enhanced by PDGF-AB. This result was consistent with those of the previous studies on lung fibroblast,6,30 canine anterior cruciate ligament cell,8 human periodontal ligament (PDL) cells36 and human anterior cruciate ligament cell (ACL).33 PDGF increased the number of fibroblasts available to enter the cell replication cycle by stimulating termination of the G0 phase of the cell cycle. The results obtained in this study are similar to those reported by Letson27 and Schmidt46 which showed that PDGF would be an effective growth factor that stimulates resting cells to advance from G0 to G1 of the cell cycle.\nAlthough the addition of PDGF-AB resulted in significant increase in cell proliferation on all the substrates, the highest increase was found on gelatin coated silicon sheet culture. This result may be explained by the physical properties of the substrate surface. High cell proliferation was observed for the substrates with contact angles of 60\u201380\u00b0 as well as on protein coated culture plates such as gelatin.18 Park et\u00a0al. have demonstrated that mescenchymal stem cells (MSCs) cultured on collagen I or gelatin coated membranes expressed similar levels of smooth muscles (SM) markers.41 Hori et\u00a0al. reported that the morphology and the proliferation of MSCs were influenced by the type of substrate and culture medium.18 The use of gelatin coated silicon sheet as a substrate allows to enhance the cell adhesion, the differentiation and the proliferation.\nIn addition, the higher response was found to 10\u00a0ng\u00a0ml\u22121 PDGF on gelatin coated silicon sheet culture but not to the maximal dose as 50\u00a0ng\u00a0ml\u22121 PDGF-AB. This finding indicates that it exist a dose-dependant effect of PDGF-AB on cell proliferation in this culture model and 10\u00a0ng\u00a0ml\u22121 PDGF might be the optimal concentration. Indeed, our result was consisting with the previous experiment which demonstrated that 10\u00a0ng\u00a0ml\u22121 of PDGF promoted fibroblast attachment to collagen coated plates and proliferation.21,36\nSecondly, we investigated the effect of TGF-\u03b21 on collagen synthesis. TGF-\u03b21 is the prototype of a large family of growth factors regulating cell growth, development, differentiation, immune system and tissue repair. On the one hand, TGF-\u03b21 mostly inhibited the cell growth both in vivo and in vitro.2 It could arrests cell cycle progression at a point in late G1 phase.12,19,43 Furthermore, stimulatory effects of PDGF-AB have been demonstrated to be blocked by the presence of TGF-\u03b21.20,23 On the other hand, it has been shown that TGF-\u03b2 regulates production of the ECM, including collagen, fibronectin and tenascin.11,16,40,44\nTherefore, in the second step of our works, we study the effect of TGF-\u03b21 on the ECM synthesis. Our data showed that the exogenous TGF-\u03b21 influenced the expression of type I and type III collagen in a substrate- dependant manner.\nIn our study, the mRNA expression of collagen type III was higher than that of collagen type I when the cells were cultured on glass and gelatin coated silicon sheet for 72\u00a0h. During the early phases of ligament healing and remodelling more type III collagen is produced than type I collagen.1,10,49 Therefore the greater upregulation of type III collagen as compared to type I collagen is a positive indicator that the cells are secreting signals for the healing and remodelling process.\nHowever, on glass, we observed the highest expression of type III collagen mRNA in the control group. In the present work, we used culture medium containing 10% FBS. Although, we did not add any TGF-\u03b21 in the medium, it seemed that this increase was due to the presence of growth factors in the serum and which normally stimulate protein synthesis and other different biological processes.\nFurthermore, we found that TGF-\u03b21 effected the expression of type I and type III collagen synthesis by fibroblasts with a dose dependant manner. The expression of type III collagen mRNA on gelatin coated silicon sheet was significantly increased at 0.1 and 10\u00a0ng\u00a0ml\u22121 TGF-\u03b21. The results of this study are consistent with the previous work which demonstrated that 10\u00a0ng\u00a0ml\u22121 of TGF-\u03b21 may promote fibroblast attachment.21 In 2D cell culture, TGF-\u03b21 has been demonstrated to have a dose-dependent effect on collagen production.4,7,28,29,32,34\nIn summary, our results demonstrated that PDGF-AB promoted the cell proliferation, which is a basic step in the process of ligament repair. Our data also suggest that TGF-\u03b21 is an important cytokine during the early phases of ligament healing process for its role in the promotion of extra cellular matrix formation. These results allowed us to determine the main biochemical conditions of in vitro construction of ligament\/tendon tissues.\nOur results implied that it is better to use protein-coating substrate to improve the proliferation and the synthesis of collagen for later mechanical stretching study. Further studies are needed to determine the combination of these two growth factors and their effect on the biological processes.","keyphrases":["substrates","growth factors","cell proliferation","collagens synthesis","fibroblasts"],"prmu":["P","P","P","P","P"]}
{"id":"Pediatr_Radiol-4-1-2292492","title":"Gastroenteropancreatic neuroendocrine tumors in children and young adults\n","text":"We review the imaging findings of pediatric gastroenteropancreatic neuroendocrine tumors (GEP-NETs) using contemporary anatomic and molecular imaging techniques. A low index of suspicion can result in significant delays in diagnosis of pediatric GEP-NETs. A multimodality imaging approach, using both anatomic and functional imaging, is essential in the diagnosis, staging, and surveillance of these potentially malignant tumors.\nIntroduction\nNeuroectodermal tumors arising from the neural crest, such as neuroblastomas, form a large proportion of childhood neoplasms. Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are a small subgroup of neural crest tumors whose imaging findings are not well described in children. These tumors arise from neuroendocrine cells found in the pancreas, the gut, and its derivatives including the bronchial tree. The term \u201cneuroendocrine tumors\u201d encompasses carcinoid tumors, islet cell tumors, and amine precursor uptake and decarboxylation (APUD) tumors. Although these tumors can produce distinct clinical syndromes because of their secretory capacity, they are underdiagnosed in children, resulting in delays in detection [1, 2]. The small size of the primary tumor and its tendency to occur anywhere along the gastroenteropancreatic (GEP) system can make detection a challenge. Advances in anatomic imaging and incorporation of metabolic imaging techniques have led to improved detection of these tumors.\nSince 1990, 13 patients 21\u00a0years of age or younger were seen at our hospital with a diagnosis of a GEP-NET. The primaries were located in the pancreas (n\u2009=\u20094), lung (n\u2009=\u20094), appendix (n\u2009=\u20091), thymus (n\u2009=\u20091) and middle ear (n\u2009=\u20091), and in two patients the tumor was primary unknown. Six patients had functional tumors and were symptomatic for several months to 5\u00a0years (mean 2\u00a0years) with symptoms including diarrhea, peptic ulcer disease, abdominal pain, and weight loss. Metastases were present in 50% of these patients by the time a GEP-NET was diagnosed. Two of the three patients with pulmonary NET tumors presented with recurrent pneumonia. One patient with an unsuspected NET developed carcinoid crisis after resection of the tumor and required intensive care management. One patient had a history of a familial disorder, von Hippel-Lindau (VHL) disease, predisposing to NET.\nDiscussion\nClassification\nGEP-NETs have traditionally been classified into two groups: (1) carcinoids, which are subdivided by their origin into tumors of the foregut (lung, thymus, stomach, duodenum), midgut (jejunum, ileum, appendix, right colon), and hindgut (left colon, rectum); and (2) pancreatic tumors [3]. A more prognostically useful classification was proposed by the World Health Organization in 2000: well-differentiated NETs, which show benign behavior (group 1a); well-differentiated neuroendocrine carcinomas (NEC), which are characterized by low-grade malignancy (group 1b); and poorly differentiated NEC of high-grade malignancy (group 2) [4].\nBased on their clinical features, these tumors can be functional or nonfunctional. Functional tumors have clinical symptoms caused by hypersecretion of hormones such as gastrin, while nonfunctional tumors can produce clinically silent hormones such as pancreatic polypeptide. These tumors often produce more than one hormone, and the functional tumors are named according to the hormone responsible for the clinical syndrome, for example gastrinoma causing Zollinger-Ellison syndrome and insulinoma causing hypoglycemic syndrome. NETs can either be sporadic or occur as part of familial syndromes such as multiple endocrine neoplasia (MEN) I and II, VHL syndrome and neurofibromatosis type I (NF-I).\nImaging approach\nThere is limited information in the pediatric literature regarding the imaging findings of NETs. Most of the information regarding imaging of these tumors is obtained from adult studies. The quoted diagnostic performance of different imaging modalities in various studies is highly variable depending on local expertise, different stages of technology development, and the imaging protocols used. A multimodality imaging approach using both anatomic and molecular imaging is essential for detection, staging, and surveillance of these tumors [5].\nAnatomic imaging\nMultidetector CT Multidetector CT (MDCT) has been shown to have an accuracy of 92% and 96% for the detection of primary tumor and metastasis, respectively [6]. The advantages of MDCT include high temporal and spatial resolution, acquisition of isotropic data resulting in high-quality reformatted images, and bolus-tracking capability resulting in precise timing of the scan. However, it is imperative that the radiologist select appropriate imaging parameters (tube current, gantry rotation time, kilovoltage, table speed and detector configuration) to achieve a balance between image quality and radiation exposure [7]. Although in general multiphase examinations should be avoided in children to keep radiation exposure as low as is reasonably achievable (ALARA), optimal evaluation of NETs requires dual-phase imaging. Images are obtained during arterial and portal venous phases of enhancement after administration of intravenous contrast agent at a rate of 3\u20135\u00a0ml\/s (Fig.\u00a01). The appropriate delay after contrast agent injection and before scanning will vary with the size of the patient. In an average teenager, there will be a delay of approximately 25\u00a0s for the arterial phase and a delay of 60 to 70\u00a0s for visualization of the portal vein. This aids detection of NETs, which often demonstrate early enhancement with washout during the portal venous phase because of their hypervascular nature. Visualization of mural masses can be improved by using water as an oral contrast agent. MDCT can also be used to perform small-bowel enteroclysis to detect small mural and luminal lesions of gut NETs [8].\nFig.\u00a01A 6-year-old girl with history of abdominal pain and diarrhea for several months. Contrast-enhanced axial CT image in the arterial phase (a) demonstrates multiple enhancing liver lesions (arrows) that are difficult to discern on the portal venous phase (b) consistent with hypervascular metastases. Also note the thickening of the gastric folds due to elevated gastrin levels. Fine-needle aspiration of a liver lesion confirmed metastatic gastrinoma\nMRI MRI has the highest sensitivity in detecting liver metastasis [9]. Arterial phase images after contrast agent administration and fat-suppressed fast spin echo T2-W images are the most sensitive in detecting liver lesions because of their hypervascular nature (Fig.\u00a02). For the detection of pancreatic lesions, MRI has been shown to have a sensitivity of 94%, with the tumor being most conspicuous on fat-saturated T1-W images [10].\nFig.\u00a02A 14-year-old boy after resection of a bronchial NEC. Axial T1-W image (a) demonstrates a hypointense liver lesion that is moderately hyperintense on the T2-W image (b). Arterial phase image after gadolinium administration (c) shows intense early enhancement with wash-out to isointensity on the portal venous phase images (d), consistent with hypervascular metastasis\nSonography Although traditional transabdominal US examinations have low sensitivity in detection of small tumors, contrast-enhanced, endoscopic, and intraoperative US examinations have been reported to have higher sensitivities in the detection of small lesions [9]. Endoscopic US with color Doppler imaging has been shown to have sensitivities in the range of 79\u2013100% for the detection of small lesions in the pancreas head [11]. Intraoperative US examinations have also been shown to improve the accuracy of lesion detection to 97% in the pancreatic head (Fig.\u00a03) [12].\nFig.\u00a03A 21-year-old man with severe gastroesophageal reflux disease for 5\u00a0years and elevated gastrin levels of 5,258\u00a0pg\/ml (normal range 0\u2013100\u00a0pg\/ml). Intraoperative US image shows a 3\u00d73\u00a0cm hypoechoic mass in the pancreas (arrow) closely abutting the splenic vein (V). Surgical resection confirmed pancreatic gastrinoma\nMolecular imaging\nThe overexpression of somatostatin receptors and the presence of amine uptake and storage mechanisms allows targeted molecular imaging of NETs using somatostatin receptor scintigraphy (SRS) and metaiodobenzylguanidine (MIBG), respectively. The sensitivity of octreotide and MIBG scintigraphy is known to range between 78% and 100% and 36% and 85%, respectively [13]. 111Indium diethylenetriaminepentaacetate (DTPA) octreotide is the most widely used radiopharmaceutical for SRS of NETs and is currently the modality of choice in the evaluation of these tumors (Fig.\u00a04) [14]. Octreotide has high affinity for somatostatin receptors, especially subtype 2, resulting in good correlation between scintigraphic findings and the expression of somatostatin receptors on these tumors as determined by immunostaining. These receptors can also be targeted for radiotherapy with radiolabeled somatostatin analogues [14].\nFig.\u00a04A 6-year-old girl with a history of abdominal pain and diarrhea for several months (same patient as in Fig.\u00a02). SRS SPECT image (a) obtained 24\u00a0h after intravenous administration of indium 111 pentatreotide in the coronal plane shows multiple foci of increased uptake in the liver (arrowhead) with an intense focus inferior to the liver (arrow), which corresponds to the exophytic lesion arising from the pancreatic neck (arrow) seen on the coronal reformatted CT image (b). This is a surgically proven gastrinoma. c Photomicrograph of immunohistochemistry slide shows positive staining (brown) for somatostatin receptors\nPositron emission tomography (PET) has the advantage of high sensitivity, reasonable resolution, and the ability to perform whole-body scans (Fig.\u00a05). 18Fluorodeoxyglucose (FDG) is the most widely used tracer but has limited sensitivity in detection of well-differentiated and slow-growing tumors [15]. PET using novel tracers such as 6-[fluoride-18] fluoro-levodopa (18F-DOPA), a catecholamine precursor, shows promise in the evaluation of NETs [16].\nFig.\u00a05A 21-year-old male with history of metastatic neuroendocrine carcinoma with unknown primary. a, b 18-FDG PET images in the coronal plane demonstrate multifocal uptake in the left humerus, bilateral ribs, liver, iliac wings, and both femurs consistent with widespread metastatic disease\nFusion imaging\nCoregistration of molecular and anatomic modalities with SPECT-CT or PET-CT can aid in accurate localization of disease and areas of physiologic uptake (Figs.\u00a06 and 7). Fusion imaging with SPECT-CT has been shown to be more accurate than either SPECT or CT alone and resulted in management changes in 28% of patients in one series [17].\nFig.\u00a06A 19-year-old male after resection of bronchial NEC 7\u00a0years previously. a Surveillance CT demonstrates expansion of the left sacral neural foramen by a soft-tissue mass (arrow). b FDG-PET demonstrates the lesion to be FDG avid raising the suspicion of tumor. Surgical resection confirmed metastasis to the left S1 nerve sheathFig.\u00a07A 16-year-old boy after resection of pancreatic gastrinoma 7\u00a0years previously with recurrent symptoms of gastroesophageal reflux disease. a, b Coregistered SRS SPECT and contrast-enhanced CT images allow accurate localization of radionuclide uptake to a paracaval lymph node (arrow) and a perigastric (arrowhead) lymph node, both of which are less than 1\u00a0cm in short axis. Also note multiple hepatic metastases with intense octreotide uptake\nCommon pediatric GEP-NETs by location\nAppendiceal NETs\nAppendiceal NETs are the most common pediatric GEP-NETs [2]. While appendiceal NETs in adults are generally incidentally detected, 78\u201397% of appendiceal NETs in children are associated with appendicitis (Fig.\u00a08) [18\u201320]. In a series of 36 children with appendiceal carcinoids, the median age at diagnosis was 12.3\u00a0years, and three-fourths presented with acute appendicitis while the rest presented with chronic abdominal pain [21]. Although carcinoid syndrome has been reported in association with appendiceal NETs in adults, the largest series of these tumors in children failed to show any children with symptoms of carcinoid syndrome [18, 20]. These tumors occur primarily at the appendix tip and are generally less than 2\u00a0cm in size. The long-term prognosis is good, with metastatic disease being unusual in children with appendiceal NETs, as shown in two large series with a median follow-up of 10\u201326\u00a0years [18, 21]. However, appendiceal carcinoids with synchronous and metachronous adenocarcinoma of the colon have been reported, even in children [2, 18].\nFig.\u00a08A 21-year-old female with a history of right lower quadrant pain for 6\u00a0days. a Contrast-enhanced axial CT image demonstrates a distended (13\u00a0mm) non-opacified appendix (arrow) with minimal periappendiceal stranding. b Gross specimen of the appendix demonstrates obliteration of the lumen by the pale tumor that extends through the wall into the serosa near the tip (arrow)\nGastrointestinal NETs\nGEP-NETs in other parts of the gastrointestinal tract are less common in children. Duodenal NETs arise from gastrin cells and tend to produce Zollinger-Ellison syndrome [22]. Duodenal carcinoids are most common in the proximal duodenum and present as early enhancing intraluminal polyps or mural masses [23]. Unlike duodenal NETs, jejunal and ileal NETs arise from the serotonin-producing enterochromaffin cells and can cause carcinoid syndrome. Serotonin production results in imaging findings of mesenteric desmoplasia, bowel kinking, elastic vascular sclerosis, and intestinal ischemia [8]. Ileal NETs might present with abdominal pain or diarrhea secondary to increased intestinal motility or obstruction secondary to intussusception. In the hindgut, the most common location of NETs is the rectum. Most of these tumors are nonfunctional, with rectal NETs having a better prognosis than NETs in other parts of the colon.\nHepatic NETs\nThe liver is a common site of extra-appendiceal NETs in children. In a series of 13 children with extra-appendiceal NETs, the liver was the second most common site of disease (5 of 13 children) [1]. It is unclear whether these tumors represent true primary hepatic neoplasms or metastases from asymptomatic occult NETs. Like the primary NETs, liver lesions are hypointense on T1-W images, hyperintense on T2-W images, and demonstrate arterial phase enhancement.\nPancreatic NETs\nPancreatic NETs account for approximately 30% of pancreatic tumors in patients younger than 20\u00a0years. While insulinomas are the most common in adults, gastrinomas are more common in children. Hypersecretion of gastrin by gastrinomas causes recurrent or ectopic peptic ulcers (Zollinger-Ellison syndrome), or malabsorption diarrhea. Most gastrinomas are solitary lesions located in the gastrinoma triangle, formed by the junction of the pancreatic neck and body, the second and third parts of the duodenum, and the junction of the cystic and common bile ducts. Nongastrinoma neuroendocrine tumors can occur in any part of the pancreas [24]. Metastases are present in 60\u201380% of patients at initial diagnosis of gastrinoma, with the liver and lymph nodes being the most common sites of metastatic disease [25]. Most neuroendocrine tumors of the pancreas are hypointense on fat-suppressed T1-W images, moderately hyperintense to the pancreas on T2-W fat-suppressed images, and show moderately intense early enhancement [24]. Patients with familial NETs, such as MEN and VHL disease, might present with multiple tumors with the majority being nonfunctional (Fig.\u00a09).\nFig.\u00a09A 21-year-old female with history of VHL disease. a, b Contrast-enhanced axial CT images during arterial phase demonstrate an enhancing mass in the body of the pancreas (arrow). A second mass located in the uncinate process (arrowhead) adjacent to the portal vein was missed on initial reading. c, d FDG-PET axial images demonstrate two metabolically active foci in the pancreas, confirmed to be nonfunctional NETs at surgery\nPulmonary NETs\nPulmonary NETs are the most common extra-appendiceal NETs in children [1]. They are also the most common primary pulmonary neoplasm of childhood, accounting for between 16% and 80% of malignant lung tumors in this population [1, 26]. Children with these tumors tend to present with wheezing and atelectasis, in addition to the classic adult triad of cough, hemoptysis, and obstructive pneumonitis [27]. Most present in late adolescence, though these tumors have been reported in children as young as 3\u00a0years [28]. Patients might be symptomatic for approximately a year prior to diagnosis [27]. Bronchial carcinoids in children tend to arise in the main, lobar, or segmental bronchi, in regions of bronchial bifurcation. They typically present as hilar or perihilar masses, with associated findings of endobronchial obstruction such as air-trapping, atelectasis, mucus-plugging, and obstructive pneumonitis (Fig.\u00a010). The mass is usually a well-defined round or ovoid homogeneously enhancing lesion with slightly lobulated margins. The intense enhancement of the tumor is helpful for differentiating the mass from adjacent postobstructive parenchymal changes (Fig.\u00a010). Calcification might be present in up to 30% at histologic analysis. Carcinoid syndrome is unusual with bronchial NETs unless they have metastasized to the liver or are large in size with systemic drainage [29]. Intraoperatively, these tumors can release vasoactive mediators such as 5-hydroxyindoleacetic acid (5-HIAA), resulting in carcinoid crisis characterized by bronchospasm, hypotension, acidosis, and ventricular tachycardia. If the diagnosis of a NET is considered preoperatively, the life-threatening complication of carcinoid crisis can be prevented by pretreatment with octreotide [30].\nFig.\u00a010A 20-year-old man with a history of \u201casthma\u201d and recurrent pneumonia for 2\u00a0years. a Chest radiograph demonstrates right base consolidation and right pleural effusion with an in-dwelling chest tube. b Contrast-enhanced axial CT image demonstrates an enhancing right hilar mass (arrow) with right lower lobe obstructive pneumonitis. c Lung section at lobectomy demonstrates a fleshy circumscribed mass (arrow) with a small endobronchial and larger extraluminal component. The lung parenchyma is filled with cheesy purulent material (arrowhead) secondary to obstructive pneumonitis\nThymic NETs\nThymic NETs tend to be more aggressive than other foregut tumors and often have metastatic disease at initial presentation [9]. Not much is known about thymic carcinoids in children; adult studies have shown that while bronchial carcinoids are malignant in about 26% of patients, thymic carcinoids can be malignant in about 80% of patients [31]. They can present with local symptoms (such as superior vena cava syndrome), can be incidentally detected on radiographs, and can produce Cushing syndrome (Fig.\u00a011).\nFig.\u00a011A 17-year-old female with an incidentally detected anterior mediastinal mass. a, b Axial contrast-enhanced CT images after biopsy demonstrate an infiltrating soft-tissue mass in the anterior mediastinum with enlarged pretracheal, subcarinal and hilar lymph nodes, and pleural studding (arrow). c Bone window image demonstrates multiple sclerotic lesions in the vertebral body, sacrum, and iliac wings consistent with metastatic disease. d Photomicrograph of H&E-stained section shows round cells with regular nuclei and salt-and-pepper chromatin. e, f Immunostaining is positive for the neuroendocrine tumor markers chromogranin (e) and synaptophysin (f)\nMiddle ear NETs\nNETs can also occur in the middle ear cavity, which is a derivative of the first pharyngeal pouch of the foregut (Fig.\u00a012). Typical presenting symptoms of middle ear NETs include hearing loss, aural fullness, and tinnitus. Physical examination might reveal a mass behind the tympanic membrane. The imaging and histologic characteristics of these tumors can mimic those of paragangliomas. Although middle ear NETs are typically low-grade malignancies that have an indolent course, there is a 22% risk of recurrence and 9% risk of metastatic disease [32].\nFig.\u00a012A 13-year-old boy with a history of a mass behind the left tympanic membrane. a, b Axial CT images through the middle ear cavity demonstrate a soft-tissue mass centered posterosuperior to the ossicles. Surgical resection led to a diagnosis of well-differentiated NET\nConclusions\nA low index of suspicion for GEP-NETs in children combined with nonspecific clinical complaints can result in delay in the detection of these rare but treatable malignancies. A multimodality imaging approach using both anatomic and functional imaging is essential in the diagnosis, staging, and surveillance of these potentially malignant tumors in children.","keyphrases":["neuroendocrine tumors","tumors","children","carcinoid","islet cell","somatostatin receptor imaging"],"prmu":["P","P","P","P","P","R"]}
{"id":"Appl_Microbiol_Biotechnol-4-1-2279157","title":"The state of the art in the analysis of two-dimensional gel electrophoresis images\n","text":"Software-based image analysis is a crucial step in the biological interpretation of two-dimensional gel electrophoresis experiments. Recent significant advances in image processing methods combined with powerful computing hardware have enabled the routine analysis of large experiments. We cover the process starting with the imaging of 2-D gels, quantitation of spots, creation of expression profiles to statistical expression analysis followed by the presentation of results. Challenges for analysis software as well as good practices are highlighted. We emphasize image warping and related methods that are able to overcome the difficulties that are due to varying migration positions of spots between gels. Spot detection, quantitation, normalization, and the creation of expression profiles are described in detail. The recent development of consensus spot patterns and complete expression profiles enables one to take full advantage of statistical methods for expression analysis that are well established for the analysis of DNA microarray experiments. We close with an overview of visualization and presentation methods (proteome maps) and current challenges in the field.\nIntroduction\nThe last decade in life sciences was deeply influenced by the development of the \u201cOmics\u201d technologies (genomics, transcriptomics, proteomics, and metabolomics), which aim for a global view on biological systems. With these tools at hand, the scientific community is striving to build functional models to develop a global understanding of the living cell.\nThe analysis of the proteome as the final level of gene expression started out with techniques based on 2-D gel electrophoresis (O\u2019Farrell 1975; Klose 1975) and extended its reach with semi-gel-free and shot gun gel-free liquid chromatography\u2013mass spectrometry (LC\u2013MS)-based techniques in recent years.\nA comprehensive evaluation of the relative merits and weaknesses of gel-based and gel-free methods is beyond the scope of our review. Studies that compare the performance on similar samples (Wolff et al. 2006, 2007) indicate that the methods are complementary, i.e., their analytical windows overlap, but each has an exclusive set of proteins or modifications that were not identified with the other. Quantitative analysis based on LC\u2013MS techniques is still in an early stage when considering available software and algorithms. Here, we focus on the computerized analysis of 2-D gels which are widely used in the scientific community. 2-D gels may separate up to 10,000 protein spots on one gel (Klose and Kobalz 1995). In a suitably equipped and experienced lab environment, 2-D gels are easy to handle, and they can be produced in a highly parallelized way. The software has meanwhile reached a level that allows for routine analysis of a large amount of samples with an investment of time that is much smaller than the efforts needed for the wet lab work.\nThe routine application of potent and easy-to-use software systems has been enabled by several improvements in 2-D gel image acquisition and analysis technology over the last decades (Aittokallio et al. 2005; Dowsey et al. 2003). Among these milestones, one should mention the introduction of the first computer-based analysis systems, still without a graphical user interface, in the late 1970s, among them ELSIE (Bossinger et al. 1979; Vo et al. 1981), GELLAB (Lemkin and Lipkin 1981a, b, c; Lemkin 1989), TYCHO (Anderson et al. 1981), HERMeS (Tarroux et al. 1989; Vincens and Tarroux 1987), GESA (Rowlands et al. 1988), and LIPS (Skolnick et al. 1982). This first generation of 2-D image analysis programs was followed by Elsie-4 (Olson and Miller 1988), Melanie (Appel et al. 1991; based on Elsie-4), and QUEST (Garrels 1989) since the mid-1980s. These programs used X-Windows-based graphical user interfaces on computer workstations. In the following years, the QUEST, TYCHO, and Melanie programs evolved to the commercially available PDQuest, Kepler, and Melanie III. While in the beginning such software needed exceptionally equipped workstations, in 1989, Nonlinear (Newcastle, UK) introduced Phoretix, the first 2-D gel analysis software running on desktop PCs. With the dropping hardware prices, the Melanie and PDQuest systems were also ported to PCs.\nWhile until then none of the available systems gave visual feedback on the quality of spot matching, Melanie II introduced image adjustment based on a global polynomial transformation of the image\u2019s geometry. This simplified the comparison of the raw images but only incompletely addressed the problem of positional variations caused by the electrophoretical separation process. Horgan et al. (1992) used for the first time a superimposing of false-colored 2-D images to simplify the finding of differences in spot patterns. Unl\u00fc et al. (1998) and Bernhardt et al. (1999) suggested different approaches that use superimposed and congruent false color images for the comparison of 2-D gels. This technology was improved by establishing positional correction by image warping of the raw 2-D gel images and commercialized with the first version of the Delta2D software from DECODON (Greifswald, Germany), coinciding with Compugen\u2019s Z3 software in 2000 (Smilansky 2001). Both systems were able to completely remove distortions from the gel images and bring the spot patterns into congruency. See Table\u00a01 for a list of current software.\nTable\u00a01Current commercial software products for 2-D gel image analysisCompanyProductsBio-Rad, Hercules, CA, USA, www.biorad.comPDQuest, ProteomWeaverCompugen, Tel Aviv, Israel, www.compugen.comZ3 (discontinued)DECODON, Greifswald, Germany, www.decodon.comDelta2DGE Healthcare, www.gelifesciences.comDecyder 2D, ImageMaster Platinum*Genebio, Geneva, Switzerland, www.genebio.com*Melanie (ImageMaster Platinum)Nonlinear Dynamics, Newcastle, UK, www.nonlinear.comProgenesis, SameSpotsSyngene, Cambridge, UK, www.syngene.comDymension\nWith the ever-increasing capacity of available hardware, more advanced image processing methods became feasible. They use all of the available image information instead of condensing it to spot boundaries before processing. This simplifies image comparison and speeds up analysis dramatically but still produces expression profiles with gaps which significantly impede reliable gene expression analysis. The most recent milestone introduced by us in 2003 addressed this problem: An algorithm to combine the information of several gels into a so-called fusion gel makes it possible to generate a proteome map that is representative for the whole experiment (Luhn et al. 2003). On a proteome map, one can detect all spots of a whole experiment in a single gel image, whereas the average images proposed earlier suffer from dilution effects for weak and rare spots. The spots detected there can serve as a spot consensus pattern that is valid for the whole gel set of the experiment. The consensus spot pattern is then transferred according to the warping transform and used on all gels. This allows for 100% matching spots and, in turn, complete expression profiles for reliable statistical analysis (Voigt et al. 2006; H\u00f6per et al. 2006).\nDepending on the workflow, one can put 2-D gel image analysis software into two broad categories:\nSpot detection first: these are the classical packages where the image information is first condensed into a set of spot centers, boundaries, and possibly spot volumes for each image. Spot matching and subsequent creation of expression profiles are done based on the data about spot geometry and volumes.Image warping first: these are packages where image warping is applied to remove running differences between gels, based on the whole image information. Spot detection is a separate and independent step. The creation of expression profiles is critically informed (and improved) by the data about positional differences between gels that were gained in the first step.\nHistorically, the \u201cspot detection first\u201d workflow was the only feasible way to proceed due to the limitations of available hardware. Software based on the second \u201cimage warping first\u201d workflow, including Compugen\u2019s Z3, DECODON\u2019s Delta2D, and, most recently, Nonlinear\u2019s SameSpots, is able to overcome many of the difficulties in spot matching that are hard to deal with when different migration positions add to the uncertainty of multiple separate spot detections. With the subsequent introduction of consensus spot patterns (in Delta2D 2003 and SameSpots 2006), one is able to virtually eliminate matching problems by using consistent spot patterns throughout the experiment. Recent comparisons between separate spot detection and using a consensus spot pattern (Eravci et al. 2007) show that the latter approach is able to find substantially more differentially expressed proteins, in a much shorter time. In other words, valuable information is lost due to spot matching problems that are inevitable when using the classical approach. We expect other vendors to evolve their products to use a workflow that is based on image warping and consensus spot patterns over the following years.\nThe typical workflow of a 2-D-gel-based proteomics analysis using the \u201cimage warping first\u201d approach and consensus spot patterns can be described as follows (Fig.\u00a01):\nPerforming a biological experiment or selecting a biological object of interest. The first sample preparation step is freezing the sample in the current state. This includes inactivation of all cellular processes that may change the proteome composition, preventing protease action, disintegration of the cell material, keeping or bringing the proteins into solution, removing or destroying macromolecules that may disturb the subsequent steps of the 2-D protocol (RNase and\/or DNase treatment and centrifuging for cell debris removal). Alternatively or in combination with radiolabeling, covalent fluorescent labeling of proteins can be applied here.Bringing the proteins into the gel and performing the 2-D separation by combining isoelectrofocussing in the first and sodium dodecyl sulfate (SDS) electrophoresis in the second dimension. An alternate 2-D approach uses the combination of two detergent treatments that resolve the protein molecules differently resulting in a scattered diagonal spot pattern (2D-16-BAC- or 2D-CTAB\/SDS-polyacrylamide gel electrophoresis). A variety of staining techniques can be applied before or after separation to enable spot detection.Capturing the gel images by using scanners, charge-coupled device (CCD) camera-based, or laser imaging devices. Depending on the protein labeling or staining techniques, a compatible imaging device has to be chosen. The capturing process results in one or more digitized computer images per gel that can be displayed with common image analysis software. The image capture step transforms the quantitative information of the gel into computer-readable data.Correction of positional spot variations by image warping. 2-D electrophoresis results in spot patterns with variations in the spot positions between gels. Therefore, gel images are positionally corrected by a combination of global and local image transforms (image warping). The information about differences in spot positions that was gained in this step is reused later for image fusion and for the transfer of the consensus spot pattern.Image fusion and proteome maps condense the image information of the whole experiment into one fusion image, also called a proteome map. The proteome map contains the information of all protein spots ever detected in the experiment.Spot detection is performed on the proteome map. As a result, a consensus spot pattern is generated, which is valid for all gels in the experiment. It describes the position and the general shape of all protein spots from the experiment.For spot quantitation and building expression profiles, the consensus spot pattern is applied to all gel images of the experiment (Fig.\u00a02). The image transformation (step 4) assures that all spots of the consensus pattern arrive at their correct position. A remodeling step makes sure that the predetermined spot boundaries from the consensus are adapted to the real gray levels observed on the target image. All boundaries of the consensus pattern can be found on every gel.Expression profile analysis identifies interesting spots which will be marked for further analysis, protein identification, and interpretation.Fig.\u00a01Analysis work flow of a 2-D-gel-based proteomics experiment in Delta2D. 1 Sample preparation; 2 2-D gel electrophoresis; 3 2-D gels are stained\/detected and digitized; 4 spot positions are aligned across gel images by warping; 5 a proteome map\/fusion gel image is generated by combining the images using a union fusion; 6 the union fusion image serves as basis for constructing the consensus spot pattern for the whole experiment; 7 the consensus spot pattern is transferred to all images and subsequently remodeled; 8 expression profiles are extracted and analyzed to find relevant proteinsFig.\u00a02Consensus spot pattern applied to four gel images (a\u2013d), before remodeling of spot shapes. The consensus spot pattern is generated by spot detection on the synthetic fusion image (e) which was computed from the original images\nProtein staining\nVarious techniques are available to make the separated spots detectable (Fig. 3). A direct method that suggests itself is the detection of the UV-induced autofluorescence of proteins (Roegener et al. 2003). Unfortunately, it is still not available for routine analysis, although it generates promising results.\nFig.\u00a03Protein labeling, staining, and tagging techniques for the selective detection of proteins. By multiplexing detection approaches, image analysis may relate different subsets of the proteome such as phosphorylated or glycosylated proteins\nIdeally, a dye should bind noncovalently to the protein after a linear response curve. It should allow for a detection of very low protein amounts because protein concentrations in biological systems may vary by six or more orders of magnitude (Corthals et al. 2000). At the same time, saturation effects have to be avoided because they impede normalized quantitation. In practice, depending on the stain that was used, a 2-D gel image analysis may give quantitative or only qualitative results for most or only a subfraction of the most intense spots. Common approaches for the detection of protein amounts use dyes that ideally bind to proteins much stronger than to the gel matrix or other compounds accompanying the 2-D electrophoresis process.\nMost fluorescent dyes are more sensitive than quantitative visible stains (Table\u00a02). Like most absorbing dyes, fluorescent stains give a measure of the accumulated amount of proteins within the sample. There are ruthenium-based dyes (ASCQ_Ru) available that covalently bind to the already separated proteins (Tokarski et al. 2006). They also give a measure of the amount of protein, and they can be used like noncovalently binding fluorescent dyes, but after 2-D separation.\nTable\u00a02The most commonly used dyes in 2-D gelsDyePrincipleSensitivityQuantitationAmount\/signalCoomassie Brilliant BlueAbsorptionVery lowAfter calibrationNonlinearColloidal Coomassie BlueAbsorption(very) highAfter calibrationNonlinearSilver StainingAbsorptionVery highImpossibleLogisticSypro RubyFluorescenceHighYesLinearRuthenium II tris (bathophenanthroline disulfonate)FluorescenceHighYesLinearFlamingoFluorescenceHighYesLinearLava PurpleFluorescenceHighYesLinearKryptonFluorescenceVery highYesLinear\nThe most striking advantage of applying covalently binding dyes before separation is the possibility to run multiple samples in one gel. Provided that the dyes induce the same positional shift in a protein\u2019s position, samples separated in parallel will give exactly congruent 2-D patterns. As long as the number of samples equals the number of available dyes, 2-D patterns with perfect positional identity can be generated. Difference gel electrophoresis (DIGE) was the first approach that used such a sample multiplexing in Unl\u00fc et al. (1998) by simultaneously separating samples labeled with Cy2, Cy3, and Cy5 within the same gel. If the amount of samples exceeds the number of available dyes (3 dyes\u2009=\u2009max 3 samples per gel), more than one gel have to be used, giving rise to positional variation between gels just like in the traditional setups. Dye multiplexing allows for a quantitative normalization over several gels by using an internal standard, i.e., a mixture of equal aliquots of every sample under analysis (Alban et al. 2003). The internal standard is separated together with Cy3\/Cy5-labeled sample pairs on every gel and serves as quantitative reference.\nProtein stainings give a measurement of the current protein amount. They are well suited for knock out experiments or analyses of long-term experiments in which treatment or stimulus effects have time to manifest themselves as observable changes in protein levels. Staining techniques that measure the accumulated quantity of proteins can overlook minor changes in protein quantities because they disappear within noise or systematic errors. CVs between 25 and 45% for the same spot\/same sample separated on different gels were reported by different authors (e.g., Nishihara and Champion 2002; Eravci et al. 2007). This means that a 1.3-fold change of a protein species cannot be reliably detected. That is why for short-term stimulus response experiments, in vivo labeling techniques can give a more detailed picture of a changing proteome.\nCharacterizing specific protein properties\nThere is a broad range of techniques available that are able to detect specific protein features (Fig.\u00a03), such as phosphorylation. The general approach is to combine a conventional staining method that displays all separated proteins with a specialized stain that highlights protein features, giving separate images that can be recombined using software. The Diamond ProQ (Steinberg et al. 2003) and Emerald ProQ (Patton and Beechem 2002) stains bind specifically but noncovalently to phosphorylated and glycosylated proteins, respectively. Multiplexing the phosphoprotein or glycoprotein pattern with the total protein clearly indicates the modified protein species. For a quantitative analysis of phosphoproteins in a bacterial system, see Eymann et al. (2007). For the redox state detection of proteins, two assays for oxidized thiols based on a radiolabeled tag (Leichert and Jakob 2004) or a fluorescent tag (Hochgr\u00e4fe et al. 2005) have been suggested. In addition to visualizing the protein\u2019s thiol state, image multiplexing allows the calculation of the degree of oxidation. An assay for the detection of carbonyl groups which are an indicator for irreversible protein oxidation has been successfully applied by Dukan and Nystr\u00f6m (1999) or Mosterz and Hecker (2003). This approach uses immunodetection of 2,4-dinitrophenylhydrazone derivated carbonyl groups.\nProtein synthesis at a given point in time can be detected by using in vivo labeling with radioisotopes. Radiolabeling is perfectly suitable for the detection of changes in protein synthesis during stimulus response studies. Especially 35S radioactively labeled amino acids like methionine or cysteine or 14C-labeled compounds are applicable. After 2-D separation, the incorporated material is analyzed by phosphorimaging (Amemiya and Miyahara 1988; Johnston et al. 1990). This delivers data with a linear correlation between radioactivity and measured signal over nearly five orders of magnitude. The striking advantage of radiolabeling in stimulus\/response experiments is the ability to detect fast but relatively small changes in protein quantities (Bernhardt et al. 1999; Fig.\u00a04).\nFig.\u00a04Protein amount (green) and protein synthesis (red) in a heat shock experiment of Bacillus subtilis 168. The synthesis patterns can differ dramatically between different stimuli but can be easily related using the protein amount patterns\nThe spot patterns may be very different when only special protein subsets are imaged, so they are hard to compare. Fortunately, the combination of autoradiography with total protein staining can help to solve this problem. Autoradiograph and densito-\/fluorograph from the same gel can be aligned very easily (Fig.\u00a04) because, normally, only minor gel distortions can occur due to staining and washing steps. Images with the total protein amount may then help in finding correspondences between gels because total protein patterns change only to a small extent, so they can be easily aligned.\nProtein degradation has been measured by using in vivo pulse chase radiolabeling as well (Kock et al. 2004). The degradation of a radiolabeled subfraction of cellular proteins is observed in a time course experiment by using a series of 2-D gels and looking for proteins whose signals disappear with time.\nRadiophosphate 32\/33P labeling can be used for in vivo detection of short-time effects in protein phosphorylation in the cell. Eymann et al. 2007 suggested to support the analysis with a Diamond ProQ (Invitrogen) stained pattern. The Diamond ProQ stain binds highly specific to phosphorylated proteins but also to a lower extent to nonphosphorylated ones. This allows for the determination of landmarks between stained and radiophosphate-labeled protein patterns that can be used to superimpose 33P-labeled and stained protein patterns (Fig.\u00a05).\nFig.\u00a05Flamingo-stained protein amount (green), Diamond ProQ Phosphoprotein staining (red), and 33P in vivo phosphoprotein labeling (blue) in an exponentially growing B. subtilis 168 sample. While the green and blue subimages seem to be almost complementary, the red subimage highlights spots from the protein level pattern as well as from the phosphate autoradiograph, so it can be used to find correspondences\n2-D Western blots are used for the detection of immunogenic proteins as suggested by Haas et al. 2002. Western blots are also suitable for finding modified forms of known proteins (using protein-specific antibodies) or for the detection of protein features (phosphoaminoacid antibodies). Again, superimposing the stained gel and the Western blot can highlight the relevant spots. For a more detailed overview on protein detection techniques, see Patton (2002).\nRecording and preparation of raw image data\nDigital images of 2-D gels are acquired using scanners or CCD-camera-based systems. Scanners produce an image by moving a light source and sensor element with CCDs over the 2-D gel. The resolution of the final image is defined by the density of sensors within the CCD element, the speed, and the frequency of the measurements. In CCD-camera-based systems, the 2-D gel is projected onto a CCD sensor array through a photographic lens so the 2-D gel is measured as a whole. Because the photographic lens is an optical system based on refraction, removal of distortions is part of the system\u2019s image preparation. Techniques compensating for lightfield variations are often applied because the central CCD sensors collect more light than those at the borders. The number of single CCD elements on the camera chip determines the resolution of the final gel image. Hybrid systems aim to combine the speed of a camera with the resolution of a scanner by moving a camera over the 2-D gel to produce image tiles that are later assembled using image processing. The challenge is to accurately remove illumination differences and distortions caused by the photographic lens to avoid discontinuities in adjacent tiles. Some scanners have the ability to measure white light, fluorescence, and radiation within one device, as well as simultaneous scanning for different wavelengths. Scanners usually provide higher resolution than CCD cameras while consuming more processing time per image. For technical information, consult Miura (2003) and Tan et al. (2007).\nThe general rule in 2-D gel image analysis is that the quality of the raw data has a significant impact on the final result. Therefore, it is essential to avoid experimentally caused artifacts and to configure the scanning devices in the best possible way. Background, artifacts, and noise influence the spot detection and quantitation process. Gel disruptions may truncate spots, speckles may mislead the spot detection or distort quantitation, noise can cover low intensity spots, background increases quantity and reduces dynamic range, etc. Background may be caused by insufficiently erased imaging plates (phosphorimaging), insufficient destaining, fluorescing glass plates, gel coverings, and backings. Furthermore, misusing optical filters for fluorescence imaging may cause background. Noise can be produced by high photo-multiplier tube voltages, which leads to the amplification of random signals. Phosphor screens that have not been used for a longer time accumulate noise.\nMany software packages allow for postscan image manipulations. One has to distinguish between image manipulations that do not change the quantitative information and those that do, incurring some loss of data in the process. All operations that leave pixels intact do not change the measured data, e.g., rotations in 90\u00b0 steps, mirrorings, and cropping (removing areas from the images that do not contain information of interest). Linear enhancements of resolution and gray levels can be undone without data loss and do not influence quantitative data because normalization is used in spot quantitation. On the other hand, many operations that are used for image enhancement cause minor changes in spot detection and spot quantitation and should be avoided if possible, e.g., free rotations or free scaling change gray level distribution of the manipulated image.\nImage warping leads to changed quantitative data, so quantitation should be done on the original images, or the warping should incorporate a factor for volume compensation (Dowsey et al. 2006) to minimize quantitative side effects. Several types of image filtering algorithms are used by 2-D gel image analysis software to remove background and noise (Fig.\u00a06). These filters are applied within the software for correct quantitation and for optimizing the appearance of the image on the computer screen.\nFig.\u00a06Decomposition of the raw image into background, noise, and cleaned images. Image filters can be used to determine background and noise, leaving the quantitative protein spot information in the cleaned image. a Raw image, b speckles and noise, c background, d cleaned image\nFinally, there are operations that should definitely be avoided because they result in data loss: for example, gamma correction changes the gray levels nonlinearly, blurring, and converting to JPEG format loose data, etc. Another hazard lies in the application of general purpose image manipulation software to special purpose file formats. In the process of, for example, cropping a gel file in Photoshop, essential calibration information can be lost in the resulting file. Therefore, it is advisable to use specialized software (e.g., the software that came with the scanner, or a 2-D gel image analysis program) that understands the characteristics of the file format.\nRemoving variations in spot positions\u2014warping\nUnfortunately, the position of a protein on a 2-D gel fluctuates from separation to separation. Even a very experienced experimenter will not be able to produce \u201cperfect\u201d gels whose spot patterns show exact congruency (Fig.\u00a07a). Reasons for changing spot positions may be variations in the pH value of the running buffer, problems of incomplete polymerization of the gel matrix, current leakage (Gustafsson et al. 2002), air bubbles in the gel, or highly abundant proteins that may influence the pH gradient in the IPG gel by their own locally concentrated buffer capacity. Some of these problems can be mitigated by using the DIGE setup or similar techniques (see below) that let multiple samples comigrate on the same gel. However, differing spot positions will still occur in any nontrivial experiment that includes more than one gel. Differences in spot positions are a major challenge in image processing because they impede accurate spot matching and thus the construction of expression profiles.\nFig.\u00a072-D gel image registration by warping. Two images are combined pixel by pixel using a false color display (a). Vectors connecting corresponding points (spots) on both images are determined automatically (b). Transforming the image geometry (warping) according to the vectors produces an exact overlay (c). Corresponding spots (black color) as well as differences in spot patterns can be easily identified. Data about differences in spot position are used in later image analysis steps (image fusion, transfer of consensus spot pattern)\nIt turns out that variations in spot positions are localized, i.e., spots that are close together on the gels will have similar gel-to-gel variations in their position (Fig.\u00a07b). This suggests the possibility of eliminating the positional variations by applying an image processing technique called warping. In a more general context, the image processing task is known as image registration; it is, for example, used to combine satellite images from the same region that were taken at different times and angles. In a sense, image registration compensates for variations in the lab process that could not be controlled otherwise. During image registration, similar regions or corresponding spots are searched on both gels in a more or less automated way. This results in pairs of landmark coordinates that link corresponding regions of an image pair to each other. The actual transformation of one image to fit another is not linear (i.e., no simple rotation or scaling suffices), and it can vary considerably between regions. It is important to note that automatic image registration uses the entire available image information and can be done independently of spot detection. Artifacts like gel disruptions, finger prints, and speckles may disturb the finding of the correct warp transform and should be experimentally avoided or removed by the analysis software. Image registration techniques have been introduced by Compugen\u2019s Z3 and DECODON\u2019s Delta2D in 2000 and are meanwhile adopted by other software packages. For surveys of image registration as applied to 2-D gels, see Aittokallio et al. (2005) and Dowsey et al. (2003).\nOnce suitable warpings between gel pairs have been produced and checked using, e.g., dual channel images (Fig.\u00a07b,c), they can be combined to allow for the registration of every gel in the experiment to every other gel. By knowing the necessary transforms between the images, the software can essentially remove all differences in spot positions as needed for, e.g., precise spot matching or fusion images (see below). In rare cases, no suitable warp transform can be found, e.g., if spots switched their relative positions or if the patterns are so different that no obvious landmark pairs can be found (e.g., stimulus\/in vivo labeling experiments, dye multiplex experiments). Under these circumstances, alternative experimental setups should be used. One example is the linking of gel images via comigration of differently labeled samples in the same gel (DIGE) or by using helper gels containing mixtures of two samples A and B to find an implicit warp transform between two gel images via a helper image (Eymann et al. 2007).\nSpot detection and quantitation\nThe goals of this step are to find the spot positions, find their surrounding boundary, and determine their quantities. There are two basic approaches that are used in current software: image segmentation and model-based quantitation. The segmentation approach partitions the image into nonoverlapping segments, essentially classifying each pixel as belonging to a certain spot, or as being part of the background between spots. Spot boundaries and quantities are then derived from the spot\u2019s pixels. The segmentation of the image can take various characteristics of the image into account: raw intensity, slope, and classification of pixels in the surrounding region. The advantage of this approach is that the image is clearly separated into spots and \u201cnonspot\u201d areas which are easy to assess by a user. If the software allows editing of spot boundaries, then any desired spot shape can, in principle, be obtained. Model-based approaches try to model a spot\u2019s intensity as a Gaussian normal distribution or some variant thereof. A spot\u2019s quantity and boundaries are then derived from the model (Fig.\u00a08). The use of a Gaussian is motivated by the \u201c3-D shape\u201d of spots (Fig.\u00a08) and by general considerations on diffusion processes in the gel. Model-based approaches limit the range of possible spot shapes, thus leading to more \u201cnatural\u201d outlines. On the other hand, irregular spot shapes are poorly represented by simple models. Spot models can be used in the subsequent quantitation, with overlapping spots being represented as the sum of multiple single-spot models. Delta2D offers a hybrid between segmentation and modeling: starting with segmentation, spots are modeled as Gaussians, and their nonoverlapping boundaries are derived from the models (Fig.\u00a09).\nFig.\u00a08Spot boundaries for high (a) and low abundance (b) spotsFig.\u00a09Spot boundaries produced by segmentation (a) and subsequent modeling (b)\nThe particulars of the methods used for spot detection are often proprietary information of the software vendors, and thus not publicly available. For a survey of published work, see Dowsey et al. (2003).\nThe spot detection process can be controlled by setting software-specific parameters, such as expected size of a spot in pixels, or even expected number of spots. Due to ambiguities in the gel images (merged spots, weak spots, noise), automated spot detection can only be a heuristic process in some areas. The user will, therefore, sometimes want to change the spot pattern by removing spots, splitting spot clusters, or joining spots. Manual intervention has a downside as well: Individual users have different perceptions about the \u201ccorrect\u201d spot shapes, so reproducibility between different operators of the software suffers. It is, therefore, advisable to reduce the necessary manual interventions to a minimum, e.g., by defining points as \u201cmarkers\u201d for the creation of new\/splitting of existing spots and letting the software determine the adapted boundaries.\nIn the simplest case, one assumes that gray values found in the image file are directly proportional to image intensities and, by extension, protein quantity in the small gel area corresponding to the pixel. However, more advanced imaging equipment utilizes calibration information that should be used to arrive at correct quantities:\nCalibration inherent in the file format (Fig.\u00a010). Some imaging devices can measure more intensity values than what fit into the available image file formats. For example, if the imaging device can measure a range of 120,000 intensity values, this range cannot be stored into a 16-bit TIFF image because the file format only provides 65,536 possible gray levels per pixel. One way to deal with this is to transform the measured intensity values linearly into the range of gray levels in the image file. Alternatively, because, for lower signal intensities, higher accuracy is desirable, a nonlinear function (e.g., square root transform as illustrated in Fig.\u00a010) is applied by some imaging devices. This provides a more precise representation of lower-intensity pixels at the price of lower accuracy for high intensities. The corresponding vendor-specific file formats, e.g., Fuji\u2019s IMG\/INF format (Fujifilm, D\u00fcsseldorf, Germany) and the GEL format used in devices by Molecular Dynamics (Sunnyvale, CA, USA) and GE Healthcare (Munich, Germany), have to be interpreted accordingly by the analysis software.\nFig.\u00a010Example of a gray level calibration curve that is used in special image file formats. Gray levels found in the image file have to be interpreted according to the curve before being summed up for quantitation. The curve has lower slope in the low intensity range resulting in better quantitative resolution for weak signals\nCalibration of the device. Another type of calibration may have to be applied to eliminate variance between imaging devices of the same model. While most of the laser scanners in the market have a built-in autocalibration, many flatbed scanners need to be calibrated manually. This can be done by using calibration wedges which are offered by several resellers, e.g., Stouffer Industries (Mishawaka, IN, USA), Danes-Picta (Praha, Czech Republic), UVP (Upland, CA, USA). Secondary calibration can be applied if the user knows about the relationship of protein amount and measured signal.\nFinally, one can take into account the dye-specific response curve for protein staining. Protein concentration wedges may help to find a fluorescent or absorptive stain-specific transfer function that may help to derive the protein amount from the emitted light or the measured absorption, respectively. It is to be expected that even different protein species have different response curves resulting from their biochemical properties.\nImage background is generated by material that is stained but not part of a protein spot. The method of background subtraction can have a significant influence on spot quantities; therefore, it is important that background quantities are made explicit by the software instead of being silently subtracted from a spot\u2019s quantity. Background levels can vary considerably between regions on a gel and between gels. Some background subtraction methods are based on the gray levels at the spot boundaries, other approaches are based on the entire available image data. One example for a background estimation that is based on the spot boundary is DeCyder\u2019s (GE Healthcare) rule: Background is determined by the tenth percentile value of all intensity values on the boundary. A background model based on local minima was used by Tyson et al. (1986). The Melanie II software (Appel et al. 1997) calculated background based on a polynomial that is fitted to image intensities. A related approach is the rolling ball method (Skolnick 1986) that determines background levels by fitting a sphere into the 3-D \u201clandscape\u201d of the image (see Fig.\u00a011). The sphere needs to be large enough such that the ball will not go too deep into the spots. Background levels are then determined relative to the center of the ball when it touches the image surface.\nFig.\u00a011Background subtraction using the rolling ball approach\nNormalization of spot quantities\nNormalization procedures aim to mitigate systematic differences between images. Such variation can occur in protein loading, imaging exposure times, and dye\/staining efficiency. Normalization starts with raw spot volumes and relates them to spot volumes on the same or on other gels. The simplest common procedure is to normalize for total volumes. The corresponding normalization rule is to set the total spot quantity to 100% on each gel image. For every spot, its proportion of total quantity is then computed. This rule results from the assumption that there is the same total protein amount on all gels. With this procedure, errors such as different protein loads, differences in staining times, scanner exposure time, or detector sensitivity can be compensated. For some instances, the total spot intensity is distorted by one or a few very strong spots leading to a skewing in the scatter plot (Fig. 12a). A countermeasure is to exclude some of the strongest spots (e.g., the 10% strongest spots) from the calculation of total spot intensity, i.e., removal of outliers.\nFig.\u00a012Scatter plot (a) of logarithmic spot quantities on two gels from different samples. Spots were normalized based on total spot quantity. b The quantile\u2013quantile plot (QQ plot) of the same data. Spots are sorted by quantity separately on each gel; spots of corresponding ranks are plotted. The QQ plot makes it easier to compare the spot volume distributions; in an ideal experiment, all points would lie on the diagonal line. The diagram shows that the quantity distributions on both gels are nearly equal, indicating a successful normalization\nA more general approach is to postulate that the spot intensity distributions (or some of the distribution\u2019s parameters) are equal between gels\/gel images. Therefore, e.g., DeCyder (GE Healthcare 2007) tries to match the distributions by adapting parameters of a normal distribution. Another approach is to use only a subset for normalization, e.g., by declaring that certain spots belong to \u201chousekeeping\u201d genes. It is hard to justify a subset like this, given that their being unaffected by the phenomenon under study is just a hypothesis. In principle, one could also spike the gel with a defined mixture of proteins with known quantities. We are, however, not aware of any published work that used this approach. More advanced normalization methods that were originally developed for the analysis of microarray data have been successfully applied in the context of 2-D gel analysis (Fodor et al. 2005). Spatial bias, i.e., a systematic increase or decrease in spot intensities in a gel region, can be observed in experiments and could be corrected by incorporating it in an error model analogous to those used in microarray analysis (Kreil et al. 2004).\nThe implicit assumption in all normalization procedures is that the majority of proteins are not affected by the phenomenon under study, or the technical variations in the process. Therefore, the global intensity distributions should be equal. The similarity of two distributions can be assessed using a quantile\u2013quantile (QQ) plot (Fig. 12b). It is a two-dimensional plot where spots are compared based on their rank, i.e., the strongest spot on one image is plotted against the strongest spot on another image, etc. For identical distributions, the QQ plot is a diagonal line. Systematic deviations like an overall higher intensity are easily visible.\nNormalization procedures that take into account only intensities on one image might be called \u201cvertical\u201d (using columns in an imagined spot intensity table); they modify values based on values in the same column (i.e., image). A different class of normalizations uses intensities from other gels\/images; these might be called \u201chorizontal\u201d normalizations. The most prominent example is the DIGE setup using a common internal standard on all gels, usually in the Cy2 channel (Alban et al. 2003). The internal standard is composed by mixing all samples from the experiment in equal amounts. Spot intensities are then normalized by dividing them by the intensity of the corresponding spot on the standard image (Cy2 channel on the same gel). In addition to the gel-to-gel variations, these normalization methods have to cope with differing backgrounds and differing distributions of spot quantities between dyes. The resulting statistics are very similar to the relative expression levels that are produced in competitive hybridization microarray experiments. For a critique of the then-current DIGE normalization used in DeCyder and an alternative normalization method, see Karp et al. (2004).\nAs a result of the spot detection and quantitation step, the user gets a variety of data on each gel spot, including normalized spot quantity, background, spot outline, position of the spot center, and spot quality measures.\nOf course, it would be desirable to relate spot intensities that are obtained from 2-D gel image analysis directly to protein molecule counts in the original sample. However, the relation between original protein quantity in the sample and measured spot intensity is influenced by various intervening factors:\nloss of sample during entry into the IEF gel,efficiency of transfer from first to second dimension,protein loss during staining,staining efficiency,a protein\u2019s staining curve over time,staining curve over concentration, anddye bleaching.\nGiven the biochemical diversity of protein molecules, it is to be expected that there are some proteins with a nonlinear relation between concentration and intensity. As a result, one expects to obtain only quantitative results of a relative nature, referring to the same protein species, similar to \u201cthis protein is two times stronger in sample A than in sample B.\u201d It is essential to track the protein across gels for this relative quantitation to work. Although the limiting factors mentioned above may seem a bit discouraging, recent controlled experiments (Eravci et al. 2007) show that even a 25% change in quantity can be reliably detected for a substantial fraction of the proteins provided that one controls experimental variation and software-related problems that have a diminishing effect on reproducibility (mainly spot matching, see below).\nBuilding expression profiles\nFor comparing the spot intensities over a whole experiment, each spot on a certain gel has to be mapped to the corresponding spots on the other gels in a process called spot matching. The quality of the matching depends on the quality and the reproducibility of electrophoretic separation and spot detection as well as on the methods employed by the software. In the ideal analysis, spot matching would produce exactly one expression profile for every protein species that is visible on any gel.\nThe challenges in spot matching lie in the differences in migration positions between gels, changes in the spot pattern itself (i.e., proteins that are missing or very weak in one of the samples), ambiguities in the gel data (e.g., more or less well-resolved spot clusters). Due to these difficulties, spot matching has been, alongside with spot editing, the most time-consuming step in the analysis when using traditional software. The traditional approach consists of doing separate spot detections for each individual gel image of the experiment. This results in differing spot patterns, even for replicate samples. In a subsequent step, the user has to revise these results and, if necessary, to correct missing or false positive spots. Furthermore, regions that were detected as separate spots on one gel but as a single spot on another have to be split or joined by hand. Unfortunately, these problems grow with the number of gels (Voss and Haberl 2000), severely limiting the statistical benefits that come with larger sample numbers. For example, in a study that used plasma from five different individuals, taken at six time points (Fodor et al. 2005), there were fewer than 150 spots matched across at least 40 of the total 45 DIGE gels, out of 2,385 spots total. Although it seems possible with enough manual work to improve expression profiles, both the inherent ambiguity in the images and the sheer amount of work necessary result in the acceptance of mismatches and gaps in expression profiles with classical packages. Spot-matching-related problems affect the statistical analysis: Gaps in expression profiles have to be treated as missing values. Mismatches are the equivalent of substituting a random value into the expression profile. Both effects decrease the statistical confidence substantially.\nTo overcome these problems, a different approach to construct expression profiles has been suggested and implemented in recent years. The approach is based on a spot consensus pattern that is derived from all gel images of an experiment and then applied to each gel image of the analysis set (Figs.\u00a01 and 2). The consensus pattern is produced by spot detection on a fused image that essentially contains all spots in the experiments (\u201cunion fusion,\u201d Luhn et al. 2003). Image fusion is a method to combine multiple images into a new, synthetic image where each pixel is a function of corresponding pixels in the input images. The resulting image looks like a real gel image, and, more importantly, all spots from the experiment are represented on it. Thus, spots that are present only on a few of the gels can be located in the fused image, and properly separated from those surrounding them. When the detection and editing of the consensus spot pattern is finished, spot boundaries are transferred back to the original images. This transfer uses the transformations that were produced from the warping step and used earlier to create the fused image. The spot sizes and intensities differ from gel to gel, so a spot remodeling step is applied to make the spot boundaries fit to the gray level distributions of the original gel images. The spot quantities are then calculated as usual by summing the pixel intensities within the spot boundaries. Because each gel has the same spot pattern (modified by the warping transform) a matching of the spots is quite easy and results in 100% spot matching and complete expression profiles. For spots that are absent on some gels, the method will nevertheless produce a spot quantity which will be near zero after background is subtracted. In essence, one could describe the spot transfer approach as quantitation of corresponding gel image regions. The resulting expression profiles were shown to be superior to those produced by the classical method (separate spot detections on each gel) both in terms of quality as well as time needed for the analysis (Eravci et al. 2007).\nAnalyzing gene expression\nHaving expression profiles at hand, we can now proceed to address the important biological questions of the phenomenon under study. Overall, the task is to distinguish those proteins that are relevant or interesting from those that are relatively unchanged. It is often the case that 2-D gel studies are exploratory in nature, i.e., that one wants to generate hypotheses and find proteins whose role can later be validated using other methods. An example of this is the discovery of protein biomarkers where one searches proteins that are consistently associated with a phenotype. Another group of applications is concerned with elucidating and interpreting subsets of proteins that are involved in a biological process, e.g., coregulated proteins induced by an external stimulus (Bernhardt et al. 2003). Methods ranging from the classical statistical tests to machine learning are applied to address these questions.\nAs in all experimental work, the design of the experiment is of crucial importance to its success. It is expedient to ask for statistical advice early to be able to make optimal use of resources and limited amounts of sample material. For all but the most exploratory experiments, one will want to use replicates: biological replicates to address genetic and environmental variability and maybe technical replicates (i.e., multiple separations of the same sample) to address the variability in the gel electrophoresis process itself. General guidelines concerning the use of replicates and statistical analyses accompanying published data have been put forward in Wilkins et al. (2006). Of course, a higher number of replicates increase the number of proteins that can be identified as being relevant with statistical confidence. A recommendation for the simplest possible case (two sample groups) is worked out in Hunt et al. (2005). Karp and Lilley (2005) present a power and cost-benefit analysis for DIGE experiments and DeCyder software that can be used as a model for other experimental setups. The type of replicate affects the statistical model that should be used: When applying statistical tests, technical replicates cannot necessarily be viewed as independent observations, so a more refined statistical analysis, e.g., nested analysis of variance (ANOVA), can give better results (Karp et al. 2005). Pooling of samples should be kept to a minimum because it diminishes (or prevents) the ability to assess biological variation within a sample type. In Molloy et al. (2003), the effects of technical and biological variance are investigated for samples of different types (bacteria, cell lines, primary cultures, human samples). For guidance on experiment design, see also Hunt et al. (2005). Of course, experimental variability is different between laboratories, and the software and analysis method used can have a crucial influence on the outcome\u2019s quality. The studies cited here use separate spot detections on each gel and often bias results by selecting only spots that were matched across a sufficient number of gel images.\nAt the level of expression profiles, 2-D gel analysis is very similar to the analysis of DNA microarrays, so a lot of the methods applied there can be used with minor adaptations. In fact, some of the work on machine learning on 2-D gel data predates the microarray technology (e.g., Appel et al. 1988). Let us highlight the main differences between 2-D gel data and microarray data before we proceed to describe the methods in detail:\nRunning differences between gels add a source of errors for spot matching, whereas in microarray data, matching is trivial because every gene is spotted at a known row and column.Spot detection in 2-D gels is much harder because spots may not be distinct.Gene information is not readily available for spots, so it is harder to correlate or cross-validate expression profiles with gene annotations.\nThe first two characteristics have the greatest impact on the quality of statistical analysis. Running differences and spot shape ambiguities (e.g., distinct spots on one gel vs overlapping spots on another) create spot matching problems that can lead to faulty assembly of expression profiles and gaps (missing values). By using consensus spot patterns (see above) that are transferred onto all images, one obtains consistent expression profiles. Of course, the ambiguities inherent in electrophoresis, e.g., masking of weak spots by strong ones in a nearby position (Pietrogrande et al. 2002; Campostrini et al. 2005), as well as operator-related variance, are still potential sources of errors and variation.\nCurrently, all 2-D gel analysis software packages come with some basic internal statistical analysis facilities. The advantage of using these facilities as opposed to external programs is that the analysis of expression profiles is tightly integrated with image analysis. For example, it is easy to see a section of all gels around a given spot that was flagged as being differentially expressed. All packages support the export of expression data in tabular form so more advanced methods can be used. Beyond the image analysis packages, there are a few commercial and noncommercial options for the statistical analysis of 2-D gel data. Genedata\u2019s Expressionist (GeneData, Basel) and DeCyder EDA (GE Healthcare) are products that offer multivariate statistics tailored for 2-D gel image data. General-purpose statistics packages like the free and open-source R (www.r-project.org) have extensive facilities for higher-level methods such as principal component analysis (PCA) and clustering. The R-based BioConductor package (www.bioconductor.org) provides access to a wide variety of data analysis methods and graphics facilities that were developed for microarray data. While these command-line-oriented packages offer great flexibility and control as well as some of the latest methods in the field, their learning curve can be steep. A more interactive and visual approach to data analysis is offered by the open-source TIGR Multiple Experiment Viewer MeV (Saeed et al. 2003; http:\/\/www.tigr.org\/software\/tm4\/mev.html). MeV combines interactive visualization of microarray data with a wide choice of analysis methods such as hierarchical clustering, self-organizing maps, and PCA (Fig.\u00a013).\nFig.\u00a013By using a consensus spot pattern in Delta2D (a), complete expression profiles (b) are generated. Profiles can be imported into DNA array analysis software (here: TIGR MultiExperiment Viewer, TMEV). With appropriate data transformations and normalization, many approaches for data analysis known from DNA arrays can be used for 2-D-gel-based proteome data. Hierarchical clustering (c) and self-organizing maps (d) group proteins by similarity of their expression profiles. Template matching (e) can be used to find proteins that conform to an expression pattern given by the user. Terrain maps (f) can give a high level overview of a data set where correlations of protein expression profiles are shown as distances in two dimensions, and protein density is shown in the third dimension (height)\nHypothesis-driven methods\nIn the simplest case, the experiment is a comparison of two samples A and B (e.g., treated vs control, mutant vs wild type, etc.) The task then is finding those proteins that show significant differences in expression levels. Normally, one will have several replicates per sample, and a statistical test is employed to find differentially expressed proteins. Certainly, the most popular test in this area is Student\u2019s t test, where the null hypothesis is that the means of expression levels in samples A and B are the same. Rejecting the null hypothesis then means that the protein under test is differentially expressed. One has to keep in mind that the t test makes the assumption that spot quantities within replicates follow a normal distribution which should in principle be tested separately. Additional tests are in use (ProteomWeaver, Bio-Rad, Hercules, CA, USA) that do not depend on the normality assumption (nonparametric tests) like the two-sample Wilcoxon test, also known as the Mann\u2013Whitney U test (Conover 2001). In addition to statistical tests, often a fold-change criterion is imposed on the spots. The reasoning behind this is that even true changes in intensity are hard to verify independently if they are small, and that, at least intuitively, a high fold-change in a protein\u2019s intensity is unlikely to be the result of mere chance. For small numbers of replicates, the fold-change criterion is sometimes the only one used.\nWhen applying statistical tests to 2-D gel data, one is faced with the so-called multiple hypothesis testing problem: For each expression profile, a separate test is done. Each test has a certain probability of giving a false positive result, i.e., a protein spot is declared to be differentially expressed, while the difference was due to pure chance. The large number of tests can produce a high number of false positives. For example, in an experiment with 2,000 spots per gel, an accepted false-positive rate alpha of 5% will result in 100 proteins that are found to be \u201cdifferentially expressed,\u201d although the difference is the result of mere chance. Various procedures try to overcome this problem by adjusting alpha in an adequate way. Bonferroni correction (Weisstein 2007a) controls the probability that there is one single error made in the whole analysis (family-wise error rate), i.e., that there is at least one false-positive protein. However, this method is usually too conservative because, in practice, it is acceptable to have some false positives, depending on the cost of repeating or validating the corresponding results. The proportion of false positives in the result set is controlled by the False Discovery Rate (FDR) approach (Benjamini and Hochberg 1995; Pawitan et al. 2005). The false discovery rate is the rate of false positive results among all profiles that were tested positive. While it is difficult to estimate the false discovery rate, the approach in Benjamini and Hochberg (1995) gives a simple procedure to control it, i.e., make sure the false discovery rate is below a given bound. Overall, the FDR approach allows one to strike a balance between the need to find statistically valid proteins of interest and the additional cost that is associated with following up on false positives. For background and applications of FDR and related methods, the reader is advised to consult the reviews in Manly et al. (2004) and Pounds (2006).\nFor more complicated experiment designs involving multiple factors, ANOVA can be used (Weisstein 2007b; Karp et al. 2005). The basic idea of the method is to find out to what extent the observed variances between different samples can be explained by the experimental parameters, as opposed to biological or technical variation.\nHypothesis-independent methods\nHypothesis-independent methods were developed for the discovery of patterns in large quantities of possibly high-dimensional data, in the fields of data mining and machine learning. As we expect a small number of fundamental biological processes to be reflected in the expression patterns of a large number of proteins, it makes sense to apply these methods to the analysis of 2-D experiments. Again, much of the work on microarray analysis can be transferred easily because the fundamental unit of data is an expression profile. When using separate spot detections on every gel, the missing values will have to be dealt with by the statistical method, for example, by missing value imputation. A large percentage of missing values decreases the utility of all statistical methods, that is why we recommend using the consensus spot pattern approach described above.\nHierarchical clustering refers to a group of methods that aim to group expression profiles or gels by similarity, forming separate clusters that can be further analyzed. Hierarchical clustering of gels can be used to detect outliers and to identify structures in the experiment. Ideally, the cluster composition will reflect the structure of the experiment, e.g., replicates and images from the same sample should end up in the same cluster (Fig.\u00a014). Clustering of images is a good first step in assessing the quality of the quantitative data. Clustering of expression profiles is done to identify proteins with similar behavior, implying that they are coregulated or at least correlated. Again, it is hoped that the cluster structure maps to functional groups or coregulated proteins. The global nature of the cluster display allows for a broad overview and the forming of hypotheses that can then be tested. However, in contrast to the situation in microarray data with 2-D gels, biological annotations of proteins are not available until after protein identification, making it harder to correlate expression behavior to function. The methods and software tools applied in the microarray analysis are applicable here, and the choices a user has to make are essentially the same (Meunier et al. 2007). The first choice is the normalization method, e.g., to standardize expression profiles to be of mean zero and variance one. Then a similarity measure between expression profiles has to be defined, e.g., correlation, or the Euclidean distance. Taken together with further choices such as using single, average, or complete linkage to connect clusters, these combine to create a variety of possible clusterings.\nFig.\u00a014Section of a heat map of a hierarchical clustering of an experiment consisting of 11 individuals with 5 replicate gels each, and 1 average fusion image per individual. Clustering was done for gels (columns) and expression profiles (rows) simultaneously. Gels are color coded by sample, replicates have the same color, sample A is colored in shades of blue, sample B is colored in shades of red. The clusterdendrogram for gels shows that replicates were clustered together, and samples are roughly grouped in the higher level clusters. The clustering did not use any sample or replicate information. The left-most replicate group is probably an outlier, as it branches off early in the dendrogram. Notice also the cluster structure in the rows, grouping proteins with similar expression profiles (row dendrogram not shown). Expression profiles were generated by spot transfer, hence the absence of missing values. Only about 20% of all expression profiles are shown\nAnother method that has been applied with great success in a variety of areas like semantic text analysis (Deerwester et al. 1990) and face recognition (Turk and Pentland 1991) is PCA or the related singular value decomposition. The basic idea is to find a projection from a high-dimensional space into a low-dimensional space (e.g., the plane) such that the structure, especially the variation in the data, is preserved. The principal components are the directions along which the variation is maximal. They can be interpreted as \u201chidden parameters\u201d of a process or experiment. The tutorial Shlens (2005) provides an accessible introduction, the book chapter Wall et al. (2003) gives background and motivation for microarray analysis.\nAs with clustering, PCA can be done for gels or expression profiles. In the first variant, each gel image is considered as a vector with coordinates given by the spot intensities on that gel. For example, an experiment with 24 gels from sample A and 24 gels from sample B and 1,500 spots on each gel would be modeled as a set (or point cloud) of 48 vectors in 1,500-dimensional space. The goal of PCA is then to find a projection of the point cloud in two- or three-dimensional space such that as much as possible the variation of the point cloud is preserved. One hopes that the gels from different samples will be in separate regions of the resulting diagram (Fig.\u00a015). The principal components can then be interpreted as \u201ctypical spot patterns\u201d or \u201ceigengels.\u201d Their coordinates can be analyzed to determine which spots are contributing most to the variance, making them candidates for protein identification and biological interpretation.\nFig.\u00a015PCA of 54 gels from 11 patients. Gels are color coded according to sample (sample a: shades of blue; sample b: shades of red). Notice how replicate gels are grouped closely together. We have chosen the projection onto the second and third principal components because it shows a good separation between samples\nWhen PCA is applied to the expression profiles, in our example, we would consider a point cloud of 1,500 vectors (one vector for each expression profile) with 48 dimensions (the expression levels on the 48 gels). The result is a display of the proteins where (hopefully) proteins with close positions are biologically related. Consider a time series experiment, where proteins are switched on and off in stages. If there is a \u201chidden parameter,\u201d such as a stage in the cell cycle, it will have a systematic influence on the expression levels, and thus increase the variance for the genes taking part in it. This increased variance will then become part of the directions that are used for the projection (the principal components). The principal components were also called \u201ceigengenes,\u201d they can be seen as \u201ctypical expression profiles,\u201d see, for example, Alter et al. (2000) and Holter et al. (2000).\nPresentation and visualization: from spots to proteome maps\nFor the presentation of an analysis, as well as throughout the whole process, going back to original images is often useful. Showing whole images or sections thereof in combination with the processed spot data (Fig.\u00a016) is therefore a feature offered by all analysis packages. Let us start with the display of a single gel image. The gray level resolution of current imaging devices (usually 16 bit for 65,536 different values) is much higher than what a computer screen can display. Therefore, the 2-D gel images have to be adapted or enhanced by histogram manipulations that cause a certain loss of information.\nFig.\u00a016Gel image tiles before (a) and after (b) multiway histogram equalization. After the equalization, the difference in the highlighted spot (middle row, left and right images) is clearly visible as shown in the expression profile (c)\nThe next more complex display is for comparison of a pair of gel images, e.g., treated vs untreated or wild type vs knockout. There are two possibilities: The gels can be shown side by side, or the gels are shown superimposed in a dual channel image (see Fig.\u00a05). For both types, a histogram equalization is necessary to give the user a visual estimate of relative spot quantities. Background can be removed before display, depending on the method that is used for background detection. Even after equalization, the visual impression of spot volumes can be misleading, for example, when comparing a small dark spot with a spot that is less intense but larger. Before gel images are superimposed in false colors, histograms should be equalized, and the chosen colors should be of similar apparent intensity. This makes sure that one image does not dominate the others and negatively influence the estimation of quantities. Ideally, the chosen colors are of similar luminance and located on opposite sides of the color wheel. Triple channel images are sometimes used (red-green-blue channels; see Fig.\u00a06) but difficult to interpret for the untrained eye.\nFor the display of multiple gels, one often shows tiles containing spots of interest in a side-by-side display. Here, a multiway equalization of histograms is necessary to give suitable visual impressions of spot intensities. Figure 16 shows the effect of multiway equalization on the display of a series of 2-D image tiles. With proper equalization quantity, the visual impression closely corresponds to the normalized quantities shown in the expression profile chart (Fig.\u00a016c).\nPresentations of large gel series, for example, for time course experiments, or large projects containing a lot of replicates, will require too much screen space for a side-by-side display. Image warping can help in making an animation of 2-D gel series from time courses by assuring that spot positions do not jump between frames. Bernhardt et al. (2003) used a combination of dual channel images showing protein amount (stained proteins\u2014false colored green) and current protein synthesis (35S methionine labeling\u2014false colored red) to monitor the fate of each protein during the development of a bacterial culture along a time line (http:\/\/microbio1.biologie.uni-greifswald.de\/starv\/movie.htm).\nEspecially for the collection of protein identifications and the presentation of proteome maps, it is very useful to condense spot pattern information from a multitude of gels into a single reference image. This can be done by collecting spot identifications on a single representative gel (Eymann et al. 2004). When using image fusion with the union or max intensity combination function, a proteome map can be generated that shows all proteins that were observed in the experiment. The map shows realistically looking spot shapes and does not ignore differentially expressed spots or dilute rarely occurring spots like it would be the case with average images (Tam le et al. 2006).\nA related feature was implemented in Proteomweaver (Bio-Rad) that allows for the combination of different narrow pI-gradients into a global proteome map. This composite map utilizes the much better resolution of narrow pH-gradient strips and supports image analysis as if the data came from a single, very wide gel.\nA proteome map normally serves as basis for further, especially physiologically oriented research and is comparable with a DNA array layout. The proteome map defines at which positions a protein spot was identified and can be recovered during gel analysis. A variety of proteome maps of many kinds of samples is available. Most of them show about a thousand different identified protein spots. Additional data can be attached to spots using labels, e.g., protein identification or functional information. Especially in gel regions with a very dense spot pattern, it is a big challenge to display the protein information without obscuring image information with spot labels.\nIn proteome maps, color can be used for encoding the association of a protein to a regulatory group (Voigt et al. 2006). In Fig.\u00a017a, proteins above a defined induction factor were grouped by showing them in the same color. If several induction groups are displayed in parallel, Venn diagrams for defining the protein set\u2019s color are used. The presented stress proteome map shows 15 combinations of expression behavior in response to four analyzed stimuli. In general, color coding of protein subsets can be applied for any way of allocating spots to (possibly overlapping) categories.\nFig.\u00a017Proteome maps with spot color coding. a Stress proteome map of B. subtilis 168 (compare Tam le et al. 2006). Spots were color coded according to their induced expression in response to different stress factors. b Proteome map of B. subtilis 168 in a glucose starvation time course experiment. Spots were color coded according to the growth phase\nColor coding also works for highlighting proteins that show elevated expression levels at certain points in a time course experiment. Also here, the information of a time-course-associated gel series is condensed in a proteome map. For each time point, a color is defined in the proteome map, which is then assigned to spots that have their maximal expression level at that time point (Fig.\u00a017b).\nIn contrast to a tabular display of spot quantities, a proteome map retains that spatial relations between spots as well as typical spot forms. It is, thus, easier to relate visually to newly produced gel images. The color coding of spots allows for easy visual identification of interesting subsets of the proteome.\nConclusions\nAdvances in software, algorithms, and experimental methods have kept 2-D gels competitive with and complementary to other methods for proteome analysis. While still not fully automatic, the software-based analysis is not the time-consuming bottleneck in a proteomics experiment anymore. With complete expression profiles, one is able to take full advantage of statistical methods (e.g., hierarchical clustering, PCA) that were established in the analysis of DNA microarray data. We see three main directions for future improvements of the available software: Firstly, the basic image processing algorithms, i.e., spot detection and image registration (warping), should be improved to a point where the need for a human operator is limited to checking the results. Ongoing research in automated registration and the potential for even greater processing power with grid computing (Dowsey et al. 2006) are promising steps towards this goal. The proteomics community can advance the state of the art by freely sharing raw data from a wide variety of experiments, and by establishing benchmarks. Standardization efforts of data formats and reporting requirements are being established for the gel electrophoresis process (MIAPE GEL, currently open for public feedback before publication at http:\/\/www.nature.com\/nbt\/consult); a similar standard for image processing and documentation of results is under development. Both are coordinated by the HUPO Proteomics Standards Initiative (www.psidev.info). The second direction for improvement is defined by the further adoption of advanced statistical methods for the analysis of large 2-D gel experiments. It seems plausible that large numbers of images from multiple studies could be aggregated to give richer \u201cfunctional profiles\u201d of proteins that show expression levels across a wider range of samples and conditions. The third direction is putting results from 2-D gel analysis into a larger context by combining spot data with functional annotations and data from other experimental techniques. Visualizing the result in biological terms (metabolic pathways, functional categories, etc.) will make it possible to gain new insights from the growing amounts of data accumulated by the \u201cOmics\u201d technologies.","keyphrases":["image analysis","statistics","warping","proteome maps","2-d gel electrophoresis"],"prmu":["P","P","P","P","P"]}
{"id":"Ann_Surg_Oncol-4-1-2277454","title":"Isolated Limb Perfusion and External Beam Radiotherapy for Soft Tissue Sarcomas of the Extremity: Long-Term Effects on Normal Tissue According to the LENT-SOMA Scoring System\n","text":"Background With the combined treatment procedure of isolated limb perfusion (ILP), delayed surgical resection and external beam radiotherapy (EBRT) for locally advanced soft tissue sarcomas (STS) of the extremities, limb salvage rates of more than 80% can be achieved. However, long-term damage to the healthy surrounding tissue cannot be prevented. We studied the late effects on the normal tissue using the LENT-SOMA scoring system.\nIn the 1960s and 1970s, the effectiveness of (neo)adjuvant isolated limb perfusion (ILP) with various cytostatics was explored in the limb salvage treatment of extremity sarcomas.1 The practice of performing extremity perfusions for sarcomas ended after Rosenberg showed the effectiveness of adjuvant radiation treatment in limb salvage. Amputations or exarticulations of the affected limb did not result in higher survival rates.2,3\nThere was a renewed interest in neoadjuvant ILP in the early 1990s, when Lejeune and co-workers added tumor necrosis factor alpha (TNF\u03b1) to Melphalan in the ILP treatment of locally advanced extremity melanomas and sarcomas. This resulted in high local response rates and high limb salvage rates with an acceptable local and systemic toxicity.4,5\nCurrently, an established limb salvage strategy for primarily irresectable soft tissue sarcomas (STSs) of the limbs consists of induction treatment with hyperthermic ILP with TNF\u03b1 and Melphalan, followed by delayed surgical resection and, in case of marginal or non-radical resection, adjuvant external beam radiotherapy (EBRT). With this combined treatment modality, short-term limb salvage rates of more than 80% are achieved, with long-term rates of 60%.5,6 However, as the number of long-term cancer survivors increases, late complications of therapy will become an increasingly important concern.7 Long-term damage to the healthy surrounding tissue cannot be prevented. Toxic effects of pre- or postoperative EBRT and intra-arterial chemotherapy have been extensively described in previous literature and include edema, fibrosis, neuropathy, limited mobility, impaired wound healing and, less commonly, pain, bone fractures and second tumors.3,8\u201311 Moreover, during ILP, normal tissue in the limb\u2014skin, subcutaneous tissue, muscle, nerves, blood vessels, bone and cartilage\u2014is exposed to the same concentrations of cytostatic agents active against the tumor, causing regional toxic effects, which lead to comparable late complications.12 Recently, we described the late vascular complications that occur after the TNF ILP treatment procedure.13 Functional morbidity and symptoms such as edema and pain can also be extremely invalidating, sometimes necessitating aggressive treatment. To evaluate all toxic long-term effects, we used the uniform, generally accepted toxicity scoring system \u201cLENT-SOMA\u201d. This system not only allows late damage to be classified in different gradations, but also allows data acquisition using three different methods: (1) subjective, the injury is recorded as perceived by the patient; (2) objective, morbidity is assessed during a clinical examination; and (3) management, indicates the active steps made to ameliorate the symptoms.14 In order to provide an overview of all late toxic effects on the limb treated with ILP, delayed surgical resection and adjuvant high-dose EBRT, a retrospective study was conducted to evaluate long-term morbidity using the LENT-SOMA scoring system.\nPatients and Methods\nBetween 1991 and 2003, 73 patients with locally advanced STSs underwent 77 perfusions with a combination of TNF\u03b1 and Melphalan, with (n\u00a0=\u00a019) or without (n\u00a0=\u00a058) interferon-gamma (IFN\u03b3), at the Division of Surgical Oncology of the University Medical Center Groningen (UMCG). The perfusion technique has been previously described extensively.15,16 Currently, 39 patients are still alive and in follow-up. There were 7 patients who were unable to participate because of severe or advanced (co-)morbidity (n\u00a0=\u00a02) or non-medical reasons (n\u00a0=\u00a05). Consequently, 32 patients, 14 males (44%) and 18 females (56%), median age 47 (range 14\u201371) years, could be evaluated (response rate 82%). Of all the 32 patients, 5 had STSs of the upper extremity (16%), treated with an axillary perfusion. In the remaining 27, they were located in the lower limb (84%) and were treated with iliacal (n\u00a0=\u00a013, 41%), femoral (n\u00a0=\u00a05, 16%) or popliteal (n\u00a0=\u00a09, 28%) perfusion. There were 27 primary (84%) and 5 recurrent (16%) STSs.\nAfter a median post-perfusion duration of 8 (range 6\u201312) weeks, all patients underwent a delayed local resection of the tumor remnant. Of the patients, 25 had clear microscopic surgical margins (R0 resection, 78%) and 7 had histologically positive microscopic margins (R1 resection, 22%). No patient had macroscopic residual disease. A total of 24 patients (75%) with marginal (\u22641\u00a0cm, n\u00a0=\u00a017) or microscopically positive resection (n\u00a0=\u00a07) margins received adjuvant high dose (60\u201370\u00a0Gy) postoperative EBRT in 25 fractions of 2\u00a0Gy, followed by a boost of 10\u201320\u00a0Gy. One patient with a recurrent STS received EBRT at the time of treatment of the primary tumor, but not after ILP and resection of the recurrence. Adjuvant systemic chemotherapy was given to 7 patients: 2 due to the definite histopathological classification of the tumor (one embryonal rhabdomyosarcoma and one extraosseal osteosarcoma); 3 as part of an EORTC trial (EORTC 62931);17 and 2 in a palliative setting because of distant metastasis.\nAll patients received a tailored post-surgical rehabilitation program, during and after the radiation treatment, until a final result was achieved. All patients were treated after informed consent was obtained according to institutional guidelines. A total of 12 different histological types of STS were distinguished. The pathological grade and stage of the tumor were scored according to the criteria of Coindre et\u00a0al. and the American Joint Committee on Cancer (AJCC), respectively.18,19 Patient and tumor characteristics are summarized in Table\u00a01.\nTable 1Characteristics of patients. PNET primitive neuroectodermal tumor, PUS pleiomorphic undifferentiated sarcoma, MPNST malignant peripheral nerve sheath tumor, FU follow-up in months, na not availableAge (years)SexHistologyGradeAJCC stageLevelEBRTAmputationFU18FemalePrimaryEmbryonal rhabdomyosarcoma33IliacalNoNo15944FemaleRecurrent Myxoid liposarcoma11IliacalYesNo15243MalePrimarySynovial sarcoma33IliacalYesNo15118MalePrimaryMyxoid chondrosarcoma23Popliteal YesYes14948FemalePrimaryWell-differentiated liposarcoma11IliacalYesNo14356FemalePrimaryPNET33IliacalYesYes13750FemaleRecurrent PUS33Popliteal NoNo13525FemalePrimarySynovial sarcoma23Popliteal YesNo13244MalePrimaryMyxoid liposarcoma11IliacalYesNo13124MaleRecurrent Synovial sarcoma23Popliteal YesNo12937FemalePrimaryMPNST23Axillary YesNo12148MalePrimaryMyxoid liposarcoma23IliacalYesNo11163FemaleRecurrent PUS23Popliteal NoYes10737MalePrimaryMyxoid liposarcoma11IliacalYesNo10558MalePrimaryPUS23IliacalYesNo9871FemalePrimaryLeiomyosarcoma11Femoral NoNo9445FemalePrimaryPUS33IliacalNoNo8256FemalePrimaryPUS33Popliteal YesNo6163MalePrimaryMyxoid chondrosarcomana3Femoral YesNo5037FemalePrimaryLeiomyosarcoma11Femoral YesNo3628MalePrimarySynovial sarcoma23Femoral YesNo3657FemalePrimaryPUS33Femoral YesNo3542FemalePrimarySynovial sarcoma23IliacalYesYes3247FemalePrimaryPUS33Axillary YesNo3058FemalePrimaryPUS33Popliteal YesNo2627MalePrimaryEpithelioid sarcoma34Axillary YesNo2671MaleRecurrent Myxoid fibrosarcoma11Axillary NoNo2456FemalePrimaryPUS33Axillary NoNo2214MalePrimaryPUS33Popliteal YesNo2065FemalePrimarySynovial sarcoma23Popliteal NoYes1863MalePrimarySynovial sarcoma33IliacalYesNo1771MalePrimaryRhabdomyosarcoma33IliacalYesNo17\nLocal status of the treated limb was first globally assessed using a checklist including the following items: lymphedema, color, stiffness, hypesthesia, paresthesia, muscle atrophy, pain, scar status, function and use of aids.\nLate effects on surrounding tissue were graded according to the subjective, objective and management portions of the LENT-SOMA scales, as proposed by the EORTC and RTOG Late Effects Working Group in 1995.14,20,21 Four LENT-SOMA scales were used: muscle\/soft tissue, peripheral nerves, skin\/subcutaneous tissue and vessels.22 Not all patients could be scored on all four items, since in 5 patients (16 %) the affected limb was amputated.\nResults\nAfter a median follow-up of 88 (range 17\u2013159) months, 14 patients (44%) mentioned one or more locoregional complaints in the treated leg, mainly consisting of pain, spasm and limitation of mobility. Scoring the local status using a checklist, deviating symptoms and signs were documented and are summarized in Table\u00a02. Only 15 patients (47%) did not use any aids; the remaining 17 (53%) needed elastic compression stockings, braces, prostheses or crutches.\nTable\u00a02Local status in 32 patients: deviating symptoms and signsSymptomNo.%Lymphedema1444Discoloration1856Stiffness1650Atrophy2784Pain516Hypesthesia1753Paresthesia1341Function\u00a0\u00a0- Limited1856\u00a0\u00a0- Severely limited413\nLENT-SOMA Scales\nResults of scoring late tissue effects according to the LENT-SOMA scoring system are listed below and illustrated in Table\u00a03. Grade 1 represents the most minor symptoms, which do not require treatment. Grade 2 represents moderate symptoms, requiring only conservative treatment. Grade 3 represents severe symptoms, which have a significant negative impact on daily activities and require more aggressive treatment. Grade 4 represents irreversible functional damage, necessitating major therapeutic intervention. SOMA stands for: S, subjective complaints; O, objective symptoms; and M, management and therapy.\nTable\u00a03Modified LENT-SOMA scoresMaximum grade of toxicity01234Muscle\/ soft tissue (n\u00a0=\u00a031)\u00a0\u00a0Subjective619%929%26%1445%00%\u00a0\u00a0Objective00%00%1548%1652%00%\u00a0\u00a0Management2065%13%929%13%00%Peripheral nerves (n\u00a0=\u00a030)\u00a0\u00a0Subjective620%310%930%1240%00%\u00a0\u00a0Objectivea621%1243%725%311%00%\u00a0\u00a0Management2377%13%413%27%00%Skin\/subcutaneous tissue (n\u00a0=\u00a027)\u00a0\u00a0Subjective830%1556%27%27%00%\u00a0\u00a0Objective14%622%1556%519%00%\u00a0\u00a0Management2385%00%00%27%27%Vessels (n\u00a0=\u00a025)\u00a0\u00a0Subjective1664%416%416%14%00%\u00a0\u00a0Objective1352%832%28%14%14%\u00a0\u00a0Management1872%416%00%14%28%a\u00a0n\u00a0=\u00a028.\nMuscle\/Soft Tissue\nOf the patients, 6 experienced no subjective toxic effects (19%), 9 scored maximum grade 1 (29%), 2 scored grade 2 (6%) and 14 had a maximum of grade-3 toxicity (45%). When scored objectively, a maximum of grade-2 toxicity was found in 15 patients (48%) and grade 3 in 16 patients (52%). No patient had only minor or no symptoms. No treatment was required in 20 patients (65%), 1 patient (3%) scored grade 1 (occasional physiotherapy), 9 scored grade 2 (29%) and 1 (3%) needed continued medical intervention (grade 3) for edema and extremity malfunction. A detailed overview of all items is provided in Table\u00a04.\nTable\u00a04LENT-SOMA scores for muscle\/soft tissue (n\u00a0=\u00a031)Grade of toxicity01234NASubjective\u00a0\u00a0- Pain2065%723%26%26%00%00%\u00a0\u00a0- Function619%929%26%1445%00%00%Objective\u00a0\u00a0- Edema1445%1032%413%26%00%13%\u00a0\u00a0- Mobility and extremity function619%929%1135%516%00%00%\u00a0\u00a0- Fibrosis13%310%1548%1032%00%00%\u00a0\u00a0- Atrophy00%310%1652%1032%00%26%\u00a0\u00a0- Contraction1858%00%1239%00%00%13%Management\u00a0\u00a0- Pain2581%310%310%00%00%00%\u00a0\u00a0- Edema2477%00%516%13%00%13%\u00a0\u00a0- Mobility and extremity function2787%13%26%13%00%00%\u00a0\u00a0- Fibrosis2994%13%00%00%00%13%\u00a0\u00a0- Atrophy2994%00%13%00%00%13%\nPeripheral Nerves\nNo subjective toxic effects were observed in 6 patients (20%), 3 scored maximum grade-1 toxicity (10%), 9 scored grade 2 (30%) and 12 grade-3 toxicity (40%). When scored objectively, 6 patients experienced no toxicity (21%), and 12 scored grade-1 (43%), 7 grade-2 (25%) and 3 grade-3 (11%) toxic effects. Of the patients, 23 did not require any treatment (77%), 1 (3%) scored grade-1 (occasional non-narcotic), 4 (13%) grade-2 (regular non- narcotic) and 2 (7%) grade-3 (physical or medical intervention because of severe motor dysfunction) toxicity. A detailed overview is provided in Table\u00a05.\nTable\u00a05LENT-SOMA scores for peripheral nerves (n\u00a0=\u00a030)Grade of toxicity01234NASubjective\u00a0\u00a0- Pain2067%723%13%27%00%00%\u00a0\u00a0- Strength723%13%1240%827%00%27%\u00a0\u00a0- Sensory1033%1137%27%620%00%13%\u00a0\u00a0- Motor paresis1447%827%517%27%00%13%Objective\u00a0\u00a0- Motor dysfunction723%1137%723%27%13%27%\u00a0\u00a0- Sensory dysfunction1240%1240%310%13%00%27%\u00a0\u00a0- Reflex1757%723%310%00%00%310%Management\u00a0\u00a0- Pain2273%310%413%00%00%13%\u00a0\u00a0- Motor dysfunction2583%00%00%27%00%310%\u00a0\u00a0- Sensory dysfunction2790%00%00%00%00%310%\u00a0\u00a0- Sensory2790%00%00%00%00%310%\nSkin\/Subcutaneous Tissue\nNo subjective symptoms were observed in 8 patients (30%), 15 scored maximum grade-1 toxicity (56%), 2 grade 2 (7%) and 2 (7%) grade 3 (scaliness\/roughness requiring constant attention). Only 1 patient experienced no objective toxicity (4%), while 6 scored grade 1 (22%), 15 grade 2 (56%) and 5 grade 3 (19%). No treatment was required in 23 patients (85%), but 2 (7%) required medical intervention because of edema and dryness (grade-3 toxicity) and 2 (7%) scored grade-4 toxicity\u2014surgical intervention due to massive fibrosis and a persistent ulcer requiring surgical therapy. A detailed overview is provided in Table\u00a06.\nTable\u00a06LENT-SOMA scores for skin\/subcutaneous tissue (n\u00a0=\u00a027)Grade of toxicity01234NASubjective\u00a0\u00a0- Scaliness\/ roughness933%1659%00%27%00%00%\u00a0\u00a0- Sensation2074%519%27%00%00%00%Objective\u00a0\u00a0- Edema1141%830%519%27%00%14%\u00a0\u00a0- Alopecia (scalp)933%1348%311%14%00%14%\u00a0\u00a0- Pigmentation change622%830%1141%14%00%14%\u00a0\u00a0- Ulcer\/ necrosis2593%14%14%00%00%00%\u00a0\u00a0- Teleangiectasia1659%726%311%00%00%14%\u00a0\u00a0- Fibrosis\/ scar27%1970%622%00%00%00%\u00a0\u00a0- Atrophy\/ contraction27%1556%622%27%00%27%Management\u00a0\u00a0- Dryness2385%14%14%14%00%14%\u00a0\u00a0- Sensation2696%00%00%00%00%14%\u00a0\u00a0- Ulcer2696%00%00%00%14%00%\u00a0\u00a0- Edema2593%00%00%27%00%00%\u00a0\u00a0- Fibrosis\/ scar2696%00%00%00%14%00%\nVessels\nNo subjective symptoms were observed in 16 patients (64%), while 4 scored maximum grade-1 (16%), 4 grade-2 (16%) and 1 grade-3 (4%) toxicity: clinical symptoms of ischemia at rest. Objective symptoms were absent in 13 patients (52%), but 8 patients scored grade-1 toxicity (32%), 2 scored grade 2 (8%), 1 grade 3 (intense ischemia) (4%) and 1 grade 4 (necrosis) (4%). No form of management was needed in 18 patients (72%). However, 4 patients scored grade 1 (16%), 1 (4%) grade 3 (conservative surgery because of arterial disease) and 2 (8%) required amputation (grade-4 toxicity) because of critical leg ischemia. A detailed overview is provided in Table\u00a07.\nTable\u00a07LENT-SOMA scores for vessels (n\u00a0=\u00a025)Grade of toxicity01234NASubjective\u00a0\u00a0- Arterial2080%14%312%14%00%00%\u00a0\u00a0- Venous2080%416%14%00%00%00%Objective\u00a0\u00a0- Arterial2288%00%14%14%14%00%\u00a0\u00a0- Venous1664%832%14%00%00%00%Management\u00a0\u00a0- Arterial2288%00%00%14%28%00%\u00a0\u00a0- Venous2184%416%00%00%00%00%\nOverall, outcomes from the Subjective and Objective symptoms are different from those of the Management part of the LENT-SOMA scales. According to the subjective and objective parts of the scale, two-thirds of patients had serious late toxic side effects concerning any of the surrounding normal tissue. A grade-3 toxicity was scored by 17 patients (53%) on one or more items of the subjective parts of the four used scales. They experienced their long-term morbidity as severe and with a negative impact on daily activities. Grade-3 or -4 toxicity was scored by 18 patients (56%) on the objective parts of the scoring scales, showing severe or irreversible damage during a clinical examination. However, according to the management parts, 16 patients (50%) scored no toxicity (grade 0) on all management items of the four used LENT-SOMA scales and did not require any treatment. Moreover, 9 patients (28%) scored a maximum grade-1 or -2 toxicity and only needed conservative treatment. In less than a quarter of patients (n\u00a0=\u00a07, 22%), aggressive treatment, e.g., continuous medical intervention or surgery, was necessary due to long-term effects of the combined treatment procedure.\nDiscussion\nThe limb-sparing treatment strategy of ILP using TNF and Melphalan\u2014with or without adjuvant EBRT\u2014is increasingly being pursued for locally advanced STS of the extremities after the publication of the results of a European multi-center trial performed in the 1990s. Using this combined treatment modality, excellent short-term limb salvage rates of more than 80% and long-term rates of 60% can be achieved with no negative impact on survival.5,6 While, at first, selected groups of patients were often excluded from undergoing this procedure, limb preservation has now become a major challenge for all patients with a locally advanced STS.\nIt seems to be a safe and highly effective procedure in elderly patients, for whom every attempt to avoid an amputation that may end their independence must be considered.23 It can also be used in a palliative setting for patients who present with distant metastasis at the time of diagnosis, as better local control improves the quality of life.24 In patients with multifocal primary or multiple recurrent extremity sarcomas, ILP also provides excellent local control and limb salvage rates of more than 80% are achieved.25\nHowever, acute toxicity and long-term morbidity due to the combined treatment procedure of ILP, surgical resection and EBRT cannot be prevented. In ILP, normal tissue in the limb is exposed to the same concentrations of cytostatic agents as the tumor. The effects of the perfusate on normal tissue vary widely among individuals and long-term morbidity also varies widely in severity.\nAs far as we know, this is the first study to assess late functional morbidity with the LENT-SOMA scales in patients treated for STS with ILP, surgical resection and EBRT.\nNeurotoxicity\nNeuropathy is particularly important and is mainly seen after the use of specific cytostatic perfusion agents, such as cisplatin, carboplatin and doxorubicin.26\u201328 Furthermore, nerve damage can be caused by initial tumor swelling or pressure from the tight tourniquet.29,30 A prophylactic fasciotomy can prevent the occurrence of a persistent neurotoxicity with motor-sensory neuropathy and is routinely performed in our series.31 Radiotherapy also plays an important role, especially when patients have received a boost.32 All our patients received a boost of 10\u2013 20\u00a0Gy EBRT. Despite the fact that neurotoxicity was subjectively found to be severe in 40% of patients, only in 11% could it be objectified, and in only 7% was physical or medical intervention needed. None of the patients had paralysis or complete anesthesia or needed surgical intervention.\nFunctional Morbidity\nFunctional outcome after limb-sparing treatment with ILP has been analyzed previously, but mostly in melanoma patients and not using a uniform scoring system. Other studies show limitations in ankle joint mobility in 25\u201340% of patients, compared with no significant objective limitations in previous investigations in our study population.12,33 The routinely performed fasciotomy, combined with an intensive physiotherapy program, might explain these better outcomes because the regional toxic effects of ILP are hereby reduced.34 In this study, we found muscle atrophy in almost all patients (96%), which may be due to an extensive surgical resection having been performed after ILP for deep-seated STSs, in contrast to the mostly superficial resection in melanoma patients. Only 4 patients (13%) still needed medical intervention: occasional or regular physiotherapy. However, 14 patients (44%) found that their limb function interfered with daily activities. In only 6 patients (19%) could no functional impairment be objectified.\nScoring Systems\nMost previous studies evaluating functional outcome after treatment for STS included no ILP, but consisted of wide local excision with or without postoperative EBRT and\/or adjuvant chemotherapy. Gerrand et\u00a0al. found a significant decrease in functional scores when the revised Musculoskeletal Tumor Society Rating Scale (MSTS) and Toronto Extremity Salvage Score (TESS) were used after treatment of deep-seated tumors35. In two studies, the LENT-SOMA scales were used to assess functional morbidity after wide local excision and EBRT sometimes combined with chemotherapy for STS. The LENT-SOMA scales were found to be time-consuming, but reliable for a detailed description of late toxicity.32,36\nTo evaluate the long-term morbidity in our study, we used this generally accepted LENT-SOMA scoring system. According to Denekamp et\u00a0al., no overall LENT score is calculated but all criteria are separately described in order to prevent the high scores being filtered out, thus giving a falsely optimistic picture.37 Although primarily developed to analyze late radiation effects, it can also be used to score the toxicity of chemotherapy.21 In this study, 25 of the 32 patients analyzed (78%) received EBRT. The 3 patients who scored grade-4 toxicity on one or more items all received EBRT. Overall, there was no patient who did not experience any late toxic effect.\nQuality of Life\nAll functional complaints can have a substantial negative impact on daily functioning, which brings into question whether expectations of the time-consuming technically demanding limb-sparing treatment are upheld. Previous studies showed no improved quality of life with limb-sparing surgery compared with that after amputation.38,39 The long rehabilitation period is a disadvantage of a limb-sparing treatment strategy, but the impact of a prosthesis should not be underestimated. Thijssens et\u00a0al. recently showed a worse physical functioning and more role limitations in amputated patients in this study population. Attention to collaborative decision making and communication with the patient seem very important.40\nFuture Perspectives\nEvery effort should be made to prevent long-term morbidity in the future. As long-term morbidity seems to be correlated to acute regional toxic reactions, these acute reactions should be reduced as far as possible.30 Improved perfusion technique and enhanced technical insights have led to the following suggestions: reduce the doses of TNF\u03b1, decrease systemic leakage, reduce maximum temperatures and strictly regulate perfusion pressures.15,30,41 To prevent the significant toxicity related to muscle and nerve damage, a prophylactic fasciotomy should be performed in all cases and the isolating tourniquet should not be applied too tightly.30,31 Furthermore, all patients should receive an individualized rehabilitation program to achieve an optimum functional situation.\nFinally, a lot of long-term morbidity after EBRT is subscribed to the occurrence of radiation-induced fibrosis, which was thought to be irreversible.7 Thanks to the pioneering work of Delanian et\u00a0al., there is growing knowledge that radiation-induced fibrosis is at least partially reversible by administering drugs with antioxidant and anti-fibrotic properties, such as pentoxifylline and vitamin E. Large randomized trials are, however, needed to confirm these findings.32\nIn conclusion, the Subjective, Objective, and Management parts of the LENT-SOMA scoring system confirm late toxic effects on surrounding normal tissue in all patients treated with a combined treatment modality for locally advanced STS of the extremity. Two-thirds of the patients have severe Subjective or Objective damage, but half of all patients do not require any Management for their symptoms. To prevent long-term morbidity in the future, efforts should be made to reduce acute toxic reactions after ILP, and a fasciotomy and a customized rehabilitation program are recommended. Furthermore, anti-fibrotic therapies might be considered.","keyphrases":["perfusion","sarcoma","lent-soma","radiation","complications"],"prmu":["P","P","P","P","P"]}
{"id":"Biochim_Biophys_Acta-2-1-2225445","title":"DNA-PK contributes to the phosphorylation of AIRE: Importance in transcriptional activity\n","text":"The autoimmune regulator (AIRE) protein is a key mediator of the central tolerance for tissue specific antigens and is involved in transcriptional control of many antigens in thymic medullary epithelial cells (mTEC). Mutations in the AIRE gene cause a rare disease named autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). Here we report using GST pull-down assay, mass-spectrometry and co-immunoprecipitation that a heterotrimeric complex of DNA-Dependent Protein Kinase (DNA-PK), consisting of Ku70, Ku80 and DNA-PK catalytic subunit (DNA-PKcs), is a novel interaction partner for AIRE. In vitro phosphorylation assays show that the residues Thr68 and Ser156 are DNA-PK phosphorylation sites in AIRE. In addition, we demonstrate that DNA-PKcs is expressed in AIRE positive mTEC cell population and that introduction of mutations into the AIRE phosphorylation sites decrease the capacity of AIRE to activate transcription from reporter promoters. In conclusion, our results suggest that phosphorylation of the AIRE protein at Thr68 and Ser156 by DNA-PK influences AIRE transactivation ability and might have impact on other aspects of the functional regulation of the AIRE protein.\n1\nIntroduction\nAIRE (autoimmune regulator) is a defective gene in APECED (autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy, OMIM #240300), the genetic autoimmune disease, which manifests as autoimmunity to multiple endocrine glands. In human tissues, AIRE is expressed in the thymus, spleen, and lymph nodes [1\u20133].\nThe AIRE protein (57.5\u00a0kDa) has several features which indicate that it might function in transcriptional regulation. AIRE shares similar domain architecture with the Sp100 family proteins (Sp140, Sp110 and Sp100C), which all have HSR (for homogenously staining region), SAND (for Sp100, AIRE-1, NucP41\/75 and DEAF-1) and PHD (for plant homeodomain) domains. HSR is central for AIRE di- or oligomerization [4,5]. The SAND domain is a DNA binding domain found in several mammalian nuclear proteins. NMR spectroscopy studies of the Sp100B SAND domain have shown that a conserved amino acid motif KDWK is essential for DNA recognition [6]. Although AIRE lacks the KDWK motif, its SAND domain has also been shown to bind DNA in vitro\n[4]. The PHD fingers are found in nuclear proteins that are often involved in chromatin-mediated transcriptional regulation and appear to mediate protein\u2013protein interactions. The zinc-binding cysteine-rich regions of the PHD finger form two flexible loops and two protein ligands have been proposed to bind one PHD finger simultaneously [7]. It has been suggested that one of the ligands can be specific for each PHD. For example, the PHD finger of ING2 can bind phosphoinositides whereas PHD of the KAP-1 repressor binds to Mi2\u03b1, a component of the NuRD chromatin remodelling complex [8,9]. Another ligand, which might be common for all PHD fingers, has been long suggested to be chromatin, and recently, PHD domains of ING2 and BPTF proteins were reported to bind tri-methylated lysine 4 of histone H3 [10,11]. However, despite the determination of its 3-D structure, no conclusive ligand of the AIRE PHD finger has yet been identified [12].\nWe and others have shown that AIRE can activate reporter genes from the interferon beta promoter or in a system where AIRE is tethered via heterologous GAL4 DNA binding domain to the reporter [5,13]. The transcriptional activation region of AIRE has been initially mapped to the two C-terminal PHD fingers as the mutations in these domains severely decreased the transactivation capacity [13,14]. More recent reports have shown that several other APECED-causing mutations in the HSR or SAND domain also decrease the AIRE transactivation ability [15,16].\nSo far, the only known protein partner for AIRE is a general transcriptional co-regulator and histone acetyltransferase, CREBP binding protein (CBP). CBP has been reported to bind directly to AIRE in vitro, it colocalizes with AIRE into the nuclear bodies in monocytic cell cultures and it enhances the transcription of reporter genes in collaboration with AIRE [5,16]. The mechanisms how AIRE in collaboration with CBP activates its target genes are largely unknown.\nDNA-dependent protein kinase (DNA-PK) is a Ser\/Thr kinase that belongs to the phosphatidylinositol 3-kinase (PI3K) family. It is formed as holoenzyme in the presence of DNA by two Ku regulatory subunits, Ku70 (70\u00a0kDa) and Ku80 (86\u00a0kDa), and a large catalytic subunit DNA-PKcs (465\u00a0kDa). With very few exceptions, DNA-PKcs is active only when it is in hetero-trimeric complex with Ku proteins and in interaction with DNA or RNA [17,18]. The main function of DNA-PK is to recognize double stranded DNA breaks and to catalyze a repair process known as non-homologous end joining (NHEJ). In a similar way, DNA-PK is crucial for V(D)J recombination in developing T and B cells. Concordantly, DNA-PKcs or Ku deficient mice are severely immunodeficient, with elevated radiosensitivity and susceptible for tumor development [17,19].\nIn addition to the role of DNA-PK in chromatin repair, DNA-PK has been shown to phosphorylate many proteins involved in cell cycling and transcriptional regulation, such as p53, Sp1, Myc, Fos, Jun, TBP, TFIIB, RNA Polymerase II [17] and several nuclear receptors, for example, the glucocorticoid [20], progesterone [21] and androgen receptors [22]. DNA-PK can positively and negatively modulate transcription. As a repressor, DNA-PK binds directly to a specific DNA sequence element within the mouse mammary tumor virus (MMTV) promoter [20,23]. Similarly, the whole DNA-PK complex has been shown to bind to the E-box\/TATA like elements and to suppress the human xanthine oxidoreductase (hXOR) gene expression [24]. At the same time, the Ku proteins have been described as transcriptional recycling co-activators of androgen receptor [22]. In addition to these examples, DNA-PK can modulate gene expression via RNA-dependent phosphorylation of hnRNP proteins [18].\nIn order to further define molecular mechanisms of AIRE functioning, we searched for novel protein partners of AIRE using the GST pull-down approach combined with mass spectrometry. We found that AIRE interacts with DNA-PK complex proteins DNA-PKcs, Ku70 and Ku80. Further in vitro phosphorylation assays demonstrated that DNA-PK phosphorylates the AIRE protein in vitro at the residues Thr68 and Ser156. In addition, we show that the mutation of either of the phosphorylation sites significantly decreases AIRE transcriptional activity.\n2\nMaterials and methods\n2.1\nPlasmids\nPlasmid pGST-PHD1 was generated by insertion of PCR-amplified sequence encoding human AIRE-1 amino acids 293\u2013354 into the NcoI and KpnI restriction sites of the pETM-30 vector (EMBL-Heidelberg protein expression and purification laboratory). To generate pGST-AIRE-SPP (SAND\/PHD1\/PHD2), the sequence encoding AIRE amino acids 178\u2013482 was cloned into EcoRI and XhoI sites of pGEX1\u03bbT-SH3 vector (a gift from K. Saksela, University of Tampere, Finland). pGST AIRE-T68A, pGST-AIRE-S156A and pGST-AIRE-V80L mutation constructs were generated by altering amino acid Thr68 or Ser156 to alanines and V80 to leucine in pGST-AIRE [5] constructs by PCR-based site-directed mutagenesis. The control plasmid pGEX-2T-p53 was a gift from T. Punga (Uppsala University, Sweden). pSI-AIRE-T68A and pSI-AIRE-S156A were prepared by further cloning the mutated AIRE fragments from pGST-AIRE-T68A and pGST-AIRE-S156A into EcoRI and SalI sites of pSI vector (Promega). To generate luciferase reporter plasmids pBL-INV and pBL-LOR, the promoter areas of involucrin (3737 nt) and loricrin (2217 nt) genes were cut out from the plasmids pTZhINV-nlbgal and pTZhLOR1 (gifts from A. M\u00e4nnik, FitBiotech, Estonia) and cloned into the HindIII site of pBL-KS (a gift from K. Saksela, University of Tampere, Finland). The cloned plasmids were verified by sequencing.\n2.2\nExpression and purification of GST fusion proteins\nThe GST-tagged proteins were expressed either from Section 2.1. or previously described [5] AIRE GST-fusion constructs, pGST-AIRE (1\u2013545), pGST-R257X (1\u2013256), pGST-AIRE293 (1\u2013293), pGST-AIRE348 (1\u2013348), pAIRE-SAND (175\u2013298) in Escherichia coli BL21-DE3 strain by induction with 0.4\u00a0mM IPTG for 4\u00a0h at room temperature. Full-length GST-AIRE and PHD-containing fusion proteins were expressed in the presence of 0.1\u00a0mM ZnCl2. The proteins were purified using Glutathione Sepharose 4B (AmershamBiosciences) according to the manufacturer's instructions except that 1% N-laurylsarcosine and 3% Triton X-100 were added to increase solubility of the proteins and 50\u00a0\u03bcM ZnCl2 was included when GST-AIRE and PHD-containing fusion proteins were purified. The purified proteins were verified on SDS-PAGE and Coomassie Blue staining.\n2.3\nGST pull-down and mass spectrometry\n50\u2013100\u00a0\u03bcg of nuclear extract, prepared from human monocyte cell line THP-1 according to [25], was incubated with 20\u201325\u00a0\u03bcg of GST-fusion proteins bound to 25\u00a0\u03bcl of packed sepharose beads in buffer B1 (10\u00a0mM HEPES pH 8.0, 150\u00a0mM NaCl, 0.7\u00a0mM MgCl2, 12.5% glycerol, 0.1\u00a0mM EDTA, 25\u00a0\u03bcM ZnCl2, 0.5\u00a0mM DTT and proteinase inhibitor mix) over-night at 4\u00a0\u00b0C. The beads were washed intensively with buffer B1, the bound proteins were eluted and separated on SDS-PAGE. The specific protein bands were cut out and analyzed with mass spectrometry.\nMass mapping of the peptides generated was performed with an Ultraflex\u2122MALDI-TOF\/TOF mass spectrometer (Bruker-Daltonics, Bremen, Germany) equipped with a nitrogen laser in a positive ion reflector mode using \u00e1-cyano-4-hydroxycinnamic acid as the matrix. The MALDI spectra were externally calibrated with the standard peptide mixture from Bruker-Daltonics (Bremen, Germany). In case of a peptide fragmentation analysis, a peptide from above mass mapping analysis was selected as a precursor ion and subjected for further MS\/MS fragmentation in the MALDI-TOF\/TOF lift-mode. Database searches were carried out by either Mascot peptide map fingerprint or Mascot MS\/MS ions search (http:\/\/www.matrixscience.com\/).\n2.4\nTransfections and immunoprecipitations\nThe transfections were carried out with ExGen500 in vitro reagent (Fermentas) according to the manufacturer's protocol. For immunoprecipitations, approximately 2\u00a0\u00d7\u00a0106 HEK293 cells were transfected with 10\u00a0\u03bcg of pcAIRE [3] or either pcDNA3.1B-myc\/his (Invitrogen) or pdEYFP-N1 (Clontech) vector. After 46\u00a0h, a whole-cell extract was prepared by lysis with 0.25\u00a0ml of lysis buffer (20\u00a0mM Tris pH 7.5, 1% NP-40, 0.3\u00a0M NaCl, 0.1\u00a0mM ZnCl2, 1.5\u00a0mM MgCl2, 0.5\u00a0mM DTT, 0.2\u00a0mM EDTA, 25% glycerol, protease inhibitors) for 30\u00a0min on ice. The lysates were treated five times with the 25G syringe to shear genomic DNA, centrifuged at 13 500\u00a0rpm, 15\u00a0min at 4\u00a0\u00b0C. The supernatants were diluted 5 times with the lysis buffer lacking NP-40 and immunoprecipitations were performed with mouse monoclonal anti-DNA-PK 4F10C5 (BD Pharmingen), anti-AIRE 6.1 [3], anti-HuR 3A2 (a gift from J. Steitz, Yale University, USA), anti-HA sc-7392 (SantaCruzBiotech) anti-GAPDH 6C5 (Ambion), anti-c-myc 9E10 (Sigma) and rabbit polyclonal anti-GST sc-459 (SantaCruzBiotech) antibodies. 1.5\u00a0\u03bcg of each antibody was added to 700\u2013900\u00a0\u03bcg of whole-cell extracts and incubated for 4\u00a0h at 4\u00a0\u00b0C, then 15\u00a0\u03bcl of packed protein G-Sepharose beads (Amersham Pharmacia Biotech) were included and incubation was continued for 1\u00a0h at 4\u00a0\u00b0C. The immunoprecipitates were washed 4 times with the IP buffer (20\u00a0mM Tris pH 7.5, 0.2% NP-40, 0.3\u00a0M NaCl, 0.1\u00a0mM ZnCl2, 1.5\u00a0mM MgCl2, 0.5\u00a0mM DTT, 0.2\u00a0mM EDTA, 25% glycerol, protease inhibitors) and eluted from the beads with 2\u00d7 SDS loading buffer, separated on SDS-PAGE and subjected to Western blot with anti-AIRE 6.1 or DNA-PK antibody (1:1000) and ECL detection. Transfer DNA-PK was carried out with 0.01\u00a0M CAPS buffer, pH 11.0, supplemented with 20% methanol in semi-dry blotting system for 90\u00a0min at 1\u00a0mA\/cm2.\nWhere indicated, RNaseA\/RNaseT mix (Fermentas) with final concentrations 4\u00a0\u03bcg\/ml and 10\u00a0u\/ml, respectively, was added. Ethidium bromide (EtBr) and micrococcal nuclease (MNase) treatments were performed as demonstrated earlier [26]. Either 0.4\u00a0mg\/ml EtBr was added to the extract or 1\u00a0u of MNase (Fermentas) in 50\u00a0\u03bcl of digestion buffer (10\u00a0mM HEPES pH 7.0, 4\u00a0mM CaCl2, 50\u00a0mM NaCl, 0.1\u00a0mM ZnCl2) was used. Digestion efficiency of MNase was tested in the separate control experiments, using the same conditions. On agarose gels, only the mononucleosomal fraction of DNA was detected when whole nuclei were treated and no DNA was left when free DNA was digested.\n2.5\nComputer prediction of AIRE phosphorylation\nScansite 2.0 program at http:\/\/scansite.mit.edu\/cgi-bin\/motifscan_seq\n[27] and NetPhos 2.0 program at http:\/\/www.cbs.dtu.dk\/services\/NetPhos\n[28] were used to predict AIRE phosphorylation sites.\n2.6\nPhosphorylation assays\nThe kinase assays were performed with SignaTECT DNA-PK Kinase Assay System kit (Promega) essentially according to the manufacturer's protocol. The phosphorylation reactions were carried out in the presence of \u03b3ATP-32P for 7\u00a0min at 30\u00a0\u00b0C and either 25\u00a0\u03bcg of biotinylated-p53 peptide (EPPLSQEAFADLWKK, Promega) or 4\u20135\u00a0\u03bcg of GST fusion proteins were used as the substrates. Efficiencies of 32P labeling of the substrates were measured by radioactivity counter (1414 Guardian, Wallac). Nuclear extracts and immunoprecipitations were made as described in Sections 2.3 and 2.4. Either 600\u00a0\u03bcg (Fig. 3A) or 15\u201320\u00a0\u03bcg (all other figures) of nuclear extracts were used per reaction. Alternatively, 10\u00a0U of purified DNA-PK complex (Promega) in the presence of 250\u00a0ng of calf thymus DNA was used. 50\u00a0\u03bcM LY294002 hydrochloride (Sigma) was added when indicated. In assays using GST fusion proteins as the substrates, the GST proteins bound to 10\u00a0\u03bcl of packed glutathione beads were pre-washed with the kinase reaction buffer (SignaTECT kit buffer supplemented with 0.1\u00a0mM ZnCl2 and 0.2\u00a0mM Na3VO4). After the kinase reaction, the beads were washed three times with ice cold washing buffer (25\u00a0mM HEPES pH 7.5, 150\u00a0mM NaCl, 0.1% BSA, 0.2% NP-40, 0.1\u00a0mM ZnCl2 and 0.2\u00a0mM Na3VO4) before measuring the radioactivity.\n2.7\nMice, thymic stromal cell isolation, cell sorting and RT-PCR\nC57BL\/6J background wild type mice were maintained at the mouse facility of the Institute of Molecular and Cell Biology, Tartu University. Thymuses from 4- to 6-week-old mice were used. Thymic stromal cell isolation, cell sorting and RT-PCR were carried out as described previously [29].\n2.8\nLuciferase reporter assays\nLuciferase assays were performed using Luciferase Assay System kit (Promega) according to the manufacturer's protocol. 4\u00a0\u00d7\u00a0104 HEK293 cells in 24-well plates were transfected with 0.1\u00a0\u03bcg of INV-pBL or LOR-pBL and 0.3\u00a0\u03bcg pSI-AIRE, pSI-AIRE-T68A or pSI-AIRE-S156A for 46\u00a0h. In Western blot, anti-AIRE 6.1 antibody was use and 0.3\u00a0\u03bcg of pSI-AIRE, pSI-AIRE-T68A or pSI-AIRE-S156A were transfected for 46\u00a0h.\n3\nResults\n3.1\nDNA-PK complex proteins co-purify with AIRE PHD domain\nIn order to find new AIRE interacting partners, we expressed the first PHD domain (PHD1) of AIRE as a GST-fusion protein (Fig. 1A) and performed the GST pull-down using the nuclear extract prepared from monocytic THP-1 cells. The interacting proteins were separated on SDS-PAGE and visualized by Coomassie staining. Three protein bands with molecular weight of about 70, 80 and over 250\u00a0kDa were consistently seen after the SDS-PAGE analyses (Fig. 1B). Next, the protein bands were excised from the gel and analyzed by mass spectrometry. The MALDI-TOF mass map analysis demonstrated that the two bands with molecular masses of about 70 and 80\u00a0kDa belong to Ku70 and Ku80, respectively, whereas the protein with the mass over 250\u00a0kDa was presumed to be the catalytic subunit of DNA-PK (DNA-PKcs) (Table 1). Mass map identifications with low sequence coverage were further verified by MALDI-TOF\/TOF fragmentation of three tryptic peptides (Table 1). Thus, these results identified a trimeric complex of DNA-PK as a candidate AIRE interacting partner.\n3.2\nFull-length AIRE interacts with DNA-PK\nTo further confirm the pull-down results and to demonstrate that the full-length AIRE protein is able to interact with DNA-PK, we carried out co-immunoprecipitation experiments. So far, no AIRE endogenous expression on protein level has been detected in immortalized cell-lines. As subcellular localization of transfected AIRE in immortalized cell lines is similar to the AIRE localization in thymus in vivo, as well as AIRE is able to activate transcription in those cells, we transfected HEK293 cells with the plasmid expressing full-length AIRE or empty vector as a negative control. The whole-cell extracts were prepared from transfected cells and used in co-immunoprecipitations with anti-DNA-PKcs, anti-AIRE or various control antibodies. The precipitates were separated on SDS-PAGE and immunoblotted with anti-AIRE antibody. As seen in Fig. 2A, anti-DNA-PKcs and anti-AIRE antibodies (as a positive control) were able to precipitate AIRE protein whereas control antibodies against GST, GAPDH and HuR (a nuclear mRNA-binding protein) could not. Similarly, AIRE and DNA-PKcs interaction was detectable when the immunoprecipitation with anti-AIRE antibody followed by the Western with anti-DNA-PKcs antibody was carried out (Fig. 2B).\nSince the whole-cell extracts used in immunoprecipitations contained remarkable amounts of chromatin and RNA (data not shown), we next wanted to test whether the interaction between AIRE and DNA-PK is DNA and RNA dependent or independent. Previously, a DNA intercalator ethidium bromide (EtBr) has been shown to efficiently disrupt the binding of Ku proteins to Oct2 and DNA-PK [26,30], whereas the micrococcal nuclease has been widely used for the disruption of chromatin as well as for the digestion of contaminating DNA from cell lysates and immunoprecipitates [26,31]. Consequently, we transfected the HEK293 cells with the plasmid expressing full-length myc-tagged AIRE or empty vector as a negative control and performed co-immunoprecipitations with anti-DNA-PKcs antibodies in the presence of EtBr, RNaseA\/T mix or treated the immunoprecipitates with the micrococcal nuclease (Fig. 2C, top panel). As a positive control, to show that the same level of the AIRE protein was present in each lysate, the immunoprecipitations were also performed with the anti-myc antibodies (Fig. 2C, bottom panel). Fig. 2C top panel reveals that DNA-PKcs still co-immunoprecipitates AIRE from the cell extracts treated with EtBr or RNaseA, which indicates that AIRE does not require DNA or RNA to interact with DNA-PKcs. However, when we treated the co-immunoprecipitates with micrococcal nuclease, we observed a weaker interaction between AIRE and DNA-PKcs (Fig. 2C, top panel), which suggests that in the cell lysates containing DNA and chromatin, a fraction of AIRE binds DNA-PK through the DNA and\/or chromatin. In conclusion, the co-immunoprecipitation results together confirm that the AIRE protein interacts with DNA-PK.\n3.3\nDNA-PK phosphorylates the AIRE protein\nDNA-PK is a nuclear serine\/threonine protein kinase that has been reported to phosphorylate many nuclear targets. To study whether the AIRE protein is one of the targets of DNA-PK, we used several different assays which enabled quantitation of DNA-PK kinase activity. We first studied whether the AIRE protein co-immunoprecipitates DNA-PK kinase activity. Co-immunoprecipitations from nuclear extracts prepared from HEK293 cells transfected with myc-tagged AIRE were carried out with antibodies recognizing DNA-PKcs, myc tag and GAPDH (as a negative control). The co-immunoprecipitates were further incubated with biotinylated p53 derived peptide as a DNA-PK specific substrate [32] in the presence of DNA-PKcs activating dsDNA and 32P-\u03b3ATP. As a positive control, HEK293 nuclear extract was used instead of the co-immunoprecipitates. Fig. 3A shows that a similar amount of 32P was incorporated into the p53 peptide when AIRE and DNA-PKcs co-immunoprecipitates were applied, whereas no activity was detectable with GAPDH co-immunoprecipitate. The relatively high phosphorylation efficiency achieved with AIRE co-immunoprecipitate was most likely due to the reason that the co-immunoprecipitation with anti-myc antibody is more efficient than that of with DNA-PK antibody. Since the p53 peptide used in the assay is a DNA-PK specific substrate [32], these data show that the AIRE\/DNA-PK complex has kinase activity and suggest that AIRE, too, might be phosphorylated by DNA-PK.\nTo further investigate whether AIRE can be phosphorylated by DNA-PK, we carried out three different in vitro kinase assays where recombinant GST-AIRE, GST-p53 and GST alone were used as the substrates. First, we performed the experiments using the nuclear extract from HEK293 cells as a source of kinases in the absence or presence of a specific inhibitor for PI3 family kinases, LY294002 (Fig. 3B). Second, we did the kinase assay using the nuclear extracts either from DNA-PKcs positive (MO59K) or DNA-PKcs negative (MO59J) cell lines (Fig. 3C). Third, we carried out kinase reactions using a purified HeLa DNA-PK complex, again, in the absence or presence of LY294002 (Fig. 3D). As a result, we observed that the phosphorylation level of the AIRE protein reached up to 50\u201380% of that of the p53 protein in all three experimental approaches, whereas only background phosphorylation of GST alone was detected (Fig. 3B\u2013D). In concordance, the kinase activity was decreased, although moderately, when LY294002 inhibitor was added (Fig. 3B) or when the nuclear extract from DNA-PKcs negative MO59J cells was used (Fig. 3C), indicating that besides DNA-PKcs other kinases can phosphorylate the AIRE protein.\nIn order to identify which region of the AIRE protein is phosphorylated, we expressed different AIRE domains as GST fusion proteins and tested their phosphorylation using the nuclear extracts as a source of kinases. The domain structure of the AIRE protein is given in Fig. 4A. Fig. 4B shows that the HEK293 nuclear extract phosphorylated more efficiently the GST-fusions of N-terminal AIRE containing the first 256 (1\u2013256), 293 (1\u2013293) or 348 (1\u2013348) amino acids (Fig. 4B) while about 2 times less phosphorylation was seen with the GST-fusions of AIRE C-terminal fragments containing SAND (175\u2013298) or SAND-PHD1-PHD2 (178\u2013482) regions. A similar pattern of AIRE phosphorylation was observed when the phosphorylation assay was performed with DNA-PKcs competent MO59K or DNA-PKcs deficient MO59J cell extracts (Fig. 4C). Thus, although in pull-down experiments, the first PHD of AIRE alone was able to interact with DNA-PK, the main DNA-PK target seems to be the N-terminal part of AIRE indicating that AIRE and DNA-PK make multiple contacts. Taken together, these results demonstrate that DNA-PK can phosphorylate AIRE in vitro and that the N-terminal region of AIRE is the predominant target of DNA-PK.\n3.4\nT68 and S156 are the phosphorylation sites on the AIRE protein\nThe N-terminal region of AIRE contains the HSR domain that mediates di- or tetramerization of the protein [4,5]. Interestingly, previous in vitro experiments have indicated that AIRE dimerization may occur as a result of phosphorylation [4]. We next aimed to find the DNA-PK specific phosphorylation sites on AIRE. Using the Scansite 2.0 and NetPhos 2.0 programs, we found two putative DNA-PK phosphorylation sites: T68 within and S156 close to the HSR domain of the AIRE protein (Figs. 4A and 5A). To verify the prediction, we changed either Thr68 or Ser156 to alanines, both in wild type GST-AIRE and truncated GST-AIRE (1\u2013256) constructs, and tested the influence of these mutations on the phosphorylation efficiency. Fig. 5B demonstrates that compared to GST-AIRE, the phosphorylation levels of the mutant proteins were decreased about 30\u201350% when the cell extracts from either HEK293 or MO59K cells were used as source of cellular kinases. Finally, we performed the phosphorylation assays using purified DNA-PK complex (Fig. 5C). In agreement with the previous experiments, a reduction of about 30% in the phosphorylation levels of the T68A and S156A mutants was detected, whereas the APECED patient mutation V80L did not negatively affect the AIRE phosphorylation level. Thus, we conclude that amino acids Thr68 and Ser156 of the AIRE protein are the phosphorylation sites of DNA-PK.\n3.5\nDNA-PK is expressed in thymic medullary epithelial cells (mTEC)\nDNA-PK is a ubiquitously expressed protein which levels are enhanced by induction of DNA double strand brakes [17]. To study whether the DNA-PK and AIRE interaction might occur in vivo, we studied whether DNA-PK is also present in thymic medullary epithelial cells (mTEC): the only subset of cells where the AIRE protein expression is detected thus far [1\u20133]. Thus, we purified the thymic mTEC based on the cell surface marker EpCAM [29] and analyzed the expression of Aire, DNA-PK and two AIRE target genes; involucrin and loricrin. The involucrin and loricrin genes are highly expressed during the terminal differentiation of epidermal keratinocytes [33] and are downregulated in mTEC of Aire knock-out mouse [34]. Fig. 6, right panel demonstrates that DNA-PKcs mRNA is highly expressed in thymic epithelial cells, including the AIRE-enriched mTEC fraction. As characteristic to Aire-regulated genes in the thymus, the expression levels of involucrin and loricrin were relatively low, remarkable lower than those of DNA-PKcs and Aire in mTEC subpopulation (Fig. 6, left panel).\n3.6\nT68A and S156A mutations downregulate the transactivation activity of AIRE\nPreviously, it has been demonstrated that several missense mutations in the HSR domain affect the dimerization, cellular localization and the transactivation ability of AIRE [13\u201315,35]. Thus, we next tested whether the mutation of AIRE phosphorylation sites influences the AIRE transactivation activity. We cloned AIRE with T68A and S156A mutations into mammalian expression vector and performed luciferase assays using the lysates from HEK293 cells transfected with wild type AIRE or the mutant constructs. As AIRE target genes, human loricrin and involucrin gene promoters fused to the luciferase gene were used. Fig. 7A demonstrates that when the HEK293 cells were transfected with AIRE T68A and AIRE S156A mutation constructs, significantly lower transactivation activity compared to the wild type AIRE was seen. Western blot of the transfected lysates with anti-AIRE antibody shows that both mutants are expressed even in higher level than the wild type AIRE construct (Fig. 7B). In conclusion, the luciferase assays suggest that the phosphorylation status of AIRE at T68 and S156 is important for the transactivation activity.\n4\nDiscussion\nIn this study, we demonstrate that AIRE interacts with and is phosphorylated by the DNA-PK complex. The best DNA-PK substrates are the DNA associated proteins and the most effective phosphorylation occurs when DNA-PK is colocalized with the same DNA molecule as its target proteins [36]. Binding to the Ku proteins improves the affinity of DNA-PKcs for DNA ends about 100-fold [37]. This is in keeping with the finding showing that AIRE is able to bind DNA via its SAND domain [4]. In addition to the double strand brakes, the DNA-PK and\/or the Ku proteins are known to bind telomeres and sequence-specific promoter elements [17,24], single stranded DNA [38], nucleosomes [39], RNA [18] and base unpairing regions (BURs) which typically are found in nuclear matrix attachment regions (MARs) [40]. Ku70 and Ku80 also colocalize to RNA polymerase II elongation sites [41,42]. Interestingly, AIRE has been reported to associate with nuclear matrix [43] and has been proposed to regulate gene clusters [44]. In addition, the AIRE protein contains PHD domains, which recently have been shown to recognize specifically modified histones within the chromatin [10,11]. Thus, AIRE appears to be a chromatin and DNA-binding protein. In this study, we observed that although AIRE is able to bind DNA-PK without DNA, most likely the DNA or chromatin is contributing to the AIRE and DNA-PK interaction (Fig. 2C). This is in concordance with DNA-PK ability to phosphorylate its substrates more efficiently at the presence of DNA [17]. At least two possibilities can be considered how DNA-PK, AIRE and DNA or chromatin may interact. First, DNA or chromatin, or even certain DNA sequence elements or specifically modified chromatin, may increase the affinity of AIRE for DNA-PK. Second, posttranslationally modified AIRE subpopulations may bind DNA-PK in different conditions. As we cannot exclude that this type of experiments can be influenced by the overexpression of AIRE, further studies are needed to find out the role of chromatin in AIRE and DNA-PK interaction in vivo, especially in the thymic environment.\nPhosphorylation of AIRE by cAMP dependent protein kinases A (PKA) and C (PKC) has been reported earlier [4], though, the phosphorylated domain of AIRE remained unknown. We here demonstrate that in vitro the AIRE protein is mainly phosphorylated at the N-terminal region and define two residues, Thr68 and Ser156, as DNA-PK specific target sites. Thr68 resides within and Ser156 is located not far from the HSR domain. Both these phosphorylation sites are located outside the PHD1 finger, the domain of AIRE that was initially shown to pull-down DNA-PK, indicating that multiple different contacts can occur between AIRE and the DNA-PK complex. However, as previously reported [4], our in vitro phosphorylation studies also indicate that other kinases may phosphorylate the AIRE protein, and that phosphorylation sites other than Thr68 and Ser156 should exist within AIRE.\nThe HSR domain of AIRE has been shown to be responsible for the dimerization and tetramerization of the protein [4,5] and several, but not all, APECED mutations within the HSR decrease AIRE transactivation activity and influence AIRE cellular localization [15,16]. In addition, previous in vitro experiments have indicated that AIRE dimerization may occur as a result of phosphorylation [4]. As shown in this study, conversion of Thr68 and Ser156 amino acids to alanines suppresses AIRE transactivation activity. However, we did not observe any influence of these mutations to AIRE dimerization and cellular localization (data not shown). Thus, since the mutations that do not impact AIRE dimerization and the dot formation ability can downregulate AIRE transactivation activity, the N-terminal part of AIRE appears to be involved in transcriptional regulation not only via its dimerization activity. It is possible that status of AIRE phosphorylation at N-terminus influences AIRE interactions with other protein partners or with DNA.\nIn response to ionizing radiation, DNA-PK can have indirect effects on transcriptional regulation. It has been shown that phosphorylation of interferon regulated factor 3 (IRF-3) and pancreatic duodenal homeobox-1 (PDX-1) protein by DNA-PK in response to ionizing radiation directs these proteins to degradation by the proteasome and therefore downregulates IRF-3 and PDX-1 target genes [45,46]. Alternatively, phosphorylation of transcription factor Oct-1 by DNA-PK in response to ionizing radiation leads to stabilization but disrupts transactivation activity of Oct-1. As a result, the expression of Oct-1 target genes U6 and Histone H2B is downregulated [47]. We also studied whether UV irradiation changes AIRE cellular localization and expression level in various cell cultures but we could not detect remarkable differences (data not shown). Thus, it seems that DNA-PK phosphorylates AIRE independently of its function in DNA repair.\nBased on research on AIRE deficient mice, it is evident that AIRE upregulates the expression level of many genes in thymus [48,49]. Studies in tissue culture cells confirm that AIRE indeed activates several promoters [5,13,15,16]. Thus, although in vivo the AIRE protein expression is only detected in thymic medullary epithelial cells [1\u20133], the transfected AIRE protein has similar transactivating properties, and nuclear localization pattern as AIRE in the thymus [14,16]. Despite these studies, the mechanisms of how AIRE regulates its target genes are still unclear. Posttranscriptional modifications, like phosphorylation, acetylation and ubiquitination, are widely used to regulate activity of transcription factors and regulators. In this study, we first describe the phosphorylation sites of AIRE and propose that its phosphorylation status is important for its transactivation activity. However, several questions remain, including what are the signals that trigger AIRE phosphorylation and how AIRE phosphorylation is connected with its function in the thymus. The differentiation of AIRE positive thymic epithelial cells was recently shown to be dependent on RANK-mediated signaling [50], which is known to activate several protein kinases [51]. As we showed in this study, DNA-PK is expressed in medullary epithelial cells (Fig. 6). Whether the thymic epithelial differentiation process correlates with the activation of DNA-PK and other possible AIRE-specific kinases needs further studies.","keyphrases":["phosphorylation","apeced","phd finger","hsr domain"],"prmu":["P","P","P","P"]}
{"id":"J_Gastrointest_Surg-3-1-1852377","title":"Does Microvascular Invasion Affect Outcomes After Liver Transplantation for HCC? A Histopathological Analysis of 155 Consecutive Explants\n","text":"Macroscopic vascular invasion (macroVI) is associated with poor outcomes after liver transplantation (LT) for hepatocellular carcinoma (HCC). Whether microvascular invasion (microVI) is associated with the same adverse prognosis is unclear. One hundred and fifty-five consecutive patients with confirmed HCC after LT from March 1991 to 2004 at our institution were reviewed. Patients had to satisfy Milan criteria to be accepted for LT. They were followed with surveillance images every 3 months while on the waiting list. Disease-free survival (DFS) and overall survival (OS) were evaluated by Kaplan\u2013Meier analysis. Demographic, tumor, and histopathologic characteristics were tested for their prognostic significance. Median follow-up after LT was 30 months. Overall graft survival rates were 87, 74, and 65% at 1, 3, and 5 years, respectively. All recurrences (22\/155, 14%) developed within 4 years after LT with an overall 5-year DFS of 79%. Vascular invasion, either microVI or macroVI, was more likely in patients with multicentric HCC (n \u2265 3, p < 0.001) and larger tumor size >4 cm (p = 0.04). Tumor size >5 cm (p = 0.04), advanced pathological TMN stage (p = 0.007), microVI (p = 0.001), and macroVI (p < 0.001) predicted poor tumor-free survival on univariate analysis, but only macroVI was significant in multivariate analysis (hazard ratio 54.2, 95% confidence interval 11, 266). Furthermore, only macroVI was a significant predictor of mortality after LT (p = 0.01). Macrovascular invasion is strongly associated with high rates of recurrence and diminished survival after LT whereas microVI is not an independent risk factor.\nIntroduction\nIn 1996, Mazzaferro et al.1 documented excellent survival results after liver transplantation (LT) in a highly selected group of patients with hepatocellular carcinoma (HCC) with a single tumor <5\u00a0cm or as many as three tumors, each smaller than 3\u00a0cm. Recent studies have shown that selected patients who do not meet these criteria can still be cured with a transplant; the challenge now is to decide which factors, other than size and number, carry a sufficiently poor prognosis to deny transplantation.2\u20135\nGross vascular invasion or radiological evidence of tumor invasion in major veins is a known determinant of poor outcome after resection or transplantation for HCC and is an absolute contraindication to LT.4\u201313 Macrovascular invasion (macroVI) or gross vascular invasion of major portal or hepatic veins evident visually at time of transplant or on pathological evaluation may also be a predictor of recurrence after LT.14 Whether microvascular invasion (microVI), defined as microscopic tumor invasion in smaller intrahepatic vessels identified on pathologic analysis, or macroVI should also be considered a contraindication to LT is controversial. Both types of tumor invasion are difficult to determine pre-LT; therefore, their significance remains uncertain.\nSeveral published studies have found histopathological factors, namely, poorly differentiated grade, microVI, and macroVI, to be independent predictors of poor survival after LT.4,11,15\u201317 We have previously shown that microVI is an independent predictor of early recurrence after resection for HCC.5 The purpose of this study was to determine if microVI, macroVI, and other pathological factors are associated with tumor recurrence after LT and examine the outcomes thereafter. If these factors are predictors of poor tumor-free survival, then how can we attempt to identify these variables before transplant?\nPatients and Methods\nOne hundred fifty-nine consecutive patients with confirmed HCC on liver explant pathology after LT from October 1991 to October 2004 at our institution were reviewed from a prospective database. The cohort includes 32 patients (20%) with incidental tumors. Patients had to satisfy Milan criteria and have no radiologic evidence of gross vascular invasion to be accepted for LT. Four patients were omitted from analysis for recurrent hepatitis B virus (HBV) before antiviral therapy; after 1991, hepatitis B immune globulin, lamivudine, or combination therapy was used. Patients with known HCC were followed every 3\u00a0months while on the waiting list with either an ultrasound or triphasic computed tomography (CT). Patients with HCC were eligible for deceased donor whole organs (n\u2009=\u2009149) and split (n\u2009=\u20090) or living donor LT (n\u2009=\u20096).\nPatient, tumor, operative, and treatment characteristics were evaluated using a prospective clinical database and review of all pathological explants. The stage, size, and histopathology of the tumor were determined by analysis of the liver explant. Patients were staged based on the explanted specimens using the TNM staging classification of the American Liver Tumor Study Group18. Tumor size was measured as the largest diameter of the major tumor in centimeters. MacroVI was defined as gross vascular invasion into major portal vessels or hepatic veins identified either intraoperatively or on pathologic explant, whereas microVI was determined on pathologic analysis as microscopic vascular invasion of small vessels within the parenchyma of the liver. Pretransplant therapy was used in selective cases; if waiting time was determined to be longer than 6\u00a0months, patients commonly underwent radio frequency ablation as a bridge to LT. This was performed in an attempt to keep patients within Milan criteria while on the LT waiting list. Postoperative immunosuppression was similar in all patients and consisted of cyclosporine or tacrolimus and steroids.\nAfter LT, patients were followed with Q3 monthly alpha-fetoprotein (AFP) and CT along with standard post-LT evaluation. Recurrences were defined as new nodules diagnosed by CT with confirmed biopsy in most cases. Overall survival (OS) was death as a result of any cause after LT. Patients were followed until death or study closure (arbitrarily denoted as October 1, 2004). Data was collected until May 1, 2005 to ensure at least 6\u00a0months of follow-up for all patients.\nStatistical Package for Social Sciences software (SPSS, Inc., Chicago, IL) was used for data analysis. Statistical comparison of categorical and continuous variables was performed using the \u03c72 test and Mann\u2013Whitney U test, respectively. All data was reported as median with range, mean \u00b1 SD or interquartile range (IQR) when appropriate. Analysis of patient OS and disease-free survival (DFS) was performed according to the Kaplan\u2013Meier method. Patient survival in different groups was compared using the log-rank test. All variables with a p value less than 0.1 were then included in a multivariate analysis applying the Cox multiple backward stepwise model.\nResults\nPatient Characteristics\nOf the 1,070 LTs performed during the 13-year period from 1991 to 2004, 159 patients (14%) had HCC. After omission of 4 patients because of recurrent HBV before the antiviral therapy era, 155 patients were included in this analysis (Table\u00a01). The average waiting time for LT was 7\u00a0months. There was no perioperative mortality. The median age was 57\u00a0years (range 28\u201370) and the majority of patients were men (79%). The median age of all patients who underwent LT for any cause was 51\u00a0years (range 16\u201371). The most common causes of end-stage liver disease and HCC were hepatitis C virus (HCV) (n\u2009=\u200979; 51%), alcoholic liver disease (n\u2009=\u200934; 22%), and HBV (n\u2009=\u200925; 16%). Selected patients underwent pre-LT therapy, most commonly radio frequency or percutaneous ethanol ablation (ablation, 23%), but the majority of patients received no treatment before LT (72%). Use of ablation did not result in any adverse outcomes in this series of patients who underwent eventual LT. \nTable\u00a01Pretransplant Demographics of 155 Patients with HCC Who Underwent LTPretransplant CriteriaN\u2009=\u2009155p ValueMedian age (range)57 (28\u201370)0.48Sex0.38\u00a0Male123 (79%)\u00a0Female32 (21%)Cause of liver disease0.10\u00a0HCV79 (51%)\u00a0HBV25 (16%)\u00a0Alcohol34 (22%)\u00a0Cryptogenic7 (4.5%)\u00a0Alpha-1 antitrypsin6 (3.9%)\u00a0NASH2 (1.3%)\u00a0PBC2 (1.3%)Pretransplant therapy0.67\u00a0Ablation36 (23%)\u00a0Resection6 (3.9%)\u00a0TACE1 (0.6%)\u00a0EBRT1 (0.6%)\u00a0None111 (72%)The p values were determined by log-rank test as predictor of DFS after Kaplan\u2013Meier analysis.NASH = nonalcoholic steatohepatitis, PBC = primary biliary cirrhosis, EBRT = external beam radiation therapy\nHistopathological Analysis\nThe pathologic features for the 155 explants are shown in Table\u00a02. The median number of tumors was 2 (range 1\u201320) and 18% were bilobar. The median size of the largest tumor was 2.6\u00a0cm and most tumors were graded as well or moderately differentiated (74%). Gross macroVI was evident in 3.9% (6\/155) of the explants on pathologic examination, whereas 21% (33\/155) of explants had the presence of microVI. Using the pathological TNM classification, 31 patients (20%) had stage I tumors, 69 patients (44%) had stage II tumors, 26 patients had stage III tumors (17%), and 29 patients had stage IV tumors (19%). Patterns of advanced stage were most often because of multifocal HCC or three or greater in number (40%). \nTable\u00a02Pathologic Characteristics of 155 Liver ExplantsCharacteristicN\u2009=\u2009155p ValueHR (95% CI)No. of tumors\u00a0Median (range, cm)2 (1\u201320)0.23\u00a0<3126 (81%)\u00a0>329 (19%)Bilobar32 (21%)0.15Size\u00a0Median (range; cm)2.6 (.1\u201316)0.040.47 (0.14,1.61)\u00a0<5\u00a0cm145 (94%)\u00a0>5\u00a0cm15 (10%)Stage\u00a0I31 (20%)0.0071.17 (0.39,3.50)\u00a0II69 (44%)\u00a0III26 (17%)\u00a0IV29 (19%)Positive lymph nodes3 (2.0%)0.31Vascular invasiona\u00a0Microscopic33 (21%)0.0013.16 (0.92,10.93)\u00a0Macroscopic6 (4%)<0.00154.2 (11.03,266.4)\u00a0None121 (78%)b1.0cGrade\u00a0Well\/mod115 (74%)0.36\u00a0Poor13 (9%)\u00a0N\/Ad27 (17%)Margins\u00a0Positive2 (1%)0.25\u00a0Negative153 (99%)Incidental tumor34 (22%)0.19The p values were determined by chi-square test or log-rank test of variables after Kaplan\u2013Meier analysis (univariate). HR (95% CI) represents multivariate analysis of factors affecting recurrence after resection.aFive of six patients had characteristics of both microvascular and macrovascular tumor invasion.bReference category for comparisoncReference category for each categorical variable is assigned HR\u2009=\u20091.0.dNot available in the analysis\nVascular invasion was associated with both the number and size of tumors. Liver explants, with either microVI or macroVI, had more tumors (3.8 vs 2.0, p\u2009<\u20090.001) and were larger in size (3.6\u00a0cm vs 2.8\u00a0cm, p\u2009=\u20090.04) compared to those without vascular invasion. Specifically, if microVI or macroVI was present on histopathology, 64 and 100% of the tumors, respectively, were outside of the Milan criteria or were TNM stage III or IV. Histological grade was not associated with either type of vascular invasion; 85% of patients with either type of vascular invasion had favorably differentiated tumors. Ablation did not affect the rate of vascular invasion, either microVI or macroVI, in this series. Eleven of 33 patients (33%) with vascular invasion on explant analysis had undergone ablation of their tumor.\nPredictors of Recurrence and Survival\nThe 5-year disease-specific survival was 79% (Fig.\u00a01a). All recurrences developed within 44\u00a0months after LT. The 1-, 3-, and 5-year overall graft OS rates were 87, 74, and 65%, respectively (Fig.\u00a01b). Only 22 patients (14%) developed tumor recurrence after LT with a median follow-up of 30\u00a0months (range 6\u2013144\u00a0months). Eighty-six percent of patients (122\/155) are currently alive and free of cancer. There was no difference in OS in patients with incidental tumors compared to those with known HCC (data not shown), but a significant difference was observed in DFS (5-year OS rate 94 vs 74%, p\u2009=\u20090.02).\nFigure\u00a01(A) Disease-free survival after LT for HCC of 155 patients. (B) Overall graft survival after LT for HCC of 155 patients.\nThe median time to recurrence for the 22 patients who developed recurrent HCC was 16.3\u00a0months (IQR 8.0\u201328). Tumor size >5\u00a0cm (p\u2009=\u20090.04), pathological TMN stage (p\u2009=\u20090.007), microVI (p\u2009=\u20090.001), and macroVI (p\u2009<\u20090.001) were found to be independent predictors of tumor-free survival. Patients with tumor size of 5\u00a0cm or larger had a 5-year DFS of 48 vs 82% for those with tumors smaller than 5\u00a0cm. Advanced TMN stage was also associated with poor recurrence-free survival after LT (Fig.\u00a02). Twenty-one of the 22 patients (95%) who developed HCC recurrence were found to have either microVI (n\u2009=\u200915) or macroVI (n\u2009=\u20096) on pathological analysis. This accounts for 68% (15\/22) of all patients with microVI and 100% (6\/6) of patients with macroVI in the entire study. Patients with macroVI had a median DFS of only 7.1\u00a0months compared to a more favorable DFS in patients with microVI or no vascular invasion (median not achieved) (Fig.\u00a03). No significant differences in DFS based on age, sex type, hepatitis status, tumor grade, bilobar disease, tumor number, ablative therapy, or type of transplant were found on univariate analysis. Pre-LT therapy did not result in any improvement in DFS or OS after LT (data not shown).\nFigure\u00a02The effect of TNM stage on DFS after LT for HCC.Figure\u00a03The effect of vascular invasion on recurrence-free survival after LT for HCC.\nIn a multivariate analysis including factors with an influence on DFS, only macroVI (hazard ratio [HR] 54.2, 95% CI 11.03\u2013266.4) was identified to be predictive (Table\u00a02). This was confirmed with a Cox backward stepwise model of multivariate analysis (data not shown).\nDiscussion\nIn this study, we report our experience with LT for HCC with a specific focus on pathological factors affecting long-term outcomes. Overall survival rates of LT for HCC were 87, 74, and 65% at 1, 3, and 5\u00a0years, respectively, with a median follow-up time of 30\u00a0months. The overall recurrence rate was 14% with 79% 5-year disease-specific survival. Patients with incidental tumors had similar OS rates as those with known tumors consistent with previous reports,8,9,14,15,19 but in this study, a significant difference was found in DFS. Our studies suggest that large tumor size (>4\u00a0cm) and multiple tumors (\u22653) correlate with an increased incidence of vascular invasion and may provide a surrogate marker for entities that are often difficult to detect before LT.\nRecurrence rates after LT may not simply reflect only size and number as suggested in the initial Milan series, but may be a complex interplay of host- and tumor-related factors, which are still largely unknown.20 This report suggests that tumor grade, size, number, and microVI do not influence outcome after LT for HCC; only the presence of macroVI appears to be associated with poor outcomes on multivariate analysis. MacroVI and microVI were more commonly found in multicentric (three or more) and large (>4\u00a0cm) HCC. Furthermore, 21 of 22 recurrences had evidence of either microVI or macroVI on pathologic examination. Because we are unable to identify these biologic factors preoperatively, markers of histopathologic or biologic variables that predict poor outcomes are extremely important.14,20\u201322\nMacroVI was shown to be an independent predictor of tumor recurrence after LT in some studies.4,9,10,14 The Pittsburgh group found that microVI and major vascular invasion was associated with increased risk of recurrence by multivariate analysis.4,8 In a report of 344 patients with HCC treated by LT, microVI and macroVI were associated with 4.4- and 15-fold increased risk of recurrence, respectively.8 Shetty et al.14 found that macroVI, but not microVI, was a significant predictor of DSF and OS after LT for HCC. In this study, microVI is associated with higher stage and recurrent tumors, but does not appear to be an independent factor for survival.\nThe role of microVI on posttransplant recurrence and survival outcomes for HCC still remains unclear. Several published studies have found poorly differentiated histological grade or microVI to be independent predictors of impaired survival after LT.11,15\u201317 Jonas et al.11 found vascular invasion and histological grade to be the only statistically significant independent predictors of poor survival after LT in 120 patients. In their study, only poorly differentiated tumors larger than 5\u00a0cm predicted the presence of microVI. But other studies involving LT and HCC were not able to corroborate poor results regarding microVI.3,14,23,24 The close relationship between histological grade and microVI may explain why microVI is often eliminated in multivariate models for analyzing tumor recurrence prognostic factors that include histological grading and vice versa.4,8,11,15,23,25,26 Multiple tumors, larger tumors, and higher grade of differentiation have all been shown to be associated with microVI after resection for HCC. Esnaola et al.13 reported that tumor size greater than 4\u00a0cm and poorly differentiated\/undifferentiated histopathologic grade increased the odds of microVI by 3 and 6.3-fold, respectively, but these tumors were primarily in Child class A cirrhotic.\nThe degree of fibrosis and scarring of the liver may play a significant role in the biological behavior and significance of microVI and macroVI. We have previously shown that microVI was a significant predictor of early recurrence and death after resection for HCC in cirrhotic patients.5 Most recurrences were intrahepatic and away from the staple line, suggesting that liver mobilization and manipulation may cause progression of microVI or a new tumor has developed in the presence of ongoing oncogenic stimulus from cirrhosis. For these reasons, prognostic factors for LT from resection studies should be interpreted with caution and a possible rationale why microVI is so important after resection but not after LT.12,13 Lack of manipulation of the liver and intrahepatic dissection may be a potential explanation for the lack of importance of microVI with LT.\nBecause of the importance of histological features of HCC, some have advocated pre-LT biopsy to examine grade, vascular invasion, and genetic typing.13,15,20 Complications of needle biopsy such as tumor tract seeding and lack of sensitivity have made routine biopsy unfavorable.27 In our experience, needle biopsy was a poor predictor of microVI or macroVI, and therefore was not considered in our pre-LT work-up for HCC (data not shown).\nThere are several limitations to this study and therefore some of the results should be interpreted with caution. The need to standardize grading systems for HCC has long been recognized and would allow us to determine if tumor grade is indeed an important prognostic marker for recurrence and survival. Few tumors in this study were graded as poorly differentiated; moreover, in 27 patients, their grade was not determined. Results from histopathological analysis are often met with inherent biases from the pathologist and comprehensive evaluation of the whole liver explant may vary among pathologists and institutions. Finally, with very few tumors containing macroVI, strong conclusions about prognostic characteristics concerning macroVI cannot be made in this report. Whether microVI is a harbinger of macroVI or in some way correlated with a more aggressive form of HCC remains unclear.\nThe use of pathological and biological features of the tumor may allow us to identify those patients who are at increased risk of recurrence; then these patients should be considered for adjuvant therapy before evidence of a recurrence. Because vascular invasion is more common in multicentric (>3) HCC or large tumors (>4\u00a0cm), we propose shortening the interval of pre-LT imaging in patients with these tumors to identify vascular invasion or rapid growth of the tumor before LT. Genetic testing of tumors before LT may be a novel method to predict other prognostic factors affecting recurrence.20 Several studies have reported improved posttransplant survival in HCC patients with transcatheter arterial chemoembolization (TACE) or systemic therapy,28\u201331 but overall results for adjuvant chemotherapy post-LT were disappointing.32 Because of small numbers, this would be best done in a multicenter-randomized trial. We are currently evaluating the role of tumor size and number in our allocation system in LT for HCC to determine their respective predictive value for prognosis.\nIn summary, LT for HCC can be performed with acceptable survival outcomes. A single tumor characteristic alone does not appear to determine prognosis or outcome. In the present study, macroVI alone was associated with very poor outcomes after LT. Extending criteria of LT for advanced HCC is possible only with better patient selection using improved pre-LT staging and identification of histopathological biological markers such as macroVI that would preclude LT.","keyphrases":["microvascular invasion","vascular invasion","liver transplantation","hepatocellular carcinoma","recurrence"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_Spine_J-2-2-1602199","title":"Endovascular treatment of an open cervical fracture with carotid artery tear\n","text":"The dilemma of how to treat penetrating wound injuries to the neck, which involve a combination of a common carotid artery rupture and a cervical spinal fracture, is presented in this case report.\nIntroduction\nPenetrating injuries of the carotid artery can pose a significant challenge to the vascular surgeon. This type of vascular trauma accounts for only 3% of patients studied retrospectively in most trauma centers [1, 3, 4, 6, 9, 10, 13]. The severity of injury varies from complete transection to formation of a pseudoaneurysm or initimal flap formation with or without occlusion. Traditionally, the treatment of these lesions has been surgical. However, limitations to obtain satisfactory vascular control led to the search for other alternative treatment options [1, 4, 13]. Recently, the management armamentarium has expanded with the advent of more sophisticated endovascular techniques [3, 9].\nPseudoaneurysm of the common carotid artery after gunshot injury is a relatively infrequent finding. We present a case of a common carotid artery pseudoaneurysm managed by a commercially flexible covered stent.\nCase history\nA 16-year-old adolescent was transferred to our institution from a different hospital for stabilization of a cervical spinal fracture. Six hours prior to his transfer he had sustained an isolated gunshot wound to his neck. He was quadraplegic and unconscious on his admission to the referring hospital and was therefore intubated and ventilated. CT scan of the cervical spine showed a blow-out fracture of the body of C6 with involvement of the left vertebral foramen (Fig.\u00a01). CT scans of the brain and chest were within normal limits.\nFig.\u00a01CT scan at the C6 level demonstrating a vertebral body fracture extending into the left vertebral foramen\nUpon admission to our institution his hemodynamic condition was stable with a blood pressure of 115\/70\u00a0mmHg and pulse of 60\u00a0beats\/minute. He was ventilated and had a SaO2 of 100% with FiO2 of 0.4. Physical examination of his neck area demonstrated an entry wound in the posterior neck to the left of the midline and an exit wound in the left side of his neck. A large hematoma on the left side of the neck was demonstrated and palpated. A duplex scan of the left carotid artery was performed and was interpreted as a probable false aneurysm of common carotid artery. CT angiogram of the neck demonstrated anterior and posterior leak of contrast from the common carotid artery, 1\u00a0cm proximal to the bifurcation (Fig.\u00a02). The left vertebral artery was shown to be intact. The left internal jugular vein was compressed by the hematoma. The patient was transferred to the angiography suite and the presence of false aneurysms was confirmed. Under local anesthesia, through a transfemoral approach a catheter was inserted into the aneurysmatic area. The aneurysms were treated by the insertion of a covered stent (Jostent 8\u00a0(\u00d7\u00a038\u00a0mm2), which also occluded the origin of the external carotid artery (Fig.\u00a02). No change in his neurological status after the insertion of the stent was seen (Fig.\u00a03). No anticoagulation was given. His neck fracture was stabilized by an external fixation device and he was transferred back to the referring hospital.\nFig.\u00a02Selective left common carotid artery angiography shows traumatic pseudoaneurysm 1\u00a0cm below bifurcationFig.\u00a03Selective common carotid artery angiography demonstrates endovascular stent\u2014extending proximal and distal to the borders of pseudoaneurysm with complete exclusion of pseudoaneurysm and external carotid artery\nOne year later, duplex US follow-up showed good hemodynamics without stenosis. Clinically, no cranial lesions were found.\nDiscussion\nThe use of endovascular stent-grafts to address arterial tears was first introduced by Dotter in 1969 [2]. Over the past decade, endovascular grafting has been evaluated as an alternative to conventional surgical repair in the management of aneurysms and other vascular lesions. The application of this form of therapy to the management of vascular trauma offers many potential advantages. Angiography assists with the diagnosis of traumatic lesions such as intimal dissections [12]. In addition, endovascular techniques can be utilized to help with vascular control, as an adjunct to surgery in regions where obtaining proximal or distal control is technically difficult. The use of endovascular stent-grafting for the definitive repair of traumatic lesions has been shown to be associated with a decrease in anesthetic requirement, blood loss and extent of dissection [7]. High-velocity missiles can produce a significant amount of tissue damage, even if not suspected on initial examination [11]. The inherent problems of graft placement in the setting of severe tissue damage, gross contamination and scar tissue formation make the use of a remote insertion site particularly advantageous in the trauma patient [5, 8].\nInjury to the carotid artery constitutes only 3% of all arterial injuries [1, 3, 4, 9, 10, 13]. In spite of the rarity of carotid injuries, this trauma presents significant challenges to the vascular surgeon. Gunshot wounds account for approximately half the injuries [9]. Injury of the carotid artery is frequently associated with other organ injuries [1, 3, 9]. Clinical examination may be unreliable due to confounding factors such as destructive injuries of spine, chest, abdomen, fractures [1, 3, 4, 6, 9, 10, 13]. Therefore, imaging evaluation is essential for the identification and localization of carotid trauma [1, 3, 9].\nIn contrast to blunt carotid injury, treatment of penetrating injuries of the carotid artery has been primarily surgical [4, 10, 13]. Depending on the described clinical scenario the surgeon is left with several options, which are observation, anticoagulation, ligation of the carotid artery, balloon occlusion of the artery, resection with graft interposition or bypass procedure. However, the best definitive management remains controversial, as no prospective studies regarding the management of penetrating carotid injuries have been reported [1, 3, 4, 6, 9, 10, 13]. Only few case reports on internal carotid artery aneurysm and pseudoaneurysm treated with endovascular stent and coiling procedure have been published [3, 9]. In case of carotid trauma, surgical treatment would be difficult because of problems of exposing the bleeding carotid and to get enough vascular access.\nThe combined evidences from all cases published in the medical literature support endovascular intervention for pseudoaneurysm of the carotid artery as a good clinical treatment choice. In our patient, we chose to use device graft to ensure complete exclusion of the pseudoaneurysm and external carotid artery without the need for endovascular coil placement. Further studies to investigate the role of endovascular treatment for penetrating carotid artery injury are indicated.","keyphrases":["endovascular treatment","cervical fracture","carotid artery tear","penetrating injury"],"prmu":["P","P","P","P"]}
{"id":"Naunyn_Schmiedebergs_Arch_Pharmacol-3-1-1915601","title":"Validation of a rapid, non-radioactive method to quantify internalisation of G-protein coupled receptors\n","text":"Agonist exposure can cause internalisation of G-protein coupled receptors (GPCRs), which may be a part of desensitisation but also of cellular signaling. Previous methods to study internalisation have been tedious or only poorly quantitative. Therefore, we have developed and validated a quantitative method using a sphingosine-1-phosphate (S1P) receptor as a model. Because of a lack of suitable binding studies, it has been difficult to study S1P receptor internalisation. Using a N-terminal HisG-tag, S1P1 receptors on the cell membrane can be visualised via immunocytochemistry with a specific anti-HisG antibody. S1P-induced internalisation was concentration dependent and was quantified using a microplate reader, detecting either absorbance, a fluorescent or luminescent signal, depending on the antibodies used. Among those, the fluorescence detection method was the most convenient to use. The relative ease of this method makes it suitable to measure a large number of data points, e.g. to compare the potency and efficacy of receptor ligands.\nIntroduction\nWhen chronically exposed to an agonist, G-protein coupled receptors (GPCRs) can undergo desensitisation, i.e. the response to a given level of agonist exposure declines, and\/or more agonist is required to maintain the same response. Various mechanisms can be involved in desensitisation, some of which directly affect receptor responsiveness. One of these mechanisms involves phosphorylation of activated receptors by GPCR kinases (GRKs). This enables binding of arrestins to the receptor, leading to uncoupling from the G-protein and hence impaired signaling. Another, not necessarily mutually exclusive mechanism of desensitisation is internalisation of the receptor, which can be either homologous (because of activation of the receptor) or heterologous (because of activation of other receptors or to other stimuli). Receptor internalisation can be arrestin-dependent or independent (B\u00fcnemann and Hosey 1999) and involves translocation of the receptor from the cell membrane to the inside of the cell. There, it is entrapped inside clathrin-coated pits and can undergo either degradation or resensitisation.\nSeveral approaches have been used in the past to detect and quantify GPCR internalisation. One classical method involves homogenisation of tissues or cells, differential centrifugation to yield a plasma membrane and a microsomal fraction followed by electrophoresis and autoradiographical analyses of the receptor of interest in both fractions (Simpson et al. 1984). Another classical method involves labeling of all receptors in intact cells with a lipophilic radioligand and competition by a hydrophilic ligand, which will replace the radioligand from sites at the cell surface but not from those entrapped intracellularly (Staehelin et al. 1983). More recently, genetic approaches have been used. An example is the use of a C-terminal green fluorescent protein (GFP; Barak et al. 1997; Kallal et al. 1998) to monitor receptor localisation after ligand stimulation sometimes combined with an algorithm to discriminate between internalised and membrane-bound receptors (Conway et al. 1999; Fukunaga et al. 2006; Schlag et al. 2004; Hirasawa et al. 2005). This latter method uses a cell-based fluorescence imaging system ArrayScanII, where the amount of internalised receptor\/GFP spots are counted and normalised against the number of nuclei stained with Hoechst 33342. However, a GFP, or other fluorescent tag, is relatively large (200\u2013300 amino acids) and can influence receptor function (McLean and Milligan 2000). Other methods use a combination of techniques, like quantitative flow cytometry analysis of the cell surface receptors and immunocytochemical confocal microscopy analysis to visualise the sub-cellular localisation of the receptor of interest (Hirasawa et al. 1998).\nDetailed quantitative studies of internalisation are of interest as this process can be the mechanism of underlying physiological and pharmacological effects. For example, receptor internalisation is thought to underlie the modulation of immune function by sphingosine-1-phosphate (S1P; Matloubian et al. 2004). This also appears to be the mechanism of action of the immunosuppressive drug FTY720 (fingolimod; Chiba et al. 2006; LaMontagne et al. 2006), which is undergoing clinical trials for the treatment of multiple sclerosis and previously also for renal graft rejection (Budde et al. 2002; Kappos et al. 2006).\nIn our search to quantify internalisation of membrane receptors in a fast and easy way, we have setup a method, which uses an N-terminal HisG (6xHis+Gly)-tagged S1P1 receptor. Because the HisG-tag is N-terminal, it is extracellular. Therefore, when immunocytochemistry is performed without permeabilisation, only membrane-bound, tagged receptors will be detected. This way, internalisation is quantified as a difference in membrane-fluorescence between unstimulated and agonist-stimulated cells.\nThe use of small, N-terminal tags has been described before (Daunt et al. 1997; Vicentic et al. 2002; Wozniak and Limbird 1996; Von Zastrow et al. 1993; Miller 2004). Of these, Daunt et al. 1997 described a method where cells were plated onto 24 well tissue culture dishes. After stimulations with an agonist, incubations with first and second antibodies followed. Measurements were done after adding alkaline phosphatase substrate, resulting in a colorimetric reading. In this paper, we now describe the validation of a similar method by testing various antibody combinations and detection methods to quantify internalisation of N-terminal HisG-tagged receptors. This resulted in immunocytochemical method using a specific anti-HisG first antibody combined with a fluorescent second antibody (AlexaFluor\u00ae 488) as our preferred choice. The signal can either be visualised under the fluorescence microscope or measured in a fluorescence microplate reader. The latter results in quantitative measurement of the decrease in signal caused by receptor internalisation, in a direct, sensitive and fast way.\nMaterials and methods\nMaterials\npcDNA3.1 containing the entire coding region of the human S1P1 receptor was purchased from UMR cDNA Resource Center (Rolla, MO).\nCell culture media, hygromycine B, Lipofectamine\u21222000, pcDNA3.1\/His C, pOG44, CHO-FlpIn cells, Alexa Fluor\u00ae 488 goat anti-mouse (IgG), anti-HisG (mouse monoclonal IgG2a), anti-HisG-horse radish peroxidase (HRP; mouse monoclonal IgG2a), pcDNA5\/FRT\/TO, NuPAGE\u00ae System, NuPAGE\u00ae Novex 4\u201312% Bis\u2013Tris gel, MES running buffer, NuPAGE\u00ae transfer buffer and Invitrolon\u2122 PVDF blotting membranes were obtained from Invitrogen (Breda, The Netherlands). BCA\u2122 Protein Assay Kit was obtained from Pierce (Etten-Leur, The Netherlands). Formaldehyde solution was obtained from Sigma (Zwijndrecht, The Netherlands). Restriction enzymes (BamHI, XhoI, EcoRI and HindIII) were obtained from Fermentas Life Sciences (St. Leon-Rot, Germany). Black, clear bottom 96 well plates were obtained from Greiner Bio One (Alphen aan den Rijn, The Netherlands). S1P was obtained from Avanti-Polar Lipids (via Instruchemie B.V., Delfzijl, The Netherlands). Chamber slides were obtained from Nunc (Amsterdam, The Netherlands). BM Chemiluminescence Blotting Substrate [POD] and ABTS [2,2\u2032-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)] were obtained from Roche (Mannheim, Germany). Hyperfilm ECL was obtained from Amersham Biosciences (Diegem, Belgium). SEW2871 (5-(4-Phenyl-5-trifluoromethylthiophen-2-yl)-3-(3-trifluoromethylphenyl)-1,2,4-oxadiazole) was obtained from Calbiochem (via VWR, Amsterdam, The Netherlands). Goat anti-mouse-HRP was obtained from Jackson ImmunoResearch Europe (via Sanbio B.V., Uden, The Netherlands). RIPA buffer was obtained from Boston bio products (Worcester, UK). Halt protease inhibitor cocktail was obtained from Pierce (via Perbio Science Nederland B.V. Etten-Leur, The Netherlands).\nMolecular cloning and transfection\nAn N-terminal HisG-tag was added to the S1P1 receptor via cloning into pcDNA3.1\/HisC using BamHI and XhoI. A second cloning step was done using HindIII and XhoI to clone the HisG-tagged S1P1 receptor into the expression vector pcDNA5\/FRT\/TO. Transfection of plasmids into CHO-FlpIn cells and cell culture was done as described previously (Jongsma et al. 2006).\nWestern blot\nCells were washed and scraped in PBS, spun down and lysed in RIPA buffer with protease inhibitor. Protein concentration was determined using the BCA kit according to the manufacturer\u2019s protocol. Ten micrograms of protein were loaded onto a 4\u201312% NuPAGE\u00ae Bis\u2013Tris Gel. Electrophoresis was carried out at 200\u00a0V for 40\u00a0min in NuPAGE\u00ae MES running buffer. Protein was transferred to Invitrolon\u2122 PVDF blotting membranes at 30\u00a0V for 1\u00a0h. The membrane was blocked for 1\u00a0h at room temperature in 50\u00a0mg\/ml non-fat dry milk in PBS followed by overnight incubation at 4\u00b0C with a dilution of 1:5,000 for mouse anti-HisG. After washing with PBS-Tween (0.1%), the membrane was incubated with a 1:20,000 dilution of goat anti-mouse-HRP IgG at room temperature. Detection was done using BM Chemiluminescence Blotting Substrate and Hyperfilm ECL.\nImmunocytochemistry\nCells (40,000 cells\/well) were plated on day\u00a01 in 96 wells black, clear bottom plates or eight well chamber slides. The next day, the medium was changed to serum-free medium. After incubation overnight, cells were stimulated with the indicated ligand in serum-free medium for 30\u00a0min at 37\u00b0C, unless otherwise indicated. The stimulation solutions were removed, and cells were fixed by applying 4% v\/v formaldehyde in PBS for 15\u00a0min at room temperature. After washing three times with PBS, cells were incubated with the first antibody for 1\u00a0h at room temperature. Cells were washed again three times with PBS and, if necessary, incubated with the second antibody for 1\u00a0h at room temperature. Cells were washed three times with PBS followed by measurements on a Victor2 (Wallac, Perkin Elmer). The following antibody combinations were used: mouse anti-HisG 1:200 with AlexaFluor\u00ae 488 goat anti-mouse 1:500, mouse anti-HisG-HRP 1:200 and mouse anti-HisG 1:200 with goat anti-mouse-HRP 1:500. In case an HRP-bound antibody was used, either ABTS solution (50\u00a0\u03bcg\/ml) or BM Chemiluminescence Blotting Substrate was added to generate an absorbance or luminescence signal, respectively. For the absorbance signal, a 405-nm filter was used; for the fluorescent signal, a 490-nm excitation and a 535-nm emission filter were used.\nData analysis\nConcentration response curves were analysed by fitting sigmoidal functions to the experimental data using Prism 4 (Graphpad Software, San Diego, CA). Data are expressed as means\u2009\u00b1\u2009SEM. Differences between groups were compared using a one way analysis of variance with a Dunnett\u2019s correction or a Student\u2019s t test where appropriate. P\u2009<\u20090.05 was considered significant.\nResults\nCharacterisation of the HisG-tagged S1P1 receptor\nThe presence of HisG-S1P1 receptor in stably transfected CHO-FlpIn cells was confirmed in Western blot analysis as a band of approximately 50\u00a0kDa (Fig.\u00a01). The HisG-S1P1 receptor was also visualised in the cell membrane by fluorescence microscopy using AlexaFluor\u00ae 488 goat anti-mouse as a secondary antibody (Fig.\u00a02). CHO-FlpIn cells stably expressing the untagged S1P1 receptor were used as a negative control for both detection methods (Figs.\u00a01 and 2). Addition of the N-terminal HisG-tag to the S1P1 receptor did not influence signaling via this receptor as it did not significantly alter the potency of S1P to inhibit forskolin-stimulated cAMP accumulation (pEC50 S1P1 9.0\u2009\u00b1\u20090.1 vs HisG-S1P1 8.9\u2009\u00b1\u20090.1; n\u2009=\u20096), measured as described previously (Jongsma et al. 2006).\nFig.\u00a01Western blot of CHO-FlpIn cells stably expressing the S1P1 receptor (lane 1) and HisG-S1P1 receptor (lane 2) using a mouse anti-HisG first antibody and a goat anti-mouse HRP second antibody. HisG-S1P1 is detected at approximately 50\u00a0kDa as indicated by the arrow. The band at 80\u00a0kDa is nonspecific. The blot shown is from a typical experiment out of fourFig.\u00a02Fluorescence microscope pictures of CHO-FlpIn cells stably expressing the HisG-S1P1 receptor (a) and the S1P1 receptor (b) using a mouse anti-HisG first antibody and AlexaFluor\u00ae 488 goat anti-mouse second antibody. Images shown are from one typical experiment out of nine\nInternalisation of the S1P1 receptor\nA 30-min incubation with 1\u00a0\u03bcM of S1P or 10\u00a0\u03bcM of the selective S1P1 agonist SEW2871 (Sanna et al. 2004) at 37\u00b0C decreased fluorescence (Fig.\u00a03a, c, e). No such decrease in fluorescence was seen when cells were incubated with either agonist at 4\u00b0C (Fig.\u00a03b, d, f). S1P concentration dependently internalised the HisG-S1P1 receptor as visualised with a fluorescence microscope (Fig.\u00a04). Such decrease in fluorescence was also measured using a fluorescence microplate reader. The fluorescent signal of cells stably expressing the HisG-S1P1 receptor was significantly higher than for cells stably expressing the untagged S1P1 receptor. Stimulation with S1P (1\u00a0\u03bcM) for 30\u00a0min significantly decreased this signal (Fig.\u00a05).\nFig.\u00a03Fluorescence microscope pictures of CHO-FlpIn cells stably expressing the HisG-S1P1 using a mouse anti-HisG first antibody and AlexaFluor\u00ae 488 goat anti-mouse second antibody. Cells were stimulated for 30\u00a0min with 1\u00a0\u03bcM S1P or 10\u00a0\u03bcM SEW2871 at either 37\u00b0C (left panel) or 4\u00b0C (right panel). Images shown are from one typical experiment out of fourFig.\u00a04Fluorescence microscope pictures of CHO-FlpIn cells stably expressing the HisG-S1P1 receptor using a mouse anti-HisG first antibody and AlexaFluor\u00ae 488 goat anti-mouse second antibody. Cells were stimulated with S1P at the indicated concentrations at 37\u00b0C for 30\u00a0min. Images shown are from one typical experiment out of four experimentsFig.\u00a05Quantitative measurements of cells stably expressing HisG-S1P1 using a mouse anti-HisG first antibody and AlexaFluor\u00ae 488 goat anti-mouse second antibody. Cells were stimulated at 37\u00b0C for 30\u00a0min at the indicated S1P concentrations. The first bar represents cells stably expressing the S1P1 receptor without a HisG-tag. The second bar represents cells stably expressing HisG-S1P1 incubated without the first antibody. Statistical significance was shown between \u2018vehicle\u2019 and \u20181\u00a0\u03bcM S1P\u2019 as well as between \u2018no HisG\u2019 and \u2018vehicle\u2019. Data are shown as relative fluorescent units (RFU)\u2009\u00b1\u2009S.E.M. (n\u2009=\u20093)\nChoice of detection method\nInstead of the antibody combination mouse anti-HisG and AlexaFluor\u00ae 488 goat anti-mouse (Fig.\u00a06a), other combinations were tested to optimise the method. The use of a first antibody mouse anti-HisG-HRP (Fig.\u00a06b) or a first antibody mouse anti-HisG with a second antibody goat anti-mouse-HRP (Fig.\u00a06c) combined with the BM Chemiluminescence Blotting Substrate (POD) resulted in a luminescence signal, which proved to be very sensitive. However, the signal was very unstable over time, which required the addition of the substrate by the microplate reader. The combination of a first antibody mouse anti-HisG-HRP (Fig.\u00a06d) or a first antibody mouse anti-HisG with a second antibody goat anti-mouse-HRP (Fig.\u00a06e) combined with ABTS resulted in an absorbance signal. This signal however, had less sensitivity compared to the other two detection methods. The use of a first antibody mouse anti-HisG-HRP resulted in a loss of sensitivity compared to the combination of a first antibody mouse anti-HisG with a second antibody goat anti-mouse-HRP. When the combination of a first antibody mouse anti-HisG and a second antibody AlexaFluor\u00ae 488 goat anti-mouse was used, we measured a concentration-dependent internalisation of HisG-S1P1 after stimulation with either S1P or SEW2871 (pEC50 S1P\u2009=\u20097.4\u2009\u00b1\u20090.1 [n\u2009=\u20098] and pEC50 SEW2871\u2009=\u20095.7\u2009\u00b1\u20090.2 [n\u2009=\u20093], respectively; Fig.\u00a07).\nFig.\u00a06Quantitative measurements of HisG-S1P1. Ligand stimulations are carried out at 37\u00b0C for 30\u00a0min at the indicated concentrations (n\u2009=\u20093\u20134). Antibody and detection combinations used are mouse anti-HisG first antibody and AlexaFluor\u00ae 488 goat anti-mouse second antibody (a); mouse anti-HisG-HRP first antibody\u2009+\u2009POD (b); mouse anti-HisG first antibody and goat anti-mouse-HRP second antibody\u2009+\u2009POD (c); mouse anti-HisG-HRP first antibody\u2009+\u2009ABTS (d); mouse anti-HisG first antibody and goat anti-mouse-HRP second antibody\u2009+\u2009ABTS (e). Data are shown as percentage of unstimulated\u2009\u00b1\u2009S.E.M. An asterisk indicates significant difference compared to unstimulated cells; a pound sign indicates significant difference compared to S1P (10\u00a0nM) stimulated cellsFig.\u00a07Effect of increasing concentrations of S1P (closed squares) or SEW2871 (open squares) on CHO-FlpIn cells stably expressing the HisG-S1P1 receptor, using a mouse anti-HisG first antibody and AlexaFluor\u00ae 488 goat anti-mouse second antibody. Stimulations were carried out at 37\u00b0C for 30\u00a0min. The data shown represent the mean\u2009\u00b1\u2009S.E.M. of six independent experiments performed in triplicate\nDiscussion\nPreviously reported methods to study the internalisation of GPCRs were very labor intensive, required specific equipment, added bulky tags and\/or were poorly quantitative. We have developed and validated a method that uses stably transfected cells expressing a receptor with a minor modification only, i.e. an N-terminal HisG-tag, which did not influence receptor signaling. Several antibody combinations and detection methods were tested. The luminescent approach was very sensitive but highly dependent on strict adherence to a specific time interval between substrate addition and actual signal measurement. The absorbent signal obtained using another detection substrate resulted in a loss of sensitivity. The antibody combination of a first HisG specific antibody and a second AlexaFluor\u00ae 488 fluorescent antibody proved the most convenient, resulting in a sensitive and stable signal. Compared to previously published methods using a similar approach (Daunt et al. 1997; Vicentic et al. 2002; Wozniak and Limbird 1996; Von Zastrow et al. 1993), this immunocytochemical detection of the N-terminal HisG-tag is suitable to assess a large number of samples, e.g. to construct detailed concentration response curves and\/or compare multiple ligands. In this regard, we showed that S1P was approximately 50 times more potent than SEW2871 in internalising the HisG-S1P1 receptor. This is in good agreement with the previously reported potency difference between the two agonists for activating the receptor (Sanna et al. 2004). As comparable methods have been described for adrenergic receptors as mentioned above (Daunt et al. 1997; Vicentic et al. 2002; Wozniak and Limbird 1996; Von Zastrow et al. 1993), it is likely that the method we described here is also suitable for receptors other than our model receptor, S1P1.\nWhile our technique does not allow following internalisation in real time, the ability to process a large number of samples compensates for that by studying multiple time points. The only limitation to our method is the inability to measure internalisation of endogenous receptors. However, this would be possible when specific receptor antibodies are available, which is not yet the case for our model receptor.\nIn conclusion, we have validated a method to quantitatively measure receptor internalisation in which we use an N-terminal HisG-tag combined with immunocytochemistry. It proves to be sensitive enough to discriminate between different receptor ligands in a fast and non-radioactive way.","keyphrases":["internalisation","g-protein coupled receptor","hisg-tag","sphingosine-1-phosphate receptors","quantification","cellular trafficking"],"prmu":["P","P","P","R","U","M"]}
{"id":"Pediatr_Surg_Int-4-1-2248216","title":"Congenital cardiovascular defects in children with intestinal malrotation\n","text":"Intestinal malrotation (IM) and cardiovascular defects (CCVD) are both common congenital defects. We investigated the prevalence and types of CCVD in a 25-year IM population, and its association with post-IM-operative morbidity and mortality. Data on the type of CCVD, other congenital defects, syndromes, associations, post-IM-operative morbidity and mortality were retrospectively reviewed from the records of IM patients born between 1980 and 2005. Data were analyzed on (significant) differences between CCVD subgroups, and risk factors for both morbidity and mortality were calculated. Seventy-seven of 284 IM patients (27.1%) were diagnosed with a major or minor CCVD (37 and 40 patients, respectively). Syndromes and associations were more frequently diagnosed in patients with major than with a minor CCVD (67.6 vs. 40%, respectively). Post-IM-operative complications, although frequently observed (61%), did not differ between patients with major and minor CCVD. Physical CCVD signs before IM surgery increased post-IM-operative morbidity significantly (OR 4.0, 95% CI 1.4\u201311.0). Fifteen patients died (19.5%), seven due to cardiovascular cause. Mortality risk was increased by intestinal ischemia and post-IM-operative complications and by major CCVD after correction for age at weight at the time of IM operation. Congenital cardiovascular defects in children with intestinal malrotation are common, with high morbidity and mortality rates after IM operation. Elective IM surgery in young patients with CCVD should be performed in a centre with adequate paediatric cardiac care. Benefits of laparoscopic intervention need further study.\nIntroduction\nIntestinal malrotation (IM) is refers to all abnormalities of intestinal position and attachment and includes the concept of atypical malrotation or malrotation variant [1]. It is often accompanied by a narrow base of the small bowel mesentery. The incidence of IM is 1:500\u20131:3,500 living births [2, 3]. Volvulus, a rotation of the gut along its mesenteric stalk occurs in 60\u201370% of neonates diagnosed with IM, the ultimate strangulation in about 15% [4]. Eighty percent of the patients with symptomatic IM present in their first month of life [5] and require surgery. Delay in diagnosis may lead to intestinal ischemia, intestinal necrosis, septicaemia and short bowel syndrome. The surgical outcome is negatively influenced by the presence of intestinal necrosis [6], and the need for operation during the first 3\u00a0months of life [6\u20139]. To keep mortality and morbidity low, all asymptomatic children with intestinal malrotation are advised to undergo semi-elective surgery (Ladd\u2019s procedure) in childhood and adolescence [10, 11].\nIntestinal malrotation has been described in almost 100 syndromes and combinations. Associated serious abnormalities increase the mortality risk in patients with IM of about 22-fold according to Messineo [6]. Commonly associated congenital malformations are duodenal atresia, abdominal wall defects or diaphragmatic hernia [3, 12]. It is more frequently observed in patients with situs inversus [13] or heterotaxia, a syndrome associated by congenital cardiovascular defects [14].\nCongenital cardiovascular defects (CCVD) are among the most common congenital birth defects occurring in about 1 in 100 living births [15]. Congenital malformations of the gastrointestinal, musculoskeletal and urinary systems are the most frequently seen extra cardiac malformations in patients with CCVD [16]. Intestinal malrotation has been observed in 2.8\u20134.1% of all the patients with CCVD, mainly in patients with single ventricle [17]. However, the incidence of CCVD in IM patients has not been precisely quantified in the recent literature and the full array of the associated cardiac anomalies has not been well described.\nThe purpose of our study was (1) to delineate and accurately describe the types and incidence of associated cardiac lesions in our most recent 25-year experience of patients with IM, and (2) to study the influence of a co-existing CCVDs on IM surgery morbidity and mortality.\nMaterials and methods\nPopulation\nA retrospective review at a single university centre was conducted on all the paediatric patients diagnosed with IM born and      between September 1980 and September 2005. Patients with CCVD were identified for further analysis. Patients demographics recorded included gestational age, weight and gender. Data on the type of CCVD, other congenital defects, known syndromes or associations, post-operative complications, morbidity and mortality were collected. The diagnosis of CCVD was based on clinical, ECG, echocardiography, heart catheterization and peri-operative findings. The IM diagnosis was based on radiological upper gastro-intestinal examination, or during acute surgery.\nData analysis\nCollected data were analyzed by calculating prevalence percentages. Analysis of nominal variables was performed  using cross tables for calculating relative risk (RR) with the corresponding 95% confidence interval (95% CI). Fisher extact test was used for comparison of percentages of both CCVD groups, expressed as P values. For the comparison of continuous variables, Mann\u2013Whitney U test was used to calculate P value. The P values are two-sided, and a value of P\u00a0=\u00a00.05 was considered statistical significant. For the calculation of risk factors for both post-operative morbidity and mortality, (adjusted) odds ratios (OR) with consequent 95% CI were calculated by uni- and multi-variate regression analysis. Statistical analysis was performed using SPSS software (version 12.0.1; SPSS Inc., Chicago, IL, USA).\nResults\nDemographics\nDuring a period of 25\u00a0years, 284 patients were diagnosed in our centre with IM. Ninety-three of the 284 patients were identified with CCVD as well. Fifteen patients had persistent pulmonary hypertension (PPHT), all but one with congenital diaphragmatic hernia. One patient had a solitary pre-duodenal portal vein. The records of these 16 patients without structural CCVD were excluded, leaving 77 patients with both IM and CCVD (27.1% of the total IM population in the last 25\u00a0years) for further analysis. The gestational age range was 26\u201342\u00a0weeks with a median of 39\u00a0weeks. Median birth weight was 2.86\u00a0kg (range 0.72\u20134.53). A female predominance was seen with 47 girls (61%) and 30 boys (39%). Median follow-up period after IM diagnosis was 2.8\u00a0years (range 0\u201324\u00a0years). The overall survival at the end of follow-up was 80.5% (15 out of 77 patients died).\nCongenital cardiovascular defects\nThe CCVDs were divided into the subgroups, major and minor CCVDs (see Table\u00a01), varying from small ventricular septal defect (VSD) to common arterial trunk (CAT). Thirty-seven out of 77 patients (48.1%) were diagnosed with major CCVD and 40 patients (51.9%) with minor CCVD.\nTable\u00a01Types of major and minor congenital cardiovascular defectsMajor CCVDn\u00a0=\u00a037 (100%)Minor CCVDn\u00a0=\u00a040 (100%)Complex cardiovascular defectsn\u00a0=\u00a018 (48.6%)Ventricular septal defectn\u00a0=\u00a016 (40.0%)Tetralogy of Fallotn\u00a0=\u00a07 (18.9%)Atrium septal defectn\u00a0=\u00a07 (17.5%)Hypoplastic left heart syndromen\u00a0=\u00a03 (8.1%)Peripheral pulmonary stenosisn\u00a0=\u00a04 (10.0%)Atrioventricular septal defectn\u00a0=\u00a03 (8.1%)Persistent ductus arteriosusn\u00a0=\u00a03 (7.5%)Pulmonary valve atresian\u00a0=\u00a01 (2.7%)Pulmonary valve stenosisn\u00a0=\u00a03 (7.5%)Transposition of the great arteriesn\u00a0=\u00a01 (2.7%)Aortic valve stenosisn\u00a0=\u00a02 (5.0%)Tricuspid valve atresian\u00a0=\u00a01 (2.7%)Aortic coarctationn\u00a0=\u00a01 (2.5%)Common arterial trunkn\u00a0=\u00a01 (2.7%)Hypertrophic cardiomyopathyn\u00a0=\u00a01 (2.5%)Aortic arch hypoplasian\u00a0=\u00a01 (2.7%)Subvalvular aortic valve stenosisn\u00a0=\u00a01 (2.5%)Dextrocardian\u00a0=\u00a01 (2.7%)Left superior caval veinn\u00a0=\u00a01 (2.5%)Arteria lusorian\u00a0=\u00a01 (2.5%)CCVD Congenital cardiovascular defects\nThe characteristics of the whole group (n\u00a0=\u00a077) as well as that of the subgroups are described in Table\u00a02. Forty-two patients underwent cardiac surgery, of which 20 patients had two or more cardiac surgical interventions. Patients with major CCVDs needed cardiac surgery more often. Minor CCVDs that needed surgical intervention most frequent were VSD, atrial septal defects (ASD) and aortic valve stenosis (AS). Six patients with severe CCVD did not undergo correction of their heart disease before IM operation: three patients died (n\u00a0=\u00a02 due to post IM surgery complications, n\u00a0=\u00a01 because of other congenital malformations). One patient with dextrocardia did not need cardiac correction. The two patients with Tetralogy of Fallot (TOF) underwent cardiac correction 6\u00a0months after IM surgery.\nTable\u00a02Characteristics of studied population and its subgroups of congenital cardiovascular defectsCharacteristicAll CCVDMajor CCVDMinor CCVDRelative risk (95% CI)\/P value100% (n\u00a0=\u00a077)100% (n\u00a0=\u00a037)100% (n\u00a0=\u00a040)Boys39% (n\u00a0=\u00a030)35.1% (n\u00a0=\u00a013)42.5% (n\u00a0=\u00a017)0.85 (0.5\u20131.4)Syndromes or associations53.2% (n\u00a0=\u00a041)67.6% (n\u00a0=\u00a025)40% (n\u00a0=\u00a016)1.8 (1.1\u20133.1)Other congenital defects84.4% (n\u00a0=\u00a065)83.8% (n\u00a0=\u00a031)85% (n\u00a0=\u00a034)0.95 (0.5\u20131.8)Cardiac surgery54.4% (n\u00a0=\u00a042)83.8% (n\u00a0=\u00a031)27.5% (n\u00a0=\u00a011)4.3 (2.0\u20139.1)CCVD diagnosed before IM 57.1% (n\u00a0=\u00a044)78.4% (n\u00a0=\u00a029)37.5% (n\u00a0=\u00a015)2.7 (1.4\u20135.2)Age at CCVD diagnosis in days [median (range)]4.5 (0\u2013210)4 (0\u201398)7 (0\u2013210)P\u00a0=\u00a00.12Birth weight in kilograms [median (range)]2.86 (0.72\u20134.53)2.96 (0.96\u20134.53)2.84 (0.72\u20134.30)P\u00a0=\u00a00.4Age at first CCVD surgery in days [median (range)]102 (2\u20133,759)43 (2\u20133,759)117 (24\u20131,705)P\u00a0=\u00a00.15Values in bold represent statistical significant resultsCCVD Congenital cardiovascular defects, IM Intestinal malrotation\nCongenital malformations, syndromes and associations\nCongenital malformations other than IM and CCVD were observed in 65 of the 77 (84.4%) patients, see also Table\u00a02. Most frequently observed congenital malformations included omphalocele (n\u00a0=\u00a07), congenital diaphragmatic hernia (n\u00a0=\u00a07), ano-rectal malformations (n\u00a0=\u00a06) and heterotaxia (n\u00a0=\u00a06).\nSyndromes and associations were recognized in 41 (53.2%) patients, of which Down syndrome (n\u00a0=\u00a07), heterotaxia (n\u00a0=\u00a06), VACTERL (n\u00a0=\u00a05), Ivemark (n\u00a0=\u00a05) and lateralization abnormalities (n\u00a0=\u00a04) were most frequently recognized. Kartagener syndrome occurred in two patients. Several other syndromes all occurred once. Syndromes and associations were more frequent in patients with major CCVD.\nIntestinal malrotation surgery\nCharacteristics of IM-operated patients and their major and minor CCVD subgroups are described in Table\u00a03. Five patients did not undergo IM surgery yet; in three patients the surgery was about to take place in the near future, in one patient no surgical intervention was planned and one patient died of an unknown cause before IM surgery could take place. A Ladd procedure was performed for IM correction. Twenty-eight out of the 72 operated patients needed acute IM surgery, at a median age of 4.0\u00a0days (range 0\u2013455). Only 4 out of these 28 patients had previously undergone surgical correction of the CCVD. Five patients had ischemic bowel disease at time of acute IM intervention; three of these patients died, one due to massive intestinal necrosis (one patient died due to cardiovascular cause, one patient died because of severe infection). At the time of IM surgery, signs and symptoms of CCVD, e.g., hepatomegaly, central cyanosis, cardiac murmurs and respiratory insufficiency, were more often observed twice in patients with a major CCVD (62.2 vs. 30.0%, respectively, P\u00a0=\u00a00.004). Most common complications were intestinal (Platzbauch, leakage of the bowel anastomosis resulting in sepsis, feeding difficulties with long-term necessity of nasogastrical tube feeding), infections and cardiovascular complications (e.g. bradycardia, thrombosis, arrhythmia) and they did not differ between both subgroups. Thirteen patients needed relaparotomy because of leakage of the bowel anastomosis (n\u00a0=\u00a04), Platzbauch (n\u00a0=\u00a02), suspected bowel obstruction with feeding difficulties (n\u00a0=\u00a01) and necrotising enterocolitis (n\u00a0=\u00a01). Five patients were reoperated for an adhesion ileus, all but one more than 1\u00a0month after surgery.\nTable\u00a03Characteristics of general population, major and minor congenital cardiovascular defects groups operated on intestinal malrotationCharacteristicAll CCVDMajor CCVDMinor CCVDP valueIM surgery100% (n\u00a0=\u00a072) 100%  (n\u00a0=\u00a034)100%   (n\u00a0=\u00a038)\u2212Physical signs CCVD prior to IM surgery48.6%  (n\u00a0=\u00a035)67.6%  (n\u00a0=\u00a023)30.0%   (n\u00a0=\u00a012)0.004Post-IM-operative complications61.1%  (n\u00a0=\u00a044)67.6%  (n\u00a0=\u00a023)55.3%  (n\u00a0=\u00a021)0.3Post-IM-operative cardiovascular complications18.1%   (n\u00a0=\u00a013)20.6%    (n\u00a0=\u00a07)15.8%   (n\u00a0=\u00a06)0.8Relaparotomy18.1%     (n\u00a0=\u00a013)17.6%  (n\u00a0=\u00a06)18.4%  (n\u00a0=\u00a07)1.0Age at IM surgery in days [median (range)]17 (0\u20131,293)104 (0\u20131,196)6 (0\u20131,293)0.006Weight at IM surgery in kilograms [median (range)]3.21 (1.20\u201311.4)4.84 (1.20\u201311.4)2.88 (1.24\u201310.6)0.02Death in days after IM surgery [median (range)]28 (0\u20131,814)33 (0\u20131,814)3 (3\u201359)0.2Age at death in days [median (range)]40.5 (3\u20133,010)46 (22\u20133,010)8 (3\u201360)0.1Values in bold represent statistical significant resultsCCVD Congenital cardiovascular defects, IM Intestinal malrotation\nPatients suffering from a post-operative IM surgery complication were younger (5.5 vs. 94.5\u00a0days, P\u00a0=\u00a00.031) and had a lower body weight (2.86 vs. 4.84\u00a0kg, P\u00a0=\u00a00.003) at time of IM surgery. Major CCVD, co-occurrence of a syndrome, or other congenital malformations did not significantly increase the risk on post-IM-operative morbidity. Correction for age and weight at the time of IM surgery in both CCVD groups showed, although not significant, a clear trend in post-IM-operative morbidity risk in patients diagnosed major CCVD (OR 2.9, 95% CI 0.9\u20139.6). Patients with pre-operative signs of CCVD had a four times increased risk on post-operative complications (95% CI 1.4\u201311.0). This risk factor was more prominent after correction for age and weight at the time of IM surgery (OR 8.2, 95% CI 2.1\u201330.7).\nMortality\nFifteen (19.5%) out of 77 patients died, 9 with major CCVDs. One patient with a minor CCVD died prior to IM surgery at the age of 1.4\u00a0years; cause of death remained unclear. Known causes of death after IM surgery were: cardiovascular complications (n\u00a0=\u00a07), massive intestinal necrosis (n\u00a0=\u00a02), sepsis (n\u00a0=\u00a02), respiratory failure (n\u00a0=\u00a02) and abstention of medical care because of unsuccessful surgical intervention on malformed trachea (n\u00a0=\u00a01). In both CCVD groups, death due to a cardiovascular cause remained the most frequent one. Median age at time of death was 40.5\u00a0days (range 3\u20133,010), with a median of 28.5\u00a0days after IM surgery (range 0\u20131,814). Of the 14 deceased patients, 9 (64.3%) had a major CCVD. Median age at time of death in the major CCVD group was 46\u00a0days (range 22\u20133,010) and in the minor CCVD group 8\u00a0days (range 3\u201360\u00a0days), although this difference was statistically not significant (P\u00a0=\u00a00.4). Twenty percent of the patients (7\/35) with pre-operative physical signs of CCVD died, only one due to a cardiovascular cause. Three out of five patients with ischemic bowel at the time of IM surgery died post-operatively. The risk factors for mortality are shown in Table\u00a04.\nTable\u00a04Risk factors (expressed as odds ratios and 95% confidence interval) for mortality in the whole study population (n\u00a0=\u00a077)CharacteristicDeceasedAliveOdds ratio (95% CI)Adjusting for weight and age at time of IM surgery OR (95% CI)Major CCVD9281.8 (0.6\u20135.7)5.0 (1.2\u201320.7)Minor CCVD634Syndrome\/association7340.7 (0.2\u20132.2)1.0 (0.3\u20133.4) No syndrome or association828Other congenital defects13521.3 (0.2\u20136.4)1.1 (0.2\u20135.9)No other congenital defects210Physical CCVD signs prior to IM-surgery7281.1 (0.3\u20133.4)1.3 (0.4\u20134.4)No physical CCVD signs prior to IM-surgery730Intestinal ischemia327.6 (1.1\u201351.2)5.9 (0.9\u201342.4)No intestinal ischemia1156Post-IM-operative complications12711.3 (1.4\u201392.3)8.7 (1.0\u201375.7)No post-IM-operative complications1331Post-IM operative cardiovascular complications765.5 (1.5\u201320.5)4.5 (1.1\u201318.4)No post-IM-operative cardiovascular complications851Values in bold represent statistical significant resultsCCVD Congenital cardiovascular defects, IM Intestinal malrotation\nDiscussion\nThis study addresses the co-existence of malrotation with different types of congenital cardiovascular defects. We found 27.1% of all the patients registered in the last 25\u00a0years for IM to have a major or minor CCVD. Major CCVDs were seen in almost half of our population. Stewart et al. [2] found only 12% in a smaller IM population to have any CCVD. Complex CCVDs (23% of our population, 6.3% of all patients with IM in the last 25\u00a0years) and VSDs (21% of our population, 5.6% of all patients with IM in the last 25\u00a0years) were most frequently diagnosed. Pradat et al. [17] reported the co-occurrence of IM in patients with single ventricle to be as high as 8.5%. Further data on co-occurrence of CCVD in IM patients are missing.\nThe high prevalence of the CCVD in our IM cohort needs some thoughts on common aetiology. The gut is developed at a later stage in embryogenesis than the heart does. Abnormal abdominal development, e.g. by early development of an intra-abdominal cyst in embryogenesis, is known to enhance the chance of developing IM [18]. Changes in intra-abdominal space caused by a CCVD might also increase the risk on IM development during embryogenesis [4]. Another explanation might be a common genetic cause. The gut and the heart are both organs that show a characteristic left\u2013right asymmetry, caused by a specific biochemical cascade during embryogenesis [19]. Two signalling molecules inevitable for this cardiac and gastro-intestinal asymmetry are nodal and pitx2c [20]. Reduced, diffuse or absent expression of pitx2c in the embryo cause abnormal looping from both heart and gut in animal studies [21\u201324], resulting mainly in abnormal outflow tract orientation (e.g. unseptated and misaligned great artery trunks, symmetrical outflow tract cushions, double outlet right ventricle, common atrioventricular trunk) [23, 24]. This disturbed expression of pitx2c could cause a spectrum of isomerism [25]. More research is needed to reveal a possible common genetic cause for both CCVD and IM.\nOur study group explores a high percentage of syndromes or associations. Although IM has been recognized in many syndromes and associations, this high prevalence should make us aware of co-occurrence of CCVD in these patients, as well as the possibility of a syndrome or association in patients diagnosed with both IM and CCVD. The post-operative morbidity after IM surgery in the general paediatric population is between 0 and 16.7% [9, 26]. A higher morbidity rate (47%) has been reported by Murphy et al. [27], yet the surgical intervention was acute in 65% of their study population. Consistent with earlier reports [6\u20138], we found post-operative morbidity and mortality to be associated with younger age at the time of surgery. However, ischemic bowel (at the time of IM surgery) did not increase post-IM-operative morbidity as reported by others [2, 6, 8, 28]. Re-laparotomy in our population was consistent with earlier reports in the general population [26, 27]. The current study showed that patients with a CCVD had a relatively high morbidity rate after IM surgery (61.1%). Syndromes, associations or the presence of other congenital malformations were not found to be risk factors for post IM morbidity. Cardiovascular complications were seen in 18.1% of our population. There are neither data on prevalence of cardiovascular complications after IM surgery in the general paediatric population, nor on influence of physical condition prior to IM surgery on the morbidity after the operation. Although the type of CCVD was not significantly associated with an increased risk on morbidity, there was a clear trend (towards increased risk on post-operative morbidity) in patients with major CCVD. Furthermore, patients with pre-operative physical signs of CCVD had a high post-operative morbidity rate (77%).\nThe overall mortality rate in literature after IM surgery varies between 3 and 9% [6, 27\u201329]. Il\u00e7e et al. [8] found a mortality rate varying between 0 and 36% in patients mainly younger than 1\u00a0year, having almost all emergency intervention. A recent study of Murphy et al. [27] reported only 1 post-operative death in 46 patients due to cardiovascular consequences in a patient with congenital cyanotic heart disease. In the current study, the mortality rate was higher: almost one-fifth of the patients died. Patients who died were significantly younger at the time of IM surgery. Other risk factors for death were major CCVD (after correction for age and weight at the time of IM surgery), ischemic bowel at the time of IM surgery, post-IM-operative complications in general and post-IM-operative cardiovascular complications.\nDiagnosis and treatment of cardiac diseases have incredibly improved in the last two decades. Associated congenital abnormalities, including IM, have increased in significance. However, since mortality rates after surgical intervention for IM in patients with an associated CCVD are high, intervention should be well thought over. Recent studies discuss the advantages of laparoscopic inspection for IM, also in acute paediatric abdomen [30]. No data on laparoscopy in IM patients with CCVD are available. Possible advantages of laparoscopy for IM diagnosis, followed by operation, even in acute cases, might improve the outcome [30], even in patients with major CCVD. Referral to a centre equipped with paediatric cardioanesthesiological care is imperative.\nStudy limitations\nThis is a retrospective study of all consecutive live-born IM patients referred to a single university centre over a 25-year period. There are asymptomatic IM patients who never consult a doctor. Patients with IM and a very mild CCVD might have been missed. Screening for CCVD in IM patients could give us more information on the exact prevalence of CCVD in IM patients. Furthermore, since CCVD are very common congenital defects, a prospective study on the prevalence of IM in all patients with minor and major CCVD would also be of great interest.\nEach retrospective study has its limitations, e.g. some variables were not (yet) available; e.g. syndromes and associations were common in our study population. Yet, the actual prevalence of syndromes might have been higher than reported. Certain syndromes and associations are quite new and nowadays there might be more attention on searching for a fitting syndrome or association diagnosis than 20\u00a0years ago.\nAs regards treatment, we perform 3\u00a0years laparoscopic inspection, followed by correction of the anomaly by laparoscopy or laparotomy in very young children [31].\nConclusion\nThe prevalence of CCVD in patients with IM is high (27.1%). Major and minor CCVDs are both frequently seen. Major CCVD is a risk factor for death after IM operation. Since morbidity and mortality rates in IM patients with CCVD are much higher than previously reported, a pre-operative screening for co-existing CCVD is indicated. Elective IM surgery in young patients with major CCVD should be conducted in a centre with adequate paediatric cardiac care. The benefit of laparoscopic intervention needs further follow-up.","keyphrases":["congenital cardiovascular defect","intestinal malrotation","post-operative morbidity","post-operative mortality"],"prmu":["P","P","P","R"]}
{"id":"Breast_Cancer_Res_Treat-4-1-2217623","title":"Estrogen receptor \u03b1 polymorphisms and postmenopausal breast cancer risk\n","text":"Background The estrogen receptor alpha (ESR1) is a mediator of estrogen response in the breast. The most studied variants in this gene are the PvuII and XbaI polymorphisms, which have been associated to lower sensitivity to estrogen. We evaluated whether these polymorphisms were associated with breast cancer risk by means of an association study in a population of Caucasian postmenopausal women from the Rotterdam study and a meta-analysis of published data.\nIntroduction\nFamily history is one of the strongest risk factors for breast cancer [1]. It has been shown that the heritability of this disease is \u223c30% [2]. The most important determinants of risk for breast cancer are related to endogenous hormone levels and major reproductive events [3], thus, suggesting that genes in the estrogen pathway may influence breast cancer risk.\nThe estrogen receptor alpha (ESR1) is one of the most important mediators of hormonal response in estrogen-sensitive tissues such as the breast [4] and plays a crucial role in breast growth and differentiation as well as in the development of cancer [5]. The human ESR1 gene is localized on chromosome 6q24-q27 [6], it extends more than 140\u00a0kb and includes eight exons [7]. The most studied variants in this gene are the PvuII (C\/T) and XbaI (G\/A) polymorphisms in intron 1, 397 and 351\u00a0bp upstream of exon 2 respectively [8, 9]. These variants have been implicated in gene expression by influencing transcription [10]. While some studies have found an increased risk for the A and T alleles of the XbaI and PvuII polymorphisms [4, 9, 10], others have found an increased risk only for the X (G) allele of XbaI [11, 12]. In addition, other studies found no effect at all for either of these polymorphisms [4, 13]. These alleles were correlated with high bone mineral density and height in other studies, including one performed in our study population, [14, 15], suggesting a stronger estrogenic effect in P(C) and X(G) allele carriers [14].\nThe aim of our study was to evaluate the effect of these polymorphisms on breast cancer risk by performing an association analysis in a population based study of Caucasian postmenopausal women. Further, we performed meta-analyses of all available published data on these polymorphisms and the risk of breast cancer.\nMaterials and methods\nStudy population and measurements\nOur study population is part of the Rotterdam study [16]. Inhabitants of the suburb of Ommoord aged 55 or older were invited to participate and 7983 agreed to do so (response rate 78.1%). Study participants signed an informed consent and the Medical Ethics Committee of the Erasmus Medical Center approved the study. Our study group was composed of 4,878 postmenopausal women. Information on risk factors such as age at entry, age at menarche, age at menopause, parity, body mass index (BMI), waist hip ratio (WHR) and hormone replacement therapy use (HRT) was retrieved at baseline through a questionnaire. BMI was calculated by dividing the weight in kilograms by the height (in meters) squared [17].\nCase identification and validation\nThree different databases were used for patient identification. First, cases diagnosed by general practitioners in the research area (Ommoord) were collected (International Classification of Primary Care (code X76)). Second, the Dutch National Registry of all hospital admissions (LMR) was consulted to detect all malignancy related hospital admissions for study participants. Finally, regional pathology databases were linked to the Rotterdam Study to identify patients. Subsequently, breast cancer cases were validated by a physician on the basis of medical records of the general practitioner, discharge letters and pathology reports. Only pathologically confirmed cases were considered in the analysis. The index date was defined as the earliest date found in the pathology report.\nGenotyping & data analysis\nOut of the 4,878 women participating in our study, 3,893 (80 %) were successfully genotyped for the PvuII and XbaI polymorphisms. The genotyping procedures have been described previously [14]. Loss to follow up was assessed to verify it was independent of genotype. Categorical variables, such as parity and HRT, were compared between genotype groups using the chi-squared test. Continuous variables, (age at entry, age at menopause, BMI and WHR) were compared using the independent sample Mann\u2013Whitney test. We used logistic regression to study the risk of breast cancer by ESR1 genotype. This analysis was performed using SPSS version 11, since there is no clear risk allele from the literature, we took the TT (PvuII) and AA (XbaI) genotypes as reference because they have been associated to lower sensitivity to estrogen in our population [14]. We also performed a trend test to evaluate if the number of risk alleles carried had an effect on disease risk. Hardy-Weinberg equilibrium (HWE) was assessed for both polymorphisms using Markov-Chain Monte-Carlo approximation of the exact test implemented in the GENEPOP package V 3.3 [18].\nMeta-analysis\nWe searched PubMed until October 2006 for all case-control studies on the association of the PvuII and XbaI polymorphisms in the ESR1 gene and breast cancer. Our search strategy was based on the keyword \u201cbreast cancer\u201d combined with \u201cestrogen receptor\u201d and \u201cpolymorphism\u201d. To verify that all studies were retrieved, the reference lists of all publications were searched for additional studies. We excluded studies from our analyses if the genotype frequencies in the control population were out of Hardy-Weinberg or if their data had been previously used in another study. To quantify the strength of association, pooled odds ratios (ORs) and 95% confidence intervals (CI) were calculated using the random-effects model of the DerSimonian and Laird method [19]. The degree of heterogeneity between the study results was tested by the inconsistency statistic (I2). Funnel plots were used to evaluate publication bias [20]. Data were analysed using Review Manager, version 4.2 (Cochrane Collaboration, Oxford, UK).\nResults\nThe total loss of follow-up for the genotyped participants was 8.4% and it was not dependent on ESR1 genotype (P\u00a0=\u00a00.51). The genotype frequencies of both polymorphisms were in HWE proportions (X2 P\u00a0=\u00a00.33 for PvuII and X2P\u00a0=\u00a00.31 for XbaI). In Table\u00a01 we show the baseline characteristics of our study population. We found that all cases (incident and prevalent) were significantly younger at entry than controls (P\u00a0<\u00a00.001) and also died earlier during follow-up (P\u00a0<\u00a00.001), when using incident cases only we found the same significant differences (P for age at entry <0.0001, P for age at death <0.0001). We also found that cases had significantly fewer children than controls (P\u00a0=\u00a00.04). We did not find any significant differences in these baseline characteristics between genotype groups (data not shown).\nTable\u00a01General characteristics of the study populationCasesControlsTotalTotal studied (%)190 (4.7%)3513 (95.3)3703Mean age of entry (SD)*67.80 (7.7)70.36 (9.6)70.24 (9.6)Mean age at death (SD)*77.30 (8.6)84.46 (8.7)84.12 (8.8)Mean age at menarche (SD)13.57 (1.7)13.68 (1.8)13.67 (1.8)Mean age at menopause (SD)*49.51 (4.8)52.19 (13.6)52.07 (13.3)Mean number of children (SD)*1.77 (1.6)2.12 (1.7)2.10 (1.7)Parity (SD) (\u22651 child)*121 (71.6)2640 (79.4)2761 (79)Hormone replacement therapy(%)27 (21.1)504 (19.5)531 (19.6)Mean BMI (SD)27.10 (3.9)26.67 (4.1)26.69 (4.1)Mean WHR (SD)0.87 (.09)0.87 (.09)0.87 (.09)*\u00a0P-value\u00a0<\u00a00.05\nThere were 38 women with previously diagnosed postmenopausal breast cancer who entered the study. During follow-up, 152 were additionally diagnosed. There were no significant differences in the number of cases between the PvuII (P\u00a0=\u00a00.43 for overall cases) and XbaI genotypes (P\u00a0=\u00a00.33 for overall cases).\nWe carried out a logistic regression analysis adjusting for age at entry, age at menopause, BMI, WHR and HRT for both polymorphisms separately (Table\u00a02). Since the T and A alleles of these polymorphisms have been correlated to lower estrogenic effects, we used the TT and AA genotypes as our reference categories in the analyses. There were no significant differences in risk for breast cancer among carriers of the different genotypes of the PvuII or XbaI polymorphisms in the ESR1 gene. There was a non-significant tendency of the C allele of PvuII (P-for trend\u00a0=\u00a00.22) and G allele of the XbaI (P-for trend 0.26) to be over represented in patients.\nTable\u00a02Odds ratios for breast cancer risk for PvuII and XbaI genotypesOverallIncidentPrevalentPvuIITTRefRefRefTC0.9 (0.6\u20131.4)1.0 (0.6\u20131.6)0.8 (0.3\u20132.1)CC1.4 (0.8\u20132.2)1.4 (0.8\u20132.5)1.2 (0.4\u20133.3)XbaIAARefRefRefGA1.2 (0.8\u20131.7)1.3 (0.8\u20132.0)0.8 (0.4\u20131.9)GG1.3 (0.7\u20132.2)1.5 (0.8\u20132.8)0.5 (0.2\u20132.4)\nTo evaluate our data together with those in the literature we performed meta-analyses. We identified nine articles studying the relation between XbaI and PvuII polymorphisms and the risk of breast cancer [4, 9\u201312, 21\u201324]. We excluded from our analyses one study [11], since the data was used in another study [4]. Furthermore, two studies were excluded since genotype frequencies of controls were out of HWE proportions [9, 10]. Using the random effects model we did not find any difference in risk among XbaI and PvuII genotypes (Figs.\u00a01 and\u00a02). High inter-study heterogeneity can render the interpretation of the results of a meta-analysis difficult and although we found high heterogeneity in the G\/A versus GG comparison there was no significant heterogeneity in the other three comparisons. Additionally, the evaluation of the funnel plots did not suggest evidence for publication bias.\nFig.\u00a01Meta-Analyses ESR1 XbaI polymorphism and breast cancer riskFig.\u00a02Meta Analysis ESR1 PvuII polymorphism and breast cancer risk\nDiscussion\nWe performed an association study to evaluate the relationship of two well-studied polymorphisms in the ESR1 gene and the risk of breast cancer in Caucasian postmenopausal women from the Rotterdam Study. Using logistic regression analysis, we found no evidence of effect, with only a non-significant increase in breast cancer risk for AA carriers of the XbaI polymorphism (overall OR\u00a0=\u00a01.3, 95% CI\u00a0=\u00a00.7\u20132.2) and for TT carriers of the PvuII variant (overall OR\u00a0=\u00a01.4, 95% CI\u00a0=\u00a00.8\u20132.2). Additionally we performed meta-analyses of published data to examine the effect of both polymorphisms. These meta-analyses also suggest there are no differences in risk among genotype groups of these two ESR1 variants.\nThe XbaI and PvuII polymorphisms are situated in intron 1 and their functionality has not yet been demonstrated. Moreover, it has been suggested their effects could be the result of high linkage disequilibrium with functional variants that affect sensitivity to estrogen [13].\nOne of the limitations of our study is the limited number of breast cancer cases present in our population. Nevertheless, we have sufficient power (\u03b2\u00a0=\u00a00.8) to detect effects of 1.6 or higher. We further conducted meta-analyses off all studies conducted to date. Our data suggests that these two polymorphisms do not play a role in the susceptibility of breast cancer in elderly Caucasian women.","keyphrases":["estrogen receptor","polymorphism","breast cancer"],"prmu":["P","P","P"]}
{"id":"J_Biol_Inorg_Chem-3-1-2099460","title":"Active-site structure, binding and redox activity of the heme\u2013thiolate enzyme CYP2D6 immobilized on coated Ag electrodes: a surface-enhanced resonance Raman scattering study\n","text":"Surface-enhance resonance Raman scattering spectra of the heme\u2013thiolate enzyme cytochrome P450 2D6 (CYP2D6) adsorbed on Ag electrodes coated with 11-mercaptoundecanoic acid (MUA) were obtained in various experimental conditions. An analysis of these spectra, and a comparison between them and the RR spectra of CYP2D6 in solution, indicated that the enzyme\u2019s active site retained its nature of six-coordinated low-spin heme upon immobilization. Moreover, the spectral changes detected in the presence of dextromethorphan (a CYP2D6 substrate) and imidazole (an exogenous heme axial ligand) indicated that the immobilized enzyme also preserved its ability to reversibly bind a substrate and form a heme\u2013imidazole complex. The reversibility of these processes could be easily verified by flowing alternately solutions of the various compounds and the buffer through a home-built spectroelectrochemical flow cell which contained a sample of immobilized protein, without the need to disassemble the cell between consecutive spectral data acquisitions. Despite immobilized CYP2D6 being effectively reduced by a sodium dithionite solution, electrochemical reduction via the Ag electrode was not able to completely reduce the enzyme, and led to its extensive inactivation. This behavior indicated that although the enzyme\u2019s ability to exchange electrons is not altered by immobilization per se, MUA-coated electrodes are not suited to perform direct electrochemistry of CYP2D6.\nIntroduction\nHeme proteins are a large class of proteins having a substituted iron porphyrin as a cofactor, with functions ranging from electron carriers to oxygen transport to catalysis. Heme enzymes such as cytochromes P450 (CYPs) and other oxygenases, being able to bind several substrates and to use electrons from their heme iron to drive a catalytic reaction, seem to be promising for nanobiotechnological applications in biosensing and biocatalysis [1, 2].\nThe immobilization of enzymes on solid supports or electrodes is a commonly used approach for constructing biosensors or biocatalytic devices [3, 4]. In principle, electrodes can be used as a source of electrons to drive a catalytic reaction, or as elements of biosensors which translate a molecular event such as substrate binding into a measurable electric signal.\nThe question arises whether immobilization preserves the protein function or rather induces losses of enzymatic activity, and what structural changes cause the dysfunction [1]. Therefore, techniques capable of probing the activity and the structure of enzymes immobilized on surfaces are valuable.\nSurface-enhanced resonance Raman scattering (SERRS) has proven to be a sensitive and selective spectroscopic technique to study heme proteins immobilized on metal surfaces, and it has been used to characterize the active site of proteins such as cytochromes c, cytochrome c oxidase and CYPs [5]. This technique is based on the inelastic scattering of laser radiation whose wavelength is in resonance with an electronic transition of the molecule studied [6\u20138], leading to a signal enhancement denoted as the resonance Raman (RR) effect. In the case of proteins having a heme as a chromophore, the porphyrin vibrational modes are intensified over the other protein vibrations if the laser wavelength falls within an associated electronic absorption band. The heme moiety can thus be selectively investigated without a dominating interference due to the protein matrix. Additional sensitivity is gained through surface enhancement (or surface-enhanced Raman scattering effect) if the protein is close to a metal surface with particular characteristics. Depending on the molecule studied and on the experimental conditions applied, the synergy between the two signal-enhancing effects (i.e., SERRS) sometimes reaches single-molecule sensitivity [9\u201312], therefore allowing the study of enzymes immobilized on surfaces, typically consisting of very low amounts of sample (i.e., from a few layers to submonolayers of protein).\nTo fulfill the requirements to achieve both resonance enhancement and surface enhancement, silver is the surface-enhancing metal of choice [7]. Recently, heme proteins such as cytochromes c [5, 13\u201319] and CYP101 [20] have been investigated using SERRS on roughened Ag electrodes coated with self-assembled monolayers (SAMs) of several different alkanethiols and their derivatives. SAMs of functionalized alkanethiols are among the most versatile coatings for both electrostatic and covalent immobilization of proteins to metal surfaces, having the additional advantage of preventing possible negative consequences of the direct contact between the protein and the metal [5, 21]. Besides technological applications, SERRS on SAM-coated electrodes can become an important tool in fundamental studies about protein\u2013membrane and protein\u2013protein interactions. In fact, because of their structure and characteristics, SAMs can be thought of as a rough model for biological membranes [5, 13], or can be used to mimic the binding domain of a partner protein, since surface properties such as polarity and charge can be selected by choosing the appropriate alkanethiol derivative [13, 22]. Moreover, electrochemically roughened Ag electrodes coated with SAMs were recently shown to be excellent for studying immobilized proteins with voltammetric techniques, so both SERRS and voltammetry can be performed on the same sample [19].\nIn the work reported here, we exploited SERRS to study the behavior of the human enzyme CYP2D6 electrostatically immobilized on a Ag electrode coated with a SAM of 11-mercaptoundecanoic acid (MUA). CYPs are heme\u2013thiolate monooxygenases capable of oxidizing and reducing a broad range of endogenous and exogenous substrates and can be found in all kinds of tissues in virtually all organisms [23]. CYP2D6 is a peripheral membrane-bound protein and is one of the most important CYPs in humans, being involved in the metabolism of about 30% of the currently marketed drugs, including \u03b2-blockers, neuroleptics, antidepressants and antiarythmics [24, 25]. Its characteristics make this enzyme not only attractive for technological applications as a biocatalyst or biosensor, but also from a toxicological and clinical point of view. In fact, a better understanding of its mechanisms of catalysis could help in developing more efficient drugs [26, 27].\nUsing a home-built SERRS spectroelectrochemical flow cell [28], we investigated the CYP2D6 active-site structure under diverse experimental conditions in order to check the functionality of the immobilized enzyme, its redox activity and its ability to bind reversibly a substrate or an exogenous heme ligand such as imidazole. Besides confirming the retention of the enzyme\u2019s function upon immobilization, SERRS spectra yield information about structural changes in the active site associated with substrate binding and electron transfer, the initial two steps of a CYP catalytic cycle.\nMaterials and methods\nChemicals\nThe pSP19T7LT plasmid containing bicistronically human CYP2D6 with a C-terminal His6-tag and human NADPH-CYP reductase,was kindly provided by Magnus Ingelman-Sundberg. Dextromethorphan (DX), imidazole, MUA and sodium dithionite were purchased from Sigma-Aldrich (St Louis, USA). Emulgen 911 was obtained from KAO Chemicals (Tokyo, Japan). Nickel nitrilotriacetic acid (NTA) agarose was from Sigma. Phosphate buffer solutions (pH 7.4, 40\u00a0mM) were prepared from K2HPO4 and KH2PO4 (J.T. Baker, Deventer, The Netherlands). All other chemicals were of analytical grade and were obtained from standard suppliers.\nExpression and purification of CYP2D6\nThe pSP19T7LT plasmids containing wild-type CYP2D6 were transformed in Escherichia coli, strain JM109. Expression, membrane isolation and purification were carried out as previously described [29]. In short, membranes were resuspended in 0.5% of the original culture volume of potassium phosphate\u2013glycerol buffer (50\u00a0mM potassium phosphate buffer, pH 7.4, 10% glycerol) and CYP contents were determined by CO difference spectra [30]. The enzyme was solubilized by stirring in potassium phosphate\u2013glycerol supplemented with 0.5% Emulgen 911 for 2\u00a0h at 4\u00a0\u00b0C. Insoluble parts were removed by centrifugation (60\u00a0min, 120,000g at 4\u00a0\u00b0C). The supernatant was mixed, while gently rocking it, with nickel nitrilotriacetic acid agarose for 60\u00a0min at 4\u00a0\u00b0C. The nickel nitrilotriacetic acid agarose was then applied to a polypropylene tube with a porous disk (Pierce, Perbio Science, Etten-Leur, The Netherlands), washed with potassium phosphate\u2013glycerol buffer containing 2\u00a0mM histidine. Subsequently, bound CYP2D6 was eluted with 0.2\u00a0M histidine. After overnight dialysis in potassium phosphate\u2013glycerol buffer the sample was concentrated on a Vivaspin 20 filtration tube (10,000 MWCO PES, Sartorius, Nieuwegein, The Netherlands) to a final concentration of 60\u00a0\u03bcM CYP2D6.\nSpectroelectrochemical flow cell\nSERRS measurements were performed using a modified version of a linearly moving low-volume spectroelectrochemical cell previously described [28]. The cell was originally designed to acquire potential-dependent SERRS spectra from proteins adsorbed on roughened Ag electrodes using a Raman microscope in a backscattering configuration. The modified version of this cell used in the present work has the possibility to flow a solution through the cell via two small holes (0.75-mm diameter each; Fig.\u00a01). By using a valve, a liquid-chromatography pump (Gilson, Middleton, USA) and flexible PEEK tubing (VICI International, Schenkon, Switzerland), the thin layer (0.3\u00a0mm) of solution in contact with the Ag electrode surface can be changed by flowing different solutions though the spectroelectrochemical cell, without the need to disassemble the cell itself (Fig.\u00a01). Moreover, by degassing the solutions with argon and by using oxygen-proof PEEK tubing, the presence of oxygen, which can interfere with electrochemical and spectroscopic measurements, can be minimized in the cell. It should be noted that small internal diameter of the liquid-chromatography tubing (0.75\u00a0mm) and the low internal volume of the cell (approximately 50\u00a0\u03bcL) allows the use of relatively small amounts of solutions. Fig.\u00a01The surface-enhanced resonance Raman scattering (SERRS) spectroelectrochemical flow system. The degassed solutions are pumped though PEEK liquid-chromatography tubing into the cell and then to the waste. A switching valve is used to select which solution has to be pumped. A scheme of the cell is shown in detail, with the inlet and outlet for the flowing solution and the three electrodes: the Ag working electrode (WE), the Pt counter electrode (CE) and the connection though a porous frit to a saturated calomel electrode (SCE) reference electrode (RE). The laser for Raman excitation is focused by the microscope objective onto the Ag electrode through a glass cover and a layer of solution\nRaman spectroscopy instrumentation\nSpectroscopic measurements were conducted by placing the spectroelectrochemical cell under a home-built Raman microscope in a backscattering configuration: a Zeiss microscope (D-7082 with a \u00d740 objective, numerical aperture 0.60, working distance 2\u00a0mm) was coupled to an Andor Shamrock SR-303i-A single monochromator (Andor Technologies DV-420OE, Belfast, UK) with a mounted 2,400\u00a0g\u00a0mm\u22121 holographic grating and an Andor Newton DU970N CCD camera. The 413.1\u00a0nm line of a continuous-wave Kr ion laser (Innova 300C, Coherent, Santa Clara, CA, USA) was used for excitation. The Rayleigh scattered light was removed using third millennium edge long pass filter. Laser powers of 5\u00a0mW at the sample and an accumulation time of 180\u00a0s were used throughout the experiments. To avoid sample photodegradation, the cell was put in a moving holder which ensured a constant linear movement of the metal electrode surface under the microscope objective, while keeping the focus approximately constant [28]. The monochromator slit was set to 120\u00a0\u03bcm, yielding a resolution of approximately 4\u00a0cm\u22121, with an increment of approximately 0.8\u00a0cm\u22121 per data point.\nThe sloping background of the spectra was subtracted using a baseline fitted to the experimental data with the Andor CCD camera software, while the fitting of the CYP2D6 spectrum at \u22121.0\u00a0V with Lorentzian functions was performed with PeakFit\u00ae version\u00a04.12 (SeaSolve Software, Richmond, CA, USA).\nElectrochemical instrumentation\nPolycrystalline silver disk electrodes (IJ Cambria Scientific, Burry Port, UK) of 2-mm diameter (geometric area\u00a00.0314\u00a0cm2) were used as working electrodes; a platinum ring and a saturated calomel electrode (AMEL Instruments, Milan, Italy), were used as the counter electrode and the reference electrode, respectively. The reference electrode was kept in a glass tube filled with a 40\u00a0mM potassium phosphate buffer solution and separated from the working solution by a PEEK porous frit. It was kept at constant room temperature (20\u00a0\u00b1\u00a00.1\u00a0\u00b0C) during all the experiments. The three-electrode system was controlled with a \u03bcAutolab potentiostat (Eco Chemie, Utrecht, The Netherlands). All potentials are referred to a saturated calomel electrode, previously calibrated against the methyl viologen (MV) MV2+\/MV+ couple.\nSample preparation\nSERRS measurements were performed on electrochemically roughened Ag electrodes coated with MUA, prepared as follows. Ag electrodes were treated as previously described [31] with water on aluminum oxide lapping film sheets (261\u00d7 and 262\u00d7, 3M\u2122) from 5- to 1-\u03bcm grain size until a mirrorlike appearance of the surfaces was obtained. After they had been polished, the electrodes were roughened ex situ with an oxidation\u2013reduction cycle procedure similar to that described by Roth et al. [32] and then kept overnight in 2\u00a0mM ethanol solution of MUA. The oxidation\u2013reduction cycles (approximately 0.67\u00a0C\u00a0cm\u22122 of anodic charge passed per step) were performed in an ordinary glass electrochemical cell (5\u00a0mL) filled with a 0.1\u00a0M KCl solution using the three-electrode system described already.\nThe MUA-coated Ag electrodes were rinsed with ethanol to eliminate the excess of MUA, dried by gently blowing nitrogen on them and then immersed into 50\u00a0\u03bcL of a 3\u00a0\u03bcM solution of protein in potassium phosphate buffer (40\u00a0mM, pH 7.4, 10% glycerol) for at least 3\u00a0h at 4\u00a0\u00b0C, to induce adsorption of the protein on the electrode. Then, the coated electrodes with the adsorbed protein were rinsed with buffer to eliminate possible excess of loosely bound protein, and were put into the spectroelectrochemical cell described already.\nDepending on the required experimental conditions, before every SERRS measurement the appropriate solution was flowed for 1\u20132 min into the cell: for measurements in the absence of DX or imidazole, a potassium phosphate buffer (40\u00a0mM, pH 7.4) was used, while for measurements in the presence of DX or imidazole, solutions of DX (5\u00a0mM, 0.02% of methanol) or imidazole (10\u00a0mM) in buffer were used. The solutions of DX were prepared by diluting with buffer a 2\u00a0M stock solution of DX in methanol. To chemically reduce the protein in the absence or presence of substrate, sodium dithionite solutions (5\u00a0mg\u00a0mL\u22121) were prepared using buffer solutions or DX solutions (5\u00a0mM) in buffer, and were flowed for 1\u20132 min into the cell before every SERRS measurement. Prior to use, all the solutions flowed into the cell were deoxygenated for at least 3\u00a0h with argon.\nRR measurements were performed using buffered CYP2D6 solutions in spinning glass capillaries as previously described [33]. To chemically reduce the enzyme in solution prior to RR measurements, solid sodium dithionite (to a final concentration of 5\u00a0mg\u00a0mL\u22121) was added to a buffered 3\u20135\u00a0\u03bcM protein solution in presence of 5 mM DX, previously degassed with argon.\nResults and discussion\nThe resting state\nIntense SERRS spectra (Fig.\u00a02, spectra\u00a0a) were observed from CYP2D6 immobilized via electrostatic interaction [34] on MUA-coated Ag electrodes at open circuit (i.e.. no external potential is applied), in the presence of phosphate buffer. These experimental conditions should reproduce the \u201cresting state\u201d of the enzyme (i.e.. the starting point of its catalytic cycle) when no substrate is present in the active site and no reaction is taking place [35, 36]. A MUA-SAM was chosen as the coating because of its structural stability, its compatibility with CYP2D6 [34] and its use in recent SERRS studies on bacterial CYP101 [20] and on cytochrome c [5, 13]. Fig.\u00a02A SERRS spectra of cytochrome\u00a0P450 (CYP) 2D6 on an 11-mercaptoundecanoic acid (MUA)-coated Ag electrode obtained by consecutively flowing different solutions in the spectroelectrochemical cell (at open circuit), in the following order: a buffer, b dextromethorphan (DX) buffered solution, c buffer (rinse), d imidazole-buffered solution, e buffer (second rinse), f dithionite and DX solution in phosphate buffer. The 1,300\u20131,450-cm\u22121 regions of spectra b--e are identical to that of spectrum\u00a0a and are omitted for visual clarity. B Detailed view of the 1,450\u20131,660-cm\u22121 region of the CYP2D6 SERRS spectra in presence of a buffer, b DX and d imidazole. Bands corresponding to the \u03bd2 and \u03bd3 vibrational modes for the six-coordinated low-spin and five-coordinated high-spin heme species are indicated. Experimental details are given in \u201cMaterials and methods\u201d\nThe most intense band of the SERRS spectra is observed at 1,371\u00a0cm\u22121, and a group of bands are detected between 1,450 and 1,650\u00a0cm\u22121. The frequencies of the vibrational modes above 1,300\u00a0cm\u22121 are correlated with the oxidation, the spin and the coordination state of the iron atom and therefore they are referred to as \u201cmarker bands\u201d for such properties [37]. The most relevant marker bands in CYP2D6 SERRS spectra are assigned to the corresponding vibrational modes on the basis of former RR studies of CYP2D6 [33] and other CYPs [37] in buffered solution (Table\u00a01). According to the frequencies observed, the heme iron in the enzyme active site is oxidized (Fe3+), mainly six-coordinated low-spin (6cLS) state, as is usually expected from a CYP in its resting state [36, 37]. An extensive amount of experimental data from other CYPs as well as theoretical studies showed that in the 6cLS state the Fe3+ atom has the four pyrrolic nitrogens of the porphyrin macrocycle as equatorial ligands, and a cysteine and a water molecule (or hydroxyl ion) as axial ligands [35, 36]. Table\u00a01Vibrational frequencies (cm\u22121) and relative intensities of most relevant oxidation- (4) and spin\/coordination- (\u03bd3, \u03bd2) marker bands in SERR spectra of CYP2D6 adsorbed on MUA-coated electrodes, under different experimental conditionsModeFe3+Fe2+6cLS6cLS\/5cHSWithout substrate\/inhibitorWith inhibitor (imidazole)With substrate (dextromethorphan)5cHSa\u03bd21,582 (0.19)1,582 (0.14)1,582 (0.20)\/1,567 (NA)b1,561 (0.91)\u03bd31,501 (0.08)1,501 (0.10)1,501 (0.09)\/1,486 (0.05)1,466 (0.65)\u03bd41,371 (1.00)1,371 (1.00)1,371 (1.00)1,344c (1.00)Relative intensities are reported after the corresponding frequencies between parentheses; values are relative to the band with an intensity of 1.006cLS six-coordinated low spin, 5cHS five-coordinated high spin, NA not availableaReduced state is achieved by adding dithionite in presence of substratebBand not sufficiently resolved, frequency estimated by spectral difference [33]cTogether with this band, in spectra of CYP2D6 another one is observed at 1,360\u00a0cm\u22121, characteristic of reduced inactive cytochrome\u00a0P420 species\nThe observation of an oxidized 6cLS state is in agreement with spectroscopic data on CYP2D6 previously reported. In particular, the SERRS spectrum of CYP2D6 under these conditions is identical to the RR spectrum of the same enzyme in buffer [33, 38] (Fig.\u00a03 spectra\u00a0b and b\u2032). An analogous correspondence with RR data was recently reported for SERRS spectra of CYP2D6 adsorbed on Ag nanoparticles coated with MUA [34]. This striking similarity in the position and relative intensity of SERRS and RR spectra suggests that, differently from what was recently reported for CYP101 [20], CYP2D6 retains its active-site structure upon adsorption on the SAM-coated electrode, or at least that there are no structural changes inducing significant alterations in heme properties. Fig.\u00a03Comparison between SERRS (a, b) and resonance Raman (a\u2032, b\u2032) spectra of reduced (a, a\u2032) and oxidized (b, b\u2032) CYP2D6. SERRS spectra were obtained from MUA-coated Ag electrodes in the presence of buffer alone (b) and a dithionite and DX solution in phosphate buffer (a), while resonance Raman spectra were obtained from CYP2D6 in solution under the same conditions. Marker bands for oxidation (\u03bd4) and spin (\u03bd3, \u03bd2) states are indicated. Experimental details are given in \u201cMaterials and methods\u201d\nReversible binding of a substrate\nAlthough the active-site structure is likely to be unaffected by the protein\u2013surface interaction, the functioning of the adsorbed enzyme still has to be verified. According to the accepted scheme of the catalytic cycle common to most CYPs, the heme iron may experience changes in its coordination sphere and its spin as a consequence of the presence of substrates in the active site [36]. RR spectra of CYP2D6 in the presence of DX, a substrate of this enzyme, showed the appearance of marker bands for a five-coordinated high-spin (5cHS) heme besides the 6cLS heme already present in the resting state [33]. In agreement with those RR data, SERRS spectra of CYP2D6 adsorbed on coated electrodes, upon flowing a DX solution into the cell (Fig.\u00a02, spectra\u00a0b), exhibit distinct differences from the spectra of the enzyme in the resting state, showing additional bands characteristic of a 5cHS heme (\u03bd3 at 1,486\u00a0cm\u22121 and \u03bd2 at 1,567\u00a0cm\u22121; Fig.\u00a02, spectra\u00a0b, Table\u00a01).\nThis partial transition from 6cLS to 5cHS is explained by the displacement of a water molecule as the heme\u2019s distal axial ligand by DX, which is accommodated in the active site on the distal side above the porphyrin macrocycle [33]. Water molecules, which may still be present in the active site, can then rebind to the heme iron, yielding an equilibrium between the 5cHS and 6cLS states [33, 37, 39].\nThe spectral changes taking place owing to the presence of DX therefore clearly indicate that the adsorbed enzyme retains its ability to accommodate a substrate into its active site, confirming similar results reported for CYP2D6 adsorbed on coated Ag nanoparticles [34]. Unfortunately, neither RR nor nanoparticle-based SERRS allowed the investigation of the reversibility of the substrate-binding process, since it is extremely difficult to rapidly eliminate the substrate from the enzyme-containing sample. Such problems are not encountered in the present spectroelectrochemical cell setup. The substrate is easily removed from the enzyme-coated electrode surface by flowing buffer through the cell, reestablishing the resting state conditions. Once the buffer had completely replaced the DX solution in the cell, 5cHS marker bands disappeared from SERRS spectra of CYP2D6, which recover the shape characteristic of the 6cLS heme in the resting state (Fig.\u00a02, spectrum\u00a0c). The exchange between the two solutions can be operated many times, showing every time the concomitant spectral changes, and indicating an intrinsic stability of the CYP2D6\u2013SAM\u2013electrode system. The similarity between the SERRS spectra of the same enzyme sample before the introduction of DX (Fig.\u00a02, spectra\u00a0a) and after its removal (Fig.\u00a02, spectrum\u00a0c) clearly demonstrates that the adsorbed CYP2D6 is able to reversibly bind a substrate, implying that the interaction with the coated electrode surface does not obstruct the substrate access routes to the enzyme active site.\nReversible binding of imidazole as an exogenous heme axial ligand\nImidazole and several imidazole derivatives are believed to form a relatively strong bond via their nitrogen with the heme iron of ferric CYPs, replacing the water molecule as a distal axial ligand, and thereby preventing the binding of oxygen to the heme in a subsequent step of the catalytic cycle [37, 40\u201343]. Since oxygen binding is essential for monooxygenase activity, often these compounds are effective inhibitors of CYPs [44].\nThe SERRS spectra of CYP2D6 collected upon flowing an imidazole solution over the enzyme-coated electrode are shown in Fig.\u00a02, spectra\u00a0d, displaying marker bands characteristic of a 6cLS state (Table\u00a01), as expected from a CYP heme having an imidazole as the sixth axial ligand. Although the frequencies of the imidazole complex are identical to those reported for the resting state, the relative intensity ratio between the \u03bd3 and \u03bd2 bands is visibly increased with respect to the 6cLS resting state from 0.4 to 0.7 (Fig.\u00a02spectra\u00a0a and d, Table\u00a01), in agreement with RR data of CYP2D6 in solution (see supplementary material) and with RR spectra previously reported for other CYP\u2013imidazole complexes [41, 45\u201347]. This difference in relative intensities has been observed in all the RR spectra of CYP\u2013imidazole complexes reported so far, and is generally regarded as the characteristic spectral feature which allows one to discriminate between the two 6cLS states having either imidazole or water as the heme axial ligand in CYPs. The explanation given for these characteristic relative intensity changes observed in the presence of imidazole in RR spectra of CYPs invokes the different resonance conditions due to redshift of the Soret absorption induced by imidazole in CYPs [37].\nAn additional but less pronounced feature observed in the SERRS spectrum of the CYP2D6\u2013imidazole complex is the intensity decrease in the region around 1,630\u00a0cm\u22121, where three overlapping bands are expected: two stretching modes of the heme vinyl substituents around 1,620 and 1,630\u00a0cm\u22121, and the \u03bd10 porphyrin mode around 1,635\u00a0cm\u22121 [37]. The presence of another vinyl stretching band, otherwise invisible because of its overlap with the more intense neighboring bands, can be substantiated by fitting the data with Lorentzian functions. A qualitative analysis of the fitting supports the hypothesis of a decrease in relative intensity of a vinyl band around 1,630\u00a0cm\u22121 for the imidazole complex (see supplementary material), suggesting a possible perturbation of the vinyls upon imidazole binding [48].\nThe observed change in relative intensity could be due to a variation in the rotational conformation of the vinyls [48]. Such variation might also cause a shift in the stretching frequencies of the vinyls [49, 50], although in SERRS spectra of CYP2D6 no significant shifts are detected. On the other hand, similar changes in relative intensities in the 1,610\u20131,640-cm\u22121 spectral region have been previously reported for the imidazole complexes of bacterial and microsomal CYPs [41, 45\u201347]. The fact that imidazole binding induces similar changes in vinyl rotational conformation in enzymes having rather different active-site structures is somewhat surprising. Since vinyl stretching modes in SERRS spectra are thought to be enhanced via an electronic coupling with the porphyrin [37], an alternative explanation of the changes detected upon imidazole binding could be the difference in resonance conditions due to the shift of the Soret band, similarly to what has been proposed for the \u03bd3\/\u03bd2 intensity ratio. However, in absence of further experimental data from other techniques, the SERRS spectra presented cannot reliably give more detailed structural information about conformational changes of vinyls occurring upon imidazole binding.\nSimilarly to what was observed for DX binding, SERRS spectra obtained by flowing buffer through the cell in place of the imidazole solution (Fig.\u00a02, spectrum\u00a0e) have the \u03bd3\/\u03bd2 relative intensity ratio restored to 0.4, and are identical to the spectra of the enzyme in the resting state (Fig.\u00a02, spectra\u00a0a), including the relative intensity pattern around 1,630\u00a0cm\u22121. Therefore, despite the imidazole being expected to form a relatively strong bond with the heme iron, the nonequilibrium situation due to the constant flow of buffer over the CYP\u2013imidazole complex eventually causes the complete dissociation of the imidazole from the enzyme and the concomitant restoration of a water molecule as sixth ligand, demonstrating that the CYP\u2013imidazole complex formation is reversible.\nRedox activity\nThe catalytic activity of CYP2D6 relies on its ability to receive electrons from NADPH-dependent CYP reductase to drive the catalysis and to activate molecular oxygen to oxidize the substrate [36]. In particular, the first of the two electrons transferred during a catalytic cycle reduces the iron from Fe3+ to Fe2+, facilitating oxygen binding to the ferrous heme. In their resting state, CYPs usually have a formal reduction potential (E\u00b0\u2032) lower than that of their electron-donor protein, so heme reduction does not occur [36]. However, the E\u00b0\u2032 of some CYPs, including CYP2D6, is shifted to more positive values by the presence of a substrate in the active site, facilitating the reduction of the enzyme heme by the redox partner [1, 36, 51]. Therefore, the electron transfer reaction is believed to be triggered by substrate binding and is usually considered the third step of the catalytic cycle [36].\nIn absence of reductase, immobilized CYP2D6 might receive electrons from a reducing agent present in solution or directly from the electrode, by applying a proper voltage. The electron transfer process from the alternative electron source to the enzyme can be monitored by examining the relative presence of reduced heme species in SERRS spectra, using solutions previously deoxygenated with argon. This requirement is crucial since dioxygen rapidly binds to the ferrous heme to form a complex, which upon reaction with another electron quickly turns into an instable peroxo-ferric intermediate, continuing the catalytic cycle and hampering the observation of reduced species [36].\nUpon flowing over the adsorbed enzyme a deoxygenated and buffered DX solution containing sodium dithionite as a reducing agent, the SERRS spectrum shown in Fig.\u00a02, spectrum\u00a0f is observed. It mainly presents bands with frequencies characteristic of a reduced 5cHS heme (Table\u00a01), which are markedly different from all the spectra of oxidized CYP2D6 obtained so far (Fig.\u00a02, spectra\u00a0a\u2013e). A noticeable feature is the coexistence of two \u03bd4 oxidation marker bands at 1,344 and 1,360\u00a0cm\u22121, originating from the downshift and the splitting of the corresponding \u03bd4 band observed at 1,371\u00a0cm\u22121 in the oxidized enzyme, and indicating the presence of two different heme species. While the \u03bd4 band at 1,344\u00a0cm\u22121\u2014consistent with the other bands observed\u2014is typical of a ferrous CYP in its active form, the band at 1,360\u00a0cm\u22121 is characteristic of a ferrous inactive form called P420 [20, 37, 52, 53]. This strongly suggests that upon reduction a significant fraction of the immobilized enzyme undergoes structural changes leading to its inactivation. However, according to the RR spectra of the ferrous active and inactive forms previously reported for other CYPs, the active enzyme still contributes to a significant extent to the observed CYP2D6 SERRS spectrum, whereas no marker bands for the inactive species other than the one at 1,360\u00a0cm\u22121 can be detected. In fact, in all the RR or SERRS spectra of P420 species reported so far for various CYPs, the \u03bd4 band is the strongest, with relative intensity much higher than for the other bands, whereas in spectra of active enzymes the intensity pattern is different, with many other bands having intensities comparable to that of the \u03bd4 band [20, 37, 52\u201355]. In the spectra of reduced CYP2D6, the bands in the 1,400\u20131,650-cm\u22121 region have relative intensities comparable to that of the \u03bd4 band of the active enzyme (at 1,344\u00a0cm\u22121), whereas the bands of the P420 species are likely to have relative intensities much smaller than that of the P420 \u03bd4 band at 1,360\u00a0cm\u22121, and are therefore buried under the more intense bands due to the active enzyme.\nNotably, this partial inactivation is not due to the immobilization as such, since every attempt to obtain reduced free CYP2D6 in solution by dithionite addition led to samples containing inactive enzyme besides the active one, yielding RR spectra which closely resemble the SERRS spectrum of the adsorbed enzyme (Fig.\u00a04, spectra\u00a0a). The inactivation might be due to laser-induced photodegradation, since laser illumination is common to both RR and SERRS. However, the use of laser powers lower than 5\u00a0mW yielded identical spectra (data not shown), and small amounts of inactive enzyme were previously detected in dithionite-reduced CYP2D6 samples using nonlaser techniques as well, such as UV\u2013vis electronic absorption spectroscopy [38]. Fig.\u00a04SERRS spectra of CYP2D6 on MUA-coated Ag electrodes in the presence of buffered solution with dithionite and DX (a) and without DX (b). The inset shows the oxidation marker band regions in detail, together with their spectral difference. Experimental details are given in \u201cMaterials and methods\u201d\nWhen reduction of CYP2D6 with dithionite is attempted in the absence of a substrate, the resulting SERRS spectrum presents some differences with respect to that of the substrate-bound enzyme (Fig.\u00a04). In particular, the presence of DX in the dithionite solution increases the amount of reduced active enzyme at the expense of the oxidized and reduced inactive forms, as deduced from the increase in intensity of the band at 1,344\u00a0cm\u22121 and the concomitant decrease of the bands at 1,360 and 1,371\u00a0cm\u22121. Thus, the presence of DX in the heme pocket appears to affect the redox activity of the enzyme, possibly by causing a shift of its E\u00b0\u2032 to more positive values, as previously observed for CYP2D6 on a polyaniline-doped glassy carbon electrode in the presence of fluoxetine [51].\nOn the other hand, in the electrochemical approach the control of the CYP2D6 oxidation state is attempted by applying a constant potential to the Ag working electrode, during which a SERRS spectrum is recorded to monitor the relative abundance of oxidized and reduced species characteristic for that voltage. Complete oxidation or reduction of the protein should in principle be obtained by applying a potential which is sufficiently more positive or more negative with respect to its E\u00b0\u2032, respectively.\nBesides the advantage of controlling the enzyme oxidation state and therefore, under adequate conditions, of supplying the electrons and driving the catalytic reaction, an additional benefit of this approach is the possibility to study the electron transfer process quantitatively. In stationary SERRS spectroelectrochemistry the analysis of a set of spectra acquired at various potentials can assess the Nernstian character the electron transfer process, yielding its E\u00b0\u2032 value [5, 14, 19]. Moreover, a time-resolved approach based on potential-jump experiments can give information about the electron transfer kinetic parameters [5, 56].\nHowever, despite the broad potential window investigated (from +0.15 V to \u22121.0 V) the electrochemical approach appeared to be unsuccessful in reducing the immobilized CYP2D6 completely, and failed to generate any reduced active enzyme at all. In fact, although there is some variability in the spectral data obtained from distinct electrodes, a common feature is the persistence of an intense marker band for the oxidized enzyme at 1,371\u00a0cm\u22121 even at potentials as negative as \u22121.0\u00a0V, together with the appearance of the marker band for a reduced inactive enzyme at 1,360\u00a0cm\u22121 (Fig.\u00a05). An additional feature in the SERRS spectra of reduced CYP2D6 is the lack of any influence of DX (see supplementary material), contrary to what is observed with chemical reduction. Fig.\u00a05Oxidation marker band (\u03bd4) regions of SERRS spectra of CYP2D6 on MUA-coated Ag electrodes at \u22121.0\u00a0V (a), \u22120.8\u00a0V (b), \u22120.4\u00a0V (c) and \u22120.1\u00a0V (d) (vs. SCE), in the presence of buffer. The spectrum at \u22121.0\u00a0V is fitted with Lorentzian functions for the reduced and oxidized forms, represented as dotted lines and labeled with their Raman shift (1,371\u00a0cm\u22121 for the oxidized form and 1,360\u00a0cm\u22121 for the reduced inactive P420 form). Experimental details are given in \u201cMaterials and methods\u201d\nAlthough the E\u00b0\u2032 of CYP2D6 on MUA-coated Ag electrodes might differ significantly from the E\u00b0\u2032 reported for a polyaniline-doped glassy carbon electrode (\u22120.12\u00a0V) [1, 51], the persistence of a consistent amount of oxidized enzyme at \u22121.0\u00a0V suggests an incomplete electron transfer. Despite the large variability of the E\u00b0\u2032 values found in the literature, depending on the CYP and on the method employed, no E\u00b0\u2032 is reported below \u22120.6\u00a0V [1]. Consistent with this fact, CYP2D6 is effectively and completely reduced by a 5\u00a0mg\u00a0mL\u22121 dithionite solution having a calculated redox potential of approximately \u22120.7\u00a0V [57]. Therefore, on a pure thermodynamical basis, the application of a potential more negative than \u22120.7\u00a0V to the Ag electrode should lead to a reduction of the adsorbed enzyme. The marked difference in the amount of reduced species obtained with chemical and electrochemical reduction (Fig.\u00a06) might therefore be due to slower kinetics of the electron transfer process when using the second method. Fig.\u00a06Comparison between SERRS spectra of CYP2D6 on MUA-coated Ag electrodes a chemically reduced with dithionite (open circuit) and b at \u2212800\u00a0mV (vs.SCE). Both spectra were recorded in the presence of DX. Marker bands for oxidation (\u03bd4) and spin (\u03bd3, \u03bd2) states are indicated. Experimental details are given in \u201cMaterials and methods\u201d\nThe efficiency of the electrochemical reduction via the electrode is likely to be dependent on both the distance and the orientation of the adsorbed protein with respect to the electrode surface [1, 3], whereas these two factors probably do not influence the dithionite reduction process. In vivo, electron transfer between the electroactive heme center, deeply buried inside the protein structure, and the electron-donating reductase occurs via an interaction involving specific domains on the protein surfaces [1, 3], with a strictly defined orientation. Conversely, the electron transfer from dithionite in solution will not be sensitive to the protein orientation, considering the capacity of the reducing agent to approach the enzyme from more directions. Moreover, while the minimum distance between the enzyme active site and the electrode surface is equivalent to the thickness of the MUA coating (approximately 19\u00a0\u00c5 [22]) plus the heme\u2013protein surface distance, dithionite is free to diffuse closer to the enzyme redox center. Therefore, an unfavorable enzyme orientation with respect to the coated metal surface or a too large heme\u2013electrode distance would be consistent with experimental data, since it would slow down or suppress the electron transfer from the electrode, while dithionite would still be able to effectively reduce the CYP2D6 heme. Moreover, the inability of the electrochemical approach to generate any amount of reduced enzyme in its active form, which is otherwise observed in dithionite-reduced protein samples, suggests that CYP2D6 inactivation is triggered by the application of potentials between \u22120.4 and \u22121.0\u00a0V during the enzyme-reduction attempts.\nThe intense electric field present at the interface of the SAM-coated Ag electrodes, which has recently been indicated as the main reason for the extensive conversion of the immobilized bacterial CYP101 to its inactive P420 forms [20], might be the cause of this potential-induced inactivation. However, according to the theory originally proposed by Smith and White [58] and later adapted by Murgida and Hildebrandt [22] and Lecomte et al. [59] to explain the behavior of c-type cytochromes adsorbed on electrodes, the intensity of this interfacial electric field is expected to decrease as the applied potential approaches the potential of zero charge (\u22120.97\u00a0V for a polycrystalline Ag electrode) [13, 60\u201362]. Therefore, supposing that this theoretical model could be extended to CYPs (as it has been assumed in the case of CYP101 [20]), the interfacial electric field should be more intense at \u22120.1\u00a0V than at \u22121.0\u00a0V, whereas CYP2D6 inactivation occurs at more negative potentials, suggesting that in this case the interfacial electric field intensity as such is not responsible for the enzyme conversion to the P420 form upon electrochemical reduction. This hypothesis would be compatible with the electric-field-induced inactivation reported for CYP101 upon immobilization [20], considering that CYP101 is a soluble cytosolic enzyme, while CYP2D6 is a peripheral membrane protein, and the same interfacial electric field might have a very different effect on their respective active-site structures.\nBesides tentatively excluding the interfacial electric field intensity as a cause, SERRS data alone cannot support any explanation for the CYP2D6 potential-induced inactivation. Additional detailed structural data on the secondary and tertiary structures of the adsorbed protein and on the nature and position of the amino acid residues interacting with the MUA monolayer would be highly valuable, and they might be retrieved in further studies using SERRS in combination with surface-enhanced infrared absorption spectroscopy [63] as well as other techniques [64\u201366].\nConclusions\nUpon immobilization on MUA-coated Ag electrodes, the human enzyme CYP2D6 retains its active-site structure, its ability to reversibly bind a substrate (DX) and an exogenous heme ligand (imidazole), and its capacity to exchange electrons, demonstrating the efficiency of the coating to prevent major protein conformational changes or denaturation. However, despite its success in preserving an intact protein structure, the MUA coating appears to be unsuitable for performing direct electrochemistry experiments on immobilized CYP2D6, since its reduced state could not be adequately achieved by varying the electrode potential. The application of potentials from \u22120.4 to \u22121.0\u00a0V appears to induce enzyme inactivation instead. Among other factors influencing the redox activity, a more favorable enzyme orientation or distance with respect to the electrode surface might be achieved using other coatings, an approach currently under study in our group.\nIn general, SERRS spectroscopy on coated Ag surfaces was proven to be successful in monitoring the active-site structure of immobilized CYP2D6, enabling the detection of reversible substrate and ligand binding using only very low amounts of protein. Besides its technological relevance as an optical biosensing tool, this technique might have a significant potential for fundamental research on the mechanisms of catalysis for CYPs, as well as for other heme enzymes.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material. \nSupplementary Material (PDF 58 kb)","keyphrases":["ag electrodes","surface-enhanced resonance raman scattering","cytochrome p450 2d6","enzyme immobilization"],"prmu":["P","P","P","P"]}
{"id":"Naturwissenschaften-4-1-2270370","title":"Non-breeding habitat preference affects ecological speciation in migratory waders\n","text":"Models of ecological speciation predict that certain types of habitat should be more conducive to species diversification than others. In this study, I test this hypothesis in waders of the sub-order Charadrii using the number of morphological sub-species per species as an index of diversity. I classified all members of this clade as spending the non-breeding season either coastally or inland and argue that these represent fundamentally different environments. Coastal mudflats are characterised by high predictability and patchy worldwide distribution, whilst inland wetlands are widespread but unpredictable. The results show that migratory species that winter coastally are sub-divided into more sub-species than those that winter inland. This was not the case for non-migratory species. I argue that coastal environments select for more rigid migratory pathways, whilst inland wetlands favour more flexible movement patterns. Population sub-division could then result from the passive segregation of breeding sites or from the active selection for assortative mating of ecomorphs.\nIntroduction\nModels of ecological speciation show that the propensity of a species to diverge is affected by the shape of the fitness landscape generated by its environment (Gavrilets 2004). This predicts that some environments should be more conducive to speciation than others. Recent comparative studies have supported this prediction (Funk et al. 2006; Phillimore et al. 2007). However, these studies do not discuss aspects of habitats that promote or inhibit speciation.\nIn this study, I test this prediction for wading birds of the sub-order Charadrii. This group was chosen because its species can be divided into those that are adapted to coastal habitats and those that feed mostly inland (Piersma 1997, 2003). On a year-to-year basis, coastal habitats offer a more predictable environment than inland wetlands (Roshier et al. 2001). Inter-tidal mudflats and ocean beaches will be located in the same place each year and offer a highly predictable pool of food resources. They are distributed patchily around the globe, and patches differ in their food resources and other conditions (Piersma et al. 1993a). On the other hand, the inland wetlands favoured by other waders are unpredictable and include waterbodies in arid regions that flood infrequently. Flooded areas are often large, but may be located in different areas between years and shift within a single non-breeding season, promoting extensive movements of birds that rely on these habitats (Roshier et al. 2002). The suitability of grasslands in semi-arid regions varies from year to year due to the differences in rainfall level. Little is known about the composition of the food resources used by waders in these inland environments, but it seems likely that these may be similar on broad spatial scales. I thus predict that coastal habitats should be more favourable to species diversification than inland habitats.\nMany of the wader species that utilise coastal or inland habitats for the greater part of the year migrate to very different habitats, such Arctic tundras or boreal swamps, to breed. Whilst the reasons for this are beyond the scope of this study (but see Piersma 1997), it means that in these species reproduction is spatially separated from the feeding habitat to which they are adapted. Red knot Calidris canutus illustrate this point. For 10\u00a0months of the year, this species feeds on bivalves on mudflats around the world. The species has evolved a range of adaptations to feeding on bivalves, including a muscular stomach and sensitive bill tip (Piersma et al. 1993b, 1998). Nevertheless, it breeds exclusively on high Arctic tundras where feeding on abundant insect larvae and berries require few special adaptations. In such cases, local adaptation to, for example, temperate or tropical inter-tidal mudflats requires that birds from the same non-breeding area mate assortatively on the breeding grounds (Webster et al. 2002).\nInvestigating ecological speciation in this group of birds thus adds two new dimensions to the general pattern found by Funk et al. (2006) and Phillimore et al. (2007). First, as argued above, I have a priori reasons to expect that one type of habitat (coastal) should be more conducive to speciation than the other (inland). Second, many of the species investigated reproduce away from the feeding grounds to which they are adapted and speciation thus requires differential migration to achieve assortative mating. To test these ideas, I use the number of sub-species per species as a measure of diversification. Thus, I treat sub-species as \u2018incipient species\u2019. Morphological sub-species are considered useful in estimating the patterns of divergence among populations (Phillimore et al. 2007). Evidence that wader sub-species represent phylogenetically distinct groups is available for Dunlin Calidris alpina (Wenink et al. 1993), and to a lesser extent, for Red knot (Buehler and Baker 2005). Because older species could have differentiated into more sub-species than younger species, I investigated whether the number of sub-species was correlated to species age.\nMaterials and methods\nData collection\nI collated data on the number of sub-species, migratoriness, breeding and non-breeding habitat from Del Hoyo et al. (1996) for all 215 species of the sub-order Charadrii (see Table 2 of S1). Crude estimates of species age were obtained from Thomas et al. (2004). Due to high levels of polytomy in parts of the phylogeny, some of these are likely to be over-estimates; however, no better estimates are available at present. I adopted the number of sub-species from one reputable source rather than search for the more recent updates because some of these revisions are still controversial. Migratoriness was scored as non-migrant, partial migrant or migrant (>80%, between 20% and 80% and <20% of non-breeding range overlaps with breeding range, respectively). Breeding habitat was scored as one of seven classes: tundra, boreal\/temperate, mountains, steppe, (sub)tropical wetlands (either inland or coastal), (sub)tropical forest or oceanic. Non-breeding habitat was scored as coastal, inland or pelagic. For species that regularly use both coastal and inland habitats, I scored the habitat considered by Del Hoyo et al. (1996) to be used by the largest part of the population.\nStatistical analysis\nI first investigated whether the dependent variable (number of sub-species) showed phylogenetic auto-correlation (i.e. whether closely related species resembled each other more in their tendency to form sub-species than expected by chance) using the phylogenetic topology from Thomas et al. (2004). I used runs test as implemented in the programme Phylogenetic Independence (Abouheif 1999) to compare the observed pattern to that generated by randomly redistributing the data over the tips of the phylogeny 1,000 times. For this analysis, the number of sub-species per species was dichotomised as 1 (i.e. only the nominate form) or >1. Finding no evidence for phylogenetic autocorrelation (see the \u201cResults\u201d section), I treated species as independent data points in further analysis.\nAs the number of sub-species per species followed a Poisson distribution, I analysed the data using generalised linear models with a Poisson error distribution and a log link function. GLMStat (Beath 2001) was used to generate glms. Significance was tested by dropping each term from the fuller model and comparing the resulting change in deviance to a chi-square distribution.\nResults\nThe results of runs test showed that there was no evidence for auto-correlation in the phylogenetic distribution of the number of sub-species (runs test P\u2009=\u20090.46). In fact, 461 of the 1,000 randomly generated runs averages were greater than that of the real data set, indicating a non-significant tendency towards over-dispersion.\nA glm containing migratoriness, non-breeding habitat, breeding habitat and species age and their interactions showed no significant effects of three- and four-way interactions. However, there was a significant two-way interaction between migratoriness and non-breeding habitat (Table\u00a01), indicating that the pattern of covariance between non-breeding habitat and sub-species richness differed between migrants and non-migrants. Individual models revealed a significant effect of non-breeding habitat on the number of sub-species in migrants, but not in non-migrants or partial migrants (Table\u00a01). These results are illustrated in Fig.\u00a01: species wintering coastally are on average sub-divided into more sub-species than inland wintering species, but only in migrants. The results are not confounded by differences in species age (Table\u00a01). Whilst there was a significant association between breeding and non-breeding habitat among migrants (, P\u2009=\u20090.009), this did not result in an association between breeding habitat and the number of sub-species (Table\u00a01).\nFig.\u00a01Mean number of sub-species per species for migratory and non-migratory waders. Pelagic wintering species (N\u2009=\u20092) and partial migrants (N\u2009=\u200931) not shown. Error bars represent the Poisson standard errorsTable\u00a01Results of the generalised linear model with the number of sub-species per species as independent variable\u00a0Estimate\u2009\u00b1\u2009SEDeviance (df\u2009=\u20091)P valueMinimal adequate model\u00a0\u00a0\u00a0\u00a0Migratoriness\u22120.10\u2009\u00b1\u20090.08\u00a00.23\u00a0Non-breeding habitat\u22120.16\u2009\u00b1\u20090.19\u00a00.39\u00a0Breeding habitat0.06\u2009\u00b1\u20090.04\u00a00.17\u00a0Species age\u22120.01\u2009\u00b1\u20090.007\u00a00.11Migratoriness\u2009\u00d7\u2009non-breeding habitat0.27\u2009\u00b1\u20090.124.920.03Separate models\u00a0\u00a0\u00a0\u00a0Non-breeding habitat\u00a0\u00a0\u00a0\u00a0\u00a0Migrants0.42\u2009\u00b1\u20090.157.390.007\u00a0\u00a0Partial migrants0.13\u2009\u00b1\u20090.100.070.79\u00a0\u00a0Non-migrants\u22120.21\u2009\u00b1\u20090.211.070.30Non-significant interaction terms were removed. P values for the main effects were obtained by using the parameter estimate divided by its standard error as the test statistic. Also shown are the results of separate models for each category of migratoriness.\nDiscussion\nThe prediction that coastal habitats should be more conducive to diversification in waders than freshwater habitats is supported, but only for migratory species. This is remarkable, given that the reduction in gene flow between locally adapting populations requires an extra step in these species. In non-migrants, all that is needed to mate with a locally adapted partner is not to disperse. In migrants, however, all individuals migrate away from the non-breeding grounds to breed, often to the other end of the globe. To mate with a partner from the same non-breeding grounds, the non-breeding populations must either have segregated breeding grounds or have a very sophisticated individual recognition system. There is good evidence for a number of coastally wintering migrants that the former is true (Wennerberg 2001; Atkinson et al. 2005; Buehler et al. 2006), whilst the latter has not been investigated.\nI suggest that the predictability of coastal non-breeding grounds favours rigid migratory pathways to and from the non-breeding grounds. Returning to the same non-breeding ground each year would save individuals from wasting time on searching for suitable non-breeding areas and allow them to benefit from learned aspects of local conditions (such as food distribution). The rigidity of the migratory pathways could then result in spatial segregation on the breeding grounds because individuals would be genetically predisposed to favour the same breeding grounds each year. The formation of distinct sub-species would then be a by-product of the spatially discontinuous distribution of non-breeding grounds. By contrast, many of the species that spend the non-breeding season on inland habitats have to track spatially and temporally variable resources (Roshier et al. 2001, 2002), which should select for flexible movement patterns during the non-breeding season. This could translate into flexible migration patterns, perhaps through a genetic correlation between migratory and non-migratory movements, and flexible movement patterns on the breeding grounds. If true, this would promote gene flow and inhibit diversification.\nAlternatively, the divergent ecology of different coastal non-breeding grounds may actively select for assortative mating of corresponding ecomorphs. For example, it may be costly for waders wintering on tropical mudflats to pair with those that winter on temperate mudflats because the resultant hybrids would be poorly adapted to either habitat. Support for this interpretation comes from the fact that many sub-species of coastal waders differ from each other in bill length, which is an ecologically relevant trait (Engelmoer and Roselaar 1998). By contrast, the unpredictability of inland wetlands would not allow specialisation to the specific conditions presented by any particular area of habitat.\nThe question remains why the difference in diversification between species occupying coastal and inland habitats is not reflected in the non-migratory species that breed there? Given that migrant species often greatly outnumber resident species at individual feeding areas during the non-breeding season, migrant species may have greater effective population sizes per non-breeding area than non-migrants. This would allow more efficient response to selection and increase the potential for local adaptive change in migrants relative to non-migrants. Given the arguments above, such change is most likely in coastal areas.","keyphrases":["non-breeding habitat","speciation","waders","sub-species"],"prmu":["P","P","P","P"]}
{"id":"Ann_Hematol-4-1-2324127","title":"Simultaneous expression of Oct4 and genes of three germ layers in single cell-derived multipotent adult progenitor cells\n","text":"Future application of adult stem cells in clinical therapies largely depends on the successful isolation of homogeneous stem cells with high plasticity. Multipotent adult progenitor cells (MAPCs) are thought to be a more primitive stem cell population capable of extensive in vitro proliferation with no senescence or loss of differentiation capability. The present study was aimed to find a less complicated and more economical protocol for obtaining single cell-derived MAPCs and understand the molecule mechanism of multi-lineage differentiation of MAPCs. We successfully obtained a comparatively homogeneous population of MAPCs and confirmed that single cell-derived MAPCs were able to transcribe Oct4 and genes of three germ layers simultaneously, and differentiate into multiple lineages. Our observations suggest that single cell-derived MAPCs under appropriate circumstances could maintain not only characteristics of stem cells but multi-lineage differentiation potential through quantitative modulation of corresponding regulating gene expression, rather than switching on expression of specific genes.\nIntroduction\nBone marrow mesenchymal stem cells (MSCs) are the most-studied adult stem cells and have been widely used owing to their advantages in autografting and multi-lineage differentiation potential, which is also referred to as the plasticity of stem cells [1\u20134]. However, some studies in recent years indicated that MSCs might have no plasticity or only have a limited plasticity [5, 6] and that the in vivo multi-lineage differentiation of MSCs might be a result of their fusion with the host cells [7, 8]. That is why, recently, people have been suspicious about the plasticity of MSCs. Some researchers think that the previously separated MSCs are actually a mixed cell population containing several kinds of stem cells with different differentiation potentials [9]. Therefore, how to isolate homogeneous MSCs with high plasticity becomes a ready task of researchers.\nIsolation of MAPCs from adult bone marrows was firstly reported by Verfaillie\u2019s team [10\u201312]. Compared with MSCs, these cells were thought to be a more primitive cell population with partially overlapping ontogeny. Moreover, MAPCs have been proven to be capable of extensive in vitro proliferation with no senescence or loss of differentiation capability. A few researchers held that when embryonic stem cells differentiate into different somatic germ layers and tissue cells, some pluripotent cells remain in specific tissues for future use [13]; judging from their differentiation property, MAPCs should belong to this type of cell. Although the above hypothesis needs to be further verified, this subpopulation of pluripotent stem cells provides a new thought and may serve as a new cell source for tissue engineering.\nBased on the above understanding, we started our experiment with isolation of MAPCs from bone marrow of adult rats and then fully studied their differentiation potentials. MAPCs were isolated using a modified protocol of Verfaillie\u2019 team [10]. By screening for cells highly expressing Oct4 from clone-like clusters, we successfully obtained a comparatively homogeneous population of MAPCs with multi-lineage differentiation potential. The present study was aimed to find a less complicated and more economical protocol for obtaining single cell-derived MAPCs and understand the molecule mechanism of multi-lineage differentiation of MAPCs. Our observations confirmed that single cell-derived MAPCs were able to transcribe Oct4 and genes of three germ layers simultaneously, and differentiate into multiple lineages.\nMaterials and methods\nCulture and clone expansion of MAPCs\nBone marrow was obtained from 4-week-old male Sprague\u2013Dawley (SD) rats. With fat and red blood cells removed, the remaining cells were seeded at a density of 1\u2009\u00d7\u2009106 cells\/cm2 in 100\u00a0mm dishes coated with 10\u00a0ng\/ml fibronectin (FN; Sigma-Aldrich, USA) and incubated at 37\u00b0C with 5% CO2 in a fully humidified atmosphere. The expansion medium of MAPCs consisted of 60% DMEM-LG (Gibco-Invitrogen, USA) and 40% MCDB-201 (Sigma), with 2% fetal bovine serum (FBS; Gibco-Invitrogen), 1\u00d7 insulin\/transferring\/selenium (ITS), 1\u00d7 linoleic acid bovine serum albumin (LA-BSA), 10\u22129M dexamethasone (Dex), 10\u22124M ascorbic acid 2-phosphate (all from Sigma-Aldrich), 100\u00a0U\/ml penicillin, 1,000\u00a0U\/ml streptomycin (Gibco-Invitrogen), 10\u00a0ng\/ml EGF (Sigma-Aldrich), 10\u00a0ng\/ml PDGF-BB (R&D Systems), and 10\u00a0ng\/ml LIF (Chemicon International, USA). The nonadherent cells were removed by medium change at 24\u00a0h and every 3\u00a0days thereafter until 100% confluence was reached. Cells were passaged once with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA; Gibco-Invitrogen) and the passage 1 (P1) cells were replated at a 1:2 dilution under the same culture condition. After being passaged for at least four generations (P4), cells were seeded on a 96-well plate coated with 10\u00a0ng\/ml FN at a density of 0.5 cell\/well by limited-dilution method in expansion medium. The wells containing more than one cell under a phase-contrast microscope were discarded. When cells grew to 40\u201350% confluence, they were pipetted from one well, seeded to one of the 24 wells, and then serially reseeded to a six-well plate, 60\u00a0mm dish, and 100\u00a0mm dish in the presence of 0.25% trypsin-EDTA. When cells reached a density of 4\u20138\u2009\u00d7\u2009103 cells\/cm2 in 100\u00a0mm dish, they were replated at a 1:3 dilution, and the fastest-growing clone was selected after being passaged for 20 times.\nTotal RNA isolation and RT-PCR analysis\nTotal RNA was extracted from the clonal cells using RNA Mini kit (Huashun, China). First-strand cDNA was synthesized with total RNA using a mixture of oligo (dT)12\u201318 and random hexamers (Roche Diagnostics, Germany) primers with AMV (Promega, USA) and Superscript II RNase H Reverse Transcriptase (Invitrogen, Germany). Polymerase chain reaction (PCR) was conducted with approximately 50\u00a0ng cDNA for amplification of the following marker genes: pluripotent marker Oct-4, endodermal marker lanosterol-14a-demethylase (CYP 51), mesodermal marker SM22\u03b1 and GATA-4, and ectodermal marker N-methyl-d-aspartic acid (NMDA) glutamate binding protein. The housekeeping gene encoding 18S was used as an internal control. The PCR primers are listed in Table\u00a01. After initial denaturation at 98\u00b0C for 3\u00a0min, PCR amplification was performed with Sawady Taq-DNAPolymerase (Takara Bio, Japan) at 95\u00b0C for 40\u00a0s followed by 28\u201335 cycles, with each cycle consisting of annealing at 55\u201360\u00b0C for 40\u00a0s, and a final extension at 72\u00b0C for 40\u00a0s. PCR products were subjected to 1.5% agarose gel electrophoresis, and the bands were visualized by ethidium bromide and photographed with Chemi Doc XRS (Bio-Rad Laboratories, Fudan, China). Each experiment was performed in triplicate.\nTable\u00a01Specific marker genes of three germ layers [14\u201316]GenesPrimers(5\u2019-3\u2019)Product size(bp)LocationLayerOct-4Forward: CGCCAGAAGGGGAAAAGA Reverse: CAGGAAAAGGGACCGAGTAG176\u00a0\u00a0CYP 51Forward:ATCCTGACCGCTACCTACA Reverse: GGCTTCCCTGAAATCCTA461LiverEndodermSM22\u03b1Forward:GTCCACAAACGACCAAGC Reverse: GTTCTCAGGCACCTTCACT279MuscleMesodermNMDAForward: TGAGCCCACCAGAAAAGG Reverse: ACTGCTTTGCCCTCCACC625BrainEctodermGATA-4Forward: TCCCCACAAGGCTATCCA Reverse: CCGAAGAAGGTCACGAGGT321HeartMesoderm18SForward: CAAGAACGAAAGTCGGAGGTTCG Reverse: TTATTGCTCAATCTCGGGTGGCTG460\u00a0\u00a0\nImmunocytofluorescence and fluorescence-activated cell sorter analysis\nClonal cultured MAPCs were fixed with 4% paraformaldehyde for 4\u00a0min at room temperature. After being blocked with phosphate-buffered saline (PBS) containing 2% BSA, cells were permeabilized with 0.1%Triton-X100 for 10\u00a0min. Slides were incubated sequentially overnight at 4\u00b0C with the following primary antibodies: CD71 (1:50, Santa Cruz, USA), Vimentin (1:200, DAKO, USA), \u03b1-SMA (1:200, Boster Biotechnology, China), and SSEA-1(1:50, Santa Cruz, USA); then cells were incubated for 40\u00a0min at room temperature with fluorescein isothiocyanate (FITC) or Cy3-coupled IgG secondary antibody (1:500, Jackson), stained with DAPI (Sigma\u2013Aldrich), and observed under a fluorescence microscope (BX41TB, Olympus, Japan).\nFor fluorescence-activated cell sorter (FACS) analysis, cells pelleted were incubated on ice for 30\u00a0min with FITC- or PE-conjugated monoclonal antibodies against CD34, CD44, CD45 (Becton\u2013Dickinson, CA), and MHC-I (Abcam, UK). After two washes with cold PBS, the labeled cells were analyzed by a FACStar flow cytometer (Becton Dickinson, CA, USA).\nIn vitro differentiation of MAPCs\nTo induce differentiation, clonal cultured MAPCs were cultured in the media containing no FCS, EGF, PDGF-BB, or LIF unless otherwise indicated. All the experiments were repeated at least three times.\nAdipogenic differentiation of MAPCs MAPCs were seeded at a density of 3\u2009\u00d7\u2009104 cells\/cm2 in six-well plates, allowed to grow confluence, and cultured for another 3\u00a0days. Subsequently, cells were allowed to differentiate for 14\u00a0days in the expansion medium of MAPCs containing 0.5\u00a0mM 3-isobutyl-1-methylxanthine (Serva Electrophoresis, Germany), 100\u00a0nM Dex, 60\u00a0\u03bcM indomethacin, and 10\u00a0\u03bcg\/ml insulin. At the end of the culture, cells were fixed with 10% formaldehyde (Shenggong, China) for 10\u00a0min and were stained with fresh Oil Red O (Sigma-Aldrich) for 2\u00a0h.\nOsteogenic differentiation of MAPCs MAPCs, at a density of 2\u2009\u00d7\u2009104 cells\/cm2, were induced with osteogenic medium containing 10\u20138M Dex, 2\u2009\u00d7\u200910\u22124\u00a0M ascorbic acid and 7\u2009\u00d7\u200910\u22123\u00a0M glycerolphosphate for 2\u00a0weeks. NBT\/BCIP staining was used to analyze alkaline phosphatase (AKP; all from Sigma-Aldrich) expression during MAPCs differentiation. Meanwhile, cells were immunocytochemically stained with osteopontin to detect osteogenic differentiation of MAPCs.\nNeuroectodermal differentiation of MAPCs MAPCs were plated at a density of 1\u2009\u00d7\u2009104 cells\/cm2 on FN in a basal medium containing 100\u00a0ng\/ml basic fibroblast growth factor (bFGF; R&D Systems). The cells were allowed to differentiate for 14\u00a0days and then immunofluorescently stained for GFAP (1:100) and TAU (1:100, both from Boster Biotechnology, China).\nHepatocyte differentiation of MAPCs MAPCs were plated at 2.2\u2009\u00d7\u2009104 cells\/cm2 on plastic chamber slides coated with 2% Matrigel in a basal medium supplemented with 10\u00a0ng\/ml FGF-4 and 20\u00a0ng\/ml HGF (both from R&D Systems). After 14\u00a0days, immunofluorescent staining for albumin (1:500, DAKO, USA) was performed as described above.\nResults\nStable in vitro proliferation and expansion of clonal cultured MAPCs\nOn the third day of primary culture, very few triangle-like cells adhered to the culture surface under a phase contrast microscope, coexisting with plenty of hematopoietic cells, which were round in shape and small in size, shining under the microscope (Fig.\u00a01a, P0-d3). During the fourth to seventh day of culture, the triangle-like cells gradually increased and had a typical colony growth, with an obvious three-dimensional appearance and growth circle. The colony cells grew confluent and became morphologically homogenous after 7\u201310\u00a0days (Fig.\u00a01a, P0-d10). After treatment with trypsin, the cultured cells no longer grew in clusters, but in a homogeneous manner (Fig.\u00a01a, P4-d3).\nFig.\u00a01Morphology of isolated MAPCs. a Phase-contrast micrographs of MAPCs at day \u00a03 (d3), day\u00a010 (d10) of primary culture (P0) and P4 at day\u00a03 (d3) after passages (original magnification 100\u00d7). b MAPCs cloned by limiting dilution. A single cell adherent to the plate in one well proliferated in the primary culture for 2\u00a0days (D2), 4\u00a0days (D4), and 8\u00a0days (D8; original magnification \u00d7400)\nSingle-cell clonal culture was performed by infinite dilution technique. Thirty percent of single cell-derived MAPCs formed larger clones during culture (Fig.\u00a01b), and six cell clones (7F#, 4G#, 6E#, 4F#, 7C#, and 10D#) were obtained in the following expansion. When being cultured at an extremely low density (250 cells\/cm2), this six-cell population still demonstrated strong capability of proliferation, with the doubling time being about 24\u201336\u00a0h. P20 cells still possessed the typical properties of MAPCs: polygonal-shaped, with larger nuclei, vacuoles around the nuclei, and sparse cytoplasm (Fig.\u00a01b, D2, D4).\nExpression of Oct4 and genes of three germ layers in single cell-derived MAPCs\nThe expression of self-renewal gene Oct4 was detected in the six cell clones after being passaged for more than 20 times. All the six cell clones were found to have different levels of Oct4 expression (Fig.\u00a02a). Specific markers of the three germ layers were detected in the three cell clones (7F#, 4G#, and 10D#), which strongly expressed Oct4. The specific markers included CYP51 (present in the liver of adults) for endoderm, SM22\u03b1 and GATA4 (present in the skeleton muscle the heart of adults) for mesoderm, and NMDA (present in the brain of adults) for neuroectoderm. As for the markers for the three germ layers, 4G# clone had a very weak expression of CYP51 and all the rest clones had high expression of their corresponding markers (Fig.\u00a02b), especially for single cell-derived MAPCs-10D#. Therefore, we subjected single cell-derived MAPCs-10D# to further experiments.\nFig.\u00a02RT-PCR analysis of expression of Oct-4 and specific markers for three somatic germ layers. a Oct-4 was highly expressed in six cell clones. b CYP 51, SM22\u03b1, GATA4, and NMDA were positive in three cell clones. NEG Negative controls, POS positive controls for CYP 51 (fetal liver), SM22\u03b1 (fetal smooth muscles), GATA4 (fetal heart), and NMDA and Oct-4 (fetal brain tissues; n\u2009=\u20093)\nIdentification of molecular markers in single cell-derived P20 MAPCs-10D#\nSingle cell-derived P20 MAPCs-10D# was chosen for immunofluorescence assay. It was found that it expressed CD71 (a surface antigen of stem cells), Vimentin (a stromal cell skeleton protein), \u03b1-SMA (a marker of early smooth muscle cells), and SSEA-1 (a marker for more primitive stem cells; Fig.\u00a03a), suggesting that it still harbored the surface antigen of stem cells, the stromal cell skeleton protein, and the marker of early smooth muscle cells.\nFig.\u00a03a Expression of cell markers in the cloned MAPCs. Immunofluorescence analyses of single cell-derived P20 MAPCs-10D# were positive for CD71, Vimentin (with nuclear counterstained by Hochest33342), \u03b1-SMA and SSEA-1 (with nuclear counterstained by DAPI, all original magnification \u00d7200). b FACS analysis: P4 MAPCs were negative for CD34, CD45, CD44, and MHC-I antigen\nThe results of flow cytometry showed that 10D# clone did not express CD45 or CD34 (markers for hematopoietic stem cells). For the expression of CD44, the stromal cell marker, and MHC I, the major histocompatibility complex class I antigens, MAPCs were all negative (Fig.\u00a03b).\nDifferentiation potential of single cell-derived P20 MAPCs-10D# into three somatic germ layers consisting of osteoblasts, adipocytes, neurons, and hepatocytes\nSingle cell-derived P20 MAPCs were seen to have strong refractive oil drops around the nuclei on as early as the eighth day under the adipogenic condition, and the oil drops gradually increased and fused into larger ones with the prolongation of the induction time (Fig.\u00a04a, 1). Twenty-one days after induction with osteoblastic medium, AKP staining of MAPCs showed purple-bluish deposits in the cytoplasm (Fig.\u00a04a, 2), and immunocytochemistry staining was positive for osteopontin (Fig.\u00a04a, 3). Fourteen days after MAPCs were directed to differentiate into neuronal cells in vitro, cell morphology underwent obvious changes: the cytoplasm was shrinking, and bipolar or multipolar processes were seen growing out from the spindle-shaped cells (Fig.\u00a04b, 1). Immunofluorescence staining showed the presence of Tau protein, the specific marker of neurons (Fig.\u00a04b, 2), and GFAP, the specific marker of glia cells (Fig.\u00a04b, 3) in the cells after neurogenic induction. Fourteen days after MAPCs were programmed to differentiate into hepatocytes, the spindle-shaped cells (Fig.\u00a04c, 1) turned into round or oval shape and became wider and larger, with narrowed intercellular junctions and appearance of cell patches (Fig.\u00a04c, 2). Immunofluorescence analysis showed that the induced cells expressed albumin, the marker of hepatocytes (Fig.\u00a04c, 3). The above findings suggested that clonal cultured MAPCs were able to differentiate into osteoblasts, adipocytes, neuronal cells, and hepatocytes under proper culture conditions.\nFig.\u00a04Differentiation of cloned 10D# MAPCs into three somatic germ layers. a Differentiation into adipogenic-like cells and osteogenic-like cells. 1 differentiated MAPCs were positive in oil red O staining counterstained with hematoxylin; 2 and 3 differentiated MAPCs were positive for alkaline phosphatases (AKP staining) and osteopontin (immunocytochemical staining). b Differentiation into neuronal-like cells. 1 Under phase microscope, apparent branches were observed to grow out from the spindle-shaped cells. 2 and 3 Immunofluorescence staining showed the presence of GFAP and Tau protein. c Differentiation into hepatocytes-like cells.1 DAPI counterstaining showed that the MAPCs untreated with hepatogenic medium retained the spindle shape. 2 MAPCs turned from spindle into round or oval after hepatogenic induction. 3 Immunofluorescence analysis showed expression of albumin in differentiated MAPCs (all original magnification \u00d7200)\nDiscussion\nThere is increasing evidence that isolation strategies of stem cells differ for different source tissues and different stem cell types; even though the tissue is from the same source, different isolating protocols may obtain different stem cell types [17]. For example, stromal stem cells screened out with different molecular markers demonstrated certain varieties in growth and plasticity, and were therefore given different names, including MSCs [18\u201319], MAPCs [10\u201312], MPCs (mesodermal progenitor cells) [20], and marrow-isolated adult multi-lineage inducible cells (MIAMI) [21]. In the present study, we used culture conditions similar to those of Verfaillie\u2019s team and successfully obtained a comparatively homogeneous population of MAPCs through the following protocol: isolating mononuclear cells from rat total bone marrow by direct adherence, eliminating non-adherent cells after three to four passages, subjecting the remaining cells to single cell clonal culture by limiting dilution technique, plating the cells at a low density of 250 cells\/cm2 for further passage, and finally screening for clone-like cells. We found that single cell-derived MAPCs did not express CD34, CD45, CD44, and MHC-I, which was consistent with the results of Verfaillie\u2019s team, who found that MAPCs were able to reserve their pluripotency when plated at a low density, and unable to do so when plated at a much higher density. With pluripotency decreasing, CD44 and MHC I turned from negative to strongly positive in MAPCs, and therefore could serve as a marker for the loss of pluripotency. It should be notified that our protocol for MAPC isolation, which involved direct adherence, passage selection, single cell clonal culture and low density passage, is less complicated and more economical than that of Verfaillie\u2019s team, who employed the technique of immunomagnetic microbeads. The reliability of our protocol is verified by its results. Single cell-derived MAPCs retained their multi-potential differentiation capability even after being passaged for 20 generations, and could not only give rise to adipocytes and osteoblasts (mesoderm) but to neurons, glia cells (ectoderm), and hepatocytes (endoderm).\nIt has been demonstrated that the isolation method and cultivation condition are vital to the isolation of bone marrow stem cells and the maintenance of stem cell property [16]. The latest research showed that in vitro culture of human MSCs with autologous serum is superior to FBS for the amplification and for maintaining the pluripotency of MSCs [22], and the original culture strategy might lead to the loss of differentiation potential of MSCs. Moreover, some researchers found that serum-free culture could upgrade the differentiation potential of MSCs [23]. So recently, researchers have made persistent efforts to optimize the isolation method of MSCs. For example, people have isolated another population of pluripotent cells, called marrow-isolated adult multilineage inducible (MIAMI) cells, from human bone marrow by plating whole bone marrow cells initially in media containing 5% FBS and subsequently maintaining them in the medium containing 2% FBS in fibronectin-coated dishes. These cultures were then maintained in hypoxic conditions at 1,300\u20131,400 cells\/cm2 [20]. MIAMI cells still expressed telomerase, Oct-4, and Rex-1 even after being expanded for more than 50 population doublings. In the present study, our medium contained a low level of serum, but was supplemented with growth factor and LIF, which promoted the proliferation of MAPCs to some extent and retained their dedifferentiated state. Single cell clonal culture and low density passage may help to isolate a more homogenous cell population with strong proliferation capability.\nAs the marker for pluripotent stem cells, Oct-4 is closely linked with the pluripotency of MAPCs [24]. Our results showed that single cell-derived MAPCs not only highly expressed Oct-4 but expressed the early markers of endoderm, mesoderm, and ectoderm: CYP51, SM22\u03b1A [14], GATA4 [25], and NMDA [26], which is the molecular basis for their differentiation into three germ layers. Woodbury\u2019s team [16] demonstrated that MSCs expressed germline, ectodermal, endodermal, and mesodermal genes before neurogenesis. The present research extended their observations. Our observations confirm that single cell-derived MAPCs transcribe Oct4 and genes of the three germ layers simultaneously, and could differentiate into multiple lineages. It may be concluded that under appropriate circumstances, single cell-derived MAPCs can maintain not only characteristics of stem cells but multi-lineage differentiation potential which has expressed corresponding regulating genes, and single cell-derived MAPCs could differentiate into multiple lineages through quantitative modulation of regulating gene expression, rather than switching on expression of specific genes.","keyphrases":["multipotent adult progenitor cell","differentiation","pluripotency","oct 4"],"prmu":["P","P","P","M"]}
{"id":"J_Neurol-3-1-1915612","title":"Neck pain in chronic whiplash syndrome treated with botulinum toxin. A double-blind, placebo-controlled clinical trial\n","text":"Objectives Neck pain in chronic whiplash syndrome is a major burden for patients, healthcare providers and insurance companies. Randomized data on treatment of botulinum toxin in chronic whiplash syndrome are scarce. We conducted a randomized, placebo-controlled clinical trial to prove efficacy of botulinum toxin for neck pain in chronic whiplash syndrome.\nIntroduction\nIntractable chronic neck pain after a whiplash trauma is a major burden for patients, healthcare providers and insurance companies. It is estimated that 14\u201342% of patients develop chronic symptoms after an acute whiplash trauma [2].\nThese patients often have reduced mobility of the neck with tender muscles and increased muscle activity [25]. Pathophysiology is still unknown, but myofascial triggerpoints can develop in cervical muscles from excessive neck and shoulder muscle contraction due to an overt injury such as acceleration of the neck in a motor vehicle accident [5]. Botulinum toxin is used to treat a variety of neurological disorders associated with pathologically increased muscle tone.\nTwo previous studies have reported that botulinum toxin might be successful in patients with chronic neck pain following whiplash injuries [6, 12]. These studies were uncontrolled and the follow-up period was relatively short. In a randomized placebo- controlled trial involving 26 patients with chronic neck complaints after whiplash injury a significant improvement on total pain was suggested [7, 8].This study however has some methodological problems, such as a relatively short follow-up period of 4\u00a0weeks and a small sample size [7, 8]. Based upon the present evidence botulinum toxin cannot be recommended as standard treatment for chronic neck pain after whiplash injury.\nWe therefore conducted a double blind placebo-controlled randomized trial to confirm if botulinum toxin in treatment of neck pain is indeed effective in chronic whiplash syndrome.\nMethods\nPatients\nPatients were eligible if they had a whiplash type neck distortion defined as a soft tissue injury of the neck following a vehicle collision, with symptoms lasting longer than 6\u00a0months. They also met criteria of the Quebec Task Force grade 1 or 2 whiplash-associated disorders (WAD). WAD grade 1 means only neck pain and stiffness and grade 2 requires also musculoskeletal signs, e.g. a decreased range of motion [24]. Exclusion criteria were: age\u00a0<\u00a018; pregnancy; neuromuscular disorders; previous use of other investigational new drugs in the past 30\u00a0days prior to the screening visit; previous use of botulinum toxin; traumatic abnormalities on radiological examinations. The institutional ethics committee approved the study protocol, and patients gave informed consent before inclusion.\nStudy design\nThe trial had a double-blind, placebo-controlled parallel design. After patient\u2019s consent was obtained, we recorded demographics (age, gender). During a baseline-period of 4\u00a0weeks, patients used a diary to record the presence of neck pain, number of neck pain hours per day, number of days on which medication was taken, and number of tablets taken per day. At the end of this baseline-period (week 0) patients recorded their mean neck pain intensity scored on 100\u00a0mm Visual Analogue Scale of the previous 4\u00a0weeks (week \u20134 to 0, VAS-pre-treatment). At week 0, patients were randomly assigned to receive 100 units botulinum toxin (Botox\u00ae) or placebo (saline) in 2 cc syringes. At 12\u00a0weeks follow-up they recorded again their mean neck pain intensity of the previous 12\u00a0weeks (week 0 to 12, VAS-post-treatment). The local trial pharmacist prepared the drug, coded the syringes, and kept treatment codes. The pharmacist had no further participation in the trial. An experienced clinical neurophysiologist (D.T.) performed all injections. Injection sites were selected individually (i.e. \u2018follow the pain\u2019 approach) in muscles with clinically increased muscle tone or muscle tenderness, and included m. occipitofrontalis, m.temporalis, m. masseter, m. sternocleidomastoideus, m. splenius capitis, m. trapezius and m. semispinalis.\nOutcomes\nAfter patients were injected at week 0 in one single treatment-session, clinical characteristics were recorded using diaries during the 12\u00a0weeks post-treatment. The primary outcome measure was the intensity of the neck pain of the total study period, week 0\u201312, scored on the VAS at week 12 (one single measurement) compared with baseline (VAS week \u20134 to 0). Patients were asked to rate (in a single value) severity of neck pain which at times may be mild and perhaps at others severe, by \u201cintegrating severity over time\u201d.\nSecondary outcome measures were average number of neck pain days, average number of neck pain hours per day, average number of days on which symptomatic treatment was taken, mean number of symptomatic tablets per day at 12\u00a0weeks compared with baseline. Additionally, a blinded investigator asked patients whether there was any improvement in their condition after 4, 8 and 12\u00a0weeks in the two treatment groups (five-point scale: great worsening, any worsening, no improvement, any improvement and great improvement). Range of cervical motion was measured with a cervical range of motion (CROM) device to measure the six conventional movements of the spine at week 12 compared with baseline. For measurements of CROM, the patients were seated in a chair with their shoulders supported on a backrest. Patients were instructed to assume a comfortable position and then to perform each required movement and return to the start position. The movements were assessed in this order: right rotation, left rotation, right flexion, left flexion, extension, flexion and then summated to determine the mean total range of motion.\nWe also divided all patients in responders and non-responders at week 12. We considered at least 45% reduction in the VAS compared with baseline to be clinically relevant (e.g. responder). All adverse events during the 3\u00a0months follow-up were recorded by the investigators.\nSample size determination\nAt baseline we expected mean neck pain intensity on the VAS of 40\/100\u00a0mm. The estimated placebo effect was a 8\u00a0mm reduction to a VAS intensity of 32\/100 at 12\u00a0weeks. We hypothesized a 18\u00a0mm reduction in the treatment group to a VAS intensity of 22\/100, although there are no earlier data on treatment effect of BTX in these patients. To detect this clinically relevant effect (8 versus 18\u00a0mm improvement) at a 5% level of statistical significance (two-tailed), with a power of 80%, a total of 40 patients was needed.\nStatistical analysis\nWe calculated mean differences between baseline VAS data and VAS data after 12\u00a0weeks, using the t-test; statistical uncertainty was expressed in 95% confidence intervals. The responders at week 12 in each treatment group and the differences in improvement scores were compared by use of the Chi-square test. All these analysis were done with SPSS (version 10.0).\nResults\nBetween November 1999 and March 2003 40 patients were included at Leyenburg hospital The Hague. Of the 59 patients assessed for eligibility, 19 patients were excluded. Four patients did not meet the inclusion criteria, eight patients refused to participate and seven patients withdrew in the baseline period. No patient was lost to follow-up.\nIn 1 patient the neck pain diary was incomplete during the whole study period (missing data on neck pain duration, days on which medication was taken and mean number of tablets per day). In 7 patients the neck pain diary for the post-treatment period was partly incomplete (missing data on neck pain duration, days on which medication was taken and mean number of tablets per day). There were no differences in baseline characteristics between the two treatment groups. Neck pain intensity, VAS at baseline was 64.5\u00a0mm (SD\u00a0=\u00a014.8) in the botulinum group and 62.1 (SD\u00a0=\u00a020.3) in the placebo group (Table\u00a01).\nTable\u00a01Baseline characteristicsCharacteristicBotulinum toxin (n\u00a0=\u00a020)Placebo (n\u00a0=\u00a020)Mean age, years (range)39 (20\u201358)34 (20\u201349)Female sex (%)14 (70%)13 (65%)Ongoing claims15 (75%)18 (90%)Mean duration of pain, months (SD)40 (71)37 (20)Neck pain severity, mean VAS (SD)64.5 (14.8)62.1 (20.3)Mean number of neck pain days, % (SD)97% (11) *196% (12)Mean neck pain duration per day, hours (SD)12.7 (4.0) *311.7 (4.5)Mean number of medication days, % (SD)50% (41) *136% (40)Mean number of analgesics per day, tablets (SD)1.4 (1.9)1.3 (2.1)Mean total cervical range of motion, degrees (SD)266 (73) *1266 (60) *1*1\u00a0=\u00a0one missing data; *3\u00a0=\u00a0three missing data\nAt 12\u00a0weeks the VAS scores were 52.0\u00a0mm (SD\u00a0=\u00a029.2) in the botulinum group and 56.7\u00a0mm (SD\u00a0=\u00a029.6) in the placebo group. Mean differences between VAS pre- and post-treatment was 12.5 in the botulinum group and 5.5 in the placebo group. The therapeutic gain (\u00a0=\u00a0difference in improvement between the two treatment groups measured in mm) was \u20137.0 (p\u00a0=\u00a00.31; 95% CI (\u201320.7 to + 6.7)). Six patients in the botulinum group were responders as defined, versus four in the placebo group (NS, p\u00a0=\u00a00.5).\nPatients\u2019 assessment of improvement\/worsening showed no significant difference between the two groups at week 4 (p\u00a0=\u00a00.9), week 8 (p\u00a0=\u00a00.4) and 12 (p\u00a0=\u00a00.4) (Table\u00a02).\nTable\u00a02Improvement scaleImprovementAt 4\u00a0weeksAt 8\u00a0weeksAt 12\u00a0weeksBotulinum toxin (n\u00a0=\u00a020)Placebo (n\u00a0=\u00a020)Botulinum toxin (n\u00a0=\u00a020)Placebo (n\u00a0=\u00a020)Botulinum toxin (n\u00a0=\u00a020)Placebo (n\u00a0=\u00a020)Great worsening400212Any worsening241423No improvement688768Any improvement555454Great improvement236363Total19*12020202020*1\u00a0=\u00a0one missing data\nThere were no significant differences between the two treatment groups in mean number of neck pain days, mean number of neck pain hours per day, mean number of days on which medication was taken, and mean number of tablets taken per day pre- and post-treatment (Table\u00a03).\nTable\u00a03Secondary outcome measuresOutcomeBotulinum toxin (n\u00a0=\u00a020)Placebo (n\u00a0=\u00a020)Therapeutic gain (95% CI)Neck pain days (%\u00a0\u00b1\u00a0SD)\u00a0\u00a0Baseline (4\u00a0weeks)97% (11) *196% (12)\u00a0\u00a0Post-treatment (12\u00a0weeks)89% (23) *289% (25) *2\u00a0\u00a0Mean difference8% (18)7% (22)\u22121% (\u221215% to\u00a0+\u00a013%)Neck pain duration (hours\/day\u00a0\u00b1\u00a0SD)\u00a0\u00a0Baseline (4\u00a0weeks)12.7 (4.0) *311.7 (4.5)\u00a0\u00a0Post-treatment (12\u00a0weeks)12.2 (4.6) *711.0 (5.1) *2\u00a0\u00a0Mean difference0.86 (2.2)0.72 (4.7)\u22120.14 (\u22123.0 to\u00a0+\u00a02.7)Days on which analgesics were taken (%\u00a0\u00b1\u00a0SD)\u00a0\u00a0Baseline (4\u00a0weeks)49% (42%)35% (40%)\u00a0\u00a0Post-treatment (12\u00a0weeks)42% (38%) *324% (32%) *2\u00a0\u00a0Mean difference6.4% (16%)5.7% (24%)0.7% (\u221215% to\u00a0+\u00a013%)Number of analgesics taken per day (tablets\/day\u00a0\u00b1\u00a0SD)\u00a0\u00a0Baseline (4\u00a0weeks)1.4 (1.9) *11.3 (2.1)\u00a0\u00a0Post-treatment (12\u00a0weeks)1.2 (1.8) *20.95 (1.9) *1\u00a0\u00a0Mean difference0.32 (0.8)0.18 (0.6)\u22120.14 (\u22120.6 to\u00a0+\u00a00.4)Total cervical range of motion (degrees\u00a0\u00b1\u00a0SD)\u00a0\u00a0Baseline266 (73) *1266 (60) *1\u00a0\u00a0Post-treatment (week 12)293 (59) *2277 (62) *3\u00a0\u00a0Mean difference22 (43)11 (36)\u221211 (\u221240 to\u00a0+\u00a017)*1 = one missing data; *2\u00a0=\u00a0two missing data; *3\u00a0=\u00a0three missing data *7\u00a0=\u00a0seven missing data\nAt baseline all patients showed a reduced cervical range of motion (CROM) in all directions [9]. There was a slight improvement in total CROM in favour of the botulinum group. However, CROM was not significantly different between BTX group and placebo, nor between responders and non-responders.\nTwenty-seven patients reported minor side-effects, 15 in the botulinum toxin group and 12 in the placebo group. The main complaint was \u201cshort-lasting pain at the injection sites\u201d.\nDiscussion\nOur study could not demonstrate botulinum toxin to be more effective than placebo in treatment of neck pain in patients with chronic whiplash syndrome.\nPrevious open label studies with botulinum toxin in patients with chronic whiplash syndrome suggested some beneficial effect on neck pain [12]. Our study is the second published randomized-controlled trial to determine efficacy of botulinum toxin in patients with chronic whiplash syndrome. The other randomized placebo-controlled trial in 26 patients with chronic whiplash syndrome reported a beneficial effect of botulinum toxin [7, 8].\nThis study, however, has some methodological shortcomings. Primary outcome was improvement of headache intensity scored on the VAS after 4\u00a0weeks compared with baseline. The post treatment difference between the two treatment groups was reported to be significant. However, baseline-characteristics were significantly different, with a significant higher headache intensity in the botulinum group at baseline. (VAS 6.5 (range 2\u20139) versus 3.0 (0\u20138) in the placebo group (p\u00a0<\u00a00.01)). The reported improvement may mainly have been caused by the unequal distribution of the patients at baseline. In their second publication ( on the same trial) the mean total pain score was reported, ranging from 0\u201330, expressing the total pain experience for neck pain, headache and shoulder pain. After 4\u00a0weeks the mean total pain was 10.0 (SE 1.3) in the botulinum group and 14.1(SE 21) in the placebo group, which was also reported to be significant. These data are difficult to interpret without individual scores. Calculation of the standard deviation could suggest that some patients even had a negative mean total pain score. In any case, the distribution was truncated and skew to the right.\nThere are some points of discussion in our study. We used injections at trigger points on an individual basis, instead of following a standardized protocol. This might seem subjective and arbitrary, but resembles clinical practice and is usually recommended for botulinum toxin (BTX) treatment in pain syndromes [10, 16]. We used doses of 100\u00a0U Botox\u00ae, which should be sufficient for a single treatment session. There was only slight, not significant, improvement of cervical range of motion in the botulinum group, suggesting some, if any, effect on muscle tenderness. Also, none of our patients reported muscular weakness as a side effect. Therefore, further increase of the dosage of botulinum toxin might be possible. There is some evidence that multiple treatment sessions are needed for sufficient clinical benefit [20]. In our study there was not even a trend for a positive treatment effect after this single treatment session of BTX. Still, a positive effect of multiple treatment injections cannot be entirely ruled out [16].\nPathophysiology of chronic pain in whiplash syndrome is still a matter of debate. There is evidence that myofascial pain is a major factor in chronic pain syndromes like chronic neck pain and chronic tension-type headache [14]. The hypothesis of BTX being effective in chronic whiplash syndrome could be pain reduction by decreasing muscle tenderness and\/or activity. If increased muscle tenderness is a key feature in chronic neck pain after a whiplash injury, a more beneficial effect of BTX would have been expected. However, in other chronic pain syndromes where muscle tenderness is thought to play a role BTX was also proven ineffective [17, 23]. BTX might only be effective in neck pain in combination with cervical dystonia [26]. In our study were no patients with cervical dystonia.\nPatients with chronic whiplash syndrome also form a very heterogeneous group, with a wide spectrum of symptoms, varying from neck pain to headache, with pain of different degrees. Many different factors seem to play a role in the development of acute and chronic pain after a whiplash injury.\nThere is ample evidence that central mechanisms might play a more important role in the development of chronic neck pain in whiplash patients [4], as in other chronic pain syndromes. Damaged peripheral tissue releases inflammatory mediators (like bradykinine and prostaglandins) that changes the sensitivity of high-threshold nociceptors (peripheral sensitization). This, in turn, sets up changes in the responsiveness of neurons in the central nervous system (CNS), ultimately increasing the excitability of neurons within the CNS, representing central sensitization [21]. Patients with chronic whiplash syndrome display exaggerated pain after sensory stimulation as a consequence of this state of hyperexcitability of the central nociceptive system [1, 13]. In other words, an alteration in function of the central nervous system, the way it responds to normal inputs, seems present in these patients.\nThis central hypersensivity is probably independent of the nociceptive input arising from the painful and tender muscles [4], and possibly a consequence of deficient pain inhibitory systems.\nMany somatic, psychological or cultural factors may influence central sensitization. Different studies show that prognosis of chronic whiplash syndrome is also dependent on cultural factors [15, 18, 22]. It has even been shown that presence or absence of compensation claims is one of the most important predictors of bad outcome in patients after whiplash injury (also present in our group in 75\u201390% of the patients) [11, 19]. The etiological contribution of all different psychological factors, like for example coping style [3], to central sensitization is not yet clarified. It is not to be predicted that BTX will improve all different factors, especially psychological and cultural factors, which may contribute to development of this chronic whiplash syndrome.\nBased on present evidence BTX cannot be recommended as treatment for neck pain in chronic whiplash patients. Future studies directed on possible central mechanisms of this complicated chronic pain syndrome are warranted.\nContributors\nThe trial was designed by S.F.T.M. de Bruijn, D.L.J. Tavy and M. Padberg. Statistical analysis was performed by M. Padberg.\nConflict of interest statement\nNon declared.","keyphrases":["neck pain","chronic whiplash syndrome","botulinum toxin"],"prmu":["P","P","P"]}
{"id":"Eur_Arch_Otorhinolaryngol-3-1-2042030","title":"Re-irradiation of a second localization of idiopathic midline destructive disease in the head and neck area\n","text":"Idiopathic midline destructive disease is a rare disease, characterized by a progressive ulceration and destruction of midline facial structures. We report a case with localization on the palate for which she received radiotherapy. Later she developed a second localization on the posterior pharyngeal wall for which she was re-irradiated, without severe sequels. Twice a complete regression was observed.\nIntroduction\nIdiopathic midline destructive disease is a rare, which is characterized by a progressive ulceration and destruction of midline facial structures, including the nasal cavity, the paranasal sinuses and the palate. In most cases, radiotherapy is an effective treatment [1]. We describe a woman with twice a localization of idiopathic midline destructive disease, which were both treated with irradiation.\nCase report\nA woman, 46\u00a0years of age, was referred with complaints of nasal discharge while drinking. She was otherwise healthy and there was no history of substance use. Physical examination revealed an almost complete destruction of the membranous nasal septum and perforations of the soft and hard palate of 3 and 5\u00a0mm, respectively. Further examination of the head and neck region and general examination showed no abnormalities. Computed tomography showed extensive destruction of the hard palate. Biopsy showed a non-specific inflammation and fibrosis (Fig.\u00a01). There were no signs of Wegener\u2019s granulomatosis, malignant lymphoma, tuberculosis or Epstein-Barr virus infection. Serum tests showed negative results for anti-neutrophil cytoplasmic antibodies (ANCA) and syphilis. A diagnosis of idiopathic midline destructive disease was made.\nFig.\u00a01Histological picture of idiopathic midline destructive disease detail (40\u00d7 objective, Hematoxilin & Eosin stain) showing predominantly small irregular lymphocytes admixed with histiocytes and some granulocytes. The lymphoid cells do not appear obviously malignant. Low power view of biopsy (5\u00d7 objective, Hematoxilin & Eosin stain) show an ulcerative surface (left) and the infiltrate (right) with prominent vessels with swollen endothelial cells\nThe patient was then treated with radiation therapy to a total dose of 52\u00a0Gy in 26 fractions. At the end of the radiation course, a grade 1 mucositis and grade 1 erythema were observed. A complete regression of the disease occurred. After 4\u00a0years, the defect in the palate was closed surgically using a temporalis muscle flap.\nFour years later she developed a sore throat and swallowing complaints. On examination, a tumor-like lesion with a central ulcer was seen on the posterior pharyngeal wall. A CT-scan of the head and neck region showed a soft tissue mass on the posterior pharyngeal wall with extension from the level of the soft palate and nasopharynx to the level of the epiglottis. Biopsy showed the same histologic appearance as previously. The site of the original lesion was without any signs of abnormalities.\nIt was decided to treat this new localization with re-irradiation. During treatment, the patient experienced grade 1 erythema and grade 2 mucositis. She experienced swallowing complaints for 4\u00a0months. Complete regression occurred again.\nFour years after re-irradiation the patient presented again with progressive swallowing complaints and nasal speech. On physical examination, an ulcer was seen on the posterior pharyngeal wall with a diameter of 1-cm, which had a different clinical appearance than the former two lesions. Therefore, the differential diagnosis included a radiation-induced ulcer or recurrence midline destructive disease. Biopsy was not taken because it was most likely that pathological examination of this lesion could not distinguish between these two entities and because of an increased probability of wound-healing problems after biopsy in a highly irradiated area. We decided to treat this lesion as a radiation ulcer and the patient was proposed to undergo hyperbaric oxygen therapy, which she refused. Therefore, as an alternative, we decided to treat her with pentoxyfilline and high dose vitamin E, which she has used for one year. In the subsequent 12\u00a0months, the ulcer as well as her complaints gradually decreased.\nDuring the last follow-up visit (more than 5\u00a0years after re-irradiation), there were no signs of recurrent tumor or other severe radiation sequels, while a small ulcer still persists. Besides a mild xerostomia, she had no other complaints.\nDiscussion\nIdiopathic midline destructive disease (IMDD), also known as lethal midline granuloma (LMG), is a rare disease which is clinically characterized by a progressive, unrelenting ulceration and necrosis of the nasal cavity or the midline facial structures. This disorder has to be distinguished from other progressive, ulcerative disorders of the midline facial tissues, including cocaine use, sarcoidosis, infections like tuberculosis and syphilis, various neoplasms like vestibular or septal carcinoma, Wegener\u2019s granulomatosis, and polymorphic reticulosis (PMR) [2]. In contrast to all the other diseases, which have distinct histopathological features that distinguish them from IMDD, the histopathological findings of IMDD are nonspecific. It is essentially a diagnosis per exclusionem.\nSome authors believe that IMDD is an early phase of PMR [2]. Others state that all cases referred to as IMDD or PMR are in fact nasal T-cell (natural killer cell) lymphomas (NTL) [1, 3], with a possible association with the Epstein-Barr virus [4]. However, Barker and Hosni recently reported a case of IMDD in which no other cause could be diagnosed by current techniques and they proposed that IMDD remains a valid description in a small number of cases [5]. In our case the clinical course (with only localized disease) and histology (no atypia or Epstein-Barr virus in the tumor) argue strongly against lymphoma and for IMDD.\nUntreated IMDD is eventually fatal. No more than approximately 100 cases have been reported, of which only part is characterized as IMDD and the other cases as PMR or NTL. Therefore outcome parameters, such as survival and local control, are difficult to interpret. The preferred treatment of IMDD is radiation therapy to a dose of at least 50\u00a0Gy [1, 3]. A 5-year overall survival after radiation therapy has been reported from 15 to 75%. Local control varies between 63 and 77% [1\u20134]. Cases reported as NTL do worse compared to IMDD [1]. Some authors administer chemotherapy in case of NTL, although no difference in survival in patients with or without the addition of chemotherapy could be established [3].\nIn our case, a total dose of 52\u00a0Gy in 26 fractions was used which is generally considered to be sufficient. The margins taken during the first treatment were adequate according to the clinical tumor extension. Also because of the long time-interval, the second lesion can be considered to be a new localization of the disease.\nIt was decided to treat this new localization with re-irradiation for the following reasons. The largest part of the new localization was outside the initial treatment field implying that only part of the previously irradiated area would be treated again with the second course of radiotherapy. Second, the interval between the initial course of radiotherapy and relapse was more than 8\u00a0years and the total dose administered was 52\u00a0Gy, which may imply that a substantial amount of initial radiation damage to the normal tissues could have recovered [6\u20138].\nData on re-treatment of IMDD after local failure are scarce. Both Chen et al. [1] and Sakata et al. [3] mention a case with local relapse with successful re-irradiation without giving any detailed information. One patient with a local relapse of IMDD benefited from salvage chemotherapy [3], whereas two other patients, who were diagnosed as NTL, failed on a combination of re-irradiation and chemotherapy [1]; also on these patients no detailed information regarding radiotherapy was provided.\nIn conclusion, radiation therapy is the treatment of choice for idiopathic midline destructive disease. In case of a second localization in the irradiated area re-irradiation should be considered.","keyphrases":["idiopathic midline destructive disease","radiotherapy","midline granuloma"],"prmu":["P","P","P"]}
{"id":"Med_Biol_Eng_Comput-4-1-2329737","title":"Alterations in vasomotor control of coronary resistance vessels in remodelled myocardium of swine with a recent myocardial infarction\n","text":"The mechanism underlying the progressive deterioration of left ventricular (LV) dysfunction after myocardial infarction (MI) towards overt heart failure remains incompletely understood, but may involve impairments in coronary blood flow regulation within remodelled myocardium leading to intermittent myocardial ischemia. Blood flow to the remodelled myocardium is hampered as the coronary vasculature does not grow commensurate with the increase in LV mass and because extravascular compression of the coronary vasculature is increased. In addition to these factors, an increase in coronary vasomotor tone, secondary to neurohumoral activation and endothelial dysfunction, could also contribute to the impaired myocardial oxygen supply. Consequently, we explored, in a series of studies, the alterations in regulation of coronary resistance vessel tone in remodelled myocardium of swine with a 2 to 3-week-old MI. These studies indicate that myocardial oxygen balance is perturbed in remodelled myocardium, thereby forcing the myocardium to increase its oxygen extraction. These perturbations do not appear to be the result of blunted \u03b2-adrenergic or endothelial NO-mediated coronary vasodilator influences, and are opposed by an increased vasodilator influence through opening of KATP channels. Unexpectedly, we observed that despite increased circulating levels of noradrenaline, angiotensin II and endothelin-1, \u03b1-adrenergic tone remained negligible, while the coronary vasoconstrictor influences of endogenous endothelin and angiotensin II were virtually abolished. We conclude that, early after MI, perturbations in myocardial oxygen balance are observed in remodelled myocardium. However, adaptive alterations in coronary resistance vessel control, consisting of increased vasodilator influences in conjunction with blunted vasoconstrictor influences, act to minimize the impairments of myocardial oxygen balance.\nIntroduction\nHeart failure constitutes a major cardiovascular disorder of which the incidence and prevalence are increasing, principally due to an increased survival of acute myocardial infarction (MI) in conjunction with an ageing population. The mechanism underlying the progressive deterioration of left ventricular (LV) dysfunction towards overt heart failure remains incompletely understood, but may involve (1) loss of cardiomyocytes through apoptosis [75], (2) a primary reduction in contractile function of the surviving myocardium [97], and\/or (3) alterations in extracellular matrix leading to progressive LV dilation [87]. In addition, myocardial blood flow (MBF) abnormalities, resulting in impaired myocardial O2 delivery to the non-infarcted regions (leading to secondary contractile dysfunction and\/or enhanced apoptosis), have been suggested to contribute to the progression of LV dysfunction after MI [99]. For example, in vivo studies in rats [54, 55] and swine [108] indicate a reduction in MBF reserve of up to 35% in the surviving remodelled LV myocardium, 3\u20138 weeks after infarction. Furthermore, in patients with overt heart failure [94], but also in patients with only asymptomatic LV dysfunction [95], flow reserve is reduced in the non-stenotic myocardial regions. In line with these clinical observations, we observed in a porcine model of post-infarct remodelling that during increased O2-demand induced by exercise, the increase in coronary blood flow (CBF) is impaired resulting in perturbations in oxygen delivery [43, 70]. The reduction in flow reserve and the perturbed oxygen delivery during exercise are caused, at least in part, by insufficient growth of the coronary vasculature to maintain flow capacity commensurate with myocardial hypertrophy, in conjunction with a decrease in diastolic pressure time index resulting from the elevated heart rate and particularly elevated LV diastolic pressures [43]. In addition, coronary vasomotor tone may also be increased secondary to neurohumoral activation and endothelial dysfunction, further adding to the perturbations in CBF. However, little is known about the alterations in vasomotor control in coronary resistance vessels within remodelled myocardium (Fig.\u00a01). For this reason we undertook a series of studies to determine whether neurohumoral (autonomic nervous system and renin-angiotensin system), local metabolic, and endothelial control mechanisms of coronary resistance vessel tone are altered in swine with remodelled myocardium produced by a recent MI.\nFig.\u00a01Alterations in determinants of oxygen supply and demand in remodelled myocardium in swine with a 3-week-old myocardial infarction (MI). The net effect of these alterations is a decrease in oxygen supply\/demand ratio\nCharacteristics of LV remodelling and dysfunction after MI in swine\nLeft ventricular remodelling was produced by permanent ligation of the left circumflex coronary artery. This ligation results in a circumscribed transmural infarction of the lateral LV wall, comprising 20\u201325% of the total LV [82, 96]. LV dysfunction in awake resting MI swine is characterised by 20\u201330% decreases in cardiac output, stroke volume and LVdP\/dt\nmax and a tripling of LV filling pressure. This difference between LVdP\/dt\nmax, cardiac output and stroke volume in normal and MI swine remained constant between \u223c10 and \u223c32\u00a0days, indicating that the degree of LV dysfunction and the circulatory adaptations in MI were stable during this observation period [43]. Similarly, we observed that already during the first week after infarction significant LV remodelling occurs, consisting of LV dilation and hypertrophy that remain fairly stable between 1 and 6\u00a0weeks after infarction [98]. During exercise, cardiac output, LV systolic pressure and LVdP\/dt\nmax increased almost in parallel in MI and normal animals up to 3\u00a0km\/h, after which curves diverged (Fig.\u00a02); 4\u00a0km\/h was also the maximally attainable exercise level for most MI swine.\nFig.\u00a02Cardiovascular responses to exercise in normal swine and swine with a \u223c3-week-old MI. 0L\u00a0=\u00a0lying, 0S\u00a0=\u00a0standing. Data are mean\u00a0\u00b1\u00a0SEM; *P\u00a0<\u00a00.05 versus 0L, \u2020P\u00a0<\u00a00.05 MI versus normal a corresponding exercise level. Data are from Haitsma et al. [43]\nLeft ventricular dysfunction produced by MI results in neurohumoral activation, characterized by a trend towards elevated plasma levels of catecholamines, but normal circulating levels of renin, angiotensin II and aldosterone at rest. The latter may have been due to the increments in atrial natriuretic peptide (ANP) and endothelin, which can suppress renin and aldosterone release [83]. In resting MI swine, ANP doubled within 24\u00a0h, recovered to 50% above normal values within 2\u00a0weeks and remained stable between 2 and 6\u00a0weeks after infarction, while renin and norepinephrine levels remained normal under resting conditions [98]. In contrast to the discrete neurohumoral activation in resting swine with MI, exercise resulted in exaggerated increases in catecholamines and ANP and increases in endothelin, angiotensin II, and aldosterone (Fig.\u00a03). While resting circulating levels of norepinephrine were still normal and the relative sympathetic drive in response to exercise was preserved in MI, the cardiac responsiveness to exercise (both heart rate and LVdP\/dt\nmax) was already blunted 3\u00a0weeks after infarction [43], likely due to \u03b2-adrenoceptor desensitization and\/or downregulation [100].\nFig.\u00a03Neurohumoral responses in exercising swine with a \u223c3-week-old MI. In the norepinephrine, epinephrine and dopamine panels, data shown for N represent 0L, 1, 2, 3, 4 and 5\u00a0km\/h and data for MI represent 0L, 1, 2, 3 and 4\u00a0km\/h. In all other panels, data shown for N represent 0L, 1, 3 and 5\u00a0km\/h and data for MI represent 0L, 1, 3 and 4\u00a0km\/h. Mean data points were fitted with second order curves. VO2\u00a0=\u00a0O2-consumption. Data are mean\u00a0\u00b1\u00a0SEM; *P\u00a0<\u00a00.05 vs 0L, \u2020\nP\u00a0<\u00a00.05 MI versus Normal. Data are from Haitsma et al [43]\nMyocardial O2 balance in remodelled myocardium\nMarked decreases in myocardial perfusion occur in pacing-induced severe heart failure in swine [88] and dogs [85, 90], especially in the more vulnerable subendocardial layers. Although one study in dogs indicated that the lower MBF is principally the result of a lower myocardial O2 consumption (MVO2) [90], studies in swine suggest that the impaired perfusion is, at least in part, responsible for the deterioration of LV function because the interstitial edema and disruption of collagen fibers in the subendocardium resemble the ultrastuctural changes that occur with recurrent ischemia [49, 89]. In animal models of severe pressure-overload induced LV hypertrophy, selective underperfusion of the subendocardium can produce myocardial ischemia during exercise and result in post-exercise myocardial stunning [2, 102]. The contribution of perfusion abnormalities in the remote surviving myocardium to LV dysfunction after a MI remains unclear. Studies in rats demonstrated a 25\u201340% reduction in coronary flow reserve in the surviving myocardium at four [55] and eight [54] weeks after MI. Similarly, maximum subendocardial blood flow was blunted by 40% in anesthetized swine with heart failure 3\u00a0weeks after a MI [108]. We hypothesized that the decreased flow reserve could limit the increase in MBF to the hypertrophied myocardium during exercise when hemodynamic abnormalities and neurohumoral activation are exacerbated, thereby impairing myocardial O2-supply.\nThree weeks after infarction, blood flow per gram of myocardium in the (remote) LV anterior wall of resting swine with MI was similar to that in normal animals (Fig.\u00a04), confirming previous studies in rats and swine [54, 55, 108]. Interestingly, we observed a trend towards slightly higher blood flows in the outer two layers (P\u00a0=\u00a00.09), suggesting that despite hypertrophy of the surviving myocardium, metabolic demand in the outer, but not the inner, layers was still slightly elevated, 3\u00a0weeks after infarction [43]. During exercise, MBF increased but was redistributed in favor of the subepicardium in MI compared to normal swine. These perturbations were most likely due to increased extravascular compressive forces, resulting from a reduction in diastolic time fraction (secondary to impaired relaxation and increased heart rate) and elevated LV filling pressures [43, 97], that impede MBF, particularly in the subendocardial layers. The decreased MBF necessitated a small increase in O2 extraction that resulted in a slightly lower coronary venous O2 tension (Fig.\u00a04), which actually may have been underestimated as the lower myocardial capillary density in swine with MI possibly prevented a greater increase in O2 extraction [43, 70]. The observation that O2 extraction was forced to increase indicates that increases in extravascular forces are not fully compensated by a concomitant lowering of coronary vasomotor tone in remodelled myocardium during exercise. These observations prompted us to further investigate the control of coronary vasomotor tone in remodelled myocardium during exercise.\nFig.\u00a04Myocardial blood flow and O2-balance in the left ventricular anterior wall of N and MI \u223c3\u00a0weeks after myocardial infarction. Epi subepicardial, OM outer mid; IM inner mid; Endo subendocardial. MVO2\u00a0=\u00a0myocardial O2-consumption; MEO2\u00a0=\u00a0myocardial O2 extraction; CVPO2\u00a0=\u00a0coronary venous O2 tension In the top left panel data myocardial blood flow data are shown for resting (Rest, lying) conditions, and during maximum exercise (Ex, 5\u00a0km\/h in N and 4\u00a0km\/h in MI). Data are mean\u00a0\u00b1\u00a0SEM; *P\u00a0<\u00a00.05 versus 0L, \u2020\nP\u00a0<\u00a00.05 MI versus Normal. Data are from Haitsma et al [43]\nVasomotor control of the coronary microcirculation in remodelled myocardium\nNeurohumoral control\nCardiac dysfunction is accompanied by a hemodynamic defense reaction consisting of salt and water retention, peripheral vasoconstriction and cardiac stimulation, which serves to partially restore cardiac output and to increase systemic vascular resistance in order to maintain arterial pressure [56]. An integral part of this defense reaction involves alterations in autonomic balance, consisting of an increase in sympathetic activity and a decrease in parasympathetic activity [56].\nSympathetic control\nIn patients with advanced heart failure plasma noradrenaline levels are already increased under resting conditions [11, 36]. These increased levels result principally from increased sympathetic nerve activity although impaired reuptake may also contribute [56]. Prolonged exposure to elevated noradrenaline levels results in desensitization and downregulation of the \u03b2-adrenergic receptors [8, 32, 101]. During exercise, the increases in catecholamine levels are exaggerated in patients with heart failure as compared to healthy controls [35], which is aimed at maintaining chronotropic and inotropic responses to exercise [34].\nAlso in swine with LV dysfunction produced by a 2\u20133-week-old MI, we observed exaggerated increases in arterial and coronary venous catecholamine levels during treadmill exercise [23, 43], at a time when resting catecholamine levels were still in the normal range [43, 98]. The exaggerated exercise-induced increases in catecholamine levels reflect increased sympathetic activity, which acts to maintain the chronotropic and inotropic responses to acute exercise. This concept is supported by the observation that \u03b2-adrenoceptor blockade produced slightly greater decreases in the chronotropic response during exercise in MI as compared to normal swine. In contrast, \u03b2-adrenoceptor blockade in MI swine resulted in a smaller decrease in global LV contractility compared to normal swine, in particular during exercise. The latter findings are consistent with a reduced left ventricular myocardial \u03b2-adrenoceptor responsiveness [100].\nIn normal swine and dogs, \u03b2-adrenergic receptor activation contributes to coronary vasodilatation during exercise [27, 39]. The \u03b2-adrenergic coronary vasodilatation results in an increase in myocardial oxygen delivery that is commensurate with the increase in oxygen consumption, so that myocardial oxygen extraction and hence coronary venous O2 tension remain constant (Fig.\u00a05). In MI swine, the net \u03b2-adrenergic vasodilator influence on the coronary circulation is maintained. However, in view of the exaggerated increases in catecholamine levels during exercise, these findings suggest a diminished \u03b2-adrenergic responsiveness of the coronary resistance vessels after MI.\nFig.\u00a05Effect of saline, \u03b1-adrenoceptor blockade (phentolamine, 1\u00a0mg\/kg iv) and\/or \u03b2-adrenoceptor blockade (propranolol, 0.5\u00a0mg\/kg iv) and of muscarinic receptor blockade (atropine, 30\u00a0\u03bcg\/kg\/min, iv) and\/or \u03b2-adrenoceptor blockade (propranolol, 0.5\u00a0mg\/kg iv) on the response of coronary venous O2 tension (CVPO2) plotted as a function of myocardial O2 consumption (MVO2) in seven normal and 7 MI swine. *P\u00a0<\u00a00.05 versus corresponding Control; \u2020\nP\u00a0<\u00a00.05 Atropine\u00a0+\u00a0Propranolol versus corresponding Atropine or Phentolamine\u00a0+\u00a0Propranolol versus corresponding Phentolamine; Data are mean\u00a0\u00b1\u00a0SEM. Data are from Duncker et al. [23]\nIn dogs, \u03b1-adrenoceptor activation limits the exercise-induced increase in CBF, thereby necessitating an increase in myocardial O2 extraction, which leads to a decrease in coronary venous O2 tension [39, 51]. In contrast, \u03b1-adrenoceptors do not contribute to regulation of coronary blood flow in normal swine during exercise [27]. We found that, in accordance with our findings in normal swine, administration of the \u03b1-adrenoceptor blocker phentolamine had also no effect on coronary venous O2 tension of MI swine (Fig.\u00a05). These findings indicate that even in the presence of exaggerated increments in catecholamine levels in MI swine during exercise, \u03b1-adrenoceptors do not contribute to regulation of tone in porcine coronary resistance vessels [23].\nParasympathetic control\nThe shift in the sympathovagal balance, with an increased sympathetic activity [12, 33] and a blunted parasympathetic activity is reflected in reduced heart rate variability and reduced baroreceptor reflex sensitivity [6, 12, 30, 76] in patients with advanced heart failure. We observed that a maximal dose of the muscarinic receptor blocker atropine produced a similar increase in resting heart rate in swine with MI as compared to normal swine, suggesting preserved parasympathetic activity under resting conditions [23]. In contrast, the atropine-induced increase in heart rate during exercise (particularly at higher exercise levels) was blunted in MI swine, while the increase in LVdP\/dt\nmax was abolished [23]. These results are consistent with the concept that gradual inhibition of parasympathetic influence on the heart during exercise was more pronounced in swine with MI compared to normal swine. Importantly, these findings suggest that after MI, at a time when parasympathetic tone under basal resting conditions is normal, a more pronounced inhibition of parasympathetic tone occurs with increasing exercise intensity. Since parasympathetic activity can presynaptically modulate sympathetic activity [1], it is likely that the greater degree of withdrawal of parasympathetic tone during exercise contributed to the exaggerated increase in sympathetic activity during exercise. This is also supported by the observation that in the presence of propranolol, the effects of atropine were no longer different between MI and normal swine [23].\nIn resting dogs, parasympathetic activity exerts a direct vasodilator influence on coronary resistance vessels that is mediated via nitric oxide [109]. In contrast, in resting swine parasympathetic activity exerts an indirect vasoconstrictor effect on the coronary resistance vessels (which wanes at increasing exercise intensity) that is mediated via inhibition of \u03b2-adrenergic vasodilatation [27]. In contrast to the loss of the inhibitory influence of the parasympathetic system on \u03b2-adrenoceptor mediated cardiostimulation, we observed that its effects on the coronary circulation were maintained in MI compared to normal swine (Fig.\u00a05) [23]. These findings indicate that at this stage of LV dysfunction, parasympathetic control of \u03b2-adrenoceptor-mediated coronary vasodilatation is unimpaired.\nAngiotensin II\nThe renin-angiotensin system plays an important role in cardiovascular homeostasis by contributing to the regulation of blood volume, blood pressure and vascular tone. Angiotensin II (ANG-II) exerts its effects on vascular tone through binding to the AT1-receptor, resulting in vasoconstriction, as well as binding to the AT2-receptor, evoking vasodilation [14]. Both receptor subtypes have been identified in the coronary microcirculation [4, 107], suggesting that endogenous ANG-II may contribute to the regulation of coronary vascular tone and to the regulation of myocardial perfusion. Under pathological circumstances, i.e. after MI, the renin-angiotensin system is activated, resulting in increased plasma levels of ANG-II, particularly during exercise [34, 43]. Moreover, there is evidence that AT1-receptor density in the viable region of the myocardium is increased early after MI [62, 98], suggesting that its vasoconstrictor influence on the coronary vasculature could be increased, which may limit myocardial perfusion thereby exacerbating LV dysfunction.\nContrary to our hypothesis, we observed a loss of ANG-II induced vasoconstrictor influence, reflected by the lack of increase in coronary venous O2 tension (Fig.\u00a06), despite increased plasma ANG-II levels and maintained AT1 receptor densities in coronary arterioles isolated from remodelled myocardium of MI swine [68]. It is unlikely that a generalized loss of vasodilator capacity in the remote myocardium contributed to the blunted vasodilator response to the AT1 receptor blocker irbesartan, as we have previously shown that vasodilation produced by nitroprusside is unperturbed [44]. Although an increased AT2-receptor expression could have acted to limit ANG-II induced vasoconstriction [84] this is unlikely as AT2-mRNA was not altered in coronary arteries from patients with ischemic heart disease [103]. Moreover, the dramatic increases in ANG-II levels that we observed after irbesartan did not result in enhanced, but rather blunted, coronary vasodilation [68]. Therefore, the observation of a reduced vasoconstrictor influence of endogenous ANG-II is best explained by AT1-receptor-desensitization, which is in accordance with studies in dogs with pacing-induced heart failure [78], in rats with pressure-overload LV hypertrophy [63], and in rats with LV remodeling after MI [84], that demonstrated blunted vasoconstrictor responses to exogenous ANG-II.\nFig.\u00a06Effect of AT1-receptor blockade with irbesartan (1\u00a0mg\/kg iv) on the relation between myocardial O2 consumption (MVO2) and coronary venous O2 tension (CVPO2) in normal swine and swine with a 2\u20133-week-old myocardial infarction. Data are means\u00a0\u00b1\u00a0SE; *P\u00a0<\u00a00.05 versus corresponding control relation, \u2020\nP\u00a0<\u00a00.05 effect of irbesartan different in MI versus Normal animals. Data are from Merkus et al. [68]\nLocal metabolic control\nAdenosine\nAdenosine has been proposed to be a metabolic messenger that regulates coronary resistance vessel tone in response to changes in metabolic needs of the myocardium [5]. However, adenosine receptor blockade with 8PT and\/or augmenting adenosine catabolism with intracoronary adenosine deaminase had either no effect [3] or produced a small decrease [28, 67, 93] in basal coronary venous O2 tension (reflecting vasoconstriction), but did not interfere with the normal exercise-induced increase in CBF and O2 delivery [3, 28, 67, 93], indicating that adenosine is not critical for the exercise-induced coronary vasodilation or that loss of adenosine-mediated vasodilation can be compensated for by increased contribution of other vasodilator pathways to maintain adequate metabolic vasodilation.\nIn contrast to the lack of evidence for an essential role of adenosine in regulation of CBF under physiological conditions, endogenously released adenosine does contribute to coronary vasodilation when there is insufficient supply of O2 [61]. Similarly, adenosine production could be increased in the remodelled myocardium after MI as a result of the perturbations in the myocardial O2 balance [43, 70, 108]. However, we found no evidence for an increased contribution of adenosine to regulation of coronary resistance vessel tone in remodelled myocardium of swine with a recent MI [71], as adenosine receptor blockade with 8PT caused a similar decrease in coronary venous O2 tension in post-infarct remodelled hearts and normal hearts both at rest and during exercise (Fig.\u00a07). These findings are in agreement with observations in dogs with pressure-overload LV hypertrophy, in which adenosine receptor blockade did not affect CBF either at rest or during exercise [65].\nFig.\u00a07Effect of the adenosine receptor antagonist 8-phenyltheophylline (8PT, 5\u00a0mg\/kg iv), the KATP channel blocker glibenclamide (Glib, 3\u00a0mg\/kg iv) or their combination on myocardial O2 balance in the LV anterior free wall of normal swine and swine with a recent MI. MVO2\u00a0=\u00a0myocardial O2 consumption; CVPO2\u00a0=\u00a0coronary venous O2 tension; Data are mean\u00a0\u00b1\u00a0SEM; Data are mean\u00a0\u00b1\u00a0SEM; *P\u00a0\u2264\u00a00.05 versus corresponding control; \u2020\nP\u00a0\u2264\u00a00.05 effect of Glib was blunted at higher MVO2 levels (Glib\u00a0\u00d7\u00a0MVO2); \u2021\nP\u00a0\u2264\u00a00.05 effect of Glib different after MI (Glib\u00a0\u00d7\u00a0MVO2\u00a0\u00d7\u00a0MI). Data are from Merkus et al. [71]\nSeveral reasons could be forwarded for the failure to observe a larger contribution of adenosine to regulation of coronary resistance vessel tone. First, it is possible that the perturbations in the myocardial O2 balance were too mild to increase adenosine production. Second, the activity of enzymes that regulate tissue adenosine levels may have been altered [16, 17]. For example, the activity of adenosine deaminase, the enzyme responsible for breakdown of adenosine to inosine, was found to be elevated in LV hypertrophy [10, 13, 15]. Furthermore, there is evidence that the activity of cytosolic 5\u2032 nucleotidase, which converts 5\u2032AMP to adenosine, is lower in certain models of pressure-overload [13] and volume-overload [10] induced LV hypertrophy, which could be related to intermittent hypoperfusion of the hypertrophic myocardium [41]. Together these enzymatic alterations, which act to decrease myocardial levels of adenosine may have prevented a significant increase in myocardial adenosine levels in the post MI remodelled hearts.\nFinally, it is possible that an increased role of adenosine in hypertrophied myocardium is masked by an increased contribution of other vasodilator systems during adenosine receptor blockade, as the process of metabolic vasodilation is thought to be mediated through multiple parallel or redundant pathways. Thus, KATP channel activity may have increased in response to adenosine receptor blockade to compensate for the loss of adenosine-mediated vasodilation. Hence, we evaluated the interactions between these vasodilator pathways.\nKATP channels\nIn addition to the role of KATP channels in the regulation of CBF under physiological conditions [24, 25, 31], there is evidence for an increased KATP channel activity in the coronary circulation of remodelled hearts. For example, in anesthetized dogs with pacing-induced severe heart failure [106], the KATP channel blocker glibenclamide resulted in an exaggerated vasoconstrictor response as compared to normal dogs. Interestingly, in dogs subjected to only 1\u00a0week of pacing (when LV function was still normal), KATP channel activity was not different from normal dogs [106], suggesting that KATP channel activity in the basal resting state was only enhanced in the presence of overt heart failure. Similarly, in awake dogs with compensated pressure-overload induced LV hypertrophy, the reduction in CBF produced by glibenclamide was similar to that in normal dogs [65]. During exercise, however, glibenclamide produced a greater reduction in CBF in hypertrophied hearts, indicating increased KATP-channel contribution to coronary vasodilation when O2 requirements of the hypertrophied heart were augmented [65].\nIn swine with MI-induced moderate LV remodeling and dysfunction, glibenclamide caused a marked decrease in coronary venous O2 tension in remodelled left ventricle under resting conditions, that was similar to the decrease in coronary venous O2 tension in normal hearts (Fig.\u00a07; [71]). Although the vasoconstriction under resting conditions in response to KATP channel blockade was similar in normal and post-MI remodelled hearts, the responses to exercise were different. Thus in normal swine, the effects of KATP channel blockade waned during exercise, suggesting that other vasodilator systems compensated for the loss of KATP channels during exercise. In contrast, in the post-MI remodelled hearts the effects of KATP channel blockade were maintained during exercise. Our findings, which are consistent with the observations in dogs [65], support the hypothesis that KATP channel opening is of greater importance in resistance vessel dilation during exercise in hypertrophied than in normal hearts. It is likely that with the progression from LV dysfunction to overt heart failure, increased KATP channel activity may also become important under resting conditions [106].\nInteraction between KATP channels and adenosine\nIn contrast to the canine heart in which adenosine can act as a back-up system [29, 81], adenosine and KATP channels appear to exert additive vasodilator influences on coronary vasomotor tone in the normal porcine heart [67]. Thus, the coronary vasoconstriction that occurs in response to combined adenosine receptor blockade and KATP channel blockade equalled the sum of the vasoconstriction induced by blockade of the individual pathways. Adenosine mediates its vasodilator effect on porcine coronary resistance vessels via KATP, KCa and Kv channels [9, 46\u201348] It is therefore possible that following KATP channel blockade adenosine maintained its vasodilator influence via KCa and\/or Kv channels.\nIn contrast to the normal porcine heart, the magnitude of the constriction induced in remodelled hearts by combined blockade of adenosine receptors and KATP channels was virtually identical to that produced by blockade of KATP channels alone [71]. These findings could be interpreted to suggest that in remodelled myocardium the vasodilator influence of endogenous adenosine was entirely mediated through opening of KATP channels, observations that are corroborated by findings in pressure-overload hypertrophied canine hearts [65]. Taken together these observations in the porcine and canine coronary circulations suggest that, although the magnitude of the vasodilator influence exerted by endogenous adenosine was similar in normal and remodelled hearts, its effector pathway was different.\nEndothelial control\nSeveral studies have indicated that endothelial dysfunction, in particular a decreased production of NO and an increased production of endothelin, could aggravate LV dysfunction due to the peripheral vasoconstriction-induced increase in LV afterload, coronary vasoconstriction, and increased myocardial O2 consumption [26, 59, 80, 105].\nNitric oxide\nClinical studies indicate that chronic heart failure is accompanied by blunted vasodilator responses to endothelium-dependent receptor mediated vasodilators (particularly acetylcholine) in the microcirculation of the LV myocardium [92], leg [45, 57], and forearm [19, 52, 57, 64]. In the canine model of pacing-induced end-stage congestive heart failure, attenuated vasodilator responses of resistance vessels to acetylcholine in vivo have also been observed in the microvasculature of the hindleg circulation [22, 64] and the coronary circulation [105]. In swine with a 2\u20133-week-old MI, we observed reduced vasodilator responses in the systemic and coronary microvasculature to ATP, in doses which we have previously shown to be completely abolished by pretreatment with the eNOS-inhibitor NLA [26]. The findings of a blunted ATP-induced vasodilation are in agreement with the hypothesis that agonist-induced eNOS-mediated NO production is blunted 2\u20133\u00a0weeks after MI.\nA loss of NO-mediated vasodilation could enhance the progression of LV dysfunction to heart failure. This is supported by studies in dogs with pacing-induced dilated cardiomyopathy, in which the loss of basal NO production in the LV myocardium coincides with the progression from LV dysfunction to overt heart failure [80, 105]. However, in swine with a 2\u20133-week-old MI, we did not find any evidence of a reduced coronary vasodilator influence of endogenous NO as decreases in coronary venous O2 tension produced by NLA in resting and exercising MI swine were similar to those of normal swine (Fig.\u00a08). In heart failure patients, studies on the contribution of NO to basal microvascular tone in the forearm, leg, or total systemic bed have yielded equivocal results with responses varying from blunted [45, 58, 64], to maintained [60], and even enhanced [20, 42] increases in vascular tone following NO synthase inhibition. It is possible that a maintained or increased NO production as observed in some studies, was the result of increased iNOS expression [21, 79], as part of a generalized inflammatory response in end-stage heart failure, that occurred in the presence of either a decreased [21, 86] or increased [37, 50] eNOS expression. Since NLA can block all three isoforms of NOS [7], we performed additional experiments, in which we blocked iNOS with aminoguanidine, to determine whether an upregulation of iNOS-mediated NO production masked a reduction in eNOS activity. iNOS blockade had no effect on coronary vasomotor tone, demonstrating that NO production via iNOS does not contribute significantly to vascular tone in MI swine, and, consequently, that basal and exercise-induced endothelial NO production is maintained early after MI [44].\nFig.\u00a08Effect of inhibition of NO synthase by NLA (20\u00a0mg\/kg iv) on myocardial O2 extraction and coronary venous PO2 at rest (lying) and during treadmill exercise in MI and N. Data are mean\u00a0\u00b1\u00a0SEM. *P\u00a0<\u00a00.05 NLA versus corresponding Control; there were no significant differences in the responses to NLA between MI and N either at rest or during exercise. Data are from Haitsma et al. [44]\nThe reason for the maintained basal and exercise-induced NO production in the presence of a blunted vasodilator response to ATP is unclear. However, Traverse et al. [91] have shown that the amount of NO, produced after stimulation with an agonist is larger than the amount of NO produced during moderate exercise (60% increase in heart rate). Hence, the maximal capacity of NO production may be reduced, whereas the capacity of eNOS is sufficient to maintain basal and exercise-induced NO production. This explanation is unlikely since agonist-induced dilation is already affected at the lowest dose of administered ATP (which probably releases less NO than strenuous exercise). Moreover, higher doses of ATP still produce more dilation. Another explanation for the divergent results between ATP and exercise-induced increases in NO-production may be that ATP activates eNOS through a different mechanism than shear stress. ATP-induced activation of eNOS is mediated through a calcium-calmodulin dependent pathway [74] whereas shear stress activates eNOS through Akt-mediated phosphorylation [18], resulting in calcium-independent activation of eNOS. Hence, it is possible that perturbations in the calcium homeostasis of endothelial cells contributed to the selective impairment of ATP-induced vasodilation in swine with MI.\nEndothelin\nDespite the increased plasma levels of endogenous ET in swine with a 2\u20133\u00a0week old MI, its vasoconstrictor influence on the coronary circulation was reduced (Fig.\u00a09 [69, 70]). To determine whether this was the result of blunted receptor responsiveness or reduced local ET-production, we studied the vasoconstriction induced by exogenous ET. The coronary vasoconstrictor influence to exogenous ET-1 in vivo was reduced after MI, indicating a reduced coronary vascular responsiveness to ET. Paradoxically, a recent study showed that ischemic heart disease results in upregulation of ETA and ETB receptor mRNA in human coronary arteries [104]. This is in accordance with our measurements in isolated coronary arterioles obtained from sham-operated swine and swine with a MI, which showed that the ET responsiveness in vessels from animals with a MI was actually increased [70]. The discrepancy between the in vivo and the in vitro findings suggests that ET receptor sensitivity is modulated in vivo, and that this modulation is apparently lost in vitro. Possible modulators of ET-receptor sensitivity are adenosine and NO, which have been shown to desensitize ET-receptors on the coronary vasculature [72, 73], and which may have been lost in the in vitro set-up due to lack of surrounding myocardium and intravascular blood flow. However, since we observed similar vasoconstrictor responses to blockade of adenosine receptors and NO production in MI and normal swine, these vasodilators would seem unlikely explanations for the observed reduced ET receptor sensitivity in MI swine in vivo.\nFig.\u00a09Effect of ETA receptor blockade with EMD (3\u00a0mg\/kg iv) on myocardial oxygen balance in normal swine and swine with a recent MI. Data are means\u00a0\u00b1\u00a0SE; *P\u00a0<\u00a00.05 versus corresponding Control relation, \u2020\nP\u00a0<\u00a00.05 effect of EMD waned during exercise. Data are from Merkus et al. [70]\nIn conclusion, our observations suggest that when additional coronary vasodilation is required in the hypertrophied myocardium after MI, withdrawal of the ET-mediated vasoconstrictor influence contributes to a shift in vasomotor tone towards vasodilation. These findings may also explain in part why clinical trials of ET-receptor antagonists in heart failure have failed to show therapeutic value of these compounds [40, 77].\nConclusions and physiological relevance\nUnder physiological circumstances, the heart matches its blood supply to the demand of the myocardium by altering the balance of vasodilator and vasoconstrictor influences, i.e. an increase in myocardial O2 demand results in an increased influence of vasodilators (opening of KATP channels, NO, adenosine [53], \u03b2-adrenergic vasodilation [27]) and a decreased influence of the potent vasoconstrictor endothelin-1 [66, 69]. Studies from our laboratory performed during the past 10\u00a0years have shown that following MI myocardial O2 balance in the remodelled LV is perturbed, necessitating vasodilation and recruitment of coronary flow reserve, as evidenced by increased opening of KATP channels [71]. Importantly, however, not only an increased KATP channel vasodilator influence, but also blunting of the coronary vasoconstrictor influences of endogenous endothelin [70] and angiotensin II [68], aid in preventing a more significant impairment of oxygen supply to the remodelled myocardium (Figs.\u00a010, 11). Our studies suggest that generalized blunting of vasoconstrictor influences is one of the first adaptive mechanisms to reduce coronary resistance vessel tone and increase myocardial blood supply. This adaptation, which occurs in the healthy porcine heart during acute exercise, also appears operative in remodelled myocardium. Blunting of vasoconstrictor influences may be a physiologically favorable strategy, since it is more energy-efficient to blunt vasoconstrictor influences than to synthesize vasodilators.\nFig.\u00a010Myocardial oxygen balance in normal and MI swine. Shown are the actual relations between MVO2 and CVPO2 in 30 normal swine (open circles) and 20 MI swine (open triangles) under control conditions. In addition, we have depicted the computed relations in MI swine if the ET (solid diamonds) and ANG II (solid squares) vasoconstrictor influences (which were both attenuated in MI swine) and the KATP (solid triangles) vasodilator influences (which were enhanced in MI swine) would have been identical to those in normal swine. The graph clearly illustrates that the adaptations in coronary vasomotor control act to blunt perturbations in oxygen balance in remodelled myocardium of swine with a recent MI\nFig.\u00a011Alterations in vasomotor balance in the coronary resistance vessels within remodelled myocardium in swine with a 2\u20133-week-old myocardial infarction\nAlthough the shift in coronary tone towards vasodilatation acts to blunt the flow perturbations in the remodelled heart, it does not appear to fully restore myocardial oxygen balance. This is also supported by the observation that KATP channel activation is increased, consistent with the presence of metabolic distress. Whether these perturbations in myocardial oxygen delivery blood are sufficiently severe to contribute to the progressive deterioration of contractile function of the remodelled left ventricle remains to be established, but it is of interest to note that we previously found that troponin I proteolysis occurred in remodelled myocardium, which was associated with a loss of myofilament force development [97]. Intermittent myocardial ischemia, as may occur in remodelled hearts during exercise or excitement, has been shown to be able to promote troponin I proteolysis [38] and could thereby mediate the flow perturbation-induced progressive deterioration of LV function after MI. Definitive proof of our hypothesis must await future studies demonstrating that prevention of such flow perturbations will indeed prevent progressive loss of contractile function of the remodelled porcine left ventricle.","keyphrases":["swine","myocardial infarction","coronary blood flow","myocardial oxygen balance","exercise"],"prmu":["P","P","P","P","P"]}
{"id":"Pharm_World_Sci-3-1-1915627","title":"Simultaneous prescribing of atypical antipsychotics, conventional antipsychotics and anticholinergics\u2014a European study\n","text":"Objective The aim of this study was to investigate to what extent atypical antipsychotics, conventional antipsychotics and anticholinergics are prescribed simultaneously in daily clinical practice in Europe.\nIntroduction \nAll antipsychotics are solely registered for use as monotherapy. The prescription of two or more antipsychotics simultaneously, also called antipsychotic polypharmacy, has no empirical foundation [1]. However, in the past antipsychotic polypharmacy with two conventional (\u2018early generation\u2019) antipsychotics was frequently practised with reported frequencies ranging from approximately 10\u201369% [2]. In the last years the conventional antipsychotics are increasingly replaced by the atypical (\u2018second generation\u2019) antipsychotics. Few studies from the UK and the US have focused on antipsychotic polypharmacy with atypical antipsychotics and reported frequencies ranging from 13% to 68% [3\u20137].\nWe carried out a pharmaco-epidemiological study to atypical antipsychotic polypharmacy and included data of patients of several psychiatric hospitals in various European countries. Additionally, we also focused on the co-prescription of anticholinergic drugs. There is considerable evidence that atypical antipsychotics cause fewer extrapyramidal side effects than conventional antipsychotics [8]. Therefore, there should be less need for anticholinergic co-medication to be prescribed with the atypicals.\nThis European study was conducted to investigate to what extent atypical antipsychotics, conventional antipsychotics and anticholinergics are being prescribed simultaneously in daily clinical practice.\nMethod\nThe study was carried out with the co-operation of members of the \u2018European Pharmacists for Psychiatry and Neurology\u2019 (EPPN), an organisation of hospital pharmacists working in psychiatry and neurology. All participating hospital pharmacists were working in psychiatric hospitals. Data were collected between November 1998 and October 1999. In that period the hospital pharmacists screened their hospital pharmacy database on one single day for patients who had been using an atypical antipsychotic for a minimum of 6\u00a0weeks. The 6-week period was chosen so that titration to the atypical antipsychotic should have been completed. No restrictions were imposed with regard to age and diagnosis.\nThe following data were collected for each patient included: age, sex, type and dose of the atypical(s) and, if prescribed, type and dose of conventional antipsychotics (maximum of four) and the type of anticholinergics (maximum of four). \u201cAs needed\u201d (PRN) medication was considered as \u201cused\u201d medication. The doses of antipsychotic depot-medications were recalculated to daily oral doses [9]. All data were checked for improbable doses, duplication of patients and other errors.\nMedian doses of the antipsychotics are given, because of positively skewed distributions. The frequencies with which antipsychotic polypharmacy was practised and anticholinergics were prescribed are calculated for the total group of patients and per individual atypical antipsychotic.\nConventional antipsychotics with a chlorpromazine equivalent dose of more than 40\u00a0mg (compared to 100\u00a0mg chlorpromazine) are considered to be low potency drugs [10, 11]. The frequencies with which antipsychotic polypharmacy was practised are calculated with and without the low-potent antipsychotics.\nResults\nHospital pharmacists from six West European countries participated: Belgium, Denmark, France, Germany, The Netherlands and Scotland. In total, data were received for 2,765 patients. Of these, 40 patients (1.5%) were excluded for the following reasons: patients mentioned twice (n\u00a0=\u00a010), no atypical antipsychotic prescribed (n\u00a0=\u00a08), improbably low clozapine doses (n\u00a0=\u00a05) and other errors (n\u00a0=\u00a017).\nTable\u00a01 shows details of the participating countries and patients. Most patients came from Belgium (40.9%), followed by The Netherlands (26.2%) and France (16.5%).\nTable\u00a01Number of hospitals and patients included in the participating countries with the mean age of patients and the percentages of male patientsCountryNumber of hospitals (%)Number of patients (%)Mean age (sd)% of malesBelgium19 (42.2%)1115 (40.9%)47.9 (15.6)59%The Netherlands5 (11.1%)715 (26.2%) 46.1 (17.9)54%France10 (22.2%)449 (16.5%)42.3 (14.5)61%Denmark4 (8.9%)217 (8.0%)48.1 (16.4)52%Scotland6 (13.3%)183 (6.7%)45.8 (17.7)51%Germany1 (2.2%)46 (1.7%)38.5 (13.9)60%Total45 (100%)2725 (100%)46.2 (16.4)57%\nTable\u00a02 shows the frequencies with which the atypical antipsychotics were prescribed, the median dose, the frequencies with which of antipsychotic polypharmacy (with and without the low-potent conventional antipsychotics) was practised, the frequencies with which anticholinergics were administrated and the frequencies of no co-prescription of antipsychotics or anticholinergics.\nTable\u00a02.Details of the atypical antipsychotics prescribed: their frequencies; median dose; the frequencies of patients prescribed antipsychotic polypharmacy (with and without low-potent antipsychotics) and anticholinergics; the frequencies of no co-prescription of antipsychotics and anticholinergics (n\u00a0=\u00a02,725)Atypical antipsychoticNumber of patients (%)Median dose (mg) Antipsychotic polypharmacy (%) (including low-potent antipsychotics)Antipsychotic polypharmacy (%) (excluding low-potent antipsychotics)*Anticholinergic prescription (%)No co-prescription of antipsychotics or anticholinergics (%)Risperidone1095 (40.2%)4.045.124.736.542.0Clozapine683 (25.1%)400.031.218.924.557.7Olanzapine562 (20.6%)15.037.022.120.356.2Sulpiride133 (4.9%)300.049.225.831.141.7Amisulpride132 (4.8%)600.054.123.345.928.6Sertindole37 (1.4%)16.062.224.327.029.7Quetiapine16 (0.6%)550.050.012.512.543.8Zotepine2 (0.1%)225.0000100Two atypicals65 (2.4%)********40.0****Total2725 (100%)\u201342.1%24.4%30.1%47.1%* See Table\u00a03 for the classification of the low-potent antipsychotics** Total median dose not calculated*** By definition 100%**** By definition 0%\nRisperidone was prescribed most often (40.2%), followed by clozapine (25.1%), olanzapine (20.6%) and others (in total 14.1%). For 65 patients (2.4%) 2 atypical antipsychotics were prescribed; the combinations used most were clozapine\/risperidone (n\u00a0=\u00a018), olanzapine\/risperidone (n\u00a0=\u00a012) and sulpiride\/risperidone (n\u00a0=\u00a011). In our population of patients using an atypical, antipsychotic polypharmacy was prescribed in 42.1% of cases. Of patients receiving antipsychotic polypharmacy 31.7%, 8.5%, 1.3% and .6% were prescribed two, three, four, and five or more antipsychotics respectively.\nTable\u00a03 shows the antipsychotics prescribed concurrently with the atypicals. The conventional antipsychotics prescribed most often were levomepromazine, haloperidol, prothipendyl, cyamemazine and zuclopentixol. When the low-potent conventional antipsychotics are not included in the analysis, 24.4% of the patients was prescribed antipsychotic polypharmacy.\nTable\u00a03The frequencies and median doses antipsychotics prescribed concurrently for patients using atypical antipsychoticsAntipsychoticAntipsychotic prescribed concurrently (%) Median dose (mg)Levomepromazine*16.6%50.0 Haloperidol11.2%10.0 Prothipendyl*9.5%80.0 Cyamemazine*7.1%100.0 Zuclopenthixol7.6%28.5 Pipamperon*5.6%80.0 Dehydrobenzperidol4.7%10.0 Thioridazine*4.4%100.0 Clotiapine4.4%40.0 Chlorpromazine*3.1%200.0 Flupenthixol2.7%5.9 Fluphenazine2.4%9.0 Bromperidol2.4%7.5 Chlorprothixeen*1.8%100.0 Perphenazine1.5%16.0 Pimozide1.4%4.0 Other conventional antipsychotics9.2%\u2013Second atypical4.2%\u2013Total100.0%\u2013* Considered as low-potent antipsychotic\nIn total 30.1% of the patients were prescribed an anticholinergic drug. The anticholinergics prescribed most often were biperiden (28.3%), procyclidine (19.9%), trihexyphenidyl (16.7%) and tropatepine (13.4%).\nIn total 47.1% of the patients are prescribed the atypical antipsychotic without any other antipsychotic or anticholinergic, 22.8% are prescribed an atypical plus one or more other antipsychotics, 10.8% are given an atypical plus an anticholinergic and 19.3% an atypical plus an anticholinergic and one or more other antipsychotics.\nDiscussion\nThis European study clearly shows that it is common clinical practice to prescribe an atypical antipsychotic and one or more other antipsychotics simultaneously. Low-potent antipsychotics may be added to an atypical antipsychotic mainly because of their sedative, anxiolytic and anticholinergic properties, rather than because of their antipsychotic properties. Even when the low-potent conventional antipsychotics are not included in the analysis, we found that antipsychotic polypharmacy was prescribed for almost a quarter of the patients. Additionally, we did not expect to find that almost a third of the patients were prescribed an anticholinergic in addition to the atypical drug. The results of this study imply that more than half (52.9%) of the patients are not being treated in the way according to the psychiatric handbooks and guidelines: The atypical is frequently combined with another antipsychotic and\/or an anticholinergic.\nSeveral studies have shown that antipsychotic polypharmacy with two conventional antipsychotics was in frequent use in clinical practice [2]. Others have shown that fairly large numbers of patients (ranging from 18.5% to 56%) are being prescribed a therapy that combines clozapine with a conventional antipsychotic [12\u201314]. Recently, a few studies have focused on antipsychotic polypharmacy with the atypical antipsychotics, with reported frequencies ranging from 13% to 68% [3\u20137]. There could be several reasons to explain this large variation in co-prescribing such as the definition of antipsychotic polypharmacy, whether inpatients or outpatients were included, stage of the disease, the differences in prescription patterns between countries and hospitals and different use of guidelines.\nWe consider several hypotheses for the relatively high rate of antipsychotic polypharmacy involving atypicals. First of all, it could be that the patients with an inadequate treatment response to one antipsychotic are treated with combinations of antipsychotics, including the atypical antipsychotics. However, there are hardly any studies that show that antipsychotic polypharmacy is effective in patients who do not respond to one antipsychotic [1].\nSecondly, it is also possible that antipsychotic polypharmacy is continued although the patient shows no improvement because psychiatrists are hesitant to discontinue any medication in patients with persistent psychotic symptoms.\nThirdly, it could be that in some patients the recommended doses of the atypical antipsychotics are too low to be effective and that adding a second antipsychotic is in fact a dose-increase strategy. In such cases, however, a higher dose of one particular atypical might be just as effective.\nFourthly, when switching between two antipsychotics it is common to titrate the first drug downwards while simultaneously titrating the second drug upwards. If the patient responds halfway through the titrating process the clinician might decide to continue both antipsychotics\u2014a situation referred to as \u201cthe cross-titration trap\u201d [15]. Our study included only patients who had been using an atypical antipsychotic for more than 6\u00a0weeks, so the titrating process would normally have been completed.\nIt is remarkable that the potential risks of antipsychotic polypharmacy have received limited research attention [16]. However, Waddington et\u00a0al. [17] found that antipsychotic polypharmacy is associated with reduced survival among patients with schizophrenia. Centorrino et\u00a0al. [16] reports that the risk of adverse effects is 56% higher with antipsychotic polypharmacy.\nThere have been previous reports of anticholinergics being added to atypical antipsychotics. Of patients using clozapine 19% [12] and 37% [14] were also prescribed anticholinergics. Anticholinergics were added to 54.9% of patients using atypical antipsychotics [3]. These numbers are in line with our finding (30.1%). The prescription of this combined therapy could be attributed to the use of a second, almost always conventional, antipsychotic (19.3%), which might induce extrapyramidal symptoms. But nevertheless, 10.8% of patients are prescribed an atypical antipsychotic in combination with an anticholinergic without any other (conventional) antipsychotics. Minzenberg et\u00a0al. [18] studied the effects of anticholinergic properties of psychiatric medications and found clinically significant effects on memory and complex attention.\nThe frequencies of atypical polypharmacy in the separate countries range between 26.1% (Germany) and 49.1% (Belgium), without the low-potent antipsychotics it varies between 17.1% (Denmark) and 31.6% (Belgium). However, these numbers should be appraised cautiously because this study was not primary set up to detect differences between the various countries (some countries are under represented).\nFurthermore, the study does not allow to draw inferences about differences between the various atypicals in antipsychotic polypharmacy and anticholinergic use. These differences could easily be biased as a result of the different introduction data of the atypicals in the diverse countries. For example, at the moment of data-collection olanzapine and sertindole had been introduced very recently in most countries, quetiapine was available only in Scotland and zotepine was used only in a clinical trial.\nNevertheless, the similarity between the frequencies with which antipsychotic polypharmacy is prescribed for the various atypicals (excluding quetiapine and zotepine, on account of their low frequencies) is remarkable: Excluding low-potent antipsychotics the frequencies range from 18.9% to 25.8%.\nThe study however has its limitations.\nFirst of all, the population included is not a random sample from Western European countries that participated. Therefore, hypothetically it could be argued that the results were biased because only hospitals practising extremely high rates of antipsychotic polypharmacy were included. However, it is unlikely that only such hospitals were selected.\nSecondly, we estimate, on the basis of the information obtained from the participating hospitals, that 90% of the data are from inpatients. The symptomatology of inpatients may be more severe than that of outpatients, and such patients may be prescribed antipsychotic polypharmacy more frequently than outpatients [3].\nThirdly, we did regard PRN-medication as \u201cused\u201d medication. This may have heightened our results somewhat but cannot explain the frequent prescription of antipsychotic polypharmacy. A major advantage of this study is the large number of patients included and the differentiation made between high- and low-potent antipsychotics. This makes extrapolation more feasible.\nConclusion\nIt is common practice to prescribe a combination of atypical and conventional antipsychotics. Furthermore, the use of atypical antipsychotics does rule out the use of anticholinergics. On the contrary, atypical antipsychotics are often prescribed in combination with anticholinergics. Apparently, monotherapy involving the atypicals is not considered to be an effective therapy for a substantial number of patients in clinical practice.\nPossible conflicts of interest\nP.N. van Harten gives lectures on symposia and congresses that are sponsored by pharmaceutical companies.","keyphrases":["anticholinergics","europe","polypharmacy","pharmaco-epidemiology","antipsychotic agents","drug therapy combination","drug use study"],"prmu":["P","P","P","P","M","R","R"]}
{"id":"Pediatr_Nephrol-3-1-1766475","title":"Prevention and treatment of renal osteodystrophy in children on chronic renal failure: European guidelines\n","text":"Childhood renal osteodystrophy (ROD) is the consequence of disturbances of the calcium-regulating hormones vitamin D and parathyroid hormone (PTH) as well as of the somatotroph hormone axis associated with local modulation of bone and growth cartilage function. The resulting growth retardation and the potentially rapid onset of ROD in children are different from ROD in adults. The biochemical changes of ROD as well as its prevention and treatment affect calcium and phosphorus homeostasis and are directly associated with the development of cardiovascular disease in pediatric renal patients. The aims of the clinical and biochemical surveillance of pediatric patients with CRF or on dialysis are prevention of hyperphosphatemia, avoidance of hypercalcemia and keeping the calcium phosphorus product below 5 mmol2\/l2. The PTH levels should be within the normal range in chronic renal failure (CRF) and up to 2\u20133 times the upper limit of normal levels in dialysed children. Prevention of ROD is expected to result in improved growth and less vascular calcification.\nIntroduction\nChronic renal failure is associated with specific abnormalities of skeletal homeostasis, commonly called renal osteodystrophy (ROD), which if not treated appropriately during the critical phases of skeletal growth can result in bone deformities and a disturbed growth pattern. The main factors for the development of ROD are disturbances in the calcium phosphate homeostasis, in vitamin D and parathyroid hormone metabolism as well as alterations in the somatotroph axis, i.e., on the endocrine and paracrine levels. In recent years it has been recognized that the spectrum of renal bone disease covers \u2018high-\u2019 as well as \u2018low-turnover\u2019 conditions. As a consequence of chronic renal failure itself and of the treatment of renal bone disease, high plasma phosphate levels and an elevated calcium phosphorus product are common. These are important risk factors for the development of vascular calcification and cardiovascular morbidity and mortality in young adults who have been on renal replacement therapy since childhood [1, 2]. Because aluminium-containing phosphate binders are no longer indicated in children, aluminium-related osteopathy is not considered in these recommendations.\nThe European Pediatric Peritoneal Working Group (EPPWG) was established in 1999 by pediatric nephrologists with a major interest in peritoneal dialysis and has, among others, published guidelines on chronic and acute peritoneal dialysis [3, 4, 5, 6]. The group incorporates pediatric nephrologists from 12 European countries. One of the functions of the group is to establish expert guidance in important clinical areas associated with chronic renal failure and dialysis [now the European Pediatric Dialysis Working Group (EPDWG)] in conjunction with other members of the multidisciplinary team. These guidelines were initiated and discussed at meetings of the group and developed by e-mail discussion to develop consensus of opinion based upon cumulative clinical experience and reported studies.\nRecommendations\nRecommendation 1\nClinical, biochemical and radiological markers of renal bone disease should be monitored regularly. The clinical markers of renal bone disease to be prevented are signs of overt rickets, slipped femoral epiphysis and disturbances of growth. The biochemical markers are plasma phosphate, calcium, alkaline phosphatase, bicarbonate and intact parathyroid hormone (PTH). The minimal frequency of measurements (and target ranges) for biochemical markers in a stable phase vary according to renal function (Table\u00a01). If the patient has active ROD, additional blood samples may be required. Table\u00a01 Frequency of measurements for biochemical and radiological markers of renal osteodystrophyMarker1Frequency of measurement (every x month)TargetGFRGFRGFR <15, dialysis59\u20133029\u201315(Evidence)Calcium or631Normal range (corrected calcium)2Ionized calciumNormal range2Phosphate631Normal range for age bandCalcium phosphorus product631\u22645.0\u00a0mmol2\/l2Target range 3.3\u20134.4\u00a0mmol2\/l2, 3Alkaline phosphatase631Normal range for age bandSerum bicarbonate\/base excess631Normal range, at least: bicarbonate >22\u00a0mmol\/lBase excess >\u22125\u00a0mmol\/lIntact PTH\/whole PTH631Normal range in moderate CRF (GFR>29\u00a0ml\/min\/1.73m2)Up to 2\u20133 times upper limit of normal in advanced CRF or on dialysis25-(OH) vitamin D3As indicated4As indicated4As indicated4>20\u00a0ng\/lLeft hand and wrist X-ray6\u201312No radiological signs of hyperparathyroidismNo Looser zones or osteopeniaCalcium: mmol\/l in mg\/dl: \u00d74, phosphate mmol\/l in mg\/dl: \u00d73.0969; calcium phosphorus product: mmol2\/l2 in mg2\/l2: \u00d712.387. 2Corrected calcium (mg\/dl) = measured calcium concentration (mg\/dl) + 0.8 \u00d7 [4-measured albumin concentration (g\/dl)]; corrected calcium (mmol\/l) = measured calcium concentration (mmol\/l) +0.2 \u00d7 [4-measured albumin concentration (g\/dl)]. 3Depending on age. 4Only in patients with suspected vitamin D deficiency\nPlasma calcium must be adjusted for albumin levels (= corrected calcium) or by measuring ionized (free) calcium, in patients with hypoalbuminemia or acid-base disorders. Ionized calcium values are often available on blood gas analysis systems with calcium-sensitive electrodes, but correct values are only obtained in validated conditions. Alkaline phosphatase as a marker for osteoblast activity has particular importance in both low and high-turnover bone disease with elevated or low alkaline phosphatase serum levels, respectively.\nPTH should be monitored monthly in advanced CRF (GFR <15\u00a0ml\/min\/1.73m2) because of its rapid changes. Different assays are available for the measurement of PTH. In principle, intact PTH(1\u201384) (iPTH) should be measured because it is believed to represent the active hormone. For this purpose, two-site assays were developed at the end of the 1980s [7]. However, PTH fragments accumulating in end-stage renal disease may also show biological activity. Furthermore, recent research using plasma samples from healthy and renal patients analyzed by HPLC and the iPTH assay demonstrated cross-reactivity of the intact PTH-assay with a PTH-fragment (PTH7\u201384). Therefore, new assays were developed that detect only PTH molecules with a complete N-terminal end, called \u201cwhole-PTH\u201d assays [8]. In adult and pediatric dialysis patients, these \u201cwhole PTH\u201d assays yielded 30\u201360% lower PTH levels than the intact PTH assay. Despite theoretical considerations, no significant improvement in the distinction between the different forms of renal bone disease using the \u201cwhole PTH\u201d assays has yet been demonstrated in clinical practice [9, 10, 11, 12].\nRadiological signs of renal bone disease, which alone are not sensitive enough to indicate therapy-adaptations, include signs of hyperparathyroidism and growth zone lesions. Periosteal resorption zones and metaphyseal changes are the most obvious signs. In late adolescent and young adult patients, Looser zones as specific signs of osteomalacia may be found. The sites and expression of bone lesions are age-dependent according to the age-dependent growth and remodeling at different sites of the skeleton. In pre-school children, the metaphysis of the distal radius and ulna may present only minor abnormalities, whereas more severe lesions may be seen at the upper and lower femoral epiphysis. Adynamic bone disease shows no specific radiological changes. However, extraosseous calcifications, fractures or osteopenia may indicate adynamic bone disease.\nHistological evaluation of bone biopsy specimens remain the \u201cgold standard\u201d in assessment of renal osteodystrophy. However, due to its invasive nature, bone biopsies are not performed in clinical practice, but they remain an important tool for research [12].\nRecommendation 2\nMetabolic acidosis should be corrected (evidence). Chronic metabolic acidosis leads to increased bone resorption and inhibits endochondral bone formation in animal experiments [13, 14]. Infants with isolated chronic metabolic acidosis show growth retardation [14]. In adult hemodialysis patients, negative aspects of chronic metabolic acidosis on metabolism have been reported [15]. Therefore, metabolic acidosis should be corrected by a stepwise approach: The first step is optimization of the dialysis regimen both for HD- and PD-patients [4, 15]. Since the introduction of HCO3-based PD solutions [16], metabolic acidosis is a less frequent problem. In combination with a daytime-dwell, patients may even be at risk for alkalosis [17]. However, if these adaptations do not result in correction of the metabolic acidosis, sodium bicarbonate should be administered orally. Whenever possible, formulations that dissolve in the small intestine should be used. The use of sodium citrate increases the risk of aluminium absorption [18, 19]. Acidosis should be corrected to the normal range of the local laboratory.\nRecommendation 3\nThe plasma-phosphate level should be kept within the normal age-specific range (evidence). In children with chronic renal failure, hyperphosphatemia is observed at GFR levels below 40\u00a0ml\/min\/1.73m2 [20] and almost always in children on dialysis. Hyperhosphatemia has several deleterious effects on PTH secretion [21, 22], parathyroid cell proliferation [23] and soft tissue (vascular) calcification [1, 2].\nIncreased phosphate levels stimulate PTH secretion in vitro [24] and in vivo [21, 25] independent of calcium and 1,25(OH)2 vitamin D3 levels. This effect of phosphate on PTH secretion occurs via post-transcriptional processes by regulating pre-pro-PTH mRNA stability [26]. Furthermore, a reduction of serum-phosphate results in a parallel reduction of PTH levels without change in serum calcium [27]. Hyperphosphatemia accelerates parathyroid cell proliferation [23], which can result in nodular hyperplasia and severe hyperparathyroid bone disease necessitating parathyroidectomy (see below).\nIncreased plasma phosphate levels have a profound effect on soft tissue and vascular calcification, which are often observed in young patients on or even after dialysis [1, 2, 28], increasing the risk of cardiovascular morbidity and mortality. Block et al. [29] found an increased risk of death for serum phosphate exceeding 2.09\u00a0mmol\/l in adult patients; other studies described a much lower phosphate level (~1.4\u20131.6\u00a0mmol\/l) at which a higher incidence of calcifications was found [1, 30, 31]. The histology of the calcification pattern is quite distinct from the process of atherosclerosis. In renal patients, extensive calcification of the tunica media occurs even after short periods of dialysis, as shown by investigations of the epigastric artery in adult patients [32]. Experimental work gave evidence for an active role of phosphate in this process as phosphate induces osteoblastic marker followed by calcification in cultured smooth muscle cells [33]. Dialysis patients are at risk of heart failure. Although this is a multifactorial process, it was demonstrated in animal experiments that high plasma phosphate levels per se accelerated cardiac fibrosis [34].\nRecommendation 4\nIf plasma phosphate is elevated, phosphate intake should be limited to the recommended levels. Dietary intake of protein and as a consequence of phosphate in western countries usually exceeds the recommended intake at least in adolescent dialysis patients. Therefore, dietary counseling should be performed by a trained dietician [35]. If the dietary records show a protein intake above the recommended level for pediatric dialysis patients, the patients and their parents should be trained to reduce their phosphate intake. This may start with the reduction of dairy products and\/or substitution with special low-phosphorus products and a reduction of meat intake.\nRecommendation 5\nIn case of hyperphosphatemia, the dialysis efficacy should be optimized (evidence). Phosphate is slowly transported via the peritoneal membrane into the dialysate [36]. The dialysate to plasma ratio (D\/P) for phosphate is volume and time related; after 4\u00a0h in standard peritoneal equilibration tests, this ratio reaches the value of 0.5\u20130.6, which is a much lower ratio than for urea, which is usually nearly 1. Therefore, an increase in phosphate removal should consider not only an increase in dwell volume to 1,000\u20131,400\u00a0ml\/m2 BSA, but also dwell time optimization, avoiding a too short dwell time. A daytime dwell should be added. The details of a dialysis regimen optimization has been described elsewhere [5]. However, due to the low clearance of phosphate achievable even with an optimized PD regimen, dietary phosphate restriction and the use of oral phosphate binders are almost always necessary.\nThe dialytic phosphate removal kinetic during a hemodialysis session differs from urea kinetic: after an early initial drop the phosphatemia decrease only slowly [37, 38]. Therefore, it is admitted that the hemodialysis phosphate purification capacity is directly impacted by the duration of the dialysis session, because the phosphate shift from the intracellular to the vascular compartment is time-dependent. Therefore, longer dialysis sessions [39] or daily dialysis sessions [40, 41, 42] offer a unique, powerful dialysis method for optimal phosphate purification. The hemodialysis modality, HD, HF or HDF, and the type of the synthetic membrane used have only a limited impact on dialytic phosphate removal [38], whereas the dialysate composition in terms of glucose and bicarbonate concentration are presumed to be of importance [42, 43, 44], modulating a shift of intracellular phosphate trapped in the cells, where it is not available for dialysis exchanges.\nRecommendation 6\nFor control of hyperphosphatemia, aluminium-free phosphate binders should be administered (evidence). Phosphate binders are necessary to reduce phosphate absorption from the gut. Ca-containing phosphate binders, i.e., calcium carbonate (CaCO3, elemental calcium content 40%) or calcium acetate (CaAc, elemental calcium content 25%) should be used as the first line. No data are published on the efficacy and safety of calcium acetate\/magnesium carbonate compound phosphate binders in pediatric patients. The upper intake level of elemental calcium is suggested to be 2,500\u00a0mg\/day for healthy children above 4\u00a0years of age [19]. Whereas for adult dialysis patients the DOQI guidelines suggest to limit the elemental calcium intake to 2,000\u00a0mg\/day, no safe upper level can be given for the pediatric age band. However, a positive calcium balance in the range of +200\u2013+300\u00a0mg in the growing skeleton should be maintained. In contrast, a too high calcium load should be avoided, because one of the identified risk factors for soft tissue calcification in pediatric CRI patients was cumulative calcium intake [2, 24].\nCalcium-containing phosphate binders are started at approximately 500\u00a0mg per 200\u00a0mg phosphate content of the diet (0\u20131\u00a0years, 1\u20132\u00d7500\u00a0mg; 1\u20134\u00a0years, 2\u20133\u00d7500\u00a0mg\/day; 5\u20138\u00a0years, 3\u20134\u00d7500\u00a0mg\/day; 9\u201318\u00a0years, 5\u00d7500\u00a0mg). An alternative approach is a start dose of approximately 50\u00a0mg\/kg\/day, which is well below the doses given in clinical studies. Phosphate binders are then adjusted to normalize serum-phosphate and calcium. CaCO3 can be crushed to fine powder, or a 10% solution can be used for administration in infants, often via a feeding tube. The phosphate binders should be taken with meals, because fecal excretion of phosphate was higher when calcium acetate was given with meals instead of in between meals [45]. Biochemically, CaAc has a higher phosphate binding capacity, which is independent of the pH. The higher efficacy of CaAc, calculated on a weight basis compared to CaCO3, for phosphate control was also shown in clinical practice in adult and pediatric patients [46, 47]. Unfortunately, compliance of the patients with the intake of phosphate binders is often poor and should be checked regularly. It may be helpful if the patient is involved in selecting the flavor and size of the phosphate binder and has regular contact with the dietician at clinic appointments. Snacks during the day are often a source of additional phosphate and require additional phosphate binders.\nPatients treated with Ca-containing phosphate binders and active vitamin D metabolites are particularly at risk for the development of hypercalcemia. Therefore, in case of hypercalcemia, active vitamin D metabolites should be stopped (see recommendation 12). If hypercalcemia, the most common side-effect of Ca-containing phosphate binder therapy, persists or the calcium phosphorus product exceeds 5.0\u00a0mmol2\/l2 with the use of Ca-containing phosphate binders, the calcium content in the dialysis fluid should be reduced [5]. Furthermore, the dose of the calcium-containing phosphate binders should be reduced whenever possible or they should be replaced by Ca- and aluminium-free phosphate binders, because epidemiological studies have shown a direct relationship between serum phosphate and calcium phosphorus product on mortality [29, 48]. The only commercially available aluminium- and calcium-free phosphate binder is Sevelamer. This compound is usually taken orally as capsules, but anecdotally can be delivered via enteral tubes by dissolving the capsule in 5\u00a0ml water (instruction Fa genzyme). Aluminium-containing phosphate binders or calcium citrate increase intestinal aluminium absorption and should not be used in pediatric dialysis patients. An alternative phosphate binder that is available is lanthanum carbonate, which has a high affinity for phosphate, and is minimally absorbed in the intestine. In a randomized study in adult patients, lanthanum carbonate controlled plasma phosphate levels well and induced less adynamic bone disease than CaCO3 [49]. However, no long-term data on the effect of lanthanum on bone, on which surface lanthanum can accumulate [50], and its safety profile for use in children are available yet.\nRecommendation 7\nVitamin D deficiency should be avoided (evidence). In early renal failure, an increase of PTH correlates positively with 25(OH)-vitamin D3 levels [51]. Renal synthesis of 1,25(OH)2 D3 is impaired in chronic renal disease. However, extrarenal cells, i.e., macrophages and osteoblasts, are also capable of 1,25(OH)2 D3 production [52]. In contrast to the kidney, the extra-renal synthesis is strictly substrate dependent. It has been shown that supplementation with vitamin D3 in elderly CRF patients with 25(OH)-vitamin D3 levels between 20 and 50\u00a0pg\/ml decreased PTH serum concentrations [53]. Furthermore, 25-OH-vitamin D3, but not 1,25(OH)2 D3, improved muscular function and phosphate content [54]. Therefore, vitamin D deficiency should be prevented.\nRecommendation 8\nMarked hyperparathyroidism should be prevented in children with CRF prior to dialysis (evidence). Due to the risk of persisting bone disease and the development of parathyroid adenoma, an increase in PTH above normal to slightly elevated levels should be prevented in children with CRF. Therefore, low doses of active vitamin D metabolites should be given in time. In adult patients this treatment regimen has resulted in controlled iPTH without negative aspects [55]. In children with moderate renal failure (GFR >30\u00a0ml\/min\/1.73\u00a0m2), normal levels of PTH in association with strictly controlled phosphate levels are associated with a normal ratio of intact PTH to \u201cwhole\u201d PTH and normal levels of alkaline phosphatase, indicating a physiological PTH secretion and unremarkable bone turnover [10]. In such patients, slight catch-up growth with PTH levels at the upper limit of normal was reported [56]. In a sub-group analysis, improved growth was restricted to patients with enteral feeding tubes.\nRecommendation 9\nPTH levels should be kept at two to three times the upper limit of the normal range in end-stage renal disease (evidence). Grossly elevated PTH concentrations (>four times the upper normal range) in the presence of normal or high serum calcium and high alkaline phosphatase are almost always associated with high-turnover bone disease [56, 57]. The exception is intermittent therapy with high-dose active vitamin D metabolites, because 1,25(OH)2 vitamin D3 directly inhibits osteoblastic function and proliferation. Hyperphosphatemia can occur in hyperparathyroid bone disease because of excessive bone resorption.\nHowever, in pediatric dialysis patients low and even normal levels of PTH are reported to be associated with low turnover bone disease [57]. A PTH concentration higher than in healthy subjects is needed to stimulate bone turnover due to resistance of the skeleton to PTH in advanced renal failure. If PTH falls into the (low) normal range, the risk of hypercalcemia increases [58]. Hyperphosphatemia and hypercalcemia can develop in low turnover bone disease because the skeleton is unable to take up enough phosphate and calcium. Low bone turnover or adynamic bone disease is increasingly observed in adult [59] as well as in pediatric PD patients [56]. Risk factors for adynamic bone disease are high calcium intake (Ca-containing phosphate binders), therapy with high doses of active vitamin D metabolites, peritoneal dialysis using dialysis fluid with high calcium content and age (adolescents after the growth spurt). Biochemically, hypercalcemia and\/or PTH levels within or below the normal range are indicative of low turnover bone disease [56, 58], which may adversely affect growth in dialysed children [60]. Therefore, active vitamin D therapy should be reduced or stopped if PTH falls below the low normal range [57, 59, 61]. If PTH remains below normal despite normal calcium and phosphate levels, the dialysate should be changed to low calcium solutions (1.0 or 1.25\u00a0mmol\/l) to stimulate PTH secretion [61], if this is not already used as the standard solution [5].\nRecommendation 10\nIf PTH is elevated in children with CRF or if PTH is elevated more than two to three times normal in the presence of Pi <2\u00a0mmol\/l in dialyzed children, active vitamin D metabolites should be administered orally (evidence). Treatment with active vitamin D metabolites results in biochemical and\/or histological improvement in patients with high-turnover bone disease. Doses of 1,25(OH)2 vitamin D3 or 1\u03b1-(OH) vitamin D3 usually range from 0.1\u00a0\u00b5g\/day to 0.75\u00a0\u00b5g\/day, with a starting dose of 20\u201340\u00a0ng\/kg. High doses often induce hypercalcemia, necessitating the reduction or discontinuation of vitamin D therapy (see hypercalcemia).\nAs to the mode of administration of the active vitamin D metabolites, intravenous or oral therapy, have the same efficacy [63]. Therefore, the oral route should be preferred in children on dialysis. Doses should be given in the evening, because fewer episodes of hypercalcemia have been reported compared to taking the vitamin D metabolites in the morning [64]. Intermittent therapy was shown to be effective to suppress elevated PTH serum levels [59]. However, recent data indicate that large intermittent 1,25(OH)2 vitamin D3 doses adversely affect bone turnover [57, 61, 65] and chondrocyte activity [66] resulting in low turnover bone disease and in reduced growth [65, 66, 67, 68]. In addition, episodes of hypercalcemia occur with similar frequency with the intermittent or oral administration of vitamin D metabolites [63, 69]; furthermore, the intermittent mode of administration was not shown to be more effective in suppressing PTH levels in adult [70] and pediatric [63] patients in prospective, randomized trials. For the above mentioned reasons, intermittent high-dose vitamin D therapy should be avoided in pediatric patients.\nThree newer vitamin D analogs, so called \u201cnon-hypercalcemic\u201d vitamin D analogs (doxercalciferol, paricalcitol and 22-oxa-calciferol), have been introduced in clinical use in adult patients with secondary hyperparathyroidism due to CRF. Despite their ability to induce less hypercalcemia in animal experiments, only one clinical study with a head-to-head comparison of the new vitamin D analogue (paricalcitol) is available demonstrating a borderline reduction of hypercalcemia or a decrease in calcium phosphorus product [71]. No data are available for their use in children.\nAgents that specifically enhance the sensitivity of the calcium-sensing receptor, called calcimimetics, used in combination with active vitamin D metabolites in adult dialysis patients resulted in a persistent decrease of PTH levels without elevated calcium phosphorus product [72]. If plasma Ca or Pi is high in the presence of elevated PTH, the calcimimetics may become first choice. However, no data for pediatric patients are available.\nRecommendation 11\nTreatment with growth hormone should not be started in the presence of severe hyperparathyroid bone disease. It is well established that the uremic growth retardation is at least in part due to disturbed pulsatile secretion of growth hormone as well as a peripheral growth hormone resistance [73]. After correction of metabolic acidosis and normalization of caloric intake, administration of recombinant human growth hormone increases growth velocity resulting in catch-up growth and in improved final height [74]. Growth hormone directly stimulates growth cartilage proliferation and metabolism and osteoblast activity [75, 76]. This growth promoting effect of GH was suspected to increase the metaphyseal instability induced by severe hyperparathyroidism. In hyperparathyroid bone disease, i.e., osteitis fibrosa, a fibrous layer is formed at the metaphyseal junction of the growth plate with the bone tissue. This can lead to epiphyseal slipping resulting in gross deformities of the affected long bones [77]. Therefore, parathyroid hormone, as well as calcium and phosphate levels, should be treated towards the recommended normal ranges prior to administration of GH. However, in a recent prospective study, no increased frequency of epiphyseal slipping associated with the use of growth hormone was reported [78]. After the start of GH administration, an increase of PTH levels was observed [79, 80].\nRecommendation 12\nIn case of hypercalcemia, active vitamin D metabolites and calcium-containing phosphate binders should be stopped and dialysate changed to low calcium solutions (evidence). Hypercalcemia and elevated calcium phosphorus product (>5.0\u00a0mmol2\/l2) should be corrected promptly (Fig.\u00a01). Vitamin D metabolites should be discontinued and in case of persisting hypercalcemia the dialysate calcium decreased to low calcium dialysate. Laboratory values should be controlled weekly until improvement is seen. One exception is hypercalcemia due to severe hyperparathyroidism and high-turnover bone disease. In addition, in patients with residual renal function, loop diuretics, i.e., furosemide may increase calcium excretion. If hypercalcemia is not corrected despite the discontinuation of active vitamin D metabolites, calcium-containing phosphate binders should be replaced by calcium-free phosphate binders to reduce the calcium load. Hypercalcemia is reported in up to 25% of patients taking calcium-containing phosphate binders ([1, 29], see recommendation 6). Fig.\u00a01 Clinical algorithm for treatment of elevated calcium phosphorus product in children with CRF. Ca-cont calcium containing; Ca-free calcium-free; phos-binder phosphate binder; vit D vitamin D; ADBD adynamic bone disease\nRecommendation 13\nThe calcium phosphorus product should be kept within the normal range, at least below 5.0\u00a0mmol2\/l2 (60\u00a0mg2\/dl2) (evidence). One of the risk factors for cardiovascular morbidity was shown to be an elevated calcium phosphorus product above 5.0\u00a0mmol2\/l2 (see also recommendation 2). If the calcium phosphorus product is elevated above this level, phosphate and\/or calcium levels should be reduced. This may be achieved by an increase in phosphate binders, reduction\/stopping of active vitamin D metabolites and use of low calcium (Ca++ 1.0\u20131.25\u00a0mmol\/l) dialysate.\nRecommendation 14\nParathyroidectomy has to be considered in case of severe, therapy-refractory hyperparathyroidism with radiological signs in combination with hypercalcemia and\/or elevated calcium phosphorus product (evidence). Severe hyperparathyroidism may no longer react to even high doses of 1,25(OH)2 vitamin D3 and reduction of phosphate levels. The clinical observations in adults show that patients with at least one parathyroid gland larger than 0.5\u00a0cm3 or 1.0\u00a0cm in diameter usually do not respond to active vitamin D metabolites [81, 82]. Therefore, persisting grossly elevated PTH levels in the presence of high-dose active vitamin D metabolites, radiological signs of hyperparathyroidism on wrist X-ray and\/or high serum calcium and normal phosphate and\/or elevated calcium phosphorus product indicate the need for surgical parathyroidectomy. Medical parathyroidectomy by alcohol injection was described in adult patients [83], but to our knowledge not in pediatric patients. Parathyroid hormone stimulates bone turnover and regulates calcium homeostasis, both of which are of utmost importance in the growing skeleton. Therefore, subtotal parathyroidectomy or autotransplantation of parathyroid tissue is recommended in children or adolescents, avoiding hypoparathyroidism ([84], review). However, these procedures carry the risk of recurrence of severe parathyroid tissue hyperplasia. The use of the new calcimimetic agents [85] may help to prevent the most severe hyperparathyroidism and therefore modify the approach to that condition.","keyphrases":["chronic renal failure","vitamin d","parathyroidectomy","secondary hyperparathyroidism","calcium-phosphate product"],"prmu":["P","P","P","P","M"]}
{"id":"Anal_Bioanal_Chem-3-1-2117333","title":"Modification and re-validation of the ethyl acetate-based multi-residue method for pesticides in produce\n","text":"The ethyl acetate-based multi-residue method for determination of pesticide residues in produce has been modified for gas chromatographic (GC) analysis by implementation of dispersive solid-phase extraction (using primary\u2013secondary amine and graphitized carbon black) and large-volume (20 \u03bcL) injection. The same extract, before clean-up and after a change of solvent, was also analyzed by liquid chromatography with tandem mass spectrometry (LC\u2013MS\u2013MS). All aspects related to sample preparation were re-assessed with regard to ease and speed of the analysis. The principle of the extraction procedure (solvent, salt) was not changed, to avoid the possibility invalidating data acquired over past decades. The modifications were made with techniques currently commonly applied in routine laboratories, GC\u2013MS and LC\u2013MS\u2013MS, in mind. The modified method enables processing (from homogenization until final extracts for both GC and LC) of 30 samples per eight hours per person. Limits of quantification (LOQs) of 0.01 mg kg\u22121 were achieved with both GC\u2013MS (full-scan acquisition, 10 mg matrix equivalent injected) and LC\u2013MS\u2013MS (2 mg injected) for most of the pesticides. Validation data for 341 pesticides and degradation products are presented. A compilation of analytical quality-control data for pesticides routinely analyzed by GC\u2013MS (135 compounds) and LC\u2013MS\u2013MS (136 compounds) in over 100 different matrices, obtained over a period of 15 months, are also presented and discussed. At the 0.05 mg kg\u22121 level acceptable recoveries were obtained for 93% (GC\u2013MS) and 92% (LC\u2013MS\u2013MS) of pesticide\u2013matrix combinations.\nIntroduction\nFor monitoring and control of pesticide residues, multi-residue methods are very cost-effective and are used in many laboratories. The pesticides are usually first extracted with an organic solvent of high or medium polarity. Typical solvents used for this purpose are acetone [1\u20134], ethyl acetate [5\u201326] (Table\u00a01), and acetonitrile [26\u201331]. With all three options, pesticides are partitioned between an aqueous phase and an organic phase. With acetone and acetonitrile this is done in two successive steps, with ethyl acetate in one step. With regard to extraction efficiency, ethyl acetate has been shown to be equivalent to the water-miscible solvents for both polar and non-polar pesticides in vegetables, fruit, and dry products (after addition of water) [6, 7, 26, 32]. It is also suitable for products with a high fat content\u2014because of the solubility of fat in ethyl acetate, pesticides are released and extracted efficiently. The extract obtained is compatible with gel-permeation chromatography (GPC), the clean-up procedure most suitable for this type of sample. Ethyl acetate is very suitable for GC analysis. It has good wettability in GC (pre)columns; this is of benefit for solvent trapping of the most volatile analytes, which is required for refocusing after injection. Its vapor pressure and expansion volume during evaporation also favor large-volume injection. Finally, it is compatible with all GC detectors. The same extract can also be used for LC analysis, after a solvent change into, e.g., methanol [11, 15\u201318, 26], as is done for acetone-based methods also [33].\nTable 1Examples from literature. Conditions typically used in ethyl acetate-based multi-residue analysisSample (g)AdditionEtAc (mL)Na2SO4 (g)Extr.Phase separationRe-extr.Evap.\/reconst. (aliquot\/to mL)Clean-upEvaporation (from\/to mL)Final extr. g mL\u22121Inj. (\u03bcL)Analysis\u00a050\u201310050B\u20135\u21921GPC None0.1910GC\u2013NPD\/ECD1987 [5]75\u201320040TF\/Na2SO4\u2013100\u21925GPCEluate\u219251.5?GC\u2013NPD, FPD1991 [6](dilute)0.3?GC\u2013ECD5\u20132010TLet settle\u201310\u219212.51\u20135GC\u2013FPD\/NPD1992 [7]15 mL H2O (wheat)0.550\u201310050TF\/Na2SO4\u20130.52\u20138GC\u2013MS\/FPD\/ECD1996 [4]50\u2013250100BF\/Na2SO4\u2013All\u2192100GPCEluate\u2192111GC\u2013NPD\/ECD1998 [8]75\u201320040TF\/Na2SO4\u2013100\u21925SPE (ENV+)3 mL1.252GC\u2013ITD\/ NPD\/ECD1999 [9]20\u2013100TSee clean-up\u2013Cartridge water abs. Polymer+ GCB\/Na2SO450\u2192dry\u21922 ace\/hex12GC\u2013MS, GC\u2013NCI-MS2001 [10]GC\u2013FPD LC\u2013PCR-Flu82 g NaHCO35070TFYesAll\u219220 MeOH0.45\u201310LC\u2013MS\u2013MS2002 [11]25\u201310075TF (vac)\u2013All\u219225 +25 cyclohexaneGPCEluate\u2192111GC\u00d7GC\u2013TOF-MS, GC\u2013TOF-HRMS2003 [12]F\/Na2SO4Rinse2004 [13]305\u20136 g NaHCO36030\u201340T (30 \u00b0C)F\/cotton wool\u20131+0.1 IS\u21921\u20130.510GC\u2013TOF-MS (DMI)2003 [14]25\u20135025TLet settle or centrifuge\u20131\u21921 H2O\u20130.520LC\u2013MS\u2013MS2003 [15]75NaOH if pH\u2009<\u20094.520040TF\/Na2SO4\u2013100\u21925\u20135\u2192MeOH2.510LC\u2013MS\u2013MS2004 [16]151 mL 6.5 mol L\u22121 NaOH9013TF\/Na2SO4RinseAll\u219215 MeOH\u2013110LC\u2013MS\u2013MS2004 [17]151 mL 6.5 mol L\u22121 NaOH90TF\/Na2SO4Yes 2\u00d7All\u219215 MeOH\u2013150LC\u2013TOF-MS2005 [18]10\u201350a10BF\u2013All\u21925GPCEluate 35\u21922210GC\u2013MS\u2013MS2006 [19]6\u2013503BCentr.Yes All\u21925GPCEluate 84\u21921b51GC\u2013MS2006 [20]20\u20138050\u2013100TFYesAll\u2192ace\/hexSPE SAX\/PSA All\u219232.42GC\u2013ECD2006 [21]50\u201310075TF\/Na2SO4RinseAll\u219210\u201352GC\u2013NPD\/MS2006 [22]5\u201310TF\/Na2SO4RinseAll\u21921\u2013510GC\u2013MS\u2013MS2006 [23]2.5\u201352TF (syringe)\u2013\u20130.550GC\u2013NPD2006 [24]305\u20136 g NaHCO36030\u201340T (30 \u00b0C)F\u20131\u21920.9 +0.1 IS\u20130.520GC\u2013FPD2006 [25]510 mL H2O (barley)5015SF\/Na2SO4\u201325\u21921GPCEluate\u219210 ACN 0.2525GC\u2013TOF-MS, LC\u2013MS\u2013MS2006 [26]252 mL 4 mol L\u22121 phosphate buffer4025TCentrifuge\u2013GC: -GCB\/PSA disp0.520GC\u2013 MSThis workLC: 0.48\u21921.5 (MeOH\/water)\u20130.210LC\u2013MS\u2013MSaEthyl acetate\u2013cyclohexane, 1:1bAdditional SPE clean-up step with Florisil EtAc\/Hex 1:1 5 mL evap. to 1 mLT, Turrax; B, blender; S, shaking; F, filtration; MeOH, methanol; ACN, acetonitrile; ace, acetone; hex, hexane\nAlthough multi-residue methods based on ethyl acetate extraction have been used for more than 20\u00a0years, and continue to be used in many laboratories (they are, for example, the official methods in Sweden and Spain and are also commonly used in the Netherlands, UK, Czech Republic, Japan, and China), the methods described in the literature frequently include steps that make them, in our opinion, unnecessary laborious. Such steps include repeated extraction, filtration, clean-up steps involving GPC for non-fatty matrices, column chromatography or solid phase extraction (SPE) manifolds and evaporative concentration. Typical examples are given in Table\u00a01. It will be shown in this paper that most of the laborious steps can be replaced by more efficient alternatives\u2014repeated extraction is not required, an aliquot is taken after settling or centrifugation rather than filtration, use of GCB instead of GPC for removal of chlorophyll, use of dispersive SPE instead of classical SPE for clean-up (analogous to an acetonitrile-based method [29]), and injection of larger volumes into the GC instead of manual evaporative concentration.\nThe objective of the work discussed in this paper was to update and improve the ethyl acetate-based multi-residue method for pesticides in vegetables and fruit in respect of straightforwardness, robustness, and ease and speed of sample and extract handling. Aspects studied include dispersive clean-up using combined GCB\/PSA, the possibility of preventing unacceptable adsorption of \u201cplanar\u201d pesticides by GCB, by addition of toluene, and large-volume (20\u00a0\u03bcL) injection in GC. The method has been validated for 341 pesticides and degradation products which are analyzed by GC\u2013MS or LC\u2013MS\u2013MS. For the latter the initial raw extract was used and injected after a solvent change to methanol\u2013water. The suitability of the method as a multi-residue, multi-matrix method is evaluated by use of analytical quality-control data generated during 15\u00a0months for 271 pesticides and degradation products for over 100 different matrices, including less common and exotic crops. Results obtained for proficiency test samples during three years are also presented.\nExperimental\nChemicals and reagents\nPesticide reference standards were obtained from C.N. Schmidt (Amsterdam, The Netherlands). For GC\u2013MS a mixed stock solution containing 135 pesticides (Table\u00a07; concentration 50\u00a0mg L\u22121 for each pesticide) was obtained from Alltech\u2013Grace (Breda, The Netherlands). The full chemical names of the metabolites of phenmedipham and pyridate are methyl N-(3-hydroxyphenyl)carbamate and 3-phenyl-4-hydroxy-6-chloropyridazine, respectively. Solvents were from J.T. Baker (ethyl acetate, Resi-analysed; Deventer, The Netherlands), Labscan (toluene, Pestiscan), and Rathburn (methanol). Anhydrous sodium sulfate, ammonium formate, potassium dihydrogen phosphate, disodium hydrogen phosphate, acetic acid, and diethylene glycol (all p.A. quality) were from Merck. Water was purified by use of a MilliQ reagent-water system (Millipore).\nBondesil primary secondary amine (PSA, 40\u00a0\u03bcm) was obtained from Varian (Middelburg, The Netherlands) and GCB (graphitized carbon black) was purchased as Supelclean ENVI-carb (120\u2013400 mesh, Supelco, Zwijndrecht, The Netherlands).\nFor GC\u2013MS, in addition to the mixed stock solution, individual stock solutions of other pesticides were prepared in ethyl acetate. From these, additional mixed solutions were prepared in ethyl acetate. For LC\u2013MS\u2013MS analysis, individual stock solutions were prepared in methanol. Mixed solutions were prepared from the individual stock solutions and diluted with methanol. The mixed solutions were used for fortification of samples and for preparation of matrix-matched standards.\nThe extraction solvent was a solution of internal standard (0.05\u00a0mg L\u22121 antor (diethatyl-ethyl)) in ethyl acetate. Matrix-matched standards were prepared by addition of mixed solutions to control sample extracts. Dilution of the sample extract with mixed solution was never more than 10%.\nInstrumentation\nGC\u2013MS analysis\nGC\u2013MS analysis was performed with a model 8000 Top GC equipped with a Best PTV (programmed temperature vaporizer) injector, an AS800 autosampler, and a Voyager mass spectrometer (Interscience, Breda, The Netherlands). The instrument was controlled by Masslab software. The injector was equipped with a 1\u00a0mm i.d. liner with porous sintered glass on the inner surface. The GC was equipped with a 30\u00a0m\u2009\u00d7\u20090.25\u00a0mm i.d., 0.25\u00a0\u03bcm film, HP-5-MS column and a 2.5\u00a0m precolumn (same as the analytical column, connected by means of a press-fit connector).\nFor PTV injection in solvent-vent mode 20\u00a0\u03bcL was injected at 5\u00a0\u03bcL s\u22121. The solvent was vented at 50\u00b0C in 0.67\u00a0min using a split flow of 100\u00a0mL min\u22121. The split valve was then closed and the analytes retained in the liner were transferred to the GC column by ramping the temperature at 10\u00b0 s\u22121 to 300\u00b0C. Total transfer time was 2.5\u00a0min after which the split was re-opened.\nHelium was used as carrier gas at constant flow (1.5\u00a0mL min\u22121). The oven temperature was maintained at 90\u00b0C for 2\u00a0min after injection then programmed at 10\u00b0\u00a0min\u22121 to 300\u00b0C which was maintained for 10\u00a0min. The transfer line to the MS was maintained at 305\u00b0C.\nMass spectrometry was performed with electron-impact (EI) ionization (electron energy 70\u00a0eV) at a source temperature of 200\u00b0C. Data were acquired in full-scan mode (m\/z 60\u2013400), after a solvent delay of 5.5\u00a0min, until 30\u00a0min. Scan time and inter-scan delay were 0.3 and 0.1\u00a0s, respectively, resulting in 2.5 scans s\u22121. The detector potential was 450\u00a0V.\nMasslab software (Interscience, The Netherlands) and an Excel macro developed in-house were used for data handling and quantitative data evaluation.\nLC\u2013MS\u2013MS analysis\nLC was performed with an Agilent, model 1100 instrument comprising degas-unit, pump, autosampler, and column oven. A 4\u00a0mm\u2009\u00d7\u20092\u00a0mm i.d. C18 guard column (Phenomenex) and a 150\u00a0mm\u2009\u00d7\u20093\u00a0mm i.d. LC column (Aqua, 5\u00a0\u03bcm C18, Phenomenex) were coupled to a triple-quadrupole mass spectrometer (model API2000 or API3000, Applied Biosystems, Nieuwerkerk a\/d Yssel, The Netherlands). Analyst 1.2 and, later, 1.4 were used for instrument control and data handling. Additional data processing was performed using an Excel macro developed in-house.\nCompounds were separated by elution with a gradient prepared from methanol\u2013water\u20131\u00a0mol L\u22121 ammonium formate solution, 20:79.5:0.5 (component A) and methanol\u2013water\u20131\u00a0mol L\u22121 ammonium formate solution, 90:9.5:0.5 (component B). The composition was changed from 100% A to 100% B in 8\u00a0min and was then isocratic until 24\u00a0min. The composition was then changed back to 100% A in 1\u00a0min and the column was re-equilibrated for 10\u00a0min before the next injection. The flow rate was 0.3\u00a0mL min\u22121 which was introduced into the MS without splitting. The injection volume was 20\u00a0\u03bcL and 10\u00a0\u03bcL for the API2000 and API3000, respectively.\nData were acquired in multiple-reaction-monitoring (MRM) mode. Electrospray ionization (ESI) (called turbo ion spray for the instruments used) mass spectrometry was performed in positive-ion mode. For the API2000 the nebulizer gas, turbo gas, and curtain gas were 20, 50, and 40 arbitrary units (a.u.), respectively. The ion-spray potential was 5000\u00a0V. Nitrogen was used as collision gas (4 psi). For the API3000 the nebulizer gas and curtain gas were 12 and 10 a.u. and the turbo gas was 7.5\u00a0L min\u22121. The ion spray potential was 2000\u00a0V. Nitrogen was used as collision gas (4 psi). For both instruments, the pause time was 5\u00a0ms. The dwell times for the pesticide transitions varied between 10 and 25\u00a0ms. The precursor and product ions and the collision energy (data for API3000) for each pesticide or degradation product are listed in Table\u00a08. In the acquisition method one transition for each pesticide was measured. All transitions were acquired in one time window. The total cycle time was 2.24\u00a0s resulting in 8\u201310 data points across the peak. To measure the second transition a second method was created and run if confirmation was needed.\nSample preparation\nVegetable and fruit samples were taken from batches of samples as received from the food industry and trade for routine multi-residue analysis. After removal of stalks, caps, stems, etc., as prescribed by 90\/642\/EEC Annex I [34], an amount corresponding, at least, to the minimum size of laboratory samples (usually 1\u20132\u00a0kg [35]) was homogenized in a large-scale Stephan food cutter. A subsample (25\u00a0g) was weighed into a centrifuge tube. Fortification was performed at this stage. Phosphate buffer (pH 7, 4\u00a0mol L\u22121, 2\u00a0mL) and extraction solution (ethyl acetate with internal standard, 40\u00a0mL) were then added. Just before Turrax extraction anhydrous sodium sulfate (25\u00a0g) was added. After Turrax extraction (1\u00a0min) the tubes were centrifuged (sets of four).\nFor GC\u2013MS analysis, Eppendorf cups were prefilled with 25\u00a0mg PSA and 25\u00a0mg GCB. To avoid a weighing step, scoops were made in-house for this purpose. Their accuracy was established to be 25\u2009\u00b1\u20092\u00a0mg (n\u2009=\u200910). For clean-up, 0.8\u00a0mL extract and 0.2\u00a0mL toluene were added to the cup with the SPE materials. The cups were then closed and the samples were vortex mixed for 30\u00a0s and centrifuged (up to 24 at one time). One aliquot was transferred to an autosampler vial with insert, and a second aliquot was transferred to an autosampler vial and stored under refrigeration as back-up extract. The calculated amount of initial sample in the final extract was 0.5\u00a0g mL\u22121.\nFor LC\u2013MS\u2013MS analysis the initial extract (3.2\u00a0mL for the API2000 and 0.48\u00a0mL for the API3000) was transferred to a disposable glass tube. After addition of a solution of diethylene glycol in methanol (10%, 200\u00a0\u03bcL) the extract was evaporated to \u201cdryness\u201d under a gentle flow of nitrogen gas at 35\u00b0C (up to 36 tubes in a heater block). The residue was reconstituted in methanol (1\u00a0mL and 0.75\u00a0mL for the API2000 and API3000, respectively), by use of vortex mixing and ultrasonication (5\u00a0min). The extract was then diluted 1:1 with component A. After centrifugation one aliquot was transferred to an autosampler vial with insert, and a second aliquot was transferred into an autosampler vial and stored under refrigeration as back-up extract. The final extract concentration was 1\u00a0g mL\u22121 and 0.2\u00a0g mL\u22121 for the API2000 and API3000, respectively.\nFor dry products (e.g. cereals) 5\u00a0g was weighed and 20\u00a0mL water was added. After soaking for 2\u00a0h samples were processed as described above. A larger amount of extract was taken for evaporation to compensate for the reduced amount of sample processed and to bring the final extract concentration to 0.2\u00a0g mL\u22121.\nWith the final method, one person can process 30 samples in eight hours. Here processing includes specific preparation before homogenization (i.e. removal of caps from strawberries, etc.), homogenization of the samples, extraction, cleaning the Turrax between samples, clean-up for GC\u2013MS, and solvent switch for LC\u2013MS\u2013MS, i.e. from laboratory sample to ready-to-inject solutions in autosampler vials.\nQuantification\nGC\u2013MS\nFor each pesticide the concentrations were calculated for two diagnostic ions. In previous validation work (not published) using the same software it was found that for most pesticides automatic integration and repeatability of response were better when peak height, rather than area, was used. Peak height was therefore used, with few exceptions (e.g. pesticides prone to tailing, for example 2-phenylphenol). All responses were normalized to the response of the internal standard (antor). One-point calibration was performed using a fixed matrix-matched standard (tomato, see Results and discussion section) at a level corresponding to five times the LOQ. The linearity of the plot of MS response against concentration was verified periodically over the range 0.01 to 1\u20135\u00a0mg kg\u22121. For most pesticides linearity was adequate (relative response within 20% of the calibration standard) up to at least 1\u00a0mg kg\u22121.\nLC\u2013MS\u2013MS\nThe internal standard (antor) was evaluated qualitatively only to confirm injection of the sample extract. Because of unpredictable and varying matrix effects for several of the matrices included in this work, normalization against the internal standard was not considered feasible. For each sample matrix that was fortified, a matrix-matched standard was also prepared by spiking the final extract of the corresponding control sample. Peak area was used for quantification. One-point calibration was performed using the matrix-matched standard at a level corresponding to five times the LOQ. Linearity of the MS response against concentration was verified periodically over the range 0.01 to 1\u00a0mg kg\u22121. For most pesticides, the relationship was linear (relative response within 20% of the calibration standard) up to at least 0.5\u00a0mg kg\u22121.\nValidation\nInitial method validation was performed in accordance with EU guidelines [36, 37]. Two times five portions of the homogenized sample were spiked with a mixture of pesticides at a low level (0.01\u00a0mg kg\u22121 or lower) and at a level ten times higher. Together with two unfortified control portions of the sample, they were processed and analyzed as outlined above.\nAdditional method-performance data were acquired by analyzing fortified samples concurrently with each batch of samples. The spike level (0.05\u00a0mg kg\u22121 for most pesticides) was five times the LOQ. With each batch different products were selected as much as possible. In the compilation the emphasis was on products which are less frequently reported in the literature to challenge the applicability of the method as a \u201cmulti-matrix method\u201d. For this purpose samples were not pre-screened for absence of pesticides and, consequently, occasionally recoveries could not be determined, because of the relatively high levels incurred. Such results were eliminated from the data set.\nSpectrophotometric measurement of removal of chlorophyll\nFor evaluation of the removal of chlorophyll by GCB and comparison with GPC, a lettuce extract was prepared by extracting 25\u00a0g lettuce with 40\u00a0mL ethyl acetate after addition of 25\u00a0g anhydrous sodium sulfate. As a reference, 0.8\u00a0mL ethyl acetate was added to 3.2\u00a0mL of this extract to bring the extract concentration to 0.5\u00a0g mL\u22121. For dispersive SPE, 100\u00a0mg GCB was added to sets of duplicate tubes and 3.2\u00a0mL extract was added to all tubes. Solvent was then added to four sets of tubes: set one 0.8\u00a0mL ethyl acetate, set two 0.4\u00a0mL ethyl acetate and 0.4\u00a0mL toluene (i.e. 10% toluene), set three 0.8\u00a0mL toluene (20% toluene), and set four 0.8\u00a0mL xylene (20% xylene). The extracts were vortex mixed and centrifuged.\nFor GPC clean-up, 2.5\u00a0mL lettuce extract was injected on to a 40\u00a0cm\u2009\u00d7\u200928\u00a0mm i.d. Biobeads SX3 column with 1:1 ethyl acetate\u2013cyclohexane as eluent. The fraction collected was such that at least 50% of the pyrethroids were recovered (fraction from 105\u2013200\u00a0mL). The eluate was first concentrated, by rotary evaporation at 40\u00b0C, to approximately 5\u00a0mL, then transferred to a tube for further concentration, under nitrogen gas, to 2.5\u00a0mL.\nFinal extract concentration before and after clean-up was always 0.5\u00a0g mL\u22121. Aliquots of the extracts were transferred to a cuvet for spectrophotometric analysis at 450\u00a0nm. If required, the extracts were diluted with ethyl acetate to bring absorption within the linear range. The amount of chlorophyll in the uncleaned extract was defined as 100%. For calibration purposes the uncleaned extract was diluted 10, 20, 40, 50 and 100 times with ethyl acetate and a calibration plot was constructed. Chlorophyll remaining after clean-up was determined from the decrease in absorption at 450\u00a0nm compared with the absorption of the uncleaned lettuce extract.\nResults and discussion\nMonitoring of residues in fresh produce for the food industry, especially trade and retail, calls for rapid turnaround, preferably within one or two days. This means sample preparation must be rapid and straightforward. With regard to cost and waste, consumption of solvents and reagents should be low. At the same time, EU directives with regard to sample definition (90\/642\/EEC, [34]) and laboratory sample size (2002\/63\/EC [35]) for residue analysis should be respected. This means, for example, that that a total of 2\u00a0kg grapes (after removal of stalks), five whole melons, or 1\u00a0kg strawberries (after removal of caps) must be processed. The actual analysis is performed on a subsample of the laboratory sample, after appropriate comminution. The more thorough the comminution, the smaller the subsample can be and the lower the amount of solvent needed for extraction. It has, furthermore, been reported that for well homogenized samples extraction by vortex mixing or shaking, instead of high-speed blending (Turrax) suffices for effective extraction [29], although there is still some debate on this matter [38].\nHomogenization\nFor homogenization there are several possibilities. Food choppers or kitchen blenders are often used. Very thorough homogenization can be achieved with the latter, but it is not possible to process the entire laboratory sample at once. For this reason, large-scale food choppers are more suited. With such devices, homogeneity is not always optimum, as can be observed with, e.g., tomatoes, for which small pieces of skin drift in the \u201csoup\u201d obtained after homogenization. Subsampling of very small amounts is, therefore, not acceptable after this procedure, because the subsample would be insufficiently representative of the original sample. More thorough homogenization can be achieved after addition of dry-ice or liquid nitrogen (cryogenic homogenization). This procedure is recommended when reducing the subsample for analysis to 10\u00a0g. This procedure is more laborious, however, because it involves cutting the sample into pieces, freezing the sample (usually overnight), cryogenic comminution, then dissipation of the dry-ice or liquid nitrogen before further processing or storage. It also puts higher demands on the cutter (blades) and requires additional precautions for the operators (protection against low temperatures and noise). Cryogenic comminution has been recommended for some pesticides because it reduces their degradation during this step [39].\nIn recent years the food trade and retail have been intensifying their residue-monitoring programs and require analytical data before harvest, before accepting an assignment, or before releasing their products from distribution centers to supermarkets. For fresh produce this means there is a much pressure on laboratories for rapid turnaround (24\u201348\u00a0h). This is difficult to achieve when the analysis involves overnight freezing for cryogenic comminution. Thus, for reasons of ease and speed, it was decided to retain the current procedure\u2014ambient homogenization of the entire laboratory sample by use of a large scale food cutter (thus accepting the consequence that for a limited number of pesticides the concentration found might be an underestimate). Because of non-optimum homogenization with the food cutter, subsamples should not be too small, and further comminution is required for efficient extraction of systemic pesticides. This can be achieved during extraction by use of an Ultra Turrax. We have previously established the minimum size of subsample that did not negatively affect the repeatability of the analysis. This was done with samples which contained residues. For subsamples (n\u2009=\u20097) of 50 and 25\u00a0g, the relative standard deviation (RSD%) was below 8% for several pesticide\u2013matrix combinations. For pear leaves (regarded as a difficult matrix to homogenize) containing bromopropylate, phosalone, and tolylfluanide it was observed that the RSD increased from <8% to 14\u201318% when the amount of subsample was reduced from 25\u00a0g to 12.5\u00a0g. From this it was concluded that, with our procedure, 25\u00a0g was the minimum required amount of subsample.\npH adjustment\nIn the ethyl acetate-extraction procedure analytes are extracted and partitioned between water (from the matrix itself, or added water for dry crops) and ethyl acetate in one step. For basic and acidic compounds the partitioning can be affected by pH, which can vary substantially with the matrix. Because the same extract is to be used not only for GC\u2013MS but also for LC\u2013MS\u2013MS (after changing the solvent to methanol) which, preferably, should also include analysis of basic and acidic pesticides, control of pH was regarded as necessary. A pH of approximately 6 was chosen as compromise for efficient extraction of basic and acidic compounds. Although acidic pesticides were not included in this work, data in the literature (for barley without pH adjustment, i.e. non-acidic conditions [26]) indicate they are extracted into ethyl acetate.\nFor pH adjustment others have used sodium hydroxide [16\u201318] or sodium hydrogen carbonate [11, 14, 25] (Table\u00a01). A disadvantage of this is that the amount of salt needed depends on the acidity of the sample. Addition of too much will result in a high pH and possible degradation of base-sensitive pesticides. To keep the method as straightforward as possible the pH was adjusted using a solution of concentrated phosphate buffer (4\u00a0mol L\u22121, 2\u00a0mL). A solution was preferred over addition of solid salts because this enabled use of a dispenser and eliminated additional weighing of the salts. The buffer resulted in appropriate pH adjustment for most matrices, although there were exceptions, for example lemon and lime.\nExtraction\nThe two conditions most relevant to extraction efficiency are the sample-to-solvent ratio and addition of salt, which in ethyl acetate-based multi-residue methods has always been sodium sulfate.\nThe amount of ethyl acetate (in mL) relative to the amount of sample (in g) is, typically, at least 2:1. This ratio has been used for many years (Table\u00a01). It results in good extraction efficiency and is practical with regard to achieving phase separation and avoidance of emulsions. To avoid sacrificing decades of method history no attempts were made to reduce the ratio; to do so might also adversely affect recovery and\/or complicate phase separation. Larger amounts (as used by several other laboratories; Table\u00a01) result in greater solvent consumption and more dilute extracts. In previous work [15] it has been shown that the efficiency of extraction of polar pesticides improves with the amount of salt added. When 50\u00a0mL ethyl acetate and 25\u00a0g sample were used, 25\u00a0g sodium sulfate was sufficient to obtain recoveries of 80% or better, even for very polar and highly water-soluble compounds, for example acephate and methamidophos. Because these recoveries were obtained with a single extraction it was found unnecessary to perform repeated extraction, as some laboratories are doing [11, 18, 20, 21]. For addition of the sodium sulfate an automatic salt-dispenser coupled to a balance, as is used in our laboratory, or a scoop, was found to be very convenient.\nThe extraction procedure involves successive addition of buffer, extraction solution (ethyl acetate with internal standard), and sodium sulfate to the centrifuge tube containing the sample, after which the pesticides are extracted and partitioned in one step using a Turrax. During this step the subsample is further comminuted for efficient extraction of the pesticides from the matrix. Vortex mixing, shaking or sonication were regarded as less efficient for subsamples that were homogenized in a large-scale food cutter under ambient conditions, but this was not investigated, partly because a variety of samples containing residues would be required to do so in an appropriate manner.\nIt was noted from the literature that filtration is often performed to separate the solid pellet from the liquid. Again, there is no real need for this step, which involves additional glassware and, occasionally, rinsing (diluting) of the extract. For many samples a clear ethyl acetate extract is obtained after settling; if not the tubes can be centrifuged. This is no more laborious than filtration and does not involve additional glassware.\nBecause the same Turrax is used for several samples, carry-over is an aspect to be considered. Between samples the Turrax is cleaned first by rinsing with water, by means of a flow-through beaker, then by brief immersing in two beakers containing ethyl acetate. Using this procedure, carry-over was tested by analyzing a blank after a sample that had been fortified at 5\u00a0mg kg\u22121. Carry-over was less then 0.1%, indicating that the straightforward cleaning procedure was sufficient to avoid cross-contamination up to 5\u00a0mg kg\u22121 when setting reporting limits not lower than 0.01\u00a0mg kg\u22121.\nGC\u2013MS analysis\nClean-up\nIn ethyl acetate-based multiresidue methods either no clean-up or GPC clean-up is performed. This has hardly changed over the years (Table\u00a01). In contrast with acetone and acetonitrile-based methods, in which SPE is commonly employed, this has been reported only occasionally for ethyl acetate-based methods. Obana et al. [10] used a cartridge packed with layers of water-absorbing polymer and GCB. Sharif et al. [21] described a clean-up using SAX\/PSA but the scope of the method was restricted to organochlorine and organophosphorus pesticides. Zhang et al. [20] used a clean-up based on Florisil and achieved adequate recovery of many pesticides but not the more polar organophosphorus pesticides. It has been stated that in GC analysis with use of highly selective detectors, for example MS\u2013MS no clean-up is required, even when injecting 15\u00a0mg equivalent of matrix (green bean, tomato, pepper, cucumber, marrow, egg plant, and water melon [40]). Other laboratories experienced problems with contamination of the GC inlet and tried to solve this by automatic exchange of liner inserts [14, 41]. This is in agreement with our experience that injection of 10\u00a0mg matrix equivalent, especially for leafy vegetables, does result in rapid deterioration of system performance because of accumulation of non-volatile material in the inlet. This makes the system less robust, and frequent exchange of the liner (daily) and GC\u2013pre column (weekly) is required. Another problem encountered with injection of the uncleaned extracts was a shift in the retention times of pesticides relative to that of the calibration standard for some sample extracts. This shift was insufficiently corrected by automatic adjustment of retention times relative to that of the internal standard. Typically, shifts were in the range 0.05\u20130.20\u00a0min and were most abundant for the \u201cazole\u201d pesticides. Such shifts can complicate automatic peak assignment during data-handling. When data acquisition is performed in a non-continuous mode (e.g. selected-ion monitoring or MS\u2013MS) such shifts also increase the risk of pesticides shifting from their acquisition window. For injection of relatively large amounts of matrix (e.g. 10\u00a0mg) in GC analysis clean-up for removal of bulk co-extractants is therefore regarded as a prerequisite for robust analysis of a wide variety of vegetable and fruit matrices.\nFor vegetables and fruit matrices, chlorophyll (MW \u223c900) and other pigments, for example carotenoids (e.g. \u03b2-carotene, MW 537) are typical bulk co-extractants. Most of these compounds are of low volatility and are not apparent as interferences in the chromatograms; they do, however, accumulate in the liner of the GC and eventually have an adverse effect on transfer of analytes to the column and\/or on peak shape. Because of its high molecular weight, chlorophyll can be removed by GPC. A disadvantage is that the extract is strongly diluted and reconcentration by rotary evaporation is almost inevitable when LODs of 0.01\u00a0mg kg\u22121 are required. Such a step would contribute substantially to overall sample-preparation time. Although a very efficient on-line combination of GPC and GC\u2013MS was described recently [42], avoiding GPC whenever possible would be even more straightforward. Solid-phase extraction is an alternative clean-up procedure which involves less dilution and is less laborious. Even more efficient is SPE in the so-called dispersive mode, as described by Anastassiades et al. [29]. Here the solid phase is simply added to the extract, thereby avoiding typical SPE procedures such as conditioning, sample transfer, elution, and evaporative reconcentration. The pesticides partition between the solid phase and the solvent and after vortex mixing and centrifugation the supernatant is ready for analysis.\nTwo stationary phases, graphitized carbon black (GCB) and phases with amino functionality, have been shown to be particularly effective for removing co-extracted material from the raw extract while not removing most of the pesticides; this makes them very suitable for wide-scope methods [28, 29, 31, 38, 43\u201345].\nInitially, a method was envisaged using SPE column clean-up with GCB, because for leafy vegetables this was found to be the only sufficiently effective alternative to GPC. After the publication on dispersive SPE [29] it was decided to investigate this approach, thus sacrificing some clean-up potential (as has been reported in the literature [31]) for ease and speed.\nGCB is well known to adsorb planar molecules, including chlorophyll and other pigments but also pesticides with planar functionality. In acetonitrile-based methods, toluene (typically 25%) is often added to the eluent to desorb these pesticides also from the SPE column [28, 38, 43, 45]. One of the objectives of this work was to investigate the possibility of using GCB in a dispersive clean-up step without unacceptable losses of planar pesticides. First we investigated which pesticides, dissolved in ethyl acetate, are adsorbed by GCB. A somewhat arbitrary, 25\u00a0mg mL\u22121 GCB phase was added to standard solutions. After vortex mixing and centrifugation the solution was analyzed by GC\u2013MS (165 pesticides) and, after changing the solvent to methanol, by LC\u2013MS\u2013MS (another 70 pesticides), and the responses were compared with those from untreated standard solutions. For 35 pesticides (15%) adsorption was observed (Table\u00a02). In addition to the pesticides included in this test, it is known from the literature [44] that chinomethionate, furametpyr, and pyraclofos are also adsorbed by GCB (from acetone\u2013cyclohexane, 1:4).\nTable\u00a02Pesticides adsorbed by GCBaStrong adsorption (rec. 0\u201350%)Medium adsorption (rec. 50\u201370%)Not consistentMeasured by GC\u2013MS\u00a0ChlorothalonilAzinphos-ethylPhosmet\u00a0CyprodinilAzinphos-methylProchloraz\u00a0FenazaquinChlorpyrifos-methylPyrazophos\u00a0HexachlorobenzeneDicloranTrifluralin\u00a0MepanipyrimEPN\u00a0PentachloroanilineFenamiphos\u00a0PhosalonePhorate\u00a0PyrimethanilQuintozene\u00a0QuinoxyfenMeasured by LC\u2013MS\u2013MS\u00a0CarbendazimFenpyroximate\u00a0ClofentezineFlufenoxuron\u00a0DesmediphamTricyclazole\u00a0DiflubenzuronTriflumuron\u00a0FlucycloxuronThiophanate-methyl\u00a0Hexaflumuron\u00a0Phenmedipham\u00a0Pymetrozine\u00a0ThiabendazoleaPesticides in ethyl acetate, 25\u00a0mg GCB mL\u22121 solventrec., recovered\nTo investigate how much toluene is required to prevent adsorption of planar pesticides by GCB in dispersive SPE, the partitioning experiment was repeated with standard solutions of 10, 20, or 30% toluene in ethyl acetate. This was done for the GC\u2013MS pesticide mixture only.\nAs is apparent from Fig.\u00a01, even 10% toluene dramatically improved recovery. With 20% toluene recovery of all pesticides was higher than 65%. It should be noted that this experiment with standard solutions is the worst case. For real samples chlorophyll and carotenoids will also affect the distribution in favor of the pesticides in solution. Use of 30% of toluene further improved recovery only slightly. Twenty percent was regarded as optimum with regard to distribution and ease of solvent elimination in large-volume injection (see below). In addition to toluene, two alternative analogues, benzene and xylene, were also considered. Benzene, was not tested because it could not be used in routine practice because of its carcinogenic properties (although it would have been favorable with regard to solvent elimination). Xylene was tested in a similar way as toluene. Results obtained for hexachlorobenzene and chlorothalonil by use of the two solvents are compared in Fig.\u00a02. Slight but consistently better recovery was obtained with xylene\u2014>70% recovery could now be obtained for all pesticides. Because of its greater volatility, however, toluene was finally selected.\nFig.\u00a01Effect of the amount (%) of toluene in ethyl acetate on recovery of pesticides adsorbed by GCB (25\u00a0mg mL\u22121). hcb, hexachlorobenzene; pca, pentachloroaniline; ctn, chlorothalonil; mep, mepanipyrim; cypr, cyprodinil; pyri, pyrimethanil; fena, fenazaquin; quin, quinoxyfen; pyra, pyrazophos; epn, EPNFig.\u00a02Comparison of toluene and xylene as additives for preventing adsorption of planar pesticides by GCB in dispersive SPE\nObviously, toluene is also likely to affect adsorption of chlorophyll and\/or carotenoids and might reduce the effectiveness of clean-up. To investigate this, a lettuce extract was prepared, the dispersive clean-up experiments were performed with different amounts of toluene, and removal of chlorophyll was verified. Visually it was clearly apparent that, despite addition of toluene, the intense green color turned light yellow, indicating that chlorophyll was removed to a large extent. To enable more quantitative evaluation, the extracts were also measured with a spectrophotometer at 450\u00a0nm. For comparison, the same extracts were also cleaned by GPC. The results are presented in Table\u00a03. Without toluene, chlorophyll was very effectively removed. Absorption at 450\u00a0nm was reduced by 94%. Toluene, as expected, reduced adsorption of chlorophyll, but removal was still 87% or 78%, after addition of 10% or 20% toluene in ethyl acetate, respectively. Similar to observations with the planar pesticides, adsorption was reduced slightly more by use of xylene than by use of toluene. With GPC, chlorophyll removal was 60%. It should be noted here that the elution window was relatively wide, to include pyrethroids within the scope of the method. The elution windows for chlorophyll (and carotenoids) partially overlap those for pyrethroids, as has also been reported by others [44]. From these experiments it can be concluded that chlorophyll has more affinity than the planar pesticides for GCB. In dispersive SPE toluene effectively prevents unacceptable adsorption of planar pesticides while to a large extent maintaining its cleaning properties in respect of chlorophyll. Dispersive GCB not only enables much faster chlorophyll removal, it is also more effective when including pyrethroids in the scope of the method. For non-fatty vegetable and\/or fruit matrices, therefore, GPC is not required and dispersive GCB clean-up is a much faster alternative without sacrificing scope.\nTable\u00a03Removal of chlorophyll by dispersive SPE (GCB) and GPCClean-up procedureChlorophyll removal (%)Dispersive SPE, 100% ethyl acetate94Dispersive SPE, 10% toluene in ethyl acetate87Dispersive SPE, 20% toluene in ethyl acetate78Dispersive SPE, 20% xylene in ethyl acetate71GPC (fraction incl. pyrethroids)60Sample extract: lettuce 0.5\u00a0g mL\u22121. Dispersive SPE: 25\u00a0mg GCB mL\u22121. GPC: wide scope elution window, i.e. including pyrethroids.\nThe GCB clean-up enabled continuous injection of extracts of leafy vegetables without rapid system deterioration. With some matrices, however (e.g. plums, grapefruit), retention time shifts were still observed. In addition, depending on the matrix, quite intensive interferences could be observed in the GC\u2013MS TIC chromatograms. Further clean-up by PSA, complementing the GCB clean-up by removing compounds such as organic acids and sugars by hydrogen bonding, was therefore investigated. To keep sample clean-up as straightforward and rapid as possible focus was on a combined dispersive GCB\/PSA clean-up.\nAfter the outcome of the GCB experiments, partitioning of the pesticides and co-extractants will be between PSA and ethyl acetate\u2013toluene, 8:2. Because no information was available about the distribution of pesticides between these two phases, this was obtained by analyzing pesticide standards in ethyl acetate\u2013toluene, 8:2, with and without PSA. Preliminary experience with dispersive PSA clean-up revealed that with some matrices (e.g. cereals) 25\u00a0mg mL\u22121 did not result in complete elimination of interfering compounds (e.g. fatty acids) typically removed by PSA. Partitioning with a much larger amount of adsorbent (200\u00a0mg mL\u22121) was, therefore, also studied.\nWith 25\u00a0mg mL\u22121 losses of 30\u201340% were observed for sixteen pesticides, most probably as a result of adsorption, although the possibility of degradation induced by the basic nature of the PSA material could not be fully excluded. The findings were confirmed by the experiment with 200\u00a0mg PSA mL\u22121 (Table\u00a04). The pesticides for which interaction with PSA was observed all had a C=O or P=O group in common (except for chlorothalonil). Our findings are not in full agreement with those of Anastassiades et al. [29] who did not observe losses as a result of using PSA. For this there can be two explanations. In our experiment adsorption was tested with standard solution rather than matrix. Co-extractants in matrix are likely to compete with the pesticides during adsorption. Second, with our method the organic phase (ethyl acetate\u2013toluene, 8:2) is less polar than the acetonitrile phase; this could result in a stronger interaction between the polar functionality of the pesticides and amino functionality of PSA. From our results it became clear that with regard to the amount of PSA \u201cthe more, the better\u201d does not apply. Another observation was that a hump appeared in the TIC chromatogram after a 20-\u03bcL injection of solvent mixed with 200\u00a0mg PSA mL\u22121. This hump, which eluted between 6 and 12\u00a0min, consisted of many peaks and a variety of masses. Cleaning of the PSA by washing with ethyl acetate (3\u2009\u00d7\u200920\u00a0mL for 1\u00a0g), then drying by rotary evaporation, eliminated this contamination without affecting the clean-up properties. To keep the method straightforward, 25\u00a0mg PSA mL\u22121 was used as default, and the material was not cleaned before use.\nTable\u00a04Adsorption of pesticides by PSAPesticideRecovery (%)Acephate43aAcrinathrin41bAsulam0aCarbaryl56bChlorothalonil17bCycloxidim39aDichlorvos33bDimethoate62bHymexazol0aMevinphos62bPhosmet25bPhosphamidon63bProfenofos56bPyridate40aPyridate-metabolite7aSethoxydim48aaAfter partitioning with ethyl acetate, 25\u00a0mg mL\u22121 and LC\u2013MS\u2013MS analysisbAfter partitioning with ethyl acetate\u2013toluene, 8:2, 200\u00a0mg PSA mL\u22121 and GC\u2013MS analysis\nThe clean-up proved effective at reducing retention time shifts. As an example, for a plum extract without clean-up, the retention times of 24 pesticides (out of 140) were shifted by more than 0.05\u00a0min compared with the calibration standard. After clean-up this occurred for three pesticides only. With other matrices also shifts were reduced, but for some matrices (herbs, e.g. parsley) deviations were still quite common.\nAs an illustration of the removal of co-extractants from the ethyl acetate extract (or, in fact, from the ethyl acetate\u2013toluene, 8:2, extract) by dispersive GCB\/PSA clean-up, GC\u2013MS total ion current chromatograms of extracts obtained with and without clean-up are shown in Fig.\u00a03. The most apparent differences are indicated. Several abundant matrix peaks are removed or strongly reduced. For lettuce, the overall background level between 15 and 25\u00a0min was also reduced. This clearly visible clean-up was mainly caused by the PSA material. With GCB alone differences between cleaned and uncleaned were much less apparent. The main benefit of GCB was prevention of rapid build up of non-volatile material (chlorophyll) in the liner, which enables prolonged use of the system without maintenance. Experience with method for more than three years and analysis of over 15,000 vegetable and fruit samples shows that, on average, the liner must typically be replaced weekly (after 150\u2013200 injections; iprodion, dimethipin, and chlorfenapyr are the first for which response is lost). Further GC\u2013MS maintenance consists in replacement of pre-column once of twice a month. The GC column is replaced approximately twice a year. The source of the MS is cleaned once a month.\nFig.\u00a03GC\u2013MS chromatograms. Overlay total ion chromatograms (TICs) obtained after 20\u00a0\u03bcL injection of an extract of mandarin (top) and lettuce (bottom) without (higher peaks) and with clean-up\nIn a continuing search for even further simplification of sample preparation, the possibility of combined extraction and dispersive SPE clean-up in one step was investigated. For two matrices (lettuce and mandarin, fortified with 140 pesticides, triplicate experiments) the solid phase materials (GCB\/PSA, relative amounts similar to previous experiments) were added directly to the centrifuge tube containing the sample, sodium sulfate, and the extraction solvent (to which 20% toluene had been added). After Turrax extraction and centrifugation, the extract was ready for injection into the GC. Recovery was compared with that obtained by use of dispersive clean-up after separation of the ethyl acetate extract from the sample mixture. As could be seen from the color of the extract (the lettuce extract was almost colorless) the GCB remained effective. Adsorption of chlorophyll is based on planarity (shape) rather than polarity and, therefore, this will occur from both the aqueous and the organic phases. As was to be expected, the same was not true for PSA. The presence of water prevented adsorption of co-extractants with a hydroxyl group, i.e. almost identical GC\u2013MS total-ion chromatograms were obtained from extracts which were not cleaned and from those cleaned in the centrifuge tube. Pesticide recovery obtained after use of successive or simultaneous dispersive SPE clean-up was very similar, although recovery of some pesticides in the combined approach was too high, because of co-elution of interferences. The final method therefore used successive extraction and dispersive SPE clean-up.\nLarge-volume injection\nGC\u2013MS analysis of sample extracts was performed in full-scan mode. This enables detection of any GC\u2013amenable pesticide. Because system LOQ for a quadrupole mass spectrometer in full-scan mode is limited, conservatively estimated at 100\u00a0pg, 10\u00a0mg matrix equivalent must be introduced into the GC to reach a target LOQ of 0.01\u00a0mg kg\u22121. With an extract concentration of 0.5\u00a0g mL\u22121, this means 20\u00a0\u03bcL must be introduced into the GC. Off-line tenfold evaporative concentration and then 2\u00a0\u03bcL injection could also be performed, but this would involve clean-up of larger volumes of extract, the risk of loss of the volatile pesticides (e.g. dichlorvos), and an additional step in sample preparation. Although large-volume injection in GC is a well established technique [47, 48], many routine laboratories are still reluctant to apply it; if they do, the volume is often restricted to 5\u201310\u00a0\u03bcL. Such volumes can be accommodated in liners with a frit or even in empty (baffled) liners when injection speed is carefully adjusted. For larger volumes there is a risk of flooding [46], i.e. that extract is lost as liquid through the split exit. To prevent this, liners can be packed with a variety of materials. Packing materials often have the disadvantage of a large surface area with active sites, however, resulting in degradation and\/or adsorption of thermo labile and\/or polar pesticides; problems can also be encountered with splitless transfer of higher boiling pesticides (e.g. deltamethrin) from the liner to the GC column. Other disadvantages can be a pressure drop over the liner (slows down solvent elimination) and liner-to-liner variability requiring re-optimization of the solvent-elimination process after liner replacement. A means of by-passing the disadvantages of packed liners while still achieving accommodation of 20\u201350\u00a0\u03bcL of liquid was described in 1993 by Staniewski and Rijks [49]. They developed a liner with a sintered porous glass bed on the inner surface wall of the liner. The liquid is retained in the porous glass bed. The potentially active glass surface area is relatively small compared with the materials in packed liners. The gas flow is not obstructed, because the centre of the liner is empty. This enables efficient solvent vapor removal during solvent elimination and efficient transfer of analytes to the analytical column during splitless injection after solvent elimination. Since the early 2000s such liners have been commercially available for PTV injectors from several suppliers, and since then our laboratory has implemented 20\u00a0\u03bcL as default injection volume for ethyl acetate.\nAfter the development of the dispersive GCB clean-up, the solvent to be introduced into the GC contained 20% toluene, which might effect the processes involved in large-volume injection differently from 100% ethyl acetate. Because toluene does not evaporate azeotropically with ethyl acetate and is less volatile, it will be the main solvent left at the end of the evaporation process. Injection of 20\u00a0\u03bcL 20% toluene in ethyl acetate means that 4\u00a0\u03bcL toluene is introduced. The PTV used in this work was equipped with a 1\u00a0mm i.d. porous glass bed liner that could hold approximately 30\u00a0\u03bcL within the zone that is appropriately heated during splitless transfer. Up to this volume there is no need for optimization of injection speed. To obtain information about splitless transfer of the last few microliters of toluene after solvent elimination, cold splitless injections of 1, 2, and 3\u00a0\u03bcL of standards in 100% toluene were performed. Even with 2-\u03bcL volumes peak distortion (fronting peak shape) was observed for pesticides of medium volatility. With 1\u00a0\u03bcL injections peak shape was good and for several pesticides even better than for ethyl acetate. On injection of 20\u00a0\u03bcL standard in ethyl acetate\u2013toluene, 8:2, in the solvent-vent mode, no peak distortion was observed, indicating that less then 2\u00a0\u03bcL toluene remained in the injector after the solvent-vent step. As observed earlier with large-volume injection of ethyl acetate, the vent time (here set at 40\u00a0s using an initial PTV temperature of 50\u00b0C) was not at all critical, even for the most volatile pesticide (dichlorvos). Venting for 35 or 50\u00a0s did not dramatically affect responses or peak shape of the pesticides. In our experience, this phenomenon is typical for porous glass bed liners and contributes to the robustness of the method.\nValidation of GC\u2013MS method\nIn the past a method based on simple ethyl acetate extraction followed by direct GC\u2013MS analysis of the raw extract [4] had been validated for concentrations in the range 0.05\u20130.5\u00a0mg kg\u22121. The modified method described here involved a dispersive clean-up step, large-volume injection, and injection of ten times more matrix into the GC. Re-validation was therefore required, and focused on method performance at low concentrations. This was done using lettuce as matrix. The validation set consisted of two control samples, five fortifications at 0.001\u20130.05\u00a0mg kg\u22121 and five fortifications at a level ten times higher. Over 200 pesticides were included in the validation procedure. The results are presented in Table\u00a05. For the 0.01\u20130.5\u00a0mg kg\u22121 concentration range the EU criteria (recovery 70\u2013110%, RSD 30%, 20%, or 15% for \u22640.01, >0.01\u20130.1, and >0.1\u20131\u00a0mg kg\u22121, respectively [37]) were met for 184 of the 201 pesticides included in the validation. At a level a factor of ten lower (fortification in the 0.001\u20130.01\u00a0mg kg\u22121 range for most pesticides) 147 pesticides could still be detected and for most (78%) of these recovery and RSDs were acceptable. For many pesticides S\/N ratios were surprisingly good and background-corrected mass spectra often contained sufficient diagnostic ions (or were even recognizable mass spectra) to enable identity confirmation, as is illustrated in Fig.\u00a04. The limits of detection, defined as S\/N\u2009=\u20093 for one favorable diagnostic ion for each pesticide, were determined on the basis of the signals from the low fortification levels and the average noise observed in duplicate control samples. The LOD was at or below 0.001\u00a0mg kg\u22121 for 78 pesticides, between 0.001 and 0.005\u00a0mg kg\u22121 for 73 pesticides, between 0.005 and 0.01\u00a0mg kg\u22121 for 29 pesticides, between 0.01 and 0.05\u00a0mg kg\u22121 for 16 pesticides, and higher for four pesticides.\nTable\u00a05GC\u2013MS re-validation data for pesticides in lettuce\u00a0PesticidetR (min) m\/z (quant)Level (mg kg\u22121)Rec. (%)RSD (%)Level (mg kg\u22121)Rec. (%)RSD (%)LOD (mg kg\u22121)1Acephate10.451360.0263540.2575890.0062Acrinathrin22.062890.018118150.1789490.0033Aldrin16.582650.003139250.0319420.0024Atrazine14.172150.00291210.0189870.0025Azinphos-methyl21.641600.0111990.09811070.0096Azoxystrobin25.803440.018280.0999250.0037Benalaxyl19.821480.0058590.0479080.0028Benzoylurea (deg)a8.9014111350.02511069Bifenthrin20.911810.0078490.0688913\u22640.00110Biphenyl9.811540.00697100.0631015\u22640.00111Bitertanol22.891700.0038390.0319040.00212Bromophos17.023310.0039970.0321052\u22640.00113Bromopropylate20.943430.003103130.0328950.00114Bromuconazole20.861730.002109120.024916\u22640.00115Bupirimate18.722730.0036180.0329150.00116Buprofezin18.681720.00285140.0199280.00117Cadusafos13.461580.002117180.02192110.00118Carbaryl15.841150.0049390.049380.00219Carbofuran14.101640.0038870.0339330.00220Chlordane, alpha-17.813730.001**0.0159240.00221Chlordane, gamma-18.123730.0028470.0159640.00122Chlorfenvinphos17.473230.0038460.039750.00123Chloroaniline, 3-7.491270.002**0.02525460.00324Chlorobenzilate19.102510.005**0.059540.01025Chlorothalonil15.052640.004146150.0421369\u22640.00126Chlorpropham13.081710.006**0.0599560.01527Chlorpyrifos16.673140.003102160.03410250.00228Chlorpyrifos-methyl15.702860.00110550.0151026\u22640.00129Chlorthal-dimethyl16.773010.0059070.0519140.00130Cinerin-118.671500.0538430.5289360.04131Clofentezine22.453040.014**0.14101140.05032Cyfluthrin I23.332260.0419170.4079360.02333Cyfluthrin II23.602260.04110080.4078880.01634Cyhalothrin-lambda21.911810.003110100.0299360.00235Cypermethrin-I23.651630.018107290.1849650.00836Cypermethrin-II23.831810.01894160.1849750.00637Cypermethrin-III24.071810.01896100.1849660.01338Cyproconazole18.972220.00672200.0598870.00139Cyprodinyl17.192240.005105250.0518510\u22640.00140Cyromazine14.471660.013**0.1382560.04041DDE, o,p\u2032-17.902480.002**0.0159230.00942DDE, p,p\u2032-18.502480.001110110.0151005\u22640.00143DDT, o,p\u2032-19.322350.00110290.0159470.00144DDT, p,p\u2032-20.282350.00286110.0169580.00145Deltamethrin25.442530.02211490.22310650.01446Demeton-S-methyl-sulfone16.111690.0371150.3029190.00447Desmethylpirimicarb15.421520.003**0.0267670.00548Diazinon14.701370.00298140.0199430.00149Dichlofluanid16.412240.0047990.044988\u22640.00150Dichlorvos8.001850.00210760.018927\u22640.00151Dicloran13.962060.00396160.02910620.00352Dicofol (as DCBP)16.752500.005**0.049126330.01053Dieldrin18.562630.004**0.0419560.00554Diethofencarb16.532670.0059850.0469660.00155Difenoconazole-I25.123230.02994100.2889530.00656Difenoconazole-II25.363230.0299190.2889930.00357Diflubenzuron (deg)6.631530.00512490.0510720.00258Dimethoate13.971250.009**0.0919140.01759Dimethomorph25.883010.0219570.2078750.00260Diniconazole19.542680.002**0.01889120.00361Diphenylamine12.761690.00386100.0287215\u22640.00162Disulfoton14.81880.00510150.059630.00263DMSA13.192000.0058790.0529270.00264DMST14.372140.005**0.05373320.01965Dodemorph16.951540.00567260.0469170.00266Edifenfos18.073100.00596100.059480.00167Endosulfan-alpha18.08239+1970.005**0.0479350.01068Endosulfan-beta19.19195+2410.005**0.0468710.02069Endosulfan-sulfate19.98274+2370.00582100.0479740.00470Endrin20.942450.005**0.0519080.00671EPN20.571690.01103230.0999470.00172Epoxiconazole20.551940.007**0.0669210.01073Esfenvalerate24.771250.004**0.0369850.00874Ethion19.362310.003**0.039730.00775Ethoprofos12.861580.00388170.0269350.00176Etofenprox23.851640.005100110.0499350.00477Etridiazole10.742110.0149580.1389840.00178Etrimfos15.012920.0039640.025935\u22640.00179Famoxadone25.903300.019790.19650.00380Fenamiphos18.233030.0159760.1549111\u22640.00181Fenarimol22.131390.004**0.03810140.00882Fenazaquin21.221600.003152120.02711480.00183Fenbuconazole23.301290.003**0.039230.00684Fenhexamid20.101770.003**0.0269070.00485Fenitrothion16.252600.001**0.0159580.00386Fenoxycarb20.891160.01511780.1549440.00287Fenpiclonil20.782380.0078850.0719280.00388Fenpropathrin21.051810.00577130.0592130.00189Fenpropimorph16.631280.001**0.019320.00290Fenthion16.632780.0029970.023995\u22640.00191Fenvalerate24.541670.004**0.03610380.00692Fipronil17.573670.0028160.024949\u22640.00193Flucythrinate-I23.771990.01793110.1749210.00494Flucythrinate-II18.511990.0179460.1749340.00495Fludioxonil19.052480.003113130.0279730.00196Flufenoxuron (deg)14.793310.012104130.118118190.00597Flusilazole18.702330.0066880.055876\u22640.00198Flutolanil18.303230.0038190.025868\u22640.00199Fluvalinate, tau-24.802500.02595110.2459550.004100Folpet17.651470.01696160.15991150.009101Fonofos14.552460.0059460.0499270.001102Formetanate15.271220.05**0.498102620.188103Formothion15.271700.005102130.0498940.004104Fuberidazole15.791840.00583290.05155170.001105Furalaxyl17.592420.00595100.05110190.002106Heptachlor12.192720.001**0.0149250.003107Heptachlorepoxide-I17.453530.003**0.03397120.004108Heptachlorepoxide-II17.363530.00196130.015948\u22640.001109Heptenophos12.241240.0039550.03933\u22640.001110Hexachlorobenzene18.332840.00575280.04996150.001111Hexaconazole18.322160.002**0.028770.003112Imazalil18.372150.00579500.0577140.002113Iprodione20.753160.01210870.129540.004114Isofenphos17.462130.005**0.0519330.010115Jasmolin-I19.361230.053**0.5287750.100116Kresoxim-methyl18.732060.0149560.1399190.005117Lindane14.411830.00286180.029960.001118Linuron16.352480.005**0.0487990.010119Lufenuron (deg)11.481760.011123200.11476340.004120Malathion16.431730.003**0.0349850.005121Mecarbam17.493290.003**0.0299350.004122Mepanipyrim18.072220.001**0.0139280.002123Mepronil19.542690.002**0.02387100.005124Metalaxyl15.952060.00392100.0289750.002125Metaldehyde8.87890.005**0.05111620.021126Methacrifos11.281800.00397170.029854\u22640.001127Methamidophos7.751410.02636240.25847150.005128Methidathion17.821450.00381200.0310150.001129Methiocarb16.261680.002109590.0277460.001130Methoxychlor21.032280.002**0.02590100.003131Metoprene17.56730.0110450.1039330.003132Mevinphos10.361920.003104160.03991\u22640.001133Monocrotophos13.431920.0468480.4568870.021134Myclobutanil18.661500.006**0.0559750.012135Nuarimol20.283140.005**0.0498970.008136Omethoate12.391560.00557190.05453140.002137Oxadixyl19.381630.012**0.1249240.038138Oxydemeton-methyl (deg)6.631100.005**0.0527970.010139Paclobutrazole18.112380.007197280.07906\u22640.001140Parathion16.692910.011106260.1069160.004141Parathion-methyl15.712630.0028870.021942\u22640.001142Penconazole17.352480.00390100.03944\u22640.001143Permethrin-cis22.651830.00510170.0499870.003144Permethrin-trans22.771830.001**0.0119870.001145Phenothrin-I21.401830.0059780.059290.001146Phenothrin-II21.511230.0059360.0593100.004147Phenthoate17.532740.00510380.0489150.001148Phenylphenol, 2-11.561700.0059660.0529540.001149Phorate13.562600.0059860.059250.001150Phosalone21.611820.00111750.0091015\u22640.001151Phosmet20.901600.005123160.0521004\u22640.001152Phosphamidon-I14.751270.01193160.1059030.002153Phosphamidon-II15.491270.0118990.1059120.005154Piperonyl butoxide20.361760.004**0.03789100.010155Pirimicarb15.251660.00210190.02955\u22640.001156Pirimiphos-methyl16.262330.002**0.0168720.004157Prochloraz22.971800.004**0.03810160.007158Procymidone17.682850.003104150.0299170.001159Profenofos18.423370.0059780.05295100.001160Propargite20.313500.01**0.1029670.020161Propham10.731790.0059750.0499450.001162Propiconazole-I19.892590.0149250.1418990.003163Propiconazole-II20.022590.0149050.1418790.002164Propoxur12.621100.0029660.02927\u22640.001165Propyzamide14.581750.00576390.0469920.001166Prothiofos18.372670.00385190.03210190.001167Pyrazophos22.172210.003137110.031454\u22640.001168Pyrethrins19.621230.053**0.52899130.087169Pyridaben22.821470.0059690.0519430.001170Pyridaphenthion20.801990.00599100.0489350.003171Pyrifenox-I17.392620.0118470.1069560.003172Pyrifenox-II14.682640.011**0.1069060.170173Pyrimethanil14.651980.002135140.021234\u22640.001174Pyriproxyfen21.651360.002119180.024916\u22640.001175Quinalphos17.551460.0047090.0418780.002176Quinoxyfen19.902720.001113130.01410513\u22640.001177Quintozene14.502370.005106100.04610820.003178Simazine16.172010.0049190.0399570.002179Spiroxamine15.671980.01899170.1768120.009180TDE, o,p\u2032-18.672350.0039950.028954\u22640.001181TDE, p,p\u2032-19.362350.00186100.014907\u22640.001182Tebuconazole20.282500.009**0.0899190.031183Tebufenpyrad21.121710.00592170.0528770.001184Tecnazene12.562030.00510860.0489960.002185Teflubenzuron (deg)8.121970.003174250.025124250.002186Tefluthrin14.911970.001**0.01489140.002187Terbufos14.462310.00510080.052953\u22640.001188Tetraconazole16.853360.0039530.026886\u22640.001189Tetradifon21.443560.003**0.039480.010190Thiometon13.78880.0059350.0551003\u22640.001191Tolclofos-methyl15.802650.0019160.011025\u22640.001192Tolylfluanid17.422380.00385170.0319620.002193Triadimefon16.752080.00790140.0659760.005194Triadimenol17.851680.005**0.0538520.029195Triazamate17.952420.003**0.02890100.010196Triazophos19.622570.005109370.05489200.001197Trifloxystrobin19.921160.00691130.05588110.002198Triflumizole17.702780.007102150.06680150.001199Trifluralin13.333060.00292190.019948\u22640.001200Vamidothion17.95870.019**0.18710050.045201Vinclozolin15.711980.00597160.0479370.003aBenzoylurea(deg) = 2,4-difluorobenzamideLOD: Amount for which S\/N\u2009=\u20093, or in the event of an interfering peak, the average peak height for fortified sample (n\u2009=\u20095) should be 3.3 times the average peak height for control sample (n\u2009=\u20092)*Fortification level below LOD as defined aboveUnderlined values are outside EU criteria for method validationFig.\u00a04GC\u2013MS extracted-ion chromatograms obtained from lettuce with (upper traces) and without fortification with pesticides, and the corresponding mass spectra (upper, reference spectra; lower, background-corrected spectra from the sample). a, b, 0.005\u00a0mg kg\u22121 disulfoton (m\/z 88); c, d, 0.002\u00a0mg kg\u22121 fipronil (m\/z 367); e, f, 0.006\u00a0mg kg\u22121 biphenyl (m\/z 154)\nThis initial validation clearly showed it is possible to introduce 10\u00a0mg of matrix equivalent of generic extracts obtained after ethyl acetate extraction of leafy vegetables. Adequate quantitative data are obtained for most of the pesticides at levels of 0.01\u00a0mg kg\u22121 or even below. Detection limits were usually well below 0.01\u00a0mg kg\u22121 after full-scan acquisition with a single-quadrupole MS. This means that for most pesticides at the target LOQ of 0.01\u00a0mg kg\u22121 (i.e. the lowest maximum residue limit set in the EU for vegetables and fruit), the signal-to-noise ratio is adequate for reliable automatic integration of peaks and that confirmation of identity of the pesticide is possible from its mass spectrum or at least one or two other diagnostic ions.\nPesticides that did not meet the EU criteria for quantitative analysis, and\/or for which relatively high LODs were obtained, included many compounds known to be troublesome in GC analysis because of to their high polarity or thermal lability. Typical examples are acephate, cyromazine, dicofol (screened for as its degradation product dichlorobenzophenone), dimethoate, imazalil, metaldehyde, methamidophos, methiocarb, omethoate, and the benzoylureas (measured as one common and one compound-specific degradation product). The relatively low recovery of the polar organophosphorus pesticides (acephate, methamidophos, and omethoate) can be attributed to the GC measurement and not to poor extraction efficiency, as was apparent from LC\u2013MS\u2013MS analysis of samples using the same extraction technique (see section LC\u2013MS\u2013MS analysis). For several other polar or labile pesticides adequate quantitative data were obtained during this initial validation, but from previous experience and the results obtained after implementation of the method it was clear that for such compounds LC\u2013based analysis is more robust than GC\u2013MS analysis. Typical examples include carbaryl, carbofuran, clofentezin, monocrotophos, and oxydemeton-methyl.\nAnalytical quality-control data from routine GC\u2013MS analysis\nThe initial validation data are continuously being supplemented by performance data generated as part of the analytical quality-control during routine analysis of the samples, to gain insight into reproducibility, robustness, recovery, and selectivity with other matrices. For this, with each analytical batch, one of the samples submitted for routine analysis was spiked with 135 pesticides at five times the target LOQ level (i.e. samples were spiked with 0.05\u00a0mg kg\u22121 of most of the pesticides). A compilation was made of recovery data from a period of 15\u00a0months which included analysis of approximately 100 different vegetable and fruit commodities. Given the wide variety of commodities, matrix-matched calibration is quite tedious and would substantially increase the number of standard solutions to be analyzed in the GC sequence. It was therefore decided to select one relatively simple matrix (tomato) as default for matrix-matched calibration, i.e. recoveries for all commodities were calculated against the tomato-matrix standard. For each pesticide, calculations were performed for two diagnostic ions. All together this resulted in approximately 30,000 values.\nAccording to the current EU guideline on quality control in pesticide residue analysis [37], the recovery obtained during routine analysis should be within 60\u2013140%. An overview of the percentage of recovery values within or outside the 60\u2013140% criterion for a wide variety of matrices is presented in Table\u00a06. With such large number of pesticides (or, actually, diagnostic ions) and matrices, one failing combination or more occurred for most matrices. There are several causes for this. Main reasons for recovery below 60% could be poor extraction efficiency or incomplete transfer of the pesticides to the GC column (e.g. adsorption and\/or degradation in a contaminated inlet). Higher recovery may occur when a compound from the matrix generates the same diagnostic ion as a pesticide and co-elutes with that pesticide (i.e. detection was not selective). Another reason could be that the matrix effect induced in the GC inlet [50] for a pesticide in a particular matrix is more pronounced than that in the tomato-based calibration standard.\nTable\u00a06Overview of percentage of recovery valuesa within or outside the EU 60\u2013140% criterion [37] after GC\u2013MS analysis\u00a0MatrixPercentage of all recovery valuesa60\u2013140%<60%>140%1Beetroot100002Cucumber (1\/2)100003Mint (1\/2)100004Sharonfruit (1\/2)100005Witloof100006Asparagus99107Bean sprouts99018Corn syrup99009Fennel leaves990110Grape990111Kohlrabi (1\/3)991012Lima bean990113Pak choi (1\/2)990114Pear concentrate990115Pumpkin990116Salsify990017Sharonfruit (2\/2)990118Strawberry990119Sugar pea991020Taro990121Bitter cucumber980222Cucumber (2\/2)981123Egg plant980224Kidney bean981125Kohlrabi (2\/3)981126Mushroom980227Pineapple981128Sweet pepper980229Tomato puree (processed)980230Turnip 981031Turnip tops (1\/2)980232Alfalfa971233Cauliflower971234Cherry970335Chestnut972136Endive970337Fig970338Kangkung (1\/2)971239Kangkung (2\/2)972140Ladies\u2019 fingers970341Mango970342Pear puree (processed)970343Sorrel973044Soybean sprouts970345Asparagus bean961346Orange962247Potato leaves962248Rhubarb962249Artichoke950550Tangelo952351Tarrragon953252Wine (red)951453Apricot940654Chives (1\/3)943355Chives (2\/3)944256Dill leaves944257Melon puree (processed)941558Mineola941659Pak choi (2\/2)942460Sugar water946061Broad bean931662Celery leaves (1\/4)933463Chervil935264Dates937065Sweetcorn (1\/3)934366Carrot921767Haricot bean920868Oregano925369Parsnip922670Fennel910971Green pea (1\/2)914572Passion fruit (1\/2)912773Celery leaves (2\/4)906474Green pea (2\/2)901975Lemon puree908276Mint (2\/2)905577Pomegranate901978Purslane901979Water cress902880Lettuce897481Chili pepper (1\/2)886682Chinese cabbage8701383Passion fruit (2\/2)8731084Bamboo shoots8601485Celery leaves (3\/4)867786Honey8614087Potato puree (processed)8614088Sugar pea8501589Turnip tops (2\/2)8501590Lime8441291Blueberry8321692Potato8315293Celery leaves (4\/4)8231594Green pea8211795Apple pulp (processed)8161396Cassava8191097Chives (3\/3)8171298Kohlrabi (3\/3)7802299Parsley (1\/2)78616100Thyme (1\/3)78220101Kale77617102Chili pepper (2\/2)76159103Coriander leaves76186104Sweetcorn (2\/3)75187105Sweetcorn (3\/3)74917106Parsley (2\/2)73207107Thyme (2\/3)73324108Rocket72325109Thyme (3\/3)66295110Golden berry (physalis)65134aRecoveries at 0.05\u00a0mg kg\u22121 (0.10\u20130.30\u00a0mg kg\u22121 for 22 pesticides). Calculated for 135 pesticides, two diagnostic ions each, against a standard prepared in blank tomato extract. The pesticides included are listed in Table\u00a07\nFailing pesticide\u2013matrix combinations were most abundant for herbs, kale, sweetcorn, and golden berry, for which up to 35% of recovery values (calculated using the two diagnostic ions for each pesticide) were outside the 60\u2013140% range. These products contain larger amounts of co-extractants than most other vegetables and fruits, which may result in insufficient detection selectivity, enhanced response as a result of a matrix effect (more shielding of active sites in the inlet), and contamination of the inlet. For this type of product more selectivity, e.g. by use of MS\u2013MS would be beneficial. Such detection is also more sensitive than single quadrupole full-scan detection and would enable reduction in the amount of matrix introduced, thus reducing build up of contamination. Overall, when data for all 110 QC samples were included, recovery was acceptable for 91% of the diagnostic ions measured.\nOn the basis of the same data, an overview by pesticide is presented in Table\u00a07. For each pesticide two diagnostic ions from the full-scan data were integrated and concentrations were calculated. In routine practice, however, the most convenient way of reviewing the data is by using one and the same diagnostic ion for each pesticide, irrespective the matrix. On the basis of the data set obtained (nearly 14,700 pesticide\u2013matrix combinations) the most favorable of the two diagnostic ions, i.e. the ion for which the highest number of recoveries within 60\u2013140% was obtained, was assigned as the Quan ion (default quantification ion). By using this ion, acceptable recoveries were obtained for 93% of pesticides\u2013matrix combinations. This also means that 7% or, in absolute figures, 1008 of the pesticide\u2013matrix combinations did not meet the criterion. 40% of these failing combinations could be accepted after use of the alternative ion, for which calculations were also performed automatically during data processing. Low recoveries (<60%) for both diagnostic ions were obtained for 2.7% of pesticide\u2013matrix combinations. High recoveries (>140%) were obtained for 2% of the combinations. For this latter group manual evaluation of other ions, if available and sufficiently abundant, could further increase the number of acceptable recoveries. Because this is a time-consuming process, it was not done routinely. In the event of deviating recovery, assessment of the results to be reported was based on visual evaluation of the extracted ion chromatograms of the two diagnostic ions at least. On the basis of on the findings it was then concluded the pesticide could not be determined in that specific matrix, or only at higher levels.\nTable\u00a07Recoveries over all matrices (GC\u2013MS analysis)PesticideQuan. ion m\/zQual. ion m\/zFortification level (mg kg\u22121)# QCs matrices (see Table\u00a06)Both diagn. ions 60\u2013140%One of diagn. ions 60\u2013140%Both diagn. ions >140%Both diagn. ions <60%Average recov. (%) Quan. ionRSD (%)Acrinathrin 2082890.10110107107309716Azaconazole1732170.05110107107219714Azoxystrobin3883440.0510897102089615Benalaxyl2061480.051101081090110013Bifenthrin1811660.051091091100010213Biphenyl1541530.051109394799820Boscalid1121400.1310998100289616Bromopropylate3413430.051101001019010914Bromuconazole2951730.051101001054110218Bupirimate2732080.02110108109019615Buprofezin1721050.051091051082010212Cadusafos1581590.051101051071210413Chlorfenapyr3643280.041101031062210216Chlorfenvinphos3232670.051101031037010316Chlorpropham2131270.051081011062210514Chlorpyrifos3142860.051091071090110114Chlorpyrifos-methyl2882860.051081011044210216Chlorthal-dimethyl3323010.051101101100010114Cinerin-11231500.111101041054110115Cyfluthrin2261990.201101021060410017Cyhalothrin, lambda-2081810.05108104109109916Cypermethrin1631810.15105991072010214Cyproconazole2222240.051101031051410216Cyprodinil2242250.05109101102088515DDE, p,p\u2032-2463180.061101101100010113DDT, o,p\u2032-2352370.051101061072110314DDT, p,p\u2032-2372350.0511082909119820Deltamethrin2532550.101109198489517Diazinon1791370.051091081100010113Dichlorvos1851090.051109096869920Dicloran2061600.0510896102359915Dieldrin263790.051101091090110414Diethofencarb1682670.051101071081110015Difenoconazole3232650.10107101106049616Dimethipin118760.05110951045110416Dimethomorph3873010.10110981000108916Dimoxystrobin2051160.051101081090110012Diniconazole2702680.15645862119717Diphenylamine1691670.051101071070310116Dodemorph2381540.05110109109019615Endosulfan-alpha195+241239+1970.501109510010010712Endosulfan-beta195+241237+1600.101101071073010214Endosulfan-sulfate272+229274+2370.051091021072110416EPN1573230.051101031063110317Epoxiconazole1921380.05110106108119814Esfenvalerate1671250.151101021034310615Ethion2311530.051101061064010314Ethoprophos1582000.051101071081110413Etofenprox3761640.05110102104249715Etridiazole2111830.0510980822179721Fenarimol2191390.051101061081110316Fenazaquin1601450.05110105105148816Fenbuconazole1291980.05110105107129917Fenitrothion2772600.05108991027110616Fenoxycarb1861160.05110891018110517Fenpiclonil2381740.051101011063110217Fenpropathrin1811410.051091011046010313Fenpropimorph1281290.051101081091010114Fenvalerate1671250.25110102103259815Fipronil3673690.05110101100379918Flucythrinate1991570.051101021063110315Fludioxonil2481820.05109105107129817Flusilazole2332060.05110104107129715Flutolanil3232810.051101071091010013Flutriafol2191230.041101021045110314Fluvalinate, tau-2502520.151109799569915Furalaxyl242950.051101061073010113Heptenophos1241260.05109971046010218Hexaconazole2162140.051101061081110214Iprodione3163140.10103798881310020Jasmolin-11641230.0411092104429715Kresoxim-methyl1162060.051091061090110015Lindane1832190.05110107110009915Malathion1731270.051081031073010417Mecarbam3291310.051101091100010115Mepanipyrim2232220.0511088917128519Mepronil2691190.10110109110009715Metalaxyl2061600.051071051082010312Methidathion145850.05109858919210715Metrafenone3953930.05110104106229414Mevinphos1921270.05110889017310417Myclobutanil1791500.05110102107219815Nitrothal-isopropyl2362540.05110108108119913Nuarimol2352030.051101081100010115Oxadixyl1631320.15110106107129913Parathion2911090.051101051091010515Parathion-methyl2632470.05109861028010717Penconazole1592480.051091081100010015Pentachloroaniline2672650.1111096970138115Pentachlorothioanisole2962460.0511087890217716Permethrin-cis1831630.051101081100010114Permethrin-trans1831630.051101061073010013Phenylphenol, 2-1701410.05109102107309813Phosalone1821840.05110909213510119Phosmet1611600.05109769016410022Phosphamidon2641270.05110919413310319Picoxystrobin3351450.051101051091010312Piperonyl-butoxide1761770.051071061091010013Pirimiphos-methyl2763050.051101091091010213Procymidone2832850.051081061081110014Profenofos3372060.05108931028010417Propargite1731350.331091041091010316Propiconazole2592610.05109106107219914Propyzamide1731750.051101071082010212Prothiofos3092670.05110108109109913Pyrazophos2212320.051109999389118Pyrethrins1231600.36110871037010518Pyridaben1471480.05110107107129914Pyridaphenthion3401990.05110961017210217Pyrifenox2622640.051101081100010015Pyrimethanil1991980.05110107106139014Pyriproxyfen2261360.051101041072110316Quinalphos1571460.051101041054110414Quinoxyfen3072720.05110106106049214Quintozene2371420.05110107107129316Silafluofen1792860.05110106106049814Spirodiclofen3123140.251109596689619Spiromesifen2722540.05110105108119616Spiroxamine1001980.10110107109019613TDE, p,p\u2032-2352370.05110971005510314Tebuconazole2502520.15676667019715Tebufenpyrad1713180.051101071081110013Tebupirimfos2343180.051101081091010114Tefluthrin1771970.051101061073010313Tetraconazole3363380.05110109109109914Tetradifon3562290.15109109110009914Thiometon881250.051101081100010415Tolclofos-methyl2652670.051081071072010113Tri-allate2682700.051101041054110413Triazamate2422270.051101071073010214Triazophos2852570.05109951008210418Trifloxystrobin1311160.051101081091010314Triflumizole2782870.03110105107039915Trifluralin2643060.051101071072110114Vinclozolin2121980.051071061091010311Total 146961368814057402300% of # QCs93.195.22.72.0\nIt should be noted that the above evaluation applies to a level five times the reporting level, which was set at 0.01\u00a0mg kg\u22121, or the LOQ if higher than 0.01\u00a0mg kg\u22121. At lower levels interferences may have a larger effect and, consequently, more frequent deviations from the 60\u2013140% criterion (most probably >140%) may be observed. For higher levels, the opposite would be true.\nPesticides for which low recoveries (<60%) were frequently obtained (10\u201321 of 110 QC samples) included iprodione and p,p\u2032-DDT (degradation in inlet), dimethomorph (polar, relatively non-volatile, could be troublesome in splitless transfer), pentachloroanisole, pentachloroaniline, and mepanipyrim (no clear explanation, but probably related to the dispersive SPE clean-up). There were no indications for poor extraction efficiency.\nHigh recovery (>140%) frequently occurred for etridiazole, methidathion, mevinphos, phosmet, phosalone, phosphamidone, and endosulfan-alpha (10\u201321 times out of 110 QC samples, often in herbs and peas). This was attributed to matrix effects and interferences.\nOverall, the pesticides that failed most frequently (11\u201328 times out of 110) during routine analytical quality control were (in descending order) etridiazole, iprodione, methidathion, pentachlorothioanisole, mevinphos, phosmet, p,p\u2032-DDT, mepanipyrim, phosalone, phosphamidon, biphenyl, dichlorvos, spirodiclofen, pentachloroaniline, deltamethrin, tau-fluvalinate, and pyrazophos. These would be the most relevant for inclusion in alternative methods, for example GC\u2013MS\u2013MS or LC\u2013MS\u2013MS.\nAverage recovery and RSD were calculated for pesticide\u2013matrix combinations that passed the acceptable recovery criterion. The results are included in Table\u00a07. Average recovery was usually close to 100% and RSDs approximately 15%. For the pesticides known to be adsorbed by GCB systematically lower average recovery (77\u201390%) was obtained, which is in agreement with the results obtained during method development.\nThese comprehensive data show that with a relatively inexpensive single-quadrupole MS detector in full-scan mode it is possible to obtain reliable quantitative data down to the 0.01\u00a0mg kg\u22121 level, or even lower, for a wide range of pesticides in a wide variety of matrices after generic rapid sample preparation based on extraction with ethyl acetate. Unified calibration based on a tomato-matrix standard is, furthermore, a feasible approach. One should, however, be aware there are also limitations and that some pesticide\u2013matrix combinations cannot be determined in the 0.01\u20130.1\u00a0mg kg\u22121 range, and that for other pesticides calibration against the corresponding matrix instead of tomato is required to bring quantitative results within the AQC criteria, especially for MRL violations, when more stringent criteria apply. The data also reveal that the only way to gain full insight into analyte recovery and method selectivity with a wide variety of matrices is by performing analytical quality control on all pesticides which are reported, rather than on a subset, as is suggested in the EU guideline [37]. A subset will suffice for demonstration of adequate sample preparation and injection but will not reveal limitations in the selectivity of GC\u2013MS.\nGC single-quadrupole MS remains an effective tool for routine GC analysis of pesticide residues. For many vegetable and fruit matrices there is no real need to change to more advanced (and expensive) MS techniques, for example MS\u2013MS (which has limited scope) or accurate mass TOF-MS (which has a limited dynamic range). Use of such equipment would be justified for more complex matrices and when low \u03bcg kg\u22121 LOQs are required\u2014for example analysis of some pesticides in baby food.\nLC\u2013MS\u2013MS analysis\nClean-up\nThe ethyl acetate extraction procedure is also appropriate for many pesticides not amenable to GC analysis [11, 15, 16, 18, 26]. Typically no clean-up is performed (Table\u00a01). One reason for this is that with regard to chromatographic performance LC columns tend to be more tolerant of injection of bulk matrix than GC columns. In our experience, continual injection of 20\u00a0mg equivalent of vegetable and fruit extracts does not result in deterioration of chromatographic performance or unacceptable contamination of the ion source (the system used here was an API2000). In LC\u2013MS co-extracted matrix does have an effect on the response, however, by interfering with the ionization process. This results in suppression (sometimes enhancement) of the response to a pesticide in a matrix compared with that in a solvent standard [51] and complicates quantification of pesticides in the samples. The possibility of reducing matrix effects by use of dispersive SPE clean-up was investigated in a similar way as for GC. First, the effectiveness of the clean-up step was investigated by addition of 25\u00a0mg GCB and 25\u00a0mg PSA to 1\u00a0mL raw extract of a mixed spinach\u2013grape\u2013onion sample (1:1:1, 1\u00a0g ml-1). Seventy pesticides (the ones in Table\u00a08 with API2000 in the MS-MS column) were added after clean-up and analyzed by LC\u2013MS\u2013MS. The response was compared with that of solutions of equal concentration in the raw extract and a solvent standard. Clean-up increased the number of pesticides for which no pronounced matrix effect (less than 20% suppression or enhancement) was observed from 38 to 84%. Several of the pesticides (Tables\u00a02 and 4) were adsorbed by the SPE material, however. Although adsorption by the GCB could have been avoided or reduced by addition of toluene (although less practical when changing from extraction solvent to methanol\/water), it was concluded that PSA was not compatible with a generic method for pesticides amenable to LC\u2013MS\u2013MS. It was therefore decided not to include a clean-up step for LC\u2013MS\u2013MS analysis and to use the initial raw ethyl acetate extract. Another reason for not further pursuing clean-up in LC\u2013MS\u2013MS analysis was that the sensitivity of current triple-quadrupole instruments enables injection of only small amounts of matrix into the LC\u2013MS\u2013MS system (e.g. 2\u00a0mg) while still achieving the desired limits of quantification. Experiments showed that tenfold dilution of 1\u00a0g mL\u22121 extracts increased the number of pesticides for which no pronounced matrix effect occurred from 65 to 82% and from 10 to 65% for cucumber and cabbage, respectively.\nTable\u00a08LC\u2013MS\u2013MS settings and performance-validation characteristicsPesticidetr (min)Precurs.Prod. ion 1DPFPCECXPProd. ion 2CECXPVegetablesn0.01\u00a0mg kg\u221210.1\u00a0mg kg\u22121Fruitsn0.01\u00a0mg kg\u221210.1\u00a0mg kg\u22121MS\u2013MSMatrixRec. (%)RSD (%)Rec. (%)RSD (%)MatrixRec. (%)RSD (%)Rec. (%)RSD (%)Abamectinea,c21.78913054634033221454910Cuc\/lett466186815Apple\/grape41593915546API2000Acephate5.518414331150111295336Cuc\/lett48021759Apple\/grape48087610API2000Acetamiprid10.52231269127029101771114Cuc\/lett4993967Apple\/grape41174986API2000Aldicarba11.72081161611011889216Cuc\/lett4103209112Apple\/grape4991310913API2000Aldicarbsulfon7.922386322002112148136Cuc\/lett41049834Apple\/grape41205913API2000Aldicarbsulfoxide7.22071324630091089196Cuc\/lett410912894Apple\/grape41094863API2000Asulam3.623115641260151292336Cuc\/lett435282938Apple\/grape413231030API2000Azamethiphos12.1325183362202314112538Cuc\/lett41016944Apple\/grape4106119110API2000Azinfos-methyl13.53181324160236160156Lettuce59316884Orange56911799API3000Bendiocarb12.22241671610013101092518Lettuce510210966Orange58681086API3000Bifenazate13.93011981611013161702714Lettuce5359337Orange5937835API3000Bitertanol15.233826921120132099218Lettuce59610817Orange59310818API3000Butocarboximb11.621375413002141561712Lettuce5101239312Orange572168919API3000Butoxycarboxim7.7223106362501381661110Cuc\/lett4116910415Apple\/grape41184957API2000Carbaryl12.520214510137013121273710Cuc\/lett41004954Apple\/grape41111210010API2000Carbendazim11.41921604623023121324310Cuc\/lett4104210210Apple\/grape212211051API2000Carbofuran13.32221654629017121232910Cuc\/lett41241211113Apple\/grape410411934API2000Carbofuran, 3-OH10.4238220312109161631912Lettuce5918944Orange51007916API3000Carboxin12.62361431135021293512Lettuce5879802Orange5899846API3000Chlorbromuron14.0295206413502712182254Lettuce510019865Orange58330846API3000Chlorfluazuron18.15423854027029301582912Lettuce5797895Orange57421868API3000Clofentezin15.5303138512802110102618Cuc\/lett493177610Apple\/grape41272410116API2000Clomazone13.62401253119025889676Lettuce59741046Orange5905899API3000Clothianidin10.2250132367023101691710Lettuce599111002Orange511041003API3000Cycloxydim14.93262804626019221802914Cuc\/lett418118829Apple\/grape438457028API2000Cymoxanil11.1199128181201310111258Cuc\/lett48313957Apple\/grape4908992API2000Cyromazine7.116785402402661252510Cuc\/lett496107811Apple\/grape4967813API2000Demeton13.625989261801361981116Lettuce59714854Orange57615769API3000Demeton-S-methyl12.523189315021461374Lettuce5935864Orange5816818API3000Dem-S-meth-sulfone8.826316941350236109414Lettuce510412926Orange51002974API3000Desmedipham13.13011825134013141542512Cuc\/lett48610883Apple\/grape495228315API2000Diafenthiuron18.13853294126027222784518Lettuce50\u20130\u2013Orange51049927API3000Dichlofluanidec14.1333224462701718123378Cuc\/lett42111636116Apple\/grape433825468API2000Dicrotophos9.5238112412701781931316Cuc\/lett41105993Apple\/grape4100129310API2000Diflubenzuron14.53111584627019121414710Cuc\/lett47915841Apple\/grape4101610212API2000Dimethirimol13.1210715129045498378Lettuce5997975Orange591101055API3000Dimethoate10.623019911350134125292Lettuce5987964Orange510917956API3000Diniconazole15.6326705631063141594516Lettuce57810936Orange59416965API3000Disulfotonc15.7275891190276614110Lettuce5536647Orange58516864API3000Disulfoton-sulfone12.830797311503981531714Lettuce5113101057Orange58161068API3000Disulfoton-sulfoxide12.82911852614017162131514Lettuce5111101156Orange59251015API3000Diuron13.3233723621037446356Lettuce511161017Orange5947946API3000DMSA11.620192261502561371310Lettuce510213974Orange58513877API3000DMST12.3215106261602181511310Lettuce5975956Orange58413855API3000Ethiofencarb12.822610736220218169914Cuc\/lett48130945Apple\/grape499179420API2000Ethiofencarbsulfon9.7258107362402162011116Cuc\/lett4120101055Apple\/grape410189711API2000Ethiofencarbsulfoxide9.9242107311802381851314Cuc\/lett411413972Apple\/grape4127101077API2000Ethirimol13.321014051370311298376Cuc\/lett4963886Apple\/grape486268126API2000Famoxadonea14.63923311113015222382518Lettuce59015801Orange5889801API3000Fenamiphos14.530421741350294234214Lettuce5878874Orange5937935API3000Fenamiphos-sulfone12.23363088136023222662920Lettuce51028945Orange58116868API3000Fenamiphos-sulfoxide12.13201715623027142333514Lettuce511410944Orange59781085API3000Fenhexamid14.2302975129035855598Lettuce58415824Orange5856845API3000Fenpyroximate19.34223666136021261354310Cuc\/lett4988959Apple\/grape4111910410API2000Fensulfothione13.03092814626021222532518Lettuce5967893Orange510123838API3000Fensulfothion-sulfone13.03252693612021181913312Lettuce510310988Orange58561006API3000Fenthion13.9279231261302116Lettuce511131818Orange53822748API3000Fenthion-sulfone12.5311125513202982792522Lettuce5956904Orange51011896API3000Fenthion-sulfoxide12.4295280462302520109458Lettuce5932946Orange5948876API3000Fipronil14.14373686637023262903716Lettuce570248811Orange592289012API3000Flucycloxuron17.34842896636015201324910Cuc\/lett411341043Apple\/grape41633812126API2000Flufenoxuron17.148915810136027121416510Cuc\/lett410717908Apple\/grape4172501028API2000Formetanate 12.2222165361901914120378Lettuce5100141036Orange5956957API3000Fosthiazate12.7284104312002362281522Lettuce59981026Orange5842986API3000Furathiocarb16.53831957637025162521918Cuc\/lett455325538Apple\/grape48717847API2000Hexaflumuronc15.24611585130027101416110Cuc\/lett49124827Apple\/grape41711511416API2000Hexythiazox17.43531684127035122282118Cuc\/lett499198415Apple\/grape4120268411API2000Hymexazolc5.8100546636021444292Cuc\/lett476345049Apple\/grape445152220API2000Imazalil15.02971594629033122012916Cuc\/lett49047612Apple\/grape411179013API2000Imidacloprid10.02561754124025142092118Cuc\/lett49998111Apple\/grape412112897API2000Indoxacarb15.15282494124023181503510Lettuce56032735Orange5846786API3000Iprovalicarb14.13211193116029102031318Lettuce510851047Orange59749010API3000Isoxaflutole12.93602514627019222205522Lettuce576189015Orange58618985API3000Linuron13.82491604629025121822114Cuc\/lett4103168611Apple\/grape490261016API2000Metamitron10.7203175512902314104316Cuc\/lett480118717Apple\/grape49717959API2000Methabenzthiazuron13.32221653120021121504512Lettuce51064986Orange58481079API3000Methamidofos4.61429441240216125198Cuc\/lett483167919Apple\/grape48611815API2000Methiocarb13.82261694630013141212510Cuc\/lett4949954Apple\/grape41015941API2000Methiocarbsulfon10.7258122563702582011316Cuc\/lett4109129911Apple\/grape4949876API2000Methiocarbsulfoxide10.12421854629019141703114Cuc\/lett411651013Apple\/grape412681042API2000Methomyl8.81638821130136106138Cuc\/lett41532213619Apple\/grape4125141037API2000Methoxyfenozide13.83693132420013241333410Lettuce5937914Orange59113913API3000Metobromuron13.12591704628025121482112Cuc\/lett411219996Apple\/grape49699912API2000Metoxuron11.6229723119037446352Lettuce510481004Orange59581024API3000Monocrotofos9.22241274124021101931116Cuc\/lett41085904Apple\/grape411189810API2000Monolinuron12.8215126412602381481912Cuc\/lett41047986Apple\/grape411171078API2000Omethoate6.52141253623029101831514Cuc\/lett498138513Apple\/grape41025862API2000Oxamyla8.0237722116023490116Cuc\/lett410731907Apple\/grape412814979API2000Oxamyl-oxim6.6163723623017490256Cuc\/lett41006853Apple\/grape411831019API2000Oxycarboxin10.92681752617019141473510Lettuce5986964Orange58522785API3000Oxydemeton-methyl8.5247169412301914109358Cuc\/lett49811897Apple\/grape41045964API2000Paclobutrazole13.829470363204541255110Lettuce5969878Orange57767696API3000Pencycuron15.43291255634035102182318Cuc\/lett41005773Apple\/grape41184929API2000Phenmedipham13.23011685129013141362910Cuc\/lett4997965Apple\/grape4108118411API2000Phenm.-metabolite10.0168136312001410108268Cuc\/lett410791035Apple\/grape4101149617API2000Phorate15.5261752615021447458Lettuce596279111Orange51042886API3000Phorate-sulfone12.92931712615017101153710Lettuce511410959Orange58361044API3000Phorate-sulfoxide12.82771994127017697454Lettuce5998963Orange5986914API3000Phosphamidon11.73001744125019141273310Lettuce510151075Orange59871007API3000Picolinafen16.43772385622041142562920Lettuce5818966Orange51038995API3000Pirimicarb13.023972263603141822312Lettuce5996964Orange5899923API3000Pirimicarb, desmethyl 11.62257221360334168216Lettuce51034983Orange531144215API3000Prochloraz15.4376308463101322704116Cuc\/lett490157813Apple\/grape484389464API2000Profoxydim16.24662806614027201803512Lettuce53325306Orange54934555API3000Propamocarb8.5189102311902561441912Lettuce5754724Orange52214188API3000Propoxur12.2210111312101981681114Cuc\/lett411431005Apple\/grape41183985API2000Prothiocarb7.4191146462402112Cuc\/lett485266337Apple\/grape410658310API2000Pymetrozine9.021810556370278201916Cuc\/lett46526858Apple\/grape4477717API2000Pyraclostrobin15.13881941350196163336Lettuce57213776Orange5874837API3000Pyridate metabolite10.42077756340456104318Cuc\/lett410012874Apple\/grape4899755API2000Rotenone14.739521310137031161923314Cuc\/lett49313938Apple\/grape494169430API2000Sethoxydim15.23281784626025142201918Cuc\/lett46739883Apple\/grape459349628API2000Spinosyn A22.073314296280431298836Lettuce5959936Orange5974922API3000Spinosyn D24.174714296110471298894Lettuce5863936Orange5997925API3000Tebuconazole14.83087061140516125538Lettuce5806933Orange5958964API3000Tebufenozide14.53531332618023102971322Cuc\/lett4103168611Apple\/grape4106427833API2000Temephos16.34671257132039104193532Lettuce56227816Orange5927959API3000Tepraloxydim12.73422503118019281662912Lettuce54419607Orange57315624API3000Terbufos16.728910311120131057378Lettuce57327758Orange580248112API3000Terbufos-sulfone13.5321171211301912115396Lettuce5108410111Orange59969310API3000Terbufos-sulfoxide13.53051876110171097598Lettuce510631035Orange5985979API3000Thiabendazole12.22021755637035121314510Cuc\/lett487121013Apple\/grape4982927API2000Thiacloprid11.025312641210278905316Lettuce59791023Orange510261167API3000Thiametoxam9.02922114627019241323310Lettuce5944974Orange51019996API3000Thiocyclamd12.6182137211602112732914Lettuce59611896Orange5100158211API3000Thiodicarb12.73558820130316108218Cuc\/lett4371154298Apple\/grape4834794API2000Thiofanox12.921957119019661154Lettuce5nd819321Orange5nd\u20138430API3000Thiofanox-sulfone10.2251571635026276214Lettuce5110161015Orange58525858API3000Thiofanox-sulfoxide9.82351043132017457272Lettuce511021053Orange510911886API3000Thiometonc13.0247891611023661458Lettuce596171009Orange587111002API3000Thiophanate-methyl12.13431513021025123111723Cuc\/lett46687516Apple\/grape441593798API2000Tolylfluanidea14.73642383121019181374110Cuc\/lett43111642115Apple\/grape475932481API2000Triadimefon14.02941973118023122251918Lettuce59210866Orange5897787API3000Triadimenol14.1296701613031499218Lettuce51017876Orange5897829API3000Triazoxide13.5248685632047495376Lettuce5991027619Orange5431076910API3000Trichlorfon10.6257109462602782211518Cuc\/lett41161610422Apple\/grape41148994API2000Tricyclazole11.51911365636039101633112Cuc\/lett41055926Apple\/grape49611833API2000Triflumuron14.93591563020023121394710Cuc\/lett4949927Apple\/grape4118121098API2000Triforine13.24353901210013302154015Cuc\/lett498131016Apple\/grape49710939API2000Vamidothion10.4288146463001912118318Cuc\/lett411116963Apple\/grape4119111047API2000Cuc, cucumberLett, lettuceaNH4 adductbNa adductcLOQ level 0.05\u00a0mg kg\u22121dLOQ level 0.02\u00a0mg kg\u22121\nRoutine experience with LC\u2013MS\u2013MS analysis for over four years, both with the API2000 (20\u00a0mg matrix) and the API3000 (2\u00a0mg matrix) has shown that injection of uncleaned extracts does not result in special maintenance requirements. The source is cleaned with a tissue daily. The LC column typically lasts for 6\u00a0months.\nChanging the solvent\nBecause ethyl acetate is less suitable for direct injection in reversed phase LC, the solvent was changed. Because only small amounts of the raw extract need to be evaporated (less than 0.5\u00a0mL in the final method) and evaporation blocks enable simultaneous evaporation of many (typically 24\u201336) extracts, this step adds little to the overall sample-preparation time. Changing the solvent was even regarded as advantageous. It resulted in more freedom in selection of the final solvent to be injected into the LC, which can be critical for very polar compounds (e.g. in acetonitrile-based extraction methods, injection of 100% acetonitrile easily leads to band-broadening for methamidophos). It is also easier to compensate for the smaller amount of sample processed for dry crops (because of the need for addition of water) by evaporating a larger amount of the ethyl acetate extract.\nIn previous work [15] a small amount of a diethylene glycol (added as solution in methanol) was added, because this was found to facilitate reconstitution, thereby improving recovery for some pesticide\u2013matrix combinations. It was also shown that the evaporation step did not require special attention and that continuing the process for another half hour after completion of evaporation of the solvent did not affect recovery. The same procedure was therefore used here without re-evaluating the real need for it. Reconstitution was performed by first dissolving in methanol (ultrasonication) and then dilution with LC mobile phase component A.\nValidation of LC\u2013MS\u2013MS method\nThe LC\u2013MS\u2013MS method was validated in three separate studies, one using the API2000 with injection of 20\u00a0mg matrix equivalent and the other two using the API3000 with injection of 2\u00a0mg matrix equivalent. A total of 140 pesticides and degradation products were included. In contrast with the full-scan acquisition in GC\u2013MS, in LC\u2013MS\u2013MS data were acquired for a fixed, limited, set of pesticides. Although many pesticides from the GC\u2013MS method can also be analyzed by LC\u2013MS\u2013MS, emphasis was on pesticides that were not, or less, amenable to GC analysis.\nRecovery, based on matrix-matched calibration, and repeatability were evaluated at the 0.01 and 0.1\u00a0mg kg\u22121 level for vegetable and fruit matrices; the results are listed in Table\u00a08. Although acceptable performance data were obtained for most of the pesticides, low recovery and\/or high variability were observed for some. Among these were compounds that were also reported as troublesome by other workers using alternative multi-residue methods, e.g. asulam [30]. Low recovery could be partly attributed to poor extraction efficiency (asulam, hymexazol, and, in orange, propamocarb) or degradation during sample preparation (cycloxydim, sethoxydim, profoxydim, tepraloxydim, dichlofluanide, tolylfluanide, thiodicarb, thiophanate-methyl, and, in lettuce, disulfoton and furathiocarb). The degradation seems to be related to the change of solvent, as is apparent from comparison of GC\u2013MS and LC\u2013MS\u2013MS validation data for dichlofluanide, tolylfluanide, and disulfoton. Fortunately, for many of these the degradation products formed are also part of the residue definition and are included in the method. Indeed, elevated recovery was observed for the degradation products when determined in the same validation set as the parent compound. In the analysis, therefore, degradation is not necessarily a problem, because the results (expressed as defined in the residue definition) have to be summed. In routine analytical quality control (see below) the data were evaluated this way.\nAnalytical quality-control data from routine LC\u2013MS\u2013MS analysis\nIn the same way as for GC\u2013MS analysis, the initial validation data are continually being supplemented by performance data generated as part of analytical quality control during routine analysis of samples. With each set of analytical samples at least one was fortified with the full quantitative suite (i.e. 136 pesticides and degradation products) at the 0.05\u00a0mg kg\u22121 level. A compilation was made from all the data generated over a period of 12\u00a0months, which included data for more than one hundred vegetable and fruit matrices. A limited number of dry matrices (flour, milk powder) were also included in the set. The data were evaluated for one transition for each pesticide, using the API3000 and injection of 2\u00a0mg equivalent of matrix (10\u00a0\u03bcL of a 0.2\u00a0g mL\u22121 extract). Examples of typical extracted ion chromatograms are shown in Fig.\u00a05.\nFig.\u00a05Typical extracted ion chromatograms obtained by LC\u2013MS\u2013MS analysis of vegetable and fruit extracts (calibration standard in mango matrix, 10\u00a0pg \u03bcL, corresponding to 0.05\u00a0mg kg\u22121)\nFor all fortified samples the matrix effect was also established by analyzing the corresponding matrix-matched standard, at the same level as in the extract of the fortified sample, against a solvent standard. Suppression (or enhancement) of up to 20% was regarded as acceptable for quantification. The number of compounds for which the response in matrix relative to that in solvent was between 80 and 120% is given in Table\u00a09 for each matrix. Whereas for beetroot, asparagus, and kangkung little or no matrix effects exceeding 20% were observed, such effects were much more common for herbs and citrus fruits.\nTable 9Overview of matrix effects and recoverya within or outside the EU 60\u2013140% criterion [37] after LC\u2013MS\u2013MS analysis\u00a0NMatrix effectsn*Recovery# Pesticides# PesticidesRel. resp. 80\u2013120%>20% suppr.>20% enhanc.Calc. using solvent stdCalc. using matrix-matched std60\u2013140%<60%>140%60\u2013140%<60%>140%Corn syrup (2\/2)1351340110497439941Beetroot135133111041013010130Corn syrup (1\/2)13513221104984210022Kangkung13513221104911129482Green pea1351313110497529932Asparagus1351304110497709860Coco nut135130411046341059450Papaya1351303210496449842Cauliflower135129151041012110211Fennel135129421041003110130Cherry (2\/3)135128701041004010040Cherry (1\/3)1351277110492849824Ladies\u2019 fingers1351278010497709770Mango (1\/2)1351276210497349824Cherry (3\/3)135126811041004010220Mango juice135126361041011210400Mushroom135126721041022010301Taro1351267210496449914Plum (3\/3)13512582104957210040Fennel leaves (2\/2)1351245610499239923Milk powder135124651045845159450Grape1351239310498339833Spinach13512312010494829653Tamarind135123841046737079250Cassava135122761048716178260Raspberry (1\/3)1351221211048420092120Sweet pepper1341221021031001210012Apple puree1351215910499509770Corn flour13512111310495639572Courgette135121771041002210031Tomato puree1351211041041013010310Raspberry (2\/3)135120150104985110031Broccoli135119142104901049383Flour (2\/2)13511921410495279734Peach (1\/2)135119160104995010040Mango (2\/2)134117125103966110030Milk\/flour mix1351171261044360155490Bitter cucumber13511617210499239914Melon puree135116181104995010310Tomato13511613610493839653Lettuce, crinkley 13411419110397339724Pear13411414610397609940Flour (1\/2)1351131481047328385190Plum (1\/3)13511313910493659824Celery leaves (1\/3)135112221104901229734Purselane13511223010496629842Apricots135111231104901319761Artichoke135111177104911219581Cucumber1351101510104995010130Horseradish powder1351101510104881159752Tarrragon (2\/2)13511081710496449464Avocado (1\/2)1351092241048121290131Haricot bean1351092511048320190131Kiwi1351091016104976110022Peach (12\/2)135108243104881429392Raspberry (3\/3)1351072621048022290113Blackberry1331061710102911019192Diced pumpkins135106272104958110031Plum (2\/3)1351062361048618085181Yam13510612810497619680Avocado (2\/2)1341032921036834180221Dill leaves135103151710494739392Honey106103308282008200Chervil13510229410495909851Parsley13510229410495459914Nectarine13410129410392839841Bean sprouts106100518276607840Sweetcorn (1\/2)10699618276517732Beetroot leaves13598325104851909950Chestnuts10698178276427930Pomegranate (1\/2)135973711048420010040Pomegranate (2\/2)135973711048420010040Pear syrup10695388279308020Alfalfa106941118275707840Fennel leaves (1\/2)10692868274537624Chili pepper13591404104958110112Turnip tops106901518276247804Blueberry135894331026636091110Litchi13588452104782609941Salak13588425104822029941Pepper powder10687163825427170111Celery leaves (2\/3)135854191049310110022Lemon13484473104782069734Physalis13583484104713309950Maize (feed)1358153110495639338Sweetcorn (2\/2)13580505104792239860Coriander (1\/2)13579560104683429563Mangostan1357640191044654469350Celery leaves (3\/3)13475581103861619922Laos13573575104703319941Chives13571577104985110211Coriander (2\/2)135656010104832109860Tea (black)136656921046043187143Lemon puree135538021046836010310Ginger13546863104683429833Grapefruit (1\/2)13346870102435909813Grapefruit (2\/2)13546881103614119733Oregano1354675141045250287161Kumquat13538952104475619464Lime13438942103485239643Tarrragon (1\/2)135389521044163090131Italian herb mix135331011104544919581Total QC results1349710488256644310395861816131649533708154Percentage of total results78193831629271aRecovery at 0.05 mg kg\u22121 (higher for seven pesticides). The pesticides included are listed in Table 10N is the total number of individual compounds (pesticides and metabolites) added to the matrixn* is the total number of pesticides added to the matrix. Compounds belonging to the same residue definition counted as one\nIn contrast with GC, for which matrix effects are mainly caused by shielding of active sites in the inlet and were, to some extent predictable (in relation to the matrix load injected and the lability and\/or polarity of analyte), in LC\u2013MS\u2013MS matrix effects are much less predictable. Although they do depend on the amount of matrix introduced into the system, and also tend to be more abundant in complex (\u201caromatic\u201d) matrices, it cannot be readily predicted for which pesticides the effects occur. For this reason use of one matrix-matched standard as representative calibrant for a whole range of commodities, which worked reasonably well in GC\u2013MS analysis, was not feasible in LC\u2013MS\u2013MS analysis. Consequently, critical evaluation of the matrix effect was required; if unacceptable suppression occurred there was no alternative to quantification by use of the appropriate matrix-matched calibration standard or, when not available, by standard addition.\nRecovery of the pesticides from the fortified samples was calculated relative to that from a solvent standard and a matrix-matched standard and tested against the 60\u2013140% criterion for evaluation of routine analytical quality-control samples [37]. A total of more than 10,000 recovery values were evaluated. Without matrix-matched calibration, acceptable recovery was obtained for 83% of the pesticides. Deviating recoveries were usually too low, mainly because of ion suppression, as is apparent from the results obtained from determination of recovery using matrix-matched calibration, for which 92% met the criterion.\nConcentrating on performance at the pesticide level (Table\u00a010) enables easy identification of troublesome pesticides. All compounds belonging to the same residue definition were summed (according to the residue definition) and counted as one, thereby compensating for possible conversion during sample pretreatment. This way the low recovery of dichlofluanide and the corresponding high recovery of DMSA were acceptable for most matrices because recovery for the sum met the criterion. Pesticides for which multi-matrix analysis under fixed conditions was less favorable included asulam, bifenazate, cyromazine, furathiocarb, propamocarb, pymetrozine, and thiocyclam (low recovery because of varying extraction efficiency and\/or degradation). As already observed during validation, the method was also less suitable for cycloxydim, profoxydim, sethoxydim, and tepraloxydim. For these compounds recovery was too high, possibly because of degradation in the calibration standard used for preparation of the matrix-matched standards.\nTable\u00a010Recovery over all matrices (LC\u2013MS\u2013MS)\u00a0\u00a0# ACQ samples# Recov. 60\u2013140%# Recov. <60%# Recov. >140%Average recov. (%)aRSD (%)a1Abamectin1021002086172Acephate102939078133Acetamiprid10297509011Aldicarb102101019113Aldicarb-sulfone102102009212Aldicarb-sulfoxide102966084134Asulam1026932185175Azamethiphos1021020089126Azinfos-methyl102965187157Bendiocarb93930088128Bifenazate986037185189Bitertanol10298408415Butocarboxim102101108814Butoxycarboxim10210110911210Carbaryl102100118713Carbendazim10097219314Carbofuran102100119212Carbofuran,3-hydroxy-10210200931111Carboxin1029750841312Chlorbromuron1029840861413Chlorfluazuron1029381871514Clofentezine10289130801515Clomazone938931851216Clothianidin939120911217Cycloxydim1026811231041918Cymoxanil10210200911519Cyromazine10249530741220Demeton102102008914Demeton-S-methyl102100208714Demeton-S-methylsulfone10210110911221Desmedipham10296608314Dichlofluanid10236660801922Dicrotophos10210020891423Diflubenzuron1029840821524Dimethirimol9390308911Dimethoate10210110901225Diniconazole9384818616Disulfoton93672517513Disulfoton-sulfone9393008812Disulfoton-sulfoxide938904961626Diuron9392108714DMSA1024106110917DMST102961510416Ethiofencarb10299308614Ethiofencarb-sulfone102102009013Ethiofencarb-sulfoxide10210110921527Ethirimol1029840881228Famoxadone10295708314Fenamiphos102100208914Fenamiphos-sulfone102102009112Fenamiphos-sulfoxide939210901129Fenhexamid1029660851230Fenpyroximate102921008713Fensulfothion102102008811Fensulfothion-sulfone9391208512Fenthion10299308714Fenthion-sulfone10299218815Fenthion-sulfoxide10210200931431Flucycloxuron1029480881532Flufenoxuron1029390871433Fosthiazate939300901234Furathiocarb10279203841635Hexaflumuron10290102851836Hexythiazox10291110851537Imazalil1019290831438Imidacloprid1029930901439Indoxacarb1019650861640Iprovalicarb939210871341Isoxaflutole9383100821442Linuron1029741851243Metamitron1029750881544Methabenzthiazuron939300881345Methamidophos102901207512Methiocarb102100208513Methiocarb-sulfone102841807815Methiocarb-sulfoxide1029921881246Methomyl102890131011447Methoxyfenozide10210110851448Metobromuron1029741871249Metoxuron939300891250Monocrotophos10210110901251Monolinuron102101108614Omethoate10299308312Oxamyl102100208912Oxamyl-oxime10210110881252Oxycarboxin102102009112Oxydemeton-methyl1029750861353Paclobutrazole10210110871254Pencycuron10296608114Phenmedipham10294718314Phenmedipham-metabolite102100209315Phorate102683407419Phorate-sulfone9393008812Phorate-sulfoxide10210110901255Phosphamidon939300891056Picolinafen9386618415Pirimicarb102101018912Pirimicarb, desmethyl-10210011901257Prochloraz1019470831458Profoxydim99543213992159Propamocarb1019920701560Propoxur10210020881661Pymetrozine10273290892062Pyraclostrobin1029570851463Pyridate-metabolite1029291861564Rotenone1029390811565Sethoxydim102723271061966Spinosyn-A9388508217Spinosyn-D9382110831567Tebuconazole939030861668Tebufenozide1029930861469Temephos1029480871670Tepraloxydim1026204011414Terbufos93623017715Terbufos-sulfone9390308613Terbufos-sulfoxide939210881271Thiabendazole989251861372Thiacloprid939030881273Thiametoxam939120891374Thiocyclam93642907816Thiodicarb102624008216Thiofanox10298318514Thiofanox-sulfone102102009013Thiofanox-sulfoxide10210110921475Thiometon9388418716Thiophanate-methyl102831907712Tolylfluanid101366507622Triadimefon10299308513Triadimenol1029831871276Triazoxide1029093841677Trichlorfon10210101871278Tricyclazole1029660871279Triflumuron10189102841880Triforine1029732871581Vamidothion10210110891182Sum aldicarb10210110881183Sum butocarboxim10210110901184Sum carbendazim1019740831285Sum carbofuran10210200921086Sum dimethoate10210020861087Sum dichlofluanid102891121071788Sum disulfoton938940861389Sum ethiofencarb10210200891190Sum fenamiphos10210110901191Sum fensulfothion10210200861192Sum fenthion10210200891293Sum methiocarb10210020831294Sum methomyl10210020871295Sum oxamyl10210110881096Sum oxydemeton-methyl10210110881197Sum phenmedipham10210110881398Sum phorate1029750811299Sum pirimicarb102101109012100Sum terbufos9388508113101Sum thiofanox102102008911102Sum tolylfluanid10195608015103Sum triadimefon10299308613aAverage and RSD for recoveries within 60\u2013140% rangeMatrix-matched calibration, API3000Level\u2009=\u20090.05\u00a0mg kg\u22121 for most pesticides\/metabolitesBold indicates pesticides, including metabolites that are part of residue definition, if appropriate\nAveraging acceptable recoveries reveals there is some bias, because the values are mostly approximately 87% (in contrast with the GC\u2013MS data, for which the average was approximately 100%). It was noted that for dry crops relatively low recovery (typically between 60\u201370%) was obtained for all pesticides. The cause is not clear. This bias can also be seen in tables in other papers (barley [26], soya grain [33]).\nIndependent evaluation of method performance by proficiency testing\nFrom results obtained over the years from participation in proficiency tests, an additional and independent verification of method performance could be made. The data are summarized in Table\u00a011 and clearly show that good quantitative data were consistently obtained from both GC\u2013MS and LC\u2013MS\u2013MS, with method performance good (Z-score<2) 54 times, doubtful (2\u2009<\u2009Z\u2009<\u20093) three times, and never poor. It also shows that the calibration approach (one-point calibration, tomato-matrix standard for GC and matrix-matched standard for LC) is fit-for-purpose.\nTable\u00a011Results from the analysis of Fapas (series 19) proficiency test samples (2003\u20132005)SamplePesticideMRMSpike level added (\u03bcg kg\u22121)Inter-lab. result (\u03bcg kg\u22121)TNO result (\u03bcg kg\u22121)Z-score TNO#53 AppleFenpropathrinGC\u2013MS5004055281.7Parathion-methylGC\u2013MS705947\u22120.9TetradifonGC\u2013MS14011591\u22120.9TriazofosGC\u2013MS14011974\u22121.7VinchlozolinGC\u2013MS6053530.0#52 CucumberIprodioneGC\u2013MS1009489\u22120.3MethomylLC\u2013MS\u2013MS2825280.5ThiabendazoleLC\u2013MS\u2013MS50128113\u22120.5#51 PearCarbendazimLC\u2013MS\u2013MS15011660\u22122.2Dodinenot in MRM6059**ImazalilLC\u2013MS\u2013MS4002372730.8#49 MelonChlorprophamGC\u2013MS109111.0ChlorpyrifosGC\u2013MS887\u22120.7DimethoateLC\u2013MS\u2013MS151915\u22120.9PirimicarbLC\u2013MS\u2013MS201916\u22120.7#48 TomatoAzoxystrobinGC\u2013MSNot given201166\u22120.9BifenthrinGC\u2013MSNot given83990.9BuprofezinGC\u2013MSNot given1081311Chlorpyrifos-methylGC\u2013MSNot given319281\u22120.6ProcymidoneGC\u2013MSNot given712668\u22120.4#47 GrapefruitDiazinonGC\u2013MSNot given2622940.6HeptenophosGC\u2013MSNot given1682341.9MalathionGC\u2013MSNot given715690\u22120.2MethidathionGC\u2013MSNot given567540\u22120.3#46 LettuceBromopropylateGC\u2013MS806751\u22121.1DimethoateLC\u2013MS\u2013MS3002853160.6OxadixylGC\u2013MS1201271340.3PenconazoleGC\u2013MS1008251\u22121.7Tolclofos-methylGC\u2013MS16013775\u22122.1#42 AppleChlorfenvinphosGC\u2013MS907150\u22121.3ChlorpyrifosGC\u2013MS400259241\u22120.3MethamidophosLC\u2013MS\u2013MS604431\u22121.3MonocrotophosLC\u2013MS\u2013MS805856\u22120.1OmethoateLC\u2013MS\u2013MS150108103\u22120.2TrifluralinGC\u2013MS10059620.2#41 BasilKresoxim-methylGC\u2013MS1509486\u22120.4ProcymidoneGC\u2013MS1208778\u22120.5PropyzamideGC\u2013MS1008159\u22121.2VinclozolinGC\u2013MS604744\u22120.3#38 TomatoAzoxystrobinGC\u2013MS150137132\u22120.2BupirimateGC\u2013MS1008362\u22121.1Chlorpyrifos-methylGC\u2013MS807253\u22121.2QuinalphosGC\u2013MS140124105\u22120.7#37 LemonDiazinonGC\u2013MS8042420.0FenitrothionGC\u2013MS10078800.1MetalaxylGC\u2013MS12094930MethidathionGC\u2013MS1501091541.9#35 LettuceCarbendazimLC\u2013MS\u2013MS805331\u22121.9lambda CyhalothrinGC\u2013MS806654\u22120.8MetalaxylGC\u2013MS1209486\u22120.4#34 AppleDiphenylamineGC\u2013MS503929\u22121.2Pirimiphos-methylGC\u2013MS5041420.1PropargiteGC\u2013MS2001621720.3TetradifonGC\u2013MS1008338\u22122.5#29 Sweet pepperDichloranGC\u2013MS2001792000.6MecarbamGC\u2013MS100901201.5MethamidophosLC\u2013MS\u2013MS6051540.3\nConclusions\nThe ethyl acetate-based multi-residue method has been modified to meet today\u2019s demands in respect of ease and speed of sample preparation. For GC\u2013MS analysis, combined GCB\/PSA dispersive clean-up enables prolonged injection of vegetable and fruit extracts (10\u00a0mg matrix equivalent) without maintenance. Retention time shifts induced by some matrices compared with the calibration standard are reduced by the clean-up procedure. Interferences are partially removed, resulting in cleaner (extracted ion) chromatograms. The last two benefits aid correct automatic peak assignment and confirmation. Addition of toluene during dispersive clean-up prevented unacceptable adsorption of planar pesticides by GCB yet removal of chlorophyll and other pigments was still sufficient. Use of liners with a sintered porous glass bed on the inner wall makes 20\u00a0\u03bcL injection non-critical and robust. In GC, use of a universal matrix-matched standard (tomato) is a feasible means of compensating for the matrix effects of many other vegetable and fruit samples. For most pesticides, LOQs of 0.01\u00a0mg kg\u22121 can be obtained by GC\u2013MS with full-scan acquisition.\nThe same initial extract (i.e. without any clean-up) can be used for LC\u2013MS\u2013MS analysis, after changing the solvent to methanol\u2013water. LC\u2013MS\u2013MS is relatively tolerant of injection of matrix\u2014despite the absence of any clean-up no special maintenance was required. Matrix-induced suppression was observed for several matrices, however, especially herbs and citrus, and must be evaluated for all pesticide-matrix combinations. In contrast with the GC\u2013based method, use of a universal matrix-matched standard to compensate for matrix effects was not feasible.\nEvaluation of analytical quality control data for 271 pesticides and degradation products in over one hundred matrices showed that, at the 0.05\u00a0mg kg\u22121 level, recovery was acceptable for 92% (LC\u2013MS\u2013MS) and 93% (GC\u2013MS) of all pesticide\u2013matrix combinations. It also revealed that the method fails in the other 7\u20138% because of lack of specificity (mostly in GC\u2013MS) or because of poor extraction efficiency and\/or degradation (LC\u2013MS\u2013MS). The only way to identify these limitations is by thorough and continual evaluation of the quantitative performance of the method for all the pesticides (rather then a \u201crepresentative subset\u201d) in all the matrices.","keyphrases":["pesticides","multi-residue analysis","foods\/beverages","gc-ms","lc-ms\/ms"],"prmu":["P","P","M","U","U"]}
{"id":"Biochim_Biophys_Acta-1-5-2212780","title":"The role of mitochondria in protection of the heart by preconditioning\n","text":"A prolonged period of ischaemia followed by reperfusion irreversibly damages the heart. Such reperfusion injury (RI) involves opening of the mitochondrial permeability transition pore (MPTP) under the conditions of calcium overload and oxidative stress that accompany reperfusion. Protection from MPTP opening and hence RI can be mediated by ischaemic preconditioning (IP) where the prolonged ischaemic period is preceded by one or more brief (2\u20135 min) cycles of ischaemia and reperfusion. Following a brief overview of the molecular characterisation and regulation of the MPTP, the proposed mechanisms by which IP reduces pore opening are reviewed including the potential roles for reactive oxygen species (ROS), protein kinase cascades, and mitochondrial potassium channels. It is proposed that IP-mediated inhibition of MPTP opening at reperfusion does not involve direct phosphorylation of mitochondrial proteins, but rather reflects diminished oxidative stress during prolonged ischaemia and reperfusion. This causes less oxidation of critical thiol groups on the MPTP that are known to sensitise pore opening to calcium. The mechanisms by which ROS levels are decreased in the IP hearts during prolonged ischaemia and reperfusion are not known, but appear to require activation of protein kinase C\u03b5, either by receptor-mediated events or through transient increases in ROS during the IP protocol. Other signalling pathways may show cross-talk with this primary mechanism, but we suggest that a role for mitochondrial potassium channels is unlikely. The evidence for their activity in isolated mitochondria and cardiac myocytes is reviewed and the lack of specificity of the pharmacological agents used to implicate them in IP is noted. Some K+ channel openers uncouple mitochondria and others inhibit respiratory chain complexes, and their ability to produce ROS and precondition hearts is mimicked by bona fide uncouplers and respiratory chain inhibitors. IP may also provide continuing protection during reperfusion by preventing a cascade of MPTP-induced ROS production followed by further MPTP opening. This phase of protection may involve survival kinase pathways such as Akt and glycogen synthase kinase 3 (GSK3) either increasing ROS removal or reducing mitochondrial ROS production.\n1\nIntroduction\nThe heart is one of the most energy demanding tissues in the body and is totally dependent upon oxidative phosphorylation to supply the large amount of ATP required for beat-by-beat contraction and relaxation. If the blood flow to the heart is impaired (ischaemia), as occurs when a blood clot occludes a coronary artery (coronary thrombosis) or where cardiac surgery requires the heart to be disconnected from the blood supply, the source of oxygen is removed leading to the cessation of oxidative phosphorylation. This causes tissue ATP and creatine phosphate concentrations to decrease with a concomitant rise in ADP, AMP and Pi concentrations. Although glycolysis is activated, it is unable to meet the demand of the beating heart for ATP. Consequently, the heart rapidly ceases to beat as the contractile machinery is inhibited by elevated Pi and ADP, combined with the decreasing pH that accompanies the accumulation of glycolytic lactic acid [1\u20133]. The heart can usually survive a short period of ischaemia and then recover upon reperfusion. Although the performance of the heart may be impaired initially (stunning), given time recovery is complete. However, if the period of ischaemia is too long, the tissue becomes irreversibly damaged. Hence, if the heart is to be salvaged, it is important to restore the blood flow as soon as possible. Yet, paradoxically, such reperfusion can exacerbate the damage occurring during the ischaemic period. This is known as reperfusion injury and is accompanied by enzyme release and morphological changes characteristic of necrosis [1\u20133]. The extent of damage can be visualised as an area of necrotic tissue known as the infarct whose area can be determined to provide a quantitative measure of injury. Quantification of damage may also be provided by measuring the release of intracellular proteins such as lactate dehydrogenase or troponin I [4]. In addition to the necrotic cell death that represents the major damage to the reperfused heart there is also evidence that some myocytes around the periphery of the infarct die by apoptosis [5,6]. Understanding the causes of reperfusion injury and devising ways of preventing it is of major clinical importance in cardiac surgery and the treatment of coronary thrombosis.\nThere is increasing evidence that mitochondrial dysfunction plays a central role in mediating both the necrotic and apoptotic components of reperfusion injury, and that one of the most effective ways of protecting hearts from such injury, known as ischaemic preconditioning (IP), acts to attenuate this [1,3,7\u201310]. This review will address the causes of reperfusion injury, emphasising the role of the mitochondrial permeability transition pore (MPTP) and prevention of its opening by IP. The proposed signalling mechanisms through which IP may exert its effects will be discussed, including the proposed role of mitochondrial potassium channels which has been a major but controversial area of research (see [11\u201313]. However, first it is necessary to explain the phenomenon of preconditioning.\n2\nThe phenomenon of ischaemic preconditioning\nIschaemic preconditioning (IP) involves exposing the heart to brief periods of ischaemia (typically 2\u20135\u00a0min) interspersed with periods of normal perfusion prior to the prolonged ischaemia initiated within an hour of the preconditioning protocol. This protocol was first shown to offer strong protection against reperfusion injury in the dog heart by Murry et al. in 1986 [14] and has since been confirmed in all species investigated, including humans [15\u201317]. However, it should be noted that significant reduction in infarct size only occurs when the optimal duration for the test period of ischemia is chosen and is lost if the ischaemic period is too long. Preconditioned hearts exhibit a smaller infarct size and intracellular enzyme release (indicators of necrotic cell death) and fewer arrhythmias [14,18], whilst contractile function is preserved [19]. Typically, 2\u20133 brief (2\u20135\u00a0min) cycles of ischaemia and reperfusion are used in IP, there being little advantage of additional cycles [15]. If ischaemia is initiated more than 1\u20132 h after the preconditioning protocol, protection is lost but re-emerges again after about 24 h and lasts for up to 3 days. This is termed the second window of preconditioning [15,20\u201322]. A wide variety of drugs that activate different signalling pathways thought to be involved in mediating IP (see Section 6) can also induce protection and thus preconditioning is often used as a generic term to encompass any protocol applied before prolonged ischaemia that protects the heart during reperfusion. Not to be confused with such preconditioning is a more recently described phenomenon known as post-conditioning. Here hearts are given several very brief ischaemic interludes (10\u00a0s) during the early phase of reperfusion which also improves the recovery of the heart and reduces infarct size [23\u201325]. This protocol has considerable clinical potential since intermittent reperfusion can be induced during angioplasty prior to permanent restoration of blood flow. It is beyond the scope of this review to consider the similarities and differences between preconditioning and postconditioning in any detail. However, many of the mechanisms and signalling pathways mediating protection are thought to be the same [25] and where effects on mitochondria have been demonstrated in postconditioning they will be noted.\n3\nCauses of reperfusion injury\nIncreases in cellular [Ca2+] and reactive oxygen species (ROS), initiated in ischaemia and then amplified upon reperfusion, are thought to be the main causes of reperfusion injury. Mitochondria are involved both in the production of ROS and as targets for the damaging action of both ROS and calcium [1,3,9,26].\n3.1\nDamage occurring in ischaemia (Reviewed in [1;3;26])\nDuring ischaemia, the increased rate of glycolysis causes lactic acid to accumulate and the intracellular pH (pHi) to drop rapidly. This activates the Na+\/H+ antiporter as the cell endeavours to restore pHi. However, the rapidly declining ATP concentrations cause inhibition of the Na\/K ATPase and lead to a rise in intracellular [Na+]. This in turn reduces the ability of the cell to restore its pHi and increases intracellular [Ca2+] since the Na+\/Ca2+ antiporter that usually pumps Ca2+ out of the cell, is inhibited or reversed. The conversion of ATP to ADP and AMP is rapid and reversible. AMP is slowly converted into adenosine and then inosine and xanthine through a purine degradation pathway. These nucleosides leak out of the cell (and may have vasodilator effects through purinergic receptors) and lead to a gradual depletion of adenine nucleotide which may contribute to the reduced cardiac performance (stunning) seen on reperfusion.\nAnother feature of ischaemia that is thought to contribute to the damage seen during reperfusion is the production of reactive oxygen species (ROS). There is a small increase in ROS production immediately the heart becomes ischaemic which remains stable for 20\u201325 min but then increases dramatically [27]. This large increase is thought to play a major role in damaging the heart during ischaemia and sensitising it to reperfusion as described below. However the source of the ROS is unclear and might involve complex 1 and complex 3 of the respiratory chain or perhaps more likely xanthine oxidase acting on xanthine formed from the degradation of adenosine as noted above [26,28\u201331]. The depletion of ATP combined with elevated [Ca2+] and ROS leads to a gradual decline in cellular integrity as degradative enzyzmes are activated and ATP-dependent repair processes are unable to operate [1,3,26,32]. If the tissue remains ischaemic for only short periods and the mitochondria remain sufficiently intact to generate the ATP, tissue damage is slight and can be repaired by ATP-dependent processes upon reoxygenation. However, a critical point is reached when recovery is not possible and reperfusion actually causes further damage to the heart, causing extensive necrosis with associated enzyme release [1,3].\n3.2\nDamage occurring during reperfusion\nReperfusion is associated with a burst of reactive oxygen species (ROS) production [27], but here too the source of the ROS is debated. Although some may be produced by xanthine oxidase and NADPH oxidase, it is probable that most is formed by complex 1 and complex 3 of the respiratory chain [26,28\u201330]. When the respiratory chain is inhibited by lack of oxygen and then re-exposed to oxygen, ubiquinone can become partially reduced to ubisemiquinone. This can then react with the oxygen to produce superoxide that is reduced to hydrogen peroxide by superoxide dismutase. Hydrogen peroxide is removed by glutathione peroxidase or catalase, but if ferrous ions (or other transition metals such as copper) are present it will form the highly reactive hydroxyl radical through the Fenton reaction [28]. Mitochondrial proteins are especially susceptible to ROS induced damage and this is reflected in the impaired respiratory chain activity of mitochondria isolated from ischaemic hearts (see [26,31]). Thus ROS have direct effects on several respiratory chain components, most especially on complex 1 but also on complex 3, and other iron sulphur proteins such as aconitase. ROS can also cause thiol oxidation and inhibition of the ATPase and adenine nucleotide translocase. In addition, ROS cause oxidation of glutathione that may then form mixed disulphides with proteins. Such protein modification is thought to have inhibitory effects on ion pumps and therefore exacerbate the effects of ATP deprivation on ionic homeostasis [33\u201335]. ROS also cause peroxidation of the unsaturated fatty acid components of the phospholipids, and especially cardiolipin of the inner mitochondrial membrane, and this leads to further inhibition of respiratory chain activity [36,37]. Furthermore, lipid peroxidation causes the release of reactive aldehydes such as 4-hydroxynonenal that can modify membrane proteins [38]. Overall, it is thought that the combined effects of ROS and elevated [Ca2+] play a critical role in the transition from reversible to irreversible reperfusion injury, and that mitochondria are the major target of these agents. In particular, they lead to the opening of the mitochondrial permeability transition pore, that is now widely accepted to play a critical role in reperfusion injury [1,3,10,26,39].\n4\nThe mitochondrial permeability transition pore (MPTP)\n4.1\nProperties of the MPTP\nUnder normal physiological conditions, the mitochondrial inner membrane is impermeable to all but a few selected metabolites and ions; this is essential to maintain the membrane potential and pH gradient that together drive ATP synthesis through oxidative phosphorylation. However, under conditions of high matrix calcium, especially when this is accompanied by oxidative stress, high phosphate and low adenine nucleotide concentrations, a non-specific pore opens in the inner mitochondrial membrane known as the mitochondrial permeability transition pore (MPTP). The properties and regulation of the MPTP are reviewed extensively elsewhere [3,40,41]. Once open, this pore allows free passage of any molecule of <\u00a01.5\u00a0kDa and so disrupts the permeability barrier of the inner membrane. This has two major consequences. First, it allows unrestricted proton movement across the inner membrane, causing oxidative phosphorylation to be uncoupled. Not only does this prevent ATP synthesis but it also enables the proton-translocating ATPase to reverse direction and so actively hydrolyse ATP rather than synthesise it. Under such conditions, intracellular ATP concentrations rapidly decline, leading to the disruption of ionic and metabolic homeostasis and activation of degradative enzymes such as phospholipases, nucleases and proteases [3,9,26]. Unless pore closure occurs, these changes will cause irreversible damage to the cell resulting in necrotic death.\nIt should be noted that opening of just a single pore in one mitochondrion is likely to cause its immediate depolarisation. This will then activate further pore opening in the same mitochondrion since MPTP opening of calcium loaded mitochondria is activated by depolarisation [42]. Thus mitochondria are either fully open or closed, and it is the fully open state that leads to the second consequence of MPTP opening; the mitochondria swell extensively [43]. This occurs as all small molecular weight solutes equilibrate across the inner membrane, leaving the high concentration of matrix proteins to exert a colloidal osmotic pressure that leads to the uptake of water and matrix swelling. Although unfolding of the cristae allows the matrix to expand without rupture of the inner membrane, the outer membrane will break and lead to the release of proteins in the intermembrane space such as cytochrome c and other factors that play a critical role in apoptotic cell death [44,45].\n4.2\nThe molecular identity of the MPTP\nThe molecular identity of the mitochondrial permeability transition pore remains uncertain [40,41,46], but it is generally accepted that an inner membrane component undergoes a calcium-triggered change in conformation that is facilitated by cyclophilin D (CyP-D), a peptidyl-prolyl cis-trans isomerase [46,47]. The role of CyP-D was first suggested by the discovery that cyclosporin A (CsA) acts as a potent inhibitor of pore opening [48]. Further studies revealed that the potency of different CsA analogues to inhibit pore opening correlates with their ability to inhibit the peptidyl-prolyl cis-trans isomerase activity within the matrix [49,50] that was subsequently identified as CyP-D [51,52]. Extensive work from many laboratories confirmed the critical role of CyP-D and this was finally put beyond doubt by the demonstration that MPTP opening in liver mitochondria from CyP-D knockout mice is much less sensitive to calcium than normal mitochondria, and is no longer inhibited by CsA [53\u201355].\nThe identity of the membrane component of the MPTP is less certain. However, the most widely accepted view is that the adenine nucleotide translocase (ANT) normally fulfils this role and extensive circumstantial data supports this view (see [9,56]). Thus opening of the MPTP is inhibited by adenine nucleotides with a similar concentration dependence and specificity as they exhibit when acting as substrates for the ANT, and this inhibition is overcome by the specific inhibitor of the ANT, carboxyatractyloside (CAT) that traps the ANT in its \u201cc\u201d conformation. By contrast, another inhibitor of the ANT, bongkrekic acid, that causes the carrier to take up the alternative \u201cm\u201d conformation, inhibits pore opening [57]. The ANT can also account for the sensitisation of the MPTP to calcium by oxidative stress and the vicinal thiol reagent phenylarsine oxide (PAO) [57]. Thus cysteine residues 160 and 260 of rat ANT2 can be cross-linked by oxidative stress or PAO, with modification of Cys160 alone being sufficient to prevent the inhibition of MPTP opening by adenine nucleotides, so stimulating pore opening [58]. Further evidence for an important role for the ANT is the ability of the ANT to bind to CyP-D in a CsA-sensitive manner [58,59]. In addition, when the purified ANT is reconstituted into proteoliposomes high calcium concentrations can induce the formation of non-specific channels [60] and this process is sensitised to [Ca2+] by the addition of purified cyclophilin [61]. Nevertheless, despite the strong evidence in favour of the ANT being the critical membrane component of the MPTP, it is unlikely to be essential. Thus in an elegant study, that has yet to be confirmed by others, mitochondria from mouse livers in which ANT1 and ANT2 had been knocked out were found to exhibit MPTP opening that was inhibited by CsA [62]. However, pore opening in the ANT-knockout mitochondria required much higher calcium concentrations than did wild-type mitochondria, and was no longer sensitive to adenine nucleotides confirming that the ANT is at the very least playing a critical regulatory role. One possibility is that the ANT is the normal membrane component of the MPTP but that other less abundant members of the mitochondrial carrier family can fulfil this role in the absence of the ANT [63]. An alternative possibility, proposed by Lemasters, is that unfolded membrane proteins form the MPTP and that the ANT is the most abundant of these [64].\nMany other proteins have been proposed to be components of the MPTP, including the peripheral benzenediazipine receptor, creatine kinase and the voltage dependent anion channel (VDAC) [3,40,41,65]. Of these perhaps the strongest candidate is VDAC since an interaction between VDAC and the ANT has been demonstrated and there is evidence that the MPTP may be associated with contact sites between the inner and outer mitochondrial membranes [3,40,65]. Furthermore, initial reports suggested that VDAC might be the locus for inhibition by ubiquinone analogues [66]. However, the recent demonstration that liver mitochondria from mice lacking VDAC1 exhibit normal MPTP opening and inhibition by ubiquinone analogues [67] make an essential role for VDAC1 unlikely. See note added in proof.\n5\nThe MPTP opening plays a central role in reperfusion injury\n5.1\nThe MPTP opens during reperfusion but not ischaemia\nWe have developed a technique to demonstrate that the MPTP is kept firmly closed in the heart under normal physiological conditions, but opens upon reperfusion following a period of ischaemia. This technique involves measuring the extent of [3H]-2-deoxyglucose entrapment in mitochondria that have undergone the permeability transition [68]. Others have used fluorescence microscopy to measure MPTP opening in isolated cardiac myocytes and have also confirmed that the pore opens under conditions of simulated ischaemia and reperfusion [69]. It might be expected that the pore would also open after prolonged ischaemia and some have reported this to be the case [70,71]. However, these studies relied on cytochrome c release as a measure of MPTP opening, but it is known that this can occur independently of the MPTP as a result of Bax translocation to mitochondria during ischaemia [72]. By contrast, our own direct measurements [68] and those of others [73] do not support MPTP opening in ischaemia. Since the pore has been shown to be powerfully inhibited by low pH (<\u00a07) [74] and the time course of opening during reperfusion correlates with the return of pH to normal [75], we have suggested that it is the low pH accompanying ischaemia that prevents pore opening despite the conditions of oxidative stress, low adenine nucleotide and elevated phosphate and calcium [75]. Indeed, as would be predicted, maintaining a low pH during the initial phase of reperfusion is known to protect hearts from reperfusion injury [76\u201378] and may play a role in the cardioprotective effects of inhibitors of the Na+\/H+ exchanger such as amelioride and cariporide (see [3,79,80]). A similar mechanism has also been implicated recently in the protection offered by postconditioning [81,82].\nIt has also been proposed that the rapid energisation of mitochondria at reperfusion will lead to electrogenic calcium uptake into the mitochondria. Since the calcium has accumulated in the cytosol during ischaemia, this might be expected to induce mitochondrial calcium overload and hence MPTP opening [1,9]. However, measurements of mitochondrial [Ca2+] in isolated cardiac myocytes imply that it is the mitochondrial [Ca2+] at the end of ischaemia rather than during reperfusion that correlates better with cell injury [83]. Another factor that will reinforce MPTP opening at reperfusion is the surge of ROS production that is known to occur [27,84] and this may well be the most important factor in determining the outcome of reperfusion [73]. Indeed, there is increasing evidence that the extent of MPTP opening is a critical factor in the transition from reversible to irreversible reperfusion injury [1,3,9,65]. More accurately, it is the extent to which pores remain in an open state that correlates with damage, since we have demonstrated that some mitochondria in which the MPTP opens initially, subsequently undergo pore closer as reperfusion continues [75,85]. It is possible that myocytes in which the majority of mitochondria have undergone such reversible MPTP opening will have swollen sufficiently to release pro-apoptotic proteins such as cytochrome c and thus initiate apoptosis as noted in Section 4.1. These myocytes are likely to be at the periphery of the infarct where the ischaemic insult was incomplete and thus would account for the ring of apoptotic myocytes that have been observed to surround the necrotic core of the infarct [5,6]. It should be noted that mice in which CyP-D had been knocked out demonstrate normal development and cells from them respond to a range of apoptotic stimuli in the same manner as wild-type mice [54,55]. Thus MPTP opening cannot the mechanism by which apoptosis is mediated in the healthy animal, but this does not preclude apoptosis being induced in this way under pathophysiological situations such as those described above [46].\n5.2\nInhibition of MPTP opening protects hearts from reperfusion injury\nIf the opening of the MPTP is a critical event in reperfusion injury then it would be predicted that inhibition of pore opening should protect hearts from injury. Many studies have confirmed that this is the case. Protection was first demonstrated with CsA which, when added prior to global ischaemia, was shown to improve haemodynamic function and ATP levels on reperfusion and to decrease necrotic damage as reflected in intracellular enzyme release [86]. Subsequent studies have shown that other CsA analogues, and the alternative CyP-D inhibitor sanglifehrin A (SfA) also provide protection under these conditions [68,87,88] whilst mitochondria isolated from these hearts showed less pore opening in response to a given calcium load [88,89]. In addition, CsA and SfA can reduce the infarct size of hearts in which a coronary artery is occluded and then re-opened to mimic the treatment of a coronary thrombosis. In this model of reperfusion injury, protection is observed even when the drug is added only at reperfusion [90,91].\nLess direct approaches to inhibit MPTP opening are also effective at protecting the heart from reperfusion injury. Thus free radical scavengers are well known to be protective and in the case of one such agent, the anaesthetic propofol, we have used the 2-deoxyglucose entrapment technique to confirm inhibition of the MPTP directly [92]. In addition, we have demonstrated that propofol provides protection from reperfusion injury in a pig model of open heart surgery [93]. Inhibition of the sodium proton exchanger with cariporide is also known to protect the heart from reperfusion injury [94] and here too inhibition of MPTP opening correlates with this protection [80]. This protection probably reflects less sodium and hence calcium loading of the cardiac myocytes during ischaemia, coupled with a lower pH during the initial phase of reperfusion, both of which will reduce MPTP opening. The most powerful agent for inhibiting MPTP opening and protecting the heart is pyruvate [75,95]. The presence of 10\u00a0mM pyruvate during ischaemia and reperfusion can almost totally protect rat hearts from 30\u00a0min global ischaemia and this is accompanied by greatly reduced MPTP opening followed by total pore closure [75]. Pyruvate has three beneficial effects that may contribute towards its inhibition of MPTP opening: it is a free radical scavenger, it maintains a lower intracellular pH during the early phase of reperfusion and it is an excellent fuel for ATP synthesis that bypasses the ATP-requiring steps of glycolysis [75,96].\n5.3\nProtection by preconditioning involves inhibition of MPTP opening\nIn view of the critical role of the MPTP in reperfusion injury and the proven ability of inhibitors of the MPTP to protect from injury it might be predicted that ischaemic preconditioning (IP) would inhibit pore opening. Both direct and indirect techniques have been used to confirm this. Our own experiments have used the 2-deoxyglucose entrapment technique to demonstrate that less MPTP opening occurs at reperfusion in preconditioned hearts and that as reperfusion continues the majority of mitochondria that originally opened close again in the IP hearts but not in control hearts [85]. Others have used confocal microscopy to monitor mitochondrial membrane potential in an isolated cardiac myocyte model of preconditioning and shown that IP desensitises the mitochondria to pore opening induced by oxidative stress [69,97]. Recent experiments performed with two-photon microscopy in the perfused heart have confirmed these data [98]. Yet other studies have investigated the sensitivity of isolated mitochondria from control and IP hearts to pore opening in response to added calcium [85,99,100]. Both light scattering and mitochondrial calcium retention techniques demonstrated that when mitochondria were isolated immediately after the preconditioning stimulus, those from IP hearts were not any less sensitive to calcium-induced pore opening than those from control hearts. Indeed, if anything they were more sensitive [85,100]. When isolated at the end of ischaemia or during the first few minutes of reperfusion mitochondria were more sensitive to MPTP opening than those isolated prior to ischaemia but at these time points mitochondria from IP hearts were considerably less sensitive to calcium-activated pore opening than those from control hearts [99\u2013101]. These data clearly show that the IP protocol alone is not having a direct effect on the MPTP but suggest that IP inhibits a process occurring during ischaemia and reperfusion that is responsible for sensitising the MPTP to calcium. This is an important fact to bear in mind when evaluating the extensive literature surrounding the mechanisms by which preconditioning exerts its protective effects as will be discussed further below (Section 6).\nTemperature preconditioning, where hearts are exposed to three brief hypothermic (26\u00a0\u00b0C) episodes prior to normothermic ischaemia is also thought to protect through inhibition of the MPTP [101] as is post-conditioning [102]. Thus, mitochondria isolated from such hearts during reperfusion are less sensitive to MPTP than those isolated from control hearts with temperature preconditioning being even more protective than IP [101].\n6\nSignalling pathways linking preconditioning to inhibition of the MPTP\nThere is a plethora of data implicating many different signalling pathways in preconditioning and the relevant role of each remains hotly debated (see [16,30,103\u2013107]). Many of these studies have utilised pharmacological agonists and antagonists of components of the different signalling kinase cascades and their potential targets. The complex interactions that exist between different signalling pathways makes interpretation of these data complicated and this is made worse by the questionable specificity of some of the pharmacological interventions. The reader is referred elsewhere for a detailed consideration of the many potential pathways that have been proposed [30,103\u2013107]. Here we will only provide a brief overview before focussing on how they may exert their effects on the mitochondria.\n6.1\nThe role of protein kinase C\nThere is extensive evidence that protein kinase C (PKC) plays a central role in preconditioning since inhibition of PKC has been shown to block the protection afforded by IP and pharmacological activators of PKC are cardioprotective (see [105]). There remains some controversy over which of the many PKC isoforms may be involved in IP, whether they translocate to the particulate fraction and how they exert their effects [103]. Nevertheless, there is a large body of evidence to implicate PKC\u03b5 as an important player in IP [105]. Thus hearts from PKC\u03b5 knockout mice do not exhibit IP [108] whereas transgenic mice with cardiac-specific over-expression of PKC\u03b5 or expression of an activator of PKC\u03b5 are protected from reperfusion injury [109\u2013111]. Some studies have reported PKC\u03b5 translocation to the particulate fraction, including mitochondria [112\u2013114] and have suggested a direct inhibition of the MPTP by PKC\u03b5 involving phosphorylation of components of the MPTP such as the voltage dependent anion channel (VDAC) [113\u2013115]. However, in our own studies we were unable to detect PKC\u03b5 translocation to the mitochondria of IP hearts (Fig. 1A) whilst others have observed that PKC translocation to the particulate fraction following IP is transient and lost during progressive brief cycles of ischaemia and reperfusion [116].\nAlthough a role for PKC\u03b5 in IP seems established, the mechanism(s) by which it exerts its effects are less clear as will become apparent below (see sections 7\u201310). It is also uncertain how ischaemic preconditioning activates PKC\u03b5, although several pathways may be involved. Factors released during the brief ischaemic periods such as adenosine, bradykinin, noradrenaline and opioids may bind to their G protein-coupled receptors to stimulate phospholipase C, producing diacylglycerol that activates PKC. Indeed, all of these factors can pharmacologically precondition the heart [104]. Accumulating evidence supports a role for the modest increase in ROS that occurs during IP protocol in the activation of PKC [27,117,118]. Thus ROS are known to activate PKC in the isolated heart [118\u2013120] and IP can be prevented if free radical scavengers are present during the preconditioning phase [100,101,118,121] Oxidation of critical cysteine residues on PKC isoforms is known to cause their activation [35,122] and thus provides a mechanism by which ROS could activate PKC.\n6.2\nThe role of nitric oxide and cyclic GMP dependent protein kinase\nData from several laboratories have implicated activation of cyclic GMP dependent protein kinase (PKG), perhaps by nitric oxide, in the signalling pathway for IP. Thus it has been reported that nitric oxide donors and PKG activators can induce preconditioning whilst NO scavengers and PKG inhibitors prevent preconditioning [106,123,124]. Pharmacological studies from Garlid's laboratory have led them to conclude that PGK and PKC\u03b5 work in concert to induce IP through an effect on the putative mitochondrial ATP-dependent potassium channel (mitoKATP \u2014 see Section 9 below). They propose that cGMP activates PKG localized at the cytosolic surface of the mitochondrial outer membrane and that this phosphorylates some target protein which in turn can somehow activate PKC\u03b5 residing in the intermembrane space of mitochondria. This PKC\u03b5 then would phosphorylate the mitoKATP channel to mediate preconditioning [123]. In a subsequent paper they proposed that activation of the mitoKATP channel increased ROS formation and that this ROS activated a second pool of PKC\u03b5 that inhibited the MPTP [125]. Our own data do not support this (see Sections 7). It should be noted that an alternative or additional mechanism has been proposed by which nitric oxide may protect the heart when present during the initial stages of reperfusion (see [128]). Cytochrome oxidase is competitively inhibited by nitric oxide and this would lead to an inhibition of respiration and hence a reduction in the mitochondrial membrane potential. It is suggested that one consequence of this would be less mitochondrial calcium accumulation and hence less MPTP opening. However, another consequence of a lower mitochondrial membrane potential would be impaired ATP production which would not be predicted for hearts showing improved recovery. Similar arguments have been made for the proposed depolarisation caused by mitochondrial KATP channel opening and these will be considered in more detail in Section 10.2.\n6.3\nThe role of pro-survival kinases\nSeveral laboratories have presented evidence to implicate activation of pro-survival kinases such as Akt (protein kinase B) in preconditioning [107,126]. It is proposed that tyrosine kinases are activated during IP through some ill-defined interaction with G-protein coupled receptors such as the adenosine and bradykinin receptors and that this causes activation of phosphatidyl inositol 3 phosphate kinase (PI-3-kinase). The resulting phosphatidylinositol 3,4-bisphosphate then activates phosphoinositide-dependent kinase 1 (PDK1) that in turn phosphorylates and activates Akt. However, it is also possible that activation of tyrosine kinases or inhibition of tyrosine phosphatases and the phosphatidyl inositol 3,4,5,-trisphosphate phosphatase PTEN (Phosphatase and TENsin homolog) may be mediated by the ROS produced during the preconditioning stimulus [107,127]. In support of this mechanism it has been demonstrated that phosphorylation of Akt is enhanced by IP, especially during reperfusion, and that pharmacological inhibitors of either Akt or PI-3-kinase prevent preconditioning [126,128\u2013130]. It has further been proposed that following phosphorylation, activated Akt phosphorylates endothelial nitric oxide synthase to produces NO that acts via PKG as noted above [106,107].\nSollott et al. [97] have suggested that all these kinases may converge to phosphorylate and inhibit glycogen synthase kinase 3\u03b2 (GSK3\u03b2) that they report translocates to the mitochondria. Inhibition of GSK3\u03b2 by Akt-induced phosphorylation would then mediate protection. Although it is known that GSK3\u03b2 is pro-apoptotic and that its phosphorylation inhibits its activity and stimulates cell survival [131], the mechanism of any mitochondrial effects remain to be elucidated. However, it may be significant that GSK3\u03b2 has been reported to phosphorylate VDAC, and that this phosphorylation is associated with a sensitisation to cytotoxic drugs that stimulate pore opening [132]. VDAC was widely regarded as an integral component of the MPTP, but recent data showing that mitochondria from the VDAC1 knockout mouse have a normal MPTP cast doubt on this [53]. See note added in proof.\n6.4\nThe role of AMP-activated protein kinase\nYet another protein kinase, AMP-activated protein kinase (AMPK), is activated by a large variety of cellular stresses that deplete ATP and increase AMP, including glucose deprivation, hypoxia, ischaemia, oxidative stress and hyperosmotic stress [133]. It could be argued that the many pharmacological agents known to induce preconditioning, from KATP channel openers, to uncouplers and respiratory chain inhibitors (see Section 10), might all represent insults to the heart that increase AMP and so activate AMPK as the focus of a common signalling mechanism to protect the heart [133]. Indeed, transgenic mice expressing a kinase dead form of AMPK in the heart have been shown to exhibit greater myocardial necrosis and apoptosis after ischaemia\/reperfusion [134] and cannot be preconditioned [135]. Furthermore, Nishino et al. [136] have shown that PKC inhibitors abolished AMPK activation by ischaemic episodes in the myocardium, suggesting that AMPK might be activated by IP in a PKC-dependent manner. However, our own data fail to show any abrogation of the IP-induced protection by the inhibitor of AMPK, compound C, under conditions where the inhibitor of PKC, chelerythrine, did overcome the protection [101].\nTaken together, all these data suggest that several signalling pathways may interact or act in parallel to induce IP, but the ultimate target of their action remains unclear. Since IP involves inhibition of the MPTP the signalling pathway must ultimately inhibit MPTP opening, but this could be achieved either by direct phosphorylation of a component of the MPTP or indirectly by influencing factors that enhance pore opening such as by reducing oxidative stress or calcium overload.\n7\nMechanism of inhibition of the MPTP by IP\n7.1\nIs there evidence for regulation by phosphorylation?\nAs noted above, there are several reports that protein kinases may migrate to the mitochondria in response to IP suggesting that phosphorylation of a component of the MPTP may be responsible for its inhibition. Baines et al. have reported that the MPTP of isolated heart mitochondria was inhibited following incubation for 15\u00a0min with purified PKC\u03b5 plus phorbol ester [115]. However, such experiments are hard to interpret since incubating isolated mitochondria in vitro can change the sensitivity of MPTP opening to calcium, especially if ATP is added since this is an inhibitor of MPTP opening in its own right [3,40]. Similar criticisms could be levelled against experiments in which incubation of isolated rat liver mitochondria with a cGMP analogue in the presence of a cytosolic extract and ATP caused inhibition of the calcium-induced mitochondrial permeability transition [137], although here reversal of the effect by the PKG inhibitor KT5823 makes the data more convincing. However, no data are available to show changes in phosphorylation of any mitochondrial protein following IP that might account for inhibition of the MPTP. Furthermore, when we and others measured MPTP opening in mitochondria isolated immediately after the preconditioning stimulus we observed no reduction in sensitivity to calcium as might have been predicted if a component of the MPTP had been phosphorylated [85,100]. Indeed, it is unclear how activated cytosolic protein kinases could cross the outer membrane to phosphorylate and inhibit components of the MPTP, let alone cross the inner membrane into the matrix. Although there are reports to suggest that this occurs [97,113,114], our own attempts to demonstrate the translocation of PKC\u03b5 or GSK3\u03b2 to mitochondria have consistently failed as illustrated in Fig. 1. A key aspect of our experiments was the use of Percoll gradient centrifugation to remove plasma membrane contamination which we have shown is significant in less pure mitochondrial fractions. This was demonstrated by the presence of monocarboxylate transporter 1 (MCT1) in the crude but not the purified mitochondria.\nOur inability to detect protein kinase translocation into the mitochondrial matrix is entirely consistent with extensive work from this laboratory more than 20\u00a0years ago in which high specific activity 32P labelling was used to investigate the phosphorylation of mitochondrial matrix proteins. Such studies demonstrated the presence of two proteins within the mitochondrial matrix that could be phosphorylated in Percoll-purified mitochondria from both heart and liver. These were pyruvate dehydrogenase (PDH) and branched chain 2-oxoacid dehydrogenase (BCDH) [138]. This was true whether the mitochondria were from control or glucagon-treated (raised cyclic AMP) rats [139], yet mitochondria from glucagon-treated rats were found to exhibit enhanced calcium retention characteristic of reduced sensitivity of the MPTP to calcium-induced opening [140,141].\nAlthough these data do not support a role for protein kinases within the mitochondrial matrix in the inhibition of the MPTP following preconditioning, they do not exclude the possibility of regulation by phosphorylation of proteins exposed to cytosolic kinases, such as those in the outer mitochondrial membrane or bound to it using scaffolding proteins [142,143]. Indeed, work from this laboratory many years ago demonstrated that when mitochondria were isolated from hepatocytes incubated with high specific activity 32P, or isolated mitochondria were incubated with \u03b3-[32P]-ATP, additional phosphorylated proteins were observed. Two of these proteins, both 30\u201335\u00a0kDa, demonstrated an increase in phosphorylation with glucagon treatment [139,144]. However, a weakness of the 32P labelling technique is that it will not detected proteins whose phosphorylation turnover is small and this may account for the larger number of phosphorylated proteins within mitochondria detected using a phosphoprotein-specific dye [145]. Nevertheless, a lack of turnover might suggest that such a phosphorylation does not play an important role in signalling.\nThere have been an increasing number or reports in which proteomic and immunodetection methods have been used to detect a variety of other kinases associated with the mitochondria [142,146]. However, the extent to which these represent real mitochondrial kinases rather than non-specific mitochondrial contamination of the mitochondrial fraction is hard to assess. We have recently used a proteomics approach to identify any mitochondrial proteins whose phosphorylation might be altered by preconditioning. Mitochondria were isolated from control and IP hearts immediately after the preconditioning stimulus as well as the end of ischaemia and after three min reperfusion and phosphor-proteins detected using 2-D gel electrophoresis and staining with ProQ Diamond phospho-protein fluorescent dye. Although a significant number of phospho-proteins were detected in addition to the predominant spots representing PDH and BCDH, no consistent changes were detected in any of these in response to IP (unpublished data of S.J. Clarke, I. Khaliulin, Kate Heesom and A.P. Halestrap). Typical data are shown in Fig. 2. Overall, our data do not support a role for mitochondrial protein phosphorylation in mediating the inhibition of MPTP opening by IP. Rather, our data suggests that the inhibition of the MPTP by preconditioning may be secondary to either diminished ROS production or calcium overload as described below.\n7.2\nEffects of IP on ROS production and calcium loading during ischaemia and reperfusion as potential mediators of MPTP inhibition\nAs noted above, opening of the MPTP is triggered by calcium overload especially when this is accompanied by oxidative stress and these two parameters appear critical determinants of the recovery of isolated heart cells from simulated ischaemia. Indeed, the extent of recovery has been reported to correlate inversely with mitochondrial matrix calcium concentrations at the end of ischaemia [83,147] and to be triggered by the rise in ROS during reperfusion [73]. It is well established that preconditioning reduces ROS production both at the end of ischaemia and during reperfusion [27,84,148,149] and decreases mitochondrial calcium overload [150\u2013153]. Thus it is quite possible to account for the IP-mediated inhibition of MPTP opening at reperfusion merely through these indirect effects without having to invoke phosphorylation of any mitochondrial protein.\nOur own data are entirely consistent with this mechanism. Opening of the MPTP in mitochondria isolated immediately following the preconditioning stimulus showed the same sensitivity to calcium as in control mitochondria [85,100]. Mitochondria isolated at the end of ischaemia, or during reperfusion, were more sensitive to calcium-induced MPTP opening than those isolated prior to ischaemia. However, at these times the mitochondria from preconditioned hearts were less sensitive to MPTP opening than were mitochondria isolated from control hearts at the same time [99\u2013101]. This difference in sensitivity to calcium correlated with the exposure of the mitochondria to oxidative stress as reflected in the carbonylation of mitochondrial proteins [100,101], a surrogate marker for oxidative stress [154]. These data confirm that the oxidative stress mitochondria experience at the end of ischaemia and during reperfusion is significantly attenuated in preconditioned hearts. As such they provide strong evidence in favour of IP exerting its primary effect on the reduction of ROS levels during ischaemia and reperfusion with the secondary consequence that MPTP opening is diminished. If this is the case, the key signalling pathways in preconditioning will be those that mediate the decrease in ROS levels at the end of ischaemia and during reperfusion. This could be the result of decreased ROS production or of improved ROS removal, but the ability of preconditioning to protect against oxidative stress mediated by exogenous hydrogen peroxide [155,156] argues in favour of the latter. However, an additional effect of IP to reduce ROS production cannot be ruled out.\n8\nThe role of mitochondrial potassium channels\nPlasma membrane ATP-dependent potassium channels (KATP) are strongly expressed in the heart and have long been associated with cardioprotection [12,157]. It was originally proposed that opening of the plasma membrane KATP channel might hyperpolarize the cell leading to a shorter action potential duration (APD) and hence less calcium entry during ischaemia which would protect the hearts from calcium overload. In support of this, mouse hearts whose KATP channel Kir 6.2 had been knocked out exhibited greater calcium overload and ischaemia\/reperfusion injury than control hearts [146]. A role for the sarcolemmal KATP channels in the protective mechanism exerted by preconditioning was first suggested by the observation that KATP channel blockers such as glibencamide prevent preconditioning (see [12,157]). Further support came from the demonstration that hearts from the Kir 6.2 knockout mice were insensitive to either ischaemic or pharmacological preconditioning [158\u2013160]. However, despite this convincing evidence in favour of a role for the sarcolemmal KATP channel in preconditioning, other data argue against such a role. In particular conditions were found in which cardioprotection could be observed in the absence of APD shortening whilst a range of KATP channel openers were reported to show a poor correlation between their effect on APD and their protective effects (see [11,12,161\u2013163]). These data led to a change of emphasis, away from the sarlcolemmal KATP channel towards a similar KATP channel proposed to reside in the mitochondrial inner membrane.\n8.1\nThe identity of mitochondrial K+ channels and their physiological roles\nThe presence of an electrogenic mechanism for K+ entry into mitochondria (K+ channel) together with a K+\/H+ antiporter to pump K+ out again has been known for more than 30\u00a0years, and their relative activities are thought to play a key role in the regulation of matrix volume (see [43,164]). What is less clear is the identity of these channels and transporters since none have been purified sufficiently to allow sequencing. Nor has genomic analysis revealed any plasma membrane K+ channel isoform or spliced variant with a mitochondrial targeting sequence [12]. Rather the presence and properties of the K+ channels and transporters have been surmised from their functional characterisation (see [165,166]).\n8.2\nTechniques used to characterise mitochondrial K+ channels\nIn isolated mitochondria, potassium ion movements can be measured directly using isotopes or K+-sensitive fluorescent dyes, or indirectly by determining matrix volume changes (see [167\u2013171]). For mitoplasts patch clamping has been employed [172\u2013174] whilst studies have been performed on proteoliposomes containing reconstituted inner mitochondrial membrane proteins using electrophysiological measurement of single channel current or fluorescent techniques to measure net K+ transport [168,175\u2013179]. A major concern with the patch clamping and reconstitution techniques is the possibility that any channels detected in the mitochondrial membranes may actually represent a small number of K+ channels present in contaminating sarcolemmal membranes. It is known that most preparations of isolated mitochondria are extensively contaminated with plasma membranes unless further purified by density gradient centrifugation, and even then it is difficult to achieve complete removal of contaminating membranes [180,181]. However, in the majority of published work no data were presented to discount such contamination. Thus it is important to confirm that the activity of any channels identified by such techniques can be demonstrated in mitochondria, and this can be achieved by measurements of mitochondrial matrix volume.\nIt is widely agreed that the matrix volume is regulated by the relative activity of the K+ channels mediating membrane potential driven potassium entry into mitochondria, and the K+\/H+ antiporter catalysing proton-linked K+ extrusion activity [43,164,165,182]. Thus activation or inhibition of any K+ channel should lead to an increase or decrease in matrix volume respectively. This can be measured either directly, using an isotopic technique based around the permeability of 3H2O into the matrix and the exclusion of [14C]-sucrose, or by light scattering (see [165,182]). The latter provides a very sensitive real-time measurement of matrix volume since as the mitochondria swell, their refractive index decreases and they scatter less light. A wavelength of 520\u00a0nm is frequently employed since this represents the isosbestic wavelength at which changes in the redox state of the mitochondrial cytochromes cause no change in absorbance [182]. However, mitochondria can exhibit light-scattering changes that are independent of changes in matrix volume, reflecting rather a change in morphology or shape. It has been known for many years that such morphological changes, often referred to in terms of orthodox and condensed conformations of mitochondria, can be induced by ligands of the adenine nucleotide translocase (ANT) that switch the carrier from the \u201cc\u201d to the \u201cm\u201d conformation [44,49,170,183,184]. Thus externally added ATP, ADP or bongkrekic acid induce the \u201cm\u201d conformation of the ANT and cause a contraction of the mitochondria detected as an increase in light scattering whilst carboxyatractyloside, that induces the \u201cc\u201d conformation, exerts the opposite effect. Yet these light scattering responses all occur without changes in matrix volume measured isotopically [44,49,170,171].\nGarlid and Paucek dismiss these data, stating that \u201cthis claim cannot be supported by any theoretical argument\u201d, but the basis on which they make this assertion is unclear. First, the claim is backed up by experimental data from two different laboratories [44,49,170,171]. Second, the curvature of the mitochondria relative to the wavelength of light determines its refractive index and hence light scattering properties [182]. Thus a shape change without a change in volume (e.g. from a sphere to a cylinder) will affect the light scattering signal. Since the ANT represents some 25% or more of the inner membrane protein in heart mitochondria [185], it is not surprising that when it switches its conformation it can cause a change in mitochondrial morphology and hence light scattering. Third, Garlid and Paucek argue that changes in light scattering induced by adenine nucleotides must reflect mitochondrial volume changes because they did not observe them in potassium free medium. They go on to say \u201cIf Das et al. had carried out these simple control experiments they would have avoided spurious claims about conformational changes\u201d. However, we [170] and Brustovetsky et al. [171] have reported other controls that confirm that the adenine nucleotide-induced light scattering changes do not reflect changes in matrix volume. Thus additions of very low concentrations of valinomycin (0.4 to 1 pmol per mg mitochondrial protein) induced changes in matrix volume of 10\u201325% that were readily detectable using radioisotopes, yet the magnitude of the light scattering changes were similar to those induced by ADP or ATP for which no detectable changes in matrix volume were detected (see Fig. 3 and [170,171]). We have also performed experiments in potassium free media as suggested by Garlid and Paucek, which we report in Fig. 3. These data clearly show that such adenine nucleotide induced changes in light scattering do occur in potassium free medium and similar results were obtained by Brustovetsky et al. [171]. We suggest that experiments directed towards investigating the activity of K+ channels in intact mitochondria that rely entirely upon light scattering changes for measurement of matrix volume should be treated with caution. Parallel isotopic measurements of matrix volume provide an essential confirmation that such light scattering changes do reflect changes in volume rather than morphology.\n8.3\nEvidence for the mitochondrial KATP channel\nSome 20\u00a0years ago we showed that adenine nucleotides could inhibit electrogenic mitochondrial K+ entry into mitochondria and that this was probably mediated by their binding to the ANT [49,186,187], in agreement with earlier proposals [188]. More recent data obtained with brain mitochondria have confirmed this proposal [171]. However, the existence of a mitochondrial KATP channel similar to that found in the plasma membrane was first formally proposed as a result of data obtained using patch clamping of giant fused mitoplasts from liver mitochondria [172] and light scattering experiments in isolated mitochondria [189]. Subsequently this channel has been studied extensively in several other laboratories (see [165,166]). Experiments using reconstituted proteoliposomes reported the characterisation of these channels with respect to the specificity for nucleotide inhibition and the activity of a range of pharmacological channel openers and blockers. In many cases the properties determined are similar to those of the plasma membrane KATP channel lending support to the possibility that it is actually these channels present in contaminating plasma membranes that are being characterised, rather than true mitochondrial channels (see Section 8.2). However, there are a number of differences in the reported properties of mitochondrial and sarcolemmal KATP channels that argue against this. For example, mitochondrial KATP channels have been reported to require the presence of Mg2+ for ATP to inhibit them, which is not true of the plasma membrane KATP channel. In addition, ADP acts like ATP to inhibit the mitochondrial channel but opens the sarcolemmal channel, whilst GTP has been reported to open the mitochondrial KATP channel but not the plasma membrane one [176,177]. Furthermore, their measured conductances were reported to be distinct [165] and different dose response curves for a range of pharmacological openers and blockers have been described [165,168,190]. This is especially true of the KATP channel opener diazoxide that has been reported to show a sensitivity towards the mitochondrial KATP channel at least three orders of magnitude greater than for the sarcolemmal KATP channel whilst 5-hydroxydecanoate (5HD) blocks the mitochondrial channel whilst having little effect on the sarcolemmal one [167,168]. However, there are other data claiming that diazoxide will open and 5HD block plasma membrane KATP channels at the concentrations frequently used to modulate mitochondrial KATP channels when investigating their possible role in cardioprotection [191\u2013194].\nAlthough no molecular identity has been ascribed to the mitochondrial KATP channel at the genomic level, similarities with the components of the plasma membrane KATP channels have been proposed. These contain one of two channel components, Kir6.1 (48\u00a0kDa) or Kir6.2 (44\u00a0kDa) and the sulphohylurea receptors, SUR1 (177\u00a0kDa) or SUR 2 (174\u00a0kDa) [12]. Two studies reported that antibodies against Kir6.1 and Kir6.2 detected proteins of the right size (about 45\u00a0kDa) in heart mitochondria, but no rigorous steps were taken to remove sarcolemmal contamination [166,195]. In heart mitochondria SUR2 antibodies detected a protein of about 25\u00a0kDa in one study [195] but no protein in the other study [166], whilst a protein of about 28\u00a0kDa was labelled by [125I]-glibencamide [196]. Overall, the available data provide no strong evidence for the presence of a KATP channel in mitochondria that is closely related to the well-characterised plasma membrane KATP channel. Garlid's laboratory found proteins of 55\u00a0kDa and 63\u00a0kDa in a partially purified preparation from brain mitochondrial inner membranes that showed KATP channel activity when reconstituted into proteoliposomes. The 63-kDa protein showed labelling by a fluorescent glibencamide derivative [197] leading them to surmise that the 55-kDa protein represented the channel forming component, but no sequence data were presented to identify either component. Furthermore, because only Coomassie blue staining of proteins was used, it was not established that there were not other minor components present in the partially purified preparation used and the high activity of channel proteins would only require a small contamination to produce significant channel activity. Indeed, it should be noted that mitochondrial preparations used in this study were unpurified on density gradient and therefore unavoidably would have contained significant contamination by fragments of plasma membrane and thus plasmalemmal KATP channels. This might explain why Garlid and co-workers found 6 to 7 times larger amounts of KATP channel in their preparations of isolated brain mitochondria than reported in liver or heart mitochondria.\nIn view of the problems associated with the reconstitution and patch-clamping studies discussed above, and the absence of a molecular identity for the mitochondrial KATP channel, the best evidence for the activity of mitochondrial KATP channels should come from the measurement of changes in matrix volume of isolated mitochondria in response to channel openers and blockers. In this context, Garlid's laboratory has performed extensive experiments using light scattering to detect matrix volume changes in response to adenine nucleotides and a variety of KATP channel openers and blockers. However, as noted above (Section 8.2), we believe that without direct measurements of matrix volume these data must be treated with caution. Thus, although data from both this laboratory and that of Brustovetsky confirmed effects of adenine nucleotides on light scattering that are consistent with the presence of a KATP channel, neither laboratory was able to detect volume changes isotopically despite demonstrating with valinomycin that the technique was quite sensitive enough to do so should they occur [170,171]. Furthermore, neither we nor Brustovetsky et al. were able to detect any significant changes in light scattering with a variety of KATP channel openers (diazoxide, nicorandil, cromakalim, pinacidil, minoxidil) or blockers (5HD and glibencamide) [170,171]. Our own data have led us to conclude that if mitochondrial KATP channels exist, there is no strong evidence for them being active in isolated mitochondria. However, this does not preclude such channels being active within the intact cell, since we might be losing a factor during mitochondrial isolation that is required for their activity. We will consider if there is any evidence for this in the context of studies investigating the role of mitochondrial KATP channels in preconditioning (Section 9.1).\n8.4\nEvidence for a mitochondrial calcium-activated K+ channel\nOur own laboratory was the first to suggest that calcium might regulate potassium entry into mitochondria and hence matrix volume (see [182,198]). We demonstrated that K+ entry into liver mitochondria was stimulated by hormones that activated gluconeogenesis, such as glucagon, adrenaline and vasopressin, and that this led to an increase in matrix volume that was important for the stimulation of mitochondrial respiration and ATP production [182,198]. The mechanism was shown to involve a rise in matrix calcium that inhibits pyrophosphatase leading to a rise in matrix pyroposphate (PPi). Indeed, elevating matrix [PPi] independently of Ca2+, for example by provision of butyrate, also caused modest mitochondrial swelling [186]. Since PPi was known to bind to the ANT [199\u2013201] and adenine nucleotide depletion had been reported to increase mitochondrial K+ uptake [202,203], we proposed that the ANT could act as a potassium channel when ATP was displaced by PPi [186]. In support of this we confirmed earlier data [188] that the permeability of the inner membrane to K+ was increased in the presence of atractyloside which inhibits the ANT by trapping it in the \u201cc\u201d conformation [186]. This mechanism has recently received support from studies on brain mitochondria where it was shown that carboxyatractyloside could enhance potassium loss from de-energised mitochondria [171].\nOur own data were unable to demonstrate calcium mediated increase in either matrix [PPi] or volume in isolated rat heart mitochondria [180,204]. However, data from O'Rourke's laboratory [174] have suggested the presence of a calcium-activated potassium channel (KCa) in these mitochondria. These workers employed patch-clamped mitoplasts to characterise mitochondrial KCa channels that, like the sarcolemmal KCa channels were blocked with charybdotoxin. Furthermore, antibodies against sarcolemmal KCa channels detected immunoreactive proteins in the mitoplasts. One problem with these data is that, as noted above, it is well established that isolated mitochondria are contaminated with plasma membrane and thus it is possible that the immunological and patch clamping data were actually detecting the sarcolemmal KCa.\n9\nIs there good evidence for mitochondrial K+ channels being involved in the protective mechanism of preconditioning?\n9.1\nThe use of pharmacological agents is hampered by lack of specificity\nThere are numerous studies reporting protection of hearts from ischaemia reperfusion injury by drugs that are claimed to be specific openers of the mitochondrial putative mitochondrial KATP channel, most notably diazoxide, and the prevention of this effect by supposed specific blockers of the channel, in particular 5HD (see [11,12,161\u2013163]). However, when haemodynamic performance of the heart is used to assess recovery after ischaemia\/reperfusion, the effect of 5HD to reverse the protective effects of diazoxide is not universally observed [205,206]. O'Rourke et al. have suggested a role for the putative mitochondrial KCa channels in preconditioning, in addition to mitochondrial KATP channels. This proposal was based on the observation that the KCa agonist NS-1619 decreased the infarct size upon reperfusion after ischaemia, and this was blocked by paxilline, a KCa blocker [13,174]. However, as noted above (Section 8.3), a major problem associated with such studies is the assumption that the pharmacological agents used are only exerting their effects on the mitochondrial KATP or KCa channels. In the absence of independent verification of this, the conclusions drawn must be treated with caution. This is especially so in the light of increasing evidence that these agents have other non-specific effects on mitochondria including inhibition of the respiratory chain and uncoupling as is discussed below.\nDiazoxide has been shown by many laboratories including our own to inhibit succinate dehydrogenase at concentrations usually used (50\u00a0\u03bcM) to open the mitochondrial KATP channel [169,206\u2013209]. Because inhibiting succinate dehydrogenase will also block the citric acid cycle in an intact cell, this may well perturb cellular energy metabolism leading to mild depolarisation of the mitochondria and flavoprotein oxidation. It has also been reported that both diazoxide and pinacidil can uncouple mitochondria although the concentrations (>\u00a050\u00a0\u03bcM) required to produce a significant depolarisation are at the high end of the spectrum used in in vivo studies [210,211]. Interestingly these effects appear to require an interaction with the ANT [210] and modelling studies based around the published structure of the ANT have shown potential drug binding sites on this membrane protein [212].\nThe specificity of mitochondrial KATP blockers is also doubtful, especially for the most commonly used 5\u00a0HD which is a substituted fatty acid and has the potential to be metabolised. Indeed, our own work from and that of others has confirmed that 5\u00a0HD can be activated to its CoA derivative and then further metabolised through the \u03b2-oxidation pathway [206,209,213]. As such it can act as a poor respiratory substrate. In addition, the slow kinetics of 3,5-dihydroxydecanoyl-CoA oxidation by l-3-hydroxyacyl-CoA dehydrogenase, the penultimate step of the pathway, can produce a bottleneck that can inhibit normal fatty acid oxidation [206,214]. Interestingly, the other commonly used mitochondrial KATP channel blocker, glibencamide, also has the potential to decrease fatty acid oxidation by inhibition of palmitoyl carnitine transferase [215\u2013217].\nOverall, the specificity of the mitochondrial KATP channel openers and blockers is poor and the ability of several of the KATP channel openers used to act as uncouplers or inhibitors of respiration is especially worrying. This is because it is well established that bona fide respiratory chain inhibitors [31,218\u2013221], succinate dehydrogenase inhibitors [222] and uncouplers [223,224] are all cardio-protective when applied before or during ischaemia even though they have no effects on mitochondrial KATP channels. Thus, in order to establish a role for mitochondrial potassium channels in ischaemic preconditioning more direct evidence for their opening during IP must be provided.\n9.2\nDirect measurements of mitochondrial KATP channel activation during preconditioning\nTwo approaches can be taken to investigate whether mitochondrial K+ channel opening is occurring during preconditioning. The first is the determination of matrix volume in situ and the second is measurement of other effects that an increase in electrogenic potassium flux into the matrix might have on mitochondrial function within the cell.\n9.3\nMeasuring matrix volume in situ\nAlthough measurement of mitochondrial volume in situ in the perfused heart is not possible we have determined matrix volume in mitochondria isolated very rapidly from the perfused heart [206]. Our data showed a significant increase in matrix volume induced by both IP (35%) and by 50\u00a0\u03bcM diazoxide (50%) which is consistent with the opening of a mitochondrial KATP channel. However, 100\u00a0\u03bcM 5HD, the putative mitochondrial KATP channel blocker, also induced an increase in matrix volume (50%) and failed to prevent the increase induced by IP. Indeed, we found no correlation between matrix volume (pre-ischaemic, end ischaemic or during reperfusion) and the haemodynamic recovery of the heart [206]. Sollott et al. [97] have used a sophisticated Fourier analysis of transmitted optics linescan imaging with a 633-nm laser in isolated cardiac myocytes to estimate changes in mitochondrial diameter from which they calculate changes in matrix volume. They report a 2.5\u20134% increase in matrix volume with 30\u00a0\u03bcM diazoxide and 50\u00a0\u03bcM pinacidil consistent with opening of mitochondrial KATP channels. However, their approach makes the assumption that changes in diameter necessarily mean a change in matrix volume, but as noted above (Section 8.2), this is not necessarily the case. Furthermore, Sollott et al. report that a wide variety of other agents known to protect hearts from reperfusion injury through quite distinct mechanisms, including cyclosporin A, the sarcolemmal sodium proton antiport inhibitor Hoe694 and ligands of G protein\u2013coupled receptors such as bradykinin and Tyr-D-Ala-Gly-Phe-D-Leu also cause similar increases in matrix volume [97]. They propose that such an increase in matrix volume is critical for providing a memory that allows protection even after removal of the primary stimulus. Their explanation as to how this is achieved is that swelling increases the threshold at which ROS induce the permeability transition. However, why this should be the case is unclear since measurements on isolated mitochondria have demonstrated that MPTP opening is enhanced at higher matrix volume [225]. Overall, the published data on measurements of mitochondrial volume in situ provide no strong evidence either for or against the opening of mitochondrial K+ channels.\n9.4\nMeasuring flavoprotein oxidation\nAnother approach used to investigate the activity of mitochondrial K+ channels in cardiac myocytes is based on the reasoning that if a K+ channel is opened the entry of K+ would cause depolarisation of the mitochondrial membrane potential. Marban and colleagues reported that with diazoxide this can be detected indirectly in rabbit cardiac myocytes by an oxidation of mitochondrial flavoproteins that is reversed by 5HD [226]. Similar effects have been reported in guinea pig myocytes using NS1619 to open the putative mitochondrial KCa channel with reversal by paxilline [227]. However, other workers have failed to reproduce the effects of diazoxide and 5HD in guinea pig and rat cardiac myocytes leading to the suggestion that the effects observed may reflect substrate deprivation [209,228]. In addition, there are theoretical reasons to question whether opening of the mitochondrial KATP channel would cause a significant depolarisation. Indeed, Garlid has argued convincingly that the fluxes of K+ predicted to occur if the channel opens would allow minimal perturbation of the membrane potential even when giving a significant increase in matrix volume [169,229]. In fact, such an increase in matrix volume might even increase membrane potential as a result of stimulating the respiratory chain itself [182]. Direct measurements of membrane potential with the fluorescent dyes TMRE or JC-1 failed to detect either an increase or decrease [228,230]. There are, however, other mechanisms by which changes in mitochondrial membrane potential and thus flavoprotein oxidation could be induced by putative mitochondrial K+ channel openers and blockers that reflect other non-specific sites of their action. For example, in substrate deprived myocytes where diazoxide has been shown to cause flavoprotein oxidation, this could be caused by the drug inhibiting succinate dehydrogenase and thus the citric acid cycle whilst the ability of 5\u00a0HD to overcome this effect might be the result of the drug acting as a respiratory substrate as noted above (Section 9.1 and [12]).\n10\nProposed mechanisms by which opening of mitochondrial K+ channels might protect hearts from ischaemia reperfusion injury?\nFor mitochondrial K+ channels to play a role in preconditioning there would have to be a mechanism by which their opening might be linked to improved recovery. Several possible mechanisms have been proposed.\n10.1\nEnhanced mitochondrial ATP production\nIt is known that an increase in matrix volume can stimulate respiration and oxidative phosphorylation [182,198] and so might improve ATP production during reperfusion leading to enhanced haemodynamic recovery [161,231]. Although we were able to measure an increase in mitochondrial volume and rates of ADP-stimulated respiration in mitochondria isolated from hearts immediately following ischaemic preconditioning or diazoxide treatment, the effects were largely lost during reperfusion when they are predicted to be most important [206]. Furthermore, 5HD-treatment was shown to exhibit a similar increase in matrix volume but was not cardioprotective [206]. Garlid and colleagues have proposed an alternative, but not unrelated mechanism in which it is the maintenance of the intermembrane space and the close association between the outer an inner membrane that is the critical factor resulting from KATP channel opening [11]. They argue that during ischaemia the loss of membrane potential would decrease electrogenic K+ entry into mitochondria and that this would cause contraction of the matrix. It is proposed that that this would disrupt the interaction between VDAC in the outer membrane, octomeric creatine kinase (CrK) in the intermembrane space and the ANT in the inner membrane leading to faster permeation of ATP through VDAC into the intermembrane space. Further translocation of this ATP into the matrix through the ANT would lead to its hydrolysis, that would be detrimental to the heart. It is suggested that opening of the mitochondrial KATP channel would prevent this matrix condensation and so decrease ATP breakdown during ischaemia leading to less damage. There is some evidence that ATP decline during ischaemia is slowed by ischaemic preconditioning or diazoxide treatment, although the mechanism originally proposed was through inhibition of the F1Fo ATPase by enhanced binding of the ATPase inhibitor protein [232,233]. Furthermore, other data report the opposite effect of preconditioning, with the decline in ATP and the development of contracture being faster than in control hearts [232\u2013235]. Garlid and colleagues further propose that during reperfusion, keeping the mitochondrial KATP channel open maintains the VDAC, CrK, ANT complex which is vital to export ATP rapidly from the mitochondria to the cytosol where it is used to drive contraction. Although this may be true, our own data showed that the slight increases in matrix volume of diazoxide-treated and ischaemic preconditioned hearts compared to control hearts was not significant at reperfusion [206]. The more probable determinant of the efficiency of myocardial ATP production during reperfusion is the extent to which the mitochondria are damaged.\n10.2\nMild uncoupling leads to less calcium overload and ROS production\nIt has been proposed that opening of K+ channels would depolarise the mitochondria sufficiently during reperfusion to reduce ROS production and calcium accumulation and hence prevent MPTP opening [12,151,152,162,163,227,236]. However, as noted above, the amount of depolarisation predicted from K+ channel opening is unlikely to cause much if any depolarisation [169,229] and this is confirmed by direct measurements of mitochondrial membrane potential that failed to detect any depolarisation [228,230]. Furthermore, should significant depolarisation occur at reperfusion, ATP synthesis would be compromised leading to poorer rather than better recovery of the heart. Yet NMR spectroscopy measurements have shown that the bioenergetic state of the preconditioned heart improves during reperfusion, consistent with the better haemodynamic function [232,237,238]. Nevertheless, it is not possible to rule out the possibility that opening of mitoKATP channels might cause a minor depolarisation during reperfusion, or slightly reduce the repolarisation, sufficient to reduce ROS formation without having a significant effect on ATP production.\nAlthough significant uncoupling during the reperfusion phase seems unlikely to be protective, it is known that adding low doses of uncoupler prior to ischaemia can precondition hearts [223,224]. Indeed, any interference with oxidative phosphorylation during the preischaemic phase seems able to exert a similar protective effect whether it is brought about by a brief ischaemic episode as in IP, by addition of a respiratory chain inhibitor [218\u2013221] or a succinate dehydrogenase inhibitor [222]. Since many of the putative mitochondrial K+ channel openers have also been shown to have direct uncoupling effects [209\u2013211,239] or to inhibit components of the respiratory chain including succinate dehydrogenase [169,206\u2013209,240], this provides a common mode of action for these agents. How this might be translated into a protective effect at reperfusion is not clear, although a signalling pathway involving AMP-activated protein kinase provides one possibility as discussed above (Section 6.4). Another potential mechanism would be through increased levels of ROS production causing PKC activation and respiratory chain inhibitors such as antimycin are known to increase ROS production which is critical in mediating its protective effects [218,241]. It has also been reported that low doses of uncoupler can increase ROS production in isolated myocytes [224], although how this might occur is unclear since in isolated mitochondria it is well established that even very modest uncoupling greatly decreases ROS production [242]. In the case of antimycin, PKC\u03b5 translocation has been demonstrated and its protective effects shown to be abolished in PKC\u03b5 knockout mice supporting a role for ROS and PKC\u03b5 activation in its protective mechanism [241].\n10.3\nProduction of ROS as a signalling mechanism\nSome groups have shown that diazoxide and other putative mitochondrial potassium channel openers cause an increase in ROS production by isolated heart mitochondria, cardiac myocytes and perfused hearts and that this can be blocked by 5HD [240,243\u2013248], but this has not been reproduced in other studies [249,250] or in our own unpublished experiments. Since the protective effects of diazoxide and nicorandil can be overcome by free radical scavengers such as N-(2-mercaptopropionyl) glycine [12,100,240,245] it has been concluded by many workers that opening of the KATP channel causes ROS production and that this signals preconditioning through the pathways discussed above. Attractive though this hypothesis may be, it is difficult to formulate a mechanism by which this might occur. As noted above, even if there were modest uncoupling, this would be predicted to decrease ROS production rather than increase it. Another possible mechanism would be via an increase in matrix volume causing a stimulation of electron flow from complex 1 into complex 3 [182,251]. Indeed, there are reports that low doses of valinomycin can increase ROS formation in isolated mitochondria [248] and myocytes [11,163,247], whilst pretreatment of the perfused rabbit heart with valinomycin substantially decreased the infarct size on reperfusion [252]. However, in our own unpublished experiments (H. Buckman and A.P. Halestrap, unpublished data) we have used Amplex Red to measured ROS production by isolated heart mitochondria incubated with a variety of respiratory substrates in State 4 and were unable to detect an increase in ROS production with any mitochondrial potassium channel opener tested, or when matrix volume was increased by decreasing the osmolarity of the incubation medium. Indeed, in the latter case we actually detected a decrease ROS production. This is consistent with the major locus of ROS production being at a highly reduced site on complex 1 that is oxidised by an increase in matrix volume as electron flow out of complex 1 and into complex 3 is stimulated [182,251].\nAnother consequence of opening a mitochondrial K+ channel would be an increase in the mitochondrial pH gradient coincident with a decrease in membrane potential [253]. Whereas the latter would be predicted to decrease ROS formation as outlined above, there is evidence to suggest than an increase pH gradient or matrix pH can increase ROS formation from complex 1 [254]. However, in view of the ability of many of the mitochondrial KATP channel openers to inhibit components of the respiratory chain [169,206\u2013209,240], its is perhaps more likely that their ability to increase ROS is through a direct interaction with a redox centre in one of the respiratory complexes.\n11\nOther proposed mechanism for inhibiting the permeability transition pore in preconditioning\n11.1\nThe role of connexin 43\nRecently, it has been proposed that connexion 43 (Cx43) may play a critical role in linking preconditioning to the mitochondria [255\u2013258]. Cx43 is the major protein of the gap junction with hexameric assemblies forming connexons on adjacent cardiomyocytes. Its traditional role is to provide a route for intercellular communication through the propagation of action potential, signalling molecules and metabolites [258\u2013260]. The permeability of the connexons can be regulated through many mechanisms including changes in [Ca2+], pH and phosphorylation by several protein kinases [258,259], including PKC\u03b5 and PKG, both of which have been implicated in IP as noted above (see Section 6). Cx43 is normally partially phosphorylated with low conductance, but progressive dephosphorylation occurs during ischemia causing increased conductance. It has been proposed that this is important in propagating injury from one cell to another, although hearts from Cx43-deficient mice showed no difference in their sensitivity to reperfusion injury [261]. Studies in both pig and rat hearts demonstrated that the dephosphorylation of Cx43 and electrical uncoupling during ischaemia were prevented by IP in a PKC-dependent manner and that IP increased the co-localization of several protein kinases with Cx43 during ischemia [262,263]. Furthermore, hearts from Cx43-deficient mice showed no protection from reperfusion injury by either IP [261] or diazoxide [247] and in the latter case the ability of diazoxide to increase ROS was also abolished.\nThese data imply a link between Cx43 and preconditioning and thus that Cx43 can in some way influence the opening of the MPTP, perhaps by a signalling mechanism involving ROS. Recent work has suggested that this may be through a translocation of Cx43 to the mitochondria following preconditioning [255\u2013257]. A small fraction of Cx43, primarily in its phosphorylated form, was detected in purified mitochondria from rat, mouse, pig and human hearts and this was increased following preconditioning. In these studies, considerable efforts were taken to show that this Cx43 was not just the result of sarcolemmal contamination of the mitochondrial fraction and further evidence for this was provided by immunofluorescence confocal microscopy and immuno-gold electron microscopy. Furthermore, sub-fractionation of mitochondrial compartments led the authors to conclude that Cx43 is translocated to the inner mitochondrial membrane, although some Cx43 may remain associated with the outer membrane [257].\nAlthough we have been able to detect Cx43 in purified mitochondria, and in preliminary experiments show that this was increased following preconditioning, we have expressed reservations about the likelihood of Cx43 translocation to the inner mitochondrial membrane [256]. Furthermore, following more extensive experiments in which we determined the extent of sarcolemmal contamination of mitochondria by measuring the presence of MCT1, we were unable to confirm any consistent increase in the Cx43 associating with the mitochondrial fraction following IP (Fig. 4). Thus the relevance of Cx43 translocation to the mitochondria remains uncertain, although the loss of preconditioning in Cx43-deficient hearts from mice argues strongly for some pool of Cx43 playing a critical role [247,261]. Since myocytes from the Cx43-deficient mice did not show any diazoxide-induced ROS formation [247], this is likely to involve a ROS-mediated signalling pathway, although the mechanism linking Cx43 to ROS production is unclear. From their data, Garcia-Dorado and colleagues propose that recruitment of Cx43 to the mitochondria in IP might be required for opening of the mitochondrial KATP channel leading to ROS production that triggers preconditioning [255,257]. However, when they prevented this recruitment with geldanamycin, a blocker of heat shock protein 90-dependent protein translocation across the outer mitochondrial membrane, the cardioprotective effects of IP were not lost [257]. Furthermore the cardioprotective effects of both diazoxide and isoprenaline were not accompanied by translocation of Cx43 to the mitochondria [257]. Thus the balance of evidence suggests that Cx43 translocation to mitochondria is not essential for preconditioning [256].\n11.2\nTransient MPTP opening\nHausenloy and colleagues have reported that when the MPTP inhibitors CsA or SfA are present during IP or preconditioning induced by uncoupler and diazoxide, cardioprotection is abolished [121]. They conclude that transient opening of the MPTP may play a critical role in triggering preconditioning and that this might be caused by the well-documented rise in ROS that occurs during preconditioning (see Section 6.1). However, we have challenged these conclusions both on theoretical and experimental grounds [264]. First, our own data using the mitochondrial 2-deoxyglucose entrapment technique failed to detect any increase in MPTP opening immediately following the preconditioning stimulus [68,75]. Second, although the authors suggest that transient MPTP opening during the preconditioning phase prevents mitochondrial calcium overload and that this mediates protection, it is the calcium overload at the end of ischaemia and during reperfusion that mediates the MPTP opening responsible for reperfusion injury. Third, the authors argue that uncoupler also preconditions hearts by opening the MPTP, but uncouplers will depolarize mitochondria independently of MPTP opening. Thus any protection mediated by uncoupler that involves a decrease in mitochondrial calcium loading should be insensitive to both SfA and CsA, but this is not what the authors observed [121]. Hence it seems probable that CsA and SfA are overcoming IP independently of any effects on MPTP opening. This might involve roles for cyclophilins in the preconditioning signalling pathway similar to those responsible for the immunosuppressive actions of these drugs [264].\n12\nConclusions and future directions\nThe ability of preconditioning (and post-conditioning) to inhibit the opening of the MPTP during reperfusion and so provide cardioprotection seems to be well established. What is less clear is the mechanism(s) by which this is achieved. We end this review by presenting our own views on how preconditioning is most likely to be mediated. These are summarised schematically in Fig. 5.\nThe observation that mitochondria isolated immediately after the preconditioning protocol show no protection from MPTP opening [85,100] argues against a signalling pathway that causes phosphorylation of some regulatory component of the MPTP as an early event. Nor have we been able to observe any consistent migration of protein kinases to the mitochondria or changes in phosphorylation of a mitochondrial protein (see Sections 6 and 7.1). By contrast, mitochondria isolated at the end of ischaemia or during reperfusion do show a decreased sensitivity to calcium-mediated MPTP opening [99\u2013101]. Here too we were unable to detect consistent migration of protein kinases to the mitochondria or changes in phosphorylation of a mitochondrial protein, arguing against this protection being mediated by phosphorylation (see Section 7.1). However, the changes in sensitivity of the MPTP do correlate with the extent of protein carbonylation, a surrogate indicator of the oxidative stress the mitochondria have experienced [100,101]. Since it is well established that oxidative stress sensitises the MPTP to calcium, this would seem to provide an adequate explanation as to how preconditioning inhibits MPTP opening at reperfusion (see Section 7.2). Furthermore, this is entirely consistent with the decrease in ROS production late in ischaemia and during reperfusion that has been observed directly in preconditioned hearts [27,265].\nIf this explanation is correct then the focus of research should shift towards investigating the mechanisms underlying the decreased levels of ROS seen in preconditioned hearts at the end of ischaemia and during reperfusion. This will involve consideration of both the sources of ROS, which may include mitochondria, NAD(P)H oxidase or xanthine oxidase, and the pathways that remove them including superoxide dismutase, glutathione peroxidase and catalase [26,28]. The many signalling pathways identified as potential mediators of preconditioning, including ROS, PKC\u03b5 and PKG (see Section 6) may converge on one or more of these steps to decrease ROS levels and hence reduce activation of the MPTP by thiol oxidation. The pharmacological agents used to implicate these pathways may interact specifically with their proposed target, but they may also have less specific effects such as on mitochondrial respiratory chain components (see Section 9.1) to produce a modest increase in ROS levels that stimulate signalling pathways rather than cause damage. Indeed, it does seem that any stress to the myocyte's energy status, be it through respiratory chain inhibition, uncoupling or ischaemia, causes preconditioning. This may also implicate the energy sensing protein kinase, AMP-activated protein kinase (AMPK) [133,266], in the signalling pathway of preconditioning, and there are data supporting such a role for AMPK in both the heart [101,135] and the liver [267].\nIn addition to the protection seen early in reperfusion that could be explained through the mechanisms described above, there is good evidence for ongoing protection against MPTP opening being important as reperfusion progresses. Thus inhibition of MPTP opening with CsA or SfA provides effective cardioprotection, as measured by decreased infarct size, even if the drug is not present at the start of reperfusion, provided it is added within the first 15\u00a0min [90,91]. Furthermore, there is a considerable body of evidence to suggest that activation of survival kinase pathways during reperfusion is critical for protection during this phase of the insult, and that these may converge on Akt and GSK3 to mediate their effects [97,107,126]. It is possible to bring these observations together by invoking the established phenomenon of MPTP-induced ROS production [268,269]. Once MPTP opening has occurred during the initial phase of reperfusion, the resulting ROS production would lead to a progressive increase in pore opening in other mitochondria and thus an escalating number of necrotic cells as reflected in the ongoing LDH release and increased infarct size. CsA or SfA will prevent this ongoing opening, but so too might the survival kinase cascades by reducing the ROS production or increasing their removal as described below.\nIt is well established that MPTP opening causes mitochondrial swelling and rupture of the outer mitochondrial membrane [44]. This leads to release of pro-apoptotic factors including cytochrome c, that activates caspase 9 and hence caspase 3 [45]. In addition, it has been shown that the pro-apoptotic factor Bax can translocate to the mitochondria during ischaemia [72] and this, in conjunction with cleaved Bid (tBid), might cause the cytochrome c release observed during prolonged ischaemia [70] despite there being no evidence of MPTP opening [68,98]. The resulting loss of cytochrome c will slow electron transfer out of complex 3 and thus potentially cause increased ROS production in either complex 3 or in complex 1. An additional mechanism that may lead to increased ROS production during ischaemia and reperfusion is through caspase 3 mediated cleavage of the p75 component of complex 1 [270,271]. Protection from the rise in ROS that accompanies such cytochrome c release could be mediated by survival kinase cascades in two ways. They might stimulate ROS removal as described above for the ischaemic phase or they could reduce the Bax-induced cytochrome c release. Indeed, it is well established that survival kinases can block apoptosis by inhibiting cytochrome c release [272]. This is brought about by Akt-mediated phosphorylation of the pro-apoptotic Bcl-2 family member Bad [273,274] and, via GSK3 phosphorylation and stabilisation of the anti-apoptotic Bcl-2 family member Mcl-1 [275].\nNote added in proof\nSince submission of this article it has been reported that the properties of the mitochondrial permeability transition pore in mitochondria devoid of all VDAC isoforms are the same as in mitochondria from wild-type mitochondria (C.P. Baines, R.A. Kaiser, T. Sheiko, W.J. Craigen, J.D. Molkentin, Voltage-dependent anion channels are dispensable for mitochondrial-dependent cell death, Nat Cell Biol. 9 (2007) 550\u2013555). This confirms that VDAC is not an essential component of the MPTP.","keyphrases":["ros","ischaemia","reperfusion","mitochondrial permeability transition pore","calcium","pkc","katp channel","5hd, 5-hydroxydecanoate","ampk, amp activated protein kinase","ant, adenine nucleotide translocase","apd, action potential duration","bcdh, branched chain 2-oxoacid dehydrogenase","crk, creatine kinase","csa, cyclosporin a","cyp, cyclophilin","cx43, connexin43","gsk3, glycogen synthase kinase 3","ip, ischaemic preconditioning","katp, atp-dependent potassium channels","mitokatp, mitochondrial atp-dependent potassium channels","mct1, monocarboxylate transporter 1","mptp, mitochondrial permeability transition pore","pdh, pyruvate dehydrogenase","pdk1, phosphoinositide-dependent kinase 1","pi-3-kinase, phosphatidyl inositol 3 phosphate kinase","pkc, protein kinase c","pkg, cyclic gmp-dependent protein kinase","ppi, pyrophosphate","ppiase, peptidyl-prolyl cis-trans isomerase","pten, phosphatase and tensin homolog","ros, reactive oxygen species","sfa, sanglifehrin a","sur, sulphohylurea receptor","vdac, voltage activated anion channel"],"prmu":["P","P","P","P","P","P","P","R","R","R","R","R","R","R","M","M","R","R","R","R","R","R","R","R","M","R","M","M","M","R","R","R","R","R"]}
{"id":"Psychopharmacologia-2-2-1705539","title":"A comparison of nicotine dose estimates in smokers between filter analysis, salivary cotinine, and urinary excretion of nicotine metabolites\n","text":"Rationale Nicotine uptake during smoking was estimated by either analyzing the metabolites of nicotine in various body fluids or by analyzing filters from smoked cigarettes. However, no comparison of the filter analysis method with body fluid analysis methods has been published.\nIntroduction\nA number of studies were published over the last 30\u00a0years that attempted to determine the amount of tar and\/or nicotine that smokers receive from their cigarettes (reviewed in Stephen et al. 1989; Pritchard and Robinson 1996; Scherer 1999). The methodology used falls into three broad categories: (1) the analysis of biomarkers in human body fluids or expired breath; (2) the measurement of smoking behavior (puff volume, duration, and frequency) followed by a smoking machine set to duplicate human puffing conditions; and (3) the analysis of spent cigarette filters and the calculation of smoke yields from the filter efficiency.\nTypically, biomarker measurements in blood\/plasma and smoking behavior measurements require that sampling or measurements be made in a laboratory environment. There is a possibility that smoking behavior becomes atypical in this type of environment (Comer and Creighton 1978; Ossip-Klein et al. 1983). Urine, saliva, and spent cigarette filters can be collected in a smoker\u2019s everyday environment. The analysis of 24-h urine samples for nicotine and major metabolites can provide quantitative data regarding uptake of smoke constituents as the product is used in a smoker\u2019s everyday environment (see Byrd et al. 1998 and references therein for examples). Subject compliance can be an issue when trying to determine actual cigarette yields because in an unmonitored environment the subject must be relied upon to smoke only a given brand, not use any other nicotine-containing products, provide an exact accounting of every cigarette smoked during the collection period, and collect all urinary output.\nThe filter analysis method is one of the least invasive of the methods mentioned above. The smokers can use their product in their normal environment and the only deviation from normal behavior is to save the filters. Compliance is not an issue when trying to determine the subject\u2019s cigarette yield because the filters from the actual cigarettes smoked are analyzed. In most cases, the returned filter can be compared to those of the subject\u2019s stated brand to assure brand compliance. The primary issue with this method has been that smoking behavior can produce changes in the filtration efficiency of the filter. The filtration efficiency can vary according to the velocity of the smoke passing through the filter and, to some extent, the length of the tobacco rod smoked (Overton 1973; Dwyer and Abel 1986; Norman et al. 1984). The method used for this study was developed to minimize the effects of smoking behavior on filter efficiency by measuring only the portion of the filter downstream of the ventilation holes (i.e., the mouth end) (St.Charles 2001; Shepperd et al. 2006). This results in relatively constant filtration efficiency over a wide range of smoking behavior.\nThe objective of this study was to compare the nicotine yield of human-smoked cigarettes (mouth exposure) as measured by the filter analysis method and human smoke uptake as measured by biomonitoring under strictly controlled conditions. The biomonitoring measurements included salivary cotinine and 24-h urinary nicotine, cotinine, 3\u2032-hydroxycotinine (3-HC), and their respective glucuronide conjugates. With a good correlation between the methods, future studies on smoker exposure can use the simpler filter analysis method rather than resorting to human biomonitoring techniques.\nMaterials and methods\nStudy design\nThe clinical portion of this study was conducted by an independent contract research organization in 2003 (Covance Clinical Research Unit, Madison, WI, USA). The analysis of salivary cotinine and urinary nicotine metabolites was performed at Covance Laboratories (Harrogate, North Yorkshire, UK). Filter analysis was performed by the study sponsor (Research and Development, Brown & Williamson Tobacco Company, Macon, GA, USA). The study was approved by Covance\u2019s Institutional Review Board and performed in accordance with applicable federal regulations. Subjects who participated in the study gave their informed consent, were told of the purpose of the study, and could withdraw at any time.\nSubject selection\nHabitual smokers were recruited by Covance. Enrollment criteria included males or nonpregnant, nonlactating females, between 21 and 65\u00a0years of age, within \u221220% to +30% of their ideal body weight, who smoked at least 15 cigarettes a day of the same cigarette brand during the previous year. Subjects were excluded if they were under 21\u00a0years of age, were pregnant or lactating, participated in any other clinical study within 30\u00a0days before study entry, had a history or showed signs of a significant medical or psychiatric condition, used prescription medications within 14\u00a0days before study entry, had a history of alcoholism or drug addiction within a year of study entry, or used alcohol or any nonprescription preparations within 72\u00a0h of study entry. A few subjects deviated from the enrollment criteria: underweight (1), overweight (5), medication or alcohol usage before study entry (4), low cigarette consumption (2), shortened brand loyalty duration (4), elevated clinical chemistry (2), abdominal\/hernia surgery (7), and positive drug screen before study entry (2). Because these deviations were considered minor and not expected to interfere with the study objectives, the subjects were allowed to participate in the study.\nSubjects were assigned into one of four tar yield groups, which span the range of Federal Trade Commission (FTC) tar yields found in commercially available USA filtered cigarettes: 1\u20133\u00a0mg (ULL or ultralights\/low), 5\u20136\u00a0mg (ULH or ultralights\/high), 9\u201312\u00a0mg (LTS or lights), and 13\u201319\u00a0mg (FF or full flavor). The purpose was to cover a wide range of human nicotine exposure to allow robust correlations between the methodologies. The goal was to enroll 20 smokers per group; however, even with additional recruitment attempts, only 15 smokers enrolled in the ULL group (market share <2%). One subject in the FF group withdrew from the study due to illness. Table\u00a01 summarizes the subjects\u2019 demographics and their respective brand characteristics by tar band. \nTable\u00a01Subjects\u2019 demographics and cigarette brand characteristics by tar bandSubjectsULLULHLTSFFTotalNumber1520201974Sex (M\/F)8\/712\/810\/1014\/544\/30Height (cm)173 (159\u2013204)174 (157\u2013194)174 (161\u2013187)177 (165\u2013190)175 (157\u2013204)Weight (kg)77 (57\u2013104)75 (42\u201398)78 (56\u201398)77 (60\u2013100)77 (42\u2013104)Age35 (21\u201354)32 (21\u201347)34 (21\u201364)32 (21\u201353)33 (21\u201364)Menthol smokers335718Brands\u00a0Menthol\/nonmenthol2\/53\/64\/84\/613\/25\u00a0Tar (mg\/cig)1.4 (0.7)5.4 (0.5)10.1 (0.9)15.0 (1.7)8.3 (5.1)\u00a0Nicotine (mg\/cig)0.15 (0.05)0.46 (0.04)0.77 (0.06)1.07 (0.14)0.64 (0.34)\u00a0CO (mg\/cig)1.9 (0.9)6.8 (0.6)11.6 (1.4)13.9 (2.0)8.9 (4.6)\u00a0Puffs\/cig7.6 (0.7)8.0 (1.1)7.9 (1.0)8.2 (0.9)7.9 (1.0)Height, weight, and age are shown as the mean (range). Cigarette yields are shown as the mean (SD) for the FTC smoking method.\nSubjects were confined to the clinic for six calendar days to give five consecutive 24-h periods. Nine confinement periods were staggered and limited to ten subjects or less (generally of the same tar range group). During confinement period number 8, one LTS smoker and two FF smokers were allowed to participate with six ULL smokers to complete the LTS and FF cells. Subjects were fed a standardized bulk diet that excluded grilled, smoked, or barbecued food items. Consumption of water and other nonalcoholic beverages was unrestricted. Subjects refrained from strenuous exercise. During confinement, subjects smoked their usual cigarette brand ad libitum in a dedicated smoking room equipped with ventilation and air filtration. Use of any form of nicotine other than the subject\u2019s declared own brand was prohibited. Cigarettes were purchased locally.\nUrine and saliva collection and analysis\nTwenty-four-hour urine samples were collected from each subject for five consecutive days. Collections started at approximately 0800 hours (first void excluded) and ended at approximately 0800 hours the following day (first void included). Urine was collected in 3-l amber plastic containers and kept refrigerated throughout the collection period. No chemical preservatives were used. After each 24-h sample collection, volume and pH measurements were recorded, a sample was taken for creatinine analysis, and 2\u2009\u00d7\u20095-ml and 4\u2009\u00d7\u2009500-ml aliquots were taken and stored frozen at \u221270\u00b0C until shipped. Aliquots were shipped under dry ice to the analytical laboratory and stored at \u221270\u00b0C until analysis.\nUrinary nicotine, nicotine-N-\u03b2-glucuronide, cotinine, cotinine-N-\u03b2-glucuronide, trans-3\u2032-hydroxycotinine, and trans-3\u2032-hydroxycotinine-O-\u03b2-glucuronide were analyzed using solid phase extraction (SPE) and liquid chromatography with tandem mass spectrometric detection using a method developed and validated at the analytical laboratory (Analytical Procedure Covance no. 2002-010). Nicotine-d3, cotinine-d3 (Sigma-Aldrich, Poole, UK), and trans-3\u2032-hydroxycotinine-d3 (Toronto Research Chemicals, North York, ON, Canada) were used as internal standards. Nicotine, cotinine (Sigma-Aldrich), trans-3\u2032-hydroxycotinine, and the three corresponding glucuronides (Toronto Research Chemicals) were used as reference standards. Samples were incubated for 18\u201322\u00a0h at 37\u00b0C with \u03b2-glucuronidase, which enzymatically deconjugated the aglycones from their respective glucuronides. The free and cleaved aglycones and their respective deuterated internal standards were then extracted and analyzed.\nSaliva samples were collected in sterile Salivette tubes (Sarstedt, Newton, NC, USA) from each subject for five consecutive days at approximately 1830 hours. On day 4, two additional saliva samples were collected at approximately 0830 and 1330 hours. Clinical staff supervised and timed the process to assure compliance. Collected saliva samples were immediately stored at \u221220\u00b0C, shipped under dry ice to the analytical laboratory, and stored at \u221220\u00b0C until analysis. Salivary cotinine was analyzed by a method developed and validated at the analytical laboratory (Analytical Procedure Covance no. HB-02-061) based on a previously reported method (Bentley et al. 1999).\nFilter collection and analysis\nTo control cigarette brand accessibility and document cigarette consumption, cigarettes were issued by clinical staff individually. The used filter had to be returned before a subject received their next cigarette. Used filters were processed under the supervision of clinical staff by having the subject remove any tobacco particles from the used filter before depositing it in an individually labeled glass container. Used filters were collected for five consecutive days with each day starting\/ending at the same time as urine samples. The filters generated daily by each subject were shipped by overnight carrier at ambient temperature to the analytical laboratory. Immediately upon receipt, a 10.0-mm portion of the mouth end (tip) was cut from the filter using a specially designed jig, which kept the cut-length constant and kept a single edge razor blade perpendicular to the filter. Tip-to-tip length variation using the jig was within 3%. The tips were stored in labeled 30-ml glass jars with Teflon-lined lids at \u221220\u00b0C until analyzed.\nNicotine yields from the human-smoked cigarettes were estimated by analyzing the tips for nicotine. Five separate machine smoking regimes were used to provide calibration curves for the filter tips of each brand style tested. Cigarettes from the same batches that the subjects smoked were used for calibration smoking. The smoking regimes were chosen to give a wide range of cigarette yields of approximately equal spacing. Table\u00a02 shows the calibration puffing conditions for each tar band. These machine puffing regimes proved to cover the spread of human smoking results for all but 7% of the subject-days tested (5 ULH, 1 LTS, and 6 FF out of 370 total). Six of the 12 involved extrapolating by less than 0.1\u00a0mg of nicotine\/cigarette and the maximum extrapolation was from 2.3 to 2.6\u00a0mg\/cigarette. \nTable\u00a02Calibration smoking conditions by tar bandULLULHLTSFF35\/60\/T\u2009+\u20093\/O35\/60\/T\u2009+\u20093\/O35\/60\/T\u2009+\u20093\/O35\/60\/T\u2009+\u20093\/O35\/60\/T\u2009+\u20093\/B35\/60\/T\u2009+\u20093\/B35\/60\/T\u2009+\u20093\/B35\/60\/3 P\/O70\/60\/T\u2009+\u20098\/O35\/60\/4 P\/O35\/60\/3 P\/O35\/60\/6 P\/O70\/60\/T\u2009+\u20098\/B70\/60\/T\u2009+\u20098\/B70\/60\/T\u2009+\u20093\/O70\/60\/T\u2009+\u20093\/O70\/40\/T\u2009+\u20093\/B70\/40\/T\u2009+\u20093\/B70\/40\/T\u2009+\u20093\/B70\/40\/T\u2009+\u20093\/OData are presented as puff volume (ml)\/puff interval (s)\/length smoked\/ventilation holesO Open, B blockedT + # indicates overtipping plus distance (mm); # P indicates number of puffs.\nCalibration equations were calculated using a linear regression of nicotine yield as a function of tip nicotine. These equations were then used to estimate human-smoked cigarette yield from the measured tip nicotine. This method was validated in a separate study using duplicated human puffing profiles (Shepperd et al. 2006). Aging tests by the study sponsor have shown that tip nicotine values were constant when whole filters were stored in glass jars at ambient temperature for up to 31\u00a0days. For this study, tips were cut from the whole filters within 5\u00a0days.\nOne to nine tips per sample were extracted using either 20 or 40\u00a0ml of methanol containing 0.038\u00a0mg\/ml decanol internal standard. Tips were extracted for 40\u00a0min using a flatbed orbital shaker at 200\u00a0rpm. All available tips were divided into at least three separate batches, which were extracted and analyzed on different days to average analytical variation. For samples from days 2, 4, and 5, extract absorbance at 310\u00a0nm was also measured to audit the nicotine analysis using an independent method. Absorbance gives a measure of the tar deposited on the filter (Sloan and Curran 1981; Shepperd et al. 2006) and we have found that absorbance per tip correlates linearly with nicotine per tip. For days 1 and 3, replicate vials of the extract were stored at \u221220\u00b0C and subsequently analyzed for nicotine to audit the nicotine analysis further. The root mean square difference between the nicotine replicates was 5.5\u00a0\u03bcg\/ml (7.3%). A backup set of replicate vials was kept for use when outlying data points were identified by either of the auditing methods. Samples were retested when they were outside of the 95% confidence interval of the absorbance per tip vs nicotine per tip correlation (days 2, 4, and 5) or when nicotine replicates differed by more than 20% (days 1 and 3).\nThe extract was analyzed for nicotine by gas chromatography using an Agilent 5890 Series II with a flame ionization detector and a J&W Scientific 30\u00a0m Megabore\u00ae 0.53\u00a0mm ID, DB-Wax (1.0\u00a0\u03bcm film) fused silica capillary column (Agilent, Palo Alto, CA, USA). The UV absorbance of the extract was measured using an Ocean Optics (Dunedin, FL, USA) PC2000 spectrometer equipped with a fiber optic dip probe with a 2-mm path length. This gave absorbance values within the linear range of less than 2 without further dilution. Absorbance was measured on the same day the tips were extracted because it was found that extract absorbance declines with even overnight storage.\nResults\nMeasured urinary concentrations for each metabolite were multiplied by their respective daily urine volumes and converted based on molecular weights to yield recovery results in nicotine equivalents. The sum of the nicotine equivalents for nicotine, cotinine, and 3-HC were calculated for each subject per day to give total daily urinary nicotine equivalents (UNE) in milligrams per day. Nicotine yield per cigarette was calculated from the mean nicotine per tip values for each subject-day and the appropriate calibration equation for the brand smoked. This was multiplied by the number of cigarettes smoked by the subject that day to give nicotine yield in milligrams per day.\nThis study design allowed us to achieve a wide range of nicotine exposure suitable for the correlation of the three methods of nicotine uptake estimation. Nicotine yield from the human-smoked cigarettes (mouth exposure) as measured by filter analysis ranged from 3.7 to 67.1\u00a0mg\/day, UNE ranged from 3.1 to 48.4\u00a0mg\/day, and saliva cotinine ranged from 70 to 866\u00a0ng\/ml. The mean (SD) proportions of urinary metabolites, including the glucuronides, were 22% (9%) for nicotine, 35% (6%) for cotinine, and 43% (13%) for 3-HC of the total UNE measured. When expressed as a percentage of nicotine entering the mouth, the proportions were 19% (9%) for nicotine, 31% (11%) for cotinine, 39% (17%) for 3-HC, and 89% (25%) for total nicotine equivalents.\nStatistical correlations\nObserved values from the three methodologies were correlated with each other. Figure\u00a01 shows the three plots and correlations for all data points. Figure\u00a01a is a graph of the UNE vs daily nicotine yield estimated from filter analysis; Fig.\u00a01b shows UNE vs saliva cotinine; and Fig.\u00a01c shows daily nicotine yield from filter analysis vs saliva cotinine. Linear regressions were significant for both the slope and intercept (p\u2009<\u20090.001) for all three correlations. The best correlation was obtained with UNE vs daily nicotine yield (Fig.\u00a01a, R2\u2009=\u20090.66). The slope indicates that 67% of the variation in nicotine mouth exposure calculated from filter analysis appeared as variation in the six urinary compounds measured. The standard error of the regression (SER) was 4.9\u00a0mg nicotine\/day. Saliva cotinine did not correlate as well with either UNE (Fig.\u00a01b, R2\u2009=\u20090.49, SER\u2009=\u20095.9\u00a0mg\/day) or nicotine yield (Fig.\u00a01c, R2\u2009=\u20090.45, SER\u2009=\u20097.4\u00a0mg\/day). Because the amount of nicotine entering the mouth was greater than the sum of the UNE, the standard error of the nicotine yield would be greater than that of the UNE due to scaling even if the correlations were equivalent. To allow an equal comparison, the standard error of the percent of the difference between calculated (using the regression equation) and measured values was calculated for the three correlations. The standard error calculated in this manner was 32 and 42% for UNE vs nicotine yield and saliva cotinine, respectively, and 41% for nicotine yield vs saliva cotinine. Summary statistics for all correlations are in Table\u00a03.\nFig.\u00a01Correlations of the three methodologies (individual measurements)Table\u00a03Summary of the statistics for linear correlationsCorrelation (y : x)\u00a0Intercept (95% CI)Slope (95% CI)R2Standard error (%)Urine : filterIndividual4.3 (1.2)0.67 (0.05)0.664.9 (32)mg\/day : mg\/dayAverage2.7 (2.0)0.74 (0.08)0.833.2 (21)Urine : salivaIndividual5.8 (1.6)0.042 (0.004)0.495.9 (42)mg\/day : ng\/mlAverage5.4 (3.2)0.043 (0.009)0.555.2 (36)Filter : salivaIndividual6.9 (2.0)0.048 (0.006)0.457.4 (41)mg\/day : ng\/mlAverage5.6 (4.0)0.052 (0.011)0.546.5 (33)Filter : urine (creatinine normalized)Individual8.7 (1.9)10.8 (1.3)0.427.6 (41)mg\/day : mg\/day\/mmol creatinineAverage7.1 (3.9)12.1 (2.7)0.526.7 (32)Urine : FTCmg\/cig : mg\/cigAverage0.49 (0.13)0.66 (0.19)0.410.27 (42)Filter : FTCmg\/cig : mg\/cigAverage0.52 (0.17)0.85 (0.24)0.410.35 (38)Saliva : FTCng\/ml : mg\/cigAverage211 (58)184 (81)0.22119 (46)Urine Urinary nicotine equivalents, Filter daily nicotine yield estimated from filter analysis, Saliva saliva cotinine concentration, FTC FTC nicotine yield, CI confidence interval\nThe means of the five daily data values per subject are shown in Fig.\u00a02 using the same format as Fig.\u00a01. Again, for all correlations, the slopes and intercepts were significant (p\u2009<\u20090.01). The correlation for UNE vs nicotine yield (Fig.\u00a02a) improved significantly as shown in Table\u00a03. The slope increased to 0.74 while the intercept moved closer to zero. The saliva cotinine correlations (Fig.\u00a02b,c) also improved, but not as dramatically as the urinary nicotine. The correlation for UNE vs saliva cotinine (Fig.\u00a02b) had an R2 value of 0.55 with a standard error of 5.3\u00a0mg\/day (36%). The correlation for nicotine yield vs saliva cotinine (Fig.\u00a02c) had an R2 value of 0.54 with a standard error of 6.5\u00a0mg\/day (33%).\nFig.\u00a02Correlations of the three methodologies (5-day average per subject)\nOther findings\nAlthough the primary purpose of this study was to determine the statistical correlations between the three methods of estimating nicotine uptake, data collected during the experiment allowed for additional observations.\nCreatinine normalization Normalization by urinary creatinine is useful when only a single or partial daily urine collection is taken. To test the effect of creatinine normalization, observed daily UNE were divided by their respective millimole creatinine. A correlation was calculated with nicotine yield as the dependent variable and UNE\/mmol creatinine as the independent variable. The results are summarized in Table\u00a03. Both the intercept and slope are significant (p\u2009<\u20090.001) but normalization with creatinine clearly degrades the correlation compared to the correlation without normalization. The findings suggest that normalization with creatinine adds another factor of variability, which has a degrading effect on R2, and therefore indicates that the use of 24-h urine samples without normalization is expected to provide more accurate results than the analysis of partial (<24\u00a0h) urine samples with normalization. This is in agreement with Heavner et al. (2006), who showed mechanistically that creatinine normalization may be appropriate for some, but not all, of the urinary metabolites of nicotine and that other methods of normalization may be more appropriate for spot urine samples.\nFTC smoke yields Because this was a confined clinical study, subjects would not necessarily be expected to behave as if they were in their normal environment. However, because a wide range of FTC cigarette yields were tested, it was considered worthwhile to test the correlation between the measured FTC yields and the three methods of nicotine estimation. Correlation results are summarized in Table\u00a03 for the 5-day mean values for the three methods vs the FTC values on a per subject basis. All correlations gave significant slopes and intercepts (p\u2009<\u20090.001).\nDifferences between tar band groupings The results for mouth exposure to nicotine (from filter analysis), UNE, saliva cotinine, and cigarettes smoked per day were grouped by tar band and the mean, standard deviation, and one-way analysis of variance (ANOVA) were calculated on a per subject basis (Altman and Bland 1997). The results are shown in Table\u00a04. Group means increased in rank order with increasing group yield except for saliva cotinine and cigarettes smoked per day. All measurements except cigarettes smoked per day gave a significant (p\u2009<\u20090.05) effect of group by ANOVA. For each measurement, results with different letter assignments are significantly different at the 95% confidence level (Fisher test). Normalizing urinary nicotine metabolites with creatinine resulted in an increase in p value for effect of group and no significant differences between the FF, LTS, and ULH groups. All three groups were significantly higher than the ULL group. This again shows that creatinine normalization degrades the discriminating power. \nTable\u00a04Mean\u2009\u00b1\u2009SD for measurements grouped by tar band.\u00a0ULLULHLTSFFANOVA pNo. of subjects15202019\u2013Mouth exposure (mg nicotine\/cigarette)0.63\u2009\u00b1\u20090.18a0.94\u2009\u00b1\u20090.29b1.19\u2009\u00b1\u20090.33c1.37\u2009\u00b1\u20090.58c<0.0005Mouth exposure (mg nicotine\/day)14.7\u2009\u00b1\u20095.6a22.0\u2009\u00b1\u20097.1b24.4\u2009\u00b1\u20096.4bc28.3\u2009\u00b1\u200912.6c<0.0005Urinary nicotine (mg\/cigarette)0.59\u2009\u00b1\u20090.19a0.84\u2009\u00b1\u20090.27b0.96\u2009\u00b1\u20090.21b1.20\u2009\u00b1\u20090.41c<0.0005Urinary nicotine (mg\/day)13.6\u2009\u00b1\u20095.7a 19.3\u2009\u00b1\u20096.8b19.8\u2009\u00b1\u20095.9b24.5\u2009\u00b1\u20098.9c<0.0005Creatinine normalized urinary nicotine (mg\/day\/mmol)0.93\u2009\u00b1\u20090.58a1.31\u2009\u00b1\u20090.51ab1.43\u2009\u00b1\u20090.58b1.45\u2009\u00b1\u20090.52b0.040Saliva cotinine (ng\/ml)216\u2009\u00b1\u2009107a347\u2009\u00b1\u2009119bc314\u2009\u00b1\u200988b411\u2009\u00b1\u2009149bc<0.0005Cigarettes\/day22.8\u2009\u00b1\u20094.7a23.4\u2009\u00b1\u20093.6a21.2\u2009\u00b1\u20094.5a20.8\u2009\u00b1\u20094.2a0.190Cigarettes\/day (self-reported minus measured)1.6\u2009\u00b1\u20095.1\u22121.0\u2009\u00b1\u20098.0\u22120.9\u2009\u00b1\u20095.4\u22120.1\u2009\u00b1\u20095.9Results with different letters are significantly different at the 95% confidence level\nSelf-reported vs actual cigarette usage Table\u00a04 also shows the difference in self-reported and measured cigarette usage. Self-reported cigarette usage was questioned and compliance with respect to truthfulness can be a concern (Byrd et al. 1998). In this study, subjects were asked to estimate their cigarette usage as part of the recruiting process. This gave a basis to compare with the measured usage during the study (overall mean\u2009=\u200922\u00a0cigarettes\/day, SD\u2009=\u20094.2, range 15.8 to 32.0). The mean value for the (5-day average\u2212 self-reported) usage per subject was \u22120.2\u00a0cigarettes\/day (SD\u2009=\u20095.9, range \u221219.4 to +11.7). Even when broken down to the smaller tar band groups of 15 to 20 subjects, the mean difference in measured and reported usage was less than 2\u00a0cigarettes\/day.\nWithin-subject variation Because the trial took place over five consecutive days, within-subject, day-to-day variation was calculated for each of the measured variables. In addition, on day 4, saliva samples were taken at approximately 0830, 1330, and 1830 hours for measurement of saliva cotinine to estimate within-day variation. Results are summarized in Table\u00a05 and are expressed as a pooled (root-mean-squared) coefficient of variation. The pooled day-to-day variations for each variable were similar, ranging from 15.2% for cigarettes smoked per day to 18.1% for UNE per day. The within-day variation of 8.1% for saliva cotinine was approximately half the day-to-day variation of 15.7%. When a single factor ANOVA was calculated using saliva cotinine for the three time periods sampled on day 4, the variation between time periods was insignificant (p\u2009=\u20090.58). However, this could have been overwhelmed by the subject-to-subject variation in nicotine uptake. This factor was removed by dividing the individual measurement by the daily average per subject for all three time periods. A single factor ANOVA was significant for time of day (p\u2009<\u20090.001) with the mean (95% confidence interval) saliva cotinine values being 1.008 (0.015), 0.96 (0.012), and 1.03 (0.013) times the average daily value per subject for the 0830, 1330, and 1830 hour samples, respectively. Even though the time of day had a statistically significant effect, the practical differences were small. \nTable\u00a05Within-subject variation expressed as a pooled coefficient of variation (CV)AnalyteTypePooled CV (range)%Filter (nicotine yield\/day)Day-to-day16.7 (6\u201336)Urine (nicotine equiv.\/day)Day-to-day18.1 (4\u201354)Saliva cotinine concentrationDay-to-day15.7 (4\u201347)Saliva cotinine concentrationWithin-day8.1 (0.8\u201316)Cigarettes\/dayDay-to-day15.2 (3\u201336)\nDiscussion\nAll three of the estimation methods correlated significantly with each other, but the best overall correlation was between the filter analysis method and UNE. The slopes for the correlations of UNE as a function of mouth exposure to nicotine were 0.67 and 0.74 for the individual and 5-day average regressions, respectively, implying that about 70% of the difference in the UNE measured in this study was due to a difference in mouth exposure to nicotine. However, the mean of the total UNE expressed as a percentage of the nicotine entering the mouth was 89%. The mean value falls within the reported range of 80% (Benowitz et al. 1994) to 90% (Curvall et al. 1991) but the slopes fall below this range. The difference between the two methods is due to the significant intercept calculated using the linear regression. The intercept using the 5-day average results was lower than the intercept using the individual results and the slope was greater. The intercept could represent compartmental nicotine and\/or metabolite storage with subsequent carryover from storage before entering the study. This influence should diminish with a 5-day average compared to using single day results. An example of this is demonstrated in Fig.\u00a01a where there is a single circled data point that appears to be an outlier. Urinary output of nicotine equivalents was approximately two times the mouth exposure of nicotine for that day. However, this was a day 1 measurement for a single subject. For subsequent days, the data points for this subject are buried within all the other data points. One possibility is that the subject had a much larger exposure to nicotine before participating in the study and the unusually high urinary output was due to clearance of the prior exposure. Averaging the results over 5\u00a0days would take out much of the metabolic influence and result in a much better correlation as demonstrated with these results.\nFor the saliva cotinine correlations, the R2 values only improved slightly with averaging. Part of the reason for the improvement with all correlations can be attributed to averaging out measurement variation. This should be similar for all correlations. However, the additional improvement in the filter vs urine correlation can be explained by metabolism. Nicotine has a relatively short serum elimination half-life of 2.2\u20132.9\u00a0h (Scherer et al. 1988; Benowitz and Jacob 1993; Benowitz et al. 1999, 2002, 2004), which means that the urinary nicotine should be from the nicotine taken in that day. Cotinine has a serum elimination half-life typically reported at 16\u201318\u00a0h (Scherer et al. 1988; Benowitz et al. 1999, 2002, 2004; De Schepper et al. 1987) and a similar urine elimination half-life (Benowitz and Jacob 1993; De Schepper et al. 1987). 3-HC, which is downstream metabolically from cotinine, has a serum elimination half-life of 5.9\u20136.6\u00a0h with a similar urine elimination half-life (Scherer et al. 1988; Benowitz and Jacob 2001). Given the pharmacokinetic information, these urinary metabolites must have originated from nicotine exposure on multiple days as has been demonstrated directly using nicotine infusion (Scherer et al. 1988).\nBecause measurements were taken over sequential days of input (filter analysis), the temporal source of urinary and salivary metabolites could be estimated by multiple regression of the metabolites vs the daily nicotine exposure. This analysis was performed using the nicotine exposure for the current day and the two previous days according to the equation: \nwhere (N), (N-1), and (N-2) equals the nicotine exposure for the current day, 1\u00a0day before, and 2\u00a0days before the urine or saliva sample, respectively. Metabolite measurements from days 3, 4, and 5 were used. None of the coefficients for nicotine exposure from 2\u00a0days before the urine collection were significant (p\u2009>\u20090.15). For saliva cotinine, the coefficient for 2\u00a0days before was not significant at the 95% confidence but the p value of 0.07 was small enough to warrant consideration. Given this, the regressions were recalculated using (N) and (N-1) and including the urinary metabolite measurements from day 2 as well. The results of this regression are shown in Table\u00a06. \nTable\u00a06Multiple regression results for source of urinary and salivary metabolites as a function of nicotine exposure from current and previous dayCoefficientSum of urinaryUrinary nicotineUrinary cotinineUrinary 3-HCSalivary cotinineR20.720.400.640.420.50Intercept3.51 (<0.001)0.77 (<0.01)0.85 (<0.01)1.89 (<0.001)97.9 (<0.01)Previous day0.24 (<0.001)\u22120.004 (0.89)0.08 (<0.01)0.16 (<0.001)5.7 (<0.01)Current day0.46 (<0.001)0.16 (<0.001)0.18 (<0.01)0.12 (<0.01)4.2 (<0.01)Results expressed as regression coefficient (p value)\nFor the sum of all urinary metabolites, the coefficients were significant for the current day and prior day\u2019s exposure. This implies that the total urinary metabolites originated from nicotine exposure over 2\u00a0days. The only coefficient that was significant for the urinary nicotine regression was for the current day\u2019s exposure as would be expected. The coefficient of 0.16 implies that 16% of the nicotine entering the mouth appeared as urinary nicotine plus the glucuronide. This agrees closely with the estimates of 13.8% (Benowitz et al. 1994) and 10\u201315% (Curvall et al. 1991). For the urinary cotinine regression, coefficients for the current day and previous day were significant with the current day being about twice that of the previous day. The two coefficients imply that 26% of the nicotine entering the mouth appears as urinary cotinine plus the glucuronide. This also agrees closely with estimates of 25.6% (Benowitz et al. 1994) and 20\u201325% (Curvall et al. 1991). For the 3-HC regression, the coefficients for the current day and previous day were significant with the current day being slightly smaller than the previous day. The sum of the coefficients (0.28) was smaller than the reported 41 to 60% of the total urinary metabolites (Benowitz et al. 1994; Curvall et al. 1991). It is possible that because 3-HC is the third step in the metabolism of nicotine, there was too much of a smoothing effect for the change in daily mouth exposure to fully capture.\nFor saliva cotinine, all coefficients were significant, suggesting that saliva cotinine results are an amalgam of at least 2\u00a0days of nicotine exposure. Saliva cotinine correlations suffer because results are expressed as concentration rather than an absolute value, and as such can be influenced by body size. In addition, other variables come into play that are not easily explained. An example of this is shown in Figs.\u00a01 and 2 by the data points with boxes around them. These were from one subject that appeared to have unusually low saliva cotinine values for both UNE (Figs.\u00a01b and 2b) and mouth exposure of nicotine (Figs.\u00a01c and 2c). This subject had the highest mouth exposure of nicotine of all the subjects studied, yet the saliva cotinine values were only slightly above midrange. There was nothing unusual about this subject and average urinary output was within 1 SD (1,100\u00a0ml) of the average for all subjects (2,400\u00a0ml). It is unlikely that nicotine yields were overestimated because Fig.\u00a01a shows that the data points are scattered about the regression line for UNE vs nicotine yield. It is also unlikely that the saliva cotinine concentrations were in error because the saliva cotinine for each day was analyzed in separate batches along with other samples. The subject did not rapidly metabolize cotinine to 3-HC because cotinine accounted for 47% of the urinary metabolites measured for this subject compared to an average of 35% for all subjects indicating the converse. Therefore, it must be concluded that this is an anomaly characteristic of this individual.\nAs shown in Table\u00a03, with many of the estimation methods, the correlations are only slightly better than simply correlating with FTC nicotine yield of the cigarette. The average nicotine mouth exposure, as measured by the filter method, correlated with the FTC nicotine yield with a standard error of 38% and an R2 value of 0.41. The correlation of nicotine mouth exposure with saliva cotinine gave a standard error of 33% and creatinine-normalized urinary metabolites gave a standard error of 32% with R2 values approximately 0.1 higher.\nCorrelations of FTC nicotine with biomarkers in this study were stronger than that reported in other studies (Byrd et al. 1998; Jarvis et al. 2001; Ueda et al. 2002; Hecht et al. 2005; Bernert et al. 2005). We believe that there are valid reasons for this. One is that the correlations were performed using the 5-day averages per subject instead of a single sample per subject. In addition, exact compliance to brand and cigarette consumption was assured in the current study, whereas all but one (Bernert et al. 2005) of the referenced studies used self-reported brand identification and cigarette consumption. Self-reported brand information was reported to have about a 25% error rate when compared with packs returned (Peach et al. 1986) or in a test\u2013retest comparison (Eisenhower et al. 1993). This and the potential for use of alternate brands during a study can further confound a correlation with biomarkers. Other confounding factors are the use of creatinine normalized spot urine samples instead of 24-h urine samples and analyzing for a subset of the nicotine metabolites used in the current study. Simple creatinine normalization is only biologically valid for xenobiotics that have the same excretion mechanism and urinary flow rate dependence as creatinine. Heavner et al. (2006) have shown that 1-HC (free and glucuronide) and cotinine glucuronide have urinary flow rate dependence similar to creatinine while nicotine (free and glucuronide), free cotinine, 1-hydroxypyrene, and the free and glucuronide forms of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol do not.\nIn conclusion, two methods stand out as superior. One is the filter method, which estimates mouth level exposure directly on a per cigarette basis. Filter collection need not be quantitative, the filter returned can be compared to the brand it is supposed to be and it can be readily determined if it was smoked or not. Thus, brand compliance and smoking status can be assured even if the subject happened to occasionally use a different brand or other form of nicotine during the study. With self-reported daily cigarette use from groups of at least 15\u201320 subjects, the exposure per cigarette can be converted accurately to daily exposure. In addition, mouth exposure to tar can also be estimated using the filter method (Shepperd et al. 2006). The other measurement, which is considered by many to be the \u201cgold standard,\u201d is the measurement of urinary nicotine and metabolites from 24-h urine samples without creatinine normalization. This method appears to reflect the mean daily nicotine uptake of the last 2\u00a0days.","keyphrases":["nicotine","filter","saliva","urine","metabolites","smoking behavior","cigarette smoking"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Purinergic_Signal-3-4-2072911","title":"Lack of evidence for direct phosphorylation of recombinantly expressed P2X2 and P2X3 receptors by protein kinase C\n","text":"P2X3 and P2X2+3 receptors are present on sensory neurons, where they contribute not only to transient nociceptive responses, but also to hypersensitivity underlying pathological pain states elicited by nerve injuries. Increased signalling through P2X3 and P2X2+3 receptors may arise from an increased routing to the plasma membrane and\/or gain of function of pre-existing receptors. An obvious effector mechanism for functional modulation is protein kinase C (PKC)-mediated phosphorylation, since all P2X family members share a conserved consensus sequence for PKC, TXR\/K, within the intracellularly located N-terminal domain. Contradictory reports have been published regarding the exact role of this motif. In the present study, we confirm that site-directed elimination of the potential phosphor-acceptor threonine or the basic residue in the P+2 position of the TXR\/K sequence accelerates desensitization of P2X2 receptors and abolishes P2X3 receptor function. Moreover, the PKC activator phorbol 12-myristate 13-acetate increased P2X3 (but not P2X2) receptor-mediated currents. Biochemically, however, we were unable to demonstrate by various experimental approaches a direct phosphorylation of wild-type P2X2 and P2X3 receptors expressed in both Xenopus laevis oocytes and HEK293 cells. In conclusion, our data support the view that the TXR\/K motif plays an important role in P2X function and that phorbol 12-myristate 13-acetate is capable of modulating some P2X receptor subtypes. The underlying mechanism, however, is unlikely to involve direct PKC-mediated P2X receptor phosphorylation.\nIntroduction\nP2X receptors constitute an abundant class of ligand-gated ion channels, which respond to extracellular ATP and related nucleotides with the opening of an intrinsic pore permeable to Na+, K+ and Ca2+ [1, 2]. A family encompassing seven P2X subunit genes, designated P2X1\u20137, has been identified in rodents and mammals. Six of the seven subunit isoforms (P2X1-P2X5 and P2X7) are able to assemble into homotrimeric receptor channels [3\u20138] with distinct pharmacological and electrophysiological phenotypes [9]. According to their sensitivity to the synthetic ATP analogue \u03b1,\u03b2-methylene adenosine 5\u2032-triphosphate (\u03b1\u03b2-meATP) and the rate of current desensitization, P2X receptors are generally subgrouped into at least two categories: (1) rapidly desensitizing and \u03b1\u03b2-meATP sensitive (P2X1 and P2X3) and (2) slowly or non-desensitizing and \u03b1\u03b2-meATP insensitive (P2X2, P2X4 and P2X7) receptors. The term \u201cnon-desensitizing\u201d means that the currents are maintained for at least a few seconds in the continuous presence of agonist. A peculiar phenotype is inherent to heteromeric P2X2\/P2X3 (P2X2+3) receptors, which feature \u03b1\u03b2-meATP sensitivity combined with a non-desensitizing current response.\nP2X receptors are found on the surface of a large variety of cells, where they are involved in numerous sensory processes including nociception under both physiological and pathological processes [10\u201315]. On nociceptive sensory neurons, extracellular ATP acts as a pain-producing neurotransmitter predominantly through homotrimeric P2X3 receptors or heterotrimeric P2X2+3 receptors. Conditions and mediators that facilitate P2X3 and P2X2+3 receptor-mediated signalling, apparently by potentiating ionic currents, include (1) substance P and bradykinin [16], (2) nerve injury [17] and (3) calcitonin gene-related peptide (CGRP), a potent vasodilator and proinflammatory agent [18]. How this increase in receptor function occurs has not been fully solved. One obvious candidate mechanism is protein kinase C (PKC)-mediated phosphorylation, since all P2X family members share a conserved consensus sequence for PKC-mediated phosphorylation (TXR\/K) within the intracellularly located N-terminal domain (Fig.\u00a01a, b). Indeed, results obtained with pharmacological activators and inhibitors are consistent with PKC being involved in P2X3 receptor potentiation by substance P and bradykinin [16], as well as the calcitonin gene-related peptide [18]. Direct PKC-mediated phosphorylation has so far been demonstrated only for the P2X2 receptor, with 18T of the 18TPK20 sequence as the phospho-acceptor site [19]. Mutational analysis has further shown that the TXR\/K sequence of the P2X1, P2X2 and P2X3 receptors plays an important role in controlling the rate of receptor desensitization [16, 19, 20]. Changes of the rate of desensitization could profoundly influence the efficiency of synaptic transmission and thus contribute to pathological pain states. Overall, there is significant interest in a better understanding of the mechanisms involved in the short-term and long-term regulation of P2X receptors.\nFig.\u00a01Effect of site-directed modification of a putative PKC motif on currents mediated by ATP-activated P2X2 and P2X3 receptors. a Schematic model of the transmembrane topology of the rat P2X3 subunit illustrating the N-terminal position of the 12TTK14 sequence. b Alignment of intracellular N-terminal amino acid sequences of the seven P2X subunit isoforms reveals a highly conserved consensus motif, TXR\/K. c Typical current traces elicited by applying 10-s pulses of 100\u00a0\u03bcM ATP to oocytes expressing the indicated wild-type or mutant P2X2 receptors. d Typical current traces elicited by applying 10-s pulses of 100 \u03bcM ATP to oocytes expressing the indicated wild-type or mutant P2X3 receptors. Gray areas indicate the duration of ATP application. e All the P2X2 and P2X3 receptors and receptor mutants were expressed efficiently at the cell surface. Intact, healthy oocytes expressing the indicated proteins for 2\u00a0days were surface-labelled with the membrane impermeant reactive Cy5 dye and then extracted with dodecylmaltoside. Recombinant proteins were isolated by Ni2+ chelate chromatography and resolved by reducing SDS-PAGE. Shown is a fluorescence scan of an SDS-PAGE gel\nThe aim of this study was to assess biochemically the conditions in which P2X2 and P2X3 receptors become directly phosphorylated by PKC in a heterologous system. While this work was in progress, a similar study was published for the P2X3 receptor that arrived at essentially the same conclusion, namely that the P2X3 receptor is unlikely to be a direct PKC substrate [21]. Our results complement and extend this finding by demonstrating that the P2X2 receptor also does not serve as a substrate for direct PKC-mediated phosphorylation.\nMaterials and methods\nMaterials\nThe sources of antibodies, enzymes or peptide substrates are specified at the appropriate places in the text. Chemicals not otherwise specified were purchased in the highest available quality from Sigma-Aldrich (Taufkirchen, Germany) or Merck (Darmstadt, Germany).\nPeptide-specific antibodies to P2X1, P2X2 and P2X3 subunits\nPeptides ATSSTLGLQENMRTS (residues 385-399 of the P2X1 subunit), QQDSTSTDPKGLAQL (residues 458\u2013472 of the P2X2 subunit) and VEKQSTDSGAYSIGH (residues 383\u2013397 of the P2X3 subunit) were selected based on their lack of similarity with known proteins and predicted hydrophilicity for raising polyclonal antibodies against the C-terminal end of the rat subunits P2X1, P2X2 and P2X3, respectively. Rabbit polyclonal antibodies were ordered through the Custom Antibody Production Services of Eurogentec (Seraing, Belgium). After 3\u00a0months and a total of four immunizations, rabbits were sacrificed by complete bleeding. Peptide-specific antibodies were purified from the serum by affinity columns coupled with the peptides used for immunization. Immunoblotting showed that the antibodies recognized only the receptor against which they were raised, thus demonstrating isoform specificity of these antibodies (results not shown). The antibodies were aliquoted at 0.12\u20130.16\u00a0mg\/ml in phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) and 0.01% thimerosal and stored at \u221280\u00b0C.\ncDNA constructs\nPlasmids encoding wild-type and N-terminal hexahistidine-tagged (His-tagged) versions of rat P2X1 subunits (rP2X1, GenBank accession no. X80477), P2X2 subunits (rP2X2, GenBank accession no. U14414) or rat P2X3 subunits (rP2X3, GenBank accession no. X90651) in the oocyte expression vector pNKS2 [22] were available from previous studies [3, 8]. Insertion and replacement mutations were introduced by QuikChange site-directed mutagenesis (Stratagene, La Jolla, CA, USA). For protein expression in mammalian cells, the receptor cDNAs were subcloned from the oocyte expression vector pNKS2 into pcDNA3.1. The plasmid His-rP2X2-EGFP-pcDNA3.1 with a C-terminal enhanced green fluorescent protein (EGFP) fusion was generated by eliminating the stop codon in His-rP2X2 and adding the coding sequence of EGFP (Clontech, Palo Alto, CA, USA) in frame. All constructs were verified by restriction analysis and nucleotide sequencing. A pEGFP-C1 plasmid harbouring the N-terminally EGFP-tagged splicing factor SF3B1 was kindly provided by our colleague Dr. Walter Becker [23].\nP2X receptor expression in Xenopus laevis oocytes\nCapped cRNAs were synthesized from plasmids linearized downstream to the 100\u00a0bp long polyA tail provided by the oocyte expression vector pNKS2 [22] and injected at 0.5\u00a0\u03bcg\/\u03bcl in 50-nl aliquots into collagenase-defolliculated X. laevis oocytes using a Nanoliter 2000 injector (WPI, Sarasota, FL, USA), as detailed previously [24]. Oocytes were maintained at 19\u00b0C in sterile oocyte Ringer\u2019s solution [ORi: 90\u00a0mM NaCl, 1\u00a0mM KCl, 1\u00a0mM CaCl2, 1\u00a0mM MgCl2 and 10\u00a0mM hydroxyethylpiperazine ethanesulfonic acid (HEPES), pH 7.4] supplemented with 50\u00a0\u03bcg\/ml of gentamicin.\nHeterologous expression in HEK293 cells\ncDNAs encoding P2X subunits in pcDNA3.1 vector (Invitrogen, Karlsruhe, Germany) or the splicing factor SF3B1 in EGFP-C1 vector were transiently transfected into HEK293 cells using Lipofectamine LTX (Invitrogen, Karlsruhe, Germany). Cells were cultured at 37\u00b0C in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) (Invitrogen) supplemented with 10% (v\/v) fetal calf serum (FCS) (PAA Laboratories, Linz, Austria), 100\u00a0U\/ml of penicillin G and 100\u00a0\u03bcg\/ml of streptomycin.\nMetabolic [35S]methionine labelling of oocytes\nFor metabolic [35S]methionine labelling of oocyte-expressed recombinant proteins, cRNA-injected oocytes and non-injected controls were incubated overnight with L-[35S]methionine (>40 TBq\/mmol; PerkinElmer, Boston, MA, USA) at about 100\u00a0MBq\/ml (0.4\u00a0MBq per oocyte) in ORi at 19\u00b0C, and then chased for 24\u00a0h.\nSurface fluorescence labelling of oocytes\nSelective fluorescence labelling of plasma membrane-bound P2X receptors was achieved by using the amino-reactive fluorescent Cy5 dye (GE Healthcare, Freiburg, Germany), which is membrane impermeant due to its two sulfonic acid groups. Two days after cRNA injection, oocytes were washed with oocyte-PBS pH 8.5 (20\u00a0mM Na phosphate, 110\u00a0mM NaCl, 1\u00a0mM MgCl2), and then incubated for 30\u00a0min at ambient temperature with Cy5 dye, which was diluted 200-fold to a final concentration of 50\u00a0\u03bcg\/ml from a dimethyl sulfoxide (DMSO) stock solution. The reaction was terminated by washing the cells with oocyte-PBS, followed by membrane protein extraction with dodecylmaltoside and receptor purification (see below).\nIsolation of recombinant P2X receptors from X. laevis oocytes or HEK293 cells\nHis-tagged receptors were purified by Ni2+ nitrilotriacetic acid (NTA) agarose (Qiagen, Hilden, Germany) affinity chromatography from non-ionic detergent extracts of oocytes or HEK293 cells in the presence of iodoacetamide essentially as described [3, 8, 25]. Slight modifications were that buffers were supplemented with ethylenediaminetetraacetate (EDTA)-free Halt\u2122 protease inhibitor cocktail (Pierce, Rockford, IL, USA), 50\u00a0mM NaF and 50\u00a0mM Na4P2O7 for phosphatase inhibition, and that digitonin was replaced by 0.2% and 0.05% dodecylmaltoside for membrane protein extraction and repetitive washing of resin-bound proteins, respectively. Proteins were released from the washed Ni2+ NTA agarose beads with non-denaturing elution buffer consisting of 250\u00a0mM imidazole\/HCl (pH 7.4) and 0.02% dodecylmaltoside, and then kept at 0\u00b0C until analysed on the day of purification.\nIn addition, immunoprecipitation assays were performed using either cRNA-injected Xenopus oocytes or transiently transfected HEK293 cells. Cells were lysed in ice-cold immunoprecipitation lysis buffer containing 150\u00a0mM NaCl, 25\u00a0mM Tris\/HCl pH 7.4, 20\u00a0mM NaF, 20\u00a0mM Na4P2O7, 10\u00a0\u03bcl\/ml Halt\u2122 protease inhibitor cocktail without EDTA (Pierce, Rockford, IL, USA), 1% sodium dodecyl sulfate (SDS) (w\/v) and 50\u00a0mM iodoacetamide. For efficient solubilization, lysates were incubated for 30\u00a0min on ice with occasional vortexing, and then cleared by centrifugation for 15\u00a0min at 16,100\u00a0g and 4\u00b0C. The cleared SDS extract was diluted 1:1 with immunoprecipitation binding buffer [150\u00a0mM NaCl, 20\u00a0mM Tris\/HCl pH 7.4, 0.4\u00a0mM phenylmethylsulfonyl fluoride (PMSF), 0.5% Triton X-100\u00a0v\/v and 25\u00a0mM iodoacetamide], supplemented 1:1,000 with the desired peptide-specific P2X2 or P2X3 antibodies or a polyclonal goat anti-EGFP antibody (Rockland, Gilbertsville, PA, USA) and incubated for 2\u00a0h on ice. Immunocomplexes were bound to the protein A Sepharose by overnight head-over-head rotation at 8\u00b0C, then washed 5 times with immunoprecipitation binding buffer containing 0.1% Triton X-100 and finally eluted by incubating the beads for 15\u00a0min at 56\u00b0C in reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer.\nIn vitro PKC phosphorylation assay\nIn vitro phosphorylation assays were performed by using P2X receptors as substrates, purified as detailed above. In addition, a selective peptide PKC substrate (RRGRTGRGRRGIYR, Calbiochem-Merck, Darmstadt, Germany, final concentration 150\u00a0\u03bcM) corresponding to the amino acid sequence 1487-1500 of the hepatitis C virus polyprotein [26] was used as a positive control. The proteins or the peptide were incubated for 30\u00a0min at 30\u00b0C with 0.02 unit (0.02\u00a0\u03bcg) of purified catalytic PKC subunit from rat brain and 50\u00a0\u03bcM [\u03b3-32P]ATP (10\u00a0mCi\/ml, Amersham Biosciences GE Healthcare, Freiburg, Germany) in phosphorylation buffer containing 6\u00a0mM CaCl2, 10\u00a0mM MgCl2, 20\u00a0mM Tris\/HCl pH 7.5, 1\u00a0mM EDTA, 1\u00a0mM mercaptoethanol, 0.05% Triton X-100 (v\/v) and 0.1\u00a0mg\/ml BSA. The reaction was terminated by boiling in reducing SDS-PAGE sample buffer for 30\u00a0s. Proteins were resolved by reducing SDS-PAGE and visualized by immunoblotting or phosphorimaging as detailed below.\nIn vivo phosphorylation assay\nTransiently transfected HEK293 cells were metabolically labelled by a 3-h incubation with 500\u00a0\u03bcCi\/ml of [32P]orthophosphate (Amersham Biosciences GE Healthcare, Freiburg, Germany) in phosphate-free oocyte Ringer solution or in phosphate-free DMEM (Invitrogen, Karlsruhe, Germany) as appropriate. Cells were treated with or without 100\u00a0nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, Taufkirchen, Germany) or 100\u00a0nM calyculin A (Tocris Bioscience, Ellisville, MO, USA) for 10\u00a0min at ambient temperature. Non-incorporated radioactivity was removed by rinsing the cells twice with tracer-free medium. Affinity purification or immunoprecipitation was performed as described above followed by SDS-PAGE and immunoblotting or phosphorimaging.\nSDS-PAGE\nSamples for SDS-PAGE were denatured by incubating in reducing SDS-PAGE sample buffer for 15\u00a0min at 56\u00b0C and then electrophoresed on a Laemmli SDS-PAGE gel (10% acrylamide) in parallel with [14C]-labelled molecular mass markers (Rainbow\u2122, Amersham Biosciences GE Healthcare, Freiburg, Germany). Cy5-labelled proteins were visualized by scanning the wet SDS-PAGE gel with a fluorescence scanner (Typhoon, GE Healthcare, Freiburg, Germany). For visualization of radiolabelled or non-labelled proteins, SDS-PAGE gels were either fixed, dried and exposed to a PhosphorImager screen, and scanned using a Storm 820 PhosphorImager (Amersham Biosciences GE Healthcare, Freiburg, Germany) or\/and blotted to polyvinylidene fluoride (PVDF) membranes.\nImmunoblotting\nProteins separated by SDS-PAGE were electroblotted onto PVDF membranes. Membranes were blocked for 1\u00a0h with 5% non-fat milk in PBS (or, alternatively, with 5% BSA if the phosphothreonine monoclonal antibody was used for immunoblotting), washed 3\u2009\u00d7\u20095\u00a0min with PBST (0.1% Tween 20 in PBS) and incubated for 2\u00a0h at 4\u00b0C in PBS containing the appropriate primary antibody. Phosphorylated proteins were detected by immunoblotting with a phosphothreonine monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA). Alternatively, the peptide-specific antibodies to P2X2 and P2X3 subunits were used to verify receptor expression levels. Following washing in PBST, an appropriate horseradish peroxidase-conjugated secondary antibody (goat anti-mouse or goat anti-rabbit, Pierce, Rockford, IL, USA) was applied for 1\u00a0h in PBS supplemented with 1% non-fat milk. After another 3\u2009\u00d7\u20095\u00a0min washes with PBST, immunoreactive bands were visualized by chemiluminescence imaging using a Fujifilm LAS-3000 system (Tokyo, Japan) and the AIDA Image Analyser Software Version 4.08 (Raytest, Straubenhardt, Germany).\nTwo-electrode voltage clamp current recordings and electrical capacitance measurements\nTwo-microelectrode voltage clamp measurements of X. laevis oocytes were performed 2\u00a0days after cRNA injection using a Turbo TEC-05 amplifier (npi electronics, Tamm, Germany) interfaced by an INT-20X AD\/DA converter (npi electronics, Tamm, Germany) to a personal computer running the CellWorks Lite 5.1 software (npi electronics, Tamm, Germany), as detailed previously [27\u201329]. Current traces were elicited by 100\u00a0\u03bcM ATP at 1- to 5-min intervals (Sigma\u2013Aldrich, Taufkirchen, Germany) at a holding potential of \u221260\u00a0mV in nominally Ca2+-free bath medium (designated Mg-ORi) to avoid activation of endogenous Ca2+-activated Cl\u2212 channels. ATP was prepared in Mg\u2212ORi at its final concentration and applied by gravity-fed perfusion at a flow rate of 10\u00a0ml\/min to an oocyte held by the two microelectrodes in the bath chamber (volume \u223c10\u00a0\u03bcl). Switching between different perfusion solutions was achieved with electromagnetic valves controlled by the CellWorks Lite 5.1 software. Electrophysiological recordings were analysed using the Origin 6.0 software (Microcal Software Inc., Northampton, MA, USA). Single exponentials were fitted to the decay phase of the macroscopic currents with a nonlinear least squares method. The time constant \u03c4 was taken as an estimate of the rate of receptor desensitization.\nTo monitor PMA-induced changes in the oocyte surface area, depolarizing voltage steps of \u0394V\u2009=\u200910\u00a0mV were applied from the holding potential to elicit a capacitive transient Icap, the integral of which yields the charge movement Qcap and thus the membrane capacitance Cm\u2009=\u2009Qcap\/\u0394V [30\u201332]. PMA was dissolved in DMSO to a stock solution of 10\u00a0mM and stored at \u221220\u00b0C. On the day of the experiment, the stock solution was pre-diluted to 100\u00a0\u03bcM in DMSO and then further diluted with Mg-ORi to the final concentration of 100\u00a0nM. Vehicle control experiments with a final concentration of 0.1% DMSO did not reveal any effects on P2X2 receptor-mediated currents measured with X. laevis oocytes (results not shown). All measurements were made at room temperature (21\u201323\u00b0C).\nData analysis\nData are shown as means\u00b1SEM. The paired Student\u2019s t-test (two-tailed) was used to compare current amplitudes with statistical significance set to p\u2009<\u20090.05.\nResults\nSite-directed modification of a putative N-terminal PKC site affects the electrophysiological phenotype of P2X2 and P2X3 receptor channels\nFirst, we tested each of the non-mutated and mutant receptor constructs for expression of an ATP-gated inward current in cRNA-injected X. laevis oocytes. Application of 100\u00a0\u03bcM ATP induced typical non-desensitizing currents through oocyte-expressed P2X2 receptors (Fig.\u00a01c, left current trace). The P2X2 subunit shares with other P2X subunit isoforms a conserved consensus site for PKC phosphorylation (TXR\/K, where T is the phosphorylation site and X denotes any amino acid; see Fig.\u00a01a, b). Disruption of the conserved N-terminal PKC site by mutating the phosphor-acceptor 18T to A resulted in a rapidly desensitizing channel (Fig.\u00a01c, middle current trace). Also, replacement of 20K by T resulted in a desensitizing phenotype (Fig.\u00a01c, right current trace). Since PKC preferentially phosphorylates threonine (or serine) residues that are close to a C-terminal basic residue, replacement of 20K in the P+2 position by a non-basic residue such as T will destroy the putative PKC site despite preserving the phosphor-acceptor 18T. Virtually identical findings had previously been reported by Boue-Grabot et al. [19]. The average half-times of desensitization (\u03c4) of the 18A and 20T P2X2 mutants were quite similar, with mean \u03c4 values (\u00b1SEM) of 4.5\u2009\u00b1\u20090.4\u00a0s and 7.3\u2009\u00b1\u20090.5\u00a0s, respectively, of eight oocytes from two females per value.\nATP applied to oocytes expressing the P2X3 receptor elicited a rapidly desensitizing inward current (Fig.\u00a01d, left current trace) as is typical for this P2X receptor subtype. Incorporation of similar mutations, as in the P2X2 subunit in the analogous positions of the P2X3 receptor T12A or K14T, completely abolished P2X3 receptor function (Fig.\u00a01d, middle and right current traces). Visualization of plasma membrane-bound fluorescence-labelled P2X receptors by affinity purification combined with SDS-PAGE and fluorescence scanning verified that all the receptors were efficiently exported to the oocyte surface (Fig.\u00a01e).\nThe PKC activator PMA augments P2X3 receptor-mediated currents, yet leaves P2X2-mediated currents unchanged\nSince the mutagenesis data for the P2X2 and P2X3 receptor were consistent with the possibility of a regulatory role for PKC in P2X receptor function, we examined whether the PKC activator PMA was capable of affecting the electrophysiological phenotype of P2X2 or P2X3 receptors. In planning the experiments, we considered that PMA treatment of X. laevis oocytes is known to induce a pronounced endocytotic reduction of the cell surface area, as evidenced microscopically and electrophysiologically by the disappearance of virtually all microvilli and a concomitant decrease of the electrical capacitance of the plasma membrane, respectively [31, 32]. Associated with oocyte surface reduction is a proportional internalization of membrane proteins, such as endogenous Na+-K+ pumps, suggesting that PMA treatment would stimulate internalization of recombinant P2X receptors as well. Re-examination of the time course of the PMA-induced reduction of the oocyte surface area showed that the electrical capacitance did not start to decline significantly before 10\u00a0min after PMA application (Fig.\u00a02a). Accordingly, all current recordings were done within a time window of 10\u00a0min after PMA application to prevent any contribution of receptor internalization to the PMA effect on P2X receptor-dependent currents.\nFig.\u00a02Effect of PMA on ATP-induced currents mediated by expressed P2X2 and P2X3 receptors in X. laevis oocytes. a The electrical membrane capacitance was monitored as a measure of the oocyte surface area during sustained stimulation with PMA. Each bar represents the mean Cm\u00b1SEM calculated from the areas under the current transients elicited in 10-s intervals by five consecutive 10-mV depolarizing steps. b, c Representative current traces elicited by 10-s lasting pulses of 100\u00a0\u03bcM ATP applied in 10-min intervals to oocytes expressing the indicated P2X receptors or P2X receptor mutants. Where denoted by a black bar, oocytes were pre-incubated with 100\u00a0nM PMA before ATP was co-applied. PMA was without effect on the P2X2 receptor-mediated current (b), but induced a marked increase of the current amplitude mediated by P2X3 receptors (c). Gray areas indicate the duration of ATP application\nFigure\u00a02b and c show typical current traces elicited by ATP from P2X2 or P2X3 receptor-expressing oocytes before and after a 10-min exposure to PMA. PMA neither affected the shape nor the amplitude of P2X2 receptor-mediated current (Fig.\u00a02b). However, PMA produced a marked increase of the current amplitude mediated by the P2X3 receptor (Fig.\u00a02c). In one experiment with four oocytes, a statistically significant increase of the P2X3-mediated current amplitude by PMA of 155\u2009\u00b1\u200942% (SEM) was determined.\nP2X2 and P2X3 receptors exist in a non-phosphorylated state in both X. laevis oocytes and HEK293 cells\nTo explain the accelerated desensitization upon site-directed perturbation of the 18TXK20 motif, it has been suggested that the wild-type P2X2 receptor is constitutively phosphorylated at 18T and that this phosphorylation is responsible for the slow desensitization rate [19]. To address this issue, we next examined biochemically whether P2X3 receptors also exist in a constitutively phosphorylated state; the P2X2 receptor was also analysed to allow for a direct comparison of the results. Both parent P2X receptors and mutants were purified by Ni2+ chelate affinity chromatography from dodecylmaltoside extracts of [35S]methionine-labelled oocytes, resolved by SDS-PAGE, and blotted onto a PVDF membrane. However, probing with a phosphothreonine-specific monoclonal antibody revealed no discernible signal (Fig.\u00a03a) despite the presence of significant amounts of [35S]methionine-labelled P2X2 and P2X3 protein in the SDS-PAGE gel (Fig.\u00a03b).\nFig.\u00a03Immunoblots show no constitutive phosphorylation of oocyte-expressed P2X2 or P2X3 receptors. The indicated wild-type or mutant P2X subunits were purified by Ni2+ chelate chromatography from [35S]methionine-labelled X. laevis oocytes and resolved by denaturing SDS-PAGE. a Shown is a representative immunoblot probed with a phosphothreonine-specific monoclonal antibody and a peroxidase-conjugated secondary antibody. No phosphorylation signal could be detected at the SDS-PAGE migration position of P2X2 and P2X3 subunits, which are indicated by the adjacent PhosphorImager scan in panel b. b Direct PhosphorImager visualization of metabolically [35S]methionine-labelled parent and mutant P2X subunits resolved by SDS-PAGE of the same samples as in panel a. cLeft panel: representative immunoblot of P2X2 subunits tagged with either an N-terminal or C-terminal hexahistidine sequence (designated His-P2X2 or P2X2-His, respectively) and probed with a phosphothreonine-specific monoclonal antibody and a peroxidase-conjugated secondary antibody. No phosphorylation signal could be detected at the SDS-PAGE migration position of P2X2 subunits, which were visualized by immunoblotting with the P2X2 subunit polyclonal antibody (middle panel) or PhosphorImager scanning of incorporated [35S]methionine (right panel)\nTo exclude the possibility that the apparent lack of receptor phosphorylation resulted from an interference between the N-terminal His tag and the PKC site of P2X3 or P2X2 receptor subunits, which are separated by only 12 or 20 residues, respectively, we performed analogous experiments with a C-terminally His-tagged P2X2 receptor, P2X2-His. As apparent from Fig.\u00a03c, neither N-terminally nor C-terminally His-tagged P2X2 receptor became phosphorylated, as evidenced by immunoblotting with a phosphor-specific anti-threonine antibody (Fig.\u00a03c). The presence of P2X2 receptor protein was verified by immunoblotting and metabolic labelling with [35S]methionine (Fig.\u00a03c, middle and right panel).\nBoue-Grabot et al. [19] have demonstrated phosphorylation of P2X2 receptors expressed in HEK293 cells. To address the possibility that P2X receptor phosphorylation is host cell specific, we analysed HEK293 cells transiently transfected with P2X2 or P2X3 expression plasmids. P2X receptor proteins were isolated by immunoprecipitation and resolved by reducing SDS-PAGE. The efficient expression of the P2X3 receptor or the C-terminally EGFP-tagged P2X2 receptor in HEK293 cells was verified by immunoblotting with subunit bands migrating at 54\u00a0kDa or 83\u00a0kDa (27\u00a0kDa contributed to the P2X2 subunit by the EGFP tag), respectively (Fig.\u00a04b). However, phosphorylated P2X2 or P2X3 subunits could not be detected by immunoblotting with a phosphothreonine-specific antibody (Fig.\u00a04a). In contrast, the PKC-phosphorylated splicing factor SF3B1, which was expressed in HEK293 cells as a positive control substrate and immunoprecipitated with an EGFP antibody, was efficiently phosphorylated under identical conditions, assuring that the assay was able to detect phosphorylated proteins (Fig.\u00a04a, lane 1). Taken together, these results suggest that both P2X2 and P2X3 receptors exist constitutively in a non-phosphorylated state in both X. laevis oocytes and HEK293 cells.\nFig.\u00a04Immunoblotting reveals no constitutive phosphorylation of HEK293 cell-expressed P2X2 and P2X3 receptors. The indicated proteins were isolated by immunoprecipitation using the peptide-specific P2X3 subunit polyclonal antibody or an EGFP-specific polyclonal antibody as appropriate and resolved by reducing SDS-PAGE. a Shown is an immunoblot probed by phosphothreonine-specific monoclonal antibody and a peroxidase-conjugated secondary antibody. Note that phosphorylation of the P2X3 receptor or the EGFP-tagged P2X2 receptor is not detectable, whereas a positive control\u2014the EGFP-tagged splicing factor SF3B1 (migration mass 97\u00a0kDa [23])\u2014was detected by the antibody. b The same samples as in panel a were immunoblotted using peptide-specific P2X2 and P2X3 subunit polyclonal antibodies to verify the expression of the transfected genes\nPMA-activated PKC does not drive the phosphorylation of P2X2 or P2X3 receptors\nA majority of protein kinases are quiescent unless their activity is stimulated by specific stimuli and effectors. To drive phosphorylation by active endogenous PKC, we incubated non-denatured recombinant His-tagged P2X1, P2X2 and P2X3 receptors isolated by Ni2+ NTA agarose affinity chromatography from dodecylmaltoside extracts of X. laevis oocytes with PMA-stimulated lysates of X. laevis oocytes (Fig.\u00a05a) or HEK293 cells (Fig.\u00a05b) in the presence of [\u03b3-32P]ATP. As a positive control, a specific peptide PKC substrate RRGRTGRGRRGIYR was incubated with the lysates. This peptide was equally well phosphorylated by addition of either cell lysate or a commercial preparation of the rat brain PKC subunit (Fig.\u00a05a, b). In contrast, however, no 32P incorporation could be detected in the SDS-PAGE migration position of the P2X1, P2X3 or P2X2 receptor subunit at 56\u00a0kDa, 54\u00a0kDa or 66\u00a0kDa, respectively. The presence of purified P2X1, P2X2 and P2X3 receptor subunit protein in this assay was verified by immunoblotting using P2X1-, P2X2- or P2X3-specific antibodies (Fig.\u00a05c). The phosphorylation of the synthetic peptide clearly demonstrates that both X. laevis oocytes and HEK293 cells contain sufficient endogenous PKC to drive at least the phosphorylation of a synthetic peptide substrate.\nFig.\u00a05Lysates of X. laevis oocytes or HEK293 cells contain endogenous PKC, but do not support PMA-driven phosphorylation of P2X2 or P2X3 receptors. The indicated wild-type and mutant P2X receptors were purified by non-denaturing Ni2+ chelate chromatography from X. laevis oocytes, supplemented with [\u03b3-32P]ATP, 100\u00a0nM PMA and oocyte lysate (a) or HEK293 cell lysate (b), and incubated for 30\u00a0min at ambient temperature. In parallel, a peptide PKC substrate (final concentration 0.5\u00a0\u03bcg\/\u03bcl) was incubated with [\u03b3-32P]ATP, PMA and either of the two cell lysates or the purified rat brain PKC catalytic subunit as indicated. The proteins were resolved by reducing SDS-PAGE and visualized by phosphorimaging. Note that the band pattern was similar irrespective of whether protein of mock-injected oocytes or P2X receptor-expressing oocytes was used as a substrate, arguing against specific labelling of either P2X receptor. Arrows indicate migration positions of P2X1, P2X2 and P2X3 subunits. Arrowhead in a, dominant background band that is present in all oocyte samples. The peptide PKC substrate was similarly labelled by lysates or purified rat brain PKC, confirming the presence of endogenous PKC in X. laevis oocytes and HEK293 cells. c The same samples as in panels a and b were resolved on a separate SDS-PAGE gel. The indicated P2X subunits were detected by immunoblotting using the appropriate P2X subunit-specific polyclonal antibodies. A sample of mock-transfected cells was simultaneously probed with the P2X2 and P2X3 subunit-specific polyclonal antibodies\nDiscernible phosphorylation of P2X2 and P2X3 receptors might be obscured by a low level of endogenous PKC. Therefore, an analogous experiment was performed, in which the purified P2X2 and P2X3 receptors were incubated with purified rat brain PKC catalytic subunit rather than cell lysate in the presence of [\u03b3-32P]ATP. Again, the specific peptide PKC substrate was efficiently phosphorylated as demonstrated both by direct phosphorimaging (Fig.\u00a06a) and immunoblotting with the phosphothreonine monoclonal antibody (Fig.\u00a06b). Phosphorylated P2X2 or P2X3 subunits could not be detected by either method (Fig.\u00a06a, b) despite the presence of abundant amounts of the respective P2X subunits in the assays (Fig.\u00a06c).\nFig.\u00a06PKC phosphorylates a specific PKC substrate in vitro, but does not phosphorylate P2X2 or P2X3 receptors. The immunoprecipitated recombinant P2X2 or P2X3 receptor from X. laevis oocytes or a synthetic peptide (1 or 10\u00a0\u03bcg) derived from the non-structural protein 3 of hepatitis C virus, which serves as a specific substrate for PKC, were incubated for 30\u00a0min with purified rat brain PKC catalytic subunit (0.02\u00a0\u03bcg corresponding to 0.02\u00a0U\/\u03bcl) in the presence of 50\u00a0\u03bcM [\u03b3-32P]ATP (10\u00a0mCi\/ml), and then subjected to SDS-PAGE and phosphorimaging. The rat brain PKC catalytic subunit does not require Ca2+ and phosphatidylserine for activity [42]. 32P was incorporated into the PKC substrate (left panel) and was also detected by immunoblotting with an anti-phosphothreonine antibody (middle panel). However, there was no detectable 32P incorporation at a migration position of \u223c66\u00a0kDa or \u223c54\u00a0kDa of the P2X2 or P2X3 subunit, respectively, the presence of which could be verified by immunoblotting (right panel)\nPMA or calyculin A do not enhance phosphorylation of P2X3 receptors in intact HEK cells\nIn a final set of experiments, we attempted to directly demonstrate phosphorylation in vivo by using metabolic [32P]orthophosphate labelling combined with immunoprecipitation analysis as a read out. Mock-transfected and P2X3 receptor-transfected HEK293 cells were incubated with [32P]orthophosphate in phosphate-free DMEM, followed by incubation with the PKC activator PMA and\/or the phosphoserine\/phosphothreonine phosphatase inhibitor calyculin A. The PhosphorImager scan revealed an identical pattern of phosphorylated bands almost irrespective of the experimental conditions, with no evidence for a significant 32P-labelling of the P2X3 subunit (Fig.\u00a07, top panel). P2X3 receptor expression was verified by immunoblotting (Fig.\u00a07, bottom panel).\nFig.\u00a07PMA and calyculin A do not induce phosphorylation of P2X3 receptors in intact HEK cells. Mock- and P2X3 receptor-transfected HEK293 cells were incubated with [32P]orthophosphate (500\u00a0\u03bcCi\/ml) for 3\u00a0h in phosphate-free DMEM, followed by a 10-min incubation with 100\u00a0nM of each, the PKC activator PMA and\/or the phosphatase inhibitor calyculin A. Triton X-100 extracts were then prepared from the cells and subjected to immunoprecipitation using the peptide-specific P2X3 subunit polyclonal antibody, followed by reducing SDS-PAGE and phosphorimaging. The migration position of the P2X3 subunit is marked by an arrow, indicating the absence of 32P incorporation into the P2X3 subunit. P2X3 receptor expression was verified by immunoblotting with the P2X3 subunit polyclonal antibody (bottom panel)\nDiscussion\nBoth P2X3 and P2X2+3 receptors are present on sensory neurons, where they play an important role in nociception. In addition to mediating transient nociceptive responses, these receptors contribute critically to the hyperexcitability that underlies abnormal pain states elicited by nerve injuries. One mechanism for the increased responsiveness to ATP is sensitization of P2X3 and P2X2+3 receptors arising from a significant enhancement in the trafficking of P2X3 subunit-containing receptors and\/or an increase in function of pre-existing receptors [17]. An obvious candidate mechanism for functional modulation is phosphorylation, which is believed to occur universally in all ligand-gated ion channels as reviewed by Levitan [33].\nEvidence for a control of P2X receptor channel function by PKC-mediated phosphorylation\nSite-directed elimination of the putative N-terminal PKC site has already previously been shown to severely affect the electrophysiological phenotype of several P2X receptor subtypes, including P2X1, P2X2 and P2X3 receptors from rat and\/or human [16, 19, 20, 34] and P2X5 receptors from frog [35]. Replacing the putative phospho-acceptor residue 18T or the basic residue in the P+2 position (20K) by A or a non-basic residue, respectively, imposed rapidly desensitizing properties onto the non-desensitizing wt P2X2 receptor without grossly affecting the current amplitude. In contrast, when analogous mutations were introduced into the rapidly desensitizing receptor subtypes P2X1 or P2X3, peak currents declined to low or undetectable levels, respectively [16 ,20, 34]; the same observation was also made with the slower desensitizing frog P2X5 receptor [35]. Overall, the results obtained by site-directed elimination of the N-terminal TXR\/K motif can be plausibly reconciled with the hypothesis that the rate of P2X receptor channel desensitization is controlled by a phosphorylation-dephosphorylation mechanism, with phosphorylation at this site being associated with a decreased rate of desensitization.\nAdditional evidence for a possible involvement of PKC-dependent phosphorylation comes from experiments with the PKC activator PMA, which was consistently found to elicit a severalfold increase in currents mediated by the P2X1 receptor [36] or the P2X3 receptor [16, 18, 21] without changing the rate of desensitization. A PMA-induced change of the current desensitization was noted for C-terminally truncated P2X2 receptors, which are known to exhibit a desensitizing phenotype in contrast to the parent full-length P2X2 receptors [19, 37]. PMA treatment converted the rapidly desensitizing phenotype of truncated P2X2 receptors to slowly desensitizing, but did not affect the kinetic properties or current amplitudes of the full-length P2X2 receptors. The same lack of effect of PMA has been observed with wild-type P2X2 receptors stably expressed in HEK293 [38]. It has been inferred from these data that P2X2 receptors are constitutively phosphorylated and that this phosphorylation is responsible for their non-desensitizing phenotype. Consistent with this view, immunoblot analysis with a phosphothreonine-proline-specific monoclonal antibody efficiently detected wild-type P2X2 receptors with an intact PKC site, but not P2X2 receptor mutants with a disrupted PKC site [19]. Constitutive phosphorylation has been also demonstrated for the P2X1 receptor expressed in HEK293 cells [34]. Further studies demonstrated that exogenously added uridine triphosphate (UTP) potentiated the current mediated by endogenous and expressed P2X3 receptors in response to \u03b1\u03b2-meATP as agonist. Substitution of phospho-acceptor residues of PKC consensus sites located in the ectodomain of the P2X3 receptor abolished this UTP-induced current potentiation, suggesting a role of ecto-protein kinase C in P2X3 receptor regulation [39, 40].\nEvidence against a control of P2X receptor channel function by PKC-mediated phosphorylation\nIn direct contrast to the results of Boue-Grabot et al. [19], we were unable to demonstrate a direct phosphorylation of wild-type P2X2 receptors expressed in X. laevis oocytes and HEK293 cells. The various experimental approaches for phosphorylation detection that we applied included immunoblot analysis of purified P2X2 receptors. In the experiments shown, we used a phosphothreonine-specific monoclonal antibody instead of a phosphothreonine-proline-specific antibody, because the TXK motif of the P2X3 receptor contains a threonine in the -X-position instead of the proline found in the amino acid sequence of the P2X1 and P2X2 subunit (cf. Fig.\u00a01b). However, even when we used the same phosphothreonine-proline-specific antibody as Boue-Grabot et al. [19], we could not demonstrate phosphorylation of P2X2 receptors in our experiments (data not shown).\nRecently, it has been shown that a proline in the P+1 position strongly disfavours substrate recognition by PKC [41], suggesting that our negative results with P2X2 receptors may not result from technical limitations, but reflect the inherent inability of P2X2 receptors to serve as substrates for PKC. Notably, P2X3 receptors, despite harbouring a T in the P+1 position, could also not be demonstrated to become phosphorylated by PKC in our experiments, as well as in a recently published paper [21]. Further evidence against a direct PKC-mediated P2X receptor phosphorylation comes from co-expression studies of P2X1 and G protein-coupled receptors. Co-expression of the metabotropic glutamate receptor mGluR1\u03b1 with P2X1 receptors produced a 2.5-fold increase of the P2X1 receptor-mediated current that was blocked by the PKC inhibitor staurosporine and could be mimicked by PMA treatment of singly expressed P2X1 receptors. Although the pharmacological data are basically consistent with PKC-mediated receptor phosphorylation being involved, mutant P2X1 receptors with a disrupted N-terminal PKC site (T18V) still showed PMA-mediated and mGluR1\u03b1-mediated potentiation. Also, PMA treatment did not lead to an increase in the (rather weak) basal phosphorylation of the P2X1 receptor [36].\nFunctional data supporting a role of PKC in P2X receptor regulation have been detailed above. There are, however, also functional data that are inconsistent with the view that the TXR\/K motif represents a true PKC site: (1) replacement of the phospho-acceptor threonine by an acidic residue to mimic incorporation of a negatively charged phosphate group by constitutive phosphorylation was unable to restore the function of P2X1 receptors [34] and P2X3 receptors [16]; (2) the PMA-induced potentiation of hP2X1 receptor currents could be blocked by the PKC inhibitor staurosporine, but surprisingly, not by site-directed elimination of the PKC site [36]; thus it is much more likely that PMA exerts its effect on the P2X1 receptor (and the P2X3 receptor) through a yet to be identified accessory protein.\nTaken together, there is no doubt that the TXR\/K motif plays an important role in the functioning of P2X receptors, and that PMA is capable of modulating P2X1- and P2X3-mediated receptor currents. The underlying mechanism, however, is unlikely to involve direct PKC-mediated phosphorylation of P2X receptors. The existence of a proline in the P+1 position in several P2X subunit isoforms (P2X1, P2X2, P2X4) might be one causative factor preventing direct phosphorylation by PKC. Additional steric constraints may explain why P2X3 receptors also do not serve as PKC substrates, despite lacking the proline in P+1. The exact role of the TXR\/K motif awaits further clarification.","keyphrases":["phosphorylation","p2x2","p2x3","protein kinase c","p2x receptors"],"prmu":["P","P","P","P","P"]}
{"id":"Behav_Genet-3-1-1914288","title":"Assessment and Etiology of Attention Deficit Hyperactivity Disorder and Oppositional Defiant Disorder in Boys and Girls\n","text":"Attention deficit hyperactivity disorder (ADHD) and oppositional defiant disorder (ODD) are more common in boys than girls. In this paper, we investigated whether the prevalence differences are attributable to measurement bias. In addition, we examined sex differences in the genetic and environmental influences on variation in these behaviors. Teachers completed the Conners Teacher Rating Scale-Revised:Short version (CTRS-R:S) in a sample of 800 male and 851 female 7-year-old Dutch twins. No sex differences in the factor structure of the CTRS-R:S were found, implying the absence of measurement bias. The heritabilities for both ADHD and ODD were high and were the same in boys and girls. However, partly different genes are expressed in boys and girls.\nIn both clinical and population samples, children diagnosed with attention deficit hyperactivity disorder (ADHD) and oppositional defiant disorder (ODD) are predominantly male (Gaub and Carlson 1997; Biederman et\u00a0al. 2002; Loeber et\u00a0al. 2000). These sex differences could either be the result of a higher liability for these disorders in boys than girls, or could be attributable to a sex effect in the actual measurement of the phenotype. For example, boys and girls with similar levels of problem behavior may receive systematically different rating scores if the items of the instrument do not reflect the problem behaviors in the same manner in boys and girls. This may conceptualized as bias with respect to sex. In this study, we will investigate this issue in the measurement of ADHD and ODD by means of the Conners Teacher Rating Scale-Revised:Short (CTRS-R:S) version.\nThe presence of sex differences in the prevalence of ADHD and ODD also raises the question whether there are sex differences in the etiology of these disorders. Sex differences in etiology can only be interpreted if the measurement of a disorder is not biased with respect to sex. Lubke et\u00a0al. (2004) discussed in detail the importance of establishing unbiasedness with respect to sex for the correct interpretation of any sex differences in the results of genetic modeling. The aim of this paper is, therefore, to investigate measurement bias in teacher ratings of ADHD and ODD with respect to sex. If unbiasedness can be shown to be tenable to reasonable approximation, we shall, as the second goal of this paper, estimate the genetic and environmental contributions to the phenotypic variance in ADHD and ODD in boys and girls.\nMellenbergh (1989) defined unbiasedness, or equivalently, measurement invariance (MI), with respect to group to mean that the distribution of the observed test score, conditional on the latent construct that the test measures, is identical over groups (e.g., boys and girls). In more simple terms, this means that the instrument is measuring the same construct in boys and girls (Mellenbergh 1989; Meredith 1993). If this is the case, we expect the score of a given person to depend on that person\u2019s score on the latent construct, but not on that person\u2019s sex. If MI does not hold, a boy and a girl, who are characterized by the same degree of problem behavior, may obtain systematically (i.e., regardless of measurement error) different scores on the instrument. This is undesirable, because we wish our measurements to reflect accurate and interpretable differences between cases in different groups.\nAnalyses aimed explicitly at establishing MI with respect to sex, according to the approach outlined by Meredith (1993), have yet to be conducted with respect to ADHD and ODD. Although MI has not been investigated, there have been some studies, which addressed sex differences in the factor structure in teacher ratings of ADHD. Fantuzzo et\u00a0al. (2001) examined the factor structure of the 28-item version of the CTRS with exploratory factor analyses. They reported a three-factor solution, which accounted for 58% of the variance. The factors admitted interpretations in terms of conduct, hyperactivity, and passivity. The invariance of the factor structure was established by comparing results from random subgroups with the results from subgroups based on sex. The results supported the similarity of the factor structure across sex. A concern in this study is that the subjects in this study were 580 children from 33 classrooms located in low-income neighborhoods. It is, therefore, unclear how representative the sample is of the general population. Furthermore, because each teacher rated more than one child, and children were clustered into classes, the assumption of independent observations, which is important in statistical inference, might not hold.\nIn this study, we conducted confirmatory factor analyses (CFA) of data from a large general population sample of 7-year-old twins, rated by their teachers on ADHD and ODD. Two questions are addressed. First is the measurement model that relates differences in the latent constructs of ADHD and ODD to the observed behavior problem scores identical in boys and girls, i.e., is MI tenable? Second, do the magnitudes of the genetic and environmental influences differ, or do different genes play a role in boys and girls?\nMethods\nSubjects and procedure\nThis study is part of an ongoing twin study of development and psychopathology in the Netherlands. The subjects were all registered at the Netherlands Twin Registry (NTR; Boomsma et\u00a0al. 2002, 2006). We assessed a sample of Dutch twins from the birth cohorts 1992\u20131996. These twins were assessed by their teachers when they were 7\u00a0years old.\nThe twins at age 3 are representative of Dutch 3-year-old children with respect to their scores on measures such as the Child Behavior Checklist (CBCL; van den Oord et\u00a0al. 1995). The socioeconomic status of the parents of the twins was somewhat higher than the level in the general Dutch population (Rietveld et\u00a0al. 2004). When twins reached the age of 7\u00a0years, parents were asked to provide informed consent to approach the teacher. Consent was given by 80.1% of the parents. Teachers of these pairs received a questionnaire by mail, and were asked to return it to the NTR by mail. The response rate of the teachers was 80.0%. CTRS data were available for at least one twin in 1,651 twin-pairs (1,511 complete pairs).\nThe maternal CBCL-AP scores at age 7\u00a0years were not significantly different between families in which parents provided permission to approach the teachers (mean\u00a0=\u00a02.95, SD\u00a0=\u00a02.93) and families in which parents did not do so (mean\u00a0=\u00a03.15, SD\u00a0=\u00a03.18; t(3,063)\u00a0=\u00a02.0, p\u00a0=\u00a00.133). However, mean maternal AP ratings were significantly higher in twins whose teachers did not return the questionnaire (mean\u00a0=\u00a03.34; SD\u00a0=\u00a03.13) than in twins whose teachers did return the questionnaire (mean\u00a0=\u00a02.78; SD\u00a0=\u00a02.81; F(1)\u00a0=\u00a016.82, p\u00a0<\u00a00.001).\nTo avoid biased test results due to statistical dependency of the twin data, we randomly included the score from only one of the members of a twin-pair in the CFA. The resulting sample for CFA consists of 1,651 individual twins (800 boys and 851 girls). In the genetic analyses, we included all complete twin-pairs. Data were available for both members of a twin-pair in 248 MZ male, 251 DZ male, 294 MZ female, 234 DZ female, and 484 DZ opposite sex pairs. Some twins were rated by the same teacher (877 pairs, 58%) while the remaining twins were rated by different teachers (634 pairs, 42%). The genetic analyses accounted for potential differences between same and different teacher ratings by using the model developed by Simonoff et\u00a0al. (1998). Incomplete twin-pairs were excluded from the genetic analyses.\nMeasure\nThe CTRS-R is a widely used instrument to assess behavior problems (Conners 2001; Conners et\u00a0al. 1998). The CTRS-R was developed by factor analyzing a large set of items, and including items that load highly on interpretable common factors. In addition to the scales that were derived based on factor analysis, an ADHD index was included. This index comprises the best 12 items for distinguishing children with ADHD from children without ADHD as assessed by the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV; American Psychiatric Association 1994; Conners 2001). The short version of the CTRS-R, which was used in this study, contains 28 items from the following scales: oppositional (ODD, five items); cognitive problems-inattention (IN, five items); hyperactivity (HI, seven items); and the ADHD index (ADHD-I, 12 items). One of the items (item 27; excitable, impulsive) is included in both the hyperactivity scale and the ADHD index. The items are rated on a 4-point Likert scale for symptom severity (i.e., 0\u00a0=\u00a0\u201dnot true at all\u201d; 1\u00a0=\u00a0\u201djust a little true\u201d; 2\u00a0=\u00a0\u201dpretty much true\u201d; 3\u00a0=\u00a0\u201dvery much true\u201d). The internal consistency and stability of the CTRS-R:S version are good, as the Cronbach\u2019s alpha coefficients range from 0.88 to 0.95, and 6\u20138-week test\u2013retest correlations range from 0.72 to 0.92 (Conners 2001).\nDistribution of the items\nBecause of the categorical nature of the item scores, Pearson product moment correlations underestimate the correlation of the underlying latent trait, and consequently the parameter estimates obtained in factor analyses or principal component analyses based on the Pearson correlation matrices are biased (Dolan 1994). We, therefore, adopted an approach that is suitable for factor analyzing discrete item scores. Polychoric correlations between items were obtained based on the liability threshold model (Lynch and Walsh 1998). In the case of a 4-point Likert scale, three thresholds divide the latent liability distribution into four categories.\nCriteria of MI\nThe criteria of MI are empirically testable in the common factor model (Meredith 1993). MI criteria are: (1) equality of factor loadings over groups; (2) equality of item intercepts over groups (i.e., differences in item means are the result of differences in factor means), and (3) equality of residual variances (i.e., variance in the observed variables, not explained by the common factor) over groups. When satisfied, these restrictions ensure that any differences in the mean and variance of the observed variables are due to differences in the mean and variance of the common factor.\nFor ordinal data, MI can be tested by constraining the thresholds and factor loadings, and residual variances to be equal in boys and girls. These constraints allow estimation of group differences in the means and variances of the common factor. To this end, the mean and variance of the common factor are fixed at 0 and 1, respectively, in an arbitrary reference group. We chose to estimate the mean and variance in girls, and to use boys as the reference group. In doing so, we are modeling the observed group differences as a function of the differences in the latent liability.\nStatistical analyses\nThe polychoric correlation matrices of the items of the four subscales were calculated using Prelis (J\u00f6reskog and S\u00f6rbom 1996). All CFA were performed on raw data using Mx (Neale et\u00a0al. 2003). In principle, factor analysis of p ordinal items can be carried out using full information maximum likelihood (FIML) estimation. However, this requires repeated integration of the p-variate normal distribution, which can become computationally demanding even with as few as 12 items. We, therefore, estimated the model parameters using marginal maximum likelihood estimation (MML; Bock and Aitkin 1981). MML maximizes the likelihood of the data conditional on the latent trait, in contrast to FIML, which maximizes the unconditional likelihood. The advantage of MML compared to FIML is that it is computationally much less demanding.\nTo test if the Conners scales are MI with respect to sex, the factor structure was constrained to be identical in boys and girls. The ODD, IN, and HI scales resulted from factor analyses, and a single factor was fit to these scales. In contrast, the ADHD-I contains items related to problems with inattention and hyperactivity, and thus a two-factor model was fitted. To detect prevalence differences in ADHD and ODD across sex, the means and variances of the latent factors were constrained to be equal in boys and girls.\nThe fit of the models was compared by \u03c7\u00b2 tests, with a type I error probability set at \u03b1\u00a0=\u00a00.01. Browne et\u00a0al. (2002) noted a complication of the \u03c7\u00b2 test. Specifically, they showed that \u03c7\u00b2 is influenced by the unique variances of the items. If a trait is measured reliably, the inter-correlations of the items are high, the unique variances are small, and the \u03c7\u00b2 test may suggest a poor fit even when the differences between the expected and observed covariance matrices are trivial. The standardized root mean square residual (SRMR; Bentler 1995) is a fit index that is not sensitive to the size of the correlations. To avoid the rejection of a simpler model due to high inter-item correlations, we only reject a model if a significant \u03c7\u00b2 test is accompanied by large residuals (SRMR\u00a0>\u00a00.08; Hu and Bentler 1999).\nAfter investigating MI with respect to sex, we look at sex differences in the genetic and environmental influences on the individual differences in the sum scores of the scales, given that MI is tenable (Lubke et\u00a0al. 2004). The polychoric correlations of the four scales were calculated by sex and zygosity in PRELIS (J\u00f6reskog and S\u00f6rbom 1996). The genetic analyses were performed on the raw data using Mx (Neale et\u00a0al. 2003).\nWith data from MZ and DZ twins, the variance in behavior can be attributed to genetic and environmental factors. In our sample, 58% of the twins were in the same classroom and 42% of the twins were in different classrooms. Correlations between twins may be higher when children are rated by the same teacher. Therefore, a correlated error model developed by Simonoff et\u00a0al. (1998) was used to analyze the data. In this model, individual differences in behavior are explained by additive genetic factors (A), common environmental factors that are shared between two twins of a pair (C), and unique environmental factors (E).\nThe unique environmental factors are allowed to correlate in twins who are placed into the same classroom, and do not correlate in twins who are placed into different classrooms. For the genetic analyses, the items of each subscale were summed, and the sum-score was recoded so that three thresholds divide the latent liability distribution into four categories. The thresholds were chosen in such a way that the categories contain more or less equal numbers of subjects. We preferred this procedure to the analysis of the raw sum scores, because these are skewed, and therefore cannot be analyzed with maximum likelihood approaches based on the assumption of normality (Derks et\u00a0al. 2004).\nSex differences in genetic and environmental influences were examined in two ways. First, we investigated if the estimates of the genetic and environmental variances are equal in boys and girls. Secondly, we investigated if the same genes influence phenotypic variation in boys and girls. These qualitative sex differences were evaluated by constraining the genetic correlation in opposite sex twins at 0.5 (similar as in same-sex DZ twins). If different genes play a role in boys and girls, the genetic correlation is expected to be lower than 0.5 in opposite sex twins.\nResults\nMeasurement invariance\nWe tested for MI by constraining the factor loadings, thresholds, and residual covariance matrices to be equal for boys and girls while allowing the factor means and variances to be different. The factor structure of ODD was MI with respect to sex (\u03c7\u00b2(18)\u00a0=\u00a016.66, p\u00a0>\u00a00.10; SRMR\u00a0=\u00a00.01 and 0.06 in boys and girls, respectively). MI was also tenable for the ADHD-I (\u03c7\u00b2(55)\u00a0=\u00a070.41, p\u00a0>\u00a00.05; SRMR\u00a0=\u00a0.03 and 0.05 in boys and girls, respectively). Both IN and HI showed statistically significant different factor structures in boys and girls (\u03c7\u00b2(18)\u00a0=\u00a098.45, p\u00a0<\u00a0.001, and \u03c7\u00b2(26)\u00a0=\u00a057.99, p\u00a0<\u00a00.001, respectively). However, the residuals between expected correlation matrices under the constrained and the non-constrained model were small (SRMR\u00a0=\u00a00.01 in girls and SRMR\u00a0=\u00a00.02 in boys, for both IN and HI). Apparently, the lack of fit was the result of the high inter-correlations between the items (Browne et\u00a0al. 2002), and not of large residuals between the expected covariance matrices. Therefore, we tentatively accept MI with respect to sex. Table\u00a01 provides the factor loadings and thresholds of the best-fitting models. We also included the factor loadings as reported by Conners (2001) to facilitate the comparison of our sample with the sample that was used to create the scales. Note that the factor loadings, as reported by Conners (2001), are generally much lower, as these estimates are based on the assumption of a continuous, normal distribution of the item scores, an assumption that is obviously violated in the instance of a four-category Likert scale.\nTable\u00a01Promax rotated factor loadings and thresholds (T) of the best-fitting factor model for the four subscales of the CTRS-R:SItemsLoadings factor 1Loadings factor 2Loadings as reported by Conners (2001) (N\u00a0=\u00a01,897)T1T2T3Oppositional2. Defiant0.91\u20140.480.711.652.456. Defies0.88\u20140.501.022.002.7410. Spiteful0.69\u20140.301.492.323.1315. Argues0.84\u20140.510.821.802.5220. Explosive0.78\u20140.411.201.923.12Cognitive problems-Inattention4. Forgets things0.93\u20140.550.341.291.828. Poor spelling0.81\u20140.500.270.951.4413. Poor reading0.71\u20140.450.390.871.2118. Lacks interest0.70\u20140.471.011.652.2722. Poor arithmetic0.82\u20140.490.551.221.68Hyperactivity3. Restless0.73\u20140.620.100.941.517. Always on the go0.79\u20140.570.661.311.8711. Leaves seat0.82\u20140.580.621.371.8317. Difficulty waiting0.83\u20140.650.120.911.5821. Runs about0.79\u20140.441.211.892.5124. Difficulty playing0.87\u20140.520.341.172.0327. Excitable0.86\u20140.660.401.121.84ADHD indexAP1. Inattentive0.99\u22120.09\u2014\u22120.360.761.4714. Attention span0.95\u22120.03\u20140.030.871.5516. Only pays attention0.450.23\u20140.361.201.8819. Distractibility0.94\u22120.01\u20140.591.542.2525. Fails to finish0.720.09\u20140.241.271.9926. Not follow instructions0.680.09\u20140.381.301.86HI5. Disturbs0.050.80\u20140.201.031.529. Remain still0.050.88\u20140.921.682.2412. Fidgets0.160.67\u20140.120.941.5323. Interrupts\u22120.050.85\u20140.541.372.0727. Excitable\u22120.090.91\u20140.461.151.8328. Restless\u22120.060.97\u20140.501.191.81AP attention problems; HI hyperactivityThe thresholds are constrained to be equal in boys and girls. The means and variances of the latent factors in boys are constrained at 0 and 1, respectively. The means and variances of the latent factors are freely estimated in girls. The variances in girls were not significantly different from 1. The means of the latent factors are estimated at \u22120.60 (oppositional), \u22120.86 (hyperactivity), \u22120.42 (ADHD-I AP), and \u22120.52 (ADHD-I-HI). The mean of the cognitive problems-inattention factor is not significantly different between boys and girls\nGenetic analyses\nHaving established MI of the CTRS-R:S scales with respect to sex, we estimated the twin-correlations and carried out a genetic analysis of the data. Twin correlations are shown in Table\u00a02, for same and different teachers, respectively. The genetic model fitting results of the four scales are reported in Table\u00a03. All correlations are higher in MZ twins than in DZ twins, suggesting the presence of genetic influences on individual differences. The correlations are higher in twin pairs rated by the same teacher than for twin pairs rated by different teachers. This was taken into account by using a correlated error model (Simonoff et\u00a0al. 1998). The lower correlations in opposite-sex DZ twins than in same-sex DZ twins suggest that different genes play a role in boys and girls.\nTable\u00a02Polychoric twin-correlations of the CTRS rated by same teachers (ST) versus different teachers (DT)OppositionalCognitive problems-inattentionHyperactivityADHD indexSTDTSTDTSTDTSTDTMonozygotic males0.860.530.900.760.810.590.850.59Dizygotic males0.500.490.640.290.420.140.490.19Monozygotic females0.870.430.920.640.830.470.870.52Dizygotic females0.660.100.600.420.340.250.450.24Opposite-sex twins0.370.130.440.230.300.170.370.27Table\u00a03Genetic model fitting results on CPRS-R:S ratingsModelParameters-2 LLWith model\u0394df\u0394\u03c7\u00b2pOppositional behavior in 7-year-old children1. Fully saturated844779.36\u2014\u2014\u2014\u20142. Equal correlations same and different teachers784812.261632.91<0.0013. ACE model, estimates of ACE are allowed to be different between same and different teachers444817.9414038.580.5344. ACE, ACE same teacher\u00a0=\u00a0ACE different teacher404853.843435.90<0.0015. Correlated errors ACE model424819.8014240.440.5406. Correlated errors AE model404823.93524.130.1277. Correlated errors AE model, AE boys\u00a0=\u00a0AE girls384829.76625.830.0548. Correlated errors AE model, AE boys\u00a0=\u00a0AE girls, opposite-sex genetic correlation-free374821.86717.900.005Cognitive problems-inattention in 7-year-old children1. Fully saturated846785.90\u2014\u2014\u2014\u20142. Equal correlations same and different teachers786822.571636.67<0.0013. ACE model, estimates of ACE are allowed to be different between same and different teachers446815.9414030.040.8744. ACE, ACE same teacher\u00a0=\u00a0ACE different teacher406843.873427.93<0.0015. Correlated errors ACE model426815.8514229.950.9186. Correlated errors AE model406820.01524.160.1257. Correlated errors AE model, AE boys\u00a0=\u00a0AE girls386821.99621.980.3728. Correlated errors AE model, AE boys\u00a0=\u00a0AE girls, opposite-sex genetic correlation-free396817.50714.490.034Hyperactivity in 7-year-old children1. Fully saturated846880.07\u2014\u2014\u2014\u20142. Equal correlations same and different teachers786906.971626.90<0.0013. ACE model, estimates of ACE are allowed to be different between same and different teachers446914.5914034.420.7154. ACE, ACE same teacher\u00a0=\u00a0ACE different teacher406942.353427.76<0.0015. Correlated errors ACE model426924.1214244.050.3856. Correlated errors AE model406926.76522.640.2677. Correlated errors AE model, AE boys\u00a0=\u00a0AE girls386929.06622.300.3178. Correlated errors AE model, AE boys\u00a0=\u00a0AE girls, opposite sex genetic correlation-free376919.99719.070.003ADHD in 7-year-old children1. Fully saturated847420.21\u2014\u2014\u2014\u20142. Equal correlations same and different teachers787458.101637.89<0.0013. ACE model, estimates of ACE are allowed to be different between same and different teachers447472.5914052.380.0914. ACE, ACE same teacher\u00a0=\u00a0ACE different teacher407507.403434.81<0.0015. Correlated errors ACE model427477.7814257.570.0556. Correlated errors AE model407479.14521.360.5077. Correlated errors AE model, AE boys\u00a0=\u00a0AE girls387480.72621.580.4548. Correlated errors AE model, AE boys\u00a0=\u00a0AE girls, opposite-sex genetic correlation-free377475.93714.790.029\nModel fitting analyses showed that variation in all four scales could be explained by additive genetic and unique environmental effects. The influences of the shared environment were not statistically significantly. The magnitude of the influences of genes and environment did not differ between boys and girls. The standardized estimates of genetic and environmental influences are shown in Table\u00a04. Genetic effects explained 56\u201371% of the variation in the CTRS subscales. Unique environmental effects explained the remaining 29\u201344% of the variation. For all four scales, the genetic correlation was significantly lower than 0.5 in opposite-sex twins. This implies that different genes are expressed in males and females. The genetic correlation in opposite-sex twins was 0.16 for oppositional behavior, 0.35 for cognitive problems-inattention, 0.21 for hyperactivity-impulsivity, and 0.32 for the ADHD index.\nTable\u00a04Standardized estimates of the genetic and environmental effects on problem behavior% Additive genetic effects (A)% Non-shared environmental effects (E)Genetic correlation opposite-sex twinsOppositional56440.16Cognitive problems-inattention71290.35Hyperactivity58420.21ADHD index61390.32\nDiscussion\nThe purpose of this study was twofold. First, we investigated if teacher ratings on ADHD and ODD are measurement invariant with respect to sex. Secondly, genetic and environmental influences on variation in ADHD and ODD were compared between boys and girls.\nMeasurement invariance\nTeacher ratings on ADHD and ODD were found to be measurement invariant with respect to sex. In other words, teacher assessments of these behavior problems relate to the same latent variables in boys and girls. Sex differences in observed scores on ADHD and ODD can, therefore, be interpreted as differences with respect to the latent construct. This supports the contention that the reported sex differences in ADHD and ODD (Gaub and Carlson 1997; Loeber et\u00a0al. 2000; Maughan et\u00a0al. 2004) are due to a higher liability for the disorder in boys than girls and not to measurement bias.\nQuantitative and qualitative differences in the heritability among boys and girls\nMore than half the variance in ADHD and ODD in boys and girls is attributable to genetic influences. The remaining variance is attributable to unique environmental influences. The magnitude of the influences of genes and environment is the same in boys and girls. However, part of the variance in ADHD and ODD is attributable to different genes in boys and girls. We base this on the fact that the genetic correlation between DZ opposite-sex twins was significantly lower than 0.5, which is the theoretical value (in the absence of assortative mating), if the same genes influence behavior in boys and girls. We observed a genetic correlation lower than 0.5 in DZ opposite-sex twins for oppositional behavior, cognitive problems-inattention, hyperactivity, and the ADHD index.\nFew studies have addressed quantitative and qualitative sex differences in heritability estimates from teacher ratings. Saudino et\u00a0al. (2005) reported qualitative sex differences in heritability of teacher ratings of hyperactive behavior in 7-year-old twins. They did not report any quantitative sex differences, which is in agreement with the current findings. Vierikko et\u00a0al. (2004) report lower correlations in opposite-sex twins than in same-sex DZ twins of teacher ratings of hyperactivity-impulsivity in 12-year-old twins. However, both genetic and shared environmental effects were found to contribute to the phenotypic variance in these data. It was not possible to determine if the lower opposite-sex correlations were the result of sex-specific genetic influences or sex-specific shared environmental influences, although the presence of the latter appeared more likely. These findings disagree with the current results in the sense that we did not find any evidence for shared environmental influences. However, both studies suggest that teacher ratings are influenced by partly different etiological factors in boys and girls.\nThe finding of different genetic influences in boys and girls in teacher ratings stands in contrast with results based on parental reports. In parent ratings, qualitative sex differences are not found for attention problems (Rietveld et\u00a0al. 2004) or ODD (Hudziak et\u00a0al. 2005). The different findings in parent and teacher ratings may be explained by the fact that the behavior of children depends on the context in which they are observed. Apparently, inattentive, hyperactive, and oppositional behavior of boys and girls are influenced by partly non-overlapping factors at school, while this is not true for these behaviors at home.\nThe finding of sex-specific genetic variation has implications for gene-finding studies of ADHD and ODD. The fact that the genes which influence the behavior of boys and girls do not completely overlap indicates that some quantitative trait loci may explain variation in boys but not in girls and vice versa. Therefore, the data from boys and girls cannot be collapsed when studying genetic effects in teacher ratings.\nIn the NTR, teacher data are collected at the ages 7, 10, and 12\u00a0years. The sample sizes at the ages 10 and 12 are currently relatively small. In the future, we plan to address the issue of qualitative sex differences in teacher ratings in a longitudinal framework. The results of such a study will reveal if the finding of sex differences in the specific genes that play a role is also present in older children. Another important issue that may be addressed is the MI of ADHD with respect to age.\nThe results of this study should be interpreted in the light of the following limitations. First, we did not replicate the factor structure of the CTRS-R:S by means of exploratory factor analyses of the 28 items. To take the ordinal nature of the data into account, we used the liability threshold model (Lynch and Walsh 1998). We limited the number of common factors to keep the computational burden manageable. Therefore, we performed CFA, in which we assumed that the items are correctly assigned to the four scales and that cross-loadings are absent. Second, teacher ratings were shown to be measurement invariant with respect to sex, but this finding may not generalize to parent ratings. The correlations between Conners parent and teacher ratings are small to moderate with a range 0.18\u20130.52 (Conners 2001). It has been shown that parents and teachers rate partly different aspects of the child\u2019s behavior (Derks et al. in press; Martin et\u00a0al. 2002). Future studies will reveal if MI is also tenable in parent ratings.\nAssessment of ADHD and ODD symptoms, through teacher reports on the CTRS-R:S, provides a solid starting point for measuring sex differences in mean scores or in heritabilities. Variation in teacher ratings of children\u2019s problem behavior is mainly influenced by genetic factors. The size of the genetic influences does not depend on the child\u2019s sex, but partly different genes are expressed in boys and girls. Future studies should reveal if these findings generalize to children from different age groups.","keyphrases":["adhd","odd","sex differences","measurement invariance","conners teacher rating scale"],"prmu":["P","P","P","P","R"]}
{"id":"Bioinformation-1-5-1891680","title":"A web server for transcription factor binding site prediction\n","text":"Promoter prediction has gained increased attention in studies related to transcriptional regulation of gene expression.We developed a web server named PMSearch (Poly Matrix Search) which utilizes Position Frequency Matrices (PFMs) to predict transcription factor binding sites (TFBSs) in DNA sequences. PMSearch takes PFMs (either user-defined or retrieved from local dataset which currently contains 507 PFMs from Transfac Public 7.0 and JASPAR) and DNA sequences of interest as the input, then scans the DNA sequences with PFMs and reports the sites of high scores as the putative binding sites. The output of the server includes 1) A plot for the distribution of predicted TFBS along the DNA sequence, 2) A table listing location, score and motif for each putative binding site, and 3) Clusters of predicted binding sites. PMSearch also provides links for accessing clusters of PFMs that are similar to the input PFMs to facilitate complicated promoter analysis.\nBackground\nTranscription factors play a pivotal role in the regulation of gene expression by sequence specific binding to the promoters of target genes. \nPrediction of putative transcription factor binding sites has become an important resource to explore genome organization and regulatory mechanisms. \nThe binding specificity of transcription factors are usually represented by known sequence motifs (consensus sequences) or matrices (PFM (Position \nFrequency Matrix) or PWM (Position Weight Matrix)). [1] High throughput analyses using SELEX (Systematic \nEvolution of Ligands by Exponential Enrichment) and CHIP-Chip (Chromatin Immunoprecipitation-Microarray) along with computational sampling methods have \ngenerated thousands of PFMs. These data together with the related transcription factor information are curated in online databases such as Transfac [2], JASPAR [3], etc.\nOnline applications, such as MatInspector [4,5], \nMATCH [6] and ConSite [7], have been built to utilize \nPFMs to predict transcription factor binding sites (TFBSs) embedded in promoter sequences. Many of the servers are comprehensive but lack the information on transcription \nfactors whose binding specificities are similar to the input PFMs, which could effectively assist regulatory module finding. To provide such information and to meet the \nneeds for efficient prediction tasks in the study of gene regulation network, we developed a server named PMSearch (Poly Matrix Search) for predicting TFBSs in DNA sequences \nwith the novel functions of fetching similar PFMs and processing multi-forms of input motifs. PMSearch made the following improvements: 1) It has a more succinct and friendly \nuser-interface. 2) It provides user with the convenience of customizing any set of PFMs from the local dataset. 3) It generates a resizable plot that shows the distribution \nand scores of predicted binding sites in variable scales. 4) It provides clusters of PFMs similar to the input PFMs.\nMethodology\nWe have implemented a scoring scheme adapted from\nprevious algorithms. [4,6] The uninformative nucleotides (ambiguous letters: N, B, \nD, H, and V) on either end of an input PFM are discarded before searching to enhance efficiency and specificity. We downloaded 507 PFMs from JASPAR and Transfac Public 7.0 to construct \na local dataset from which the user may take any subset of PFMs, or along with userdefined PFMs, for a prediction task. In addition to PFMs, the server could also accept sequence motifs \n(modeled in the format of consensus sequences), which will then be converted into pseudo PFMs and applied in subsequent prediction. We have set a default global cutoff value of 0.85 previously \nadopted by TFSEARCH. [9] The user may adjust this cutoff if required.\nUser interface\nThe web interface of PMSearch helps in promoter analysis. The inputs are composed of PFMs and sequences under analysis. The user may submit PFMs, motifs or retrieve desired \nPFMs from our dataset using keywords (e.g., name of the transcription factor), its source database accession number (e.g. Transfac Matrix Accession number M00001) or our local \naccession number (e.g. X00001). Sequences in FASTA format or Genbank format are acceptable and will be auto-parsed. When the user initiates a predication task, each sequence will \nfork an independent task in which corresponding results can be retrieved respectively. In the output, PMSearch plots a figure that illustrates binding sites that are scored above \nthe cutoff value (Figure 1). The user may specify a sub-region on the sequence that will be plotted for more detailed view, such as a specific promoter region in a sequence file. \nOther results including scores, sequence motifs and closely located putative binding sites (clusters) are also listed. In addition, the server provides a hyperlink for the user to \nfetch PFMs that are related to the input PFMs by implementing a gap-allowed alignment algorithm. A comprehensive instruction for users is available online.\nConclusion\nPMSearch is an easy-to-use and efficient tool that utilizes PFMs to predict transcription factor binding sites in DNA sequences. It offers user the flexibility to search for\nputative TFBS with any set of PFMs. PMSearch outputs a plot demonstrating distribution of predicted binding sites and a table of the locations, scores, motifs and clusters of predicted binding \nsites. In addition, PMSearch provides the user access to PFMs that are relative to an input PFM for more sophisticated promoter analysis, as the predicted binding sites of one transcription \nfactor could also be bond with other transcription factors that share the similar binding specificity. Such information may give hints to untangle the composite transcription regulatory network. \nWe propose to update PFMs in our local dataset regularly. The source codes are available from the authors upon request.\nCaveats\nIt has been proposed that many of the predicted TFBSs lack biological function in vivo. [10] This could result from 1) The \npredicted site is located in a context which is insufficient to facilitate transcription factor binding, 2) The low specificity of the input PFM gives rise to large portion of false \npositive predictions. We suggest the users prepare a list of certain transcription factors that are suspected to regulate the target gene to specify a prediction task.","keyphrases":["web server","transcription factor binding site","motif","position frequency matrix"],"prmu":["P","P","P","R"]}
{"id":"J_Digit_Imaging-3-1-2039858","title":"Mastering DICOM with DVTk\n","text":"The Digital Imaging and Communications in Medicine (DICOM) Validation Toolkit (DVTk) is an open-source framework with potential value for anyone working with the DICOM standard. DICOM\u2019s flexibility requires hands-on experience in understanding ways in which the standard\u2019s interpretation may vary among vendors. DVTk was developed as a clinical engineering tool to aid and accelerate DICOM integration at clinical sites. DVTk is used to provide an independent measurement of the accuracy of a product\u2019s DICOM interface, according to both the DICOM standard and the product\u2019s conformance statement. DVTk has stand-alone tools and a framework with which developers can create new tools. We provide an overview of the architecture of the toolkit, sample scenarios of its utility, and evidence of its relative ease of use. Our goal is to encourage involvement in this open-source project and attract developers to build off and further enrich this platform for DICOM integration testing.\nINTRODUCTION\nDigital Imaging and Communications in Medicine (DICOM)1 plays a major role in the health care information technology (IT) field as the standard for medical images and communication throughout the hospital. With the organization of the DICOM Standards Committee in 1996 and the support of major medical groups and imaging vendors worldwide, DICOM has become a dominant integration mechanism in the hospital enterprise. The DICOM standard version 3.0 contains 16 parts (more than 2,000 pages), and the standard itself is constantly evolving as new software and imaging technologies are developed. Mastering a technology like DICOM can be a daunting task, but the successful student will be on the right path with the DICOM Validation Toolkit (DVTk) from DVTk.org. Consider DVTk the lab kit that is handed out with the DICOM standard on the first day of DICOM 101 class.\nAs with any other technology, true learning and understanding require hands-on experience.2 Although the standard itself does not define or identify testing or validation procedures to assess conformance, a number of third-party tools have been developed to fill that role. DVTk is a powerful tool for mastering the intricate DICOM file format and transfer syntax. The mantra of DVTk is to make DICOM easy. If a picture archive and communication system (PACS) adminsitrator, for example, is having a problem with imaging device integration, DVTk can be the first line of defense in tracking the problem. When vendors are pointing fingers at one another, DVTk can help them past recriminations and on to real solutions. For medical software developers, integrators, and testers, DVTk can help to more quickly produce robust systems.\nThe DICOM Validation Tool (DVT) test framework is a flexible architecture and uses service\u2013object pair (SOP) class definition files, making it adaptable as the DICOM standard evolves. It includes a graphical user interface (GUI) and a command line interface, DICOM media validation, service class user (SCU) and service class provider (SCP) emulators, and a rich scripting language. For more advanced testing, VBScript (Microsoft Visual Basic Scripting edition; Microsoft; Redmond, WA) or JScript (Microsoft) can be executed by DVT, and a set of .NET assemblies is available for developing stand-alone test tools with languages such as Visual Basic.NET (Microsoft) and C# (Microsoft).\nIf a validation tool is to be taken seriously, it must be vendor neutral. With the release of DVT 2.1 in 2005, DVTk now exists as an open-source community project. Not only does this encourage more vendors to contribute (because of the incentive of reduced development and integration costs), but the adoption and proliferation of standards make it easier for individual developers to contribute to open-source projects such as DVTk. Independent software developers are sometimes discouraged from proceeding because of the chance that their work will provide a \u201cone-off\u201d solution only and not be widely used. DVTk, with the backing of the mature DICOM standard, is attracting talented developers with a desire to create something useful and lasting. This success assures that time devoted to learning this toolkit will not be wasted.\nThis article focuses primarily on the DVT main application. We provide background on the toolkit and specific examples about the two intended roles of DVT: service and development. Current efforts within the DVTk project and future directions are also highlighted.\nDVTk HISTORY AND ARCHITECTURE\nIn 2000, Agfa HealthCare (Mortsel, Belgium) and Philips Medical Systems (Eindhoven, The Netherlands) decided to coordinate activities around DICOM validation testing by bringing together efforts already started by both companies under a joint DVT project. The intention was to produce a DVT that could not only be used internally by both companies to test their own products but also made freely available to other original equipment manufacturers (OEMs) as a means of testing their products to the same level of detail. The ultimate aim was to reduce the time spent integrating proprietary systems by first exposing these systems\u2019 equipment to tests run using DVT.\nDVT project has a steering committee with responsibility for guiding legal, technical, and commercial aspects. The steering committee meets every 6\u00a0months to discuss past progress, current issues, and future requirements. A project manager was elected by the steering committee to manage the DVT project on a daily basis and report back to the committee. Development tasks were divided up based on the available skills of developers who report to the project manager. In its first years, Agfa and Philips provided the personnel to staff the DVT project.\nIn September 2005, DVT was made into an open-source project (http:\/\/www.dvtk.org, DICOM Validation Toolkit; last accessed May 2007) under SourceForge (http:\/\/sourceforge.net\/projects\/dvt, SOURCEFORGE.NET ; last accessed May 2007) as DVTk and is licensed under the GNU Lesser General Public License (LGPL). The steering committee decided that the time had come to begin promoting DVTk to a wider audience, with the aim of attracting other companies who might join and supply development resources. Around this time, ICT Healthcare (Eindhoven, The Netherlands), which had already been supplying development resources to the project, joined as a full member with representation on the steering committee. The goal was to make DVTk the independent gold standard for DICOM validation and thereby improve the interoperability of all vendor products using the DICOM interface.\nThe dvtk.org Web site is now the location for the latest downloads, defect tracking details, and forums on using DVTk. Interested individuals and companies may join the project through the Web site. A weekly project telephone conference coordinates the activities of the development team.\nDVTk-based applications include DVT, the main application, which together with the core forms what is now referred to as the DICOM Validation Framework (Fig.\u00a01). The core includes a DICOM testing data model and object-oriented class structure. The DVTk and DVTkData libraries provide access to this data model and class structure via the managed code adapter. The core includes the DICOMScript language that is supported by DVT. This language can be separated into basic programming, which includes the SEND and RECEIVE commands for simulating DICOM SCPs and SCUs, and advanced programming. The advanced programming language includes commands such as SYSTEM, for executing operating system native applications, and a number of commands for working with the Data Warehouse feature. The Data Warehouse is a run-time memory structure in which the user can store Association Control Service Element (ACSE) requests and responses, DICOM commands, and DICOM objects for reuse across test scripts and sessions.\nFig\u00a01.DICOM Validation Framework.\nFor more advanced testing scenarios, DVTk includes the Script Support library and the High-Level Interface (HLI) library. The HLI library is a newer abstraction built on top of the core that makes it easy to write multithreaded tests. The application program interface (API) exposed by this library encapsulates many of the low-level core API classes and methods that make writing VBScripts similar to writing scripts in DVTk\u2019s native DICOMScript language. VBScripts can be executed entirely within the DVT GUI application or command-line executable or debugged in Visual Studio .NET. Other DVTk-based applications built on the core and currently available on dvtk.org include: DICOM Network Analyzer, a network sniffer and DICOM protocol analyzer; DICOM Editor, for displaying and editing DICOM files; DICOM Compare Tool, for comparing the attributes and values of two DICOM files; DICOM Attribute Validator, for validating DICOM files, including Structured Report objects, against definition files; DICOM File Stripper, for removing all but mandatory attributes from a DICOM file; and DICOM File Anonymizer, for removing patient and physician information from a DICOM file.\nTo date, the following contributions have been made to the DVTk project by outside parties, companies, and\/or institutions:\nMedical Communications (UK) provided the underlying Transmission Control Protocol\/Internet Protocol (TCP\/IP) Capture File to the DICOM protocol data unit (PDU) conversion utilities used by the DICOM Network Analyzer application.The National Institute of Standards and Technology (NIST; Gaithersburg, MD) provided the Health Level Seven (HL7)3 validation Web services used in the latest DVTk HL7 validation components.\nGETTING STARTED WITH DVTk OUT OF THE BOX\nThe latest version of DVT can be downloaded from http:\/\/www.dvtk.org . It comes packaged as a Microsoft Windows InstallShield application. The installation subfolder includes a user\u2019s guide and Windows help files for the extensible .NET assemblies (available only if MS Visual Studio .NET is installed). A large number of examples on how to use the many features of DVT are included in the subfolder.\nStarting DVT presents the user with an empty workspace. DVT is designed to work on a single project at a time, although multiple views of the project may be opened from which multiple tests may be simultaneously executed. A DVT project is a container for one or more test sessions. A session is a container for the configuration of one or more tests to be performed against a system under test (SUT). Project and session configuration properties are stored in flat files. The DVT GUI exposes most of the configuration properties, although some of the more advanced settings, such as STRICT-VALIDATION, are available only by directly editing the session file. Some settings can also be modified in script files. For example, the STRICT-VALIDATION script command overrides the value of the STRICT-VALIDATION session property. Some settings, such as CALLED_AE_TITLE, also have built-in default values that are assumed if the property is not defined in a script or session file.\nSession files come in three types: emulator, script, and media. Emulator sessions are used when DVT should act as an SCU or SCP emulator to test the DICOM transfer syntax. The emulators have support for Verification SCU\/SCP, Storage SCU\/SCP, and Print SCP. Script sessions are used when DVT is used to execute a DICOMScript, DICOMSuperScript, or VBScript. A DICOMSuperScript is simply a script that calls one or more DICOMScripts. Script sessions have support for network SCP\/SCU message exchange and DICOM media file creation. Media sessions are used to validate the DICOM file format as a DICOMDIR and\/or DCM media files. When validating a DICOMDIR file, any referenced files are also validated.\nAn easy way to gain familiarity with DVT is go through the examples in the subfolder using DVT as both the test tool and the SUT. Opening up the example project displays a tree view of all test sessions defined in the project and provides details on the session currently selected in the tree. Clicking on the Window menu and selecting the New Project View and Tile item displays two views of the project.\nThe next example will walk through the Modality Worklist (WLM) SCP\/SCU test script sessions. Selecting the WLM_SCP session in the top view and the WLM_SCU session in the bottom view presents a view from which two test sessions may be executed simultaneously (Fig.\u00a02).\nFig\u00a02.DVT-Worklist Management SCP and SCU test sessions.\nAt the top of the Session Information tab are general settings, including the session type, session ID (used in uniquely naming results files), and location of files used\/created by the test session. The DVT Roles Settings section defines the Application Entity (AE) title and some connection settings that DVT assumes during the test session. The SUT Settings section defines the AE title and some connection settings, including the TCP\/IP address, of the system the session is testing. Because this example uses DVT as both the testing system and the SUT, the DVT role settings in the top view are synchronized to the SUT settings in the bottom view, and the SUT settings in the top view are synchronized to the DVT role settings in the bottom view.\nLocations of the SOP class definition files required for the test session are selected under the Specify SOP Classes tab. The selected definition files are loaded into memory when the test session starts, and DVT uses these to validate the DICOM messages and objects it sends and receives. A definition file describes a single DICOM SOP class in terms of the combination of DICOM Message Service Element (DIMSE) commands and Information Object Definitions (IODs) that make up the SOP class. In this case, both test sessions specify the Modality Worklist Information Model\u2013FIND SOP Class item. The standard definition files that come with DVT in the definitions subfolder are taken directly from the DICOM standard parts 3 and 4. Private definition files can be made that extend the validation capabilities of DVT by copying one of these standard files and modifying it with private user IDs (UIDs), modules, and attributes. A typical customization might specify that when a particular SUT is known to always send a value for the Patient Name (0010,0010) attribute, the corresponding definition file can be modified to define the Patient Name attribute as a type 1 (mandatory, nonzero-length) attribute.\nOpening the WLM_SCP tree node in the top view and selecting the DICOMScript, 1.ds, displays a Script tab in the right side of the view. This script defines the test steps for the SCP. Simple DICOMScripts such as this can be written using only the SEND and RECEIVE commands. A large number of additional commands are available for more advanced testing scenarios. The Script tab is read-only, but Windows Notepad can be launched from a context menu on the script name in the tree view. A handy DVT DICOM script reference (dvtDICOMscript.hlp) file is in the docs folder. This script first executes a RECEIVE command for an association request message that specifies a single presentation context, consisting of the Modality Worklist Information Model\u2013FIND SOP Class and three possible transfer syntaxes. The SCU script in the bottom view SENDs an association request message. The only required parameter for the ASSOCIATE-RQ message is the presentation context. For the SCP\u2019s RECEIVE command, the ASSOCIATE-RQ message is referred to as the reference or expected object. This will be compared to the received ASSOCIATE-RQ message. DVT will perform validation in two steps:\nThe received object is first validated against any loaded definition files. This step ensures that the correct attributes are present in the object and that they are encoded correctly.This step is optional and applied only if a reference object is present in the script. Checks made here are that the expected number of attribute values has been received and that each attribute value matches the corresponding reference object attribute value.\nOften, it is not known with what values an SUT will respond, in which case no attributes can be specified in the reference object, so that only step one validation occurs (http:\/\/www.dvtk.org \u201cDVT User Guide\u201d, version 2.1, August 2005). The VALIDATION script command determines how the DICOM objects are validated by DVT. By default, validation is ENABLED, meaning full validation will occur. The value of the STRICT-VALIDATION session property determines how the result of the validation is handled by DVT. If STRICT-VALIDATION is enabled, the presence of attributes in the received message must match the definitions exactly. If they do not match, then DVT reports a FAILED validation and aborts further DICOMScript interpretation. If STRICT-VALIDATION is disabled and attributes do not match, then DVT reports a WARN message. By default, STRICT-VALIDATION is disabled.\nFollowing a successful association validation and negotiation, the SCU script SENDs a C-FIND-RQ message of type Modality Worklist\u2013FIND. The SCP script RECEIVEs the C-FIND-RQ, validates it, and proceeds to SEND three hard-coded C-FIND-RSP messages. The SCU script was written to mirror the SCP script, so the three reference C-FIND-RSP messages it defines match exactly with those sent by the SCP. These scripts also demonstrate two DVT scripting features: Value Mapping and Value Representation (VR) Keywords. Value mapping allows the user to substitute a label for a value defined in a script, generated by DVT, or received from the SUT, and then refer to that value with the label further down in the script. The LABEL: keyword is used to map an attribute value defined in the script or received from the SUT. The NEW: keyword tells DVT to generate a new value and assign it to the given label. An example of this is in the SCP script, where the Study Instance UID attribute (0020,000D) is assigned a new value generated by DVT and named with the label StudyInstanceUid1. The generated UID value can be referred to subsequently in the script as StudyInstanceUid1 (although this was not done in this example). VR keywords, such as the AUTOSET keyword used in both the SCU and SCP scripts, tell DVT to generate a value and assign it to the corresponding attribute. In the example case, AUTOSET is used in the Scheduled Procedure Step Start Date attribute to ensure that values match between the two scripts. Both Value Mapping and VR Keywords are sensitive to the type of attribute to which they are applied.\nThis test is begun by right clicking on the SCP script and selecting execute. When DVT is executing a test session, all tabs except the Activity Logging tab are hidden, the session tree in the corresponding view is disabled, and the test stop button in the toolbar is enabled. The SCP\u2019s Activity Log will indicate that it is waiting for a connection on port 104. Executing the SCU script in the lower window will result in a number of messages in the Activity Logs, followed by the termination of the test sessions. Comparing and correlating the SCP and SCU activity log entries provides a good visual reference for the DICOM message flow between two AEs.\nBy default, the AUTO-TYPE-2-ATTRIBUTES session property is set to true in the session file, which means that DVT will automatically add any zero-length type 2 (type 2 attributes must be included; however, they may be encoded with a zero-length value or no value) attributes from the definition file to the dataset before sending to the SUT. This behavior is recorded in the SCU\u2019s activity column by the \u201cAutomatic Type 2 Attributes population...\u201d statement. This feature means that only type 1 (required, nonzero-length) attributes must be explicitly stated in the scripts.\nAfter completion of the scripts, each view displays the Validation Results tab, where the results of the test sessions are displayed. DVT stores the results of each test in a _res.xml file in the test session\u2019s configured results directory. The file name takes the form <Detail\u2223Summary>_nnn_<scriptName>_res.xml, where nnn is the session ID from the session properties. Varying the session ID from one test execution to the next allows storage of multiple sets of results in the results directory. Two session Boolean properties define whether summary or detailed results are generated: SUMMARY-VALIDATION-RESULTS and DETAILED-VALIDATION-RESULTS. If the test session\u2019s STORAGE_MODE property is set to as-media or as-dataset, any media files received by DVT during the test are also stored in the results directory, again using the session ID to help uniquely name the files. The generated results files are listed in the session tree under the script name. Selecting one of the results files displays it in the Validation Results tab. The Validation Results tab is a Hypertext Markup Language viewer allowing navigation between the summary results, detailed results, and any generated media files using hypertext links. DVT does not include a DICOM file viewer. To automatically view a generated .dcm file by clicking on a link in the Validation Results tab, a DICOM image viewer must be installed and associated with the .dcm file type.\nThe SCP\u2019s Validation Results tab will show a test result of PASSED, and a .dcm media file is created that contains the Modality Worklist\u2013FIND dataset received in the C-FIND-RQ message sent from the SCU. The SCU\u2019s Validation Results tab also shows a result of FAILED, with nine errors reported. The Summary Results File lists the errors; in this case, three type 1 attributes are missing from each C-FIND-RSP message sent from the SCP. Clicking on a Link to Detailed Result link displays the errors in the context of the complete C-FIND-RSP message. The detailed results file contains the complete contents of each message sent and received in chronological order. Any comment lines in the DICOMScript that begin with ## are copied directly to the detailed results file. This is a good way to insert additional test information directly into the results. For additional troubleshooting output in the Activity Logging tab and detailed results file, the LOG-RELATION, LOG-DEBUG, LOG-DULP-STATE, and PDU-DUMP test session properties can be enabled. In the example described here, it is left as an exercise for the user to add the missing type 1 attributes to the SCP script so that the SCU test session result becomes PASSED.\nDICOMSuperScripts (script files with a .dss extension) enable the reuse of DICOMScripts in various test scenarios. The storage example script sessions included with DVT demonstrate the benefits of DICOMSuperScripts.\nDVTk FOR SERVICE TROUBLESHOOTING\nAs demonstrated in the previous section, DVTk can be a useful tool when troubleshooting modality worklist problems. Another common use case would occur when adding new modalities to a PACS network. It can be frustrating to ensure that AE titles, ports, and IP addresses match between the modality and PACS configuration. This section will illustrate ways in which DVT can be used to troubleshoot a PACS-modality interface problem and describe another, more service-orientated tool available from DVTk.org.\nBecause DVT can act as both an SCP and SCU, it is an excellent starting point for troubleshooting PACS-modality problems. One of the most common and most frustrating failures is when a modality does not send images to the PACS. Most modalities provide little or no error information when image storage problems occur with the PACS. The PACS system is often equally unhelpful. Without an error message of some kind, PACS administrators have no guidance on where to begin in addressing the problem. DVT can simulate the modality or the PACS to pinpoint the source of the problem while obtaining hard evidence that can be used to engage the involvement of additional service layers, such as the hospital networking group or modality vendor. The Emulator_1 test session in the example project that comes with DVT installation can be used to emulate the problem modality. The problem modality\u2019s AE title and PACS connection properties must be copied to the Emulator_1 session\u2019s properties. Although not absolutely necessary, the modality being emulated should be taken off the network to prevent any AE title conflicts during troubleshooting. The first test to perform is a simple Verification to ensure that there is DICOM network communication between the modality AE and the PACS. The test is started by opening the Emulator_1 session\u2019s tree node, right clicking on the Storage SCU Emulator node, and selecting the Execute menu item. All tabs except the Activity Logging tab are hidden, and a dialog box is displayed. Clicking on the Echo button tells DVT to send a C-ECHO message to the SUT. The activity logging tab will display the operations performed. For the purposes of this article, the tests are run against the open-source DICOM PACS DCM4CHEE (http:\/\/www.dcm4che.org, dcm4chee-2.x DICOM Clinical Data Manager system; last accessed May 2007).\nIf the verification test succeeds, then one can assume the patency of DICOM network connectivity between the modality and PACS. The next test is to attempt image storage to the PACS. This test requires a set of DICOM test images, preferably from the modality that is experiencing the problem. The images should contain no names, IDs, or UIDs that will conflict with real-world data. It is important, however, that the data used to perform the test matches the real-world data in structure as closely as possible, including any private data elements the modality creates. A copy of the modality\u2019s DICOM conformance statement comes in handy here. This will help simulate the behavior of the modality as closely as possible. Under the Specify Transfer Syntax (TS) button on the Session Information tab, all transfer syntaxes supported by the modality as a storage SCU can be selected. For example, to test a computed tomography (CT) modality, one would select the CT Image Storage SOP Class (1.2.840.10008.5.1.4.1.1.2). For this test, the storage SOP class and transfer syntax that match the DICOM test images that will be stored to the PACS should be selected. Note that this step is not actually necessary; when emulating a storage SCU, DVT will automatically add to the association negotiation the transfer syntaxes and SOP Classes from the DICOM media files being stored. Selecting the correct transfer syntaxes and SOP Classes is necessary when emulating a storage SCP, however. Executing the Storage SCU Emulator opens the Storage SCU Emulator dialog box. In the dialog box, the Add button is used to add the test images to the list that will be sent to the SCP. Selecting the Validate before export option tells DVT to validate the media files against the corresponding definition file prior to sending them. The number of associations the modality uses when sending multiple images is also specified here (most modalities would send multiple images on a single association). Finally, clicking the Send button will tell DVT to execute the storage test and close the dialog. DVT\u2019s detailed results will show the verification of each media file followed by each storage transaction. The Storage SCU emulator does not automatically do storage commitment as the SCP emulator does. To simulate the modality performing a storage commitment request, one could create a DICOMScript session similar to the Commit_SCU example session, modifying the SOP instance UIDs to match the images stored. This test should be performed immediately after the storage test.\nTo demonstrate some of the more advanced DICOMScript features and the Data Warehouse in DVT, one can imagine a scenario where a CT modality has recently undergone a software upgrade. Since the upgrade, technicians have been reporting that although they can successfully postprocess images on the modality before sending them to the PACS, processing of images retrieved from the PACS back to the modality fails. No errors have been reported by the PACS or modality on storage or retrieval. One possible reason is that the PACS is doing something to the stored images that is preventing the modality from processing them. To evaluate, one can perform what is called a PACS transparency test. The idea is to make what the PACS is doing (if anything) to the images transparent by comparing the before and after images. This test will require a DICOM CT image file from the modality on the DVT workstation and a DVT project with two sessions. A script session playing the role of SCU is used to put the image in the DVT Data Warehouse, store it to the PACS, and initiate a C-MOVE from the PACS back to DVT. An Emulator SCP session is used to receive the image moved back from the PACS and for performing the validation of the received image against the original image in the Data Warehouse.\nThe first commands the script performs are to reset the Data Warehouse and read the test image into the Data Warehouse (Fig.\u00a03). The first READ command loads the DICOM image into the Data Warehouse and uses the value of attribute 0\u00d700080018, the SOP Instance UID, as a reference value. This will allow the SCP Emulator that subsequently receives the image back from the PACS to automatically compare it to the image in the Data Warehouse based on the fact that the SOP Instance UID values are the same. The second READ command loads the same image again into the Data Warehouse but this time references it with CTIMAGE1. This reference will be used to export the image directly from the Data Warehouse to the PACS. As an alternative to READing the image into the Data Warehouse, one could use the CREATE and SET commands to build an image object directly in the Data Warehouse. DVT has the ability to generate pixel data patterns for VRs of type other byte (OB), other float (OF), and other word (OW). For the purposes of this example, it is best to use an image from the problem CT modality. The next operation the script performs is to establish a C-STORE CT Image association with the PACS. After establishing the association, the script creates a C-STORE-RQ command object in the Data Warehouse and exports the CT Image referenced by CTIMAGE1 using the C-STORE-RQ command referenced by CSTOREREQID, over the established association (Fig.\u00a04). After closing the storage association, the script creates a new association on which to perform the C-MOVE, sends the request to move the image from the PACS to the DVT AE, and then closes the association (Fig.\u00a05). Note that the Patient ID (0\u00d700100020) and SOP Instance UID (0\u00d700080018) were manually copied from the CT Image test file. Also, before executing this test, the SCP Emulator must already be started and waiting for the C-STORE from the PACS in response to the C-MOVE request. If the test executes successfully, the statement \u201cReference Dataset with identifier \u20181.3.46.670589.10.900123.19970114.35042000040\u2019 found in Warehouse\u201d should appear in the Activity Log indicating that DVT found the same CT image previously loaded into the Data Warehouse and will use it to perform a comparison of the dataset attributes and values.\nFig\u00a03.PACS transparency test using DICOMScript-reading image into the Data Warehouse.Fig\u00a04.PACS transparency test using DICOMScript-exporting image from the Data Warehouse.Fig\u00a05.PACS transparency test using DICOMScript-moving the image back to DVT.\nThis test requires that the PatientRootQueryRetrieve-MOVE.def definition file is loaded in the script session and the CTImageStorage.def definition file is loaded in the Emulator SCP session. If, as suspected, the PACS is modifying the CT Image object before returning it to the modality, the Emulator SCP results data will identify where these modifications occurred. In this case, the hypothetical tester notices that DVT is indicating that a set of private attributes in the Data Warehouse copy of the image are missing from the image returned from the PACS. Upon further investigation, it turns out that the software upgrade performed on the modality included the addition of a set of new private attributes required by the modality to perform new postprocessing algorithms on the images. The PACS does not support these new private attributes for some reason and is stripping them from the image objects.\nInstead of using a combination of a DICOMScript test session and Emulator test session, this test could have been implemented with a single VBScript session using the HLI library. In this case, separate threads would be created for the SCU and the SCP parts. This approach is left up to the user as an exercise.\nAlthough DVT can analyze all aspects of DICOM interfacing, it tends to be overloaded for field service issues. DVTk.org has a number of service-orientated tools also based on the DVTk framework. One of these is the DICOMSniffer & Analyzer Tool. The tool is a GUI-based network sniffer and DICOM protocol analyzer. This tool uses the WinPcap open-source network library for packet capturing and filtering and includes a user guide. This tool can sniff a live DICOM network stream given two endpoints, such as a modality and PACS. The troubleshooting power of this tool should not be underestimated. It can perform message validation between two devices similar to DVT but does not require replacing an SUT device with the testing tool. Figure\u00a06 shows the tool capturing a modality worklist query\/response communication between two IP addresses.\nFig\u00a06.DICOM Sniffer & Analyzer tool in capturing mode.\nAfter the capture process, the user is presented with a per-association analysis of the DICOM control and datasets that were captured. The Association Overview tab shows the requested and accepted services and each field of the association PDUs. In the Service Elements Overview tab (Fig.\u00a07), one can see all of the DIMSE messages that were transmitted during the association, save a copy, and view the PDUs. The summary and detailed validation results are similar to those produced by DVT, including message validation against SOP Class definition files. The tool also has the option of saving the captured data to a capture file for latter analysis or for sending on to additional service personnel. The DICOM Sniffer & Analyzer is a tool every PACS administrator should have on his or her workstation.\nFig\u00a07.DICOM Sniffer & Analyzer tool in analyzing mode.\nTwo more tools built on the DVTk framework that should be in the imaging professional\u2019s toolbox in working with DICOM on a regular basis are DICOM Compare and DICOM Editor. DICOM Compare can compare the attributes and values of two DICOM files. It can also filter out of the compare process any attributes and sequences to make it easier to identify offending elements. A known \u201cgood\u201d DICOM object can be compared to one causing a problem that was captured from the network with DICOM Sniffer.\nWith the DICOM Editor, one can add\/delete\/modify any attribute or sequence and sequence item and save the modified DICOM file to any location. This tool goes hand-in-hand with the DVT, DICOM Sniffer, and DICOM Compare tools. After those tools have identified the DICOM elements that may be causing a DICOM object storage problem, the editor can be used to manually fix the DICOM object and then resend it to the system where the failure is occurring. If the store succeeds, chances are the source of the problem has been identified.\nDVT AS A DEVELOPMENT TEST TOOL\nAnother use of DVT is automated unit or system testing in a software development environment. Most modern source code management systems include automated build-and-test subsystems. Scripts can be created to test the DICOM interfaces of a vendor\u2019s product, and the command line version of DVT can then be launched to execute those scripts against the product as part of the normal automated build-and-test processes. The DVT results and output files can then be saved along with the rest of the build\/test artifacts. The command line version of DVT can be called on a DICOMScript as follows: dvtcmd Modality_System_Test.ses Modality_System_Test.ds. The summary and detailed results Extensible Markup Language files output by DVT make it easy for an automated test system to determine the results of the test, generate a report, and take any other appropriate action, such as e-mailing the development team.\nA tool as programmable and rich in output as DVT is valuable to the entire iterative development process. Software validation teams can link test session files, scripts, and result files to software defect issues, so that a developer can then use them to reproduce the defect. A validator can subsequently use those same scripts to test the fixed software. Maintaining a repository of DVT scripts is also an excellent way to run regression tests.\nIf the DICOM software being tested is in the .NET language family, tighter testing integration with DVTk can be achieved via NUnit,4 the open-source unit testing framework for .NET. This integration uses a DVTkInvoker class to invoke the command line version of DVT with a session file, script file, and results file as arguments. One can use DVT Clients and Servers within NUnit to handle the various DICOM SOP Classes, starting and stopping DVT as necessary and obtaining validation results information. One future goal is to be able to start up and test an entire workflow scenario from NUnit using DVT.\nDVTk makes it quick and easy to perform repeatable, detailed testing of DICOM interfaces. In the following example, we posit a stress test to run against a storage SCP service. Because this is a stress test, the test application must hit the SCP simultaneously from multiple SCUs, so that multiple threads will be needed. Writing multithreaded Visual Basic (VB) test applications is a breeze with the new HLI library. In fact, it looks quite similar to a DICOMScript, with high-level abstractions for creating and releasing associations and for sending and receiving messages. This example using the HLI requires DVTk alpha version 2.1.007.006 or greater , which can be downloaded from dvtk.org after registering as a Plus User. Plus Users have access to early releases and draft documents, such as the draft version of the HLI API help file.\nIn Visual Studio .NET, a VB console project is created and references to the Dvtk.dll, DvtkData.dll, and DvtkHighLevelInterface.dll libraries are added. This simple stress test application takes three arguments: the number of modalities to create (each modality runs on a separate thread), the number of images each modality will store to the SCP under test, and the location of the DVTk definition files. Figure\u00a08 shows the imports required and the Sub Main(ByVal CmdArgs() As String) routine in module Module1. The first operation this application entry point method performs is to call Dvtk.Setup.Initialize(). This must be performed before any calls to the DVTk libraries, matched by the last call in the application, which should be Dvtk.Setup.Terminate(). Then it creates and initializes the MainDicomThread object, attaches it to the activity logging form, starts the main DICOM thread, and finally waits for the threads to complete. After the threads are complete, it logs selected performance data gathered during the stress test.\nFig\u00a08.Multithreaded stress test using VB.NET-main subroutine and imports.\nFigure\u00a09 shows the MainDicomThread class that inherits from the DvtkHighLevelInterface.Dicom.Threads.DicomThread class. A single instance of this class is started from the entry point method. This class\u2019s thread execute method starts all of the modalities and returns. As it creates each modality (instances of CtStorageScu), it generates unique identifying information for each SCU, such as AE title. Some of the options are analogous to test session properties. This test application could have loaded a script session configuration file and initialized some of the options from it, but in this case, all of the necessary session options are set directly in the code. After an SCU is created, it is immediately started by calling its thread Start() method.\nFig\u00a09.Multithreaded stress test using VB.NET-MainDicomThread class.\nFigure\u00a010 shows a CtStorageScu class that also inherits from DvtkHighLevelInterface.DICOM.Threads.DICOMThread. The purpose of the execute method is to C-STORE a specified number of images to the SCP under test. It first creates unique patient, study, and series level identifiers for the images that will be stored. It then creates the C-STORE CT Image association; then loops to store the images, cloning a CT image dataset that was read from a DICOM CT image file in the constructor; creates unique image identifiers for each image; sends the C-STORE-RQ message; receives the response message; and checks the response status value. Finally, it logs its total storage time for later analysis and closes the association. This application could have been created and executed from the DVT GUI, but developing it in Visual Studio allows the application to be debugged and compiled to an executable file. This application is executed from the command line as: Storage_SCP_Stress_Test.exe 20 1000 \u201cC:\\Program Files\\DVT\\Definitions\u201d.\nFig\u00a010.Multithreaded stress test using VB.NET-CtStorageScu class.\nFigure\u00a011 is an excerpt from the Hl1Form that receives all of the logging from the DicomThread objects. Notice the multithreading capabilities of the HLI in this output. While modality 10 is validating an A-ASSOCIATE-AC message it received, modality 3 is handling a CSTORERSP message. This test created 20 CT modalities that are each simultaneously sending a 1,000-image series to the PACS.\nFig\u00a011.Multithreaded stress test-activity logging.\nRECENT EXTENSIONS AND FUTURE WORK\nDVTk has been extended recently to support HL7 validation in addition to DICOM validation. This is currently used by the DVTk Integrating the Healthcare Enterprise (IHE)5 Actors framework where it is possible to configure DVT to emulate the role of certain actors in the IHE integration profiles. The idea is that the IHE Actors necessary to allow the SUT to be tested are emulated by DVTk. DVTk will then validate the DICOM and HL7 transactions taking place between actors and, in addition, compare the values of certain attributes such as Patient ID, Patient Name, etc., between messages to ensure consistent use.\nIn the future, the aim is to enhance the capabilities of the DVTk IHE Actors framework by supporting additional functionality in the existing actors and supporting other actors needed in the various IHE Integration Profiles. Support for other protocols is also envisioned.\nThe DVTk development team is also involved in the new IHE Connectathon Toolkit (code-named \u201cGazelle\u201d), which is being developed as a successor to the MESA Toolset. It is hoped that the DVTk DICOM validation engine can be wrapped as a Web service for use in Gazelle.\nVarious new tools will be developed using the DVTk frameworks. A GUI for the DVT-IHE framework; various new emulators, such as a radiology information system emulator and modality emulator; and stand-alone validation applications are likely candidates. The current number of development resources limits what can be done\u2014anyone wishing to contribute can do so via the Web site.\nThe aim for future IHE extensions is to integrate any other validation services into the DVTk framework as Web services. This may include Cross-Enterprise Document Sharing validation, for example. It is hoped that the Gazelle cooperation will result in web services that can be reused for this purpose.\nCONCLUSION\nOriginally developed by Agfa and Philips to test their products, DVTk has grown into a professionally managed, open-source, vendor neutral, DICOM Validation Framework. Flexible for the changing standard, programmable, and extensible, DVTk is a powerful tool for anyone working with the DICOM standard. The toolkit includes GUI and command line versions of the main validation application, DVT, and a collection of .NET libraries for creating new validation and test tools. The large collection of example validation sessions that come with the toolkit are a great place to start for understanding how the DICOM standard works in practice.\nHospital IT staff can use DVTk for simple tasks, such as pinging the network for the existence of modality AE titles, or for more detailed troubleshooting, such as checking for the presence of specific DICOM attributes and values in messages and media files. Other DVTk-based tools are available for tasks such as editing or comparing DICOM files or for capturing and analyzing messages on a live DICOM stream. The XML-structured test results and media files created by the validation framework provide great evidence for IT staff when discussing a problem with vendors. Developers and testers can create scripts for testing their products DICOM conformance, or build on the framework by creating new standalone test tools. NUnit integration and the command line version of DVT make it possible to incorporate DVTk and the generated test results into automated build-and-test systems.\nCurrent and future efforts include support for IHE actor validation, including HL7 validation, and new device emulators, such as a Radiology Information System (RIS) emulator. As the digital hospital enterprise continues to grow, DVTk is well positioned to take on these new validation roles.\nWe encourage the reader to continue working with DVTk as a means to master the changing DICOM environment. With the move to an open-source community approach, DVTk is well on its way to becoming the independent gold standard for DICOM interface testing.","keyphrases":["dicom","ihe","systems integration","pacs dicom ihe conformance","health level 7"],"prmu":["P","P","R","R","R"]}
{"id":"Bioprocess_Biosyst_Eng-2-2-1705497","title":"Genetic algorithm for multi-objective experimental optimization\n","text":"A new software tool making use of a genetic algorithm for multi-objective experimental optimization (GAME.opt) was developed based on a strength Pareto evolutionary algorithm. The software deals with high dimensional variable spaces and unknown interactions of design variables. This approach was evaluated by means of multi-objective test problems replacing the experimental results. A default parameter setting is proposed enabling users without expert knowledge to minimize the experimental effort (small population sizes and few generations).\nIntroduction\nIn many technical problems scientists face the problem of identifying optimal process conditions like pH, temperature, concentrations or other variables. A typical example is fermentation medium development. The composition of a fermentation medium consisting of carbon sources, nitrogen sources, mineral salts, trace elements, amino acids and\/or peptides, vitamins and other growth factors determines the chemical and nutritional environment of cells in a bioreactor and is thus vital for the effective manufacturing of bioproducts.\nDue to the large number of process variables and the metabolic complexity of microorganisms or cells methods of experimental design need to be applied in order to identify values of the relevant variables resulting in an improved performance. Considering several objectives to be optimal there is per definition not one optimal solution but a set of efficient solutions. Mathematically such a multi-objective optimization problem (MOP) can be described in terms of Eq. 1. \nThe functional relation between the m design variables and the objective functions \u03c6 does not necessarily need to be known, as y will be obtained from n experimental observations.\nAn important criteria for MOPs is that of Pareto optimality, shortly saying that a member of the efficient set is not dominated by any other. In Fig.\u00a01 the principle of dominance is exemplified in the objective space, where for the case of maximizing two objectives the efficient solutions are indicated.\nFig.\u00a01Efficient points (black) are not dominated by any other point. For a dominated point (white) there exist at least one other point that has greater values in both objectives y1 and y2\nClassical statistical experimental design methods (Plackett\u2013Burman Design, Response Surface Method) have drawbacks like screening for principal components and assuming an unimodal objective function, reviewed in [1].\nMore sophisticated stochastic search strategies like genetic algorithms (GAs) have grown in popularity since Rechenberg [2] and Holland [3] first published their work on this subject. GAs are based on evolutionary principles, encoding several sets of design variables on binary strings (individuals in a population) which are processed by GA operators (crossover and mutation) throughout several generations. The principle \u201csurvival of the fittest\u201d assures a convergence towards optimal values in the design variables with proceeding generations.\nThe first GA dealing with multiple objectives was the Vector Evaluated Genetic Algorithm (VEGA) proposed by Schaffer [4]. This multi-objective optimization strategy has already been applied successfully for experimental medium optimization in many cases [1]. The most recent published multi-objective GAs are the Non-Dominated Sorting Genetic Algorithm-II [5] and the Strength Pareto Evolutionary Algorithm (SPEA) [6]. The SPEAs main feature is processing two populations: besides a normal population an external population serves as a kind of archive, keeping track of the efficient set. Especially in the case of experimental design with a limited number of experiments the SPEA is supposed to have advantages compared to VEGA with respect to the experimental effort.\nThe new software tool with a genetic algorithm for multi-objective experimental optimization making use of SPEA will be outlined. The performance of GAME.opt will be evaluated with the help of special test-functions to achieve appropriate parameter settings for experimental design.\nMethods\nGAME.opt was developed with LabVIEW 7 (National Instruments). The Application Builder was used to create a stand alone application running under Windows platforms. Data handling is based on spreadsheet files, which can be accessed via a text editor or Microsoft Excel. GAME.opt will be made available on request by the Technical University of Munich.\nAs the core algorithm is based on the SPEA, fitness assignment, selection and clustering is described in detail by Zitzler and Thiele [6]. To abstract the algorithm: fitness assignment is either based on the strength of an individuals dominance or on the degree an individual is dominated by others. A mating pool equal to the population size is filled by selecting individuals using binary tournament without replacement [7]. Crossover is performed between two parental binary strings from the mating pool at the specified number of crossover points in order to create two off-springs. Each bit of these new strings is inverted with a probability which is defined by the mutation rate. Clustering is used in order to bound the external population to a maximal value Nextern. Each time the external population exceeds this value it is pruned to a size of Ncluster by means of average clustering.\nThe first step when using GAME.opt is to create a new project where all information on the experimental design is defined. For this purpose the user is led through the steps explained together with some recommendations in the following.\nDesign variables\nThe design variables considered in the problem (x in Eq. 1) need to be defined initially. Alternatively to choosing the amount of bits encoding a variable GAME.opt gives the opportunity to choose the amount of levels, resulting from the variable\u2019s bounds and its increment. The choice of levels can be necessary for practical purposes but causes problems in the coding of a decision variable as follows. A general decoding function linearly transforms an n-bit-long binary string (a1...an) into a bounded real number x\u00a0\u2208\u00a0[xL, xU]: where a1 is the most significant bit and an is the least significant bit [8]. This kind of transformation is just unique if the amount of levels equals (2n\u00a0\u2212\u00a01). In other words, like its natural paradigm, the binary code is degenerated if there are more bit combinations than levels (the opposite case will not occur as the program computes the minimal amount of required bits).\nConsidering the above mentioned the following points are important when defining the design variables:If the experimental setup prevents an adequate choice of levels the coding can be made visible in GAME.opt. In order to assure equal processing of all variables the amount of levels among the variables should not vary too much.From the amount of levels results the length of the binary string that is needed to encode the design variables, which is shown in GAME.opt in terms of bits for each variable. From a set of M binary strings with the length l each point in the search space (X in Eq. 1) can be reached with a probability p. The relation is given in Eq. 3 and GAME.opt will display p when selecting the number of individuals (M) [9].The value of p should be higher than 0.99.As each individual corresponds to one experiment, the experimental effort will be correlated to the chosen amount of levels.The increment should be higher than the possible precision in the experiment.\nExperimental results\nThe experimental observations y have to be specified in GAME.opt just in terms of their names and units. The following items should be considered when planning the experiments:All parameters different from the design variables have to remain fixed in all experiments throughout the optimization procedure.The experimental error should be lower than the differences that are expected to result from different levels in the design variables.\nObjective functions\nThe objective functions in GAME.opt can be any linear combination of the experimental results and the decision variables. GAME.opt will maximize all objective values. If for example one objective is minimizing a certain experimental result, the objective function is \u20131 times the according result. When defining the objective functions it is important to make sure that they are independent from each other. For example it is no use maximizing simultaneously the amount of a reaction\u2019s product and minimizing the amount of non-converted reactants at the end of a batch process, because a high product concentration is correlated to low resting concentrations of reactants. A reasonable combination is for example maximization of an experimental result while minimizing the sum of decision variables, possibly reducing the costs of a process.\nOptimization procedure\nOnce the MOP is defined by the above mentioned steps a random generated initial population is available. This set of design variables can be exported from GAME.opt and the according experiments are performed. After the experimental results are entered in GAME.opt the next population is generated and checked for individuals that have already appeared in order to prevent repeated experiments. This iterative process is performed until satisfying results are obtained. The external population contains the efficient set recovered so far and thus constitutes the result of the optimization process. The experimental results in the external population are already assigned because their members originate from former populations.\nResults\nA previous version of GAME.opt was used for experimental design for media optimization (13 medium components, 20 individuals\/generation, 91\u00a0bits\/binary string, eight generations) and gave comparable results to another GA making use of VEGA with half the number of experiments [10]. Further process optimizations considering media composition together with process parameters are currently performed in our laboratory. In order to evaluate the software for a proper processing and its general ability to solve MOPs it was tested by means of several multi-objective test problems in such a way that the experimental results are replaced by evaluation of mathematical functions. An exemplary MOP with three objectives and six design variables is given in Eq. 4. This MOP is based on Deb et al. [11], where the development of test problems is described. The MOP in Eq. 4 was solved with GAME.opt, therefore the six design variables have been defined in the program together with the bounds, being 0 and 1 for each variable. As mentioned above the choice of the increment and thus the amount of levels is crucial for a proper processing of the variables. An increment of 0.1 will give 11 Levels, namely [0, 0.1, 0.2, ..., 1] encoded with 4\u00a0bits giving 16 levels. According to Eq. 2 an adequate increment for a given number of bits n results from: \nTable\u00a01 confronts a proper coding with 4\u00a0bits with a degenerated coding for a variable range from 0 to 1.\nTable\u00a01Four bit binary coding of the design variables in Eq. 4 for different increments (incr)Binary codeDegenerated codingDecoded value (incr\u00a0=\u00a00.1)Proper codingDecoded value (incr\u00a0=\u00a01\/15)0 0 0 0000 0 0 10.11\/150 0 1 00.12\/150 0 1 10.23\/150 1 0 00.34\/150 1 0 10.35\/150 1 1 00.46\/150 1 1 10.57\/151 0 0 00.58\/151 0 0 10.69\/151 0 1 00.710\/151 0 1 10.711\/151 1 0 00.812\/151 1 0 10.913\/151 1 1 00.914\/151 1 1 111\nUsing the proper coding shown in Table\u00a01 the GA was run eight generations with 20 individuals in the normal population and 10 individuals in the external population. Two crossover points and a mutation probability of 1% was chosen what has been identified as a reasonable default setting in other problems. As a rule of thumb the size of the external population should be half of the normal population, for which a minimal number can be estimated by Eq. 3. Table\u00a02 summarizes the default parameter setting.\nTable\u00a02Default parameter setting for the genetic algorithmParameterValueCrossover points2Mutation rate1%Individuals in normal population (N)Eq. 3Individuals in external population (Nextern)N\/2Individuals in external population after cluster analysis (Ncluster)N\/2\nThe results are shown in Fig.\u00a02, by means of the objective values f1, f2, f3 of the ten individuals in the external population after eight generations.\nFig.\u00a02The objective space of the test problem lies between the surfaces. Optimal solutions are located on the unit sphere in the first quadrant. The black dots indicate the efficient set after eight generations of GAME.opt (external population). The two views of the plot point out the closeness of the efficient set to the Pareto front (a) and its equal distribution (b)\nThe two spherical surfaces in Fig.\u00a02 constitute the objective space of Eq. 4.\nFor the minimization problem the first quadrant of the unit sphere constitutes the Pareto optimal frontier where g(x)\u00a0=\u00a00 and it is already approximated well by the ten members of an external population after eight generations (Fig.\u00a02a). Beside the closeness to the Pareto frontier the distribution of the solutions is an important performance criteria. Figure\u00a02b reveals the equal distribution of the ten solutions resulting amongst other things from the cluster analysis. Optimal solutions are characterized by xi\u00a0=\u00a00.5 for i\u00a0=\u00a01, 2, 3, 4, whereas x5 and x6 can take any value in the given range. Splitting the design variables in such a way allows to test the algorithm for a crucial feature of GAs, the populations diversity. On the one hand the algorithm is expected to converge fast to the optimal solutions but on the other the populations diversity must be high enough to ensure exploration of the whole Pareto optimal frontier. This fact is reflected in the box plots of the initial and the eighth generation together with their corresponding external populations in Fig.\u00a03.\nFig.\u00a03Box plot of the initial population (a), the first external generation (b), the eighth generation (c) and the eighth external generation (d). The boxes contain the middle of 50% of the data. The line in the box indicates the median. The bold dashed line separates variables with an optimal solution (x1, x2, x3, x4\u00a0=\u00a00.5) from variables without optimal value (x5, x6)\nInitially the individuals are randomly distributed, what is reflected by the box plots of the six design variables in Fig.\u00a03a. A trend towards 0.5 is already visible in Fig.\u00a03b where box plots are shown for the first external population. In Fig.\u00a03c, d, depicting the box plots of the corresponding populations after eight generations, a clear distinction between the variables with optimal value and those without can be made. After eight generations the diversity of x5 and x6 is still high in both populations. The remaining variables show the expected divergence towards 0.5 in the external population but remain in parts distributed over a wider range in the normal population. From this result it gets obvious that GAME.opt performs well with a small amount of individuals and a short evolution on a MOP considered a benchmark of evolutionary computing.\nConclusions\nIn order to keep the experimental effort low, GAME.opt will use small population sizes and fewer generations compared to other GA applications, where the amount of objective function evaluations is mostly limited by the computational costs. For this reason the external population is expected to be only a rough approximation of the true Pareto optimal frontier. Nevertheless in all test problems a satisfying performance of GAME.opt was achieved in spite of the relatively low number of experiments (160 compared to 166 possible design variable combinations with the test problem presented).\nIn the near future, efficient parallel search strategies for experimental design are becoming more and more important in bioprocess optimization due to the availability of new parallel stirred-tank bioreactor technologies [12].","keyphrases":["genetic algorithm","software tool","experimental design","multi-objective optimization"],"prmu":["P","P","P","P"]}
{"id":"Apoptosis-4-1-2423418","title":"Hypertonicity-induced cation channels rescue cells from staurosporine-elicited apoptosis\n","text":"Cell shrinkage is one of the earliest events during apoptosis. Cell shrinkage also occurs upon hypertonic stress, and previous work has shown that hypertonicity-induced cation channels (HICCs) underlie a highly efficient mechanism of recovery from cell shrinkage, called the regulatory volume increase (RVI), in many cell types. Here, the effects of HICC activation on staurosporine-induced apoptotic volume decrease (AVD) and apoptosis were studied in HeLa cells by means of electronic cell sizing and whole-cell patch-clamp recording. It was found that hypertonic stress reduces staurosporine-induced AVD and cell death (associated with caspase-3\/7 activation and DNA fragmentation), and that this effect was actually due to activation of the HICC. On the other hand, staurosporine was found to significantly reduce osmotic HICC activation. It is concluded that AVD and RVI reflect two fundamentally distinct functional modes in terms of the activity and role of the HICC, in a shrunken cell. Our results also demonstrate, for the first time, the ability of the HICC to rescue cells from the process of programmed cell death.\nIntroduction\nIt has become increasingly evident in recent years that the mechanisms of cell volume regulation are employed in a variety of physiological processes, in addition to just the maintenance of cell homeostasis. Among these processes are the coordination of transport across the apical and basolateral membranes in epithelia, the locomotion of cells, the regulation of metabolic processes in the liver and, most notably, the control of cell proliferation and apoptosis [1\u20133].\nSince the rates of ion transport through channels are some 4\u20135 orders of magnitude higher than those achieved by transporters (carriers and pumps), any modulation of ion channel activity may serve as a rapid and efficient mechanism of osmolyte transport and cell volume regulation. Accordingly, hypertonicity-induced cation channels (HICCs) were found to be the main mediators of the \u201cregulatory volume increase (RVI)\u201d of a shrunken cell, whenever a system was analysed in a quantitative fashion [3\u20135]. Despite the low selectivity of HICCs for Na+ over K+, activation of these channels leads to an actual net uptake of cations, which is mainly due to the membrane voltage, which is negative intracellularly. Anions and osmotically obliged water follow, allowing the cell to complete the process of RVI [3\u20135].\nPersistent isotonic shrinkage of a given cell, as opposed to hypertonicity-induced shrinkage, is one of the hallmarks of apoptosis. In many instances, this \u201capoptotic volume decrease (AVD)\u201d employs the activation of K+ channels, which serve as the main routes of K+ exit [6\u20139]. Cl\u2212 channels also should open, with this parallel transport strongly depending, however, on whether or not the electro-chemical driving forces actually favour conductive Cl\u2212 export [1, 2, 10\u201312].\nThe question is thus raised of how hypertonic stress might interfere with AVD. Would osmotic shrinkage facilitate the process? Or would the activation of HICCs or RVI oppose the induction of apoptosis?\nMaterials and methods\nCell culture\nHuman cervix HeLa cells were grown as monolayers in minimum essential medium supplemented with 10% fetal bovine serum, 100\u00a0\u03bcg\/ml streptomycin and 40\u00a0IU\/ml penicillin, under 95% air\/5% CO2 at 37\u00b0C. For the experiments, cells were mechanically detached from the plastic substrate of the culture flasks with a jet of culture medium and then kept in suspension for 15\u2013120\u00a0min.\nDetermination of cell volume\nChanges of HeLa cell volume were quantified by means of electronic cell sizing on a Coulter-type analyzer (CDA-500; Sysmex, Kobe, Japan), as previously described [13]. The experimental solution contained (in mM): NaCl, 95; KCl, 4.5; MgCl2, 1; CaCl2, 1; NaHCO3, 10; HEPES, 5. Osmolality was adjusted to values from 300 to 600 mosmol\/kg-H2O by adding mannitol under osmometric control (OM802; Vogel, Giessen, Germany). Measurements were performed at room temperature.\nPatch-clamp experiments\nMembrane currents were recorded in the fast whole-cell mode of the patch-clamp technique using 2\u00a0M\u03a9 borosilicate pipettes. Currents were recorded with an Axopatch 200B amplifier (Molecular Devices, Union City, CA), filtered at 5\u00a0kHz with a four-pole Bessel filter and digitized at 20\u00a0kHz. pClamp 9.02 software (Molecular Devices) was used for control of the pulse protocol, as well as for data acquisition and analysis. Series resistance was <5\u00a0M\u03a9 and was compensated (by 70\u201380%) to minimize voltage errors. In the experiments, voltage ramps from \u221280 to +20\u00a0mV and of 1\u00a0s duration were applied every 10\u00a0s; holding voltage was \u221230\u00a0mV.\nThe bath solution (pH 7.5) contained (in mM): NaCl, 94; Na-gluconate, 6; MgCl2, 1; CaCl2, 2; TEA-Cl, 2; HEPES, 10; d-glucose, 10. Osmolality was adjusted to 310 (isotonic) and 400\u00a0mosmol\/kg-H2O (hypertonic) by the addition of mannitol.\nThe pipette solution (pH 7.3, 300\u00a0mosmol\/kg-H2O) contained (in mmol\/l): NaCl, 26; Na-gluconate, 69; MgCl2, 1; TEA-Cl, 2; Na2-ATP, 2; Na2-GTP, 0.5; HEPES, 10; EGTA, 1. With these ion gradients, the equilibrium potentials for Na+ and Cl\u2212 (as the only permeant ions) are set at 0 and \u221230\u00a0mV, respectively [13]. Experiments were performed at room temperature.\nMonitoring apoptotic cell death\nCell viability and caspase-3\/7 activity were determined by use of a calorimetric MTT assay (Cell Counting Kit-8; Dojindo, Kumamoto, Japan) and a fluorometric probe (Apo-ONE Homogeneous Caspase-3\/7 Assay; Promega, Madison, WI), respectively.\nFragmentation of DNA was determined by means of a photometric enzyme immunoassay (Cell Death Detection ELISAPLUS, Roche Applied Science, Diagnostics, Mannheim, Germany) with HeLa cells plated on 96-well plates. Following exposure to the various experimental conditions, cells were lysed and centrifuged at 200g. The cytoplasmic (DNA containing) fraction was then transferred to streptavidin-coated microtiter plates that had been incubated with a biotinylated monoclonal anti-histone antibody. Finally, the amount of the fragmented DNA on bound nucleosomes was determined with a peroxidase-conjugated monoclonal anti-DNA antibody and the substrate ABTS [2,2\u2032-azino-di-(3-ethylbenzthiazoline sulfonate)], which was quantified photometrically at 405\u00a0nm. In all measurements, cells were incubated for 6\u00a0h at 37\u00b0C.\nTesting for necrosis and late apoptosis\nNecrosis and late apoptosis of cells were quantified fluorometrically on the basis of a propidium iodide staining of nuclei (excitation\/emission at 530\u00a0nm\/620\u00a0nm) performed with reference to an overall staining of cells by Hoechst 33342 (excitation\/emission at 350\u00a0nm\/460\u00a0nm).\nChemicals\nAll reagents were purchased from Sigma\u2013Aldrich (Tokyo, Japan).\nStatistical analysis\nData are presented as mean values\u00a0\u00b1\u00a0SEM with n denoting the number of observations. Student\u2019s t tests for paired and unpaired data were used as appropriate and P\u00a0<\u00a00.05 was considered significant.\nResults and discussion\nAs was determined by means of electronic cell sizing, increasing osmolality from 300\u00a0mosmol\/kg-H2O to 330, 400, 500 and 600\u00a0mosmol\/kg-H2O led to an initial shrinkage of HeLa cells to 96.6\u00a0\u00b1\u00a00.4, 82.6\u00a0\u00b1\u00a01.9, 72.5\u00a0\u00b1\u00a00.2 and 66.2\u00a0\u00b1\u00a00.6% of the control level, respectively (n\u00a0=\u00a010; Fig.\u00a01a). When plotted as a function of 1\/osmolality (which is equivalent to a \u2018Boyle-van\u2019t Hoff graph\u2019 [3, 14]), these data could readily be fitted by a straight line; its intercept with the y-axis yields the osmotically active versus inactive space of the cells, with the latter amounting to 30.6% of total cell volume (Fig.\u00a01a, Insert). This osmometric behaviour of HeLa cells was then followed by an active volume recovery to 101.7\u00a0\u00b1\u00a00.4, 92.6\u00a0\u00b1\u00a00.5, 89.7\u00a0\u00b1\u00a00.8 and 88.4\u00a0\u00b1\u00a00.9%, in 120\u00a0min of hypertonic stress (Fig.\u00a01a, b). The process of \u201cregulatory volume increase (RVI)\u201d occurred at essentially constant rates that equalled 0.4\u00a0\u00b1\u00a00.2, 0.9\u00a0\u00b1\u00a00.2, 1.6\u00a0\u00b1\u00a00.2 and 1.8\u00a0\u00b1\u00a00.2%\/10\u00a0min, respectively, in the range of 5\u2013120\u00a0min after application of hypertonicity.\nFig.\u00a01Hypertonicity-induced HeLa cell shrinkage and RVI determined by electronic cell sizing. (a) The volume response of cells to increases in osmolality (from 300\u00a0mosmol\/kg-H2O) to 330, 400, 500 and 600\u00a0mosmol\/kg-H2O at time zero (arrow) are depicted. The insert gives the initial (passive) amount of cell shrinkage as a function of 1\/osmolality (equivalent to a \u2018Boyle-van\u2019t Hoff plot\u2019 of these data; see text for details). 3.33\u00a0kg-H2O\/osmol\u00a0=\u00a01\/(0.300\u00a0osmol\/kg-H2O) is the 100% value under isotonic conditions. (b) The volume of HeLa cells at 120\u00a0min of various levels of hypertonic stress, after partial volume recovery (RVI) had been achieved. n\u00a0=\u00a010 for each experimental condition\nIt has become increasingly evident in recent years that persistent cell shrinkage is one of the hallmarks of apoptosis [6, 15]. Also, it has recently been found that after 2-h stimulation with staurosporine (STS), TNF\u03b1 or Fas ligand, HeLa cells lose their ability to undergo RVI (12). To test whether HeLa cell shrinkage and\/or RVI interferes with apoptotic volume decrease (AVD), 4\u00a0\u03bcM STS was used to induce apoptosis in HeLa cells [6], and a series of volume measurements was made. This was done under isotonic conditions (300\u00a0mosmol\/kg-H2O) and under hypertonic conditions in which osmolality was increased to 330, 400, 500 and 600\u00a0mosmol\/kg-H2O (n\u00a0=\u00a010). Within an experimental time frame of 120\u00a0min, STS was found to reduce cell volume to 65.8\u00a0\u00b1\u00a01.6% that of the control, under isotonic conditions (Fig.\u00a02a: diamonds). 5\u00a0min after osmolality was increased to 330, 400, 500 and 600\u00a0mosmol\/kg-H2O, cell volume equalled 89.6\u00a0\u00b1\u00a00.7, 81.4\u00a0\u00b1\u00a00.2, 71.4\u00a0\u00b1\u00a00.9 and 64.5\u00a0\u00b1\u00a00.8% of control, respectively. These values are virtually identical to the values of purely passive or physical responses to hypertonic stimulation in the absence of STS (cf. to Fig.\u00a01a). Moreover, an osmotically inactive space of 35.4% could be calculated from these measurements; this value is very similar to that obtained in the control experiments (see above). Of note, no RVI followed hypertonicity-induced cell shrinkage when STS was present. This indicates that the RVI response was completely inhibited by STS (or surpassed by the STS-induced AVD process), a phenomenon which clearly holds for the full 2\u00a0h period after STS application. Using the respective values for volume at 5\u00a0min as passive reference values, AVD was computed to be 30.8\u00a0\u00b1\u00a00.7, 22.2\u00a0\u00b1\u00a00.7, 12.1\u00a0\u00b1\u00a00.5 and 7.2\u00a0\u00b1\u00a00.7% for 330, 400, 500 and 600\u00a0mosmol\/kg-H2O, respectively (calculated as the volume at 5\u00a0min minus the volume at 120\u00a0min). So as summarized in Fig.\u00a02b, a decrease of AVD was observed with increasing hypertonic stress. On the one hand, then, hypertonic stress appears to suppress STS-induced AVD; on the other hand, STS seems to abolish hypertonicity-induced RVI. Also of note, in this respect, is a recent study on HeLa cells in which tyrosine kinases, PKC, and p38 MAPK were identified as part of the signalling machinery employed in the activation of HICC and RVI [16]. Given the non-specificity of STS with respect to its inhibition of various protein kinases, it is not surprising that RVI is inhibited in the presence of the compound, while AVD is induced.\nFig.\u00a02Effects of hypertonic stress on apoptotic volume decrease (AVD). (a) At time zero (arrow), 4\u00a0\u03bcM staurosporine (STS) was added to induce apoptotic shrinkage of HeLa cells under isotonic conditions (\u25c6) or together with an increase of osmolality to 330 (\u25a0), 400 (\u25b2), 500 (\u25cf) and 600 (\u25bc) mosmol\/kg-H2O. Hypertonic cell shrinkage, at 5\u00a0min, was very similar in the presence and absence (see Fig.\u00a01) of STS. (b) AVD under isotonic conditions (volume at 0\u00a0min minus volume at 120\u00a0min) and hypertonic conditions (volume at 5\u00a0min minus volume at 120\u00a0min). n\u00a0=\u00a010 for each experimental condition\nTo study the interference of STS-induced AVD with hypertonicity-induced RVI in more detail, experiments were performed in which osmolality was increased from 300 to 500\u00a0mosmol\/kg-H2O, at time zero (see Fig.\u00a03a: arrow). In addition, 4\u00a0\u03bcM STS was applied either 2\u00a0h before (t\u00a0=\u00a0\u2212120\u00a0min), 1\u00a0h before (t\u00a0=\u00a0\u221260\u00a0min), at the same time as (t\u00a0=\u00a00) or 1\u00a0h after (t\u00a0=\u00a060\u00a0min) hypertonic stimulation (see Fig.\u00a03a: arrowheads). AVD was then computed from the overall changes in cell volume (i.e. the volume at 0, \u221260 or \u2212120\u00a0min, respectively, minus the volume at 120\u00a0min) minus the amount of passive cell shrinkage (i.e. the volume at 0\u00a0min minus the volume at 5\u00a0min). Hypertonic stress (500\u00a0mosmol\/kg-H2O) significantly reduced AVD from the control value of 34.2\u00a0\u00b1\u00a00.5% (Fig.\u00a02b: 300; also marked as STS-ISO, in Fig.\u00a03b) to 12.1\u00a0\u00b1\u00a00.5 or 12.5\u00a0\u00b1\u00a00.5% (Fig.\u00a03b: STS-HYPER or HYPER\/1hSTS) when given simultaneously with, or 1\u00a0h before, application of STS (n\u00a0=\u00a010, P\u00a0<\u00a00.001 each), respectively. Also apparent from these experiments is that no RVI occurred in the presence of STS. It is noteworthy that increasing the osmolality to 500\u00a0mosmol\/kg-H2O after 1-h pretreatment with STS still led to a markedly reduced AVD of 25.9\u00a0\u00b1\u00a00.7% (P\u00a0<\u00a00.001; Fig.\u00a03b: STS1h\/HYPER). A hypertonic stimulation after 2-h pretreatment with STS, however, was not effective at suppressing AVD (Fig.\u00a03b: STS2h\/HYPER). These results suggest that hypertonic stimulation of HeLa cells is somehow interfering with the process of AVD when applied in the time range of 1\u00a0h before to 1\u00a0h after the application of STS.\nFig.\u00a03Interference of AVD with RVI. (a) Osmolality was increased from 300 to 500\u00a0mosmol\/kg-H2O at time zero (arrow), with 4\u00a0\u03bcM STS applied (at arrowheads) either 2\u00a0h before (STS2h\/HYPER: \u25a0), 1\u00a0h before (STS1h\/HYPER: \u25cf), at the same time as (STS-HYPER:\u25bc) or 1\u00a0h after (HYPER\/1hSTS: \u25b2) the start of hypertonic stress. (b) Total AVD computed from the overall volume changes shown in (a) minus the initial amount of passive cell shrinkage in the first 5\u00a0min of hypertonic stress (i.e. volume at 0\u00a0min minus volume at 5\u00a0min). STS-ISO refers to the AVD observed under isotonic conditions. n\u00a0=\u00a010 for each experimental series\nNext, the effects of STS on activation of the HICC were tested. This was done by increasing osmolality at various times relative to STS application. As shown in Fig.\u00a04 (STS-HYPER), STS had no significant short-term effect on activation of the HICC when given together with hypertonic stress, i.e. the peak activation of HICC current was virtually unaffected for 5\u20137\u00a0min by STS applied simultaneously with hypertonic stress. This finding fits with the observation that HICC activation appears to occur on a considerably faster time scale than the initiation of AVD does. In this respect, it is also of note that even with STS applied 1\u00a0h ahead of hypertonicity (STS1h\/HYPER), the HICC was still activated to a level approximately half of the control value, i.e. to 1.59\u00a0\u00b1\u00a00.39 pA\/pF. With STS given 2\u00a0h before hypertonic stress (STS2h\/HYPER), HICC currents were almost completely inhibited (0.62\u00a0\u00b1\u00a00.20\u00a0pA\/pF, n\u00a0=\u00a05\u201333).\nFig.\u00a04Effects of STS on HICC activation. (a) HICC currents in the absence (Control) or presence of STS, which was applied simultaneously with hypertonic stress (STS-HYPER), 1\u00a0h ahead of hypertonic stress (STS1h\/HYPER) or 2\u00a0h ahead of hypertonic stress (STS2h\/HYPER). Representative traces of 5 to 33 experiments. (b) Typical I\/V-relationships under these conditions. (c) Summary of HICC current densities obtained under the four different conditions shown in (a)\nWhenever studied in a quantitative way, HICCs have been proven to be the main mechanism of the RVI of a given system [3\u20135]. This also holds for HeLa cells, in which the relative contribution of HICC activity and Na+\/H+ antiport to RVI is approximately 2:1 (with no detectable contribution of Na+-K+-2Cl\u2212 symport at all) [5, 13]. This raises the question of whether the apparent interference of hypertonic stress with AVD may occur via activation of the HICC. In the experiments shown in Fig.\u00a05a, osmolality was increased from 300 to 500\u00a0mosmol\/kg-H2O, at time zero (arrow). Following 60\u00a0min of hypertonic stress, 300\u00a0\u03bcM flufenamic acid (FFA, an established blocker of the HICC in HeLa cells [4, 5, 13]), STS (4\u00a0\u03bcM) or FFA plus STS were then applied (arrowhead). As expected, FFA significantly blunted the progress of RVI, presumably by inhibition of the HICC. The volume change from 60 to 120\u00a0min (volume at 120\u00a0min minus volume at 60\u00a0min) was \u22122.0\u00a0\u00b1\u00a00.6% in the presence of FFA; in the control experiments, on the other hand, the 60-to-120\u00a0min volume change due to RVI equalled 10.2\u00a0\u00b1\u00a00.9% (n\u00a0=\u00a010, P\u00a0<\u00a00.001; Fig.\u00a05b: Control). Of note, STS led to a pronounced decrease in cell volume of \u221212.5\u00a0\u00b1\u00a01.6% (Fig.\u00a05b: STS), and the effects of FFA plus STS were additive, resulting in a decrease in cell volume of \u221219.0\u00a0\u00b1\u00a01.0% (P\u00a0<\u00a00.01; Fig.\u00a05b: STS+FFA). Experiments with 1\u00a0mM 2-aminoethoxydiphenyl borate (2-APB) as an alternative blocker of the HICC [17] yielded virtually identical results. With 2-APB, the 60-to-120\u00a0min volume change equalled 2.9\u00a0\u00b1\u00a01.1% (Fig.\u00a05b: 2-APB), and again, the effects of HICC blockage and STS (volume change of \u221216.3\u00a0\u00b1\u00a00.6%; P\u00a0<\u00a00.05) were additive (Fig.\u00a05b: STS+2-APB).\nFig.\u00a05Effects of HICC channel blockers and STS on RVI and AVD. (a) Osmolality was increased from 300 to 500\u00a0mosmol\/kg-H2O at time zero (arrow) for all groups. 300\u00a0\u03bcM flufenamic acid (FFA), STS or FFA plus STS were applied at t\u00a0=\u00a060\u00a0min of hypertonic stress (arrowhead) as indicated. (b) Summary of the volume changes from 60 to 120\u00a0min (volume at 120\u00a0min minus volume at 60\u00a0min) observed in the experiments shown in (a) and two additional experiments in which 1\u00a0mM 2-aminoethoxydiphenyl borate (2-APB) was used as a blocker of HICC channels, alone or in combination with STS. n\u00a0=\u00a010 for each experimental condition. ***\u00a0Significantly different from Control with P\u00a0<\u00a00.001; #,##\u00a0significantly different from STS with P\u00a0<\u00a00.05 and P\u00a0<\u00a00.01, respectively\nIt may be argued that, due to the limited specificity of FFA and 2-APB, the effects of both compounds described above may be due to an inhibition of volume-sensitive outwardly rectifying (VSOR) Cl\u2212 channels (see [2, 18] for review), rather than blockage of the HICC. In an additional series of measurements, however, it was found that even with application of 10\u00a0\u03bcM DCPIB (4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid), an effective blocker of the VSOR Cl\u2212 channel [19], an RVI value of 10.6\u00a0\u00b1\u00a00.6% (when added at t\u00a0=\u00a060\u00a0min; n\u00a0=\u00a010) was obtained; this is virtually identical to the value of 10.2\u00a0\u00b1\u00a00.9% observed under control conditions (see above). Furthermore, 10\u00a0\u03bcM DCPIB inhibited VSOR currents in HeLa cells by 67.6\u00a0\u00b1\u00a03.6% (n\u00a0=\u00a06) whereas it did not exhibit an effect on HICC currents at all (n\u00a0=\u00a08; Wehner and Numata, unpublished results).\nThese data strongly imply that, even in early-stage apoptosis, HICC activation is an effective mechanism of RVI in HeLa cells. Furthermore, they provide additional strong evidence for the functional interplay of AVD and RVI. In this regard, the apparent lack of RVI in HeLa cells undergoing AVD (Fig.\u00a02a) could be accounted for as follows: In early-stage apoptosis (say, within 1\u00a0h after STS application) the AVD process surpasses the RVI process, which involves HICC activity, whereas in the later stage of apoptosis, the RVI process is suppressed because of inhibition of the HICC by STS.\nFinally, we characterized the interplay between HICC activation and STS-induced cell death and apoptosis. As determined by an MTT assay (Fig.\u00a06a), a caspase-3\/7 activity assay (Fig.\u00a06b), and an apoptosis assay measuring nucleosome-bound DNA (Fig.\u00a06c), increasing osmolality from 300\u00a0mosmol\/kg-H2O to 400, 500, and 600\u00a0mosmol\/kg-H2O led to significant and parallel reductions of both cell death and apoptosis observed 6\u00a0h after STS application; the maximal effect was observed at 500\u00a0mosmol\/kg-H2O and above. This is a surprising finding because, based on what has been reported so far concerning the interplay of cell volume and apoptosis, one might expect a synergistic effect of cell shrinkage on the induction of programmed cell death [15]. Furthermore, FFA and 2-APB almost completely blocked these effects, the fact providing strong further evidence that it is the activation of the HICC that is actually rescuing HeLa cells from apoptosis.\nFig.\u00a06Rescue from STS-induced apoptosis by hypertonic stimulation and inhibition of rescue by HICC blockers. (a) Cell death observed 6\u00a0h after STS treatment at various osmolalities, and at 500\u00a0mosmol\/kg-H2O with 300\u00a0\u03bcM FFA or 1\u00a0mM 2-APB as indicated (n\u00a0=\u00a012\u201334). (b) Apoptosis monitored as caspase-3\/7 activity induced by 6-h exposure to STS at various osmolalities, and when FFA or 2-APB was also applied, at 500\u00a0mosmol\/kg-H2O (n\u00a0=\u00a012\u201326). (c) DNA fragmentation analyzed with a nucleosomal ELISA assay after a 6-h exposure to STS at various osmolalities, and when FFA or 2-APB was also applied, at 500\u00a0mosmol\/kg-H2O (n\u00a0=\u00a06)\nTo insure that the above measurements were not corrupted by the necrosis of cells, propidium iodide staining was performed. Propidium iodide is known to selectively bind to cell nuclei, and because it does not permeate intact cell membranes, it provides a reliable measure for the necrosis and late apoptosis of cells. Following a 6-h incubation, the percentages of necrotic cells were 0.1\u00a0\u00b1\u00a00.1% (for 300\u00a0mosmol\/kg-H2O), 1.8\u00a0\u00b1\u00a00.3% (for 300\u00a0mosmol\/kg-H2O\u2014STS), 0.8\u00a0\u00b1\u00a00.1% (for 400\u00a0mosmol\/kg-H2O\u2014STS), 0.6\u00a0\u00b1\u00a00.1% (for 500\u00a0mosmol\/kg-H2O\u2014STS), and 0.5\u00a0\u00b1\u00a00.1% (for 600\u00a0mosmol\/kg-H2O\u2014STS; n\u00a0=\u00a08\u201310, for each condition). It is concluded from these experiments that STS, at 4\u00a0\u03bcM, is an effective and reliable tool for the induction of apoptosis in HeLa cells, as reported also in an earlier study from this laboratory [6].\nIt may be argued that hypertonic stress (and HICC activation) just delays, rather than prevents, STS-induced cell death in HeLa cells. As propidium iodide staining after 24\u00a0h of STS exposure revealed, however, the necrosis or late apoptosis of cells was 94.7\u00a0\u00b1\u00a00.7% for 300\u00a0mosmol\/kg-H2O, and 76.1\u00a0\u00b1\u00a02.8% for 500\u00a0mosmol\/kg-H2O. Clearly, even under these harsh experimental conditions (i.e. stimulation with STS for 1\u00a0day), hypertonicity rescued HeLa cells from death to a significant degree (P\u00a0<\u00a00.001; n\u00a0=\u00a08).\nIn the last series of measurements, the timing of apoptosis induction by STS versus hypertonic stress was analysed. As depicted in Fig.\u00a07, increasing osmolality from 300 to 500\u00a0mosmol\/kg-H2O rescued HeLa cells from apoptosis induced by 6-h application of STS if this maneuver was performed 2\u00a0h before (HYPER2h\/STS), 1\u00a0h before (HYPER1h\/STS) or at the same time as the application of STS (STS-HYPER). Moreover, even with STS given 1\u00a0h ahead of hypertonic stress, apoptotic cell death was reduced to some 50% of the isotonic control value (Fig.\u00a07). Of note, the overall profiles of the effects of the timing of STS application versus hypertonic stress on cell death and apoptosis, as shown in the figure, are undistinguishable from those observed on AVD (see Fig.\u00a03) as well as on HICC activation (Fig.\u00a04).\nFig.\u00a07Interference of HICC activation with STS-induced apoptosis. For the control, STS was applied for 6\u00a0h under isotonic conditions (STS-ISO). For the other groups, it was applied 2\u00a0h before (STS2h\/HYPER), 1\u00a0h before (STS 1h\/HYPER), at the same time as (STS-HYPER), 1\u00a0h after (HYPER1h\/STS) or 2\u00a0h after (HYPER2h\/STS) osmolality was increased from 300 to 500\u00a0mosmol\/kg-H2O. Apoptosis was monitored as a decrease of cell viability (a, n\u00a0=\u00a012 to 42), caspase-3\/7 activity expressed as relative fluorescence units (FU; b, n\u00a0=\u00a06 and 12), and DNA fragmentation measured in an enzyme-linked (ELISA) assay as absorbance values (OD, optical density; c, n\u00a0=\u00a06)\nIt has long been known that AVD occurs in parallel with a loss of cellular K+ and a gain of cellular Na+ [20\u201322]. To some extent, these effects might reflect reduced activity of the Na+\/K+-ATPase [23]. Nevertheless, if the activation of the HICC were triggered as a consequence of conductive K+ export and AVD, this would lead to very similar results. Also of interest in this respect is a recent study on human leukemia U937 cells reporting two distinct phases during STS-induced apoptosis: an initial phase in which cellular K+ and Cl\u2212 decreased in parallel, followed by a second phase in which there was a further decrease in cellular K+ and an enormous increase in cellular Na+ (accompanied by a rise in cellular Cl\u2212) [24]. Of note, U937 cells were shown to express HICC currents [25]. Thus, the ionic scenario described could also reflect the interplay of HICC activation and AVD, as proposed in the present study.\nIn rat hepatocytes, 405\u00a0mosmol\/kg-H2O was insufficient to induce apoptosis but it sensitized cells to CD95-triggered cell death, whereas 505\u00a0mosmol\/kg-H2O functioned as an effective apoptosis inducer per se [26\u201329]. Since rat hepatocytes express (amiloride-sensitive) HICC currents [4, 30\u201332], it would be interesting to examine the degree to which blockage of the HICC might interfere with the induction of cell death in this system.\nConclusions\nTaken together, our results indicate that hypertonic stress caused a significant reduction in the staurosporine (STS)-induced apoptosis of HeLa cells. When analysing the functional interplay between apoptotic volume decrease (AVD) and regulatory volume increase (RVI) through the use of different activation protocols and effective blockers, the activation of hypertonicity-induced cation channels (HICCs) could be identified as the molecular mechanism by which HeLa cells are actually rescued from STS-induced apoptosis in hypertonic conditions. The precise mechanism by which HICC activation inhibits AVD and apoptosis remains to be elucidated.\nNevertheless, the results reported herein demonstrate a novel role of the HICC in the induction of programmed cell death. They are also in perfect agreement with a recent study in which inhibition of the HICC was found to sensitize HeLa cells to shrinkage-induced apoptosis [33]. We have only begun to understand the role of the HICC in these processes [4, 5], and the general relevance of the channel in cell proliferation and apoptosis remains a question of considerable interest.","keyphrases":["cation channel","apoptosis","cell shrinkage","hela cells","volume regulation"],"prmu":["P","P","P","P","P"]}
{"id":"Knee_Surg_Sports_Traumatol_Arthrosc-3-1-1950586","title":"Femoroacetabular impingement in 45 professional athletes: associated pathologies and return to sport following arthroscopic decompression\n","text":"Femoroacetabular impingement (FAI) occurs when an osseous abnormality of the proximal femur (cam) or acetabulum (pincer) triggers damage to the acetabular labrum and articular cartilage in the hip. Although the precise etiology of FAI is not well understood, both types of FAI are common in athletes presenting with hip pain, loss of range-of-motion, and disability in athletics. An open surgical approach to decompressing FAI has shown good clinical outcomes; however, this highly invasive approach inherently may delay or preclude a high level athlete\u2019s return to play. The purpose of this study was to define associated pathologies and determine if an arthroscopic approach to treating FAI can allow professional athletes to return to high-level sport. Hip arthroscopy for the treatment of FAI allows professional athletes to return to professional sport. Between October 2000 and September 2005, 45 professional athletes underwent hip arthroscopy for the decompression of FAI. Operative and return-to-play data were obtained from patient records. Average time to follow-up was 1.6 years (range: 6 months to 5.5 years). Forty two (93%) athletes returned to professional competition following arthroscopic decompression of FAI. Three athletes did not return to play; however, all had diffuse osteoarthritis at the time of arthroscopy. Thirty-five athletes (78%) remain active in professional sport at an average follow-up of 1.6 years. Arthroscopic treatment of FAI allows professional athletes to return to professional sport.\nIntroduction\nIn publications dating back several decades, irregularities in the morphology of the femoroacetabular articulation have been implicated as a possible source of hip pain in young, active athletes. A French article published in 1979 described hip pain associated with structural abnormalities of the proximal femoral neck in athletes participating in hockey, football, soccer, rugby, martial arts, and tennis [5]. More recently, developments pioneered by open hip surgeons have shown that morphologic abnormalities of both the femur and acetabulum underlie a large number of labral and chondral injuries in the hip [1, 29]. Wenger et al. [29] noted the presence of osseous abnormalities, including dysplasia and femoroacetabular impingement (FAI), in the majority of patients with labral tears. This suggests that isolated treatment of soft tissue pathologies may not be adequate without concomitantly addressing underlying structural abnormalities.\nThe concept of FAI has been defined by Ganz and colleagues [1, 8, 27]. In this condition, a structural or spatial abnormality of the femur (cam) or acetabulum (pincer) damages the chondrolabral structures during normal joint movement. The most common situation is a mixed cam and pincer pathology, occurring along the anterior femoral neck and the anterior\u2013superior acetabular rim. In high flexion and internal rotation movements, abutment and impingement of the labrum and cartilage occurs. The precise etiology of FAI is not well understood. Sub-clinical slipped capital femoral epiphyses [15, 25], mal-union of femoral neck fractures [6], and decreased femoral anteversion [26] are described causes of cam impingement. Relative posterior opening of the acetabulum (acetabular retroversion) or global (coxa profunda) overcoverage of the femur by the acetabulum are described causes of pincer impingement [23, 24]. Cam and pincer lesions lead to distinct patterns of labral and chondral damage [1, 10] and long-standing impingement is likely a significant cause of previously described idiopathic hip joint degeneration [1].\nGanz et al. developed an open surgical dislocation approach to decompress FAI [7, 13]. This technique has demonstrated good results of FAI decompression in a general population [2]. However, we believe that an arthroscopic approach involves less post-operative morbidity and allows patients, including professional athletes, to return to high-functioning lifestyles [28]. The purpose of this study was to define associated pathologies and determine if professional athletes could return to high-level athletics following arthroscopic decompression of FAI.\nMethods\nPatient selection\nA retrospective chart review was performed of all professional athletes presenting for arthroscopic treatment of FAI by the senior author between October 2000 and September 2005. All athletes presented with debilitating hip pain and an inability to participate in their sport. All were diagnosed with FAI based on physical and radiographic examination. Physical examination criteria included a positive impingement test [14] or flexion\u2013abduction\u2013external rotation (FABER) test. A positive impingement test was defined as groin pain with 90\u00a0degrees of hip flexion and maximal internal rotation. A positive FABER test was defined as asymmetry in the distance between the lateral knee and the exam table between the injured hip and the non-injured hip. Radiographic criteria included decreased anterior and superior femoral head\u2013neck offset, acetabular retroversion (as defined by a positive cross-over sign), or coxa profunda (as defined by the medial acetabular border overlapping the ilioischial line).\nFor these athletes with documented physical and radiographic evidence of FAI, conservative treatment was limited to only 6\u00a0weeks. Based on evidence described by Beck and colleagues [1], our thought was that conservative treatment could not address the underlying bony abnormalities and any further treatment delay in these highly active patients would likely precipitate further irreversible damage to the articular cartilage. Inclusion criteria for this study included professional athletes with at least one positive physical exam finding, at least one positive radiographic finding, and failure of at least 6\u00a0weeks of conservative therapy. Forty-five professional athletes from various sports met the inclusion criteria and were included in the study (Table\u00a01). There were 42 male and 3 female patients.\nTable\u00a01Pre-operative sports activities of the 45 professional athletesSportNo. of patientsHockey24Golf6Football5Soccer3Dance2Baseball2Martial arts1Tennis1Jockey1Total45\nArthroscopic procedure\nThe modified supine approach to hip arthroscopy was used [3]. Two portals (anterior, anterolateral) were established and a third portal (distal lateral accessory) was established as needed [12]. At the time of arthroscopy, the central compartment of the hip was first inspected.\nPincer lesions were typically identified in the superior acetabular quadrant. For the treatment of small pincer lesions (<2 to 3\u00a0mm), osteoplasty of the proximal femoral neck was occasionally sufficient to relieve the impingement [28]. Patients with moderate or large-sized lesions underwent acetabular rim trimming, using an arthroscopic osteotome or 5.5\u00a0mm round motorized burr [22].\nAcetabular labral tears were arthroscopically identified, and based on previous research on the vascularity of the labrum [9, 11], detached and peripheral midsubstance tears were typically repaired [12, 22]. Care was taken to preserve as much healthy, viable labrum as possible, however, degenerative or frayed tissue was debrided to a stable remnant.\nLabral repair was performed with suture anchors to repair detached labral tears or to re-fix the labrum following iatrogenic detachment for complete resection of pincer lesions. The previously described technique involved placement of the anchors high on the acetabular rim in the area of detachment [12]. Typically, one bioabsorbable anchor (BioRaptor, Smith+Nephew, Andover, MA, USA) was placed at the 12:00 acetabular position, and re-inforcement was placed either anteriorly or posteriorly of this area as needed. One limb of suture (Ultrabraid, Smith+Nephew, Andover, MA, USA) was passed between the labral tissue and the rim, and was retrieved through the substance of the labral tissue. In cases of inadequate availability of labral substance or in patients with highly degenerative, friable tissue, the suture was passed around the labral tissue. Standard arthroscopic knots fixed the repair to the rim. For midsubstance labral tears, a suture passer looped an 0-Vicryl around the torn tissue to approximate the edges of the midsubstance split.\nThe condition of the articular cartilage was evaluated according to the Outerbridge Classification System [18]. For this study, the Outerbridge Classification System was defined as follows: grade 0, normal cartilage; grade 1, cartilage with softening and swelling; grade 2, a partial thickness defect with fissures on the surface that do not reach the subchondral bone or exceed 1.5\u00a0cm in diameter; grade 3, fissuring to the level of the subchondral bone in an area with a diameter more than 1.5\u00a0cm; and, grade IV, exposed subchondral bone [18]. Chondral procedures performed in this group included chondroplasty and microfracture of lesions of the femoral head and acetabulum as needed.\nFollowing inspection of the central compartment, traction was released and the peripheral compartment was evaluated for cam lesions. A dynamic examination of the hip in all motion planes allowed for direct visualization of impingement at the femoroacetabular interface. For the treatment of cam lesions, an osteoplasty was performed with a 5.5\u00a0mm round motorized burr, restoring the anatomic concavity of the femoral head\u2013neck junction [28]. Burring was performed from superior to inferior along the anterior femoral head\u2013neck junction, to a depth of approximately 5\u20137\u00a0mm and a width of approximately 8\u201312\u00a0mm. Care was taken to avoid resecting too distally along the femoral neck to avoid the lateral epiphyseal vessels. A final dynamic examination was performed to confirm adequate decompression, and a lack of entrapment of the labrum during joint motion.\nIn general, the above-described procedure took approximately 2\u20133\u00a0h, depending on the degree of labral and chondral pathology. Traction time was limited to less than 2\u00a0h to reduce the risk of neurovascular compromise.\nPost-operative management\nPost-operative medical prophylaxis included an aspirin or a low-molecular weight heparin, a non-steroidal anti-inflammatory, and an antibiotic. Each of the 45 athletes underwent post-operative rehabilitation according to the following generalized protocol that was devised based on personal experience. For basic procedures involving decompression of FAI and labral treatment, weightbearing was restricted to 20\u00a0pounds, flatfoot for 4\u00a0weeks. A brace was prescribed to be worn for 10\u00a0days to protect the hip and limit abduction and rotation. Continuous passive motion (CPM) was used to apply 0\u201390\u00a0degrees of hip flexion for up to 8\u00a0h per day for 4\u00a0weeks. Night boots were worn for ten post-operative days to limit rotation during sleep. If capsular modification procedures (thermal capsulorrhaphy or capsular plication) were performed, rotation precautions were extended for a total of 21\u00a0days to avoid capsular stress. If microfracture was performed, weightbearing restrictions and CPM use were extended for a total of 8\u00a0weeks.\nPhysical therapy exercises were implemented within four post-operative hours. It is our belief, based on clinical observation, that this, in addition to an early emphasis on passive hip motion (particularly internal rotation) reduces the incidence of adhesion formation. Active hip flexion was limited for 4\u00a0weeks, based on clinical observation, to minimize the risk of hip flexor tendonitis.\nAthletes were typically allowed to return to full competitive activity between 12 and 16 post-operative weeks. Return-to-play statistics were determined by retrospective chart review and personal follow-up communication with the athletes.\nResults\nThe average age of the athletes at the time of surgery was 31\u00a0years (range: 17\u201361). Eleven athletes (24%) previously underwent hip arthroscopy by multiple primary surgeons for the treatment of labral and chondral pathologies and experienced a recurrence of hip symptoms. The average time to follow-up was 1.6\u00a0years (range: 6\u00a0months to 5.5\u00a0years).\nTwenty-two athletes (49%) were treated for an isolated cam lesion and three athletes (7%) were treated for an isolated pincer lesion. Twenty-one athletes (47%) had a mixed pathology of both cam and pincer lesions.\nAll of the athletes had acetabular labral tears. Twenty-five patients (56%) underwent either labral repair or re-fixation following rim trimming with suture anchors (average 1.3 anchors per patient, range: 1\u20133). Twelve patients (27%) had intra-substance labral repair. Five patients had labral debridement; one patient had a detached tear, one had frayed labral tissue, and three patients had complex tears consisting of detached, frayed, and flap components. Two patients were labral deficient from a previous arthroscopic debridement. They underwent labral grafting using an iliotibial band autograft.\nTwenty-one patients (47%) had a grade IV acetabular chondral defect; 14 underwent arthroscopic microfracture, 5 underwent thermal chondroplasty, and 2 patients had no treatment due to the diffuseness of their disease. Three patients (7%) had a grade IV femoral head chondral defect; one was treated with microfracture, one with chondroplasty, and one patient had no treatment due to the diffuseness of his disease. Seventeen (38%) had a grade I\u2013III acetabular chondral defect (13 treated with chondroplasty and 1 treated with microfracture) and 11 (24%) had a grade I\u2013III femoral head chondral defect (all 11 were treated with chondroplasty). Four patients (one baseball player, one football player, one hockey player and 1 golfer) had extensive diffuse OA at the time of arthroscopy, but opted to delay arthroplasty.\nTwenty-six patients (58%) had a partial tear of the ligamentum teres and 3 patients (7%) had a complete ligamentum teres avulsion. Thirteen (29%) patients underwent thermal capsulorrhaphy and 9 (20%) underwent plication for capsular redundancy. Seventeen patients (38%) had loose bodies in the joint and two patients required excision of myositis ossificans of the rectus femoris. Three patients (7%) underwent cheilectomy of a stenotic cotyloid fossa and 9 patients (20%) underwent arthroscopic release of a tight iliopsoas.\nFigure\u00a01 illustrates restoration of femoral head\u2013neck concavity in an athlete treated for cam impingement (Fig.\u00a01) and Fig.\u00a02 illustrates successful removal of the anterior acetabular overhang in an athlete treated for pincer impingement (Fig.\u00a02).\nFig.\u00a01A 27-year-old professional hockey player was evaluated for right hip pain. a A pre-operative cross-table lateral radiograph demonstrated convexity at the anterior femoral head\u2013neck junction. b The presence of a cam lesion was verified arthroscopically. The femoral head (FH) and acetabulum (Ac) are visualized in the peripheral hip compartment with the camera in the anterior portal. c A motorized burr was used to restore anterior concavity at the head\u2013neck junction. This was visualized at the superior acetabular (Ac) position (12:00) with the camera in the anterior portal. d A post-operative film verified successful decompression of the cam impingement and restoration of the femoral head\u2013neck junction concavityFig.\u00a02A 22-year-old professional football player was evaluated for right hip pain. a A pre-operative AP radiograph demonstrated a cross-over sign of the right acetabulum, indicating acetabular retroversion. b The presence of a pincer lesion was verified arthroscopically with the camera in the anterolateral portal with excessive bone along the anterior\u2013superior acetabulum (Ac). c A motorized burr in the anterior portal resected the lesion with the burr shield placed against the labrum (L) for protection. d The labrum was fixed back to the rim using two suture anchors. e A post-operative film verified the successful removal of pincer impingement and lack of retroversion in the superior region of the acetabulum\nForty-two athletes (93%) returned to professional sport following hip arthroscopy. Three players (1 football player, 1 hockey player, and 1 baseball player) did not return to play following arthroscopy. Each of these patients had diffuse osteoarthritis at the time of arthroscopy.\nFive athletes (11%) required re-operation. Three underwent lysis of adhesions and two had symptomatic treatment of extensive osteoarthritis. All of the patients who underwent revision surgery for lysis of adhesions returned to professional play and the two with extensive osteoarthritis did not return to play.\nThirty-five of the 45 athletes (78%) remained active at the professional level at an average of 1.6\u00a0years after hip arthroscopy.\nDiscussion\nIntra-articular hip injuries are a recognized cause of pain, mechanical symptoms, and disability in athletes. Traumatic intra-articular injury results from acute injury, including hyperabduction injuries, direct hip contact, and joint dislocation or subluxation. Atraumatic hip injury, however, is less well understood. Various motions exerted during sport, particularly flexion combined with internal rotation (hockey-goalie stance), have been suggested as potential causes of overuse hip injury [19, 21]. In this position, a cam lesion extending anteriorly or superiorly from the proximal femoral neck would impinge at the anterosuperior acetabular rim. Combining this understanding with recent reports by Wenger et al. [29] and Ganz et al. [1, 2, 8, 13], morphological abnormalities of the femoroacetabular joint have been closely examined as possible underlying sources of atraumatic hip injuries in athletes. Ganz and colleagues described FAI resulting from structural abnormalities of the proximal femoral neck (cam), acetabular rim (pincer), or most commonly, a combination of the two pathologies (mixed). Damage to the articular cartilage and acetabular labrum results from this pathologic bony contact and FAI is a likely trigger of early hip joint degeneration. In our experience, FAI is a common problem that has only recently been recognized in athletes with a primary hip complaint.\nAn open surgical dislocation approach was first described to treat hip impingement [7]. This technique showed good midterm results in a general population [2]. However, increasing recognition of FAI in a high-demand, competitive athletic population encouraged the development of a less invasive arthroscopic approach to facilitate prompt return to competitive play.\nVarious studies have demonstrated that hip arthroscopy is a safe and effective intervention in athletes with intra-articular injuries [4, 17, 19, 20]. Philippon reported on a series of ten elite athletes who underwent hip arthroscopy for labral debridement with thermal capsulorrhaphy [20]. All of the athletes returned to high-level athletic activities. McCarthy et al. [17] reported 80% excellent results following hip arthroscopy in elite athletes (average follow-up of 23.6\u00a0months). Byrd et al. [4] reported a series of 44 hip arthroscopies in 42 recreational, high school, collegiate, elite, and professional athletes. Post-operative improvement, as quantified by the modified Harris Hip Score, was present in all classes of athletes and in athletes undergoing any arthroscopic procedure (removal of loose bodies, debridement of ligamentum teres, excision of osteophyte, labral excision, microfracture, chondroplasty). Byrd et al. reported better results in athletes who recalled a traumatic onset to their hip symptoms, when compared to those with an acute or insidious onset. The authors suggested that an unaddressed pre-disposition to injury might have had a negative impact on self-reported outcomes in athletes [4].\nFAI may be the unaddressed pre-disposition to which Byrd and colleagues were alluding. While these above articles demonstrated successful outcomes following debridement procedures in athletes, there are a few caveats that must be addressed. First, it is likely that simple excision of damaged or diseased tissue, like with meniscal tears or loose bodies, can provide short-term abatement of pain and mechanical symptoms, and allow an athlete to return to professional athletics. However, recent studies have shown that simple hip debridement procedures failed to address the primary pathology in a vast majority of cases [1, 29]. A recent unpublished study performed by the senior author showed that a major cause of revision hip arthroscopy in all patients was treatment of labral and chondral re-injury and decompression of previously unaddressed FAI. Future studies examining the clinical and radiographic long-term outcomes of athletes treated with debridement procedures need to be compared to those athletes treated for FAI at an early stage of this disease. The purpose of this study was to simply state that despite the fact that FAI surgery is a more extensive procedure than debridement, professional athletes can still return to play following this procedure.\nAn arthroscopic approach to treat FAI was recently reported [28]; however, early outcome studies are lacking. The results of this study show that professional athletes with FAI can return to high-level competitive sport following this procedure. Ninety-three percent of athletes returned to their previous level of sport. Three athletes did not return to sport (1 hockey player, 1 football player, and 1 baseball player), however all had diffuse hip osteoarthritis present at the time of arthroscopy. One patient (a senior PGA golfer) was able to return to sport following arthroscopy, despite his diffuse osteoarthritis. Based on this information, it seems that arthroscopic treatment of FAI in the presence of OA can allow patients to return to low-impact, however, not likely high-impact professional sports. A larger cohort of patients is needed to test this hypothesis.\nAvascular necrosis (AVN) and femoral neck fracture are recognized risks of osteoplasty. A recent study showed that up to 30% of the femoral neck may be resected before it is structurally compromised [16]. In this series of patients, no post-operative cases of AVN or femoral neck fracture were diagnosed. Additional potential complications include adhesion formation at either the capsulolabral margin or between the capsule and the site of bony resection. In this series of 45 athletes, 3 patients (7%) required revision arthroscopy for the lysis of adhesions (LOA). All athletes returned to competitive sport following revision arthroscopy for LOA. Two additional athletes had extensive OA at the time of primary arthroscopy and subsequently underwent revision arthroscopy for symptomatic treatment of OA, including osteophyte debridement. Both did not return to professional athletics due to the extent of their disease. There were no reported cases of heterotopic ossification or neurovascular compromise, including lateral femoral cutaneous, sciatic, and pudendal neuropraxia, reported, although this statistic may have been underestimated due to the nature of this professional athlete cohort.\nA limitation of this study was the inherent selection bias involved with the study of professional athletes. This patient sub-group was financially motivated to return to play and may have been less likely to report post-operative symptoms and complications than the rest of patients treated for FAI. Despite this, we believed that a return-to-play analysis was critical to assess the outcomes of FAI treatment in a high-demand population. This cohort of professional athletes was debilitated prior to hip arthroscopy, and following intervention, was able to perform in physically intense professional sport activities. Also, the athletes\u2019 precise reason for retirement and whether it was related to their hip injuries was difficult to discern. For this reason, given the relatively short span of all professional athletes\u2019 careers, we chose to include all athletes at a minimum of 6\u00a0months post-operative. This time period was selected as an ample time period for completion of post-operative rehabilitation and return to sport. Another limitation of this study was the lack of follow-up subjective and objective data. Current outcomes instruments (modified Harris Hip score and the Non-Arthritic hip score) have not been validated for use in high-level athletes. It is our belief that these scoring systems fail to address the activities that are most limiting to athletes, and hence, underestimate the degree of debilitation in professional athletes. Evaluating the applicability of current scoring systems in athletes, and developing new outcomes instruments for athletes will be the focus of future studies. Other limitations of this study included unequal distribution of gender (42 males and 3 females) and professional sports. Because different sports place different demands on the hip, we cannot draw conclusions among the various sports.\nThe arthroscopic treatment of FAI represents the evolution of hip arthroscopy. In the past, soft tissue pathologies were treated in isolation, without addressing underlying impinging osseous abnormalities. Recent developments, particularly by Ganz and colleagues, have enlightened us to the pathology of FAI and the associated treatment options. In order to treat high-demand patients with FAI, an arthroscopic technique was developed. This study has demonstrated that full return to professional competitive sport is possible following arthroscopic treatment of FAI. Additional studies are needed to determine the effect on long-term joint degeneration of early surgical intervention to treat FAI.","keyphrases":["femoroacetabular impingement","cam","pincer","hip arthroscopy","osteoplasty"],"prmu":["P","P","P","P","P"]}
{"id":"Immunogenetics-2-2-1592254","title":"A novel family of diversified immunoregulatory receptors in teleosts is homologous to both mammalian Fc receptors and molecules encoded within the leukocyte receptor complex\n","text":"Three novel and closely related leukocyte immune-type receptors (IpLITR) have been identified in channel catfish (Ictalurus punctatus). These receptors belong to a large polymorphic and polygenic subset of the Ig superfamily with members located on at least three independently segregating loci. Like mammalian and avian innate immune regulatory receptors, IpLITRs have both putative inhibitory and stimulatory forms, with multiple types coexpressed in various lymphoid tissues and clonal leukocyte cell lines. IpLITRs have an unusual and novel relationship to mammalian and avian innate immune receptors: the membrane distal Ig domains of an individual IpLITR are related to fragment crystallizable receptors (FcRs) and FcR-like proteins, whereas the membrane proximal Ig domains are related to several leukocyte receptor complex encoded receptors. This unique composition of Ig domains within individual receptors supports the hypothesis that functionally and genomically distinct immune receptor families found in tetrapods may have evolved from such ancestral genes by duplication and recombination events. Furthermore, the discovery of a large heterogeneous family of immunoregulatory receptors in teleosts, reminiscent of amphibian, avian, and mammalian Ig-like receptors, suggests that complex innate immune receptor networks have been conserved during vertebrate evolution.\nIntroduction\nImmune receptors that bind similar ligands often form clusters that are encoded on distinct chromosomes in the human genome. For example, receptors that bind to the Fc portion of Ig [fragment crystallizable receptors (FcRs)] are found on chromosome 1q21-23 and consist of the high-affinity IgG and IgE receptors (Fc\u03b3RI and Fc\u03b5RI) and the related low- or moderate-affinity Ig receptors (Fc\u03b3RII, Fc\u03b3RIII, and Fc\u03b3RIV) (Hullet et al. 1991; Ravetch and Kinet 1991; Hullet and Hogarth 1994; Daeron 1997; Falk et al. 2005; Falk and Ravetch 2006). Alternatively, immune receptors involved in the recognition of major histocompatability class 1 (MHC I) antigens, such as killer cell Ig-like receptors (KIRs) and leukocyte Ig-like receptors (LILRs), are clustered together on chromosome 19q13.4 in a region known as the leukocyte receptor complex (LRC) (Kremer et al. 1992; Wagtmann et al. 1997; Wende et al. 1999; Martin et al. 2002). Recently, comparative studies have suggested that FcRs and LRC-encoded receptors, although genomically and functionally distinct, appeared to have evolved from a common ancestor. This contention is supported in part by recent attempts to find LRC regions in other species.\nMouse chromosome 7 is syntenic to the human LRC and encodes the paired Ig-like receptors (PIRs) that were originally discovered in an attempt to find a murine IgA receptor ortholog (CD89; Kubagawa et al. 1997; Hayami et al. 1997). Subsequently, searches for PIR relatives resulted in the identification of the chicken Ig-like receptors (CHIRs) in birds (Dennis et al. 2000), representing a multigene family encoded within an avian LRC-like region (Nikolaidis et al. 2005a; Viertlboeck et al. 2005). More importantly, CHIR Ig domain sequences are not only related to the mammalian PIRs and KIRs, but also to various FcR Ig domains (Dennis et al. 2000). This previously unrecognized relationship was supported by phylogenetic and comparative structural analysis and was the first evidence suggesting that certain genomically and functionally diverse mammalian and avian immune receptor families probably evolved from a common ancestor (Dennis et al. 2000; Nikolaidis et al. 2005b).\nCompared to mammals and birds, bony fish are among the most phylogenetically distant vertebrates with characteristic innate and adaptive immune systems that have mammalian-like B and T cells (Yoder 2004). Furthermore, due to the establishment of clonal lymphoid cell lines, catfish are known to also possess natural killer (NK)-like cells capable of killing xenogeneic targets by an antibody-dependent cell-mediated cytotoxicity (ADCC) process (Shen et al. 2002\u20132004). This functional evidence suggested that catfish NK cells possess a putative Fc\u03bcR (Shen et al. 2003). Additionally, catfish NK cells can kill allogeneic targets in an ADCC-independent manner, indicating that target recognition may also involve KIR and\/or LILR-like receptors (Shen et al. 2002, 2004). However, to date, FcR and\/or KIR\/LILR homologs have not been described in ectothermic vertebrates, making it difficult to elucidate the evolutionary history of these important receptor families. Large families of putative activatory and inhibitory novel immune-type receptors (NITRs) also belonging to the Ig superfamily (IgSF) have been described in teleosts. Pufferfish (Spheroides nephelus), zebrafish (Danio rerio), catfish, and rainbow trout (Oncorhynchus mykiss) all express NITRs (Strong et al. 1999; Yoder et al. 2001, 2002; Hawke et al. 2001). Because some NITRs are also expressed by catfish NK cells (Hawke et al. 2001), it has been suggested that these receptors may represent the \u201cfunctional orthologs\u201d of the mammalian KIRs and\/or LILRs (Yoder et al. 2001; Hawke et al. 2001). However, NITRs do not exhibit any sequence identity or phylogenetic relationship to such receptors (Yoder et al. 2001) and have only been identified in bony fishes.\nHerein is described a novel set of IgSF receptors present in the channel catfish, and possibly other teleosts, that are composed of Ig domains related to functionally diverse immunoreceptor families present in mammals. Detailed sequence analysis supports that individual catfish leukocyte immune-type receptors (IpLITRs) contain a unique composition of Ig C2-like domains sharing homology with both FcRs and receptors encoded within the LRC. Thus, IpLITRs provide further evidence supporting the common evolutionary origins of genomically and functionally distinct immunoregulatory receptor families present in mammals.\nMaterials and methods\nExperimental animals and cell lines\nCatfish (1\u20132\u00a0kg) were obtained from a commercial source (ConAgra, Isola, MS, USA) and maintained in individual tanks as described previously (van Ginkel et al. 1992). Catfish peripheral blood leukocytes (PBL) were isolated from heparinized blood by centrifugation on a cushion of Ficoll-Hypaque (Lymphoprep, Accurate Chemical, Westbury, NY, USA) as described previously (Miller et al. 1994a). Catfish cells lines were grown at 27\u00b0C in AL-3 medium consisting of equal parts AIM-V and L-15 (Life Technologies, Gaithersburg, MD, USA) adjusted to catfish tonicity with 10% (v\/v) deionized water and supplemented with 1\u00a0\u03bcg\/ml NaHCO3, 50\u00a0U\/ml penicillin, 50\u00a0\u03bcg\/ml streptomycin, 20\u00a0\u03bcg\/ml gentamicin, 50\u00a0uM 2-ME, and 3% heat-inactivated, pooled, normal catfish serum (Miller et al. 1994a,b). 3B11 is a cloned autonomous B cell line generated from an outbred catfish by mitogen stimulation (Wilson et al. 1997). G14D is an automonous T cell line derived from a gynogenetic catfish (Hogan et al. 1999), whereas 42TA is a macrophage cell line derived from a different outbred fish (Miller et al. 1994b). TS32.15 and TS32.17 are clonal nonautonomous antigen-dependent cytotoxic T cell lines developed from an alloantigen immunized outbred fish (Stuge et al. 2000). 1F3 is a clonal nonautonmous NK-cell line developed from an alloantigen-stimulated culture of PBL from a naive outbred fish (Shen et al. 2004). The nonautonomous cytotoxic T cell and NK cell lines require weekly restimulation with irradiated allogeneic cells for continuous proliferation. The channel catfish ovary fibroblast cell line (CCO; Browser and Plumb 1980) was provided by Dr. V.G. Chinchar (University of Mississippi Medical Center). CCO cells were maintained in Dulbecco\u2019s modified Eagle\u2019s medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FCS.\nChannel catfish LITRs and sequence analysis\nIn a search for IgSF receptors expressed by alloantigen stimulated catfish PBL and various catfish clonal cell lines, several expressed sequence tag (EST) libraries were analyzed. Subsequently, IpLITR1 and IpLITR3 cDNAs were identified by single pass sequencing of a cDNA library generated from a catfish 7-day-old mixed lymphocyte culture (MLC) enriched in NK-like cells, MLC52-1 (Shen et al. 2004; Stuge et al. 1997). IpLITR2 was obtained from the catfish 42TA macrophage cell line (Miller et al. 1994b) cDNA library. The cDNAs were sequenced on both strands using universal forward and reverse primers and gene-specific primers. Nucleotide and amino acid sequences were analyzed using DNASTAR software (Madison, WI, USA) and aligned using CLUSTALW (Thompson et al. 1997). Neighbor-joining (NJ) trees with pairwise gap deletions were drawn using MEGA v3.0 (Kumar et al. 2001). Maximum parsimony and maximum likelihood trees were drawn with essentially the same results (data not shown). Similarity searches were performed with the extracellular Ig domains of IpLITR1, IpLITR2, and IpLITR3 using position-specific iterative (PSI) and Basic Local Alignment Search Tool (BLAST) analysis (Altschul et al. 1990) against the National Center for Biotechnology Information (NCBI) nonredundant database. SOSUI (signal) beta version, Simple Modular Architecture Research Tool (SMART) (Letunic et al. 2004), Pfam databases (Bateman et al. 2004) and the three-dimensional position-specific scoring matrix server (Kelly et al. 2000) were used for predictions of signal peptides, Ig domains, transmembrane (TM) segments, and secondary structure. Sequence decorations were performed using GeneDoc (http:\/\/psc.edu\/biomed\/genedoc).\nSouthern and Northern blot analyses\nGenomic DNA was prepared from erythrocytes of 16 catfish siblings. These siblings were representatives of a 78-member family obtained from the Catfish Genetics Research Unit, United States Department of Agriculture, Agricultural Research Service (Stoneville, MS, USA; designated family 1). The sibling catfish 1\u201316 used were A10, A5, A32, A22, A38, A36, A16, A29, A18, A49, A159, A15, A23, A25, A186, and A37, respectively (Quiniou et al. 2005). The DNA (10\u00a0\u03bcg) was digested to completion with Pst1 or EcoRI, separated on 1% agarose gels and transferred by capillary action onto Hybond-N+ membranes (Amersham Pharmacia Biotech, Arlington Heights, IL, USA) using standard techniques. Hybridizations were performed in Rapid-hyb buffer (Amersham Pharmacia Biotech) at 65\u00b0C and membranes were washed at high stringency (65\u00b0C with 0.1\u00d7 saline-sodium citrate, 0.1% sodium dodecyl sulfate). Specific probes (Table\u00a01) were amplified by PCR using IDPol DNA polymerase (ID Labs Biotechnology, London, Ontario, Canada) according to the manufacturer\u2019s recommended protocol. Typical parameters were: 1\u00a0min 94\u00b0C, followed by 29 cycles of 94\u00b0C 30\u00a0s, 62\u00b0C 30\u00a0s, 72\u00b0C 1\u00a0min, then extension at 72\u00b0C for 10\u00a0min. Probes were random primed labeled with [32P] 2\u2032-deoxycytidine 5\u2032-triphosphate using a Megaprime labeling kit (Amersham Pharmacia Biotech). \nTable\u00a01IpLITR PCR primers used for generating Ig domain-specific probes and gene-specific primers used in RT-PCR experimentsPrimer\u00a0\u00a0IpLITR1 D1Forward5\u2032 GTCGGACAAGGTCAAGTTCTG 3\u2032Reverse5\u2032 GGCTTTTGGTCTCTCTATCAC 3\u2032IpLITR1 D4Forward5\u2032 AATCCTGATGAACAGGTGTACC 3\u2032Reverse5\u2032 GTGTTTACAGTGGTAGAAACC 3\u2032IpLITR2 D3Forward5\u2032 AGTCGTGAAGGAGCTGTACTGA 3\u2032Reverse5\u2032 TTCACTGCCAGAATGATGGTCAC 3\u2032IpLITR3 D6Forward5\u2032 CTGAGGGACATCCTCTGACCTT 3\u2032Reverse5\u2032 GTGTTTACAGTGGTAGAAACC 3\u2032IpLITR1 GSPForward5\u2032 GTCGGACAAGGTCAAGTTCTG 3\u2032Reverse5\u2032 CTGCAGACATGATGAACTTCT 3\u2032IpLITR2 GSPForward5\u2032 GTCGGACAAGCTCAAGAGTTT 3\u2032Reverse5\u2032 TGTGTAGTAGAGTGGGTTTCC 3\u2032IpLITR3 GSPForward5\u2032 GTCGGACAAGGTCAAGTTCTG 3\u2032Reverse5\u2032 GCTTTTGGGTGACTCTCCTCT 3\u2032IpEF1\u03b1 GSPForward5\u2032 GACTGCCACACTGCTCACATTG 3\u2032Reverse5\u2032 TTAGTTACTCAGCAGCTTTCTTCCGSP gene-specific primers\nTotal RNA from catfish PBL and various tissues was prepared using RNA-Bee (Tel-test, Friendswood, TX, USA); 10\u00a0\u03bcg of each sample were electrophoresed on 1.5% formaldehyde-agarose gels and transferred by capillary action onto Hybond-N+ (Amersham Pharmacia Biotech), hybridized, and washed as above.\nReverse transcription (RT)-PCR\nTotal RNA from PBL, MLC, various tissues, and catfish clonal cell lines were prepared as described above, and treated with DNase I (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer\u2019s recommended protocol. One \u03bcg RNA was reverse transcribed using an oligo-T primer and 200 units of Superscript III reverse transcription (RT) (Invitrogen Life Technologies). RT-PCR was performed using specific primers for IpLITR and the housekeeping gene elongation factor 1 alpha (IpEF1\u03b1; Table\u00a01). Typical parameters were: 3\u00a0min 94\u00b0C, followed by 30 cycles of 94\u00b0C 45\u00a0s, 58\u00b0C 45\u00a0s, 72\u00b0C 2\u00a0min, then extension at 72\u00b0C for 10\u00a0min. PCR products were visualized on 1.2% Tris-acetate-EDTA agarose gels. PCR products were also cloned into pCR4-TOPO\u00ae (Invitrogen Life Technologies) and verified by sequencing. The Genbank accession number for IpEF1\u03b1 is CB938718.\nResults\nCatfish LITR sequences\nIn a search for IgSF receptors expressed by alloantigen-stimulated catfish PBL and various catfish clonal cell lines, several EST libraries were analyzed. Three IpLITR sequences were subsequently identified that encoded type I TM proteins with extracellular C2-like Ig domains, which were predicted using SMART (see Electronic Supplementary Material). Even though these sequences vary in length, they are highly similar (Fig.\u00a01a,b). All three receptors have identical signal peptides and IpLITR1 and IpLITR3 have almost identically encoded Ig domains D1, D2, D3, and D4 (Fig.\u00a01a). Comparatively, IpLITR2 D1 and D2 are 77.2 and 86.8% identical at the amino acid level to their IpLITR1 and IpLITR3 counterparts (Fig.\u00a01c). IpLITR1 encodes a 346 amino acid extracellular region consisting of four Ig domains, a 23-amino acid TM segment and a 116-amino acid cytoplasmic tail (CYT). The IpLITR1 CYT contains two immunotyrosine-based inhibition motifs (ITIMs) centered at Y439 and Y461 and an ITIM-like motif (SEYTTE) centered at Y479 (Fig.\u00a01b) (Daeron and Vivier 1999; Ravetch and Lanier 2000; Billadeau and Leibson 2002). An overlapping immunotyrosine-based switch motif (ITSM; TVYSQL) centered at Y465 is also present in the CYT of IpLITR1 (Shlapatska et al. 2001). In contrast, the smaller IpLITR2 and the longer IpLITR3 transcripts encode for molecules with identical 25-amino acid TMs containing a lysine residue and very similar charged CYTs (Fig.\u00a01b). Overall, the individual IpLITRs are composed of a membrane distal to membrane proximal ordering of Ig domains, each containing similar D1s and D2s. However, the membrane proximal domains vary, i.e., IpLITR3 D5 and D6 are only 15.7\u201324.7% and 15.2\u201325% identical, respectively, to all other IpLITR Ig domains, and IpLITR2 D3 is only 17.9\u201339.3% identical to other IpLITR domains (Fig.\u00a01c).\nFig.\u00a01Predicted amino acid sequence, domain comparisons, and schematic representation of IpLITR1, IpLITR2, and IpLITR3. a Alignment of the extracellular and b TM\/CYT regions of IpLITR1, IpLITR2, and IpLITR3. Signal peptide (SP), and immunoglobulin (Ig) domains are labeled; cysteine residues predicted to be involved in intrachain disulfide bonds are marked with asterisks; gray shaded residues represent differences from IpLITR1 in a and differences from IpLITR2 in b. TMs are underlined, ITIM-like motifs are boxed, an overlapping ITSM within the CYT of IpLITR1 is indicated by a bracket, and TM charged residues are shaded black and marked (+). c Phylogenetic analysis of Ig domains in IpLITRs. NJ trees with pairwise gap deletions were drawn using MEGA v3.0 (Kumar et al. 2001) with 10,000 bootstrap replications, and bootstrap values >50% are shown. Branch lengths were measured in terms of amino acid substitutions and a scale bar are shown below the trees. The predicted SP, Ig domains, TM, and CYT are indicated. ITIM-like motifs are shown as boxes, N-linked glycosylation sites are marked as ballpoint lines. Individual IpLITR domains are shaded according to their relatedness between IpLITRs and percent amino acid identity with IpLITR1 Ig domains indicated to the left of IpLITR2 and IpLITR3. IpLITR2 D3 and IpLITR3 D5 and D6 are 15.2\u201339.3% identical to all IpLITR1 Ig domains\nIpLITRs belong to polymorphic and polygenic gene families\nSibling restriction fragment length polymorphism (RFLP) patterns using domain-specific probes for the various IpLITRs show a large number of hybridizing bands with each probe used, as well as differences in the banding patterns among siblings (Fig.\u00a02). These findings indicate that the IpLITR gene complex is both polygenic and polymorphic. However, it is presently unknown whether these polymorphic differences are due to variability within the exons encoding these receptors and\/or within the introns. Some of the high-intensity bands found in each of the Southern blots may reflect multiple IpLITR copies, further increasing the possible number of genes. Importantly, the Southern blot segregation patterns show that there is more than one IpLITR locus. For example, among the 16 sibling fish used there are at least nine, perhaps more, different RFLP patterns for IpLITR D1 (Fig.\u00a02b). If these genes were closely linked then only four RFLP patterns (barring cross-over events) would be observed, as shown for the MHC class II\u03b2\u00a0RFLP patterns (Fig.\u00a02a). When all Southern blots are compared, at least three distinct linkage groups (marked by \u25cf, \u25b2, and \u25a0 ) are observed, indicating the presence of multiple independently segregating loci, and none of these are linked to the MHC II RFLP (Fig.\u00a02b\u2013d). Similar results were also obtained using the IpLITR2 D3 probe (data not shown). Mining of the zebrafish genome for IpLITR relatives [whole genome shotgun database of the NCBI (Sanger Genomic Institute, Cambridge, UK)] further supports that these receptors are present on different chromosomes because five contigs were identified with IpLITR-like homologs on zebrafish chromosomes 3, 7, and 8 (Table\u00a02).\nFig.\u00a02Southern blot analyses. Genomic DNA from 16 sibling catfish was digested, separated, transferred to nylon membranes, and hybridized with a MHCII\u03b2, b IpLITR1 D1, c IpLITR1 D4, and d IpLITR3 D6-specific probes. Letters a\u2013i indicate different segregation patterns (b\u2032, e\u2032, and h\u2032 represent variant segregation patterns that may be the result of recombination). Representative RFLP bands illustrating different linkage groups are indicated by \u25cf, \u25b2, and \u25a0. Kilobase size markers are indicated to the left of each blotTable\u00a02Identification of IpLITR-like genes in the zebrafish genomeAccession numbersChromosomeBX0051568BX0005248BX5111763BX6496273CAAK01000490.17Search (04\/05) of the zebrafish whole genome shotgun database of the NCBI (Sanger Genomic Institute)\nIpLITR expression\nNorthern blot analyses using a cross hybridizing IpLITR1 D1 probe revealed that LITR message is highly expressed in the hematopoietic pronephros and mesonephros, with predominant transcript sizes of \u223c6.6, \u223c4.0, and \u223c3.8\u00a0Kb (Fig.\u00a03a). Low levels of IpLITR D1 expression were detected in the spleen, with little to no message detected in the heart, liver, gill, or muscle. The use of the more specific IpLITR2 D3 probe revealed message expression not only in the pronephros and mesonephros, but also in the spleen, gill, and perhaps heart with transcripts of \u223c6.0, \u223c3.7, \u223c2.7, and \u223c1.9\u00a0Kb (Fig.\u00a03b). RNA load levels for each of the tissues tested were similar based upon hybridization with a catfish EF1\u03b1 probe. RT-PCR, using primers specific for the putative inhibitory IpLITR1 and the putative activating IpLITR2 as representative IpLITRs, was also used to assess tissue expression. As shown in Fig.\u00a03c, IpLITR1 and IpLITR2 are in most cases coordinately expressed in a variety of different tissues and confirm the Northern blot data. In addition, multiple PCR products were amplified using the IpLITR1 and IpLITR2 specific primers, which is reminiscent of the multiple transcripts observed following RNA blotting. Because the predicted sizes of PCR products for the prototype IpLITR1 and IpLITR2 are 1,461 and 780\u00a0bp, respectively, at least two additional IpLITR1 products (1,545 and 2,392\u00a0bp) and one additional IpLITR2 product (1,298\u00a0bp) were amplified (Fig.\u00a03c).\nFig.\u00a03Northern blot and RT-PCR analyses of IpLITR tissue expression. Catfish LITR tissue expression was performed by Northern blot analysis using a an IpLITR1 D1-specific probe, and b an IpLITR2 D3-specific probe. Total RNA from pronephros (head kidney), mesonephros (trunk kidney), spleen, heart, liver, gill, and muscle were examined. Kilobase markers are on the left margin and arrows indicate the major hybridizing bands observed. RNA integrity and load levels were determined by hybridization using a catfish EF1\u03b1 probe as a housekeeping gene. c RT-PCR analyses of IpLITR1 and IpLITR2 in various catfish tissues. The sizes of the IpLITR bands verified by sequencing are indicated at the right margin and base pair sizes are at the left margin\nTo further examine the expression of IpLITR1 and IpLITR2, RT-PCR was performed using RNA from catfish clonal macrophage, B, T, and NK-like cell lines, as well as a MLC (Fig.\u00a04a). RT-PCR products of 1,461, 1,545, and 2,392\u00a0bp were obtained using IpLITR1-specific primers, and these products were detected variably among the cell lines examined. It was also found that three RT-PCR products were also variably amplified from the different catfish leukocyte cell lines using IpLITR2-specific primers, the expected 780-bp product, a 1,298-bp product (previously observed in tissues; see Fig.\u00a03c), and an additional 1,060-bp product (Fig.\u00a04a). In contrast, no IpLITR message was detected in the fibroblast CCO cell line (data not shown), indicating that these genes are primarily expressed in cells of immune function.\nFig.\u00a04a RT-PCR analyses of IpLITR1 and IpLITR2 expression in various catfish clonal cell lines and a polyclonal MLC. Total RNA was obtained from the catfish 42TA macrophages, 3B11 B cells, TS32.15 and TS32.17 nonautonomous cytotoxic T cells, autonomous G14D T cells, a MLC, and 1F3 NK-like cells. RT-PCR was performed using primers specific for IpLITR1, IpLITR2, and IpEF1\u03b1. The sizes of the IpLITR bands verified by sequencing are indicated at the right margin and base pair sizes are at the left margin. b Schematic representation of IpLITR-types identified by sequencing of RT-PCR products obtained using IpLITR1 and IpLITR2-specific primers. The originally identified prototype IpLITRs are boxed, and arrows indicate the relative positions of the primer pairs used in RT-PCR reactions. Sizes in base pairs corresponding to the bands observed in a are indicated above each schematic. The predicted SP, Ig domains, TM, and CYT are indicated. ITIM-like motifs are shown as hatched boxes and noncanonical immunotyrosine-based activation motif-like motifs shown as white boxes within the CYT. Individual Ig domains are shaded according to relatedness as described for Fig.\u00a01, and percentages above each receptor represent percentage of amino acid identities of the predicted extracellular region of the IpLITR-like sequences when compared with the prototype IpLITRs\nThe sequencing of the various IpLITR1 and IpLITR2 RT-PCR products from all sources (both tissues and cell lines) resulted in the identification of multiple different IpLITR-related sequences (Stafford et al. manuscript in preparation). Schematic diagrams of representative IpLITR1 and IpLITR2 variant forms are shown in Fig.\u00a04b. These highly related IpLITR1- and IpLITR2-like sequences differed in the number of Ig-like domains present, and in the case of some IpLITR1-variants, also differed in the length of the CYT.\nHomology between IpLITRs, LRC, and FcR genes\nWhen the extracellular region of IpLITR1 was used in BLASTP analysis, various adhesion molecules, FcRs, FcR-like proteins (FCRLs), LILRs, and KIR gene family members were identified with significant homology (i.e., E values <10\u22127), albeit with moderate amino acid identities ranging from \u223c25\u201330% (Table\u00a03). Examples include the adhesion molecules CD22 and carcinoembryonic antigen-related cell adhesion molecule 5, Xenopus leukocyte FCRLs, XFL, PIRA1, LILRA2, CHIRA2, KIR3DL1, FCRL5, and Fc\u03b3RIIB. When the Ig domains of IpLITR2 were used for screening, most matches were members of the FcR gene family, e.g., Fc\u03b3RI, Fc\u03b3RII, Fc\u03b3RIII, and Fc\u03b5RI\u03b1, from humans, primates, and rodents, as well as various FCRL proteins (Table\u00a03). Searches using the extracellular region of IpLITR3 were essentially the same as the IpLITR1 BLASTP results, and PSI-BLAST analysis further reinforced the evolutionary relationships between IpLITRs and mammalian FcRs, FCRLs, KIRs, and LILRs, as well as avian CHIRs (data not shown). \nTable\u00a03Representative IpLITR BLASTP resultsReceptor\u00a0AccessionScoreE-valueChromosomeIpLITR1CD22HumanNP_001762884e-1619q13.1XFL1.4XenopusAAQ56585845e-15?CEACAM5HumanCAA34474845e-1519q13.1PIRA1RatAAD50905731e-11?LILRA2 (ILT1)ChimpanzeeNP_001009044722e-1119FCRL5 (FcRH5)MouseNP_899045716e-113XFL1.3XenopusAAQ63874716e-11?LILRA2 (ILT1)HumanAAD50364701e-1019q13.4KIR3DL1HumanAAC83928693e-1019q13.4KIRChimpanzeeAAF86243684e-1019CHIRA2ChickenCAG33731661e-9?KIR3DL1OrangutanAAM78465661e-919FCRL5 (FcRH5)HumanCAH71428662e-91q21KIR3DS1HumanAAV32446662e-919q13.4Fc\u03b3RIIBRatAAL29888631e-813q24XFL1.6XenopusAAQ63873631e-8?XFL1.2XenopusAAQ56587622e-8?CHIRB3ChickenCAG25957623e-8?LILRA3 (ILT6)HumanAAB87661608e-819q13.4LIRBovineXP_586587601e-7?PIRA2MouseAAC53213592e-77IpLITR2IRTA2BovineXP_595289761e-12?Fc\u03b3RIIGuinea pigA34636613e-8?FCRL5 (FcRH5)MouseNP_899045606e-83FCRL3 (FcRH3)MouseAAS91578606e-83Fc\u03b3RIMacaqueAAL92095591e-7?FCRL5 (FcRH5)HumanNP_112571574e-71q21Fc\u03b3RI\u03b1HumanCAI12557575e-71q21Fc\u03b3RIIBMouseAAH19758569e-71XFL1.4XenopusAAQ56585569e-7?IRTA1BovineXP_614632552e-6?Fc\u03b5RI\u03b1HumanAAH05912538e-61q23FREB2HumanAAX11390538e-61q23.3Fc\u03b3RIHumanAAA58414538e-61q21.2Similarity search (BLASTP) of the NCBI\u2019s nonredundant protein database using the four Ig domains of IpLITR1 and the three Ig domains of IpLITR2CEACAM5 carcinoembryonic antigen-related cell adhesion molecule, XFLXenopus leukocyte FCRL\nPhylogenetic analyses and alignments\nThe IpLITR D1 and D2 Ig domains are related to those of mammalian FcRs and FCRLs (Fig.\u00a05a) in that these domains clustered with the FcR\/FCRLs and away from catfish NITRs and from representative LRC members in a phylogenetic tree. In contrast to the D1\/D2 sequence analyses, the membrane proximal D3 and D4 sequences of IpLITR1 and IpLITR3 resulted in a significant shift within the phylogenetic tree; IpLITRs now clustered with the LRC-encoded genes (Fig.\u00a05b). The LRC relationship is supported by high bootstrap values when comparing IpLITR1 and IpLITR3 D3 and D4 sequences with D2 and D3 sequences of representative LILRs and KIRs, as well as with D1 and D2 sequences of NKp46 and CHIRs. The D5 and D6 sequences of IpLITR3 are related to the D1 and D2 domains of the FcR family (Fig.\u00a05c). Thus, IpLITR3 is the best example of the unique domain structure of these teleost receptors, as it encodes for combinations of Ig domains that are phylogenetically related to the FcRs and FCRLs (D1 and D2; D5 and D6), as well as LRC encoded receptors (D3 and D4).\nFig.\u00a05Phylogenetic analyses reveal unique Ig domain composition within individual IpLITRs. The Ig domains compared are indicated by gray shading in the schematics to the left of each phylogenetic tree. a Comparisons of IpLITR, representative FcR and FCRL, and human LRC D1 and D2 sequences. b Comparisons of IpLITR1 D3 and D4 with representative FcR, FCRL, KIR, LILR, and PIRA1 D2 and D3 sequences, as well as NKp46 and CHIRA2 D1 and D2 sequences. c Comparison of IpLITR3 D5 and D6 with D1 and D2 of representative mammalian FcRs, FCRLs, and LILRs\/KIRs. The accession numbers for the various Ig domain sequences used are: human (hu)Fc\u03b3RI(CAI12557), huFc\u03b3RII (CAA35642), huFc\u03b3RIII (CAA36870), huFc\u03b5RI (AAH05912), huFCRL3 (AAH28933), huFCRL4 (AAK93970), huFCRL5 (NP_112571), huFc\u03b1R\/CD89 (AAH27953), huLILRB1 (AAH15731), huLILRB2 (AAB87662), huKIR3DL1 (AAC83928), huKIR3DS1 (AAV32446), huSIRP (CAA71404), human NKp46 (AJ001383), mouse (mo)Fc\u03b3RI (AAD34931), moFc\u03b3RII (AAA37608), moFc\u03b3RIII (NP_034318), moFc\u03b5RI (NP_034314), moFCRL3 (AAS91578), moNKp46 (AJ223765), rat (ra) Fc\u03b3RIII (AAA42050), raPIRA1 (XP_341773), raNKp46 (AF082533), guinea pig (gp) Fc\u03b3RII (D13692), macaque (mac)Fc\u03b3RI (AAL92095), bovine (bo)FCRL5 (XP_595289), boKIR3DS1 (AAP33626), boSIRP (CAA71943) chimpanzee (cp)LILRA2 (NP_001009044), orangutan (or)KIR (AAM78473), monkey (mk)KIR3DL20 (AAU50562), CHIRA2 (CAG33731), catfish (Ip)NITR1 (AF397454), and IpNITR2 (AF397455). For all analyses, SIRPs and\/or IpNITRs were included as outgroups to root the trees. NJ trees with pairwise gap deletions were drawn using MEGA v3.0 (Kumar et al. 2001) with 10,000 bootstrap replications, and bootstrap values >50% are shown. Branch lengths were measured in terms of amino acid substitutions, and scale bars are shown below the trees\nSubsequently, the D1 and D2 Ig domain sequences of IpLITR2 (as a representative) were aligned with various mouse and human FcRs and three representative human FCRLs. A majority of the predicted \u03b2-strands in IpLITR2 is present in similar positions as the \u03b2-strands found in FcR\/FCRL family members (Fig.\u00a06a), indicating a predicted degree of secondary structural conservation between IpLITRs and these mammalian immune receptors. In most cases, the predicted IpLITR2 \u03b2-strands were present in regions of high amino acid identity\/similarity; however, none of the FcR residues known to contact the Fc portion of Ig were present (Fig.\u00a06a). The D3 of IpLITR2, a domain specific to this IpLITR type, appears to be related to the membrane distal domain (D1) of several CHIRs (Fig.\u00a06b), and alignment of IpLITR2 D3 with representative CHIR D1s indicated a degree of amino acid identity\/conservation between these Ig domains (Fig.\u00a06c). To reinforce the unique nature of domains present within this receptor, IpLITR2 D1 did not cluster with the CHIRs but was related to mammalian FcR D1 domains (Fig.\u00a06b).\nFig.\u00a06IpLITR2 contains Ig domains related to both FcRs\/FCRLs and CHIRs. a Amino acid alignment of IpLITR2 D1 and D2 sequences with representative mammalian FcRs and FCRLs. Accession numbers are as in Fig.\u00a05. Gray shading: residues similar\/identical to IpLITR2. Boxed residues: contacts for Ig Fc in huFc\u03b5RI. Black and gray arrows represent the predicted \u03b2-strands for IpLITR2 and huFc\u03b5RI, respectively. Hatched boxes indicate conserved cyteines and dashes represent gaps. b Phylogenetic analysis of IpLITR2 D1 and D3 sequences (gray shaded) compared with representative mammalian FcR sequences (as in Fig.\u00a05) and D1 sequences of representative CHIRs. The accession numbers for the various Ig domain sequences used are: CHIRB2 (XP_422905), CHIRB1 (CAH55757), CHIRAB2 (CAG33733), CHIRA2 (CAG33731), CHIRB5 (AJ879908), CHIR A1 (AF306851), CHIRB4 (XP_428342), CHIRB6 (CAI53861), and CHIRAB3 (AJ879909). NJ trees with pairwise gap deletions were drawn using MEGA v3.0 (Kumar et al. 2001) with 10,000 bootstrap replications, and bootstrap values >50% are shown. Branch lengths were measured in terms of amino acid substitutions, and scale bars are shown below the trees. c Alignment of IpLITR2 D3 and CHIR D1. Gray shading indicates residues that are similar\/identical to IpLITR2, black and gray arrows represent predicted \u03b2-strands for IpLITR2 and CHIRA2, respectively; \u201cdotted\u201d lines indicate predicted helices and hatched boxes indicate conserved cyteines. Gaps in alignment are indicated by dashes\nAttempts to model IpLITR Ig domains were unsuccessful as no sufficient modeling templates were identified using SWISS-Model. However, similarity searches against the NCBI\u2019s protein data bank (PDB) indicated a significant match between D1 and D2 of IpLITRs and the Ig domains of human Fc\u03b5RI\u03b1 (E value <10\u22128). Conversely, the membrane proximal D3 and D4 Ig domains of IpLITR1 and IpLITR3 had significant alignment scores (E value <10\u22127) with the first two Ig domains of the human natural cytotoxicity receptor NKp46. Taken together, these PDB search results further support that IpLITRs D1 and D2 Ig domain sequences are related to mammalian FcRs and FCRLs, and that the D3 and D4 Ig domains are related to LRC encoded receptors.\nMining of the zebrafish genome identified five contigs (Table\u00a02) containing sequences encoding IpLITR-like proteins predicted to contain several Ig domains and, in some cases, signal peptides, TM segments, and CYTs with signaling motifs (Fig.\u00a07). These findings indicate that IpLITR-like molecules are not unique to the catfish, but are likely common in other teleost species. Because IpLITR3 was identified as the closest match from the NCBI\u2019s database to the zebrafish (Zf) LITR-like proteins, it was used for further comparisons. Phylogenetic analysis of the various Ig domain sequences indicated that the ZfLITR-like proteins were closely related to those of IpLITRs (Fig.\u00a07a). In most cases, the membrane proximal domains of ZfLITRs are highly related to their IpLITR3 counterparts with >60% amino acid identity (Fig.\u00a07b). However, one of the ZfLITRs (XP_692267) contains two Ig domains similar to IpLITR3 D6 in the membrane distal region. This zebrafish receptor also contains three Ig domains that are phylogentically related (Fig.\u00a07a) with high amino acid identity (\u223c50%) to IpLITR3 D4 (Fig.\u00a07b). The other ZfLITR-like proteins are also composed of combinations of Ig domains that are related to IpLITR3 Ig domains with varying degrees of amino acid identities (e.g., 25\u201366%). However, none of the predicted ZfLITR-like molecules contained membrane distal Ig domains that were related to IpLITR D1 and D2, which are shared among all the IpLITRs identified to date. This suggests either that LITR membrane distal domains may have coevolved with distinct ligands within individual fish species or that ZfLITRs with D1 and D2 domains similar to IpLITRs have yet to be identified. It should be noted, however, that these zebrafish domains were obtained from genomic sequence and that they do not accurately represent the precise organization of the Ig domains likely to be present in encoded ZfLITRs, which will only be determined from cDNA sequences. Nonetheless, the genomic segments are useful for domain-to-domain comparisons and reinforced the presence of IpLITR-like sequences in another teleost species.\nFig.\u00a07Annotation of zebrafish LITR-like proteins. a Phylogenetic analysis of ZfLITR and IpLITR3 Ig domains. Ig domains were predicted using SMART (Letunic et al. 2004) and the amino acid sequences aligned using CLUSTALW (Thompson et al. 1997). NJ trees with pairwise gap deletions were drawn using MEGA v3.0 (Kumar et al. 2001). Only branch values >50 are shown, and the major Ig domain groups are designated by brackets and shaded according to their relatedness with IpLTR3 Ig domain. b Schematic representation of ZfLITR-like proteins compared with IpLITR3. Domains are shaded according to their phylogenetic relationship with IpLITR3 domains. Hatched bars on IpLITR3 and Zf XP_692267 indicate signal peptides, TM segments are solid black lines, and ITIMs are shown as gray boxes for XP_694305. Amino acid identity vs corresponding IpLITR3 Ig domains are indicated as percentage value above each domain, and only IpLITR3 D3, D4, D5, and D6 were related to the Ig domains of the representative ZfLITR analyzed, as indicated by the dashed box. Predicted size in amino acids for each molecule is indicated on the left\nDiscussion\nThe novel IpLITRs described herein represent a polymorphic and polygenic gene complex that are primarily expressed in hematopoietic tissues. The polymorphic nature of this gene complex was indicated by RFLP analysis, which revealed differences in where various restriction enzymes cut the genomic DNA among the sibling catfish. However, it should be noted that only detailed sequence analyses of these IpLITR genes in a number of individuals would be required to determine if any polymorphic differences are also found within the coding regions. In addition, segregation analyses suggested that IpLITR genes are encoded within multiple independently segregating but homologous loci, similar to the situation seen with the ZfLITR homologs identified by genome mining. These paralagous relationships strongly suggest that IpLITRs are products of multiple gene duplication and translocation events from a common ancestoral gene. Whether or not the receptors encoded by the different IpLITR loci are functionally redundant remains to be determined. However, IpLITRs are related to functionally and genomically distinct mammalian FcRs and LRC encoded receptors, suggesting that such paralogous loci could have evolved to give rise to an array of immunoregulatory receptors with different functions.\nThe coordinate expression of IpLITRs by myeloid and lymphoid cell lines in combination with their potential activating and inhibitory signaling capabilities suggests that they play an important role in immune cell function(s). This possible importance of IpLITRs in immune responses is highlighted by the RT-PCR expression studies where a large number of highly related, but unique, IpLITR sequences were identified. These variant IpLITR sequences differed in the number of Ig domains present, and in some cases in the length of the CYT. Therefore, in addition to the prototype IpLITR1, IpLITR2, and IpLITR3 described here, other IpLITR family members are coexpressed by catfish myeloid and lymphoid cells. Whether or not some of the variously sized transcripts represent messages from different IpLITR genes or are the result of alternative splicing is unknown. However, the large number of unique IpLITRs found expressed suggests that IpLITRs represent a complex immune receptor family that can potentially generate a large number of IpLITR types.\nThe prototypical IpLITR1, IpLITR2, and IpLITR3 encode receptors with four, three, and six Ig domains, respectively. Pairwise comparisons and phylogenetic analysis of these domains suggest a high degree of conservation and a conserved membrane distal to membrane proximal ordering. Each of these three receptors has very similar D1 and D2 sequences, and IpLITR1 and IpLITR3 have almost identical D3 and D4s. However, variation does occur between predicted membrane proximal Ig domains. The IpLITR2 D3 sequence is unique to this receptor and IpLITR3 D5 and D6 are not similar to any other of the prototypical IpLITR Ig domain sequences. Identification of LITR-like sequences on different zebrafish chromosomes and comparisons with IpLITRs supports the notion that these polymorphic and polygenic immune receptors are not unique to catfish. Like IpLITRs, the predicted ZfLITRs are also composed of unique combinations of Ig domains. Such differences in the Ig domain compositions of IpLITRs and ZfLITRs may influence their ligand binding capabilities, formation of homo- or heterodimers and overall immunomodulatory functions.\nIpLITR1 has a long CYT with two consensus ITIMs and a third ITIM-like motif, whereas both IpLITR2 and IpLITR3 have shorter CYTs devoid of recognizable signaling motifs. However, these latter two receptors each contain a lysine residue within their TM segments, indicating that associations with an adaptor molecule(s) would be necessary for them to initiate intracellular signaling (Daeron and Vivier 1999; Ravetch and Lanier 2000; Billadeau and Leibson 2002). Within the CYT of IpLITRI was also an overlapping ITSM suggesting that multiple motifs (ITIMs and ITSM) within the CYT of this receptor may participate in downstream signaling cascades through recruitment of various adaptor molecules (Sidorenko and Clark 2003). Similarly, predicted ZfLITR family members were shown to either contain long CYTs with ITIM motifs or short CYTs with charged TM segments. Genes for immunotyrosine-based-activation-motif-bearing adaptor molecules (e.g., FcR\u03b3 chain, CD3\u03b6, and DAP) have been identified in several ectothermic vertebrates, including frogs and fish (Guselnikov et al. 2003a,b). Hence, it seems likely that IpLITR2 and IpLITR3 would each associate with such molecules and function in a similar manner as mammalian receptors with charged TM segments. Thus, the paired expression pattern of IpLITRs with activatory and inhibitory signaling potential is very similar to that shown for several mammalian and avian families of immune receptors, including PIRs, LILRs, KIRs, FcRs, and CHIRs.\nSimilarity searches for IpLITR relatives revealed a striking dichotomy. The majority of matches were members of two distinct mammalian receptor families. While IpLITR2 appeared to be more closely related to FcRs and FCRLs, IpLITR1 and IpLITR3, exhibiting very similar D1\u2013D4 sequences, were related to both FcR\/FCRLs and LRC-encoded receptors. Thus, it is postulated that the D1 and D2 domains common to all IpLITRs are evolutionarily more closely related to domains encoded by the mammalian FcR and FCRLs, a contention supported by the retrieval of the human Fc\u03b5RI alpha chain as the most similar structure from a PDB search. Phylogenetic analyses also supported this hypothesis because the IpLITR D1 and D2 clustered with members of the FcR and FCRL receptor families and away from representative LRC gene family members. Although the D1D2 of IpLITRs are phylogenetically related to FcR and FcRLs, there is no evidence to support that IpLITRs bind Ig. It appears more likely that the relatedness of these membrane distal Ig domains, as also demonstrated for FCRLs, PIRs, and CHIRs, are only evident at the phylogenetic level and that they are not functional homologs. Furthermore, during the preparation of this manuscript, a \u201cbona fide\u201d Ig-binding catfish FcR homolog, which is not a member of the IpLITR receptor family, was identified and characterized (Stafford et al. 2006). In contrast to the D1 and D2 domains, IpLITR1 and IpLITR3 D3 and D4 Ig domains are related to several members of the LRC (human chromosome 19q13.4). As above, these two domains clustered with one family of receptors (the LRC) and not the other (i.e., FcR\/FcRLs). The identification of NKp46 as a structural relative from the PDB also supports this notion. Interestingly, D5 and D6 of IpLITR3 are related to FcR and FCRLs, but their membrane proximal location suggests that they are unlikely to be involved in ligand recognition (i.e., Ig). Finally, the third domain of IpLITR2 clustered with the Ig domains of several representative CHIRs. Taken together, IpLITR extracellular domain sequences appear reminiscent of both FcR\/FCRLs and members of the mammalian and avian LRC, implying that catfish LITRs are distant relatives to each of these genomically and functionally diverse receptor gene families. At present, the functional significance of these novel teleost immune receptors is unknown.\nRecently, key insights into the evolution of the FcR and LRC gene families and their potential common origin has been presented: (1) human chromosomes 1 and 19 are believed to be in paralogous regions of the genome due to an entire genome duplication event (Kasahara 1999; Shiina et al. 2001); (2) attempts at cloning rodent Fc\u03b1R (CD89) orthologs identified the PIR gene family located on a mouse chromosome, which is syntenic to the human LRC (Kubagawa et al. 1997; Hayami et al. 1997); (3) the subsequent search for distant PIR relatives resulted in the discovery of the CHIRs, providing the first molecular evidence for a common ancestor of LRC and FcR gene families (Dennis et al. 2000); (4) sequencing of genomic segments encoding the CHIR gene family identified the chicken equivalent of an LRC that appears to have also been expanded by gene duplication events (Nikolaidis et al. 2005a); and (5) the identification of Ig-like domains in chickens, Xenopus, and bony fish that are related to Ig domains of mammalian FcRs and members of the LRC (Nikolaidis et al. 2005b). Taken together, these findings suggest that immune receptors encoded by human chromosome 1q21 and 19q13 most likely evolved from a common ancestor that has since undergone multiple rounds of duplication and homologous recombination events.\nAlthough there is functional evidence for the existence of FcR- or KIR\/LILR-like homologs in ectothermic vertebrates (Coosemans and Hadji-Azimi 1986; Shen et al. 2003, 2004), their genetic identification has not been previously reported and the inability to clone these receptors has made it difficult to elucidate their evolutionary history. The recent discovery of mammalian FCRL genes located on chromosome 1q21-22, however, has expanded the view of immune receptor evolution (Davis et al. 2001; Hatzivassiliou et al. 2001; Miller et al. 2002; Guselnikov et al. 2002). For example, FCRL3, FCRL4, and FCRL5 not only encode D1, D2, and D3 Ig domains homologous to the corresponding domains of Fc\u03b3RI, but also contain variable numbers of unique membrane proximal Ig domains (Davis et al. 2001; Hatzivassiliou et al. 2001). Homology searches demonstrated that the FCRLs, also termed IFGP (for IgSF, FcR, and gp42) exhibited a \u201cchimeric\u201d Ig domain compositions and consequently these receptors were proposed to be a phylogenetic link between the FcRs, rat NK cell-specific gp42 antigen, and adhesion molecules (Guselnikov et al. 2002). This feature of similar Ig domains encountered in homologous but functionally diverse receptors is also a striking feature of the IpLITRs and supports the hypothesis that certain mammalian immune receptor families (i.e., FcRs and KIRs) have evolved from a common ancestor. Notably, although the IpLITRs D1 and D2 domains are phylogenetically and structurally similar to the mammalian FcRs and FCRLs, the IpLITR ligand(s) is unknown. This is similar to the situation with mammalian FCRL molecules, whose membrane distal Ig domains are clearly related to the classic FcRs, but are unlikely to be functional homologs because they do not bind Ig (Hatzivassiliou et al. 2001; Davis et al. 2005). Therefore, although certain Ig domains encoded by several receptor gene families identified in humans, rodents, birds, and fish (i.e., FCRLs, PIRs, CHIRs, and IpLITRs, respectively) are related to FcRs, they appear to have evolved to recognize different ligands. Recently, it was shown that rodent PIRs recognized MHC class 1 and \u03b22M (Takai 2005), raising the possibility that related immunoregulatory receptors in other species (i.e., CHIRs and IpLITRs) could also be involved in MHC recognition.\nIn summary, the discovery of individual catfish receptors that encode Ig domains related to both FcR and LRC gene families provides further support for the common origin of these important mammalian immune receptors. Identification of LITR-like sequences in the zebrafish genome also demonstrates that these receptors are likely found in all teleosts. Annotation of these genomic segments will be necessary to determine if zebrafish LITR homologs exhibit conserved genomic features and syntenic genomic location with mammalian 1q21-23 and\/or 19q13. Thus, the discovery of IpLITRs is an important step towards further understanding the common origin and evolutionary history of the different families of mammalian immunoregulatory receptors. The investigation of the functional significance of these novel teleost receptors is underway and will be aided by the availability of channel catfish cell lines that coordinately express IpLITRs and the eventual production of IpLITR specific antibodies.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nElectronic Supplementary Figure\u00a01\n52 kb","keyphrases":["teleosts","innate immunity","evolution","molecular immunology","inhibitory receptors"],"prmu":["P","P","P","M","R"]}
{"id":"Hum_Reprod-1-1-2387217","title":"Predicting the FSH threshold dose in women with WHO Group II anovulatory infertility failing to ovulate or conceive on clomiphene citrate\n","text":"BACKGROUND The objective of this investigation was to establish independent predictors of follicle-stimulating hormone (FSH) threshold dose in anovulatory women undergoing ovulation induction with FSH preparations.\nIntroduction\nApproximately 20\u201330% of the women seeking fertility treatment present with anovulation (Healy et al., 1994). Clomiphene citrate has been used as the first line treatment for inducing ovulation in anovulatory women. In most women who failed to ovulate or conceive with clomiphene citrate, ovarian stimulation with gonadotrophins leads to ovulation (Coelingh-Benning et al., 1998; Yarali et al., 1999; Platteau et al., 2006; Balen et al., 2007). The use of low-dose step-up protocols has been shown to be successful in producing mono-follicular development in more than half of the women undergoing stimulation with gonadotrophins, and thereby minimizing the risk of multiple follicular development and the associated risks of ovarian hyperstimulation syndrome (OHSS) and multiple pregnancy (Homburg and Howles, 1999).\nA typical low-dose step-up protocol starts with 50\u201375 IU\/day of follicle-stimulating hormone (FSH) or menotrophins for \u223c7 to 14 days, and the dose is then increased by 37.5\u201350 IU\/day if adequate response is not obtained. For some patients, the dose of gonadotrophin needed to provide adequate response is relatively high and the use of low-dose regimens will in these cases lead to stimulations of long duration. A long ovarian stimulation is associated with more monitoring visits and would be expected to result in increased costs and inconvenience for the patients. Selection of the most optimal starting dose of gonadotrophin for each individual patient should minimize these disadvantages, while ensuring adequate response.\nImani et al. (1998) have identified clinical and laboratory parameters such as body mass index (BMI), menstrual cycle history, mean ovarian volume and free androgen index (FAI) which can predict ovulation in WHO type II anovulatory women undergoing clomiphene citrate treatment. A similar investigation in anovulatory women undergoing ovulation induction with gonadotrophins suggested BMI, clomiphene citrate resistance, serum insulin-like growth factor-1 (IGF-1) and serum FSH as significant predictors of FSH threshold dose (Imani et al., 2002). The clinical application of this latter result is complicated by the fact that the IGF-1 assay is not part of most clinics standard battery for laboratory screening of anovulatory patients. Therefore, further data are needed to identify simple parameters which can predict the FSH threshold dose in women who will undergo low-dose protocols with gonadotrophins.\nThe purpose of the present prospective investigation was to evaluate the influence on the FSH threshold dose of clinical, sonographic and endocrine parameters available at the start of ovarian stimulation as well as type of FSH preparation to be used in a low-dose step-up protocol. The data from this investigation form the basis for constructing a nomogram which provides an indication of the expected FSH threshold dose for the individual patient, facilitating the selection of the most appropriate starting FSH dose for ovarian stimulation.\nMaterials and Methods\nStudy population and design\nThis investigation included 151 women with anovulatory WHO Group II infertility who were included in a randomized, open label, assessor-blind, parallel group, multicentre, multinational, non-inferiority study comparing two different FSH preparations (highly purified (HP)-FSH, BRAVELLE, Ferring Pharmaceuticals A\/S, Copenhagen, Denmark and recombinant FSH (rFSH), follitropin alfa, GONAL-F, Serono, Geneva, Switzerland) with respect to ovulation rate. Patients were recruited in 22 fertility centres (12 in Belgium, 7 in Denmark and 3 in the UK) that on average each included seven patients, ranging from 2 to 20. A detailed description of the study population, all inclusion and exclusion criteria, all study procedures, as well as the main outcome of the study, is described in detail in a separate publication (Balen et al., 2007).\nPatients were to be 18\u201339 years of age with BMI 19\u201335 kg\/m2, have been diagnosed with infertility for \u22651 year, have chronic anovulation (amenorrhea, oligomenorrhea or anovulatory cycles based on progesterone levels in patients with cycle lengths of 21\u201335 days) and have failed to ovulate with clomiphene citrate doses of at least 100 mg\/day for at least 5 days or failed to conceive after three cycles of ovulation induction with clomiphene citrate. Women with a history of \u226512 unsuccessful ovulation induction cycles, persistent ovarian cysts (\u226515 mm) for >1 cycle or ovarian endometrioma on ultrasound and any significant systemic disease, endocrine or metabolic abnormalities were excluded. The study was carried out in accordance with the declaration of Helsinki on good clinical practice and ethical committee approval was obtained in all participating centres.\nAll patients underwent screening assessments prior to start of ovarian stimulation. These included evaluation of demographics (age), physical characteristics (body weight, height, waist and hip circumference), obstetric history (previous pregnancies, previous live births), infertility history (duration of infertility, number of previous ovulation induction cycles with clomiphene citrate and in total, failure to ovulate with clomiphene citrate, failure to conceive with clomiphene citrate) and menstrual cycle history. Just prior to the first dose of gonadotrophin, patients underwent a transvaginal ultrasound and blood sampling for endocrine evaluation. The sonographic recordings comprised dimensions of right and left ovary, number of follicles (antral follicles >2 mm) in right and left ovary and endometrial thickness. The endocrine evaluation included the following parameters: luteinizing hormone (LH), FSH, estradiol (E2), prolactin, total testosterone, sex hormone binding globulin (SHBG), FAI, glucose and insulin. Chemiluminescent immunometric assays were used for LH, FSH, prolactin, total testosterone and SHBG, and radioimmunoassays were used for E2 and androstenedione, whereas a hexokinase assay was used for glucose and immulite technology was applied for analysis of insulin. All endocrine analyses were conducted by a central laboratory (Quest Diagnostics\u2014Quest Diagnostics Limited, Heston, UK; Quest Diagnostics Limited, Van Nuys, CA, USA; Nichols Institute, San Juan Capistrano, CA, USA). FAI was calculated as (total testosterone\/SHBG) \u00d7 100.\nAll patients underwent ovarian stimulation following a low-dose step-up protocol. Stimulation was started 2\u20135 days after a spontaneous or progesterone-induced menstrual bleed. The starting dose of gonadotrophin was 75 IU daily, which was maintained for 7 days. After the first 7 days, the gonadotrophin dose was either maintained or increased by 37.5 IU increments according to individual response. Patients were maintained on any specific dose level for at least 7 days. The maximum allowed daily dose was 225 IU and patients were treated with gonadotrophin for a maximum of 6 weeks. Gonadotrophin stimulation was maintained until at least one of the following criteria for human chorionic gonadotrophin (hCG) administration was met: one follicle with a diameter of 17 mm or greater, or 2\u20133 follicles with a diameter of 15 mm or greater. Patients were not given hCG in either of the following situations: no follicular response after 6 weeks of gonadotrophin treatment, \u22654 follicles with diameters of \u226515 mm or serum E2 levels >2000 pg\/ml. Patients who reached the hCG criteria received a single s.c. or i.m. injection of hCG (PROFASI, Serono) at a dose of 5000 IU to trigger ovulation. At least one blood sample was taken during the midluteal phase (6\u20139 days after hCG administration) and analysed for progesterone by a central laboratory using competitive immunoassay using direct chemiluminometric technology with a sensitivity of 0.48 nmol\/l (Quest Diagnostics Limited, UK). Ovulation was defined as a mid-luteal serum progesterone concentration of \u226525 nmol\/l (\u22657.9 ng\/ml).\nThe FSH threshold dose was defined as the dose of FSH that was given when the criteria for hCG was reached. The analysis involved the influence of the type of gonadotrophin preparation (i.e. HP-FSH or rFSH) as well as of clinical, sonographic and basal endocrine parameters on the FSH threshold dose. All potential predictive variables were investigated: age, BMI, waist\u2013hip ratio, previous pregnancy, duration of infertility, failure to ovulate on clomiphene citrate, menstrual cycle history (amenorrhea if the patient had absent menstruation, oligomenorrhea if menstruation intervals were longer than 35 days or cycle length of 21\u201335 days in which case anovulation had been documented by progesterone levels), total number of follicles, mean ovarian volume (based on length, width and depth of both right and left ovary), LH\/FSH ratio, androstenedione, total testosterone, SHBG, FAI, prolactin, estradiol, glucose, insulin and insulin\/glucose ratio.\nStatistical analysis\nThe primary end-point of the study was ovulation rate after one cycle of gonadotrophin treatment, and these data have been reported elsewhere (Balen et al., 2007). The influence of the selected clinical, sonographic and endocrine parameters as well as type of gonadotrophin on FSH threshold dose were assessed in univariate and multivariate analyses using proportional odds polytomous logistic regression models (an extension of the model and method for binary response). Multivariate analyses were performed including characteristics that had statistically significant influence on the FSH threshold dose in the univariate analysis first by entering all selected characteristics into the polytomous logistic model in a forward selection approach and secondly by entering all selected characteristics into the polytomous logistic model using a backward elimination approach. P-values of <0.05 were regarded as statistically significant. Score tests were used to evaluate the proportional odds assumption and Wald tests were used to assess the influence of the baseline characteristics on the FSH threshold dose. For the present analysis, all parameters were analysed as continuous variables, except for type of gonadotrophin, previous pregnancy, failure to ovulate on clomiphene citrate and menstrual cycle history (glucose and insulin were categorical variables as normal\/abnormal and insulin\/glucose ratio was a continuous variable).\nIn addition to the above, the model was evaluated on the subset of patients with mono-follicular development (one follicle \u226517 mm and no follicles of 15\u201316 mm). In this evaluation, the same set of variables was used as selected for the main model according to the above procedure. The predictive performance of the model was assessed by using the \u2018leave-one-out\u2019 cross-validation procedure. In addition, the residual standard error after a standard multiple regression analysis was evaluated.\nFinally, ROC curves were produced and AUC under the ROC curves were calculated. For the standard situation where ROC curves are used, the classification is binary, whereas the threshold dose had four possible outcomes (87.5, 112.5, 150 and 187.5) and the prediction model therefore also predicts into four classes. A consensus approach on how to handle multiclass ROC has not been reached (Landgrebe and Duin, 2007), but when the outcome classes are ranked, ROC curves can be made for each of the possible levels (Waegeman et al., 2006).\nResults\nThe clinical, sonographic and endocrine characteristics of the 151 patients included in the study are shown in Table\u00a0I. The mean age of the study population was 28.9 years, ranging from 19 to 39 years. The BMI spanned from 18 to 39 kg\/m2, with 54% of the patients having a normal BMI, 32% being overweight and 14% being obese and with the mean BMI being 24.8 kg\/m2. On average, patients had been infertile for \u223c3 years and had undergone four previous ovulation induction cycles with clomiphene citrate, with 34% of the patients failing to ovulate on clomiphene citrate. Regarding menstrual cycle history, 56% of the patients were oligomenorrheic and 15% amenorrheic. An LH\/FSH ratio above 1 was noted for 59% of the patients. Among the 151 patients who started stimulation with FSH (HP-FSH 73, rFSH 78), 132 patients met the hCG criteria (HP-FSH 65, rFSH 67) and contributed with data to the analysis of FSH threshold dose. There were 19 patients who initiated gonadotrophin therapy but did not contribute to this analysis; 10 discontinued from the study during ovarian stimulation (five because of excessive response), 2 had spontaneous ovulation and 7 received hCG despite either not meeting or exceeding the hCG criteria.\nTable\u00a0I.\nClinical, sonographic and endocrine parameters just prior to starting ovarian stimulation with FSH (Day 1 of stimulation).\nTotal (n = 151)\nClinical parameters\nAge (years)\n28.9 \u00b1 3.7\nBody weight (kg)\n68.6 \u00b1 13.2\nBMI (kg\/m2)\n24.8 \u00b1 4.5\nPrimary infertility, n (%)\n97 (64%)\nDuration of infertility (years)\n2.8 \u00b1 1.7\nPrevious cycles of ovulation induction (all)\n4.9 \u00b1 2.4\nPrevious cycles of ovulation induction (with clomiphene citrate)\n4.1 \u00b1 2.4\nClomiphene citrate non-responders, n (%)\n\u2003Failure to ovulate on clomiphene citratea\n51 (34%)\n\u2003Failure to conceive on clomiphene citrateb\n100 (66%)\nMenstrual cycle history, n (%)\n\u2003Amenorrhea\n23 (15%)\n\u2003Oligomenorrhea\n84 (56%)\n\u2003Anovulatory cycles with cycle length 21\u201335 days\n44 (29%)\nSonographic parameters\nMean ovarian volume (cm3)\n7.1 \u00b1 3.5\nAntral follicles >2 mm\n14.8 \u00b1 12.5\nEndometrial thickness (mm)\n4.1 \u00b1 2.2\nEndocrine parameters\nLH (IU\/l)\n7.1 \u00b1 4.6\nFSH (IU\/l)\n5.5 \u00b1 2.4\nLH\/FSH ratio\n1.4 \u00b1 0.8\nProlactin (\u03bcg\/l)\n16 \u00b1 13\nAndrostenedione (nmol\/l)\n6.74 \u00b1 3.08\nTotal testosterone (nmol\/L)\n1.8 \u00b1 0.6\nSex hormone binding globulin (nmol\/l)\n61 \u00b1 40\nFree androgen index\n4.45 \u00b1 3.47\nEstradiol (pmol\/l)\n172 \u00b1 98\nGlucose (mmol\/l)\n\u2003Fasting\n5.1 \u00b1 0.6\n\u2003Non-fasting\n5.2 \u00b1 1.5\nInsulin (pmol\/l)\n\u2003Fasting\n66.4 \u00b1 43.1\n\u2003Non-fasting\n143.9 \u00b1 192.6\nAll data are mean \u00b1 SD or number of patients (percentage of patients).\naOn at least 5 days of 100 mg\/day.\nbAfter three cycles.\nTable II provides the outcome of the univariate and multivariate analysis for the predictors of FSH threshold dose. Eight parameters were statistically significant predictors of FSH threshold dose (P < 0.05): age, BMI, failure to ovulate with clomiphene citrate, menstrual cycle history (amenorrhea, oligomenorrhea or anovulatory cycles with cycle length 21\u201335 days), mean ovarian volume, LH\/FSH ratio, testosterone and FAI. The type of gonadotrophin preparation, HP-FSH or rFSH, did not predict FSH threshold dose. All eight significant parameters were included in a multivariate analysis which showed that only three of the parameters were independently statistically significant (P-values < 0.001) predictors of FSH threshold dose: BMI, mean ovarian volume and menstrual cycle history (Table III). The odds for needing >75 IU FSH were 1.18 [95% confidence interval (CI): 1.07\u20131.29] for BMI and 1.22 [95% CI: 1.09\u20131.37] for mean ovarian volume; thus the higher the BMI and ovarian volume prior to start of stimulation, the higher the FSH threshold dose. Regarding the menstrual cycle history, the odds for needing a FSH dose >75 IU were 11.9 [95% CI: 3.35\u201342.0] and 2.57 [95% CI: 0.97\u20136.79] for amenorrhea and oligomenorrhea, respectively, compared with anovulatory cycles with a length of 21\u201335 days. The observed data for these three independent predictors are presented in Table III.\nTable II.\nStatistically significant predictors of FSH threshold dose (univariate and multivariate analysis).\nUnivariate analysis\nMultivariate analysis\nOR (95% CI)\nP-valuea\nOR (95% CI)\nP-valuea\nAge (years)\n0.91 (0.83\u20131.00)\n0.040\n\u2014\n\u2014\nBMI (kg\/m2)\n1.10 (1.02\u20131.19)\n0.010\n1.17 (1.07\u20131.29)\n<0.001\nFailure to ovulate on clomiphene citrate: yes versus no\n2.27 (1.14\u20134.52)\n0.020\n\u2014\n\u2014\nMenstrual cycle history\n<0.001\n<0.001\n\u2003Amenorrhea versus cycle length 21\u201335 days\n8.33 (2.81\u201324.7)\n11.88 (3.35\u201342.1)\n\u2003Oligomenorrhea versus cycle length 21\u201335 days\n3.48 (1.51\u20138.03)\n2.57 (0.97\u20136.79)\nMean ovarian volume (cm3)\n1.18 (1.06\u20131.31)\n0.002\n1.22 (1.08\u20131.37)\n<0.001\nFree androgen index (nmol\/l)\n1.25 (1.13\u20131.38)\n<0.001\n\u2014\n\u2014\nTotal testosterone (nmol\/l)\n2.14 (1.14\u20134.03)\n0.018\n\u2014\n\u2014\nLH\/FSH ratio (IU\/l)\n1.59 (1.03\u20132.45)\n0.038\n\u2014\n\u2014\naWald test for effect of factor\/covariate.\nTable III.\nBMI, mean ovarian volume and menstrual cycle history by observed FSH threshold dose.\nBMI (kg\/m2)\nMean ovarian volume (cm3)\nMenstrual cycle history (%) Amenorrhea \/ oligomenorrhea \/ cycle length 21\u201335 days\n75 IU (n = 67)\n23.8 \u00b1 4.2\n6.3 \u00b1 2.9\n8% \/ 48% \/ 45%\n112.5 IU (n = 46)\n25.5 \u00b1 4.8\n7.3 \u00b1 3.4\n22% \/ 63% \/ 15%\n150 IU (n = 15)\n25.8 \u00b1 4.0\n9.5 \u00b1 4.8\n33% \/ 47% \/ 20%\n187.5 IU (n = 4)\n28.1 \u00b1 4.6\n10.2 \u00b1 2.3\n25% \/ 75% \/ 0%\nAll data are mean \u00b1 SD or percentage of patients.\nThe model predicted the FSH threshold dose to be the same as the dose observed in the study for 59% of the patients. The AUC under the ROC curve was 0.78 for 75 IU (Fig.\u00a01a) and 0.79 for 112.5 IU (Fig.\u00a01b). In a multiple regression analysis with the same three variables as in the logistic model, the FSH threshold dose was estimated on a continuous scale. From this estimation, the residual mean square error was 25 IU and the R2 = 25%.\nFigure 1:\nROC curves for threshold dose (a) 75 IU versus <75 (AUC = 0.78) and (b) \u2264112.5 IU versus >112.5 IU (AUC = 0.79).\nA simple nomogram can be constructed based on the two clinical and one sonographic parameters that significantly predict the FSH threshold dose. Figure 2 displays three nomograms\u2014one for each type of menstrual cycle history\u2014where the shaded areas indicate the predicted FSH threshold dose, based on the BMI displayed on the x-axis and mean ovarian volume on the y-axis. For example, the expected threshold FSH dose for a patient with amenorrhea (Fig. 2a), mean ovarian volume of 10.5 cm3 and a BMI of 30 kg\/m2 will be 150 IU. In the case that the patient presents with oligomenorrhea (Fig. 2b), but similar mean ovarian volume and BMI, the expected threshold FSH dose would be 112.5 IU. As another example, a threshold FSH dose of 75 IU will be expected for a patient with anovulatory cycles of 21\u201335 days, mean ovarian volume of 8.5 cm3 and BMI 27 kg\/m2, whereas it will be 112.5 IU for a patient with oligomenorrhea and similar mean ovarian volume and BMI.\nFigure 2:\nNomograms for prediction of individual FSH threshold dose in anovulatory patients undergoing ovulation induction with FSH preparations, according to menstrual cycle history, BMI and mean ovarian volume. (a) Amenorrhea, (b) oligomenorrhea and (c) anovulatory cycles with cycle length 21\u201335 days.\nThe accuracy of the model was 60% when applied to the subset of patients with mono-follicular development (80 patients), and thus similar to that achieved for the overall study population.\nDiscussion\nThe main finding in the present study was identification of the three variables: menstrual cycle history, BMI and mean ovarian volume, as independent predictors of the FSH threshold dose during FSH treatment of anovulatory infertility. For clinical use, such data could be implemented and used as screening predictors to determine the individual FSH threshold dose of any patient before start of gonadotrophin treatment, in order to select the most appropriate starting dose. For this purpose, we have used the three independent predictors to develop the FSH dose nomograms presented in Fig. 2. The proposed model had an accuracy of 59\u201360%, which is well above the 25% that would be expected by random allocation to one of the four dose levels evaluated in this study.\nOur findings are partly in line with those obtained by Imani et al. (2002) who studied 90 anovulatory women, also treated with either urinary or rFSH, and showed that BMI, clomiphene resistance, free IGF-1 and basal FSH were independent predictors of the FSH threshold dose. Imani et al. (2002) made an equation showing that the predicted dose was: [4 BMI (in kg\/m2)] + [32 clomiphene citrate resistance (yes = 1 or no = 0] + [7 initial free IGF-I (in ng\/ml)] + [6 initial serum FSH level (in IU\/l)] \u2212 51. Similar to our study, it was found that menstrual bleeding interval as well as clomiphene resistance were significant predictors in the univariate analysis, but among the possible clinical predictors only clomiphene resistance and BMI remained significant in the multivariable analysis. The population studied by Imani et al. (2002) seems very similar to the present study, even though the latter included patients if they failed to conceive after three clomiphene cycles, whereas the study by Imany et al. (2002) only included women with six failed ovulatory clomiphene citrate cycles, but it seems unlikely that this would explain the difference in predictive factors. A more plausible explanation could be that the study by Imani et al. (2002) included, in the forward step-wise multivariate analysis, some endocrine factors, such as free-IGF-I and its binding proteins, that may alter the predictors that remained in the final model during the analysis.\nIt is worth briefly discussing a statistical methodological difference between the present model and that suggested by Imani et al. (2002). The polytomous logistic regression model used on the present data set predicts FSH threshold doses that correspond to actual dose levels applied in the clinical trial, in contrast to a multiple regression where the predicted dose is on the continuous scale, as the equation published by Imani et al. (2002). In our model, the residual mean square error was 25 IU and therefore the individual prediction is at least as good as in the model suggested by Imani et al. (2002) where the residual mean square error was 35 IU. The R2 value of 25% in the present model is explained by the relatively few patients in the two highest dose levels in our trial compared with a more even distribution of patients in the trial conducted by Imani et al. (2002) yielding an R2 of 39% in their model.\nOur finding that the menstrual status was important is in line with expectations. Patients with amenorrhea may need more FSH to reach the threshold than a patient with oligomenorrhea, who may also need more FSH compared with patients with anovulatory cycles, indicating that the severity of the ovarian dysfunction may well be reflected in the degree of menstrual cycle disturbance. The relative resistance to FSH in amenorrheic patients is supported by the finding that these patients also have a relative resistance to endogenously released FSH during clomiphene citrate treatment (Imani et al., 1998), as patients with clomiphene resistance do release a similar amount of endogenous FSH as the clomiphene sensitive women (Polson et al., 1989). This supports the concept that it is indeed the threshold of the ovarian sensitivity that is altered.\nA high BMI was associated with a higher FSH threshold dose; an observation that is supported by the findings that the total dose of gonadotrophins needed to induce ovulation is increased in parallel with body weight (Hamilton-Fairly et al., 1992; Balen et al., 2006). The cause of the association with BMI may be that the endocrine disturbance is related to the degree of body fat and hyperinsulinaemia, but it may also be due to a lower bioavailability of exogenous gonadotrophins in obese women (Chan et al., 2003; Steinkampf et al., 2003).\nThe type of gonadotrophin used for stimulation was not a significant predictor of FSH threshold dose, and thus of limited relevance compared with the patient's clinical characteristics.\nIn theory, the threshold is defined as the lowest dose that induces growth of the single most sensitive follicle. In this clinical trial, a dual criterion for hCG administration was used, covering not only the development of a single dominant follicle, defined as one follicle of 17 mm or greater, but also the presence of 2\u20133 follicles of 15 mm or greater. Therefore, our threshold is based on the stimulation in those 132 (87%) patients who reached either of these criteria, including those patients with up to 3 follicles \u226515 mm. The threshold dose in the study and subsequently the model could also be termed the \u2018response dose\u2019 and can be viewed as a surrogate for the real threshold. It should be considered that serum FSH may accumulate over time so the theoretically ideal threshold should be defined as the dose that caused growth of a single dominant follicle to around 9\u201310 mm, where dominance is normally established. The exact timing of dominance was not determined in the present study, but it is important to note that the model for threshold dose predictions also applied in the subset of patients with mono-follicular development defined as only one follicle \u226517 and no follicles of 15\u201316 mm. The average number of follicles \u226515 mm was 1.5 (Balen et al., 2007) indicating that some patients did indeed receive FSH doses slightly above the threshold for single dominant follicle development. However, the analysis restricted to patients with mono-follicular development showed that the accuracy of the model was the same for this subset of patients as for the overall study population. On the basis of the intention to treat population in the present study, 92% of the patients received hCG and the ongoing pregnancy rate was 19%, of which 79% accounted for singletons and 21% for multiple pregnancies (Balen et al., 2007). These figures are much in line with literature data on 7 day step-up protocols (Homburg and Howles, 1999), but it could be argued that the multiple pregnancy rate is somewhat high, compared with a number of recent studies using starting doses of FSH <75 IU\/day (Balasch et al., 2000; Calaf Alsina et al., 2003). Indeed, multiple pregnancy rates as low as 6% have been reported in a large clinical trial using starting doses of 50 IU\/day, even though up to 3 follicles >16 mm before hCG administration was accepted (Calaf Alsina et al., 2003). An alternative approach is to restrict hCG administration to those patients who have only one or two mature follicles, but this would increase the cancellation rates. As published (Balen et al., 2007), 3.3% (5\/151) of the patients had their cycle cancelled before hCG, due to development of multiple follicles. Identification of these patients with a very low threshold would have necessitated a starting dose of <75 IU\/l.\nOvarian volume was another significant parameter predicting the FSH threshold dose. The larger the ovaries, the more FSH was needed to obtain adequate ovarian response. This finding is consistent with earlier clinical studies of ovulation induction with gonadotrophins, where it has been shown that patients with larger ovaries need longer duration of stimulation and higher doses of gonadotrophins (Lass et al., 2002).\nVan den Meer et al. (1994) studied three consecutive cycles in 16 patients during gradually increased doses of intravenously infused urinary FSH and elegantly documented how the inter-individual variability in FSH threshold dose was far greater than the variability from cycle to cycle. The key problem is therefore to define the appropriate dose for any individual patient and the present study provides a suggestion for nomograms that may be used clinically. A major concern, if patients were dosed according to the nomograms, would be that 13% would receive a higher starting dose that the observed threshold dose. The implications of an excessive starting dose are likely to be the same as when step-down protocols are used, i.e. risk of multiple follicular development. Indeed, van Santbrink et al. (2002) have used the prediction model developed by the same group of investigators (Imani et al., 2002), and retrospectively evaluated the doses used in a step-down regimen. In that study, even though the average excessive dose was 28.5 IU\/day above the threshold, the step-down approach resulted in an overall multiple pregnancy rate of 17% and not a single case of OHSS (van Santbrink et al., 2002).\nThe expected main advantage of dosing according to the nomogram would be a shortening of the stimulation phase. It is important to note that the suggested dosage nomograms could be easily used clinically because they only include simple characteristics that are routinely recorded in all anovulatory patients. Before using this model in clinical practice, a randomized controlled trial should be conducted to compare the presently used low-dose step-up protocol with a flexible protocol using this model.\nFunding\nThe study was sponsored by Ferring Pharmaceuticals A\/S, Copenhagen, Denmark. Funding to pay the Open Access publication charges for this article was provided by Ferring Pharmaceuticals A\/S, Copenhagen, Denmark.","keyphrases":["threshold dose","anovulation","predictors","follicle-stimulating hormone","efficiency"],"prmu":["P","P","P","P","U"]}
{"id":"Eur_Radiol-4-1-2270358","title":"Dual-source CT for chest pain assessment\n","text":"Comprehensive CT angiography protocols offering a simultaneous evaluation of pulmonary embolism, coronary stenoses and aortic disease are gaining attractiveness with recent CT technology. The aim of this study was to assess the diagnostic accuracy of a specific dual-source CT protocol for chest pain assessment. One hundred nine patients suffering from acute chest pain were examined on a dual-source CT scanner with ECG gating at a temporal resolution of 83 ms using a body-weight-adapted contrast material injection regimen. The images were evaluated for the cause of chest pain, and the coronary findings were correlated to invasive coronary angiography in 29 patients (27%). The files of patients with negative CT examinations were reviewed for further diagnoses. Technical limitations were insufficient contrast opacification in six and artifacts from respiration in three patients. The most frequent diagnoses were coronary stenoses, valvular and myocardial disease, pulmonary embolism, aortic aneurysm and dissection. Overall sensitivity for the identification of the cause of chest pain was 98%. Correlation to invasive coronary angiography showed 100% sensitivity and negative predictive value for coronary stenoses. Dual-source CT offers a comprehensive, robust and fast chest pain assessment.\nIntroduction\nComprehensive CT angiography protocols for a complete assessment of the thoracic vessels, often referred to as \u201ctriple rule out\u201d protocols, are used in the differential diagnosis of chest pain increasingly often [1\u20134]. These protocols aim to opacify pulmonary and coronary arteries as well as the aorta simultaneously to rule out pulmonary embolism, coronary artery disease and aortic aneurysm or dissection in a single exam. With ECG gating, the acquired images of the coronary arteries should have a similar diagnostic accuracy as a specific coronary CT angiography. Quite a few studies have meanwhile shown the feasibility of a simultaneous evaluation of these vascular territories in one single breathhold scan with a good sensitivity in the identification of the cause of chest pain [5]. Also, recent studies indicate that coronary CT angiography can be helpful for a fast and cost-effective triage of chest pain patients [6, 7]. So far, a major limitation of these studies especially in acutely ill patients is the restricted image quality of the coronary arteries in high heart rates [8]. The administration of beta-blockers to lower heart rates is general practice in 16- and 64-slice CT [9]. However, this approach is time consuming and limited by contraindications, and a sufficient reduction of the heart rate cannot be achieved in many acutely ill patients. Initial studies of dual-source CT (DSCT) cardiac imaging have shown a robust image quality and a very good diagnostic accuracy of coronary CT angiography even in high heart rates [10, 11]. Additionally, a more comprehensive cardiac assessment including wall motion and valve function is possible with DSCT [10]. The aim of this study was to assess the diagnostic accuracy of a specific dual-source CT protocol for chest pain assessment, regarding a 6-month follow-up for other findings and coronary angiography in patients in whom coronary artery disease could not be reliably excluded as standard of reference.\nMaterials and methods\nPatients\nOne hundred nine consecutive patients [31 women, 78 men; median age 64 (59\u201367) years] were prospectively enrolled in the trial. The study was approved by the institutional review board, and informed written consent was obtained from every patient prior to the examination. All patients with acute chest pain were eligible for the study if referred by a colleague after initial diagnostic workup including physical examination, ECG and serum levels of creatinine and TSH. Exclusion criteria were positive ECG changes or troponine test, severe ventricular arrhythmia, a history of severe allergoid reaction to iodinated contrast material, renal insufficiency and young age below 30\u00a0years. Also, severe dyspnea with inability to hold the breath for at least 15 s was regarded as exclusion criterion. The patients were asked to hold their breath for approximately 15 s prior to the examination. If the breathhold could not be maintained, the patient was excluded from the study, and seven otherwise eligible patients had to be excluded for this reason. Heart rates were 68\u2009\u00b1\u200914, ranging from 58 to 118\u00a0bpm. Beta-blockers were not administered in preparation of the scan. Eighteen patients had known coronary artery disease and 14 were on continuous oral beta blocker medication. One patient had a known chronic aortic dissection.\nExamination\nExaminations were acquired on a DSCT Somatom Definition (Siemens, Forchheim, Germany) in supine position. A coronal topogram was scanned to plan the spiral acquisition, which included the whole chest from the first ribs to the diaphragm. The level of the carina was defined as the trigger point where the second tube is switched on additionally. The volume and flow rate of the contrast material (Ultravist 370, Schering, Germany) were adapted to the patient\u2019s body weight as described elsewhere [5] according to Table\u00a01. A bolus tracking technique was used to assess the transit time of the contrast agent. For this purpose, a region of interest was defined in the ascending aorta to detect the arrival of the contrast material bolus in images scanned at 2-s intervals, and the scan was initiated with a delay of 4 s if the density increased more than 100 Hounsfield units (HU). The patient was then instructed to hold his breath at a mild inspiratory position, and the spiral scan was started. The parameters for the acquisition in craniocaudal direction were 0.33-s gantry rotation time, 120-kVp tube voltage, 560-mAs effective tube current with 0.6-mm collimation and double Z-sampling. The pitch and the tube current modulation were adapted automatically to the heart rate of the patient. Pitch values ranged between 0.2 and 0.48, whereas the full tube current was only applied at 70% of the cardiac cycle for heart rates below 70 bpm and between 30 and 70% for higher heart rates. Scan duration was 15\u2009\u00b1\u20093 s. \nTable\u00a01Body-weight-adapted contrast material injection protocolWeight (kg)Volume (ml)Flow (ml\/s)501063.5551113.7601163.9651214.0701254.2751304.3801344.5851384.6901424.7951464.91001505.01051545.11101575.21151615.41201645.5\nImage reconstruction\nContinuous ECG-gated axial slices and coronal images of the whole chest were reconstructed with a temporal resolution of 330\u00a0ms using a soft kernel (B30f). Slice thickness of 3\u00a0mm and 2.5-mm increment were applied to avoid artificial inhomogenities in the lung parenchyma due to undersampling with gating. Additionally, axial slices of the heart were reconstructed for a limited range from the carina to the diaphragm with a 200-mm field of view, 0.75-mm slice thickness and 0.5-mm increment using a B26f convolution kernel. If stents had been implanted, an additional reconstruction was performed using a B46 kernel to reduce blooming and enhance the depiction of the lumen. For ECG gating, a single segment reconstruction algorithm utilizing quarter scan segments from both detectors was applied to achieve a temporal resolution of 83\u00a0ms, and delays were set to 300\u00a0ms and 70% of the cardiac cycle for the initial reconstructions. Also, axial slices with 1-mm thickness and increment were reconstructed with a B26f kernel for the whole cardiac cycle at 10% intervals for dynamic evaluation.\nDiagnostic evaluation\nThe examinations were read by two radiologists in consensus immediately after the exam, including a review of axial and coronal slices for evaluation of pulmonary embolism and aortic disease (Figs.\u00a03 and 4). Additionally, a complete cardiac workup (Figs.\u00a01 and 2) was done using a 3D workstation (Multimodality Workplace, Siemens, Forchheim, Germany) to interactively browse multiplanar reconstructions and volume-rendered images if necessary. Multiphase reconstructions were reviewed as cine loops of the heart with interactive browsing of dynamic short- and long-axis sections. The coronary artery tree was extracted using region-growing algorithms, and curved multi-planar reformats were created for review (Fig.\u00a01a,b). In case of motion artifacts, additional reconstructions with an optimized delay were available. If these still did not resolve motion artifacts sufficiently to make a sufficient diagnostic evaluation feasible, the depiction of the respective coronary artery segment was rated as non-diagnostic and excluded from further analysis. Additionally, the multiphase reconstructions were reviewed to assess wall motion abnormalities and valvular disease (Fig.\u00a02a-d).\nFig.\u00a01High grade stenosis (arrows) of the left anterior descending coronary artery. a Curved multiplanar reconstruction along the centerline of the vessel. b Orthogonal curved multiplanar reconstruction. c Left anterior oblique-cranial projection at invasive angiography. d Respective right anterior oblique projectionFig.\u00a02Subendocardial scar (arrows) from a myocardial infarction in the territory of the left anterior descending coronary artery. a Diastolic mid-ventricular short axis view. b Systolic short axis view. c Diastolic left two-chamber view. d Systolic two-chamber view. Note the dyskinesia bulging out of the anterior wall. e Multiplanar reconstruction of the left anterior descending coronary artery. Note the stent (white arrow) in the proximal vessel, which had been implanted after interventional re-perfusion of the occluded vessel\nIndication for invasive angiography was evaluated by a cardiologist based on CT and clinical findings. Invasive angiography was always initiated if significant coronary artery disease could not be reliably excluded in CT and there was no other evident pathology explaining the symptoms. Selective catheterization and coronary angiography were performed by an experienced cardiologist using a transfemoral arterial approach and standard Seldinger technique. At least three standard projections including a 45\u00b0 left anterior oblique, a 30\u00b0 right anterior oblique and a 45\u00b0 left anterior and 30\u00b0 cranial projection were acquired for both coronary arteries. For quantitative coronary assessment (QCA) analysis, a densitometrical analysis of two projection images was performed using the Quant-Cor QCA software (Siemens, Erlangen). Conventional coronary angiograms of 29 patients were available for correlation. A third reader correlated the findings in the reports to invasive coronary angiography and performed dose calculations, which were based on the dose length product given in the patient protocol of the CT scanner. Also, he reviewed the files of patients with initially negative results for further diagnoses in the subsequent 6 months. The readers of the initial CT images were unaware of any findings from conventional angiography, and most invasive angiographies were performed to further evaluate or treat lesions detected in CT. Thus, the cardiologists performing the coronary angiography were mostly aware of the CT findings.\nStatistical analysis\nContinuous variables are given as median and range. Ninety-five percent confidence intervals were calculated using MedCalc software (MedCalc Software, Mariakerke, Belgium). Diagnostic accuracy was calculated as sensitivity, specificity, positive and negative predictive values based on standard contingency tables.\nResults\nTechnical success\nThe scans were acquired from all 109 patients without severe adverse events. Although previously tested, three patients were unable to hold their breath for the duration of the scan, which resulted in artifacts in the lower part of the chest, rendering the coverage and depiction of the coronary arteries insufficient for diagnostic evaluation. All examinations showed an adequate contrast enhancement of the pulmonary arteries, the coronary arteries and the aorta except for six examinations. In two, the bolus was too short due to hemodynamic parameters, making the assessment of pulmonary embolism only possible in the central arteries. In three examinations an erroneous administration of a smaller volume of contrast material caused a weak opacification of the pulmonary arteries, and in one examination overall opacification was too weak (220\u2013245\u00a0HU) for coronary artery assessment.\nPatient findings\nThe diagnoses in the patient population were spread over the different organs and vascular territories quite evenly (Table\u00a02). Ten patients had pulmonary embolism (Fig.\u00a03) and three had morphological signs of chronic pulmonary hypertension. Twelve patients showed other, non-vascular pulmonary findings including pneumonic consolidation in four patients and edema from left heart failure in four cases. Thirty-seven patients had coronary atherosclerosis, and in 21 of these patients, stenoses were rated as hemodynamically significant (>50%) (Fig.\u00a01). Three coronary arteries were occluded. Fifteen patients had had bypass surgery, and five bypass graft vessels were occluded. One bypass was rated as stenotic, but invasive angiography showed a patent lumen and a very slow runoff. There were 20 non-coronary cardiac findings including valvular disease in seven patients. Among those, there were four cases of aortic stenosis, one bicuspid aortic valve and one with vegetations from endocarditis. In one patient an insufficiency of tricuspid valve leaflets was diagnosed. Three patients had had aortic valve replacement, and the valve prostheses worked normally. Seven patients showed an abnormal global or regional wall motion with pulmonary congestion in four cases. Areas of myocardial infarction were noted in three patients (Fig.\u00a02). Other non-coronary cardiac findings were left ventricular hypertrophy, scars from myocarditis and ventricular thrombi. Among the aortic pathologies there were seven aneurysms, five dissections (Fig.\u00a04), one case of intramural hematoma and one acute rupture with active bleeding into the mediastinum. Incidental vascular findings were a lusorian artery and a right descending aorta with Kommerell\u2019s diverticulum (Fig.\u00a05). Another frequent finding was neoplastic lesions, partially with unknown primary cancer. Three patients had disseminated pulmonary metastases, one patient had a previously known lymphoma and one an esophageal cancer. In 26 patients there were no findings that were regarded as the main cause of chest pain. The review of the files of those patients in a 6-month follow-up showed additional diagnoses in two patients: One had intermittent arrhythmia that was identified as the cause of chest pain. The other patient developed pleural effusion and was diagnosed to have pleuritis. In the other 24 patients there was no evidence of the cause of chest pain in follow-up. Considering those results as correct-negative, the sensitivity of the protocol in the identification of the cause of chest pain amounts to 98%. Specificity can be estimated at 92% based on these data, although the exact significance of the findings for chest pain is frequently unclear and may therefore be regarded as arbitrary.\nFig.\u00a03Curved multiplanar reconstruction of the lower lobe pulmonary arteries showing emboli in the lobar and segmental arteriesFig.\u00a04Dissection of the descending aorta. a Multiplanar reconstruction. Arrows indicate the depiction of the left coronary artery in the gated reconstruction (black arrow), the dissection membrane (white arrow) and the junction between the gated scan and the non-gated scan, which had been continued in this case to include the whole extent of the dissection (outlined arrow). b Angulated para-coronal reconstruction showing the topographyFig.\u00a05Kommerell\u2019s diverticulum (arrows). a Volume-rendered reconstruction showing the origin from the aortic arch and the course of the subclavian artery. Note the median-right course of the descending aorta ventral to the spine. b Curved multiplanar reconstruction showing the proximity to the tracheaTable\u00a02Diagnoses found in the study populationTerritoryTypeDiagnosisnPulmonary19%Vascular62%Embolism77%10Pulmonary hypertension23%3Non-vascular38%Pneumonia50%4Edema50%4Cardiac40%Coronary84%Atherosclerosis100%37Relevant stenosis57%21Occlusion8%3Bypass grafts18%Patent75%15Occluded25%5Non-coronary13%Valvular50%7Myocardial50%7Aortic13%Aneurysm50%7Dissection43%6Rupture7%1Other7%Vascular variants25%2Neoplastic75%6Negative24%26Sum of patients100%109\nCorrelation to invasive angiography\nThe 29 conventional coronary angiograms that were available for correlation of the coronary findings were evaluated in a segment-based and a patient-based analysis. Thirty-two coronary artery segments (7%) of these patients were excluded from analysis due to small size or insufficient visualization in CT. Of the 29 patients, 25 had evidence of coronary atherosclerosis in CT. Nineteen stenoses were rated as potentially hemodynamically relevant with more than 50% stenosis in CTA, but invasive angiography confirmed only 15 of those, whereas the other 4 were overrated. There were no false-negative results, i.e., all the stenoses had been cited in CTA, although it has to be taken into account that the indication for invasive angiography was frequently derived from CTA findings. Thus, sensitivity, specificity, positive and negative predictive value amounted to 100%, 99%, 79% and 100%, respectively, in the segment-based analysis.\nIn the patient-based analysis, all 13 patients with significant stenoses were identified. There were two patients with completely negative CTA and symptoms very suggestive of coronary ischemia who had additional invasive angiography, and both of them remained negative. The overrated stenoses occurred in patients with other, significant stenoses. Thus, CTA achieved full diagnostic accuracy in correlation to invasive angiography for this patient-based analysis. The database of the coronary angiography laboratory was screened for those patients who had not initially been examined invasively, and there were no readmissions for angiography within 6 months after inclusion of the last patient.\nRadiation exposure\nAs the patient protocols had not been archived in all patients, there were only 51 dose-length products available for calculation. Based on a conversion factor of 0.012 for chest exams, the estimated equivalent dose ranged between 9.2\u00a0mSv in low heart rates with strict dose modulation to 19.5\u00a0mSv in tall patients with heart rates around 70\u201380 bpm, which resulted in a wide pulsing interval of 30\u201370% with a still quite low pitch below 0.3. Overall, the median radiation exposure was 15.1\u00a0mSv (95% CI 9.9\u201318.8).\nDiscussion\nCT angiography can be regarded as the modality of choice for the assessment of pulmonary embolism and aortic disease in acute care [12]. The fast volume coverage that has become available with recent CT technology has triggered efforts for a combined assessment of these pathologies, and the additional evaluation of the coronary arteries would make the protocol a universal diagnostic tool for chest pain assessment [2, 4, 12]. Quite a few studies showed the feasibility of this approach based on 64-slice CT technology with temporal resolution in the area of 165\u2013200\u00a0ms, but also indicated that coronary CTA still represented the limitation of the method due to the high incidence of high heart rates that made coronary assessment unreliable [1, 3]. Recent studies have shown that diagnostic images of the coronary arteries can be obtained reliably with dual-source CT even in very high heart rates [10, 11]. Thus, dual-source CT was expected to greatly improve the diagnostic accuracy of the combined CT protocol, and our results indicate that this assumption was correct: There were no examinations rated as non-diagnostic because of blurring of the coronary arteries due to high heart rates. There were six exams with partially insufficient contrast opacification, which mainly affected pulmonary artery evaluation and three examinations with breathing artifacts that impeded coronary assessment, but otherwise the main coronary artery segments were reliably assessable. This is reflected in the detailed analysis of the coronary CT angiography in relation to invasive angiography, in which only 7% of coronary artery segments were excluded, mainly due to very small size in distal vessel segments and side branches. Regarding the diagnostic accuracy, the excellent performance in the patient-based analysis confirms that the method can be very helpful for fast patient triage. However, it has to be acknowledged that stenoses were quite frequently rated as \u2018potentially relevant\u2019 in CTA so that invasive angiography was not only necessary for intervention, but also for definite diagnosis. Also, some false-positive results would have to be expected in larger patient groups.\nStill, it has to be emphasized that a valid, relevant diagnosis could be made in 62% of the patients, and in 25% relevant diseases were ruled out. Regarding sensitivity for the cause of chest pain, the value of 98% in the present study is somewhat higher than the 93% observed in a study on ECG-gated 64-slice thoracic CTA [1] and compares well with an initial DSCT study that reported a sensitivity of 96% [13]. In 13% of patients there were findings such as coronary atherosclerosis or vascular variants that were not immediately relevant as a reason for chest pain, but that are still valuable information for further patient management.\nComparing the results of coronary CTA correlation in this study, sensitivity, specificity, positive and negative predictive values of 100%, 99%, 79% and 100% are comparable to an initial study by Scheffel et al. [11]. They observed respective values of 96%, 98%, 86% and 99% for dual-source CT coronary CTA without heart rate control in a high pretest-probability group, although the rate of 1.4% non-assessable segments was remarkably low compared to 7% in our study. The somewhat higher negative and lower positive prediction may be attributed to a more sensitive diagnostic reading in our group of acutely ill patients. In a similar 64-slice CT study by Nikolaou et al. [9], respective values of 97%, 79%, 86% and 96% with 10% non-assessable segments were recorded for an intermediate likelihood population with strict heart rate control. These values suggest that a similar accuracy can be achieved with DSCT without beta blockers.\nDue to the variability in body size and habitus, the equivalent doses calculated from dose length products are only correct for less than half of the population and therefore can only serve as rough estimations. The median value of 15.1\u00a0mSv is about twice that of coronary artery CTA with dual-source CT, but only moderaltely higher than that for coronary CTA alone with single-source 64-slice CT [14, 15]. The increase is mainly related to the high heart rates in this acute patient group that require a wider pulsing interval for the reconstruction of systolic images. The longer coverage including the upper chest contributes rather little to the dose because it is acquired normally with only one tube switched on. Therefore, performing coronary CTA alone without coverage of the upper chest would save only little of the dose in this patient group. Thus, the additional dose from the longer coverage is well invested considering the diagnostic value. Also, comparing DSCT to single source CTA, the high diagnostic accuracy of DSCT can be regarded as well invested dose compared to single source scanners in which the dose can be estimated to be higher for this patient group because the pitch would have to be reduced to apply multi-segment reconstruction algorithms [16, 17], and pulsing would mostly have to be switched off to yield diagnostic examinations with optimized reconstructions [8]. Also, considering alternative diagnostic approaches for the evaluation of chest pain, for example, including pulmonary CTA, aortic CTA and invasive coronary angiography, this combination would result in a higher total patient dose [1]. Magnetic resonance imaging would be an alternative modality for the assessment of pulmonary embolism and aortic pathology, and an MR exam of myocardial wall motion and perfusion could serve as a method to rule out hemodynamically relevant coronary pathology without radiation exposure [18, 19]. However, the limited availability and the long examination times currently let such an approach seem less applicable in a clinical setting [20].\nSeveral limitations of this study have to be acknowledged. One limitation is the exclusion of patients with the evidence of an acute coronary syndrome based on ECG or serum markers, which causes a bias in the assessment of the diagnostic accuracy of coronary CTA. Another limitation is the lacking correlation to other diagnostic approaches in the evaluation of pulmonary embolism and aortic disease. Of course, additional ventilation\/perfusion scintigraphy and aortic MRI would be desirable for study purposes, but cannot be justified considering the necessary time and the encroachment of acutely ill patients and the involved radiation exposure. Also, additional invasive coronary angiography of the patients without coronary findings would be helpful to assess the actual negative predictive value, but the application of this invasive procedure involving radiation exposure to patients with other pathologies would of course not be acceptable. The follow-up of the coronary angiography database that showed no readmissions at least confirms the unlikelihood of relevant coronary artery stenoses in these patients.\nThe fact that immediately therapy-relevant findings in all vascular districts were represented and quite evenly distributed in our patient population emphasizes that a combined protocol can be a valuable tool for diagnostic workup of acute, unclear chest pain. In conclusion, the specific dual-source CT protocol for chest pain assessment proved to be a very helpful tool offering a fast diagnostic workup and patient triage.","keyphrases":["chest pain","ct angiography","pulmonary","embolism","coronary artery disease","aortic dissection"],"prmu":["P","P","P","P","P","P"]}
{"id":"Pediatr_Nephrol-3-1-2100430","title":"Cardiovascular complications of pediatric chronic kidney disease\n","text":"Cardiovascular disease (CVD) mortality is a leading cause of death in adult chronic kidney disease (CKD), with exceptionally high rates in young adults, according to the Task Force on Cardiovascular Disease. Recent data indicate that cardiovascular complications are already present in children with CKD. This review summarizes the current literature on cardiac risk factors, mortality and morbidity in children with CKD.\nLearning objectives:\nTo review recent data on the epidemiology of CVD in children with CKD\nTo understand the mechanisms of cardiovascular abnormalities in pediatric CKD\nTo review recent advances in the diagnosis and clinical presentation of cardiovascular complications in children with CKD\nTo outline current understanding in the strategies to prevent progression of CVD in children with CKD.\nCardiovascular mortality and morbidity\nThe survival of children with CKD in the U.S. remains low: for children on dialysis the lifespan is 40\u201360\u00a0years less and for transplant patients, about 20\u201325\u00a0years less than that of an age- and- race-matched US population [1, 2]. The most likely cause of this is increased cardiovascular mortality due to the development of accelerated ischemic heart disease and premature dilated cardiomyopathy. The evidence comes from studies of young adults who developed renal failure during childhood. Oh et al. [3] analyzed the outcome of 283 young adults with childhood onset CKD between 1970 and 1997. Fifty percent of the deceased patients had died of cardiovascular or cerebrovascular events. Groothoff et al. [4] conducted a national retrospective and prospective cross-sectional study to evaluate the late physical, social and psychological effects of renal insufficiency (LERIC) in all Dutch children who started renal replacement therapy between 1972 and 1992. Of 381 patients, 85 had died. Cardiovascular disease was the most common cause of death and accounted for 41% of all deaths. Cerebrovascular accident, congestive heart failure, myocardial infarction and cardiac arrest (respectively) were the most common causes of cardiac death. Similar analysis of long-term survival from the Australia and New Zealand Dialysis and Transplant Registry [5] of all children and adolescents who were under 20\u00a0years of age when renal replacement therapy commenced (study period was 40\u00a0years) showed mortality rates 30 times higher than in the age-matched general population; CVD was the most common cause of death (45%).\nCardiovascular death happens not only in later life, but also in childhood CKD. In the general pediatric population, the incidence of annual death due to cardiac disease is less than 3%. Yet annual reports from the United States Renal Data System (USRDS) indicate that over the last decade CVD has remained the second most common cause of death in children on chronic dialysis or after transplantation, accounting for approximately 20\u201325% of all deaths. Parekh et al. [6], using the USRDS database, performed a detailed cross-sectional analysis to evaluate the risk of a cardiac death in children and young adults (age 0\u201330\u00a0years) and to identify factors potentially associated with CVD mortality. A total of 1,380 deaths between 1990 and 1996 were analyzed. There were 311 cardiac deaths (22.5% of the total). Cardiac deaths in children and young adults in whom ESRD developed during childhood were approximately 1,000 times more frequent than in the general pediatric population. Of the specific categories of cardiovascular deaths, cardiac arrest was the most common cause in each of the age groups, followed by arrhythmia and cardiomyopathy. These causes of cardiac death are different from those of adults. In adults, coronary artery disease and chronic congestive heart failure are the leading causes of CVD mortality and, as shown by Parekh et al. [6], these causes are extremely rare in children and adults younger than 30\u00a0years of age. The incidence of cardiac arrest in the youngest age group (0\u20134\u00a0years) was five to ten times higher than in other age groups, perhaps, as noted by the authors, a reflection of the difficulty of ascertaining the true cause of death in young children. Some of these young children might have died from other co-morbid conditions such as congenital disorders that are not included in the USRDS database.\nThe high rate of sudden death in children, especially in infants with ESRD, is poorly understood and warrants further investigation. In adults, sudden death is often a result of fatal arrhythmias due to acute ischemia of preexisting atherosclerotic disease. It is believed that arrhythmias are also the likely cause of most cases of sudden cardiac death (SCD) in children. However, the origin of acquired malignant arrhythmias in children is unlikely to be an atherosclerotic lesion. Dilated, especially hypertrophic, cardiomyopathies are a leading cause of SCD in children [7]. The macroscopic and microscopic structural abnormalities in cardiomyopathies involve fibrosis and cellular hypertrophy prone to produce an electrical instability with resultant arrhythmias. Ischemia of small coronary vessel disease secondary to medial hypertrophy might result in dispersion of repolarization properties and arrhythmia from re-entrant or autonomic mechanisms. As we discuss in more detail below in this review, children with CKD develop left ventricular hypertrophy (LVH), which is frequently severe, especially in children on prolonged dialysis therapy [8, 9]. It is currently unknown if LVH in young patients with CKD is characterized by structural abnormalities similar to familial or idiopathic hypertrophic cardiomyopathies associated with SCD. Whether LVH of children with ESRD can contribute to increased SCD is also not known. Another possibility for deadly arrhythmias in children with ESRD is acute changes in the cardiac extra- or intracellular ionic milieu, especially involving abnormalities of sodium- and potassium-based repolarization currents.\nBecause the causes of cardiac death in children and adults are different, it is not surprising that none of the traditional or uremia-related risk factors for adult atherosclerotic CVD predicted cardiac death in the study by Parekh [6]. One of the examples presented in this paper is the effect of race on CVD death. In adults with ESRD, white males are at higher risk for CVD mortality. In contrast, black children as shown by Parekh et al. [6] appear to have an increased risk for cardiac death. Another important observation of this study is that transplant recipients had 78% lower risk of cardiac death than dialysis patients. However, the authors pointed out that the CVD mortality rate in transplanted patients was still significantly higher (approximately ten times) than in the general pediatric population.\nThe analysis of cardiac morbidity in children on chronic dialysis performed by Chavers et al. [10] has confirmed that cardiac disease in children is different from adults. A total of 1,454 Medicare incident pediatric (0\u201319\u00a0years) dialysis patients were identified from 1991 to 1996. Among them, 452 (31.2%) developed a cardiac-related event. Arrhythmia was most common (19.6%), followed by valvular disease (11.7%), cardiomyopathy (9.6%) and cardiac arrest (3%). Ischemic heart disease was extremely rare in these children.\nRisk factors for cardiovascular disease\nThe risk factors and pathogenic mechanisms of development of CVD in young adults who had onset of CKD in childhood are better understood than are those producing cardiac morbidity and mortality in children. The conventional thinking is that two groups of risk factors are responsible for accelerated CVD in adults with CKD (Table\u00a01). \nTable\u00a01Cardiovascular risk factors in chronic kidney disease in adultsTraditionalCKD-relatedOlder ageDecreased GFRWhite raceProteinuriaMale genderPeripheral renin-angiotensin-aldosterone activityHypertensionAbnormal calcium and phosphorus\u2191 LDL CholesterolDyslipidemia\u2193 HDL CholesterolHypoalbuminemiaDiabetes mellitusHemodynamic overloadTobacco useAnemiaPhysical inactivityThrombogenic factorsPsychosocial stressHyperhomocysteinemiaFamily history of CVDOxidative stressLVHInfection (Chlamydia pneumoniae)ObesityChronic inflammation\nFirst, as compared to the non-uremic population, there is an over-representation in uremic patients of classical risk factors, e.g., diabetes, hypertension and hyperlipidemia. A majority of the adults who develop ESRD do so as a complication of diabetes or generalized atherosclerosis. Often cardiac disease antedates the onset of CKD in these patients. Unfortunately, children with CKD share with adults a similar high prevalence of risk factors for adult-type atherosclerotic CVD. It is also troubling that the frequencies of these traditional risk factors have not changed over last decade. The North American Pediatric Renal Trials Collaborative Studies (NAPRTCS) data demonstrate that hypertension develops at early stages of CKD (48%) and persists (50\u201375%) in uremic children [11\u201314]. Dyslipidemia is found in 70 to 90% during chronic dialysis [15\u201318]. Successful renal transplantation leads to a dramatic improvement in renal function and elimination of many risk factors for atherosclerotic CVD that were present while on dialysis. However, transplant recipients are not free from multiple complications, and transplantation may amplify some of the traditional risk factors. Indeed, the prevalence of hypertension in pediatric renal allograft recipients is between 50\u201380% [19\u201321]. Also, hyperlipidemia may not disappear after renal transplantation; the reported prevalence is above 50% [22\u201324]. The NAPRTCS data also show that the rate of obesity, another CV risk, is increasing in children with ESRD at the time of transplantation (12.4% after 1995 vs. 8% prior to 1995) [25]. Single-center study data indicate that the number of obese patients can double at 1\u00a0year after transplantation [26].\nSecond, there is a multitude of uremia-related risk factors for atherosclerotic CVD. In adults with CKD, an increased homocysteine level appeared to be an independent risk factor for CVD morbidity and mortality [27\u201329]. Anemia has been linked to negative CVD outcome [30]. Hyperphosphatemia with increased calcium-phosphorus product constitutes a risk for cardiovascular calcification, cardiac ischemia and adverse cardiovascular outcomes [31, 32]. Elevated serum C-reactive protein (CRP), a marker of systemic inflammation, has been found to be a strong predictor of cardiac morbidity and cardiac death in CKD patients [33]. Another marker of inflammation, IL-6, has been associated with increased cardiac morbidity in adults with CKD [34, 35]. The role of systemic inflammation in CKD has been reviewed recently elsewhere [36]. Inflammation is directly linked to oxidative stress, which is now considered as a hallmark of uremia [37]. Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase and a marker of oxidative stress, has been extensively evaluated in patients with CKD by Zoccali et al. [38]. The authors demonstrated that elevated ADMA per se was responsible for a 52% higher risk of death and for a 34% higher risk of cardiovascular events in dialysis patients [39]. Another subset of novel CVD risk factors is adipokines. Adipokines, leptin and adiponectin, are the products of adipose tissue involving in regulation of lipid and glucose metabolism. Abnormal adipokines are strongly linked to insulin resistance, a known CV risk in CKD [40]. It has been determined that lower plasma adiponectin concentration independently predicts increased CVD morbidity and mortality in adults with ESRD [41]. Clinical Practice Guidelines for Cardiovascular Disease in Dialysis Patients extensively reviewed the current literature on CVD biomarkers, and the reader is referred to this publication for more information [42]. As in adults, in children with CKD many of these risk factors are very prevalent (Table\u00a02). \nTable\u00a02Prevalence of risk factors for CVD in children with CKDRisk factorsCRIa (%)Dialysisb (%)Transplantc (%)ReferencesHypertension4852\u20137563\u20138111a, 12\u201314b, 19\u201321cDyslipidemia25\u20135333\u20138755\u20138415a, 15\u201318a, b, 22\u201324cAnemia4840\u20136732\u20136443a, 44\u201347b, 48\u201351c,Hyperparathyroidism32.6\u201343.758\u201343Hyperhomocysteinemia87\u20139225\u201310052c, 53b, c, 54c, 55b\u2191 CRP761656b,57cHypoalbuminemia\u201340\u201360\u201343b, 44b, 58\u201359b\nRecent studies in adults on chronic hemodialysis place a malnutrition-inflammation complex at the center of a debate about the role of traditional and non-traditional risk factors for poor cardiovascular outcome. This issue emerged after publication of a series of articles, summarized in the review by Nurmohamed and Nube [60], describing the phenomenon of \u201creverse epidemiology.\u201d The studies have shown that in adults on chronic hemodialysis low blood pressure, low body mass index (BMI), low serum cholesterol and low serum homocysteine are often correlated with an unfavorable clinical outcome. Thus, whereas traditional risk factors of CVD are correlated with an unfavorable outcome in the general population and patients with CKD not yet on dialysis, in hemodialyzed patients, mild hypertension, hypercholesterolemia and being overweight appear to be protective and associated with an improved survival. It has been speculated that a malnutrition-inflammation-atherosclerosis complex underlies, at least partly, the phenomenon of reverse epidemiology, since malnutrition causes a low BMI, hypocholesterolemia and low serum homocysteine levels.\nMechanisms of cardiovascular disease in chronic kidney disease\nThere are two parallel processes involved in the development of CVD in CKD patients (Fig.\u00a01).\nFig.\u00a01Cardiovascular disease in chronic kidney disease\nThe first is cardiac remodeling leading to hypertrophy of the left ventricle (LV) as a response to mechanical or hemodynamic overload. Two different patterns of LV remodeling can produce increase in LV mass (LVM) [61]. The concentric LV remodeling and hypertrophy may be the results of pressure overload as occurs with hypertension, whereas eccentric LVH may be related to volume and sodium retention, anemia and arteriovenous shunt. The patterns of sarcomere formation induced by pressure or volume overload are distinct. Pressure-induced concentric LVH is characterized by a parallel addition of sarcomeres resulting in the increase of cross-sectional area and diameter of the myocytes. Increase in LVM in this case is obtained by a marked increase in wall thickness with a less evident increase in the LV cavity that yields an elevated relative wall thickness and concentric LVH. From the physiological view, increased systolic blood pressure (BP) and pulse pressure, due to increased peripheral resistance and arterial stiffness, are the principal factors opposing LV ejection and leading to an increased LV workload and concentric LVH. An increase in LVM can also be obtained by an increase in the LV cavity with a symmetric increase in wall thickness to maintain the ratio between the wall thickness and LV transversal radius (relative wall thickness) normal, producing eccentric LVH. In this case, the addition of sarcomeres occurs mainly in series resulting in longitudinal cell growth. In the transition to maladaptive LVH, LV dilatation becomes disproportional to wall thickness, with myocytes elongated without an increase in diameter.\nExperimental models of cardiac hypertrophy support the theory that mechanical stress due to either pressure or volume overload is a trigger for activation of other multiple mechanisms leading to myocardial remodeling [62]. These factors include a local overexpression of the renin-angiotensin-aldosterone system (RAAS), adrenergic system, inflammatory cytokines and other autocrine and paracrine mechanisms. In patients with CKD, these mechanisms might be activated independently of hemodynamic overload since uremia per se is associated with an alteration in multiple humoral factors [63, 64]. With time, a maladaptive phase of LVH develops, characterized by decreased capillary density, decreased coronary reserve and subendocardial perfusion, a tendency to arrhythmia, and the development of myocardial fibrosis. All this leads to myocyte death and, finally, to diastolic and systolic dysfunction.\nThe second process involves vascular injury. Exposure to CV risk factors results in vascular changes, including atherosclerotic and arteriosclerotic processes and vascular calcification.\nAtherosclerosis refers to the process of plaque formation or atheroma development. The process of atheroma formation begins with an accumulation of lipid-containing foam cells (macrophages) in the vascular intima and evolves into successive structures that penetrate the vascular wall and include lipids, smooth muscle cells and collagen fibers [65]. Calcification is an intrinsic part of the process and generally involves the intima. Atherosclerotic lesions have a patchy distribution along the length of the artery and cause local stenoses and occlusions. Recently, endothelial progenitor cells (EPCs) have been identified as contributing to angiogenesis [66]. The EPC number has been shown to be reduced in patients with CVD, leading to speculation that atherosclerosis may be caused by a consumptive loss of the endothelial repair capacity. Animal experiments have shown that EPCs reendothelialise injured vessels and that this reduces neointimal formation, confirming that EPCs have an atheroprotective effect.\nArteriosclerosis is arterial stiffening involving the entire arterial tree, although it principally affects the elastic arteries. Unlike atherosclerosis, arteriosclerosis involves both intimal and medial thickening. In CKD, arteriosclerosis can occur in the absence of significant atherosclerotic disease [67\u201369]. Arteriosclerosis is associated with vascular remodeling characterized by increased wall thickness, lumen enlargement and increased length of arteries. This leads to an increase of systolic BP and pulse pressure and arterial stiffening.\nThe triggers for vascular calcification are complex and include metabolic, mechanical, infectious and inflammatory injuries. Increased calcium x phosphate ion product or hyperphosphatemia may be the key promoter of vascular calcification [70]. The mechanisms include either stimulation of the uptake and precipitation of calcium and phosphate into the vessel or a decrease of the inhibitory process that prevents these ions from precipitation. Fetuin-A, the anti-inflammatory protein, is a highly potent inhibitor of serum calcium-phosphate complex formation [71, 72]. The serum concentrations of Fetuin-A are decreased in ESRD patients [73]. Another promoter of vascular calcification is 1,25(OH)2D3, which may have a direct effect on the calcium deposition in vascular smooth muscle cells [74]. In a comprehensive review, Vattikuti and Towler [75] defined four major histo-anatomic variants of vascular calcification, including classic atherosclerotic (fibrotic) calcification related to disorders of lipid metabolism, medial arterial calcification, vascular calcifilaxis and cardiac valve calcification.\nSpectrum of cardiovascular abnormalities in children with CKD\nOver the last decade, abnormalities of the LV such as LVH and LV dysfunction, abnormalities of the large arteries such as stiffness and increased intima-medial thickness (IMT) of the carotids, and calcification of the coronaries have been accepted as early markers of cardiomyopathy and atherosclerosis. They constitute strong independent predictors of cardiac morbidity and mortality both in the general population and in adults with CKD. In children and young adults with CKD, recent studies have proven that these abnormalities are also present and that risk factors for cardiac and vascular injury in children with CKD are similar to those for adults.\nLeft ventricular hypertrophy\nLVH develops when renal insufficiency, is mild or moderate in children and progresses as renal function deteriorates. About one third of children with mild to moderate renal insufficiency have an increased left ventricular mass (LVM) index [76\u201378]. In a 2-year prospective longitudinal study of 31 pediatric subjects with CKD stage 2\u20134, Mitsnefes et al. [79] showed that a substantial proportion of children had a significant increase in the LVM index, with many of the children developing LVH. Indeed, 32% of the patients who initially had a normal LVM index have developed incident LVH. At initiation of maintenance dialysis, 69\u201382% of pediatric patients have evidence of LVH [80, 81]. LVH persists (40\u201375%) during long-term dialysis [8, 76, 82\u201385], with both concentric and eccentric geometric patterns of LVH present in these patients. Post-mortem studies confirmed high rate (more than 50%) of LVH in children with ESRD [86]. Small retrospective studies also suggest that with a better BP and volume control, LVH regression might be achieved in young patients on dialysis [80, 81]. On the other hand, a recent retrospective study demonstrated that LVH remains very prevalent and severe in a selected group of children who remained on maintenance dialysis for at least 2\u00a0years [9].\nAs in children prior to transplantation, most pediatric studies indicate that LVH remains common post transplant (48\u201382%) [87\u201391]. In contrast, a significantly lower frequency of LVH was found in a study by Englund et al. [92], who reported the results of a longitudinal analysis of children receiving renal transplants 10\u201320\u00a0years ago. Of 53 children who received a renal transplant between 1981 and 1991, 47 survived and were observed for 10 to 20\u00a0years. At the 10-year follow-up, echocardiography showed minor LVH in only two children with hypertension. No child without hypertension at 10\u00a0years post transplant had LVH.\nThe factors associated with cardiac hypertrophy in children are similar to those in adults with CKD. As in adults, most pediatric studies of patients with pre-terminal, terminal renal failure and post transplant found significant relationships between low hemoglobin and an increased LVM index [8, 78, 79, 90]. However, recent adult studies in mild to moderate CKD or in chronic dialysis determined that correction of anemia was not associated with regression of LVH [93\u201396]. Authors suggested that relationships between anemia and LVH might not be causal. Of note, the above studies enrolled subjects with relatively mild degrees of baseline anemia and could not answer the question whether treatment of patients with initially significantly decreased hemoglobin levels might lead to a reduction of LVM. In contrast, Morris et al. [97] observed a significant reduction in the LVM index with the correction of severe anemia in seven children on chronic dialysis.\nThere are several studies on the association between parathyroid hormone (PTH) levels and LVH in adults with CKD [98, 99]. In children, elevated PTH is associated with progression of LVH in stages 2\u20134 CKD [79]. The possible mechanisms of parathyroid-induced cardiac hypertrophy in CKD include a direct effect of PTH on cardiomyocytes and an indirect effect via elevated BP [99]. A support for a causal relationship comes from in vitro studies showing that PTH appears to have chronotropic, inotropic as well as hypertrophic effects on cardiomyocytes [100, 101].\nIn adults with CKD, hypertension is directly linked to the development of LVH [39]. The relationships between BP and LVH in pediatric CKD are unclear. Consistent correlations of LVM and BP are limited to children with ESRD [8, 85]. However, a detailed cross-sectional analysis of BP characteristics by ambulatory blood pressure monitoring (ABPM) in children from the ESCAPE trial did not demonstrate any relationship between office BP or ABPM parameters and LVM, suggesting only a minor role of hypertension in the pathogenesis of LVH in early CRI [78]. In contrast, analysis of longitudinal data suggests that ABPM might be an important tool to assess the risk of development of LVH in children with CKD [79]. In this study, authors determined that an increase in the nighttime systolic BP load (number of BP measurements above the 95th percentile BP value) were independently associated with the increase in the LVM index over time, arguing that persistent and chronic elevation of BP might be more important in the development of LVH [79].\nLeft ventricular function\nIn contrast to adults, in whom systolic dysfunction is frequently associated with early cardiac failure and decreased survival, in children with CKD systolic LV function is usually preserved [76, 102\u2013104]. On the other hand, diastolic dysfunction, often the initial abnormality of cardiac function, is already present in children with CKD. Doppler measurement of mitral inflow velocity has been the most widely used method to assess LV diastolic function. Using this method, Goren et al. [103] showed that LV relaxation (E\/A ratio) was impaired in dialyzed children as compared to controls. Johnstone et al. [76] also found a reduction in the E\/A ratio in children on chronic peritoneal dialysis and with pre-terminal renal failure, although none of these patients had an E\/A ratio <1.0, which is considered to be abnormal. Unfortunately, the transmitral Doppler velocities and, therefore, the E\/A ratio, are affected by several factors, including left atrial pressure and preload. This is particularly important for patients with advanced chronic renal failure, since many of them are hypervolemic. Recently, new indices were introduced to evaluate diastolic function using tissue Doppler imaging (TDI). In contrast to E\/A, the TDI indices may be less load dependent and provide a more accurate measure of diastolic function. Recent studies employing TDI determined that children with CKD might have abnormal diastolic function [105, 106]. In these studies, children on chronic dialysis had significantly worse diastolic dysfunction than children with mild-to-moderate CRI or post transplant. Poor diastolic function in patients on dialysis was associated with anemia, hyperphosphatemia, increased calcium-phosphorus ion product and LVH. The clinical significance of diastolic dysfunction in pediatric patients with CKD is not known. Longitudinal studies are necessary to determine if abnormal diastolic function predicts the development of systolic dysfunction and congestive heart failure in these patients.\nArterial structure and compliance\nStudies of young adults who developed ESRD during childhood found a high prevalence of abnormal carotid IMT, diminished arterial wall compliance and coronary artery calcification (CAC). These vascular abnormalities are accepted as markers of asymptomatic atherosclerosis and predictors of future symptomatic CVD in the general population and in adults with CKD [107, 108]. Groothoff et al. [109] found increased arterial stiffness and showed that systolic hypertension was the main determinant of abnormal arterial wall compliance. Goodman et al. [110] showed that among 23 patients on chronic dialysis who were younger than 20\u00a0years of age, none had evidence of CAC; in contrast, 14 of the 16 patients who were 20 to 30\u00a0years of age had evidence of CAC on CT scanning. Oh et al. [3] screened for coronary and carotid artery disease in 39 patients, aged 19 to 39\u00a0years, with childhood onset ESRD. Coronary artery calcification was present in 92% and carotid IMT was significantly increased compared to matched controls. Carotid IMT was correlated with cumulative dialysis and serum Ca x P product in their study. Milliner et al. [111], in an autopsy study of pediatric patients with ESRD who died in 1960\u20131983, showed a high prevalence of soft tissue and vascular calcinosis. In their study, CAC was present in 28%. Peak Ca x P product, peak serum P and cumulative dose of calcitriol were significantly associated with the severity of the calcinosis. Civilibal et al. [112] screened 53 children with ESRD for the presence and predisposing factors of CAC. Coronary artery calcification was present in 15% of patients (three hemodialysis patients, three peritoneal dialysis patients and two renal transplant recipients). The patients with CAC had a longer duration of total dialysis, had higher time-integrated serum phosphorus, calcium-phosphate product, intact parathyroid hormone, vitamin B12 levels and the amount of cumulative calcium-containing oral phosphate binders. In a study by Briese et al. [113] of 40 young adults (mean age 23.6\u00a0years) who developed ESRD at a mean age of 11.5\u00a0years, carotid artery IMT was similar to healthy controls and only 4 (10%) patients had evidence of coronary calcification. Authors noticed that a relatively low rate of cardiac calcification compared to other studies might be explained by a significantly lower amount of prescribed calcium-containing phosphate binders and vitamin D preparations in their patients. Of note, these patients had decreased vascular reactivity and a high rate of LVH (68.2%). As in adults, cardiac valve calcification was also described in children on maintenance dialysis [114].\nEvidence of early atherosclerotic changes was found in the study by Nayir et al. [115], who reported on the histopathology of internal iliac artery samples obtained at the time of kidney transplantation in 12 children. Five arteries had fibrous or fibroelastic intimal thickening, medial mucoid ground substance and disruption of the internal elastic lamella. Two of these had microcalcification in the intimal layer; another two demonstrated atheromatous plaques. These abnormalities were associated with longer duration of renal failure.\nVascular abnormalities in children develop in parallel with cardiac abnormalities early in the course of CKD and become more severe as end-stage disease is reached [116]. Mitsnefes et al. [54] showed that carotid arteriopathy is present in children after successful renal transplantation and is associated with hypertension. Litwin et al. [117], as a part of the ESCAPE trial, investigated vascular structure and function in children with chronic renal failure and after renal transplantation. The authors demonstrated vascular abnormalities in all patient groups with the most marked changes in the dialysis patients. The degree of arteriopathy in their study was correlated with conventional CVD risk factors such as hypertension and dyslipidemia in pre-dialytic CKD, while in children on dialysis and after transplantation, hyperphosphatemia, hyperparathyroidism and treatment with calcium-containing phosphate binders were determinants of arterial abnormalities. Another important observation in this study was a significantly lower carotid IMT in children post transplant, suggesting that the vascular abnormalities partially regress. Epidemiological studies should determine if vascular abnormalities detected during childhood CKD are associated with future accelerated coronary artery disease.\nEndothelial dysfunction as a marker of early atherosclerosis in children with CKD\nEndothelial function can be evaluated by the assessing of endothelial vasodilatation. Healthy vascular endothelium will respond to the release of nitric oxide by vasodilatation. With endothelial injury, the response to nitric oxide is diminished [118], and endothelial-mediated vasodilatation is impaired. Flow-mediated dilation (FMD) of the brachial artery is currently used to measure endothelial function. This test consists of applying obstruction to the flow of the brachial artery by placing the tourniquet or inflated BP cuff for few minutes. The artery dilates due to post-obstruction reactive hyperemia. FMD is calculated based on the difference in the diameter of the brachial artery before obstruction and after it released. Impaired (decreased) FMD predicts CVD morbidity and mortality in adults with ESRD [119]. It has been shown that endothelial dysfunction as determined by impaired FMD is also present in children with advanced renal failure, on chronic dialysis and after renal transplantation [120\u2013122].\nEvaluation and treatment recommendations\nThe overall strategy in the prevention of cardiovascular complications in children with CKD is avoidance of long-term dialysis. The goal is to prevent the development and delay the progression of cardiomyopathy and atherosclerosis. The identification of modifiable risk factors and markers of CVD and early intervention should be initiated at the time of mild-to-moderate renal insufficiency, prior to the need for dialysis. Even though kidney transplantation poses a continuous CV risk (hypertension, hyperlipidemia and allograft dysfunction), it eliminates many uremia-related risks, reduces the risk of cardiac death by approximately 80 percent and prolongs the life span by 20\u201330\u00a0years. Thus, kidney transplantation should be the ultimate goal to minimize cardiovascular morbidity and mortality in patients with advanced CKD. For those patients who must have long-term dialysis, the strategy is directly linked to achievement of optimal dialysis outcomes, which include aggressive monitoring and management of hypertension, dyslipidemia, calcium-phosphorus metabolism, anemia, nutrition, systemic inflammation and other dialysis complications.\nCurrent recommendations on evaluation of CV abnormalities and treatment of modifiable risk factors in children are based mostly on clinical experience and adult data. Recent K\/DOQI Clinical Practice Guidelines for Cardiovascular Disease in Dialysis Patients [42] recommend echocardiographic evaluation for the presence of cardiac disease in children (cardiomyopathy and valvular disease) at the time of initiation of dialysis therapy along with the screening for dyslipidemia, hypertension, anemia and increased Ca\u00d7P product. Management of modifiable risks in children with CKD should follow recommendations from the K\/DOQI guidelines for treatment of anemia [123] and dyslipidemia [15] and from the Fourth Report on Blood Pressure in Children for Management of Hypertension [124]. Current K\/DOQI guidelines for treatment of anemia recommend keeping the hemoglobin level above 11\u00a0g\/dl by using an appropriate iron therapy and recombinant erythropoietin. In the opinion of the Working Group, there is insufficient evidence to recommend routinely maintaining hemoglobin levels above 13\u00a0g\/dl or more.\nK\/DOQI recommends evaluation of dyslipidemia in adolescents upon presentation with CKD stage 5 (GFR <15\u00a0ml\/min\/1.73\u00a0m2 or on dialysis), at 2\u20133\u00a0months after a change in treatment or other conditions known to cause dyslipidemia and at least annually thereafter. Reasons to repeat lipid measurements after 2\u20133\u00a0months include changes in the kidney replacement therapy modality, treatment with diet or lipid-lowering agents, immunosuppressive agents that affect lipids (e.g., prednisone, cyclosporine or sirolimus) or other changes that may affect plasma lipids. The assessment of dyslipidemia should include a complete fasting lipid profile with total cholesterol, LDL, HDL and triglycerides. The definition of dyslipidemia differs in children and adults. Hyperlipidemia in children is defined as lipid levels greater than the 95th percentile for age and gender. The normative data for lipids in children and adolescents currently used are from the Lipid Research Clinics Program from the NIH published in 1980 and can be found in the 2003 K\/DOQI guidelines for management of dyslipidemia in chronic kidney disease [15].\nFor adolescents with stage 5 CKD and a level of LDL \u2265130\u00a0mg\/dl, K\/DOQI recommends treatment to reduce LDL to <130\u00a0mg\/dl. If LDL is <130\u00a0mg\/dl, fasting triglycerides \u2265200\u00a0mg\/dl and non-HDL cholesterol (total cholesterol minus HDL) \u2265160\u00a0mg\/dl, treatment should be considered to reduce non-HDL cholesterol to <160\u00a0mg\/dl. All children with dyslipidemia should follow the recommendations for therapeutic lifestyle changes (TLC), which include diet modification with a reduction in saturated fat intake and increase in fiber intake, and moderate physical activity. Adolescents should be counseled about avoiding smoking. Unfortunately, non-compliance with TLC is one of the major problems in the management of dyslipidemia in adolescents. Pediatric nephrologists must also recognize that appropriate caloric intake, including calories from fat, should be emphasized to avoid malnutrition and ensure normal growth and development, especially in young children. If LDL cholesterol is \u2265160\u00a0mg\/dl and non-HDL cholesterol \u2265190\u00a0mg\/dl, statin therapy is recommended in children older than 10\u00a0years.\nTarget blood pressure in children should be lower than the 90th percentile for normal values adjusted for age, gender and height or less than 120\/80\u00a0mm Hg, whichever is lower. Ambulatory blood pressure monitoring is recommended to assess the circadian rhythm. Angiotensin-converting enzyme inhibitors or angiotensin receptor blockers may be the preferred antihypertensive agents to slow the progression of CKD in children and possibly for regression of LVH. The Working Group on Cardiovascular Disease in Dialysis Patients recommends maintaining calcium and phosphorus levels within the normal range and the Ca\u00d7P product <55\u00a0mg2\/dl2 in children on chronic dialysis [42].\nThere are no data available to make evidence-based recommendations on management of hyperhomocysteinemia, chronic inflammation or other potential CVD risk factors.\nQuestions(Answers appear following the references) \nA 14-year-old African-American boy with ESRD secondary to FSGS has been treated with thrice-weekly hemodialysis for 1\u00a0year. His pre-dialysis BP is 132\/85, hemoglobin is 9.8\u00a0g\/dl, serum calcium is 10.6\u00a0g\/dl, serum phosphorus is 8.2\u00a0g\/dl and iPTH is 412\u00a0pg\/ml. He is taking a calcium channel blocker to control his hypertension, erythropoietin and iron to control his anemia and calcium carbonate and a vitamin D IV preparation to control his renal bone disease.Which ONE of the following statements would be the BEST therapeutic intervention in this child to minimize the risk of future CV complications?Aggressive treatment of hypertensionMaximize dialysis treatment to achieve dry weightCorrection of anemiaKidney transplantationReduction of Ca-P productThe patient from the previous question was diagnosed with eccentric LVH.With regard to treatment of his LVH, which ONE of the following actions is MOST LIKELY to lead to reduction of LVH?Adding ACE inhibitor as a second antihypertensive agentIncrease the dose of erythropoietin and assure appropriate iron status to treat anemiaAchievement of dry weightSwitch from hemodialysis to peritoneal dialysisMaximize treatment of secondary hyperparathyroidismThe annual evaluation showed that this patient has LDL cholesterol of 170\u00a0mg\/dl. You would like to start this patient on atorvastatin.With regard to the use of \u201cstatin\u201d in this patient, which ONE of the following statements is correct?Statins are not approved by the United States Food and Drug Administration (USFDA) for use in children and adolescentsGrapefruit is contraindicatedThe dose of atorvastatin should be reduced by 50% compared to dosage in adult patients on dialysis to prevent adverse effects on growth and developmentThe target level for LDL cholesterol in children with CKD should be below 100\u00a0mg\/dlAtorvastatin therapy will reduce mortality risk in this patientWhich ONE of the following statements concerning development of cardiovascular complications in a child with CKD is NOT correct?Cardiovascular disease is the cause of mortality in approximately 25% of chronically dialyzed childrenCardiac arrest is the most common cardiac cause of death in children with ESRDCardiac and vascular remodeling might develop in children during early stages of CKDCorrection of anemia reduces concentric LVHLVH is the most common cardiac abnormality in children on maintenance dialysisThe patient described in the previous questions is at risk for development of vascular calcification.Which ONE of the following choices does NOT correctly describe the mechanisms of vascular calcification in dialyzed patients?Histo-anatomic variants of calcification include classic atherosclerotic (fibrotic) calcification related to disorders of lipid metabolism, medial arterial calcification, vascular calcifilaxis and cardiac valve calcificationIncreased Ca-P product and hyperphosphatemia are the key drivers of vascular calcification in young patients with CKDInflammatory mechanisms are involved in mediating all stages of atherosclerosis including calcificationIncreased serum level of Fetuin-A promotes Ca-P ion product precipitation1, 25 (OH)2D3 increases calcium deposition in the vascular wall\nAnswers\ndcbdd","keyphrases":["chronic kidney disease","cardiovascular disease","children","esrd"],"prmu":["P","P","P","P"]}
{"id":"Intensive_Care_Med-3-1-2039828","title":"Management of invasive pulmonary aspergillosis in non-neutropenic critically ill patients\n","text":"During recent years, a rising incidence of invasive pulmonary aspergillosis (IPA) in non-neutropenic critically ill patients has been reported. Critically ill patients are prone to develop disturbances in immunoregulation during their stay in the ICU, which render them more vulnerable for fungal infections. Risk factors such as chronic obstructive pulmonary disease (COPD), prolonged use of steroids, advanced liver disease, chronic renal replacement therapy, near-drowning and diabetes mellitus have been described. Diagnosis of IPA may be difficult and obtaining histo- or cytopathological demonstration of the fungus in order to meet the gold standard for IPA is not always feasible in these patients. Laboratory markers used as a non-invasive diagnostic tool, such as the galactomannan antigen test (GM), 1,3-\u03b2-glucan, and Aspergillus PCR, show varying results. Antifungal therapy might be considered in patients with persistent pulmonary infection who exhibit risk factors together with positive cultures or sequentially positive GM and Aspergillus PCR in serum, in whom voriconazole is the drug of choice. The benefit of combination antifungal therapy lacks sufficient evidence so far, but this treatment might be considered in patients with breakthrough infections or refractory disease.\nIntroduction\nInvasive pulmonary aspergillosis (IPA) has emerged as an important cause of morbidity and mortality in patients receiving intensive chemotherapy, allogeneic stem cell transplantation, and solid organ transplantation. However, during recent years, several reports have described a\u00a0rising incidence of IPA in critically ill patients admitted to the intensive care unit (ICU), even in the absence of an apparent predisposing immunodeficiency\u00a0[1\u20136]. The incidence of IPA in the ICU ranges from 0.3% to as much as 5.8%\u00a0[2, 3, 6] and it carries an overall mortality rate exceeding 80%, with an attributable mortality ofalmost 20%\u00a0[4, 5]. This high mortality is at least partially related to difficulties in timely diagnosis, caused by insensitive and non-specific clinical signs and lack of unequivocal diagnostic criteria.\nIn this review, we will describe the pathophysiological mechanisms and risk factors for IPA in non-neutropenic critically ill patients, limitations and advances in the diagnostic process, and alterations in treatment with antifungal therapy. A\u00a0Medline\/PubMed search was performed for all articles about IPA in critically ill patients in relation to risk factors, diagnosis and antifungal therapy. All publication types of human studies in the English language were searched and an extraction of relevant articles was made for the purpose of this narrative review.\nPathophysiological mechanisms and risk factors\nAspergillus spp. are opportunistic moulds that cause both allergic and invasive syndromes. The genus Aspergillus contains approximately 175 species, only a\u00a0minority of them have been associated with human disease. Infections are caused mostly by Aspergillusfumigatus; next in line are Aspergillus flavus, Aspergillus terreus, Aspergillus niger andAspergillus nidulans\u00a0[7]. Aspergillus is found in soil, water, food, and in the air and grows on a\u00a0wide variety of organic material, such as decaying vegetation. The conidia (spores) are easily aerosolised. The route of transmission is by air. Although exposure is universal, invasive infection occurs almost exclusively in immunocompromised individuals. Infections have frequently been described in patients with haematological malignancies and solid organ transplant recipients, but also in patients undergoing chronic intermittent haemodialysis in whom these infections were associated with hospital construction and\/or ventilation systems contaminated with Aspergillus spp.\u00a0[8]. Even hospital water is a\u00a0frequently overlooked source of nosocomial aspergillosis\u00a0[9, 10].\nNatural antifungal defence in humans is based on normal mucosal barriers and an intact macrophage and neutrophil function. Alveolar macrophages form the first line of defence against inhaled Aspergillus conidia that reach the alveoli. Macrophages normally are capable of killing the conidia and preventing germination, by releasing cytokines such as tumour necrosis factor (TNF)-\u03b1 and macrophage inflammatory protein (MIP)-1\u03b1\u00a0[11]. During neutropenia, TNF-\u03b1 and MIP-1\u03b1 synthesis is reduced and the conidia can germinate to form hyphae. T-cell mediated acquired immunity also has an important role in protecting against fungal infections, as Aspergillus antigens are able to induce T-helper (Th)-1 and Th-2 type reactivity\u00a0[12, 13]. Th-1 reactivity is displayed by an increase of interferon-\u03b3 and interleukin (IL)-12 and has protective effects against infection. In contrast, Th-2 reactivity is characterised by production of IL-4 and IL-10 and leads to disease progression, at least in a\u00a0murine model of IPA\u00a0[12, 14, 15].\nCritically ill patients in ICU exhibit a\u00a0complex change in immune function characterised by deactivation of macrophages and an altered cellular response due to the severity of illness which is also termed \u201cimmunoparalysis\u201d\u00a0[16, 17]. This immunologic derangement might explain why Aspergillus infections are able to develop in critically ill patients who do not display the predisposing classical risk factors\u00a0[17, 18]. Many other factors will negatively influence the immune function during critical illness, such as (acute) hyperglycaemia\u00a0[19] and the use of corticosteroids\u00a0[20\u201323]. Corticosteroids have profound effects on the distribution and function of neutrophils, monocytes, and lymphocytes and they directly stimulate the growth of Aspergillus fumigatus in vitro possibly via sterol binding proteins in the fungus\u00a0[21]. In particular intravenous corticosteroids treatment in patients with chronic obstructive pulmonary disease (COPD), is associated with a\u00a0rising incidence of IPA\u00a0[1, 24\u201326]. Also broad-spectrum antibiotics, which affect the distribution of normal flora, have been described as a\u00a0risk factor\u00a0[27]. However, not every critically ill patient in the ICU is at risk for developing invasive fungal infections. Apparently, other specific \u2013 patient-related \u2013 predisposing conditions seem to be associated with the development of IPA, in which COPD and other chronic lung disease\u00a0[1\u20134, 28\u201332], diabetes mellitus\u00a0[2, 31, 33], acute liver failure\/advanced liver cirrhosis\u00a0[2\u20134, 34], chronic renal failure\u00a0[8, 35], and near drowning\u00a0[4, 36\u201338] have been described (Table\u202f1). In these, mainly retrospective, studies a\u00a0mean in-hospital mortality of 80% was found in patients with highly suspected or proven IPA in the presence of at least one of these underlying conditions, despite antifungal therapy. Remarkably, patients who were suspected to be colonised only with Aspergillus spp. (i.\u202fe. no signs of pulmonary infection) demonstrated even a\u00a0high in-hospital mortality rate\u00a0[3, 31], which might suggest that colonisation should be considered as a\u00a0potentially important finding.\nTable\u00a01Risk factors for IPA in non-neutropenic critically ill patients in the ICURisk factorReferenceCOPD in combination with prolonged corticosteroid use\u00a0[1, 24\u201326]High-dose systemic corticosteroids >\u202f3\u202fweeks (e.\u202fg. prednisone equivalent\n>\u202f20\u202fmg\/day)\u00a0[2, 31, 33]Chronic renal failure with RRT\u00a0[8, 35]Liver cirrhosis\/acute hepatic failure\u00a0[2\u20134, 34]Near-drowning\u00a0[4, 36\u201338]Diabetes mellitus\u00a0[2, 3, 31, 33]COPD, Chronic obstructive pulmonary disease; RRT,\nrenal replacement therapy\nDiagnostic features\nA\u00a0few years ago, a\u00a0consensus for standard definitions and diagnosis for invasive fungal infections in immunocompromised patients with cancer and recipients of haematopoietic stem cell transplants was established by the European Organization for Research and Treatment of Cancer and the Mycoses Study Group (EORTC\/MSG) in which three levels of probability for invasive fungal infections are proposed: \u201cproven\u201d, \u201cprobable\u201d and \u201cpossible\u201d\u00a0[39]. However, these guidelines are intended for use in clinical trials and for epidemiologic research and, moreover, are focused on patients with classical risk factors such as neutropenia, malignancies and after haematopoietic stem cell transplantation, and it may not be possible to extrapolate them to the non-neutropenic critically ill patient.\nThe diagnosis of IPA in non-neutropenic critically ill patients is difficult because signs and symptoms are non-specific, and the initiation of additional diagnostic examinations is often delayed because of a\u00a0low clinical suspicion. For the timing of suspicion of IPA in these patients, the combination of persistent or rapid developing infiltrative abnormalities on thoracic imaging and\/or a\u00a0persistent pulmonary infection despite broad spectrum antibiotics accompanied by one or more predisposing conditions (Table\u202f1) might be the moment for triggering further diagnostic exploration. Although histopathological evidence of IPA is defined as the gold standard due to a\u00a0very high tropism for blood vessels\u00a0[39], trans-bronchial biopsy or surgical lung biopsy via mini-thoracotomy may not justify the risk of this invasive procedure in critically ill patients on mechanical ventilation with, sometimes, severe bleeding diathesis. Therefore, the diagnostic process will contain thoracic imaging and microbiological examination by means of direct microscopy and culture of sputum or broncho-alveolar lavage fluid (BALF). Excluding the possibility of contamination during the pre-analytical phase of a\u00a0sample, isolation of Aspergillus spp. in the respiratory tract may represent three clinical situations: (1) evidence of current disease, (2) true colonisation, or (3) a\u00a0marker for the future development of invasive disease. In the immunocompetent host, cultures of Aspergillus in respiratory secretions are usually a\u00a0result of colonisation\u00a0[32]; in the immunocompromised host, however, they may indicate invasive disease\u00a0[6, 7, 39]. The positive predictive value of cultures in general is as high as 80\u201390%\u00a0[2, 6, 40], although in some groups of patients (e.\u202fg. after lung transplantation) specificity is much lower\u00a0[41]. Sensitivity of cultures in the diagnosis of IPA is poor\u00a0[2, 6, 40]. Direct microscopic examination of sputum or BAL, stained with specific fluorescent stain for chitin (a fungal cell wall component) is easy to perform, rapid to read, and improves the sensitivity of microbiological examination\u00a0[40, 42]. Direct visualisation of the hyphae makes it possible to discriminate between septate (e.\u202fg. Aspergillus, Fusarium and Scedosporium) and non-septate (e.\u202fg. Mucorales) moulds. Ubiquitous moulds of the order Mucorales cause serious infection in immunocompromised patients and, in contrast to Aspergillus, Fusarium and Scedosporium spp., they are not susceptible to voriconazole, which is an important factor when choosing a\u00a0pre-emptive antifungal drug. The extra value of a\u00a0positive culture is that growing of the fungus enables identification and susceptibility testing to antifungal drugs. This is important in view of the antifungal resistance of \u2013 for example \u2013 Aspergillus fumigatus isolates to voriconazole\u00a0[43]. Nonetheless, reliance on microscopy and\/or culture alone results in substantial underdiagnosis due to the low sensitivity. Fibre-bronchoscopy with inspection of the tracheobronchial tree, sampling of deep airway secretions and BAL can be helpful; the macroscopic finding of ulcerative lesions and\/or pseudomembranes together with a\u00a0positive microscopy and\/or culture is highly suggestive for Aspergillus-related tracheobronchitis.\nChest computerised tomography (CT) has proved to be an important tool for the diagnosis of IPA in neutropenic, severely immunocompromised patients, even in the absence of evident lesions on a\u00a0conventional chest X-ray. Radiological findings might include nodules with rapid growth and\/or cavitations. A\u00a0`halo sign' (a pulmonary mass surrounded by a\u00a0zone of lower attenuation with ground-glass opacification produced by adjacent haemorrhage) and\/or the `air crescent sign' (crescentic radiolucencies around a\u00a0nodular area of consolidation) may be present\u00a0[44\u201350]. The frequency of the halo sign in patients with IPA is relatively high in the early stages of the disease, but becomes progressively lower with the passage of time\u00a0[51]. Combining the halo sign and the air crescent sign, the sensitivity for IPA is more than 80% as specificity reaches 60\u201398%\u00a0[47]. However, thoracic imaging in mechanically ventilated ICU patients is less helpful due to many confounding factors such as atelectasis and, sometimes major, pleural effusions. A\u00a0lower sensitivity (5\u201324%) of the halo sign and air crescent sign in non-neutropenic patients has been reported in the literature\u00a0[3, 6, 52]. Due to the high tropism for blood vessels, IPA might be complicated by localisations in the central nervous system. Therefore, in patients with documented or highly suspected IPA, CT scanning or magnetic resonance imaging (MRI) of the brain should be considered to exclude dissemination to the brain.\nIn the past decade, non-invasive diagnostic tests, serological and molecular, have focused on the detection of surrogate markers for Aspergillus spp., such as the galactomannan (GM) antigen, 1,3-\u03b2-glucan and the detection of Aspergillus DNA by PCR. GM is a\u00a0major Aspergillus cell-wall component that is released during the growth phase of the fungus, and detection of GM would be indicative for invasive disease\u00a0[53]. Many studies have been done in order to investigate the value of the commercial Platelia Aspergillus assay (BioRad\u2122, Marnes-La-Coquette, France) as a\u00a0diagnostic tool for IPA, but mainly in patients with haematological malignancies\u00a0[54\u201357]. The specificity of the GM assay for diagnosing IPA is at least 85%, as demonstrated by these studies, but the sensitivity of the assay varied considerably between 29% and 100% depending on the cut-off value. The most important finding of these studies was that in around two-third of patients, circulating antigen could be detected at a\u00a0mean of 8 days before a\u00a0probable diagnosis was made by a\u00a0combination of radiographic findings and Aspergillus isolation\u00a0[53, 54]. In a\u00a0recent meta-analysis, 27 studies were included regarding the value of the GM serum assay for surveillance of IPA in high-risk patients. The median sensitivity for proven cases was 71% (specificity 89%); for proven or probable cases, median sensitivity and specificity were 61% and 93%, respectively\u00a0[58]. Specificity increased to 95% using a\u00a0cut-off value of 1.5 in cases with proven or probable IPA. Because GM is a\u00a0water-soluble carbohydrate, it can also be detected in BALF. Although the Platelia ELISA (enzyme-linked immunosorbent assay) is not validated for detection of GM in this fluid, there is an increasing tendency to use these samples for diagnosis of IPA. In small clinical studies among patients with haematological malignancies and in solid organ transplant recipients, the sensitivity of the GM EIA (enzyme immuno-assay) applied to BALF ranges from 85% to 100% with a\u00a0high index cut-off (>\u202f1.5) to define positivity\u00a0[59\u201362]. However, there are several clinical circumstances that might influence the diagnostic performance of the GM antigen test in either serum or BALF. First, the false-positive reactivity which might be caused by gastro-intestinal translocation of fungal GM from contaminated food or drink, as demonstrated in small children\u00a0[63], and the use of the intravenous antibiotics piperacillin\u2013tazobactam and amoxicillin\u2013clavulanic acid, which is associated with serum ELISA reactivity in patients without evidence of IPA\u00a0[64\u201367]. Second, an important factor that affects the release of GM antigens is antifungal drug therapy. Different animal and human studies have shown decreased sensitivity of the GM assay when (prophylactic) antifungal drugs were used\u00a0[63, 68, 69]. In several prospective studies that assessed the performance of antigen detection, patients received antifungal prophylaxis with itraconazole, which might have a\u00a0significant effect on the sensitivity of the assay\u00a0[53, 54]. Third, when the Platelia Aspergillus ELISA kit was launched in Europe about a\u00a0decade ago, another cut-off serum ratio was recommended than at present. Over the past years, several studies suggested lower cut-off values, ranging from 0.5 to 1\u00a0[39, 53, 54, 70\u201372]. It is clear that alterations in cut-off level will change the performance of the assay. Finally, the studies show that monitoring GM levels is crucial in order to diagnose (and eventually monitor treatment outcome) correctly, which means that the assay has to be performed twice weekly, preferably on receipt of the specimen. In critically ill patients without classical risk factors for IPA, the diagnostic value of the GM assay has been investigated only in one retrospective study and demonstrated a\u00a0sensitivity of only 53% in patients with proven or probable IPA (cut-off value 1.0)\u00a0[3]. Thus, it has to be stressed that the available data from patients with (haematological) malignancies and after solid organ transplantation can not be extrapolated to the critically ill patient in general. In the meantime, due to lack of more reliable, non-invasive diagnostic tests, the GM assay could be used as an additive tool in the diagnostic work-up of\u00a0IPA.\nThe 1,3-\u03b2-glucan is a\u00a0cell wall component of many filamentous fungi and yeasts, including Aspergillus spp. and Candida spp. Reproducible assay results, with high specificity and a\u00a0high positive predictive value, demonstrated that use of an assay to detect serum 1,3-\u03b2-glucan derived from fungal cell walls is a\u00a0useful diagnostic adjunct for invasive fungal infection\u00a0[73]. In addition, false-positive tests have been found in patients after haemodialysis, cardiopulmonary bypass surgery, high-dose immunoglobulin treatment, and after exposure to glucan-containing gauze\u00a0[33]. Furthermore, in a\u00a0recent small prospective study among ICU patients, serum glucan levels did not appear to be specific for fungal infections, as serum glucan levels were also elevated in bacterial infections\u00a0[74]. Hence, the usefulness of 1,3-\u03b2-glucan in the diagnosis of IPA has to be further evaluated.\nAmplification of nucleic acid by PCR technology for the diagnosis of IPA is being increasingly studied. It can be applied to serum and BAL specimens\u00a0[75\u201379]. Experience is limited to patients with haematological malignancies. White et al. evaluated the performance of a\u00a0real-time PCR in whole blood in a\u00a0group of patients with haematological malignancies and showed sensitivity of 92.3% and specificity of 94.6% for the diagnosis of IPA with good agreement of the GM ELISA\u00a0[80]. They concluded that a\u00a0negative PCR obtained twice weekly allowed a\u00a0wait-and-see approach concerning starting antifungal treatment. However, comparable to the GM antigen test, there are a\u00a0number of factors that potentially have an impact upon the clinical sensitivity of PCR. The magnitude of the quantitative PCR signal falls with antifungal therapy, thereby causing false-negative PCR results\u00a0[81] while the (transient) colonising presence of Aspergillus in the respiratory tract may suggest a\u00a0low positive predictive value\u00a0[78]. Furthermore, patients at risk for IPA are often prescribed a\u00a0multitude of drugs and fluids, all of which may act as non-specific inhibitors of the PCR. For example, anticoagulants inhibit PCR, thereby limiting its sensitivity\u00a0[82].\nOne might conclude that the use of GM antigen test, 1,3-\u03b2-glucan, and Aspergillus PCR as serological and molecular markers cannot be advocated for routine use in critically ill patients, and caution is warranted in the interpretation of positive test results in patients without a\u00a0clinical suspicion of pulmonary infection as well as negative test results in patients with persisting pneumonia. However, the finding of sequentially positive GM tests in serum or BALF \u2013 using higher cut-off values \u2013 together with a\u00a0positive Aspergillus PCR, in a\u00a0patient with persisting pulmonary infection who carries one or more risk factors, is highly indicative for IPA and might justify treatment with antifungal therapy.\nAntifungal therapy\nThere have been important developments in antifungal drugs in the past few years, although amphotericin B deoxycholate has been the standard therapy for IPA for decades. However, multiple studies have now established not only its lack of efficacy due to an increasing antifungal resistance but also demonstrated unacceptable toxicity of this compound, in particular nephrotoxicity\u00a0[83, 84]. Continuous infusion of amphotericin B deoxycholate over 24\u202fh may reduce its nephrotoxicity\u00a0[85, 86], although the efficacy may be reduced due to lower peak serum levels\u00a0[87]. The use of lipid formulations may also reduce toxicity and have been studied extensively for empirical use in febrile neutropenia with the same efficacy rate as conventional amphotericin B\u00a0[88]. However, lipid formulations are more expensive, and the initial use of higher doses does not improve efficacy and is associated with greater toxicity than lower doses, which suggests that high doses may not be routinely warranted.\nAmong the triazoles, itraconazole has activity against Aspergillus, but its clinical utility in critically ill patients with IPA has been limited by drug interactions and toxicity as well as erratic bioavailability of the oral suspensions\u00a0[89]. Furthermore, strains of Aspergillusfumigatus resistant to itraconazole have already been described\u00a0[90]. A\u00a0large multicentre randomised trial established that voriconazole provides higher response rates and better survival than amphotericin B in the treatment of \u201cprobable or proven\u201d IPA among patients with haematological diseases with fewer drug-related adverse events\u00a0[91]. As a\u00a0result of this study, voriconazole is increasingly recommended as initial therapy for IPA\u00a0[92]. Voriconazole is available for intravenous and oral use. It is rapidly absorbed within 2\u202fh after oral administration and the oral bioavailability is over 90%\u00a0[93]. Clearance is hepatic via N-oxidation by the hepatic cytochrome P450 (CYP) isoenzymes, CYP2C19, CYP2C9 and CYP3A4\u00a0[93], which makes the potential for drug interactions considerable. For instance, voriconazole considerably reduces the clearance of intravenous midazolam\u00a0[94], and fatal interactions with highly active antiretroviral therapy (HAART) have been described\u00a0[95]. As voriconazole has limited aqueous solubility, the intravenous form includes the solvent vehicle sulfobutylether beta cyclodextrin sodium\u00a0[96]. The clearance of sulfobutylether beta cyclodextrin sodium is linearly related to creatinine clearance and accumulation has been described in subjects with moderate to severe renal impairment\u00a0[96], although in animal experiments the frequency of acute toxicity of sulfobutylether beta cyclodextrin sodium is low. Target organs for toxic effects are the kidney and liver, causing obstruction of renal tubules and necrosis in the liver respectively\u00a0[96]. Because of this potential toxicity, it is recommended to treat patients with moderate to severe renal failure and who are on renal replacement therapy only with the oral form of voriconazole, if feasible. However, in critically ill patients safe oral administration of drugs is difficult to accomplish as, for instance, gastric reflux, gastro-intestinal bleeding and impaired function of the intestine are frequent co-morbidities, leading to potentially insufficient intestinal absorption of the drug. Furthermore, it is important to recognise that some patients may have inadequate levels of the oral drug, particularly if a\u00a0standard dose of 200\u202fmg twice daily is used rather than the recommended 4\u202fmg\/kg twice daily dose which has been studied for the intravenous formulation\u00a0[97]. At present, no clinical data are available regarding the bioavailability of the oral form of voriconazole in critically ill patients. It might be considered that the fear of potential adverse effects from intravenous solutions does not justify the risk of insufficient treatment by oral solutions. Moreover, limited clinical data showed no obvious toxicity in patients undergoing intermittent haemodialysis who were treated with daily 400\u2013800\u202fmg of intravenous voriconazole during 2 weeks\u00a0[96], and the pharmacokinetics of voriconazole appears not to be affected by continuous renal replacement therapy (CRRT). On the basis of pharmacokinetics, dose reduction is not recommended in patients receiving CRRT\u00a0[98, 99]. Regarding the efficacy of voriconazole, there is a\u00a0potential concern with (long-term) voriconazole therapy as occasional breakthrough infections with yeasts and moulds, with decreased susceptibility to voriconazole, have been reported\u00a0[43, 100, 101].\nPosaconazole is a\u00a0promising new triazole with broad-spectrum antifungal profile and has shown activity for salvage treatment of IPA in patients who are refractory to or intolerant of conventional therapy. A\u00a0recent multicentre, prospective study among haematological and non-neutropenic patients with refractory IPA demonstrated a\u00a042% overall success rate for posaconazole recipients versus 26% for control subjects\u00a0[102]. Posaconazole is, however, only available for oral administration, which makes it probably less applicable in critically ill patients who are susceptible to impaired drug absorption in the digestive tract.\nEchinocandins are a\u00a0novel class of parenterally administered semi-synthetic lipopeptides with a\u00a0pathogen-specific mechanism for non-competitive inhibition of biosynthesis of the fungus cell-wall enzyme complex 1,3-\u03b2-D-glucan\u00a0[103]. The echinocandins have documented in vivo activity against Candida spp. and Aspergillus spp. At present, there are three approved echinocandins, caspofungin, anidulafungin and micafungin, of which particularly caspofungin has demonstrated efficacy for the treatment of IPA. The first clinical trial to document the efficacy of caspofungin was among patients with \u201cproven or probable\u201d IPA who had treatment failure with (liposomal) amphotericin B, itraconazole or voriconazole, or who were intolerant to these antifungal drugs\u00a0[104]. Caspofungin seems to be as effective as and generally better tolerated than liposomal amphotericin B when given as empirical antifungal therapy in patients with persistent fever and neutropenia\u00a0[105]. However, prospective randomised clinical trials aimed at the treatment of IPA in (non-neutropenic) critically ill patients are lacking.\nIn general, there is no conclusive evidence that extended-spectrum triazoles are superior to echinocandins or polyenes, or vice versa, for monotherapy of IPA in ICU patients. We need prospective randomised controlled trials to solve this issue in critically ill patients. Table\u202f2 gives an overview of treatment options with antifungal drugs for IPA.\nTable\u00a02Treatment options with antifungal drugs for IPA in critically ill patients in the ICUSettingFirst choiceAlternativesPrimary therapy of IPAVoriconazole 6\u202fmg\/kg q\u202f12\u202fh i.\u202fv. on day\u202f1, then 4\u202fmg\/kg\nq\u202f12\u202fh i.\u202fv.Liposomal amphotericin B 3-5\u202fmg\/kg\/day i.\u202fv.ororVoriconazole 400\u202fmg q\u202f12\u202fh oral on day\u202f1, then 200\u202fmg q\u202f12\u202fh\noral aAmphotericin B deoxycholate 1\u202fmg\/kg\/day i.\u202fv.orCaspofungin 70\u202fmg i.\u202fv. on day\u202f1, then 50\u202fmg\/day i.\u202fv. ba Oral administration is recommended only in patients with intact intestinal\nabsorption; b In patients with moderate to severe hepatic failure, dose reduction\nis recommended to 35\u202fmg\/day i.\u202fv.\nCombination therapy\nBecause the efficacy of antifungal therapy for IPA is poor, with more than 50% of all patients experiencing failure of first-line therapies\u00a0[106, 107] empirical administration of combination antifungal regiments for proven or probable IPA may be an important strategy to improve outcome.\nTheoretically, there are several foreseeable advantages of combination therapy, such as a\u00a0widened spectrum and potency of drug reactivity, more rapid antifungal effect, synergy, and a\u00a0reduced risk of antifungal resistance\u00a0[108]. The available antifungal drugs target four different cell functions: cell membrane integrity (polyenes), ergosterol biosynthesis (azoles, allylamines), DNA synthesis (pyrimidine analogues) and cell-wall integrity (echinocandins). Although antifungal drugs are targeted against specific cell functions, many drugs also have pleiotropic effects that may inhibit other elements of fungal homeostasis\u00a0[108]. For instance, azoles inhibit many cytochrome-dependent enzymes of the fungal respiration chain and amphotericin\u00a0B generates oxidative species that damage fungal mitochondrial function and enhance macrophage fungal killing\u00a0[109]. These subtle effects could be enhanced when one antifungal is applied together with a\u00a0second drug, resulting in synergy. Conversely, the combination could act antagonistically, e.\u202fg. when one antifungal agent affects the targets of the other one.\nOnly a\u00a0few clinical studies with small numbers of patients have tried to address the need for combination antifungal therapy\u00a0[110\u2013113] . These studies have important limitations because they lack a\u00a0control group and because of many other uncontrolled factors including the choice of combination, the duration of therapy, concomitant antibacterial or antiviral treatment, the lack of adequate follow-up to estimate relapses, and no discrimination between primary and sequential therapy. Results from those studies should therefore be viewed with caution. Large, adequately powered, prospective clinical trials are needed but they might be difficult to perform because of inadequate enrolment, differences in providing benefits, difficulty in documenting fungal infection, controversy regarding endpoints, the lack of surrogate markers to correlate in-vitro evidence to outcome prognosis, and the associated costs\u00a0[114]. Nevertheless, combination antifungal therapy might be considered for certain clinical conditions such as refractory disease or breakthrough infections\u00a0[115, 116].\nConclusion\nRecent data indicate that IPA may be an underestimated opportunistic fungal infection in critically ill patients, even in the absence of severe pre-existent immunological disorders, and carries a\u00a0high mortality rate. A\u00a0decrease in immune function or dysregulation of the immune system due to the severity of illness, together with specific underlying risk factors such as COPD, diabetes mellitus, chronic renal replacement therapy, advanced liver disease, and long-term use of steroids, might explain the relatively high occurrence of IPA among these patients. The high mortality rate is partially related to difficulties in timely diagnosis because of non-specific signs and symptoms, low clinical suspicion, and time delay due to high risks for invasive procedures to obtain histopathological evidence for diagnosing IPA. The presence of a\u00a0persistent pulmonary infection despite broad-spectrum antibiotics or abnormal thoracic imaging by CT scanning together with one of these risk factors should trigger further diagnostic exploration by collecting respiratory secretions and\/or laboratory markers. Meeting the gold standard alone should not be the threshold for starting antifungal therapy, considering the high mortality rate. Invasive infection in patients with negative cultures might be supported by positive serological and molecular markers such as galactomannan antigen testing and Aspergillus PCR, which requires at least two sequentially positive samples. Antifungal therapy might be considered when persistent pneumonia with positive cultures for Aspergillus spp. or sequentially positive GM and Aspergillus PCR are present, accompanied with one of those risk factors for which voriconazole appears to be the first-line treatment. Combination therapy might be considered in breakthrough infections with moulds or yeasts or in refractory disease, although clear evidence is lacking.","keyphrases":["invasive pulmonary aspergillosis","non-neutropenic critically ill","antifungal therapy","serological markers"],"prmu":["P","P","P","R"]}
{"id":"Eur_J_Pediatr-4-1-2254659","title":"What is new in iron overload?\n","text":"Children with severe chronic hemolytic anemia or congenital erythroblastopenia are transfusion dependent. Long-term transfusion therapy prolongs life but results in a toxic accumulation of iron in the organs. The human body cannot actively eliminate excess iron. Therefore, the use of a chelating agent is required to promote excretion of iron. So far, iron chelation has been done by subcutaneous infusion of deferoxamine given over 10 h, 5\u20136 days per week. Compliance is poor and chelation often insufficient. Ferritin measurements and sometimes liver biopsies are used to evaluate the iron burden in the body. At the present time, new iron chelators that can be given orally are available. Furthermore, magnetic resonance imaging (MRI) assessment of tissue iron is a noninvasive and highly reproducible method, which is able to quantitate organ iron burden. In conclusion, iron overload can be measured more accurately with noninvasive methods such as MRI. Deferasirox is a once-daily oral therapy for treating transfusional iron overload, which improves patient compliance and quality of life.\nIntroduction\nChildren suffering from severe chronic hemolytic anemia such as \u03b2-thalassemia, or congenital erythroblastopenia are at risk of developing severe iron overload. Regular blood transfusions are potentially life saving. However, every single transfusion of one unit of blood brings 200\u00a0mg of iron into the body of the recipient. Patients receiving 3 U every 4\u00a0weeks (39 U per year) will accumulate 7.8\u00a0g of iron by the end of the year, in addition to the amount absorbed by the gut. The human body has no mechanism for active excretion of excess iron. Repeated transfusions lead to elevated iron levels that result in major organ damage. Excess iron is deposited largely in the liver, spleen, myocardium, and several endocrine organs. In young patients, growth and maturation is slowed, and in all age groups, liver disease, diabetes mellitus, and other complications develop. Once the body\u2019s storage capacity is exceeded, free iron catalyses the formation of highly reactive hydroxyl radicals, which cause membrane damage and protein denaturation. This process leads to tissue damage and ultimately to significant morbidity and mortality [3].\nMeasuring iron levels\n(Table\u00a01)\nTable\u00a01Comparison of methods for evaluating iron overload\u00a0InvasiveCostLimitationValidationFerritinNoLowInfection\/inflammationYesLICYesMedianFibrosis\/cirrhosisYesSQUIDNoHighNot easily availableYesMRINoHighDedicated personYesLIC liver iron content, SQUID superconducting quantum interference device, MRI magnetic resonance imaging\nThere are several methods of assessing body iron load. As 90% of excess iron is deposited in the liver, most techniques focus on measuring liver iron levels, and it is widely accepted that liver iron content (LIC) provides an accurate measure of whole-body iron concentration. The invasive nature of the liver biopsy means that other markers such as serum ferritin levels are frequently employed. Other approaches using biomagnetic susceptometry and magnetic resonance imaging (MRI) are also being assessed in order to identify an accurate, low-risk, and convenient approach to assessing patient iron status.\nFerritin\nSerum ferritin is easily measured using a commercially available kit. A ferritin constantly below 2,500\u00a0mg\/l has been shown to reduce the risk of cardiac complications, but a target value of 1,000\u00a0mg\/l is recommended. Factors such as inflammation, ascorbate status, and hepatitis can affect serum ferritin levels. Therefore, results should be interpreted with caution and a trend in the evolution of serial measurements is a better index than day-to-day variation. In patients with transfusion-dependent diseases, chelation should be initiated after 10\u201320 blood transfusions or when ferritin level rises above 1,000\u00a0mg\/l.\nLiver biopsy\nLIC measurement is the most accurate method for assessing body iron (Fig.\u00a01). It also provides information about the severity of the liver disease [8]. Liver samples can be obtained by percutaneous or transjugular access. LIC measurement from a needle biopsy has been associated with a coefficient of variation of less than 10% in livers with no advanced disease. However, it has been shown that variability can be much higher in fibrotic and cirrhotic livers. Values of LIC are expressed as milligrams of iron per gram of dry liver weight.\nFig.\u00a01Liver biopsy (Perls stain): iron deposits are in blue. Courtesy of B. Turlin, Pathology Department, University Hospital of Rennes\nLiver biopsy is an invasive technique that is associated with some pain and at risk of hemorrhage and infection. It is not indicated for routine assessment.\nSuperconducting quantum interference device (SQUID)\nSQUID is capable of measuring very small changes in magnetic flux. Iron stored as ferritin and hemosiderin is the only relevant paramagnetic material in the human body. The magnitude of paramagnetic response is directly related to the amount of iron in a certain volume of tissue. This is a noninvasive method, with a linear correlation with LIC assessed by biopsy. However, equipment availability is extremely limited [6].\nMagnetic resonance imaging (MRI)\nMRI measures tissue iron concentration indirectly by detecting the paramagnetic influences of storage iron (ferritin and hemosiderin) on proton resonance behavior. With MRI, the transmitted signal is a microwave, which excites water protons in the body to higher magnetic energy states. As these water protons relax back to the unexcited state, they emit microwaves that are received and interpreted by the scanner. Iron deposits act like little magnets when placed in a strong magnetic field, disrupting coherence among the protons and darkening the image more quickly (Fig.\u00a02) [13]. LIC determined using MRI shows excellent correlation with that obtained from liver biopsy. Furthermore, MRI has the ability to evaluate the entire organ and gives more accurate measurement of LIC, particularly in patients with heterogeneous iron content (Fig.\u00a03). It requires a dedicated imaging method and equipment operator [13].\nFig.\u00a02Gradient echo images of liver collected by J. Wood [13] at four different echo times. The top four images were collected from a patient having a liver iron of 6\u00a0mg\/g. The bottom four images were collected from a normal volunteer. All images darken as the echo time (TE) lengthens, but the iron-heavy tissue darkens faster. The half-life of this process is called T2* and the rate is called R2* (R2*\u2009=\u20091,000\/T2*)Fig.\u00a03Gradient echo imaging illustrating discordant iron loading of the liver and heart. a Heavy liver iron loading (dark tissue) with heart sparing image. b Heavy cardiac iron loading with no liver deposition. From [13]\nIron chelation therapy\nWithin 1\u20132\u00a0years of initiation of regular blood transfusions, iron starts to accumulate in the body. The use of a chelating agent then becomes mandatory [6]. At the present time, three different therapies are available (Table\u00a02).\nTable\u00a02Comparison between iron chelators (adapted from [4])CharacteristicsDeferoxamine (Desferal)Deferiprone (Ferriprox)Deferasirox (Exjade)Route of administrationSubcutaneous or intravenousOralOralHalf-life20 min2\u20133 h8\u201316 hRoutes of iron excretionUrine\/stoolUrinestoolDose range20\u201360\u00a0mg\/kg per day50\u201360\u00a0mg\/kg per day20\u201330\u00a0mg\/kg\/dayGuidelines for monitoring therapyAudiometry and eye exams annuallyComplete blood count weekly; ALT level monthly for first 3\u20136\u00a0months then every 6\u00a0monthsSerum creatinine level monthly; ALT level monthlySerum ferritinSerum ferritinSerum ferritinAssessment of liver iron annuallyAssessment of liver iron annuallyAssessment of liver iron annuallyAssessment of cardiac iron annually after 10\u00a0years of ageAssessment of cardiac iron annually after 10\u00a0years of ageAssessment of cardiac iron annually after 10\u00a0years of ageAdvantagesLong-term experienceOrally activeOrally activeEffective in maintaining normal or near-normal iron storesSafety profile well establishedOnce-daily administrationReversal of cardiac disease with intensive therapyEnhanced removal of cardiac ironDemonstrated equivalency to deferoxamine at higher dosesMay be combined with deferiproneMay be combined with deferoxamineTrials in several hematologic disordersDisadvantagesRequires parenteral infusionMay not achieve negative iron balance in all patients at 75\u00a0mg\/kg per dayLimited long-term dataEar, eye, bone toxicityRisk of agranulocytosisNeed to monitor renal functionPoor complianceMay not achieve negative iron balance in all patients at highest dose\nDeferoxamine mesylate (Desferal) is the current standard for iron chelation therapy. The long-term efficacy of this approach has been demonstrated, limiting organ damage and preventing premature death. Deferoxamine has a very short half-life in plasma (5\u201310 min). Therefore, deferoxamine is administered parenterally, either subcutaneously 8\u201312 h a day at least 5\u00a0days a week, or intravenously, through a Port-a-Cath for continuous infusion. Low compliance with the prolonged subcutaneous administration is the main reason of ineffective treatment. Toxic side effects such as auditory, ocular, and neurotoxic abnormalities are also drawbacks in the therapy.\nDeferiprone (Ferriprox) is one of the hundreds of oral chelators that have been tested. It was shown to have comparable efficacy to subcutaneous deferoxamine. It has been used as a monotherapy or in combination with deferoxamine [7, 9]. It increases urinary excretion of iron and is more effective than deferoxamine in the removal of excess iron from the heart, as shown from MRI T2* studies [1, 9, 10, 11]. Deferiprone and its iron complex are cleared from the plasma with a half-life of 47\u2013134 min. Therefore, it should be taken three times a day. The major toxic side effects are agranulocytosis, musculoskeletal and joint pains, gastric intolerance, and zinc deficiency. Those side effects are considered to be reversible. The combination of deferoxamine and deferiprone is most effective when used in the following way: deferiprone (80\u2013110\u00a0mg\/kg per day) during the day and deferoxamine (40\u201360\u00a0mg\/kg per day) at least 3 nights a week. No new toxic effects have been reported for the combination.\nDeferasirox (Exjade) has been tested recently in clinical trials [2, 3, 12]. Its long half-life makes it suitable for once-daily oral administration. Efficacy at a dose of 20\u201330\u00a0mg\/kg per day is at least equivalent to that of deferoxamine (\u226535\u00a0mg\/kg per day 5\u00a0days weekly) in a subgroup of patients with higher hepatic iron burdens. The most common toxic side effects are skin rash, increase in transaminases and creatinine, and gastrointestinal symptoms such as nausea, vomiting, diarrhea, and abdominal pain. The skin rash and gastrointestinal symptoms tend to decrease spontaneously with time and an increase in creatinine may require a dose adjustment. The increase in transaminases is usually linked to an insufficient dose of deferasirox and an increase in the hepatic iron overload. A higher dose of deferasirox will result in an improvement in transaminases.\nDiscussion\nChronic iron overload due to blood transfusions leads to significant morbidity and early mortality unless adequate chelation therapy is administered. Deferoxamine is the reference chelation therapy, but compliance to the treatment is often poor because it must be administered by prolonged subcutaneous or intravenous infusion. Oral chelators are now available: Deferasirox has the advantage of being administered once daily, with a low toxicity profile. Furthermore, its cost effectiveness has also been demonstrated [5]. MRI has emerged as the dominant technique by which to evaluate iron overload because of its sensitivity, reproducibility, and ability to image multiple organs in the body during a single imaging session.","keyphrases":["iron overload","chelators","diagnosis"],"prmu":["P","P","U"]}
{"id":"Ann_Biomed_Eng-4-1-2413127","title":"Biomechanics of Traumatic Brain Injury: Influences of the Morphologic Heterogeneities of the Cerebral Cortex\n","text":"Traumatic brain injury (TBI) can be caused by accidents and often leads to permanent health issues or even death. Brain injury criteria are used for assessing the probability of TBI, if a certain mechanical load is applied. The currently used injury criteria in the automotive industry are based on global head kinematics. New methods, based on finite element modeling, use brain injury criteria at lower scale levels, e.g., tissue-based injury criteria. However, most current computational head models lack the anatomical details of the cerebrum. To investigate the influence of the morphologic heterogeneities of the cerebral cortex, a numerical model of a representative part of the cerebral cortex with a detailed geometry has been developed. Several different geometries containing gyri and sulci have been developed for this model. Also, a homogeneous geometry has been made to analyze the relative importance of the heterogeneities. The loading conditions are based on a computational head model simulation. The results of this model indicate that the heterogeneities have an influence on the equivalent stress. The maximum equivalent stress in the heterogeneous models is increased by a factor of about 1.3\u20131.9 with respect to the homogeneous model, whereas the mean equivalent stress is increased by at most 10%. This implies that tissue-based injury criteria may not be accurately applied to most computational head models used nowadays, which do not account for sulci and gyri.\nIntroduction\nThe brain is often one of the most seriously injured parts of the human body in case of a road traffic crash situation.1,21,41 The incidence rate and mortality rate in Europe are estimated to be 235 and 15.4 per 100,000 of the population per year, respectively.41 Traumatic brain injury (TBI) is therefore considered as a widespread problem. Understanding the mechanisms inducing TBI is necessary for reducing the number of occurrences, e.g., by developing more appropriate protective systems and diagnostic tools.\nBrain injury criteria are used for the assessment of the probability of TBI for certain mechanical loading conditions. The most commonly used injury criterion in the automotive industry is the Head Injury Criterion (HIC).16,43 It is developed to predict TBI resulting from a translational acceleration of the head. One of the drawbacks of the HIC is that it is based on global kinematic data to predict TBI, whereas actual brain damage is caused at the cellular level as a consequence of tissue strains and stresses.35 Furthermore, it is based on experimental data, in which only anterior\u2013posterior contact loading has been applied to human cadavers, not accounting for angular accelerations of the head. For a better approximation of the relation between TBI and a mechanical load, more advanced methods have been developed. For instance, three-dimensional finite element (FE) head models have been developed to predict brain injury.2,6,7,9,17,22,38,42,45,47 With these numerical head models, different injury mechanisms and loading conditions can be distinguished. However, in these models, the heterogeneous anatomy of the cerebrum is usually represented by a relatively homogeneous geometry. A comparison between the homogeneous geometry of a typical finite element head model and the complex structure of a real brain is given in Fig.\u00a01. The main function of the heterogeneous morphology is to increase the cortical surface in order to obtain a more complex level of the brain functions.34 The most recent numerical head models include ventricles and the invaginations of the dura mater, but none include the convolutions of the cerebral cortex. Consequently, the stresses and strains that are predicted from these models likely do not represent actual tissue stresses and strains, at least in the cortex. Therefore, although tissue-based injury criteria may be used, their accuracy is expected to be limited. This might prohibit the direct use of tissue-based injury criteria. Such criteria predict injury at the tissue level and are based on in\u00a0vitro and in\u00a0vivo experiments.3,4,10,11,13,31,32 For a direct application of tissue-based injury criteria in a computational head model, a more detailed description of the biomechanical behavior of the cerebrum may be required, which can be achieved by including its morphologic heterogeneities in these models. A few two-dimensional FE models of the brain containing the convolutions of the cerebral cortex have been described in literature. Miller et\u00a0al.30 compared different modeling techniques for the relative motion between the brain and the cranium. Nishimoto and Murakami33 developed a model to investigate the relation between brain injury and the HIC. However, these models have not been developed with the purpose of investigating the local biomechanics at the level of these convolutions. No conclusions have been drawn from these studies on the biomechanical influence of the heterogeneities of the cerebral cortex, due to the limited spatial resolution of the mesh.Figure\u00a01(a) Numerical head model developed by Claessens.6,7 (b) Lateral view of the human brain. Adapted from Welker et\u00a0al.44\nPhysical experiments have been conducted in several studies to investigate the biomechanical consequences of the heterogeneities of the cerebrum.14 In a study by Bradshaw et\u00a0al.,5 a gel-filled chamber that represented the brain and skull in a coronal plane including the falx cerebri and the sulci of the cerebral cortex was subjected to a rotation with a peak acceleration of approximately 7800\u00a0rad\u00a0s\u22122. An increase of the maximum principle strain in the cerebral cortex due to the sulci was found.\nThe aim of this study is to investigate the biomechanical influences of the morphologic heterogeneities in the cerebral cortex. To achieve this, several two-dimensional FE models with detailed geometries of a part of the cerebral cortex have been developed. Also, an FE model with a homogeneous morphology of the cortex has been made. The loading conditions are based on simulations with a computational head model as used by Brands et\u00a0al.6 The results of the simulations of the heterogeneous models will be compared to those of the homogeneous model.\nMethods\nIn this study, plane strain models of small sections of the cerebrum are made using the FE code Abaqus 6.6-1 (HKS, Providence, USA). An explicit time integration is used, anticipating a dynamic load with a high magnitude and a short duration. The time increments are limited by the stability condition, which is determined in the global estimator function in Abaqus.\nGeometries\nTo investigate the influence of the heterogeneities of the cerebral cortex, a homogeneous model and three heterogeneous models have been developed. The heterogeneous models, which are shown in Figs.\u00a02a, 2c, and 2d have detailed geometries of a small part of the cerebrum, including also a part of the cerebrospinal fluid (CSF). The cranium is modeled by a boundary constraint, as will be detailed further on. Since the dura mater and the arachnoid are connected to the inside of the cranium in the region that is modeled,29,34 it is assumed that they can be ignored for this situation. The pia mater, which is a thin and delicate membrane covering the brain,29,34 is also not included, since it is expected to have no mechanical influence for the used loading conditions. The same assumption is used for the arachnoid trabeculae, which extend from the arachnoid to the pia mater and are less existent inside the sulci.34Figure\u00a02(a) Heterogeneous geometry 1 and (b) its spatial discretization. (c) Heterogeneous geometry 2. (d) Heterogeneous geometry 3. (e) Homogeneous geometry\nThe first geometry has one narrow sulcus on the right hand side and a small part of a sulcus on the left hand side. The second geometry contains two deeper and wider sulci than the other two geometries. The third geometry consists of one vertical sulcus and one partly horizontal sulcus, where horizontal and vertical refer to the x- and y-direction, respectively. These geometries, which represent typical stylized shapes of the cerebral cortex, are based on the topological studies by Mai et\u00a0al.27 The left and right boundaries of the models are chosen to be periodic, i.e., the internal geometries near the opposite boundaries match. The periodicity of the boundary conditions will be explained further on. The models do not distinguish between gray (cerebral cortex) and white matter. In Fig.\u00a02e, the homogeneous model is shown. Similar to the heterogeneous models, it also consists of CSF and brain tissue, but it does not contain any gyri and sulci. The outer dimensions of each model are 32\u00a0mm by 24\u00a0mm. The meshes consist of bi-linear, quadrilateral, reduced integration elements with hourglass control. The heterogeneous models also contain a small number of triangular elements. The total number of elements of the heterogeneous models ranges from 4243 to 4533 elements. The homogeneous geometry consists of 3072 elements.\nMaterial Properties\nFor the material properties of the CSF, a nearly incompressible, low shear modulus elastic solid has been assumed, since the shear stress in the brain tissue due to the applied loading conditions is estimated to be about a factor 104 higher than that in the CSF. The material properties are listed in Table\u00a01. The shear modulus of CSF is estimated from the loading conditions that are described further on by using  in which G is the elastic shear modulus, \u03b7 is the viscosity, \u03b3 is the estimated shear strain, and  is the estimated shear rate. Because two different loading conditions have been used, also two different estimates for the CSF shear modulus have been used. However, with these shear moduli being much lower than that of the brain tissue, the exact values of these estimates do not affect the outcome of this study. The bulk modulus is obtained from literature.2,47Table\u00a01Linear material parameters\u00a0Bulk modulus (GPa)Shear modulus (Pa)Time constant (s)CSF2.20.036a\u221e0.12b\u221eBrain tissue2.5182.9\u221e98840.00013835.50.012231.20.3567.14.623.6112.12.7954.3aShear modulus in case of loading condition AbShear modulus in case of loading condition B\nThe material properties of the brain tissue are described by a non-linear viscoelastic constitutive model that has been developed by Hrapko et\u00a0al.19 This model was found to accurately describe the response of brain tissue for large deformations in both shear and compression. This model is extended here to account for compressibility.\nThe constitutive model consists of an elastic part, denoted by the subscript \u2018e\u2019, and a (deviatoric) viscoelastic part, denoted by the subscript \u2018ve\u2019, with N viscoelastic modes. The total Cauchy stress tensor  is written asin which the superscripts \u2018h\u2019 and \u2018d\u2019 denote the hydrostatic and the deviatoric part, respectively. For simplicity, the subscript i indicating the number of the viscoelastic mode will be omitted from this point on. The hydrostatic part of Eq. (1) is defined aswhere K is the bulk modulus and  is the volume change ratio.\nThe deviatoric elastic mode describes a non-linear response to the deformation gradient tensor F, which is given bywhere G\u221e is the elastic shear modulus,  is the isochoric part of the Finger tensor B, and  and  are the first and second invariant of the isochoric Finger tensor  respectively. A, C, and b are fitting parameters describing the non-linearity of the elastic response.\nThe third term on the right hand side of Eq. (1) consists of the summation of the viscoelastic modes. The deformation gradient tensor F is partitioned into an elastic deformation gradient tensor  and a viscous deformation gradient tensor  by assuming multiplicative decomposition25,36:\nThe decomposition involves a fictitious intermediate state, which could exist after application of merely the viscous deformation gradient tensor  This is the stress-free state, which after application of the elastic deformation tensor  transforms into the final state. The third term on the right hand side of Eq. (1) describes the viscoelastic contribution to the stress as follows:with G the shear modulus,  the isochoric part of the elastic Finger tensor  and a a fitting parameter.\nThe viscous deformation  is assumed to be volume-invariant, i.e.,  and  The viscous rate of deformation tensor is calculated from the flow rule aswhere the dynamic viscosity \u03b7 is a function of the scalar equivalent stress measure  for which the Ellis model is adopted:with subscripts 0 and \u221e denoting the initial and infinite values, respectively. The initial value for the viscosity is defined as \u03b70\u00a0=\u00a0G\u03bb, whereas the infinite viscosity is defined as \u03b7\u221e\u00a0=\u00a0k\u03b70. Here, \u03bb refers to the time constant.\nAlthough differences between the material properties of the gray and white matter may exist, these differences are not well characterized. Therefore, no distinction between gray and white matter has been made in this study, except for the investigation of the influence of varying the material properties of gray matter with respect to those of white matter (see the Discussion and Conclusions). For simulating a head impact situation representative of road traffic accidents, an extra viscoelastic mode with a smaller time constant has been added to the behavior as characterized by Hrapko et\u00a0al.19 The extra mode18 is based on the experimental data from Hrapko and co-workers in combination with the data by Shen et\u00a0al.39 The linear material properties are listed in Table\u00a01. The values of the non-linear viscoelastic parameters are shown in Table\u00a02.Table\u00a02Non-linear material parameters for brain tissueElasticViscousA\u00a0=\u00a00.73\u03c40\u00a0=\u00a09.7\u00a0PaC\u00a0=\u00a015.6n\u00a0=\u00a01.65a\u00a0=\u00a01k\u00a0=\u00a00.39b\u00a0=\u00a01\nBoundary Conditions\nThe boundary conditions have been chosen such that they represent the biomechanical influences of the surroundings on the cerebral cortex model. Figure\u00a03 shows the labeling of the corner nodes and the boundaries. The symbols x and y denote the components of position vector  with respect to a Cartesian vector basis  whereas u and v are the components of the displacement vector  with respect to this basis.Figure\u00a03Labeling of corner nodes and boundaries\nThe Young\u2019s modulus of the cranium is much higher than that of brain tissue.19,46 Still, in a contact loading situation of the head the deformation of the skull is important, because it initiates strain waves in the brain tissue. In this study, however, only inertial loading of the head is considered and therefore the cranium is assumed to be rigid. The cranium is incorporated in the boundary condition at \u03933. Because of the low shear modulus of the CSF, the influence of the rigid constraint associated with the cranium at boundary \u03933 in the x-direction can be neglected. Provided no rotation of the model occurs, the constraint equation for all nodes on boundary \u03933 iswith vs the vertical displacement of the skull.\nThe boundaries \u03932 and \u03934 are subjected to periodic boundary conditions23:\nThese constraints imply that throughout the deformation process the shapes of the opposite boundaries, \u03932 and \u03934, remain identical to each other, while the tractions on opposite boundaries are opposite to satisfy stress continuity, which can be written aswith  the Cauchy stress tensor and  the unit outward normal vector of boundary \u0393i.\nThe lower boundary, \u03931, of the brain tissue in the model lies adjacent to brain tissue in neighboring regions. Therefore, boundary \u03931 has to be constrained accordingly. The applied constraint on \u03931 is obtained by tying all nodal displacements on \u03931 to a linear interpolation between the displacements of corner nodes C1 and C2. For any node on boundary \u03931, this results inwith the subscript 0 denoting the initial configuration. The displacements of corner nodes C1 and C2 are prescribed and calculated from the applied loading conditions.\nThe loading conditions of the cerebral cortex model (micro-level in Fig.\u00a04) are based on the loading conditions that have been used by Brands et\u00a0al.6 for a three-dimensional numerical head model (macro-level in Fig.\u00a04). In that model, an eccentric rotation has been applied to the skull to simulate an angular head acceleration around the neck-shoulder joint in the sagittal plane in the anterior\u2013posterior direction. The eccentricity has been chosen to represent a typical neck length. The axis of rotation has been positioned at 155\u00a0mm below the anatomical origin, i.e., the ear hole projected to the sagittal plane. The rotation of the head model consists of two successive sine functions that describe the angular acceleration:Figure\u00a04The loading conditions of the cerebral cortex model (micro-level) are derived from the region of interest in a parasagittal cross-section (15\u00a0mm offset from the midsagittal plane) of the head model (macro-level). Shown at the macro-level is the equivalent stress field of the head model at 10\u00a0ms\nIn Eqs. (12) and (13), the angular acceleration  is given in rad\u00a0s\u22122.\nThe loading conditions are applied to the cerebral cortex model by means of body forces. In all integration points of the elements in the model, a non-uniform body force is imposed that reversely simulates the inertial forces:in which  represents the distributed load per unit of volume, \u03c1 is mass density, t is time, and  refers to the acceleration in the x-direction that is represented by these body forces. Note that for the head model the loading conditions contain an angular component, whereas the cerebral cortex model uses translational loading conditions. Because only a small part of the head is modeled and because of the small rotation of the head model with a maximum of 4\u00b0, the loading of the cerebral cortex model is assumed to be translational in x-direction only.\nThe loading conditions of the cerebral cortex model, i.e., the representative accelerations  are calculated from the head model (from the region indicated in Fig.\u00a04) in two different approaches: In the first approach, the input accelerations of the head model are used to define the loading condition of the cerebral cortex model. This approach will be referred to as loading condition A.The translational acceleration  can be calculated usingwith  the angular acceleration, which is defined by Eqs. (12) and (13), and r the radius from the axis of rotation (neck-shoulder) in the head model to a point in the region of interest. The radius r is a function of the y-position in the cerebral cortex model. It varies between r(0)\u00a0=\u00a00.251\u00a0m at boundary \u03931 to r(0.024)\u00a0=\u00a00.275\u00a0m at \u03933. The accelerations  at \u03931 and \u03933 are depicted in Fig.\u00a05a. All other accelerations are interpolated linearly between these two boundaries, thereby creating a gradient across the height of the model. The acceleration gradient is important for the resulting shear stresses. Figure\u00a05b shows the acceleration profile of the cerebral cortex model. The accelerations are used to calculate the body forces as a function of both time and y-position.The disadvantage of this loading condition is that a spatially constant acceleration gradient is assumed and therefore it does not account for the influence of the geometry of the cranium. To account for the geometry of the head, another loading condition has been developed that is described next.The second approach, loading condition B, uses output accelerations from a global head model simulation as the input of the cerebral cortex model. For this, a modified version of the head model, as used by Brands et\u00a0al.,6 has been employed in the simulation code Madymo, in which the constitutive model for brain tissue by Hrapko et\u00a0al.19 has been implemented. The accelerations obtained from the region inside the box in Fig.\u00a04 from the head model are imposed on the cerebral cortex model. Hence, the influence of the geometry of the head is modeled indirectly by means of an acceleration profile that is obtained from the head model.The displacements of the brain tissue in the head model in the field of interest are almost entirely in the x-direction justifying the assumption of inertial loading (of the cerebral cortex model) in the x-direction only. In Fig.\u00a06, the acceleration profiles as a function of the y-position are shown at 5, 10, and 20\u00a0ms. Similar to loading condition A, the accelerations are used to calculate the body forces as a function of both time and y-position.Figure\u00a05Loading condition A. (a) Acceleration at the upper and lower boundary of the cerebral cortex model. (b) Acceleration profiles at different timesFigure\u00a06Loading condition B: displacement (top) and acceleration (bottom) profiles derived from the output of the head model\nIn order to quantify the influence of the morphologic heterogeneities, the equivalent stress  is used, in which  is the deviatoric part of the Cauchy stress tensor  The equivalent stress is chosen, because the simulations are based on an angular acceleration of the head, in which deviatoric stresses are considered to be the most important.30 The maximum principal strain is considered important as well with respect to diffuse axonal brain injury.3,30 Therefore, also the maximum principal logarithmic strain is used to quantify the influence of the morphologic heterogeneities.\nResults\nFigure\u00a07 depicts the development in time of the equivalent stress fields for the homogeneous model (top row) and the heterogeneous models from the simulation with loading condition A. Stress concentrations are present in the heterogeneous models at the surface of the brain tissue between two gyri at 5, 10, and 20\u00a0ms. Near boundary \u03931, all heterogeneous models have lower equivalent stresses compared to the homogeneous model at 20\u00a0ms.Figure\u00a07The equivalent stress fields as a result of loading condition A\nIn order to obtain a good comparison of the results for all geometries during the complete simulation time, the maximum and mean equivalent stress from the simulations with loading condition A are shown in Fig.\u00a08 as a function of time. It shows the stresses in the brain tissue only. It can be noticed that the heterogeneous models have a higher maximum equivalent stress than the homogeneous model. Among the heterogeneous configurations, geometry 1 causes a noticeably lower maximum equivalent stress of 112\u00a0Pa compared to geometries 2 and 3, with a maximum equivalent stress of approximately 156\u00a0Pa. The large maximum equivalent stress in heterogeneous geometry 2 lasts longer than the stresses of the other geometries. The maximum equivalent stress of the homogeneous model reaches a value of 80\u00a0Pa. The mean equivalent stresses are nearly the same for all geometries.Figure\u00a08Maximum and mean equivalent stress for the heterogeneous and homogeneous models as a result of loading condition A\nTo investigate the influence of the heterogeneities, the equivalent stress of the cerebral cortex in the heterogeneous models is taken relative to that of the homogeneous model. For the maximum equivalent stress, this will be done by taking the maximum values, whereas for the mean equivalent stress, this will be done by taking the time averaged values. The maximum equivalent stress of the heterogeneous models 1, 2, and 3 is 1.31, 1.84, and 1.83 times higher than the homogeneous model, respectively. The mean equivalent stress of the heterogeneous models 1, 2, and 3 with respect to the homogeneous model is 1.09, 1.08, and 1.10, respectively.\nThe equivalent stress fields obtained with loading condition B are displayed in Fig.\u00a09. During the beginning of the simulation, the equivalent stress fields in the brain tissue are comparable for all models. When the field of higher equivalent stress moves downwards, the heterogeneities result in local peak stress concentrations, which can be seen at 10\u00a0ms for geometries 1 and 3. Later on, at 20\u00a0ms, the heterogeneous geometries 1 and 3 have less influence on the equivalent stress fields. The differences of geometry 2 with respect to geometries 1 and 3 are a consequence of the deeper sulci in geometry 2.Figure\u00a09The equivalent stress fields as a result of loading condition B\nThe maximum and mean equivalent stress of the cerebral cortex as a function of time obtained with loading condition B is shown in Fig.\u00a010. The maximum equivalent stress is higher for the heterogeneous models than for the homogeneous models, but not for the complete duration of the simulation. After about 10\u201315\u00a0ms, the maximum equivalent stress of the heterogeneous models drops to approximately the same magnitude as the one obtained for the homogeneous model. For the heterogeneous models, the maximum equivalent stress reaches values of approximately 470, 565, and 624\u00a0Pa for geometries 1, 2, and 3, respectively. The homogeneous model has a maximum equivalent stress reaching 325\u00a0Pa. Also, the moment in time at which the maximum occurs differs from one geometry to the other. The mean equivalent stress values of all the geometries are similar.Figure\u00a010Maximum and mean equivalent stress for the heterogeneous and homogeneous models as a result of loading condition B\nTo quantify the influence of the heterogeneities, the equivalent stress of the brain tissue of the heterogeneous models is taken relative to the homogeneous model in the same manner as described previously for loading condition A. The maximum equivalent stress of the heterogeneous models 1, 2, and 3 has increased by 1.44, 1.74, and 1.92 with respect the homogeneous model, respectively. The mean equivalent stress of the heterogeneous models 1, 2, and 3 is 0.97, 0.99, and again 0.99 relative to the homogeneous model, respectively.\nThe distribution of maximum principal strains for loading condition B at 10\u00a0ms is shown in Fig.\u00a011. One can notice that a concentration of maximum principal strains at 10\u00a0ms occurs in the same location as the equivalent stress concentration at 10\u00a0ms (Fig.\u00a09), both in case of loading condition B. The same method for the quantification of the influence of the heterogeneities is used, but with the maximum principal strain instead of the equivalent stress. For the simulations with loading condition A, the peak maximum principal strain in the brain tissue of the heterogeneous models 1, 2, and 3 has increased with respect to the homogeneous model by 1.22, 1.92, and 1.80, respectively. If loading condition B is used, the increases are 1.43, 1.84, and 1.90, respectively.Figure\u00a011The maximum principal logarithmic strain field as a result of loading condition B at 10\u00a0ms\nDiscussion and Conclusions\nIn this study, the influences of the heterogeneities in the cerebral cortex were investigated. This was done with FE models of several different geometries from small detailed parts of the cortex. In a preliminary study, the boundary constraints were tested. The loading conditions were derived from a numerical head model.\nIn order to determine which constraints on the boundaries would represent the surroundings best, a preliminary study was conducted in which several different constraints were applied to boundaries \u03931 and \u03933. The different conditions on boundary \u03933, i.e., rigid constraint or slip-condition in the x-direction, did not contribute to differences in the results of the brain tissue. This was probably caused by the low shear modulus of the CSF. For boundary \u03931, several different boundary conditions were tested and compared to the results of models with the same width, but with twice the height of the models in this study. By comparing the \u2018normal\u2019 and the \u2018high\u2019 models, the boundary condition at boundary \u03931 that represented adjacent brain tissue could be determined. The periodic boundary condition that was applied to boundaries \u03932 and \u03934, was used because of the assumed periodicity in the cerebral cortex.\nSince the loading conditions were dynamic they could not be applied to the model by directly imposing a deformation, which would induce boundary effects. For this reason, an indirect deformation was imposed by means of body forces. This approach worked well for the first 20\u00a0ms of the simulation, which had a total duration of 30\u00a0ms. After 20\u00a0ms the deformations of the brain tissue differed from the deformations derived from the head model that were indirectly applied to the model (Figs.\u00a06 and 9). Therefore, only the first 20\u00a0ms of the simulation are considered to be realistic.\nLoading condition A was derived from the acceleration pulse that has been applied to a head model.6,12 The equivalent stress fields of the homogeneous models caused by loading condition A (Fig.\u00a07) showed no similarities to the equivalent stress field of the head model (Fig.\u00a04). Provided that the equivalent stress field of the head model is realistic, loading condition A can be considered unrealistic. Loading condition B was obtained from the resulting accelerations in the region of interest of the head model. The equivalent stress fields of the homogeneous models from simulations with loading condition B (Fig.\u00a09) were approximately similar to the stress fields in the corresponding region of the head model (Fig.\u00a04) during the first 20\u00a0ms. Nevertheless, differences in the equivalent stress fields existed at the surface of the cortex. This is due to the CSF layer in the cerebral cortex model being described elastically with a low shear modulus, as opposed to the head model, which contains a relatively stiff CSF\/dura layer. In spite of all these obvious differences, by comparing these two loading conditions, it was shown that the different loading conditions have hardly any effect on the relative mean and maximum equivalent stress and the relative peak maximum principal logarithmic strain. Hence, the mechanical influences of the heterogeneities of the cerebral cortex seem to be independent of the loading conditions.\nThe constitutive model for brain tissue was based on experiments on porcine white matter.19 This was used as a substitute for human brain tissue, as it was readily available and it allowed to conduct experiments with a shorter post-mortem time.12,26 Experiments conducted by Prange et\u00a0al.37 on fresh human brain tissue indicated that its mechanical properties are approximately 30% stiffer than those of fresh porcine brain tissue. Although the constitutive model for brain tissue in the cerebral cortex model was not based on human brain tissue, the geometries of the models were based on the human cerebrum. This assumption is expected to have more effect on the absolute equivalent stress than on the relative equivalent stress, which is dominated by the heterogeneities of the model. Furthermore, no distinction between the mechanical properties of white and gray matter was made, so that the material properties of the cerebral cortex, which consists of gray matter, were based on experiments on white matter, as well. In their results, Prange et\u00a0al.37 found differences between the material properties of the corpus callosum, i.e., the white matter that connects the two cerebral hemispheres, and the cerebral cortex, but not between the corona radiata, i.e., the white matter that lies inferior to the cerebral cortex, and the cerebral cortex. Nevertheless, in several other studies of head and brain models, different material properties were used for gray and white matter based on the assumption that white matter was more fibrous than gray matter.2,28,30,47 Therefore, simulations of the cerebral cortex model with shear moduli for gray matter ranging from 75% to 125% with respect to the shear moduli white matter were conducted as well. The results of these simulations indicated that the material properties had an effect on the magnitude of the equivalent stress, but not on the regions in which the peak equivalent stress was observed. The equivalent stress of the heterogeneous model relative to the homogeneous model was hardly affected by the different material properties.\nIn this model, the meninges and the blood vessels were considered to have no mechanical influence on the cerebral cortex for the used loading conditions. Although Jin et\u00a0al.20 suggested that the pia-arachnoid complex can have a mechanical influence on the brain tissue during an impact, it is still not clear if it would affect the influence due to the gyrification of the cerebral cortex. In a study performed by Ho and Kleiven,17 it was found that the vasculature of the brain can be neglected, as far as the mechanical influences are concerned in a head model not containing the gyrification of the cortex.\nIn order to validate this model, a comparison with physical experiments is required. However, data of physical experiments at a typical length scale of this level is rather limited. Parallel to this study, the results of physical experiments, in which brain slices have been accelerated, showed increased equivalent strains near the sulci.24 Furthermore, some studies showed that angular accelerations of the head induce high stress concentrations in and near the cerebral cortex.8,15 Another method of validation can be performed by comparing the results of the model to clinically observed injury. It has been shown that small cortical infarcts exist in diffuse brain injury at the bottom of the sulci.40 This is in accordance with the locations of the high stress and strain regions in the cerebral cortex model.\nThe two loading conditions and the different geometries resulted in different equivalent stress fields. The simulations with loading condition A resulted in a lower mean and maximum equivalent stress compared to the simulations with loading condition B. However, relative to the homogeneous model, it was observed that the equivalent stress was almost independent of the different loading conditions used in this study. The differences between the several heterogeneous geometries had more influence on the relative mean and maximum equivalent stress. The morphologic heterogeneities of the cerebral cortex led to an increase of the maximum equivalent stress by a factor of about 1.3\u20131.9, depending mostly on the geometry, whereas the relative mean equivalent stress values of all the geometries were 1.1 and 1.0 for loading condition A and B, respectively. Furthermore, the peak maximum principal logarithmic strain was increased by a factor of about 1.2\u20131.9 due to the morphologic heterogeneities of the cerebral cortex. This is a strong indication that predictions of brain injury obtained from head models with a homogeneous cerebrum should be interpreted with care. To obtain a more accurate assessment of injury, the influence of the morphologic heterogeneities in the cerebral cortex should be accounted for.","keyphrases":["traumatic brain injury","cerebral cortex","finite element model","cerebrum","brain tissue","inhomogeneities"],"prmu":["P","P","P","P","P","U"]}
{"id":"Ann_Biomed_Eng-2-2-1705524","title":"Three-dimensional Numerical Modeling and Computational Fluid Dynamics Simulations to Analyze and Improve Oxygen Availability in the AMC Bioartificial Liver\n","text":"A numerical model to investigate fluid flow and oxygen (O2) transport and consumption in the AMC-Bioartificial Liver (AMC-BAL) was developed and applied to two representative micro models of the AMC-BAL with two different gas capillary patterns, each combined with two proposed hepatocyte distributions. Parameter studies were performed on each configuration to gain insight in fluid flow, shear stress distribution and oxygen availability in the AMC-BAL. We assessed the function of the internal oxygenator, the effect of changes in hepatocyte oxygen consumption parameters in time and the effect of the change from an experimental to a clinical setting. In addition, different methodologies were studied to improve cellular oxygen availability, i.e. external oxygenation of culture medium, culture medium flow rate, culture gas oxygen content (pO2) and the number of oxygenation capillaries. Standard operating conditions did not adequately provide all hepatocytes in the AMC-BAL with sufficient oxygen to maintain O2 consumption at minimally 90% of maximal uptake rate. Cellular oxygen availability was optimized by increasing the number of gas capillaries and pO2 of the oxygenation gas by a factor two. Pressure drop over the AMC-BAL and maximal shear stresses were low and not considered to be harmful. This information can be used to increase cellular efficiency and may ultimately lead to a more productive AMC-BAL.\nIntroduction\nAcute liver failure (ALF) is a severe disease with high mortality rates (60\u201390%). At present, the only efficient therapy is orthotopic liver transplantation (OLT).11 To bridge ALF patients to liver transplantation or to regeneration of the native liver, liver support systems are needed. The most promising bridging method for the treatment of ALF patients are bioartificial liver (BAL) support systems. These systems are extracorporeal devices that are generally comprised of a bioreactor in which living hepatocytes are seeded. In a clinical setting, toxic plasma of ALF patients is perfused through a BAL system and detoxified by viable hepatocytes. A major advantage of BAL systems, as compared to other non-biological liver support systems, is the capacity to provide a full range of metabolic functions to compensate for the complex metabolic disorders seen in ALF. Although many BAL systems have demonstrated their detoxifying capacity or liver-specific functions in in vitro, ex vivo and in phase I clinical studies, no BAL system has shown to significantly improve survival in ALF patients bridged to transplantation in a controlled trial (for review:40).\nImportant issues for the development of an effective BAL system are local plasma flow and oxygen transport. Optimal cell function, i.e. detoxifying capacity, is only obtained when a BAL system is adequately perfused with plasma to enable efficient mass transfer. In this situation, sufficient nutrients (e.g. glucose, fatty acids) and oxygen are supplied, whereas unwanted metabolites (e.g. ammonia) are efficiently detoxified and metabolic end products (e.g. urea, CO2) subsequently removed. In principle, oxygen (O2) transfer is the main limitation in the efficiency of a BAL,3 because of the low solubility of oxygen in plasma and high demand for oxygen of functionally active hepatocytes.\nThe AMC-Bioartificial Liver (AMC-BAL; Academic Medical Center, Amsterdam, The Netherlands, patent No: WO 97\/12960) was developed with a special design for on-site oxygenation of hepatocytes and direct contact of plasma to the cells to enhance bidirectional solute exchange. The AMC-BAL has shown promising results in in vitro set-ups, small and large animal ALF models, and finally in a phase I clinical study with ALF patients.13,39 But to increase the efficiency of the AMC-BAL, further optimization of plasma perfusion and oxygenation of hepatocytes may be useful.\nNumerical techniques and Computational Fluid Dynamics (CFD) simulations are useful tools to gain insight in local flow field and oxygen transport in hollow fiber BALs19,27,30 as well as in other types of hepatocyte systems.16,23,25 These numerical techniques can also be used to determine the optimal working parameters and to further optimize the design of a BAL system. In this respect, we used CFD simulations as a methodological approach to analyze the configuration of the AMC-BAL. We constructed three-dimensional computer models of two representative unit volumes \u2013 micro models \u2013 of the laboratory-scale AMC-BAL. Fluid flow and oxygen transport and consumption were simulated in these micro models to gain insight in the flow field and local cellular oxygen availability. Numerical parameter studies were performed to assess possible improvements in local oxygen availability of hepatocytes. Several parameters were tested: (1) effect of the internal oxygenator system, (2) pO2 of the oxygenation gas, (3) culture medium flow rate, (4) incorporation of an external oxygenator in the extracorporeal circuit, (5) the number of oxygen capillaries, (6) the effect of plasma perfusion versus normal culture medium, and (7) the effect of changes of hepatocyte oxygen consumption characteristics in time. Finally, we discuss the implication of this information for the optimization of the AMC-BAL into a more efficient bioartificial liver.\nMaterials and methods\nThe internal geometry of the laboratory-scale AMC-BAL is schematically drawn in Fig.\u00a01 in a longitudinal and transverse cross-section. The bioreactor is built from a cylindrical polycarbonate housing (Fig.\u00a01l) in which two pieces of three-dimensional (3D) non-woven polyester matrix mat (Fig.\u00a01e, g) are spirally wound around a massive inner core (Fig.\u00a01i). A space between the two mat segments (Fig.\u00a01f) is left open for an additional hepatocyte seeding port (not shown). In the hydrophilic matrix, high-density hepatocyte culture is possible. Between the matrix windings, hydrophobic gas capillaries (Fig.\u00a01m) are positioned in parallel along the entire bioreactor as an internal oxygenator system to supply additional oxygen to the hepatocytes. Culture gas (95% air, 5% CO2) is perfused through these capillaries. Plasma enters the bioreactor through an inlet port (Fig.\u00a01b) and flows through the inflow zone (Fig.\u00a01j) further passes the hydrophilic matrix mat and through the void inter-capillary space (Fig.\u00a01n). Plasma exits the bioreactor via the outflow zone (Fig.\u00a01k) through the outlet port (Fig.\u00a01d). Plasma and culture gas are counter-current flows.\nFigure\u00a01.Longitudinal (left) and transverse (right) view of the internal geometry of the AMC-BAL with a \u2013 gas outlet; b \u2013 gas inlet; c \u2013 plasma inlet port; d \u2013 plasma outlet port; e \u2013 first mat segment; f \u2013 interspace; g \u2013 second mat segment; h \u2013 polyurethane potting to separate gas and fluid compartment; i \u2013 inner core; j \u2013 inflow zone; k \u2013 outflow zone; l \u2013 polycarbonate housing; m \u2013 gas capillaries; n \u2013 inter-capillary space through which plasma flows. The inline and triangular micro models are designated.\nComputer Model\nSince the entire BAL geometry is too large and complex to model, three-dimensional unit volumes \u2013 micro models \u2013 were isolated from the geometry (Fig.\u00a01). Each three-dimensional micro model (Fig.\u00a02) consists of 2 pieces of mat (thickness\u00a0=\u00a0400\u00a0\u03bcm, height\u00a0=\u00a01563\u00a0\u03bcm, length\u00a0=\u00a050\u00a0mm; Fig.\u00a01e, g) separated by a void interspace section (length\u00a0=\u00a06\u00a0mm; Fig.\u00a01f). At both sides of the mat, gas capillaries (outer diameter\u00a0=\u00a0380\u00a0\u03bcm) and an inter-capillary space are present. The two micro models differ only in their gas capillaries\u2019 position. Either the capillaries are in a rectangular (inline micro model \u2013 Fig.\u00a02) or in a triangular (triangular micro model \u2013 Fig.\u00a02) pattern. These two configurations are the most distinct that could be isolated as the position of the capillaries relative to each other changes continuously along the course of the spiral mat (Fig.\u00a01). These two micro models were therefore considered to be representative for the entire AMC-BAL. Simulations on both models will allow us to assess the influence of the capillary arrangement.\nFigure\u00a02.AMC-BAL micro models. Upper left, inline micro model; upper right, triangular micro model (c \u2013 capillary wall, M \u2013 non-woven matrix mat, f \u2013 inter-capillary space); lower left, inline micro model with double number of capillaries; lower right, triangular micro model with double number of capillaries.\nBoth models were created in the modeling software Gambit 2 (Fluent Inc., Sheffield, UK). All dimensions were derived from a laboratory-scale AMC-BAL or were supplied by the manufacturer. The curvature of the mat was neglected and the inflow and outflow zones before and after the mat segments in the AMC-BAL were not included in the micro models. Each standard micro model contains the equivalent capillary wall surface of 1 whole capillary. Since the entire laboratory-scale AMC-BAL contains 300 capillaries, each micro model can thus be considered as 1\/300th part of AMC-BAL. Consequently, one AMC-BAL can be regarded as a combination of 300 separate micro models in parallel.\nModeling Fluid Flow\nTheoretical Model\nTo simulate fluid flow, the commercial CFD package Fluent 6.2 (Fluent Inc., Sheffield, UK) was used to numerically solve the steady-state Navier\u2013Stokes equations. Standard pressure discretization scheme and second order upwind momentum discretization scheme were used. Pressure\u2013velocity coupling scheme was SIMPLE. Fluid properties were set to those of culture medium to allow future validation with in vitro experiments. Culture medium was modeled as an incompressible, isothermal, Newtonian fluid composed of 90% water (\u03c1\u00a0=\u00a0998\u00a0kg\/m3) and 10% serum (\u03c1\u00a0=\u00a01030\u00a0kg\/m3) with a resulting density of 1001.2\u00a0kg\/m3. Dynamic viscosity \u03bc of culture medium with 5\u00a0g\/l bovine serum albumin (BSA) at 37\u00b0C was set to 0.691\u00a0mPa\u00a0s.26 Plasma viscosity at 37\u00b0C was set to 1.3\u00a0mPa\u00a0s.31\nResistance to Flow of the Non-woven Polyester Mat\nThe non-woven polyester matrix was modeled as an isotropic homogeneous porous zone. A measure for the viscous resistance of the non-woven polyester matrix used in Fluent 6.2 was determined in an experimental set-up by establishing the pressure drop \u2013 flow rate relationship. A sample of this matrix fabric sheet was clamped between two connecting tubes. These tubes were placed in a closed circuit with an overflow reservoir, which provided a constant static pressure load over the matrix sample. Pressure difference over the matrix sheet was measured using a calibrated differential pressure transducer (Fuji Electrics FCX, Japan). Flow rate was measured gravimetrically. De-ionized water was used. Static pressure load was set so average velocities in the experiment were in the same order of magnitude as could be expected in the AMC-BAL (1\u20137\u00a0mm\/s). A viscous resistance factor of 2.7\u00a0\u00b1\u00a00.2\u00a0\u00d7\u00a01010\u00a0m\u22122 was calculated as an average of 10 measurements. Resistance to flow of hepatocyte cell layers (section \u201cModeling oxygen transport and consumption\u201d) was modeled using the same viscous resistance factor.\nBoundary Conditions\nWe assumed that total flow rate (15\u00a0ml\/min) inside the research scale AMC-BAL is homogenously distributed. In this way, each micro model has the same flow rate of 0.05\u00a0ml\/min, i.e. 1\/300th of the total flow rate. Pressure inlet boundary conditions were used; outlet boundary condition was a zero pressure outflow; the capillary walls were \u2018no-slip\u2019 walls and symmetry boundary conditions were used at the side walls of the model.\nModeling Oxygen Transport and Consumption\nTheoretical Model\nTo simulate oxygen transport and consumption, Fluent 6.2 solves the steady-state convection\u2013diffusion-reaction equation (Eq. 1). Discretization scheme was set to QUICK. \nThe transported scalar \u03c6 is the local oxygen concentration (vol.%), which is also the product of the oxygen solubility \u03b1 and the local oxygen partial pressure (pO2) according to Henry\u2019s Law (\u03c6\u00a0=\u00a0\u03b1*pO2). Oxygen solubility \u03b1 is 3.1385\u00a0\u00d7\u00a010\u22125\u00a0ml O2\/mmHg*ml fluid in culture medium and 2.855\u00a0\u00d7\u00a010\u22125\u00a0ml O2\/mmHg*ml fluid in plasma. D is the oxygen diffusion coefficient, which is 2.92\u00a0\u00d7\u00a010\u22129\u00a0m2\/s in culture medium and 2.18\u00a0\u00d7\u00a010\u22129\u00a0m2\/s in plasma.2,17,33,44 Oxygen consumption by the primary porcine hepatocytes in the AMC-BAL was modeled by an additional source term S\u03c6, which was only implemented in regions that were designated to contain hepatocytes. \nThis source term (Eq. 2) was based on the Michaelis\u2013Menten kinetics of O2 consumption by hepatocytes. As such, O2 uptake is dependent on the local hepatocyte density \u03c1cell and in a non-linear way dependent on the local O2 availability.\nMichaelis\u2013Menten parameters of primary porcine hepatocytes were obtained from Balis et\u00a0al.1 Values of culture day 2 were taken as they are characterized by a highly demanding O2 uptake (VM\u00a0=\u00a00.7286\u00a0nmol\/s*106 cells; KM\u00a0=\u00a02\u00a0mmHg). These values were measured in 2D culture, whereas hepatocyte culture in the AMC-BAL is three-dimensional and at high density. For this reason, a lower O2 consumption may be expected as the specific O2 uptake rate per cell is reported to decrease at increasing cell densities.30 On the other hand, O2 consumption may rise significantly when a metabolic load is applied to the cells.4 As such, the proposed consumption characteristics are justified by assuming these values to represent the most limiting case for oxygen availability. Consequently, attention must be paid when comparing simulation results to other numerical studies.19,23,25\nHepatocyte Distribution in the Micro Models\nIn an in vitro setting, the laboratory scale AMC-BAL is seeded with 1\u00a0billion porcine hepatocytes via three different loading ports, while gently rotating the bioreactor to assure a homogeneous cell distribution. As we assumed that one micro model is one 300th part of an entire laboratory scale AMC-BAL, one micro model contained 3.33\u00a0million cells.\nSince no detailed information on the internal hepatocyte distribution in the AMC-BAL is available, two cell distributions are hypothesized. In cell distribution 1, all hepatocytes are homogeneously distributed in the non-woven matrix (resulting hepatocyte density \u03c1cell of 53.7\u00a0\u00d7\u00a0106\u00a0cells\/ml). In previous macroscopic and microscopic studies of an early prototype AMC-BAL, the majority of the hepatocytes were immobilized within the three-dimensional matrix.14 However, given the undifferentiated method of hepatocyte seeding, it is possible that hepatocytes also adhere to the surface of gas capillaries. Therefore, in cell distribution 2, 50% of the hepatocytes are located in the matrix (\u03c1cell\u00a0=\u00a031.7\u00a0\u00d7\u00a0106\u00a0cells\/ml) and 50% in a hepatocyte layer around the capillaries. The hepatocyte layer was set to be 122\u00a0\u03bcm, with a \u03c1cell of 81.7\u00a0\u00d7\u00a0106\u00a0cells\/ml. This cell layer thickness is in the range of what is used in other numerical studies19,20 and is consistent with the size of primary porcine hepatocyte 3D cell clusters found in vitro.15 All proposed cell densities were also considered to be realistic.8,21,37\nThe complete AMC-BAL cannot be modeled by using only one cell distribution or only one micro model. Therefore, each cell distribution was applied to both micro models, leading to four basic configurations in total. All four micro model configurations were assessed independently. With a combination of these micro models, the entire AMC-BAL can be modeled in future studies.\nO2 Diffusion Constant Through Non-woven Matrix\nFree O2 diffusion through the mat zone is hindered by the non-woven arrangement of hydrophilic polyester fibers, which are considered to be impermeable to O2. To compensate for this hindered diffusion, a correction factor for the O2 diffusion coefficient in the mat volume was determined.\nOn a microscopic view of the non-woven matrix, the mat porosity was determined to be 91%. A modified two-dimensional inline micro model was constructed with 250 polyester fiber circles randomly located within the boundaries of the mat volume. On this modified inline micro model, the pO2 distribution was simulated using hepatocyte distribution 1 (Fig.\u00a03A). Standard boundary conditions were used and culture flow rate was zero. O2 diffusion coefficient in the mat zone of the modified micro model was the same as for free culture medium, since culture medium occupies the void spaces between the polyester fibers. Subsequently, an analogous simulation was performed on a regular inline micro model, i.e. with the mat modeled as homogeneous medium, with an adjusted O2 diffusion coefficient in the mat zone to match the results of the \u2018modified\u2019 model (Fig.\u00a03B). A correction factor of 0.85 (Dmat\u00a0=\u00a02.48\u00a0\u00d7\u00a0109\u00a0m2\/s) for O2 diffusion was necessary to take into account the presence of the non-woven polyester matrix mat. This correction factor is in accordance with the equation of Rayleigh (correction factor \u223c\n), which has been experimentally verified for various sheets of porous media.29,32,34Figure\u00a03.Determination of the correction factor for O2 diffusion through the non-woven polyester mat. (A) pO2 distribution [mmHg] on modified inline micro model (polyester fibers drawn) under standard boundary conditions, no medium flow; (B) identical pO2 distribution [mmHg] obtained with identical simulation settings, but with mat as homogeneous medium with an adjusted diffusion constant (with a factor of 0.85) to account for hindered diffusion.\nO2 Diffusion Constant Through Zones Containing Hepatocytes\nOxygen can diffuse through both continuous fluid spaces as well as through hepatocytes. In regions seeded with hepatocytes, the resulting \u201ceffective\u201d diffusion coefficient Deff will be lower than in free medium, since O2 diffuses more slowly through hepatocytes. The extent to which the diffusivity is reduced will logically depend on the local hepatocyte density. Since experimental data on O2 diffusion constants through hepatocyte cell layers with different cell densities is not readily available, a theoretical approach was chosen.\nRiley et\u00a0al.35 developed an empirical relation (Eq. 3) that relates the effective diffusion coefficient Deff to the local free diffusion coefficient D0, the intracellular diffusion coefficient Dcell and the cell volume fraction \u03c6. This equation, based on Monte Carlo simulations, shows a good agreement with available data throughout a wide range of cell volume fractions (0.04\u00a0<\u00a0\u03c6\u00a0<\u00a00.95). \nDcell was set to 0.25\u00a0\u00d7\u00a010\u22129\u00a0m2\/s, which is an average of intracellular O2 diffusion constants in hepatocytes reported by Jones.22 Cell volume fraction \u03c6 is the local volume percentage of space that is occupied by hepatocytes and was calculated as the product of the local hepatocyte cell density and the average hepatocytes cell volume.9 Local free diffusion coefficient D0 is dependent on the perfusion fluid, e.g. culture medium or plasma, and on the location of hepatocytes in the model; either in the inter-capillary space or in the mat zone. O2 diffusivities in different regions of both micro models are presented in Table\u00a01.\nTABLE\u00a01.O2 diffusion constants in different zones of the micro models.Region in the micro modelD0 [10\u22129\u00a0m2\/s]\u03c1cell [106 cells\/ml]\u03c6Deff [10\u22129\u00a0m2\/s]Cult. med. PlasmaCult. med.PlasmaNo hepatocytes\u00a0\u00a0\u00a0\u00a0Inter-capillary space2.922.180012.922.18\u00a0\u00a0\u00a0\u00a0In mat2.481.850012.481.85Cell distribution 1\u00a0\u00a0\u00a0\u00a0In mat2.481.8553.70.1720.751.871.41Cell distribution 2\u00a0\u00a0\u00a0\u00a0In mat2.481.8531.70.1020.852.111.59\u00a0\u00a0\u00a0\u00a0In hepatocyte cell layer around capillaries \u2013 part in mat zone2.481.8581.70.2620.641.591.22\u00a0\u00a0\u00a0\u00a0In hepatocyte cell layer around capillaries \u2013 part in inter-capillary space2.92 2.1881.70.2620.641.861.41The ratio of the effective diffusion constant to the free diffusion constant Deff\/D0 is calculated from the local free oxygen diffusion constant D0 and the local cell volume fraction \u03c6 using Eq. 3. The local effective diffusion coefficients Deff in culture medium and plasma per region are presented in the last column.\nThe obtained local diffusion coefficients are in the range of reported values for hepatocytes and other cell types.6,16,18,41,43\nBoundary Conditions\nOxygen partial pressure (pO2) of incoming culture medium was set to 146.5\u00a0mmHg, which was an average of three culture medium pO2 measurements using an ABL505 blood-gas analyzer (Radiometer Copenhagen). Axial and radial O2 transport resistances in gas capillaries were neglected, since the culture gas flow rate through the gas capillaries and their O2 permeability are high.5 As such, O2 supply through gas capillaries was modeled by imposing a constant 150\u00a0mmHg pO2 on the capillaries\u2019 outer walls, which corresponded to the pO2 of culture gas used in vitro (95% air, 5% CO2). All other boundary faces have no-flux boundary conditions.\nGrid Dependency\nOne micro model mesh contained approximately 3.75\u00a0million finite volume mesh elements. Further increase in the number of cells rendered identical simulation results.\nSimulations Overview and Assessment\nAs previously stated, four micro model configurations were used: (a) the inline micro model with cell distribution 1, (b) the inline micro model with cell distribution 2, (c) the triangular micro model with cell distribution 1, and finally (d) the triangular micro model with cell distribution 2. Fifteen case studies (Table\u00a02) were performed on each configuration.\nTABLE\u00a02.Overview of simulations.CaseFluidpO2 gas (mmHg)pO2 medium (mmHg)Qmedium (ml\/min)# capillariesKM (mmHg)Reference case\u00a0\u00a0\u00a0\u00a0(1) Standard boundary conditionsCulture medium150146.50.0512Effect of the internal oxygenator\u00a0\u00a0\u00a0\u00a0(2) pO2 gas\u00a0=\u00a00Culture medium0146.50.0512\u00a0\u00a0\u00a0\u00a0(3) pO2 gas\u00a0=\u00a00; pO2 medium\u00a0\u00d7\u00a02Culture medium02930.0512Increasing oxygen availability\u00a0\u00a0\u00a0\u00a0(4) pO2 gas\u00a0\u00d7\u00a02Culture medium300146.50.0512\u00a0\u00a0\u00a0\u00a0(5) pO2 gas\u00a0=\u00a0carbogenCulture medium722146.50.0512\u00a0\u00a0\u00a0\u00a0(6) pO2 medium\u00a0\u00d7\u00a02Culture medium1502930.0512\u00a0\u00a0\u00a0\u00a0(7) pO2 medium\u00a0=\u00a0carbogenCulture medium1507220.0512\u00a0\u00a0\u00a0\u00a0(8) Fluid flow rate Qmedium\u00a0\u00d7\u00a02Culture medium150146.50.1012\u00a0\u00a0\u00a0\u00a0(9) Fluid flow rate Qmedium\u00a0\u00d7\u00a010Culture medium150146.50.5012\u00a0\u00a0\u00a0\u00a0(10) No. capill. \u00a0\u00d7\u00a02Culture medium150146.50.0522\u00a0\u00a0\u00a0\u00a0(11) No. capill. \u00a0\u00d7\u00a02; pO2 gas\u00a0\u00d7\u00a02Culture medium300146.50.0522Clinical versus experimental setting\u00a0\u00a0\u00a0\u00a0(12) PlasmaPlasma150146.50.0512\u00a0\u00a0\u00a0\u00a0(13) Plasma; pO2 gas\u00a0\u00d7\u00a02Plasma300146.50.0512Changes of hepatocyte O2 consumption in time\u00a0\u00a0\u00a0\u00a0(14) KM day 4Culture medium150146.50.0514.75\u00a0\u00a0\u00a0\u00a0(15) KM day 5Culture medium150146.50.0517.5Each case is applied to the four basic micro model configurations (inline and triangular micro model each with cell distribution 1 or 2).\nThe first case study was simulated using standard boundary conditions as described in previous paragraphs. This \u2018reference case\u2019 study (1) is the internal control for all other case studies with the same micro model configuration. The effect of the internal oxygenation system was assessed by disabling culture gas flow (2), and by assessing the effect of an external oxygenator that oxygenates incoming medium to a doubled pO2 as an alternative for internal oxygenation (3). To increase oxygen availability to the hepatocytes, different strategies were assessed. First, by using a more oxygen rich culture gas, i.e. a doubled pO2 gas (4) or carbogen gas (95% O2) (5). Second, by incorporating an external oxygenator to increase incoming medium pO2 to a doubled (6) or carbogen (7) level. Third, by changing culture medium flow rate to a doubled (8) or 10-fold (9) flow rate. Finally, by doubling the number of capillaries (10) or by combining the double number of capillaries with a doubled culture gas pO2 (11). In cases with a doubled number of capillaries, cell density in the hepatocyte layer around the capillaries was kept constant. This lead to a cell layer thickness of 68\u00a0\u03bcm, keeping the number of hepatocytes around all capillaries equal to 50% of total, as was initially proposed. In micro models with double number of capillaries the distance between the capillaries is halved (see Fig.\u00a02).\nIn the next case studies, we studied the effect of plasma versus culture medium, since this is the main difference in perfusion in a clinical setting as compared to an experimental setting. Standard boundary conditions (12) and a doubled pO2 gas (13) were used and compared with cases (1) and (4). Finally, time-related changes in oxygen consumption characteristics of hepatocytes were investigated by varying KM values. KM values of culture day 4 (14) and 5 (15) were compared to the reference case (1). According to Balis et\u00a0al.,1KM values change remarkably throughout the first 5\u00a0days, whereas VM values remain constant. We therefore kept VM constant in these simulations.\nThe proposed simulation cases on the four different micro model configurations were assessed by examining pO2 distributions. However, not every simulated pO2 profile can be shown due to brevity reasons. To evaluate the effect of the parameters discussed in the parameter study, the local effective hepatocyte utilization ratioVratio (Eq. 4) was studied.30\nVratio is the ratio of the observed oxygen consumption rate to the maximal hepatocyte oxygen consumption rate, i.e. VM, and ranges between 0 and 1 (asymptotically). A threshold for Vratio of 0.9 was chosen as introduced by Patzer30 and corresponds with a minimal pO2 level of 18\u00a0mmHg when KM\u00a0=\u00a02\u00a0mmHg. Consequently, oxygen availability in the AMC-BAL was quantified by determining the percentage of hepatocytes with Vratio\u00a0>\u00a00.9.\nResults\nFluid Flow and Shear Stress Distribution\nFigure\u00a04 represents an example of colorimetric contour plots of fluid flow and shear stress distributions in two different micro models with different hepatocyte distributions. In the upper part, velocity magnitudes (m\/s, left legend) in a transverse plane midway through the first mat segment are shown for the reference case (case 1) of an inline micro model with hepatocyte distribution 1 (Fig.\u00a04A1) and for a triangular micro model with hepatocyte distribution 2 and with a double number of capillaries (case 10\u201311) (Fig.\u00a04B1). The contour plots can be mirrored with respect to the horizontal axis as micro models are symmetrical.\nFigure\u00a04.Colorimetric contour plot of velocity magnitudes (m\/s, left legend, upper part 1) and shear stress levels (Pa, right legend, lower part 2) in a transverse plane midway through the first mat segment, in the reference case (case 1) of an inline micro model with hepatocyte distribution 1 (A1 and A2 resp.) and in a triangular micro model with hepatocyte distribution 2 and with double number of capillaries (case 10\u201311) (B1 and B2 resp.).Figure\u00a05.Colorimetric contour plot of pO2 (mmHg, left legend, upper part 1) and effective hepatocyte utilization ratio Vratio (dimensionless, right legend, lower part 2) in a transverse plane midway through the first mat segment, in the reference case (case 1) of an inline micro model with hepatocyte distribution 1 (A1 and A2 resp.) and in a triangular micro model with hepatocyte distribution 2 and with double number of capillaries and doubled culture gas pO2 (case 11) (B1 and B2 resp.) (Note: B1 different scale compared to A1).\nFluid flows in the non-woven matrix mat zone and in the hepatocyte cell layers were orientated axially and are uniform in size (approx. 8\u00a0\u03bcm\/s in Fig.\u00a04A1 and 37\u00a0\u03bcm\/s in Fig.\u00a04B1). In the inter-capillary space, flow velocities have a poiseuille-like (parabolic) profile with maximal velocities of approx. 3.6\u00a0mm\/s in Fig.\u00a04A1 and 7.8\u00a0mm\/s in Fig.\u00a04B1. In the interspace between two mat segments, flow lines expand radially due to the sudden increase in local cross-sectional area. However, these flow lines reconverge when entering the second mat segment, where the velocity profile was identical compared to the first mat segment (not shown). Static pressure drop over the entire micro model was 15.7\u00a0Pa in the reference case of the inline micro model with hepatocyte distribution 1 (Fig.\u00a04A) and 69.3\u00a0Pa for the triangular micro model with hepatocyte distribution 2 and double number of capillaries (Fig.\u00a04B). The lower parts of Fig.\u00a04 (A2 and B2) show corresponding shear stress distributions (Pa, right legend). Maximal shear stresses were generally located near the mat side surfaces and also \u2013 in case of cell distribution 2 \u2013 at the boundary between the hepatocyte cell layer and inter-capillary space.\nIn Table\u00a03, an overview is given of maximum velocity magnitudes in the inter-capillary space, uniform velocity magnitudes in the mat\/hepatocyte layer zone, as well as static pressure losses over the entire micro model and maximal local shear stresses for all simulation cases. Maximum velocities in the inter-capillary space ranged between 3.6 and 45\u00a0mm\/s, whereas uniform velocities in the mat\/hepatocyte layer ranged between 8 and 110\u00a0\u03bcm\/s. Simulation results showed that velocities in the mat zone and in hepatocyte layers were consistently two orders of magnitude smaller than in the inter-capillary space. Velocity profiles and shear stress profiles were similar for the different simulation cases, but differed in magnitude between cases when flow rate was altered or when the internal geometry had been changed, e.g. due to additional gas capillaries and\/or hepatocyte layers around the capillaries. Static pressure loss over the entire micro model and local shear stress levels were additionally influenced by the type of fluid, e.g. plasma or culture medium. Static pressure loss over the entire micro model ranged from approx. 16 to 203\u00a0Pa. Maximum shear stress ranged between approx. 0.03 and 0.40\u00a0Pa. Largest maximum values were reached in hepatocyte distribution 2 with a 10-fold flow rate.\nTABLE\u00a03.Overview of the maximum velocity in the inter-capillary space, the uniform velocity in the mat\/hepatocyte layer zone, the static pressure loss over the entire model and the maximal shear stress for all simulation cases and for different hepatocyte distributions.CasesHepatocyte distr. 1Hepatocyte distr. 2Velocity in mat\/hepatocyte cell layer (mm\/s)\u00a0\u2013\u00a0Maximum velocity in inter-capillary space (mm\/s)\u00a0\u00a0\u00a0\u00a0(1,2,3,4,5,6,7,14,15) Standard flow rate0.0085\u00a0\u2013\u00a03.59 0.011\u00a0\u2013\u00a04.60 \u00a0\u00a0\u00a0\u00a0(8) Qmedium\u00a0\u00d7\u00a020.017\u00a0\u2013\u00a07.170.022\u00a0\u2013\u00a09.20\u00a0\u00a0\u00a0\u00a0(9) Qmedium\u00a0\u00d7\u00a0100.084\u00a0\u2013\u00a035.70.11\u00a0\u2013\u00a045.0\u00a0\u00a0\u00a0\u00a0(10,11) No capillaries\u00a0\u00d7\u00a020.0195\u00a0\u2013\u00a05.690.037\u00a0\u2013\u00a07.75\u00a0\u00a0\u00a0\u00a0(12,13) Plasma0.0085\u00a0\u2013\u00a03.590.011\u00a0\u2013\u00a04.60Static pressure loss over micro model (Pa)\u00a0\u00a0\u00a0\u00a0(1,2,3,4,5,6,7,14,15) Standard flow rate15.720.6\u00a0\u00a0\u00a0\u00a0(8) Qmedium\u00a0\u00d7\u00a0231.441.1\u00a0\u00a0\u00a0\u00a0(9) Qmedium\u00a0\u00d7\u00a010155.7203.3\u00a0\u00a0\u00a0\u00a0(10,11) No capillaries\u00a0\u00d7\u00a0236.169.3\u00a0\u00a0\u00a0\u00a0(12,13) Plasma30.039.3Maximum shear stress (Pa)\u00a0\u00a0\u00a0\u00a0(1,2,3,4,5,6,7,14,15) Standard flow rate0.0320.041\u00a0\u00a0\u00a0\u00a0(8) Qmedium\u00a0\u00d7\u00a020.0640.083\u00a0\u00a0\u00a0\u00a0(9) Qmedium\u00a0\u00d7\u00a0100.310.40\u00a0\u00a0\u00a0\u00a0(10,11) No capillaries\u00a0\u00d7\u00a020.0560.083\u00a0\u00a0\u00a0\u00a0(12,13) Plasma0.0570.073Values for the inline and triangular micro model are identical.\nImportantly, both the inline and triangular micro model had the same flow field, static pressure loss and shear stress distributions within a simulation case with certain boundary conditions and hepatocyte distribution (comparison not shown).\nOxygen Transport and Consumption\nResults of oxygen transport and consumption simulations are presented by means of two contour plots of pO2 and Vratio distributions (Fig.\u00a05). Also, Table\u00a04 presents detailed information for each simulated case and micro model configuration on the percentage of hepatocytes that consume oxygen at a minimal Vratio level of 0.9.\nTABLE\u00a04.Percentages of total hepatocyte cell amount that attain a Vratio\u00a0>\u00a00.9.% Hepatocytes with Vratio\u00a0>\u00a00.9CaseHepatocyte distribution 1Hepatocyte distribution 2Inline (%)Triangular (%)Inline (%)Triangular (%)Mat (%)Capill. (%)Mat (%)Capill. (%)Reference case(1) Standard boundary conditions15.715.828.828.63.354.13.353.5The effect of the internal oxygenator\u00a0\u00a0\u00a0\u00a0(2) pO2 gas\u00a0=\u00a001.71.71.71.73.10.43.10.4\u00a0\u00a0\u00a0\u00a0(3) pO2 gas\u00a0=\u00a00; pO2 medium\u00a0\u00d7\u00a026.26.26.35.911.01.710.31.7Increasing oxygen availability\u00a0\u00a0\u00a0\u00a0(4) pO2 gas\u00a0\u00d7\u00a0230.430.250.350.35.195.15.094.9\u00a0\u00a0\u00a0\u00a0(5) pO2 gas\u00a0=\u00a0carbogen62.464.580.788.361.2100.076.3100.0\u00a0\u00a0\u00a0\u00a0(6) pO2 medium\u00a0\u00d7\u00a0220.120.234.533.911.857.011.056.5\u00a0\u00a0\u00a0\u00a0(7) pO2 medium\u00a0=\u00a0carbogen35.535.154.253.440.667.737.569.1\u00a0\u00a0\u00a0\u00a0(8) Qmedium\u00a0\u00d7\u00a0217.918.131.631.46.656.46.556.0\u00a0\u00a0\u00a0\u00a0(9) Qmedium\u00a0\u00d7\u00a01028.428.645.745.622.268.922.168.9\u00a0\u00a0\u00a0\u00a0(10) No capillaries\u00a0\u00d7\u00a0231.831.856.756.814.1100.014.3100.0\u00a0\u00a0\u00a0\u00a0(11) No capillaries\u00a0\u00d7\u00a02; pO2 gas\u00a0\u00d7\u00a0261.059.987.584.875.3100.069.9100.0Clinical versus experimental setting\u00a0\u00a0\u00a0\u00a0(12) Plasma12.012.022.923.02.643.02.643.0\u00a0\u00a0\u00a0\u00a0(13) Plasma; pO2 gas\u00a0\u00d7\u00a0222.822.940.840.92.978.42.978.3Changes in hepatocyte O2 consumption in time\u00a0\u00a0\u00a0\u00a0(14) KM day 48.78.718.918.91.136.41.136.4\u00a0\u00a0\u00a0\u00a0(15) KM day 55.05.112.913.00.325.30.325.5In the case of hepatocyte distribution 2, distinction is made between the percentage of the total number of hepatocytes in the mat and the percentage of total number of hepatocytes in the cell layers around the capillaries.\nReference Case Simulations (Case 1)\nFigure\u00a05A1 illustrates the pO2 distribution in a transverse plane midway through the first mat segment in the reference case (case 1) for the inline micro model with hepatocyte distribution 1. Highest pO2 (150\u00a0mmHg) was found close to the capillary wall. A large pO2 gradient extended radially from the capillary and was steeper at the side of the capillary in the mat zone. At a radial distance of 86\u00a0\u03bcm from the capillary, pO2 in the mat zone dropped already below 20\u00a0mmHg. A region where oxygen was depleted (0\u00a0mmHg) is present in the center of the hepatocyte-seeded mat throughout both mat zones. Analogous pO2 distributions were found in cross-sections of the micro model further downstream to the plane of Fig.\u00a05A1, but with overall decreasing average oxygen level. Oxygen content of the perfused culture medium in the inter-capillary space dropped from its initial value of 146.5\u00a0mmHg to approx. 60\u00a0mmHg midway the first mat segment and further decreased downstream to 28\u00a0mmHg at the end of the first mat segment. At the start of the second mat segment, culture medium pO2 in the inter-capillary space was increased to 40\u00a0mmHg and again decreased to 22\u00a0mmHg at the end of the mat segment.\nAs a standard for cellular oxygen availability, the effective hepatocyte utilization ratio Vratio was calculated (Fig.\u00a05A2). Complying with pO2 distribution, hepatocytes in the center of the mat zone cannot consume oxygen at all (Vratio\u00a0\u223c\u00a00), whereas hepatocytes closest to the capillaries have enough oxygen available to consume oxygen at near maximum capacity (Vratio\u00a0\u223c\u00a01). Approximately 16% of all hepatocytes in the model consumed oxygen with Vratio\u00a0>\u00a00.9 (Table\u00a04). A change to hepatocyte distribution 2 was characterized by a larger radial pO2 gradient around the capillaries and a more extended zone of zero oxygen content in the mat segments. Due to the additional presence of a dense hepatocyte layer around the capillary, pO2 dropped below 20\u00a0mmHg from a radial distance of 65\u00a0\u03bcm from a capillary (compare to 86\u00a0\u03bcm in hepatocyte distribution 1). Nevertheless, 29% of all hepatocytes attained a Vratio of more than 0.9. Fifty four percent of the hepatocytes in cell layers around the capillaries reached the threshold of Vratio\u00a0>\u00a00.9, whereas 3% of the hepatocytes in the mat zone.\npO2 and Vratio distributions for the reference case of the other three micro model configurations are not shown for brevity reasons.\nSimulation results also showed that inline and triangular micro models, using the same hepatocyte distribution, showed qualitative and quantitative identical pO2 distributions.\nThe Effect of the Internal Oxygenator (Case 2,3)\nDisabling culture gas flow (\u223cpO2 gas\u00a0=\u00a00 \u2013 case 2) led to a reduction of cells with Vratio\u00a0>\u00a00.9 to approximately 2% for all micro model configurations. Replacing the internal oxygenator by an external oxygenator, which doubles culture medium pO2 (case 3), led to a \u201cVratio\u00a0>\u00a00.9\u201d fraction of approx. 6% for all micro model configurations. More specifically, in cell distribution 2, this was true for only 1% and 2% of the hepatocytes around the capillaries, and for 3% and11% of the hepatocytes in the mat zone, for case 2 and 3, respectively.\nIncreasing Oxygen Availability (Case 4\u201311)\nDoubling culture gas pO2 (case 4) almost doubled \u201cVratio\u00a0>\u00a00.9\u201d percentages (Table\u00a04) from approximately 16% to 30% in micro models with hepatocyte distribution 1; an increase of 93%. A 75% increase (29\u201350%) was obtained in micro models with cell distribution 2. This increase mainly occurred in the hepatocyte layer around capillaries, i.e. 55% in case 1 to 95% in case 4. A further pO2 gas increase to carbogen level (case 5) led to an approximate quadruple and triple \u201cVratio\u00a0>\u00a00.9\u201d fraction compared to the reference case for distribution 1 and 2, respectively. In the latter, hepatocytes in both the mat zone as well as in the cell layers have increased threshold percentages. Furthermore, about 7% more hepatocytes attained the threshold in the triangular capillary arrangement compared to inline when using hepatocyte distribution 2. In distribution 1, inline and triangular configurations rendered quasi-identical results.\nDoubling culture medium pO2 (case 6) led to a 28% and 20% increase in Vratio percentages for cell distribution 1 and 2, respectively. In the latter, the relative increase was higher in the mat zone as compared to the hepatocyte layers. Further increase of pO2 medium to carbogen level (case 7) led to a 125% and 88% increase in \u201cVratio\u00a0>\u00a00.9\u201d percentages compared to the reference case for distribution 1 and 2, respectively. Inline and triangular models rendered identical results under the same hepatocyte distribution.\nDoubling culture medium flow rate (case 8) resulted in a 14% and 10% increase in \u201cVratio\u00a0>\u00a00.9\u201d fraction for distribution 1 and 2, respectively. Further increase of culture medium flow rate up to a 10-fold (case 9) resulted in an 81% and 59% increase for distribution 1 and 2, respectively. Typically in cases with increased flow rate in cell distribution 2, Vratio percentages in the mat zone changed remarkably, whereas in the hepatocyte layers there was only a minor increase. Again, inline and triangular models rendered identical results under the same hepatocyte distribution.\nA 2-fold increase in the number of capillaries (case 10) resulted in a doubled percentage of cells that attain the Vratio\u00a0>\u00a00.9 threshold in cell distribution 1 (+102%) and 2 (+98%). In the latter, this increase occurred in the mat zone as well as in the cell layer around the capillaries. The combination of a double number of gas capillaries and a doubled pO2 gas (case 11) increased the threshold percentages to almost quadruple (+280%) for cell distribution 1 and triple (+200%) for cell distribution 2. Again, only minor differences existed between inline and triangular micro models.\nFigure\u00a05B1 shows the pO2 distribution in a transverse plane midway through the first mat segment in the triangular micro model with cell distribution 2 (case 11). In this case, highest pO2 levels (\u223c300\u00a0mmHg) were only found close to the capillary walls. A very steep radial gradient was noted in the hepatocyte cell layer around the capillaries. pO2 in the center of the mat zone was 7\u00a0mmHg. As in the reference case, pO2 distributions of cross-sections further downstream showed analogous results as compared to Fig.\u00a05A1, but with overall decreasing average oxygen level. The distribution of Vratio corresponding to the pO2 distribution of case 11 is depicted in Fig.\u00a05B2. The entire hepatocyte layer (100%) around the capillaries and large parts of the mat zone (70%) had a Vratio of at least 0.9. Consequently in case 11, 85% of all hepatocytes had a Vratio higher than 0.9 (Table\u00a04).\nClinical Versus Experimental Setting (Case 12,13)\nChanging fluid properties from culture medium to plasma resulted in a 24% decrease in Vratio\u00a0>\u00a00.9 percentages in micro models with cell distribution 1 and a 20% decrease in cell distribution 2 (case 12). Doubling pO2 gas in the clinical setting (case 13) showed a relative rise of 91% and 78% compared to the clinical reference case (case 12) in cell distribution 1 and 2, respectively. Compared to the analog case in in vitro settings (case 4), the change of fluid properties also corresponds with a respective 25% and 19% decrease of the \u201cVratio\u00a0>\u00a00.9\u201d fraction.\nChanges in Hepatocyte O2 Consumption in Time (case 14,15)\nChanging KM values to the situation on day 4 (case 14) or 5 (case 15) causes slight increases in absolute pO2 level. Also, pO2 gradients are less steep and the oxygen depleted zone in the center of the matrix is slightly smaller (not shown). However, \u201cVratio\u00a0>\u00a00.9\u201d fractions decreased 45% and 68% for cell distribution 1 and 34% and 55% for distribution 2 compared to day 2 (case 1) for day 4 and 5, respectively.\nDiscussion\nFluid Flow and Shear Stress Distribution\nPerfusion of a micro model was largely influenced by the presence of the non-woven mat. Although the cross-sectional area of the inter-capillary space and the non-woven mat are roughly the same size, fluid flow in the mat zone was generally two orders of magnitude smaller as compared to the flow in the inter-capillary space. This effect is caused by the higher resistance to flow of the non-woven mat, forcing the majority of fluid flow through the inter-capillary spaces, which have a negligible flow resistance. This also causes the overall pressure loss to be minimal. Since the same hydraulic permeability is used for the hepatocyte layers, fluid velocities there are also in the range of micrometer per second. Consequently, apart from the incorporation of additional gas capillaries (case 10\u201311), also the presence of hepatocyte layers around the capillaries (cell distribution 2) increased flow velocities and pressure loss in the model as the free cross-sectional area of the inter-capillary space is reduced. Fluid flow simulations for the inline and triangular variant of the different case studies render identical results for velocity profiles, pressure loss and shear stress distributions. This was expected as the micro models consist of the identical geometrical entities, which are only changed in location relative to each other. From a fluid dynamical point of view, we conclude that the change in capillary arrangement along the course of the spiral mat in the AMC-BAL does not influence fluid flow, pressure drop or shear stress distribution.\nThe static pressure loss over one micro model can be regarded as the total pressure drop over the entire AMC-BAL without the inflow and outflow zone (Fig.\u00a01e\u2013g), since the AMC-BAL can be represented by 300 micro models in parallel. This pressure drop, i.e. max. 200\u00a0Pa\u00a0\u223c\u00a01.5\u00a0mmHg, is negligible when compared to the pressure losses in the extracorporeal circuit. These low pressure gradients are in accordance with the in vivo situation in the liver lobule, in which pressure drops of approximately 3\u00a0mmHg over the sinusoids are normal.7,28 This is considered advantageous for cell culture inside the AMC-BAL.\nShear stress was also assessed as it is a possible determinant of cellular damage and reduced metabolic function. Shear stress is directly proportional to the local velocity gradient and the fluid viscosity. Consequently, shear stresses are generally more elevated in cases with higher velocity magnitudes in the inter-capillary space (e.g. in case of increased flow rate, doubled number of capillaries, cell distribution 2) and where fluid viscosity is increased (e.g. when plasma was used instead of culture medium \u2013 \u2018clinical setting\u2019 \u2013 case 12\u201313). Results show that only hepatocytes located at the side of the mat and at the border of the hepatocyte layers with the inter-capillary space are subjected to a certain level of shear stress.\nIn vivo values of hepatocyte wall shear stress are difficult to obtain, and comparison is therefore difficult. In human, liver sinusoid wall shear stress \u03c4 can be calculated as \n\u00a0\u223c\u00a01Pa (with static pressure drop \u0394p\u00a0\u223c\u00a03\u00a0mmHg; sinusoid diameter D\u00a0\u223c\u00a09\u00a0\u03bcm; length L\u00a0\u223c\u00a01\u00a0mm;7,28 and considering laminar flow and a cylindrical shape of the sinusoid). In mice sinusoids, shear stress was calculated to be 0.55\u00a0Pa.24,27 However, in vivo, hepatocytes are not directly subjected to this shear stress as they are shielded by the sinusoid endothelial lining. As a result, the maximal tolerable level of shear stress is lower than 1\u00a0Pa. Tilles et\u00a0al.38 showed in in vitro studies on rat hepatocytes that hepatocyte function, measured as albumin and urea synthesis rates, was significantly decreased (resp. 2.6- and 1.9-fold) when hepatocytes were directly exposed to shear stresses higher than 0.5\u00a0Pa compared to low shear stresses (<0.033\u00a0Pa). Under standard conditions, maximum shear stress values in the AMC-BAL do not exceed 0.04\u00a0Pa. Additionally, the majority of the hepatocytes are not subjected to significant shear stresses. Small zones with increased shear stress exist but maximal values are considered to be acceptable and not detrimental for the viability and function of hepatocytes. However, shear stresses may increase up to 0.4\u00a0Pa when flow rate is increased 10-fold (case 9). Moreover, should plasma be used in this case, instead of culture medium, maximal shear stress values may then reach up to 0.71\u00a0Pa (not simulated; obtained by data extrapolation), which is in the critical range (>0.5\u00a0Pa38). Additionally, very high flow rates, e.g. 10-times the normal culture flow rates, may cause detachment of hepatocytes and therefore should not be used in spite of any possible increase in local O2 availability. Subsequently, case 9 (culture medium flow rate\u00a0\u00d7\u00a010) can already be discarded in the search for optimal O2 availability, based on fluid dynamical grounds.\nOxygen Transport and Consumption\nWe assessed the oxygen availability in different case studies and in different micro model configurations. In this paragraph, the results are discussed per case.\nReference Case Simulations (Case 1)\nHighest oxygen concentrations in the mat zone are located near the gas capillaries and near the border of the mat and the inter-capillary space. In zones with limited flow velocities, cellular oxygen supply is clearly dependent on diffusion, which causes large radial pO2 gradients. On the other hand, diffusive O2 transport from the culture medium towards the hepatocytes also causes an axial gradient in culture medium oxygen content. As a consequence, culture medium only oxygenates the mat near the very start of the first mat segment and near the side of the mat. The depth of O2 penetration into the mat decreases rapidly further downstream, since as the majority of oxygen content in the convective flow is already consumed in the first part of the micro model. Nevertheless, oxygen level in the culture medium is not totally depleted at the end of the micro model as the fluid transit time is not sufficiently long for all oxygen to diffuse to the hepatocytes.\nIn contrast, the O2 concentration in the gas capillaries remains constant along the micro model axis, so the zone around the capillaries, which is oxygenated has a constant radius throughout the entire micro model. This pattern of oxygenation leads to about 80% higher Vratio percentages in hepatocyte distribution 2 as compared to 1, since more hepatocytes are close to the gas capillaries in cell distribution 2. So despite steeper pO2 gradients near the gas capillaries and consequently a larger zone of depleted oxygen in the mat zone in case of hepatocyte distribution 2, this cell distribution leads to significantly higher Vratio percentages. However, this effect is related to the maximal oxygen diffusion distance, which is limited by the Michaelis\u2013Menten oxygen consumption parameters, local hepatocyte density and local diffusion coefficient in general and by the specific local gas capillary and culture medium pO2 in this reference case.\nSince regions which are oxygenated by one gas capillary in particular do not overlap or influence each other in the reference case simulations, diffusive oxygen supply by the capillaries is independent of relative capillary location. Convective oxygen transport is also identical as flow distribution is irrespective of capillary placement (section \u201cFluid flow and shear stress distribution\u201d). Consequently, inline and triangular capillary pattern give identical results concerning oxygen transport and consumption in these reference cases when the same hepatocyte distribution is used, as is confirmed by simulation results.\nConcerning oxygen transport, we can conclude that the oxygen supply in the AMC-BAL is not fully adequate to provide all hepatocytes with sufficient oxygen to maintain O2 consumption at minimally 90% of the maximal uptake rate (Vratio\u00a0>\u00a00.9). Given that the AMC-BAL can be modeled as a combination of micro model configurations, 16\u201328% of the hepatocytes in the AMC-BAL are adequately oxygenated. In other words, between 72% and 84% of the hepatocytes are subjected to a pO2 of less than 18\u00a0mmHg under the current conditions. Normal physiological pO2 values in the liver sinusoids are between 70\u00a0mmHg in the periportal area and 20\u00a0mmHg in the pericentral area.42 But it is difficult to determine the effect on viability and function of hepatocytes below this threshold. Studies indicate that hepatocyte respiration becomes impaired below 1\u20132\u00a0mmHg pO2.10 With respect to these values, further investigation of simulation results showed that under current conditions 67% and 52% of the hepatocytes in cell distribution 1 and 2 have a pO2 below 2\u00a0mmHg and will suffer from hypoxia. The remaining 17\u201320% of the hepatocytes are likely to be characterized by shifts to more anaerobic metabolic processes.36 However, it should be noted that the low \u201cVratio\u00a0>\u00a00.9\u201d percentages are largely influenced by the very stringent hepatocyte oxygen consumption ratios used in this numerical model. Also, hepatocytes which are subjected to low oxygen levels for a prolonged period of time might show changes in oxygen consumption characteristics (VM, KM) to compensate for the lack of oxygen. This could account for account for higher oxygen availability in vitro. The use of different (VM, KM)-values throughout the model to address this behavior can be implemented in future work. Nevertheless, further increase in O2 availability may prove useful to increase the cell viability and subsequently the efficiency of the AMC-BAL.\nThe Effect of the Internal Oxygenator (Case 2,3)\nDisabling the internal oxygenator leads to detrimental results and reduces the percentage of adequately oxygenated hepatocytes to virtually zero. This confirms the importance of the internal oxygenator and illustrates the small contribution of the incoming convective oxygen flux by culture medium. Incoming culture medium flow adds only 0.17\u00a0nmol O2 per second to the micro model, while maximal oxygen uptake VM equals 0.7286\u00a0nmol\/s*106 cells. As such, theoretically no more than 0.2\u00a0\u00d7\u00a0106 cells, i.e. 6% of the total amount of hepatocytes per micro model, would be able to consume at maximal uptake rate. In reality, only 1.7% of the hepatocytes can consume at a Vratio\u00a0>\u00a00.9 due to the presence of pO2 gradients. Replacing the internal oxygenator by an external oxygenator, which doubles the flow pO2 and thus the convective oxygen flux, will only increase the \u201cVratio\u00a0>\u00a00.9\u201d percentage to about 6%. Since this is still considerably less than in the reference cases, external oxygenation \u2013 even at elevated pO2 levels \u2013 is a not suitable replacement for the internal oxygenation system as the gas capillaries constitute the main oxygen supply. As such, the internal oxygenator is an essential part of the AMC-BAL.\nIncreasing Oxygen Availability (Case 4\u201311)\nIn the reference case models, merely 16\u201328% of the hepatocytes are adequately supplied with oxygen to consume at a Vratio\u00a0>\u00a00.9, depending on hepatocyte distribution. We therefore assessed several strategies to increase overall oxygen availability.\nCulture medium flow rate (case 8,9) does not have a considerable effect on cellular oxygen availability. A 2-fold increase in flow rate resulted in an increase in regions with Vratio\u00a0>\u00a00.9 of only 14%. This was expected as the convective oxygen supply is only a minor contributor to the overall oxygen supply. Consequently, doubling the medium pO2 (case 6) does not greatly increases the amount of regions with sufficient oxygen either. As already discussed, changes in culture medium properties are reflected in a small increase of regions with Vratio in the mat zone, and more specifically in the first mat segment as oxygen content of the fluid is rapidly decreased downstream. It is interesting to note that doubling the flow rate and doubling the medium pO2 both supply exactly the same amount of oxygen to the micro model: in the latter, oxygen is supplied at increased pO2, whereas in the former oxygen is supplied at standard level but with a higher flow rate. Nevertheless, doubling pO2 medium is preferred compared to increasing flow rate when aiming at a better effective hepatocyte utilization ratio since the doubled flow rate does not give the oxygen enough time to diffuse to the hepatocytes as the residence time before oxygen exits the micro model is cut in half. However, a disadvantage of increasing pO2 in the medium is the requirement of an external oxygenator, which makes the extracorporeal circuit more complex and costly and increases the extracorporeal plasma volume in a clinical setting.\nDoubling the pO2 of the oxygenation gas (case 4) is much easier to achieve in practice. Importantly, the \u201cVratio\u00a0>\u00a00.9\u201d percentage increases significantly to almost a doubled level as compared to the reference case. In comparison, culture medium flow rate has to be increased more than 10-fold (case 9) to achieve similar results as with a 2-fold increase in pO2 of the oxygenation gas. This illustrates how oxygen supply is only minor dependent on fluid flow rate. However, saturation of \u201cVratio\u00a0>\u00a00.9\u201d percentages with increased flow rate is not yet achieved at 10-fold flow rate. Nevertheless, a 10-fold flow rate is not preferred because of possible detachment of the hepatocytes and an increased risk of possible shear stress damage to the hepatocytes.\nThe effective hepatocyte utilization ratio can even be further improved. Culture medium pO2 can be set to carbogen level (case 7) or the number of capillaries can be doubled (case 10). On average and for both hepatocyte distributions, these two methods lead to a 2-fold increase in the amount of hepatocytes consuming oxygen at Vratio\u00a0>\u00a00.9 as compared to the reference case. When both approaches are compared for hepatocyte distribution 1, the results are slightly better when using the carbogen medium pO2. But for hepatocyte distribution 2, the double number of capillaries leads to better results. An increase in the number of capillaries is preferred over carbogen medium perfusion, since carbogen medium perfusion leads to extremely high and possible toxic12pO2 values of almost 720\u00a0mmHg. Carbogen medium perfusion also requires an additional external oxygenator. Therefore, increasing the number of capillaries by 2-fold is a more easy and safe method to increase the effective hepatocyte utilization ratio.\nThe results obtained with double capillary numbers are also better when compared to the doubling of the oxygenation gas pO2. In cell distribution 2, this is mainly caused by the redistribution of the hepatocytes located around the capillaries. By increasing the number of capillaries, the hepatocyte layer thickness decreased (section \u201cModeling oxygen transport and consumption\u201d). As a consequence, the region that a gas capillary can sufficiently oxygenate now spans the entire hepatocyte layer so 100% of the cell layer (Table\u00a04) is sufficiently oxygenated. In cell distribution 1, the total region with Vratio\u00a0>\u00a00.9 has also increased by two, as each additional capillary supplies an additional (constant) volume of hepatocytes with Vratio\u00a0>\u00a00.9. In contrast, doubling the oxygenation gas pO2 increases the region surrounding a single capillary with Vratio\u00a0>\u00a00.9 with a factor of less than two.\nThe ultimate results in improving cellular oxygen availability and effective hepatocyte utilization ratio are found when oxygenation gas is set to carbogen level (case 5) or when a double number of capillaries is used in combination with a doubled oxygenation gas pO2 (case 11). Vratio\u00a0>\u00a00.9 percentages have quadrupled for cell distribution 1 and tripled for cell distribution 2 as compared to the reference case for both approaches.\nSimulation results show that in case 11, the respective gains in Vratio\u00a0>\u00a00.9 regions are cumulated when the case of a double number of capillaries and a doubled pO2 gas are combined. It can also be noted that there is a small but significant difference between the inline and triangular capillary pattern in case 5. When carbogen oxygenation gas is used, the region with Vratio\u00a0>\u00a00.9 surrounding a capillary has grown to such an extent that it could overlap with the region of surrounding capillaries. If a capillary is present straight opposed to another (inline pattern), this overlap is present. Consequently, the intersection of the two regions only contributes once to the amount of region with Vratio\u00a0>\u00a00.9. In contrast, when capillaries are in a triangular pattern, the region surrounding the capillary can extent to its fullest, thus increasing the percentage Vratio\u00a0>\u00a00.9 more compared to the inline micro model. This effect is more pronounced in the cell distribution 2 compared to distribution 1.\nGiven the possible toxicity of the hyperoxic oxygen levels when carbogen oxygenation gas is used (case 5), the preferred method to optimally and maximally increase oxygen availability in the AMC-BAL is to double the number of gas capillaries together with a 2-fold increase in culture gas pO2. We note, however, that the calculated improvements in O2 availability and Vratio\u00a0>\u00a00.9 percentages do not necessarily translate into identical increases in cell viability or functional activity. Nevertheless, similar trends between simulation results and the in vitro situation are expected, but have to be validated.\nClinical Versus Experimental Setting (Case 12,13)\nAn important 20\u201325% decrease in the amount of hepatocytes with Vratio\u00a0>\u00a00.9 is caused by the lower diffusion coefficient (\u22129%) and oxygen solubility (\u221225%) in plasma as compared to culture medium. As in reference case 1, results are independent of capillary pattern and are higher in cell distribution 2 as compared to cell distribution 1. Doubling the oxygenation gas pO2 in the clinical setting leads to approximately the same relative increase in Vratio percentages as it does in the experimental setting. As such, we conclude that any relative increase in oxygen availability found in the experimental setting by any possible strategy can also be applied to the clinical setting, taking into account that the absolute Vratio\u00a0>\u00a00.9 fractions are decreased with a constant percentage depending on hepatocyte distribution.\nChanges in Hepatocyte O2 Consumption in Time (Case 14,15)\nHigher KM values of day 4 and 5 cause the decrease in oxygen consumption to start at higher oxygen levels compared to lower KM values of day 2. Consequently, pO2 gradients are less steep and oxygen penetrates further into the mat zone or into hepatocyte layers because of the reduced local O2 uptake. Paradoxically, \u201cVratio\u00a0>\u00a00.9\u201d fractions have decreased. This is understood when converting the Vratio threshold to the minimal cellular pO2 to which the hepatocytes must be subjected to, using Eq. 4. Whereas in the reference case, hepatocytes are considered to be sufficiently oxygenated when cellular pO2 reaches at least 18\u00a0mmHg, threshold values are now approximately 43 and 68\u00a0mmHg for day 4 and 5, respectively. Since the overall pO2 level in the latter cases is not equally elevated, Vratio\u00a0>\u00a00.9 fractions are strongly reduced. Conversely, when applying an identical pO2 threshold instead of Vratio, hepatocyte percentages in case 14 and 15 are increased with 3% and up to 20% compared to day 2. As such, an increase in KM causes average cellular pO2 to increase, but whether there is also an increase in the number of hepatocytes that are sufficiently oxygenated is difficult to determine based on computer simulations, as this depends on the criteria, i.e. minimal Vratio versus minimal pO2, used.\nConclusions\nA numerical model to investigate fluid flow and oxygen transport and consumption in the AMC-BAL was developed and applied to two representative micro models of the AMC-BAL. Two different gas capillary patterns, i.e. \u2018inline\u2019 and \u2018triangular\u2019, were used and combined with two proposed hepatocyte distributions, leading to four basic configurations in total. Fifteen case studies were performed on each of the configurations in order to gain insight in the fluid flow, shear stress distribution, oxygen availability and effective hepatocyte utilization ratio Vratio of the AMC-BAL and to assess possible strategies to further improve cellular oxygen availability. We found that the AMC-BAL does not provide sufficient oxygen to all hepatocytes to allow them to consume oxygen at 90% of their maximal uptake rate under standard operating conditions. The internal oxygenator is an essential part of the AMC-BAL. Doubling the number of gas capillaries together with a 2-fold increase in the oxygenation gas pO2 was found to be the optimal method to maximally increase O2 availability. Additionally, pressure drop over the AMC-BAL and cellular shear stress levels were found to be low and advantageous to cell culture. The developed model also allowed us to assess the effect of the transition from the in vitro to the clinical setting and the effect of the change of hepatocyte O2 consumption characteristics in time. Since large variations in simulation results between hepatocyte distributions are shown, an assessment of the in vitro hepatocyte distribution in the AMC-BAL is useful. Subsequently, an attempt to validate the numerical model with in vitro experiments should be made. Eventually, adoption of this information may lead to a more efficient and productive AMC-BAL in the near future.","keyphrases":["computational fluid dynamics","fluid flow","shear stress","internal oxygenation","external oxygenator","acute liver failure","oxygen partial pressure","effective hepatocyte utilization ratio","michaelis\u2013menten oxygen consumption","hepatocyte bioreactor"],"prmu":["P","P","P","P","P","P","P","P","P","R"]}
{"id":"Dig_Dis_Sci-3-1-1914226","title":"Rectal GIST Presenting as a Submucosal Calculus\n","text":"This case report presents an incidental finding of a rectal GIST (gastrointestinal stromal tumor) presenting as a submucosal calculus, not previously reported. A 53-year-old man without a significant medical history presented with abdominal pain in the left lower quadrant, and with constipation. Upon rectal examination, a hard submucosal swelling was palpated 4 cm from the anus, at 3 o\u2019clock, in the left rectum wall. X-ray photos, computerized tomography (CT)-scan and a magnetic resonance imaging (MRI) scan clearly showed a calculus. Excision revealed a turnip-like lesion, 3.1\u00d72.3\u00d71.8 cm. Analysis showed it was a rectal GIST, a rare mesenchymal tumor of the gastrointestinal tract, which expressed CD117 (or c-kit, a marker of kit-receptor tyrosine kinase) and CD34. Calcification is not a usual clinicopathological feature of GISTs [1\u20133], and although a number of rectal GISTs have been reported [4\u20139], we have found no cases so far of rectal GIST presenting as a submucosal calculus.\nCase Report\nA 53-year-old man without significant medical history presented with abdominal pain in the left lower quadrant and constipation. He got up that morning with a cramping abdominal ache that lasted over the day. He was nauseous, had vomited once, and ructus was present. There had been a small amount of hard stool and discomfort during defecation initially, but after the general practitioner had administered laxatives, he had liquid diarrhea. There was no complaint of constipation on a regular basis; this was a new finding. Tenesmus was absent. There was no bleeding or mucous discharge. He was feverish, with a temperature of 38.2\u00b0C. On examination, the abdomen was slightly distended. Auscultation showed diminished intestinal peristalsis, without borborygmi, and percussion was tympanic. Upon palpation, the abdomen was supple: there was no reflex rigidity nor guarding, and no rebound tenderness. Upon rectal examination, a hard submucosal swelling was palpated 4 cm from the anus, at 3 o\u2019clock, in the left rectum wall. At the time, this was considered an accidental finding that was to be investigated later.\nThere were no specific deviations in the lab results. Ultrasound investigation displayed some hydronephrosis of the left kidney and a stone (size: 0.13\u00d70.7\u00a0cm) of the left ureter. X-ray photos displayed a spherical lesion, projecting in the pelvis and situated in the rectum, and some phleboliths projecting on the left colon, but not the ureter stone (Fig. 1). Proctoscopy was not conclusive, so further investigation was performed. A computerized tomography (CT) scan and a magnetic resonance imaging (MRI) scan also clearly showed the submucosal calculus (Figs. 2 and 3).\nFig. 1X-ray photo showing the rectal calculus projecting in the pelvis (thick arrow) above the pubic symphysis and the phleboliths projecting on the left colon (thin arrow)Fig. 2Computerized tomography (CT) scan showing the rectal calculus in the center (arrow)Fig. 3Magnetic resonance imaging (MRI) scan. The arrow shows the rectal calculus in the rectum wallFig. 4Excision of the rectal calculus in the operating roomFig. 5Excision revealed a turnip-like lesion, dimensions 3.1\u00d7 2.3\u00d71.8\u00a0cm\nOur patient was diagnosed with abdominal pain and constipation based on a ureter stone and slight hydronephrosis of the left kidney, with coincidental finding of a rectal tumor. He was sent home with adequate analgesia and spontaneously released the ureter stone. An appointment was made for elective excision of the rectal calculus. Transanal excision was performed in the operating room (Fig. 4) and revealed a turnip-like lesion, dimensions 3.1\u00d72.3\u00d71.8 cm (Fig. 5). No normal tissue margins were excised because the tumor was not connected to the surrounding tissues. After surgery, the patient went home and recovered well, without any further complaints or complications.\nThe calculus was analyzed and consisted of fibroid and bony tissue, together with bundles of spindle-shaped cells with cigar-like nuclei. A partially calcified leiomyoma was considered at first, but supplementary immunoperoxidase analysis showed that the spindle-shaped cells were positive for CD117 (c-kit) and CD34. Unfortunately, histopathologic slides showing CD117 staining are not available to us. There was some focally positive smooth muscle actin, and MIB1 expression appeared negative. The mitotic count was low: fewer than five mitoses per 50 HPF. Only then did this analysis lead to the conclusion of rectal GIST, with extended calcification and ossification. It was classified as low-risk GIST because of the size (between 2 and 5\u00a0cm) and low mitotic count (Table 1).\nTable IPrognosis of primary GISTRiskSize (cm)Mitotic count (per 50 HPF)Very low risk<2<5Low risk2\u20135<5Intermediate risk<56\u2013105\u201310<5High risk>5>5>10>Any mitotic rateAny tumor>10Note. from Fletcher et al (13). Abbreviations: HPF, high-power field.\nConclusion\nCalcification is not a usual clinicopathologic feature of GISTs [1\u20133], and although a number of rectal GISTs have been reported, we have found no cases so far of rectal GIST presenting as a submucosal calculus. Furthermore, in the literature we found no cases of calcified GIST in other sites of the body. Expression of CD117 (c-kit) and CD34 proved that our calculus was in fact a rectal GIST. Calcification might be explained by internal bleeding of the tumor. The abdominal pain of our patient could be explained entirely by the ureteral stone. The rectal tumor might only explain the constipation. Because of the unpredictable biological behavior of gastrointestinal stromal tumors, the prognosis for our patient is uncertain, in spite of the low-risk character. For this reason, at multidisciplinary oncological deliberation, an expectant policy with regular follow-up was arranged.","keyphrases":["calculus","gastrointestinal stromal tumors","rectal tumor"],"prmu":["P","P","P"]}
{"id":"Lipids-3-1-2039812","title":"Arachidonic Acid but not Eicosapentaenoic Acid (EPA) and Oleic Acid Activates NF-\u03baB and Elevates ICAM-1 Expression in Caco-2 Cells\n","text":"In patients with inflammatory bowel disease (IBD), intestinal activation of the transcription factor NF-\u03baB as well as intercellular adhesion molecule (ICAM)-1 expression, which is involved in recruiting leukocytes to the side of inflammation is increased. Moreover, colonic arachidonic acid (ARA) proportions are increased and oleic acid (OA) proportions are decreased. Fish oils are protective in IBD patients however, a side-by-side comparison between effects of fish oils, ARA and OA has not been made. We therefore, compared effects of eicosapentaenoic acid (EPA) versus ARA and OA on ICAM-1 expression in Caco-2 enterocytes. To validate our model we showed that dexamethasone, sulfasalazine and PPAR\u03b1 (GW7647) or PPAR\u03b3 (troglitazone) agonists significantly lowered ICAM-1 expression. ICAM-1 expression of non-stimulated and cytokine stimulated Caco-2 cells cultured for 22 days with ARA was significant higher as compared to EPA and OA. Furthermore, ARA increased NF-\u03baB activation in a reporter cell-line as compared to EPA. Antibody array analysis of multiple inflammatory proteins particularly showed an increased monocyte chemotactic protein (MCP)-1 and angiogenin production and a decreased interleukin (IL)-6 and IL-10 production by ARA as compared to EPA. Our results showed that ARA but not EPA and OA activates NF-\u03baB and elevates ICAM-1 expression in Caco-2 enterocytes. It suggests that replacement of ARA by EPA or OA in the colon mucosa might have beneficial effects for IBD patients. Finally, we suggest that the pro-inflammatory effects of ARA versus EPA and OA are not related to PPAR\u03b3 activation and\/or eicosanoid formation.\nIntroduction\nEpidemiological studies have shown a low incidence of inflammatory bowel disease (IBD) in Eskimo\u2019s as compared to West-European populations [1] and increasing incidences of IBD in Japan [2]. These findings suggest that an increased dietary intake of n-6 polyunsaturated fatty acids (PUFA) and a lower intake of n-3 PUFAs contribute to the development of IBD. Thus, n-3 fish oil PUFAs may have anti-inflammatory effects as compared to n-6 PUFA [3]. Indeed, elevated proportions of the n-6 PUFA arachidonic acid (ARA) in colon mucosa of both ulcerative colitis (UC) and Crohn's disease (CD) patients as compared to those of control subjects have been shown [4\u20137]. Although the proportion of the n-3 PUFA eicosapentaenoic acid (EPA) in the mucosa was in most studies not statistically different between IBD patients and healthy controls, one study reported a tendency towards lower EPA proportions in IBD patients [4]. In contrast to EPA, the proportion of the fish oil PUFA docosahexaenoic acid (DHA) was elevated in colonic mucosa of IBD patients as compared to control subjects [4, 5, 7]. Interestingly, the proportion of the n-9 monounsaturated fatty acid (MUFA) oleic acid (OA) was lower in colon mucosa of IBD patients [4, 7]. Because of these observations, and because EPA is a more important precursor of eicosanoids than DHA [8], we decided to compare the effects of EPA versus ARA and OA in an in vitro model of intestinal inflammation. OA is already the most abundant fatty acid present both in our diet [9] as well as in colon mucosa [4]. Therefore, and because of differences in ability to compete with ARA for incorporation in tissue phospholipids [10, 11], it is probably easier to lower mucosal ARA levels by increasing fish oil intake than by increasing OA intake. Indeed, by increasing their intake of fish oil, ARA in the colon mucosa of IBD patients was replaced by EPA and DHA [12], which was associated with significantly reduced corticosteroid requirements [12] and lower relapse rates [13]. It should be noted however, that not all intervention studies using fish oils were that positive, although the overall conclusion is that fish oil supplementation shows at least minor protective effects [14].\nThe intercellular adhesion molecule (ICAM)-1 plays an important role in the pathology of IBD. In IBD patients intestinal ICAM-1 expression [15] and plasma levels of soluble ICAM-1 (sICAM) are increased [16], and IBD is associated with polymorphisms in the gene encoding for ICAM-1 [17]. Moreover, animal models [18, 19] and a human intervention study [20] have shown that ICAM-1 blocking inhibited intestinal inflammation. The transcription factor NF-\u03baB is a key regulator of the inflammatory response and activation of NF-\u03baB seems to play a critical role in the initiation and perpetuation of intestinal inflammation in IBD [21, 22]. NF-\u03baB activity in the colon is increased during active episodes in IBD patients and certain anti-inflammatory drugs commonly used for IBD appear to inhibit NF-\u03baB [23\u201327]. In animal models, NF-\u03baB blockade abolished experimental colitis [28, 29]. A side-by-side comparison of the n-3 PUFA EPA, the n-6 PUFA ARA and the n-9 MUFA OA on ICAM-1 expression and NF-\u03baB activation of intestinal cells has, as far as we are aware of, never been performed. Therefore the aim of the present study was to compare the effects of EPA, ARA and OA on ICAM-1 expression and NF-\u03baB activation in the human intestinal epithelial Caco-2 cell line in vitro.\nMaterials and Methods \nReagents\nBovine serum albumin (BSA; endotoxin and fatty acid-free), sulfasalazine, dexamethasone, GW7647, oleic acid (OA), arachidonic acid (ARA), eicosapentaenoic acid (EPA) and indomethacin were obtained from Sigma Chemical Company (St Louis, MO). Troglitazone was purchased from Biomol (Plymouth Meeting, PA). Recombinant human IL-1\u03b2 and interferon (IFN)\u03b3 were purchased from Roche Molecular Biochemicals (Mannheim, Germany). DMEM, trypsin, penicillin streptomycin (PS), sodium pyruvate (SP) and non-essential amino acids (NEAA) were obtained from Invitrogen Corporation (Paisley, UK). Fetal calf serum (FCS; South-American) was obtained from Greiner Bio-one (Frickenhausen, Germany).\nIntestinal Cell Cultures \nThe human cell line Caco-2 was purchased from the American Tissue Type Collection (ATTC). Caco-2 cells were cultured in DMEM supplemented with 10% heat-inactivated FCS and 1% penicillin streptomycin (PS), 1% sodium pyruvate (SP) and 1% non-essential amino acids (NEAA). Cells were cultured at 37\u00a0\u00b0C in a 5% CO2 humidified atmosphere, refreshed every second day and separated by trypsin\u20130.03% EDTA, when they had reached 70\u201390% confluence. To evaluate the immune-modulating effects of different interventions, Caco-2 cells were plated in six well tissue culture plates at an initial density of 0.5\u00a0\u00d7\u00a0106 cells\/mL in a total volume of 1.5\u00a0mL. Medium was replaced every other day for 24\u00a0days. After 24\u00a0days Caco-2 wells were fully differentiated into small intestinal enterocytes [30]. First, effects of immune-suppressive pharmacological compounds (sulfasalazine, dexamethasone and troglitazone) were tested to validate the model. Although these pharmacologic compounds have known immune-suppressive effects, effects on ICAM-1 expression in Caco-2 cells have\u2014as far as we know\u2014not been reported before. Therefore, after Caco-2 cells were fully differentiated, medium was replaced by medium containing the compound of interest in combination with an inflammation-inducing cocktail consisting of the cytokines IFN\u03b3 (100\u00a0U\/mL) and IL-1\u03b2 (50\u00a0U\/mL). The compounds of interest were pre-incubated 30\u00a0min (sulfasalazine) or 2\u00a0h (dexamethasone, troglitazone or GW7647) before stimulation with the cytokine cocktail. After 16\u00a0h of cytokine stimulation, cells were used to determine cell surface ICAM-1 protein expression.\nFatty Acid Experiments \nThe effects of various fatty acids were evaluated using the same Caco-2 cell model. For this, various fatty acids were added at indicated concentrations 2\u00a0days after plating the cells and again for the following 22\u00a0days each time when the medium was refreshed. We used OA as a control n-9 MUFA because this is the most abundant fatty acid in the diet [9]. We further compared the effects of the n-6 PUFA ARA versus the n-3 PUFA EPA. We have explicitly chosen to use EPA instead of DHA since the proportion DHA in the colon mucosa of IBD patients was already higher as compared to control subjects [4, 5, 7]. In addition, EPA is most likely a more important eicosanoid precursor [8]. The fatty acids were dissolved in ethanol up to a final ethanol concentration in the medium of maximal 0.5% (v\/v). To prevent cytotoxicity of the fatty acids the FA were bound to albumin, by pre-incubating the fatty acids dissolved in ethanol for 30\u00a0min at 37\u00a0\u00b0C in full culture medium together with 10% FCS, which also contained 0.1% BSA. Caco-2 cells were cultured with respectively 160\u00a0\u03bcM OA [C18:1(n-9)] versus 130\u00a0\u03bcM ARA [C20:4(n-6)] plus 30\u00a0\u03bcM OA (in total 160\u00a0\u03bcM fatty acids) or 6\u00a0\u03bcM EPA [C20:5(n-3)] plus 154\u00a0\u03bcM OA (in total also 160\u00a0\u03bcM fatty acids). By this approach the total molarity of fatty acids supplied was similar in all experiments, while supplying different amounts of the fatty acid of interest (i.e., 130\u00a0\u03bcM ARA or 6\u00a0\u03bcM EPA). These relatively low concentrations of OA (160\u00a0\u03bcM), ARA (130\u00a0\u03bcM) and EPA (6\u00a0\u03bcM) were chosen because they are four\u00a0times higher than normally present in culture medium of Caco-2 cells containing 10% FCS. We have deliberately chosen for this low EPA concentration since the EPA concentration is very low in FCS. However, the EPA concentration used is\u2014as for all fatty acids used in these experiments\u2014already four times higher than normally present in culture medium. We cultured the cells for 22\u00a0days with these relatively low concentrations of fatty acids to simulate a realistic long-term in vivo change in dietary fatty acid intake. After 22\u00a0days culture with fatty acids, medium was replaced by medium enriched with the different fatty acids plus the cytokine cocktail [IFN\u03b3 (100\u00a0U\/mL) and IL-1\u03b2 (50\u00a0U\/mL)]. After 16\u00a0h stimulation, ICAM-1 expression on living cells was measured and culture medium was collected to determine inflammatory protein expression profiles. To evaluate the effects of the fatty acids on NF-\u03baB activity and the role of cyclooxygenase (COX)-enzymes, the experiments with the fatty acids ARA and EPA were repeated but now in our NF-\u03baB reporter Caco-2 cell line with and without indomethacin (20\u00a0\u03bcM) added 2\u00a0h before and during cytokine stimulation. Prostaglandin PGE2 levels in the supernatant were quantitated using a PGE2 Biotrak enzyme-immunoassay (EIA) system (Amersham Biosciences Ltd, Buckinghamshire, UK) according to the high sensitivity enzyme immunoassay protocol 2.\nFlow Cytometry Analysis of ICAM-1\nIn order to quantify cell surface ICAM-1 protein expression on living Caco-2 cells, we developed a flow cytometry assay. After 16\u00a0h of stimulation, the cells were washed three times with PBS and detached with trypsin\u20130.03% EDTA. Next, medium was added and cell suspensions were centrifuged for 5\u00a0min at 1,200\u00a0rpm at room temperature, followed by resuspending the pellets in 500\u00a0\u03bcL PBS-1% BSA. Cells were counted and diluted to 106\u00a0cells\/mL in PBS-1% BSA. Recombinant-phycoerythrin (R-PE)-conjugated mouse-anti-human CD-54 monoclonal antibody (anti-ICAM-1) or isotype-matched control antibody (Becton Dickinson Biosciences, San Diego, CA; 20\u00a0\u03bcL\/106 cells) was added and incubated for 30\u00a0min on ice in the dark. Next, cell suspensions were centrifuged for 5\u00a0min at 1,500\u00a0rpm and pellets were resuspended in 500\u00a0\u03bcL PBS-1% BSA. The amount of fluorescence of 10,000 living cells was counted and analyzed with the FACSort and CellQuest analysis software (Becton Dickinson, Franklin Lakes, NJ).\nStable Transfection of NF-\u03baB in Caco-2 Cells\nFor evaluating the effects of the various interventions on transcriptional activity of NF-\u03baB, a stable NF-\u03baB reporter Caco-2 cell line was created. The 6\u03baB-TK-luciferase (NF-\u03baB reporter) plasmid and neomycin resistance plasmid were both kindly provided by Dr. R.C. Langen (Department of Pulmonology, Maastricht University, The Netherlands). Cells were transfected using Lipofectamine 2000 (Invitrogen Corporation, Paisley, UK) according the manufacturers\u2019 instructions. Positive clones were selected by culturing with geneticin (1\u00a0mg\/mL). To determine luciferase activity, non-stimulated and 3\u00a0h cytokine (100\u00a0U\/mL IFN\u03b3 and 50\u00a0U\/mL IL-1\u03b2) stimulated cells were lysed in luciferase lysis buffer (Promega, Madison, WI) and stored at \u221280\u00a0\u00b0C. Luciferase (Promega) activity was measured according to the manufacturers\u2019 instructions and expressed relative to total protein (Bio-rad assay; Bio-rad, Hercules, CA).\nPeroxisome Proliferator-Activated Receptor (PPAR)\u03b3 and PPAR\u03b1 mRNA Expression of Differentiated Caco-2 Cells\nTotal RNA was extracted from differentiated Caco-2 cells with Trizol according to the manufacturers\u2019 instructions (Gibco BRL, Gaithersburg, MD). Next, cDNA was made as described [31], and mRNA expression of PPAR\u03b3 and PPAR\u03b1 was determined using commercially available Taqman gene expression assays (Applied Biosystems, Foster city, CA). Data were normalized against \u03b2-actin as housekeeping gene.\nFatty Acid Composition of Caco-2 Cells\nFatty acid incorporation into the Caco-2 cells was evaluated using extraction and analysis procedures as previously described [32]. Briefly, total lipids were extracted from 500\u00a0\u03bcL cell suspension in PBS-1% BSA according to the method of Bligh and Dyer [33]. Aminopropyl-bonded silica columns (Varian, Harbor City, CA) were used to separate phospholipids from the total lipid extract [34]. The phospholipids were then saponified, and the resultant fatty acids were methylated into their corresponding fatty acid methyl esters (FAMEs) [35]. Fatty acids were separated on an Autosystem (Perkin-Elmer, Norwalk, CT) gas chromatograph that was fitted with a silica-gel column (Cp-sil 88 for FAME, 50\u00a0m \u00d7 0.25\u00a0mm, 0.2-\u03bcm film thickness; Chrompack, Middelburg, The Netherlands) with helium gas (130\u00a0kPa) as the carrier gas. Both the injection and detection temperatures were set at 300\u00a0\u00b0C. The starting temperature of the column was 160\u00a0\u00b0C. Ten min after injection, the temperature was increased up to 190\u00a0\u00b0C at a rate of 2.5\u00a0\u00b0C\/min. After 20\u00a0min at 190\u00a0\u00b0C, the temperature was increased up to 230\u00a0\u00b0C at a rate of 4\u00a0\u00b0C\/min. The final temperature of 230\u00a0\u00b0C was maintained for 10\u00a0min. Data were analyzed by using CHROMCARD software (version 1.21; CE Instruments, Milan, Italy). The fatty acid compositions of the Caco-2 cells are expressed in relative amounts (% of total fatty acids identified; w\/w).\nInflammatory Protein Expression Profiles Using an Antibody Array\nProtein expression patterns of multiple cytokines, chemokines and growth factors, were detected simultaneously in Caco-2 cell culture media with the human cytokine antibody array III (Ray Biotech Inc., Norcross, GA) according to the manufacturers\u2019 instructions. First, duplicates of cell culture media of Caco-2 cells cultured with ARA and EPA after cytokine stimulation were pooled. One millilitre of the pooled samples was added to the array membranes. After incubating and washing, the protein-bound membrane was incubated with a cocktail of biotin-labeled antibodies, followed by the addition of horseradish peroxidase-conjugated streptavidin. Array spot intensity was detected by using a LAS-3000 Lite Image reader (Raytest GmbH, Straubenhart, Germany) based on chemiluminecence imaging. Intensity of the spots was quantified in arbitrary units (a.u.) by densitometry using Aida software version 3.50 (Raytest GmbH), thereby correcting for background staining of the gel. Comparison of protein expression profiles was possible after normalization of each spot on an array using the positive controls, provided by the manufacturer. The sensitivity of the array is not the same for the various proteins. Differences in heights of bars from different proteins do therefore not necessarily represent differences in concentrations. The cytokines used for stimulation (IFN\u03b3 and IL-1\u03b2) were excluded from analysis.\nDetection of ICAM-1 on Caco-2 Frozen Sections\nTo determine the localization of ICAM-1 in our in vitro Caco-2 cell model, Caco-2 cells were cultured and differentiated into small intestinal enterocyte on collagen-coated polyfluoroethylene transwell membrane inserts with a 0.4\u00a0\u03bcm membrane pore size (Corning Costar, Cambridge, MA). Differentiated Caco-2 cells were stimulated with IFN\u03b3 (100 U\/mL) and IL-1\u03b2 (50 U\/mL) for 16 h, embedded in Tissue-Tek (Sakura Finetek, Zoeterwoude, The Netherlands) and rapidly frozen in 2-propanol (Fluka, Zwijndrecht, The Netherlands), dry-ice-cooled and stored at \u221280\u00a0\u00b0C. Serial cryosections (10\u00a0\u03bcm) were obtained using a Leica CM3050 cryostat (Leica Microsystems GmbH, Wetzlar, Germany) and thaw mounted on uncoated glass slides. Before processing or storage at \u221280\u00a0\u00b0C, the samples were air dried overnight. To detect ICAM-1 the sections were incubated 30 min in the dark at room temperature with recombinant-phycoerythrin (R-PE)-conjugated mouse\u2013anti-human CD-54 monoclonal antibody or isotype-matched control antibody (Becton Dickinson Biosciences, San Diego, CA) 1:50 diluted in PBS-1% BSA. To detect cytokeratin (CK)-19 the sections were simultaneously incubated with a monoclonal antibody directed to CK-19, kindly obtained from Dr. E.B. Lane (University Dundee, Dundee, UK) 1:10 diluted. Then the sections were washed three times for 5 min in PBS. After that the secondary antibody goat anti-mouse IgG1 (ALEXA555) (Molecular Probes Europe, Leiden, The Netherlands) (1:500) against anti-CD54 (to evade fast quenching of the PE-label) and goat anti-mouse IgG2b (FITC) (Southern Biotech, Sanbio BV, Uden, The Netherlands) (1:50) against CK-19 diluted in PBS-1% BSA was added to the sections and incubated for 30\u00a0min. Again the sections were washed three times for 5\u00a0min with PBS. Finally, sections were mounted in Mowiol-TRIS pH 8.5 (Calbiochem, Omnilabo International, Etten-Leur, The Netherlands) containing 0.5 g\/mL 4\u20136-diamino-2-phenylindole (DAPI; Molecular Probes Europe) to stain the nuclei. All sections were examined using a Nikon E800 fluorescence microscope (Uvikon, Bunnik, The Netherlands) coupled to a Basler A101C progressive scan colour CCD camera. By just a simple shift in filters, images were grabbed in fluorescence using the ALEXA excitation filter (540\u2013580\u00a0nm), the FITC excitation filter (465\u2013495\u00a0nm) and DAPI UV excitation filter (340\u2013380\u00a0nm) in the red, green and blue channel, respectively. The images acquired were merged to examine the cellular localisation and level of expression of ICAM-1.\nStatistical Analysis \nData were expressed as means and standard deviations (SD) for the non-stimulated condition, the cytokine stimulated condition and cytokine stimulated\u2013non-stimulated (called net stimulated) condition. To determine statistical significance unpaired t-tests (comparison between two interventions) or ANOVA (comparison between three interventions) with a Bonferonni post-hoc test when differences between interventions were significantly different, were performed. First non-stimulated values between interventions were tested to examine differences in basal values. Then, net stimulated values between interventions were tested to examine differences in cytokine-induced changes. Furthermore cytokines stimulated values were tested to examine differences in \u201cend\u201d values. All statistical analyses were performed using SPSS 11 for Mac Os X (SPSS, Chicago, IL). P-values of less than 0.05 were considered statistically significant.\nResults\nModel Validation\nStimulation with IL-1\u03b2 and IFN\u03b3 increased cell-surface ICAM-1 protein expression on Caco-2 cells, which resulted in a net stimulated ICAM-1 expression of about 50 a.u. (Fig.\u00a01, panels a and b). After pre-treatment of the cells for 2\u00a0h with dexamethasone, a corticosteroid with known therapeutic effects in IBD patients, the cytokine stimulated ICAM-1 expression was significantly decreased as compared to control (P\u00a0=\u00a00.044). Also the net stimulated ICAM-1 expression (25 a.u.) was significantly reduced as compared to control (P\u00a0=\u00a00.019). Next, we examined the effect of another frequently used therapeutic drug for IBD patients, sulfasalazine. Effects of sulfasalazine were comparable to those observed for dexamethasone. However, also ICAM-1 expression without cytokine stimulation (P\u00a0=\u00a00.004) (Fig.\u00a01, panel a) was lowered. Altogether, these results show that in our cell model ICAM-1 expression is related to the clinical outcomes of drugs proven to treat IBD and could therefore be used as the main outcome parameter in the following experiments. To further validate characteristics of our in vitro model, we also localized the site of ICAM-1 expression on the Caco-2 cells by means of immunohistochemistry on frozen sections. As shown in Fig.\u00a01 (panels c\u2013f), ICAM-1 was expressed on the apical (lumen) side of the polarized Caco-2 cells. Besides a low constitutive expression (panel d), there was a clear increase after stimulation with the cytokine cocktail (panel e). This localisation is in line with the apical ICAM-1 expression, as found in intestinal biopsies from IBD patients [36].\nFig.\u00a01Model validation. a ICAM-1 expression (in arbitrary units a.u.) on living control Caco-2 cells and after 2\u00a0h pre-treatment with 1\u00a0\u03bcM dexamethasone or 30\u00a0min 5\u00a0mM sulfasalazine with and without cytokine stimulation for 16\u00a0h (100\u00a0U\/mL IFN\u03b3 and 50\u00a0U\/mL IL-1\u03b2) and b net stimulated (stimulated\u2013non-stimulated) ICAM-1 expression. Results represent means\u00a0\u00b1\u00a0SD; n\u00a0=\u00a02. Representative representation of two independent experiments. aP\u00a0<\u00a00.05 versus control non-stimulated, bP\u00a0<\u00a00.05 versus control cytokine stimulated, cP\u00a0<\u00a00.05 versus control net stimulated. (c\u2013f) Immunohistochemistry of ICAM-1 (red) on frozen sections of Caco-2 cells on a transwell showed increased apical expression after cytokine stimulation (100\u00a0U\/mL IFN\u03b3 and 50\u00a0U\/mL IL-1\u03b2). c Hematoxylin staining d Non-stimulated Caco-2 cells e Apical cytokine stimulated Caco-2 cells f Isotype control staining of ICAM-1. Red staining ICAM-1, green staining cytoskeleton (cytokeratin-19), blue staining nucleus. Magnification 40X\nEffects of PPAR Agonists on ICAM-1 Expression and NF-\u03baB Activation \nSince PPARs are known modulators of inflammation and fatty acids are natural ligands for PPARs, we first examined PPAR expression in our differentiated Caco-2 cells. We found that PPAR\u03b1 and PPAR\u03b3 mRNA are expressed in equal amounts (data not shown). Next we examined the effects of the PPAR\u03b3 agonist troglitazone [a thiazolidinedione (TZD)] and the PPAR\u03b1 agonist GW7647 on ICAM-1 expression. Although both troglitazone and GW7647 significantly lowered the cytokine stimulated ICAM-1 expression as compared to control (P\u00a0<\u00a00.001 and P\u00a0=\u00a00.009, respectively), only troglitazone significantly (P\u00a0=\u00a00.008) reduced the net stimulated ICAM-1 expression (Fig.\u00a02, panels a and b). Troglitazone and GW7647 were used at a concentration where they are selective for their receptor subtypes [37, 38]. It is known that PPARs suppress inflammation by inhibiting the transcription factor NF-\u03baB, which is a key regulator of inflammation [39]. Therefore, we also examined effects of troglitazone on NF-\u03baB transactivation in our Caco-2 NF-\u03baB reporter cell line. These results showed that the cytokine cocktail induced after already 3\u00a0h a more than 200% increase in NF-\u03baB transactivation. This increase of NF-\u03baB transactivation was significantly (P\u00a0=\u00a00.008) lower after 2\u00a0h pre-treatment with the PPAR\u03b3 agonist troglitazone (Fig.\u00a02, panels c and d). Moreover, the inhibitory effect of pre-treatment with the PPAR\u03b3 agonist troglitazone on cytokine induced NF-\u03baB transactivation was significantly (P\u00a0=\u00a00.001) larger as compared to pre-treatment with the PPAR\u03b1 agonist GW7647 (data not shown).\nFig.\u00a02a, b ICAM-1 expression (in arbitrary units a.u.) on living control Caco-2 cells and after pre-treatment with PPAR agonists (2\u00a0h; PPAR\u03b3 troglitazone 100\u00a0\u03bcM and PPAR\u03b1 GW7647 1\u00a0\u03bcM) and c, d NF-\u03baB transactivation measured by luciferase activity (in RLU\/mg) in a NF-\u03baB reporter Caco-2 cell-line of control cells and after troglitazone pre-treatment with and without cytokine stimulation for 3\u00a0h (100\u00a0U\/mL IFN\u03b3 and 50\u00a0U\/mL IL-1\u03b2) and net stimulated (stimulated\u2013non-stimulated). Results represent means \u00b1 SD; n\u00a0=\u00a02. Representative representation of two independent experiments. bP\u00a0<\u00a00.05 versus control cytokine stimulated, cP\u00a0<\u00a00.05 versus control net stimulated\nFatty Acid Incorporation in Phospholipids\nTable\u00a01 shows that the fatty acids kept for 22\u00a0days in the culture medium were incorporated into the phospholipid fraction (% of total fatty acids) of the Caco-2 cells. Fatty acid incorporation into phospholipids showed the same pattern as the fatty acid composition of total lipids (data not shown), although changes in the total lipids were more pronounced. Moreover, the fatty acid profiles of phospholipids (Table\u00a01) and total lipids (data not shown) were similar in non-stimulated and stimulated Caco-2 cells. Cells cultured with ARA showed in particular an increase in the proportion of long chain n-6 PUFAs (mainly ARA) and a decrease in n-9 MUFAs (mainly OA) as compared with cells cultured with only OA. The cells cultured with EPA showed an increase in the proportion of n-3 fatty acids (mainly EPA) whereas the proportions of n-9 MUFAs and n-6 PUFAs did not change much as compared with cells cultured with only OA.\nTable\u00a01Fatty acid composition in phospholipids of Caco-2 cells without (\u2212) and with (+) cytokine stimulation (IL-1\u03b2 and IFN\u03b3) supplemented with different fatty acids (% of total fatty acids)Fatty acidsOA (160\u00a0\u03bcM)ARA\u00a0+\u00a0OA (130\u00a0+\u00a030\u00a0\u03bcM)EPA\u00a0+\u00a0OA (6\u00a0+\u00a0154\u00a0\u03bcM)\u2212+\u2212+\u2212+16:0 (PA)16.016.118.719.516.315.818:0 (SA)5.96.19.610.56.76.518:1 trans 3.03.16.15.72.72.718:1 (n-7)3.43.42.62.53.33.318:1 (n-9) (OA)47.146.314.413.845.245.018:2 (n-6) (LA)1.71.71.11.11.71.620:1 (n-9)2.12.10.30.31.71.720:4 (n-6) (ARA)9.39.429.328.98.98.920:5 (n-3) (EPA)0.40.40.00.02.12.222:1 (n-9)0.80.81.51.30.80.824:1 (n-9)2.02.10.91.02.02.122:4 (n-6)0.40.47.77.80.30.322:5 (n-3)0.50.50.40.41.31.322:6 (n-3) (DHA)1.11.10.60.60.70.6\u221193.793.593.293.493.792.8\u2211 SAFA21.922.228.330.023.022.3\u2211 MUFA58.457.825.824.655.755.6\u2211 PUFA12.312.438.538.214.314.3\u2211 n-32.02.01.01.04.14.1\u2211 n-611.411.538.137.810.910.8\u2211 n-952.051.317.116.449.749.6Data are representative for two independent experiments and the data are derived from pooled samplesARA arachidonic acid, EPA eicosapentaenoic acid, LA linoleic acid, MUFA monounsaturated fatty acids, OA oleic acid, PA palmitic acid, PUFA polyunsaturated fatty acids, SA stearic acid, SAFA saturated fatty acids, \u03a3 sum, \u2212 non-stimulated, + cytokine stimulated\nEffects of Fatty Acids on ICAM-1 Expression and NF-\u03baB Activation\nICAM-1 expression was analyzed on living cells. FACS analysis did not show significant changes in cell populations cultured with the various fatty acids, suggesting no significant cell death. As shown in Fig.\u00a03 (panel a), ARA significantly elevated ICAM-1 expression as compared to OA or EPA (non-stimulated P\u00a0<\u00a00.001 ARA versus OA and ARA versus EPA, and stimulated P\u00a0=\u00a00.010 ARA versus OA, and P\u00a0=\u00a00.013 ARA versus EPA). Interestingly, stimulated and non-stimulated ICAM-1 expression on cells cultured with EPA did not significantly differ from those cultured with OA (Fig.\u00a03, panel a). Despite these clear differences in non-stimulated and stimulated ICAM-1 expression between ARA and EPA or OA, the net stimulated ICAM-1 expression did not differ between the groups (ANOVA P\u00a0=\u00a00.134). Since dietary interventions aiming at lowering mucosal ARA content are most likely more successful by dietary EPA enrichments than by OA enrichment\u2013because OA is the most abundant fatty acid present in our diet [9], making higher intakes not realistic\u2013in further experiments we have focused on effects of ARA versus EPA. First, we examined the effects on NF-\u03baB activation in the stable NF-\u03baB reporter Caco-2 cell line (Fig.\u00a04). NF-\u03baB transactivation in stimulated Caco-2 cells cultured with ARA was significantly increased as compared to that in stimulated cells cultured with EPA (Fig.\u00a04, panels a and b) (stimulated P\u00a0=\u00a00.017 and net stimulated P\u00a0=\u00a00.001). To evaluate whether the effects were related to the production of different families of eicosanoids we measured PGE2 production and examined the effects of the different fatty acids in the presence of the cyclooxygenase (COX)-inhibitor indomethacin. As expected, PGE2 production by Caco-2 cells cultured with ARA was significantly higher than by cells cultured with EPA (data not shown). Stimulation of the cells with the cytokine cocktail did however, not influence PGE2 production. Since cytokine stimulation enhanced NF-\u03baB activation, this suggests that PGE2 is not directly involved in NF-\u03baB activation. Indomethacin decreased PGE2 production of Caco-2 cells by approximately 70% (data not shown), but had no effect on NF-\u03baB transactivation (Fig.\u00a04, panels c and d) as compared to untreated Caco-2 cells. Also ICAM-1 expression was not affected (data not shown). However, as shown in Fig.\u00a04, the effects of ARA versus EPA in the presence of indomethacin treatment (panel e) showed an identical pattern as observed without indomethacin (panel a). Also in the presence of indomethacin the net cytokine stimulated NF-\u03baB transactivation was significantly higher after ARA as compared to EPA (P\u00a0=\u00a00.047) (Fig.\u00a04, panel f). This indicates that the increased basal PGE2 production by ARA cannot explain the different effects of ARA and EPA on NF-\u03baB activation.\nFig.\u00a03a\u2013b ICAM-1 expression (in arbitrary units a.u.) on living Caco-2 cells cultured for 22\u00a0days with 160\u00a0\u03bcM oleic acid (OA) or 130\u00a0\u03bcM arachidonic acid (ARA) plus 30\u00a0\u03bcM OA or 6\u00a0\u03bcM eicosapentaenoic acid (EPA) plus 154\u00a0\u03bcM OA with and without cytokine stimulation for 16\u00a0h (100\u00a0U\/mL IFN\u03b3 and 50\u00a0U\/mL IL-1\u03b2) and net stimulated (stimulated\u2013non-stimulated). Results represent means \u00b1 SD; n\u00a0=\u00a02. Representative representation of two independent experiments. ANOVA between groups, non-stimulated P\u00a0<\u00a00.001, stimulated P\u00a0=\u00a00.006 and net stimulated P\u00a0=\u00a00.134. aBonferroni P\u00a0<\u00a00.05 versus OA and EPA non-stimulated, bBonferroni P\u00a0<\u00a00.05 versus OA and EPA cytokine stimulatedFig.\u00a04NF-\u03baB transactivation with and without cytokine stimulation for 3\u00a0h (100 U\/mL IFN\u03b3 and 50 U\/mL IL-1\u03b2) (a, c, e) and net stimulated (stimulated\u2013non-stimulated) (b, d, f) in a NF-\u03baB reporter Caco-2 cell line measured by luciferase activity (in RLU\/mg). a\u2013b Reporter Caco-2 cells cultured for 22\u00a0days with 130\u00a0\u03bcM arachidonic acid (ARA) plus 30\u00a0\u03bcM oleic acid (OA) or 6\u00a0\u03bcM eicosapentaenoic acid (EPA) plus 154\u00a0\u03bcM OA. c\u2013d Control reporter Caco-2 cells and after 2\u00a0h pre-treatment with the COX-inhibitor indomethacin (20\u00a0\u03bcM). e\u2013f Reporter Caco-2 cells cultured for 22\u00a0days with 130\u00a0\u03bcM arachidonic acid (ARA) plus 30\u00a0\u03bcM OA or 6\u00a0\u03bcM eicosapentaenoic acid (EPA) plus 154\u00a0\u03bcM OA and 2\u00a0h pre-treatment with the COX-inhibitor indomethacin (20\u00a0\u03bcM). Results represent means \u00b1 SD; n\u00a0=\u00a02. bP\u00a0<\u00a00.05 versus EPA cytokine stimulated, cP\u00a0<\u00a00.05 versus EPA or EPA + indomethacin net stimulated\nEffects of Fatty Acids on Inflammatory Proteins Expression Profiles\nFinally, to explore the effects of ARA and EPA on a broader scale, we evaluated protein expression profiles consisting of various inflammatory mediators by using antibody arrays. Figure\u00a05 shows that ARA treatment particularly increased expression of monocyte chemotactic protein (MCP)-1 and angiogenin, while EPA treatment increased IL-10, IL-6, macrophage inflammatory protein (MIP)-1\u03b4, and growth regulated protein (GRO) expression.\nFig.\u00a05Protein expression profile in the culture medium of Caco-2 cells cultured for 22\u00a0days with 130\u00a0\u03bcM arachidonic acid (ARA) plus 30\u00a0\u03bcM oleic acid (OA) or 6\u00a0\u03bcM eicosapentaenoic acid (EPA) plus 154\u00a0\u03bcM OA after 16\u00a0h cytokine stimulation (100\u00a0U\/mL IFN\u03b3 and 50\u00a0U\/mL IL-1\u03b2) measured with an antibody array. Results are represented as mean \u00b1 SD; n\u00a0=\u00a02. CSF colony stimulating factor, ENA epithelial-derived neutrophil activating protein, MCP monocyte chemotactic protein, MDC, macrophage derived chemokine, MIG monokine induced by gamma interferon, MIP macrophage inflammatory protein, SDF stromal cell-derived factor, GRO growth regulated protein, MCSF Macrophage colony stimulating factor, Tpo thrombopoietin, EGF epidermal growth factor, OSM oncostatin M, VEGF Vascular endothelial growth factor, SCF stem cell factor, IL interleukin, TNF tumor necrosis factor, Ang angiogenin\nDiscussion\nICAM-1 seems important in the pathology of IBD [15]. Whether fish oils, which have been shown to protect against relapses in IBD patients on remission [13], have effects on ICAM-1 expression, is however, unknown. In addition, a direct side-by-side comparison of fish oils with ARA, which is postulated to have pro-inflammatory effects [40] and is elevated in the colon mucosa of IBD patients [4, 7], has never been made. The transcription factor NF-\u03baB is important in regulating intestinal inflammation and is elevated in IBD patients [21, 22, 26]. We have now shown that the n-3 PUFA EPA, as compared to the n-6 PUFA ARA, clearly reduced cytokine stimulated NF-\u03baB activation and ICAM-1 expression in enterocytes in vitro. Moreover, effects of OA on ICAM-1 expression were comparable to those of EPA. Because EPA and OA resulted in comparable changes in the proportions of ARA in the phospholipids of the enterocytes, the reported effects may be ascribed to the increased ARA proportion in the ARA cultured cells. Thus, decreasing cellular ARA levels seems to be a crucial step. Since OA is already the most abundant fatty acid present in our diet [9] and in the colon mucosa [4], decreasing mucosal ARA levels may be easier by increasing fish oil intake than by increasing OA intake. Moreover, EPA does compete with ARA for incorporation into tissue phospholipids [10, 11].\nOur data showed that replacing ARA for EPA or OA decreased ICAM-1 expression and NF-\u03baB activation in Caco-2 enterocytes. In line with our observations in enterocytes, n-6 PUFAs also increased NF-\u03baB activation as compared to n-3 PUFAs in monocytes [41] and macrophages [42]. Also, earlier in vitro studies have demonstrated that fish oils reduced cytokine stimulated ICAM-1 expression in endothelial cells [43] and monocytes [44] as compared to conditions without addition of fatty acids. Moreover, in vivo ICAM-1 expression (surface and mRNA) on peritoneal macrophages was reduced in mice fed fish oils compared to that in mice fed coconut oil [45]. In humans, dietary fish oil supplementation lowered expression of ICAM-1 on ex vivo stimulated monocytes as compared to no supplementation [46]. However, our study is the first that examined effects of EPA versus ARA on ICAM-1 expression and NF-\u03baB activation in enterocytes. We however, realize that, although enterocytes play an important role in intestinal inflammation, immune modulating effects of fatty acids in CD patients will be influenced not only by enterocytes, but also the interaction with other intestinal immune and non-immune cells is important. Therefore, in future experiments effects on other cell types, e.g. isolated from mucosal biopsies of CD patients should be evaluated. Moreover, to validate if the effects of EPA versus ARA are also applicable in the pathogenesis or even the treatment of CD patients, these effects should be confirmed in appropriate animal models of IBD.\nWe used an approach of supplying different amounts of the fatty acids of interest (i.e., 130\u00a0\u03bcM ARA or 6\u00a0\u03bcM EPA), while the total molarity of fatty acids supplied was similar in all experiments by adding OA. These different concentrations of ARA and EPA were deliberately chosen because they were both four times the amount, in which the cells grow normally (i.e., the fatty acid composition of culture medium with 10% FCS). Since the EPA concentration is very low in FCS, we have deliberately chosen for a low EPA concentration. Higher concentrations of EPA would be interesting to examine, however, are difficult to be achieved with dietary interventions in vivo. Using iso-molaric total concentrations of fatty acids is essential, because an increase in total fat can be immune suppressive [47]. Therefore, we used OA as a reference fatty acid to make total fatty acid concentrations between experimental fatty acid conditions iso-molaric, i.e. OA was exchanged for ARA or EPA. In addition, we evaluated the condition of OA only. The results of this latter condition showed that decreasing ARA levels in the mucosa seems to be more important than increasing EPA levels.\nRegarding the pathways underlying the anti-inflammatory effect of fish oils, several suggestions have been made. As compared to n-6 PUFAs, n-3 PUFAs may have different effects on (I) signal transduction pathways, and (II) the types and levels of eicosanoids synthesized [40]. Regarding the first mechanism, our finding that EPA lowered NF-\u03baB activation and ICAM-1 expression as compared to ARA indeed showed that EPA and ARA differently affected the NF-\u03baB signal transduction pathway. In this respect, two peroxisome proliferators-activated receptors (PPARs), PPAR\u03b1 and PPAR\u03b3, seem relevant, since fatty acids are natural ligands for these PPARs. Both PPARs seem to have anti-inflammatory effects by inhibiting NF-\u03baB activation [48, 49]. In our in vitro model, the PPAR\u03b3 agonist troglitazone inhibited NF-\u03baB activation, and cytokine and net stimulated ICAM-1 expression, while the PPAR\u03b1 agonist GW7647 only inhibited cytokine-stimulated ICAM-1 expression. This suggests that both PPAR\u03b3 and PPAR\u03b1 have anti-inflammatory effects on enterocytes, in which effects of PPAR\u03b3 seem more pronounced. This is in agreement with the finding that PPAR\u03b3 agonists but not a PPAR\u03b1 agonist inhibited IL-8 expression of Caco-2 cells and HT-29 cells [50]. Differences between fatty acids in activation of PPARs might explain the activation of NF-\u03baB by ARA and not by EPA in our Caco-2 cells. However, our findings are not supported by the PPAR\u03b3 binding affinities of EPA and ARA, which are about the same [51], while we used much lower EPA concentrations than ARA concentrations. Secondly, not only EPA but also OA lowered intestinal ICAM-1 expression while OA is a poor PPAR\u03b3 ligand [51]. Therefore, although PPAR\u03b3 activation certainly protects against inflammation, our results do not suggest that the protective effects of EPA and OA as compared to ARA on intestinal inflammation are PPAR\u03b3-mediated. Therefore, there should be another explanation why effects of OA and EPA were comparable but different from those of ARA.\nThe mechanism underlying a second possible explanation relates to the incorporation of fatty acids into cell membrane phospholipids and eicosanoids synthesis. Eicosanoids can modulate the intensity and duration of the inflammatory response. [8, 40] Replacement of ARA by EPA in the culture medium of the cells resulted in a decrease in the proportion of ARA in cell membranes and an increase in the EPA proportion and as expected the PGE2 production of Caco-2 cells cultured with EPA was also lower than PGE2 production of cells cultured with ARA. However, PGE2 production was not different between non-stimulated and stimulated cells, while stimulation resulted in an abundant elevation of NF-\u03baB activation. To our opinion this rules out the role of PGE2 in the NF-\u03baB activation of ARA and EPA. Moreover, although in our experiments indomethacin\u2013a blocker of COX\u2013indeed inhibited PGE2 production, it did not lower NF-\u03baB activation. This is in line with findings of De Catherina et al. [43] showing that in endothelial cells the effects of DHA on VCAM-1 expression could not be inhibited by indomethacin, although prostacyclin production was completely suppressed. Therefore, we fully agree that in an in vivo situation pro-inflammatory effects of ARA are probably mediated by eicosanoids synthesized from ARA, however, these effects are not derived from a direct eicosanoid mediated activation of NF-\u03baB as shown in our model.\nFinally, we evaluated inflammatory protein signatures of Caco-2 cell culture medium after 22\u00a0days treatment with EPA versus ARA. Since these signatures were analyzed in pooled material we cannot draw conclusions based on statistical analysis. However, this array was intended to generate hypotheses about differences in immune modulating effect of ARA versus EPA based on the protein expression profiles. These profiles indicate, that both fatty acids induced specific changes in various inflammation mediators. Most of these proteins, but not all, are regulated by NF-\u03baB. For example, IL-6 protein expression was higher after EPA as compared to ARA, whereas NF-\u03baB activation was higher after ARA. It should however, be considered that although highly important, NF-\u03baB is not the only transcription factor involved in the regulation of IL-6 and other inflammatory proteins. Thus, expression of individual proteins cannot be predicted by the level of NF-\u03baB activation solely. In addition, we found clear differences between EPA and ARA, i.e., that some proteins were expressed higher after EPA (IL-6, IL-10, MIP-1\u03b4 and GRO), while others were higher expressed after ARA (MCP-1, and angiogenin). The precise role of these individual proteins in the process of intestinal inflammation is not clear. However, the increased MCP-1 expression after ARA treatment is in line with the finding that MCP-1 expression is upregulated in the mucosa of IBD patients and correlates with disease activity [52]. The increased expression of the Th2 cytokine IL-10 after EPA treatment fits with the anti-inflammatory role for IL-10 in IBD pathology. In this respect, IL-10 knockout mice develop chronic intestinal inflammation and delivery of recombinant IL-10 to the intestinal mucosa by the bacterium L. lactis attenuated mucosal inflammation in two mouse models [53]. In humans, a pilot study using these bacteria showed also promising effects [54].\nIn conclusion, we have shown that ARA but not EPA and OA activates NF-\u03baB and elevates ICAM-1 expression in Caco-2 enterocytes and we hypothesize that the effects are not related to PPAR\u03b3 activation or eicosanoid formation.","keyphrases":["caco-2 cells","intestinal inflammation","prostaglandins","peroxisome proliferator-activated receptor (ppar)"],"prmu":["P","P","P","R"]}
{"id":"Eur_J_Pediatr-3-1-2151778","title":"Hepatitis C virus infection acquired in childhood\n","text":"Hepatitis C virus (HCV) infection occurs less frequently in children than in adult patients, and the natural history, prognosis, and clinical significance of HCV infection in children are poorly defined. We report here a descriptive follow-up of the clinical course, biochemical data, and viral markers observed in 37 children with anti-HCV. Ten patients included in the study tested persistently negative for serum HCV-RNA (group 1) and 27 patients tested persistently positive (group 2). In group 1, serum alanine aminotransferase (ALT) was normal in all patients, while two patients had non-organ-specific autoantibodies. In group 2, serum ALT was elevated in 13 of 27 patients, and five patients had non-organ-specific autoantibodies. HCV genotype 1a and 1b were the most prevalent among HCV-RNA-positive patients. Twenty liver biopsies were carried out on 17 patients in our series (mean evolution time, 11.2 years; range, 3\u201321 years). The liver specimens showed mild necroinflammatory changes in most patients, and fibrosis was absent or low grade. Two HCV-RNA-positive patients became persistently HCV-RNA negative. Of the 26 children investigated, 7 (one in group 1, six in group 2) had a co-infection with hepatitis G virus. Conclusion Most children chronically infected with HCV were asymptomatic and presented only mild biochemical evidence of hepatic injury. Autoimmunity in the form of non-organ-specific autoantibodies was common. HCV in children induced mild changes in the liver with a low level of fibrosis and at a low rate of progression.\nIntroduction\nHCV (hepatitis C virus) infection occurs less frequently in children than in adult patients. The natural history, prognosis and clinical significance of HCV infection are poorly defined in childhood [11]; in contrast, HCV infection in adults presents a high degree of chronicity, with up to 50% of all HCV-infected adults developing progressive liver disease [21]. Results from prospective studies show that 20\u201330% of chronically infected adults develop compensated and eventually decompensated cirrhosis or hepatocellular carcinoma, or both, within 20\u00a0years of the initial infection [1].\nThe lower prevalence of HCV infection in children and the fact that most patients undergo antiviral drugs treatment result in very limited knowledge of the natural outcome of chronic HCV infection acquired at early ages of life. Thus, immediate goals in the investigation of HCV infection should be to characterize current epidemiology and to describe the pathogenesis and course of hepatitis C in children and adults [20]. To understand the evolution of HCV infection could be itself an important surrogate end-point for the evaluation of infected young patients with prognostic implications and therapeutical consequences.\nThe aim of this study was to determine the clinical features and long-term evolution of HCV infection in a group of children who had never received treatment with antiviral drugs.\nPatients and methods\nThirty-seven children (16 females, 21 males) with positive antibodies to hepatitis C (anti-HCV) were investigated retrospectively. These patients were followed-up for a period of 5\u00a0years. None had received treatment with antiviral drugs for viral hepatitis or had a history of intravenous drug abuse. All subjects made regular visits to our outpatient clinic, and the serum levels of alanine aminotransferase (ALT), albumin, prothrombin time, antinuclear antibodies (ANA), anti-mitochondrial antibodies (AMA), anti-smooth muscle antibodies (SMA), liver-kidney anti-microsomal antibodies type I (LKM), anti-gastric parietal cells antibodies (GPCA), rheumatoid factor, thyroxine (T4), thyroid stimulating hormone (TSH), anti-thyroid antibodies, anti-HCV and HCV-RNA were determined at least on five consecutive occasions at 1-year intervals. Hepatitis B virus (HBV) surface antigen (HBsAg), antibodies to HBV surface antigen (anti-HBs), antibodies to HBV core antigen (Anti-HBc), human immunodeficiency virus (HIV) and antibodies to hepatitis E (anti-HEV) were investigated in all patients during one visit. Hepatitis G virus-RNA (HGV-RNA) and antibodies to HGV (anti-HGV) were determined in 26 patients. Genotypes of HCV were performed in 21 viremic children.\nThe duration of infection was calculated as the interval between the presumed date of infection and the date of the last visit to the clinic. Liver biopsies were obtained in 17 patients. Repeated biopsies were performed in three patients.\nViral markers for HBV, HCV and HIV were tested by third generation ELISA (Axsym; Abbott Diagnostics, Chicago, Ill.). Anti-HEV was detected by a commercially available ELISA (Bioelisa HEV IgG; Biokit, Barcelona, Spain). HGV-RNA and anti-HEV antibodies were detected using commercial tests (Roche Diagnostics, Mannheim, Germany). HCV-RNA was detected by PCR (Amplicor HCV PCR test, Roche Diagnostics), and HCV genotyping was performed by a second generation line probe assay (INNO-LIPA HCV; Innogenetics, Ghent, Belgium).\nLiver biopsies were performed percutaneously and under the ultrasound guidance of an experienced operator. All liver biopsy specimens (>12\u00a0mm long) were fixed in formalin, embedded in paraffin stained with hematoxylin-eosin and Masson\u2019s trichrome and then observed by a single experienced pathologist (AM). Histological necroinflammatory activity and fibrosis were scored separately. The degree of histological necrosis with inflammatory activity was scored using the three parameters of the histological activity index (HAI) developed by Knodell et al. [12]; these consist of: (1) piecemeal necrosis and bridging necrosis (score: 0\u201310), (2) lobular cytolysis (score: 0\u20134) and (3) portal inflammation (score: 0\u20134). Fibrosis was scored independently using the following system: F0, absence of fibrosis; F1, fibrous expansion of portal areas; F2, portal to portal bridging fibrous tracts; F3, portal-central bridging fibrous septa; F4, cirrhosis (bridging fibrous septa with parenchymal nodules). This score is a modification of the fibrosis scoring system of Knodell\u2019s method and introduces one additional degree of fibrosis. Macrovesicular steatosis was graded semiquantitatively using a modified score from Brunt et al. [5]. Alternative and additional diagnoses identified in the liver biopsy were also recorded.\nResults\nMean chronological age for the 37 patients (21 males and 16 females) at the last follow-up evaluation was 20.1\u00a0years (range: 9\u201330\u00a0years). Most of the subjects were diagnosed among patients who were screened for HCV infection after receiving transfusions of blood products for heart surgery, hematological diseases or casual injuries before 1992. Thus, most of the patients included in the study presented with other diseases that were unrelated to HCV infection: 16 patients had a congenital heart disease, seven had various hematological disorders, three had neurological diseases, one had vesicoureteral reflux and one had Turner syndrome associated to IgA deficiency.\nTen patients were anti-HCV-positive and HCV-RNA-negative (Group 1). The presence of HCV-RNA was demonstrated in the other 27 patients (Group 2). Demographic characteristics and the main results obtained in these children are shown in Table\u00a01.\nTable 1Characteristics and biologic data of children with HCV infection\u00a0Patients negative for HCV-RNA (n\u2009=\u200910)Patients positive for HCV-RNA (n\u2009=\u200927)Age (years) at the follow-up evaluationa18 (9\u201326)20 (9\u201330)Duration of the HCV infection (years) in 36 patients at the follow-up evaluationa14 (6\u201323)16 (5\u201330)Route of infection\u00a0Parenteral1024\u00a0Vertical02\u00a0Unknown01Alanine aminotransferase (U\/l)First visitLast visit\u00a0Normal (n\u2009)10714\u00a0\u22642 \u00d7 UNL (n\u2009)b067\u00a02\u20134 \u00d7 UNL (n\u2009)055\u00a0>4 \u00d7 UNL (n\u2009)091Non-organ specific antibodies\u00a0Antinuclear antibody02\u00a0Anti-mitochondrial antibodies10\u00a0Anti-smooth muscle antibodies01\u00a0Liver-kidney anti-microsomal antibody type 111\u00a0Gastric parietal cell antibodies01HCV genotype\u00a01a8\u00a01b12\u00a02a1\u00a0Unknown6HBV00HIV10HGV-RNA\/Anti-HGV (n\u2009=\u200926)1\/04\/2Anti-HEV00aValues are given as the mean, with the range presented in parenthesisbUNL, Upper normal level\nGroup 1\nThe once-yearly PCR analysis revealed that ten patients had positive anti-HCV and negative viremia, which reflected an ongoing or past infection with HCV. These patients were asymptomatic, and the liver function tests remained normal in all but two who had been diagnosed with congestive heart failure; the liver abnormalities of these two patients were normalized after their cardiac condition had improved.\nOne female patient presented persistent positive AMA for 3\u00a0years, which then normalized and remained negative during the subsequent 9-year follow-up period. Throughout this period the patient was asymptomatic, and liver tests were normal. Two years after the laboratory tests were negative for antibodies, the patient presented with psoriasis. Another patient had persistent high titers (1:620) of anti-LKM. Fifteen years after acquiring the infection this patient lost the anti-HCV but the anti-LKM remained positive for two more\u00a0years. During the follow-up this patient did not present liver abnormalities or other diseases.\nDuring the follow-up span of this study, another female patient became persistently anti-HCV negative 12\u00a0years after she was infected. At the time of writing, eight patients remain anti-HCV-positive and HCV-RNA-negative, and they show no evidence of liver disorders.\nGroup 2\nHCV-RNA was detectable in the blood of 27 patients, two of whom became HCV-RNA-negative. The age of exposure to the infection and the type of exposure could not be established in one patient; following the detection of HCV in the blood serum, this patient was followed for 11\u00a0years and was positive for HCV up to and including the last follow-up visit.\nAll patients, with one exception, were asymptomatic. The exception had been diagnosed with leukemia in 1987 and received transfusions of blood products on several occasions. She underwent chemotherapy and had a cholestatic hepatitis; liver tests revealed persistent abnormalities. Anti-HCV and HCV-RNA were detected in 1991. The patient presented portal hypertension and progressive liver failure, and when she was 8\u00a0years old, in 1993, she received a liver transplant.\nThe evolution of serum ALT levels is shown in Table\u00a01. Serum levels of albumin and prothrombin time were abnormal only in the patient who developed hepatic failure.\nThe presence of non-organ-specific autoantibodies (NOSAs) was detected in five patients. One patient (genotype1a) diagnosed with a single ventricle and Fontan surgery developed persistent anti-LKM (titers 1:160\u20131:620) and the rheumatoid factor throughout the evolution of the disease. Liver biopsy could not be obtained in this patient. Another male patient (genotype 1b) had positive ANA for 2\u00a0years, which subsequently normalized. This patient had increased ALT, and the liver biopsy showed minimal necrosis with inflammatory changes (HAI: 2) and no fibrosis. A third patient (genotype 1b) had persistent PGCA during her follow-up period (10\u00a0years), with slightly elevated ALT levels, mild necrosis with inflammatory changes (HAI: 4) and minimal fibrosis (stage 1). A fourth patient (genotype1b) with AST levels greater than fourfold the upper normal level presented SMA during the last 2\u00a0years of her follow-up (HAI: 6). The patient diagnosed with Turner syndrome and IgA deficiency (genotype 1b) developed throughout the years a seronegative poli-articular rheumatoid arthritis, psoriasis and celiac disease. She had intermittently positive ANA.\nThe levels of T4 and TSH were normal in all patients, and no patient developed antithyroid antibodies.\nAbdominal ultrasounds did not show significant abnormalities. Only the patient who presented cirrhosis had changes associated to portal hypertension.\nLiver biopsies were performed in viremic patients who presented altered hepatic function tests and for whom parental consent had been obtained. Seventeen patients underwent 20 liver biopsies. Mean evolution time of the infection at the time of biopsy was established in 16 patients (mean: 11.2\u00a0years; range: 3\u201321\u00a0years). The results on necroinflammatory activity and fibrosis are shown in Table\u00a02.\nTable\u00a02Histopathological features of patients positive for HCV-RNALiver biopsyPatients (n)Necroinflammatory activity\u00a0\u00a01\u201326\u00a0\u00a03\u201347\u00a0\u00a05\u201364\u00a0\u00a06\u2013180Fibrosis\u00a0\u00a0F08\u00a0\u00a0F16\u00a0\u00a0F22\u00a0\u00a0F30\u00a0\u00a0F4 (Cirrhosis)1Steatosis0\nA second hepatic biopsy was performed in three patients after 5, 12 and 14\u00a0years, respectively. The minimal necroinflammatory activity observed in the first biopsy specimens of these three children remained unchanged in the second biopsy. One of the patients progressed from F0 to F1 fibrosis. A second patient never presented with fibrosis, and both samples of the third patient had minimal fibrosis (F1).\nDuring the follow-up period, 2\/27 patients became persistently HCV-RNA-negative (for 9 and 10\u00a0years, respectively). Both patients showed minimal necrosis and inflammatory activity with minimal fibrosis (F1) in the biopsy performed before HCV-RNA clearance. One patient of Group 1 was HIV-positive, while one patient of Group 1 and six patients of Group 2 were HGV-positive.\nViral coinfections observed in both groups are expressed in Table\u00a01.\nDiscussion\nIn this study, chronic hepatitis C infection was silent in most of the children, and there was little biochemical evidence of liver disease in these patients. Only one girl with portal hypertension and liver failure developed symptoms related to the infection. Consequently, our data suggest that HCV infection may be currently underdiagnosed in children and, moreover, that young patients could become a potential source of infection. We therefore strongly recommend that children falling in risk groups be screened for HCV infection.\nOf the 37 pediatric patients in this series, seven (19%) had NOSAs. These antibodies are highly prevalent in subjects exposed to the HCV, and a positive test result for SMA, GPCA and ANA is part of the natural course of chronic HCV infection in adults and children [7]. Lenzi et al. [13] reported a higher prevalence of NOSAs in anti-HCV-positive adult patients than in normal controls (25 vs. 6%). Stroffolini et al. [22] found positive NOSAs in 36.9% of 502 subjects with HCV-RNA-positive chronic hepatitis. Muratori et al. [16] detected positive NOSAs in 16 of 47 children (34%) with chronic hepatitis C, and anti-LKM was present in 11% of the children.\nOf the 37 children participating in our study who manifested past or active HCV infection, two had serum anti-LKM. This incidence is lower than that observed by Muratori et al. (11%) [16] and Bortolotti et al. (10.3%) [4] in children with HCV infection. A possible explanation for this finding is the higher age and longer mean duration of HCV infection in the patients included in our series in comparison to the children in the other two studies. The prevalence reported by Lenzi et al. (1.3% [13]), Stroffolini et al. (2.25% [22]) and Reddy et al. (0% [18]) in adults support this hypothesis. Anti-LKM is an immunomarker of type 2 autoimmune hepatitis [2] that tends to present at younger ages and to affect mainly children. Our data suggest that age may be a factor contributing to the presence of anti-LKM1 in children infected with HCV. Moreover, we observed that one of our non-viremic patients had persistent antibodies after the test for anti-HCV became negative.\nLiver specimens from the children with hepatitis C showed mild necroinflammatory changes and a low level of fibrosis. In most patients, fibrosis was absent or low grade. Only one patient, as mentioned above, developed cirrhosis and liver failure over a period of 6\u00a0years. This leukemic patient had other additional risk factors for liver damage that may have affected this outcome. In this respect, Hoshiyama et al. [9] observed that chronic hepatitis C is more frequent among children with hepatitis C infection following blood transfusions for malignant disease than in patients with hepatitis C following blood transfusions for open heart surgery.\nWe have compared our results with those obtained from other pediatric series [6, 8, 9, 23]. Table\u00a03 summarizes the data on fibrosis obtained from liver biopsies of 210 pediatric patients (above-mentioned studies). Fibrosis was not detected or was low grade in 200 patients (95%), and severe fibrosis or cirrhosis was detected in ten patients (5%). Minola et al. [15] and Poynard et al. [17] demonstrated an inverse correlation between age at HCV infection and progression to cirrhosis that suggests that chronic HCV infection in childhood induces mild changes in the liver with a low level of fibrosis and a low rate of progression. However, it has been observed that mild chronic hepatitis C can be a progressive disease in adult patients [3, 19] and that the long evolution of infection acquired in childhood could increase the risk of fibrosis. Thus, some children infected early in life, in whom chronic disease has a mild liver expression, might develop fibrosis in adulthood.\nTable\u00a03Hepatic fibrosis in biopsies from children with HCV infection\u00a0Vogt et al. [23]Hoshiyama et al. [9]Guido et al. [8]El-Raziky et al. [6]This reportNumber of patients173811226a17Mean duration of the HCV infection (year)21.2\u2009\u00b1\u20094.67.1\u2009\u00b1\u20092.8 (n\u2009=\u200923)12.9\u2009\u00b1\u20093.1 (n\u2009=\u200915)8.04\u2009\u00b1\u20095.3Non-determined11.2\u2009\u00b1\u20095,6FibrosisNo\/low grade14\/2b231525\/8120\/47\/9Severe\/cirrhosis0\/1c005\/11\/1d0\/1eaSome patients were co-infected with HBV and \/or had secondary iron overloadbTwo patients with congestive heart failurecPatient with Anti-HBs, anti-HBc and anti-HEV antibodiesdThis 12-year-old cirrhotic patient was not thalassaemic and HBV markers were negativeeThis leukemic patient was infected with HCV and was also receiving chemotherapy concurrently\nIt is noteworthy that 6\/27 of the children with HCV infection in our series had a co-infection with HGV (HGV-RNA-positive) or had a past HGV infection (anti-HGV-positive). This prevalence of HGV infection is higher than that found in healthy children (6%) in Spain [10]. These data suggest that HCV-infected children must be considered to be a risk group for HGV infection since HCV and HGV are both parenterally transmitted. Moreover, no differences were observed in biochemical and histological findings in HCV-infected children with and without a concomitant infection with HGV, which suggests that superimposed HGV infection does not influence the course of HCV infection.\nConversely, our data do not support that HEV, which can be transmitted by a parenteral route [24], is prevalent in children who had undergone blood transfusions (prevalence rate: 0%). However, this low value may result from the low prevalence of HEV infection in Spain (2.8%) [14].\nConclusion\nMost children chronically infected with HCV are asymptomatic and present only mild biochemical evidence of hepatic injury. Spontaneous clearing of the virus occurs occasionally. Autoantibodies are common in HCV patients. The natural history of chronic hepatitis C in children differs from that in adults since HCV infection is relatively benign, induces mild changes in the liver with a low level of fibrosis and a low rate of progression and is rarely associated with severe or decompensate liver disease.","keyphrases":["hepatitis c virus","anti-liver\/kidney microsomal antibody","infectious hepatitis","liver fibrosis","nonorgan-specific autoantibodies"],"prmu":["P","M","M","R","M"]}
{"id":"Clin_Oral_Investig-2-2-1544376","title":"Bone apposition to titanium implants biocoated with recombinant human bone morphogenetic protein-2 (rhBMP-2). A pilot study in dogs\n","text":"The aim of the present study was to investigate bone formation to recombinant human bone morphogenetic protein-2 (rhBMP-2)-biocoated and rhBMP-2-nonbiocoated titanium implants after implantation in dogs. Implantation of sand-blasted and acid-etched (C), chromosulfuric acid surface-enhanced (CSA), and rhBMP-2-biocoated CSA [BMP-A: noncovalently immobilized rhBMP-2 (596 ng\/cm2), BMP-B: covalently immobilized rhBMP-2 (819 ng\/cm2)] implants was performed in both the mandible and tibia of dogs. After 4 weeks of healing, the percentage of direct bone to implant contact (BIC) and the induced bone density (BD) at a distance of less than and greater than 1 mm adjacent to each implant was assessed. Histomorphometric analysis of implants inserted in the mandible and tibia revealed that BIC values appeared to be highest in the BMP-B group, followed by BMP-A, CSA, and C. BD as measured at a distance of <1 mm revealed obvious differences between groups: BMP-B>BMP-A>CSA>C. However, no differences between groups were observed at a distance of >1 mm. Within the limits of the present study, it may be concluded that rhBMP-2 immobilized by covalent and noncovalent methods on CSA-treated implant surfaces seemed to be stable and promoted direct bone apposition in a concentration-dependant manner.\nIntroduction\nThe adhesion of plasma proteins on the surface of titanium implants has been reported to play an essential role in the process of osseointegration [2, 4, 8, 9]. Each surface of a material is characterized by a unique composition of adsorbed proteins, which influences the type of cells that may adhere. Subsequently, the specific pattern of adsorbed proteins determines the type of tissue that will develop at the interface between the implanted material and the host [32, 41, 42]. In recent years, several modifications of specific surface properties such as structure, chemistry, surface charge, and wettability have been investigated to improve osseointegration of titanium implants [1]. Additionally, several growth factors and cytokines have also been suggested to stimulate a deposition of cells with the capacity of regenerating the desired tissue [27, 39, 46]. In case of endosseous titanium implants, an enhanced proliferation and differentiation of undifferentiated mesenchymal cells, osteoprogenitor cells, and preosteoblasts into osteoblasts may improve bone response and subsequently osseointegration [5]. One particular growth factor, bone morphogenetic protein (BMP), has shown considerable potential to stimulate bone formation both in extraskeletal sites [47, 48] and in defect models in different species [38, 50]. BMPs originate from the transforming growth factor-\u03b2 family, including at least 18 proteins [29]. BMP-2, which has been described as an anthelix structure, seems to possess the highest osteoinductive potential among the BMPs [25]. In recent years, the regenerative potential of recombinant human BMP-2 (rhBMP-2) has been demonstrated in various experimental animal studies, including sinus floor augmentation, alveolar ridge preservation, bone augmentation procedures, and periodontal repair [3, 11\u201315, 31, 33, 49]. Most recently, the effects of rhBMP-2 on the osseointegration of titanium implants have also been investigated in experimental animal studies [14, 26, 33\u201337, 45]. Experimental titanium plasma-sprayed hollow cylinder implants were filled with a solution of rhBMP-2 soaked on an absorbable type-I collagen sponge before insertion. The histomorphometric analysis revealed a significantly increased bone regeneration in rhBMP-2-treated defects compared to controls. Furthermore, the level of osseointegration, as measured by direct bone-implant contact, was significantly higher for the rhBMP-2 implants compared to controls [34]. Recently, biologically active rhBMP-2 has also been covalently immobilized on metal surfaces [16, 19\u201321, 44]. Osseointegration of rhBMP-2-biocoated plasma spray-coated titanium\u2013alloy cylinders, as evaluated histomorphometrically 4\u00a0weeks after implantation in the distal femur condylus in a gap healing model in sheeps, was predominantly characterized by circumferential bone formation and integration with minimal residual gaps. In contrast, control specimens generally exhibited a wide gap surrounding the implant cylinder [18]. The rationale for BMP immobilization was to avoid ectopic bone formation by a limited and targeted release of rhBMP-2 from the implant surface. Recently, treatment of titanium with chromosulfuric acid (CSA) has been reported to result in ultrahydrophilic bioadhesive surfaces, which in turn improves biocoating with rhBMP-2 [19]. However, there are currently no histological data evaluating osseointegration of CSA-modified and rhBMP-2-biocoated titanium implants.\nTherefore, the aim of the present study was to investigate histomorphometrically bone formation to CSA-enhanced and rhBMP-2-biocoated titanium implants after implantation in the mandibula and tibia of dogs in comparison to control titanium implants.\nMaterials and methods\nAnimals\nTwo 3-year-old male mongrel dogs (approximate weight 25\u00a0kg) were used in the study. Both animals exhibited a fully erupted, healthy, permanent dentition. During the experiment, the dogs were fed ad libitum with soft-food diet and water. Animal selection, management, and surgery protocol were approved by the Animal Care and Use Committee of Belgrade University (ref. no. 2179). The experimental segment of the study started after an adaptation period of 4\u00a0weeks.\nStudy design\nThe study was performed in two surgical phases. In the first phase, extraction of the mandibular second, third, and fourth premolar and first molar (P2\u2013M1) was performed bilaterally. After 4\u00a0months of healing, surgical implantation of rhBMP-2-biocoated and rhBMP-2-noncoated screw-typed implants was performed in a submerged healing procedure during the second phase. Throughout the study period, oral hygiene procedures were performed three times a week including tooth and implant brushing. Radiographs were obtained before and immediately after tooth extraction as well as immediately after implant installation. Both animals were killed after a healing period of 4\u00a0weeks.\nImplant preparation\nRhBMP-2 was prepared as previously described [21]. The biological activity of soluble rhBMP-2 was assessed with MC3T3-E1 cells by the induction of the de novo synthesis of alkaline phosphatase (AP) [43]. The half-activation constants (K0.5) were in the range of 20\u201375\u00a0nM [18]. Twenty-four screw-type implants (Camlog Screw Line, Wimsheim, Germany) were manufactured from commercially pure titanium. The core diameter of the implants was 3.3\u00a0mm and the total length was 11\u00a0mm. A total of six (n=6) implants were sand-blasted and acid-etched according to a standardized procedure (Promote, Altatec, Wimsheim, Germany) (C), while a total of 18 (n=18) implants were surface-enhanced by a novel procedure with CSA [21]. The treatment of metals with CSA (CSA\u2013Ti\u2013alloy) [21] leads to ultrahydrophilic (contact angles 0\u201310\u00b0, no hysteresis) bioadhesive surfaces [17]. A total of 12 (n=12) surface-enhanced implants were divided into two subgroups (A and B) and biocoated with rhBMP-2 [BMP-A: noncovalently immobilized rhBMP-2 (596\u00a0ng\/cm2), BMP-B: covalently immobilized rhBMP-2 (819\u00a0ng\/cm2)] [40]. RhBMP-2 was immobilized by covalent and noncovalent methods on these CSA-treated surfaces [18, 21, 40]. In brief, the implants were assigned to the following test and control groups: BMP-A (n=6), BMP-B (n=6), CSA (n=6), and C (n=6).\nTo control the produced surface, the following \u201csibling method\u201d was employed: Parallel to the preparation of the above dental implants for in vivo experiments, miniplates (10\u00d75\u00d71\u00a0mm) with identical Promote surfaces were surface-enhanced with CSA and coated with 125I-rhBMP-2 under identical conditions as the dental implants. In this way, the corresponding contact angles, the amount of immobilized rhBMP-2, and the in vitro biological activity [6] could be tested before the implants were placed into the animals. Only those dental implants were released for implantation, whose sibling miniplates reached the standard as mentioned above and whose surfaces showed an intense in vitro bioactivity by fluorescence microscopy [6].\nSurgical procedure for both phases\nThe dogs were anesthetized with 1\u00a0mg\/kg sodium pentobarbital. To maintain hydration, both animals received a constant rate infusion of lactated Ringer\u2019s solution while being anesthetized. Prophylactic antibiotics were administrated intraoperatively with a combination of 20,000\u00a0IU penicillin and 1.0\u00a0g streptomycin\/10\u00a0kg body weight. In the first surgery, P2\u2013M1 were carefully removed after reflection of full thickness mucoperiosteal flaps and tooth separation. After wound closure by means of mattress sutures, the sites were allowed to heal for 4\u00a0months. In the second surgery, the test and control implants were randomly allocated to both sides of the mandible (left and right sides, one implant each) and one implant each to either the posterior left or right tibia. In brief, bilateral vestibular incisions were made, and full thickness mucoperiosteal flaps were elevated to expose the respective sites for implant placement in the mandible. Surgical implant sites were prepared bilaterally, at a distance of 10\u00a0mm apart, according to the protocol suggested by the manufacturer. All implants in the mandible were sealed with cover screws (Camlog, Wimsheim, Germany). After irrigation, mucoperiosteal flaps were repositioned, and primary wound closure was achieved with consecutive polyglycolic acid 5.0 Polyester sutures (Resorba, N\u00fcrnberg, Germany). An area of approximately 15\u00a0cm in length and 4\u00a0cm in width was depilated on the respective side of the tibia using an electric shaver and a razor blade. After disinfection with polyvidone iodine (Betaisodona, Mundipharma, Limburg\/Lahn, Germany), a skin incision was made and a flap was elevated to expose the respective sites of the tibia for implant placement. On the inner-posterior side of the tibias, implants were inserted at a distance of 30\u00a0mm apart. All titanium implants were inserted with good primary stability according to a low-trauma surgical technique under copious irrigation with sterile 0.9% physiological saline. After implant placement, the periosteum and fascia were sutured using 3.0 Polyester sutures (Resorba, N\u00fcrnberg, Germany).\nAnimal killing and retrieval of specimens\nThe animals were killed (overdose of sodium pentobarbital, 200\u00a0mg\/kg i.v.) after 4\u00a0weeks. The jaws as well as the posterior tibias were dissected, and blocks containing the experimental specimens were obtained. Block sections of the anterior tibia served as additional control (UC). All specimens were fixed in 10% neutral buffered formalin solution for 4\u20137\u00a0days. The specimens were dehydrated using ascending grades of alcohol and xylol, and infiltrated and embedded in methylmethacrylate (MMA, Technovit 7200, Heraeus Kulzer, Wehrheim, Germany) for nondecalcified sectioning. After 18 to 24\u00a0h, the specimens were completely polymerized. Each implant site was cut in the mesiodistal direction and along with the long axis of the implant using a diamond wire saw (Exakt, Apparatebau, Norderstedt, Germany), resulting in four sections of approximately 500\u00a0\u03bcm in thickness [7]. Subsequently, all specimens were glued with acrylic cement (Technovit 7210 VLC, Heraeus Kulzer, Wehrheim, Germany) to opaque Plexiglas and ground to a final thickness of approximately 40\u00a0\u03bcm. All sections were stained with toluidine blue.\nHistological and histomorphometric analysis\nHistomorphometrical analyses as well as microscopic observations were performed by one experienced investigator masked to the specific experimental conditions. For histomorphometrical measurements, images were obtained using a light microscope (BX50, Olympus, Hamburg, Germany) at a magnification of 100\u00d7, associated with a video camera (SIS Color View3, Soft imaging System GmbH, M\u00fcnster, Germany). Digital images were evaluated using a software program (SIS analySIS Auto Software 3.2, Soft imaging System GmbH, M\u00fcnster, Germany). The percentage of direct contact between mineralized bone and the titanium surface (bone to implant contact\u2014BIC) was measured at every thread on both sides of the implant [23]. BIC was subdivided into crestal and apical values (upper and lower halves of the intraosseous implant length, respectively). Furthermore, in the tibias, induced bone density (BD) adjacent to the titanium surfaces was measured at distances of <1 and 1\u00a0mm by assessing the ratio of mineralized bone vs bone marrow within the respective areas [28].\nResults\nClinical observations\nThe postoperative healing was uneventful in both dogs. No complications such as allergic reactions, abscesses, or infections were observed throughout the study period.\nHistological and histomorphometric analysis\nBIC and BD values for each group and respective anatomical sites (mandible and tibia) are presented in Fig.\u00a01a\u2013c. In particular, all test and control implants inserted in the mandible generally exhibited new bone formation in direct contact with the implant interface. The formation of organized trabeculas of woven bone, recognized by osteon formation, could be observed in all groups. However, new bone formation in direct contact with the implant interface appeared to be higher in the BMP groups (Fig.\u00a02a\u2013d). In particular, BIC values appeared to be highest in the BMP-B group, followed by BMP-A, CSA, and C groups. No differences in BIC with respect to crestal and apical values were observed within or between groups (Fig.\u00a01a).\nFig.\u00a01Boxplots with outliners for the medians and Q1\u2013Q3 quartiles of BIC and BD (%) in different groups after 4\u00a0weeks of healing with respective values in the crestal and apical portion of the implant (BIC) and at a distance of less than and greater than 1\u00a0mm adjacent to the implant surface (BD). Lines below and above box plots min, max. control c. a BIC mandibula. b BIC tibia. c BD tibiaFig.\u00a02Histology of representative titanium implants after 4\u00a0weeks of healing in the mandible (original magnification \u00d740). BIC appeared to be highest for BMP-B, followed by BMP-A, CSA, and C. a C. b CSA. c BMP-A. d BMP-B\nIn comparison to the organized trabeculas of woven bone noted in the mandible, bone formation in the tibia seemed to be of a cancellous type, mainly characterized by tiny trabeculas (Fig.\u00a03a\u2013d). BIC values appeared to be highest in the BMP-B group, followed by BMP-A, CSA, and C groups. Again, with respect to crestal and apical BIC values, no differences were observed within groups (Fig.\u00a01b). Histomorphometric analysis of BD in the tibia at a distance of <1\u00a0mm revealed obvious differences between groups. In particular, highest values were noted for both BMP groups (BMP-A=BMP-B). This was followed by CSA, also exhibiting higher BD values than C. No differences between groups were found at a distance of >1\u00a0mm (Fig.\u00a02c).\nFig.\u00a03Histology of representative titanium implants after 4\u00a0weeks of healing in the tibia (original magnification \u00d710). BIC and BD appeared to be highest for BMP-B, followed by BMP-A, CSA, and C. a C. b CSA. c BMP-A. d BMP-B\nDiscussion\nThe present histological study was designed to evaluate bone formation and direct bone apposition to rhBMP-2-biocoated, CSA surface-enhanced, and C titanium implants after implantation in the mandible and tibia of dogs. In particular, rhBMP-2 was immobilized by covalent and noncovalent methods on CSA surface-enhanced titanium implants [18, 21, 40]. Within its limits, histomorphometrical analysis of implants inserted in both mandible and tibia after 4\u00a0weeks revealed that BIC values appeared to be highest in the BMP groups, followed by CSA and C groups. Furthermore, it was observed that BD as measured at a distance of <1\u00a0mm in the tibia was obviously highest in the BMP-B group, followed by BMP-A, CSA, and C groups. However, no differences between groups were observed at a distance of >1\u00a0mm. In this context, it is important to realize that the present pilot study does not have the statistical power to rule out the possibility of a difference between groups. Further experimental studies of higher power are needed to support equivalence or superiority [10]. On the other hand, it needs also to be pointed out that these are the first histological data evaluating bone formation and apposition on rhBMP-2-biocoated and CSA surface-enhanced titanium implants in the mandible and tibia. However, the present findings corroborate, to a certain extent, previous results observed in an ectopic bone formation model [40]. Electropolished titanium miniplates were surface-enhanced by CSA and coated with a total amount of 150\u2013200\u00a0ng rhBMP-2. Periosteal flaps were prepared from the anterior surface of the tibias of adult rabbits and wrapped around the titanium specimens. Additionally, some titanium miniplates were inserted to which nonimmobilized soluble rhBMP-2 was added. After 28\u00a0days of healing, noncoated specimens revealed bone formation in 2\/12 implants, rhBMP-2-coated implants in 6\/8, and implants with free rhBMP-2 in 8\/8 cases. However, in the case of rhBMP-2-coated implants, the induced bone had direct contact to the implant in all cases. In contrast, titanium miniplates inserted with free administered rhBMP-2 revealed direct BIC in just six cases, whereas in two cases, the titanium surface was separated by a fibrous capsule [40]. The finding that rhBMP-2 may promote periimplant bone regeneration and osseointegration of titanium implants is in accordance with the previous studies [14, 26, 33\u201337, 45]. In all of these studies, however, rhBMP-2 was admixed with a carrier (i.e., collagen, calcium-phosphate cement carrier), acting as a slow delivery system, for instillation with the implant due to a rapid diffusion of BMP after implantation in vivo. In particular, Sigurdsson et al. [33] evaluated rhBMP-2- (2\u00d70.43\u00a0mg\/ml in a type-I bovine collagen carrier) induced bone regeneration and osseointegration in a supraalveolar periimplant defect model in dogs. At 16\u00a0weeks after healing, bone regeneration (height) was significantly larger for rhBMP-2 than control defects. However, the large amount of BMP-induced bone was poorly adapted to the implant surface. In contrast, the smaller amount of new bone in the control group seemed to be well adapted [33]. In contrast, Wikesj\u00f6 et al. [45] reported similar BIC values 16\u00a0weeks after implantation of rhBMP-2 (0.4 and 0.75\u00a0mg\/ml in a calcium-phosphate cement carrier) or carrier alone subsequent to a vertical alveolar ridge augmentation procedure and simultaneous implant installation in dogs. Furthermore, Howell et al. [14] applied rhBMP-2 using a collagen sponge carrier to stimulate bone formation in artificially created defects around endosseous implants in the canine mandible. Nonresorbable expanded tetrafluoroethylene (e-PTFE) membranes served as controls. Histological analysis revealed that the addition of rhBMP-2 resulted in a significantly greater amount of new bone area and BIC after 4 and 12\u00a0weeks of healing than e-PTFE. Although membrane-treated sites were reported to have less new bone formation after 4\u00a0weeks of healing, this difference seemed to be equalized after 12\u00a0weeks [14]. Similar results were also reported by Sykaras et al. [34]. RhBMP-2-induced bone regeneration and osseointegration was evaluated in mandibular bony defects created within the hollow chamber of endosseous dental implants in dogs. Before insertion, hollow chambers were filled with 20\u00a0\u03bcg of rhBMP-2 soaked on an absorbable type-I collagen sponge (0.4\u00a0mg\/ml). Histological observation revealed statistically significant higher BIC values in the rhBMP-2 group at 8 and 12\u00a0weeks after implantation [34]. There might be several explanations for the discrepancies noted in these studies. First of all, it must be emphasized that little information is available on the interaction between rhBMP-2 and the individual carriers. Furthermore, the high concentrations of rhBMP-2 used in these studies (in the milligram per milliliter range) strongly indicate that an optimal method is still lacking. In this context, it is important to point out that BMP-2 has the potency to induce or modulate apoptosis [24], and that in vivo application of high doses of BMP-2 may inhibit bone formation [22, 30]. Furthermore, the results of a recent cell culture study have shown that the dose-dependent effect of rhBMP-2 on AP induction in MC3T3-E1 cells plateaus out into a maximal response at 300\u20131,000\u00a0nM BMP-2 (i.e., 8\u201325\u00a0\u03bcg\/ml) [43]. Indeed, the results of the present study have shown that BIC and BD values seemed to be ameliorated after application of rhBMP-2 at far lower concentrations. Furthermore, it must also be noted that the effects of rhBMP-2 were limited to a range of 1\u00a0mm, outlining that both covalent and noncovalent methods of immobilization seemed to be stable. This finding may also be supported by the observation that no differences with respect to BIC values were observed in crestal and apical areas of the implant surface in all groups. In accordance, it might be hypothesized that from a clinical point of view, both methods of immobilization are suitable to avoid ectopic bone formation due to a limited and targeted release of rhBMP-2 from the implant surface. However, further studies are needed to clarify this issue.\nWithin the limits of the present study, it may be concluded that rhBMP-2 immobilized by covalent and noncovalent methods on CSA-treated implant surfaces seemed to be stable and promoted direct bone apposition in a concentration-dependent manner.","keyphrases":["titanium implant","rhbmp-2","animal study","bioactive surface","ultrahydrophilic surface"],"prmu":["P","P","P","R","R"]}
{"id":"Skeletal_Radiol-4-1-2271083","title":"Use of FDG-PET in differentiating benign from malignant compression fractures\n","text":"Objective The objective was to evaluate the use of fluorodeoxyglucose positron emission tomography (FDG-PET) in differentiating benign from malignant compression fractures.\nIntroduction\nDifferentiating between malignant and benign vertebral compression fractures can represent a diagnostic challenge and is particularly difficult in elderly patients, who frequently have a history of malignancy and are also predisposed to benign compression fractures from osteoporosis or treatment-related changes [1\u20133]. Differentiating between benign and malignant compression fractures has important therapeutic and prognostic implications.\nComputed tomography (CT) and magnetic resonance imaging (MRI) are routinely used in the evaluation of compression fractures; however, these imaging modalities do not always permit definite diagnosis [2\u20135]. In contrast to MRI or CT, fluorodeoxyglucose positron emission tomography (FDG-PET) can yield metabolic information that is based on increased glucose metabolism of malignant and inflammatory lesions. Tumor cells typically accumulate FDG, while traumatic fractures are not expected to significantly accumulate FDG. Therefore, FDG-PET may allow differentiation between malignant and benign compression fractures [6\u20139].\nThe purpose of our study was to evaluate the use of FDG-PET in differentiating benign from malignant compression fractures.\nMaterials and methods\nThis study was approved by the institutional review board of our institution, which waived the need for informed consent. The study was compliant with the Health Insurance Portability and Accountability Act. A retrospective search was performed using Boolean operators (Folio; Open Market, Proto, UT, USA) to identify all patients who had undergone whole-body FDG-PET at our institution from 2003 to 2006 and had the finding of a compression fracture mentioned in the PET report. Imaging studies and reports, medical records, and pathology reports of selected cases were reviewed.\nPatients\nWe identified 33 patients with compression fractures who underwent whole-body FDG-PET. There were 23 women and 10 men, aged 48\u201393\u00a0years, with a mean age of 72\u00a0years. Twenty-nine patients had a history of malignancy (1 had leukemia, 5 lung cancer, 1 ovarian cancer, 2 breast cancer , 5 colon cancer,  9 lymphoma, 1 sarcoma, 1 pancreatic cancer, 1 Klatskin tumor, 1 gastrointestinal stromal tumor,  1 laryngeal cancer, and 1 had esophageal cancer). The 4 patients without history of malignancy underwent whole body PET for work-up of indeterminate lung nodules detected on prior CT or radiographs. Sixteen patients underwent FDG-PET and 17 patients underwent FDG-PET\/CT at time of evaluation. Nine patients underwent CT and 14 patients underwent MRI of the spine within 4\u00a0weeks of FDG-PET. Seventeen patients were followed up with serial FDG-PET studies. Nine patients underwent biopsy and the pathologic results were used as a standard of reference. Twenty-four patients who did not undergo biopsy were followed up clinically and with repeat imaging, with MRI, CT, or FDG-PET for a period of 1\u20133\u00a0years and findings at clinical follow-up and imaging studies were used for lesion verification. In these patients, the development of new lesions and\/or progression of existing lesions on imaging were used as criteria for malignant compression fractures.\nImage acquisition\nWhole-body PET was performed using an ECAT HR+ scanner (CTI Molecular Imaging, Knoxville, TN, USA). All patients fasted for at least 6\u00a0h prior to image acquisition, and blood glucose levels were measured prior to the injection of FDG. A dose of 15\u201320\u00a0mCi (555\u2013740\u00a0MBq) of FDG was administered intravenously 45\u00a0min to 1\u00a0h prior to scanning.\nPatients were positioned supine on the scanner and emission images were acquired in 6\u20137 bed positions from the mandible to the mid-thigh or to the level of the ankles in the case of lower extremity lesions. Transmission images obtained with a rotating germanium 68-rod source were used for attenuation correction. Images were reconstructed using the ordered-subset expectation maximization (OSEM) algorithm.\nCombined PET\/CT studies were performed with a 16-section hybrid PET\/CT gantry (Biograph Sensation 16; Siemens, Erlangen, Germany), which comprises a 16-section high-performance multi-detector row CT scanner with a lutetium oxyorthosilicate-based PET scanner. The PET image spatial resolution was 5.0\u00a0mm full width at half maximum, with a section thickness of 3.5\u00a0mm. Patients fasted for at least 6\u00a0h prior to image acquisition, and blood glucose levels were measured prior to the injection of FDG. Two 10-oz cups of water were administered as negative contrast material 1\u00a0h prior to scanning. A dose of 15\u201320\u00a0mCi (555\u2013740\u00a0MBq) of FDG was administered intravenously 45\u00a0min to 1\u00a0h prior to scanning. Patients were positioned supine on the scanner and emission images were acquired in 6\u20137 bed positions from the mandible to mid-thigh or to the level of the ankles in the case of lower extremity lesions. Images were reconstructed with Fourier rebinning and attenuation-weighted ordered-subsets expectation maximization. A low-dose CT scan was performed prior to PET imaging primarily for attenuation correction, with patients holding their breath mid-expiration, and included an area from the mandible to the mid-thigh or to the level of the ankles in the case of lower extremity lesions. Slice thickness was 5\u00a0mm. A diagnostic contrast-enhanced CT was performed subsequent to the PET\/CT following the administration of 100\u00a0mL intravenous contrast material (Isovue 300; Bracco Diagnostics, Princeton, NJ, USA) at an injection rate of 2\u00a0mL\/s using 2.5-mm sections.\nImage analysis\nSemiquantitative and qualitative evaluation of PET images was performed on a high-resolution workstation (Reveal-MVS; Mirada Solutions, Oxford, UK) by one investigator, who was blinded to the clinical and pathological results (MAB). The images were displayed in rotating maximum intensity projections and in axial, coronal, and sagittal planes. Semiquantitative analysis of FDG uptake was performed by creating a region of interest over the area of maximal radiotracer activity.\nMaximum standardized uptake values (SUVs) were automatically generated according to the following equation: SUVmax (bw)\u2009=\u2009Ctis\/Dinj\/bw, where SUVmax (bw) is maximum SUV normalized for body weight; Ctis, tissue concentration expressed as megabecquerels per milliliter; Dinj, injected dose expressed as megabecquerels; and bw, body weight expressed as kilograms. Lesions with SUV greater than 3.0 were considered malignant and less then 3.0 were considered benign. However, lesions with SUV between 2 and 3 are often indeterminate and do not always allow definite diagnosis, and also the pattern of uptake has to be considered. Qualitative assessment was made with the specific aim of establishing whether the lesion was benign or malignant. The radiotracer uptake by the lesion was compared with the liver, and those lesions with uptake greater than the liver were classified as malignant.\nStatistical analysis\nThe recorded data were analyzed using JMP statistical database software (SAS Institute, Cary, NC, USA). The findings on the PET images as well as SUV of benign and malignant compression fractures were correlated with the final diagnosis of the lesion and determined either malignant or benign. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were calculated. The Student\u2019s t test was used to determine whether there was a statistically significant difference between the SUV for benign and malignant compression fractures. A difference with p\u2009<\u20090.05 was considered to be statistically significant.\nResults\nForty-three compression fractures were identified in 33 patients. Twenty-two fractures involved the thoracic and 21 fractures the lumbar spine. Nine patients underwent biopsy and 24 patients were followed up clinically and with repeat imaging. In the 9 patients who underwent biopsy, there were 3 benign and 6 malignant compression fractures. Of the 24 patients who were followed clinically and with repeat imaging, 6 patients were thought to have malignant and 18 patients were thought to have benign compression fractures. Overall, there were 21 patients with benign and 12 patients with malignant compression fractures. Out of the 43 compression fractures, there were 29 benign and 14 malignant fractures.\nBased on clinical history (acute onset of back pain often after minor trauma) and imaging characteristics on MRI (bone marrow edema) there were 5 acute benign compression fractures. Three patients were on bone marrow-stimulating therapy at the time of FDG-PET. Five patients were unable to undergo MRI because of pacemakers (3) or severe pain.\nQualitative PET analysis\nForty-three compression fractures were identified in 33 patients. Visual inspection of suspected lesions on FDG-PET characterized 26 compression fractures as benign and 17 fractures as malignant. The malignant lesions demonstrated intense radiotracer uptake (Figs.\u00a01, 2), while benign compression fractures showed only mildly increased or no increased uptake on FDG-PET (Figs.\u00a03, 4). Histological and clinical follow-up data were analyzed. By FDG-PET, 12 lesions were correctly classified as malignant (true-positive) and 24 lesions were correctly classified as benign (true-negative). Two malignant tumors were incorrectly classified as benign (false-negative) and 5 benign tumors were incorrectly classified as malignant (false-positive). In the 2 false-negative cases there was moderately increased uptake of the compression fractures, which was thought to represent acute benign fractures prospectively, but was found to be metastatic disease on subsequent biopsy (Fig.\u00a05). SUV in these patients were 2.5 to 2.8 respectively, and the primaries in these cases were esophageal and lung cancer.\nFig.\u00a01a Malignant compression fracture in a patient with lymphoma. Sagittal FDG-PET image demonstrates intense radiotracer uptake in the L1 vertebral body (SUV\u2009=\u20097.1; arrow). b Axial FDG-PET image demonstrates tumor infiltration of the L1 vertebral body with associated extra-osseous component (arrows). c Sagittal T1-weighted MRI demonstrates diffuse low signal intensity of the bone marrow of the lumbar spine with compression fracture of L1 (arrow). d Sagittal fat-suppressed T1-weighted MRI following the administration of gadolinium demonstrates diffuse enhancement of the L1 compression fracture (arrow). e Axial image from a CT-guided biopsy demonstrates large extra-osseous soft tissue component (white arrow) and infiltration of the L1 vertebral body (black arrows)Fig.\u00a02a Malignant compression fracture from metastatic colon cancer. Sagittal fused FDG-PET\/CT image demonstrates intense radiotracer uptake in the T7 compression fracture (SUV\u2009=\u20096.3; arrow). b Axial fused FDG-PET\/CT image demonstrates focal increased radiotracer uptake in the T7 vertebral body (arrow). c Sagittal T1-weighted MRI demonstrates compression fracture of T7 with low signal marrow infiltration (arrow). d Sagittal STIR image demonstrates mild hyperintensity of the T7 vertebral compression fracture (arrow). e Sagittal fat-suppressed T1-weighted MRI following the administration of gadolinium demonstrates diffuse enhancement of the T7 compression fracture (arrow). Malignancy was indeterminate on MRIFig.\u00a03Benign compression fracture in a patient with lymphoma. Sagittal fused FDG-PET\/CT image demonstrates compression fracture of T9 (arrow) without significant radiotracer uptake (SUV\u2009=\u20090.7)Fig.\u00a04a Benign subacute compression fractures in a patient without history of malignancy. Sagittal FDG-PET image demonstrates mildly increased radiotracer uptake in the upper thoracic spine (SUV\u2009=\u20091.8; arrow). b Axial FDG-PET image at the level of T4 demonstrates mildly increased radiotracer uptake (arrow), consistent with subacute compression fracture. c Sagittal CT image demonstrates compression fracture of T4 (arrow) and multiple chronic osteoporotic compression fractures throughout the thoracic spineFig.\u00a05a False-negative compression fracture on FDG-PET in a patient with metastatic esophageal cancer. Sagittal fused FDG-PET\/CT image demonstrates mild to moderately increased radiotracer uptake in the L2 compression fracture (arrow; SUV\u2009=\u20092.5) that was thought to represent a benign compression fracture but was found to be malignant on subsequent biopsy. Note the mildly increased radiotracer uptake in the T11 compression fracture (arrowhead), which was thought to represent a benign fracture. b Axial fused FDG-PET\/CT image demonstrates increased radiotracer uptake in the L2 vertebral body (arrow). c Sagittal CT image demonstrates L2 and T11 compression fractures (arrows), for which malignancy was indeterminate\nThree of the false-positive patients were on bone marrow-stimulating agents (Fig.\u00a06). One of the false-positive patients had an acute compression fracture (Fig.\u00a07). Except for that patient, there was no significant difference in uptake pattern of benign acute and benign chronic compression fractures (Fig.\u00a08).\nFig.\u00a06a False-positive compression fracture in a patient on bone marrow-stimulating agents. Sagittal FDG-PET image demonstrates diffuse increased radiotracer activity throughout the spine. Note the compression fractures with focal kyphotic angulation at L1\u2013L2 (arrow). The patient was thought to have diffuse metastatic disease with malignant compression fracture. b Sagittal FDG-PET image performed 2\u00a0months later demonstrates resolution of diffuse radiotracer uptake. Kyphotic angulation from compression fractures remains (arrow). The patient was off bone marrow-stimulating agents for 6\u00a0weeks at the time of the studyFig.\u00a07a False-positive compression fracture in a patient with laryngeal cancer. Sagittal fused FDG-PET\/CT image demonstrates increased radiotracer uptake in the T12 compression fracture (SUV\u2009=\u20094.9; arrow). b Sagittal T1-weighted MRI demonstrates compression fracture of T12 with linear low signal of the superior endplate and preservation of the normal marrow signal (arrow). Note compression fracture of L1 with relative preservation of normal marrow signal (arrowhead). c Sagittal STIR image demonstrates hyperintensity of the superior endplates of the T12 and L1 compression fractures, suggesting acute fractures (arrows). d Sagittal fat-suppressed T1-weighted MRI following the administration of gadolinium demonstrates enhancement of the T12 compression fracture (arrow). Mild enhancement is noted, involving the L1 compression fracture (arrowhead). Findings were thought to be benign on MRI and on follow-up imaging, there was resolution of bone marrow edema and enhancementFig.\u00a08a Acute and chronic benign compression fractures in a patient without history of malignancy. Sagittal fused FDG-PET\/CT image demonstrates multiple compression fractures throughout the thoracic and lumbar spine with mild increased radiotracer uptake at T11 (SUV\u2009=\u20092.1; arrow). b Sagittal T1-weighted MRI demonstrates multiple chronic compression fractures (arrowheads). Mild decreased signal intensity of the T11 compression fracture suggests edema (arrow). c Sagittal STIR image demonstrates mild edema in the T11 compression fracture suggestive of an acute fracture (arrow). There is no evidence of marrow edema in the remaining compression fractures\nQuantitative PET analysis\nStandardized uptake values were measured in all 43 compression fractures. SUV for benign compression fractures ranged from 0.7 to 4.9 with a mean of 1.94\u2009\u00b1\u20090.97 standard deviation (SD) on FDG-PET. Malignant compression fractures showed SUV from 2.2 to 7.1 with a mean of 3.99\u2009\u00b1\u20091.52\u00a0SD on FDG-PET. The difference between the SUV values of benign and malignant compression fractures was statistically significant (p\u2009<\u20090.001, Student\u2019s t test; Fig.\u00a09).\nFig.\u00a09Mean standardized uptake value (SUV) of benign and malignant compression fractures. The top of the boxes represent the mean and error bars represent the range of SUV. There is a statistically significant difference between the SUV values of benign and those of malignant compression fractures (p\u2009<\u20090.001, Student\u2019s t test)\nStatistical analysis\nSensitivity, specificity, positive and negative predictive values, and accuracy of FDG-PET in differentiating benign from malignant compression fractures were 86%, 83%, 84%, 71%, and 92% respectively.\nDiscussion\nThe correct diagnosis of benign and malignant compression fractures can be problematic, but has important prognostic and therapeutic implications. MRI, CT, or bone scintigraphy are commonly used in the diagnostic work-up of patients with compression fractures. However, in some cases, these imaging techniques do not permit definite diagnosis of the cause of the compression fracture. Diffusion-weighted MRI has been used successfully to differentiate benign from malignant fractures [10\u201313], but benign and malignant compression fractures can show significant overlap on quantitative assessment with apparent diffusion coefficient maps [12]. Also, some patients are unable to undergo MRI due to pacemakers, claustrophobia, or severe pain. This is especially true in the elderly population. Five of our 33 patients were unable to undergo MRI.\nIn this context, metabolic imaging modalities such as FDG-PET might be used as a management problem solver. FDG-PET has been successfully used to differentiate benign from malignant neoplasms and in the evaluation of metastatic disease [14\u201316]. Preliminary studies and case reports have shown that FDG-PET might be helpful in differentiating benign from malignant compression fractures [8, 9]. Since elderly patients often have a history of malignancy and are also predisposed to benign compression fractures due to osteoporosis, we thought that FDG-PET might be used as a diagnostic problem solver in this patient population in cases of equivocal MRI or CT findings or if the patient was unable to undergo MRI.\nIn our study, malignant compression fractures demonstrated significantly increased FDG uptake compared with benign fractures. Mean SUV of malignant and benign fractures were 3.99\u2009\u00b1\u20091.52\u00a0SD for malignant lesions and 1.94\u2009\u00b1\u20090.97\u00a0SD for benign lesions. There were 2 false-negative results. In these cases there was moderately increased uptake of the compression fractures, which were thought to be acute benign fractures prospectively, but were found to represent metastatic disease on subsequent biopsy. SUV in these patients were 2.5 to 2.8 respectively.\nThere were 5 false-positive results; 3 of those patients were on bone marrow-stimulating agents, which mimicked tumor involvement. This effect on bone marrow FDG uptake has been described in the literature [17]. One to 2\u00a0months after cessation of bone marrow-stimulating therapy, FDG uptake returned to normal in our patients. The false-positive results occurred because the investigator was blinded to the clinical history. This is less likely to occur in a clinical setting where this history should be actively sought when interpreting positive findings, especially in cases of diffuse osseous uptake, mimicking a diffuse marrow infiltrative process as was seen in our cases. In fact, when we reviewed the original reports, these cases were reported to be consistent with the known use of bone marrow-stimulating agents.\nOnly mildly increased or no increased uptake was seen in chronic benign fractures and mild to moderately increased uptake was seen in acute benign fractures, which was less than in malignant fractures. This is in contrast to bone scintigraphy, in which increased uptake persists for many months [18].\nPositron emission tomography and PET\/CT were equally sensitive in differentiating benign from malignant compression fractures. However, the CT portion of the PET\/CT improved the exact fracture localization and was able to provide additional information on fracture morphology, which can be helpful in diagnosing benign vs. malignant fractures.\nOur study had several limitations. The first is the retrospective nature of the study. Second is the lack of histologic correlation in all cases. Only 9 patients underwent biopsy of the spine. However, we obtained clinical follow-up and serial imaging studies, including serial FDG-PET for a period of 1\u20133\u00a0years to evaluate for benign or malignant fractures. Also, there were no patients with osteomyelitis\/discitis, which can mimic malignancy [8, 19, 20]. Another limitation is the lack of inter-observer variability, since only one observer interpreted the images. However, we compared our results with the reports of the original interpreter, who had access to all data. On the original reports, the 3 cases of patients on bone marrow-stimulating agents were interpreted as being consistent with the known use of bone marrow-stimulating agents. Malignancy was said to be indeterminate in the two false-negative reports. However, in our review, we categorized each fracture as either benign or malignant without the option of an indeterminate lesion.\nIn summary, FDG-PET is a useful method of differentiating between benign and malignant compression fractures and can serve as a problem solver in cases of equivocal MRI or CT findings, and in patients who are unable to undergo MRI. We do not recommend FDG-PET as a screening test, but rather as an additional imaging modality in problem cases, particularly in elderly patients with osteoporosis and a history of malignancy. In these patients, FDG-PET has the additional advantage of being able to evaluate the entire skeletal system and screen for metastatic disease.","keyphrases":["fdg-pet","benign","malignant","compression fractures","spine"],"prmu":["P","P","P","P","P"]}
{"id":"Int_J_Legal_Med-3-1-2039830","title":"Variables affecting the probability of complete fusion of the medial clavicular epiphysis\n","text":"In this study, we have combined data on clavicle fusion from different studies and applied a binomial logistic regression analysis. As such, we aimed to assess whether or not variables such as sex, socioeconomic status, and ethnicity influence the probability of having mature, i.e., completely fused clavicles at a given age. We further explored whether the method of clavicle examination, i.e., diagnosis from either a dry bone specimen, an examination of X-rays, or an examination of computed tomography scans, affects the probability of being diagnosed with mature clavicles. It appeared that only ethnicity did not significantly affect this probability. Finally, we illustrated how the logit model may be used to predict the probability of being diagnosed with mature clavicles.\nIntroduction\nThe medial (or sternal) clavicular epiphysis matures relatively slowly. In the human skeleton, it is generally the last long bone epiphysis to fuse. Its developmental stage is therefore useful when estimating age at death in the post-pubertal period [1] or the age of living persons involved in criminal proceedings [13, 15]. In the Netherlands, a radiological clavicle examination may be applied to verify the age of young asylum seekers without valid identification documents.\nVarious authors have studied clavicle development to determine the age interval in which fusion of the clavicular epiphyses occurs. Most of the published studies were based on the examination of dry bone specimens [3, 6, 9, 10, 17, 18]. When dealing with living individuals, examiners analyzed either X-rays [4, 13] or computed tomography (CT) scans [5, 15, 16]. In a number of studies, the results for different ethnic groups were evaluated or the results for males were compared with those for females. In most studies, such comparisons were, however, hampered by a relatively small sample size.\nIn this study, we aim to assess from a large sample whether or not variables such as sex, socioeconomic status, and ethnicity affect the probability of having completely fused clavicles at a certain age. We further aim to compare results from X-ray and CT scan examinations with those from studies based on dry bone specimens. To do so, we have combined data from different studies and performed a binomial logistic regression analysis. We then illustrate how the resulting model can be applied to estimate the probability of completely fused clavicles at a given age.\nData\nPublished and unpublished data on the individual subjects, taken from various studies on clavicle development [3\u20136, 8, 10, 13, 15, 18] were combined. For each individual, information on the calendar age in years was combined with data on the fusion status of the clavicle, i.e., exhibiting a complete bony fusion of epiphysis and diaphysis (i.e., mature) or not. When data for both left and right clavicles were published, we used the side for which most records were provided. Data on the sex and ethnicity of the individuals were copied when available. In the context of this study, we defined ethnicity as being either of European (Caucasian), Asian, or Afro-American descent. It was further recorded which method was used for determining the fusion status, i.e., using dry bone specimens, X-rays, or CT scans.\nThe socioeconomic status of individuals was estimated from the human development index (HDI) associated with the country in which the study took place. The HDI is a summary composite index that measures a country\u2019s average achievements in three basic aspects of human development: longevity, knowledge, and the standard of living [19]. The HDI is used by the United Nations Development Programme, and data are available from 1975 onward. To accommodate data from two American studies that took place before this year [6, 18], we extrapolated the HDI by first calculating the linear regression of the HDI on time.\nIn total, data of 3,552 individuals aged 15\u201333\u00a0years were combined. Figure\u00a01 shows the age distribution within the sample. Table\u00a01 provides further information on the number of individuals per category.\nFig.\u00a01Age distribution of individuals in the sampleTable\u00a01Number of individuals per categoryCategoryValuesTotal number of individuals3,552SexMales2,133Females1,123Unspecified296EthnicityaEuropean descentb1,316Afro-American descent287Asian descent695Unspecified1,254MethodDry bone specimen1,374X-ray1,326CT scan852aA small number of individuals in McKern and Stewart [6] were of Afro-American descent. As no individual data were provided, all 374 individuals are classified \u201cof European descent\u201d.b\u201cOf European descent\u201d includes Caucasian and Latino individuals from North America.\nStatistical analysis\nAnalysis of the data was performed using the SPSS (version 10.0) software package for statistical analysis. We carried out a binomial logistic regression analysis to assess the effect of various variables on the probability of mature clavicles. In logistic regression, the dependent variable is transformed into a logit variable, i.e., the natural log of the odds of the dependent occurring or not. This transformation ensures that the estimated probabilities are between 0 and 1. A logit model is a form of the generalized linear model.\nAge, sex, ethnicity, and socioeconomic status were considered as potential predictor variables for the probability of mature clavicles. The method of clavicle examination was further considered as a factor influencing the probability of being diagnosed with mature clavicles. Age and socioeconomic status (i.e., HDI) were analyzed as covariates. Sex, ethnicity, and the method of clavicle examination were analyzed as fixed factors. Possible interaction between these fixed factors and the covariate age was also investigated. The initial (exploratory) logit model may be described by the following equation:\nwhere p denotes the probability of the dependent, \u03b1 the intercept, \u03b2 the estimated effect of a covariate or factor-by-covariate interaction term on the logit of the probability, and \u03b3 the estimated effect of a fixed factor.\nVariables to be kept in the model were selected by means of backward elimination using the Wald test. Insignificant interaction terms were successively dropped. After each alteration to the exploratory model, possible significance of the remaining variables was reassessed. A p value lower than 0.05 was considered to be statistically significant.\nNot all studies that were used as a source for our data provided individual data on sex or ethnicity. Consequently, the described model (M1) could be tested on the data of a subgroup of 2,002 individuals only. No data from CT scans were available for this subgroup. To be able to include data from all available studies, we therefore also tested simplified models. For the first of these simplified models (M2), we omitted the variable ethnicity and the associated interaction term from the initial model. This model could be fitted to the data of a subgroup of 3,256 individuals. For this subgroup, data resulting from the evaluation of CT scans were drawn from a single study [15], as the second available study [5] did not provide details on the sex of individuals. As a next step, we therefore further omitted the variable sex and the associated interaction term from the initial model. The resulting model (M3) could be fitted to the data of all 3,552 individuals. As a final step, we assessed the significance of the change in \u22122 log likelihood between the resulting model and a nested model including the main terms only. We further compare the Nagelkerke pseudo R2. As such, we compare the difference in model fit.\nResults\nTable\u00a02 provides the results for the three different models. Model M1 resulted in a p value higher than 0.05 for ethnicity after all non-significant interaction terms had been removed. Tested for a subgroup of 2,002 individuals, ethnicity therefore did not appear to affect the probability of having mature clavicles. Including this variable in the model does not improve the model fit. Exploring model M1 therefore effectively resulted in model M2, which could be fitted to the data of 3,256 individuals.\nTable\u00a02Estimated parameters of logistic regression models for the logit of the probability of mature claviclesModelIndependent variablesBStandard errorp valueM1 N\u2009=\u20092,002Ethnicitya0.355M2 N\u2009=\u20093,256Age0.6290.0340.000Sex (ref: male)0.026\u00a0Female0.3680.166Human development index2.7460.5900.000Method (ref: dry bone specimen)0.000\u00a0X-ray\u221221.0463.055\u00a0CT scan\u22127.6242.346Method \u00d7 age0.000\u00a0X-ray1.0290.135\u00a0CT scan0.4230.104Constant\u221218.6381.0020.000M3 N\u2009=\u20093552Age0.6300.0340.000Human development index3.0120.5840.000Method (ref: dry bone specimen)0.000\u00a0X-ray\u221220.7293.060\u00a0CT scan\u22127.5261.928Method \u00d7 age0.000\u00a0X-ray1.0230.135\u00a0CT scan0.4010.083Constant\u221218.8030.9980.000aThe remaining parameters of model 1 (M1) are omitted from this section in the table, as final results for a model without the variable ethnicity are provided for model 2 (M2).\nFrom model M2, it appeared that age, the HDI (i.e., socioeconomic status), sex, and the method of clavicle examination all significantly affected the probability of having completely fused clavicles. Exploring possible interaction between sex and age did not indicate a significant effect. The interaction term method \u00d7 age, however, yielded a p value of 0.000. The subsequent assessment of the significance of the difference in \u22122 log likelihood between the resulting model and a nested model omitting the interaction term yielded a p value of 0.000. The Nagelkerke pseudo R2 reduced from 0.815 to 0.795 after omission of the interaction term. All indicates a better model fit when the interaction term is included.\nModel M3, which could be fitted to all available data on clavicle fusion, resulted in comparable model parameters for the dummy variable for CT scans. Confounding by an observer effect is therefore reduced.\nApplication of the model\nThe most appropriate model to predict the probability of being diagnosed with mature clavicles, i.e., providing the best model fit to the currently available data, appeared to be model M2. The resulting model\u2014i.e., after the removal of non-significant main and interaction terms and using the parameters for covariates and the intercept\u2014may be described by:\nParameters for the appropriate dummies for the categorical variables may be found in Table\u00a02. From this equation, it follows that the predicted probability may be calculated using the following equation:\nBased on the currently available data on clavicle fusion, we can now predict the probability of having mature clavicles for different individuals. Here, we provide a few examples.\nThe predicted probability of mature clavicles is 0.016 (1.6%) for a contemporary 19-year-old German male (HDI is 0.932). For a 20- or 21-year-old German male, this probability increases to 0.029 (2.9%) and 0.054 (5.4%), respectively. For females, the predicted probability values are increased to 0.023 (2.3%), 0.042 (4.2%), and 0.076 (7.6%), respectively. However, if the individuals have a lower socioeconomic status, these values are decreased. For contemporary 19-, 20-, and 21-year-old males from India (HDI is 0.611), for instance, they are 0.007 (0.7%), 0.012 (1.2%), and 0.023 (2.3%), respectively. When diagnosed from X-ray or CT scan, the values increase. For the 21-year-old Indian male, it would be 0.040 (4%) if diagnosed from an X-ray and 0.076 (7.6%) if diagnosed from a CT scan (composed using a slice thickness that is far from optimal).\nIn Figs.\u00a02 and 3, we show the differences in predicted probabilities at different ages between the various categories. In Fig.\u00a02, we compare the predicted probability values for a German male with that for a German female and an Indian male. Figure\u00a03 illustrates the difference in predicted probability for a German male depending on whether dry bone specimens, X-rays, or CT scans are examined.\nFig.\u00a02Predicted probability of having completely fused clavicles: a comparison between German males, German females and Indian malesFig.\u00a03Predicted probability of being diagnosed with mature clavicles: a comparison between examination by means of dry bone specimens, X-rays and CT scans\nDiscussion\nEthnicity did not appear to significantly influence the probability of having completely fused clavicles, which is in line with the results of Schmeling et al. [12]. We should, however, keep in mind that the current database is not well balanced. In addition, a small number of individuals taken from McKern and Stewart [6] were misclassified in our analysis (see footnote Table\u00a01). Additional data from future studies may contribute to the knowledge on the possible effect of ethnicity, if not strengthen our conclusion that there is no significant effect.\nThe effect of sex on the probability of having completely fused clavicles appeared significant. The estimated model parameter for the dummy variable for females being greater than zero indicates a positive effect on the logit of the probability. The predicted probability of mature clavicles is therefore greater for females than for males of similar ages. This would point to a generally earlier onset of fusion in females. A similar disparity has been reported for epiphysial closure of all other long bones [11]. There was no indication of significant interaction between sex and age. We may therefore assume similarly shaped probability curves for males and females, indicating an equal number of years during which complete fusion may generally be achieved.\nA higher HDI, and therefore a higher socioeconomic status, appears to increase the probability of having mature clavicles. This would mean that under less preferable socioeconomic conditions, the process of fusion is generally delayed. This is in concordance with conclusions drawn by Schmeling et al. [12, 14], and was summarized before by Eveleth and Tanner [2].\nThe method of clavicle examination appeared to also be significant. The significant interaction term method \u00d7 age indicates that we should assume a difference in the estimated effect of age depending on the method applied. For the age interval in which mature clavicles have been observed, the predicted probability of being diagnosed with mature clavicles is greater when X-rays or CT scans are used instead of dry bone specimens. It is noteworthy that the difference in the predicted probability is initially very small, but becomes increasingly greater with increasing age during a limited age interval. When keeping in mind that the curve for the predicted probability will be s-shaped, one may image that the difference is again only minimal for the older individuals.\nA number of theories can be postulated to explain a difference in the predicted probabilities between methods of examination.\nInappropriate slice thickness Regarding the results from CT scans, one could postulate that the slice thickness affected the diagnosis. M\u00fchler et al. [7] concluded that reconstructions composed with a slice thickness of 1\u00a0mm should be used. The vast majority of the analyzed data were, however, obtained from reconstructions with a greater slice thickness (up to 8\u00a0mm) [5, 15]. One can foresee that an almost completely fused clavicle may be diagnosed as being mature when details are lost due to the greater slice thickness. Schulze et al. [16]\u2014from whom no data were included in our analysis, as no individual data were provided\u2014observed two 19-year-olds with completely fused clavicles. Both observations were done from reconstructions with a slice thickness of 7\u00a0mm (Schulze, personal communication). We therefore think there is a realistic possibility of these cases having been misdiagnosed.\nPersistent small grooves or notches between diaphysis and epiphysis When comparing the results from their study based on CT scans with two studies based on dry bone specimens [6, 9], Kreitner et al. [5] hypothesized that persistent small grooves or notches seen in dry bone specimens may be classified as partial fusions of the epiphysis with the clavicular shaft. This would reduce the probability of dry bone specimens being classified as completely fused.\nReduced visibility of the epiphyseal disc on X-rays X-rays used in the published studies have not been made with the purpose of examining the clavicles. Possibly, this has hindered a correct classification of the clavicle at times.\nInterobserver variability As the sample included results from two studies based on X-rays and two studies based on CT scans, we hold it unlikely that what we perceive to be an effect of the applied method is in fact an observer effect.\nExpectation bias In some X-rays, the (nearly fused) epiphyseal disc will have been more difficult to judge than in others. In such cases, knowing the actual age of the individuals could have affected the interpretation of the observation. Clavicles of relatively young individuals would then more often be classified as \u201cnot completely fused\u201d, while those of relatively old individuals would be classified as \u201ccompletely fused\u201d more often. We know, however, that in at least two out of four studies, one based on X-rays [13] and one on CT scans [15], the examiner was not aware of the age of the subjects when classifying the images. We therefore do not believe that expectation bias could have played a role of any great importance.\nStatistical artifact In theory, it may be possible that due to an unfortunate coincidence, there were no relatively late maturing individuals examined by X-ray or CT scan.In theory, all of these aspects could have contributed to the significant effect of the variable method. However, if significance of the interaction between method and age is not explained as a statistical artifact, only expectation bias would have resulted in a significant change in the shape of the probability curve. In case of misclassifications of nearly fused clavicles or differences in the classification system among studies, we would have expected a higher number of individuals with completely fused clavicles in the relatively young subjects, even below the age of 20.If we, however, were to conclude that significance of the interaction term is in fact a statistical artifact, the term should be removed from the model, and model parameters should be recalculated. It is our ambition to investigate the matter further by comparing high quality CT scans, expected to allow a correct diagnosis, with X-rays taken from the same person.\nConclusions\nThe probability of having mature clavicles at a certain age is affected by the sex and socioeconomic status of the individual. The probability of being diagnosed with mature clavicles further appears affected by the method of clavicle examination. Predicted probabilities from the model may be used to illustrate acceptance or rejection of individual age claims.\nPlease note that the odds on having mature clavicles given a certain age should not be confused with the (posterior) odds of having reached a certain age given that the clavicles have matured. These probabilities are only equal if we assume a prior odds of 1.","keyphrases":["clavicle","age estimation","medial epiphysis","epiphyseal fusion"],"prmu":["P","R","R","R"]}
{"id":"Arch_Dermatol_Res-2-2-1705535","title":"The incidence of arthropathy adverse events in efalizumab-treated patients is low and similar to placebo and does not increase with long-term treatment: pooled analysis of data from Phase III clinical trials of efalizumab\n","text":"A large-scale, pooled analysis of safety data from five Phase III clinical trials (including open-label extensions of two of these studies) and two Phase III open-label clinical trials of efalizumab was conducted to explore whether arthropathy adverse events (AEs) were associated with efalizumab treatment in patients with moderate-to-severe chronic plaque psoriasis. Data from patients who received subcutaneous injections of efalizumab or placebo were stratified for analysis into phases according to the nature and duration of treatment. These included: the \u2018first treatment\u2019 phase (0\u201312-week data from patients who received either efalizumab, 1 mg\/kg once weekly, or placebo in the five placebo-controlled studies); the \u2018extended treatment\u2019 phase (13\u201324-week data from seven trials for all efalizumab-treated patients); and the \u2018long-term treatment\u2019 phase (data from efalizumab-treated patients who received treatment for up to 36 months in two long-term trials). Descriptive statistics were performed and the incidence of arthropathy AEs per patient-year was calculated using 95% confidence intervals (CIs). During the first treatment phase, a similar proportion of patients had an arthropathy AE in the efalizumab group (3.3%; 58\/1740 patients) compared with the placebo group (3.5%; 34\/979 patients); the incidence of arthropathy AEs per patient-year was 0.15 in the efalizumab group (95% CI 0.11\u20130.19) and 0.16 in the placebo group (95% CI 0.11\u20130.22). Analysis of first treatment phase data from one study (n = 793) showed that the incidence of psoriatic arthropathy per patient-year was lower in efalizumab-treated patients (0.10; 95% CI 0.05\u20130.18) than in those given placebo (0.17; 95% CI 0.08\u20130.30). During the extended treatment phase, the incidence of arthropathy remained low (0.17; 95% CI 0.14\u20130.22). Data from two long-term studies showed that there was no increase in the incidence of arthropathy AEs over time in patients treated with efalizumab for up to 36 months. Patients who had an arthropathy AE during treatment with efalizumab appeared to be more likely to have a history of arthropathy prior to treatment. Efalizumab does not appear to increase the risk of arthropathy AEs compared with placebo.\nIntroduction\nPsoriasis is a chronic, immune-mediated, inflammatory skin disorder that is currently incurable. Consequently, the majority of people with psoriasis require long-term treatment to maintain disease control. Traditional immunosuppressive systemic treatments, such as acitretin, methotrexate, cyclosporine, hydroxyurea, and thioguanine, may be effective in controlling psoriasis in some patients but significant toxicity and the need to closely monitor patients limit the viability of these treatments for long-term, continuous use [23]. Recently developed systemic therapies that selectively target specific pathways in the inflammatory cascade of psoriasis generally have a much improved safety profile compared with traditional therapies [26].\nEfalizumab (anti-CD11a; Raptiva\u00ae) is a recombinant humanized monoclonal IgG1 antibody that has been approved for the treatment of moderate-to-severe chronic plaque psoriasis. It interferes with the pathogenesis of psoriasis via multiple mechanisms, including inhibition of T-lymphocyte trafficking and T-lymphocyte activation and reactivation [1, 10, 11, 21, 25]. The safety and efficacy profile of efalizumab has been established in numerous clinical trials, in which more than 3,500 patients were enrolled and treatment was assessed for up to 3\u00a0years [4\u20136, 12\u201317, 22].\nAlthough psoriasis can be associated with the co-morbidity of psoriatic arthritis, a minority of patients with psoriasis (7\u201330%) will develop this joint disease [27]. Nevertheless, psoriatic arthritis constitutes a major consideration in patients who are receiving long-term treatment for their psoriasis. A Nordic study of more than 5,000 patients with psoriasis showed that patients with arthritis exhibited greater impairment of psoriasis-related quality of life (QoL), longer disease duration, and greater self-reported disease severity, compared with patients who had psoriasis but no co-morbid arthritis [27].\nA low incidence of arthropathy adverse events (AEs; any form of joint disease) associated with efalizumab treatment has been reported in both clinical studies and routine clinical practice [8, 12]. However, anecdotal reports of arthropathy in routine clinical practice have expressed concern that efalizumab may be associated with exacerbation of arthropathy [8]. To address this concern, we conducted a large-scale pooled analysis of safety data from five Phase III clinical trials (including open-label extensions of two of these studies) and two Phase III open-label clinical trials of efalizumab to explore whether arthropathy AEs were associated with efalizumab treatment in patients with psoriasis.\nMethods\nThe primary objective of this pooled safety analysis was to assess the incidence of arthropathy AEs in patients who had received either efalizumab or placebo. Safety data were pooled from five randomized, double-blind, placebo-controlled clinical trials (including data from two open-label extension studies of two of these trials) and two open-label clinical trials of efalizumab [4\u20136, 12\u201317, 22]. Patients included in these Phase III studies were aged \u226518\u00a0years and had moderate-to-severe chronic plaque psoriasis, a psoriasis area and severity index (PASI) score of \u226512 at screening, and plaque psoriasis covering \u226510% of body surface area. All patients were candidates for either systemic anti-psoriatic therapy or had received systemic anti-psoriatic therapy. Patients included in these trials received subcutaneous injections with efalizumab, 1\u20134\u00a0mg\/kg once weekly or 2\u00a0mg\/kg once-every-other week, or placebo. Details of individual study methodologies are described in other publications [4\u20136, 12\u201317, 22].\nArthropathy AEs were defined according to the Coding Symbols for Thesaurus of Adverse Reaction Terms (COSTART) [3] preferred terms \u2018arthritis\u2019 and \u2018arthrosis\u2019, or the Medical Dictionary for Regulatory Activities (MedDRA, http:\/\/www.meddramsso.com\/NewWeb2003\/index.htm) preferred terms \u2018arthritis not otherwise specified (NOS)\u2019, \u2018psoriatic arthropathy\u2019, \u2018arthropathy NOS\u2019, \u2018monoarthritis\u2019, \u2018polyarthritis\u2019, and \u2018osteoarthritis NOS\u2019.\nTreatment groups analyzed\nDue to the variety of study designs, five analyses were considered: \u2018first-treatment phase\u2019, \u2018first exposure phase\u2019, \u2018extended treatment phase\u2019, \u2018re-treatment phase\u2019, and \u2018long-term treatment\u2019 (see Table\u00a01).\nTable\u00a01Summary of the Phase III data from five placebo-controlled clinical trials (including data from two open-label extension studies of two of these trials) and two open-label clinical trials of efalizumab included in the pooled safety analysisPublication (protocol number)Study designNumber of patients in each analysisFirst treatment (0\u201312\u00a0weeks)Efalizumab sc 1\u20134\u00a0mg\/kg qw or 2\u00a0mg\/kg qowFirst exposure \u00a0Extended treatment (13\u201324\u00a0weeks)Long-term treatmenta (\u226436\u00a0months)Re-treatmentPlaceboEfalizumab 1\u00a0mg\/kgEfalizumab 2\u00a0mg\/kgLeonardi [13] (ACD2058g)Randomized, double-blind, parallel-group, placebo-controlled170162166462123\u201355Lebwohl [12] (ACD2059g)Randomized, double-blind, parallel-group, placebo-controlled 122232243579289\u2013\u2013Gordon [4] (ACD2390g)Randomized, double-blind, parallel-group, placebo-controlled 187368\u2013368\u2013\u2013\u2013Papp [17] (ACD2600g)Randomized, double-blind, parallel-group, placebo-controlled 236449\u2013449\u2013449\u2013Sterry [22] (IMP24011)Randomized, double-blind, parallel-group, placebo-controlled264529\u2013772308\u2013145Papp [16] (ACD2062g)Open-label\u2013\u2013\u201334137\u2013365Gottlieb [5, 6] (ACD2243g)Open-label\u2013\u2013\u2013339b290339\u2013Menter [14] (ACD2391g)Open-label extensionc of study ACD2390g [4]\u2013\u2013\u2013174342\u2013\u2013Menter [15] (ACD2601g)Open-label extensionc of study ACD2600\u00a0g [17]\u2013\u2013\u2013217622635d\u2013Pooled analysis 97917404093,3942,111n.a.e565qow once-every-other week, qw once weekly, sc subcutaneousaNumber of patients at startbPatients received combined therapy with fluocinolone acetate (n\u00a0=\u00a0169) or petrolatum (n\u00a0=\u00a0170) for weeks 9\u201312; for months 3\u201315, the dose of efalizumab could be escalated to 4\u00a0mg\/kg per week for up to 4\u00a0weeks if clinically indicatedcSome patients are included in the analyses more than once because patients in the open-label extension studies are also included in analyses of the parent studiesdIncluded patients who received either efalizumab or placebo in the parent study [17]eNot applicable because data are analyzed and reported separately for the study by Gottlieb et al. [6] and the study published by Papp [17] and Menter [15]\nIt is worth noting that most of the studies included in this pooled analysis were designed and conducted before efalizumab had received regulatory approval and before it was known that doses of more than 1\u00a0mg\/kg once weekly (the approved dose) did not confer additional treatment benefit (EMEA, Raptiva Summary of Product Characteristics; FDA US, FDA Prescribing Information for Raptiva). For this reason, only the efalizumab 1\u00a0mg\/kg once-weekly dose data are reported for the \u2018first treatment phase\u2019 of the analysis. Due to the wide variety of study designs included in the pooled analysis, data for patients receiving any dose of efalizumab are combined for all other treatment phases analyzed.\nThe \u2018first treatment phase\u2019 analysis included 0\u201312-week data from patients in the five placebo-controlled studies who received either efalizumab 1\u00a0mg\/kg once weekly or placebo. This analysis allows a comparison between the efalizumab and placebo treatment groups.\nThe \u2018first exposure phase\u2019 included 12-week data from all studies in patients who had their first exposure to any dose of efalizumab and, thus, did not include placebo data. This analysis was conducted to include the maximum number of patients who received efalizumab for their first 12\u00a0weeks of treatment (i.e., it included those patients who first received efalizumab treatment after crossing over from a placebo group, as well as the patients who first received efalizumab during weeks 0\u201312).\nThe \u2018extended treatment phase\u2019 analysis included 13\u201324-week data in patients given any dose of efalizumab who had already received efalizumab during the first treatment phase.\nThe \u2018long-term treatment phase\u2019 analysis included all patients who received continuous long-term treatment (up to 36\u00a0months) with any dose of efalizumab. Data were analyzed in 12-week segments to assess change in the incidence of arthropathy AEs over time. This analysis included data from two long-term studies [6, 15, 17], which were analyzed separately due to differences in study design.\nThe \u2018re-treatment phase\u2019 analysis included all patients who re-started treatment with efalizumab following a treatment-free observation period.\nStatistical analyses\nDescriptive statistics were used to explore the association between efalizumab and the occurrence of arthropathy AEs. Results are expressed as point-estimates of the incidence rates (ratio of the number of patients with an arthropathy AE to the total number of patient-years at risk of an arthropathy AE) with their 95% confidence intervals (CIs). Descriptive comparisons are provided; no formal statistical tests were performed.\nAnalyses were also conducted to explore the relationship between onset of arthropathy AEs during efalizumab treatment and a previous history of arthropathy (reported as a narrative by patients at the baseline visit) and the incidence of arthropathy AEs and clinical response to efalizumab treatment [measured at 12\u00a0weeks using the Physician Global Assessment (PGA) and PASI scales]. Differences in patient and psoriasis characteristics at baseline were also compared between patients who had arthropathy AEs and those who did not. An additional analysis of data from patients included in the first treatment phase of the study by Sterry et al. [22] (Table\u00a01) was conducted to assess the incidence of psoriatic arthropathy in these patients. This was the only study to define arthropathy AEs according to MedDRA; other studies used the COSTART, which did not include \u2018psoriatic arthropathy\u2019 specifically as a preferred term.\nBaseline demographics and psoriasis characteristics and the proportion of patients with a previous history of arthropathy (as reported by patients at the baseline visit) were tabulated by presence\/absence of an arthropathy event.\nResults\nThe number of patients included in each of the pooled safety analyses from each of the seven trials and two open-label extensions is summarized in Table\u00a01. Up to 3,394 patients received at least one dose of efalizumab. A total of 2,719 patients were included in the first treatment phase analysis, of whom the majority (64%; 1,740 patients) received efalizumab 1\u00a0mg\/kg per week; 979 patients (36%) received placebo. Efalizumab 2\u00a0mg\/kg per week regimen was given to 409 patients (15%) in two of the five studies [12, 13]; consequently, these patients were not included in the first treatment analysis. Patient demographics and baseline psoriasis characteristics were similar between treatment groups in the first treatment phase (Table\u00a02).\nTable\u00a02Baseline demographic and disease characteristics for patients in the placebo-controlled first treatment phaseCharacteristicsPlacebo (n\u00a0=\u00a0979)Efalizumab 1\u00a0mg\/kg per week (n\u00a0=\u00a01,740)Efalizumab 2\u00a0mg\/kg per week (n\u00a0=\u00a0409)Mean age (years), mean (SD)45 (12)45 (12)45 (13)Weight (kg), mean (SD)90.0 (20.0) 89.4 (19.6)93.6 (20.5)Mean BMIa (kg\/m2)30.4 (6.4)30.2 (6.3)31.4 (6.6)Race, n (%)\u00a0Caucasian891 (91)1,569 (90)356 (87)\u00a0Other88 (9)171 (10)53 (13)Duration of psoriasis, mean number of years (SD)19.2 (11.4)19.1 (11.4)17.6 (11.7)History of arthritis, n (%)286 (29.2)529 (30.4)141 (34.5)BMI body mass indexaDue to missing height data, BMI was calculated for 971 patients in the placebo group, 1,719 patients in the efalizumab 1\u00a0mg\/\/kg per week group and 404 patients in the efalizumab 2\u00a0mg\/kg per week group\nFirst treatment phase (weeks 0\u201312)\nDuring the first 12\u00a0weeks of treatment a similar proportion of patients had an arthropathy AE in the efalizumab 1\u00a0mg\/kg group (3.3%) and the placebo group (3.5%; Fig.\u00a01a). Correspondingly, the incidence of arthropathy AEs per patient-year was 0.15 in the efalizumab 1\u00a0mg\/kg group (95% CI 0.11\u20130.19) and 0.16 in the placebo group (95% CI 0.11\u20130.22; Fig.\u00a01b). The majority of the arthropathy AEs was mild-to-moderate in intensity in both the efalizumab (41\/58 events; 71%; 95% CI 57\u201382%) and placebo groups (31\/34 events; 91%; 95% CI 76\u201398%).\nFig.\u00a01a Proportion of patients who had arthropathy adverse events (AEs) during each phase of the safety analysis and b incidence of arthropathy AEs per patient-year for each phase\nThe additional analysis of data from the study by Sterry et al. [22] demonstrated that the incidence of psoriatic arthropathy per patient-year was lower in the group treated with efalizumab 1\u00a0mg\/kg per week (0.10; 95% CI 0.05\u20130.18) than in the placebo group (0.17; 95% CI 0.08\u20130.30); the proportion of patients with psoriatic arthropathy was 2.3% (12\/529 patients) in the efalizumab group and 3.8% (10\/264 patients) in the placebo group.\nFirst exposure phase\nIn total, 3,394 efalizumab-treated patients were included in this analysis. A small proportion of patients had an arthropathy AE (3.6%; Fig.\u00a01a) and the incidence of arthropathy AEs per patient-year was also low (0.16; 95% CI 0.14\u20130.19; Fig.\u00a01b).\nThe incidence of arthropathy AEs in this group of patients was similar to that in the placebo group in the first treatment phase, as indicated by the overlap in CIs.\nExtended treatment phase (weeks 13\u201324)\nIn total, 2,111 patients were included in the extended treatment phase analysis. During this phase, a low proportion of patients had an arthropathy AE (3.8%; Fig.\u00a01a) and the incidence of arthropathy AEs per patient-year was also low (0.17; 95% CI 0.14\u20130.22; Fig.\u00a01b). Overlap in the CIs indicates that the incidence of arthropathy AEs in this group of patients was also similar to that in the placebo group in the first treatment phase.\nLong-term treatment phase\nThe results of two long-term studies were analyzed separately to assess the incidence of arthropathy AEs in patients treated with efalizumab. In both of these studies (Fig.\u00a02), there was no overall increase in the incidence of arthropathy AEs over time. Furthermore, the incidence of arthropathy remained similar to that of the placebo group in the first treatment phase and stable between 12-week periods.\nFig.\u00a02Incidence of arthropathy AEs in long-term studies of patients treated with efalizumab a for up to 36\u00a0months and compared indirectly with pooled placebo data from the first treatment (FT) phase [5, 6] and b for up to 15\u00a0months and compared with the study\u2019s placebo group during month 0\u201312 [15, 17]. *Following the first 3-month double-blind, placebo-controlled phase of this study, patients in the placebo group who continued were switched to open-label treatment with efalizumab. Consequently, the month 6, 9, 12 and 15 results included patients who had received placebo during the initial 3\u00a0months of the study\nIn total, 339 patients were included in the analysis of the study by Gottlieb et al. [6]. These patients received continuous treatment with efalizumab 2\u00a0mg\/kg once weekly for weeks 1\u201312 (fluocinolone acetate or petrolatum was co-administered during weeks 9\u201312), followed by continuous maintenance treatment with efalizumab 1\u00a0mg\/kg once weekly for up to 36\u00a0months in patients who had a\u00a0\u2265\u00a050% improvement in PASI score. For months 3\u201315, the dose of efalizumab could be escalated to 4\u00a0mg\/kg per week for up to 4\u00a0weeks if clinically indicated, then maintained at 2\u00a0mg\/kg per week. During the entire study period, there was little variation in the incidence of arthropathy AEs (range 0.06\u20130.19; Fig.\u00a02a). Reasons for discontinuation were diverse and were representative of the overall population; refer to Gottlieb et al. [6, 7] for details of discontinuations.\nFor the other long-term study, the analysis included 3-month data from 449 efalizumab-treated patients in the placebo-controlled first treatment phase of the study [17] and data from 635 patients who entered the open-label extension phase and received efalizumab treatment [15]; 218 of the 635 patients included in the open-label extension had switched from placebo to efalizumab after completing the first treatment phase. Patients who entered the open-label extension phase continued to receive, or initiated treatment with, efalizumab 1\u00a0mg\/kg once weekly for up to 15\u00a0months continuously. As in the long-term study by Gottlieb et al. [6], there was little variation in the incidence of arthropathy AEs during the entire study period (range 0.06\u20130.12; Fig.\u00a02b).\nRe-treatment phase \nIn total, 565 efalizumab-treated patients were included in the re-treatment phase of the analysis. In this phase, a lower proportion of patients had an arthropathy AE (2.7%; Fig.\u00a01a) compared with the first treatment phase, and the incidence of arthropathy AEs per patient-year was also lower (0.12; 95% CI 0.07\u20130.19; Fig.\u00a01b). The incidence of arthropathy AEs in this group of patients was lower than in the placebo group in the first treatment phase.\nBaseline characteristics and previous history of arthropathy\nThere were no differences in baseline demographics or disease characteristics between the patients who had arthropathy AEs and those who did not. Patients who experienced an arthropathy AE during treatment with efalizumab appeared to be more likely to have a history of arthropathy prior to treatment. Of the patients who never developed an arthropathy AE during efalizumab treatment, 27% reported a previous history of arthropathy compared with 59% in patients who did have an arthropathy AE.\nDuring the first treatment phase, 88% (n\u00a0=\u00a034) and 76% (n\u00a0=\u00a079) of patients who developed an arthropathy AE had a history of arthropathy prior to receiving placebo or efalizumab 1\u00a0mg\/kg once weekly, respectively.\nArthropathy AEs and clinical response to efalizumab\nArthropathy AEs appeared to be less likely to occur in patients who had a good clinical response to treatment (\u226575% improvement in PASI score; 2.3% of patients had events) than in patients who had a partial response (50\u201374% improvement in PASI score; 3.5% of patients had events; Fig.\u00a03) and non-responders (<50% improvement in PASI score; 4.5% of patients had events). The corresponding incidences of arthropathy AEs per patient-year were 0.10 in patients with a good clinical response (95% CI 0.05\u20130.18), 0.17 in patients with a partial clinical response (95% CI 0.11\u20130.25), and 0.21 in patients who did not respond (95% CI 0.15\u20130.28).\nFig.\u00a03a Proportion of patients with an arthropathy AE by response category on the psoriasis area and severity index (PASI) and physician global assessment (PGA) scales and b incidence of arthropathy AEs per patient-year by response category on the PASI and PGA scales\nWhen assessed using the PGA scale, arthropathy AEs also appeared to be less likely to occur in patients who had better clinical responses to treatment with efalizumab (Fig.\u00a03). During the extended treatment phase, the incidence of arthropathy AEs per patient-year was 0.17 in patients with responses categorized as \u2018cleared\u2019, \u2018excellent\u2019 or \u2018good\u2019 on the PGA scale (95% CI 0.10\u20130.25) and 0.25 in patients with responses categorized as \u2018fair\u2019, \u2018slight\u2019, \u2018unchanged\u2019 or \u2018worse\u2019 on the PGA scale (95% CI 0.17\u20130.35).\nDiscussion\nThe placebo-controlled results of this large-scale pooled analysis of arthropathy data from seven clinical trials show that efalizumab does not appear to increase the risk of developing arthropathy AEs compared with placebo during the first 12\u00a0weeks of treatment. In addition, for patients treated with efalizumab, the incidence of arthropathy AEs did not appear to increase over time. The proportion of patients who had an arthropathy AE within any 12-week treatment period was low (<4.1%) through all treatment phases (first treatment, first exposure, extended treatment, re-treatment, long-term treatment).\nJoint disease has also been reported as a side-effect of other approved biological treatments for psoriasis, namely infliximab (EMEA public statement on infliximab, http:\/\/www.emea.eu.int\/pdfs\/human\/press\/pus\/444500en.pdf) [2, 18, 19], alefacept (Biogen safety presentation on Alefacept to the FDA, http:\/\/www.fda.gov\/ohrms\/dockets\/ac\/02\/slides\/3865S1_04_Biogen-Safety\/sld007.htm) [20, 24], and etanercept (EMEA Scientific discussion for the approval of Enbrel, http:\/\/www.emea.eu.int\/humandocs\/PDFs\/EPAR\/Enbrel\/014600en6.pdf). Indeed, placebo-controlled studies of infliximab and alefacept indicate that in patients with moderate-to-severe psoriasis the incidence of arthralgia (joint pain) is 7 and 5%, respectively (Biogen safety presentation on Alefacept to the FDA, http:\/\/www.fda.gov\/ohrms\/dockets\/ac\/02\/slides\/3865S1_04_Biogen-Safety\/sld007.htm) [18, 24]. Psoriatic arthritis has been reported as serious treatment-related AE in three placebo-controlled studies of etanercept in the treatment of chronic plaque psoriasis (incidence data have not been published) (EMEA Scientific discussion for the approval of Enbrel, http:\/\/www.emea.eu.int\/humandocs\/PDFs\/EPAR\/Enbrel\/014600en6.pdf). The incidence of arthropathy AEs in the current analysis of efalizumab appears to be similar to that for arthralgia in studies of infliximab and alefacept. Moreover, the term \u2018arthropathy\u2019, used in the current study encompasses a variety of joint diseases, not just a single joint condition such as arthralgia or psoriatic arthritis, and therefore has greater potential to include more patients. However, this between-study comparison is indirect and thus should be treated with caution. Moreover, no arthropathy event (defined by any of the MedDRA or COSTART preferred terms) was excluded from the analysis. Also, data from first treatment phase of the study by Sterry et al. [22] indicate that the proportion of patients with psoriatic arthropathy specifically was low (2.3%) in patients treated with efalizumab 1\u00a0mg\/kg per week\u2014in fact, lower than in the placebo group (3.8%). It should be noted, however, that psoriatic arthropathy events were not confirmed by a rheumatologist\u2014this is a potential limitation of the study. However, the umbrella term \u2018arthropathy\u2019 was designed to capture all joint diseases, including \u2018psoriatic arthropathy\u2019. Also, the incidence of psoriatic arthropathy in the study by Sterry et al. was in line with the incidence of \u2018arthropathy\u2019 in the overall pooled analysis.\nTo put the results of this pooled analysis, which by its very nature included select patient populations (determined by the inclusion\/exclusion criteria and study designs), in the context of routine clinical practice, post-marketing surveillance data were assessed. During post-marketing surveillance of efalizumab, which accounts for approximately 17,500 patient-years to date, serious arthropathies requiring hospitalization were reported with a frequency of about 4.8 per 1,000 patient-years in patients receiving efalizumab. It should be noted, however, that underreporting of AEs in routine clinical practice setting may lead to an underestimate of the true incidence of arthropathy.\nFor both the 12-week first treatment and first exposure phases of the current analyses, the proportions of patients reporting an arthropathy AE appeared to be lower in the efalizumab groups than in the placebo group in the first treatment phase. Correspondingly, the incidences of AEs per patient-year in these treatment phases were also lower in the efalizumab groups than that observed in the placebo group in the first treatment phase. However, the proportion of AEs that were moderate or severe was greater in the efalizumab groups than in the placebo groups; too few patients had events to draw any meaningful conclusions.\nDuring the extension phase (weeks 13\u201324), the incidence of arthropathy AEs in efalizumab-treated patients remained similar to the placebo group in the first treatment phase. Previous history of arthropathy and poor clinical response may potentially indicate a risk for occurrence of new arthropathy AEs during treatment. Indeed, arthropathy AEs were most frequent in patients who did not respond to therapy with efalizumab or in patients with a history of arthropathy.\nImportantly, the data from the two long-term studies of efalizumab indicate that the incidence of arthropathy AEs remains stable and low for up to 3\u00a0years of continuous treatment. These results, coupled with efficacy data showing that the clinical improvements of the skin after 3\u00a0months of efalizumab therapy are maintained throughout 36\u00a0months of continuous dosing [5], support the suitability of efalizumab for the chronic, continuous treatment of patients with psoriasis. Reasons for patients\u2019 discontinuations in the 36-month study by Gottlieb et al. [6, 7] were diverse and representative of the overall population included in this analysis and have been described previously. When considering the long-term analysis of the 36-month study (Fig.\u00a02a), it should be noted that the number of patients who remained in the study decreased over time. This discontinuation rate is not unexpected for a study that is 3\u00a0years in duration but, by month 36, there is a small number of patients on which to base comparisons with the first treatment phase. Another factor that may confound between-phase analysis comparisons was the possible use of concomitant medications for psoriasis after the first treatment phase in the study by Gottlieb et al. [6], which permitted the use of topical corticosteroids and ultraviolet B phototherapy. Accordingly, it should be noted that comparisons of the results between any of the treatment phases of this analysis are observational (i.e., not direct) but do confirm the results of the long-term treatment phase and the placebo-controlled 12-week first treatment phase studies, suggesting that the risk of joint disease is not increased with continued efalizumab treatment and that the incidence of arthropathy is low and similar to placebo. Further investigation is needed to confirm the results of this preliminary analysis of arthropathy events during long-term treatment with efalizumab.\nIn patients who re-started treatment for a further 12\u00a0weeks following an intervention-free period, the proportion of patients who had an arthropathy AE was lower than during the first treatment phase; the same was true for the incidence of arthropathy AEs per patient-year in re-treated patients. Although this scenario is likely to occur infrequently in clinical practice, these data show that if a patient needs to stop (e.g., during pregnancy) and then restart treatment, there appears to be no increased risk of arthropathy AEs.\nAlthough arthritis in patients with psoriasis has a significant impact on QoL [9, 27], it can, in most cases, be managed effectively [8]. In the small minority of patients who develop arthropathy during treatment, the symptoms can be managed successfully with non-steroidal anti-inflammatory drugs [8].\nIn conclusion, the results of this pooled analysis show that efalizumab does not appear to increase the risk of developing arthropathy AEs compared with placebo. Long-term studies of efalizumab indicate that the incidence of arthropathy AEs remains stable and low for up to 3\u00a0years of continuous treatment.","keyphrases":["arthropathy","efalizumab","psoriasis","psoriatic arthritis","arthritis"],"prmu":["P","P","P","P","P"]}
{"id":"Naturwissenschaften-4-1-2270361","title":"Mechanical analysis of infant carrying in hominoids\n","text":"In all higher nonhuman primates, species survival depends upon safe carrying of infants clinging to body hair of adults. In this work, measurements of mechanical properties of ape hair (gibbon, orangutan, and gorilla) are presented, focusing on constraints for safe infant carrying. Results of hair tensile properties are shown to be species-dependent. Analysis of the mechanics of the mounting position, typical of heavier infant carrying among African apes, shows that both clinging and friction are necessary to carry heavy infants. As a consequence, a required relationship between infant weight, hair\u2013hair friction coefficient, and body angle exists. The hair\u2013hair friction coefficient is measured using natural ape skin samples, and dependence on load and humidity is analyzed. Numerical evaluation of the equilibrium constraint is in agreement with the knuckle-walking quadruped position of African apes. Bipedality is clearly incompatible with the usual clinging and mounting pattern of infant carrying, requiring a revision of models of hominization in relation to the divergence between apes and hominins. These results suggest that safe carrying of heavy infants justify the emergence of biped form of locomotion. Ways to test this possibility are foreseen here.\nIntroduction\nAll higher primates (except humans) carry their young clinging to their fur from birth (Jolly 1972). The correlation between infant carrying and the form of locomotion of adult primates is clear, but no detailed study has focused on the mechanics of the problem. Among nonhuman primates, there is a change in the carrying pattern of infants by adults (mostly by the mother) as the infant grows (Jolly 1972; de Vore 1965; Goodall 1967; MacKinnon 1974; Fossey 1979, 1983; Tuttle and Watts 1985). Newborns are carried clinging in close ventro-ventral contact, often with additional support from the mother (Hoff et al. 1983). Change to infant support over the adult body (dorsal or lumbar clinging) occurs some months later for all nonhuman higher primates and extends for years in apes. It seems clear that safety in infant carrying imposes limits on the weight of infants.\nThis work deals with the problem of infant carrying in Hominoids, characterized by increasing body size. Accepted hominoid phylogeny places the branch to the lesser ape gibbon as the oldest. It is followed by the great apes, with the older branch to the arboreal orangutan and the branch originating terrestrial gorilla and chimpanzee coming later, followed by biped hominids (Yunis and Prakash 1982; Ruvolo et al. 1994; Lockwood et al. 2004). Mechanical analyses of constraints for safe infant carrying in the usual primate pattern are reported. Measurements of tensile and friction properties of hominoid hairs are presented and conditions for mechanical equilibrium determined. The usual pattern of primate carrying of heavy infants is shown to be incompatible with bipedalism. This brings a new perspective to the hominization process and to the basic changes at the divergence between apes and hominins.\nMaterials and methods\nSamples\nFor the experimental study of the mechanical properties of ape hairs, three pieces of skin with hairs were obtained from frozen limbs of dead animals at the Laboratoire d\u2019Anatomie Compar\u00e9e, Mus\u00e9um National d\u2019Histoire Naturelle, Paris, France (one gibbon, one orangutan, and one gorilla\u2014chimpanzee was not available). Due to difficulty in obtaining animal skins, the samples came from three individual animals, one piece of about 200\u00a0cm2 from each. The three pieces of skin were transported at room temperature immersed in formaldehyde and were treated afterwards by a taxidermist. After the skin was fixed, it became leather, and chemicals were washed out from the hairs, which looked natural. The effect of formaldehyde on hair has been tested in human head hair; results are presented in the Appendix, and evidence is that no significant change occurs in the relevant tensile parameters.\nThe skin and hair from the different animals studied here are easily recognized by appearance and texture and are species-characteristic. Gibbon hairs are smooth and silky, while orangutan hairs are harsh, hard and very long; gorilla hair has an intermediate appearance. Estimated values of skin thickness and hair length are given in Table\u00a01, with other parameters obtained.\nTable\u00a01Parameter values for apes\u00a0GorillaGibbonOrangutanSkin thickness (mm)~1.5~0.6~2.3Hair Length (cm)~6~4~10\u2205 (\u03bcm)66\u2009\u00b1\u20092 (21%)52\u2009\u00b1\u20092 (19%)120\u2009\u00b1\u20094 (24%)\u03b1 (N)17\u2009\u00b1\u20092 (25%)6.0\u2009\u00b1\u20090.6 (33%)32\u2009\u00b1\u20091 (13%)FE (N)0.38\u2009\u00b1\u20090.03 (30%)0.18\u2009\u00b1\u20090.02 (36%)0.49\u2009\u00b1\u20090.02 (12%)Fr (N)0.67\u2009\u00b1\u20090.09 (39%)0.29\u2009\u00b1\u20090.04 (47%)0.79\u2009\u00b1\u20090.04 (17%)Sr0.26\u2009\u00b1\u20090.05 (56%)0.28\u2009\u00b1\u20090.05 (52%)0.07\u2009\u00b1\u20090.01 (51%)E (GPa)5.0\u2009\u00b1\u20090.6 (42%)2.8\u2009\u00b1\u20090.3 (43%)2.8\u2009\u00b1\u20090.2 (36%)Skin thickness and hair length (estimated), hair diameter \u2205, and results obtained from the n\u2009=\u200910 tensile curves for each ape hair. The regression coefficient \u03b1 and the elastic limit FE are measured in the initial linear Hookean part of the curve, while the force Fr and strain Sr at rupture are measured at the end of the curve. Mean values are given with their standard errors. The Young modulus E is given by \u03b1 divided by the hair cross-sectional area (with error propagation). The variation coefficient (CV\u2009=\u2009100\u2009\u00d7\u2009standard deviation\/average value) is given in parenthesis.\nThe three skins provided several samples of different areas with the skin side fixed on pieces of wood, leaving the hairs exposed. The use of taxidermized specimens is the only possibility for simulating the natural situation for friction coefficient measurements. The basic data by Schultz (1931) on hair density of primates were also obtained from embalmed animals.\nMethods\nSeveral sectioned ape hair cross-sections were examined under a compound microscope (magnification up to 360), and external appearance as well as hair diameters were measured with a Zeiss Axiovert 200 microscope (several positions along the hair length). Ten single hairs of each of the three animals were cut and analyzed on an automated Instron Tensile Tester, at the standard 65\u2009\u00b1\u20092% relative humidity of the air (RH), 20\u2009\u00b1\u20092\u00b0C and speed 2.6\u00a0mm\/min. The hair was fixed on one end to a fixed grip and on the other extremity to a movable grip, connected to a pulling force. The defined distance between grips (Lo) is between 20 and 50\u00a0mm. Curves of force (given in Newton, MKS unit) versus longitudinal elongation L (mm) were obtained; the strain, or relative elongation, is given by (L\u2009\u2212\u2009Lo)\/Lo. Single human head hair (with and without immersion in formaldehyde) was also analyzed as a standard, as a comparison was possible with previous data for human hairs (Robbins 1994; Nikiforidis et al. 1993; Franbourg et al. 2003). Twenty single hairs from each of the three animals, still on the skins, were also analyzed and pulled until detachment from the skin occurred.\nFor friction measurements, the wooden pieces with the larger areas from each animal were fixed to a movable inclined plane. The other pieces (with areas in the interval 16\u201376\u00a0cm2) were used in measurements of the static friction coefficient \u03bc (hairs against hairs) using variable weights attached to the wood face. The angle of the inclined plane was carefully changed until slipping started at its critical value, and the process was repeated several times to check reproducibility. The RH and the room temperature (20\u201326\u00b0C) were measured and controlled with a dehumidifier and an air-conditioning system.\nResults\nTensile measurements\nMechanical studies of animal fibers have been extensively performed over the last decades because of interest in textile production (mainly wool, see Feughelman 1997) as well as cosmetics (human head hair, see Robbins 1994). Animal hair is composed of three parts: an external thin cuticle (protective covering with a scale structure from root to tip), a thicker cortex with fibrous proteins, and a central porous medulla, which may be absent in finer hair. Ape hair viewed under an optical microscope are similar in structure to wool and human head hair, as in Fig.\u00a01, where the external appearance with the cuticle scale structure is shown in Fig.\u00a01a and the cross-section inner structure in Fig.\u00a01b.\nFig.\u00a01Micrographs showing orangutan hair, whose light color allows better visualization; a external appearance with typical cuticle scale structure, b transversal cross section with external cuticle, thicker cortex, and inner medulla\nMechanical properties of natural fibers are defined by the cortex of the fiber and are due to the molecular structure of keratin, which constitutes the cortex, as determined many years ago (Astbury and Street 1931; Astbury and Woods 1933). Extensive work during decades evidenced the dependence of mechanical properties essentially on the cross-sectional area of the fiber, thus enabling basic research to be conducted on relatively few single fibers, eliminating the need for statistical methods on a large number of samples (Feughelman 1997).\nTensile measurements give curves of force against strain (relative elongation) for a single hair fiber. Force has an important meaning for this study; stress is obtained dividing the force by the cross-sectional area of the fiber. A typical result with standard human head hair is shown in Fig.\u00a02a and is similar to earlier data on human hair (Robbins 1994; Nikiforidis et al. 1993; Franbourg et al. 2003). The curve displays an initial elastic (Hookean) linear region, followed by a plastic yield region and a postyield region before breakage. Transition from the elastic to the plastic region corresponds to a first-order transition of the alpha-keratin molecule from the coiled helices to the stretched beta-keratin form (Bendit 1957), and the plastic region is strongly influenced by the water content (Feughelman 1997).\nFig.\u00a02Measured force\u2009\u00d7\u2009strain (relative elongation) curves for hominoid a human, b gorilla, c gibbon, and d orangutan hairs. In a are stressed the initial Hookean linear elastic region (E), the plastic plateau (P), the post-yield region (PY), and the breaking threshold (B). The different curves in b, c, and d correspond to ten hairs of each individual ape. The inset in a is a magnification of the elastic linear region, showing the slope \u03b1 and the force at the elastic limit FE\nThe linear region extends up to 1% strain, a result well established for helical alpha-keratins of all animal hairs: the cortex composite has mechanical properties of a single material (Feughelman 1997). The value of the force at the Hookean elastic limit (called FE in this paper) corresponds to the limiting force for full recovery of the fiber after being relaxed. The regression coefficient of the linear region (called \u03b1 in this paper) of the force versus strain curve divided by the cross-sectional area of the fiber gives the Young modulus of elasticity (E) of the fiber (dimension of pressure unit, GPa\u2009=\u2009109\u00a0N\/m2). The values \u03b1 and FE are determined expanding the initial elastic part of the curve (which does not, in general, start precisely at zero), as seen in the insert in Fig.\u00a02a. Further analysis of human head hair is given in the Appendix.\nApparently, no tensile studies on ape hair have been reported. Curves obtained with n\u2009=\u200910 ape hairs of each of the three apes (one gorilla, one gibbon, and one orangutan) are shown in Fig.\u00a02b\u2013d. The curves display all the classical regions of animal hair. Human hair has a larger postyield region and a larger strain at breakage, related to the usual human hair treatment (frequent washing and combing). Maximum stretching is very different in the wet (50% strain) and dry (10% strain) states and may be influenced also by hair conditioners (Robbins 1994; Colombera and Joekes 2004).\nThe initial linear region is well defined in all hair curves, and its variability among different hairs of the same ape is due to differences in hair cross-sectional area. The physical parameters \u03b1 and FE of each of the ape curves were obtained with the procedure shown in the insert of Fig.\u00a02a. Values of force (Fr) and strain (Sr) at rupture were measured directly at the end of the curves.\nTable\u00a01 gives statistical averages of parameters obtained from tensile curves (\u03b1, FE, Fr, Sr) and measured hair diameter \u2205 for each species, together with calculated Young modulus of elasticity E, with respective errors. The average values for FE are 60% (\u00b12%) of Fr for the hairs of the three ape species. Hair diameters \u2205 in Table 1 are averages over several positions measured along ten single hairs of each ape (\u223c50 measurements for each ape). One-way analysis of variance (ANOVA) test for these \u2205 measurements evidenced significant difference between apes (F2,155\u2009=\u2009177; p\u2009<\u20090.0001).\nAll tensile parameter values for gorilla hairs, with the only exception being the Young modulus E, are intermediate between gibbon and orangutan hairs. Table\u00a02 gives statistical tests for these four parameters: significant differences result from two-population t tests and one-way ANOVA test.\nTable\u00a02Statistics for tensile parameters of ape hairs\u00a0FE\u03b1FrSrGibbon \u00d7 Orangutant1011188.44.4p<0.0001<0.0001<0.00010.0003Gorilla \u00d7 Gibbont105.36.24.10.32p<0.0001<0.00010.00080.75Gorilla \u00d7 Orangutant103.17.61.34.0p0.007<0.00010.20.0009ANOVAF2,2744119209.3p<0.0001<0.0001<0.00010.0009Results from statistical tests for the n\u2009=\u200910 measured values of the four parameters obtained from the force\u2009\u00d7\u2009strain curves of hairs of the three apes. Two-population t-test (t10 values and corresponding probability p) are given for each parameter and each compared population in the upper lines. ANOVA test of the four parameters in the three ape populations and corresponding probability p in the lower line\nHominoid species differ significantly regarding hair strength, which increases from gibbon to gorilla and to orangutan. However, the Young modulus has a different behavior: E values for gibbon and orangutan are equal (2.8\u00a0Gpa), but gorilla has a higher value (5.0\u00a0Gpa). The value for human head hair from literature (Robbins 1994; Nikiforidis et al. 1993) is 3.8\u00a0Gpa, intermediate between gorilla and orangutan hair. Human head hairs measured in this paper are within reported values, as detailed in the Appendix.\nResults obtained indicate that the force strength and average thickness of hominoid hairs are species-dependent and may be evolutionary meaningful. Data for human hair in the Appendix give further indication of species dependence. Such direction of research, together with definition of the Young modulus in terms of the inner hair structure, have clear interest but outside the scope of this work.\nFor infant carrying, the really decisive parameter is FE, representative of the upper limit of the force that a single hair can safely withstand, because it is the limit for recovery after relaxing.\nIt is convenient to express FE in kilogram-force (and gram-force), corresponding to the force weight of a given mass at the earth surface. This unit is often still used for load (instead of N\u2009=\u20090.10198\u00a0kgf) because it makes the mass value of the weight clear. The important values FE are then 18\u2009\u00b1\u20092\u00a0gf for gibbon, 39\u2009\u00b1\u20093\u00a0gf for gorilla, and 50\u2009\u00b1\u20092\u00a0gf for orangutan. The value for human head hair is 29\u2009\u00b1\u20092\u00a0gf (see Appendix).\nThe force to pull hairs from the skin samples of the three ape species has also been measured and may be considered as an upper limit, in view of changes in skin due to taxidermy. They are typically near the elastic limit and lower than rupture value, as occurs with human head hair in the scalp (Robbins 1994), giving a further reason to focus on the FE values as upper limits for safe load carrying.\nA robust conclusion can be thus extracted. It is clear that to withstand the clinging infant weight, a large number of hairs held together is required. Results obtained evidence that bunches of about 100 hairs are necessary to carry infants weighing a few kilogram-force. Infant hands (and feet) can possibly grasp a bunch of hairs available in some square centimeters of skin so that safety in the usual pattern of nonhuman primate infant carrying critically depends on the density of hairs.\nInfant carrying\nValues of hair density for nonhuman primates have been obtained in the extensive work of Schultz (1931, 1968); values are not gender-dependent but vary with body region: density is highest in the vertex and smaller on the ventral than on the dorsal side of the trunk. Densities are generally compatible with the carrying requirement defined above, but the great apes have the lowest hair density among primates. Hair density values on the back of primates vary, from over 1,000 hairs\/cm2 for smaller species (monkeys and gibbons), down to about 100 hairs\/cm2 for great apes. The problem of safely carrying heavy infants requires thus particular consideration among the great apes.\nAnalysis of hair density data (Schultz 1931) in many anthropoid primate taxa yield (Schwartz and Rosenblum 1981) a negative allometry of relative hair density (rhd\u2009=\u2009hair density\/total surface area). This decrease in rhd with increase in primate body mass has been correlated to thermal constraints imposed by the decreasing ratios of surface area to body volume. However, chimpanzees apparently have a lower rhd than would be expected for their body volume (see Fig. 1 of Schwartz and Rosenblum 1981), with rhd similar to that of larger gorillas.\nThe bunch of hairs available for clinging depends also on hair length and infant hand size. Accepting that about 3\u00a0cm are necessary to roll hairs on the infant fingers, the excess in length defines the area available for clinging. Clinging area per limb may be thus estimated to be 3, 10, and 50\u00a0cm2 for, respectively, gibbon, gorilla, and orangutan. In a dynamic situation, the load should be supported instantaneously by a single limb.\nThe infant-carrying method of the arboreal Asian apes (gibbon and orangutan) is different from that of the terrestrial African apes (gorilla and chimpanzee). The arboreal apes carry their young over one side of the mothers\u2019 pelvis, not on the back (de Vore 1965). In accordance with the small size (7\u00a0kgf when adults), the lesser ape gibbon has a high hair density, over 1,000 hairs\/cm2, more than ten times the density of great apes (Schultz 1931, 1968). This ensures safety for carrying their light young in their acrobatic arboreal life. The arboreal but heavier orangutans (adult females weigh around 50\u00a0kgf and males 90\u00a0kgf) compensate the lower hair density by longer, thicker, and stronger hairs and thicker skin. Orangutan infants live with their mothers for about 7\u00a0years.\nThe gorilla is about 50% heavier than orangutan, but instead of even greater hair thickness and length, the solution for terrestrial apes is the mounting position for heavier infant support. Orangutan hair and skin are, in this paper, estimated to be able to withstand about seven times more weight than gorilla hair and skin. Gorillas are the heaviest primates and have very few chest hairs (Schultz 1931, 1968). Adult gorilla is much heavier than adult human, but their newborns have only half the weight of human babies. In the first 1 or 2\u00a0months, the infant gorilla is supported manually by its mother as she walks tripedally or bipedally (Jolly 1972; de Vore 1965; Tuttle and Watts 1985; Doran 1997). The same occurs for chimpanzee babies, unable to support their own weight by clinging prior to 2\u00a0months of age (Plooij 1984). Change from quadruped to triped or biped motion occurs systematically among all great apes when infant safety requires manual support (Jolly 1972; de Vore 1965; Tuttle and Watts 1985). Slow and careful locomotion of female chimpanzees while carrying young infants has been reported (Goodall 1967). Two primary causes of mortality have been found among infant chimpanzees: inadequacy of the mother\u2013infant bond and injuries caused by falling from the mother (Goodall 1967). It is clear that infant carrying is crucial and depends on not trivial behavior among African great apes.\nWeight limit for clinging in African apes\nThe gorilla FE value, combined with hair density and length, indicates that load up to 40\u00a0kgf could be supported by clinging. However, the weight limit for clinging depends also on several other variables. Increase in hair pull-out or break in the dynamic situation, capacity of the infant to withstand its own weight and to grasp hairs without sliding, and skin capacity to withstand the load without rupture are all variables that may decrease the infant weight limit for clinging.\nA bunch of hairs on about 1\u00a0cm2 (\u00b110%) of gorilla skin under load was observed, still on the skin, to simulate the real load effect in the actual clinging situation. Counting hairs in this bunch gave 140 hairs (\u00b110%), in agreement with the value reported by Schultz (1968) for the back side of the trunk of gorillas (145 hairs\/cm2), evidencing that there are no marked differences between the limb sample and the back-trunk hairs. This bunch of hairs supported 1\u00a0kg without problems, even with some pendulum movement. With 2\u00a0kg, several problems became evident. Besides eventual detachment of single hairs from the skin during movement, the hair bunch started to slip from the double-face adhesive tape used to attach it to the load support. It was necessary to make a new attachment system; the hair bunch was rolled several times around the weight support. Even so, loads of 2\u00a0kg and more could be observed only with the rolled hairs attached by super glue (ester cyanoacrylate) to the weight support. This indicates a possible problem also with the clinging capacity of infant fingers, and slippage may occur. Also, the skin itself began to deform and detach from the wood support, requiring a much stronger attachment of the skin to the wood base. The bunch of hairs broke at the same hair length for all under a static load of 7\u00a0kg, compatible with the values of force at rupture (Fr) obtained from the tensile measurements of single hairs (Table\u00a01). An upper limit of 1\u00a0kgf\/cm2 for clinging without problems may be deduced from this simple experiment, but the actual limit depends on the clinging capacity of infant fingers and on the mother\u2019s skin resistance. The effective weight limit for clinging must be obtained from careful observation of live apes.\nFossey (1979) describes physical and behavioral development of gorilla infants: at 4\u20136\u00a0months (weight about 5\u00a0kgf), the gorilla infant travels 60% of the time in ventral position while with 6\u201312\u00a0months (weight about 8\u00a0kgf), it travels in the dorsal position 80% of the time. A detailed investigation of the ontogeny of locomotion in the African ape (Doran 1997) revealed that up to 5\u00a0months of age, chimpanzees are slightly more precocious than gorillas. However, at 6\u00a0months, gorilla locomotor development becomes faster than that of chimpanzees, and surprisingly, much of the interspecific variation in behavior is explained by differences in body size (Doran 1997). Afterward, when the infants are of similar weights (although of widely disparate ages) gorillas and chimpanzees perform very similar locomotion activities (Doran 1997).\nIt is, in this paper, suggested that the weight level of 5\u00a0kgf may be considered the limiting value for safe support over long periods by only ventral clinging, defined probably by a combination of security factors1 related to hair strength, infant clinging capacity, and skin resistance to pressure. The limiting clinging pressure on the mother\u2019s skin for the African great apes may be thus estimated to be 0.5\u00a0kgf\/cm2 (a factor of 2 in relation to the simple test made with the skin sample). The skin is also a visco-elastic medium, reflecting properties of a strain-induced aligned-collagen network that can stretch up to 100% before permanent damage (Silver 1987). The shear modulus for the human dermis ranges from 1.2 to 3.1\u00a0MPa, while for the human hypodermis, it ranges from 3.1 to 9.7\u00a0kPa (Gennisson et al. 2004). The pressure limit 0.5\u00a0kgf\/cm2 (~50\u00a0kPa) could be a limit also for danger to the skin of the mother ape. It is known that one of the important differences between the skin of humans and of African apes is the higher elasticity of the human skin (Montagna 1982).\nThe locomotor development of the infants must therefore adapt to such an effective weight limit. This does not mean that infants over 5\u00a0kg will fall. Larger infants may travel briefly in ventral position during stressful situations (Fossey 1979) or when the chimpanzee mother swings through trees (Goodall 1967). Usual locomotion of adult African apes is terrestrial quadrupedalism (~96% of the time in gorillas and ~86% in chimpanzees, from Doran 1997), and safe infant support over longer periods clearly requires the necessity of change to a mounting position, with the infant weight supported by the adult body.\nDorsal clinging position\nIn order to analyze the mechanics of the carrying system in the African great apes, it is also necessary to consider, besides the tensile properties of hairs, the hair\u2013hair friction that prevents slipping, particularly for heavier infants in dorsal position. Fig.\u00a03 is a sketch of the mounting position in African apes, showing the angle \u03b8 of the inclined plane where the infant stands. For simplicity in the sketch, the angle \u03b8 coincides with the angle defined by the usual knuckle-walking position of African great apes, but that is not necessary. The important parameter is the inclination of the base where the infant stands clinging; its relation to the average body inclination angle does not need to be taken into account in analysis of the friction effect.\nFig.\u00a03Sketch of ape carrying infant. For simplicity, in the sketch, the angle \u03b8 of the inclined plane where the infant stands (to which Eq. 1 refers) coincides with the angle defined by the knuckle-walking position of African great apes, which is not necessarily so, as discussed in the text\nThe total infant weight, in the vertical direction, has two components, one in the inclined plane, favoring slipping, and one normal to the inclined plane, responsible for the friction force opposing slipping. Calling Wt the total infant weight carried in mounting position, Wc the effective weight limit for clinging, and \u03bc the static friction coefficient (Bowden and Tabor 1956), the equilibrium of forces on the infant requires that force components on the inclined plane direction satisfy the condition:\nIn the absence of clinging (Wc\u2009=\u20090), slipping on the body surface of contact starts for a critical value \u03b8c, given by tan \u03b8c\u2009=\u2009\u03bc. Both friction and clinging are thus essential to hold heavy infants in the mounting position. The requirements for dynamic equilibrium may be greater, so that in fact the mounting position in dynamic movement may be unsafe even when Eq. 1 is satisfied.\nIt should be stressed that Eq. 1 is a necessary condition for infant survival and therefore also for species survival. It is a very robust requirement of basic mechanics for static equilibrium. The ape hair\u2013hair friction coefficient \u03bc must be known to analyze the actual ape situation.\nFriction measurements\nApparently, no results for hair\u2013hair friction coefficients exist in the literature; only friction between human hair and other materials are reported (Robbins 1994). The skin samples were used to measure \u03bc as a function of the supported load W in known conditions of relative air humidity and temperature, in a situation analogous to that depicted in Fig.\u00a03. It was verified that \u03bc depends on the hair direction, being larger for hairs in the parallel position (as in nature, with the two animals heading in the same direction) than in the antiparallel condition. Systematic measurements have been thus made in the parallel condition.\nFigure 4a shows a typical result of \u03bc as a function of W. For hard solid surfaces, \u03bc is constant and independent of the load W and contact area A. This is clearly not the case for hairs, especially at low loads. Long ago, it was shown that a variation of \u03bc with W occurs for textiles and fibers (Gralen 1952; Makinson 1952), given by the empirical equation:\nFig.\u00a04Results for friction coefficient at 80% RH. a friction coefficient \u03bc as a function of W for a gorilla skin with smaller area (16\u00a0cm2). The line shows a fit to the data using Eq. 2. b\u03bc\u2009\u00d7\u2009W for (circles) gorilla with larger area (76\u00a0cm2); (squares) gorilla with smaller area (16 cm2), and (triangles) gibbon with intermediate area (42\u00a0cm2). Linear fits to the data are shown in lines\nThe parameter a gives the friction coefficient in the limit of high loads. It has been proposed (Gralen 1952) that the parameter b is related to the pressure inside an oil lubricating film on the fiber surface. The thickness of this film decreases due to pressure, which causes changes in the friction mechanism. Hair is a natural fiber (Howell et al. 1959) and behaves in a similar way. In fact, the data could be well-fit with Eq. 2, shown as a solid line in Fig.\u00a04a.\nMany curves were analyzed for different values of the contact area A and RH. A strong dependence of \u03bc on A was observed, probably due to shear among the several hairs in contact. The fits to the linear equation \u03bcW\u2009=\u2009aW\u2009+\u2009b are more convenient for yielding the parameters a and b, as shown in Fig.\u00a04b. Both a and b increase with A, but a is less sensitive to A, while b is roughly proportional to A, so that the term b\/W in Eq. 1 is inversely proportional to the pressure W\/A exerted on the surfaces in contact.\nResults indicate that the friction coefficient might be considered constant for pressures higher than 20\u00a0gf\/cm2. This pressure corresponds to infant weight around 1\u00a0kgf, from the relation between body surface area and weight (Schwartz and Rosenblum 1981; Kleiber 1975) and estimating the contact area as one third of the infant surface area.\nThe average value is \u03bc\u2009=\u20090.27\u2009\u00b1\u20090.03 for gorilla hairs and \u03bc\u2009=\u20090.20\u2009\u00b1\u20090.02 for gibbon hairs at 60% RH, in agreement with the expected null or small difference on friction depending on the fiber diameter (Robbins 1994). Such values are in good agreement with known values for single fibers (Bowden and Tabor 1956; Howell et al. 1959). The orangutan skin was not as perfect and homogeneous as the gibbon and gorilla skins, showing some parts without hairs, and provided only two samples, so dependence on area could not be analyzed. However, experiments of \u03bc as a function of load gave similar values to those for the other apes.\nThe hair\u2013hair friction coefficient of apes obtained is intermediate between values for friction between human hair and hard rubber with dry (\u03bc\u2009=\u20090.19) and wet (\u03bc\u2009=\u20090.38) surface conditions (Robbins 1994), indicating that results obtained can be considered of general validity. Note that Robbins (1994) mentions the difficulty of testing hair\u2013hair friction, important in hair combing, and that it should be similar to hair\u2013rubber friction.\nEquilibrium condition in African apes\nIt is possible now to focus the problem on our nearest relatives, the great apes gorilla and chimpanzee, applying Eq. 1 to their mounting condition, in which clinging and friction act together for safe infant carrying.\nThe effective weight limit for constant ventral clinging, already discussed, is estimated to be 5\u00a0kgf. As the hair density in the back is higher (30% for chimpanzees), the estimate is Wc\u2009~\u20095\u20137\u00a0kgf. The maximum value Wt can be estimated from the weight of infants when they are no longer carried by mothers. This occurs when gorillas are about 2.5\u00a0years old and chimpanzees 5\u00a0years old, both weighing around 20\u00a0kgf (Fossey 1979; Doran 1997).\nBy inserting such values in Eq. 1, and using the estimated constant friction coefficient \u03bc, it is possible to obtain the maximum inclination of the plane supporting the infant for the safe carrying system in the African great apes. The condition \u03b8\u2009<\u200929 to 34\u00b0 is obtained.\nThis basic equilibrium constraint related to friction in the mother\u2013infant contact is satisfied in ape knuckle-walking quadruped position2.\nIt should be stated that it is frequently reported that in the dorsal position, the African ape infant rides high on the mother\u2019s neck and lies stretched and prostate (de Vore 1965; Fossey 1979). This position increases the fixed base of the load and places it in a body region with a promontory and a smaller inclination angle (\u03b8\u2009~\u200920\u00b0 as estimated2 from the same Fig. 7\u201320 in Schultz 1968). Older infants ride in a jockey position; they do not normally grip hairs with their feet, but press the sides of their ankles against the mother\u2019s flank (Goodall 1967). Clearly, among terrestrial great apes, infant safety does not rely only in grasping hairs.\nIt may be concluded that slipping imposes clear limits on the maximum body angle attained by heavy ape species carrying infants, representing a hindrance against evolution towards bipedality. This might explain the persistence of knuckle-walking among the great African apes.\nEffect of humidity on friction\nThe two most relevant variables to friction in hairs (Robbins 1994) are the load pressing the two surfaces together, as analyzed previously, and the relative humidity RH. Dependence of \u03bc on RH was reported for fibers (Howell et al. 1959) and human hair (Robbins 1994), and a similar effect was verified for ape hairs.\nReduction in RH, from 80 to 38%, led to a decrease of gorilla hair \u03bc by about 30%. The friction coefficient between human hair and hard rubber is reduced by 50% between the wet and dry states (Robbins 1994).\nFriction of gorilla hairs has been also measured in wet conditions (immersion in water, followed by slight shaking of the sample). The result is \u03bc\u2009=\u20090.35\u2009\u00b1\u20090.03, in good agreement with reported values for human hair and hard rubber in the wet condition. The larger \u03bc value in wet condition is possibly due to network of hydrogen bonds connecting hairs in contact. The thin outer epicuticle membrane of hair is hydrophobic, but hydrogen bonds may occur through the thicker exocuticle and endocuticle, especially the latter, which swells much more in water (Feughelman 1997, Robbins 1994). Such structures may be accessible to external water through the ratchet scale structure of hair.\nIt should be concluded that reduction in humidity destroys the delicate balance between friction and clinging (Eq. 1) necessary for survival of infants of heavy ape species. Figure 5 shows the maximum carrying weight Wt obtained from Eq. 1 as a function of the inclination angle \u03b8 for \u03bc varying from zero up to 0.40, with Wc\u2009=\u20095\u00a0kgf.\nFig.\u00a05Curves of total weight Wt supported in dorsal position as function of the inclination angle \u03b8 of the infant base on the mother\u2019s trunk, obtained from Eq. 1, for several values of the friction coefficient \u03bc (0\u20130.4, as indicated on top), with Wc\u2009=\u20095\u00a0kgf (limit value shown with dashed line). The line with cross is for Wc\u2009=\u200910\u00a0kgf and \u03bc\u2009=\u20090.4. Vertical position corresponds to \u03b8\u2009=\u200990\u00b0\nDiscussion\nIn this section, the relevance of the above results for models of hominization is discussed. Safe infant carrying is clearly very important for species survival and has to be considered in the discussion of the transition from apes to hominins. The mechanical analysis made in this paper evidenced that habitual bipedalism, with a large \u03b8 angle, is incompatible with the usual primate type of infant carrying. Even if one considers Wc\u2009=\u200910\u00a0kgf and \u03bc\u2009=\u20090.40, the maximum attainable angle is \u03b8\u2009~\u200949\u00b0, as seen in Fig.\u00a05.\nIt seems clear that together with bipedality probably also came the characteristic human type of infant carrying in the arms and hands of their mothers, making their locomotion and foraging particularly difficult. Females would not adopt bipedality if they could avoid it, and the species would not adopt bipedalism if females with infants would not engage in it. The transition to bipedality was not trivial.\nEtkin (1954) considered the problem of the burden on females \u201calmost continuously carrying a child,\u201d just to conclude that the female could not be an effective hunter, turning to a theory of the monogamous family unit. It is clear now that such a scenario could be considered only for Homo at a much later period. Iwamoto (1985) recalled examples of facultative bipedalism among monkeys and speculated that the \u201cdecisive factor (for habitual bipedalism) may have been some everyday necessity to carry something in both hands\u201d. After criticizing proposals in which the \u201csomething\u201d is food, Iwamoto suggested that the \u201csomething\u201d could be their helpless babies. However, this proposal has been dismissed under the hypothesis that babies became helpless only with increase of brain in Homo.\nNow models for bipedalism origin based on \u201cfood acquisition and foraging strategies\u201d are considered \u201clikely\u201d but are in fact based on the premises that bipedal locomotion frees the arms and hands and does not have high costs. However, such premises just forget the fundamental fact that bipedalism frees the arms and hands only of males and juveniles, but females, on the contrary, have arms and hands occupied by infant carrying, which represents a high cost for bipedalism, not considered up to now. A review of bipedalism mentions: \u201cinfants (from early hominin biped) probably clung to their mothers\u2019 hair, as is the case for most other primates\u201d (Richmond et al. 2001).\nIt seems that the not trivial mechanical constraint of safe carrying of heavy infants has never been considered before.\nCurrent evidence suggests (Richmond et al. 2001; Kingston et al. 1994; Potts 1998) that the key adaptation to bipedalism originated in wooded environments during a drier period in East Africa. The equilibrium condition (Eq. 1) shows that reduction of friction in the dorsal clinging position would most probably lead to a decrease in infant weight and decrease in the body inclination angle \u03b8, corresponding to the solution adopted by quadruped savanna monkeys in the arid savanna. It seems clear that habitual bipedalism would not be reached by continuous increase of the body angle. However, among hominoids, reduction of friction between infant fingers and mother hairs could reduce infant grasping capacity, increasing the period of mother\u2019s manual support of newborns and eventually inducing habitual bipedalism.\nConcluding remarks and future perspectives\nAn important conclusion from this work refers to interest in systematic study of tensile properties of hairs from an evolutionary point of view. The other clear conclusion is that models on locomotion and bipedalism evolution must focus on females carrying infants, who are the target of the strongest selective pressures, and this conclusion is independent of the form of locomotion of hominid ancestors (knuckle-walkers, terrestrial or arboreal quadrupeds, or full-time arboreal climbers).\nTo disentangle the divergence between apes and hominins, several directions of research become clear from this work. Studies of living apes focused on the mechanical problem of infant carrying, as well as determination of the elastic limits of the ape skin and the limits for grasping capacity of ape infants, which are all essential to identify the critical factor responsible for emergence of bipedalism.\nIt should be stressed that in a scenario with bipedalism emerging for safe infant carrying, the selective pressure would act particularly on females. This is not a problem, as only genes in the nonrecombinant part of the Y chromosome are not transmitted to both sexes and therefore cannot be associated with species locomotion. The process correlates with increase in the mother\u2013infant bond, characteristic of the human lineage.\nOther hypotheses for emergence of bipedality (see Richmond et al. 2001 for a review) do not, in fact, explain why other primates did not follow such line. The many scenarios invoked previously for bipedalism evolution may sound plausible, but are not necessary; neither take into account the problem of infant carrying and can be considered complementary and a consequence of the selective pressure on females carrying infants. The difficulties in locomotion and food gathering for biped females carrying infants may well be at the origin of the necessity of group cooperation, which could initially have been among females, with males in their usual role of primate group protection.\nA fundamental question is the possible correlation between bipedalism and reduction of body hairs, the two basic biological modifications of hominins. It is evident that decrease in body hair as the initial modification would bring on bipedality as a necessary consequence (Amaral 1989), through the strong selective pressure of safe infant carrying, when infants could no longer cling to body hairs.\nThe timing of reduction of body hairs is very controversial, since Darwin\u2019s original (1871) emphasis on the issue. Several works indicated a very early beginning of body hair reduction, still in forested environments, based on thermoregulatory requirements (Newman 1970), rhd of Australopithecines (Schwartz and Rosenblum 1981), and comparison of human and ape skin (Montagna 1982, 1985). Bipedality preadaptive to nakedness was suggested later from water-consumption requirements under reduced humidity (Wheeler 1992). However, detailed analysis of thermal loads and water consumption for quadrupeds and bipeds in the furred and naked conditions suggested again that reduction of body hair started in a forested environment becoming drier (Amaral 1996). Savanna monkeys tolerate heat even at high levels of exercise, evidencing that the biological avenue to cope with heat stress in an open environment is to keep a hair covering, increase sweating capacity, and have a variable conductance (patas monkeys, Mahoney 1980, and baboons, Rogers et al. 1992). The only situation in which nakedness is favorable concerns dissipation of heat loads that do not come from sun absorption but from activity in a more closed forest condition at temperatures below that of the body (Amaral 1996). Furthermore, regarding water requirements, advantages for nakedness exist in dry ambients with temperatures below body temperature (Amaral 1996).\nRecent genetic work has focused on evolution of black skin as a result of naked unprotected skin under solar stress, estimated to have occurred at least 1.2\u00a0mya (Rogers et al. 2004), while clothing is a recent innovation of only about 70,000\u00a0years ago (Kittler et al. 2003). However, up to now, there is no date for the beginning of reduction of body hairs. Therefore, localization of the genetic changes responsible for reduction of body hairs in humans is necessary to settle the issue.\nThe new perspective developed in this paper evidences continuity between physical and behavioral aspects of early hominins and their ancestors. The more differentiated aspects of humans are left to the emergence of Homo, at a much later period.","keyphrases":["infant carrying","clinging","friction","bipedalism","hair strength","reduction of body hairs"],"prmu":["P","P","P","P","P","P"]}
{"id":"Arch_Orthop_Trauma_Surg-4-1-2228384","title":"Viscosupplementation in the hip: evaluation of hyaluronic acid formulations\n","text":"This study compares three different hyaluronate formulations and evaluates functionality, time of satisfactory pain relief and also the delay in performing a total hip arthroplasty. One hundred and twenty patients (126 hips) received viscosupplementation with one of the three hyaluronate formulations. All patients were candidate for surgical treatment with a total hip arthroplasty. Three different products were consecutively used: Adant\u00ae, Synocrom\u00ae or Synvisc\u00ae. Patients were assessed 6 weeks after each infiltration using Visual Analogue Scale and Harris Hip Score. The Harris Hip Score increased significantly in two of the three groups compared to baseline, but no statistical significant difference was noted between the groups. Viscosupplementation provides significant pain reduction in two of the three groups. There is no significant difference in duration of the effect of the first infiltration between the three groups. The positive effect was still ongoing at the end point of the study in 46 hips: 51% of the patients did not undergo total hip arthroplasty, 3 years after viscosupplementation.\nIntroduction\nSevere osteoarthritis (OA) of the hip is characterized by pain and reduced mobility. It affects primarily adults aged over 50\u00a0years, but the age of onset of hip OA linked complaints seems to decrease over the last decennia [12]. First-line treatment consists of pain reduction with analgesics and non steroidal anti inflammatory drugs (NSAID). Rehabilitation treatment, education and the use of walking aids can help providing pain relief and improving mobility. When those therapeutic options fail or the analgesic treatment causes intolerable side effects, joint lavage, hip osteotomy or total hip arthroplasty (THA) can be considered. This arthroplasty technique has greatly improved and patients experience pain reduction and improvement of the quality of life. However, the intervention still requires prolonged anaesthesia, and is followed by a long-term rehabilitation program. Though complications are rare and protheses have an increasing duration of life, reinterventions are still needed [4].\nViscosupplementation (VS) is the administration of hyaluronan and\/or hyaluronic acid preparations to joint synovial fluid for the treatment of OA in order to restore the biologic properties of normal hyaluronic acid (HA).\nThe use of VS with HA was first described to provide pain relief and to increase mobility of the knee joint. The VS is an effective treatment for OA of the knee with beneficial effects on pain, function and patient global assessment [14]. HA products have more prolonged effects than intra-articular corticosteroids [14]. Since 1984, this technique is also used for the management of OA of the hip joint [2]. In the available literature there is no consensus on the number of injections, the dosage per injection, the most appropriate formulation of HA, and the optimal method for controlling the needle positioning in the hip joint. Moreover, the patient selection criteria differ from one study to another. To our knowledge there is no documentation available on the possible time gain for patients treated with VS of the hip joint before they have to undergo THA.\nThe results of a trial comparing three formulations of HA were presented. All patients were candidates for THA. The time of satisfactory pain relief, improved functionality obtained with each infiltration of HA and the delay in surgery were evaluated.\nPatients and methods\nPatient selection\nThe use of VS in patients with severe OA is common practise in our hospital, thus not requiring approval of the ethical committee. Patients gave their informed consent for this prospective study. Between March 2001 and February 2005, 120 patients (126 hips), 49 males and 71 females, responding to the eligibility criteria listed below, received viscosupplementation. Three different products were consecutively used: Adant\u00ae (Tedec-Meiji Failma, Madrid, Spain) (Group 1) (Synthetic hyaluronic acid with an average molecular weight of 0.6\u20131.2\u00a0million Da), Synocrom\u00ae (Croma Pharma, Leobendorf, Austria) (Group 2) (Sodium hyaluronate with a average molecular weight of 1.6\u00a0million Da) or Synvisc\u00ae (Altana Pharma, Konstanz, Germany) (Group 3) (Hylan GF 20 with an average molecular weight of 6.0\u00a0million Da) (Table\u00a01).\nTable\u00a01Characteristics of infiltrated populationPeriod of treatmentPatientsGenderAgeAdant27-11-02 till 23-02-059135 \u2642 and 65 \u264061.8\u00a0\u00b1\u00a012.8Synocrom19-01-04 till 04-10-04207 \u2642 and 13 \u264062.1\u00a0\u00b1\u00a014.5Synvisc15-03-01 till 24-04-03157 \u2642 and 8 \u264061.9\u00a0\u00b1\u00a015.3\nEligibility criteria\nAge between 30 and 70\u00a0years and suffering idiopathic radiologically confirmed hip OA.\nVisual Analogue Scale (VAS) score for pain greater then 30 (on a 100-point scale; 0 no pain and 100 \u201cthe worst pain imaginable\u201d)\nHave persistent pain for longer than 1\u00a0month despite use of analgesics or NSAID\u2019s.\nBe candidate for surgical treatment with a THA, according to the following criteria:\ncontinuous hip pain, also during the night, requiring daily intake of NSAID\u2019s or pain medicationdisabled gait pattern and need of walking aid\nBe able to understand the information relative to viscosupplementation and to give informed consent.\nExclusion criteria\nPregnancy\nContraindications to intra-articular hyaluronic-acid preparations\nMajor hip dysplasia or congenital abnormality of the hip\nPatients with systemic corticosteroids or intra-articular corticosteroid injections in the last 6\u00a0months\nContra-lateral THA or hip arthroscopy in the last 6\u00a0months\nOral or parenteral anticoagulant therapy\nPrevious hyaluronic acid hip infiltrations\nSkin diseases or infections\nSigns of haemarthrosis\nHistory of allergy or hypersensitivity to iodated contrast\nTreatment\nPatients received an intra articular infiltration with one of the three products. The manufacturer\u2019s treatment recommendations were followed. Patients having initially experienced a satisfactory pain relief are offered a second and third infiltration or THA when the condition deteriorates. Injection of the viscosupplementation was performed under sterile conditions by the same experienced orthopaedic surgeon (MM) in all patients. After skin cleaning a lumbar puncture needle was inserted in a lateral approach. Layer by layer local anaesthesia was performed using lidocaine 1%. Iodinated contrast agent Ultravist\u00ae (Schering, Berlin, Germany) was injected. The needle positioning into the joint cavity was fluoroscopically controlled (Fig.\u00a01). Arthrocentesis was carefully performed prior to each injection to remove any effusion.\nFig.\u00a01a Needle placement for injection of the viscosupplementation. b Fluoroscopic image of the needle insertion in the hip joint space\nAfter resting for 2\u00a0h, the patient was allowed to walk and to return home. The patient was advised to rest at home until the next morning.\nOral symptomatic slow acting drugs for osteoarthritis were authorized if they were taken at a stable dose for more than 3\u00a0months prior to inclusion in the study. These analgesics were continued at a stable dose during the VS treatment.\nEvaluation\nAll patients were assessed at baseline and 6\u00a0weeks after each infiltration. During this consultation in the outpatient clinic, the pain and functionality were evaluated using the VAS pain during walking score (100-point scale) and the Harris Hip Score (HHS). The latter is a clinical scoring system on a total of 100 points whereby the following subscales are rated: function (47 points), pain (44 points), range of motion of the hip (5 points) and absence of muscle contractures and length discrepancy (4 points). All side effects and complications of viscosupplementation were noted. In April 2005, all patients were contacted for follow-up assessment over the phone VAS and HHS.\nStatistical analysis\nSoftware (MS Excel)\u00ae was used to collect the data. Statistical analyses were of two kinds, first the differences in HHS and VAS pre-infiltration and post-infiltration (in the three groups) were compared using the paired t test and with the Wilcoxon paired test in the Synvisc group because this group is too small to analyse with the paired t test.\nThe second comparison was made between the three groups (Synvisc, Synocrom and Adant). The differences in HHS and VAS evolution between the three groups were checked by the analysis of variance statistics with Tukey extension, which also was used for controlling if the groups were initially homogeneous.\nThe duration of the viscosupplementation effect of the first infiltration was analysed using the Kaplan-Meier survival curves. The delay for the need of hip surgery was also analysed using a Kaplan-Meier survival curve. The statistical significance was set on P\u00a0\u2264\u00a00.05.\nResults\nThe mean pre-infiltration HHS was comparable for the three groups and varied from 64.8 points in the Adant group to 66.8 points in the Synocrom-group. The post-infiltration HHS increased with 6.3 points in the Adant group (P\u00a0<\u00a00.001), with 10.6 points in the Synocrom group (P\u00a0<\u00a00.05) and with 6.1 points in the Synvisc-group (P\u00a0>\u00a00.05; Table\u00a02). There was no statistical significant difference in the evolution of the HHS between the three groups (Table\u00a02).\nTable\u00a02Evolution in average HHS scoreHHS preHHS postDifferencePAdant64.8\u00a0\u00b1\u00a013.871.1\u00a0\u00b1\u00a015.7+ 6.3<\u00a00.001Synocrom66.8\u00a0\u00b1\u00a013.877.4\u00a0\u00b1\u00a014.7+ 10.6<\u00a00.05Synvisc66.3\u00a0\u00b1\u00a013.572.4\u00a0\u00b1\u00a014.5+ 6.1>\u00a00.05\nViscosupplementation provided a highly significant pain reduction in the Adant-group (P\u00a0<\u00a00.0001), a significant pain reduction in the Synocrom-group (P\u00a0<\u00a00.05) and a pain reduction that did not reach significance in the Synvisc-group (P\u00a0>\u00a00.05). There was no significant difference in pain relief between the three treatment products (Fig.\u00a02, Table 3).\nFig.\u00a02Pain relief in the three treatment groups a Adant, b Synocrom and c Synvisc\nThe duration of the effect of the first infiltration in the three groups is shown in a Kaplan-Meier curve (Fig.\u00a03). The first infiltration was the starting point. Endpoints were the second infiltration or operation of the afflicted hip, or when these were not applicable the latest patient contact, which can be considered as ongoing effect.\nFig.\u00a03Kaplan-Meier survival curve of the duration of effect of the first infiltration in days for the three different treatment groups\nThere is no significant difference between the three groups (\u03c72\u00a0=\u00a00.988 and P\u00a0=\u00a00.61). Figure\u00a04 illustrates the duration of effect of the three groups globally.\nFig.\u00a04Kaplan-Meier survival curve of the duration of effect of the first infiltration in days (three treatment groups together)\nThe positive effect was still ongoing in 46 hips, while in 80 hips patients had either received a second infiltration or THA at, the end of the study (Table\u00a04).\nTable\u00a03Evolution in average VAS pain during walking scoreVAS preVAS postDifference PAdant51\u00a0\u00b1\u00a02339\u00a0\u00b1\u00a027\u2212 12<\u00a00.0001Synocrom43\u00a0\u00b1\u00a02229\u00a0\u00b1\u00a023\u2212 14<\u00a00.02Synvisc47\u00a0\u00b1\u00a02630\u00a0\u00b1\u00a029\u2212 17>\u00a00.05Pre: at first infiltrationPost: at follow-up\nThe delay in performing a hip operation is analysed using a Kaplan-Meier survival curve (Fig.\u00a04). After 3\u00a0years, 51% of the patients have not undergone surgery. Because of the comparable outcome achieved with the three HA formulations, we did not differentiate between the three products in this survival analysis (Fig.\u00a05).\nFig.\u00a05Kaplan-Meier survival curve for the delay to surgery in days for the three groups confounded\nDiscussion\nThe patients attending our outpatient clinic, who were judged eligible for THA, were offered the possibility of a VS therapy. Hundred twenty patients choose this treatment option and 126 hips were treated with Adant, Synocrom or Synvisc. All studies, published up till now, studying the effect of VS in the treatment of hip OA were performed in small patient groups (12\u201357 patients) [6, 9].\nTable\u00a04Duration of treatment effect after first infiltrationPatientsAverage duration (days)Effect of 1st infiltration still going on46 352\u00a0\u00b1\u00a0258 (range 6\u2013886)Effect of 1st infiltration terminated80174\u00a0\u00b1\u00a0163 (range 13\u2013724)\nThe three preparations provided a significant pain relief and improvement of the HHS. The isolated Synvisc group never reached statistical significance in HHS score evolution and VAS during walk test after VS treatment; possibly due to the small number of patients (N\u00a0=\u00a015) in this group. As in the study of Tikiz [13], we found no significant difference between lower and higher molecular weight hyaluronic acids.\nWe could identify two groups of responders, the ongoing responders who last on average for 352\u00a0days after the first infiltration. The second group, where patients either received a second infiltration or THA had an average duration of effect of 174\u00a0days.\nThe VS method is widely used for OA of the knee joint, but there are only a few studies about its use in OA of the hip. Most authors agree that there should be a role for viscosupplementation in the treatment of hip OA. [1\u20133, 6, 7, 9\u201311] Our findings confirm the effect of VS in patients suffering OA of the hip. To our knowledge, this is the largest series of patients with hip osteoarthritis treated with viscosupplementation. There are no guidelines regarding the optimal number of injections needed to obtain optimal clinical response, which seems to be different [1, 2, 8, 10, 11] between products and dependent on the molecular weight (MW) [7]. We evaluated the time to relapse of one infiltration.\nOther authors advocated the use of ultrasound for controlling the needle position in the hip joint. [9, 3] In our series, we performed the infiltrations under fluoroscopy, in experienced hands this approach has proven to be accurate and safe. We use a very low amount of contrast liquid to avoid dilution of the product.\nOf all the reports published, we have the longest follow-up, up to approximately 3\u00a0years. The analysis by means of the Kaplan-Meier survival curve learns that there is a 50% probability of long-term effect (more than 2\u00a0years).\nWe saw no infectious adverse events and no serious systemic reactions, but all the interventions are performed in the operating theatre under strict aseptic conditions. The adverse events rates ranged from 10 to 30% which is slightly higher than the rates reported in VS treatment of knee OA [7]. Repeated injections did not increase the risk of adverse events [7]. Some patients experienced transient hip pain after the infiltration but made a full recovery in the next days. In the study of Conrozier [6], transient hip pain was reported after 10.1% of the injections. The products used in this study were all well tolerated. Complications are rare but a single case of septic arthritis was reported after multiple injections of hyaluronate and glucocorticoid [5]. Gout, pseudogout and chondrocalcinosis have not been reported after hip infiltrations.\nIntra articular injection of hyaluronic acid can provide long-term pain relief and improvement of joint function even in patients eligible for THA. Despite the use of fluoroscopy, this technique can be performed in an outpatient clinic, allowing patients to return home on the day of the intervention. At present, viscosupplementation therapy for OA of the hip is only palliative. It can be an alternative for young THA candidates, patients with surgical contra-indications and patients in whom NSAID use is not appropriate. There is only 1 randomised, double blind, placebo controlled trial of patients with hip OA [15]. The study was designed with a three-armed parallel-group (Hyalgan vs. methylprednisolone vs. lidocaine). One hundred and one were treated. There was no statistically significant difference between the three products on any outcome measure including the primary outcome measure, i.e., \u2018Pain on walking\u2019, at three 3\u00a0months of follow-up.\nThe results of our study should be considered in the light of the limitations of the design of this study. It is a non placebo controlled non randomised prospective study. It is known from experience with knee OA that the placebo effect of VS tends to be substantial [8]. The dimension of the groups treated with the three different products differs from 15 to 91 patients.\nViscosupplementation with hyaluronic acid seems to be a valuable technique for the management of painful OA of the hip that may delay the need for surgical intervention. Further prospective randomised placebo controlled studies are necessary to draw definite conclusions.","keyphrases":["viscosupplementation","hyaluronic acid","hyaluronan","intra-articular","injections","hip osteoarthritis","non-operative treatment"],"prmu":["P","P","P","P","P","P","M"]}
{"id":"Exp_Brain_Res-4-1-2257995","title":"The effect of age on task-related modulation of interhemispheric balance\n","text":"Normal aging is associated with less lateralised task-related activation of the primary motor cortices. It has been hypothesized, but not tested, that this phenomenon is mediated transcallosaly. We have used Transcranial Magnetic Stimulation to look for age-related changes in interhemispheric inhibition (IHI). Thirty healthy individuals (aged 19\u201378 years) were studied using a paired-pulse protocol at rest and during a low-strength isometric contraction with the right hand. The IHI targeting the right motor cortex was assessed at two intervals, 10 ms (IHI10) and 40 ms (IHI40). The corticospinal excitability of the left hemisphere was assessed by means of input\u2013output curves constructed during voluntary construction. Age was not correlated with IHI10 or IHI40 at rest. During muscle contraction IHI tended to increase at both intervals. However, this increase in IHI during the active condition (changeIHI) was less evident with advancing age for the 40 ms interval (r = 0.444, P = 0.02); in fact a degree of disinhibition was often present. There was no correlation between age and changeIHI10. Age was negatively correlated with the area under the recruitment curve (r = \u22120.585, P = 0.001) and the size of the maximum MEP collected (r = \u22120.485, P = 0.007). ChangeIHI and measures of corticospinal excitability were not intercorrelated. In conclusion, task-related increases in interhemispheric inhibition seem to diminish with advancing age. This phenomenon is specific for long-latency IHI and may underlie the age-related bihemispheric activation seen in functional imaging studies. The mechanism underlying changes in IHI with advancing age and the association with changes in corticospinal excitability need further investigation.\nIntroduction\nPhysical performance declines with increasing age (Hackel et al. 1992; Smith et al. 1999). Although age-related changes in muscular (Vandervoort 2002) and neural architecture (Dorfman and Bosley 1979; Haug and Eggers 1991; Madden et al. 2004) are an important cause of this decline, there is increasing interest in the role of functional alterations occurring throughout the motor system. Excitation\u2013contraction uncoupling (Delbono et al. 1995), motor unit remodelling (Wang et al. 1999), changes in the agonist\u2013antagonist activation pattern (Hortobagyi and Devita 2006) and reorganization of the central motor pathways (Kido et al. 2004; Ward and Frackowiak 2003; Minati et al. 2007) are all areas currently under study. Understanding the mechanisms underlying these phenomena will aid differentiation between inevitable functional failure and potential compensatory strategies which in turn may allow the development strategies to combat this age-related decline (Delbono 2003).\nFunctional imaging has been used to assess age-dependent changes in the cerebral motor system in humans (for review see Ward 2006). In general, motor task-related brain activation is seen in a wider network with advancing age. The exact pattern of age-related change depends on the task being performed. However, it has been repeatedly shown that in older individuals there is increased bihemispheric activation during the execution of motor tasks that in younger individuals involve mainly lateralized processing. In particular, with advancing age there is less de-activation of the primary motor cortex (M1) ipsilateral to the moving hand (Ward and Frackowiak 2003; Naccarato et al. 2006; Ward et al. 2007). It has been suggested, but not tested, that the underlying mechanism for this phenomenon involves changes in interhemispheric connections between the motor cortices.\nTranscranial magnetic stimulation (TMS) can be used to study interhemispheric connectivity between the motor cortices. At rest, this connectivity consists primarily of an inhibitory effect (interhemispheric inhibition, IHI) with a latency of 6\u201350\u00a0ms (Ferbert et al. 1992; Di Lazzaro et al. 1999; Daskalakis et al. 2002; Chen 2004). This tonic IHI is modulated and follows a specific time course during preparation and execution of voluntary movement (Murase et al. 2004; Duque et al. 2005, 2007). During the execution of an isometric muscle contraction, the IHI (at 10\u00a0ms latency) targeting the M1 ipsilateral to the moving hand has been shown to increase (Ferbert et al. 1992). This \u201cextra\u201d inhibition is in keeping with the primarily unilateral activation pattern seen in imaging studies of movement and has been interpreted as a means of minimizing mirror activity (Leocani et al. 2000; Duque et al. 2005). Task-related changes in longer latency IHI, i.e. 40\u00a0ms, have not been studied in detail but there is some evidence that they may be distinct to the ones seen in short latency IHI (Chen et al. 2003).\nPrevious TMS studies have shown that there is reduction of activity in the intracortical inhibitory circuits in the elderly (Peinemann et al. 2001; Hortobagyi et al. 2006). It has been suggested that reduced inhibition represents a mechanism to compensate for the deleterious effects of aging on several levels of the motor system including the primary motor cortex (Pitcher et al. 2003; Oliviero et al. 2006) but these notions remain mainly hypothetical (Wassermann 2002).\nThe main purpose of this study was to investigate whether normal aging has an effect on the task-related modulation of IHI measured at two different latencies, 10 and 40\u00a0ms. We hypothesized that with increasing age there would be less extra activation in the inhibitory circuits targeting the M1 ipsilateral to the moving hand. In order to gain some insight of the mechanisms and the physiological meaning of these changes we also performed measures of corticospinal excitability on the side contralateral to the moving hand and looked for correlations with age and IHI measures.\nMethods\nSubjects\nThirty healthy right-handed volunteers (mean age 42.9\u00a0years, range 19\u201378; 30% female) participated in the study after giving informed consent. They reported no history of neurological illness, psychiatric history, vascular disease or hypertension and they were not taking regular medication. The study was approved by local Ethics Committee.\nTranscranial magnetic stimulation\nSubjects were seated in an armchair with their eyes open. EMGs were recorded via Ag\/AgCl electrodes placed over the First Dorsal Intersosseus (FDI) bilaterally, using a belly-tendon montage. Signals were filtered (30\u00a0Hz to 10\u00a0kHz), amplified using a Digitimer 360 (Digitimer Ltd, Welwyn Garden City, Herts., UK) and stored on computer via a Power 1401 data acquisition interface (Cambridge Electronic Design Ltd, Cambridge, UK). Analysis of data was carried out using Signal Software (Cambridge Electronic Design).\nTwo figure-of-eight coils connected to two monophasic Magstim 200 stimulators were used for the experiments (all Magstim Co., UK). A 70-mm coil was used for motor hot spot identification and threshold measurements on both sides of the brain. The motor hotspot was defined as the scalp location where TMS consistently resulted in the largest MEP. The resting motor threshold (RMT) was defined as the lowest intensity needed to evoke an EMG response of 50\u00a0\u03bcV in 50% of the trials with the FDI relaxed; the active motor threshold (AMT) was defined as the intensity which evoked a 200\u00a0\u03bcV EMG response in 50% of the trials with a background FDI contraction of 10\u201315% of the maximum voluntary contraction (MVC). For all studies requiring activation of the FDI, visual feedback was provided using an oscilloscope.\nA recruitment curve (RC) for the active MEP amplitude elicited in the left FDI was obtained using the 70\u00a0mm coil while the subjects maintained a background FDI contraction of 15\u201320% MVC. Ten MEPs were collected and averaged at the following stimulus intensities: 90, 100, 110, 120, 140, 150, 160 and 170% AMT. The contraction level was tested by measuring the mean value of the rectified EMG in the 80\u00a0ms preceding the TMS pulse. The averaged peak-to-peak amplitude of the unrectified MEPs was then expressed as a ratio of the maximum peak-to-peak amplitude of the unrectified compound action potential (CMAP) evoked by supramaximal electrical stimulation of the ulnar nerve at the wrist using a Digitimer pulse stimulator (model DS7).\nInterhemispheric inhibition (IHI) was measured using a standardised paired-pulse paradigm (Ferbert et al. 1992). IHI is expressed as the reduction in the response to a suprathreshold TMS pulse (test) delivered to the M1 when another suprathreshold pulse is delivered to the contralateral M1 6\u201350\u00a0ms earlier (conditioning). Here, we concentrated on the IHI targeting the right M1. Hence the conditioning pulse was given over the left M1 using the same 70-mm coil. For the test pulse a smaller 50\u00a0mm figure-of-eight coil was held over the right M1; this allowed us to accommodate two coils on the head of all the subjects without compromising the exact positioning of the coils over the motor hot-spot (Ferbert 1992). MEPs were measured from the left FDI. Ten single (test) and ten paired-pulse (conditioning\u00a0+\u00a0test) trials were randomly intermingled and averaged. IHI was defined as the conditioned\/test MEP amplitude ratio, smaller values reflecting stronger IHI. Interstimulus intervals (ISIs) of 10 and 40\u00a0ms were studied in different blocks. IHI was initially measured at rest (restIHI10 and restIHI40). The stimulation intensity for both the conditioning and the test stimuli was adjusted to evoke an MEP of 1\u20131.5\u00a0mV in the contralateral FDI muscle. For the active condition (activeIHI10 and activeIHI40) the subjects were instructed to contract the right FDI to 15\u201320% of their MVC in response to an auditory cue preceding the conditioning stimulus by 600\u00a0ms. In this way, we made sure that the subject had reached the target level of background contraction for at least 200\u00a0ms before the conditioning pulse. This was to avoid measuring changes in IHI that are associated with the preparation of the movement rather than a steady isometric contraction (Duque et al. 2005, 2007). EMG activity in the left FDI was also recorded and contaminated trials were rejected to avoid the confounding effect of mirror activity. The stimulation intensity both for the conditioning and test stimulus was the same as in the resting state, as in previous studies (Ferbert et al. 1992). The absolute values of activeIHI were then expressed as a ratio to the values at rest for the respective ISI (changeIHI10 and changeIHI40). ChangeIHI therefore reflects the change seen in the IHI targeting the right motor cortex when the right hand is active. Values <1 reflect stronger inhibition, while values >1 reflect less inhibition.\nStatistical analysis\nAnalysis was performed using SPSS v.14 (SPSS Inc., USA). Exploratory plotting and statistical tests (Shapiro-Wilk) confirmed normal distribution for most variables. Some of the variables were skewed by a single outlier and this could not be corrected by logarithmic transformations; the outlier was therefore discarded from the dataset to allow use of parametric tests.\nData obtained during the RC paradigm were plotted against the stimulation intensity. In most subjects the resulting curves were sigmoidal in shape. Two representative examples, one from a young and one from an old subject, are shown in Fig.\u00a01. The area under the RC (AUC) was calculated using the method of trapezoid integration to provide a summary measure of the corticospinal output across all stimulation intensities. Data points were then fitted in the Boltzmann sigmoidal model based on the modified Levenberg\u2013Marquardt nonlinear least-mean-squares algorithm (Press et al. 1986). This function is often used to provide parameter estimates for RCs (Capaday et al. 1999; Devanne et al. 1997). The data were best fitted by a 4-parameter equation in which the amplitude of the MEP at a given stimulation intensity (I) is estimated as:  MEPmax and MEPmin represent the maximum and minimum MEP amplitude, respectively; I50 represents the stimulation intensity required to get a response 50% of the maximum. The inverse of the slope parameter (1\/slope) is directly proportional to the maximal steepness of the curve, which occurs at I50 (Devanne et al. 1997). The R2 co-efficient of determination was greater than 0.89 in all subjects.\nFig.\u00a01Representative examples of the recruitment curves plotting the amplitude of the active MEPs against the stimulation intensity. The circles represent the collected data. The line represents the relation predicted by the Boltzmann model. The area under the curve (grey area) was calculated with the method of trapezoid integration using the actual data collected during the construction of the curves. CMAP, compound motor action potential, AMT, active motor threshold\nPair-wise comparisons between different TMS measures were performed using paired t tests. Correlations with age and between TMS measures were assessed by computing Pearson\u2019s product-moment correlation coefficient. We have also looked for correlations between these parameters and gender. Partial and part correlations were employed as appropriate. Significance level was set at P\u00a0<\u00a00.05.\nResults\nThe mean and range of the values for all TMS measures are summarized in the Table\u00a01.\nTable\u00a01TMS measures of corticospinal excitability and interhemispheric inhibitionCorticospinal excitabilityLeft hemisphere Right hemisphereMotor thresholds (% stimulator\u2019s intensity)Resting (RMT)37.6 (28\u201365)36.4 (29\u201350)Active (AMT)28.4 (19\u201345)28 (22\u201339)RC of active MEP amplitude(CMAP corrected)Mean MEP amplitude recorded\u00a090% AMT0.02 (0.01\u20130.06)\u2013\u00a0100% AMT0.03 (0.01\u20130.07)*\u2013\u00a0110% AMT0.06 (0.02\u20130.18)*\u2013\u00a0120% AMT0.09 (0.03\u20130.35)*\u2013\u00a0140% AMT0.25 (0.06\u20130.51)*\u2013\u00a0150% AMT0.30 (0.10\u20130.57)*\u2013\u00a0160% AMT0.34 (0.13\u20130.56)*\u2013\u00a0170% AMT0.22 (0.39\u20130.62)\u2013Maximum MEP amplitude recorded0.38 (0.13\u20130.62)*\u2013AUC12.8 (1.8\u201323.4)*\u2013Parameter estimates (Boltzman model)\u00a0MEPmax\/CMAP0.4 (0.13\u20130.72)*\u2013\u00a0I50 (%AMT)137 (115\u2013168)\u2013\u00a01\/slope0.14 (0.05\u20130.37)\u2013IHI (targeting the right M1)10 ms40 msrest IHI0.61 (0.32\u20130.9)0.6 (0.28\u20131.1)activeIHI0.54 (0.1\u20130.89)0.57 (0.2\u20131)changeIHI (activeIHI\/restIHI)0.96 (0.42\u20132.47)0.97 (0.5\u20131.49)*CMAP, compound motor action potential; AUC, area under the curve; MEPmax, maximum MEP amplitude estimated; I50, stimulation intensity needed to get a response of 50% of the maximal MEP; IHI, interhemispheric inhibition; restIHI, IHI measured with both hands relaxed; activeIHI, IHI measured during a tonic contraction of the dominant hand at 15\u201320% MVC; changeIHI, values <1 indicate stronger IHI at the active condition*\u00a0Significant correlation with advancing age (P\u00a0<\u00a00.05)\nCorticospinal excitability\nThreshold values were not different between the left and right M1. Age was not correlated with RMT or AMT on either side or the amplitude of the CMAP. The mean amplitude (CMAP corrected) of the active MEPs recorded during the construction of the recruitment curves was negatively correlated with age at all stimulation intensities between 100\u2013160%AMT (100%AMT: r\u00a0=\u00a0\u22120.431, P\u00a0=\u00a00.02; 110%AMT: r\u00a0=\u00a0\u22120.397, P\u00a0=\u00a00.03; 120%AMT: r\u00a0=\u00a0\u22120.481, P\u00a0=\u00a00.007; 140%AMT: r\u00a0=\u00a0\u22120.556, P\u00a0=\u00a00.001; 150%AMT: r\u00a0=\u00a0\u22120.457, P\u00a0=\u00a00.11; 160%AMT: r\u00a0=\u00a0\u22120.463, P\u00a0=\u00a00.01). The same was true for the amplitude of the maximum MEP recorded (Pearson\u2019s r\u00a0=\u00a0\u22120.485, P\u00a0=\u00a00.007) (Fig.\u00a02a). Age was negatively correlated with the total area under the RC (Pearson\u2019s r\u00a0=\u00a0\u22120.585, P\u00a0=\u00a00.001) (Fig.\u00a02b). From the parameters estimated using the Boltzmann model, MEPmax showed a weak correlation with age (r\u00a0=\u00a0\u22120.397, P\u00a0=\u00a00.04); age was not correlated with the maximum slope or I50.\nFig.\u00a02Correlations between age and TMS parameters. There was a significant negative correlation between the amplitude of the maximum MEP recorded during the construction of the RC (a) and the total area under the RC (b). Age was negatively correlated with changeIHI at 40\u00a0ms (d) but not at 10\u00a0ms (c). CMAP, compound motor action potential, RC, recruitment curve constructed during tonic contraction of the target muscle, IHI, interhemispheric inhibition, restIHI, IHI measured with both hands relaxed, activeIHI, IHI measured during a tonic contraction of the dominant hand at 15\u201320% MVC, changeIHI, values <1 indicate stronger IHI at the active condition\nGender was not correlated with any of the TMS measures of corticospinal excitability.\nInterhemispheric inhibition\nMost subjects showed some amount of IHI at rest; only in one subject could restIHI40 not be elicited. Paired-samples t test did not show any difference between restIHI10 and restIHI40 (P\u00a0=\u00a00.44). Because the amount of IHI depends on the intensity used for both the conditioning and the test stimulus (Ferbert et al. 1992; Chen et al. 2003) we looked for correlations between these values and age. Age was not correlated with the intensities used to stimulate either the left M1 (70-mm coil; 45.7\u00a0\u00b1\u00a07.1 for IHI10 and 45.8\u00a0\u00b1\u00a07.2 for IHI40) or the right M1 (50-mm coil; 46.2\u00a0\u00b1\u00a08.6 for IHI10 and 46.9\u00a0\u00b1\u00a08.1 for IHI40). The amplitude of the MEPs elicited by the test stimulus and by the conditioning stimulus was slightly higher for the restIHI40 but the values fell within the target range of 1\u20131.5\u00a0mV (see below).\nDuring activation of the right hand the MEP elicited by the test stimulus in the resting left FDI tended to increase (from 1.23\u00a0\u00b1\u00a00.53 to 1.44\u00a0\u00b1\u00a00.67 for IHI10 and from 1.48\u00a0\u00b1\u00a00.58 to 1.64\u00a0\u00b1\u00a00.70 for IHI40), but this was not significant across the group, as reported earlier for low-strength isometric contractions (Liepert et al. 2001). As expected, the amplitude of the MEP elicited by the conditioning pulse in the right FDI increased significantly during activation of the muscle (from 1.27\u00a0\u00b1\u00a00.40 to 5.95\u00a0\u00b1\u00a01.69, P\u00a0<\u00a00.001 for IHI10 and from 1.5\u00a0\u00b1\u00a00.47 to 6.17\u00a0\u00b1\u00a02.57, P\u00a0<\u00a00.001 for IHI40).\nThere was an overall tendency for stronger IHI during activation of the right FDI at both ISIs, but this did not reach significance. In fact, changeIHI was quite variable ranging from more inhibition to less inhibition compared to the resting condition. Because changeIHI might be biased by the amount of IHI at rest we looked for correlations between changeIHI and restIHI; there was none for either ISI.\nThere was no correlation between age and changeIHI10 (Fig.\u00a02c). However, we found a significant positive correlation between age and changeIHI40 (r\u00a0=\u00a00.444, P\u00a0=\u00a00.02) (Fig.\u00a02d). In other words, when younger individuals activated their right hand the amount of inhibition targeting the ipsilateral (right) motor cortex increased; with increasing age this phenomenon was gradually attenuated and often reversed. Because age showed a significant correlation with the amplitude of the active MEPs we performed semi-partial correlations correcting for the change seen in the amplitude of the conditioning MEP between the resting and the active condition (changeMEP\u00a0=\u00a0activeMEP\/restingMEP). Again no correlation was found between age and changeIHI10; the correlation between age and changeIHI40 remained and was in fact slightly stronger (r\u00a0=\u00a00.553, P\u00a0=\u00a00.006).\nGender was not significantly correlated with any of the IHI measures; in line with a previous report (De Gennaro et al. 2004) there was a tendency for a positive correlation between female gender and the amount of restIHI10 targeting the right hemisphere, but this finding did not reach significance in our group of subjects (r\u00a0=\u00a0345, P\u00a0=\u00a00.07).\nFinally, changeIHI was not correlated with any of the measures of corticospinal excitability elicited from the stimulation of the left M1.\nDiscussion\nThe purpose of this study was to investigate the effect of age on the interhemispheric balance between the two primary motor cortices and in particular the modulation of inhibition targeting the right M1 during activation of the right hand (changeIHI). We found that the amount of tonic inhibition (restIHI) was very variable across the group but age could not explain this variability. However, age was significantly correlated with changeIHI. As reported previously, when young individuals perform a low strength grip, the inhibition targeting the ipsilateral M1 tends to increase (Ferbert et al. 1992). Our data show that with increasing age this extra inhibition is not as strong and some degree of disinhibition may be present instead. Interestingly, the effect of age was significant for the 40\u00a0ms ISI (changeIHI40) but no correlation was found between age and changeIHI10.\nOur results on changeIHI are in keeping with the findings from functional imaging studies. A number of studies have consistently shown that task-related activation is less lateralised in older individuals (for review see Ward 2006). This bilateral recruitment includes primary and non-primary motor areas; activation of the latter seems to characterise more complex tasks. Regarding the primary motor cortices, reduced deactivation of the M1 ipsilateral to the moving hand has been a common finding among studies using different scanning paradigms (Hutchinson et al. 2002; Ward and Frackowiak 2003; Naccarato et al. 2006; Ward et al. 2007). It has been hypothesized that this phenomenon could be mediated transcallosally. In support of this hypothesis, we have directly demonstrated that there is task-related reduction in interhemispheric inhibition with advancing age at least when the right hand is activated.\nThe differential effect of age on changeIHI10 and changeIHI40 is an interesting finding that adds to the current knowledge of the cortical circuits that these measures reflect. So far, it is thought that both short latency IHI (8\u201312\u00a0ms) and long latency IHI (40\u201350\u00a0ms) are mediated through excitatory callosal projecting neurons that are distinct from corticospinal neurones (Lee et al. 2007). In accordance with this view, corticospinal output and IHI measures in this study did not correlate. The callosal neurons are thought to activate inhibitory interneurons in the contralateral M1 (Daskalakis et al. 2002; Chen 2004), but these connections may differ for short and long latency IHI (Chen et al. 2003; Kukaswadia et al. 2005). The neurotransmitters involved are not entirely understood but there is evidence of significant involvement of GABAB-ergic activity. However, Irlbacher et al. (2007), have recently demonstrated that exogenous enhancement of GABAB activity strengthens long IHI but has little effect on short IHI at rest. Long IHI was also sensitive to GABAA agonists (Irlbacher et al. 2007). Finally, Chen et al. (2004), has shown that activation of the target muscle results in a reduction in the IHI targeting the active M1 (the opposite direction than the one studied here) measured at 8\u00a0ms but has little effect on the IHI40. We have shown that advancing age has an effect on the modulation of IHI during voluntary muscle contraction at 40\u00a0ms but not at 10\u00a0ms. We have thus provided additional evidence that IHI10 and IHI40 may respond differently to dynamic changes within the motor system and thus may represent physiologically distinct phenomena. This evidence could be strengthened in the future by testing the effect of age on IHI in both directions, i.e. the IHI targeting the motor cortices ipsilateral and contralateral to the moving hand.\nWhy is IHI40 not enhanced during voluntary contraction in older individuals? One possible explanation is age-related failure of the involved pathways. MRI studies have reported age-related reduced fractional anisotropy within the corpus callosum suggesting callosal fibre degeneration (for review see Minati et al. 2007). However, for callosal degeneration to be the cause of our finding, one would expect some effect on IHI10 as well. Another possibility is that the inhibitory circuits with which the callosal fibres synapse are less excitable. Previous studies have provided some evidence of age-related reductions in cortical inhibition. For example, GABAA-ergic short-interval intracortical inhibition (SICI) assessed with a biphasic stimulator (Peinemann et al. 2001) and cortical reciprocal inhibition (Hortobagyi et al. 2006) are reduced in older individuals. However, Wasserman et al. (2002) failed to show a correlation between SICI and advancing age in a large series of normal subjects. Others have found increased SICI in middle-aged adults (Kossev et al. 2002). Further experiments, which include active conditions are needed to study these associations in the aging brain. Finally, it should be noted that although TMS-measured IHI is thought to reflect mainly transcallosal connections, Gerloff et al. (1998) has demonstrated a role for other pathways involving subcortical and\/or spinal structures, the exact nature of which is unclear. Age related changes in these pathways may therefore also contribute to the correlations that we have observed.\nAnother question is whether the age-related reduced activity reported in multiple inhibitory systems, including IHI is an inevitable consequence of neurodegeneration or in fact represents a compensation strategy of the aging brain. Similar changes have been described in response to brain injury and have been interpreted as markers of cortical reorganization (Ward and Cohen 2004; Talelli et al. 2006). The relative preservation of changeIHI10 could suggest that the release from inhibition is not uncontrolled or at least it occurs in a hierarchical manner. For example, IHI10 may be important for direct suppression of mirror activity in the contralateral M1 (Duque et al. 2005). Longer latency IHI40, on the other hand, may reflect polysynaptic pathways extending even beyond the primary motor cortices. In that case relatively less inhibition in an active state could allow bilateral recruitment if that was necessary. In this study, there was no correlation between changeIHI40 (or any other IHI measure) and any of the measures of corticospinal excitability despite the fact that the latter appeared to be compromised in older individuals. This finding could mean that changeIHI40 does not reflect an adaptation to the failing corticospinal pathways. However, it should be kept in mind that if compensatory mechanisms are indeed operating with increasing age (or decreasing corticospinal output) TMS may not measure the real deficit but the compensated deficit. The next reasonable step could be to study a series of elderly individuals known to have different levels of manual dexterity and look for correlations between electrophysiological and behavioural measures, including mirror movements during performance of complex motor tasks.\nWe found variable effects of age on parameters relating to corticospinal excitability. Motor thresholds were not affected in keeping with previous reports (Wassermann 2002; Oliviero et al. 2006; Hortobagyi et al. 2006). We have found negative correlations between age and the amplitude of the active MEP at most stimulation intensities, between age and the amplitude of maximum MEP and finally between age and the total area under the recruitment curve (AUC). These results are in agreement with previous findings of reduced amplitude of the MEPs elicited during an isometric contraction (Oliviero et al. 2006; Sale and Semmler 2005). The values derived from the Boltzmann model did not add extra information, since there was no correlation between age and the maximal slope of the curve or the stimulation intensity required to obtain 50% MEPmax (I50). Pitcher et al. (2003) have previously reported that I50 significantly increased with increasing age. In other words, the recruitment of the MEPs was slower at low stimulation intensities. The authors discussed that this finding may reflect reduced excitability or asynchronous activation of spinal motorneurons. In this study we did not perform measures of spinal excitability mainly due to time limitations. We have, however, constructed our RCs during background activation of the target muscle; that means that some a-motorneurons are already activated and the resulting measures are thus less subject to changes in spinal cord excitability (Day et al. 1989). We propose that increasing age does have a detrimental effect on the total output of primary motor cortex and that the mechanisms underlying (or compensating for) this effect are variable; thus a summary variable such as the AUC might be more suitable as a surrogate marker of overall corticospinal output.\nIn conclusion, this study has shown for the first time that there is an age-related reduction in the extra inhibition targeting the right hemisphere during an isometric handgrip with the right hand. These changes may underlie the bihemispheric pattern of activation seen in functional imaging studies of older individuals performing a unimanual hand task. Should our findings be confirmed, studies of interhemispheric balance may prove to be a useful marker of reorganization in the aging brain.","keyphrases":["aging","transcranial magnetic stimulation","interhemispheric inhibition","motor system"],"prmu":["P","P","P","P"]}
{"id":"Anal_Bioanal_Chem-4-1-2226000","title":"Characterization of cellular chemical dynamics using combined microfluidic and Raman techniques\n","text":"The integration of a range of technologies including microfluidics, surface-enhanced Raman scattering and confocal microspectroscopy has been successfully used to characterize in situ single living CHO (Chinese hamster ovary) cells with a high degree of spatial (in three dimensions) and temporal (1 s per spectrum) resolution. Following the introduction of a continuous flow of ionomycin, the real time spectral response from the cell was monitored during the agonist-evoked Ca2+ flux process. The methodology described has the potential to be used for the study of the cellular dynamics of a range of signalling processes.\nIntroduction\nThe advance of miniaturized microfluidic systems for chemical and\/or biochemical applications based on so-called Lab-on-a-Chip technology has demonstrated that such micro systems represent the ability to \u201cshrink\u201d conventional bench systems to the size of a few square centimetres with major advantages of speed, performance, integration, portability, reduced sample\/solvent quantity, automation, hazard control and lower cost [1\u20136]. These merits are important for a variety of applications in analytical chemistry, biochemistry, clinical diagnosis, medical chemistry and industrial chemistry [1, 2]. Consequently, numerous micro total analysis systems (\u03bc-TAS) and micro reactor systems have been developed, and many more are currently under investigation [2].\nFor the study of cellular and subcellular systems, a wide range of analytical methods have been used with fluorescence techniques being the most common. In addition, fluorescence-based imaging is a highly attractive methodology for the study of organelle dynamics, identifying subcellular compartments and monitoring biological kinetics [7]. Over the last two decades, Raman spectroscopy has become an increasingly important technology with ability to study the biophysics and biochemical processes involving cells [4, 8\u201312]. Since Raman spectroscopy is based on vibrational transitions where frequency shifts are associated with specific molecular vibrations within the sample of interest, it enables the identification of polarizable bio\/chemical species, the elucidation of molecular structure and the investigation of interface reactions, all in a non-destructive manner. In addition, unlike fluorescence-based techniques, Raman spectroscopy does not require labelling dyes and since water is almost Raman \u201ctransparent\u201d, the technique is ideally suited for analysing cell-based biological systems. Coupling a Raman spectrometer with a confocal microscope enables the acquisition of full spectral information with a high spatial (<1\u00a0\u03bcm) resolution in three dimensions. Raman spectroscopy, however, suffers from an inherent poor level of sensitivity compared for example to fluorescence, which can be 12\u201314 orders of magnitude more sensitive. As a consequence, despite advances in detector technologies, the technique can be less than ideal for the direct detection of intracellular components present at low concentrations, which may take from a few tens of seconds to a few minutes for spectral acquisition [12]. With the unexpected discovery of surface-enhanced Raman scattering (SERS) by Fleischman et al. [13], Raman intensity can now be dramatically increased (a factor of up to 105\u20131010) with the inclusion of metallic nano structures either on a substrate surface or in a colloidal solution [8]. For cellular and subcellular analysis with SERS, colloid nanoparticles (e.g. silver or gold) are normally loaded into cells by different means such as general incubation (fluid-phase uptake) or ultrasonication-assisted uptake [14, 15]. Due to its chemical inactivity, gold nanoparticles are generally regarded to be more suitable for incorporation within living cells [14].\nTo analyse living cells using Raman spectroscopy, the cells are usually fixed at a specific location. The most commonly used method for cell fixation is to seed or grow cells on a substrate, e.g. a microscope cover slide. Other approaches such as optical tweezers have also been reported [9]. However, the conventional batch operation in a static system limits the in situ Raman analysis, especially when a series of reagent treatments are required. With those limitations in mind, the development of microfluidic and associated Lab-on-a-Chip technologies provides unique opportunities for delivering and immobilizing cells on a microchannel surface, prior to introducing different reagents with a continuous flow in a given sequence [3, 4] whilst under spectroscopic investigation.\nIn this study, we report the successful integration of a range of techniques including microfluidics, surface-enhanced Raman scattering (SERS) and confocal microspectroscopy which enables in situ characterization of single living CHO cells with high spatial and temporal resolution. By using the microfluidic methodology, cells and reagents were introduced into the chip in a continuous flow as a series of plugs in a given sequence where agonist (ionomycin) evoked intracellular Ca2+ fluxes and the cell\u2019s real time spectral response was recorded.\nMaterials and methods\nConfocal Raman microspectroscopy\nAll Raman spectra were acquired with a LabRam inverted microscope spectrometer, manufactured by Jobin Yvon Ltd. Figure\u00a01 shows the schematic of the experimental setup. The spectrometer was equipped with dual laser sources at wavelengths of 780\u00a0nm (diode laser, 70\u00a0mW) and 633\u00a0nm (He\u2013Ne laser, 20\u00a0mW), confocal optics, a holographic transmission grating, and a charge coupled device (CCD) detector with 1,024\u2009\u00d7\u2009256\u00a0pixels. The instrument included a precision motorized X\u2013Y sample stage for automated mapping at spatial resolution down to less than 1\u00a0\u03bcm and extensive software support (LabSpec 4.18) for data processing. In this study, an objective lens of \u00d750 magnification, 17-mm working distance and numerical aperture (NA) of 0.45 was used (L Plan SLWD 50, Nikon, Japan). This objective lens was mounted on a PI-721.10 piezo actuator (Physik Instrumente, Germany) for automatic focussing of the microscope objective at different depths in the Z direction enabling 3D mapping. A grating with 1,800\u00a0grooves\u00a0mm\u22121, a confocal aperture of 300\u00a0\u03bcm and an entrance slit of 150\u00a0\u03bcm were selected for the experiments. The Raman spectrometer wavelength range was calibrated using the centre frequency of the silicon band from a silicon sample (520.2\u00a0cm\u22121). Using these conditions, a typical acquisition time of 1\u00a0s was used to collect SERS spectra from cells within the microchannel.\nFig.\u00a01Schematic of experimental setup\nMicrofluidic device fabrication\nThe microfluidic device was constructed using a manifold clamping method according to published procedures with some adaptations [16\u201318]. The assembly of the device is illustrated in Fig.\u00a02a. Briefly, the microchip consisted of a PARAFILM\u00ae sheet (thickness 130\u00a0\u03bcm, American National Can Company, US) with a channel network and two glass plates which sandwiched the polymer film. The Y-shaped channel network (Fig.\u00a02b) cut through the film was 500-\u03bcm wide. The top glass plate (B-270, 25\u2009\u00d7\u200925\u2009\u00d7\u20093\u00a0mm) had three holes (diameter 1.5\u00a0mm) drilled through at appropriated positions in order to link the ends of the channels with inlet\/outlet tubing. The bottom glass plate was a thin quartz coverslip (22\u2009\u00d7\u200922\u00a0mm, Agar Scientific Ltd, UK) which had a thickness of 250\u00a0\u03bcm in order to minimise the glass background during Raman measurements. This sandwich chip was then clamped using two aluminium frames with screws. The windows on the frames were designed for tubing connections (through top frame) and for optical passage (through bottom frame).\nFig.\u00a02(a) Assembly of microfluidic device and (b) Y-shaped channel network (channel depth 100\u00a0\u03bcm, width 500\u00a0\u03bcm) with cells loaded (c) for examination\nTwo KDS 200 syringe pumps (KD Scientific Inc., USA) were used to deliver cells in suspension and test solutions into the microchip channel (Fig.\u00a02c). Ethylene tetrafluoroethylene (ETFE) polymer tubing with an inner diameter of 250\u00a0\u03bcm, on\u2013off valves, and appropriate fittings and connectors, all obtained from Upchurch (Upchurch Scientific Inc., USA), were used for plumbing to link the chip and the syringes.\nCell culture and assay reagents\nCHO-K1 (Chinese hamster ovary, Cricetulus griseus) cells were supplied by ATCC\/LGC Promochem (ATCC\u00ae No. CCL-61\u2122, LGC Promochem, UK). The cells were cultured routinely in DMEM\/F-12 medium without L-glutamine (Invitrogen Ltd, UK) which was supplemented with fetal bovine serum (Invitrogen Ltd) to a final concentration of 10%, and L-glutamine (Invitrogen Ltd) to a final concentration of 4\u00a0mM. An incubator was used at 37\u00a0\u00b0C supplying 5% CO2. The concentration of cells used in loading the chips during this experiment was in the range of 7.5\u2009\u00d7\u2009106\u00a0cells\u00a0mL\u22121.\nA wash solution consisting of a modified Tyrodes buffer was used to wash the cells and to prepare the test solution. The Tyrodes buffer was composed of 145\u00a0mM NaCl, 2.5\u00a0mM KCl, 10\u00a0mM HEPES, 10\u00a0mM D-glucose and 1.2\u00a0mM MgCl2. CaCl2 (99.5%, BDH AnalaR, 150\u00a0mM dissolved in Tyrodes buffer) and probenecid (98%, Sigma, 0.834\u00a0M dissolved in 1\u00a0M NaOH aqueous solution) were then added to the Tyrodes buffer giving final concentrations of 1.5\u00a0mM and 2.5\u00a0mM for CaCl2 and probenecid, respectively. Ionomycin test solutions were made by adding ionomycin stock (1\u00a0mM in DMSO) into wash solutions for a concentration of 50\u00a0\u03bcM. Ionomycin was obtained from Calbiochem (Calbiochem of EMD Biosciences, Inc., USA), and DMSO from Sigma\u2013Aldrich (99%, D2650). The flow rate for the introduction of ionomycin test solution into the microchip was 2\u00a0\u03bcL\u00a0min\u22121.\nGold colloid with a particle size of 50\u00a0nm (EM.GC50), suspended in water, was supplied by BBinternational Ltd, UK. The gold nanoparticles were introduced into cells by a passive uptake method where the cells were incubated with gold colloid solution at desired concentration (20% colloid solution, v\/v) and room temperature for 50\u00a0mins. Prior to loading into the microfluidic chip, the cells were washed with wash solution to remove the culture media and gold nanoparticles outside cells. The cells were re-suspended in the wash solution for measurements.\nResults and discussion\nSERS effects\nAfter loading cells into the microfluidic channel, Raman spectra were taken from a selected single cell (Fig.\u00a02c). By using the near-infrared laser excitation (780\u00a0nm) the optical and thermal effects of laser illumination on the living cells were minimised [19, 20]. In addition, using a near-infrared laser can significantly reduce the fluorescence interference background and light scattering from the quartz base plate. It has been reported that silver or gold nanoparticles with an individual size in the range of 20\u201360\u00a0nm can yield significant enhancement of Raman scattering for cellular analysis when nanoparticles are introduced into cells [14]. This has been confirmed in this study by using gold nanoparticles with a size of 50\u00a0nm.\nFigure\u00a03 shows a typical spectrum (upper) taken from a cell incubated with gold colloid solution. The spectrum was recorded in the range from 300 to 2,800\u00a0cm\u22121 with an acquisition time of 1\u00a0s. We observed that with the introduction of gold nanoparticles the Raman spectra were clearly detectable under those conditions. For comparison, a spectrum obtained from a cell incubated in medium without gold colloid solution is also shown in Fig.\u00a03 (lower). It can be seen that even though the spectra acquisition time was increased to 10\u00a0s it was still unable to obtain a good quality spectrum without gold nanoparticles present. This observation confirmed the significant enhancement of Raman scattering with the presence of gold nanoparticles which also enabled a fast spectra acquisition although the exact mechanism of the SERS enhancement of Raman signals is not fully understood [21]. The introduction of gold nanoparticles therefore significantly shortened signal acquisition times mainly owing to the increase in sensitivity which in turn allowed an array of spectra to be obtained in a relatively short time period.\nFig.\u00a03Raman spectra from cells following incubation in medium with gold colloid (upper spectrum) and without gold colloid (lower spectrum)\nCharacterization of single living cells by 3D mapping\nUsing the SERS technique described above it was possible to obtain spectra from a single living cell which represented a \u201cfingerprint\u201d from which various chemical constituents in the cell can be assigned. Combined with an automated microscope stage which offers spatial resolution (i.e. 1\u00a0\u03bcm), mapping of an entire cell area at a specific plane was carried out. It has been suggested from previous studies that most of the spectral bands associated with living cells occur in the range 800\u20131,700\u00a0cm\u22121 [11, 22, 23], hence this range was used in this study for mapping. Figure\u00a04a shows an \u201cimage\u201d of Raman spectra obtained from the middle layer across the single cell by mapping an area of 21\u2009\u00d7\u200921\u00a0\u03bcm2 with a spatial step of 1\u00a0\u03bcm in both X and Y directions. The mapping provided information on the distribution of selected bands, as seen in Fig.\u00a04b in the range from 1,290 to 1,370\u00a0cm\u22121, which represent most of the significant bands associated with DNA and proteins within a cell\u2019s nucleus and cytoplasm [11, 23].\nFig.\u00a04a Spectral mapping of a single CHO cell on an X\u2013Y plane and b corresponding spectra from three positions in the area of nucleus, cytoplasm and membrane, respectively\nIn general, the Raman spectra of single CHO cells showed contributions from all its cellular components including nucleic acids, proteins, lipids and carbohydrates. Table\u00a01 summarises the band assignment for the Raman spectra taken from CHO cells based on the published data [8, 9, 12, 23\u201327]. Comparison of the spectra taken from different positions across the cell on an X\u2013Y plane (Fig.\u00a04) indicated that strong peaks from the nucleus spectrum corresponding to DNA sugar\u2013phosphate backbone (895 and 1,142\u00a0cm\u22121), and bases G (1,320 and 1,487\u00a0cm\u22121), A (1,420 and 1,578\u00a0cm\u22121), T (1,176 and 1,376\u00a0cm\u22121) and C (1,420\u00a0cm\u22121) were noticeably reduced in the cytoplasm and membrane spectra (Table\u00a01). This change was expected as the nucleus contains high densities of DNA, whilst the cytoplasm also had significant quantities of RNA contributing to the corresponding peaks. As expected, the spectrum taken from membrane area showed significant peaks corresponding to lipids (1,068 and 1,453\u00a0cm\u22121).\nTable\u00a01Band assignment for Raman spectra of CHO cellsBands (cm\u22121)AssignmentsDNA\/RNAProteinsLipids830Phosphodiester BkB [12, 23, 24]Tyr [12, 23, 24]895Phosphodiester BkB, deoxyribose [12, 23, 24]940\u03bd (C\u2013C), \u03b1-helix [9, 12, 23]1,004Phenylalanine [9, 12, 23]1,065\u03bd (C\u2013O) [12, 23]\u03bd (C\u2013C) chian [25]1,126\u03bd (C\u2013N) BkB [9, 12, 23]\u03bd (C\u2013C) chain [25]1,144Ribose\u2013phosphate [12, 23]1,157Ribose\u2013phosphate [20, 26]1,176T, C, G [9, 12, 23, 24]Phenylalanine [12, 23]1,230C [12, 23, 24]1,266Amide III [25]\u03b4 (C=CH2) [25]1,295\u03b4 (CH2) [25]1,320G [24]1,342A [12, 23, 24]1,376T, A, G [9, 12, 23, 24]1,448\u03b4 (CH) [9, 12, 23]\u03b4 (CH) [9, 12, 23]1,482A, G [12, 23, 24]1,566Hemoglobin [25]1,578A, G, purine [12, 23, 24]1,603A, C [24]1,643Amide I [27]Abbreviations: BkB DNA sugar\u2013phosphate backbone, Tyr tyrosine, A adenine, T thymine, G guanine, C cytosine, \u03bd stretching vibrations, \u03b4 deformation vibrations\nUsing the confocal optics of the microscope system, the chemical concentration distribution in the Z axis at three levels in the nucleus was examined (Fig.\u00a05) and indicated that the main peaks positions were generally identical but the peak heights were markedly different, indicating a concentration difference. From Fig.\u00a05a significant a peak was observed at 1,320\u00a0cm\u22121 on the spectrum taken from Z\u2009=\u20093\u00a0\u03bcm (near to the bottom of the cell) which was assigned to DNA bases G [23], whilst the peak at 1,450\u00a0cm\u22121 from Z\u2009=\u20096\u00a0\u03bcm indicated a strong deformation from a C\u2013H stretch in proteins [8, 12, 23]. Thus, by collecting spectra from different spatial positions, it provided an approach to build a 3D mapping of the distribution of chemicals within a single cell.\nFig.\u00a05Raman spectra taken at three levels along the Z direction in the nucleus area\nIt should be noted that the spectra taken from different positions are distinct in terms of both Raman band amplitudes and band positions. This spectral variation can be attributed to the native chemical inhomogeneity within a cell. However, the possibility of non-uniform distribution of gold nanoparticles within the cell cannot be excluded [15]. In addition, this non-uniform distribution can also take place during the time-resolved monitoring because dynamic processes inside the cell can lead to local fluctuations of the particle densities and hence changes of the spectra. To further develop this spectroscopic technique for both qualitative and quantitative analysis, technologies need to be explored in order to deliver and position nanoparticles within cells in a controllable format for a uniform distribution. Nevertheless, it is still possible using this promising methodology to examine a specific point within a cell in a dynamic way with time, especially when applying stimulation, e.g. an agonist, under microfluidic control conditions.\nIn situ monitoring of cellular chemical dynamics\nFinally, we monitored the chemical dynamics of the cell when exposed to the agonist ionomycin [3, 28]. After loading cells into the chip channel, a 40-min period of settlement was allocated before introducing the ionomycin solution at a constant flow rate of 2\u00a0\u03bcL\u00a0min\u22121. Whilst focussed on a region close to the cell nucleus, a series of spectra were taken at an interval of 1\u00a0s in the range from 800 to 1,700\u00a0cm\u22121 (Fig.\u00a06a). It can be seen from the spectra (Fig.\u00a06b) that most of the peaks corresponding to nucleic acids, proteins and lipids remain visible with time but the peak heights vary noticeably, indicating the concentration change of these compounds. One of the most significant changes is the appearance of a peak at 1,643\u00a0cm\u22121 which can be assigned to amide I [27]. The time profile of this peak (Fig.\u00a06c) reveals the concentration change of amide I within the cell, showing a similar trend to that of the Ca2+ flux evoked by the agonist ionomycin [3] which is commonly used in biomedical research to stimulate the intracellular production of proteins such as interferon [29]. Since amide I contributes spectrally to the Raman spectrum of interferon, it follows that the in situ monitoring of such molecules could in future be used to produce characterization of protein expression dynamics at subcellular levels. Clearly, more studies are required in order to understand the mechanism and cause of the concentration change of amide I within the cell when exposed to agonist ionomycin.\nFig.\u00a06In situ monitoring of cellular dynamics by recording a series of Raman spectra (a) from a specific area at an interval of 1\u00a0s. The first spectrum, at time 0, was taken when no agonist was introduced (b); (c) shows the time profile of the peak at 1,643\u00a0cm\u22121 corresponding to the change of amide I\nConclusions\nThe SERS technique has been used for characterisation of single CHO (Chinese hamster ovary) cells with a microfluidic device where gold nanoparticles were introduced into cells for Raman enhancement. The use of an inverted microscope optical systems in combination with a charge coupled device (CCD) detector allows the measurement of Raman spectra for simultaneous analysis of bio\/chemical species within cells. In addition to a precision automated X\u2013Y sample stage, the confocal optics provides discrimination between points of different depths within the cell, enabling chemical mapping in three dimensions. By using microfluidic methodology, the cell manipulation and reagent delivery were performed in a controllable manner. By introducing cells and test reagent into the chip in a continuous flow as a series of plugs in a given sequence, it enabled the in situ chemical characterization of single CHO cells with a high degree of spatial and temporal resolution. This allows the real time monitoring of the dynamics of the agonist-evoked Ca2+ flux response. The approach described has the potential to be used for the study of the spatial dynamics of a range of intercellular processes.","keyphrases":["microfluidics","confocal microspectroscopy","surface-enhanced raman scattering (sers)","chinese hamster ovary (cho) cells","dynamic monitoring"],"prmu":["P","P","P","R","R"]}
{"id":"J_Med_Internet_Res-6-2-1550592","title":"Setting the Public Agenda for Online Health Search: A White Paper and Action Agenda\n","text":"Background Searches for health information are among the most common reasons that consumers use the Internet. Both consumers and quality experts have raised concerns about the quality of information on the Web and the ability of consumers to find accurate information that meets their needs.\nIntroduction\nSearches for health information are among the most common reasons that consumers use the Internet. The Pew Internet & American Life Project (Pew) reported in 2003 that 80% of Americans with Internet access have used the Web to get health or medical information [1]. The Internet has transformed the ability of consumers to find health information and to connect with other individuals with similar interests. The Internet has been recognized as an important source of health information by the federal government, which established a series of goals relating to access and quality of information on the Internet in the Healthy People 2010 action plan [2].\nHealth information on the Internet can dramatically improve consumers' health-care and lifestyle choices. However, increased access to Web-based information has also raised concerns about the quality of information consumers are using, and the impact of this information [3]. Disparities in access to information have also become apparent. These factors suggest the need to better understand how consumers find health information on the Web, how to evaluate the quality of information retrieved, and how to help consumers to critically evaluate and manage information. These factors suggest the need to better understand how consumers find health information on the Web, how they evaluate the quality of information retrieved, and how to they could be helped to critically evaluate and manage information.\nResearch on health Web sites raised concerns about the quality of information on the Web [4]. A 2001 study by RAND for the California Healthcare Foundation showed that information on health Web sites is often incomplete or out of date [5]. This might be of little concern if consumers routinely consulted health-care professionals about the information. However, Pew found that 69% of consumers did not discuss the information they found with a doctor or nurse.\nMany people use search engines to find the information they use to help make personal health decisions. Search engines and the Internet have vastly improved access to health information for many consumers. However, search processes and results vary considerably among search engines, and are not transparent to consumers. The criteria used to identify and rank health-related Web sites vary among search engines, and often is not apparent to consumers. Search results may be affected by the structure of content on health Web sites, consumer search terminology, and the use of paid placements by the search engine.\nIn short, research on health searches suggests that the process by which consumers locate health information on the Internet, and the evaluations they make regarding which Web sites to review are important variables in the quality of information they ultimately view and use. Improved understanding of factors influencing online searches will facilitate technical and educational approaches for maximizing quality and benefit of health searches.\nMethods\nIn 2003, URAC and Consumer WebWatch (CWW), a project of Consumers Union, carried out a project funded by the Robert Wood Johnson Foundation to examine factors influencing the results of online health searches and to develop an agenda for future research and development that would improve the results of health searches. We reviewed published literature and industry reports, and convened two stakeholder groups consisting of consumers, quality experts, search engine experts, researchers, health-care providers, informatics specialists and others.\nLiterature Review Method\nOur literature review was not exhaustive: its purpose was to provide a baseline understanding of consumer, Web site, quality measurement, and search engine factors that influence the results of searches for health information. We conducted a search of key terms in the Cumulative Index of Nursing and Allied Health Literature (CINAHL), Medline, PubMed, Expanded Academic ASAP, Lexis-Nexis, Proquest, Ingenta, and related databases in health care, information science, and computer science. The initial searches took place in early 2003, but citations were added as they were identified.\nWhere initial searches revealed poor topic coverage, associated reference lists, books and other media that were considered to inform the topic were included. The following search terms were included: Web-based, Web site, information quality, Web search, consumer health, eHealth, health information, search engine, information retrieval, information seeking. We also examined bibliographies of articles retrieved by electronic searches and solicited recommendations from members of the project advisory committee. We discontinued searching in specific topic areas when project staff believed we had adequately described current understanding of key issue areas.\nMethods for Convening Stakeholder Meetings\nAn open announcement about the project and recommendations from industry leaders helped identify interested stakeholders, and participants were selected by URAC and CWW with guidance from a project advisory committee. Not everyone invited was available to attend. We attempted to achieve a balance of different stakeholders at each meeting. Meetings were held in California and Washington, DC to facilitate participation.\nThe purpose of each stakeholder meeting was to review existing knowledge about results of consumer searches for health information, and to develop recommendations for additional research, technical improvements, and educational approaches needed to improve the results of online consumer searches for health information. Participants reviewed the summary recommendations presented in this article after the meeting and had the opportunity to comment, but were not asked to vote on or endorse the recommendations.\nFor the purposes of this project, we assumed that most searchers would prefer information that is accurate and reliable. These attributes are also components of effective health communication [2]. This was our working definition for quality Web sites. The perception of other elements that might be used to define quality, such as the site's reading level and comprehensiveness, will vary depending on the user and the user's needs at a given time.\nResults\nResults of Literature Review\nHow Consumers Use the Internet to Locate Health Information\nAn April 2003 report from the Pew Internet & American Life (Pew) report provided an overview of the US Internet consumer population [6]. The study found that Internet access has grown across-the-board, but that demographic gaps remain. A variety of factors continue to separate Internet users from non-users. Internet users tend to be younger and more affluent, and are more likely to be employed, white, well-educated, and to be suburban or urban residents.\nPew noted that consumers often overestimate their knowledge of the Internet and their ability to locate information. A 2002 analysis by Houston et al using Pew data noted a need to educate patients about searching for health information online and for tools to help them identify high quality information [7]. They also found that chronically ill Internet users were often relatively new to the Internet, but noted that they were more likely than those in good health to discuss findings with their physicians.\nConsumer Search Strategies\nA 2002 Pew Internet & American Life Project poll found that the typical health-information seeker usually starts searching for medical information at a general search site, not a medical site. Eighty-one percent of online health seekers start at a search engine or use the search function of a general portal such as the Yahoo home page, MSN, or AOL. Consumers visit two to five sites during an average visit and typically spend at least thirty minutes on a search [8].\nSeveral studies have investigated behaviors consumers exhibit in retrieving and health information on the Internet and in assessing its quality. Eysenbach and K\u00f6hler, examining Web searchers in Germany, found that although search technique was often suboptimal, Internet users found the health information they were looking for relatively quickly [9]. A search optimization firm, iProspect, reports that users generally use the same search engine for all types of search requests. Users look at up to three pages of search results to determine relevance, and abandon a search if they do not find appropriate results in the first three pages. Users usually modify their query after abandoning an initial search, and may at that point change search engines [10]. These findings illustrate the importance of search engines to the process of retrieving health information. They imply a business rationale for search engines to ensure that health searchers locate what they want, since they may otherwise lose search traffic.\nComprehension, Literacy, and Access Issues\nSearches are heavily influenced by the search terms used, even when the terms used are considered to be synonyms. Use of lay terminology for a health subject can result in unrelated or misleading information [11]. Berland et al concluded that accessing health information using search engines and simple search terms was not efficient because Web sites are inconsistent in their provision of key information, and because high reading levels are required to comprehend Web-based health information [4]. Also, the relevance of information located was often of limited value, which may have been due to terminology used in the original search phrase. Non-English speakers face challenges finding and reviewing information on the Internet. One Internet accessibility study for people with disabilities found that there are significant access barriers. Governmental and educational health-information Web sites were more accessible than other categories, such as Web portals and community sites [12].\nPhysician Responses to Internet Information\nA study of physician views on online information found that physicians increasingly encounter patients who have conducted health searches. Use of the Internet by patients does appear to affect treatment processes: for example, many physicians reported having changed the treatment protocols they had initially planned as a result of consumer requests. Although most physicians believe the information their patients find is accurate, many believe that having to discuss this information with their patients decreases their efficiency and challenges their authority. Some are also concerned that the information may be inaccurate. The study concluded that quality of information on the Internet is critical, as it does influence both patient requests and physician treatment choices [13]. In an effort to steer patients to credible Web sites, some health-care organizations have begun to suggest ( \"prescribe\") credible Web sites to their patients in the course of their consultations [14].\nConsumer Evaluation of Web Site Credibility\nExperts and consumers use different criteria for evaluating the quality of Web sites. Eysenbach found that consumers assessing the credibility of a Web site primarily looked for the source, a professional design, a scientific or official touch, language, and ease of use. Study participants never checked any \"about us\" sections of Web sites, disclaimers, or disclosure statements. Very few participants noticed and remembered which Web sites visited [9]. A Consumer WebWatch (CWW) study of consumers reported findings similar to Eysenbach's: once people get to a site, they do not use rigorous criteria to assess the site's credibility. For example, they almost never referred to a site's privacy policy. The average consumer paid far more attention to the superficial aspects of a site, such as visual cues, than to its content. Nearly half of all consumers in the CWW study assessed the credibility of sites based in part on the appeal of the overall visual design, including layout, typography, font size, and color schemes. In comparison, a parallel group of health and finance experts were far less concerned about the surface aspects of these industry-specific types of sites and more concerned about the breadth, depth, and quality of a site's information [15].\nHow Web Sites Influence Availability of Quality Health Information\nTechniques for Conveying Information about Web Site Content\nThe structure of a Web site influences how information can be retrieved from the site by a search engine, as well as the usability of the site for consumers. Coding and structure of Web sites can facilitate retrieval by search engines or can pose a barrier to information retrieval. Coded information on a Web site is processed through the search engine algorithm, and determines whether and how the site is ranked in search returns. The same tags and codes that can be used to highlight information on a legitimate Web site may also be used by \"spoofers\" who try to lure traffic onto the site.\nIn general, Web sites can support retrieval of information on their pages by using metadata, metatags and keywords to guide search crawlers to important content. These codes provide a means for relaying information directly to the search engine. Keywords are recognized indicators of specific services or products that can be used to increase specificity of searches and help Web sites attract \"qualified\" traffic. One strategy for enhancing search rankings of quality Web sites is to code certain types of information for consistent retrieval by the search engine. Efforts are under way to implement metadata codes to support a \"semantic Web.\" The semantic Web uses code to establish relationships between words to enable search engines to effectively understand intent, rather than simply identifying the presence of a search term [16].\nQuality Indicators for Web Site Content\nEysenbach et al found wide-ranging differences in studies of the quality of health Web sites. There are significant variations in study methods and rigor, quality criteria, study population, and topic chosen. Operational definitions of quality are often inconsistent. As a result, the conclusions on quality of health-related Web sites vary widely. Eysenbach found that the most frequently used quality criteria include accuracy, completeness, readability, design, disclosures, and references provided [16].\nGriffiths and Christensen evaluated the quality of Web-based information on treatment of depression to identify potential indicators of content quality, and to establish whether accountability criteria are indicators of quality [17]. They found that although the sites examined contained useful information, their overall quality was poor. Sites typically did not cite scientific evidence in support of their conclusions.\nResearchers have also studied the correlation between popularity of Web sites and quality of content. Meric et al found that more popular breast cancer-related Web sites were more likely than less popular ones to contain information on ongoing clinical trials, results of trials, and opportunities for psychosocial adjustment. These characteristics were also associated with a higher number of links. More popular sites were more likely to provide updates on other breast cancer research, information on legislation and advocacy, and a message board service. Measures of quality such as display of authorship, attribution or references, currency of information, and disclosure did not differ between popular and less popular sites [18]. In similar findings, Kunst et al found that while there is a correlation between credibility features and accuracy of information, the association is relatively weak [19].\nThese findings suggest that additional research is needed to identify indicators of content quality, and to correlate consumer preferences to quality indicators. Sites that include content correlated with popularity may best meet the public's desire for health information. Current search algorithms may not be in agreement with quality clinical indicators and performance measures currently used throughout the health-care industry.\nCodes of Conduct\nA wide range of tools has been developed to assist site developers to produce good quality sites and for consumers to assess the quality of sites. Adherence to accepted codes could theoretically be used as a factor in searches. Ratings instruments include codes of conduct, quality labels, user guides, filters, and third party certification. The value of these tools is unclear: studies have demonstrated that consumers do not routinely seek out information on certifications or adherence to voluntary codes. However, it is assumed by many that such codes benefit consumers indirectly by influencing Web site behaviors and practices. For example, most standards require sites to implement privacy protections and disclosure of site information as consumer protections. No research has been done on the effect of compliance to a code of conduct on Web sites.\nA number of organizations have developed quality criteria for health-related Web sites, some with verification and some completely voluntary. Voluntary, self-certifying standards have been developed by the eHealth Code of Ethics of the Internet Health Coalition [20], the American Medical Association [21] and the Health On the Net (HON) Foundation [22]. URAC has developed a health Web site accreditation program that involves independent verification of compliance with its standards. URAC accreditation includes review of the Web site by an external auditor [23].\nWeb Site Rating Instruments\nWeb site ratings could be potentially be used to inform searchers and search engines as well, if ratings could be clearly correlated to quality. Two common approaches to rating Web sites include expert ratings, and user (consumer) ratings. Gagliardi and Jadad conducted two evaluations of Web site rating tools, published in 1998 and 2002 respectively [24,25]. They concluded that ratings instruments tend to proliferate and disappear, and that few have been validated for direct correlation between standards and quality. Few provide details on how they were developed, or provide instructions for use, or information about the inter-observer reliability and construct validity of the measurements.\nKim et al reviewed published criteria for evaluating health-related information on the Web, and identified areas of criteria-based consensus [26]. They identified 29 published rating tools and journal articles that had explicit criteria for assessing health-related Web sites. The most frequently cited criteria were those dealing with content, design and aesthetics of site, disclosure of authors, sponsors or developers, currency of information (includes frequency of update, freshness, maintenance of site), authority of source, ease of use, and accessibility and availability.\nA number of tools are available to guide users in evaluating information on the Web. Interactive user guidance systems can be used to assess characteristics of Web sites. Tools such as DISCERN and QUICK allow Web site users to assess Web site credibility by responding to a series of questions [27]. Other organizations such as the National Library of Medicine, which operates MEDLINEplus, and the Medical Library Association, have developed guidelines and tips for consumers to evaluate health Web site content [28]. The European Union sponsored a collaborative project called MedCERTAIN to develop a rating system to enable consumers and professionals to rate quality information on the Web. The MedCERTAIN project evolved in a project called MedCIRCLE, which has developed a metadata coding language to mark quality indicators on health Web sites [29].\nDiscussion\nSearch Engines and Mediators of Health Information\nElectronic and Human Mediation\nSearch engines serve an essential function in enabling users to find relevant information on the Internet. Recognizing the challenges of sorting the enormous amount of information on the Internet, many organizations are augmenting or mediating the results of electronic searches. Mediation can be either electronic or human-augmented techniques for reviewing information and making a pre-selected set of information available to consumers. One challenge to search engines and human mediators is making access to personalized information as effortless as possible, as consumers rarely use even the advanced search features currently available to them [30].\nHow Search Engines Work\nSearch engines and Web directories play a central role in facilitating access to health information. Web directories are organized Web site listings put together by human reviewers. Search engine listings are put together by automated systems and lack a navigable structure. Directories usually concentrate on indexing Web sites, while search engines typically index individual Web pages. Consumer searches for keywords will result in a valid match only if the keyword appears in the Web site's description. Hybrid models of search engines and directories are common.\nSearch Engine Indexing and Retrieval Methods\nVirtually all commercial search engines rely on large powerful databases that utilize automated search agents called robots (\"bots\"), crawlers, or spiders. Search agents crawl the Web continuously to index information on Web sites. Crawlers capture metadata, page titles and textual content, and add them to the search engine's index or main database. The search engine's algorithm compares indexed data to the user term to process a search. Search engine algorithms are quite complex and scientific. They make frequent use of complementary directories aimed at optimizing and positioning Web sites in the right categories. Search algorithms are closely guarded as proprietary corporate information [31].\nCurrent metrics for evaluating search engines include initial page retrieval capacity and the ability to revisit Web sites to update information. Currency of information, as demonstrated by elimination of non-working links to Web sites is also a performance metric. These criteria are features of business performance, not necessarily the content relevance or quality of the sites returned by a search.\nContent and format of Web sites determine how they are indexed by search engines. Some search engines use keyword location, frequency, phrasing, and density as indexing and ranking factors. Type and number of links associated with a Web site are common indexing factors. Web sites also use tags to identify certain types of information. Search engine databases include only Web sites that have been registered with or indexed by the search engine-hence the importance of Web site developers making their sites accessible to automated agents, or becoming known to directory developers.\nRanking and Ratings\nRanking of sites in the final display of search results is of great importance to Web sites, users, and search engines. Ranking effectively drives the likelihood of particular sites being recognized and visited because, as noted, consumers rarely look at more than three pages of results. A poorly designed or executed search may produce an unwieldy list of Web site results that is difficult to navigate. Alternatively, searches that are too narrowly drawn may omit important sites.\nPaid preference and placement by search engines also affects which sites are retrieved in a given search [32]. A study by CWW demonstrated that consumers experience considerable confusion about paid listings, and may not distinguish them from other returned listings [33]. The Federal Trade Commission has also expressed concern about how paid placement is disclosed to consumers, and has warned search engines to clearly distinguish advertising from search returns. Search engines may operate their own paid placement programs or obtain search results from third parties, who in turn operate paid placement programs.\nMediated Searches\nMediated searches may be as simple as having a librarian assist with a search, or they may be based on much more complex algorithms. Participants in the URAC\/CWW stakeholder group noted that medical and general librarians play an important role in helping large segments of the population retrieve online information and learn effective search strategies. More complex mediated search strategies employ both human mediation and electronic queries to interface with users and focus a search.\nMany search engines offer filters that allow users to exclude unwanted search results, most typically pornographic sites. Users, including libraries, can also install blocking software to prevent unauthorized use. However, this electronic mediation may unintentionally block desired health information and create an access barrier. For example, because pornography-blocking software and filters cannot perfectly distinguish between pornographic and non-pornographic Web sites, such products may block access to legitimate health-information sites, particularly those related to sexuality [34].\nGateways employ filters, either electronic or human, to accept or reject types of sites of information based on preset criteria. Gateways are used to organize information on the Internet through selection of resources based on quality and relevance of information to a particular audience. Internet resources are reviewed, classified, and stored with descriptive information. In the US, healthfinder.gov\u00ae, is a widely used gateway to selected consumer health and human services information resources provided by US government agencies and other organizations serving the public interest [35].\nParticipants in the stakeholder meetings noted that domain name extensions such as .com or .org could be used as a distinguishing feature of Web sites for the purpose of focusing search efforts. The World Health Organization is considering the feasibility of requesting a \"dot health (.health)\" extension for a pre-selected set of trusted Web sites [36]. In informal proposals describing the .health domain name, the extension is proposed for health information, services and organizations under a framework promoting minimum standards of conduct. Oversight of Web sites would be delegated to independent verifying organizations. The advantage to sites for adhering to standards of content quality would be more ready identification of sites by search engines as a result of the .health domain name.\nStakeholder Discussion of Literature Review\nResearch Needs to Address Consumer Evaluation of Web Quality\nThere is great variation in how consumers seek information via the Internet, and in how successful they are in searching for health information. Since there is significant consumer-level variation in how consumers search for health information, search algorithms that support variation and still return expected results will meet consumer needs most effectively. Additional research is needed on information needs of different consumer segments and how to effectively educate differing consumer segments to improve the results of their health searches. Research is needed on how to efficiently validate the quality of Web sites and communicate this information to consumers.\nResearch Needs for Web Site Quality Indicators\nThere is a need for tools to enhance recognition of quality Web sites by consumers and search engines. Such tools may be implemented by Web sites themselves, for example through increasingly sophisticated coding to highlight quality indicators. The MedCERTAIN project has been created precisely to address this issue, and has developed the HIDDEL vocabulary to mark features of Web sites [29]. Technical tools can be used to direct consumers more effectively to relevant, high quality information. In addition, since there are currently multiple tools for either self-evaluation or third party evaluation of Web sites, future research should be undertaken to validate these tools.\nAs noted, gateways filter information to increase its relevance to consumers and provide expert assessment regarding validity of sources is available. It may also be useful to develop more sophisticated search models for providing useful and relevant information to consumers via customization approaches. Such approaches could potentially be embedded in search algorithms. In addition, more research is needed on the impact of Internet-based health information on outcomes. The benefits and risks of health information, both from a health outcome and a system outcome (quality, cost), are poorly understood and should be examined further.\nResearch Needs for Search Factors Influencing Search Results\nSearch engines are increasingly important as a tool for locating and organizing information from the vast Internet resource. The volume of information on the Web is so significant that consumers may need different types of mediators, such as search engines or librarians, to help manage the volume of information. Human assistance is also helpful to counteract electronic spoofing and to help consumers overcome limitations in their search strategies.\nTo effectively improve health searches, more information is needed about search algorithms and how quality factors are identified in the algorithms. Search engines are also developing technology to search for synonyms, which may enhance health searches conducted by laypersons. It may also be helpful for search engines to develop methods to distinguish health related searches from other types of searches, rather than using a simple word match. Search technology to intuit consumer needs more effectively and learn from repeated searches could help search engines steer consumers to quality results. New technologies may ultimately be more effective than electronic filtering, requiring consumers to apply filters, or modifying their search strategies.\nWith technology advances, search engines may be able to identify quality proxies that could improve page rankings of high-quality Web sites. Search engines could, for example, give higher ranking to \"official sites\" for diseases. They could also piggyback onto credibility assessments provided by groups such as healthfinder.gov, or give higher ranking to sites listed in directories from trusted independent sources. Ultimately, adoption of technological solutions depends on the ability of researchers to understand the relationship between electronic proxies for quality and actual quality of content.\nDiscussion of Stakeholder Recommendations for Next Steps\nThe URAC and CWW expert panels discussed consumer, Web site, and search engine factors that influence the outcomes of health searches. In the course of discussion, they developed a number of recommendations for future research and development (Textbox 1). Their recommendations fell into several categories: needs for health services research, consumer and provider education, technological improvements, and development of tools and information to improve the results of health searches. For some recommendations, the evidence base for implementation is strong; for others, not. Implementation of some recommendations will be enhanced by creation of a national research agenda for health information and targeted funding to study and improve consumers' ability to locate and retrieve quality health information on the Internet. Other recommendations could be embraced at any time by researchers, educators or technology organizations as a business need becomes increasingly evident.\nRecommendations of the Group\nLeadership for Health Search Improvement\nOrganizations concerned about the quality and accessibility of health information online should continue to collaborate to promote \"health search literacy.\"Collaborators should convene a leadership summit on health search literacy to discuss feasibility and implementation of many of the recommendations herein.Collaborating organizations shouldwork with funding organizations to develop a comprehensive long-term research agenda to improve health searches and increase access to quality health information;develop enhanced research methodologies to evaluate the quality, impact, and effectiveness of online health information.\nConsumer-directed Tools\nCreate tools to support consumer health-information needs, including preset, prescreened health bookmarks and more guidance on how to reach health gateways and portals containing trusted health content.Develop and circulate a public domain brochure on health search strategies that could be branded and distributed by physicians, employers, health plans, and others to educate consumers.Develop public domain interactive, validated search strategy content pages that could be branded and used by health Web sites.\nResearch Needs\nIdentify the search needs and capabilities of diverse populations of searchers, including culturally diverse users and searchers with health needs of differing intensity and severity.Develop more understanding about how consumers interpret online health information, assess its credibility, and make health-related decisions.Research the relationship between consumer search strategies and consumer expectations for results to determine effective approaches for conveying information on the Internet.Research factors affecting physician assessments of Web-based information and how quality content affects physician recommendations to patients about online health-information resources.Assess the relationship between expert accreditation, quality seals, ratings and content quality, as well as the impact of such endorsements on both consumer behavior and Web site behavior.Research the correlation between Web site traffic volume and consumer satisfaction, particularly for health Web sites where there is variation in dimensions of quality such as accuracy, comprehensiveness, ease of navigation, and reading level.Evaluate content quality of Web sites in different domains, (eg, .gov, .edu, .com, and .org) to identify similarities and differences related to quality within and across categories of Internet domain namesEvaluate the impact of Internet-based health information on health outcomes: utilization, behavior change, knowledge, burden of illness and disease, or other measures.Research the relative effect of each component of a search algorithm (word frequency and placement, links, etc) for finding health information.Validate elements of some search algorithms, such as link frequency, as indicators of value\/quality.Conduct periodic studies to monitor changes in accuracy and quality of content over time, including updating findings from the California HealthCare Foundation \/RAND study [5].\nEducation Agenda\nDevelop models for offering health search education at teachable moments and in diverse consumer settings.Promote dissemination of existing educational tools and resources to assist consumers in evaluating health information on the Web more effectively.Develop user-appropriate tools and approaches to assist Internet users with special needs. High priority user groups may include disability, low literacy, and non-English speaking groups.Urge provider organizations to educate provider members on the value of offering Internet information and interactive learning recommendations as part of the therapeutic intervention.Educate health Web site developers on how to make information easy to find and how to meet the content-level of their intended users.Urge education organizations, in collaboration with health organizations, to develop a school-based or publicly available health search curriculum.\nTechnology Improvement Agenda\nContinue to develop interactive features on search engines and sites to customize and personalize health searches.Develop more functionality for search engines to enhance selected health queries by offering additional relevant information.Develop technological markers or indicators that could be uniformly applied by Web site developers to indicate accuracy and comprehensiveness of health Web sites.Develop codes to indicate when information on a Web site supercedes previous information.Develop collaborations between health quality and search engines experts to develop codes for validated quality proxies.Develop search technology similar to that used in the commercial sector to direct consumers to related, relevant information based on both searching and viewing behaviors.Enhance personalized searches by building search engine capability to \"learn\" from repeated searches and user behavior.\nExpanding the Market for Quality\nDevelop a health equivalent of \"BizRate\" or \"eBay\" surveys that can be used by consumers to evaluate Web sites after viewing. Existing models for such a survey could be adapted and disseminated.Sponsor a competition for individuals or organizations to design a search algorithm that returns the most credible health results as evaluated by experts. Design a separate contest for the most effective business plan to make the business case for building quality factors into health searches.\nConclusion\nThe Internet has opened a vast library of information to consumers of health information and made that information more accessible than ever before. This represents a significant step forward for consumers. However, the volume of information and the variable quality of information has created new interpretive challenges. Now, one great challenge is helping consumers find the information they want that is also accurate, reliable, and presented in an accessible format. Searches for health information rely on a complex interplay of search algorithms, Web site content and coding, and consumer behaviors. The recommendations presented here address each of those factors with ideas for further research as well as more immediate recommendations for action. This agenda is a start at maximizing the potential of the Internet to deliver high-quality health information for diverse users.","keyphrases":["internet","ehealth","health services research","information management","quality of health care","consumer participation","patient education"],"prmu":["P","P","P","R","R","R","R"]}
{"id":"Neuropsychologia-2-1-2265793","title":"Free recall in autism spectrum disorder: The role of relational and item-specific encoding\n","text":"Autism spectrum disorders (ASDs) are characterised by a relatively specific pattern of typical and atypical memory functioning. Convergent behavioural and neuroscientific evidence indicates that this pattern of functioning may be the result of specific impairments in hippocampally mediated relational memory processes, whilst brain-mechanisms mediating item-specific memory processes remain intact. In the current paper we draw on a behavioural paradigm developed by Hunt and Seta [Hunt, R. R., & Seta, C. E. (1984). Category size effects in recall\u2014The roles of relational and individual item information. Journal of Experimental Psychology: Learning, Memory and Cognition, 10, 454\u2013464], which not only allowed us to determine whether individuals with ASD did indeed experience selective difficulties in relational processes, but in addition enabled us to gain insights into the severity of this impairment. Our results suggest that whilst individuals with ASD employ relational memory processes atypically, this impairment seems restricted to situations in which such processes need to be deployed spontaneously to facilitate memory. Under situations that provide environmental support for the processing of relational information, individuals with ASD did demonstrate the ability to employ such processes relatively effectively. These findings provide further support for the \u2018Task Support Hypothesis\u2019 and suggest that relational memory processes may in principle be functionally intact despite not being triggered by the same environmental situations as in typical development.\n1\nIntroduction\nAutism spectrum disorders (ASDs) are clinically defined by difficulties in reciprocal social behaviour and communication and the presence of stereotyped patterns of behaviour and restricted interests (ICD-10: World Health Organisation, 1992; DSM IV-TR: American Psychiatric Association, 2000). In addition to this unique combination of symptomatology, the condition is also characterised by a relatively specific combination of typical and atypical functioning within the domain of memory. Since this patterning of memory functioning cannot be accounted for by the varying degree of language or general intellectual disability that often accompanies the core clinical features of ASD, it is thought to reflect a facet of the broader phenotype characterising the disorder. We propose that a cognitive framework that distinguishes between item-specific and relational memory processes may not only provide a suitable explanation for available behavioural evidence, an idea that we test in the present study, but may also prove useful in guiding future neuroscientific work relating to medial temporal lobe (MTL) functioning in ASD.\nOn the basis of currently available evidence the patterning of memory functioning in ASD may be summarised as follows. Procedures such as recognition, priming and cued recall generally tend to yield typical levels of performance in ASD (Boucher & Lewis, 1989; Bowler, Gardiner, & Grice, 2000a; Bowler, Matthews, & Gardiner, 1997; Gardiner, Bowler, & Grice, 2003; Minshew, Goldstein, Muenz, & Payton, 1992; Minshew, Goldstein, Taylor, & Siegel, 1994; Tager-Flusberg, 1991). By contrast, free recall paradigms generally lead to diminished performance in this population especially when semantic, syntactic or phonological information is available to aid recall (Bowler, Gardiner, Grice, & Saavalainen, 2000b; Hermelin & O\u2019Connor, 1967; Smith, Gardiner, & Bowler, 2007; Tager-Flusberg, 1991; but see L\u00f3pez & Leekam, 2003 for contrary evidence). These free recall difficulties parallel findings from the typical aging literature (e.g. Craik & Anderson, 1999; Craik, Morris, Morris, & Loewen, 1990) and led Bowler et al. (1997) to posit a \u2018Task Support Hypothesis\u2019 according to which procedures that provide cues to the remembered material at test attenuate the memory difficulties experienced by individuals with ASD. Bowler, Gardiner, and Berthollier (2004) demonstrated that this framework could account for conflicting results regarding source memory capacities in individuals with ASD where previous studies had observed impaired performance on tests of source recall but undiminished performance on tests of source recognition (e.g. Bennetto, Pennington, & Rogers, 1996; Farrant, Blades, & Boucher, 1998).\nAlthough the task support hypothesis can account for the patterning of performance by individuals with ASD across a variety of memory paradigms, the causes for this greater reliance on support for the retrieval of previous experiences remain to date unknown. Earlier attempts to account for the pattern of intact and impaired memory processes in ASD have often invoked encoding as the source of difficulty. The most influential of these accounts is based on the seminal work of Hermelin and O\u2019Connor (1970) who demonstrated that compared to non-ASD children who demonstrate superior recall for semantically and syntactically organised word sequences, children with autism do not tend to draw on such semantic and syntactic features to aid recall. On the basis of this evidence Hermelin and O\u2019Connor (1970) argued that individuals with ASD do not encode stimuli meaningfully. Although several investigations have supported this hypothesis (e.g. Bowler et al., 2000b; Tager-Flusberg, 1991), three strands of more recent evidence indicates that the encoding difficulties seen in ASD may be more subtle than general problems with processing semantic information per se. We will briefly consider each of these in turn.\nFirst, individuals with ASD have been found to be subject to semantically induced memory illusions when Roediger and McDermott's (1995) procedure is used. In such paradigms individuals are asked to try to remember a series of words that include the strongest semantic associates of one non-presented target word (e.g. bed, dream, night, etc. for the target word \u2018sleep\u2019). Bowler et al. (2000b) and Beversdorf, Smith, Crucian, Anderson, and Keillor (2000) showed that individuals with ASD like typical individuals are more likely to falsely remember the semantically related target words than semantically unrelated words. Although the findings by Beversdorf and colleagues suggested that individuals with ASD may be better at discriminating the illusory target words from actually studied items, the finding that individuals with ASD did experience illusory memories shows that they are sensitive to the semantic associations of the studied words at least to some extent.\nThe second strand of evidence concerns the observation that individuals with ASD exhibit relatively typical levels of performance following deep levels of encoding (Bowler et al., 1997; Gardiner et al., 2003; Mottron, Morasse, & Belleville, 2001; Toichi & Kamio, 2002). Deep levels of encoding generally involve the processing of semantic aspects of material (e.g. thinking about category membership of words), which typically leads to enhanced memory in comparison to shallower levels of encoding that involve the processing of non-semantic features of material (e.g. counting the number of syllables of words) (Craik & Lockhart, 1972). The finding of typical levels of performance following deep levels of encoding in ASD thus again suggests that under some circumstances such individuals encode semantic aspects of stimuli relatively effectively. Interestingly, studies employing levels of processing paradigms have also tended to note superior performance of individuals with ASD following shallow levels of encoding (e.g. Toichi & Kamio, 2002). This pattern has led Mottron et al. (2001) to suggest that rather than being deficient in processing semantic or \u2018higher-level\u2019 conceptual information, individuals with ASD may be superior at processing \u2018low-level\u2019 perceptual information and that this processing style may interfere with higher-level conceptual processes in some circumstances. We will return to this argument again later.\nThe third and final strand of evidence regards a recent set of studies from our laboratory. In this set of experiments we asked participants to study a list of words, each of which was accompanied by a semantically related or semantically unrelated context word (e.g. \u2018Wood\u2019 in the context of \u2018Tree\u2019 vs. \u2018Stone\u2019 in the context of \u2018Motor\u2019). Whilst individuals with ASD failed to benefit from the semantic relatedness of to-be-remembered words and simultaneously presented context items on a test of free recall, their performance on a test of recognition was enhanced by such semantic relationships to a similar extent as found in typically developed comparison participants (Bowler, Gaigg, & Gardiner, 2008b). Again this finding suggests that individuals with ASD are sensitive to semantic features of stimuli, at least when test procedures support retrieval.\nThe apparent contradiction between diminished use of semantic relations to aid free recall and relatively typical use of semantic features of stimuli under certain circumstances may be resolved by means of a closer analysis of what each of the paradigms described above requires of the participant. Experiencing an illusory memory on the basis of studying strong associates of a non-studied word implicates the relation between each studied word and the participant's existing knowledge base (e.g. Item A is associated with Concept X, Item B is associated with Concept X, etc.) and does not rely heavily on processing the relations amongst the studied items (Roediger, Watson, McDermott, & Gallo, 2001). Deeper levels of encoding equally do not necessitate relating studied items to one another but rather require enhanced attention to the semantic properties of each studied item. Finally, performance on tests of recognition has been found to rely more heavily on the ability to draw on information specific to individual items, including their semantic properties, rather than relationships among items (e.g. Anderson & Bower, 1972). In contrast, making efficient use of semantic features of stimuli during free recall tasks relies not only on the ability to process the semantic properties of each item but in addition on the ability to make use of these semantic features to establish associations amongst the items (i.e. Item A is associated to Item B because they are both associated with Concept X).\nThe foregoing analysis leads us to speculate that individuals with ASD may have specific difficulties in using semantic information that emerge as a result of the relationships between items, whilst their capacity to draw on semantic information that is specific to individual items appears to be intact. This distinction between relational and item-specific processing has been widely applied to account for a variety of memory phenomena (Anderson & Bower, 1972; Hunt & McDaniel, 1993) and specific difficulties in processing relational information would explain why individuals with ASD rely on greater task support during retrieval. Such difficulties would also explain why individuals with ASD experience fewer episodically defined recollective experiences but somewhat more familiarity based experiences on tests of recognition that employ the \u2018Remember\/Know\u2019 procedure (Bowler et al., 2000a; Bowler, Gardiner, & Gaigg, 2007; see Gardiner, 2001 for further details on the \u2018Remember\/Know\u2019 procedure). Recollective experiences require that information be encoded and stored in relation to spatial and temporal contextual information whilst familiarity based recognition judgments can be mediated on the basis of available item-specific information alone (see Gardiner, 2001; Tulving, 1985, 2002 for further details). Furthermore inefficient use of organisational strategies such as semantic clustering (e.g. Minshew & Goldstein, 2001) or subjective organisation (Bowler, Gaigg, & Gardiner, 2008a) to facilitate memory in ASD also indicate that this population experiences difficulties in using relationships amongst items to organise their retrieval in free recall.\nThe suggestion that ASD may be characterised by relatively specific difficulties in relational memory processes has recently also emerged on the basis of neuroscientific evidence (Nicolson, DeVito, Vidal, Sui, & Hayashi, 2006). Since the first direct examinations of the brains of individuals with ASD (Bauman & Kemper, 1985), atypicalities in areas associated with memory processes have repeatedly been documented (see Bachevalier, 1994; Kemper & Bauman, 1998; Palmen, van Engeland, Hof, & Schmitz, 2004 for reviews). Although the findings remain somewhat inconsistent, morphological abnormalities of the hippocampus are relatively well documented in ASD (see Nicolson et al., 2006). Areas surrounding the hippocampus, such as perirhinal, entorhinal and parahippocampal areas have less often been the focus of investigation but the observations by Bauman and Kemper (1985) suggest that at least the entorhinal cortex seems to be less affected than the hippocampus in ASD individuals.1 Until recently it has been difficult to relate these pathological findings to the memory difficulties experienced by individuals with ASD because the precise role of distinct medial temporal lobe areas in mediating memory processes was only vaguely understood. Accumulating evidence, however, now demonstrates that relational and item-specific processes are mediated by distinct sub-systems of the medial temporal lobe (MTL) memory system. More specifically, the hippocampus has been identified as the site of domain-general relational memory processes where individual features of an episode are integrated and organised (e.g. Eichenbaum, 2004; Holdstock, Mayes, Gong, Roberts, & Kapur, 2005; Squire, 1992). Areas outside the hippocampus, such as perirhinal, entorhinal and parahippocampal areas, on the other hand, seem to mediate more domain-specific item and contextual processes (e.g. see Davachi, 2006; Mayes, Montaldi, & Migo, 2007 for comprehensive reviews). Of particular interest in relation to ASD is the finding that episodically based recognition judgements that involve the recollection of contextual information (and are impaired in ASD) are primarily mediated by hippocampal processes whilst familiarity based recognition judgements (which are intact in ASD) are mediated by perirhinal processes (Brown & Aggleton, 2001; Davachi, Mitchell, & Wagner, 2003; Davachi & Wagner, 2002; Holdstock et al., 2005). This dissociation, together with the wider memory and neuropathological literature in ASD suggests that the item-specific\/relational distinction may provide a useful heuristic device to guide further neuroscientific investigations of MTL pathology in ASD. For such an endeavour to be successful, however, it is necessary to test whether this framework provides an adequate explanation for the behavioural manifestations of memory difficulties in ASD.\nIn the current paper we test the hypothesis that individuals with ASD are characterised by specific behavioural difficulties in relational memory processes whilst item-specific memory processes are spared. The current paradigm is based on a study by Hunt and Seta (1984), who argued that the efficiency of recalling items from a list of categorised words depended on the availability of both item-specific and relational information. Item-specific information, they suggest, is important in order to effectively distinguish amongst items from within a given category whilst relational information is important in order to recall the category per se. In order to test this hypothesis, they asked participants to study a list of words that included varying instances of items belonging to different categories (e.g. 2 Items of Fruit, 4 Professions, 8 Countries, 12 Animals, 16 Furniture). Hunt and Seta (1984) argued that because the relational nature of the items from the relatively small categories in such a list is relatively unobvious, effective recall of these categories depends disproportionately on the availability of relational information. By contrast, effective recall of items from the relatively large categories depends disproportionately on the availability of item-specific information because such information facilitates the differentiation of items within these categories. In support of their hypotheses, they showed that participants who encoded words through a relational orienting task (i.e. sorting words into categories) recalled items from the less obvious categories that were represented relatively infrequently in the study list significantly better than participants who encoded the words through an item-specific orienting task (i.e. rating words on pleasantness). In addition, the relational orienting task facilitated the recall of at least one item from each of the categories (particularly the relatively small categories) supporting the view that relational information is important for the recall of the categories per se. By contrast, participants who encoded words through the item-specific orienting task exhibited superior recall of items from the categories that were represented relatively frequently in the study list. In short, whilst the encoding of relational information disproportionately benefits the recall of words from relatively small (relative to other categories in the list) and therefore not very obvious categories, the encoding of item-specific information is disproportionately beneficial for the recall of items from relatively large categories.\nIn the current experiment we asked participants to study two lists of words that, following Hunt and Seta (1984), consisted of varying instances of members from different categories. For the first list, individuals were simply asked to try to remember as many words as possible for an upcoming free recall test. Following this baseline condition, participants studied a second list whilst carrying out either the item-specific or relational encoding tasks employed by Hunt and Seta (1984). On the basis of evidence showing that typical individuals consistently benefit from semantic and categorical relationships to facilitate recall (e.g. Bousfield, 1953; Bower, Clark, Lesgold, & Winzenz, 1969) we predicted that during the baseline condition, the typical group would tend to rely on relational memory processes. In contrast, and on the basis of the evidence outlined above, we hypothesised that ASD individuals would rely more heavily on item-specific memory processes. Since relational information is particularly important for effectively recalling relatively small categories, we therefore predicted that during the baseline condition, the ASD group would exhibit disproportionate recall difficulties for items from relatively small categories whilst their recall of items from relatively large categories would not be as seriously compromised. In relation to performance following the relational orienting task, our prediction is less specific. If relational memory processes in ASD are impaired to such an extent that they cannot be deployed even when environmental support would facilitate such processes, the disproportionate recall impairment for smaller categories would persist. If, on the other hand, the impairment in relational memory processes is restricted to circumstances in which such processes would need to be deployed spontaneously, the task support hypothesis would predict that a relational orienting task would alleviate the recall difficulties in ASD thereby resulting in a relatively typical level of performance across category sizes. Finally, based on the evidence that individuals with ASD employ item-specific memory processes effectively, we predicted no recall impairment of this group following the item-specific orienting task. To the contrary individuals with ASD may outperform typical individuals in this condition because they may have developed superior skills in item-specific processing in order to compensate for their difficulties in relational processes. The finding that individuals with ASD tend to outperform typical individuals following shallow encoding tasks (e.g. Mottron et al., 2001) would be in line with this suggestion.\n2\nMethod\n2.1\nParticipants\nTwenty individuals with autism spectrum disorder (7 female, 13 male) and 20 typical individuals (7 female, 13 male) took part in this experiment. Participants were individually matched on Verbal IQ as measured by the WAIS-R or WAIS-IIIUK (The Psychological Corporation, 2002) and groups did not differ on Performance IQ, Full scale IQ or age. Ten participants from each group were randomly allocated to each of the two orienting task conditions (described below) with the constraint that IQ scores and age were similarly distributed across the two conditions. Table 1 summarises these data. All individuals with ASD were diagnosed by local health authorities and\/or experienced clinicians, and met DSM-IV-TR (American Psychiatric Association, 2000) criteria for Asperger's disorder or Autistic disorder. The Comparison group was recruited via local newspaper advertisements. Brief interviews ensured that no participant had a history of neurological or psychiatric illness. Individuals gave their informed consent to take part in the study and were paid standard University fees for their participation.\nAll but four individuals with ASD (two from each orienting task condition), who had been prescribed low doses of antidepressant medication, were free of psychotropic medication. Since the exclusion of these participants and their matched typical individuals did not alter the results significantly, all participants were included in the analysis.\n2.2\nDesign and materials\nOn the basis of Hunt and Seta's (1984) first experiment study lists were constructed from a master pool of words that consisted of 16 words from each of 10 categories selected from the Battig and Montague (1969) category norms. The frequency of the words ranged from 1 to 25 and the average category rank of words was 10 (see Battig & Montague, 1969 for details). The categories of Sports, Clothing, Weapons, Countries and Animals served as set A and the categories of Birds, Kitchen Utensils, Parts of the Body, Fruits and Vehicles served as set B. From each set, 5 study lists were constructed consisting of a total of 42 target items and 8 buffer items to counter primacy and recency effects (4 at the beginning and 4 at the end). Within each list relative category size was manipulated by selecting 2, 4, 8, 12 or all 16 items from the 5 categories (e.g. 2 Sports, 4 Clothing, 8 Weapons, 12 Countries and all 16 Animals). Across the 5 lists each category appeared at each category size once. Buffer items were selected from the categories of Professions and Parts of a Building for set A and Earth Formations and Alcoholic Drinks for set B.\nWords were printed in bold, arial font (size 36; Microsoft Word for Windows) in the centre of 8.2\u00a0cm\u00a0\u00d7\u00a07.6\u00a0cm, laminated cards. The 42 cards constituting the target items were ordered pseudo randomly with the constraint that the average lag between items from the same category be as close to 2 as possible (ranging from 0 to 5).2 The buffer items in the beginning and end of the target list were also randomised so that no more than two consecutive words were from the same category. The orders of items in the 5 lists from sets A and B were equivalent in terms of the list position of words from the differently sized categories.\n2.3\nProcedure\nUnlike Hunt and Seta (1984) the current experiment included a baseline condition during which participants were presented with one of the study lists (in the form of a deck of cards) from either set A or B and simply asked to try to remember as many words as possible. Participants were allowed to go through the cards at their own pace and the total amount of time they required to do so was recorded. Participants were instructed to put each card face down in front of them after they had tried to remember it and to not look at a card again once it was placed on the table. Immediately after the last word, oral free recall was requested.\nFollowing a 5\u201310-min break, individuals were given the respective deck of cards from the set not used during baseline, and given instructions for either the item-specific or the relational orienting task employed by Hunt and Seta (1984). For the relational orienting task printed category labels were placed on the table and participants were asked to sort the word-cards into their respective categories. In the case of uncertainty participants were asked to guess what category a word belonged to. For the item-specific orienting task, labels representing a 5-point pleasantness rating scale (very pleasant, a little pleasant, neutral, a little unpleasant, very unpleasant) were placed on the table, and individuals were asked to rate each of the words on this scale orally and not sort the cards underneath the labels. Regardless of orienting task individuals were asked to try to remember as many words as possible and following the last word all materials were cleared from the table and oral free recall was again requested.\n3\nResults\nA 2 (ASD vs. Comparison)\u00a0\u00d7\u00a02 (Baseline vs. Orienting Tasks condition) mixed ANOVA of the time participants spent looking through the decks of cards revealed a significant (F(1, 36)\u00a0=\u00a07.96, p\u00a0<\u00a0.01) effect of condition with participants spending an average of 323\u00a0s (S.D.\u00a0=\u00a0404) looking through the cards whilst carrying out the orienting tasks compared to 244\u00a0s (S.D.\u00a0=\u00a0254) during the baseline condition. Neither the main effect of group nor the interaction between group and condition were significant. An analysis of the time participants spent looking through the cards during the two orienting tasks revealed no main effects or interaction of the factors group (ASD vs. Comparison) and orienting task (Rate vs. Sort). Since the time participants spent looking through the cards correlated highly (r\u00a0>\u00a0.65) with overall recall levels for both groups in all conditions, encoding time was entered as a covariate in all subsequent analysis of the recall data (Miller & Chapman, 2001).\n3.1\nBaseline condition\nThe free recall data for the baseline condition are illustrated in Fig. 1, which gives the average proportion of items recalled from the smaller (i.e. size 2, 4 and 8) and larger (i.e. size 12 and 16) categories for the ASD and Comparison groups. Overall, the ASD group recalled fewer words than the Comparison group (F(1, 37)\u00a0=\u00a08.08, p\u00a0<\u00a0.01) which was most marked with smaller categories resulting in a significant group by category size interaction (F(1, 37)\u00a0=\u00a06.89, p\u00a0<\u00a0.05). Thus, in line with our prediction that individuals with ASD would exhibit a recall decrement that would be indicative of specific difficulties in relational memory processes, the ASD group recalled significantly fewer words from the small categories (t(38)\u00a0=\u00a03.37, p\u00a0<\u00a0.01; Cohen's d\u00a0=\u00a00.96) but not the large categories (t(38)\u00a0=\u00a01.09, ns; Cohen's d\u00a0=\u00a00.30). We note that we have collapsed the recall data into \u2018small\u2019 and \u2018large\u2019 categories for simplicity and in order to facilitate the calculation of effect sizes. An analysis of the data across the five levels of category size yielded the same significant main effects and interactions (or lack thereof) as those reported above and in the analysis of the data from the orienting tasks below.\nAs indicated earlier, Hunt and Seta (1984) suggest that the recall of at least one item from any given category (i.e. category availability\u2014CA) represents the availability of relational information during recall, as does the amount of category clustering individuals employ during retrieval. Category clustering, as indexed by the Modified Ratio of Repetition is a simple ratio of the number of category repetitions (i.e. two consecutive items are recalled from the same category) to the total number of items recalled across all categories. As Hunt and Seta (1984) point out, more sophisticated measures of clustering are unsuitable for obtaining measures of organisation for each category size because they are mathematically undefined for a single category. In contrast to these indices of relational information, the number of items participants recall within a particular category (i.e. items per category\u2014IPC) depends on the availability of item-specific information since such information aids the differentiation of individual instances of a particular category. In order to provide further insights into the use of item-specific and relational information to facilitate memory in ASD we computed these measures which are set out in Table 2.3\nA 5 (category size) by 2 (group) mixed ANCOVA on the number of categories recalled (category availability) revealed a significant main effect for category size (F(4, 34)\u00a0=\u00a015.22, p\u00a0<\u00a0.001) with larger categories being nearly perfectly recalled whereas the smallest 2-item category was only recalled by 35% of participants. As expected, the ASD group recalled significantly fewer categories (F(1, 37)\u00a0=\u00a010.69, p\u00a0<\u00a0.01) and this effect was again characterised by a significant interaction between category size and group (F(4, 34)\u00a0=\u00a012.25, p\u00a0<\u00a0.001). Post hoc nonparametric comparisons showed that the ASD group recalled the small 2 and 4 item categories less often than typical participants (z\u00a0=\u00a02.82, p\u00a0<\u00a0.01, one-tailed) whilst both groups recalled the larger 12 and 16 item categories nearly perfectly. Although this result needs to be interpreted with caution due to the ceiling performance on larger categories, further evidence for the attenuated use of relational information to facilitate recall in ASD stems from the analysis of the category clustering data. Again this measure increased with category size (F(4, 34)\u00a0=\u00a010.13, p\u00a0<\u00a0.001) and again individuals with ASD clustered words into their respective categories less than the comparison group (F(1, 37)\u00a0=\u00a05.66, p\u00a0<\u00a0.05). Again the interaction between group and category size needs to be interpreted with some caution due to the floor performance on smaller categories. However, as the data set out in Table 2 indicate, clustering scores increased linearly with category size for typical participants whilst for the ASD group clustering only increased notably with a category size of 12. This quadratic trend is significant (F(1, 37)\u00a0=\u00a05.36, p\u00a0<\u00a0.05). In contrast, an analysis of the IPC data revealed no significant main effects of group (F(1, 37)\u00a0=\u00a00.04, ns) or category size (F(1, 37)\u00a0=\u00a01.25, ns) and no interaction between these factors (F(4, 34)\u00a0=\u00a00.23, ns). Thus, our findings from the baseline condition confirm that without any support, participants with ASD use relational information to aid recall to a lesser extent than typical individuals, whereas their use of item-specific information to help their recall appears similar to that of the Comparison group.\n3.2\nOrienting tasks\nPrior to analysing the recall performance following the orienting tasks, we assessed whether groups may have completed these tasks differently. During the category sorting task, participants in both groups performed at ceiling with only 3 ASD and 2 Comparison individuals committing either 1 or 2 errors. During the rating condition, ASD participants provided average ratings of 3.04 (S.D.\u00a0=\u00a00.41), which did not differ significantly from the average rating of 2.88 (S.D.\u00a0=\u00a00.27) given by the Comparison group. Similarly, an inspection of the frequency distributions of the ratings given by individuals revealed no differences between the groups. Taken together with the observation that groups did not differ significantly in terms of the time they spent looking through the deck of cards whilst they completed the orienting tasks these findings suggest no group differences in fulfilling the requirements of the orienting task instructions.\nOur analysis of the recall data following the orienting tasks (illustrated in Fig. 2) paralleled that of the baseline condition and encoding time was again entered as a covariate. A 2 (category size) by 2 (orienting task) by 2 (group) mixed ANCOVA of the recall data revealed a main effect of orienting task (F(1, 35)\u00a0=\u00a06.88, p\u00a0<\u00a0.05) indicating that recall following the relational encoding task (i.e. sorting words into categories) was superior to recall following the item-specific encoding task (i.e. rating words on pleasantness). The only other significant effect was an interaction between category size and orienting task (F(1, 35)\u00a0=\u00a012.79, p\u00a0<\u00a0.01), which replicates the findings reported by Hunt and Seta (1984). Post hoc comparisons showed that recall of items from the small categories was superior following the relational compared to the item-specific encoding task (t(38)\u00a0=\u00a04.15, p\u00a0<\u00a0.001; equal variance not assumed) whereas recall of items from the large categories was similar following either type of encoding task (t(38)\u00a0=\u00a01.51, p\u00a0=\u00a0.13). The lack of any interactions involving the group factor (Fs\u00a0<\u00a01) and the absence of a main effect of group (F(1, 35)\u00a0=\u00a00.67, ns) suggests that the provision of support in the form of orienting tasks attenuated the free recall difficulties seen in ASD. One may criticise this latter conclusion on the grounds that the reduced group sizes during the two encoding conditions decreased the statistical power of the analysis of these data in comparison to the baseline condition. In relation to this issue three aspects of our data are worth further comment. Most important amongst these is the observation that unlike performance during the baseline condition, recall following the orienting task conditions was not characterised by interactions between group and category size for either the relational orienting task (F(1, 17)\u00a0=\u00a00.36, ns) or the item-specific orienting task (F(1, 17)\u00a0=\u00a00.13, ns). Thus the patterning of performance as a function of category size did no longer differ as a function of group. Second, Cohen's d effect sizes for the between group differences in recall of items from small categories were reduced from 0.96 during the baseline condition to 0.57 following the relational orienting task and 0.08 following the item-specific orienting task (respective effect sizes for larger categories were 0.44 and 0.21). Third, although order confounds and differences in encoding time (i.e. time spent looking through deck of word cards) make analyses across baseline and orienting task conditions problematic, inspection of the data set out in Figs. 1 and 2 show that performance of ASD individuals following the relational orienting task was nearly identical to the comparison groups\u2019 performance during the baseline condition. Interestingly the item-specific orienting task reduced performance of comparison participants to the level of ASD individuals\u2019 baseline performance. We will return to the implications of these results in more detail in our discussion.\nTable 3 summarises the category availability, clustering and IPC data as a function of orienting task. As the category availability data suggest, overall recall of categories is generally better for larger categories (F(4, 32)\u00a0=\u00a024.97, p\u00a0<\u00a0.001) and following the relational orienting task (F(1, 35)\u00a0=\u00a012.04, p\u00a0<\u00a0.01). Furthermore, a significant interaction between category size and orienting task (F(4, 32)\u00a0=\u00a04.47, p\u00a0<\u00a0.01) indicates that the main effect of orienting task is mostly due to the increased availability of smaller categories following relational as compared to item-specific processing. Again the lack of a main effect of group or interactions involving the group factor (Fs\u00a0<\u00a01) suggests that the effect of item-specific and relational orienting tasks on the recall of categories was similar for the two participant groups. An analysis of the clustering data revealed a main effect of category size (F(4, 32)\u00a0=\u00a08.54, p\u00a0<\u00a0.001) and a marginally significant orienting task by category size interaction (F(4, 32)\u00a0=\u00a02.58, p\u00a0=\u00a0.056), which follows Hunt and Seta's observation of larger differences in clustering between the item-specific and relational encoding conditions for the smaller as compared to the larger categories. Again the group factor did not yield a main effect (F(1, 35)\u00a0=\u00a01.39, p\u00a0=\u00a0.25) or interactions with the other factors (Fs\u00a0<\u00a02). An analysis of the IPC data as a function of category size, group and orienting task, did not reveal any significant main effects or interactions (Fs\u00a0<\u00a01.1), thus not replicating Hunt and Seta (1984) who reported higher IPC scores following the item-specific orienting task, especially for larger categories. In summary, these analyses are in line with the suggestion that recall performance in ASD is no longer characterised by disproportionate difficulties in drawing on relational information when orienting tasks constrain the processes by which information is encoded.\n4\nDiscussion\nIn the current experiment we drew on a procedure developed by Hunt and Seta (1984) in order to evaluate the hypothesis that individuals with ASD are characterised by specific difficulties in relational memory processes. Furthermore we hoped to gain insights into the severity of such difficulties by assessing whether environmental support in the form of a relational orienting task could help individuals with ASD to employ such relational processes.\nOur results from the baseline condition support previous demonstrations (e.g. Bowler et al., 1997; Smith et al., 2007; Tager-Flusberg, 1991) of reduced recall in individuals with ASD when categorical information is available to aid recall. The finding that the ASD group showed selectively reduced recall of smaller but not larger categories confirms our prediction that ASD is characterised by relatively specific difficulties in relational but not item-specific memory processes. Further support for this view stems from the finding that the ASD participants recalled overall fewer categories and were less likely than typical participants to cluster items into their respective categories during recall. In contrast, the ASD participants recalled as many items per category (IPC) as the Comparison group indicating that they make as much use of item-specific information to facilitate memory as typical individuals. Together these results strongly suggest that, in the absence of any support, individuals with ASD employ relational memory processes to facilitate recall to a lesser degree than typical individuals whilst their ability to draw on item-specific information to aid recall seems relatively intact.\nSolely on the basis of the results from the baseline condition it is difficult to determine the severity of the relational memory difficulty evident in individuals with ASD. Our results from the supported encoding conditions shed some light on this issue. These results revealed that following item-specific and relational orienting tasks, overall recall performance between ASD and comparison participants were comparable. As noted in our results, we concede that this conclusion may be criticised on the basis of the reduced group sizes for each of the orienting task conditions, particularly because the ASD group's performance was numerically (if not significantly) worse than the typical group following the relational orienting task. What is crucial to note, however, is that unlike performance during the baseline condition the patterning of recall as a function of category size following the orienting tasks was very similar for the two groups as were the indices of relational and item-specific encoding. In other words, individuals with ASD no longer exhibited the disproportional difficulties with relational memory processes that characterised their performance during the baseline condition. In this context it is particularly noteworthy that the overall level of recall and the pattern of recall across category sizes of individuals with ASD following the relational orienting condition were almost identical to that of typical individuals during the baseline condition. Conversely, the comparison groups\u2019 performance following the item-specific orienting task was nearly identical to that of the ASD group during the baseline condition. Thus, whilst the relational orienting task allowed individuals with ASD to achieve a level of performance comparable to that of typical individuals\u2019 unsupported performance, the item-specific orienting task seems to have created a learning situation for typical individuals that mimics that experienced by individuals with ASD under normal circumstances.\nA possible limitation of our observations from the orienting task conditions is the fact that all participants first completed the baseline condition. On the basis of this order confound it may be argued that individuals with ASD simply required more practice in order to employ relational memory processes successfully. Although problematic to some extent, our conclusions would not be altered even if the improvement in performance by individuals with ASD is to some extent attributable to disproportionate practice effects. In relation to the task support hypothesis (Bowler et al., 1997), the findings from the orienting task suggest that support in the form of an orienting task (and perhaps increased practice) helps individuals with ASD to overcome difficulties in deploying relational memory processes effectively. Thus our main conclusion is that rather than lacking the capacity to process relational information sufficiently to aid recall, individuals with ASD experience difficulties in spontaneously deploying them in a way that fosters effective learning and memory in novel and unsupported situations. This conclusion is in line with an argument developed by Mottron and colleagues (Mottron, 2004; Mottron, Dawson, Souli\u00e8res, Hubert, & Burack, 2006) on apparent conceptual difficulties in ASD. Rather than accepting the view that higher-level conceptual processes are impaired in this population, these authors contend that enhanced low-level perceptual processes compete with higher-level integrative functions. In the domain of memory this competition may occur between item-specific and relational encoding processes.\nAs we have highlighted in our introduction, the distinction between item-specific and relational memory processes may not only prove useful in terms of understanding the behavioural pattern of memory functioning in individuals with ASD but it may also provide a fruitful heuristic framework for more direct investigations regarding the neuropathological correlates underlying memory functioning in this group. Since our observations are purely behavioural, we can only speculate about the neural underpinnings of the specific difficulties in relational memory processes that characterised performance of individuals with ASD in the current study. Given the evidence regarding morphological abnormalities of the hippocampus in ASD (e.g. Kemper & Bauman, 1998) and the growing evidence implicating this structure in relational memory processes, an appealing possibility is that the memory difficulties experienced by individuals with ASD stem from relatively specific functional atypicalities of hippocampally mediated memory processes (see also Nicolson et al., 2006). Although more direct neuroscientific investigations will be needed in order to specify the nature of this functional abnormality further, we would argue that at least two hypotheses may be generated on the basis of the current literature. Based on evidence suggesting that areas surrounding the hippocampus may under some circumstances mediate relational memory processes (see Eichenbaum, 2004 for a review), one possibility is that in ASD these adjacent areas are able to compensate for deficits in hippocampally mediated relational processes if environmental circumstances invite this level of processing. If environmental support is absent on the other hand, cortical areas adjacent to the hippocampus may simply perform their \u2018default\u2019 operations and mediate item-specific processes. Another possibility is that hippocampally mediated relational memory processes are principally intact but limited to such an extent that they are ineffectively deployed under spontaneous learning conditions. When environmental circumstances emphasise relational processes, however, this functional limitation may be sufficiently supported to permit a relatively typical behavioural expression of relational memory capacities. These two hypotheses are most likely not the only ones that may be put forward but we include them here to reinforce the point that the framework of item-specific versus relational memory processes provides a useful heuristic to generate future research to further specify the neural underpinnings of memory difficulties in ASD.\nIn summary, our observations provide strong support for the view that individuals with ASD exhibit relatively specific difficulties in the spontaneous deployment of relational memory processes. We stress the term spontaneous because we think it important to distinguish between an impairment in the ability to engage in otherwise normally functioning processes and processes that are so impaired that they cannot function normally under any circumstances. Our finding that individuals with ASD no longer exhibit disproportionately attenuated memory for smaller categories following a relational orienting task illustrates that supporting learning environments promote relational processes in this group. Future studies will be needed in order to determine whether the relational processes employed by individuals with ASD under supported conditions are mediated by the same hippocampal-based neural mechanisms as in typical individuals or whether adjacent brain areas which typically mediate item-specific memory processes compensate for atypical hippocampal functioning.","keyphrases":["autism spectrum disorder","task support hypothesis","hippocampus","medial temporal lobe memory system"],"prmu":["P","P","P","R"]}
{"id":"Doc_Ophthalmol-4-1-2244701","title":"Functional characteristics of patients with retinal dystrophy that manifest abnormal parafoveal annuli of high density fundus autofluorescence; a review and update\n","text":"Purpose To examine the presence and functional significance of annular fundus autofluorescence abnormalities in patients with different retinal dystrophies. Methods Eighty one patients were ascertained who had a parafoveal ring of high density on fundus autofluorescence imaging. Sixty two had had a clinical diagnosis of retinitis pigmentosa (RP) or Usher syndrome with normal visual acuity. Others included a case of Leber congenital amaurosis and genetically confirmed cases of cone or cone-rod dystrophy (GUCA1A, RPGR, RIMS1), \u201ccone dystrophy with supernormal rod ERG\u201d (KCNV2) and X-linked retinoschisis (RS1). International-standard full-field and pattern electroretinography (ERG; PERG) were performed. Some patients with rod-cone or cone-rod dystrophy underwent multifocal ERG (mfERG) testing and photopic and scotopic fine matrix mapping (FMM). Results In patients with RP, the radius of the parafoveal ring of high density correlated with PERG P50 (R = 0.83, P < 0.0005, N = 62) and encircled areas of preserved photopic function. In the other patients, AF rings either resembled those seen in RP or encircled an area of central atrophy. Ring radius was inversely related to the PERG P50 component in 4 of 18 cases with a detectable response. FMM showed that arcs of high density were associated with a gradient of sensitivity change. Conclusions Parafoveal rings of high density autofluorescence are a non-specific manifestation of retinal dysfunction that can occur in different retinal dystrophies. Electrophysiology remains essential for accurate diagnosis. The high correlation of autofluorescence with PERG, mfERG and FMM demonstrates that AF abnormalities have functional significance and may help identify suitable patients and retinal areas amenable to future therapeutic intervention.\nIntroduction\nLipofuscin is derived from the shed and degraded photoreceptor outer segments and normally accumulates in the retinal pigment epithelium (RPE) with age [1\u20134]. An abnormal increase or depletion of lipofuscin manifests as high or low density areas respectively in fundus autofluorescence (AF) images [5] and such changes may aid in the detection and characterisation of a wide range of inherited retinal disorders, either by accentuating the visibility of fundus abnormalities or by revealing changes not accessible by routine examination or fluorescein angiography [5\u201313]. Lipofuscin has been shown to fragment when exposed to light [14] and its presence suggests continuing metabolic demand [5]. Absence of autofluorescence suggests either blocking of the incident radiation, photoreceptor cell death and RPE atrophy [5, 6] or disruption of the vitamin A cycle [15, 16].\nSome patients with genetically different forms of retinitis pigmentosa manifest a ring of high density AF representing abnormal parafoveal accumulation of lipofuscin which usually encircles preserved foveal areas [17\u201320]. Similar parafoveal rings have recently been documented in other retinal dystrophies including some patients with Leber congenital amaurosis [21], Best disease [13], X-linked retinoschisis [22] and cone-rod dystrophy consequent upon mutation in GUCA1A [23], GUCY2D [24], RIMS1 [25, 26], or RPGRORF15 [26, 27]. Abnormal annular AF has also been described in some cases of \u201ccone dystrophy with supernormal rod ERG\u201d consequent upon mutation in KCNV2 [28, 29]. Unlike RP, these disorders often result in atrophic macular changes, manifesting as low density AF within the ring.\nThe main aims of the current study were to review the electrophysiological phenotypes associated with annular AF abnormalities, and to examine two heterogeneous groups of patients in more detail by comparing indices of macular function associated with abnormal macular AF in rod-cone and cone-rod dystrophies.\nMaterials and methods\nAutofluorescence imaging was performed using a scanning laser ophthalmoscope according to previously described techniques [5, 30]. Eighty one patients with abnormal AF of the posterior pole in the form of a high density parafoveal ring were reviewed. Sixty two had a clinical diagnosis of retinitis pigmentosa or Usher syndrome with a visual acuity of 6\/9 or better and included 30 cases described previously [17]. Nineteen other individuals were ascertained from previous studies [21\u201323, 25\u201329] including 14 with cone-rod or cone dystrophy consequent upon mutation in RPGR ORF15 (4 cases), RIMS1 (8 cases) or GUCA1A (2 cases). Two subjects had \u201ccone dystrophy with supernormal rod ERG\u201d consequent upon mutation in KCNV2, two had RS1mutations and manifested rings in one or both eyes. One patient had Leber congenital amaurosis.\nFull-field ERGs were performed according to extended testing protocols incorporating the ISCEV minimum standard [31] in order to assess generalised retinal function. A stimulus 0.6 log units greater than the ISCEV maximum was also used, to demonstrate better the a-wave under conditions of dark adaptation. Pattern ERGs evoked by high contrast checkerboard reversal were recorded according to ISCEV recommendations [32] using standard parameters; checkerboard size 12\u00a0\u00d7\u00a015 degrees, check size 45\u2032, Michelson contrast 0.98. The PERG P50 component was used as an index of macular function. Additional PERG testing was performed using a range of circular checkerboard fields ranging from 3 to 18 degrees in diameter presented in a random order [17]. Check size was constant at 45\u2032. International-standard mfERGs [33, 34] and fine matrix mapping were performed in some cases. Fine matrix mapping measures rod and cone system sensitivity and has been described previously [18, 35, 36]. In brief, detection thresholds for a spot of light were determined at 1-degree intervals over selected 9\u00a0\u00d7\u00a09-degree retinal areas. Data were presented both as sensitivity contours, illustrating the position and orientation of tested retinal locations and as three-dimensional threshold profiles, plotted using interpolated values at 0.25-degree intervals, obtained by Gaussian filtering.\nResults\nAll patients were selected according to fundus autofluorescence that was characterised by an abnormal high density parafoveal ring (Figs.\u00a01, 2, 4\u20138). Sixty two with a clinical diagnosis of retinitis pigmentosa or Usher syndrome and a visual acuity of 6\/9 or better had evidence of preserved RPE within the ring. Nineteen others had either cone or cone-rod dystrophy, \u201ccone-dystrophy with supernormal rod ERG\u201d, X-linked retinoschisis or Leber congenital amaurosis. Many of the older non-RP cases had evidence of macular RPE atrophy within the ring (Figs.\u00a02b\u2013d, 7c, 8b\u2013c). Conversely, AF imaging in some RP patients showed mild to moderate atrophic RPE changes within the vascular arcades but external to the ring (Figs.\u00a04d and 5e).\nFig.\u00a01Full-field ERGs, PERGs and AF in 3 patients with rod-cone dystrophy (a\u2013c) including a patient with RP18 (a) and Usher syndrome (b). Row D shows full-field ERGs and AF in a case of Leber congenital amaurosis; ERGs and the AF image were obtained in the presence of nystagmus and are consequently noisy. Normal examples are shown for comparison (e). LU indicates log units greater (+) or less (\u2212) than the ISCEV standard flashFig.\u00a02Full-field ERGs, PERGs and AF in cone rod-dystrophy consequent upon mutation in RPGR (a), RIMS1 (b), \u201ccone dystrophy with supernormal rod ERG\u201d (c) and in a case of X-linked retinoschisis (RS1; d). Normal examples are shown for comparison (e). LU indicates log units greater (+) or less (\u2212) than the ISCEV standard flash\nFigures\u00a01 and 2 show representative full-field ERGs and PERGs in eight individuals with different retinal dystrophies. Figure\u00a01 shows data from 3 subjects with rod-cone dystrophy and normal visual acuity including a case of autosomal dominant RP18 (Fig.\u00a01a) and Usher syndrome (Fig.\u00a01b). Figure\u00a01d shows undetectable ERGs in a patient with Leber congenital amaurosis; ERGs are contaminated by the effects of nystagmus but no ERG is detectable, in keeping with severe generalised photoreceptor dysfunction. Figure\u00a02 shows representative examples of cone-rod dystrophy consequent upon mutations in RPGR (Fig.\u00a02a), RIMS1 (Fig.\u00a02b) and KCNV2 (\u201ccone dystrophy with supernormal rod ERG\u201d; Fig.\u00a02c). Figure\u00a02d shows the ERG and AF findings in a patient with X-linked retinoschisis.\nIn the 62 RP cases PERG P50 components varied between normal (>2\u00a0uV) and undetectable, consistent with varying degrees of macular involvement. There was no significant correlation with the bright flash ERG a-wave amplitude or 30\u00a0Hz flicker ERG amplitude. Figure\u00a03 shows high positive correlation between PERG P50 and mean ring radius (r\u00a0=\u00a00.83, P\u00a0<\u00a00.0005). Data are shown from one RP patient that was tested comprehensively (Fig.\u00a04); PERGs were normal to the smallest diameter checkerboard but minimal enlargement was seen as the stimulus field size was increased (Fig.\u00a04b). Multifocal ERGs showed widespread reduction with relative preservation of the response associated with the central stimulus element (Fig.\u00a04a), consistent with a central island of visual field preservation (Fig.\u00a04c). The internal edge of visual field constriction corresponded closely with the ring of high density, as shown by photopic FMM (Fig.\u00a04d). Scotopic fine matrix mapping revealed rod sensitivity losses that were severe and that encroached upon the central macula within the ring. Additional examples of mfERGs, visual fields and fine matrix mapping in RP patients are shown in Figs.\u00a05 and 6.\nFig.\u00a03Comparison of mean ring radius with PERG P50 in 62 patients with rod-cone dystrophy (RP; broken linear regression line) and normal visual acuity and in 19 patients with other retinal dystrophies, including 4 cone or cone-rod dystrophy cases in which there was a detectable PERG (solid regression line)Fig.\u00a04Multifocal ERGs (a), small field PERGs (b), Humphrey visual field (c) and photopic (d) and scotopic (e) fine matrix mapping in a patient with a clinical diagnosis of RP. Diamonds and error bars in (b) show mean values and standard deviations for 8 normal subjects; triangles and squares show patient data from right and left eyes. Contour plots (d and e) show sensitivity gradients over tested retinal locations; corresponding 3-D plots show retinal location (abscissa, degrees) and thresholds (ordinate, log units). Labelling (x) shows correspondence between the orientation of contour and threshold plots. Normal photopic and scotopic values are plotted in Fig.\u00a05a and dFig.\u00a05Contour sensitivity plots (rows 1 and 3) and 3-D threshold plots (rows 2 and 4) obtained in representative normal subjects (a, d) and in 3 RP patients (b, e and c, f). Subjects were tested under photopic (a\u2013c) and\/or scotopic conditions (d\u2013f). Labelling (x) shows correspondence between the orientation of contour and threshold plots. Abscissa shows retinal location (degrees), ordinate axes show threshold (log units). Corresponding photopic FMM in individual (e) has been published elsewhere [18]. Normal 3-D plots show averaged data from 14 (a) or 12 (d) normal subjectsFig.\u00a06AF images, mfERGs and corresponding Humphrey visual fields in 3 patients with RP and normal visual acuity\nPattern ERGs were detectable in only 4 patients with cone or cone-rod dystrophy (GUCA1A, RPGR or RIMS1, Fig.\u00a03). Pattern ERG P50 was inversely related to ring size in these subjects (Fig.\u00a03). Data from one subject with cone-rod dystrophy (RIMS1) are shown in Fig.\u00a07. Multifocal ERG showed widespread reduction with only relative preservation of the central response (Fig.\u00a07a). Standard Humphrey visual fields showed a central scotoma and some superior field loss (Fig.\u00a07b). Fine matrix mapping revealed severe threshold elevation across the macula but with relatively preserved photopic sensitivity over a central island of preserved RPE AF (Fig.\u00a07c). Threshold values inside the ring are maximally elevated over a concentric atrophic area and show a gradient of increasing sensitivity over the arc of high density (Fig.\u00a07c and e). The PERGs in this patient were undetectable (data not shown). Additional examples of fine matrix mapping in RIMS1 and RPGR patients are illustrated in Fig.\u00a08. Central RPE atrophy was not always present (Figs.\u00a02a, 8a).\nFig.\u00a07Multifocal ERGs (a), Humphrey visual field (b) and photopic (c, d) and scotopic (e, f) fine matrix mapping in a patient with cone-rod dystrophy consequent upon RIMS1 mutation. Labelling (x) shows correspondence between the orientation of contour and 3-D threshold plots. Abscissa shows retinal location (degrees), ordinate axes show threshold (log units). Threshold values for half the tested area have been removed from c and e, to expose the foveal values that would otherwise be obscuredFig.\u00a08Contour sensitivity plots (rows 1 and 3) and 3-D threshold plots (rows 2 and 4) obtained in 3 patients with cone-rod dystrophy consequent upon RPGR (column 1) or RIMS1 mutations (columns 2 and 3). Subjects were tested under photopic (a\u2013c) and scotopic conditions (d\u2013f). Labelling (x) shows correspondence between the orientation of contour and threshold plots. Abscissa shows retinal location (degrees), ordinate axes show threshold (log units)\nDiscussion\nThis study reviews a heterogeneous group of 81 patients with genetically-determined retinal diseases that manifest a common feature on fundus autofluorescence imaging in the form of a parafoveal ring of high density. Patients with RP and normal visual acuity had rings that encircled preserved central AF. Mild to moderate atrophic changes were occasionally detected within the vascular arcades but eccentric to the ring (Figs.\u00a04d, 5e). In the other retinal dystrophies that were examined in this and in previous studies [25\u201327], the ring could also encircle preserved central AF but in older subjects there was often central RPE atrophy within the annulus and preserved AF at more eccentric locations.\nAs patients with RP or different forms of retinal dystrophy can have indistinguishable AF abnormalities, the AF appearance cannot be used to establish a diagnosis in such cases. Non-specific annular increases in AF may be associated with a wide variety of distinctive or pathognomonic full-field ERG changes that are essential for accurate diagnosis and functional phenotyping (see below).\nThe data from 62 patients with RP and normal visual acuity show a high positive correlation between ring size and the PERG P50 component extending and confirming findings in a cohort of 30 of these cases [17], demonstrating the robust nature of this relationship. The findings are corroborated by high spatial resolution fine matrix mapping, visual field data and mfERG testing that shows preserved photopic sensitivity within central areas bordered by the ring, consistent with previous reports [18\u201320, 37]. Scotopic sensitivity losses encroach upon central macular areas suggesting that rod-system dysfunction precedes abnormal parafoveal accumulation of lipofuscin and progressive visual field loss [18]. Serial data indicate that the rings may vary greatly in terms of their stability; to date only 3 cases have been reported in which progressive ring constriction has occurred [37]. The rate of AF ring constriction may prove to be of prognostic value in predicting retention of visual acuity and visual field preservation, but further monitoring is required.\nIn addition to RP, the parafoveal ring of high density may occur in cone or cone-rod dystrophy consequent upon mutation in GUCA1A [23], GUCY2D [24], RPGR [25, 26], RIMS1 [26, 27], in \u201ccone dystrophy with supernormal rod ERG\u201d (KCNV2) [28], in X-linked retinoschisis (RS1) [22] and in Leber congenital amaurosis [21]. Similar AF findings have also been documented in Best Disease [13] and other maculopathies [10]. In young patients with cone-rod dystrophy, small rings may have preserved central AF and are similar to those seen in RP cases [26]. Older individuals tend to manifest central atrophic changes that are encircled by the ring and there may be a central island of RPE preservation. Fine matrix mapping suggests that the abnormal accumulation of lipofuscin in cases of cone-rod dystrophy represents a transitional stage between relatively preserved parafoveal function and severe central dysfunction that is likely to precede central atrophy [26, 27]. In patients with cone-rod dystrophy consequent upon RPGR or RIMS1 mutations, serial studies have recently demonstrated ring expansion [26], suggesting an expanding front of macular photoreceptor dysfunction. This contrasts with RP where the opposite occurs [37]. It is possible that rings associated with other causes of maculopathy may also expand with time as lesions become larger with age. It is noted that RPGR mutations are more commonly associated with X-linked retinitis pigmentosa [38] with visual acuity reduction [39]; in the current study not all patients underwent genetic screening but none were known to have X-linked RP.\nLipofuscin accumulation in the RPE is likely to reflect metabolic activity which is largely determined by outer segment renewal. There is evidence of light-induced degradation of lipofuscin [14], so that its presence would depend upon continuing metabolic demand. Evidence from RCS rats indicates that failure of RPE cells to phagocytose outer segments results in reduced lipofuscin levels [40]. Lipofuscin formation is almost completely dependent on a normal visual cycle and on the availability of dietary vitamin A [41]. In the RPE65 knockout mouse, the visual cycle is impaired and fluorescence is reduced [42]. Similarly, patients with RPE65 mutations can have reduced autofluorescence in the presence of near-normal fundi and OCT findings that do not suggest reduction in the photoreceptor layer thickness [15, 16]. It is widely accepted that photoreceptor degeneration results in atrophy and reduced rather than stable levels of autofluorescence and it is likely that autofluorescence is lost within a few weeks of photoreceptor loss. The presence of foveal or parafoveal AF in the photoreceptor dystrophies outlined in the current study, suggest that photoreceptors are likely to be intact and thus may be amenable to functional rescue. Recent developments, for example in the field of gene therapy, make early identification of candidate patients increasingly important.\nPatients with RP classically present with impaired night vision and visual field constriction, consistent with generalised retinal dysfunction involving rod more than cone photoreceptors. In the early stages the fundi may be near normal and rod-cone dystrophy is established by full-field ERG testing (Fig.\u00a01). In patients with normal visual acuity, the degree of macular sparing cannot be predicted from the severity of peripheral dysfunction. The cone and cone-rod dystrophies are typically characterised by progressive worsening of visual acuity, dyschromatopsia, and photophobia with eventual central scotomata and peripheral field abnormalities. Ophthalmoscopic abnormalities, when present, are generally confined to the macula. Photopic full-field ERGs are typically delayed and subnormal with milder scotopic ERG abnormalities (Fig.\u00a02a and b). A notable exception is the autosomal dominant cone dystrophy consequent upon mutation in GUCA1A; cone-mediated ERGs show amplitude reduction without significant implicit time delay [23] and although not diagnostic, may enable focussed mutational screening. Patients with \u201ccone dystrophy with supernormal rod ERG\u201d exhibit a wide range of fundus AF abnormalities including ring-like or bull\u2019s eye changes, central atrophy or increased foveal AF [28]. Full field ERGs are pathognomonic in this disorder (Fig.\u00a02c), have recently been shown to be consequent upon mutation in KCNV2 and have been described in detail [28, 29]. X-linked retinoschisis is usually associated with an electronegative bright flash ERG in keeping with inner retinal dysfunction (Fig.\u00a02d); two cases (aged 47\u00a0years and 49\u00a0years) with annular AF abnormalities have been documented within a series of seven atypical but genetically confirmed cases [22]. The rings of high density in these individuals surround areas of central atrophy and may represent an outer boundary of macular photoreceptor dysfunction in addition to generalised inner rather than outer retinal disease. Commonly, younger individuals with X-linked retinoschisis manifest stellate macular lesions that are also visible in AF images [43].\nConclusions\nA parafoveal ring of high density autofluorescence is a non-specific manifestation seen in different retinal dystrophies. Electrophysiology remains essential for accurate diagnosis. The high correlation of autofluorescence with PERG, mfERG and FMM demonstrates that AF abnormalities have functional significance and may be an important parameter in the monitoring of these patients. Autofluorescence may be of prognostic value and may help identify suitable patients and retinal areas amenable to future therapeutic intervention.","keyphrases":["autofluorescence imaging","retinitis pigmentosa","usher syndrome","cone-rod dystrophy","electroretinography","genotype-phenotype correlation"],"prmu":["P","P","P","P","P","M"]}
{"id":"J_Med_Internet_Res-8-2-1550702","title":"Who\u2019s Using PDAs? Estimates of PDA Use by Health Care Providers: A Systematic Review of Surveys\n","text":"Background Personal digital assistants (PDAs) find many uses in health care. Knowing rates of collective PDA use among health care providers is an important guiding step to further understanding those health care contexts that are most suited to PDA use and whether PDAs provide improved health outcomes.\nIntroduction\nA handheld computing device, also commonly known as a personal digital assistant (PDA), is a mobile computer about the size of the palm of the hand. More modern devices can access external networks or the Internet through a wireless connection. Since 1993, when Apple launched the first PDA (Newton MessagePad), use of PDAs has increased worldwide, with global PDA sales projected to surpass 17 million in 2008. This represents a compounded annual growth rate of 17.8% between 2002 and 2008 [1].\nHealth care has not been immune to this technological advance in handheld computing. In fact, PDAs find many applications in health care. Family physicians and specialists have been using PDAs for general medical reference, such as drug interactions, pharmacopeias, and cardiac risk [2-4]. Other important applications of PDAs are those involving data collection and management, as in patient tracking, electronic Case Report Forms in clinical trials, patient diaries, and infection surveillance [4-9]. However, the suitability of PDAs across all health care contexts and whether they benefit health outcomes remain open questions.\nMany of us would agree that it is necessary to evaluate a technology before its adoption to allow health care providers to make informed decisions. However, given that technology is a moving target, a common problem with evaluation is that practice frequently precedes research. By the time researchers have obtained funding, completed a study, and published it, the technology is either in widespread use or has been abandoned [10]. As well, the appropriate type of evaluation is not independent of the stage of adoption of the technology. For example, if 90% of the target users have already adopted a technology, then studies evaluating its general utility will no longer inform the adoption decision. In this case, research should focus on optimization of the technology in use. This is a familiar scenario in information technology research, and it underscores the importance of understanding the rates of adoption in helping direct approaches to research [10].\nIn a general overview article, Fischer et al (2003) summarized the current literature covering the use of handheld devices in medicine, primarily related to PDA functionality [4]. While implementation issues were discussed, rates of adoption were not addressed. Further, a recent review of PDA use in health care by Baumgart (2005) examined operating systems, basic functionality, security and safety, and limitations of PDA use [11]. It is a thorough overview of studies published since 2000 that addresses applications of handheld computers for health care professionals, but it touches only briefly on the prevalence of handheld use. Therefore, to our knowledge, there has not been any structured review conducted to date that specifically addresses the extent of use of handheld devices and estimated adoption rates. As such, this paper aims to systematically summarize all available survey data on health care providers\u2019 use of PDAs with the view of presenting the best available estimates of current PDA use. This paper also aims to project expected future adoption based on established technology diffusion models. From this information we draw implications for research and practice.\nMethods\nFor the purposes of this systematic review of surveys, the term PDA is used synonymously to refer to any handheld device. Some examples include the following: Blackberry; Palm operating system devices, which include Palm Tungstens, Handspring Visor, and Sony Clie; and Pocket PC devices, which include the Compaq iPAQ and HP Jordana.\nData Sources\nSurveys were identified as a subset selected from a broader systematic review examining all studies related to handheld devices in health care settings. Thus, initial search strategies and retrieved articles reflected this more extensive focus.\u00a0This comprehensive literature search was conducted in consultation with an information specialist. The searched bibliographic databases covered both medical and engineering disciplines, including the following eight databases: Medline, Current Contents, Inspec, BA\/RRM, Biotechnology, Biological Abstracts, EI Compendex, and EMBASE. The search was restricted to English-language literature published January 1993 (corresponding to the development of the first palm device) to February 2005. An updated search of Medline (PubMed) and EI Compendex (EI Village 2) was run near the project\u2019s completion (January 30, 2006).\nFurthermore, the reference lists from included studies were examined in an effort to identify additional surveys not captured in the reference databases. In addition, surveys identified from Google searches and those known to the authors to have been conducted by private market research firms as well as physician groups were nominated for inclusion in our screening.\nElectronic Search Strategy\nThe intent of searching the biomedical databases was to retrieve all studies related to handheld devices in health care. It is for this reason that the word survey was not included as a specific term in the original search strategy. The search did include the sample search terms detailed in Appendix 1. The search strategy for engineering databases limited retrievals to those articles relating to both handheld computing and health. All bibliographic databases were searched using subject headings tailored to each database and free-text terms in the titles and abstracts.\nEligibility Criteria\nSurveys were included for this present review if they met the following initial criteria: related to an application in human health care and involved the use of a PDA device; contained original data; written in English (not including abstract or conference proceedings); published after 1993; and specifically reported handheld usage rates (prevalence of PDA use as a metric) in populations of health care professionals who were surveyed about the extent of their PDA use. Although conference proceedings were excluded, if deemed potentially relevant, a cross-check was conducted to see if there was an ensuing journal publication. A survey was not included if the handheld device being evaluated had undergone extensive custom modifications. A final set of unique references was identified and posted to the proprietary Web-based screening system SRS (Systematic Review Software).\nSelection Process\nThe selection process for this present survey review consisted of two phases. First, it began with a screen of full-text articles that had already been retained because their title, abstract, or keywords suggested they contained relevant information on PDA use in health care settings. Therefore, for assessment of relevance, surveys were included if they appeared to contain pertinent study information and if there was no unequivocal reason for exclusion. Second, upon updating the searches, authors returned to the screening of the title, abstract, and keywords for each citation strictly to identify potentially relevant and most recent PDA usage surveys. Eligibility criteria were applied to the full-text surveys, which were reviewed independently by two reviewers (CG and KE). Disagreements were resolved by consensus. Figure 1 provides a modified QUOROM flow chart outlining the process for selecting identified PDA usage surveys.\nFigure 1\nModified QUOROM Flow Chart for Identified PDA Usage Surveys\nData Abstraction\nThe contents of each included survey were abstracted by one reviewer (CG), with an additional research assistant providing verification (TR).\nAnalysis\nThe data from all included surveys were extracted in a predefined, standardized fashion with abstraction verified by a second person and assessed descriptively (Appendix 2). Quality assessment methods for descriptive study designs such as surveys have not been established. Although some assessment frameworks exist for assessing survey research [12,13], none of them have been validated or empirically shown to include criteria that are associated with the reduction of bias in empirical surveys. Therefore, survey quality was not formally assessed.\nTable 1\nIncluded surveys\nYear of Survey\/Publication\nAuthor\nPrevalence of PDA Use\nHealth Care Professional Group\n1\n1999\/2000\nHucko [18]\n15% (use in clinical work)\nPhysicians\n2\nNS\/2001\nACP-ASIM [19]\n47% (use in clinical work)\nSpecialists (Internists)\n3\n2001\/2001\nVersel* [20]\n60% (use in practice)\nPhysician Executives (organizational survey)\n4\n2001\/2001\nMartin [21]\n19.3% (use in clinical practice)\nPhysicians & Specialists\n5\n2001\/2001\nTaylor [22]\n26% (use in practice)\nPhysicians\n6\n2001-2002\/2002\nAAP [23]\n38% (NS)\nSpecialists (Pediatricians)\n7\n2000-2001\/2002\nCriswell* [24]\n67% (use in practice)\nResidents (Family Medicine) (organizational survey)\n8\n2001\/2004\nMiller [25]\n26.2% (office-based use)\nPhysicians\n9\n2001\/2004\nBalen [26]\n33% (use at work or home)\nPharmacists\n10\n2001-2002\/2004\nBarrett [27]\n75% (use in practice)\nMedical Residents\n11\n2002\/2002\nMartin [2]\n27.9% (use in clinical practice)\nPhysicians & Specialists\n12\n2002\/2002\nVersel* [28]\n33% (use in physician offices)\nPhysician Executives (organizational survey)\n13\n2002\/2003\nMcCleod [29]\n46% (use at medical institutions)\nSpecialists, Medical Residents, & Fellows (Internists)\n14\n2002\/2004\nCarroll [30]\n35% (use at work)\nSpecialists (Pediatricians)\n15\n2002\/2004\nDeGroote [31]\n61% (use on an academic health science campus)\nHealth Sciences Faculty & Medical Residents\n16\n2003\/2003\nMartin [32]\n32.9% (use in clinical practice)\nPhysicians & Specialists\n17\nNS\/2003\nVincent [33]\n36% (use alone or in conjunction with log-card procedure in documenting)\nMedical Residents (Family Practice)\n18\n2003\/2003\nVersel* [34]\n75% (carry & use PDAs)\nPhysician Excutives (organizational survey)\n19\n2004\/2005\nAMA\/Forrester [14]\n57% (use regularly in a work week)\nPhysicians, Specialists (Surgeons), & Medical Residents\n20\n2004\/2005\nWilden [35]\n91% own; 85% use on daily basis; 9% weekly; 215% monthly\nSpecialists (Anestheologists)\n21\n2001\/2005\nStromski* [36]\n64% of programs report \u201cmost or all\u201d residents use for clinical purposes\nMedical Resident Programs (Emergency Medicine) (organizational survey)\n22\nNS\/2005\nStroud [37]\n67% (NS)\nNurse Practitioners & Students\n23\nNS\/2005\nBoonn [38]\n45.1% (own or use daily)\nSpecialists (Radiologists)\nNS\/2004\nJoy\u2020 [17]\nDifficult to interpret the prevalence numbers among the resident respondents\nMedical Residents (Obstetrics & Gynecology)\n2004\/2005\nNational Physician Survey (Canada)\u2020 [15]\nUnable to establish overall prevalence due to way data have been presented;48.6% of medical students have a PDA (although unable to infer use)\nPhysicians, Specialists (various), & Medical Students\nNote: An excerpt from the \u201cTaking the Pulse\u201d study published in October 2004 by Manhattan Research [16] reports that 40% of all US physicians currently use a PDA, increasing from 35% in 2003. However, for this present review, the authors were unable to obtain a full copy of the report in spite of having contacted Manhattan Research on two separate occasions (February 2006).\nNS = not specified\n*Survey conducted at organizational level (vs individual level responses)\n\u2020Survey of PDA use but prevalence data could not be established (referred to descriptively only)\nResults\nFrom a total of 816 full-text articles that underwent relevance assessment for a systematic review of the literature examining broad-ranging PDA use in health care, a subset of 18 surveys reporting PDA prevalence rates were identified (see Figure 1). Additionally, upon updating the search, an additional 959 records were retrieved and screened, from which 5 additional unique surveys were included. Furthermore, a total of 8 surveys were reviewer nominated, 3 of which were identified upon updating. Unfortunately, the authors were not able to obtain access to one Internet market research report. Prevalence numbers from 2 surveys were found too difficult to interpret, and, therefore, these data could not be utilized further in our results; however, we refer to both studies descriptively.\nIt is from this pool of literature that a total of 23 unique surveys were identified (Table 1):15 were published articles in scientific journals, and 8 were nonacademic, reviewer-nominated citations that were either reports available for purchase, press releases, or trade magazine articles and thus not subject to formal peer review. Of these 8 surveys, 5 were conducted by Internet market research firms, 2 were conducted by physician groups, and 1 was conducted by a market research firm in conjunction with a physician group (American Medical Association).\nSurvey Characteristics\nThe included surveys were published between 2000 and 2005, with survey data collected between 1999 and 2004. One survey had a four-year lag between data collection and publication, three surveys had a lag of three years, and three surveys had a lag of two years. We were unable to determine publication lag in four surveys as no data collection dates were provided. Surveys were from the United States (16), Canada (4), Australia (1), both the United States and Puerto Rico (1), and both the United States and Canada (1). Survey methodology reflected the following: self-administered questionnaires distributed solely by mail (11); telephone interviews (2); Web-based online surveys (4); and combined distribution by electronic or postal mail as determined by the recipient (4). Two studies did not report the methodology used. Response rates ranged from 5.7% to 92.6% across 13 of the included surveys; 10 surveys did not report such rates.\nPDA Use\nIn presenting the results, we group the PDA users by type of health care provider and personal characteristics (eg, age).\nIn terms of PDA use, physician specialists were surveyed exclusively in five surveys. Three surveys examined practicing physicians, three included physicians and specialists combined, two included medical residents exclusively, while two surveyed an amalgam of physicians, specialists, medical residents, and\/or students. Three surveys targeted physician executives and organizational practice leaders. One survey was directed at directors of family practice residency programs, while a further survey targeting individual PDA use in emergency medicine resident programs was completed at the organizational level.\nIn addition to physicians as users of technology, one survey targeted practicing hospital pharmacists and another targeted a national sample of nurse practitioner students and faculty. One survey included faculty and residents across several health science disciplines, including medicine, dentistry, nursing, public health, pharmacy, and applied health science.\nTo more accurately reflect handheld use across time, reported surveys were examined, when possible, from the timepoint when survey data were collected versus when published. When not possible, the publication date was the reported timepoint used. Collectively, the included surveys do indicate that PDA use is high, albeit somewhat variable, across studies. The reported prevalence rates of PDA use lend themselves well to an estimation of trend over time (Figure 2), and, as such, since 1999, there is evidence of an increase in PDA usage. Results do not include surveys completed at the organizational level. Surveys are presented according to data collection dates, with the exception of the American College of Physicians study (2001) [19], Stroud (2005) [37], and Boonn (1995) [38], which report publication dates only. The noted drop in 2003 is due to the paucity of surveys conducted in that year. Based on the most recent survey statistics (2004\/2005), the current overall adoption rate varies between 45% and 85%, as derived from individual level survey data. In addition, of the five surveys completed at the organization level (eg, physician executives or medical program directors speaking on behalf of their individual members), the PDA use of their group members was estimated to be 60% (2001) [20], 67% (2001) [24], 64% (2001) [36], 33% (2002) [28], and 75% (2003) [34].\nFigure 2Range plots of PDA usage by health care providers (n = 17); middle points represent range medians\nTo elaborate on the percentage of overall adoptions rates, a US survey of 769 practicing physicians conducted in 1999 found that only 15% of physicians use a PDA in practice [18]. In a 2000\/2001 survey of directors of family practice residency programs in the United States and Puerto Rico, use of handheld computers by either an individual or group was reported in 67% of the residency programs [24]. In 2001, 47% of 489 US-based internists surveyed were using a PDA [19]. A subsequent 2001 survey of 834 practicing physicians found that the proportion using PDAs had increased to 26% [22]. If we only look at professional use, then the increase is from 10% in 1999 to 18% in 2001 [22]. Among a national sample of practicing physicians surveyed in 2001, 26% reported using PDAs for office-based work [25]. In 2001\/2002, 38% of 696 office-based physicians indicated that they used a PDA in their practice [23]. Of practicing hospital pharmacists surveyed in 2001, 33% reported using a PDA at work or home, with 28% using one on a daily basis [26]. These numbers reflect both types of use: personal and professional (ie, as an integral part of everyday practice). In 2001, 75% of residents in a teaching hospital reported using their PDA on a daily basis [27]. In 2002, 35% of US pediatricians were using a PDA at work, and 40% had one for personal use [30], and 46% of internal medicine physicians and residents were reporting PDA use [29].\nIn Canada, similar PDA use data have been collected since 2001 as part of the annual Physician Resource Questionnaire conducted by the Canadian Medical Association. PDA use among physicians increased from 19% in 2001 [21] to 28% in 2002 [2] and to a third in 2003 [32]. These data conclude that, in 2003, a third of Canadian physicians were using PDAs, which marked a 73% increase from 2001. Further, more than 50% of Canadian medical doctors under 35 years of age reported that they were using a PDA or wireless device in clinical practice [32]. The data did not differentiate type of professional use.\nIn a PriceWaterhouseCoopers survey in 2001, 60% of the physician executives who responded indicated that their organization had at least one physician with a PDA [20]. Reportedly, this represented an upward trend from 26% in a similar 2000 survey. Further, in 2003, the trend continued, and 75% of respondents reported that their organization\u2019s physicians were using PDAs. This increase in PDA use came after a steep decline to 33% in 2002 [28,34]. A sample of health science faculty and medical residents was surveyed in 2002 about their PDA use. Combined results from the various faculties and residents indicated that 61% used a PDA [31].\nIn 2004, 57% of a sample of US physicians indicated that they regularly used a handheld computer in a typical work week [14]. Results obtained in 2004 from a survey of members of the Austalian Society of Anaesthetists indicated that 91% of respondents owned a PDA; 85% reported using it on a daily basis, and 66% were reportedly \u201cdependent\u201d upon the handheld device, although the term dependent was not defined [35]. In 2005, when physician members of the Radiologicial Society of North America were surveyed, 45.1% reported owning or using a PDA on a daily basis [38]. However, the survey authors suggested use among this group of specialists appeared to be lower than for other physicians because a radiologist often works in front of a full workstation in clinic and therefore relies less on a mobile device. Further, PDAs are not yet well equipped to handle the tasks radiologists need to perform. In 2005, Stroud et al became the first group of researchers to address the use of PDAs in the field of nursing. Survey results concluded that the majority (67%) of participants used this technology [37].\nWhile PDA use has clearly increased since 1999, it appears as though only a handful of studies have examined the prevalence and usage patterns of such technology outside of physician groups. Furthermore, when comparing the included surveys in depth, distribution of use is not uniform across selected characteristics of surveyed health care professionals. Therefore, further subgroup analyses from the included surveys are provided below. Patterns of handheld use are also briefly examined.\nPatterns of PDA Usage\nAge\nBased on a survey of 250 family physicians, as far back as 1995, younger physicians (less then 40 years of age) were more likely to consider carrying a handheld computer than older physicians (94% vs 84.5%) [39]. More recent data from this present review also suggest an age differential in usage patterns. A 2001 survey of 834 US practicing physicians found that use of handheld devices was higher among doctors under age 45 (33%) than among older doctors (21%) [22]. Another study found that pediatricians graduating from medical school in the last five years were more likely to use a PDA in practice than those who graduated more than five years ago [30]. According to a survey conducted by the American Academy of Pediatricians in 2001, PDA use was highest among those members under 30 years of age, with a reported usage rate of 75% [23]. Another study found that 60% of US internists below 40 years of age used a PDA, while only 34% older than 51 years did [19]. McLeod et al (2003) also found that PDA usage captured in 2002 among a sample of internal medicine physicians and residents under 30 years was much higher (68%) versus those over 40 years of age (37%) [29]. In Canada, 2003 usage was highest among younger physicians, with more than half of those under the age of 35 years (53%) using a PDA, compared with 15% of physicians aged 65 or older [32]. According to the American Medical Association\/Forrester Research 2005 Physician and Technology Study, more doctors under the age of 40 years were reportedly using PDAs (55%) than those over 40 years (45%) [14]. In 2005, the mean age of nurse practitioners and students who reported using a PDA was 42 years [37].\nStudents and Medical Residents\nResidents tend to be younger, therefore it follows that they are more likely to use PDAs. This is also substantiated by direct evidence. A survey of directors of family practice in the United States and Puerto Rico conducted in November 2000 (306 responses) found that use of handhelds in residency programs, either by an individual or group, was 67% [24]. A 2001 survey of residents in a teaching hospital reported that more than 75% used their PDA on a daily basis [27]. Stromski et al (2005) surveyed emergency medicine residency programs in 2001 to identify the methods of procedure documentation to examine the number of programs transitioning to more advanced information technology systems (eg, PDA use). Their results indicated that 13% of the residency programs required the use of PDAs, 15% of programs purchased PDAs for their residents, and a similar proportion reported that PDAs were used by \u201cmost or all\u201d of their residents to document procedures. Further, 64% of programs reported that \u201cmost or all\u201d of their residents utilized PDAs for clinical purposes. DeGroote et al found that, in 2002, 71% of medical residents reported using PDAs versus 56% of faculty members [31]. In a 2002 survey, McLeod et al noted that the percent of frequent PDA users among internal medicine residents and fellows in training exceeded 70%, compared to only 50% of attending physicians [29]. From a survey of the experiences of family resident graduates in obtaining hospital privileges and in documenting procedures and deliveries, Vincent et al (2003) concluded that 36% of the respondents used a PDA alone or in conjunction with a log-card, paper-based system. Unfortunately, this study did not present any other prevalence data on PDA use [33]. However, from survey data captured in 2004, the handheld technology gap between residents and physicians began to close: a US study concluded that 73% of residents regularly used a handheld computer in a typical work week, followed closely by 71% of family\/general practitioners [14]. In a survey of PDA use by nurse practitioner students and faculty, Stroud et al found that of the total respondents who reported PDA use, 73% were nursing students [37].\nOne survey by Joy et al (2004) met our initial criteria but could not be incorporated into the results analysis. Although this study did examine PDA use in obstetrics and gynecology residency programs, it was difficult to interpret the prevalence numbers among the resident respondents. Likewise, the National Physician Survey (2004) did not present overall PDA prevalence rates but did ask Canadian medical students if they had a PDA or wireless device [15]. Of the 2721 respondents, 24% in first year, 40.6% in second year, 70.6% in third year, and 71.6% in fourth year reported having a PDA, representing an overall average of 48.6% among students [15]. Unfortunately, these 2004 figures provide no information on how medical students were using this technology and in what contexts.\nGender\nPDA usage among men and women was equal in a 2001 survey of internists [19]. Similarly, McLeod et al (2002) found no significant gender difference in PDA users among a 2002 sample of internal medicine physicians and residents [29]. However, pediatrician PDA users were most likely male, as reported in 2002 [30]. As well, the 2003 Physician Resource Questionnaire analysis concluded that male physicians were somewhat more likely to use a PDA in their practice than were females (35% vs 30%) [32]. More recent data from a 2004 survey of PDA use among US physicians, specialists, and medical residents suggested that male clinicians were slightly more likely than their female counterparts to regularly use handhelds (53% vs 47%) [14]. On the other hand, nurse practitioner data from 2005 show that men (82%) were notably more likely than women (64%) to use a PDA (P < 0.05) [37]. However, the authors cautioned that they were unable to determine the significance of this finding given that the actual survey sample of men (n = 38) as opposed to women (n = 188) was small. The authors suggested that if ease with PDA technology is less common in women, then the nursing profession, dominated by females, may need elevated momentum to adopt PDA technology across nursing practice [37].\nFamily Physicians versus Specialists\nThe most recent Physician Resource Questionnaire (2003) analysis concluded that Canadian family physicians were just as likely to use a PDA (33%) when compared to medical (34%) and surgical (32%) specialists [32]. This was the third consecutive year these figures rose consistently across all physician groups in Canada [2,21,32]. However, according to a US survey of physicians published in 2005, the biggest adopters of PDAs in professional practice were family and general practitioners (71%) when compared to surgical specialists (54%) [14]. The above mentioned studies are the only survey data available directly comparing general physician use to that of specialists.\nLarge and Hospital-Based Practices\nA US survey of practicing physicians found that use was higher among those who were wholly or partly hospital-based (33% and 29%, respectively) than among those who were office-based (23%) [22]. Usage was also higher among physicians in large practices (33%) than in solo practice (16%) [22]. Carroll et al (2004) also found that PDA users tended to not be in private practice [30]. Additional survey data from 2004 indicated that of US physicians practicing in primary practice offices with fewer than 10 physicians, 49% reported regular use of a handheld computer [14]. Miller et al (2004), reporting on a national sample of practicing physicians, found that in a group practice consisting of an average of nine physicians, handheld use was approximately 56% [25].\nUrban versus Rural Physicians\nFrom a random sample of US pediatricians in 2002, PDA users were most likely from urban communities [30]. Similarly, results from Canada\u2019s Physician Resource Questionnaire in 2001 indicated PDA use to be higher among physicians practicing in urban centers (19.9%) than in rural centres (13.4%) [21]. However, by 2002, rural use (29.6%) surpassed urban use (27.7%) among physicians [2]. In Canada, this trend continued in 2003, with 36.9% of rural respondents indicating PDA use versus 32.5% of urban respondents [32].\nProfessional Use\nFive surveys considered PDA use in both a professional and personal context; 17 studies exclusively captured professional use. One study reported general prevalence rates for PDA use among pediatricians; however, it did not specify if use was in clinical practice or outside of work.\nIn order to discern professional use more closely, we explored administrative PDA uses versus direct use in clinical patient care. We found that of the surveys that concern PDA use within a health care setting, 17 of 23 studies (74%) reported use pertaining to administrative or organizational tasks, while 14 of 23 studies (61%) addressed PDA use in patient care. Billing and coding were the most frequently performed administrative PDA functions in 50% of the surveys reporting administrative uses. This was followed by 44% reporting calendar scheduling, 31% reporting Web and email access, 25% reporting address book use, and 25% stating use in charting patient details into an electronic health record. Other reported administrative tasks included the following: word processing, calculator, charge capture, procedure documentation, outpatient tracking, resident hours, telephone message tracking, general time management\/personal organizer, patient referrals, procurement of supplies, patient census, order entry, dictation, and passwords and pins.\nIn terms of patient care, access to drug information was reported in 93% of the surveys reporting clinical PDA use, while 50% reported prescribing, 43% stated accessing patient records, 43% described medical calculator use, and 36% indicated use in reference to laboratory values. Other reported clinical PDA uses included access to medical references, patient tracking and patient reminders, clinical decision pathways and managed care applications, telemedicine, and diagnostic imaging or radiology applications.\nOnly one survey reported PDA use for patient education, and one referred to PDA use for research purposes.\nDiscussion\nThis paper summarizes the results from surveys examining adoption of PDA use. These survey data are in reasonably good agreement and suggest a sizable proportion of physicians use handheld devices. However, most of the sources of survey data did not distinguish well between types of applications being used most often and whether the PDAs were being used professionally for administrative purposes or for direct clinical work. It is encouraging to note that our findings are similar to those of an analysis of online registrations and downloads of a PDA drug reference guide, which concluded that approximately one fifth of US physicians (150000) and half of medical students in the United States (33000) were PDA users [40].\nOur grouped survey data suggest that there is little information on the PDA usage rates among nonphysician health care providers. However, collectively, these data suggest that use of handheld devices has become a subject that health care professionals need to know about. By systematically gathering this usage information, it is difficult to deny the prevalence of PDAs in health care. With this basic understanding of current handheld usage patterns, we need to consider the impact of this development of mobile handheld technology on both practice and research.\nAccording to a commonly accepted descriptive model of the diffusion of innovations developed by Rogers, when the cumulative rate of users of a new invention is plotted versus time, the result is an S-shaped curve [41]. Interestingly, this appears to be true of most technological innovations, irrespective of the technology. For example, Hall and Khan (2003) reviewed the S-shape adoption patterns of a variety of 20th century consumer products (eg, washing machines, video cassette recorders) [42], while Teng et al (2002) developed historical diffusion curves for information technologies (eg, personal computers, email) [43]. Variations in diffusion slopes do exist given that some technologies will diffuse more rapidly than others.\nHealth care information technologies have also been examined within this diffusion framework. England et al (2000) studied organizational and technological factors determining the rate at which innovations diffuse in the health industry [44]. In 2005, RAND Health completed a report characterizing the diffusion of electronic health records along an S-shaped adoption curve [45].\nTechnologies typically go through multiple phases during their adoption life cycle, which may last for many years [41,46]. The characteristics of the adopters change over time and so does the nature of suitable evidence to inform their adoption decisions. For example, innovators (the first 2.5% who adopt a new technology) do not need evidence to make an adoption decision. Early adopters (the next 13.5%) are satisfied with case studies and examples of successful adoption and benefits [41]. Examining the typical technology adoption curve for handheld devices (Figure 3) based on the adoption percentage of PDAs thus far from the most recent available data (2004\/2005), it can be concluded that we are now at the steepest stage in the adoption S-curve, with a transition from the early majority to the late majority.\nFigure 3\nThe S-shaped diffusion of technology curve [41]\nThe increase in PDA adoption means a potential reduction in hardware and training costs when using handheld devices in the provision of care and in research. Because of the high probability that target health care professionals may already have a handheld device and will already know how to use one, the overall hardware purchase costs could be reduced, and the end user will not necessarily have to be trained from scratch.\nTo date, use of PDAs in health care appears to have preceded extensive evaluative research. PDA adoption rates, already high, continue to be a moving mark with projections for rapid growth in the short term. By comparing handheld device diffusion to other health information innovations, and by placing PDA use within existing diffusion models, we are able to better predict the future of handheld growth in health care and therefore develop more timely and appropriate evaluative research to accompany such growth.\nUnfortunately, we were unable to include information from two national physician surveys. The first report entitled \u201cTaking the Pulse\u201d was published in October 2004 by Manhattan Research [16]. Information gleaned from a report excerpt stated that 40% of all US physicians surveyed in 2004 were using a PDA, marking an increase from 35% in 2003. Reported top activities performed on a PDA by all US physicians (in order) were personal scheduling, professional scheduling, accessing a drug reference database, accessing online information, writing\/entering clinical notes, and mobile email access [47]. These report findings are similar to our overall findings in this present review.\nThe second national physician survey not incorporated into our analysis was the Canadian National Physician Survey (NPS) (2004), which provides valuable insight into what information technology, including PDAs, physicians and specialists have in their main patient care settings [15]. However, overall prevalence rates could not be determined from the data provided given the manner in which they were presented. Nonetheless, in reviewing the national data, we can descriptively draw some conclusions. First, it appears as though male physician PDA use is higher than that of females. This appears to be consistent across all tasks involving PDA use although differences do appear to be small. This is consistent with our general findings in which males are only marginally more likely to use a PDA than are females. Interestingly, when examining age-related data from the NPS, it appears as though the age factor may in fact be PDA task-specific. For example, electronic health record usage appears to decrease as the age of physician users decreases. However, PDA use for drug interaction information increases when the age of the physician user decreases. This appears contrary to most other surveys that show younger age is associated with higher general PDA use. Perhaps what this information tells us is that handheld use may be more complex when broken into task-specific strata.\nIt is worthy to note that, with the exception of one survey focusing on nurse practitioner students, little mention was made in the surveys of PDA use by students across health care disciplines, including medicine. Several universities in Canada and the United States now mandate use of PDAs for medical undergraduate students and residency programs; therefore, it is assumed this could potentially affect prevalence rates. However, because none of the included surveys examined mandated use, we are unable to infer if this is responsible for recent increases. However, this raises an important issue to be considered in future studies related to students and rates of handheld adoption.\nTo better understand the prevalence rates among the included surveys, it became important to categorize the drivers for PDA use as either professional or personal. We therefore attempted to discern what specific PDA tasks the respective health care professionals were performing. This was done by classifying, whenever possible, the use as administrative versus care. On the surface, it would appear that administrative and organizational tasks on a PDA exceed those related to patient care, perhaps signaling where the growth in adoption is most likely to occur.\nIn this present review, we can only speak broadly to rates of adoption and patterns of use. Drawing inferences from the survey data was often limited by lack of, or differences in, operational definitions in aspects of handheld use being measured. For example, the term use was often not defined by frequency (eg, specific units of time\u2014day, week, month). Taking these issues into consideration would be a useful exercise for future surveys as well as information technology prevalence studies in health care.\nIn conclusion, physicians are increasingly accustomed to using a PDA, and, therefore, technology expertise will not likely be a barrier to deploying handheld applications. There is an urgent need to evaluate the effectiveness and efficiency of specific tasks using PDA technology (eg, implementation, searching, reference, data entry, reporting) to inform those persons developing and those using handheld applications. Furthermore, it is not clear why there is a paucity of evidence on the extent of adoption of PDAs by other health care providers: is it that they lag in the use of this technology or is it simply that they have yet to be studied?\nLimitations\nThis review has a number of limitations. Issues around response bias and inability to draw causal inferences weaken survey methodology. It may be the case that those surveyed feel a stronger affinity to the survey sponsor, who has a greater interest in the questions asked, or are in complete disagreement with the topic at hand. This can skew results in difficult-to-measure ways. Quite possibly, the nonrespondents are the least committed (ie, nonusers of PDAs). As a result, the critical objective of drawing a true random sample of the populations that are the focus of the survey is compromised and the findings somewhat impure.\nThe reported methodologies across these surveys appear to be heterogeneous, which limits their comparability. As noted, the quality of the included surveys could not be determined given the absence of validated quality assessment instruments, and, therefore, there was no adequate way to assess the influence of bias. A related issue is that some of the included surveys did not go through a rigorous peer-review process. These combined issues made judging the strength of the evidence not possible. One would assume surveys identified from scientific journals would be a source of less biased information. However, in defense of the nonacademic surveys, there is a consistency in results between those peer-reviewed versus those that were not. This may suggest that our main conclusions regarding adoption rates are fairly robust and not disconnected even with the inclusion of non\u2013peer-reviewed evidence.\nConclusions\nThe objective of this study was to determine the adoption rates of PDAs in health care settings, and to project expected adoption in the future based on established technology diffusion models. Our findings from a systematic review indicate the current overall adoption rate for professional use of PDAs among health care providers, namely physicians, is 45% to 85%. Younger physicians, residents, and those working in large and hospital-based practices are more likely to use a PDA. Professional use in health care settings appears to be more focused on administrative tasks when compared to those related to patient care, although this requires further study. The adoption rate is now at its highest rate of increase according to a commonly accepted diffusion of innovations model. Additionally, the impact of PDA use on practice appears to be immediate in terms of costs and training. Familiarity will not likely be a barrier to deploying handheld applications in health care. However, there is a critical need to evaluate the effectiveness and efficiency of specific tasks using handheld technology within the health care system and across health care provider PDA user groups.","keyphrases":["health care","systematic review","survey","personal digital assistant","health technology adoption"],"prmu":["P","P","P","P","R"]}
{"id":"Int_Arch_Occup_Environ_Health-4-1-2254471","title":"Sickness absence due to depressive symptoms\n","text":"Objective There is no information on the duration of absence of depressed Dutch workers. The aim of this study was to determine the duration of sickness absence due to depressive symptoms in the working population.\nIntroduction\nMental illness is an important cause of disability and its impact on job performance and productivity is substantial. Depression is one of the most common mental disorders. According to the World Health Organization (2002), depression will be the leading cause of morbidity by the year 2010. Depression was found to be related to long absence from work (Harrison and Martocchio 1998; Nieuwenhuijsen et al. 2006) and to be a significant predictor of work disability (Kouzis and Eaton 1994; Skodol et al. 1994; Kessler et al. 1999). Depressed employees had a 28\u00a0times higher risk of absence as compared to employees who did not suffer from depression (Kouzis and Eaton 1994). Respondents with depression reported a mean of 35\u00a0days (95% CI 27\u201344) in the past year when they were totally unable to work or carry out their normal activities because of their depression (Kessler et al. 2003). When depressed employees stayed at work, they had a lower than normal productivity (Kessler and Frank 1997).\nDepressive episodes were reported to vary widely in their duration, with a median ranging from 2 to 12\u00a0months and with rates of chronicity (i.e. a duration of 12\u00a0months or more) between 15% and 50% (Keller et al. 1982; Coryell et al. 1994; Mueller et al. 1996; Eaton et al. 1997; Solomon et al. 1997; Spijker et al. 2002; Furukawa et al. 2005). Estimates of the mean duration of depressive episodes varied from 13 to 27\u00a0weeks (Blazer et al. 1994; Eaton et al. 1997; Kendler et al. 1997; Kessler et al. 2003).\nIn international research the duration of sickness absence due to depressive disorder was 142\u00a0days in 213 adult psychiatric outpatients (Sorvaniemi et al. 2003). In Norway, the mean sickness absence duration in psychiatric patients amounted to 110\u00a0days in men and 103\u00a0days in women (median 57 and 52\u00a0days, respectively), with depression as the most common diagnosis (Hensing et al. 2000). In a Swedish sick-leave database a mean sickness absence duration due to combined depression and anxiety disorders was 73\u00a0days in men and 75\u00a0days in women (Hensing et al. 1996). In a reference population consisting of working people, the mean duration of a major depression was 52\u00a0days for a single episode (with 4.1% lasting longer than 6\u00a0months) and 74\u00a0days for a recurrent episode (with 9.2% lasting longer than 6\u00a0months) according to the Medical Disability Advisor (2006). There is no information on the duration of absence of depressed workers in The Netherlands (Raddjoe and van der Hoek 2005). The results of international studies are not comparable to the Dutch situation, because of differences in social legislation and sickness absence regulations.\nAs it is very likely that the results of international studies are not comparable to the Dutch situation, the aims of this study were: (a) to investigate the mean (and median) duration of absenteeism due to depressive symptoms in the Dutch working population by sector and company size, and (b) to investigate gender and age influences on the mean (median) duration of absenteeism due to depressive symptoms.\nStudies on sickness absence because of depression revealed gender differences, with men having a higher risk of sickness absence when depressed (Laitinen-Krispijn and Bijl 2000). Women had a higher incidence of sickness absence due to mental illness (North et al. 1993; Hensing et al. 1996, 2000), but the duration of sick-leave spells due to mental illness were longer for men (Hensing et al. 1996, 2000). Based on Dutch national statistics (Central Statistical Office of the Netherlands 2006), in which women have longer absence durations and higher disability figures than men, we hypothesize that depressed women have longer absence durations than men (H1). We also hypothesize that older employees have longer absence durations than younger employees (H2) based on Dutch national statistics and studies of Hensing et al. (1996, 2000). Furthermore, we hypothesize that employees in small companies have longer absence durations as compared to employees in large companies (H3), because of less reintegration opportunities in small companies. Finally, we hypothesize that employees in educational and health care services have longer absence durations as compared to employees in other sectors (H4). Employees in the education and care sectors have a substantially increased risk of being at work when sick, because of difficulties in replacement (Aronsson et al. 2000) and therefore, when they take absence the duration may be longer.\nMethods\nData\nThe prevention, supervision and medical examination of sickness absence is a task of the employer for which he can choose to engage an occupational health department. ArboNed (the second largest occupational health department in the Netherlands) employs an absenteeism registration system for their affiliated companies. We register sickness absence and its causes in about 15% of the total Dutch working population. In our population, commercial services are over-represented (57% vs. 41%) and non-commercial services (e.g. health care, civil servants) are under-represented (17% vs. 34%) as compared to the total working population in the Netherlands.\nAll absence periods that started between April 2002 and November 2005, and were diagnosed as depression were selected from the registration system. Absence periods were encoded as depression by the occupational physician when symptoms of depressed mood (such as feelings of profound sadness or emptiness) and\/or reduced interest in activities that used to be enjoyed lasted for at least two weeks in combination with 3 or more of the following symptoms: decreased energy, decreased motivation, appetite changes, disturbed sleep pattern, agitation or inhibition, guilt feelings, low sense of self-worth, impaired concentration, or self-destructive thoughts. The symptoms must be accompanied by evident suffering and adverse effects on personal as well as social functioning.\nThe age of the employees was registered at the moment of sick-leave. Absent men had a mean age of 41.6\u00a0years (SD\u00a0=\u00a09.9) and women of 38.2\u00a0years (SD\u00a0=\u00a09.9) (T\u00a0=\u00a017.2; df\u00a0=\u00a09,864; P\u00a0<\u00a00.01). The company size in which the employee worked was divided into four categories: <75 employees, 75\u2013500 employees, 500\u20135,000 employees and >5,000 employees. In 95% of cases the company size was known. The companies were categorized into the following sectors (mean age\u00a0\u00b1\u00a0standard deviation): construction industry (40.2\u00a0\u00b1\u00a010.2), health care (39.6\u00a0\u00b1\u00a010.1), trade (38.1\u00a0\u00b1\u00a010.3), catering industry (37.5\u00a0\u00b1\u00a010.0), industry (40.9\u00a0\u00b1\u00a09.8), education and public sector (43.9\u00a0\u00b1\u00a09.9), transportation and communication (41.0\u00a0\u00b1\u00a09.7), commercial services (38.6\u00a0\u00b1\u00a09.9), Other\/unknown (39.9\u00a0\u00b1\u00a09.9). In 72% of cases the sector was known.\nAbsence duration\nBetween April 2002 and November 2005 a total of 9,910 newly originating episodes of absence due to depressive symptoms were registered. The period (in calendar days) between the first day of sick-leave and the date of return to work or disability pension was computed. In The Netherlands an employee can benefit a disability pension after one year of work incapacity1. Absence periods of 365 or more calendar days were considered to reach this limit, and therefore the maximal duration of an absence episode was 365\u00a0days.\nAbsence duration was not corrected for part-time return to work, unless mentioned otherwise. When the number of absence days is corrected for part-time return to work, this is done by dividing the number of absence days by 1\/reintegration percentage. For example, when the employee returns to work for 50% the number of absence days is divided by 2.\nWhen estimating the duration of absence spells, it is important to censor absences which have not ended by the end of the observation period or at the dismissal date (Blossfeld and Rohwer 2002). Therefore, we censored absences that did not end before the retrieval date (November 1, 2005). Absences which ended, because the employee resigned, were also censored.\nStatistical methods\nThe data were analyzed using SPSS for Windows, version 13. Kaplan\u2013Meier survival curves were computed in order to obtain mean and median absence duration. The Kaplan\u2013Meier method (Kaplan and Meier 1958) calculates the risk set at every point in time where at least one recovery occurred. The risk set also includes episodes that are censored at this point in time. A censored episode contains the information that there was no return to work or reaching the one-year absence limit at the end of the study period or the dismissal date. The survival functions were plotted using life tables. The mean number of absence days corrected for part-time return to work in the different groups was compared using analysis of variance.\nIf the main effect was significant, the Tukey post hoc test was performed in order to determine which groups differed from each other.\nResults\nIn the period April 2002 to November 2005, 9,540 employees were absent with depressive symptoms. This is about 1% of the population covered by the occupational health department. Of these employees 9,196 (96.4%) had one absence episode, 321 (3.4%) had two absence episodes and 23 (0.2%) had three or more episodes of absence due to depressive symptoms. In Table\u00a01, the course of sick-leaves with depressive symptoms is presented. Men more often returned to work and women more often reached disability pension (\u03c72\u00a0=\u00a013.6; df\u00a0=\u00a03; P\u00a0<\u00a00.01).\nTable\u00a01Number and course of depressive episodesMenWomenTotaln%n%n%Number of employees4,5225,0189,540Number of absences4,735100.05,175100.09,910100.0Returned to work within a year3,20567.73,35564.86,56066.2One year of incapacity for work71915.290317.41,62216.4Left the employment during the illness3076.53807.36876.9Not returned to work at end of observation period50410.653710.41,04110.5\nMen and women of age groups <35\u00a0years, 35\u201344\u00a0years, 45\u201354\u00a0years and \u226555\u00a0years were distinguished. Figure\u00a01 shows the life table survival function of episodes with depressive symptoms in each of these subgroups.\nFig.\u00a01Survival function of the duration of absence due to depression in men and women by age\nWomen with depressive symptoms had a lower return to work rate than men. After 26\u00a0weeks 53.0% were still absent as compared to 48.8% in men. More women (25.5%) than men (22.3%) reached the disability pension date. In Table\u00a02 the results of the Kaplan\u2013Meier estimation are presented by age, company size and sector. The estimation of the mean duration of absence due to depressive symptoms (with a maximum of one year) was 200 (median 179) days in men and 213 (median 201) days in women.\nTable\u00a02Mean (95% CI) and median (95% CI) duration of depressive episodes by age, company size and sector in men and womenNumber of absencesMean (95% CI)Median (95% CI)MenWomenMenWomenMenWomenAge\u00a0\u00a0<35\u00a0years1,3012,105184 (177\u2013191)204 (198\u2013209)153 (143\u2013163)182 (171\u2013193)\u00a0\u00a035\u201344\u00a0years1,6101,729200 (194\u2013206)217 (211\u2013223)181 (170\u2013192)206 (193\u2013219)\u00a0\u00a045\u201354\u00a0years1,3571,081212 (205\u2013219)223 (215\u2013230)199 (184\u2013214)223 (205\u2013241)\u00a0\u00a0\u226555\u00a0years456248205 (193\u2013217)222 (205\u2013238)182 (163\u2013201)207 (163\u2013251)Company size\u00a0\u00a0<75 employees1,3491,214214 (207\u2013221)226 (218\u2013233)198 (182\u2013214)229 (210\u2013248)\u00a0\u00a075\u2013500 employees1,1741,225200 (192\u2013207)210 (203\u2013217)181 (167\u2013195)201 (187\u2013215)\u00a0\u00a0500\u20135,000 employees1,4212,032196 (189\u2013203)213 (207\u2013218)174 (163\u2013185)200 (188\u2013212)\u00a0\u00a0>5,000 employees531423188 (177\u2013198)208 (195\u2013220)170 (157\u2013183)179 (145\u2013213)Sector\u00a0\u00a0Construction industry33839206 (192\u2013220)206 (174\u2013239)182 (151\u2013213)189 (133\u2013245)\u00a0\u00a0Health care1961,218212 (194\u2013230)214 (207\u2013221)202 (163\u2013241)200 (184\u2013216)\u00a0\u00a0Trade524406205 (194\u2013216)212 (199\u2013224)183 (163\u2013203)200 (171\u2013229)\u00a0\u00a0Catering industry176345197 (177\u2013216)203 (189\u2013217)168 (136\u2013200)174 (144\u2013204)\u00a0\u00a0Industry1,077341189 (182\u2013197)205 (192\u2013219)163 (149\u2013177)192 (167\u2013217)\u00a0\u00a0Education and public sector165196232 (213\u2013251)242 (224\u2013260)236 (199\u2013273)272 (223\u2013321)\u00a0\u00a0Transportation and communication417332196 (183\u2013208)208 (194\u2013221)182 (159\u2013205)177 (151\u2013203)\u00a0\u00a0Commercial services645682213 (203\u2013223)219 (209\u2013229)196 (176\u2013216)212 (187\u2013237)\u00a0\u00a0Other\/unknown1,1971,616194 (186\u2013201)212 (206\u2013218)171 (160\u2013182)201 (188\u2013214)Total4,7355,175200 (196\u2013204)213 (210\u2013217)179 (172\u2013186)201 (193\u2013209)\nIn all age categories, women with depressive symptoms were absent longer than men. Elderly employees had a longer duration of absence and a higher risk of reaching disability. Employees in companies with less than 75 employees were absent for a longer period than employees working in large sized companies. Employees in the educational and public sector had the longest absence duration, followed by employees in commercial services and health care. Men working in the industrial sector had the shortest duration of absence due to depressive symptoms.\nTable\u00a03 shows the mean number of absence days corrected for part-time return to work. Men with depressive symptoms had significantly less absence days than women: 154 and 165 absence days, respectively (T\u00a0=\u00a0\u22125.3; df\u00a0=\u00a09,844; P\u00a0<\u00a00.01). Because the mean percentage of part-time return to work was almost equal in men and women, this was due to a shorter duration of absence episodes in men. Elderly employees with depressive symptoms had more absence days than younger employees (F(3, 9,862)\u00a0=\u00a016.5; P\u00a0<\u00a00.01), because of a longer duration of the absence episodes and lower part-time return to work percentages. The post hoc Tukey test revealed that the difference was significant in employees aged 35\u00a0years and more as compared to younger employees.\nTable\u00a03Mean (95% CI) number of absence days corrected for partial work resumption of depressive episodes by age, company size and sector in men and womenMenWomenTotalMean (95% CI)Mean (95% CI)Mean (95% CI)Age \u00a0\u00a0<35\u00a0years140 (134\u2013146)156 (151\u2013160)150 (146\u2013153)\u00a0\u00a035\u201344\u00a0years153 (148\u2013158)169 (164\u2013174)161 (158\u2013165)\u00a0\u00a045\u201354\u00a0years164 (158\u2013170)176 (169\u2013183)169 (165\u2013174)\u00a0\u00a0\u226555\u00a0years163 (152\u2013173)178 (163\u2013192)168 (160\u2013176)Company size\u00a0\u00a0<75 employees166 (160\u2013172)176 (169\u2013182)171 (166\u2013175)\u00a0\u00a075\u2013500 employees154 (148\u2013161)166 (159\u2013172)160 (156\u2013165)\u00a0\u00a0500\u20135,000 employees150 (144\u2013156)163 (158\u2013168)158 (154\u2013161)\u00a0\u00a0>5,000 Employees134 (125\u2013142)157 (146\u2013168)144 (137\u2013151)Sector\u00a0\u00a0Construction industry164 (152\u2013176)160 (132\u2013189)164 (152\u2013175)\u00a0\u00a0Health care168 (152\u2013185)167 (161\u2013174)168 (162\u2013173)\u00a0\u00a0Trade158 (149\u2013168)166 (155\u2013177)162 (154\u2013169)\u00a0\u00a0Catering industry153 (137\u2013170)163 (151\u2013176)160 (150\u2013170)\u00a0\u00a0Industry143 (137\u2013150)157 (145\u2013169)147 (141\u2013152)\u00a0\u00a0Education and public sector181 (162\u2013199)196 (179\u2013213)189 (177\u2013201)\u00a0\u00a0Transportation and communication141 (130\u2013151)151 (139\u2013163)145 (138\u2013153)\u00a0\u00a0Commercial services162 (154\u2013171)164 (155\u2013172)163 (157\u2013169)\u00a0\u00a0Other\/unknown151 (145\u2013158)166 (161\u2013172)160 (156\u2013164)Total154 (150\u2013157)165 (162\u2013169)160 (158\u2013162)\nAmong employees working in companies with more than 5,000, we found more part-time return to work than in smaller companies, resulting in a relatively low mean number of corrected absence days (F(3, 9,344)\u00a0=\u00a014.7; P\u00a0<\u00a00.01). The post hoc Tukey test revealed that three subsets based on decreasing number of absence days could be distinguished: companies with less that 75 employees, companies with 75\u20135,000 employees and companies with more than 5,000 employees. The difference in mean number of absence days between the sectors was significant (F(8, 9,880)\u00a0=\u00a08.2; P\u00a0<\u00a00.01). According to the post hoc Tukey test, employees in the educational and public sector had the highest number of absence days corrected for part-time return to work. Employees in the transportation and communication sector had a significantly lower number of absence days as compared to the other sectors.\nDiscussion\nOur study showed that depressed women were absent longer from work than men, which confirmed our first hypothesis (H1). The mean duration of absenteeism due to depressive symptoms was 213 (median 201) days in women and 200 (median 179) days in men. This could be due to gender differences in the severity of the depressive symptoms. However, no gender differences were found in the duration of depressive episodes in a population sample (Eaton et al. 1997) and in the severity or symptomatology of depressive disorders in general practice (Hildebrandt et al. 2003).\nIn studies on absence and depression, reverse gender differences are found. For sickness absence in general, women have more sickness absence days than men (Harrison and Martocchio 1998; Kivim\u00e4ki et al. 2003; Central Statistical Office of the Netherlands 2006). According to Laitinen-Krispijn and Bijl (2000), depression was a stronger risk factor for absence in men than in women. However, Hensing et al. (1996, 2000) found a higher incidence of absence due to psychiatric disorders in women as compared to men. Hensing et al. (1996, 2000) even found longer absence durations for men with psychiatric disorders as compared to women. They reported about neurosis, and we investigated depressive symptoms, which might explain part of the difference.\nHensing et al. (1996, 2000) reported that the longer duration of absence among men could be explained by gender differences in co-morbidity with alcohol problems and other psychiatric disorders. They also mentioned gender differences in seeking health care, diagnostic procedures and treatment of psychiatric disorders. The social consequences of having a psychiatric disorder might be worse for men than for women, leading to under-reporting among men. Men might postpone the use of health care and get into a poorer mental condition. As a consequence, they need a longer period of recovery and are absent longer from work. Gender differences in the (assessment of) severity of psychiatric problems might also explain the different duration of sick-leave. However, if these mechanisms do play a role in our study, they are more unfavourable to depressed women than to depressed men.\nDewa et al. (2003) reported that depressed women returned to work more often than men, rather than leaving employment. They defined the end of the absence period as returning to work either part-time or fulltime. In our study men returned more often to work than women. We regarded fulltime return to work as the end of an absence period, because we did not have the exact dates of part-time return to work. It is possible that women returned more often to work part-time than men.\nDepressed elderly workers are absent longer than young employees with depressive symptoms, confirming our second hypothesis (H2). Sickness absence increases with age, regardless of diagnosis (North et al. 1993; Marmot et al. 1995). Hensing et al. (2000) found higher rates of absence with psychiatric diagnoses in older ages. Like in our study, the increase they found is less linear than in general absence studies. This can be explained by the fact that sickness absence with a psychiatric diagnosis differs from sickness absence due to other diagnoses, since the duration of absence is long in the youngest age groups as well. Age differences in the severity of psychiatric disorder, in help-seeking behavior, recognition and\/or treatment may influence the duration of sick-leave due to depression. An alternative factor, which may contribute to the longer duration, is that elderly employees prefer to retire from the labour force. In contrast with Hensing et al. (1996) we found that men in all age groups, especially in the youngest age groups, had fewer days of sick-leave than women in the corresponding age group. Further study is necessary to investigate these gender differences by age.\nIn accordance with our third hypothesis (H3), the periods of absence due to depressive symptoms were shorter in large-scale companies than in smaller ones. The reintegration percentage is higher in large companies, resulting in a lower number of absence days. Probably, large companies have more opportunities for part-time return to work. Moreover, large companies have structured protocols as to how to deal with long-term absence from work. In the transportation and communications sectors, part-time return to work was most often observed. In our sample, these are mostly large companies in the postal and telecommunications sector, offering more reintegration possibilities. Although blue-collar workers generally have higher sickness absence than white-collar workers, depressed employees in industrial settings had shorter absence durations. A possible explanation might be that employees in the industrial sector are more susceptible to stigmatization of having mental problems, and return to work earlier. Also, in blue-collar workers, depressive symptoms may more often be masked by or diagnosed as somatic diseases.\nEmployees in the educational and public sectors had longer duration of depressive episodes, resulting in more absence days, confirming our fourth hypothesis (H4). An explanation is that these employees find it harder to report themselves sick because of the difficulty to substitute their work and the personal ties with their clients and pupils (Aronsson et al. 2000). Probably they have more severe depressive symptoms at the moment of sick-leave. Another explanation for their longer absence might be the lesser reintegration opportunities in these sectors. Finally, the mean age of employees in the educational and public sectors was higher than average, and it is known from Dutch national sickness absence surveys that elderly employees are absent longer than younger ones. More research is needed to examine these possible explanations.\nDuration of absence due to depression compared to other absenteeism studies\nThe duration of absence due to depressive symptoms is longer than reported in international research (Hensing et al. 1996, 2000; Sorvaniemi et al. 2003) and the figures according to the Medical Disability Advisor (2006). Differences in definitions and methods may account for this finding. Hensing et al. (1996) reported on episodes of combined depression and anxiety disorder, exceeding 7\u00a0days and without maximum duration. In contrast to our study, they did not censor for the end of the observation period. Hensing et al. (2000) investigated sickness absence due to neurosis with a minimum duration of 15\u00a0days until the end of the absence. We investigated absence due to depressive symptoms with a minimum duration of 1\u00a0day and maximized at 365 calendar days. In our study, 98% of cases had a minimum absence duration of 15\u00a0days. The code depressive symptoms was given in the second or third week of absence, when the absent employee consults the occupational physician.\nSorvaniemi et al. (2003) studied sick-leaves with depression according to DSM-III-R in psychiatric outpatients. They used exact beginning and ending dates of sick-leaves, resulting in durations ranging from 2 to 374\u00a0days and a mean duration of 142\u00a0days. At this point, it should be reminded that depressive symptoms were not strictly assessed according to the DSM criteria, limiting the comparability of results.\nBesides, the occupational physician can only give one diagnosis per absence episode. Comorbidity could not be coded. Approximately two-thirds (64%) of respondents with 12-month major depressive disorder meet the criteria for at least one other mental disorder, with anxiety disorders (58%) more common than substance abuse (9%) or impulse control (17%) (Kessler et al. 2003). Comorbidity may substantially prolong the duration of an absence episode. The long duration of absence due to depressive symptoms might be explained by comorbid features. Alternatively, it is possible that depressive symptoms were reactive to somatic disease. In case the symptoms of somatic disease appeared first, the occupational physician encoded the somatic disease and not the reactive depressive symptoms. During sick-leave, the occupational physician evaluates the medical situation and recovery every 6\u00a0weeks. Bias can only be expected when the somatic disease was not known in the sick-leave period of the depressed worker. Further investigation of the influence of comorbidity by more detailed research of the medical files is necessary, as Chisholm et al. (2003) reported that the economic consequences of depression (e.g. number of lost work days) were influenced to a greater extent by the presence of medical comorbidity than by symptom severity alone.\nDuration of a depressive episode compared to the duration of absence\nThe median duration of absence due to depression in our study exceeds the estimations of duration of depressive episodes in the general Dutch population (Spijker et al. 2002). In a population survey, Kessler et al. (2003) found a mean duration of a depressive episode of 16\u00a0weeks using the composite international diagnostic interview (CIDI). This is about half of our estimated duration of absence. Population studies may underestimate the duration of a depressive disorder, because adults with severe forms of depression are less likely to participate in such studies (Centers for Disease Control and Prevention 2004). Studies on the duration of a depressive episode using the additional life chart interview showed a mean duration of 26\u00a0weeks in men and 27\u00a0weeks in women (Eaton et al. 1997). This corresponds to the mean absence durations we found: 28.6\u00a0weeks in depressed men and 30.4\u00a0weeks in depressed women. Blazer et al. (1994) found an overall mean duration of 22.6\u00a0weeks by means of a diagnostic interview. For the purpose of their analysis, however, 3% of the participants who had had very long episodes were excluded.\nWhen chronic cases are considered, our estimates were comparable to the study of Spijker et al. (2002) in the general population. They found a chronic course (duration 12\u00a0months or more) in 24% of cases. Our survival analysis also indicated a chronic course in 24% of cases. It is important to investigate which employees are at risk for a chronic course.\nThe duration of a depressive episode in The Netherlands is comparable to other countries, whereas the duration of absenteeism is longer. It could be hypothesized that Dutch employees return to work when they are completely recovered, while in other countries employees start working (part-time) during recovery. We think that differences in social legislation and benefits contribute to these differences. In most other European countries compensation schemes apply to work-related diseases (\u201crisque professional\u201d). In The Netherlands, however, the social insurance system does not take into account the cause of the disease (\u201crisque social\u201d). Moreover, social benefits could have contributed to the relatively long absence duration. Dutch employers are obliged to pay the employee on sick-leave at least 70% of his\/her salary. In almost all cases the full salary is paid during the first year of sick-leave, which does not motivate employees to return to their work.\nLimitations of the study\nOur population is not a random sample from the total Dutch working population, because employers voluntarily engage our occupational health department. Therefore, results are not representative of the whole Dutch working population. However, the considerable sample size and the use of registered, rather than self-rated information on sickness absence, are strong points of this study.\nA depressive episode was diagnosed in employees who visited the occupational physician. Persons with lower grade depressive moods might not have reported complaints, and stayed at work. If absent, they might have returned to work before they consulted the occupational physician. Alternatively, they might have called off a visit to the occupational physician, because they intended to resume their work soon. Therefore, more severe cases of depression were included in our sample. The duration of absence due to depressive symptoms is related to the severity of depression (Dewa et al. 2003).\nWe investigated the interaction between depression and work sectors, but could not examine the influence of work content and working conditions. Rugulies et al. (2006) reported work environment to influence the risk of developing severe depressive symptoms. Work factors also influence the probability of returning to work once absent (North et al. 1996; V\u00e4\u00e4n\u00e4nen et al. 2003; Nielsen et al. 2004).\nClinical implications\nBecause of the risk of chronicity, it is important that occupational physicians recognize depressive symptoms in an early stage of the absence episode\nIt is recommended to develop and apply tools for recognizing employees at risk for chronic depression\nSpecial attention should be given to employees in educational and public services, commercial services and health care as they are at risk for longer absence duration in case of depressive symptoms.\nStudy limitations\nDiagnosis of depressive symptoms in absent employees may be biased towards absences with a longer duration. Mildly depressed employees, who did not report ill or returned to work before consulting the occupational physician, were not included\nDepressive symptoms were not strictly assessed according to DSM criteria, limiting the comparability to other studies\nOur absence registration system does not account for comorbidity.","keyphrases":["depression","sector","company size","duration of sick-leave","mental health","gender and age differences"],"prmu":["P","P","P","P","R","R"]}
{"id":"Pediatr_Radiol-3-1-1950216","title":"Magnetic resonance imaging protocols for paediatric neuroradiology\n","text":"Increasingly, radiologists are encouraged to have protocols for all imaging studies and to include imaging guidelines in care pathways set up by the referring clinicians. This is particularly advantageous in MRI where magnet time is limited and a radiologist\u2019s review of each patient\u2019s images often results in additional sequences and longer scanning times without the advantage of improvement in diagnostic ability. The difficulties of imaging small children and the challenges presented to the radiologist as the brain develops are discussed. We present our protocols for imaging the brain and spine of children based on 20 years experience of paediatric neurological MRI. The protocols are adapted to suit children under the age of 2 years, small body parts and paediatric clinical scenarios.\nIntroduction\nThe use of imaging protocols allows the standardization of procedures and workflow as well as consistency of image quality. With appropriate training, radiographers can select and implement imaging protocols with relatively little requirement for radiological involvement. This use of skill mix provides radiographers with an expanded role and greater responsibility that increases job satisfaction and staff retention.\nImaging protocols can also provide guidance for radiologists and radiographers for patients in shared care between secondary and tertiary care institutions. The use of protocols set up by the referring centre will prevent repeat MR scanning with the additional burden on MR scanner time. These advantages are offset by the minor disadvantage that radiologists\u2019 personal preferences and the intricacies of individual scanners are not taken into consideration.\nAt Great Ormond Street Hospital (GOSH), MRI protocols have been implemented for nearly 20\u00a0years and have been modified as scanners are replaced and new sequences developed. Our success is measured by the minimal number of patients recalled (less than 1%). Increasingly, we are asked about our MR protocols by departments throughout the United Kingdom and have set them out here to make them freely available to those who may find them beneficial to patient care. The protocols provided are confined to conventional imaging techniques and diffusion-weighted imaging (DWI), and do not include the advanced imaging techniques of perfusion imaging, diffusion tensor imaging and MR spectroscopy that remain largely within the research domain.\nPractical issues of MRI in children\nOne of the biggest challenges of paediatric neuroimaging is the acquisition of high-quality diagnostic images, as it requires the infant or child to keep still for a long period of time (sometimes nearly 1\u00a0h). Children over the age of 7\u00a0years are scanned, where possible, without sedation and with recourse to play specialists and videos. Neonates under 2\u00a0months old (corrected age, if premature) are scanned during natural sleep induced by food, comfort and warmth (feed and wrap), often best after a period of sleep deprivation. If this fails then light oral sedation, e.g. chloral hydrate, may be used. The majority of our patients (approximately 70%) require sedation or a general anaesthetic. When sedation is required, it is usually heavy and heavier than that required for CT or nuclear medicine studies. Sedation is applied by specially trained nurses who are trained to decide when and how much to administer and monitor its effects, and who are skilled in the recovery of the child [1]. Details can be found in a review of the management of painless imaging in children [2].\nAll MR sequences may disturb the sleeping infant or child and ear protection such as earplugs and baby earmuffs should be used. Some motion can be avoided by swaddling infants, keeping them warm, and by placing moulded foam or airbags around the baby\u2019s or child\u2019s head. Cardiorespiratory monitoring with MR-compatible equipment is essential in all infants, whether sedated or not, and all sedated or anaesthetized children [2].\nCommercial MR machines are designed for adult practice and few manufacturers make provision for the issues encountered in paediatric practice. Currently, most children are imaged in units that have a wider adult practice with only a handful of institutions throughout the world having dedicated paediatric MR facilities. The structures we need to examine in children are generally small and we aim for a maximum slice thickness of 5\u00a0mm in the brain and 3\u00a0mm in the spine. The slice thickness is reduced to 3\u00a0mm when acquiring images of the pituitary gland and orbits.\nWe do not recommend ultrafast \u2018breath-hold\u2019-type T2-weighted (T2-W) sequences that have reduced contrast to noise as an alternative to sedating a child, as achieving high contrast to noise is of utmost importance given the small size of the paediatric head. Advances in coil design have improved signal-to-noise and we now use multichannel head and spine coils.\nPractical consideration of the changing brain with age\nChanges in normal values of measured parameters in very young children occur as the brain undergoes very rapid development during the first 2\u20133\u00a0years of life. This necessitates the creative modification of pulse sequence parameters in order to optimize the soft-tissue contrast between anatomical and pathological structures. The two most important physiological changes to take place within the first 2\u20133\u00a0years are reduction in the total brain water content and T1 and T2 shortening largely as a consequence of the maturation of myelin [3].\nFrom birth to 6\u00a0months of age the maturation of myelin is best assessed using T1-weighted (T1-W) images. From 6\u00a0months until the completion of myelination at around 2\u00a0years of age, T2-W images are more useful. During the process of myelination on T1-W images the signal from white matter goes from dark to bright. For a variable period the subcortical white matter becomes isointense with grey matter. This loss of grey\/white matter differentiation obscures structural detail and makes identification of subtle abnormalities such as polymicrogyria difficult to detect within the first 6\u00a0months of life. The white matter shows maturity later, between 6\u00a0months and 2\u00a0years, on T2-W images and at this stage T1-W images are essential to evaluate structural abnormalities [4].\nTo overcome the difficulty of increased brain water in those under 2\u00a0years of age, we use a dual-echo short-tau inversion recovery (STIR) sequence that has improved contrast resolution in this age group (Fig.\u00a01) [5]. Typical values for a dual-echo STIR sequence are TE 30\/128\u00a0ms, TR 5,400\u00a0ms and TI 130\u00a0ms. We prefer this sequence to the more conventional T2-W fast (or turbo) spin-echo sequences, for reasons of increased lesion conspicuity. However, we recognize that similar tissue contrast may be achieved if such fast spin-echo sequences are modified using increased TR and TE to compensate for the increased water content in this age group. The additional brain water is difficult to suppress and we do not find the fluid-attenuated inversion recovery (FLAIR) technique useful in this age group as lesion conspicuity is poor.\nFig.\u00a01Axial images acquired through the bodies of the lateral ventricles of a 6-month-old child. First (a) and second (b) echo (TE 30\/120\u00a0ms, TR 5,500\u00a0ms, TI 130\u00a0ms) of a dual-echo short-tau sequence shows the increased contrast between grey and white matter on the second echo compared to (c) the T2-W (TE 90\u00a0ms, TR 3,500\u00a0ms) sequence that would be used in those over 2\u00a0years of age. The slice thickness (5\u00a0mm) and matrix size (512\u2009\u00d7\u2009192) were the same for both sequences\nDiffusion measurements in the brain of newborns and infants show that there is more movement of water (high apparent diffusion coefficient, ADC) with less directionality (low anisotropy) within the immature and unmyelinated brain than in the mature and myelinated adult brain [6, 7]. The most dramatic decrease in ADC values occurs within the first few months of life and the greatest changes are seen in the frontal and parietal white matter [8]. Failure to appreciate the normal changes with age of the ADC will result in the erroneous reporting of white matter abnormality particularly in the neonatal period.\nFactors influencing sequence choice\nFor any imaging protocol, it is the specific combination of sequences that determines the diagnostic efficacy of the examination. It is clear that imaging protocols vary between institutions. This is largely because rapid advances in imaging technology and variations between manufacturers, applied in the context of investigating relatively rare disorders, precludes an effective evidence-based approach to sequence choice.\nThe main advantages that MRI offers over alternative imaging modalities is the ability to demonstrate different tissue contrasts (principally T1-W, T2-W and spin density, but also flow and diffusion) in multiple imaging planes (principally sagittal, coronal and axial). The disadvantage of MRI is the artefacts that it generates in almost every image.\nThe choice of sequence combination should reflect the multicontrast and multiplanar capabilities of MRI. We have found that applying the generic principle of combining T2-W images in two planes, supported by T1-W imaging in two planes, as the basis of our imaging protocols, serves to optimize the benefits of MRI whilst minimizing the impact of artefacts.\nStandard MRI studies\nBrain\nOur standard brain sequence (>2\u00a0years of age) is composed of an axial T2-W, coronal FLAIR, and coronal and sagittal T1-W images. Sagittal T1-W images allow the assessment of the midline structures, particularly the corpus callosum and cerebellum, which are frequently abnormal in congenital abnormalities. Coronal T1-W images are valuable in the diagnosis of abnormalities of the anterior visual pathway, schizencephaly and holoprosencephaly. Axial T2-W and coronal FLAIR images provide T2-W images in two different planes and have been shown to be complementary in children [9]. DWI is acquired in all children unless artefacts from, for example, dental braces or a ventriculoperitoneal shunt, preclude it, and an ADC is calculated using automated computer software and provided for reporting.\nIn those under 2\u00a0years of age, the T2-W sequence is replaced by a dual-echo axial STIR sequence (see above). In some cases, a T2*-W gradient-echo (GE) sequence (\u201csusceptibility-weighted\u201d sequence), sensitive to changes in local field inhomogeneity caused by the breakdown products of haemoglobin, is added. The sequence is particularly useful in trauma and vascular malformations such as multiple cavernomas.\nSpine\nMRI is the modality of choice for imaging the intraspinal components of the paediatric spine. Prior to ossification of the posterior elements of the spinal column, US has been shown to be a valuable screening tool; however, infants with an abnormal sonogram or who have a neurological abnormality in the context of a normal sonogram, still require MR imaging [10].\nOur standard spine imaging includes sagittal, fast spin-echo T1- and T2-W sequences (3-mm-thick slices). Both axial T1-W and T2-W images are acquired through any abnormality. Unlike most adult spine imaging protocols, groups of axial images through disc levels are not applied because degenerative disc disease is rare. Children with scoliosis and\/or suspected spinal dysraphism routinely have axial T1-W images through the conus and filum terminale to detect lipomas of the filum terminale that may not be visible on sagittal imaging.\nContrast medium\nThe use of contrast medium is a personal choice and in the majority of units is requested on a case-by-case basis. At GOSH the radiographers or sedation nurses administer MR contrast medium according to guidelines set up by the neuroradiologists (Table\u00a01). In the event of an unexpected finding the radiographers are encouraged to make a decision on contrast administration without discussion with a radiologist.\nTable\u00a01Indications for contrast medium administrationVarious indicationsAcute inflammation\u00a0Acute disseminated encephalomyelitis (ADEM)\u00a0Optic neuritisAcute infection\u00a0Abscess\u00a0Cerebritis\u00a0Discitis\u00a0Empyema\u00a0Encephalitis\u00a0Meningitis\u00a0Transverse myelitisNeurocutaneous disorders\u00a0Congenital melanocytic naevus\u00a0Neurofibromatosis type IITumours\u00a0Benign and malignant\u00a0Intracranial\u00a0IntraspinalWhite matter disordersaVascular anomalies\u00a0Cavernomas\u00a0Developmental venous anomaliesVascular disorders\u00a0Intraparenchymal haemorrhage\u00a0Sturge-Weber syndrome\u00a0VasculitisaAt presentation and at follow-up if necessary\nMRI protocols for specific areas\nThe strategies applied to the brain are also applied to small parts such as the orbits and pituitary gland. The orbit is scanned using a STIR sequence to benefit from the fat saturation properties of the sequence and improve the conspicuity of lesions within the orbit. The slice thickness is reduced to 3\u00a0mm and, to increase the signal-to-noise, the matrix size is reduced, thereby slightly reducing the in-plane resolution.\nA heavily T2-W 3-D volume sequence of the petrous temporal bones is used to image the membranous labyrinth and is reconstructed in the coronal plane of the petrous temporal bone, and the axial and sagittal plane of the internal auditory meati (IAM). The sagittal plane is used to view the vestibulocochlear nerve in cross section. A constructive interference steady state sequence (CISS), available on Siemens scanners, is acquired and reconstructed at a slice thickness of 0.7\u00a0mm. The GE equivalent is the FIESTA sequence and the Philips equivalent is DRIVE.\nMRI protocols for specific neurological or neurosurgical presentations\nThe majority of children undergoing an MRI brain scan will have epilepsy, stroke or a brain tumour and are often referred to specialists with an interest in these areas.\nTumours of the brain and spine\nBrain\nImaging of brain tumours is performed using our MRI brain with contrast enhancement protocol. We add contrast-enhanced T1-W imaging of the spine to establish the presence of any distant metastases in accordance with the United Kingdom Children\u2019s Cancer Study Group (UKCCSG) guidelines for the imaging of brain tumours [11]. One of the commonest reasons for repeat scanning of a child with a brain tumour is the acquisition of contrast-enhanced imaging of the spine in a child who has had only a brain scan at the referring centre.\nThe purpose of preoperative imaging of brain tumours is to assess tumour location and type, establish whether there are single or multiple lesions, define its relationship with vital structures and look for any complications such as hydrocephalus. Contrast-enhanced T1-W volumetric acquisitions are also acquired for image-guided surgery.\nImaging of the spine is performed in all children with intracranial tumours (not only posterior fossa tumours) and although this requires a change of coil and an additional sequence, we find it beneficial as tumour histology is often not certain at the time of the initial imaging. Some tumours unexpectedly metastasize to the spine and assessment of the postoperative spine is made more difficult in the presence of blood products.\nPostoperative MRI is acquired using our MRI brain with contrast enhancement protocol to assess the degree of tumour clearance. We aim to do this within 72\u00a0h of surgery to reduce enhancement due to postoperative changes that can complicate the assessment of tumour clearance, in accordance with guidelines proposed by the UKCCSG [11]. If preoperative spinal imaging has not been acquired, the spine is imaged with both unenhanced and contrast-enhanced T1-W imaging to enable differentiation between haematoma and tumour metastases.\nSurveillance imaging (SI) during adjuvant treatment and follow-up is a major part of the management of childhood tumours and scanning frequency has important implications to anaesthetic and radiology service provision. In children with ependymoma and medulloblastoma, studies at GOSH have shown that SI reveals a substantial number of asymptomatic recurrences and survival appears to be improved in these patients compared with those patients in whom the recurrence is identified by symptoms [12, 13]. Benefits of SI following incomplete resection of posterior fossa low-grade pilocytic astrocytoma have also been shown [14, 15]. SI schedules have been written based on this work (Table\u00a02).\nTable\u00a02Proposed protocols for surveillance imaging in children with ependymoma, medulloblastoma and pilocytic astrocytomaTumourTiming of postoperative imagingFrequency of studyMacroscopically complete excisionIncomplete excisionCranial studySpinal studyCranial studySpinal studyEpendymoma24\u201348\u00a0hFirstNoneFirstNone1st year3\u20136\u00a0months6\u00a0months3\u00a0months3\u20136\u00a0months2nd\u20135th years6\u00a0months6\u201312\u00a0months6\u00a0months6\u201312\u00a0monthsMedulloblastomaa24\u201348\u00a0hFirstNoneFirstNone1st year3\u20134\u00a0months3\u20134\u00a0months3\u20134\u00a0months3\u20134\u00a0months2nd\u20136th year6\u20138\u00a0months6\u20138\u00a0months6\u20138\u00a0months6\u20138\u00a0monthsCerebellar low-grade astrocytomab24\u201348\u00a0hFirstNoneFirstNone1st year6\u00a0monthsNone6\u00a0monthsNone2nd yearAt 24\u00a0monthsNone6\u00a0monthsNone3rd yearAt 3.5\u00a0yearsNone6\u00a0monthsNone4\u20135th yearAt 5\u00a0yearsNone1\u00a0yearNone6th year onwardsNoneNone2\u00a0yearsNoneaAll imaging studies in children with medulloblastoma should include the entire neuroaxis.bThe surveillance imaging protocol following complete resection should be applied to children who have radiotherapy with the postoperative scan omitted\nSpine\nImaging of spinal tumours is performed using our MRI spine with contrast enhancement protocol (Table\u00a03) with dedicated axial imaging through the tumour. We add a standard brain scan with contrast enhancement to the initial or immediate postoperative scan to stage the tumour. If the brain scan is normal, further brain imaging is not acquired as part of SI and acquired only when clinically indicated (Table\u00a04).\nTable\u00a03Protocols for specific areasSpecific areasProtocolsOrbits (3-mm slices)\u00a0Coronal and axial dual-echo STIR\u00a0Coronal and axial T1-W spin-echoOrbits with contrast enhancement (3-mm slices)\u00a0Coronal dual-echo STIR\u00a0Coronal and axial T1-W spin-echo\u00a0Contrast-enhanced coronal and axial T1-W images with fat saturationPituitary (3-mm slices)\u00a0Sagittal and coronal T1-W spin-echo\u00a0Coronal T2-W spin-echoPituitary with contrast enhancement (3-mm slices)\u00a0Pituitary protocol\u00a0Contrast-enhanced coronal and sagittal T1-W spin-echoInternal auditory meati\u00a03-D volume axial CISS\u00a0Brain MRIFace and neck MRI\u00a0Coronal and axial dual-echo STIR\u00a0Coronal and axial T1-W spin-echo\u00a0Fat-saturated contrast-enhanced coronal and axial T1-W spin-echoMidline facial lesions\u00a0Axial dual-echo STIR from floor of anterior cranial fossa to hard palate\u00a0Sagittal T1-W and T2-W spin-echo (3-mm slices)\u00a0Coronal T1-W spin-echo from nose to brainstemTable\u00a04Standard MRI brain and spine protocolsTypes of brain and spine MRIProtocolsMRI brain (under 2 years old)\u00a0Axial and coronal dual echo STIR\u00a0Coronal and sagittal T1-W spin-echo\u00a0DWI in three planes and calculated ADC map\u00a0Axial \u201csusceptibility-weighted\u201d GE sequenceaMRI brain (over 2 years old)\u00a0Axial T2-W fast spin-echo\u00a0Coronal FLAIR\u00a0Coronal and sagittal T1-W spin-echo\u00a0DWI in three planes and calculated ADC map\u00a0Axial \u201csusceptibility-weighted\u201d GE sequenceaMRI brain with contrast enhancement (under 2 years old)\u00a0Axial and coronal dual-echo STIR\u00a0Coronal T1-W spin-echo\u00a0DWI in three planes and calculated ADC map\u00a0Contrast-enhanced axial, coronal and sagittal T1-W spin-echo with magnetization transfer\u00a0Axial \u201csusceptibility-weighted\u201d GE sequenceaMRI brain with contrast enhancement (over 2 years old)\u00a0Axial T2-W fast spin-echo\u00a0Coronal FLAIR\u00a0Coronal T1-W spin-echo\u00a0DWI in three planes and calculated ADC map\u00a0Contrast-enhanced axial, coronal and sagittal T1-W spin-echo with magnetization transfer\u00a0Axial \u201csusceptibility-weighted\u201d GE sequenceaMRI spine\u00a0Sagittal T1-W and T2-W fast spin-echo\u00a0Axial T1-W and T2-W fast spin-echo through target area and conus\u00a0(Coronal T1-W spin-echo for scoliosis, if patient compliant)MRI spine with contrast enhancement\u00a0Sagittal T1-W and T2-W fast spin-echo\u00a0Contrast-enhanced sagittal T1-W fast spin-echo\u00a0Axial T1-W spin-echo through target areaaOptional sequence\nEpilepsy\nUK guidelines for the imaging of epilepsy include children who have either multiple or focal seizures (NICE guidelines) [16]. In children with epilepsy we are aiming to detect focal cortical abnormalities; the majority are extratemporal and in a smaller proportion they are mesial temporal in origin. The majority of cortical abnormalities are easily diagnosed using conventional brain imaging and our standard brain protocol is used to investigate these children. The acquisition of a coronal T2-W sequence (either STIR, T2-W or FLAIR) allows the visualization of the mesial temporal lobe, and is a sequence often not acquired in referring centres.\nThere are a group of children with intractable epilepsy, i.e. seizures not controlled by three drugs, who are considered for epilepsy surgery. In this subset of children a more rigorous epilepsy protocol is carried out that includes a 3-D volume T1-W acquisition and hippocampal T2 relaxometry (Table\u00a05). To identify mesial temporal abnormalities the coronal studies are planned on a parasagittal scout image to lie perpendicular to the hippocampi. The 3-D, T1-W gradient-echo dataset is acquired isometrically in the sagittal plane (this saves time) and reconstructed in all three planes with the axial and coronal images tilted to follow the plane of the hippocampus. More recently a 3-D FLAIR sequence reconstructed along the same lines has been added. A sequence is acquired that measures the true T2 values of the hippocampi (T2 relaxometry), which is only of value in suspected mesial temporal sclerosis (MTS). It has been shown that T2 values are more sensitive to the presence of MTS than visual inspection and T2 relaxometry is of particular value in bilateral disease [17]. Normal values will vary between scanners, so normative data should be acquired. The radiographers carry out the postprocessing of the 3-D dataset and the T2 relaxometry.\nTable\u00a05Protocols for particular clinical indicationsTypes of clinical indicationProtocolsBrain tumours\u00a0MRI brain with contrast enhancement\u00a0Contrast-enhanced sagittal T1-W images of whole spine\u00a0Contrast-enhanced image-guided images when requiredStrokeAcute\u00a0Axial T2-W fast spin-echoa\u00a0Coronal FLAIRb\u00a0Sagittal T1-W spin-echoDWI in three planes and calculated ADC mapIntracerebral 3-D TOF MRAAxial dual-echo STIR and T1-W spin-echo through the neckExtracerebral 2-D TOF MRA of the neck down to the aortic rootNon-acuteAcute stroke protocol without imaging of the neckEpilepsy\u00a0Axial T2-W fast spin-echoa\u00a03-D T1-W volume acquisition reconstructed in three planes\u00a0Coronal T2-W fast spin-echob\u00a0Coronal FLAIR (or 3-D FLAIR if available)\u00a0Hippocampal T2-relaxometry (see text)Intraparenchymal haemorrhage\u00a0MRI brain with contrast enhancement\u00a0Intracerebral 3-D TOF MRA\u00a0MRVNon-accidental head injury\u00a0Standard MRI brain\u00a0Axial GE \u201csusceptibility-weighted\u201d sequence\u00a0Sagittal T2-W spin-echo and GE \u201csusceptibility-weighted\u201d sequence of the cervical spineMPRAGE magnetization prepared rapid acquisition gradient echo, MRV magnetic resonance venographyaReplaced by dual echo STIR sequence in the under 2 age groupbReplaced by a dual echo STIR coronal in the under 2 year age group.\nNon-traumatic intraparenchymal haemorrhage\nThe aim of imaging children with intraparenchymal haemorrhage (IPH) is to detect the underlying cause and we advocate the use of CT angiography as a matter of urgency in those children who have a depressed level of consciousness or whose clinical condition is deteriorating. MRI is used to investigate clinically stable children, and we perform brain MRI with contrast enhancement and a 3-D, time-of-flight (TOF) intracranial MR angiography (MRA). In our experience nearly two-thirds of children presenting with nontraumatic IPH have an underlying cause diagnosed by MRI and MRA, with aetiologies including tumour, arteriovenous malformation (AVM), venous sinus thrombosis and cavernous angioma [18]. An attempt is made to perform MRI within 72\u00a0h of the ictus to overcome the problem of the visibility of blood products (T1 shortening due to methaemoglobin) on 3-D TOF sequences that may obscure the underlying pathology. The value of intravenous contrast agents in identifying underlying vascular causes has not been proven. We have a largely anecdotal view that it is helpful in identifying small AVMs and venous anomalies.\nStroke\nThe aims of conventional MRI are not only to detect the infarct, but also to provide information to establish the cause of the stroke and to exclude other causes (such as tumour or infection). The majority of children with stroke have a combination of risk factors including sickle cell disease, congenital heart disease, anaemia, prothrombotic disorders and infections such as varicella-zoster [19]. The rationale for the inclusion of intracranial MRA in all children with stroke is that cerebral arteriopathy is found in up to 80% of these children and most commonly affects focal areas of large intracranial arteries [19]. Specific entities such as moyamoya disease may also be diagnosed. The commonest abnormality identified is occlusion or stenosis, of unknown aetiology, affecting the terminal internal carotid artery (ICA) or proximal middle cerebral artery (MCA).\nExtracranial MRA and, either a dual-echo STIR or fat-saturated axial T1-W imaging, through the neck can detect arterial dissection, particularly in children with posterior circulation infarcts [20]. The inversion time of the STIR sequence is selected to suppress the fat within the neck. The fat saturation provided by both these sequences improves the conspicuity of the haematoma within the extracranial vessel wall.\nA 3-D TOF MRA sequence is used for the imaging of the intracranial vessels and a 2-D TOF MRA sequence for the extracranial vessels. The TOF scan times are shorter than phase-contrast (PC) MRA and there is lack of dependence on the choice of correct velocity encoding with obvious advantages when scanning ill children. Intracranial 3-D TOF MRA is also included in the investigation of children with IPH, although its sensitivity to T1 shortening may obscure the underlying abnormality (see above).\nAs CT is often the first-line investigation in children with stroke, the potential of DWI to detect hyperacute cerebral infarction prior to changes on T2-W MRI is not realized. However, inpatients (e.g. cardiac patients, patients with recent-onset stroke) can be imaged early, and in these children DWI can be used to detect infarcts of different ages.\nNon-accidental head injury\nAlthough CT is the primary imaging tool in the evaluation of nonaccidental head injury (NAHI), we perform MRI in children with an abnormal CT or neurological symptoms and signs on day 3\u20134 in accordance with the protocol proposed by Jaspan et al. [21]. The rationale for performing MRI is that it is more sensitive than CT to both hypoxic\u2013ischaemic injury, particularly when DWI is used, and subdural haematomas adjacent to the calvarium or in the posterior fossa. MRI is repeated at 2\u20133\u00a0months in those children with parenchymal brain injury or a persistent neurological abnormality.\nChildren with suspected NAHI are imaged using our standard brain protocol. In addition axial GE (susceptibility-weighted) sequences are acquired, as they are more sensitive to blood products than standard sequences. Sagittal T2-W and GE images are acquired of the cervical cord, including the brainstem, particularly to identify shear injuries within the brainstem that have been proposed as a mechanism of hypoxic\u2013ischaemic injury in children who have been shaken as the mechanism of injury [22, 23]. The aim of the sagittal T2-W sequence of the cervical cord is to look for any evidence of trauma as a result of violent shaking in an attempt to establish the true, albeit rare, incidence of pathology in the upper cervical spine [21].\nNeonatal imaging\nConventional imaging can detect patterns of regional brain injury in the neonatal period that can help time the injury, determine underlying mechanisms and ultimately provide some prognostic information. MRI is useful in detecting hypoxic\u2013ischaemic injury, and germinal matrix and intraventricular haemorrhage, and can be useful to distinguish other pathologies that may mimic hypoxic\u2013ischaemic encephalopathy in the neonatal period such as venous infarction, metabolic disease, infection, and congenital developmental abnormalities.\nOur neonatal imaging protocol is the same as the under-2-year brain protocol. Achieving high signal-to-noise is of utmost importance given the small size of the infant head and is improved by decreasing the slice thickness to 4\u00a0mm whilst decreasing the matrix size from 512\u2009\u00d7\u2009512 to 256\u2009\u00d7\u2009256 in a field-of-view of 180\u00a0mm. Although specific neonatal head coils have been developed, most nonpaediatric centres are unlikely to have access to them and improved results can be obtained by using an adult knee coil.\nDevelopmental delay\nDevelopmental delay, without other clinical features such as epilepsy or dysmorphic features, is not considered an indication for an MRI scan at our institution. This decision was made in conjunction with our neurologists as it was recognized that there is an extremely low yield of clinically relevant abnormalities seen in patients with developmental delay alone.\nThe future\nThe future of paediatric neuroimaging lies in the incorporation of research techniques, such as perfusion imaging and diffusion tensor imaging, into standard imaging protocols once they have been demonstrated to be of value within the clinical arena. The use of higher field strength magnets will have the advantage of increasing signal-to-noise and reducing scanning time, which will be of benefit to the child who is both small and liable to move.","keyphrases":["protocols","mri","children"],"prmu":["P","P","P"]}
{"id":"Eur_Spine_J-2-2-1602198","title":"Vascular complications of prosthetic inter-vertebral discs\n","text":"Five consecutive cases of prosthetic inter-vertebral disc displacement with severe vascular complications on revisional surgery are described. The objective of this case report is to warn spinal surgeons that major vascular complications are likely with anterior displacement of inter-vertebral discs. We have not been able to find a previous report on vascular complications associated with anterior displacement of prosthetic inter-vertebral discs. In all five patients the prosthetic disc had eroded into the bifurcation of the inferior vena cava and the left common iliac vein. In three cases the aortic bifurcation was also involved. The fibrosis was so severe that dissecting out the arteries and veins to provide access to the relevant disc proved impossible. Formal division of the left common iliac vein and artery with subsequent repair was our solution. Anterior inter-vertebral disc displacement was associated with severe vascular injury. Preventing anterior disc displacement is essential in disc design. In the event of anterior displacement, disc removal should be planned with a Vascular Surgeon.\nKey points\nAnterior prosthetic disc displacement frequently involves major arteries and veins.\nRemoval of an L45 disc prosthesis is associated with vascular injury.\nA Vascular Surgeon should be involved in disc removal.\nIntroduction\nReplacement of the inter-vertebral disc is designed to relieve symptomatic back pain secondary to disc degeneration by preserving segmental function. A successful artificial disc replacement would avoid the significant long-term problems associated with spinal fusion [9]. We report five consecutive cases of anterior displacement of prosthetic inter-vertebral discs resulting in erosion into the bifurcation of the vena cava and aorta, from a series of only 13 patients. One patient suffered recurrent back pain. These patients had no preoperative vascular symptoms to warn of this vascular involvement and all of these discs were removed in order to prevent further vascular injury. The complications associated with removal are described together with our suggestions on an approach to this challenging surgical problem.\nProsthetic disc replacements (AcroFlex disc, DePuy Acromed, Raynham, MA, USA) were carried out as part of a clinical trial with ethical approval, regulatory approval and with informed patient consent. All patients had incapacitating back pain. Leg pain was not an exclusion criteria unless directly attributable to a prolapsed inter-vertebral disc. All patients in this study had magnetic resonance imaging evidence of degenerative disc disease. All patients had an Oswestry disability index (ODI) greater than 30% and all had discographically established concordant pain at the target level\/s. Inclusion criteria, the follow-up protocol and disc design for this study have been described previously [5].\nCase 1\nA 33-year-old woman presented with a 5-year history of low back and right leg pain. A two level artificial inter-vertebral disc replacement was performed at L45 and L5S1 without complication through an anterior transperitoneal approach (L45 31.5\u00a0\u00d7\u00a043\u00a0\u00d7\u00a09\u00a0\u00d7\u00a05\u00b0 AcroFlex disc and L5S1 34\u00a0\u00d7\u00a046\u00a0\u00d7\u00a09\u00a0\u00d7\u00a010\u00b0 AcroFlex disc) (Fig.\u00a01a\u2013c).\nFig.\u00a01Postoperative X-ray with satisfactory placement of L45 and L5S1 AcroFlex disc, lateral view (a) AP view (b and c) with anterior disc displacement at 6\u00a0months (d)\nThe patient made an uneventful recovery with improved symptoms at 6\u00a0weeks. However on X-ray the prosthesis at L45 had moved 5\u00a0mm anteriorly. At 6\u00a0months there was further L45 prosthesis displacement; however, mobility was preserved with full lumber flexion and extension at video fluoroscopy (Fig.\u00a01d).\nThe L45 artificial disc was replaced at 15\u00a0months with a size 2 SB Charit\u00e9 III disc containing an 8.5-mm spacer (Waldemar Link GmbH & Co., Hamburg, Germany). At surgery the disc had stretched and eroded into the left common iliac vein. The distal vein was occluded with thrombus. An injury to the medial wall of the left common iliac artery was repaired with 5\/0 Prolene and the occluded vein was oversewn with 4\/0 Prolene. This provided access and permitted removal of the disc. Postoperative recovery was complicated by a paralytic ileus only. The patient has an L5S1 prosthesis which remains in an acceptable position. She has subsequently become pregnant and had a normal delivery without the need for Caesarian section.\nCase 2\nA 40-year-old woman presented with a 3-year history of lumber back pain. She underwent L45 disc replacement with a 29\u00a0\u00d7\u00a040\u00a0\u00d7\u00a09\u00a0\u00d7\u00a010\u00b0 AcroFlex disc. This surgery was complicated only by a temporary loss of bladder filling sensation. At 6\u00a0week follow-up her back and leg symptoms were significantly improved. Unfortunately at 6\u00a0months her back pain had returned and she was now suffering pain and tingling in the left L5 dermatome. On X-ray the prosthesis had moved anteriorly. At 1\u00a0year the patient agreed to have the prosthesis removed. At operation the prosthesis was eroding into the left common iliac artery and vein. Both vessels were mobilised with difficulty due to dense scar tissue, the inevitable venous and arterial injuries were repaired with 4\/0 Prolene. The disc was replaced with a Stabilis cage (Stryker, UK) containing artificial bone graft which was secured with a 3.2\u00a0\u00d7\u00a034\u00a0mm screw and washer. Postoperatively the patient wore class II compression hosiery and was prescribed 40\u00a0mg Clexane twice daily. Unfortunately, despite this prophylaxis, she suffered left common iliac artery and iliofemoral deep vein thrombosis. Full anticoagulation was achieved with Tinzaparin and subsequently Warfarin. Swelling is controlled by class II compression hosiery. Transluminal angioplasty has been performed for a stenosis of the left common iliac artery at the site of repair. Her claudication has improved and is now thought to be venous.\nCase 3\nA 51-year-old man presented with bilateral leg pain and no motor or sensory loss. He had undergone laminectomy 12\u00a0years previously for a disc prolapse. A prosthetic inter-vertebral disc (29\u00a0\u00d7\u00a040\u00a0\u00d7\u00a09\u00a0\u00d7\u00a010\u00b0 AcroFlex disc) was placed in the L45 inter-vertebral space, without complication, using an anterior transperitoneal approach. The postoperative X-ray showed the prosthesis to be in an acceptable position, i.e. centrally placed in the disc space on antero-posterior projection and posteriorly placed on the lateral view (Fig.\u00a02a,\u00a0b). The patient\u2019s symptoms were improved but the prosthesis had migrated 2\u00a0mm anteriorly at 6\u00a0weeks and 5\u00a0mm by 12\u00a0months (Fig.\u00a02c).\nFig.\u00a02Satisfactory placement of L45 AcroFlex disc, lateral view (a) and AP view (b) with anterior displacement at 12\u00a0months (c) and subsequent spinal stabilisation with plate and pedicle screws (d)\nAt operation the disc had migrated into the left common iliac vein posterior to the aortic bifurcation. A tear was made in the left iliac vein while mobilising these vessels. The left common iliac vein was divided transversely to facilitate removal of the disc. A 14-mm carbon fibre cage filled with iliac crest bone graft was inserted and the iliac vein re-anastomosed with 4\/0 Prolene. Postoperative duplex scanning showed the left common iliac vein and artery to be patent. The spine was stabilised with a flat plate and pedicle screws 12\u00a0days later (Fig.\u00a02d).\nCase 4\nA 47-year-old man presented with lumbar back pain radiating to his thigh and buttock. He underwent L45 and L5S1 disc replacement with a 31.5\u00a0\u00d7\u00a043\u00a0\u00d7\u00a011\u00b0 AcroFlex disc and a 31.5\u00a0\u00d7\u00a043\u00a0\u00d7\u00a09\u00b0 AcroFlex disc respectively without postoperative complication. At 1\u00a0month follow-up he was well with no pain.\nAnterior displacement of the L45 disc was first seen on plain X-ray at 2\u00a0years and the disc was removed 11\u00a0months later. Having learned from previous bitter experiences, the vascular surgeon formally divided the left common iliac artery and vein so that there was good access to remove the disc and replace it with a Stabilis cage. The left common iliac artery and vein were then re-anastomosed. Despite this approach the right common iliac vein was injured and required repair. Full deep vein thrombosis prophylaxis was undertaken with pneumatic compression in theatre, peri-operative low molecular weight Heparin, postoperative compression stockings in addition to anticoagulation with Warfarin. Both iliac vessels were widely patent with no thrombus and normal blood flow velocity on duplex imaging at 1\u00a0month. Postoperatively he has continued to suffer pain after prolonged standing with occasional sleep disturbance. The L5S1 disc was stable on X-ray at 6\u00a0months with fusion progressing between L45.\nCase 5\nA 44-year-old man who had long standing low back pain had a single level degenerate disc at L5S1. The L5S1 disc was replaced with a 29\u00a0\u00d7\u00a040\u00a0\u00d7\u00a010\u00b0 11\u00a0mm AcroFlex disc. Immediately postoperatively he was well with an improvement in his symptoms. Gradual displacement of the prosthesis occurred from 3\u00a0months. The prosthesis was removed at 19\u00a0months and fusion performed using a Stabilis cage and cancellous bone graft. During surgery a tear of the left common iliac vein occurred during mobilisation this was repaired with 2.0 Prolene. Formal division of the iliac vein and artery was not necessary as the disc was removed from L5S1. Preoperatively the ODI was 48% after fusion it had fallen to 10%.\nDiscussion\nThe five patients reported here gained early symptomatic relief from low back and leg pain with an artificial inter-vertebral disc. However, in each case, revision following surgery for benign disc degeneration was associated with a major vascular injury. Anterior displacement of the L45 disc caused pressure on the left common iliac vein and artery with stretching of the vessels and erosion into the vessel wall in three cases. It was impossible to mobilise the stretched vessels from the firmly adherent surrounding dense scar tissue without vascular injury. In one case the left common iliac vein was thrombosed while in the others repairs were necessary after almost impossible mobilisation of the vessels to expose the prosthesis. Damage to the left common iliac vein and artery in one case led to both venous and arterial thrombosis. It is likely that this complication will lead to longstanding symptoms of venous insufficiency.\nThe only safe solution for adequate exposure of an anteriorly displaced visco-elastic artificial L45 disc is the formal division and subsequent re-anastomosis of the left common iliac vein and if necessary the left iliac artery; this strategy was successfully employed in cases 3 and 4. An anterior transperitoneal approach is essential to allow proximal and distal vessel control before division. A retroperitoneal approach in this type of revision surgery would be extremely hazardous and potentially life threatening due to an inability to control the right common iliac vein or vena cava when there is bleeding from the left common iliac vein. The aortic bifurcation would also be difficult to control.\nThe Link SB Charit\u00e9 disc is currently the most widely implanted inter-vertebral disc. This articulating disc consists of an ultra high molecular weight polyethylene spacer between two separate cobalt\u2013chromium\u2013molybdenum alloy plates. Griffith et al. reported displacement or dislocation of the prostheses in 6 of 139 implants with an average follow-up of 11\u00a0months. There were six reported vein injuries although it is not clear whether these injuries were associated with displacement or revision surgery or at what level the implant was placed [6]. Cinotti et al. reported only one anterior displacement of 46 SB Charit\u00e9 III prostheses and no vascular injury, with a minimum follow-up of 2\u00a0years [3]. In a series of 105 patients with 154 SB Charit\u00e9 III discs five vascular problems were reported (two phlebitis, two pulmonary embolism and one acute leg ischaemia) although no information on the rate of revision or anterior displacement was presented in this series [10]. Zeegers et al. reported a prospective study of 50 patients where 75 SB Charit\u00e9 III prosthesis were inserted. None of 38 patients with full 2\u00a0year radiographic follow-up had migration of the prosthesis. One patient lost to follow-up had malposition and some slip but was lost to follow-up. Of 12 patients who underwent reoperation only one aortic injury was reported during failed disc re-positioning and subsequent fusion [12]. These reports are all non-randomised series without any control and in one case retrospective. The only published randomised study of inter-vertebral disc replacement compares the Charit\u00e9 disc with spinal fusion. This study showed that pain and disability scores are equivalent at 24\u00a0months follow-up. There was a 4.4% incidence of venous injury in the arthroplasty group and device failure in 5.1% compared to 9.1 and 2% respectively in the spinal fusion group [2].\nThis first design of the AcroFlex disc contained a hexane based polyolefin rubber core vulcanised between two titanium plates. A retrospective review of six patients who had AcroFlex discs, with a mean follow-up was 3.5\u00a0years, reported a poor outcome in two patients with fracture of the rubber component and persistent symptoms in a second. There were no reported cases of disc displacement or of vascular injury during one revision although the prosthesis was at L5S1 [4]. Fraser et al. reported a series of 28 patients who had the third generation AcroFlex disc implanted, 17 patients had the same disc design used in this study. Eight of 28 required revision surgery for deterioration of symptoms and failure of the prosthesis. Two of the four patients who had removal of the prosthesis suffered left iliac vein injury during surgery. Fraser et al. inserted the discs through a retroperitoneal approach and suffered one anterior displacement in this case series [5]. It should be noted that there were two pilot studies. In the first series all patients were implanted in Adelaide. Displacement occurred in series 2 at both test centres but only in Manchester was displacement severe enough to warrant removal for the reasons stated. Disc displacement due to placement of the disc too far anteriorly has been reported with the PRODISC [1]. The high incidence of disc displacement in this series (5 of 13) is thought to be associated with an anterior transperitoneal approach and more extensive removal of the annulus rather than malposition. Design is also an important factor in preventing displacement since it occurred in both centres. On revision surgery no osteointegration of these implants was apparent and displacement occurred despite re-design of the endplates, as a result of the series 1 failure, to a domed surface with 4\u20136 tapered fins on each surface. This redesign was also inadequate.\nVan Ooij has described a series of 27 patients with anterior displacement of four Charit\u00e9 discs one of which led to compression of the iliac artery causing symptoms of ischaemia [11]. A systematic review of total disc replacement examined the results from 564 arthroplasties in 411 patients. The majority of the cases considered were the Charit\u00e9 disc and only eight cases of vascular injury were identified [8]. The authors of both papers concluded that removal of artificial discs is potentially dangerous and no reliable strategy for revision surgery exists for a procedure without proven long-term reliability.\nThe five cases described in this report highlight failure of three design criteria for a disc prosthesis: (i) the rapid fixation to bone, (ii) fail-safe design to prevent damage to surrounding structure in case of failure and (iii) medium-term osteointegration [7]. If a disc does fail and needs to be removed, surgery can be hazardous with associated vascular injury [8]. Consequently, we advise that a Vascular Surgeon should be available to try and mobilise or divide and re-anastomose the left common iliac vessels when needed for adequate exposure of a L45 disc. Furthermore, this revision disc surgery is usually difficult and should be planned to include autologous blood transfusion.","keyphrases":["vascular injury","disc design","artificial inter-vertebral disc","inter-vertebral disc replacement","inter-vertebral arthroplasty"],"prmu":["P","P","P","P","R"]}
{"id":"Evid_Based_Complement_Alternat_Med-4-1-1810360","title":"Randomized Controlled Trials of Pediatric Massage: A Review\n","text":"The existing reviews of massage therapy (MT) research are either limited to infants, adults, or were conducted prior to the publication of the most recent studies using pediatric samples. Randomized controlled trials (RCTs) of pediatric MT are reviewed. A literature search yielded 24 RCTs of pediatric MT, defined as the manual manipulation of soft tissue intended to promote health and well-being in recipients between 2 and 19 years of age. Because RCTs of pediatric MT varied considerably in the amount and types of data reported, quantitative and narrative review methods were both used. Single-dose and multiple-dose effects were examined separately. Among single-dose effects, significant reductions of state anxiety were observed at the first session (g = 0.59, P < 0.05) and the last session (g = 1.10, P < 0.01) of a course of treatment. Effects for salivary cortisol (g = 0.28), negative mood (g = 0.52) and behavior (g = 0.37) were non-significant. Three of eleven multiple-dose effects were statistically significant. These were trait anxiety (g = 0.94, P < 0.05), muscle tone (g = 0.90, P < 0.01) and arthritis pain (g = 1.33, P < 0.01). Results of studies not permitting effect size calculation were judged to be generally consistent with quantitative results. MT benefits pediatric recipients, though not as universally as sometimes reported. Numerous weaknesses endemic to MT research (e.g. low statistical power, frequent failure to report basic descriptive statistics) are identified, and recommendations for future pediatric MT research are discussed.\nIntroduction\nBackground\nMassage therapy (MT) is one of the most widely used complementary and alternative medicine (CAM) therapies in the United States according to National Health Interview Survey data. It is estimated that consumers spend between 2 and 4 billion dollars on 75 million visits to massage therapists annually (1). Studies indicate that parents are making increasing use of CAM therapies, including MT, for their children. One study found that 33% of parents reported using CAM for their child within the past year, with MT being one of the most popular therapies (2). Another study shows that families of children with special health care needs are almost twice as likely to have used CAM for their child (3). CAM use is now prevalent, even in many traditional medical settings [e.g. 49% of university-affiliated pain management centers in the US and Canada offer MT (4)], yet pediatricians and other health care professionals are often not informed about the CAM therapies that are being used by their patients (2). This may be especially true for pediatric CAM, where survey results indicate that 81% of parents currently using CAM for their child wanted to discuss it with their pediatrician, but only 36% did (5). For CAM to truly be integrated into the health care system, it needs to be openly discussed and recognized for its value, particularly in the area of palliative care (6). A scientific understanding of CAM therapies, such as pediatric MT, will permit a greater understanding of the value of this type of therapy.\nNumerous studies of MT for children and young adults have been conducted. These include randomized controlled trials (RCTs), evaluation studies, descriptive case studies and reviews. However, the existing reviews of MT research are either limited to adult participants, limited to infants, were conducted prior to the publication of the most recent studies using pediatric samples, or neglected to quantify results. Ottenbacher and colleagues (7) quantitative review of tactile stimulation for infants and young children, published nearly 20 years ago, found statistically significant beneficial outcomes for five of the six categories examined (these were motor\/reflex, cognitive\/language, social\/personal, physiological and overall development; visual\/auditory was non-significant). Field (8) conducted a narrative review, including research on persons of all ages, and concluded that MT had a multitude of condition-specific effects in addition to consistently providing reductions of anxiety, depression and levels of stress hormones (most notably cortisol) that were observed across studies. The two most recent reviews are quantitative reviews that focus specifically on either infants or adults. A Cochrane review systematically examines MT for preterm and low birth-weight infants, and reaches the negative conclusion that \u2018there is insufficient evidence of effectiveness to warrant wider use of preterm infant massage\u2019 (9). The quantitative review of MT performed on adults confirmed (and quantified) some of Field's conclusions while disputing others (10). Substantial reductions resulting from multiple sessions of MT were noted for depression (g = 0.62, P < 0.01) and trait anxiety (g = 0.75, P < 0.01), while MT's effect on cortisol levels was not statistically significant (g = 0.14). While this review supported the value of MT by quantifying its ability to substantially reduce symptoms of psychological distress in adults, taken together this set of findings called into question some of the theories most often invoked to explain the benefits of MT, especially the theory that MT benefits recipients primarily by activating the parasympathetic nervous system (10).\nCurrent Review\nWhile the latest quantitative reviews increase our understanding of MT and indicate new directions for research, they neglect MT performed on pediatric samples. The current review examines MT's effects in pediatric samples (defined here as studies where the mean age of participants was between 2 and 19 years), with a focus on RCTs. While some have argued against using RCTs to understand CAM modalities such as MT (11), RCTs represent the state-of-the-art for establishing cause and effect relationships in treatment research because they are the most effective study design for ruling out alternate (i.e. non-treatment) explanations for observed effects, including spontaneous recovery, placebo effects and statistical regression (12).\nThough there have been some new pediatric MT studies published since the reviews by Ottenbacher et al. (7) and Field (8), the number of RCTs that examine pediatric MT is still quite small. In addition, a weakness of existing MT research is that, more often than not, studies in this area do not include the minimal statistical detail necessary to calculate effect sizes. For these reasons, a full meta-analytic treatment of MT research with pediatric samples is not possible. However, consistent with Rosenthal's observation that a narrative literature review can only be improved by the addition of a simple, descriptive quantitative analysis (13), we quantified results where possible, and include these results in combination with a narrative review of other RCTs. In some cases, the present findings are being compared with analogous findings in the adult literature. When these comparisons are made, we are referring to the adult meta-analysis conducted by Moyer et al., unless otherwise stated (10).\nOperational Definition\nA notable challenge in reviewing MT studies is that there are many forms of MT in practice. The American Massage Therapy Association (AMTA) defines massage as \u2018manual soft tissue manipulation, [including] holding, causing movement, and\/or applying pressure to the body\u2019 (14). As written, this very broad definition includes numerous MT approaches commonly used in clinical practice that are relevant to the current review, but could also include rare forms of medical massage (e.g. optic nerve massage (15), light compressive massage for congenital dacryocystocele (16), cardiac massage (17)), that are outside the intended scope of this review. For this reason, we focus on forms of MT that are consistent with traditional Swedish styles of massage. Swedish massage uses five main strokes to stimulate the circulation of blood through the body; petrissage (kneading), effleurage (stroking), friction, tapotement (tapping) and vibration. For the purposes of this review, MT is typified by the manual manipulation of soft tissue, performed by a person other than the recipient, intended to promote health and well-being. This operational definition allows a range of MT styles to be included in this review. Studies vary on many details, including the amount of clothing worn by recipients, whether a massage chair or massage table was used, whether MT took place in a clinical setting or at home, and whether MT was performed by a person with full, partial, or no training as a massage therapist. Studies also vary in which anatomical regions are massaged. Despite all these variations, it is reasonable to expect that there will be some consistent outcomes that result from MT. Eventually, as a scientific understanding of MT grows, studies that examine the importance of these variations will be advisable, but currently the questions of greatest interest are at a more fundamental level.\nTypes of Effects\nMT effects can be divided into single-dose and multiple-dose. Single-dose effects include MT's influence on psychological or physiological states that are transient in nature and that might reasonably be expected to be influenced by a single session of MT. Multiple-dose effects are restricted to MT's influence on variables that are considered to be more enduring, or that would likely be influenced only by a series of MT sessions performed over a period of time, as opposed to a single dose. Frequently, both single- and multiple-dose effects are examined in the same study. One example is a study of MT for autistic children that examined the single-dose effect of MT on salivary cortisol (immediately prior to, and immediately following, an individual session of MT) and the multiple-dose effect of MT on depression (at the beginning of, and at the conclusion of, a sequence of MT sessions over time) (18). A second example is a study that evaluated children's distress during burn treatment, which included the single-dose effect of MT for state anxiety and the multiple-dose effect of MT for depression (19). Typically, studies include the terms \u2018short-term effect\u2019 and \u2018long-term effect\u2019 to indicate single- and multiple-dose effects, respectively. Our decision to use the single-dose and multiple-dose terminology is motivated by the desire to prevent any confusion that may arise related to how long an effect may last following the termination of treatment. None of the studies in the current review examine whether any MT effects last beyond the final day on which a participant receives treatment, making the use of the term \u2018long-term effect\u2019 potentially misleading.\nThe potential benefits of MT can be further classified according to whether they are primarily affective, physiological or behavioral in nature. Affective refers to effects most closely associated with the recipients' feelings and emotions. Physiological effects are those concerned with recipients' vital organismic processes. Behavioral effects are those related to the recipients' observable responses to their environment. Study results reviewed here will first be separated by the single-dose versus multiple-dose distinction, then further categorized into affective, physiological and behavioral dimensions.\nMethods\nLiterature Search\nA literature search was conducted by the first author (S.B.) using the keywords massage, child and pediatric to search the MEDLINE, LexisNexis, CINAHL and PsycInfo databases. We checked the NIH CRISP Database to search for other publicly funded studies currently in progress. MT researchers were also contacted to obtain studies that were unpublished, in press or otherwise not found by means of database searches. With the introduction of the first Massage Therapy Research Conference in Albuquerque, New Mexico (43), leaders in the field of massage research were readily contacted. We used this opportunity to access current unpublished pediatric massage studies. Studies obtained by these methods were inspected to ensure that they examined a form of MT consistent with the present study's operational definition of MT performed on a pediatric sample. Application of these criteria yielded 24 RCTs of MT with a pediatric sample. These studies, along with important details, are listed in Table 1.\nStatistical Analysis\nFor studies that provided sufficient data, between-groups comparisons on variables of interest were converted to Hedges' g effect size by the second author (C.A.M.). Hedges' g, calculated as (Group mean 1 \u2212 Group mean 2)\/Pooled SD, estimates the number of standard deviations that the average member of a treatment group differs from the average member of a comparison group for a given outcome. Hedges' g was selected over Cohen's d, a similar standardized mean difference effect size. This choice was made for two reasons. First, in some cases the original study data could only be converted to g. Second, using g makes the results of the current review as consistent as possible with the existing meta-analysis of MT effects for adults.\nIn cases where a study employed more than one measure to examine the same outcome variable, results of multiple measures completed by participants or by blinded observers (but not those completed by non-blind observers) were standardized and then averaged, yielding one effect size per variable for each study. Similarly, if a study examined the immediate effects of more than one application of treatment, the results of the multiple applications or assessments were standardized and, when similar in magnitude, averaged in order to calculate a single effect size for that study. In the single case where these assessments clearly differed in a systematic way (i.e. state anxiety, where the effects of a final session were always larger than the effects of the initial session; paired sample t(3) = 4.46, one-tailed P < 0.02), separate effects were calculated for each timepoint. Effect sizes were coded such that positive values, for any variable, indicate a more desirable outcome (e.g. a reduction in anxiety) for the participants who received MT. Individual study effect sizes were subjected to a correction for small sample bias, then weighted by their inverse variance and averaged to generate a mean effect size for each outcome variable (20). All effect sizes were calculated according to a random effects model of error estimation. Statistical significance of the mean effect sizes was assessed by calculating the 95% confidence interval for the population parameter. A significance level of 0.05 or better is inferred when zero is not contained within the confidence interval.\nResults\nOf the 24 RCTs, only 9\u2014accounting for a total of 200 participants\u2014provided sufficient data for their results to be systematically quantified. Table 2 lists mean effect sizes for 16 outcome variables (g), as well as the number of studies contributing to each effect size (k), the total number of participants contributing to each effect size (N) and 95% confidence intervals. Five of these sixteen effect sizes were statistically significant. For the single-dose effects category, these included state anxiety at the first session (g = 0.59, P < 0.05) and at the last session (g = 1.10, P < 0.01) of a course of treatment. Effects for salivary cortisol (g = 0.28), negative mood (g = 0.52) and behavior (g = 0.37) were non-significant. Only 3 of the 11 multiple-dose effect sizes were statistically significant. These were trait anxiety (g = 0.94, P < 0.05), arthritis pain (g = 1.33, P < 0.01) and muscle tone (g = 0.90, P < 0.01).\nOf the 24 RCTs, 15 (accounting for 458 research participants) do not report sufficient data to permit effect size calculation, a frequent problem in MT research that makes objective interpretation of results difficult. Nevertheless, by judiciously comparing the scant data presented in this subset of studies with the objective data previously summarized, it should be possible to see if there are any dramatic contrasts among the findings. These interpretations, within the context of the more objective findings, appear in the results categories that follow.\nSingle-Dose Effects\nAffective Dimension\nState anxiety. Field et al. have conducted several pediatric MT studies where anxiety is an outcome measure. These studies date back to 1992 when MT was applied to a group of pediatric psychiatric patients (21). Four studies with reportable effect sizes (No.'s 6, 14, 17 and 21 in Table 1), using a total of 81 participants, compared MT with either relaxation therapy or a reading comparison group with state anxiety\u2014a momentary emotional reaction consisting of apprehension, tension, worry and heightened autonomic nervous system activity (22)\u2014as a dependent variable. Three of the studies (No.'s 14, 17 and 21) used the State Trait Anxiety Inventory (two specify the child version). One study (No. 6) of MT for children with juvenile rheumatoid arthritis used a behavior observation of the child's anxiety level performed by a blinded rater. MT consistently reduced state anxiety in these studies. This single-dose effect was unique in that it is significantly larger at the second timepoint, so we examined these separately. Results of a first session of MT yield a statistically significant effect, g = 0.59 (95% CI = 0.15, 1.04). The effect at the last session is even greater, g = 1.10 (95% CI = 0.64, 1.57). These results are depicted graphically in Fig. 1. Possibly, the substantially larger effect occurring at the last session of treatment may be the result of participants' increasing comfort with MT (or with the massage therapist) over the course of time, or the effect may be related to MT's potential to reduce trait anxiety over a course of treatment (8). These pediatric results are consistent with the same effect found for adults, where this effect has been estimated as g = 0.37 (95% CI = 0.14, 0.59). Possibly, the larger effect for the pediatric samples examined here reflects a greater treatment aptitude for this population; however, with such wide confidence intervals, this is only speculation.\nStudies where state anxiety was a dependent variable but effect sizes could not be calculated include samples of children who experienced Hurricane Andrew (23), depressed adolescent mothers (24), and children and adolescents with a range of illnesses including diabetes (25), atopic dermatitis (26), asthma (27), bulimia (28) and leukemia (29). The most commonly used measures across these studies were the State Trait Anxiety Inventory for Children (STAIC) and the State Trait Anxiety Inventory (STAI) for adolescents. A study of children who experienced Hurricane Andrew assessed both the single-dose effects of state anxiety and the multiple-dose effects of trait anxiety. These studies are consistent with the previously reported effect sizes, in that MT appears to decrease state anxiety. The MT in all of these studies was conducted either daily or twice weekly over a treatment period of 30\u201345 days. MT sessions generally lasted between 20 and 30 min, the one exception being the leukemia study where MT sessions were 15 min in duration. Four of these studies trained parents to provide MT directly to their child and the remaining three either used trained massage therapists or massage students.\nMood. Mood, which may be defined as \u2018transient episodes of feeling or affect\u2019 (30), has frequently been an outcome measure in pediatric MT studies. Study populations have included depressed adolescent mothers, and children and adolescents with cystic fibrosis, leukemia and bulimia. Two studies (No.'s 13 and 14 in Table 1), using a total of 50 participants, compared MT with either a reading or wait-list control to examine MT's effect on mood. One of these studies (No. 14) used the Profile of Mood States (POMS) (31) depressed mood subscale. The second study (No. 13) used a faces scale and a modification of the Children's Pain\/Fear Thermometer Rating Scale. Taken together, these studies yield a non-significant effect, g = 0.52 (95% CI = \u22120.05, 1.10) that is consistent with the results found for adult recipients (g = 0.34, 95% CI = \u22120.08, 0.76). These positive but non-significant results suggest at least four possibilities. One is that the tools that have been used to measure mood in these studies are not precisely capturing overall mood, but are being affected by correlates of mood such as depression, pain or fear. A second possibility is that MT's modest effect on mood has not been examined with sufficient statistical power, resulting in wide confidence intervals. The third is that MT's positive effect on mood is affected by a moderator variable that has not yet been examined (e.g. recipient's comfort with MT; the existence of a therapeutic bond between the recipient and provider, etc.). Finally, the possibility that MT does not specifically have a positive effect on mood cannot be definitively ruled out.\nPhysiological Dimension\nSalivary cortisol. Two studies (No.'s 6 and 13 in Table 1), using a total of 50 participants, compared MT with either relaxation training or a wait-list control to examine MT's single-dose effect on cortisol\u2014a stress hormone associated with activation of the sympathetic nervous system in response to certain kinds of stressors. In both studies, salivary cortisol [but not urinary cortisol, which less accurately captures short-term stress responses (32) most likely to respond to single-dose MT] was sampled pre- and post-MT session, with a delay of 20\u201330 min after the session, because salivary cortisol samples reflect responses to stimulation occurring \u223c20 min prior to collection. Both studies also took into account the diurnal cortisol cycle, characterized by an increase in secretory activity following awakening and a declining trend over the course of the day (33). Combined, these studies yield a non-significant reduction, g = 0.28 (95% CI = \u22120.27, 0.84) of salivary cortisol for the participants receiving MT in comparison to controls. This finding contrasts Field's assertion that reductions in cortisol level are one of MT's most reliable effects, but is consistent with meta-analytic findings based on adult samples, where cortisol effects were small and non-significant (g = 0.14, 95% CI = \u22120.10, 0.38) (10). Based on the available evidence, MT's single-dose effect on cortisol levels appears to be small, and possibly zero.\nBehavioral Dimension\nDistress behaviors. One study (No. 20 in Table 1) compared MT with standard care for children receiving burn treatment, and examined the children's distress behaviors before and during this painful procedure. The Children's Hospital of Eastern Ontario Pain Scale (34) was used to code distress behaviors before and during a dressing change (35). Six behavior categories were assessed including cry, facial, verbal, torso, touch and legs. The MT group showed only an increase in torso movements during the dressing change, whereas the control group showed an increase in five out of the six distress behaviors. Combined observational ratings made by nurses, who were blind to the group to which the children were assigned, favored MT, g = 0.37 (95% CI = \u22120.43, 1.17). Though this effect favors MT, the wide confidence interval, which may be partially or wholly attributable to the small sample (n = 24), makes this result difficult to interpret.\nIn several other studies with behavioral outcomes where effect sizes could not be calculated, researchers reported improvements in fidgetiness, activity, vocalization and cooperation. The most commonly used instrument in these studies is a Behavior Observation Scale that was first used to assess behavior after relaxation therapy classes (36). Using this measure, behavior is observed three times during the 30 min prior to MT, during MT itself and during the 30 min after MT. Because the effect size data are inconclusive and researchers report positive behavioral improvements resulting from MT, further study on distress behaviors may be warranted.\nMultiple-Dose Effects\nAffective Dimension\nDepression. Depression, including motivational and cognitive deficits, vegetative signs, and disruptions in interpersonal relationships beyond those expected from ordinary unhappiness or poor mood (37), has been examined in pediatric MT studies, albeit with small samples. Two studies (No.'s 13 and 17 in Table 1), using a total of 54 participants, examined whether MT would reduce depression in comparison to either a wait-list control or to progressive muscle relaxation. The first study (No. 13) used the Children's Depression Inventory-Short Form, and was the only study reviewed to use that measure. It is an abbreviated version of a widely used self-report measure of depression for children and adolescents. The second study (No. 17) used the Center for Epidemiological Studies-Depression scale, which was also used in several studies that did not permit effect size calculation. Though the mean effect favored MT, g = 0.48, this effect is not statistically significant (95% CI = \u22120.06, 1.02). This result is inconclusive, but promising given that the confidence interval approaches significance, and also because the estimated effect is not greatly different from statistically significant reductions of depression found in adult samples (g = 0.62, 95% CI = 0.37, 0.88). It is also generally consistent with the conclusions reached by the authors of studies that did not permit effect size calculation. Still, the possibility that MT does not reduce depression for pediatric recipients to the extent it does in adults cannot be ruled out. At this point, further study on MT for pediatric depression is needed.\nTrait anxiety. One study (No. 13 in Table 1), using a sample of children and adolescents with attention-deficit hyperactivity disorder, examines MT's effect on trait anxiety, a dispositional, internalized proneness to be anxious (38), with enough detail to permit effect size calculation. This study, which also appears in the adult MT meta-analysis, uses the Revised Children's Manifest Anxiety Scale to compare MT with a wait-list condition and yields a large, statistically significant effect (g = 0.94, 95% CI = 0.20, 1.68) that is consistent MT's anxiolytic effects demonstrated in numerous studies with adult recipients. This result is consistent with author claims in the \u2018Hurricane Andrew\u2019 study, where improvements in children's trait anxiety at the end of the MT treatment are reported. Reduction of trait anxiety, resulting from a course of MT sessions, is certainly worthy of further study in pediatric populations.\nPain. Four pediatric studies have been conducted that include pain as an outcome measure. Three of these studies used the Happy Faces Scale while only one study, a study of children with juvenile rheumatoid arthritis (and the only study with sufficient data for calculating effect sizes), used the Varni\/Thompson Pediatric Pain Questionnaire. This study (No. 6 in Table 1) assessed pain in three ways\u2014child self report, parent report and physician report. The children and a pediatric rheumatologist, who was blind to group assignment, generated reports of pain reduction that were consistent; parents' ratings were omitted from effect size calculation due to their non-blind status and proneness to bias. In comparison to a relaxation therapy group, MT yielded a large, statistically significant pain reduction (g = 1.33, 95% CI = 0.37, 2.29). This very large effect, in contrast with other studies that have shown mixed results for pain reduction resulting from MT, suggests that MT may be particularly well-suited to pain reduction for children with this condition.\nThree RCT's that do not permit effect size calculation looked at MT's impact on pain, and concluded that children experienced reductions of pain resulting from MT. Samples in these studies included children who experienced Hurricane Andrew (23), adolescents with attention-deficit hyperactivity disorder (39) and children with atopic dermatitis (26). These reports, combined with the findings in the juvenile rheumatoid arthritis study, indicate that MT for pain reduction in pediatric recipients is worthy of further examination.\nPhysiological Dimension\nMuscle tone. Two recent studies that assess muscle tone (No.'s 23 and 24 in Table 1) yield g = 0.90 (95% CI = 0.23, 1.57). The first examines MT for children with cerebral palsy who received 30 min of MT 2 times per week for 12 weeks. This study also assesses spasticity, motor functioning, facial expressions and limb activity. The second study, of children with Down syndrome provided 30 min of MT 2 times per week for 8 weeks. Children's development, and fine and gross motor functioning, was evaluated. Both studies used the Arms, Legs and Trunk Muscle Tone Scale (ALT Muscle Tone Scale), which was designed during the pilot phase of the Down syndrome study. There is modest support that MT improves muscle tone, though it must be pointed out that the two studies diverge greatly in their individual results. Study number 23 in Table 1 had virtually no effect, while study number 24 had a huge effect. It must be noted that because these results come from a newly developed measure, the validity of the measure is not yet well-established. This may account for the divergent results across studies.\nRange of motion. One study (No. 23 in Table 1), with a sample of children suffering from cerebral palsy, yields non-significant improvements in range of motion, g = 0.31 (95% CI = \u22120.57, 1.19). The study notes that right and left hip extension, but not abduction, improved. Study authors note that increased muscle tone may have led to an increase in range of motion, but this result, based on a single study with 20 participants, is inconclusive.\nImmune measures. Two studies (No.'s 17 and 22 in Table 1) examined markers of immune system functioning in HIV+ adolescents or children. Though study authors are quick to point out that there were some within-group effects for those who received MT, a between-groups analysis that compares MT recipients with controls yields no effect (g = 0.06, 95% CI = \u22120.52, 0.63). In light of this result, specific MT effects on CD4 count were then examined separately, to account for the fact that the numerous immune system markers reported in those studies, and combined in the process of effect size calculation, might be obscuring this most important measure of disease progression in HIV+ persons. When an effect size for CD4 count only is generated from these two studies, the effect is only marginally larger and still non-significant (g = 0.24, 95% CI = \u22120.33, 0.82). Though these studies, and another study of MT for children with leukemia (29), make much of within-groups MT effects on immune system markers, the available between-groups data show that MT's effect on immune system function is probably small and possibly zero.\nPulmonary function. One study (No. 14 in Table 1) examined whether MT might generate an improvement in peak air flow for a sample of 20 children with cystic fibrosis. In comparison to a reading control group, the children receiving MT had higher peak air flow, g = 0.47 (95% CI = \u22120.41, 1.35), indicating MT may be of specific value to children with this condition. Another study (27) that does not permit effect size calculation reports multiple measures of breathing improvement for asthmatic children ages 6\u20138 years, though older children (ages 9\u201314 years) showed fewer improvements. Overall, evidence for MT effects on pulmonary function is promising, and further study is warranted.\nSkin condition. Two RCT's examine MT's effect on skin condition. Children with atopic dermatitis were studied to determine the effect of MT on redness, scaling, lichenification, excoriation and pruritus (26). Though effect sizes cannot be calculated, study authors indicate some improvement following MT. Pediatric patients with hypertrophic scarring (HTS) were also studied and vascularity, pliability and height of the HTS revealed no appreciable effects. There is some evidence that pruritis decreased in certain patients with mature burn scars following MT. There is insufficient evidence to validate MT effects on skin condition in pediatric recipients.\nGlucose level. (No. 7 in Table 1) One study of children with diabetes examined the effect of MT on blood glucose levels (25). The authors conclude that MT lowers mean blood glucose levels and that compliance for insulin and food regulation improved, though data necessary for effect size calculation are not reported.\nBehavioral Dimension\nDevelopmentally appropriate functioning. Two studies (No.'s 23 and 24 in Table 1) used the Developmental Programming for Infants and Young Children (DPIYC) scale to assess developmentally appropriate functioning in recipients following MT. The average effect was small and non-significant (g = 0.24, 95% CI = \u22120.38, 0.86), though it must be pointed out that the studies had divergent results. Study number 24 yields a modest improvement in developmentally appropriate functioning in a sample of children with Down syndrome. Study number 23, with a sample of cerebral palsy sufferers, yields no effect. Possibly, these divergent results are the result of the different populations sampled within the studies. Taken together these results are inconclusive, but the encouraging results of the study with children who have Down syndrome indicate that further studies should be done with children having this or a related condition.\nSpasticity. One study (No. 23 in Table 1), with a sample of children suffering from cerebral palsy, examined whether MT had an effect on spasticity by using the Spasticity scale\/modified Ashworth scale. The small, non-significant effect (g = 0.26, 95% CI = \u22120.62, 1.14) does not support an MT effect on spasticity. Because this study was very small (n = 20), further testing in this area may be beneficial.\nHostility. One study (No. 21 in Table 1), with a small sample of aggressive adolescents, examined whether MT might reduce hostility compared with relaxation therapy. This study yields g = \u22120.85 (95% CI = \u22121.85, 0.15), a non-significant effect, but one that almost reaches significance in the wrong direction. The most tenable conclusion from this finding, a result based on a very small sample, is that MT has no effect on hostility beyond that provided by relaxation therapy to which it was compared. Though participants were randomly assigned, the MT group was approximately three points higher on the SCL-90R Hostility subscale prior to treatment. While scores decreased in the MT group during the treatment period, they also did in the relaxation therapy group, such that the MT group is still three points higher than the relaxation therapy group at the end of the treatment period. The authors' assertion that \u2018by the end of the study [massaged adolescents] reported feeling less hostile\u2019 obfuscates the fact that there was no between-groups effect. Related measures used in this study, such as the Overt Aggression Scale, were completed not by study participants, but by their legal guardians who were not blinded to group assignment, making their ratings prone to bias. Nevertheless, when all such measures included in the study are used to calculate an effect size, regardless of blinding, the result still converges on no effect.\nClassroom behavior. One study (No. 13 in Table 1) examined the classroom behavior of students with attention-deficit hyperactivity disorder using the Conners Teacher Rating Scale. Six factors of this scale were used including hyperactivity, conduct, emotional-indulgent, anxious-passive, asocial and daydream\/attention problems. In comparison with a wait-list control, MT yielded g = 0.66 (95% CI = \u22120.07, 1.39), an effect that, while non-significant, is encouraging. Further studies of MT for this population are needed to confirm whether it might be of value in improving behavior, an outcome that seems possible given MT's relatively well-established potential to reduce anxiety. Studies that do not permit effect size calculation also suggest some improvements in classroom behavior following a course of MT. Improved classroom behavior and social relatedness are reported in studies of preschool children (40) and children with autism (41).\nCognitive performance. (No. 11 in Table 1) A study of MT for children in preschool concludes that MT impacts cognitive performance (42). This study used three subtests of the Wechsler Preschool and Primary Scale of Intelligence\u2014Revised, but does not report sufficient data to quantify the effect.\nSleep and relaxation. (No.'s 1, 2 and 18 in Table 1) Though none permit effect size calculation, three studies have examined the effect of MT on sleep and relaxation. In one study, parents of children with autism recorded their children's sleep behavior in sleep diaries that included 5-point Likert scales (41). It is reported that the MT group showed decreases in fussing\/restlessness, crying, self-stimulation behavior and getting out of bed. Nighttime sleep recordings were conducted for a previously mentioned study of MT for child and adolescent psychiatric patients (21). A video camera was set up on a tripod in the participant's room. The video tapes were subsequently coded for quiet sleep, active sleep, awake and lying quietly, and awake and active. Percentage of time asleep increased significantly from the first to last day of MT and the percentage of nighttime wakefulness decreased over the same time frame. Finally, children who experienced Hurricane Andrew were rated by an observer on a visual analogue scale (VAS), based on the child's apparent relaxation level (23). The VAS relaxation score increased significantly from the first to the last day of MT for the MT group. Possibly, MT promotes sleep and relaxation, but current studies do not permit this effect to be quantified.\nDiscussion\nAvailable data reveals that MT provides benefit to pediatric recipients, though not as universally as has sometimes been reported. Benefits from both single-dose and multiple-dose sessions are evident. Most of the statistically significant effect sizes were observed for affective outcomes; findings for the behavioral and physiological dimensions were less consistent. These results parallel known MT effects in adult recipients, where multiple-dose reductions of depression and trait anxiety are the largest effects.\nIn reviewing MT for pediatric recipients, we encountered several weaknesses endemic to the MT research literature that should be addressed in subsequent studies. These included (i) low statistical power, (ii) frequent failure to report basic descriptive statistics, (iii) descriptions of results that do not logically follow study designs, and (iv) lack of replication. We discuss these in turn.\nLow statistical power. Most pediatric MT studies were conducted with fewer than 30 participants. When studies are this small, only the largest effects have any likelihood of being uncovered. As researchers engaged in our own clinical MT studies, we are sensitive to the expense and difficulty involved in recruiting participants, but our empathy, unfortunately, does nothing to change the mathematics of the situation. The statistical power of MT studies must be increased (primarily by conducting studies with larger samples) if we wish to discover anything beyond the largest effects.\nFailure to report basic descriptive statistics. Many pediatric MT studies have appeared in journals with lax standards for the reporting of data; there is no other explanation for why studies would fail to report the most basic descriptive statistics. When a report does not include the standard deviations that describe the spread of the data, that report has almost no value as scientific evidence. This must not be allowed to continue in MT research, because it represents an enormous waste of resources. Simply put, researchers who take the time, effort and expense to perform a study should not allow the results of that study to be published without the statistics that permit an understanding of the outcome. If a journal does not require them, then the onus is on the researcher to ensure that the statistics are reported.\nResults that do not logically follow study designs. The importance of between-groups designs in MT has been noted (12). Why, then, do so many MT studies that employ a between-groups design emphasize within-group comparisons? The likely answer is that planned between-groups comparisons were non-significant (possibly due to low statistical power), so study authors may given in to the temptation to report the statistically significant, but misleading, within-group effect. This problem is rampant in MT research, and represents a real threat to the way this research will be perceived in the future. Many of the consumers of MT research are committed practitioners who have not had training in statistics and research design (though it must be noted that research literacy among MT practitioners is increasing). As such, they may be inclined to believe that coming across the sacrosanct \u2018P < 0.05\u2019 is proof of an MT effect, without realizing that this \u2018effect\u2019 may be nothing more than the effect of time, a placebo effect and\/or regression to the mean. Knowledgeable consumers of MT research must learn to distinguish within-group effects from between-groups effects, and MT researchers must clearly present between-groups findings when their studies employ a between-groups design.\nLack of replication. All but two of the pediatric MT studies we reviewed were conducted by the Touch Research Institute at the University of Miami. Their contribution to MT research has been considerable; however, scientific understanding is hampered when one laboratory is responsible for almost all of the results in an area of inquiry. Replication of results is a foundation of scientific progress, so it is necessary that other researchers contribute to this field. Given recent interest in CAM modalities, the small number of studies from other laboratories is surprising. We hope that this is about to change, and the success of the recent Highlighting Massage Therapy in CAM Research Conference (43) suggests that it will. Three pediatric MT RCT's from other research groups, currently in progress, support our optimism. Two of these examine MT for children and adolescents with cancer. While the results of a study that will examine pain, mood, stress and relaxation outcomes from MT and heat therapy for terminal children (44) cannot yet be reported, preliminary results of another study examining anxiety, fatigue, pain and nausea outcomes from MT in a sample of children with cancer (45) are promising; in particular, reductions of anxiety are expected to be consistent with what has been reported in this review. The third study, conducted by the first author of this review (S.B.) and researchers at the UCLA Pediatric Exercise Laboratory (46), evaluates pre-exercise MT for children with and without heart disease. Preliminary results show significant improvement in VO2 (oxygen consumption) for children who have an MT session prior to performing an exercise regimen on a stationary bicycle. If these results are validated in final analyses, they will be generally consistent with the finding that MT can improve pulmonary function. There are two other studies worth mentioning that have received IRB approval from Children's Memorial Hospital in Chicago and will be enrolling participants shortly. These studies are an evaluation of MT for reduction of pain, nausea and anxiety in pediatric cancer patients (47) and an evaluation of MT on immune function and heart rate variability in HIV-infected pediatric patients (48). The outcome measures for the first study are self-report ratings of pain, nausea and anxiety, whereas the second study evaluates T-Helper cells (CD4+), T-Suppressor cells (CD8+), Total B cells (CD3, CD19), Natural Killer cells (CD56+), viral load and heart rate variability.\nConclusion\nCurrent research indicates that MT is not a panacea for conditions studied in the pediatric population. In contrast to what has sometimes been claimed, there is little to no evidence to date to support effects such as improved immune system functioning, reduction of spasticity, or amelioration of hostility. In addition, there is currently scant evidence that MT provides benefits by first reducing cortisol, as MT's effect on this stress hormone is seen to be small when analyzed correctly (i.e. in between-groups as opposed to within-group comparisons). There is, however, a set of MT effects that have been shown to have real value to the pediatric population. MT shows a considerable impact on the state and trait anxiety levels of children. Because these effects are strong, and also because they are consistent with the findings in adults, future research on the anxiolytic effects of MT on pediatric recipients does not need to simply replicate previous studies. The greatest progress can now be made by focusing on the mediators and moderators of MT effects on anxiety, and on testing explanatory theories of these outcomes. MT effects on arthritis pain and muscle tone also appear to be strong, but these results do need to be replicated, as they are based on single studies. Other pediatric outcomes that are promising, but in need of further study, include MT's effects on depression, negative mood, certain types of behavior (likely due to reductions of anxiety) and air flow in those suffering from pulmonary disorders such as cystic fibrosis. As increased statistical power in the form of additional studies is brought to bear on these potential benefits, it is likely that some will be quantitatively validated.\nFinally, it has been noted that prior MT research has not accounted for the communication that inevitably takes place between massage therapists and their recipients, nor has it examined the likelihood that therapists and recipients develop a therapeutic relationship during the course of MT (10). This is also true in pediatric MT studies. MT has important parallels (in both process and outcomes) to psychotherapy (10), a treatment that relies on communication and therapeutic relationship to provide effects. It seems likely that MT effects, especially those belonging to the affective category, are mediated or moderated by these previously unexamined factors. These should not be neglected in subsequent pediatric MT research.\nAs adult consumers continue to explore and utilize all of their health care options, children will increasingly be recipients of MT. With this in mind, it is essential that we continue to study the benefits of MT for children, and the explanatory models that underlie them, so children's health and wellness can be maximized. The value of MT has been examined for many specific conditions that afflict children. It is our hope that this review has consolidated those findings, indicated areas that require further study, and led to an increased scientific understanding of pediatric MT.","keyphrases":["cam","child","health care","pain management","comfort","touch","integrative medicine","kid","quality of life"],"prmu":["P","P","P","P","P","P","R","U","M"]}
{"id":"Purinergic_Signal-4-2-2377320","title":"Purinergic signaling in the lumen of a normal nephron and in remodeled PKD encapsulated cysts\n","text":"The nephron is the functional unit of the kidney. Blood and plasma are continually filtered within the glomeruli that begin each nephron. Adenosine 5\u2032 triphosphate (ATP) and its metabolites are freely filtered by each glomerulus and enter the lumen of each nephron beginning at the proximal convoluted tubule (PCT). Flow rate, osmolality, and other mechanical or chemical stimuli for ATP secretion are present in each nephron segment. These ATP-release stimuli are also different in each nephron segment due to water or salt permeability or impermeability along different luminal membranes of the cells that line each nephron segment. Each of the above stimuli can trigger additional ATP release into the lumen of a nephron segment. Each nephron-lining epithelial cell is a potential source of secreted ATP. Together with filtered ATP and its metabolites derived from the glomerulus, secreted ATP and adenosine derived from cells along the nephron are likely the principal two of several nucleotide and nucleoside candidates for renal autocrine and paracrine ligands within the tubular fluid of the nephron. This minireview discusses the first principles of purinergic signaling as they relate to the nephron and the urinary bladder. The review discusses how the lumen of a renal tubule presents an ideal purinergic signaling microenvironment. The review also illustrates how remodeled and encapsulated cysts in autosomal dominant polycystic kidney disease (ADPKD) and remodeled pseudocysts in autosomal recessive PKD (ARPKD) of the renal collecting duct likely create an even more ideal microenvironment for purinergic signaling. Once trapped in these closed microenvironments, purinergic signaling becomes chronic and likely plays a significant epigenetic and detrimental role in the secondary progression of PKD, once the remodeling of the renal tissue has begun. In PKD cystic microenvironments, we argue that normal purinergic signaling within the lumen of the nephron provides detrimental acceleration of ADPKD once remodeling is complete.","keyphrases":["signaling","kidney","adenosine","atp","nucleotides","nucleosides","polycystic kidney disease"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Ann_Biomed_Eng-3-1-2040176","title":"Occupant Dynamics in Rollover Crashes: Influence of Roof Deformation and Seat Belt Performance on Probable Spinal Column Injury\n","text":"Motor vehicle crashes are the leading cause of death in the United States for people ages 3\u201333, and rollover crashes have a higher fatality rate than any other crash mode. At the request and under the sponsorship of Ford Motor Company, Autoliv conducted a series of dynamic rollover tests on Ford Explorer sport utility vehicles (SUV) during 1998 and 1999. Data from those tests were made available to the public and were analyzed in this study to investigate the magnitude of and the temporal relationship between roof deformation, lap\u2013shoulder seat belt loads, and restrained anthropometric test dummy (ATD) neck loads.\nIntroduction\nMotor vehicle crashes are the leading cause of death in the United States for persons of every age from 3 through 33.18 Rollover crashes, in particular, have a higher fatality rate than any other crash mode. Of the 6,159,287 police reported crashes in 2005 in the United States, only 4.1% involved a rollover. Yet, rollovers accounted for 34.4% (10,816) of all passenger vehicle fatalities. The fatality rate (defined as deaths per 100,000 registered vehicles) in rollover crashes is more than two and one-half times higher in sport utility vehicles (SUVs) than in passenger cars (8.28 vs. 3.22, respectively). Another 149,406 individuals sustained serious injuries in rollover crashes in 2005, approximately 30% of whom were occupants of SUVs.18\nMeasurement tools are required to evaluate appropriate intervention strategies to reduce the mortality and morbidity associated with rollover crashes. The Abbreviated Injury Scale (AIS) and Harm value (HARM) are two such measures, which are used to compare injuries of different types and severities.9 The AIS reflects \u201cthreat to life\u201d (or risk of death) and stratifies injuries into six categories (AIS 1\u20136) with AIS 6 defined as an \u201cuntreatable\u201d injury. The maximum AIS (MAIS) score refers to the most severe injury sustained in a specific individual. The highest rate of seriously injured (MAIS 3+) children 4\u201312\u00a0years old occurs in rollovers, more than double any other crash mode.22 One study reported that despite the increased weight of SUVs, the risk of injury for children in SUVs was similar to that for passenger cars, which may be due to the increased tendency of SUVs to roll over.6 HARM is a mathematical measure that applies a weighting factor to each injured, yet surviving occupant, which includes both monetary costs of the injury (i.e., direct costs associated with the loss of wages) and comprehensive costs (i.e., monetary costs plus non-monetary costs).9,14 Importantly, the non-monetary costs include such measures as loss of functional capacity and quality of life. In rollover crashes, the highest HARM is reflected in the population of occupants who survive the crash, yet sustain catastrophic injury, including permanent brain and\/or spinal cord injury.9\nA major source of HARM in rollover crashes results from occupant contact with the vehicle roof, support pillars and side headers.9 According to Digges et\u00a0al. \u201cCountermeasures to reduce rollover casualties include increased belt use, and technological interventions to prevent ejection and reduce the severity of body contacts with the vehicle interior.\u201d9 Hugh DeHaven outlined principles for designing vehicles for human transport in 1952, and stated that \u201cthe package (the passenger compartment) should not collapse under expected conditions of force, thereby exposing objects (and people) inside it to damage.\u201d7 Closely related to this principle, DeHaven stated that \u201cthe packaging structures which shield the inner container must not be made of brittle or frail materials; they should resist force by yielding and absorbing energy applied to the outer container so as to cushion and distribute impact forces and thereby protect the inner container.\u201d\nFranchini11 reported the importance of maintaining the integrity of the occupant compartment (referred to as \u201csurvival space\u201d) for belted occupants during a crash event. Few studies, however, have experimentally evaluated the influence of dynamic roof deformation with concomitant loss of occupant survival space as a potentially preventable cause of head and cervical spine injuries in rollover crashes. Bahling et\u00a0al.1 conducted rollover and drop tests of late 1980s model Chevrolet Malibu passenger cars using lap\u2013shoulder belted anthropomorphic test dummies (ATDs) to evaluate the influence of roof strength on injury mechanics. In several studies, Moffatt and other investigators reported on a customized, dynamic test procedure with controlled roof impacts of passenger vans and sedans.3,5,15 Both groups concluded that \u201cpotentially injurious impacts\u201d (defined by these authors as 2000\u00a0N recorded by the ATD upper neck sensor) occurred prior to significant roof crush and that enhanced roof strength made no difference in the likelihood of serious neck injury to restrained occupants. These conclusions have been contradicted in other literature, albeit without the benefit of scientific data from comparable full-scale, dynamic rollover crash tests.12,13\nRecently, the raw data from a series of full-scale, dynamic rollover tests of 1998\u20131999 model year Ford Explorer SUVs, using fully instrumented ATDs, were made publicly available to the scientific community.2 These tests were conducted using a standardized test methodology specified by Federal Motor Vehicle Safety Standard (FMVSS) 208 of the U.S. Department of Transportation (FMVSS 208 49 CFR Ch. V (10-01-04 Edition)). These data provide the first opportunity to analyze the dynamics of belted occupants within a contemporary SUV in a standardized rollover test environment.\nThe authors used the described data in the present study. The study\u2019s objective was to investigate both the magnitude of and the temporal relationship between dynamic roof deformation, lap\u2013shoulder seat belt loads, and restrained ATD neck loads in these FMVSS 208 dolly rollover tests of Ford Explorer SUVs.\nMaterials and Methods\nThree full-scale dolly rollover tests of 1998\u20131999 Explorer SUVs were sponsored by Ford Motor Company and conducted at the North American Autoliv test facility1 according to the standardized FMVSS 208 test protocol. This standard, issued by the National Highway Transportation Safety Administration (NHTSA) in 1971, outlines vehicle crash performance requirements designed for improving occupant protection. Described within FMVSS 208 is a voluntary, dolly rollover test used to evaluate occupant protection during a lateral rollover. This standardized test procedure was used for all three SUV rollover tests.\nThe FMVSS 208 test procedure involves accelerating a rollover dolly carrying a test vehicle to a set velocity and then rapidly decelerating the dolly, causing the vehicle to release, trip, and enter a lateral roll. The SUVs were first placed on a dolly fixed with a rigid, angled platform positioned 9\u00a0in. from the ground and 23\u00b0 from the horizontal. To assure that the roll was driver-side leading (i.e., that the driver\u2019s side contacted the ground first), each vehicle was situated on the platform so that its driver\u2019s side tires rested along a 4\u00a0in. lip at the forward edge of the test platform, as shown in Figs.\u00a01a and 1b.\nFigure\u00a01(a) FMVSS 208 dynamic rollover pre-test setup of SUV test B190043 on a dolly rollover cart; (b) critical dimensions of FMVSS 208 dolly\nThe vehicle and dolly were then accelerated along a straight path perpendicular to the longitudinal axis of the vehicle. The dolly was then decelerated rapidly (minimum deceleration rate of 20\u00a0g for 0.04\u00a0s), resulting in vehicle trip and subsequent rollover. The target speed for all Explorer tests was 30\u00a0mph. The vehicle identification number (VIN) and actual dolly launch velocity for each of the three tests are summarized in Table\u00a01.\nTable\u00a01Test conditionsAutoliv test numberTest descriptionData sampling rateSampling durationTest dateAccelerometerCoordinates (mm)XYZB190042FMVSS 208 Rollover (VIN1FMCU24E5VUC19292) (23\u00b0; 30.5\u00a0mph)20,000\u00a0Hz\u223c13,000\u00a0ms8\/10\/99Center of gravity2073.10\u221224.50975.00Driver\u2019s rail at A-pillar2038.20\u2212596.101770.0Driver\u2019s rail at B-pillar2776.40\u2212571.301863.30Driver\u2019s rail at C-pillar3209.70\u2212580.601871.40Passenger\u2019s rail at A-pillar2011.80592.101758.60Passenger\u2019s rail at B-pillar2786.60532.601857.10Passenger\u2019s rail at C-pillar3234.40523.301868.20B190043FMVSS 208 Rollover (VIN1FMDU34E6VUB99290) (23\u00b0; 30.4\u00a0mph)20,000\u00a0Hz\u223c13,000\u00a0ms8\/11\/99Center of gravity2231.0\u22124.1761.3Driver\u2019s rail at A-pillar2077.2\u2212543.71626.8Driver\u2019s rail at B-pillar2637.2\u2212535.01644.0Driver\u2019s rail at C-pillar3436.0572.41598.0Passenger\u2019s rail at A-pillar2077.9573.51564.7Passenger\u2019s rail at B-pillar2636.5569.71625.5Passenger\u2019s rail at C-pillar3436.3\u2212543.01617.6B180220FMVSS 208 Rollover (VIN1FMDU35P5VUC14510) (23\u00b0; 30.9\u00a0mph)12,500\u00a0Hz\u223c8000\u00a0ms12\/10\/98Center of gravity2231.0\u22124.1761.3Driver\u2019s rail at A-pillar2077.2\u2212543.71626.8Driver\u2019s rail at B-pillar2637.2\u2212535.01644.0Driver\u2019s rail at C-pillar3436.0572.41598.0Passenger\u2019s rail at A-pillar2077.9573.51564.7Passenger\u2019s rail at B-pillar2636.5569.71625.5Passenger\u2019s rail at C-pillar3436.3\u2212543.01617.6\nVehicle and ATD Instrumentation\nTriaxial and biaxial accelerometers were installed in each SUV to measure vehicle structural dynamics during the rollover tests. Accelerations were recorded at each of the following vehicle structure locations in coordinate systems consistent with SAE J211 guidelines (SAE J211-1 REV DEC03), as shown in Figs.\u00a02a and 2b, and Table\u00a01.Center of Gravity (CG) (x, y, z)Left (driver) roof rail at\nA-pillar (y, z)B-pillar (y, z)C-pillar (y, z)Right (passenger) roof rail at\nA-pillar (y, z)B-pillar (y, z)C-pillar (y, z)\nFigure\u00a02\n(a) Exterior view of test vehicle indicating A, B, and C-pillars; (b) interior view of test vehicle\u2014accelerometers were mounted at the roof rail-to-pillar junction at the A, B, and C-pillars for both driver and passenger sides of the Ford Explorer SUVs\nIn each test, two Hybrid III 50th percentile male, instrumented ATDs were placed in the front seats of Ford Explorer SUVs and restrained with lap\u2013shoulder belts that were affixed with load cells. Time synchronized with all other sensors and cameras in the experimental setup, these load cells continuously recorded tensile forces in the webbing of the lap and shoulder belts. Both ATDs were positioned in their respective seats according to reference FMVSS 208 Dummy Positioning Procedure with the seats located in the mid-track position. Given that the vehicle was positioned to roll driver-side leading, the driver ATD represented the \u201cnear-side\u201d occupant and the passenger ATD, the \u201cfar-side\u201d occupant in this test series. The following sensor outputs were recorded continuously throughout each rollover test for both driver and passenger ATDs:Head CG acceleration (x, y, z)Upper Neck\nForce (x, y, z)Moment (x, y, z)Lower Neck\nForce (x, y, z)Moment (x, y, z)Chest\nAcceleration (x, y, z)DeflectionPelvis Acceleration (x, y, z)Femur Force (Fz, right and left)\nDynamic motion of the SUVs was visually recorded by up to 10 high speed external cameras, which were time synchronized with the cart release trigger and imaged at 500\u00a0frames\/s. (Fig.\u00a03) Clocks that were positioned along the test track were visible in the external camera views throughout the roll sequence. Targets were affixed to the rear aspect of the driver and passenger ATD heads and were visible by the same camera monitoring the onboard time clock. Up to three onboard, high speed cameras recorded ATD kinematics as a function of time using the onboard clock, which was also time synchronized with the cart release trigger, external cameras, and electronic sensors affixed to both ATDs, the seat belts, and the SUV structure.\nFigure\u00a03External camera setup\nWith the cart positioned according to FMVSS 208 specifications, the data acquisition system was set up and armed for trigger. Time zero for all data acquisition was provided by a contact switch at both shock absorbers to indicate the start of cart deceleration. The data-sampling rates for each test are provided in Table\u00a01.\nData Analysis\nThe raw ASCII sensor data retrieved from the ATD and vehicle-mounted transducers were filtered according to the SAE J211 channel filtering class (CFC) requirements to remove the effects of crash related noise on the sensor readings. Each CFC specifies that the channel frequency response lies within limits detailed in SAE J211. Neck forces, neck moments, and vehicle data were filtered at CFC 1000, CFC 600, and CFC 60 accordingly. Subsequently, the filtered vehicle acceleration data were transformed into a vehicle-fixed, center-of-gravity, coordinate system using MATLAB\u00ae (registered trademark of The Mathworks, Inc.). Roof rail accelerations were transformed by subtracting the center of gravity accelerations from each measurement as a function of time. The result of the transformations was the acceleration of the roof rail relative to the vehicle\u2019s center of gravity. The transformed roof rail acceleration values provided insight into when and in what direction the rail was moving. Transformed data was then compared to the ATD neck and seat belt loads as a function of time. The accelerations recorded by the rail sensors were influenced by both vehicle rotation (angular velocity) and localized pillar deformation. The contribution due to vehicle rotation was made at a low frequency given that the peak roll rate of the vehicle at any time during the test interval (0\u20131000\u00a0ms) was approximately 350\u2013400\u00b0\/s (i.e., less than 2\u00a0Hz). Thus, all higher frequency accelerations were necessarily due to localized structural deformation.\nThe sensor outputs from the driver and passenger roof rail accelerometers positioned at the B-pillar were used as a quantitative definition of roof intrusion (i.e., \u201cobjective roof crush\u201d) into the occupant survival space. The mathematically transformed driver and passenger roof rail acceleration tracings in the present study provided objective evidence of the time interval(s) when roof\/pillar deformation occurred, and the time of deformation was corroborated through careful scrutiny of the high speed video images (\u201cobservable\u201d roof crush). Objective roof crush in our study was defined under the following necessary, contemporaneous conditions:Vertical and\/or lateral rail acceleration peak(s) \u201cdownward\u201d and\/or inboard toward the restrained ATDExternal camera video images consistent with SUV roof-to-ground contactOnboard camera video images consistent with a compromise in occupant survival space (i.e., reduced headroom)\nRoof rail accelerations, seat belt loads and peak ATD axial neck force (Fz, axial compression\/tension) and moments (My, chin-to-chest flexion, and Mx, ear-to-shoulder lateral bending) were compared to the high-speed video data to validate the correlation in time between the presence of objective roof crush and the development of peak ATD neck and seat belt loads.\nResults\nResults are presented and discussed for the first full second of the roll sequence for each of the three SUV tests, which includes the initial driver-side-to-ground contact and full roof contact, followed by the first passenger-side-to-ground contact.\nIn all three tests, the external high-speed cameras recorded continuous roof-to-ground contact from the first contact of the driver\u2019s roof rail with the ground until the end of the 1000\u00a0ms period, which corresponded to approximately 5\/8 roll. Onboard high speed video cameras recorded the front passenger compartment of each SUV, capturing the kinematics of the ATDs as well as the deformation of the roof header and side roof rails during the rollover event. These cameras recorded inboard displacement of roof rails (\u201cobservable\u201d roof crush) of both driver and passenger-side roof rails during the respective roof rail-to-ground contact for all three tests. The onboard clock, which was recorded by the interior cameras, allowed for a time-synchronized comparison of this data to the sensor output of the ATD neck transducers and roof rail accelerometers.\nIn all three tests, the head of each lap\u2013shoulder belt restrained ATD (driver and passenger) contacted the roof two or more times during the first 1000\u00a0ms roll interval as indicated by increased neck compression loads (negative Fz values) recorded by the ATDs\u2019 upper neck sensor (Fig.\u00a04). The time of occurrence of each peak neck load for the driver and passenger ATD head-to-roof contacts (the \u201cLocal Maximum\u201d as well as the \u201cAbsolute Maximum\u201d or \u201cPeak\u201d neck loads) was consistent with the onboard high speed video data. For each of the three tests, the time recorded by the onboard camera during observable ATD head-to-roof contact was within 3\u00a0ms of the time recorded by the neck sensors for peak neck loads. Peak axial neck force data (Fz) were measured by the ATDs\u2019 upper neck sensors and the moment loads (My and Mx) were measured by the ATDs\u2019 lower neck sensors (Table\u00a02).\nFigure\u00a04Passenger upper neck Fz vs. time (Test B190042)Table\u00a02Magnitudes of Local and Absolute maximum (Peak) neck loads (during time interval of continuous roof-to-ground contact)Test parameterDriverPassengerB190042B190043B180220B190042B190043B180220Max peak Fz (N)a\u2212958\u22121962\u22121920\u22125933\u22123245NonebLocal peak (s), Fz\u2212200\u2212295\u2212223\u2212361\u221250200\u2013260Max peak My (N\u00a0m)5811094304178261Local peak (s), My2112\u20135412\u20132220\u20132410Max peak Mx (N\u00a0m)\u2212106\u2212124\u2212167689841Local peak (s), Mx\u221211 to \u221218n\/a\u221220 to \u22124691219\u201321aFz was measured at the dummy upper neck load cell; My and Mx measured at the lower load cellbIn Test 180220, no absolute maximum Fz was identified as all peak neck compression loads were within the range of 200\u2013260\u00a0N\nThe near-side (i.e., the driver) ATD recorded Peak axial neck compression (Fz) loads of \u22122000\u00a0N or less for all three tests. Peak neck flexion (My) loads for the driver ATD resulted in chin-toward-chest head motion and were within a range of 58\u2013110\u00a0N\u00a0m for all three tests. In contrast, the far-side (i.e., the passenger) ATD recorded Peak neck Fz values in the range of \u22123244 to \u22125933\u00a0N for two out of three tests. In Test B180220, no Absolute Maximum Fz value was identified as all Local Maxima compression loads were within the range of \u2212200 to \u2212260\u00a0N. The Peak neck flexion (My) loads, recorded by the passenger ATDs, resulted in chin-toward-chest head motion and ranged from 178 to 304\u00a0N\u00a0m for all three tests. The Peak lateral neck bending ear-to-shoulder (Mx) loads ranged from 41 to 98\u00a0N\u00a0m for the passenger ATD, and the driver experienced Mx load magnitudes in the range of \u2212106 to \u2212167\u00a0N\u00a0m, for all three tests.\nThe time of occurrence of objective vertical and lateral roof crush was identified by the roof rail acceleration peak, for both the passenger and driver side roof rails in all three tests (Table\u00a03). Objective roof crush occurred prior to the Absolute Maximum dummy neck loads (Fz, My, Mx) in each of the three tests for both driver and passenger ATDs (Table\u00a03, Fig.\u00a05). The far-side, passenger ATD recorded Peak neck loads within 10\u00a0ms following the time of occurrence of objective roof crush for all three tests. Peak neck loads for the near-side, driver ATD occurred up to 65\u00a0ms following the time of occurrence of objective roof crush for all three tests.\nTable\u00a03Time of occurrence (ms) of roof\/pillar deformation and absolute maximum neck loadsTest parameterDriverPassengerB190042B190043B180220B190042B190043B180220Objective roof\/pillar deformation (vertical acceleration peaks)497513510730\u223c600\u2013800a742Objective roof\/pillar deformation (lateral acceleration peaks)497513494496512495Peak Fzc533540516730764n\/abPeak My533541517729764751Peak Mx537548540783774760aNo isolated spike in acceleration was noted; however, a sustained level of roof deformation is reflected in the roof rail tracings and the video footage for this testbNo isolated absolute maximum Fz value was noted, which differed significantly from the other \u201clocal\u201d maximums. This was consistent with the very high peak My in this test, however, which was noted at 751\u00a0ms (see Table\u00a02)cFz was measured at the upper neck load cell, whereas My and Mx were measured at the lower neck load cellsFigure\u00a05Driver upper neck Fz vs. vertical acceleration of the driver rail at B-pillar (Test B190043)\nLap and shoulder belt loads were recorded for both driver and passenger ATDs in all three tests, with the exception of the driver\u2019s shoulder belt for test B190043 and passenger shoulder belt for test B180220, both of which experienced sensor failures (Table\u00a04). The Peak lap belt loads for the near-side, driver ATD occurred within 1\u00a0ms of the time of Peak Fz and My neck loads for two out of three tests (Figs.\u00a06, 7). In the remaining test (B190042), the Peak lap belt load occurred 22\u00a0ms prior to the time that peak neck Fz and My neck loads occurred in the driver ATD. In contrast, the far-side, passenger ATDs\u2019 Peak lap belt loads occurred 65\u2013131\u00a0ms prior to the time of Peak neck compression (Fz) for two out of three tests and 65\u2013145\u00a0ms prior to the time of Peak neck flexion (My) for all three tests (Fig.\u00a08). In Test B180220, the passenger\u2019s Peak lap belt load occurred 6\u00a0ms after the time of Peak neck compression, yet 145\u00a0ms before the Peak neck flexion load (My).\nTable\u00a04Peak belt loads compared to belt loads at time of absolute maximum (peak) neck loadsTest parameterDriverPassengerB190042B190043B180220B190042B190043B180220Peak lap belt (N)548 (511\u00a0ms)795 (540\u00a0ms)940 (515\u00a0ms)797 (599\u00a0ms)705 (699\u00a0ms)779 (606\u00a0ms)Peak shoulder belt (N)395 (379\u00a0ms)N\/Aa410 (373\u00a0ms)899 (587\u00a0ms)953 (689\u00a0ms)N\/AaPeak FzLap belt (N)398 (533\u00a0ms)789 (540\u00a0ms)937 (516\u00a0ms) 359 (730\u00a0ms)115 (764\u00a0ms)771 (600\u00a0ms)Shoulder belt (N) 231 (533\u00a0ms)N\/Aa236 (516\u00a0ms)337 (730\u00a0ms)426 (764\u00a0ms)N\/AaPeak My(N\u00a0m)Lap belt (N)393 (533\u00a0ms) 785 (541\u00a0ms)937 (517\u00a0ms)361 (729\u00a0ms)115 (765\u00a0ms) 218 (751\u00a0ms) Shoulder belt (N) 227 (533\u00a0ms)N\/Aa236 (517\u00a0ms)341 (729\u00a0ms)417 (764\u00a0ms)N\/AaaData not available due to transducer failureFigure\u00a06Driver lap belt and neck load vs. time (Test B190043)Figure\u00a07Inverted 50th percentile Hybrid III Driver ATD \u201cdiving\u201d into roof with lap and shoulder belt providing restraining forces, which reduces neck loadFigure\u00a08(a) Roof crush\u2014passenger lap belt and neck load vs. time (Test B190043); (b) Roof crush\u2014passenger shoulder belt and neck load vs. time (Test B190043)\nThe driver ATDs\u2019 Peak lap belt loads ranged from 548 to 940\u00a0N for the three tests, and the shoulder belt loads were 395\u2013410\u00a0N for two tests (the driver\u2019s shoulder belt sensor failed on Test B190043) (Table\u00a04). The passenger ATDs\u2019 Peak lap belt loads were similar to those of the driver ATDs\u2019, ranging from 705 to 797\u00a0N for all three tests; the shoulder belt loads were 899\u2013953\u00a0N for two of three tests (the passenger\u2019s shoulder belt sensor failed on test B180220).\nThe near-side, driver ATD lap belt loads at the time of occurrence of the Peak Fz and My neck loads were 72\u201399% of the ATDs\u2019 respective Peak lap belt load for all three tests (Table\u00a04). In contrast, the far-side, passenger ATD lap belt loads at the time of occurrence of the Peak neck compression (Fz) were less than 45% of the passenger ATDs\u2019 respective Peak lap belt load recorded for two of the three tests. At the time of Peak neck flexion (My), the lap belt loads were less than 45% of the Peak lap belt load for all three tests. Shoulder belt loads for both driver and passenger ATDs at the time of occurrence of the Peak neck Fz and My were 37\u201358% of the ATDs\u2019 respective Peak shoulder belt loads for two of three tests (load cells malfunctioned in the driver\u2019s shoulder belt in test B190043 and passenger\u2019s shoulder belt in test B180220).\nDiscussion\nThe three rollover tests of 1998\u20131999 Ford Explorer SUVs analyzed in the present study represent a unique dataset evaluating occupant dynamics in rollover crashes as represented by Hybrid III ATDs. To the authors\u2019 knowledge, this study represents the first published test series of full-scale rollover crashes of a contemporary SUV with time synchronized sensor output from ATD neck transducers, roof rail accelerometers, an onboard high speed clock, and high speed external and internal video cameras.\nATD Biofidelity\nThe biofidelity of the Hybrid III ATD in rollover crash conditions is a significant limitation of this study as well as other published rollover studies1,15; however, ATD sensor data may be carefully evaluated within such limitations to yield objective insights. The ATDs used in this study were originally designed as a measurement tool in frontal crash tests, not rollovers. Thus, the Hybrid III ATD neck was designed and tested primarily to simulate dynamic head motion in the sagittal plane (neck flexion and extension).8,10 The Hybrid III ATD neck was not specifically designed to produce a biofidelic response under axial compression loading, typical of the neck loads resulting from the head-to-roof contacts observed in this study. Researchers have shown that with an axially directed impact to the crown of the ATD head, the ATD lower neck sensor records essentially the same magnitude axial force as the upper neck load cell, whereas the human cadaver neck lower load cell records a much reduced load magnitude compared to the upper load cell.24,29\u00a0Also, the Hybrid III ATD necks are neither frangible nor viscoelastic; therefore, they cannot accurately simulate individual vertebral displacement and\/or fracture.\nNotwithstanding the biomechanical differences between the necks of human cadavers and ATD mechanical necks, the Fz measurements of the upper load cell of a restrained Hybrid III dummy may provide a reasonable estimate of the initial input load to a restrained occupant\u2019s head when it contacts the roof in a rollover crash under similar dynamic conditions (e.g., impact velocity, impact duration). Given that the mass of a Hybrid III dummy head reasonably mimics that of the comparable human (50th percentile male in the present study), the inertial effect of the dummy head should not confound Fz readings from its upper neck load cell.\nIn all three tests within the present study, Fz values for ATD axial neck load were evaluated solely from the upper neck load cells, thereby taking into consideration, as much as possible, the significant differences in axial stiffness between the ATD and cadaver human neck (the lower neck Fz sensor was not evaluated). These upper neck Fz sensor readings were interpreted relative to the head impact loads in cadaver tests under similar dynamic conditions (described below). Given that the Hybrid III ATD neck was specifically designed to be biofidelic in dynamic, sagittal plane bending (flexion and extension) loads, neck flexion loads (My) were evaluated in this study from the lower neck load cell of driver and passenger ATDs. Lateral bending loads (Mx) were recorded and reported, but not interpreted in this study due to the lack of available biomechanical tolerance data for the human spine when subjected to lateral bending. Additional research in this important area, for both rollover and side impact crashes, is needed.\nInjury Tolerance of the Human Cervical Spine\nSemantic confusion exists in the published literature regarding cervical spine tolerance or \u201cfailure load.\u201d Some investigators have reported cervical spine \u201cfailure loads\u201d resulting from impacts to cadaver heads as the output force measured at the lower cervical spine, thereby neglecting the kinetic energy dissipation associated with vertebral fracture and\/or subluxation above the position of the sensor as well as the viscoelastic response of the intervertebral discs.19,20 Other studies have reported both the input and output loads in cadaver tests of the cervical spine.23\u201326 These data provide a quantitative comparison of the significant differences between the input loads measured at the cadaver head versus what might be more appropriately referred to as the residual force magnitude that is recorded at the base of the cervical spine segment. No experimental cadaver work has measured and\/or estimated the input loads at the superior aspect of the human neck, which is the site of the upper neck load cell in the Hybrid III dummy, when the head is subjected to an axial impact on the crown of the head. Additional research in this area is needed.\nThe confusion of terms regarding cervical spine failure loads in the published literature has a significant bearing on the proper interpretation of Hybrid III ATD axial neck load sensor output in controlled rollover tests such as the present study. Bahling et\u00a0al. utilized 2000\u00a0N as a neck sensor Fz threshold for \u201cpotentially injurious impacts (PII)\u201d in rollovers, which is in the range of the published residual force magnitude recorded at the lower cervical spine following an injurious impact to the cadaver head.1,19 In contrast, other investigators reported the actuator (input) force at the head that was required for cervical spine injury in axial compression.24 In interpreting the ATD neck sensor data in this study, an Fz value of 7000\u00a0N was utilized as a threshold of \u201cprobable (spinal) column injury,\u201d which is in the range of the published head impact loads in cadaver studies. A value of 150\u00a0N\u00a0m was used as the threshold for probable column injury for interpretation of My readings from the lower neck sensor. In reality, the true axial compressive failure load of the human cervical spine is likely slightly lower than that measured by the head input loads in cadaver tests (due to inertial effects of the cadaver head), yet substantially greater than the lower cervical spine force data reported in the same cadaver tests. Thus, the real world relevance of threshold values used for both PII and \u201cprobable column injury\u201d as a predictor of human injury risk must be tempered, consistent with the known biofidelity limitations of the ATD neck (described previously) and the wide variation in injury tolerance of the human spine, as described below.\nThe biomechanical literature suggests that the tolerance of the human cervical spine to serious injury is influenced by several mechanical factors, including individual tolerance variations due to age and gender, load magnitude and loading rate, pre-alignment (initial head-to-neck position), and end conditions of the cervical spine. Pintar et\u00a0al.24 reported that serious (AIS 3+) cervical spine injury under axial compression loading conditions occurred at 5856\u00a0N with a loading rate of 3\u00a0m\/s (for a 67-year-old female cadaver) and 11,242\u00a0N with a loading rate of 8\u00a0m\/s (for a 50-year-old male cadaver). In the present study, none of the near-side, driver ATD sensors recorded upper neck Fz values exceeding even 2000\u00a0N neck compression, and only one far-side passenger dummy (Test B190042) exceeded a neck sensor reading of 5856\u00a0N compression. Controlling for loading rate, age, and gender in a sample of 25 human cadaver head-neck compression tests, Pintar and Yoganandan27 reported that compressive tolerance varied from 7\u00a0kN in the young (third decade) to 2\u00a0kN in the very old (ninth decade). \u201cFailure loads\u201d (recorded at the lower cervical spine) ranged from 2\u00a0kN at quasi-static loading rates to 5\u00a0kN at dynamic loading rates (8\u00a0m\/s). Failure tolerance for the male population was 25% higher than the female population (without regard to age and rate of loading). The present study recorded roof rail-to-dummy head contacts in the range of 5\u00a0m\/s (11.2\u00a0mph) in the first ground strike of the SUV rollovers.4 Thus, the roof rail-to-head impact speeds in the present study were within the range of loading rates to cadaver and Hybrid III ATD heads in the studies by Pintar et\u00a0al.24,27\nPre-alignment of the head and neck complex when struck by (or when it strikes) the roof is a key variable in determining whether catastrophic injury will occur, and if so, the specific type of injury that will be sustained. Published biomechanical studies suggest that cervical spine pre-flexion yields a greater incidence of lower cervical compression and burst fractures than neutrally positioned spines.24,25 Restrained occupants in rollover collisions are typically pre-flexed, due to the initial locking of their belt restraint system, and thus are at higher risk of cervical spine injury in the presence of an intruding roof structure when compared to unrestrained occupants. This higher risk of roof impact for restrained vs. unrestrained occupants in rollovers was confirmed with U.S. field accident data.9 The degree of pre-flexion of the ATD necks prior to head-to-roof contact in the present study was not determined because the onboard cameras were positioned behind the ATDs during the rollover tests. The seat belt load cells (i.e., excluding the two sensors described previously that malfunctioned) recorded continuous tensile loads in both lap and shoulder belts throughout the test interval for both driver and passenger ATDs in all three tests. This belt load data indicated that the lap\u2013shoulder belts locked and remained locked during the first 1000\u00a0ms test interval for all ATDs in all three tests.\nNightingale et\u00a0al.20 reported on the influence of boundary or end conditions of the cervical spine relative to specific injury types. When the head was unconstrained and free to translate and\/or rotate away from the applied load, no cervical injury was sustained, despite high input loads to the head. In \u201cfull constraint\u201d conditions, with the head pre-flexed along its stiffest axis (i.e., the removal of the normal cervical lordosis), buckling and burst fractures were noted. With rotational constraint (e.g., when the neck flexes forward to the point where the chin is against the sternum), bilateral locked facets typically occurred. Using cadaver spine specimens, Pintar et\u00a0al.26 determined that the average peak My magnitude resulting in \u201cmajor\u201d neck injury was 97\u00a0N\u00a0m at the specific site of injury in the cervical spine (i.e., not positioned as inferior as the lower neck load cell of the Hybrid III ATDs in the present study). The peak axial head impact force ranged from 3000 to 9700\u00a0N, with the peak flexion bending moment at the injured level ranging from 19 to 169\u00a0N\u00a0m. Thus, the degree of constraint imposed by the contacting surface, such as an intruding roof, can be a major determinant for cervical spine injury. In all three tests of the present study, the far-side, passenger ATDs recorded lower neck flexion (My) loads that exceeded 175\u00a0N\u00a0m. None of the near-side, driver ATD lower neck sensors recorded peak My loads exceeding 110\u00a0N\u00a0m. No dynamic test data for cervical spine tolerance in lateral bending was identified; therefore, no interpretation of the peak Mx sensor outputs for any of the ATDs was made.\nThe biomechanical impact environment to which the restrained ATDs were subjected in the present study was shown to be comparable to that of published cadaver studies on the basis of the following: head impact load magnitude and direction, loading rate, pre-alignment of the ATD head and neck, and degree of rotational head constraint. Thus, a comparison of the ATD sensor outputs for upper load cell Fz and lower load cell My values to probable column injury values of 7000\u00a0N and 150\u00a0N\u00a0m, respectively appears scientifically reasonable, subject to the limitations described above.\nRoof Crush as a Correlate or Cause of Injury\nTwo opposing views exist in the published literature regarding whether roof crush causes serious injury or whether it is simply associated or correlated with serious injury. One group of investigators has concluded that restrained occupants receive catastrophic head and neck injury from diving into the roof and making head-to-roof contact while the top of the vehicle\/roof is striking\/hitting the ground during a rollover.1,3,5,15,16 Other investigators have concluded that serious neck injuries to restrained occupants are directly caused by the dynamic intrusion of the roof structure into the occupant\u2019s survival space during a rollover crash.12,13,28 The present study reports quantitative evidence of the temporal relationship between dynamic roof deformation, lap\u2013shoulder seat belt loads, and restrained ATD neck loads, which provides further clarification in this scientific debate.\nObjective roof\/pillar deformation occurred prior to the occurrence of Peak neck loads (Fz, My, Mx) for both driver and passenger ATDs in all three rollover tests. Prior to the occurrence of Peak neck loads, the driver and passenger ATD heads contacted the roof one or more times in all three tests; the magnitude of neck sensor responses ranged from 3 to 57% of the respective dummies\u2019 Peak neck loads. Thus, the restrained driver and passenger ATDs did, indeed, \u201cdive\u201d into the roof while the roof was in contact with the ground, resulting in Local Maximum neck load values, which in no instance exceeded 57% of the Absolute Maximum (Peak) neck load. In particular, the \u201cdiving\u201d neck loads (Local Maxima) for the far-side, passenger ATDs were only 2\u201313% of the Peak neck loads (Fz and My) for all three tests (Table\u00a02, Fig.\u00a04).\nIn each test, the Peak neck loads of the far-side, passenger ATD occurred within 10\u00a0ms following the time of maximum objective roof crush for all three tests, which is consistent with published inertial effects of the ATD head in transmitting an axial force to the lower neck sensors.24 In contrast, Peak neck loads for the near-side, driver ATD occurred up to 65\u00a0ms following the time of occurrence of objective roof crush for all three tests. The onboard cameras also revealed observable roof crush into the far-side versus the near-side occupant compartment during the time of the peak neck loads, which was completely consistent with the timing and higher neck load magnitudes of the passenger, compared to driver, ATD for all three tests.\nThe lap and shoulder belt load profiles for both driver and passenger ATDs provide further insights in the scientific debate regarding roof crush as a correlate versus a cause of serious injury. During the time interval of the Peak neck loads in the passenger ATDs for all three tests, the shoulder belt load, which should increase in magnitude as it resists torso augmentation, instead consistently decreased by 65\u201385% to only 44\u00a0N in some cases. The lap belt showed the same reduction in load during the time period that Peak neck load was recorded; it was reduced by 58% to approximately 156\u00a0N (Table\u00a04, Figs.\u00a08a and 8b). The belt load reductions occurred at the same time that on-board cameras clearly recorded the passenger ATD hanging upside down in the lap\u2013shoulder belt and the roof crushing into the occupant space. This time of \u201cobservable\u201d roof crush also occurred contemporaneously with the \u201cobjective\u201d roof crush predicted by the accelerometers mounted on the SUV roof rails. The belt load that was recorded during each Peak neck load event for the far-side, passenger ATD was less than 50% of the Peak belt load in all three tests (Table\u00a04). The shoulder and lap belt load cells indicated that the belts were off-loaded (i.e., loading decreased) as the roof crushed down on the ATD head and pushed the ATD back toward the seat cushion, away from the shoulder and lap belts, at the time of Peak neck loads (Fig.\u00a09). Additionally, the deformation of the passenger (far-side) B-pillar lowered the upper attachment point (i.e., D-ring) for the belt, causing slack in the shoulder belt with a concomitant reduction in shoulder belt load.\nFigure\u00a09The shoulder and lap belts are off loaded (i.e., load decreases) as the roof crushes down on the passenger head and pushes the dummy back toward the seat cushion, away from the shoulder and lap belts, at the time of injurious neck loads\nRepeatability and Reliability\nThe FMVSS 208 dolly rollover test methodology has been criticized for its alleged lack of reliability and\/or repeatability.15 The results of this study, however, demonstrated that the 208 rollover tests are very reliable when viewed from a vehicle-based, occupant protection frame of reference. Test-to-test comparisons of the FMVSS 208 rollover results revealed remarkable similarity in predicting the time of occurrence of Peak neck loads for both driver and passenger ATDs. The Peak neck Fz occurred in all three tests at 530\u00a0\u00b1\u00a015\u00a0ms for the driver ATD (Fig.\u00a010 and Table\u00a03) and 730\u00a0\u00b1\u00a015\u00a0ms for the passenger ATD (Table\u00a03). The Peak neck My occurred in all three tests at 530\u00a0\u00b1\u00a018\u00a0ms for the driver ATD (Fig.\u00a011 and Table\u00a03) and 750\u00a0\u00b1\u00a021\u00a0ms for the passenger ATD (Table\u00a03). The Peak value for Mx occurred in all three tests at 530\u00a0\u00b1\u00a018\u00a0ms for the driver ATD and 770\u00a0\u00b1\u00a013\u00a0ms for the passenger ATD. These small variations in time of occurrence of roof\/pillar deformation and Peak neck loads are particularly remarkable given that (1) the data came from six different dummies and three different vehicles tested on three different days, (2) the differences in the time of occurrence of the Peak neck loads was \u226420\u00a0ms in an overall time interval of 1000\u00a0ms, and (3) each 1000\u00a0ms time interval included either a sampling rate of 20,000 data points (B190043 and B190042) or 12,500 (B180220).\nFigure\u00a010The absolute maximum value for upper neck Fz occurred in all three tests at 530\u00a0\u00b1\u00a015\u00a0ms for the driver dummyFigure\u00a011The absolute maximum value for lower neck My occurred in all three tests at 530\u00a0\u00b1\u00a018\u00a0ms for the driver dummy\nQuantitative analyses were undertaken to determine the significance probability of each pairwise comparison. Restated, the probability that the similarity of the maximum and minimum values of any two tests were related to something other than chance was analyzed (e.g., B190042-Fz vs. B190043-Fz, B190042-Fz vs. B190220-Fz, and B190043-Fz vs. B190220-Fz). Appendix 1 examines this issue and computes the probability for each pair of test metrics. This analysis revealed that there is a 93.1\u201398.6% probability that the differences in time occurrence of Peak neck loads between these results are NOT due to random chance alone. Stated more simply, there is a less than 7% probability that these differences are coincidental.\nValidity\nThe constellation of driver vs. passenger neck loads measured in this test series of SUV rollovers was consistent with certain incidence trends of catastrophic head and neck injuries observed in real-world rollovers with restrained front seat occupants.9,21,28 None of the peak neck loads recorded in the near-side, driver ATD neck sensors exceeded the threshold values for probable column injury, which were used in this study. In contrast, the far-side, passenger ATDs recorded Peak neck loads that consistently exceeded the threshold for probable column injury due to neck flexion, My. These findings are consistent with 1992\u20131998 data obtained from NHTSAs National Accident Sampling System (NASS) database, which showed serious spinal injuries were more frequent for the far-side occupants (compared to the near-side), where the source was most often coded as roof, windshield, and interior.21\nA probability of two common catastrophic spinal column injuries was predicted by the passenger ATD neck sensors in all three rollover tests. A combination load of compression and of flexion is associated with burst fractures and wedge compression fractures. This load profile was recorded by the upper and lower neck sensors in the far-side passenger ATD in B190042. Bilateral facet dislocation injuries have been associated with sagittal plane flexion.17 The flexion moments recorded by the passenger ATD lower neck sensors in tests B180220 and B190043 exceeded the threshold values for probable spinal column injury by 40\u2013240%. Thus, the results of this rollover test series suggest that the FMVSS 208 dolly rollover test may be a valid predictor of serious spinal injuries to restrained occupants in real-world rollovers.\nThe results of this study provide a unique data set that furthers understanding of probable spinal column injury mechanisms within a rollover crash environment. Such information may assist the scientific community and automotive engineers in recommending and designing appropriate intervention strategies to mitigate morbidity and mortality in rollover crashes. Moreover, these data may inform government agencies in formulating appropriate public safety policy to improve rollover crash protection for restrained occupants.\nConclusions\nDuring each of the three FMVSS 208 dolly rollover tests of Ford Explorer SUVs, the far-side, passenger ATDs exhibited Peak neck compression and flexion loads, which indicated a probable spinal column injury in all three tests. In those same tests, the near-side, driver ATD neck loads never predicted a serious injury. In all three tests, objective roof\/pillar deformation occurred prior to the occurrence of Peak neck loads (Fz, My) for far-side, passenger ATDs, and Peak neck loads were predictive of probable spinal column injury. The production lap and shoulder seat belts in the SUVs, which restrained both driver and passenger ATDs, consistently allowed ATD head contact with the roof while the roof was contacting the ground during this 1000\u00a0ms test series. Local peak neck forces and moments were noted each time the far-side, passenger ATD head contacted (\u201cdived into\u201d) the roof while the roof was in contact with the ground; however, the magnitude of these local maxima was only 2\u201313% of Peak neck loads in all three tests. \u201cDiving-type\u201d neck loads were not predictive of spinal column injury for either driver or passenger ATD in any of the three tests. When viewed from a vehicle-based, occupant protection frame of reference, the existing FMVSS 208 dynamic rollover test is a repeatable, reliable, and valid test method to evaluate the risk of probable spinal column injury in rollover crash environments.","keyphrases":["suv","roof crush","crash test","restraint system","spinal injury"],"prmu":["P","P","P","P","P"]}
{"id":"Breast_Cancer_Res-5-6-314403","title":"Transforming growth factor beta-regulated gene expression in a mouse mammary gland epithelial cell line\n","text":"Background Transforming growth factor beta (TGF-\u03b2) plays an essential role in a wide array of cellular processes. The most well studied TGF-\u03b2 response in normal epithelial cells is growth inhibition. In some cell types, TGF-\u03b2 induces an epithelial to mesenchymal transition (EMT). NMuMG is a nontransformed mouse mammary gland epithelial cell line that exhibits both a growth inhibitory response and an EMT response to TGF-\u03b2, rendering NMuMG cells a good model system for studying these TGF-\u03b2 effects.\nIntroduction\nTransforming growth factor beta (TGF-\u03b2) is the prototype of a large family of signaling molecules with more than 40 members. TGF-\u03b2 signals through the type I receptor (T\u03b2RI) and the type II receptor (T\u03b2RII), and the Smad transcriptional regulators, as well as many other signaling pathways [1]. TGF-\u03b2 plays an essential role in a wide array of cellular processes, including early embryonic development, cell growth, differentiation, motility, and apoptosis. In nontransformed epithelial cells, the most studied TGF-\u03b2 response is growth inhibition. In some cell types, TGF-\u03b2 induces an epithelial to mesenchymal transition (EMT). TGF-\u03b2 plays an important, albeit complex, role in tumorigenesis. In many tumors of epithelial origin, cells become resistant to TGF-\u03b2-mediated growth inhibition. In parallel, TGF-\u03b2 induces EMT in tumor cells, and increases tumor invasion and metastasis [2]. NMuMG is a nontransformed mouse mammary epithelial cell line that exhibits both a growth inhibitory and EMT response to TGF-\u03b2 [3]. These properties render NMuMG cells a suitable model system for studying these TGF-\u03b2 actions.\nPrevious gene expression profiling studies of TGF-\u03b2 action on cell lines utilized cDNA microarrays to characterize extracellular matrix-related genes in human dermal fibroblasts. The upregulation of these genes by TGF-\u03b2 was Smad mediated [4]. Another study examining the genetic programs of epithelial cell plasticity directed by TGF-\u03b2 utilized the human keratinocyte HaCaT cell line [5]. In this study, HaCaT cells were treated for up to 4 hours, and microarray gene expression profiling was performed using an array of 16,580 human cDNAs. The expression of 728 known genes (10% of those analyzed) was regulated by TGF-\u03b2 within 4 hours of treatment.\nIn the present study, we examined TGF-\u03b2 regulation of gene expression in the NMuMG epithelial cell line. NMuMG cells were chosen as a model system because the results are more relevant to our studies employing transgenic mouse mammary tumor models, and because NMuMG cells are a well-established model system for studying both growth inhibition and EMT induced by TGF-\u03b2 [3]. EMT represents a complex physiological process that includes dissolution of adherens junctions, a change to spindle-like cell morphology, cytoskeletal reorganization, increased cell motility, loss of epithelial markers, and induction of mesenchymal markers. EMT thus includes several cellular responses that may occur through different mechanisms and at different time points after TGF-\u03b2 treatment. We consequently examined changes in gene expression at 1, 6, and 24 hours after TGF-\u03b2 treatment.\nIn agreement with previous studies with HaCaT cells [5], approximately 10% of the genes examined were regulated by TGF-\u03b2 in our microarray studies that queried more than 15,000 mouse cDNAs. Our study demonstrates that by 1 hour, TGF-\u03b2 suppresses the expression of multiple genes that play key roles in regulating cell cycle progression. At 6 and 24 hours, TGF-\u03b2 enhances the expression of multiple genes involved in regulating cell shape and cell adhesion, and therefore may mediate TGF-\u03b2-induced EMT. Additionally, several novel TGF-\u03b2-regulated genes were identified that may play important roles in TGF-\u03b2 responses.\nMaterials and methods\nCell culture and treatment\nNMuMG cells were cultured in Dulbecco's modified Eagle's medium containing 10% serum and 10 \u03bcg\/ml insulin, until reaching 70\u201380% confluency. The cells were treated with 4 ng\/ml TGF-\u03b21 for 1, 6 or 24 hours. Untreated cells were used as reference samples at each time point, and were referred to as controls in the analyses. Four independent replicates of each experiment were performed. Cell morphology was examined under phase contrast using an Olympus CK40 microscope (Melville, NY, USA).\nGrowth inhibition assay\n3H-thymidine incorporation assays were performed using cells that had been treated for 24 hours with TGF-\u03b2. The cells were pulsed with 3H-thymidine for the final 2 hours of the treatment period. 3H-thymidine incorporation was quantitated using a LS 6500 Multi-Purpose Scintillation Counter (Beckman). Four replicates of each experiment were performed, and each 3H-thymidine incorporation experiment was performed in triplicate.\nRNA sample preparation\nNMuMG cells were lysed and homogenized in Trizol reagent (Invitrogen, Carlsbad, CA, USA), and total RNA was prepared according to the manufacturer's instructions. Two rounds of extraction were performed to isolate pure, high-quality total RNA. Thirty micrograms of total RNA from each sample were used for reverse transcription and cyanine (Cy)-dye incorporation.\ncDNA microarray hybridization\nThe National Institutes of Aging (NIA) 15,000 cDNA microarray was printed on three contiguous slides by the Vanderbilt Microarray Shared Resource. The mouse 15,000 NIA microarray contains 15,247 genes of known and unknown function, developed at the NIA. Detailed descriptions of the gene array list, microarray hardware and procedures are available online .\nThe reference RNA samples from nontreated NMuMG cells were labeled with Cy5, and the test RNA samples from NMuMG cells treated with 4 ng\/ml TGF-\u03b2 for 1, 6, and 24 hours were labeled with Cy3. The Cy3-labeled and Cy5-labeled samples were hybridized simultaneously to the same array. Four independent replicates of TGF-\u03b2 treatment, RNA isolation and labeling, and microarray hybridization were performed. To estimate 'system noise' that may be due to differences in Cy-dye labeling between samples, we performed a 'self-to-self' hybridization, where the same control sample was labeled separately with Cy3 and Cy5, and then hybridized.\nOligo dT for reverse transcription was synthesized by Invitrogen. SuperScript II reverse transcriptase (Invitrogen) was used. A Qiagen PCR purification kit was used for purification of probes (Qiagen, Valencia, CA, USA). Slides were prehybridized to eliminate nonspecific interactions in prehybridization solution: 1% BSA, 5 \u00d7 sodium chloride\/sodium citrate buffer (SSC), 0.1% SDS at 65\u00b0C for 45 min. Hybridizations were performed at 65\u00b0C for 14\u201316 hours in a humidified hybridization chamber (Corning, Acton, MA, USA). After hybridization, the slides were washed once each in solutions 1\u20133 (wash solution 1, 2 \u00d7 SSC, 0.1% SDS; wash solution 2, 1 \u00d7 SSC; wash solution 3, 0.1 \u00d7 SSC) for 5 min at 55\u00b0C with gentle stirring\/agitation. Washed slides were centrifuged in a conical tube for 5 min at 1600 rpm to dry them. Microarray slides were scanned using a Genepix 4000 B scanner (Axon Instruments, Union City, CA, USA) at a resolution of 10 \u03bcm, and the original data files were generated by GenePixPro software (version 4.0; Axon Instruments).\nData analysis\nA merged 15,000 data file was constructed by combining the three original 5000 data files generated from the three contiguous slides on which the total 15,000 gene clones were printed. The GenBank accession number and the full annotation gene name for each gene were incorporated. Data analyses were performed using the GeneSpring 4.1.5 software package (Silicon Genetics, Redwood City, CA, USA).\nThe normalization for the two-color cDNA microarray data was designed as follows. The net signal intensity in each channel (Cy3 or Cy5) was determined by subtracting the local background from signal intensity values. Each gene's measured intensity was divided by its control channel value (the reference RNA sample channel of Cy5). A Cy3\/Cy5 ratio represents the relative abundance of a target transcript in TGF-\u03b2-treated and nontreated samples respectively. When the control channel value was below 1000.0, the data point was considered too weak and was discarded. Each sample was normalized to the 50th percentile of all measurements. The bottom 10th percentile was considered background, and was subtracted from all the other values.\nEach experimental result was interpreted as the average of the four replicates of each time point. Time-course gene expression profiles were required to be present in two of the four datasets, and to be present in at least two of the three time points. A signal to control channel ratio > 2.5 and a probability t test P < 0.05 was defined as upregulation by TGF-\u03b2. A signal to control channel ratio < 0.4 and a probability t test P < 0.05 was defined as downregulation. The annotated gene lists were constructed from all genes represented in the normalized dataset. The differentially expressed genes were classified into functional subgroups using Simplified Ontology in GeneSpring. For each pathway, pathway images were saved as GIF files and loaded into GeneSpring. Each pathway element was identified in the normalized data.\nNorthern hybridization\nAll cDNAs of the NIA mouse 15,000 microarray were cloned into the NotI\/SalI sites of the ampicillin-resistant pSPORT1 vector (Invitrogen). The insert sizes ranged from 0.5 to 3 kb, with an average insert size of 1.5 kb. The Escherichia coli host strain used was DH10B. Plasmid DNA was isolated using the High Pure Plasmid Isolation Kit (Roche, Indianapolis, IN, USA). The inserts were verified for their correct annotation by DNA sequencing. The reverse pSPORT1 primer was used as the sequencing primer. To prepare cDNA probes, cDNA inserts were amplified using pSPORT1 primers: forward, GTTTTCCCAGTCACGACGTTG; reverse, TGA-GCGGATAACAATTTCACACAG.\nPlasmid DNA was used as the template for PCR amplification under the following conditions: initial denaturation at 95\u00b0C for 1 min; then 40 cycles of denaturation at 94\u00b0C for 30 s, annealing at 54\u00b0C for 30 s, and extension at 72\u00b0C for 3.5 min. A final extension was performed at 72\u00b0C for 7 min. To examine the quality and quantity of PCR products, they were separated on 1.2% agarose gels. Specific cDNA bands were recovered from the gels using QIAEX II Gel Extraction Kits (Qiagen). Seventy-five nanograms of cDNA was labeled with 32P using a Random Primed DNA Labeling Kit (Roche).\nFor northern blot analysis, 10 \u03bcg total RNA was separated by electrophoresis on a 1% agarose, 0.66 M formaldehyde gel and transferred to Hybrid Nylon transfer membranes (Amersham Biosciences, Piscataway, NJ, USA). After transfer, the membrane was UV cross-linked (Stratalinker; Stratagene, La Jolla, CA, USA). All cDNA probes were hybridized overnight at 42\u00b0C in ULTRAhyb solution (Ambion, Austin, TX, USA). Each filter was washed twice for 5 min in 2 \u00d7 SSC and 0.1% SDS at 42\u00b0C, followed by two 15 min washes each in 0.5 \u00d7 SSC and 0.1% SDS at 42\u00b0C, followed by two 15 min washes in 0.1 \u00d7 SSC, 0.1% SDS at 42\u00b0C. Equal loading of gel lanes was confirmed by hybridization with the house keeping gene 1B15. Results were visualized and quantitated using a FUJIFILM-FLA-5000 Phosphoimager (Fuji Photo Film Co. Ltd, Stamford, CT, USA).\nResults\nBiological and gene expression responses of NMuMG cells to TGF-\u03b2\nNMuMG cells react to TGF-\u03b2 treatment with both an EMT response and a growth inhibitory response (Fig. 1). These characteristics render NMuMG cells an appropriate model system to study the signaling pathways mediating these TGF-\u03b2 effects. To examine TGF-\u03b2 regulation of genes involved in EMT and cell cycle control, we treated NMuMG cells with TGF-\u03b2 for 1, 6, or 24 hours, isolated the total RNA, and performed gene expression profiling by cDNA microarray. The relative transcript abundance was expressed as Cy3\/Cy5 ratios of signal intensities after background subtraction in each channel. Data analysis and quality control procedures are described in detail in Materials and methods. TGF-\u03b2 treatment resulted in the upregulation or downregulation of 939 genes out of the 15,247 genes examined. Subtracting redundancy or signals too weak to score, this represents approximately 10% of the genes analyzed.\nThe time course of TGF-\u03b21-dependent changes in gene expression demonstrates a progressively greater alteration of gene expression over time (Fig. 2). In general, TGF-\u03b21 regulation of cell cycle-related genes occurred with an onset of less than 1 hour, while TGF-\u03b21 regulation of EMT-related genes occurred with a later onset, with changes first becoming apparent at 6 or 24 hours (see later).\nFigure 3 and Table 1 present the distribution of downregulated genes and of upregulated genes, and the sum of upregulated and downregulated genes at each time point. The total number of differentially expressed genes at each time point does not differ significantly. The 6 and 24 hour time points shared more TGF-\u03b21-regulated genes than were shared between the 1 and 6 hour time points, or the 1 and 24 hour time points. Full lists of TGF-\u03b2-regulated genes at each time point can be accessed on our website .\nFunctional class distribution of the novel and known TGF-\u03b21-regulated genes\nOf the 939 genes regulated by TGF-\u03b21, approximately one-third (350 genes) can be classified into functional groups. The classified genes were further separated into two tables of genes previously reported or not previously reported to be regulated by TGF-\u03b2. This was determined by performing a Medline search using the gene name and TGF-\u03b2 as keywords. Tables of the complete dataset with each gene in its functional category are presented on our website . Tables 1 and 2 present some selected examples of known or unknown TGF-\u03b2-regulated genes, respectively. The genes involved in cell cycle regulation and cell adhesion (see later) are included.\nNorthern analysis verification of microarray data\nTo validate the microarray results, we subjected nine of the novel TGF-\u03b2-regulated genes to northern analysis. Of the nine genes examined, seven exhibited TGF-\u03b21-induced changes in gene expression that matched those observed in the microarray experiments. The seven novel TGF-\u03b21-regulated genes verified by northern analysis include Akt, mCalpain, RhoB, PR53, actinin 3, Ikki (Fig. 4) and the IQ motif-containing GTPase-activating protein 1 (IQGAP1). IQGAP1 showed upregulation in NMuMG cells after 1, 6 and 24 hours of treatment with TGF-\u03b2, supporting the microarray data. As reported by other investigators [6], we detected multiple IQGAP1 transcripts. The IQGAP1 northern blot is thus not shown in Fig. 4. Akt and RhoB were reported previously to be regulated by TGF-\u03b2 at the translational level [7,8], but not at the transcriptional level as we show here. Some of the TGF-\u03b21-induced changes in gene expression were also verified by real-time RT-PCR (data not shown). In further support of the reliability of the microarray experiments, genes that were represented multiple times in the array as different clones exhibited identical expression profiles (examples can be found in the supplementary tables at our website: . In addition, our expression profiling results are consistent with previously reported expression patterns of TGF-\u03b21-responsive genes [1,5].\nExamination of cell cycle control and cell adhesion-related genes by microarray\nThere are multiple ways to explore the biological significance of results obtained in microarray experiments. One may organize the genes into functional groups or signaling pathways to examine the regulation of clusters of genes. We were interested in identifying TGF-\u03b2-regulated genes involved in the induction of EMT and cell cycle arrest. In our study in NMuMG cells, the expression of many cell cycle regulators was altered by TGF-\u03b2. One hour after TGF-\u03b2 treatment, cyclin D2, cdk4, E2F5, cyclin A, and c-myc were downregulated. Six hours after TGF-\u03b2 treatment, cyclin D2, p107, E2F5, c-myc, and Id2 were downregulated. Twenty-four hours after TGF-\u03b2 treatment, cyclin D2, p107, E2F5, cyclin A, cyclin B, cyclin H, and Id2 were downregulated (Fig. 5). These data are consistent with these genes serving as mediators of TGF-\u03b2-induced growth arrest.\nThe microarray results also revealed upregulation of a number of genes that play a role in cell\u2013cell and cell\u2013matrix adhesion and EMT. At 1 hour, c-Jun, \u03b2-integrin, and IQGAP were upregulated. At 6 hours, actin, calpain, \u03b2-integrin, fibronectin, collagen, myosin light chain, \u03b1-actinin, \u03b2-catenin and IQGAP were upregulated. At 24 hours, c-Jun, actin, calpain, \u03b1-integrin, \u03b2-integrin, fibronectin, collagen, \u03b1-actinin, \u03b2-catenin and IQGAP were upregulated. The only gene in this category observed to be downregulated was E-cadherin at 6 hours (Fig. 6). The results are consistent with these genes playing a role in TGF-\u03b21-mediated changes in cell\u2013cell and cell\u2013substratum adhesion.\nOur results are consistent with previous studies, but we identified several novel TGF-\u03b2-regulated genes that could play important roles in both EMT and cell cycle regulation, including RhoB, mCalpain, actinin 3, IQGAP1, Ikki and protein phosphatase 2A (PP2A)-PR53.\nDiscussion\nThe mechanisms by which TGF-\u03b2 signals are not fully understood. In this regard, gene expression profiling has been used successfully by a variety of investigators to explore genetic events involved in TGF-\u03b2 signaling. Zavadil and colleagues detected genetic programs of epithelial cell plasticity directed by TGF-\u03b2 [5]. Verrecchia and colleagues identified novel TGF-\u03b2\/Smad gene targets in dermal fibroblasts [4]. Chen and colleagues found defective repression of c-myc in breast cancer cells, suggesting that this event is at the core of the TGF-\u03b2 growth arrest program [9]. Shen and colleagues discovered that the activity of the guanine exchange factor NET1 is essential for TGF-\u03b2-mediated stress fiber formation [10].\nIn the present study, we have selected the NMuMG nontransformed mouse mammary gland epithelial cell line as a model system to identify TGF-\u03b2-regulated target genes involved in the control of growth inhibition and EMT. To identify TGF-\u03b2-regulated genes, we employed a cDNA microarray that represents 15,247 genes of known and unknown function. We found that the expression of 939 of the genes was altered by TGF-\u03b2 regulation. This represents about 10% of the genes examined. Many of the genes identified were previously determined to mediate key biological responses to TGF-\u03b2. In addition, several novel TGF-\u03b2-regulated genes were identified. The present report provides a comprehensive view of the coordinated regulation of genetic programs induced by TGF-\u03b2 during the processes of cell cycle arrest and EMT. A remarkably large number of genes undergo rapid changes in expression after TGF-\u03b2 treatment. This report adds to the database of hundreds of genes that are regulated by TGF-\u03b2 [5]. In addition, we have identified several novel TGF-\u03b2-regulated genes, and have verified these results in northern blotting experiments.\nTable 1 presents previously identified TGF-\u03b2-regulated genes, many of which are known to play important roles in TGF-\u03b2 induction of EMT and cell cycle arrest. Briefly, in the cellular communication category, platelet-derived growth factor was upregulated by TGF-\u03b2. Multiple genes involved in cell adhesion were upregulated by TGF-\u03b2, including integrins (integrin \u03b13A, integrin \u03b2, integrin \u03b25), integrin-linked kinase, and the integrin ligand fibronectin. These results are consistent with TGF-\u03b2 activating integrin-dependent cell adhesion. TGF-\u03b2 upregulation of \u03b1-actinin, \u03b2-tropomyosin 2, and p120 catenin may play a role in the induction of EMT. RhoB was upregulated at 6 and 24 hours. RhoB is an endosomal small GTPase, and it functions as a negative regulator of integrin and growth factor signals [11]. Our previous results [8] and those of other workers [12] suggest a novel mechanism of tumor suppressive regulation by TGF-\u03b2, and implicate RhoB as a negative regulator of TGF-\u03b2 signal transduction.\nTGF-\u03b2 downregulation of the helix\u2013loop\u2013helix DNA binding protein Id2 and of the inhibitor of DNA binding Idb3 were previously identified as important events in TGF-\u03b2-regulated signaling. These proteins belong to a family of helix\u2013loop\u2013helix proteins that, in general, function as positive regulators of cell growth and as negative regulators of cell differentiation in many cell types [13].\nTGF-\u03b2 also regulates many genes involved in the progression of cancer. The proto-oncogene Akt is upregulated at 1 hour at the mRNA level. The Gro1 proto-oncogene is downregulated by TGF-\u03b2 at all the time points examined. The roles that these gene products play downstream of TGF-\u03b2 during tumorigenesis have not been extensively studied [14,15]. Well-established TGF-\u03b2 responses such as downregulation of c-myc and upregulation of the Jun oncogene were also observed.\nTGF-\u03b2 is a potent growth inhibitor of epithelial cells. TGF-\u03b2-mediated growth inhibition has been associated with effects on G1 phase cyclins, cyclin-dependent kinases (Cdks), Cdk-activating kinase, Cdc25A, and Cdk inhibitors. TGF-\u03b2 potently inhibits the activities of both the cyclin D\u2013Cdk4\/Cdk6 and cyclin E\u2013Cdk2 complexes, resulting in the hypophosphorylation of pRbs and in the decreased transcriptional activity of E2Fs. TGF-\u03b2 does not affect the expression levels of Cdk4, Cdk6, or Cdk2 in exponentially proliferating cells. TGF-\u03b2 decreases the expression levels of cyclin A, which is probably the result of cell cycle arrest in the late G1 phase. TGF-\u03b2 has been associated with upregulation of Cdk inhibitors p15Ink4B, p21Cip1, and p27Kip1. The c-myc proto-oncogene has been implicated in TGF-\u03b2-mediated growth inhibition. TGF-\u03b2 rapidly inhibits c-myc expression in a wide variety of cell types [1]. c-Myc downregulation is a key event in the TGF-\u03b2 program of growth inhibition [9]. Additional mechanisms mediating TGF-\u03b2-induced cell cycle arrest continue to be uncovered. In our study, examination of cell cycle genes demonstrated TGF-\u03b21-induced downregulation of cyclin D2, c-myc, Id2, p107, E2F5, cyclin A, cyclin B, and cyclin H. These data are consistent with these genes serving as mediators of TGF-\u03b2-induced growth arrest.\nOf the genes that could be placed into functional categories, the expression of the majority of the genes was not previously reported to be regulated by TGF-\u03b2. Table 2 presents some examples of genes that we have identified in this study to be regulated by TGF-\u03b2 that are involved in cell signaling. Gadd45g was upregulated. Gadd45g specifically interacts with the Cdk1-cyclin complex, B1 resulting in inhibition of kinase activity [16]. The IQGAP1 gene is a target molecule of Cdc42 and Rac1. IQGAP negatively regulates the E-cadherin\u2013catenin complex-based cell\u2013cell adhesion by dissociating \u03b1-catenin and\/or \u03b2-catenin, a key molecule that links the E-cadherin-catenin complex to the actin cytoskeleton. These data suggest that TGF-\u03b2 upregulation of IQGAP may be one of the mechanisms by which TGF-\u03b2 regulates cell\u2013cell adhesion [17]. Calpains are a family of Ca2+-dependent intracellular cysteine proteases, including the ubiquitously expressed micro-calpains and m-calpains. Calpain activity may be regulated by targeting to specific adhesion-related substrates [18].\nOur results also demonstrate that TGF-\u03b2 regulates multiple genes involved in regulation of the actin cytoskeleton. Some of these components include actinin \u03b13, the Arp2\u2013Arp3 complex, and myosin light chain 2. Four different isoforms of tropomyosin were upregulated. Actinin \u03b13 is a calcium-dependent cytoskeletal protein with an actin-binding domain. Actinin \u03b13 is associated with adherens junctions and desmosomes, together with E-cadherin, \u03b1-catenin, \u03b2-catenin and \u03b3-catenin, vinculin, \u03b1-actinin, and polymerized actin. The genes \u03b1-actinin, myosin light chain 1\/myosin light chain 2, and tropomyosin are components of myofilaments involved in cell contractility and motility.\nDuring our studies we identified several genes not previously reported to be regulated by TGF-\u03b2 that are regulated by TGF-\u03b2 in NMuMG cells. These novel TGF-\u03b2-regulated genes include IQGAP1, mCalpain and actinin \u03b13, which were discussed earlier, and also Ikki and PP2A-PR53. For the first time, we show in the present study that Ikki is downregulated by TGF-\u03b2 within 1 hour of treatment, and that downregulation persisted for at least 24 hours. NF-\u03baB activation depends on the phosphorylation and degradation of its inhibitor protein, IkB. The phosphorylation of IkB\u03b1 is initiated by an IkB kinase complex that includes a catalytic heterodimer composed of IkB kinase 1 (IKK-1) and IkB kinase 2 (IKK-2) [19]. A novel inducible IkB kinase (IKKi) has recently been described. IKKi is functionally and structurally distinct from the constitutively expressed IKK-1 and IKK-2. In contrast to TGF-\u03b2, tumor necrosis factor alpha stimulated IKKi expression, an effect that persisted for at least 24 hours [20]. The biological significance of TGF-\u03b2 downregulation of IKKi is currently under investigation. PP2A is a multifunctional serine\/threonine phosphatase that is critical to many cellular processes, including cell cycle regulation and signal transduction. PR53 belongs to one of the families of PP2A regulatory subunits. The significance of TGF-\u03b2 regulation of PR53 expression is unknown [21].\nAltogether, our results are consistent with the important role of TGF-\u03b2 in regulating focal adhesions, integrin-based adhesion, actin cytoskeletal architecture, and cell motility. TGF-\u03b2 plays a profound role in the dedifferentiation of epithelial cells, causing depolarization, disruption of epithelial interactions, altered expression of extracellular matrix proteins, rearrangement of the cytoskeleton, and formation of actin stress fibers. These TGF-\u03b2 responses provide cells with increased metastatic potential and with increased motility.\nOver the past few years, tremendous progress has been made in identifying signal transduction pathways activated by TGF-\u03b2 family members. In the present study, we have combined microarray analysis with the examination of specific signaling pathways. Because of the important role that TGF-\u03b2 plays in controlling cell proliferation and cell adhesion, we used the signaling elements involved in these processes as a template for the analysis of our microarray results. Many of the genes that we identified as being regulated by TGF-\u03b2 were previously reported to be downstream targets of TGF-\u03b2. We also discovered several novel TGF-\u03b2 target genes known to play roles in regulating cell adhesion, and thus EMT, and also cell cycle regulation. Examining the biological functions of these novel TGF-\u03b2 target genes will increase our knowledge of the mechanisms by which TGF-\u03b2 mediates its cellular effects. Furthermore, our results indicate several possible points of convergence between TGF-\u03b2 signaling and other major intracellular and intercellular signaling systems.\nConclusion\nA 15,247 cDNA microarray was used to examine TGF-\u03b2-regulated gene expression in mouse mammary NMuMG cells. Expression of 10% of the genes examined (939) was altered by TGF-\u03b2 treatment. We have reported a comprehensive analysis of the coordinated regulation of genetic programs induced by TGF-\u03b2 in mammary epithelial cells during the processes of cell cycle arrest and EMT. In addition, several genes previously not known to be regulated by TGF-\u03b2 at the transcriptional level were identified, including Akt, RhoB, IQGAP1, mCalpain, actinin \u03b13, Ikki and PP2A-PR53.\nCompeting interests\nNone declared.\nAbbreviations\nBSA = bovine serum albumin; Cdk = cyclin-dependent kinase; Cy = cyanine; EMT = epithelial to mesenchymal transition; IKKi = inducible IkB kinase; IQGAP1 = IQ motif-containing the GTPase-activating protein 1; NF = nuclear factor; NIA = National Institutes of Aging; PCR = polymerase chain reaction; PP2A = protein phosphatase 2A; RT = reverse transcriptase; SSC = sodium chloride\/sodium citrate (buffer); TGF-\u03b2 = transforming growth factor beta.","keyphrases":["mammary gland","epithelial","cdna microarray","adhesion","cell cycle control"],"prmu":["P","P","P","P","P"]}
{"id":"Histochem_Cell_Biol-4-1-2248609","title":"Clathrin-independent endocytosis: from nonexisting to an extreme degree of complexity\n","text":"Today it is generally accepted that there are several endocytic mechanisms, both the clathrin-dependent one and mechanisms which operate without clathrin and with different requirements when it comes to dynamin, small GTP-binding proteins of the Rho family and specific lipids. It should be noted that clathrin-independent endocytosis can occur even when the cholesterol level in the membrane has been reduced to so low levels that caveolae are gone and clathrin-coated membrane areas are flat. Although new investigators in the field take it for granted that there is a multitude of entry mechanisms, it has taken a long time for this to become accepted. However, more work needs to be done, because one can still ask the question: How many endocytic mechanisms does a cell have, what are their function, and how are they regulated? This article describes some of the history of endocytosis research and attempts to give an overview of the complexity of the mechanisms and their regulation.\nIntroduction\nThe existence of different endocytic mechanisms is becoming of increasing interest, for instance in relation to growth and differentiation, cell adhesion, regulation of the activity of signalling receptors, drug delivery, and entry of pathogens. The most well-studied endocytic mechanism involves clathrin-coated pits and vesicles, and impressive amounts of details are now known about clathrin-dependent endocytosis (Schmid and McMahon 2007; Benmerah and Lamaze 2007; Ungewickell and Hinrichsen 2007). Initially, the idea was that the formation of the clathrin basket was the driving force during membrane internalization, and quantitative studies suggested that the pathway could account for everything endocytosed (Doxsey et al. 1987). With basic ideas such as \u201cbiology is simple\u201d and questions like, \u201chow can a vesicle form without clathrin?\u201d, it was difficult and it took time to get acceptance for the existence of alternative endocytic pathways (Montesano et al. 1982; Sandvig et al. 1985, 1987; Moya et al. 1985; Payne et al. 1988; Sandvig and van Deurs 1991, 1994; Damke et al. 1995). Clathrin-dependent endocytosis can be inhibited both by removing clathrin from the plasma membrane by a treatment involving hypotonic shock and depletion of cytosolic potassium (Larkin et al. 1983), and by \u201cfreezing\u201d the clathrin coat at the surface by acidification of the cytosol (Sandvig et al. 1987, 1988). The finding that endocytosis of for instance the protein toxin ricin continued under both conditions, suggesting that clathrin-independent endocytosis was responsible for this uptake, was apparently difficult to accept for a number of investigators during a 10\u00a0year period (1985\u20131995). New techniques in molecular biology have been crucial for the elucidation of endocytic processes, and when in 1995 it was published that the dynamin-mutant K44A inhibited clathrin-mediated endocytosis and that the cells still endocytosed (Damke et al. 1995), the complexity of endocytosis started to become apparent to the community in general. Not only did clathrin-independent endocytosis exist, it could also be regulated (Damke et al. 1995). We now know that a number of signalling pathways can regulate clathrin-independent endocytosis (for review, see Sandvig and van Deurs 2005; Marsh and Helenius 2006; Mayor and Pagano 2007). Despite accumulating evidence for several endocytic mechanisms, partly obtained by studies of toxins and viruses (Marsh and Helenius 2006; Sandvig and van Deurs 2002), many text-books in cell biology still operate with only two endocytic mechanisms, uptake from clathrin-coated pits and caveolae. The aim of this brief review is to outline the extreme complexity of clathrin-independent endocytosis as it appears today.\nClathrin-independent endocytosis: expansion of the field\nThe number of clathrin-independent endocytic pathways revealed has been increasing with time. Mayor and Pagano (Mayor and Pagano 2007) recently subdivided the different mechanisms into four types: RhoA-regulated, cdc42-regulated, and Arf6-dependent mechanisms, and caveolar uptake. Of these, the RhoA-regulated mechanism and caveolar endocytosis also require the GTP-binding protein dynamin (Fig.\u00a01a). However, there are already reports of mechanisms that may not necessarily fit into any of these four categories. For instance, clathrin-independent internalization of proteoglycans seems to depend not only on dynamin, but also on flotillin (Payne et al. 2007), and internalization of Herpes Simplex Virus Protein VP22 is independent of clathrin, but dependent on dynamin and Arf6 (Nishi and Saigo 2007). Also, studies of virus and toxin uptake indicate that there could be even more mechanisms (Marsh and Helenius 2006; Sandvig 2001; Sandvig and van Deurs 2005). Since cells may compensate for loss of one endocytic pathway by upregulating another (Damke et al. 1995), one can of course also question to which extent interfering with the endocytic machinery of physiologically relevant pathways in different ways, might create new pathways or cause upregulation of those normally playing only a minor role. Today, more specific methods such as RNA interference (RNAi) are used to interfere with a given process. However, also RNAi-mediated removal of a given molecule can induce upregulation of related molecules (Utskarpen et al. 2006). Moreover, upon removal of a membrane-associated molecule one may ask: To which extent do we actually know that the endocytic process is blocked? Could the rest of the machinery allow uptake to continue with different kinetics and be interpreted as a \u201cnew mechanism\u201d? Similarly, an old objection to the notion that removal of clathrin by potassium-depletion (Larkin et al. 1983; Moya et al. 1985; Madshus et al. 1987; Sandvig et al. 1985) revealed endocytosis normally operating in parallel to the clathrin-dependent pathway, was that the rest of the endocytic machinery originally belonging to the clathrin-dependent machinery was still operating. A similar critical attitude should be applied on today\u2019s interpretations. Importantly, both toxins and viruses can modify endocytic events, in some instances by inducing signalling, even though they may bind to glycolipid receptors (Marsh and Helenius 2006; Lauvrak et al. 2006; R\u00f6mer et al. 2007).\nFig.\u00a01An overview of a factors involved in different types of endocytosis, and b schematic drawing of polarized MDCK cells where some proteins\/enzymes involved in regulation of apical clathrin-independent endocytosis have been listed. It should be noted that apical clathrin-independent endocytosis may consist of more than one mechanism and that polarized MDCK cells have caveolae only at the basolateral surface. For references, see the text\nInternalization of membrane components and ligands by endocytosis thus have different requirements when it comes to dependency of dynamin, caveolin, flotillin, and small GTPases, and in addition, cholesterol and other specific lipids play a role (see below). To which extent the different endocytic mechanisms vary depending on cell type, growth conditions and the extent of cell polarization is to a large extent not known, and has to be addressed in future studies. It should be stressed that most of our knowledge about clathrin-independent endocytic mechanisms derives from cells in culture.\nThe work of combining different results into clearly defined models is far from finished. In 2005 it was published that CtBP3\/BARS drives membrane fission in processes where dynamin is not involved (Bonazzi et al. 2005). Interfering with the action of this protein reduces fluid phase uptake, as previously reported for cdc42-dependent uptake (Mayor and Pagano 2007). However, whether cdc42 and CtBP3\/BARS affect the same process does not seem to be known. The possibility exists that CtBP3\/BARS might indirectly affect endocytosis (Mayor and Pagano 2007). Furthermore, when inhibiting endocytic uptake of a ligand, it can be difficult to measure whether it is movement of the ligand into the membrane invagination giving rise to the endocytic vesicle that is reduced or whether it is the formation of the vesicle that is the step affected. To classify the different mechanisms properly, more information is required (see Fig.\u00a01a, b). It is important to note that polarized cells have a differential regulation of clathrin-independent endocytosis at the two poles, and regulators of apical clathrin-independent endocytosis have been indicated in Fig.\u00a01b (separate chapter below). Interestingly, caveolae are only present on the basolateral side in the polarized epithelial cell line MDCK (Verkade et al. 2000; Vogel et al. 1998). In the following sections we will describe various aspects of the clathrin-independent mechanisms in more detail.\nMembrane lipids required for clathrin-independent endocytosis\nIn addition to a large number of proteins, also membrane lipids are important for clathrin-independent endocytosis. Interfering with sphingolipid synthesis may affect different types of clathrin-independent endocytosis by mechanisms poorly understood (Cheng et al. 2006). Importantly, sphingomyelin seems to be required both for recruitment of RhoA and cdc42 to the membrane and for endocytosis involving these GTP-binding proteins (Cheng et al. 2006). Cdc42-dependent uptake is independent not only of RhoA, but also of Rac, clearly separating it from macropinocytosis which requires Rac activity (for review, see (Hanzal-Bayer and Hancock 2007; Chadda et al. 2007). Interestingly, sphingolipid synthesis, or rather the production of glycosphingolipids was found to be required for transport of caveolin to the plasma membrane and for caveolae formation (Cheng et al. 2006).\nA change in the fatty acid composition of membrane lipids can also be expected to change the properties of the membrane and its interaction with cytosolic proteins of importance for endocytosis. It was recently found that incubation of cells with unsaturated fatty acids affect the uptake of the glycolipid-binding Shiga toxin which is internalized both by clathrin-dependent and clathrin-independent endocytosis (Spilsberg et al. 2007). A possible explanation for this finding is that there is a change in the amount and\/or size of rafts or their ability to become associated with the endocytic machinery. Lipid rafts play a central role in membrane structure and function (Rajendran and Simons 2005), and it is therefore not surprising that the fatty acid composition may affect endocytosis. Raft-dependent uptake have recently been reviewed (Marsh and Helenius 2006; Lajoie and Nabi 2007; Hanzal-Bayer and Hancock 2007).\nOne can often see the generalized statement: clathrin-independent endocytosis is cholesterol-dependent (Hanzal-Bayer and Hancock 2007). This is true for processes such as macropinocytosis (Grimmer et al. 2002), uptake from caveolae, Rho-dependent uptake of IL2 (a recent review has been written by Lajoie and Nabi 2007), and cdc42-dependent endocytosis where it was reported that there is a cholesterol-dependent activation of cdc42 which in turn mediates actin polymerization required for endocytosis via this pathway (Chadda et al. 2007). However, a normal concentration of cholesterol does not seem to be required for uptake of all ligands taken in by clathrin-independent endocytosis (Rodal et al. 1999). When enough cholesterol has been extracted from the plasma membrane for the caveolae to disappear and the clathrin-coated pits to flatten, endocytosis of the plant toxin ricin still occurs (Rodal et al. 1999). Furthermore, when Cholera toxin uptake into HeLa cells is reduced by expression of the dynamin-mutant K44A or by induction of antisense to clathrin heavy chain in BHK cells, endocytosis of this toxin can be increased by extraction of cholesterol with methyl-\u03b2-cyclodextrin (Sandvig et al. 2004). Similarly, uptake of Shiga toxin is increased by treatment with methyl-\u03b2-cyclodextrin after block of clathrin-dependent uptake by induced expression of antisense to clathrin heavy chain in BHK cells (Fig.\u00a02). One possible explanation is that these toxins are retained at the cell surface by being associated with rafts, and that release from these structures by treatment with methyl-\u03b2-cyclodextrin actually permits endocytosis of the toxins. Also, uptake of proteoglycan-binding ligands, reported to be clathrin- and caveolin-independent, but dynamin- and flotillin-dependent, were unaffected by filipin and nystatin, drugs known to disrupt lipid rafts (Payne et al. 2007). It should be noted that cholesterol-depletion can inhibit the formation of invaginated clathrin-coated pits by a so far unknown process (Rodal et al. 1999; Subtil et al. 1999), and cholesterol-dependency is therefore not necessarily a tool to easily distinguish between endocytic processes.\nFig.\u00a02Shiga toxin endocytosis is strongly increased upon methyl-\u03b2-cyclodextrin-treatment of BHK cells induced for expression of antisense to clathrin heavy chain. BHK cells with inducible expression of antisense to clathrin heavy chain was preincubated with methyl-\u03b2-cyclodextrin (10\u00a0mM) for 30\u00a0min. Then biotin- and TAG-labeled Shiga toxin (25\u00a0ng\/ml) was added and the cells were incubated for 20\u00a0min. Surface-bound and endocytosed Shiga toxin was quantified as previously described for Cholera toxin (Torgersen et al. 2001). The data are presented as internalized toxin as percent of total cell-associated toxin (mean\u00a0\u00b1\u00a0SD, n\u00a0=\u00a04). Endocytosis of transferrin was performed in parallel to verify the inhibition of clathrin-dependent endocytosis upon induction of antisense to clathrin heavy chain. Upon induction, transferrin uptake was reduced by 95%\nRole of actin in endocytosis\nRecruitment of actin to clathrin-coated pits and subsequent actin-driven formation and internalization of endocytic vesicles and the appearance of actin \u201ccomet tails\u201d on such vesicles is well-established and appears to be a highly complex process involving several protein systems (McPherson 2002; Kaksonen et al. 2005, 2006; Smythe and Ayscough 2006). Interestingly, a crucial role of actin in clathrin-independent endocytosis is now becoming evident as well (Kaksonen et al. 2006; Yarar et al. 2007; Lanzetti 2007), and it seems likely that actin will turn out to be involved in all clathrin-independent endocytic mechanisms. However, in some cases actin might function to facilitate the process and not be strictly required. It has for instance been reported that there is a difference between cells growing in monolayers and cells in suspension when it comes to actin requirement for clathrin-dependent endocytosis (Fujimoto et al. 2000). One of the most studied examples of a clathrin-independent endocytic mechanism that is based on actin polymerization, is macropinocytosis (see below), a major fluid-phase uptake pathway.\nMacropinocytosis \nAn increasing complexity of the formation of macropinosomes is becoming evident. Thus, it has long been known that GTP-bound Rac stimulates accumulation of actin filaments at the plasma membrane and that it is involved in plasma membrane ruffling induced by growth factors (Ridley et al. 1992) (Fig.\u00a01a). Rac in turn activates the Arp2\/3 protein complex and WAVE, a process involved in ruffling (Gao et al. 2007). In addition to Rac, also Ras, Src and phosphoinositide (PI) 3-kinase promote macropinocytosis (Gao et al. 2007), and a role of N-WASP and the sorting nexin 9 (SNX9) in assembly and organization of actin during ruffle formation and macropinocytosis has recently been shown (Yarar et al. 2007). Also, histone deacetylase 6 (HDAC6) is associated with plasma membrane ruffles and formation of macropinosomes upon stimulation with growth factors, processes that were inhibited in HDAC6-deficient cells. Interestingly, a substrate of HDAC6, the heat shock protein Hsp90, seemed to be involved in ruffling and macropinocytosis as well (Gao et al. 2007). Moreover, a clathrin-independent process reminiscent of macropinocytosis has recently been reported (Orth et al. 2006). Here, actin-dependent circular dorsal ruffles or dorsal membrane waves are formed upon growth factor stimulation, leading to significant receptor internalization. Like in ordinary macropinocytosis, PI 3-kinase is involved, but interestingly, also dynamin, which is a part of the actin-polymerizing Arp2\/3-N-WASP-cortactin system and a large number of other proteins are required for this internalization mechanism (Orth et al. 2006; Orth and McNiven 2006; McNiven 2006).\nRhoA-, cdc42- and CTBP3\/BARS-dependent uptake\nThe RhoA-dependent uptake of IL-2 receptors, \u03b3c-cytokine receptor and IgE receptor is a dynamin-dependent process (Mayor and Pagano 2007) (Fig.\u00a01). Whether RhoA is required for the correct sorting of ligand or whether it is involved in the endocytic process as such is however being discussed (Mayor and Pagano 2007). Data from polarized cells showing that ricin uptake and fluid phase endocytosis at the apical side of polarized MDCK cells are also dependent on RhoA (Garred et al. 2001), suggest that it is the endocytic mechanism itself that is RhoA-dependent.\nA cdc42-regulated process, which operates independently of dynamin, has been reported to be involved in uptake of glycosylphosphatidylinositol (GPI)-anchored proteins as well as being responsible for a major fraction of fluid-phase uptake in the cell (Kalia et al. 2006). GPI-anchored proteins were found to be internalized into CHO cells by a cdc42-dependent, but Arf6-independent mechanism. The internalized membrane ended up in vesicles without EEA1, Rab4, or Rab5, but these vesicles were found to fuse with normal endosomal compartments via a Rab5\/PI3-kinase dependent machinery. That a GPI-anchored protein and its ligand can enter independently of caveolae and dynamin and reach an acidic compartment, was first shown in studies of a GPI-anchored diphtheria toxin receptor (Skretting et al. 1999). The finding that diphtheria toxin bound to this GPI-linked receptor intoxicates cells, demonstrates that the toxin has been transferred to an acidic compartment facilitating low pH-induced conformational change of diphtheria toxin and translocation of the toxin to the cytosol.\nThe finding that the cdc42-dependent mechanism is responsible for most fluid uptake (Kalia et al. 2006) is surprising, considering all the other uptake mechanisms now reported, not least macropinocytosis. It was recently shown that cdc42 is associated with the recycling endosome and that also Par proteins play a role in the endocytic process (Balklava et al. 2007). To which extent there is an indirect effect on the uptake from the plasma membrane when mutants of cdc42 are studied, is not quite clear. Also, as mentioned earlier, since CtBP3\/BARS is involved in dynamin-independent endocytosis (Bonazzi et al. 2005), and also endocytosis regulated by this protein can account for a considerable amount of fluid phase uptake, it should be elucidated to which extent cdc42 and CtBP3\/BARS regulated processes are related.\nArf6-dependent uptake \nADP-ribosylation factor 6 (Arf6) has been identified as a key regulator of a clathrin- and dynamin-independent endocytic pathway mainly studied in HeLa and COS-7 cells, and responsible for uptake and recycling of a collection of integral plasma membrane proteins devoid of conventional endocytic signals (see below). The number of cargo proteins reported to be internalized by Arf6-regulated endocytosis in these cells is increasing and includes the plasma membrane proteins Major Histocompatibility Complex Class I (MHCI) and interleukin-2 receptor \u03b1-subunit\/Tac (Radhakrishna and Donaldson 1997), \u03b21-integrin (Brown et al. 2001), M2 muscarinic acetylcholine receptor (Delaney et al. 2002) and certain GPI-anchored proteins, such as CD59 (Naslavsky et al. 2004). Internalized cargo can recycle to the plasma membrane via the tubular Arf6-positive recycling compartment (Radhakrishna and Donaldson 1997) or, alternatively, be delivered to Rab5 and EEA1 positive early endosomes for further transport to late endosomes and lysosomes for degradation (Naslavsky et al. 2003). Recently, rafts were reported to be recycled by an Arf6-dependent process (Balasubramanian et al. 2007). Interestingly, the Herpes Simplex Virus protein VP22 was in CHO-K1 and HeLa cells shown to be endocytosed by a mechanism dependent on Arf6, dynamin and lipid rafts, but independent of Rho-GTPases (Nishi and Saigo 2007).\nA new class of trafficking motifs in non-clathrin-dependent cargo proteins have been identified and suggested to prevent internalization and\/or facilitate recycling of proteins carrying these, via the Arf6-regulated pathway by enhancing their association with the Arf6-specific guanine nucleotide exchange factor EFA6 (Gong et al. 2007). In certain cell types, rather than defining a separate endocytic pathway, Arf6 regulates clathrin-dependent uptake. In Hek293 cells, for example, Arf6 regulates uptake of several G-protein coupled receptors by clathrin-dependent endocytosis (Houndolo et al. 2005), and there is also evidence for complex formation between Arf6, the \u03b2-subunit of AP2 and the heavy chain of clathrin (Poupart et al. 2007). The data furthermore suggest that Arf6 regulates the angiotensin AA type 1 receptor by recruiting AP2 and clathrin. Arf6 has also in HeLa cells been proposed to have a direct function in clathrin coat recruitment to the plasma membrane. In fact, Arf6-GTP has recently been demonstrated to interact with AP-2 both in vitro and in vivo, and a clathrin-interacting Arf6-specific GTPase activating protein (called SMAP), affecting clathrin-dependent endocytosis only, has also been identified (Paleotti et al. 2005; Tanabe et al. 2005). Interestingly, in polarized MDCK cells, Arf6 associated with the guanine nucleotide exchange factor ARNO regulates endocytosis exclusively at the apical surface by recruiting actin to clathrin-coated pits (Hyman et al. 2006).\nThe present picture is complicated as many of the roles ascribed to Arf6 within the endocytic system seem to be not only cell type dependent, but also determined by the exchange factor associated with Arf6 (Kalia et al. 2006). The Arf6 GTP exchange factor ARNO has previously been shown to be dependent on inositol-lipids for its membrane recruitment. However, the process seems to be even more complex as small G proteins of the Arl 4 family can function in recruitment of the Arf6 GEF cytohesin to the plasma membrane (Hofmann et al. 2007). Perhaps this might explain some of the apparent differences between cell types.\nCaveolae and endocytosis\nCaveolae are characteristic, small (50\u2013100\u00a0nm), regular invaginations of the plasma membrane as seen in the EM (Fig.\u00a03). Since caveolae have this distinct, easily recognizable ultrastructural morphology, they have been of particular interest in relation to clathrin-independent endocytosis. The distinct shape of caveolae early led to the assumption that caveolae were endocytic structures comparable to clathrin-coated pits and vesicles. Also, in thin sections for EM which are not cut perpendicular to the plasma membrane, one might get the impression of several free, i.e. not surface-connected, caveolar profiles, supporting the idea of caveolar endocytosis and a front of internalized caveolae moving into the cell (Fig.\u00a03). Interestingly, more careful EM examination\u2014for instance using Ruthenium Red as a surface marker\u2014reveal that most if not all such vesicular profiles are indeed surface-connected caveolae (Fig.\u00a04). Also, the observation that e.g. Cholera toxin localized to caveolae supported the idea that caveolae were responsible for the endocytic uptake of Cholera toxin, a notion that is certainly an oversimplification (Torgersen et al. 2001; Shogomori and Futerman 2001; Nichols et al. 2001; see discussion in Hommelgaard et al. 2005; see also Kirkham et al. 2005).\nFig.\u00a03Appearance of caveolae in myoepithelial cells. In (a) is seen a single caveola (Cav) and for comparison a clathrin-coated pit (Cp). b shows a group of caveolae at the plasma membrane; as the section is largely perpendicular to the membrane, most of the caveolae are clearly seen to be surface-connected. In (c) is shown an example of a section which is not perpendicular to the plasma membrane, and here many of the caveolae appears as free (not surface-connected) vesicles. If such \u201cfree\u201d vesicles should be unequivocally established as caveolae, they can be immunogold-labeled using an antibody against caveolin, as shown on the ultracryosection in (d) (Cav). An unlabeled, Cp is also seen. Bars 500\u00a0nmFig.\u00a04Caveolae are surface-connected structures. a\u2013d show filter-grown MDCK cells postfixed with the electron-dense cell surface marker Ruthenium Red from the basolateral side. In (a) is seen a number of caveolae connected to the basolateral membrane (arrowheads). Also note that three caveolae (arrows) are connected to the plasma membrane (open arrow) via a larger membrane invagination (asterisk). b\u2013d show clusters of caveolae (arrows) associated with elongated \u201cvacuolar\u201d structures (asterisks) apparently freely localized in the cytoplasm. However, the labelling with Ruthenium Red clearly demonstrates that these structure are indeed connected to the basolateral membrane, as revealed in (a). En, early and later endosomes; Nu, nucleus. Bar 500\u00a0nm\nHowever, fluorescence recovery after photobleaching (FRAP) analysis of cells expressing GFP-caveolin revealed that caveolae under normal conditions are not involved in endocytosis to any significant degree (Thomsen et al. 2002). Similarly, more recently live cell imaging of cells expressing GFP-caveolin showed that even high concentrations of EGF, which had been reported to lead to internalization of the EGFR via caveolae (Sigismund et al. 2005), do not mobilize caveolae above a low background level (Kazazic et al. 2006). Also, in a careful EM study where the setup allowed to discriminate between caveolin-associated and other vesicles as well as between surface-connected and free vesicles, Kirkham et al. (2005) showed that only about 2% of caveolae bud from the plasma membrane per minute. For comparison, it takes 50\u2013150\u00a0s for a clathrin-coated pit to assemble and pinch off to form a free, clathrin-coated vesicle (Ungewickell and Hinrichsen 2007). This means that the entire population of coated pits present at a given time point will have been internalized within approximately 1\u20132.5\u00a0min. Thus, today it appears well-established and generally accepted that under normal, non-stimulating conditions, caveolae are quite stable structures at the plasma membrane that are not involved in endocytosis to any significant degree (van Deurs et al. 2003; Pelkmans et al. 2004; Hommelgaard et al. 2005; Parton et al. 2006; Kirkham et al. 2005; Lajoie and Nabi 2007).\nOn the other hand, it is also evident from the above mentioned studies that a few caveolae may pinch off from the plasma membrane (Thomsen et al. 2002; Kirkham et al. 2005). This could simply be due to a basal turnover of caveolae at the plasma membrane. Normally, caveolae seem to be stabilized at, or anchored to, the plasma membrane by actin filaments (Stahlhut and van Deurs 2000; Hommelgaard et al. 2005; Thomsen et al. 2002; Pelkmans et al. 2002). The phosphatase inhibitor okadaic acid stimulates caveolar clustering and internalization in an actin-dependent manner (Parton et al. 1994; Thomsen et al. 2002). One could therefore also imagine that caveolae might be involved in a very slow, regulated endocytic uptake which under certain circumstances can be speeded up, for instance by controlled reorganization of the actin cytoskeleton. However, this remains speculative.\nInterestingly, it was recently reported that coassembly of flotillin-1 and -2 induced membrane curvature and formation of membrane invaginations morphologically similar to caveolae but different from these by not containing caveolin, the major caveolar protein. These flotillin-positive but caveolin-negative structures became internalized in contrast to \u201cclassical\u201d caveolae (Frick et al. 2007).\nCaveolae perform short range \u201ckiss-and-run\u201d motility very close to the plasma membrane. The process most likely involves continuous internalization and recycling of caveolae immediately beneath the plasma membrane (probably in the narrow 100\u2013200\u00a0nm zone between the plasma membrane and the stabilizing actin cytoskeleton) (Thomsen et al. 2002, Fig.\u00a05, online movie; Pelkmans and Zerial 2005). The significance of this process is unclear but it may explain the widely accepted role of caveolae in endothelial transcytosis. Thus, since endothelial cells are often very flat (100\u2013200\u00a0nm), caveolae that have pinched off from e.g. the luminal endothelial plasma membrane, could fuse immediately after with the ablumenal plasma membrane or, in cases where the endothelial cell is very flat, the fusion may take place even before the pinching has been completed, and temporary transendothelial channels may arise (van Deurs et al. 2003).\nWhen SV40 virus is added to cells, the virus particles are trapped in caveolae which subsequently become internalized by a process which requires tyrosine phosphorylation, reorganization of the cortical actin cytoskeleton, and recruitment of dynamin (Pelkmans et al. 2001, 2002). Characteristically, the caveolar internalization is a single wave phenomenon and it takes hours before caveolin\/caveolae reappear at the plasma membrane. More recently, it was shown that SV40 virus is actually internalized much more efficiently by a caveolin-independent mechanism (Damm et al. 2005). In any case it is questionable whether the downregulation of plasma membrane caveolae induced by SV40 virus represents any normally occurring, inducible clathrin-independent endocytic mechanism. Rather, it might reflect clearance of the plasma membrane for caveolae occupied by cross-linked or aggregated receptors or ligands. Such downregulation of caveolae stimulated by receptor cross-linking is discussed in more detail elsewhere (see Hommelgaard et al. 2005).\nIn this context, it is important to consider the many non-endocytic functions that have been associated with plasma membrane caveolae (see van Deurs et al. 2003). Thus, endothelial nitric oxide synthase (eNOS) is associated with caveolin-1, and caveolae play an important role in NO signaling, and are also involved in e.g. calcium signalling (Murata et al. 2007). Moreover, caveolae is important for cholesterol transport and homeostasis in adipocytes, and it was recently found that cholesterol-induced transport of caveolin to lipid droplets requires dynamin and PKC and very likely involves caveolar endocytosis (Le et al. 2006). Therefore, rather than considering caveolae in general as structures specialized for clathrin-independent endocytosis, they should be considered multifunctional membrane domains with physiological roles depending on cell type and situation (van Deurs et al. 2003). In addition, a recent report suggests that caveolins may even regulate cell function independently of caveolae (Head and Insel 2007).\nRegulation of endocytosis in polarized epithelial cells\nStudies of endocytic mechanisms in cell lines do not necessarily reflect the uptake mechanisms operating in vivo. Both cell density, cell adhesion and polarization can be regulators of endocytosis (del Pozo and Schwartz 2007; Sandvig 1978; Sandvig and van Deurs 2005; Eker et al. 1994; Holm et al. 1995; Llorente et al. 2000; Sandvig et al. 2000; Llorente et al. 1996). An early argument against clathrin-independent endocytosis was, not unexpectedly, that this could be a process occurring only in unpolarized cell lines grown on plastic support. However, investigations of polarized cells, such as MDCK I cells grown on filters, revealed that clathrin-independent endocytosis contributed to the endocytic uptake both on the apical and basolateral side (Eker et al. 1994). When using cytosolic acidification to block clathrin-dependent endocytosis (Sandvig et al. 1987), there was still uptake of the plant toxin ricin at both poles (Eker et al. 1994). The fact that there was a larger reduction of uptake at the apical side than at the basolateral side, does, however, not necessarily provide information about the fraction of clathrin-independent endocytosis at the two poles, since the low pH might affect the remaining uptake at the two poles differently. Importantly, apical clathrin-independent endocytosis turns out to be under complex regulation, and can be regulated independently of uptake at the basolateral side. Using either cells permeabilized at the basolaterial side or membrane permeable compounds (for review, see Sandvig and van Deurs 2005), we have shown that apical clathrin-independent endocytosis is regulated by protein kinase A (Eker et al. 1994), protein kinase C (Holm et al. 1995), phospholipase D (Sandvig et al. 2000), cyclooxygenase (Llorente et al. 2000) and calmodulin (Llorente et al. 1996). Interestingly, a Rho family member, apparently RhoA, seems to regulate apical endocytosis in a manner which can be supported by GTP\u03b3S (Garred et al. 2001). The RhoA-mediated mechanism is able to mediate uptake of both gold particles, ricin-HRP and HRP.","keyphrases":["endocytosis","caveolae","ricin","flotillin","shiga toxin","cholera toxin","rho proteins"],"prmu":["P","P","P","P","P","P","R"]}
{"id":"Biochem_Pharmacol-2-1-2279149","title":"Mouse N-acetyltransferase type 2, the homologue of human N-acetyltransferase type 1\n","text":"There is increasing evidence that human arylamine N-acetyltransferase type 1 (NAT1, EC 2.3.1.5), although first identified as a homologue of a drug-metabolising enzyme, appears to be a marker in human oestrogen receptor positive breast cancer. Mouse Nat2 is the mouse equivalent of human NAT1. The development of mouse models of breast cancer is important, and it is essential to explore the biological role of mouse Nat2. We have therefore produced mouse Nat2 as a recombinant protein and have investigated its substrate specificity profile in comparison with human NAT1. In addition, we have tested the effects of inhibitors on mouse Nat2, including compounds which are endogenous and exogenous steroids. We show that tamoxifen, genistein and diethylstilbestrol inhibit mouse Nat2. The steroid analogue, bisphenol A, also inhibits mouse Nat2 enzymic activity and is shown by NMR spectroscopy, through shifts in proton peaks, to bind close to the active site. A three-dimensional structure for human NAT1 has recently been released, and we have used this crystal structure to generate a model of the mouse Nat2 structure. We propose that a conformational change in the structure is required in order for ligands to bind to the active site of the protein.\n1\nIntroduction\nArylamine N-acetyltransferases (NATs, EC 2.3.1.5) have traditionally been identified as drug-metabolising enzymes responsible for the metabolism of arylamines, arylhydroxylamines and arylhydrazines. NATs were first identified in man and a range of eukaryotes and have had an important part to play in the early years of identification of pharmacogenetic variation in response to drug treatment [1].\nThe hydrazine drug isoniazid is polymorphically N-acetylated in humans to its therapeutically inactive form by the human isoenzyme now known as human NAT2. There are two human NAT isoenzymes: human NAT2, which metabolises isoniazid, and human NAT1, which does not metabolise isoniazid or other arylhydrazines [2], but does catalyse the acetylation of a distinct but overlapping series of arylamines [2]. The pattern of expression of the two human NAT genes also differs. As a result of genome-wide microarray [3\u201312] and proteomic studies [13], it is clear that human NAT1 is highly expressed in oestrogen receptor positive breast cancer [14]. There is also evidence that the level of expression of human NAT1 affects the growth of cultured breast cancer cells [13].\nThe role of the human NAT1 enzyme in breast cancer has not been extensively explored, although it has previously been demonstrated that the anti-oestrogen compound tamoxifen is an inhibitor of human NAT1 [15\u201317]. It has also been demonstrated that both human NAT1 and the oestrogen receptor are down-regulated in tissues in which p53 is mutated [5]. It appears that human NAT1 is a marker for oestrogen receptor positive breast cancer, although information is still accumulating on the relationship of this marker to others, including the oestrogen receptor itself [14]. However, if the human NAT1 enzyme is to be considered as a target for breast cancer therapy, it is essential that there is a suitable in vivo animal model for testing [18,19]. Mice represent the most convenient animal models of disease. At present, the status of mouse Nat2, the equivalent of human NAT1, in a model of breast cancer has not been explored. We have previously demonstrated that mouse Nat2 is expressed in the epithelial cells lining the mammary ducts [20]. This is the same location as the human NAT1 enzyme in normal breast tissue [13,21].\nIn order to provide a firm foundation for establishing such a model, we have generated pure recombinant mouse Nat2 and investigated its activity with a wide range of substrates and the effects of a range of potential inhibitors, including endogenous and exogenous steroids.\n2\nMaterials and methods\n2.1\nChemicals\nAll chemicals were purchased from Sigma\u2013Aldrich and all molecular biology reagents were purchased from Promega, unless otherwise stated.\n2.2\nCloning and expression studies of mouse Nat2\nThe mouse Nat2 open-reading frame [22] was sub-cloned into pET28b(+) (Novagen) using the compatible restriction enzyme sites, NdeI and EcoRI. This allowed the production of recombinant mouse NAT protein with an N-terminal His-tag, for ease of downstream purification. The pET28b(+) plasmid containing mouse Nat2 was transformed into Escherichia coli strain JM109 and further transformed into Rosetta(DE3)pLysS (Novagen) after confirming the correct insert sequence (DNA Sequencing Facility, Biochemistry, University of Oxford). The positive transformant was grown in LB to mid-log phase at 37\u00a0\u00b0C with shaking (180\u00a0rpm) and stored at \u221280\u00a0\u00b0C with 10% (v\/v) glycerol. Thawed glycerol stock (100\u00a0\u03bcL) was used to inoculate fresh LB media (100\u00a0mL) supplemented with kanamycin (30\u00a0\u03bcg\/mL) and chloramphenicol (34\u00a0\u03bcg\/mL) and the culture was incubated at 37\u00a0\u00b0C for 16\u00a0h with shaking (180\u00a0rpm). The starter culture was then diluted 50-fold into fresh LB media (2\u00a0L) supplemented with kanamycin (30\u00a0\u03bcg\/mL), which was then incubated at 27\u00a0\u00b0C with shaking (180\u00a0rpm). When the absorbance at 600\u00a0nm reached 0.7, the expression of mouse Nat2 was induced by addition of isopropyl-\u03b2-d-thiogalactopyranoside (IPTG) to a final concentration of 0.1\u00a0mM. The culture was grown for a further 16\u00a0h and the cells were harvested by centrifugation (6000\u00a0\u00d7\u00a0g, 4\u00a0\u00b0C, 20\u00a0min). The cell pellet was resuspended in 20\u00a0mL lysis buffer (300\u00a0mM NaCl, 20\u00a0mM Tris\u2013HCl (pH 8.0) containing 1\u00d7 EDTA-free Complete Protease Inhibitor (Roche)) and then stored at \u221280\u00a0\u00b0C.\nFor expression of 15N-labelled mouse Nat2, the starter culture was diluted 50-fold in 15N-labelled Standard-E. coli-OD2\u00a0N medium (Silantes) supplemented with kanamycin (30\u00a0\u03bcg\/mL) and grown at 37\u00a0\u00b0C until the absorbance at 600\u00a0nm reached approximately 0.6. The cells were further grown at 27\u00a0\u00b0C until the absorbance reached 0.7, when the expression was induced with 0.1\u00a0mM IPTG. After induction for 17\u00a0h, the cells were harvested and stored as described for unlabelled mouse Nat2.\nThe resuspended frozen cells were thawed at 37\u00a0\u00b0C and sonicated on ice (25 cycles of 30\u00a0s on, 45\u00a0s off at 10\u00a0\u03bcm). The soluble lysate was isolated from the cell debris by centrifugation (12,000\u00a0\u00d7\u00a0g, 4\u00a0\u00b0C, 20\u00a0min) and incubated (4\u00a0\u00b0C, 5\u00a0min) with 5\u00a0mL of Ni-NTA resin (Qiagen). The resin was sequentially washed with lysis buffer containing increasing imidazole concentrations in a step gradient at 4\u00a0\u00b0C (2 washes each of 10\u00a0mL of 0\u00a0mM, 10\u00a0mM, 20\u00a0mM, 50\u00a0mM and 100\u00a0mM imidazole). Fractions containing hexa-histidine tagged Nat2 (His-Nat2), were pooled and thrombin (5\u00a0U\/mg NAT) was added to remove the His-tag. After 16\u00a0h incubation at 4\u00a0\u00b0C, the protein sample was dialysed against 20\u00a0mM Tris\u2013HCl (pH 8.0), 1\u00a0mM dithiothreitol (DTT), 1\u00a0mM EDTA buffer, and concentrated to 1\u00a0mg\/mL by centrifugation in a 15\u00a0mL concentrator (Amicon) after addition of 5% (v\/v) glycerol. Protein samples in 100\u00a0\u03bcL aliquots were frozen in liquid nitrogen and stored at \u221280\u00a0\u00b0C.\nRecombinant hamster Nat2 was expressed and purified as previously described [23]. 15N-labelled hamster Nat2 was grown and expressed as described for 15N-labelled mouse Nat2 and purified by the same method as for the unlabelled hamster Nat2.\nThe expression levels and activities of soluble Nat2 were monitored by visualizing the levels of recombinant protein expression on 12% acrylamide Tris\u2013Glycine SDS-PAGE as well as by measuring the rate of acetylation of 4-aminobenzoic acid (PABA) as a substrate (see method below) [24].\n2.3\nEnzymic assays\n2.3.1\nAcetylation of arylamines\nThe rates of arylamine acetylation by NATs were determined colorimetrically as previously described [25], but with minor modifications. Each assay contained the enzyme, arylamine substrate (see each figure legends for concentrations) and AcCoA (400\u00a0\u03bcM) in a total volume of 100\u00a0\u03bcL in assay buffer (20\u00a0mM Tris\u2013HCl (pH 8.0), 1\u00a0mM DTT). Initially, the enzyme and substrate mixtures were pre-incubated at 25\u00a0\u00b0C for 5\u00a0min and AcCoA was added to start the reaction. The reaction was quenched using 20% (w\/v) trichloroacetic acid (TCA) at different time intervals. The stopped reaction was centrifuged (16,000\u00a0\u00d7\u00a0g\u00a0rpm, 10\u00a0min) to pellet the precipitated proteins. The stopped reaction mixture (200\u00a0\u03bcL) was added to 800\u00a0\u03bcL of 5% (w\/v) 4-(N,N-dimethylamino)benzaldehyde (DMAB) in 9:1 acetonitrile:water to develop the colour. The absorption at 450\u00a0nm was measured with a Hitachi U-2001\u00a0UV\u2013vis spectrophotometer, with the amount of residual substrate in the reaction was determined by comparison with a standard curve.\n2.3.2\nHydrolysis of AcCoA (free CoA production)\nThe rate of production of the free thiol Coenzyme A by NAT, in the presence of a range of known NAT acetyl-acceptor substrates, was determined by using Ellman's reagent, 5,5\u2032-dithio-bis(2-nitrobenzoic acid) (DTNB) as previously described [2,26]. The substrate (500\u00a0\u03bcM) and purified recombinant NAT were pre-incubated (25\u00a0\u00b0C, 5\u00a0min) in 96-well flat-bottomed polystyrene plates (Costar\u00ae, Corning Inc.) in 20\u00a0mM Tris\u2013HCl (pH 8.0). AcCoA (400\u00a0\u03bcM) was added to start the reaction in a final volume of 100\u00a0\u03bcL. The reaction was quenched with the addition of 25\u00a0\u03bcL of guanidine\u2013HCl solution (6.4\u00a0M guanidine\u2013HCl, 0.1\u00a0M Tris\u2013HCl (pH 7.3)) containing 5\u00a0mM DTNB. The absorbance at 405\u00a0nm was measured on a plate-reader (Sunrise, Tecan). Assay buffer was used to replace substrate, AcCoA or NAT for control reactions. The amount of CoA produced in the assay was determined in comparison with a CoA standard curve.\nSubstrate abbreviations used for the substrate selectivity assays are as follows: aniline (ANL), 4-aminobenzoic acid (PABA), 4-aminosalicylic acid (4AS), 5-aminosalicylic acid (5AS), 4-chloroaniline (CLA), 4-bromoaniline (BRA), 4-iodoaniline (IOA), 4-methoxyanline (ANS), 4-ethoxyaniline (EOA), 4-butoxyanline (BOA), 4-hexyloxyaniline (HOA), 4-phenoxyaniline (POA), 4-aminoveratrole (4AV), 2-aminofluorene (2AF), 4-aminobenzoyl-l-glutamate (pABGlu), sulfamethazine (SMZ), procainamide (PRO), 4-aminopyridine (APY), isoniazid (INH), hydralazine (HDZ) and phenylhydrazine (PHZ). The stock concentration of substrate was 100\u00a0mM dissolved in DMSO, such that the final concentration of DMSO was less than 5% in the assays. DMSO only controls were carried out.\n2.4\nNMR spectroscopy\nThe 2D 1H-15N HSQC spectrum of mouse Nat2 was collected using a 750\u00a0MHz NMR spectrometer (Department of Biochemistry, University of Oxford). The spectrum was collected at 20\u00a0\u00b0C for a sample containing 30\u00a0mg\/mL of uniformly 15N-labelled mouse Nat2 in 95% H2O\/5% D2O 10\u00a0mM Tris buffer at pH 7.0. Spectral widths of 12,500 and 2500\u00a0Hz were used in F2 and F1, respectively. The dataset contained 1024 and 128 complex points in t2 and t1, respectively.\n1D 1H NMR spectra for hamster and mouse Nat2 were collected at 20\u00a0\u00b0C using a jump-return sequence. This sequence enabled the observation of HN peaks which exchange to a significant extent with H2O. Spectra were collected using a 600\u00a0MHz spectrometer using a sweep width of 12,500\u00a0Hz. Unlabelled hamster and mouse Nat2 were used at a concentration of 10\u00a0mg\/mL in the buffer described above. The titration of mouse Nat2 with bisphenol A was carried out by stepwise addition of small volumes (5\u201320\u00a0\u03bcL) of a concentrated solution of bisphenol A (100\u00a0mM in deuterated DMSO) to the mouse Nat2 solution in the NMR tube. The highest concentration of DMSO in the sample used was 2.8%. DMSO alone had no effect on the NMR spectrum of mouse Nat2 using concentrations up to 5%.\n3\nResults\n3.1\nCloning and expression of mouse Nat2\nThe mouse Nat2 was cloned into E. coli expression vector pET28b(+) and expressed in E. coli Rosetta (DE3)pLysS with an N-terminal hexa-histidine tag. The recombinant mouse His-Nat2 was purified using immobilised metal affinity chromatography, using a Ni-NTA column (Novagen), and eluted (Fig. 1a). Pure Nat2 predominantly eluted in the 50\u00a0mM and 100\u00a0mM imidazole washes. The hexa-histidine tag was readily removed by thrombin digestion (Fig. 1b). The purified recombinant Nat2 was active, readily catalysing the acetylation of PABA, a well-studied arylamine substrate of mouse Nat2 [27] (Table 1). The enzyme was relatively stable, and the enzymic activity was maintained (>85%) after incubation at temperatures ranging between 4\u00a0\u00b0C and 25\u00a0\u00b0C over a 72\u00a0h period.\n3.2\nSubstrate specificity of mouse Nat2\nTo further characterise the purified recombinant mouse Nat2, a panel of known NAT substrates (20 compounds) was tested to determine the substrate selectivity of recombinant mouse Nat2. In order to allow direct comparison with substrate selectivity profile for human NAT1 and NAT2, the assay for mouse Nat2 was performed under the same conditions as previously described [2].\nMouse Nat2 shows high specific activities with a broad range of arylamines, including PABA, 5AS, 4AS and 2AF (Fig. 2 and Supplementary Table 1). In contrast, mouse Nat2 shows low specific activities against arylhydrazine substrates such as HDZ, INH, and PHZ, and against arylamine drugs procainamide and sulfamethazine (Fig. 2). This overall trend is in agreement with the substrate selectivity observed in previous studies with impure recombinant mouse Nat2 using a subset of these compounds [22,27,28], although it is much less than the activity observed towards these substrates with human NAT2 (Fig. 2). There was low, yet measurable activity using arylhydrazine substrates with purified mouse Nat2, which has not been observed in previous studies using recombinant mouse Nat2 expressed in a transient expression system [28]. Pure recombinant mouse Nat2 also readily acetylates 2-aminofluorene, the substrate which has been used to identify mouse strains carrying an active form of mouse Nat2\u2014the fast acetylating C57Bl\/6 strain, rather than the inactive form as is found in the slow acetylating A\/J strain which is unstable and likely to be subject to rapid degradation [29].\nThe specific activity pattern of human NAT1 [2] overlaps with the mouse Nat2 profile (Fig. 2), reconfirming that mouse Nat2 is very similar to human NAT1 in substrate specificity. Mouse Nat2 shows differences from the activity profile of human NAT1, however. It appears that mouse Nat2 generally has higher inherent specific activity than human NAT1 and also acetylates arylhydrazines, when the acetylation of these compounds by human NAT1 is not detected under the same conditions [2].\n3.3\nMouse Nat2 inhibition by steroidogenic compounds\nIt had been demonstrated, with extracts of tissues and cells now known to express the human NAT1 gene, that tamoxifen is an inhibitor of human NAT1 [15\u201317]. In view of the relationship between the expression of human NAT1 and oestrogen receptor positivity in breast cancer [14], we wished to investigate the effects of steroidogenic compounds and xenobiotic oestrogenic compounds, including tamoxifen, on the activity of mouse Nat2.\nFrom the inhibition study using a broad range of steroidogenic compounds, it has been demonstrated that mouse Nat2 is inhibited selectively by oestrogenic compounds, with 17-hydroxy-\u03b2-estradiol identified as the most potent inhibitor (Table 2 and Supplementary Fig. 1). This was substantiated by the inhibition of mouse Nat2 activity by xenobiotic oestrogens, such as tamoxifen, and the IC50 values for these inhibitors are shown in Table 3. The phytoestrogens genistein, alpha-zearalenol and the synthetic oestrogen diethylstilbestrol show similar potency of mouse Nat2 inhibition to 17-hydroxy-\u03b2-estradiol, tamoxifen and 4-hydroxy tamoxifen. Bisphenol A was also shown to be an inhibitor of mouse Nat2, albeit with lower potency than some of the other compounds tested. One key practical advantage of using bisphenol A as an inhibitor is the improved solubility of this compound relative to the other phytoestrogens and steroids.\n3.4\nStructure of mouse Nat2\nUntil recently, there has been no structural information on eukaryotic NAT enzymes. However, recently the three-dimensional structure of a human NAT1 mutant (Phe\/Ser substitution at position 125) was determined by X-ray crystallography (PDB accession code: 2IJA [30]). A non-mutated human NAT1 crystal structure has also been deposited in which the active-site cysteine residue was modified to S-(2-anilino-2-oxoethyl)-cysteine (PDB accession code: 2PQT). It is considered likely that both the mutation of residue 125 and the modification of the active-site cysteine residue stabilise the structure of the human enzyme, because thousands of attempts to generate a crystal from the native human NAT1 protein have been unsuccessful (A. Kawamura, unpublished results). The structure of human NAT1 is illustrated in comparison with the NAT from M. smegmatis (Fig. 3). The eukaryotic proteins contain a loop between the second and third domains. This inter-domain loop is not present in their prokaryotic counterparts [31], as highlighted in Fig. 3. It is interesting to note that in the structure of the human NAT1 enzyme, this inter-domain loop is folded back over the active site. Likewise, the C-terminus is shown to be folded over the active-site cleft. These observations indicate that a conformational change in the protein may be required to allow the substrates to access the active-site cysteine residue. However, a structure of human NAT2 with coenzyme A bound has also been deposited (PDB accession code: 2PFR), and the position of the coenzyme A is such that the C-terminus and inter-domain loops regions are not significantly changed. This is in contrast to the recently reported structure of Mycobacterium marinum NAT in complex with coenzyme A [32]. In this bacterial NAT, the C-terminus is shorter than in the eukaryotic structures, and there is no inter-domain loop, and the ligand is found in the space where these regions are located in the eukaryotic structure.\nWe have generated a structural model of the mouse Nat2 protein, whose amino acid sequence is over 80% identical to human NAT1, by using the program Modeller 8v2 [33]. Fig. 4 shows the structural model of mouse Nat2 and a comparison of the mouse Nat2 and human NAT1 protein structures. The two structures share 1191 equivalent atoms, over which the root mean squared deviation is 0.75\u00a0\u00c5. In mouse Nat2, as in human NAT1, the inter-domain loop and the C-terminus occlude the active site. Despite the evidence for coenzyme A binding to human NAT2 without major conformational rearrangement of the C-terminus and inter-domain loop regions, it has not been possible to perform ligand docking into the mouse Nat2 or human NAT1 structures without disturbing these regions of the protein structure. Therefore, it remains possible that the inter-domain loop regions and C-termini of the eukaryotic NAT proteins are conformationally flexible, in order to accommodate substrate or inhibitor binding.\n3.5\nNMR studies\nNMR studies are ideal for investigating protein interactions with substrates and inhibitors in solution. Therefore, we have generated mouse Nat2 uniformly labelled with 15N to allow 2D NMR investigations of the protein. The 2D 1H-15N HSQC spectrum of mouse Nat2 is shown in Fig. 5; this spectrum contains a peak for each backbone amide (1HN-15N) and additional peaks from the side chains of Asn, Gln and Trp. The HSQC spectrum is well resolved, as expected for a compact globular structure, and some cross peaks are relatively sharp for a 30\u00a0kDa protein, this may indicate some flexibility between the domains of mouse Nat2 or the presence of mobile loops. The HSQC spectrum of mouse Nat2 has been compared with the corresponding spectrum from hamster Nat2 for which a full assignment is available [34]. The pattern of peaks in the HSQC spectra is extremely similar for the two homologous proteins; this allows some peaks observed in the mouse Nat2 spectrum to be assigned on the basis of the hamster Nat2 spectrum.\nThe downfield region of the 1H 1D spectra, collected with a jump-return sequence, of mouse and hamster Nat2 also show a similar pattern of peaks (Fig. 6A and B). The four peaks are compared in Table 4, together with their assignments. Two of the peaks (2 and 3) have been assigned previously for hamster Nat2 and can be assigned in the mouse spectrum by homology; these correspond to strongly hydrogen bonded tryptophan indole groups: the tryptophan associated with the P-loop (Trp132, see Fig. 7) and Trp67, which is located adjacent to the active-site Cys68, and appears to have a structural role. The strong hydrogen bonds are likely to be responsible for the large downfield shifts of these tryptophan peaks compared to random coil values (\u223c9.5\u00a0ppm). The two remaining peaks (1 and 4) are assigned to the imidazole 1HN of histidine on the basis of 15N decoupling experiments; these peaks have not been assigned previously for hamster Nat2. The side chain 1HN groups of histidine are usually not observed by NMR, particularly at pH 7, due to their rapid exchange with solvent protons. The observation of these peaks indicates that the histidine residues are probably buried within the protein and that the side chain 1HN are involved in hydrogen bonds. Analysis of the homology model of mouse Nat2 suggests that at least one of these peaks might arise from the active site histidine, His107. The homology model of mouse Nat 2 indicates that the side chain of His107 is involved in two hydrogen bonds in the active site and is the least accessible to solvent. In addition, recent enzymological studies with hamster Nat2 have confirmed that the active-site cysteine and histidine residues exist as a thiolate-imidazolium ion pair [35], in which the histidine has two tightly bound and H-bonded protons.\n3.6\nInteraction of mouse Nat2 with exogenous steroid inhibitors\nIn order to probe the molecular interactions of mouse Nat2 and the exogenous steroid inhibitors, we have investigated the effect of bisphenol A on the 1D 1H NMR spectrum of mouse Nat2 (Figs. 8 and 9). The observation of changes in chemical shift for particular amino acid residues which result from the addition of a ligand are usually interpreted as an indication that these residues are located in close proximity to the ligand-binding site. When bisphenol A was titrated into a solution of mouse Nat2, the two downfield histidine peaks (1 and 4, Table 4) were observed to shift by more than 0.05\u00a0ppm (Fig. 9); peak 1 shifts downfield by \u223c0.12\u00a0ppm and peak 4 shifts upfield by \u223c0.08\u00a0ppm in the presence of 8 equivalents of bisphenol A. Additional small shifts were also observed for the side chain HN of Trp67 and Trp132 (Fig. 9). The observation of progressive changes in chemical shift upon addition of bisphenol A indicates fast exchange on the NMR timescale; this would be expected given the relatively weak affinity of mouse Nat2 for this ligand (IC50\u00a0=\u00a0290\u00a0\u00b1\u00a010\u00a0\u03bcM). We have postulated above that at least one of the histidine peaks arises from the active-site histidine, His107; therefore, these results show that binding of bisphenol A is likely to take place in close proximity to the active site of mouse Nat2.\n4\nDiscussion\nThe possibility that human NAT1 may serve as a marker for sub-dividing populations of different breast cancers is intriguing. It may also be that human NAT1 will find a role as a target for breast cancer therapies. In order to exploit these findings fully, it will be essential to have an animal model. There have been, for a long time, suggestions that human NAT1 has an endogenous role. The mouse equivalent of human NAT1 is mouse Nat2. Genetic deletion of mouse Nat2 has not created an overt phenotype; however, some of these studies in mice indicate that Nat2 deletion gives rise to skewed sex ratios both on mixed [36] and pure [37] genetic backgrounds, indicating a gender-dependent phenotype, discernable at the population level, that is consistent with a role in endocrine function. There is now also clear evidence that mouse Nat2 has a role in vivo in folate catabolism [38]. These studies, together with the data on human NAT1 and breast cancer, are intriguing and are likely to provide clues as to the endogenous role of NAT1. The data presented increases the knowledge of mouse Nat2 in comparison with human NAT1 and reinforce the notion that mouse Nat2 is a good homologue of human NAT1. Mouse Nat2 has a similar substrate specific activity profile to human NAT1.\nThe inhibition studies show that mouse Nat2 is inhibited by endogenous steroids and, like human NAT1, is inhibited by tamoxifen. Bisphenol A, which is used for making polycarbonates [39], has been reported to activate the oestrogen receptor [40]. Like tamoxifen, it is also an inhibitor of mouse Nat2. We have used bisphenol A to look at the binding of an oestrogenic stimulatory compound to mouse Nat2 and show by NMR that bisphenol A interacts with mouse Nat2 close to the active site region. These studies could not be done directly with tamoxifen because of the high concentrations of protein (and hence ligand) required for NMR experiments under which conditions tamoxifen is insoluble. Both endogenous and xenobiotic oestrogens have been shown to modify breast cancer risk, and tamoxifen is widely used for breast cancer therapy (reviewed in [41]). The effect of different synthetic or natural oestrogens on cell proliferation has been analysed directly, in breast cancer cell lines, and in gene expression studies [41\u201344]. Although the dose\u2013response curves for different oestrogenic compounds vary with the assay system, bisphenol A is generally less active than tamoxifen, genistein or diethylstilbestrol, which reflects the order of potency of mouse Nat2 inhibition. Circulating plasma concentrations of the phytoestogen genistein have been found to be as high as 18\u00a0\u03bcM after a soy-based meal [45], and intratumoral levels of tamoxifen can reach micromolar concentrations [46]. Whether the effects on inhibition of Nat2 activity in vitro may be physiologically relevant for breast cancer prevention or therapy will require further analysis.\nThese studies pave the way for investigation of the role of mouse Nat2, the human NAT1 homologue, in studies of oestrogen receptor positive breast cancer. This is particularly relevant in view of the observation that mouse Nat2 is located histologically in the mammary gland epithelial cells [20]. The structural studies also provide a rational basis for identification of mouse Nat2 inhibitors. We have recently screened a 5000-strong compound library using human NAT1, human NAT2, mouse Nat1 and mouse Nat2; from these studies, several compounds been have identified which are potent and specific inhibitors of human NAT1 and its mouse homologue, Nat2. An accumulation of information on the eukaryotic NAT proteins at a structural level will allow further development of chemical tools for investigation of the role of this marker in breast cancer in humans, and its potential in animal models.","keyphrases":["breast cancer","steroid","xenobiotic","arylamine n-acetyltransferase\/nat","enzyme inhibition","selective estrogen receptor modulator\/serm"],"prmu":["P","P","P","M","R","M"]}
{"id":"Acta_Neuropathol-4-1-2386160","title":"Cellular distribution of vascular endothelial growth factor A (VEGFA) and B (VEGFB) and VEGF receptors 1 and 2 in focal cortical dysplasia type IIB\n","text":"Members of the vascular endothelial growth factor (VEGF) family are key signaling proteins in the induction and regulation of angiogenesis, both during development and in pathological conditions. However, signaling mediated through VEGF family proteins and their receptors has recently been shown to have direct effects on neurons and glial cells. In the present study, we immunocytochemically investigated the expression and cellular distribution of VEGFA, VEGFB, and their associated receptors (VEGFR-1 and VEGFR-2) in focal cortical dysplasia (FCD) type IIB from patients with medically intractable epilepsy. Histologically normal temporal cortex and perilesional regions displayed neuronal immunoreactivity (IR) for VEGFA, VEGFB, and VEGF receptors (VEGFR-1 and VEGFR-2), mainly in pyramidal neurons. Weak IR was observed in blood vessels and there was no notable glial IR within the grey and white matter. In all FCD specimens, VEGFA, VEGFB, and both VEGF receptors were highly expressed in dysplastic neurons. IR in astroglial and balloon cells was observed for VEGFA and its receptors. VEGFR-1 displayed strong endothelial staining in FCD. Double-labeling also showed expression of VEGFA, VEGFB and VEGFR-1 in cells of the microglia\/macrophage lineage. The neuronal expression of both VEGFA and VEGFB, together with their specific receptors in FCD, suggests autocrine\/paracrine effects on dysplastic neurons. These autocrine\/paracrine effects could play a role in the development of FCD, preventing the death of abnormal neuronal cells. In addition, the expression of VEGFA and its receptors in glial cells within the dysplastic cortex indicates that VEGF-mediated signaling could contribute to astroglial activation and associated inflammatory reactions.\nIntroduction\nThe vascular endothelial growth factor (VEGF) family includes seven members which are structurally homologous, but display molecular and functional diversity [58, 81]. VEGFA, the most well known member of the VEGF family, is a crucial regulator of angiogenesis and vascular permeability in both physiological and pathological conditions such as tumor growth, chronic inflammation, and ischemia [10, 16, 19]. In addition to the unquestioned role in angiogenesis, it has recently been shown that VEGFA has direct trophic effects on neuronal and glial cells in the central nervous system [10, 22, 25, 53].\nVEGFB is most closely related to VEGFA [46, 81]; however, the biological function of VEGFB is less well characterized than the function of VEGFA. VEGFB is expressed early during development and appears to have prominent expression in the central nervous system [1, 36]. Additionally, VEGFB has been shown to function as an angiogenic and neuroprotective protein [29, 63, 68] and recent evidence suggests a role for VEGFB in neurogenesis [69, 70].\nThe diverse functions of VEGF proteins can be explained by their differential binding to signaling VEGF receptors [VEGFRs; VEGFR-1 (Flt-1), VEGFR-2 (Flk1\/KDR), and VEGFR-3 (Flt-4) [50, 81]]. VEGFA binds to VEGFR-1 and VEGFR-2, whereas VEGFB binds specifically to VEGFR-1 and not to VEGFR-2 [81].\nThe VEGF-signaling pathway, involving both neuronal and glial cells, has been implicated in several neurological disorders, including neurodegeneration, stroke, and cerebral and spinal trauma [10]. In addition, expression of VEGFA is upregulated in neuronal and glial cells after epileptic seizures in rats [13], suggesting a role for VEGFA in seizure disorders. A recent study points to a neuroprotective role for VEGFA following status epilepticus [45]. The relevance of these findings in animal models to human epileptic disorders is uncertain. Using serial analysis of gene expression (SAGE), we recently identified the VEGFB gene to be upregulated in human tissue from a patient with focal cortical dysplasia (FCD) and intractable epilepsy compared to control cortex (Boer et al., unpublished observations). Upregulation of VEGFA and its receptor has also been recently shown in the hippocampus of cases of human temporal lobe epilepsy (TLE) [56]. However, the distribution of VEGFA, VEGFB, and VEGFRs in epilepsy-associated human malformations of cortical development has not yet been defined.\nIn the present study, we investigated the expression of both VEGFA and VEGFB and their receptors (VEGFR-1 and VEGFR-2) in patients with FCD, which is a developmental disorder known to be a major cause of intractable epilepsy [73]. We report the specific cellular distribution, including both the neuronal and the glial components of the dysplastic cortex, and we discuss the potential role of VEGFA, VEGFB, and their receptors in the histogenesis and epileptogenesis of this developmental lesion.\nMaterials and methods\nSubjects\nThe cases included in this study were obtained from the databases of the Departments of Neuropathology of the Academic Medical Center (University of Amsterdam; UVA) in Amsterdam and the University Medical Center in Utrecht (UMCU). We examined surgically resected tissue from nine patients undergoing epilepsy surgery for focal cortical dysplasia. Informed consent was obtained for the use of brain tissue and for access to medical records for research purposes. The tissue was obtained and used in a manner compliant with the Declaration of Helsinki.\nThe classification system proposed by Palmini et al. [48] was used for grading the degree of FCD and only patients with FCD type IIB located in the temporal lobe were included. The clinical characteristics derived from the patient\u2019s medical records are summarized in Table\u00a01. The predominant type of seizure pattern was that of complex partial seizures, which were resistant to maximal doses of antiepileptic drugs (AEDs; carbamazepine, valproic acid, phenytoin, levetiracetam, oxcarbazepine, and clonazepam). Information concerning the exact time of last seizure occurrence prior to surgical resection was not available. However, all the patients included in our series did not have seizure activity in the last 24\u00a0h before surgery. The patients underwent presurgical evaluation [74]. Intraoperative ECoG was performed routinely in all operations for tailoring of surgery and we classified the post-operative seizure outcome according to Engel [17]. Follow-up period ranged from 1 to 9\u00a0years.\nTable\u00a01Summary of clinical findings of patients with focal cortical dysplasiaPatient\/sex\/age (years)DiagnosisDuration of epilepsy (years)Seizure typeEngel class1\/M\/11FCD IIB11CPSI2\/M\/31FCD IIB20CPSI3\/F\/25FCD IIB9CPSI4\/F\/22FCD IIB21CPS\/SGSI5\/M\/18FCD IIB14CPSI6\/M\/17FCD IIB10CPSI7\/F\/16FCD IIB11CPSI8\/M\/29FCD IIB21CPSI9\/M\/28FCD IIB21CPSIFCD focal cortical dysplasia, CPS complex partial seizures, SGS secondary generalized seizures\nNormal-appearing control cortex\/white matter from temporal region was obtained at autopsy from five adult control patients (male\/female: 2\/3; mean age 42, range 17\u201355) without history of neurological diseases. All autopsies were performed within 12\u00a0h after death (post mortem delay: 11, 11.5, 9, 8.5, 6). The cause of death was represented by acute myocardial infarction. In addition, four of the nine FCD cases contained sufficient amount of perilesional zone (normal-appearing cortex\/white matter adjacent to the lesion), for comparison with the autopsy specimens. This material represents good disease control tissue, since it is exposed to the same seizure activity, drugs, fixation time, and the age and gender are also the same.\nTissue preparation\nTissue was fixed in 10% buffered formalin and embedded in paraffin. Two representative paraffin blocks per case (containing the complete lesion or the largest part of the lesion resected at surgery) were sectioned, stained, and assessed. Paraffin-embedded tissue was sectioned at 6\u00a0\u03bcm, mounted on organosilane-coated slides (Sigma, St Louis, MO) and used for histological and immunocytochemical reactions as described below. Frozen tissue from control cortex and FCD tissue, stored at \u221280\u00b0C, was used for western blot analysis.\nAntibody characterization\nTo document the presence of a heterogeneous population of cells, we used the following antibodies: glial fibrillary acidic protein (GFAP; polyclonal rabbit, DAKO, Glostrup, Denmark; 1:4,000; monoclonal mouse, DAKO; 1:50), vimentin (mouse clone V9, DAKO; 1:1,000), MAP2 (polyclonal rabbit; Chemicon; 1:500), neuronal nuclear protein (NeuN; mouse clone MAB377, Chemicon, Temecula, CA, USA; 1:2,000), non-phosphorylated neurofilament (SMI311; Sternberger monoclonals, Lutherville, MD; 1:1,000), human leukocyte antigen (HLA)-DP, -DQ, -DR (CR3\/43; monoclonal mouse, DAKO; 1:400), CD68 (mouse clone PG-M1, DAKO; 1:200) and CD31 (mouse clone JC70A, DAKO; 1:100).\nFor the detection of VEGFA, VEGFB, and their receptors, the following antibodies (Abs) were used: VEGFA (G153-694, monoclonal mouse; recognizing VEGF 165 and 189 [51], Pharmingen, CA, USA; 1:100), VEGFA (A-20, SC-152, polyclonal rabbit; raised against the N-terminus of VEGFA, recognizing VEGF 121, 165 and 189, Santa Cruz Bio., CA, USA; 1:100), VEGFB (H-70, SC-13083, polyclonal rabbit; raised against amino acids 1\u201370 of human VEGFB, Santa Cruz Bio.; 1:20), Flt-1 (VEGFR-1; C-17, SC-316, polyclonal rabbit, Santa Cruz Bio.; 1:100), Flk-1 (VEGFR-2; A-3, SC-6251, monoclonal mouse; Santa Cruz Bio.; 1:100). To allow comparative analysis, we used on frozen specimens of normal (n\u00a0=\u00a03) and FCD tissue (n\u00a0=\u00a02), two additional antibodies (which are not suitable for staining paraffin-embedded, formalin-fixed tissue): VEGFR-1 (clone Flt-19, 1:400; developed against the recombinant human extracellular domain of VEGFR-1) and VEGFR-2 (clone KDR-1, 1:400; developed against the recombinant human extracellular domain of VEGFR-2), kindly provided by Dr. H. A. Weich (National Research Center for Biotechnology, Braunschweig, Germany), and previously characterized on human tissues [49, 64, 80]. Similar immunoreactivity patterns were observed on paraffin-embedded and frozen tissue.\nThe specificity of the antibodies used for immunocytochemistry on paraffin-embedded, formalin-fixed tissue (VEGFA, VEGFB, VEGFR-1, VEGFR-2; Santa Cruz Bio.), was further tested by performing western blot analysis of total homogenates of human control cortex. We also include one FCD case of which sufficient frozen material for blot analysis was available (Fig.\u00a01). VEGFR1 and VEGFR-2 receptor proteins were detectable as a band of approximately 180 and 200\u00a0kDa, respectively; VEGFB was detectable as a band of approximately 40\u00a0kDa; VEGFA labeled a prominent band at approximately 48\u00a0kDa and a light band at 21\u00a0kDa (reducing conditions), as recently reported in human brain tissue (control hippocampus and FCD; [56]). All immunoreactive bands disappeared after preadsorption with the corresponding peptide.\nFig.\u00a01Representative immunoblot of VEGFA, VEGFB, VEGFR-1, and VEGFR-2 in total homogenates from control cortex and FCD tissue. Expression of \u03b2-actin (as reference protein) is shown in the same protein extracts\nFor immunoblot analysis, human normal cortex (n\u00a0=\u00a03) and FCD (n\u00a0=\u00a01) samples were homogenized in lysis buffer containing 10\u00a0mM Tris (pH 8.0), 150\u00a0mM NaCl, 10% glycerol, 1% NP-40, Na-orthevanadate (10.4\u00a0mg\/ml), 5\u00a0mM EDTA (pH 8.0), 5\u00a0mM NaF, and protease inhibitor cocktail (Boehringer Mannheim, Germany). Protein content was determined using the bicinchoninic acid method [65]. Non-reducing conditions were used to improve the detection of the VEGFA antibody, as previously reported [8]. For electrophoresis, equal amounts of proteins (30\u00a0\u03bcg\/lane) were separated by sodium dodecylsulfate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis in 7.5\u201312.5% gels. Separated proteins were transferred to nitrocellulose paper for 1\u00a0h and 30\u00a0min, using a semi-dry electroblotting system (BioRad, Transblot SD, Hercules, CA, USA). Blots were incubated overnight in TTBS (20\u00a0mM Tris , 150\u00a0mM NaCl, 0.1% Tween, pH 7.5)\/5% non-fat dry milk, containing the primary antibody (VEGFA, VEGFR-1, and VEGFR-2, 1:1,000; VEGFB, 1:200). After several washes in TTBS, the membranes were incubated in TTBS\/5% non-fat dry milk\/1% BSA, containing the goat anti-rabbit coupled to horseradish peroxidase (1:2,500; Dako, Denmark) for 1\u00a0h. After washes in TTBS, immunoreactivity was visualized using an enhanced chemiluminescence kit (Amersham, Buckinghamshire, UK). Expression of \u03b2-actin (monoclonal mouse, Sigma, St Louis, MO; 1:50,000) was used as reference protein. Because of the limited availability of frozen material from FCD cases, a complete analysis with statistical comparison between control and FCD by immunoblot could not be performed.\nImmunocytochemistry\nSections were deparaffinized, re-hydrated, and incubated for 20\u00a0min in 0.3% H2O2 diluted in methanol to quench the endogenous peroxidase activity. Antigen retrieval was performed by incubation for 10\u00a0min at 121\u00b0C in citrate buffer (0.01\u00a0M, pH 6.0); sections were washed with phosphate-buffered saline (PBS), and incubated for 30\u00a0min in 10% normal goat serum (Harlan Sera-Lab, Loughborough, Leicestershire, UK). We incubated the sections with the primary antibodies overnight at 4\u00b0C. Hereafter, sections were washed in PBS and we used the ready-for-use Powervision peroxidase system (Immunologic, Duiven, The Netherlands) and 3,3\u2032-diaminobenzidine as chromogen to develop the staining. Sections were counterstained with hematoxylin, dehydrated, and coverslipped. Sections incubated without the primary antibody and excess of the antigenic peptide were essentially blank.\nFor double-labeling studies, after incubation overnight at 4\u00b0C with the primary antibodies, sections were incubated for 2\u00a0h at RT with Alexa Fluor\u00ae 568-conjugated anti-rabbit IgG and Alexa Fluor\u00ae 488 anti-mouse IgG (1:1,000; Molecular Probes, The Netherlands). The VEGFR-2 antibody (monoclonal mouse) could only be combined with MAP2 and GFAP (polyclonal rabbit). Sections were analyzed by means of a laser scanning confocal microscope (Bio-Rad, Hercules, CA, USA; MRC1024) equipped with an argon-ion laser.\nEvaluation of immunostaining\nSemi-quantitative evaluation of immunoreactivity\nAs previously reported [3, 54], a semi-quantitative analysis was done using an Olympus microscope and examining in each section, high-power non overlapping fields (of 0.0655\u00a0\u00d7\u00a00.0655\u00a0mm width, each corresponding to 4.290\u00a0\u03bcm2), defined in the center of the lesion using a square grid inserted into the eyepiece. A total microscopical area of 858.050\u00a0\u03bcm2 was assessed per case. Neuronal cell bodies were differentiated from glia and glia-neuronal balloon cells on the basis of morphology. Balloon cells have eccentric nuclei and ballooned opalescent eosinophilic cytoplasm. The staining intensity was evaluated using a semi-quantitative three-point scale where immunoreactivity was defined as: \u2212 absent (0); + moderate (1); ++ strong staining (2); intensity score (Table\u00a02). This score represents the predominant cell staining intensity found in each section for the different cell types (neurons, astrocytes, microglial cells, and balloon cells) as averaged from the selected fields (as previously described [3, 54]).\nTable\u00a02VEGFA, VEGFB, VEGFR-1, and VEGFR-2 distribution in different cellular types in cases of FCD (% of cases with immunoreactive cells)Focal cortical dysplasia (n\u00a0=\u00a09)NeuronsAstrocytesBalloon cells\u2212+++\u2212+++\u2212+++VEGFA022%78%011%89%011%89%VEGFB033%67%78%22%078%22%0VEGFR-1055%45%022%78%033%67%VEGFR-2011%89%55%45% 022% 45%33%FCD focal cortical dysplasia; immunoreactivity: \u2212 not present, + moderate, ++ strong\nFrequency of cell staining\nIn each slice, we assessed the number of neurons and astrocytes labeled by a specific Ab on the total number of each cell type within the lesion using an ocular grid [4]. This frequency score was assigned using three distinct categories: (1) <10%, rare; (2) 11\u201330%, sparse; (3) >30%, high. The product of the intensity and the frequency scores was taken to give the total immunoreactivity score, as previously reported [23, 54].\nFor statistical analysis of data, SPSS for Windows was used. Data were compared using a non-parametric Kruskal\u2013Wallis test followed by a Mann\u2013Whitney test to assess the difference between groups. P\u00a0<\u00a00.05 was taken as the level of significance.\nResults\nHuman material and histological features\nAll nine patients had chronic pharmacoresistant epilepsy and were all seizure-free postoperatively (Engel\u2019s class I; Table\u00a01). The FCD cases included in this study have all the histopathological features of severe (type IIB) FCD, according to the classification of Palmini et al. [48]. The resected specimens consisted of disorganized neocortex containing immature neurons, giant neurons, dysmorphic neurons, and balloon cells. Neurons and balloon cells were also observed in the subcortical white matter and there was a prominent population of reactive astrocytes. Cells of the microglia\/macrophage lineage were also observed within the dysplastic cortex, suggesting activation of inflammatory processes in FCD [6].\nExpression of VEGF and VEGFR in normal temporal cortex and FCD\nCellular distribution of VEGFA\nVEGFA staining was observed within the histologically normal cortex (Fig.\u00a02a, b). The staining was strongest in pyramidal neurons, which displayed somatic staining and staining of the apical dendrites (Fig.\u00a02b). Neuropil staining was weak and resting glial cells did not show VEGFA immunoreactivity (IR). Weak staining was observed in endothelial cells. Autopsy material and the perilesional cortex showed similar IR.\nFig.\u00a02VEGFA immunoreactivity in focal cortical dysplasia type IIB. a VEGFA immunoreactivity (IR) within the histologically normal adult cortex. b High magnification showing somatic staining in pyramidal neurons (inset: vascular staining). c VEGFA in focal cortical dysplasia (FCD; type IIB) showing strong IR within the dysplastic cortex. d High magnification showing VEGFA IR in dysplastic neurons (arrows). e A binucleated VEGFA positive dysplastic neuron. f VEGFA IR within the subcortical dysplastic region. g VEGFA expression in a balloon cell. h, i VEGFA expression in reactive astrocytes (arrows in i indicate perivascular astrocytic end-feet) j\u2013l double-labeling of GFAP (green, j) with VEGFA (red, k) shows co-localization (yellow, l) in astrocytes. m\u2013o Double-labeling of non-phosphorylated neurofilament (SMI311; NF, green, m) with VEGFA (red, n) shows co-localization (yellow, o) in dysplastic neurons. p Merged image showing co-localization of CD68 (green) with VEGFA (red) in macrophages. Scale bar in a a, c, f 400\u00a0\u03bcm; b, d 120\u00a0\u03bcm; e, g\u2013i 35\u00a0\u03bcm; j\u2013o 40\u00a0\u03bcm; p 18\u00a0\u03bcm\nIn the majority of FCD cases, strong VEGFA immunoreactivity (IR) was observed in dysplastic neurons located throughout the dysplastic cortex (Fig.\u00a02c\u2013e; Table\u00a02; Fig.\u00a06). Strong staining was also detected in balloon cells, reactive astrocytes, and in perivascular astrocytic end-feet (Fig.\u00a02f\u2013i; Table\u00a02; Fig.\u00a06). Endothelial IR was weak. Double-labeling experiments confirmed expression in reactive astrocytes, neurons, and in CD68+ macrophages (Fig.\u00a02p). Immunocytochemistry using two different antibodies to VEGF (Pharmingen and Santa Cruz Bio.) demonstrated similar patterns.\nCellular distribution of VEGFB\nHistologically normal cortex displayed only weak VEGFB IR (Fig.\u00a03a). Both autopsy and surgical specimens showed light staining in pyramidal neurons and in endothelial cells. Glial cells did not show VEGFB IR.\nFig.\u00a03VEGFB immunoreactivity in focal cortical dysplasia type IIB. a Histologically normal adult cortex, showing neuronal distribution of VEGFB with weak immunoreactivity (IR) in pyramidal cell neurons (high magnification of a pyramidal neuron is shown in the inset aI); weak staining was also observed in blood vessels (inset aII). b VEGFB in focal cortical dysplasia (FCD; type IIB) showing strong IR within the dysplastic cortex. c Strong VEGFB IR in dysplastic neurons of different size and shape (arrows). d Undetectable VEGFB IR in balloon cells (arrows). e\u2013g Absence of co-localization between GFAP (green, e) with VEGFB (red, f) in astrocytes (g, merged image). h\u2013j Double-labeling of non-phosphorylated neurofilament (SMI311; NF, green, h) with VEGFB (red, i) shows co-localization (yellow, j) in dysplastic neurons. k Merged image showing co-localization of CD68 (CD68; green) with VEGFB (red) in macrophages. Scale bar in a a, b 200\u00a0\u03bcm; c\u2013j 40\u00a0\u03bcm; k 18\u00a0\u03bcm\nIn FCD specimens, moderate to strong VEGFB IR was observed within the dysplastic cortex (Fig.\u00a03b; Table\u00a02; Fig.\u00a06) with strong VEGFB IR in dysplastic neurons (Fig.\u00a03c). In the majority of cases (seven out of nine), balloon cells and reactive astrocytes did not express VEGFB (Fig.\u00a03d; Table\u00a02; Fig.\u00a06). Double-labeling experiments confirmed the absence of VEGFB IR in GFAP-positive cells (astrocytes), whereas co-localization was found with neurofilament in dysplastic neurons (Fig.\u00a03h\u2013j). VEGFB IR was also observed in CD68+ macrophages (Fig.\u00a03k).\nCellular distribution of VEGFR-1\nHistologically normal cortex (autopsy and surgical specimens) displayed only weak VEGFR-1 IR, which was restricted to pyramidal neurons (Fig.\u00a04a). IR in blood vessels was weak (Fig.\u00a04b). Glial cells did not show VEGFR-1 IR.\nFig.\u00a04VEGFR-1 immunoreactivity in focal cortical dysplasia type IIB. a, b Histologically normal adult cortex (a) and white matter (b) showing weak immunoreactivity (IR) in neurons (a) and blood vessels (b; arrowheads). c, d VEGFR-1 in focal cortical dysplasia (FCD; type IIB) showing strong IR in dysplastic neurons (c; arrows) and in blood vessels (d; arrows). Inset in c Co-localization between non-phosphorylated neurofilament (SMI311; NF, green) with VEGFR-1 (red) in dysplastic neurons. Inset in d Co-localization between CD31 (endothelial marker; green) with VEGFR-1 (red) in blood vessels. e\u2013g Strong IR in balloon cells of different size (arrows) and glial cells (arrowheads in e). h\u2013j Co-localization between GFAP (green, h) with VEGFR-1 (red, i) in astrocytes (j, merged image). k\u2013m Co-localization of CD68 (CD68; green, k) with VEGFR-1 (red, l) in macrophages (m, merged image). Scale bar in a a\u2013d and h\u2013m 40\u00a0\u03bcm; e\u2013g 35\u00a0\u03bcm\nIn FCD specimens, moderate to strong VEGFR-1 staining was observed within the dysplastic cortex (Fig.\u00a04c\u2013g; Table\u00a02; Fig.\u00a06). VEGFR-1 IR was observed in different cell types, including dysplastic neurons, astrocytes, and endothelial cells. Double-labeling experiments confirmed the co-localization of VEGFR-1 IR with neuronal (inset in Fig.\u00a04c), endothelial (inset in Fig.\u00a04d), and glial (Fig.\u00a04h\u2013j) markers. Additionally, expression of VEGFR-1 was observed in CD68+ macrophages (Fig.\u00a04k\u2013m).\nCellular distribution of VEGFR-2\nVEGFR-2 staining was observed within the histologically normal cortex (autopsy and perilesional zone) in pyramidal neurons (Fig.\u00a05a). IR in blood vessels was weak (Fig.\u00a05b). Glial cells did not show VEGFB IR.\nFig.\u00a05VEGFR-2 immunoreactivity in focal cortical dysplasia type IIB. a, b Histologically normal adult cortex (a) and white matter (b) showing moderate immunoreactivity (IR) in pyramidal cells (a); in the white matter, endothelial IR was weak and glial IR was under detection level (b). c, d VEGFR-2 in focal cortical dysplasia (FCD; type IIB) showing strong IR in dysplastic neurons (arrows in c), but weak IR in blood vessels (arrowheads in d). e, f Strong IR in a dysplastic neuron (arrow in e) and in a balloon cell (arrow in f), but weak IR in glial cells (arrowheads in e, f). Inset in e Co-localization between non-phosphorylated neurofilament (SMI311; NF, green) with VEGFR-2 (red) in a dysplastic neuron. g\u2013i Co-localization between MAP-2 (green, g) with VEGFR-2 (red, h) in balloon cells (i, merged image). Scale bar in a a\u2013d and g\u2013i 40\u00a0\u03bcm; e, f 35\u00a0\u03bcm\nIn the large majority of FCD cases (eight out of nine), VEGFR-2 was strongly expressed in dysplastic neurons (Fig.\u00a05c, e; Table\u00a02; Fig.\u00a06). VEGFR-2 IR was also detected in balloon cells, but only three out of nine cases displayed strong staining for VEGFR-2 (Fig.\u00a05f; Table\u00a02). Endothelial expression was weak. In many FCD cases (five out of nine), expression of VEGFR-2 was undetectable in reactive astrocytes (Table\u00a02). Double-labeling experiments confirmed the co-localization of VEGFR-2 IR with neuronal markers (neurofilament or MAP2, Fig.\u00a05e, g\u2013i) within the dysplastic cortex.\nFig.\u00a06Distribution of immunoreactivity scores (total score; see details in \u201cMaterials and methods\u201d section) in neurons and astrocytes of control, perilesional, and FCD specimens. a, e VEGFA; b, f VEGFB; c, g VEGFR-1; d, h VEGFR-2. a\u2013d Neurons; e\u2013h astrocytes. IR scores of VEGFs and VEGFRs in neurons of FCD were greater than IR scores of VEGFs and VEGFRs of control and perilesional cortex; scores of VEGFA and VEGFRs in astrocytes of FCD were greater than IR scores of VEGFA and VEGFRs of control and perilesional cortex (P\u00a0<\u00a00.05). There were no significant differences in IR scores of VEGFs and VEGFRs between control and perilesional cortex\nDiscussion\nIn addition to their role in angiogenesis, VEGF proteins and their receptors have been implicated in several neurological disorders, including epilepsy [13, 44, 56]. In the present study, we demonstrate a prominent expression of VEGFA, VEGFB, and their signaling receptors in FCD type IIB, a malformation of cortical development associated with intractable epilepsy. This is particularly interesting in view of the recently proposed role of VEGFs and their signaling pathways during development and in epilepsy-associated pathologies [13, 26, 33, 69, 76].\nExpression of VEGFA and VEGFB in normal temporal cortex\nIn histologically normal temporal cortex (autopsy and perilesional zone), we have shown weak expression of both VEGFA and VEGFB in cortical neurons. Expression of VEGFA and VEGFB, including both mRNA and protein, has been demonstrated in neurons in adult rodent brain [39, 41, 42, 72]. In human adult brain, only few studies have described neuronal expression of VEGFA in control tissue [8, 78], which was similar to our observed staining pattern in the control temporal specimens.\nTo our knowledge, previous studies of VEGFB protein expression in human control cortex have not been described. However, VEGFB mRNA was detected in human hippocampal cortex [24], and VEGFB mRNA and protein expression has been described in adult rodent brain [36, 42]. In control rat brain, VEGFB was constitutively expressed in endothelial cells [42]. In our study, we observed only weak endothelial VEGFB IR in blood vessels. In agreement with previous studies [8, 36, 78], immunoreactivity (IR) for both VEGFs was not observed in glial cells within control specimens.\nDifferential cellular distribution of VEGFA and VEGFB in FCD\nIn the present study, we provide evidence for a consistent expression of both VEGFA and VEGFB within the dysplastic cortex of patients with FCD. Both VEGFs are highly expressed in dysplastic neurons; however, only the VEGFA protein is prominently expressed in reactive astrocytes. Expression of VEGFA in astrocytes has been shown in several other pathologies associated with reactive gliosis, such as ischemic stroke, traumatic brain injury, neurodegenerative disorders, and the hippocampus following entorhinal deafferentation [8, 27, 59, 78, 79]. In addition, we previously reported upregulation of both neuronal and glial VEGFA expression in patients with hemimegalenchephaly, an epilepsy-associated malformation of cortical development [5, 7]. In the present study, we also observed expression of VEGFA in balloon cells, which are characteristic cell types of severe FCD [48]. Whether these cells are glial or neuronal in nature is still controversial [12].\nInduction of both neuronal and astroglial VEGFA expression has been shown in different experimental models of seizures and human temporal lobe epilepsy (TLE) [13, 44, 56]. Rigau et al. [56] showed increased levels of VEGFA in the hippocampus of several cases of TLE, including two cases of focal dysplasia. All TLE cases showed VEGFA expression in pyramidal neurons and granule cells of the hippocampus [56]. However, immunocytochemical analysis of the temporal cortex and the FCD cases was not performed.\nThe molecular mechanism underlying the induction of VEGFA expression after seizures remains unclear. One possible mechanism, which has been proposed to explain the association between seizure activity and the induction of VEGFA expression, is represented by the stabilization of the hypoxia inducible factor-1\u03b1 (HIF-1\u03b1). HIF-1\u03b1 is a transcription factor which upregulates VEGFA transcription under hypoxic conditions [21, 38, 61]. Hypoxia may occur during seizures, representing an important trigger in the induction of VEGFA expression, particularly in case of long lasting seizures, such as in status epilepticus models. However, VEGFA expression is already induced after acute seizures [44] and the mechanisms that regulate VEGFA expression are complex. Several transcription factors, including AP-1, HIF-1\u03b1, and NF-\u03baB, have been identified to regulate VEGFA expression [31], and recently it has also been shown that inflammatory cytokines, such as interleukin-1\u03b2 (IL-1\u03b2), activate HIF1\u03b1 and VEGFA gene expression in primary human astrocytes [2]. Interestingly, increased expression of proinflammatory cytokines and related molecules has been reported in both animal models and human epilepsy-associated pathologies, including FCD [3, 15, 54, 77]. In addition, VEGFA has been demonstrated to be a key mediator of the inflammatory process [14, 35]. Thus, we might speculate that the prominent expression of VEGFA within the dysplastic cortex could be a critical component of the complex cascade of events leading to a chronic inflammatory state and the sustained seizure activity [76, 77]. With respect to inflammation, inflammatory cells, such as macrophages, can also release various angiogenic cytokines including VEGFs [71]. Accordingly, we observed expression of both VEGFA and VEGFB in macrophages (CD68-positive cells), as previously shown in animal models of brain ischemia [11, 42, 52].\nVEGFA effects can also compromise the integrity of the blood\u2013brain barrier (BBB; [60]). Interestingly, alterations of the BBB permeability have been recently observed in both human and experimental temporal lobe epilepsy with positive correlation between the increased vascular permeability and the occurrence of spontaneous seizures in chronic epileptic rats [55, 56, 75].\nIn contrast, several studies highlight a dichotomous function of VEGFA, also demonstrating a neuroprotective role [13, 47]. Administration of VEGFA and neuronal expression of VEGFA have been shown to stimulate neurogenesis in vitro and in vivo [9, 28]. In addition, it has been suggested that the neuroprotective effects of VEGFA are mediated by the neuronal VEGFR-2 and the subsequent activation of the PI3K\/Akt survival pathway [32, 67].\nVEGFB expression is not induced by hypoxia or several transcription factors known to regulate VEGFA expression [18], as the promoter region of VEGFB lacks HIF-1 and AP-1 sites [43, 62]. The regulation of the expression of VEGFB remains unknown. Since all cases examined were associated with epilepsy, we cannot exclude that chronic seizure activity could also contribute to the VEGFB expression in FCD specimens. Alternatively, since VEGFB expression has been shown to be prominent during early brain development [36], the strong neuronal expression of VEGFB could represent an intrinsic and immature feature of the dysplastic neuronal cells that could contribute to their survival. Recent studies using VEGFB knock-out mice demonstrate a neurotrophic and neuroprotective activity of VEGFB, exerting a direct action on neurons, and promoting neurogenesis [68, 69].\nThis is an observational study and we were, therefore, not able to investigate the spatio-temporal regulation of the VEGF system. Further research in animal models of cortical dysplasia is clearly needed to elucidate the role of VEGFs and their signaling pathways in the histogenesis or epileptogenesis of developmental disorders.\nExpression of VEGF receptors in normal temporal cortex\nIn histologically normal temporal cortex (autopsy and perilesional zone), VEGFR-1 and VEGFR-2 showed a similar pattern of expression, with weak to moderate immunostaining in pyramidal neurons. Neuronal expression of VEGFRs\u2019 mRNA and protein has been reported in adult human and rodent brain, with strong expression in the hippocampus [8, 11, 78, 79]. In agreement with these studies, we did not detect glial VEGFR expression in histologically normal cortex and only weak VEGFR expression was observed in endothelial cells.\nDifferential cellular distribution of VEGFR-1 and VEGFR-2 in FCD\nConsistent expression of both VEGFR-1 and VEGFR-2 was detected within the dysplastic cortex of patients with FCD. Both receptors were upregulated in dysplastic neurons. Increased expression of the VEGFRs and, in particular VEGFR-1, was observed in reactive astrocytes. Upregulation of VEGFRs in neurons and reactive astrocytes has been shown in several other pathological conditions including ischemia, neurodegenerative diseases, and trauma [8, 11, 37, 39, 66, 79]. Recently, increased expression of VEGFR-2 has been shown in several cases of TLE, including two cases of cortical dysplasia [56]. Immunocytochemical analysis demonstrated expression only in endothelial cells, whereas neuronal VEGFR-2 IR was not detected in either control hippocampus or TLE specimens [56]. Differences in the phenotypes of cells expressing VEGFRs have been observed in several other studies [8, 30, 52]. These discrepancies may be caused by differences in experimental methods, tissue processing, or the use of different antibodies that recognize different epitopes.\nOur results support the neuronal expression of VEGFR-2 recently reported in human brain [8]. The similar expression pattern of VEGFA and VEGFR-2, with prominent neuronal IR, suggests autocrine\/paracrine effects on dysplastic neurons, supporting the hypothesis of a mechanism to protect abnormal neurons from cell death associated with seizures. Autocrine and\/or paracrine effects of VEGFA are supported by the observation that administration of VEGFA has been shown to induce mRNA and protein expression of both receptors in adult rat brain [34, 57]. A protective mechanism of VEGFA has been suggested in epileptic rats, showing that VEGFA may reduce spontaneous discharges in epileptic rats [40]. Therefore, upregulation of VEGFA could represent an endogenous compensatory mechanism to reduce excitability and to prevent cell loss after severe seizures. Accordingly, infusion of VEGFA into the hippocampus has been shown to protect against neuronal cell loss after pilocarpine-induced status epilepticus [45].\nIn the present study, we also provide evidence for the expression of VEGFR-1 in activated cells of the microglia\/macrophage lineage, which have been shown to be present in FCD specimens [6]. This is in agreement with previous in vitro and in vivo studies [11, 20] showing VEGFR-1 expression in activated microglial cells. These observations suggest that the microglia\/macrophage lineage is also a target for VEGF, which may affect chemotaxis and proliferation of these cells, contributing to the inflammatory state in the epileptic brain.\nThere is substantial information about the function and the signaling through VEGFR-2; in contrast, signaling through VEGFR-1 remains poorly understood and has been a matter of discussion. A decoy role has been proposed for VEGFR-1, but more recently, functional signaling via VEGFR-1 has been reported (for reviews see [43, 67]). These observations may give rise to new therapeutic strategies focusing on VEGFR-1 specific ligands, such as VEGFB [43].\nAlthough a rapid induction of VEGF and its receptors has been shown in different experimental models of seizures [13, 44, 56], seizures alone cannot account for changes in neuronal and glial expression in FCD since perilesional tissue was exposed to seizures but did not show significant upregulation of VEGFs and\/or VEGFRs. Therefore, the lesion per se or the concomitant presence of the lesion and the epileptic activity, are likely to play a role in modulating the VEGF system in these developmental disorders.\nConclusions\nOur observed cellular distribution of VEGFA, VEGFB, and their signaling receptors indicate that different cellular components of FCD are involved in VEGF-signaling. In this context, future studies, using both in vivo and in vitro models, will be important to achieve a better understanding of the role of the VEGF-mediated pathways in the histogenesis and epileptogenesis of developmental lesions associated with intractable chronic epilepsy. Presently, signaling via VEGF receptors is not targeted by existing therapies in epileptic patients, but it can be potentially useful in view of its involvement in the regulation of neurogenesis, inflammation, and BBB integrity. However, an effective therapeutic intervention based on modulation of the VEGF system has to take into consideration the specific role of VEGFA and VEGFB and the multiple effects (protective and\/or detrimental) reported for VEGFA.","keyphrases":["vascular endothelial growth factor","receptors","epilepsy","dysplastic neurons","immunocytochemistry","astrocytes"],"prmu":["P","P","P","P","P","P"]}
{"id":"Arch_Dermatol_Res-4-1-2254658","title":"Lymphocyte subsets in peripheral blood of patients with moderate-to-severe versus mild plaque psoriasis\n","text":"In several studies peripheral blood T-cells have been quantified, yet few data are available on lymphocyte subsets in moderate-to-severe psoriasis (in terms of extent and activity of lesions) versus mild psoriasis. The objective is to compare lymphocyte subsets in peripheral blood of patients with moderate-to-severe disease (PASI-score \u226512) to patients with mild disease (PASI-score <12) and to healthy subjects. By means of flow cytometry method, lymphocytes in peripheral blood of 27 patients with psoriasis and 10 healthy controls were characterized. The absolute number of total lymphocytes was markedly decreased in patients with moderate-to-severe psoriasis as compared to patients with mild disease and normal subjects. Cellcounts of all analysed subsets were found to be increased in more severe psoriasis, except for CD8+CD45RO+ cells. The under-representation of CD8+CD45RO+ cells is compatible with the dynamics of acquired immunity, which requires a time log after the relapse of the lesions to differentiate from CD45RA+ naive cells.\nIntroduction\nPsoriasis is a common inflammatory skin disease characterized by hyperproliferation of keratinocytes. It is a well established fact that T-cells play an important role in the pathogenesis of psoriasis [6, 9]. Indeed, treatments such as anti-CD4 [5, 15, 19], anti-CD11 [5, 13] and anti-CD25 [13], targeting at specific T-cells, have shown to be effective in psoriasis. Several T-cell subsets seem to play a primary role. Major classes are CD4+, CD8+, CD45RO+ and CD45RA+ T-cells. Recently, NK-T cells have been suggested to play an additional role in the regulation of immunity, through the release of cytokines [3, 14, 16]. NK-T cells are cells bearing both T cell receptors as well as natural killer-cell specific receptors such as CD161. It has been shown that circulating NK-T cells are significantly reduced in some autoimmune diseases [8, 17].\nThe large body of research on the role of T-cells in psoriasis has been focused on lesional T-cells. This has resulted in the hypothesis that there is a final common pathway responsible for the development of chronic plaque psoriasis, which may involve specific antigen recognition by T-cells that results in stimulation of keratinocyte proliferation. In psoriasis, CD4+ cells seem to be important mainly in the early phase of plaque development. These cells are found predominantly in the upper dermis, whereas CD8+ cells have proven to be relevant during chronic phases and are found predominantly in the epidermis [21], although others have found a more early involvement of CD8+ T-cells in the development of psoriatic plaques [22]. A large number of activated T-cells have been shown in clinically involved skin of psoriatic patients, but also uninvolved skin contains a significant number of T-cells. In healthy skin, however, T-cells can hardly be found. It has been suggested that circulating T-cells are activated and subsequently recruited from the circulation during the development of psoriatic plaques [2, 10].\nSeveral studies have investigated peripheral blood T-cells of psoriatic patients [1\u20133, 7, 10, 12, 14, 20, 21]. These studies indicated that the total amount of T-cells in patients is comparable or slightly increased as compared to that found in normal subjects. No relevant differences have been shown with respect to total T-cell counts and T-cell subsets including CD4+, CD8+, CD45RO+ and CD45RA+ cell counts. Only few studies comprise a comparison between mild and more severe forms of psoriasis, using clinical severity indicators such as the Psoriasis Area and Severity Index (PASI-score) [4, 18].\nThe present study compares specific circulating lymphocyte subsets in patients with mild psoriasis, patients with moderate-to-severe psoriasis and normal subjects.\nMaterials and methods\nPatients and controls\nFifteen patients with moderate-to-severe psoriasis vulgaris (12 male and three female aged 19\u201366, mean age 46.2\u00a0years) and 12 patients with mild psoriasis (seven male and five female, aged 34\u201372, mean age 52.8\u00a0years) from our outpatient department participated in this study. Mean PASI-scores in both groups were 20.97\u00a0\u00b1\u00a02.55 (mean\u00a0\u00b1\u00a0SEM) and 6.11\u00a0\u00b1\u00a01.27, respectively. Patients were classified into one of both groups based on their PASI-score. We considered all patients with a PASI-score <12 as having a \u201cmild\u201d, and all patients with a PASI >12 as having a \u201cmoderate-to-severe\u201d psoriasis [4, 18].\nPatients were free of any systemic therapy for at least 4\u00a0weeks and did not use any topical therapy in the last 2\u00a0weeks. Peripheral blood was obtained from all subjects with their written informed consent.\nControl samples were collected from 10 healthy volunteers without any history or signs of skin disease (four male and six female aged 24\u201349, mean age 33.8\u00a0years).\nPreparation of PBMC\u2019s\nFor each patient, the exact amount of blood withdrawn was determined by measuring the height of the column of blood in the tube, which was subsequently converted to the corresponding volume in microliter.\nPBMC\u2019s were isolated from heparinized blood by density centrifugation on polyester gel (Becton Dickinson Vacutainer\u2122 CPT\u2122, Franklin Lakes NJ, USA). After filtering through a 70\u00a0\u03bcm cellstrainer, cells were washed twice.\nFor flow cytometry, single-cell suspensions (concentration 5\u00a0\u00d7\u00a0105\u00a0cells\u00a0ml\u22121) were stained in 1% fetal calf serum in phosphate-buffered saline (PBS) at concentrations recommended by the manufacturer. Additionally, 450\u00a0\u03bcl propidium iodide (PI) in PBS was added to each sample in order to exclude non-viable cells from analysis.\nThe following monoclonal antibodies were used: fluorescein isothiocyanate (FITC)-conjugated anti-CD4, FITC-conjugated anti-CD8, phycoerythrin (PE)-conjugated anti-CD4, PE-conjugated anti-CD8, PE-conjugated anti-CD94 and PE-conjugated anti-CD161 (all from Immunotech, Marseille, France), as well as FITC-conjugated anti-CD45RA (Becton Dickinson, San Jose, CA, USA) and FITC-conjugated anti-CD45RO (DAKO, Glostrup, Danmark).\nFlowcytometric analysis\nCells were analyzed with an EPICS Elite flow cytometer (Coulter, Luton, UK), using the forward scatter as a discriminator. Lymphocytes were identified by gating on CD45 and side and forward scatter properties. All samples were processed within 18\u00a0h of phlebotomy.\nThe following (combinations of) PE and FITC-conjugated reagents were used to determine the expression of each antigen or antigen combination on lymphocytes derived from peripheral blood: CD4+ (marker for T-helper cells), CD8+ (marker for cytotoxic T cells), CD45RA+ (marker for na\u00efve T-cells), CD45RO+ (marker for memory T-cells), CD94+ (marker for NK-cells, T cells), CD161+ (marker for NK-cells, T-cells with memory phenotype), CD4+ CD45RA+, CD4+ CD45RO+, CD8+ CD45RA+, CD8+ CD45RO+, CD8+ CD94+ and CD8+CD161+.\nDetermination of absolute numbers of lymphocytes\nEnumeration of positive cells was performed by adding Flow Count Beads (Beckman Coulter, Fullerton, CA, USA) to the cell suspension of PBMCs. An automatic stop after a defined amount of beads was programmed on the flow cytometer. Absolute counts of lymphocyte subsets per blood sample were calculated by determining the ratio of the beads to the cell population and then multiplying this ratio by the number of beads in the tube. By dividing this count by the amount of microliters in the tube, a relative absolute lymphocyte count in cells\u00a0\u03bcl\u22121 blood withdrawn was obtained. Analysis was performed with Verity software.\nRatios for CD4+\/CD8+ cells, CD45RO+\/CD45RA+ cells, CD4+CD45RO+\/CD4+CD45RA+ cells and CD8+CD45RO+\/CD8+CD45RA+ cells were calculated afterwards, with data derived from this analysis.\nStatistical analysis\nData-entry and analysis was performed using Statistica 6.0 software. Means and standard deviations were calculated for each parameter and were tested with one-way ANOVA. Differences were considered statistically significant at p\u00a0<\u00a00.05.\nResults\nIn total, 27 patients with psoriasis and 10 normal subjects without signs or symptoms of skin disease were included in the present study. Table\u00a01 summarizes the demographic details and psoriasis related characteristics of the patients with moderate-to-severe psoriasis, patients with mild psoriasis and normal subjects. Four out of 12 patients with mild psoriasis had shown a minimal increase in the extent of lesions; in the others the skin abnormalities were stable. With respect to the 15 patients with moderate-to-severe-psoriasis, 12 had an increase of the extent of lesions during the previous 4\u00a0weeks, whereas the other three had been stable.\nTable\u00a01Demographic and psoriasis related characteristics of patient and subjects participating in the study (mean\u00a0\u00b1\u00a0SEM)Moderate-to-severe psoriasisMild psoriasisNormal subjectsDemographicsAge (years)46.2\u00a0\u00b1\u00a03.4552.8\u00a0\u00b1\u00a03.4933.8\u00a0\u00b1\u00a03.1Male:female ratio 12:37:54:6Psoriasis related characteristicsPASI-score 20.97\u00a0\u00b1\u00a02.556.11\u00a0\u00b1\u00a01.27NADuration (years)16.7\u00a0\u00b1\u00a02.722.2\u00a0\u00b1\u00a02.5NA\nTotal cell counts\nThe total amount of gated cells (per microliter blood withdrawn) in normal subjects was 2,220\u00a0\u00b1\u00a0301\u00a0cells\u00a0\u03bcl\u22121 (mean\u00a0\u00b1\u00a0SEM). The cell-count in mild psoriasis was 1,923\u00a0\u00b1\u00a0230\u00a0cells\u00a0\u03bcl\u22121. In contrast, patients with moderate-to-severe psoriasis had cell counts of 498\u00a0\u00b1\u00a095\u00a0cells\u00a0\u03bcl\u22121. No statistically significant difference was observed between normal subjects and patients with mild psoriasis. However, a highly significant difference (p\u00a0<\u00a00.0001) was observed between mild and severe psoriasis. The CD4+\/CD8+ ratios in all three groups were in the same range, meaning no shift occurred in the distribution of these subgroups of peripheral blood lymphocytes.\nThe total number of lymphocytes, and the CD4+ and CD8+ counts of the patients with moderate-to-severe disease were also routinely measured at the GLP certified Central Haematology Laboratory in our hospital, in which the whole blood lysing method is the standard. Comparison of both methods reveals an acceptable resemblance in results, with lower counts of maximum 10%.\nLymphocyte subsets\nLymphocyte subsets as a percentage of total gated cells are summarized in Fig.\u00a01. CD4+, CD8+, CD45RA+ and CD45RO+ cells are increased in patients with severe psoriasis as compared to patients with mild psoriasis and normal subjects (p\u00a0<\u00a00.0001 in all cases).\nFig.\u00a01CD4, CD8, CD45RO and CD45RA subpopulations, expressed as a percentage of total lymphocyte counts. Comparison between normal subjects, patients with mild- and patients with moderate-to-severe psoriasis. (*: p\u00a0<\u00a00.0001)\nNo significant differences were observed between patients with mild psoriasis and normal subjects.\nDouble labeling of lymphocytes revealed that the percentages of CD4+CD45RO+, CD4+CD45RA+ and CD8+CD45RA+ cells were significantly increased in moderate-to-severe versus mild psoriasis (p\u00a0<\u00a00.001 in all cases), whereas the percentage of CD8+CD45RO+ cells was not increased significantly (Fig.\u00a02). Cell ratios for CD4+\/CD8+ cells, CD45RO+\/CD45RA+ cells, CD4CD45RO+\/CD4CD45RA+ cells and CD8CD45RO+\/CD8CD45RA+ cells are summarized in Fig.\u00a03.\nFig.\u00a02Expression of CD4+CD45RO+, CD4CD45RA+, CD8+CD45RO+ and CD8CD45RA+ cells, as percentage of the total amount of lymphocytes, in three subgroups: moderate-to-severe psoriasis, mild psoriasis and normal subjects (*: p\u00a0<\u00a00.001)Fig.\u00a03Ratios of CD4\/CD8, CD45RO+\/CD45RA+, CD4CD45RO+\/CD4CD45RA+ and CD8CD45RO+\/CD8CD45RA+ in normal subjects, mild and moderate-to-severe psoriasis (*: p\u00a0<\u00a00.05)\nLymphocytes expressing NK cell receptors\nCD94+ and CD161+ cells, expressed as a percentage of total cells were increased in patients with moderate-to-severe psoriasis, as compared to patients with mild psoriasis and normal subjects (p\u00a0<\u00a00.05 and p\u00a0<\u00a00.001, respectively). Figure\u00a04 summarizes these observations. Again no statistically significant differences were observed between normal subjects and patients with a mild form of psoriasis.\nFig.\u00a04CD94 and CD161 subpopulations, expressed as a percentage of total lymphocyte counts. Comparison between normal subjects, patients with mild- and patients with moderate-to-severe psoriasis (*: p\u00a0<\u00a00.05 and p\u00a0<\u00a00.001 respectively)\nWith respect to double stained cells, we observed an increase in the expression of both CD8+CD94+ cells as well as CD8+CD161+ cells (markers for NK T subpopulations) in patients with a more extensive form of psoriasis, expressed as a percentage of the total amount of cells, as compared to patients with mild psoriasis and normal subjects (p\u00a0<\u00a00.008 and p\u00a0<\u00a00.009, respectively, results shown in Fig.\u00a05).\nFig.\u00a05Cellcounts of CD8+CD94+ and CD8+CD161+ cells as percentage of total lymphocytes (*: p\u00a0<\u00a00.009)\nCorrelation between PASI-score and lymphocyte subsets and cells expressing NK receptors\nLinear regression analysis revealed no significant correlation between the individual PASI-scores and the quantifications of lymphocyte subsets or cells with NK-cell receptors. Correlation analysis between disease activity and peripheral lymphocytes did not reveal a difference between patients with spreading lesions and those with stable plaque psoriasis.\nDiscussion\nThe results of this study show marked differences in absolute cellcounts between the three patient groups. Our main goal was to investigate whether there is a difference in the occurrence of biomarkers on circulating lymphocytes in mild psoriasis versus more severe forms of the disease. One of the criteria to qualify for a treatment with major systemic therapies such as a biological is often a minimum PASI-score of 12 [4, 18]. We were interested whether this cut-off point also implies a differentiation between mild versus moderate\/severe psoriasis in terms of T-cell and NKT-cell subsets. The results of the present study have shown us that there are indeed marked differences between mild psoriasis and the more severe forms of disease defined by cut-off point PASI 12.\nA decrease of total lymphocyte counts, as percentage of PBMC, and an increase in the percentage of CD4+CD45RO+ cells in patients with moderate-to-severe psoriasis has been reported before [10]. These patients (32 subjects) had an average PASI of 25.7 (10\u201348), and all had had an exacerbation following infection. Patients had a mixed morphology, from guttata psoriasis to chronic plaque psoriasis. In the same study, however, a decrease in the amount of CD4+ cells was described, which is in contrast with the findings in our study. Other authors have reported no significant difference in total T-cells as percentage of PBMC counts between patients with psoriasis and normal subjects [3]. These patients (14 subjects) had an average PASI of 23.5\u00a0\u00b1\u00a010.6. However, no information was given whether these patients had relapsing psoriasis. In the present study, the patients with severe psoriasis had an extension of lesions during the wash out period (average increase in PASI: 4.9). Therefore, the severity in our patients not only comprises the extent of lesions but also activity of lesions with progression and therefore, approaches the characteristics of the patients reported by Lecewicz-Tor\u00fan et al. [10], who also reported a decrease of T-cells in peripheral blood in psoriasis (expressed as percentage of total PBMC). The decrease of lymphocyte counts, expressed as a percentage, in peripheral blood in patients with moderate-to-severe psoriasis is the reverse of the substantial accumulation of lymphocytes in psoriatic skin. The most likely explanation for this paradox is an increased influx of cells into the skin of patients with severe disease. Further studies, in which simultaneous measurements of lesional and circulating T cells will be performed, need to be carried out to support this hypothesis.\nA striking observation is the increased percentage of both CD4+ cells and CD8+ cells in patients with moderate-to-severe psoriasis as compared to mild psoriasis and normal subjects, without a change in CD4\/CD8 ratio. So far, reports on the CD4+\/CD8+ ratio in psoriasis have been at variance [10, 11].\nWith respect to the double labeled lymphocytes, both the percentage of CD4+CD45RO+ cells as well as the percentage of CD4+CD45RA+ cells is increased in patients with moderate-to-severe psoriasis, in contrast to the absolute decrease of total lymphocytes. The relative count of both subsets is increased, whereas their ratio shows a tendency to decrease (p\u00a0=\u00a00.0509). Thus, with increasing severity of disease, the increase in CD4+CD45RA+ cells seems to exceed the increase in CD4+CD45RO+ cells. An explanation for this phenomenon is the preferential migration of CD4+CD45RO+ cells into lesional skin in extensive disease, resulting in a relative decrease of this subset of CD4+ lymphocytes in peripheral blood. This hypothesis supports earlier studies where CD4+CD45RO+ cells have been shown to be the major players in psoriatic plaques. Indeed, CD4+ cells appear in a very early phase of the psoriatic process. In a previous study, CD4+ cells have been detected in the distant uninvolved skin of patients with active psoriasis [22]. The percentage of CD8+CD45RO+ cells in patients with moderate-to-severe psoriasis approached the percentages in patients with mild psoriasis and normal subjects, in contrast with the CD8+CD45RA+ cells, which did show a significant increase in peripheral blood. The impressive under-representation of CD8+CD45RO+ lymphocytes as compared to CD8+CD45RA+ lymphocytes in moderate to severe psoriasis suggests a preferential influx of CD8+CD45RO+ cells into the skin. Both CD8+ cells, as well as CD45RO+ cells are abundantly present in psoriatic lesional skin. Previously, we have shown that in the outer margin of the spreading psoriatic plaque the number of CD8+ cells and CD45RO+ cells were increased [22]. Therefore, the relative under-representation in peripheral blood of CD45RO+ cells, in particular the CD8+ CD45RO+ cells, most likely reflect the influx of these cells into the skin in patients with moderate-to-severe psoriasis in an active phase of disease. However, further studies are needed to support this hypothesis. Lymphocyte counts, preferably T-cells, in skin as well as in the peripheral blood should be monitored during a longer period of time, e.g., during exacerbation and during disease-free intervals. The CD8+CD94+ cells and CD8+CD161+ cells show a marked increase in patients with moderate-to-severe psoriasis as compared to mild psoriasis and normal subjects. The increases of these lymphocytes expressing NK receptors in peripheral blood are accompanied with increases of these cells in the psoriatic lesion.\nThe results of the present study suggest that in peripheral blood of patients with moderate-to-severe psoriasis, in contrast to the vast presence of T-cells in skin lesions, the absolute lymphocyte counts are profoundly decreased, without any change of CD4\/CD8 ratio. The relative under-representation of CD45RO+ cells, as compared to CD45RA+ cells, in peripheral blood most likely results from a massive influx of memory effector T-cells into the psoriatic lesions in an active phase of disease. Both in blood and skin of patients with moderate-to-severe psoriasis the percentages of CD94+ and CD161+ cells are increased. These results are compatible with the functions of these cells. CD45RO+ cells differentiate from CD45RA+ cells and represent acquired immunity, whereas NK-T cells are able to act without prior sensitization, representing innate immunity [3]. In further research, more selective lymphocyte markers are required in order to investigate the role of peripheral blood T-cells in the pathogenesis of psoriasis, and correlation studies between peripheral blood T-cells and T-cells in skin are required in order to understand the dynamics of compartmentalization of T-cell subsets belonging to either the acquired or the innate immunity system in psoriasis.","keyphrases":["lymphocytes","peripheral blood","psoriasis","t cells","severity of disease"],"prmu":["P","P","P","P","P"]}
{"id":"Pituitary-3-1-1915635","title":"Progressive improvement of impaired visual acuity during the first year after transsphenoidal surgery for non-functioning pituitary macroadenoma\n","text":"Objective Improvement of visual field defects continues even years after the initial surgical treatment. Because this process of continuing improvement has not been documented for visual acuity, we audited our data to explore the pattern of recovery of visual acuity until 1 year after transsphenoidal surgery for non-functioning pituitary macroadenoma.\nIntroduction\nNon-functioning pituitary macroadenomas (NFMA) are the most prevalent macroadenomas [1, 2]. Patients with NFMA mainly present with decreased visual acuity, visual field defects and hypopituitarism, caused by mass effects of the tumor [3\u20135]. Transsphenoidal surgery is the treatment of choice, resulting in improvement of visual field defects in 75\u2013100% of all patients [3\u20135]. Accordingly, visual acuity improves in the majority of patients [6\u201310], although visual field defects and visual acuity may worsen in a limited number of patients after surgery [6, 7, 9, 10].\nThe process of visual fields recovery starts directly after surgery and can already be documented on the second postoperative day [11]. This process of recovery is probably due to restoration of the velocity of conduction. However, improvement of visual field defects continues even years after the initial surgical treatment [12, 13]. Although a reduced visual acuity is correlated with the extent of visual field defects [13, 14], postoperative changes in visual acuity do not parallel changes in visual fields in every case [6].\nTo our knowledge, the process of gradual visual improvement, has only been observed for visual field defects, but not for visual acuity [12]. This is, however, clinically relevant, since improvement of minor visual field defects can occur unnoticed, whereas improvement in visual acuity is almost invariably noticed, and can potentially overcome thresholds to previous impairments in daily life. We therefore audited our data to explore the pattern of recovery of visual acuity until 1\u00a0year after transsphenoidal surgery for non-functioning pituitary macroadenoma.\nPatients and methods\nPatient selection\nForty-three patients, mean age 56\u00a0\u00b1\u00a014\u00a0years, were included in this retrospective analysis. In order to obtain a homogenous cohort for assessment, the inclusion criteria were the following:Compression of the optic chiasm on MRI by the pituitary tumor prior to surgeryTranssphenoidal surgery for non-functioning pituitary macroadenoma (diameter\u00a0>\u00a01\u00a0cm) for decompression of the optic chiasmAssessment of visual acuity at least once before surgery, and at least twice in the first year after surgeryThe availability of two postsurgical MR scans, within a time frame of maximal 3\u00a0months of the ophtalmological assessments\nWe did not restrict the inclusion criteria to patients with visual field defects, but also included patients with clear compression of the optic chiasm without decreased visual function, for two reasons. First, we were interested in the pattern of change in visual function prior to surgery. Second, it is, at least theoretically, possible that in patients with compression of the optic chiasm, surgical treatment may decrease visual function. To overcome the potential effect of postoperative tumor-regrowth on visual outcome, patients were excluded from analysis if the 1\u00a0year post-operative scans revealed growth of residual tumor.\nFor complete assessment, we reviewed the patient records of all departments involved in the treatment of NFMA (Endocrinology, Neurosurgery, Ophthalmology). Endocrine (pituitary function) and ophthalmologic data (visual acuity and visual fields) were assessed before surgery, 3 and 12\u00a0months after surgery. An MRI was performed before surgery, 3\u20136\u00a0months and 12\u201315\u00a0months after surgery. Transsphenoidal surgery was performed by one of two neurosurgeons\nCorrected visual acuity was determined by the Snellen chart [15, 16] and was scored for both eyes. Visual fields were assessed by Humphrey perimetry in all patients. Goldman perimetry was used as an additional tool to assess peripheral visual field defects. To increase the reliability of the visual field testing, assessment of the visual fields, and thereby also visual acuity, was preferably performed twice prior to surgical treatment. However, surgical treatment was not delayed due to double ophthalmologic testing in patients with severe impaired visual acuity.\nDefinitions\nThe diagnosis of non-functioning pituitary macroadenoma was based on two criteria: the presence of a pituitary macroadenoma (>1\u00a0cm) on MRI, and the absence of overproduction of any of the pituitary hormones. In all cases the diagnosis was histologically confirmed. Tumor size was classified according to Hardy [17]. Tumor extension was classified as suprasellar (Hardy II0, A, B, C) or parasellar\/infrasellar (Hardy IID,E, Hardy III0-E and Hardy IV0-E).\nIn all patients, visual acuity was scored on a scale between 0 and 1.25. Visual field defects were scored semi-quantitative and classified as mild, moderate or severe. Visual field defects were classified as mild, if there were peripheral defects in only one quadrant. Defects were classified as moderate if the upper quadrants were affected, whereas in combined upper and lower quadrant field defects, these were classified as severe.\nGrowth hormone (GH) deficiency was defined as an insufficient rise in GH levels (absolute value\u00a0<\u00a03\u00a0\u03bcg\/l) after stimulation during an insulin tolerance test (ITT). When secondary amenorrhoea was present for more than 1\u00a0year premenopausal women were defined as LH\/FSH deficient. Postmenopausal women were defined as LH\/FSH deficient, when gonadotropin levels were below the normal post-menopausal range (LH\u00a0<\u00a010 U\/l, FSH\u00a0<\u00a030 U\/l). In men, LH\/FSH deficiency was defined, as a testosterone level below the reference range (8.0\u00a0nmol\/l). TSH deficiency was defined as a total or free T4 level below the reference range. ACTH deficiency was defined as a basal cortisol level at 8.00\u00a0AM of <0.12\u00a0\u03bcmol\/l and\/or an insufficient increase in cortisol levels (absolute value\u00a0<\u00a00.55\u00a0\u03bcmol\/l) after an ITT.\nStatistical analysis\nThe paired t-test was used for paired samples. SPSS software version 12.0 (SPSS Inc., Chicago, IL, USA) was used. A P-value of <0.05 was considered statistically significant.\nResults\nPreoperative patient characteristics (Table\u00a01)\nForty-three patients, 51% male, were included in this study. Radiological imaging by MRI revealed a macroadenoma in all patients, with suprasellar extension in 100% and parasellar\/infrasellar extension in 42% of cases. Hypopituitarism was present in 79% of all patients, and panhypopituitarism in 23%.\nTable\u00a01Patient characteristics before transsphenoidal surgeryMale\/female22\/21Age (years\u00a0\u00b1\u00a0SD)56\u00a0\u00b1\u00a014Visual acuity Right eye (mean\u00a0\u00b1\u00a0SD)0.65\u00a0\u00b1\u00a00.37Left eye (mean\u00a0\u00b1\u00a0SD)0.60\u00a0\u00b1\u00a00.32Visual field defectsSevere60%Moderate17%Mild14%None9%Pituitary functionGH deficiency76%LH\/FSH deficiency69%ACTH deficiency43%TSH deficiency36%Panhypopituitarism23%MRI characteristicsSuprasellar extension100%Infrasellar\/parasellar extension42%\nPre-operative assessment of visual function (Table\u00a01)\nVisual fields were normal in 4 patients. Transsphenoidal surgery in these patients was performed because compression of the optic chiasm was evident on the MR scan (n\u00a0=\u00a04). In these 4 patients visual acuity was at least 1.0 for both eyes. Of all patients with visual field defects 60% were classified as severe, 17% as moderate, and 14% as mild. Mean visual acuity was 0.65\u00a0\u00b1\u00a00.37 for the right eye, 0.60\u00a0\u00b1\u00a00.32 for the left eye.\nVisual acuity was assessed twice before surgery in 34 patients (79% of total). The median time between the first and the second pre-surgical assessment was 4\u00a0weeks (range 1\u201345). No significant decrease in visual acuity was observed within this time period. The mean visual acuity for the two pre-surgical assessments was: 0.65\u00a0\u00b1\u00a00.37 vs. 0.66\u00a0\u00b1\u00a00.38 (right eye), and 0.60\u00a0\u00b1\u00a00.32 vs. 0.62\u00a0\u00b1\u00a00.33 (left eye). Moreover, no decrease in visual acuity was observed in patients (n\u00a0=\u00a013) with a time interval of >8\u00a0weeks between the first and the second pre-surgical assessment. In 4 of these patients there were no visual field defects.\nSurgical treatment\nAll patients were treated by transsphenoidal surgery. Repeat surgery within 6\u00a0months after initial treatment was performed twice (large residual tumor mass (n\u00a0=\u00a01) and persisting liquor leakage (n\u00a0=\u00a01)). Repeat surgery was performed by transcranial approach.\nTranssphenoidal surgery was followed by radiotherapy in 10 patients in order to prevent recurrence. Patients received 40\u00a0Gy (n\u00a0=\u00a05) or 46\u00a0Gy (n\u00a0=\u00a05).\nThree months postoperative assessment of visual function\nAfter transsphenoidal operation in patients with preoperative visual field defects improvement was observed in 60% and normalization of the visual fields in 30%. In 1 patient there was a slight increase in visual field defects.\nVisual acuity improved significantly 3\u00a0months after transsphenoidal surgery (Fig.\u00a01). The mean visual acuity increased from 0.65\u00a0\u00b1\u00a00.37 to 0.75\u00a0\u00b1\u00a00.36 (P\u00a0<\u00a00.01) (right eye), and from 0.60\u00a0\u00b1\u00a00.32 to 0.82\u00a0\u00b1\u00a00.30 (P\u00a0<\u00a00.01) (left eye).\nFig.\u00a01The pattern of improvement of visual acuity after transsphenoidal surgery for non-functioning pituitary macroadenomas (n\u00a0=\u00a043)\nOne-year postoperative assessment of visual function\nOne year after initial surgical therapy visual field defects showed continuous improvement in 36% of patients, compared to early postoperative results. In 80% of them, this improvement was accompanied by continuous improvement of visual acuity.\nIn 56% of all patients (n\u00a0=\u00a024) visual acuity shows continuing improvement until 1\u00a0year after surgery. In 8 of them, this improvement was not accompanied by further improvement of visual field defects. Visual acuity was improved 1\u00a0year after transsphenoidal surgery compared to the 3\u00a0months postoperative values (Fig.\u00a01). The mean visual acuity increased from 0.75\u00a0\u00b1\u00a00.36 to 0.82\u00a0\u00b1\u00a00.34 (P\u00a0<\u00a00.05) (right eye), and from 0.82\u00a0\u00b1\u00a00.30 to 0.88\u00a0\u00b1\u00a00.27 (P\u00a0<\u00a00.05) (left eye).\nDiscussion\nThe main aims of surgery in non-functioning pituitary macroadenoma are restoration of visual acuity and visual field defects by decompression of the optic chiasm. Nonetheless, our data indicate that there is no necessity for immediate decompression, since postponement of surgery for several weeks did not result in deterioration of visual acuity. Moreover, in this series of 43 patients, we demonstrated a continuing improvement of visual acuity until 1\u00a0year after transsphenoidal surgery.\nThere is a significant correlation between the severity of visual loss prior to surgery and persisting visual field defects [9, 13, 14]. In our patients in whom visual acuity was assessed twice before surgery, no decrease of visual acuity was observed with a median time interval between two measurements of 4\u00a0weeks. This indicates that postponing surgery for 1\u00a0month does not negatively influence visual outcome, which is in line with the slow growth pattern of non-functioning adenomas [4]. Nonetheless, the delay of surgery should not unnecessarily be prolonged because a significant, inverse, correlation between visual outcome and the prolonged duration of symptoms has been reported [9]. Moreover, especially in patients with rapid loss of visual function or decreased visual acuity due to apoplexy, urgent surgical intervention is indicated.\nThe initial event in the pathogenesis of decreased visual function in pituitary macroadenomas is compression of the optic chiasm. Nerve compression leads to decreased conduction and to demyelination. In an experimental setting, the process of demyelination after nerve compression has been observed even after 2\u00a0days [18, 19]. In case of continuous nerve compression remyelination can be observed after several weeks, although remyelinated fibers do not seem to reach normal thickness and organization structure, and complete demyelinated fibers co-exist [18, 19]. Re-myelinated fibers restore conduction, at least partially, even if the causative nerve lesion is still existing [20, 21]. The improvement of visual dysfunction after surgical treatment is supposed to consist of two, or probably even three, phases [12]. There is an early phase, comprising the first hours and days after surgery. In this early fast phase, the improvement is caused by decompression of the visual pathways, leading to a restoration of signal conduction. Visual recovery has been demonstrated in the first days after surgical treatment [11, 12]. The second phase, i.e., delayed recovery, is pathophysiologically caused by restoration of axonal transport and remyelination, and based on remyelination of the optic nerve vessels. This phase of delayed recovery may last for several years [12, 13]. A precise boundary between the end of the fast phase of recovery and the start of the delayed recovery seems to be artificial, because these two phases reflect different pathophysiological mechanisms, which may co-exist for a certain time-period. The contribution of the first phase of recovery might be larger, given the fact that more than 50% of eventual recovery takes place within the first 3\u00a0months after surgery [13].\nIt is already known that visual acuity improves in the first months after surgical treatment [6\u201310] and that the improvement of visual field defects is a continuing process for at least 1\u00a0year [12, 13]. Kerrison et\u00a0al. [12] showed progressive improvement of visual fields even more than 2\u00a0years after surgical decompression of the optic chiasm. However, they did not demonstrate this same pattern of recovery for visual acuity. This might be due to the relative small number of patients during prolonged follow-up. In the present study we demonstrate that also improvement of visual acuity continued 1\u00a0year after surgical treatment.\nThe clinical consequences of the delayed phase of recovery for both visual field defects and visual acuity are obvious. Follow-up of patients after surgical treatment for pituitary macroadenomas should include ophthalmologic assessment within several weeks after surgery, as well as subsequent assessments after one and 2\u00a0years, in order to estimate the final effect of surgery on visual function. Moreover, patients should be told that visual function can continue to improve, at least until 1\u00a0year after surgery. The relevance of these findings are obvious and is of importance to all patients, given the impact of a decreased visual acuity as an independent predictor for a decreased quality of life [22, 23]. Moreover, these data are essential in order to evaluate potential effects of recurrent pituitary adenomas on visual function.\nTen patients in our series received postoperative radiotherapy. However, it is unlikely that this treatment affected the results of our study. In a series reported by Gnanalingham et\u00a0al. [13], in which 34% of all patients received postoperative radiotherapy, persistent improvement of visual field defects was documented even years after surgical therapy. In another series of 21 patients, 2\u00a0years after pituitary irradiation, there were no cases of radiation-induced visual field or visual acuity deterioration [24].\nIn conclusion, this study demonstrates that the improvement of visual acuity, after transsphenoidal treatment for non-functioning pituitary macroadenomas, consists of both an early and a delayed phase of recovery. After initial post-surgical recovery, a progressive delayed improvement of visual acuity at least until 1\u00a0year after transsphenoidal surgery, is likely to occur.","keyphrases":["visual acuity","surgery","non-functioning macroadenoma"],"prmu":["P","P","R"]}
{"id":"Evid_Based_Complement_Alternat_Med-4-2-1876616","title":"Effectiveness of Massage Therapy for Chronic, Non-malignant Pain: A Review\n","text":"Previous reviews of massage therapy for chronic, non-malignant pain have focused on discrete pain conditions. This article aims to provide a broad overview of the literature on the effectiveness of massage for a variety of chronic, non-malignant pain complaints to identify gaps in the research and to inform future clinical trials. Computerized databases were searched for relevant studies including prior reviews and primary trials of massage therapy for chronic, non-malignant pain. Existing research provides fairly robust support for the analgesic effects of massage for non-specific low back pain, but only moderate support for such effects on shoulder pain and headache pain. There is only modest, preliminary support for massage in the treatment of fibromyalgia, mixed chronic pain conditions, neck pain and carpal tunnel syndrome. Thus, research to date provides varying levels of evidence for the benefits of massage therapy for different chronic pain conditions. Future studies should employ rigorous study designs and include follow-up assessments for additional quantification of the longer-term effects of massage on chronic pain.\nIntroduction\nMassage therapy has been defined as soft-tissue manipulation by trained therapists for therapeutic purposes (1). Massage therapy has a long history, being first described in China during the second century B. C. and soon thereafter in India and Egypt (1). More recently, massage therapy has been administered using mechanical devices in addition to hands-on treatment by therapists. Massage can be applied to single or multiple body parts or to the entire body. There are many different types of massage therapy including Swedish massage, Shiatsu, Rolfing, reflexology and craniosacral therapy. Most of the published trials on massage therapy have utilized Swedish or Swedish-type massage.\nDespite the growing popularity of massage, there is inconsistent empirical support for its effectiveness in chronic pain. Although the effects of massage therapy on chronic pain has been the subject of prior reviews (described subsequently), most of these reviews have focused on a single chronic pain condition and the level of supportive evidence appears to vary greatly, depending on the particular pain condition investigated. This review aims to bring together the existing data on the effectiveness of massage therapy for a wide range of chronic, non-malignant pain conditions. (Evidence regarding the effects of massage on acute pain is reviewed under \u2018Meta-Analysis\u2014Massage Therapy Effects on Pain. Chronic pain has been defined by the International Association for the Study of Pain (IASP) as continuous or recurrent pain that persists for longer than the normal time of healing, generally about 3 months (2). It is hoped that by providing an overview of the field, gaps in extant research may be identified in order to inform future clinical trials.\nTypically, massage is viewed as adjunctive therapy to help prepare the patient for exercise or other interventions, and is rarely administered as the main treatment (3). Following the methodology of the Cochrane Collaboration, the focus of this review will be on those studies in which massage for pain relief is delivered alone rather than as part of a treatment package, since it is difficult to draw conclusions regarding the effectiveness of massage when multiple treatments are involved. In addition, the emphasis of this review will be on randomized, controlled trials (RCTs) or quasi-RCTs of massage therapy. As noted above, the main purpose of this article is to provide a broad overview of extant literature on the application of massage to a wide range of chronic pain conditions. Prior reviews have typically focused on a single pain complaint, even though many chronic pain patients present with multiple pain conditions. Thus, the current review summarizes the findings of existing reviews and meta-analyses as well as key individual studies that have appeared since the publication of these comprehensive reviews. Although this approach is limited as it depends heavily on the methodology used in extant reviews and the quality of the methodology likely varied across reviews, it was considered the most feasible approach in order to synthesize the large number of studies examining massage for a broad array of chronic pain conditions.\nThis review is organized as follows. First, empirical findings of effectiveness are presented according to the type of chronic pain condition examined; each of these sections concludes with a summary statement of the level of evidence for the specific pain condition studied. (A summary table of the overall findings is also presented in Table 6.) It should be noted that a review of the findings from the handful of studies on mixed chronic pain problems is also included. Second, the results of a meta-analysis of massage therapy for pain complaints are discussed. This study was unusual in that it examined massage therapy effects across a number of pain complaints. The review concludes with a summary of the findings across the various chronic pain conditions, together with a discussion of putative mechanisms, clinical implications and recommendations for future trials.\nMethods\nThe PubMed, PsychInfo, CINAHL, and Cochrane Library databases searched up to July 2006 using the keywords \u2018massage\u2019, \u2018pain\u2019, \u2018analgesia\u2019 and \u2018analgesics\u2019. As noted above, the focus of this article is to provide a comprehensive overview of the evidence regarding massage therapy for chronic, non-malignant pain. Thus, due to the large number of trials uncovered using the present search strategy, reviews by the Cochrane Collaboration and other authors were used where applicable to identify relevant trials. Primary studies that were excluded by the Cochrane group or others due to methodological or other limitations were therefore not included in the present review. However, findings from relevant primary studies that had been published since these reviews appeared were included. Only those chronic, non-malignant pain conditions that had been examined by at least one controlled trial were included in this review. The studies included in this review focused on adult participants; the application of massage therapy for chronic pain in children has been discussed in a prior review (4,5). The study findings are summarized in Tables 1\u20136. Mean reductions in the main pain outcome measure are shown in the Tables. Since the most commonly used pain outcome measure was a visual analog scale or VAS rating of pain intensity, means for this outcome are reported whenever possible.\nTable 1.Summary of results for studies included in the Cochrane Review on massage therapy (MT) for low back painStudynPain durationControl conditionsOutcomesFindings (Mean reduction in pain)Hsieh et al. (1992) (6)**63Not statedSpinal manipulation (SM); Corset (CT);TranscutaenousOswestry Low Back Pain Q\u2019aireSM (20) > MT (9.2)\u2003\u2003\u2003\u2003muscular stimulation (TMS)Roland-Morris Activity Scale(SM = CT) > MT; SM > TMSCherkin et al. (2001) (11)**26261%=Acupuncture (AC); Self-care education (SC)Symptom scale (0\u201310)MT (2.6) > SC (1.5)>1 year1 year F\/UMT (3.0) = SC (2.3); MT > AC (1.7)Roland Disability ScaleMT > SC; MT > AC1 year F\/UMT = SC; MT > ACUse of Medications (1 year F\/U)MT > SC; MT > ACSF-12 (Physical Health)MT > SC1 year F\/UMT = SC = ACSF-12 (Mental Health)MT > SC; MT > AC1 year F\/UMT = SC = ACHernandez-Reif et al.24Not stated but >6\u2009mProgressive muscle relaxation (PMR)VAS pain intensity (0\u201310)MT (3.9) > PMR (1.6)\u2003\u2003\u2003\u2003(2001) (10)*McGill Pain QuestionnaireMT = PMRRange of Motion (ROM)MT > PMRHoehler et al. (1981) (13)*9548\u201352% < 1\u2009mSMPatient-rated pain (unspecified scale)MT = SM17\u201329% > 6\u2009mStraight-leg raiseMT = SMPope et al. (1994) (7)**16429% < 6\u2009mSM; TMS; CTVAS pain intensity (0\u201310)MT (17.2) = SM (24.1) = TMS (9.6) = CT (15.9)35% = 6\u201312\u2009mROM (flexion\/extension)MT = SM = TMS = CT36% > 2 yearsMaximum voluntary extension effortMT = SM = TMS = CTSorensen fatigue testMT = SM = TMS = CTGodfrey et al. (1984) (12)*81Not stated but <14 daysSM; Low-level electrical stimulation (LES)Pain, stiffness, tenderness (0\u20134)MT = SM = LESDaily activities (0\u20134)MT = SM = LESSelf-reported limitations due to painMT = SM = LESFingertip flexion testMT = SM = LESMelzack et al. (1983) (14)**41Mean = 36.2 wksTranscutaneous electrical nerve stimulation (TENS)McGill Pain Questionnaire (PRI)TENS (85%) > MT (38%) (>50% reduction on PRI)Straight-leg raiseTENS > MTBack FlexionTENS = MTPreyde et al. (2000) (9)**98Mean range = 12.0\u201314.8 wksSoft-tissue manipulation (STM); Exercise (EX);Roland Disability QuestionnaireCMT (5.9) > EX (0.3), SL (0.4); STM (5.2) > EX, SLSham laser therapy (SL)1 month F\/UCMT (6.8) > EX (1.5), SL (0.7); (STM (5.7) = EX) > SLNote: Above were compared with Comprehensive Massage Therapy (CMT) which included STM and EXLumbar ROMCMT = STM = EX = SLNote: **denotes high quality study per Cochrane review (3); *denotes low quality study per Cochrane review (3). The Hsieh and Pope studies reported findings from the same trial.\nTable 2.Summary of findings for studies on massage therapy (MT) for headache painStudy and type of headachenPain durationComparison conditionsOutcomesFindings (Mean reduction in pain)Nilsson et al. (1997) (17)*Cervicogenic Headaches54Not statedSpinal manipulation (SM)\u2003\u2003\u2003\u2003but \u22653\u2009mNOTE: The above was compared with MT plus placeboVAS pain intensity (0\u201310)SMT (17.0) > MT (4.2)LaserHeadache durationSMT > MTWylie et al. (1997) (18)Migraine and67Mean = 10.2AcupuncturePain total index (hours \u00d7 severity)\u2003\u2003\u2003\u2003Tension-type\u2003\u2003\u2003\u2003yearsNOTE: The above was comparedMigraineMT (171.7) > AC (128.0)\u2003\u2003\u2003\u2003with MT plus relaxationTension-typeMT (217.0) = AC (119.5)Both groups combinedMT = ACHeadache index (number \u00d7 severity)MigraineMT > ACTension-typeMT = ACBoth groups combinedMT = ACMigraine DaysMT = ACHanten et al. (1999) (20)65Not statedResting position (RP); No treatment (CON)Tension-typeNOTE: The above was compared with CV-4 techniqueVAS pain intensity (0\u201310)MT (19.3) > [RP (11.2) = CON (7.8)]VAS pain affect (0\u201310)MT > (RP = CON)Note: *this study was included in the Cochrane review of non-invasive physical treatments for chronic\/recurrent headache (15).\nTable 3.Summary of findings for studies on massage therapy (MT) for neck and shoulder pain and carpal tunnel syndromeStudy and type of painnPain durationComparison conditionsOutcomesFindings (Mean reduction in pain)Irnich et al. (2001) (28)VAS pain intensity (0\u2013100)AC (17.3) > MT (3.1); AC = SLA (11.4)Neck pain17721.5\u201322.6% > 5 yearsAcupuncture (AC);3 month F\/UAC (15.0) = MT (8.1); AC = SLA (11.2)Sham laserRange of Motion (ROM)AC > MT; AC = SLA\u2003\u2003\u2003\u2003acupuncture (SLA)3 month F\/UAC = MT; AC = SLAPressure pain threshold (algometer)AC = MT; AC = SLA3 month F\/UAC = MT; AC = SLASpontaneous pain (7 point scale)AC = MT; AC = SLA3 month F\/UAC > MT; AC = SLAMotion-related pain (7-point scale)AC > MT; AC > SLA3 month F\/UAC > MT; AC = SLAGlobal complaints (7-point scale)AC > MT; AC > SLA3 month F\/UAC > MT; AC = SLAHealth-related quality of life (SF-36)AC = MT; AC = SLA3 month F\/UAC = MT; AC = SLADyson-Hudson et al. (2001) (24)Shoulder pain18Mean range = 13.4\u201316.2 yearsACWheelchair user's shoulder pain indexAC (23.3) = MT (21.7)ROMAC = MTMok and Woo (2004) (25)Shoulder pain102Not statedNo treatment control (CON)VAS pain intensity (unspecified scale)MT (14.6) > CON (0.2)State anxiety (STAI)MT > CONHeart rateMT > CONSystolic blood pressureMT > CONDisastolic blood pressureMT > CONvan den Dolder (2003) (27)Shoulder pain29Mean range = 26\u201330 weeksCONMcGill Pain QuestionnaireVAS pain intensity (0\u201310)MT (26.6) > CON (0.1)Present Pain Intensity (PPI) scaleMT = CONVerbal descriptorsMT > CONFunctional DisabilityMT > CONROMMT > CONField (2004) (32)Carpal Tunnel16Mean = 6.7 yearsCONVAS pain intensity (0\u201310)MT (3.2) > CON (.08)\u2003\u2003\u2003\u2003SyndromeGrip StrengthMT > CONState anxiety (STAI)MT > CONProfile of Mood States \u2014 DepressionMT > CONPhysician assessed CTS symptomsMT > CON\nTable 4.Summary of findings for studies on massage therapy (MT) for fibromyalgiaStudynPain durationComparison conditionsOutcomesFindings (Mean reduction in pain)Sunshine et al.30Not statedTranscutaneous electricalImmediate post-session\u2003\u2003\u2003\u2003(1996) (33)\u2003\u2003\u2003\u2003stimulation (TENS); Sham TENS (STENS)State anxiety (STAI)MT = TENS; TENS, MT > STENSProfile of Mood States (POMS) \u2014 DepressionMT = TENS; TENS, MT > STENSSalivary cortisolMT = TENS; TENS, MT > STENSPre- Post TreatmentSelf-Report Interview \u2014 Pain (unspecified scale)MT (3.3) > TENS (0.2); STENS (1.6)StiffnessMT > TENS; STENSFatigueMT > TENS; STENSSleepMT > TENS; STENSDolorimeter testMT > TENS; STENSPhysician assessment of conditionMT = TENS; TENS, MT > STENSDepression (CES - D)MT = TENS = STENSField et al.20Mean years inProgressive muscleImmediate post-session(2002) (34)\u2003\u2003\u2003\u2003treatment = 9.2\u2003\u2003\u2003\u2003relaxation (PMR)STAIMT = PMRPOMS \u2013 DepressionMT = PMRPre- Post TreatmentVAS pain intensity (0\u201310)MT (2.3) > PMR (1.4)VAS fatigue (0\u201310)MT > PMRVAS stiffness (0\u201310)MT > PMRPhysician assessment of conditionMT > PMRNumber of tender pointsMT > PMRCES - DMT > PMRBratttberg (1999) (35)4886% > 5 years;No treatment control (CON)VAS pain intensity (0\u2013100)MT (26.3) > CON (1.4)50% > 10 yearsDisability rating indexMT = CONSleep disturbance (0\u20135)MT = CONHospital anxiety and depression scaleAnxietyMT = CONDepressionMT > CONAlnigenis et al. (2001) (36)37Mean range = 5\u201310.4 yearsUsual care (UC);Arthritis impact measurement scaleUC plus phone calls (UCP)Pain (0\u201310)MT (0.9) = UC (1.0) = UCP (0.3)Rheumatology attitudes indexMT = UC = UCPCES-DMT = UC = UCPQuality of well being scaleMT = UC = UCP\nTable 5.Summary of findings for studies on massage therapy (MT) for mixed chronic painStudynPain durationComparison conditionsOutcomesFindings (Mean reduction in pain)Walach et al. (2003) (37)29Not stated but > 6 monthsUsual care (UC)Pain intensity (1\u20139 point scale)MT (1.0) > UC (0.1)Profile of Mood States (tiredness)MT > UCCES-DMT > UCState anxiety (STAI)MT > UCFrankfurt body concept scalesMT = UCHasson et al.129Not statedProgressive MusclePain (unspecified scale)(2004) (38)but > 3 monthsRelaxation (PMR)Post-treatmentMT (14.5) > PMR (2.1)3 month F\/UMT (.03) = PMR (1.3)Mental energy (unspecified scale)Post-treatmentMT > PMR3 month F\/UMT = PMRSelf-perceived health status (5-point scale)Post-treatmentMT > PMR3 month F\/UMT = PMRPlews-Ogan et al. (2005) (39)30Not stated but > 3 monthsMindfulness-based stress reductionVAS pain unpleasantness ratings (0\u201310)MT (2.9) > UC (.13); MBSR (.7) = UC(MBSR); UC1 month F\/UMT = MBSR = UCSF-12 physical healthMT = MBSR = UC1 month F\/UMT = MBSR = UCSF-12 mental healthMT = MBSR = UC1 month F\/UMBSR > UC; MBSR = MT\nTable 6.Summary of overall findingsType of painNumber of studiesResults (number of studies)Quality ranking (global assessment of quality)Low back pain8MT > relaxation (1), education (1), acupuncture (1)1 (good)MT = corsets (2), exercise (1)MT < spinal manipulation (4), TENS (1)Shoulder pain3MT > acupuncture (1), no treatment (2)2 (moderate)Headache pain3MT > rest for migraine (1) and tension-type (1)3 (moderate)MT < spinal manipulation for cervicogenic (1)Fibromyalgia4MT > TENS (1), relaxation (1)4 (preliminary)MT = no treatment (1), usual care (1)Mixed chronic pain3MT > usual care (1)5 (preliminary)MT = relaxation (1), meditation and usual care (1)Neck pain1MT = acupuncture (1)6 (preliminary)Carpal tunnel1MT > no treatment (1)7 (preliminary)Note: Quality Ranking refers to the overall ranking of the quality of the literature on the effectiveness of massage therapy for each chronic pain condition relative to the other chronic pain conditions discussed in this review.\nResults\nEmpirical Findings\nMassage Therapy for Low Back Pain\nThe Cochrane Collaboration recently published a report on the use of massage therapy for non-specific low back pain (LBP) (3). Their comprehensive review included studies published until May 2001 and was substantively amended at the end of January 2002. The review included randomized or quasi-randomized trials testing the use of any type of massage (using hands or mechanical device) as an intervention for LBP. The Cochrane review identified nine publications which reported the results of eight randomized trials (Table 1 for detailed information). Note that the studies by Hsieh et al. (6) and Pope et al. (7) listed in Table 1 reported results from the same trial. One study was in German (8) (results not shown in Table 1) and the remainder were in English. Numerous studies were excluded from the review; many studies were excluded because massage was tested within a treatment package combined with various other therapies. Standardized criteria were applied to the included studies to assess methodological quality. For the eight trials, five were judged to be of high methodological quality (denoted as ** in Table 1) and three were deemed to be of low quality (denoted * in Table 1).\nIn one study (9), massage was compared with a placebo (sham laser). Massage was found to be superior to the placebo treatment. In the other seven trials, massage was compared with various active treatments. These studies showed that massage was superior to relaxation (10), acupuncture (11) and education (11); massage was equal to corsets (6,7) and exercises (9); massage was inferior to spinal manipulation (6,7,12,13) and transcutaneous electrical nerve stimulation (TENS) (14). The single German study showed that acupressure\/pressure point massage techniques provided more pain relief than classical (Swedish) massage (8).\nBeneficial Effects for Subacute and Chronic Non-specific Low Back Pain\nThe Cochrane review concluded that massage therapy may be beneficial for patients with subacute and chronic non-specific LBP, particularly when combined with exercises and education. They also noted that the results of one high quality study showed that the benefits of massage last as long 1 year following the end of active treatment (11). The benefit obtained from massage exceeded that achieved from relaxation, education or acupuncture. However, the beneficial effects may be less than that provided by spinal manipulation or TENS. The Cochrane Review noted that there is insufficient evidence regarding the effects of massage on acute back pain and on specific forms of massage for chronic LBP.\nAlthough the Cochrane review represents a synthesis of the most rigorous trials to date examining massage for LBP, it should be noted that the review based their conclusions on a relatively small number of studies. For example, their conclusions regarding the superiority of massage to relaxation and acupuncture were based on only a single study each, and therefore await further confirmation in future trials. On the other hand, the studies included in the review demonstrated therapeutic effects for massage that exceeded or equaled those obtained from various active treatment conditions. This level of evidence is more encouraging than that obtained from trials showing that massage is superior to no treatment or waitlist control.\nPain Management for Headaches from Massage Therapy\nThe Cochrane Collaboration also published a recent review of non-invasive physical treatments for chronic\/recurrent headache (15). This review included studies published until November 2002 and was substantively updated in May 2004. The review studied five types of headaches (i.e. migraine, tension-type, cervicogenic, mixed tension-type and migraine and post-traumatic headache), and a broad range of treatments were examined. Of the 22 studies that met inclusion criteria, only one trial, reported in two studies (16,17) examined the impact of massage on headaches. In this trial, massage plus placebo laser was compared with spinal manipulation for cervicogenic headache. The findings of this study indicated that spinal manipulation was superior to massage for headache pain intensity, headache duration and medication use (Table 2). The Cochrane review concluded that there is moderate evidence that massage plus placebo laser is inferior to spinal manipulation for pain intensity and duration in relation to cervicogenic headache.\nThe Cochrane review did not include the results of Wylie and colleagues (18) compared massage and relaxation to acupuncture for headache pain in patients with migraine or combined headache and patients with tension-type headaches. Patients received six sessions lasting 45\u2009min. For the massage and relaxation condition, it is unclear how much of each session was devoted to relaxation which included muscle and breathing exercises as well as visualization techniques. All patients exhibited significant decreases in pain total index (PTI; monthly number of headache hours multiplied by severity) and headache index (HI; monthly number of attacks multiplied by severity). For patients with tension-headaches (n = 40), there was no difference in pain outcomes based on intervention type. However, for patients with migraines (n = 27), those who received massage had significantly lower PTI and HI scores than those who received acupuncture (Table 2). The number of migraine days did not vary across treatments. These findings suggest that massage may be superior to acupuncture for migraine headaches. But, because massage was combined with relaxation and other self-help techniques, it is not possible to draw definitive conclusions regarding the specific effects of massage based solely on these findings.\nCraniosacral therapy is based on the notion that movement restrictions in the cranial structures of the skull adversely impact rhythmic impulses conveyed through the cerebral spinal fluid from the cranium to the sacrum (19). Thus, craniosacral therapy is a form of massage that uses gentle pressure on the plates of the patient's skull. Few controlled studies have been conducted on craniosacral therapy and a recent review concluded that there is insufficient evidence to support the effectiveness of this approach (19). One study, not included in the review, examined the CV-4 craniosacral technique on tension-type headaches (20). The CV-4 technique moves with the narrowing and widening of the skull, or the cranial rhythm; the basis of the technique is the compression of the fourth ventricle. Sixty patients were randomly allocated to one of three conditions: (i) a 10\u2009min session during which multiple still points were induced by the CV-4 technique; (ii) a 10\u2009min session during which the head and neck were positioned according to the resting position technique; (iii) a 10\u2009min no treatment control. Immediately following treatment, the CV-4 group reported less pain intensity and pain affect than the control group; there were no differences between the resting position group and controls.\nModerate Evidence for Cranioscaral Massage in Managing Tension-Type Headache Pain\nThere have been surprisingly few published RCT's on massage therapy for headache pain. The single study included in the Cochrane review of non-invasive treatments for recurrent\/chronic headache found that spinal manipulation resulted in greater pain reduction than massage plus sham laser for cervicogenic headache. One other study reported that massage may be more beneficial than acupuncture for migraine headaches but that both approaches were equally effective for tension-type headaches. A single study found that craniosacral therapy led to superior pain reduction compared with rest positioning and no intervention for tension-type headaches. This latter study using CV-4 technique was included in a recent review of manual therapies for tension-type headaches (21). The review authors gave this study a score of 6 out of 10 possible points for methodological quality, suggesting that there is moderate evidence from this trial that the CV-4 technique exerts a beneficial effect on pain related to tension-type headaches. As noted in the review, given that headaches are among the most common problems seen in medical practice (22), there is an urgent need to establish the effectiveness of manual therapies, including massage in the treatment of headache pain. The review also noted that not only is tenderness of the pericranial myofascial tissues one of the prominent features of tension-type headache, myofascial tissues may play an important role in the genesis of such headaches (23). Because the aim of soft tissue manipulation is to alter mechanical stress caused by myofascial tissue disorders, the review concluded that massage techniques may therefore be an effective therapy for tension-type headaches. However, further large-scale studies are needed before conclusions regarding the effectiveness of massage for tension-type or other types of headaches can be drawn.\nShoulder Pain \u2014 Moderate Support for the Use of Massage Therapy\nThree analyses examined the effects of massage for shoulder pain. The first study compared acupuncture to Trager Psychophysiological Integration (a form of massage) in 18 patients with chronic shoulder pain who used manual wheelchairs as their primary means of mobility (24). Each patient received 10 treatments over 5 weeks; the acupuncture sessions lasted approximately 20\u201330\u2009min and the Trager sessions lasted approximately 45\u2009min. By 5-week follow-up, both groups exhibited improvements in pain and range of motion (see Table 3); there were no significant differences between groups. The study authors concluded that both acupuncture and Trager were effective for shoulder pain in wheelchair users. In their meta-analysis of massage therapy effects (discussed subsequently) this study was included as evidence supporting the benefits of massage on the delayed assessment of pain (i.e. pain that is assessed following a period after which no treatment is delivered).\nMok and Woo (25) analyzed hospitalized stroke patients with shoulder pain who were randomly assigned to receive slow-stroke back massage (SSBM) (26) or no intervention control. SSBM was administered for 10\u2009min before bedtime for seven consecutive days. Patients who received SSBM experienced decreases in pain, anxiety, heart rate and blood pressure, compared with no such changes in controls (see Table 3). Another study (27) compared patients with shoulder pain who received six 15\u201320\u2009min sessions of massage over two weeks (n = 15) to a waitlist control (n = 14). Patients who received massage improved significantly in functional disability, pain, and range of motion, whereas the control group evidenced no changes on these outcomes (Table 3).\nThese results provide moderate support for the use of massage for shoulder pain. However, the patient characteristics varied greatly across studies (e.g. stroke patients; wheelchair users) suggesting that generalizability of the findings may be limited. Moreover, with the exception of Mok and Woo (25) these studies had small sample sizes and only the study by Dyson-Hudson et al. (24) included a follow-up assessment. Finally, two of these studies compared massage with no treatment control. Additional studies are needed to determine whether massage therapy leads to improvements in shoulder pain when controlling for non-specific effects of treatment (e.g. increased clinician time\/attention). Replication of these findings of these three studies in larger samples and with longer follow-up evaluation periods is warranted.\nComparison with Acupuncture for Neck Pain\nAnother group (28) examined patients with chronic neck pain randomly assigned to one of the following conditions: acupuncture (n = 56), massage (n = 60), \u2018sham\u2019 laser acupuncture (n = 61). Patients received five 30-min treatments over 3 weeks. For the main outcome, pain VAS (0\u2013100) ratings, acupuncture was superior to massage, but no different than sham laser immediately after treatment. However, by 3-month follow-up, there were no differences between acupuncture and massage or between acupuncture and sham laser (Table 3). Among the secondary measures, acupuncture showed more improvement than massage across most measures assessed immediately post-treatment. Nevertheless by 3-month follow-up, acupuncture was superior to massage only on spontaneous pain, global complaints and motion-related pain.\nEffectiveness of Massage Therapy for Neck Pain Remains Unclear\nThe authors of the previous work concluded that acupuncture appears to be effective in the short-term for chronic neck pain, it should be noted that by the 3-month follow-up, acupuncture was no more effective than massage or sham laser acupuncture on most outcome measures. Unfortunately, the study did not include direct comparisons between the massage and sham laser conditions. Therefore, it is not possible to determine whether massage was superior to a placebo condition. An unpublished master's thesis (29) has been cited in a recent meta-analysis as providing support for the longer-term effects of massage on pain (30). These studies were included in a very recent Cochrane Review on massage for mechanical neck disorders (31), which also included a broad array of interventions such as traditional Chinese massage, ischemic compression, self-administered ischemic pressure using a J-knob cane, and occipital release, among others. The review authors noted that many of these approaches were of questionable value. The review concluded that no practice recommendations could be made since the effectiveness of massage for neck pain remains unclear.\nPreliminary Support for Pain Relief in Carpal Tunnel Syndrome\nPatients with carpal tunnel syndrome (CTS) were randomly assigned to either a 4-week course of massage therapy or usual care (32). The massage group received a 15-min massage once a week from a massage therapist and were also taught self-massage to be done daily at home prior to bedtime. The massage group evidenced improvements in pain, grip strength, anxiety and depression compared with no such improvements in the control group (Table 3). Physician assessments of carpal tunnel symptoms also indicated significant improvements in the massage group versus no change in the control group.\nThe findings of this single study provide preliminary support for the application of massage to CTS. However, the sample size was very small, and it is unclear whether the physicians assessing the patients were aware of group assignment. Moreover, it is unclear to what extent patients practiced self-massage at home and whether the amount of massage administered was related to treatment response. Further work with larger samples and more rigorous study methodology are needed to determine the effectiveness of massage therapy for CTS.\nEquivocal Support for Analgesic Effects in Fibromyalgia\nThe effects of massage therapy on fibromyalgia have been examined in four investigations. Two of these studies were conducted by Tiffany Field and colleagues. In the first study (33), women with fibromyalgia were randomly assigned to receive massage, TENS or sham TENS for 30\u2009min twice a week for 5 weeks. Immediately following treatment on the first and last days, the massage therapy group evidenced reductions in anxiety, depression and salivary cortisol. The TENS group experienced the same improvements but only on the last day. The sham TENS groups demonstrated no such improvements. By the end of treatment, the massage therapy group reported less pain, stiffness, fatigue and difficulty sleeping (as assessed via interviews), as well as improvements in dolorimeter test value and physician's assessment of clinical condition (Table 4). The TENS group improved on this latter measure only. Limitations of this study include inadequate information regarding the interview items. Moreover, it was unclear whether interviewers and physicians were blind to patient group assignment.\nThe Field group (34) also randomly assigned fibromyalgia patients to receive either massage therapy or progressive muscle relaxation (PMR) for 30\u2009min twice a week for 5 weeks. Both groups reported reductions in anxiety and depression immediately following treatment on the first and last days. By the end of treatment, the massage group evidenced significant reductions in self- and physician-assessed pain and symptoms, as well as reductions in the number of tender points and substance P levels (Table 4). No significant improvements were seen in the PMR group.\nBrattberg (35) randomly allocated patients with fibromyalgia to massage therapy or no treatment control. The massage group received 15 massages of indeterminate length over 10 weeks. Comparisons immediately post-treatment revealed greater improvements in pain, depression and quality of life in the massage group relative to controls, but no differences in disability, sleep disturbance or anxiety (Table 4). Brattberg maintained that there was a 37% reduction in pain following the massage treatment but that 30% of the improvement in pain had disappeared by 3-month follow-up and 90% of the reduction in pain was gone by the 6 month follow-up. Thus, it appears that the benefits of massage for fibromyalgia do not persist over the longer-term after the termination of active treatment. Brattberg recommended that following an initial treatment of 15 sessions, maintenance therapy may be instituted (e.g. once or twice per month).\nAnother study randomly assigned 37 patients with fibromyalgia to one of three conditions: massage, usual care, usual care with follow-up phone calls from a nurse (36). The massage group received 10 treatments of indeterminate length over 24 weeks.\nUnfortunately, only 16 patients completed the full study protocol (six patients in the each of the two usual care groups and four patients in the massage group). Although the massage group showed a trend towards greater improvement in pain and self-efficacy for managing their condition, there were no between-group differences by the end of treatment (Table 4), likely due to the small cell sizes.\nSummary \u2014 Massage Therapy for Fibromyalgia\nThe evidence supporting the use of massage for fibromyalgia is mixed. Whereas the Field group has found that massage leads to improvements in pain and symptoms compared with relaxation (33) or TENS (34), their work suffers from methodological limitation such as small sample size, inadequate blinding of assessors and an absence of follow-up assessments. The two other studies found either no benefits for massage (36) or only short-term benefits that eroded over time (35). Replication of the positive results reported by the Field group in an independent research group would increase confidence in their findings.\nApplication to Mixed Chronic Pain Conditions\nWhereas the majority of existing trials of massage therapy have examined patients with discrete chronic pain syndromes, three studies have investigated the impact of massage on patients with a variety of chronic pain complaints. Walach and colleagues (37) conducted a randomized controlled trial comparing massage therapy (10\u201320\u2009min sessions administered twice weekly for 5 weeks) to usual care for patients with various chronic pain symptoms (i.e. lower back, neck, shoulders, headaches). By 3-month follow-up, patients who received massage reported less pain, depression, anxiety and tiredness relative to controls (Table 5). However, the study authors noted that their study was limited due to lack of equivalence across groups on demographic characteristics. Moreover, it is unclear whether the groups were similar in terms of the type of pain complaints represented or important clinical characteristics such as the duration and\/or severity of pain.\nAnother study randomly assigned patients with chronic pain to receive either massage or relaxation (listening to a PMR tape recording) (38). The massage group received 6\u201310 sessions lasting 30\u2009min each; patients were treated 1\u20133 times per week. The relaxation group listened to the audiotape twice a week for 5 weeks. Although the massage group evidenced improvements in pain, mental energy and self-perceived health status compared with the relaxation group immediately following treatment, by 3-month follow-up, there were no differences between groups.\nIn a third analysis, patients with chronic musculoskeletal pain were randomly assigned to mindfulness-based stress reduction (MBSR), massage or usual care (UC) (39). MBSR involved 8 weekly 2.5-h sessions in a group format, with audiotaped meditation exercises assigned as daily home practice. The massage group received 1-h massage once per week for 8 weeks. At post-treatment, the massage group reported less pain unpleasantness and improved mental health compared with the usual care group. However, by 1-month follow-up, there were no differences among the groups in pain intensity or pain unpleasantness (Table 5).\nModerate Support for Short-term Benefits of Massage Therapy for Mixed Chronic Pain Conditions\nThese reports provide modest support for the immediate benefits of massage for a variety of chronic pain complaints. However, it appears that these treatment gains were not maintained following the end of active treatment. A potential difficulty with such studies including heterogeneous pain complaints is ensuring that treatment and control conditions are equivalent on key clinical and demographic characteristics. On the other hand, evidence of therapeutic effects across a variety of pain conditions supports the generalizability of the findings to a potentially broader group of patients. Future studies incorporating samples with mixed chronic pain conditions may also examine which types of pain conditions may benefit most from massage therapy in order to promote a more targeted approach to treatment.\nMeta-Analysis \u2014 Massage Therapy Effects on Pain\nAs noted above, the majority of primary analyses and review articles have focused on the application of massage to discrete pain conditions. Moyer and colleagues (30) have published a recent meta-analysis of massage therapy research that examined the effects of massage for a variety of both chronic and acute pain symptoms. Data across trials were aggregated to investigate these effects across pain conditions on the immediate and delayed assessment of pain. According to Moyer et al. (30), immediate assessment of pain pertains to \u2018single dose effects\u2019 or short-term effects observed on the same day following massage therapy. The delayed assessment of pain reflects \u2018multiple dose effects,\u2019 which refers to outcomes that were assessed at various time points after treatment has been discontinued and following multiple sessions of massage. It should be noted that several of the studies included in this meta-analysis were also included in the Cochrane reviews discussed earlier in this article.\nMoyer and colleagues (30) concluded that massage therapy did not exhibit a significant effect on the immediate assessment of pain. The studies included in this category examined a wide range of pain problems ranging from back pain, neck pain, cancer pain, headaches and fibromyalgia, as well as acute pain in relation to surgery and other procedures (e.g. amniocentesis; cardiac catheterization). However, Moyer et al. (30) did conclude that massage evidenced a significant effect for the delayed assessment of pain. They maintained that patients who received a course of massage and were evaluated several days or weeks after the end of active treatment exhibited levels of pain that were on average 62% lower than controls. This conclusion was based on the results of 5 studies\u2014two of these were conducted with patients experiencing LBP (9,11), one was conducted with patients with tendonitis (40), one was conducted with patients experiencing shoulder pain (24) (discussed earlier in this article), and one was an unpublished master's thesis (29) examining patients with neck pain.\nIt should be noted that the Moyer et al. (30) analysis of the delayed assessment of pain did not include three of the trials discussed earlier in this article that did not find long-term benefits for fibromyalgia (35) and mixed chronic pain (38,39). The latter two studies were published after the Moyer review was completed; it is unclear why the study on fibromyalgia was not included in the meta-analysis. Nevertheless, the rigorous approach employed by Moyer and colleagues, which expressly included the calculation of between-group effect sizes and the aggregation of data across numerous trials lends confidence to their overall conclusion that multiple applications of massage therapy appears to confer lasting benefits on pain. Their work did not however, speak to the time period over which such analgesic effects are maintained, or the rate at which such effects decay. These considerations warrant further examination in additional studies.\nDiscussion\nThe existing literature provides varying levels of support for the effectiveness of massage therapy for chronic pain. The most abundant and rigorous evidence was found for the effects of massage on non-specific LBP. The Cochrane Collaboration (3), concluded that massage therapy may be beneficial for patients with subacute and chronic non-specific LBP, especially when combined with exercises and education. Whereas the evidence supporting the application of massage for LBP is fairly robust, there is less support for the use of massage for the other chronic pain conditions reviewed. This review suggests that the level of evidence for massage therapy effects by pain condition is (in order from most to least): LBP, shoulder pain, headache pain, fibromyalgia, mixed chronic pain, neck pain and CTS.\nAlthough shoulder pain has been the subject of only three studies, all of the studies yielded positive outcomes for pain and were methodologically rigorous, with one study including over 100 patients (25). Moreover, one of these studies was cited by Moyer et al. (30) in their meta-analysis indicating that massage therapy demonstrates significant effects on the long-term assessment of pain. Like shoulder pain, massage for headache pain has only been the subject of three studies but the data are somewhat weaker. The Cochrane Collaboration (15) concluded that there is moderate evidence that spinal manipulation is superior to massage plus placebo laser for pain related to cervicogenic headache, although these conclusions were based on the results of a single trial reported in two studies (16,17). Two additional studies provided preliminary evidence for the benefits of massage and craniosacral therapy in the treatment of pain related to migraine headaches (18) and tension-type headaches (20), respectively. However, in the migraine study (18), massage was combined with relaxation and other self-help techniques, making it difficult to draw conclusions regarding the specific effects of massage.\nThere is considerably less support for the effectiveness of massage therapy in treating the remaining chronic pain conditions. Of the four studies examining massage therapy for fibromyalgia, only two studies, both by the same research group, revealed therapeutic effects (33,34), whereas the other two studies found no benefits (36) or improvements that attenuated over time (35). Thus, there is only modest evidence for the effectiveness of massage for pain related to fibromyalgia. For mixed chronic pain, the three studies to date provide somewhat conflicting findings. Whereas one study found that massage was superior to usual care (37), two other studies found that by follow-up, massage was no better than relaxation (38), mindfulness meditation or usual care (39). Taken together, these studies provide fairly weak support for the application of massage to mixed chronic pain. For neck pain, one trial using conventional massage techniques found that massage was similar to acupuncture by 3-month follow-up. A recent Cochrane review which included a broad array of massage techniques, many of which were considered questionable, reported that no firm conclusions could be drawn regarding the effectiveness of massage for neck pain (31). Only one published trial has investigated massage therapy effects on CTS; this study found that massage was superior to no treatment. Based on these findings, there is only preliminary evidence to support the effectiveness of massage for both neck pain and CTS.\nPutative Mechanisms of Massage Therapy for Chronic Pain\nThe precise mechanism of action in massage therapy is not known. It has been proposed that increased parasympathetic activity (41) and a slowed-down physiological state may underpin the behavioral and physiological processes associated with massage. As discussed by Wright and Sluka (42), massage is thought to induce a variety of positive physiological effects that may contribute to tissue repair, pain modulation, relaxation, and improved mood. For example, these authors point to research showing that massage has beneficial effects on arterial and venous blood flow and edema (43). In addition, they note that vigorous massage has been shown to increase local blood flow and cardiac stroke volume (44), as well as improve lymph drainage (45); massage also appears to have an anticoagulant effect (46). Finally, Wright and Sluka maintain that massage may activate segmental inhibitory mechanisms to suppress pain and that some techniques may activate descending pain inhibitory systems (43), as suggested by gate theory (discussed subsequently).\nThe main theories regarding the analgesic effects of massage include gate theory, the serotonin hypothesis, and the restorative sleep hypothesis (47). According to gate theory (48), pressure receptors are longer and more myelinated than pain fibers, and thus pressure signals from massage are transmitted faster, closing the gate to pain signals. The serotonin hypothesis maintains that massage increases levels of serotonin, a neurotransmitter that modulates the pain control system (49). The restorative sleep hypothesis holds that because substance P, a neurotransmitter associated with pain is released in the absence of deep sleep, the ability of massage to increase restorative sleep reduces substance P and consequent pain (50). There is little definitive data to support these major theories concerning the mechanisms underlying the analgesic benefits of massage.\nClinical Implications: The Application of Massage Therapy for Chronic Pain\nThe existing literature suggests that massage therapy may be a useful approach for pain relief in a number of chronic, non-malignant pain conditions, particularly musculoskeletal pain complaints (e.g., shoulder pain, low back pain). Massage is typically administered as adjunct therapy to help prepare the patient for exercise or other interventions and is rarely administered as the main treatment (3). Thus, massage is not usually considered a first line treatment, but rather as a complement to other conventional first line approaches (e.g., physical therapy; medications). It should be noted that the studies reviewed above did not specifically report on findings regarding possible interactions of massage therapy with other CAM or conventional medicine approaches. Nevertheless, the increasing popularity of massage and the fact that it is typically used as an adjunctive approach with other established treatments suggests that massage may be successfully integrated into the treatment of a variety of chronic or recurrent non-malignant pain conditions. The paucity of data on negative side effects pertaining to massage does not necessarily mean that such effects do not exist. Future work should focus on systematically characterizing those patients for whom massage is not indicated.\nFuture Directions: Critical Issues for Studies on Massage Therapy for Chronic Pain\nThis review highlights the need for continued rigorous research on the effectiveness of massage therapy for chronic, non-malignant pain conditions. Somewhat surprisingly, this review indicated that very few studies to date have focused on massage for pain related to chronic\/recurrent headaches and chronic neck pain. Given that massage promotes relaxation, it would appear to be a particularly appropriate therapy for tension-type headaches as well as migraine related to increased stress. Moreover, at pointed out above, massage therapy may alter the mechanical stress caused by myofascial tissue disorders (21) which have been implicated in tension-type headaches (23). In light of the contradictory findings noted above, future work may also continue to examine massage therapy effects on pain related to fibromyalgia which involves wide-spread, diffuse pain that is often not responsive to traditional approaches.\nNumerous methodological problems were noted in the studies reviewed including small sample sizes, lack of equivalence across treatment and control groups, and inadequate blinding of assessors. However, one of the most notable limitations of the literature as a whole is that very few studies included follow-up assessments. As indicated by Moyer et al. (30) in their meta-analysis, the beneficial effects of massage therapy on pain are predominately evidenced after the end of active treatment. They concluded that such delayed effects on pain were substantial, with patients who were evaluated several days\/weeks after treatment cessation exhibiting on average 62% less pain than controls and one study on LBP revealed significant benefits from massage persisting 1 year after the cessation of active treatment (11). It should be noted however, that the conclusions by Moyer et al. (30) were based on only 5 studies, suggesting that future trials of massage therapy should include follow-up assessments in order to further quantify such delayed effects. Moreover, additional studies may focus on examining the optimal time periods for the scheduling of \u2018booster\u2019 sessions to maintain treatment gains. Previous work has suggested that psychological treatment delivered according to a schedule with increasing time intervals between sessions (e.g. 1, 4, 10 intervening days) is more effective over the long-term compared to a uniform schedule (e.g. 5, 5, 5 intervening days) of treatment delivery (51). Thus, future research may also examine the optimal treatment schedule for delivery of massage therapy with a view to enhancing longer-term analgesic effects.\nLimitations of the Current Review and Concluding Statements\nThe main limitation of the current study is its reliance on existing reviews and meta-analyses. Thus, many of the conclusions drawn in this article are based on the findings of other authors. Relatedly, the soundness of the methodological approach of these existing reviews may have been limited (e.g. due to improper exclusion of specific studies) as well as highly variable across reviews. Nevertheless, the reliance on extant reviews was considered necessary in order to synthesize a vast and diverse literature examining a broad array of chronic pain conditions. Another limitation of the present study is that only those pain conditions that were the subject of at least one controlled trial of massage therapy were included. Thus, not all chronic pain problems were examined in this review. It is possible that positive effects for massage therapy on other chronic pain conditions may have been reported in uncontrolled trials and\/or case studies.\nIn sum, this review identified important areas for future research on the effectiveness of massage therapy for chronic, non-malignant pain. Whereas there is fairly robust support for the analgesic effects of massage for non-specific LBP, there is only moderate support for such effects on shoulder pain and headache pain. Extant literature provides only modest, preliminary support for massage in treating fibromyalgia, mixed chronic pain, neck pain and CTS. One of the most important methodological considerations that should be addressed in future trials is the inclusion of follow-up assessments in order to allow further quantification of the longer-term effects of massage therapy on pain. Another key methodological consideration is the inclusion of comparison conditions that control for non-specific effects including physical contact and therapist time and attention. Moyer et al. (30) in their meta-analysis maintained that their positive findings for delayed assessment of pain are consistent with the notion that massage may promote pain reduction by enhancing restorative sleep. However, they note that data on sleep patterns was not included in the studies reviewed and therefore, this possibility remains to be tested. Thus, the careful consideration of potential mechanisms may inform future research, particularly with respect to the inclusion of key outcome variables as well as the examination of possible moderators and mediators of treatment response. Additional rigorous research is needed to establish massage therapy as a safe and effective intervention for the treatment of chronic, non-malignant pain.","keyphrases":["low back pain","headache pain","recurrent pain","musculoskeletal pain","fibroymalgia"],"prmu":["P","P","P","P","U"]}
{"id":"Pediatr_Nephrol-3-1-1766474","title":"Hemodialysis in children: general practical guidelines\n","text":"Over the past 20 years children have benefited from major improvements in both technology and clinical management of dialysis. Morbidity during dialysis sessions has decreased with seizures being exceptional and hypotensive episodes rare. Pain and discomfort have been reduced with the use of chronic internal jugular venous catheters and anesthetic creams for fistula puncture. Non-invasive technologies to assess patient target dry weight and access flow can significantly reduce patient morbidity and health care costs. The development of urea kinetic modeling enables calculation of the dialysis dose delivery, Kt\/V, and an indirect assessment of the intake. Nutritional assessment and support are of major importance for the growing child. Even if the validity of these \u201curea only\u201d data is questioned, their analysis provides information useful for follow-up. Newer machines provide more precise control of ultrafiltration by volumetric assessment and continuous blood volume monitoring during dialysis sessions. Buffered bicarbonate solutions are now standard and more biocompatible synthetic membranes and specific small size material dialyzers and tubing have been developed for young infants. More recently, the concept of \u201cultrapure\u201d dialysate, i.e. free from microbiological contamination and endotoxins, has developed. This will enable the use of hemodiafiltration, especially with the on-line option, which has many theoretical advantages and should be considered in the case of maximum\/optimum dialysis need. Although the optimum dialysis dose requirement for children remains uncertain, reports of longer duration and\/or daily dialysis show they are more effective for phosphate control than conventional hemodialysis and should be considered at least for some high-risk patients with cardiovascular impairment. In children hemodialysis has to be individualized and viewed as an \u201cintegrated therapy\u201d considering their long-term exposure to chronic renal failure treatment. Dialysis is seen only as a temporary measure for children compared with renal transplantation because this enables the best chance of rehabilitation in terms of educational and psychosocial functioning. In long term chronic dialysis, however, the highest standards should be applied to these children to preserve their future \u201ccardiovascular life\u201d which might include more dialysis time and on-line hemodiafiltration with synthetic high flux membranes if we are able to improve on the rather restricted concept of small-solute urea dialysis clearance.\nIntroduction\nThe European Paediatric Dialysis Working Group was established in 1999 by pediatric nephrologists from different European countries with a major interest in dialysis. The group has already published guidelines, mainly on peritoneal dialysis\u00a0[1, 2]. Hemodialysis practices for children have improved over the ensuing 20 years, especially because of technological developments and the evolution from \u201cminimum\/adequate\u201d to \u201coptimum\/maximum\u201d dialysis prescription\u00a0[3]. Therefore, new general recommendations seem necessary.\nThese guidelines were initiated and discussed at meetings of the group and refined by e-mail discussion to develop a consensus of opinion, on the basis of cumulative clinical experience and reported studies. This paper will discuss the main factors affecting hemodialysis prescription and management in children.\nIn some European countries hemodialysis (HD) is often preferred for children over the age of five years\u00a0[3]. In contrast, peritoneal dialysis (PD) is offered to the younger children especially under the age of two years or weighing less than 10\u00a0kg. A multicenter European study has, however, found that 73% of 189 children were older than five years when peritoneal dialysis was started\u00a0[4]. Factors ranked as first priority for choice of therapy\u00a0[4] included age of the child (30%), parent choice (27%), distance from unit (14%), patient choice (11%), social condition (7%), and unable to do one mode (6%). Nevertheless important differences appear in the individual countries. Usually, however, HD is not offered to children less than 5 years old unless there are important contra-indications for PD\u00a0[1]. For older children HD is applied for drop-outs from the PD program or if there are medical (rare) or psychosocial (more often) reasons for not performing PD.\nChoosing a mode of dialysis, either HD or PD, for a child requires consideration, among other factors, of the probable impact of either mode of dialysis on the maintenance of residual renal function (RRF), because of its specific impact on patient outcome. Although there is no general consensus, peritoneal dialysis has been associated with less risk of RRF loss\u00a0[5, 6]. Overall the choice of the mode of dialysis is just a part of the integrated care model, each child should be considered for a combined dialysis-transplantation program.\nProvision of adequate vascular access remains the single greatest obstacle to successful HD, especially in infants. Unlike in the USA, where patients frequently use a central catheter for vascular access\u00a0[7], in Europe an arteriovenous fistula is the most common vascular access for chronic\/long term dialysis\u00a0[8]. According to the K-DOQI guidelines, the percentage of catheters in a dialysis unit for adults should be less than 10%, although many pediatric centers do not meet this standard, because of the difficulty of creating fistulas in smaller children, especially in children less than 2 years of age.\nDuring the past two decades there have been many improvements in the technology\u00a0[3]: bicarbonate used as buffer in the dialysis solution, volumetrically controlled ultrafiltration, smaller dialysis lines and synthetic membranes useful even for babies, modeling of ultrafiltration rate and dialysate composition, on line hemodiafiltration and the concept of ultrapure dialysate, i.e. sterile and pyrogen free. Non invasive technologies to assess patient target dry weight and access flow offer a potential decrease in dialysis morbidity and costs\u00a0[9]. Recently marketed medications to improve anemia, for example erythropo\u00efetin even darbepoietin, and iron infusion, contribute to the clinical improvement of the hemodialysis session\u00a0[10].\nDialysis adequacy quantification by urea kinetic modeling enables a more specific approach to dialysis dosing and indirect assessment of protein intake, despite the limited value of small-solute clearance\u00a0[11]. Nevertheless, it has been widely accepted that clinical results depend at least in part on the dialysis dose delivered\u00a0[12, 13, 14, 15]. In fact, a single center experience shows the beneficial impact of longer dialysis duration\u00a0on clinical outcome in children\u00a0[13]. However, it is now becoming more and more evident that increasing the dialysis dose when delivered only three times weekly is an unphysiological strategy, self limited by the potential increased risk of hemodynamic and electrolytic disturbances\u00a0[16, 17, 18]. In adult care, there is a growing interest in the use of daily dialysis, because long term experience has shown good results\u00a0[16, 17, 18]. In children only a pilot study in one center supported the positive impact of daily dialysis in very non-compliant adolescents\u00a0[19].\nIn children the hemodialysis prescription should be individualized. Choice of the mode of hemodialysis should take into account the presumed waiting time before kidney transplantation as a \u201c justification\u201d for the use of \u201c the best available\u201d mode having the highest cost and, conversely, being supported by very limited\/preliminary studies only\u00a0[13, 19].\nThe importance of the choice of material used for dialysis and its application should not obviate the need for management of the entire child with ESRF, especially regarding optimum nutrition\u00a0[20]. Because dialysis per se is not able to correct completely the numerous functions of the kidney lost during ESRF, medications and dietary recommendations are needed in children on hemodialysis\u00a0[20]. Recombinant growth hormone is often needed considering the growth velocity rate of children on chronic dialysis\u00a0[3].\nGuideline 1: the dialysis unit\nhemodialysis should be delivered in a \u201cpediatric\u201d dialysis center with a multidisciplinary support team which supports individualized and integrated therapy\nnutrition, growth, and educational support are of major importance\nBecause of the specific needs of children, hemodialysis should be delivered at the best, and probably only, in a pediatric dialysis unit\u00a0[3, 4, 7] This includes the treatment of adolescents up to the age of 18 years and beyond depending upon their physical and psychological development and transition arrangements to adult units\u00a0[21]. Taking care of a child with ESRF necessitates an engaged team consisting of doctors, nurses, dietician, psychologist, school teacher, play therapist, and social worker\u00a0[22]. This \u201csecond family or support team\u201d should be multidisciplinary and immediately available to the chronically ill child, both close and distant enough to stimulate normal family life, supporting a proper (school) education, leaving all possibilities open for \u201cfull\u201d integration into society in the future.\nHemodialysis, in contrast with peritoneal dialysis, is usually performed in an hospital setting, with a frequency of three times per week for most patients. This frequency may be increased to address the specific needs of babies and\/or adolescents requiring \u201cmore dialysis\u201d\u00a0[3, 13, 19].\nGuideline 2: water quality\nadequate in terms of biochemical composition\nfree from microbiological contamination\nThe dialysis machine needs water for dialysate production adequate in terms of biochemical composition and free from microbiological contamination, i.e. germs and endotoxins (Table\u00a01). Water purification depends on the disposable water quality. Usually filtration with charcoal and the small sieving coefficient associated with reverse osmosis produces water for dialysis in accordance with the recommendations\u00a0[23] (Table\u00a02). Currently, all new dialysis machines have the ability to filter the dialysate through a high flux membrane, which increases microbiological purity.Table\u00a01 Water contaminants and associated complicationsaDissolved organic materialComplicationsContaminants:- Pesticides, herbicidesNo documentation during dialysis- Chloramines, chlorine compoundsSevere hemolytic anemiaBacteria and pyrogens:- BacteriaBacteremia or septicemiaFever, chills, shakingHypotension and death- PyrogensPyrogenic reaction-feverChills, uncontrollableShaking, vomiting, hypotensionaNot exhaustiveTable\u00a02 Definitions of water and dialysate quality (levels given as an upper limit for water-quality definition\u00a0[70])Bacterial growth (cfu\u00a0mL\u22121)Endotoxin (EU\u00a0mL\u22121)Cytokine-inductionAAMI, water2005+European pharmacopoeiaRegular water1000.25+Ultra-pure0.010.03\u2013Sterile10\u221260.03\u2212\nIn hemodiafiltration using an on-line technique\u00a0[24, 25] with direct production from the dialysate of the hemofiltration substitution fluid, the dialysate benefits from double ultrafiltration, producing an ultrapure dialysate which is sterile and endotoxin free, at least at detectable levels. This ultrapure dialysate should limit the risks related to microbiological contamination, i.e. inflammatory process induction with both acute and chronic consequences\u00a0[23]. This level of ultrapure dialysate is also required for synthetic high-flux membrane use even or especially when used in a conventional hemodialysis mode. Decontamination or sterilization by chemical agents or by heating should be performed in line with water, before final dialysate production by the dialysis machine, by filtration and osmosis installation and water distribution, without any break between sterilization and final dialysate. Quality control of the water for the dialysate should be performed regularly with regard to chemical composition (at least once per year), and final dialysate purity should be assessed with regard to bacteria and endotoxins (more regularly, depending in part on the mode of dialysis, weekly for high-flux membrane use) (Table\u00a02)\u00a0[23].\nGuideline 3: the dialysis machine\nvolumetric ultrafiltration control\noption for both single and double-needle dialysis\nIn the last decade numerous innovations in equipment have been developed by different manufacturers\u00a0[3]. But the relevance to child outcome remains unknown, because of the absence of sufficient controlled study results. Nevertheless the following innovations seem essential: dialysate production by double dilution pumps using volumetric ultrafiltration control and blood pumps with double pumps available for single-needle dialysis.\nOther \u201chigh-tech\u201d innovations only deserve mention because of their limited application in \u201cexpert\u201d centers: individual modeling of the dialysis session with monitoring of ultrafiltration and dialysate solute concentration (i.e. sodium, bicarbonate); polyvalency machine which enables not only conventional dialysis but also hemofiltration and hemodiafiltration providing the highest standard in terms of tolerance and efficiency\u00a0[24, 25]. Newer dialysis machines provide monitoring of hematocrit variation as a major promising innovation\u00a0[9, 26] and direct urea kinetic monitoring\u00a0[27]. There is a restricted offer for blood thermal monitoring to avoid loss of calories to the dialysate or to prescribe cooled dialysate\u00a0[28].\nAll these innovations enable individualized hemodialysis for the children, but their regular application should take into consideration the balance between the expected benefits and the costs.\nGuideline 4: blood lines\navailable in infants\/babies size\nbiocompatible material\nA range of blood lines are available for dialysis of babies to dialysis of the largest adolescent. They should be considered for their biocompatibility, type of sterilization (ethylene oxide-free), and the blood volume required\u00a0[3].\nGuideline 5: principles of blood purification\nsmall solute clearance and more, from diffusion process (urea) to convection (other uremic toxins \u201cmiddle molecules\u201d) mass transport\nhemodiafiltration is an option to consider to obtain \u201cmaximum\u201d dialysis efficiency\nUremic toxin extraction in dialysis\u00a0[3, 24] is related to a combination of the diffusion process and convection mass transport (Table\u00a03). In hemodialysis (HD), blood purification depends mostly on a diffusion process secondary to a concentration gradient, which ensures the best elimination of small molecules (urea). HD clearance (KHD) correlates directly with blood flow rate. In hemofiltration (HF), uremic toxin extraction is mostly dependent on convection mass transport secondary to a pressure gradient, which optimizes the elimination of both low and middle-molecular-weight compounds. HF clearance (KHF) directly correlates with ultrafiltration flow rate which is limited by the blood flow rate. In the post dilution mode, i.e. replacement fluid in the venous line chamber located after the dialyzer membrane, maximum filtrate flow rate is less than half the blood flow rate; it is usually one third, to limit the risks of excessive hemoconcentration. In the predilution mode, i.e. replacement fluid perfusion in the arterial line chamber, which is situated before the dialyzer membrane, maximum filtrate flow rate should be two thirds of or equal to the blood flow rate. Hemodiafiltration (HDF) combines HD and HF simultaneously, which enables blood purification by both a diffusive process and convective mass transport. HDF clearance (KHDF) in post-dilution mode is measured by use of the Granger formula\u00a0[24]:Table\u00a03 Dialyzer membrane permeability: diffusion and convectionDiffusion processConvection mass transportMembrane areaUltrafiltrate flow (QUF)Mass-transport coefficientHydraulic permeabilityConcentration gradientTransmembrane pressure (TMP; mmHg)Blood flow\u00d7extraction coefficientMembrane areaSieving coefficient (S)*ci and co\u00a0are inlet and outlet solute concentrationsMolecular permeability*CUF\u00a0is the ultrafiltrate solute concentration(postdilution)(predilution)KHD is hemodialysis clearance and\nKHF is hemofiltration clearance\nOn replacement of QUF\u00d7S by KHF and Qb by Kmax (maximum achieved clearance) the formula for KHDF becomes:\nThus it is clear that in terms of blood purification KHDF enhances the clearance of a uremic toxin if HF or HD clearances are lower than the Kmax (equal to the blood flow rate). HDF with a highly permeable membrane is as efficient as HD for low-molecular-weight compounds, but is more efficient than HF for low-molecular-weight compounds\u00a0[29]. Moreover, HDF, besides its blood purification efficiency, is associated with a lower intradialytic morbidity rate\u00a0[30, 31], as is HF\u00a0[3]. On-line HDF\u00a0[24, 25], in which filtered dialysate free of toxins and pyrogens is used as replacement fluid, enables use of an elevated convection fluid rate, especially in predilution mode, and facilitates a dialysis dose increase without a cost increase. The use of ultrapure dialysate, i.e. sterile and pyrogen free, as used for on-line HDF dialysate should reduce the diseases possibly associated with chronic inflammation related to contaminated dialysate\u00a0[23], e.g. \u03b22 microglobulin amyloidosis, hypercatabolism with loss of lean body mass, decreased growth rate, fibrosis and cardiovascular diseases. A high flux membrane\u00a0[32], with an elevated ultrafiltration coefficient of permeability enabling backfiltration from the dialysate to the blood compartment, which is called retrofiltration, increases these risks, especially with contaminated dialysate\u00a0[23].\nGuideline 6: extracorporeal blood access and circulation\nfistula vascular access is preferred for long-term chronic hemodialysis\nin young children, less than 15\u00a0kg, the time needed to develop a fistula before it can be used could be some months\nthe double-needle technique is the standard, but single needle with double pump system is an alternative\na single lumen catheter with clamps offers for small children an acceptable compromise between a very low extracorporeal blood volume and valuable dialytic efficacy\ntotal extracorporeal blood volume (needles, tubing and dialyzer) should, approximately, be less than 10% of patient total blood volume\nanticoagulation in the extracorporeal circuit is achieved either with conventional heparin or with low-molecular-weight heparin\nan extracorporeal blood flow rate (QB) of 150\u2013200\u00a0mL\u00a0min\u22121\u00a0m\u22122 or 5\u20137 mL\u00a0min\u22121\u00a0kg\u22121 is often sufficient\nThe success of chronic hemodialysis depends on good vascular access: internal arteriovenous fistulae (AVF), shunt (AVS), graft (AVG) or central venous catheter. The type of access used is variable depending on factors in different units and countries, for example surgical experience, patient age and size, the time available before dialysis must be started, and the presumed waiting time before transplantation. Patient choice plays a major part, especially with adolescents.\nA catheter is more commonly used in the USA than in Europe\u00a0[7, 8]. A catheter can be a primary access particularly in acute renal failure or chronic renal failure with acute presentation, in small children and in the case of a presumed short period on chronic hemodialysis. Internal jugular vein catheter access is superior to subclavian vein; it admittedly preserves the future arteriovenous fistula implantation on the arm. Femoral catheter access should be used only for \u201crescue and transient\u201d access if intensive care is needed: it is easy to perform but with a higher risk of infection and thrombosis. A double lumen cuffed catheter, at least 8 French, is mostly preferred for children and has been reported to have a survival rate as high as 60 to 85% in one year\u00a0[33], or as low as 30%\u00a0[34]. Nevertheless in small infants a single lumen catheter used with the alternative clamps technique offers an acceptable compromise between recirculation and both the amount of extracorporeal blood volume and the achieved blood flow\u00a0[35]. Thrombosis, a major cause of catheter failure, is reported to be between 9 and 46%\u00a0[34]. Thrombosis causing poor flow can be corrected to salvage the catheter by different methods: catheter replacement over guidewire, systemic oral anticoagulation and local urokinase or tissue plasminogen activator instillation\u00a0[36]. Loss of catheter access related to infection has decreased during the last decade; the aggressive use of antibiotics and perhaps antibiotic lock therapy, although not universally accepted, account for this lower rate of infection related catheter loss\u00a0[34, 36, 37].\nMicrosurgery enables creation of a functional AVF at the wrist in most children, even small ones\u00a0[8] but only a few surgeons are trained for vascular microsurgery, which therefore is rarely used. Creation of a fistula at the elbow is a second-choice vascular access. With a non functional cephalic vein, a basilic vein transposition, i.e. superficialization, is possible\u00a0[38]. Synthetic grafts should be reserved for children who have exhausted autologous veins and should be used in children only very rarely. For all these reasons preoperative evaluation of the vessels to determine the correct choice of vein before the operation is mandatory. The non-dominant arm should be regarded as first choice of fistula implantation. The survival rate for a AVF is higher than the survival rate for a catheter, with more than two thirds of the children having a functioning AVF at four years\u00a0[8]. With a basilic vein superficialization the fistula should not be used before full healing (2 to 6 weeks) to avoid a dissecting hematoma. Otherwise the time needed for venous development before use depends on the age of the patient and the place of the AVF (distal or proximal). In small children this period of time is often a delay of months. Before surgery it is essential to avoid venopuncture of the selected arm in the weeks before AVF creation. It is of interest to protect the dominant arm from the beginning of taking care of a child with \u201cchronic dialysis risk\u201d to enable, if necessary, implantation of a fistula. Such venoprotection should not be forgotten for peritoneal dialysis children, even babies\/infants. For a period of time before surgery, especially for small children,\u00a0[8] dilatation of the veins by immersion of the forearm in hot water is advantageous, a maneuver enhanced by placement of a tourniquet. A proximal AVF with a high blood flow, usually close to 1000\u00a0mL\u00a0min\u22121\u00a0m\u22122 , is a risk factor for cardiac failure. Nevertheless, the major complication is thrombosis, consequent to local stenosis. Therefore, follow up of the access flow is essential, on the one hand clinically: auscultation (the sound of the AVF is maximum at the surgical site and decreases with distance from the fistulae), observation\u00a0(elevation of the forearm should induce emptying of the previous dilated veins, and on the other hand by Doppler ultrasound or vascular access flow monitoring\u00a0[9]. Application of regular access flow monitoring can be used to detect vascular stenosis before complete AVF thrombosis\u00a0[9]. But it should be remembered that \u201cTransonic\u201d access flow monitoring can only be performed with double-pump dialysis and is not available for pediatric-sized blood lines.\nThe extracorporeal blood flow rate is achieved through venous puncture, most often via two needles, one for blood aspiration called the arterial needle, one for venous reinjection called the venous needle. The distance between the needles should be sufficient to limit recirculation, which is best prevented by opposite orientation of the needles: the arterial one toward the fistula, the venous one in the opposite direction. Usually the needle size is 17-gauge at initiation of dialysis; thereafter considering patient need and fistula development 16 or 14-gauge needles, particularly in adolescents, can be used to achieve a sufficiently high blood flow rate. Pain related to the puncture should be prevented by anesthetic cream (Emla or Amelop); this advance is important for both the children and nurses\u00a0[39].\nAn extracorporeal blood flow rate (QB) of 150\u2013200\u00a0mL\u00a0min\u22121\u00a0m\u22122 , 5\u20137 mL\u00a0min\u22121\u00a0kg\u22121, is often sufficient to achieve the targeted goals with double needle dialysis; in small children QB is determined using body weight (BW, kg): (BW+10)\u00d72.5=QB (mL\u00a0min\u22121). The arterial blood aspiration pressure should be monitored if possible and kept between 150\u2013200\u00a0mmHg to limit endothelial trauma.\nFor single-needle dialysis in children the highest blood flow rate is obtained with a double pump system (venous flow higher than arterial flow) monitored by the pressure, system called time pressure regulation. The risk of recirculation is important with the latter; some machines limit this risk more than others, especially with the addition of clamps. Conversely for small infants a single lumen catheter used with the alternative clamps technique is an acceptable compromise between recirculation and both the extracorporeal blood volume and the achieved blood flow\u00a0[35].\nThe total extracorporeal blood volume (needles, tubing, and dialyzer) should preferably be less than 10 % of patient total blood volume. This is essential for small children; however, the relative normal hemoglobin level obtained with erythropo\u00efetin therapy enables this volume to be exceeded slightly without significant hypotension at the end of dialysis session when the patient reaches dry body weight. Nevertheless, it should be kept in mind that the higher the extracorporeal blood volume, the higher the volume of returned fluid, which will load the patient with fluid at the end of the dialysis session. (In very small children the substitution by air may be necessary to limit blood loss on one side and high substitution volume on the other side, but is very dangerous and should be strictly monitored.) System priming with saline, albumin, and sometimes blood should be applied in the first dialysis sessions with babies or small infants.\nAnticoagulation of the extracorporeal blood volume is performed either by use of conventional, heparin with continuous infusion of 20 to 30\u00a0IU\u00a0kg\u22121\u00a0h\u22121, or with low-molecular-weight heparin at 1\u00a0mg\u00a0kg\u22121 as a bolus at the beginning of the dialysis session. If the hematocrit is over 35%, the risk of clotting is increased. Regional citrate anticoagulation is sometimes used especially when acute dialysis is needed\u00a0[2]. Predilution treatment, feasible in either hemofiltration or hemodiafiltration, reduces the risk of clotting and even enables dialysis without anticoagulation in some circumstances. In the presence of thrombopenia heparin-toxicity is to be suspected.\nThe venous blood line has a pediatric size air-trap chamber to limit extracorporeal blood volume. The dialysis membrane is protected by an arterial chamber of expansion which in small children is often not incorporated in the line to reduce the extracorporeal blood volume. Prevention or treatment of ethylene oxide allergy is possible by using steam sterilization of needles, lines, and membranes; this is becoming the preferred option throughout Europe.\nGuideline 7: which dialyzer membrane to \u201cchoose\u201d\nsynthetic membrane, low flux, capillary configuration\nhigh-flux membrane use requires use of ultrapure dialysate\nremoval of urea and other uremic toxins dialytic should be considered, especially in chronic, long-term dialysis\nThree general types of membrane are available at present\u00a0[32]: unmodified cellulose (low flux and so-called bioincompatible membranes), modified\/regenerated cellulose (low flux or high flux; so-called relatively biocompatible), synthetic (low flux or high flux; so called relatively biocompatible).\nThe choice of a dialyzer membrane should take into account the following (Table\u00a04):Table\u00a04 Dialyzer membranes: practical parameters of choice- Type of membrane: biocompatibility toward complement system- Initial blood volume needed, i.e. area-related, quality of restitution- Molecular permeability: maximum clearance for urea and the other uremic toxins, e.g. phosphate, related to potential patient osmotic risk- Hydraulic permeability: possibility of use for HF or HDF procedure, but related to back filtration risk, high flux membranes need ultrapure dialysate- Adsorption capacity on to the membrane (a characteristic of synthetic membranes)- Costthe biocompatibility of the material towards leucocytes and complement activationthe blood volume priming requirement, which is membrane area-relatedthe permeability, determined in the most simple way by two characteristics:hydraulic permeability (CUF) measured in mL per mmHg of transmembrane pressure achieved per hour, i.e. either low permeability, CUF under 5\u00a0mL\u00a0mmHg\u22121\u00a0h\u22121 (low-flux membrane), and high permeability, CUF over 15 to 20\u00a0mL\u00a0mmHg\u22121\u00a0h\u22121 (high-flux membrane)molecular permeability determined at least by the molecular weight of the molecule considered, usually between 0.8 and 0.9 for urea and lower for the other uremic toxins with a cut off of zero for albumin. In practice this cut off is often under a molecular weight of 20,000 Daltons. The profile of this molecular permeability\u00a0[40, 41] is a specific characteristic of each manufactured dialysis membrane. Highly permeable membranes give the theoretical potential for middle-molecular-weight (Babb theory; 500 to 2,000 Daltons) uremic toxins being removed during dialysis. In adult dialysis patients the clinical benefits of improved removal of middle molecules by high flux, large pore, biocompatible membranes, more or less established, are\u00a0[41]: reduction of uremia related amyloidosis, maintenance or residual renal function, and reduction of inflammation, malnutrition, anemia, dyslipidemia, and mortality.The absorption capacity on to the membrane, (IL1, TNF, IL6, \u03b22 microglobulin):a characteristic of synthetic membranes\nFor conventional dialysis low-flux membranes are suitable, but to achieve hemofiltration or hemodiafiltration high-flux membranes are necessary. The higher the hydraulic permeability, the higher is the backfiltration risk; this process could be limited both by permanent convective flow from the blood compartment to the dialysate compartment, as ultrafiltration (HF, HDF, or at least weight loss) and by use of ultrapure dialysate. Synthetic membranes seem the best theoretical choice but clinical justification of the relatively higher cost is uncertain\u00a0[32]. Justification for use of high-flux synthetic membranes, as used in on-line HDF, remains a matter of debate for children on dialysis for short periods only while waiting for their kidney transplant.Reuse of the membrane is not applied in practice for children.\nGuideline 8: the dialysate\nbicarbonate buffered,\nlow calcium level (1.25\u00a0mmol\u00a0L\u22121) becomes the standard,\nglucose concentration at physiological level,\ndialysate quality control (germs and endotoxins) is required\nThe dialysate is prepared as a dilution of concentrate with water, ideally with ultrapure water. The composition of the dialysate has changed over the last two decades\u00a0[42]. Acetate as buffer has been replaced by bicarbonate, with the development of machines with two separate dilution pumps, one for bicarbonate concentrate free from calcium, often as a powder, and one for the acid concentrate containing residual levels of acetate and the electrolytes (Na, K, Cl, Ca). The current use of oral calcium carbonate as a phosphate binder has mandated the need to decrease the calcium concentration of the dialysate, usually at a low rate, 1.25 mmol\u00a0L\u22121 Ca2+, less often at a normal rate, 1.5\u00a0mmol\u00a0L\u22121, avoiding the \u201chistorically\u201d high level of 1.75\u00a0mmol\u00a0L\u22121 Ca2+. In fact, the use of calcium carbonate combined with a high concentration of calcium in the dialysate, often led to an elevated Ca\u00d7P serum product, compared with the current recommendation of a product less than 5 mmol2\u00a0m\u22122 (60\u00a0mg2\u00a0dL\u22122)\u00a0[43, 44]. This Ca\u00d7P serum product seems to be an important factor implicated in the vascular calcifications seen in the dialyzed patients\u00a0[43], affecting even the dialyzed children\u00a0[44, 45]. The need for glucose in the dialysate is of importance\u00a0[46] and should be near the physiological concentration. Higher glucose concentrations or the introduction of parenteral feeding during dialysis will drive the potassium into the cells, leading to ineffective potassium-extraction\u00a0[41].\nPotassium-free dialysate is rarely used because of the theoretical risk of hypokalemia\u00a0[42]. Therefore \u201clow\u201d (1\u20131.5\u00a0mmol\u00a0L\u22121), \u201cnormal\u201d (2\u20132.5\u00a0mmol\u00a0L\u22121), and \u201chigh\u201d (3\u20133.5\u00a0mmol\u00a0L\u22121) potassium dialysate are available enabling individual adaptation and prevention of the arythmogenic potential of dialysis\u00a0[42]. Nevertheless special attention should be devoted to avoiding any confusion among the \u201cpotassium charged\u201d dialysates. Sodium concentrations have increased from the previous classical level of 132\u00a0mmol\u00a0L\u22121 to a more physiological level of 138 to 144\u00a0mmol\u00a0L\u22121. Newer machine capabilities enable dialysate profiles to change during a dialysis with respect to sodium and ultrafiltrate profiles\u00a0[47, 48] to increase tolerated weight loss; and bicarbonate profiles\u00a0[49], to enhance phosphate removal. Intermittent ultrafiltration rates, enabling better plasma refilling is the most common profile used. Similarly, the dialysate flow rate can be adapted to need, usually in the range 300 to 800\u00a0mL\u00a0min\u22121. In general practice, 500\u00a0mL\u00a0min\u22121 is used. The dialysate flow is usually in the opposite direction of the blood flow, separated by the membrane of the dialyzer. Dialytic thermal exchanges seem of importance especially for babies and\/or high-flow dialysate use, leading to a risk of patient hypothermia. Control of thermal exchanges during a dialysis session is therefore available on a new machine\u00a0[3, 28].\nGuideline 9: post-dialytic dry weight assessment and adjustment\nparticularly difficult to define in growing children\nno \u201cunique\u201d optimum method, importance of a clinical \u201cpediatric\u201d experience\nneed for regular assessment in a growing child\nclose collaboration with pediatric renal dietician\nPatient dry weight is defined as the weight at the termination of a regular dialysis session, below which the patient will become symptomatically hypotensive. Incorrect estimation of dry weight will lead either to chronic fluid overload or chronic dehydration. Estimation of dry weight is particularly difficult in children for many reasons. First, the hypotensive tendency during a dialysis session is multifactorial and not only related to the ultrafiltration rate but also to the plasma refilling rate capacity\u00a0[47, 48]. Second, body composition, i.e. total body water ratio to total body mass, is variable with age, especially during infancy and puberty. In infants and in adolescents dry weight must be assessed almost monthly to follow rapid body composition changes during a rapid growth period. This is also important under anabolic conditions such as with growth hormone treatment, and conversely under catabolic conditions such as the ill child with intercurrent infections or reduced food intake.\nClinical criteria used to assess hydration status are important but not always reliable. Therefore, different approaches have been proposed: assessment of total body water by bioelectrical impedance analysis\u00a0[50], continuous measurement of hematocrit variations by non-invasive methods during dialysis\u00a0[9, 26], plasma atrial natriuretic peptide or cyclic guanosine monophosphate determination\u00a0[51], and, last, by echography of the inferior vena cava (IVC)\u00a0[52, 53, 54, 55]. Measurement of the diameter of the IVC (IVCD) by ultrasound, expressed as an index to body surface-area in mm\u00a0m\u22122, and the decrease on deep inspiration, called the collapse index, expressed as a percentage (%) seems to be an accurate non-invasive method easily performed serially. An IVCD between 8.0 and 11.5\u00a0mm\u00a0m\u22122 and a collapse index between 40 and 75 % is considered as representing normovolemia\u00a0[52, 53, 54, 55]. However, unlike body impedance, interstitial volume and sodium balance are not reflected by IVCD\u00a0[55]. In fact all these approaches have to be balanced by clinical judgment and experience and combined with nutritional support.\nAchievement of dry weight during ultrafiltration is associated with a drop of the hematocrite level. Ultrafiltration is well tolerated until a certain level of decrease of initial hematocrite, called \u201ccrash hematocrite\u201d a patient individual characteristic, usually over 10% blood volume reduction over a 3-h session\u00a0[56]. If the hematocrite curve is flat over time during a dialysis session, the patient could be considered as being over his optimum dry weight\u00a0[9, 56]. In practice, monitoring of hematocrit (or blood volume) and guided ultrafiltration should avoid both fluid overload and hypotensive \u201ccrash hematocrit\u201d and consequently approach more precisely the patient dry weight\u00a0[9, 56].\nGuideline 10: urea dialytic kinetic, dialysis dose, and protein intake assessment (nutrition)\nUrea kinetic modeling (UKM) has been widely accepted as a method for dialysis dose assessment despite its limited value as a unique measure of dialysis adequacy. Does small solute clearance really matter?\u00a0[11]. In adult patients the HEMO study suggested that increasing urea clearance above currently accepted target ranges does not lead to improved patient outcome\u00a0[11]. Although urea per se is not toxic in concentrations normally encountered in dialysis patients, it may serve as a marker of unknown toxins of uremia, some of which are called \u201cmiddle-molecular-weight\u201d uremic toxins\u00a0[11, 41].\nUKM facilitates identification of underdialyzed patients and recognition of dietary compliance. The measures most widely used to gauge dialyzer treatment are Kt\/V, that is dialyzer urea clearance (K) multiplied by duration (t) of the dialysis session and divided by urea volume (V) of distribution, and the normalized protein catabolic rate (nPCR)\u00a0[57, 58, 59]. Urea dialytic reduction rate (URR) is derived from the pre and post-dialysis serum urea values and quantitates urea removal by dialysis. URR expressed as the ratio post\/pre should be at least equal to or lower than 0.35 and when expressed as the difference between pre and post-urea, divided by the predialysis value, should at least equal to or higher than 0.60\u00a0[60]. URR is proportional to dialysis efficiency, and thus to urea dialytic clearance. URR is inversely proportional to the urea refilling rate of the blood compartment and the extracellular space (EC) from the intracellular space (IC), called the transcellular urea mass transfer coefficient (Kie). URR is also correlated with the amount of urea dialytic removal (Kt) compared to the amount of urea body content (V) and thus to Kt\/V. Usually urea dialytic clearance in children is low in comparison with the high Kie which is between 200 to 1000\u00a0mL\u00a0min\u22121 (6 to 12\u00a0mL\u00a0min\u22121\u00a0kg\u22121 BW)\u00a0[58, 61]. Nevertheless, after dialysis the concentration of urea in plasma increases rapidly in an initial period, usually until 60\u00a0min postdialysis\u00a0[62]. This postdialytic urea rebound (PDUR) is a multifactorial event\u00a0[63, 64]. Vascular access and cardiopulmonary recirculation occurs within the first 2 to 3\u00a0min of discontinuing hemodialysis and account for 60 to 70% of total PDUR. Subsequently, tissue rebound occurs, because of intercompartmental, i.e. IC versus EC, urea dysequilibrium at the end of the dialysis session and tissue re-equilibration which is usually complete within one hour postdialysis, reaching the equilibrated postdialytic plasma urea concentration. For highly diffusible substances such as urea, distribution in total body water (TBW) seems to be limited by cardiovascular flow rather than diffusion\u00a0[64]. The apparent IC\u2013EC two-pool model should perhaps be the result of a regional blood flow distribution system in which approximately 80% of TBW (and thereby urea) is located in muscle, bone, and skin, with organs receiving only 20 to 30% of the cardiac output, i.e. low-flow system. The remaining organs contain only 20% of TBW (hence urea) but receive 70\u201380% of the cardiac output, high-flow system. One would expect the urea concentration in these organs to fall quite rapidly during dialysis. This flow system, and the vascular resistance model could explain the great variability of the PDUR among patients for which the IC\u2013EC two-pool model is not accurate. Do some patients have thicker cell walls than others? By contrast the possible causes of vascular resistance variability, i.e. hypovolemia, hypertension, heart failure, hematocrit, alkalosis or acidosis, low-temperature dialysate, can explain PDUR variability. The URR variability could also be explained by vascular resistance changes over the dialysis session, at least for urea\u00a0[64].\nKt\/V calculation based on a single pool urea model neglects compartmental urea distribution in the body, hence PDUR, resulting in overestimation of actual Kt\/V. Therefore, a two pool model should be applied, using instead of the urea plasma concentration at the end of the dialysis, the equilibrated urea, i.e. 60\u00a0min postdialysis\u00a0[65, 66]. Other improvements from the initial formula are proposed to provide a more accurate Kt\/V calculation: weight loss (UF\/BW) and urea generation during the dialysis session (0.008td), leading to the Daugirdas and Schwartz formula proposed in 1994\u00a0[60]:where td is the dialysis time (h), Cpre and Ceq are, respectively, the predialysis and equilibrated postdialysis urea concentrations, and UF\/BW is the ultrafiltrate-to-body weight ratio (L\u00a0kg\u22121)\nThe predialysis blood sample should be taken from the arterial line, before any rinsing. Because of the practical difficulty in obtaining the postdialysis equilibrated urea sample 60\u00a0min after the end of the dialysis, different indices have been proposed to estimate Ceq, for example using a 6\u00a0min\u00a0[67] or a 15\u00a0min\u00a0[68, 69] post treatment sample. The most important rule of the urea end dialysis sample should be the use of the \u201cstop dialysate flow method\u201d\u00a0[67], measuring urea 6\u00a0min after angio access was removed and cardiopulmonary recirculation completed.\nThe other major cause of error for the Kt\/V calculation is determination of V. The V, hence the TBW, could be calculated from a formula (Table\u00a05)\u00a0[71] or determined by bioimpedance measurements\u00a0[50].Table\u00a05 Formulas enabling calculation of the volume of distribution of urea in liters (total body water) using height, weight, sex and age (from Ref.\u00a0[67])Boys:Ht<132.7\u00a0cmV=1.927+0.465\/BW (kg)+0.0045\/ht (cm)Ht>132.7\u00a0cmV=\u221221.1933+0.406\/BW (kg)+0.209\/ht (cm)Girls:Ht<110.8\u00a0cmV=0.076+0.507\/BW (kg)+0.013\/ht (cm)Ht>110.8\u00a0cmV=-10.313+0.252\/BW (kg)+0.154\/ht (cm)\nGuideline 11: dialysis dose and outcome\nonly \u201csmall solute urea clearance\u201d prescription?\na minimum Kt\/V urea level of 1.2\u20131.4 is thought to be desirable; adequacy tests should be performed monthly\ndialysis and residual renal small-solute clearance are not equivalent\ndialysis prescription should be adequate before being optimum, not only a \u201curea dialysis dose\u201d\nAlthough the optimum level of Kt\/V required is matter of debate, a minimum Kt\/V level of 1.2\u20131.4 is now thought to be desirable\u00a0[11]. Overall, this Kt\/V as an index of dialysis dose should only be analyzed in comparison with the nPCR, hence the diet, protein and caloric intake (Fig.\u00a01). Because of the mathematical relationship between Kt\/V and nPCR\u00a0[72, 73] the real impact of these variables for a given patient would determine the therapy necessary for a patient to achieve an \u201curea dialysis dose\u201d. Nevertheless increasing dialysis dose seems to have a direct impact on nutrition\u00a0[74] and the combination of increased dialysis dose and adequate nutrition can promote normal growth in children treated with long-term hemodialysis\u00a0[75]. Therefore malnutrition should be avoided\u00a0[76] by using a diet survey, anthropometric measurements, and perhaps IGF1 determination\u00a0[77].Fig.\u00a01 Dialysis prescription balance\nDoes small solute clearance, i.e. urea, really matter?\u00a0[11]. Because of the limited number of children on chronic dialysis the relationship between optimum urea dialysis dose and patient outcome will be \u201cdifficult\u201d to establish. It is, however, known that blood purification, dialysis and residual renal small solute clearance, are not equivalent\u00a0[11, 78] giving more importance to other uremic toxins, whose removal is enhanced using high-flux dialyzer membranes with on-line hemodiafiltration\u00a0[3, 24, 32, 41].\nEven if the pediatric data only seem to be unique center experiences, the case for a greater urea dialysis dose\u00a0[12] could be correlated with both growth rate enhancement\u00a0[12, 13, 19, 78] and improved cardiac function\u00a0[14, 19]. The duration of each hemodialysis session is also matter of debate, long duration being able to induce regression of left ventricular hypertrophy in adult patients\u00a0[16] and being able in children\u00a0[13] to promote growth and well being. In the same way daily dialysis seems related to better clinical results both in adults\u00a0[17, 18, 77, 79] and in adolescents\u00a0[19].\nDialysis prescription should be adequate before being optimum (Table\u00a06)\u00a0[80]. In long term chronic dialyzed children the individualized prescription should consider all the available new strategies to fully preserve at the best \u201cthe life chances\u201d\u00a0[13, 14, 19].Table\u00a06 Hemodialysis prescription for children: adequate, before optimum- Dialysis modality should enable achievement of blood pressure control (without antihypertensive medications for most children), normal myocardial morphology and function- Dialysis dose prescription should not only be an urea dialysis dose. Removal of the other uremic toxins should be considered, not only middle molecules but overall phosphate- Dialysis frequency and duration must be adjusted to the tolerance of ultrafiltration to reach the dry weight. Ultrafiltration rate should not exceed 1.5\u00b10.5% of body weight per hour (in theory no more than 5% BW loss per whole session ). Blood volume (hematocrite) guided ultrafiltration secure- A regular diet survey is essential to maintain adequate protein and calorie intakes. Urea kinetic assessment enables not only urea dialysis dose calculation, i.e. Kt\/V, but also estimation of protein intake by use of the PCRn calculation (protein catabolic rate). Fasting to enable a short duration three times a week dialysis schedule is inadequate care management- Too fast ultrafiltration can induce hypotension and cramps during dialysis, usually during the second half time session, and fatigue and\/or hang over after dialysis- A small solute, e.g. urea, clearance which is too high is a factor of disequilibrium syndrome occurring during dialysis, usually after the first half\/or one hour session time with headache, even seizures, nausea, vomiting, sleepiness or a hypertensive tendency with a narrow range between systolic and diastolic pressure values. Symptoms usually disappear a few hours after the end of the dialysis\nGuideline 12: the dialysis session, prescription, and monitoring\nindividual prescription is required: babies\/infants\/children specificities\nassessment and adjustment is needed regularly in small\/growing children\npsychological preparation of the child and his family is needed, pain prevention is essential\nThe first dialysis session is of importance to induce child and parent confidence, therefore appropriate preparation is needed. The site of the puncture of the fistula, most often with a double needle, size gauge 17, is carefully chosen and determined so that the needles are sufficiently separated to limit recirculation. Pain prevention is essential by application of a xylocaine ointment (Emla) one hour before needle insertion\u00a0[39]. Psychological preparation of the child and family is also needed to limit \u201canxious stress\u201d\u00a0[22]. An aseptic procedure is essential. The extracorporeal circulation is adapted to the level of arterial aspiration pressure if measurable by the machine to prevent endothelial vascular trauma (not less than \u2212150\u00a0mmHg). The venous return pressure should not be more than +200\u00a0mmHg to prevent endothelial vascular trauma.\nDuring the first dialysis session, the blood flow rate is maintained at a low level to prevent the dysequilibrium syndrome secondary to too efficient solute removal during this first session. Therefore, the blood flow rate should be approximately 3\u00a0mL\u00a0kg\u22121 BW (or 90\u00a0mL\u00a0m\u22122), or even less, so that urea clearance will be less than 3\u00a0mL\u00a0min\u22121\u00a0kg\u22121 BW, which is usually well tolerated even in small children and limits the development of the dysequilibrium syndrome. The duration of the first dialysis session should be short, no more than 3 h, or adapted to the ultrafiltration need. The dysequilibrium syndrome is most often only symptomatic after one to two hours of dialysis, with variable symptoms such as headache or seizure, vomiting, fatigue, sleepiness, or a hypertensive tendency with a narrow range between systolic and diastolic pressure values. If needed, mannitol infusion (1\u00a0g\u00a0kg\u22121 BW over 1 to 2\u00a0h during dialysis) is effective in preventing the syndrome. Symptoms usually disappear a few hours after the end of the dialysis.\nThe extracorporeal blood flow rate, the duration of the session, and the number of sessions a week is progressively increased to individual patient need. Usually a blood flow rate of 150 to 200\u00a0mL\u00a0min\u22121\u00a0m\u22122 and three sessions per week for 3 to 4\u00a0h per session achieve the minimum target prescription of 1.2 to 1.4 Kt\/V\u00a0[11].\nThe duration of a dialysis session is often prescribed to reach the anticipated dry weight at the end of the session. The total amount and the rate of ultrafiltration needed must be tolerable. A weight loss per hour of 1.5 to 2% of the BW is standard\u00a0[3, 80] and most often well tolerated. Intermittent ultrafiltration with bicarbonate buffered dialysate which is not too warm (so called \u201ccooled dialysate\u201d), a normal \u201chigh\u201d level of sodium (140 to 144\u00a0mmol\u00a0L\u22121), which is not more than the normal concentration of sodium per liter plasma water, a normal hematocrit over 30% and optimally near 35% but not higher\u00a0[78], and a mode of dialysis based on hemofiltration, i.e. (optimally HDF) are some of the major \u201ctricks\u201d used to enhance ultrafiltration tolerance\u00a0[3, 48]. Intolerance of weight loss usually becomes symptomatic at the end of the dialysis session, when the patient is near his dry weight. Continuous blood volume monitoring during the session should become a real clinical support to enable optimum ultrafiltration tolerance (notion of crash hematocrit)\u00a0[26, 56]. This information is limited to blood compartment changes. The interstitial space, which is mostly sodium-dependent, is better estimated by clinical assessment of edema or body weight\u00a0[54, 55]. Rarely bed scales are used to assess more precisely the weight changes over a dialysis session.\nFor most infants and children weighing less than 10\u00a0kg the need for more than three sessions a week may become evident to enable adapted nutrition, i.e. milk that is\u00a0\u201dwater\u201d, hence 4 to 5 sessions a week are frequently prescribed\u00a0[3, 11]. The adequate number and duration of each session should avoid partial fasting to achieve the weight needed to facilitate a short dialysis duration\u00a0[7, 21, 80]. The volume of fluid used for extracorporeal blood replacement at the end of the session should be limited, and preferably a glucose solution instead of saline solution be used, especially in infants without residual renal function\u00a0[3].\nAt the beginning of the dialysis session clinical manifestations of bioincompatibility may occur. This first use reaction is related to the biocompatibility of the material in the extracorporeal circuit, i.e. membrane, lines or even the needle either during the first session, first contact with the \u201cextracorporeal\u201d material or thereafter for example, in a new dialysis center for holidays. The major positive diagnostic criteria is the onset within 20\u00a0min of starting dialysis, of the major symptoms of dyspnea, burning heat throughout the body or access site, angioedema, flushing or vascular collapse, or with minor symptoms such as itching, rhinorrhea, lacrymation, urticaria, or abdominal cramping. Even if its occurrence is rare, or underestimated in the event of intermittent minor symptoms only during the first hour of session, the risk could be substantial. Biocompatible membranes, steam-sterilized material, adequate flushing of the circuit before blood connection, are some of the most important prevention factors\u00a0[32].\nThe dialysis per se should be regarded as part of an overall strategy for care including dietary adequacy and interdialytic therapy\u00a0[1, 21]. A weight gain over 10% dry BW during the interval of two sessions is often correlated with global non-compliance\u00a0[3, 80]. In these cases, major outcomes could even occur: first acute, i.e. hyperkalemia or pulmonary edema, second chronic, i.e. hyperparathyroidism, and third long term, i.e. cardiovascular and coronary involvement\u00a0[44, 45].\nConclusions\nHemodialysis in children has benefited from major progress over the last 20 years. The morbidity of the sessions has decreased, even disappeared, seizures being exceptional, hypotensive episodes or headaches rare, and pain related to the fistula puncture effectively prevented by xylocaine ointment. The development of urea kinetic modeling enables calculation of the dialysis dose and indirect assessment of protein intake, nPCR. Even if the validity of these values is questioned their combined analysis provides an assessment and therefore is a \u201cgood thing\u201d. The patient also benefits from the technological revolution. The newer machines enable precise control of ultrafiltration volumetric assessment and continuous blood volume monitoring during the session, buffered bicarbonate has become a standard technique, synthetic more biocompatible membranes and specific material available for babies\/infants have been developed. Non invasive intervention, for example blood volume guided ultrafiltration have provided more adequate dialysis sessions and better dry weight assessment\u00a0[81]. Last, the availability of erythropoietin\u00a0[82] and of growth hormone and the promising results from enhanced dialysis dose on both growth and cardiac function\u00a0[13, 20], all give the dialyzed child a real increased quality of life. In theory, reduction of dialysis prescription to only a urea dialysis dose achieved by three short (3-h) dialysis sessions, should be abandoned for long term dialyzed children and replaced by optimum dialysis obtained with longer (4 and more hours) and\/or more frequent (daily: 5 to 6) sessions\u00a0[13, 20, 79, 80]. But for such a daily dialysis strategy all the costs must be considered. On the one hand the financial cost cannot be neglected. For the patient bearing the burden, on the other hand, such an intensive dialysis prescription is acceptable only as an integrated therapy life project, a dialysis\u2013transplantation program (HD, PD) with special regard for prevention of the vascular calcification\u00a0[83]. Daily hemodialysis is one approach, perhaps the only one, to achieve phosphate purification\u00a0[16, 17, 18, 19] and thereby maintain the calcium\u00d7phosphorus product in the optimum range of 3.3 to 4.4\u00a0mmol2\u00a0mL\u22122\u00a0[43].","keyphrases":["hemodialysis","children","guidelines"],"prmu":["P","P","P"]}
{"id":"Planta-4-1-2413075","title":"Pepper pectin methylesterase inhibitor protein CaPMEI1 is required for antifungal activity, basal disease resistance and abiotic stress tolerance\n","text":"Pectin is one of the main components of the plant cell wall that functions as the primary barrier against pathogens. Among the extracellular pectinolytic enzymes, pectin methylesterase (PME) demethylesterifies pectin, which is secreted into the cell wall in a highly methylesterified form. Here, we isolated and functionally characterized the pepper (Capsicum annuum L.) gene CaPMEI1, which encodes a pectin methylesterase inhibitor protein (PMEI), in pepper leaves infected by Xanthomonascampestris pv. vesicatoria (Xcv). CaPMEI1 transcripts are localized in the xylem of vascular bundles in leaf tissues, and pathogens and abiotic stresses can induce differential expression of this gene. Purified recombinant CaPMEI1 protein not only inhibits PME, but also exhibits antifungal activity against some plant pathogenic fungi. Virus-induced gene silencing of CaPMEI1 in pepper confers enhanced susceptibility to Xcv, accompanied by suppressed expression of some defense-related genes. Transgenic ArabidopsisCaPMEI1-overexpression lines exhibit enhanced resistance to Pseudomonas syringae pv. tomato, mannitol and methyl viologen, but not to the biotrophic pathogen Hyaloperonospora parasitica. Together, these results suggest that CaPMEI1, an antifungal protein, may be involved in basal disease resistance, as well as in drought and oxidative stress tolerance in plants.\nIntroduction\nPlant cell wall, the first barrier of defense against invading pathogens, is composed of cellulose microfibrils cross-linked by hemicellulose, pectin and extensin. Pectin comprises a highly heterogeneous group of polymers that includes homogalacturonans and rhamnogalacturonans I and II (Willats et al.2001a, b). In pectin polymers, the galacturonic acid carboxyl groups can be methylesterified by a group of pectinases. These galacturonic acid methylesters are hydrolyzed by pectin methylesterase (PME) (Hagerman and Austin 1986; Pelloux et al. 2007). Many physiological processes, such as fruit maturation, microsporangenesis, seed germination and pollen growth, are affected by the degree to which PME changes the methlyesterification of galacturonic acid (Tieman and Handa 1994; Kagan-Zur et al. 1995; Ren and Kermode 2000; Bosch et al. 2005). The number and distribution of free and unesterified galacturonate carboxyl groups along the homogalacturonan chain has a great influence on the pectin properties and cell wall firmness (Willats et al. 2001a, b).\nPlant pathogens hydrolyze the cell wall components of plants using extracellular pectinolytic enzymes (Collmer and Keen 1986), and PME is found in many plant pathogenic bacteria and fungi (Asoufi et al. 2007). The black filamentous fungus Aspergillus niger secretes a set of pectin-degrading enzymes that include PME, polygalacturonase and pectin lyase, and these decompose the plant cell wall to establish infection and absorb nutrients from the host (de Vries and Visser 2001). In soft rot disease caused by Erwinia chrysanthemi, bacterial PME activity is induced during infection of Saintpaulia plants; however, PME-deficient mutants are noninvasive to the host cells (Boccara and Chatain 1989). PMEs have also been found in higher plants; they play significant roles in physiological processes and interactions with pathogens (Micheli 2001). In tobacco plants, host cell pectin methylesterases are required for the tobacco mosaic virus movement protein to transfer the viruses between host cells (Chen et al. 2000). Furthermore, PME-degraded polygalacturonans are associated with recognition of fungal pathogens (Wietholter et al. 2003). PME is also involved in symbiosis-specific functions (Lievens et al. 2002). For example, plant PME isoenzymes may undergo organism-specific post-translational processing for structural and functional integrity during interactions with various microorganisms (Micheli 2001).\nThe PME enzyme activity is modulated specifically by inhibitor proteins such as the pectin methylesterase inhibitor (PMEI; Micheli 2001). Moreover, the PMEIs that inhibit demethylesterification of highly heterogeneous polymers (pectins) are the plant invertase inhibitor-related proteins, which are inhibitors of important metabolic enzymes (Koch 1996). Plant invertase inhibitor-related proteins play key roles in wounding, the plant defense reaction and developmental transitions (Raush and Greiner 2004), as well as during osmotic stress, senescence and seed development (Greiner et al. 1998, 1999). Investigation of gain- and loss-of-function mutants of tobacco cell wall invertase inhibitor (NtCIF) protein demonstrated that these inhibitor proteins play a role in seed development (Raush et al. 1998). Overexpression of the tobacco vacuolar invertase inhibitor protein (NtVIF) gene in transgenic potatoes is of potential use in the field of food technology (Greiner et al. 1998). However, there is little known about the in vivo functions of PMEI protein.\nProteinaceous inhibitors have been purified from kiwi (Actinidia deliciosa) (Giovane et al. 2004), Arabidopsis (Wolf et al. 2003; Raiola et al. 2004), rice (Han et al. 2005) and the jelly fig (Ficus awkeotsang cv. Makino) (Jiang et al. 2001, 2002). The kiwi PMEI is specific for PME (Balestrieri et al. 1990) and is active against PMEs from several plants, including kiwi, orange, apple, tomato, apricot, carrot, potato and banana (Ly-Nguyen et al. 2004). Four Cys residues conserved in several isoforms of PMEI are involved in the formation of disulfide bridges (Camardella et al. 2000). PME and PMEI form a stoichiometric 1:1 complex, in which the interaction between the PME and the inhibitor occurs in close proximity to the putative active site (Di Matteo et al. 2005). Since PME activity can be modulated by pH, the stability of the PME\u2013PMEI complex is also affected by pH (Den\u00e8s et al. 2000). Crystallographic work has revealed that an \u03b1-helical hairpin motif plays a structurally important role in PMEI activation (Hothorn et al. 2004). Many cDNAs encoding PMEIs have been isolated and functionally characterized from plants (Rausch and Greiner 2004); however, their role in plant defense remains relatively unknown.\nTo date, the functional analyses of genes associated with defense responses in plants have utilized reverse-genetics approaches based on loss-of-function via double-stranded RNA interference (Robertson 2004) or gain-of-function via transgenic gene expression (Clough and Bent 1998). Virus-induced gene silencing (VIGS) has been proven to be a useful method for assessing the function of target genes in Solanum species (Brigneti et al. 2004). In particular, VIGS studies have been used to investigate disease resistance signaling and defense-related genes such as SGT1 (Liu et al. 2002c; Peart et al. 2002b), EDS1 (Liu et al. 2002b; Peart et al. 2002a) and NPR1\/NIM1 (Liu et al. 2002b) in Nicotiana benthamiana. AtPGIP1 is among the genes encoding pectic enzyme-related proteins; it encodes polygalacturonase (PG)-inhibiting protein (PGIP), and an antisense AtPGIP1 gene was used to silence its expression in transgenic Arabidopsis plants (Ferrari et al. 2006). Previous studies have revealed that overexpression of two closely related genes, AtPGIP1 and AtPGIP2, conferred resistance against Botrytis cinerea infection (Ferrari et al. 2003). Silencing of AtPGIP1 resulted in enhanced susceptibility to infection, as well as reduced activity of PGIP (Ferrari 2006). However, gene-silencing techniques such as VIGS and antisense RNA have not yet been used to investigate the role played in plant defense by other PMEI-encoding genes.\nHere, we used a macroarray technique to isolate and functionally characterize a pectin methylesterase inhibitor gene, CaPMEI1, from a cDNA library of pepper (Capsicum annuum L.) leaves infected with Xanthomonas campestris pv. vesicatoria (Xcv; Jung and Hwang 2000). Local and systemic induction of CaPMEI1 was investigated in pepper plants following inoculation with pathogenic and non-pathogenic bacteria. We also examined the involvement of CaPMEI1 in defense-related signal transduction cascades via exogenous application of abiotic elicitors to pepper plants. Recombinant CaPMEI1 proteins were expressed in E. coli and exhibited antifungal activity against plant pathogenic fungi. Since it is difficult to transform pepper plants, we performed gene silencing and CaPMEI1 overexpression in pepper and Arabidopsis, respectively, to identify the cellular functions of the CaPMEI1 gene. The functional data obtained by VIGS and transgenic ectopic expression of CaPMEI1 suggest that this pepper pectin methylesterase inhibitor is involved in plant defense and abiotic stress responses.\nMaterials and methods\nPlant materials and growth conditions\nPepper (Capsicum annuum L. cv. Nockwang) plants were grown at 28\u00b0C under a 16\u00a0h day at 70\u00a0\u03bcmol\u00a0photons\u00a0m\u22122\u00a0s\u22121. Plants were seeded into a plastic tray (55\u00a0\u00d7\u00a035\u00a0\u00d7\u00a015\u00a0cm3) containing steam-sterilized soil mix (peat moss, perlite and vermiculite; 5:3:2, v\/v\/v) and loam soil (1:1, v\/v). At the two-leaf stage, seedlings were transplanted into plastic pots (5\u00a0\u00d7\u00a015\u00a0\u00d7\u00a010\u00a0cm3) containing the soil mix previously described.\nArabidopsisthaliana (ecotype Columbia) plants were grown in pots containing vermiculite, peat moss and perlite (1:1:0.5, v\/v\/v) in a growth chamber under a 12\u00a0h light\/12\u00a0h dark photoperiod (130\u00a0\u03bcmol photons\u00a0m\u22122\u00a0s\u22121) at 24\u00b0C and 60% relative humidity. Prior to sowing, the seeds were surface-sterilized using 1% sodium hypochlorite and vernalized at 4\u00b0C for 3\u00a0days to break dormancy.\nPathogens, inoculation procedures, disease rating and tissue staining\nXanthomonas campestris pv. vesicatoria (Xcv) strains Ds1 and Bv5-4a were used in this study. Bacteria were cultured overnight in yeast-nutrient (YN) broth (5\u00a0g\u00a0L\u22121 yeast extract, 8\u00a0g\u00a0L\u22121 nutrient broth) at 28\u00b0C. Prior to inoculation, bacteria were harvested by centrifugation and resuspended in sterile tap water (108\u00a0cfu\u00a0mL\u22121). Pepper plants were inoculated at the six-leaf stage by infiltrating the bacterial suspension into the abaxial side of fully expanded leaves using an atomizer. Infected plants were then incubated for 16\u00a0h at 28\u00b0C in a moist chamber with 100% relative humidity. Bacteria-infected leaves were sampled at various time intervals after inoculation. To evaluate systemic induction in the upper leaves, bacterial suspensions (108\u00a0cfu\u00a0ml\u22121) were infiltrated into the lower leaves of pepper plants at the two-leaf stage using a needless syringe. The bacterial strains used for this study included: virulent and avirulent strains of X. campestris pv. vesicatoria (Ds1 and Bv5-4a); a non-pathogenic strain (Pseudomonasfluorescens ATCC13525); and Escherichiacoli JM109.\nLeaves of 6-week-old Arabidopsis plants were infiltrated with a suspension (OD600\u00a0=\u00a00.001) of virulent Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000). The bacteria were cultured overnight at 28\u00b0C and suspended in 10\u00a0mM MgCl2. To determine bacterial growth, leaf discs were cut from infected leaves at different time intervals after inoculation. Bacterial growth was monitored by performing serial dilutions onto KB agar containing 100\u00a0\u03bcg\u00a0mL\u22121 rifampicin. Each experiment was replicated three times.\nHyaloperonospora parasitica isolate Noco2 was propagated by weekly subculturing on 7- to 10-day-old Arabidopsis seedlings. The 7-day-old seedlings were inoculated with an H. parasitica asexual inoculum (5\u00a0\u00d7\u00a0104 conidiosporangia\u00a0mL\u22121). The seedlings inoculated with H. parasitica were covered with a plastic dome to maintain a high relative humidity (80\u2013100%) and grown in a growth chamber at 17\u00b0C. Seven days after inoculation, disease rating was scored for more than 50 plants per treatment. A visual disease rating was expressed as the number of sporangiophores on each cotyledon and was divided into five classes: 0\u20135, 6\u201310, 11\u201315, 16\u201320 and >20 sporangiophores per cotyledon. The cotyledons from inoculated plants were stained with lactophenol-trypan blue (10\u00a0mL lactic acid, 10\u00a0mL glycerol, 10\u00a0g phenol and 10\u00a0mg trypan blue, dissolved in 10\u00a0mL of distilled water) to assess H. parasitica growth. At 2\u20135\u00a0days after inoculation, the infected cotyledons were boiled for 5\u00a0min in the staining solution and de-stained overnight in chloral hydrate (2.5\u00a0g chloral hydrate dissolved in 1\u00a0mL distilled water). The destained cotyledons were subsequently mounted in 70% glycerol for microscopic observation.\nIsolation and sequence analysis of pathogen-induced cDNAs\nTo construct a pathogen-induced cDNA library, pepper leaves were inoculated with the avirulent strain X. campestris pv. vesicatoria Bv5-4a. The pathogen-induced cDNA library was constructed using 5\u00a0\u03bcg poly(A)+ mRNA extracted from inoculated pepper leaves (Kim and Hwang 2000). To isolate pathogen-inducible cDNAs from the pepper cDNA library, we performed differential hybridization, as described previously by Jung and Hwang (2000). Digoxigenin (DIG)-labeled, single-stranded cDNA probes were generated from total RNA of healthy and Bv5-4a-infected leaves using RT-PCR. Nylon membranes were pre-hybridized at 65\u00b0C for 3\u00a0h in 5\u00d7 SSC, 0.1% sodium lauroylsarcosine, 0.02% SDS and 1% blocking reagent (Boehringer Mannheim, Mannheim, Germany). Hybridization was then performed overnight at 65\u00b0C in the same buffer with single-stranded cDNA probes. Hybridized membranes were rinsed twice for 5\u00a0min with 2\u00d7 SSC and 0.1% SDS at room temperature, and twice for 10\u00a0min with 0.1\u00d7 SSC and 0.1% SDS at 65\u00b0C. The hybridization signals were detected according to the manufacturer\u2019s protocol (Boehringer Mannheim).\nWe selected cDNA clones that were expressed strongly in pathogen-infected leaves, compared with those of healthy leaves. Clones were sequenced with an ABI 310 DNA sequencer (Applied Biosystems, Foster City, CA, USA) using the PRISM Big Dye\u2122 Terminator Cycle Sequencing Ready Reaction Kit (PE Biosystems, Foster City, CA, USA). Sequencing results were analyzed using BLAST (National Center for Biotechnology Information; Altschul et al. 1997).\nTreatment with abiotic elicitors and environmental stresses\nThe leaves of pepper plants at the six-leaf stage were sprayed with 5\u00a0mM salicylic acid (SA), 100\u00a0\u03bcM methyl jasmonate (MeJA) or 100\u00a0\u03bcM absicisic acid (ABA). Pepper plants treated with methyl jasmonate were sealed tightly in plastic bags. For ethylene treatment, whole plants were removed from soil, and then placed in a water-containing glass chamber, followed by injection of ethylene gas (5\u00a0\u03bcL\u00a0L\u22121). For cold stress treatment, plants were placed at 4\u00b0C in a cold room. For wounding stress, the leaves were pricked with needles. To impose drought stress, the plants were removed from the soil and then incubated at room temperature without water. H2O2 treatment was performed by spraying leaves with 100\u00a0mM H2O2 solution. Leaves treated with various elicitors and abiotic stresses were removed from the plants, frozen in liquid nitrogen and stored at \u221270\u00b0C until used for RNA isolation.\nRNA isolation and RNA gel blot analysis\nTotal RNA was extracted from pepper leaves, stems, roots, flowers and fruits using the guanidine isothiocyanate method (Chomczynski and Sacchi 1987). Frozen tissues (1\u00a0g) were ground to a powder and homogenized in 10\u00a0mL extraction buffer (4\u00a0M guanidine isothiocyanate, 25\u00a0mM sodium citrate [pH 7.0], 0.55% [w\/v] N-laurylsarcosine and 0.1\u00a0M 2-mercaptoethanol). A mixture of 2\u00a0M sodium acetate (pH 4.0), water-saturated phenol and chloroform\u2013isoamylalcohol (24:1) was added to the homogenate, followed by precipitation. Total RNA (20\u00a0\u03bcg) was separated by 1.2% formaldehyde-agarose gel electrophoresis and then blotted onto Hybond-N+ membranes (Amersham, Buckinghamshire, UK). Transferred RNA was fixed to the membrane using UV cross-linking.\nThe 3\u2032 UTR region of CaPMEI1 was amplified for use in the generation of a gene-specific probe. The primers used for amplification of the CaPMEI1 gene-specific region were 5\u2032-CATGGGTAAGTGCTGCCTTGACGGAC-3\u2032 and 5\u2032-GTTAACAAATGCATA TGGAACATTT-3\u2032. The CaBPR1 coding region was amplified with the primers 5\u2032-ATGGGACACTCTAATATTGCC-3\u2032 and 5\u2032-GACATCAGTTGGAAGTTCCAA-3\u2032. The CaSAR82A coding region was amplified using the primers 5\u2032-ATGGTTTCCAAAAGT AGTATTTTTATTT-3\u2032 and 5\u2032-TATGCTTAACAATTATTACTGAATA TAATC-3\u2032. PCR-amplified products were 32P-labeled using a random priming kit (Boehringer, Mannheim). Hybridization was performed overnight at 65\u00b0C in 5% dextran sulfate, 0.25\u00a0M disodium phosphate (pH 7.2), 7% (w\/v) sodium dodecyl sulfate (SDS) and 1\u00a0mM EDTA. Following hybridization, the membranes were washed twice for 10\u00a0min with 2\u00d7 SSC and 0.1% SDS at room temperature, and then twice for 5\u00a0min with 0.1\u00d7 SSC and 0.1% SDS at 65\u00b0C. Equal loading of RNA was confirmed by ethidium bromide-staining of ribosomal RNA.\nIn situ RNA localization\nIn situ RNA localization was performed as described previously (Lee et al. 2000). Leaf tissue was fixed for 2\u00a0h in a solution of 1\u00d7 phosphate-buffered saline (PBS), 4% paraformaldehyde and 1\u00a0\u03bcL\u00a0mL\u22121 Triton X-100 by vacuum infiltration for 10\u00a0min and shaking for 2\u00a0h at room temperature. The fixed samples were washed with 1\u00d7 PBS, dehydrated through a graded ethanol and xylol series, and then embedded in liquid paraplast at 57\u00b0C (Sherwood Medical, St. Louis, MO, USA). Paraplast-embedded sections (10\u00a0\u03bcm in thickness) were placed on glass slides coated with poly-l-lysine (Sigma, St. Louis, MO, USA) and incubated at 42\u00b0C. Sections were deparafinated using xylene and 1-propanol, followed by rehydration with serial dilutions of ethanol. Tissue samples were treated with 0.01\u00a0M Tris\u2013HCl (pH 8.0) and 1% bovine serum albumin (BSA) for 10\u00a0min, followed by incubation in 100\u00a0mM Tris\u2013HCl solution (pH 8.0) containing proteinase K (5\u00a0mg\u00a0mL\u22121) and 50\u00a0mM EDTA, for 30\u00a0min at 37\u00b0C. Sections were treated with 0.25% acetic anhydride in 100\u00a0mM triethanolamine (pH 8.0) for 10\u00a0min at room temperature to inhibit non-specific signals. Digoxigenin (DIG)-labeled probes were prepared using the Dig High Prime DNA Labeling and Detection kit, according to the manufacturer\u2019s instructions (Boehringer Mannheim). Sections were prehybridized and hybridized at 42\u00b0C in 50% formamide, 4\u00d7 SSC, 150\u00a0\u03bcg\u00a0mL\u22121 tRNA and 0.5% blocking reagent (Boehringer Mannheim). After hybridization, the sections were washed twice with 50% formamide and 4\u00d7 SSC at 42\u00b0C, twice with 4\u00d7 SSC and then once with diethyl pyrocarbonate (DEPC)-treated water. The DIG signal was detected, according to the manufacturer\u2019s instructions (Boehringer Mannheim). Color reactions were developed overnight at 37\u00b0C with nitro-blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP), and reactions were stopped with TE buffer (10\u00a0mM Tris\u2013HCl, 1\u00a0mM EDTA, pH 8.0). Sections were photographed with Kodak ISO 100 film under a bright field Olympus BH-2 microscope (Olympus, Tokyo, Japan). To demonstrate the specificity of in situ hybridization, control hybridizations were performed without DIG-labeled probes.\nPurification of recombinant CaPMEI1 protein\nThe CaPMEI1 coding region, including the stop codon, was amplified by PCR using the forward and reverse primers 5\u2032-GAATTCATGGAAGGTGGCAATTTTCT-3\u2032 and 5\u2032-CTCGAGTAGCCGTGAAGGGCAGCCAGACG-3\u2032, respectively. Amplification products were cloned into pCR2.1-TOPO (Invitrogen, Carlsbad, CA, USA), which was digested with EcoRI and XhoI and ligated into the similarly digested pET32a (Novagen, Madison, WI, USA).\nEscherichia coli BL21 (DE3) pLysS (Novagen), which is defective for thioredoxin reductase, was used as a host for recombinant protein expression. Cultures were started from single colonies, grown in LB broth at 37\u00b0C, and then diluted 1:100 at OD600\u00a0=\u00a00.6. After dilution, bacteria were grown to a density of OD600\u00a0=\u00a00.6, induced with 10\u00a0mM IPTG and grown for a further 5\u00a0h at 37\u00b0C. Cells were harvested by centrifugation for 15\u00a0min at 5,000g, extracted with denaturation buffer (8\u00a0M urea, 20\u00a0mM sodium phosphate buffer [pH 7.8], 500\u00a0mM NaCl) and disrupted by sonication. Following centrifugation at 5,000g for 15\u00a0min at 4\u00b0C, the supernatant was loaded onto a 1.5\u00a0mL column of Ni-NTA resin (Qiagen, Hilden, Germany), which was washed with an initial denaturing wash buffer (8\u00a0M urea, 20\u00a0mM sodium phosphate buffer [pH 6.0], 500\u00a0mM NaCl), followed by a second denaturing wash buffer (8\u00a0M urea, 20\u00a0mM sodium phosphate buffer [pH 5.3], 500\u00a0mM NaCl). Bound fusion protein was eluted with a final denaturing elution buffer (8\u00a0M urea, 20\u00a0mM sodium phosphate buffer [pH 4.0], 500\u00a0mM NaCl). Recombinant proteins were dialyzed against a buffer containing 10\u00a0mM Tris (pH 8.0) and 0.1% Triton X-100, according to the manufacturer\u2019s protocol. As a control, thioredoxin was purified using a native buffer that did not contain urea. The purified CaPMEI1 recombinant protein was digested for 16\u00a0h at room temperature with recombinant enterokinase (1\u00a0U\/5\u00a0\u03bcg recombinant protein; Novagen).\nSDS polyacrylamide gel electrophoresis\nCaPMEI1 was dissolved in 1\u00d7 SDS sample buffer (0.9\u00a0g glycerol, 5% SDS, 1% bromophenol blue, 0.1\u00a0mL mercaptoethanol and 1\u00a0L H2O), separated by 12% SDS-PAGE as described previously (Laemmli 1970), and stained with Coomassie Brilliant Blue R-250. Molecular weights were estimated using 6.5 to 200.5\u00a0kDa marker proteins (Bio-Rad, Hercules, CA, USA).\nPectin methylesterase enzyme inhibitor assay\nThe inhibitory effect of CaPMEI1 on the enzymatic activity of pectin methylesterase (PME) was assayed under standard conditions (Grsic-Rausch and Rausch 2004). The reaction mixture comprised 894\u00a0\u03bcL 0.4\u00a0mM NAD in 50\u00a0mM phosphate buffer (pH 7.5), 80\u00a0\u03bcL 5% (w\/v) pectin (Sigma) in H2O, 8\u00a0\u03bcL formaldehyde dehydrogenase (0.35\u00a0U; Sigma) and 8\u00a0\u03bcL alcohol oxidase (1.0\u00a0U; Sigma). After mixing, the reaction was started with the addition of 10\u00a0\u03bcL (7.8\u00a0mU) PME from orange peel (Sigma). To analyze CaPMEI1 inhibition of PME, 1\u00a0\u03bcL inhibitor solution (0.5\u00a0mg CaPMEI1 fusion protein, 10\u00a0mM Tris buffer [pH 7.5], 0.1\u00a0M NaCl) was mixed with 10\u00a0\u03bcL PME (7.8\u00a0mU) and 0.5\u00a0\u03bcL 3\u00a0M K-acetate buffer (pH 5.3), followed by incubation for 15\u00a0min at room temperature. All reaction temperatures were maintained at 25\u00b0C. Reaction rates were recorded continuously at 340\u00a0nm using a DU650 UV-visible spectrophotometer (Beckman, Fullerton, CA, USA). PME\u2013PMEI interactions were determined by measuring the rate of NADH formation per minute at pH 7.5 and 25\u00b0C.\nIn vitro antifungal activity\nTo determine the antifungal activity of CaPMEI1, we examined its effect on plant pathogenic fungi. Fusarium oxysporum f.sp. matthiole and Alternaria brassicicola were incubated on potato dextrose agar (PDA) at 28\u00b0C for 1\u20132\u00a0weeks; B. cinerea was incubated at 20\u00b0C for 1\u20132\u00a0weeks. Fungi were grown in 48-well plates (Cell Wells\u2122, Corning Glass Works, Corning, NY, USA) containing sterile 4\u00d7 potato dextrose broth (PDB, 100\u00a0\u03bcL) and a range of concentrations of purified CaPMEI1 protein (0\u2013500\u00a0\u03bcg\u00a0mL\u22121). Spore suspensions (104 spores mL\u22121) were prepared and 100\u00a0\u03bcL of inoculum was added to each microwell. Plates containing B. cinerea were incubated at 20\u00b0C; the other fungi were incubated at 28\u00b0C for 4\u20137\u00a0days.\nTo determine the inhibitory effect of CaPMEI1 on spore germination and hyphal growth, CaPMEI1 (0.1\u2013500\u00a0\u03bcg\u00a0mL\u22121) or the thioredoxin control were added to F. oxysporum f.sp matthiolae spore suspensions (105 spores mL\u22121), placed on glass slides and incubated for 12\u00a0h at 28\u00b0C. After 6\u00a0h incubation, 100 germinated spores were examined using a haemocytometer and the lengths of 50 individual hyphae were determined. The experiment was repeated three times.\nPlasmid construction and plant transformation\nThe CaPMEI1 coding region was PCR-amplified without stop codon using the following primers to generate XbaI and BamHI sites: 5\u2032-TCTAGAATGGAAGGTGGCA ATTTTCTCACA-3\u2032 and 5\u2032-GGATCCGCCGTGAAGGGCAGCCAGACGGT -3\u2032. The fragment was inserted into pCR2.1-TOPO (Invitrogen) and digested with XbaI and BamHI. The construct was confirmed by sequencing. To generate the reporter construct p35S-CaPMEI1-GFP, the fragment was inserted into the XbaI\u2013BamHI sites of the binary vector p35S-smGFP. p35S-smGFP was generated by fusing the gene encoding smGFP from the vector 326-GFP into pBIN35S digested with HindIII and EcoRI. The CaMV35S promoter-CaPMEI1-smGFP construct was introduced into the Agrobacterium tumefaciens strain EHA105 using electroporation.\nTransgenic Arabidopsis plants were generated by floral-dipping wild-type (Col-0) plants into an A. tumefaciens culture containing the appropriate construct (Clough and Bent 1998). Agrobacterium was grown at 28\u00b0C and 250\u00a0rpm in YEP medium (10\u00a0g Bacto peptone, 10\u00a0g yeast extract, 5\u00a0g NaCl) supplemented with kanamycin (25\u00a0\u03bcg\u00a0ml\u22121). Cells were harvested by centrifugation for 20\u00a0min at 5,500g and resuspended in inoculation media containing 5.0% sucrose and 0.05% Silwet L-77 (OSi Specialties, Inc., Danbury, CT, USA) to OD600\u00a0=\u00a00.8. Arabidopsis plants with 2\u201310\u00a0cm long bolts were inoculated by dipping into an Agrobacterium suspension (OD600\u00a0=\u00a00.8) and then left in the dark overnight, prior to return to a growth chamber. Seeds from the transformed Arabidopsis plants were collected and screened for kanamycin resistance.\nVirus-induced gene silencing (VIGS)\nThe TRV-based VIGS system was used for silencing of CaPMEI1 in pepper plants, as described previously (Liu et al. 2002a; Chung et al. 2004). The pepper CaPMEI1 coding region was cloned into pTRV2 to generate the construct pTRV2:CaPMEI1. The fully extended cotyledons of 2-week-old pepper seedlings were co-infiltrated with A. tumefaciens GV3101 carrying pTRV1 or pTRV2:CaPMEI1 (OD600\u00a0=\u00a00.2 for each construct). Plants were placed in a growth room at 25\u00b0C under a 16\u00a0h light\/8\u00a0h dark photoperiod, to allow for growth and viral spread. Experiments were performed 5\u20136\u00a0weeks after the induction of silencing.\nRT-PCR analysis\nThe RT reactions (20\u00a0\u03bcL) were performed at 42\u00b0C with total RNA (2\u00a0\u03bcg), oligo p(dT)15 primer (Roche, Mannheim, Germany) and AMV reverse transcriptase (Roche). Aliquots (1\u00a0\u03bcL) of the RT reaction products were used for RT-PCR analysis with the following gene-specific primers: 5\u2032-CAGGATGCAACACTCTGGTGG-3\u2032 and 5\u2032-ATCAAAGGC CGGTTGGTC-3\u2032 for CaBPR1 (accession no. AF053343); 5\u2032-TGTCGAAGGTGGTCC AATAAA-3\u2032 and 5\u2032-TAGACAGAAGGATTGGCGAGG-3\u2032 for CaPR10 (accession no. AF244121); 5\u2032-ATCTGTACCAGCTTGCACGTGT-3\u2032 and 5\u2032-CCCTCACTGTGGCCT TGG-3\u2032 for CaPOA1 (accession no. AF442387); and 5\u2032-CAGGGAGATGAATTCTGA GGC-3\u2032 and 5\u2032-CATATGAACCTCTATGGATTTCTG-3\u2032 for CaSAR82A (accession no. AF313766). RT-PCR conditions were 95\u00b0C for 10\u00a0min and 30 cycles of 95\u00b0C for 30\u00a0s, 52\u00b0C for 30\u00a0s, and 72\u00b0C for 1\u00a0min and 30\u00a0s. Single bands for PCR products were confirmed on an agarose gel.\nDrought stress treatment and evaluation\nFor germination tests, seeds from Arabidopsis wild-type and CaPMEI1-OX transgenic lines were surface-sterilized and placed on MS media (Murashige and Skoog 1962) containing 200 or 600\u00a0mM mannitol. The seeds were maintained at 4\u00b0C for 48\u00a0h under dark conditions to synchronize germination, and then transferred to a growth chamber. Experiments were repeated at least three times, using approximately 100 seeds.\nSurface-sterilized seeds were also used for examination of relative root length. Seedlings were grown on plates placed vertically in growth chambers and root length was estimated for 14\u00a0days. To assess mannitol tolerance, seedlings were grown for 7\u00a0days in 1\u00d7 MS agar media supplemented with 1% sucrose, and then transferred to wells containing 1\u00d7 MS liquid media supplemented with 100, 200, 300 or 400\u00a0mM mannitol. For drought treatment, 4-week-old soil-grown plants were deprived of water for 15\u00a0days and then re-watered on day 16. To minimize experimental variation, both the transgenic and control plants were grown in the same tray. Experiments were repeated at least three times.\nFor transpiration rate measurements, leaves of 4-week-old plants were detached and maintained at room temperature. Leaf weight was determined every 20\u00a0min for 2\u00a0h, and then every 1\u00a0h thereafter. Each measurement was performed using eight leaves. Experiments were repeated at least three times with similar results.\nOxidative stress treatment and evaluation\nFor analysis of oxidative stress tolerance, surface-sterilized seeds from Arabidopsis wild-type, vector control and CaPMEI1-OX transgenic lines were placed on MS medium containing different concentrations of methyl viologen (MV; Sigma). After synchronized germination, seeds were maintained in a growth chamber. To investigate whether or not MV treatment caused retardation of seedling development, 7-day-old seedlings were randomly transferred into well-plates containing 1\u00d7 MS liquid medium supplemented with 0.05, 0.1 or 0.5\u00a0\u03bcM MV, and then grown for a further 2\u00a0weeks. To determine leaf senescence during MV treatment, fully expanded leaves were detached from 4-week-old plants and rinsed briefly with 70% ethanol. The detached leaves were floated on MS medium containing MV for 24\u00a0h following extraction with liquid nitrogen. Their chlorophyll content was measured spectrophotometrically according to the formula (Ca\u00a0+\u00a0b\u00a0=\u00a05.24\u00a0\u00d7\u00a0A664\u00a0+\u00a022.24\u00a0\u00d7\u00a0A648), where C is the chlorophyll concentration (Ca\u00a0+\u00a0b) in micrograms per mL and A is absorption (Lichtenthaler 1987).\nResults\nIsolation and sequence analysis of CaPMEI1 cDNA in pepper\nTo identify the molecular mechanisms involved in plant defense against the invasion of microbial pathogens, we performed a differential hybridization screening of a cDNA library constructed from pepper leaves undergoing an incompatible interaction with Xcv (Jung and Hwang 2000). PR-1, PR-10, SAR8.2 and Myb transcription factor were among the pathogen-induced genes isolated by this screen. In addition, we isolated a full-length cDNA (834\u00a0bp) designated CaPMEI1 (Capsicum annuumpectin methylesterase inhibitor protein, accession no. AF477956), which contains a 54\u00a0bp 5\u2032 untranslated sequence and a 174\u00a0bp 3\u2032 untranslated region. The predicted open reading frame encodes a full-length protein of 200 amino acid residues, with an estimated molecular mass of 21.39\u00a0kDa and an isoelectric point (pI) of 6.51.\nBLAST searches were performed between the predicted amino acid sequence of CaPMEI1S (accession no. AF477956) from C. annuum and the EMBL\/GenBank database. These sequence analyses revealed that CaPMEI1 contains a conserved pectin methylesterase inhibitor (PMEI) domain of 156 amino acids (Fig.\u00a01). Four cysteine residues were conserved in all the aligned proteins (Fig.\u00a01), and these are predicted to be involved in the formation of disulfide bridges (Camardella et al. 2000). The predicted amino acid sequence of CaPMEI1 is 80% identical to that of Nicotiana tabacum DC1.2 protein (accession no. BAA95794) and has 50% identity with Arabidopsis thaliana ripening-related protein (accession no. BAA97200), 36% with Pinus radiata pectinesterase homolog (accession no. T08112) and 24% with A. thaliana invertase homolog (accession no. NP_201267).\nFig.\u00a01Amino acid sequence alignments of pepper CaPMEI1 with Nicotiana tabacum DC1.2 protein (accession no. BAA95794), A. thaliana ripening-related protein (accession no. BAA97200), A. thaliana invertase homolog (accession no. NP201267) and Pinus radiata pectinesterase homolog (accession no. T08112). The boxed amino acid sequences represent the pectin methylesterase inhibitor (PMEI) domain. The conserved cysteine residues are marked by asterisks and the shaded regions represent conserved amino acid residues\nOrgan-specific expression of CaPMEI1 in pepper tissues\nRNA gel blot analysis was performed to examine the organ-specific expression of CaPMEI1 in pepper plants. Although high levels of CaPMEI1 transcription were observed in stem tissues, only relatively low levels of expression were found in leaf, root, flower and green fruit (Fig.\u00a02a). CaPMEI1 transcripts were not detected in red fruit.\nFig.\u00a02RNA gel blot analysis of expression of CaPMEI1, CaSAR82A and CaBPR1 in pepper plants. Membranes were hybridized with probes from the 3\u2032 UTR region of pepper CaPMEI1 cDNA or full-length CaBPR1 cDNA. Equal loading (20\u00a0\u03bcg) was verified by visualizing RNA on a gel stained with ethidium bromide. H healthy, M mock-inoculated or mock-treated. aCaPMEI1 expression in various organs of pepper plants. b Expression of CaPMEI1 and CaBPR1 in pepper leaves at various time intervals after inoculation with C. coccodes or virulent strain Ds1 (compatible interactions with pepper: susceptible response) and avirulent strain Bv5-4a (incompatible interactions with pepper: resistant response) of Xcv.cCaPMEI1 expression in lower (local) infected and upper (systemic) uninfected leaves at various time intervals after inoculation with the virulent and avirulent strains Ds1 and Bv5-4a of Xcv, P.fluorescence ATCC13525 and E.coli JM109. The lower leaves of pepper plants were inoculated at the 6-leaf stage. For the mock-inoculation, the lower leaves were infiltrated with 10\u00a0mM MgSO4. d Expression of CaPMEI1 and CABPR1 in pepper leaves at various time intervals after treatment with salicylic acid (SA, 5\u00a0mM), methyl jasmonate (MeJA, 100\u00a0\u03bcM), ethylene (5\u00a0\u03bcl\u00a0L\u22121) and abscisic acid (100\u00a0\u03bcM). e Expression of CaPMEI1 and CaSAR82A in pepper leaves at various time intervals after treatment with drought, wounding, cold and H2O2\nCaPMEI1 induction by pathogen infection\nTo assess the accumulation of CaPMEI1 transcripts in pepper leaves during the compatible and incompatible interactions with pathogens, leaves were inoculated with C. coccodes and Xcv Ds1 (virulent, compatible), as well as Xcv Bv5-4a (avirulent, incompatible). CaPMEI1 transcripts were not detected in leaves mock-inoculated with sterilized water (Fig.\u00a02b, c). During fungal infection at the four- and eight-leaf stages, CaPMEI1 expression was induced at 48 and 12\u201348\u00a0h after inoculation, respectively, suggesting that the adult plant stage is more resistant to C. coccodes (Fig.\u00a02b, upper panel). CaPMEI1 transcripts increased rapidly within 30\u00a0min after infection by both the virulent (Ds1) and avirulent (Bv5-4a) strains of Xcv (Fig.\u00a02b, lower panel). High transcript levels accumulated by 18\u00a0h after infection with the virulent strain (Ds1) that causes a susceptible response; however, they started to decline rapidly by this time following infection with the avirulent strain (Bv5-4a) that causes a resistant response. CaBPR1 (Capsicum annuum basic PR1) expression was observed to confirm the success of individual inoculations. CaBPR1 transcripts were detected at 6\u00a0h after inoculation with the virulent and avirulent strains of Xcv and increased gradually during the 24\u00a0h following inoculation (Fig.\u00a02b).\nLocal and systemic expression of CaPMEI1 in infected pepper plants\nThe local and systemic CaPMEI1 expression was analyzed in pepper leaves inoculated with Xcv and the non-pathogenic bacteria Pseudomonas fluorescens ATCC13525 and E. coli JM109 (Fig.\u00a02c). In leaves infected with the virulent (Ds1) and avirulent (Bv5-4a) strains of Xcv, CaPMEI1 transcripts were detected 3\u00a0h after inoculation and levels decreased thereafter. Infection of lower (local) leaves by either Xcv strain induced systemic accumulation of CaPMEI1 transcripts in the upper leaves. The systemic induction by the avirulent strain (Bv5-4a) infection was stronger than that by the virulent strain (Ds1) infection. Local or systemic induction of CaPMEI1 transcripts was not observed 30\u00a0h after infection. In response to the non-pathogenic bacteria P. fluorescens and E. coli, CaPMEI1 transcripts were detected 3\u00a0h after inoculation and decreased subsequently in both local and systemic leaves.\nCaPMEI1 induction following treatment with plant hormones\nPlant hormones are involved in defense-related signal transduction pathways (Hammond-Kosack and Jones 1996; Chung et al. 2007). We examined whether or not CaPMEI1 expression is induced by plant hormones using pepper leaves treated with salicylic acid (SA), ethylene, methyl jasmonate (MeJA) or abscisic acid (ABA) (Fig.\u00a02d). Salicylic acid strongly induced expression of CaPMEI1 at 1 and 2\u00a0h, as well as between 18 and 24\u00a0h after treatment. Following treatment with ethylene and MeJA, CaPMEI1 expression was strongly induced for 24\u00a0h. In addition, strong CaPMEI1 induction was observed 1\u00a0h after treatment with ABA.\nIn comparison, SA only induced CaBPR1 transcription 18\u201324\u00a0h after treatment (Fig.\u00a02d), although transcripts were detected 6\u00a0h after treatment with ethylene and MeJA. In response to ethylene treatment, CaBPR1 transcripts increased rapidly between 18 and 24\u00a0h after treatment, but increased gradually between 6 and 24\u00a0h after MeJA treatment. CaBPR1 expression was induced between 2 and 12\u00a0h after ABA treatment.\nCaPMEI1 induction by abiotic stresses\nAbiotic stresses resulted in differential induction of CaPMEI1 in pepper leaves (Fig.\u00a02e). The CaPMEI1 gene was responsive to mechanical injury, and expression was observed between 15\u00a0min and 6\u00a0h after wounding, although no transcription was detected thereafter. CaPMEI1 transcription also increased between 2 and 18\u00a0h after cold treatment. CaPMEI1 transcription was induced at 1\u00a0h of drought stress, but was undetectable thereafter. In response to H2O2 treatment, CaPMEI1 transcript levels increased for the first 6\u00a0h and declined thereafter. The marker genes CaBPR1 and CaSAR82A were used for comparison, as they also exhibit differential induction by these abiotic stresses.\nIn situ localization of CaPMEI1 transcripts\nWe performed an in situ hybridization using a CaPMEI1 probe to examine spatial expression of CaPMEI1 in pepper leaf tissues (Fig.\u00a03). No hybridization signals were observed in non-treated leaves (Fig.\u00a03a, b). Intense localization of CaPMEI1 transcripts was observed in the xylem area of the vascular bundle in non-treated stems (Fig.\u00a03c). CaPMEI1 transcripts were detected in the vascular bundle of leaves inoculated with C. coccodes (Fig.\u00a03e) and in the vascular bundles and upper epidermis of leaves treated with ethylene (Fig.\u00a03g). No transcript signals were observed in any pepper tissues hybridized with the CaPMEI1 sense DIG-labeled RNA probe (Fig.\u00a03b, d\u2013f). These in situ hybridization results are supported by the temporal expression patterns observed in the RNA gel blot analysis of CaPMEI1 expression (Fig.\u00a02).\nFig.\u00a03In situ localization of CaPMEI1 transcripts in pepper leaf and stem tissues. Cross sections of leaf tissues were hybridized with CaPMEI1 antisense (a, c, e, g) and sense (b, d, f, h) DIG-labeled RNA probes, and then photographed under bright-field conditions. The transcript signal is purple. a, b Untreated leaf tissues. c, d Untreated stems. e, f Leaf tissues at 24\u00a0h after inoculation with C. coccodes. g, h Leaf tissues treated with 5\u00a0\u03bcl\u00a0L\u22121 ethylene. P phloem, X xylem, UE upper epidermis, LE lower epidermis, Vs vascular bundle, C cortical cell\nInhibition of pectin methylesterase (PME) activity by CaPMEI1 protein\nTo prepare recombinant CaPMEI1 protein, the CaPMEI1 coding region was PCR-amplified and cloned into pET32a. E. coli strain BL21 (DE3) pLysS was used as a host for the recombinant expression of the CaPMEI1 construct and an empty vector control, yielding recombinant CaPMEI1 and thioredoxin, respectively. The purified CaPMEI1 (Fig.\u00a04a, lane 5) and thioredoxin (Fig.\u00a04a, lane 3) were examined by SDS-PAGE analysis. The purified thioredoxin\u2013CaPMEI1 fusion protein formed a single band with an apparent molecular mass of 42\u00a0kDa (Fig.\u00a04a, lane 5). Following cleavage of thioredoxin by enterokinase digestion, the purified CaPMEI1 protein (Fig.\u00a04a, lane 6) was used for biological function determination. The cleaved recombinant CaPMEI1 protein was found exclusively in the insoluble fraction as inclusion bodies (data not shown). After attempting unsuccessfully to renature the cleaved protein using Triton X-100 as a detergent (Kim and Hwang 1994), we performed the PME inhibition assay using crude and purified CaPMEI1 fusion proteins.\nFig.\u00a04Inhibition of pectin methylesterase (PME) activity by the pepper pectin methylesterase inhibitor (CaPMEI). a Recombinant CaPMEI1 expression in E. coli BL21 (DE3) pLysS. Cells were grown in LB media, and recombinant protein expression was induced with 1\u00a0mM IPTG. M, molecular marker (kDa); Lane 1, Uninduced E. coli BL21 cell extracts; Lane 2, Soluble fraction of E. coli BL21 cell extracts encoding thioredoxin protein after IPTG induction; Lane 3, Purified thioredoxin; Lane 4, Crude protein extracts of E. coli cells producing the thioredoxin\u2013CaPMEI1 fusion protein after IPTG induction; Lane 5, Purified thioredoxin\u2013CaPMEI1 fusion protein; Lane 6, Cleaved CaPMEI1 and thioredoxin proteins following enterokinase digestion. Protein staining was performed using Coomassie brilliant blue. b Inhibition of PME activity following treatment with CaPMEI1. For the inhibition assay, crude CaPMEI1 (0.5\u00a0\u03bcg) was mixed with 7.8\u00a0mU of orange peel PME in a total volume of 11\u00a0\u03bcL, and then preincubated at 25\u00b0C for 15\u00a0min, followed by addition to the reaction solution\nCaPMEI1 contains a pectin methylesterase inhibitor (PMEI) domain (Fig.\u00a01) and shares similarity with a plant pectin methylesterase inhibitor related to regulation of pectin degradation (Raiola et al. 2004). In addition, PMEIs have been found to play crucial roles in plant and microbial enzyme regulation of pectin degradation by de-esterification (Raiola et al. 2004). To determine whether or not orange peel PME activity is inhibited in the presence of crude CaPMEI1 fusion protein, we used the PME enzyme assay to measure the effect of PME inhibitor on the formation of NADH (Fig.\u00a04b). Both crude and purified CaPMEI1 (ca. 0.5\u00a0\u03bcg) strongly inhibited PME enzyme activity, and a higher concentration of purified CaPMEI1 (ca. 1.0\u00a0\u03bcg) exhibited the strongest inhibition. However, thioredoxin alone did not exhibit any inhibitory effect. These results suggest that CaPMEI1 functions as an inhibitor of PME activity.\nAntimicrobial activity of CaPMEI1\nCaPMEI1 exhibited antifungal activity against the three plant pathogenic fungi examined: F. oxysporum f.sp. matthiole, A. brassicicola and B. cinerea (Fig.\u00a05a). The thioredoxin\u2013CaPMEI1 fusion protein (50\u00a0\u03bcg\u00a0mL\u22121) suppressed mycelial growth of the three plant pathogenic fungi, whereas thioredoxin alone did not. Furthermore, the purified thioredoxin\u2013CaPMEI1 (500\u00a0\u03bcg\u00a0mL\u22121) fusion protein inhibited spore germination and hyphal growth of F.oxysporum f.sp. matthiole, whereas thioredoxin alone did not (Fig.\u00a05b). Increasing concentrations of recombinant thioredoxin\u2013CaPMEI1 fusion protein increasingly inhibited both spore germination and hyphal growth (Fig.\u00a05c).\nFig.\u00a05Assay of thioredoxin\u2013CaPMEI1 fusion protein antimicrobial activity. a Inhibitory effects of the CaPMEI1\u2013thioredoxin fusion protein on mycelial growth of the plant pathogenic fungi F. oxysporum f.sp. matthiolae, A. brassicicola and B. cinerea. In each plate, the upper wells (a) were treated with purified thioredoxin and the lower wells (b) were treated with the purified thioredoxin-CaPMEI1 fusion protein. b Inhibition of germination and hyphal growth of F.oxysporum f.sp. matthiolae. Fungal spores were allowed to germinate and grow in 100\u00a0\u03bcL potato dextrose broth medium alone (top), or with 500\u00a0\u03bcg\u00a0mL\u22121 thioredoxin (middle) or 500\u00a0\u03bcg\u00a0mL\u22121 thioredoxin\u2013CaPMEI1 fusion protein (bottom). Photographs were taken after incubation for 10\u00a0h at 28\u00b0C. Bars\u00a020\u00a0\u03bcm. c Inhibition of spore germination and hyphal growth of F.oxysporum f.sp. matthiolae by CaPMEI1. The percentage of germinated spores and the length of fungal hyphae were determined by light microscopy. Data represent means\u00a0\u00b1\u00a0SD from three independent experiments\nEnhanced susceptibility of CaPMEI1-silenced pepper plants to Xcv infection\nSince inoculation with Xcv strongly induced CaPMEI1 expression in pepper plants, we performed the virus-induced gene silencing (VIGS) technique (Liu et al. 2002a; Chung et al. 2004) to examine its cellular function during pathogen infection. The full-length ORF of CaPMEI1 was used to construct pTRV2:CaPMEI1. Five to six weeks after induction of silencing, empty vector control (TRV:00) and CaPMEI1-silenced (TRV:CaPMEI1) pepper plants were inoculated with virulent and avirulent strains Ds1 and Bv5-4a of Xcv, respectively. To assess the efficiency of VIGS, CaPMEI1 transcript levels were examined by RT-PCR (Fig.\u00a06). CaPMEI1 transcripts were nearly undetectable in both non-inoculated empty vector control and CaPMEI1-silenced plants. However, at 12\u00a0h after inoculation with virulent and avirulent Xcv, we observed strong induction of CaPMEI1 in the empty vector control plants, whereas only weak or undetectable transcript levels were found in CaPMEI1-silenced plants. These results confirm the effective silencing of the target gene in pepper plants.\nFig.\u00a06RT-PCR analysis of expression of CaPMEI1 and several defense-related genes in empty vector control (TRV:00) and CaPMEI1 gene-silenced (TRV:CaPMEI1) pepper plants 12\u00a0h after inoculation with the virulent (Ds1; C, compatible) and avirulent (Bv5-4a; I, incompatible) strains of Xcv (5\u00a0\u00d7\u00a0106\u00a0cfu\u00a0mL\u22121). 18S rRNA levels were visualized as a loading control. This experiment was repeated three times with similar results. H healthy leaves, CaBPR1 basic pathogenesis-related protein 1, CaPR10 putative ribonuclease-like protein, CaPOA1 ascorbate peroxidase 1, and CaSAR82A SAR8.2\nTo determine whether or not the expression of defense-related genes is affected by bacterial infection in the silenced plants, we used RT-PCR to analyze transcript levels of some defense-related genes (Fig.\u00a06). In the empty vector control plants, both CaBPR1 (basic pathogenesis-related protein 1) and CaPR10 (putative ribonuclease-like protein) were strongly induced by virulent and avirulent Xcv infection. However, induction of these genes was slightly reduced in CaPMEI1-silenced plants infected with virulent Xcv, but was unaffected in plants infected with avirulent Xcv. Thus, CaPMEI1 gene silencing did not alter the induction of CaPOA1 (ascorbate peroxidase) or CaSAR82A (SAR 8.2) in response to Xcv infection.\nTo examine the function of the CaPMEI1 gene in basal defense or gene-for-gene resistance, empty vector control and CaPMEI1-silenced pepper plants were infected with virulent or avirulent Xcv (Fig.\u00a07). CaPMEI1 gene silencing significantly increased susceptibility to virulent Xcv infection, but not to avirulent Xcv infection. The CaPMEI1-silenced pepper leaves exhibited more severe disease symptoms 5\u00a0days after virulent Xcv inoculation than did empty vector control plants, and these symptoms were accompanied by severe chlorosis and enlarged water-soaked lesions (Fig.\u00a07a). However, we did not observe any phenotypical changes in the cell death response of CaPMEI1-silenced plants following avirulent Xcv inoculation (Fig.\u00a07a). At 3\u00a0days after inoculation with virulent Xcv, bacterial growth was tenfold greater in CaPMEI1-silenced plants than in the empty vector controls (Fig.\u00a07b). However, silencing of CaPMEI1 conferred a slightly enhanced susceptibility to avirulent Xcv. These findings suggest that CaPMEI1 may function in basal resistance of pepper plants against Xcv infection, rather than gene-for-gene resistance.\nFig.\u00a07Enhanced disease susceptibility of CaPMEI1-silenced pepper plants to infection by the virulent Xcv strain Ds1, but not the avirulent Xcv strain Bv5-4a. a Disease symptoms developed on the leaves at different time points after inoculation with the virulent Xcv strain Ds1 (5\u00a0\u00d7\u00a0106\u00a0cfu\u00a0mL\u22121) and the avirulent Xcv strain Bv5-4a (various bacterial concentrations). b Bacterial growth in leaves of empty vector control (TRV:00) or CaPMEI1-silenced (TRV:CaPMEI1) pepper plants at different time points after inoculation with the virulent Xcv strain Ds1 or the avirulent Xcv strain Bv5-4a (104\u00a0cfu\u00a0mL\u22121). Data represent the mean\u00a0\u00b1\u00a0SD from three independent experiments\nEnhanced resistance of CaPMEI1-OX plants to Pst DC3000\nThe p35S-CaPMEI1-GFP and p35S-GFP overexpression (OX) plants were generated by transformation of A. thaliana using the floral dipping method (Clough and Bent 1998). Plants transformed with the empty vector p35S-GFP were used as a control. Seeds (T1) were collected from each transformed plant and screened for resistance to kanamycin. Northern blot analysis was performed on T1 plants exhibiting kanamycin resistance to determine the integrity of the inserted transgene. Three T2 plants showing strong CaPMEI1 expression were selected for analyses in planta (Fig.\u00a08a).\nFig.\u00a08Responses of wild-type (Col-0) Arabidopsis and CaPMEI1-OX transgenic plants to infection with P. syringae pv. tomato DC3000. a RNA gel blot analysis confirming CaPMEI1 overexpression (OX) in the transgenic Arabidopsis lines. Total RNA (10\u00a0\u03bcg) was loaded into each lane. The 3\u2032 UTR region of pepper CaPMEI1 cDNA was used as a probe. b Growth of Pst DC3000 in the leaves of wild-type and transgenic plants. The mature leaves of the 6-week-old plants were infiltrated with a Pst Dc3000 suspension (105\u00a0cfu\u00a0mL\u22121), and the degree of bacterial growth was rated at 0, 2 and 4\u00a0days after inoculation. c Disease symptoms on leaves of 6-week-old plants infiltrated with virulent Pst DC3000 (105\u00a0cfu\u00a0mL\u22121). d Expression of pathogen-related (PR) genes in transgenic plants. Northern blot analyses were performed with 10\u00a0\u03bcg total RNA prepared from 5-week-old leaves of the wild-type (WT), vector control (smGFP) and transgenic (CaPMEI1::smGFP) plants. The samples were collected at 5, 15 and 25\u00a0h following pathogen infiltration with a suspension of the virulent strain Pst DC3000 (105\u00a0cfu\u00a0mL\u22121) \nTo determine the contribution of CaPMEI1 to Arabidopsis resistance, wild-type and CaPMEI1-OX plants were inoculated with virulent P. syringae pv. tomato DC3000 (Pst; 105\u00a0cfu\u00a0mL\u22121). Bacterial titers were determined 4\u00a0days after inoculation. CaPMEI1-OX plants exhibited much less growth of Pst bacteria than wild-type or empty vector control plants (Fig.\u00a08b). In the transgenic plants, this reduced bacterial multiplication was most pronounced at 4\u00a0days after inoculation. At 6\u00a0days after inoculation, wild-type (Col-0) and empty vector control plants developed typical chlorotic symptoms in the infected leaves, whereas the transgenic plants displayed few disease symptoms (Fig.\u00a08c).\nExpression of defense-related genes in CaPMEI1-OX plants\nTo gain insight into the role played by CaPMEI1 in PR gene induction, we examined the expression patterns of the well-established marker genes AtPR1a, AtPR2 and AtPR5 in the defense responses of wild-type, vector control and the CaPMEI1-OX Arabidopsis plants infected with virulent Pst DC3000 (Fig.\u00a08d). The expression of AtPR1a and AtPR2 was very similar in the CaPMEI1-OX, wild-type and vector control plants. AtPR5 expression was not induced in either un-inoculated or inoculated, wild-type or vector control plants. However, there was significant expression of AtPR5 in the transgenic CaPMEI1-OX plants at 15 and 25\u00a0h after Pst DC3000 infection. AtPR1a, AtPR2 and AtPR5 expression is known to be regulated via the SA-dependent pathway in Arabidopsis (Uknes et al. 1992), and in nahG plants, lack of SA reduces expression of these three PR genes during pathogenesis (Delany et al. 1994; Nawrath and Metraux 1999). In our experiments, AtPDF1.2 (defensin) transcripts were not detected in wild-type, vector control or CaPMEI1-OX plants (data not shown).\nResponses of wild-type and CaPMEI1-OX plants to Hyaloperonospora parasitica\nWe examined whether or not ectopic CaPMEI1 expression in Arabidopsis plants affected resistance to the virulent biotrophic oomycte pathogen H. parasitica isolate Noco2 (Fig.\u00a09). Over 100 seedlings of both wild-type (Col-0) and CaPMEI1-OX transgenic lines were inoculated with spores of H. parasitica isolate Noco2 (5\u00a0\u00d7\u00a0104 conidiosporangia mL\u22121). At 7\u00a0days after inoculation, over 50 plants in each line were sampled to estimate the disease and assess the degree of asexual sporulation, which was quantified and expressed as the mean number of sporangiophores per cotyledon.\nFig.\u00a09Responses of wild-type (Col-0) Arabidopsis and CaPMEI1-OX transgenic plants to infection with H. parasitica isolate Noco2. a Disease symptoms and trypan blue-stained pathogen structures on 7-day-old cotyledons of wild-type and transgenic plants 7\u00a0days after inoculation; dpi days post-inoculation. Bars\u00a00.5\u00a0mm. b Quantification of asexual sporangiophores per cotyledon for at least 50 cotyledons of wild-type and transgenic plants 7\u00a0days after inoculation. The average number of sporangiophores produced on the cotyledons of wild-type and transgenic lines are shown below each of the lines tested\nAs shown in Fig.\u00a09a, the cotyledons of wild-type and transgenic seedlings responded to H. parasitica infection by stimulating similar levels of asexual parasite sporulation. Their trypan blue-stained cotyledons also exhibited abundant hyphae, conidiospores and oospores (Fig.\u00a09a). Quantitative disease ratings are shown in Fig.\u00a09b. Both the transgenic and the wild-type plants showed a high level of susceptibility to H. parasitica isolate Noco2, and similar levels of heavy asexual sporulation (>20 sporangiophores per cotyledon) were observed on both lines.\nDrought tolerance of CaPMEI1-OX plants\nTo investigate a possible role for CaPMEI1 in the dehydration response, we tested seed germination and seedling growth under osmotic stress. The seeds of wild-type, vector control and CaPMEI1-OX plants were placed on MS media supplemented with various concentrations of mannitol. We observed no significant differences in seed germination between these lines (Fig.\u00a010a). However, treatment with 200 and 600\u00a0mM mannitol strongly inhibited germination in the wild-type and vector control plants compared with the CaPMEI1-OX plants.\nFig.\u00a010Transgenic ArabidopsisCaPMEI1-OX lines exhibit enhanced tolerance to drought stress. a Seed germination in wild-type, smGFP and transgenic plants on the MS media containing 0, 200 and 600\u00a0mM mannitol. The data represent the mean\u00a0\u00b1\u00a0SD of 100 seeds for each line tested. b Relative root length of wild-type, smGFP and transgenic lines in MS agar medium containing different concentrations of mannitol. Three independent experiments were performed with 40 seedlings of both wild-type and transgenic lines. c Drought tolerance test of transgenic seedlings. Wild-type, smGFP and transgenic lines were germinated and grown in 1\u00d7 MS agar medium. Each seedling was transferred to liquid medium containing 100\u00a0mM mannitol. d Wild-type, smGFP and the CaPMEI1 transgenic Arabidopsis plants after 15\u00a0days without water. e Water loss from the excised leaves of wild-type, smGFP and transgenic plants. Data represent the mean\u00a0\u00b1\u00a0SD from three independent experiments\nWe also tested the sensitivity of root growth to osmotic stress (Fig.\u00a010b). Drought tolerance was observed during post-germination growth. The root growth of wild-type and vector control seedlings was inhibited in the presence of 150 and 200\u00a0mM mannitol. However, root elongation of CaPMEI1-OX plants was less sensitive to mannitol-induced osmotic stress and in comparison with wild-type and vector control plants, mutant seedlings grew well in liquid medium supplemented with 100\u00a0mM mannitol (Fig.\u00a010c).\nIn addition, we noticed that adult CaPMEI1-OX plants exhibited enhanced drought tolerance. Following 14\u00a0days without water, wild-type and vector plants had withered severely, whereas the CaPMEI1-OX plants remained healthy. To determine the effect of CaPMEI1 on survival, these plants were rewatered on day 16; wild-type and vector control plants died, whereas the transgenic lines survived (Fig.\u00a010d). Next, we examined transpiration rates by measuring fresh weight loss in detached leaves. The leaves of CaPMEI1-OX lines exhibited slightly slower water loss than those of wild-type or vector control plants (Fig.\u00a010e). Together, these results indicate that CaPMEI1 overexpression enhanced water stress resistance.\nOxidative tolerance of CaPMEI1-OX plants\nTo investigate the response of CaPMEI1-OX plants to oxidative stresses, seeds of wild-type, vector control and CaPMEI1-OX plants were exposed to MS medium containing methyl viologen (MV) (Fig.\u00a011). Treatment with 5 or 10\u00a0\u03bcM MV significantly inhibited germination of wild-type and vector control seeds compared with CaPMEI1-OX seeds (Fig.\u00a011a). Seven-day-old seedlings were transferred to a medium containing different MV concentrations (0\u20130.5\u00a0\u03bcM) and grown for 2\u00a0weeks. Wild-type and vector control seedlings turned white and started to die after 15\u00a0days of MV treatment, but CaPMEI1-OX lines were much less affected by the treatment (Fig.\u00a011b). Furthermore, these differences in plant phenotype were also observed with respect to the higher fresh weight of transgenic seedlings compared to those of wild-type and vector control plants (Fig.\u00a011c). Detached leaves of 4-week-old plants were treated with 10\u00a0\u03bcM MV for 24\u00a0h, after which chlorophyll content was measured (Fig.\u00a011d). The leaves of transgenic lines treated with MV retained more chlorophyll than those of wild-type or vector control plants.\nFig.\u00a011Transgenic ArabidopsisCaPMEI1-OX lines exhibit tolerance to oxidative stress. a Effects of methyl viologen on the seed germination of transgenic lines. Seeds from wild-type, vector control and transgenic lines were plated on media with or without methyl viologen (MV, 5 and 10\u00a0\u03bcM) and incubated for 3\u00a0days. The data represent mean\u00a0\u00b1\u00a0SD of 100 seeds for each line tested. b Phenotypes of wild-type, vector control and transgenic line seedlings treated with different concentrations of MV. c Fresh weights of seedlings grown in the indicated concentrations of MV for 2\u00a0weeks. The results are presented as the average fresh weight per seedling. Data represent mean\u00a0\u00b1\u00a0SD from three independent experiments. d Chlorophyll content of MV-treated leaves of wild-type, vector control and transgenic plants, which were floated on 0, 0.05, 0.1 and 0.5\u00a0\u03bcM MV in MS medium and then incubated for 24\u00a0h in a growth chamber\nDiscussion\nPlants possess a diverse range of cell wall-modified enzymes, which are post-transcriptionally regulated by numerous inhibitor-related proteins (Tymowska-Lalanne and Kreis 1998; Raush and Greiner 2004). Several invertase inhibitor-related proteins have been isolated from higher plants such as kiwi, tobacco and Arabidopsis (Greiner et al. 1998; Wolf et al. 2003; Giovane et al. 2004; Raiola et al. 2004). Attempts to characterize the activity of invertase inhibitor proteins from plant species other than kiwi or Arabidopsis have been either unsuccessful or resulted in the isolation of invertase inhibitors that share structural similarities with pectin methylesterase inhibitor proteins (PMEI), but which represent completely different target enzymes (Greiner et al. 1998; Scognamiglio et al. 2003). In this study, we identified and functionally characterized a novel pepper CaPMEI1 gene encoding a PMEI. This CaPMEI1 protein contains the four cysteine residues that are conserved among other PMEI proteins (Camardella et al. 2000; Sato et al. 2000). These residues are expected to be engaged in two disulfide bridges, which constitute a common structural motif within the PMEI domain (Camardella et al. 2000).\nAt the molecular level, CaPMEI1 expression was induced in pepper leaves by infection with bacterial pathogens and treatment with plant hormones such as SA, ethylene, MeJA and ABA. In particular, these hormone treatments strongly induced CaPMEI1 transcription, suggesting that this gene may be involved in the early stages of the active defense responses to bacterial pathogen infection and exogenous treatment with plant hormones.\nIn enzymatic assays, purified CaPMEI1 proteins significantly inhibited activity of plant pectin methylesterase (PME). In addition, CaPMEI1 exhibited antifungal activity against a broad range of plant pathogenic fungi, including F. oxysporum f.sp. matthiolae, A. brassicicola and B. cinerea. To penetrate the cuticular layer, plant fungal pathogens produce plant cell wall-degrading enzymes such as polygalacturonase, pectin lyase and cellulase (Collmer and Keen 1986). An aggressive Phaeosphaeria nodorum isolate was shown to produce high amounts of xylanase, cellulase, polygalacturonase and butyrate esterase in vitro (Lalaoui et al. 2000). Recently, it was found that plant PMEIs do not inhibit the PMEs produced by plant pathogens (Giovane et al. 2004; Di Matteo et al. 2005). However, Arabidopsis plants expressing either AtPMEI-1 or AtPMEI-2 showed reduced infection by B. cinerea (Lionetti et al. 2007). This finding suggests that the increased level of pectin methylesterification caused by overexpression of AtPMEI-1 results in the inhibition of fungal endopolygalacturonase activity. Thus, the increase in PMEI activity resulted in reduced accessibility for fungal pectin degrading-enzymes and hence provided increased resistance to pathogens (Boudart et al. 1998; Lionetti et al. 2007). Consistent with these findings, our results suggest that CaPMEI1 may function as part of a new group of plant pectin methylesterase inhibitors, which restrict fungal pathogen infection in plants.\nWe used virus-induced gene silencing (VIGS) to investigate the effect of CaPMEI1 loss-of-function in pepper plants during Xcv infection. The CaPMEI1-silenced plants were susceptible to Xcv infection, and in particular to infection with the virulent strain, which resulted in enhanced bacterial growth and reduced PR1 and PR10 gene expression. Basal resistance is activated during the compatible bacterial interaction which restricts the spread of pathogens in the host plants to a certain extent (Glazebrook 2001). In addition, basal resistance is also effective in retarding proliferation of a wide range of microbial pathogens (Chisholm et al. 2006), but it is dependent upon SA accumulation (Cao et al. 1994, 1997; Kinkema et al. 2000). Therefore, we conclude that CaPMEI1 expression may be involved in basal resistance by triggering downstream PR gene induction in pepper plants.\nTo determine the effect of CaPMEI1 gain-of-function inplanta, we generated the CaPMEI1-OX Arabidopsis transgenic lines and investigated their response to P. syringae and H. parasitica infection, because these well-known model pathogens have been used extensively for the study of disease resistance mechanisms in Arabidopsis (Quirino and Bent 2003; Slusarenko and Schlaich 2003). The CaPMEI1-OX lines were resistant to Pst DC3000 infection, but not to infection by the biotrophic oomycete H. parasitica, which uses living cells as a nutrient source during the infection cycle (Alfano and Collmer 1996; Heath 2002). Moreover, as CaPMEI1 transcripts localize intensively in the xylem of vascular bundles in leaf tissues, they may not affect the accessibility of host plant cells to H.parasitica. In contrast, we hypothesize that intercellular growth of P. syringae may be restricted by the extracellular secretion of CaPMEI1 into host cells, resulting in triggering of the basal resistance response.\nWe found that fungal growth was inhibited by treatment with the recombinant CaPMEI1 protein in vitro, suggesting that CaPMEI1 may interfere directly with pathogen infection of host plants. Plant pathogenic microorganisms have been shown to produce a variety of pectinolytic enzymes that macerate and kill plant tissues (Collmer and Keen 1986). Cell wall fragments released by these pectinolytic enzymes may elicit the plant defense response (D\u2019Ovidio et al. 2004). The polygalacturonase-inhibiting protein (PGIP) plays an important role in the recognition and inhibition of fungal polygalacturonase (PG). Overexpression of PGIPs in Arabidopsis not only significantly reduces disease symptoms, but also enhances defense gene activation during pathogen infection (Ferrari et al. 2003). Thus, we suggest that CaPMEI1 can disrupt invading pathogenic microorganisms by inhibiting pectin methylesterases produced by these pathogens.\nThe reduced bacterial growth observed in CaPMEI1-OX Arabidopsis lines may result from the expression of SA-inducible genes and CaPMEI1 overexpression. The SA-inducible genes such as PR1, PR2 and PR5 are activated in the SA defense pathway (Uknes et al. 1992). PR5 proteins are similar to thaumatin, which is a sweet-tasting protein from Thaumatococcus daniellii (Hu and Reddy 1997), and several pathogens can induce these proteins in a wide range of plant species (Ward et al. 1991; Hu and Reddy 1997; Reuber et al. 1998). Members of the PR5 group have been shown to exhibit antifungal activity against a broad spectrum of fungal pathogens (Coca et al. 2000) and to participate in the coordinated induction of systemic acquired resistance (SAR) against TMV (Ward et al. 1991). Therefore, the concomitant induction of PR genes may contribute to the enhanced resistance of CaPMEI1-OX Arabidopsis plants to bacterial pathogens.\nThe CaPMEI1-OX Arabidopsis lines showed a strong tolerance to drought stress, and CaPMEI1 overexpression resulted in reduced transpiration and enhanced root elongation. In contrast, transgene overexpression did not cause any obvious phenotypic differences under optimal growing conditions. Dehydration factors such as a mannitol and polyethyleneglycol (PEG) have been used to evaluate the effects of decreased water availability and simulate drought conditions in wild-type and CaPMEI1-OX Arabidopsis plants (Gupta and Kaur 2005; Verslues et al. 2006). Other plant invertase inhibitor-related protein genes such as NtCIF and NtVIF are also strongly induced by treatment with PEG or ABA (Rausch and Greiner 2004). However, the molecular and genetic roles played by PMEIs in drought and osmotic stress tolerance remain poorly understood.\nSince plant responses to different abiotic stresses may be related to the accumulation of ROS and the mechanisms for their detoxification, the role played by ROS in stress signaling has been studied extensively (Apel and Hirt 2004). Methyl viologen binds to the thylakoid membranes of chloroplasts and in the presence of light, transfers electrons to O2 in a chain reaction causing continuous formation of superoxide radicals and oxidative stress (Asada 1996). The ArabidopsisCaPMEI1-OX lines exhibited tolerance to oxidative stress, both during seed germination and seedling growth. This tolerance to oxidative stress may reduce the damage caused by other stresses via the antioxidizing system, which suggests that CaPMEI1 overexpression results in detoxification of endogenous superoxide.\nHere, we have determined that CaPMEI1 from pepper plays a role as an antifungal protein and has an inhibitory effect on PME. Furthermore, we have shown that Arabidopsis CaPMEI1-OX lines are resistant to bacterial pathogens. In addition, they exhibit tolerance to drought and oxidative stress. In conclusion, these multivariate functions of CaPMEI1 provide valuable insights into understanding the physiological significance of PMEIs in plant disease resistance and abiotic stress tolerance.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nS1 (DOC 19 kb)","keyphrases":["pectin methylesterase inhibitor protein","antifungal activity","disease resistance","capsicum annuum","oxidative stress tolerance","drought tolerance"],"prmu":["P","P","P","P","P","P"]}
{"id":"J_Gastrointest_Surg-3-1-1852386","title":"Bromodeoxyuridine Labeling Index as an Indicator of Early Tumor Response to Preoperative Radiotherapy in Patients with Rectal Cancer\n","text":"Purpose Assessment of tumor proliferation rate using Bromodeoxyuridine labeling index (BrdUrdLI) as a possible predictor of rectal cancer response to preoperative radiotherapy (RT).\nIntroduction\nIn specialized centers, a refined surgical technique has resulted in high local control figures in rectal cancer. However, local recurrence rates after \u201cstandard\u201d surgery are generally high, with figures ranging between 20 and 40%1,2, although after adopting the total mesorectal excision (TME) concept they fell down to 10\u201312%3,4. Radiotherapy in addition to surgery significantly diminishes the risk of local failure by more than half, from 8 to 2% after 2\u00a0years3. Therefore, combined treatment: radiotherapy (RT) and surgery in the treatment of patients with resectable rectal cancer has been proposed in many trials using either preoperative5,6 or postoperative irradiation7,8. Better results of preoperative RT for 5\u00a0days (25\u00a0Gy in five fractions) in comparison with postoperative 60\u00a0Gy in 30 fractions were achieved by a Swedish group4,9,10, with respect to the local recurrence rate11 and overall survival11,12.\nA corresponding improvement in overall survival has not been demonstrated after postoperative radiotherapy alone13. Graf\u2019s12 study provided a clinically significant biologic effect of a short preoperative course of radiotherapy on the tumor size and on the incidence of nodal metastases; however, this effect was minimized if surgery was performed immediately after radiotherapy. The effect is most likely caused by death of tumor cells in the primary tumor and in the involved nodes. A short treatment course of radiotherapy, i.e., 5\u2009\u00d7\u20095\u00a0Gy is desirable, and this regimen is currently considered as the gold standard in many centers. However, using this schedule it is difficult to observe a down-staging and\/or downsizing of the tumor, which is of importance for the selection of patients for sphincter-preserving surgery (anterior resection).\nIn clinical practice there are no certain methods able to predict tumor response to preoperative radiotherapy (RT). The optimal timing of surgery after preoperative radiotherapy in rectal cancer is unknown. However, it was shown that a long interval (6\u20138\u00a0weeks) between preoperative radiotherapy (39\u00a0Gy in 13 fractions) and surgery was associated with a significantly greater clinical tumor volume reduction than a short interval (2\u00a0weeks)14. On the other hand, it was shown that subclinical pelvic deposits of rectal cancer could grow rapidly during preoperative radiation therapy and during the radiotherapy\u2013surgery interval, with an adverse influence on the rate of pelvic tumor control from protracting the overall treatment time15. Graf et al12 showed that low doses in short RT only offer clinically relevant reduction in the risk of pelvic relapses if the overall radiation treatment time is short. Thus, the rate of cancer cell proliferation seems to be a very important prognostic factor.\nThe aim of this study is to evaluate BrdUrd LI and S-phase fraction (SPF) as the possible indicators of tumor proliferation rate and predictors of the tumor response to neoadjuvant RT in patients with rectal cancer, and to suggest an optimal interval between short RT course and surgery.\nMethods and Materials\nPatients\nBetween November 2003 and January 2006 we recruited 92 patients with resectable rectal carcinoma for whom curative surgery was planned. Patients were eligible for the trial if they were less than 75\u00a0years old, had a histopathologically proved adenocarcinoma (T2\/T3)16 situated less than 12\u00a0cm from the verge of the anus, and gave informed consent for their participation. The protocol was approved by the Ethical Committee of the Center of Oncology, and each patient gave written consent.\nThe criteria for exclusion were: locally nonresectable tumor; plan to perform only local tumor excision; known metastatic disease; previous radiotherapy of pelvis region; other malignant disease; and patient\u2019s refusal.\nPreoperative Radiotherapy\nThe patients assigned to preoperative radiotherapy received a total tumor dose of 25\u00a0Gy. The treatment was given in five fractions over 5\u00a0days, one posterior and two lateral wedged fields were irradiated with photons of maximum 6\u00a0MV energy. According to the random selection surgery was performed the following week (schedule I) or after longer interval of 4\u20135\u00a0weeks (schedule II).\nSurgery\nAnterior resection of rectum or abdominoperineal excision was performed within a week or a month after the completion of RT. Type of surgery was resection of the rectum and lower sigmoid with involved adjacent tissue and regional lymph nodes up to or above the origin of inferior mesenteric artery. A minimal touch technique was used with high tight ligation of the inferior mesenteric artery. The decision whether the patient should have an abdominoperineal resection or a sphincter-preserving surgery was made by the surgeon during the operation.\nAn abdominoperineal resection of rectum was performed in 41 (44.6%) of the patients, and sphincter preserving surgery was performed in 51 (55.4%).\nBiological Assessment of Tumor Response\nTumor samples were taken twice: before radiotherapy (through a rectoscope) and during surgery from the same place, i.e., at the lowest edge of the tumor mass. Each biopsy was divided into two parts: one was used for BrdUrd LI assessment, and the second was used for immunohistochemical analysis (these results will be the subject of a separate study).\nBromodeoxyuridine Labeling Index\nIncorporation of BrdUrd in tumor samples from a biopsy (0.3\u20130.5\u00a0cm3) was carried out in vitro according to the high-pressure oxygen method. The BrdUrd staining procedure and flow cytometry have been described in detail elsewhere17. The stained preparations were analyzed with a FACS Calibur flow cytometer (Becton Dickinson Immunocytometry Systems, Sunnyvale, CA, USA) by one coauthor (AG) and 20\u2009\u00d7\u2009103 events were collected in each histogram. The BrdUrdLI was calculated as a percentage of BrdUrd-labeled cells in a sample, which incorporated BrdUrd during 1\u00a0h of incubation at 37\u00b0C (with discrimination of diploid subpopulation in aneuploid tumors). The tumor ploidy and SPF were calculated from the DNA profile with ModFit software running on a MacIntosh computer. Apoptotic cells were identified as objects with a fractional DNA content not less than 20% of the 2n DNA content. Cell death was calculated as the sum of apoptosis and debris. The tumor ploidy was estimated by evaluating the DNA index, i.e., the ratio of the modal DNA fluorescence of abnormal to normal G1\/0 cells. Aneuploidy was assessed in cases in which the normal and neoplastic cell populations gave two separate peaks. Human lymphocytes were used for the reference peak. Tumors with BrdUrdLI >8.5% (median value) were considered as fast, and those with BrdUrdLI \u22648.5% were considered as slowly proliferating tumors.\nClinical Assessment of Tumor Response\nTumor size before RT was assessed basing on measures taken during rectoscopy, and endorectal sonography. Tumor regression after RT was assessed at the time of operation by surgeons according to the following Response Evaluation Criteria in Solid Tumors (RECIST)18:\nComplete response (CR): 100% disappearance; partial response (PR): 30\u201399% decrease; stable disease (SD): neither CR, PR or PD criteria met; progression of disease (PD): 20% increase in sums of tumor longest diameters.\nPathological Assessment of Tumor Response\nTumor regression after RT was evaluated by a pathologist on the excised tumor mass. The following criteria of tumor regression assessed by Dworak et al.19 were applied:\nD0\u2014no regression; D1\u2014dominant tumor mass with obvious fibrosis and\/or vasculopathy; D2\u2014dominantly fibrotic changes with few tumor cells or groups; D3\u2014very few (difficult to find microscopically) tumor cells in fibrotic tissue with or without mucous substance; D4\u2014no tumor cells, only fibrotic mass (total regression or response).\nStatistical Methods\nStatistical analysis was performed with STATISTICA vs.5. Intergroup differences in the ordinal data were tested with ANOVA test or Student\u2019s t test. P values of less than 0.05 were considered to indicate statistical significance. Linear regression was applied for assessing differences between fast and slowly proliferating tumors in relation with OTT, and its significance was determined by testing the difference between two correlation coefficients. Stratification by BrdUrd LI level was introduced and tested by the inclusion of dummy variable in the regression model.\nResults\nPatients\nA total of 92 patients were included in the study. Twenty-eight (23.3%) out of 120 patients initially qualified for this study were excluded from the analysis because of discontinuation of treatment, metastatic tumor noticed at operation, or no tumor samples taken for biological assessment during surgery. Mean age for the entire group of patients was 61.6\u00a0years (range 30\u201375). There were 68 men and 24 women. There were no statistical differences between the two groups at the time of recruitment for prognostic factors such as: sex, age, histologic grade, or tumor stage (Table\u00a01). \nTable\u00a01Selected Characteristics of Patients and Treatment ParametersCharacteristicsSchedule ISchedule IITotalAge mean (\u00b1SD) years(38)a 61.2\u2009\u00b1\u200912.0(54) 61.9\u00b1\u20099.5(92) 61.6\u2009\u00b1\u200910.6Sex\u00a0Male303868\u00a0Female81624Histological grade\u00a0G162026\u00a0G2293463\u00a0G3303Tumor stage\u00a0T181927\u00a0T2253055\u00a0T35510PTNM\u00a01162541\u00a028614\u00a03131730\u00a04123Interval between RT and surgery\u00a0Mean (range) days(38)a 8.8 (2\u201314)(54) 32.9 (17\u201345)(92) 22.9 (2\u201345)\u00a0OTT mean (range) days(38) 13.8 (7\u201319)(54) 37.9 (22\u201350)(92) 27.9 (7\u201350)Surgery\u00a0Sphincter-preserving20 (52.6 %)31(57.4 %)51\u00a0Abdominoperineal resection182341aNumber of patients\nIn our series of patients, there were 27 stage 1 (29.3%), 55 were T2 (59.8%), and 10 were T3 (10.9%). In 26 patients, tumor cells well differentiated (G1), 63 moderately differentiated (G2), and three poorly differentiated (G3) (Table\u00a01). Thirty-eight patients were treated according to schedule I, in which time interval between end of irradiation and surgery averaged 8.8\u00a0days (range 2\u201314; Table\u00a01). In 54 patients, schedule II was applied, in which mean break was 32.9\u00a0days (range 17\u201345). Because the interval between RT and surgery appeared to be longer than planned, overall treatment time (OTT), e.g., time from the beginning of RT to surgery, was calculated and it appeared to be 7\u201350\u00a0days (Table\u00a01).\nBiologic, Pathologic, and Clinical Assessment of Tumor Response\nMean BrdUrd LI before RT was 8.5% (range 1.0\u201324.2%) and SPF was 22.0% (range 3.8\u201349.9%) and the mean values did not differ between the two schedules (Table\u00a02). Poorly differentiated tumors showed statistically significant higher BrdUrd LI than grades 1 and 2 tumors (P\u2009=\u20090.015; Table\u00a03). After RT, tumors treated according to both schedules showed statistically significant growth inhibition (reduction of BrdUrd LI and percentage of SPF cells) in comparison with the values obtained before RT (Table\u00a02). Radiation induced inhibition of tumor proliferation was expressed as a percentage of the after RT to before RT BrdUrd LI, and SPF as after\/before RT percentage. This ratio ranged from 2.5 to 514% for BrdUrd LI (Fig.\u00a01) and from 5.8 to 522.2% for SPF. When we stratified patients into two groups according to their biological RT response, those radioresponsive with reduction of pretreatment values after radiotherapy above 50% and those less responsive with reduction below 50%, it appeared that the mean values (of the after\/before RT ratios of BrdUrd LI and SPF) for the more radioresponsive tumors were significantly higher than for the less responsive ones. Therefore, these ratios were presented separately for fast (BrdUrd LI >8.5%, SPF >22.0%) and slowly (BrdUrd LI \u22648.5%, SPF \u226422.0%) proliferating tumors. Mean BrdUrd LI value after RT for fast proliferating tumors (41 cases) showed statistically significant (P\u2009=\u20090.027) reduced pretreatment percentage (46.8%) in comparison with slowly proliferating tumors (85.3%, 51 cases). The same was true for SPF of fast (56.4%, 55 cases) and slowly (113.8%, 37) proliferating tumors (P\u2009=\u20090.006). \nTable\u00a02Status of Biological Parameters Before and After RTGroupBrdUrd LI (%) Mean (range)S-phase fraction (%) Mean (range)Apoptosis (%) Mean (range)All patientsBefore RT8.5 (1.0\u201324.2)22.0 (3.8\u201349.9)5.9 (0\u201352.8)After RT4.1* (0.4\u201318.3)16.8** (1.5\u2013101.0)9.8*** (0\u201345.9)RT schedule IBefore RT8.4 (1.1\u201324.2)21.5 (6.1\u201349.2)6.6 (0\u201332.4)After RT3.8*(0.8\u201312.6)14.1**** (1.5\u201347.9)10.5 (0\u201343.3)RT schedule IIBefore RT8.6 (1.0\u201320.0)22.3 (3.8\u201349.9)5.4 (0\u201352.8)After RT4.5* (0.4\u201318.3)17.2 (2.6\u2013101.0)9.5***** (0\u201345.9)*P\u2009=\u20090.000**P\u2009=\u20090.015***P\u2009=\u20090.010****P\u2009=\u20090.002*****P\u2009=\u20090.042Table\u00a03The Relationship Between Tumor Biological Parameters and Histological GradeHistological gradeNBrdUrd LI (%) Mean (range)S-phase fraction (%) Mean (range)Apoptosis (%) Mean (range)G1268.5 (1.1\u201317.1)23.7 (5.8\u201349.9)3.4 (0\u201332.4)G2618.2 (1.0\u201320.0)21.0 (3.8\u201345.6)7.2 (0\u201352.8)G3316.2*, ** (9.5\u201324.2)27.7 (18.9\u201334.7)2.1 (0.4\u20134.8)*P\u2009=\u20090.015, difference between G1 and G3**P\u2009=\u20090.013, difference between G2 and G3Figure\u00a01The association between biological tumor response for slowly (BrdUrd LI\u2009\u2264\u20098.5%; closed symbol) and faster proliferating tumors (BrdUrd LI\u2009>\u20098.5%; open symbol) and overall treatment time. Insert shows linear regression performed separately for each of the tumor subgroups for OTT\u2009>\u200930\u00a0days. P value shows difference between two correlation coefficients.\nNext, the after\/before RT ratios for BrdUrd LI and SPF were correlated with OTT. For SPF, statistical difference between linear regression coefficients for fast and slowly proliferating tumors was not obtained (P\u2009=\u20090.446), therefore the data for BrdUrd LI only are shown (Fig.\u00a01). Insert on Fig.\u00a01 shows a significant (P\u2009=\u20090.033) difference in proliferation rate between fast and slowly proliferating tumors treated within OTT >30\u00a0days. At that time slowly proliferating tumors, contrary to fast proliferating ones, show no inhibition but accelerated proliferation of tumor cells. This phenomenon was also confirmed by increased fraction of S-phase cells in tumors treated with longer RT schedule (Table\u00a02). The influence of BrdUrdLI level has been also tested by the extended regression model between OTT and the percentage of after\/before RT BrdUrd LI. BrdUrd LI level higher than 8.5% has been coded as dummy variable. It appeared to be significant (P\u2009=\u20090.025) in the relation between OTT and the percentage of after\/before RT BrdUrd LI. The partial regression coefficient indicates that the average decrease of the percentage after\/before RT BrdUrdLI for fast proliferating tumors (BrdUrd LI >8.5%) equals 39%.\nAll 92 irradiated rectal tumors were reviewed by the same pathologist (KN). The tumors were classified according to the World Health Organization classification of intestinal carcinoma16 and staged according to the TNM classification20. Of the 92 rectal tumors four had no pTNM classification, 41 were pT1 (46.6%), 14 were pT2 (15.9%), 30 were pT3 (34.1%), and three were pT4 (3.4%). Regional lymph node metastases were found in 27 (30.7%) patients, and 27 (30.7%) patients had their tumor down-staged.\nPathologic assessment of tumor regression after RT according to classification described by Dworak19 was performed in 90 out of 92 patients (for two patients the assessment was impossible). The analysis showed no tumor regression (D0) in 18 (20.0%) tumors, dominant tumor mass (D1) in 46 (51.1%) tumors, a few tumor cells in fibrotic mass (D2) in 18 (20.0%) tumors, single tumor cells (D3) in four (4.4%), and no tumor cells were observed in four (4.4%) of the examined tumors (Fig.\u00a02a). In 25 (27.8%) out of 90 patients marked pathologic down-staging (no residual tumor confined to the rectal wall) was visible. Pretreatment BrdUrdLI and SPF were not correlated with early clinical and pathologic tumor response. However, patients having tumors with LI >8.5% were more radioresponsive (showed significant reduction in proliferative rate after radiotherapy) than patients with BrdUrdLI \u22648.5% tumors, although statistically significant difference between the two tumor subgroups was seen only for D0\u2013D1 grade (Fig.\u00a02b).\nFigure\u00a02Association between biological and pathological assessment (Dworak classification) of early tumor regression for (A) total group of patients and (B) for slowly (BrdUrdLI\u2009\u2264\u20098.5%) and fast proliferating (BrdUrdLI\u2009>\u20098.5%) tumors. Mean values\u2009\u00b1\u2009SE are shown. For stages D0\u2013D1, statistically significant lower inhibition of tumor cell proliferation after RT was observed for slowly than fast proliferating tumors.\nIn the clinical assessment of tumor mass resected during surgery, 34 (36.9%) tumors showed stable disease, 12 (13.0%) showed progressive disease, 41 (44.6%) showed partial response, and four (4.3%) showed complete response (Fig.\u00a03a). And again, in fast proliferating tumors, greater inhibition in tumor proliferation rate (reduction of pretreatment BrdUrd LI value >50%) was observed in fast than in slowly proliferating tumors; however, this difference was not statistically significant (Fig.\u00a03b). As the observed correlation between clinical assessment and SPF was weaker than for BrdUrd LI, the data were not shown. Partial and total tumor regression was observed in 45 (48.9%) tumors. However, tumor proliferation status was not in agreement with the kind of surgery. Sphincter-preserving surgery was performed in 51 out of 92 patients: in 22 (23.9%) fast proliferating and in 29 (31.5%) slowly proliferating tumors.\nFigure\u00a03Association between biological and clinical assessment of early tumor regression after RT for all tumors (A) and separately for slowly and fast proliferating tumors (B). Mean value\u2009\u00b1\u2009SE are shown.\nDiscussion\nThis study provides evidence of a clinically significant biological effect of a short preoperative course of RT on tumor proliferation rate. The impact of irradiation on biological tumor response was assessed by BrdUrd LI, SPF, and the degree of subsequent pathologic and clinical down-staging of the tumors after surgery. The study showed differences in the pretreatment proliferation rate of the tumor. Mean BrdUrd LI before RT was equal to 8.5% and ranged from 1 to 24.2%. Mean SPF was 22.0% and ranged from 3.8 to 49.9%. The proportion of cells in S-phase as estimated by the DNA content overestimates the labeling index determined by the uptake of BrdUrd. This may be so because the exposure time is quite short and there may be subpopulations in the tumors that are synthesizing DNA at a very slow rate, or there may indeed be cells with an S-phase DNA content that are not synthesizing DNA (as a result of nutrient or oxygen supply, lack of growth factors, inadequate vascularity).\nMean value of the BrdUrd LI obtained in this study was lower than the one estimated by Bergstrom et al.21, Palmqvist et al.22, and Terry et al.23, and can be explained by a different method used by these authors: in vivo incorporation of iodouridine\/bromodeoxyuridine, which can cause longer exposure of the tracers to S-phase cells. The differences in the LI value might be caused also by heterogeneity in proliferation within the tumor. It was shown by Bergstrom et al.21 that rectal tumors are polarized, having the superficial surface toward the lumen of the gut and the other toward deep structures facing totally different environments. Apart from Bergstrom et al., none of the above-mentioned authors gave account of site from where the tumor samples were taken. In each tumor analyzed by us, all the samples were taken from the same region, i.e., the bottom part of the mass.\nIn our study, pretreatment BrdUrd LI or SPF was not predictive for early clinical and pathologicl tumor response, probably because of different tumor microenvironment. However, BrdUrd LI after\/before RT ratio gave information on the different significant biological processes that take place after irradiation, and have impact on cell death like redistribution, repopulation, and reoxygenation.\nBrdUrd LI after RT decreased to mean 4.1% independently of the time interval between RT and surgery. Magnitude of LI reduction after RT was correlated with tumor proliferation rate. Greater reduction of BrdUrd LI value was observed in fast proliferating (LI >8.5%) tumors (to mean 46% of the pretreatment value) than in slowly (LI \u22648.5%) proliferating tumors (to mean 85.3% of pretreatment value). What then is the justification for better RT response of fast proliferating tumor cells? According to current knowledge on tumor proliferation, radiation therapy should preferentially inactivate rapidly dividing cells, leaving behind a population biased toward slow proliferation. However, recruitment is a known effect of cytotoxic treatment, and new cells from quiescent cell populations are recruited into active proliferation after irradiation. Probably, slowly proliferating tumors might have greater propensity to recruit cells into rapid cycle in response to treatment than fast proliferating tumors, which might have little reserve capacity for further accelerating their cell cycle24. That might be why we observed acceleration of proliferation rate in slowly proliferating tumors from 5\u00a0weeks after RT (basing on after\/before RT BrdUrd LI ratio), which followed temporary reduction of the number of DNA-synthesizing cells, 4\u20135\u00a0weeks after the start of RT. Accelerated proliferation was confirmed by increased S-phase fraction. However, better biological tumor responsiveness of fast proliferating tumors on cellular level did not find confirmation on tissue level that is in surgery because a fewer number of sphincter saving resections were performed in patients with fast (22) than those with slowly proliferating tumors (29).\nRegression of rectal carcinoma after preoperative irradiation varies, likely reflecting differences in the physical and biologic properties of these tumors. Apart from biological characteristics discussed here, tumor down-staging depends on the total irradiation dose, the fractionation, and the interval between irradiation and surgery25. We showed association of tumor proliferation rate after RT with tumor response basing on BrdUrd LI. SPF, considered as a less sensitive method of tumor proliferation, did not show such a correlation. The after\/before radiotherapy BrdUrd LI ratios correlated, however nonsignificantly, with the degree of pathologic and clinical down-staging, which indicates that more radiation-induced cell death occurred in tumors that expressed high levels of BrUrd LI, or that an increased rate of tumor clearance occurred in more rapidly proliferating tumors. This effect was reflected by significantly higher incidence of apoptosis observed after RT only in fast proliferating tumors (4.1% vs 11.1%; P\u2009=\u20090.000). However, patients having tumors with LI >8.5% did not show higher rate (11.2%) of tumor pathological down-staging (D2\u2013D4) than patients with BrdUrd LI \u22648.5% (16.8%) tumors, which may be suggestive of significant impact on tumor response also by biological processes other than proliferation. In the Spanish study26, high proliferative activity of rectal cancer, as determined by PCNA immunostaining, was predictive of response to preoperative chemoradiotherapy. Willett et al.27, in the same tumor type treated with higher RT dose (47\u201352\u00a0Gy) and 5\u00a0FU, showed that patients having tumors with extensive Ki-67 staining had also a higher rate of tumor down-staging (36%) 4\u20136\u00a0weeks after treatment than patients with minimal to moderate Ki-67 staining tumors (22\u201323%). These authors show that elevated postirradiation tumor proliferative activity correlated strongly with improved survival28. These authors, in contrast to our study, did not consider the proliferation profile of pre- and postirradiation for individual patients. The correlation of down-staging and higher survival rates was also found by other authors29,30.\nIn our study, even in totally regressed tumors (D4), the percentage of the after\/before radiotherapy BrdUrd LI was about 50%, which may not indicate tumor but normal cell proliferation, mainly a fraction of activated fibroblasts or cycling endothelial cells in capillaries high in colorectal carcinoma31. Our study showed complete pathologic response (D4) similar to that in a Norwegian study (4.5%)32, where histological tumor slides were analyzed after treatment with a dose of 31.5\u00a0Gy in 18 fractions and 2\u20133\u00a0weeks interval between RT and surgery. However, it should be stressed that in this study, a high incidence (31.3%) of recurrences was observed at late follow-up. Our analysis showed that patients having fast proliferating tumors, as assessed by BrdUrdLI, experienced higher rates of regression than patients with slowly proliferating tumors, which could suggest a more frequent possibility of performing sphincter-preserving procedures in these tumors. However, this was not confirmed in surgical procedures. Therefore, we do not know yet if pretreatment BrdUrd LI assessment will be a good predictor for a locoregional failure. Berger et al.25, analyzing tumor sterilization after preoperative RT for rectal cancer, did not find a predictive factor for complete pathological response among such factors as age, sex, tumor stage, and pathologic grade. However, they found favorable influence of higher doses (>44\u00a0Gy) on pathologic stage.\nThere is no known optimal time for the interval between RT and surgery. The Swedish group keeps the interval at about a week; however, in other institutions, using longer RT treatments and higher total dose, longer intervals\u20144 to 6\u00a0weeks were adopted14,25. The main reason for a longer interval is tumor regression, which makes sphincter preservation possible. Similar to Francois et al14, we observed higher clinical and pathologic response rate after longer interval between RT and surgery. However, these authors14 showed nonsignificantly better overall survival for patients treated with shorter interval. Withers and Haustermans33 estimated the interval between long course of fractionated RT (40\u201354\u00a0Gy) and surgery and stated that the interval is not critical to either local recurrence or distant metastases. The authors offered the following arguments: the tumor cells do not disseminate until the primary tumor is large enough to be clinically detectable (probably 80% of patients whose rectal tumors have not metastasized to lymph nodes will be free of metastases). Irradiation with a dose of 40\u00a0Gy in 2\u00a0Gy fractions (equivalent to 25\u00a0Gy in five fractions) reduces tumor cell survival by about six decades, e.g., from 1010 to 104 cells. However, we have to remember that although the short overall treatment duration in the 25\u00a0Gy in five-fraction regimen provides a radiobiological advantage, this is a relatively low dose34, which causes about a 66% reduction in the rate of local recurrence11. A retrospective analysis of published results of preoperative radiation therapy for rectal cancer showed that local control probability curves were displaced toward higher doses as the overall duration of preoperative radiation therapy was increased15. Therefore, longer intervals between short RT schedule (25\u00a0Gy) and surgery may be inappropriate in case of patients with incomplete resection (cut-through) of primary tumor, in whom the average subclinical cancer cell burden increases during long interval. Also, subclinical disease beyond the future surgical margins, may be a potential target for future recurrences. Longer intervals after short RT can be dangerous because of potential subclinical tumor, which may grow more quickly than primary tumor15,33, the and risk of developing distant metastases. If we imply that moderately differentiated adenocarcinoma cells have different metastatic and proliferative activities from poorly differentiated cancer cells, which was shown by Taniyama et al35, then we could have an indication to adjuvant chemotherapy for patients with differentiated tumors. The authors35 indicated that moderately differentiated cancer cells are associated with hematogenous metastases to the liver, and the loss of tubular formation of cancer cells in poorly differentiated tumors may be fundamentally related to lymph node metastases and infiltrative growth. Therefore, particularly in patients with moderately differentiated and slowly proliferating tumors, adjuvant chemotherapy could be suggested after OTT shorter than 4\u00a0weeks, to prevent developing metastases to the liver.\nIn conclusion, our study shows that pretreatment BrdUrd LI or SPF were not predictive for early clinical and pathologic tumor response. After\/before BrdUrd LI ratios showed inhibition of proliferation in responsive tumors, but this was not reflected in the number of sphincter preserving procedures performed. As 1\u00a0month after RT, accelerated proliferation of tumor cells is observed only in slowly proliferating tumors, we think that longer interval between RT and surgery is inadvisable.\nIf late tumor response confirms that patients having tumors with increased proliferative activity have statistically significantly less recurrences and improved survival rates compared with patients with less proliferative tumors, then we will be able to suggest a prognostic factor for individual rectal cancer patient, and a basis for selection to postoperative adjuvant chemotherapy.","keyphrases":["early tumor response","radiotherapy","rectal cancer","proliferation rate","brdurdli"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_J_Pediatr-4-1-2254655","title":"Pneumomediastinum in the neonatal and paediatric intensive care unit\n","text":"The incidence, aetiology and pathophysiology of pneumomediastinum (PM), an uncommon and potentially serious disease in neonates and children, were evaluated. A retrospective chart review of all patients diagnosed with PM who were hospitalised in the intensive care unit of the University Children\u2019s Hospital Z\u00fcrich, Switzerland, from 2000 to 2006, was preformed. We analysed the incidence, severity and causes of PM and investigated the possible differences between neonatal and non-neonatal cases. Seven children and nine neonates were identified with PM. All patients had a good outcome. Six cases of PM in the group of children older than 4 weeks were deemed to be caused by trauma, infection and sports, whereas one case was idiopathic. All nine neonatal cases presented with symptoms of respiratory distress. We were able to attribute four cases of neonatal PM to pulmonary infection, immature lungs and ventilatory support. Five neonatal cases remained unexplained after careful review of the hospital records. In conclusion, PM in children and neonates has a good prognosis. Mostly, it is associated with extrapulmonary air at other sites. It is diagnosed by chest X-ray alone. We identified mechanical events leading to the airway rupture in most children >4 weeks of life, whereas we were unable to identify a cause in half of the neonates studied (idiopathic PM).\nIntroduction\nPneumomediastinum (PM) is defined as a mediastinal air leak. The experimental works of Macklin and Macklin provided insights into its pathophysiology [4, 5]; alveolar rupture occurs because of a pressure gradient between the alveolus and the surrounding tissues. This gradient develops either through overinflation of the alveolus or a reduction of interstitial pressure. The air that subsequently leaks into the interstitial tissue diffuses toward the peribronchial and perivascular tissue, and then towards the mediastinum, the neck and into the subcutaneous tissue. However, due to pressure equalisation between the affected and adjacent alveoli in the lungs, the interalveolar walls remain intact and the lungs inflated.\nThe diagnosis of PM is confirmed by frontal chest roentgenogram, including the cervical region. Typical radiological signs of PM include the continuous diaphragm sign (interposition of air between the pericardium and the diaphragm, which becomes visible in the central mediastinal part) and linear bands of mediastinal air parallelling the left side of the heart and the descending aorta (pleura is shown as a fine opaque line) with extension superiorly along the great vessels into the neck. In infants, the \u201cspinnaker sign\u201d (an upwards and outwards deviation of thymic lobes) can be seen when the thymus is raised above the heart by pneumomediastinal air that elevates the thymus and separates it from the cardiac silhouette beneath [2].\nVarious causes of PM are found in the literature, such as airway obstruction (e.g. foreign body aspiration), iatrogenic (e.g. mechanical ventilation), infections (e.g. pneumonia), obstructive lung disease (e.g. asthma), toxic effects (e.g. smoking), trauma (e.g. chest trauma), Valsalva manoeuvres (e.g. vomiting) and the weakness of tissue (e.g. anorexia nervosa). In spontaneous PM, the underlying lung is healthy and the air leak is thought to be atraumatic [3]. In neonates, known predisposing factors are mixed lung diseases, such as pneumonia or meconium aspiration syndrome, with coexisting atelectasis and airway obstruction [1]. However, only scarce literature is found about neonates with PM.\nIn this study, we retrospectively analysed the incidence, severity and causalities of PM in neonates and children >4 weeks of life admitted to our intensive care unit, and we investigated the possible differences between the groups.\nMaterial and methods\nWe retrospectively reviewed all records of children diagnosed with PM who were hospitalised in the interdisciplinary neonatal and paediatric intensive care unit of the University Children\u2019s Hospital in Z\u00fcrich, Switzerland, between January 2000 and September 2006. The patients were divided into two groups according to their age: neonates (under 4\u00a0weeks of age) and children (over 4\u00a0weeks of age). We were interested in the causes of PM as documented by the treating physicians, the types and results of radiologic investigations performed, any invasive interventions used to treat PM, the severity of the PM and the length of stay in the intensive care unit.\nResults\nAbout 1,200 children are admitted to our intensive care unit per year. The incidence of PM in our intensive care unit was 0.08% for children >4\u00a0weeks of age and 0.1% for neonates. In all patients, PM was diagnosed by chest X-ray and all had a positive outcome related to the PM. All five patients with pneumopericardium (PP) did not suffer from any complications (e.g. pericardial tamponade).\nSeven children were >4\u00a0weeks of age (Table\u00a01). Their mean age was 7.1\u00a0years (range 1.3\u201315.8\u00a0years). In addition to PM, two children of this group had subcutaneous emphysema (SE), two a pneumothorax (PT) and two a PP. Different causes were found for the air trapping. There were two traumatic aetiologies (rib fracture after a severe car accident, lesion in the hypopharynx after a fall). Two children were diagnosed with obstructive bronchitis and in one child, barotrauma occurred intraoperatively due to a clamped expiratory tube during mechanical ventilation (Fig.\u00a01). One child had exercised vigorously three days before hospitalisation, which may have caused the PM. In one adolescent, PM occurred spontaneously. All children were hospitalised in the intensive care unit for one to seven days, depending on the severity of their underlying disease. Only two children required pleural drainage and intubation. All other children were treated for their underlying conditions and received oxygen therapy. Diagnostics for the PM other than chest X-rays were performed in four patients. All of these had received a thoracic CT scan. In one child, who also had a huge subcutaneous emphysema and dysphagia, the reason for the air trapping could only be found by means of a laryngotracheoscopy, which showed a traumatic lesion in the hypopharynx. The patient\u2019s history revealed that she had fallen onto a piece of wood by her neck.\nTable\u00a01Results for the group of children >4\u00a0weeks of lifeAir leakAetiologyAge (years)Intubation (after diagnosis)Pleural drainageDiagnostic testsDays in ICUPM, PP, SESpontaneous\/3 days earlier intensive sport15.8NoNoChest X-ray 3\u00d72PM, PTTraumatic7.53\u00a0daysYesCT\/chest X-ray 3\u00d75PM, SE Traumatic (lesion in hypopharynx)1.3NoNoCT\/chest X-ray 3\u00d7, oesophagogram with contrast medium, laryngotracheoscopy4PM, PPIatrogenic: equipment failure with barotrauma during mechanical ventilation2.3NoNoChest X-ray 2\u00d72PMSpontaneous15.4NoNoChest X-ray\/CT1PM Obstructive bronchitis5.6NoNoChest X-ray 2\u00d7\/CT2PM, PTObstructive bronchitis1.97\u00a0daysYesChest X-ray 7\u00d77Fig.\u00a01Pneumomediastinum (PM), subcutaneous emphysema (SE) and pneumopericardium (PP) in a 2-year-old intubated patient\nThe group of children older than 4\u00a0weeks stayed in the intensive care unit for a mean of 3.2\u00a0days (range 1\u20137\u00a0days), depending on the severity of the PM and the underlying disease. Compared to the neonatal group, the length of stay in the intensive care unit was shorter. However, most neonates stayed in the intensive care unit longer, primarily because of comorbid conditions and not because of the PM.\nWe found nine neonates who were diagnosed with PM (Table\u00a02); two premature and seven term infants, all of whom presented with signs of respiratory distress. Three neonates were also diagnosed with a PP, one with SE and five with a PT. Birth weight ranged from 2,150\u00a0g to 4,140\u00a0g (mean 3,340\u00a0g). Five children were born vaginally and four by caesarean section. All children were vigorous at birth and none required resuscitation with bag mask ventilation or surfactant. Before arriving in the intensive care unit, where the diagnosis of PM was confirmed by chest X-ray, two infants had received ventilatory support by CPAP and one of the premature infants had to be intubated for respiratory failure. During hospitalisation in the intensive care unit, two children deteriorated and required mechanical ventilation for three and four days, respectively, and two other children needed CPAP for a few hours. Only one child received pleural drainage. All children received oxygen therapy and specific therapy for their underlying disease. The age at admission to the intensive care unit ranged from a few hours to four days. One neonate was admitted to the intensive care unit due to convulsions and developed a PM on day six of life. The treating physicians felt that the PM may have been associated with a Valsalva manoeuvre, which occurred during the seizure. Other causes of PM were a pulmonary infection due to maternal infection and a possible barotrauma due to peak inspiratory pressure of 25\u00a0cm H2O in a mechanically ventilated premature neonate. Two newborns had PM related to CPAP and four neonates were diagnosed with spontaneous PM. Neonates stayed in the intensive care unit for 3\u201313\u00a0days (mean 5.6\u00a0days), depending on the severity of the underlying diseases.\nTable\u00a02Results for the group of neonatesAir leakAetiologyBirth weightGestational age (weeks)Mode of deliveryMechanical ventilation before diagnosisDuration of ventilatory support after diagnosisPleural drainageDays in ICUPMSpontaneous4,140\u00a0g40 0\/7VaginalNoNoNo4PM, PP, SEPremature lungs, barotrauma2,150\u00a0g34 4\/7Caesarian section Pip max. 25\u00a0cm H203\u00a0days (intubation)No4PM, PT, PPPremature lungs, spontaneous or CPAP2,480\u00a0g35 6\/7VaginalCPAP4\u00a0days (intubation)Yes6PM, PTSpontaneous3,485\u00a0g38 1\/7Caesarian sectionNoNoNo3PM, PTSpontaneous3,440\u00a0g37 5\/7VaginalNo6\u00a0hours (CPAP)No3PM, PPSpontaneous2,830\u00a0g39 1\/7Caesarian sectionNoNoNo13PM, PTSpontaneous3,970\u00a0g38 5\/7Caesarian sectionNoNoNo9PM, PTPulmonary infection due to maternal infection3,440\u00a0g38 5\/7VaginalCPAP1\u00a0day (CPAP)No5PMConvulsions or spontaneous4,130\u00a0g40 5\/7VaginalNoNoNo4\nDiscussion\nAll children with PM had a good outcome without any complications due to air trapping.\nIn the group of children older than 4\u00a0weeks, only two children developed a respiratory insufficiency, leading to mechanical ventilation. In both of them, respiratory failure was related to their underlying condition (polytrauma with haematothorax and severe obstructive bronchitis, respectively). These were also the children who stayed longest in the intensive care unit. All other children were treated with oxygen only and stayed in the intensive care unit until they improved clinically and radiographically. Regarding radiologic diagnostics, four patients of the group of children >4 weeks of life had CT scans (three of them had been done in outside clinics from where the patients had been admitted to our intensive care unit). Retrospectively, the utility of the CT scans was put into question, as these scans did not change the patient management. The only patient in whom the CT scan changed management was the child with polytrauma. In this patient, other intrathoracic injuries needed to be ruled out.\nIn the group of neonates, it was much more difficult to find the aetiology of the PM, since all neonates had presented with respiratory disease, and radiologic investigations were partially performed only after the use of CPAP or tracheal intubation. Five of the nine neonates had a spontaneous PM without risk factors, such as mechanical respiratory support (bag mask ventilation after birth, CPAP, mechanical ventilation) or restrictive lung disease. Three of these five babies were delivered by caesarean section. In the remaining four newborns, possible mechanical incidents leading to the air leak could be revealed: mechanical ventilation with high inspiratory pressure, CPAP, pulmonary infection and convulsion. Further investigations are needed to find the aetiology of spontaneous PM in healthy, term neonates.\nIn conclusion, PM in children and neonates has a good prognosis. Mostly, it is associated with extrapulmonary air at other sites. It is diagnosed by chest X-ray alone. Whereas in older children mechanical events leading to the airway rupture can be revealed in most cases, about half of the neonates in our series suffered from PM without obvious reason.","keyphrases":["pneumomediastinum","air leak","pneumopericardium","subcutaneous emphysema","respiratory distress syndrome"],"prmu":["P","P","P","P","R"]}
{"id":"Law_Hum_Behav-4-1-2175020","title":"Policy Forum: Studying Eyewitness Investigations in the Field\n","text":"This article considers methodological issues arising from recent efforts to provide field tests of eyewitness identification procedures. We focus in particular on a field study (Mecklenburg 2006) that examined the \u201cdouble blind, sequential\u201d technique, and consider the implications of an acknowledged methodological confound in the study. We explain why the confound has severe consequences for assessing the real-world implications of this study.\nOne of the most interesting products of the first wave of wrongful convictions exposed by DNA has been a vigorous debate over potential changes in the design and execution of the lineups and photographic arrays, familiar to every television viewer, that police rely on to probe memory in eyewitness cases, the category that dominates the exoneration lists. All of the current proposals for change in investigative practice derive from extensive laboratory inquiry, and they have at their cores the novel \u201cdouble-blind, sequential\u201d technique for conducting eyewitness identification procedures. In this technique the law enforcement personnel conducting an identification procedure are \u201cblind\u201d concerning which person in the lineup or photo array is the police suspect, and they present the \u201cfillers\u201d and the suspect to the witness individually (\u201csequentially\u201d) rather than in a group (\u201csimultaneously\u201d), as in the traditional practice. The changes from current procedure are designed to ensure that witnesses discern no inadvertent cues as to which individual they should or should not identify, to encourage witnesses to compare each individual they see to the remembered image of the criminal (rather than to make a relative, \u201clooks-most-like,\u201d judgment comparing the individuals displayed to each other), and to eliminate unnecessary \u201cfeedback\u201d to witnesses who have made a selection and might look to the lineup administrator for confirmation or contradiction.\nEveryone agrees that proposed changes in investigative practice should be tested in the field, but moving from the laboratory to the field has always been problematic. The proper design of field studies used to evaluate new procedures in the field has become an important issue. Two recent efforts at field-testing the \u201cdouble-blind, sequential\u201d option have taken place. The first, conducted by several departments in Hennepin County, Minnesota, produced results consistent with those predicted by the laboratory scientists, but made no explicit comparison to traditional practices, and it has not been controversial. (Klobuchar et\u00a0al. 2006). The second field study, conducted in three Illinois jurisdictions under the direction of the general counsel for the Chicago Police, Sheri Mecklenburg, and documented at length in a report (usually referred to as \u201cThe Mecklenburg Report,\u201d after its author) appeared to contradict both the laboratory scientists\u2019 predictions and the sparse existing field data on eyewitness performance (Mecklenburg 2006). The Mecklenburg report stated that in two of the three jurisdictions reporting, the traditional method of an aware, \u201cnot-blind\u201d detective displaying the suspect and \u201cfillers\u201d in a group to the witness produced a lower rate of identifications of innocent fillers and a higher rate of identifications of suspects than did the lab-generated \u201cdouble-blind, sequential\u201d technique. The recommendation of the Mecklenburg Report, in other words, was that the system should not institute changes on the basis of the laboratory science. The Mecklenburg Report was vigorously publicized, and it immediately drew both determined support and sharp criticism from psychologists who had long been interested in the issue of eyewitness investigative procedures.\nUnfortunately for criminal justice practitioners who must decide whether procedures should be changed, the early scientific commentaries on the Mecklenburg Report generally aligned with the views on the potential of these particular procedural innovations that the commentators had announced throughout their long careers of involvement with the issue of eyewitness memory. Seizing on this, partisans on both sides of the debate over procedures have unfairly dismissed some criticism and praise of the Mecklenburg Report as reflecting nothing more than the scientific commentators\u2019 stubborn loyalty to their own pre-existing beliefs. A standoff has arisen. Although everyone agrees that further field studies are required, practitioners considering future field studies have been left to wonder whether they should simply repeat the Illinois Study described in the Mecklenburg Report, or attempt to find a new design.\nWe have read the materials related to the Mecklenburg study, including the Mecklenburg Report, its Addendum and Appendices, the supportive comments of Dr. Roy Malpass (2006) and Dr. Ebbe Ebbesen (2006), and the critical comments of Dr. Gary Wells (2006) and Dr. Nancy Steblay (2006). The Report indicates, and all commentators seem to agree, that the study does contain a confound: a non-blind simultaneous procedure is compared with a blind sequential procedure. The bottom line issue here, or at least the one that drew our group\u2019s attention, concerns the importance of the confound.\nIt is easy to understand the sentiment expressed by Mecklenburg in her Addendum that not all variables can be controlled in a field study such as the one she designed and describes in the Report. Confounds can occur in laboratory studies as well as field studies. The issue that always arises in such cases concerns the implications of the confound: Is it critically related to interpreting the major outcome of the study? Or is the confound incidental to the main conclusion, such that even though the confound is acknowledged, the major results of the study are still interpretable?\nThe Mecklenburg Report asserts that \u201cThe Illinois Pilot Study was properly designed to answer the question: how do the current procedures compare with the proposed procedures, both in terms of identification rates and implementation?\u201d From this perspective, the confound between blind\/non-blind and sequential\/simultaneous would not be critical, because non-blind simultaneous reflects the current procedure to which the blind\/sequential procedure is compared. Unfortunately, this perspective seems seriously problematic.\nOur reading of the materials forces us to conclude that the confound has devastating consequences for assessing the real-world implications of this particular study.\nIf it is the case that the better outcome from the non-blind\/simultaneous procedure is partly or entirely attributable to subtle, unintentional cues provided by the administrator, then the Illinois results may simply underscore that the present procedure produces a biased outcome that may ultimately result in the increased conviction of innocent individuals. Stated slightly differently, it is critical to determine whether the seemingly better result from the simultaneous procedure is attributable to properties of the simultaneous procedure itself, or to the influence of the non-blind administrator.\nWe should note that under these testing conditions, if the results had shown the sequential lineup to be superior, one would not know whether it was really the use of the sequential lineup or the use of a blind investigator conducting the lineup that produced the result. Of course, any difference between conditions could be due to some combination of the factors. Even if no difference in outcome occurred between the procedures, one could not safely conclude there is no difference between them if the detectives were informed in one condition and not in the other. Thus, although the conditions used in the study made some sense from a practical standpoint, the design guaranteed that most outcomes would be difficult or impossible to interpret. The only way to sort this out is by conducting further studies including, at a minimum, a blind\/simultaneous condition (it would also be desirable to include a non-blind\/sequential condition to fill out the design, but it is not an absolutely necessary condition for the present purposes).\nIn the materials we have reviewed, the Mecklenburg Report\u2019s detractors (including Wells) and its advocates (including Mecklenburg herself) disagree on whether the misidentification rates in two of the three participating jurisdictions (a zero rate of \u201cfiller\u201d identifications) are suspiciously low. However, Wells cites enough evidence that they may be low to justify the concern that administrator bias is operating, either consciously or unconsciously; either by failing to count tentative \u201cfiller\u201d choices, or in steering witnesses away from fillers, or toward suspects.\nThe problem is that we cannot know on the basis of the Mecklenburg study whether such bias is operating, even though the entire interpretation of the significance of the study for real-world practices hinges on this issue.\nMecklenburg states in her Addendum that the question of how blind administrators affect simultaneous lineups is one of several questions to be addressed in future studies. We certainly hope so. But the statement that follows is problematic: \u201cHowever, the Illinois Pilot Program was not intended to answer those questions and any attempt to discredit the Illinois study on that basis is misguided.\u201d\nIf the Illinois study was not designed to address the question of what happens in a blind\/simultaneous line-up, given its centrality to the issue, then our assessment is that the Illinois study addressed a question (comparing blind\/sequential and non-blind\/simultaneous) that is not worth addressing, because the results do not inform everyday practice in a useful manner.\nNo single field study can produce a final blueprint for procedural reform; we will need many. The design of these studies, however, will be crucial. A well-designed field study that avoids the flaw built into the Illinois effort, can be an important first step toward learning what we need to know about the best practices in identification procedures.","keyphrases":["eyewitness identification","field studies","double blind sequential procedure"],"prmu":["P","P","R"]}
{"id":"Matern_Child_Health_J-2-2-1592248","title":"The National Summit on Preconception Care: A Summary of Concepts and Recommendations\n","text":"The Centers for Disease Control and Prevention (CDC) and 35 partner organizations have engaged in developing an agenda for Preconception Health. A summit was held in June 2005 to discuss the current state of knowledge regarding preconception care and convene a select panel to develop recommendations and action steps for improving the health of women, children, and families through advances in clinical care, public health, and community action. A Select Panel on Preconception Care, convened by CDC, deliberated critical related issues and created refined definition of preconception care. The panel also developed a strategic plan with goals, recommendations, and action steps for improving preconception health. The recommendations and action steps are specific to the implementation of health behavior, access, consumer demand, research, and surveillance activities for monitoring and improving the health of women, children and families. The outcome of the deliberations is the CDC publication of detailed recommendations and action steps in the Morbidity and Mortality Weekly Report series, Recommendations and Reports.\nIntroduction\nIn June 2005 the Centers for Disease Control and Prevention (CDC) and the March of Dimes, in collaboration with 35 professional and governmental organizations, convened a 3-day summit to discuss an agenda for preconception care programs, research, and policy. The summit was the result of internal workgroup discussions at CDC and an initial meeting with external stakeholders in November 2004. Prior to the November meeting, subject matter experts in 19 programs from 8 centers throughout CDC conducted a detailed review of the relevant literature. The 3-day summit was divided evenly into two components: a presentation series from preconception care practitioners, followed by a select panel meeting. More than 60 presentations on preconception care research and programs were made and subsequently discussed by the select panel to generate recommendations for improving maternal and child health outcomes through better preconception care. The goals of the select panel discussions were to define current scientific knowledge, to identify best practices, and to highlight key issues needing further attention that could be used as the basis for formulating recommendations and action steps.\nDeliberations of the select panel\nThe select panel consisted of nationally recognized experts from a variety of disciplines representing different perspectives on preconception care services designed to promote women's health and reduce adverse pregnancy and perinatal outcomes. The panel included experts in the fields of obstetrics, family practice, pediatrics, public health, nursing, reproductive health, toxic exposures, and chronic and infectious disease. The initial conceptual framework for deliberations and discussions was based on the acronym \u201cACT\u201d (Accessible health care, Comprehensive care, and Timely provision of care) proposed by Dr. Jennifer Howse, president of the March of Dimes. It was then expanded to include six \u201cA\u2019s\u201d: access, availability, affordability, acceptability, accuracy, and appropriateness. Five organizing themes (clinical practice, social marketing and health promotion, public health and community, public policy and finance, data and research) served as the basis for discussion.\nOne key topic of debate was the potential conflict in characterizing preconception care as preparation for pregnancy, as opposed to the broader promotion of women's health. Some panelists believed strongly that the focus must be on both women and infant health outcomes. In moving forward, the panel was conscientious not to carve preconception care out separately from good routine primary care; however, panel members also asserted that even though the focus should be on preconception care, providers should also recognize that the scope of care should include comprehensive women's health services. As one panel member said, \u201cPreconception care should be happening at every interaction with a woman or man of reproductive age\u2026. It's part of what we\u2019re already doing in primary care. We\u2019re just trying to get providers to reframe their thinking so we\u2019re achieving preconception health.\u201d Some, while agreeing, argued that the ultimate goal was to improve perinatal outcomes, making this a part of, but distinct from, all well-woman's health care.\nThe status of current research emerged as a central issue. Discussions often centered around the concern that insufficient scientific evidence currently exists for many preconception care interventions, for the best methods of integrating them into primary care, and for effectively delivering interventions as a package\u2014or even if they work. Many panelists cited the need for intervention trials. In contrast, other members believed that existing research findings were sufficient, and that moving in the direction of translation and action should be the next step. This theme was captured in one member's comment, \u201cResearch is important; there's no doubt about it. But instead of just creating more new knowledge, let's ask ourselves: Why is it that the knowledge we created before isn't getting translated into action?\u201d Ultimately, the panel's recommendations reflected both areas where sufficient evidence exists and those where more research is needed.\nMany panel members supported recommendations that would advocate provider assistance for a woman and her partner in developing a reproductive life plan and in communicating her\/their intentions through the implementation of that plan. As one panel member described it, \u201c[Primary care providers can] develop the ability to help a woman write her reproductive health plan and facilitate her ability to carry this out, including the upgrading of this plan throughout her reproductive years.\u201d Some participants suggested that the recommendations should strongly encourage all providers not only to ask a woman about her reproductive intentions, but to ask in a manner that conveyed to the woman that her decisions should be based on her personal preferences. This would allow the provider to help her choose the best contraceptive for meeting her life goals and to provide appropriate preconception awareness information.\nPanelists generally agreed that the recommendations should focus on feasible actions that could have the greatest positive impact on health outcomes. This resulted in much discussion of fundamental issues, including 1) who gets preconception care\u2014women at high risk for adverse pregnancy outcomes or all women; 2) what would yield the greatest return on investment (e.g., focus on the preconception period or the interconception period; focus on all women or high-risk women); 3) whether interventions could be packaged and targeted to reach specific populations or address specific needs of women; and 4) how finance case and services, as well as the potential cost effectiveness and cost benefit of the proposed interventions?\nDefining preconception care\nThe select panel worked to refine the definition of preconception care. The participants agreed that it is important to recognize what preconception care is and what it is not. Preconception care is not a single visit but a continuum of care designed to meet the needs of a woman throughout the various stages of her reproductive life. The goal of the preconception care process is to make sure that the woman is healthy as she attempts to become pregnant [1\u20136], and to promote her health and the health of her children throughout her reproductive lifespan. Based on the Select Panel deliberations and its subsequently published recommendations, preconception care is defined here as a set of interventions that aim to identify and modify biomedical, behavioral, and social risks to a woman's health or pregnancy outcome through prevention and management, emphasizing those factors that must be acted on before conception or early in pregnancy to have maximal impact. Thus, it is more than a single visit and less than all well-woman care. It includes care before a first pregnancy or between pregnancies (commonly known as interconception care) [7].\nPreconception care and interventions are designed to reduce perinatal risk factors and, for optimal effectiveness, must be successfully implemented before the start of pregnancy [4]. The components of preconception care, including an array of interventions to address medical, psychosocial, and environmental risks associated with childbearing, augment routine well-woman care. Current Guidelines for Perinatal Care (jointly issued by the American Academy of Pediatrics and American College of Obstetricians and Gynecologists) recommend that \u201cAll health encounters during a woman's reproductive years, particularly those that are a part of preconception care, should include counseling on appropriate medical care and behavior to optimize pregnancy outcomes\u201d [8, 9]. There is scientific evidence that certain preconception interventions are effective, yet at present they are not routinely and systematically applied.\nFig. 1Ecological model\nThe framework for preconception care recommendations\nThe framework for the recommendations and action steps to improve preconception health is based on two well-established models for community health and behavior change, the ecological and the lifespan models. The ecological model for medical care is constructed with the woman being the central player, while recognizing the significant roles of the larger community, the providers, and the institutions in ensuring the best outcomes for her (Fig. 1). The four levels of this model interact and are not distinct. Each level has primary responsibility for different aspects of health and well-being; however, all levels are integrated and each plays a supportive role in the success of the other levels and the system as a whole. If one level is not fully engaged, the system will weaken, and the result may be poor health for the individual woman. In many regards the community and institution levels overlap; institutions operate within the community, just as the community determines what services the institutions provide. Using this framework, the proposed recommendations outline actions and responsibilities at each level and demonstrate the role that all sectors have in improving preconception care and ultimately the health of women and their families. The recommendations and action steps described here aim to address needs and gaps at each level.\nThe second model influencing the framework for preconception care is the lifespan perspective [10]. This framework recognizes that health is determined by factors across the lifespan and is intergenerational. Improving preconception health will require, first, that the woman herself is achieving optimal health throughout her life. With women living in a healthy state, healthy children are more likely to result. As one panel member said, \u201cWe should build on the pediatric model of anticipatory guidance\u2026 [and] have consistent messages and care for women through the lifespan that will address women's and preconception health before, between and beyond.\u201d Furthermore, good health in infancy is likely to carry through to childhood, adolescence, young adulthood and older adulthood so that healthy infants become healthy adults and achieve their reproductive goals. This lifespan framework extends beyond the individual to promote health in the family and community. Thus, the recommendations and action steps both address health needs across the lifespan and recognize that the individual, family, community, and institutions each have roles in ensuring that all women have lifelong optimal health.\nChallenges for implementing the proposed frameworks\nMaximizing the potential impact of preconception interventions will require integration across the various levels of care and areas of expertise [5, 11\u201313]. Changes in individual behavior, clinical practice, community programs, and public policies are recommended.\nIn the context of clinical care, much can be done by primary care providers, but evidence suggests that many women have two primary care providers\u2014one with expertise in obstetrics and gynecology and one with general medical training. In addition, the U.S. health-care system is fragmented into highly specialized components. Nevertheless, bundling the various preconception interventions has the potential to improve perinatal outcomes and reduce costs associated with adverse outcomes. These recommendations call for more integrated delivery of health promotion and care services for women rather than for greater fragmentation of the health-care system. Integration is critical to moving forward, In consideration of competing priorities and limited resources, integration may achieve health-care economies of scale that produce more efficient delivery of services and more effective results in improving preconception health. For example, primary care providers potentially could routinely and efficiently use the tools available to screen for smoking, alcohol abuse, genetic risks, and occupational hazards; endocrinologists, geneticists, and nutritionists could become an integral part of a preconception care strategy. Moreover, settings such as family planning or sexually transmitted disease clinics must become part of an integrated approach in order to reach many women at high risk for adverse pregnancy outcomes. New models of care and well-designed quality-improvement efforts could foster the integration of preconception interventions.\nAt the same time, preconception care must be tailored to meet the needs of the individual woman. Given that preconception care is a process, not a visit, some recommendations will be more relevant to women at specific stages in their lives or those with varying levels of health risks. Health promotion efforts, risk screening, and interventions would be different for a young woman who has never experienced pregnancy than for a 35-year-old woman who has had three children. Women with chronic diseases, prior pregnancy complications, or behavioral risk factors may need more intensive interventions [5, 6, 11, 12]. At the clinical level, it is especially important that individual interventions be tailored to the specific needs of the woman. Such variations in interventions also place constraints on how interventions can and should be bundled or prepackaged.\nThis special issue of the Maternal and Child Health Journal, as well as the MMWR Recommendations and Reports, emphasizes components of preconception care supported by research and an evidence base that documents the efficacy of specific interventions [7]. For example, that increasing pre-pregnancy intake of folic acid reduces neural tube defects, and controlling diabetes prior to pregnancy yields better outcomes for mothers and babies [14, 15]. As with prenatal care or well-child care, however, evidence may not be forthcoming that one regimented process of preconception care significantly improves outcomes. Rather, the process of providing individualized screening, health education, and necessary interventions that can yield better outcomes.\nFinally, implementing these recommendations to improve preconception health will take time. Diffusion of innovation theory demonstrates how concepts and best practices tend to be disseminated and adopted [16]. The innovators who lead current efforts to advance preconception care and the early adopters who run model programs are making only the first steps toward larger societal change. Whereas the select panel's action steps were designed to be short-term efforts, better implementing professional standards of care, modifying physician behaviors, developing effective health promotion messages, and adjusting payment mechanisms all will take time.\nA strategic plan for improving women's health and pregnancy outcomes\nBased on the June summit presentations and subsequent expert panel deliberations, 10 recommendations were developed (Table 1). These recommendations were based on four overarching goals that were identified as critical to help women reach optimal health and realize their reproductive goals. These four broad goals are 1) to improve both men's and women's knowledge, attitudes, and behaviors related to preconception health; 2) to ensure that all U.S. women of childbearing age receive preconception care services\u2014screening, health promotion, and interventions\u2014that will enable them to begin a pregnancy in optimal health; 3) to reduce risks indicated by a prior adverse pregnancy outcome through interventions during the interconception (inter-pregnancy) period that can prevent or minimize health problems for a mother and her future children; and 4) to reduce the health disparities in adverse pregnancy outcomes.\nTable 1.Panel Recommendations and ActionsRecommendation 1. Individual responsibility across the lifespan. Each woman, man and couple should be encouraged to have a reproductive life plan.Action Steps\u2022 Develop, evaluate, and disseminate reproductive life planning tools for women and men in their childbearing years, respecting variations in age; literacy, including health literacy; and cultural\/linguistic contexts\u2022 Conduct research leading to development, dissemination, and evaluation of individual health education materials for women and men regarding preconception risk factors, including materials related to biomedical, behavioral, and social risks known to affect pregnancy outcomesRecommendation 2. Consumer awareness. Increase public awareness of the importance of preconception health behaviors and preconception care services by using information and tools appropriate across various ages; literacy, including health literacy; and cultural\/linguistic contextsAction Steps\u2022 Develop, evaluate, and disseminate age-appropriate educational curricula and modules for use in school health education programs\u2022 Integrate reproductive health messages into existing health promotion campaigns (e.g., campaigns to reduce obesity and smoking)\u2022 Conduct consumer-focused research necessary to develop messages and terms for promoting preconception health and reproductive awareness\u2022 Design and conduct social marketing campaigns necessary to develop messages for promoting preconception health knowledge and attitudes, and behaviors among men and women of childbearing age\u2022 Engage media partners to assist in depicting positive role models for lifestyles that promote reproductive health (e.g., delaying initiation of sexual activity, abstaining from unprotected sexual intercourse, and avoiding use of alcohol and drugs)Recommendation 3. Preventive visits. As a part of primary care visits, provide risk assessment and educational and health promotion counseling to all women of childbearing age to reduce reproductive risks and improve pregnancy outcomesAction Steps\u2022 Increase health provider (including primary and specialty care providers) awareness regarding the importance of addressing preconception health among all women of childbearing age\u2022 Develop and implement curricula on preconception care for use in clinical education at graduate, postgraduate, and continuing education levels\u2022 Consolidate and disseminate existing professional guidelines to develop a recommended screening and health promotion package\u2022 Develop, evaluate, and disseminate practical screening tools for primary care settings, with emphasis on the 10 areas for preconception risk assessment (e.g., reproductive history, genetic, environmental risk factors)\u2022 Develop, evaluate, and disseminate evidence-based models for integrating components of preconception care to facilitate delivery of and demand for prevention and intervention services\u2022 Apply quality improvement techniques (e.g., conduct rapid improvement cycles, establish benchmarks and brief provider training, use practice self-audits, and participate in quality improvement collaborative groups) to improve provider knowledge and attitudes, and practices and to reduce missed opportunities for screening and health promotion\u2022 Use the federally funded collaboratives for community health centers and other FQHC to improve the quality of preconception risk assessment, health promotion, and interventions provided through primary care\u2022 Develop fiscal incentives for screening and health promotionRecommendation 4. Interventions for identified risks. Increase the proportion of women who receive interventions as follow-up to preconception risk screening, focusing on high priority interventions (i.e., those with evidence of effectiveness and greatest potential impact)Action Steps\u2022 Increase health provider (including primary and specialty care providers) awareness concerning the importance of ongoing care for chronic conditions and intervention for identified risk factors\u2022 Develop and implement modules on preconception care for specific clinical conditions for use in clinical education at graduate, postgraduate, and continuing education levels\u2022 Consolidate and disseminate existing professional guidelines related to evidence-based interventions for conditions and risk factors\u2022 Disseminate existing evidence-based interventions that can be used in primary care settings (e.g., brief interventions for alcohol misuse and smoking)\u2022 Develop fiscal incentives (e.g., pay for performance) for risk management, particularly in managed care settings\u2022 Apply quality improvement techniques and tools (e.g., conduct rapid improvement cycles, establish benchmarks, use practice self-audits, and participate in quality improvement collaborative groups)Recommendation 5. Interconception care. Use the interconception period to provide additional intensive interventions to women who have had a previous pregnancy which ended in adverse outcome (e.g., infant death, fetal loss, birth defects, low birthweight or preterm birth)Action Steps\u2022 Monitor the percentage of women who complete postpartum visits (e.g. using HEDIS measures for managed care plans and Title V Maternal Child Health Block Grant state measures), and use these data to identify communities of women at risk and opportunities to improve provider follow-up\u2022 Develop, evaluate, and replicate intensive evidence-based interconception care and care coordination models for women at high social and medical risk\u2022 Enhance the content of postpartum visits to promote interconception health\u2022 Use existing public health programs serving women in the postpartum period to provide or link to interventions (e.g., family planning, home visiting, and WIC)\u2022 Encourage additional states to develop preconception health improvement projects with funds from the Title V Maternal Child Health Block Grant, Prevention Block Grant, and similar public health programsRecommendation 6. Prepregnancy check up. Offer, as a component of maternity care, one prepregnancy visit for couples and individuals planning pregnancyAction Steps\u2022 Modify third party payer rules to permit payment for one prepregnancy visit per pregnancy, including development of billing and payment mechanisms\u2022 Consolidate existing professional guidelines to develop the recommended content and approach for such a visit\u2022 Educate women and couples regarding the value and availability of prepregnancy planning visitsRecommendation 7. Health insurance coverage for women with low incomes. Increase public and private health insurance coverage among women with low incomes to improve access to preventive women's health, preconception, and interconception careAction Steps\u2022 Improve the design of family planning waivers by permitting states (by federal waiver or by creating a new state option) to offer interconception risk assessment, counseling, and interventions along with family planning services. Such policy developments would create new opportunities to finance interconception care\u2022 Increase health coverage among women who have low incomes and are of childbearing age by using federal options and waivers under public and private health insurance systems and the State Children's Health Insurance Program\u2022 Increase access to health-care services through policies and reimbursement levels for public and private health insurance systems to include a full range of clinicians who care for womenRecommendation 8. Public health programs and strategies. Integrate components of preconception health into existing local public health and related programs, including emphasis on interconception interventions for women with previous adverse outcomesAction Steps\u2022 Use federal and state agency support to encourage more integrated preconception health practices in clinics and programs\u2022 Provide support for CDC programs to develop, evaluate, and disseminate integrated approaches to promote preconception health\u2022 Analyze and evaluate the preconception care activities used under the federal Healthy Start program and support replication projects\u2022 Convene or use local task forces, coalitions, or committees to discuss opportunities for promotion and prevention in preconception health at the community level\u2022 Develop and support public health practice collaborative groups to promote shared learning and dissemination of approaches for increasing preconception healthRecommendation 9. Research. Increase the evidence base and promote the use of the evidence to improve to preconception healthAction Steps\u2022 Prepare an updated evidence-based systematic review of all published reports on science, programs, and policy (e.g., through the Agency for Healthcare Research and Quality)\u2022 Encourage and support evaluation of model programs and projects, including integrated service delivery and community health promotion projects\u2022 Conduct quantitative and qualitative studies to advance knowledge of preconception risks and clinical and public health interventions, including knowledge of more integrated practice strategies and interconception approaches\u2022 Design and conduct analyses of cost-benefit and cost-effectiveness as part of the study of preconception interventions\u2022 Conduct health services research to explore barriers to evidence-based and guidelines-based practice\u2022 Conduct studies to examine the factors that results in variations in individual use of preconception care (i.e., barriers and motivators that affect health-care use)\u2022 Support activities to translate research into clinical practice and public health actionRecommendation 10. Monitoring improvements. Maximize public health surveillance and related research mechanisms to monitor preconception healthAction Steps\u2022 Apply public health surveillance strategies to monitor selected preconception health indicators (e.g., folic acid supplementation, smoking cessation, alcohol misuse, diabetes, and obesity)\u2022 Expand data systems and surveys (e.g., PRAMS and NSFG) to monitor individual experiences related to preconception care\u2022 Use geographic information system techniques to target preconception health programs and interventions to areas where high rates of poor health outcomes exist women of reproductive age and their infants\u2022 Use analytic tools (e.g., PPOR) to measure and monitor the proportion of risk attributable to the health of women before pregnancy\u2022 Include preconception, including interconception, health measures and population-based performance monitoring systems (e.g., in national and state Title V programs)\u2022 Include a preconception measure in the Healthy People 2020 objectives\u2022 Develop and implement indicator quality improvement measures for all aspects of preconception care. For example, use HEDIS measures to monitor the percentage of women who complete postpartum visits\nFor each of the 10 recommendations, the panelists identified specific actions. For each action step, those persons primarily responsible for implementation as well as those who had supportive roles were listed. The action steps were designed as feasible and practical activities that could be undertaken in the near future and could result in change in the next 2 to 5 years.\nThese goals, recommendations, and action steps form a strategic plan for improving preconception health and pregnancy outcomes. This can be illustrated as a pyramid (Fig. 2), building from the smaller attainable actions steps to fulfilling a recommendation, to attainment of the four goals, and finally to the combination of the goals to reach the pinnacle of the pyramid\u2014a vision for better women's health and improved pregnancy outcomes.\nFig. 2Preconception care pyramid\nThe recommendations presented here focus on individual health knowledge and behavior, clinical care, public health programs, and health-care policies, improving preconception health will require the involvement of and changes in other sectors, including education, housing, urban planning, and environmental health. Moving forward, these sectors should be included as part of the comprehensive solution to improving women's health and, by extension, the health of families. In all of the action steps, women and their families have a critical role to play in engaging the system and developing the consumer demand and culture to normalize preconception care as part of women's health promotion and routine care.\nThe focus of the recommendations\nThe 10 recommendations outlined in Table 1 address individual, provider, system, and research needs that mirror the different levels described in the ecological model. Under each of the recommendations there are multiple actions steps that describe specific activities geared towards achieving the recommendations.\nThe first two recommendations focus on individual responsibility for preconception health. Under these recommendations, action steps are focused on the development, evaluation, and dissemination of tools to help women and their partners make decisions regarding their reproductive health across the lifespan. The action steps clearly highlight the need to ensure that these tools are age-appropriate and culturally relevant and cover both general health topics and specific risk behaviors. The action steps recognize the importance of being able to integrate preconception health-care messages with existing health promotion activities whenever possible. This is especially important in an environment with limited resources for health promotion activities. Both recommendations 1 and 2 call for action to be taken at both the individual and community level so that societal norms shift toward supporting optimal preconception health behaviors.\nRecommendations 3, through 6 call for actions to improve health-care services and are particularly focused on changing provider knowledge, attitudes, and behaviors. Because the existing knowledge base has not been widely disseminated, the action steps call for new and continuing education activities to help all providers improve their skills and coverage of preconception care services. The use of quality improvement tools and techniques are suggested as a means of changing professional practices. In the current market-driven environment, it is clear that incentives such as pay for performance and risk management activities also must be developed to help encourage the provision of these services.\nRecommendations 5 and 6 particularly focus more on improving preconception care and health for specific groups of women. Recommendation 5 focuses on interconception care and the opportunity for the prevention of adverse pregnancy outcomes among those who are at risk for or have previously had pregnancy complications. In addition to action steps aimed at modifying provider practices, others encourage public health programs to do more identification and follow-up of women at risk. Recommendation 6 suggests pre-pregnancy checkups or visits for couples to focus on their health and risks when they are trying to conceive. The action steps include consumer education and consolidated professional guidelines, as well as better third-party health insurance coverage of such visits as a part of prenatal and maternity coverage.\nAction steps for recommendation 7 call for increased health-care coverage among uninsured, low-income women, specifically through Medicaid. Currently, most low-income women do not qualify for Medicaid unless they are pregnant; unfortunately, this is often too late for some health promotion and therapeutic interventions to have maximum impact on the health of the woman and her child.\nRecommendation 8 describes a series of action steps that public health and community programs can take to improve preconception health by increasing the access to and use of preconception care services. These steps include using publicly funded women's and children's health programs to promote preconception health, to screen for health risks, and to refer women at risk to appropriate clinicians. For example, preconception health promotion messages are complementary to the purposes of family planning and HIV\/STD clinics. However, the use of publicly funded women's and children's health programs for these purposes will require that federal and state public health agencies minimize the categorical restrictions that often prevent true integration at the community level. The second two action steps encourage the adaptation, implementation, and evaluation of community-based programs that provide preconception services. There are model programs currently being implemented that could be modified and used to meet the unique needs of specific populations. A key component of these actions is to engage the community to develop the best methods for development, implementation, and evaluation of local preconception care programs. Without community support and designs that truly represent the needs of the communities, programs will be ineffective.\nRecommendations 9 and 10 include action steps focused on the continuous quality improvement and planning feedback loop that supports excellence in health-care systems. Research and evaluation are critical to examining progress toward achieving goals. Currently, a need exists for updating the systematic reviews of existing literature on preconception care interventions as well as increasing the knowledge base through qualitative and quantitative research projects. Economic analyses of preconception care interventions are not often conducted, and a great deal of work must be done to understand the impact of interventions. Limitations exist in many of the interventions currently available; and effective interventions have not been developed in some areas. Meeting the need for research and evaluation is a key factor in the ability to continue moving the field forward. In addition to research activities, ongoing monitoring systems\u2014typically managed by public health agencies\u2014are critical to program planning, development, and evaluation. This recommendation includes the use of surveillance systems such as the Pregnancy Risk Assessment and Monitoring System (PRAMS) and the Health Employee Data and Information Set (HEDIS) measures.\nConclusion\nImproving preconception health requires changes in the knowledge, attitudes, and behaviors of individuals, families, communities, and institutions (e.g., government, health-care settings). The purpose is to improve the health of each woman before any pregnancy and to thereby affect the future health of the woman, her child, and her family. Through the CDC Preconception Health Initiative and the deliberations of the Select Panel on Preconception Care, a set of goals, recommendations, and specific actions steps have been developed to form a strategic plan. The framework incorporates both an ecological model and a lifespan perspective on health. This work also recognizes the unique contributions and challenges faced by individual women and their families, communities, and institutions. As one Select Panel member put it, \u201cIf you raise the level of health of women in a society, you\u2019ve raised the level of health of families and the community at the same time.\u201d The successful implementation of the recommendations will help achieve the vision for preconception health and pregnancy outcomes where\nAll women and men of childbearing age have high reproductive awareness (e.g., understand risk factors related to childbearing).All women have a reproductive life plan (e.g., whether or when they wish to have children, how they will maintain their reproductive health).All pregnancies are intended and planned.All women of childbearing age have health-care coverage.All women of childbearing age are screened prior to pregnancy for risks related to the outcomes of pregnancy.Women with a prior pregnancy loss (e.g., infant death due to very low birthweight or preterm birth) have access to intensive interconception care aimed at reducing their risks.","keyphrases":["preconception care","health behavior","maternal health","research and surviellance","access to care"],"prmu":["P","P","R","M","R"]}
{"id":"Matern_Child_Health_J-2-2-1592148","title":"Preconception Care Between Pregnancies: The Content of Internatal Care\n","text":"For more than two decades, prenatal care has been a cornerstone of our nation\u2019s strategy for improving pregnancy outcomes. In recent years, however, a growing recognition of the limits of prenatal care and the importance of maternal health before pregnancy has drawn increasing attention to preconception and internatal care. Internatal care refers to a package of healthcare and ancillary services provided to a woman and her family from the birth of one child to the birth of her next child. For healthy mothers, internatal care offers an opportunity for wellness promotion between pregnancies. For high-risk mothers, internatal care provides strategies for risk reduction before their next pregnancy. In this paper we begin to define the contents of internatal care. The core components of internatal care consist of risk assessment, health promotion, clinical and psychosocial interventions. We identified several priority areas, such as FINDS (family violence, infections, nutrition, depression, and stress) for risk assessment or BBEEFF (breastfeeding, back-to-sleep, exercise, exposures, family planning and folate) for health promotion. Women with chronic health conditions such as hypertension, diabetes, or weight problems should receive on-going care per clinical guidelines for their evaluation, treatment, and follow-up during the internatal period. For women with prior adverse outcomes such as preterm delivery, we propose an internatal care model based on known etiologic pathways, with the goal of preventing recurrence by addressing these biobehavioral pathways prior to the next pregnancy. We suggest enhancing service integration for women and families, including possibly care coordination and home visitation for selected high-risk women. The primary aim of this paper is to start a dialogue on the content of internatal care.\nFor more than two decades, prenatal care has been a cornerstone of our nation\u2019s strategy for improving pregnancy outcomes. In recent years, however, the effectiveness of this strategy has been called into question [1]. Increasing use of early and adequate prenatal care has not led to a significant decline in low birthweight (LBW) or prematurity, which are leading causes of infant mortality in the U.S.. Nor has closing the access gap in prenatal care led to a significant reduction in racial-ethnic or socioeconomic disparities in these adverse birth outcomes. There is now a growing consensus that further reduction in this nation\u2019s LBW and prematurity rates cannot be achieved solely by improving access to prenatal care [2].\nA limitation on the effectiveness of prenatal care may be its timing. Many of the pathophysiological pathways leading up to adverse birth outcomes may have their onset early in pregnancy, possibly at or even before implantation. By the time prenatal care is initiated, it may be too little too late to significantly alter the course or outcome of the pregnancy. Indeed, there is now a growing recognition that birth outcomes are the end product of not only the nine months of pregnancy, but the entire life course of the mother leading up to the pregnancy [3]. Improving birth outcomes, therefore, takes promoting maternal health not only during pregnancy, but before and between pregnancies and, indeed, across the woman\u2019s lifespan.\nThe recognition of the importance of maternal health before pregnancy has led to a growing interest in preconception care [4]. A special subtype of preconception care is internatal care. Internatal care refers to a package of healthcare and ancillary services provided to a woman and her family from the birth of one child to the birth of her next child. It is, in essence, preconception and prenatal care for a subsequent pregnancy. We prefer the term \u201cinternatal care\u201d over the more popular term \u201cinterconception care\u201d (from the conception of one pregnancy to the conception of the next pregnancy) because 1) internatal care represents an extension of prenatal care with which the public is familiar; 2) the point of entry into care is the birth of one child; and 3) the endpoint of care is the birth of the next child. And while in this paper we will focus primarily on the interpregnancy interval (from the termination of one pregnancy to the conception of the next pregnancy), we prefer the term \u201cinternatal care\u201d over \u201cinterpregnancy care\u201d because internatal care suggests a continuity of care into a subsequent pregnancy.\nDespite the growing interest, for most people it remains unclear what is meant by internatal care. For example, many Healthy Start programs now offer internatal care, and yet the content of care varies greatly across sites. There have also been a few intervention studies on internatal care, most notably in Denver [5], Atlanta [6], and Philadelphia [7], with vastly different approaches. The lack of some standards of care makes it difficult to move forward research, practice, and policy, or even discussion, on internatal care. The purpose of this paper is to begin to define the content of internatal care.\nFor healthy mothers, internatal care offers an opportunity for wellness promotion between pregnancies. Presently many women lack such opportunity, particularly among low-income and minority women. In most states, Medicaid programs terminate pregnancy-related healthcare coverage for most low-income mothers at 60 days postpartum. Other than the one recommended postpartum visit, many of these women will have little or no access to healthcare between pregnancies. Internatal care can help close the gap in healthcare for these women. Even for women with healthcare coverage, it is unclear how much and what internatal care they are currently getting. In this paper, we begin to define the core contents of universal internatal care that all women of childbearing age should receive between pregnancies.\nWhile we believe that internatal care should be universally available to all women between pregnancies, it may be particularly beneficial for high-risk mothers, such as women with chronic diseases or prior adverse pregnancy outcomes. Many such outcomes (e.g. prematurity, fetal death) carry a high recurrence risk in a subsequent pregnancy, and the biobehavioral risk factors are often carried from one pregnancy to the next [8]. For these mothers, internatal care offers an opportunity for risk reduction before their next pregnancy. In this paper, we also begin to describe the content of enhanced internatal care for high-risk mothers. We will use women with chronic health conditions (hypertension, diabetes, or weight problems) and women with a prior preterm delivery as examples of what more can be done for high-risk mothers during internatal care than under current prevailing practice which, for many women, consists of the one recommended six-week postpartum visit, or none at all.\nTwo decades ago, the U.S. Public Health Service assembled an expert panel to define the contents of prenatal care. In its report Caring for Our Future: The Content of Prenatal Care, the Expert Panel identified four basic components of prenatal care consisting of 1) early and continuing risk assessment, 2) health promotion, 3) clinical interventions, and 4) psychosocial interventions [9]. We now use the same framework to outline the contents of internatal care, based on our search of the literature for clinical standards, best practices, and proven or promising strategies. The focus of this paper is on the content of internatal care, particularly during clinical visits. Other important topics, such as financing, organization, delivery, motivation, and community interventions, are beyond the scope of this paper and will be addressed in a series of papers to follow.\nIt is not our intention in this paper to propose a complete and final model of internatal care. Instead, we present here the start of a framework for internatal care, to be filled in through more dialogues and consensus-building which we hope this paper will help move forward.\nRecommended core contents of universal internatal care for all women following a pregnancy\nMost pregnant women in the United States are healthy and experience healthy pregnancy and birth outcomes. For these women, internatal care is still important because it offers an opportunity for wellness promotion. In this section, we describe the core contents of internatal care, including risk assessment, health promotion, clinical interventions, and psychosocial interventions (Table 1). These components should be offered to every woman following a pregnancy, regardless of her risk status. We recommend expanding the current six-week postpartum visit to three or more internatal visits, at 2 weeks, 6 weeks, and 6 months postpartum, with annual follow-up beginning at one year postpartum. We will expand on the rationale for the recommended schedule of visits, and address issues related to systems integration later in this section.\nTable 1.Recommended content and schedule of internatal care, and organizations recommending these practice standardsRisk assessmentOrganizationsaSchedulebCore content of internatal care for all womenFamily violenceJACHO, AMA1, 2, 3\u2003Infections\u2003\u2003ChlamydiaUSPSTF, ACPM2\u2003\u2003PeriodontalADA2\u2003Immunizations\u2003\u2003Diptheria-tetanus boosterUSPTSF, ACPM, HHS2\u2003\u2003Hepatitis BUSPTSF, ACPM, HHS2\u2003\u2003Measles and mumpsUSPTSF, ACPM, HHS, ACIP2\u2003\u2003RubellaUSPTSF, ACPM, HHS, ACIP2\u2003\u2003VaricellaUSPTSF, ACPM, HHS, ACIP2\u2003NutritionUSPSTF1,2, 3\u2003DepressionUSPSTF, ACOG1, 2, 3\u2003Stress1, 2, 3Health promotion\u2003BreastfeedingAAP, HP20101, 2, 3\u2003Back-to-SleepAAP, NICHD, HP20102\u2003ExerciseNHLBI, HHS, HP20102\u2003Exposures\u2003\u2003Smoking, alcohol, drug useHHS, ACOG1, 2, 3\u2003\u2003MercuryFDA\/EPA2, 3\u2003\u2003LeadCDC, AAP2, 3\u2003\u2003DioxinsIOM2, 3\u2003\u2003Indoor\/outdoor pollutants & allergensNHLBI2, 3\u2003Family planningACOG\/AAP1, 2, 3\u2003Folate supplementationCDC, AAP3Clinical interventions\u2003Height and weight measurementsICSI, ACOG1, 2, 3\u2003Blood pressureICSI, ACOG2\u2003Total skin examinationACS, ACPM2\u2003Clinical breast examinationACS, ACOG3\u2003Papanicolau smear and pelvic examinationACS, ACOG2\u2003Screening mammographyACS, ACOG3Psychosocial interventions\u2003Social services1, 2, 3\u2003Clinical support1, 2, 3\u2003Parenting support1, 2, 3Enhanced content of internatal care for high-risk womenChronic hypertension\u2003Risk assessment, health promotion, clinical & psychosocial interventionsNHLBIGestational hypertension\u2003Risk assessment, health promotion, clinical & psychosocial interventionsNHLBI, ACOGPregestational diabetes\u2003Risk assessment, health promotion, clinical & psychosocial interventionsADAGestatational diabetes\u2003Risk assessment, health promotion, clinical & psychosocial interventionsADA, ACOGOverweight\/obesity\u2003Risk assessment, health promotion, clinical & psychosocial interventionsNHLBI, HHS USPSTFPreterm birth\u2003Risk assessment\u2003\u2003Reproductive historyACOG\u2003\u2003Family historyACOG\u2003\u2003Medical assessmentACOG\u2003\u2003Nutritional assessmentACOG\u2003\u2003Social assessmentACOG\u2003Health Promotion\u2003\u2003Smoking cessationHHS\u2003\u2003Substance abuse treatmentACOG\u2003\u2003Optimal prepregnancy BMIIOM\u2003\u2003Ensure adequate omega-3 fatty acids intakeISSFAL, IOM\u2003\u2003Family planningACOG\/AAP\u2003Clinical interventions\u2003\u2003Progesterone use in subsequent pregnancyACOG\u2003Psychosocial Interventions\u2003\u2003Services for children with special health care needsAAP\u2003\u2003Home visitationAAPaReferences to these recommendations available through the authors. Abbreviations: JACHO, Joint Commission on Accreditation of Healthcare Organizations; AMA, American Medical Association; USPSTF, United States Preventive Services Task Force; ACPM, American College of Preventive Medicine; ADA, American Dental Association; HHS, Department of Health and Human Services; ACIP, Advisory Committee on Immunization Practices (CDC). ACOG, American College of Obstetricians and Gynecologists; AAP, American Academy of Pediatrics; HP2010, Healthy People 2010; FDA, Food and Drug Administration; EPA, Environmental Protection Agency; CDC, Centers for Disease Control and Prevention; IOM, Institute of Medicine; NHLBI, National Heart, Lungs, and Blood Institute; ICSI, Institute for Clinical Systems Improvement; ACS, American Cancer Society; ADA, American Diabetes Association.bRecommended schedule for each component of core content of internatal care; 1= 2-week visit, 2= 6-week visit; 3= 6-month visit . Schedule for enhanced content varies according to need\nRisk assessment\nThe primary objective of risk assessment is to identify on-going problems that need to be addressed. Most pregnant women in the U.S. are healthy and thus should have no major problems postpartum. However, some medical, behavioral or psychosocial problems may have been overlooked by their prenatal care providers. Other problems may develop during the internatal period. Providers of internatal care should pay particular attention to five problems that are now commonly missed during prenatal or postpartum care: family violence, infection\/immunization, nutrition, depression and stress (FINDS).\nFamily violence\nThe experience of family violence may be quite common among pregnant and postpartum women in the U.S. Most studies of the prevalence of physical abuse during pregnancy report estimates in the range of 4 to 8 percent, though higher estimates (around 20%) have been reported in some populations [10]. Less is known about the prevalence of violence after pregnancy, with estimates at 3 months postpartum ranging from 3.2% in a population-based survey to 21% in a prospective study of adolescent mothers [11, 12]. In the latter study [12], three of four women who reported intimate partner violence (IPV) had not reported IPV prior to delivery. Family violence puts maternal health and child development at great risk. The literature contains solid links between intimate partner violence and child abuse [13]. Because violence is frequently missed by healthcare providers including prenatal care providers, we recommend routine screening for family violence during every internatal visit. Several brief screening instruments, such as the 4-item Hurt Insulted Threatened or Screamed at (HITS), the 3-item Partner Violence Screen (PVS), and a 3-item Abuse Assessment Screen (AAS), can be effectively used to screen for IPV in a clinical setting [13].\nInfection\/immunization\nSome infections may go unrecognized or untreated during pregnancy. Chronic, untreated infections, such as periodontal disease, could pose a threat not only to the next pregnancy [14], but also to the mother\u2019s long-term health as well [15]. Most women are not screened for periodontal disease during prenatal care, and many women lack access to oral health services following pregnancy. While presently there is insufficient evidence to justify universal screening for asymptomatic infections among \u201clow-risk\u201d women in the internatal period (other than the U.S. Preventive Services Task Force (USPSTF) recommendation for Chlamydia screening in women under the age of 25) [16], in some populations routine screening for periodontal, sexually-transmitted and some urogenital tract infections may be warranted.\nInternatal care also offers an important opportunity to update women\u2019s immunization status. The USPSTF and other national advisory committees recommend that 1) all adults receive periodic diptheria-tetanus toxoids booster every 10 years, 2) all young adults receive a series of Hepatitis B vaccines, 3) all adults born after 1956 who lack evidence of immunity receive vaccination against measles and mumps, and 4) all women of childbearing age be screened for rubella and varicella susceptibility; susceptible nonpregnant women of childbaring age should be offered vaccination and avoid conception for at least 28 days after vaccination; susceptible pregnant women should be vaccinated immediately after delivery (Table 1). Immunization status should be assessed and updated at the six-week internatal visit.\nNutrition\nNutrition can play an important role in promoting maternal health and child development, and yet women\u2019s nutritional needs are often overlooked by prenatal or postpartum care providers. One recent population-based survey in California found that nearly one in three pregnant women who were income-eligible for Women, Infants, and Children (WIC) program reported being food-insecure; of these, one in four reported not being enrolled in WIC [17]. Nutritional screening should address the ABCD\u2019s of nutritional risks: anthropometric (e.g. low or high body mass index), biochemical (e.g. for anemia or folate deficiency in some populations), clinical (e.g. eating disorder) and dietary (e.g. content, pattern, food insecurity). An abbreviated 6-item version of the Household Food Security Scale can be used to screen for food insecurity. Inquiry should be made about folate or multivitamin supplementation (see under Health Promotion). We recommend routine screening of nutritional risks at every internatal visit.\nDepression\nMaternal depression affects a large number of women and their children. A recent systematic review reported combined estimates of the point prevalence of major and minor depression ranging from 8.5 to 11 percent during pregnancy, and 6.5 to nearly 13 percent during the first year postpartum [18]. Maternal depression can have negative long-term impact on both maternal health and child development, but is often missed by prenatal and postpartum care providers. We recommend routine screening for maternal depression at every internatal visit. Fairly accurate and feasible screening measures are available, including the Postpartum Depression Screening Scale (PDSS), Edinburgh Postnatal Depression Scale (EPDS), Beck Depression Inventory (BDI), and the Center for Epidemiologic Studies Depression Scale (CES-D) [18]. The evidence available, albeit limited, suggests that providing some form of psychosocial support to pregnant and postpartum women at risk of having a depressive illness may decrease depressive symptoms [18].\nStress\nMaternal stress is perhaps the hardest to screen; clinicians are often at a loss as to how to screen or what to do with a positive screen. Yet chronic stress can pose significant risks to maternal health and child development. For the mother, chronic stress can cause wear and tear to her body\u2019s adaptive systems, what McEwen refers to as allostatic load, which could lead to more rapid deterioration in her health and function over time [19]. For the infant (or the fetus in a subsequent pregnancy), maternal stress could result in the re-programming of the child\u2019s basic autonomic rheostat and alteration of immune regulation, leading to increased vulnerability to disease and dysfunction later in life [20]. There is an important need for the development of a multi-dimensional screening tool for stress that can be feasibly implemented in a clinical setting. For now, clinicians need to ask about major stressors in women\u2019s lives (e.g. homelessness, unemployment, family violence, social isolation) at every internatal visit, and find resources and supports to help the family deal with these stressors.\nThus even for healthy mothers with healthy infants, much can be done to reduce health risks and promote well-being during internatal care. In addition to the five areas we highlighted (FINDS\u2013family violence, infections\/immunization, nutrition, depression, and stress), a battery of reproductive, medical, family, genetic and psychosocial risks should be routinely assessed during internatal care. Other areas of risk assessment can be added to the contents of internatal care as the model becomes more fully developed.\nA word of caution about risk screening\nPresently a major limitation on the effectiveness of risk screening is the lack of available or accessible services for those with a positive screen. The benefit of depression screening is limited when there are no mental health services available or accessible to refer mothers who screen positive for depression. And a lack of support services (and coordination thereof) for abuse victims is identified by many providers as a major deterrent to screening for family violence. For risk screening to be effective, it needs to be followed up by effective interventions. This means having all the necessary resources and service capacity (e.g. oral health services for those with periodontal disease, nutritional support for those who are food-insecure, housing assistance for homeless mothers and families) to assist those with a positive screen.\nHealth promotion\nThe primary objective of health promotion is to promote the health and wellbeing of the mother, infant, and family. We will highlight six components health promotion during internatal care: breastfeeding, back-to-sleep, exercise, exposures, family planning and folic acid (BBEEFF).\nBreastfeeding\nDespite well-known benefits of breastfeeding for both mother and infant [21], at least one-third of mothers in the U.S. never initiate breastfeeding [22]. Among women who initiated, nearly one in five will stop nursing by one month, and nearly two-thirds will stop by six months [22], despite current recommendation to breastfeed for at least one full year [21]. Internatal care offers an important opportunity to promote breastfeeding, particularly the initial two-week visit to address nascent problems, the six-week visit to anticipate problems related to transition back to work, and the six-month visit to encourage continued breastfeeding with introduction of solid foods.\nBack-to-sleep\nWith the introduction of the Back-to-Sleep campaign by the American Academy of Pediatrics in 1992, the overall incidence of sudden infant death syndrome (SIDS) has decreased by almost 50% [23]. Despite this success, SIDS continues to be a leading cause of infant death in the U.S., as well as racial disparity in infant mortality [23]. SIDS rate among black infants remains more than twice that of white infants, even after the implementation of the Back-to-Sleep campaign [23]. Black infants are more likely than white infants to be placed in the prone position and soft sleep surfaces for sleep [23]. Providers of internatal care need to discuss the infant\u2019s sleep environment with the parents. Because the risk of SIDS peaks at 2 to 4 months of age, the six-week visit offers an important opportunity to talk about back-to-sleep.\nExercise\nHealth education about physical activities should be part of an on-going effort for wellness promotion during internatal care. The 1988 National Maternal and Infant Health Survey revealed that 25 percent of white women and 45 percent of black women were heavier by 4.1\u00a0kg (nearly 9 pounds) or more at 10 to 18 months post partum than they were before pregnancy [24]. Clinicians can help women develop a weight loss program that consists of healthy dieting, physical activities, and behavioral modifications if needed. The National Heart, Lung, and Blood Institute recommends that all adults should set a long-term goal to accumulate at least 30\u00a0min or more of moderate-intensity physical activity on most, and preferably all, days of the week [25]. For women in the postpartum period, prepregnancy exercise routines may be resumed gradually as soon as it is physically and medically safe. Clinicians need to routinely discuss physical activity with the woman, particularly at the six-week and six-month visits.\nExposures\nCigarette smoking poses a significant threat to the health of the mother, her infant, and her subsequent pregnancy. Approximately one in eight (11.2%) U.S. women who gave birth in 2002 reported smoking cigarettes during pregnancy [26]. Furthermore, up to 70% of women who quit smoking during pregnancy will relapse within 6 months of delivery [27]. Effective models of clinical intervention for smoking cessation (e.g. 5A\u2019s) and relapse prevention have been developed and should be incorporated into internatal care [28]. Prenatal exposure to alcohol and other substances like cocaine and amphetamines have been associated with birth defects as well as adverse pregnancy and neurodevelopmental outcomes. According to the Behavioral Risk Factor Surveillance System, in 1999 the prevalence of any alcohol use and binge drinking among pregnant women in the U.S. was 12.8 and 2.7%, respectively [29]. Postpartum use and recidivism rates are less well described. Several screening questionnaires have been developed to detect problem drinking which may also prove helpful in detecting substance abuse, include T-ACE (tolerance, annoyed, cut down, eye opener) questions, the CAGE (cut-down, annoyed, guilty, eye-opener) questionnaire, and the brief MAST questionnaire [30]. All women should be screened at the time of their first internatal visit, and referral for evaluation and treatment should be offered to those who screen positive. Additionally, mothers whose infants are at risk for neurobehavioral problems from prenatal exposures should be provided resources for early developmental evaluation and interventions for their infants [30].\nA large number of environmental exposures during the internatal period can affect maternal health and child development. For example, chronic exposures to indoor and outdoor pollutants and allergens, including household molds and dust mites, may increase the child\u2019s future risk for atopy, allergies, and asthma. Less is known about the long-term effects of such exposures to maternal health and subsequent pregnancy outcomes. Lead is a potent developmental neurotoxicant and immunotoxicant, and household exposures can come from old paint, leaching from plumbing, and ceramic dinnerware and glazes. Certain measures can be taken to reduce household exposures to pollutants, allergens and lead. Exposure to mercury can be reduced by adherence to the EPA\/FDA joint advisory related to consumption of fish and shellfish, and exposure to dioxins can be reduced by decreasing dietary fat consumption (Table 1). Clinicians need to assess and discuss with their patients avoidance of exposures that could adversely impact on maternal health and child development at every internatal visit, but particularly at the 6-week and 6-month visits.\nFamily planning\nFamily planning is vital to the health and wellbeing of women and their families [31]. Unintended pregnancy and short interpregnancy intervals are associated with increased risk for adverse birth outcomes [31, 32]; family planning can reduce the risk by promoting effective contraceptive use and optimal birth spacing. An unintended pregnancy also puts future maternal health and social wellbeing at risk; family planning can protect maternal health and choice [31]. Discussion of family planning should begin prenatally and prior to discharge from the hospital; it should be addressed again at the two-week and six-week internatal visits, including risks, benefits and side effects of available contraceptive options and plans for future childbearing. Adherence to method and side effects should be reassessed at the six-month visit.\nFolate supplementation\nFolate is important to both maternal and child health. Maternal folate deficiency has been linked to future risk for cardiovascular and other chronic adult diseases, as well as neural tube and other birth defects in the offspring. Periconceptional (preconceptional and in early pregnancy) use of folic acid has been demonstrated to reduce the occurrence and recurrence of neural tube defects [33]. Thus the U.S. Public Health Services recommends that all women of childbearing age consume 400 micrograms of folic acid daily [34]. Even though there are several ways of getting this amount of folic acid everyday, two-thirds of U.S. women of childbearing age still do not consume enough folic acid, and surveys continue to find substantial knowledge gaps regarding the benefits of folic acid. Folic acid should be promoted at every internatal visit, or at the minimum during the six-month visit. We feel that the recommendation for folic acid can be broadened to include daily multivitamin supplementation given the potential benefits of a number of other vitamins (e.g. B12) and elements to maternal health and subsequent pregnancy outcomes, particularly for women with nutritional deficiencies.\nTable 1 summarizes the core contents of health promotion during internatal care, with an emphasis on BBEEFF (breastfeeding, back-to-sleep, exercise, exposures, family planning and folic acid). Other topics can be added to health promotion as the model of internatal care becomes more fully developed.\nClinical interventions\nMost women in this category are, by definition, healthy and require little in the way of clinical interventions during internatal care, other than the standard preventive health services recommended for all women of childbearing age (Table 1). These include periodic height, weight and blood pressure measurements, total skin examination, clinical breast examination, Pap smear and bimanual pelvic examination every 1 to 3 years. Mammography should also be performed in women ages 40 and above. We will address clinical interventions for women with chronic conditions in the next section.\nPsychosocial interventions\nMany healthy women who gave birth to a healthy infant will nonetheless need some psychosocial support. There are three types of social support that need to be made available as part of the core package of internatal care services: 1) social services, 2) clinical support, and 3) parenting support (Table 1). Social services may include assistance with obtaining certain public insurance (e.g. SCHIP) or benefits (e.g. TANF, EITC). Some women may not be aware of their eligibility for child care subsidies (e.g. CCDF) or availability of early childhood education programs. Women experiencing intimate partner violence may need multiple medical, legal, and social services and could use some help with service coordination. The internatal visits provide a platform for accessing these social services.\nWomen who experience postpartum depression or other affective disorders may benefit from some forms of psychological support and therapy during the internatal period. Couples who experience problems with marital or sexual relationship in the internatal period may benefit from counseling or interventions. Women with alcohol or drug problems could also use clinical support and treatment. These and other professional clinical support services should be made available and accessible as part of standard internatal care services.\nMany parents could benefit from some parenting support, such as mother support groups or parenting classes. A current innovative idea in prenatal care is \u201ccentering\u201d in pregnancy, which provides social support to pregnant women through group prenatal care [35]. While this idea remains to be tested, we believe that group internatal care\u2013a \u201ccentering\u201d in internatal care model\u2013may provide additional support for some women, particularly around issues related to parenting practices and the stress of parenting.\nA note on psychosocial interventions\nThroughout this paper, the readers will notice an imbalance in the levels of details we used to describe clinical and psychosocial interventions. That is, clinical recommendations are, for the most part, described with much greater specificity than psychosocial interventions. It is certainly not our intention to propose a predominantly biomedical model of internatal care. Rather, the lack of specificity in our description of psychosocial interventions reflect both the complexity of psychosocial issues that families\u2013particularly low-income families\u2013face for which there are often no easy solutions, as well as the dearth of well-designed and evaluated intervention programs addressing these psychosocial issues. For example, mental health services alone are unlikely to be effective in treating the postpartum depression of a homeless mother, and housing assistance alone is unlikely to find her stable housing without sustainable income and employment, but few well-designed intervention studies have critically evaluated the impact of a comprehensive package of mental health services, housing assistance, work- and life-skills training and childcare on maternal and family health. Our model of internatal care is unlikely to be effective without effective psychosocial interventions, but we will not know what these are as long as we keep looking for quick biomedical fixes. Future intervention studies on internatal care need to take a more integrative, comprehensive approach to improving family and women\u2019s health.\nService coordination and integration\nAs aforementioned, presently some referral services (e.g. mental health services, oral health services) are unavailable or inaccessible to many women between pregnancies. But even if the services are available, they are often poorly coordinated. Fragmentation in service delivery can deter access to care, particularly for low-income women with many other competing needs. Providers of internatal care need to consider how to better coordinate and integrate services in order to improve access. This can be accomplished by establishing a well-developed referral network. In some populations, care coordination or case management provided by a nurse or social worker may be needed. Ideally these services should be provided at one location to increase service coordination and integration. For example, the two-week internatal visit can be provided at the same time and location as the two-week well-baby check-up, with on-site WIC, health education, and social services that allow for \u201cone-stop shopping.\u201d We will take up issues related to the organization and delivery of internatal care that will maximize access and utilization of services in a series of papers to follow.\nSchedule of visits\nIn developing a schedule of visits for internatal care, we had to first wrestle with two questions. First, shouldn\u2019t the schedule of visits be individualized according to individual needs? While we support this view, we nonetheless feel that it may be useful to recommend some minimum standards to start a dialogue about this topic. For healthy women with healthy infants, we propose expanding the current six-week postpartum visit to three or more internatal visits, at 2 weeks, 6 weeks, and 6 months postpartum, with annual exams beginning at one year postpartum. For some activities, the six-week postpartum visit may be too little too late. For example, one in five mothers who initiated breastfeeding will quit nursing within the first month [22]; a 2-week postpartum visit may offer more timely encouragement and interventions for maintenance of breastfeeding than a 6-week visit could. The 2-week visit also offers an opportunity to follow-up on wound care for cesarean delivery. Whenever possible, this visit should be coordinated with the two-week well-baby check-up, preferably at the same location, to increase motivation for attendance. We feel that it is important to keep the current six-week postpartum visit as the second internatal visit. This is a well-established visit in obstetrics, with well-defined contents. This visit, however, can be made more useful by expanding its contents. We feel that a third internatal visit at 6 months is important for follow-up on a number of issues, such as breastfeeding and family planning. The standard annual visits should begin at one year postpartum (Table 1).\nSecond, shouldn\u2019t any routine visit by a woman who may, at some time, become pregnant again be viewed as an opportunity to emphasize the importance of internatal health and habits [36]? While we support this view, we nonetheless feel that it would be preferable to recommend a schedule of internatal visits rather than relying on \u201cany routine visit.\u201d Several components of internatal care are time sensitive (e.g. counseling about breastfeeding and family planning at 2 weeks or about SIDS at 6 weeks) which should not wait for any routine visit. Scheduled internatal visits may be particularly important for women who are not making routine visits, or who are seeking care through the emergency department or urgent care clinics which lack the resources and capacity to provide comprehensive internatal care. We would also like to see internatal care become reimbursable visits, with standardized contents and schedule (rather than as an add-on to a visit for mastitis or cystitis), which may increase provider incentives to provide internatal care. Issues related to patient motivation and provider reimbursement will be addressed in a series of papers to follow.\nRecommended contents of enhanced internatal care for high-risk women\nIn this section, we begin to define the contents of enhanced internatal care for high-risk women (Table 1). We focus on four groups of high-risk women: 1) women with chronic hypertension or history of hypertensive disorders during pregnancy, 2) women with pregestaional or gestational diabetes mellitus, 3) women who are underweight, overweight or obese, and 4) women with prior preterm birth. We call attention to the first 3 groups because they are often underserved in the internatal period, especially if they gave birth to a healthy infant. Their continued health needs may not be met because they had a good birth outcome. Some women will lose healthcare coverage (e.g. pregnancy-related Medicaid), and will have little on-going care despite their chronic conditions. We call attention to the 4th group because of their high recurrence risk [8] and the significance of recurrence. The goals of internatal care for these women are to provide ongoing care for their health problems and optimize their health prior to next pregnancy. In addition to the core contents of universal internatal care recommended above for all women of childbearing ages, we will examine what enhanced contents may benefit their long-term health, as well as the outcome of their subsequent pregnancy.\nWomen with chronic hypertension or hypertensive disorders during pregnancy\nHypertensive disorders are the most frequently reported medical risk factor during pregnancy [11]. Among the 4 million U.S. women who gave a live birth in 2002, more than 150,000 had \u201cpregnancy-associated\u201d (gestational) hypertension, more than 3,000 had chronic hypertension, and nearly 13,000 had eclampsia [11].\nAmong women with chronic hypertension (i.e. hypertension diagnosed prior to twentieth week of gestation and probably antedates the pregnancy) that persists after pregnancy, guidelines for evaluation and treatment are well established and can be incorporated into the contents of internatal care [37]. Risk assessment has three major objectives: 1) to assess lifestyle and identify other cardiovascular risk factors or concomitant disorders that may affect prognosis and guide treatment 2) to reveal identifiable causes of hypertension, and 3) to assess the presence or absence of target organ damage and cardiovascular disease. Initial evaluation should include medical history, physical examination (including fundoscopic and cardiovascular examinations), laboratory testing and other diagnostic procedures (including an electrocardiogram and testing for renal function and lipoprotein profile). Health promotion should promote weight reduction, DASH (Dietary Approaches to Stop Hypertension) eating plan, dietary sodium reduction, physical activities, moderation of alcohol, and smoking cessation. Clinical interventions should include drug therapy for all women with hypertension, as well as prehypertensive women with compelling indications (e.g. chronic kidney disease or diabetes). Follow-up should be monthly until the blood pressure goal is achieved, and then at 3\u20136 months interval thereafter during the internatal period. Psychosocial interventions should address chronic stressors that may exacerbate the chronic hypertension, as well as barriers (e.g. financial, motivational) to adherence with treatment protocol.\nThe contents of internatal care for women with gestational hypertension, pre-eclampsia, and eclampsia are less well defined, especially if the hypertension does not persist following pregnancy. In most women with gestational hypertension, the blood pressure will become normal during the first week postpartum; for women with preeclampsia the hypertension will take longer to resolve. If the hypertension has not remitted by the 6-week visit, the women should be reexamined 6 weeks later, when persisting pathology will probably be chronic [38]. During internatal care, providers need to discuss with their patients recurrence risk in the subsequent pregnancy. They also need to be aware that preeclampsia has been associated with increased risk of maternal cardiovascular diseases later in life [39], and that attention to cardiovascular risk factors is required in all subsequent internatal care and wellness visits. Presently there is no proven internatal clinical intervention for the prevention of recurrent gestational hypertension or preeclampsia.\nDuring a subsequent pregnancy, antihypertensive therapy should be used only for pregnant women with severe hypertension, as pharmocologic treatment of women with uncomplicated mild chronic hypertension has not been found to be beneficial [38]. Several studies failed to demonstrate a benefit for low-dose aspirin in preventing preeclampsia among women with chronic hypertension [38]. When chronic hypertension is complicated by intrauterine growth restriction or preeclampsia, fetal surveillance is warranted. For women with preeclampsia during a previous pregnancy, low-dose aspirin started in the second trimester of pregnancy did not reduce the recurrence risk of preeclampsia [38]. Further studies are needed to identify effective interventions during the internatal period and subsequent pregnancy to prevent recurrent gestational hypertension or preeclampsia.\nWomen with pre-gestational or gestational diabetes mellitus\nDiabetes complicating pregnancy is the second most frequently reported medical risk factor during pregnancy [11]. In 2002, more than 130,000 women reported having diabetes during pregnancy [11]. For women with pre-gestational diabetes, they are at increased risk for microvascular, neuropathic and cardiovascular diseases, especially if the diabetes is long-standing and poorly controlled. Their risks are reduced with tight glycemic control. Additionally, their fetuses are at increased risk for stillbirth and congenital anomalies. Preconception glycemic control among women with pre-gestational diabetes has been shown to reduce the risk of congenital anomalies [40]. Women who had gestational diabetes have 30\u201370% chance of recurrence during their next pregnancy [41] and an increased risk of developing Type II diabetes later in life [42]. Gestational diabetes increases the risk of fetal macrosomia, birth trauma, newborn hypoglycemia and hyperbilirubinemia. Evidence from animal models suggests that gestational diabetes, especially if poorly controlled, may also program insulin and leptin resistance in the fetus, leading the greater susceptibility for diabetes and obesity in the offspring [43].\nFor women with pre-gestational diabetes, internatal care should follow the guidelines issued by the American Diabetes Association on preconception care of women with chronic diabetes [44]. At the initial office visit, risk assessment should include a complete history, physical examination (including cardiovascular, neurological, and dilated retinal exams), and laboratory evaluation (including glycosylated hemoglobin (HbA1c), serum creatinine and 24-h urinary excretion of total protein and\/or albumin, and measurement of thyroid stimulating hormone and\/or free thyroxine level in women with type 1 diabetes because of the 5 to 10% coincidence of hyper- or hypothyroidism) and electrocardiogram if the diabetes has been long-standing. Health promotion should emphasize practical self-management skills essential for glycemic control and preparation for pregnancy including the use of an appropriate meal plan, self-monitoring of blood glucose (SMBG), self-administration of insulin and self-adjustment of insulin doses, treatment of hypoglycemia (patient and family members), incorporation of physical activity, and development of stress reduction techniques. Patients should be counseled about fetal and neonatal complications (and possibly long-term health consequences) of maternal diabetes, including the risk of congenital malformations, and the need for effective contraception and optimal glycemic control prior to the next pregnancy. Clinical interventions include dietary control and pharmacological treatment using oral hypoglycemics or insulin. Glycemic control can be self-monitored at home, and glycosylated hemoglobin levels should be evaluated at 1- to 2-month intervals until stable. Follow-up visits should be at 1- to 2-month intervals, and frequent phone contact for adjustment of insulin doses and other aspects of the treatment regimen is advised. Psychosocial interventions should address barriers to adherence with treatment protocol. Referral to and coordination with a dietician and programs such as W.I.C. or Sweet Success in California may enhance treatment efficacy. Stress management should be emphasized because stress can affect dietary control as well as glucose homeostasis.\nFor women with gestational diabetes in a previous pregnancy, guidelines for internatal care are less well established. The American Diabetes Association recommends that a 75-g oral glucose tolerance test (GTT) be performed 6 to 8 weeks after delivery [41]. The extent to which this recommendation is adhered is presently unknown. We believe that much more can be done in the internatal period to promote maternal health (as well as fetal health in a subsequent pregnancy) among women with gestational diabetes. Because these women are at increased risk for developing type II diabetes, risk assessment should include a fasting glucose once a year. If clinical assessment suggests polycystic ovarian syndrome and\/or metabolic syndrome, periodic testing for lipoprotein profiles, fasting glucose, and possibly glucose tolerance test may be warranted [45]. Patients should be evaluated for glucose intolerance before their next pregnancy. For health promotion, all women who have had gestational diabetes should be encouraged to exercise and lose weight if they are obese. Dietary counseling is recommended given their increased risk for future type II diabetes. Presently there are no specific clinical interventions recommended in the internatal period for women with a history of gestational diabetes.\nDuring a subsequent pregnancy, women with pregestational diabetes using oral hypoglycemic agents should be switched to insulin because the safety and efficacy of currently available oral hypoglycemics have not been evaluated for use in pregnancy among women with pregestational diabetes. For women with gestational diabetes in a prior pregnancy, the use of a second-generation oral hypoglycemic agent (i.e. glibenclamide or glyburide) may be considered if gestational diabetes recurs in a subsequent pregnancy. The agent does not cross the placenta and has been shown to be comparable to insulin in improving glucose control for women with gestational diabetes [46]. Weight gain during pregnancy should be closely monitored.\nWomen who are underweight, overweight, or obese\nA growing number of women of reproductive age in the U.S. are overweight or obese. According to the 1997 National Health Interview Survey, one-third of women ages 18 to 24 and 43% of women ages 25 to 44 are overweight [47]. The survey also found one in eight women ages 18 to 24 and one in six women ages 25 to 44 are obese [47]. Based on the CDC 2003 Pregnancy Nutrition Surveillance System (PNSS) of nearly 728,000 low-income pregnant women, 43% had a prepregnancy body mass index (BMI) that is considered overweight or obese, up from less than 30% in 1989 [48]. Maternal obesity poses a threat to not only subsequent pregnancy outcomes, but also long-term maternal health. Pregnancy itself is a risk factor for maternal obesity. According to the 2003 PNSS, more than 44% had gained more than the ideal weight gain [48] and, as previously cited [24], weight retention following pregnancy is common. Weight retention needs to be addressed during internatal care, particularly for women who are overweight or obese.\nAccording to the 2003 PNSS, one in eight pregnant women were underweight before pregnancy, and one in four had gained less than the ideal weight gain during pregnancy [48]. Both low pre-pregnancy BMI and poor pregnancy weight gain have been shown to be risk factors for preterm delivery and low birth weight [49].\nFor women who are underweight, overweight, or obese, an important goal of internatal care is to optimize their weight prior to the next pregnancy. For risk assessment, the BMI can be used because it provides an acceptable approximation of body fat and fair correlation with disease outcomes. Potential causes of underweight (e.g. eating disorders, food insecurity), overweight or obesity (e.g. polycystic ovarian syndrome) need to be evaluated. For health promotion, women who are underweight should be encouraged to gain weight through healthy nutrition; for women who are overweight or obese, clinical guidelines have been established for the identification, evaluation, and treatment of overweight and obesity [50]. These guidelines should be followed for internatal care. The initial goal of weight loss therapy should be to reduce body weight by approximately 10 percent from baseline, and weight loss should be about 1 to 2 pounds per week for a period of about 6 months. This amount of postpartum weight loss in overweight women does not appear to affect the growth of their infants, even if they are exclusively breastfed [51]. Weight loss and weight maintenance therapy should employ the combination of low-calorie diets, increased physical activity, and behavior therapy. Clinical interventions may include weight loss drugs as an adjunct to diet and physical activity for patients with a BMI of \u226530, as well as for patients with a BMI of \u226527 with concomitant risk factors or diseases. After successful weight loss, the likelihood of weight loss maintenance is enhanced by a program consisting of dietary therapy, physical activity, and behavior therapy which should be continued until the next pregnancy. Psychosocial interventions should address barriers to healthy nutrition and coordination with a dietician, WIC and other community programs. The schedule of internatal visits should be individualized; the literature suggests that frequent contacts between professional counselors and patients help promote healthy weight maintenance. In a subsequent pregnancy, the Institute of Medicine guidelines for weight gain during pregnancy should be promoted based on prepregnancy BMI [52].\nWomen who had a prior preterm birth\nPresently this group of women is most often targeted for internatal care. Preterm birth is a leading cause of infant mortality and long-term disabilities in children in the U.S.. Prior preterm birth is the strongest and most consistent predictor of a subsequent preterm birth. This may be due to the fact that many of the biobehavioral risk factors for preterm birth are carried from one pregnancy to the next. The goal of internatal care for these women is to prevent recurrence of preterm birth by addressing these continuing biobehavioral risks prior to their next pregnancy.\nRisk assessment\nWomen who had a preterm birth should undergo a comprehensive risk assessment, starting with a complete review of their obstetrical history at the two-week visit. Distinction should be made between spontaneous and induced preterm deliveries. Details about the preterm delivery should be obtained. Work-up should be guided by known etiologic pathways (e.g. infectious-inflammatory, vascular, neuroendocrine and pathologic uterine over-distension) [53]. To identify infectious-inflammatory causes, medical records should be reviewed for untreated infections (e.g. asymptomatic bacteriuria), physical examination should be performed to uncover on-going, chronic infections (e.g. periodontal or reproductive tract infections), and placental pathology should be reviewed for evidence of chorioamnionitis [54]. To identify vascular causes, placental pathology should be reviewed for evidence of uteroplacental ischemia and thromboses [54]. The presence of these findings, in the absence of known causes, may indicate work-up for hereditary or acquired thrombophilia [55]. To identify neuroendocrine causes, inquiries should be made about psychosocial stress and support during pregnancy. Special attention should be paid to FINDS (family violence, infections, nutrition, depression, and stress), since these risk factors may be involved in one or more etiologic pathways leading to the occurrence or recurrence of preterm birth. Review of past medical history should focus on conditions that could increase the risk of preterm deliveries (e.g. systemic lupus erythematosus or renal disease). Review of family history should identify other preterm deliveries in the family (particularly among first-degree relatives), as well as other conditions associated with preterm deliveries. A thorough review of reproductive history, including other preterm births or pregnancy losses, sexual history including sexually transmitted infections, past and current contraceptive use, and plans for future childbearing and child spacing should be conducted.\nHealth promotion\nThe goal of health promotion is to promote protective factors and reduce risk factors for recurrent preterm births. We will highlight three areas of health promotion: smoking cessation, healthy nutrition, and family planning. First, exposure to cigarette smoking significantly increases the risk of preterm delivery. Smoking cessation (by women and their partners) should be encouraged during internatal care. As previously discussed, effective models of clinical interventions (e.g. 5A\u2019s) have been developed [28]. Second, healthy nutrition should be promoted. Low pre-pregnancy BMI has been associated with increased risk for spontaneous preterm birth, while high pre-pregnancy BMI has been associated with increased risk for indicated preterm birth [49]. For women with a history of preterm birth, achieving a healthy weight prior to their next pregnancy may reduce the risk of recurrent preterm birth. A number of programs have demonstrated effectiveness in helping women achieve and maintain healthy weight [56]. Additionally, nutrition may also play an important role in modulating several of the etiologic pathways leading to preterm birth. For example, increased dietary intake of omega-3 polyunsaturated fatty acids may help modulate inflammation [57]. Women should be encouraged to increase consumption of foods rich in omega-3 (the International Society for the Study of Fatty Acids and Lipids recommends that during pregnancy and lactation women must ensure a minimum daily intake of 300\u00a0mg of Docosahexaenoic Acid or DHA, an important source of omega-3 fatty acids [58]), and decrease consumption of foods high in saturated fatty acids, trans-fats, and partially-hydrogenated oils. Increasing dietary intake of fruits and vegetables rich in anti-oxidants and certain phytochemicals should also be encouraged. These recommendations could arguably be adopted on the basis of promoting women\u2019s health alone, even in the absence of data supporting their effectiveness in preventing recurrent preterm birth. Third, the importance of family planning cannot be over-emphasized, particularly for women with a history of preterm birth. Both unintended pregnancies and short interpregnancy intervals are associated with increased risk for preterm delivery. Family planning has been shown to reduce unintended pregnancies and promote optimal child spacing. Other BBEEFF topics, such as breastfeeding and back-to-sleep, are also important to address with mothers of preterm babies.\nClinical interventions\nTo our knowledge, there has been only one randomized controlled trial for the prevention of recurrent preterm birth in which a specific clinical intervention is initiated during the interpregnancy period [59]. In that study, women with a prior spontaneous preterm birth at less than 34 weeks of gestation were randomized at 3 months postpartum to a course of oral azithromycin and sustained-release metronidazole or placebo. The regimen was repeated every 4 months until conception of another pregnancy. The investigators found no significant difference in the rates of preterm birth between the study and control groups. In fact, women in the treatment group, on average, had lower birth weight babies and delivered 2.4 weeks earlier [59]. In the absence of evidence of effectiveness for preventing recurrent preterm birth, presently no specific clinical intervention can be recommended for women with prior preterm birth in the interpregnancy period.\nSeveral studies have examined the effectiveness of clinical interventions applied to women with prior preterm birth during a subsequent pregnancy for the prevention of recurrent preterm birth. Perhaps the most promising is the use of progesterone. A large randomized controlled trial in the U.S. [60] and two recent meta-analyses [61, 62] found evidence of effectiveness of progestational agents (e.g. 17-hydroxyprogesterone caproate) for the prevention of preterm or recurrent preterm birth. However, \u201cbecause unresolved issues remain, such as optimal route of drug delivery and long-term safety of the drug,\u201d the American College of Obstetricians and Gynecologists (ACOG) has issued a committee opinion that \u201cwhen progesterone is used, it is important to restrict its use only to women with a documented history of a previous spontaneous birth at less than 37 weeks of gestation\u201d [63]. Another promising clinical intervention is the use of fish oil supplementation for the prevention of recurrent preterm birth. In the Fish Oil Trials in Pregnancy (FOTIP) study, fish oil supplementation in a subsequent pregnancy significantly reduced recurrence risk of preterm delivery [64]. There is also emerging evidence that scaling and root planing in pregnant women with periodontitis may reduce preterm birth [65], though no published studies have evaluated the effectiveness of periodontal screening in women with prior preterm birth. Further studies are needed to demonstrate the effectiveness of these interventions.\nPerhaps the most controversial clinical intervention is antibiotic treatment of asymptomatic bacterial vaginosis among women with a history of preterm birth [66, 67]. While some studies have shown a benefit, others have not. Efficacy of treatment may depend on the agent used, the route of administration, the timing of treatment, test of cure, and a number of host factors. In at least four antibiotic trials in pregnancy involving women with prior preterm birth [59, 65, 68, 69], antibiotic treatment (of bacterial vaginosis, Trichomonas vaginalis, or periodontal infection) was associated with higher, not lower, incidence of recurrent preterm birth. Clearly there is a need for further intervention studies, guided by better understanding of pharmacokinetics and pharmacogenetics, disease mechanisms and host response, as this remains a potentially important area of intervention for the prevention of recurrent preterm birth.\nPsychosocial interventions\nA growing body of evidence suggests that maternal psychosocial stress is an important risk factor for preterm delivery, and that risk can be mitigated by reducing stress and\/or increasing support. However, most extant studies of social support during pregnancy suffer from methodological flaws [70], and there is a paucity of research on the effectiveness of preconception or internatal support. The provision of social, clinical and parenting support during the internatal period, as previously described, may be quite valuable to mothers of a preterm infant, who often has special healthcare and other developmental needs that require additional support services. Such services may also be particularly beneficial to low-SES mothers, who may face greater burdens with less personal and social resources to deal with them. More research is needed to identify effective models of internatal psychosocial interventions for women with a history of preterm delivery.\nService coordination and integration\nWomen who have had a preterm birth may require multiple services during the internatal period, particularly if their children have significant disabilities and special healthcare needs. Providing integrated services, including linkages to early intervention or other early child development programs for preterm or LBW infants, should be a primary objective of care for these women. Several models of care coordination and service integration have been developed [71, 72]. One of the most successful models is the Olds nurse home visitation program [73]. The program provided nurse home visitation during pregnancy and the first two years postpartum. Evaluation of the Memphis\/Shelby County program [73] found women who received home visits by nurses had fewer closely spaced subsequent pregnancies, longer internatal intervals, and fewer months of using public assistance programs, compared to a control group of women. There were, however, no statistically significant program effects on LBW or stillbirth in the subsequent pregnancy. The program is technically not an \u201cinternatal care\u201d program and not targeted specifically to women who had a preterm birth, but this model could potentially be adapted by Healthy Start or other public health or community-based programs to provide service coordination and integration for internatal care using community nurses. We recommend that all women with prior preterm birth be offered care coordination and\/or home visitation during internatal care.\nSchedule of visits\nThe schedule of internatal visits for women with prior preterm birth needs to be individualized (with a minimum of three visits plus annual visits, as previously described), depending on the clinical and psychosocial needs of the woman and family. During their subsequent pregnancy, these women should be supervised by a maternal fetal medicine specialist, either directly or indirectly through consultations.\nIn sum, given the large impact of preterm birth on infant mortality and childhood disabilities and the high rates of recurrence in a subsequent pregnancy, women with prior preterm birth could be one of the most critical target groups for enhanced internatal care. Other high-risk women (e.g. women with prior fetal death), could also benefit from such program; we are unable to describe the content of their care in this paper due to space limitation and will do so in a series of papers to follow. The goal of such program is to prevent recurrence of preterm birth by addressing known biobehavioral pathways (e.g. infections\/inflammation, neuroendocrine, vascular, etc) prior to the next pregnancy. There is a great need for more comprehensive and systematic risk assessment and health promotion during internatal care, as well as more basic, clinical and intervention research to identify effective clinical and psychosocial interventions for the prevention of recurrent preterm birth.\nConclusion\nIn this paper, we began to define the contents of internatal care. We recommended expanding the current six-week postpartum visit to three or more internatal visits (at two weeks, six weeks, and six months postpartum, and annual visits beginning at one-year postpartum). We suggested some core contents that all women should receive during internatal care, including risk assessment (e.g. FINDS), health promotion (e.g. BBEEFF), clinical and psychosocial interventions. For women with chronic health conditions such as hypertension, diabetes, or weight problems, we identified clinical guidelines for their evaluation, treatment, and follow-up during the internatal period. For women who had a preterm birth, we proposed an internatal care model based on known etiologic pathways, with the goal of preventing recurrence by addressing these biobehavioral pathways prior to the next pregnancy. We suggested enhancing service integration for women and families, including possibly care coordination or home visitation for women with prior preterm birth.\nWe were limited in our effort to define the contents of internatal care by the paucity of research on the internatal period. Many pre-disease pathways leading to recurrence of adverse birth outcomes have not yet been elucidated. More importantly, even less is known about the trajectories to long-term women\u2019s health or child health, or how to alter these pathways and trajectories. There is a need for more intervention studies evaluating the effectiveness of the various components of internatal care. More importantly, we need more research on mechanisms of disease to guide the design of internatal interventions. Our proposal will need to be updated and revised continually as new research data emerge.\nWe were also limited by the paucity of research on community-level or public health interventions during the internatal period. While our paper focused on the contents of the clinical visits, we believe that provision of internatal care should not be limited to the clinician\u2019s office. For example, clinical interventions to control hypertension may be more effective if they are supported by community-level or public health interventions, such as implementation of the American Public Health Association resolution that the food manufacturers and restaurants reduce sodium in the food supply by 50 percent over the next decade [37]. The 5A\u2019s of smoking cessation may be more effective if they are supported by multilevel interventions at the interpersonal (e.g. targetting partner smoking), community and institutional (e.g. billboards and marketing of tobacco products, access to smoking cessation programs), and policy levels (e.g. ban on smoking in public space, promotion of smoke-free environments, and increased taxation on tobacco products) [3]. We believe that the ultimate success of internatal care may hinge on the development of a much broader and more integrated conception of internatal care than currently prevails with prenatal care [2].\nPresently there are numerous public health programs offering some components of internatal care, including many Healthy Start programs. They could provide the ideal social laboratory settings for implementing and evaluating various community-level interventions. While there are significant challenges to this type of research, it needs to be done. Identifying what internatal interventions work in the community may be just as important as identifying what works in the clinical setting. Such research needs to be guided by the principles and methods of community-based participatory research (CBPR) [74] and be held to scientifically rigorous standards.\nAlthough more research is needed to identify effective internatal interventions, we believe we cannot wait for the perfect studies to be done. We must begin to act. Most of our recommendations can be justified on the basis of providing good preventive and primary care for women and children, even in the absence of conclusive evidence supporting their effectiveness in improving subsequent pregnancy outcomes. For the most part they reflect current practice standards recommended by major national organizations (Table 1); most of these are considered standards of care or best practices in women\u2019s and children\u2019s health care. The only recommendations without the endorsement of a national organization\u2013screening for psychosocial stress and provision of psychosocial support\u2013are based on our collective opinions; we strongly believe these are important to reduce allostatic load on women\u2019s health and improve subsequent pregnancy outcome. In this paper we did not attempt to address all the institutional components and challenges in developing a successful model of internatal care, only the content of such a program. This paper reflects our effort to start the process of defining the contents of internatal care. We hope that this paper can be used to stimulate further dialogues about internatal care. Such dialogues, we believe, are needed to move forward research, practice, and policy on internatal care, and to move this nation one step closer toward the provision of a more universal, longitudinally-integrated continuum of women\u2019s healthcare.","keyphrases":["preconception care","content","internatal care","interpregnancy","preterm birth"],"prmu":["P","P","P","P","P"]}
{"id":"Mycorrhiza-4-1-2245993","title":"Tricholoma matsutake 1-Ocen-3-ol and methyl cinnamate repel mycophagous Proisotoma minuta (Collembola: Insecta)\n","text":"Two major volatiles produced by the mycelia and fruiting bodies of Tricholoma matsutake (1-octen-3-ol and methyl cinnamate) repel a mycophagous collembolan, Proisotoma minuta. Aggregation of the collembolans on their diet was significantly inhibited by exposure to 1 ppm methyl cinnamate or 10 to 100 ppm 1-octen-3-ol. The aggregation activity decreased dose-dependently upon exposure to 1-octen-3-ol at concentrations higher than 0.01 ppm. Aggregation in the presence of methyl cinnamate exhibited three phases: no significant effect at concentrations ranging from 0.001 to 0.1 ppm, significant inhibition from 1 to 100 ppm, and strong inhibition at 1,000 ppm. These results may explain why certain collembolan species do not prefer T. matsutake fruiting bodies.\nIntroduction\nFungal fruiting bodies are more nutritious than their mycelia (Stark 1972; Cromack et al. 1975; Vogt and Edmonds 1980) and are exploited by various arthropods, including dipteran larvae, collembolans, and acarine and oribatid mites (Hammond and Lawrence 1989). Collembolans are the most abundant group of insects found on agaric fruiting bodies (Yamashita and Hijii 2003), and numbers often reach hundreds to thousands on a suitable fruiting body (Sawahata et al. 2000, 2001, 2002; Nakamori and Suzuki 2005a). Collembolans attack the hymenial area, often consuming 50% or more of it in suitable fruiting bodies (Sawahata 2006). Additionally, collembolans are able to break the hyaline and thin-walled basidiospores of some fungi (Ponge and Charpentie 1981; Sawahata et al. 2001; Nakamori and Suzuki 2005b). Therefore, collembolans are likely to adversely affect spore production by fungal species with these types of spores.\nThe fruiting body of the ectomycorrhizal fungus Tricholoma matsutake (S. Ito and Imai) Sing., which has the common name matsutake, is one of the favorite and most valuable food items produced by forests in Japan because of its characteristic odor and texture, but artificial cultivation of T. matsutake has not yet succeeded. Sawahata and Narimatsu (2006) reported that collembolan populations on the fruiting bodies of T. matsutake are smaller than those on other mushrooms growing in the same forest. The gut contents of collembolans collected from the fruiting bodies suggested that they fed on the fungal tissue on the gill surface (Sawahata and Narimatsu 2006). In our preliminary studies, Folsomia candida Willem and Proisotoma minuta Tullberg fed on sliced samples of the fruiting body of T. matsutake in a laboratory experiment (data not shown). These findings suggested that the mushroom is not poisonous to collembolans and that it is simply avoided as a source of food when alternatives are available.\nCollembolans use fungal volatiles to recognize potential sources of food (Bengtsson et al. 1988; Hedlund et al. 1995). Bengtsson et al. (1991) suggested that the volatile compounds 1-octen-3-ol and 1-hepten can aid the collembolans in their search for a palatable fungus. The major volatiles produced by T. matsutake are 1-octen-3-ol and methyl cinnamate (Ohta 1983; Terashita et al. 1991). The former is a primary volatile produced by many species of fungi (Kaminski et al. 1974; Pyysalo 1976), and the latter determines the characteristic odor of this fungus (Terashita et al. 1991). Hence, methyl cinnamate may prevent aggregation and feeding on the fruiting body by some collembolans. Further, high concentrations of 1-octen-3-ol can repel fungivores (Pfeil and Mumma 1993; Wood et al. 2001). The fruiting body of T. matsutake contains 1-octen-3-ol at concentrations ranging from 2 to 186\u00a0ppm on a fresh-weight basis (Ohta 1983; Terashita et al. 1991), but it is unknown whether 1-octen-3-ol repels collembolans at high concentrations.\nThe present study aimed at investigating the effects of the major volatile components of T. matsutake (1-octen-3-ol and methyl cinnamate) at different concentrations on the aggregation of mycophagous collembolans by biological assay (feeding test). Our goal was to provide an explanation for why collembolans aggregate in relatively small numbers on T. matsutake fruiting bodies.\nMaterials and methods\nProisotoma minuta was extracted from forest soils near the Matsunoyama Museum of Natural Science in Niigata Prefecture (307\u00a0m above sea level, 37\u00b005\u2032N, 138\u00b036\u2032E), in central Japan. This species was used because it fed on various ectomycorrhizal fungi in a choice experiment (Schultz 1991; Hiol Hiol et al. 1994), and it is amenable to laboratory culture. The collembolans were reared on dry yeast at 20\u00b0C in a cylindrical chamber (11\u00a0cm in diameter, 7\u00a0cm in height) whose bottom (1.0 to 1.5\u00a0cm in thickness) was covered by a mixture of plaster of Paris and charcoal (10:1\u00a0v\/v). We followed the methods used in previous food choice experiments (e. g., Schultz 1991; Kaneda and Kaneko 2004). Each assay (with six replications) used 70 individuals (1\u00a0month after eclosion from eggs, 0.8 to 1.1\u00a0mm in body size) at a time in the same type of chamber.\nThe major volatile compounds of T. matsutake have already been determined to be 1-octen-3-ol and methyl cinnamate by several researchers (e.g., Ohta 1983; Terashita et al. 1991). We substituted laboratory-grade versions of these chemicals for the natural volatile compounds. 1-Octen-3-ol (Wako Pure Chemical Industries, Osaka, Japan) was diluted with distilled water to prepare solutions of 0.001, 0.01, 0.1, 1, 10, 100, and 1,000\u00a0ppm. Methyl cinnamate (Wako) was diluted similarly but with dichloromethane. Controls were created using distilled water and dichloromethane, respectively. Into each diluted solution, three pieces of filter paper (3\u00a0cm in diameter) were dipped for long enough that the paper became completely saturated. The wet 1-octen-3-ol filter papers were placed 1\u00a0cm apart on the bottom of the assay chamber to form a triangle (Fig.\u00a01). The wet methyl cinnamate filter papers were kept in air for 15\u00a0min to evaporate the solvent and then moistened with distilled water before bioassay by the same procedure.\nFig.\u00a01Bioassay method used to assess the ability of 1-octen-3-ol and methyl cinnamate to repel the collembolan Proisotoma minuta (n\u2009=\u200970). Plugs of potato-dextrose agar (PDA) were placed at the center of each sample-treated filter paper on a mixture of plaster of Paris and charcoal (10:1\u00a0v\/v) at the bottom of the cylindrical chamber\nOn each sample of filter paper, including the control, a plug (1\u00a0cm in diameter) of 3% potato dextrose agar medium (a PDA plug) was provided as food (Fig.\u00a01). Seventy insects were introduced into the chamber and held for 90\u00a0min in the dark at 20\u00b0C; then, the individuals on each PDA plug were counted without disturbance. The results represent means of 18 samples (six replicates of three PDA plugs per chamber) among the dilution series (eight levels, including the control) for the two test compounds. The mean numbers of collembolans in each treatment were compared by F test, one-way analysis of variance, and a multiple-range test (Fisher\u2019s PLSD). Statistical analyses were performed with Stat View 5.0J software (SAS Institute, Cary, NC, USA).\nResults\nBoth compounds at 1,000\u00a0ppm almost completely inhibited the aggregation of P. minuta on the PDA plugs (Figs.\u00a02 and 3), leaving means of fewer than 1.0 and 0.5 individuals per PDA plug on methyl cinnamate and 1-octen-3-ol, respectively. Statistically significant reductions in aggregation were also observed at several lower concentrations.\nFig.\u00a02Influence of the concentration of methyl cinnamate on the number of Proisotoma minuta aggregated to feed on PDA plugs. Gray bars indicate the means of 18 samples (six replicates of three PDA plugs per dish), and the error bars indicate the standard error (SE) of the mean. Means labeled with different letters differ significantly (Fisher\u2019s PLSD test, P\u2009<\u20090.05)Fig.\u00a03Influence of the concentration of 1-Octen-3-ol on the number of Proisotoma minuta aggregated to feed on PDA plugs. Gray bars indicate the means of 18 samples (six replicates of three PDA plugs per dish), and the error bars indicate the standard error (SE) of the mean. Means labeled with different letters differ significantly (Fisher\u2019s PLSD test, P\u2009<\u20090.05)\nDiscussion\nMethyl cinnamate exhibited three ranges of concentrations with different abilities to inhibit the aggregation of P. minuta (Fig.\u00a02): no significant effect (a mean of 13.3 to 16.1 individuals per plug) from 0.001 to 0.1\u00a0ppm, incomplete but significant inhibition (a mean of 1.2 to 8.2 individuals per plug) from 100 to 1,000\u00a0ppm, and strong inhibition at 1,000\u00a0ppm. Thus, methyl cinnamate repels collembolans at high concentrations.\nMethyl cinnamate has previously been shown to be a principal volatile component of the fruiting body of T. matsutake (Terashita et al. 1991), and its content increases during fungal development to reach 22 to 154\u00a0ppm on a fresh weight basis by the time the veil breaks (Ohta 1983). High levels of the compound (150 to 360\u00a0ppm, with a mean of 236\u00a0ppm) are localized in the lamellae and spores (Ohta 1983), where collembolans are frequently observed feeding on the spores and hyphae of the hymenia (Sawahata 2006). The high content of methyl cinnamate in the lamellae may thus prevent aggregation and grazing on gill surfaces by collembolans.\n1-Octen-3-ol has previously been shown to be another principal volatile component of the fruiting body of T. matsutake, at concentrations in the fruiting bodies ranging from 2 to 186\u00a0ppm on a fresh weight basis (Ohta 1983; Terashita et al. 1991). This volatile component exhibited a clear dose\u2013response relationship (Fig.\u00a03): As the concentration of 1-octen-3-ol increased from 0.001 to 1000\u00a0ppm, aggregation of the insects decreased continuously, and the difference from the control became significant at concentrations of 10\u00a0ppm and higher. At concentrations of 100 and 1,000\u00a0ppm, 1-octen-3-ol significantly reduced aggregation of P. minuta (to an average of 1.2 individuals per PDA plug). These results suggest that Collembola show a different response pattern at different concentrations, with a slight and nonsignificant increase in aggregation compared with the control at concentrations ranging up to 0.01\u00a0ppm; at higher concentrations, aggregation decreased significantly (to less than 50% of the control), and at 1000\u00a0ppm, aggregation was almost completely eliminated. Bengtsson et al. (1991) also found a weak but nonsignificant attractive effect of 1-octen-3-ol on collembolans (individuals of Onychiurus armatus Tullb.) in soil fungi. Further studies with new methods are needed to explain the attractive effect of low levels of 1-octen-3-ol among a range of collembolans.\nThe present study provides an explanation of why certain collembolans avoid aggregation on T. matsutake fruiting bodies in the field. Both major components of the volatiles present in the fruiting bodies of this species (methyl cinnamate and 1-octen-3-ol) defend against collembolan attack at high concentrations. Wood et al. (2001) reported that 1-octen-3-ol prevents feeding by banana slugs at a concentration of 28\u00a0ppm. Pfeil and Mumma (1993) reported that 1-octen-3-ol and 3-octanone seemed to deter gravid females of Megaselia halterata Wood (Phoridae) at high concentrations. Therefore, some fungal species may produce high concentrations of 1-octen-3-ol to defend against mycophagous insects.","keyphrases":["matsutake","methyl cinnamate","collembola","1-octen-3-ol","repellent activity of tricholoma."],"prmu":["P","P","P","P","R"]}
{"id":"Eur_J_Epidemiol-4-1-2190784","title":"Rise in seroprevalence of herpes simplex virus type 1 among highly sexual active homosexual men and an increasing association between herpes simplex virus type 2 and HIV over time (1984\u20132003)\n","text":"Objectives Herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) are both highly prevalent. The rate of genital HSV-1 transmission is reportedly increasing over time. HSV-2 is considered to be an important risk factor for HIV transmission. We therefore studied changes in the HSV-1 and HSV-2 prevalence in a large cohort of men who have sex with men (MSM) over a 20-year time period. Methods Among 1847 HIV-infected and HIV-uninfected MSM participating in the Amsterdam Cohort Studies, seroprevalence of HSV-1 and HSV-2 was determined and prevalence rate ratios (PRR) and 95% confidence intervals were calculated. Results Between 1984 and 2003 the HSV-1 and HSV-2 prevalence decreased among HIV-uninfected MSM (P < 0.001), but remained stable among HIV-infected MSM. HSV-1 prevalence increased among men with at least 200 sexual partners over lifetime (PRR: 1.49, P < 0.001). The association between HIV infection and HSV-2 became stronger over time (PRR: 3.45, P < 0.001). Conclusions Seroprevalence of HSV-1 and HSV-2 remained high among HIV infected MSM from 1984 to 2003. The association of HIV and HSV-2 increased during the HIV epidemic. Since the proportion of sexual transmission of HSV-1 is rising, it is important to study the potential role of HSV-1 as risk factor for HIV acquisition.\nIntroduction\nHerpes simplex virus type 1 (HSV-1) is widespread in the general population, while herpes simplex virus type 2 (HSV-2) is more restricted to risk groups such as men who have sex with men (MSM). HSV-1 prevalence is around 70% in the general population [1, 2]. Transmission usually occurs during childhood through oral contact and normally causes oropharyngeal infection. Childhood HSV-1 transmission has declined in industrialised countries, resulting in a lower prevalence of HSV-1 and leaving a larger population of adolescents at risk for sexual transmission of HSV-1. Earlier studies have reported sexually related risk factors for HSV-1 infection in women [3]. Among those persons attending the STD clinic and blood donors, HSV-1 infection is associated with younger age of first intercourse [4]. However, less is known about sexually related risk factors for HSV-1 infection among MSM [5]. HSV-2 infection is usually transmitted sexually and is considered as a marker for sexual risk behaviour in populations [6]. In HIV-infected MSM, the prevalence of HSV-2 is as high as 61% [1], while being 15\u201325% in the general population [7, 8]. HSV-2 is a risk factor for HIV acquisition, especially in the African setting [9] and in MSM [10]. HSV-2 infected persons are more susceptible for HIV [11, 12]. Moreover, HIV-infected persons are more likely to have subclinical reactivation of HSV-2 and are therefore more likely to transmit the virus [13].\nWe previously demonstrated a decline in the prevalence of HSV-2 among MSM in Amsterdam between 1984\u20131997, which could be explained by a decrease in sexual risk behaviour [5]. However, in the second half of the 1990s their sexual risk behaviour increased after effective HIV therapy became generally available. This may have caused an increase in the prevalence of HSV-2 and possibly also in HSV-1 since 1996.\nWe here studied the trend in HSV-1 and HSV-2 prevalence among homosexual men over a 20-year time period (1984\u20132003) and whether risk factors for infection changed during this period.\nMethods\nStudy population\nIn 1984 an open and prospective cohort study on HIV seroconversion and AIDS among sexually active HIV-negative and positive homosexual men was started. The Amsterdam Cohort Study (ACS) is still ongoing, although entry criteria with respect to HIV status and age have changed over time. From 1984 until May 1985, both HIV-positive and HIV-negative men were included. From May 1985 until February 1988, only HIV-negative men were allowed in the study. From February 1988 through 1994, HIV-positive and HIV-negative men could enter the study, but since 1995, they must be \u226430\u00a0year of age.\nAt an ACS visit, a standardised questionnaire is administered regarding demographics, sexual behaviour, and medical history for sexually transmitted infections (STI). Blood samples are collected for immunologic and virologic testing and for storage. For this study, stored sera (collected at the first cohort visit) taken from ACS participants with at least two cohort visits (1847\/2100 (88%)) were tested for HSV-1 and HSV-2.\nLaboratory methods\nSensitive and specific FDA approved serological assay for HSV-1&2 was used (HerpeSelect by FOCUS technologies, USA). Its manufacturer recommends an index value\u00a0>\u00a01.1 as positive. However, there is evidence that using this cut-off value in HSV-2 studies yields a high rate of false positive results in populations with multiple infections, such as those in Africa [14]. Raising the positive cut-off will increase the specificity [14]. Since the optimal cut-off for our target population has not been established, 100 samples with results in the range of 0.9 and 3.5 were re-tested with a highly specific Western blot. HSV-2 ELISA and Western blot results were concordant for 80\/100 samples. The proportion of samples that were positive with both ELISA and Western blot increased with increasing index value (Fig.\u00a01). Based on these results, we consider a cut-off value of \u22652.1 as being positive and an index value <2.1 is classified as negative. The HSV-2 index value <2.1 had 36% concordance with Western blot results, while the HSV-2 index value of \u22652.1 showed 93% concordance. There were no differences in Western blot outcomes between HIV-infected and HIV uninfected MSM.\nFig.\u00a01Comparison of HSV-2 serology by ELISA and Western blot. 100 samples were tested with HSV-2 ELISA and re-tested with the Western blot to identify discrepancies between the two assays\nBlood samples were tested for HSV-1 and HSV-2 at the Public Health Laboratory of the Health Service of Amsterdam. The Western blot was conducted at the Institute for Pathology and Medical Research (ICPMR) in Sydney, Australia. Blood samples are also tested for HIV antibodies by enzyme linked immunosorbent assay (ELISA) (Abbot Laboratories, North Chicago, Illinois, USA; Vironostika, Organon, Teknika, Boxtel, the Netherlands), and when positive, are confirmed by Western blot.\nVariables and statistical analyses\nThe statistical analyses were based on the data collected at entry of the cohort. Variables used in this study were calendar year of ACS entry, HIV-status, age, nationality, education, age of first homosexual contact, lifetime sexual partners, and self-reported history of syphilis and gonorrhoea in the past 5\u00a0years.\nVariables concerning sexual practices included orogenital, anogenital and oroanal contact in the prior 6\u00a0months. Some changes in the questions were made in the questionnaires between 1984 and 2003 regarding the sexual practices. Oroanal contact was not asked for from 1889 to 1994, resulting in approximately 25% missing values on this variable. From 1995 onward, the most important difference was that orogenital contact with ejaculation was asked, while in the previous years orogenital contact in general was asked. The percentage of participants not having orogenital contact was somewhat higher for the years 1995 and 1996.\nThe prevalence of HSV-1 and 2 at the ACS entry was determined and risk factors for HSV-1 and 2 were assessed by calculating prevalence rate ratios (PRR), with their 95% confidence interval (CI). Odds ratios could not be interpret as relative risks, since the rare event assumption was not reached. Therefore PRR\u2019s were directly estimated, using a modified Poisson regression approach [15]. This approach provided a correctly estimated standard error for the estimated relative risk. Since inclusion criteria with respect to age and HIV-status changed over time, all risk factor analyses were adjusted for age and HIV-status. Variables that were statistically significant were included in the multivariate model, using a stepwise forward approach forcing age and HIV-status in the model.\nWe tested whether risk factors changed over time by testing for interaction between variables under investigation and calendar time in the multivariate model. Calendar time therefore was categorised as 1984\u20131986, 1987\u20131991, 1992\u20131996, 1997\u20132003.\nConfounding was defined to be present when the included variable caused a change of the prevalence ratio by more than 10%. Interaction was defined to be present when the addition of an interaction term improved the original model and the P-value was <10%. Statistical significance was defined as a P-value <0.05. To reduce residual confounding when measuring the association between HSV and sexual practices, three variables measuring sexual practices over the prior 6\u00a0months were included in the model at the same time, together with the lifetime sexual partners. We modelled time trends in the HSV-1 and 2 prevalence with calendar time as a continuous variable using restricted cubic splines with four knots, resulting in a smoothly varying curve.\nFor 77 MSM the HSV-1 index value was missing and 91 MSM had a missing HSV-2 index value. Participants with a missing index value for both HSV-infections were not included in the analyses. MSM with a missing index value for one HSV-infection, but with a known HSV status of the other HSV-infection were included in the analyses for the known HSV serostatus.\nFinally, sensitivity analyses for HSV-2 were conducted by using the cut-off value of 1.1, as recommended by the manufacturer and by using the cut-off value of 3.5, excluding those with an index value in the grey area (between 0.9\u20131.1 and 0.9\u20133.5). However, time trends in prevalence and risk factors found were comparable to when 2.1 was the cut-off value (data not shown).\nResults\nGeneral characteristics\nBetween 1984 and 2003, a total of 1847 MSM had at least two visits. General characteristics of the total study group are presented in Table\u00a01. Of the 1847, 1207(65%) MSM were HSV-1 antibody positive, while 759\/1847(41%) of the men were HSV-2 antibody positive. Of the total group, 558(30%) were positive for both. Participants were predominantly of Dutch nationality (86%) and had a median age of 29\u00a0years (interquartile range: 25\u201336).\nTable\u00a01Demographic and sexual characteristics of 1847 homosexual men, for the total study group, and for HSV-1-infected participants and HSV-2-infected participants separately, between 1984 and 2003, with the prevalence ratios for HSV-1 and HSV-2 with their 95% confidence intervalsaCharacteristicsTotalHSV-1-infectionPRR (95% CI)Overall P-valueHSV-2-infectionPRR (95% CI)Overall P-valueTotal18471207 (65)759 (41)Year of study entry<0.0001<0.00011984\u20131986943675 (72)1461 (49)11987\u20131991165113 (68)0.87 (0.78\u20130.97)92 (56)0.84 (0.72\u20130.98)1992\u20131996222138 (43)0.88 (0.79\u20130.96)80 (36)0.66 (0.56\u20130.77)>1997517281 (54)0.76 (0.70\u20130.84)126 (24)0.47 (0.40\u20130.55)Index value<0.9\u20135358530.9\u20131.1\u201329321.1\u20132.1\u2013100112\u22652.1\u20131107759Missing7791Age<0.0001<0.0001<30\u00a0years100257012521\u226530\u00a0years8456371.20 (1.13\u20131.29)5071.98 (1.75\u20132.23)Nationality:0.0060.62Dutch1586 947 (60)1615 (39)1Northern\/central Europe116 72 (62)1.04 (0.92\u20131.18)54 (47)1.08 (0.90\u20131.38)Non-European145 117 (81)1.17(1.07\u20131.28)75 (52)1.04 (0.91\u20131.23)Education0.0050.007Low9677 (80)157 (59)1Middle669408 (61)0.85 (0.76\u20130.95)239 (36)0.72 (0.59\u20130.87)High971574 (59)0.81 (0.73\u20130.81)387 (40)0.79 (0.66\u20130.94)Missing11577 (67)54 (47)Sexual partners in lifetime1\u201320860497 (58)1<0.0001277 (32)10.5121\u2013200531360 (68)1.12 (1.04\u20131.22)212 (40)2.30 (0.93\u20131.21)>200443339 (77)1.24 (1.15\u20131.34)262 (59)1.07 (0.95\u20131.20)Age of first homosexual contact (median, IQR)18 (15\u201320)17 (15\u201320)1.01(1.01\u20131.02)^<0.000117 (15\u201320)0.99 (0.99\u20131.01)c0.32HSV co-infection5685681.29 (1.14\u20131.47)<0.00015681.167 (1.08\u20131.24)<0.0001HIV infection (%)513 367 (72)1.11 (1.03\u20131.18)0.007312 (61)1.12 (1.00\u20131.24)0.05History of gonorrhoea in the past 5\u00a0years1053 666 (63)0.88 (0.83\u20130.93)0.0005424 (40)0.88 (0.79\u20130.97)0.02History of syphilis in the past 5\u00a0years278 219 (79)1.15 (1.07\u20131.24)0.001197 (71)1.51 (1.36\u20131.69)<0.0001Orogenital contact in the past 6\u00a0monthsb1363892 (65)0.99 (0.82\u20131.22)0.46565 (41)0.69 (0.55\u20130.87)0.01Anogenital contact in the past 6\u00a0monthsb1222819 (67)1.11 (1.14\u20131.60)0.002551 (45)1.36 (1.16\u20131.60)0.0002Oroanal in the past 6\u00a0monthsb1081700 (65)1.00 (0.91\u20131.09)0.99438 (41)0.98 (0.85\u20131.12)0.80aSince ACS inclusion criteria have changed over times all analyses were adjusted for age (per 10-year increase) and HIV status at cohort entrybAnalyses are also adjusted for the sexual techniques and number of partners to exclude residual confoundingcPer 10\u00a0year of increase\nPrevalence of HSV-1 and HSV-2 over time\nThere was an overall decline in the prevalence of both HSV-1 and 2 between 1984 and 2003 (Tables\u00a01, 2). To investigate time trends in HSV seroprevalence we included an interaction term between time and HIV-status (Fig.\u00a02a, b). Among HIV-negative MSM, the HSV-1 prevalence decreased significantly over time, P\u00a0<\u00a00.001 (Fig.\u00a02a). Among HIV-positive MSM, the HSV-1 prevalence remained stable over time, P\u00a0=\u00a00.35(Fig.\u00a02a).\nTable\u00a02Multivariate model of risk factors associated with HSV-1 infectionHSV-1Overall P-value(a) HSV-1 infectionYear of study entry<0.00011984\u2013198611987\u201319910.91 (0.70\u20131.18)1992\u201319960.83 (0.66\u20131.02)>19970.75 (0.63\u20130.90)Age1.13(1.07\u20131.18)<0.0001HIV serostatusNegative10.01Positive1.10 (1.02\u20131.18) NationalityDutch10.0006Northern or Central European1.05 (0.92\u20131.20)Non European1.62 (1.12\u20131.36) Education 0.25Low1Middle 0.90 (0.79\u20131.00)High0.84 (0.76\u20131.06)Sexual partners in lifetime1\u20132010.00321\u20132001.13 (1.05\u20131.25)>2001.13 (1.04\u20131.23)History of Gonorrhoea in the past 5\u00a0years0.97 (0.90\u20131.03) 0.11History of Syphilis in the past 5\u00a0yearsOrogenital contact in the past 6\u00a0months1.12 (0.88\u20131.43)0.42Anogenital contact in the past 6\u00a0months1.08 (0.97\u20131.20) 0.20Oroanal contact in the past 6\u00a0months1.02 (0.70\u20131.13)0.81HSV-2Overall P-value(b) HSV-2 infectionYear of study entry<0.00011984\u2013198611987\u201319910.86 (0.70\u20131.06)1992\u201319960.58 (0.48\u20130.71)>19970.47 (0.39\u20130.56)HIV serostatusNegative1<0.0001Positive1.50 (1.37\u20131.68)HSV coinfection1.15 (1.02\u20131.30)0.02History of Syphilis in the past 5\u00a0years1.21 (1.08\u20131.36)0.001Orogenital contact in the past 6\u00a0months0.69 (0.56\u20130.84)<0.0001Anogenital contact in the past 6\u00a0months1.20(1.08\u20131.42)0.02Oroanal contact in the past 6\u00a0months1.00(0.87\u20131.15)0.72Fig.\u00a02(a) HSV-1 prevalence in the Amsterdam Cohort Study among MSM, according to the HIV status (1\u00a0=\u00a0HIV positive, 0\u00a0=\u00a0HIV negative) and the 95% confidence interval. (b) HSV-2 prevalence in the Amsterdam Cohort Study among MSM, according to the HIV status (1\u00a0=\u00a0HIV positive, 0\u00a0=\u00a0HIV negative) and the 95% confidence interval\nThe HSV-2 prevalence significantly decreased among HIV-negative men, P\u00a0<\u00a00.001(Fig.\u00a02b). Results from the regression models showed that, after adjustment for changes in age, nationality, education and changes in sexual risk behaviour, the decline in HSV-2 prevalence among HIV negative MSM remained significant (PRR adjusted 0.92, P\u00a0<\u00a00.001). In contrast, the HSV-2 prevalence remained stable over time for men infected with HIV (P\u00a0=\u00a00.12). Again, this result was observed after controlling for age, demographic characteristics and sexual behaviour.\nRisk factors\nRisk factors for HSV-1 and HSV-2 infection, adjusted for age and HIV-status, are presented in Table\u00a01.\nIn the final model calendar year, HIV-status, nationality, and number of lifetime sexual partners remained independent predictors for HSV-1 infection (Table\u00a02). For HSV-2 infection, earlier year of study entry, positive HIV-status, HSV-1 co-infection, a history of syphilis and sexual behaviour remained independent predictors (Table\u00a02).\nChanging risk factors over time\nDifferent interaction terms were included in the model. It appeared that the effect of calendar year differed between HIV-infected and HIV-uninfected MSM for both HSV-1 and HSV-2.\nAs shown in Fig.\u00a02a, the association between HIV and HSV-1 became stronger over time. This was due to the decline in HSV-1 prevalence over time among HIV-negative MSM but not among HIV-positive MSM (Fig.\u00a02a).\nFor HSV-2 the association with HIV infection increased with calendar year and was highest after 1996 (Fig.\u00a02b).\nFor HSV-1, also the effect of calendar time differed with respect to nationality, number of lifetime sexual partners, and with HSV-2 co-infection. As shown by the regression model, a decrease in HSV-1 infection over time was observed only among MSM with Dutch or Northern\/Central-European nationality. The association between HSV-1 and having non-European origin became stronger over time. The adjusted PRR was 1.08 (P\u00a0=\u00a00.3) before 1986 and the adjusted PRR became 1.58 (P\u00a0<\u00a00.0001) for the time period after 1996.\nAlso the association between HSV-1 and a higher number of lifetime sexual partners became stronger after 1996. Figure\u00a03a shows the prevalence of HSV-1 infection over time according to the number of sexual lifetime partners. A decrease in the HSV-1 prevalence was seen among MSM with fewer than 21 partners (P\u00a0<\u00a00.0001), while among MSM with more than 200 partners, the HSV-1 prevalence increased between 1988 and 2003 (P\u00a0=\u00a00.01).\nFig.\u00a03(a) Prevalence of HSV-1 and the 95% confidence interval over time among HIV negative MSM, according to number of lifetime sexual partners; 1\u00a0=\u00a01\u201320, 2\u00a0=\u00a021\u2013200, 3>200 partners. (b) Prevalence of HSV-2 and the 95% confidence interval over time among HIV negative MSM, according to number of lifetime sexual partners; 1\u00a0=\u00a01\u201320, 2\u00a0=\u00a021\u2013200, 3\u00a0>\u00a0200 partners\nFor HSV-2, the effect of calendar time differed with respect to the number of lifetime sexual partners and with HSV-1 co-infection. A large number of lifetime partners also was strongly associated with HSV-2. However, a decrease in the HSV-2 prevalence was seen among all categories of lifetime sexual partners (Fig.\u00a03b), but this decrease was stronger for MSM with fewer than 21 partners and for those MSM with 21\u2013200 partners.\nDiscussion\nIn the present study, we demonstrated an overall decrease in HSV-1 and HSV-2 prevalence among HIV-negative MSM, but not among HIV-positive MSM. In the 1984\u20132003 period, the association between HSV-2 and HIV among MSM became stronger over time, and HSV-1 prevalence increased in highly sexually active HIV-negative MSM. To our knowledge, this is the first study based on almost 20\u00a0years of HSV-1 and HSV-2 prevalence data among MSM.\nThe decrease seen in the seroprevalence of HSV-1 and HSV-2 over time, could not be explained by changes in demographic characteristics or sexual behaviour. The decrease in HSV-1 is likely to reflect a decrease in childhood transmission by the oropharyngeal contact of HSV-1. Since fewer individuals are infected in childhood with HSV-1, there is a growing population of persons at risk at the time they become sexually active, resulting in a larger proportion of sexual transmission of HSV-1. Two risk factors for HSV-1 infection that could be important in its sexual transmission were identified by this study. First, HIV infection is associated with HSV-1 seropositivity. Second, the prevalence of HSV-1 was higher among highly sexual active MSM (at least 200 lifetime sexual partners). An association also shown earlier by others [3].\nHIV infection in this respect may reflect an epidemiological marker for sexual risk behaviour for HSV-1 transmission. HSV-1 prevalence did not decrease in those infected with HIV, and we consider that genital HSV-1 infection has a growing role in the acquisition of HIV.\nLikewise, HSV-2 prevalence did not decline over time among those infected with HIV, whereas a decline was noted among HIV uninfected MSM.\nAlthough HSV-2 is sexually transmitted, we did not find an association between a higher number of lifetime partners and HSV-2 infection. HSV-2 was also highly prevalent among MSM with 1\u201320 lifetime partners. Mainly between 1984 and 1995, there were no major differences in the proportion of MSM infected with HSV-2. This suggests that HSV-2 is highly sexual transmissible and when having a low number of life time partner the risk of receiving a HSV-2 infection is still very high.\nThe results of this study show a protective effect of orogenital contact for HSV-2 infection, which might be explained by the fact that anogenital contact is a stronger predictor for HSV-2 infection. All variables measuring sexual practices are included in the analyses at the same time. Since most MSM practised all the techniques during the same time period these practices could not be analysed as independent risk factors. The stronger effect of anogenital contact might have overruled the effect of orogenital contact, resulting in a protective effect of orogenital contact.\nThe overall prevalence of HSV-2 in this study is similar to that among MSM in San Francisco in 1989, but higher than the prevalence found in more recent studies in the US [16\u201318]. The lower prevalence in those more recent studies probably reflects the decline in HSV-2 prevalence over time, as found in our study.\nRussell et\u00a0al., found high prevalence rates of HSV-2 among HIV-infected MSM in Australia [1]. The HSV-2 seroprevalence was more than twice as high as among HIV-uninfected MSM; there was no significant difference in HSV-1 prevalence between HIV-infected and HIV-uninfected MSM.\nSeveral epidemiological studies have described an association between HIV and HSV-2 [10]. HSV-2 is recognised as a risk factor for HIV acquisition in MSM. In addition, HSV-2 may up-regulate HIV and increase local HIV replication on mucosal surfaces, leading to an increased risk of HIV transmission. Our study is the first to show an increase in the association between HSV-2 and HIV since 1996, suggesting that HSV-2 may play a growing role in driving the HIV epidemic in MSM. If this is the case, prevention of HSV-2 may well contribute to the prevention of HIV among highly sexually active MSM. Although serological screening for HSV-2 among MSM is still under debate, the increasing association between HSV-2 seropositivity and HIV is an argument in its favour. Several reasons against screening have been raised, such as the lack of a reliable serological test. We are aware of the low specificity of the various HSV-2 serological assays. However, these serological assays might be useful as a screening tool, when used with an increased cut-off value less individuals will be classified as false positive. A second argument against serological screening is that HSV-2 infection is largely asymptomatic and condom use appears only partially protective against HSV transmission. These factors complicate the prevention of HSV-2 and genital HSV-1 infection. However, it has been shown that half of the patients, initially unaware of their HSV-2 infection are able to recognise symptoms after being educated to do so [19]. Also, knowledge of the HSV-2 status of a sexual partner has been associated with a reduced risk of HSV-2 transmission [20]. Antiviral drugs used as suppressive therapy will lower the frequency of recurrences by 70\u201380% [21, 22]. A combined approach of offering serological screening to highly sexual active MSM together with encouraging condom use to reduce the risk of HSV transmission and using suppressive therapy among those with recurrent lesions might eventually play an effective part in controlling the HIV epidemic among MSM.\nOne limitation of our study is its cross-sectional design. As a consequence, we cannot reveal the relation between HIV and HSV infection, being unable to determine which occurred first. As both HIV and HSV are sexually transmitted diseases, their association may well reflect shared sexual behavioural practices leading to transmission as well as a biological relation. Longitudinal studies, in which incident HIV and HSV cases are captured are therefore needed to give more insight into the relationship between HIV and HSV as affected by changes in sexual risk behaviour.\nThe results of this study have two implications for HIV and HSV research among highly sexually active MSM. First, it appears that HSV-2 and HIV are now more strongly related than in the early days of the HIV epidemic. As a vaccine against HSV-2 for MSM is not yet available, a determination of the extent to which the prevention of HSV-2, specially aimed for MSM at high risk for HIV, can contribute to controlling the HIV epidemic is needed. Second, since the extent of sexual transmission of HSV-1 is rising, we need to clarify its potential role as a risk factor for HIV acquisition in longitudinal studies.","keyphrases":["prevalence","hiv","hsv-1","hsv-2","msm"],"prmu":["P","P","P","P","P"]}
{"id":"Bioprocess_Biosyst_Eng-2-2-1705492","title":"Dynamics of amino acid metabolism of primary human liver cells in 3D bioreactors\n","text":"The kinetics of 18 amino acids, ammonia (NH3) and urea (UREA) in 18 liver cell bioreactor runs were analyzed and simulated by a two-compartment model consisting of a system of 42 differential equations. The model parameters, most of them representing enzymatic activities, were identified and their values discussed with respect to the different liver cell bioreactor performance levels. The nitrogen balance based model was used as a tool to quantify the variability of runs and to describe different kinetic patterns of the amino acid metabolism, in particular with respect to glutamate (GLU) and aspartate (ASP).\nIntroduction\nBioreactor technology for extracorporeal liver support using primary human liver cells has been developed within the last decade [1]. The design of a bioreactor for maintaining the hepatocyte\u2019s full functionality is of great importance. The used multi-compartment bioreactor consists of three interwoven, independent capillary membrane systems. Two of them (compartments 1 and 2, in the following aggregated to the \u2018perfusion compartment\u2019) provide decentralized plasma flow and the third one (compartment 3) provides oxygen supply to the cells, which are localized in the extracapillary space (compartment 4, \u2018liver cell compartment\u2019). This three-dimensional spatial structure represents an artificial equivalent of the hepatic vasculature at the lobular level. The bioreactor is integrated into a perfusion system that enables monitoring and control of system conditions (see Fig.\u00a01). Previous studies have shown that primary liver cells reconstitute to tissue-like structures after inoculation into the bioreactor and that they maintain metabolic activities over several weeks [2\u20134]. This bioreactor therefore provides a valuable tool to analyze the dynamics and network structures of the physiological and molecular interactions of liver cells under standardized conditions that closely reflect the situation in the natural organ.\nFig.\u00a01Scheme of the liver cell bioreactor with the perfusion circuit, the two inflow streams and the outflow stream. Measured data were acquired from the waste\nRecently, data from this bioreactor system has been analyzed statistically as well as by fuzzy cluster and rule based data mining and pattern recognition methods in order to identify early performance predictors for the bioreactor\u2019s long-term performance [5\u20137]. With respect to the prominent role of hepatocytes in the amino acid metabolism, the analysis and modeling of metabolic pathways of amino acids could provide important information on the functional state of hepatocytes cultured in vitro. In initial studies the role of amino acid regulation in extracorporeal liver support systems was analyzed [8]. More recently, three different modeling paradigms, i.e. correlation networks, Bayesian networks and systems of differential equations were applied to characterize the concentration profiles of six amino acids and related nitrogen-containing compounds in bioreactor cultures of primary human liver cells [6]. The present work reports on the model based analysis of the measured kinetics of 18 amino acids as well as ammonia (NH3) and urea (UREA) of 18 liver cell bioreactor runs.\nMaterials and methods\nCells and bioreactors\nPrimary human liver cells were isolated according to a method described elsewhere [9] from human donor livers (n=18) that were not suitable for transplantation due to organ injury. After isolation, cells were cultured within bioreactors over 7\u201334\u00a0days. (This broad variability in duration is caused by their use for liver support from the seventh day onwards and by the performance. Low performance runs were finished earlier. High performance runs were continued for a longer period of time. However, only the first 6\u00a0days of the runs were analyzed in this study.) Concentrations of free amino acids in the culture perfusates were determined by an automated reversed phase high performance liquid chromatography system (RP-HPLC) with precolumn derivatization using the ophthaldialdehyde method [10]. NH3 and UREA concentrations were determined using routine clinical analyzers (Roche Diagnostics, Heidelberg, Germany).\nWith respect to the model based analysis, two compartments of the multi-compartment capillary membrane bioreactor system were considered: The \u2018liver cell compartment\u2019 with the volume V2=600\u00a0mL contains the liver cells in the inter-capillary space. The \u2018perfusion compartment\u2019 with the volume V1=900\u00a0mL supplies a stream through the inside of the capillaries. This perfusion stream carries the concentrations of the compounds that are supplied to or removed from the bioreactor. Decentralized mass exchange at low gradients is achieved by independent perfusion of two medium capillary systems, enabling different perfusion modes [10]. In this study, counter-directional medium flow was performed to facilitate rapid matter distribution. Due to the high flow rate of the perfusion stream (250\u00a0mL\u00a0min\u22121), an almost ideal mixing within the perfusion compartment can be assumed (Fig.\u00a01). To this perfusion compartment two time-variant inflow streams are added with the flow rates FA(t) and FB(t) as defined by Eq. (1). FA(t) follows a step function from FA1=150\u00a0mL\u00a0h\u22121 down to FA2=50\u00a0mL\u00a0h\u22121 switching at tA(=1\u00a0day). FB(t) switches from FB1=0 up to FB2=1\u00a0mL\u00a0h\u22121 at the time tB (see Table\u00a04). The outflow rate F0(t) to the waste equals the sum of both inflow rates. (This is realized by overflow keeping a constant pressure in the perfusion compartment).  The inflow rate FA(t) carries the 18 amino acids and NH3 at the concentrations cAi (see Table\u00a01). The inflow rate FB(t) carries only the amino acid aspartate (ASP) at the concentration cB15=1,500\u00a0\u03bcmol\u00a0L\u22121, i.e. cBi=0 for all i\u226015. The time courses of the concentrations c0i(t) in the outflow stream may be considered to describe the response of the medium to the inoculation of the bioreactor with cells. These concentrations are in steady-state at c0i(0)=cAi prior to inoculation. Due to the almost ideal mixing in the perfusion compartment, the concentrations c0i(t) in the perfusion stream are the same as in the outflow stream.\nTable\u00a01Model variables ci and c0,i for the liver cell compartment and the perfusion compartment, respectively, their initial values ci(0) and c0,i(0) before inoculation of the bioreactor with liver cells, the concentration cAi in the inflow stream fed with the flow rate FA(t) according to Eq. (1) and the stoichiometric coefficients si for nitrogenNameModel variables ci and c0,Ici(0)=c0,i(0)=cAi [\u03bcmol\u00a0L\u22121]Stoichiometric coefficient siLEUc1, c0,122581HISc2, c0,29403ARGc3, c0,326044VALc4, c0,49871TRPc5, c0,54642PHEc6, c0,613001ILEc7, c0,75001ALAc8, c0,818251TYRc9, c0,922151LYSc10, c0,103272METc11, c0,112591SERc12, c0,128611GLYc13, c0,1319571THRc14, c0,148591ASPc15, c0,152841ASNc16, c0,161682GLUc17, c0,172651GLNc18, c0,186872NH3c19, c0,19411UREAc20, c0,2002PROTc21, c0,210\u2013\nData\nA data set with the elements ci,j,k (i=1,...,20; j=1,...,18; k=1,...,K) for 20 kinetic variables and 18 bioreactor runs at K time points tk was analyzed. The kinetics over the first 6\u00a0days of the bioreactor runs were analyzed here (tk=6\u00a0days). Concentrations of NH3 and UREA were measured daily (tk=0\u20136\u00a0days). Amino acid concentrations were determined up to the third day daily and every third day afterwards (tk=0, 1, 2, 3, 6\u00a0days). The samples for the measurement of the ci,j,k were taken from the waste of the liver cell bioreactor system (see Fig.\u00a01) which was emptied daily after accumulation of the outflow stream over the period of 24\u00a0h.\nThe model based analysis described focuses on the amino acid metabolism as quantified by the measured time series of the concentrations of 18 amino acids (i=1,..., 18; see Table\u00a01) as well as of NH3 (i=19) and UREA (i=20).\nEach run was labeled by Lj \u2208 {\u2018low\u2019, \u2018medium\u2019, \u2018high\u2019} describing the performance with respect to the long-term maintenance of the functionality of the liver cells within the bioreactor. Seven runs were labeled \u2018high\u2019 (j=1,..., 7), seven runs \u2018medium\u2019 (j=8,..., 14) and four runs \u2018low\u2019 (j=15,..., 18). This performance labeling was provided by an expert based on his assessment of altogether 99 variables that were measured to quantitatively characterize the system.\nDifferential equation system\nThe differential equation system (2.1\u20132.10) was developed to describe the measured kinetics of the 18 amino acids as well as of NH3 and UREA (Table\u00a01). This model takes into account two compartments as described in Cells and bioreactors, the \u2018perfusion compartment\u2019 and the \u2018liver cell compartment\u2019.\nThe Eq. (2.1) describe the dynamics of the components i in the perfusion compartment by four terms: The first and second term represent the fresh medium inflow with the volumetric rates FA(t) and FB(t), respectively, from the reservoir into the perfusion compartment with the volume V1. The reservoir concentrations cAi of the components i are specified in Table\u00a01. The volumetric rates FA(t) and FB(t) are specified by Eq. (1). The third term in Eq. (2.1) denotes the outflow from the perfusion compartment into the waste with the volumetric rate F0(t). The last term describes the diffusion between the perfusion and the liver cell compartment with its rate being proportional to the difference of the concentration c0,i in the perfusion compartment and the concentration ci in the liver cell compartment. The parameter p0 is proportional to the diffusion coefficient and assumed to be the same for all compounds to simplify parameter identification.\nThe Eqs. (2.2\u20132.9) formulate the changes of the concentrations ci in the liver cell compartment with the volume V2 due to the exchange with the perfusion compartment (the first term on the right side of each differential equation) and the metabolic reactions (the following term(s) on the right side of each differential equation) as drawn schematically in Fig.\u00a02.  The model drawn in Fig.\u00a02 and its Eqs. (2.1\u20132.10) were constructed based on the mean kinetics of 20 measured variables averaged over the seven high-performance runs H1\u2013H7 as shown in Fig.\u00a03. The kinetics of these seven individual runs are highly correlated with their correlation coefficients being greater than 0.8 for at least six of the seven high-performance runs and the 11 amino acids MET, SER, THR, ARG, HIS, GLY, TRP, LEU, VAL, PHE, and ILE [6]. The dynamics of these amino acids are characterized by monotonously, almost exponentially decreasing kinetics [6]. Their dynamics were therefore described by first order reactions as formulated by the second term in Eq. (2.2). The observed decreasing kinetics of amino acid concentrations is the net result of anabolic and catabolic reactions, where the catabolism of amino acids (which was considered here only) surpasses the amino acid synthesis and release by proteolytic activities (which were neglected here). The second term with the parameters p1,..., p14 in Eq. (2.2) can be interpreted mainly as amino acid uptake by transamination and oxidative deamination (see Table\u00a02). In addition to the 11 amino acids with highly correlated and monotonously decreasing kinetics, the time courses of the concentrations of the amino acids ALA, LYS, and TYR were, for simplification, also modeled using Eq. (2.2). The kinetics of the amino acids ASP, ASN, GLU, and GLN were modeled in greater detail by Eqs. (2.3\u20132.6). Glutamate (GLU) is known to be the central compound of amino acid metabolism. Its kinetics were found in [6] to be highly correlated with ASP. ASP was fed by the inflow rate FB(t) (see Cells and bioreactors) to avoid or compensate the exhaustion of ASP which is essential for NH3 elimination via the urea cycle. The degradation of amino acids is interpreted in a simplified form by transamination to GLU for the amino acids LEU, HIS, ARG, VAL, TRP, PHE, ILE, ALA, TYR, and LYS (denoted by \u2018AAg\u2019 in Fig.\u00a02). The amino acids MET, SER, GLY, and THR (denoted by \u2018AAn\u2019 in Fig.\u00a02), however, are not transaminated forming GLU, but deaminated via different pathways forming NH3. The parameters with the indices 15\u201318, 21\u201323 as shown in Fig.\u00a02 and Table\u00a02 describe the metabolic fluxes between ASP, ASN, GLU, GLN, and NH3. They can be interpreted in a simplified way by reactions catalyzed, e.g., by the enzymes asparaginase, aspartate aminotransferase (AST), glutamate oxalacetate transaminase (GOT), glutamine synthetase (GS) and glutamate dehydrogenase (GLDH). In general, the biochemical reactions were assumed to be either of linear or bilinear nature (first or second order). In two cases, however, additional assumptions were required to obtain sufficient model fits: (1) For the aggregated modeling of the NH3 elimination via the urea cycle in the presence of ASP a non-linear Michaelis-Menten-type kinetics was used (see Eq. 2.8). This reflects the observation that the UREA formation rate does not significantly increase any more at strongly elevated ASP concentrations (see ASP and UREA in Fig.\u00a03). (2) Protein synthesis, i.e. anabolism, seems to be switched on or is strongly increased after the third day. This was modeled using Eq. (2.10). The total protein (PROT) was not measured, but a corresponding variable c21 was hypothetically introduced by Eq. (2.9) to improve the model fit of the other 20 variables to the measured data for t>3\u00a0days.\nFig.\u00a02Structure of the model Eqs. (2.2\u20132.10). The numbers within the circles denote the indices i of the corresponding concentrations ci and the numbers at the arrows denote the indices m of the corresponding model parameters pm. (The diffusion process modeled by the parameter p0 is not shown here.) \u2018AAg\u2019 denotes the amino acids LEU, HIS, ARG, VAL, TRP, PHE, ILE, ALA, TYR, and LYS; \u2018AAn\u2019 denotes the amino acids MET, SER, GLY, and THRFig.\u00a03Mean kinetics (\u00b1\u00a0SD) of 20 measured variables (18 amino acids, NH3, UREA) averaged over the kinetics of the n=7 high performance runs H1\u2013H7 (n=7)Table\u00a02Parameters pm of the model (2) with interpretation, estimated values and confidence intervals [pmlow, pmhigh]; enzyme activities: AST aspartate aminotransferase, GOT glutamate oxalacetate transaminase, GS glutamine synthetase, GLDH glutamate dehydrogenase; values and units of the parameters pm of the model (2) as identified by the model fit to the mean kinetics averaged over the seven high performance runs (Fig.\u00a03); PC_1, PC_2: first and second principal componentmInterpretation\/enzymespm [pmlow, pmhigh]UnitPC_1PC_20Diffusion49628 [34681, 132980]mL\u00a0h\u221210.18820.35301Aminotransferases (Transamination)LEU1.816 [0.000, 4.629]day\u221210.2679\u22120.01872HIS16.29 [3.42, 620.16]day\u221210.0251\u22120.29483ARG100.00 [88.75, 2655]day\u22121\u22120.14300.22534VAL1.147 [0.428, 23.112]day\u221210.2506\u22120.06175TRP15.99 [7.42, 833.88]day\u221210.2022\u22120.10636PHE4.148 [1.740, 9.520]day\u221210.2595\u22120.03647ILE3.723 [0.000, 6.213]day\u221210.2491\u22120.02688ALA1.573 [0.000, 6.213]day\u221210.25140.01359TYR1.135 [0.559, 3.450]day\u221210.1866\u22120.273010LYS0.192 [0.000, 2.164]day\u221210.23580.005311Other specific reactionsMET34.53 [7.27, 284.76]day\u221210.25870.117112SER10.80 [3.34, 102.71]day\u221210.26980.039913GLY10.76 [2.69, 251.25]day\u221210.26740.084914THR9.326 [3.16, 420.70]day\u221210.2543\u22120.054615Asparaginase0.0009 [0.0000, 0.002]day\u22121\u00a0\u03bcmol\u22121\u00a0L0.18250.4175163.593 [1.215, 8.505]day\u221210.22540.297117AST50.08 [39.25, 59.37]day\u22121\u22120.06500.034118GOT56.73 [50.35, 71.85]day\u221210.18190.145619Urea Cycle Km (ASP)225.76 [187.7, 378.9]day\u22121\u00a0\u03bcmol\u22121\u00a0L0.2000\u22120.3185205.00 [0.9375, 6.17]\u03bcmol\u00a0L\u22121\u22120.10230.181321GS0.0033 [0.001, 0.007]day\u22121\u00a0\u03bcmol\u22121\u00a0L0.1198\u22120.1839220.0205 [0.001, 0.039]day\u221210.1017\u22120.391723GLDH, Protein Synthesis40.68 [24.12, 50.38]day\u221210.11210.0330240.5475 [0.465, 0.813]\u2013\u22120.0057\u22120.1114\nThe model parameters were identified by fitting the model to the measured data ci,j,k minimizing the scaled mean square error (mse) as defined by Eq. (3) for run j. The model fitting error mse was scaled by the square of the maximum of the respective measured variable. According to Eq. (3), the kinetics c0,i(t) obtained from the simulation of Eqs. (2.1\u20132.10) with the initial values listed in Table\u00a01 were averaged over 24\u00a0h (i.e. over the time interval of the accumulation of the bioreactor outflow in the waste where the samples for the measurements were taken from daily) and then compared with the measured data ci,j,k.  The differential equations were solved using a Runge-Kutta fourth order algorithm. The parameter identification by model fitting minimizing the mse was performed using a simplex search method. MATLAB tools (The MathWorks, Inc., Natick, MA, USA) were used for all calculations.\nResults and discussions\nThe mean time profiles of the 20 measured variables each averaged over the high performance runs H1\u2013H7 are shown in Fig.\u00a03. The results of the model fitting to these averaged data are shown in Fig.\u00a04 and the identified model parameters p0,..., p24 are listed in Table\u00a02 (third column). The confidence intervals for the parameter values (fourth and fifth column in Table\u00a02) were identified by repeated model fitting to the randomly disturbed time series data with means and standard deviations as shown in Fig.\u00a03. The fit of the modeled kinetics to the measured data is acceptable for 18 of the 20 variables with the exception of lysine (LYS) and alanine (ALA). The initial increase of the LYS kinetics could be hypothetically explained by proteolytic activities. The final increase of ALA can be explained by the ALA aspartate transferase activity. The decreasing kinetics of 11 out of the 18 amino acids is explained by transamination and oxidative deamination forming GLU and NH3. The feeding of ASP after the third day results not only in an increase of the concentration of ASP but also of asparagine (ASN), GLU, and glutamine (GLN) due to the activities of the enzymes AST, GOT, GS, and asparaginase (alternatively or additionally the activity of asparagine synthetase could be included in the model).\nFig.\u00a04Measured and simulated kinetics of the concentrations of 18 amino acids, NH3 and UREA. The measured kinetics (dots) are those of the mean profiles shown in Fig.\u00a03 (n=7). The simulated kinetics c0i(t): (thin lines) are those obtained from the model (2). The simulated kinetics c0i(t) were averaged according to Eq. (3) over the past 24\u00a0h (thick lines) in order to use them for model fitting to the measured data that were acquired from the waste (outflow accumulated over 24\u00a0h)\nTable\u00a03 lists the model parameter values pm with the respective model fitting error mse as identified by individual model fitting to the seven high performance runs. Figs.\u00a05\u20137 show the results of the model fitting to the data for the individual high performance runs H1, H5, and H7, respectively. The kinetics of run H2 (not shown here) is very similar to the mean kinetics of the seven high performance runs shown in Fig.\u00a04. The quasi-stationary concentrations of several amino acids, such as MET and LEU, are lower in run H5 (see Fig.\u00a06) than in run H2 (compare Fig.\u00a04). This results in a p0 value, which is about 6.5\u00d7 higher for run H5 than for run H2 (see Table\u00a03). While the variability of the parameter values p1,..., p24 can be explained by differences in the liver cell material obtained from different individual donors (having different age, weight, liver damage, etc.), it is not entirely clear at this stage how to explain such different values of the parameter p0 that represents diffusion and potentially other phenomena taking place across and beyond the membrane between the perfusion and the liver cell compartment. These issues are addressed in greater detail in [11] as well as in studies described in [12] to elucidate the dynamics of electrolyte distribution in the bioreactor without liver cells using tracer experiments and input\/output systems analysis.Table\u00a03Values of the parameters pm of the model (2) with the values of the scaled mean square error (mse) as identified by individual model fits to the seven high performance runs H1\u2013H7mH1H2H3H4H5H6H70937624859915419060636318770949398101012.43642.11022.75503.75785.85762.930202224.49445.133536.905850.803938.03622.913921.94173342.2608285.530379.992797.785355.361589.3504278941.50421.94421.33832.24653.23482.2254058.391812.479319.586688.639644.309819.81934.866662.86253.79438.639110.571211.08434.07560.834574.83146.49384.00106.018610.17706.62230.010283.54400.91921.77712.03255.82352.7174092.13891.56292.44631.57461.29080.854801000.02.21511.52131.69980.470801110.800524.378383.028053.8904116.586825.381312.99081210.07679.470921.121816.373531.182010.25363.38151310.05735.139727.421322.631545.111610.22233.25321411.66507.21708.506922.044225.209312.49543.0131150.00080.00070.00180.00070.01290.00080.0007165.15803.47423.85547.125130.91683.79471.20831740.468559.405837.907663.556960.347343.376069.34681869.917457.329772.578270.4225109.786752.520163.280919291.5727192.9329341.5933334.0218238.6410263.7827111.2798205.56698.42293.25044.82613.82513.78088.6594210.00140.00.01140.00260.00060.00520.0034220.05130.00.00310.02800.00040.01570.00002352.597035.600151.898249.646767.233839.360326.0489240.57070.68330.19630.59330.61320.68230.6408mse0.01620.01430.02020.01150.01350.01450.0321Fig.\u00a05Measured data of the 20 variables of bioreactor run H1 with the simulated kinetics of the model fitted to the data of H1Fig.\u00a06Measured and simulated kinetics as in Fig.\u00a05, but for run H5\nThe model, which was developed using the kinetic patterns of high performance runs was also fitted to the data of the 11 runs with medium and low performance. The aim of these model studies is to elucidate causes of low liver cell bioreactor performance. The fit of the model with 25 parameters to 18 bioreactor runs results in an array of 25\u00d718 parameters. Three selected parameters with their values as identified by fitting the model to the individual 18 runs are listed in Table\u00a04. Considering a tolerable model fitting error mse of about 2% or less, the model fit proved satisfactory for six of the seven high performance runs (with the exception of run H7) and for two of the seven medium performance runs (M8 and M10). However, the model fit was not acceptable for the high performance run H7, the medium performance runs M9, and M11\u2013M14 as well as all four low performance runs L15\u2013L18. The main reason for the insufficient model fits is that the measured kinetics of leucine (LEU), isoleucine (ILE), valine (VAL), ALA, and other amino acids are not decreasing in these runs. This behavior is shown in Fig.\u00a07 for run H7. In these cases some parameter values, e.g., for p1, were found to be zero (negative parameter values were set to zero; see Table\u00a04). These zero parameter values cause that the medium and low performance runs displayed in Fig.\u00a08 are artificially close to each other. Run H7 labeled as a high performance one appears to be a medium performance run according to the model simulation results (see Figs.\u00a07 and 8).Table\u00a04Parameters for the 18 liver cell bioreactor runs: tB as used in Eq. (1); the star (*) for run 13 denotes that the feed FB(t) was switched on during the period from the second to the fourth day; the scaled mean square error (mse) and the parameters p1, p2, and p22 as used in Eq. (2) were identified by model fitting to the data of the high performance runs H1\u2013H7 (averaged) and of the individual high performance runs H1,..., H7, medium performance runs M8,..., M14 and low performance runs L15,..., L18Run No.tB[d]mse [-]p1 [d\u22121]p2[d\u22121]p22*1,000 [d\u22121]H1-H7 (averaged)30.00991.81616.2920.5H130.01622.4364224.494451.2761H230.01432.11025.13350H330.02022.755036.90583.1020H430.01153.757850.803928.0101H530.01355.857638.03620.4057H630.01452.93022.913915.7285H730.0321021.94170.0186M830.01471.03678.77420.0497M950.033002.86110.0381M1050.01990.000811.29470.0578M1130.03130.001032.36580.2347M1230.02590.9387168.22990.0722M132\u20134*0.15590.000311.64390.1391M1430.04920.000310.97870.1615L1500.2747039.92250.0572L1660.080302.59350.0268L1730.08640137.27210.0131L1830.132101.26130.0560Fig.\u00a07Measured and simulated kinetics as in Fig.\u00a05, but for run H7Fig.\u00a08Biplot of the first and second principal components of the model parameter matrix {p0,...,p24} as identified for the 18 liver cell bioreactor runs (high performance runs H1,...,H7, medium performance runs M8,...,M14, low performance runs L15,...,L18)\nFigure\u00a08 displays the identified model parameters of the 18 liver cell bioreactor runs after principal component analysis (PCA) of the parameter array for the first and second principal components in a biplot [13]. The first principal component represents 53% of the total parameter variance and the first and second principal components together represent almost 64% of this variance. Most of the parameters p1\u2013p14 (with the exception of p2, p3, p9) representing transamination and oxidative deamination activities mainly constitute the first principal component of the parameter array (see Table\u00a02: PC_1>0.2 for these parameters). These 11 parameters are correlated: The 55 correlation coefficients calculated between the 11 parameters estimated over the 18 runs have values between 0.54 and 0.987 with the median of 0.87 and the mean of 0.84. The values of these 11 parameters are high for six of the seven high performance runs and low for run H7 as well as for the medium and low performance runs as shown in Table\u00a04 for parameter p1. The value of the parameter p1 is strongly correlated with the bioreactor performance (p-value 0.00017 by the two-sided t-test of pm values of high versus medium or low performance runs). The mean value of the parameter p1 averaged over the high performance runs equals 2.84 (\u00b11.77). The parameter is zero for all low performance runs. In Fig.\u00a08 the medium and low performance runs are displayed in the left part describing low transamination and oxidative deamination activities whereas six of the seven high performance runs (with the exception of run H7) are displayed in the right part representing high transamination and oxidative deamination activities.\nSome parameters are highly correlated over the 18 runs with a correlation coefficient r of more than 0.98, e.g., the parameters p11 and p13 (representing the catabolism of methionine and glycine) as well as p4 and p7. Also, the parameter p1 is highly correlated (r>0.96) with the parameters p4 and p7. The high correlation of the parameters p1, p4, and p7 representing the transamination of LEU, ILE and VAL can be hypothetically explained by the fact that LEU, ILE and VAL are branched-chain amino acids which cannot be transaminated in hepatocytes. To transaminate these amino acids, the activity of non-parenchymal liver cells is required.\nAmong the high performance runs there are further individual differences that are displayed in Fig.\u00a08 by a low second principal component PC_2 (e.g., high values of the parameters p2 and p22 for run H1) and a high PC_2 (e.g., low values of the parameters p2 and p22 for runs H2, H5, and H7). The parameters p2 and p22 represent the uptake of histidine and glutamine (see Table\u00a02). As shown in Fig.\u00a05 for run H1, the high parameter value p2 results in a low stationary concentration of HIS and the high parameter value p22 results in high NH3 and low GLU levels. These effects could be related to the amino acid transport system N (SN1) that mediates specifically the uptake of histidine and glutamine [14]. The parameter p21 that represents the activity of GS was found to be very low for the high performance runs H2 and H5 (see Table\u00a03). GS plays an important role in the spatial organization (zonation) of the liver and is exclusively expressed in pericentrally located hepatocytes [15]. The low GS activity in the high performance runs H2 and H5 could therefore be caused by a high proportion of periportal versus pericentral hepatocytes in the cell preparations for these two runs.\nConclusion\nThe kinetics of 18 amino acids and the related nitrogen-containing compounds NH3 and UREA in a primary human liver cell bioreactor were analyzed and modeled using a differential equation system. The model focuses on the kinetics of GLU and ASP as well as on the formation and elimination of NH3 and the synthesis of UREA. It describes the degradation of amino acids by transamination, oxidative deamination and other specific reactions. In addition, the activities of selected enzymes such as AST, GOT, GS, and GLDH as well as, in a more aggregated form, the activities of urea cycle enzymes are included. The differential equation system does not represent a fully mechanistic but rather a phenomenological model since essential metabolic activities had to be neglected because they cannot be identified based on the measured data.\nThe differential equation system allows the analysis of a number of representative liver cell functions in terms of their kinetic behavior. The identification of the model parameters by fitting the model responses to the measured data was used to generate hypotheses about the causes of specific differences between the bioreactor runs. The model fits were found to be very satisfactory for eight high and medium performance runs. The model is however inadequate for low performance runs and with respect to the LYS kinetics also for high performance runs. This is probably caused by the neglection of proteases activities in the model that appear to be relevant for low and medium performance runs. Both, protein synthesis and degradation could not be modeled in detail due to the lack of representative protein measurements.\nThe applied model based analysis of data obtained from the bioreactor system can be used to quantitatively evaluate the functional state of liver cell cultures under high performance conditions intended for clinical application in extracorporeal liver support systems. The approach can also be used to study the effect of several exogenous factors, e.g., of hormones or drugs, on hepatocyte metabolism in vitro. The model based analysis methods applied here therefore provide suitable tools for in silico studies supplementing in vitro studies of hepatocyte functions in a systems biological way.","keyphrases":["bioreactor","liver support","systems biology","metabolic network"],"prmu":["P","P","P","R"]}
{"id":"Osteoporos_Int-4-1-2267486","title":"Development and application of a Japanese model of the WHO fracture risk assessment tool (FRAX\u2122)\n","text":"Summary The present study estimated the 10-year probability using the Japanese version of WHO fracture risk assessment tool (FRAX\u2122) in order to determine fracture probabilities that correspond to intervention thresholds currently used in Japan and to resolve some issues for its use in Japan.\nIntroduction\nFractures related to osteoporosis have become a major health and economic burden in Asian countries just as they have in North America and Europe. An estimated 117,900 cases of hip fracture occurred in 2002 [1], and the incidence in Japan has increased in the past 10\u00a0years [1, 2]. Asia will be expected to have the highest absolute increase in fracture number because it has the largest population. Early detection of individuals with high fracture risk using clinical risk factors would have a substantial impact on reducing the burden of fractures in Asia.\nA series of meta-analyses on prospective population-based cohorts has identified a number of clinical risk factors that contribute to fracture risk independently of BMD at the femoral neck [3]. The integration of these risks would, therefore, enhance the predictive value of BMD [4]. The risk factors comprise age, sex, bone mineral density, body mass index (BMI), long-term use of glucocorticoids, parental history of hip fracture, history of fragility fracture, smoking, alcohol consumption (3 or more units\/day), and secondary osteoporosis such as rheumatoid arthritis. A WHO scientific group has proposed that the 10-year probability for fracture is used to express fracture risk for clinical assessment [5] and to determine intervention thresholds [3] The aim of this study was to create a fracture probability model based on the methodology of the WHO risk assessment tool (FRAX\u2122) [6] calibrated to the epidemiology of Japan.\nIn addition, several problems need to be resolved before the FRAX\u2122 model is applied to Japan. First, the FRAX\u2122 tool inputs femoral neck BMD and the Z-score or T-score is based on the NHANES III reference data base. In Japan, BMD at the lumbar spine is widely used clinically because the physical size of Japanese people is smaller than that of Western people, giving rise to a view based on little evidence that the reproducibility of measurements at the femoral neck BMD would be poorer than that at the lumbar spine. Furthermore, data on the young adult mean (YAM) and the mean at each age are installed in the DXA systems in Japan, and programmed to calculate T- and Z-scores from Japanese reference data. In addition, the Japanese Society for Bone and Mineral Research [7, 8] provide recommendations for the diagnosis of osteoporosis and intervention based on YAM, and these are widely used in clinical practice.\nAgainst this background, additional aims of the present study were to provide fracture probabilities based on the FRAX\u2122 tool that were equivalent to currently accepted intervention thresholds, explore the impact of using Japanese-specific normative data for femoral neck BMD, and reassess the respective performance characteristics of BMD at the femoral neck and lumbar spine.\nMethods\nModels were constructed to compute the 10-year probability of hip fracture and a major osteoporosis-related fracture in Japan. A major osteoporosis-related fracture was defined as a clinical spine, hip, proximal humeral and forearm fracture. Poisson modelling was used to calculate the hazard functions. The relationship between probability and hazard functions were used to calculate the 10-year probability or fracture for a combination of the risk factors. The mortality estimates for Japan were those published by the World Health Organization for 1999, which accord with estimates from Japan [9]. The incidence of hip fractures was taken from previously published sources [1] as was the incidence of fractures at the proximal humerus and distal forearm [10]. Since the incidence of a clinical vertebral fracture was not known in Japan, we assumed that the ratio of clinical vertebral fracture incidence to that of a vertebral fracture diagnosed by radiographic surveys [11] would be the same in the Japan as it was for Sweden [12].\nThe relationship of clinical risk factors to fracture outcomes was assumed to be the same as that determined in a large meta-analysis of risk factors of 190,000 patient years from nine prospectively studied population-based cohorts from Europe, Australia, North America and Asia [3]. The relationship has been validated in a further 11 cohorts of population-based samples with 1.2 million patient years of observation from the same regions [4]. The independent contribution of each risk factor was used to compute probabilities of fracture in the absence of clinical risk factors or in the presence of any combination [13, 14].\nIn Japan, the criteria for the diagnosis of osteoporosis prepared by the Japanese Society for Bone and Mineral Research [7] are based on BMD measurements expressed as a percentages of the young adult mean (YAM) for women. In patients with no prior fragility fracture a diagnosis of osteoporosis is made where the BMD is less than 70% of YAM. In patients with a previous fracture, osteoporosis is diagnosed where the BMD is less than 80% of YAM. These diagnostic thresholds, derived by maximising sensitivity and specificity for fracture detection, are also used as intervention thresholds. In order to compare intervention thresholds using YAM with probabilities derived from the FRAX\u2122 algorithm, T-score equivalents were used. The T-score equivalent to 70% and 80% of YAM for Japanese people is \u22122.7 SD and \u22121.8 SD, respectively, using the NHANES III reference for BMD at the femoral neck in Caucasian women aged 20\u201329\u00a0years [15].\nThe relative performance characteristics of BMD at the lumbar spine and femoral neck were examined in a population-based prospective study in Hiroshima. The Hiroshima cohort comprised 2,596 men and women (69% female, 9,803 person years, mean age 65.1\u00a0years). Details of the cohort have been previously published [11]. In brief, the participants received measurement of lumbar spine and femoral neck BMD using dual X-ray absorptiometry (DXA, QDR-2000, Hologic) during the period from 1994 to 1995 and were followed for a mean period of 4\u00a0years. Information about hip fracture, fracture of the distal radius, proximal humeral fracture and clinical spinal fracture was collected at interview by trained nurses and physicians during the biennial health examinations. One hundred eighty-six fractures were detected during the follow-up period, of which 89 were categorized as osteoporotic fractures and 31 were hip fractures. The gradient of fracture risk (increase in fracture risk per SD change in Z-score for BMD and increase in fracture risk per 0.1\u00a0g\/cm2 change in BMD) at the two sites was determined by the use of Poisson models [16]. The fracture outcomes were calculated for hip fracture, a major osteoporosis-related fracture (femoral neck, distal radius, proximal humerus and clinical spine fracture) and all fractures.\nThe mean height and body weight for Japanese women in the Hiroshima cohort was 150\u00a0cm and 52.6\u00a0kg, respectively, giving a BMI of 23.4\u00a0kg\/m2. The calculation of fracture probability was made at this BMI, but differences in BMI have little effect on predictive value for fracture risk assessment in the presence of BMD [17].\nJapanese-specific T- and Z-scores and those derived from NHANES III were compared using the database of the Japanese Society for Bone and Mineral Research [7]. Data using both methods of calculation were entered into the FRAX\u2122 tool.\nResults\nTen-year probability of fracture\nThe 10-year probability of a major osteoporotic fracture for individuals without clinical risk factors is shown in Fig.\u00a01 grouped by sex, age and T-score. The 10-year probability increased with age and with decreasing T-score. At younger ages, the fracture probability was similar in men and women. With advancing age, probabilities rose as expected, but the increase was greater in women than in men. In men aged 80\u00a0years, the 10-year probability for osteoporosis-related fracture exceeded 10% at a T-score of \u22123 SD, whereas in women of the same age, fracture probabilities exceeded 10% with T-score of \u22121 SD.\nFig.\u00a01Ten-year probability (%) of osteoporotic fracture (hip, clinical spine, humerus, forearm) in Japanese men and women without clinical risk factors according to age and T-score for BMD at the femoral neck\nThe contribution of clinical risk factors to fracture probability is shown in Fig.\u00a02 for women aged 65\u00a0years with a BMI of 23.4\u00a0kg\/m2. In women without clinical risk factors, the 10-year probability for an osteoporosis-related fracture was 7.5%. The 10-year probability was higher in the presence of clinical risk factors. Smoking and alcohol were relatively weak risk factors, the use of long-term glucocorticoids of intermediate weight, and a parental history of hip fracture or a prior fragility fracture were associated with the highest risks. For example, the 10-year probability was 8.1% for smokers and 14.5% for individuals with a prior fracture. The 10-year probability for hip fracture was 1.1% in women without a clinical risk factor, 1.6% in smokers and 2.7% in women with a previous fracture (see Fig.\u00a02).\nFig.\u00a02Ten-year probability for osteoporotic (hip, clinical spine, humerus, forearm) and hip fracture (%) according to the presence of a clinical risk factor, in women at the age of 65\u00a0years and with a BMI of 23.4\u00a0kg\/m2\nFracture probabilities were computed in women at the diagnostic threshold recommended in Japan. Thus, the cut-off level of BMD was set at 70% of YAM in women without a previous fracture and at 80% of YAM in those with a previous fragility fracture. In women aged 50, 60, 70 and 80\u00a0years without clinical risk factors and with BMD equivalent to 70% of YAM, the 10-year probability was 5.4%, 8.7%, 13.8% and 23%, respectively. In women having BMD equivalent to 80% of YAM and existing fracture but no other clinical risk factors, the 10-year probability was 7.1%, 10.5%, 14.7% and 23.4% at the same ages, respectively. Thus, at each age, the fracture probability was similar using the two diagnostic criteria. In contrast, the fracture probability equivalent to the diagnostic threshold in Japan rose with age, and at the age of 80\u00a0years was about four times higher than that at age 50\u00a0years (Fig.\u00a03). Similar findings were apparent for hip fracture probability in that probabilities equivalent to the diagnostic threshold in Japan rose with age. The increase with age was more marked than for all major fractures and at the age of 80\u00a0years was about 6\u201340 times higher than that at age 50\u00a0years depending on the threshold used (see Fig.\u00a03).\nFig.\u00a03Ten-year probability of osteoporotic (hip, clinical spine, humerus, forearm) and hip fracture based on women at the threshold for the diagnosis of osteoporosis using the criteria of the Japanese Bone Mineral Metabolism Association\nComparison of lumbar spine and femoral neck BMD\nThe gradient of fracture risk for spine BMD and femoral neck BMD in the Hiroshima cohort indicated that lumbar spine measurements predicted all fractures, osteoporosis-related fracture and hip fracture with approximately equal gradients of risk that ranged from 1.25\/SD for all fractures to 1.17\/SD for hip fractures. There was no difference in the gradient of risk between men and women. In the case of hip fracture risk, the gradient of risk in men and women combined was not statistically significant with BMD measured at the lumbar spine. BMD at femoral neck had a similar or slightly higher gradient of risk for fractures compared with spine BMD, particularly in the case of hip fracture (Table\u00a01). There was no significant difference in gradient of risk between lumbar spine BMD and femoral neck BMD with the exception of that for hip fracture where the gradient of risk was significantly higher for measurements made at the femoral neck. When gradient of risk was standardized to a constant denominator (i.e., RR\/0.1\u00a0g\/cm2) the findings remained unchanged (see Table\u00a01).\nTable\u00a01Predictive ability of spine and femoral neck BMD for any, osteoporotic and hip fracture in men and women from Japan\u00a0RR\/SDRR\/0.1\u00a0g\/cm2RR95% CIRR95% CIa. Any fractureFemoral neck1.451.23\u20131.701.431.22\u20131.68Lumbar spine1.251.13\u20131.391.381.19\u20131.61b. Osteoporosis-related fracturesFemoral neck1.401.09\u20131.781.381.09\u20131.74Lumbar spine1.201.04\u20131.401.301.05\u20131.61c. Hip fractureFemoral neck2.08b1.34\u20133.222.111.38\u20133.23Lumbar spine1.170.91\u20131.501.250.87\u20131.80a Hip, clinical spine, forearm and proximal humerusb Significantly higher than lumbar spine (\u2009=\u20090.049)\nJapanese reference values\nThe reference mean in women aged 20\u201329\u00a0years at the femoral neck was 0.858\u00a0g\/cm2 (SD\u2009=\u20090.120\u00a0g\/cm2) using the NHANES III data. When young normal values were computed from the Japanese population the mean BMD was 0.786\u00a0g\/cm2 (SD\u2009=\u20090.107\u00a0g\/cm2). Thus the threshold for osteoporosis using the NHANES III data was 0.558\u00a0g\/cm2 and that derived from the Japanese data was 0.519\u00a0g\/cm2. The thresholds for osteopenia (WHO definition) were 0.738\u00a0g\/cm2 and 0.679\u00a0g\/cm2, respectively. Thus there were systematic differences in the T-score derived from the two data sets. A comparison of fracture probabilities computed from the z-scores using the two approaches is shown in Fig.\u00a04 for different combinations of risk factors. The differences in probabilities were relatively modest, but as expected, the use of Japanese reference values overestimated fracture probabilities.\nFig.\u00a04Correlation between 10-year fracture probabilities (%) in women without clinical risk factors computed from normative data using NHANES III reference values and Japanese-derived reference values for femoral neck BMD. BMI is set at 23.4\u00a0kg\/m2\nDiscussion\nThis paper describes the development of the WHO fracture risk assessment tool calibrated to the epidemiology of Japan. The incidence of hip fracture, distal radius fracture and proximal humeral fracture in Japan is lower than that in North America or Northern Europe [1, 10]. However, the prevalence and incidence of spinal fracture are higher in Japan [11, 18]. A minority of all cases of morphological spinal fracture are assumed to be clinical spine fractures in the Japanese FRAX\u2122 models. The multiplier is age and sex specific. For men the multiplier goes from approximately 33% at age 50 to 48% at age 85. For women the corresponding figures are 19% and 24%. These estimates, derived from the epidemiology of fracture in Sweden [12], have been shown to hold true for Japan [19].\nThe FRAX\u2122 algorithm is suitable for assessment in men and women from the age of 40\u00a0years and calculates the 10-year probability for both hip fracture and a major osteoporosis-related fracture. One of its strengths is that it can capture the independent contribution of several clinical risk factors to fracture risk and can be used with or without information on femoral neck BMD. A more detailed account of the properties of the FRAX\u2122 models is provided elsewhere [13]. In this paper, we focused on its application to decision-making in clinical practice with the estimation of intervention threshold i.e., the fracture probability at which intervention is currently considered to be worthwhile. The approach should be distinguished from intervention thresholds that are based on health economic analysis.\nThe WHO makes no specific recommendation concerning intervention thresholds, since these depend on many local factors [6]. Rather, it is suggested they should be determined by each country, based on the local healthcare situation and cost-effectiveness of the treatment of osteoporosis. Intervention thresholds, based on cost-effectiveness have been formulated in the UK, the USA and in Sweden [20\u201322]. In Japan, diagnostic thresholds are used as intervention thresholds. When the probabilities of osteoporosis-related fracture were determined at these thresholds, they varied with age (see Fig.\u00a03), ranging from approximately 5% at the age of 50\u00a0years to more than 20% at the age of 80\u00a0years. Against this background, a 10-year probability of 10% for osteoporosis-related fracture may be an acceptable intervention threshold for Japan, though an optimization should take account of health economic consequences for individuals and for the health care budget.\nThe FRAX\u2122 tools are designed to be extensively used in the world as a means of identifying individuals with elevated risk for fracture and aid in the determination of the threshold for therapeutic intervention, but there will be hurdles to be faced in the ease of its acceptance. Such hurdles are likely to differ from country to country. In Japan, the choice of clinical risk factors is not at issue since the risk factors adopted in the FRAX\u2122 algorithms included the data from the Hiroshima cohort, Japan [11, 23, 24], and the validation included the Japanese Miyama cohort [13]. More problematic is the inclusion of femoral neck BMD, since the lumbar spine measurement is the most widely used in Japan. Asian physiques are smaller than those of Caucasians, and the geometric characteristics of the femoral neck in Japanese differ from those in American women, in that the femoral neck length is shorter than in the Japanese [25, 26]. Because of uncertainty regarding measurement of femoral neck BMD, measurement of lumbar BMD is widely used in Japan. The present study indicates that concerns over the use of femoral neck BMD are unfounded.\nFemoral neck BMD was superior in its ability to predict hip fractures compared with spine BMD, and BMD at the spine and femoral neck had similar predictive value for fractures other than hip fracture. Fujiwara et al. [11] have also shown that the ability to predict the risk for morphological spinal fracture was similar between femoral neck BMD and lumbar spine BMD. These data on gradients of fracture risk, derived in Japan, did not differ from those reported in Western countries. Indeed, the meta-analysis used to inform the FRAX\u2122 tool showed no evidence for heterogeneity in gradients of risk between cohorts [16]. Thus the evidence suggests that fracture risk assessment is not disadvantaged by the use of femoral neck BMD. Indeed, the converse may be true.\nA further hurdle, unique to Japan, is that diagnostic thresholds differ from the WHO description of osteoporosis which defines osteoporosis on the basis of a fixed T-score threshold (\u2264 \u22122.5 SD) using an international reference standard for young (aged 20\u201329\u00a0years) Caucasian women [15]. In Japan, diagnostic thresholds are also derived by reference to a young population, but differ from the WHO in that a local (i.e., Japanese) standard is used and that the criteria differ in patients with or without previous fracture. In view of the widespread use of data derived from the Japanese population, the question arises whether T-scores or Z-scores derived from Japanese databases could be used in the FRAX\u2122 tool, rather than those derived from the international reference base. In the present study, mean BMD at the femoral neck was lower in Japanese women than in the NHANES III sample from the USA, as previously shown [27]. The difference was not, however, large (approximately a half a SD). There were also differences in the SD which was smaller in Japanese women than in the NHANES III sample (0.107 and 0.120\u00a0g\/cm2, respectively). Not surprisingly, the DXA-based T-score obtained from Japanese and USA populations differed as did the computed probabilities. Although the differences were small at low T-scores, when applying the FRAX\u2122 model to Japan, it is preferable to program the system so that the Japanese T- and Z-scores are converted into the appropriate T- and Z-scores based on NHANES III.\nIn conclusion, a FRAX\u2122 tool has been developed to compute fracture probabilities calibrated to the epidemiology of Japan. The tool has been used to determine possible thresholds for therapeutic intervention, based on equivalence of risk with current guidelines. The approach will need to be supported by appropriate health economic analyses. The present study indicates that the femoral neck BMD is suitable for prediction of the risk for fracture among Japanese people. However, when applying the FRAX\u2122 model to Japan, T-scores and Z-scores should be converted into those derived from the international reference.","keyphrases":["japan","fracture","fracture risk assessment tool","fracture probability","intervention thresholds","bone mineral density"],"prmu":["P","P","P","P","P","P"]}
{"id":"Matern_Child_Health_J-2-2-1592243","title":"Do We Practice What We Preach? A Review of Actual Clinical Practice with Regards to Preconception Care Guidelines\n","text":"Objectives: To review what past studies have found with regard to existing clinical practices and approaches to providing preconception care. Methods: A literature review between 1966 and September 2005 was performed using Medline. Key words included preconception care, preconception counseling, preconception surveys, practice patterns, pregnancy outcomes, prepregnancy planning, and prepregnancy surveys. Results: There are no current national recommendations that fully address preconception care; as a result, there is wide variability in what is provided clinically under the rubric of preconception care. Conclusions: In 2005, the Centers for Disease Control and Prevention sponsored a national summit regarding preconception care and efforts are underway to develop a uniform set of national recommendations and guidelines for preconception care. Understanding how preconception care is presently incorporated and manifested in current medical practices should help in the development of these national guidelines. Knowing where, how, and why some specific preconception recommendations have been successfully adopted and translated into clinical practice, as well as barriers to implementation of other recommendations or guidelines, is vitally important in developing an overarching set of national guidelines. Ultimately, the success of these recommendations rests on their ability to influence and shape women's health policy.\n\u00a0\nThere is a strong body of evidence to demonstrate that preconception care can modify behavioral, medical, and other health risk factors known to impact pregnancy outcomes. For example, preconception care efforts and interventions have been shown to improve folate status among women planning to conceive and to reduce the risk of fetal alcohol syndrome [1, 2]. Controlling known medical problems prior to pregnancy not only helps to optimize the mother's health, it may also improve neonatal and pediatric outcomes [3]. Preconception care efforts and interventions have been shown to lower the risk for both major and minor birth defects among the children of women with pre-existing diabetes [4]. Women with epilepsy and their offspring benefit from preconception care counseling that manages medications, optimizes seizure control and prescribes folic acid for neural tube defect (NTD) prevention [5, 6].\nWhile there is a burgeoning knowledge base on how to improve pregnancy outcomes, there are few studies examining the effects of this knowledge base on actual practice patterns. It is not well known to what extent health care providers are translating preconception care knowledge into practice for all women capable of becoming pregnant (whether planning pregnancy or not).\nMaterial and methods\nA literature review between 1966 and September 2005 was performed using Medline to examine the impact of preconception care trials and recommendations on the clinical practice patterns of health care providers. Key words included preconception care, preconception counseling, preconception surveys, practice patterns, pregnancy outcomes, prepregnancy planning, and prepregnancy surveys. Relevant review articles regarding preconception care were also included. All studies had to be done in the United States. Studies that examined existing clinical practices and approaches directly to providing preconception care were eligible for inclusion. Studies that used indirect methods of measurement, e.g., interviews with women as a means of assessing preconception care practices of providers, were not included. A total of 11 studies were located and included analyses of practice patterns of various health care providers and specialties (see Appendix).\nResults\nStudies researching health care provider awareness, knowledge and practices regarding preconception care in the United States are infrequently performed, so it is difficult to fully assess health care provider approaches and practices related to preconception care. A small pilot study involving family practice nurse practitioners was done in 1987 to determine what preconception care issues were discussed with women of childbearing age during a well woman visit. Audiotapes of the visits were compared to an investigator-developed model for preconception counseling. Based on the results, the authors concluded that current obstetrical care had not expanded to include preconception counseling [7].\nA study among family physicians conducted in 1991 using the Comprehensive Prevention Knowledge and Applications Survey Instrument found that only 37% of providers reported counseling women of childbearing age about preconception behaviors >75% of the time [8]. When asked how often they felt ready to counsel women on preconceptional factors, about 70% of providers said they were prepared at least three-fourths of the time. However, perceived preparedness did not equate to counseling success. Among providers who did counsel, only about one-third reported being successful at helping their patients change preconceptional behaviors at least 75% of the time.\nA 1991 study conducted among family practice and internal medicine residents in an inner-city public hospital found that both groups of residents reported low levels of knowledge and management decision skills regarding preconception care when compared with standard recommendations, despite their favorable attitudes towards preconception care [9]. In this survey, close to 50% of the residents did not mention family planning during counseling sessions with women of reproductive age and 74% would not raise the issue of congenital defects in their diabetic patients seeking to become pregnant. While both family practice and internal medicine residents had favorable attitudes towards preconception care, the family practice residents scored higher in positive attitude about preconception care. There was no difference between the groups in terms of management skills, and there was no sustained difference between groups in the area of knowledge. Previous rotation through a high-risk perinatal clinic increased scores in areas of management and knowledge compared to family practice or internal medicine residents who had not rotated through such a clinic, but these differences were not significant [9].\nIn an effort to assess the practices of genetic counseling and screening for consanguineous couples, their pregnancies, and their children, 1,582 surveys were mailed to board certified genetic counselors and medical geneticists in the United States in 1999 [10]. While the response rate was very poor (only 20%) there was wide variability in suggested screening practices for consanguineous couples before conception, during pregnancy, after birth, and for children placed for adoption. Respondents seemed to generally agree to do screening based on ethnicity but there was no consistency regarding which genetic disorders to include in these screening efforts.\nA mailed survey of obstetrician-gynecologists in 2000 focused on issues around folate for the prevention of NTDs [11]. Almost 97% of participants knew that when taken early enough, folic acid reduced the incidence of NTDs. While two-thirds of respondents said they routinely screened their pregnant patients for folate intake, only 53% screened nonpregnant women of childbearing age. The authors of the study concluded that most obstetrician\/gynecologists are aware of the link between folate intake and NTDs although the data showed that physicians who routinely screened for folate intake correctly answered more survey questions than those who did not.\nAs part of a larger effort to improve the documentation and delivery of preconception care, Bernstein and colleagues evaluated the knowledge base and awareness of preconception care for women in their childbearing years seeking care in an inner city outpatient gynecology clinic in 2000 [12]. Providers in the clinic included physicians and nurse practitioners. A pre-intervention chart review found that many of the providers were not addressing family planning services, domestic violence, nutrition and medical risk factors, medication use, appropriate counseling and use of referral services during gynecologic visits. The authors concluded that the providers did not take the opportunity to discuss preconception care during routine gynecologic visits.\nA series of studies looking at provider knowledge and practice regarding preconception and prenatal screening for cystic fibrosis (CF) was conducted during 2001\u20132004 using the Collaborative Ambulatory Research Network (CARN) [13]. Results showed that almost one-half of the responders did not ask nonpregnant women of childbearing age about their family history of CF, nor did they provide information on CF screening. Close to 90% of respondents did offer CF screening or inquire about a family history of CF among pregnant women. Of those Ob\/Gyns who did selectively screen for CF among their pregnant patients, about 25% utilized all of the selection criteria noted in the American College of Obstetricians and Gynecologists (ACOG) recommendations.\nIn 2002 and 2003 the March of Dimes examined folic acid knowledge and practice patterns of health care providers in obstetric\/gynecology and family\/general practice settings [14]. Survey results found that, while knowledge about the need for supplementation and timing of folic acid was high, increased knowledge about unintended pregnancy rates and correct doses of folic acid for prevention of occurrence and recurrence of NTDs is needed. A little more than half of all providers knew the correct dose of folic acid (400\u00a0mcg daily) for the prevention of NTDs. This percentage dropped dramatically when providers were asked the correct dose of folic acid for a woman with a history of a previous NTD-affected pregnancy. Almost two-thirds of providers did not know the folic acid dose for recurrence prevention (4\u00a0mg daily).\nIn the surveys, the vast majority of providers reported always recommending folic acid supplementation for women who expressed interest in becoming pregnant, approximately 60% of respondents who provide prenatal care reported seeing less than one fifth of their prenatal patients for a preconception care visit [14]. At annual gynecologic or well-woman examinations as well as other types of patient visits, less than 60% of respondents always or usually addressed supplementation. Lack of knowledge (39%) and lack of time (30%) during a busy exam schedule were the most cited reasons providers gave for not always recommending folic acid.\nIn 2004 a survey was sent to Florida health care providers to establish their baseline knowledge and practice behavior regarding folic acid and NTDs [15]. At baseline, 97% of providers reported awareness of the protective nature of folic acid during preconception and early in the first trimester; however, less than half of providers reported that they discuss folic acid consumption with all women of child-bearing age. After a statewide educational program, the same providers were surveyed again to see if there had been any change in knowledge base or practice patterns. The survey done after the educational outreach efforts did demonstrate an increase in awareness and an increase in the percentage of providers who recommended peri-conceptional use of folic acid to prevent NTDs. The authors note, however, that despite these modest gains, the need for continued education and modification of provider practice patterns remains.\nA recent survey by ACOG was done to assess the opinions and practices of obstetrician-gynecologists regarding preconception care, and their perception of patients\u2019 receptiveness to preconception services [16]. The vast majority of physicians (97.3%) stated that they provide preconception care for their patients, although obstetrician-gynecologists were more likely to do so than providers who only practiced gynecology. The majority of physicians (87%) agreed with the definition of preconception care as \u201cSpecialized pre-pregnancy care that focuses on issues not typically addressed during a routine exam which are specific to ensuring an optimal pregnancy outcome.\u201d Most of the respondents indicated that women are more likely to present for preconception care to ensure a healthy pregnancy than because of an elevated risk for a birth defect or developmental disorder. The majority of physicians agreed that preconception care is an important issue and that it has a positive effect on pregnancy outcomes, though only 21% agreed that it was a high priority in their workload. Half of the physicians said there was not enough time to provide preconception care to all women with reproductive potential, nor were there reimbursement incentives to do so.\nDiscussion\nPreconception care- care that is initiated before pregnancy- is advocated to help women reduce their risk for adverse pregnancy outcomes and make informed decisions regarding their readiness for and timing of pregnancy [17]. Appropriate preconception care improves pregnancy outcomes and has several components. These include, but are not limited to: the systematic identification of preconceptional risks through an assessment of the woman's reproductive, family, and medical history; the family and medical history of the father; the woman's nutritional status, social concerns, and any drug or substance exposures she or the father may have (had); discussions regarding possible effects of any existing medical problems and potential interventions; screening for infectious diseases with treatment and immunization where indicated; discussions about environmental exposures, both occupational and household; a review of the circumstances of the woman's life and behavioral patterns; counseling and discussion about birth spacing including real and perceived barriers to achieving her desires; and inquiry and education regarding contraceptive use.\nTraditionally, preconception care endeavors have focused on women who have a chronic medical condition, a history of a poor outcome in a previous pregnancy, or who are planning to become pregnant in the near future. For women with chronic medical conditions, the effect(s) of their illness(es) range from minimal to limited activity to hospitalization. Drawing on information for the 1996 National Health Interview Survey (NHIS), 9.9% of women ages 18\u201344 years had some limitations placed on their activity levels due to chronic disease [18]. There is clear evidence that the initiation of preconception counseling and care for women with some chronic health conditions, e.g., diabetes, will positively impact pregnancy outcomes [19]. For women with a history of a poor pregnancy outcome in the past, pre- or interconceptional intervention strategies have been demonstrated to be effective [20]. Limiting preconception care endeavors to women actively planning pregnancy, however, reaches a limited audience as almost 50% of pregnancies in the U.S. are unintended [21]. Comprehensive preconception care, however, encompasses much more than just these risk categories and is inclusive of all women with the potential to become pregnant.\nWomen of childbearing age visit their physician an average of about three times per year; these visits represent opportunities to deliver preconception care and messages related to preconception care [22]. In reviewing the literature related to the clinical practice of preconception care, it is clear that most of these opportunities are either missed or foregone. In an article focusing on preconceptional health promotion, Moos proposes 5 categorical reasons for why providers are inconsistent in applying proven prevention strategies for poor pregnancy outcomes: lack of knowledge regarding the incidence of unintended pregnancy, inadequate provider education, lack of confidence that preconceptional health counseling is valuable, a belief that women will \u2018know\u2019 to seek the care appropriate to their needs, and concerns over lack of reimbursement coverage for preconceptional visits [23].\nChanging behavior patterns is no small feat, but understanding the \u2018hows\u2019 and \u2018whys\u2019 of change(s) in provider practice patterns is becoming better understood. For example, in diffusion theory, diffusion is the process through which an innovation is adopted for use or application by a community [24]. One proposed method for facilitating diffusion of innovation utilizes an expert opinion leader process whereby a designated (or identified) local physician leader communicates information about new innovation(s) or knowledge to colleagues and then follows up with them about their personal experiences with the information [25]. This approach has demonstrated effectiveness for eliciting changes in practice patterns in certain situations and settings, but it is not uniformly effective in all instances [26\u201329].\nIn his assessment of how research findings get translated into \u201cbest practices\u201d, Green postulates that there are three areas representing barriers to the adoption of best practice: 1) accessibility gap, i.e., practitioners do not have the same resources available as the researchers; 2) credibility gap, e.g., a comparison of differences in practice settings or populations that might \u2018explain\u2019 why the research is not applicable to the general practitioner; 3) expectations gap, e.g., the practitioner views the research findings as unnecessary goals to set for their own practices [30]. These barriers are not insurmountable, and there is evidence that different approaches to changing provider behavior patterns are effective [26, 27]. In an article by Cullum, several model programs are reviewed that have demonstrated changes in the practices of health care providers related to preconception care [31].\nStudies evaluating effective preconception care interventions and strategies are numerous, and continue to grow. There is also an increasing amount of information about how patients assimilate and act upon recommendations regarding preconception care. There is little data, however, that analyzes the impact and integration of preconception care innovations on daily clinical practice(s). One limitation of this literature review is that articles with secondary goals of evaluating actual clinical practices may have been overlooked. Efforts were made to include all relevant search terms but it is possible that articles written before 1985 may have been inadvertently excluded if different keywords were used at that time. Nonetheless, there is a large need for further research into the contrast between the science of preconception care and the reality of actual clinical practice.\nConclusion\nPreconception care seeks to promote the health of women of childbearing age prior to conception and to improve pregnancy-related outcome. Various authors propose the integration of preconception care into routine wellness care for all women of reproductive potential [23, 32\u201334]. The question is how do we raise the level of importance and prioritization of preconception care during individual encounters as well as on a national health policy level? Proving the efficacy of a practice does not guarantee an actual change in practice and simply prompting providers to incorporate preconception care will not suffice. Creating the expectation that preconception care is an ongoing process during a woman's reproductive life span will require a myriad of efforts concurrently directed at providers, patients, payors, and policy makers.\nIn 2005, the Centers for Disease Control and Prevention sponsored a national summit regarding preconception care with a broad cross-section of stakeholders. Efforts are currently underway to develop a uniform set of national recommendations and guidelines for preconception care. Understanding how preconception care is presently incorporated and manifested in current medical practices should help in the development of these national guidelines. Knowing where, how, and why some specific preconception recommendations have been successfully adopted and translated into clinical practice, as well as barriers to implementation of other recommendations or guidelines, is vitally important in developing an overarching set of national guidelines. Ultimately, the success of these recommendations rests on their ability to influence and shape women's health policy.","keyphrases":["preconception care","preconception counseling","preconception surveys","practice patterns","pregnancy outcomes","prepregnancy planning","prepregnancy surveys"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Eur_Radiol-3-1-2039780","title":"MRI of the kidney\u2014state of the art\n","text":"Ultrasound and computed tomography (CT) are modalities of first choice in renal imaging. Until now, magnetic resonance imaging (MRI) has mainly been used as a problem-solving technique. MRI has the advantage of superior soft-tissue contrast, which provides a powerful tool in the detection and characterization of renal lesions. The MRI features of common and less common renal lesions are discussed as well as the evaluation of the spread of malignant lesions and preoperative assessment. MR urography technique and applications are discussed as well as the role of MRI in the evaluation of potential kidney donors. Furthermore the advances in functional MRI of the kidney are highlighted.\nIntroduction\nThe role of computed tomography (CT) and magnetic resonance imaging (MRI) in the evaluation of renal abnormalities is ever increasing. Although multidetector helical CT has taken the largest leap, MRI can be used in case of compromised renal function, severe contrast allergy, or in case radiation exposure is a problem, such as in children and pregnant women. Furthermore, MRI can be used as a problem-solving modality when the CT findings are nondiagnostic. Attempts are being made to use MRI for imaging of renal function, including perfusion [1, 2], glomerular filtration rate [1, 3, 4] and intrarenal oxygen measurement [1, 5].\nMRI technique\nIn MRI of the kidneys, fast imaging techniques are essential because of respiratory motion of the kidneys [6]. When possible the scan should be performed within one breath-hold. The patient should get clear instructions on breath-hold technique. If the patient has difficulty with breath-holding, a short period of hyperventilation before breath-holding may be helpful. The scan should be performed during expiration because the kidney position is more constant in expiration than in inspiration. If the sequence is too long to perform in one breath-hold, respiratory triggering can be used [6]. Another technique of respiratory motion control is respiratory gating by use of a navigator pulse. In this technique the movement of the diaphragm is monitored by a very fast 1D MRI sequence. If breath-holding is not possible, signal averaging can be used, but the quality of the images will be limited. The use of a phased array body coil is preferable because of the improved signal-to-noise ratio. To prevent aliasing in coronal imaging, the patient\u2019s arms should be raised above the head, or the arms may be supported by cushions, anterior to the coronal plane through the kidneys.\nThe imaging protocol for evaluation of the kidney at our institution on a 1.5-Tesla (T) MRI system consists of the following sequences: \nCoronal T2-weighted half Fourier single-shot turbo spin echo sequence (HASTE) (TR infinite, TE 120\u00a0ms, flip angle 90\u00b0, breath-hold), serving as a localizer, but also supplying valuable T2-weighted information. The limitation of this sequence is a relatively low signal-to-noise ratio.Axial T2-weighted turbo spin echo sequence with fat suppression (TR 2,000\u00a0ms, TE 100\u00a0ms, flip angle 90\u00b0, respiratory triggering). This sequence provides for more detailed T2-weighted information. The T2-weighted sequence is especially helpful in characterizing cysts and intraparenchymal abscesses and in evaluating hydronephrosis. Furthermore, the T2-weighted sequence is helpful in detecting solid lesions.Axial T1-weighted gradient echo sequence, in-phase and opposed-phase (TR 180\u00a0ms, TE 2.3\u00a0ms\/4.6\u00a0ms, flip angle 90\u00b0, breath-hold), preferably as a dual-echo sequence. Many solid renal lesions are hypointense compared to the renal parenchyma on T1-weighted images, but lesions with hemorrhage, lesions with macroscopic fat, melanin-containing lesions and cysts with high protein content may show hyperintense signal [6]. Opposed-phase T1-weighted gradient echo sequences can be used to prove the presence of small amounts of fat.Axial T1-weighted gradient echo sequence for dynamic imaging (TR 130\u00a0ms, TE 1.0 ms, flip angle 90\u00b0), using 30 ml intravenous gadolinium contrast, immediately followed by three breath-hold periods with four scan series per breath-hold. In this way pre-contrast and post-contrast images in arterial and nephrographic phase are obtained. Gadolinium-enhanced images are used for lesion detection and characterization.Coronal 3D fast gradient echo with fat suppression, obtained immediately after the dynamic series for delayed contrast-enhanced images (TR 3\u00a0ms, TE 2\u00a0ms, flip angle 15\u00b0). This sequence can be used for renal venous anatomy, for the analysis of (tumor) thrombus and for evaluation of extent of the tumor in the perinephric fat.\nCurrently 1- to 1.5-T systems are generally used for abdominal imaging, but the advent of 3-T MRI systems brings a twofold increase in the signal-to-noise ratio (SNR). The increase in SNR can be spent on higher resolution or on even faster imaging. When combined with parallel imaging techniques such as sensitivity encoding (SENSE), the speed of any sequence can be increased by up to a factor of four or higher. However, although 3-T MRI is promising, only a limited amount of research has been published on 3-T MR imaging for renal lesions, and its value has still to be established [7, 8].\nRenal lesions\nThe main goal in the evaluation of renal lesions is to differentiate surgical lesions from nonsurgical lesions. Most simple cysts are easily recognized and don\u2019t need further analysis. Complicated or multiloculated cysts need more attention in order to differentiate them from cystic carcinomas. In most solid renal lesions, neither CT nor MRI is able to reliably distinguish benign from malignant. Some solid lesions, however, may be identified as benign with high confidence, like angiomyolipomas. In general, if a lesion cannot be characterized as benign or malignant, it should be considered malignant [9].\nMalignant renal lesions\nRenal cell carcinomas\nRenal cell carcinomas account for 3% of all malignancies in adults. Almost 50% are detected incidentally. As many as 85% of suspicious renal lesions are malignant [10]. Features indicating potential malignancy of a renal lesion are size of the lesion, the presence of calcifications, the distribution of the calcifications within the lesion, wall thickness and the presence of septa in case of a cystic lesion (Fig.\u00a01), inhomogeneity of the lesion, extension of the tumor beyond Gerota\u2019s fascia, and last but not least, enhancement after contrast administration. Concerning calcifications in cystic lesions, recent research suggests that the importance of these calcifications as a determinant of malignancy is relatively low [11].\nFig.\u00a01Gradient echo images with intravenous gadolinium at baseline (a), 8\u00a0months later (b) and 15\u00a0months later (c). The complicated cortical cyst (arrow) in the left kidney on image (a) in a patient with Von Hippel Lindau disease progresses into a frank renal cell carcinoma with multiple enhancing internal septations (c). Several simple cysts are visible. Images courtesy of Roy S. Dwarkasing\nDuchene found in 186 renal tumors that all tumors greater than 7\u00a0cm (n\u2009=\u200948) were malignant. About 80% of tumors smaller than 3\u00a0cm were malignant [10]. The differential diagnosis of solid renal lesions smaller than 7\u00a0cm consists of oncocytoma, angiomyolipoma, hemangioma, leiomyoma, and focal xanthogranulomatous pyelonephritis. Of these lesions, only cysts and angiomyolipomas can often be positively identified as benign lesions.\nGenerally, MRI is performed only after a renal lesion has been detected by ultrasound or CT. CT may be followed by MRI if the enhancement at CT imaging is indeterminate (10\u201320 Hounsfield Units) [12] or in case of suspected pseudo-enhancement. At CT examination, simple cysts may show pseudo-enhancement after intravenous contrast administration, which is an increase in attenuation of more than 10 Hounsfield Units, not caused by administered contrast or by partial volume effect but by technical factors [13].\nAssessment of enhancement\nThe main MRI feature indicating potential malignancy of a renal tumor is enhancement after intravenous gadolinium administration, differentiating the lesion from a cyst. However, enhancement at MRI cannot be measured as easily as enhancement at CT. The MR signal is not calibrated, in contrast to density at CT imaging. The MRI signal depends not only on tissue characteristics, but also on the size of the patient, the gain setting of the MR system, the pulse sequence and the coils. The presence of enhancement can be assessed subjectively, by subtraction imaging, and by quantitative assessment. Subjective assessment of enhancement has been shown to be accurate in detecting renal cell carcinomas [14]. However, in a cystic lesion with only a small solid component, subjective assessment may be difficult and subtraction images may be used to better assess the presence of enhancement. It is important to realize that renal cell carcinomas may be hypovascular and therefore may show less enhancement than the surrounding renal parenchyma [15]. Also in these hypovascular lesions and in lesions that are hyperintense on T1-weighted imaging, subjective assessment may be difficult, and subtraction images may be of particular use [16].\nSome investigators have studied quantitative assessment of enhancement by calculating the relative enhancement defined as the signal intensity increase after contrast administration compared to the signal intensity before contrast administration. Ho used relative signal intensity enhancement to differentiate cysts from malignant lesions. Using a threshold of 15% relative signal intensity enhancement after administration of intravenous gadolinium, Ho found in 74 patients with renal lesions a sensitivity of 100% and a specificity of 94% in the detection of renal cell carcinomas [17]. The relative enhancement peak was maximum between 2 and 4\u00a0min after injection of gadolinium contrast. It is worthwhile to note that cysts also showed a mean enhancement change of up to 5%. This pseudo-enhancement may be attributed to motion artifacts and volume averaging [14].\nSubtypes of renal cell carcinoma\nSeveral histological subtypes of renal cell carcinoma are recognized. The most frequent subtype is the conventional or clear cell carcinoma, which comprises 88% of all renal cell carcinomas, followed by papillary carcinoma (10%) and chromophobe carcinoma (2%). Collecting-duct carcinoma is very rare. The first three subtypes do not differ significantly in prognosis and show a 5-year survival rate of 73\u201388% [18]. Several attempts have been made to distinguish the subtypes of renal cell carcinoma by imaging features using CT characteristics [19, 20]. There are only a few studies using MRI to differentiate the subtypes of renal cell carcinoma. Outwater et al. described that clear cell carcinomas may show loss of signal intensity on opposed-phase images compared to in-phase images, due to intracellular lipid [21]. This effect is caused by the presence of fat and water protons in the same voxel, resulting in cancellation of the signal on the opposed-phased sequence. The intracellular lipid contributes to the histological appearance of the clear cell. Because oncocytoma and transitional cell carcinoma do not contain diffuse lipid, the loss of signal on opposed-phase images may allow differentiation between clear cell carcinoma on the one hand and oncocytoma and transitional cell carcinoma on the other hand. However, this distinction is of little clinical use. If the tumor does not show signal loss, it can still be a clear cell carcinoma [21].\nFat-containing renal cell carcinomas\nAlthough rare, it has been claimed that renal cell carcinomas may contain macroscopic fat. Since intralesional fat has long been considered diagnostic for angiomyolipoma, these rare fat-containing renal cell carcinomas may easily be confused with angiomyolipoma. A few cases of fat-containing renal lesions, suggestive of angiomyolipoma, that appeared to be renal cell carcinoma have been reported [22\u201327]. With a few exceptions [25, 27], most of the reported cases also contained intratumoral calcifications [22\u201326], which is very rare in angiomyolipoma. Therefore, the presence of calcification in a lesion with macroscopic fat should be a warning that the lesion may very well be a carcinoma [28]. Because the accuracy of MRI in detecting calcifications is relatively low, it is important always to assess earlier ultrasound or CT examinations of the patient, which will likely have been made in most cases.\nLess than 5% of renal cell carcinomas are cystic [9] (Fig.\u00a02). On CT imaging, cystic renal lesions are classified using the Bosniak classification system [29]. This system was developed for CT, but recently its use has been evaluated in MRI by Israel et al. [12]. The CT and MRI findings in this study were similar in 81% of 69 renal masses. In the remaining 19% of cases, MRI showed more septa, increased wall thickness or increased enhancement compared to CT. This resulted in an upgrade of the Bosniak classification in seven cases (10%), of which two cases were upgraded to category 3 and two cases to category 4. The latter two cases appeared to be malignant at surgery. One of the cases upgraded to category 3 appeared to be benign, and the second one showed progression (patient refused surgery). The authors state that the Bosniak grading system is appropriate for use in MRI, but acknowledge that further research is necessary [12].\nFig.\u00a02T2-weighted turbo spin echo sequence (a) showing a cystic multiloculated lesion in the left kidney (arrow) with high signal intensity content. The pre- and post-gadolinium images (b and c) show enhancement of the septations. Pathologic examination showed a cystic renal cell carcinoma\nStaging\nThere are two staging systems for renal cell carcinoma, both based on the degree of tumor spread beyond the kidney. The Robson staging system [30] (Table\u00a01) is still in use, but it is being replaced by the TNM staging system, developed by the American Joint Committee on Cancer (AJCC) [31] (Table\u00a02). The TNM staging system is similar to the Robson system, but provides a more detailed description. Staging is usually performed using CT. Hallscheidt compared the performance of CT and MRI in the TNM system and found a similar accuracy in the staging of renal cell carcinoma [32]. \nTable\u00a01Robson staging system for renal cell carcinoma [30]StageTumor extentITumor confined to the kidneyIITumor extension through the capsule of the kidney in the perirenal fat including the adrenal gland, no involvement of Gerota\u2019s fasciaIIIaTumor extension into the renal vein or inferior vena cavaIIIbInvolvement of regional lymph node(s)IIIcInvolvement of regional lymph node(s) and extension into the renal vein or inferior vena cavaIVaTumor extension beyond Gerota\u2019s fascia into adjacent organsIVbDistant metastasisTable\u00a02AJCC TNM staging system for renal cell carcinoma (2002, sixth edition) [31]StageDescriptionTxNo information on primary tumor availableT0No evidence of primary tumorT1aTumor size 4\u00a0cm or less, limited to the kidneyT1bTumor size more than 4\u00a0cm but no more than 7\u00a0cm, limited to the kidneyT2Tumor size more than 7\u00a0cm, limited to the kidneyT3aTumor extension into the perinephric fat and\/or renal sinus fat or the adrenal gland, but not beyond Gerota\u2019s fasciaT3bTumor grossly extends into the renal vein or its segmental (muscle-containing) branches, or inferior vena cava below the diaphragmT3cTumor grossly extends into the inferior vena cava above the diaphragm or invasion of the IVC wallT4Tumor extension beyond Gerota\u2019s fasciaNxNo information on regional lymph nodes availableN0No regional lymph node metastasisN1Metastasis in a single regional lymph nodeN2Metastasis in more than one regional lymph nodeMxNo information on distant metastases availableM0No distant metastasesM1Distant metastases\nDetermination of the extent of the tumor into the renal vein and the inferior vena cava (IVC) (TNM stage T3b and T3c) is important for the surgical approach. Involvement of the IVC is reported to occur in 4\u201310% of renal cell carcinoma patients [33]. If the tumor extends into the large veins, the upper level of the tumor thrombus dictates the surgical approach. If the extension is limited to the renal vein, it is necessary to know the distance to the IVC, in order not to dissect the renal vein through the intraluminal tumor. Extension into the IVC requires cavotomy with clamping of the IVC and the contralateral renal vein (Fig.\u00a03). If a tumor thrombus extends above the hepatic veins, the liver should be mobilized to control the IVC above the hepatic veins. Extension into the right atrium generally requires cardiopulmonary bypass during surgery with hypothermic cardiac arrest [33].\nFig.\u00a03T1-weighted gradient echo sequence after intravenous contrast of a large renal cell carcinoma in the upper pole of the left kidney with tumor thrombus extending into the IVC up to the level of the liver\nFor differentiation between bland thrombus and tumor thrombus, the use of gadolinium contrast is indicated, since the enhancement of the thrombus indicates a tumor, whereas the lack of enhancement indicates a clot [34]. Before the introduction of multidetector CT scanners, MRI was considered to be superior to CT in the assessment of intravenous tumor extension, especially at the level of the intrahepatic IVC. However, multislice helical CT scanners are nowadays able to generate multiplanar reconstructions in any direction at high resolution. Recent studies comparing multidetector CT (MDCT) and MRI showed no difference in accuracy of MDCT and MRI in assessing the extension of thrombus in the IVC [35, 36].\nInvasion of the IVC wall (TNM stage T3c) is important to detect because in that case the surgeon has to partially resect and reconstruct the IVC. The accuracy of MRI in IVC wall invasion has not yet been well documented. One study reported a sensitivity of 100% and a specificity of 89% in the detection of IVC wall involvement, however, this study consisted of only 12 patients and no inclusion criteria were reported [33]. In this study it was noted that altered signal in the vessel wall and wall enhancement were nonspecific. The most reliable sign of IVC wall invasion was tumor signal both inside and outside the vessel wall [33].\nThe detection of lymph node metastasis by CT mainly relies on the size of the lymph nodes. In recent years, several studies have been published on the use of ultra-small superparamagnetic iron oxide particles (USPIO) as a negative contrast agent for the detection of small lymph node metastasis. The USPIO particles consist of an iron-oxide core covered with a low-molecular-weight dextran coating. After intravenous injection, the USPIO particles accumulate in healthy lymph nodes. The USPIOs are ingested by macrophages through phagocytosis and cause a decrease in signal intensity on T2- and T2*-weighted images. Gradient echo sequences are the most sensitive for these susceptibility effects. Lymph node metastases displace the macrophages in the lymph node and therefore do not show the loss in signal intensity seen in normal lymph nodes. To our knowledge no studies have been published on the use of USPIOs in renal cell carcinoma. However, a recent meta-analysis by Will et al. [37] describes the pooled results of several studies on USPIOs for several types of metastasis. In these studies, MRI with and without ferumoxtran, a first-generation USPIO, was compared with histology. MRI with ferumoxtran significantly improved the diagnostic precision compared to MRI without ferumoxtran. It was shown to be both sensitive and specific, especially in the detection of abdominal and pelvic metastasis.\nMRI in nephron-sparing surgery\nIn recent years, interest in nephron-sparing surgery has been growing. At first, partial nephrectomy was mainly performed in case of a solitary kidney or diminished renal function, in order to preserve as much function as possible. Due to improving techniques and to increasing application of modern imaging modalities, the number of small, incidentally detected renal tumors is increasing [38]. This development has encouraged surgeons to use nephron-sparing surgery also for patients with normal renal function. The long-term follow-up data suggest that survival after partial nephrectomy of small renal cell carcinomas is comparable to total nephrectomy [39]. The ideal tumor for partial nephrectomy is smaller than 3\u00a0cm, is confined to the parenchyma of the kidney and has a peripheral location [40]. The presence of a pseudocapsule around a renal tumor is a sign of lack of perinephric fat invasion and therefore a favorable sign for partial nephrectomy [38, 41]. A pseudocapsule consists of compressed renal tissue and fibrous tissue. The sensitivity of CT in depicting a pseudocapsule is low (10\u201326%), whereas MRI shows a moderate to high sensitivity in depicting the pseudocapsule (54\u201393%) [41, 42]. On MRI, a pseudocapsule presents as a hypointense rim around the tumor on both T1-weighted and T2-weighted images, but can be best seen on the T2-weighted images and sometimes on gadolinium-enhanced GRE images [40].\nTransitional cell carcinoma\nTransitional cell carcinoma of the kidney is usually evaluated by intravenous urography, CT and endoscopy. However, MRI may play a role if CT and endoscopy are not feasible. CT may not be possible in case of poor renal function, and in case of ureteral obstruction, contrast excretion may be too limited to allow tumor detection. The ureter may not be accessible for endoscopy because of fibrosis and stricture of the ureter and ureter ostium. This may especially be the case in patients who have been treated for bladder cancer. These patients are at particularly increased risk for upper urinary tract transitional cell carcinoma [43]. Transitional cell carcinoma of the pyelum or ureter will generally show as an irregular mass projecting in the lumen. In the collecting system, transitional cell carcinoma is usually confined to the lumen (Fig.\u00a04), but infiltrative growth into the renal parenchyma occurs and typically does not distort the renal contour. Chahal et al. applied MR urography (MRU) in 23 patients with high clinical suspicion of upper tract transitional carcinoma and hydronephrosis that could not be explained with other imaging modalities. MRU showed five renal pelvic transitional cell carcinomas and eight ureteral transitional cell carcinomas, confirmed by histology. In the remaining patients, no sign of transitional cell carcinoma was observed during 1-year follow-up [43]. Although these results are promising, the number of publications on MRI for transitional cell carcinoma is limited and more research is required to determine the value of MRI for the detection of transitional cell carcinoma.\nFig.\u00a04T1-weighted gradient echo images before (a) and after (b) gadolinium administration. A mass in the renal pelvis (arrow) shows moderate enhancement after gadolinium administration. A transitional cell carcinoma was suspected, which was confirmed after nephrectomy\nBenign renal lesions\nOncocytoma\nOncocytomas are benign, most often asymptomatic renal tumors. They represent 2\u201312% of renal masses. On MRI, oncocytomas show variable low signal intensity on T1-weighted images (Fig.\u00a05) and heterogeneous high signal intensity on T2-weighted images. In 33\u201354% a central scar with low signal intensity on both T1- and T2-weighted images is visible. After contrast enhancement a spoke-wheel-like pattern may be observed [44]. Both a central scar and spoke-wheel pattern, however, may also be seen in renal cell carcinomas [38, 45] and are therefore not specific for oncocytomas. Oncocytomas may show a pseudocapsule, consisting of compressed renal parenchyma and fibrous tissue. One should be aware that a pseudocapsule is not specific for oncocytoma, since renal cell carcinomas can also be surrounded by a pseudocapsule. Because the characteristics of oncocytomas show considerable overlap with the characteristics of renal cell carcinomas, the therapy for a suspected oncocytoma is usually surgical [38].\nFig.\u00a05Post-contrast fat-suppressed T1-weighted gradient echo images, arterial phase (a) and nephrographic phase (b) of a central oncocytoma (arrow). The tumor shows a hypointense central scar in the arterial phase. In the nephrographic phase, the central scar is slightly hyperintense\nAngiomyolipoma\nAngiomyolipomas are benign hamartomatous tumors, consisting of fat, smooth muscle and blood vessels. Angiomyolipomas are the only solid renal tumors that can be positively characterized using MRI [46]. Angiomyolipomas are identified by demonstrating macroscopic fat in the lesion (Fig.\u00a06). The ability to differentiate angiomyolipomas is especially urgent in patients with tuberous sclerosis, since angiomyolipomas develop in about 80% of these patients, and at the same time these patients are at an increased risk of developing renal cell carcinomas. Macroscopic fat in a renal lesion can be detected by CT using density measurement and by MRI using fat-suppression techniques. On the opposed-phase gradient echo images, macroscopic fat is demonstrated by a hypointense rim surrounding the fat (India ink artifact) [46]. It must be noted that the India ink artifact also occurs at the interface between tumors that don\u2019t contain fat and the surrounding perinephric fat, if the tumor extends into the perinephric fat.\nFig.\u00a06T1-weighted gradient echo image (a), post-contrast fat-suppressed T1-weighted gradient echo image (b), and T2-weighted HASTE (half Fourier single-shot turbo spin echo) image of the left kidney (c). The hyperintense parts of the tumor in the lower pole (black arrow) on the T1-weighted image show a drop in signal intensity on the post-contrast fat-suppressed T1-weighted image, proving the presence of fat, while the hypointense parts of the tumor enhance after gadolinium. The fatty portions are hyperintense on the T2-weighted sequence, but not as high as the cyst (white arrow) in the midportion of the kidney. The MRI characteristics of the tumor in the lower pole are consistent with angiomyolipoma\nIf the amount of intralesional fat is small, the differentiation between angiomyolipoma and renal cell carcinoma may be difficult (Fig.\u00a07). Kim et al. showed for CT that homogeneous enhancement and a prolonged enhancement pattern were significantly more prevalent in angiomyolipoma with minimal fat than in renal cell carcinoma [47]. Using both CT findings as a criterion for the differentiation of angiomyolipoma with minimal fat from renal cell carcinoma, they found a positive predictive value of 91% and a negative predictive value of 87%. It is likely that this is also the case in contrast-enhanced MRI, however this has not yet been proved.\nFig.\u00a07Lesion in the upper pole of the right kidney in a patient with tuberous sclerosis (arrow). The lesion shows low signal intensity on T2-weighted turbo spin echo images (a) and intermediate signal intensity on in-phase T1-weighted gradient echo images (b). No signal loss is observed on the out-of-phase T1-weighted gradient sequence (c). The lesion shows moderate enhancement after intravenous gadolinium administration (d). Pathologic examination after resection of the lesion showed an angiomyolipoma. In this unusual case, no macroscopic fat was detected on MRI\nIt has been shown that clear cell carcinomas may show signal loss on opposed-phase images compared to in-phase images, due to intracellular lipid [21]. This loss of signal intensity should be distinguished from the signal loss at the interface of macroscopic fat and surrounding tissue in angomyolipomas on opposed-phase images, especially if the amount of macroscopic fat is small. If in doubt, the in-phase gradient echo images are often helpful because the fatty portion of angiomyolipomas will be hyperintense, whereas renal clear cell carcinomas are generally hypo- or isointense [6, 46]. Unfortunately, clear cell carcinomas are incidentally hyperintense on T1-weighted images [48]. In these cases spectral fat-suppression images should be used to prove the presence of macroscopic fat in the angiomyolipoma.\nAttention should be paid to the possibility that carcinomas sometimes contain hemorrhage, causing high signal intensity on in-phase T1-weighted images. In these cases, opposed-phase images and spectral fat suppression will not show a drop in signal intensity.\nXanthogranulomatous pyelonephritis\nXanthogranulomatous pyelonephritis is a rare chronic pyelonephritis, which may result in severe renal impairment. It is most common in middle-aged women, but it may also occur in children. Often Proteus or E. coli species are involved and the pyelonephritis is often accompanied by calculi, most typically staghorn calculi. It may be accompanied by calyx obstruction and parenchymal abscesses. In xanthogranulomatous pyelonephritis, the affected renal parenchyma is replaced by lipid-laden macrophages, resulting in the typical yellow-gray appearance of the lesion at macroscopy. It may involve the whole kidney, or it may be focal. Especially when it is focal, xanthogranulomatous pyelonephritis may be mistaken for a renal carcinoma. On T1-weighted images, the solid component of the lesion may be isointense or hyperintense, which can be attributed to the fatty component. On T2-weighted images, the signal intensity of the solid component is isointense to slightly hypointense. The parenchymal cavities filled with fluid and pus show high signal intensity on T2-weighted images and low signal intensity in T1-weighted images, varying according to the protein concentration in the cavity [49, 50]. In xanthogranulomatous pyelonephritis, the perirenal fascia may be thickened and show enhancement after gadolinium administration [49]. The absence of hyperintense signal on T2-weighted images of the solid components may be helpful in the differentiation between xanthogranulomatous pyelonephritis and renal tumor [50].\nMR urography\nIn MR urography, the pyelocalyceal system and the ureters are visualized using heavily T2-weighted images or T1-weighted images with gadolinium contrast. On the heavily T2-weighted images, the urine in the pyelocalyceal system and ureters is hyperintense because of its long T2 relaxation time, whereas the surrounding tissue is hypointense. The HASTE or single-shot fast spin echo (SSFSE) is very suitable for this purpose; they are very fast with sufficient in-plane resolution [51]. The thin sections can be used for detailed evaluation; a maximum intensity projection is useful for overview of the urinary tract. For T2-weighted MR urography, it is essential that the urinary tract is sufficiently filled with urine. Therefore it is often necessary to use a diuretic if the urinary tract is not dilated [51]. Hagspiel et al. evaluated whether MRI of the urinary system of potential renal donors was feasible without diuretic stimulation or compression and found sufficient visualization of the urinary collecting system in only 14% of the rapid acquisition with relaxation enhancement (RARE) urograms and in 26% of the gadolinium-enhanced 3D fast low-angle shot (FLASH) urograms [52].\nIn contrast-enhanced MR urography, intravenous gadolinium can be combined with a T1-weighted 3D gradient echo sequence. In case the patient is unable to hold his breath, a fast 3D GRE EPI sequence can be used that offers the additional advantage of reduced ghost artifacts caused by ureteral peristalsis [53]. However, the conventional GRE images provide better high resolution images compared to the EPI images [53]. In contrast-enhanced MR urography, the images are acquired in the excretory phase, typically 5\u20138\u00a0min after intravenous gadolinium injection [51]. Additional use of a diuretic is advisable to increase excretion and to dilute the excreted contrast: if the excreted gadolinium is too concentrated, the T2* effect may cause signal loss [51]. The accuracy of MR urography in assessing renal obstruction is similar to CT urography [54, 55]. MRI has the advantage that it is better able to detect perirenal edema as a secondary sign of obstruction [54]. In a study by Sudah et al., gadolinium-enhanced MR urography showed renal calculi with considerably higher sensitivity than T2-weighted MR urography [55]. On MR urography, a calculus appears as a signal void, which is nonspecific: blood clots, gas, sloughed papilla and tumors may also appear as a low signal within the bright signal of urine [51]. If a signal void is not clearly detached from the wall of the pelvis or ureter, additional T1-weighted and contrast-enhanced images are necessary to further characterize the lesion.\nMRI of potential donor kidneys\nDue to the increasing demand for donor kidneys and the relative shortage of cadaver kidneys, the importance of living donors is increasing. Nephrectomy is increasingly performed by laparoscopic surgery to keep the burden for the donor as low as possible. A thorough pre-operative evaluation of the donor kidney is essential to keep the risks as low as possible. Especially for endoscopic nephrectomy, it is important for the surgeon to be informed about the arterial and venous vasculature of the kidney, about the presence of accessory vessels and about abnormal vessel location, like extrahilar branching and retrocaval position of vessels. Moreover, the surgeon needs to be informed about the presence of an abnormal collecting system and the presence of cysts or tumors [56].\nIn the imaging protocol, special attention should be focused on arterial and venous imaging, in addition to the standard parenchymal imaging. For the arterial MR angiography, a 3D fast GRE with intravenous gadolinium after timing bolus can be used (TR 4.7\u00a0ms, TE 1.4\u00a0ms, flip angle 30\u00b0), with coronal thin-section reconstructions. A relatively large flip angle (up to 40\u00b0) can be used to minimize background signal around the high signal of the renal arteries [56]. The venous angiography sequence should follow the arterial angiography immediately. Due to the excretion of gadolinium by the kidneys, the concentration of gadolinium in the renal veins is lower than in the renal arteries, causing lower contrast of the veins compared to the background. A lower flip angle (15\u00b0) can be used to compensate for the lower gadolinium concentration, at the expense of more background signal [56]. The 3D dataset can be used for the reconstruction of thin 2D sections for detailed evaluation, as well as for maximum intensity projection (MIP) reconstructions of the vessels (Fig.\u00a08).\nFig.\u00a08Pre-operative imaging of potential donor kidneys. a Gradient echo image after intravenous contrast, arterial phase, TR 3\u00a0ms, TE 1\u00a0ms, slice thickness 2.2\u00a0mm, flip angle 27\u00b0, coronal. b Gradient echo image after intravenous contrast, nephrographic phase, TR 4\u00a0ms, TE 1\u00a0ms, slice thickness 2.2\u00a0mm, flip angle 15\u00b0, coronal. The lower flip angle in the depiction of the renal veins was chosen to compensate for the lower gadolinium concentration in the renal veins\nAlthough the time\u2013of-flight technique can be used to evaluate renal arteries, it is not recommended for the detection of accessory renal arteries of small caliber [57]. It may be used to clarify intraluminal filling defects potentially caused by flow artifacts on gadolinium-enhanced MR angiography [6]. Phase contrast imaging can be used for detection and grading of renal artery stenosis [58, 59] but has limitations in evaluating potential kidney donors. The problem of motion artifacts in phase contrast imaging caused by the long acquisition time can be overcome by using interleaved gradient echo-planar technique, shortening the acquisition time from minutes to about 30\u00a0s [59]. However, it is difficult to select the proper velocity encoding gradient [6], and tortuous venous anatomy and low flow limit the use of phase contrast MRI in potential kidney donors [56].\nMost studies on the accuracy of MR angiography (MRA) in the evaluation of renal vessels compared to digital subtraction angiography and CT angiography show similar or even better results [60\u201363], although not all studies confirm these findings [64]. In a recent study on 111 MRA examinations for donor nephrectomy, nine accessory arteries were missed, requiring anastomosis in four arteries. Of 14 kidneys with more than one vein, only 4 were identified by MRA, requiring anastomosis in 1 case [65].\nMRI for imaging of renal function\nIn recent years, progress has been made in the use of MRI for the evaluation of renal function. Renal disease often causes impairment of renal function. Measurement of renal function can be used as an indicator of severity of disease and can direct therapy. The most simple tests of renal function are serum creatinine and creatinine clearance. However, these tests do not provide information about the function of each individual kidney. This information can be important in case of a living renal kidney donor, prior to nephrectomy or in case of renal artery stenosis. Renal scintigraphy provides information about the function of each kidney, but this test lacks anatomic detail. CT has the disadvantage of ionizing radiation and the use of potentially nephrotoxic contrast, which generally isn\u2019t a problem in healthy kidneys, but may cause deterioration of renal function in diseased kidneys. MRI has the potential to combine the functional and anatomic information about each kidney individually [66].\nPerfusion\nMeasurement of renal perfusion may be a tool to assess the significance of renal artery stenosis and to assess ischemic nephropathy, and it may be used in renal transplant assessment. Several techniques have been studied to measure renal perfusion [1]. The maximum slope Gd-DTPA technique uses the maximum slope of the Gd-DTPA enhancement curve in relation to the maximum Gd-DTPA concentration in the aorta to calculate renal blood flow. The Gd-DTPA concentration in the aorta can be calculated using pre-contrast and post-contrast relaxation times measured in the aorta, assuming a linear relationship between gadolinium concentration and the inverse of the pre- and post-contrast T1 difference. Calibration of the signal intensity is performed using phantoms with increasing gadolinium concentration [67].\nFurthermore, attempts have been made to assess renal perfusion by arterial spin labeling. To prevent leakage of the contrast medium into the extravascular space, albumin-bound contrast agent is used [68]. However, the different MRI renal perfusion techniques still need to be validated so more research is needed to assess the clinical usefulness.\nGlomerular filtration rate\nThe glomerular filtration rate (GFR) is a parameter that is used to assess renal function. The serum creatinine level is a rough indicator of the glomerular filtration rate, it is easy and cheap to obtain, but it provides no information about each individual kidney. For the measurement of the GFR by MRI, several techniques have been investigated. The first technique used MR spectroscopy to measure the T1 relaxation times of urine and serum samples taken at intervals after intravenous gadolinium administration. Calculation of the glomerular filtration rate was based on the linear relationship of 1\/T1 to the serial dilution measurements of the serum and urine samples [69]. This technique still had the disadvantage of measuring the GFR of both kidneys together.\nAnother MR technique assessing each individual kidney was developed by calculating the extraction fraction (EF) of Gd-DTPA, which is the difference between renal arterial and renal venous Gd-DTPA concentration, normalized to the arterial Gd-DTPA concentration:  The gadolinium concentrations can be calculated after measurement of the T1 relaxation time of arterial and venous blood before and after intravenous gadolinium administration, using the formula  where R is the relaxivity of gadolinium, and T1pre and T1post are the T1 relaxation times before and after intravenous gadolinium. The glomerular filtration rate can then be calculated according to the formula where RBF is renal blood flow, which can be measured by phase contrast flow quantification [3], and Hct is hematocrit [66].\nAn alternative MRI technique to measure glomerular filtration rate also uses contrast-enhanced dynamic MRI and is based on a the time-dependent concentrations of Gd-DTPA in the cortex and the medulla\u2014a two compartment model [1]. The different techniques to measure glomerular filtration rate still need to be validated, and their role in clinical practice needs to be established.\nDiffusion-weighted MRI\nIn diffusion-weighted MR imaging, the image contrast is influenced by the Brownian motion of water molecules. The signal intensity is high if water molecules are restricted in their motion, which can be caused by cell membranes or, in the case of free fluid, by high viscosity. The MR signal intensity is low if water molecules can diffuse freely. Diffusion-weighted imaging has found its place in neuroradiology, especially for the early detection of ischemic brain lesions. Diffusion MRI can also be used for fiber tract mapping in cerebral white matter, by measuring the directional components of the diffusion. Because diffusion-weighted images are inherently T2-weighted, the images are influenced by the T2-shine-through effect. This is the presence of high signal intensity in restricted water, caused by the T2 effect. To cope with this effect, the apparent diffusion coefficient (ADC) may be calculated from two images acquired with different gradient duration and amplitude (b-values) and used for ADC mapping.\nIn renal MR imaging, the role of diffusion MRI is not yet as clear as in neuro-imaging. Fukuda et al. showed that diffusion in the kidney is anisotropic, due to the radial orientation of the tubules in the pyramids and the blood vessels in the renal cortex [70] (Fig.\u00a09). Diffusion-weighted MRI has been applied in patients with solid renal masses as well as in pyelonephritis and renal failure [71, 72]. ADC mapping showed differences between the lesions and normal tissue, but more research is needed to know whether diffusion-weighted MRI can actually help in characterizing different abnormalities. Diffusion-weighted imaging has been applied to differentiate between hydronephrosis and pyonephrosis. Chan et al. found in a limited group of patients that diffusion-weighted imaging showed a hypointense pyelocalyceal system in hydronephrosis and a hyperintense pyelocalyceal system in pyonephrosis [73]. The hyperintensity in pyonephrosis is thought to be due to the high viscosity of the pus, whereas the free-moving molecules in hydronephrosis cause low signal intensity. The results of diffusion-weighted MRI in the kidney are still preliminary, and more research should reveal the value of its clinical application.\nFig.\u00a09Diffusion-weighted tensor image of the right kidney on a 3T system. The renal pyramids show lower signal intensity than the surrounding parenchyma because of the radial orientation of the tubules in the pyramids, restricting the Brownian motion of the water molecules to one direction\nConclusion\nThe role of MRI in renal imaging is still mainly in differentiating benign lesions versus malignant lesions in patients who cannot undergo CT scanning with intravenous iodinated contrast media, or in cases with nondiagnostic CT results. MRI and CT show comparable accuracy in detection and characterization of most renal lesions. MRI can have additional diagnostic value in the evaluation of lesions with minimal amounts of fat or with intracellular fat. Data suggest that MRI has a higher sensitivity in evaluating complicated cysts, however, the clinical implications still have to be studied. There is evidence to suggest that MRI has a higher accuracy than CT in the evaluation of early lymph node spread. MRI is a suitable tool in the preoperative work-up of potential kidney donors. Functional MRI of the kidney has not yet found broad clinical application, but it has great potential. Through the ongoing development of functional MRI techniques, we may expect an increasing role for functional MRI in the management of patients with renal disease.","keyphrases":["magnetic resonance imaging","humans","kidney diseases","kidney neoplasms"],"prmu":["P","U","R","M"]}
{"id":"Matern_Child_Health_J-2-2-1592159","title":"Oral Health in Women During Preconception and Pregnancy: Implications for Birth Outcomes and Infant Oral Health\n","text":"The mouth is an obvious portal of entry to the body, and oral health reflects and influences general health and well being. Maternal oral health has significant implications for birth outcomes and infant oral health. Maternal periodontal disease, that is, a chronic infection of the gingiva and supporting tooth structures, has been associated with preterm birth, development of preeclampsia, and delivery of a small-for-gestational age infant. Maternal oral flora is transmitted to the newborn infant, and increased cariogenic flora in the mother predisposes the infant to the development of caries. It is intriguing to consider preconception, pregnancy, or intrapartum treatment of oral health conditions as a mechanism to improve women's oral and general health, pregnancy outcomes, and their children's dental health. However, given the relationship between oral health and general health, oral health care should be a goal in its own right for all individuals. Regardless of the potential for improved oral health to improve pregnancy outcomes, public policies that support comprehensive dental services for vulnerable women of childbearing age should be expanded so that their own oral and general health is safeguarded and their children's risk of caries is reduced. Oral health promotion should include education of women and their health care providers ways to prevent oral disease from occurring, and referral for dental services when disease is present.\nIntroduction\nA woman's preconception and pregnancy experience with the two most prevalent diseases of the mouth\u2014periodontal disease and dental caries\u2014not only influences her own oral health status but also may increase her risk of other diseases such as atherosclerosis [1\u20134], rheumatoid arthritis [5], and diabetes [6], impact pregnancy outcome [7\u20139], and her offspring's risk of developing early and severe dental caries [10\u201313]. Although largely preventable through evidence-based interventions, both periodontal disease and caries in women of childbearing age are highly prevalent, particularly among low-income women and members of racial and ethnic minority groups. In addition, both periodontal disease and caries are typically asymptomatic for long periods of time with only intermittent painful exacerbations. The combination of high prevalence, insufficient treatment rates, missed preventive opportunities, and intermittent symptoms led the US Surgeon General to publish a report in 2001 on oral health in America characterizing dental and oral disease as a \u201csilent epidemic\u201d [14]. Socioeconomic factors, lack of resources to pay for care, barriers to access to care, and lack of public understanding of the importance of oral health and effective self-care practices all represent underlying reasons cited for observed inadequacies in oral health.\nAn important oral condition affecting many pregnant women is periodontal disease. Periodontal disease is a destructive inflammatory condition of the gingiva and bone that supports teeth. It is most commonly associated with a gram-negative anaerobic infection of these structures. Fluid that bathes the tooth at the gingival margin, known as gingival crevicular fluid, often contains inflammatory mediators and oral pathogens associated with periodontal disease.\nThe mechanisms underlying this destructive process involve both direct tissue damage resulting from plaque bacterial products, and indirect damage through bacterial induction of the host inflammatory and immune responses [15]. Destructive periodontal disease affects up to 15% of the population of childbearing age, with a relatively high proportion of pregnant women demonstrating some degree of periodontal disease [7, 8, 16]. Advancing age, smoking, and diabetes are risk factors for the development of periodontal disease [17]. Whereas periodontal disease is a chronic, local oral infection, systemic inflammation may also occur [15].\nThe second oral disease important to women of childbearing age because of its maternal-child health associations is dental caries. Dental caries is the pathologic process by which teeth \u201cdecay\u201d and develop \u201ccavities.\u201d It occurs when acid is produced at the tooth surface by cariogenic bacteria in the dental plaque that metabolize dietary carbohydrates. Acquisition of these cariogenic bacteria, dietary practices that govern the caries process, use of fluorides that dampen the caries process, and utilization of dental care all link mothers\u2019 and children's experience with tooth decay through biological, behavioral, and social pathways.\nIs maternal oral health linked to pregnancy outcome?\nPreterm birth\nPreterm birth, delivery at less than 37 weeks\u2019 gestation, occurs in approximately 12% of all births [18, 19]. Prematurity is the leading cause of neonatal morbidity and mortality in non-anomalous infants [20]. There are numerous and heterogeneous factors associated with preterm birth, such as low maternal body mass index, maternal smoking, and maternal infections [21]. In 1996, Offenbacher and colleagues [7] first reported a potential association between maternal periodontal disease and delivery of a preterm\/low birthweight infant. In a case-control study of 124 pregnant women, they observed that women who delivered at less than 37 weeks\u2019 gestation or an infant <2500\u00a0g had significantly worse periodontal disease than control women. The adjusted odds ratio for delivery of a preterm, low birth weight infant was \u223c7; these data led the authors to conclude that periodontal disease may represent a previously unrecognized and clinically significant risk factor for delivery of a preterm low birth weight infant [7]. Extrapolation from these data suggested that 18% of the preterm, low birth weight infants born annually might be attributable to periodontal disease, and thus account for a significant proportion of the $5.5 billion annual hospital costs associated with the care of preterm\/low birthweight infants. In a subsequent case-control study, Dasanayake et\u00a0al. studied 55 pairs of women. Logistic regression indicated that mothers with \u2018healthy gingiva\u2019 were at lower risk for low birth weight infants [22]. Women in both of these case-control studies were examined at the end of pregnancy or after delivery, which does not convincingly prove an antecedent exposure and thus causality. Despite this limitation, these early studies led to the hypothesis that periodontopathic bacteria, primarily Gram-negative anaerobes, may serve as a source for endotoxin and lipopolysaccharides, which then increases local inflammatory mediators including PGE2, and cytokines, and that this increases systemic inflammatory mediators that can then lead to preterm birth [23]. Additionally, Jeffcoat et\u00a0al. [9] examined the relationship between maternal periodontal disease and spontaneous preterm birth among 1313 pregnant women, and found that moderate\/severe maternal periodontal disease identified early in pregnancy was associated with an increased risk for spontaneous preterm birth, independent of other traditional risk factors [9].\nDespite these compelling data, it is important to recognize that other studies have failed to demonstrate any association between maternal periodontal disease and preterm birth. In a case control study conducted in London, Davenport et\u00a0al. [24] examined 236 infants born at <37 weeks\u2019 gestation or <2500\u00a0g and compared them to a random sample of 507 control infants born at \u226538 weeks\u2019 gestation and weighing \u22652500\u00a0g. The authors found no evidence for an association between delivery of a preterm, low birth weight infant and periodontal disease and somewhat surprisingly, found that deeper mean tooth pocket depths at delivery was associated with a reduction in the risk of delivery of a preterm, low birth weight infant [24]. The authors surmised that these discrepant findings might be due at least in part to racial differences in study populations. In a follow-up longitudinal study of 3738 women, Moore et\u00a0al. [25] found no association between maternal periodontal disease and preterm birth. However, there was an increase in second trimester fetal loss rates among women with periodontal disease [25].\nIn an effort to better understand the possible mechanism behind the association between periodontal disease and preterm delivery, Offenbacher and colleagues [23] measured gingival crevicular levels of PGE2 and IL-1\u00df in 48 mothers who delivered preterm, low birth weight infants compared to control women and discovered that gingival crevicular fluid levels of PGE2 were significantly higher in case compared to control women. Furthermore, among the primiparous women delivering preterm, low birth weight infants, a significant inverse association was demonstrated between birthweight and gestational age and gingival crevicular PGE2 levels [23].\nIt is not yet clear whether the relationship between periodontal disease and adverse pregnancy outcomes is causal or is a surrogate for another maternal factor. As further evidence to support the concept that maternal oral health is important for normal pregnancy outcome, other investigators have examined the effect of antepartum treatment of periodontal disease on preterm birth risk. Three published studies of antepartum versus delayed (postpartum) treatment of maternal periodontal disease demonstrate promise for this intervention for preterm birth prevention. The effect of periodontal interventions on pregnancy outcome was assessed in a prospective study designed to examine the relationship between periodontal disease and preterm low birthweight infants in a cohort of young, minority, pregnant and postpartum women. Of 164 women for whom birth outcome data were available, 74 were subjected to oral prophylaxis during pregnancy, and 90 received no periodontal treatment. The preterm\/low birthweight rate was lower among women who received periodontal treatment compared to those who did not (13.5% vs. 18.9%) [26]. Lopez et\u00a0al. conducted a randomized clinical trial to assess the impact of periodontal treatment initiated during pregnancy versus delayed until postpartum on preterm low birthweight infant rates. The incidence of preterm\/low birthweight infants in the antepartum treatment group was 1.8% (3\/163) and in the delayed\/postpartum group was 10.1% (19\/188), (odds ratio [OR] 5.5, 95% confidence interval [CI] 1.7\u201318.2, P=0.001). Multivariable logistic regression analysis showed that periodontal disease was the strongest factor related to delivery of a preterm\/low birthweight infant (OR 4.7, 95% CI 1.3\u201317.1) [27]. The data from these two studies suggest that treatment of periodontal disease during pregnancy could reduce preterm\/low birthweight infant rates [26, 27].\nIn a pilot intervention trial designed to assess the feasibility of conducting a trial to determine whether treatment of periodontal disease reduces the risk of spontaneous preterm birth, Jeffcoat et\u00a0al. found that among women at high risk for preterm birth and presence of periodontal disease, scaling and root planning therapy initiated during pregnancy is tolerated by pregnant women and may reduce spontaneous preterm birth [28].\nOther adverse pregnancy outcomes\nPreeclampsia is a hypertensive disorder of pregnancy responsible for significant, maternal and fetal morbidity and mortality. Preeclampsia affects up to 5% of pregnant women [29]. The etiology of preeclampsia remains elusive. The underlying pathology may be related to a generalized intravascular hyperinflammatory state [29]. Some investigators have hypothesized a potential role for maternal periodontal disease as a risk factor for preeclampsia. In a retrospective analysis of data collected as part of the Oral Conditions and Pregnancy Study, Boggess et\u00a0al. reported that women were at higher risk for preeclampsia if they had severe periodontal disease at delivery (adjusted odds ratio 2.4, 95% confidence interval 1.1, 5.3), or if they had periodontal disease progression during pregnancy (adjusted odds ratio 2.1, 95% confidence interval 1.0, 4.4) [30]. In a case-control study, Canakci et\u00a0al. found that pre-eclamptic patients were 3.5 (95% CI=1.1\u201311.9) times more likely to have periodontal disease than normotensive patients [P < 0.01) [31]. In a study of 30 pregnant women, significantly higher periodontal probing depth and clinical attachment level scores were found among preeclamptic women compared with non-preeclamptic women. Gingival crevicular fluid levels of PGE2, TNF-\u03b1, and IL-1\u03b2 levels were all significantly higher in the preeclamptic group [32]. Further study on the maternal and fetal inflammatory responses to chronic oral infection and on placental pathology in women with periodontal disease is needed to determine whether the relationship between periodontal disease and preeclampsia is causal or simply associative. If the relationship between maternal periodontal disease and preeclampsia risk proves causal in nature, then prevention of periodontal disease before pregnancy or treatment of periodontal disease during pregnancy may represent a novel approachs to the prevention of preeclampsia.\nIs maternal oral health linked to children's experience with tooth decay?\nCariogenic bacteria are typically acquired by young children through direct salivary transmission from their mothers [10]. Factors influencing transmission are the levels of these bacteria in maternal salivary reservoirs, frequency and efficiency of transmission, and the child's receptivity to implantation, which is largely diet dependent. Additional factors include timing of transmission, which is affected by the window of infectivity and the age of the child, and the composition and flow of the child's saliva. The earlier the transmission and the more caries-supportive the diet, the earlier and more substantial the transfer will be. For this reason, mothers who have themselves experienced extensive tooth decay and therefore most likely harbor high titers of mutans streptococci in their saliva will more effectively transmit this infection vertically, thereby putting their young children at elevated risk for early childhood caries. Although maternal cariogenic bacteria can be isolated in the pre-dentate infant's mouth [11], these organisms become established in the dental plaque on the tooth surface only after teeth first appear at around six months of age. Because oral flora tends to remain stable over time, a woman's cariogenic flora before and during pregnancy anticipates her flora during the child's first years of life as well as the likelihood of transmitting infection early to her offspring. The lag time between infection and expression of a discernable cavity in a tooth depends upon additional factors, including the frequency of simple carbohydrate exposure in a child's diet, oral hygiene, and exposure to fluorides. The evidence that caries is frequently established as a pathologic process in the mouths of very young children is strong, as 28% of US children, over 4 million toddlers and preschoolers, experience one or more frank cavities by ages 2\u20135 years [33].\nGiven the biological and behavioral pathways that govern intergenerational transmission of caries activity, disease management, and use of dental care, it is not surprising that disparities in dental caries among adults are mimicked among their children. As with adults, children of color and children of low-income families experience substantially more extensive and severe disease and less treatment than their peers without these risk factors [33]. Fortunately, despite the high prevalence of caries in women and children, this disease is readily preventable or manageable though early and regular dental care, exposure to fluoridated water, use of appropriate topical fluorides including those in toothpastes, application of sealants to primary teeth, and adoption of a health-promoting diet like that suggested in the Dietary Guidelines for Americans [34].\nIs preconception preventive oral health care the answer?\nIt is intriguing to consider preconception, pregnancy, or intrapartum treatment of oral health conditions as a mechanism to improve women's oral and general health, pregnancy outcomes, and their children's dental health. Evidence is currently weakest for interventions that seek to reduce the incidence of preterm low birth weight through oral care. The mechanism of periodontal disease-associated adverse pregnancy outcomes is as yet unclear, and althoughit is hypothesized that if the \u2018insult\u2019 occurs early (either at conception or implantation) the risk is greater, no direct evidence to confirms that this is the case. However, given the strong relationship between oral health conditions and periodontal disease and general health and well-being, oral health care should be a goal in its own right for all individuals. If treatment of periodontal disease is going to impact pregnancy outcomes, then it is likely that the therapy will be of greatest benefit before or in very early pregnancy.\nThe science supporting interventions before, during, and after pregnancy to reduce caries transmission is much stronger. Educational and behavioral interventions that reduce caries activity through appropriate use of fluorides, dietary guidelines, chlorhexidine gels and varnishes, and xylitol [35], can reduce a woman's caries activity and salivary cariogenic flora, thereby improving her own oral health and, at the same time, also reducing the risk of transmission to her offspring. In two landmark Swedish studies [12, 13], children of mothers who had their cariogenic oral flora suppressed were less likely to experience cavities, more likely to develop cavities later if they were affected, and had fewer cavities than children of control mothers. Pregnancy is itself often regarded as an opportune time for anticipatory guidance and oral health education, and is a suitable time, particularly during the second trimester, for dental repair.\nAccess to oral health care during pregnancy\nThe CDC's Pregnancy Risk Assessment Monitoring System (PRAMS) reported that only 23\u201343% of pregnant women received dental care during their pregnancies [36, 37]\u2014a rate only half to two-thirds of US women's overall use of dental services (67%) (14). The PRAMS data revealed that overall, pregnant women covered by Medicaid were 24%\u201353% less likely to obtain a dental visit during pregnancy than women who are privately insured. Similarly, women who initiated prenatal care later than the first trimester, who did not intend the pregnancy, and who were poor were also less likely to obtain care [36]. In contrast, the Behavioral Risk Factor Surveillance System (BRFSS) revealed that 70% of pregnant women in the years 1999 and 2002 had received a dental visit in the prior 12 months [38]. One possible explanation for the higher level reported by BRFSS is that it includes three or more months of pre-pregnancy time, during which dental care utilization would be expected to reflect the national norm for women. However, in contrast to recognized disparities in dental care utilization [33], race and ethnicity were not significantly associated with dental care during pregnancy in the BRFSS study. The authors suggest that, \u201cthe prevalence of dental visits \u2026 probably reflects factors such as prevailing attitudes toward dental care, provider availability and practice norms, and salient features of medical and dental care delivery within the state.\u201d Overall, women covered by Medicaid were 24%\u201353% less likely to obtain a dental visit during pregnancy than women who are privately insured. Similarly, women who initiated prenatal care later than the first trimester, who did not intend the pregnancy, and who were poor were also less likely to obtain care.\nAn important additional consideration is that dentists are reportedly reluctant to provide care to pregnant women because of concern about possible risks [39]. Current practice typically limits non-urgent dental treatment of pregnant women to the second trimester, as there is concern about possible teratogenic consequences during the first trimester and about the woman's comfort in the dental chair during the third trimester. A single study relating antepartum dental radiography with full term low birth weight raised concern about the safety of dental care during pregnancy [40], but was criticized for its methodology. Neither professional associations nor government agencies have promulgated any authoritative guidance regarding dental care of pregnant women, although multi-center NIH clinical trials are underway that will determine the impact of dental care for periodontal disease during pregnancy on preterm low birth weight outcomes. Currently, the New York State Department of Health is developing \u201cGuidelines for Prenatal Care, Oral Health, and Child Health Professionals\u201d that promotes routine use of dental care during pregnancy.\nIndependent of pregnancy, the presence and source of dental insurance coverage is an important predictor of dental care utilization with publicly insured adults experiencing higher levels of oral diseases but less access to dental care. Medicaid is particularly significant to dental care of pregnant women as this program covers approximately 1\/3rd of births in the US. However, states vary widely in adult Medicaid dental coverage, and at present only 7 jurisdictions providing comprehensive care to eligible adults. In contrast, low income pregnant women seeking dental services find themselves with no coverage in 8 states, coverage for only relief of pain or infection in 18 states or eligible for a limited range of services in 18 states. Three states (UT, LA, CA) have recently expanded dental benefits specifically to pregnant women in anticipation of reduced rates of unfavorable pregnancy outcomes. Pregnancy may be the only time that some low-income woman can readily obtain dental care as some state Medicaid programs provide adult dental coverage only to pregnant women or enhanced coverage during pregnancy.\nConclusions and future directions\nData are emerging to support a role for maternal periodontal disease as an infectious risk factor for preterm birth and other adverse outcomes of pregnancy. The prevalence of periodontal disease and the possibility of preterm birth prevention by treatment of oral infection make this a novel approach to improve the health and well being of our mothers and their soon-to-be born children. Further studies to better understand the mechanism of periodontal disease-associated preterm birth will enable us to tailor treatment to those women who might benefit the most.\nData on the relationship between maternal and child experience with dental caries is well established. Therefore, regardless of the potential for improved oral health to improve pregnancy outcomes, public policies that support comprehensive dental services for vulnerable women of childbearing age should be expanded, so not only their own oral and general health is safeguarded but also so that their children's risk of caries is reduced. Particularly if NIH trials confirm that treating pregnant women for periodontal disease reduces the incidence of unfavorable birth outcomes, the Centers for Medicare and Medicaid Services should build on its September 2004 coverage expansions for pregnant women by stimulating the states to similarly expand oral health services for pregnant women.\nThe power of prevention needs to be brought to bear, as both periodontal disease and caries are overwhelmingly preventable through well recognized strategies including regular and effective home care for periodontal disease and use of fluorides and sealants for caries. To the degree that pregnancy provides a \u201cteachable moment\u201d in self-care and future child-care, prenatal education should universally adopt an oral health component. This educational intervention should prioritize those mothers who have suffered significantly from dental caries so that they can learn to effectively prevent transfer of this disease to their children.\nTo be effective, oral health promotion must first seek to educate women and their health care providers about the importance of oral health and must promote an understanding of their ability to prevent and manage both periodontal disease and caries and to thereby limit the personal and intergenerational consequences of both conditions.","keyphrases":["oral health","pregnancy","infant caries"],"prmu":["P","P","R"]}
{"id":"Behav_Processes-2-1-2396234","title":"The exploratory behaviour of rats in the hole-board apparatus: Is head-dipping a valid measure of neophilia?\n","text":"The exploratory behaviour of laboratory rodents is of interest within a number of areas of behavioural pharmacology. However, how best to measure exploratory behaviour in rodents remains a contentious issue. Many unconditioned tests, such as the open field, potentially confound general locomotor activity with exploration. The hole-board apparatus appears to avoid this confound, as head-dipping into holes in the floor is assumed to be a valid measure of the subject's attraction towards novelty (neophilia). This study aimed to investigate whether head-dipping should be considered a valid measure of neophilia by comparing performance of adult male and female Lister hooded rats on the hole-board task (a) over repeated sessions and (b) when novel objects were absent or present underneath the holes. The results show that head-dipping initially decreased across repeated exposures, while time spent in the aversive central area increased. No change in head-dipping was seen in response to objects being placed underneath the holes. Rather than being a measure of neophilia, these results support the hypothesis that head-dipping represents an escape response, which declines as the subject becomes less fearful. These results are compared with previous studies of repeated exposure to other novel environments.\n1\nIntroduction\nWhen faced with an unfamiliar environment or object, animals often exhibit behaviour patterns that broadly can be termed exploration, such as locomoting around the environment, orientating towards novelty, and touching or sniffing novel objects (Berlyne, 1950, 1960; Glickman and Sroges, 1966; Welker, 1957). Exploration potentially provides an animal with new information about food sources, shelters or mating opportunities. However, by entering a new environment or attending to a novel stimulus, an animal might also increase it's risk of predation, aggression from conspecifics or other hazards. Whether an animal investigates or avoids novelty has been described as the outcome of an approach\u2013avoidance conflict (Montgomery, 1954, 1955; Montgomery and Monkman, 1955) or as a balance between neophilic and neophobic tendencies (Greenberg, 2003). In motivational terms, neophilia can be defined as the attraction that an animal displays towards an object or place simply because it is novel, while neophobia is the aversion that an animal shows towards approaching a novel object or place (Greenberg, 2003). In behavioural terms, neophilia and neophobia can be considered respectively as curiosity-based approach to, and fear-based avoidance of, a novel stimulus (Hughes, 2007).\nThe exploratory behaviour of rodents has gained recent interest within a number of areas of behavioural pharmacology. For instance, researchers studying drug addiction are interested in the neural mechanisms underlying neophilia due to the apparent overlap with the neural mechanisms involved in the rewarding effects of drug-taking (Bardo et al., 1996). However, considerable controversy still surrounds the question of how best to measure neophilic and neophobic responses in laboratory animals. One of the most commonly used behavioural tests for laboratory rodents is known as the open field. Originally, the open field apparatus consisted of a flat, raised platform (Hall, 1934, 1936), although the term open field is now commonly used to refer to any enclosed arena that can range in size from a small box to a large playing field (Crawley, 1985; Whishaw et al., 2006). In such an arena, the overall level of locomotion and time spent in the centre of the arena (which is assumed to be aversive to rodents) are often interpreted as measures of exploratory behaviour.\nHowever, some researchers have argued that forcing an animal to be in an enclosed area, or on an open platform, does not allow the animal to exhibit its \u2018motivation\u2019 to explore an unknown environment, as the task evokes a strong fear response (Birke and Sadler, 1986; Denenberg, 1969; Renner, 1990; Walsh and Cummins, 1976). Corticosterone levels have been found to rise in rodents on exposure to a novel open field environment (e.g. Marin et al., 2007; Matzel et al., 2006; Rees et al., 2006), and open field behaviour is influenced by some anxiolytic (anxiety-reducing) substances (Prut and Belzung, 2003). In particular, benzodiazepines and serotonin receptor agonists, which have anxiolytic effects in human beings, generally increase the proportion of entries into the centre of the open field in rodents (Prut and Belzung, 2003). Together, these data suggest that the open field may provide valid behavioural measures of fearfulness, but may have limited value for researchers interested in measuring neophilia. Another common concern with interpreting open field tests is that differences in the performance of animals in the open field may result simply from differences in overall locomotor activity, which could be unrelated to differences in exploratory behaviour (Berlyne, 1960; Birke and Archer, 1983).\nIn the 1970s, researchers began to use the hole-board apparatus, which consists of an enclosed arena with holes in the floor into which an animal can poke it's head, referred to as head-dipping (e.g. File and Wardill, 1975a,b; Nolan and Parkes, 1973). The frequency and duration of head-dipping are assumed to provide measures of neophilia (or directed exploration) that are independent from the general locomotor activity of the animal (File and Wardill, 1975a; Ljungberg and Ungerstedt, 1976). This apparatus has been argued, therefore, to avoid the difficulties of interpreting general locomotion that prove problematic in the open field, and a number of studies have shown that head-dipping and locomotion can vary independently of each other (e.g. Abel, 1995; Durcan and Lister, 1989; File, 1977; Lister, 1987; Rogers et al., 1999). In general, high levels of head-dipping are interpreted as indicative of neophilia, while low levels are assumed to result from a lack of neophilia or are assumed to reflect a high anxiety-like state in the animal (Crawley, 1985; Takeda et al., 1998). The hole-board task is currently being used as a test of neophilia in many areas of behavioural pharmacology (Kliethermes and Crabbe, 2006a).\nResearchers have attempted to validate head-dipping as a measure of neophilia by administering different classes of drugs and by comparing different genetic strains of rodents in their performance on the hole-board task. For example, if head-dipping is a neophilic response that is suppressed by an anxiety-like response, treatment with anxiolytic agents is predicted to increase head-dipping. Such studies have produced conflicting evidence; for instances, treatment of rodents with anxiolytic benzodiazepines has been reported to increase (rats: File, 1977; mice: Nolan and Parkes, 1973; Takeda et al., 1998), decrease (rats: Pellow et al., 1985) or have no effect (rats: Sayin et al., 1992) on the frequency of head-dipping. A recent review has suggested that the effects of anxiolytic compounds on head-dipping behaviour are generally confounded by changes in overall locomotion, despite the claims that head-dipping is unrelated to locomotor activity (Kliethermes and Crabbe, 2006a). Similarly, a study of several inbred mouse strains reported that head-dipping and locomotion are highly correlated (Kliethermes and Crabbe, 2006b). Whether head-dipping can be interpreted as a valid measure of neophilia remains unresolved (Bilkei-Gorz\u00f3 and Gyerty\u00e1n, 1996; Renner, 1990).\nThe aim of this study was to investigate whether head-dipping behaviour should be considered a valid measure of neophilia by comparing performance of rats on the hole-board task (a) over repeated sessions and (b) when objects are placed underneath the holes. Repeated exposure to a novel apparatus is expected to produce a reduction in exploration as the animal becomes familiar with the environment, a process commonly referred to as habituation (Leussis and Bolivar, 2006). If head-dipping behaviour is a measure of neophilia, the frequency of head-dipping is therefore predicted to decrease over repeated sessions. In early study by Nolan and Parkes (1973), head-dipping by young mice (21\u201325 postnatal days) was reported to be lower on a second exposure to the hole-board apparatus compared to the first exposure. Two recent studies have also provided evidence that head-dipping by mice and rats decreases on repeated exposure to the hole-board apparatus (Gagliano et al., 2008; Mayeux-Portas et al., 2000). If this behaviour is a valid measure of neophilia, head-dipping is also predicted to be higher in the presence, than in the absence, of objects. Although an early study reported that rodents head-dip more frequently when objects are present (File and Wardill, 1975a), this finding has not received recent replication. Given that male and female rodents are reported to exhibit behavioural differences on the hole-board task (e.g. Aguilar et al., 2003; Ray and Hansen, 2004), subjects of both sexes were included.\n2\nMaterials and methods\n2.1\nSubjects and housing\nThe subjects of this experiment were eight male and eight female adult Lister Hooded rats (supplied by Harlan, U.K.). The animals were housed in a single room, which was controlled for temperature and humidity and was maintained on a 12-h light:dark cycle (lights on at 07:00). The animals were housed in same-sex pairs in plastic and wire mesh home-cages (measuring 25\u00a0cm\u00a0\u00d7\u00a045\u00a0cm\u00a0\u00d7\u00a015\u00a0cm) with ad libitum access to rodent pellets and water. All guidelines and requirements set out in the Principles of Laboratory Animal Care (National Institutes of Health, U.S.A., Publication No. 86-23, revised 1985) and the U.K. Animals (Scientific Procedures) Act 1986 were followed.\n2.2\nApparatus and experimental design\nThe hole-board apparatus consisted of a wooden, grey box, measuring 68\u00a0cm\u00a0\u00d7\u00a068\u00a0cm. The walls were 40\u00a0cm high, and the box was raised 28\u00a0cm above the ground on a metal stand. Four holes (4\u00a0cm in diameter) were cut into the floor of the apparatus; each hole was 28\u00a0cm from a corner of the box along the diagonal from the corner to the centre. The floor of the box was marked out into four outer areas and one central area using black masking tape. The central area was delineated by four lines of tape each 20\u00a0cm from one of the walls, while the four outer areas were marked out by diagonal lines of tape running from the corners of the floor to the corners of the central square. The four holes were thus located at the corners of the central square. The apparatus was located in a small testing room with dimmed white lighting. The stand of the apparatus was open on all sides, allowing the floor or objects to be dimly lit.\nEach subject was tested ten times in the hole-board apparatus, once per day during two sets of five consecutive days (Monday\u2013Friday and the following Monday\u2013Friday). During the first set of five trials, no objects were present underneath the holes of the apparatus; during the second set of five trials, an object was placed on the floor under each of the four holes prior to the start of the trial, approximately 20\u00a0cm below each hole. The objects were all distinct from each other but were similar in size (approximately 10\u00a0cm in length or diameter: a black-and-white rubber ball, a purple plastic star, a red-and-white rubber pet toy, and a yellow, rubber dumb-bell shaped pet toy).\nAll trials were carried out between 09:00 and 17:00\u00a0h, and trials on males and females were alternated throughout the day. At the beginning of each trial, a subject was placed in one corner of the apparatus (always the corner closest to the door of the room), facing the centre of the arena. Each trial lasted 10\u00a0min. At the end of the trial, the subject was immediately placed into a carrying box and returned to the home cage. Between each trial, the floor and walls of the apparatus and the novel objects, if present, were cleaned with 70% alcohol solution.\n2.3\nBehavioural measurements\nDuring each 10-min trial, behavioural data were recorded by the observer (C.N.) onto a spreadsheet that was divided into 60 10-s time blocks. Inter-observer reliability between two independent observers (C.N. and G.R.B.) was over 80%. The following behaviour patterns were recorded:(i)enter a new area: the animal moves from one area of the open field to another (all four paws had to be placed on the floor of a new area);(ii)head-dip: the animal places it's head into one of the holes, to a minimum depth such that the ears were level with the floor of the apparatus (a new bout of head-dipping was recorded if the animal raised it's head fully out of the hole before resuming);(iii)rear: the animal is stationary on it's backpaws and raises it's forepaws off the ground, extending it's body vertically.\nThe data on entries into a new area were used to calculate the total amount of locomotion (number of entries into all areas summed together) and the percentage of entries that were in made into the central area. The location of the animal during each of the 10-s time intervals was used to estimate the percentage of time spent in the central area.\n2.4\nStatistical analyses\nThe data were analysed using repeated-measure ANOVAs, with \u2018objects\u2019 (with or without) as a within-subject variable, \u2018trial\u2019 (trials 1\u20135) as a within-subject repeated measure and \u2018sex\u2019 (male or female) as a between-subject variable. Significant interactions were analysed further using simple effects post hoc tests (Howell, 2007).\n3\nResults\n3.1\nTotal locomotion\nThe total amount of locomotion did not vary across trials (F4,56\u00a0=\u00a00.096, n.s.) or vary with the presence or absence of objects (F4,56\u00a0=\u00a00.071, n.s.). Females locomoted more on average than males (F1,14\u00a0=\u00a04.871, p\u00a0=\u00a00.045; Table 1). There were no interactions between trial and sex (F4,56\u00a0=\u00a01.433, n.s.), trial and object (F4,56\u00a0=\u00a02.615, n.s.) or object and sex (F4,56\u00a0=\u00a00.559, n.s.), and the three-way interaction between these variables was not significant (F4,56\u00a0=\u00a01.174, n.s.).\n3.2\nLocomotion into the central area\nThe percentage of entries that were into the central area differed significantly across trials (F4,56\u00a0=\u00a014.842, p\u00a0<\u00a00.001) and varied with the presence or absence of objects (F1,56\u00a0=\u00a084.240, p\u00a0<\u00a00.001). There was also a significant interaction between trials and the presence or absence of objects (F4,56\u00a0=\u00a07.662, p\u00a0<\u00a00.001). Post hoc analyses indicated that the proportion of entries into the centre increased over the first five trials and remained steady thereafter (Fig. 1a). The percentage of entries into the central area varied between the sexes (F1,14\u00a0=\u00a05.209, p\u00a0=\u00a00.039), with females exhibiting an overall greater proportion of entries into the centre than males (Table 1). There were no interactions between trial and sex (F4,56\u00a0=\u00a00.761, n.s.) or object and sex (F4,56\u00a0=\u00a01.956, n.s.), and the three-way interaction between these variables was not significant (F4,56\u00a0=\u00a02.567, n.s.).\n3.3\nTime spent in the central area\nThe percentage of time spent in the central area differed significantly across trials (F4,56\u00a0=\u00a08.085, p\u00a0<\u00a00.001) and varied with the presence or absence of objects (F1,56\u00a0=\u00a035.393, p\u00a0<\u00a00.001). There was also a significant interactions between trials and the presence or absence of objects (F4,56\u00a0=\u00a03.493, p\u00a0=\u00a00.013). Further analyses indicated that the time spent in the centre increased over the first five trials and remained steady thereafter (Fig. 1b). The percentage of time spent in the central area varied between the sexes (F1,14\u00a0=\u00a05.032, p\u00a0=\u00a00.042), with females exhibiting an overall greater percentage of time in the centre than males (Table 1). There were no interactions between trial and sex (F4,56\u00a0=\u00a00.083, n.s.) or object and sex (F4,56\u00a0=\u00a00.076, n.s.), and the three-way interaction between these variables was not significant (F4,56\u00a0=\u00a01.001, n.s.).\n3.4\nFrequency of head-dipping\nThe frequency of head-dipping differed significantly across trials (F4,56\u00a0=\u00a02.626, p\u00a0=\u00a00.043), and there was a significant interaction between trials and the presence or absence of objects (F4,56\u00a0=\u00a04.482, p\u00a0=\u00a00.013). Further analyses indicate that head-dipping decreased over the first five trials and slightly increased towards the end of the experiment (Fig. 2). There was no main effect of the presence or absence of objects (F1,14\u00a0=\u00a00.142, n.s.). The frequency of head-dipping varied significantly between the sexes (F1,14\u00a0=\u00a015.401, p\u00a0=\u00a00.002), with females exhibiting an overall greater frequency of head-dipping than males (Table 1). There were no interactions between trial and sex (F4,56\u00a0=\u00a00.324, n.s.) or object and sex (F4,56\u00a0=\u00a00.553, n.s.), or a three-way interaction between these variables (F4,56\u00a0=\u00a00.779, n.s.).\n3.5\nFrequency of rearing\nThe frequency of rearing differed significantly across trials (F4,56\u00a0=\u00a03.204, p\u00a0=\u00a00.019), with frequency increasing slightly across sessions (Fig. 3). There was no main effect of the presence or absence of objects (F1,14\u00a0=\u00a03.898, n.s.) or sex (F1,14\u00a0=\u00a01.332, n.s.; Table 1). There were no interactions between trials and the presence or absence of objects (F4,56\u00a0=\u00a00.457, n.s.), between trial and sex (F4,56\u00a0=\u00a00.111, n.s.) or object and sex (F4,56\u00a0=\u00a00.006, n.s.), or a three-way interaction between these variables (F4,56\u00a0=\u00a01.708, n.s.).\n4\nDiscussion\nThe aim of this study was to investigate whether head-dipping behaviour should be considered a valid measure of neophilia by comparing performance on the hole-board task (a) over repeated sessions (trials 1\u201310) and (b) when no objects were present (trials 1\u20135) and when objects were placed underneath the holes (trials 6\u201310). The results show that head-dipping was high during the first test, decreased over the following two trials and remained relatively stable during the rest of the experiment. The initial drop in head-dipping following the first trial could be interpreted in two ways. First, head-dipping could be indicative of a neophilic response that decreases as the animal becomes familiar with the apparatus, i.e. head-dipping represents directed exploratory behaviour that drops as the apparatus loses its novelty. If this interpretation is correct, we would also predict that head-dipping would be greater in the presence of objects; however, there was no evidence of an increase in head-dipping behaviour when objects were present underneath the holes. These results do not support the hypothesis that head-dipping is a valid measure of neophilia.\nThe second interpretation of the initial drop in head-dipping frequency is that head-dipping could represent a fearful, neophobic response, such that, on first exposure to the apparatus, the animal actively attempts to find an escape route (Renner, 1990). Adult male rats have been shown to exhibit an increase in circulating corticosteroid levels following a single exposure to the hole-board apparatus (M\u00e1rquez et al., 2005, 2006), suggesting that testing in this apparatus is a stressful event. If this interpretation of head-dipping is correct, we would also predict that, as head-dipping behaviour declines, fearfulness would also decline. In favour of this interpretation, while head-dipping frequency declined over the first few tests, the amount of locomotion into the central area of the hole-board, and the time spent in the central area, greatly increased over these trials. Therefore, as fearfulness apparently decreased, head-dipping also decreased. If we assume that the fear experienced on exposure to a novel apparatus can be equated to normal or \u2018state\u2019 anxiety (Belzung and Griebel, 2001), these results contradict the assumption that head-dipping behaviour is suppressed by an anxiety-like response, in which case we might have expected head-dipping to vary in the opposite direction to anxiety-like behaviour.\nA small number of previous studies on rodents have also presented evidence that the frequency of head-dipping decreases on second exposure to the hole-board apparatus (Gagliano et al., 2008; Mayeux-Portas et al., 2000; Nolan and Parkes, 1973). The early study by Nolan and Parkes (1973) investigated whether previous experience with the hole-board apparatus with a solid floor inserted would influence frequency of head-dipping on second exposure to the apparatus. The results indicated that prior exposure to the plain board reduced the frequency of head-dipping on subsequent exposure as much as prior exposure to the board with holes (Nolan and Parkes, 1973). Again, these results do not support the idea that head-dipping provides a valid measure of neophilia, rather than a neophobic response induced by an unfamiliar test situation.\nIn our study, head-dipping did increase in frequency towards the end of the experiment, after eight or more exposures to the apparatus. Therefore, we cannot reject the possibility that, as the subjects became very familiar with the apparatus, they engaged in a greater level of visual exploration through the holes. Rearing behaviour is also commonly interpreted as an activity by which an animal obtains information about distal environmental cues (Lever et al., 2006). Given that rearing behaviour gradually increased in frequency across the sessions, as the subjects presumably became less fearful, the amount of visual assessment of the testing room, both above and below the apparatus, may have increased. This interpretation is supported by an experiment carried out by Bilkei-Gorz\u00f3 and Gyerty\u00e1n (1996). This study reported that the effects of the anxiolytic benzodiazepine chlordiazepoxide on head-dipping behaviour in rats varied with the light intensity during testing, such that treatment decreased head-dipping in very bright light and increased head-dipping under normal light. The authors argued that head-dipping during the aversive testing condition (bright light) represents an attempt by the subject to find an escape route from the apparatus, while head-dipping in less aversive conditions represents visual exploration of the apparatus. Future studies could investigate whether head-dipping increases with extended familiarisation to the apparatus even in the absence of novel objects, and whether head-dipping increases when novel objects are brought closer to the holes, when new novel objects are placed under the holes for each test, or when objects are re-located beneath different holes.\nOur results show that repeated exposure of subjects to a novel hole-board apparatus greatly affects the behavioural response, and that the neophobic response experienced by subjects during the first exposure to an apparatus apparently declines over further exposures. Repeated exposure to another commonly used behavioural test, the elevated maze, has been reported to result in a decrease in total locomotion and a decrease in the amount of time spent on aversive open sections of the maze (e.g. Cook et al., 2002; Dawson et al., 1994; Rodgers et al., 1996). In contrast, in our study, no change of overall locomotion was found on repeated exposure to the hole-board, and the amount of time spent in the aversive central area of the hole-board apparatus increased over sessions. The reason for the differences in the behavioural effects of repeated exposure to these two types of novel environments may be related to differences in the design of the two types of apparatus. Repeated exposure to an elevated maze, which consists of open areas and closed areas, may have resulted in the subjects retreating into the darker, closed arms once an initial investigation had found no potential escape routes from the apparatus; such a response was not an option in the hole-board test. If a shelter were added to the hole-board apparatus, locomotion might be predicted to decrease over repeated exposure, with subjects choosing to remain under a shelter once potential escape routes had been investigated.\nIn support of previous reports (e.g. Aguilar et al., 2003; Ray and Hansen, 2004), our results indicate that female rats head-dip more frequently, locomote more and spend more time in the centre of the hole-board apparatus than males. These sex differences in behaviour were not affected by repeated exposure to the apparatus, or by the presence of novel objects. Given that the locomotor patterns of female rodents are influenced by the stage of the estrus cycle and by experimental manipulation of hormones such as estradiol (e.g. Garey et al., 2001; Morgan and Pfaff, 2002), researchers have questioned whether gonadal hormones might influence general levels of arousal that might impact on how females respond to novelty compared to males (Morgan et al., 2004). However, while reports of sex differences in hole-board performance have been interpreted as showing that females are more exploratory than males, females have been reported to locomote more than males in familiar, as well as novel, environments (e.g. Cortright et al., 1997; Eckel and Moore, 2004), and, as discussed above, head-dipping may not represent neophilic behaviour. Therefore, the conclusion that females are more exploratory or neophilic than males should not be drawn from such data.\nThe results of this study indicate that the assumption that head-dipping on the hole-board task represents a neophilic response is not necessarily accurate. At least on first exposure to the apparatus, head-dipping is likely to result from an attempt by the subject to find a potential escape routes. This issue highlights a more general problem with unconditioned behavioural tests of exploratory behaviour, in that high levels of locomotion around an environment should not immediately be interpreted as neophilia or a positively rewarded aspect of novelty-seeking (Welker, 1957). Any behaviour in a novel environment will be influenced by both neophilia and neophobia, such that a single behavioural measure is unlikely to be purely indicative of either neophilic or neophobic tendencies. Rather than being at polar ends of a continuum, neophilia and neophobia should be thought of as two orthogonal factors that can vary independently (Greenberg, 2003). For researchers interested in rodent exploration, simple measures, such as head-dipping, are unlikely to expose by themselves the complex interaction between these two factors.","keyphrases":["exploration","rat","hole-board","head-dipping","neophilia","neophobia"],"prmu":["P","P","P","P","P","P"]}
{"id":"J_Urban_Health-3-1-2134844","title":"Quality of Water the Slum Dwellers Use: The Case of a Kenyan Slum\n","text":"As a result of rapid urbanization in a context of economic constraints, the majority of urban residents in sub-Saharan Africa live in slums often characterized by a lack of basic services such as water and sewerage. Consequently, the urban poor often use inexpensive pit latrines and at the same time may draw domestic water from nearby wells. Overcrowding in slums limits the adequate distance between wells and pit latrines so that micro-organisms migrate from latrines to water sources. Sanitary practices in these overcrowded slums are also poor, leading to contamination of these wells. This study sought to assess sanitary practices of residents of a Kenyan urban slum and fecal contamination of their domestic water sources. This cross-sectional study involved 192 respondents from Langas slum, Kenya. Forty water samples were collected from the water sources used by the respondents for laboratory analysis of coliforms. Of these 40 samples, 31 were from shallow wells, four from deep wells, and five from taps. Multiple-tube fermentation technique was used to enumerate coliform bacteria in water. The study found that most people (91%) in the Langas slum used wells as the main source of domestic water, whereas the rest used tap water. Whereas most people used pit latrines for excreta disposal, a substantial percentage (30%) of children excreted in the open field. The estimated distance between the pit latrines and the wells was generally short with about 40% of the pit latrines being less than 15 m from the wells. The main domestic water sources were found to be highly contaminated with fecal matter. Total coliforms were found in 100% of water samples from shallow wells, while 97% of these samples from shallow wells were positive for thermotolerant coliforms. Three out of the four samples from deep wells were positive for total coliforms, while two of the four samples were positive for thermotolerant coliforms. None of the samples from taps were positive for either total or thermotolerant coliforms. Because the presence of thermotolerant coliforms in water indicates fecal contamination, facilitated by the proximity between the wells and pit latrines, the study suggests that the pit latrines were a major source of contamination of the wells with fecal matter. However, contamination through surface runoff during rains is also plausible as indiscriminate excreta disposal particularly by children was also common. Owing to the fecal contamination, there is a high possibility of the presence of disease pathogens in the water; thus, the water from the wells in Langas may not be suitable for human consumption. To address this problem, treatment of the water at community or household level and intensive behavioral change in sanitary practices are recommended. Efforts should be made to provide regulated tap water to this community and to other slums in sub-Saharan Africa where tap water is not accessible. However, more sampling of different water sources is recommended.\nINTRODUCTION\nRapid urban growth in a climate of economic constraints has resulted in the majority of residents in Africa\u2019s large cities, and an increasing proportion of Africans overall, living in overcrowded slums and shantytowns. In these slums and shantytowns, health conditions and livelihood opportunities are poor.1\u20133 Available evidence indicates that the poor urban residents of Africa exhibit higher morbidity, have poor access to health services, and consequently exhibit higher mortality rates than residents of other areas including rural residents.4\u20138\nThe situation in Kenya is similar to other situations in Africa. The proportion of urban population in Kenya nearly doubled between 1980 and 1998, increasing from 16 to 31%.9 Rapid urbanization amid economic degradation in Kenya has resulted in an increased proportion of people living in absolute poverty in the urban areas.10 Therefore, poverty has increasingly become a crucial urban problem in Kenya leading to mushrooming of informal settlements in the urban parts of Kenya where the urban poor find shelter. This has overwhelmed the environmental health resources in urban areas. Because of their illegal status, residents of informal settlements in Kenya do not receive government services such as water, drainage, sewerage, and rubbish collection. Consequently, informal settlements are characterized by poor environmental conditions that predispose their inhabitants to poor health outcomes.4 Evidence shows that children of poor families in urban areas of Kenya exhibit poorer health conditions than their rural counterparts. According to a report by African Population and Health Research Center (APHRC) in 2002,4 infant and child mortality risks are particularly higher in the slums of Nairobi than those observed in other urban areas and in rural Kenya. For instance, the under five mortality was 35% higher among slum residents in Nairobi than among the rural population in Kenya. The report attributes these patterns to poor water and sanitation in these slum settlements.4\nAn adequate supply of safe drinking water is universally recognized as a basic human need. Yet millions of people in the developing world do not have ready access to an adequate and safe water supply. By 1996, the number of people without access to safe water in urban areas was rising sharply in developing countries as a result of  rapid urbanization, much of which was occurring in peri-urban and slum areas.11 Because the United Nations projects a rapid population growth in urban areas between 2000 and 2030,12 access to safe drinking water and adequate sanitation in urban areas is likely to worsen unless there is a drastic policy change to cater to the needs of the urban poor.\nHuman excreta and the lack of adequate personal and domestic hygiene have been implicated in the spread of many infectious diseases including cholera, typhoid, hepatitis, polio, cryptosporidiosis, ascariasis, and schistosomiasis. It is estimated that one-third of deaths in developing countries are caused by the consumption of contaminated water and on average as much as one-tenth of each person\u2019s productive time is sacrificed to water-related diseases.13 The World Health Organization estimates that 2.2 million people die annually from diarrhea diseases and that 10% of the population of the developing world are severely infected with intestinal worms related to improper waste and excreta management.14,15 In Kenya, diarrheal diseases are among the major illnesses affecting children of the slum residents. According to the report by APHRC in 2002, prevalence of diarrhea was 32% among children below 5\u00a0years of age in the slums, which is double the rate for Nairobi and the national average.4\nWhere ground water is used as a source of domestic water, use of pit latrines is not recommended because the two are incompatible unless the water table is extremely low and soil characteristics are not likely to contribute to contamination of ground water. Where they coexist, although it is difficult to give a general rule for all soil conditions, the commonly used guideline is that the well should be located in an area higher than and at least 15\u00a0m from the pit latrines and should be at least 2\u00a0m above the water table. Available evidence shows that increased lateral separation between the source of pollution and groundwater supply reduces the risk of fecal pollution.16 Coexistence of on-site sanitation and use of underground water has in the past been mainly confined to the rural areas where there is adequate land to allow for adequate distance between pit latrines and shallow wells. With the rapid urbanization and rapid expansion of slum settlements in sub-Saharan Africa, on-site sanitation and underground water are used in some urban areas because they are affordable options in the absence of government-supplied services. However, the congestion in the urban slums does not allow for adequate distance between the wells and the pit latrines, which allows micro-organisms to migrate from fecal contents into the underground water sources. Furthermore, poor sanitary practices (for example, disposal of human excreta) in these slum areas lead to contamination of water and consequently water-borne diseases. It is in this context and in the context of high levels of diarrheal diseases in the urban slums in Kenya that this study sought to assess the sanitary practices and the fecal contamination of domestic water sources in an urban slum in Eldoret, Kenya.\nSTUDY CONTEXT\nThe study was conducted between January and June 1999 in Langas, an urban slum in Eldoret municipality, Kenya, less than 10\u00a0km from Eldoret town. Eldoret town is located in the Rift Valley Province, about 330\u00a0km north west of Nairobi. Eldoret, the headquarter of Uasin Gishu District, is one of the fastest growing urban areas in Kenya. Langas falls under high density, low-income areas of the Eldoret municipality. It is divided into four administrative blocks that are further subdivided into about 2,500 plots. Each plot (1\/8 of an acre) has between one and 30 households each with an average of six occupants. Settlement in Langas began in 1965 and at the time there were no basic services. Early settlers dug shallow wells for their water needs.17 The water table is high, and this raises the possibility of ground water contamination where on-site sanitation systems are in use.\nMETHODOLOGY\nA cross-sectional study design was used and a sample of 192 households1 was selected through multistage sampling technique as follows: two out of the four administrative blocks were randomly picked and from the two blocks, 192 plots were picked. From each of the selected plots, one household was selected to participate. The 192 households were selected as follows: starting from one corner of each of the blocks and walking across the two blocks in a more or less a diagonal line, about every plot on the diagonal line was selected until the sample of 192 was reached. From each of these plots, one household was selected to represent the plot (this depended mainly on availability of respondents and their willingness to participate), and the first household to be contacted in a selected plot was considered for recruitment into the sample. From the household, a credible respondent was interviewed. A credible respondent was a resident of the selected household who was aged 18\u00a0years or above.\nFor water sampling, 20 samples were taken from water points in each of the two blocks to make a total of 40 samples. This number for water samples was determined by financial and logistical feasibility. In most circumstances, residents of a plot shared one water source and there was roughly one water point per plot. In a few cases, several plots shared a water point. To get the 40 samples, every fourth household2 of the 192 households included in the interview sample was asked their source of water and a water sample was taken from this source so long as it had not already been taken. In the event that a previous fourth household shared the source with the current household, the source for the immediate next household in the study sample was considered. Of the 40 water samples, 31 were from shallow wells (defined as a hand-dug well), four from deep wells (defined as a drilled well) and five from taps (referring to tap water from the municipal council distribution system). Using the above described criteria, only one deep well was selected and purposive sampling was then used to get three other such wells, consequently including all the deep wells that were used by the study sample.\nQuestionnaires were administered to the 192 selected households to obtain information on the type of toilet facility used, major source of domestic water, method of human waste disposal, whether drinking water was boiled, and the perceptions of possible sources of water contamination in the area. For the method of excreta disposal and water source, the main method and source were considered in instances where there was more than one method or source, respectively. The distance between the pit latrines used by the 192 households and the wells (in cases where they used wells) was estimated. We also observed sanitation practices.\nWater samples were collected aseptically with sterile sampling bottles. The samples were transported within 2\u00a0hours of collection in a cool box containing ice packs to the Faculty of Health Sciences, Moi University microbiology laboratory for analysis. Fecal contamination of the water was determined through isolation of indicator organisms, total coliforms, and then thermotolerant (fecal) coliforms, through multiple-tube fermentation (MTF) technique. Probability tables (McCrady tables) were used to determine the Most Probable Number (MPN) estimates of the coliform organisms per 100\u00a0ml of water. Analysis of data was generally descriptive, involving determination of frequencies. Stata statistical package was used to analyze the data.\nRESULTS\nMethod of Excreta Disposal\nThe majority of respondents (98%) said that adults used pit latrines, whereas the rest said adults defecated indiscriminately. Similarly, a majority of respondents (70%) said that children used pit latrines, whereas 30% said children used open field\/defecated indiscriminately. Most of the pit latrines (95%) in the community were traditional, whereas the rest were ventilated improved pit latrines (VIP latrines). A walk through the community confirmed the report that some people excreted indiscriminately as human excreta was observed strewn all over the compounds.\nSource of Domestic Water\nMost people (89%) said they used shallow wells as the major source of domestic water, whereas 2% said they used water from deep wells and the rest said they used tap water from the municipal council (Table\u00a01). The shallow wells often had no concrete slab and often the aperture was not covered at all or was poorly covered with a loose lid that was not lockable, whereas the deep wells had a piped system. Those who used deep wells were mainly the more affluent people in the community who often owned the plot in which the well was situated. Tap water was mainly from water kiosks where water was being sold to the slum residents. Respondents who did not use the tap water said that the water from water kiosks was expensive and unaffordable to be used for domestic purposes.3 Problems of unreliability were mentioned as hindering use of tap water from the kiosks as some respondents said that sometimes the kiosk near their house could remain closed for a whole day or more. Some of the respondents reported that the nearest water kiosk was too far from their homes.\nTABLE\u00a01Source of domestic water among 192 sampled households in Langas Slum, KenyaSource of domestic waterNumberPercentTap178.9Shallow well17189.1Deep well42.1Total192100\nDistance between Pit Latrine and Well\nThe wells were very close to the pit latrines. In many circumstances (38%), the distance between the wells and the pit latrines was estimated to be less than 15\u00a0m (the commonly used guideline is that the distance should be at least 15\u00a0m). Most wells (about 59%) were estimated to be at a distance between 15 and 30\u00a0m from the pit latrines (Table\u00a02). The distance between pit latrines and wells for the wells from which water samples were taken was similar to that of other wells (Table\u00a03).\nTABLE\u00a02Distance between pit latrine and wells for all wells in the study in Langas Slum, KenyaDistanceNumberPercent1\u201315\u00a0m6738.315\u201330\u00a0m10358.930\u00a0m and above52.9Total175*100*This total reflects only wells, and thus excludes taps.TABLE\u00a03Distance between pit latrine and well for wells whose water was sampled in Langas Slum, KenyaDistanceNumberPercent1\u201315\u00a0m144015\u201330\u00a0m195430\u00a0m and above26Total35*100*This total reflects only wells whose water was sampled, and thus excludes taps.\nBoiling of Drinking Water\nDespite the short distance estimated between the pit latrines and the wells and the poor sanitary practices like indiscriminate excreta disposal, only 42% of those who reported using wells said they boiled their drinking water, when asked if they did.\nResidents\u2019 Perception of Contamination\nRespondents pointed out various possible sources of contamination of the water sources in the area. These included children dipping dirty objects into water source (34%) as the main source of contamination, drawing water from the source with dirty containers (27%), domestic animals defecating around water sources (19%), and people washing clothes (5%) at the water source, among others. Interestingly, no one mentioned closeness of the well to the pit latrines as a possible source of contamination.\nWater Analysis Results\nForty water samples were analyzed: 31 from shallow wells, four from deep wells, and five from taps.\nWells\nShallow Wells\nAll the samples (31) taken from shallow wells were positive for total coliforms. The most probable number of total coliforms for most of the samples (71%) was 1,100+ per 100\u00a0ml. The minimum number of total coliforms was 63, whereas the maximum was 1,100+ coliforms per 100\u00a0ml water.\nOnly one of the samples had no thermotolerant coliforms at all, whereas most had 1,100+ thermotolerant coliforms per 100\u00a0ml.\nDeep (Drilled) Wells\nOf the four samples taken from the deep wells, three were positive for total coliforms, whereas one was negative. The highest number of total coliforms was 240. For thermotolerant coliforms, two samples were negative. The maximum number of thermotolerant coliforms was 23.\nTaps\nFor the five samples taken from taps (municipal tap water), all of them were negative for total coliforms and consequently thermotolerant coliforms.\nDISCUSSION\nThis paper provides evidence on the extent of contamination of main domestic water sources in the Langas slum and suggests the most probable sources of this contamination. The evidence reveals that the most probable sources of contamination are hardly mentioned among the many sources perceived to contaminate the water sources by the residents of the slum, which impacts how interventions ought to be developed.\nThe results indicate that the majority of the community members used pit latrines and at the same time used wells as the major source of domestic water. The conditions found in Langas do not fulfill the recommendations given for coexistence of onsite sanitation and use of ground water for domestic purposes, which indicate that there should be an adequate lateral separation between the pit latrine and the well to reduce chances of fecal contamination of the ground water.16 The distance between the wells and the pit latrines was estimated to be generally short with nearly 40% of the pit latrines estimated to be at a distance of less than 15\u00a0m from the wells. This raises the risk of contamination of the water sources as coliforms migrate from the pit latrines to the wells.\nThe presence of indicator organisms (Escherichia coli or thermotolerant coliform bacteria) in water indicates recent contamination of the water source with fecal matter and hence possible presence of intestinal pathogens. According to World Health Organization (WHO) guidelines, E. coli or thermotolerant coliform bacteria should not be detectable in any water intended for drinking.18 The laboratory analysis results of water samples in this study show that fecal matter heavily contaminated the water sources and especially the shallow wells. None of the shallow wells met the WHO requirements for water intended for drinking. The presence of indicator organisms in the water samples collected from the wells indicates the coliforms migrated from fecal matter in the pit latrines through the soil to the water sources, facilitated by the very short distance between the pit latrines and the wells. Poor sanitary practices are also likely sources of pollution of the water sources. Sanitary practices were found to be generally poor from observation and from responses from the respondents. Thirty percent of children excreted indiscriminately and consequently there were a lot of indiscriminately disposed excreta observed. Rains are likely to wash off indiscriminately disposed excreta into shallow wells particularly if the wells are not protected. Therefore, this may have also contributed to the contamination of the generally open shallow wells with fecal matter. Other studies have also attributed contamination of water sources to wet seasons.19 Despite the contamination of water, it was evident that it was not a common practice for the slum dwellers to boil the water, as only 42% of those who used water from wells said they boiled drinking water.\nThe illegal status of the slum areas in Kenya has hindered the expansion of municipal services to serve them. This has resulted to the poor being denied access to safer drinking water and proper sanitation. The results of this study suggest that water from the tap (which was from the municipal council) was safe for human consumption according to WHO guidelines.18 However, it is important to note that although the presence of thermotolerant coliforms indicates the presence of fecal contamination and potential presence of pathogens, absence of the same does not necessarily mean absence of pathogens, and further investigations would be worthwhile.\nLangas is not the only area with the problem of safe drinking water; other urban centers of the developing world have experienced similar problems.19\u201322 Similar to Langas, in the slums of Nairobi, Kenya, although wells are not a common source of water, slum residents are forced to buy tap water at exorbitant prices from vendors who operate without regulatory mechanisms.23,24 If the Millennium Development Goals of reducing by half the proportion of people without sustainable access to safe drinking water by 2015 and achieving a significant improvement in lives of at least 100 million slum dwellers by 202025 are to be met, there is a dire need for reconsideration of the slum areas in the developing world as far as water supply is concerned, as these slums are home to about 70% of all urban residents in sub-Saharan Africa.26\nHowever, improving the water quality at source alone may not be the ultimate solution because improving water quality at source may not always ensure a reduction in the transmission of water-related diseases. Studies27\u201330 have shown significant deterioration in water quality between the source and the point of use. Esrey31 concluded that improving water had no health impact if the sanitation was not improved and that improving both water and sanitation together were synergistic in producing larger impacts than either alone. Although the results of this study suggest the need for provision of safer water sources, in this community and in many other slum communities with evident poor sanitary practices, intensive behavior change communication on sanitary practices is also paramount as this has been found effective in the reduction of water-borne diseases elsewhere.32\u201335\nWhereas the findings from this study are worthwhile and act as an eye opener for the situation of quality of water in the rapidly growing informal settlements in the urban areas in Kenya and in the rest of Africa, more sampling of different water sources is highly recommended.\nCONCLUSION\nIt is evident that most of the sources of domestic water in Langas slum are contaminated with fecal matter and do not meet the WHO guidelines for drinking water quality. This poses a health hazard to the residents of the slum as they are at risk of water-borne diseases. The results of this study also suggest that tap water may be safer, but additional sampling is needed. The ideal intervention in the long-run may therefore be provision of adequate piped water to all slum dwellers. However, this may take sometime, and simpler interventions could be put in place in the mean time. Basic sanitary improvement may be worthwhile at the moment. Covering the shallow wells and possibly installing hand pumps or mechanical pumps at the wells could improve the situation. Basic treatment of the water at the community or household level by chemical disinfection using chlorine, filtration using simple household filters, and boiling should also be promoted. These interventions may have a great impact on the health of the slum dwellers as access to safe drinking water and basic sanitation services for populations at risk would result in 200 million\/year fewer diarrheal episodes and 2.1 million\/year fewer deaths caused by diarrhea.35","keyphrases":["water","slums","kenya","urbanization","africa","urban poor","sanitation","sanitary practices","contamination","coliforms","poverty","water quality"],"prmu":["P","P","P","P","P","P","P","P","P","P","P","P"]}
{"id":"Environ_Health_Perspect-113-12-1314901","title":"The Tobacco Industry and Pesticide Regulations: Case Studies from Tobacco Industry Archives\n","text":"Tobacco is a heavily pesticide-dependent crop. Because pesticides involve human safety and health issues, they are regulated nationally and internationally; however, little is known about how tobacco companies respond to regulatory pressures regarding pesticides. In this study we analyzed internal tobacco industry documents to describe industry activities aimed at influencing pesticide regulations. We used a case study approach based on examination of approximately 2,000 internal company documents and 3,885 pages of U.S. Environmental Protection Agency documents obtained through Freedom of Information Act requests. The cases involved methoprene, the ethylene bisdithiocarbamates, and phosphine. We show how the tobacco industry successfully altered the outcome in two cases by hiring ex-agency scientists to write reports favorable to industry positions regarding pesticide regulations for national (U.S. Environmental Protection Agency) and international (World Health Organization) regulatory bodies. We also show how the industry worked to forestall tobacco pesticide regulation by attempting to self-regulate in Europe, and how Philip Morris encouraged a pesticide manufacturer to apply for higher tolerance levels in Malaysia and Europe while keeping tobacco industry interest a secret from government regulators. This study suggests that the tobacco industry is able to exert considerable influence over the pesticide regulatory process and that increased scrutiny of this process and protection of the public interest in pesticide regulation may be warranted.\nTobacco is a pesticide-intensive crop. With nearly 27 million pounds of pesticides (including insecticides, herbicides, fungicides, and suckercides) applied to the U.S.-grown crop from 1994 to 1998, it ranks sixth in terms of the amount of pesticides applied per acre [U.S. Government Accounting Office (GAO) 2003]. The tobacco industry regards pesticides as essential to tobacco production, stating that \u201cthe crop could not be produced economically without them\u201d (Davis 1989; Philip Morris 1990b). According to industry documents, government-imposed limitations on pesticide use \u201cmay present a serious impediment\u201d to the international tobacco trade (Hill 1989).\nInternal tobacco industry documents provide a window into the tobacco industry\u2019s activities regarding pesticide regulations. These case studies drawn from industry documents describe the tobacco industry\u2019s responses to pesticide regulatory action. The documents also provide insight into the relationships between the tobacco industry and pesticide regulatory agencies and tensions between business and public health interests.\nThe Tobacco Industry Documents\nLitigation against the tobacco industry has resulted in the release of nearly 7 million previously secret tobacco industry documents (Bero 2003; Malone and Balbach 2000). Scanned PDF versions of original handwritten, typed, or printed documents have been archived at the University of California, San Francisco, library in electronic repositories, searchable using basic keywords (http:\/\/legacy.library.ucsf.edu). Between July 2003 and February 2004, we searched the archives using a \u201csnowball\u201d sampling strategy, beginning with broad search terms (\u201cpesticide\u201d and \u201ccrop protection agent\u201d) and using retrieved documents to identify more specific search terms (such as names of specific pesticides, people, and regulatory agencies). Table 1 provides examples of keyword searches and the number of documents yielded. This process produced nearly 300,000 documents relating to many different pesticides. The first author reviewed these documents\u2019 index entries and excluded duplicates and documents unrelated to pesticide regulatory issues. The final sample size was approximately 2,000 documents, spanning 1974\u20132001.\nWe also filed Freedom of Information Act (FOIA) requests with the U.S. Environmental Protection Agency (EPA) on pesticide issues raised by information in the industry documents, resulting in 3,885 pages of government documents. Finally, we reviewed public health agency reports based on industry documents (Zeltner et al. 2000). We analyzed the industry, government, and public health agency documents by assembling chronologically constructed case studies, a method common to sociology, political science, and anthropology (e.g., analyses of a corporation\u2019s organizational structure, a social movement, or a tribe) (Hill 1993; Yin 1994) (Table 2). The pesticides chosen for inclusion [methoprene, the ethylene bisdithiocarbamates (EBDCs), and phosphine] were those for which sufficient information related to regulatory activities was available in the archives to construct a case study.\nPesticides and Tobacco\nPesticides used on tobacco are also used regularly on food crops. As with food crops, trace amounts of pesticides remain on tobacco leaves after treatment; typically, residue levels decline during the drying and manufacturing process, although additional pesticides may be applied to the finished product (U.S. GAO 2003). Although pesticides increase production of tobacco and food crops, pesticide exposure may harm humans; thus, regulatory agencies such as the U.S. EPA may set limits on the amount of pesticide residue permitted in or on food and tobacco and establish standards for workers handling pesticides. Because tobacco is burned and the smoke inhaled, active and passive smokers are exposed to pyrolyzed pesticide residues (U.S. GAO 2003). The U.S. EPA has concluded that this exposure poses no short-term risk, but little is known about the long-term health effects (U.S. GAO 2003).\nMethoprene\nIn 1974, Philip Morris formed a partnership with the chemical company Zoecon to market a new insecticide (Manzelli 1975). The insecticide\u2019s active ingredient, methoprene, acted as an endocrine disruptor in cigarette beetles and tobacco moths, preventing their larvae from maturing into adult insects (Manzelli 1975). Philip Morris anticipated that methoprene would replace phosphine, a common warehouse fumigant (Philip Morris 1988) and pledged to assist Zoecon in introducing methoprene \u201cin as many countries as we can\u201d (Seligman 1982).\nSome countries have regulations that require the establishment of maximum residue limits (MRLs) for pesticides on crops; however, Philip Morris determined that MRLs were not required in all countries, especially for pesticides on nonfood crops such as tobacco (Ryan 1991). Philip Morris asked Zoecon \u201cto not force this issue and submit for MRLs when not required\u201d (Lindahl 1992b). In April 1991 Zoecon alerted Philip Morris\u2019s director of research that the Malaysian pesticide board had recently set an MRL of 1.0 ppm for methoprene on tobacco (Hutney 1991). Zoecon considered 1.0 ppm too low to enable the effective use of methoprene; the level supported by the labeled application rate was 10 ppm (Ryan 1992). Philip Morris requested that Zoecon ask for an even higher MRL of 15 ppm to allow for application errors (Greenberg and Transon 1992; McCuen 1992). Zoecon representatives met with government authorities and requested a change to 15 ppm (Hutney 1991).\nA Zoecon representative informed Philip Morris that \u201cin order to avoid surprises of this nature in the future,\u201d he had directed Zoecon\u2019s pharmaceutical group to obtain information from health authorities in other countries regarding the commodities for which methoprene tolerances were assigned (which could include foods such as rice and mushrooms as well as tobacco) (Hutney 1991). Assigning this task to the pharmaceutical group instead of the pesticide group, the Zoecon representative wrote, \u201cwill not arouse the curiosity of the health directorates and will allow us to keep our promise to the tobacco industry, namely, that we won\u2019t initiate queries that may cause the health authorities to direct attention to tobacco\u201d (Hutney 1991).\nIn April 1992, George Lindahl of Zoecon faxed a letter to Bob McCuen, head of Philip Morris\u2019s biochemical research, outlining some of his concerns about Philip Morris\u2019s approach to establishing MRLs for methoprene on tobacco (Lindahl 1992b). In regard to Zoecon\u2019s effort to establish an MRL of 15 ppm in Malaysia, Lindahl explained that\nI know we simply argued this case without any data to support our request. In more advanced countries, this tactic will not succeed. \u2026 All our data demonstrate the need for a 10 ppm MRL. If a higher value is desired then we will require data from real field operations showing that a worse [sic] case scenario for faulty application will result in a 15 ppm residue, and hence the need for this value. (Lindahl 1992b)\nIn a fax following this one, Lindahl asked Philip Morris to provide such data; a handwritten comment from a Philip Morris employee who reviewed the fax noted that \u201cdata doesn\u2019t [sic] exist\u201d (Lindahl 1992a). Initially, the Malaysian authorities agreed to increase methoprene\u2019s MRL to 10 ppm (Lindahl 1992c); subsequently, it was raised to 15 ppm (Mueller and Ward 1998). Philip Morris continued to advocate (through Zoecon) for MRLs of 15 ppm in Italy and Germany (Greenberg and Transon 1992).\nIn the meantime, anticipating the creation of a single European market with uniform pesticide regulations, Philip Morris had asked the longtime tobacco industry law firm, Shook, Hardy, and Bacon, to prepare a document with MRL recommendations for possible submission to the European Community (Kemna 1991). Philip Morris first provided a draft of recommended MRLs to the Scientific Working Group of the Confederation of European Community Cigarette Manufacturers (CECCM) (Philip Morris 1991c). At their June 1991 meeting, members of this group (including representatives of Philip Morris, British American Tobacco, R.J. Reynolds, Gallaher, and Rothmans) recommended that the document be rewritten as a voluntary code of practice \u201cto be used pre-emptively \u2026 in advance of any EC [European Community] initiative\u201d to impose formal regulations on pesticide residue limits on tobacco (Philip Morris 1991a). A meeting participant reported, \u201cIt is hoped that, by implementing this Code, the EC Commission would not any longer see the need to develop a formal EC regulation on pesticide residues in tobacco (products)\u201d (Mueller 1991). Manuel Bourlas, Philip Morris\u2019s director of research and development, was appointed chair of a subgroup of tobacco company representatives who were to assist in preparing the code (Philip Morris 1991a).\nThis voluntary code underwent numerous revisions throughout 1991 and 1992 (CECCM 1991, 1992a, 1992b, 1992c, 1992d, 1992e; Philip Morris 1991b). Although 236 regulated and unregulated tobacco pesticides were in use at the time (Mitchell 1991b), the voluntary code proposed MRLs for only 25\u201327 pesticides [including chlordane, dichlorodiphenyltrichloroethane (DDT), lindane, dithiocarbamates, methoprene, and maleic hydrazide]. According to British American Tobacco\u2019s Terry Mitchell, \u201cmany of the substances in the list are no longer recommended for tobacco production\u201d (e.g., DDT) (Mitchell 1991a). Moreover, this list did not impose \u201cany constraint automatically on non-specified substances\u201d (Mitchell 1991a). Mitchell noted that this lack of limits was \u201chighly desirable\u201d (Mitchell 1991a).\nIn December 1992, Walter Russell, a legal assistant, reported that the code \u201chas undergone two more revisions (by SHB) [Shook, Hardy, and Bacon] and it [is] currently watered down, but still causing much agitation\u201d (Philip Morris 1992). Russell pointed out that the code set MRLs that Philip Morris \u201cmight have trouble complying with\u201d if they were to become international standards (Philip Morris 1992). In addition, \u201cfailure to comply with tolerances written by the tobacco industry which might come up during litigation would put the tobacco industry at great disadvantage\u201d (Philip Morris 1992). He indicated that Philip Morris had decided to withdraw its support from the voluntary code (Philip Morris 1992). In 1993, the tobacco companies suspended work on the document due to \u201cprinciple disagreements both within and between participating companies\u201d (R.J. Reynolds 1993). Throughout the 1990s, the tobacco industry continued to anticipate European Union harmonization of tobacco pesticide MRLs (Philip Morris 1995); as of April 2004, the European Union had established community-level MRLS for 150 pesticides, but none specifically applied to tobacco (European Union 2004).\nEBDC Fungicides\nIn 1987, the U.S. EPA initiated a review of EBDC fungicides, prompted by the agency\u2019s determination that a breakdown product of EBDCs, ethylene thiourea (ETU), was a probable human carcinogen (U.S. EPA 1987). Anticipating the U.S. EPA\u2019s cancellation of many EBDC uses, U.S. manufacturers voluntarily withdrew EBDC registrations for all but 13 food crops in 1989, including wheat and corn (U.S. EPA 1989). At least one company continued to hold registrations for EBDCs on tobacco, but only for seed bed use, not plants (Arce 1989).\nIn internal documents, the tobacco industry expressed concern that the U.S. EPA\u2019s action could result in the \u201cimposition of potentially crippling product residue tolerances\u201d in Europe [Centre de Coop\u00e9ration pour les Recherches Scientifiques Relatives au Tabac (CORESTA) 1989b; Mitchell 1990]. EBDCs were regarded as vital to control blue mold outbreaks in Europe (Philip Morris 1990a). In October 1989, members of CORESTA, an international tobacco research organization with members drawn largely from the tobacco industry, established a subcommittee to \u201cprovide regulatory agencies with a sound basis for the development of tobacco agro-chemical regulations\u201d (CORESTA 1989a, 1989b).\nAs discussed in a larger World Health Organization (WHO) report on tobacco industry influence at that agency, the subcommittee hired a consultant, Gaston Vettorazzi, to provide advice on influencing regulation (CORESTA 1990b; Zeltner et al. 2000). Vettorazzi was a former WHO toxicologist and former technical secretary of the Joint Food and Agriculture Organization\/WHO Meeting on Pesticide Residues (JMPR), an international meeting of scientists whose decisions often formed the basis of international law (Zeltner et al. 2000). Selected partly for his \u201cold boys\u2019 contacts\u201d (Reif 1991b), Vettorazzi\u2019s initial duties were to provide a review and analysis of toxicologic data on EBDCs and ETU (CORESTA 1990a).\nSome CORESTA members were concerned that Vettorazzi\u2019s review might conclude that EBDCs were unsafe (Beuchat 1990). However, according to one member\u2019s notes, at his first meeting with the subcommittee in April 1990, Vettorazzi stated that \u201csomeone has to lay the red carpet for [me], otherwise [I] can spoil more than help\u201d (Reif 1990).\nVettorazzi\u2019s initial review concluded that ETU was neither carcinogenic nor genotoxic (Vettorazzi 1991a). Some of the tobacco industry scientists commented that this statement was \u201ctoo strong in light of the NTP feeding studies\u201d\u2014a reference to the U.S. National Toxicology Program\u2019s conclusion that animal studies showed clear evidence of ETU\u2019s carcinogenicity (Reif 1991a). Vettorazzi subsequently revised his conclusions, stating that ETU\u2019s \u201ctoxicity, including carcinogenicity, can be explained by the known mechanisms of action characteristic of thyroid-function inhibiting agents\u201d (Vettorazzi 1991b). Thus, he stated, a threshold could be set below which ETU did not cause thyroid tumors (Vettorazzi 1991b).\nCORESTA authorized the distribution of Vettorazzi\u2019s revised report to his former colleagues at WHO, once all references to tobacco and CORESTA were removed (CORESTA 1992). WHO\u2019s JMPR was scheduled to review EBDCs\/ETU in 1993; if this review were favorable, the tobacco industry would be assured continued access to EBDCs in Europe (Zeltner et al. 2000).\nWith CORESTA funding ($100,000 a year) and approval, Vettorazzi offered to assist J. Herrman, of the JMPR WHO Secretariat, with JMPR toxicologic reviews, without disclosing his tobacco industry ties (Herrman 1991; Vettorazzi 1991c, 1992a). Vettorazzi wrote and reviewed several working papers on compounds to be discussed at the 1992 JMPR, including the EBDC thiram (Herrman 1992; Vettorazzi 1992b). One outcome of that meeting was the reestablishment, at a higher level, of the previously cancelled Acceptable Daily Intake (ADI) for thiram (Vettorazzi 1992b).\nVettorazzi continued his work with WHO in 1993, supplying his CORESTA-funded reviews to the adviser responsible for drafting the working paper that would form the basis of the September JMPR on EBDCs\/ETU without revealing their sponsor (Zeltner et al. 2000). Vettorazzi also attended the September meeting as an invited \u201ctemporary adviser\u201d (Zeltner et al. 2000). The meeting\u2019s outcome reflected Vettorazzi\u2019s conclusions. In contrast to the U.S. EPA, JMPR determined that ETU was not genotoxic, and thus raised the ADI level from 0.002 to 0.004 mg\/kg body weight (Black 1993). CORESTA considered this \u201ca very positive result for the industry,\u201d since it \u201cclearly indicates that the \u2018carcinogenicity\u2019 of [ETU] is not really a burning issue any longer\u201d (CORESTA 1994; Mueller 1993). JMPR\u2019s safety standard became part of international trade law, preserving tobacco industry access to EBDCs (Zeltner et al. 2000). Soon after the JMPR meeting, CORESTA extended Vettorazzi\u2019s contract for 18 months, listing one of his duties as providing \u201cinformation about the activities of pesticide action groups\u201d (CORESTA 1993). He was to be paid another $100,000 (CORESTA 1993). Vettorazzi continued working for CORESTA until at least 2001, when the organization paid him $30,000 to monitor international activities related to tobacco pesticide residues and registrations (CORESTA 2001).\nPhosphine\nPhosphine is a fumigant used on stored commodities, including nuts, seeds, grains, coffee, tobacco, and finished cigarettes to kill insects. Because of the risks it poses, applicators are advised to wear respirators and protective clothing, and warehouses must be sealed to prevent leaks that contribute to air pollution and endanger nearby residents (U.S. EPA 1998b). By the early 1990s, several case reports had been published noting sometimes fatal phosphine poisoning among workers and community members (Garry et al. 1989, 1993; Heyndrickx et al. 1976; Schoonbroodt et al. 1992; Wilson et al. 1980).\nIn December 1998, the U.S. EPA proposed a series of 15 risk mitigation measures (RMMs) for phosphine. The U.S. EPA\u2019s primary concern was the risk that phosphine posed to applicators and community residents (U.S. EPA 1998b). Thus, the RMMs included a threshold limit value of 0.03 ppm of phosphine during fumigation (reduced from the existing 0.3-ppm standard), the establishment of a 500-foot buffer zone around all fumigated structures, and prior notification of all residents living within 750 feet of a fumigated structure (U.S. EPA 1998a).\nThe Tobacco Association of the United States, in a letter to the U.S. EPA, stated that the economic burdens imposed by the RMMs would \u201cmake it virtually impossible for our industry to continue to fumigate stored tobacco\u201d (Ward 1999). The Tobacco Association, R.J. Reynolds, Philip Morris, and > 150 other organizations with a stake in the continued use of phosphine formed a lobbying group, the Commodity Industry Coalition for Phosphine Fumigation (Harrell 1999).\nR.J. Reynolds, represented primarily by toxicologist Joel Seckar, took an active role in the Commodity Industry Coalition (Seckar 1999c). The company calculated that complying with the U.S. EPA\u2019s buffer zone requirement would cost approximately $50 million in new land and warehouse purchases (R.J. Reynolds 1999a). Increasing the time required to aerate warehouses before employee reentry to comply with the worker exposure limit of 0.03 ppm would increase costs, as would the possibility of liability suits brought by nearby residents notified of phosphine use (Degesch America 1998; R.J. Reynolds 1999d).\nCoalition members lobbied Congress, released media statements, worked closely with the U.S. Department of Agriculture, and attended U.S. EPA-sponsored stakeholder meetings (Goldman 1998; Lyon 1999; R.J. Reynolds 1999b, 1999c). Their message was that the proposed RMMs were overly conservative, based on \u201canecdotal information and hypothetical risk\u201d rather than on \u201csound science\u201d (Lyon 1999; Ong and Glantz 2001). To challenge the scientific basis of the U.S. EPA\u2019s proposals, the coalition decided to hire an expert whose research would support existing standards (Seckar 1999h). They chose Sciences International, a consulting firm specializing in health and environmental risk assessment. It was headed by Elizabeth Anderson, a former director of the Carcinogen Assessment Group and the Office of Health and Environmental Assessment at the U.S. EPA (Sciences International 2005). She was also an experienced expert defense witness, having served in that capacity in a number of environmental lawsuits brought against corporations (Anderson 1999c).\nTo support the Commodity Industry Coalition\u2019s assertion that the proposed exposure level of 0.03 ppm was too conservative, Sciences International focused on the inter-species uncertainty factor. The U.S. EPA had first determined from a published subchronic toxicity study of rats that there were no observed effects attributable to inhaled phosphine at 3 ppm (Seckar 1999a). To extrapolate to humans, the U.S. EPA had then used two 10-fold uncertainty factors, one for intraspecies variability and one for interspecies variability, to arrive at a maximum exposure level of 0.03 ppm (Sciences International 1999c). Documents indicate that Sciences International\u2019s strategy was to convince the U.S. EPA that the interspecies uncertainty factor was unnecessary, showing that because a number of animal species reacted in the same manner to phosphine, humans were similar enough that the interspecies uncertainty factor could be removed (Seckar 1999a, 1999b). This would leave only the intraspecies factor of 10, which would result in a maximum exposure level for humans of 0.3 ppm, the existing standard.\nIn April 1999, the U.S. EPA representatives met with a small group of Commodity Industry Coalition members, including R.J. Reynolds\u2019s Seckar and Sciences International\u2019s Anderson (Seckar 1999a). Anderson questioned the U.S. EPA\u2019s interspecies uncertainty factor, citing several animal studies and an epidemiologic study to suggest that the U.S. EPA\u2019s calculations were too conservative (Seckar 1999a). In an e-mail, Seckar noted that Anderson\u2019s presentation was very effective, as evidenced by the fact that U.S. EPA representatives were now informing coalition members that the 0.03 ppm standard \u201cwas not \u2018set in stone,\u2019\u201d a direct contradiction of earlier statements to the U.S. Department of Agriculture (Bair 1999; Seckar 1999d). (Despite Freedom of Information Act requests, we were unable to obtain U.S. EPA documents related to its meetings with the coalition.)\nSoon after, Sciences International asked the Commodity Industry Coalition for additional funding to turn its phosphine report into a peer-reviewed journal article (Turim 1999). In a memo to Seckar, Anderson (1999b) explained that\nMy experience is that consultant reports funded by those being regulated, and written expressly for the EPA, are easily and frequently ignored or dismissed by the Agency, no matter how scholarly. However, a paper or article that is peer-reviewed and published, or in the peer review process for publication, in an accepted scientific journal can neither be ignored nor dismissed.\nAnderson suggested that since she was editor-in-chief of Risk Analysis, \u201cperhaps the peer review process could be expedited if we decide that it is the journal of choice\u201d (Anderson 1999b). R.J. Reynolds, Brown and Williamson, and several other tobacco companies agreed to fund most of the cost of this work (Seckar 1999e). The paper was published in Risk Analysis in 2004, with the acknowledgment that \u201cThis work was supported by the Phosphine\/Metal Phosphide Coalition, consisting of the producers and users of phosphine and metal phosphides for the control of insects in stored commodities\u201d (Pepelko et al. 2004).\nCoalition members also pursued other strategies. At a meeting with U.S. EPA representatives in March 1999, the Commodity Industry Coalition proposed that the U.S. EPA participate in a series of small, coalition-sponsored focus groups to \u201ceducate [EPA] on the issues involved with \u2026 fumigations\u201d (Seckar 1999g). One such group met in May 1999, when tobacco companies demonstrated a tobacco warehouse fumigation (Ward and Cowan 1999). The following month, several companies conducted additional emissions tests to show that the proposed 500-foot buffer was unnecessary (Bridges 1995). However, an e-mail message from a Philip Morris employee indicated that Philip Morris\u2019s test coordinator had \u201csome reservations regarding the quality of the test design\/data generation\u201d and that he himself believed that \u201cthe test plan and methods will provide, literally, no information, so it won\u2019t hurt us to do it\u201d (Bridges 1995).\nIn June 1999, Sciences International submitted a first draft of its phosphine toxicity review to some coalition members (Sciences International 1999a). A reviewer from the coalition\u2019s lobbying firm pointed out that the animal studies cited did little to support the idea that the interspecies uncertainty factor should be eliminated \u201csince most [of the animals] appear to be rat or mouse strains with similar breathing characteristics\u201d (Wilkinson 1999). Instead, the studies cited by Sciences International seemed to support the idea that phosphine called for a conservative standard, as they indicated that \u201cphosphine is a very toxic material to most species tested\u201d (Wilkinson 1999). Another reviewer noted that the uncertain and tentative tone of the report \u201cwill trigger concerns by EPA and they will say \u2018if [an] expert in the field states that there remains great uncertainty, maybe we are on solid ground by being very conservative\u2019\u201d (Barolo 1999a). Sciences International staff revised the report, removing tentative statements and asserting that their work to date supported reducing the interspecies uncertainty factor to 1 (effectively eliminating it), thus preserving the existing exposure standard of 0.3 ppm (Sciences International 1999b). They submitted this revised interim report to the U.S. EPA in July 1999 (Sciences International 1999b). At a Commodity Industry Coalition meeting that same month, coalition consultant Dan Barolo, former director of the U.S. EPA\u2019s Office of Pesticide Programs (OPP), reportedly urged members to speed their efforts because\nphosphine is quite hazardous when used improperly. The more the Coalition slows the process, the greater the chance for an accident with possible fatalities, which would send EPA back into conservative mode and make it far more difficult for them to publish reasonable RMMs. (Seckar 1999f)\nIn August, John Whalan, a toxicologist at the U.S. EPA\u2019s Health Effects Division, summarized in a memo his analysis of Sciences International\u2019s interim report (Whalan 1999). He noted that\nthere is no precedent for using an [interspecies uncertainty factor] of 1 when establishing \u2026 an inhalation regulatory value in the Health Effects Division. The only time an interspecies [uncertainty factor] is not applicable is when human data are used. The available data do not support deviating from Agency policy, and the Coalition did not provide any new data. (Whalan 1999)\nHe also pointed out that Sciences International\u2019s review of animal studies, intended to show that phosphine toxicity was relatively constant across species, was largely \u201cirrelevant\u201d because it did not include a comparison of toxicity for a small versus large mammal.\nIn September 1999, phosphine registrants and several coalition members again met with U.S. EPA officials to discuss alternative RMMs proposed by the coalition (Seckar 1999i). Instead of a 500-foot buffer and a 750-foot neighbor notification requirement, the coalition recommended a \u201csite management plan\u201d that required companies to develop emergency preparedness measures. The U.S. EPA asked the Commodity Industry Coalition to reword its proposals to specify how and when workers and bystanders would be informed of danger (Seckar 1999i). On the exposure limit for workers, the U.S. EPA now proposed a 0.1-ppm standard (reflecting a reduction from 10 to 3 in the interspecies uncertainty factor) based upon Sciences International\u2019s interim report (despite the weaknesses noted by Whalan) (Seckar 1999i). (The U.S. EPA failed to provide memos or notes regarding this decision.)\nIn several fall 1999 memos to Seckar, Sciences International staff explained that they thought it would be difficult to convince the U.S. EPA to drop the interspecies uncertainty factor without human exposure studies (Anderson 1999a; Gray 1999). Commodity Industry Coalition members expressed reluctance to commit to human studies without confirmation that this would convince the U.S. EPA to \u201cgive up\u201d the uncertainty factor (Barolo 1999b). Barolo commented to Seckar, \u201cI do not believe it will be easy for OPP to abandon both safety factors. There are too many unknowns from children to endocrine to reliability of studies to absence of dog\/monkey study. \u2026 Some day they are going to figure out there is a 0.1 ppm standard in other countries and the door will close\u201d (Barolo 1999c).\nAlthough Sciences International had not yet submitted to the U.S. EPA its full report on phosphine, in December 1999, the U.S. EPA made its final decision (Sharp 1999). (This decision was published in the Federal Register in February 2001 [U.S. EPA 2001]). The U.S. EPA now mandated a \u201cfumigation management plan\u201d like that proposed by the Commodity Industry Coalition (U.S. EPA 2000). The agency also eliminated the inter-species safety factor and left the old 0.3-ppm standard in place, on condition that phosphine registrants conduct additional research if Sciences International\u2019s review was found to be inadequate (U.S. EPA 2000). A coalition member noted that \u201cit is important to point out that this additional work will take years and that the current 0.3 ppm threshold will stay in place during that time\u201d (Sharp 1999). R.J. Reynolds credited its leadership on the scientific issues with saving the company \u201cmany millions of dollars\u201d (R.J. Reynolds 2000).\nConclusion\nAlthough others have charged that agencies responsible for protecting human health and the environment are unduly influenced by the industries they regulate (Abraham 2002; Huff 2002), it is rare to be able to study this process from the perspective of the regulated industry. This study provides documentation of the behind-the-scenes activities of an industry as it attempts to influence the regulatory process on matters that have a direct bearing on public health.\nOur analysis has limitations. Given the sheer volume and limited indexing of the documents, it is impossible to ensure that we located all potentially relevant documents. Some may have been destroyed or concealed by the tobacco companies (Liberman 2002); others may have never been obtained in the legal discovery process. In addition, we had no access to pesticide company documents, except those in the tobacco documents archives. Finally, despite properly filed Freedom of Information Act requests, we were unable to obtain from the U.S. EPA documentation of its meetings with the industry\u2019s Commodity Industry Coalition. All minutes of meetings with stakeholders should be part of the public record.\nDespite these limitations, the case studies discussed here provide insight into tactics that the tobacco industry applies to a regulatory agency when trying to influence the outcome of a decision. These tactics go significantly beyond the usual approaches\u2014such as participation in public comment periods and public meetings\u2014to influence scientific and regulatory decision making. Tobacco industry tactics described in these cases include:\nEncouraging a chemical company (Zoecon) to advocate for high MRLs without any supporting data and directing that same company to gather information about international regulatory efforts on methoprene in a manner designed to hide the interest of the tobacco industry in this chemical;\nAttempting to forestall regulatory efforts on tobacco pesticides in the European Community by creating voluntary industry MRLs for a subset of chemicals;\nHiring an ex-WHO scientist to participate (without disclosing his funding source) in the WHO regulatory effort on EBDCs;\nHiring several ex-U.S. EPA scientists to influence the U.S. EPA\u2019s regulatory decision making on phosphine;\nHiring scientific consultants with instructions to marshal data to support the tobacco industry\u2019s a priori arguments and funding consultants to publish a report supporting these arguments in a journal over which the consultants had influence;\nStaging fumigations for the U.S. EPA with the knowledge that the methodology was flawed and the results would show no emissions problem.\nYet, as the case of European MRLs showed, the tobacco industry does not always work together effectively to influence regulations. Tobacco companies may disagree about regulatory strategies or conclude that inaction is preferable to action that might have unintended consequences. Moreover, the fact that even voluntary, industry-friendly pesticide guidelines posed significant problems for Philip Morris underscores tobacco industry motivation for resisting or influencing more stringent, government-imposed regulations.\nThis study also raises questions about industry influence over regulatory agencies. In the case of WHO deliberations on EBDCs, the tobacco industry coordinated covert actions, hiding the financial ties and involvement of CORESTA. Rigorous disclosure requirements and oversight might have allowed the WHO\u2019s agencies to judge more accurately the potential for bias related to conflicts of interest. In the case of the U.S. EPA\u2019s review of phosphine, a regulatory agency appears to have been quite willing to cooperate with the industry and its consultants. This is a reminder of why regulatory processes were designed to be transparent and open to the public, and why \u201cclosed-door\u201d meetings between regulators and industry have been ruled illegal (Federal Advisory Committee Act 1972; Registration Standards 2004; Special Review Procedures 2002).\nProtection of the public interest hinges on an open process and regulatory agencies\u2019 willingness to stand up to pressure from regulated industries. When these are in doubt, public confidence in the fairness and efficacy of regulations may be unwarranted. The resource disparities between powerful industries and public health organizations may also make it difficult to ensure that the public interest is fairly represented, particularly when discussions occur behind closed doors, as apparently occurred at the U.S. EPA. Increased public and media scrutiny of these processes could help ensure that public health considerations are weighed at least as heavily as commercial ones.\nFinally, given the deadly epidemic of tobacco-caused disease, which kills an estimated 5 million people annually worldwide (WHO 2004), is it in the public interest for regulatory agencies today to continue facilitating standards that make it easier and less costly to grow, transport, store, and manufacture tobacco products?","keyphrases":["tobacco industry","pesticide regulation","environmental protection agency","methoprene","ethylene bisdithiocarbamates","phosphine","world health organization"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Purinergic_Signal-4-2-2377325","title":"Purinergic signalling in the subretinal space: a role in the communication between the retina and the RPE\n","text":"The retinal pigment epithelium (RPE) is separated from the photoreceptor outer segments by the subretinal space. While the actual volume of this space is minimal, the communication that occurs across this microenvironment is important to the visual process, and accumulating evidence suggests the purines ATP and adenosine contribute to this communication. P1 and P2 receptors are localized to membranes on both the photoreceptor outer segments and on the apical membrane of the RPE which border subretinal space. ATP is released across the apical membrane of the RPE into this space in response to various triggers including glutamate and chemical ischemia. This ATP is dephosphorylated into adenosine by a series of ectoenzymes on the RPE apical membrane. Regulation of release and ectoenzyme activity in response to light-sensitive signals can alter the balance of purines in subretinal space, and thus coordinate communication across subretinal space with the visual process.\nIntroduction\nThe retinal pigment epithelium (RPE) lies between the outer segments of the photoreceptors and the choroidal blood supply (Fig.\u00a01). The RPE combines the functions of epithelial and glial cells, providing a barrier while also supporting the neural photoreceptors and modulating their function. Tight communication between photoreceptors and the RPE is critical to coordinate the multiple levels of interaction, and the purinergic contribution to this communication is becoming apparent. The relevance of this purinergic input is emphasized by the many functional effects of P1 and P2 receptor stimulation and by the multiple mechanisms in place to regulate subretinal levels of purine agonists. As the dynamics of ATP release and extracellular conversion into adenosine will modify agonist availability, the modulation of these processes can exert a temporal control on purinergic signaling. The following review will outline the main interactions between the RPE and photoreceptors, describe the effects of stimulating purinergic receptors on both sides of subretinal space, and summarize how levels of ATP, ADP, and adenosine are manipulated in this microenvironment.\nFig.\u00a01Schematic illustration of the key components of purinergic signaling in the subretinal microenvironment. Stimulation of P2 receptors on the RPE can enhance transepithelial fluid absorption while P1 receptors can modulate phagocytosis. ATP released through CFTR and other Cl\u2212 channels can stimulate P2 receptors or be converted to ADP, AMP, and adenosine (Ado) by a series of ectonucleotidases present on the apical membrane of the RPE. By controlling the balance of extracellular purines available to stimulate these receptors these mechanisms can control levels of endogenous purines available to activate the receptors. While theoretically possible, it remains to be determined whether these subretinal purines can actually stimulate photoreceptors\nPurines and subretinal space\nRPE-photoreceptor interactions across the subretinal space\nThe outer segments of the rods and cones are responsible for the initial stages of vision, converting photon energy into a series of enzymatic reactions that close the light-sensitive channels on the photoreceptor plasma membrane, hyperpolarize the cells, and reduce the release of glutamate from the synaptic terminals [1, 2]. Efficient photoreceptor function depends upon both short-term and long-term support from the RPE. The critical nature of these interactions is evident from the rapid degeneration of photoreceptors in the absence of a healthy RPE layer and by the RPE localization of defective gene product in some forms of hereditary photoreceptor degeneration [3].\nThe apical membrane of the RPE is separated from the plasma membrane of the outer segments by an extracellular space of only 10\u201320\u00a0nm [4]. Although small, this subretinal space contains a highly structured matrix which ensheathes the outer segments and extends to the RPE [5, 6]. The presence of enzymes within this interphotoreceptor matrix emphasizes that this extracellular space itself is functionally active [7, 8]. This intimate anatomical relationship between photoreceptors and the RPE reflects multiple functional interactions. For example, the RPE accepts, recycles, and exports central components of the phototransduction process [9]. The outer segments are continuously resynthesized, and the phagocytosis, degradation, and processing of shed outer segment tips by the RPE cells is central to this renewal [10]. The ion channels and transporters on the apical membrane of the RPE help regulate the ionic composition of the subretinal space [11]. As extracellular levels of ions can modify the ionic driving forces across the photoreceptor plasma membrane, these RPE transporters can influence the state of neural activity. The transport of fluid and ions from the apical membrane to basolateral membrane of the RPE is also one of the main forces keeping the retina attached [12].\nThe control of photoreceptor activity by light gives a rapid temporal dependence to some interactions between the photoreceptors and the RPE. The volume of subretinal space increases in response to light [13], linking small changes in the ionic composition of the subretinal space with activity of the RPE transport mechanisms which maintain this volume [14, 15]. Other processes are controlled on a diurnal cycle. The shed tips of the outer segments are phagocytosed by the RPE soon after the onset of light [16, 17]. These processes can both be modulated by purine levels in subretinal space, indicating purinergic regulation is important over multiple time scales.\nPurinergic receptors on photoreceptors\nA2 adenosine receptors were localized to both the inner and outer segments of photoreceptor outer segments over a decade ago by Blazynski and colleagues [18], with more recent reports emphasizing their functional role. A2 agonists inhibit the L-type Ca2+ channel on rod outer segments [19] and can inhibit the synaptic release of glutamate from rods, suggesting changes in adenosine levels in subretinal space could modulate light sensitivity [20]. The A2 agonist DPMA and the adenosine deaminase inhibitor EHNA reduce mRNA for opsin in rods, suggesting that endogenous levels of adenosine can downregulate opsin message at night [21]. EHNA and the A2A receptor agonist CGS21680 also increase the survival of chick embryonic photoreceptors in culture [22], indicating a long-term neuroprotective role for adenosine.\nP2 receptors are also present in the photoreceptors. mRNA for the P2X2 receptor is expressed in the photoreceptor cell bodies, with immunohistochemical localization of the protein to outer segments [23]. In situ hybridization indicates the photoreceptor layer has the highest level of P2Y2 receptor of any region in the rabbit retina, although staining was not pronounced in monkey [24]. P2X7 receptors have recently been localized to photoreceptor synaptic terminals, with evidence for ecto-ATPase activity in the synapse, and functional evidence suggesting ATP augments transmission of the light response by rods [25]. It was suggested that ATP might be co-released from photoreceptors with glutamate, although this remains to be tested directly.\nPurinergic receptors on the RPE\nStimulation of P1 receptors can have a considerable impact on RPE cells. A2 receptors have been recognized on cultured and fresh RPE cells for some time [26, 27], with in situ hybridization confirming the presence of A2A receptors in rat RPE [28]. Stimulation of A2 receptors reduces the rate of rod outer segment phagocytosis by RPE cells [29], while application of adenosine to the apical membrane of chick RPE cells increases the basolateral Cl- conductance, the transepithelial potential, and the c-wave, and decreases the hyperpolarization of the basal membrane in response to light [30]. Although adenosine alone does not increase intracellular Ca2+ levels [31], adenosine acts synergistically with ATP to elevate Ca2+ levels in human RPE cells by stimulating both A1 and A2A receptors [32, 33]. Stimulation of A1 receptors with high doses of NECA increases the active transport of fluorescein across the RPE, while activation of A2A receptors decreases this transport, and by extension, transport of the ions that underlie fluid movement [34]. Stimulation of A1 and A2A receptors produces analogous increases and decreases, respectively, in the absorption of subretinal fluid blebs. This is consistent with the negative coupling of the A1 receptor and the positive coupling of the A2 receptors to adenylate cyclase, as increasing cAMP inhibits the transport of fluid across the RPE towards the choroid [35\u201337]. The agonist 2-Cl adenosine reverses the deficit in phosphoinositide metabolism found in diabetic RPE cells [38], suggesting effects on metabolism in addition to transport and phagocytosis.\nMultiple P2 receptors have been localized to the RPE. The P2Y2 receptor was initially characterized in cultured human RPE [31], with subsequent reports localizing transcript for P2Y1, P2Y2, P2Y4, and P2Y6 in the rat RPE\/choroid [39] and for P2Y1 and P2Y12 receptors in ARPE-19 cells [40], and functionally identifying a P2X receptor in rat RPE cells [41]. ATP, ADP, and UTP induce numerous effects on RPE physiology [32, 33, 42, 43]. While these effects likely involve multiple different receptor types, the contributions of the P2Y2 receptor have been explored in most detail to date. The P2Y2 receptor has been specifically localized to the apical membrane of fresh bovine RPE cells, and addition of ATP to this membrane transiently elevates Ca2+, activates a basolateral Cl- conductance, inhibits an apical K+ conductance, and increases the apical to basolateral flow of fluid [43]. This increased absorption of fluid from the subretinal space suggests P2Y2 receptor stimulation could reduce the excess fluid that accumulates in retinal edemas, and several reports have supported this theory. ATP, UTP, and the P2Y2 receptor agonist INS37217 decrease the size of subretinal fluid blebs when injected into subretinal space of rats [44]. In both normal and rds +\/- mice with experimentally induced detachment, INS31217 improves the ERG recovery and decreased cell death [45]. INS37217 also reduces subretinal blebs in rabbits [46]. Injection of another P2Y2 agonist, INS542, increases the active transport of fluorescein across the RPE, consistent with this upregulation of ion and fluid transport across the tissue [47]. Together these experiments emphasize the clinical potential of treating retinal edema with P2Y2 agonists.\nRegulation of purine levels in subretinal space\nWhile synthesized purinergic agonists may prove useful in treating some ocular disorders, the endogenous activation of P1 and P2 receptors in the subretinal microenvironment will ultimately be determined by availability of agonists. These levels are largely controlled by the release of ATP into the subretinal space, its conversion into other purines including adenosine, and the manipulation of adenosine by enzymes or transporters. Recent work has increased our understanding of both the stimuli that initiate changes in subretinal purine levels and the mechanisms that mediate these changes.\nRelease of ATP by the RPE\nAt least some of the ATP capable of stimulating the purinergic receptors on RPE cells is released from the RPE itself. The resulting autocrine stimulation ensures local delivery, and control, of purines to initiate the physiologic changes in the RPE. The release of ATP by RPE cells is triggered by multiple stimuli including osmotic stress, bFGF, UTP, NMDA, glutamate, and ATP [39, 40, 48\u201351]. The ATP release following activation of NMDA receptors by glutamate may have the most interesting implications for communication across subretinal space, given that glutamate confers the light signal from photoreceptors to the rest of the visual system. Glutamate and the specific receptor agonist NMDA triggers ATP release from ARPE-19 cells, with the release inhibited by NMDA antagonist MK-801, and by DCKA, which inhibits the glycine B binding site on NMDA receptors [51, 52]. Although NMDA raises intracellular Ca2+ levels, this increase is prevented by eliminating ATP with apyrase, indicating autostimulation through released ATP is responsible for this Ca2+ signal. NMDA also triggers a release of ATP when applied to the intact bovine RPE eyecup [51]. The NMDA receptors and the ATP release sites have been functionally identified to the apical membrane of the bovine RPE, suggesting the neurotransmitter interactions could amplify the signal from any glutamate reaching subretinal space.\nThe ability of both UTP and ATP to stimulate release of ATP from the RPE supports the theory that the system acts to amplify signals. When applied at greater than 1 \u03bcM, ATP triggers a secondary release of ATP peaking 10\u00a0min after the initial stimuli [40]. UTP also initiates a release in extracellular ATP with a similar delay [48]. The rise in ATP triggered by UTP is inhibited by the Cl- channel blocker NPPB, and UDP is much less effective at triggering release than UTP; both observations suggest the P2Y2 receptor contributes to the increase in ATP more than diphosphokinase, although influence from the enzyme cannot be ruled out [53].\nRecent evidence suggests that ischemia may lead to the release of ATP from RPE cells. Chemical ischemia triggers a substantial ATP release from cardiac myocytes [54], while changes in oxygen levels trigger ATP release in central chemoreceptors [55]. We found that exposure to sodium cyanide led to a rapid release of ATP from ARPE-19 cells (Fig.\u00a02). As hypoxic and\/or ischemic challenge may lead to changes in the expression of growth factors in RPE cells during certain ocular disorders such as macular degeneration [56], and as purines can induce expression of VEGF in other cells [57], this ATP release may contribute to growth factor signaling by the diseased RPE.\nFig.\u00a02Chemical ischemia triggers ATP release from ARPE-19 cells. ATP release was measured in the bath directly from cells plated in 96-well plates to which the luciferin- luciferase reaction mixture was added [51]. Left Levels of ATP in the bath after addition of 5\u00a0mM NaCN to the cells. Measurement began 1\u00a0min after addition of NaCN or control solution to wells. Right Levels of ATP measured at the peak, 3\u00a0min after addition of NaCN (n\u2009=\u200912). Levels were normalized to concurrent levels in control (n\u2009=\u200914). Symbols and bars represent mean \u00b1 SE, *p\u2009<\u20090.001\nThe particular conduit for ATP release varies with the stimuli. The release in response to hypotonic challenge is largely dependent upon CFTR, as it was prevented by the specific CFTR inhibitor CFTR172 in addition to the more general blocker glybenclamide [50]. While the precise mechanisms by which CFTR contributes to this release are not yet known, a role for CFTR in ATP release into subretinal space is consistent with the reduction of certain ERG components in cftr -\/- mice [58] and with the ability of apical ATP to activate conductances associated with these ERG components [43]. The release of ATP is also largely blocked by the vesicular transport inhibitor brefeldin A, suggesting the two processes occurred in series whereby ATP efflux follows the insertion of vesicles containing CFTR into the plasma membrane. Although the Ca2+ chelator BAPTA blocks this ATP release [50], raising Ca2+ alone with ionophore ionomycin does not itself initiate release [48]. This necessary but not sufficient contribution of Ca2+ also supports a role for vesicular insertion.\nIn contrast to the release following hypotonic challenge, the ATP release in response to NMDA does not involve CFTR [51]. Release is blocked by NPPB, however, suggesting another type of anion channel could serve as a conduit for ATP release. The presence of parallel mechanisms coexisting on the same cell for ATP release triggered by either agonists or by cell swelling has also been reported in astrocytes [59] and may reflect the multiple roles of purinergic signaling within a given tissue. As both stimuli lead to release across the apical membrane into subretinal space, both are expected to influence signaling in the microenvironment.\nInterconversion of purines in subretinal space\nThe interconversion of nucleotides and nucleosides each capable of stimulating distinct receptors makes the purinergic signaling system of particular interest in a confined region such as the subretinal space. The main enzymes responsible for dephosphorylating extracellular ATP on the RPE cells have been analyzed and a basic understanding of their regulation has begun. This section first describes the enzymes that act on ATP and ADP, followed by enzymes which convert AMP into adenosine.\nThe dephosphorylation of extracellular ATP by RPE cells involves enzymes from multiple families [40], as found in airway epithelial cells [60]. Degradation of ATP by the apical membrane of the fresh bovine eyecup and by ARPE-19 cells is inhibited by ARL67156 or \u03b2\u03b3mATP. Message for eNPP1, eNPP2, and eNPP3 is present in ARPE-19 cells, consistent with the preference of \u03b2\u03b3mATP for members of the eNPP family [61]. The cells also express NTPDase2, and NTPDase3, although the intermittent presence of NTPDase1 likely reflects a regulated process [40]. Ecto-alkaline phosphatase has no effect on ATP degradation in RPE cells, in contrast to its considerable contribution in airway epithelium [62]. The putative contribution from diphosphokinases to interconversion of subretinal purines is presently unknown.\nExtracellular AMP is rapidly dephosphorylated into adenosine in subretinal space. The production of adenosine from ATP at the apical membrane of the bovine RPE eyecup is inhibited by the ecto-5\u2032-nucleotidase inhibitor \u03b1\u03b2mADP, confirming a role for this enzyme [63]. The enzyme is localized to rat RPE and ARPE-19 cells immunohistochemically. Degradation of 5\u2032AMP is highest near the subretinal space of rat retina [63], although localization in mouse indicated larger amounts of ecto-5\u2032-nucleotidase at the tips of adjacent M\u00fcller cells [64]. Levamisole does not inhibit the dephosphorylation of 5\u2032AMP by the RPE, consistent with the absence of substantial ecto-alkaline phosphatase in subretinal space.\nThe presence of light may alter the levels of adenosine in subretinal space. Epinephrine is released at the onset of light [65] and stimulation of the RPE with epinephrine can decrease activity of ecto-5\u2032-nucleotidase [63]. While norepinephrine and phenylephrine lead to similar decreases in enzyme activity, prazosin and corynanthine block the effects of norepinephrine, implicating the \u03b11 epinephrine receptor in the inhibition of ecto-5\u2032-nucleotidase [63]. The kinetics of inhibition are consistent with cleavage of the nucleotidase from its GPI anchor. The phagocytosis of rod outer segments is maximal shortly after light onset [16], and this phagocytosis is inhibited by adenosine [29]. The ability of epinephrine released by the illuminated retina to reduce ecto-5\u2032-nucleotidase activity and consequently adenosine levels may relieve this inhibition and enhance the rate of phagocytosis at light onset.\nPhysiologic effects of subretinal purines on the RPE and photoreceptors\nThe number of purinergic receptors on both photoreceptor and RPE membranes suggests purines make multiple contributions to the physiology of the outer retina. Our increased understanding of how agonist levels in subretinal space are controlled has begun to indicate how and when this contribution may occur. Future research will involve applying these findings from isolated systems to intact RPE-photoreceptor models, and pursuing the role of defective purinergic regulation in ocular disease. While it is unlikely that ATP released across the apical membrane of the RPE can diffuse to these P2 receptors in the outer plexiform layer given the ecto-ATPase activity in the synaptic clef [25], stimulation of receptors elsewhere on the photoreceptor membrane is possible. It would be interesting to determine whether ATP released from the RPE and converted to adenosine by ecto-nucleotidases can actually modulate the response to light by stimulating the A2A receptors on photoreceptor outer segments. The impact of purinergic signaling on chronic ocular diseases is also of interest, such as the role of ischemia-driven ATP release in VEGF production. While the small size of subretinal space can complicate pharmacologic manipulation within the intact RPE-photoreceptor complex, molecular approaches may provide new insight into how endogenous purines in subretinal space affect the physiology, and pathophysiology, of both RPE and photoreceptors.","keyphrases":["rpe","photoreceptors","microenvironment","glutamate","atp release","cftr","ecto-5\u2032-nucleotidase","ntpdase"],"prmu":["P","P","P","P","P","P","P","U"]}
{"id":"Pediatr_Radiol-4-1-2292498","title":"Voiding urosonography with ultrasound contrast agents for the diagnosis of vesicoureteric reflux in children\n","text":"Voiding urosonography (VUS) encompasses examination of the urinary tract with intravesical administration of US contrast agent (UCA) for diagnosis of vesicoureteric reflux (VUR). The real breakthrough for US examination of VUR came with the availability of stabilized UCAs in the mid-1990s. This article presents a comprehensive review of various procedural aspects of VUS. Different US modalities are available for detecting the echogenic microbubbles: fundamental mode, colour Doppler US, harmonic imaging and dedicated contrast imaging with multiple display options. The reflux is graded (1 to 5) in a similar manner to the system used in voiding cystourethrography (VCUG). The most commonly used UCA for VUS, Levovist, is galactose-based and contains air-filled microbubbles. The recommended concentration is 300 mg\/ml at a dose of 5\u201310%, or less than 5%, of the bladder filling volume when using fundamental or harmonic imaging modes, respectively. There are preliminary reports of VUS using a second-generation UCA, SonoVue. Here the UCA volume is less than 1% of the bladder filling volume. There is no specific contraindication to intravesical administration of UCA. The safety profile of intravesical Levovist is very high with no reports of side effects over a decade of use in VUS.\nIntroduction\nDiagnostic imaging for vesicoureteric reflux (VUR) is a common procedure in children. Currently, three modalities are available for reflux diagnosis, namely voiding cystourethrography (VCUG), radionuclide cystography (RNC) and voiding urosonography (VUS). The last of these is carried out using US in combination with intravesical administration of US contrast agent (UCA). This allows the use of ionizing radiation to be avoided, which is not the case in RNC and VCUG. However, in VCUG a marked reduction in radiation dose has been achieved with the introduction of digital pulsed fluoroscopy.\nThe first attempts at the implementation of US for the diagnosis of VUR began in the mid-1970s. A comprehensive account of the evolution of this undertaking over the subsequent two decades has been presented by Darge [1]. The indirect methods for reflux diagnosis were based on US of the urinary tract, without administration of any kind of substance into the bladder. These included depicting various sonomorphological changes of the urinary tract as a result of VUR, detecting newly appearing or an increase in existing ureteral or pelvicalyceal dilatation during voiding and assessing ureteric jet changes with duplex and colour Doppler US. The direct means used to diagnose VUR required instilling different substances intravesically. The most frequently administered fluid was physiological saline solution. Ballooning of the renal pelvis during the filling of the bladder was the criterion for diagnosis of VUR. Application of air bubbles, by shaking the normal saline before administration or adding carbon dioxide, were also tried. US studies were also carried out, in which the empty bladder was solely filled with air. In addition to low diagnostic accuracy, all the above methods had major procedural drawbacks making them impractical for widespread integration into routine imaging.\nThe intravesical use of a UCA consisting of sonicated albumin (Albunex; Molecular Biosystems, San Diego, Calif.) for VUS in a child was first reported in 1994 [2]. Another UCA used in the past was Echovist (Schering, Berlin, Germany), which is composed of galactose with incorporated microbubbles [3]. Its very short imaging window of approximately 5\u00a0min, however, prevented its routine application. The breakthrough in US diagnosis of VUR in children came about the mid-1990s with the availability of UCAs containing stabilized microbubbles. Levovist (Levograf, Schering Spain, Madrid, Spain; SHU-508-A, Schering, Berlin, Germany) was the first such UCA to become available for clinical use in Europe. This opened the door for rapid development of VUS and its introduction as part of the routine diagnostic imaging option of VUR.\nA number of different names and acronyms have been put forward to denote US examination for the diagnosis of VUR using intravesical UCAs. These include simply \u201csonography\/ultrasound\u201d [4\u20136], \u201creflux sonography\u201d [7], \u201ccystography\u201d [8\u201310], \u201ccystosonography\u201d [11\u201315], \u201ccystourethrosonography\u201d [16] and \u201curosonography\u201d [17\u201323]. They are more often used in combination with one or more of the following terms: \u201cecho-enhanced\u201d, \u201ccontrast-enhanced\u201d and \u201cvoiding\u201d. Depending on the US technique employed further descriptions such as \u201ccolour Doppler US\u201d or \u201charmonic imaging\u201d are added. The most widely applied name \u201cvoiding urosonography\u201d with the abbreviation \u201cVUS\u201d was proposed for the first time in 2000 [24]. The selection of this particular designation was based on careful consideration of various factors. The prefix \u201curo-\u201d is used to denote the bladder, ureters and kidneys. \u201cSonography\u201d was chosen rather than \u201cultrasonography\u201d as it is a shorter form. Thus \u201curosonography\u201d correctly describes the fact that in this examination US of the bladder, ureters and kidneys takes place independent of the absence or presence of VUR. \u201cVoiding\u201d was selected instead of \u201cmicturating\u201d as it is used more commonly in the medical literature. Despite the fact that for VCUG x-ray contrast agent is necessary, this is not directly added to the name VCUG in the form of, for example, contrast-VCUG. Likewise, the terms \u201ccontrast\u201d or \u201cecho-enhanced\u201d were not added by default to the basic term \u201cvoiding urosonography\u201d. The abbreviation \u201cVUS\u201d is not only similar to the most common acronym used for the radiological reflux examination, namely VCUG, but had also not been used in the medical literature to denote something else [25]. For the sake of minimizing the confusion of names and facilitating communication, literature search and procedural standardization, the use of one name for the same procedure, \u201cvoiding urosonography\u201d (\u201cVUS\u201d) is recommended.\nThis article is part I of a comprehensive review of all currently available literature on VUS and prepares the ground for objective evaluation and decision-making. In this part (part I) a detailed procedural description is presented, including discussion of the pros and cons of the various examination steps and imaging modalities for VUS. The measures undertaken to optimize VUS are elaborated. Furthermore, studies dealing with adverse events of intravesical administration of UCAs are reviewed. In part II a review of comparative studies between VUS and RNC\/VCUG is presented [26].\nUS contrast agent\nThe most widely used UCA for VUS is Levovist. This first-generation UCA was introduced for intravenous (IV) use in the mid-1990s and from 1999 started to obtain approval for intravesical application in children successively in 13 European countries and Australia. Levovist consists of dry granules made of galactose and palmitic acid [27]. The granules easily disintegrate into microparticles upon preparation. They form porous structures, which are necessary for the formation of bubbles. Gas bubbles are formed within the pores during suspension of the granules, while dissolution of the galactose particles begins simultaneously. The Levovist bubble is a microbubble of air\u201465% nitrogen and 35% oxygen\u2014stabilized by palmitic acid. One gram of Levovist granules contains 999\u00a0mg d-galactose and 1\u00a0mg palmitic acid. The concentrations of microbubbles in freshly prepared batches (300\u00a0mg\/ml) are within the range of approximately 1\u20132\u00d7108 microbubbles per millilitre of suspension. The Levovist suspension should always be freshly prepared prior to intravesical administration. The steps of preparation, as outlined by the manufacturer, must be strictly adhered to in order to avoid any reduction of contrast enhancement due to improper handling [28]. The administration of freshly prepared UCA has to be carried out within 30\u00a0min [29].\nConcentration\nThe recommended concentration of Levovist for VUS is 300\u00a0mg\/ml [28]. This concentration has been used for VUS in most studies. A concentration of 200\u00a0mg\/ml seems to result in faster dissipation of microbubbles, as found in in-vitro experiments (unpublished data). In only three studies has a concentration of 200\u00a0mg\/ml been used [13, 15, 30]. Bosio initially used this concentration, but later changed to the higher one as the conspicuity of refluxing microbubbles in fundamental mode was inadequate [13]. In another study in adults with transplanted kidneys this concentration in combination with an increased volume, i.e. 10\u201315% of bladder filling, was used [30]. The combination of colour Doppler US with the burst technique administering 200\u00a0mg\/ml selectively in infants has been reported, but the results using just this concentration were not analyzed separately [15]. The utilization of the higher concentration of 400\u00a0mg\/ml has only been reported once without any additional advantages being mentioned [31]. Probably, the disadvantage would be an increase in UCA volume leading to an unwarranted increase in cost.\nDosage\nThe volume of UCA administered has been rather variable. Some have applied a fixed dose for all, independent of the patient\u2019s age, weight and bladder volume [4, 5]. In one study the volume was calculated in terms of body weight [13]. In most studies the volume of bladder filling is taken as the decisive factor [1, 12, 28]. This is very logical when considering intravesical administration of UCA and contrast enhancement of bladder content. It is possible to directly measure the bladder volume using the formula for an ellipse (length\u00d7width\u00d7height\u00d70.5], note the total volume of normal saline administered in the bladder and also calculate in a simple way the maximum bladder capacity: volume in millilitres = (age in years +2)\u00d730 [32]. Furthermore, using the bladder volume for calculation of UCA dose makes comparison between studies much easier.\nNakamura et al. [21] carried out simultaneously VCUG and VUS in 56 children. The fundamental (i.e. conventional) imaging modality was used for VUS. They initially administered Levovist into an almost empty bladder followed by continuous infusion of the radiographic contrast agent. They simultaneously monitored the appearance of reflux during VUS, relating it to the concentration of the UCA in the bladder. The concentration of UCA in the bladder at one point in time ranged form 1.8% to 23%. All false-negative results in the VUS were associated with a Levovist volume of <5% of the total bladder filling. Thus a volume of UCA between 5% and 10% of the bladder filling is required when performing VUS using fundamental imaging. As there is a clear correlation between the experience of the sonographer and the sensitivity of VUS, it is advisable for beginners to start with the highest recommended volume of 10% and with increasing experience to reduce the amount, but without going below 5% of the bladder filling when using fundamental imaging. It seems possible to reduce the UCA volume when adding colour Doppler US [15]. There is clear evidence that when using harmonic imaging, 5% or less of UCA with respect to the bladder filling will suffice [18, 33]. Preliminary experience with dedicated contrast imaging modalities such as cadence agent detection imaging (ADI) (Acuson; Siemens, Mountain View, Calif.) point to the potential of further dose reduction [34].\nPhysicochemical properties\nUCAs were primarily developed for IV use and, consequently, preclinical studies were aimed at elucidating problems that could potentially have been encountered during administration via this route. Intravesical use is different and has its own peculiarities one must be aware of. An in-vitro study was carried out with Levovist to measure how variations in US machine setting, transducer choice, mode of application and bladder content may affect the microbubbles [35]. The effects of power output, transducer frequency and injection rate were found to be comparable to those during IV administration. The physicochemical properties of particular relevance for intravesical use are described below.\nInteraction with normal saline solution\nUnlike in VCUG and direct RNC, normal saline plays an important role in VUS. It is the actual bladder filling medium in which the microbubbles are injected. In an in-vitro study the median contrast duration when the UCA was mixed with normal saline solution from glass containers was 30\u00a0s, whereas when the same UCA was added to normal saline solution from a plastic container the median contrast duration increased to 11\u00a0min [36]. None of the plastic containers were sealed under vacuum. In contrast, most of the glass containers were sealed under vacuum. The mean pO2 of the normal saline solutions from vacuum-sealed containers was found to be 50% less than the pO2 of the normal saline solutions from plastic containers. The air-filled microbubbles of Levovist are permeable to air, and gas is exchanged with the atmosphere until equilibrium is reached [27]. Thus, in normal saline solution with low air saturation the microbubbles tend to collapse promptly [37]. Collapse of the microbubbles means that echo enhancers are not available. In practice, the use of normal saline solution from plastic containers is preferred.\nInteraction with urine\nContrast duration of the UCA has been found to be more than four times longer in urine than in normal saline. It has been shown that simply adding urea to buffer the solution significantly increases contrast duration [38]. It is postulated that urea affects the bonding between water molecules and facilitates the formation of more microbubbles. Thus, having some urine in the bladder when performing VUS actually has a positive effect on imaging.\nAscent of microbubbles in the ureter\nOnce reflux of microbubbles takes place from the bladder into the distal ureter, the question as to whether the microbubbles can ascend passively to the proximal ureter and renal pelvis while the refluxed fluid stays in the distal ureter has been raised [39]. Given constancy of gravity, the density difference between gas and fluid and the viscosity of fluid, the velocity of ascent is proportional to the square of the microbubble radius. The typical microbubble radius of Levovist is 1\u20132.5\u00a0\u03bcm. Calculation of velocity of ascent indicates that for all practical purposes passive ascent can be excluded. Furthermore, in an in-vitro simulation of VUS with a set-up imitating the worst-case scenario (90\u00b0, glass tube (no adsorption), constant stirring of fluid suspension, and absence of counter-flow) the possibility of passive ascent was evaluated using an ultraviolet spectrometer for the detection of microbubbles. The result of this experiment was also negative. These findings suggest that the microbubbles in the ureter do not ascend passively and that reflux pressure is necessary for propagation, particularly in vivo with a constant counter-flow of urine from the renal pelvis to the bladder.\nProcedural details\nVUS entails four major basic steps: (1) scan of the urinary tract before contrast agent administration, (2) intravesical administration of prewarmed physiological saline solution and UCA, (3) scan of the urinary tract after administration of UCA, and (4) scan of the urinary tract after administration of UCA during and after voiding [28, 40]. Optionally, a transperineal US of the urethra and\/or cyclical filling of the bladder may be added. Various US modalities are available for performing VUS. The main differences between the modalities are in the degree of conspicuity of the microbubbles and sensitivity of reflux detection.\nUS scan modalities\nFundamental imaging\nThis conventional modality in B-mode is the earliest and most widespread modality employed for VUS. In the case of fundamental imaging it is important to use the same scan planes and magnification of the ureters and renal pelves before and after UCA administration in order to facilitate comparison of the images (Fig.\u00a01). This is particularly important when the reflux is not obvious.\nFig.\u00a01Scans in fundamental mode before (a, c) and after (b, d) contrast agent administration of a dilated left distal ureter (a, barrow) and pelvicalyceal system (c, d). In the postcontrast scans echogenic microbubbles fill the distal ureter (b) and are also detected in the pelvicalyceal system (d)\nColour Doppler US\nThe microbubbles increase the backscattered signal from the urine giving it a characteristic chromatic effect, e.g. a set of blue and red punctiform colour collection [32]. This makes recognition of the urine flow direction easier and enhances the visualization of the microbubbles in the pelvicalyceal system. It is recommended that the Doppler US settings be optimized to perform VUS [31, 41]. Currently, there are five reports in which colour Doppler US alone or in combination with fundamental imaging have been used for VUS [11, 14, 31, 41, 42]. The diagnostic accuracy of colour Doppler US was compared with fundamental imaging in only one study [41]. In the same group of patients VUS without and with colour Doppler US followed by VCUG was carried out. With VCUG as the reference method, the diagnostic accuracy was found to be 96% with colour Doppler US and 90% without. In another study, no significant increase was found in the sensitivity and specificity of VUS when adding colour Doppler US [42]. In one study a modified colour Doppler US examination was used employing a high mechanical index (MI) burst contrast technique based on stimulated acoustic emission (SAE), in which the microbubbles are made to burst creating strong acoustic signals [15]. It was possible to detect more reflux with the burst colour Doppler US compared to regular colour Doppler US or fundamental imaging and even VCUG. No systematic evaluation is available regarding the application of power Doppler US for VUS. Overall, adding optimized colour Doppler US to fundamental imaging seems to have some advantage and should be tried when other contrast-specific modalities are not available.\nHarmonic imaging\nThe nonlinear propagation property of US waves is the basis of harmonic imaging [43]. This may be optimized to receive harmonics from either tissue or microbubbles, thus creating \u201ctissue-specific harmonic imaging\u201d or \u201ccontrast-specific harmonic imaging\u201d, respectively. Some US machines only use the second harmonic for imaging (narrow band), whereas others are capable of implementing a wider range of harmonics (wide band). There may be some degree of difference in image quality between these different technical approaches, in general the latter being of higher quality.\nHarmonic imaging increases contrast and spatial resolution and also results in a reduction of artefacts [43]. The resulting images are clearer and crisper. The advantage of this modality compared to fundamental imaging for scanning the urinary tract in children has already been shown [44]. Due to attenuation, at a certain depth the positive effect of harmonic imaging will disappear. Regarding contrast-enhanced imaging, there is distinctly increased conspicuity of the microbubbles with harmonic imaging (Fig.\u00a02). Air-filled bowel can be a disturbing factor, particularly when imaging the retrovesical space, requiring adjustment of the gain. Darge et al. [17] compared fundamental and harmonic imaging options in 54 children undergoing VUS. In all cases the conspicuity of the microbubbles was much higher with harmonic imaging than with the fundamental modality. Moreover, there was an increase in the reflux detection rate of 30% (from 19 to 27 pelvic-ureter units, PUUs). In this study the volume of Levovist used was 10% with respect to bladder filling. Even reducing the volume of UCA, more reflux episodes were detected in VUS with harmonic imaging than in VCUG [18, 33]. If available, harmonic imaging should be chosen in preference to the fundamental modality for VUS [17, 18, 33].\nFig.\u00a02Scans after contrast agent administration in fundamental mode (a, c) and with harmonic imaging (b, d) of the bladder and right dilated ureter (a, bdotted circle) and a duplex kidney (c, d) with a multicystic dysplastic upper moiety. Reflux in the right ureter and in the lower moiety of the duplex kidney (grade II, arrow) are much more conspicuous with harmonic imaging (b, d). Note also the crisper depiction of the cysts in the upper moiety with harmonic imaging\nDedicated high-MI contrast imaging with multiple display options\nIn recent years, there have been major developments in US technologies for contrast-enhanced US. One such US modality uses high-MI imaging resulting in destruction of microbubbles and depicts the bubble destruction signature as a colour overlay with the possibility of visualizing the grey-scale image alone, the grey-scale image together with the microbubbles with colour overlay or just the microbubbles with colour overlay alone. Additional features may be real-time dual imaging in which two of the above modalities can be used in parallel and the possibility to switch between the three different presentations after having acquired an image in just one option (Figs.\u00a03 and 4). This modality is named differently by different manufacturers: for example, agent detection imaging (ADI; Sequoia, Acuson Siemens), contrast tissue enhancement imaging (CTEI; Technos MPX, Esaote), rate subtraction imaging (RSI; Aplio, Toshiba), true agent detection (TAD; Logiq 9, GE), etc. [45]. The increase in microbubble conspicuity with this modality is so striking that even a beginner should find the examination easy to perform. The potential for further reduction of the dose of UCA and duration of examination are enormous and require future evaluation. If available, this US technique is preferred for VUS using Levovist. This feature is also available with low-MI imaging, which is important when using newer generation UCAs.\nFig.\u00a03VUS with the application of dedicated contrast imaging modality using high-MI (agent detection imaging, ADI). Reflux into the right ureter (arrow) and right renal pelvis (grade II). a Using grey-scale display alone the refluxing microbubbles are not easily demonstrated but the renal parenchyma is seen well. b Using \u201cgrey scale + contrast\u201d visualization of both the reflux with a colour overlay and the bladder and kidney are noticeably improved. c Using \u201ccontrast only\u201d the tissue part has been subtracted and only the microbubbles are seenFig.\u00a04VUS with the use of a dedicated contrast imaging modality using high-MI (agent detection imaging, ADI). Note the time and number of the images (arrow). Once just one image has been documented it is possible as a postprocessing option to switch between the different modalities for display: a \u201cgrey-scale + contrast\u201d and b \u201ccontrast only\u201d options. In this case of grade III reflux marked intrarenal reflux is present in the upper pole (arrowhead)\nProcedural steps\nPrecontrast scans of the bladder, ureters and kidneys\nThe necessity to perform a detailed scan of the urinary tract arises when the VUS is combined with a follow-up US of the urinary tract, for example after pyelonephritis or when there is a need for comparison with the postcontrast images. The scan before contrast agent administration should be carried out in accordance with standard US of the urinary tract in the supine position (and\/or the prone position), in both the transverse and longitudinal planes [28]. Particular attention should to be paid to documentation of subtle changes in the retrovesical region, the vesicoureteric junction and any dilated ureter. The renal pelves (and\/or the calyces) are imaged with maximum magnification. A scan in the supine position may suffice if adequate visualization of the renal pelves on both sides is possible, even though scanning the kidneys with the child in the prone position definitely allows better demonstration of the pelvicalyceal system. It should always be kept in mind that not performing a precontrast scan reduces the duration of the whole examination. This should be the case whenever harmonic or other dedicated contrast imaging is used.\nIntravesical administration of normal saline and US contrast agent\nThe UCA and normal saline are administered via a transurethral catheter, but suprapubic puncture is also possible. The administration of UCA is carried out under US monitoring. The aim should be to have homogeneous strong contrast agent density of the bladder contents. Early studies advocated filling the bladder to the maximum prior to injecting the UCA [13, 28]. This makes the calculation of UCA volume to be administered easier, but has the disadvantage that low-pressure reflux may be obscured. Moreover, in neonates and infants due to repeated voiding at small bladder filling volume, there is insufficient time to administer UCA and scan the urinary tract. Increasingly, a more fractional approach to administering UCA is being practised [21, 40]. It is important to note that emptying the bladder for the sake of UCA administration is not mandatory as explained above. It is helpful to inject UCA into a bladder that is not completely empty in order to have better visualization of the microbubbles and avoid a strong dorsal acoustic shadow. Using a three-way valve it is easy to alternately administer normal saline and UCA adjusting the volume of UCA to the bladder filling volume and in the end reaching the recommended dose. The Levovist suspension should be administered slowly for two reasons: first, to minimize the destruction of the microbubbles and second, to reduce settling of the suspension at the bottom of the bladder that would create an acoustic shadow that temporarily obscures the retrovesical region [28]. In such a case, turning the child several times from side to side hastens the homogeneous distribution of the UCA in the bladder. The administration of normal saline is continued until the child has the urge to micturate or there is the first slight sign of back pressure to the infusion or injection.\nPostcontrast scan of the bladder, ureters and kidneys\nThe US scan after contrast agent administration is basically carried out in the same manner as that before contrast agent administration. There are minor procedural variations depending on the type of US modality utilized. The diagnosis of reflux is made when echogenic microbubbles appear in a ureter or renal pelvis. When a ureter is distinctly visible behind the bladder, the microbubbles render the echo-free lumen echogenic. When the ureter is not clearly visible as a round echo-free structure, VUR can be demonstrated if microbubbles are seen entering the vesicoureteric junction or if just behind this junction, one can depict a round echogenic spot in transverse section, which can be differentiated from its surroundings by its strong echogenicity and possibly constant movement of microbubbles [28]. When the reflux reaches the kidney the echogenic microbubbles can be detected in the pelvicalyceal system. In a recent study by Kopac et al. [23] the need for UCA to demonstrate reflux was again emphasized. They compared indirect non-contrast VUS with contrast-enhanced VUS in 47 children with 93 PUUs. In the former there was no catheterization and bladder filling and any increase in renal pelvic and proximal ureteric width during voiding was considered as a sign of VUR. Compared to VUS with UCA this was found to have low diagnostic accuracy of only 59%, which is not sufficiently reliable for routine use.\nPostcontrast scan of the bladder, ureters and kidneys during and after voiding\nThe US examination is continued during and after voiding in a similar manner as above, always scanning the right and left kidneys alternately, and also the bladder if the patient\u2019s position allows. If cyclic filling is not planned, the catheter can be removed prior to micturition. Most children can void around a thin catheter on the examination table while lying supine, prone or on the side [28]. Other alternatives to consider are to have the child sit on a potty and to scan the kidneys from the back. A \u201cmusic potty\u201d that signals voiding with music is helpful. In older boys the option to micturate into a urine bottle while standing and being scanned from the back may be offered. At the end the bladder is checked for residual urine.\nPostcontrast scan of the urethra during voiding (urethrosonography)\nThe main focus in VUS has been on detection of reflux. In the past, transperineal imaging of the urethra was not given as much consideration during VUS. The lack of urethral imaging in VUS was regarded as a drawback compared to VCUG [46, 47]. In recent years there has been an increased interest in the inclusion of urethral imaging. To date, six studies are available that deal exclusively or partly with contrast-enhanced voiding US of the urethra [16, 19, 48\u201351]. In these studies the urethra was examined in a total of 847 children comprising 647 (76%) boys and 200 (24%) girls (age range 1\u00a0day to 15\u00a0years). Levovist in combination with fundamental imaging was used in all children. In half of the studies, both boys and girls were included and in the remainder only boys [19, 48, 49]. An unbiased comparison of all cases with VCUG was carried out in only two studies [48, 49]. In three studies just those children with pathological findings of the urethra by voiding urethrosonography underwent VCUG [19, 50, 51]. In the remaining one study the selection for comparison was not clearly presented [16]. The diagnosis of posterior urethral valve was made in 22 boys (3.3%) using transperineal contrast-enhanced voiding urethrosonography (Fig.\u00a05). This was also found to be suitable for assessment of the urethra after valve resection [48, 49]. Other diagnoses in boys were anterior urethral valve and urethral stenosis [48]. All diagnoses were confirmed by VCUG. However, the urethral pathologies detected were not only too few, but also the range of pathologies encountered was small [47]. Other urethral pathologies such as paraurethral cysts, diverticula, double urethra, urethral fistula, and complex anorectal and cloacal malformations are still missing in these series. Urethrovaginal reflux was evaluated in two studies and was found to be present in 67 of 165 girls (40.6%) [19, 49].\nFig.\u00a05Transperineal voiding urethrosonography (a) as part of VUS in comparison with (b) VCUG. To facilitate the comparison the US image (a) is presented upside down. Note in the transperineal US (a) the microbubbles in the bladder (B) and in the massively dilated posterior urethra (pU). The anterior urethra (aU) is depicted as very thin in the presence of a posterior urethral valve (arrow). The finding was confirmed on VCUG (b) (courtesy of Dr. M. Bosio, Milan, Italy)\nThe most important study comparing contrast-enhanced voiding urethrosonography and VCUG is that by Berrocal et al. [49]. In this prospective study a total of 146 children (87 boys, 59 girls) with a mean age of 3.3\u00a0years (8\u00a0days to 14\u00a0years) were recruited. In the voiding part of VUS, primarily transperineal US of the urethra was performed. During voiding the catheter was removed. The diameters of the anterior and posterior parts of the urethra were measured at maximum dilatation during voiding. In boys the normal value was found to be 6.1\u2009\u00b1\u20090.8\u00a0mm (2.8\u20137.1\u00a0mm) and 6.3\u2009\u00b1\u20090.67\u00a0mm (3.7\u20137.2\u00a0mm), respectively. In girls the urethral diameter was 4.2\u2009\u00b1\u20091.0\u00a0mm (2.5\u20137.8\u00a0mm). This was the basis for evaluation of pathological changes in the urethra. In all children the US study was followed by VCUG. There were three boys with posterior urethral valves detected on US with dilated posterior urethra, reduced diameter of the anterior urethra and delay in UCA flow at the valve. Furthermore, urethral stenosis was diagnosed in two boys. Seven children were evaluated after resection of the posterior urethra valves and were found to have a normal posterior urethral diameter in the presence of dilatation of the anterior part without any flow delay. In all children these findings were confirmed on VCUG. All girls and 75 boys showed a normal urethra at both transperineal US and VCUG. Thus sensitivity and specificity were each 100%. The approximate mean duration for VUS including transperineal voiding US was 30\u00a0min. In conclusion, transperineal contrast-enhanced VUS of the urethra has not only been shown to be possible but also to be a potential adjunct to VUS in routine examinations.\nCyclical filling of the bladder\nVUR is an intermittent phenomenon. Differences in presence and degree of reflux on repeated examinations have been reported in both VCUG and RNC [52, 53]. In general, cyclical filling results in an increase in reflux detection rate. Repeated filling of the bladder may be necessary when there is marked discrepancy in kidney size and\/or intermittent dilatation of the ureters on US and a negative result during the initial examination. VUS has a specific advantage in this regard as it is not associated with repeated exposure to radiation. Three studies have evaluated the value of cyclical filling in VUS [54\u201356]. In two of the studies, two cycles of bladder filling including Levovist administration were carried out with scanning in fundamental mode [54, 55]. One study was performed in 27 patients with transplanted kidneys [54]. This group comprised eight children (age range 12.2\u201318.3\u00a0years) and 19 adults (age range 19.2\u201354.8\u00a0years). The reflux detection rate in the first and second cycles was 17 and 16 of 27, respectively, and thus not very different, but there were significantly higher reflux grades in the second cycle. So the conclusion was that cyclical filling in VUS with fundamental imaging did not result in increased reflux detection rate. In another study with a greater number and wider spectrum of patients, exclusively children, the opposite was found. Novljan et al. [55] evaluated cyclical filling in 49 children (age range 1.4\u201315.8\u00a0years, mean 4.1\u00a0years) with 98 PUUs. A total of 35 PUUs with reflux were detected when taking both cycles into account. In the first cycle 28 of 35 and in the second 33 of 35 of the PUUs were positive. In the first cycle only just 2 of 35 refluxes (both grade II) were detected. The VUR diagnosed in the second cycle alone comprised six PUUs with grade II and one\u00a0PUU with grade III reflux. Not only were 25% more refluxing units detected in the second cycle but also 50% more grade III refluxes.\nWhen the bladder is completely emptied one can often still find residual echogenic UCA lining the bladder mucosa which together with the hypoechoic wall resembles the mouth part of a \u201csmile sign\u201d (Fig.\u00a06). This so-called \u201csmile sign\u201d indicates that there is still enough UCA in the bladder to facilitate repeat filling with normal saline alone and allow another cycle of VUS. Papadopoulou et al. [56] found that in 112 of 117 children (96%) undergoing cyclical harmonic VUS the residual UCA in the bladder was sufficient to allow a second filling with normal saline only. In the 112 children (224\u00a0PUUs) they compared the results of the first cycle with Levovist and normal saline administration with those of a second cycle with administration of only normal saline. VUR was detected in 57\u00a0PUUs in the first cycle, and of the remaining 167 nonrefluxing PUUs, 12 showed VUR in the second cycle (one grade I, nine grade II, two grade III). Only one PUU reflux (grade II) was diagnosed in the first cycle but missed in the second cycle. Considering all refluxing units as true positives, the sensitivities of the first and second cycles were 87% and 98% and the negative predictive values 84% and 98%, respectively. Thus a second cycle of harmonic VUS with no addition of UCA reveals significantly more PUUs with VUR at almost no additional cost for the examination. From the current standpoint of available data, cyclical filling in VUS, particularly when using harmonic imaging, seems to be a very promising adjunct.\nFig.\u00a06At the end of VUS and bladder emptying there can still be a \u201clayer\u201d of UCA lining the mucosa of the empty bladder. The configuration of the bladder in transverse section in combination with the remaining echogenic UCA resembles the mouth part of a \u201csmile sign\u201d. This sign indicates that there is still sufficient UCA in the bladder and if considered necessary a second filling with normal saline only would suffice to carry out cyclical VUS\nGrading of reflux\nVUR grading is necessary because the severity of reflux correlates with prognosis and consequently serves as a basis for therapeutic decision making. It is also essential to have a uniform grading system for comparison between different research studies. The reflux gradings are based on the degree of pelvicalyceal and ureteral dilatation. Intrarenal reflux is not taken into consideration for grading purposes. It is to be noted that it is possible to depict intrarenal reflux in VUS, particularly when using harmonic imaging and other high-MI modalities [13, 57]. No systematic evaluation of intrarenal reflux in VUS is available. The first grading system for VUS was proposed in 1985 by Beyer et al. [58]. Here grading was for VUS with administration of normal saline only into the bladder. This sonographic grading has five levels based on the extent of dilatation of the renal pelvis, calyces and ureters. Taking this grading system and in conformity with the international reflux grading for VCUG, a five-level grading system was also adapted for contrast-enhanced VUS [59, 60]. The diagnosis of reflux is based on the presence of microbubbles and the severity of the reflux determined by taking primarily the pelvicalyceal and secondarily the ureteric dilatation into account (Table\u00a01). This grading system for VUS has gained widespread acceptance. With the severity of reflux being a continuum, no sonographic measurements have been proposed for differentiating between the grades. Unavoidably, as in VCUG, there is a certain element of subjectivity in the grading system and also some reflux episodes do not fall precisely within one of the five grades. The attempt to differentiate between reflux in dilated and nondilated systems has not become widely used, probably due to lack of immediate consequence of such an addition [60].\nTable\u00a01Reflux grading in contrast-enhanced VUSGradeDefinitionIMicrobubbles only in the ureterIIMicrobubbles in the renal pelvis; no significant renal pelvic dilatationIIIMicrobubbles in the renal pelvis + significant renal pelvic dilatation + moderate calyceal dilatationIVMicrobubbles in the renal pelvis + significant renal pelvic dilatation + significant calyceal dilatationVMicrobubbles in the renal pelvis + significant renal pelvic dilatation and calyceal dilatation + loss of renal pelvis contour + dilated tortuous ureters\nDuration of VUS examination\nIn four comparative studies, in which VUS and VCUG were carried out successively, the duration of each procedural step was recorded [12, 28, 31, 42]. In two of the studies VUS was carried out using fundamental mode [12, 28]. The other two studies incorporated colour Doppler US [31, 42]. In these studies the durations of VUS, including catheterization, were in the range 26\u201334\u00a0min with the precontrast US taking up almost one-third of the time. The durations of VCUG, adding the time for catheterization, were in the range 13\u201320\u00a0min. In one other study VUS and VCUG were carried out simultaneously and here the duration of one such examination including catheterization was 9\u00a0min [61]. Novljan et al. [55] performed VUS with cyclical filling (two fillings) and the mean duration of the VUS was 24\u00a0min (range 15\u201330\u00a0min). Overall, these studies show that the duration of VUS shows a wide range and is longer than that of VCUG. As mentioned above the precontrast US may not be necessary when using harmonic or other dedicated contrast imaging modalities, resulting in a reduction of the duration of VUS by almost one-third. This would put the duration of VUS equivalent to that of VCUG.\nVUS with a second-generation US contrast agent (SonoVue)\nIn 2001 a second-generation UCA\u2014SonoVue (Bracco, Milan, Italy)\u2014was approved in the European Union for IV use in adults. SonoVue is now widely used, the main application being for the evaluation parenchymal abdominal lesions [62]. Even though it has not been approved yet for use in children one in-vitro and four clinical studies point out potential advantages compared to the use of the first-generation UCA, Levovist [63\u201367]. SonoVue is composed of a stabilized aqueous suspension of sulphur hexafluoride (SF6) microbubbles with a phospholipid shell and is available as a package comprising one vial with granules and a 5-ml syringe prefilled with normal saline [62]. In an in-vitro comparative study with Levovist (5% volume) the contrast duration with SonoVue was seven times longer at a dose that was 80% lower [63]. It was found that UCA at 0.25\u20131.0% of the filling volume would provide adequate contrast. Moreover, the contrast duration of a freshly prepared suspension of SonoVue was stable over 6\u00a0h, whereas Levovist showed a significant reduction after 30\u00a0min.\nThe first clinical studies [64\u201367], in total comprising 210 children, have shown that low-MI imaging is the most favourable US modality for VUS when using SonoVue [67] (Fig.\u00a07). Although it is possible to use all other modalities with this UCA, the highest contrast difference between tissue and microbubbles seems to be achieved with low-MI imaging, in which the tissue is suppressed and the microbubbles become more conspicuous. During intravesical administration of SonoVue there is a minor difference compared with Levovist. With the patient in the supine position at the beginning of injection the UCA accumulates at the roof of the bladder and thus does not reach the vesicoureteric junctions [65]. Only after starting normal saline infusion can a homogeneous distribution of the microbubbles be observed. Not much acoustic shadowing of the retrovesical space is encountered. The intravesical dosages that have been successfully used in clinical studies are 1% of bladder filling [65] and 1\u00a0ml per bladder filling [67]. This implies that with a 5-ml suspension from one vial that is stable over 6\u00a0h there is the potential for performing several studies from the one vial. If one vial can be used for more than one patient, the cost of the UCA, a major obstacle to the widespread use of VUS, could be reduced. It is important to note that in some countries there may be restrictions regarding the use of one vial for more than one patient.\nFig.\u00a07VUS using the second-generation UCA, SonoVue. The scan before contrast agent administration (a) uses the tissue harmonic imaging (THI) modality and the scan after contrast agent administration (b) uses echo contrast imaging (ECI). In the former the MI (arrow) is high (1.1) while in the latter a low-MI (0.2) technique is applied. In THI the kidney and particularly the renal pelvis are very well depicted. The refluxing microbubbles of the second-generation UCA are displayed most conspicuously using the low-MI imaging modality\nContraindications and adverse events from intravesical administration of US contrast agents\nThere are no specific contraindications as such to the intravesical administration of Levovist. There are five clinical studies including a total of 626 patients with an age range of 2\u00a0days to 20\u00a0years, in which systematic monitoring for possible adverse events related to intravesical administration of Levovist were carried out [12, 20, 28, 41, 42]. The evaluation incorporated all or some of the following: various levels of vital sign monitoring [12, 28], assessment for possible signs and symptoms during and after the procedure, observation for up to 12\u00a0h as inpatient [42], and request to the patient and parents to report any symptoms in the next 24\u00a0h and an active 24-h follow-up by phone. This evaluation was carried out within the context of comparative studies, in which VCUG was performed following VUS. All patients were catheterized for the examinations. There were 15 patients with transient visible haematuria during or at completion of voiding at either VUS or VCUG and one patient each with transient mild abdominal and urethral pain. For these observed adverse events the catheterization was primarily to blame rather than the UCA [68]. No substance-specific adverse events definitely related to the intravesical administration of Levovist were observed.\nThe osmolality of Levovist in solution is about five times higher than that of blood [37]. This osmolality corresponds to that of previously used ionic radiographic contrast agents, which were likewise instilled into the bladder. No adverse events attributable to the osmolality were observed with these agents. Apart from this, the amount of UCA administered is small and in the bladder there is usually urine and\/or normal saline, resulting in dilution of the hyperosmolar solution. Hyperosmolality of the UCA can, therefore, be ignored in VUS, particularly as it lies approximately in the physiological range of osmolality of urine. No animal study has been published on the potential effect of insonation of the urothelium in the presence of microbubbles, particularly at high MI settings. Potential alterations of the urothelium cannot be excluded with absolute certainty. Over the last 10\u00a0years, predominantly in Europe, it is roughly estimated that some 20,000 VUS examinations have been conducted in children (publications, reports and personal communications). There have been no reports of clinical adverse effects related to the intravesical administration of Levovist.\nExperience with the new UCA, SonoVue, for VUS is still very limited. Contraindications for the IV administration are known hypersensitivity to sulphur hexafluoride and certain cardiopulmonary disorders [29]. Recently, a large-scale retrospective analysis showed that IV SonoVue has a good safety profile in abdominal applications [69]. In the small number of VUS studies with a total of 210 children no adverse events directly related to the UCA have been reported [64\u201367]. There are, as yet, no detailed studies regarding any possible interaction with urine and the urothelium.\nConclusion\nIt is important to realize that a breakthrough to implement US for reflux diagnosis, which had began in the late 1970s, only came with the availability of a stable UCA. Advances in US technology resulting in marked improvement in the depiction of microbubbles have facilitated many procedural aspects in addition to the diagnostic accuracy of the modality. Furthermore, the first results of VUS with the use of a more stable second-generation UCA point to further procedural and diagnostic progress. Like VCUG and direct RNC, it is still necessary to catheterize the bladder or perform suprapubic puncture for VUS. This is a major drawback of all direct reflux examinations and one that makes them invasive. Attempts to generate bubbles exogenously using high-intensity focused US would solve this problem, but have not yet moved beyond the stage of animal studies [70].","keyphrases":["voiding urosonography","ultrasound contrast agent","vesicoureteric reflux"],"prmu":["P","P","P"]}
{"id":"Acta_Neuropathol-3-1-2080348","title":"Gray matter injury associated with periventricular leukomalacia in the premature infant\n","text":"Neuroimaging studies indicate reduced volumes of certain gray matter regions in survivors of prematurity with periventricular leukomalacia (PVL). We hypothesized that subacute and\/or chronic gray matter lesions are increased in incidence and severity in PVL cases compared to non-PVL cases at autopsy. Forty-one cases of premature infants were divided based on cerebral white matter histology: PVL (n = 17) with cerebral white matter gliosis and focal periventricular necrosis; diffuse white matter gliosis (DWMG) (n = 17) without necrosis; and \u201c\nIntroduction\nPeriventricular leukomalacia (PVL), a major disorder of the immature cerebral white matter, has long been considered the underlying neuropathologic substrate of cerebral palsy in premature infants who survive into childhood [41]. The substrate of the cognitive impairments in these children, however, is less certain, given that cognition is typically attributed to gray matter (neuronal cell body), as opposed to white matter (oligodendrocyte), function, and the incidence of gray matter injury in PVL has historically been considered minimal [1, 4, 8]. Understanding the neuroanatomic basis of impaired cognition is of increasing importance in the care of premature infants: currently, in extremely premature infants (born at <1,500\u00a0g), the most common neurological disabilities involve cognition and learning, affecting 20\u201350% of such infants, compared to cerebral palsy which affects 10% [39, 42]. Attempts have been made to correlate neuropathologic findings in autopsied brains with neurologic sequelae in survivors of premature birth, however, much of this work has focused on the role of white matter injury and hemorrhages and not gray matter injury [13]. In the seminal paper of the neuropathology of PVL in 1969, Banker and Larroche reported only \u201cmild neuronal injury\u201d in the cerebral cortex, hippocampus, subiculum, basis pontis, and cerebellar dentate nucleus and Purkinje cells in the majority of PVL brains [4]. Subsequent neuropathologic studies reported only rare \u201canoxic neuronal injury\u201d in association with PVL [1, 8], or downplayed the importance of observed gray matter lesions and did not emphasize their potential importance to neurologic sequelae in survivors. Thus, the conventional teaching has been that cerebral white matter is especially vulnerable to hypoxia-ischemia in the premature brain, with relative sparing of the gray matter, and that gray matter injury dominates only in older infants, children, and adults [23, 24], even though recent evidence shows that neuronal\/axonal injury is common in the perinatal brain [5, 25]. This teaching is increasingly challenged by modern quantitative volumetric MRI studies of premature infants who exhibit reduced volumes of the cerebral cortex [35], thalamus [17, 18], basal ganglia [17, 18], and hippocampus [19, 34]. The anatomic substrate for the gray matter volumetric deficits associated with the apparent white matter disease remains unknown.\nIn the following study of the neuropathology of premature infants autopsied at this institution in the modern era of neonatal intensive care, we aimed to determine: (1) whether gray matter abnormalities in premature infants are more common in the presence of PVL than in the absence of this lesion; and (2) whether the constellation of any gray matter abnormalities provides insight into the basis of the cognitive impairments in living premature infants. In this study, we stratified the cases according to three patterns of cerebral white matter histology: (1) PVL (n\u00a0=\u00a017), defined as diffuse cerebral white matter gliosis combined with focal (macro- and\/or microscopic) periventricular necrosis and; (2) diffuse white matter gliosis (DWMG) (n\u00a0=\u00a017), defined as diffuse gliosis without focal necrosis; and (3) \u201cNegative\u201d (n\u00a0=\u00a07), without diffuse gliosis and focal necrosis. While the pathogenesis of the white matter gliosis in the DWMG group is unknown, we considered it in a separate category from PVL (gliosis combined with focal necrosis) because the cerebral white matter pathology is distinct, without macro- and\/or microscopic periventricular cysts. In the following study, we analyzed the three groups independently of each other. We hypothesized that PVL cases have a significantly greater incidence and degree of gray matter injury than non-PVL, i.e., DWMG and Negative cases; that this injury involves structures critical for cognition and learning, i.e., deep gray nuclei, cerebral cortex, and hippocampus; and that the pattern of gray matter injury mimics the pattern of volume reduction in the deep gray nuclei and cerebral cortex detected by neuroimaging studies in long-term survivors.\nMaterials and methods\nCase selection criteria\nThe neuropathology of all premature infants (<37 gestational weeks at birth) autopsied between 1997\u20131999 at Children\u2019s Hospital Boston was retrospectively reviewed. This time-frame was selected because it represents a modern era of intensive care management of premature infants in a Level 3 neonatal intensive care nursery. It also represents a period at our hospital when the brain and spinal cord were extensively sampled in a relatively standardized fashion, whether or not macroscopic lesions were apparent, thereby permitting a systematic neuropathologic survey in a large dataset. Parental authorization of the use of autopsy human tissue for research was given in each case.\nMicroscopic slide review\nA median of 15 (range 9\u201320) hematoxylin\u2013eosin (H&E) or H&E\/Luxol-fast-blue stained sections was examined from each case. These sections included cerebral cortex from all lobes, thalamus (at the level of lateral geniculate nucleus and including the dorsomedial and lateral posterior nuclei), hypothalamus, caudate, putamen, globus pallidus, hippocampus (level of lateral geniculate nucleus), amygdala, cerebellar dentate nucleus, cerebellar cortex (including Purkinje cells and granule cell layer), midbrain, pons, and medulla.\nWe scored the density of neuronal necrosis, neuronal loss, and gliosis in the most severely affected high power fields of each of the gray matter sites. Two of three neuropathologists (CRP, RDF, HCK) reviewed the slides together at any one time, with a three-way consensus achieved on difficult cases. The brainstem tegmentum was graded separately from the basis pontis and inferior olive in recognition of their susceptibility to injury in the perinatal period [23]. We defined neuronal necrosis as hypereosinophilic neurons with pyknotic nuclei, or, in cases with neuronal immaturity and scant cytoplasm, karyorrhexis [2]. We used the following scale: 0, no necrosis; 1, a few scattered necrotic neurons in a high-powered field (hpf; 400\u00d7); 2, isolated clusters of necrotic neurons\/hpf; and 3, larger, confluent areas of necrotic neurons\/hpf. We scored neuronal loss as: 0, no neuronal loss\/hpf; 1, mild, scattered neuronal dropout\/hpf; 2, moderate, focal areas of neuronal dropout\/hpf; and 3, severe, confluent areas of neuronal dropout\/hpf. We interpreted neuronal necrosis as an acute or agonal change, occurring within 24\u201348\u00a0h of death [2], while neuronal loss and gliosis were interpreted as markers of subacute or chronic injury, indicative of insult occurring 3\u20135\u00a0days or more prior to death [2]. We combined cases for analysis that had grades 2\/3 and 3\/3 of neuronal loss because these grades are unequivocally recognized by standard microscopic examination, and therefore represent, in our opinion, a substantial degree of injury. We scored gliosis as: 0, no reactive astrocytes\/hpf; 1, 1\u201310 reactive astrocytes\/hpf; 2, 11\u201320 reactive astrocytes\/hpf; and 3, >20 reactive astrocytes\/hpf. Reactive astrocytes were defined as stellate configured cells with abundant (\u201chypertrophic\u201d) eosinophilic cytoplasm and an enlarged, often eccentrically placed nucleus with delicate chromatin. The cytoplasm of these cells was immunopositive for glial fibrillary acidic protein (GFAP), the well-established astrocytic marker. In scoring neuronal necrosis, neuronal loss and gliosis, we evaluated the entire available region of each gray matter structure in the section, and assessed the most severely injured region, which was virtually always representative of all fields.\nWe assessed white matter in the cerebral lobes, corpus callosum, posterior limb of the internal capsule, and cerebellum for PVL and diffuse white matter gliosis (DWMG). PVL is defined by the combined presence of: (1) focal necrosis in the periventricular region; and (2) diffuse reactive gliosis in the surrounding white matter [24]. DWMG is defined by the presence of gliosis in the cerebral white matter unaccompanied by periventricular foci of necrosis [24]. Both PVL and DWMG are characterized by reactive astrocytes throughout the white matter, and are thus associated with a pattern of \u201cdiffuse\u201d white matter injury. The density of white matter gliosis was scored according to the same scale used for gray matter gliosis (grades 0\u20133).\nGlial fibrillary acidic protein (GFAP) immunohistochemistry\nFour-micron thick formalin-fixed paraffin-embedded sections of frontal cortex could be cut from 27 of the 41 cases and were immunostained with mouse anti-GFAP antibody (1:500, #SM1-22R, Covance, Berkeley, CA). Negative controls were performed without primary antibody. Scoring of GFAP stained sections was performed by counting positive cells\/hpf, in the most intensely immunopositive region of frontal cortex after a survey of all fields. Reactive astrocytes were defined as those cells with substantial cytoplasmic GFAP staining around a nucleus. Other non-reactive cortical astrocytes with limited cytoplasmic staining had GFAP positive processes that were generally perpendicular to the glial limitans were counted separately. The grading system was: 0, no staining; 1, 1\u201310 cells\/hpf; 2, 11\u201320 cells\/hpf; and 3\u00a0>\u00a020 cells\/hpf. Two observers (CRP, HCK) scored each case without knowledge of the white matter group.\nO4 and GFAP double-labeling immunofluorescence\nMyelination gliosis is commonly encountered in the newborn brain and must be discerned from reactive gliosis. So-called myelination glia are oligodendrocyte precursors that form during myelination; these cells have large nuclei with chromatin intermediate in density between oligodendrocytes and astrocytes and large slightly basophilic cell bodies so they can potentially be mistaken for reactive astrocytes [36]. To help discern reactive gliosis from myelination gliosis fresh tissue that was immediately fixed in 4% paraformaldehyde and sectioned at 40\u201350\u00a0\u03bcm was available from 4 PVL, 1 DWMG and 4 of the Negative cases. Double labeling was performed sequentially beginning with the mouse anti-O4 monoclonal antibody (1:750; gift from Dr. Steven Pfeiffer) to detect developing oligodendrocytes and followed with rabbit anti-GFAP antibodies (1:200, Z0334, Dako) to detect astrocytic differentiation. Relevant secondary antibodies conjugated with FITC or Texas Red were used and sections were visualized with Nikon Eclipse E800 microscope (Nikon, Melville, NY) outfitted with Spot image capture software (Diagnostics Instruments Incorporated, Sterling Heights, MI).\nStatistical analysis\nThe 41 cases were divided into three groups according to cerebral white matter histology: (1) a PVL group; (2) a DWMG group; and (3) a \u201cNegative\u201d white matter group with no diffuse gliosis or focal periventricular necrosis in the cerebral white matter [23, 24]. Demographic characteristics were compared between the PVL, DWMG and Negative groups using Wilcoxon rank sum tests for continuous variables and \u03c72 tests for categorical variables (Table\u00a01). The number of gray matter sites involved with an injury, i.e., acute neuronal necrosis, neuronal loss, and gliosis, was counted for each case in each group, and plotted relative to postconceptional age (i.e., gestational age plus postnatal age, PCA; in weeks) to graphically depict the total number of gray matter sites that were injured in each case. To test the hypothesis that the incidence and severity (grade 2\u20133\/3) of neuronal necrosis, neuronal loss, and gliosis in all gray matter sites analyzed varies significantly among PVL, DWMG, and Negative cases, Fisher exact tests were used. To control for the potential impact of age on these analyses, analysis of covariance of diagnosis on lesion severity were performed, controlling for PCA. Nonsignificant interaction effects between diagnosis and age were subsequently eliminated from the models. In all analyses, P\u00a0<\u00a00.05 was considered significant.\nTable\u00a01Clinicopathologic variables of the three white matter study groups for comparison of gray matter injuriesMean\u00a0\u00b1\u00a0SD; median, (range) or percentP valuePVL group n\u00a0=\u00a017DWMG group n\u00a0=\u00a017Negative group n\u00a0=\u00a07Three-wayPVL versus DWMGDemographics\u00a0Gestational age (weeks)32.8\u00a0\u00b1\u00a03.1; 34, (26\u201336)31.6\u00a0\u00b1\u00a03.8; 33, (24\u201336)26.5\u00a0\u00b1\u00a02.3; 27, (23\u201330)0.0030.282\u00a0Postnatal age (weeks)3.7\u00a0\u00b1\u00a04.1; 2.3, (0.1\u201315)3.4\u00a0\u00b1\u00a04.0; 1.2, (0.1\u201312.0)0.8\u00a0\u00b1\u00a01.2; 0.1, (0.1\u20133.0)0.141\u00a0Postconceptional age (weeks)36.5\u00a0\u00b1\u00a05.4; 35.5, (26\u201352)34.8\u00a0\u00b1\u00a04.5; 35.0, (28.4\u201348.0)27.3\u00a0\u00b1\u00a02.8; 28.3, (23.1\u201330.3)0.0010.293\u00a0Length of ICU stay (days)13.8\u00a0\u00b1\u00a015.9; 9.5, (0\u201356) 23.8\u00a0\u00b1\u00a034.6; 5.0, (1\u201396) 5.0\u00a0\u00b1\u00a08.9; 1.5, (0.04\u201323.0) 0.419\u00a0Race: Caucasian8\/17; 47%9\/17; 53%5\/7; 71%0.801\u00a0African-American2\/17; 12%2\/17; 12%1\/7; 14%\u00a0Unknown7\/17; 41%6\/17; 35%1\/7; 14%\u00a0Percent male9\/17, 53%7\/17, 41%4\/7, 57%0.702Postmortem interval (h)16.0\u00a0\u00b1\u00a07.7; 18, (2\u201330)23.4\u00a0\u00b1\u00a019.6; 17, (2\u201372) 36.3\u00a0\u00b1\u00a042.4; 22, (16\u2013132)0.364Birth weight (g)1576\u00a0\u00b1\u00a0717; 1380, (780\u20132700)1832\u00a0\u00b1\u00a01048; 2100, (610\u20133800)925\u00a0\u00b1\u00a0408; 930, (440\u20131400)0.232Brain weight (g)245.0\u00a0\u00b1\u00a081.1; 257.5, (110\u2013340)255.0\u00a0\u00b1\u00a072.1; 245, (124\u2013380) 127.6\u00a0\u00b1\u00a040.7; 140.0, (63.8\u2013178.0)0.0030.905Body weight (g)2423.1\u00a0\u00b1\u00a01165.0; 2225, (760\u20134875)3276.2\u00a0\u00b1\u00a04706.0; 2290, (1150\u201321300)947.9\u00a0\u00b1\u00a0359.2; 915, (440\u20131400)0.0020.815Body length (cm)42.0\u00a0\u00b1\u00a07.8; 41.5, (26\u201356)43.2\u00a0\u00b1\u00a04.4; 44.0, (36\u201351)34.7\u00a0\u00b1\u00a04.0; 32.5, (30\u201341)0.0070.589Twin or other multiple gestation3\/17, 18%4\/17, 24%2\/7, 29%0.824Multiple congenital anomaliesa4\/17, 24%2\/17, 12%3\/7, 43%0.242Cesarean section10\/17, 59%12\/17, 71%3\/7, 43%0.436Cardiorespiratory factorsApgar score at 1\u00a0min4.1\u00a0\u00b1\u00a02.4; 4, (1\u20138) 4.7\u00a0\u00b1\u00a02.9; 5, (0\u20139) 3.5\u00a0\u00b1\u00a02.2; 3, (1\u20137) 0.691Apgar score at 5\u00a0min5.2\u00a0\u00b1\u00a02.9; 6, (1\u20139) 6.6\u00a0\u00b1\u00a02.9; 7, (0\u201310) 5.0\u00a0\u00b1\u00a02.8; 5.5, (1\u20138) 0.405Acute respiratory distress syndrome7\/17, 41%10\/17, 59%5\/7, 71%0.344Mechanical ventilation13\/17, 77%13\/17, 77%7\/7, 100%0.359Length of mechanical ventilation (days)8.4\u00a0\u00b1\u00a011.6; 3.0, (0\u201338)3.5\u00a0\u00b1\u00a06.3; 1.0, (1\u201319) 4.4\u00a0\u00b1\u00a08.2; 1.0, (0.04\u201323.0) 0.383Extracorpeal membrane oxygenation1\/17%, 6%0\/17%, 0%0\/7, 0%0.485Continuous positive airway pressure0\/17, 0%3\/17, 18%1\/7, 14%0.202Cardiopulmonary resuscitation any time during hospitalization5\/17, 29%7\/17, 41%5\/7, 71%0.165Congenital heart diseasea2\/17, 12%2\/17, 12%1\/7, 14%0.983Infectious\/inflammatory factorsPneumoniaa4\/17, 24%2\/17, 12%0\/7, 0%0.303Maternal fever at delivery0\/15, 0%2\/9, 22%0\/4, 0%0.103Maternal history of urinary tract infection 0\/15, 0%0\/10, 0%1\/4, 25%0.039NAChorioamnionitis1\/15, 7%2\/10, 20%1\/5, 20%0.562Necrotizing enterocolitisa1\/17, 6%4\/17, 24%2\/7, 29%0.415Clinical diagnosis of sepsis8\/17, 47%8\/17, 47%3\/7, 43%0.980If a three-way P value was significant (P\u00a0<\u00a00.05), a two-way P value between the PVL and DWMG groups was performed. NA, not applicable since the incidence was 0 in the PVL and DWMG groups NS, not significanta diagnosed at autopsy\nResults\nClinical and autopsy data\nSeventeen cases (41%) fulfilled the criteria for PVL, while 17 cases (41%) had DWMG, and there were 7 so-called Negative cases (17%).\nPregnancy, labor and delivery\nThe Negative group was significantly younger in terms of gestational age (GA) than the PVL and DWMG groups, but there was no significant difference in GA between the PVL and DWMG groups (Table\u00a01). In terms of PCA, the PVL and DWMG groups were significantly older than the Negative group (Table\u00a01). The Negative group had significantly lower brain weight and body weight, and shorter body length, but not birth weight, than the PVL and DWMG groups. The low somatic and brain measurements in the Negative group compared to the PVL and DWMG groups reflect the early gestational age at birth and younger postnatal age at death (Table\u00a01). The incidence of various clinical variables, e.g., chorioamnionitis, maternal fever at delivery, history of Cesarean section, and congenital anomalies were not significantly different among the three groups. The mean Apgar scores were less than seven at 1 and 5\u00a0min in all three groups (Table\u00a01).\nSyndromes\nTwenty-four percent of cases had a constellation of findings classified as a genetic\/developmental syndrome, e.g., Treacher-Collins syndrome, Potter\u2019s sequence, and osteogenesis imperfecta in three PVL cases; Down\u2019s syndrome in a DWMG case; and Fryns syndrome, Beckwith-Wiedemann syndrome, and VACTERL association in three Negative cases. Excluding these cases from the analysis had no significant effect on the results for the different acquired lesions analyzed semi-quantitatively, i.e neuronal loss and gliosis (data not shown), and thus, their data were combined with that of the non-syndromic cases in the complete analysis reported below.\nPostnatal period\nAlthough there were no statistically significant differences in postnatal age (PNA) among the three groups, the median PNA was only 0.1\u00a0week in the Negative group in contrast to 2.3\u00a0weeks in the PVL group and 1.2\u00a0weeks in the DWMG group (Table\u00a01). The younger PNA and significantly different PCA and GA lead us to limit our gray matter comparisons to those between the PVL and DWMG groups, although the data from the Negative group is reported in all of the tables for completeness. Cardiorespiratory disorders were common in all three groups, with no significant differences (Table\u00a01). Acute respiratory distress syndrome was noted in 41% of PVL cases, 59% of DWMG cases, and 71% of Negative cases. Seventy-seven percent of PVL and DWMG cases, and 100% of the Negative cases required ventilation (Table\u00a01). The duration of ventilation and incidence of cardiopulmonary resuscitation was not significantly different among the three groups. Infectious and inflammatory disorders e.g., pneumonia, necrotizing enterocolitis, and sepsis, occurred in all groups, and did not differ significantly among the groups (Table\u00a01).\nWhite matter findings\nEighty-five percent of all cases studied had diffuse gliosis in the cerebellum, and 82% in the cerebral hemispheres. Forty-one percent of the cases had PVL, and 41% had DWMG. Macroscopically evident periventricular cysts (<5\u00a0mm in diameter) were noted in one PVL case, while chalky-white necrotic foci (2\u20133\u00a0mm) were visible in two cases. In all of the other PVL cases (82%), necrotic foci were only detected microscopically, and were <1\u00a0mm in diameter. Necrotic foci were typically found within a few millimeters from the ventricles. All cerebral lobes demonstrated a similar incidence of PVL (23\u201328%), except for the temporal lobe (12%). DWMG particularly involved the internal capsule (67%) and corpus callosum (64%) (Table\u00a02). Necrotic foci were also identified in nonperiventricular regions, notably the internal capsule (19%) and corpus callosum (9%) (Table\u00a02). The incidence of PVL increased with age, but only significantly so in the frontal lobe (Table\u00a02). In contrast, the incidence of DWMG significantly increased in almost all sites with increasing gestational and postnatal ages (Table\u00a02). By 37+ weeks, DWMG was present in 100% of the cases in the fronto-parieto-temporal lobes, 90% of the cases in the cerebellum, and 80\u201386% of the cases in the internal capsule and corpus callosum (Table\u00a02). The degree of severity of DWMG significantly increased with both GA and PNA in all cases in the frontal, parietal, temporal, occipital, and cerebellar white matter (P\u00a0<\u00a00.04; data not shown). Moreover, the coefficients in the regression models for GA and PNA were similar, indicating that the degree of gliosis increased the same amount for each extra week of gestation and for each extra week of postnatal life (data not shown). In effect, the degree of gliosis increased constantly with PCA (gestational age plus postnatal age).\nTable\u00a02Distribution of white matter lesions in 41 autopsied premature infants by anatomic site and postconceptional age (weeks)White matter siteOverall incidenceIncidence by postconceptional age (weeks) P value Logistic Regression of incidence and postconceptional weeks23\u20132930\u20133637+Frontal lobePVL11\/40 (28%)0\/8 (0%)6\/22 (27%)5\/10 (50%)0.025DWMG31\/40 (78%)2\/8 (25%)19\/22 (86%)10\/10 (100%)0.004Temporal lobePVL4\/33 (12%)0\/6 (0%)3\/22 (14%)1\/5 (20%)NSDWMG22\/33 (67%)1\/6 (17%)16\/22 (73%)5\/5 (100%)0.006Parietal lobePVL7\/28 (25%)0\/6 (0%)6\/16 (38%)1\/6 (17%)NSDWMG20\/28 (71%)1\/6 (17%)13\/16 (82%)6\/6 (100%)0.010Occipital lobePVL8\/35 (23%)1\/5 (20%)5\/23 (22%)2\/7 (29%)NSDWMG28\/35 (80%)3\/5 (60%)19\/23 (83%)6\/7 (86%)NSCorpus callosumPVL2\/22 (9%)0\/3 (0%)1\/14 (7%)1\/5 (20%)NSDWMG14\/22 (64%)0\/3 (0%)10\/14 (71%)4\/5 (80%)0.049Internal capsulePVL5\/27 (19%)0\/2 (0%)3\/18 (17%)2\/7 (29%)NSDWMG18\/27 (67%)0\/2 (0%)12\/18 (67%)6\/7 (86%)NSCerebellumPVL3\/40 (8%)0\/8 (0%)2\/22 (9%)1\/10 (10%)NSDWMG34\/40 (85%)4\/8 (50%)21\/22 (95%)9\/10 (90%)0.012P values denote significant differences in the incidence of DWMG with postconceptional age.NS, not significant; PVL, periventricular leukomalacia; DWMG, diffuse white matter gliosis in the cerebral and cerebellar hemisphere\nTo exclude the possibility that myelination glia were scored along with reactive astrocytes in these infants immunofluoresence staining was performed to co-localize O4 a marker of the developing oligodendrocyte, and GFAP, an astrocytic marker. No co-localization of these proteins was identified in of the three groups of cases studied (Fig.\u00a01).\nFig.\u00a01Immunoflourescence images of parieto-occipital white matter from a PVL case at 39 postconceptional weeks. O4 labeling (a) is shown in red and GFAP labeling (b) is shown in green. Merged images (c) show no co-localization of O4 and GFAP suggesting two distinct cell populations, i.e. GFAP-positive astrocytes and O4- positive oligodendrocyte precursors, are present. The scale bar represents 50\u00a0\u03bcm\nGray matter lesions associated with PVL in the premature infant\nAcute neuronal necrosis\nAcute neuronal necrosis, which is considered a marker of terminal\/agonal injury, was common, and occurred diffusely across gray matter regions in all three groups. Sixty-six percent of PVL, 59% of DWMG, and 43% of Negative cases had two or more gray matter sites with acute neuronal necrosis (data not shown). Significant differences in the incidence of acute neuronal necrosis between the three groups were noted only in the cerebellar cortex (53%, PVL; 13%, DWMG; 0%, Negative; P\u00a0=\u00a00.008) and frontal cortex (56%, PVL; 41%, DWMG; 0%, Negative; P\u00a0=\u00a00.039). The incidence of neuronal necrosis was not significantly different at any gray matter site when adjusted for GA and PNA (data not shown).\nNeuronal loss and gliosis\nNeuronal loss and gliosis, considered markers of subacute and chronic injury, were more prevalent and of greater severity in PVL cases compared to non-PVL cases (DWMG and Negative groups) (Figs.\u00a02 and 3, Tables\u00a03 and 4). PVL cases showed more damage to the deep nuclear structures than was encountered in non-PVL cases. In PVL cases, the thalamus and globus pallidus had significantly higher incidences of neuronal loss (38 and 33%, respectively) and more severe neuronal loss (38 and 33%, respectively) than did the DWMG and Negative groups (both, 0%) (Fig.\u00a02, Table\u00a03). The incidence of gliosis was also significantly higher in the thalamus (56%), caudate (60%), putamen (50%) and globus pallidus (60%) in PVL than in DWMG (12\u201347%) and Negative cases (0\u201314%). The cerebellar dentate nucleus showed a significantly higher incidence of neuronal loss in PVL (29%) compared to the DWMG (6%) and Negative (14%) groups. PVL cases (29%) had significantly more severe neuronal loss in the cerebellar dentate compared to the DWMG and Negative groups (both, 0%) (Table\u00a03). Gliosis of the basis pontis was seen in 100% of PVL cases and only 79% of DWMG and 29% of Negative cases (P\u00a0=\u00a00.001; Table\u00a04). The hippocampus also had substantial neuronal loss and gliosis (Tables\u00a02 and 3). PVL cases showed relatively mild cerebral cortical neuronal loss, compared to other neuroanatomic sites, while the incidence of gliosis ranged from 20% (temporal cortex) to 31% (frontal cortex) (Tables\u00a03 and 4). By contrast, the cerebral cortex in all lobes from DWMG and Negative cases was totally free of neuronal loss and was infrequently gliotic (all <10%) (Tables\u00a03 and 4).\nFig.\u00a02Photomicrographs from thalami illustrating neuronal loss scores of 0 (a), 1 (b), 2 (c) and 3 (d). The asterisk in panel b denotes a focal area of neuronal loss. The scale bar represents 20\u00a0\u03bcmFig.\u00a03Photomicrographs from the inferior olivary nuclei depicting gliosis scores of 0 (a), 1 (b), 2 (c) and 3 (d). Arrows in panels b and c indicate some of the reactive astrocytes that are present. The scale bar represents 20\u00a0\u03bcmTable\u00a03Incidence and severity of neuronal loss in PVL, DWMG and Negative casesOverall incidenceIncidence of severity 2\u20133PVLDWMGNegativeP valuePVLDWMGNegativeP valueNeuronal lossCerebral cortexFrontal cortex13% (2\/16)0% (0\/17)0% (0\/7)0.4776% (1\/16)0% (0\/17)0% (0\/7)0.575Temporal cortex0% (0\/15)0% (0\/13)0% (0\/6)1.0000% (0\/15)0% (0\/13)0% (0\/6)1.000Parietal cortex8% (1\/13)0% (0\/11)0% (0\/6)1.0008% (1\/13)0% (0\/11)0% (0\/6)1.000Occipital Cortex0% (0\/15)0% (0\/16)0% (0\/4)1.0000% (0\/15)0% (0\/16)0% (0\/4)1.000Deep gray nucleiThalamus38% (6\/16)0% (0\/17)0% (0\/7)0.00538% (6\/16)0% (0\/17)0% (0\/7)0.005Hypothalamus20% (2\/10)0% (0\/10)0% (0\/2)0.56710% (1\/10)0% (0\/10)0% (0\/2)1.000Caudate13% (2\/15)0% (0\/16)0% (0\/7)0.32913% (2\/15)0% (0\/16)0% (0\/7)0.329Putamen13% (2\/16)0% (0\/17)0% (0\/7)0.47713% (2\/16)0% (0\/17)0% (0\/7)0.477Globus pallidus33% (5\/15)0% (0\/15)0% (0\/6)0.02833% (5\/15)0% (0\/15)0% (0\/6)0.028Cerebellum and relay nucleiBasis pontis21% (3\/14)0% (0\/14)0% (0\/7)0.20614% (2\/14)0% (0\/14)0% (0\/7)0.341Inferior olive15% (2\/13)8% (1\/13)20% (1\/5)0.8078% (1\/13)8% (1\/13)20% (1\/5)0.549Cerebellar cortex24% (4\/17)6% (1\/16)14% (1\/7)0.44924% (4\/17)6% (1\/16)14% (1\/7)0.449Dentate29% (4\/14)0% (0\/15)0% (0\/6)0.03129% (4\/14)0% (0\/15)0% (0\/6)0.031Limbic structuresHippocampus33% (5\/13)0% (0\/14)14% (1\/7)0.05533% (5\/15)0% (0\/14)14% (1\/7)0.055Amygdala0% (0\/6)0% (0\/3)0% (0\/2)1.0000% (0\/6)0% (0\/3)0% (0\/2)1.000Substantia inominata29% (2\/7)0% (0\/3)0% (0\/1)1.00029% (2\/7)0% (0\/3)0% (0\/1)1.000Brainstem14% (2\/14)0% (0\/14)0% (0\/5)0.62914% (2\/14)0% (0\/14)0% (0\/5)0.629PVL, periventricular leukomalacia; DWMG, diffuse white matter gliosis in the cerebral and cerebellar hemisphere. P values denote differences in the incidence or severity of neuronal loss at these neuroanatomic sites between PVL and DWMG groups with postconceptional ageTable\u00a04Incidence and severity of gliosis in PVL, DWMG and Negative casesOverall incidenceIncidence of severity 2\u20133PVLDWMGNegativeP valuePVLDWMGNegativeP valueGliosisCerebral cortexFrontal cortex31% (5\/16)6% (1\/17)0% (0\/7)0.10213% (2\/16)0% (0\/17)0% (0\/7)0.477Temporal cortex20% (3\/15)8% (1\/13)0% (0\/6)0.4950% (0\/15)0% (0\/13)0% (0\/6)1.000Parietal cortex23% (3\/13)9% (1\/11)0% (0\/6)0.4998% (1\/13)9% (1\/11)0% (0\/6)1.000Occipital cortex27% (4\/15)0% (0\/16)0% (0\/4)0.05413% (2\/15)0% (0\/16)0% (0\/4)0.395Deep gray nucleiThalamus56% (9\/16)18% (3\/17)14% (1\/7)0.03119% (3\/16)0% (0\/17)14% (1\/7)0.161Hypothalamus40% (4\/10)10% (1\/10)50% (1\/2)0.26420% (2\/10)0% (0\/10)50% (1\/2)0.130Caudate60% (9\/15)19% (3\/16)14% (1\/7)0.02813% (2\/15)6% (1\/16)14% (1\/7)0.659Putamen50% (8\/16)12% (2\/17)14% (1\/7)0.04419% (3\/16)0% (0\/17)0% (0\/7)0.130Globus Pallidus60% (9\/15)47% (7\/15)0% (0\/6)0.04020% (3\/15)7% (1\/15)0% (0\/6)0.492Cerebellum and relay nucleiBasis pontis100% (14\/14)79% (11\/14)29% (2\/7)0.00136% (5\/14)21% (3\/14)14% (1\/7)0.684Inferior olive92% (12\/13)92% (12\/13)80% (4\/5)0.54962% (8\/13)54% (7\/13)20% (1\/5)0.400Cerebellar cortex29% (5\/17)6% (1\/16)14% (1\/7)0.25912% (2\/17)6% (1\/16)0% (0\/7)1.000Dentate43% (6\/14)13% (2\/15)17% (1\/6)0.17721% (3\/14)0% (0\/15)0% (0\/6)0.125Limbic structuresHippocampus47% (7\/15)7% (1\/14)29% (2\/7)0.05620% (3\/15)0% (0\/14)29% (2\/7)0.143Amygdala50% (3\/6)0% (0\/3)0% (0\/2)0.32730% (0\/6)0% (0\/3)0% (0\/2)1.000Substantia inominata29% (2\/7)0% (0\/3)0% (0\/1)1.0000% (0\/7)0% (0\/3)0% (0\/1)1.000Brainstem43% (6\/14)20% (3\/14)20% (1\/20)0.5187% (1\/14)0% (0\/14)0% (0\/5)1.000PVL, periventricular leukomalacia; DWMG, diffuse white matter gliosis in the cerebral and cerebellar hemisphere. P values denote differences in the incidence or severity of gliosis at these neuroanatomic sites between PVL and DWMG groups with postconceptional age\nGlial fibrillary acidic protein immunohistochemistry performed on sections of frontal cortex from PVL (n\u00a0=\u00a010), DWMG (n\u00a0=\u00a013) and Negative cases (n\u00a0=\u00a04) showed astrocytes of two different general morphologies (Fig.\u00a04). Greater numbers of reactive astrocytes with abundant GFAP positive cytoplasm tended to occur in the frontal cortex of PVL cases (mean score 0.75\u00a0\u00b1\u00a00.22), compared to DWMG cases (0.48\u00a0\u00b1\u00a00.19) or (0.59\u00a0\u00b1\u00a00.38); however, when controlled for PCA this trend was not statistically significant. Non-reactive astrocytes were found in similar frequency among PVL, DWMG and Negative cases, and no such trend, as that noted for reactive astrocytes appeared (data not shown).\nFig.\u00a04GFAP immunohistochemical staining of frontal cortex illustrating non-reactive astrocytes (a) with a linear GFAP-positive process that is perpendicular to glial limitans, which is at the right of this image (not depicted) and a reactive astrocyte (b), with abundant GFAP-positive cytoplasm, and an eccentrically placed, enlarged nucleus. The scale bar represents 20\u00a0\u03bcm\nDiscussion\nThis neuropathologic analysis shows clearly that gray matter abnormalities are more common in the presence of PVL than in its absence (summarized in Fig.\u00a05). Moreover, and remarkably, neuronal loss and gliosis in the cerebral cortex and deep nuclear structures are essentially confined to those infants with PVL. Thus, not a single infant with DWMG exhibited neuronal loss in the cerebral cortex, hippocampus, and deep gray nuclei. Similarly, gliosis was very unusual in these areas in the infants with DWMG. Although the incidence and severity of lesions in this autopsy series may not be completely representative of the brain pathology in premature infants who survive beyond the perinatal period, the findings are nevertheless important to understand the neuroanatomic substrate and pathogenesis of the neurological sequelae in long-term survivors.\nFig.\u00a05Summary diagram comparing gray matter sites with a significantly higher incidence (percentages) of neuronal loss (a) and gliosis (b) in PVL (right of panel) and DWMG (left of panel) cases. Gliosis of the cerebral and cerebellar white matter, basis pontis, brainstem tegmentum and inferior olives is depicted by small red dots, and focal, periventricular necrosis in the cerebral white matter (PVL) is denoted by a large red periventricular circle\nThe pathogenesis of the gray matter lesions in PVL is likely due to the same phenomena implicated in the white matter lesion. The pathogenesis of PVL likely involves cerebral ischemia-reperfusion in the respiratory compromised preterm infant, in combination with one or more infectious\/inflammatory and other, yet to be defined, derangements [24]. Thus, the topographic patterns of gray and white matter damage in the premature brain likely reflect a complex interplay of the differential vulnerabilities of the regions to glutamate, free radical, and cytokine toxicity. These differential vulnerabilities appear to be based upon the maturational stage of neurons and oligodendrocytes [3, 23, 40], i.e., the targeted cell types in gray and white matter injury, respectively, and upon the developmental profiles of glutamate and cytokine receptors [9, 23] and antioxidant systems [9], as well as multiple related factors [23, 40]. The term \u201cperinatal panencephalopathy\u201d best describes, in our opinion, the combined gray and white matter injury delineated in this study that is typical of perinatal neuropathology of prematurity. We regard PVL as a major part of this disorder that should now be considered, we believe, in the context of total brain injury. Since the majority of patients in the PVL, DWMG, and Negative groups required mechanical ventilation of comparable durations, and showed substantial involvement by inflammatory\/infectious processes, it is difficult to decipher the factors responsible for the substantial brain injury in the PVL group. It is very likely that there are specific clinical factors at work that we do not yet know, or were not analyzed in this study, e.g., lowest oxygen levels, alterations in acid-base status, and dysfunction in cerebral autoregulation, which are difficult to analyze in a meaningful way from complicated neonatal records. Thus, this study is hypothesis-generating for a prospective analysis of the key clinical factors involved in the pathogenesis of perinatal panencephalopathy.\nThe presence of isolated hypertrophic astrocytes in the cerebral white matter of premature infants, as reported in this series in the DWMG group, has been recognized for decades, but its significance remains unknown. Focal necrosis and diffuse hypertrophic astrocytes that are associated with \u201cglobules\u201d and \u201cacutely damaged glia\u201d have been considered histological manifestations of the same disorder of immature cerebral white matter for which the term acquired perinatal telencephalopathy (PTL) has been coined [27]. Yet, hypoxic\u2013ischemic white matter injury may follow a continuum of damage, from mild (gliosis [hypertrophic astrocytes] alone) to severe (periventricular necrosis combined with gliosis) [27]. Astrocytes, however, may also normally undergo hypertrophy in the late fetal and perinatal white matter as an obligatory developmental change, potentially due to the \u201cphysiological oxidative stress\u201d of active myelin sheath synthesis, and thus may not be a marker of pathology at all [15]. These so-called myelination glia are immature oligodendrocytes that express markers such as O4, and are morphologically similar to GFAP positive reactive astrocytes. Coimmunofluoresence studies show no overlap in the expression of O4 and GFAP. The significant differences in age and survival encountered among the PVL, DWMG and Negative groups in this study precluded using the Negative group as a control representing \u201cno white matter injury\u201d. The Negative group consisted of infants who were born after significantly shorter gestational periods and who survived for significantly shorter time-periods postnatally than those in the PVL or DWMG groups. Thus, it is possible that the infants in the Negative group showed no white matter gliosis because the white matter is not vulnerable to injury at this early age, immature astrocytes are not capable of mounting a hypertrophic reaction to injury at this early time-point, and\/or the patients did not survive long enough for astrocytic hypertrophy to develop. Further studies are needed to examine the significance of astrocytic hypertrophy in developmental pathology. The challenge is heightened by the unavoidable fact that live-born infants dying during the last half of gestation are not \u201cnormal\u201d, but rather, typically die in intensive care units with multiple complications of prematurity that are known to adversely affect the brain.\nThis study suggests that neuronal loss and\/or gliosis in the perinatal period in gray matter sites critical to cognition, memory, and learning, i.e., thalamus [7, 38], basal ganglia [33], hippocampus [7, 26], and cerebellum [21, 37], play a role in cognitive defects in long-term survivors of prematurity. The neuroanatomic structures involved with neuronal loss and\/or gliosis correlates well with the neuroimaging data, which has shown volumetric deficits in the thalamus and basal ganglia [17, 18], and to a lesser degree, the hippocampus [19, 34], and cerebral cortex [35] in survivors of prematurity. This thalamic damage could be important in the pathogenesis of subsequent cognitive impairments. Of note, afferent thalamocortical axons fail to reach the cortex when the subplate neurons are ablated and abnormal cortical lamination results [10\u201312, 14, 22, 30]. Selective subplate neuronal loss occurs in hypoxic-ischemic injury in neonatal rats [31], underscoring the possibility of homologous injury in human premature infants [16] and the need for in depth studies of the subplate-thalamic-cortical unit in humans. Premature infants are also at high risk for cerebellar injury [6, 20, 28, 29, 32], given the mounting evidence that the cerebellum plays a role in cognition [21, 37], our finding of substantial damage in this structure and its brainstem relay nuclei suggests that it could contribute to cognitive defects in survivors. In addition, injury to the cerebellum, as well as the thalamus and basal ganglia (globus pallidus), may contribute to the motor deficits of prematurity. Traditionally, the spastic motor deficits, i.e., cerebral palsy, in preterm infants has been attributed to damage to axons in the necrotic foci in PVL that are coursing through the periventricular white matter from the motor cortex to the spinal cord [41]. Our data suggest that at least some of the common, less severe motor deficits are due to gray, as well as white, matter damage.\nIn conclusion, this study draws attention to the combination of white and gray matter injury in the brains of preterm infants dying in the perinatal period by the term \u201cperinatal panencephalopathy\u201d. Our findings suggest that future treatment strategies should target both white and gray matter damage to prevent the neurologic deficits in survivors of prematurity.","keyphrases":["white matter gliosis","thalamus","basal ganglia","brainstem","perinatal panencephalopathy","neurodevelopmental disability","perinatal hypoxia\u2013ischemia"],"prmu":["P","P","P","P","P","M","M"]}
{"id":"Ann_Biomed_Eng-2-2-1705490","title":"Automatic Regulation of Hemodynamic Variables in Acute Heart Failure by a Multiple Adaptive Predictive Controller Based on Neural Networks\n","text":"Automated drug-delivery systems that can tolerate various responses to therapeutic agents have been required to control hemodynamic variables with heart failure. This study is intended to evaluate the control performance of a multiple adaptive predictive control based on neural networks (MAPCNN) to regulate the unexpected responses to therapeutic agents of cardiac output (CO) and mean arterial pressure (MAP) in cases of heart failure. The NN components in the MAPCNN learned nonlinear responses of CO and MAP determined by hemodynamics of dogs with heart failure. The MAPCNN performed ideal control against unexpected (1) drug interactions, (2) acute disturbances, and (3) time-variant responses of hemodynamics [average errors between setpoints (+35 ml kg\u22121 min\u22121 in CO and \u00b10 mmHg in MAP) and observed responses; 6.4, 3.7, and 4.2 ml kg\u22121 min\u22121 in CO and 1.6, 1.4, and 2.7 mmHg (10.5, 20.8, and 15.3 mmHg without a vasodilator) in MAP] during 120-min closed-loop control. The MAPCNN could also regulate the hemodynamics in actual heart failure of a dog. Robust regulation of hemodynamics by the MAPCNN was attributable to the ability of on-line adaptation to adopt various responses and predictive control using the NN. Results demonstrate the feasibility of applying the MAPCNN using a simple NN to clinical situations.\nINTRODUCTION\nHemodynamic variables in a critical care patient with heart failure must be monitored during and after cardiac surgery. The hemodynamic conditions must then be regulated using infusion of several drugs. In particular, cardiac output (CO) and mean arterial pressure (MAP) are primary target variables because increased CO is required with suppressing myocardial oxygen consumption and MAP must be kept at the lowest level that can adequately maintain coronary circulation with decreasing systemic vascular resistance (SVR).14 Combined infusion of an inotropic agent such as dobutamine (DBT) or dopamine and a vasodilator such as sodium nitroprusside (SNP) or nitroglycerin has proven effective for patients with heart failure to regulate hemodynamics.12,13,25 Inotropic agents increase the force and velocity of cardiac muscle contraction and result in enhancing CO. Vasodilators reduce SVR and result in the decrease of the afterload of the heart, which thereby decreases MAP and increases stroke volume secondarily.3,14\nMultivariable automated drug-delivery systems have been developed to help busy physicians or anesthesiologists use several drugs in many critical tasks to regulate the various hemodynamics that occur during heart failure.11,21,22,24,28,32,33 In simulation and animal studies undertaken during early system development, adaptive controllers demonstrated the feasibility of application of a multivariable drug-delivery system to simultaneously control CO and MAP using a combination of an inotropic agent and a vasodilator.11,24,28 In subsequent stages, multiple models and adaptive predictive controllers have been developed to adequately adjust the hemodynamic parameters in the presence of nonlinear physiological responses5,32 and drug interactions.21,22,33 Fuzzy controls have also shown optimal performance in regulation of multivariable hemodynamics with heart failure.6,7,31 However, patients with heart failure may have nonlinear and time-variant responses under unexpected variability to drugs and drug interactions with disturbances. Under such unknown conditions, the model-based controllers might require the preparation of numerous linear models to describe the various heart-failure patient responses to drugs,21,22,33 and the fuzzy controllers might require many rules based on the expert knowledge and heuristics of physicians or anesthesiologists for drug therapy of heart failure.6\nThe controllers that can adjust adaptively to unexpected responses with heart failure are required in actual clinical situations. Because neural networks (NN) might be one of the simple tools used for nonlinear and time-variant system responses in the presence of the unknown response variability and interactions with exogenous perturbation,15,27 the application of the NN to drug-delivery systems has been desired.8,17 To our knowledge, the multivariable controller using the NN for hemodynamic variables with heart failure has not been tested whereas the NN controllers have engendered the MAP controls such as post-operative hypertension1 and during acute hypotension9. Therefore, this study is intended to develop a multiple adaptive predictive control based on NN (MAPCNN) for hemodynamic variables and to evaluate its control performance under unexpected responses to drugs during CO and MAP regulation using SNP and DBT in heart failure. To be assured of a control algorithm before animal experiments, the MAPCNN was tested under largely unexpected (1) drug interactions, (2) acute disturbances, and (3) time-variant hemodynamic responses which can be freely operated. Finally, the performance of the MAPCNN was tested by actual hemodynamics of a canine left heart failure.\nMATERIALS AND METHODS\nThis section fundamentally includes three parts: (i) \u2018Modeling of Pharmacological Response\u2019, which explains the method for the development of a computer response model that allows testing of the MAPCNN, (ii) \u2018Development of Controller\u2019, and (iii) \u2018Evaluation of Controller\u2019, which is the actual test of the MAPCNN. In addition, the animal study was divided into two parts. First, to model the pharmacological response in (i), animal experiments were performed using dogs with heart failure. Five of eleven dogs were used for every drug infusion. Then, for additional validation of the developed controller after computer simulations, an animal experiment was performed using one dog with heart failure.\nModeling of Pharmacological Response\nTo produce the response models to therapeutic agents in acute heart failure, the following animal study, which conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health, was performed. Microsphere embolization of the left main coronary artery induced acute ischemic heart failure in dogs (n\u00a0=\u00a05, 26\u201332\u00a0kg) that were anesthetized with pentobarbital sodium and ventilated artificially. A double-lumen catheter was introduced into the right femoral vein for administration of pharmaceutical agents using a computer-controllable infusion pump (CFV-3200; Nihon Kohden, Tokyo, Japan). An in-line electromagnetic flow probe (MFV-2100; Nihon Kohden) was used to measure CO; MAP was measured through a fluid-filled catheter and a pressure transducer (DX-200; Nihon Kohden). The CO and MAP were digitized at a 10-Hz sampling rate through a 12-bit digital-to-analog converter connected to a laboratory computer.\nThe total number of all animals used for modeling was 11. The orders of drug infusions were the following four: (i) 3, 6, and 9\u00a0\u03bcg\u00a0kg\u22121\u00a0min\u22121 in DBT and 1, 2, and 4\u00a0\u03bcg\u00a0kg\u22121\u00a0min\u22121 in SNP (n\u00a0=\u00a02 of 11 animals); (ii) 3 and 6\u00a0\u03bcg\u00a0kg\u22121\u00a0min\u22121 in DBT (n\u00a0=\u00a03); (iii) 9\u00a0\u03bcg\u00a0kg\u22121\u00a0min\u22121 in DBT (n\u00a0=\u00a03); and (iv) 1, 2, and 4\u00a0\u03bcg\u00a0kg\u22121\u00a0min\u22121 in SNP (n\u00a0=\u00a03). Specifically, five of all animals were used for each drug infusion rate. The hemodynamic variables [n\u00a0=\u00a09 except for the two animals in the above case (iii), the 9\u00a0\u03bcg\u00a0kg\u22121\u00a0min\u22121 in DBT (n\u00a0=\u00a03), because of no measurements] were changed to \u221260.2\u00a0ml\u00a0kg\u22121\u00a0min\u22121 in CO (p\u00a0<\u00a00.01, paired t-test) and +4.9\u00a0mmHg in MAP (not significant) before (CO, mean\u00a0\u00b1\u00a0S.E.M\u00a0=\u00a0132.0\u00a0\u00b1\u00a011.2\u00a0ml\u00a0kg\u22121\u00a0min\u22121; and MAP, 92.8\u00a0\u00b1\u00a03.2\u00a0mmHg) and immediately after (CO, mean\u00a0\u00b1\u00a0S.E.M\u00a0=\u00a071.8\u00a0\u00b1\u00a06.2\u00a0ml\u00a0kg\u22121\u00a0min\u22121; and MAP, 97.7\u00a0\u00b1\u00a04.3\u00a0mmHg) the heart failure; for hemodynamics immediately before each drug infusion, see Fig.\u00a01A.). The period between two trials of drug infusions was 10\u00a0min in each animal. To prevent the washout process of a catheter in the above case (i), both the drug infusions of DBT and SNP (n\u00a0=\u00a02), a double-lumen catheter was used.\nFIGURE\u00a01.Single-drug dose responses in canine left heart failure (n\u00a0=\u00a05). A. Step responses of the change of cardiac output (\u0394CO, left) and mean arterial blood pressure (\u0394MAP, right) from the baseline after induced acute heart failure during 10-min infusion of (a) dobutamine (DBT) at 3, 6, and 9\u00a0\u03bcg\u00a0kg\u22121\u00a0min\u22121 and (b) sodium nitroprusside (SNP) at 1, 2, and 4\u00a0\u03bcg\u00a0kg\u22121\u00a0min\u22121. Data digitized at 10\u00a0Hz were averaged every 30\u00a0s. Data are mean\u00a0\u00b1\u00a0S.E.M. B. Unit impulse responses of \u0394CO (left) and \u0394MAP (right) calculated from data of (a) DBT infusion at 3, 6, and 9\u00a0\u03bcg\u00a0kg\u22121\u00a0min\u22121 and (b) SNP infusion at 1, 2, and 4\u00a0\u03bcg\u00a0kg\u22121\u00a0min\u22121 in dogs. CO response of SNP at 1\u00a0\u03bcg\u00a0kg\u22121\u00a0min\u22121 was eliminated because of impossible fit to step response.\nIn this study, component models comprising a first order dynamic system cascaded with a nonlinear sigmoidal function were used to model the responses of CO and MAP with heart failure. After pharmacodynamics for the evaluation of controllers was represented by a linear first-order transfer function,18 a sigmoidal function was applied to the linear model to express the nonlinear characteristic with a component model approximating the positive step response.4,30 The procedure to produce the model responses can be described as follows.\nFirst, simple models for responses to therapeutic agents were produced from experimental data in canine left-heart failure. Figure\u00a01A shows the step responses of CO and MAP changed (\u0394CO and \u0394MAP) from baseline values immediately before each drug infusion after inducing the acute heart failure during 10-min (a) DBT and (b) SNP infusions. The step responses of \u0394CO and \u0394MAP during infusion of DBT at 3, 6, and 9\u00a0\u03bcg\u00a0kg\u22121\u00a0min\u22121 or SNP at 1, 2, and 4\u00a0\u03bcg\u00a0kg\u22121\u00a0min\u22121 were averaged every 30\u00a0s. Then, each single-input single-output response [\u0394\u0177(t)] of the four step responses (DBT-CO, DBT-MAP, SNP-CO, and SNP-MAP loops as input\u2013output relationships) was approximated to the linear first-order delay system with a pure time delay in the continuous-time domain, as  where K is a proportional gain, L is a pure time delay, and Tc is a time constant. If t\u00a0<\u00a0L then \u0394\u0177(t)\u00a0=\u00a00. The fitted parameters to the averaged step responses (n\u00a0=\u00a05) in the single infusion of DBT or SNP were acquired by least squares method and used for calculation of the following unit impulse response (i.e., the response to the infusion of 1\u00a0\u03bcg\u00a0kg\u22121\u00a0min\u22121 of a given drug).\nSecond, the linear-model response [\u0394y*(t)] was calculated by the convolution integral in the discrete-time domain:  where .\nIn those equations, u(t) is the drug infusion rate, \u0394T is the sampling interval, and Nm is the finite number of terms in the model for the unit impulse response. The unit impulse response is g(t), as calculated from the derived values of the step response of Eq. (1). The proportional gain of a unit impulse response is Ku; Tc and L are the same values as Eq. (1). For the simulation study, \u0394T and Nm were set, respectively, to 30\u00a0s and 20. According to this method, the unit impulse responses of \u0394CO and \u0394MAP during infusions of DBT at 3, 6, and 9\u00a0\u03bcg\u00a0kg\u22121\u00a0min\u22121 and SNP at 1, 2, and 4\u00a0\u03bcg\u00a0kg\u22121\u00a0min\u22121 were calculated from the fitting parameters to the average step responses (Fig.\u00a01B, the CO response to SNP at 1\u00a0\u03bcg\u00a0kg\u22121\u00a0min\u22121 was excepted because of the impossible fit to the step response). Although some differences existed among unit impulse responses at those infusion rates, as shown in Fig.\u00a01B, in the present study, median values (i.e. 6\u00a0\u03bcg\u00a0kg\u22121\u00a0min\u22121 in DBT and 2\u00a0\u03bcg\u00a0kg\u22121\u00a0min\u22121 in SNP) were used for the following simulations because they characterized the effects of drugs on hemodynamics well. Numerical data in Ku, Tc, L, and g(t) used for the simulation study are shown in Table\u00a01 (left).\nTABLE\u00a01.Model parameters in linear-fitting and nonlinear-fitting functions.Drug-responseLinearNonlinefsarProportional gain (Ku)Time constant (Tc)Pure time delay (L)R2Response range (p1)Coefficient of gain (p2)R2DBT-CO15.8164.3300.98105.30.0280.99DBT-MAP4.465.2300.7522.80.1450.98SNP-CO3.040.6600.2837.70.0510.80SNP-MAP\u221212.5209.4600.96-26.2\u22120.0850.99Fitting parameters (Ku, Tc, L, p1, and p2) in a single-input single-output relationship. The Ku [ml\u00a0\u03bcg\u22121 in DBT-CO and SNP-CO loops or mmHg (\u03bcg\u00a0kg\u22121\u00a0min\u22121)\u22121 in DBT-MAP and SNP-MAP loops], Tc (s), and L (s) are the parameters of Eq. (2). The p1 and p2 are the parameters of Eq. (3). R2 shows a multiple coefficient of determination.\nThird, to express the nonlinear response to a single drug infusion, the \u0394y*(t) in Eq. (2) as the linear model response was modified through a sigmoidal function:30 in which p1 is the parameter of the response range, which shows the difference between the maximum and minimum values of \u0394y\u2032(t) as the nonlinear model response, and p2 is the parameter of the coefficient of gain. Parameters p1 and p2 were determined by nonlinear least-squares method for the simulation study [Table\u00a01 (right) and Fig.\u00a02A]. Figure\u00a02A contains the average values in \u0394CO and \u0394MAP responses of the final 30\u00a0s during 10-min infusions of DBT at 3, 6, and 9\u00a0\u03bcg\u00a0kg\u22121\u00a0min\u22121 and SNP at 1, 2, and 4\u00a0\u03bcg\u00a0kg\u22121\u00a0min\u22121 (Fig.\u00a01A). The \u0394CO and \u0394MAP responses tested in simulations were over the ranges of the averaged responses in actual hemodynamics of dogs with heart failure.\nFIGURE\u00a02.A. Nonlinear responses of \u0394CO and \u0394MAP in infusion of DBT or SNP. Circles (\u2022) show experimental data for dogs with heart failure; solid lines are curves fitted to the averaged data. Dashed lines show the limits of the tested range [one-third (a1\u00a0=\u00a0a2\u00a0=\u00a0b1\u00a0=\u00a0b2\u00a0=\u00a01\/3) to three-times (a1\u00a0=\u00a0a2\u00a0=\u00a0b1\u00a0=\u00a0b2\u00a0=\u00a03) responses compared with the averaged data (a1\u00a0=\u00a0a2\u00a0=\u00a0b1\u00a0=\u00a0b2\u00a0=\u00a01) in Eq. (4)] in simulations. B. Multi-input multi-output model responses for \u0394CO and \u0394MAP. System inputs are the infusion rates of DBT and SNP; outputs are the \u0394CO and \u0394MAP. The outputs were determined by the first-order dynamic systems cascaded with nonlinear sigmoidal functions. The proportional gains of patient sensitivities to drugs and drug interactions are shown as a1, a2, b1, and b2.\nFinally, the model responses containing patient sensitivities to therapeutic agents and drug interactions are expressed as , where \u0394COmod(t) is the model response containing sensitivities to drugs and drug interactions of DBT and SNP; \u0394CO1\u2032(t) and \u0394CO2\u2032(t), respectively, indicate the nonlinear responses to single infusions of DBT and SNP; and a1 and a2, respectively, represent the proportional gain of patient sensitivity to DBT and SNP. The model response containing sensitivities to drugs and drug interactions of SNP and DBT is \u0394MAPmod(t). The respective nonlinear responses to single infusions of SNP and DBT are \u0394MAP1\u2032(t) and \u0394MAP2\u2032(t). The respective proportional gains of patient sensitivity to SNP and DBT are denoted as b1 and b2 (see Fig.\u00a02B). In particular, a2 and b2 can be defined as the strength of the drug interaction when the two treatments for DBT-CO and SNP-MAP loops are performed.\nDevelopment of Controller\nControl Design\nFigure\u00a03 portrays a block diagram of a MAPCNN system for adaptation to various patient responses with heart failure. Treating the multiple loops in the therapy for heart failure separately allows setting of a clear goal of NN learning for the various patient responses during closed-loop control. On the other hand, in the completely separated controllers, the total control performance will be late because one controller performs the drug therapy after detection of the drug interaction disturbance induced by performing an action taken by the other controller. Therefore, the MAPCNN in this study includes two module controllers for the DBT-CO loop considering the effects of SNP on CO and the SNP-MAP loop considering the effects of DBT on MAP.\nFIGURE\u00a03.A block diagram showing multiple adaptive predictive control using neural networks (MAPCNN) to regulate CO and MAP. The r is a target value, and e(t) is the error between the target value and observed value. The value e(t\u00a0+\u00a0i) represents the error between the target value and output predicted by the NN. Thick lines show the learning loop in the NN; dotted lines show the prediction loop using the NN.\nFigure\u00a04A depicts one of the two NN structures in MAPCNN tested for the simulation study. The MAPCNN is a control system in which the NN recursively learns patient characteristics using their observed responses to drug infusions only once every 30\u00a0s during closed-loop control (Learning Loop). It subsequently determines the future outputs using the learned NN (Prediction Loop). A multilayer feed-forward NN with two hidden layers (\u0394yNN) emulated the nonlinear responses in \u0394COmod and \u0394MAPmod. The \u0394yNN is predicted through NN as  where \u0394ymod(t\u22121) and \u0394ymod(t\u22122) are model responses of past \u0394CO or \u0394MAP; u1(t\u22121),..., and u1(t\u22126) or u2(t\u22121),... and u2(t\u22126) represent the past 3-min infusion rates of DBT or SNP. Here, to determine the length of the history of model response and infusion rates as inputs to the NN, the accuracy of NN learning was tested under various lengths of components (for a detailed protocol, refer to the following paragraph \u201cLearning of Initial Weights in NN\u201d). Figure\u00a04B shows the average values (final 500 points) in the absolute error between the \u0394COmod and \u0394CONN responses (left) or \u0394MAPmod and \u0394MAPNN responses (right) from the trained NN [100,000 times, a1\u00a0=\u00a0a2\u00a0=\u00a0b1\u00a0=\u00a0b2\u00a0=\u00a01 in Eq. (4)] under various input\u2013output components to the NN input. The numbers of the input units (m) to a single NN component and of the units in the first (n1) and second (n2) hidden layers of the NN were set to the same as that of components to the NN input. For example, if the inputs to the NN were 14 (the past 3\u00a0min in both the two drug inputs and the past 1\u00a0min in the model response), then m\u00a0=\u00a0n1\u00a0=\u00a0n2\u00a0=\u00a014. Learning rates of the two NN in both the CO and MAP controls were set to Kn1\u00a0=\u00a0Kn2\u00a0=\u00a00.2. The starting weights of NN were given at random every trial. In the past history of infusion rates, the error between the NN and model responses in the past 2-min infusion rates (eight components in Fig.\u00a04B: four in DBT and four in SNP) showed adequate accuracy, although that in the past 1-min infusion rates (four components) was not demonstrably accurate in both the CO and MAP responses. Here, it was predicted that the pure time delays to drug inputs were different among patients and that the NN was required to express the characteristics of the transient response to a drug input adequately considering the effect of the other drug input. Therefore, to adjust the NN response to the changes of hemodynamics with noise and disturbances during the real-time control, the infusion rates of the past 3\u00a0min in both drugs (total 12 components: 6 components in each drug) were, on the safe side, selected for both the CO and MAP controls in the present study. In the past history of model response, the accuracy of NN learning was the almost equal among the cases of the past 1, 1.5, and 2\u00a0min (2, 3, and 4 components in Fig.\u00a04B) in both the CO and MAP responses, whereas that in the past 30\u00a0s (1 component) showed inadequate accuracy. Accordingly, the length of the past history of model response was determined as 1\u00a0min (2 components) in both the CO and MAP controls.\nFIGURE\u00a04.A. A single component of a four-layer feed-forward NN with two hidden layers in MAPCNN to emulate the characteristics of a patient. The number of units in each hidden layer of the NN was set to 14 (numerically equal to the input units). A hyperbolic tangent function was used as the output of each unit. B. Average values (final 500 points) in the absolute error between the \u0394COmod and \u0394CONN responses (left) or \u0394MAPmod and \u0394MAPNN responses (right) from the trained NN [100,000 times, a1\u00a0=\u00a0a2\u00a0=\u00a0b1\u00a0=\u00a0b2\u00a0=\u00a01 in Eq. (4)] under various lengths of input\u2013output components to NN input. C. The absolute error between the model response and the predicted response by NN in (a) \u0394CO (left) or (b) \u0394MAP (right).\nIn the simulation study, the number of input units to a single NN component was m\u00a0=\u00a014; the numbers of units in the first and second hidden layers of the NN were n1\u00a0=\u00a0n2\u00a0=\u00a014, being equal to the input units. The weights in the single NN were 435: 196 in the input to first hidden layer, 196 in the first to second hidden layer, 14 in the second hidden layer to output layer, and 29 for biases (Fig.\u00a04A). The two NN for the controls of DBT-CO and SNP-MAP loops had identical structures.\nIn the learning loop, a single NN was trained by the output of \u0394COmod or \u0394MAPmod to the random inputs of DBT and SNP using the backpropagation algorithm in an on-line mode, showing that the error function is calculated after presentation of an input. The prediction loop in the MAPCNN determines the optimal DBT or SNP infusion rate that minimizes the cost function using the updated NN through the learning loop every 30\u00a0s. \nTherein, the cost function [J1(t), J2(t)] comprises the weight of input change (q1, q2), the prediction range (Np1, Np2), and the setpoint (r1, r2) in the controllers for the DBT-CO loop or the SNP-MAP loop. The physiological responses predicted by the NN are indicated as \u0394CONN and \u0394MAPNN. The controller based on the NN for each loop predicts future outputs using past inputs of infusion rates of DBT and SNP. The optimization of the infusion rates [u1(t), u2(t)] was performed using a Nelder\u2013Mead Simplex algorithm.16,27\nDetermination of Control Parameters\nLearning of Initial Weights in NN\nThe two NN, respectively, learned \u0394COmod response in the DBT-CO loop and \u0394MAPmod response in the SNP-MAP loop to determine the initial weights in the NN for the MAPCNN. The starting weights in the NN before learning the model response were given at random between \u22121 and 1. Subsequently, the infusion rates of DBT and SNP were given at random between \u22124 and 6\u00a0\u03bcg\u00a0kg\u22121\u00a0min\u22121. Here, both the plus and minus signs as drug inputs (artificial infusions) to the NN were used because the learning of NN was inferred to be more effective than that under a plus sign alone as the drug input to the NN in the trial and error and previous studies.9,27 Learning of the NN for \u0394COmod or \u0394MAPmod responses was repeated 100,000 times. The \u0394COmod was divided by 200, and \u0394MAPmod was divided by 100 for normalization during NN learning.\nFigure\u00a04C shows the absolute error between the \u0394COmod and \u0394CONN responses (left) or \u0394MAPmod and \u0394MAPNN responses (right) from the trained NN in the average patient sensitivity [a1\u00a0=\u00a0a2\u00a0=\u00a0b1\u00a0=\u00a0b2\u00a0=\u00a01 in Eq. (4)]. Learning rates of the two NN for the following simulation study were set to Kn1\u00a0=\u00a0Kn2\u00a0=\u00a00.2 showing a suitable number by trial and error in both the \u0394COmod and \u0394MAPmod. Learning results of the NN, respectively, showed errors of 2.5\u00a0ml\u00a0kg\u22121\u00a0min\u22121 in the DBT-CO loop and 1.5\u00a0mmHg in the SNP-MAP loop.\nController Tuning\nOptimal values of the range of prediction (Np1, Np2) and the weight of input (q1, q2) in the cost function (6) of the controller were explored using the model patient responses, \u0394COmod and \u0394MAPmod, to determine the initial controller parameters. The prediction range was set to Np1\u00a0=\u00a0Np2\u00a0=\u00a04, 8, or 12, and the weight of input was set to q1\u00a0=\u00a0q2\u00a0=\u00a00.01, 0.1, or 1. The learning rates of \u0394COmod and \u0394MAPmod were fixed at Kn1\u00a0=\u00a0Kn2\u00a0=\u00a00.2. To regulate the control speed and stability simultaneously, the setpoint of \u0394CO was guided by the linear function r1\u00a0=\u00a035t\/600\u00a0ml\u00a0kg\u22121\u00a0min\u22121 during the 10\u00a0min following the start of the closed-loop control. Thereafter, it was maintained at r1\u00a0=\u00a035\u00a0ml\u00a0kg\u22121\u00a0min\u22121. The setpoint of \u0394MAP was set to r2\u00a0=\u00a0\u00b10\u00a0mmHg. The duration of the closed-loop control was set to 40\u00a0min.\nFigure\u00a05 shows simulation results of the MAPCNN using average responses of \u0394COmod and \u0394MAPmod [a1, a2, b1, and b2 in Eq. (4) were set to unity]. The controller suppressed a control speed instead of facilitating stable control with the increase of the weight of input (q1\u00a0=\u00a0q2\u00a0=\u00a00.01\u21921) at each range of prediction (Np1\u00a0=\u00a0Np2\u00a0=\u00a04, 8, or 12). On the other hand, when the weight of the input is small (q1\u00a0=\u00a0q2\u00a0=\u00a00.01), the controller performed a slightly aggressive control with the decreased range of prediction (Np1\u00a0=\u00a0Np2\u00a0=\u00a012\u21924). The relationship at the large weight of input (q1\u00a0=\u00a0q2\u00a0=\u00a01) was opposite because of the strong effects of the input weight at the small range of prediction (Np1\u00a0=\u00a0Np2\u00a0=\u00a04). For subsequent simulations, the parameters (Np1, Np2, q1, q2) in the MAPCNN were set to Np1\u00a0=\u00a0Np2\u00a0=\u00a012 and q1\u00a0=\u00a0q2\u00a0=\u00a00.01 considering the settling time which reflects control speed and stability. In this case, the settling time within \u00b13\u00a0ml\u00a0kg\u22121\u00a0min\u22121 in \u0394COmod was 660\u00a0s; its time within \u00b12\u00a0mmHg in \u0394MAPmod was 990\u00a0s. The average absolute value of error between a setpoint and model response in \u0394COmod or \u0394MAPmod over the entire control period (average error) for 40\u00a0min was 2.8\u00a0ml\u00a0kg\u22121\u00a0min\u22121 in \u0394CO or 0.5\u00a0mmHg in \u0394MAP.\nFIGURE\u00a05.Simulation results of the MAPCNN under average responses of \u0394COmod and \u0394MAPmod [a1\u00a0=\u00a0a2\u00a0=\u00a0b1\u00a0=\u00a0b2\u00a0=\u00a01]. The input weight was changed to q1\u00a0=\u00a0q2\u00a0=\u00a00.01, 0.1 or 1 fixing the range of prediction at Np1\u00a0=\u00a0Np2\u00a0=\u00a0(A) 4, (B) 8, or (C) 12. The NN learning rate was set to Kn1\u00a0=\u00a0Kn2\u00a0=\u00a00.2 under all conditions.\nEvaluation of Controller\nSimulations\nTo evaluate the control performance, a simulation study in MAPCNN, which expressed the repeatability and freely operated physiological parameters such as nonlinearity and interaction,22,30 was performed under unexpected changes of patient responses to therapeutic agents with acute disturbances using the model response based on experimental data of canine heart failure. Increased CO to more than 95\u00a0ml\u00a0kg\u22121\u00a0min\u22121, while keeping MAP within the normal range (80\u2013100\u00a0mmHg) is desirable to treat acute heart failure.6,7 Therefore, the control objectives in this study were to increase the low CO (mean\u00a0\u00b1\u00a0S.E.M.\u00a0=\u00a067.5\u00a0\u00b1\u00a03.4\u00a0ml\u00a0kg\u22121\u00a0min\u22121, Fig.\u00a01A) at the setpoint of +35\u00a0ml\u00a0kg\u22121\u00a0min\u22121 (\u0394CO\u00a0=\u00a0+35t\/600 within 10\u00a0min after the start of closed-loop control) using DBT infusion and to simultaneously maintain the normal MAP (mean\u00a0\u00b1\u00a0S.E.M.\u00a0=\u00a096.7\u00a0\u00b1\u00a02.0\u00a0mmHg, Fig.\u00a01A) at the setpoint (\u0394MAP\u00a0=\u00a0\u00b10\u00a0mmHg) using SNP infusion when hypertension is induced by treatment for DBT-CO loop in acute heart failure. The infusion rates were basically bounded as 0\u00a0\u2264\u00a0u1(t)\u00a0\u2264\u00a010 in DBT and 0\u00a0\u2264\u00a0u2(t)\u00a0\u2264\u00a06 in SNP to avoid an overdose or drug toxicity.5,22 Hemodynamic control was simulated under the following cases: drug interactions between DBT and SNP, acute disturbances, and time-variant changes of physiological parameters.\nDrug Interactions\nGrasping and estimating the acts of drug interactions are difficult for hemodynamic control using multiple drugs.6,30 To examine the controller\u2019s robustness for wide ranges of patients\u2019 sensitivity to drugs and drug interactions, parameters (a1, a2, b1, b2) in Eq. (4) were changed to 2, 1\/3, or 3 every 40\u00a0min for 120-min control (Fig.\u00a06A). Hemodynamic responses were unknown to the NN because the NN learned only the average model responses (a1\u00a0=\u00a0a2\u00a0=\u00a0b1\u00a0=\u00a0b2\u00a0=\u00a01). The controller was also tested under various combinations for sensitivities to drugs and drug interactions. The model parameters, a1, a2, b1, and b2 in Eq. (4), were set to one of 1\/3 (Low), 1 (Mid.), and 3 (High), and all combinations were tested with or without the limitations of drug infusion rates. The control duration was 40\u00a0min.\nFIGURE\u00a06.Simulation results of MAPCNN with unexpected patient sensitivities and drug interactions. A in the top graph displays changes of parameters (a1, a2, b1, b2) in Eq. (4). B(a) and C(a) in the graph display setpoints, \u0394COmod and \u0394MAPmod (solid lines), and predicted outputs by NN (dashed lines). B(b) and C(b) are the time courses of weights changed from the baseline at the starting time in the NN for controllers in the DBT-CO and SNP-MAP loops: weights between input and first hidden layers (top), first and second hidden layers (middle), and second hidden and output layers (bottom). D. Infusion rates of DBT (solid line) and SNP (dashed line).\nTo know the limitation of the control performance, the MAPCNN was tested under very low (a1\u00a0=\u00a0a2\u00a0=\u00a0b1\u00a0=\u00a0b2\u00a0=\u00a01\/5 or 1\/10) and high (a1\u00a0=\u00a0a2\u00a0=\u00a0b1\u00a0=\u00a0b2\u00a0=\u00a05 or 10) sensitivities to drugs and drug interactions. The limitations of infusion rates of drugs were eliminated to emphasize the control performance. The control duration was 40\u00a0min.\nAcute Disturbances\nBolus infusion of drugs during and after cardiac surgery often introduces severe disturbances to a controller. Hemorrhage, patient-position changes, and changes in anesthesia levels will modulate patient response characteristics.10,20 To examine the controller\u2019s performance during acute disturbances, the MAPCNN against acute hypertension was simulated using the \u0394COmod and \u0394MAPmod responses for 120\u00a0min. Exogenous perturbations were added to the patient responses ranging within \u00b110\u00a0ml\u00a0kg\u22121\u00a0min\u22121 in \u0394COmod and +20\u00a0mmHg in \u0394MAPmod. To mimic physiological variation, random noises within \u00b13\u00a0ml\u00a0kg\u22121\u00a0min\u22121 in \u0394COmod and \u00b12\u00a0mmHg in \u0394MAPmod were also added [Fig.\u00a09A(a) and B(a)].\nTo implicate the limitation of the control performance, the MAPCNN was tested under very severe situations with random noise and exogenous perturbations over physiological responses: (i) very huge amplitudes of random noise, (ii) very large and (iii) acute disturbances. First, (i) three levels of huge amplitudes of random noise (level 1: \u00b15\u00a0ml\u00a0kg\u22121\u00a0min\u22121 in \u0394COmod and \u00b15\u00a0mmHg in \u0394MAPmod, level 2: \u00b125\u00a0ml\u00a0kg\u22121\u00a0min\u22121 and \u00b125\u00a0mmHg, and level 3: \u00b150\u00a0ml\u00a0kg\u22121\u00a0min\u22121 and \u00b150\u00a0mmHg) were added to model responses (a1\u00a0=\u00a0a2\u00a0=\u00a0b1\u00a0=\u00a0b2\u00a0=\u00a01) during drug treatment for 40\u00a0min [Figs.\u00a010A(a) and B(a)]. The random noises among the three levels had the same pattern except for the amplitude. Next, (ii) the MAPCNN was evaluated under very huge disturbances (level 1: \u221220\u00a0ml\u00a0kg\u22121\u00a0min\u22121 in \u0394COmod and +20\u00a0mmHg in \u0394MAPmod, level 2: \u221250\u00a0ml\u00a0kg\u22121\u00a0min\u22121 and +50\u00a0mmHg, and level 3: \u2212100\u00a0ml\u00a0kg\u22121\u00a0min\u22121 and +100\u00a0mmHg; a1\u00a0=\u00a0a2\u00a0=\u00a0b1\u00a0=\u00a0b2\u00a0=\u00a01; Fig.\u00a011A). Finally, (iii) the MAPCNN was tested under very acute disturbances with model responses in high sensitivities to drugs and drug interactions (a1\u00a0=\u00a0a2\u00a0=\u00a0b1\u00a0=\u00a0b2\u00a0=\u00a03). At 20\u00a0min after the start of control for 80\u00a0min, the acute disturbances of three levels [10 (level 1), 5 (level 2), and 0 (level 3) min to reaching the offset values of \u221250\u00a0ml\u00a0kg\u22121\u00a0min\u22121 in \u0394COmod and +50\u00a0mmHg in \u0394MAPmod after the start of the disturbances] were added (Fig.\u00a011B). In those simulations, the limitations of infusion rates of drugs were eliminated to elucidate the limitations of the control performance.\nTime-variant Changes\nPatient responses to therapeutic agents have a nonlinear and time-variant nature. If the delay of a plant, which reflects the infusion rate of drugs through the catheter, internal patient circulation and perfusion delay, and the drug-recirculation characteristics of a patient,2,26,30 is not known accurately or changes during drug infusions, infusion delays will engender an unstable condition. Therefore, the controller robustness was tested under the change of the infusion delay in this study. The pure time delays [L in the unit impulse response of Eq. (2)] of patient responses to therapeutic agents were varied from 30 to 90\u00a0s in CO responses and 60 to 120\u00a0s in MAP responses to DBT and SNP infusions during the closed loop control [Fig.\u00a012A(a)]. The parameters (a1, a2, b1, b2) in Eq. (4) were varied from 1\/3 to 3 to examine the controller performance under time-variant and wide ranges of patient sensitivities to drugs and drug interactions [Fig.\u00a012A(b)]. The exogenous perturbations were added to the time-variant patient responses ranging within \u00b110\u00a0ml\u00a0kg\u22121\u00a0min\u22121 in \u0394COmod and +20\u00a0mmHg in \u0394MAPmod. Random noises within \u00b13\u00a0ml\u00a0kg\u22121\u00a0min\u22121 and \u00b12\u00a0mmHg were added to \u0394COmod and \u0394MAPmod responses [Fig.\u00a012B(a) and C(a)]. The control duration was 120\u00a0min.\nAnimal Study\nTo evaluate the control performance in MAPCNN under the unknown physiological responses such as nonlinearity and drug interaction, the simultaneous control of CO and MAP was performed using a dog with acute heart failure. Acute ischemic heart failure in an anesthetized and ventilated dog (23\u00a0kg) was induced by microsphere embolization of the left main coronary artery. A double-lumen catheter was introduced into the right femoral vein for administration of drugs using an infusion pump (CFV-3200; Nihon Kohden, Tokyo, Japan). CO was measured by an electromagnetic flow probe (MFV-2100; Nihon Kohden), and MAP was measured through a pressure transducer (DX-200; Nihon Kohden) at a 10-Hz sampling rate through a 12-bit digital-to-analog converter.\nThe control objective was to increase the low CO (62.4\u00a0ml\u00a0kg\u22121\u00a0min\u22121) at the setpoint (+40%) using DBT infusion and to maintain the MAP (73.9\u00a0mmHg) at the setpoint (\u0394MAP\u00a0=\u00a0\u00b10\u00a0mmHg) using SNP infusion in acute heart failure. The closed-loop control duration was 60\u00a0min. The infusion rates were bounded as 0\u00a0\u2264\u00a0u1(t)\u00a0\u2264\u00a010 in DBT and 0\u00a0\u2264\u00a0u2(t)\u00a0\u2264\u00a06 in SNP.\nRESULTS\nSimulations\nDrug Interactions\nFigure\u00a06 shows simulation results of closed-loop control by MAPCNN (Np1\u00a0=\u00a0Np2\u00a0=\u00a012, q1\u00a0=\u00a0q2\u00a0=\u00a00.01, and Kn1\u00a0=\u00a0Kn2\u00a0=\u00a00.2) under unknown patient sensitivities to drugs and drug interactions. The \u0394CO in the DBT-CO loop converged on the setpoint (+35\u00a0ml\u00a0kg\u22121\u00a0min\u22121) within approximately 15\u00a0min, according to the guided setpoint during the first 40-min control period, regardless of unanticipated patient sensitivities and drug interactions (a1\u00a0=\u00a0a2\u00a0=\u00a0b1\u00a0=\u00a0b2\u00a0=\u00a02). The control for the \u0394MAP in the SNP-MAP loop minimally suppressed the hypertension (+4.2 vs. +15.0\u00a0mmHg with or without SNP infusion) induced by the DBT infusion. At 40 and 80\u00a0min of the closed-loop control, patient sensitivities and drug interactions were widely changed (a1\u00a0=\u00a0a2\u00a0=\u00a0b1\u00a0=\u00a0b2\u00a0=\u00a02 to 1\/3 and 1\/3 to 3). The \u0394CO converged on the setpoint robustly, whereas the \u0394CO showed transient and large changes (+35.0 showing the setpoint to +14.9 and to +99.3\u00a0ml\u00a0kg\u22121\u00a0min\u22121 at approximately 50 and 85\u00a0min). Although \u0394MAP was decreased acutely by the change of patient sensitivity and drug interaction (\u00b10 to \u221220.7\u00a0mmHg at approximately 85\u00a0min), it returned robustly to a normal level. The average errors between setpoints and observed responses were 6.4\u00a0ml\u00a0kg\u22121\u00a0min\u22121 in CO and 1.6\u00a0mmHg (10.5\u00a0mmHg without SNP infusion) in MAP during 120-min closed-loop control. Weights and biases of two NN were adjusted to optimal values [Fig.\u00a06B(b) and C(b)] when the unexpected changes occurred. The infusion rates of DBT and SNP were adjusted smoothly to optimal levels corresponding to the unknown patient responses to drugs.\nTable\u00a02 shows average errors between setpoints and model responses in \u0394CO and \u0394MAP during closed-loop control by MAPCNN (Np1\u00a0=\u00a0Np2\u00a0=\u00a012, q1\u00a0=\u00a0q2\u00a0=\u00a00.01, and Kn1\u00a0=\u00a0Kn2\u00a0=\u00a00.2) under the various sensitivities to drugs and drug interactions. The control performance was overall accurate and a tendency excited for dependence on the sensitivity of CO to DBT (i.e., parameter a1); the control performance was increased as the sensitivity of CO to DBT increased (a1\u00a0=\u00a01\/3\u21923). However, in some cases, great hypertension was observed during control, as displayed in Fig.\u00a07A; although the CO control was smoothly completed under such cases, the great hypertension was induced because of the high interaction of DBT infusion used for CO treatment (i.e. b2\u00a0=\u00a03). The maximum values of the hypertension in cases 1 (a1\u00a0=\u00a0\u2018Low\u2019, a2\u00a0=\u00a0\u2018Low\u2019, b1\u00a0=\u00a0\u2018Low\u2019, and b2\u00a0=\u00a0\u2018High\u2019), 2 (a1\u00a0=\u00a0\u2018Low\u2019, a2\u00a0=\u00a0\u2018Low\u2019, b1\u00a0=\u00a0\u2018Mid.\u2019, and b2\u00a0=\u00a0\u2018High\u2019), and 3 (a1\u00a0=\u00a0\u2018Low\u2019, a2\u00a0=\u00a0\u2018Mid.\u2019, b1\u00a0=\u00a0\u2018Low\u2019, and b2\u00a0=\u00a0\u2018High\u2019) were 60.3, 39.4, and 33.8\u00a0mmHg, respectively. Here, \u2018Low\u2019\u00a0=\u00a01\/3, \u2018Mid.\u2019\u00a0=\u00a01, and \u2018High\u2019\u00a0=\u00a03. In case 2, the infusion rates of DBT were slightly disturbed and the estimation error between the NN and \u0394CO responses oscillated. However, the \u0394CO in case 2 was unaffected by the oscillation of DBT infusion because of the low sensitivity of CO to DBT (a1\u00a0=\u00a0\u2018Low\u2019). Infusion rates of SNP in all cases were saturated at around 8\u00a0min because of the limitation of drug infusion. After the saturation, the infusion rate of SNP in case 1 was decreased at around 12\u00a0min.\nTABLE\u00a02.Average errors between setpoints and model responses in CO and MAP under various sensitvities to drugs and drug interactions.CO (a1):LowMid.HighCO (a2):MAP (b1)MAP (b2)LowMid.HighLowMid.HighLowMid.HighLowLow5.5 (0.5)4.8 (0.4)3.4 (0.3)2.9 (0.3)2.7 (0.3)2.2 (0.2)1.3 (0.1)1.2 (0.1)1.2 (0.1)Mid.5.3 (10.3)4.1 (4.8)3.0 (1.0)3.3 (1.9)2.6 (1.0)2.0 (0.7)1.3 (0.5)1.2 (0.5)1.2 (0.4)High5.7 (45.0*)3.8 (25.1*)4.2 (3.0)3.5 (17.3)2.9 (6.1)2.2 (1.6)4.2 (3.4)3.9 (3.5)1.5 (1.2)Mid.Low6.0 (1.2)5.7 (1.2)5.8 (1.1)3.2 (0.6)2.9 (0.6)2.4 (0.5)1.5 (0.4)1.4 (0.4)1.3 (0.3)Mid.5.5 (0.6)4.7 (0.5)3.5 (0.4)3.2 (0.5)2.8 (0.5)2.1 (0.4)1.6 (0.4)1.4 (0.4)1.3 (0.3)High5.3 (29.6*)3.8 (12.2)3.4 (1.9)3.3 (3.9)2.7 (1.4)2.0 (1.0)1.9 (1.1)1.7 (0.9)1.3 (0.7)HighLow6.0 (2.5)5.9 (2.5)6.2 (2.3)2.8 (0.7)2.8 (0.6)2.7 (0.7)1.3 (0.6)1.3 (0.5)1.3 (0.4)Mid.6.0 (4.1)6.2 (3.9)7.0 (3.4)2.9 (0.6)2.8 (0.6)2.5 (0.6)1.4 (0.6)1.3 (0.5)1.3 (0.4)High5.6 (1.0)4.8 (0.7)6.4 (4.3)3.2 (0.8)2.9 (0.8)2.3 (1.3)2.0 (1.6)1.8 (1.6)1.5 (1.5)The numbers show the average errors in CO (left) and MAP (right). \u2018Low\u2019\u00a0=\u00a01\/3, \u2018Mid.\u2019\u00a0=\u00a01, and \u2018High\u2019\u00a0=\u00a03 in the parameters in Eq. (4). The sign of * shows the cases of large hypertension (for detailed results, see Fig.\u00a07).FIGURE\u00a07.Simulation results of MAPCNN under cases of great hypertension (A) with and (B) without the limitation of drug infusion rates. Parameters (a1, a2, b1, b2) in Eq. (4) are a1\u00a0=\u00a0\u2018Low\u2019, a2\u00a0=\u00a0\u2018Low\u2019, b1\u00a0=\u00a0\u2018Low\u2019, and b2\u00a0=\u00a0\u2018High\u2019 in case 1, a1\u00a0=\u00a0\u2018Low\u2019, a2\u00a0=\u00a0\u2018Low\u2019, b1\u00a0=\u00a0\u2018Mid.\u2019, and b2\u00a0=\u00a0\u2018High\u2019 in case 2, and a1\u00a0=\u00a0\u2018Low\u2019, a2\u00a0=\u00a0\u2018Mid.\u2019, b1\u00a0=\u00a0\u2018Low\u2019, and b2\u00a0=\u00a0\u2018High\u2019 in case 3. \u2018Low\u2019, \u2018Mid.\u2019, and \u2018High\u2019 are 1\/3, 1, and 3, respectively. The hemodynamic responses (top), the error between the model response and the predicted response by NN during control (middle), and infusion rates of DBT and SNP (bottom) in \u0394CO (left) or \u0394MAP (right).\nTo elucidate the control performance without the limitation of infusion rates of drug inputs, the MAPCNN was also tested under the same cases 1, 2, and 3 (Fig.\u00a07B). However, great hypertension was not improved by SNP infusion without the drug input limitation (63.8, 39.8, and 38.4\u00a0mmHg in cases 1, 2, and 3) compared with those with the limitation of the drug input. In cases 2 and 3, the drug infusion rates in DBT were disturbed and the estimation errors between the NN and \u0394CO responses had some oscillations. In addition, in all cases, the infusion rates of SNP were decreased after increasing to around 10\u00a0\u03bcg\u00a0kg\u22121\u00a0min\u22121, irrespective of the remaining the hypertension.\nFigure\u00a08 shows the closed-loop control by MAPCNN (Np1\u00a0=\u00a0Np2\u00a0=\u00a012, q1\u00a0=\u00a0q2\u00a0=\u00a00.01, and Kn1\u00a0=\u00a0Kn2\u00a0=\u00a00.2) under (A) very low (a1\u00a0=\u00a0a2\u00a0=\u00a0b1\u00a0=\u00a0b2\u00a0=\u00a01\/5 or 1\/10) and (B) high (a1\u00a0=\u00a0a2\u00a0=\u00a0b1\u00a0=\u00a0b2\u00a0=\u00a05 or 10) sensitivities to drugs and drug interactions. Under very low sensitivities to drugs and drug interactions, the \u0394CO response was not able to reach the setpoint (\u00b135\u00a0ml\u00a0kg\u22121\u00a0min\u22121) because of the slight sensitivity of CO to DBT (a1\u00a0=\u00a01\/5 or 1\/10), but it finally converged on a stable value (Fig.\u00a08A, left). Estimation errors between the NN and model responses in \u0394CO had oscillations under such conditions. On the other hand, under very high sensitivities to drugs and drug interactions, both the \u0394CO and \u0394MAP responses oscillated depending on the degree of sensitivities to drugs [maximum amplitudes between observed values and setpoints: (+)9.2\u00a0ml\u00a0kg\u22121\u00a0min\u22121 and (\u2212)18.8\u00a0mmHg in a1\u00a0=\u00a0a2\u00a0=\u00a0b1\u00a0=\u00a0b2\u00a0=\u00a05 or (+)35.9\u00a0ml\u00a0kg\u22121\u00a0min\u22121 and (\u2212)54.1\u00a0mmHg in a1\u00a0=\u00a0a2\u00a0=\u00a0b1\u00a0=\u00a0b2\u00a0=\u00a010, Fig.\u00a08B]. However, the MAPCNN had a tendency to suppress oscillations in the \u0394CO and \u0394MAP responses gradually. The estimation error between the NN and model responses and the infusion rates of drugs were also disturbed.\nFIGURE\u00a08.Simulation results of MAPCNN with (A) very low (a1\u00a0=\u00a0a2\u00a0=\u00a0b1\u00a0=\u00a0b2\u00a0=\u00a01\/5 or 1\/10) or (B) very high (a1\u00a0=\u00a0a2\u00a0=\u00a0b1\u00a0=\u00a0b2\u00a0=\u00a05 or 10) patients\u2019 sensitivities to drugs and drug interactions. The hemodynamic responses (top), the error between the model response and the predicted response by NN during control (middle), and infusion rates of DBT and SNP (bottom) in \u0394CO (left) or \u0394MAP (right). The limitations of drug infusion rates were eliminated.\nAcute Disturbances\nFigure\u00a09 shows closed-loop control by MAPCNN (Np1\u00a0=\u00a0Np2\u00a0=\u00a012, q1\u00a0=\u00a0q2\u00a0=\u00a00.01, and Kn1\u00a0=\u00a0Kn2\u00a0=\u00a00.2) under unexpected acute disturbances with background noise. The \u0394CO converged on the setpoint (+35\u00a0ml\u00a0kg\u22121\u00a0min\u22121) within approximately 12\u00a0min according to the guided setpoint during the first 40-min control period, regardless of the added random noise (\u00b13\u00a0ml\u00a0kg\u22121\u00a0min\u22121) with expected patient responses (a1\u00a0=\u00a0a2\u00a0=\u00a0b1\u00a0=\u00a0b2\u00a0=\u00a01). The appropriate infusion of SNP for control of \u0394MAPmod with random noise (\u00b12\u00a0mmHg) suppressed hypertension (+3.5 vs. +15.4\u00a0mmHg with or without SNP treatment) induced by DBT infusion. At 40 and 80\u00a0min of the closed-loop control, acute disturbances were added to the \u0394COmod (+10 and \u221210\u00a0ml\u00a0kg\u22121\u00a0min\u22121) and \u0394MAPmod (+20 and +10\u00a0mmHg) responses. The MAPCNN suppressed the transient change of CO minimally and the CO converged on the setpoint as quickly as possible. The induced transient hypertension was suppressed robustly by optimal infusion of SNP (+6.7 vs. +31.0\u00a0mmHg with or without SNP treatment in the added disturbance at 40\u00a0min). Average errors between setpoints and observed responses were 3.7\u00a0ml\u00a0kg\u22121\u00a0min\u22121 in CO and 1.4\u00a0mmHg (20.8\u00a0mmHg without SNP) in MAP during 120-min closed-loop control. Weights and biases of two NN were adjusted to the optimal values when the unexpected changes occurred [Fig.\u00a09A(c) and B(c)]. Changes of biases were linked to those of the acute disturbances and physiological variation. The infusion rates of DBT and SNP were adjusted smoothly to optimal levels corresponding to the unknown disturbances.\nFIGURE\u00a09.Simulation results of MAPCNN with unknown disturbances. In the graph, A(a) and B(a) show changes of acute disturbances and random noise added to \u0394COmod and \u0394MAPmod responses. A(b) and B(b) represent setpoints, \u0394COmod and \u0394MAPmod (solid lines), and predicted outputs by NNs (dashed lines). A(c) and B(c) indicate weight changes in NN for the controllers in DBT-CO and SNP-MAP loops: weights between the input and first hidden layers (top), first and second hidden layers (middle), and second hidden and output layers (bottom). C. Infusion rates of DBT (solid line) and SNP (dashed line).\nFigure\u00a010 shows the closed-loop control by MAPCNN (Np1\u00a0=\u00a0Np2\u00a0=\u00a012, q1\u00a0=\u00a0q2\u00a0=\u00a00.01, and Kn1\u00a0=\u00a0Kn2\u00a0=\u00a00.2) under very large amplitudes of random noise [levels 1 (\u00b15 in both \u0394COmod and \u0394MAPmod), 2 (\u00b125), and 3 (\u00b150), Fig.\u00a010A(a) and B(a); a1\u00a0=\u00a0a2\u00a0=\u00a0b1\u00a0=\u00a0b2\u00a0=\u00a01]. Although the \u0394CO and \u0394MAP responses [Figs.\u00a010A(b) and B(b)] approached the setpoints, they depended directly on the appearance patterns and the amplitudes in large random noise. The infusion rates in DBT and SNP and the estimation errors between the NN and model responses were also reflected by changes of random noise.FIGURE\u00a010.Simulation results of MAPCNN with large random noise. A(a) and B(a) show changes of large random noise added to \u0394COmod and \u0394MAPmod responses. Noise level 1 (\u00b15 in \u0394COmod and \u00b15\u00a0mmHg in \u0394MAPmod), level 2 (\u00b125\u00a0ml\u00a0kg\u22121\u00a0min\u22121 and \u00b125\u00a0mmHg), and level 3 (\u00b150\u00a0ml\u00a0kg\u22121\u00a0min\u22121 and \u00b150\u00a0mmHg). A(b) and B(b) represent setpoints, \u0394COmod and \u0394MAPmod responses (a1\u00a0=\u00a0a2\u00a0=\u00a0b1\u00a0=\u00a0b2\u00a0=\u00a01). A(c) and B(c) indicate the error between the model response and the predicted response by NN during control. A(d) and B(d) are Infusion rates of DBT and SNP. The limitations of drug infusion rates were eliminated.\nFigure\u00a011A shows the closed-loop control by MAPCNN (Np1\u00a0=\u00a0Np2\u00a0=\u00a012, q1\u00a0=\u00a0q2\u00a0=\u00a00.01, and Kn1\u00a0=\u00a0Kn2\u00a0=\u00a00.2) under huge disturbances [levels 1 (\u221220\u00a0ml\u00a0kg\u22121\u00a0min\u22121 in \u0394COmod and +20\u00a0mmHg in \u0394MAPmod), 2 (\u221250\u00a0ml\u00a0kg\u22121\u00a0min\u22121 and +50\u00a0mmHg), and 3 (\u2212100\u00a0ml\u00a0kg\u22121\u00a0min\u22121 and +100\u00a0mmHg); a1\u00a0=\u00a0a2\u00a0=\u00a0b1\u00a0=\u00a0b2\u00a0=\u00a01]. In the disturbance of level 1, the \u0394CO and \u0394MAP responses converged on the setpoints, whereas the estimation error between the NN and model responses in \u0394CO exhibited some oscillations around 60\u201365\u00a0min. At level 2, the CO control was completed well. However, the MAP did not reach the setpoint because of the large disturbance and the interaction from DBT used for the CO control. In addition, the infusion rate of SNP was decreased at around 30\u00a0min irrespective of the hypertension of approximately 40\u00a0mmHg. The MAP response did not oscillate and converged on a steady state being far from the setpoint. At level 3, neither the \u0394CO nor \u0394MAP responses reached the setpoints. The infusion rate of SNP was increased instantaneously by receiving a great disturbance at 20\u00a0min, and it was decreased at around 25\u00a0min regardless of remaining the large hypertension of approximately 120\u00a0mmHg. The estimation error between the NN and model responses in \u0394CO showed oscillation at approximately 40\u00a0min.FIGURE\u00a011.Simulation results of MAPCNN with (A) very huge and (B) acute disturbances. A: huge disturbance level 1 (\u221220\u00a0ml\u00a0kg\u22121\u00a0min\u22121 in \u0394COmod and +20\u00a0mmHg in \u0394MAPmod), level 2 (\u221250\u00a0ml\u00a0kg\u22121\u00a0min\u22121 and +50\u00a0mmHg), and level 3 (\u2212100\u00a0ml\u00a0kg\u22121\u00a0min\u22121 and +100\u00a0mmHg). B: acute disturbance level 1 (10\u00a0min to the added disturbance of \u221250\u00a0ml\u00a0kg\u22121\u00a0min\u22121 in \u0394CO and +50\u00a0mmHg in \u0394MAP), level 2 (5\u00a0min), and level 3 (0\u00a0min); a1\u00a0=\u00a0a2\u00a0=\u00a0b1\u00a0=\u00a0b2\u00a0=\u00a03 in Eq. (4). The hemodynamic responses (top), the error between the model response and the predicted response by NN during control (middle), and infusion rates of DBT and SNP (bottom) in \u0394CO (left) or \u0394MAP (right). The limitations of drug infusion rates were eliminated.\nFigure\u00a011B shows the closed-loop control by MAPCNN (Np1\u00a0=\u00a0Np2\u00a0=\u00a012, q1\u00a0=\u00a0q2\u00a0=\u00a00.01, and Kn1\u00a0=\u00a0Kn2\u00a0=\u00a00.2) under very acute disturbances with high sensitivities to drugs and drug interactions [levels 1 (10\u00a0min to the added disturbances of \u221250\u00a0ml\u00a0kg\u22121\u00a0min\u22121 in \u0394CO and +50\u00a0mmHg in \u0394MAP), 2 (5\u00a0min), and 3 (0\u00a0min); a1\u00a0=\u00a0a2\u00a0=\u00a0b1\u00a0=\u00a0b2\u00a0=\u00a03]. At levels 1 and 2, although the \u0394CO and \u0394MAP responses showed small oscillations because of the acute disturbances, they converged on the setpoints by 80\u00a0min. In level 3 (step input of disturbance), the \u0394CO and \u0394MAP received the effects of the step input of the very acute disturbances at 20\u00a0min directly. Although \u0394CO and \u0394MAP responses were disturbed until 70\u00a0min, those values tended to converge on the setpoints eventually. In particular, the infusion rate of SNP at level 3 was increased acutely to 20\u00a0\u03bcg\u00a0kg\u22121\u00a0min\u22121 against the large hypertension of 50\u201360\u00a0mmHg induced by the very acute disturbance with high amplitude of 50\u00a0mmHg at 20\u00a0min; in turn, the excessive hypotension after the acute hypertension was induced by the high sensitivity of MAP to the SNP used for the MAP control (b1\u00a0=\u00a03). However, the infusion rate of SNP was adjusted to the optimal value at approximately 70\u00a0min.\nTime-variant Changes\nFigure\u00a012 shows the closed-loop control by MAPCNN (Np1\u00a0=\u00a0Np2\u00a0=\u00a012, q1\u00a0=\u00a0q2\u00a0=\u00a00.01, and Kn1\u00a0=\u00a0Kn2\u00a0=\u00a00.2) under the unknown time-variant responses containing time delays to therapeutic agents with acute disturbances. The \u0394CO converged on the setpoint (+35\u00a0ml\u00a0kg\u22121\u00a0min\u22121) within approximately 10\u00a0min according to the guided setpoint during the first 40-min control, irrespective of time-variant patient sensitivities and drug interactions with added noise; it showed only slight oscillation (\u00b15\u00a0ml\u00a0kg\u22121\u00a0min\u22121). The SNP infusion for the control of \u0394MAP suppressed the hypertension (+6.1 and +22.8\u00a0mmHg with and without SNP treatment) induced by DBT infusion beforehand. At 40\u00a0min of the closed-loop control, acute disturbances (\u221210\u00a0ml\u00a0kg\u22121\u00a0min\u22121 in CO and +20\u00a0mmHg in MAP) were added to the patient responses. The \u0394CO and \u0394MAP quickly converged on the setpoint within approximately 10\u00a0min, whereas the transient hypertension was induced (+9.4 vs. +38.9\u00a0mmHg with or without SNP). The average errors between setpoints and observed responses were 4.2\u00a0ml\u00a0kg\u22121\u00a0min\u22121 in CO and 2.7\u00a0mmHg (15.3\u00a0mmHg without a vasodilator) in MAP during 120-min closed-loop control. The weights and biases of two NN were adjusted to optimal values when the unexpected changes occurred [Fig.\u00a012B(c) and C(c)]. Changes of biases were linked to those of the time-variant responses and acute disturbances. The infusion rates of DBT and SNP were adjusted to optimal levels corresponding to the unknown time-variant responses.\nFIGURE\u00a012.Simulation results of MAPCNN under unknown time-variant responses with disturbances. A(a) in the graph displays changes of the parameters of the time delays (L) in the unit impulse response of Eq. (2). A(b) indicates changes of the parameters (a1, a2, b1, b2) in Eq. (4). B(a) and C(a) show changes of acute disturbances and random noise added to \u0394COmod and \u0394MAPmod responses. B(b) and C(b) denote setpoints, \u0394COmod and \u0394MAPmod (solid lines), and predicted outputs by NN (dashed lines). B(c) and C(c) are the weight changes in NN for controllers in DBT-CO and SNP-MAP loops: weights between input and first hidden layers (top), first and second hidden layers (middle), and second hidden and output layers (bottom). D. Infusion rates of DBT (solid line) and SNP (dashed line).\nAnimal Study\nFigure\u00a013 shows results of closed-loop control by MAPCNN (Np1\u00a0=\u00a0Np2\u00a0=\u00a08, q1\u00a0=\u00a0q2\u00a0=\u00a00.3, and Kn1\u00a0=\u00a0Kn2\u00a0=\u00a00.2) under the actual response of canine left heart failure. The CO in the DBT-CO loop converged on the setpoint within approximately 10\u00a0min regardless of the unknown response containing nonlinear, drug interaction, and the effects of arterial baroreflex with physiological variation in actual heart failure. The control for the \u0394MAP in the SNP-MAP loop suppressed the acute hypertension (+20\u00a0mmHg) induced by the DBT infusion. Whereas the large disturbance like arrhythmia was induced at around 38\u00a0min during the control, the CO and MAP were appropriately controlled by the MAPCNN adjusting the infusion rates of DBT and SNP to the optimal levels. The average errors between setpoints and observed responses were 7.3\u00a0ml\u00a0kg\u22121\u00a0min\u22121 in CO and 6.4\u00a0mmHg in MAP during 60-min closed-loop control.\nFIGURE\u00a013.Results of hemodynamic regulation by means of MAPCNN under canine left heart failure. Raw data of CO (top) and MAP (middle) responses during DBT and SNP infusions. Bottom in the graph is the Infusion rates of DBT (solid line) and SNP (dashed line).\nDISCUSSION\nThe development of automatic drug-delivery systems requires a controller that can adapt to the various patient responses in clinical situations. The MAPCNN was confirmed to be robust with respect to uncertainty in drug interactions, acute disturbances, time-variant responses containing time delays to therapeutic agents (Figs.\u00a06, 9, and 12), and the actual response of a dog with heart failure (Fig.\u00a013) because of its ability to learn nonlinear and time-variant changes of the system during the on-line control.\nThe infusion of DBT increased MAP as well as CO in acute left heart failure of dogs [Fig.\u00a01A(a)]. The DBT infusion does not generally act on the SVR whereas both the CO and SVR affect MAP.14 Therefore, the increase of MAP during DBT infusion in this study would have resulted mainly from increasing the CO induced by the actions of the beta receptors (\u03b21, \u03b22) of cardiac smooth muscle30 rather than the SVR. On the other hand, the SNP infusion resulted in the decrease of MAP and the increase of CO between middle and high doses [Fig.\u00a01A(b)] because of the decrease of SVR (afterload of a heart) by SNP and secondarily increased CO.6 In addition, the SNP treatment might have suppressed the increase in preload through the decrease of SVR because of increasing venous compliance for retaining the blood in the veins and lowering the venous return to the heart in case of congestive heart failure. In the simultaneous regulation of CO and MAP, the control for CO induced hypertension (Figs.\u00a06, 9, 12, and 13) because the primary control target in this study was the increase of low CO in acute heart failure. The MAPCNN was able to suppress the hypertension using optimal infusion of SNP as well as increasing CO using DBT to an optimal target value because the combined infusion of an inotropic agent and a vasodilator would have acted effectively.12,13,25\nApplication of MAPCNN to a multiple hemodynamic control accomplished the regulation of CO and MAP under various changes of the patient\u2019s responses to drugs and disturbances (Figs.\u00a06, 9, 12, and 13). In particular, regardless of the large change of patient sensitivities as shown in Fig.\u00a06, the MAPCNN robustly adjusted the acute and large changes to generate a stable condition. Similarly to the previous controllers,7,10 the MAPCNN suppressed those disturbances performed stabilized control under unexpected acute disturbances (Figs.\u00a09 and 12). Under time-variant patient responses with pure time delays, which are a crucial obstacle to stable control,2,26 the MAPCNN provided sufficient control performance (Fig.\u00a012). Regardless of actual nonlinear response, drug interaction, and partial disturbances with arrhythmia, the MAPCNN could regulate the CO and MAP simultaneously (Fig.\u00a013). The superior control performance resulted from the function to adjust the weights and biases of the NN to optimal points during the on-line control (Figs.\u00a06, 9, and 12).\nOnly the two-NN models of average responses with heart failure were considered in the calculation of the appropriate multiple drug infusion rates of DBT and SNP (Fig.\u00a03) to mitigate the enormous number of trials associated with the control design. Model predictive controllers or fuzzy controllers might require an extremely lengthy set-up stage to prepare the model banks as linear models of patients\u2019 responses to drugs or to provide the experienced rules describing various cases in clinical settings whereas the controllers are an effective means of adjusting to various patients\u2019 sensitivities to drugs21 and describing nonlinear responses to drugs5. A controller based on NN solves those problems because it decreases the number of models required for the control design of the various changes of hemodynamics clearly.\nIrrespective of the wide range over physiological responses (Fig.\u00a02) in simulations and actual response of a dog with heart failure containing the effects of baroreflex and the full renin\u2013angiotensin system induced by long-term control,14 CO and MAP in the MAPCNN promptly approached the setpoints because of the optimization of both the stability and speed for the MAPCNN (Figs.\u00a06, 9, 12, and 13). Therefore, the designed MAPCNN will be feasible for application to automatic drug therapy in heart failures. However, when rapid treatment using drugs against more acute and large disturbances is required during hemodynamic controls, another supplemental system might be required.9 Diagnoses of characteristics of patients\u2019 responses to drugs or tuning weights of NNs during closed-loop controls1 may also be effective for hemodynamic controls to accelerate the NN learning speed. In addition, because the fluid infusion, blood transfusion, anesthesia, and muscular blockade as well as the therapeutic agents controlled in this study are common in clinical practice,22,29 the controllers that can adjust physiological responses to further multiple drugs will be desired. The MAPCNN tested herein can be extended simply to multivariate control systems under such clinical conditions for drug therapy with heart failure.\nThe MAPCNN was tested under various conditions over physiological responses to elucidate the limitations of the control performance (Figs.\u00a07, 8, 10, and 11). Regardless of such severe conditions, the hemodynamics during MAPCNN learning very large changes of the sensitivities to drugs and drug interactions and the disturbances using NN tended to converge on the setpoints with some oscillations observed, insofar as those responses were within the possible range of the control using DBT and SNP (e.g., Figures\u00a08B and 11B). On the other hand, there existed cases for which it was obviously impossible for MAPCNN to control the hemodynamics (Figs.\u00a07, 8A, and 11A). For example, when the interaction from DBT for the CO control to MAP was very large (b2\u00a0=\u00a03) and the sensitivity of MAP to SNP was small (b1\u00a0=\u00a01\/3 or 1, Fig.\u00a07), the MAPCNN was actually incapable of attenuating the induced hypertension. Those cases resulted from nonlinear model responses to drugs in the present study; the hemodynamic responses, therefore, would have saturated because of the range of nonlinear model responses, regardless of the increase of drug infusion rates.\nThe NN in the MAPCNN under the severe conditions seems to have tried to learn and adapt to the situations during real-time control. In cases of large error between actual responses and predicted responses where the NN in the MAPCNN learned the average responses before the control (e.g. the CO responses of case 2 in Fig.\u00a07A, cases 2 and 3 in Fig.\u00a07B, and cases 1 and 3 in Fig.\u00a011A), the hemodynamics during the control exhibited oscillation and the NN in the MAPCNN would have tried to learn the severely changed situation again. In addition, when the hemodynamic response showed incorrect or opposite response to the drug input compared with the learned average response by the NN before the control [e.g. cases of not reaching the setpoint and converging on a stable state regardless of the DBT infusion for CO control because of very low sensitivity to the drug (a1\u00a0=\u00a01\/5 or 1\/10, Fig.\u00a08A) and of the opposite effect of SNP on MAP, speciously, because of the very large disturbances (levels 2 and 3 in Fig.\u00a011A) compared with the learning response], the drug infusion rate of the MAPCNN was decreased despite the remainder of the low CO or hypertension during the closed-loop control with real-time NN learning. Therefore, these simulation results suggest the following two points. First, in those cases such as the high interaction over the drug effect on the target physiological parameter and the incorrect or opposite responses to drugs compared with those of previously learned NN, improvement of the strategy in drug treatment would be required; alternatively, the MAPCNN would fall into a situation of control impossibility. Second, the physiological variations related with responses to anesthesia, antiarrhythmic drug, and muscle relaxant, the external disturbances, and the artificial background noise must be set to the smallest possible values to bring out the best performance of the controller.\nSeveral limitations are apparent in present study. First, the modeling for the CO and MAP responses to drugs might depend on the protocols of animal experiments such as the order and washout period of drug infusions. The protocol in the present animal study was, therefore, described in detail (reference \u2018Modeling of Pharmacological Response\u2019 in the \u2018MATERIALS AND METHODS\u2019 section). Second, one (low CO and normal MAP) of the heart-failure conditions was tested using the present animal study. The kinds of heart failures are various in actual patients. Therefore, further animal studies will be required. Finally, an electromagnetic flow probe was used for CO measurement in the present study. However, in an actual clinical setting, the common technique for CO measurement (e.g., the thermodilution technique using a pulmonary artery catheter19) has a slower response than that of an electromagnetic probe. The high accuracy of a flow probe, such as the time resolution (at least 30\u00a0s as used for this experiment), and the pure time delay from an actual response (as few response delays as possible) would be required to acquire the results that were obtained in the present study.\nCONCLUSIONS\nThe MAPCNN was designed and evaluated in simulation and animal studies to regulate the nonlinear responses of CO and MAP in acute heart failure using DBT and SNP under unexpected changes of patient sensitivities to drugs, drug interactions, acute disturbances, and time-variant responses to therapeutic agents. The MAPCNN showed robust control performance irrespective of various unexpected responses to drugs over actual physiological responses (Fig.\u00a02) and actual response of a dog in heart failure. Flexibility of a NN coupled with an adaptive control mechanism will enable the regulation of various physiological responses to drugs with heart failures.","keyphrases":["therapeutic agents","cardiac output","mean arterial pressure","an automated drug infusion system"],"prmu":["P","P","P","R"]}
{"id":"Ann_Surg_Oncol-4-1-2277442","title":"The Microanatomic Location of Metastatic Breast Cancer in Sentinel Lymph Nodes Predicts Nonsentinel Lymph Node Involvement\n","text":"Background The majority of sentinel node (SN) positive breast cancer patients do not have additional non-SN involvement and may not benefit from axillary lymph node dissection (ALND). Previous studies in melanoma have suggested that microanatomic localization of SN metastases may predict non-SN involvement. The present study was designed to assess whether these criteria might also be used to be more restrictive in selecting breast cancer patients who would benefit from an ALND.\nAxillary nodal status is among the most important prognostic factors in breast cancer patients. SN biopsy with an intensive pathological assessment of selectively removed lymph nodes is currently a highly accurate, minimally invasive technique to assess nodal status.1,2 It reduces the morbidity of breast cancer surgery by avoiding unnecessary axillary lymph node dissection (ALND) in patients with negative sentinel nodes (SNs).3 The optimal treatment of patients with a positive SN is however less clear. The few SNs can be cost-effectively analyzed by multiple-level evaluation and immunohistochemistry (IHC) which increases the likelihood of detecting small metastases.4 The decision to proceed with an ALND in patients with macrometastatic SN involvement does not pose a major clinical dilemma. The need for routine ALND in patients with minimal SN involvement however continues to be debated since only a minority of these patients (10\u201315%) show non-SN involvement.5 Predicting the chance of involvement of the non-SN would facilitate the selection of patients with a potential therapeutic benefit of ALND.\nSeveral features of the primary tumor and the involved SNs have been investigated as potential predictors for non-SN involvement. Primary tumor size, palpability, presence of peritumoral lymphovascular invasion, number of tumor-involved SNs, size of the SN metastases, and extracapsular extension (ECE) correlate with non-SN status.6\u201310 However, none of these factors are sensitive and reproducible enough to reliably identify a subgroup of patients who might be spared ALND.\nSeveral melanoma studies have reported that the microanatomic pattern of SN involvement and the penetrative depth (defined as the maximum distance of cancer cells from the inner margin of the SN capsule) predict non-SN involvement.11\u201314 In breast cancer, this has been studied less extensively.15We therefore set out to study the predictive value of microanatomic location and penetrative depth of SN metastatic deposits for non-SN metastases, accurately assessed by morphometry, in a large series of SN-positive breast cancer patients.\nPatients And Methods\nPatients\nA retrospective database was analyzed, including patients with invasive breast cancer and a tumor positive axillary SN followed by ALND, treated at the University Medical Center Utrecht or the St Antonius Hospital in Nieuwegein from January 2000 to May 2007 (n\u00a0=\u00a0357), including patients from our previous study.16 Exclusion criteria were multicentric tumors, neoadjuvant chemotherapy, and a total of fewer than six lymph nodes examined.\nSN Biopsy Technique\nThe technical aspects used for the SN procedure are described in detail elsewhere.16 Briefly, before surgery, SN identification was performed by peritumoral injection of 120 MBq 99mTc-Nanocolloid (Amersham Cygne, Eindhoven, The Netherlands) in a maximal volume of 0.5\u00a0mL. Dynamic and static scintigraphic images were subsequently obtained. On the same day, immediately preoperatively 0.5\u00a0mL Patent blue dye (Guerbet, Aulnay-sous-Bois, France) was injected intradermally and intra\/peritumorally. The SN was identified after careful dissection of blue lymphatic channels and detection of radioactivity with a handheld gamma ray detection probe. Palpation of the open axilla was performed to detect enlarged non-SN metastases.\nHistopathological Evaluation\nThe SNs were processed according to the protocols described previously.16\u201318 SNs were lamellated according to their size, fixed in neutral buffered formaldehyde, and completely embedded. Step sections 5 \u03bcm thick were cut at five levels with 250\u00a0\u03bcm intervals for staining with haematoxylin and eosin (H&E). In the absence of apparent metastases by H&E examination, immunohistochemistry was performed with CAM 5.2 (Beckton Dickinson, Franklin Lakes, New Jersey, USA) or CK AE1\/3 (Dako, Glostrup, Denmark) at each level. All non-SNs were identified visually or by palpation, dissected, processed routinely, and examined at one level with H&E staining. All SNs and non-SNs were examined initially by multiple pathologists at the two institutions, reviewed histologically, and reclassified according to the current 6th edition of the American Joint Committee on Cancer (AJCC) staging system by one observer (CHMvD). All cases were evaluated without knowledge of non-SN involvement.\nClinicopathological Features\nClinicopathological features recorded included age, pT (TNM system of the AJCC), histological subtype (according to the WHO), histological grade (defined according to the Nottingham modified Bloom\u2013Richardson score based on the percentage of tubule formation, nuclear pleomorphism and mitotic activity), mitotic activity index (MAI),19 steroid receptor and HER-2\/neu status (not routinely determined before 2005).\nSN and Non-SN Characteristics\nSN characteristics included metastatic size according to the 6th edition of the AJCC staging system [isolated tumor cells (ITC) (\u22640.2\u00a0mm), micrometastases (>0.2\u00a0mm and \u22642\u00a0mm), macrometastases (>2\u00a0mm)], the number of SNs, ECE, maximal diameter of the largest metastases, microanatomic location of the metastatic deposit, and the penetrative depth. If multiple but distinct deposits were identified in the same SN, the largest metastasis was recorded. If single tumor cells, cluster of nests were continuous, or separated by a few cells distance, they were measured as one focus.20 If more than one SN was involved in an individual patient, the most extensive and\/or deepest metastatic deposit was recorded. In case both axillary and internal mammary SNs were involved, the features were measured in the axillary SN.\nThe microanatomic location of metastatic deposits within each SN was classified as subcapsular, combined subcapsular and parenchymal, parenchymal or extensive. Extensive SN involvement, as defined in the study of Ruiter et\u00a0al.,21 was a deposit >5\u00a0mm in diameter (Fig.\u00a01). The centripetal depth was, according to Starz et\u00a0al.,11 defined as the maximal depth at which tumor cells have infiltrated the SN, as measured from the inner margin of the capsule (Fig.\u00a02), further denoted tumor penetrative depth according to the proposal of Scolyer et\u00a0al.12 Multifocality was defined as two or more separated metastatic deposits at some distance from each other. All measurements were calculated microscopically in the plane of the tissue sections using interactive video morphometry systems (Q-PRODIT, Leica, Cambridge, UK or Research Video Assistant, Baarn, The Netherlands).\nFIG.\u00a01.Pattern of distribution of metastatic breast cancer deposits in SNs.FIG.\u00a02.The tumor penetrative depth of metastases was defined as the maximal distance of breast cancer cells from the inner margin of the SN capsule (arrow) (H&E, original magnification\u00a0\u00d7\u00a010).\nNon-SN characteristics included the total number of non-SNs, maximal tumor diameter, AJCC classification, and ECE. If more than one non-SN was involved, the largest diameter was recorded.\nStatistical Analysis\nStatistical analysis was performed using SPSS 13.0 for Windows. Patients were divided into groups with and without non-SN involvement. The Pearson chi-square test was used to determine the relationship between categorical variables (histological type and grade, steroid receptor and HER-2\/neu status, number of SNs, AJCC classification, ECE) on the one hand and non-SN status on the other. Continuous data (age, diameter primary tumor, MAI, SN tumor diameter) were analyzed using the Mann\u2013Whitney U-test. P-values\u00a0<0.05 were considered significant. All relevant variables that were associated with the presence of positive non-SNs were included in a multivariate logistic regression model. SN metastatic characteristics (diameter and penetrative depth) were further compared by receiver operating characteristic (ROC) analysis, calculating the area under curve (AUC) as a measure of discriminative value.\nResults\nThe median age was 53\u00a0years (range 22\u201386\u00a0years) and the mean histological invasive tumor size was 2.4\u00a0cm (range 0.3\u20139.0\u00a0cm). Overall, 571 SNs were obtained (mean 1.6 SN per patient) of the 357 patients with a positive SN as well as 4939 non-SNs (mean 14 per patient). The metastatic deposits were subcapsular in 167 patients (47%), combined subcapsular and parenchymal in 40 patients (11%), parenchymal in 11 patients (3%), and extensive in 139 patients (39%) (Fig.\u00a01). Of the 357 patients, 24 (7%) patients had ITC only in the SN, whereas 112 (31%) had micrometastases and 221 (62%) had macrometastases. Localisation of ITCs was subcapsular (96%) or parenchymal (4%). SN micrometastases were located subcapsularly (81%), parenchymally (5%) or had combined localisation (13%). The overall prevalence of non-SN involvement was 38% (136\/357 patients). Other descriptive characteristics of the study population are listed in Tables\u00a01 and\u00a02.\nTABLE\u00a01.Clinicopathological characteristics of 357 invasive breast cancer patients with a positive SN and subsequent axillary lymph node dissectionFeatureNo.%Mean age (range)54 y (22\u201386)Primary tumorMean tumor size (range)2.4 cm (0.3\u20139.0)\u00a0\u00a0pT118752\u00a0\u00a0pT215042\u00a0\u00a0pT3195\u00a0\u00a0pTx10Histological subtype\u00a0\u00a0Invasive ductal cancer29884 \u00a0\u00a0Invasive lobular cancer3610\u00a0\u00a0Others236Histological grade (B&R)\u00a0\u00a017421\u00a0\u00a0216145\u00a0\u00a0312234MAI, mean\/2\u00a0mm2 (range)13 (0\u2013102)Steroid receptor status\u00a0\u00a0ER \u2013 positive \u202132290\u00a0\u00a0ER \u2013 negative3410\u00a0\u00a0ER \u2013 unknown 1\u00a0\u00a0PR \u2013 positive \u202128480\u00a0\u00a0PR \u2013 negative7020\u00a0\u00a0PR \u2013 unknown3HER-2\/neu status \u00a0\u00a0Positive2615\u00a0\u00a0Negative15085\u00a0\u00a0Unknown181\u2021 \u226510% immunoreactive neoplastic cells.TABLE\u00a02.SN and non-SN characteristics of 357 invasive breast cancer patients with a positive SN and subsequent axillary lymph node dissectionFeature No. %SNTotal number of SNs571Mean number of SNs 1.6Total number of positive SNs419Mean diameter SN metastases 4.7\u00a0mmAJCC classification of SN metastatic size \u00a0\u00a0ITC247\u00a0\u00a0Micrometastasis11231\u00a0\u00a0Macrometastasis22162Extracapsular extension \u00a0\u00a0No25772\u00a0\u00a0Yes10028Microanatomic location\u00a0\u00a0Subcapsular16747\u00a0\u00a0Parenchymal 113\u00a0\u00a0Combined4011\u00a0\u00a0Extensive13939Mean penetrative depth2.9\u00a0mmNon-SNsTotal number of non-SNs4939Mean number of non-SNs (range)14 (5\u201338)Total number of positive non-SNs474Total number of negative non-SNs4465Size of non-SN metastases\u00a0\u00a0ITC32\u00a0\u00a0Micrometastases3324\u00a0\u00a0Macrometastases10074\nThe following factors were significant predictors of non-SN metastases by univariate analysis: primary tumor size, number of involved SNs, ECE, AJCC classification, diameter, and penetrative depth of the SN metastatic deposit (Tables\u00a03 and\u00a04). None of the other classic variables (age, histological subtype and grade, steroid receptor and HER-2\/neu status, MAI) of the primary tumor correlated significantly with non-SN involvement.\nTABLE\u00a03.Comparison of categorical clinicopathological and SN characteristics in invasive breast cancer patients without and with non-SN metastases by Pearson chi-square testWithout non-SN metastasesWith non-SN metastasesP valueFeatureNo (%)No (%)Total 221 (62)136 (38)Histological subtype0.914\u00a0\u00a0Ductal183 (51)115 (32)\u00a0\u00a0Lobular22 (6)14 (4)\u00a0\u00a0Others16 (5)7 (2)Histological grade (B&R)0.798ER positive198 (56) 124 (35)0.715\u00a0\u00a0Negative22 (6)12 (3)PR positive179 (50)106 (30)0.589\u00a0\u00a0Negative41 (12)29 (8)HER-2\/neu positive16 (9)10 (6)0.670\u00a0\u00a0Negative83 (47)67 (38)No. of SN0.246\u00a0\u00a01131 (37)89 (25)\u00a0\u00a0>190 (25)47 (13)No. of involved SN 0.050\u00a0\u00a01198 (55)112 (31)\u00a0\u00a0>123 (6)24 (7)AJCC SN metastases<0.001\u00a0\u00a0ITC21 (6)3 (1)\u00a0\u00a0Micrometastasis86 (24)26 (7)\u00a0\u00a0Macrometastasis114 (32)107 (30)Extracapsular extension<0.001\u00a0\u00a0No175 (49)82 (23)\u00a0\u00a0Yes46 (13)54 (15)Microanatomic location SN metastases<0.001\u00a0\u00a0Subcapsular126 (35)41 (11)\u00a0\u00a0Combined23 (6)17 (5)\u00a0\u00a0Parenchymal8 (2)3 (1)\u00a0\u00a0Extensive64 (18)75 (21)Multifocality0.087 \u00a0\u00a0No176 (49)118 (33)\u00a0\u00a0Yes45 (13)18 (5)TABLE\u00a04.Comparison of continuous clinicopathological and SN characteristics in invasive breast cancer patients without and with non-SN metastases by Mann\u2013Whitney U-testWithout non-SN metastasesWith non-SN metastasesP valueFeatureTotal no. (%)221 (62)136 (38)Age (years, mean)54530.249Primary tumor diameter, cm (mean)2.32.50.019MAI, 2mm2 (mean)13120.693SN tumor diameter, mm (mean) 3.66.5<0.001SN tumor penetrative depth, mm (mean) 2.23.9<0.001\nThe primary tumor features histological grade and diameter were associated with multiple SN tumor deposits (P\u00a0=\u00a00.03 and 0.05, respectively).\nFrequency of non-SN metastases in patients with SN ITC (N\u00a0=\u00a024), micro- (N\u00a0=\u00a0112) and macrometastases (N\u00a0=\u00a0221) was 12.5%, 23%, and 48%, respectively (P\u00a0<\u00a00.001, Fig.\u00a03). Of those three patients with SN ITC and involved non-SNs, two had a non-SN micro- and one had a non-SN macrometastasis. One of these ITC was located in the parenchyma, the other two patients had a subcapsular location. Two of these three patients with SN ITC and non-SN involvement showed multiple small cell clusters and single cells in the SN.\nFIG.\u00a03.Flow chart showing distribution of SN AJCC classification according to non-SN involvement.\nThe microanatomic localization of SN metastatic deposits correlated with non-SN involvement. Patients with subcapsular (N\u00a0=\u00a0167), combined subcapsular and parenchymal (N\u00a0=\u00a040), parenchymal (N\u00a0=\u00a011) and extensive (N\u00a0=\u00a0139) tumor deposits showed non-SN involvement in 25%, 42%, 27%, and 54% of cases, respectively (Table\u00a03). Morphometrically assessed penetrative depth of SN metastases was also associated with non-SN involvement. Frequency of non-SN metastases in patients with a SN tumor penetrative depth <0.155\u00a0mm (N\u00a0=\u00a029), 0.155\u20132.7\u00a0mm (N\u00a0=\u00a0181) and >2.7\u00a0mm (N\u00a0=\u00a0147) was 10%, 28%, and 56%, respectively (P\u00a0<\u00a00.001, Table\u00a05). In ROC analysis, diameter (AUC\u00a0=\u00a00.686) and the penetrative depth (AUC\u00a0=\u00a00.680) of the SN tumor deposit had comparable discriminative value (Fig.\u00a04).\nTABLE\u00a05.Predictive value of penetrative depth of SN metastases for non-SN involvement in patients with invasive breast cancerSN penetrative depth (mm)NNo. of patients with non-SN involvement (%)<0.155293 (10)0.155\u20132.718151 (28)>2.714782 (56)FIG.\u00a04.ROC curves showing the sensitivity and specificity of SN tumor diameter and penetrative depth as predictors of non-SN involvement. The larger the area below the curve, the more accurate the prediction of non-SN involvement (P\u00a0<\u00a00.001).\nIn multivariate analysis, SN tumor diameter (P\u00a0=\u00a00.032) and SN tumor penetrative depth (<0.155\u00a0mm versus 0.155\u20132.7\u00a0mm versus >2.7\u00a0mm) (P\u00a0=\u00a00.015) were significant in predicting non-SN involvement. Cutoff values were interactively statistically as those best discriminating between low and high risk of non-SN metastases. Risk stratification by combining these features identified a low-risk group, an intermediate-risk group, and a high risk group for non-SN metastases with frequency of non-SN metastases in 11%, 29%, and 56% of patients, respectively (Table\u00a06). Frequencies of non-SN involvement in patients with SN micrometastases and a subcapsular (N\u00a0=\u00a091), combined subcapsular and parenchymal (N\u00a0=\u00a015), and parenchymal location (N\u00a0=\u00a06) were 21%, 40%, and 17%, respectively. Frequencies of non-SN involvement in patients with SN macrometastases and a subcapsular (N\u00a0=\u00a054), combined subcapsular and parenchymal (N\u00a0=\u00a025), parenchymal (N\u00a0=\u00a04) and extensive location (N\u00a0=\u00a0138) were 39%, 44%, 25%, and 54%, respectively.\nTABLE\u00a06.Predictive value of a combination of AJCC classification of SN metastases and penetrative depth of SN metastases for non-SN involvement in patients with invasive breast cancerNNo. of patients with non-SN involvement (%)ITC\/SN penetrative depth < 2.7\u00a0mm or Micrometastases\/penetrative depth\u00a0<\u00a00.155\u00a0mm384 (11)Micro- or macrometastases\/penetrative depth 0.155\u20132.7\u00a0mm17250 (29)Micro- or macrometastases\/penetrative depth >2.7\u00a0mm14782 (56)\nDiscussion\nThe lymphatic spread of breast cancer cells has been shown to follow an orderly progression via the SN to non-SNs, which implies that the risk of spread of tumor from the SN to the non-SN may depend on the extent of SN involvement. While many studies have focused on the maximal diameter of the metastatic tumor deposit22, 23 we investigated the microanatomic location and the penetrative depth of the metastatic deposits as a putative predictor of non-SN involvement. This was based on the concept that within the SN, tumor cells also follow an orderly route, arriving in the subcapsular sinuses through an afferent lymph vessel. Later, there is subcapsular outgrowth of malignant cells in the marginal sinuses and into the cortical parenchyma. Finally, these cells extend to the deeper zones of the lymph node parenchyma, frequently following the medullary sinuses to efferent lymph vessels.24 Consistent with this concept we found that the microanatomic location, the size, and the penetrative depth of SN tumor deposits were correlated significantly with non-SN involvement. This finding is consistent with recent melanoma studies. Startz et\u00a0al.11 proposed a micromorphometric classification, based on the depth of the metastasis from the capsule and the number of 1-mm slices containing melanoma. This classification was a highly significant predictor for distant metastases and overall survival. Dewar et\u00a0al.13 also recorded that the microanatomic location of melanoma SN metastases predicts non-SN involvement, and proposed that it would be possible to safely avoid a lymph node dissection in patients with subcapsular deposits only. Indeed, subcapsular location and small tumor penetrative depth correlated with less non-SN involvement in our breast cancer study, although no subgroup of patients could be selected without non-SN involvement.\nThe measurement of the SN tumor penetrative depth was difficult in many cases, especially in case of extensive tumor deposits. In these cases the tumor deposits frequently extended beyond the center of the lymph node, making it difficult to determine which edge of the SN capsule should be used to measure the tumor penetrative depth. Similar difficulties were encountered when the SN had a lobulated outline. Further studies which more rigorously define the tumor penetrative depth may strengthen its predictive power and reproducibility.\nIn conclusion, patients with breast carcinoma and SN involvement can be stratified into subgroups at significantly different risk for non-SN involvement, according to microanatomic localization of the SN metastatic deposits and penetrative depth into the SN. However, based on these features no subgroup of patients could be selected without non-SN involvement.","keyphrases":["breast cancer","sentinel node","morphometry","axillary lymph node metastases"],"prmu":["P","P","P","R"]}
{"id":"Int_J_Colorectal_Dis-4-1-2225996","title":"NF-\u03baB-dependent synergistic regulation of CXCL10 gene expression by IL-1\u03b2 and IFN-\u03b3 in human intestinal epithelial cell lines\n","text":"Background and aims Little is known about the intestinal epithelial expression and secretion of CXCL10 (IP-10), a chemokine involved in recruiting T cells and monocytes. We aimed to study CXCL10 gene expression and regulation by the pro-inflammatory cytokines interleukin (IL)-1\u03b2, interferon (IFN)-\u03b3 and tumour necrosis factor (TNF)-\u03b1 in intestinal epithelial cell lines.\nIntroduction\nIntestinal epithelial cells (IECs) form the first immunologic barrier of the organism while the villus surface is continuously in contact with various agents. IEC gene expression is tightly regulated to induce appropriate responses to pathogens and to avoid deleterious reactions to microbial flora. When stimulated with pro-inflammatory cytokines or infected with microbial pathogens, IECs may secrete chemokines, a set of low-molecular weight cytokines, directing migration and activation of leucocytes, which play a major role in the perpetuation of inflammatory processes [1\u20135]. The final composition of leucocytes present in the inflamed intestine is most likely due to both secreted chemokines and the relative expression of chemokine cell surface receptors on different cell types. Chemokine production by colonic epithelial cells is thought to contribute to the characteristic increased infiltration of selected populations of leucocyte cells in inflammatory bowel disease (IBD) [6, 7].\nCXCL10 is a CXC chemokine, known to be secreted by endothelial cells [8], keratinocytes [9], eosinophils [10] and neutrophils [11]. It is also constitutively expressed by normal human colon epithelium [12] and chemoattracts activated T cells as well as monocytes by binding to the CXCR3 receptors present on the respective cells [13, 14]. While CXCL10 up-regulation may occur in acute inflammation in response to pathogens, it is also known to be permanently over-expressed in IBD patients [15, 16]. Blocking CXCL10 could prevent mice from acute colitis, suggesting this chemokine to play an important role in IBD pathogenesis [17]. Moreover, CXCL10 is believed to possess anti-tumoural properties by promoting anti-tumour T cell immunity [18, 19]. Knowledge of its regulation in colonic cell lines may therefore give also insight into the pathobiology of colorectal cancer.\nMost previous studies on chemokine expression in IEC concentrated on CXCL8 (interleukin [IL]-8), a neutrophil chemoattractant [20, 21], but there were only few studies regarding the expression and secretion of CXCL10 in IECs [12]. Although it is well known that interferon (IFN)-\u03b3 is a major inducer of CXCL10 and that CXCL10 expression may be enhanced by pathogens and other inflammatory cytokines, the relative contribution of cytokines on CXCL10 induction, their synergistic action and the kinetics of cytokine-induced CXCL10 expression in intestinal epithelial cells has not been fully elucidated. Especially, the role of IL-1\u03b2 in CXCL10 gene regulation in IECs and also in other cell types has not been fully evaluated so far. Intestinal mucosa is composed of cell populations in continuous change from a proliferative and undifferentiated stage in the basal parts of crypts to mature surface villus epithelial cells [22, 23]. Little is known about the alteration of immunological functions as IECs mature. It has been shown that cellular differentiation affects IEC responsiveness to IL-1\u03b2 [24].\nIn the present study, we aimed to elucidate the role of the pro-inflammatory cytokines IL-1\u03b2, tumour necrosis factor (TNF)-\u03b1 and IFN-\u03b3, which have established role in IBD pathogenesis, in inducing CXCL10 gene expression in IEC lines. Especially, the role of IL-1\u03b2, one of the most abundantly expressed cytokines, both in normal and inflamed mucosa [25], in inducing CXCL10 expression by IECs and its possible signalling mechanisms was given importance. To consider possible influences of cellular differentiation on cytokine responsiveness, we performed studies on three different colonic epithelial cell lines. Our findings will get an insight into the role of intestinal epithelium in immune responses and in evaluating pathways, which might be targets of present and future pharmacotherapy of IBD and possibly colorectal cancer.\nMaterials and methods\nBiological reagents\nThe recombinant cytokines IL-1\u03b2, TNF-\u03b1 and IFN-\u03b3 were purchased from Roche applied science (Mannheim, Germany). An enzyme-linked immunosorbent assay (ELISA) kit for CXCL10 was purchased from RnD systems (Weisbaden, Germany). The nuclear factor (NF)-\u03baB inhibitor BAY11-7082 was purchased from Calbiochem (San Diego, CA).\nCell culture and stimulation protocols\nThe human colon adenocarcinoma cell lines Caco-2, HT-29 and DLD1 were obtained from DSMZ (Braunschweig, Germany). Caco-2 cells were grown in Eagle\u2019s minimal essential medium (BioWhittacker, Germany) containing 20% foetal calf serum (FCS) supplemented with 100\u00a0U\/ml each of penicillin and streptomycin and 1% non-essential amino acids at 37\u00b0C and 5% CO2. HT29 and DLD1 were grown in Roswell Park Memorial Institute medium containing 10% FCS and 100\u00a0U\/ml penicillin and streptomycin at 37\u00b0C and 5% CO2. Caco-2, HT29 and DLD-1 cells were plated into six-well plates at a density of 5\u2009\u00d7\u2009105 cells per well for real-time polymerase chain reactions (PCRs), ELISA, Northern blotting and electrophoresis mobility shift assay (EMSA) experiments unless until stated and grown till they reached 70\u201380% confluence. These cells were then stimulated with IL-1\u03b2 (0.1, 0.5, 1, 2, 10 and 50\u00a0ng\/ml), TNF-\u03b1 (2, 10 and 50\u00a0ng\/ml) and IFN-\u03b3 (2, 10 and 50\u00a0ng\/ml) based on the type of experiments.\nRNA isolation, real-time PCR\nRibonucleic acid (RNA) was isolated by using Qiagen RNeasy mini kit according to the manufacturer\u2019s protocol. The RNA concentrations were determined photometrically using a Gene Quant RNA\/deoxyribonucleic acid (DNA) calculator (Pharmacia, Freiburg, Germany). RNA was subsequently used for real-time PCR and Northern blot analysis. Reverse transcription of messenger RNA (mRNA) was performed using 1\u00a0\u03bcg of total cellular RNA. To determine the mRNA expression of CXCL10, real-time PCR was carried out using gene-specific primers for human CXCL10 (Invitrogen GmbH, Karlsruhe) forward 5\u2032-CCA GAA TCG AAG GCC ATC AA-3\u2032, reverse 5\u2032-CAT TTC CTT GCT AAC TGC TTT CAG-3\u2032 and \u03b2-actin (Invitrogen GmbH) forward 5\u2032-CTG GCA CCC AGC ACA ATG-3\u2032, reverse 5\u2032-CCG ATC CAC ACG GAG TAC TTG-3\u2032 in an ABI Prism 7000 sequence detection system. PCR reaction was set up with Sybr\u00ae Green PCR Master mix (Invitrogen) containing 0.3\u00a0\u03bcmol\/l primers each and 1\u00a0\u03bcl of RT-product in 25\u00a0\u03bcl volume. A two-step amplification protocol was chosen consisting of initial denaturation at 95\u00b0C for 10\u00a0min followed by 45 cycles with 15\u00a0s denaturation at 95\u00b0C and 30\u00a0s annealing\/extension at 60\u00b0C. Finally, a dissociation protocol was performed to control specificity of amplification products.\nRelative expression of CXCL10 was then calculated using the comparative threshold-cycle (CT) method. The amount of target mRNA in each sample was normalized to the amount of \u03b2-actin mRNA designated as calibrator, to give \u0394CT (CT \u03b2-actin\u2009\u2212\u2009CT CXCL10). The relative expression of CXCL10 was calculated as the . MRNA expression is presented as fold increase calculated in relation to unstimulated cells after normalization to \u03b2-actin.\nTransient transfections\nCaco-2, HT29 and DLD1 cells were plated in 24-well plates, 24\u00a0h before transfection (Costar, Corning, NY), at a density of 50,000\/well in 1\u00a0ml medium. After 24\u00a0h, the cells were transfected with 250\u00a0ng of respective plasmids along with 20\u00a0ng of renilla plasmid using the non-liposomal formulation FuGENE (Roche Molecular Biochemicals, Mannheim, Germany). On the day of transfection, fresh medium was added. FuGENE was added to the plasmid DNA at a ratio of 3\u00a0\u03bcl\/\u03bcg DNA. FuGENE was prediluted in 100\u00a0\u03bcl serum-free medium and added drop-wise to the concentrated plasmid DNA. After 15\u201325\u00a0min at room temperature, the mixture was added to the cells. The plasmids TGL-IP10, TGL-IP10 \u03baB2 Mut were kind gifts from Dr. Richard M. Ranshoff (Cleveland, OH) and were described elsewhere [26].\nLuciferase assays\nTwenty-four hours after transfection, cells were stimulated with IL-1\u03b2 (0.1,1, 2, 10 and 50\u00a0ng\/ml), TNF-\u03b1 (2, 10 and 50\u00a0ng\/ml), IFN-\u03b3 (2, 10 and 50\u00a0ng\/ml), IL-1\u03b2 (1\u00a0ng\/ml)\u2009+\u2009TNF\u03b1 (50\u00a0ng\/ml), IL-1\u03b2 (1\u00a0ng\/ml)\u00a0+\u00a0IFN-\u03b3 (50\u00a0ng\/ml) and TNF\u03b1 (50\u00a0ng\/ml)\u2009+\u2009IFN-\u03b3 (50\u00a0ng\/ml) for 24\u00a0h, harvested and lysed, and luciferase was assayed by using a luciferase kit (Promega). Values were normalised to renilla luciferase.\nEnzyme-linked immunosorbent assay\nTo study the synergistic effects of cytokines on CXCL10 secretion, supernatants were collected at 0, 3, 6, 12 and 24\u00a0h in Caco-2 and HT29 cells stimulated with IL-1\u03b2 (1\u00a0ng\/ml), TNF-\u03b1 (50\u00a0ng\/ml), IFN-\u03b3 (50\u00a0ng\/ml), IL-1\u03b2 (0.5\u00a0ng\/ml)\u2009+\u2009TNF\u03b1 (50\u00a0ng\/ml), IL-1\u03b2 (0.5\u00a0ng\/ml)\u2009+\u2009IFN-\u03b3 (50\u00a0ng\/ml) and TNF\u03b1 (50\u00a0ng\/ml)\u2009+\u2009IFN-\u03b3 (50\u00a0ng\/ml) and stored at \u221270\u00b0C until measured. CXCL10 protein secretion was measured by ELISA according to the manufacturer\u2019s protocol.\nPreparation of nuclear extract\nCaco-2 cells were grown for 24\u00a0h, pre-treated for 1\u00a0h with BAY 11-7082 (10\u00a0\u03bcM) and then stimulated with respective cytokines for 30\u00a0min. Cells were washed with 10\u00a0ml cold phosphate-buffered saline (PBS) twice, harvested, centrifuged at 1,000\u2009\u00d7\u2009g for 5\u00a0min and resuspended in 1\u00a0ml of cold PBS. Centrifuged briefly, excess PBS was removed, and the pellet was resuspended in ice-cold extraction buffer (20\u00a0mM hydroxyethyl piperazineethanesulfonic acid [HEPES] pH\u00a07.8, 10\u00a0mM KCL, 0.1\u00a0mM ethylenediamine tetraacetic acid [EDTA], 1\u00a0mM dithiothreitol and 0.5\u00a0mM phenylmethanesulphonylfluoride [PMSF]) by gentle pippeting. Cells were then kept on ice for 15\u00a0min, and 30\u00a0\u03bcl of 10% Nonidet NP-40 was added and vortexed briefly for 10\u00a0s. The lysed cell suspension was centrifuged for 30\u00a0s, and the supernatant was discarded. The pellet was resuspended in 50\u00a0\u03bcl extraction buffer containing 400\u00a0mM NaCl, agitated vigorously for 15\u00a0min at 4\u00b0C. Then, the cell suspension was centrifuged for 5\u00a0min at 4\u00b0C, and the supernatant containing the nuclear extract was collected and stored at \u221280\u00b0C until further use.\nElectrophoretic mobility shift assay and super-shift assay\nFor binding reactions, 5\u00a0\u03bcg of nuclear extracts were incubated in 20\u00a0\u03bcl reaction mixture containing 40\u00a0mM HEPES pH\u00a07.5, 50\u00a0mM NaCl, 1\u00a0mM EDTA, 1mM DithitreitolHSCl, 1\u00a0mM PMSF, 1.28\u00a0\u03bcg\/ml pBluescript vector DNA as half of the reaction and the rest being nuclear extract plus other components. Oligonucleotide duplex probes (Promega) were end-labelled with T4 polynucleotide kinase and [\u03b332P] adenosine triphosphate; 1\u2009\u00d7\u2009104\u00a0cpm were then added to the reaction mixtures and incubated overnight at 4\u00b0C. Reaction products were analysed by non-denaturing electrophoresis in a 5% polycralamide gel with 0.5\u00d7 Tris\u2013borate\u2013EDTA buffer at room temperature. Gels were then exposed directly to X-ray film at \u221270\u00b0C for autoradiography. For competition, unlabelled oligonucleotides were added in molar excess at room temperature for 15\u00a0min before adding radiolabelled probe. In super shift experiments, nuclear extracts were incubated with anti-p65 anti-body (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4\u00b0C.\nStatistical analysis\nStatistical analyses were carried out using the Prism software packet (Version 3.0, Graphpad Software, San Diego, CA). Comparisons between two or more different treatment groups were made with the non-paired t test or analysis of variance (ANOVA) where appropriate. For time kinetics experiments, a two-way ANOVA test followed by the Bonferroni post-test was performed. In case of RNA expression, a log transformation was performed before. Statistical differences were regarded significant at a p level below 0.05. Data were expressed as means\u2009\u00b1\u2009standard error of the mean (SEM).\nResults\nDifferential expression of CXCL10 mRNA in intestinal epithelial cell lines under basal conditions\nIn the present study, the basal mRNA expression of CXCL10 in three different IECs, Caco-2, HT29 and DLD1 cells, were studied first. Real-time PCR experiments using gene-specific primers for CXCL10 revealed that the basal mRNA expression of CXCL10 was highest in Caco-2 followed by HT29 and DLD1 (Fig.\u00a01).\nFig.\u00a01Basal level expression of CXCL10 in IECs. Caco-2, HT29 and DLD1 cells were plated at a density of 5\u2009\u00d7\u2009105 cells in six well plates, grown for 24\u00a0h and total RNA from the cells was isolated. First-strand complementary DNA was prepared from 1\u00a0\u03bcg of total RNA. Real-time PCR was performed for CXCL10 and \u03b2-actin was used as an internal control. The bars indicate the mean of six individual experiments. Asterisk corresponds to p\u2009<\u20090.01\nIL-1\u03b2, TNF-\u03b1 and IFN-\u03b3 dose-dependent influence on CXCL10 gene expression in IECs\nCaco-2, HT29 and DLD1 cells were tested for their ability to respond to pro-inflammatory cytokines by the induction of CXCL10 mRNA expression. Cells were stimulated for 4\u00a0h with various concentrations of cytokines, and CXCL10 expression was analysed by real-time PCR. In response to IFN-\u03b3 and TNF-\u03b1, Caco-2, HT29 and DLD1 cells showed a dose-dependent induction of CXCL10 mRNA (Fig.\u00a02). Increased expression of CXCL10 mRNA was observed when 2\u00a0ng\/ml IL-1\u03b2 was used, while a higher concentration did not further enhance expression. Further, we checked the response of IECs to lower doses of IL-1\u03b2 showing a dose response in the range of 0.1 to 1\u00a0ng\/ml in the induction of CXCL10 mRNA. IL-1\u03b2 at a concentration of 0.5\u00a0ng\/ml was able to induce maximum expression of CXCL10 mRNA at 4\u00a0h (data not shown).\nFig.\u00a02Dose-dependent expression of CXCL10 mRNA in IECs stimulated with cytokines. 5\u2009\u00d7\u2009105 cells were plated into six-well plates and grown for 24\u00a0h and then stimulated with IL-1\u03b2, TNF-\u03b1 and IFN-\u03b3 at an increasing doses of 2, 10 and 50\u00a0ng\/ml. The cells were harvested after 4\u00a0h, total RNA from the cells was isolated, and first-strand cDNA was prepared from 1\u00a0\u03bcg of total RNA. Real-time PCR was performed for CXCL10, and \u03b2-actin was used as an internal control. Graph represents the mean\u2009\u00b1\u2009SEM of four individual experiments. All cytokine-stimulated expressions were significantly different from unstimulated controls (p\u2009<\u20090.05). Asterisk, no significant difference between different IL-1\u03b2 concentrations (p\u2009<\u20090.05)\nIL-1\u03b2, TNF-\u03b1 and IFN-\u03b3 time-dependent influence on CXCL10 gene expression in IECs\nOnce we confirmed the responsiveness of IECs to pro-inflammatory cytokines, we next examined the mRNA kinetics of CXCL10 induced by the cytokines alone or in combination with each other. Each cell line had a different time course of response when stimulated with respective cytokines and their combinations (Fig.\u00a03a\u2013c). In Caco-2, stimulated with IL-1\u03b2 or TNF-\u03b1, CXCL10 mRNA was induced within the first 40\u00a0min, and the induction reached to a peak after 4\u00a0h, while IFN-\u03b3 had a later initial effect inducing CXCL10 mRNA after 2\u00a0h and reached to a maximum at 8\u00a0h. Combination of IL-1\u03b2\u2009+\u2009IFN-\u03b3 had a stronger synergistic effect in the induction of CXCL10 mRNA within 20\u00a0min and reaching to maximum after 4\u00a0h, whereas TNF-\u03b1\u2009+\u2009IFN-\u03b3 synergistically led to a later induction of CXCL10 mRNA starting at 40\u00a0min but had a strong synergistic effect after 8\u00a0h when compared to IL-1\u03b2\u2009+\u2009IFN-\u03b3 (Fig.\u00a03a). In HT29 (Fig.\u00a03b) and DLD1 (Fig.\u00a03c), CXCL10 mRNA time kinetics deviated from that in Caco-2 (Fig.\u00a03a). CXCL10 mRNA was induced after 1\u00a0h of stimulation with the respective single cytokines in DLD1. In case of HT29, either TNF-\u03b1 or IFN-\u03b3 alone led to delayed induction of CXCL10 mRNA after 1\u00a0h, whereas IL-1\u03b2 alone led to an induction starting at 2\u00a0h. HT29 and DLD1 cells treated with combinations of IL-1\u03b2\u2009+\u2009IFN-\u03b3 showed a strong synergistic induction of CXCL10 mRNA within 40\u00a0min, whereas the TNF-\u03b1\u2009+\u2009IFN-\u03b3 combination led to an even stronger induction of CXCL10 mRNA within 20\u00a0min in HT29 and within 40\u00a0min in DLD1. IL-1\u03b2\u2009+\u2009TNF\u03b1 in combination had no synergistic effect on CXCL10 mRNA induction in all the cell lines used for this study. The real-time PCR results could be confirmed by Northern blot analysis in Caco-2 and HT29 cells. However, the detection threshold was not low enough to display basal and single cytokine-induced CXCL10 mRNA (data not shown).\nFig.\u00a03Time kinetics of CXCL10 mRNA expression in IECs stimulated with cytokines. a Caco-2, b HT29 and c DLD1 cells were plated at a density of 5\u2009\u00d7\u2009105 cells in six-well plates and grown for 24\u00a0h and then stimulated with respective cytokines and their combinations. Cells were harvested at different time points as indicated, and total RNA from the cells was isolated and first-strand cDNA was prepared from 1\u00a0\u03bcg of total RNA. Real-time PCR was performed for CXCL10, and \u03b2-actin was used as an internal control. Graphs represent the mean\u2009\u00b1\u2009SEM of three to five experiments. IFN-\u03b3 led to a significant increase in CXCL10 expression from 2\u00a0h onwards. A significant increase (p\u2009<\u20090.05) from control was observed in all expect in those marked as N.S. (non-significant). Number sign indicates time points with significant differences (p\u2009<\u20090.05) of cytokine combinations or IL-1\u03b2 compared to IFN-\u03b3 alone\nIL-1\u03b2, TNF-\u03b1 and IFN-\u03b3 time-dependent influence on CXCL10 protein secretion in IECs\nTo test whether cytokine stimulation of IECs would lead not only to the induction of CXCL10 mRNA but also to the synthesis and secretion of the protein, Caco-2, HT29 and DLD1 cells were incubated with respective cytokines (from 3 to 24\u00a0h), and secretion of CXCL10 was detected by ELISA. Under basal conditions, Caco-2 cells secreted 0.012\u2009\u00b1\u20090.003\u00a0ng\/ml CXCL10. Treatment with IL-1\u03b2, TNF-\u03b1 and IFN-\u03b3 led to a time-dependent increase in CXCL10 protein, with IL-1\u03b2 being the most potent stimulus during the first 6\u00a0h (Fig.\u00a04a). Of importance, after 12\u00a0h, CXCL10 secretion was stimulated strongly by IFN-\u03b3. Co-incubation of Caco-2 with respective cytokines revealed a synergistic effect of IL-1\u03b2\u2009+\u2009IFN-\u03b3 and TNF-\u03b1\u2009+\u2009IFN-\u03b3 on CXCL10 secretion, reaching levels of 3.354\u2009\u00b1\u20090.690 and 7.808.1\u2009\u00b1\u20090.173\u00a0ng\/ml after 24\u00a0h. The combination of IL-1\u03b2\u2009+\u2009TNF-\u03b1 had no synergistic effect on CXCL10 secretion when compared to the respective cytokines alone. These time kinetic studies revealed that in Caco-2, IL-1\u03b2 alone or in combination with IFN-\u03b3 induced the secretion of CXCL10 very early compared to the combination of TNF-\u03b1\u2009+\u2009IFN-\u03b3, which induced a delayed but stronger synergistic secretion of CXCL10 protein, suggesting that in case of IL-1\u03b2\u2009+\u2009IFN-\u03b3, a synergism could take place very early, e.g., at the level of intracellular signal cascades. In HT29 cultures, CXCL10 secretion was not detectable under basal condition. TNF-\u03b1 or IFN-\u03b3 alone led to an induction of CXCL10 protein secretion within 3\u00a0h (0.0172\u2009\u00b1\u20090.005 and 0.011\u2009\u00b1\u20090.001\u00a0ng\/ml), whereas IL-1\u03b2 could not induce the secretion of CXCL10 protein. IL-1\u03b2, TNF-\u03b1 and IFN-\u03b3 led to a time-dependent increase in CXCL10 concentrations in HT29 cell supernatants reaching maximum levels of 0.039\u2009\u00b1\u20090.0057, 0.632\u2009\u00b1\u20090.0222 and 19.929\u2009\u00b1\u20090.838\u00a0ng\/ml showing that IFN-\u03b3 is the strongest inducer of CXCL10 protein secretion when compared to other two cytokines in this cell line (Fig.\u00a04b). The combinations of TNF\u03b1\u2009+\u2009IFN-\u03b3 and IL-1\u03b2\u2009+\u2009IFN-\u03b3 induced CXCL10 protein secretion to 1.822\u2009\u00b1\u20090.109 and 0.410\u2009\u00b1\u20090.027\u00a0ng\/ml within 3\u00a0h and led to a maximum of 492.728\u2009\u00b1\u200983.776 and 87.158\u2009\u00b1\u20097.913\u00a0ng\/ml after 24\u00a0h (Fig.\u00a04b).\nFig.\u00a04Time kinetics of CXCL10 protein secretion in Caco-2 and HT29 cells stimulated with cytokines. 5\u2009\u00d7\u2009105 cells were plated into six-well plates and grown for 24\u00a0h and then stimulated with IL-1\u03b2, TNF-\u03b1 and IFN-\u03b3 as indicated. Supernatants were collected at respective time points from a Caco-2 and b HT29 cells and ELISA for CXCL10 protein was performed. Data represent means of three to five experiments\u2009\u00b1\u2009SEM done in duplicates for each sample. Asterisk indicates time points with significant differences (p\u2009<\u20090.05) of cytokine combinations compared to IFN-\u03b3 alone\nCXCL10 promoter activation by cytokines\nTGL-IP10, a promoter-reporter containing 435\u00a0bp of human CXCL10 sequence upstream of the transcriptional start site, was used to evaluate the role of IL-1\u03b2, TNF-\u03b1 and IFN-\u03b3 alone or in combination with each other in the transcription of CXCL10. When TGL-IP10-transfected Caco-2 cells were stimulated with single cytokines, IL-1\u03b2 (1\u00a0ng\/ml) led to a stronger induction (6.53\u2009\u00b1\u20091.65-fold increase) than TNF-\u03b1 (50\u00a0ng\/ml) and IFN-\u03b3 (50\u00a0ng\/ml) alone (2.76\u2009\u00b1\u20090.53 and 3.14\u2009\u00b1\u20091.2-fold increase). The combination of IL-1\u03b2\u2009+\u2009IFN-\u03b3 showed a stronger synergistic induction (26.18\u2009\u00b1\u200911.68) than the combination of TNF-\u03b1\u2009+\u2009IFN-\u03b3 (13.97\u2009\u00b1\u20093.25-fold increase) (Fig.\u00a05a). In HT29 cells, when stimulated with single cytokines, IFN-\u03b3 (50\u00a0ng\/ml) led to a maximum induction (6.52\u2009\u00b1\u20090.58-fold increase), followed by TNF-\u03b1 (4.44\u2009\u00b1\u20090.16-fold increase) and IL-1\u03b2 (1.43\u2009\u00b1\u20090.05-fold increase). The combination of IL-1\u03b2\u2009+\u2009IFN-\u03b3 synergistically led to 12.76\u2009\u00b1\u20093.21-fold induction, and TNF-\u03b1\u2009+\u2009IFN-\u03b3 led to 26.70\u2009\u00b1\u20097.5-fold induction of promoter activity (Fig.\u00a05b). In DLD1 cells (Fig.\u00a05c), IFN-\u03b3 (50\u00a0ng\/ml) led to a maximum induction (8.06\u2009\u00b1\u20090.51-fold increase) of CXCL10 promoter activity followed by TNF-\u03b1 (50\u00a0ng\/ml) induction (3.59\u2009\u00b1\u20090.08-fold increase) and IL-1\u03b2 (1\u00a0ng\/ml) induction (2.32\u2009\u00b1\u20090.12-fold increase). The combination of IL-1\u03b2\u2009+\u2009IFN-\u03b3 led to a 40.72\u2009\u00b1\u200912.19-fold induction, and TNF-\u03b1\u2009+\u2009IFN-\u03b3 led to a 20.56\u2009\u00b1\u20096.27 fold induction. In all three cell lines, the combination of IL-1\u03b2\u2009+\u2009TNF-\u03b1 had no synergistic effect on CXCL10 promoter activity.\nFig.\u00a05Transient transfection analysis of TGL-IP10 in IECs. The CXCL10 promoter containing reporter construct, pTGL-IP10 and pRL-TK (renilla) plasmids, was transiently transfected into a Caco-2, b HT29 and c DLD1 cells. 24\u00a0h after transfection, cells were stimulated with cytokines as indicated for 24\u00a0h, harvested, and promoter activity was monitored by luciferase assay. Data were normalised with renilla luciferase values and included as fold increase vs control. Data represent the means\u2009\u00b1\u2009SEM of three to five independent experiments done in duplicates for each sample. Asterisk indicates an induction significantly different (p\u2009<\u20090.05) from control (C). Significant differences between combinations of cytokines and the respective single cytokines are indicated with bars with a number sign (p\u2009<\u20090.05). N.S. is not significant\nEvaluating the role of NF-\u03baB in CXCL10 gene expression\nTo evaluate the possible role of NF-\u03baB in cytokine-induced CXCL10 gene expression, experiments with a specific NF-\u03baB inhibitor were performed. The effect of BAY 11-7082 on NF-\u03baB inhibition was evaluated by reporter assays. BAY11-7082 was able to dose-dependently inhibit IL-1\u03b2-induced pNF-\u03baB-SEAP gene reporter activity in Caco-2 cells (data not shown). Based on this experiment, we could determine that 10\u00a0\u03bcM of BAY11-7082 was sufficient to inhibit the NF-\u03baB activation.\nThe induction of CXCL10 gene expression by cytokines was inhibited by addition of the NF-\u03baB-specific inhibitor BAY11-7082 in Caco-2, HT29 and DLD1 cells, with a varying degree of inhibition in the individual cell lines. Inhibition of NF-\u03baB led to almost complete inhibition (approximately 90\u201395% inhibition) of CXCL10 mRNA and protein secretion induced by cytokines in Caco-2 and DLD1 cells (Fig.\u00a06a and c). In DLD1 cells, IL-1\u03b2 (1\u00a0ng\/ml) did not have any inducing effect on CXCL10 secretion. In HT29, inhibition of NF-\u03baB had approximately 60\u201375% inhibitory effect on cytokine-induced CXCL10 mRNA expression and secretion (Fig.\u00a06b).\nFig.\u00a06Role of NF-\u03baB in cytokine-induced CXCL10 mRNA expression by IECs. 5\u2009\u00d7\u2009105 cells were plated and grown for 24\u00a0h. Then, the cells were pre-treated with 10\u00a0\u03bcM of BAY 11-7082 for 1\u00a0h and then stimulated with respective cytokines and their combinations as stated in the figure for 2 and 24\u00a0h. After 2\u00a0h, cells were harvested for RNA isolation to perform real-time PCRs, and at 24\u00a0h, cell supernatants were collected and stored at \u221270\u00b0C until ELISA was performed for CXCL10. Data presented are the means\u2009\u00b1\u2009SEM of three independent experiments done in duplicates for each sample in a Caco-2, b HT29 and c DLD1 cells. Significant CXCL10 inhibition by BAY 11-7082 is indicated by an asterisk (p\u2009<\u2009 0.05). N.S. is not significant\nTo further evaluate the role of NF-\u03baB, transient transfections were done with a CXCL10 promoter construct bearing a mutated NFkB-binding site (TGL-IP10 \u03baB2 Mut). In Caco-2 cells, TGL-IP10 \u03baB2 Mut was not responsive to any of the cytokines and their combinations, suggesting that CXCL10 gene induction is strongly dependent on NF-\u03baB in this cell line (Fig.\u00a07a). In HT29 and DLD1 cells, the combination of TNF-\u03b1\u2009+\u2009IFN-\u03b3 led to 6.49\u2009\u00b1\u20092.13 and 4.87\u2009\u00b1\u20091.43-fold increase, and the combination of IL-1\u03b2\u2009+\u2009IFN-\u03b3 led to 2.26\u2009\u00b1\u20090.63 and 4.75\u2009\u00b1\u20090.53-fold increase of the TGL-IP10 \u03baB2 Mut promoter activity (Fig.\u00a07b and c), revealing that a smaller portion of CXCL10 gene expression might be regulated independently from NF-\u03baB.\nFig.\u00a07Transient transfection analysis of TGL-IP10 and TGL-IP10 \u03baB2 mutant in IECs. TGL-IP-10 and TGL-IP10-\u03baB2 Mut plasmid was transiently transfected into a Caco-2, b HT29 and c DLD1. 24\u00a0h after transfection, cells were stimulated with cytokines for 24\u00a0h, and promoter activity was monitored by luciferase assay. Data were normalised with renilla luciferase values. Data presented are the means\u2009\u00b1\u2009SEM of three to five independent experiments done in duplicates for each sample. Significant differences vs controls (C) are indicated by an asterisk (p\u2009<\u20090.05). Significant differences of wild type vs mutant are indicated by a number sign (p\u2009<\u20090.05). N.S. is not significant\nTo confirm further our results from promoter and inhibitor studies, we did gel-shift and super shift assays to show that NF-\u03baB is activated by cytokines and that the activated NF-\u03baB complex contains the NF-\u03baB functional sub-unit p65. In the nuclear extracts of Caco-2 cells, we could clearly see a shift in the samples treated with IL-1\u03b2, TNF-\u03b1 alone or the combinations of IL-1\u03b2\u2009+\u2009IFN-\u03b3 and TNF-\u03b1\u2009+\u2009IFN-\u03b3. The protein-DNA complex was efficiently competed by unlabelled \u03baB oligonucleotides. There was no shift in the samples treated with the NF-\u03baB inhibitor BAY11-7082, stating that the shift was indeed because of NF-\u03baB binding. Furthermore, antibody super shift assay using anti-p65 antibody had clearly shown that the complex contains NF-\u03baB functional sub-unit, p65 (Fig.\u00a08).\nFig.\u00a08Gel shift and super shift analysis for NF-\u03baB. Caco-2 cells were seeded and grown for 24\u00a0h before pre-treating with BAY 11-7082 (10\u00a0\u03bcM) for 1\u00a0h and then stimulated with respective cytokines for 30\u00a0min; cells were then harvested, and nuclear extracts were prepared. 5\u00a0\u03bcg of nuclear extracts were used for gel shifts with a wild-type \u03baB oligonucleotide. Super shifts were performed with anti-p65 antibody. Similar results were obtained in two separate experiments\nDiscussion\nIn addition to its many homeostatic functions such as barrier protection, mucus production and resorption, the intestinal epithelium plays an important role in regulating local inflammation and immune responses. The intestinal epithelium participates in inflammatory responses in part through the generation of numerous cytokines and chemokines that mediate recruitment and activation of inflammatory cells. The epithelium is involved in local cytokine networks allowing first response to noxious agents as well as cross-talk with immune cells to help provide an effective inflammatory response. These bi-directional inflammatory communications are necessary for normal host defence, but they also play an important role in the pathogenesis of IBD and might be also of importance in modulating anti-tumoural immunity in colonic malignancies. In this report, we demonstrate that pro-inflammatory cytokines, which may be produced by sub-mucosal immune cells, can differentially and co-ordinately regulate CXCL10 gene expression in IECs.\nCXCL10 is one of several chemokines produced by IEC. Previous studies could show that IFN-\u03b3 in combination with TNF-\u03b1 caused induction of RANTES (regulated upon activation, normal t-cell expressed, and secreted protein) and monocyte chemotactic protein-1 in colorectal tumour cell lines, while IFN-\u03b3 had no effect on CXCL8 (IL-8) secretion [27], the latter observation being confirmed in all three cell lines in the present study (data not shown).\nDwinell et al. [12] had previously shown that stimulation of IECs with TNF-\u03b1 or IL-1\u03b1 strongly potentiated IFN-\u03b3-induced CXCL10. It is well known that among IL-1\u03b1 and IL-1\u03b2, the latter is secreted and has an active role in inflammation. Importantly, IL-1\u03b2 was shown to be over-expressed in IBD patients [25, 28]. This makes IL-1\u03b2 an interesting cytokine to study its effect on CXCL10 gene regulation. Although the synergistic effects of TNF-\u03b1\u2009+\u2009IFN-\u03b3 on CXCL10 gene expression and the signalling mechanisms involved were studied in astrocytes [26] and their synergistic effect on inducible nitric oxide synthase expression in IECs and macrophages [29] had been studied in detail, the synergistic action of these cytokines on CXCL10 gene expression and the signalling mechanisms involved have not been evaluated in IECs. Especially, the role of IL-1\u03b2 alone and its synergism with both TNF-\u03b1 and IFN-\u03b3 on CXCL10 gene expression in IECs has been given no attention.\nPreviously, it has been shown that IECs may express elevated levels of CXCL10 [12], and its expression is increased in the mucosa of patients with IBD [16]. A critical role for CXCL10 in IBD has also been shown in animal models [30, 31]. Apart from this, CXCL10 has been shown to be expressed constitutively in colorectal carcinomas and primary metastatic sites exerting anti-tumour activity by recruiting mononuclear cells via the CXCR3 receptor. However, at the same time, it has also been shown that CXCL10 promoted metastatic adhesion and tumour cell migration [32] so that care should be taken considering CXCL10 as an anti-tumoural therapy option.\nIn light of these findings, we assessed whether stimulation of IECs in culture by inflammatory mediators could induce CXCL10 gene expression. All three IEC lines we used expressed CXCL10 mRNA in a concentration-dependent manner when stimulated with IFN-\u03b3 and TNF-\u03b1 alone. These data support a vast body of evidence that demonstrates that IFN-\u03b3 can induce CXCL10 expression in a variety of cells types, including granulocytes, T lymphocytes, bronchial epithelial cells and keratinocytes [33, 34], while TNF-\u03b1 is a potent inducer for CXCL10 in hepatocytes [17]. Importantly, in addition to these data, we could also show that Caco-2 cells stimulated with IL-1\u03b2 up-regulate CXCL10 mRNA to a significant level, whereas in HT29 and DLD1 cells, induction of CXCL10 mRNA was less pronounced.\nA number of authors have reported functional synergy between TNF-\u03b1 and IFN-\u03b3 in promoting gene expression for proteins such as RANTES, IFN regulatory factor 1 (IRF-1), MIG and intercellular adhesion molecule-1[35\u201338] in a variety of cell types and have demonstrated that this synergy depends on the co-existence of TNF-\u03b1-responsive NF-\u03baB-binding sites and IFN-\u03b3-responsive signal transducers and activators of transcription (STAT) protein-binding elements within the promoters of the genes of interest [35, 36]. In our study, when IFN-\u03b3 and TNF-\u03b1 were combined, the accumulation of CXCL10 mRNA and protein was elevated compared to unstimulated cells or cells stimulated with either cytokine alone, implying a synergistic effect on transcription of the CXCL10 gene. A synergistic effect by these two cytokines on CXCL10 expression was also demonstrated in astrocytes, and this synergy has been shown to be at least in part dependent on NF-\u03baB activation and binding to a NF-\u03baB-binding site within the CXCL10 promoter [26].\nThe synergistic action of IL-1\u03b2 with IFN-\u03b3 in IECs has not been characterised so far. Recently, Takami et al. [39] showed the synergistic induction of hepatocyte growth factor in human skin fibroblasts by IL-1\u03b2\u2009+\u2009IFN-\u03b3+\u2009IFN-\u03b3 [39]. We here report for the first time that in intestinal epithelial cell lines, IL-1\u03b2 alone or in combination with IFN-\u03b3 induced CXCL10 gene expression. In Caco-2 cells, the combination led to an early synergistic induction of both CXCL10 mRNA and protein compared to the combination of IFN-\u03b3+TNF-\u03b1. This early induction of CXCL10 mRNA and protein by IL-1\u03b2+IFN-\u03b3 in Caco-2 might be due to the early convergence of signalling pathways that activates transcription factors like NF-\u03baB, IRF-1, STAT1 or other factors that mediate transcription factor interactions.\nTransient transfections with human CXCL10 promoter [26], in Caco-2, HT29 and DLD1 cells, showed that the synergistic effect of IL-1\u03b2\u2009+\u2009IFN-\u03b3 and IFN-\u03b3\u2009+\u2009TNF-\u03b1 on CXCL10 gene expression in these cell lines was indeed because of the transcriptional synergy employed at the level of the CXCL10 promoter. A number of studies had revealed that cytokines like IL-1\u03b2, TNF-\u03b1 or enteroinvasive bacteria and the bacterial cell wall product lipopolysaccharide induce the expression of several inflammatory genes through the transcription factor NF-\u03baB in IECs [40\u201343]. To find out the signalling mechanism involved in CXCL10 gene expression in IECs, we explored the role of transcription factor NF-\u03baB. Inhibition of NF-\u03baB with BAY11-7082 clearly inhibited both CXCL10 mRNA and protein induced by single cytokine alone or in combinations in Caco-2, HT29 and DLD1 cells. Gel shift in Caco-2 could confirm that NF-\u03baB is activated and binds to consensus \u03baB oligonucleotides in cells treated with either IL-1\u03b2 or TNF-\u03b1 alone and in combination with IFN-\u03b3. It was shown previously that the binding complex of NF-\u03baB to the \u03baB2-binding site of the CXCL10 promoter contains the homodimer of p65 [26]. In our study, super shift assays in Caco-2 cells treated with IL-1\u03b2 alone or in combination with IFN-\u03b3 confirmed the presence of p65 in the complexes bound to the consensus \u03baB oligo nucleotides. In addition to these experiments, transient transfection experiments with a \u03baB2 mutant of the CXCL10 promoter [26] clearly showed that in Caco-2 cells, none of the cytokines and their combinations were able to induce the CXCL10 promoter activity. In HT29 cells, the combination of IFN-\u03b3\u2009+\u2009TNF-\u03b1 led to a weaker induction of TGL-IP10\u03baB-2 mutant activity, whereas in DLD1cells, the combination of IL-1\u03b2\u2009+\u2009IFN-\u03b3 and IFN-\u03b3\u2009+\u2009TNF-\u03b1 led to a weak induction of TGL-IP10 \u03baB-2 mutant activity. Based on these data, we can conclude that NF-\u03baB plays a critical role in CXCL10 gene expression induced by cytokines in IECs, and this is mainly dependent on the \u03baB-2 site present in its promoter. Moreover, IFN-\u03b3-induced CXCL10 mRNA and protein secretion was inhibited upon NF-kB inhibition.\nAlthough the present results may reflect only a small aspect in the overall concert of chemokines, it seems apparent that the basic regulatory principles elucidated here might be shared also by a number of other chemokine genes like that of CCL19, which has recently been shown to be regulated by multiple NF-\u03baB and IRF family transcription factors in human monocyte-derived dendritic cells [44]. Recently, Hiroi M et.al. [45] showed that co-operation between IFN-\u03b3 induced STAT1, and the constitutively active or inducible NF-\u03baB is necessary for the transcriptional activity of IFN-\u03b3-inducible genes like MIG and CXCL10. Therefore, we suggest a similar mechanism for IFN-\u03b3-induced CXCL10 in IECs.\nThe responsiveness to cytokines might depend on the state of differentiation. Schlottmann et. al. [46] recently showed that in contrast to HT29 where IFN\u03b3 up-regulated CXCL8 secretion, the same was unaffected in Caco-2 cells or even down-regulated in primary colonic epithelial cells. Future studies on chemokine regulation should therefore include primary cells, keeping well in mind that the presence of contaminating immune cells, culture conditions and cell stress during isolation may influence results.\nTaken together, our data suggest that while IFN-\u03b3, TNF-\u03b1 and IL-1\u03b2 may work individually through disparate signalling pathways in CXCL10 gene regulation, a combination of these cytokines enhances transcription of the CXCL10 gene in an NF-\u03baB-dependent manner. The fact that IL-1\u03b2 via activation of NF-\u03baB is able to mimic the effect of TNF-\u03b1 in augmenting CXCL10 expression might be of importance in patients with IBD, who do not respond to anti TNF-\u03b1 antibody therapy. In those cases, an additional inhibition of IL-1\u03b2 might be desirable as a future therapy option in treating IBD.","keyphrases":["intestinal epithelial cells","inflammatory bowel disease","cytokines and chemokines","nuclear factor kappa b","colon cancer"],"prmu":["P","P","P","M","R"]}
{"id":"Graefes_Arch_Clin_Exp_Ophthalmol-4-1-2292476","title":"The prescribing of prisms in clinical practice\n","text":"The use of prisms in cases of decompensated heterophoria is an established treatment modality. The clinical literature lacks consensus upon the appropriate use of prisms, and fails to provide the necessary evidence base. While the experimental literature can guide the practitioner, the lack of double-blind, placebo-controlled clinical studies needs to be addressed.\nThe use of prisms in cases of binocular dysfunction is an important treatment modality for dealing with such patients [1]. The decision to prescribe a prism, and what value of prism to give, is subject to varying clinical opinions and practices [2]. The conservative view in clinical practice is that prisms should not be prescribed in the absence of symptoms of binocular dysfunction [1]. Furthermore, the prescribing of prisms is contraindicated in certain cases, due to the risk of exacerbating an existing condition through the process of vergence adaptation [3\u20136].\nThe control of the vergence response has been described previously using linear systems modelling [7], and can aid our understanding of the aetiology of binocular visual dysfunction, and the effect of clinical intervention. The normal vergence response operates in a closed-loop feedback system, where the total response is the sum of the outputs of two control elements with differing temporal properties; a fast (phasic) control element and a slow (tonic) adaptive control element [7].\nPrevious reports have suggested that the strength of the adaptive vergence control element is often related to the presence of symptoms in patients with binocular dysfunction, and in severe cases the output of the adaptive vergence controller may be reduced to almost zero [8, 9]. In less severe cases, vergence adaptation may be present at a sub-normal level, but sufficient to produce an adaptive response to the introduction of a prism [8]. In such patients, strengthening the adaptive vergence control element is the treatment of first choice [8, 9]. Other studies have shown that in elderly patients vergence adaptation is very limited, and that while this may often be the cause of binocular dysfunction in these patients, remedial action with prisms is clinically viable because adaptation is very unlikely [10]. Indeed, we observe in our own binocular vision clinic that treatments designed to improve the strength of the adaptive vergence controller are generally unsuccessful in elderly patients.\nTypical clinical measures of binocular function, three of which are investigated by Otto et al. [11] in the current issue, are:\nHeterophoria (or dissociated phoria).This represents the fusion-free position of the eyes, and therefore the magnitude of the deviation which has to be overcome by the vergence system.Fixation disparity.First investigated by Ogle [12], this represents a small misalignment of the visual axes during binocular viewing, normally measured in seconds of arc. Many clinicians take the view that fixation disparity is indicative of stress within the binocular system [1]. Other authors regard fixation disparity to be a purposeful error, necessary for the vergence control system [5].Associated heterophoriaMallett [13, 14] is the author associated with describing the clinical characteristics of this measurement, which measures the amount of prism required to reduce any fixation disparity (in 2 above) present to zero. In Mallett\u2019s opinion, the presence of an associated phoria in a patient with symptoms of binocular vision dysfunction is indicative of stress within the binocular system, and requires treatment [15].Fusional vergence reserves.This is a clinical measure of the overall ability of the vergence system to control heterophoria. It is generally used in the calculation of Sheard\u2019s Criterion [16], which requires the vergence reserves to be at least twice the size of any heterophoria present. There is evidence that the vergence reserves are related to the strength of the adaptive component of the vergence system [8, 9].\nEach of these measures assess one specific aspect of binocular function, and while some practitioners prefer to rely on a particular measure to assess binocular function, others may assess a combination of the measures above to decide the most appropriate clinical intervention for the patient [2].\nThere is a lack of consensus in the literature about which of these measures correlates most closely with symptoms of binocular dysfunction, and which provides the most accurate basis for prescribing a prism when this is appropriate. Previous work has shown that no single measure correlates well with patient symptoms in all types of binocular anomaly, suggesting that neither fixation disparity, nor associated phoria, provide a universal indicator of binocular dysfunction [17]. This work also found that measures of vergence reserves often showed a better correlation with symptoms [17].\nThe imperative question for the clinician remains; is there a reliable method to determine the magnitude of prism required to compensate the heterophoria in patients suitable for this method of treatment? Mallett [15] suggested that the associated heterophoria identified the uncompensated portion of the binocular anomaly, and therefore this value represented the prism required to give the patient binocular comfort and stability. Other studies have questioned this [5, 17], and indeed the presence of an associated phoria in patients without binocular visual problems casts doubt on the general applicability of this measure [18]. Sheard\u2019s criterion provides a rationale for prescribing prisms [1, 18]; however, there is still a lack of randomised controlled clinical trials to prove the efficacy of any treatment of binocular vision [2], although a recent study has begun to address this [19]. The use of a measure such as the \u201ccomfortable prism\u201d, while clinically appealing, is highly subjective in nature, and would also require a clinical evidence base to prove efficacy. There is considerable work still to be done in the field of clinical management of binocular vision dysfunction in order to provide the required evidence base.\nIn all cases of binocular dysfunction, the choice to prescribe prisms lies with the clinician, who must judge what is most appropriate for each individual patient. A relatively straightforward procedure has been described to assess the probability of vergence adaptation occurring and rendering any prescribed prism ineffective [20]. Where prism treatment is being considered, obtain a prior measurement of the associated phoria, insert the prism to be prescribed into the trial frame and measure the associated phoria with the prism in situ. Allow the patient to wear the prism for 10\u00a0minutes; if the associated phoria returns to the value measured prior to insertion of the prism, then the patient\u2019s vergence adaptation mechanism has been strong enough to overcome the prism, and it will have little clinical benefit. Conversely, if no vergence adaptation is observed, the patient is likely to derive significant clinical benefit from this treatment.","keyphrases":["prisms","binocular","vergence adaption"],"prmu":["P","P","P"]}
{"id":"Histochem_Cell_Biol-4-1-2386527","title":"Actin: its cumbersome pilgrimage through cellular compartments\n","text":"In this article, we follow the history of one of the most abundant, most intensely studied proteins of the eukaryotic cells: actin. We report on hallmarks of its discovery, its structural and functional characterization and localization over time, and point to present days\u2019 knowledge on its position as a member of a large family. We focus on the rather puzzling number of diverse functions as proposed for actin as a dual compartment protein. Finally, we venture on some speculations as to its origin.\nIntroduction\nA common incident, ubiquitous and frequently observed by the experienced teacher: a student wants to purify protein X from an eukaryotic organism Y. Sooner or later the undergraduate stares at SDS polyacrylamide gels and is surprised, disappointed, depressed, furious about those horrible actin contaminations that seem to be everywhere. The student does not care about the advisor\u2019s soothing explanations: \u201cactin is the most abundant protein\u201d, \u201cactin exists as globular monomer (G-actin) in the soluble supernatant\u201d, \u201cbut it is also sedimentable as filamentous actin (F-actin)\u201d. It will be difficult to calm the young scientist down. Looking at these copious amounts of the protein, the student cannot believethat a long time ago it was a scientific breakthrough to show that actin exists at all,that it took years and years to convince truculent groups of actin pioneers that a non-muscle cell contains actin as well,that actin is not a singular and once for all invention in evolution, but is the founder of a large family of isoforms and related proteins, andthat quite a number of these proteins can be found in different subcellular compartments. For example, it has been established as a regular component of both, the cytoplasm and the nucleus; but this perception is the result of a fight that went on for decades.\nThe dawn: actin as a muscle component\nThe first traceable report that described contractile substances in muscle goes back to 1859 and Wilhelm Friedrich K\u00fchne (Kuehne 1859). He belonged to the best known physiologists of his time and coined modern designations like \u201cenzyme\u201d or \u201cmyosin\u201d. The latter one he described as a substance that could be isolated from frog muscle and forms a contractile clot under certain conditions. Today we would call this clot a contractile pellet, consisting mainly of actomyosin. Halliburton followed shortly afterwards and was intrigued by this clot formation (Halliburton 1887). He compared it to blood clotting and the transformation of fibrinogen to fibrin. In tedious experiments he extracted mammalian muscle tissue at different time scales, temperatures, salt concentrations and found that myosin apparently needed an additional component to perform fast aggregation and contraction. Without really knowing it, Halliburton worked with actin and produced with this so-called \u201cmyosin ferment\u201d an actomyosin precipitate. In our days one reads Halliburton\u2019s publication with some amazement. The report covers 70 pages and looks with its descriptions and tables like a lab-book, e.g.: \u201cExtract #1\u201410 a.m.\u2014Diluted with an equal amount of water\u201410 a.m. next day\u2014No change\u201d. Surely, at that time editors were not as busy as they are today.\nMuscle actin comes of age\nThen, it took almost 60\u00a0years until Br\u00fan\u00f3 F. Straub in Albert Szent-Gy\u00f6rgy\u2019s laboratory at the Hungarian University of Szeged was able to purify actin in good qualities and quantities. At that time ATP was known and available, i.e. Straub could study the influence of ATP on the viscosity of myosin preparations in the presence and absence of other extractable muscle components. The decrease in viscosity upon addition of ATP was considered \u201cactivation\u201d of myosin, the protein responsible for this activation was named \u201cactin\u201d, and the complex of these two proteins was \u201cactomyosin\u201d. Straub and Szent-Gy\u00f6rgyi discovered these interrelationships 1941\/1942 in the middle of World War II and, therefore, published the data only in local periodicals (\u201cStudies from the Institute of Medical Chemistry\u201d, University of Szeged). The research on actin and myosin, the political situation and the excitement in Szent-Gy\u00f6rgi\u2019s group in the early 1940s are nicely summarized by W.\u00b7F. Mommaerts who was a witness of the original experiments (Mommaerts 1992), and by A.G. Szent-Gy\u00f6rgyi himself (Szent-Gyorgyi 2004).\nIt is far beyond the scope of this review to cover the avalanche of actin and myosin research that started after those first molecular characterizations. Of course, muscle was and still is the major source to purify actin from, and, consequently, the research during the following decades used exclusively muscle actin.\nActin conquers the cytoplasm\nBut as it is in science, one opens one door and discovers ten closed ones. Keeping that in mind a scientist should always be suspicious regarding dogmas of all sorts. For some time it was almost a law that actin exists only in skeletal muscle and that the well-known conventional myosin is the only motor protein which turns the world go round. Today it is a common place that both assumptions were wrong, although it was quite a task to convince the scientific community.\nMany minor publications pointed at the presence of actin in non-muscle cells. For example, one found actomyosin-like components and Mg2+ATP-dependent viscosity changes in extracts from sea urchin eggs (Ohnishi 1962), and an actin-like protein from calf thymus nuclei (Ohnishi et al. 1963), and it was assumed that the filaments seen in fibroblasts are analogous to filaments in muscle, responsible for cell motility (Buckley and Porter 1967). Most of these publications never had a really strong impact, especially since they argued against a rock-solid preconception. But to be fair: these tiny reports were the pebbles that paved the road for better equipped groups and more detailed molecular studies. Notably, it was a slime mould, Physarum polycephalum that was used as a model organism to prove the existence of actin and myosin in non-muscle cells. The key experiments have been performed in Japan, in Sadashi Hatano\u2019s group (Hatano and Oosawa 1966a, b; Hatano and Tazawa 1968) and the data were confirmed shortly afterwards by other laboratories. Especially the decoration of actin filaments in non-muscle cells with myosin was a breakthrough technique that unequivocally proved the existence of the actomyosin system in many non-muscle cells (for reviews see: Huxley 1973; Pollard and Weihing 1974; Pollard 1981; Tilney 1975).\nActin as the prototype of a large family\nToday conventional actin is thoroughly studied, with respect to its polymerization and depolymerization equilibrium, its function in the cytoskeleton as a morphological stabilizer, its role during motile activities of a cell, its three-dimensional structure, its binding partners, etc. But the traitorous word in this sentence is \u201cconventional\u201d. What hides behind \u201cconventional\u201d actin? And what is an actin-related protein?\nA \u201cconventional\u201d actin consists of 375 highly conserved amino acids, give or take a few. The polypeptide sequence is folded into a roughly U-shaped molecule, exposing well-characterized binding sites for many different ligands. There are several isoforms as products of different genes, coined alpha, beta and gamma actin, all of which are polymorphic proteins capable to form polymers. \u201cConventional actins\u201d display a particular fold which enables them to interact with a variety of partners, to form specific suprastructures involved in intracellular motility, adhesion and locomotion (see below). Figure\u00a01 shows the dynamics of the actin cortex in a Dictyostelium discoideum amoeba during random migration, pinocytosis, and phagocytosis of a yeast cell. An actin-related protein harbours an actin domain, but it differs from \u201cconventional\u201d actin in size and overall amino acid sequence, usually cannot form polymers and may differ from the conventional form in subcellular distribution and its physiological activity. The increasing number of completely sequenced genomes is an enormous help towards our understanding of actin and actin-related proteins. One can take any sequenced genome to analyze actin as a founder of a large protein family. We chose the recently unravelled D. discoideum genome since it represents the genome of a model organism that can locomote as a single cell and in cell assemblies, and can differentiate into simple tissues in a developmental cycle. Hence, it is a social amoeba at the evolutionary border between unicellular and multicellular organisms (Eichinger et al. 2005). Notably, the number of actin genes does not tell us very much about the complexity of an organism. Whereas the genome of budding yeast contains only one single and, not surprisingly, essential gene that codes for a conventional actin, mouse harbours 35, and the plant Arabidopsis thaliana 10 actin genes. The D. discoideum genome contains 33 genes that code for a bona fide conventional actin. Most puzzling, 17 of these actins share identical amino acid sequences, but are encoded by 17 distinct genes, and many of these are developmentally regulated. Why would evolution allow this seemingly luxurious feature? Elimination of redundant genes can only be avoided if they represent a selective advantage. If the identical actins are translated in a developmental pattern, then posttranslational modifications might play a much larger role than we are aware of today. Indeed, actin\u2019s posttranslational modifications are numerous. Actins are acetylated, acylated, arginylated, Ser\/Thr\/Tyr-phosphorylated, ubiquitinylated etc. This suggests that a developmentally regulated expression of actin genes requires a similarly regulated expression of enzymes that catalyze posttranslational modifications. Such a hypothesis opens a completely new search for our understanding of actin and its isoforms.Fig.\u00a01Dynamics of the actin cortex during amoeboid movement, pino- and phagocytosis. D. discoideum was transformed with a truncated LimE construct, tagged to green fluorescent protein (GFP). LimE binds specifically to filamentous actin (Bretschneider et al. 2004). The transformed cells are depicted in phase contrast (left panels) and fluorescence (right), which reveals the concentration of actin filaments by GFP-LimE. Upper panels: optical section through the actin cortex during random amoeboid movement. Bottom panels: during uptake of a rhodamine-labelled yeast cell the actin cortex forms a phagocytic cup around the particle. The engulfment of liquids by macropinocytosis requires the actin system as well (arrow). Size bar 5\u00a0\u03bcm\nThe picture became even more complicated after the discovery of the actin-related proteins (Arps). Calculation of the putative structures shows very nicely that actin and Arps share a common three-dimensional fold (Fig.\u00a02, taken from Muller et al. 2005). Most characteristic is the nucleotide-binding cleft which divides the protein in roughly two halves. The depicted structures all show the same orientation. For actin itself, this means that, when incorporated into a \u201cconventional\u201d actin filament, the bottom part of the folded monomer points towards the fast growing (\u201cbarbed\u201d) end, the upper part towards the slow growing (\u201cpointed\u201d) end (see also Fig.\u00a03). Figure\u00a02 shows strikingly (1) that all Arps can be moulded into a structure quite similar to the overall structure of conventional actin and (2) that they display a clearly distinct pattern of patches with conserved amino acids. As seen in the comparative scale (Fig.\u00a02, right) Arps 1, 2 and 3 are most closely related to actin, whereas all the other Arps show a much lower percentage of identity. Arps 2 and 3 have been shown to form a tight complex that, after activation through several actin binding proteins and ATP, binds to actin filaments and induces their branching (Kiselar et al. 2007). However, it should be noted that the role of the Arp2\/3 complex for branching of actin filaments in vivo is still heavily challenged (Koestler et al. 2008). It is quite amusing to read Vic Small\u2019s statement in the Research Roundup of J Cell Biol: (Feb 25, 2008): \u201cWe haven\u2019t disproven branching yet. We\u2019ll need 3D imaging to put the nail in that coffin. But what we\u2019ve seen makes the branching model unlikely\u201d. Undoubtedly, we are still on thin ice with our knowledge on Arp2\/3 function in vivo.Fig.\u00a02Structural conservation in the ARP subfamily (adapted from Muller et al. 2005, with permission). The conservation scale weights identity from 0 (blue) to 100% (red). All Arps can adopt a global actin conformation (upper left) but only Arps 1, 2 and 3 show a pronounced sequence homologyFig.\u00a03Generation of different actin polymers as a result of different subunit interactions (adapted from Jockusch et al. 2006, with permission). The subdomains of the actin monomer, as deduced from the crystal structure, are numbered 1\u20134. In the process that leads to the \u201cconventional\u201d actin filament (F-actin), three such monomers (a\u2013c) form intermolecular contact sites involving the subdomains as indicated left of the trimeric seed. Under physiological conditions, these trimers convert subsequently into the polar F-actin filaments that are depicted in the electron micrograph (top right). In a more rapid process, the actin monomers may form an antiparallel dimer which, at least in vitro, can form bipolar filaments (LD filaments) that show a strikingly different appearance in the electron microscope (bottom right)\nLet us risk some sort of a heretical view. Trivial as it may be, one has to repeat it again and again: not the three-dimensional fold, but the surface of a protein determines its function. There are curious examples for this. Hisactophilin, a histidine-rich actin-binding protein from D. discoideum, has a structure nearly identical to that of interleukin-1\u03b2 and fibroblast growth factor, despite its unrelated amino acid sequence. Luckily for the authors, these data were published in a high ranking journal (Habazettl et al. 1992), although there was no functional similarity between these three proteins whatsoever. Today, their structural similarity is not considered important at all and, like many others, they are just members of the large trefoil protein superfamily (Liu et al. 2002). Sippl and coworkers systematically analyzed novel proteins based on known structures (Koppensteiner et al. 2000). They came to the conclusion that almost 30% of the studied proteins have similar structures but different functions.\nComing back to actins and Arps. Is it possible that the name \u201cactin\u201d for all of these proteins is utterly misleading? Are we wasting time in our search for cytoskeleton-related activities just because the fold triggered a wrong name? Is the so-called actin fold another example for a successful superfamily which contains only a few proteins that play a role in the cytoskeleton, while many other members are e.g. subunits of larger protein complexes in chromatin or hexokinases or proteins with yet unknown functions?\nActin-like proteins in the nucleus: the early period\nThe first reports on actin as a nuclear protein appeared already 5\u00a0years after its acceptance as a ubiquitous cytoplasmic component. Nuclear filament bundles resembling microfilaments were observed in oocytes when transcription was inhibited by actinomycin D (Lane 1969), and biochemical analyses identified a prominent protein present in isolated nuclei of the multinucleated plasmodia of Physarum polycephalum (Jockusch et al. 1971; Jockusch et al. 1974; Lestourgeon et al. 1975). The concentration of actin in the nucleus fluctuated with the cell cycle phase of the plasmodia, leading to suggestions that nuclear actin might either have a role in the constriction of the nuclear membrane in Physarum plasmodia that exhibit an intranuclear mitosis (Jockusch et al. 1971), or with changes in transcriptional activity during the cell cycle (Lestourgeon et al. 1975), or during transition from active growth to the formation of sklerotia (spherules) from plasmodia after starvation (McAlister et al. 1977). All this remained speculation at that time, and the majority of the cell biological community dismissed these findings as an artifact, caused by a contamination of nuclei with cytoplasmic actin. However, between 1979 and 1984 three reports appeared which supported the concept of actin being involved in transcription: (1) actin was identified in a complex of RNA polymerase II from Physarum (Smith et al. 1979), (2) it stimulated markedly the transcriptional activity of RNA polymerase II purified from HeLa cell extracts at the preinitiation phase (Egly et al. 1984), and (3) antibodies specific for actin, when injected into the large amphibian oocyte nuclei, caused a dramatic collapse of lampbrush chromosome loops, concommitant with a complete stop of mRNA transcription (Scheer et al. 1984). However, these observations still did not overcome the scepticism at that time\u2014it took another two decades to recognize actin as a dual compartment protein that can execute different functions by selectively associating with ligands specific either for the cytoplasm or the nucleus.\nActin and Arps in the nucleus\nIn the last 20\u00a0years, studies on nuclear actin became fashionable, and now a new area began which firmly established actin, its relatives and binding partners as nuclear components.\nSeveral important steps served as signposts in this process:Cell biologists began to understand that proteins are not necessarily confined to a single intracellular compartment. Numerous examples proved that proteins may shuttle between intracellular regions and even adopt different, compartment-specific activities. Shuttling of actin and actin-related proteins between the nucleus and the cytoplasm cannot be considered any longer as an exceptional, exotic behaviour.Genetic studies, for example performed with yeast or Drosophila, showed that activities specific for the nucleus, like chromatin remodelling or transcription required the presence of the beta actin isoform, in a configuration that could bind to nuclear protein complexes, but were inhibited with actin mutants that failed in this respect.The development of cell biological and immunological methods over the past 20\u00a0years, like cellular expression of flurorochrome-tagged proteins in conjunction with video microscopy, and the generation of epitope-characterized monoclonal antibodies allowed to follow actin trafficking throughout the cell and its location in the nucleus.And finally, some old observations made in structural biology came back into people\u2019s mind: actin is a highly polymorphic protein, which can give rise to several different and distinct polymers (Aebi et al. 1981; Millonig et al. 1988). Thus, while there are functionally quite unrelated proteins that may share the same structure (see above), it is also conceivable that a particular protein may adopt slightly different \u201cunconventional forms\u201d, possibly induced by specific binding partners. Such \u201cdistortions\u201d may then trigger the formation of specific oligomers or polymers and complex formation with still other partners. Thus, intranuclear actins need not necessarily adopt the same structures found so abundantly in the cytoplasm, like G- or F-actin. This topic is discussed in detail in (Pederson and Aebi 2002), and this concept is supported by monoclonal anti-actins that specifically decorate nuclear actin (Gonsior et al. 1999; Schoenenberger et al. 2005). In Fig.\u00a03 (taken from Jockusch et al. 2006), we show actin in two different forms that both can form polymers. The conventional actin filament requires the interaction of three actin monomers in a specific orientation. Such a trimeric seed will then grow into the polarized F-actin filaments (Fig.\u00a03, top). However, there is also evidence for another pathway: two actin monomers can bind to each other in another orientation, and this \u201clower dimer\u201d may then lead to nonpolar, quite different filaments (Fig.\u00a03, bottom). It is of course tempting to speculate that such \u201cunconventional\u201d actin polymers may play a role in vivo, for example in the nucleus, but so far, there is no solid evidence for this assumption.\nNot surprisingly, the wealth of data on nuclear actin and its relatives stimulated numerous hypotheses on their function. The relevant findings and the conclusions on putative functions are the topic of a number of interesting reviews, and in this article, we can only refer the reader to them and to the list of original references covered there (Bettinger et al. 2004; Blessing et al. 2004; Franke 2004; Jockusch et al. 2006; Pederson and Aebi 2002; Rando et al. 2000). The brave reader who works himself through all this information will arrive at two conclusions: the actin-like proteins have definitely conquered a firm position among the group of respectable, important nuclear proteins, and they seem to be involved in a plethora of different activities. While the tasks for actin in the cytoplasm all are connected with the conventional G- and F-actin structures and the dynamic equilibrium between these two states, nuclear functions of these proteins seem associated with several different forms or configurations which are ill or even not at all defined.\nIn the following paragraphs, we will briefly mention the most important tasks proposed for nuclear actins.\nActin filaments and a caryoskeleton\nIt is generally accepted that nuclear activities require a precise topographical arrangement of chromatin, to spatially separate hetero- from euchromatin and allow for the complex mechanism of chromatin remodelling during gene activation and transcription. Hence, when electron microscopy provided elegant images of networks of intranuclear filaments, preferably in large objects like the amphibian oocyte nuclei (germinal vesicles), it seemed quite plausible to conclude that a \u201cnuclear matrix\u201d or scaffold is composed of F-actin filaments (Clark and Merriam 1977; Clark and Rosenbaum 1979; Gard 1999) that could provide mechanical stability to the nucleus and serve as a platform to anchor, at least temporarily, chromatin constituents. Indeed, there is evidence from both earlier and more recent investigations that actin associates with filamentous structures: actin antibodies and myosin subfragment 1 decorate such filaments in amphibian oocyte nuclei (Clark and Rosenbaum 1979; Gard 1999; Scheer et al. 1984), and fluorescently labelled actin is found in a polymeric form in Xenopus and HeLa cell nuclei (Kiseleva et al. 2004; McDonald et al. 2006). Yet, in general, these filaments do not stain with fluorescent phalloidin, a small drug binding with high affinity to actin filaments of the conventional \u201cF-actin\u201d type. It is only after subjecting cells to stress or fixation protocols that nuclear filaments bind phalloidin, thus, the suspicion arises that such treatments rearrange actin into conventional filaments (Gall 2006; Jockusch et al. 2006). This, of course, does not preclude the concept that actin participates in a nuclear matrix, especially in the very large, actin-rich nuclei of amphibian oocytes. There is good evidence that it engages in providing mechanical support to this cellular compartment (Bohnsack et al. 2006; Stuven et al. 2003).\nThe nuclear Arps from yeast, Arp 7 and Arp 9, have been shown to build heterodimers and have the potential to form filaments from tetramers (Szerlong et al. 2003). However, it is not clear whether they do in vivo, and whether they might coassemble with nuclear actin in polymers (Blessing et al. 2004).\nActin\/Arps and their connection with the nuclear envelope\nSeveral structural proteins that are essential for the assembly and maintenance of an intact, functional nuclear envelope and its underlying lamina, display well defined binding motifs for actin. These include spectrin, protein 4.1, the nesprins, lamin A and emerin, (Krauss et al. 2003; Shumaker et al. 2003, more references in Blessing et al. 2004; Rando et al. 2000). Hence, it has been speculated, but not proven, that at least part of the nuclear actin is involved in the structural organization of the nuclear envelope.\nActin\/Arps and the nucleo-cytoplasmic traffic\nActin-containing, intranuclear filaments were seen to connect to nuclear pore complexes in the amphibian oocyte nucleus (Hofmann et al. 2001; Kiseleva et al. 2004). In the Xenopus oocyte, but also in dipteres and mammalian cells, actin is engaged in the transport and nucleocytoplasmic export of mRNA (Hofmann et al. 2001) and RNA-protein complexes, in particular in those containing a subset of hnRNPs (Percipalle et al. 2001, 2002, reviewed in Bettinger et al. 2004; Pederson and Aebi 2005).\nActin\/Arps and chromatin remodelling\nActin and nuclear Arps were both reported as being critically involved in chromatin remodelling (reviewed in (Bettinger et al. 2004; Blessing et al. 2004; Olave et al. 2002; Percipalle and Visa 2006; Rando et al. 2000). Chromatin remodelling complexes operate as large, multiunit machines in mammals, insects, yeast and plants to reorganize the genetic material by unravelling nucleosomes and converting the genetic material into a form suitable for transcription. Many of these steps require energy, and thus many of these protein complexes contain ATPases, comprised of several subunits, that can be grouped into subfamilies. Actin was identified in complex with specific subunits of most ATPases, together with four nuclear Arps (4, 5, 6 and 8) in all organisms, with the exception of yeast. Here, the two yeast-specific nuclear Arps (7 and 9) are found in the corresponding ATPases. One of the histone acetyl transferases that also contribute to chromatin remodelling and subsequent transcription (NuA4, Doyon et al. 2004) is highly conserved from yeast to man and is also found in a complex with actin and Arp 4 (Harata et al. 2002). Structural organization and function of the actin and Arp molecules in these ATPase and acetyl transferase complexes are largely unknown, and speculations for their respective roles range from stabilizing the enzymatic and a chaperoning activity to connecting these gigantic protein complexes to nuclear filamentous scaffolds.\nActin\/Arps and transcription\nActin has been identified as a regular component of all the three nuclear RNA polymerases (Pol I, II and III), apparently interacting with two subunits that are shared among all three enzyme complexes. In vitro transcription by all three enzymes is actin-dependent (reviewed in Grummt 2006; Percipalle and Visa 2006). In the nucleus, pre-mRNA is complexed with ribonucleoproteins to form hnRNPs, and, presumably, there is co-translational recruitment of beta actin to these particles during their formation, as deduced from findings that several hnRNP components bind directly actin. Actin\u2019s association with the RNA polymerases may precede, be simultaneous with or follow the initiation of actin-dependent chromatin remodelling by ATP-dependent complexes and\/or histone acetylation, as described above (Grummt 2006; Percipalle and Visa 2006). Most of the data available today are derived from studies with Pol II, in insects and vertebrates, supporting and confirming the conclusions drawn more than two decades earlier from work on Xenopus oocyte and HeLa cell nuclei (Egly et al. 1984; Scheer et al. 1984). There is solid evidence that actin\u2019s role in Pol II-dependent transcription is executed by the beta actin isoform (Hofmann et al. 2004; McDonald et al. 2006), and no other isoform seems involved. While actin\u2019s contribution to the formation of pre-initiation complexes and subsequent transport of pre-mRNPs through the nucleus towards their export through nuclear pores is well accepted today (Bettinger et al. 2004; Franke 2004), it is completely unknown whether actin might mediate such intranuclear movements in filamentous or even contractile structures (Pederson and Aebi 2005).\nActin ligands in the nucleus\nThe nuclear constituents harbouring actin, like those of the nuclear matrix, the nuclear envelope, the nuclear pores, chromatin remodelling complexes, RNA polymerases and pre-mRNPs are all highly insoluble, gigantic structures. However, there are also actin ligands that are found in the cytoplasmic as well in the nuclear compartment, possibly in rather dynamic and temporary complexes with actin. Since the discovery of actin as a prominent component in the cytoplasm of practically all eukaryotic cells, a plethora of such actin-binding proteins has been described and characterized. They arrange actin into the various suprastructures required for cytoplasmic functions, such as intracellular motility and locomotion, by either regulating the balance between monomeric and filamentous actin, or generating networks and bundles of filaments (Winder and Ayscough 2005). Remarkably, many of these actin ligands are evolutionary well conserved, like actin itself (Korn 1982; Schleicher et al. 1988). Members of the various subfamilies, with affinity to either monomeric or filamentous actin, are dual compartment proteins that can shuttle between the nucleus and the cytoplasm, their main location frequently being dependent on the differentiation state of cells. Comprehensive lists of these proteins that have previously been described as cytoplasmic actin ligands but were also identified in the nucleus are given in (Rando et al. 2000; Bettinger et al. 2004; Pederson and Aebi 2005). Among them are several which form complexes with actin and interfere with the formation of conventional actin filaments, like profilin, binding to monomeric actin, and cofilin, a ligand for both, monomeric and polymeric actin. Nuclear profilin is apparently involved in the regulation of the level of nuclear actin, as profilin\u2013actin complexes are recognized and exported from mammalian nuclei by a specific exportin (Stuven et al. 2003), while actin free of profilin can apparently be exported by a different exportin, due to its nuclear export sequences (Wada et al. 1998). Profilin and cofilin both can sligthly change the fold of actin. Profilin induces a form which faciliates the exchange of bound ATP in G-actin (reviewed in Jockusch et al. 2007). Cofilin, a phosphoprotein, contains a nuclear location sequence, and in its dephosphorylated state can transport actin \u201cpiggy-back\u201d into the nucleus (Pendleton et al. 2003). When bound to actin polymers, it distorts their conformation such that these filaments do not bind phalloidin anymore. Thus, these proteins might be critical in creating forms specific for nuclear actin, as detected by specific antibodies (Gonsior et al. 1999; Jockusch et al. 2006; Schoenenberger et al. 2005). Among the actin ligands solely found in the nuclear compartment are a nuclear actin binding protein identified in Acanthamoeba (Rimm and Pollard 1989), an actin binding protein composed of two different subunits in mammalian cells (Ankenbauer et al. 1989), and a nucleus-specific form of myosin I (Pestic-Dragovich et al. 2000). The latter is a small, monomeric myosin that does not form filaments as would be needed for contraction of putative actin filaments in the nucleus. It is apparently involved in transcription of ribosomal genes by Pol I, where it binds to a transcription initiation factor but also to components of a chromatin remodelling complex. It has been speculated that in this location it may recruit actin associated with Pol I to the site of rRNA transcription, but so far, there is no evidence that these two proteins interact directly in vivo (reviewed in (Grummt 2006; Percipalle and Farrants 2006).\nConclusions\nSo, have we now lost the student being confronted with actin for the first time in his scientific education, or have we been able to attract his curiosity for this abundant, highly conserved, mysterious protein?\nActin has come a long way from the times of its discovery in muscle and we have learned to know it as a member of a large family of structurally related proteins, some of which are dual compartment proteins. Concomitant with this, there is an overwhelming expansion of their putative functions, and both, the fine structural conformation as well as the different functions are probably determined by the numerous actin binding proteins. As a cartoon, Fig.\u00a04 summarizes the most important players in the cytoplasmic and the nuclear compartment. It is obvious that the putative activities in the nucleus outnumber those in the cytoplasm. Taken together, these findings allow for the view that actin and its relatives were originally engaged in nuclear activities, and that their appearance in the cytoplasm is related to a specialization in functions connected solely with intracellular motility and locomotion (F-actin filament formation, actin dynamics). Further functional narrowing would then result in the special case of skeletal muscle, where a specific isoform, alpha actin, is overexpressed and knows nothing else than to allow myosin-driven contraction.Fig.\u00a04The manifold functions of nuclear actin. The G-\/F-actin equilibrium is only a highly specialized function of actin in the cytoplasm (top); in the nucleus (bottom) the actin system plays a quite different regulatory and structural role. Actin can execute its tasks as monomer or as nucleus-specific polymer. It interacts with heterogenous ribonucleoproteins (hnRNPs) and is thought to function as a track for RNA transport through NPCs. It remains to be shown how nuclear actin cooperates with nuclear myosin-1 to enhance movement of RNA polymerases or to re-organize chromatin (Nunez et al. 2008). Positioning of the nucleus in the cell is achieved by nuclear membrane (NM) proteins that, like the large nesprins, hook the nucleus to the actin cytoskeleton. Anchorage of nesprins is guaranteed by SUN proteins in the inner nuclear membrane (Worman and Gundersen 2006). The stabilization of the nucleoskeleton involves the interaction of inner nuclear membrane proteins like emerin or nesprins with lamins as well as polymeric actin (Libotte et al. 2005). It should, however, be borne in mind that the level of nuclear actin is apparently tightly regulated in mammalian cells by exportin 6 which selectively transports profilin\u2013actin complexes from the nucleus into the cytoplasm (Bohnsack et al. 2006). The nuclear functions of many dual compartment actin binding proteins are not well understood, but one has to assume that their interaction can discriminate between nuclear and cytoplasmic actin (Pederson and Aebi 2005). For example, the recently described cyclase associated protein CAP2 is nuclear in myoblasts but sarcomeric in myotubes (Peche et al. 2007). Actin-related proteins are only summarized in one symbol and are either nucleus specific subunits in chromatin remodelling complexes (Arp4, Arp8 etc.), cytoplasmic or belong also to the group of dual compartment proteins\nIf one was to speculate further, one might imagine that prokaryotic precursors of present days\u2019 actin, related to the proteins that spatially and temporally control macromolecular trafficking, chromosome segregation and cell polarity in recent bacteria (Carballido-Lopez 2006), appeared first as \u201cnuclear actin\u201d in an ancestor eukaryotic cell as part of the endosymbiontic engulfment. But, it will require several decades and lots of industrious students to decide whether this is science fiction or facts about actin.","keyphrases":["transcription","chromatin remodelling","actin history","cytoplasmic motility","nucleocytoplasmic traffick"],"prmu":["P","P","R","R","R"]}
{"id":"J_Urban_Health-2-2-1705505","title":"Respondent-Driven Sampling in a Study of Drug Users in New York City: Notes from the Field\n","text":"Beth Israel Medical Center (BIMC), in collaboration with the Centers for Disease Control (CDC) and the New York State Department of Health (NYSDOH), used respondent-driven sampling (RDS) in a study of HIV seroprevalence among drug users in New York City in 2004. We report here on operational issues with RDS including recruitment, coupon distribution, storefront operations, police and community relations, and the overall lessons we learned. Project staff recruited eight seeds from a syringe exchange in Lower Manhattan to serve as the initial study participants. Upon completion of the interview that lasted approximately 1 h and a blood draw, each seed was given three coupons to recruit three drug users into the study. Each of the subsequent eligible participants was also given three coupons to recruit three of their drug-using acquaintances. Eligible participants had to have: injected, smoked or snorted an illicit drug in the last 6 months (other than marijuana), aged 18 or older, adequate English language knowledge to permit informed consent and complete questionnaire. From April to July 2004, 618 drug users were interviewed, including 263 (43%) current injectors, 119 (19%) former injectors, and 236 (38%) never injectors. Four hundred sixty nine (76%) participants were men, 147 (24%) were women, and two (<1%) were transgender. By race\/ethnicity, 285 (46%) were black, 218 (35%) Hispanic, 88 (14%) white, 23 (4%) mixed\/not specified, and four (<1%) native American. Interviews were initially done on a drop-in basis but this system changed to appointments 1 month into the study due to the large volume of subjects coming in for interviews. Data collection was originally proposed to last for 1 year with a target recruitment of 500 drug users. Utilizing RDS, we were able to recruit and interview 118 more drug users than originally proposed in one quarter of the time. RDS was efficient with respect to time and economics (we did not have to hire an outreach worker) and effective in recruiting a diverse sample of drug users.\nBackground\nA variety of sampling methods have been used to recruit hard-to-reach populations, such as drug users, into research studies. These methods include snowball sampling, targeted sampling, and time-space sampling.1\u20133 Despite widespread use of these methods, they are not without important limitations.\nSnowball sampling begins with a set of initial study participants, called seeds, who refer other eligible respondents. Once these persons are interviewed, they are also asked to bring in or provide references for other potential respondents. This process continues until the sample size is achieved. Snowball sampling can provide easy access to hidden populations, but it is most often biased because it is usually done out of convenience rather than randomly.1\u20133\nTargeted sampling involves thoroughly collecting preliminary data to determine various characteristics such as locations where a sample congregates and demographic characteristics such as race, gender, and age.1,2,4 Similar to snowball sampling, using targeted sampling to study drug users may result in a selection bias because less-visible, isolated drug users may not be found at the sites where other drug users are sampled and, thus, may be overlooked.\nTime-space sampling involves preliminary research by ethnographers to determine when and where hidden populations congregate.3,5 Once a list of times and sites is determined, researchers then randomly choose times to visit those sites to collect data. Time-space sampling has limited use with drug users because making an exhaustive list of where drug users congregate could be a very protracted and expensive process, as drug-use sites change frequently in response to environmental factors such as police presence.\nRespondent-driven sampling (RDS),1 a relatively new method being used to draw probability samples of hidden populations, incorporates some methods of snowball sampling, such as chain referral sampling, but includes additional provisions to minimize bias. It produces population estimates that are asymptotically unbiased, which means that bias is only on the order of 1\/[sample size], so bias is minimal in samples of substantial size.6 This allows for probability-based inferences based on the social network of the sample.\nThis paper reports on the use of RDS to recruit participants for a study of risk behavior and HIV seroprevalence among drug users in New York City. The study was conducted by Beth Israel Medical Center in collaboration with the Centers for Disease Control and Prevention and the New York State Department of Health. We discuss the operational issues faced with RDS such as recruitment, coupon distribution, storefront operations, police and community relations, and the overall lessons learned.\nMethods\nIn April 2004, Beth Israel Medical Center staff recruited eight seeds from a syringe exchange in Lower Manhattan. Each seed was screened for eligibility before being given a coupon to come back for an interview. The seeds were asked about their drug use, including mode of use and drug preparation techniques. Those who claimed to be an injection drug user (IDU) were also asked to show track marks. Although not a requirement of RDS, the seeds were recruited to resemble the race and gender profile of drug users in Lower Manhattan.7 They were asked to come to a research storefront in Lower Manhattan the following day to complete a computer-assisted interviewer-administered personal interview (CAPI) and to have their blood drawn for an HIV test. Each seed, and subsequent study subject, received $20 compensation for their time.\nWhen subjects arrived at the storefront, they were questioned by the study screener to ensure eligibility. Once they were deemed eligible by the screener, each subject was assigned a unique code to serve as their study identification. This code included the following information: the first two letters of the last name, first letter of mother\u2019s first name, the last two digits of the birth year, one letter for the person\u2019s race, and one letter for gender. This code was used to identify blood work and questionnaires and could be regenerated if the subject forgot it. No names or other identifying information were asked.\nAfter being assigned a study code, subjects met with an interviewer to be consented and interviewed. The interview had three parts, an hour-long questionnaire, administered by the interviewer, HIV pre-test counseling and a blood draw for an HIV test. The interview consisted of a structured questionnaire which took approximately 1\u00a0h and asked about drug-use frequency, drug and sexual risk behavior, syringe acquisition, and knowledge of HIV and hepatitis B and C. After the interview, HIV counseling, and blood collection, each subject was given three coupons to recruit three other drug users into the study. The subjects were briefly trained on how to recruit others, with specific emphasis on the recruitment of friends and acquaintances who use drugs. Eligible respondents had to have done the following: injected, smoked, or snorted an illicit drug in the past 6\u00a0months (those who smoked only marijuana were not eligible for the study); turned age 18\u00a0years or older by the time of the interview; been able to speak English adequately to consent to the study and complete the questionnaire; and lived, bought, and\/or used drugs on the Lower East Side of Manhattan. Subjects could have, however, lived in the larger New York metropolitan area, as long as they bought or used their drugs on the Lower East Side of Manhattan.\nCoupons contained the time that the storefront opened (9:00 a.m.) and a unique number (to make each one distinct). To make them difficult to duplicate, coupons were printed on thick cardstock paper with color images. To track the coupons and payment for each respondent, we used custom-developed software for RDS called IRIS Plus. Information such as respondent\u2019s unique code, physical traits, coupon number, and the numbers of the coupons each respondent distributed were all recorded in IRIS Plus. This information enabled us to link coupons together, determine when respondents should be paid and who gave coupons to whom. This software also helped to prevent the redemption of duplicated coupons because the database would not accept duplicate coupon numbers. When respondents came in to make an appointment, the coupon was checked in the IRIS Plus database to verify that it had not been used previously. Additionally, each person\u2019s code was checked in IRIS Plus after being screened to determine whether a person with that code had previously been enrolled in the study. If another study subject had that code, the screener looked at the physical traits listed for that person for verification. Additionally, if the screener felt that a person looked familiar, she would search for the person's physical traits in IRIS Plus to see if another person with similar characteristics was previously enrolled.1\nOn July 2, 2004, we ceased coupon distribution to give potential subjects 2 weeks to redeem remaining coupons. On July 16, 2004, we concluded data collection, and the study officially concluded on July 30, 2004. The last 2\u00a0weeks of July were set aside to allow the remainder of subjects to come in for their HIV test results.\nResults\nFrom April to July 2004, 618 respondents were interviewed, including 263 (43%) current injectors (had injected drugs within the past 6\u00a0months), 119 (19%) former injectors (had injected drugs more than 6\u00a0months prior to the interview\u2014who used drugs in the past 6\u00a0months by other modes of administration), and 236 (38%) never injectors (those that had never injected any drugs but used non-injection drugs in the past 6\u00a0months). The mean age of respondents was 44\u00a0years, with a mean age of first drug use at 19\u00a0years and mean age of first injection drug use at 22\u00a0years (among current and former drug injectors). By gender, 469 (76%) were men, 147 (24%) were women, and two (<1%) were transgender. By race\/ethnicity, 285 (46%) were black; 218 (35%), Hispanic; 88 (14%), white; 23 (4%), mixed race or unspecified race; and four (<1%), native American.\nSeed Recruitment\nTo control the flow of recruitment, we recruited only eight seeds: two white men, one white woman, two Hispanic men, one Hispanic woman, one black man, and one black woman, all of whom identified themselves as active IDUs. A relatively small number of seeds was recruited so that we could assess how productive they were and then recruit more if necessary, without being too overwhelmed with respondents.\nRespondent Recruitment\nOur initial goal was to interview 500 drug users within 1\u00a0year. After 4\u00a0months, 618 drug users had been interviewed. Respondent recruitment (those people recruited after the seeds) happened much faster than we had expected. In the first couple of weeks, recruitment was slow but quickly picked up as more coupons got out into the community. Interviews were initially done on a drop-in basis, but this system changed to appointments after 1\u00a0month into the study due to the large volume of respondents coming in for interviews. As the flow of potential subjects continued to be rapid and at times overwhelming, we were also forced to post-date all coupons by 2\u00a0days. Therefore, when a respondent was given a coupon, that person could not be interviewed or make an appointment until the date written on the coupon. This system was helpful in pacing the number of people coming into the storefront for interview appointments but perhaps choosing a smaller number of seeds may have alleviated the need to implement these changes altogether.\nPolice and Community Relations\nAs previously noted, as the study progressed and more coupons were distributed in the community, the number of people coming into the storefront for interviews increased dramatically. For the first 2\u00a0weeks, the interviewers would arrive in the morning to find 10 to 20 people waiting for them. People reported arriving as early as 7:00 a.m. so that they could be interviewed that day. Once crowds began to form outside the storefront, neighboring businesses complained to our staff or called the police to file complaints. The police came to speak with staff on three separate occasions regarding complaints of noise and loitering. At that point we decided to move to an all-appointment system. Instead of telling people that their recruits could show up and be interviewed, recruits were told to first come into the storefront to be screened, and that if they passed the screener, they could make an appointment.\nLessons Learned\nWere we to do another RDS study in New York City, we would do the following:\nWe would have a phone number so that screening and appointments for potential participants could be done by telephone. This would lessen the amount of traffic in the storefront and reduce some confusion. Once participants arrived for their interviews, we would then screen them about their drug use.\nIf we were looking to recruit a large number of drug users in a short amount of time, we would again recruit eight seeds. The eight seeds recruited for this study seemed to be an appropriate number because we were trying to maximize our time in the field for data collection. Although the first 2\u00a0weeks of the study were relatively slow-paced, it gave the field staff time to adjust to their new environment and new roles and make any necessary changes before they became too overwhelmed. Drug users in New York City are generally a very overstudied population, and thus we expected a large portion of the seeds to show up for their interviews. In cities where the population is not as familiar with research, researchers may want to recruit more seeds, considering that many may not return for the initial interview. Another alternative, however, if time permits, is to recruit a smaller number of seeds, give them a few weeks to return for their interview and then recruit more seeds, if necessary. It was our intention to recruit more seeds if our initial eight were not productive, but this was not necessary.\nWe would start with an appointment-only system. We began with a drop-in system for interviews because our time to collect data was very limited and this was the most efficient use of the interviewers\u2019 time. For instance, if a person dropped in and wanted to be interviewed, we could do it on the spot if the person with a scheduled interview had not shown up. Even though this system worked for us initially, it soon became unwieldy, and we were forced to change a month into the study. People adjusted very quickly, and many people reported being relieved because they didn\u2019t have to wait in front of the storefront for 2\u00a0h in the morning before staff arrived.\nWe would be very rigid about our appointments. In this study we were very strict about appointment times and conveyed this to each participant. If persons with appointments were more than 5\u00a0min late and someone else arrived to schedule an appointment but was free to complete it then, we took the person who dropped in rather than waiting for the person with the scheduled appointment to show up. We rarely had interviewers sitting around with nothing to do. There was almost always someone who dropped in when someone else was late for an appointment.\nWe would meet with local business owners to inform them of who we are and what we were doing prior to the start of the study. We would invite them to tell us if they had any concerns. We would not disclose the type of research being done or with whom, but it would be helpful to let the surrounding business owners know that they may see an increase in the number of people in or around the storefront.\nWe would remain at the storefront 6\u00a0weeks after ending coupon distribution. Coupon distribution ended a full 4\u00a0weeks before we vacated the storefront, and data collection ended 2\u00a0weeks before. The timing of both of these allowed for a fair amount of those with coupons to redeem them, for those with outstanding incentives to collect their money, and for those with outstanding test results to come in for them. Two additional weeks would have been helpful to allow for remaining coupons to be redeemed. Because the storefront we used stayed open for other research studies, we were able to leave contact information for our interviewers in case any respondents showed up for their HIV test results. Within the month after we left the storefront, we received two additional calls for HIV test results. Our interviewers met the respondents at a park and a coffee shop to give them their results. The other storefront staff reported a handful of people coming in with coupons after the study closed, but they did not report any incidents with those people that were unable to redeem them. Finally, we did not pay out any uncollected incentives after July 30, 2004.\nSome of the lessons that we learned about conducting a study using RDS may be specific to New York City. Because there was an abundance of willing and able participants, we had to take several measures to make data collection more manageable. These measures may not be necessary or practical for researchers in cities where the drug-using population is smaller, less willing to participate in research, or both.\nConclusions\nUsing RDS, we were able to recruit and interview 118 more drug users than originally proposed in one third of the time. In our experience, RDS was efficient with respect to time and economics (we did not have to hire an outreach worker) and effective in garnering a diverse sample of drug users. We were able to interview many more drug users than expected in a short period of time with very minimal recruitment effort. In summary, RDS can be an efficient and effective form of recruitment, particularly for research involving drug users or other hidden populations.","keyphrases":["drug users","respondent-driven sampling (rds)","time-space sampling","capi","iris plus"],"prmu":["P","P","P","P","P"]}
{"id":"Pflugers_Arch-3-1-1839769","title":"The contribution of refractoriness to arrhythmic substrate in hypokalemic Langendorff-perfused murine hearts\n","text":"The clinical effects of hypokalemia including action potential prolongation and arrhythmogenicity suppressible by lidocaine were reproduced in hypokalemic (3.0 mM K+) Langendorff-perfused murine hearts before and after exposure to lidocaine (10 \u03bcM). Novel limiting criteria for local and transmural, epicardial, and endocardial re-excitation involving action potential duration (at 90% repolarization, APD90), ventricular effective refractory period (VERP), and transmural conduction time (\u0394latency), where appropriate, were applied to normokalemic (5.2 mM K+) and hypokalemic hearts. Hypokalemia increased epicardial APD90 from 46.6 \u00b1 1.2 to 53.1 \u00b1 0.7 ms yet decreased epicardial VERP from 41 \u00b1 4 to 29 \u00b1 1 ms, left endocardial APD90 unchanged (58.2 \u00b1 3.7 to 56.9 \u00b1 4.0 ms) yet decreased endocardial VERP from 48 \u00b1 4 to 29 \u00b1 2 ms, and left \u0394latency unchanged (1.6 \u00b1 1.4 to 1.1 \u00b1 1.1 ms; eight normokalemic and five hypokalemic hearts). These findings precisely matched computational predictions based on previous reports of altered ion channel gating and membrane hyperpolarization. Hypokalemia thus shifted all re-excitation criteria in the positive direction. In contrast, hypokalemia spared epicardial APD90 (54.8 \u00b1 2.7 to 60.6 \u00b1 2.7 ms), epicardial VERP (84 \u00b1 5 to 81 \u00b1 7 ms), endocardial APD90 (56.6 \u00b1 4.2 to 63.7 \u00b1 6.4 ms), endocardial VERP (80 \u00b1 2 to 84 \u00b1 4 ms), and \u0394latency (12.5 \u00b1 6.2 to 7.6 \u00b1 3.4 ms; five hearts in each case) in lidocaine-treated hearts. Exposure to lidocaine thus consistently shifted all re-excitation criteria in the negative direction, again precisely agreeing with the arrhythmogenic findings. In contrast, established analyses invoking transmural dispersion of repolarization failed to account for any of these findings. We thus establish novel, more general, criteria predictive of arrhythmogenicity that may be particularly useful where APD90 might diverge sharply from VERP.\nIntroduction\nHypokalemia exerts important clinical effects on cardiac function that in some respects resemble those seen in the congenital long-QT syndromes (LQTS). Thus, both conditions result in electrocardiographic QT prolongation [12, 23] and premature ventricular depolarizations (PVDs), which may result in the initiation of an arrhythmic activity [41, 52]. In contrast to the cardiac effects of hypokalemia, arrhythmic activity in LQTS has been extensively studied and has often been attributed to after-depolarizations occurring against a background of re-entrant substrate [2, 36, 44]. Re-entry may take place as a result of inhomogeneities producing regions of conduction block, which lead to wave-break and circus movement [21, 37] or altered repolarization gradients, which lead to wave reflection [1]. In this situation, depolarization propagates from active cells into previously active adjacent regions, establishing re-entrant circuits. These may become established either locally or over larger regions of the myocardium, such as across the thickness of the myocardial wall.\nTendencies to transmural re-entrant excitation in models of LQTS have been previously analyzed in terms of transmural dispersions of repolarization (TDR) obtained from the positive part of the difference between respective endocardial and epicardial stimulation to repolarization times [36, 44, 45]. In human LQTS, increases in the interval between the peak and full recovery of electrocardiographic precordial T waves (Tpeak to Tend), previously shown to reflect TDR [54], are indeed associated with arrhythmic activity [33]. Certainly, recent reports correlate Tpeak to Tend to arrhythmic risk more closely than more widely accepted indicators such as corrected QT interval and QT dispersion [53]. However, such re-excitation may also be limited by recovery from refractoriness; re-entrant excitation would require this to precede the return of the membrane potential to threshold [40]. Certainly, class 1 antiarrhythmic drugs such as lidocaine are known to increase ventricular effective refractory period (VERP) [28]. Yet, such use of spatial differences in action potential repolarization times to quantify arrhythmic substrate neither explicitly considers changes in VERP nor applies such criteria to potential local as opposed to transmural re-excitation.\nThis paper associates for the first time the proarrhythmic effect of hypokalemia with a significant decrease in VERP, despite contrasting prolongation of action potentials, in agreement with computer-modeling studies of action potential waveforms using established data on the various effects of hypokalemia on ionic conductivity properties of ventricular myocytes. Furthermore, it associates the antiarrhythmic effects of lidocaine with a significant increase in VERP, despite having little effect on action potential duration, in agreement with clinical observations. Analyses using TDR were insufficiently sensitive to account for any of these arrhythmogenic findings. This study accordingly established more general novel criteria that would provide necessary conditions for local and transmural and epicardial and endocardial re-excitation incorporating not only action potential duration but also VERP and conduction times that may be particularly useful when action potential duration differs sharply from VERP. These criteria successfully accounted for all the arrhythmogenic findings.\nMaterials and methods\nExperimental animals\nMice were housed in an animal facility at 21\u2009\u00b1\u20091\u00b0C with 12\u00a0h light\/dark cycles. Animals were fed sterile chow (RM3 Maintenance Diet, SDS, Witham, Essex, UK) and had free access to water. Wild-type 129\u00a0Sv mice aged 3\u20136\u00a0months were used in the experiments. All procedures complied with UK Home Office regulations (Animals [Scientific Procedures] Act 1986).\nSolutions\nAll solutions were based on bicarbonate-buffered Krebs-Henseleit solution (mM: NaCl 119, NaHCO3 25, KCl 4, KH2PO4 1.2, MgCl2 1, CaCl2 1.8, glucose 10 and Na-pyruvate 2; pH adjusted to 7.4) bubbled with 95% O2\/5% CO2 (British Oxygen Company, Manchester, UK). Hypokalemic (3.0\u00a0mM K+) solutions were prepared by reducing the quantity of KCl added. Lidocaine-containing normokalemic and hypokalemic solutions were prepared by adding lidocaine (Sigma\u2013Aldrich, Poole, UK) to a final concentration of 10\u00a0\u03bcM.\nPreparation\nA Langendorff-perfusion protocol previously adapted for murine hearts [4] was used. In brief, mice were killed by cervical dislocation (Schedule 1: UK Animals [Scientific Procedures] Act 1986), and hearts were then quickly excised and placed in ice-cold bicarbonate-buffered Krebs-Henseleit solution. A short section of aorta was cannulated under the surface of the solution and attached to a custom-made 21-gauge cannula filled with the same solution using an aneurysm clip (Harvard Apparatus, Edenbridge, Kent, UK). Fresh Krebs-Henseleit solution was then passed through 200 and 5\u00a0\u03bcm filters (Millipore, Watford, UK) and warmed to 37\u00b0C using a water jacket and circulator (Techne model C-85A, Cambridge, UK) before being used for constant-flow retrograde perfusion at 2\u20132.5\u00a0ml\/min using a peristaltic pump (Watson-Marlow Bredel model 505S, Falmouth, Cornwall, UK). Hearts were regarded as suitable for experimentation if, on rewarming, they regained a healthy pink colour and began to contract spontaneously.\nElectrophysiological measurements\nAn epicardial monophasic action potential (MAP) electrode (Hugo Sachs, Harvard Apparatus) was placed against the basal region of the left ventricular epicardium. In addition, a small access window was created in the interventricular septum to allow access to the left ventricular endocardium [9]. A custom-made endocardial MAP electrode composing two twisted strands of high-purity Teflon-coated 0.25\u00a0mm diameter silver wire (Advent Research Materials, UK) was constructed. The Teflon coat was removed from the distal 1\u00a0mm of the electrode, which was then galvanically chlorided to eliminate DC offset, inserted and placed against the septal endocardial surface. MAPs were amplified, band-pass filtered (0.5\u00a0Hz to 1\u00a0kHz: Gould 2400S, Gould-Nicolet Technologies, Ilford, Essex, UK), and digitized at a sampling frequency of 5\u00a0kHz (micro1401, Cambridge Electronic Design, Cambridge, UK). Analysis of MAPs was performed using Spike II software (Cambridge Electronic Design).\nExperimental protocol\nA bipolar platinum stimulating electrode (1\u00a0mm interpole spacing) was placed on the basal surface of the right ventricular epicardium. Square-wave stimuli (Grass S48 stimulator, Grass-Telefactor, Slough, UK) of 2\u00a0ms duration and with amplitudes of twice the excitation threshold were initially applied to hearts at a constant cycle length of 125\u00a0ms for at least 10\u00a0min and until MAPs showed stable baselines, rapid upstroke phases that reached consistent amplitudes and smooth repolarization phases [30]. Hearts were then exposed to test solutions for 20\u00a0min, during which time stimulation was continued, before subsequent recordings were made.\nIntrinsically evoked MAPs were recorded in the absence of stimulation while action potential duration (at 90% repolarization, APD90) and stimulation to depolarization latency were determined during regular stimulation at a constant interstimulus interval of 125\u00a0ms. Hearts were then subjected to an adapted form of an extrasystolic electrical stimulation procedure previously used to assess arrhythmogenicity and refractoriness in both human [43] and murine [22] studies of congenital LQTS, described in detail later. The possibility that events evoked by extrasystolic stimuli rather represented motion artifacts was excluded by their being reproducible between hearts and appearing identical in both electrodes.\nAll data are presented as means\u00b1standard errors of the means and include both the number of repetitions and the number of hearts. Comparisons were made using analysis of variance (significance threshold set at P\u2009\u2264\u20090.05).\nModeling\nThe charge-difference model of Fraser and Huang [16, 18] was adapted to permit computational modeling of the murine ventricular cardiac myocyte using ion channel equations and parameters from the model of Bondarenko et al. [6] with the Na+\/K+-ATPase model of Hernandez et al. [24]. The use of charge-difference modeling allowed the model to reach a true beat-to-beat steady state that was independent of initial intracellular ion concentrations [17], thus permitting simulation of the influence of changes in extracellular ion concentrations that are well recognized to influence Na+\/K+-ATPase activity, and hence steady-state intracellular ion concentrations.\nModel cells were studied under normokalemic (5.2\u00a0mM K+) conditions with normal ion permeabilities, hypokalemic (3\u00a0mM K+) conditions with these same ion permeabilities and hypokalemic (3\u00a0mM K+) conditions with the K+ permeabilities of channels carrying the repolarizing currents IK1 and Ito reduced by 20%, replicating the effect of such hypokalemia on transmembrane K+ permeabilities observed experimentally by Killeen et al. [29]. Stimulation was applied at a regular 125\u00a0ms interstimulus interval at an amplitude of twice the diastolic threshold, as for the experimental preparations. After beat-to-beat stability was achieved, APD90 was measured under each condition. Refractory periods were then determined using a similar protocol to that used in the experiments, every eighth (S1) stimulus being followed by an extrasystolic (S2) stimulus. S1S2 interval was initially 70\u00a0ms and was subsequently decremented by 1\u00a0ms with each successive cycle until an S2 stimulus failed to initiate an action potential.\nResults\nAfter-depolarizations initiate arrhythmic activity in bradycardic hypokalemic hearts\nIn initial experiments, isolated perfused hearts were stimulated at a constant interstimulus interval of 125\u00a0ms for 20\u00a0min after 20\u00a0min exposure to test solutions. This demonstrated stable trains of MAPs, under all normokalemic (5.2\u00a0mM K+, n\u2009=\u20097; five hearts), hypokalemic (3.0\u00a0mM K+, n\u2009=\u20098; five hearts), or lidocaine-treated (10\u00a0\u03bcM) normokalemic (n\u2009=\u20096; five hearts) or hypokalemic (n\u2009=\u20097; five hearts) conditions: After-depolarizations and arrhythmic activity were consistently absent throughout. The subsequent experiments then examined arrhythmic properties at the longer cycle lengths (between 224 and 271\u00a0ms) that occurred in the absence of extrinsic stimulation (Fig.\u00a01) and that have previously been reported to be proarrhythmic both under hypokalemic conditions and in the congenital LQTS [11]. Intrinsic cycle length did not differ significantly (P\u2009>\u20090.05) between normokalemic (250\u2009\u00b1\u200921\u00a0ms, six hearts), hypokalemic (253\u2009\u00b1\u200916\u00a0ms, seven hearts), lidocaine-treated normokalemic (248\u2009\u00b1\u200922\u00a0ms, six hearts), and lidocaine-treated hypokalemic (260\u2009\u00b1\u200910\u00a0ms, eight hearts) hearts.\nFig.\u00a01After-depolarizations and arrhythmic activity in spontaneously contracting hypokalemic hearts. Epicardial monophasic action potential recordings in the absence of extrinsic stimulation in hearts exposed to normokalemic (5.2\u00a0mM K+, a) and hypokalemic (3.0\u00a0mM K+, b) test solutions and normokalemic (c) and hypokalemic (d) test solutions containing lidocaine (10\u00a0\u03bcM) for 20\u00a0min\nEpicardial MAPs then retained morphologically consistent waveforms and were entirely free of after-depolarization and arrhythmic phenomena through 116\u00a0min of recordings over six normokalemic hearts (Fig.\u00a01a). In contrast, 46\u2009\u00b1\u20097% of MAPs showed after-depolarizations early in their repolarization phases during 140\u00a0min of recordings that led to episodes of arrhythmic activity in 52\u2009\u00b1\u20093% of cases in five out of seven hypokalemic hearts (P\u2009<\u20090.01 as compared to normokalemic hearts, Fig.\u00a01b). However, MAPs showed consistent waveforms without such after-depolarizations or arrhythmic activity during 118\u00a0min of recordings over six lidocaine-treated normokalemic hearts (Fig.\u00a01c). Finally, 40\u2009\u00b1\u20099% of MAPs showed after-depolarizations occurring late in the repolarization phase in 40\u2009\u00b1\u20099% of cases more than 98\u00a0min of recordings in six out of eight lidocaine-treated hypokalemic hearts (P\u2009>\u20090.05 as compared to hypokalemic hearts). After-depolarizations occurred more frequently in those instances where intrinsic cycle length was long. However, these events were never followed by arrhythmic activity (P\u2009<\u20090.01 as compared to hypokalemic hearts, Fig.\u00a01d).\nExtrasystolic stimulation immediately after recovery from refractoriness initiates arrhythmic activity in hypokalemic hearts\nA programmed electrical stimulation protocol recently shown to predict arrhythmogenicity in clinical LQTS [43] and previously adapted for use in murine models of LQTS [22] confirmed the above arrhythmogenic tendencies in hypokalemic hearts (Fig.\u00a02). This comprised regular (S1) stimulation at a constant interstimulus interval of 125\u00a0ms interrupted by an extrasystolic (S2) stimulus after every eighth S1 stimulus. The S1S2 interval was decremented in 1\u00a0ms steps with each successive stimulus cycle from an initial value of 120\u00a0ms until the S2 stimulus either appeared to initiate arrhythmic activity, confirmed during an imposed 250\u00a0ms pause, or failed to initiate a MAP suggesting that the VERP has been reached. Accordingly, VERP values are reported to the nearest millisecond.\nFig.\u00a02Arrhythmic activity in hypokalemic hearts after extrasystolic stimulation applied close to the refractory period. Epicardial monophasic action potential recordings resulting from application of extrasystolic (S2) stimuli at S1S2 intervals greater than the action potential duration at 90% repolarization (A), just greater than the ventricular effective refractory period (VERP; B), and just less than the VERP (C) in hearts exposed to normokalemic (5.2\u00a0mM K+, a) and hypokalemic (3.0\u00a0mM K+, b) test solutions and normokalemic (c) and hypokalemic (d) test solutions containing lidocaine (10\u00a0\u03bcM) for 20\u00a0min. Single vertical lines indicate the timing of S1 stimuli, and double lines indicate the timing of S2 stimuli\nNormokalemic hearts (Fig.\u00a02a, A\u2013C) again were consistently free from arrhythmic activity after S2 stimulation after any S1S2 interval (n\u2009=\u200911; eight hearts). This also applied to hypokalemic hearts when S2 stimuli were delivered when MAPs had reached 90% repolarization (Fig.\u00a02b, A, n\u2009=\u20097; five hearts). However, S2 stimuli delivered within the period just after recovery from refractoriness consistently initiated arrhythmic activity under these conditions (Fig.\u00a02b, B). In contrast, S2 stimuli delivered before recovery from refractoriness failed to elicit MAPs, and this was followed by the resumption of stable rhythms (Fig.\u00a02b, C). Finally, S2 stimulation did not result in arrhythmic activity in lidocaine-treated hearts whatever the S1S2 interval, whether under normokalemic (Fig.\u00a02c, A\u2013C, n\u2009=\u20098; six hearts) or hypokalemic (Fig.\u00a02d, A\u2013C, n\u2009=\u20098; five hearts) conditions. When taken together, the presence or absence of arrhythmogenicity in these experiments parallels clinical findings.\nArrhythmic tendency in hypokalemia correlates with increased local critical intervals\nOne hypothesis for the tendency towards either local or transmural re-excitation during action potential repolarization might consider the relationship between the time course of the recovery of membrane voltage and the corresponding time course of recovery of excitability from total refractoriness to a finite threshold for excitation in the myocardial regions concerned. These parameters were approximated by action potential duration at 90% repolarization (APD90) and VERP, respectively. Both these were measured during the procedures of the kind illustrated in Fig.\u00a02, allowing for the delay between endocardial and epicardial excitation where appropriate. Firstly, the risk of local reexcitation of either the epicardium or the endocardium would be reflected in a critical interval given by the relevant APD90\u2013VERP. Secondly, the risk of transmural re-excitation of either the epicardium by the endocardium (or the reverse) would require incorporation of the delay between endocardial and epicardial excitation given by the difference between endocardial and epicardial stimulation to depolarization latencies, \u0394latency. This would give critical intervals of (endocardial APD90\u2009+\u2009\u0394latency\u2212epicardial VERP) and (epicardial APD90\u2009+\u2009\u0394latency\u2212endocardial VERP), respectively.\nFigures\u00a03a and 4a show typical epicardial and endocardial action potential waveforms during regular stimulation under each of the four above conditions (A\u2013D). Figures\u00a03b and 4b show the corresponding APD90s (vertical solid lines and dense hashing), VERPs (vertical broken lines and sparse hashing), and local critical intervals (shading). Asterisks indicate values that are significantly (P\u2009<\u20090.05) larger and daggers those that are smaller than those recorded in normokalemic hearts. Neither epicardial (46.6\u2009\u00b1\u20091.2\u00a0ms) nor endocardial (58.2\u2009\u00b1\u20093.7\u00a0ms) APD90s were significantly different (P\u2009>\u20090.05) from the corresponding VERPs (41\u2009\u00b1\u20094 and 48\u2009\u00b1\u20094\u00a0ms) under normokalemic conditions (Figs.\u00a03A and 4A, n\u2009=\u200910; eight hearts). This resulted in local critical intervals taking small positive values of 5.4\u2009\u00b1\u20094.3\u00a0ms in the epicardium and 9.8\u2009\u00b1\u20095.3\u00a0ms in the endocardium. In contrast, epicardial (53.1\u2009\u00b1\u20090.7\u00a0ms) but not endocardial (56.9\u2009\u00b1\u20094.0\u00a0ms) APD90 increased significantly (P\u2009<\u20090.05), whereas both epicardial (29\u2009\u00b1\u20091\u00a0ms) and endocardial (29\u2009\u00b1\u20092\u00a0ms) VERPs decreased significantly under hypokalemic conditions (Figs.\u00a03B and 4B, n\u2009=\u20096; five hearts). This resulted in significant positive shifts (P\u2009<\u20090.01) in both epicardial (23.7\u2009\u00b1\u20091.2\u00a0ms) and endocardial (28.5\u2009\u00b1\u20094.6\u00a0ms) local critical intervals, in fitting with the occurrence of arrhythmic activity under hypokalemic conditions.\nFig.\u00a03Changes in epicardial action potential duration, ventricular effective refractory period, and local critical interval after exposure to hypokalemia and to lidocaine. Epicardial MAP waveforms during regular stimulation in hearts exposed to normokalemic (5.2\u00a0mM K+, A) and hypokalemic (3.0\u00a0mM K+, B) test solutions and normokalemic (C) and hypokalemic (D) test solutions containing lidocaine (10\u00a0\u03bcM) for 20\u00a0min comparing action potential duration at 90% repolarization; APD90 (vertical solid lines), VERP (vertical broken lines), and local critical interval (shading; a). Action potential duration at 90% repolarization, APD90 (dense hashing), VERP (sparse hashing) and critical interval (shading) under these conditions (b). Asterisks indicate values that are significantly (P\u2009<\u20090.05) larger and daggers those that are smaller than those recorded in normokalemic heartsFig.\u00a04Changes in endocardial action potential duration, ventricular effective refractory period, and local critical interval after exposure to hypokalemia and to lidocaine. Endocardial MAP morphologies during regular stimulation in hearts exposed to normokalemic (5.2\u00a0mM K+, A) and hypokalemic (3.0\u00a0mM K+, B) test solutions and normokalemic (C) and hypokalemic (D) test solutions containing lidocaine (10\u00a0\u03bcM) for 20\u00a0min comparing action potential duration at 90% repolarizationl; APD90 (vertical solid lines), VERP (vertical broken lines), and local critical interval (shading; a). Action potential duration at 90% repolarization, APD90 (dense hashing), VERP (sparse hashing), and critical interval (shading) under these conditions (b). Asterisks indicate values that are significantly (P\u2009<\u20090.05) larger and daggers those that are smaller than those recorded in normokalemic hearts\nThe opposing effects of hypokalemia on APD90 and VERP can be explained in terms of alterations in conductances of repolarizing K+-channels\nThe above MAP findings concerning APD90 and VERP were in close agreement with the predictions of established ion channel equations and parameters from the model of Bondarenko et al. [6] with the Na+\/K+-ATPase model of Hernandez et al. [24] using charge-difference modeling in simulated single cells. The model simulated the effects of regular stimulation at a 125\u00a0ms interstimulus interval until a steady state was reached, as reflected in beat-to-beat stability. Action potential characteristics were then simulated under normokalemic conditions, hypokalemic conditions with normal K+ permeabilities, and hypokalemic condition with the 20% reduction in the permeabilities of channels carrying the repolarizing K+ currents IK1 and Ito (C) as reported in recent experimental results [29] (Fig.\u00a05). Figure\u00a05a demonstrates the predicted steady-state action potential waveforms under each condition. Figure\u00a05a and b also show APD90  (vertical solid lines and dense hashing), VERP (vertical broken lines and sparse hashing), and critical intervals (shading). Under normokalemic conditions (A), the resting membrane potential was \u221283\u00a0mV. APD90 (22\u00a0ms) was shorter than recorded in the whole hearts in keeping with previous results from microelectrode studies [7, 20]. Nevertheless, APD90 was shorter than VERP (27\u00a0ms) resulting in a critical interval of \u22125\u00a0ms. Figures\u00a05B (a and b) demonstrate the consequences of an altered Nernst potential for K+  alone both upon resting membrane potential and the time course of a subsequent action potential. Hypokalemia hyperpolarized the membrane potential (\u221293\u00a0mV) and shortened both APD90 (19\u00a0ms) and VERP (25\u00a0ms), thus having little effect on the limiting criterion for re-excitation (\u22126\u00a0ms). In contrast, Fig.\u00a05C (a and b) additionally demonstrate the combined effects of hypokalemia on both the Nernst potential and K+ permeabilities of the respective channels carrying IK1 and Ito. Although there was no additional effect on the resting membrane potential (\u221293\u00a0mV), hypokalemia increased both APD90 (29\u00a0ms) and VERP (27\u00a0ms) causing a positive shift in critical interval from \u22125\u00a0ms to +2\u00a0ms (Fig.\u00a05C).\nFig.\u00a05Computational modeling of murine ventricular action potentials showing changes in action potential duration, ventricular effective refractory period, and critical interval after exposure to hypokalemia. Action potential morphologies during regular stimulation in cells under normokalemic (5.2\u00a0mM K+, A) and hypokalemic (3.0\u00a0mM K+) conditions comparing action potential duration at 90% repolarization; APD90 (vertical solid lines), VERP (vertical broken lines), and critical interval (shading; a). In B, permeabilities of ion channel are under normokalemic conditions. In C, permeabilities of ion channels carrying the repolarizing K+ currents IK1 and Ito are reduced by 20%. APD90 (dense hashing), VERP (sparse hashing), and critical interval (shading) under these conditions (b)\nThe abolition of arrhythmic tendency by lidocaine correlates with negative shifts in local critical intervals\nExposure of normokalemic hearts to lidocaine (Figs.\u00a03, C and 4C) significantly increased (P\u2009<\u20090.05) epicardial (54.8\u2009\u00b1\u20092.7\u00a0ms), although not endocardial (56.6\u2009\u00b1\u20094.2\u00a0ms), APD90 and significantly increased (P\u2009<\u20090.01) both epicardial (84\u2009\u00b1\u20095\u00a0ms) and endocardial (80\u2009\u00b1\u20092\u00a0ms) VERPs (n\u2009=\u20095; five hearts). This resulted in significant (P\u2009<\u20090.01) negative shifts in local critical intervals in both the epicardium (\u221231.7\u2009\u00b1\u20095.3\u00a0ms) and endocardium (\u221223.4\u2009\u00b1\u20094.7\u00a0ms). Lidocaine exerted concordant effects on hypokalemic hearts (Figs.\u00a03D and 4D): Epicardial APD90 increased to 60.6\u2009\u00b1\u20092.7\u00a0ms, and endocardial APD90 remained unchanged (63.7\u2009\u00b1\u20096.4\u00a0ms), whereas both epicardial (81\u2009\u00b1\u20097\u00a0ms) and endocardial (84\u2009\u00b1\u20094\u00a0ms) VERPs were significantly increased (P\u2009<\u20090.05, n\u2009=\u20096; five hearts). The resulting significant negative shifts (P\u2009<\u20090.01) in local critical intervals in both epicardium (\u221220.4\u2009\u00b1\u20097.5\u00a0ms) and endocardium (\u221220.7\u2009\u00b1\u20097.6\u00a0ms) paralleled the antiarrhythmic effect of lidocaine.\nArrhythmic tendency in hypokalemia also correlates with increased transmural critical intervals\nFigures\u00a06a and 7a show epicardial and endocardial action potential waveforms during regular stimulation under each condition. Figures\u00a06b and 7b show APD90 (vertical solid lines and dense hashing) and VERPs (vertical dotted lines and sparse hashing), as detailed above, together with \u0394latencies (horizontal arrows and horizontal shading) and transmural critical intervals (shading) under the four conditions studied. Asterisks indicate values that are significantly (P\u2009<\u20090.05) larger and daggers those that are smaller than recorded in normokalemic hearts. Figure\u00a06 thus compares endocardial APD90s with epicardial VERPs, allowing for \u0394latency to describe critical intervals for epicardial re-excitation. In contrast, Fig.\u00a07 compares epicardial APD90s with endocardial VERPs, allowing for \u0394latency to describe critical intervals for endocardial re-excitation.\nFig.\u00a06Changes in endocardial action potential duration, epicardial ventricular effective refractory period, transmural conduction time, and epicardial transmural critical interval after exposure to hypokalemia and to lidocaine. Epicardial and endocardial MAP morphologies during regular stimulation cross-comparing epicardial and endocardial waveforms and the relationship between the action potential duration at 90% repolarization; APD90 (vertical solid lines) and VERP (vertical broken lines) of one waveform and the decay of the other, indicating critical intervals (shading). Hearts were exposed to normokalemic (5.2\u00a0mM K+, A) and hypokalemic (3.0\u00a0mM K+, B) test solutions and normokalemic (C) and hypokalemic (D) test solutions containing lidocaine (10\u00a0\u03bcM) for 20\u00a0min. Horizontal arrows indicate the time taken for depolarization to spread from epicardium to endocardium (a). APD90 (dense hashing), VERP (sparse hashing), transmural conduction time (horizontal hashing), and critical interval (shading) under these conditions (b). Asterisks indicate values that are significantly (P\u2009<\u20090.05) larger, and daggers those that are smaller than those recorded in normokalemic heartsFig.\u00a07Changes in epicardial action potential duration, endocardial ventricular effective refractory period, transmural conduction time, and endocardial transmural critical interval after exposure to hypokalemia and to lidocaine. Epicardial and endocardial MAP morphologies during regular stimulation cross-comparing epicardial and endocardial waveforms and the relationship between the action potential duration at 90% repolarization; APD90 (vertical solid lines) and VERP (vertical broken lines) of one waveform and the decay of the other, indicating critical intervals (shading). Hearts were exposed to normokalemic (5.2\u00a0mM K+, A) and hypokalemic (3.0\u00a0mM K+, B) test solutions and normokalemic (C) and hypokalaemic (D) test solutions containing lidocaine (10\u00a0\u03bcM) for 20\u00a0min. Horizontal arrows indicate the time taken for depolarization to spread from epicardium to endocardium (a). APD90 (dense hashing), VERP (sparse hashing), transmural conduction time (horizontal hashing), and critical interval (shading) under these conditions (b). Asterisks indicate values that are significantly (P\u2009<\u20090.05) larger and daggers those that are smaller than those recorded in normokalemic hearts\nEpicardial (21.0\u2009\u00b1\u20090.82\u00a0ms) and endocardial (58.2\u2009\u00b1\u20093.7\u00a0ms) latencies were statistically indistinguishable in normokalemic hearts giving a \u0394latency of 1.6\u2009\u00b1\u20091.4\u00a0ms. This contributed to significant (P\u2009<\u20090.05) negative shifts in transmural critical intervals in both the epicardium and endocardium (\u221214.3\u2009\u00b1\u20096.1 and \u22123.3\u2009\u00b1\u20094.2\u00a0ms, respectively, Figs.\u00a06A and 7A). Hypokalemia had no significant effect (P\u2009>\u20090.05) on latencies or on \u0394latency (1.1\u2009\u00b1\u20091.1\u00a0ms) but resulted in significant (P\u2009>\u20090.01) positive shifts in transmural critical intervals in both the epicardium (23.6\u2009\u00b1\u20092.6\u00a0ms) and endocardium (29.2\u2009\u00b1\u20096.0\u00a0ms; Figs.\u00a06B and 7B).\nThe abolition of arrhythmic tendency by lidocaine also correlates with significantly decreased transmural critical intervals\nIn contrast, exposure of normokalemic hearts to lidocaine significantly increased both epicardial and endocardial latencies (33.8\u2009\u00b1\u20092.6 and 46.3\u2009\u00b1\u20095.6\u00a0ms, respectively) but still did not significantly alter (P\u2009>\u20090.05) \u0394latency (12.5\u2009\u00b1\u20096.2\u00a0ms). However, transmural critical intervals became significantly negative in both the epicardium (\u221215.2\u2009\u00b1\u20096.6\u00a0ms) and endocardium (\u221216.6\u2009\u00b1\u20098.8\u00a0ms; Figs.\u00a06C and 7C). This was also true when hypokalemic hearts were exposed to lidocaine: Both epicardial and endocardial latencies were significantly increased (26.3\u2009\u00b1\u20093.2 and 33.8\u2009\u00b1\u20091.3\u00a0ms, respectively) but \u0394latency was not significantly altered (P\u2009>\u20090.05, 7.6\u2009\u00b1\u20093.4\u00a0ms). Again, transmural critical intervals were significantly decreased (P\u2009<\u20090.05) in both the epicardium (\u221212.8\u2009\u00b1\u20095.9\u00a0ms) and endocardium (\u221210.4\u2009\u00b1\u20091.7\u00a0ms; Figs.\u00a06D and 7D).\nArrhythmogenesis occurs in the absence of significant alterations in the TDR\nThe analysis above thus established four critical intervals, which, when taken together, provided a clear prediction of arrhythmogenicity under these circumstances when changes in APD90 did not correspond to changes in VERP. In contrast, an analysis in terms of TDR, previously shown to predict arrhythmogenicity in both congenital and acquired forms of the LQTS [2], gave insufficiently sensitive predictions. TDR was calculated as the time between stimulation and 90% repolarization in the endocardium minus the time between stimulation and 90% repolarization in the epicardium. TDR values did not significantly alter with the presence or otherwise of arrhythmogenicity (Fig.\u00a08). Thus, in normokalemic hearts (Fig.\u00a08, A) epicardial repolarization time (67.6\u2009\u00b1\u20091.5\u00a0ms) was significantly shorter (P\u2009<\u20090.05) than endocardial repolarization time (77.6\u2009\u00b1\u20093.9\u00a0ms), giving a TDR of 10.0\u2009\u00b1\u20094.2\u00a0ms (n\u2009=\u200910; eight hearts). Epicardial and endocardial repolarization times, as well as the resulting TDR, remained unchanged under hypokalemic conditions (Fig.\u00a08, B). Finally, although treatment with lidocaine significantly increased (P\u2009<\u20090.05) epicardial and endocardial repolarization times under both normokalemic (Fig.\u00a08, C, to 86.1\u2009\u00b1\u20092.8 and 103.0\u2009\u00b1\u20097.0\u00a0ms, respectively, n\u2009=\u20096; five hearts) and hypokalemic (Fig.\u00a08, D, to 86.9\u2009\u00b1\u20094.1 and 97.6\u2009\u00b1\u20096.6\u00a0ms, respectively, n\u2009=\u20095; five hearts) conditions, in neither case did it affect TDR.\nFig.\u00a08Changes in transmural dispersion of repolarization after exposure to hypokalemia and to lidocaine. Epicardial (up-sloping hashing) and endocardial (down-sloping hashing) stimulation to repolarization times, and the difference between these values giving transmural dispersion of repolarization (open bars) in hearts exposed to normokalemic (5.2\u00a0mM K+, A) and hypokalemic (3.0\u00a0mM K+, B) test solutions and normokalemic (C) and hypokalemic (D) test solutions containing lidocaine (10\u00a0\u03bcM) for 20\u00a0min. Asterisks indicate values that are significantly (P\u2009<\u20090.05) larger than those recorded in normokalemic hearts\nThese findings indicate that despite similarities, criteria that have been established to predict arrhythmogenicity in LQTS do not necessarily apply in hypokalemia. However, explicit inclusion of refractory behavior yields novel criteria, which may constitute more sensitive general predictors of arrhythmogenicity and may prove particularly useful in situations where APD90 diverges sharply from VERP. Further, these novel criteria provide a physiological basis for the participation of refractoriness in arrhythmogenicity.\nDiscussion\nIn clinical situations, hypokalemia is associated with arrhythmogenesis initiated by PVDs [52] and accompanied by prolongation of the electrocadiographic QT interval, reflecting increased action potential duration [23]. Furthermore, class 1 antiarrhythmic agents are effective in suppressing arrhythmic activity in hypokalemic patients [42]. These features thus resemble corresponding characteristics of the congenital LQTS [12, 41], where arrhythmic activity is thought to result from re-entrant excitation [2]. This has been attributed to the propagation of depolarization from active cells to previously active adjacent regions subsequently triggering spread of excitation and thus establishing re-entrant circuits [1, 3, 37].\nWe sought to study the physiological basis for the arrhythmogenicity observed under hypokalemic conditions, particularly the extent to which this resembles or differs from the corresponding features of LQTS, in intact isolated perfused murine hearts. Epicardial and endocardial MAPs were first recorded from hypokalemic hearts, confirming that stimulation at a regular interstimulus interval of 125\u00a0ms (S1 stimulation) resulted in stable rhythms. In contrast, at the long intrinsic cycle lengths occurring in the absence of extrinsic stimulation, frequent after-depolarizations were observed and were often followed by the initiation of an arrhythmic activity. This is consistent with the known proarrhythmic effect of bradycardia [10, 13, 46, 51]. Previous studies correlating results from single-cell and whole-heart preparations have attributed such after-depolarizations to inward currents flowing through reactivated voltage-operated Ca2+ channels [27, 35], Ca2+-coupled inward Na+ currents via the Na+\u2013Ca2+ exchanger [47], or Ca2+-induced Ca2+-release from intracellular stores [26]. In the presence of the class 1b antiarrhythmic agent lidocaine, after-depolarizations persisted. These events were especially common in hearts where intrinsic cycle length was particularly long, again in agreement with the known proarrhythmic effect of bradycardia [10, 13, 46, 51]. However, in the presence of lidocaine, these events were never followed by the initiation of arrhythmic activity. Thus, the experiments demonstrated that hypokalemic murine hearts showed arrhythmogenic properties in agreement with clinical findings and established conditions in which arrhythmogenicity was and was not observed.\nA quantitative assessment of MAP waveforms and refractory characteristics associated with this arrhythmogenicity was then performed using an extrasystolic stimulation (S2) procedure previously established in the assessment of arrhythmogenicity in both clinical [43] and murine [22] studies of LQTS. S2 stimulation reproducing the effect of after-depolarizations immediately after recovery from refractoriness failed to initiate arrhythmic activity in normokalemic hearts. However, such stimulation consistently resulted in arrhythmic activity in hypokalemic hearts. This is in agreement with previous reports that after-depolarizations and S2 stimulation early during action potential repolarization are particularly arrhythmogenic [14, 38] and also parallels clinical observations that PVDs coincident with T-waves frequently initiate arrhythmic activity [48]. In contrast, S2 stimuli did not elicit arrhythmic activity in lidocaine-treated hearts, whether studied under normokalemic or hypokalemic conditions. These are consistent with after-depolarizations having occurred late in action potential repolarization, and thence, failing to initiate arrhythmic activity.\nPrevious studies in murine [30], canine [15], and human [31] ventricles have consistently reported that maneuvers, which alter action potential duration, also produce corresponding changes in refractory period, with the notable exception of exposure to class 1 antiarrhythmic drugs [39]. However, the effect of isolated reduction in [K+]o on these parameters has not been studied. Exploration of the effect of varying S1S2 interval demonstrated for the first time that although action potential duration (quantified at 90% repolarization, APD90) was increased in hypokalemia, VERP was decreased. Furthermore, exposure to lidocaine had no effect on APD90 in the epicardia or endocardia of normokalemic hearts and significantly increased APD90 only in the endocardia of hypokalemic hearts, despite significantly increasing VERP in all cases. Thus, the proarrhythmic effect of hypokalemia was associated with recovery from refractoriness occurring earlier in action potential repolarization, whereas the antiarrhythmic effect of lidocaine was associated with recovery from refractoriness occurring later in action potential repolarization.\nReduction of [K+]o might be expected to increase outward K+ currents and thereby decrease APD90. Indeed, computer modeling of single ventricular myocytes confirmed that reduction of [K+]o per se resulted in decreased APD90. Furthermore, the Nernst equation would predict that reduction of [K+]o should hyperpolarize the resting membrane potential, thereby increasing the proportion of sodium channels available for activation [19] and decreasing the VERP: our model replicated this effect. However, incorporation of recent data from our group demonstrating that reduction of [K+]o decreases the repolarizing K+ currents IK1 and Ito [29] resulted in VERP returning to its normokalemic value and APD90 being increased beyond its normokalemic value. Thus, although reduction in K+ permeability compensates for the change in VERP, it overcompensates for the change in APD90, rendering the APD90 longer than the VERP, in fitting with experimental results and with the proarrhythmic effect of hypokalemia. Previous studies have reported that although exposure to lidocaine increases VERP through an effect on the gating of fast Na+-channels [32, 34], it has a proportionately smaller effect on action potential duration [5]. Exposure to lidocaine is thus established to result in postrepolarization refractoriness [39], in fitting with experimental results and with the antiarrhythmic effect of lidocaine.\nWe then applied an analytical scheme to provide a simple physiological explanation for these findings. Subject to electrotonic coupling between cells [25], the simplest condition for local re-excitation between adjacent cells within either epicardium or endocardium would require membrane potential to exceed threshold at some point during action potential repolarization, the recovery of membrane potential lagging behind the recovery of excitability. Recovery of excitability was approximated by the VERP, measured using a standard stimulus of consistent amplitude and duration. Recovery of membrane potential was approximated by the action potential duration at 90% repolarization (APD90). Accordingly, APD90\u2212VERP gives a critical interval that reflects tendency towards local re-excitation and arrhythmogenicity. Positive shifts in this interval would reflect a relatively proarrhythmic state, whereas negative shifts in this interval would reflect an antiarrhythmic state. The corresponding analytical condition for transmural re-excitation across the thickness of the myocardial wall with a transmural conduction time given by \u0394latency [40] yields transmural critical intervals of (endocardial APD90\u2009+\u2009\u0394latency\u2212epicardial VERP) for the epicardium and (epicardial APD90\u2009+\u2009\u0394latency\u2212endocardial VERP) for the endocardium, again subject to electrotonic spread of current between cells [25]. Although exposure to lidocaine increased both epicardial and endocardial stimulation to depolarization latencies, attributable to its established effect on conduction velocity [8], it had no significant effect on \u0394latency. Hypokalemia resulted in significant positive shifts in the magnitude of all four critical intervals, whereas exposure to lidocaine resulted in significant negative shifts, in precise agreement with the presence or absence of arrhythmogenicity. Modelling of single ventricular myocytes predicted shorter APD90s than were recorded from whole-heart preparations, in agreement with previous experimental observations in such single-cell preparations [7, 20, 30]. Nevertheless, hypokalemia when similarly modeled for such single-cell preparations resulted in a positive shift in critical interval, in common with our experimental observations from whole hearts.\nAlthough changes in all four critical intervals correlated with arrhythmogenicity, TDR, previously shown to predict arrhythmogenicity in LQTS [36, 44, 45], proved an insufficiently sensitive predictor. This finding may be attributable to sharp differences between APD90 and VERP: it is possible that APD90 and VERP were in close agreement in previous studies on LQTS [49, 50]. In the latter event, the present analysis would have yielded identical results to one adopting TDR.\nThus, we establish for the first time that hypokalemia decreases VERP despite increasing APD90 and attribute this surprising finding to effects of reduced [K+]o on ion channel gating. Secondly, we establish novel indices incorporating VERP as general criteria for re-entrant arrhythmogenicity that additionally provide a physiological basis for the association between changes in epicardial and endocardial VERP and APD90 and susceptibility to arrhythmogenesis. Thirdly, we establish that such analyses provide more sensitive indications of arrhythmogenicity than previous analyses invoking TDR.","keyphrases":["action potential duration","refractory period","conduction time","transmural dispersion of repolarization","critical intervals","arrhythmia"],"prmu":["P","P","P","P","P","U"]}
{"id":"Histochem_Cell_Biol-4-1-2228382","title":"Morphogenesis of post-Golgi transport carriers\n","text":"The trans-Golgi network (TGN) is one of the main, if not the main, sorting stations in the process of intracellular protein trafficking. It is therefore of central importance to understand how the key players in the TGN-based sorting and delivery process, the post-Golgi carriers (PGCs), form and function. Over the last few years, modern morphological approaches have generated new insights into the questions of PGC biogenesis, structure and dynamics. Here, we present a view by which the \u201clifecycle\u201d of a PGC consists of several distinct stages: the formation of TGN tubular export domains (where different cargoes are segregated from each other and from the Golgi enzymes); the docking of these tubular domains onto molecular motors and their extrusion towards the cell periphery along microtubules; the fission of the forming PGC from the donor membrane; and the delivery of the newly formed PGC to its specific acceptor organelle. It is now important to add the many molecular machineries that have been described as operating at the TGN to this \u201cmorphofunctional map\u201d of the TGN export process.\nIntroduction\nThe Golgi complex serves as the central station in the biosynthetic pathway, where proteins are sorted for their different destinations, such as various domains of the cell surface and the endosomal\u2013lysosomal system. This delivery of cargo proteins from the Golgi complex to their target compartments is carried out by dynamic, membrane-bound organelles that are frequently called either \u201ctransport carriers\u201d or \u201cpost-Golgi carriers\u201d (PGCs) (Luini et al. 2005). As these PGCs have an important role in the process of intracellular transport, their morphology, living dynamics and molecular compositions have become the focus of significant interest, particularly over the last few years.\nThese PGC transport organelles were originally defined through the development of green fluorescent protein (GFP) technology and living-cell imaging (Lippincott-Schwartz et al. 2000), whereby the first fluorescent cargo protein that was followed in living cells revealed a new world of images of these relatively large and highly dynamic structures that travel from the Golgi complex to the plasma membrane (Wacker et al. 1997; Hirschberg et al. 1998; Nakata et al. 1998). Then, with time, the list of cargo molecules that could be visualized in vivo expanded, further exposing the unexpected complexity of the post-Golgi transport pathways. Finally, further technical advances resulted in the combination of video and electron microscopy (correlative light-electron microscopy, CLEM), which provided the means to define the morphogenesis of these PGCs at the ultrastructural level.\nAs a result of these studies we now know that PGCs arise from specific membrane domains of the Golgi complex that lack resident Golgi enzymes, forming what are known as the \u201cPGC precursors\u201d (Hirschberg et al. 1998; Keller et al. 2001; Polishchuk et al. 2003; Puertollano et al. 2003). The shapes and sizes of PGCs that even carry the same cargo can vary across a wide range, and under the light microscope, most of them are seen to be clearly larger that plasma-membrane associated clathrin vesicles and 100-nm fluorescent beads (Hirschberg et al. 1998). The smaller PGCs usually have a size of 300\u2013400\u00a0nm, although some of the larger ones can reach several microns in length. Video microscopy has also revealed that many of these carriers appear globular, although they frequently stretch out into tubular shapes during their translocation through the cytosol; thus PGCs have frequently been termed as \u201cpleiomorphic\u201d structures.\nIn transporting their cargoes to the correct acceptor compartments, the movement of PGCs is mediated via microtubules. However, carriers can also form and support post-Golgi transport without microtubules, although the correct targeting of their cargo proteins is usually compromised under these conditions (Rindler et al. 1987; Kreitzer et al. 2003). Finally, the life cycle of a PGC can be schematically imagined to consist of three stages: (1) its formation (which can in turn be further divided into several substages; see below); (2) its transition through the cytosol; and (3) its docking and fusion with the target membrane (Polishchuk et al. 2000). The first of these steps, the formation of the PGC, appears to be the most complex, and it also probably remains the least understood.\nSince the question of the morphogenesis of a PGC is closely intertwined with that of the structure of the organelle from which it originates, the trans-Golgi network (TGN), both of these issues will now be discussed in an integrated fashion.\nThe trans-Golgi network and the origin of PGCs\nThe process of PGC morphogenesis has been characterized in detail using video and electron microscopy, as well as with the combination of these techniques (CLEM). As indicated above, this process comprises the formation, the extrusion and the fission of the export domain from the TGN, thus generating the free carrier (Polishchuk et al. 2003; see also Fig.\u00a01). The first step (the formation of the tubular export domains) also includes the segregation of the cargo proteins from the Golgi resident enzymes. These events appear to be common to the proteins directed towards different post-Golgi compartments, such as basolateral plasma membrane (Hirschberg et al. 1998; White et al. 2001; Polishchuk et al. 2003), the apical plasma membrane (Keller et al. 2001) and the endosomal\u2013lysosomal system (Puertollano et al. 2003). These export domains usually contain TGN markers; i.e. they are part of the TGN (Polishchuk et al. 2003; Puertollano et al. 2003). Of note, these three post-Golgi compartments are the ones that have been best characterized for the PGCs leaving the TGN, with the total number and types of pathways for cargo exit from the TGN at present not known, although there are at least six, and probably more (Rodriguez-Boulan et al. 2005).\nFig.\u00a01Formation of post-Golgi transport carriers. Subsequent frames extracted from a time-lapse sequence illustrating the different stages of PGC biogenesis (arrows): a formation of the tubular domain containing the cargo VSVG-YFP and devoid of the Golgi-resident protein galatosyltransferase-CFP. b Extrusion of this tubular domain from the Golgi complex. c, d Fission of the domain from the parental Golgi membranes. e Thin section of a cell expressing the TGN38-HRP construct. Arrows indicate tubular PGC precursor that has been pulled out of the TGN area of the Golgi complex\nAlthough the existence of the TGN has been known for many years (Griffiths and Simons 1986), its precise structure and identity still remain to be ascertained. In one view, which predominated in the field until recently, the TGN was considered to consist of essentially an anastomosing tubular network that emanates from (or that results from the breakdown of) the trans-most Golgi cisterna, and that projects mainly in the trans direction (Griffiths et al. 1989, 1985; Clermont et al. 1995). Rambourg and colleagues have thoroughly described the TGN in several different cell types. In these descriptions, this trans-most cisterna also tends to \u201cpeel off\u201d from the rest of the Golgi stack, and together with the other morphological characteristics of the Golgi stack, this is a feature that is suggestive (Clermont et al. 1995) of the cisternal progression-maturation trafficking model (Bannykh and Balch 1997; Mironov et al. 1997; Bonfanti et al. 1998; Losev et al. 2006; Matsuura-Tokita et al. 2006). Thus, in a simple version of this scheme, the TGN would result from the final stages of maturation of the Golgi cisternae. This would include the partial transformation of this trans-most cisternae into a tubular network, and the various carriers that leave the TGN for their different destinations would all originate form different domains of this tubular network.\nA more complex and more recent view of the organization at the trans face of the Golgi complex comes from studies using electron microscopy tomography of cryofixed, freeze-substituted cells (Ladinsky et al. 1999, 2002). These studies have indicated that the TGN derives not only from the last trans cisterna, but instead from the three trans-most cisternae (Roth et al. 1985), from where tubules emanate into the trans space of the Golgi stacks. Remarkably, only the trans-most cisterna exhibits clathrin-coated buds, with the other buds appearing to have different, yet-to-be identified, types of coats (Ladinsky et al. 1999). Thus, only the trans-most cisterna would be responsible for the clathrin- and AP-1-dependent trafficking towards the endo-lysosomes, and although all of these three trans-most cisternae of the Golgi stacks would serve as the classical TGN, each of them could be specialized in the packaging and export of specific cargo proteins. This might have important functional and mechanistic implications for the proteins sorting and export process.\nAnother remarkable TGN feature that has been revealed by these tomographic studies is that these three trans-most cisternae can intercalate with the cisternae of the endoplasmic reticulum (ER) (Ladinsky et al. 1999, 2002). These have been proposed to have important roles in the transfer of lipids (through specialized contact sites) between the ER itself and the Golgi complex.\nIt will be important to establish whether the morphological discrepancies described above reflect fundamental differences in the organization of the export of cargo in different cell systems. Indeed, it is possible that the first model (the TGN as a network resulting from the tubular disassembly of the last trans-most cisterna) might actually only represent a special case of the more complete three-trans-cisterna model, and that both of these two different models of TGN may apply, depending on the cell type and the functional conditions.\nIn support of this possibility, it is clear that the TGN can vary significantly in both size and composition across different cell types. For example, cells with a well-developed endo-lysosomal system and without secretory granules exhibit an extensive tubular TGN, while the tubular component of the TGN is reduced in cells that are specialized in regulated secretion (Clermont et al. 1995). This appears to occur in the latter because most of the TGN membranes are used for the packaging of secretory granules, and the tubules only form the thin bridges between the granule precursors. Such an organization simplifies the release of the granules, which occurs through the rupture of these tubular elements (Clermont et al. 1995). Similarly, human fibroblasts have large collagen-containing distensions that are connected to tubular elements in the trans-Golgi area (Polishchuk et al. 2003). Thus, the morphology and size of the TGN apparently depend on the predominant type and amount of cargo protein departing from the Golgi complex.\nThe morphology of the TGN depends also on its secretory status. When exit from the TGN is blocked by a lowering of the temperature to 20\u00b0C in the presence of abundant cargo (Matlin and Simons 1983), the volume and surface areas of the TGN increase greatly (Griffiths et al. 1989); while in the absence of cargo, the 20\u00b0C block results in all three of the trans-most cisternae producing bulging exit domains (Ladinsky et al. 2002). At physiological temperatures, three-dimensional (3D) analyses of the Golgi stacks have revealed well developed, tubular\u2013reticular, TGN-like membranes at the trans side of the Golgi complex in actively secreting cells (Trucco et al. 2004), while the TGN is nearly absent in quiescent cells (Trucco et al. 2004). Thus, the TGN is a very dynamic structure, the shape and size of which is potently regulated by the extent of traffic flowing through it. To discover the regulatory mechanisms is an important challenge for the future.\nAnother challenge will be to define whether the three-cisterna organization really underlies a functional specialization of these cisternae in the sorting and packaging of different cargo classes, as suggested by the presence of clathrin buds only on the last cisterna of the Golgi stack. So far, the lack of immunolabeling studies does not allow determining whether different trans cisternae really do contain different transport proteins.\nIn addition to being an organelle of the biosynthetic pathways, the TGN is involved in endocytic transport routes (Griffiths and Simons 1986; Mellman and Simons 1992; Pavelka et al. 1998). Several studies with different cell types showed that the TGN and Golgi stacks can contain plasma-membrane constituents and internalized materials (Stoorvogel et al. 1988; van Weert et al. 1997). These exchanges with the endocytic systems could well introduce a further level of structural complications, since the recycling endosomes, in particular, reside in the Golgi area and are tubular in nature. In conclusion, both the dynamics and the structure of the TGN are variable and incompletely defined, thereby presenting an additional layer of difficulty to our understanding of the biogenesis of the carriers that depart from this organelle.\nFormation of PGC precursors at the TGN\nThere are two key aspects to the formation of the PGC precursors: their morphogenesis at the TGN; and the segregation between different types of cargo proteins that are targeted for different destinations, as well as between the Golgi-resident proteins, such as the Golgi enzymes.\nRegarding PGCs morphogenesis, in principle, the tubular carrier precursors might form through the mechanical pulling force exerted by microtubule-based motors on a flatter, parent, membrane domain (e.g., a Golgi cisterna). This has been shown to be mechanistically possible for both artificial and natural membranes (Roux et al. 2002). If this is the case, the PGC precursors would be expected to be essentially simple linear tubular structures (Roux et al. 2002). The second possibility is that these precursors are actually formed from the tubular subdomains of the TGN, which are generated prior to docking and extrusion by microtubules. In this case, the GPCs should reflect the morphologically complex structure of their parent membranes.\nRecent studies have provided compelling support in favor of the latter model (Polishchuk et al. 2003). First, both free PGCs and their precursors at the TGN comprise complex tubulo-reticular structures, which have often been described as the main component of the TGN in many cell types (Clermont et al. 1995). For example, PGCs carrying VSVG show a mostly tubular morphology, and can have a complex structure that even contains clearly visible fenestrae (Polishchuk et al. 2003); this would be expected of membranes that derive from protrusions of the TGN. Indeed, PGC precursors that have been visualized using CLEM, appear to be comprised of tubular segments that are interconnected with complex branching and fenestrated membranes; they are also seen to be continuous with the parent membranes of the Golgi stack (Polishchuk et al. 2003). Similarly, carriers containing the apical cargo protein hemagglutinin (HA) frequently have a tubular morphology as well as HA-positive domains at the TGN (Puertollano et al. 2001). Thus structural similarities between PGC precursors and the TGN appear to be a common feature of different types of PGCs. This strongly suggests that PGCs form via the fission of an entire precursor domain (or a large part thereof) from the rest of the TGN membranes.\nThus the question regarding the mechanism by which the originally flat Golgi membranes are converted into highly bent, tubular\u2013reticular TGN structures arises. Essentially, this can be achieved by one of two mechanisms: either by the action of proteins that can bend these membranes into tubules, in a manner that is possibly similar to that of amphiphysin, endophilin, sorting nexins and others (Antonny 2006), or via the alteration of the lipid composition of these trans-Golgi membranes, which can be mediated, in turn, by several processes. For instance, changes in lipid content, and hence membrane curvature, can be modulated via the lipid-metabolizing enzymes that reside at the Golgi complex (for review, see De Matteis and Godi 2004; Luini et al. 2005). The transmembrane or inter-organelle transfer of lipids can contribute to the generation of particular lipid environments in the membranes of the TGN. In this respect, it is important to note that numerous contact sites between the ER and the trans-cisternae of the Golgi complex have been detected by electron microscopy tomography (Ladinsky et al. 1999, 2002). Such contact sites can be explored for lipid transfer between the ER and trans-Golgi that is mediated by specific lipid-transfer proteins. Also, since the cisterna-like morphology of Golgi compartments can be stabilized by large polymers of Golgi enzymes (Nilsson et al. 1996), the loss of oligomerized Golgi enzymes in the trans-Golgi should, in principle, favor cisterna-to-tubule transformation of cargo-containing membranes. Moreover, this process can be accompanied by the loss of the stacking mechanisms at the trans side of the Golgi complex. Here, GRASP65 and GRASP55 are two proteins that have been suggested to be involved in the maintenance of cisterna juxtapositioning, and they are located mainly at the cis and medial Golgi, rather than the trans-Golgi (Barr et al. 1997; Shorter et al. 1999). So this intercisternal \u201cglue\u201d may be gradually lost as a cisterna progresses towards the trans pole of the Golgi complex. This has been confirmed both in mammals and yeast by the observation that the trans-most cisterna frequently peels off from the main Golgi stack (Clermont et al. 1995; Mogelsvang et al. 2003). It is possible that more than two or more mechanisms act in synergy to provide this transition from flat Golgi cisternae to the tubulo-reticular TGN morphology.\nIn addition to the formation of tubular domains at the exit face of the Golgi complex, this is also the level at which cargo proteins that are directed to different post-Golgi destinations should be sorted. The classical view in the membrane transport field implies that sorting at the TGN (as well as throughout the whole secretory pathway) is driven mainly by the coat-adaptor-protein machinery, which interacts specifically with amino-acid signals of certain transmembrane cargo proteins; this then provides the mechanical force for budding and fission of transport vesicle (Mellman and Warren 2000).\nThis holds true for the endo-lysosome-directed carriers that have been thoroughly characterized. These carriers consist of clusters of clathrin-coated buds that are connected by tubular regions, and thus exhibit a grape-like structure. In contrast, PGCs carrying a cargo like the G-protein of vesicular stomatitis virus (VSVG) form in a coat and an AP-independent manner. Both PGCs and their precursors do not show \u03b2-COP or \u03b3-, \u03b4- and \u03b5-adaptins at their membranes (Polishchuk et al. 2003). Other adaptors, such as the GGAs, are excluded from VSVG carriers as well (Puertollano et al. 2003; Polishchuk et al. 2003). Similarly, coats and adaptors have also never been detected on PGCs that are carrying proteins to the apical surface in polarized cells (Kreitzer et al. 2003). Thus, these carriers should form either by virtue of some still-unknown adaptors that cannot yet be visualized by electron microscopy, or by their association with specific lipid microdomains that are involved in sorting (Schuck and Simons 2004). This might be the case for proteins directed to the apical surface in polarized epithelial cells, the concentration of which at the TGN appear to be through their partition into cholesterol- and sphingolipid-rich membrane domains that are known as \u201crafts\u201d (Schuck and Simons 2004).\nThe scission of PGCs from the TGN\nThe morpho-dynamics of the fission process are also fairly complex. Observations in living cells have revealed that the fission of PGCs frequently coincides with mechanical pulling of carrier precursor from the TGN along microtubules. Apparently, the pulling force which the molecular motors such as kinesin (see below) can apply to the TGN membranes is important to facilitate the extension of PGC precursors from the Golgi body and for the later fission of the PGC (Kreitzer et al. 2000; Polishchuk et al. 2003). In cell-free systems, the addition of kinesin to Golgi membranes (and even to liposomes) together with microtubules induces the formation of tubule-like membranes that are similar to PGC precursors (Roux et al. 2002), while a block of kinesin function by microinjection of an inhibitory antibody (Kreitzer et al. 2000) or expression of the headless kinesin mutant (Nakata and Hirokawa 2003) prevent PGC formation from the Golgi complex. Kinesin has been seen to be associated with the tip of PGC precursors, although it can also attach to other points along the PGC precursor membrane (Polishchuk et al. 2003). The movement of kinesins along microtubules can then create tension within the PGC precursor that will facilitate the fission process. Indeed, based on in vitro data, membranes under tension have recently been proposed to have an important role in fission (Roux et al. 2006). However, PGCs can also form when microtubules have been destroyed by nocodozole treatment; in this case, the pulling force to create membrane tension in fission-prone regions might be provided by actin motors (Warner et al. 2003; Sahlender et al. 2005).\nLive cell imaging and CLEM have also shown that fission does not take place randomly along the membranes of PGC precursors. In this case, which regions of a PGC precursor can be defined as prone to fission? First, this depends on the geometry of the precursor membranes. Our data suggest that fission usually takes place at the thinnest parts of the PGC precursor (Fig.\u00a02a), which at the electron microscopy level corresponding to thin tubular segments of membranes (Polishchuk et al. 2003). In contrast, fission does not take place at the TGN regions with a complex morphology (i.e., in those containing tubular networks and branching tubules, or in thick vacuolar regions). Obviously, the precise points of fission will define not only the compositions of the PGC carriers, but also their morphology. If fission occurs close to the tip of the TGN tubule, a carrier will be smaller in size. In contrast, larger PGCs can form by cleavage at the base of the PGC precursors (Fig.\u00a02a). Similarly, endosome-directed PGCs can apparently detach from the TGN as simple cargo-containing vesicles if the fission occurs at the neck of the clathrin-coated buds (Fig.\u00a02b). However, many clathrin-positive PGCs have a grape-like morphology (tubule with several buds), suggesting that entire chunks of TGN membranes containing 2\u20133 clathrin-coated buds can be cleaved from the Golgi complex (Polishchuk et al. 2006).\nFig.\u00a02Fission of post-Golgi transport carriers. a TGN precursors of post-Golgi carriers are pulled along microtubules by kinesin. The fission (red line) of the carriers occurs at the thinnest parts of the PGC precursor, which correspond to thin tubular segments of the TGN membrane at the electron microscopy level. In contrast, fission does not take place at the TGN regions with a complex morphology (i.e., containing tubular networks and fenestrae, or in thick vacuolar regions). If fission occurs close to the tip of a PGC precursor, the carrier will be smaller in size (1). In contrast, larger PGCs can be formed by cleavage at the bottom of a PGC precursor ( 2). b PGCs directed to endosomes detach from the TGN as simple clathrin-coated vesicles if fission (red line) occur at the neck of the clathrin-coated bud (1). Alternatively, entire chunks of the TGN membrane containing 2-3 clathrin-coated buds can be cleaved from the Golgi complex\nAnother factor that might be important for PGC shaping is the type of cargo that is embedded in the PGC. For example, procollagen-I usually forms quite large aggregates that are visible within 300\u2013400-nm-diameter membrane distensions of the Golgi membranes. As a consequence, similar distensions have been detected in most collagen-containing PGCs (Polishchuk et al. 2003; Canty et al. 2004).\nMovement of PGCs from the Golgi complex to the target membrane\nAfter fission from the TGN, PGCs move to the acceptor membrane. Different members of the kinesin superfamily (Kamal et al. 2000; Nakata and Hirokawa 2003; Teng et al. 2005), and also the other microtubule motor dynein (Tai et al. 1999), have been shown to drive post-Golgi transport of specific cargo to various destinations. This high fidelity of cargo selection by molecular motors at the TGN and their further delivery to the correct surface or intracellular domain may be regulated by interactions of motor protein directly with the cargo (Kamal et al. 2000; Teng et al. 2005) or with components of the sorting machinery at the TGN (Nakagawa et al. 2000).\nAs an example, transport of HA and annexin 13b to the apical surface in epithelial cells relies on raft-associated motor KIFC3 (Noda et al. 2001). KIF13A operates in the other post-Golgi route that is used for the transport of the mannose-6-phosphate receptor (Nakagawa et al. 2000). A number of neuronal proteins, including bAPP, GAP43 and vamp-2, require KIF5 for their correct targeting (Nakata and Hirokawa 2003), while the microtubule minus-end-directed motor dynein has been shown to support rhodopsin transport in rod photoreceptors (Tai et al. 1999; Yeh et al. 2006).\nSelection of specific cargoes by motors could be driven by various mechanisms. The simplest would use a direct interaction between the motor and a specific domain of a cargo protein, as is seen for the dynein light chain and the cytoplasmic tail of rhodopsin (Tai et al. 1999). Alternatively, adaptor proteins could serve as a bridge between a motor and its cargo. For example, KIF13A transports the mannose-6-phosphate receptor through its interaction with the AP-1 complex (Nakagawa et al. 2000). Finally, both motor and cargo could associate with the same specific lipid microdomain, as for instance, for KIFC3 and the apically targeted annexin XIIIb.\nAnother issue that needs to be addressed is whether any of these sorting processes take place in the PGCs while they are moving toward their acceptor compartment. This happens, for example, with the maturation of secretory granules when the mannose-6-phosphate receptor is concentrated and sorted from the secretory granules by clathrin-coated vesicles (Klumperman et al. 1998). So several strategies have been used to determine whether similar sorting events happen with PGCs. Mature exocytic carriers can be arrested before their fusion with the plasma membrane either by microinjection of an anti-NSF antibody or by treatment with tannic acid (which fixes the plasma membrane but does not penetrate inside the cell). In contrast to secretory granules, the comparison of mature and newly formed PGCs did not reveal significant changes in either their ultrastructure or their composition (Polishchuk et al. 2003, 2004). Similarly, mature Golgi-to-endosome carriers were accumulated in cells upon endosome inactivation. However, they did not show any significant transformation, except for a very moderate reduction in the area covered by clathrin (Polishchuk et al. 2006).\nLive-cell imaging of subconfluent MDCK cells has shown that PCGs that initially contained both a basolateral marker (VSVG-CFP) and an apical marker (GPI-YFP) did not sort out either of these cargoes into any separate structures, and instead delivered both of the proteins to the plasma membrane (Polishchuk et al. 2004). GPI-GFP was then sorted from the basolateral surface to the apical membrane through transcytosis (Polishchuk et al. 2004). On the other hand, the partitioning of two proteins from a common PGC into separate carriers has also been reported (Jacob and Naim 2001). This suggests that sorting from the PGC may exist, but that it should depend on the nature of the cargo proteins being transported.\nIntermediate stations in post-Golgi transport\nThe complexity of sorting events in the post-Golgi space appears to be even worse since the discovery that certain cargoes may pass through the endosomal compartments before their arrival at the plasma membrane. Sporadic reports in the past have suggested that some secretory proteins do not move from the Golgi complex directly to the plasma membrane, but instead pass through an endocytic intermediate on their way to the cell surface (Leitinger et al. 1995). This indirect \u201cthrough-endosome\u201d delivery of cargo to the plasma membrane might be also facilitated by the intimate association of TGN membranes with number of endocytic compartments in the perinuclear area of the cell (Marsh et al. 2001). The list of the proteins using this pathway has been recently updated, and it has now been shown that in MDCK epithelial cells, VSVG, the LDL receptor and E-cadherin can be detected in the endosomes before their exit to the plasma membrane (Ang et al. 2004; Lock and Stow 2005).\nThese findings, however, promote a number of further questions. The first is whether this transport route is ubiquitous (i.e., does it exist in different cells?). The second question is whether different cargoes move through the same endosomal compartment on their way to the cell surface in epithelial cells? A number of proteins (such as VSVG and the LDL receptor) have been reported to use a Rab8-positive sub-population of endosomes as an intermediate station on their way to the basolateral membrane in epithelial cells (Ang et al. 2003). These proteins require the AP-1B adaptor complex to be properly sorted from these Rab8 endosomes towards the basolateral surface domain (Ang et al. 2003). Other cargoes (such as E-cadherin, for example) move to the plasma membrane through a Rab11 endocytic compartment (Lock and Stow 2005). It remains to be determined, however, whether there is any cross-talk between these Rab8- and Rab11-dependent routes. This possibility apparently exists, since Rab8 and Rab11 endosomes are both accessible to transferrin (Ang et al. 2004; Lock and Stow 2005).\nFinally it is important to clarify as to what extent this through-endosome transport route is used by different cargo proteins in epithelial cells. This issue has been partially addressed by the silencing of the \u03bc1B subunit of the AP-1B adaptor complex, which resides at the endosomes and is required for the correct delivery of many basolateral proteins. The use of RNA interference has revealed, however, that a number of cargoes (such as transferrin and Fc receptors, for example) can be targeted correctly even in the absence of AP-1B, which suggests that these proteins can move directly from the Golgi complex to the basolateral surface without crossing the endocytic routes (Gravotta et al. 2007). The existence of more than one basolateral pathway has also been supported by the observation that transport of different basolateral proteins can be selectively regulated by different PKD isoforms (Yeaman et al. 2004). Thus, further efforts need to be made to understand to what extent an endocytic post-Golgi compartment is involved in the sorting and transport events of cell-surface proteins.\nConclusions\nThe extensive characterization of PGC morphology by video and electron microscopy has provided a framework for the positioning of the molecular machineries in the morpho-functional maps of various trafficking segments. At the same time, many molecular players in TGN-to-plasma membrane transport have been identified. The attribution of each of the molecular components to each of the pathways is probably now the main challenge. We believe that the development of specific assays will significantly assist in the achieving of this objective. Video microscopy of GFP-tagged cargo proteins has allowed us to evaluate the process of PGC formation, as well as the speed and directionality of PGC movement through the cytosol. Other assays use tannic acid treatment (Polishchuk et al. 2004; Jakob et al. 2006) or horse-radish-peroxidase-based endosome immobilization (Ang et al. 2004; Polishchuk et al. 2006) to prevent the fusion of PGCs with their target membranes. These conditions allow us to trap mature PGCs and to compare their compositions to newly formed carriers and the TGN membranes. Moreover, preferential sites of PGC docking and fusion can also be easily identified in this way. The combination of these experimental approaches with specific molecular inhibitors now allows us to attribute a protein of interest to one of the steps in post-Golgi transport.","keyphrases":["post-golgi transport","trans-golgi network","post-golgi carriers","golgi complex"],"prmu":["P","P","P","P"]}
{"id":"Neuroimage-2-1-2330063","title":"Tractography of the parahippocampal gyrus and material specific memory impairment in unilateral temporal lobe epilepsy\n","text":"Introduction Temporal lobe epilepsy (TLE) is associated with disrupted memory function. The structural changes underlying this memory impairment have not been demonstrated previously with tractography.\nIntroduction\nTemporal lobe epilepsy (TLE) is the most common form of refractory focal epilepsy (Crawford, 2000). Parahippocampal structures, which are critically implicated in the generation and propagation of seizures in TLE (Avoli et al., 2002; Bertram, 2006; Du et al., 1993; Plate et al., 1993; Rutecki et al., 1989; Spencer and Spencer, 1994), are essential for declarative memory (Eichenbaum, 2000). Longitudinal neuropsychological studies have shown that persisting epilepsy is associated with progressive memory impairment (Dodrill, 2002; Helmstaedter et al., 2003). Those who undergo anterior temporal lobe resection (ATLR) are at further risk of memory impairment, the nature of which depends on whether surgery is on the dominant or non-dominant side (Ivnik et al., 1987; Spiers et al., 2001).\nNeuropsychological assessment, quantitative MRI, and latterly functional MRI (fMRI) indicate the role of the medial temporal lobe structures (MTL) in sustaining material specific memory functions (Powell et al., 2005), and the reorganisation of memory that occurs with TLE (Powell et al., 2007a). These and lesion deficit studies (Frisk and Milner, 1990; Smith and Milner, 1981) have shown that memory deficit after ATLR is related to the functional integrity of the parahippocampal structures. The strength of connections, or structural connectivity, of the parahippocampal gyrus in TLE has not been evaluated, or related to function.\nTractography is a technique which uses diffusion tensor imaging (DTI) data to delineate white matter tracts, and to quantify their volume, and infer structural characteristics (Johansen-Berg and Behrens, 2006). Using one such tractography technique, Probabilistic Index of Connectivity or PICo (Parker et al., 2003; Parker and Alexander 2003), we evaluated the structural connectivity of the parahippocampal gyrus in TLE, with the hypothesis that this would be impaired ipsilaterally to the seizure focus, and that the degree of any such impairment would correlate with material specific memory function.\nMethods\nSubjects\nWe studied 18 patients (median age 33.5\u00a0years; range 22\u201347\u00a0years; 11 males) with medically refractory TLE undergoing pre-surgical evaluation at the National Hospital for Neurology and Neurosurgery, London, UK. All patients had undergone structural MRI at 1.5T (Duncan, 1997). Of the eight left TLE patients, seven had hippocampal sclerosis (HS) (one also had a ganglioglioma in the left fusiform gyrus) and one had a MTL dysembryoblastic neuroepithelial tumour (DNET). Of the ten right TLE patients, seven had HS, one had a right MTL glioma, another had a right MTL DNET, and the other had right superior temporal focal cortical dysplasia (FCD). Video-EEG had confirmed seizure onset in the MTL ipsilateral to the clinically defined seizure site, and all patients had a normal, contralateral hippocampus based on qualitative and quantitative MRI criteria (Woermann et al., 1998).\nAll patients were on anti-epileptic medication, and were fluent English language speakers. Handedness was determined using the Edinburgh handedness inventory (Oldfield, 1971), and language dominance was determined using a range of fMRI tasks which have been described previously and include the use of verbal fluency, and reading tasks (Powell et al., 2006). All patients underwent a standardised pre-surgical neuropsychological assessment (Baxendale et al., 1998). Patient demographics, neurological test results, and surgical outcome data are listed in Table 1. The ILAE classification of post-operative seizure outcome following epilepsy surgery was used (Wieser et al., 2001). We also studied 10 right-handed native English speaking, healthy volunteers (median age 29.5\u00a0years; range 23 to 50\u00a0years; 7 females). The study was approved by the National Hospital for Neurology and Neurosurgery and the Institute of Neurology Joint Ethics Committee, and informed written consent was obtained from all subjects.\nMR data acquisition\nMRI studies were performed on a 1.5-T GE Signa Horizon scanner (General Electric, Wakashua, Milwaukee, Wisconsin, USA). Standard imaging gradients with a maximum strength of 22 m Tm-\u00a01 and slew rate 120 Tm-\u00a01s-\u00a01 were used. All data were acquired using a standard quadrature birdcage head coil for both RF transmission and reception. The scanning protocol also included a coronal T1-weighted volumetric acquisition sequence with 1.5-mm-thick slices, and hippocampal volumes were determined according to a previously described method (Moran et al., 1999).\nDiffusion tensor imaging\nThe DTI acquisition sequence was a single-shot spin-echo planar imaging (EPI) sequence, cardiac gated (triggering occurring every QRS complex) (Wheeler-Kingshott et al., 2002), with TE\u00a0=\u00a095\u00a0ms. Sets of 60 contiguous 2.3-mm thickness axial slices were obtained, covering the whole brain, with diffusion sensitizing gradients applied in each of 54 non-colinear directions (maximum b value of 1148\u00a0mm2 s-\u00a01 (\u03b4\u00a0=\u00a034\u00a0ms, \u0394\u00a0=\u00a040\u00a0ms, using full gradient strength of 22\u00a0mTm-\u00a01)) along with 6 non-diffusion weighted (b\u00a0=\u00a00) scans. The field of view was 24\u00a0cm, and the acquisition matrix size was 96\u00a0\u00d7\u00a096, zero filled to 128\u00a0\u00d7\u00a0128 during reconstruction so that the reconstructed voxel size was 1.8\u00a0\u00d7\u00a01.8\u00a0\u00d7\u00a02.3\u00a0mm3. The DTI acquisition time for a total of 3600 image slices was approximately 25\u00a0min (depending on the heart rate).\nWe used the method of Parker and Alexander (Parker et al., 2003; Parker and Alexander, 2003) to reduce fibre orientation ambiguities in voxels containing fibre crossings. Voxels in which the single tensor fitted the data poorly were identified using the spherical-harmonic voxel-classification algorithm of Alexander et al. (Alexander et al., 2002). In these voxels a mixture of two Gaussian probability densities was fitted and the principal diffusion directions of the two diffusion tensors provided estimates of the orientations of the crossing fibres (Tuch et al., 2002). In all other voxels a single tensor model was fitted. For all voxels, fractional anisotropy (FA) maps were generated from the single tensor fit (Pierpaoli et al., 1996; Pierpaoli and Basser, 1996).\nTractography\nAll scans were transferred to a Unix workstation for processing. We used the PICo algorithm extended to cope with crossing fibres (Parker et al., 2003; Parker and Alexander, 2003) to track from anatomically defined regions of interest (ROIs) within the parahippocampal gyrus. This algorithm adapts the commonly used streamline approach to exploit the uncertainty due to noise in one or more fibre orientations defined for each voxel. This uncertainty is defined using probability density functions (PDFs) constructed using simulations of the effect of realistic data noise on fibre directions obtained from the mixture model (Parker and Alexander, 2003). The streamline process is repeated using Monte Carlo methods to generate maps of connection probability or confidence of connection from the chosen start region(s).\nThe anatomical definition of the ROI was based on a previously published tractography analysis of the parahippocampal gyrus in healthy subjects (Powell et al., 2004). Viewing the FA images in three orthogonal planes using MRIcro (http:\/\/www.psychology. nottingham.ac.uk), the centre of the white matter tract just anterior to the brainstem, and posterior to the cerebral peduncles was selected, such that the parahippocampal gyrus was defined at its longest in the corresponding sagittal view (Fig. 1). The corresponding coronal slice was then used to select two adjacent voxels in a left-right direction, such that they both lay within the white matter tract on axial and sagittal views. This process was repeated in one anterior and one posterior coronal slice. This method was chosen as the parahippocampal gyrus runs anterior to posterior, inferior to superior and medial to lateral within the medial temporal lobe. The principal eigenvector of each voxel, when viewed using PICo and projected on the axial plane was orientated anterior-posterior. A threshold of FA \u2265 0.1, and curvature threshold of 180\u00b0 were set for tractography.\nNeuropsychological tests\nThe list learning and design learning tests were used to assess material specific memory function (Coughlan and Hollows, 1985). In the verbal learning task the subject is read a list of 15 words five times, and on each presentation attempts to recall as many of the words as possible. The overall percentage of correct responses was used as the measure of verbal memory efficiency. For non-verbal memory we employed a design learning task; the subject is presented with a visual design on five occasions with recall being tested after each presentation. The percentage of correct responses over the five trials was used as a second measure of non-verbal memory efficiency. These tests form part of our presurgical memory assessment in TLE cases, and have proven least affected by performance anxiety, have a good test-retest reliability and are sensitive indicators of medial temporal lobe function (Baxendale et al., 1998). Neuropsychological test results are listed in Table 2.\nData analysis\nAll data were analysed using SPSS (11.0.0). It was first verified whether all parameters were normally distributed using the Kolmogorov\u2013Smirnov test for normal distribution. Group differences for age were determined by a one-way analysis of variance (ANOVA), and gender distribution was assessed using the Pearson's \u03c72 test. The age of onset of epilepsy, duration of epilepsy, and frequency of complex partial (CPS) and secondarily generalized seizures (SGS) in right and left TLE patients were compared using the Mann\u2013Whitney U test.\nEach subject's output tractography connection probability map was spatially normalised by mapping into a standard space using the MNI template provided by SPM2 (Wellcome Department of Imaging Neuroscience, London; http:\/\/www.fil.ion.ucl.ac.uk\/spm). Binary masks at a threshold connection probability value of 0.05 were then constructed. Our group has previously demonstrated in this same group of patients and control subjects, that the threshold 0.05 strikes a balance between losing non-specific low probability connections, while retaining the main body of the pathways (Powell et al., 2006). Binary masks at this threshold were therefore averaged across each group, to produce variability (or commonality) maps indicating the degree of spatial variability and overlap of the identified connections (Parker et al., 2005). A voxel commonality value C of 1.0 indicates that every individual had a connection identified in this voxel, while a C value of 0 indicates that none of them did (Parker et al., 2005). Tracts were then assessed visually in all three planes for visual symmetry and size\/extent using MRIcro.\nNormalised tract volumes were calculated for the connecting tracts from the left and right PHG of each control and patient at a threshold of 0.05 (Toosy et al., 2004). An asymmetry index for volume (AIvol) defined as AIvol\u00a0=\u00a0[100\u00a0\u00d7\u00a0(Right Volume\u00a0\u2212\u00a0Left Volume)]\u00a0\/\u00a0[(Right Volume\u00a0+\u00a0Left Volume)\/2] was calculated (Jutila et al., 2001), and the mean values between groups compared using a one way ANOVA analysis. Comparisons between the control AI values, and left and right TLE AI values were carried out using post-hoc Dunnett t-tests. Two way mixed ANOVA with one between subjects factor (group \u2013\u2014 controls or TLE (both left and right)) and one within subjects factor (hemisphere \u2014 left or right) was used to test for the effect of interaction between group and hemisphere on volume, and unpaired t-tests were used to compare the patient and control group tract volumes.\nThe mean FA of the connected volume was calculated in native space for the left and right tracts in controls and patients. This was carried out by multiplying the native, thresholded, binarised images with that subject's whole brain FA image, in order to calculate the mean intensity value of the voxels. An asymmetry index for FA (AIFA) defined as AIFA\u00a0=\u00a0[100\u00a0\u00d7\u00a0(Right FA\u00a0-\u00a0Left FA)]\u00a0\/\u00a0[(Right FA\u00a0+\u00a0Left FA)\/2] (Jutila et al., 2001) was calculated, and the mean values between groups compared using one way ANOVA analysis. Comparisons between the control AI values and left and right TLE AI values were carried out using post-hoc Dunnett t-tests. Two way mixed ANOVA with one between subjects factor (group \u2014 controls or TLE (both left and right)) and one within subjects factor (hemisphere \u2014 left or right) were used to test for the effect of an interaction between group and hemisphere on FA, and unpaired t-tests were used to compare the patient and control group tract FA values.\nPearson's correlation test was used to evaluate the evidence for a correlation of hippocampal volume with tract volume and FA, and tract volume with tract FA, ipsilateral and contralateral to the seizure focus. Performance on material specific memory measures was investigated for evidence of a correlation with tract volume and FA in right and left TLE groups, omitting patient 14 because of his atypical language dominance.\nResults\nDemographic analysis\nThere was no significant difference in the mean age or gender distribution of participants in the three groups (controls, left TLE, right TLE). There was no significant difference in the age of onset, duration of epilepsy or frequency of CPS and SGS between left and right TLE patients.\nQualitative tract analysis\nIn controls, PHG connections were visually symmetric. Connections between the para-hippocampal gyrus and anterior temporal lobe, orbitofrontal areas and posterior temporal and extrastriate occipital areas were observed as documented previously (Powell et al., 2004). There was a clear decrease in ipsilateral compared with contralateral connections in left TLE (Fig. 2), though an ipsilateral reduction was not evident in the commonality map of the right TLE group (Fig. 3).\nQuantitative tract analysis\nThere was a significant difference in AIvol between groups [F(2,25)\u00a0=\u00a03.31 p\u00a0=\u00a00.05]. The AIvol was greater in left TLE patients than in controls (p\u00a0=\u00a00.05) with a mean 22% reduction in volume on the left (Table 3). There was no significant difference between the AIvol in right TLE patients and controls. There was no significant interaction between group and hemisphere for tract volume, and no significant differences between left and right volumes in left\/right TLE patients, and controls.\nThere was a significant difference in AIFA between groups [F(2, 25)\u00a0=\u00a04.92 p\u00a0=\u00a00.02], attributable to reduced FA on the left in left TLE (p\u00a0=\u00a00.02 against controls). There was no significant difference between the AIFA in right TLE patients and controls. There was a significant interaction between group and hemisphere on tract FA [F(2, 25)\u00a0=\u00a04.35 p\u00a0=\u00a00.02], with FA being lower on the left in left TLE patients, compared with controls (p\u00a0=\u00a00.03). Although a similar trend was present in right TLE patients, with FA being lower on the right side compared with controls, this was not significant (p\u00a0=\u00a00.06).\nCorrelation of tract volume and FA with pre-surgical material specific memory\nAll but one patient were left hemisphere dominant for language. Patient 14 was left handed and right hemisphere dominant on both fMRI and intracarotid amytal testing. He was therefore omitted from the correlation analysis. In the left TLE patients, FA of the left PHG connections were correlated significantly with pre-surgical verbal learning (r\u00a0=\u00a00.88, p\u00a0=\u00a00.002), and right FA correlated significantly against pre-surgical design learning (r\u00a0=\u00a00.63, p\u00a0=\u00a00.05) (Fig. 4). Left and right tract volumes were not significantly correlated with verbal learning and design learning, respectively. In the right TLE patients, there were no significant correlations between left or right tract FA or volume, with pre-operative verbal learning or design learning respectively.\nCorrelations of hippocampal volumes with tract volumes and tract FA\nThere was no significant correlation between ipsilateral hippocampal volume and ipsilateral tract volumes or FA nor between contralateral hippocampal volumes and contralateral tract volume or FA.\nCorrelation analysis of tract volume with tract FA\nThere was a significant correlation between tract FA and tract volume (r\u00a0=\u00a00.61, p\u00a0=\u00a00.008) ipsilateral to seizure focus (Fig. 5), but not contralateral to seizure focus.\nDiscussion\nOur principal finding was that in TLE the white matter connections of the parahippocampal gyrus ipsilateral to the seizure focus had smaller volumes and decreased FA. This was statistically significant in left but not right TLE patients. Furthermore, in left TLE, decreased FA was associated with poorer performance on both material specific memory measures. These results are consistent with the hypothesis that TLE involves dysfunction and structural changes in a network that includes the parahippocampal gyrus.\nThe connections of the human parahippocampal gyrus, visualized with tractography, have been described in a control population (Powell et al., 2004). Using a different tractography algorithm (Fast Marching Tractography or FMT) connectivity was found between the parahippocampal gyrus and the anterior temporal lobe, orbitofrontal areas, posterior temporal lobe and extra-striate occipital lobe via the lingual and fusiform gyri. These findings are similar to those in this control group.\nThis is the first study to use tractography to quantitatively assess the structural changes in the parahippocampal gyrus in TLE. Several volumetric MRI (Bernasconi et al., 2003; Bonilha et al., 2003) and voxel-based morphometry (VBM) studies (Bernasconi et al., 2004; Bonilha et al., 2004; Keller et al., 2002b,a, 2004) have investigated the extra-hippocampal structural changes in unilateral TLE. The para-hippocampal gyrus or its sub-regions including the entorhinal cortex, parahippocampal cortex and perirhinal cortex have been shown to be affected in TLE. Several studies suggested that the degree of atrophy and regional distribution was more extensive in left TLE, though none were specifically designed to evaluate this (Bernasconi et al., 2004; Bonilha et al., 2004; Keller et al., 2002b). It has however been recently demonstrated that the distribution and severity of extra-hippocampal grey matter loss is more extensive in left TLE patients than right TLE patients (Bonilha et al., 2007). Our study is the first to show that the white matter connections of the parahippocampal gyrus are affected by unilateral TLE. This process appears to be more severe in left TLE compared with right TLE. A similar pattern was observed in another tractography study that assessed language pathways in TLE patients (Powell et al., 2007b), although in this case there was also asymmetry of connections in controls, with more extensive connections in the speech dominant hemisphere. Ultimately, longitudinal quantitative MRI studies will be necessary to determine the effects of underlying cause, seizures, medications, and co-morbidity on the white matter structures and connections of the brain.\nParahippocampal connections and memory in temporal lobe epilepsy\nThe memory deficits associated with TLE, particularly left TLE, can be disabling. Consequently, much attention has been focused on the effects of both chronic TLE and ATLR on memory. While the hippocampus plays a critical role in the initial formation of memories (Eichenbaum, 2000), the parahippocampal region is thought to be involved in the intersection between perception and memory, and the translation of material into a more permanent storage in the cortical association areas (Eichenbaum, 2000; Murray and Bussey, 1999). Animal models have shown that the parahippocampal region is important for recognition memory (Brown and Aggleton, 2001; Eichenbaum, 2000), and that selective lesions to the parahippocampal area can severely impair memory (Suzuki et al., 1993). Furthermore, the functions of the medial temporal lobe are highly lateralized and the classic model of material specific memory predicts that lesions in the left hippocampal system impair verbal memory retrieval (Hermann et al., 1997), while those in the right hippocampal system affect non-verbal memory, though these findings are less consistent (Alessio et al., 2004).\nIn left TLE patients there was a significant correlation between parahippocampal tract FA and material specific memory measures on both the left and right sides. No significant correlations were seen with respect to tract volume in left TLE, and neither volume nor FA in right TLE. The finding of fewer occipital connections in the left TLE group compared with right TLE patients and controls may be significant in this respect (Figs. 2 and 3). Animal models suggest that potential roles for these connections may involve the priming of mesial temporal lobe structures to facilitate the consolidation of visual memory, or enhancing the visual processing of emotionally significant stimuli (Suzuki et al., 1993; Suzuki and Amaral, 1994). Visual cues may be important not only for non-verbal memory, but may also be useful in verbal memory. This could explain the correlation of FA found with verbal and non-verbal memory in left TLE patients. In the right TLE patients the occipital connections did not seem to be as affected, and hence these patients may have been able to use visual cues to aid both verbal and non-verbal memory.\nSeveral other studies have also observed correlations between MRI findings and memory dysfunction in TLE. Some have shown a correlation between left and right hippocampal MRI volumes, and verbal and non-verbal memory respectively (Baxendale et al., 1998; Kalviainen et al., 1997; Lencz et al., 1992). Others have shown correlations between hippocampal T2-signal, or abnormalities of MR spectroscopy and material specific measures (Gadian et al., 1996; Wendel et al., 2001). Lui et al. (Lui et al., 2005) examined 18 TLE patients and found correlations between apparent diffusion coefficients (ADC) in the left and right hippocampi and parahippocampal gyri and, verbal and non-verbal memory respectively. Others have demonstrated correlations between localized diffusion measures and domains of cognitive functioning in diseases as varied as schizophrenia (Nestor et al., 2004), HIV related dementia (Ragin et al., 2005), age related cognitive decline (Charlton et al., 2006), mild cognitive impairment (Rose et al., 2006), and Alzheimer's disease (Yoshiura et al., 2002). There are also studies that have observed that the relationship between the side of pathology and memory dysfunction appears to be more evident in those with left TLE than right TLE. Alessio et al evaluated the relationship between several medial temporal lobe structures and memory in 39 patients, and found a correlation between the degree of left sided hippocampal atrophy and verbal memory deficits, but not between right sided hippocampal atrophy and visual memory deficits (Alessio et al., 2004).\nIn this study, it also appears that tractography derived parahippocampal FA is a more sensitive marker than volume of the functional integrity of tracts that form the hardwiring of circuits needed for memory (Charlton et al., 2006). Other tractography studies have shown that FA is a more sensitive and robust measure than volume of pathology in white matter tracts (Heiervang et al., 2006). In biological terms the interpretation of reduced anisotropy is complex (Beaulieu, 2002), and depends on the context or disease in which it is found (Alexander et al., 2007). In epilepsy, it may represent neuronal loss, gliosis, and structural disorganisation (Wieshmann et al., 1999).\nThe association between hippocampal volume and parahippocampal Volume\/FA\nNo association between hippocampal volume, and tract volume or FA was found in this study. Other studies have reported mixed findings. While Bonhila et al found no correlations between the severity of hippocampal atrophy and grey matter volume in the parahippocampal region (Bonilha et al., 2003), Jutila et al reported positive correlations but only in a subgroup analysis of those patients with the most severe hippocampal atrophy (Jutila et al., 2001). This suggests that despite the fact that the hippocampus and parahippocampal gyrus are significantly interconnected, the structural size relationship between the two structures is not linear. There was also a significant correlation between ipsilateral tract volume and FA in the current study.\nLimitations of Study\nOnly modest numbers of patients and controls were available for this study, as the scanner used was decommissioned and replaced with one of higher field strength, making comparison with more recent data impossible. These small numbers, particularly in the left TLE group, may have contributed to the pattern of observed changes in left but not right TLE patients. Furthermore, for this reason several morphological and functional factors that can influence cognition and memory in chronic epilepsy were not included as co-variates in the statistical analysis. The pathological basis of TLE was not homogenous throughout the group, and the degree of hippocampal atrophy was varied. Secondly the spatial resolution of the tractography was limited and we did not separately evaluate the entorhinal cortex, parahippocampal cortex, and perirhinal cortex (Duvernoy, 1998). The entorhinal cortex is considered to be the route by which data reaches the hippocampus. The perirhinal, and parahippocampal cortex on the other hand, provide the incoming connections to the entorhinal cortex, conveying information from the polymodal and unimodal cortices (Squire, 1991). A sub-regional analysis is an area that should be explored in future tractography studies.\nConclusion\nThis tractography study has shown disruption of the architecture and atrophy of the connections of the parahippocampal gyrus ipsilateral to the seizure focus in patients with refractory TLE, and these structural changes were associated with memory deficits evident on psychometric testing. This information has both diagnostic and prognostic implications. Larger, longitudinal studies at a higher resolution will enable both sub-regional analysis, and the investigation of other factors that may contribute to neuronal loss and structural changes, and subsequent memory impairment in patients with TLE.","keyphrases":["tractography","parahippocampal gyrus","memory","epilepsy"],"prmu":["P","P","P","P"]}
{"id":"Pediatr_Nephrol-3-1-1915616","title":"Interactions of Shiga-like toxin with human peripheral blood monocytes\n","text":"The cytotoxic effect of Shiga-like toxin (Stx; produced by certain Escherichia coli strains) plays a central role in typical hemolytic uremic syndrome (HUS). It damages the renal endothelium by inhibiting the cellular protein synthesis. Also, the monocyte has a specific receptor for Stx but is not sensitive for the cytotoxic effect. In this work, monocytes were studied as a potential transporter for Stx to the renal endothelium. Coincubation of isolated human monocytes loaded with Stx and target cells (vero cells and human umbilical vascular endothelial cells) were performed. Transfer was determined by measuring the protein synthesis of target cells and by flow cytometry. Furthermore, the effect of a temperature shift on loaded monocytes was investigated. Stx-loaded monocytes reduced the protein synthesis of target cells. After adding an antibody against Stx, incomplete recovery occurred. Also, adding only the supernatant of coincubation was followed by protein synthesis inhibition. Stx detached from its receptor on the monocyte after a change in temperature, and no release was detected without this temperature shift. Although the monocyte plays an important role in the pathogenesis of HUS, it has no role in the transfer of Stx.\nIntroduction\nHemolytic uremic syndrome (HUS) is a clinical syndrome consisting of three characteristic features: hemolytic anemia, thrombocytopenia, and acute renal failure [1]. In the new classification of HUS, infections due to Shiga-like toxin (Stx)-producing bacteria belong to the category \u201cetiologically advanced\u201d [2]. This work focuses on the form in which Stx-producing Escherichia coli is the most common pathogen [1]. It can produce several types of Stx, of which Stx1, Stx2, and Stx2c are most frequently associated with HUS [3, 4]. Stx plays a crucial role in the pathogenesis because of its cytotoxic effect on the renal endothelium. Both renal tubular epithelial cells and glomerular visceral epithelial cells (podocytes) are also sensitive to the toxic effect of Stx [5, 6]. It can inhibit the protein synthesis of these cells after specifically damaging the ribosomal RNA [7]. However, the question of how this toxin is targeted mainly to the kidney remains unsolved. Stx was never detected in the serum of patients, but it was detected in renal biopsy material of patients with HUS [8]. As a specific treatment for HUS is still lacking, more insight into the transport of this toxin might lead to new intervention strategies.\nAfter oral ingestion of the bacteria through contaminated food or water, the noninvasive bacteria adhere to the intestinal epithelial cells of the distal small bowel and colon. This leads to a rearrangement of the morphology of the cells and initiates inflammation [9, 10]. Bacterial flagellin plays an important role in this process [11]. Stx can probably reach the circulation because of active transport in these cells and also passively after damage to the intestinal cells [12]. Subsequently, it has to be transported in the circulation to reach its primary target, the renal endothelium.\nIt is very tempting to look at the blood cells as a carrier for the toxin. Stx can bind to a specific receptor, which is a globotriaosylceramide (Gb3, Pk Antigen, CD77) [13]. This receptor is present on renal endothelial cells but also on blood cells. Stx binding has been described on red blood cells [14], B lymphocytes [15], and platelets, which also have an additional binding possibility (glycolipid, band 0.03) [16]. Several groups showed the existence of a specific binding of Stx on monocytes [17, 18, 19]. After binding to its receptor, Stx can be internalized. Whereas in epithelial cells the toxin follows the retrograde transport route and becomes cytotoxic, in monocytes it is targeted to the lysosomes and will get degraded [19]. During this transport, the monocyte becomes activated. This will lead to an increase of transcription factors, such as nuclear factor kappa B (NF-\u03baB) and activator protein 1 (AP-1), and an upregulated production of cytokines such as interleukin (IL)-1\u03b2, tumor necrosis factor (TNF)-\u03b1, IL-6, and IL-8 [17, 20]. These events will have a pro-inflammatory effect.\nWe postulated that, as the monocyte has a specific receptor, it might also function as a carrier to transport Stx to the renal endothelium. To investigate this hypothesis, Stx was loaded to isolated monocytes from healthy donors and coincubated with target cells [vero cells and human umbilical cord venous endothelial cells (HUVEC)]. The level of transfer was determined by measuring the protein synthesis of these target cells and the transfer of fluorescein isothiocyanate (FITC)-labeled B subunit of Stx1 with flow cytometry.\nMaterials and methods\nMaterials\nStx2 was kindly provided by Dr. M. Karmali (Public Health Agency of Canada, Ontario, Canada). FITC-labeled Stx1B subunit and 125I-Stx1B subunit were a gift from Dr. L. Johannes (Institut Curie, Paris, France). Stx1B subunit is a useful tool for studying binding in monocytes [19]. It is the binding part of the toxin, whereas the enzymatic A subunit will only stimulate the uptake of the toxin and does not affect binding [21]. Vero cell medium consists of M199 (Gibco; Paisley\/UK), fetal calf serum (FCS, Greiner Bio-One; Kremsmunster\/Austria), penicillin\/streptomycin (Gibco, Paisley\/UK), and glutamine (MP Biomedicals; Eschwege\/Germany). HUVEC medium is made of M199, human serum (HS; Cambrex; Walkersville\/USA), newborn calf serum (NBCS; Gibco, Paisley\/UK), penicillin\/streptomycin, glutamine, heparine (Leo Pharma BV, Breda\/The Netherlands), and endothelial-cell growth factor [22].\nEthylenediamine tetraacetic acid (EDTA) tubes were purchased from BD Vacutainer (Alphen aan de Rijn\/The Netherlands). The MACS kit for negative selection of monocytes was provided by Miltenyi Biotec (Bergisch Gladbach\/Germany). Hank\u2019s balanced salt solution (HBSS) was ordered from MP Biomedicals (Eschwege\/Germany). Human serum albumin (HSA) from Sanquin (Amsterdam\/The Netherlands) and porcine gelatin from Fluka (Neu-Ulm, Germany) was used. Trichloroacetic acid and TNF-\u03b1 was obtained from Sigma-Aldrich Chemie B.V. (Zwijndrecht\/The Netherlands). The antibody against Stx2 (TMA-15) is well characterised [23]. It was a kind gift from Dr. Yamagami from the Department of Biomedical Research from the Teijin Institute, Tokyo, Japan. 3H-leucine and Ficoll-paque PLUS was purchased from Amersham Biosciences (Uppsala, Sweden). Culture plates were ordered from Corning Inc. (Corning, USA).\nCulture of vero cells and HUVEC\nVero cells (renal epithelial cells of the African green monkey) were grown to confluency on 24-well plates (ordered from ATCC; Middlesex, UK). These cells have a high basal expression of Stx-receptor CD77. HUVEC were isolated, and these cells were grown to confluence on gelatin-coated 24-well plates [24]. Every 2 days, fresh medium was added to the cells. In contrast, HUVEC were preincubated for 24\u00a0h with TNF-\u03b1 (10\u00a0ng\/ml) to upregulate the expression of CD77.\nIsolation of monocytes and loading with Stx2\nFresh venous blood (20\u00a0ml) from 40 healthy donors was collected into EDTA tubes. Monocytes were isolated by negative selection using antibody-labeled beads (CD3, CD7, CD16, CD56, and CD123). After centrifugation of blood over Ficoll (20\u00a0min 400\u00a0g without break at room temperature), the interphase (containing monocytes, lymphocytes, and platelets) was collected. Platelets were removed by centrifugation (200\u00a0g 10\u00a0min at room temperature) before adding the beads. The purity of monocytes after the magnetic isolation was (as determined by flow cytometry) 80\u201385%. After the isolation, the monocytes were resuspended in HBSS with 1% HSA and placed on ice. In every experiment, monocytes from one donor were used. To load the monocytes with Stx2, the toxin was added to a concentration of 10\u00a0nM during a time period of 3\u00a0h [17]. The cells remained on ice. After proper washing to remove all unbound Stx2, the cells were resuspended in vero cell or HUVEC medium (in which HS and NBCS were substituted by FCS). Monocytes stayed viable after this loading, as determined by trypan blue exclusion.\nCoincubation Stx2-loaded monocytes and target cells\nThese experiments were performed in two different experimental settings: with transfer of loaded monocytes from 4\u00b0 to 37\u00b0, and without change of temperature. To start with the first setting, the Stx2-loaded monocytes were added to a monolayer of target cells [HUVEC (n\u2009=\u200910) or vero- cells (n\u2009=\u20099)] in a concentration of 1\u2009\u00d7\u2009106 per well. This was performed during 24\u00a0h at 37\u00b0C. For comparison, also monocytes without Stx were used. To determine the specificity of the effect of Stx2, the loaded monocytes were preincubated with a well-characterized antibody against Stx2 (TMA-15, 1\u00a0\u03bcg\/ml, approximately 150\u00d7 excess). All experiments were performed in duplicate. To measure the transfer of Stx2 from the monocyte, the protein synthesis of the target cells was determined by adding 3H-Leucine (0.67\u00a0\u03bcCi\/ml). Subsequently, intracellular proteins were precipitated by treatment with trichloroacetic acid (TCA), and the radioactivity was measured in a liquid scintillation counter. In the other experimental setting, monocytes were loaded in a similar way with FITC-labeled Stx1B subunit; 1\u2009\u00d7\u2009106-loaded monocytes were coincubated with 1.25\u2009\u00d7\u2009105 vero cells in suspension for 3\u00a0h at 4\u00b0C (n\u2009=\u20095) while being continuously rotated. At this temperature, bias due to internalization of the toxin could be avoided [19, 25]. After 3\u00a0h, the presence of Stx1B subunit FITC on the vero cells was determined by flow cytometry. For every experiment, at least 1,000 cells were measured.\nStudy of supernatant Stx-loaded monocytes\nTo study the effect of the supernatant of Stx2-loaded monocytes, it was collected after 16\u00a0h of coincubation on vero cells (n\u2009=\u200911). The supernatants were centrifuged to remove possible monocytes and added again to fresh vero cells. Protein synthesis was measured by adding 3H-leucine and measuring the incorporation after 24\u00a0h of incubation at 37\u00b0C. Also, the antibody against Stx2 was used to determine a possible effect of free Stx2. Furthermore, monocytes were loaded with 125I-Stx1B subunit to investigate whether it could be released after a change in temperature from 4\u00b0 to 37\u00b0C (n\u2009=\u20093). For each experiment, between 3 and 4\u2009\u00d7\u2009106 monocytes are loaded with 200\u00a0nM Stx1B subunit (3,600\u00a0cpm\/ng protein). After loading, unbound Stx1B subunit was removed by centrifugation. Subsequently, the monocytes were placed at 37\u00b0C for 2\u00a0h. They were then centrifuged, and cells and supernatant were measured separately for the presence of 125I-Stx1B with a gamma counter. This was compared with the amount of binding to the monocytes before the temperature change. The experiment was performed in duplicate.\nStatistics\nAll data presented are expressed as a range with the median. Significance of increase or decrease of protein synthesis compared with controls was analyzed using the Wilcoxon signed ranks test. The statistical level of significance was defined as P\u2009<\u20090.05.\nResults\nCoincubation Stx2-loaded monocytes and target cells\nTo investigate whether monocytes can transfer Stx2 to target cells (vero cells and HUVECs), Stx2-loaded monocytes were added to a monolayer of these cells. After coincubation, protein synthesis of vero cells and HUVEC was measured. If the Stx2 was transferred to the target cells, there was protein synthesis inhibition, as this is the biological effect of Stx2 in both types of target cells. After 24\u00a0h of coincubation at 37\u00b0C, in both cell types, there was protein synthesis inhibition (Fig.\u00a01). In vero cells, greater protein synthesis reduction was measured than in HUVEC. Directly adding 10\u00a0nM Stx2 to vero cells reduced protein synthesis to 5.5% (data not shown). When an antibody against Stx2 was added to the monocytes before coincubation with vero cells, protein synthesis inhibition was partly restored (Fig.\u00a02). Only in one out of five experiments was there complete recovery.\nFig.\u00a01Coincubation of Shiga-like toxin (Stx)2-loaded monocytes with target cells [vero cells and human umbilical cord venous endothelial cells (HUVEC)]. To investigate whether monocytes could transfer Stx2, toxin-loaded monocytes were coincubated with vero cells (n\u2009=\u20099) and HUVEC (n\u2009=\u200910). This led to protein synthesis inhibition, as could be determined with the incorporation of 3H-leucine. The coincubation with unloaded monocytes was used as a control and set at 100%. ** P\u2009<\u20090.01Fig.\u00a02Effect of the addition of an antibody against Shiga-like toxin (Stx)2. The monocytes of five healthy donors were coincubated with vero cells (Stx2-loaded monocytes, white bar; Stx2-loaded monocytes with Stx2 antibody, black bar). Addition of the antibody against Stx2 led to partial protein synthesis recovery in four donors and complete recovery in one donor. Coincubation with unloaded monocytes was used as a control and set at 100%\nIn equal experiments performed with HUVEC (n\u2009=\u20093), TMA-15 also partly prevented  protein synthesis inhibition (data not shown).\nStudy of supernatant Stx-loaded monocytes\nBecause the addition of an antibody against Stx2 was not sufficient for complete protein synthesis recovery in four out of five tested donors, we investigated the possible presence of an additional inhibitor in the supernatant after 16\u00a0h of coincubation. This was performed by re-adding the supernatant of coincubated Stx2-loaded monocytes and a monolayer of vero cells to new vero cells. Figure\u00a03 shows that this incubation again led to an inhibitory effect on vero-cell protein synthesis in contrast to the supernatant of unloaded monocytes. But this reduction was less than with direct coincubation. The effect could be partly blocked by adding the antibody against Stx2. This means that there is unbound Stx2 present in the supernatant. Next, we investigated whether the temperature change of the Stx-loaded monocytes from 4\u00b0 to 37\u00b0C could lead to toxin release. For this reason, we loaded the monocytes with 125I-Stx1B subunit. The amount of radioactivity on or inside the monocytes was measured before and after incubation of the cells for 2\u00a0h at 37\u00b0C (Table\u00a01). Vero cells were used as a control. In vero cells as in monocytes, the toxin was released from its receptor after the incubation.\nFig.\u00a03Addition of supernatants coincubation to vero cells. After coincubation of Shiga-like toxin (Stx)-loaded and unloaded monocytes, supernatants were collected and re-added to fresh vero cells (n\u2009=\u200911). The supernatant from the toxin-loaded monocytes induced, again, protein synthesis inhibition. This inhibition could be partially restored with a Stx2 antibody (AB). The incubation of supernatant with unloaded monocytes was used as a control and set at 100%. ** P\u2009<\u20090.01Table\u00a01Amount of 125I- Shiga-like toxin (Stx)1B subunit on cells after change of temperatureStx-loaded cellsBefore 4\u219237\u00b0C (cpm)After 4\u219237\u00b0C (cpm)Vero cells1,034,383671,060Monocytes donor 11,437427Monocytes donor 21,070374Monocytes donor 31,121677cpm counts per minute\nTransfer of Stx to target cells without change in temperature\nA change in temperature from 4\u00b0 to 37\u00b0C released Stx from its receptor on the monocyte. To study again the possibility of Stx transfer from monocyte to target cells, we loaded isolated monocytes with Stx1B subunit labeled with FITC. Coincubation of these monocytes with vero cells in suspension without a change in temperature did not result in a transfer of the B subunit to the vero cells (n\u2009=\u20095, Fig.\u00a04).\nFig.\u00a04Transfer of Shiga-like toxin (Stx)-loaded monocytes to vero cells without temperature shift (white: normal monocytes; black: Stx-loaded monocytes; grey: vero cells). The x-axis represents the intensity of the fluorescein isothiocyanate (FITC) signal, the y-axis represents the number of cells. a FITC-labeled Stx1B subunit was bound to monocytes from a healthy donor, showing an increase in intensity. b Coincubation of vero cells and unloaded monocytes; note the high basal signal of vero cells. c After 2\u00a0h of coincubation with vero cells in suspension at 4\u00b0C, there was no transfer of the FITC signal. The cells remained in position\nDiscussion\nAfter coincubation of Stx2-loaded monocytes with target-cells (vero cells and HUVEC), protein synthesis inhibition could be detected. As the biological effect of Stx is protein synthesis inhibition, a transfer was expected. This effect could not be due to the presence of monocytes, because it is well described that Stx has no inhibitory effect on peripheral blood monocytes [17]. However, the addition of an antibody against Stx only partly restored the inhibition. We hypothesized that in parallel to transfer of Stx2, a possible additional inhibiting factor was present in the supernatant during the experiment. To further investigate this possibility, the supernatant of this coincubation was re-added to fresh vero cells. Again, there was protein synthesis inhibition, which could partly be decreased by blocking with the Stx2 antibody. The conclusion was made that there another inhibitory factor needed to be present (possibly released by activated monocytes), but unbound Stx2 was also present [26]. Apparently, Stx1B subunit is released from its receptor when there is shift of temperature from 4\u00b0 to 37\u00b0C, as shown in this study and also by Ramegowda and Tesh [18]. This finding stresses the caution that must be taken when drawing conclusions from in vitro experiments performed at 4\u00b0C. In Fig.\u00a05, all findings are schematically summarized.\nFig.\u00a05Schematic summary of performed experiments. a Monocytes loaded with Shiga-like toxins (Stx) at 4\u00b0C. b After shifting Stx-loaded monoytes to target cells at 37\u00b0C, Stx was released from its receptor. This led to protein synthesis inhibition of target cells. However, transport could not be excluded (but this was performed in experiments without a shift in temperature). Probably, some toxin was internalized and monocytes became activated. This could lead to cytokine and proteases production (possible cytotoxic factors). c When the supernatant is re-added to new target cells, there are released Stx and secreted products present. This induced, again, protein synthesis inhibition\nAs these in vitro experiments were not suitable to investigate a possible transfer, experiments were performed without a change of temperature. No transfer of the binding part of the toxin could be detected in this setting. Experiments could not be performed at 37\u00b0C because the toxin is internalized by the monocytes during 3\u00a0h of incubation. Because of these in vitro experiments, we believe that the monocyte cannot function as a transporter for Stx in the circulation. However, monocytes still seem to play an important role in pathogenesis. As a component of the innate immune system, they play a central role in immunity and inflammation. Fernandez et al. showed that patients in the acute period of HUS have monocytes with phenotypic (reduced expression of CD14, CD64, and CD11b) and functional [decreases lipopolysaccharide (LPS)-induced TNF-\u03b1 production and Fc\u03b3-dependent cytotoxicity] differences compared with healthy children and acute uremic children [27].\nSome unanswered questions remain. What is the inhibitory factor released by monocytes after loading with Stx? Why is there still unbound Stx2 present in the supernatant after 16\u00a0h of coincubation? It is surprising that Stx2 is not completely bound and internalized by the numerous receptors on monocytes and vero cells. Also, how Stx is transported in the circulation remains unsolved. Kimura et al. described that serum amyloid P (SAP) can bind Stx2 and function as a neutralizing factor [28]. However, in humans, there was no correlation between circulating SAP and the development of HUS [29].\nAs renal endothelial damage is already present after the occurrence of clinical symptoms of HUS, it is very important to develop efficacious early prevention. Understanding the mechanism in which the toxin is specifically targeted to the kidney can lead to novel intervention strategies.","keyphrases":["shiga-like toxin","monocyte","hemolytic uremic syndrome","vero cells","acute renal failure","huvec"],"prmu":["P","P","P","P","P","P"]}
{"id":"Eur_Arch_Otorhinolaryngol-4-1-2254469","title":"Fiberoptic endoscopic evaluation of swallowing in intensive care unit patients\n","text":"Aspiration in critically ill patients frequently causes severe co-morbidity. We evaluated a diagnostic protocol using routine FEES in critically ill patients at risk to develop aspiration following extubation. We instructed intensive care unit physicians on specific risk factors for and clinical signs of aspiration following extubation in critically ill patients and offered bedside FEES for such patients. Over a 45-month period, we were called to perform 913 endoscopic examinations in 553 patients. Silent aspiration or aspiration with acute symptoms (cough or gag reflex as the bolus passed into the trachea) was detected in 69.3% of all patients. Prolonged non-oral feeding via a naso-gastric tube was initiated in 49.7% of all patients. In 13.2% of patients, a percutaneous endoscopic gastrostomy was initiated as a result of FEES findings, and in 6.3% an additional tracheotomy to prevent aspiration had to be initiated. In 59 out of 258 patients (22.9%), tracheotomies were closed, and 30.7% of all 553 patients could be managed with the immediate onset of an oral diet and compensatory treatment procedures. Additional radiological examinations were not required. FEES in critically ill patients allows for a rapid evaluation of deglutition and for the immediate initiation of symptom-related rehabilitation or for an early resumption of oral feeding.\nIntroduction\nPulmonary aspiration is a serious cause of morbidity and mortality in patients with a depressed sensorium, patients with neuromuscular discoordination, or patients having structural disorders of the upper aerodigestive tract. It is a leading cause of nosocomial infection in the intensive care unit (ICU). The most common manifestations of pulmonary aspiration are pneumonia, pleuropulmonary infection, and acute airway obstruction [1].\nHospital-acquired pneumonia is the second most common nosocomial infection in the critically ill patient and is associated with the greatest mortality and increased morbidity and cost of care. Incidence of HAP varies in different populations of critically ill patients and generally ranges from 9 to 20%. The etiologic agents differ according to the population studied, duration of hospital stay, time after intubation, and prior antimicrobial therapy [2]. Risk factors include nonmodifiable factors like age, chronic obstructive pulmonary disease, severe head trauma, and multiple trauma, and modifiable factors like large-volume gastric aspiration, duration of mechanical ventilation, elevated gastric pH, histamine type two blocker therapy, ventilator circuit change frequency, self-extubation, and silent aspiration following scheduled extubation [2]. When patients are admitted to the ICU, they are at increased risk of more frequent aspiration events because of a variety of factors, including depressed level of consciousness (often caused by excess analgesia and sedation), forced supine position, and the presence of nasogastric or endotracheal tubes [3].\nThere is a growing body of evidence suggesting that intubation for longer than 48\u00a0h may cause at least transient injury to the larynx with a subsequent reduction in the protective mechanism and increased incidence of oropharyngeal secretions once the patient is extubated. The presence of an orotracheal tube has been shown to alter the mechanoreceptors and chemoreceptors of the pharyngeal and laryngeal mucosa, causing dysfunction of the swallowing reflex. The prevalence of swallowing dysfunction post-extubation has been reported to occur in between 20 and 83% of those patients intubated longer than 48\u00a0h. This wide range of estimate has partly been attributed to the variation in the diagnostic tools [4]. The impact that diagnosis using invasive diagnostic techniques may have on the epidemiological characteristics of HAP in critically ill patients is unknown, but may potentially improve aspiration-related specific therapy and ultimately clinical outcome.\nBased on these considerations, we prospectively evaluated a diagnostic protocol using routine fiberoptic endoscopic evaluation of swallowing (FEES) in critically ill patients at risk to develop aspiration following temporary transnasal intubation or tracheotomy and tried to define the impact of post-extubation FEES on the initiation of aspiration-related treatment.\nMaterial and methods\nWe conducted a prospective, interventional, clinical study at Klagenfurt General Hospital. The hospital is a 1,400-bed tertiary referral centre that serves a population of approximately 1,000,000 and runs seven ICUs (paediatrics and neonatology, 1; neurology, 1; neurosurgery, 1; cardiology, 1; general internal medicine, 1; and anaesthesiology, 2) with a total of 84 beds.\nWe offered a standardized endoscopy protocol for critically ill patients considered to be at risk for silent aspiration following transnasal intubation or tracheotomy for ventilation. Patient selection for endoscopic assessment of swallowing was done by ICU physicians using a protocol that we designed to help in the early recognition of risk factors for, or overt signs of, aspiration. This protocol included a taxonomic summary of symptoms and clinical signs potentially related to aspiration. Rating was applied after the stop of mechanical ventilation. Symptoms were defined as:Weight loss,unexplained fever >38.0\u00b0 centigrade,coughing,bronchitis\/pneumonitis,impaired voice,witnessed regurgitation\/aspiration event at the bedside accompanied by coughing, choking, and\/or expectoration of material,prolonged oral feeding,aversion for oral intake of liquids and solids,disturbance of bolus transport,frequent postural changes during oral intake, andregurgitation.If such conditions were observed in previously intubated patients following extubation, or in patients with tracheotomies, physicians at ICUs were encouraged to request endoscopic assessment of swallowing.\nThe FEES procedure was consequently provided by staff members of the department of Oto-Rhino-Laryngology, Head and Neck Surgery. The team consisted of a laryngologist and a nurse who had both previously gone through a specialized training in performing FEES procedures in patients with deglutition disorders.\nOver the 45-month period from 1 January 2002 to 30 September 2005, we were called to perform 913 endoscopic examinations in 553 patients treated in one of the ICUs. Four hundred and 46 patients underwent one single endoscopy, and 107 had repeated examinations. Two hundred and 95 patients had shortly before been extubated after transnasal tracheal intubation, and 258 patients had indwelling tracheotomies. For further data interpretation, patients were sub-grouped according to their route of ventilation (transoral\/transnasal intubation vs. tracheotomy). Findings and recommendations from the initial FEES procedure in all 553 patients were accumulated for this study. The results of 360 control procedures were not further analyzed.\nDetails of the FEES procedure have previously been described in detail. Therefore, only a short summary of the procedure shall be given here: The FEES procedure is a portable examination, easily taken to bedside in ICUs. Since our patients were usually bedridden, we performed the examination in a in bed with the head of bed elevated to approximately 70\u00b0 with the bend of the bed is at the patient\u2019s lower back (904\/913 patients, i.e., 99%). In four patients (0.4%), the examination was carried out with the patient in reverse Trendelenberg at 30\u00b0\u201345\u00b0, and five patients (0.6%) were able to sit on a chair during the procedure. A fiberoptic laryngoscope was passed transnasally to the oropharynx, where the larynx and surrounding structures could then be visualized. Patients were led through various tasks to evaluate the sensory and motor status of the pharyngeal and laryngeal mechanism. Stained liquid and semi-liquid boluses were then given to determine the integrity of pharyngeal deglutition. The interior larynx and airway were examined for evidence of food penetration within the laryngeal vestibule and aspiration of food below the true vocal folds before and after each swallow. In each case, the nasogastric tube was removed prior to the procedure. We assessed structural changes of the larynx and pharynx, timing and direction of movement of the bolus through the pharynx, the ability to protect the airway and to uphold airway protection for a some seconds, the capability to clear the bolus during deglutition, presence of pooling and residue of material in the hypopharynx, and timing of bolus flow and laryngeal closure.\nAspiration was defined as the entry of material into the airway below the levels of the true vocal cords. Silent aspiration was defined as aspiration occurring in the absence of acute symptoms (i.e., lack of cough or gag reflex as the food or liquid bolus passed into the trachea).\nFindings were documented in a standardized form to allow for monitoring therapeutic interventions over time, and for later data analysis and evaluation.\nResults\nOver a 45-month period, we were called to perform 913 endoscopic examinations in 553 patients. Of these, 295 patients had previously been extubated, and 258 patients had indwelling tracheotomies. Silent aspiration or aspiration with acute symptoms (cough or gag reflex as the bolus passed into the trachea) was detected in 383 (69.3%) of all patients during initial FEES. Prolonged non-oral feeding via a naso-gastric tube was initiated in 49.7% of all patients. In 13.2% of patients, a percutaneous endoscopic gastrostomy was initiated as a result of FEES findings, and in 6.3% an additional tracheotomy to prevent aspiration had to be set off. In 59 out of 258 patients (22.9%), tracheotomies were closed, and 30.7% of 553 patients could be managed with the immediate onset of an oral diet \u00b1 compensatory treatment procedures without further diagnostic evaluation. Logopedic instruction of the patient and\/or rehabilitation using compensatory strategies to reduce the risk of aspiration were an integral part of our rehabilitation program and included one or more of the following:Modifications in volume and tempo of food presentationoral feeding with consistency modificationshead rotationholding the chin down during deglutition to narrow the airway entrancesupraglottic swallow: This technique uses simultaneous swallowing and breath-holding, closing the vocal cords and protecting the airway.One hundred and seven patients were scheduled for repeated FEES, usually to assess the progress of the rehabilitation program.\nSigns and symptoms encountered during initial FEES are condensed in Table\u00a01. Table\u00a02 stratifies the route of feeding in the two subgroups of our study population. Table\u00a03 classifies the type of aspiration detected during FEES. Table\u00a04 summarizes the recommendations derived from our endoscopic findings for the further management of patients with aspiration.\nTable\u00a01Symptoms of aspiration in 553 patientsPatients after transnasal tracheal intubation (n\u00a0=\u00a0295)Patients with indwelling tracheotomies (n\u00a0=\u00a0258)Unexplained fever17 (5.8%)39 (15.1%)Coughing68 (23.1%)31 (12.0%)Bronchitis\/pneumonitis57 (19.32%)39 (15.1%)Impaired voice57 (19.32%)0 (0%)Witnessed regurgitation\/aspiration event 74 (25.1%)149 (57.8%)Others16 (5.4%)23 (8.9%)No data69 (23.3%)19 (7.4%)Since more than one symptom or sign could apply per patient, results sum up to more than 100%Table\u00a02Route of feeding for 553 patients on initial FEESPatients after transnasal tracheal intubation (n\u00a0=\u00a0295)Patients with indwelling tracheotomies (n\u00a0=\u00a0258)Nasogastric tube224 (75.9%)186 (72.1%)PEG15 (5.1%)59 (22.9%)Parenteral12 (4.1%)5 (1.9%)Oral diet44 (14.9%)8 (3.1%)PEG percutaneous endoscopic gastrostomyTable\u00a03Classification of aspiration for 553 patients on initial FEESPatients after transoral or transnasal tracheal intubation (n\u00a0=\u00a0295)Patients with indwelling tracheotomies (n\u00a0=\u00a0258)Silent aspiration51 (17.3%)95 (36.8%)Aspiration115 (39.0%)126 (48.8%)No aspiration129 (43.7%)37 (14.4%)Aspiration\u00a0=\u00a0inhalation of material into the airway below the level of the true vocal cords, with acute symptoms (cough or gag reflex as the bolus passed into the trachea), silent aspiration\u00a0=\u00a0aspiration occurring in the absence of acute symptomsTable\u00a04Recommendation for further treatment in 553 patients after initial FEESPatients after transoral or transnasal tracheal intubation (n\u00a0=\u00a0295)Patients with indwelling tracheotomies (n\u00a0=\u00a0258)Non-oral feeding (naso-gastric tube) \u00b1 logopedic (functional) therapy144 (48.8%)131 (50.8%)Indication for PEG27 (9.1%)46 (17.8%)Indication for non-oral feeding plus tracheotomy35 (11.9%)NAOral feeding \u00b1 logopedic (functional) therapy89 (30.2)22 (8.5%)Decannulation and oral feeding \u00b1 logopedic (functional) therapyNA59 (22.9%)PEG percutaneous endoscopic gastrostomy, NA not applicable\nDiscussion\nAspiration is a common event. The North American Summit on Aspiration in the Critically Ill Patient, in a consensus statement published in 2002 [5], estimates the frequency of aspiration in various patient populations is as follows:45% in normal individuals during sleep70% in patients with impaired consciousness0\u201340% in patients on ETF50\u201375% in patients with endotracheal tubesWhat determines the clinical consequences of an aspiration event are host factors (age, immune status, underlying disease process, and comorbidities) and the nature of the aspirate. The morbidity and mortality associated with aspiration (with or without subsequent pneumonia) is difficult to ascertain and may vary greatly depending on the patient population that is under study [5].\nDespite these general considerations, aspiration is generally accepted as a leading cause of nosocomial pulmonary infection in the ICU [6].\nAjemian reported a 56% (27\/48) incidence of swallowing dysfunction following prolonged intubation; 12 (25%) of 48 patients were silent aspirators [7]. In a study by Leder et al. [8], FEES demonstrated swallowing dysfunction in 33% of critically ill trauma patients after prolonged intubation, with 20% having silent aspiration. In a recent study by El Solh and co-workers, aspiration was documented in 52% of ICU patients aged over 65\u00a0years and in 36% of those up to age 65. No significant difference in the co-morbidity index and the length of mechanical ventilation was found between aspirators and non-aspirators [4].\nNon-specific methods to monitor for and detect aspiration are usually subjective, not standardized or validated, and inaccurate, and there are few outcome valid data. Blue dye has been commonly used to color the feeding solutions, but this method is insensitive and non-specific for assessing aspiration and pneumonia. Testing of pharyngeal secretions with glucose oxidase strips also is not standardized and is too insensitive and non-specific to test for aspiration of feeding solution. The best single test for aspiration remains undetermined to date. Therefore, clinical studies are needed to determine the potential role of particular tests suited for specific indications [9].\nVideo-fluoroscopy has traditionally been accepted as the \u201cgold standard\u201d for evaluation of a swallowing disorder for the comprehensive information it provides. However, it is not very efficient and accessible in certain clinical and practical situations. This is particularly true for critically ill patients treated at ICUs, who are almost invariably bedridden.\nFEES has been shown to be safe and effective for assisting in swallowing evaluation, and in therapy as a visual display to help patients learn various swallowing manoeuvres. [4, 10\u201313]. It can be used in a wide variety of settings, ranging from office evaluation of swallowing, inpatient and outpatient services to critically ill patient treated at the ICU [14\u201320].\nFEES is not just a screening procedure, it can achieve a complete assessment of the pharyngeal stage of swallowing. It includes five components: assessment of structural changes in the larynx and pharynx, assessment of movement and sensation, assessment of secretions management, direct visualization of swallowing function for food and liquid, and response to therapeutic interventions [12\u201315, 17].\nAviv and co-workers suggested combining FEES with sensory testing of the laryngeal and pharyngeal mucosa using discrete pulses of air delivered to the epithelium innervated by the internal branch of the superior laryngeal nerve (FEESST: Fiberoptic endoscopic evaluation of swallowing with sensory testing). They performed 1,340 consecutive FEESST procedures on 1,076 adult patients with dysphagia, among them 172 examinations in critically ill patients at the ICU (mostly following cardiac surgery). The authors concluded that FEESST for the purpose of swallowing function can be done safely and yields a 96.5% success rate in terms of completed examinations [19, 21].\nOur experience suggests that FEES can be performed easily at the bedside of ICU patients. It is associated with minimal complications for those patients recently liberated from mechanical ventilation, and for patients with indwelling tracheotomy treated at the ICU. However, we cannot determine from our data whether adopting FEES prevented aspiration pneumonia. Barquist et al. [22] conducted a randomized, prospective clinical trial of FEES versus routine clinical management in 70 patients after prolonged intubation. The addition of a FEES examination did not change the incidence of aspiration or postextubation pneumonia. Larger prospective randomized trials of FEES versus conventional patient management will be required to determine whether a difference in the rate of post-extubation pneumonia can be demonstrated. However, in our study, the observations recorded during FEES had immediate impact on the further treatment of patients: prolonged non-oral feeding via a naso-gastric tube was initiated in 49.7% of patients, percutaneous endoscopic gastrostomy in 13.2%, and an additional tracheotomy to prevent aspiration in 6.3%. On the other hand, 81 out of 258 pre-existing tracheotomies were immediately closed, and 30.7% of all 553 patients with endoscopy-proven aspiration could be treated with the immediate onset of an oral diet and compensatory treatment procedures.\nAfter assessing the critically ill patient for risk of aspiration, the clinician still must decide if the patient is ready to be fed. The goal is to identify critically ill patients who are likely to tolerate enteral nutrition and attempt to minimize complications [23]. FEES is certainly a versatile tool in achieving timely and appropriate clinical decisions in patients at risk for aspiration-related morbidity.\nIn contrast to most of the recently published material on FEES in critically ill patients, we report our observation in a large proportion of patients with indwelling tracheotomies (258 out of 553, i.e., 46.7%) at the time of the examination. Tracheotomy is a commonly performed procedure in ventilator dependent patients. Many critical care practitioners believe that performing a tracheostomy early in the postinjury period decreases the length of ventilator dependence as well as having other benefits such as better patient tolerance and lower respiratory dead space [24]. Table\u00a04 demonstrates that silent aspiration as well as clinically overt aspiration was more frequently encountered in patients with tracheotomies as compared to those that had previously been intubated via an oral or transnasal route. However, the clinical impressions that a tracheotomy or tracheotomy tube increases aspiration risk or that decannulation results in improved swallowing function have not been supported by a recent study. Rather, the need for a tracheotomy indicated comorbidities (e.g. respiratory failure, trauma, stroke, advanced age, reduced functional reserve, and medications used to treat the critically ill) that by themselves predispose patients for dysphagia and aspiration [25]. These data confirmed similar findings previously published by same authors: Neither the presence of a tracheotomy tube nor decannulation affected aspiration status in early, postsurgical head and neck cancer patients. In the acute care setting, no causal relationship between tracheotomy and aspiration status was exhibited [26].\nOur results allow no conclusion regarding the impact of tracheotomies on deglutition, since the initial decision to perform a tracheotomy had not been randomly assigned to patients in our two subgroups. The decision to perform tracheotomies was not consistent over different ICUs and largely depended on the anticipated length of assisted ventilation. In our study, FEES was used to determine the need for maintaining tracheotomy, and was accepted by ICU physicians as an important criterion in deciding to close tracheotomies following assisted ventilation.\nConclusion\nAfter the introduction of a standardized endoscopy protocol for critically ill patients considered being at risk for silent aspiration, ICU physicians soon requested FEES routinely for their patients.\nFEES in critically ill patients allows for a rapid evaluation of deglutition, for targeted further diagnostic procedures if needed, and for the immediate initiation of symptom-related rehabilitation or for an early resumption of oral feeding. FEES is now accepted at our institution as an important tool in achieving timely and appropriate clinical decisions for ICU-patients at risk for aspiration-related morbidity.\nLaryngologists should be encouraged to offer FEES procedures to responsible coordinators of ICUs.","keyphrases":["swallowing","aspiration","tracheotomy","deglutition","rehabilitation","endoscopy","dysphagia","laryngology"],"prmu":["P","P","P","P","P","P","P","U"]}
{"id":"Eur_J_Appl_Physiol-3-1-1914240","title":"Conventionally assessed voluntary activation does not represent relative voluntary torque production\n","text":"The ability to voluntarily activate a muscle is commonly assessed by some variant of the twitch interpolation technique (ITT), which assumes that the stimulated force increment decreases linearly as voluntary force increases. In the present study, subjects (n = 7) with exceptional ability for maximal voluntary activation (VA) of the knee extensors were used to study the relationship between superimposed and voluntary torque. This includes very high contraction intensities (90\u2013100%VA), which are difficult to consistently obtain in regular healthy subjects (VA of \u223c90%). Subjects were tested at 30, 60, and 90\u00b0 knee angles on two experimental days. At each angle, isometric knee extensions were performed with supramaximal superimposed nerve stimulation (triplet: three pulses at 300 Hz). Surface EMG signals were obtained from rectus femoris, vastus lateralis, and medialis muscles. Maximal VA was similar and very high across knee angles: 97 \u00b1 2.3% (mean \u00b1 SD). At high contraction intensities, the increase in voluntary torque was far greater than would be expected based on the decrement of superimposed torque. When voluntary torque increased from 79.6 \u00b1 6.1 to 100%MVC, superimposed torque decreased from 8.5 \u00b1 2.6 to 2.8 \u00b1 2.3% of resting triplet. Therefore, an increase in VA of 5.7% (from 91.5 \u00b1 2.6 to 97 \u00b1 2.3%) coincided with a much larger increase in voluntary torque (20.4 \u00b1 6.1%MVC) and EMG (33.9 \u00b1 6.6%max). Moreover, a conventionally assessed VA of 91.5 \u00b1 2.6% represented a voluntary torque of only 79.6 \u00b1 6.1%MVC. In conclusion, when maximal VA is calculated to be \u223c90% (as in regular healthy subjects), this probably represents a considerable overestimation of the subjects\u2019 ability to maximally drive their quadriceps muscles.\nIntroduction\nWhen a supramaximal electrical stimulus is applied to the nerve of a muscle during a voluntary isometric contraction, the contractile response of any motor units not yet (fully) recruited will lead to a force increment. Merton (1954) first reported a linear decrease of this superimposed force increment as voluntary contraction intensity increased in the adductor pollicis muscle. Hence, as voluntary activation (VA) increases, the force increment as a result of electrical stimulation decreases. For the most accurate assessment of maximal VA, Behm et\u00a0al. (1996) recommended using the maximal voluntary contraction (MVC) with a superimposed electrical stimulus, which is now common practice (Allen et\u00a0al. 1995; Babault et\u00a0al. 2003; Becker and Awiszus 2001; Behm et\u00a0al. 2001; de Ruiter et\u00a0al. 2004; Maffiuletti and Lepers 2003; Newman et\u00a0al. 2003; Oskouei et\u00a0al. 2003; Suter and Herzog 1997). Maximal VA is generally quantified by expressing the \u2018twitch\u2019 force increment on an MVC as a percentage of resting twitch force. Subtraction from 100% results in a value for maximal VA.\nGenerally, high levels of VA (\u223c90%) for the knee extensors are achieved by regular healthy subjects (e.g. Babault et\u00a0al. 2003; Behm et\u00a0al. 2001; Bulow et\u00a0al. 1993; de Ruiter et\u00a0al. 2004; Newman et\u00a0al. 2003; Oskouei et\u00a0al. 2003; Suter and Herzog 1997). An important implicit meaning of a VA level of 90% is that 90% of the maximal torque capacity (MTC) is reached, and that at most, a 10% further increase in torque is possible. In our experience, however, a greater increase in torque can be observed than the obtained VA level suggested when subjects succeed in improving their MVC in a later attempt during the same session. This suggests that VA can be overestimated using the conventional method of superimposed stimulation. This would have important consequences for conclusions drawn regarding calculated VA. The inability of patient groups to access their muscles\u2019 potential could be strongly underestimated. This has implications for the estimations made of the effects of paralytic poliomyelitis on strength and endurance for instance (Grabljevec et\u00a0al. 2005). Furthermore, many studies have been performed using target force levels expressed as %MVC (de Ruiter et\u00a0al. 2006; Grabljevec et\u00a0al. 2005; Hisaeda et\u00a0al. 2001; Kalmar and Cafarelli 1999; Ng et\u00a0al. 1994; Place et\u00a0al. 2005). For example, the %MVC at which full occlusion of blood flow of the knee extensors occurs during isometric contraction (de Ruiter et\u00a0al. 2006). Clearly, knowledge of the potential of a far greater torque increase than, for example, the 10% implied by a VA of 90% is very important.\nIn our experience, in subjects capable of consistently high levels of maximal VA (\u223c97%), the superimposed torque increment is already very small at high contraction intensities (\u223c70%MVC). Further increases in voluntary torque show only minor reductions of the superimposed torque increment. This is in accordance with the modelling study of Herbert and Gandevia (1999); for the adductor pollicis muscle they show that large increases of motoneuronal excitation near MVC will be accompanied by only minor reductions in the size of the superimposed force. At high contraction intensities, therefore, the %VA does not necessarily represent the same percentage of the muscles\u2019 maximal torque capacity. The first aim of the present study was therefore to assess the relationship between calculated VA and voluntary torque of the knee extensors at high contraction intensities. To achieve the best possible assessment of the relationship between conventionally assessed VA and voluntary torque (as %MVC), in the present study, only those individuals capable of consistently high levels of maximal VA were included.\nAn additional concern of using the ITT is the dependence of the calculated VA on the size of the resting twitch. Following a single electrical stimulus, tendon slack must first be taken up before resting peak twitch torque is reached. Therefore, during an isometric contraction, where most of the tendon slack has already been taken up, the twitch torque increment will be relatively large compared to the twitch torque at rest (Belanger and McComas 1981). By scaling the superimposed twitch to resting twitch torque, calculated VA will thus be lower than it otherwise would have been. The effect of tendon slack on the resting twitch has been reported to be of minor influence on the relationship between the superimposed response and voluntary torque, at flexed knee angles (Behm et\u00a0al. 2001; Bulow et\u00a0al. 1993; Rutherford et\u00a0al. 1986). However, tendon slack is greater at short compared to long muscle length (Muraoka et\u00a0al. 2004). Therefore, particularly at short muscle lengths, scaling the superimposed torque response to the response obtained at rest may lead to a relatively lower calculated VA at short (extended knee angles) compared to long muscle lengths (flexed knee angles).\nIn subjects with a high ability for maximal VA, a neural drive may be reached that is usually unattainable for regular healthy subjects. Moreover, during voluntary fast isometric torque development, rectified surface EMG levels of the knee extensors may be substantially higher than at the torque plateau of an MVC (de Ruiter et\u00a0al. 2004). These findings indicate a potential to increase neural drive at the torque plateau of a MVC (see also Herbert and Gandevia 1999). However, based on the shape of the stimulation frequency\u2013force (de Ruiter et\u00a0al. 1999; Deutekom et\u00a0al. 2000) and pCa\u2013force curve (Stephenson and Williams 1982) at high force levels large increases in neural drive and calcium are required for a comparatively small increase in force. As EMG is often measured to quantify the neural drive at different contraction intensities, this has important consequences for the EMG\u2013torque relationship. EMG is generally normalized to the EMG obtained during MVC, a disproportionate increase in EMG at very high force levels will consequently lead to a more curvilinear EMG\u2013torque relationship. However, this can only be demonstrated in subjects who are able to consistently reach high levels of neural drive. The shape of a subjects\u2019 EMG\u2013torque relationship would therefore be determined by the subjects\u2019 ability for VA. This would explain why both linear (Woods and Bigland-Ritchie 1983) and non-linear (Alkner et\u00a0al. 2000) EMG\u2013torque relationships have been reported. Therefore, this study further aims to investigate the EMG\u2013torque relationship of the knee extensors in subjects with high ability for VA.\nMethods\nSubjects\nSeven healthy male subjects (23\u201332\u00a0year, 69\u201383\u00a0kg, and 1.73\u20131.93\u00a0m) volunteered to be subjects for this investigation. Before participation, each subject was thoroughly informed about the procedures and provided written informed consent prior to testing. Only subjects capable of >95%VA across knee angles (see below) were included in the present study. The study was performed according to the Declaration of Helsinki and approved by the local ethics committee. Subjects did not perform any fatiguing exercise 48\u00a0h prior to measurements.\nExperimental procedures\nThe subjects visited our laboratory on two occasions with at least 1\u00a0day in between. All subjects were familiar with the set-up, electrical stimulation, and isometric knee extension from previous studies (de Ruiter et\u00a0al. 2004; Kooistra et\u00a0al. 2005). During the first experimental day, several contractions with superimposed stimulation (see below) were performed at 30\u00b0 (extended) and 60 and 90\u00b0 (flexed) knee angles. At the same time, surface EMG of the rectus femoris (RF), vastus lateralis (VL), and vastus medialis (VM) was recorded. To gain information concerning test-retest variability, the experimental procedure performed on experimental day 1 was repeated during experimental day 2.\nTorque measurements\nIsometric knee extension torque of the right leg was measured using a custom-built dynamometer (Kooistra et\u00a0al. 2005). Subjects were seated with their hips at 70\u00b0 (0\u00b0\u00a0=\u00a0full extension). Shoulders, hips, and lower thigh were strapped to the dynamometer. By very tight strapping of the hips and lower thigh, a contribution of the hip extensors to knee extension was absolutely impossible. Moreover, during contractions, subjects had to lean forward about 10\u00a0cm with their shoulders (while grasping the strap around their shoulders) pushing their lower back into the back rest. This further guaranteed that no hip extension occurred. The distal part of the shank was strapped to a force transducer (KAP, E\/200\u00a0Hz, Bienfait B.V. Haarlem, The Netherlands) that was attached to the lever arm of the dynamometer. A shin guard ensured subjects could exert maximal forces without discomfort at the shin. The compliance of the dynamometer at the position of the force transducer was 1.4\u00a0\u00d7\u00a010\u22124\u00a0deg\/Nm. The backrest, force transducer height, and its medio-lateral position were adjusted for precise alignment of the knee axis with the axis of rotation of the dynamometer arm. A crank enabled changing of the knee angle of the subject. Knee joint angles were determined with a handheld goniometer (model G300, Whitehall Manufacturing) using the greater trochanter and lateral epicondyle of the femur, and the lateral malleolus of the fibula as references. The dynamometer arm was positioned so that the indicated knee angles were angles in an active state, with subjects delivering \u223c50% of MVC. During pilot experiments using an electrogoniometer attached to the lateral side of the knee, the changes (active\u2013passive) in knee angle were found to be 3\u20137\u00b0, independent of knee angle, with minimal (<1\u00b0) changes above \u223c50% of MVC. Real-time force applied to the force transducer was displayed on line on a computer monitor and digitally stored (1\u00a0kHz) on computer disc. The force signals were automatically corrected for gravity at each angle: the average force applied by the weight of the limb to the transducer during the first 50\u00a0ms after the start of a recording, with the subject seated in a relaxed manner, was set to zero force by the computer program. Extension torque was calculated by multiplication of force with the individual lever arm and data will be presented as torque.\nElectrical stimulation\nA cathode (self-adhesive stimulation electrode, 5\u00a0\u00d7\u00a05\u00a0cm, Schwa-Medico, The Netherlands) was placed over the femoral nerve. The anode (13\u00a0\u00d7\u00a08\u00a0cm) was placed over the gluteal fold. The quadriceps femoris muscle was stimulated transcutaneously with rectangular pulses of 100\u00a0\u03bcs using a computer-controlled constant current stimulator (Digitimer DS7H, Digitimer Ltd., Welwyn Garden City, UK). Stimulation current was increased until torque measured in response to a triplet (three 100\u00a0\u03bcs pulses applied at 300\u00a0Hz) levelled off at each knee angle tested. The current (in mA) was then increased by a further 50\u00a0mA to ensure supramaximal stimulation (range: 250\u2013600\u00a0mA). At this point, we considered all muscle fibres of the knee extensors to be activated. Pilot studies had shown that triplet torque did not increase after an MVC at any knee angle, indicating that any potential potential post-activation potentiation did not result in a triplet torque increase (see also Discussion). Usually, torque generated by the triplet is about \u223c35% of the MTC (Kooistra et\u00a0al. 2005). Furthermore, in pilot studies, the stimulation current was increased far beyond supramaximal levels (up to 200%) without a subsequent decrease in triplet torque. Hence, the hamstring muscles were assumed not to be activated during supramaximal stimulation of the knee extensors (see also Discussion).\nVoluntary activation\nDuring experimental day 1, the level of maximal VA was determined in random order at 30\u00b0, 60\u00b0, and 90\u00b0 knee angles. At each knee angle, subjects were asked to maximally generate isometric torques for about 3\u20134\u00a0s to determine MVC extension torque. Real-time torque was visible on a computer monitor and subjects were vigorously encouraged to exceed their maximal value, which was also displayed. MVC torque was defined as highest torque within a stable 1,000\u00a0ms plateau of the torque signal. MVC torque was taken as the highest value, which did not exceed preceding attempts by >5%, allowing a maximum of four attempts to prevent fatigue. In the incidental case a trial had no stable plateau, the trial was repeated. Subjects then performed a series of superimposed measurements consisting of a triplet applied to a fully relaxed muscle and another triplet superimposed on the stable part of the torque signal (Fig.\u00a01). If the torque level was unstable (>1% torque fluctuation) prior (100\u00a0ms) to stimulation, the contraction was repeated. As all our subjects were very experienced at maintaining stable torque levels at all contraction intensities, it was rarely necessary to repeat a measurement. As an indication, for all subjects, less than 1 in 10 contractions (across all knee angles and intensities) were repeated. Several different contraction intensities with an emphasis toward higher intensities were chosen (25, 50, 70, 80, 90, and 100%MVC). At each knee angle, the order of the contraction intensities with superimposed triplet was randomized, and the order of the knee angles was randomized per subject. A 3-min rest period separated contractions. For each superimposed contraction, the accepted formula for the interpolated twitch torque technique (Allen et\u00a0al. 1995; Babault et\u00a0al. 2001, 2006; Becker and Awiszus 2001; de Ruiter et\u00a0al. 2005; Kooistra et\u00a0al. 2005; Maffiuletti and Lepers 2003; McKenzie et\u00a0al. 1992; Newman et\u00a0al. 2003; Oskouei et\u00a0al. 2003) was used to determine the level of voluntary activation (VA): Fig.\u00a01Typical example of a superimposed contraction. The resting triplet torque as a result of the electrical stimulation as well as the triplet torque increment on a 100%MVC contraction at the 90\u00b0 knee angle are shown. The timing of the triplet stimulation is shown by the vertical arrows and vertical dashed lines. Voluntary activation (VA) is generally calculated using the following equation: VA (%)\u00a0=\u00a0100\u2013[(triplet torque increment\/resting triplet torque)\u00a0\u00d7\u00a0100]. In this particular example, voluntary activation was calculated to be: 100\u2013[(4.2\/94.6)\u00a0\u00d7\u00a0100]\u00a0=\u00a095.6%\nWith this method, the triplet torque increment is expressed as a percentage of the resting triplet torque, and subtraction from 100% results in a value for VA (Fig.\u00a01). Maximal VA was obtained from the 100%MVC superimposed contraction. To illustrate the relationship between superimposed and voluntary torque from 90 to 100%VA, only subjects capable of an average maximal VA of >95% across knee angles were included in the present study. MVC torques as close to the muscles\u2019 MTC as possible were thus obtained. Due to our strict inclusion criteria (average VA of >95%) at each knee angle, we were able to select a contraction at a lower than maximal torque level where the superimposed triplet was \u223c10% of resting triplet. At this torque level, VA is conventionally calculated to be \u223c90%, and we expressed this torque level as a percentage of MVC. We could thus compare the theoretical 10% torque increase implied by a conventionally calculated VA level of 90% with the torque increase actually achieved by our subjects during MVC. This provides us with a good indication of the potential torque increase that may be possible in regular healthy subjects with a maximal VA level of \u223c90%. The \u223c90% is a VA level that is frequently reported in the literature for regular healthy subjects (Babault et\u00a0al. 2001, 2003; Bulow et\u00a0al. 1993; de Ruiter et\u00a0al. 2004; Newman et\u00a0al. 2003; Oskouei et\u00a0al. 2003; Suter and Herzog 1997).\nFurthermore, for each subject and for each superimposed contraction, the increment in torque as a result of the superimposed triplet was plotted as a function of the torque reached just prior to the superimposed stimulation (e.g. Figs.\u00a02, 3a). Curvilinear relationships between voluntary and superimposed torque are often reported in the literature for group data (e.g. Behm et\u00a0al. 2001; Dowling et\u00a0al. 1994; Oskouei et\u00a0al. 2003; Suter and Herzog 1997). In the present study, however, neither a linear nor a curvilinear fit closely followed the data points for any of the individual subjects at any knee angle. However, linear regression was performed on the highest four contraction intensities (70, 80, 90, and 100%), and R2 values were calculated at each knee angle for each subject. This was done to show that although superimposed torque increments were small at high contraction intensities, the torque increment continued to decrease with contraction intensity (e.g. Fig.\u00a03a, b).\nFig.\u00a02Torque increment as result of the triplet versus voluntary torque delivered at the 30\u00b0 knee angle by a normal healthy subject in a pilot study. The empty circles denote the first day of testing, and the black triangles the second day. The dashed and solid lines represent linear regression fits for the data points on experimental days 1 and 2, respectively, excluding the resting triplet torque at 0\u00a0Nm. The resting triplet torque is clearly underestimated as it is very similar to the triplet torque increment at \u223c30\u00a0Nm. The dotted lines represent backward extrapolation of the regression fits for both experimental days and illustrate the underestimation of the resting triplet torque as a result of tendon slack. For each experimental day, the respective equations and R2 values are shown. Using the interpolated twitch torque technique, voluntary activation (VA) is calculated to be quite high for experimental days 1 (84.4%) and 2 (94.7%)Fig.\u00a03a The torque increment as a result of the triplet on a voluntary contraction (y-axis) is shown as a function of the voluntarily delivered torque just before the triplet (x-axis). The data points shown are those of subject no. 1, a subject with high ability for maximal voluntary activation, at the 30\u00b0 knee angle. The open circles denote results from experimental day 1 and the filled triangles from experimental day 2. It is immediately apparent that using a linear or a curvilinear fit over all data points is incorrect for this subject. The dashed and solid lines illustrate linear regression fits that have been performed for a voluntary torque level of \u223c50\u2013150\u00a0Nm for experimental days 1 and 2, respectively. The dotted lines represent backward extrapolation of the regression fits for both experimental days and illustrate the potential effect of tendon slack on the resting triplet torque, especially when compared to the triplet torque increment at \u223c60\u00a0Nm. For clarity, and to illustrate the continued decrease of the triplet torque increment with increasing voluntary torque, the data points of torque levels above 140\u00a0Nm have been replotted in Fig.\u00a03b. b Data points from Fig.\u00a03A have been replotted for torque levels above 140\u00a0Nm. The torque increment as a result of the triplet is shown as a function of the voluntarily delivered torque just before the triplet (x-axis) for four high intensity contractions (70, 80, 90, and 100%MVC). The open circles represent data points obtained on experimental day 1 and the filled triangles experimental day 2. A linear regression line shows the continued decrease in triplet torque increment with increase in contraction intensity for each set of four data points with corresponding R2 values\nSurface electromyography\nElectromyographic activity of the RF, VL, and VM muscles was recorded using surface EMG electrodes (Blue Sensor, Ambu, \u00d8lstykke, Denmark, lead-off area: 1.0\u00a0cm2). After shaving, roughening, and cleansing the skin with 70% ethanol, electrodes were placed on the muscle belly in a bi-polar configuration in line with the muscle fibre direction with a centre to centre inter-electrode distance of 25\u00a0mm. Reference electrodes were placed on bone structures, on each patella, and on the lateral epicondyle of the femur of the right leg. Surface EMG signals were amplified (\u00d7100), digitized (1\u00a0kHz), and stored with the force signal on computer disc. All EMG signals were band-pass filtered (10\u2013400\u00a0Hz). Rectified surface EMG amplitude (rsEMG) was calculated for the RF, VL, and VM for 1,000\u00a0ms segments just before the superimposed stimulation. RsEMG values obtained during the highest torque level on each experimental day were set to 100%.\nStatistics\nAll results are presented as mean\u00a0(\u00a0SD. Knee angle effects were tested for significance with repeated measures ANOVA. If significant main effects were observed, Bonferroni tests were performed for post hoc analysis. Test-retest reliability was tested for using the intraclass correlation coefficient. The level of significance of all statistical analyses was set at P\u00a0<\u00a00.05.\nResults\nTorque levels during the superimposed contractions at the 25, 50, 70, 80, 90, and 100%MVC levels were not different from the intended values or different between experimental days. Linear extrapolation of the torque increment as a result of the triplet on the high intensity contractions (70, 80, 90, and 100%MVC) provided high R2 values at each knee angle (30\u00b0: 0.89\u00a0\u00b1\u00a00.09; 60\u00b0: 0.90\u00a0\u00b1\u00a00.10; and 90\u00b0: 0.87\u00a0\u00b1\u00a00.10 averaged over days), which were not different between knee angles or the two experimental days (e.g. Fig.\u00a03b). The continuing consistent decrement of the triplet torque increment with increase in contraction intensity was denoted by the overall negative slope (\u22120.13\u00a0\u00b1\u00a00.05 across days and angles), which was not different between days and angles. The relationship between the triplet torque increment and voluntary torque is shown in Fig.\u00a04 for the 30, 60, and 90\u00b0 knee angles.\nFig.\u00a04The torque increment as result of the triplet is shown as a function of voluntary torque for subject no. 5 on experimental day 1. Note that at the higher contraction intensities (>70%), the shape of the curve is similar at the 30\u00b0 (black triangles, solid line), 60\u00b0 (white squares, dashed line), and 90\u00b0 (grey circles, dotted line) knee angle at the higher (>70%MVC) contraction intensities\nThere was no difference in resting triplet torque or MVC between experimental days and average values across days are presented. At the 60\u00b0 knee angle, MVC was significantly (P\u00a0<\u00a00.05) greater compared to both 30 and 90\u00b0 knee angles (Table\u00a01). Resting triplet torque, however, was significantly (P\u00a0<\u00a00.05) lower at 30\u00b0 compared to 60 and 90\u00b0 (Table\u00a01). Reproducibility for both MVC and resting triplet torque was very high, which was illustrated by high ICC values (Table\u00a01).\nTable\u00a01TorqueKnee angle30\u00b060\u00b090\u00b0ICCMVC (Nm)186.1\u00a0\u00b1\u00a036.6278.4\u00a0\u00b1\u00a044.5*189.6\u00a0\u00b1\u00a019.70.99***Triplet torque (Nm)65.9\u00a0\u00b1\u00a010.0**91.6\u00a0\u00b1\u00a015.880.1\u00a0\u00b1\u00a010.80.96***All values are means\u00a0\u00b1\u00a0SD, averages across days shown, n\u00a0=\u00a07. Maximal voluntary isometric knee extension (MVC) and resting triplet torque for the 30\u00b0, 60\u00b0, and 90\u00b0 knee angle*\u00a0Significantly different from 30\u00b0 to 90\u00b0 knee angle (P\u00a0<\u00a00.05)**\u00a0Significantly different from 60\u00b0 to 90\u00b0 knee angle (P\u00a0<\u00a00.05)***\u00a0Significant ICC, the intraclass correlation coefficient (ICC) was calculated for the measurements made on experimental days 1 and 2 and is shown in the last column\nVoluntary activation\nOverall, there was no difference in maximal VA, determined by applying the ITT to the highest MVC, between experimental days and average values across days are presented. The significantly lower triplet torque at 30\u00b0 compared to the 90\u00b0 knee angle did not result in a lower maximal VA at 30\u00b0 compared to 90\u00b0 At each knee angle, maximal VA was very high and similar between knee angles (Fig.\u00a05). Furthermore, at each knee angle, at torques where the triplet increment was \u223c10% (8.5\u00a0\u00b1\u00a02.6%), VA was calculated to be \u223c90% (91.5\u00a0\u00b1\u00a02.6%, across angles). This VA level turned out to be significantly higher compared with the relative voluntary torque level at which the triplet was superimposed. The torques at which VA was calculated to be 91.5\u00a0\u00b1\u00a02.6% represented only 79.6\u00a0(\u00a06.1% of MVC (across knee angles), a finding that was similar at each knee angle (P\u00a0>\u00a00.05). Note that at this torque level, the triplet torque increments were already small (3.2\u00a0\u00b1\u00a01.1% MVC) compared to the torque generated when the triplet was applied on the resting muscle (\u223c37% MVC). The key point we want to address with our study is illustrated in Fig.\u00a06. For both subject nos. 1 and 7, a VA level of \u223c90% was calculated at the 60\u00b0 knee angle at a torque level of \u223c250\u00a0Nm. This suggests a potential further increase of maximal torque by 10%. However, subject no. 1 was able to significantly increase his torque production by more than 30%.\nFig.\u00a05Average (2\u00a0days) maximal voluntary activation (VA) per subject for the 30\u00b0 (black triangles), 60\u00b0 (white squares), and 90\u00b0 (grey circles) knee angles. Note that only 2 out of a total of 21 data points are below 95%VA. The horizontal dotted line indicates the 90%VA level that is generally obtained for the knee extensors by regular healthy subjectsFig.\u00a06The torque increment as result of the triplet is shown as a function of voluntary torque for subject no. 1 (grey squares) and 7 (open circles) at the 60\u00b0 knee angle on day 1. At the 60\u00b0 knee angle, the maximal VA level of subject no. 7 calculated at 250\u00a0Nm was 90.7%, which resembled that of a regular subject, and is exceptionally low in our study (see lowest point in Fig.\u00a05). A VA of 90.0% is also calculated at 240\u00a0Nm for subject no. 1. In both subjects, this implies a further potential increase in torque of 10%, yet a >30% torque increase (to 330\u00a0Nm) could be demonstrated in subject 1. This is most likely due to the exceptional neural drive of subject 1 (\u223c98%VA) during his best attempts at this knee angle\nEMG\nThe rsEMG values obtained during MVC were similar between days at each knee angle (P\u00a0=\u00a00.96). For all submaximal contraction intensities, normalized rsEMG values for each muscle (RF, VL, and VM) at each contraction intensity was similar between days; hence, averaged values across days are presented. Furthermore, no difference between knee angles or muscles was found, and with each increase in contraction intensity, there was a significant increase in normalized rsEMG (Fig.\u00a07a, VL shown). An increase of normalized rsEMG of \u223c21% (across angles and muscles) was seen for a 25% increase in torque from 25 to 50%MVC (Fig.\u00a07a). When contraction intensities approached MVC, there was a much larger increase in normalized rsEMG (\u223c34%) relative to the torque increase of only \u223c18% at all knee angles (Fig.\u00a07a). In regular healthy subjects, the highest MVC would have occurred at torque levels where VA would have been calculated to be \u223c90%, and consequently the EMG\u2013torque relationship would most likely have been considerably less curvilinear. This is illustrated by the two EMG\u2013torque relationships shown in Fig.\u00a07b for the VL at the 90\u00b0 knee angle. The first EMG\u2013torque relationship includes all contraction intensities measured, whereas for the second EMG\u2013torque relationship, the 90 and 100%MVC contraction intensities have been removed. In the latter case, all rsEMG values are normalized to a torque level that corresponds to the maximal VA level of \u223c90% (generally obtained by regular healthy subjects), the result being a near linear EMG\u2013torque relationship (Fig.\u00a07b).\nFig.\u00a07a Normalized rsEMG levels averaged over the two experimental days versus normalized torque for the vastus lateralis (VL) muscle at 30\u00b0 (black triangles), 60\u00b0 (white squares), and 90\u00b0 (grey circles) knee angles. *Significantly different (P\u00a0<\u00a00.05) from preceding intensity level. At the lower contraction intensities, \u0394EMG1 denotes the increase in EMG (21%, across muscles) that is accompanied by a comparatively larger increase torque (\u223c25%), which is denoted by \u0394Torque1. Conversely, \u0394EMG2 denotes the much larger increase in EMG (\u223c34%, across muscles) that is accompanied by a comparatively smaller increase in normalized torque (\u223c18%, denoted by \u0394Torque2) as the contraction intensity approaches MVC. Note that on average for \u0394Torque2, calculated voluntary activation (VA) increased from 91.5\u00a0\u00b1\u00a02.6% to 97.2\u00a0\u00b1\u00a02.3%. Thus, a 34% increase in normalized rsEMG was accompanied by an 18% increase in torque, for which only a \u223c5.7% increase in VA (denoted by \u0394VA) was calculated. As a consequence of the relatively large increase in EMG as MVC is approached, the rsEMG of contraction intensities below MVC are normalized to a relatively large value and are located well beneath the line of identity. b Normalized rsEMG levels averaged over days versus normalized torque for the vastus lateralis (VL) muscle at the 90\u00b0 knee angle. The white circles represent rsEMG values that have been normalized to the MVC of subjects with a very high ability for voluntary activation (VA). With the black circles, the EMG\u2013torque relationship for regular healthy subjects has been predicted. The black circles denote rsEMG values that have been renormalized to the rsEMG value reached at 90%VA. This is similar to the usual maximal VA in regular individuals. Note that the EMG\u2013torque relationship predicted for regular healthy subjects is linear and closer to the line of identity compared to subjects with very high ability for voluntary activation\nAs a consequence of the disproportionate increase in EMG as MVC is approached, normalized rsEMG values obtained at intensities below MVC are normalized to a relatively large value. Hence, these normalized rsEMG values are relatively smaller compared to the relative torques at which they have been obtained. For example, at 25 and 50%MVC, normalized rsEMG for the RF, VL, and VM muscle was significantly less than would be expected based on the %MVC at which they were obtained at all knee angles (Fig.\u00a07a, VL shown). By normalizing the rsEMG levels to the rsEMG values obtained at a torque level that corresponds to the VA level of 90% as found in regular healthy subjects, normalized rsEMG values are closer to the line of identity, and a more linear EMG\u2013torque relationship is seen (Fig.\u00a07b).\nDiscussion\nIn the present study, the relationship between calculated VA and voluntary torque was assessed in subjects with high ability for maximal VA at different knee angles. At the same time, the EMG\u2013torque relationship in these selected subjects was examined. The first and main finding of the present study was that when VA is calculated to be \u223c90% (as in regular healthy subjects), this probably represents a considerable overestimation of the subjects\u2019 ability to maximally drive their quadriceps muscles. An additional finding was that, although resting triplet torque was lower at 30\u00b0 versus 90\u00b0, this was of minor influence on the calculated maximal VA in our subjects. Furthermore, a relatively large increase in normalized rsEMG was observed as MVC was approached, making the shape of the normalized EMG\u2013torque relationship of the knee extensors curvilinear.\nMaximal voluntary activation and knee angle\nSubjects in the present study exhibited a consistent high level of maximal VA of the knee extensors (Fig.\u00a05), which is higher than the maximal VA levels of 90% that are generally reported for the knee extensors in regular healthy subjects when applying the ITT (Babault et\u00a0al. 2001, 2003; Bulow et\u00a0al. 1993; de Ruiter et\u00a0al. 2004; Newman et\u00a0al. 2003; Oskouei et\u00a0al. 2003; Suter and Herzog 1997). Although full activation of the knee extensors has been reported in the past (Bigland-Ritchie et\u00a0al. 1986; Rutherford et\u00a0al. 1986), in those studies a single superimposed twitch was used. Due to the declining signal-to-noise ratio with increase in contraction intensity, the detection of a single superimposed twitch is difficult, and 100% VA may mistakenly have been assumed (Dowling et\u00a0al. 1994). To improve the signal-to-noise ratio, multiple stimuli have been suggested (Gandevia and McKenzie 1988; Suter and Herzog 2001). In the present study, triplet stimulation provided considerable resting triplet torque levels at each knee angle (Table\u00a01), which ensured a good signal-to-noise ratio. Furthermore, pilot studies had shown no increase in triplet torque before and after an MVC at any knee angle. By using supramaximal and high frequency (300\u00a0Hz) triplet stimulation, we avoided the potential influence of post-activation potentiation on our data.\nA limitation of the ITT may be the scaling of the twitch increment to the resting twitch. When muscle is stimulated by a twitch at rest, all tendon slack has to be taken up; conversely, for a twitch superimposed on an ongoing isometric contraction, no further slack has to be taken up (Suter and Herzog 1997). Since calculated VA is determined by the scaling of the triplet torque increment to the resting triplet, calculated VA will be reduced by the influence of tendon slack. As the effect of tendon slack is greater at short versus long muscle length (Muraoka et\u00a0al. 2004), VA will be influenced to a greater degree at short versus long muscle length. Besides tendon slack, length-dependent Ca2+ sensitivity may also influence the size of the resting triplet by shifting the resting triplet torque\u2013length relationship to shorter muscle length compared to the MVC torque\u2013length relationship. The length-dependent effect of tendon slack and Ca2+ sensitivity were reduced by the use of a triplet (Kawakami et\u00a0al. 2001), but not abolished as triplet torque was significantly smaller at the 30\u00b0 versus 90\u00b0 knee angle (Table\u00a01), whereas MVC levels were similar. This implies that despite using triplet stimulation, compared to the MVC, resting triplet torque remains relatively lower at the 30\u00b0 versus 90\u00b0 knee angle. The latter is supported by Figs.\u00a02 and 3A, where the triplet response is similar at 0 and 25%MVC at the 30\u00b0 knee angle. It is evident that calculated VA will be influenced by tendon slack, as the resting triplet is influenced by tendon slack, especially at short muscle length, whereas the triplet torque increment is not (Suter and Herzog 1997).\nDespite a lower resting triplet torque at the 30\u00b0 compared to the 90\u00b0 knee angle, maximal VA was not different between the 30\u00b0 and 90\u00b0 knee angle in our subjects. From the literature, it is unclear whether maximal VA is knee angle-dependent. Kubo et\u00a0al. (2004) report a lower level of maximal VA at extended compared to flexed knee angles, whereas Suter and Herzog (1997) find maximal VA to be highest at the most extended knee angle (15\u00b0). Newman et\u00a0al. (2003) and Babault et\u00a0al. (2003), however, find no effect of knee angle on maximal VA, which is in line with our current findings. Note that due to our selection criteria (average maximal VA of 95%, and at least 90% at each knee angle), maximal VA was very high (\u223c97%) and close to 100%. Potential knee angle-dependent differences in maximal VA could consequently only occur within a very small range. Furthermore, the small size of the superimposed triplet torque makes the calculation of maximal VA insensitive to large differences in the size of the resting triplet torque. By hypothetically increasing the resting triplet torque at 30\u00b0 by 20%, making it similar to the 90\u00b0 knee angle, an increase in maximal VA of less than 0.5% is calculated at the 30\u00b0 knee angle. In subject populations possessing lower maximal VA levels (e.g. patient groups), a relatively greater increment will be obtained on an MVC. This greater increment is more susceptible to the muscle-length dependent influence of, for example, tendon slack on the size of the resting triplet. The influence of tendon slack would have been greater still had a twitch instead of a triplet been used (Kawakami et\u00a0al. 2001), leading to an even greater difference in resting twitch size at 30\u00b0 versus 90\u00b0. Combined with the use of a resting twitch, therefore, a muscle length-dependent effect of tendon slack is expected to significantly influence the calculation of VA in this subject population.\nCalculated voluntary activation in relation to relative voluntary torque\nFor the most accurate assessment of maximal VA, Behm et\u00a0al. (1996) recommended using the MVC with a superimposed electrical stimulus. As mentioned previously, in regular healthy subjects a maximal VA level of \u223c90% is often reported for the knee extensors using this method (Babault et\u00a0al. 2001, 2003; Bulow et\u00a0al. 1993; de Ruiter et\u00a0al. 2004; Newman et\u00a0al. 2003; Oskouei et\u00a0al. 2003; Suter and Herzog 1997) implying that the MTC has almost been reached. In the present study, with our selected subject group, we were able to demonstrate that 90%VA is already calculated at torque levels that represent only \u223c79% of MVC (across knee angles). In some subjects, this phenomenon is very pronounced. For the subject shown in Fig.\u00a03a and b, already at a torque of \u223c148\u00a0Nm, VA using the ITT was calculated to be 95.3 and 97.1% for experimental day 1 and 2, respectively. These are very high VA levels that have been calculated at 148\u00a0Nm, especially when compared with the MVC of >200\u00a0Nm that was obtained on both experimental days. Note that the higher part of the curve (in this example over 140\u00a0Nm), as shown in Fig.\u00a03b can usually not be obtained in regular healthy subjects. In our experience, we can only demonstrate the existence of this part of the curve in subjects with a very high ability for maximal VA (>99% in this extreme example, subject no. 1 in Fig.\u00a05). This finding strongly suggests that the 90%VA reported for regular healthy subjects is a large underestimation of the MTC of the muscle. A far greater torque increase therefore seems possible than the 10% implied by a calculated VA of 90% (Fig.\u00a06). The main new finding, therefore, is that the present study for the first time quantifies the difference between calculated VA and relative voluntary torque.\nExperimental and methodological factors\nWhen relating the triplet torque increment to the triplet torque obtained at rest, it is important to maintain supramaximal stimulation during both conditions. In a previous study examining superimposed stimulation of the knee extensors (Behm et\u00a0al. 1996), the authors reported significantly lower M-wave amplitudes during a superimposed stimulation on an MVC compared to during rest. They presumed that the contraction of the knee extensors resulted in a displacement of the stimulating electrode from its optimal position over the femoral nerve. In pilot studies of the present investigation, M-wave amplitude at rest and superimposed on an MVC was similar. Moreover, a substantial (50%) increase in stimulation current did not lead to an increase in the size of the triplet increment on high intensity contractions (70 and 80%MVC). This strongly suggests that even during high intensity contractions, there was supramaximal stimulation of the femoral nerve during superimposed triplet stimulation. By using multiple stimuli during superimposed stimulation, spinal reflexes may have more time to diminish the superimposed response (Herbert and Gandevia 1999; Herbert et\u00a0al. 1997). Modeling suggests that the use of twin stimuli at 100\u00a0Hz has a minimal effect on the estimates of VA (Herbert and Gandevia 1999). By applying a stimulation frequency of 300\u00a0Hz in the present study, triplet stimulation occurred within 10\u00a0ms, which is even less when compared to the use of twin stimuli at 100\u00a0Hz. The influence of spinal reflexes on the superimposed stimulation is therefore considered negligible. Furthermore, although the current passes through the sciatic nerve that innervates the hamstring muscles, its stimulation was regarded negligible. This is illustrated by the very small EMG recording of the m. biceps femoris compared to the VL muscle during stimulation of the knee extensors [Fig.\u00a08, data from a previous study of ours (Kooistra et\u00a0al. 2005)]. We accordingly assumed a marginal activation, if at all, of the hamstring muscles during superimposed stimulation and therefore no influence on our calculation of VA. In a previous study by our group (de Ruiter et\u00a0al. 2004), we measured the contribution of co-activation during isometric knee extensions during brief MVCs and found it to be very small (5\u201310%). As this finding is in accordance with previous findings (Newman et\u00a0al. 2003; Psek and Cafarelli 1993), we did not measure coactivation in the present study and considered it minimal.\nFig.\u00a08Representative EMG recordings are shown for the vastus lateralis (VL, upper panel) and biceps femoris (BF, lower panel) muscle. On the left, the M-wave for the VL as a result of supramaximal twitch stimulation applied to the n. femoralis. The simultaneous EMG recording for the BF muscle is shown in the lower left panel. On the right, the EMG recording during maximal voluntary extension (top, right) and flexion (lower, right) torque is shown to illustrate that the lower M-wave of the BF is not due a lower sensitivity of the BF recordings compared to the VL recordings\nThe EMG\u2013torque relationship\nAs expected, when MVC was approached we observed a relatively large increase in normalized rsEMG (Fig.\u00a07a). This large increase makes the shape of the EMG\u2013torque relationship of the knee extensors curvilinear. This is in contrast to the findings of Woods and Bigland-Ritchie (Woods and Bigland-Ritchie 1983) who report a linear EMG\u2013torque relationship, yet in accordance with Alkner et\u00a0al. (2000) and Pincivero and Coelho (2000) who show a non-linear relationship for the knee extensors.\nBased on a modelling study of motor-unit pools, Fuglevand et\u00a0al. (1993) indicate that the difference between muscles that exhibit the linear as opposed to the nonlinear form of the EMG\u2013torque relationship may be related to differences in firing rate behaviour rather than due to differences in recruitment organization. It is reasonable to expect that only subjects with a high ability for maximal VA (high neural drive) will achieve very high motor unit firing rates. For this large increase in firing rate, however, little gain in torque may be predicted (Herbert and Gandevia 1999). The large increase in excitation, as represented by the disproportionate increase in normalized rsEMG when MVC was approached, confirms the model study by Herbert and Gandevia (1999). However, in contrast to their model where essentially 100%MVC was achieved at 60% of maximal voluntary excitation, in the present study, we experimentally determined that the disproportionate increase in excitation lead to a more substantial increase in torque (\u223c18%). However, many factors may affect the EMG\u2013torque relationship that have not been taken into account. Those include, signal cancellation from overlapping positive and negative phases of action potentials (Day and Hulliger 2001); the sigmoid relationship between motor unit force and firing rate (Herbert and Gandevia 1999); and the nonlinear distribution of recruitment thresholds (Fuglevand et\u00a0al. 1993). Interestingly, in a review, de Luca (1997) states that the amplitude of the EMG signal should be normalized to values less than 80%MVC. Above this level, the EMG signal is said to be exceptionally unstable and, hence, is unable to provide a suitable reference point. As shown in the present study, very large increases in EMG coincide with very small increases in calculated VA (Fig.\u00a07a). This is consistent with unstable EMG measurements for similarly high torque levels.\nEMG, superimposed torque and voluntary activation\nWhen combining the non-linearity of the EMG\u2013torque relationship and non-linearity of the superimposed triplet-torque relationship, the EMG\u2013torque relationship indicates that a large amount of extra EMG produces relatively little extra torque at high intensities. The triplet given to evoke the superimposed triplet could be considered as extra EMG in that three extra action potentials are added. As voluntary EMG is reduced in its effectiveness at producing torque in strong voluntary contractions, the extra activation from the triplet might be reduced similarly. This is in accordance with a model study of the adductor pollicis motoneuron pool (Herbert and Gandevia 1999). The authors predict large increases in motoneuronal excitation for minor increases in voluntary force and VA (as shown by small decreases in the superimposed twitch, their Fig.\u00a09c). As mentioned above, this is consistent with the EMG\u2013torque data in the present study (Fig.\u00a07a). The small decreases in modelled interpolated twitch amplitude are also consistent with the current findings, where the triplet torque increment was only 3.2\u00a0\u00b1\u00a01.1%MVC at a torque level that corresponded to 79.6\u00a0\u00b1\u00a06.1%MVC and showed only a minor further reduction in size (to 1.0\u00a0\u00b1\u00a00.9%MVC).\nNote that the minor reduction in triplet increment with increasing voluntary torque is, and can only be, observed in those subjects consistently able to achieve high levels of VA, as would be expected from the model (Herbert and Gandevia 1999). This would indicate that these subjects are capable of excitation levels that (almost) evoke their MTC. Having noted this, it is important to point out that a 100% excitation may not be necessary to evoke the MTC as \u201conly\u201d 60% excitation was modelled to elicit 100% MTC (Herbert and Gandevia 1999). This is in agreement for example with the much greater excitation (EMG) levels attained during voluntary fast isometric knee extensions compared to during the plateau of an MVC (de Ruiter et\u00a0al. 2004).\nHow can we be certain that our selected subjects did indeed approach their true MTC? The only way to truly determine the MTC of a muscle is by the use of tetanic nerve stimulation. In two subjects with exceptional ability for maximal VA (>99%), supramaximal tetanic nerve stimulation (2\u00a0s, at 150\u00a0Hz) of the knee extensors was performed and torque levels showed no further increase above MVC (data not presented). Applying supramaximal tetanic nerve stimulation is not recommended, however, as it is highly unpleasant and could be harmful to certain vulnerable subject groups (e.g. subjects with an ACL deficiency).\nCurrently, the ITT is the only feasible available technique used to determine VA of the knee extensors and, as mentioned previously, it is used extensively (Allen et\u00a0al. 1995; Babault et\u00a0al. 2003; Becker and Awiszus 2001; Behm et\u00a0al. 1996, 2001; de Ruiter et\u00a0al. 2004; McKenzie et\u00a0al. 1992; Newman et\u00a0al. 2003; Oskouei et\u00a0al. 2003; Suter and Herzog 1997). The findings of the present study indicate that for the knee extensors, despite a commonly reported maximal VA of \u223c90% for regular healthy subjects, a far greater relative torque increase seems possible than the 10% implied by 90%VA. This has important implications for conclusions that are drawn regarding calculated VA. Especially because many studies have used target force levels expressed as %MVC to investigate: effects of caffeine neuromuscular function (Kalmar and Cafarelli 1999), the %MVC at which full occlusion of the blood supply occurs (de Ruiter et\u00a0al. 2006), and fatigue of the quadriceps in patients with multiple sclerosis for instance (de Haan et\u00a0al. 2000). Clearly, knowledge of the potential of a far greater torque increase than, for example, 10% implied by a VA of 90% is very important.\nIn conclusion, when maximal VA is calculated to be \u223c90% (as in regular healthy subjects), this probably represents a considerable overestimation of the subjects\u2019 ability to maximally drive their quadriceps muscles. Moreover, the effect that the length dependent size of the resting triplet has on the calculation of VA with the conventional method is only minimal when VA is greater than 95%. Furthermore, a curvilinear shape of the EMG\u2013force relationship may be caused by a disproportionately large increase in normalized rsEMG when MVC is approached in subjects with very high capacity to drive their muscles maximally.","keyphrases":["voluntary activation","twitch interpolation","emg","muscle length","neural drive"],"prmu":["P","P","P","P","P"]}
{"id":"Pflugers_Arch-3-1-2040175","title":"Dysregulation of the expression and secretion of inflammation-related adipokines by hypoxia in human adipocytes\n","text":"The effect of hypoxia, induced by incubation under low (1%) oxygen tension or by exposure to CoCl2, on the expression and secretion of inflammation-related adipokines was examined in human adipocytes. Hypoxia led to a rapid and substantial increase (greater than sevenfold by 4 h of exposure to 1% O2) in the hypoxia-sensitive transcription factor, HIF-1\u03b1, in human adipocytes. This was accompanied by a major increase (up to 14-fold) in GLUT1 transporter mRNA level. Hypoxia (1% O2 or CoCl2) led to a reduction (up to threefold over 24 h) in adiponectin and haptoglobin mRNA levels; adiponectin secretion also decreased. No changes were observed in TNF\u03b1 expression. In contrast, hypoxia resulted in substantial increases in FIAF\/angiopoietin-like protein 4, IL-6, leptin, MIF, PAI-1 and vascular endothelial growth factor (VEGF) mRNA levels. The largest increases were with FIAF (maximum 210-fold), leptin (maximum 29-fold) and VEGF (maximum 23-fold); these were reversed on return to normoxia. The secretion of IL-6, leptin, MIF and VEGF from the adipocytes was also stimulated by exposure to 1% O2. These results demonstrate that hypoxia induces extensive changes in human adipocytes in the expression and release of inflammation-related adipokines. Hypoxia may underlie the development of the inflammatory response in adipocytes, leading to obesity-associated diseases.\nIntroduction\nWhite adipose tissue has traditionally been considered as primarily a vehicle for the storage of fuel in the form of triacylglycerols, but several other functions are now recognised for the tissue. In particular, adipose tissue is a major endocrine organ secreting several key hormones, notably leptin and adiponectin [11, 26, 29, 37, 38]. These hormones are part of the large group of protein signals and factors secreted by adipocytes, termed adipokines, many of which are linked to immunity and the inflammatory response [14, 26, 29, 38]. These encompass major cytokines and chemokines, including TNF\u03b1, IL-1\u03b2, IL-6, IL-10, MCP-1 and MIF [11, 26, 38].\nObesity, which is characterised by a major expansion in adipose tissue mass, is associated with a state of chronic mild inflammation, there being increased circulating levels of inflammatory markers such as C-reactive protein, IL-6, IL-18, haptoglobin, MIF and PAI-1 [11, 14, 26, 38]. The production of a number adipokines increases during the expansion of adipose tissue mass in the obese (for example, leptin, MIF, TNF\u03b1 and PAI-1), with the result that there is an inflammatory state within the tissue [11, 14, 26, 38]. An exception is adiponectin, which has an anti-inflammatory action [25, 49]. Inflammation in WAT is considered to be causal in the development of type 2 diabetes and the metabolic syndrome linked to obesity [11, 14, 26, 29]. There has, however, been little focus on why the rise in adipose tissue mass in the obese should lead to the increased production of inflammatory adipokines. One possibility, which we have recently suggested, is that it is a response to relative hypoxia in clusters of adipocytes distant from the vasculature, inflammation serving to increase blood flow and stimulate angiogenesis [38, 39].\nHypoxia, which occurs, for example, in solid tumours and during wound healing, induces a series of adaptive responses by cells, and a key role in the transmission of the hypoxic response is played by the hypoxia-inducible transcription factors, particularly hypoxia-inducible factor-1 (HIF-1) [6, 13, 32, 33, 44]. HIF-1 is composed of two subunits, HIF-1\u03b2, which is constitutively expressed, and HIF-1\u03b1, which is recruited in response to low O2 tension to yield the functional transcription factor [6, 32, 44]. HIF-1\u03b1 is present in murine clonal adipocytes, and hypoxia has been shown to induce an increase in the expression of leptin and vascular endothelial growth factor (VEGF) in these cells [21]. Recently, hypoxia has also been reported to induce the production of PAI-1 and to inhibit the synthesis of adiponectin by 3T3-L1 adipocytes [4]; it is also reported to induce the expression of visfatin in these cells [31]. HIF-1\u03b1 has been identified in human adipose tissue and is reported to be increased in obesity [3]. However, the extent to which human adipocytes respond to hypoxia and which genes are hypoxia-sensitive are unknown.\nIn the present study, we have examined the effects of hypoxia on the expression and secretion of key adipokines linked to inflammation in human adipocytes differentiated from fibroblastic preadipocytes in culture. The results demonstrate that hypoxia, whether through low O2 tension or induced chemically by CoCl2, leads to a marked recruitment of HIF-1\u03b1 in human adipocytes and that expression of the GLUT1 facilitative glucose transporter in adipocytes is hypoxia-sensitive. Importantly, the expression and secretion of several adipokines, including FIAF\/angiopoietin-like protein 4, IL-6, leptin, MIF and VEGF, is stimulated by hypoxia in human adipocytes, while adiponectin and haptoglobin are inhibited.\nMaterials and methods\nHuman adipocyte culture\nHuman subcutaneous preadipocytes, derived from adipose tissue pooled from seven female subjects, were obtained (together with culture media) from Zen-Bio (USA). The patients had a mean body mass index of 25 (range 22.5\u201328.2) and average age of 41\u00a0years (range 27\u201351\u00a0years).\nCells were trypsinized from a 75-cm2 flask and plated at a density of 40,000\/cm2 in a 24-well plate and maintained in preadipocyte medium containing Dulbecco\u2019s modified Eagle\u2019s medium (DMEM)\/Ham\u2019s F12 (1:1, v\/v), 10% fetal calf serum (FCS), 15\u00a0mM HEPES, 100\u00a0U\/ml penicillin, 100\u00a0\u03bcg\/ml streptomycin and 0.25\u00a0\u03bcg\/ml amphotericin B at 37\u00b0C in a humidified atmosphere of 95% air\/5% CO2. Cells were induced at confluence by incubation in differentiation medium composed of adipose medium (AM) supplemented with 0.25\u00a0mM isobutyl methylxanthine and 10\u00a0\u03bcM of a PPAR\u03b3 agonist for 4\u00a0days. The cells were then cultured with AM containing DMEM\/Ham\u2019s F-12 (1:1, v\/v), 3% FCS, 1\u00a0\u03bcM dexamethasone, 100\u00a0U\/ml penicillin, 100\u00a0\u03bcg\/ml streptomycin and 0.25\u00a0\u03bcg\/ml amphotericin B. The medium was changed every 3\u00a0days.\nFully differentiated cells at day 15 post-induction were treated with CoCl2 or exposed to 1% O2 for up to 24\u00a0h. For CoCl2 treatment, wells incubated without CoCl2 were used as controls; 75\u2013200\u00a0\u03bcM CoCl2 was used in initial experiments, and 100\u00a0\u03bcM CoCl2 was employed in a time course study. For exposure to hypoxia, the cells were transferred to a MIC-101 modular incubator chamber (Billups-Rosenberg, USA), which was flushed with 1% O2, 94% N2 and 5% CO2, and sealed and placed at 37\u00b0C for up to 24\u00a0h as indicated. The control cells were cultured in a standard incubator (21% O2 and 5% CO2). The cells were harvested in 700\u00a0\u03bcl of Trizol (Invitrogen, UK) or 150\u00a0\u03bcl of lysis buffer per well at the stated time points. Media were collected and centrifuged at 1,000\u00a0rpm for 10\u00a0min to remove cell debris and the supernatant stored at \u221220\u00b0C until required for analysis. All incubations at each time point were performed in replicates of up to six wells.\nRNA extraction and cDNA synthesis\nTotal RNA was isolated from cells using Trizol, and 1\u00a0\u03bcg of RNA was treated with DNase I (Invitrogen) according to the manufacturer\u2019s instructions. RNA concentration was quantified from the absorbance at 260\u00a0nm; all samples had a 260\/280\u00a0nm absorbance ratio of 1.7\u20131.9.\nOne microgram of DNase I-treated RNA was reverse-transcribed using a Reverse-iT\u2122 1ST strand synthesis kit (Abgene, UK) in the presence of anchored oligo dT in a total volume of 20\u00a0\u03bcl.\nReal-time PCR\nQuantitative real-time polymerase chain reactions (PCRs) were carried out in a final volume of 12.5\u00a0\u03bcl consisting of 12.5\u201350\u00a0ng of reverse-transcribed cDNA mixed with optimal concentrations of primers and probe and qPCR\u2122 core kit (Eurogentec, UK) in 96-well plates on a Mx3005P detector (Stratagene, USA).\nThe primer and probe sets were designed using Primer Express software (Applied Biosystems) and synthesized commercially; the primers and TaqMan probes were from Eurogentec. TaqMan probes were labelled with a reporter fluorescent dye (FAM: 6-carboxyfluorescein) at the 5\u2032-end and a fluorescent dye quencher (TAMRA: 6-carboxy-tetramethyl-rhodamine) at the 3\u2032-end. The sequence and optimal concentrations of primers and probes, together with the size of products, are as detailed previously [41, 42], with the exception of GLUT1, FIAF, HIF-1\u03b1, MIF and POLR2A which were as follows.\nGLUT1 (93\u00a0bp): 5\u2032-ATACTCATGACCATCGCGCTAG-3\u2032 (forward), 5\u2032-AAAGAAGGCCACAAAGCCAAAG-3\u2032 (reverse) and 5\u2032-FAM-TGGAGCAGCTACCCTGGATGTCCTATCTGA-TAMRA-3\u2032 (probe);\nFIAF (117\u00a0bp): 5\u2032-GATGGCTCAGTGGACTTCAACC-3\u2032 (forward), 5\u2032-CCCGTGATGCTATGCACCTTC-3\u2032 (reverse) and 5\u2032-FAM-CCAGACCCAGCCAGAACTCGCCGT-TAMRA-3\u2032 (probe);\nHIF-1\u03b1 (75\u00a0bp): 5\u2032-TCCAGTTACGTTCCTTCGATCA-3\u2032 (forward), 5\u2032-TTTGAGGACTTGCGCTTTCA-3\u2032 (reverse) and 5\u2032-FAM-CACCATTAGAAAGCAGTTCCGCAAGCC-TAMRA-3\u2032 (probe);\nMIF (74\u00a0bp): 5\u2032-AGCCCGGACAGGGTCTACA-3\u2032 (forward), 5\u2032-GCGAAGGTGGAGTTGTTCCA-3\u2032 (reverse) and 5\u2032-FAM-CTATTACGACATGAACGCGGCCAATGT-TAMRA-3\u2032 (probe);\nPOLR2A (81\u00a0bp): 5\u2032-ATGGAGATCCCCACCAATATCC-3\u2032 (forward), 5\u2032-CATGGGACTGGGTGCTGAAC-3\u2032 (reverse) and 5\u2032-FAM-TGCTGGACCCACCGGCATGTTC TAMRA-3\u2032 (probe).\nTypically, the amplification started with 2\u00a0min at 50\u00b0C, 10\u00a0min at 95\u00b0C and then 40 cycles of the following: 15\u00a0s at 95\u00b0C and 1\u00a0min at 60\u00b0C.\nHuman POLR2A was used as an endogenous reference. This housekeeping gene was selected based on comparison with \u03b2-actin, where POLR2A demonstrated no significant changes in expression when exposed to CoCl2 and 1% O2 (data not shown). Relative quantitation values were expressed using the  method (see user bulletin no. 2, ABI Prism 7700, pp 11\u201315, Applied Biosystems), as fold changes in the target gene normalised to the reference gene (POLR2A) and related to the expression of the untreated controls. The PCR efficiency in all runs was close to 100%, and all samples were analysed in at least duplicate.\nMeasurement of HIF-1\u03b1 and adipokines by ELISA\nTotal HIF-1\u03b1 in cell lysates was measured with an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, UK) according to the manufacturer\u2019s protocol. Cells were solubilised in lysis buffer consisting of 50\u00a0mM Tris (pH 7.4), 300\u00a0mM NaCl, 10% (w\/v) glycerol, 3\u00a0mM ethylenediaminetetraacetic acid, 1\u00a0mM MgCl2, 20\u00a0mM \u03b2-glycerophosphosphate, 25\u00a0mM NaF, 1% Triton X-100, 25\u00a0\u03bcg\/ml leupeptin, 25\u00a0\u03bcg\/ml pepstatin and 3\u00a0\u03bcg\/ml aprotinin. Before measuring HIF-1\u03b1 by ELISA, the total protein content of the lysates was determined by the BCA protein assay reagent (Sigma, UK).\nAdiponectin, IL-6, leptin, MIF and VEGF were measured in cell culture media using commercial ELISA kits (R&D Systems). The assays were conducted in 96-well microplates according to the manufacturer\u2019s instructions.\nMeasurement of HIF-1\u03b1 by Western blotting\nSamples were prepared as described above for the HIF-1\u03b1 ELISA assay. Fifteen micrograms of protein were separated by sodium dodecyl sulfate\u2013polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane. The membranes were blocked and probed with polyclonal goat anti-human HIF-1\u03b1 (R&D Systems) or mouse monoclonal anti-\u03b1-tubulin (Sigma, UK) as the primary antibody, then subjected to HRP-conjugated anti-goat IgG (R&D systems) or anti-mouse IgG (Santa Cruz Biotechnology) as the secondary antibody. Specific proteins were visualised with the enhanced chemiluminescence reagent (Amersham, UK).\nStatistical analysis\nThe results are expressed as mean values\u00b1SE. Differences between groups were analysed by unpaired two-tailed Student\u2019s t tests.\nResults\nHIF-1\u03b1 expression during differentiation of human adipocytes\nWe first investigated whether human white adipocytes express the HIF-1\u03b1 gene and synthesize the encoded protein when differentiated in culture. Quantitative changes in HIF-1\u03b1 gene expression after the induction of adipocyte differentiation were analysed by real-time PCR. Although the HIF-1\u03b1 gene was expressed both before and after differentiation, there was a marked decrease in HIF-1\u03b1 mRNA level after differentiation was induced (Fig.\u00a01a): By day 2, the mRNA level fell to <25% of that seen at day 0 and remained low. The expression of a classical adipokine\u2013leptin\u2014 was determined as a reference gene. As expected, leptin mRNA was differentiation-dependent and detectable only at day 2 after the induction of differentiation, increasing progressively thereafter (Fig.\u00a01b).\nFig.\u00a01Time course of quantitative changes in HIF-1\u03b1 mRNA level (a), leptin mRNA level (b) and HIF-1\u03b1 protein (c) during the differentiation and development of human adipocytes. Confluent human preadipocytes (day 0) were differentiated and cultured for up to 14\u00a0days. Relative mRNA levels were normalised to human POLR2A at day 0 (or day 2 when no signal was evident at day 0). Total protein concentration of HIF-1\u03b1 in cell lysates was measured by ELISA. Results are means\u00b1SE (n\u2009=\u20093)\nHIF-1\u03b1 protein expression was examined at day 0 and day 14 using a specific ELISA and was detected at both time points. In parallel with the mRNA level, HIF-1\u03b1 protein was substantially higher in preadipocytes than in adipocytes (Fig.\u00a01c).\nResponse to CoCl2 treatment\nHypoxic effects can be mimicked by the divalent transition-metal ion cobalt. In the next experiments, differentiated human adipocytes (day 15 post-induction) were treated with CoCl2 at concentrations of 75 and 200\u00a0\u03bcM for 24\u00a0h. CoCl2 treatment resulted in a marked increase in total HIF-1\u03b1 protein, the level being fivefold higher at both doses compared to untreated cells (Fig.\u00a02a). In contrast, HIF-1\u03b1 mRNA level was reduced three to fivefold by CoCl2 treatment (Fig.\u00a02b).\nFig.\u00a02Effect of CoCl2 at two dose levels on HIF-1\u03b1 protein concentration (a) and on the mRNA levels of HIF-1\u03b1, GLUT1 and adipokines (b) in human adipocytes. Differentiated human adipocytes at day 15 were incubated in medium containing 75 or 200\u00a0\u03bcM CoCl2 for 24\u00a0h. Total HIF-1\u03b1 protein and mRNA level of adipokines were measured as described in Fig.\u00a01. Results are means\u00b1SE (n\u2009=\u20096). In a,**P\u2009<\u20090.01, ***P\u2009<\u20090.001 compared with controls; in b \u2020P\u2009<\u20090.001 compared with controls. Open bars, controls; shaded bars, 75 and 200\u03bcm CoCl2 (light and dark bars, respectively)\nTo assess whether human adipocytes respond to the CoCl2-induced increase in HIF-1\u03b1 protein by increasing the expression of \u2018classical\u2019 hypoxia-sensitive genes, the mRNA level of the GLUT1 facilitative glucose transporter was examined as a reference; this gene is recognised in other cells to be upregulated under hypoxic conditions. Treatment with CoCl2 induced a sixfold increase in GLUT1 mRNA level in the adipocytes (Fig.\u00a02b).\nThe expression of a series of inflammation-related adipokine genes was then examined in the CoCl2-treated adipocytes, and three distinct responses were observed. No effect was found on TNF\u03b1 or adipsin mRNA level at either of the two doses of CoCl2. There was, however, a significant reduction in mRNA level for adiponectin, MCP-1 and haptoglobin (Fig.\u00a02b). Adiponectin mRNA level was decreased three to fourfold, MCP-1 by up to fivefold and haptoglobin mRNA by less than half with both concentrations. In the case of IL-6, the high dose of CoCl2 reduced mRNA level threefold, but there was no response with the lower dose (Fig.\u00a02b). In contrast, CoCl2 treatment resulted in a marked increase in mRNA level for FIAF, leptin, MIF, PAI-1 and VEGF. The increase was six to sevenfold for VEGF and FIAF, and two to fourfold for PAI-1, MIF and leptin (Fig.\u00a02b).\nTime course of response to CoCl2\nThe previous experiment indicated that in most cases, there was little difference in the effect of 75 or 200\u00a0\u03bcM CoCl2 on HIF-1\u03b1 protein level and adipokine gene expression. However, with leptin, MIF and PAI-1 in particular, effects were noted with the lower, but not the higher, dose. This might suggest the possibility of additional, or toxic, responses to CoCl2 at higher doses. Consequently, in the subsequent time course study, a concentration of 100\u00a0\u03bcM was employed at day 15 post-induction. There was a rapid and significant accumulation of HIF-1\u03b1 protein in human adipocytes after the addition of CoCl2. The protein level was increased 4.7-fold by 2\u00a0h of CoCl2 treatment and peaked at 8\u00a0h, at which point it was increased 7.7-fold before gradually falling (Fig.\u00a03a). HIF-1\u03b1 protein level was also assessed by Western blotting, and the pattern of results was similar to that obtained by ELISA (Fig.\u00a03b).\nFig.\u00a03Time course of effects of CoCl2 (100\u00a0\u03bcM) on total HIF-1\u03b1 protein level (a and b) and GLUT1 mRNA levels (c) in human adipocytes. Cells were cultured and treated as in Fig.\u00a02. Results are means\u00b1SE (n\u2009=\u20094). **P\u2009<\u20090.01, ***P\u2009<\u20090.001 compared with 0-h controls\nGLUT1 was again used as a hypoxia-sensitive reference gene, and the pattern of the time course of the changes in GLUT1 mRNA level was similar to that of HIF-1\u03b1 protein. The mRNA level was significantly increased (2.3-fold) by 2\u00a0h, peaking at 8\u00a0h, at which point it was 14-fold higher, gradually declining thereafter (Fig.\u00a03c).\nThe time course of the effect of CoCl2 on the expression of genes encoding adipokines was next examined. A group of adipokines including FIAF, IL-6, leptin, MIF, PAI-1 and VEGF showed marked increases in mRNA level in response to CoCl2, some of which were both substantial and rapid. FIAF mRNA level was increased 30-fold by 2\u00a0h after the addition of CoCl2, peaking at 8\u00a0h, at which point it, had risen 210-fold (Fig.\u00a04a). There was a fall thereafter, but even at 24\u00a0h, FIAF mRNA was still elevated 30-fold. A similar pattern was also evident with IL-6, PAI-1 and VEGF, but the changes were not as large (Fig.\u00a04a). In comparison to the above genes, there was a more gradual response to CoCl2 with leptin and MIF, the mRNA for each reaching a peak (fourfold) at 16\u00a0h, with little change for the rest of the 24-h incubation period (Fig.\u00a04a). Intriguingly, MCP-1\u00a0mRNA level exhibited a substantial acute increase, being elevated >20-fold at 2\u00a0h, but there was a rapid decline thereafter such that at 16 and 24\u00a0h the levels were similar to the controls (Fig.\u00a04a).\nFig.\u00a04Time course of effects of CoCl2 (100\u00a0\u03bcM) on adipokine mRNA levels of FIAF, IL-6, leptin, MCP-1, MIF, PAI-1, TNF-\u03b1 and VEGF (a) and adiponectin, haptoglobin and adipsin (b) in human adipocytes. Relative mRNA levels. Cells were cultured and treated as in Fig.\u00a02. Results are means\u00b1SE (n\u2009=\u20094). *P\u2009<\u20090.05, **P\u2009<\u20090.01, ***P\u2009<\u20090.001 compared with 0-h controls\nIn contrast, to the genes described so far, no effect was found on mRNA level throughout the 24-h incubation period in the case of adipsin (Fig.\u00a04b), while TNF\u03b1 mRNA was also unchanged except for a transitory increase at 2\u00a0h only (Fig.\u00a04a). There was, however, a significant reduction in mRNA level for adiponectin and haptoglobin after 8\u201316\u00a0h of the treatment; by 24\u00a0h, adiponectin and haptoglobin mRNA was decreased by 15.7-fold and threefold, respectively (Fig.\u00a04).\nInduction of HIF-1\u03b1 protein and GLUT1 gene expression by low O2 tension\nIn the next set of experiments, the direct effects of low O2 tension were examined. Human adipocytes at day 15 post-differentiation were exposed to normoxic conditions (21% O2) or to 1% O2 for up to 24\u00a0h. Some cells were returned to normoxia for 16\u00a0h after 8\u00a0h of exposure to 1% O2 to examine the reversibility of responses. Total HIF-1\u03b1 protein level was measured by both ELISA and Western blotting. HIF-1\u03b1 concentration in cells was rapidly and substantially upregulated on exposure to 1% O2, being 7.8-fold higher by 4\u00a0h than in control cells cultured under normoxia. The HIF-1\u03b1 level fell markedly after 8\u00a0h, but at 24\u00a0h, it was still 3.8-fold higher than in control cells (Fig.\u00a05a). The transfer of adipocytes back to normoxic conditions after 8\u00a0h at 1% O2 led to a rapid fall in HIF-1\u03b1, the level of the protein at 1\u00a0h being threefold lower than in cells maintained in 21% O2 throughout (the cells were returned to normoxia after 8\u00a0h, rather than 24\u00a0h, because the high level of HIF-1\u03b1 at the earlier time point facilitated assessment of changes with normoxia). By 16\u00a0h after the return to normoxia, the level of HIF-1\u03b1 was similar to that in the control adipocytes. The pattern of HIF-1\u03b1 level as determined by ELISA was mirrored by Western blot analysis (Fig.\u00a05b).\nFig.\u00a05Time course of effects of hypoxia (1% O2) on total HIF-1\u03b1 protein level (a, b) and GLUT1 mRNA (c) levels in human adipocytes. Differentiated adipocytes at day 15 post-induction were exposed to 1% O2 for up to 24\u00a0h; some cells were transferred back to normoxic conditions (21% O2) for 1 or 16\u00a0h after 8\u00a0h of hypoxia (8- to 1- and 8- to 16-h groups). Results are means\u00b1SE (n\u2009=\u20094). *P\u2009<\u20090.05, **P\u2009<\u20090.01, ***P\u2009<\u20090.001 compared with controls\nGLUT1 mRNA level was markedly increased (fivefold) after 4\u00a0h of exposure to 1% O2 (Fig.\u00a05c) and reached a peak at 24\u00a0h, being 14-fold higher than in cells maintained under normoxia. After exposure to hypoxia for 8\u00a0h, the level of GLUT1 mRNA returned to normal after 16\u00a0h in 21% O2 (Fig.\u00a05c).\nRegulation of adipokine gene expression by low O2 tension\nThe effect of hypoxia on the expression of genes encoding key inflammation-related adipokines was next examined. The first group of adipokines, which encompassed FIAF, IL-6, leptin, MIF, PAI-1 and VEGF, exhibited rapid and marked increases in mRNA level in response to hypoxia, as with CoCl2 treatment. FIAF mRNA level was increased 3.3-fold by 4\u00a0h after exposure to 1% O2, rising to 11-fold by 24\u00a0h (Fig.\u00a06a). FIAF mRNA level was completely normalised by 16\u00a0h of reexposure to normoxia after 8\u00a0h in 1% O2. A similar pattern was also evident with five other adipokines. The changes were even more substantial in the case of leptin and VEGF. After 4\u00a0h of hypoxia, leptin and VEGF mRNA levels were increased four to fivefold and rose such that at 24\u00a0h they were 29-fold and 23-fold greater than in controls, respectively (Fig.\u00a06a). Again, after 16\u00a0h of reexposure to normoxia after 8\u00a0h in 1% O2, the mRNA level of these two adipokines had returned to normal.\nFig.\u00a06Time course of effects of hypoxia (1% O2) on adipokine gene expression in human adipocytes.Relative mRNA levels of FIAF, IL-6, leptin, MIF, PAI-1, VEGF (a) and adiponectin, haptoglobin, MCP-1, adipsin, TNF-\u03b1 (b). Cells were cultured as in Fig.\u00a05; some cells were transferred back to normoxic conditions (21% O2) for 1 or 16\u00a0h after 8\u00a0h of hypoxia (8- to 1- and 8- to 16-h groups). Results are means\u00b1SE (n\u2009=\u20094). *P\u2009<\u20090.05, **P\u2009<\u20090.01, ***P\u2009<\u20090.001 compared with controls\nIL-6, MIF and PAI-1\u00a0mRNA levels also showed significant increases in response to hypoxia, but these increases (2.5-fold to fivefold at 24\u00a0h), were less than those exhibited by FIAF, leptin and VEGF. Returning the adipocytes to 21% O2 led to a reversal by 16\u00a0h of the hypoxia-induced increases in IL-6, MIF and PAI-1\u00a0mRNA levels; in the case of IL-6, the mRNA was lower than in control cells at 16\u00a0h after the transfer from hypoxic to normoxic conditions.\nIn contrast to the genes described so far, there was a significant reduction in adiponectin and haptoglobin mRNA levels by the end of the 24-h exposure period, the decrease being two to threefold (Fig.\u00a06b). This reduction was reversed after 1\u00a0h of reexposure to 21% O2, but surprisingly was evident again after 16\u00a0h of reexposure. Adipsin mRNA level showed a significant, but very small, increase at 8 and 16\u00a0h (1.5 and 1.7-fold, respectively). There were no significant changes in the level of TNF\u03b1 mRNA during exposure to 1% O2, while MCP-1\u00a0mRNA was also unchanged after 4 and 24\u00a0h of hypoxia. For reasons that are not apparent, there was a significant reduction in MCP-1\u00a0mRNA at 8\u00a0h, and this was sustained 1\u00a0h after return to normoxia (Fig.\u00a06b).\nAdipokine secretion in hypoxia\nThe secretion into the medium of a selected group of adipokines was examined during the exposure to hypoxia using specific ELISAs. A small but statistically significant reduction in adiponectin secretion was observed after 8 and 24\u00a0h incubation under 1% O2 (Fig.\u00a07a). This reduction was not evident after reexposure to 21% O2 for 16\u00a0h (Fig.\u00a07a).\nFig.\u00a07Time course of effects of hypoxia (1% O2) on the release of adipokines from human adipocytes. Adiponectin secretion (a) and IL-6, leptin, MIF and VEGF release (b\u2013e) after exposure to hypoxia. Adipokine concentration was measured by ELISA in the medium of cells cultured as in Fig.\u00a06; some cells were transferred back to normoxic conditions (21% O2) after 8\u00a0h of hypoxia and the adipokine concentration then measured 16\u00a0h later (8- to 16-h group). Values are means\u00b1SE (n\u2009=\u20096). *P\u2009<\u20090.05, **P\u2009<\u20090.01, ***P\u2009<\u20090.001 compared with controls\nIn marked contrast to adiponectin, there was a significant increase in leptin, MIF and VEGF release after both 8 and 24\u00a0h exposure to hypoxia (Fig.\u00a07c\u2013e). The increases in secretion were modest for MIF, but substantial in the case of both leptin and VEGF. Thus, after 24\u00a0h of hypoxia, leptin release was tenfold higher, while that of VEGF was fourfold greater. Elevated rates of secretion were still evident after transfer of the adipocytes from hypoxic to normoxic conditions, indicating a long-term carryover effect of low O2 tension. The secretion of IL-6 was not altered after 8\u00a0h of exposure to hypoxia, but it was significantly increased at 24\u00a0h (Fig.\u00a07).\nDiscussion\nAn important recent development in our understanding of obesity is the emergence of the concept that it is characterised by a state of chronic low-grade inflammation [7, 9, 38]. White adipose tissue produces a number of adipokines linked to inflammation, including adiponectin, IL-1\u03b2, IL-6, TNF\u03b1, MCP-1 and MIF [11, 14, 26, 38], and their synthesis is generally substantially increased in obesity\u2014with the exception of adiponectin whose production and circulating levels fall [15, 45]. We have recently proposed that hypoxia may occur in adipocytes distant from the vasculature as adipose tissue mass expands and that this underlies the inflammatory response exhibited by the tissue [38]. The central aim of the present study was to examine the effects of hypoxia on the expression and secretion of key inflammation-related adipokines in human adipocytes.\nThe transcription factor HIF-1 is a key signal in the cellular response to hypoxia, the \u03b1-subunit of which is highly induced by hypoxia [13, 32, 44]. HIF-1\u03b1 expression was evident in human adipocytes, as noted previously in murine fat cells, and the level of both the mRNA and the protein fell after the induction of differentiation. It has been reported previously that HIF-1\u03b1 mRNA level in 3T3-L1 cells peaked at 3\u00a0h after the addition of induction medium and then rapidly decreased [16]. Immunoreactive HIF-1\u03b1 has been reported in murine adipocytes, and hypoxia results in an increase in the amount of the protein in cultured fat cells [4, 21]. We have observed that the HIF-1\u03b1 gene is expressed in human, mouse and rat WAT depots, and expression occurs in both the mature adipocytes and in the cells of the stromal vascular fraction. A previous study has suggested that HIF-1\u03b1 is predominantly expressed in the stromal vascular fraction of obese subjects [3].\nHypoxia stabilises HIF-1\u03b1 protein, which is otherwise (under normoxic conditions) degraded by an ubiquitin-dependent proteasome [32]. Total HIF-1\u03b1 protein level in human adipocytes increased rapidly and substantially under hypoxic condition, both with 1% O2 and by treatment with CoCl2. The elevated HIF-1\u03b1 protein rapidly fell on return to 21% O2, and the level was even decreased compared to controls after 1\u00a0h of reoxygenation. Thus, the accumulated HIF-1\u03b1 in human adipocytes is rapidly degraded on returning to normoxia. Indeed, it was observed in another human adipocyte system (SGBS cells) that HIF-1\u03b1 protein declined to control levels by just 10\u00a0min after return to 21% O2 (Wang, unpublished results).\nCoCl2, a known inducer of HIF-1\u03b1, has been widely employed as a hypoxia mimic and was used here to examine the effects of chemically induced hypoxia on adipokine gene expression. Previous studies have shown that at a global gene expression level, both CoCl2 and ambient hypoxia regulate a similar group of genes, and the observed similarity in gene expression appears to be dependent on functional HIF-1\u03b1 [40]. CoCl2 stabilises HIF-1\u03b1 under normoxia via inhibition of the iron-containing HIF prolyl hydroxylase enzyme, which plays a critical role in mediating normal hypoxic signalling by modifying HIF-1\u03b1 and targeting it for degradation.\nOur results demonstrate that CoCl2 rapidly induces an accumulation of HIF-1\u03b1 and influences the expression of key inflammation-related adipokines in human adipocytes. Other studies have also found that CoCl2 (100\u00a0\u03bcM) causes HIF-1\u03b1 accumulation in various cell types, including bone marrow stromal cells, cancer cells and brown adipocytes [20, 24, 27]. In contrast to protein levels, HIF-1\u03b1 mRNA level was inhibited by CoCl2 treatment. This indicates that CoCl2 modulates HIF-1\u03b1 at the posttranslational rather than the transcriptional level. There is, however, evidence for hypoxic induction of HIF-1\u03b1 mRNA in some cells [17, 43].\nUnder hypoxia, GLUT1 gene expression has been shown to increase in several cell types, including rat liver cell lines and fibroblasts [1, 5], Chinese hamster ovary cells [48] and in human cancer cells [12]. Our data demonstrate that GLUT1 expression is also substantially upregulated by hypoxic conditions in human adipocytes. Measurement by Western blotting (using a specific antibody) of GLUT1 in total tissue lysates of adipocytes exposed to 1% O2 demonstrated that the level of the protein was also increased by hypoxia; at 24\u00a0h, GLUT1 protein was eightfold higher than in the normoxic controls (Wood, unpublished results). This phenomenon presumably reflects an adaptation of glucose metabolism to hypoxic conditions, with a marked stimulation of glycolysis in the face of a fall in aerobic metabolism.\nDuring the adaptive response to hypoxia, the expression of several genes encoding glycolytic enzymes is recognised to increase [34]. To sustain higher levels of glycolysis, there is a need for an increase in glucose uptake, and this would be aided by an increase in the level of GLUT1 expression in hypoxia. GLUT1 is a key facilitative glucose transporter in white adipocytes, but other transporter isoforms such as GLUT4, GLUT8, GLUT10 and GLUT12 are also expressed in the cells [46, 47].\nThe main focus of the present study was the effects of hypoxia on the expression and secretion of adipokines, and qualitatively similar results were obtained over the same time scale with both 1% O2 and CoCl2 treatment. Given the mechanism of action of CoCl2, this is consistent with the effects of hypoxia on adipokine production being mediated by HIF-1. Of the adipokines studied, the expression of TNF\u03b1 and adipsin were not altered, and these genes would seem not to be hypoxia-sensitive. However, the expression of both adiponectin and haptoglobin (an acute phase protein) in human adipocytes was reduced by hypoxic conditions, and in the case of adiponectin, this was also evident at the level of the secretion of the protein into the medium. The reduction, albeit modest, in adiponectin production in human adipocytes by hypoxia is consistent with a recent report in murine 3T3-L1 adipocytes [4]. There was a discrepancy in the present study between the changes in the scale of alterations in adiponectin mRNA level and the amount of adiponectin secreted into the medium. This may, however, reflect the lag between changes in mRNA and alterations in secreted protein.\nIn marked contrast to adiponectin, hypoxia stimulated the expression of the genes encoding FIAF, IL-6, leptin, MIF, PAI-1 and VEGF. VEGF is involved in the development of the vascular system, promoting angiogenesis, and is well known to be activated by hypoxia [50]. In the present study, an increase in VEGF protein secretion, as well as mRNA level, was observed in human adipocytes with hypoxia. This is in agreement with studies on rat omental adipose tissue and on murine 3T3-F442A adipocytes [21, 51].\nLeptin is the most extensively studied adipokine, and adipose tissue is quantitatively the main leptin-producing organ. Activation of leptin gene expression by hypoxia resulted in a substantial increase in leptin release by human adipocytes. This would be expected to reduce food intake and stimulate energy expenditure, and as such, hypoxia could provide a mechanistic basis for the increase in leptin as adipose tissue mass expands in obesity. Leptin has been reported to be an angiogenic factor [35], and the physiological significance of a hypoxia-mediated induction of leptin secretion by adipocytes may relate to a local effect to stimulate the development of new blood vessels during adipose tissue expansion. Therefore, increased leptin production, together with that of VEGF, under hypoxic conditions could also reflect an adaptive process to promote angiogenesis, allowing appropriate oxygenation of adipose tissue. It has been previously established that leptin is a hypoxia-inducible hormone, and its production is increased by hypoxia in tissues such as the placenta, cardiac myocytes and cancer cells [8, 10, 23] as well as in murine 3T3-F442A adipocytes [21].\nFIAF (also known as angiopoietin-like protein 4) is expressed particularly in adipose tissue and the liver, and has been suggested to play a role in adipose differentiation, systemic lipid metabolism and energy homeostasis [18]. In parallel with the activation of leptin and VEGF, FIAF expression was highly activated by hypoxia in human adipocytes; FIAF expression has also been shown to be hypoxia-sensitive in cardiomyocytes through a HIF-1-dependent pathway [2]. The major upregulation of FIAF by hypoxia in adipocytes may again relate to angiogenesis, this being one of the functions suggested for this protein [22]. However, a major effect of FIAF is in the clearance of plasma triglycerides through an inhibition of lipoprotein lipase, and it also appears to stimulate lipolysis [18]. Thus, the hypoxia-induced stimulation of FIAF production may alter lipid utilisation.\nThe present study shows that the expression and release of MIF, a potent macrophage migration inhibitory factor which has recently been shown to be secreted from human adipocytes [36], was also stimulated by hypoxia. MIF has been described as a pro-inflammatory agent, and elevated expression has been seen in many inflammatory pathologies. Although MIF may play a role in the normal adaptive response to hypoxia, it is likely that exaggerated expression of MIF may contribute to the inflammatory state. It has also been proposed that MIF may play an important role in oncogenic transformation and tumour survival, and recent studies have implicated MIF in the regulation of VEGF expression [28]. In this context, during hypoxia, elevated MIF expression may play an important role in enhancing angiogenesis as well as macrophage recruitment. In contrast to MIF, no consistent pattern was observed with MCP-1, an important factor in macrophage recruitment; the acute, but transitory, increase in MCP-1\u00a0mRNA observed with CoCl2 may reflect an effect of the metal unrelated to hypoxia.\nThe expression and release of the classic inflammatory factor IL-6 was significantly upregulated by hypoxia. Thus, hypoxia can directly affect key components of the inflammatory cascade within adipose tissue. The expression of PAI-1, an acute phase protein involved in fibrinolysis, was modestly increased in human adipocytes by hypoxia, consistent with the effects observed in 3T3-L1 adipocytes [4]. Induction of PAI-1 expression by hypoxia has also been reported in HepG2 cells [19] and in vascular smooth muscle cells [30].\nOverall, the present results indicate that a number of genes are hypoxia-sensitive in human adipocytes and suggest that HIF-1 is involved in the transmission of the response to low O2 tension in these, as in other, cell types. The hypoxia-sensitive genes in adipocytes include those involved in basic metabolic processes, such as glucose transport, as well as those linked to energy balance, inflammation and angiogenesis. The stimulation of IL-6, leptin, MIF, PAI-1 and VEGF expression, together with the downregulation of adiponectin synthesis, is consistent with our recent proposal that hypoxia may underlie the inflammatory response in adipose tissue in obesity and play a causal role in the development of obesity-associated diseases [38]. There was, however, no effect of hypoxia on the expression of TNF\u03b1, a key pro-inflammatory cytokine. The hypoxia-induced changes in IL-6 and adiponectin, with its anti-inflammatory action, in particular, might be expected to impact on insulin sensitivity.","keyphrases":["inflammation","adipokines","hypoxia","obesity","adipose tissue","metabolic syndrome"],"prmu":["P","P","P","P","P","P"]}
{"id":"Knee_Surg_Sports_Traumatol_Arthrosc-4-1-2190783","title":"Effects of a contoured articular prosthetic device on tibiofemoral peak contact pressure: a biomechanical study\n","text":"Many middle-aged patients are affected by localized cartilage defects that are neither appropriate for primary, nor repeat biological repair methods, nor for conventional arthroplasty. This in vitro study aims to determine the peak contact pressure in the tibiofemoral joint with a partial femoral resurfacing device (HemiCAP\u00ae, Arthrosurface Inc., Franklin, MA, USA). Peak contact pressure was determined in eight fresh-frozen cadaveric specimens using a Tekscan sensor placed in the medial compartment above the menisci. A closed loop robotic knee simulator was used to test each knee in static stance positions (5\u00b0\/15\u00b0\/30\u00b0\/45\u00b0) with body weight ground reaction force (GRF), 30\u00b0 flexion with twice the body weight (2tBW) GRF and dynamic knee-bending cycles with body weight GRF. The ground reaction force was adjusted to the living body weight of the cadaver donor and maintained throughout all cycles. Each specimen was tested under four different conditions: Untreated, flush HemiCAP\u00ae implantation, 1-mm proud implantation and 20-mm defect. A paired sampled t test to compare means (significance, P \u2264 0.05) was used for statistical analysis. On average, no statistically significant differences were found in any testing condition comparing the normal knee with flush device implantation. With the 1-mm proud implant, statistically significant increase of peak contact pressures of 217% (5\u00b0 stance), 99% (dynamic knee bending) and 90% (30\u00b0 stance with 2tBW) compared to the untreated condition was seen. No significant increase of peak contact pressure was evaluated with the 20-mm defect. The data suggests that resurfacing with the HemiCAP\u00ae does not lead to increased peak contact pressure with flush implantation. However, elevated implantation results in increased peak contact pressure and might be biomechanically disadvantageous in an in vivo application.\nIntroduction\nIn 50% of the patients undergoing routine arthroscopy, articular cartilage defects are detected [6]. The majority of cartilage lesions are observed in patients over the age of 40\u00a0years [6, 11]. Approximately 20% of these lesions were categorized as full thickness cartilage defects (Grade IV) [6]. The average defect size in 1,000 knee arthroscopies was 2.1\u00a0cm2 with 42% of these defects being between 2 to 4\u00a0cm2 in area [11]. Location of the lesions are predominantly on the medial femoral condyle [6, 11]. Clinical and experimental studies have shown that a relationship exists between larger defect size and degeneration of the articular surface. Linden et al. [19] demonstrated that the presence of a significant chondral defect is associated with a much higher incidence of osteoarthritis than occurs in the general population. Hughston et al. [13] showed that larger osteochondral defects are associated with a poorer clinical outcome than smaller defects.\nThe treatment for articular cartilage defects offers a great variety of options depending on the severity of the damage. The main factors to be considered are the patient age, the size of the lesion, the location of the defect, and further damage to the joint. Biological repair techniques, such as autologeous chondrocyte transplantation, osteochondral transplantation (OATS, Mosaicplasty) and marrow stimulation techniques are proposed for the treatment of localized cartilage defects in younger patients with a normal weight bearing axis and no further damage to the joint [3, 8]. However, many middle aged patients are affected by localized cartilage damage and are neither fit for biological repair methods, nor for traditional resurfacing techniques such as unicompartimental or total joint replacement. A contoured articular prosthetic unicondylar femoral resurfacing prosthesis (HemiCAP\u00ae, Arthrosurface Inc., Franklin, MA, USA) was developed to offer a treatment option among the currently used modalities. It is intended for use as a partial resurfacing device of the femoral condylar surface of the knee when only one compartment is affected by posttraumatic, degenerative disease or necrosis associated with large unstable articular defects with significant subchondral bone exposure.\nHowever, it is unknown if the femoral resurfacing device leads to increased peak contact pressure to the opposing biological structures such as meniscus and articular cartilage of the tibial plateau.\nThe purpose of our study was to determine peak contact pressures in the medial compartment of human cadaveric knees under different loading conditions comparing the normal articular surface to flush and proud HemiCAP\u00ae implantation and defect. Based on the experimental study, biomechanical device safety on opposing structures can be assessed and a guideline might be given to the clinician for prosthetic implantation while avoiding potentially damaging effects.\nMaterials and methods\nKnee joints\nA total of eight fresh-frozen knee cadaver specimens (3 pairs, 2 single) were used for data collection in this study. The specimens were obtained from donors, who consented in writing during their lifetime to the use of their body for research and education. The average age of the seven male and one female specimen was 71\u00a0years (range 61\u201381) with an average weight of 71\u00a0kg (range 62\u201385). Specimens were selected after inspection of the medial compartment according to the following criteria: Intact femorotibial cartilage, intact meniscus, and intact collateral and cruciate ligaments. Thirteen specimens were excluded following these criteria. Another six specimens were excluded due to specimen failure during the testing procedure: The reason for exclusion was fracture of the femur or tibial plateau (2 specimens), rupture of the anterior cruciate ligament (2 specimens) or rupture of the patella tendon (2 specimens). All specimen failures were female human cadaver knees.\nTechnology\nA specially designed knee simulator was used for this study (Fig.\u00a01). Similar to in vivo conditions, the main system composed of artificial muscle, force transducer sensor, the joint angle detection and the ground reacting force form a closed loop. The ground reaction force is adjustable according to the donor\u2019s weight. The knee simulator consists of a loading frame (MTS 858 Bionix, MTS Systems, Eden Prairie, MN, USA) with a long stroke main actuator driven by a hydraulic pump unit (MTS 505.11 silent flow) to simulate body weight and the vertical hip displacement in the mechanical axis of the lower limb. Ankle joint simulation is performed with a hinge joint with one free motion axis. The possible rotation during the movement occurs in the artificial hip joint as if standing with fixed shoe contact. A load transducer is fixed between the mounting plate and ankle joint to detect the vertical ground reacting force (U3 load cell, Hottinger-Baldwin, Darmstadt, Germany). Two smaller actuators apply loads, which simulate the quadriceps force. The tendons of the quadriceps muscle are attached to customized curved cryo-clamps, which avoid patella tilting. These cryo-clamps are connected to a waterproof force transducer (SSM-AJ 500, Interface, Scottsdale, AZ, USA) and connected to an artificial muscle (Fluidic muscle MAS, Festo, Esslingen, Germany). The mathematical model shows that the properties of this fluidic muscle is comparable with a skeletal muscle [28].\nFig.\u00a01Schematic drawing of the knee simulator that was used for this study\nA 0.1\u00a0mm thin electronic pressure measuring sensor (K-scan 4000, Tekscan, Boston, MA, USA) was placed in the medial compartment above the menisci and fixed with sutures in a manner that no displacement was possible. The sensor consists of load-sensing regions oriented in a grid with 1.27\u00a0mm spacing between rows and columns. Each region is referred to a sensel with piezo-resistive pigments to determine the total compressive load within that region. The size of the sensor is 28\u00a0\u00d7\u00a033\u00a0mm with 62\u00a0sensels\/cm2. The K-scan sensor was successfully used in comparable applications by several authors [18, 21, 25, 31].\nThe HemiCAP\u00ae implant (Arthrosurface Inc., Franklin, MA, USA) is a contoured articular prosthetic (CAP) unicondylar femoral resurfacing prosthesis consisting of two components, a fixation component and an articular component, that mate together via a taper interlock to provide stable and immobile fixation of the implant and stress-bearing contact at the bone\/prosthetic interface. The fixation component is a modified titanium cancellous screw with a tapering distal tip, a full-length cannulation, and a proximal female taper bore. The articular component is a dome-shaped component manufactured of a Cobalt\u2013Chromium\u2013Molybdenum alloy with titanium plasma spray coverage on the underside for bony in-growth. Each diameter comes in a variety of incremental offset sizes which correspond to the superior\/inferior and medial\/lateral radius of curvatures at the implant site. The size of the implant used in this study was 20\u00a0mm in diameter with different offset sizes, matching the individual specimen joint curvature.\nTesting protocol\nThe specimens were aligned using a fixed laser beam to achieve correct alignment in the mechanical axis of the lower limb. The mechanical axis was defined by a line through the center of the head of the artificial hip joint, the center of the knee joint and the center of the hinge joint representing the artificial ankle. For calibration of the sensor the ankle hinge joint was secured with two aluminum plates perpendicular to the ground. Thereby the knee was fixed in full extension. Each sensor was individually preconditioned and calibrated intra-articulary with a two-point calibration method at 700 and 1,500\u00a0N according to manufacturer\u2019s guidelines. Definitions of the correct angles of the actual knee position were adjusted with a custom-made goniometer and by the displacement-controlled main rod. During the test cycles the cadaver were sprayed with saline solution to prevent dehydration.\nThe specimens were tested in four different conditions: (1) Untreated knee, (2) Flush HemiCAP\u00ae (20\u00a0mm) implanted in the central weight bearing area of the medial femoral condyle, (3) 1\u00a0mm proud implantation to adjacent cartilage, (4) 20\u00a0mm defect (Fig.\u00a02a\u2013d). Each knee was tested in static knee stance positions (5\u00b0, 15\u00b0, 30\u00b0, 45\u00b0) with body weight ground reaction force, 30\u00b0 flexion with two times body weight ground reaction force and during a knee-bending dynamic cycle (10\u00a0cycles) with body weight ground reaction force. The setting of ground reaction force to be achieved during the trial was adjusted to the living body weight of the donator of the cadaver (e.g. 70\u00a0kg\u223c700\u00a0N). The loading rate for exploring the dynamic contact pressures was 0.1\u00a0Hz\/s.\nFig.\u00a02Different testing conditions (a Untreated; b Flush; c 1\u00a0mm; d Defect)\nOperative technique\nAll specimens were released at their femoral fixation during preparation of a new testing condition. The exact position was marked and recorded to maintain specimen position in the knee simulator across all testing conditions. The position of the sensor did not change during the preparation. All procedures were performed by the same investigator (CB). The medial femoral condyle was exposed by a medial parapatellar incision. The knee was flexed to 90\u00b0 to expose the central weight-bearing portion. A drill guide was used to place a pin perpendicular to the joint surface representing the center of the defect. The center of the defect was determined by measuring the condylar width and bisecting the distance. The cannulated instrumentation set ensured that the vertical axis was maintained throughout the procedure. After drilling a pilot hole, the fixation component was inserted. A contact probe determined the radius of curvature in two planes. With a matching reamer, the site for the implantation was prepared and a sizing trial with corresponding offsets inserted. The selected device was oriented in the correct planes and connected to the anchoring screw with a tapered lock. In order to allow for careful removal of the articular component, the guide wire was initially drilled through the condyle and remained inside the cannulated screw during the trial. After testing with flush implantation (Fig.\u00a02b), the device was removed with the guide wire. The anchoring screw was elevated with a counter-clockwise quarter turn representing 1\u00a0mm and the device re-inserted for positioning 1\u00a0mm above the adjacent cartilage (Fig.\u00a02c). Defect condition was tested after removing the guide wire and device leaving a 20-mm osteochondral defect of 3\u20134\u00a0mm depth (Fig.\u00a02d).\nData analysis\nData were obtained using I-Scan software 4.23 (Tekscan, Boston, MA, USA). Peak contact pressure was assessed and recorded as the highest value at each stance position and during the dynamic knee-bending cycle. Mean, median and standard deviation values were evaluated using MS-Excel 2003 (Microsoft Inc., Redmond, WA, USA) and SPSS 11.0 (SPSS Inc., Chicago, IL, USA). For statistical analysis a paired sampled t test to compare means (significance, P\u00a0\u2264\u00a00.05) was used.\nResults\nContinuous data were obtained at every trial. No difference in the quality of data collection was seen comparing the stance positions or dynamic testing.\nThe highest mean peak contact pressures were noted with the implant being 1\u00a0mm proud (Fig.\u00a03; Table\u00a01). Statistically significant increase of peak contact pressures of 217% (5\u00b0 stance; P\u00a0\u2264\u00a00.004), 99% (dynamic knee bending; P\u00a0\u2264\u00a00.02) and 90% (30\u00b0 stance with 2tBW; P\u00a0\u2264\u00a00.03) compared to the untreated condition was seen. Average results of 45\u00b0 static testing demonstrated only slightly higher values compared to the other testing positions, indicating that the implant was non-weight bearing and lost contact to the sensor at this position (Fig.\u00a03; Table\u00a01).\nFig.\u00a03Mean peak contact pressures with one standard deviationTable\u00a01Peak contact pressures at different testing conditionsTesting positionTesting conditionPeak contact pressure (Mpa)Mean difference to untreated (%)P value5\u00b0Untreated3.09\u00a0\u00b1\u00a00.86 (3.05, 1.92\u20134.86)N\/AN\/AFlush3.98\u00a0\u00b1\u00a02.19 (3.59, 2.15\u20139.02)29NS1\u00a0mm proud9.80\u00a0\u00b1\u00a04.37 (9.36, 4.70\u201318.37)217\u22640.004Defect3.35\u00a0\u00b1\u00a01.39 (3.39, 1.90\u20135.83)8NS15\u00b0Untreated3.01\u00a0\u00b1\u00a00.81 (2.95, 2.01\u20134.11)N\/AN\/AFlush3.69\u00a0\u00b1\u00a01.79 (3.89, 1.38\u20137.06)23NS1\u00a0mm proud 9.19\u00a0\u00b1\u00a09.27 (5.87, 2.10\u201330.49)205NSDefect2.94\u00a0\u00b1\u00a01.07 (2.84, 1.69\u20135.09)\u22122NS30\u00b0Untreated3.18\u00a0\u00b1\u00a00.94 (3.52, 1.46\u20134.26)N\/AN\/AFlush2.89\u00a0\u00b1\u00a00.85 (3.02, 1.03\u20133.83)\u22129NS1\u00a0mm proud 7.07\u00a0\u00b1\u00a05.97 (5.24, 1.03\u201319.81)122NSDefect3.31\u00a0\u00b1\u00a01.06 (3.11, 2.05\u20135.34)4NS45\u00b0Untreated4.77\u00a0\u00b1\u00a01.85 (4.23, 2.66\u20137.90)N\/AN\/AFlush4.96\u00a0\u00b1\u00a02.02 (4.18, 2.79\u20137.92)4NS1\u00a0mm proud 5.79\u00a0\u00b1\u00a02.76 (5.54, 2.01\u201311.82)21NSDefect4.94\u00a0\u00b1\u00a01.81 (4.65, 2.05\u20137.54)4NSDynamicUntreated5.84\u00a0\u00b1\u00a02.12 (5.14, 3.85\u20139.82)N\/AN\/AFlush6.02\u00a0\u00b1\u00a02.05 (5.21, 3.46\u20138.97)3NS1\u00a0mm proud 11.61\u00a0\u00b1\u00a06.39 (10.50, 6.22\u201325.46)99\u22640.02Defect5.68\u00a0\u00b1\u00a01.76 (5.11, 3.76\u20138.24)\u22123NS30\u00b0 2tBWUntreated6.57\u00a0\u00b1\u00a02.31 (6.01, 4.09\u201311.15)N\/AN\/AFlush6.05\u00a0\u00b1\u00a01.40 (5.82, 4.68\u20138.97)\u22128NS1\u00a0mm proud 12.49\u00a0\u00b1\u00a08.02 (9.67, 5.83\u201330.49)90\u22640.03Defect7.38\u00a0\u00b1\u00a04.68 (5.31, 4.75\u201317.70)12NSValues given as mean\u00a0\u00b1\u00a0SD (median, range)NS not significant\nOn average, comparison of the untreated normal knee with flush device implantation demonstrated neither statistically significant differences in peak contact pressure during the dynamic knee-bending cycle nor static testing (5\u00b0, 15\u00b0, 30\u00b0, 45\u00b0) or two times body weight GRF at 30\u00b0 static testing (Table\u00a01). Six out of eight specimens demonstrated similar peak contact pressures in both testing conditions, two specimens showed a noticeable increase in peak contact pressures (flush device implantation) averaging 29% during 5\u00b0 static testing and 23% in 15\u00b0 static testing. Data evaluation of the two respective trials with outlying measurements demonstrated edge loading at the border of the implant to the adjacent cartilage (Figs.\u00a04b, 5b).\nFig.\u00a04Peak contact pressure at 15\u00b0 static knee stance position with single body weight of specimen No. 2. The color represents the spectrum of pressures (high pressure-red; low pressure-blue). From left to right the different testing conditions are displayed (a Untreated, b Flush, c 1\u00a0mm proud, d Defect). Marked increase of peak contact pressure at the edge of the implant to the adjacent cartilage is demonstrated (b)Fig.\u00a05Picture of specimen No. 2 before testing with flush implantation of the device in front view (a) and top view (b). The arrow indicates the area of peak contact pressure displayed in Fig.\u00a04b\nNo significant increase of peak contact pressures was evaluated with the 20-mm osteochondral defect. However, average increase by 8% (5\u00b0 stance) and 12% (30\u00b0 stance with 2tBW) were found compared to untreated-condition (Table\u00a01). Furthermore, maximum values were higher compared to untreated condition (except 45\u00b0 stance position and dynamic testing cycle).\nDiscussion\nThe patient aged over 40\u00a0years with a full thickness chondral or osteochondral defect reflects a serious problem for the orthopedic surgeon. Considered as being too old for biological repair of the defect, primarily the patients are mostly managed with conservative, non-surgical treatment including weight reduction, physical therapy to increase and support musculature, unloading braces and medications such as NSAID\u2019s, intraarticular injections (Corticosteroids, Hyaluronic acid, etc.) and dietary supplements.\nHowever, conservative treatment at best ameliorates the symptoms. Biomechanical studies have shown that untreated osteochondral defects may result in increased contact pressures [4, 7, 17]. Animal models proved that untreated osteochondral defects undergo progressive degenerative changes [5, 14]. Whereas smaller defects might have the capacity for healing [5], it was shown that larger defects resulted in resorption of the osseous walls of the defect, the formation of a large cavitary lesion, and the collapse of the surrounding articular cartilage and subchondral bone as well as to degeneration of the opposing tibial articular surface [5, 14]. A\u201cthreshold\u201d effect for localized full thickness defects was described with rim concentration around the lesion becoming a factor for defects greater than 10\u00a0mm in diameter (0.79\u00a0cm2) [7].\nDefect repair is therefore regarded crucial to prevent or delay progressive degenerative joint destruction. However, unicompartimental or total knee arthroplasty represent procedures of final resort for some of the affected patients. The HemiCAP\u00ae resurfacing prosthesis (Arthrosurface Inc., Franklin, MA, USA) offers an interim or alternative treatment strategy for the middle-aged patient with a full thickness cartilage defect. However, effects of a metallic implant articulating with intact opposing tibial articular cartilage and meniscus remain largely unanswered to date. An experimental study assessing the functional and biological response to its use in a goat model resulted in good clinical outcomes. One year after implantation, gross necropsy and histologic data implied the biocompatibility and functionality of the implant. No ongoing degenerative joint disease was apparent [15]. Macroscopic and histological analysis showed that the cartilage around the implant was largely intact although some focal fraying and erosion and limited meniscal damage was observed. The opposing tibial plateau cartilage surface was generally intact but exhibited some focal erosions of variable depth [15]. The outcome was substantially better by comparison to other reported experimental animal studies in goats with untreated full-thickness defects [14, 29]. A patellar resurfacing prosthesis resulted at an average of 8.1\u00a0years in 71% good or excellent cases with no progressive degenerative changes in the non-resurfaced apposing femoral articular cartilage as long as 16\u00a0years [9]. A successful clinical outcome with a metallic distal femoral prosthesis articulating directly against the menisci and proximal tibial plateau over 30\u00a0years was documented in a case report [12].\nOur results revealed significant increase of peak contact pressures with the implant being proud to the surrounding cartilage compared to the untreated condition with an average maximum increase of 217% and 205% at 5\u00b0 and 15\u00b0 stance compared to untreated knees. Results in a biomechanical model using osteochondral plugs for the treatment of osteochondral defects demonstrated increased contact pressures up to 57% compared to intact condition in elevated or angled plugs with an edge placed higher than the adjacent cartilage [16, 17]. Thus, increased peak contact pressures may suggest biomechanical disadvantages and may cause damage to opposing structures. Several in vitro studies have shown increased chondrocyte apoptosis and matrix deformation after peak stress loading of bovine and human articular cartilage [2, 20, 22, 23]. However, quantitative thresholds above which elevated pressure is detrimental are not defined yet.\nFlush implantation showed no statistical increase of peak contact pressures compared to untreated. However, in two specimens we found marked increase of peak contact pressures at the edge of the implant. Evaluation of the pictures of the specimen after the trial showed that the implant did not appear to be proud to the adjacent cartilage in the front view (Fig.\u00a05a). However, in the top view it seemed that the implant was in level with the adjacent cartilage or even slightly elevated at the point of increased peak contact pressure (Fig.\u00a05b). Reduced quality of the adjacent cartilage directly next to the measured peak contact pressure might have lead to less resistive cartilage capacity allowing for edge loading. Thus, it appears to be crucial to have healthy surrounding cartilage around the implant and to spend special care during the implantation process so that the implant is not elevated above the adjacent cartilage at any point. Koh et al. [16] showed in their biomechanical study, that slightly recessed osteochondral grafts with the highest edge placed flush to neighboring cartilage demonstrated nearly normal contact pressures, whereas elevated angled grafts produced increased contact pressures. Our results confirm the observation concluding that slightly recessed implantation should be considered if flush implantation cannot be accomplished.\nDefect situation in our study did not result in significant increase of peak contact pressures. Rim stress concentration has been described by several authors in their biomechanical models evaluating the contact pressures in full thickness cartilage defects [4, 5, 7, 16, 17, 26]. However, results were uncertain whether the peak contact pressure increases at the rim of the defect. Whereas Koh et al. [17] reported average increase of peak contact pressure by 23% compared to the untreated condition, Raimondi et al. [26] and Nelson et al. [24] found no elevated contact stresses at the rim of the defect. These different observations might be caused by the different deformation of defect rims under a given load, especially in larger defects than in smaller defects. The relationship of defect size to the condylar surface may be also a factor [7]. The methodology concerning the model (animal or human being cadaver), the experimental setup (loading mechanics, dynamic or static, etc.) and the pressure-sensitive sensor and performance of the trial have also to be considered.\nLimitations of this study have to be considered and include the following. (1) This is a human cadaver study and biomechanical model. Only approximation of the living system can be achieved. However, the pre-selection of the applied load by the ground reaction force gave the opportunity to load the specimens with the known body weight of the donor, and thus better approximate the forces that occur in the living system [30]. Furthermore, the knee bending dynamic measurement may have provided a more accurate reproduction of physiologic weight-bearing activity. However, the complexity of weight-bearing motions including all the muscle groups for knee motion could not be reproduced. Applied forces were too high for female human cadaver knees resulting in a high specimen\u2019s failure rate. (2) Peak contact pressures were determined by an electronic pressure sensitive sensor. The reliability of the K-scan sensor was verified in several studies [10, 18, 21, 31]. In comparison to other measuring devices (Fuji Photo Film), the Tekscan K-sensor proved to be superior [10, 27]. Limitations of the sensor include the thickness (0.1\u00a0mm), its sensitivity to temperature changes, its disposition for crinkling and the establishment of the position [1]. Although the position of the sensor was accurately secured, a small amount of displacement and crinkling could not be excluded. A certain amount of loss of sensitivity during the different testing conditions and imprecise calibration due to the different surface materials (metal on cartilage, cartilage on cartilage) have to be considered. Some drop of data point was observed at some specimen. However, peak contact values seemed not to be affected. (3) Stable, reproducible ground reaction force in full extension could not be established by the simulator. The quadriceps tendon could not be adequately tensioned in this position. (4) Implantation of the device was performed in the central weight-bearing area. Results might be different for the implantation in different areas of the medial femoral condyle or lateral condyle.\nIn conclusion, the data suggest that resurfacing with the HemiCAP\u00ae with flush implantation does not lead to significantly increased peak contact pressure. However, elevated implantation results in significantly increased peak contact pressure and might be biomechanically disadvantageous in an in vivo application. Further research is necessary to evaluate the effects of the prosthetic device on contact pressures after loss of meniscus function and in longer continuous dynamic testing.","keyphrases":["articular prosthetic device","peak contact pressure","tibiofemoral joint","osteochondral defect","knee biomechanics"],"prmu":["P","P","P","P","R"]}
{"id":"Matern_Child_Health_J-2-2-1592246","title":"Preconception Care for Improving Perinatal Outcomes: The Time to Act\n","text":"Introduction\nA healthy baby and a healthy mother are valued hopes and dreams of families of all cultural heritages. National health goals in many countries around the world prioritize infant and maternal mortality and morbidity. In the United States, as in many other industrialized countries, pregnancy outcomes and maternal and infant health indicators have improved dramatically. At the turn of the 20th century, 7.28 women and 96 infants died for every 1,000 babies born alive [1]. By the end of the year 2000, the maternal mortality rate had decreased to less than 0.08 and infant mortality had decreased to less than 7 deaths per 1,000 live births [2]. These changes were characterized as one of the ten \u201cgreat public health achievements\u201d of the 20th century [3]. Much of this progress can be attributed to changes in social and living conditions as well as the development and implementation of effective medical interventions. Despite this progress, during the last two decades of the 20th century, even in the presence of significant breakthroughs in medical technology and its application, improvements in maternal and infant pregnancy ouctomes slowed down significantly, and in some cases, outcomes deteriorated. For example, from 1960 to 1980, the maternal mortality rate decreased from 32.1 to 9.4 deaths per 100,000 live births, a decrease of 70.7%. From 1980 to 2000, the maternal mortality rate decreased only 12.8%, from 9.4 to 8.2 (Figure\u00a01) [2]. At the same time, from 1980 to 2000, the proportion of babies born preterm, very preterm, low birth weight, and very low birth weight increased by 26%, 8.2%, 14.7% and 25.9%, respectively (Figure\u00a02) [4]. However, unlike other maternal and infant health indicators, from 1980 to 2000 the infant mortality rate continued to decrease almost at a similar pace as that from 1960 to 1980, dropping by 45.2% from 12.6 to 6.9 infant deaths per 1,000 live births, compared with a drop of 51.5% from 26.0 to 12.6 per 1,000 live births for 1960 to 1980 (Figure\u00a01) [2]. Finally, it is estimated that the proportion of babies born in the United States who have a serious or major structural defect that can have adverse effects on their health or development continues to be about 3% [5].\nAssociated with this slowing rate of improvement (and, in some cases, deterioration) in pregnancy outcomes and maternal and infant health indicators is a shift in the leading causes of infant mortality. In 1960, maternal complications of pregnancy (including complications of placenta, cord, and the membranes) did not appear on the list of the 10 leading causes of infant mortality [1]. By 1980, maternal complications of pregnancy became the fifth leading cause of infant mortality, and the third leading cause of infant death in 2002, after congenital anomalies and low birth weight\/preterm delivery [6, 7]. In 2002, congenital anomalies, low birth weight, preterm delivery, and maternal complications of pregnancy accounted for 46.4% of all infant deaths in the United States (12,996 infant deaths) (Figure\u00a03) [7]. Although some of these infant deaths might have been prevented through interventions targeted at improving the health of mothers and modifying behaviors contributing to adverse pregnancy outcomes, poor maternal health, behaviors contributing to adverse pregnancy outcomes, and maternal complications of pregnancy continue to be prevalent. For example, 28.6% of women who gave birth between 1993 and 1997 were reported to have an obstetric complication, 4.1% had a preexisting medical condition, and 43.0% experienced some form of maternal morbidity (i.e., an obstetric complication, a preexisting medical condition, a cesarean section, or any combination of the three types of morbidity) [8]. In 2002, 26.1% of all deliveries were performed using cesarean section, presumably because of maternal or infant complications [8].\nFig. 1Maternal and infant mortality rates, United States, 1960\u20132002Fig. 2Percent of all live births that are preterm delivery, very preterm delivery, low birth weight, and very low birth weight, United States, 1980\u20132002\nEarly prenatal care is too late\nOne of the reasons that progress in improving pregnancy outcomes has slowed down, and in some cases reversed direction, is that we have failed to intervene before pregnancy to detect, manage, modify, and control maternal behaviors, health conditions, and risk factors that contribute to adverse maternal and infant outcomes. Although we know many interventions that, if delivered before pregnancy, could improve pregnancy outcomes, we have failed to make those services and interventions available to couples and women in need. Women of childbearing age suffer from a variety of chronic conditions that could potentially contribute to adverse pregnancy outcomes. For example, in 2002, the U.S. Department of Health and Human Services reported that 6.1% of women of reproductive age have asthma, 5% are obese, 3.4% have cardiac disease, 3.0% are hypertensive, 9.3% are diabetic, and 1.4% have thyroid disorder [9]. Moreover, a substantial proportion of women continue to enter pregnancy with risks proven to contribute to adverse pregnancy outcomes: in 2002, 11.4% of pregnant women smoked during pregnancy, a risk factor for low birth weight [4]; at the same time, 10.1% of pregnant women and 54.9% of women at risk of getting pregnant consumed alcohol, a risk factor for fetal alcohol syndrome [10]. Finally, using the Perinatal Periods of Risk approach, researchers in three cities (New York City; Tulsa, Oklahoma; and Kansas City, Missouri) concluded that racial and ethnic disparities in feto-infant mortality were largely related to maternal health, and, interventions to reduce feto-infant mortality should include preconception care and improvements in women's health [11\u201313].Fig. 3Leading causes of infant mortality, United States, 1960, 1980, and 2002. *IMR =  Infant Mortality Rate\nThe prenatal care \u201crevolution\u201d of the 1980s resulted in an increase in the proportion of women receiving early prenatal care (defined as prenatal care begun in the first trimester) [2]. After decreasing from 76.3% in 1980 to 75.8% in 1990, the proportion of women receiving early prenatal care increased to 83.2% in 2000 [2]. However, for many women, \u201cearly prenatal care is too late\u201d [14]. By the time a pregnant woman makes it to her first early prenatal visit, most fetal organs are already been formed, and many interventions to prevent birth defects or adverse maternal and infant outcomes come too late to have any effect. As a result, many national organizations now recommend routine preconception care. For example, the March of Dimes recommends that \u201cas the key physician\/primary care provider, the obstetrician\/gynecologists must take advantage of every health encounter to provide preconception care and risk reduction before and between conceptions\u2014the time when it really can make a difference\u201d [14]. The American Academy of Pediatrics (AAP) and the American College of Obstetricians and Gynecologists (ACOG) recommend that \u201call health encounters during a woman's reproductive years, particularly those that are a part of preconception care, should include counseling on appropriate medical care and behavior to optimize pregnancy outcomes\u201d [15].\nPreconception interventions work\nInterventions designed to lower preconception risks are known collectively as preconception care. These interventions are characterized by the need to start\u2014and sometimes complete\u2014a designated intervention before conception occurs. To simplify matters, ACOG and AAP have grouped the main components of preconception care under four categories of interventions: maternal assessment (e.g., family history, behaviors, obstetric history, general physical exam); vaccinations (e.g., rubella, varicella and hepatitis B); screening (e.g., HIV, STD, genetic disorders); and counseling (e.g., folic acid consumption, smoking and alcohol cessation, weight management) [15]. Many of these interventions are currently available to women and, when indicated, to couples; however, they are not systematically delivered. A comprehensive review of the literature identified a long list of risk factors suggested to be included in comprehensive preconception care (Table\u00a01). The strength of evidence supporting the efficacy of the various components of preconception care varies greatly [16]. Clinical practice guidelines (CPGs) have been developed for reducing the risk related to some of these factors. We identified 14 conditions for which CPGs exist and for which there is scientific evidence demonstrating effectiveness in improving pregnancy outcomes (Table\u00a02) [17--74].\nTable 1Risk factors (by Category) identified in the literature as needing attention during the preconception period1Chronic diseases: Diabetes; heart disease; high blood pressure; thyroid disease; asthma; anemia; kidney disease; metabolic and hematological disorders; depression and other mental disorders; autoimmune disease; and physical disability (access)2Infectious diseases: Vaccine-preventable diseases (rubella, hepatitis B, varicella, influenza, and tetanus); HIV\/AIDS; syphilis, chlamydia, and other sexually transmitted diseases; periodontal disease; toxoplasmosis, and cytomegalic inclusion virus3Reproductive concerns: Unplanned pregnancies; contraception; infertility; adverse past pregnancy outcomes (preterm delivery, birth defects, fetal\/infant death, maternal complications)4Genetic\/inherited conditions: Sickle cell anemia; thalassemia; Tay-Sachs disease; fragile X syndrome; Down syndrome; cystic fibrosis; muscular dystrophy; hearing and vision loss associated with genetic predisposition5Medications and medical treatment: Prescription medications contraindicated in pregnant women (FDA's Category X Drugs, 117 products in 2001 PDR, antiepileptic drugs, oral anticoagulants for maternal clotting disorders, and Accutane); diagnostic radiation exposures6Personal behaviors and exposures: Smoking; alcohol consumption; illicit drug use; overweight\/underweight; folic acid supplement use; domestic violence; eating disorders; exposure to infections; exposures to chemicals and other environmental toxins; consumption of over-the-counter medications; hyperthermia (e.g., from sauna use)Note. This list is not exhaustiveTable 2Selected preconception risk factors for adverse pregnancy outcomes for which clinical practice guidelines have been developed\u2022Folic acid. Daily use of vitamin supplements containing folic acid has been demonstrated to reduce the occurrence of neural tube defects by two thirds [17\u201324]\u2022Rubella seronegativity. Rubella vaccination provides protective seropositivity and prevents the occurrence of congenital rubella syndrome [25\u201327]\u2022Diabetes (preconception). The three-fold increase in the prevalence of birth defects among infants of women with type 1 and type 2 diabetes is substantially reduced through proper management of diabetes [28\u201331]\u2022Hypothyroidism. The dosages of Levothyroxine\u2122 required for treatment of hypothyroidism increases in early pregnancy. Levothyroxine\u2122 dosage needs to be adjusted for proper neurologic development [32\u201334]\u2022HIV\/AIDS. If HIV infection is identified before conception, timely treatment can be administered and women (or couples) can be given additional information that can influence the timing of the onset of pregnancy [35\u201341]\u2022Maternal phenylketonurea (PKU). Women diagnosed with PKU as infants have infants with mental retardation. However, this adverse outcome can be prevented when mothers adhere to a low phenylalanine diet before conception and continue it throughout their pregnancy [42, 43]\u2022Oral anticoagulant. Warfarin, which is used for the control of blood clotting, has been demonstrated to be a teratogen. To avoid exposure to warfarin during early pregnancy, medications can be changed to a nonteratogenic anticoagulant before the onset of pregnancy [15, 44\u201346]\u2022Anti-epileptic drugs. Certain anti-epileptic drugs are known as teratogens. Before conception, women who are on a regimen of these drugs and who are contemplating pregnancy should be prescribed a lower dosage of these drugs [15, 47\u201351]\u2022Isotretinoins (Accutane\u00ae): Use of isotretinoins in pregnancy to treat acne results in miscarriage and birth defects. Effective pregnancy prevention should be implemented to avoid unintended pregnancies among women with childbearing potential who use this medication [15, 52\u201354]\u2022Smoking. Preterm birth, low birthweight, and other adverse perinatal outcomes associated with maternal smoking in pregnancy can be prevented if women stop smoking during early pregnancy. Because only 20% of women successfully control tobacco dependence during pregnancy, cessation of smoking is recommended before pregnancy [55\u201358]\u2022Alcohol misuse. No time during pregnancy is safe to drink alcohol, and harm can occur early, before a woman has realized that she is or might be pregnant. Fetal alcohol syndrome and other alcohol-related birth defects can be prevented if women cease intake of alcohol before conception [59\u201364]\u2022Obesity. Adverse perinatal outcomes associated with maternal obesity include neural tube defects, preterm delivery, diabetes, cesarean section, and hypertensive and thromboembolic disease. Weight loss before pregnancy reduces these risks [15, 65\u201369]\u2022STD. Chlamydia trachomatis and Neisseria gonorrhea have been strongly associated with ectopic pregnancy, infertility, and chronic pelvic pain. STDs during pregnancy might result in fetal death or substantial physical and developmental disabilities, including mental retardation and blindness [70, 71]\u2022Hepatitis B. Vaccination is recommended for men and women who are at risk for acquiring hepatitis B virus (HBV) infection. Preventing HBV infection in women of childbearing age prevents vertical transmission of infection to infants and eliminates risk for infection and sequelae, including hepatic failure, liver carcinoma, cirrhosis, and death [72\u201374]\nHowever, the best evidence for the effectiveness of specific components of preconception care is seen when the focus was on a single intervention and often not in the context of improving pregnancy outcomes. For example, effectiveness documented by the U.S. Preventive Services Task Force for interventions related to smoking, alcohol misuse, and obesity are based on studies of interventions delivered in primary care settings that was not complicated by the additional delivery of other components of preconception care [56, 61, 66]. One study reported the effectiveness of comprehensive preconception care but inferences for the United States are limited because the setting of intervention delivery was in Hungary [75].\nPriorities for preconception care and preconception health\nThe immediate priority is to ensure that evidence-based preconception interventions are implemented to further improve infant and maternal pregnancy outcomes. Many national professional organizations have developed and published CPGs for specific components of preconception care. For example, the American Diabetes Association developed CPGs to be started before pregnancy for women with preconception diabetes [28]. The American Association of Clinical Endocrinologists has developed preconception CPGs for women with hypothyroidism [34]. CPGs have also been developed for women being treated with teratogenic medications to guide the transition to safer medications. CPGs for women using antiepileptic drugs or oral anticoagulants have been developed by the American Academy of Neurology [50], the American Heart Association, and the American College of Cardiologists, respectively [44, 46]. Guidelines, recommendations, and strategies for reducing alcohol misuse and prevention of fetal alcohol syndrome have been developed and published by the U.S. Public Health Service, U.S. Preventive Service Taskforce, ACOG, and the National Taskforce on Fetal Alcohol Syndrome and Fetal Alcohol Effects [55\u201359].\nHowever, moving forward towards more universal preconception care is not without its challenges. There is no national policy, nor do standard tools exist, for the delivery of these services. Some have suggested that challenges could be overcome by promoting preconception health to all women of reproductive age at each and every encounter with the health care system. The generally recommended mechanisms would include risk assessment (screening), health promotion (education and counseling), and intervention or referral. However, the existing recommendations to provide these three essential components have not been translated into practical tools for action and decision-making in the clinical setting. Stated another way, primary care providers do not have the tools they need to implement what is known to work for improving preconception health.\nChallenges and opportunities\nWhereas clinical practice and the promotion of preconception care services could potentially be shaped by guidelines, widespread adoption of the guidelines requires a more active approach [76, 77]. The literature on diffusion of innovation and translation of research into practice clearly indicates that changing primary care providers\u2019 knowledge, attitudes, and practices requires multiple steps including consolidation and distillation of guidelines, active organizational support, clinically relevant decision tools, use of quality improvement techniques, and performance monitoring [78, 79]. A growing body of research indicates that providers are more likely to engage in evidence-based practices following participation in quality improvement projects (e.g., rapid improvement cycles or collaborative groups). The federally funded Health Disparities Collaborative, the Perinatal Care and Patient Safety Collaboratives, and similar quality improvement efforts for community health centers demonstrate how such approaches are being used to better serve low-income and uninsured patients.\nThe need to develop innovative approaches to deliver and finance bundles of preconception interventions must be viewed as a high priority. However, although strong evidence exists for many preconception care interventions and shows they are effective, discussion continues regarding the benefits of delivering comprehensive preconception care [16, 80, 81], and the effectiveness or added value of \u201cpackaging\u201d or \u201cbundling\u201d these interventions remains to be determined. For example, preconception care interventions could potentially be bundled into behavioral modification services (e.g., smoking and alcohol cessation, weight control, exercise), screening and assessment (e.g., vaccines, general physical examination), and specialized care (e.g., management of diabetes and hypertension). Alternatively, interventions could be bundled into visits (e.g., do x at each well-woman visit, do y every 2 years, do z only for women at risk). Some evidence already suggests that bundling could be of added value. For example, an evaluation of the National Centers of Excellence in Women's Health found that, compared with other settings, women received better-quality primary health care at those centers having better integrated and coordinated services [82]. However, more such research, focused specifically on integration of preconception care components into primary care, is clearly warranted.\nEfforts to promote healthy behavior and improve service delivery should be accompanied by improvements in health care coverage and financing for women of childbearing age. Affordability of care is a major concern for many women [83], and more can be done to improve access to preconception care. Many women under age 65 are uninsured, including 40% of poor women, one third of near-poor women (with income between 100% and 200% of the federal poverty level), 50% of women with disabilities, and 29% of young women ages 19\u201324 years [84]. Expanding health care coverage for low-income women, through public programs such as Medicaid, Medicaid waivers, the State Children's Health Insurance Program (SCHIP), is particularly important. Medicaid offers particularly important opportunities. In 2003, 12% of all women of childbearing age (15\u201344 years) and 37% of poor women in that age group relied on Medicaid for health care coverage [83]. Many low-income women do not, however, qualify for Medicaid because they do not have children under age 18, are not over age 65, or are undocumented. As states seek to expand Medicaid coverage to low-income, uninsured adults, women of childbearing age should receive high priority.\nAlthough two thirds of women in Medicaid are of childbearing age [19\u201344, 85], many additional low-income women do not qualify for Medicaid benefits under current state eligibility rules unless they are pregnant. Without coverage before and between pregnancies, low-income women typically miss preventive visits [83]. Recognizing these gaps and missed opportunities for prevention, 21 states have developed special programs (under federal Medicaid waivers) that cover some women who do not otherwise qualify for Medicaid, particularly to offer family planning and interconception care. Specifically, these states extend eligibility to women who lose coverage after the birth of a baby or starting a job, while other states offer family planning coverage based on income status to men and\/or women [86]. An evaluation of \u201cfamily planning waiver\u201d projects prepared for the federal Center for Medicare and Medicaid Services (CMS) found that they resulted in significant savings to both the federal and state governments [87]. Greater potential savings and prevention, however, could undoubtedly result if states offered more preconception risk screening, health promotion, and interventions; to do so, however, states need permission from the federal government to include such services in their waivers or need for Congress to approve interconception care as an optional benefit.\nFinally, special attention should be given to the continuing, and sometimes increasing, racial gap in poor outcomes, especially in terms of access to services, and quality of care. These priorities can potentially complicate decision-making for public health policy. The best evidence of intervention effectiveness is often based on studies of health care systems. However, in developing programs and policies for the delivery of preconception services, one must consider the fact that poor access to health care can be a major determinant of health disparities.\nPreconception care: the next maternal and child health frontier\nThe significant improvements in infant and maternal pregnancy outcomes during the past 40 years have resulted largely from the intensified focus on family planning in the 1960s and 1970s, on child health in the 1970s and 1980s, and on prenatal care in the 1980s and 1990s. Over the past 40 years, many public health and clinical care providers and their organizations have also recognized that, although it is important to offer family planning services to prevent unplanned pregnancies, it is not enough to worry about a pregnant woman's health only during her pregnancy or a child's health only after its birth, because many of the factors that contribute to the mother's and child's health can be identified and often successfully managed before pregnancy.\nToday, the greatest opportunities for further improvement in pregnancy outcomes\u2014in improving the health of women and their children\u2014lie in prevention strategies that must be implemented prior to conception to be effective. In 1989, the U.S. Public Health Service Expert Panel identified preconception care as an essential part of prenatal care [88]. National organizations of health professionals, such as the AAP, ACOG, and the American Academy of Family Physicians (AAFP), all recommend specific components of preconception care that should be delivered within the context of health care systems [15, 89]. The time has come to move forward to the next maternal and child health frontier of \u201cprevention\u201d by acting on the recommendations of professional organizations and implementing scientifically proven interventions to further improve pregnancy outcomes.\nAction is urgently required in an array of clinical settings, in health promotion campaigns, and in academic research settings. Because of the wide range of interventions included under the umbrella of preconception care, such interventions may be delivered in both primary care and specialty care practices. However, opportunities also exist to improve preconception health through wellness care, through maintenance care for women with chronic health conditions associated with increased preconception risk (e.g., maternal diabetes), and in settings where women seek medical support for one specific risk such as smoking or obesity. In addition, more research is needed to understand how a women's motivation for healthy behavior can be used to initiate the reduction of a number of preconception risks. For example, comparison of concurrent versus sequential interventions for smoking and weight control requires additional clarification [90].\nAbove all, health care providers and health agencies must understand that early prenatal care is too late for many women and babies. In particular, some evidence-based interventions recommended for implementation during pregnancy could be more effective and beneficial if implemented before conception. For example, although smoking cessation is recommended for all pregnant women, more than one attempt to stop may be necessary to achieve success. Only about 20% of pregnant women who smoke are able to stop smoking during pregnancy [90]. Starting smoking cessation treatment \u2014 and successfully completing it\u2014should be recommended before pregnancy is begun. Similarly, the only definitive way of preventing fetal alcohol syndrome is to stop drinking even before pregnancy. In the same manner, HIV testing and genetic counseling are recommended during pregnancy, but additional options are available to women who use these interventions prior to conception, including the decision not to become pregnant.\nConclusion\nThere is ample evidence that individual elements of preconception care work. Equally important, however, is the evidence that some interventions work best and others only work if provided before pregnancy. Given these realities, to further improve perinatal outcomes, the United States must commit to improving preconception health and to providing preconception care to all women of reproductive age. Even though sufficient knowledge and evidence is present to take action now, further efforts are needed to identify best practices and the most effective means of delivering integrated preconception services.","keyphrases":["preconception care","perinatal outcomes","women's health"],"prmu":["P","P","P"]}
{"id":"Pflugers_Arch-3-1-2082651","title":"Criteria for arrhythmogenicity in genetically-modified Langendorff-perfused murine hearts modelling the congenital long QT syndrome type 3 and the Brugada syndrome\n","text":"The experiments investigated the applicability of two established criteria for arrhythmogenicity in Scn5a+\/\u0394 and Scn5a+\/\u2212 murine hearts modelling the congenital long QT syndrome type 3 (LQT3) and the Brugada syndrome (BrS). Monophasic action potentials (APs) recorded during extrasystolic stimulation procedures from Langendorff-perfused control hearts and hearts treated with flecainide (1 \u03bcM) or quinidine (1 or 10 \u03bcM) demonstrated that both agents were pro-arrhythmic in wild-type (WT) hearts, quinidine was pro-arrhythmic in Scn5a+\/\u0394 hearts, and that flecainide was pro-arrhythmic whereas quinidine was anti-arrhythmic in Scn5a+\/\u2212 hearts, confirming clinical findings. Statistical analysis confirmed a quadratic relationship between epicardial and endocardial AP durations (APDs) in WT control hearts. However, comparisons between plots of epicardial against endocardial APDs and this reference curve failed to correlate with arrhythmogenicity. Restitution curves, relating APD to diastolic interval (DI), were then constructed for the first time in a murine system and mono-exponential growth functions fitted to these curves. Significant (P < 0.05) alterations in the DI at which slopes equalled unity, an established indicator of arrhythmogenicity, now successfully predicted the presence or absence of arrhythmogenicity in all cases. We thus associate changes in the slopes of restitution curves with arrhythmogenicity in models of LQT3 and BrS.\nIntroduction\nMutations in the SCN5a gene encoding the \u03b1-subunit of the cardiac voltage-gated Na+ channel are well established to underlie hereditary arrhythmic syndromes which may result in sudden cardiac death. On the one hand, gain-of-function mutations which increase inward depolarising currents and consequently prolong the ventricular action potential are associated with arrhythmogenicity in the congenital long QT syndrome type 3 (LQT3) [23]. On the other hand, loss-of-function mutations which decrease such currents are associated with arrhythmogenicity in the Brugada syndrome (BrS) [1]. In this case, spatial heterogeneities in the degree of shortening of the ventricular action potential result in characteristic electrocardiographic changes, namely a partial right bundle branch block pattern with ST segment elevation in the right precordial leads [2].\nDespite the similar clinical outcomes of LQT3 and BrS, class 1A and 1C, anti-arrhythmic agents, exemplified by quinidine and flecainide respectively, exert contrasting effects on arrhythmogenicity in these two syndromes [31]. Thus in LQT3, quinidine is pro-arrhythmic [33] and flecainide is anti-arrhythmic [22]. In contrast, in BrS quinidine is anti-arrhythmic [24], while flecainide is pro-arrhythmic [7] and used to unmask the arrhythmic phenotype in asymptomatic patients. These contrasts are precisely reflected in results from our murine models of LQT3 [39] and BrS [38], emphasizing the translatability of results obtained from such systems. Furthermore, both quinidine and flecainide are known to have the potential for pro-arrhythmic effects [12, 30].\nThese similarities and differences have parallels in the physiological changes thought to underlie arrhythmogenicity in LQT3 and BrS. Thus circus-type re-entry [2], requiring both an initiating trigger and a re-entrant substrate, has been implicated in arrhythmogenicity in both these conditions. Of these requirements, both conditions are associated with triggering extrasystoles [3]. In LQT3, these are initiated by early after-depolarizations attributable to action potential prolongation [42], while in BrS they are initiated by phase 2 re-entry [20, 46]. Furthermore, in both LQT3 and BrS, arrhythmia occurs against a re-entrant substrate [3]. In LQT3 this is provided by increased transmural dispersion of repolarization [13, 27, 42]. While increased transmural dispersion of repolarization has also been demonstrated in some studies on BrS [3], increased epicardial dispersion of repolarization [2] and slowed conduction [29] are also implicated. Thus studies in murine hearts have associated alterations in transmural dispersion of repolarization, reflected in relationships between epicardial and endocardial action potential durations, with arrhythmogenicity in models of LQT3 but failed to do so in models of BrS [21, 33, 38, 39, 42].\nArrhythmogenicity has often been associated with increases in the slopes of restitution curves, plotting action potential duration against the preceding diastolic interval, to greater than unity [14, 17\u201319, 28, 40]. Possible mechanisms that might underlie this association have been discussed in detail on a number of occasions [11, 25, 43, 44]. Furthermore, decreases in the slopes of such curves are associated with the anti-arrhythmic effects of several clinically important agents including amiodarone, bretylium and procainamide [11, 26, 32]. Such associations have previously been demonstrated in a pharmacological model of LQT2 [45] but has not been assessed in LQT3 or in BrS.\nThe present study compares an analysis examining transmural dispersion of repolarization, assessed by comparing epicardial and endocardial action potential durations obtained over a range of heart rates and previously established to predict arrhythmogenicity in hypokalaemic Langendorff-perfused murine hearts [34], with another examining effects on restitution curve parameters. This represents the first occasion on which restitution curves have been constructed in a murine system, in relation to BrS or LQT3, or indeed in any genetic model of an hereditary arrhythmic syndrome. To this end, the monophasic action potential (MAP) technique and an established Langendorff-perfused system [15, 34\u201336] are used to study genetically modified mice modelling LQT3 (Scn5a+\/\u0394) [13] and BrS (Scn5a+\/\u2212) [29] before and after exposure to quinidine and flecainide. We proceed to demonstrate that while the former criterion fails, the latter produces predictions in full agreement with the arrhythmogenic findings in every case and thus suggest that alterations in the slopes of restitution curves are associated with arrhythmogenicity in both LQT3 and BrS.\nMaterials and methods\nExperimental animals\nWild-type (WT), Scn5a+\/\u0394 and Scn5a+\/\u2212 129 Sv mice aged 3\u20136\u00a0months were housed at 21\u2009\u00b1\u20091\u00b0C with 12\u00a0h light\/dark cycles. They were fed sterile chow (RM3 Maintenance Diet, SDS, Witham, Essex, UK) and had free access to water. All procedures complied with the UK Animals (Scientific Procedures) Act 1986.\nSolutions\nSolutions were based on bicarbonate-buffered Krebs-Henseleit solution (119\u00a0mM NaCl, 25\u00a0mM NaHCO3, 4\u00a0mM KCl, 1.2\u00a0mM KH2PO4, 1\u00a0mM MgCl2, 1.8\u00a0mM CaCl2, 10\u00a0mM glucose and 2\u00a0mM Na-pyruvate; pH adjusted to 7.4) and were bubbled with 95% O2\/5% CO2 (British Oxygen Company, Manchester, U.K). Drug-containing solutions were prepared by adding flecainide (Sigma-Aldrich, Poole, UK) to final concentration of 1\u00a0\u03bcM and quinidine (Sigma-Aldrich, Poole, UK) to final concentrations of 1 and 10\u00a0\u03bcM: these concentrations were chosen to reflect clinically therapeutic values (effective free and total plasma concentrations of 0.79 and 1.48\u00a0\u03bcM, respectively, for flecainide and 0.74 and 7.9\u00a0\u03bcM, respectively, for quinidine) [9].\nPreparation\nA Langendorff-perfusion protocol previously adapted for murine hearts [4] was used. Briefly, mice were killed by cervical dislocation [Schedule 1: UK Animals (Scientific Procedures) Act 1986] before hearts were quickly excised and placed in ice-cold bicarbonate-buffered Krebs-Henseleit solution. Short sections of aorta were cannulated under the surface of the solution and attached to a custom-made 21-gauge cannula filled using an aneurysm clip (Harvard Apparatus, Edenbridge, Kent, UK), within 90\u00a0s of excision. Fresh Krebs-Henseleit solution was passed through 200- and 5-\u03bcm filters (Millipore, Watford, UK) and warmed to 37\u00b0C using a water jacket and circulator (Techne model C-85A, Cambridge, UK). This solution was then used for constant-flow retrograde perfusion, thereby circumventing any changes in coronary vascular resistance and therefore flow that might otherwise result from the effects of the drugs studied [10], at 2\u20132.5\u00a0ml\/min, driven by a peristaltic pump (Watson-Marlow Bredel model 505S, Falmouth, Cornwall, UK). Hearts were regarded as suitable for experimentation if they regained a healthy pink colour and began to contract spontaneously on re-warming.\nElectrophysiological measurements\nAn epicardial MAP electrode (Hugo Sachs, Harvard Apparatus, UK) was placed against the basal left ventricular epicardium. A custom-made endocardial MAP electrode consisting of two twisted strands of high-purity Teflon-coated silver wire of 0.25\u00a0mm diameter (Advent Research Materials, UK) was manufactured. A small access window was created in the interventricular septum to allow access to the left ventricular endocardium [8]. The Teflon coating was removed from the distal 1\u00a0mm of the electrode; this was then galvanically chlorided to eliminate DC offset, inserted and placed against the septal endocardial surface. MAPs were amplified, band-pass filtered (0.5\u00a0Hz to 1\u00a0kHz: Gould 2400S, Gould-Nicolet Technologies, Ilford, Essex, UK) and digitised at a sampling frequency of 5\u00a0kHz (micro1401, Cambridge Electronic Design, Cambridge, UK). Analysis of MAPs was performed using Spike II (Cambridge Electronic Design, Cambridge, UK).\nExperimental protocol\nA bipolar platinum stimulating electrode (1\u00a0mm inter-pole spacing) was placed on the basal surface of the right ventricular epicardium. Square-wave stimuli (Grass S48 stimulator, Grass-Telefactor, Slough, UK) of 2\u00a0ms duration and amplitudes of twice the excitation threshold were initially applied to hearts at a constant baseline cycle length (BCL) of 125\u00a0ms until MAPs showed stable baselines, rapid upstroke phases that reached consistent amplitudes and smooth repolarization phases [16] and for at least 10\u00a0min. Before subsequent recordings were made, hearts were then exposed to test solutions for 20\u00a0min while stimulation was continued.\nIn initial experiments, MAPs were recorded during regular stimulation at a BCL of 125\u00a0ms. Arrhythmogenicity was then assessed both during regular stimulation and using an extrasystolic stimulation protocol [37] previously adapted for the study of arrhythmogenesis in murine hearts incorporating the Scn5a+\/\u0394 and Scn5a+\/\u2212 mutations [13, 38] and described below. Hearts were then subjected to an adapted dynamic pacing protocol [17] explained in detail below, and the data obtained used to construct restitution curves.\nData analysis\nAll data are presented as means \u00b1 standard errors of the means and include the number of hearts studied. Comparisons between data sets used analysis of variance (significance threshold set at P\u2009\u2264\u20090.05). Curve fitting of particular functions to data sets used a Levenberg-Marquardt algorithm (OriginPro 7.5, OriginLab, MA, USA).\nResults\nThe experiments made direct measurements of MAPs from intact Langendorff-perfused murine hearts. They compared the extents to which relationships between epicardial and endocardial action potential durations and the slopes of restitution curves are associated with arrhythmogenicity in genetic models of the congenital LQT3 (Scn5a+\/\u0394) and of the BrS (Scn5a+\/\u2212), and in wild-type (WT) controls. They began by successfully demonstrating that these experimental models recapitulated arrhythmic phenotypes observed clinically as well as confirming the anticipated effects of treatment with clinically-relevant [6] concentrations of flecainide (1\u00a0\u03bcM) and quinidine (1 or 10\u00a0\u03bcM). Hearts were exposed to solutions for 20\u00a0min while stimulated at a constant BCL of 125\u00a0ms before recording was commenced.\nArrhythmogenicity in WT, Scn5a+\/\u0394 and Scn5a+\/\u2212 hearts subject to both regular and extrasystolic stimulation recapitulates clinical observations\nFigures\u00a01 and 2 show results obtained from WT (Figs.\u00a01a, 2a), Scn5a+\/\u0394 (Figs.\u00a01b, 2b) and Scn5a+\/\u2212 (Figs.\u00a01c, 2c) hearts treated with control solution (A) and with solutions containing 1\u00a0\u03bcM flecainide (B), 1\u00a0\u03bcM quinidine (C) and 10\u00a0\u03bcM quinidine (D). Figure\u00a01 illustrates MAP waveforms recorded from the epicardia of hearts during the extrasystolic stimulation procedure showing arrhythmic waveforms in situations where arrhythmic activity was observed in >50% of cases; Fig.\u00a02 quantifies the incidence of such arrhythmic activity both during regular stimulation (i) and during the extrasystolic stimulation procedure (ii).\nFig.\u00a01Arrhythmic activity resulting from extrasystolic stimulation. Epicardial monophasic action potential (MAP) recordings illustrating the result of the application of extrasystolic stimuli to WT (a), Scn5a+\/\u0394 (b) and Scn5a+\/\u2212 (c) hearts treated with control solution (A) and with solutions containing 1\u00a0\u03bcM flecainide (B), 1\u00a0\u03bcM quinidine (C) and 10\u00a0\u03bcM quinidine (D). Traces show arrhythmic activity where this was observed in \u226550% of casesFig.\u00a02Incidences of arrhythmic activity in WT, Scn5a+\/\u0394 and Scn5a+\/\u2212 hearts subjected to the extrasystolic stimulation procedure. Percentage of WT (a), Scn5a+\/\u0394 (b) and Scn5a+\/\u2212 (c) hearts demonstrating arrhythmic activity during regular stimulation (i) and during the extrasystolic stimulation procedure (ii). In both these cases, MAPs were obtained during treatment with control solution (A) and with solutions containing 1\u00a0\u03bcM flecainide (B), 1\u00a0\u03bcM quinidine (C) and 10\u00a0\u03bcM quinidine (D). Hashing indicates hearts demonstrating arrhythmic activity, and dots indicate hearts not demonstrating such activity\nThe frequencies of arrhythmic phenomena in hearts subjected to regular stimulation at a BCL of 125\u00a0ms were relatively low irrespective of the genetic or pharmacological conditions but were nevertheless consistent with clinical expectations. First, zero out of seven WT hearts (~0%, Fig.\u00a02a, i) exposed to control solution (A) demonstrated arrhythmic activity, while treatment with flecainide (B) led to arrhythmic activity in only one out of six (~17%) cases. Furthermore, such hearts never showed arrhythmic activity during treatment with 1\u00a0\u03bcM quinidine (C, zero out of six hearts, ~0%) and again did so in only one out of six cases (~17%) with 10\u00a0\u03bcM quinidine (D). Secondly, arrhythmic activity did occur in one out of six (~17%) Scn5a+\/\u0394 hearts (Fig.\u00a02b) treated with control solution (A) and was absent in all cases (zero out of six hearts, ~0%) during treatment with flecainide (B). Arrhythmic activity was not observed (zero out of four hearts, ~0%) during treatment with 1\u00a0\u03bcM quinidine (C) but did occur in one out of five hearts (~20%) exposed to 10\u00a0\u03bcM quinidine (D). Thirdly, two out of nine Scn5a+\/\u2212 hearts (~22%, Fig.\u00a02c) exhibited arrhythmic activity during exposure to control solution (A). Such arrhythmic activity was also observed in one out of five hearts (~20%) treated with flecainide (B). Arrhythmic activity was then absent after treatment with either 1\u00a0\u03bcM (C) or 10\u00a0\u03bcM (D) quinidine (zero out of five, 0%, and zero out of six, 0%, hearts, respectively).\nIn contrast, extrasystolic stimulation (Figs.\u00a01 and 2, ii) revealed clearer trends in arrhythmogenicity in full agreement with clinical expectations. First, while zero out of seven WT hearts (0%, Fig.\u00a02a, ii) exposed to control solution (A) exhibited arrhythmic activity, treatment with flecainide (B) increased the incidence of such events to two out of six hearts (~33%). Furthermore, exposure to 1\u00a0\u03bcM quinidine (C) resulted in arrhythmic activity in four out of eight hearts (~50%), while exposure to 10\u00a0\u03bcM quinidine (D) resulted in arrhythmic activity in three out of six hearts (~50%). Secondly, one out of five Scn5a+\/\u0394 hearts (~20%, Fig.\u00a02b, ii) demonstrated arrhythmic activity during exposure to either control solution (A) or flecainide (B). However, exposure to quinidine resulted in arrhythmic activity in three out of five hearts (~60%) at 1\u00a0\u03bcM (C) and four out of four hearts (~100%) at 10\u00a0\u03bcM (D). Thirdly, Scn5a+\/\u2212 hearts (Fig.\u00a02c, ii) demonstrated such arrhythmic activity in three out of nine cases (~33%) during treatment with control solution (A) but in five out of six cases (~83%) during treatment with flecainide (B). Furthermore, arrhythmic activity was observed in only one out of six such hearts (~17%) during treatment with 1\u00a0\u03bcM quinidine (C) and in zero out of six hearts during exposure to 10\u00a0\u03bcM quinidine (D).\nGraphical analyses of arrhythmogenic tendency\nIn an effort to explore the possible basis for these arrhythmic phenomena, subsequent experiments made systematic measurements of epicardial and endocardial action potential durations (at 90% repolarization, APD90s) during a dynamic pacing protocol [17]. This consisted of cycles each comprising 100 stimuli, initially delivered at a BCL of 175\u00a0ms. BCL was then decremented in 50-ms steps with each subsequent cycle until a reproducible sequence of consistently shaped MAP waveforms was no longer obtained. Steady states were consistently reached within the first 50 responses, and therefore mean values of APD90 were obtained from the final 50 action potentials in each cycle. This approach provided data that could be used to evaluate over the full range of BCLs the relationship between arrhythmogenicity and two possible electrophysiological properties. Thus it was possible first to evaluate transmural repolarization gradients and secondly to evaluate slopes of restitution curves relating APD90 to the preceding DI, given by BCL minus APD90 [17, 25, 28, 43].\nRelationships between epicardial and endocardial action potential durations are not consistently associated with either the presence or absence of arrhythmogenicity\nAlterations in relationship between epicardial and endocardial APD90 determined at a single BCL has previously been associated with arrhythmogenicity in hearts obtained from our Scn5a+\/\u0394 mice [42]. The present study now extends this analysis from a single BCL to a range of BCLs and applies it to Scn5a+\/\u2212 hearts for the first time, plotting endocardial APD90 on the abscissa and epicardial APD90 on the ordinate over the full range of BCLs investigated (triangles, Fig.\u00a03). Such plots have previously been used to assess arrhythmogenicity in hypokalaemic WT murine hearts [34] and provided possible criteria for arrhythmogenicity. The control plot obtained from WT hearts perfused with control solution (six hearts, Fig.\u00a03a, A) gave a quadratic relationship between endocardial and epicardial APD90 (solid line), in agreement with previous work [34]. Thus the curve y\u2009=\u2009ax2\u2009+\u2009bx\u2009+\u2009c, where a\u2009=\u20090.0097\u2009\u00b1\u20090.0028, b\u2009=\u20091.46\u2009\u00b1\u20090.27 and c\u2009=\u2009\u22125.06\u2009\u00b1\u20096.34, gave a reduced \u03c72 of 0.49. Repeating the regression analysis this time fitting the line y = mx + c gave a reduced \u03c72 of 0.86, and permitted the derivation of an F statistic for the appropriate, N\u2009+\u20091\u2009=\u200917 data points, given by  in which the subscripts on the right-hand side of the equation denote the number of parameters involved [5]. This yielded a probability of exceeding F of <0.05, suggesting that inclusion of the quadratic term was justified, again in full agreement with the previous study [34].\nFig.\u00a03Relationships between epicardial and endocardial action potential durations (at 90% repolarization, APD90s) obtained over a range of baseline cycle lengths. WT (a), Scn5a+\/\u0394 (b) and Scn5a+\/\u2212 (c) hearts were exposed to control solution (A) and to solutions containing 1\u00a0\u03bcM flecainide (B), 1\u00a0\u03bcM quinidine (C) and 10\u00a0\u03bcM quinidine (D) and stimulated at baseline cycle lengths of between 80\u00a0ms and 175\u00a0ms. Broken lines indicate reference curves constructed by least-squares fitting of the equation y\u2009=\u2009ax2\u2009+\u2009bx\u2009+\u2009c to data points obtained from wild-type hearts treated with control solution\nData points obtained under the range of genetic and pharmacological conditions studied could then be compared with this reference curve (broken lines, Fig.\u00a03) with the expectation that the departure of points to the left of this curve might be associated with arrhythmogenicity [34]. However, data sets obtained before and after the pharmacological manoeuvres satisfied this expectation in WT but clearly failed to do so in the remaining genetically modified hearts.\nFirst, treatment of WT hearts (Fig.\u00a03a) with flecainide (five hearts, B), 1\u00a0\u03bcM quinidine (six hearts, C) and 10\u00a0\u03bcM quinidine (six hearts, D) all resulted in points falling to the left of the reference curve, in parallel with the occurrence of arrhythmic activity under these conditions (Fig.\u00a02a, B, C and D). Secondly, data points obtained from Scn5a+\/\u0394 hearts (Fig.\u00a03b) treated with control solution (five hearts, A) again fell to the left of the reference curve in contrast with the low incidence of arrhythmic activity in this setting (Fig.\u00a02b, A). This pattern was maintained when such hearts were exposed to flecainide (five hearts, Fig.\u00a03b, B), once again in contrast with the low frequency which arrhythmic activity was observed under these conditions (Fig.\u00a02b, B). Exposure to 1\u00a0\u03bcM quinidine (five hearts, Fig.\u00a03b, C) and 10\u00a0\u03bcM quinidine (six hearts, D) again both resulted in points falling to the left of the control curve, this time in parallel with the high incidence of arrhythmic activity in these settings (Figs.\u00a02b, C and 3b, D). Thirdly, data points obtained from Scn5a+\/\u2212 hearts (Fig.\u00a03c) treated with control solution (six hearts, A) consistently fell on the reference curve, in contrast with the occurrence of arrhythmic activity in this setting (Fig.\u00a02c, A). Furthermore, treatment with flecainide (six hearts, Fig.\u00a03c, B) failed to result in a left-shift of data points, in contrast with the large increase in the incidence of arrhythmic activity (Fig.\u00a02c, B). In addition, points obtained during treatment with either 1\u00a0\u03bcM quinidine (five hearts, Fig.\u00a03c, C) or 10\u00a0\u03bcM quinidine (seven hearts, D) again fell to the left of the reference curve, in contrast with the anti-arrhythmic effect of this agent demonstrated in Fig.\u00a02c, C and D.\nAlterations in the slopes of restitution curves correlate with the presence and absence of arrhythmic activity\nRestitution curves (data points, left ordinate) were then plotted using the APD90 (left ordinate) and DI data obtained during the dynamic pacing protocol from the epicardia (circles, Fig.\u00a04) and endocardia (squares, Fig.\u00a05) of WT (Figs.\u00a04a, 5a), Scn5a+\/\u0394 (Figs.\u00a04b, 5b) and Scn5a+\/\u2212 (Figs.\u00a04c, 5c) hearts for the first time. Hearts were studied during treatment with control solution (A) and with solutions containing flecainide (B), 1\u00a0\u03bcM quinidine (C) or 10\u00a0\u03bcM quinidine (D). Under all conditions, epicardial and endocardial APD90 both decreased as DI decreased. Increases or decreases in the slopes of such relationships were in full agreement with the presence or absence of arrhythmic activity under all conditions studied.\nFig.\u00a04Epicardial restitution curves plotting action potential duration (at 90% repolarization, APD90) against preceding diastolic interval (DI). Data were obtained from the epicardia (circles) of WT (a), Scn5a+\/\u0394 (b) and Scn5a+\/\u2212 (c) hearts exposed to control solution (A) and to solutions containing 1\u00a0\u03bcM flecainide (B), 1\u00a0\u03bcM quinidine (C) and 10\u00a0\u03bcM quinidine (D). Curves are fitted with mono-exponential growth functions obtained by least squares fitting to the experimental values of APD90 and DI (solid lines, left ordinates). Gradients were obtained by differentiation of the fitted functions (broken lines, right axes). Shaded boxes indicate ranges of DI values at which such gradients exceed unityFig.\u00a05Endocardial restitution curves plotting action potential duration (at 90% repolarization, APD90) against preceding diastolic interval (DI). Data were obtained from the endocardia (squares) of WT (a), Scn5a+\/\u0394 (b) and Scn5a+\/\u2212 (c) hearts exposed to control solution (A) and to solutions containing 1\u00a0\u03bcM flecainide (B), 1\u00a0\u03bcM quinidine (C) and 10\u00a0\u03bcM quinidine (D). Curves are fitted with mono-exponential growth functions obtained by least squares fitting to the experimental values of APD90 and DI (solid lines, left ordinates). Gradients were obtained by differentiation of the fitted functions (broken lines, right axes). Shaded boxes indicate ranges of DI values at which such gradients exceed unity\nRestitution curves yield parameters predictive of arrhythmogenicity\nTo permit quantitative analysis, simple mono-exponential growth functions, as used previously [28], were fitted to the experimental data. These took the form:\nwhere y represents APD90, x represents DI, and y0, A and \u03c4 are constants obtained by least squares fitting to the experimental values of APD90 and DI in each case. These fitted parameters, along with corresponding reduced \u03c72 values, are shown in Tables\u00a01 (epicardium) and 2 (endocardium). Curve fits performed on data sets obtained by separating successive odd- and even-numbered action potentials yielded statistically indistinguishable (P\u2009>\u20090.05) parameters in all cases (Tables\u00a03 and 4) thus eliminating the possibility of error arising as a result of averaging. The corresponding gradients were then given by:\nand take their largest values at the shortest BCLs studied.\nTable\u00a01Fit parameters for epicardial restitution curvesConditiony0 (ms)A (ms)\u03c4 (ms)\u03c72WT (6 hearts)1.6\u2009\u00b1\u20094.456.2\u2009\u00b1\u20092.458.4\u2009\u00b1\u20099.81.4WT + flecainide (5 hearts)\u221216.3\u2009\u00b1\u20099.4104.7\u2009\u00b1\u20097.182.8\u2009\u00b1\u200928.05.9WT\u2009+\u20091\u00a0\u03bcM quinidine (6 hearts)\u221238.3\u2009\u00b1\u200917.492.4\u2009\u00b1\u200914.745.1\u2009\u00b1\u20099.42.5WT\u2009+\u200910\u00a0\u03bcM quinidine (6 hearts)\u2212863.0\u2009\u00b1\u20091,387.1981.6\u2009\u00b1\u20091,369.127.1\u2009\u00b1\u200915.9113.3Scn5a+\/\u0394 (5 hearts)\u221219.6\u2009\u00b1\u20097.290.0\u2009\u00b1\u20094.453.1\u2009\u00b1\u200910.714.0Scn5a+\/\u0394 + flecainide (5 hearts)\u221216.1\u2009\u00b1\u20096.582.2\u2009\u00b1\u200912.395.3\u2009\u00b1\u200942.13.5Scn5a+\/\u0394\u2009+\u20091\u00a0\u03bcM quinidine (5 hearts)\u2212677.1\u2009\u00b1\u2009306.1748.0\u2009\u00b1\u2009304.715.2\u2009\u00b1\u20092.210.0Scn5a+\/\u2212 (6 hearts)\u2212674.8\u2009\u00b1\u2009329.9729.4\u2009\u00b1\u2009329.114.2\u2009\u00b1\u20092.18.4Scn5a+\/\u2212 + flecainide (6 hearts)\u221256,359.8\u2009\u00b1\u20092,559.752,677.3\u2009\u00b1\u20092.27.8\u2009\u00b1\u20092.514.8Scn5a+\/\u2212\u2009+\u20091\u00a0\u03bcM quinidine (5 hearts)\u2212118.3\u2009\u00b1\u200957.4202.5\u2009\u00b1\u200949.436.4\u2009\u00b1\u200910.516.7Scn5a+\/\u2212\u2009+\u200910\u00a0\u03bcM quinidine (7 hearts)\u22121.7\u2009\u00b1\u200944.7131.9\u2009\u00b1\u200962.3158.4\u2009\u00b1\u2009262.210.8Results of least-squares fits to the function  under the conditions indicatedTable\u00a02Fit parameters for endocardial restitution curvesConditiony0 (ms)A (ms)\u03c4 (ms)\u03c72WT (6 hearts)\u221211.8\u2009\u00b1\u20093.773.8\u2009\u00b1\u20091.765.2\u2009\u00b1\u20098.41.2WT + flecainide (5 hearts)\u2212769.0\u2009\u00b1\u2009244.1827.3\u2009\u00b1\u2009243.714.5\u2009\u00b1\u20091.31.4WT\u2009+\u20091\u00a0\u03bcM quinidine (6 hearts)\u221265.8\u2009\u00b1\u2009100.4113.5\u2009\u00b1\u200998.324.9\u2009\u00b1\u200912.011.7WT\u2009+\u200910\u00a0\u03bcM quinidine (6 hearts)\u221241.9\u2009\u00b1\u200971.578.2\u2009\u00b1\u200968.732.8\u2009\u00b1\u200919.313.6Scn5a+\/\u0394 (5 hearts)\u221231.9\u2009\u00b1\u20099.0109.9\u2009\u00b1\u20095.149.6\u2009\u00b1\u20099.010.3Scn5a+\/\u0394 + flecainide (5 hearts)\u2212561.1\u2009\u00b1\u20091.2631.6\u2009\u00b1\u200915.118.7\u2009\u00b1\u20090.36.7Scn5a+\/\u0394\u2009+\u20091\u00a0\u03bcM quinidine (5 hearts)\u2212499.7\u2009\u00b1\u2009143.8563.0\u2009\u00b1\u2009142.916.3\u2009\u00b1\u20091.656.0Scn5a+\/\u2212 (6 hearts)\u2212281.7\u2009\u00b1\u20099.843.5\u2009\u00b1\u20096.448.5\u2009\u00b1\u200919.04.3Scn5a+\/\u2212 + flecainide (6 hearts)\u22121,050.6\u2009\u00b1\u20091,558.51,100.1\u2009\u00b1\u20091,557.811.7\u2009\u00b1\u20094.010.0Scn5a+\/\u2212\u2009+\u20091\u00a0\u03bcM quinidine (5 hearts)\u2212261.2\u2009\u00b1\u2009117.9350.5\u2009\u00b1\u2009107.834.2\u2009\u00b1\u20099.321.7Scn5a+\/\u2212\u2009+\u200910\u00a0\u03bcM quinidine (7 hearts)\u2212220.1\u2009\u00b1\u2009322.0319.1\u2009\u00b1\u2009316.826.2\u2009\u00b1\u200912.745.5Results of least-squares fits to the function  under the conditions indicatedTable\u00a03Fit parameters for endocardial restitution curves separating successive odd- and even-numbered action potentialsConditionOddEveny0 (ms)A (ms)\u03c4 (ms)\u03c72y0 (ms)A (ms)\u03c4 (ms)\u03c72WT (6 hearts)13.4\u2009\u00b1\u20097.640.5\u2009\u00b1\u200912.950.3\u2009\u00b1\u200921.46.38.1\u2009\u00b1\u200911.258.3\u2009\u00b1\u200925.448.2\u2009\u00b1\u200932.34.6WT + flecainide (5 hearts)\u221214.7\u2009\u00b1\u20098.9105.2\u2009\u00b1\u20099.088.0\u2009\u00b1\u200931.35.8\u221244.6\u2009\u00b1\u200918.5117.4\u2009\u00b1\u200914.249.0\u2009\u00b1\u20099.91.5WT\u2009+\u20091\u00a0\u03bcM quinidine (6 hearts)\u221220.6\u2009\u00b1\u20099.975.8\u2009\u00b1\u20096.256.4\u2009\u00b1\u200913.23.8\u221234.2\u2009\u00b1\u200911.789.2\u2009\u00b1\u20099.345.9\u2009\u00b1\u20097.83.3WT\u2009+\u200910\u00a0\u03bcM quinidine (6 hearts)\u22121,391.7\u2009\u00b1\u20093,195.91,496.0\u2009\u00b1\u20093,182.021.1\u2009\u00b1\u200914.3117.9\u2212393.1\u2009\u00b1\u20095.3.0532.8\u2009\u00b1\u2009481.741.1\u2009\u00b1\u200927.6115.7Scn5a+\/\u0394 (5 hearts)\u221223.9\u2009\u00b1\u20098.392.0\u2009\u00b1\u20095.347.8\u2009\u00b1\u20099.012.4\u221224.4\u2009\u00b1\u20098.692.6\u2009\u00b1\u20095.647.6\u2009\u00b1\u20098.911.8Scn5a+\/\u0394 + flecainide (5 hearts)14.6\u2009\u00b1\u20096.377.1\u2009\u00b1\u20098.484.1\u2009\u00b1\u200932.23.4\u221233.2\u2009\u00b1\u200946.4110.1\u2009\u00b1\u200941.936.4\u2009\u00b1\u200913.12.0Scn5a+\/\u0394\u2009+\u20091\u00a0\u03bcM quinidine (5 hearts)\u2212250.2\u2009\u00b1\u200912.8483.0\u2009\u00b1\u200924.518.8\u2009\u00b1\u20097.812.4\u2212254.7\u2009\u00b1\u2009321.2328.1\u2009\u00b1\u2009318.320.0\u2009\u00b1\u20097.75.3Scn5a+\/\u2212 (6 hearts)\u2212850.4\u2009\u00b1\u2009282.7904.2\u2009\u00b1\u2009282.113.5\u2009\u00b1\u20091.34.3\u2212913.3\u2009\u00b1\u2009283.3968.6\u2009\u00b1\u2009282.713.7\u2009\u00b1\u20091.23.7Scn5a+\/\u2212 + flecainide (6 hearts)\u22121,473.8\u2009\u00b1\u20093,144.51,534.3\u2009\u00b1\u20092,141.214.7\u2009\u00b1\u20097.634.3\u22122,108.8\u2009\u00b1\u20094,489.92,171.3\u2009\u00b1\u20094,486.814.0\u2009\u00b1\u20096.735.0Scn5a+\/\u2212\u2009+\u20091\u00a0\u03bcM quinidine (5 hearts)\u221246.8\u2009\u00b1\u200951.2146.8\u2009\u00b1\u200921.359.5\u2009\u00b1\u200943.59.4\u221246.7\u2009\u00b1\u200950.6149.2\u2009\u00b1\u200920.261.0\u2009\u00b1\u200944.29.3Scn5a+\/\u2212\u2009+\u200910\u00a0\u03bcM quinidine (7 hearts)7.3\u2009\u00b1\u200934.2167.7\u2009\u00b1\u2009264.8272.8\u2009\u00b1\u200978.310.82.8\u2009\u00b1\u200944.6143.1\u2009\u00b1\u2009121.3192.2\u2009\u00b1\u2009405.812.0Results of least-squares fits to the function  under the conditions indicatedTable\u00a04Fit parameters for endocardial restitution curves separating successive odd- and even-numbered action potentialsConditionOddEveny0 (ms)A (ms)\u03c4 (ms)\u03c72y0 (ms)A (ms)\u03c4 (ms)\u03c72WT (6 hearts)\u221217.4\u2009\u00b1\u20095.373.8\u2009\u00b1\u20092.867.3\u2009\u00b1\u200913.54.7\u221238.9\u2009\u00b1\u200915.491.6\u2009\u00b1\u200913.046.5\u2009\u00b1\u20098.51.8WT + flecainide (5 hearts)\u221249.2\u2009\u00b1\u200926.2113.8\u2009\u00b1\u200923.035.7\u2009\u00b1\u20098.27.6\u2212367.3\u2009\u00b1\u2009171.3426.9\u2009\u00b1\u2009170.617.7\u2009\u00b1\u20092.41.3WT\u2009+\u20091\u00a0\u03bcM quinidine (6 hearts)\u2212393.2\u2009\u00b1\u2009368.6437.7\u2009\u00b1\u2009368.614.9\u2009\u00b1\u20094.318.1\u22121,337.3\u2009\u00b1\u20091,206.41,384.5\u2009\u00b1\u20091,205.711.5\u2009\u00b1\u20092.514.7WT\u2009+\u200910\u00a0\u03bcM quinidine (6 hearts)\u221217.7\u2009+\u2009\u221239.053.2\u2009\u00b1\u200935.042.6\u2009\u00b1\u200928.911.2\u221228.0\u2009\u00b1\u200962.766.6\u2009\u00b1\u200958.239.6\u2009\u00b1\u200929.717.3Scn5a+\/\u0394 (5 hearts)\u2212149.6\u2009\u00b1\u2009131.4221.0\u2009\u00b1\u2009128.626.4\u2009\u00b1\u20097.60.9\u221242.3\u2009\u00b1\u200910.5115.3\u2009\u00b1\u20098.240.8\u2009\u00b1\u20095.02.6Scn5a+\/\u0394 + flecainide (5 hearts)\u22121,133.8\u2009\u00b1\u2009784.21,201.4\u2009\u00b1\u2009783.115.2\u2009\u00b1\u20092.62.0\u22122,164.0\u2009\u00b1\u20091,195.72,233.3\u2009\u00b1\u20091,195.013.6\u2009\u00b1\u20091.61.4Scn5a+\/\u0394\u2009+\u20091\u00a0\u03bcM quinidine (5 hearts)\u22122,720.4\u2009\u00b1\u20093,917.82,779.7\u2009\u00b1\u20093,917.210.4\u2009\u00b1\u20092.83.4\u22123,527.2\u2009\u00b1\u20094,674.783,603.0\u2009\u00b1\u20094,674.111.1\u2009\u00b1\u20092.73.9Scn5a+\/\u2212 (6 hearts)\u2212134.6\u2009\u00b1\u200928.9195.3\u2009\u00b1\u200928.022.8\u2009\u00b1\u20092.13.3\u2212149.1\u2009\u00b1\u200933.5212.0\u2009\u00b1\u200932.622.9\u2009\u00b1\u20092.23.6Scn5a+\/\u2212 + flecainide (6 hearts)\u221213.8\u2009\u00b1\u200929.266.2\u2009\u00b1\u200927.132.5\u2009\u00b1\u200911.11.8\u221255.4\u2009\u00b1\u200955.5108.6\u2009\u00b1\u200954.623.6\u2009\u00b1\u20095.91.2Scn5a+\/\u2212\u2009+\u20091\u00a0\u03bcM quinidine (5 hearts)\u22121,979.9\u2009\u00b1\u20091,709.02,051.5\u2009\u00b1\u20091,704.615.9\u2009\u00b1\u20092.813.4\u2212108.4\u2009\u00b1\u200915,101.810,911.4\u2009\u00b1\u200915,098.311.8\u2009\u00b1\u20093.014.8Scn5a+\/\u2212\u2009+\u200910\u00a0\u03bcM quinidine (7 hearts)\u2212332.1\u2009\u00b1\u2009447.2431.4\u2009\u00b1\u2009439.825.2\u2009\u00b1\u200912.7116.0\u2212160.0\u2009\u00b1\u2009289.5262.2\u2009\u00b1\u2009281.920.1\u2009\u00b1\u200918.148.1Results of least-squares fits to the function  under the conditions indicated\nFigures\u00a04 and 5 thus superimpose optimisations of Eq.\u00a01 to the data shown in Tables\u00a01 and 2 (solid lines, left-hand ordinates). Furthermore, they show the corresponding gradients calculated using Eq.\u00a02 (broken lines, right-hand axes). Shaded regions where present indicate DIs less than the critical DI at which these gradients exceed unity: the existence of such a region is an established criterion for arrhythmogenesis [25]. Critical DI was calculated as:\nFigure\u00a06 proceeds to show these values of critical DI derived from the fitted values of A and \u03c4 for the epicardia (filled bars) and endocardia (open bars) under each of the above conditions. In contrast to the findings from the analysis of the relationships between epicardial and endocardial APD90s described above, the present analysis of epicardial and endocardial restitution properties together provided datasets in complete agreement with the observed incidences of arrhythmic activity.\nFig.\u00a06Critical diastolic intervals obtained from restitution curves. Critical diastolic intervals at which gradients equalled unity obtained from epicardial (filled bars) and endocardial (open bars) restitution curves shown in Figs.\u00a04 and 5 in WT (a), Scn5a+\/\u0394 (b) and Scn5a+\/\u2212 (c) hearts exposed to control solution (A) and to solutions containing 1\u00a0\u03bcM flecainide (B), 1\u00a0\u03bcM quinidine (C) and 10\u00a0\u03bcM quinidine (D). Asterisks indicate values that are significantly (P\u2009<\u20090.05) larger than those recorded in WT hearts exposed to control solution\nFirst, in WT hearts (Fig.\u00a06a) exposed to control solution (A) the critical DI took a value of \u221263.63\u2009\u00b1\u200914.41\u00a0ms in the epicardium and \u221215.43\u2009\u00b1\u20094.97\u00a0ms in the endocardium. Treatment with flecainide (B) significantly (P\u2009<\u20090.05) increased these values to 19.45\u2009\u00b1\u20099.39\u00a0ms in the epicardium and 58.75\u2009\u00b1\u200918.71\u00a0ms in the endocardium. This critical DI was attained in the endocardium (shaded region in Fig.\u00a05a, B), in parallel with the increased incidence of arrhythmogenesis observed during the extrasystolic stimulation procedure under these conditions (Fig.\u00a02, ii). Treatment with 1\u00a0\u03bcM quinidine (Fig.\u00a06a, C) also significantly (P\u2009<\u20090.05) increased the critical DI in both the epicardium and endocardium (32.30\u2009\u00b1\u20097.80\u00a0ms and 27.80\u2009\u00b1\u200914.53\u00a0ms respectively) in parallel with the observed arrhythmogenicity on extrasystolic stimulation under these conditions (Fig.\u00a02, ii), although such values were not attained during the dynamic pacing protocol. Furthermore, treatment with 10\u00a0\u03bcM quinidine (Fig.\u00a06a, D) again significantly (P\u2009<\u20090.05) increased these values to 97.20\u2009\u00b1\u200957.90\u00a0ms in the epicardium and to 28.50\u2009\u00b1\u200934.52\u00a0ms in the endocardium. This DI was attained in the epicardium in parallel with the observed arrhythmogenicity during the extrasystolic stimulation procedure (Fig.\u00a02, ii). Thus, increases in the critical DI are precisely paralleled with arrhythmogenicity in WT hearts treated with flecainide and quinidine.\nSecondly, critical DIs obtained from Scn5a+\/\u0394 hearts (Fig.\u00a06b) were always significantly greater than those in WT controls (P\u2009<\u20090.05 in all cases) in parallel with the increased incidence of arrhythmic activity observed during the extrasystolic stimulation procedure (Fig.\u00a02, ii). Thus, in hearts treated with control solution (Fig.\u00a06b, A) the critical DI increased to 28.04\u2009\u00b1\u20098.08\u00a0ms in the epicardium and 39.38\u2009\u00b1\u200910.28\u00a0ms in the endocardium: These values were attained in both the epicardium and endocardium in association with the increased incidence of arrhythmic activity on extrasystolic stimulation observed (Fig.\u00a02, ii) and in full agreement with clinical expectations [23]. Treatment with flecainide (Fig.\u00a06b, B) decreased the epicardial value (\u221214.09\u2009\u00b1\u20099.05\u00a0ms, P\u2009<\u20090.05 as compared to hearts treated with control solution) but did not significantly affect the endocardial value (21.41\u2009\u00b1\u20098.58\u00a0ms, P\u2009>\u20090.05 as compared to hearts treated with control solution), having no effect on the observed incidence of arrhythmic activity on extrasystolic stimulation (Fig.\u00a02, ii). However, treatment with 1\u00a0\u03bcM quinidine (Fig.\u00a06b, C) significantly (P\u2009<\u20090.05) increased both epicardial and endocardial values (59.09\u2009\u00b1\u200926.91\u00a0ms and 57.64\u2009\u00b1\u200916.68\u00a0ms, respectively) as compared to those obtained from such hearts treated with control solution, in association with the observed increases in the incidence of arrhythmic activity resulting from extrasystolic stimulation (Fig.\u00a02, ii) and again in agreement with expectations from clinical work [33]. It was not possible to obtain MAPs over a sufficiently wide range of BCLs to permit the construction of such curves in Scn5a+\/\u0394 hearts treated with 10\u00a0\u03bcM quinidine.\nThirdly, critical DI values were also increased in Scn5a+\/\u2212 hearts (Fig.\u00a06c) in association with the increased incidence of arrhythmic activity observed. Thus, in hearts treated with control solution (A), the critical DIs were significantly (P\u2009<\u20090.05) increased as compared to WT controls in both the epicardium and endocardium to 56.00\u2009\u00b1\u200927.79\u00a0ms and 52.59\u2009\u00b1\u20099.50\u00a0ms, respectively. These values were attained in both the epicardium and endocardium, fulfilling clinical expectations [1]. Treatment with flecainide (B) did not significantly alter these values (68.14\u2009\u00b1\u200924.09\u00a0ms in the epicardium and 58.56\u2009\u00b1\u200914.75\u00a0ms in the endocardium). These were again attained in both epicardium and endocardium in association with the arrhythmic activity observed on extrasystolic stimulation (Fig.\u00a02, ii). Furthermore, treatment with quinidine at a concentration of 1\u00a0\u03bcM (Fig.\u00a06c, C) had no significant effect on critical DIs in either the epicardium or endocardium (62.45\u2009\u00b1\u200929.74\u00a0ms and 79.63\u2009\u00b1\u200939.31\u00a0ms, respectively). These values were again attained in both epicardium and endocardium. However, treatment with quinidine at 10\u00a0\u03bcM significantly decreased both epicardial and endocardial critical DIs, such that they became statistically indistinguishable from WT controls (29.01\u2009\u00b1\u20099.27\u00a0ms and \u221280\u2009\u00b1\u20093.58\u00a0ms, respectively). This was associated with the complete absence of arrhythmogenesis during the extrasystolic stimulation procedure (Fig.\u00a02, ii).\nThus, while relationships between epicardial and endocardial action potential durations obtained over a range of cycle lengths are poorly correlated with arrhythmogenicity, restitution curves are, in general, predictive of arrhythmogenicity under the genetic and pharmacological conditions studied.\nDiscussion\nThe congenital LQT3 and the BrS constitute two important examples of genetic causes for ventricular arrhythmia and sudden cardiac death [3]. These conditions result from similarly targeted mutations in the SCN5a gene coding for the cardiac Na+ channel: gain-of-function mutations are characteristic of LQT3, while loss-of-function mutations are characteristic of BrS [1, 23].\nThe present study began by using the MAP technique to verify that Langendorff-perfused murine hearts modelling LQT3 (Scn5a+\/\u0394) [13] and BrS (Scn5a+\/\u2212) [29] recapitulated features of the incidence of arrhythmic activity reported clinically. These results were in full agreement with previous physiological and pharmacological findings from our LQT3 [39] and BrS [38] models. Epicardial and endocardial MAP recordings were then used to evaluate the applicability of two established criteria in predicting arrhythmogenicity in these models over the full physiological range of heart rates (quantified as baseline cycle lengths, BCLs).\nTransmural dispersion of repolarization was studied over a range of BCLs by comparing epicardial and endocardial action potential durations (at 90% repolarization, APD90s) in the two genetic models. Such an analysis had previously been demonstrated to reveal arrhythmogenicity in hypokalaemic Langendorff-perfused murine hearts [34]. Furthermore, changes in this relationship investigated at a single BCL had previously been associated with arrhythmogenicity in our Scn5a+\/\u0394 hearts [42]. Plots of epicardial against endocardial APD90 in WT hearts treated with control solution gave reference relationships best fitted by a quadratic function, in full agreement with previous work [34]. Treatment of such WT hearts with either quinidine or flecainide resulted in the data points falling to the left of the reference curves, reflecting increased transmural repolarization gradients, in association with increased incidences of arrhythmic activity and again in full agreement with a previous study [34]. However, such plots failed to consistently predict the presence or absence of arrhythmogenicity in the Scn5a+\/\u0394 and Scn5a+\/\u2212 hearts. Such left-shifting of points was observed in Scn5a+\/\u0394 hearts under all pharmacological conditions studied, despite the established anti-arrhythmic effect of flecainide in this setting. Furthermore, data points obtained from Scn5a+\/\u2212 hearts treated with control solution fell on the reference curve despite the increased incidence of arrhythmic activity observed. Treatment with flecainide failed to result in a left-shift of points, despite its established pro-arrhythmic effect; treatment with quinidine did result in such a shift, despite its anti-arrhythmic effect.\nThese discrepancies prompted an investigation for alternative criteria for arrhythmogenicity. We therefore explored the applicability of a second approach involving restitution curves plotting APD90 against the preceding DI. Arrhythmic activity occurring in a range of settings has previously been associated with increases in the slopes of such curves to greater than unity [14, 17\u201319, 28, 40]. Such an association has been demonstrated in a canine pharmacological model of the congenital long QT syndrome type 2 [45] but has not previously been studied in any models of LQT3 or BrS in any species. Furthermore, decreases in the slopes of such curves have been associated with the effects of clinically important anti-arrhythmic drugs, including the class 1A agent procainamide in a canine model [32]. However, such slopes have not been examined in relation to either quinidine or flecainide, nor have any such studies previously been attempted in murine models. This study accordingly proceeded to construct epicardial and endocardial restitution curves for the first time in a murine system and in relation to LQT3 or BrS in any species.\nIn contrast to the previous criterion, epicardial and endocardial restitution curves taken together yielded parameters in precise agreement with the presence or absence of arrhythmic phenomena demonstrated. In WT hearts, treatment with either quinidine or flecainide significantly increased the critical DI at which the slopes of the restitution curves exceeded unity, in full agreement with the increased incidences of arrhythmic activity observed. Not only did the Scn5a+\/\u0394 and Scn5a+\/\u2212 mutations result in significant increases in critical DI in association with increased rates of arrhythmic activity, but quinidine and flecainide exerted contrasting effects on this critical DI in agreement with clinical expectations in both cases. Thus, in Scn5a+\/\u0394 hearts, treatment with quinidine increased critical DIs in association with its pro-arrhythmic effect. Furthermore, in Scn5a+\/\u2212 hearts, treatment with quinidine decreased critical DIs in association with the decreased incidence of arrhythmic activity observed, while treatment with flecainide had the opposite effect. Thus class 1A agents may result in either increases (quinidine in WT and Scn5a+\/\u0394 murine hearts) or decreases (quinidine in Scn5a+\/\u2212 murine hearts and procainamide in canine hearts) [32] in the critical DI: Such changes are in full agreement with effects on arrhythmogenicity in all cases.\nTaken together, these results demonstrate that criteria considering effects on the slopes of restitution curves successfully account for arrhythmogenicity in murine models of arrhythmic syndromes resulting from mutations in the Scn5a Na+ channel, as well as for the effects of quinidine and flecainide on such arrhythmogenicity. These observations might form the basis for future clinical explorations of the utility of restitution curves obtained using the MAP technique [28, 40] in the assessment of arrhythmic propensity in hereditary arrhythmic syndromes. Such a robust means of assessing arrhythmogenicity might be particularly useful given the known variable expressivity of the arrhythmic phenotype in patients harbouring such mutations [41].","keyphrases":["heart","action potential","arrhythmia","heart rate","heart excitation","venticular muscle","ventricle"],"prmu":["P","P","P","P","R","U","U"]}
{"id":"Exp_Brain_Res-3-1-1914263","title":"Perception of limb orientation in the vertical plane depends on center of mass rather than inertial eigenvectors\n","text":"We performed two experiments to test the hypothesis that the perception of limb orientation depends on inertial eigenvectors (ei) against the alternative hypothesis that it depends on the center of mass vector (CM). Whereas ei constrains the dynamic torques involved in angular rotation, CM constrains the static torque necessary to keep the limb aloft in the gravitational field. Hence, possible effects of ei and CM on kinesthetic judgments must be related to the dynamic and static torques, respectively, involved in moving and positioning a limb. In the first experiment, blindfolded participants matched, with upper arms supported, the orientation of their forearms while the forearms\u2019 ei and CM were manipulated relative to the elbow. The manipulation of the vector CM alone induced a matching bias, as did the combined manipulation of ei and CM, whereas the manipulation of ei alone did not. In the second experiment, participants positioned their unseen and unsupported right arm at an indicated spatial configuration while ei and CM of the right forearm were manipulated as in Experiment 1. As in the first experiment, forearm positioning was affected by the independent manipulation of CM and the combined manipulation of ei and CM, but not by the independent variation of ei. Moreover, none of the manipulations affected upper arm positioning. These results refute the claim that the perception of limb orientation (in the vertical plane) is based on ei and demonstrate, for the first time, the implication of a limb segment\u2019s CM in the perception of its orientation.\nIntroduction\nHow do we perceive where our limbs are in space without having to look at them continuously? Although this question is fundamental to the understanding of both perception and motor control, it is far from resolved. Investigations of the neurophysiological basis of kinesthesis have produced many relevant findings, but they have also highlighted the need for psychophysical concepts that pertain to the role of mechanical, in particular kinetic, information in kinesthetic experiences, as is illustrated by a brief overview of the pertinent literature.\nGiven that the relative position of limb segments is specific to a particular set of joint angles, the neurophysiological basis of kinesthesis has been sought primarily in neural signals providing geometric information related to joint angles. In principle, such signals may come from mechanoreceptors in joint, skin, and muscle. The role of joint receptors proved to be modest at best as they were found to be mostly silent in the mid-range of motion and to fire only at the extremes of a joint\u2019s movement range (e.g., Burgess and Clark 1969; Clark and Burgess 1975; Grigg and Greenspan 1977). Similarly, no prominent role of cutaneous receptors in signaling joint angles was established (Gandevia 1996), leaving muscle receptors as the primary candidates for signaling limb geometry. Although skeletal muscles contain both Golgi tendon organs and muscle spindles, only the latter are currently thought to reliably signal muscle length and, hence, inform about limb geometry (Kandel et al. 2000). Goodwin and colleagues firmly established this view by showing that tendon vibrations targeted at primary and secondary spindle endings induced marked illusory joint displacements (e.g., Goodwin et al. 1972a, b). More recently, Ribot-Ciscar et al. (2003) showed that the exclusive reliance on primary muscle spindle afferents may in theory lead to the accurate perception of joint angle.\nIt is thus beyond doubt that geometrical information pertaining to joint angles, primarily signaled by muscle spindles, plays an important role in kinesthesis. Yet it has become apparent that our sense of limb position and movement is not based exclusively on geometric information. Signals of a kinetic nature (i.e., relating to force or effort), which presumably cannot be conveyed by muscle spindles, probably provide positional information as well. Rymer and D\u2019Almeida (1980) demonstrated such kinetic influences by showing that errors in perceived finger orientation occur upon the generation of isometric contractions. They attributed this effect to a central mechanism receiving muscle force information from Golgi tendon organs. Results of Worringham and Stelmach (1985) suggested that limb kinesthesis depends on gravitational torque (Ng), which would also imply that it has a kinetic component. The significance of information related to muscular force or effort was amplified further by recent findings indicating a kinesthetic effect of muscular effort (Proske et al. 2004; Walsh et al. 2004; Winter et al. 2005), motor outflow signals (Gandevia et al. 2006), and muscle activity (Prud\u2019homme and Kalaska 1994).\nGiven the accumulating evidence that signals related to muscular force or effort are involved in kinesthesis, one may wonder how information relevant to kinesthetis is extracted from such kinetic signals. This issue is far from trivial because, unlike muscle spindle activity, tendon organ activity or motor outflow corrolaries bear no direct relation to muscle length or joint angle. This fact may have contributed to the now widespread view, put forward by McCloskey (1981), that motor outflow corollaries are only used to filter out spindle discharges related to changes in muscle activity so as to obtain accurate muscle length information (see Gandevia 1996). Yet it is possible that both afferent and efferent kinetic signals contribute to kinesthesis independent of muscle spindle activity. Information about muscular force directly reflects the dynamic and static torques involved in actively moving or positioning a limb. Through Newton\u2019s laws of motion, those torques are linked to the limb\u2019s mass distribution in space. Therefore, a promising psychophysical approach may be to postulate that an independent kinetic foundation of kinesthesis resides directly in specific characteristics of the limb\u2019s mass distribution.\nTaking such an approach, Pagano and colleagues (e.g., Pagano and Turvey 1995; Pagano et al. 1996; Garrett et al. 1998) hypothesized that the perception of a limb\u2019s spatial orientation depends on its inertial eigenvectors (ei), which represent a characteristic of a limb\u2019s mass distribution related to the direction in which it resists rotation (see the appendix for a more detailed explanation of the physical meaning of inertial eigenvectors). Relative to a given point in space, any rigid object has three orthogonal eigenvectors: e1, e2, and e3. For a forearm rotating around the elbow, e1 and e2 describe a plane through the elbow, orthogonal to the forearm\u2019s longitudinal axis. The axis of minimal resistance against rotation, e3, roughly coincides with the forearm\u2019s longitudinal axis. The possible kinesthetic role of ei in general and e3 in particular resides in the fact that they reflect the forearm\u2019s spatial orientation. In studying limb kinesthesis, it is essential to recognize that information about ei is only available when a perceiver actively rotates his or her forearm. After all, ei is related to the resistance against rotation and thus exclusively affects the dynamic torques involved in rotation. When a forearm is held stationary, the required muscular torque only has a static component and is therefore independent of ei.\nThe so-called inertial eigenvector hypothesis was introduced by Pagano and Turvey (1995) and corroborated in numerous subsequent studies (Pagano et al. 1996; Garrett et al. 1998; Pagano and Turvey 1998; Turvey 1998; Pagano 2000; Riley and Turvey 2001; Riley and Pagano 2003; Bernardin et al. 2005; Riley et al. 2005). All pertinent experiments adopted a similar method in which a single load was attached to the limb at a distance from its longitudinal axis, thereby breaking its coincidence with e3. With the exception of a recent study by Craig and Bourdin (2002, but see Riley and Pagano 2003; Riley et al. 2005) it was found that the perception of limb orientation was biased towards e3, which was taken as evidence for the hypothesis in question. The studies by Pagano et al. (1996) and Garrett et al. (1998) are of particular interest for the present study because they specifically addressed the perception of limb orientation in a vertical (i.e., gravitational) plane and found that it was affected by the aforementioned manipulation of e3.\nHowever, close scrutiny of this manipulation reveals that an alternative explanation is possible. The placement of a single load off the forearm\u2019s longitudinal axis not only introduces a rotation of the vector e3 relative to the elbow, but also displaces the forearm\u2019s center of mass. Similar to e3, we view the center of mass as a vector originating at the elbow (CM), and its displacement as a rotation of this vector. Importantly, CM and e3 constitute principally different characteristics of the forearm\u2019s mass distribution. Whereas ei exclusively affects the dynamic torques involved in limb rotation, the vector CM exclusively affects the static gravitational torque (Ng). A dependence of kinesthesis on CM would thus imply a fundamentally different kinetic basis than a dependence on ei. Yet the possibility that CM, rather than e3, governed the results of Pagano et al. (1996) and Garrett et al. (1998) has never been tested experimentally. In order to disentangle the effects of both variables, and thus to test the inertial eigenvector hypothesis against the center of mass hypothesis, ei and CM must be varied independently. We accomplished such a manipulation and experimental test in the experiments reported below.\nExperiment 1\nIn the first experiment, we tested the inertial eigenvector hypothesis against the center of mass hypothesis using a forearm-matching task similar to that employed by Pagano et al. (1996) and Garrett et al. (1998). The inertial eigenvector hypothesis predicts that variation in e3 is sufficient to induce a matching bias. Hence, according to this hypothesis a matching bias should occur in all conditions in which e3 is manipulated. According to the center of mass hypothesis, a pointing bias should occur upon rotation of the vector CM, irrespective of the orientation of e3. In the present experiment, the mass distribution of the forearms was manipulated such that e3 and CM either varied independently or covaried (as was the case in the experiments of Pagano and colleagues), thus allowing for a critical test of the two hypotheses of interest.\nMethod\nTwenty healthy participants (11 female and 9 male; all right-handed; mean age 28.5\u00a0years, SD 6.0\u00a0years) participated voluntarily in the experiment. They were not familiar with the type of experiment or the rationale behind it. The experiment, which was conducted in accordance with the 1964 Declaration of Helsinki, was approved formally by the ethical committee of our faculty and carried out with the adequate understanding and written informed consent of all participants.\nParticipants were blindfolded and sat on a stool with their upper arms resting on a wooden surface. They were to match the orientation of their unseen forearms, to which carbon fiber frames with two brass loads were fixed in order to achieve the desired orientations of e3 and CM (see Fig.\u00a01).\nFig.\u00a01The experimental setup of Experiment 1. Participants were blindfolded and sat on a stool with their upper arms resting on a wooden surface oriented at a 60\u00b0 angle with the horizontal. Participants\u2019 armpits touched the upper edge of the wooden surface and their upper arms were positioned in parallel and flush with the wooden surface. The carbon fiber frames, used to attach loads to the forearms (see text for a detailed description), were to remain in parallel vertical planes while forearm orientation was matched by flexion and extension of the elbows. Two straws with a length of 5\u00a0cm extended from the upper end of the proximal crosspieces, preventing the loads from touching the upper arms and marking the maximal allowed elbow flexion. The straws are visible inside the dotted circle\nEach carbon fiber frame consisted of a stem, 30\u00a0cm in length and 1\u00a0cm in diameter, and two parallel crosspieces, both 40\u00a0cm in length and 0.6\u00a0cm in diameter, that were pierced through the stem at a 90\u00b0 angle. These crosspieces were placed 22\u00a0cm apart with the most distal crosspiece at 4\u00a0cm from the distal end of the stem. The total mass of each frame was 32\u00a0g. The frames were fixed to the ventral side of the forearm, along its longitudinal axis, and their distal tips protruded from the closed hand between the ring finger and the middle finger. The crosspieces were positioned at a distance of 11 and 33\u00a0cm, respectively, from the medial epicondyl of the humerus. A thin straw with a length of 5\u00a0cm protruded from the upper part of the proximal crosspiece (see Fig.\u00a01), preventing the upper arm from contacting the loads. Moreover, by touching the upper arm, it signaled the smallest elbow angle allowed in the experiment (approximately 80\u00b0).\nForearm matching occurred in eight experimental conditions and a control condition. In the experimental conditions, two cylindrical brass loads were attached to the frames to achieve the desired orientations of e3\u00a0and CM. The experimental manipulations are illustrated in Fig.\u00a02 and the exact masses and positions of the loads in the eight experimental conditions are reported in Table\u00a01. The magnitude and direction of e3 rotation induced by the loads was calculated by adding the loads\u2019 inertia tensors relative to the elbow to that of the unloaded forearm (using the parallel axis theorem) and subsequently calculating the new orientation of e1, e2, and e3 by diagonalizing the resulting tensor. In conditions 1 and 2, e3 was thus manipulated 5\u00b0 toward flexion in one arm (the right arm in condition 1; the left arm in condition 2) and 5\u00b0 toward extension in the other, without manipulating CM, resulting in a 10\u00b0 difference between the eigenvectors of the forearms. In conditions 3 and 4, CM was manipulated 5\u00b0 toward flexion in one arm (the right arm in condition 3; the left arm in condition 4) and 5\u00b0 toward extension in the other, without manipulating e3, resulting in a 10\u00b0 CM difference between the forearms. In conditions 5 and 6, both e3 and CM were manipulated such that there was a 10\u00b0 e3 and CM difference between both forearms. In conditions 7 and 8, one arm remained unloaded (the left arm in condition 7; the right arm in condition 8) and loads were attached to the other arm in a symmetrical way, i.e., without inducing a rotation of either e3 or CM. A ninth condition, in which both forearms remained unloaded, served as control condition. The maximal value of Ng about the elbow was equal in both arms in all conditions, except for conditions 7 and 8, which were included to explicitly test for the effect of Ng suggested by Worringham and Stelmach (1985). In conditions 1 through 6, the placement of the loads merely induced an asymmetrical mass distribution relative to the forearm\u2019s longitudinal axis.\nFig.\u00a02The experimental manipulations of the vectors e3 and CM. CM is the vector from the elbow joint to the average position of the (loaded) forearm\u2019s mass (i.e., the effective point of origin of gravitational force). The eigenvector e3 is the axis through the elbow joint about which the (loaded) forearm\u2019s rotational inertia is minimal. Panel a shows the loads as they were attached to one of the forearms in conditions 7 and 8 (in which the other forearm remained unloaded). The loads are equal in mass, are placed at equal but opposite distances from the forearm\u2019s longitudinal axis, and have equal distances to elbow. They thus do not induce a rotation of either CM or e3 relative to an unloaded arm. In panel b (conditions 5 and 6), the asymmetrical load placement causes both vectors e3 and CM to be rotated towards the loads. In panel c (conditions 1 and 2), the loads have equal mass and are placed at equal but opposite distances from the forearm\u2019s longitudinal axis, so that no rotation of CM is induced. Yet the loads have different distances to the elbow, so that they do induce a rotation of e3. Panel c thus represents the independent variation of e3. Finally, in panel d (conditions 3 and 4), the two loads are placed equidistant from the forearm\u2019s longitudinal axis and have different distances to the elbow, as in panel c, but now their masses differ with the heaviest load being closest to the elbow. This leads to CM being rotated towards the greatest load, while e3 still coincides with the longitudinal axis. Panel d thus represents the independent variation of CMTable\u00a01Masses and positions of the loads and corresponding rotations of e3 and CM in the experimental conditions of experiment 1Right forearmLeft forearmLoad 1Load 2Load 1Load 2ConditionMass (g)x, ya (cm)Mass (g)x, y (cm)Mass (g)x, y (cm)Mass (g)x, y (cm)\u0394e3b \u0394CMc 115011, \u221218.815033, 18.815011, 18.815033, \u221218.810\u00b00\u00b0215011, 18.815033, \u221218.815011, \u221218.815033, 18.8\u221210\u00b00\u00b0330011, 13.810033, \u221213.830011, \u221213.810033, 13.80\u00b010\u00b0430011, \u221213.810033, 13.830011, 13.810033, \u221213.80\u00b0\u221210\u00b0515011, 5.815033, 12.815011, \u22125.815033, \u221212.810\u00b010\u00b0615011, \u22125.815033, \u221212.815011, 5.815033, 12.8\u221210\u00b0\u221210\u00b0710033, 18.810033, \u221218.8\u2013\u2013\u2013\u20130\u00b00\u00b08\u2013\u2013\u2013\u201310033, 18.810033, \u221218.80\u00b00\u00b0ax refers to the distance of the load\u2019s center of mass from the elbow along the arm\u2019s longitudinal axis; y refers to the orthogonal distance of the load\u2019s center of mass from the arm\u2019s longitudinal axis, where a positive (negative) distance indicates a position on the radial (ulnar) side of the forearmb\u0394e3 indicates the angle between the smallest eigenvector (e3) of the left and the right forearm. A positive angle indicates that e3 of the right forearm was manipulated towards elbow flexion and e3 of the left forearm towards elbow extensionc\u0394CM indicates the angle between the center of mass vector (CM) of the left and the right forearm. A positive angle indicates that CM of the right forearm was oriented towards elbow flexion and CM of the left forearm towards elbow extension\nParticipants performed the nine conditions in nine corresponding trial blocks. Before each trial block, they assumed a position in which one arm was flexed at an elbow angle of 80\u00b0, which was achieved by letting the tip of the straw just touch the upper arm, and the other arm was fully extended. Note that, in this starting position, a perceptual reference was ensured both in the flexed arm (by the straw) and in the fully extended arm (by the end of the elbow\u2019s movement range), preventing any drift effects over trials. From this position, one of the forearms (the target arm) was moved towards the other forearm until the experimenter called out \u201cstop\u201d. The experimenter ensured that this stop signal was given at a different arm orientation in each trial. The other arm (the matching arm) was then moved towards the target arm until the participant perceived the orientation of the two forearms to be identical. At this moment, the participant stopped the movement and called out \u201cja\u201d (\u201cyes\u201d). After registering the orientation of both forearms (see below), the experimenter instructed the participant to assume the starting position for the next trial: The arm that was flexed at the start of the previous trial was now extended and vice versa. The target arm was alternately the left and the right arm. Participants were instructed to keep both arms and the crosspieces they enclosed with their hands in a vertical (i.e., sagittal) plane at all times.\nEach trial block was started with either the left or the right arm extended, and with either the extended or the flexed arm as the target arm. The four resulting starting configurations were counterbalanced across participants, with each individual participant starting all trial blocks from the same assigned configuration. Each trial block consisted of two series of eight matching trials. In one series the extended arm was the target arm and in the other the flexed arm was the target arm. All participants performed 144 matching trials in total (9 trial blocks; 16 trials per block). The duration of an experimental session was approximately 45\u00a0min.\nForearm orientation was measured using a 3D active movement registration system (Optotrak 3020, Northern Digital Inc., Waterloo, Canada), which was calibrated using a coordinate frame with one axis aligned with the gravitational vertical, and one parallel to the horizontal axis described by participants\u2019 elbow and shoulder joints. During the experiment, the position of four infrared markers was registered: two markers on the distal tip of each frame stem, and two markers on the horizontal axes described by the two elbow joints and the two shoulder joints, respectively. The latter two markers were placed on an adjustable carbon fiber frame to the left of the participants after they assumed the correct starting position. In this starting position, both armpits contacted the upper edge of the wooden board, which ensured that the elbow joints as well as the shoulder joints described a horizontal axis. The experimenter registered the position of the four markers each time a participant indicated that the orientation of the forearms was matched. For each participant and for each trial, the angle of the two elbow joints, projected onto a sagittal plane, was calculated from the position data of the four Optotrak markers. The elbow angles of both arms were subsequently averaged to obtain the angle around which matching occurred in each trial. To obtain the direction and magnitude of matching errors, the elbow angle of the right arm was subtracted from that of the left arm. A positive matching error thus indicated that elbow angle was smallest in the right arm, that is, that the right arm had a flexion bias relative to the left arm. Finally, matching errors in the control condition were subtracted from those in each experimental condition to obtain matching biases due to the eight experimental manipulations. We first examined the range of elbow angles around which matching occurred in each condition, the pattern of matching errors within conditions, and the matching errors in the control condition. Subsequently, we analyzed the matching errors according to a repeated measures analysis of variance (ANOVA) with condition (9 levels) and repetition (16 levels) as within-subject factors. Finally, we tested the matching biases due to the eight experimental manipulations using one-sample two-tailed t tests.\nResults and discussion\nThe average and the range of the elbow angles around which matching occurred in each of the nine conditions was 115.5\u00b0 (SD over conditions 1.0\u00b0) and 26.9\u00b0 (SD over conditions 1.3\u00b0), respectively. The low standard deviations indicate that both the average and the range of matching angles only differed marginally between conditions.\nBefore turning to the effects of our experimental manipulations on the matching errors, we first examined matching errors within conditions. Participants showed a significant overshoot of the target arm\u2019s orientation with the matching arm (t(19)\u00a0=\u00a02.41, P\u00a0=\u00a00.027, \u03b72\u00a0=\u00a00.23), which is in keeping with Worringham and Stelmach (1985), but not with Pagano et al. (1996) who did not find such an effect. Matching errors of a representative participant in the control condition are shown in Fig.\u00a03. The alternating pattern of errors in this figure is a manifestation of the aforementioned overshoot effect. After all, the left and the right arm alternatingly assumed a flexed starting position, so that an overshoot of the target arm would alternatingly lead to a positive and a negative matching bias. In this way, matching biases due to overshoot canceled out after averaging over trials. Accordingly, in the control condition, matching errors did not deviate significantly from zero (P\u00a0=\u00a00.53) after averaging over trials (mean 0.5\u00b0; SD across participants 3.7\u00b0). However, partly due to a tendency to overshoot the target arm, error variability across trials was considerable (SD 5.7\u00b0; see Fig.\u00a03). This trial-to-trial variability is comparable to that reported by Soechting (1982).\nFig.\u00a03Matching errors of a representative participant in the control condition of Experiment 1, in which no loads were attached to the forearms. The alternating pattern of errors over trials reflects a tendency to overshoot the target arm with the matching arm\nThe ANOVA performed on the matching errors in all conditions revealed that they were affected significantly by condition (F(8, 152)\u00a0=\u00a010.5, P\u00a0<\u00a00.001, \u03b7p2\u00a0=\u00a00.36), but neither by repetition (P\u00a0=\u00a00.80) nor by the condition\u00a0\u00d7\u00a0repetition interaction (P\u00a0=\u00a00.46). Figure\u00a04 shows the average matching biases (with 95% confidence intervals) due to the eight experimental manipulations. No significant effect of e3 was found (P\u00a0=\u00a00.72 and P\u00a0=\u00a00.69, in conditions 1 and 2, respectively). In contrast, the 10\u00b0 difference in CM orientation resulted in significant matching biases of \u22121.5\u00b0 in condition 3 (t(19)\u00a0=\u00a0\u22122.92, P\u00a0=\u00a00.009, \u03b72\u00a0=\u00a00.31) and of 2.2\u00b0 in condition 4 (t(19)\u00a0=\u00a02.70, P\u00a0=\u00a00.014, \u03b72\u00a0=\u00a00.28). The combined manipulations of e3 and CM had similar effects: A 10\u00b0 difference of e3 and CM orientation between the arms resulted in significant matching biases of \u22121.9\u00b0 in condition 5 (t(19)\u00a0=\u00a0\u22123.63, P\u00a0=\u00a00.002, \u03b72\u00a0=\u00a00.41) and of 2.5\u00b0 in condition 6 (t(19)\u00a0=\u00a04.13, P\u00a0=\u00a00.001, \u03b72\u00a0=\u00a00.47). A negative (positive) matching bias implied that the flexion of the right arm relative to the left arm was smaller (greater) than in the control condition. The direction of the significant effects of conditions 3 through 6 was consistent with the hypothesis that perceived orientation would be biased towards CM. Finally, loading one of the arms symmetrically (i.e., without rotating either e3 or CM) while leaving the other arm unloaded did not induce significant matching biases relative to the control condition (t(19)\u00a0=\u00a0\u22121.87, P\u00a0=\u00a00.077, \u03b72\u00a0=\u00a00.16 for condition 7; t(19)\u00a0=\u00a01.50, P\u00a0=\u00a00.15, \u03b72\u00a0=\u00a00.11 for condition 8), although there was a trend towards an extension bias in the loaded arm (see Fig.\u00a04). Such an extension bias would be consistent with the direction of the effect of Ng (or muscular effort) reported in the literature (Worringham and Stelmach 1985; Proske et al. 2004; Walsh et al. 2004; Winter et al. 2005). More importantly, the significant effect of CM in conditions 3 and 4, together with the absence of a significant effect of e3 in conditions 1 and 2, indicates that the results of Pagano et al. (1996) and Garrett et al. (1998) should be interpreted in retrospect as a sensitivity to CM rather than ei.\nFig.\u00a04Average matching biases relative to the control condition in the different manipulation conditions of Experiment 1. A positive bias implies that the flexion of the right arm relative to the left arm was greater in that manipulation condition than in the control condition. Error bars show the 95% confidence interval of the matching biases. Only e3 was rotated in conditions 1 and 2, only CM was rotated in conditions 3, and 4, and e3 and CM were rotated together in conditions 5 and 6. In conditions 1, 3, and 5, rotations were toward flexion in the right arm and toward extension in the left arm (and vice versa in conditions 2, 4, and 6). In conditions 7 and 8, e3 and CM were not rotated; only Ng differed between the two forearms: in condition 7 (8), it was greater in the right (left) arm\nGiven that people can perceive the orientation of their limbs in space, and not only relative to each other, one may wonder whether the present results, favoring the center of mass hypothesis, generalize to the perception of the orientation of an unsupported arm in extrinsic space. In this situation, loading the forearm not only affects the torques around the elbow joint, as was the case in the present experiment, but also the torques around the shoulder joint. In principle, this could restrict or alter the implication of CM in the perception of limb orientation. These possibilities were investigated in Experiment 2.\nExperiment 2\nIn the second experiment, we manipulated e3 and\/or CM of the forearm in the same manner as in Experiment 1, but this time to test their effects on the perception of the orientation of an unsupported arm in a vertical plane. Based on the results of Experiment 1, we hypothesized that perceived forearm orientation would be affected by CM and not by e3. We further hypothesized that the manipulations would not affect the perceived orientation of the upper arm, even though we recognized that loading the forearm does affect the torque at the shoulder when the upper arm is not supported. The latter hypothesis was motivated from the insight that the shoulder torque can only be informative about the orientation of the upper arm when the elbow torque is taken into account as well. We reasoned that manipulation of the forearm\u2019s CM would induce torques at the elbow indicating an altered forearm orientation. Given that the corresponding change in the torque pattern at the shoulder is consistent with this change in forearm orientation, we further reasoned that it too would indicate an altered forearm orientation, rather than an altered upper arm orientation.\nMethod\nTwenty healthy participants (13 female and 7 male; all right-handed; mean age 26.0\u00a0years, SD 4.3\u00a0years) participated voluntarily in the experiment. They were not familiar with the type of experiment or the rationale behind it. The experiment, which was conducted in accordance with the 1964 Declaration of Helsinki, was approved formally by the ethical committee of our faculty and carried out with the adequate understanding and written informed consent of all participants.\nThe experimental setup is shown in Fig.\u00a05. Participants were seated on a stool besides a vertical wooden board. Their right arm and shoulder were placed through a circular hole in the board between two overlapping pieces of lycra cloth that prevented them from looking through the hole to the other side of the board. Participants were wearing a T-shirt, of which the right sleeve was rolled up to just above the acromion to allow the placement of a marker on the shoulder (see below). The stool was adjusted such that the projection of the forearm on the vertical plane could coincide with line segments 1 through 3, and that of the upper arm with line segment 4. The experimental task consisted of matching the orientation of the unseen forearm with line segment 1, 2, or 3, while maintaining the upper arm parallel to line segment 4. Line segments 1, 2, and 3 were oriented at angles of 15\u00b0, 0\u00b0, and \u221215\u00b0, respectively, relative to horizontal. Line segment 4 was oriented at an angle of \u221260\u00b0 relative to horizontal, which corresponded to the orientation of the upper arm support in Experiment 1. Brass weights were attached to the forearm by means of a carbon fiber frame\u2014as in Experiment 1\u2014to achieve the desired orientations of the vectors e3 and CM (see Fig.\u00a02). The straw, used in Experiment 1 to prevent arm orientations smaller than about 80\u00b0, was not used in the present experiment because the risk that loads would contact the upper arm was much smaller than in Experiment 1.\nFig.\u00a05The experimental setup of Experiment 2. Participants sat on a stool besides a vertical wooden board. Their right arm and shoulder were placed through a circular hole in the board between two overlapping pieces of lycra cloth. Participants were wearing a T-shirt, of which the right sleeve was rolled up to uncover the shoulder. The stool was adjusted such that the projection of the right forearm on the vertical plane could coincide with line segments 1, 2 and 3 (15\u00b0, 0\u00b0 and \u221215\u00b0 relative to horizontal, respectively) and the projection of the upper arm with line segment 4 (\u221260\u00b0 relative to horizontal). The experimental task consisted of matching the orientation of the unseen forearm alternately with line segments 1, 2 and 3, while maintaining the unsupported upper arm parallel to line segment 4. The carbon fiber frame, used to attach loads to the forearm (see text for a detailed description), was to remain parallel to the board at all times\nArm positioning was required in seven experimental conditions and a control condition. Load placements in the experimental conditions were equal to those in experiment 1. The exact masses and positions of the loads can thus be found in the five leftmost columns of Table\u00a01 (i.e., those referring to the right arm). In conditions 1 and 2, e3 was independently manipulated by 5\u00b0 towards extension and 5\u00b0 towards flexion, respectively, resulting in a 10\u00b0 difference in e3 orientation between the two conditions. A 10\u00b0 difference in CM orientation was achieved between conditions 3 and 4, and a 10\u00b0 difference in the orientation of both e3 and CM was achieved between conditions 5 and 6. In condition 7, loads were attached to the forearm in a symmetrical way, i.e., without inducing a rotation of either e3 or CM. An eighth condition, in which the forearm remained unloaded, served as control condition.\nThe eight conditions were performed in eight corresponding trial blocks. Before each trial block, participants assumed a position in which the upper arm was oriented parallel to line segment 4, and the arm was either fully flexed or fully extended (counterbalanced across participants). The experimenter then called out one of the numbers 1 through 3 to indicate the target line segment for the forearm, upon which the participant rotated his or her forearm around the elbow towards the target line segment. Participants were instructed to stop moving the arm and push a button with the left hand when they perceived their forearm to be parallel to the target line segment and their upper arm to be parallel to line segment 4. If the arm was initially fully flexed (extended), participants subsequently fully extended (flexed) their arm. The experimenter then called out a new target line segment and the forearm was again rotated about the elbow (albeit in opposite direction) to match the orientation of the indicated line segment. Participants thus alternated between full elbow flexion and full elbow extension, and assumed the instructed arm configuration as a pause in each elbow flexion and extension movement. They were instructed to keep the crosspieces they enclosed with their right hand parallel to the wooden board at all times. In each trial block, participants matched the orientation of their forearm with each of the three target line segments four times, resulting in a total of 12 trials per block. Throughout each trial block, the upper arm was to remain parallel to line segment 4. All participants thus performed a total of 96 matching trials (8 trial blocks; 12 trials per block). The duration of an experimental session was approximately 30\u00a0min.\nThe configuration of the arm was measured using Optotrak. The position of six infrared markers was continuously registered at 100\u00a0Hz during each trial block: a marker on the tip of the carbon fiber frame (marker 1), a marker on either side on the distal crosspiece (markers 2 and 3), a marker on the lateral epicondyl of the humerus (marker 4), a marker just below the acromion on the deltoid muscle (marker 5), and finally a marker (marker 6) connected to the button in participants\u2019 left hand. The latter marker only emitted infrared light when the button was pushed. For each participant and for each trial, the orientation of the upper arm and forearm was calculated from the position data of markers 1, 4, and 5. The target orientations of forearm (15\u00b0, 0\u00b0, or \u221215\u00b0 relative to horizontal) and upper arm (\u221260\u00b0 relative to horizontal) were subtracted from the actual orientations that forearm and upper arm had at each moment the button was pushed. Errors in forearm and upper arm orientation were analyzed according to a repeated measures analysis of variance (ANOVA). For forearm orientation errors, we analyzed condition (8 levels), target (3 levels, corresponding to line segments 1, 2, and 3; see Fig.\u00a05), and repetition (4 levels) as within-subject factors. For errors in upper arm orientation, we analyzed only the factors condition and repetition. The difference in positioning errors between conditions 1 and 2 (e3 manipulation), 3 and 4 (CM manipulation), 5 and 6 (combined e3 and CM manipulation), and 7 and 8 (Ng manipulation without e3 and CM rotation), collapsed over targets, were subsequently compared using paired-samples two-tailed t tests. Note that in the present experiment four t tests rather than eight (as in Experiment 1) sufficed, because only one arm was manipulated rather than two.\nResults and discussion\nThe average error in aligning the forearm with line segments 1, 2, and 3 was 4.0\u00b0 in upward direction. The ANOVA performed on these errors revealed that they were affected significantly by condition (F(7, 133)\u00a0=\u00a08.4, P\u00a0<\u00a00.001, \u03b7p2\u00a0=\u00a00.31) and target (F(2, 38)\u00a0=\u00a07.0, P\u00a0=\u00a00.003, \u03b7p2\u00a0=\u00a00.27), but not by repetition (P\u00a0=\u00a00.15). The target effect indicated that a lower line segment was associated with smaller upward forearm positioning errors than a higher line segment. None of the two-way interactions was significant (all P\u2019s\u00a0>\u00a00.5), whereas the three-way interaction just reached significance (F(42, 798)\u00a0=\u00a01.4, P\u00a0=\u00a00.042, \u03b7p2\u00a0=\u00a00.07). As the latter effect had a marginal effect size and had no readily apparent origin, we abstained from seeking an account for it. The results of the paired t tests performed on forearm positioning errors are shown in Fig.\u00a06. No significant effect of adding mass in a symmetrical configuration was found (P\u00a0=\u00a00.18). The effect of e3 was also non-significant (P\u00a0=\u00a00.08). In contrast, CM rotation significantly affected forearm orientation (t(19)\u00a0=\u00a04.1, P\u00a0=\u00a00.001, \u03b72\u00a0=\u00a00.47), as did rotation of e3 and CM together (t(19)\u00a0=\u00a03.9, P\u00a0=\u00a00.001, \u03b72\u00a0=\u00a00.44). A 10\u00b0 difference of CM orientation alone was accompanied by a 2.4\u00b0 difference in forearm orientation. A 10\u00b0 difference of both e3 and CM orientation corresponded to a 2.6\u00b0 difference in forearm orientation. As expected, a more downward (upward) orientation of the forearm\u2019s CM always accompanied a more upward (downward) forearm orientation.\nFig.\u00a06Difference in forearm orientation between conditions 1 and 2 (e3 manipulation), 3 and 4 (CM manipulation), 5 and 6 (combined e3 and CM manipulation), and 7 and 8 (Ng manipulation) in Experiment 2. In conditions 1, 3 and 5 (2, 4 and 6), e3 and\/or CM were manipulated downward (upward). In condition 7, in which loads were attached to the frame symmetrically, Ng was larger than in condition 8, in which no loads were attached to the frame. For e3 and\/or CM manipulation (three leftmost bars), a positive difference indicates that downward manipulation was associated with a more upward forearm orientation. For Ng manipulation (rightmost bar), a negative difference indicates that a greater Ng was associated with a more downward forearm orientation. Error bars show the 95% confidence interval of the orientation differences\nThe average error in aligning the upper arm with line segment 4 was 7.4\u00b0 in downward direction. The ANOVA performed on these errors revealed that they were only significantly affected by repetition (F(11, 209)\u00a0=\u00a02.4, P\u00a0=\u00a00.008, \u03b7p2\u00a0=\u00a00.11). The main effect of condition and the condition\u00a0\u00d7\u00a0repetition interaction just failed to reach significance (P\u00a0=\u00a00.056 and P\u00a0=\u00a00.093, respectively). The paired t tests revealed that neither CM rotation nor e3 rotation significantly affected upper arm orientation errors (both P\u2019s\u00a0>\u00a00.7). The effect of symmetrical mass addition was also non-significant (P\u00a0>\u00a00.3). Finally, no effect of the combined e3 and CM manipulation was revealed, albeit that this effect approached significance (P\u00a0=\u00a00.071).\nIn sum, forearm orientation was affected in a very similar way as in the matching task of Experiment 1, whereas the unsupported upper arm was not affected, or only marginally at best, by the manipulation of e3 and\/or CM. The results thus corroborate both hypotheses forwarded in the introduction to the present experiment.\nGeneral discussion\nWith the overarching aim to uncover the kinetic foundation of kinesthesis, we tested the inertial eigenvector hypothesis against the alternative hypothesis that the perception of limb orientation in the vertical plane depends on CM. To this end, two experiments were conducted, one in which the orientations of the forearms had to be matched (Experiment 1) and one in which the orientation of a single arm had to be matched to an external reference configuration (Experiment 2). The results of both experiments supported the center of mass hypothesis and were inconsistent with the inertial eigenvector hypothesis. Experiment 1 revealed that the results of Pagano et al. (1996) and Garrett et al. (1998) should be interpreted in retrospect as a sensitivity to CM rather than ei. Experiment 2 generalized the results of Experiment 1 for forearm matching with the upper arm supported to orienting a single, unsupported limb with respect to extrinsic space. In particular, the results of Experiment 2 indicated that forearm manipulation in a vertical plane shifted perceived forearm orientation in that plane without causing a significant shift in the perceived orientation of the upper arm. The combined results of Experiments 1 and 2 raise the crucial question how CM might mediate limb kinesthesis. We address this question at two levels, first extensively in terms of the torques involved and then briefly and more tentatively in terms of possible neurophysiological mechanisms.\nIn both experiments, placing mass symmetrically (see Fig.\u00a02a) had no significant effect on the perception of forearm orientation, but did show a trend towards an extension bias in the heavier arm. It may be that adding mass symmetrically only had a small effect, which could explain the inconsistency of previous findings in this regard. In particular, whereas the results of Worringham and Stelmach (1985) indicate an effect of symmetrical mass placement, Soechting (1982) as well as Darling and Hondzinski (1999) failed to find such an effect. More important for the present discussion is the suggestion in the latter studies that Ng does not play a significant role in limb kinesthesis. This suggestion stands in stark contrast with the marked effects of CM found in the present experiments, which necessarily imply that Ng plays a role in limb kinesthesis, albeit in a different manner than considered previously. After all, as we already noted in the introduction, Ng is the only detectable variable affected by CM rotation. So how does CM affect Ng, and how is this different from the Ng manipulation in previous studies and our conditions with symmetrical mass addition? In an unloaded arm, Ng is a sinusoid function of arm inclination that reaches its maximum at a horizontal arm orientation and becomes zero when the arm is vertical (see solid gray curve in Fig.\u00a07). Adding loads symmetrically (as shown in Fig.\u00a02a) may be viewed as a scaling of this relationship. That is, it increases Ng at each inclination angle by a constant factor so that the maximum (minimum) of Ng still occurs at a horizontal (vertical) arm orientation (see the dotted black curve in Fig.\u00a07). Yet a rotation of CM by asymmetric mass addition (as shown in Fig.\u00a02b, d) not only scales but also horizontally shifts this relationship, so that the maximum of Ng no longer occurs at a horizontal arm orientation (see the solid black curve in Fig.\u00a07). Hence, this horizontal shift reflects the rotation of the vector CM. One may therefore conclude that Ng plays an indirect role in the perception of limb orientation, namely as a mediator of CM orientation, rather than Ng magnitude itself being informative about limb orientation.\nFig.\u00a07Schematic depiction of the relationship between Ng and arm inclination angle (i.e., the angle of the arm\u2019s longitudinal axis with the horizontal) in an unloaded arm (gray solid curve), a symmetrically loaded arm (black dotted curve) and an asymetrically loaded arm in which CM is rotated (black solid curve). Whereas symmetrical mass addition only changes the scaling of the relationship, CM rotation also induces a horizontal shift. We propose that the latter shift governs the sensitivity to CM demonstrated in the present study \nIn a vertical plane, the perception of limb orientation relative to extrinsic coordinates (e.g., the gravitational vertical) has often been found to be more accurate than perception with respect to intrinsic coordinates (e.g., joint angle, or the trunk- and head-longitudinal axes; Soechting 1982; Worringham and Stelmach 1985; Worringham et al. 1987; Darling 1991; Darling and Hondzinski 1999). It follows from the present results that the use of extrinsic coordinates in limb kinesthesis may substantially benefit from CM-related information, conveyed by a horizontal shift of Ng as a function of arm inclination, which could explain this higher accuracy in extrinsic coordinates. The issue of extrinsic versus intrinsic coordinate systems is also a relevant dimension when comparing the center of mass hypothesis with the inertial eigenvector hypothesis.\nUnlike CM, ei cannot aid the perception of limb orientation in extrinsic, earth-fixed coordinates. After all, as we noted in the introduction, the possible perceptual role of ei resides exclusively in its effect on the dynamic torques involved in limb rotation, which are independent of a limb\u2019s orientation in extrinsic space. Hence, the perception of limb orientation through the detection of inertial eigenvectors necessarily implies an intrinsic coordinate system, similar to that involved in the detection of muscle length or joint angle. This important point was overlooked by Garrett et al. (1998), who found that their manipulation of ei (and implicitly CM) affected the perception of forearm orientation relative to the gravitational vertical and interpreted this as an effect of ei. It follows from the preceding argument that this interpretation was invalid because ei, being gravity independent, cannot convey such information. Hence, irrespective of the present empirical evidence, Garrett et al. (1998) could have attributed their findings to gravity-dependent information.\nBecause the experimental tasks in the present study allow for the use of extrinsic as well as intrinsic axes, the apparent dominance of CM over ei cannot be explained in terms of their respective coordinate systems. So what did underlie the difference in perceptual effect between ei and CM in the present experiments? The answer may reside in their respective signal-to-noise ratios, or saliences (cf. van de Langenberg et al. 2007). Recall that information about ei is only reflected in the dynamic torques involved in active limb movement. In the present experiments, as well as in Pagano et al. (1996) and Garrett et al. (1998), the arms were moved at a relatively low angular velocity, implying that the contribution of dynamic torques to the muscular tension was small relative to the static torque Ng. It follows that the salience of ei must have been low relative to that of CM, which exclusively affects Ng. In general, one can say that during slow movements or stationary postures, the salience of CM will be superior to that of ei. During fast movements, the salience of ei will increase at the cost of the salience of CM, which leaves open the possibility that the perception of arm orientation in fast movements, at least in intrinsic coordinates, is affected by ei as well.\nThe results of Experiment 2 generalized the role of CM from matching the orientation of contralateral forearms to orienting a single arm in extrinsic space. They further showed that the perception of upper arm orientation was unaffected by the manipulation of the forearm\u2019s CM, even though the manipulation affected Ng at both the elbow and the shoulder due to the absence of an upper arm support. The latter finding may be explained in two ways: Participants may have exploited Ng at the shoulder to obtain information about the configuration of the whole arm in space, as we anticipated in the introduction to Experiment 2, or they may have discounted it, only taking Ng into account at the elbow. The latter alternative would suggest that the role of CM in the perception of limb orientation pertains only to the most distal rigid segment, whereas the former alternative would suggest that its role is more general, supporting the perception of the orientation of both proximal and distal limb segments. This issue may be resolved in experiments in which the center of mass vectors of both distal and proximal limb segments (e.g., both the forearm and the upper arm) are manipulated in tasks similar to that used in Experiment 2.\nIn seeking an encompassing account of the perception of limb orientation, it is important to note that we only tested the effect of CM and e3 manipulations in the vertical plane. Manipulation of CM in the horizontal plane would only affect the direction of Ng, not its magnitude, and hence cannot be mediated by the aforementioned horizontal shift in the relationship between arm orientation and Ng magnitude (see Fig.\u00a07). In a recent study (van de Langenberg et al. 2007), we tested the effect of horizontal CM and e3 manipulations in a pointing task similar to that adopted in Experiments 1 and 2 of Pagano and Turvey (1995). We found essentially the same pattern of results as in the present study: Perceived arm orientation was affected by the horizontal manipulation of CM but not by that of e3. It thus appears that humans are sensitive to changes in both torque magnitude and direction, and hence to CM rotations in 3D.\nAnother important point of discussion is that the observed perceptual biases were consistently smaller than the magnitude of CM rotation, as was the case in previous studies on the effect of vertical (CM and) e3 rotations (Pagano et al. 1996; Garrett et al. 1998). However, the relative sizes of our effects (i.e., 15\u201326% of the actual CM rotation) were smaller than those observed in those previous studies (i.e., 40\u201345% of the actual CM rotation). We suspect that this difference is related to differences in the magnitude of CM manipulation, which was 10\u00b0 in our Experiment 1 and only about 5\u00b0 in Pagano et al. (1996) and Garrett et al. (1998). In a study on the effect of horizontal e3 (and CM) rotations, Bernardin et al. (2005) adopted three manipulation magnitudes in a single experiment and indeed found a strong negative relationship between effect size and manipulation magnitude (upon an increase of CM manipulation from about 1.3\u00b0 to about 5.5\u00b0 effect size decreased from 38 to 13%). In addition to manipulation magnitude, other factors, such as manipulation direction (i.e. horizontal or vertical) and exploration style, may also influence the magnitude of CM\u2019s effect on the perception of limb orientation. These factors should be explored further in future research. Regardless of their precise contributions, however, one can already conclude that the perceptual biases introduced by manipulating CM and e3 are in general incomplete. It follows from this general observation that additional information, unrelated to either CM or e3, must have been employed. The use of information unrelated to CM is also apparent in conditions in which the arm\u2019s CM cannot be detected, such as when the limb is moved passively to a certain position (see e.g., Lee et al. 2003; Ulkar et al. 2004) or when gravity is absent (see e.g., Lackner and DiZio 2000).\nNotwithstanding the preceding qualifications, the present findings clearly underscore that there is a kinetic component to limb kinesthesis, which has important implications for its possible neural basis. More specifically, the apparent dependence of limb kinesthesis on CM points to an important role of neural signals related to muscular torque or effort. As noted in the introduction, several other psychophysical studies provided support for this view (Rymer and D\u2019Almeida 1980; Prud\u2019homme and Kalaska 1994; Proske et al. 2004; Walsh et al. 2004; Winter et al. 2005; Gandevia et al. 2006). Although these studies clearly challenge the view that limb kinesthesis relies on muscle spindle activity alone (see Ribot-Ciscar et al. 2003), they do not discard the possibility that signals related to muscular torque or effort only serve to accurately interpret spindle discharges in terms of muscle length changes, as proposed by McCloskey (1981, see also Gandevia 1996). The effect of CM on limb kinesthesis indicates that, in addition to such an indirect role, kinetic signals may convey information about the distribution of a limb\u2019s mass in space and hence directly affect kinesthesis, independent of geometric information about muscle length or joint angle. Golgi tendon organs seem particularly suited for fulfilling this function, given that their activity is closely associated with muscle force (Kandel et al. 2000). This implies that the possible implication of Golgi tendon organs in limb kinesthesis may need to be reconsidered and that a mechanism akin to that suggested by Rymer and d\u2019Almeida (1980) may indeed be possible.","keyphrases":["center of mass","inertial eigenvectors","kinesthesis","information","gravitational torque","limb position sense","proprioception"],"prmu":["P","P","P","P","P","R","U"]}
{"id":"J_Urban_Health-2-2-1705507","title":"Respondent-Driven Sampling of Injection Drug Users in Two U.S.\u2013Mexico Border Cities: Recruitment Dynamics and Impact on Estimates of HIV and Syphilis Prevalence\n","text":"Respondent-driven sampling (RDS), a chain referral sampling approach, is increasingly used to recruit participants from hard-to-reach populations, such as injection drug users (IDUs). Using RDS, we recruited IDUs in Tijuana and Ciudad (Cd.) Ju\u00e1rez, two Mexican cities bordering San Diego, CA and El Paso, TX, respectively, and compared recruitment dynamics, reported network size, and estimates of HIV and syphilis prevalence. Between February and April 2005, we used RDS to recruit IDUs in Tijuana (15 seeds, 207 recruits) and Cd. Ju\u00e1rez (9 seeds, 197 recruits), Mexico for a cross-sectional study of behavioral and contextual factors associated with HIV, HCV and syphilis infections. All subjects provided informed consent, an anonymous interview, and a venous blood sample for serologic testing of HIV, HCV, HBV (Cd. Ju\u00e1rez only) and syphilis antibody. Log-linear models were used to analyze the association between the state of the recruiter and that of the recruitee in the referral chains, and population estimates of the presence of syphilis antibody were obtained, correcting for biased sampling using RDS-based estimators. Sampling of the targeted 200 recruits per city was achieved rapidly (2 months in Tijuana, 2 weeks in Cd. Ju\u00e1rez). After excluding seeds and missing data, the sample prevalence of HCV, HIV and syphilis were 96.6, 1.9 and 13.5% respectively in Tijuana, and 95.3, 4.1, and 2.7% respectively in Cd. Ju\u00e1rez (where HBV prevalence was 84.7%). Syphilis cases were clustered in recruitment trees. RDS-corrected estimates of syphilis antibody prevalence ranged from 12.8 to 26.8% in Tijuana and from 2.9 to 15.6% in Ciudad Ju\u00e1rez, depending on how recruitment patterns were modeled, and assumptions about how network size affected an individual\u2019s probability of being included in the sample. RDS was an effective method to rapidly recruit IDUs in these cities. Although the frequency of HIV was low, syphilis prevalence was high, particularly in Tijuana. RDS-corrected estimates of syphilis prevalence were sensitive to model assumptions, suggesting that further validation of RDS is necessary.\nIntroduction\nInjection drug use is a growing problem in cities located along the U.S.\u2013Mexico border. Approximately 70% of U.S. cocaine originating in South America passes through the Central America\u2013Mexico corridor.1 Cities and towns positioned on drug trafficking routes often experience epidemics of injection drug use.2,3 Injection drug users (IDUs) are at high risk of blood-borne infections, such as hepatitis C virus (HCV) and human immunodeficiency virus type-1 (HIV-1) infection, and of acquiring HIV-1 and other sexually transmitted infections (STIs) through high rates of unprotected sex.4\u201310\nMexico is currently considered a country of low HIV\/AIDS prevalence11 (180,000 adult cases in 2005, a seroprevalence in the general population of 0.3%12), and the HIV epidemic has been mainly confined to men who have sex with men.13 Although injection drug use appears to have played only a minor role in the epidemic on a country-wide level,14 injection drug use appears to be increasingly important as a risk factor for HIV infection in some Mexican cities bordering the U.S. Viani et al.15 noted that the prevalence of HIV among pregnant women giving birth at Tijuana General Hospital rose from 0.29% in 1998 to 1.02% in 2001 and, in a subsequent study, showed that pregnant HIV-infected women were more likely to either inject drugs or to have a spouse\/partner who injected drugs.16 In 2002, Valdez et al.17 reported that 21% of female sex workers in Ciudad (Cd.) Ju\u00e1rez injected illicit drugs, whereas a study by Patterson et al.18 in 2005 showed that over half of female sex workers in Cd. Ju\u00e1rez injected drugs, suggesting increasing overlap between sexual and IDU networks.\nOverlap between injection drug use and the trade of sex for money or drugs may contribute to elevated risk of STIs other than HIV, such as syphilis. Syphilis has been associated with higher HIV seroprevalence in a number of populations and is considered a cofactor of HIV transmission.19\u201322 In contrast to HIV, syphilis has been present in Mexico since at least the time of the Spanish invasion; however, the number of reported cases has decreased from 40,607 in 1945 (190.5 per 100,000) to 2,608 in 1990 (3.2 per 100,0000).23\nAggregate figures for syphilis prevalence belie the sub-epidemics occurring within specific risk groups. Several studies have been conducted with female sex workers in Mexico and have found varying syphilis prevalence levels. In 1990, 23.7% of 1,386 sex workers in four Mexican states had a reactive syphilis test.24 In 1993, testing of 826 sex workers in Mexico City showed an overall prevalence of 6.4%, with different syphilis rates associated with different patterns of sex work: 1.3% for massage parlor workers, 4.4% for bar girls, and 9.6% for streetwalkers.25 The prevalence of syphilis among 3,100 female sex workers tested at an AIDS clinic during 1992 and 1993 was 8.2%.26 In contrast, syphilis prevalence was low (2.3 and 1.1%) among gynecological outpatients in two Mexican cities between the years 1994 and 1995.27 However, little is known about syphilis prevalence in IDU populations in Mexico.\nIn order to estimate the prevalence of HIV, HCV, and syphilis among IDUs, we conducted a cross-sectional study of IDUs in the border cities of Tijuana and Cd. Ju\u00e1rez, Mexico. Both cities are located on major drug trafficking routes and have large IDU populations (c. 6,000), with a similar sex ratio among the IDUs (c. 80% male).3 As stigma surrounding injection drug use makes it difficult to obtain a representative sample of injection drug users, we recruited individuals using respondent-driven sampling (RDS).28,29 By collecting data on individuals\u2019 personal network sizes, RDS attempts to correct for biases in the sampling process, in order to obtain unbiased estimates of parameters such as the prevalence of a disease. In this study, we report on patterns of recruitment and the prevalence of HIV, HCV, and syphilis (and hepatitis B infection, for Cd. Ju\u00e1rez) in the context of sexual risk.\nMaterials and Methods\nStudy Population\nFrom February through April 2005, IDUs were enrolled in a cross-sectional study in Tijuana and Cd. Ju\u00e1rez, Mexico. Eligibility criteria for the study included: having injected illicit drugs within the past month, confirmed by inspection of injection stigmata (\u2018track marks\u2019); aged 18\u00a0years or older; willing and able to provide informed consent; and not having been previously interviewed for the study. Subjects gave their written informed consent to participate in the study. Study methods were approved by the Institutional Review Board of the University of California, San Diego and the Ethics Board of the Tijuana General Hospital, which has one of the few federal-wide assurances in Mexico.\nRecruitment\nRDS methods were used to recruit participants.28,29 A diverse group of \u201cseeds\u201d (heterogeneous in age, gender, and geographic location) were selected to initiate the process. After providing informed consent, seeds underwent an interview, were educated on how to refer other eligible IDUs, and were given three uniquely coded coupons to refer their peers. Coupons were given to participants until approximately 150 participants were recruited in order to obtain a target sample size of approximately 200 per site.\nOn each coupon, the study name, locations where they could participate, and a brief explanation was printed. In Cd. Ju\u00e1rez, interviews were conducted at a clinic run by Programa Compa\u00f1eros, A.C., which is a trusted and well-respected non-governmental organization (NGO) that has been providing services to and conducting studies with IDUs in the city for decades. In Tijuana, staff from both COMUSIDA, the municipal HIV\/AIDS program, and the Centro de Integraci\u00f3n y Recuperaci\u00f3n para Enfermos de Alcoholismo y Drogadicci\u00f3n \u201cMario Camacho Esp\u00edritu\u201d, A.C. (CIRAD), an NGO that began working with drug users in 1991, made weekly trips to three geographically diverse \u2018colonias\u2019 (i.e., neighborhoods) in the city: Zona Norte, Grupo M\u00e9xico, and Sepanal, using a modified recreational vehicle that operated as a mobile clinic (the \u2018Prevemovihl\u2019).\nMonetary reimbursements were given to participants to cover transportation costs and to compensate them for their time. The study staff in each site proposed the incentive levels based on their experience with this population and the incentives for previous studies. Participants in Cd. Ju\u00e1rez received $20 U.S. dollars (USD) for participation in the baseline visit and $5 USD when receiving laboratory test results at a one month follow-up visit. In Tijuana, $10 USD was given at baseline and $5 for the follow-up visit. In addition, participants at both sites were given $5 for each eligible person they recruited. These levels were not regarded as high.\nData Collection\nUpon enrollment, trained staff administered quantitative surveys eliciting information on topics such as socio-economic and demographic profiles, drug use practices, sterile syringe access, barriers to sterile syringe use, experience with drug abuse treatment and incarceration history, health status, and HIV knowledge and testing history. We also asked about sexual behaviors and condom use with regular, casual and client partners of the opposite and same sex. Questions pertained to lifetime risk behaviors and those occurring in the prior six months.\nFor RDS purposes, we measured network size using the question \u201cEn los \u00faltimos 6 meses, \u00bfcu\u00e1ntas personas conoce de nombre o de apodo que se han inyectado drogas?\u201d (\u201cIn the past 6\u00a0months, how many people do you know by name or street name who have injected drugs?\u201d). To determine the relationship between recruiter and recruitee, we asked \u201c\u00bfCu\u00e1l es su relaci\u00f3n con la persona que le entreg\u00f3 el cup\u00f3n?\u201d (\u201cWhat is your relationship to the person who gave you the coupon?\u201d). Participants were given the choice of: \u201cparientes\u201d (relative); \u201cpareja sexual\u201d (sex partner\/spouse); \u201camigo(a)\u201d (friend); \u201cconocido\u201d (acquaintance); \u201cdesconocido\u201d (stranger); and \u201cotro\u201d (other). To determine the size of individuals\u2019 networks with respect to injection drug use, we asked \u201cEn los \u00faltimos 6 meses, \u00bfcon cu\u00e1ntas personas diferentes acostumbra inyectarse?\u201d (\u201cIn the last 6\u00a0months, on average how many different people did you usually inject with?\u201d). After the interview, blood was drawn for antibody testing of HIV, HCV, HBV (Cd. Ju\u00e1rez only), and syphilis. Pre- and post-test counseling, and referral to treatment where indicated, was provided to all participants.\nLaboratory Samples\nBlood samples were obtained by venipuncture and serum was stored at the municipal health clinic in Tijuana or Cd. Ju\u00e1rez before being shipped frozen to the San Diego County public health laboratory or New Mexico State Laboratory, respectively. All participants were screened on-site in Mexico for HIV with the Determine rapid test (Abbott Laboratories). For the Tijuana samples, in the event of an HIV-positive or indeterminate test, results were confirmed with a Western blot, HIV enzyme immunoassay (EIA), and HIV immunofluorescence assay. For samples from Cd. Ju\u00e1rez, the HIV EIA was conducted on all samples, and a confirmatory Western blot was performed on positive or indeterminate sample. Cd. Ju\u00e1rez samples were also tested for hepatitis B antigen (Genetic Systems HBsAg EIA 3.0, Bio-Rad Laboratories) and antibody (DiaSorin ETI-AB-COREK PLUS). All samples were tested for syphilis with the rapid plasma reagin (RPR) test (Macro-Vue, Becton Dickenson) and if reactive, confirmed by a Treponema pallidum particle agglutination assay (TPPA; Fujirebio Diagnostics).\nStatistical Methods\nObtaining estimates of population proportions of groups using RDS involves combining three kinds of data: the sample proportion of each group, the crosstabulation of groups between pairs of recruiters and recruitees, and differences in network size between groups. To estimate equilibrium proportions of different groups, and to estimate the pattern of mixing between groups, we assumed that the recruitment process followed a first order Markov process.28,29 Under this model, the relationship between the state of the recruiter and recruitee can be modeled using log-linear models applied to a two-way table of counts.30,31 We classified individuals by sex and syphilis seropositivity and fitted a series of hierarchical log-linear models of increasing complexity to the data to determine patterns of nonrandom mixing between groups along each recruitment tree, choosing the best model as that which had the lowest value of Akaike\u2019s Information Criterion.32 For the purposes of analysis, we considered all individuals with positive syphilis tests based upon RPR, and did not classify individuals further into those with TPPA titers greater than or equal to 1:8 (who may represent infectious cases) and those with titers of 1:1 to 1:4 (who may represent past infection).\nTo derive RDS-corrected estimates of syphilis seropositivity in men and women in Tijuana and Ciudad Ju\u00e1rez, we estimated recruitment weights for each group (as the ratio of the equilibrium to sample proportions of each group). We estimated the equilibrium fraction as previously described.29 We used both raw counts and predicted counts based on the best fitting log-linear model. Degree weights were estimated using linear least squares.29 We used both unadjusted and adjusted estimates of personal network size.33 An overall sampling weight was derived for each group, from which population-level estimates were obtained.\nPre-processing of the data was performed using Stata v. 8.2 (Stata Corporation, College Station, TX). Networks and trees were generated using scripts written in Python and visualized using GraphViz (AT&T Research, Florham Park, NJ). Statistical analyses and summary statistics of the recruitment network were generated in R,34 and RDS based corrections were calculated using Maxima (http:\/\/maxima.sourceforge.net). We chose to develop our own programs rather than use RDSAT (http:\/\/www.respondentdrivensampling.org) primarily to familiarize ourselves with the statistical theory underlying RDS-based corrections. All code is available from the first author on request.\nResults\nStudy Population\nTable\u00a01 summarizes some basic data relating to the Tijuana (15 seeds, 207 recruits) and Cd. Ju\u00e1rez (9 seeds, 197 recruits) study populations. Both populations were predominantly male, with participants in their early to mid-30s. Crude HCV seroprevalence was extremely high (>95%) in both cities. Hepatitis B seroprevalence was only determined for Cd. Ju\u00e1rez, where it was high (85% overall); only one individual was positive for HBV antigen. Crude HIV seroprevalence was low, but the crude prevalence of syphilis was high, especially among women.\nTable\u00a01.Summary statistics of age, parameters pertaining to risk of STI, and seroprevalence of HIV, HCV and syphilis, by city, sex, and by whether individuals were seeds, or recruitsCityTijuanaCiudad Ju\u00e1rezType of recruitSeedsRecruitsSeedsRecruitsGender (n)Male (9)Female (6)Male (194)Female (13)Male (6)Female (3)Male (184)Female (13)Age (median, range)35 (29\u201354)28 (22\u201332)34 (18\u201356)32 (24\u201348)42 (33\u201349)33 (30\u201342)33 (18\u201362)36 (20\u201348)Age at sexual debut (median, range)15 (12\u201318)16.5 (9\u201327)15 (7\u201328) [n=191]14 (11\u201315)13.5 (8\u201318)16 (15\u201318)15 (5\u201323) [n=181]14 (12\u201318)No. of lifetime male sexual partners (median, range)2 (0\u201315)8 (0\u201310)0 (0\u2013100) [n=186]20 (2\u2013500) [n=11]0 (0\u20132)5 (3\u20136)0 (0\u201315)  [n=178]5 (1\u2013100)  [n=11]No. of lifetime female sexual  partners (median, range)10 (4\u2013300)0 (0\u20131)10 (0\u2013500) [n=191]0 (0\u20134)22 (8\u2013200)5 (0\u201310) [n=2]10 (1\u2013300) [n=175]0 (0\u20137) [n=12]Prostitution as main source of income over the  last 6\u00a0months (fraction, %)0\/9 (0)1\/6 (16.7)1\/191 (0.5)6\/13 (46.2) 0\/6 (0)1\/3 (33)0\/184 (0)1\/13 (7.7)Had sex in last 6\u00a0months (fraction, %)5\/9 (55.6)4\/6 (66.7)106\/194 (54.6)12\/13 (92.3)4\/6 (66.7)2\/3 (66.7)108\/184 (58.7)10\/13 (76.9)Ever been given (bought) sex in the last 6\u00a0months  (fraction, %)1\/3 (33.3)2\/3 (66.7)24\/67 (35.8)8\/8 (100)0\/2 (0)0\/0 (N\/A)10\/57 (17.5)4\/5 (80.0)Ever given (sold) sex in the last 6\u00a0months (fraction, %)1\/3 (33.3)2\/3 (66.7)17\/67 (25.4)2\/8 (25)1\/2 (50)0\/0 (N\/A)23\/57 (40.4)3\/5 (60.0)Injected drugs with sex partner in last 6\u00a0months1\/9 (11.1)2\/6 (33.3)10\/194 (5.15)6\/13 (46.2)0\/6 (0)1\/3 (33.3)15\/184 (8.2)3\/13 (2.3)Syphilis RPR + (fraction, %)1\/7 (14.3)2\/6 (33.3)20\/194 (10.3)8\/13 (61.5)0\/4 (0)2\/3 (66.7)3\/175 (1.71)2\/11 (18.2)HIV antibody (fraction, %)1\/7 (14.3)1\/6 (16.7)4\/194 (2.1)0\/13 (0)0\/6 (0)0\/3 (0)8\/182 (4.4)0\/12 (0)HCV antibody (fraction, %)6\/7 (85.7)6\/6 (100)188\/194 (96.9)12\/13 (92.3)6\/6 (100)3\/3 (100)174\/181 (96.1)10\/12 (83.3)HBV antibody (fraction, %)N\/AN\/AN\/AN\/A6\/6 (100)3\/3 (100)154\/181 (85.1)8\/12 (66.7)\nRecruitment Dynamics\nRDS was an effective means of recruiting IDUs in both cities. The number of individuals recruited increased rapidly following the first interview, especially in Cd. Ju\u00e1rez (Figure\u00a01a), where many individuals interviewed the same day as their recruiter (Figure\u00a01b). Apart from these differences in the tempo of recruitment, patterns of recruitment were very similar between the two cities; recruitment was highest in the fourth wave of recruitment, with some individuals being recruited after eight waves, suggesting that despite rapid recruitment, good sociometric depth was obtained (Figure\u00a01c). After excluding individuals who were not given coupons, the number of recruits per recruiter showed a bimodal distribution, with many individuals either recruiting zero or three recruits (Figure\u00a01d), suggesting the presence of a mixed population of ineffective and effective recruiters. In both cities, approximately one half of participants were recruited via referral trees originating from two seeds (Figure\u00a01e). The relationship between recruiter and recruit was usually \u2018friend,\u2019 \u2018acquaintance,\u2019 or another close relationship such as a family member or a sex partner (Figure\u00a01f), which is important as RDS-based estimates assume that these relationships are reciprocal.28,29Figure\u00a01Summary of the dynamics of recruitment, by city. (a) The cumulative number of recruits over time. (b) The interval between the interview of the recruiter and that of their recruitee (omitting those individuals who did not recruit). (c) The number of recruits in each recruitment wave from the seed. (d) The number of recruits per recruiter (excluding individuals who were not given any coupons). (e) The number of recruits from each seed. (f) The relationship between recruiter and recruitee.\nRecruitment Trees\nFigure\u00a02 shows a \u2018forest\u2019 of recruitment trees for each city, with the syphilis antibody status indicated by shading and the gender of each individual indicated by different symbols. This figure illustrates that the sample prevalence of syphilis was higher in Tijuana than in Cd. Ju\u00e1rez and that syphilis cases appeared to cluster in the recruitment trees. Although there was a low frequency of women in the sample, a disproportionate number of women also had syphilis.\nFigure\u00a02Recruitment networks (strictly speaking, a forest of recruitment trees) for the RDS based samples of IDUs in (a) Tijuana and (b) Ciudad Ju\u00e1rez. Seeds are shown at the top of the figure, and arrows indicate the direction of recruitment. Syphilis serostatus is shown by shading: black- syphilis antibody positive, white- syphilis antibody negative, gray- missing data. The gender of participants is indicated by the shape of the symbol: square for female and circle for male. The size of the symbol is related to the reported network size: the larger the symbol, the larger the network size. Symbols marked with an \u2018\u00d7\u2019 denote individuals who were given coupons, but did not recruit.\nTo test whether syphilis prevalence differed by sex and whether cases of syphilis were clustered, we analyzed the relationship between the sex and syphilis status of the recruiter and recruitee in Tijuana and Cd. Ju\u00e1rez (Table\u00a02). A set of hierarchical log-linear models were used to test whether there was an association between syphilis seropositivity and sex and between the state of the recruiter and that of the recruitee, with the best fitting model chosen using Akaike\u2019s Information Criterion, AIC,32 with lower values indicating better fit (Table\u00a03). This approach is functionally equivalent to calculating the RDS homophily index, for which 1 denotes a perfect association, 0 a zero association, and \u22121 a perfect negative association. The reason for using log-linear models is that they offer a solid statistical framework with which we can compare different models of association between the characteristics of the recruiter and those of the recruitee.\nTable\u00a02Relationship between recruiter and recruitee in terms of sex and syphilis antibody status\u00a0TijuanaCiudad Ju\u00e1rezFromToMaleFemaleMaleFemaleRPR\u2212RPR+RPR\u2212RPR+RPR\u2212RPR+RPR\u2212RPR+MaleRPR\u2212145 (145.2)15 (14.66)2 (2.33)5 (4.82)147 (146.7)3 (2.30)7 (7.43)1 (1.53)RPR+15 (15.23)5 (4.91)1 (0.24)1 (1.61)1 (1.59)0 (0.2)0 (0.08)1 (0.13)FemaleRPR\u22128 (7.19)0 (0.11)1 (1.29)0 (0.41)7 (6.50)0 (0.10)0 (0.33)0 (0.07)RPR+6 (6.38)0 (0.32)1 (1.14)2 (1.16)3 (3.17)0 (0.40)1 (0.16)0 (0.27)Smoothed estimates are shown in parentheses and are based upon the best fitting log linear model for each sample (Table\u00a03).Table\u00a03.Fit of 12 log-linear models to the data shown in Table\u00a02ModelpTijuanaCiudad Ju\u00e1rezlnLAICLRT, G2lnLAICLRT, G2Symmetric(sex1,sex2) + Symmetric(rpr1,rpr2)5\u221242.0694.1240.4 (p<0.001)\u221228.8467.6829.3 (p=0.001)sex1 + rpr1 + sex2 + rpr25\u221246.27102.548.8 (p<0.001)\u221229.9169.8231.4 (p=0.001)sex1 + rpr1 + sex2 * rpr26\u221237.2786.5530.8 (p=0.001)\u221227.2566.4926.1 (p=0.004)sex1 * rpr1 + sex2 * rpr27\u221230.3274.6416.9 (p=0.05)\u221219.2152.4310.0 (p=0.35)sex1 * rpr1 + sex2 * rpr2 + sex1 * sex28\u221227.4770.9511.2 (p=0.189)\u221219.1254.249.81 (p=0.28)sex1 * rpr1 + sex2 * rpr2 + rpr1 * rpr28\u221228.3172.6112.9 (p=0.115)\u221218.1652.327.89 (p=0.44)sex1 * rpr1 + sex2 * rpr2 + sex1 * sex2 + rpr1 * rpr29\u221226.0170.028.31 (p=0.306)\u221218.1454.287.85 (p=0.35)sex1 * rpr1 + sex2 * rpr2 + sex1 * sex2 + rpr1 * rpr2 + sex1 * rpr210\u221223.8767.734.02 (p=0.674)\u221216.4852.964.53 (p=0.61)sex1 * rpr1 + sex2 * rpr2 + sex1 * sex2 + rpr1 * rpr2 + rpr1 * sex210\u221225.6371.277.56 (p=0.272)\u221216.1752.353.92 (p=0.69)sex1 * rpr1 + sex2 * rpr2 + sex1 * sex2 + rpr1 * rpr2 + sex1 * rpr2 + rpr1 * sex211\u221223.6769.343.62 (p=0.604)\u221216.6553.294.87 (p=0.56)Symmetric(sex1 * rpr1, sex2 * rpr2)10\u221225.9171.828.1 (p=0.23)\u221215.0352.061.63 (p=0.90)Saturated model (sex1 * rpr1 * sex2 * rpr2)16\u221221.8675.71NA\u221214.2160.43NAThe variables in the \u2018Model\u2019 column correspond to the sex of the recruiter (sex1) and recruitee (sex2) and the syphilis serostatus of the recruiter (rpr1) and recruitee (rpr2). The term \u2018Symmetric(a, b)\u2019 applies to a table which is symmetric about the diagonal of the table a by b. The number of parameters is denoted p, the log-likelihood of the model (higher is better) by lnL, Akaike\u2019s Information Criterion (lower is better) by AIC, and a likelihood ratio test for goodness of fit against a saturated model by LRT. The best fitting model for each city, based on the lowest AIC, is indicated by underlining.\nFor both samples, the best fitting model included a significant (positive) association between the syphilis serostatus of the recruiter and the syphilis serostatus of the recruitee and was not significantly different from a \u2018saturated\u2019 model, in which each cell in the table is modeled with a single parameter. For the Tijuana sample, there was a positive association between the sex of the recruiter and the sex of the recruit, and, independently of this association, syphilis antibody positive women were disproportionately less likely to recruit syphilis positive men than syphilis negative men.\nNetwork Size\nThere was great variation in both measures of network size, with the average number of known IDUs an order of magnitude higher than the number of injecting partners. There was no correlation in network size between recruiter and recruitee, measured using either the number of IDUs known (Spearman\u2019s rho = 0.0328 and 0.0204 for Tijuana and Cd. Ju\u00e1rez, respectively) or the number of injecting partners as a measure of network size (Spearman\u2019s rho = \u22120.006 and \u22120.0121 for Tijuana and Cd. Ju\u00e1rez, respectively; Figure\u00a03a, b). Network sizes were similar when the sample was grouped by syphilis serostatus and sex (Figure\u00a03c, d). As chain referral samples are biased towards individuals with larger network sizes, we adjusted the distribution of network size by weighting the distribution of network sizes by the inverse of the network size.33 This reweighting led to a significant drop in estimated network size, from a median of 25\u201350 to 4\u20135 in Tijuana and from 15\u201320 to 8\u201310 in Cd. Ju\u00e1rez (Figure\u00a03e).\nFigure\u00a03Summary of the network size distribution. (a) Scatterplot of the number of IDUs known by name or street name between recruiter and recruitee. (b) Scatterplot of the number of injecting partners between recruiter and recruitee. (c) Boxplot of the number of IDUs known by sex (M male, F female) and by syphilis antibody status (+ positive, \u2212 negative). (d) Boxplot of the number of injecting partners by sex (M male, F female) and by syphilis antibody status (+ positive, \u2212 negative). (e) Cumulative distribution of the number of IDUs known before (solid line) and after (dashed line) adjusting for biased sampling of individuals.\nCorrecting for Sampling Bias\nInformation on network size, and on who recruited whom, collected as part of RDS, allows population estimates to be generated from the sample, despite biases in the sampling process. To do so, we need to estimate the pattern of mixing between different groups along the recruitment network and to determine how different the network size is in each group. The seroprevalence of HIV was too low, and the seroprevalence of HCV was too high in order to obtain meaningful correction factors; hence we concentrated on obtaining population estimates of the prevalence of syphilis antibody in men and women in the two cities.\nIn order to obtain unbiased population estimates of the prevalence of syphilis antibody, we used a poststratification process, in which we calculated recruitment weights and degree weights, which can be combined to give an overall sampling weight.46 As RDS is a chain referral method, the prevalence of syphilis in the sample may have been different if recruitment had continued for further waves. Assuming that recruitment follows a first order Markov process, the \u2018equilibrium prevalence\u2019 of syphilis can be calculated from the cross-tabulations of the syphilis status of the recruiter and the recruitee. Using these estimates, we calculated recruitment weights, as the ratio of the equilibrium to sample frequencies of each group (Table\u00a04); the closer these weights are to 1, the more representative the sampling of the group. For the Tijuana sample, recruitment weights were close to 1 for three groups, except for syphilis negative women, whose recruitment weight was 0.87 (i.e., this group was oversampled). For the Cd. Ju\u00e1rez sample, recruitment weights were close to 1, with the exception of syphilis positive women (1.25), who were undersampled.\nTable\u00a04.RDS-corrected estimates of syphilis seroprevalence, using the raw transition data, and adjusted degreesCityTijuanaCiudad Ju\u00e1rezSexMaleFemaleMaleFemaleSyphilis antibodySyphilis  RPR\u2212Syphilis  RPR+Syphilis  RPR\u2212Syphilis  RPR+Syphilis  RPR\u2212Syphilis  RPR+Syphilis  RPR\u2212Syphilis  RPR+n1742058158382Sample (%)84.169.662.423.8692.41.754.681.17Equilibrium (%)84.279.82.113.8292.31.754.461.46Recruitment weight11.010.870.99110.951.25Degree54.9664.83059.1247.5419.3345.1115Adjusted degree14.638.4518.294.8914.6714.418.6213.33Adjusted degree weight0.861.670.372.750.9810.713.16Adjusted sampling weights0.861.690.322.720.9810.673.95Adjusted population (%)72.3716.350.7710.590.471.753.164.62\nSamples obtained using RDS can also be biased due to differences in network size between the groups. To compensate for this effect, we calculated degree weights, based upon the reported network size of known IDUs using the adjusted mean estimates of network size33 in each group (Figure\u00a03f). After calculating degree weights for each group, and multiplying them by recruitment weights to generate an overall sampling weight, the RDS corrected estimates of syphilis antibody prevalence were higher than those in the overall sample for both cities (Table\u00a04).\nSensitivity of RDS Estimates\nTo determine the sensitivity of point estimates of the prevalence of syphilis antibody to modeling assumptions, we also obtained estimates using unadjusted rather than adjusted network sizes and using a \u2018smoothed\u2019 transition matrix based on the best fitting log-linear model, rather than the raw counts. We found that RDS-based estimates were highly sensitive to these assumptions (Table\u00a05). Estimated syphilis seroprevalence ranged from 12.4 to 26.8% in Tijuana and from 2.9 to 15.6% in Cd. Ju\u00e1rez, depending on how the pattern of recruitment was modeled and how reported network size was assumed to affect an individual\u2019s probability of being included in the sample. However, our results suggest that syphilis seroprevalence is higher among women than men and higher in Tijuana than in Cd. Ju\u00e1rez and that sample proportions of syphilis using RDS in these populations may be underestimates of the true population seroprevalence.\nTable\u00a05.Sensitivity of the estimated population prevalence of syphilis antibody among men and women to model assumptionsModel assumptionsPopulation prevalenceTijuanaCiudad Ju\u00e1rezMaleFemaleMaleFemaleTransition  matrixNetwork  sizesSyphilis  RPR\u2212Syphilis  RPR+Syphilis  RPR\u2212Syphilis  RPR+Syphilis  RPR\u2212Syphilis  RPR+Syphilis  RPR\u2212Syphilis  RPR+UnadjustedUnadjusted84.799.342.083.880.613.763.7611.86UnadjustedAdjusted72.3716.350.7710.590.471.753.164.62SmoothedUnadjusted84.818.982.753.4587.943.974.693.41SmoothedAdjusted73.3515.941.049.6793.281.743.721.25Smoothed transition matrices are obtained by using the fitted values from the best fitting model in Table\u00a03. Adjusted network sizes are obtained as described in Salganik and Heckathorn.33\nConclusions\nRespondent driven sampling offers the promise of a probability sample of individuals from hidden and hard-to-reach populations. RDS was originally developed in the context of recruiting IDUs35\u201339 and, in our context, was an efficient method to recruit IDUs in two Mexican cities bordering the U.S. Recruitment was extremely rapid in Cd. Ju\u00e1rez compared to Tijuana, which may be due to greater access to the study site, higher monetary incentives, and that Programa Compa\u00f1eros is more established in Cd. Ju\u00e1rez than CIRAD in Tijuana and had carried out studies in the past with monetary renumeration. In contrast, Mueller et al.40 report much slower recruitment of IDUs in Las Cruces, NM using RDS, despite similar methodology and the same eligibility criteria.\nThe sample seroprevalence of HIV was relatively low in both cities. HIV-1 seroprevalence in IDUs in Tijuana recruited through RDS was similar to that found in IDUs studied by G\u00fcere\u00f1a-Burgue\u00f1o et al.41 in the early 1990s and in a study by Magis-Rodriguez et al.42 in 2003 that used time-location sampling methods, although the absolute number of HIV-positive cases was too low to perform reliable RDS corrections. In contrast to HIV, syphilis prevalence was extremely high, especially in women and in Tijuana.\nUnlike many adaptive sampling schemes43 in which the sampling process is controlled by the investigator, RDS enables the study subjects to control the sampling process. While this facilitates the recruitment process, it makes statistical inference more difficult. We found that estimates of syphilis seroprevalence were extremely sensitive to modeling assumptions. First, as recruitments between low-frequency groups are relatively rare, estimates of the recruitment rates may be biased. Smoothing these estimates using a statistical model can lead to different estimates. Although simulations and analytical results show that RDS-based estimates are unbiased in large populations, errors in RDS based estimates may be so high for small populations and\/or low frequencies of groups as to render the use of RDS impractical.33 Secondly, the estimated prevalence of syphilis was sensitive to the assumption of how inclusion probability depends on reported network size. Estimates of network size may well have been different had we asked \u201cHow many people do you currently know by name or street name that inject drugs?\u201d Estimating group-level network sizes is compromised by high variances, the small size of some of the subpopulations, and the poor ability of individuals to estimate the size of their personal networks.44 Although RDS controls for differences in network sizes, a sampling bias long known to be inherent in chain-referral samples, it is important that this information is as accurate as possible. It might be argued that prior to the advent of RDS, there was little incentive to accurately measure relative network sizes in epidemiological studies; given that this information plays a crucial role in the post-stratification process of RDS, we encourage further research to determine how best to collect this information accurately.\nRDS also has some inherent limitations in terms of inferences that can be drawn from the data45: it does not generate estimates of the absolute size of the population, only proportions, and it exploits social ties between individuals, limiting what one can conclude about sexual or drug-injecting networks from RDS data. Furthermore, without comparison of RDS to other types of sampling, we cannot conclude that obtaining a sample through RDS gives us a more representative sample than other methods. Nevertheless, RDS, or a modified version thereof, has the potential to efficiently recruit hidden populations such as IDUs, and creates avenues through which interventions can reach members of these populations. In the context of this study, prevention and treatment of syphilis is clearly an important public health concern.","keyphrases":["injection drug users","hiv and syphilis prevalence","respondent driven sampling."],"prmu":["P","P","R"]}
{"id":"Appl_Microbiol_Biotechnol-4-1-2270352","title":"Lactic acid production from lime-treated wheat straw by Bacillus coagulans: neutralization of acid by fed-batch addition of alkaline substrate\n","text":"Conventional processes for lignocellulose-to-organic acid conversion requires pretreatment, enzymatic hydrolysis, and microbial fermentation. In this study, lime-treated wheat straw was hydrolyzed and fermented simultaneously to lactic acid by an enzyme preparation and Bacillus coagulans DSM 2314. Decrease in pH because of lactic acid formation was partially adjusted by automatic addition of the alkaline substrate. After 55 h of incubation, the polymeric glucan, xylan, and arabinan present in the lime-treated straw were hydrolyzed for 55%, 75%, and 80%, respectively. Lactic acid (40.7 g\/l) indicated a fermentation efficiency of 81% and a chiral l(+)-lactic acid purity of 97.2%. In total, 711 g lactic acid was produced out of 2,706 g lime-treated straw, representing 43% of the overall theoretical maximum yield. Approximately half of the lactic acid produced was neutralized by fed-batch feeding of lime-treated straw, whereas the remaining half was neutralized during the batch phase with a Ca(OH)2 suspension. Of the lime added during the pretreatment of straw, 61% was used for the neutralization of lactic acid. This is the first demonstration of a process having a combined alkaline pretreatment of lignocellulosic biomass and pH control in fermentation resulting in a significant saving of lime consumption and avoiding the necessity to recycle lime.\nIntroduction\nLactic acid is used throughout the world in manufacturing of food, chemicals, and pharmaceutical products. Recently, there is a lot of interest in biodegradable poly-lactic acid, which is an alternative to petrochemically derived plastic (Drumright et al. 2000). Chiral pure lactic acid is produced commercially by microbial fermentation of the carbohydrates glucose, sucrose, lactose, and starch\/maltose derived from feedstocks such as beet sugar, molasses, whey, and barley malt (Narayanan et al. 2004). The choice of feedstock depends on its price, availability, and on the respective costs of lactic acid recovery and purification (Datta et al. 1995; Vaidya et al. 2005).\nAs an alternative to these traditional feedstocks, lignocellulosic biomass is an inexpensive and widely available renewable carbon source that has no competing food value. Lignocellulose consists primarily of cellulose and hemicellulose; polymers build up of mainly hexose sugars and pentose sugars, which are embedded in a matrix of the phenolic polymer lignin. The main pathway to derive fermentable sugars from lignocellulose is through enzymatic hydrolysis by cellulolytic and hemicellulolytic enzymes. A mechanical and chemical pretreatment of the lignocellulose is required to reduce particle size, to modify and\/or to remove the lignin, and with that to enhance the accessibility of the polysaccharides for enzymatic hydrolysis (Claassen et al. 1999). Various chemical pretreatments of biomass have been studied in research and development of lignocellulose-to-ethanol production technology (Mosier et al. 2005). One is the use of lime (calcium hydroxide) at relatively mild temperature conditions (Chang et al. 1998). Lime as a pretreatment agent has promising potential because it is inexpensive, safe, and its use hardly results in sugar degradation products such as furfural and hydroxymethyl furfural. Nevertheless, this alkaline pretreatment features a relatively high pH value (>10) of the treated biomass, and at these pH levels, the activity of common cellulolytic and xylanolytic enzymes, necessary for the depolymerization of (hemi)-cellulose, is negligible low. Therefore, lowering the pH is essential to achieve an efficient enzymatic hydrolysis of the polysaccharides. One approach to remove calcium hydroxide is by washing the lime-treated biomass before enzymatic hydrolysis (Chang et al. 1998); however, this leads to the use of high amounts of water. Another way to lower the pH of the pretreated material is by neutralizing calcium hydroxide with sulfuric acid. Yet, this results in the formation of the low value byproduct gypsum.\nAs an alternative improvement to these approaches, we propose to use the calcium hydroxide present in lime-treated biomass as neutralizing agent for organic acids produced in microbial fermentation processes. To examine this proposed concept, lime-treated wheat straw (LTWS) was added fed-batch-wise during a simultaneous saccharification and fermentation (SSF) process in a 20-l controlled stirred fermenter containing hydrolytic enzymes and Bacillus coagulans DSM 2314, a thermophilic bacterium capable to convert both hexoses and pentoses homofermentative to l(+)-lactic acid (Otto 2004; Patel et al. 2006). The objective of this research was to evaluate whether high alkaline-treated lignocellulosic biomass (without neutralization) can be used directly in a SSF process by (1) providing a carbon source for enzymatic hydrolysis and fermentation and (2) providing a source of alkali to control the pH in the fermentation process.\nMaterials and methods\nFeedstock and pretreatment\nWheat straw was selected as a lignocellulose model feedstock and was purchased from a farm in the Northeast of The Netherlands. The wheat straw was air dried (89.5% [w\/w] dry matter [DM]) and ground through a 2-mm screen. The lime pretreatment was performed by filling two 15-l mixers (Terlet, The Netherlands), both with 1,650\u00a0g ground wheat straw, 13\u00a0kg tap water, and 165\u00a0g calcium hydroxide. This wheat straw suspension was heated and kept at 85\u00b0C for 16\u00a0h under continuously stirring at 30\u00a0rpm. The LTWS suspension was subsequently cooled to 30\u00b0C, dehydrated by placing the LTWS in a cotton bag, and pressing the suspension using a manual piston press at pressure up to 9.7\u00a0kg\/m2. After dehydration, an amount of 11.45\u00a0kg LTWS with an average DM content of 27.0% (w\/w) and pH\u00a011.8 was obtained and served as substrate for further experiments. The chemical composition of LTWS was determined as described by van den Oever et al. (2003).\nEnzyme preparation\nThe enzyme preparation GC 220 (Genencor-Danisco, Rochester, USA) containing cellulase, cellobiase, and xylanase activity of 116, 215, and 677\u00a0U\/ml, respectively (Kabel et al. 2006), was used for this study. The preparation had a specific gravity of 1.2\u00a0g\/ml and contained 4.5\u00a0mg\/ml glucose, 2.9\u00a0mg\/ml mannose, and 0.8\u00a0mg\/ml galactose.\nMicroorganism and preculture\nThe bacterium B. coagulans strain DSM 2314 was used as the lactic acid-producing micro-organism. Bacterial cells were maintained in a 10% (w\/w) glycerol stock solution and stored at \u221280\u00b0C. Chemicals, unless indicated otherwise, were purchased from Merck (Darmstadt, Germany). Gelrite plates were prepared with a medium containing (per liter): glucose, 10\u00a0g; Gelrite, 20\u00a0g (Duchefa, Haarlem, The Netherlands); yeast extract, 10\u00a0g (Duchefa); (NH4)2HPO4, 2\u00a0g; (NH4)2SO4, 3.5\u00a0g; Bis\u2013Tris, 10\u00a0g (USB, Ohio, USA); MgCl2 6H2O, 0.02\u00a0g; and CaCl2.2H2O, 0.1\u00a0g. Glucose and Gelrite were dissolved in stock solution A (four times concentrated). The pH of this stock solution was adjusted to 6.4 with 2\u00a0M hydrochloric acid and autoclaved for 15\u00a0min at 125\u00b0C. The remaining nutrients were dissolved in stock solution B (1.33 times concentrated), which was also adjusted to pH\u00a06.4 with 2\u00a0M hydrochloric acid but was filter sterilized (cellulose acetate filter with pore size of 0.2\u00a0\u03bcm, Minisart, Sartorius). After sterilization, the medium was prepared by combining stock solutions A and B and Gelrite plates were poured. The bacteria were cultivated on Gelrite plates for 48\u00a0h at 50\u00b0C.\nAn isolated colony was used to inoculate a 100-ml broth with similar composition and preparation as described above but without the addition of Gelrite. The culture was incubated statically for 24\u00a0h at 50\u00b0C and functioned as the inoculum for a 1,400-ml broth. This culture was incubated also statically for 12\u00a0h at 50\u00b0C and served as a 10% (v\/v) preculture for the SSF experiments.\nSimultaneous saccharification and fermentation\nThe SSF of LTWS was carried out in a 20-l fermenter (Applikon, Schiedam, The Netherlands) with pH and temperature control (biocontroller ADI 1020). At the start of SSF, the fermenter was filled with 6.0\u00a0kg tap water and 1,400\u00a0g dehydrated LTWS (DM content of 27.0% [w\/w]). The following nutrients were then added to the LTWS suspension: yeast extract, 150\u00a0g (Duchefa); (NH4)2HPO4, 30\u00a0g; (NH4)2SO4, 52.5\u00a0g; MgCl2 6H2O, 0.3\u00a0g; and CaCl2 2H2O, 1.5\u00a0g. The LTWS suspension was then heated to 50\u00b0C, and the pH was adjusted to 6.0 with 101\u00a0g 3\u00a0M sulfuric acid (~30\u00a0g H2SO4).\nThe SSF process of LTWS to lactic acid consisted of three phases: (1) the prehydrolysis phase of preloaded LTWS, (2) the fed-batch phase with automatic feeding of LTWS from a screw feeder, and (3) the batch phase with pH control by a calcium hydroxide suspension and no LTWS feeding. A schematic representation of the experimental setup is shown in Fig.\u00a01. The prehydrolysis was initiated by the addition of 40\u00a0ml enzyme preparation (88\u00a0mg enzyme\/g DM substrate) to the LTWS suspension and was incubated for 2\u00a0h at 50\u00b0C under continuously stirring at 250\u00a0rpm. The fed-batch phase was initiated by the addition of 1,500-ml preculture of B. coagulans DSM 2314 to the fermenter. The lactic acid produced by the bacteria was neutralized by the automatic addition of 8,623\u00a0g dehydrated LTWS (DM of 27.0%) to the fermenter through a feeder (K-Tron Soder Feeders, Canada) and was regulated by the pH of the medium, which was set at 6.0. Throughout the fed-batch phase, an amount of 280\u00a0ml of enzyme preparation (total enzyme loading of 98\u00a0mg\/g DM substrate) was added proportional to the LTWS addition rate into the fermenter. During the batch phase, the pH was controlled at 6.0 by the addition of 20.0% (w\/v) calcium hydroxide suspension. Samples were withdrawn for DM, substrate, and (by)product analysis.\nFig.\u00a01Schematic representation of the simultaneous saccharification and fermentation of lime-treated wheat straw to lactic acid\nAnalytical methods\nFor the analysis of monomeric sugars, the fermentation broth samples were centrifuged (3\u00a0min at 17,400\u2009\u00d7\u2009g), and the pH of the supernatant was adjusted to 5.0 with barium carbonate using a pH indicator (Bromophenolblue) followed by filtration of the liquid. The analysis was performed by high-performance anion-exchange chromatography using a Carbopack PA1 column (column temperature of 30\u00b0C) and a pulsed amperometric detector (ED50; Dionex, Sunnyvale, CA, USA). Before injection, the system was equilibrated with 25.5\u00a0mM NaOH for 10\u00a0min at a flow rate of 1.0\u00a0ml\/min. For the separation of monomeric sugars, at injection, the mobile phase was shifted to deionized water for 30\u00a0min. Postcolumn addition of sodium hydroxide was used for detection of the neutral monomeric sugars.\nThe determination of soluble oligomeric sugars was performed by centrifugation for 5\u00a0min at 3,000\u00a0rpm (Centaur 2, Beun de Ronde, The Netherlands) of preweighed samples and freeze drying the supernatant overnight. Pellets were weighed and hydrolyzed with sulfuric acid, and neutral monomeric sugars were determined according to the method as described by van den Oever et al. (2003). For the calculations, an average molecular weight of oligomers from glucan and xylan of 166 and 132\u00a0g\/mol, respectively, were applied, resulting in a hydrolysis factor of 1.08 and 1.14, respectively.\nFor the analysis of insoluble polymeric sugars, samples of 25\u00a0g were centrifuged for 5\u00a0min at 3,000\u00a0rpm (Centaur 2, Beun de Ronde); the supernatant was removed, and the pellet was washed by resuspension in 25\u00a0ml fresh demineralized water followed by a centrifugation step of 5\u00a0min at 3,000\u00a0rpm (Centaur 2, Beun de Ronde). The sequence of resuspension and centrifugation was repeated three times. After the last removal of the supernatant, the pellets were freeze dried overnight. The pellets were weighed (values used for DM calculation), polymeric material was hydrolyzed with sulfuric acid, and neutral sugars were analyzed according to the method as described by van den Oever et al. (2003). For the calculations, a molecular weight of glucan and xylan of 162 and 132\u00a0g\/mol, respectively, were applied, resulting in a hydrolysis factor of polymer to monomer of 1.11 and 1.14, respectively.\nThe analysis of organic acids was performed by high-pressure liquid chromatography according to the procedure described by Maas et al. (2006).\nThe chiral purity (%) of lactic acid was determined by derivatization of all lactates using methanol, after which both enantiomers of methyl lactate were separated on a chiral gas chromatography column and detected using a flame ionization detector. The chiral purity was expressed as the area of the main enantiomer divided by the sum of areas of both enantiomers.\nCalculations\nThe theoretical maximum lactic acid (LAtheor. max. [g]) production was calculated according the following equation (Eq. 1):\nwhere DMsubstrate\u2009=\u2009the total dry matter of substrate LTWS (g), Fpolysacch.\u2009=\u2009fraction polysaccharides per substrate (g\/g), HFmonsacch.\/polysacch.\u2009=\u2009hydrolysis factor of polysaccharides, incorporation of water results in 1.11\u00a0g hexose from 1.00\u00a0g glucan and 1.14\u00a0g pentose from xylan and arabinan (g\/g), and FF\u2009=\u2009fermentation factor of 1.00\u00a0g lactic acid per gram of monomeric sugar.\nThe efficiency of the enzymatic hydrolysis (%, w\/w) was based on the amount of hydrolyzed polysaccharides (g; calculated by the difference between initial amounts and analyzed insoluble amounts) divided by the amount of polysaccharides (g) initially present in the substrate. The fermentation efficiency (%, w\/w) is expressed as the amount of lactic acid produced (g) divided by the amount of monomeric sugars consumed (g) by the bacteria. The overall efficiency of the SSF (%, w\/w) was calculated by the amount of lactic acid produced (g) divided by the theoretical maximum amount of lactic acid (g) determined as described in Eq. 1.\nResults\nSimultaneous saccharification and fermentation of LTWS to lactic acid\nThe polysaccharide composition of the LTWS consisted mainly of glucan, xylan, and arabinan of 33.0%, 19.0%, and 2.0% (w\/w), respectively, whereas the remaining mass constituted of lignin, ash, extractives, and uronic acids. Some of the soluble components in wheat straw were partially removed by the solid\/liquid separation (dehydration) of the LTWS. The focus of this study was on the conversion of glucan, xylan, and arabinan, which are the predominant polysaccharides present in LTWS and accounted for 99.8% (w\/w) of the total polymeric sugars. Previous work showed that the cellulase preparation GC 220, used for the saccharification of polysaccharides, functioned optimally at 50\u00b0C and pH\u00a05.0 (Maas et al., submitted for publication), whereas growth conditions for B. coagulans DSM 2314 were 54\u00b0C and pH\u00a06.5 (Otto 2004). In this study, both the enzymatic hydrolysis and the fermentation occurred simultaneously in the same reactor at compromising conditions, which were set at 50\u00b0C and pH\u00a06.0.\nThe SSF of LTWS to lactic acid was studied in a 20-l controlled stirred fermenter. Previous results showed that when this process was performed without a prehydrolysis of an initial amount of LTWS, the concentration of monomeric sugars was low and resulted, therefore, in relatively low lactic acid productivity. As a consequence, the fed-batch addition rate of the alkaline substrate to neutralize the produced lactic acid was low (results not shown). To start the fermentation with a substantial initial amount of fermentable sugars (>2\u00a0g\/l), a prehydrolysis of 378\u00a0g LTWS and enzyme preparation (88\u00a0mg per g DM LTWS) in approximately 6\u00a0l volume at pH\u00a06.0 for 2\u00a0h was introduced. This resulted in glucose, xylose, and arabinose concentrations of 2.0, 0.4, and 0.3\u00a0g\/l, respectively (Fig.\u00a03a).\nThe second phase (II) was initiated by introducing a 1,500-ml preculture of B. coagulans DSM 2314. A minor amount of lactic acid produced in the preculture caused a slight pH decrease and was automatically neutralized by the addition of LTWS (Fig.\u00a02a,b). After a lag phase of 4\u00a0h, the dissolved oxygen concentration decreased rapidly within 1\u00a0h from 100% to oxygen-limiting conditions of below 1% (results not shown), and lactic acid production started. At that moment, concentrations of glucose, xylose, and arabinose of 3.3, 0.7, and 0.3\u00a0g\/l, respectively, were present (Fig.\u00a03a). These sugars were consumed simultaneously where glucose was utilized faster than xylose and arabinose. Simultaneous with the consumption of these monomeric sugars, lactic acid was produced, which was neutralized by the automatic addition of alkaline LTWS. By the addition of alkaline substrate throughout the fed-batch phase, the pH was maintained accurately at 6.0\u2009\u00b1\u20090.1 (Fig.\u00a02a,b). At the end of phase II, a total amount of 10,023\u00a0g dehydrated LTWS (~2,706\u00a0g DM LTWS) and 320\u00a0ml of enzyme preparation was added to the fermenter. A lactic acid concentration of 20.5\u00a0g\/l supernatant was detected (Fig.\u00a03b), corresponding to a total of 342\u00a0g lactic acid. The chiral L(+) purity of lactic acid was determined at 99.4%, which is similar to that obtained with xylose as the sole carbon source (Otto 2004).\nFig.\u00a02Control of pH (a) during simultaneous saccharification and fermentation of lime-treated wheat straw by commercial enzyme preparation GC 220 and B. coagulans DSM 2314 (b). The areas between the dotted lines represent the prehydrolysis phase (I), the fed-batch phase (II) with pH control by addition of alkaline LTWS and enzymes, and the batch phase (III) with pH control by addition of Ca(OH)2 suspension. Extra enzyme preparation GC220 was added at the times indicated by the arrowsFig.\u00a03Profiles of glucose (empty square), xylose (empty diamonds), arabinose (empty triangles) (a) and lactic acid (filled diamonds) (b) in simultaneous saccharification and fermentation of lime-treated wheat straw by commercial enzyme preparation GC 220 and B. coagulans DSM 2314. The areas between the dotted lines represent the prehydrolysis phase (I), the fed-batch phase (II), and the batch phase (III). Extra enzyme preparation GC220 was added at the times indicated by the arrows\nAt the end of phase I, a low acetic acid concentration was detected in the medium, which increased to 1.5\u00a0g\/l throughout phase II but remained constant during phase III (results not shown). This indicates that acetic acid was most likely not a fermentation product formed by B. coagulans. Acetic acid can be released upon solubilization and hydrolysis of hemicellulose during chemical pretreatment (Palmqvist et al. 1999). By the dehydration procedure of the LTWS, part of the acetic acid was easily separated from the substrate by removing the press water. Apparently, a remaining amount of acetic acid was fed together with the substrate to the fermenter. Furthermore, minor traces of other organic acids such as succinic acid and formic acid (<0.5\u00a0g\/l) were detected in the fermentation broth.\nPhase III was initiated by changing the pH control from the addition of alkaline LTWS to a 20% (w\/v) calcium hydroxide suspension. To maintain the pH at 6.0, the addition of calcium hydroxide suspension occurred relatively fast but shifted, however, after a few hours to a lower addition rate indicating a decline of the volumetric lactic acid productivity (Figs.\u00a02b, 3b). To exclude limitation (e.g., by inactivation) of enzymes, an extra dosage of enzyme preparation (80\u00a0ml) was added to the fermenter after 23.5\u00a0h of incubation. This resulted immediately in a slight acceleration of the calcium hydroxide addition rate indicating an increased lactic acid productivity and limitation of enzymatic activity (Fig.\u00a03b). Nevertheless, after 29.7\u00a0h of incubation, a decline of the calcium hydroxide addition rate was observed again. Therefore, a second extra dosage of the enzyme preparation (240\u00a0ml) was added and resulted this time in a slight accumulation of glucose and xylose of 1.5 and 1.0\u00a0g\/l (Fig.\u00a03a), respectively, indicating that microbial conversion instead of enzymatic hydrolysis was rate limiting. After 32\u00a0h of incubation, a lactic acid concentration of 37.1\u00a0g\/l was obtained, with a chiral l(+)-lactic acid purity of 99.4%. Continuation of the SSF process to a total incubation period of 55\u00a0h resulted in a slightly increased lactic acid concentration of 40.7\u00a0g\/l supernatant (~37.8\u00a0g lactic acid\/kg fermentation broth) with an overall volumetric lactic acid productivity of 0.74\u00a0g l\u22121 h\u22121. At this stage, a chiral l(+)-lactic acid purity of 97.2% was analyzed. This slight decline in lactic acid purity is possibly a result of infection with other undesired lactic acid-producing microorganisms. Because the substrate used was not sterile and also the chemical pretreatment and fermentation occurred in an open system under nonsterile conditions, microbial contamination throughout the SSF process is possible.\nConversion efficiency\nThe efficiency of the enzymatic hydrolysis of the polymeric material present in LTWS is shown in Fig.\u00a04. The insoluble polymeric fraction was determined at various time points throughout the SSF experiment. At the end of the prehydrolysis (2\u00a0h) of 378\u00a0g LTWS, 36% of the insoluble glucan (Fig.\u00a04a), 55% of xylan (Fig.\u00a04b), and 62% of arabinan (Fig.\u00a04c) were converted to soluble saccharides including monomeric sugars and oligomeric sugars. After the fed-batch phase (13\u00a0h), 2,706\u00a0g LTWS was added and resulted in a conversion of 42% of glucan, 57% of xylan, and 63% of arabinan to products including soluble saccharides and lactic acid. Between 13 and 32\u00a0h of incubation, further hydrolysis of the polymeric sugars was observed. However, during the last 23\u00a0h of the SSF, minor hydrolysis of the polysaccharides occurred, and this corresponded with the decline in lactic acid productivity during this phase. After 55\u00a0h, 398\u00a0g of glucan, 130\u00a0g of xylan, and 11\u00a0g of arabinan was still present as insoluble polymeric material. With these values, the hydrolysis efficiency of the initial glucan, xylan, and arabinan present in LTWS were calculated as 55%, 75%, and 80%, respectively.\nFig.\u00a04Insoluble fraction (empty bars) and hydrolyzed soluble fraction (filled bars) (g; calculated by the difference between initial amounts and analyzed insoluble amounts) of the polysaccharide glucan (a), xylan (b), and arabinan (c) at various time points during the simultaneous saccharification and fermentation of lime-treated wheat straw. The figure represents also the percentage of polysaccharide hydrolyzed into soluble products (filled triangles). The error bars denote the deviation of duplicate analysis\nThe monomeric sugars, derived from the LTWS, were partly converted to lactic acid (711\u00a0g) by B. coagulans and accounted for 81% (w\/w) of the theoretical maximum, indicating the formation of other products such as microbial biomass and carbon dioxide. An overall conversion yield of 43% (w\/w) of the theoretical maximum was calculated according to Eq. 1. The fate of polysaccharides initially present in LTWS after 55\u00a0h of incubation is shown in Table\u00a01. A part of the polysaccharides present in LTWS remained as insoluble polysaccharides (37% w\/w), whereas a minor part was converted into soluble oligomeric (5% w\/w) and monomeric (3% w\/w) sugars. Another part of the initial polysaccharides present in the LTWS was not recovered in the form of saccharides or lactic acid and was therefore ascribed as \u2018unaccounted.\u2019\nTable\u00a01Fate of polysaccharidesa initially present in lime-treated wheat straw after 55\u00a0h of simultaneous saccharification and fermentationFractionPercentage (% w\/w)Polysaccharides (insoluble)b37Oligosaccharides (soluble)5Monosaccharides (soluble)3Lactic acid (soluble)43Unaccounted (insoluble\/soluble)c13Presented values are averages based on duplicate analytical measurements.aTotal of glucan, xylan, and arabinanbPart of the initial polysaccharides remained present as insoluble polysaccharides.cPart of the initial polysaccharides was not recovered and therefore denoted as \u2018unaccounted.\u2019\nNeutralization of acid by alkaline substrate\nThe lactic acid produced (342\u00a0g) during the fed-batch phase (II) was neutralized with alkaline-pretreated wheat straw. During this phase, an amount of 2,328\u00a0g LTWS was added to the fermenter. Together with this substrate, an amount of 230\u00a0g calcium hydroxide was added to the fermenter and accounted for a ratio of 0.67\u00a0g calcium hydroxide per gram of lactic acid. The lactic acid (369\u00a0g) produced during the batch phase (III) was neutralized with 163\u00a0g calcium hydroxide resulting in a ratio of 0.44\u00a0g lactic acid per gram calcium hydroxide.\nDiscussion\nLignocellulosic feedstocks are considered as potential attractive substrates for the production of bulk chemicals. Pretreatment of biomass is required to break open the lignocellulosic matrix, and an enzymatic hydrolysis is necessary for the hydrolysis of polymeric carbohydrates. The lime pretreatment has proven to enhance enzymatic digestibility of the polysaccharides present in lignocelluloses (Chang et al. 1998; Kaar and Holtzapple 2000) and results, in comparison to other pretreatment routes, in minor inhibitor formation. However, before the enzymatic hydrolysis, it is essential to adjust the pH to a level optimal for enzymatic activity. In this study, the reduction in pH by washing or neutralization was omitted by using the alkaline character of LTWS to neutralize lactic acid produced by microbial fermentation during a SSF process.\nThe results showed that the largest part of the polysaccharides in LTWS was converted enzymatically and the resulting sugars were fermented simultaneously to mainly lactic acid by B. coagulans DSM 2314. Between 10 and 30\u00a0h of incubation, the bacteria utilized the monomeric sugars, as soon as they appeared in the medium, resulting in relatively low monomeric sugar concentrations (<2\u00a0g\/l). This indicates that throughout this period, the enzymatic hydrolysis was the rate-controlling step. The highest lactic acid productivity was observed during the fed-batch phase and the initial hours of the batch phase and declined rapidly after approximately 20\u00a0h of incubation, as shown in Fig.\u00a03b. An extra addition of enzyme preparation showed a slight improvement of the volumetric lactic acid productivity but shifted within a few hours again to a relatively low production rate. A second extra enzyme addition did not affect the lactic acid productivity significantly (Fig.\u00a03b). This addition of new enzymes resulted in a modest liberation of hemicellulose sugars (xylose, arabinose), but no further hydrolysis of glucan occurred. This shows that the remaining glucan was too recalcitrant or not accessible for further hydrolysis, resulting in decreasing lactic acid productivity. Another possible explanation of the decreased lactic acid productivity is the inhibition of enzymes and\/or bacteria by the increasing lactic acid concentration.\nA lactic acid concentration of 40.7\u00a0g\/l supernatant (~37.8\u00a0g lactic acid\/kg fermentation broth) with a relatively high chiral purity was determined after 55\u00a0h of incubation, corresponding to an overall lactic acid yield of 43% of the theoretical maximum. Moreover, the efficiencies of the enzymatic saccharification and the fermentation were both determined. These calculations showed that, based on residue analysis, at the end of the SSF process (55\u00a0h), 55% of the glucan, 75% of the xylan, and 80% of the arabinan present in LTWS was enzymatically hydrolyzed, which agree well with previously obtained results from experiments aiming to convert LTWS to ethanol. To improve the yield, it is necessary to decrease the recalcitrance or improve the accessibility of polymeric sugars in the LTWS by optimization of the pretreatment procedure. The concentrations of soluble monosaccharides and oligosaccharides in the medium were relatively low, which can be expected in a SSF process. A fermentation yield of 81% was determined (related to the amount of monosaccharides released from the LTWS) and is slightly better than the results obtained by Otto (2004) who reported the production of 35\u00a0g\/l lactic acid from 50\u00a0g\/l xylose as the sole carbon source. Because no other soluble fermentation products were detected, the remaining 19% of the LTWS-derived monomeric sugars were most presumably converted to bacterial biomass and some carbon dioxide during the aerobic part of the fermentation. Several process parameters can be listed for enhancement of the overall lactic acid yield and productivity such as improving the accessibility of polysaccharides by a more severe lime pretreatment, enzyme dosage, type of enzymes, B. coagulans strain, size and growth phase of inoculum, pH gradient in SSF, and in situ product removal of lactic acid. These issues will be subject to further studies.\nDuring the fed-batch phase (II), it was possible to counterbalance the pH decrease caused by lactic acid production by the addition of the alkaline feedstock. This suggests that it is possible to combine lime treatment with the production of other organic acids from lignocellulosic biomass. Throughout this phase, the ratio of calcium hydroxide in LTWS added per produced lactic acid was determined at 0.67\u00a0g\/g. The theoretical stoichiometric neutralization of 1.00\u00a0g lactic acid requires 0.41\u00a0g calcium hydroxide. Therefore, only 61% of the calcium hydroxide initially added to the wheat straw was used for lactic acid neutralization. On the other hand, throughout the batch phase (III), an alkaline\/acid ratio of 0.44\u00a0g\/g was calculated corresponding to 93% of the added calcium hydroxide suspension used for lactic acid neutralization. The low efficiency of the calcium hydroxide added with the LTWS for lactic acid neutralization during phase II has three possible explanations. First, part of the calcium hydroxide could have been used during the chemical pretreatment of the wheat straw such as the neutralization of acetic acid or other organic acids and\/or irreversible binding to the lignin. Second, the calcium hydroxide might be released slowly from the insoluble wheat straw fibers and could therefore partly have been used for lactic acid neutralization in the fed-batch phase. Finally, besides lactic acid production, other acidification reactions could have contributed to the decrease in pH and therefore the demand of alkaline substrate, for instance, the decrease in pH caused by the consumption of ammonium as the nitrogen source by microorganisms (Guebel et al. 1992).\nThe results in this paper show that it is possible to use lignocellulosic materials for the production of lactic acid. Lignocellulosic biomass is a relatively inexpensive substrate, and this affects feedstock costs for lactic acid production positively. Nevertheless, in comparison to the traditional relatively \u2018clean\u2019 feedstocks with a well-defined composition, using heterogenic lignocellulosic substrates will require a more intensified downstream processing (DSP) to recover and purify the lactic acid from the complex fermentation broth. The costs of feedstock materials and operational costs of the DSP contribute considerably to the overall production costs of lactic acid (\u00c5kerberg and Zacchi 2000). Whether the cost decrease in using lignocellulosic feedstocks outweighs the potential increasing costs of DSP was not analyzed at the moment.\nIn summary, LTWS was converted into l(+)-lactic acid by B. coagulans throughout a SSF process at a 20-l bench scale. The pentose and hexose sugars derived from the polymeric material were utilized simultaneously by B. coagulans resulting in a final lactic acid concentration of 40.7\u00a0g\/l supernatant, which accounted for 43% (w\/w) of the theoretical yield. To our knowledge, this is the first paper demonstrating a process having a combined alkaline pretreatment of lignocellulosic biomass and pH control in organic acid fermentation resulting in a significant saving of lime consumption and avoiding the necessity to recycle lime.","keyphrases":["lactic acid","lime-treated wheat straw","ph control","calcium hydroxide","simultaneous saccharification and fermentation"],"prmu":["P","P","P","P","P"]}
{"id":"Clin_Oral_Investig-3-1-2099161","title":"Three-year survival of single- and two-surface ART restorations in a high-caries child population\n","text":"The aim of this study was to evaluate the survival of single- and two-surface atraumatic restorative treatment (ART) restorations in the primary and permanent dentitions of children from a high-caries population, in a field setting. The study was conducted in the rainforest of Suriname, South America. ART restorations, made by four Dutch dentists, were evaluated after 6 months, 1, 2, and 3 years. Four hundred seventy-five ART restorations were placed in the primary dentition and 54 in first permanent molars of 194 children (mean age 6.09 \u00b1 0.48 years). Three-year cumulative survivals of single- and two-surface ART restorations in the primary dentition were 43.4 and 12.2%, respectively. Main failure characteristics were gross marginal defects and total or partial losses. Three-year cumulative survival for single-surface ART restorations in the permanent dentition was 29.6%. Main failure characteristics were secondary caries and gross marginal defects. An operator effect was found only for two-surface restorations. The results show extremely low survival rates for single- and two-surface ART restorations in the primary and permanent dentitions. The variable success for ART may initiate further discussion about alternative treatment strategies, especially in those situations where choices have to be made with respect to a well-balanced, cost-effective package of basic oral health care.\nIntroduction\nThe concept of minimal invasive dentistry has evolved as a consequence of an increased understanding of caries and the development of adhesive restorative materials [27]. Within this concept, prevention and hard tissue preservation are the primary goals, and dentists are encouraged to prefer a more conservative and biological approach rather than a surgical approach, although the latter is sometimes unavoidable. The atraumatic restorative treatment (ART) technique is part of a minimal invasive approach and, as such, a technique that meets the specific goals mentioned above. In brief, with ART, soft demineralized carious tooth tissue is removed using hand instruments only, followed by restoration of the tooth with an adhesive restorative material, often glass ionomer cement [4, 7]. Because neither electricity nor running water is required for this treatment approach, ART can be applied in almost any setting. Although initially developed to provide restorative dental treatment in outreach or rural areas, ART or modified ART techniques are increasingly introduced into dental clinics in industrialized countries [1, 11, 14].\nSince its introduction in the mid-1980s, ART has been evaluated in several community field trials. These studies served mainly to obtain information on technical aspects of the process, handling characteristics of the restorative material, and on the survival of the restorations. They led to improvement of the technique [20] and to the development of new, more appropriate glass ionomer restoration materials, especially for ART purposes.\nStudies focussing on the survival of ART restorations have shown that the ART approach is very successful in restoring single-surface dentine lesions in the permanent dentition: 3-year survival rates of 71\u201392% have been reported [5, 6, 9, 10, 12, 20]. Regarding the survival rates of ART restorations in the primary dentition, only a few field studies were performed. They showed acceptable survival rates (65\u201396.7%) for single-surface ART restorations, but generally low success rates (31\u201376.1%) for multi-surface ART restorations, even with the newer glass ionomer materials [2, 3, 11\u201313, 19, 23, 24, 28]. Although its performance under multisurface conditions is disappointing, ART is considered a valuable approach towards the treatment of dental caries. The use of ART has resulted in the retention of many teeth that would otherwise have been extracted in a later stage. Nevertheless, there still remain some controversies towards the technique, presumably based on the inconsistency in survival results. Moreover, a recent study, investigating the influence of dental treatment on the oral health of a Surinamese child population, concluded that performing ART restorations only, did not contribute significantly to an improvement of the oral health, suggesting that ART alone is not a sufficient solution in the battle against dental decay (van Gemert-Schriks et al., submitted, 2007).\nFrencken et al. [8] described comprehensively that ART should be part of a basic package of oral care in which prevention and urgent care are also represented. However, within this package, these three components should be geared to one another as much as possible and the individual effects of all three components must be sufficient and beneficial under different circumstances. When the success of either component, particularly ART, cannot be guaranteed, its contribution in the package should be reduced. Thus, the evaluation of ART in different countries or communities, among different kinds of caries-risk populations and under diverging conditions remains useful. Therefore, the aim of this study is to evaluate the survival of both single- and two-surface ART restorations in the primary and permanent dentitions of children from a high-caries population in a field setting on a longitudinal base.\nMaterials and methods\nThis cohort study was conducted in the rainforest of Suriname, South America. It was part of a large-scale project investigating the influence of dental treatment on the oral health of children (van Gemert-Schriks et al., submitted, 2007). Within the scope of that particular project, 380 6-year-old children were divided randomly among four different treatment groups. Material presented in the current article concerns only those children who received restorative treatment, according to the ART method, either in their primary or permanent dentitions.\nThe restorative treatments were performed in accordance with the ART guidelines [4, 7] and took place in empty classrooms where four children were treated at the same time. Ketac-Molar (3M-ESPE\u00ae) was used as the restorative material of choice. The treatments were carried out by four Dutch dentists who were trained in ART during a 1-week ART course and by using ART in children from their own practices, for a period of 3\u00a0months, before the start of the treatment phase of the study. They were assisted by six Surinamese health care assistants from the Medical Mission who completed an ART course supplemented with some basic dental knowledge. The dentists were asked to note any contamination with blood and\/or saliva during the restoration of the cavity. Furthermore, the presence or absence of adjacent teeth was noted. During the treatment, one of the authors (MGS), who was not involved in the treatment phase, observed and classified the overall behavior of the child, based on a modified Venham scale [23, 29]. Before the study, this observer was trained in using the Venham behavior scale by scoring 42 videotapes of children in a dental situation. These observations were compared to the consensus score of two calibrated observers. This comparison resulted in a Cohen\u2019s Kappa of 0.87, implying an excellent agreement.\nRestorations were not assessed at the time of placement (T0). The children were revisited for evaluation of the ART restorations 6\u00a0months (T1), 1\u00a0year (T2), 2\u00a0years (T3), and 3\u00a0years (T4) after the initial treatment. The same author and dentist mentioned above (MGS), evaluated the restorations according to the ART criteria (Table\u00a01) using a CPITN probe, a mouth mirror and a headlamp. Before the study, this person was calibrated against a \u201cgold standard\u201d (Kappa 0.94). This gold standard was achieved by the consensus of two experienced dentists during the assessment of 24 extracted molars with ART restorations. Restorations scored code 00 or 10 were considered successful, codes 11\u201340 were classified as failures, and codes 50\u201390 were assigned in case the tooth was unavailable for evaluation. If a tooth or restoration showed multiple defects, a marginal defect dominated an over- or underfilled restoration (10, 11\u2009>\u200912, 13), secondary caries dominated a marginal defect (20, 21\u2009>\u200910, 11), absence of a restoration dominated secondary caries (30\u2009>\u200920, 21), and an overfilled cavity dominated an underfilled cavity (13\u2009>\u200912).\nTable\u00a01Evaluation criteria for the ART restorationsCodeEvaluation characteristics00Restoration present, correct10Restoration present, slight marginal defect\/wear of surface (<0.5\u00a0mm). No repair needed.11Restoration present, gross marginal defect\/wear of surface (>0.5\u00a0mm). Repair needed.12Restoration present, underfilled (>0.5\u00a0mm). Repair needed.13Restoration present, overfilled (>0.5\u00a0mm). Repair needed.20Secondary caries, discoloration in depth, surface hard and intact, caries within dentin. Repair needed.21Secondary caries, surface defect, caries within dentin. Repair needed.30Restoration not present, bulk fracture, moving or partial lost. Repair needed.40Inflammation of the pulp; signs of dentogenic infection (abscesses, fistulae, pain complaints). Restoration might still be in situ. Extraction needed.50Tooth not present because of extraction60Tooth not present because of shedding70Tooth not present because of extraction or shedding90Patient not present\nStatistical analysis\nStatistical analyses were performed using SPSS for Windows, version 12.0.1 (SPSS, Chicago, USA). All significant differences were detected at a 95% confidence level.\nKaplan\u2013Meier survival analyses were performed on the censored data of both single- and two-surface restorations. The significance of differences between survival curves was determined with log-rank tests. Possible confounding variables were taken into account using a Cox regression analysis.\nResults\nAs stated in the \u201cMaterials and methods\u201d section, the children in this study were derived from a larger study population of children participating in another project. The overall caries prevalence, expressed in terms of decayed, missing and filled surfaces (dmfs) among that group of children, was 11.51 (SD 10.5; range 0\u201353) in the primary dentition and 0.20 (SD 0.62; range 0\u20135) in the permanent dentition. According to the standards of the World Health Organisation [17], this denotes a high-caries child population based on the caries prevalence in the primary dentition. Within the larger group, 194 children (mean age 6.09\u2009\u00b1\u20090.48\u00a0years) received ART restorations in either their primary or permanent teeth, or both. Only these children were included in the current study. Their baseline caries prevalence was 12.75 (SD 9.88; range 0\u201353) in the primary dentition and 0.23 (SD 0.67; range 0\u20135) in the permanent dentition.\nAt baseline (T0), 475 ART restorations were placed in the primary dentition (mainly first and second molars) and 54 in the first permanent molars (predominantly mandibular). Table\u00a02 presents data for the ART restorations, performed at baseline. A Mann\u2013Whitney U test showed that children who received two-surface restorations scored higher on the Venham behavior scale (p\u2009=\u20090.005) than children that received single-surface restorations, in the primary dentition. Furthermore, dentists reported more contamination (chi-square\u2009=\u200925.02, df\u2009=\u20091, p\u2009<\u20090.001) when placing two-surface restorations than single-surface restorations.\nTable\u00a02Baseline data for the ART restorations\u00a0Primary dentitionPermanent dentitionNumber of filled surfaces121Number of restorations13334254Number of children (N)6114734Mean number of restorations per child (SD; range)3.50 (1.61; 1\u20137)3.64 (1.73; 1\u20138)2.07 (0.97; 1\u20134)Dentist143 (32.3%)74 (21.6%)12 (22.2%)241 (30.8%)84 (24.6%)16 (29.6%)334 (25.6%)89 (26.0%)7 (13.0%)415 (11.3%)a95 (27.8%)19 (35.2%)Adjacent tooth presentYes117 (88.0%)303 (88.6%)45 (83.3%)No16 (12.0%)39 (11.4%)9 (16.7%)Contamination blood\/salivaYes13 (9.8%)110 (32.2%)a5 (9.3%)No120 (90.2%)232 (67.8%)49 (90.7%)Venham behavior score050 (37.6%)78 (22.8%)8 (14.8%)144 (33.1%)137 (40.1%)27 (50.0%)226 (19.5%)82 (24.0%)13 (24.1%)313 (9.8%)33 (9.6%)6 (11.1%)4\u201312 (3.5%)a\u20135\u2013\u2013\u2013SD Standard deviationaStatistical significant difference at p\u2009=\u20090.005.\nThe lost-to-follow-up percentage of the restorations originally placed was 4.63%. After 3\u00a0years, the cumulative survival of the single-surface ART restorations in the primary dentition was 43.4% (standard error (SE) 10.9%). For the two-surface restorations, a cumulative survival of 12.2% (SE 2.99%) was observed. The survival curves, with censored data, are presented in Fig.\u00a01a and b. The cumulative survival of the single-surface ART restorations in the permanent dentition was 29.6% (SE 8.2%) after 3\u00a0years (Fig.\u00a02).\nFig.\u00a01a Survival curve single surface ART restorations, primary dentition. b Survival curve multisurface ART restorations, primary dentitionFig.\u00a02Survival curve single-surface ART restorations, permanent dentition\nTable\u00a03 represents the failure characteristics for the restorations in both primary and permanent dentitions at 3\u00a0years. The main failure characteristics of both single- and two-surface ART restorations in the primary dentition were gross marginal defects (score 11) and total or partial losses (score 30). For restorations in the permanent dentition, the main failure characteristics were secondary caries (score 21) and gross marginal defects (score 11).\nTable\u00a03Failure characteristics for the ART restorations at 3\u00a0years\u00a0Primary dentitionPermanent dentition1 surface2 surface1 surfaceRestorations baseline (N)13334254Failures (N)4225132Failure scoreGross marginal defect (11)21 (15.8%)86 (25.1%)13 (24.1%)Restoration present, underfilled (12)1 (0.8%)9 (2.6%)\u2013Restoration present, overfilled (13)2 (1.5%)11 (3.2%)2 (3.7%)Sec. caries, discoloration (20)\u2013\u2013\u2013Sec. caries, surface defect (21)5 (3.8%)1 (0.3%)14 (25.9%)Total or partial loss (30)13 (9.8%)120 (35.1%)3 (5.6%)Pulpal inflammation (40)\u201324 (7.0%)\u2013Restoration missing, extracted (50)\u2013\u2013\u2013Scores 60\u201390 were not included (censored data)\nA log-rank test indicated that there were no statistically significant differences in survival times between the four dentists regarding single-surface restorations in both primary and permanent teeth. However, regarding the two-surface restorations in the primary dentition, statistically significant differences between the four dentists appeared (log-rank statistic 11.7, df\u2009=\u20093, p\u2009=\u20090.009). The separate survival curves are presented in Fig.\u00a03.\nFig.\u00a03Survival curves per dentist, multi-surface ART restorations primary dentition\nTo detect any confounding variables on the survival of the ART restorations, a Cox regression analysis was performed. No significant relation could be found, indicating that neither the presence or absence of an adjacent tooth, nor contamination with blood and\/or saliva, nor the behavior of the child during the restorative phase of the treatment had an influence on the 3-year survival of the restorations in the primary dentition. No effect also could be found regarding the number of restorations per child.\nNeither of these variables had an effect on the survival rates in the permanent dentition, except for the presence of adjacent teeth. Restorations in teeth where no adjacent tooth was present were found to be more likely to fail (hazard ratio\u2009=\u20096.53, 95% CI 2.66\u201316.02, p\u2009<\u20090.001).\nDiscussion\nIn contrast with other studies, the results of this study show extremely low survival rates for both single- and two-surface ART restorations in the primary and permanent dentitions. An operator effect was observed for two-surface restorations only. Neither the behavior of the child during restoration, and the number of restorations per child, nor the contamination of preparations with blood or saliva had a significant influence on the survival of the restorations in this study.\nThis field study was performed correctly and the statistical power was sufficiently high to detect at least medium effects. However, because it was part of a large-scale randomized controlled clinical trial, no comprehensive criteria were formulated beforehand regarding, for example, the number of restorations per patient, and the location and the size of the cavities. This aspect is inherent to many cohort studies and it does not imply an inferior study quality, but it limits a meaningful comparison with other survival studies.\nAlthough all possible efforts were exercised to trace the participating children over the evaluation period, 22 restorations (4.63%, eight children), all in primary molars, could not be evaluated at any of the recall visits. Either the children did not show up, or the teeth concerned had exfoliated before the first evaluation. These restorations were regarded as missing data and, therefore, excluded from further analysis. Twenty-six restorations (5.47%, 21 children) were \u201clost\u201d for evaluation because the teeth either exfoliated or the child moved to another district during the course of the study, but after the first evaluation. These restorations (scores 60\u201390) were treated as censored data and not as true failures because they survived up to a certain moment.\nMany causative factors could be suggested that might explain the failure of the ART restorations, such as secondary caries, cervical margin gaps, material properties, and field conditions (outside temperature, atmospheric humidity). However, many other ART studies face these or comparable problems and, therefore, these factors cannot sufficiently explain the extremely low survival rates found in this particular study. The operator difference for the survival rates of the two-surface restorations was not unique, and not a sufficient explanation for the disappointing survival results. Operator effects are often found in ART studies [4, 9, 15, 21, 26] and, as in every profession, there will always be individual differences in technical skills. The finding that the absence of an adjacent tooth was related to a lower 3-year survival of single-surface ART restorations in permanent molars could not be explained. One can only speculate about possible reasons for this relationship, such as that these freestanding molars experience larger occlusal forces.\nA possible influence of the relatively high caries prevalence on the survival of the restorations could be hypothesized, but is very doubtful. A study in Indonesia, where the child population exhibited a much higher caries prevalence, also found disappointing survival rates for two-surface ART restorations [28], but these rates were not as extreme as those found in the current study. The survival rates for single-surface ART restorations, derived from other earlier cited studies, were all very promising regardless of the caries profile of the study populations. Furthermore, no effect on the survival rates of the restorations was found when the number of restorations per child was included in the analysis.\nThe ART protocol prescribes not to eat or drink within at least 1\u00a0h after the completion of the restorative treatment [7]. The children in the current study were not supervised after they received restorative treatment and consequently, their food intake could not be controlled. Future studies should take this aspect into account.\nOther patient-related factors that may influence the survival of the restorations are the behavior and saliva flow of the child. The survival of the ART restorations in this study was analyzed at the restoration level. This method requires independency of the restoration data and, with respect to the mentioned patient-related possible bias, this assumption could not be guaranteed. To control for this lack of independency, the survival analyses also were performed at the patient level, including only one randomly selected restoration per child. These analyses did not render higher survival rates.\nThe predominant failure characteristics for both single- and two-surface ART restorations in the primary dentition were gross marginal defects and total or partial losses. This agrees with previous studies concerning the survival of ART restorations in the primary dentition [6, 13, 25, 26]. Gross marginal defects could be induced by occlusal forces or insufficient wear resistance of the restorative material. Ketac-Molar was specifically developed for ART purposes [13], and it has shown excellent results for posterior restorations in the primary dentition [16, 22]. Glass ionomer restorations can be dislodged for a number of reasons, such as insufficient cleaning and conditioning of the cavity, and improper mixing of the material. None of these conditions was recorded at the time the tooth was restored. However, all dentists and chair-side assistants followed the ART guidelines and the manufacturer\u2019s instructions as much as possible under the given circumstances.\nThe main reasons for failure of the single-surface restorations in the permanent dentition were gross marginal defects and secondary caries. This latter finding is somewhat surprising and contrasts with earlier ART studies [5, 6, 10, 26]. Glass ionomer cement has been the restorative material of choice for the ART technique, based mainly on its fluoride releasing and, thus, caries-preventive properties [7]. Many studies underline these characteristics of glass ionomer [18, 26, 30, 31].\nThe extremely low survival of the ART restorations observed in this study remains unexplained. Circumstances that were not recognized as possible interfering factors at the start of the study might have played an important role, including cultural and seasonal dietary influences. People living in the rainforest of Suriname eat seasonal fruits such as mangos and fruits of the fiber palm (Awarra). In particular, the latter may influence the survival of the restorations, given the frequency and method in which they are consumed. The authors have seen unusual wear patterns, also in adult dentitions, which might have been caused by excessive consumption of Awarras. A possible causality between these dietary habits and the survival of the ART restorations can only be disclosed by future controlled studies.\nAlthough previous studies have suggested that ART should not be considered as a routine procedure to restore multisurface cavities [13, 24], based on the results of this study, even the ART restoration of single-surface cavities might be reconsidered. This study underlines the inconsistency and variation in the success of the treatment. Apparently, certain conditions must be fulfilled to make ART successful. These conditions can be approached, but not always achieved, under all circumstances.\nConclusion\nThe uncertain predictability for the success of ART may introduce further discussion about alternative treatment strategies, especially in those situations where choices have to be made with respect to a well-balanced, cost-effective package of basic oral health care. To gain insight into factors determining the cumulative success rate of ART restorations, future studies should focus in more detail on variables that could possibly contribute to the failure of restorations.","keyphrases":["art","primary dentition","cumulative survival","oral health"],"prmu":["P","P","P","P"]}
{"id":"Reprod_Biol_Endocrinol-1-_-153492","title":"Luteal regression: a redefinition of the terms\n","text":"The corpus luteum is a transient endocrine gland that is specialized for the production of progesterone and that plays a critical role in the establishment and maintenance of pregnancy. The life span of the corpus luteum varies between species and, within a species, can be dramatically altered by events such as mating or pregnancy. Regardless of the duration of its life span, the corpus luteum eventually enters a dynamic regression process during which it loses the capacity to produce progesterone and undergoes structural involution.\nThe overall process of luteal regression has been referred to by a variety of terms over the last several decades. In Table 1, we show the results of a cursory MEDLINE\/PubMed search on luteal regression performed in November of 2002, which reveals that the most frequently utilized term for this process is \"luteolysis,\" followed by \"luteal regression,\" and then, to a lesser extent, by \"functional luteolysis\" and \"structural luteolysis.\" Other terms, such as \"luteal involution,\" \"functional luteal regression,\" or \"structural luteal regression\" lag far behind in usage. The necessity of using several keywords to search for information on luteal regression is illustrated by the fact that in our sample MEDLINE\/PubMed search, only 47% of the manuscripts retrieved using the keyword \"luteal regression\" were also retrieved when using the keyword \"luteolysis.\" This inconsistency in terminology can result in serious under-retrieval and under-citation of papers using less popular keywords.\nTable 1\nResults of a cursory MEDLINE\/PubMed search performed on November 2002 using the terms as listed.\nRank\nTerms\nTotal Number of Articles Retrieved\nRetrieval Period\nNumber of Articles Retrieved for Period 2001\u20132002\n1\n\"luteolysis\"\n1251\n1967\u20132002\n96\n2\n\"luteal regression\"\n403\n1966\u20132002\n35\n3\n\"functional luteolysis\"\n82\n1973\u20132002\n5\n4\n\"structural luteolysis\"\n72\n1969\u20132002\n14\n5\n\"luteal involution\"\n41\n1978\u20132002\n8\n6\n\"functional luteal regression\"\n10\n1984\u20132002\n0\n7\n\"structural luteal regression\"\n5\n1988\u20132002\n1\nTo quote Rossdale and Cox in their communication entitled Terminology: a mark of scientific progress [1]: \"Scientific terminology must be as exact as is possible within the state of knowledge available.\" As our knowledge of the molecular mechanisms participating in the process of luteal regression evolves, some of the commonly used terms cited above have been rendered obsolete, inappropriate, and even incorrect. The foremost of these is \"luteolysis,\" still one of the most common terms used to define the process of luteal regression, most probably because it is listed among Medical Subject Headings (MeSH), and, therefore, used most frequently. MeSH is the U.S. National Library of Medicine's controlled vocabulary used for indexing articles in PubMed. MeSH terminology provides a consistent way to retrieve information that may use different terminology for the same concepts. In MeSH, \"luteolysis\" is defined as \"degradation of corpus luteum\" and further described as \"...characterized by the involution and cessation of its endocrine function.\" From a cell biology point of view, \"lysis\" means \"rupture of cell plasma membrane, leading to the release of cytoplasm and the death of the cell\" [2]. If interpreted with strict accordance to the Latin, lysis refers to \"disintegration,\" as recently discussed by Davis and Rueda [3]. Over the last decade, it has become clear that the involution of the corpus luteum is associated with a phenomenon of programmed cell death or apoptosis [4]. Because apoptosis is an organized process that does not involve a major inflammatory response, and because the majority of cells are removed prior to rupture, the term \"lysis\" when applied to luteal regression is largely inaccurate. Therefore, while the term luteolysis remains very popular \u2013 1251 manuscripts were retrieved using the word \"luteolysis\" and only 403 were retrieved using the terms \"luteal regression\" in the MEDLINE\/PubMed search (see Table 1) \u2013 this term no longer accurately reflects the mechanisms involved in luteal regression. In 1999, McCraken et al. [5] stated that \"the term luteolysis may be something of a misnomer;\" but also referred to the deep entrenchment of this term in the literature. However, the exponential increase in the number of scientific manuscripts published in recent years ensures that a prompt move to replace the older term with a more accurate descriptor will rapidly result in an overturn of the current ranking displayed in Table 1 and that only a minority of papers will continue to be retrieved using the older term.\nDespite the fact that a strict definition of \"luteolysis\" appears to regard only the structural disintegration of the corpus luteum, broader interpretations are rife within the literature. Rothchild [6] defined luteolysis as \"stopping the secretion of progesterone by the corpus luteum.\" Two other recent reviews take a middle road. Hoyer [7] refers to the fall in progesterone secretion as functional regression but considers structural regression, or cell death, the \"true\" regression. Niswender et al. [8], while referring to luteal regression as the structural demise of the corpus luteum, go on to describe the loss of progesterone synthesis and secretion \u2013 not a structural event \u2013 as part of the regression of the corpus luteum.\nAs the previous paragraph shows, the adjectives \"structural\" and \"functional\" cannot be overlooked when addressing terminology in the field of luteal regression. These words have had a consistent presence in literature concerning the regression of the corpus luteum since Malven and Sawyer [9] described the process of luteal regression in the rat as having two components: \"1) an initial termination of luteal secretory function and 2) a subsequent morphological regression of the nonfunctioning corpora lutea.\" A few years later Malven et al. [10] used the term \"structural luteolysis\" to describe the morphological regression of persistent, nonfunctional corpora lutea in the hypophysectomized rat. The definition of \"nonfunctional\" provided in this manuscript expanded upon the 1966 description of the first component of luteal regression in the rat by explaining that the corpora lutea \"did not secrete enough progesterone to induce deciduoma formation following endometrial traumatization.\" Thus, the corpora lutea were \"nonfunctional\" in terms of progesterone production and the potential for establishment of pregnancy.\nSince the appearance of these early papers, the terms functional and structural regression of the corpus luteum (or the more popular phrasing \"functional and structural luteolysis\") have gained relative acceptance and usage. The cursory MEDLINE\/PubMed search shown in Table 1 turned up 82 references containing the phrase \"functional luteolysis,\" of which 5 were 2001 and 2002 publications, and 72 references containing the phrase \"structural luteolysis,\" of which 14 were 2001 and 2002 publications. The prevalence of the term \"structural luteolysis\" over \"functional luteolysis\" in recently published articles may be due in part to a greater disparity in the perceived meaning of the latter term. This stems largely from studies in rats, in which it was revealed that corpora lutea which no longer secreted significant quantities of progesterone retained many functions, including active steroidogenesis [11-13].\nIt is our contention that these two frequently described components of the process of luteal regression (i.e. cessation of progesterone secretion and disappearance of the structure) merit equal attention. However, the timing and inter-relationship of these two types of regressive changes appears to vary by species. As new information on the molecular steps triggered during luteal regression is obtained, these mechanisms will need to be defined as they relate to these facets of the overall regressive process. Davis and Rueda [3] advised that the terms functional and structural used in the context of luteal regression should be adequately defined by the authors in the context of the particular species and reproductive stage being studied, with which we concur. It is not extreme to predict that with the advancement of knowledge in the process of luteal regression researchers will find increasing difficulties in establishing the limits as to what mechanisms belong to the \"functional\" facet of luteal regression, and which ones are part of the \"structural\" facet, as luteal regression is a process of synchronized events. For example, it now appears that progesterone itself is responsible for preventing the onset of apoptosis and structural disintegration [14-17].\nConclusions\nThese authors strongly recommend that the term luteal regression should be used to refer to the process of regression of the corpus luteum. Luteal regression represents a broad definition of the process of demise of the corpus luteum that is capable of accommodating all new knowledge evolved on the molecular mechanisms activated or inhibited during the process of regression of the corpus luteum regardless of species or reproductive stage. Further, according to the etymology of the word, luteolysis has a very limited meaning that no longer describes properly the complex sequence of synchronized molecular events associated with the demise of the corpus luteum. The modifying terms \"functional\" and \"structural\" can be used in conjunction with the term luteal regression to further identify aspects of the overall process but must be adequately defined within the context of the publication.","keyphrases":["luteal regression","corpus luteum","luteolysis","functional luteolysis","structural luteolysis"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_J_Clin_Pharmacol-4-1-2426925","title":"Pharmacokinetics and tolerability of artesunate and amodiaquine alone and in combination in healthy volunteers\n","text":"Objectives The WHO recommends artemisinin-based combination therapies for treatment of uncomplicated falciparum malaria. At least 15 African countries have adopted artesunate plus amodiaquine as treatment policy. As no pharmacokinetic data on this combination have been published to date, we investigated its pharmacokinetic interactions and tolerability in healthy volunteers in Africa.\nIntroduction\nSince Plasmodium falciparum has developed resistance to almost all anti-malarial drugs, the use of combinations of effective antimalarials with different mechanisms of action is required to improve cure rates and delay drug resistance. The World Health Organisation (WHO) now recommends treatment of uncomplicated falciparum malaria with one of several artemisinin-based combination therapies (ACTs), including artesunate plus amodiaquine [1]. To date at least 15 African countries have adopted artesunate plus amodiaquine as first-line malaria treatment policy [2].\nAlthough the limited data available suggest that the combination of artesunate and amodiaquine is well tolerated as short-course treatment, there are two safety concerns that have not yet been fully addressed. Neutropaenia and hepatitis curtailed the use of amodiaquine as prophylaxis in the 1980s [3\u20135]. A recent randomised, controlled trial comparing treatment with amodiaquine alone or in combination with artesunate found marked, asymptomatic neutropaenia on day 28 (NCI grade 3\u20134, absolute neutrophil counts 306\u2013900 \u03bcL\u22121) in 6% of 153 children with uncomplicated falciparum malaria, all of whom had normal baseline absolute neutrophil counts [6]. We have previously reported a case of hepatitis in this group of volunteers that developed after the second dose of amodiaquine and was considered probably related to amodiaquine [7]. A similar report of delayed-onset asymptomatic hepatitis has been described in healthy American volunteers who had received artesunate plus amodiaquine alone followed by artesunate plus amodiaquine plus efavirenz, resulting in early study discontinuation [8]. In that study, efavirenz was thought to have caused an increase in amodiaquine concentrations by competitive inhibition of the P450 cytochrome enzymes that metabolise amodiaquine. With the widespread use of artesunate plus amodiaquine treatment in Africa, these studies support the need for laboratory monitoring of the treatment\u2019s safety, particularly if antiretrovirals are used concomitantly [1].\nPharmacokinetic data are still sparse for amodiaquine. After oral administration, amodiaquine hydrochloride is rapidly absorbed and undergoes rapid and extensive metabolisation to desethylamodiaquine, the main active metabolite. Amodiaquine has a short elimination half-life (approximately 5\u00a0h), while that for desethylamodiaquine is much longer (6\u201318\u00a0days) [9, 10].\nThere are several pharmacokinetic studies on oral artesunate in healthy volunteers and malaria patients that give broadly consistent results [11, 12]. Artesunate is rapidly absorbed, rapidly converted to its main active metabolite dihydroartemisinin and rapidly cleared (elimination half-life generally <1\u00a0h). Dihydroartemisinin has similar pharmacokinetic characteristics but has a slightly longer elimination half-life. There is wide inter-individual variation in artesunate and dihydroartemisinin pharmacokinetic parameters.\nNo pharmacokinetic interaction data have yet been published on amodiaquine when used in combination with artesunate. In view of the forthcoming widespread use of this combination in Africa, our study aimed to investigate potential pharmacokinetic interactions between these antimalarials and assess their tolerability in healthy volunteers in Africa.\nMethods\nStudy subjects and study design\nMale or female healthy normal volunteers who provided written informed consent and met the following criteria were eligible to participate in the study: aged 18\u201345\u00a0years; no abnormalities on medical history, clinical examination, laboratory safety assessment (full blood count, differential white cell count, routine liver and renal function tests) or electrocardiogram; and a negative pregnancy test (for female volunteers). Volunteers were excluded if they were smokers (>5 cigarettes\/day), had taken antimalarials or been in a malarial area in the preceding 8\u00a0weeks, had malaria parasites on a thick smear, used recreational drugs, or had ingested any alcohol or any medicines (including over-the-counter preparations) in the week preceding study commencement.\nThis was a randomised three-phase crossover study. All volunteers took artesunate (4\u00a0mg\/kg as Arsumax, 50\u00a0mg tablets, Sanofi-Aventis), the drug with the shorter elimination half-life, in the first phase, prior to the administration of amodiaquine. In the second phase, 7\u00a0days later, the volunteers were randomly allocated to one of two treatment groups: group 1 received a single oral dose of artesunate (4\u00a0mg\/kg) plus amodiaquine (10\u00a0mg base\/kg as Camoquin, 200\u00a0mg tablets, Parke Davis), and group 2 received a single oral dose of amodiaquine (10\u00a0mg\/kg) alone. In phase three, 21\u00a0days later, the groups received the alternative regimen. All trial drugs were given under direct supervision with 200\u00a0ml tap water on an empty stomach after an overnight fast. No caffeinated drinks were allowed during the study period. Standardised meals were offered, commencing with breakfast at 2\u00a0h post-dose. Volunteers in phase one had blood samples collected before taking artesunate and at 0.25, 0.50, 0.75, 1.0, 1.5, 2, 3, 4, 6, 8 and 12\u00a0h after artesunate administration. In phases two and three (i.e. volunteers receiving artesunate plus amodiaquine or amodiaquine alone) venous blood samples were collected pre-dose and at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 48 and 72\u00a0h, and days 4, 5, 7, 10, 14 and 20 post-dose. At each time two 5-ml venous blood samples were collected in tubes containing sodium heparin anticoagulant, while three 5-ml samples were collected at each time point for the first 12\u00a0h from those volunteers receiving both drugs. Each sample was centrifuged within 5\u00a0min of collection and the plasma transferred to separate plastic cryotubes and frozen at \u221270\u00b0C until analysed for artesunate, dihydroartemisinin, amodiaquine and desethylamodiaquine. Volunteers remained in the study ward for the first 12\u00a0h of each phase; thereafter specimens were collected at outpatient visits.\nA clinical examination and laboratory safety tests were performed during screening and 6\u00a0days after artesunate administration and 20\u00a0days after administration of both amodiaquine and amodiaquine plus artesunate. A 12-lead electrocardiogram (ECG) was recorded for each volunteer during screening and approximately 2\u00a0h after each drug dose. The corrected QT interval (QTc) was calculated by dividing the QT interval by the square root of the R-R interval. ECGs were reviewed by C.O. and by a consultant cardiologist. At follow-up visits, the volunteers were asked to report any new symptoms. A targeted physical examination was conducted if indicated. An adverse event together with its relationship to study drugs was defined according to the WHO guidelines [13] and the severity graded using the National Cancer Institute (NCI) Common Toxicity Criteria [14]. All adverse events and concomitant medications taken were recorded. Any significant clinical or laboratory abnormalities or the use of a concomitant medication with a previously described interaction resulted in the volunteer being withdrawn from the study.\nEthical approval\nThis clinical study was conducted in accordance with the principles laid down by the World Health Assembly of 1975 on Ethics in Human Experimentation and the Helsinki Declaration. The study adhered to the standards established for good clinical practice. The protocol was approved by the University of Cape Town Research Ethics Committee and the WHO Secretariat Committee for Research Involving Human Subjects (SCRIHS). Each volunteer was informed of the objectives, nature and possible risks of the trial. Written informed consent was obtained from every volunteer participating in the study. The volunteers were informed that they were free to withdraw consent at any time.\nDrug assays\nArtesunate and dihydroartemisinin concentrations in plasma were analysed using the method of Batty and colleagues [15] with minor modifications. Solid-phase extraction of samples was performed using Bond-Elut phenyl extraction cartridges (Varian, Harbour City, CA, USA). After washing the column with 2\u00a0ml of water, samples were eluted from the column with 1.5\u00a0ml of acetonitrile, which was then evaporated to dryness under nitrogen. Samples were reconstituted in 100\u00a0\u03bcl of mobile phase (45% acetonitrile, 0.05\u00a0M acetic acid, pH 6.0), and 50\u00a0\u03bcl was injected onto the column. HPLC was carried out on a Symmetry C 8, 5\u00a0\u03bcm, 15\u00a0cm\u2009\u00d7\u20094.6\u00a0mm HPLC column (Waters, Milford, MA, USA) using a Waters 600 pump and WISP 712 auto-sampler. The post-column reagent (1.2\u00a0M KOH in 90% methanol) was introduced using a Waters reagent delivery module and the mixture passed through a 1-ml reaction coil (Waters) kept at 69\u00b0C. Detection was at 290\u00a0nm using a Waters 484 Tunable Absorbance Detector, which produced a linear response over the range of the standard curve (20\u20131,000\u00a0ng\/ml). Quality control samples of 15, 75 and 150\u00a0ng\/ml for artesunate and 75, 450 and 750 ng\/ml for DHA were included in each run. Within- and between-day coefficients of variation were below 14%. The lower limit of quantification was 24\u00a0ng\/ml for both artesunate and dihydroartemisinin.\nAmodiaquine and desethylamodiaquine were analysed by LC mass spectrometry using an Agilent 1100 Series LC\/MS system. Protein was precipitated from plasma samples (200\u00a0ul) using three volumes of acetonitrile. Supernatant (5\u00a0\u03bcl) was injected onto the HPLC. Chromatography was carried out using a 50\u2009\u00d7\u20094.6\u00a0mm C18 Xterra column (Waters). The mobile phase comprised 75% acetonitrile, 0.02\u00a0M ammonia, pH 10.2. The extracted ion for amodiaquine was m\/z 356 and for desethylamodiaquine m\/z 328. The calibration curve for amodiaquine was linear in the range 5\u2013100\u00a0ng\/ml and for desethylamodiaquine in the range 5\u2013400\u00a0ng\/ml. Quality control samples of 25, 75 and 250\u00a0ng\/ml were used for desethylamodiaquine and 2.5, 7.5 and 25\u00a0ng\/ml for amodiaquine. Within- and between-day coefficients of variation were below 12%. The lower limit of quantification was 5\u00a0ng\/ml for both amodiaquine and desethylamodiaquine.\nStatistical analysis\nPharmacokinetic parameters were calculated from the artesunate, dihydroartemisinin, amodiaquine and desethylamodiaquine concentration profiles using WinNonLin, version 3.3 (Pharsight), with concentrations outside the specified limits of quantification regarded as missing values. Clearance (Cl) was calculated as 0.693 \u00d7 Vd\/T1\/2 where Vd is the apparent volume of distribution and T1\/2 is the elimination half-life. Because there was no intravenous comparator arm in this study, equivalent bioavailability was assumed for comparisons of apparent volume of distribution (Vd\/f) and clearance (Cl\/f), where f is the oral bioavailability or fraction of the drug absorbed (unknown).\nAll four compounds were first analysed separately. Because both artesunate and dihydroartemisinin are highly parasiticidal in malaria patients, the pharmacokinetic parameters associated with therapeutic response [area under the plasma concentration time curve (AUC) and maximum clearance (Cmax)] are also reported together as dihydroartemisinin equivalents, defined as the sum of the measured dihydroartemisinin concentrations plus the artesunate concentration converted to dihydroartemisinin equivalents (using molecular weights of 384 and 284 for artesunate and dihydroartemisinin respectively) [16, 17]. As amodiaquine undergoes a rapid and extensive conversion to its active metabolite, desethylamodiaquine was considered the primary analyte [9, 10, 18].\nThe pharmacokinetic parameters following administration of artesunate and amodiaquine alone and in combination were compared using Stata, version 9.0 [Stata, College Station, TX, USA]. Data were log-transformed and then compared using the analysis of variance (ANOVA) for a cross-over design to take into account the repeated measures by study subject, treatment period and treatment groups and, for amodiaquine and desethylamodiaquine, a sequence effect. The treatment effects generated from the ANOVA were exponentiated in order to express comparisons between monotherapy and combination therapy as a ratio. Any apparent discrepancies between the difference in the group means and these ratios are due to the fact that the ratios are based on the within-patient differences in log-transformed values and not the group arithmetic means. Given the multiple testing, statistical significance of results should be interpreted with caution.\nFor the safety analysis, means and 95% confidence intervals (95% CI) for each haematological parameter (haemoglobin, haematocrit, platelets, white cell count, absolute lymphocyte and neutrophil count) were calculated at baseline and at the end of each treatment phase. Changes in these values from baseline were determined using a mixed effect regression model that took into account repeated measures within-patient and were adjusted for the period and period-treatment interaction effects.\nResults\nSubject demographics\nNineteen volunteers were screened; two were excluded because of neutropaenia and two withdrew consent. Fifteen healthy normal volunteers (10 male, 5 female) entered the study with a mean age, weight and height of 24.4\u00a0years, 67.3\u00a0kg and 171\u00a0cm, respectively. The mean dose of amodiaquine was 10.72\u00a0mg\/kg and that of artesunate was 4.26\u00a0mg\/kg. Safety analysis included all 15 volunteers; only data from the 13 volunteers who completed the study were included in the pharmacokinetic analyses. A female volunteer was withdrawn due to a new prescription of fluoxetine for depression, as this drug could potentially interact with artesunate and amodiaquine by inhibiting cytochrome P450 enzymes. One male volunteer was withdrawn due to a possible hypersensitivity reaction on his first exposure to amodiaquine.\nPharmacokinetic parameters\nEffect of amodiaquine on artesunate pharmacokinetic parameters\nData were analysed using a non-compartmental model. Figure\u00a01a shows the mean plasma concentration time curves of artesunate and dihydroartemisinin following administration of artesunate alone and artesunate plus amodiaquine. The AUC, Cmax and time to Cmax (Tmax) of artesunate were similar following monotherapy and combination therapy (Table\u00a01). Figure\u00a02a illustrates the impact of adding amodiaquine on the dihydroartemisinin AUC. Following the combination therapy, dihydroartemisinin AUC was lower [ratio 67% (95% CI 51\u201388%); P\u2009=\u20090.008], Cmax was lower [ratio 51% (95% CI 33\u201378%); P\u2009=\u20090.005], T1\/2 was longer [ratio 157% (95% CI 115\u2013213%); P\u2009=\u20090.008] and Vd was larger [ratio 192% (95% CI 133\u2013275%); P\u2009=\u20090.003], when compared with artesunate monotherapy (Table\u00a01). These ratios have been adjusted for repeated measures by subject and period effects. As a measure of the total exposure to artemisinin derivatives, the Cmax and AUC of the combined DHA-equivalents were also analysed and found to be lower following combination therapy [Cmax ratio 51% (95% CI 32\u201384%), P\u2009=\u20090.011; AUC ratio 72% (95% CI 54\u201395%), P\u2009=\u20090.023] when compared with monotherapy. Cl and Tmax of dihydroartemisinin were similar when monotherapy and combination therapy were administered (Table\u00a01).\nFig.\u00a01Profiles of mean drug concentrations over time following monotherapy (mono) and combination (combo) therapy for a artesunate (AS) and dihydroartemisinin (DHA) and b amodiaquine (AQ) and desethylamodiaquine (DEAQ)Table\u00a01Pharmacokinetic parameters for artesunate and dihydroartemisinin when artesunate was administered alone and with amodiaquine (ACT)\u00a0AUC (ng\u00b7h\/ml)Cmax (ng\/ml)Tmax (h)T1\/2 (h)Vd\/f (l)Cl\/f (l\/min)ArtesunateAS alone (mean\u2009\u00b1\u2009SD)206.4\u2009\u00b1\u2009135.5231.8\u2009\u00b1\u2009155.00.62\u2009\u00b1\u20090.28NANANAACT (mean\u2009\u00b1\u2009SD)183.3\u2009\u00b1\u2009146.5141.6\u2009\u00b1\u2009117.50.86\u2009\u00b1\u20090.67NANANAACT-to-monotherapy ratio (%) (mean, 95% CI)36 (8\u2013173)25 (6\u2013105)125 (76\u2013202)NANANA\u00a0\u00a0Significance (ANOVA)0.180.0570.33DihydroartemisininAS alone (mean\u2009\u00b1\u2009SD)2,044.4\u2009\u00b1\u2009564.2844.5\u2009\u00b1\u2009309.41.10\u2009\u00b1\u20090.951.46\u2009\u00b1\u20090.484.89\u2009\u00b1\u20091.672.46\u2009\u00b1\u20090.86ACT (mean\u2009\u00b1\u2009SD)1,410.5\u2009\u00b1\u2009543.6446.2\u2009\u00b1\u2009239.52.08\u2009\u00b1\u20091.722.20\u2009\u00b1\u20090.859.68\u2009\u00b1\u20094.163.08\u2009\u00b1\u20090.82ACT-to-monotherapy ratio (%) (mean, 95% CI)67 (51\u201388)51 (33\u201378)165 (82\u2013334)157 (115\u2013213)192 (133\u2013275)122 (96\u2013156)\u00a0\u00a0Significance (ANOVA)0.0080.0050.150.0080.0030.097Treatment effects generated from the ANOVA were exponentiated to express within-subject comparisons between monotherapy and combination therapy as a ratio, adjusted for period effectsAUC Area under the plasma concentration time curve, Cmax maximum concentration, Tmax time to Cmax, T1\/2 elimination half-life, Vd volume of distribution, f oral bioavailability or fraction of the drug absorbed, Cl clearance, AS artesunate, SD standard deviation, CI confidence interval, NA not available (too few detectable concentrations for calculation of T1\/2, Cl, Vd)Fig.\u00a02Stick plot comparing individual patient AUCs following monotherapy and combination therapy of a dihydroartemisinin (DHA) and b desethylamodiaquine (DEAQ). (Note: volunteer A was excluded from the statistical analyses)\nEffect of artesunate on amodiaquine pharmacokinetic parameters\nAmodiaquine samples for one volunteer were missing. Figure\u00a01b shows the mean plasma concentrations of desethylamodiaquine over time following administration of amodiaquine alone and in combination. Figure\u00a02b illustrates the impact of artesunate on the desethylamodiaquine AUC. The volunteer (subject A) who had the highest observed AUC for desethylamodiaquine following combination therapy [29,504\u00a0ng\u00b7h\/ml compared with the mean of 8,437 (95% CI 5,744\u201311,131) ng\u00b7h\/ml] was excluded from the pharmacokinetic analyses of desethylamodiaquine, as this was an extreme outlier and influential observation (Fig.\u00a02b; Fig.\u00a03).\nFig.\u00a03Stick plot comparing individual patients. Day 7 concentrations of desethylamodiaquine when amodiaquine was administered as monotherapy and in combination with artesunate. (Note: volunteer A was excluded from the statistical analysis)\nThe impact of adding artesunate to amodiaquine on the pharmacokinetic parameters of amodiaquine and desethylamodiaquine is reported for the remaining 11 volunteers. The pharmacokinetic parameters of amodiaquine were similar when administered as monotherapy or in combination with artesunate (Table\u00a02). The desethylamodiaquine AUC is lower [ratio 65% (95% CI 46\u201390%); P\u2009=\u20090.015], Tmax shorter [ratio 60% (95% CI 45\u201380%); P\u2009=\u20090.003] and clearance more rapid [ratio 164% (95% CI 112\u2013243%); P\u2009=\u20090.016] when amodiaquine was administered in combination with artesunate rather than as monotherapy (Table\u00a02). There was a trend towards a lower day 7 concentration of desethylamodiaquine [ratio 56% (95% CI 30\u2013104%); P\u2009=\u20090.064] when amodiaquine was administered in combination (Fig.\u00a03). These ratios have been adjusted for repeated measures by subject, period and sequence effects. The Vd and Cmax were not significantly different when monotherapy and combination therapy were administered.\nTable\u00a02Pharmacokinetic parameters for desethylamodiaquine (DEAQ) when amodiaquine was administered alone and with artesunate (ACT)\u00a0AUC (ng\u00b7h\/ml)Cmax (ng\/ml)Tmax (h)[Day 7] (ng\/ml)T1\/2 (h)Vd\/f (l)Cl\/f (l\/min)AmodiaquineAQ alone (mean\u2009\u00b1\u2009SD)162.4\u2009\u00b1\u2009101.429.2\u2009\u00b1\u200910.92.32\u2009\u00b1\u20091.16NA5.3\u2009\u00b1\u20094.1361.0\u2009\u00b1\u2009128.369\u2009\u00b1\u200959ACT (mean\u2009\u00b1\u2009SD)108.5\u2009\u00b1\u200956.022.7\u2009\u00b1\u20099.02.18\u2009\u00b1\u20091.61NA3.9\u2009\u00b1\u20091.2467.7\u2009\u00b1\u2009180.586\u2009\u00b1\u200926ACT-to-monotherapy (%) (mean, 95% CI)77 (47\u2013127)78 (58\u2013103)92 (57\u2013147)NA74 (29\u2013189)128 (93\u2013175)172 (84\u2013357)\u00a0\u00a0Significance (ANOVA)0.270.070.680.440.110.11DesethylamodiaquineAQ alone (mean\u2009\u00b1\u2009SD)12,041\u2009\u00b1\u20093,480268.7\u2009\u00b1\u200970.83.68\u2009\u00b1\u20091.8519.4\u2009\u00b1\u20097.3240.8\u2009\u00b1\u2009146.9234.1\u2009\u00b1\u200997.5768\u2009\u00b1\u2009252ACT (mean\u2009\u00b1\u2009SD)8,437\u2009\u00b1\u20094,009301.4\u2009\u00b1\u2009166.12.18\u2009\u00b1\u20091.0313.3\u2009\u00b1\u20097.3136.9\u2009\u00b1\u200983.8210.8\u2009\u00b1\u200992.91,330\u2009\u00b1\u2009735ACT-to-monotherapy ratio (%) (mean, 95% CI))65 (46\u201390)103 (73\u2013147)60 (45\u201380)56 (30\u2013104)53 (25\u2013111)88 (51\u2013149)164 (112\u2013243)\u00a0\u00a0Significance (ANOVA)0.0150.820.0030.0640.080.580.016Treatment effects generated from the ANOVA have been exponentiated to express within-subject comparisons between monotherapy and combination therapy as a ratio, adjusted for period and sequence effectsAUC Area under the plasma concentration time curve, Cmax maximum concentration, Tmax time to Cmax, [Day 7] day 7 concentration, T1\/2 elimination half-life, Vd apparent volume of distribution, f oral bioavailability or fraction of the drug absorbed, Cl clearance, AQ amodiaquine, SD standard deviation, CI confidence interval, NA not available (as too few patients had quantifiable amodiaquine concentrations)\nSafety\nTwenty-nine adverse events were reported over the course of the study by 10 (67%) of the 15 volunteers. The frequency of adverse events was similar across all three treatment arms (P\u2009>\u20090.10): 4\/15 (27%) following artesunate alone, 8\/15 (53%) following amodiaquine alone, and 5\/15 (33%) following the combination. All adverse events were consistent with the product information available, resolved spontaneously and, except for the transaminitis (see below) were mild or moderate in intensity.\nA case of asymptomatic, prolonged, severe transaminitis (NCI grade 3, AST 5\u201320\u00d7 upper limit of normal) that developed after phase 3 of the study has been previously published [7]. This was considered to be probably related to amodiaquine, rather than artesunate, before the pharmacokinetic results were available. The subsequent finding that this volunteer had the highest measured desethylamodiaquine AUC following the administration of artesunate plus amodiaquine [29,504\u00a0ng\u00b7h\/ml compared with the combination therapy mean of 8,437 (95% CI 5,744\u201311,131) ng\u00b7h\/ml] strengthened this assessment. There were no liver function abnormalities detected in any other volunteers. No renal or electrolyte abnormalities were detected in any volunteers.\nSignificant haematological changes were confined to the white cell counts. During phase 2 of the study, two volunteers (one following amodiaquine alone, one following the combination) developed asymptomatic, NCI grade 1 leucopaenia (3 to <4 \u00d7 109\/L) and either a grade 1 (1.5 to <2 \u00d7109\/L) or grade 2 (1 to <1.5 \u00d7 109\/L) neutropaenia. None of the other observed changes in haematological parameters were outside the normal range.\nThere were no significant changes following treatment seen on the ECGs; the mean (95% CI) QTc interval was 398 (390\u2013406) ms at screening, 401 (393\u2013409) ms following artesunate alone, 400 (385\u2013414) ms following amodiaquine alone and 412 (400\u2013424) ms following the combination. In two patients, the prolongation following treatment with amodiaquine or artesunate plus amodiaquine was considered of borderline clinical significance.\nDiscussion\nThe pharmacokinetic analyses in this randomised, cross-over study showed statistically significant pharmacokinetic interactions resulting in reductions in the AUC of both dihydroartemisinin and desethylamodiaquine when artesunate and amodiaquine were given in combination to healthy volunteers. Of further concern is that one healthy volunteer failed to reach quantifiable concentration of both artesunate and dihydroartemisinin throughout the initial 12\u00a0h. There is insufficient evidence currently available to explain the basis of these interactions.\nThe clinical significance of our findings is unclear because we studied healthy volunteers. In one study comparing pharmacokinetic parameters of dihydroartemisinin in patients and volunteers, patients had a significantly higher Cmax (1,948 vs. 1,192\u00a0nmol\/l) and AUC0\u201312 (4,024 vs. 1,763\u00a0nmol\u00b7h\/l), while their Vd and Cl were both lower. The Tmax and terminal elimination half-life of the patients were similar to those of the volunteers [19]. Desethylamodiaquine pharmacokinetic parameters in malaria-infected adults in Africa [20] and children with acute malaria in Papua New Guinea [21] were similar to those of normal volunteers.\nIn artemisinin-based combination therapy, the artemisinin is given to kill most of the parasites quickly, while the longer-acting partner drug (e.g. amodiaquine) is given to kill the residual parasites. For an antimalarial with a long elimination half-life like desethylamodiaquine, the key pharmacokinetic parameter for parasite killing is the AUC, which is a useful predictor of therapeutic response as it captures both the drug concentration and the duration of exposure [22, 23]. For such drugs the antimalarial concentration on day 7 is strongly correlated with AUC, and thus treatment response [23]. An association of desethylamodiaquine concentration on day 7 and amodiaquine treatment response has been found in children with uncomplicated falciparum malaria [18]. In our study, the AUC (and probably the day 7 desethylamodiaquine concentration) was reduced following the artemisinin-based combination; this may result in reduced parasite killing, particularly in areas where there is reduced amodiaquine sensitivity. Although there is a linear relationship between amodiaquine dose and the desethylamodiaquine AUC [24], the wide inter-individual variability in desethylamodiaquine levels (five-fold in our study), and the consequent potential for drug-induced toxicity, could preclude increasing the amodiaquine dose to compensate for the lower desethylamodiaquine AUC observed following co-administration with artesunate.\nExtensive clinical trial data show that artesunate plus amodiaquine is more effective (at clearing parasitaemia and preventing recrudescence) than amodiaquine alone [1, 6]. Therefore, if reductions in the Cmax and AUC of desethylamodiaquine and dihydroartemisinin occur in patients, the negative effect on parasite killing may become clinically insignificant due to the additive (or possibly synergistic) parasiticidal effects of the combination. However, this may only apply to areas where the level of amodiaquine resistance is low. Although clinically significant resistance to the artemisinins has not yet been documented, amodiaquine resistance is well documented and this is probably the principal explanation for low artesunate plus amodiaquine cure rates observed in a number of African countries [1, 6].\nDespite our small sample size, we detected the well-known amodiaquine-related adverse drug reactions. Clinically important adverse events were reported by a quarter of these volunteers, even though all were healthy and only two doses were administered 3\u00a0weeks apart (rather than the daily administration for 3\u00a0days recommended for malaria treatment). When the artesunate plus amodiaquine combination becomes widely used, monitoring of liver function tests and haematological parameters are warranted to define these risks in malaria patients, particularly in patients at special risk such as those at the extremes of age, with HIV co-infection or malnutrition [1]. However, the feasibility of this recommendation outside of the research setting is of concern.\nTo conclude, the total exposure to the main active metabolites of both artesunate and amodiaquine was significantly reduced when administered in combination to healthy African volunteers; this might be important clinically. However, because cure rates with this combination are generally higher than with amodiaquine monotherapy, artesunate and amodiaquine could remain in the armamentarium of drugs used to combat falciparum malaria, provided efficacy continues to be monitored and adequate safety precautions can be taken. Further pharmacokinetic research on artesunate plus amodiaquine, when administered concurrently or as a fixed dose combination, is urgently required in patients with malaria to establish the extent and clinical significance of these pharmacokinetic interactions.","keyphrases":["pharmacokinetics","tolerability","artesunate","amodiaquine","healthy normal volunteers"],"prmu":["P","P","P","P","P"]}
{"id":"Virchows_Arch-3-1-1888720","title":"Elevated expression of cyclooxygenase-2 is a negative prognostic factor for overall survival in intrahepatic cholangiocarcinoma\n","text":"The production of prostaglandins is regulated by cyclooxygenases (COXs), which also have a role in tumour development and progression in various human malignancies, including cholangiocarcinoma. Limited information is available of the correlation of COX-2 protein expression and prognosis in intrahepatic cholangiocarcinoma (ICC). The aim of the present study was to determine the clinical significance of COX-2 expression in ICC. In addition the correlation of COX-2 expression and apoptosis\/proliferation was analysed. COX-2 expression was determined immunohistochemically in 62 resected ICCs. Proliferation was assessed using Ki67-immunohistochemistry, and apoptosis was measured with the TdT-mediated dUTP nick-end-labelling technique. COX-2 was identified as an independent prognostic factor (P = 0.028) in resected ICC by survival analysis. High levels of COX-2 expression were found to be associated both with reduced apoptosis and increased proliferation of tumour cells. This study demonstrates the independent prognostic value of the COX-2 expression in resected ICC, thus, offering a potential additional adjuvant therapeutic approach with COX-2 inhibitors.\nIntroduction\nCholangiocarcinoma (CCC) is composed of cells resembling those of bile ducts. CCC arises either from the extrahepatic duct including the hilar bifurcation or from the intrahepatic ducts. Intrahepatic cholangiocarcinoma (ICC) is less frequent than extrahepatic bile duct carcinoma but second among primary malignant liver tumours after hepatocellular carcinoma. In Western countries, ICC accounts for about 10% of the primary liver malignancies with increasing incidence in the last two decades [24]. The identification of patients with most aggressive ICC might help to optimise surgical treatment and avoid unnecessary surgical therapy. Therefore, it is desirable to identify molecular markers that individually predict tumour behaviour and may facilitate an individualized therapy.\nAmong the potential prognostic parameters, cyclooxygenase (COX)-2 is of particular interest, as it may also offer the option of treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) [15]. COXs regulate the synthesis of prostaglandins and are, thus, the major target of NSAIDs. Its two isoforms (COX-1 and COX-2) have different expression patterns, with COX-1 being expressed in a broad variety of tissues. COX-2 has been shown to participate in tumour development and progression [11, 21]. Both elevated COX-2\u00a0messenger RNA (mRNA) and protein levels were found to be raised in ICC [3, 9]. There exist only few studies dealing with the potential prognostic value of the COX-2 expression. A small study with only 24 cases of CCC could not prove a significant association with survival; however, this study did not discriminate between extra- and intrahepatic cholangiocarcinoma [12]. Another study on extrahepatic cholangiocarcinoma failed to demonstrate a correlation of the COX-2 expression and the patients\u2019 outcome in CCC [16]. In the present study, COX-2 expression was determined by immunohistochemistry in one of the largest contemporary series of consecutive resected ICC. The findings were correlated with a broad range of clinicopathological features. Recent observations suggest that COX-2 overexpression may cause a prostaglandin E2-mediated inhibition of apoptosis in CCC. Moreover, COX-2 overexpression was shown to increase cell growth by the activation of E series of prostaglandins (EP receptors) [8, 31]. Thus, apoptosis and proliferation was determined in this series to monitor a putative imbalance between cell death and cell proliferation induced by COX-2 protein overexpression [26].\nMaterials and methods\nBetween August 1998 and August 2006, a total of 62 patients with a mean age of 58\u2009\u00b1\u200911.5\u00a0years were available for this study. The study comprised consecutive patients who underwent surgery for liver resection. Patients solely undergoing an explorative laparatomy without subsequent resection were excluded from the study. Patients with hilar cholangiocarcinoma, gallbladder carcinoma, or mixed hepato\/-CCC and liver cirrhosis were also excluded from the study. The diagnosis of ICC was based on histology by the examination of the resected liver specimen. The tumours were classified according to the pathologic TNM (pTNM) system (sixth edition) [28]. Detailed clinical data was available including preoperative therapy, operative details, and pathological findings including surgical radicability, tumour staging, and clinical follow-up. One patient suffered from primary sclerosing cholangitis without cirrhosis; hepatolithiasis was not present in any case. Data were completed by August 2006, and minimum follow-up was every six months or until death. The median length of the follow-up was 12\u00a0months.\nImmunohistochemistry\nImmunohistochemistry was performed with an automated staining device (Dako Autostainer, Glostrup, Denmark).\nCyclooxygenase-2\nIn this study, a monoclonal rabbit anti-human COX-2 antibody (DCS, Hamburg, Germany) was used. Immunohistochemistry was performed on 5-\u03bcm-thick paraffin, and antigen retrieval was carried out with 0.01-M citrate buffer at pH\u00a06.1 for 20\u00a0min in a hot water bath (95\u00b0C). The primary antibody was incubated for 30\u00a0min at 1:250 dilution. Antibody demonstration was achieved using the commercially available anti-mouse IgG detection kit (EnVision, DakoCytomation, Carpenteria, CA, USA) The replacement of the primary antibodies by mouse immunoglobin served as negative controls. Positive controls (colorectal carcinoma) were included in each staining series. In ICC, COX-2 was scored according to the amount of positive stained tumour cells. One complete tumour slide was examined for specific cytoplasmic COX-2 immunostaining. If none or less than 10% of the tumour cells showed specific COX-2 immunostaining regardless of staining intensity, the case was classified as negative. The cases with 11\u201350% of the positively stained tumour cells were classified as moderately positive and tumours with more than 50% stained tumour cells as strongly positive.\nKi67 immunostaining and TdT-mediated dUTP nick-end labelling\nKi67 immunohistochemistry was performed on 5-\u03bcm-thick paraffin sections. Dewaxed and rehydrated sections were incubated with hydrogen peroxide to block endogenous peroxidase. After the antigen retrieval in a hot water bath, the prediluted monoclonal anti-Ki67 antibody (Biogenex, San Ramon, USA) was incubated for 30\u00a0min; antibody demonstration was performed with the commercially available anti-mouse IgG detection kit (EnVision, DakoCytomation). The replacement of the primary antibodies by mouse immunoglobin served as negative controls.\nThe growth fraction was defined as the percentage of Ki67-positive, randomly chosen nuclei per 600 tumour cells. In situ DNA fragmentation was established using the terminal desoxyribonucleotide transferase TdT-mediated dUTP nick-end-labelling technique (TUNEL) in paraffin-embedded sections. We used ApoTag\u2122 plus peroxidase in situ apoptosis detection kit (Intergen). The staining procedures were performed according the manufacturer\u2019s recommendations. The percentage of the stained apoptotic tumour cells per 600 randomly chosen tumour cells was calculated. To avoid miscounting of the necrotic cells, corresponding H&E sections were examined.\nStatistical analysis\nCOX-2 immunostaining was assessed by two of the authors (K.J.S., H.R.) in a blind-trial fashion without knowledge of the clinical outcome. In case of disagreement, slides were re-evaluated by both investigators until agreement was reached. All data were converted to a PC and statistically analysed using SPSS version 12 for Windows Statistical Package for Social Sciences, Chicago, IL, USA.\nThe kappa coefficient of the inter-observer agreement was calculated for the semiquantitative COX-2 immunostaining results; the interpretation of the kappa value was performed using the commonly cited scale of Landis and Koch [19]. The interobserver agreement of COX-2 immunostaining was substantial (kappa\u2009=\u20090.73). Relationships between ordinal parameters were investigated using the two-tailed \u03c72 analysis (or the Fisher\u2019s exact test where patient numbers were small). The relationship between categorical data (e.g. COX-2) and numeric data (e.g. number on Ki-67 positive tumour cells) was determined using the ANOVA test. The overall survival (OS) curves were estimated using the Kaplan\u2013Meier method, and any differences in the survival curves were compared by the log-rank test. For multivariate analysis, the COX regression model was used. Overall, 95% confidence intervals were used throughout.\nResults\nCOX-2 immunohistochemistry\nThe COX-2 immunostaining was located in the cytoplasm of epithelial cells. The plasma cells exhibited a positive cytoplasmic immunostaining, serving as internal control. Noncancerous intrahepatic bile duct epithelial cells next to invasive cancer showed frequently a positive COX-2 immunoreactivity, whereas no COX-2 staining could be detected distant from the tumour (Fig.\u00a01). Hepatocytes near and distant from ICC revealed a positive staining in all cases. In all, 12 (19.4%) tumours were classified as negative, 25 (40.3%) as moderately positive, and 25 (40.3%) as strongly positive. Epithelial tumour cells exhibited a broad range of COX-2 immunoreactivity with a complete lack of staining or a specific and strong granular cytoplasmic COX-2 immunostaining (Fig.\u00a02). COX-2 staining was not associated with any of the investigated clinicopathological parameters (Table\u00a01).\nFig.\u00a01Immunostaining for COX-2 in noncancerous intrahepatic bile duct epithelial cells. Whereas portal bile ducts distant from the ICC consistently lacked COX-2 immunostaining (asterisk, left side), bile duct epithelial cells (asterisk, right side) adjacent to ICC (arrow) exhibited cytoplasmic immunoreactivity of varying intensity. Notice the COX-2 protein expression in normal hepatocytes. Original magnification, \u00d7400Fig.\u00a02Representative COX-2 immunostaining results in ICC. Missing COX-2 staining (left) in contrast to strong cytoplasmic COX-2 immunoreactivity in ICC tumour cells (right). Original magnification, 400\u00d7. Inset: positive control (colorectal carcinoma) with strong immunostainingTable\u00a01COX-2 expression, demographic characteristics, and clinicopathological characteristics in 62 patients of the resected ICC\u00a0AllCOX-2 negativeCOX-2 moderateCOX-2 strongP values\u00a0n (%)6212 (19.4)25 (40.3)25 (40.3)Gender (male\/female)7\/59\/1610\/150.422Mean age at diagnosis (y\u00b1SD)58.8\u2009\u00b1\u200911.552\u2009\u00b1\u200912.160.4\u2009\u00b1\u20099.8 60.6\u2009\u00b1\u200911.80.067Grading0.612\u00a0G1 (n%)2 (3.2)0 (0)2 (100)0 (0)\u00a0G2 (n%)41 (66.1)7 (17.1)15 (36.6)19 (46.3)\u00a0G3 (n%)19 (30.6)5 (26.3)8 (42.1)6 (31.6)UICC stage grouping0.277\u00a0I166 (37.5)3 (18.8)7 (43.8)\u00a0II30 (0)1 (33.3)2 (66.7)\u00a0IIIA,B,C385 (13.2)19 (50)14 (36.8)\u00a0IV51 (20)2 (40)2 (40)Staging0.390\u00a0T1 (n,%)217 (33.3)5 (23.8)9 (42.9)\u00a0T2 (n,%)70 (0)4 (57.1)3 (42.9)\u00a0T3 (n,%)284 (14.3)13 (46.4)11 (39.3)\u00a0T4 (n,%)6 1 (16.7)3 (50)2 (33.3)Lymph vessel infiltration0.121\u00a0\u00a0L0477 (14.7)18 (38.3)22 (46.8)\u00a0\u00a0L1155 (33.3)7 (46.7)3 (20)Blood vessel infiltration0.916\u00a0V0398 (20.5)15 (38.5)16 (41)\u00a0V1234 (17.4)10 (43.5)9 (39.1)Nodal statusa0.281\u00a003710 (27)15 (40.5)12 (32.4)\u00a01212 (9.5)10 (47.6)9 (42.9)Distant metastasis0.999\u00a0No5711 (19.3)23 (40.4)23 (40.4)\u00a0Yes51 (20)2 (40)2 (40)Solitary tumourb327 (21.9)11 (34.4)14 (43.8)0.545Multifocal tumour295 (17.2)14 (48.3)10 (34.5)Tumour size 8.18.75\u2009\u00b1\u20094.3 8.3\u2009\u00b1\u20092.97.4\u2009\u00b1\u20093.60.506Resection status0.627\u00a0R0399 (32.1)15 (38.5)15 (38.5)\u00a0R1\/2c233 (13)10 (43.5)10 (43.5)P values were calculated using chi-square analysis and ANOVA for continuous variablesaData available for 58 patients; bdata available for 61 patients; c20 cases were classified R1, only 3 were R2\nClinical outcome by COX-2 expression\nOS dependent on COX-2 expression was analysed using Kaplan\u2013Meier survival (n\u2009=\u200962; p\u2009=\u20090.036; Fig.\u00a03). Patients with a strong COX-2 expression exhibited a significantly decreased OS, whereas patients classified as COX-2 negative revealed the most favourable OS. The unfavourable prognostic effect of tumours with elevated COX-2 protein expression remained in the subgroup of patients with primarily R0-resected ICC (n\u2009=\u200939; p\u2009=\u20090.038).\nFig.\u00a03Kaplan\u2013Meier survival curves in resected intrahepatic cholangiocarcinoma in relation to different COX-2 expression. Tumours with elevated COX-2 levels exhibit a significantly decreased overall survival (p\u2009=\u20090.036, Log Rank test)\nThe parallel univariate survival analysis showed a significant association of the resection status (p\u2009<\u20090.001), multifocal tumour growth (p\u2009=\u20090.005), higher International Union Against Cancer (UICC) stage (p\u2009=\u20090.012), and vascular invasion (p\u2009=\u20090.011) with a reduced OS. Table\u00a02 shows the mean OS for all patients in the various subgroups. \nTable\u00a02Mean overall survival in relation to different patient subgroupsSubgroupsMean overall survival in all ICC patients (months)All patients16.5R0-resected patients19.7R1-resected patients11.4R2-resected patients9.3COX-negative patients22.8Moderate COX-2 expression18.7Strong COX-2 expression11.4\nTo clarify the independent prognostic value of the COX-2 expression in the patients with resected ICC, a multivariate analysis of the relevant parameters was performed. The COX regression analysis revealed the R-classification to be the best prognostic factor for OS followed by the COX-2 expression (Table\u00a03). \nTable\u00a03Multivariate COX regression analysis for overall survival in 62 patients with resected intrahepatic cholangiocarcinoma\u00a0HR (95% CI) P valuespTstage I\u2013II\/III\u2013IV1.63 (0.44\u20136.02)0.465UICC I\u2013II, III\u2013IV1.20 (0.62\u20132.35)0.589Multifocality, solitary\/multifocal1.26 (0.41\u20133.89)0.689Resection status, R0\/R1, R2a3.08 (1.27\u20137.44)0.012Vascular invasion, V0\/V11.98 (0.88\u20134.41)0.095COX-2, negative\/moderate\/strong1.07 (1.07\u20133.49)0.028aTwenty cases were classified R1, only three were R2\nCOX-2 and apoptosis\/proliferation\nThere were significant lower mean numbers of apoptotic tumour cells per 600 tumour nuclei in tumours with strong COX-2 expression in contrast to tumours with moderate or negative COX-2 immunoreactivity (Table\u00a04). In contrast, the proliferative activity increased in tumours with high levels of COX-2, reflected by a higher mean value of proliferating tumour cells. However, due to a substantial overlap of values, statistical significance was not reached. \nTable\u00a04Apoptosis (TUNEL) and proliferative activity (Ki67) in relation to COX-2 expression\u00a0All (mean value\/SD)COX-2 negativeCOX-2 moderate\/strongP valuesApoptosis (percentage of positive tumour cells)2.17\u2009\u00b1\u20092.403.84\u2009\u00b1\u20094.041.76\u2009\u00b1\u20091.730.028Growth fraction (percentage of Ki67 positive tumour cells)9.74\u2009\u00b1\u20097.378.55\u2009\u00b1\u20098.419.99\u2009\u00b1\u20097.340.486P values were calculated using ANOVA for continuous variables\nDiscussion\nCCC is a devastating cancer of the hepatic biliary tract. The assessment of the individual prognosis of ICC patients may be helpful to optimise decision making for surgical treatment and avoid unnecessary surgical therapy. At present, surgical therapy remains the only curative treatment for ICC; thus, novel therapeutic strategies for this aggressive cancer are necessary. This study on a large series of 62 patients with resected ICC showed the independent prognostic value of immunohistochemically determined COX-2 protein expression, thus, offering a potential additional adjuvant therapeutic approach with COX-2 inhibitors. Elevated COX-2 expression was associated with a marked reduced OS in multivariate analysis. The main advantage of our study is the large number of patients (n\u2009=\u200962) in combination with a high homogeneity of this series composed of consecutively resected ICC. A previous study on extrahepatic cholangiocarcinoma failed to demonstrate a relation of COX-2 expression with clinical outcome [16]. This suggests (genetic) differences depending on the site of origin within the biliary tract. Mutations of the RAS and TP53 genes are the most common genetic abnormalities identified both in ICC and cancer of extrahepatic bile duct (BDC) [13, 14, 16, 20, 29]. However, recent studies disclosed genetic differences regarding p53 protein overexpression among carcinomas of the proximal (ICC) and distal bile ducts (BDC), indicating different patterns of the inactivation of tumour-suppressor genes [1]. Because cell line studies suggest COX-2 both as an effector and inhibitor of the tumour suppressor p53 or DNA-damaging agents [4, 7], different genetic patterns in ICC and BDC, with varying induction of COX-2 gene\/protein expression, might explain the diverse clinical impact of COX-2 in ICC and BDC.\nA small study on 24 patients with CCC failed to show a prognostic significance of the COX-2 expression [12]. However, the power of this study is limited due to two reasons: (1) The authors did not discriminate between extra- and intrahepatic cholangiocarcinoma, and (2) the statistical power of a small series with 24 patients is very low. However, Javle et al. [12] demonstrated in their study that the median survival time of patients with low COX-2 expression was more than twice as high than that of patients with high COX-2 expression. This finding is in accordance with our results identifying COX-2 overexpression as a predictor of reduced OS. Javle et al. found COX-2 to be expressed in all 24 ICC, whereas in our study, only 81% of ICC exhibited an either moderate or strong COX-2 expression. Kim et al. [16] found COX-2 to be overexpressed in only 52% of the examined extrahepatic cholangiocarcinomas. These discrepancies are likely to be caused by differences in antigen retrieval and varying scoring systems, emphasizing the relevance of standardized protocols and immunoscores.\nPrevious studies suggest COX-2 overexpression as an early carcinogenetic event in the human biliary tract and pointed out that COX-2 expression was highest in more differentiated CCC, whereas COX-2 expression decreased with the loss of differentiation [5]. Our study did not reveal a significantly increased COX-2 expression in tumours with higher differentiation. Nevertheless, a detailed analysis of our data rather supports this notion. The group of well and moderately differentiated tumours exhibited the highest amount of cancers classified as strongly COX-2 positive (44.2%) in contrast to ICC with poor tumour differentiation (31.6%). On the other hand, we found the lowest levels of COX-2 expression to be more frequent in poorly differentiated tumours (26.3%) in contrast to well or moderately differentiated ICC (16.5%). The study by Kim et al. [16] on 102 extrahepatic cholangiocarcinoma describes no significant differences in terms of tumour differentiation. In the light of these varying data, one should be careful not to draw premature conclusions about the differential expression COX-2 protein in ICC.\nThe clinical conditions associated with ICC include parasitic liver fluke infestations, hepatolithiasis, which is frequently observed in clonorchiasis [10], nonbiliary cirrhosis, and primary sclerosing cholangitis [30]. It would be interesting to compare the levels of COX-2 expression in ICC with or without these established associated clinical risk factors. However, our series is not suitable to answer this question because all cases with liver cirrhosis, regardless of its origin, were excluded from our study to maintain the high homogeneity of our cohort and rule out cirrhosis- related influence on patient survival. As a consequence, this series comprises only one patient with associated primary sclerosing cholangitis. Patients with liver fluke infestations and associated hepatolithiasis were not present in this series because our cohort is composed from patients living in western Europe.\nInterestingly, we found the up-regulation of COX-2 protein expression not only in cancerous epithelial cells but also in noncancerous epithelial cells adjacent to invasive cancer. Noncancerous epithelial cells distant from the ICC lacked COX-2 immunostaining. This staining pattern is in accordance with the results of Hayashi et al. [9]. The elevated expression of COX-2 in bile duct epithelial cells adjacent to invasive cancer is not surprising, as inflammation often accompanies invasive cancer and inflammatory cytokines such as IL-1\u03b1 [22], IL-1\u03b2 [2], or TNF-\u03b1 are believed to induce COX-2 gene at the transcriptional level [23]. We observed a constant positive specific immunostaining in normal hepatocytes regardless of the distance to the ICC. This finding is in agreement with previous studies documenting elevated COX-2 protein and mRNA expression in nontumourous liver [3, 17].\nAgainst the background of NSAIDs, recent studies have evaluated selective COX- inhibitors for their effect on CCC cells cell growth and invasion in vitro and in nude mice [6, 18, 27, 32, 33]. The treatment with COX-2 inhibitors resulted in induced apoptosis and inhibited proliferation. The mechanisms by which COX-2 contributes to the poor prognosis in variety of human cancers have not been completely elucidated yet. COX-2 has been shown to contribute to both tumourigenesis and the malignant phenotype of tumour cells by different mechanisms, including (1) the inhibition of apoptosis by the activation of the PI3-Kinase\/Akt pathway and increased production of PGE2, leading to increased expression of Bcl-2 and attenuation of nitric oxide signalling, (2) increased angiogenesis by increased PGE2 production with subsequent vascular endothelial growth factor production, (3) increased invasiveness by the overexpression of CD44, and (4) increased cell growth by the activation of EP receptors [8, 31]. The above mentioned effect of COX-2 overexpression on apoptosis and proliferation can be confirmed in our study. COX-2 over-expressing ICC revealed a significantly decreased apoptosis and a higher mean value of proliferating tumour cells, although the latter finding did not reach statistical significance. This finding suggests an impaired balance between cell loss and cell gain, resulting in a shift towards tumour net tumour growth due to increased apoptosis. The observed decrease of apoptosis in COX-2 overexpressing ICC might be due to the PGE2-mediated induction of the antiapoptotic protein Bcl-2 and increased NF\u03baB transcriptional activity, which is a key antiapoptotic mediator [25, 26].\nIn conclusion, this study demonstrates the independent prognostic value of immunohistochemical COX-2 protein expression in resected ICC, thus, offering a potential additional adjuvant therapeutic approach with COX-2 inhibitors and facilitating an optimised therapeutic strategy. Our results suggest that COX-2 overexpression causes a shift towards increased tumour cell proliferation and decreased apoptosis contributing to the unfavourable clinical course.","keyphrases":["immunohistochem","cancer","mol path"],"prmu":["P","P","U"]}
{"id":"Mcgill_J_Med-11-1-2322923","title":"Asian wasp envenomation and acute renal failure: a report of two cases\n","text":"Acute renal failure is an unusual complication of wasp stings. We report two cases of renal failure after multiple wasp stings (Vespa affinis). Both patients had evidence of intravascular haemolysis, hepatic dysfunction, oligo-anuria and azotaemia and required dialysis. The first patient had severe hemolysis, rhabdomyolysis, pigment and venom nephropathy and died on the 8th day in hospital. The second patient, who recovered completely in 3 weeks time with steroid and antihistaminic therapy, had interstitial nephritis. Although acute renal failure after wasp stings is typically caused by acute tubular necrosis (ATN) in the setting of haemolysis or rhabdomyolysis, in some patients, acute renal failure may result from a direct nephrotoxic effect or acute interstitial nephritis from a hypersensitivity reaction.\nINTRODUCTION\nIn the Pokhara Valley, Nepal, there are many unpublished cases of wasp poisoning which take a heavy death toll annually. Wasp stings usually cause local allergic reactions but can sometimes lead to intravascular haemolysis, rhabdomyolysis, thrombocytopenia, acute tubular necrosis, acute hepatic injury (1) and even myocardial infarction (2) in addition to various respiratory and neurological (3) manifestations.\nDeath from wasp envenomation is a rare event and results from acute renal failure (ARF) involving various mechanisms. Although ARF after wasp stings is typically caused by ATN in the setting of haemolysis or rhabdomyolysis, in some patients, renal failure may result from a direct nephrotoxicity of wasp venom or acute interstitial nephritis from a hypersensitivity reaction. We here we report two cases of acute renal failure after wasp stings (Vespa affinis).\nCASE 1\nA 55-year-old farmer who had been collecting fodder from a jungle was admitted with dyspnea, hoarse voice and myalgia within 8 hours of being attacked by several wasps. The patient was given intravenous saline, oxygen, salbutamol (\u03b22-adrenergic receptor agonist), chlorpheniramine (antihistamine), cyproheptadine (antihistamine), prednisolone (corticosteroid) and ranitidine (histamine H2-receptor antagonist). He also received fluid, mannitol and furosemide. By the next morning he had haemoptysis and had produced 400 ml of dark urine. On examination, the patient was drowsy, pale, icteric and cyanosed and had approximately one hundred and fifty red and swollen sting marks all over the body. Systemic examination revealed polyphonic wheezes and crepitations at the base of the right lung. Investigations are shown in Table 1. The onset of the oliguric phase was at 12 hours postenvenomation. Chest radiograph showed right basal consolidation. He died on the 8th day following admission despite aggressive therapy with medication, blood transfusions, assisted ventilation, and 16 cycles of dialysis.\nCASE 2\nA 40-year-old forest guard was attacked by a swarm of wasps. He presented with approximately twenty-five sting marks in exposed areas of face, throat, hands and legs [Fig. 1]. The patient was treated in the primary health centre with rubbing of saliva and papaya slices over the sting marks and referred to teaching hospital almost 24 hours after being stung. He developed anuria and had not passed any urine the previous night. On examination, the patient had a rapid pulse, unrecordable blood pressure, icterus, an urticarial rash in exposed parts of the face, legs and hands, facial puffiness, and a swollen left ankle and right knee joint. The rest of the physical examination was unremarkable. Investigations are shown in Table 1. He was treated with oxygen, salbutamol, chlorpheniramine, cyproheptadine, prednisolone and ranitidine. His hepatic and renal function improved gradually with fluid challenge, furosemide, mannitol, bicarbonate infusion, dopamine, and 12 cycles of dialysis in 3 weeks\u2019 time.\nOBSERVATION\nDISCUSSION\nWasp stings are well-known causes of toxic and hypersensitivity reactions. Direct toxicity is rare, but has been reported in cases when a very large amount of venom is injected. Immediate hypersensitivity reactions, such as bronchospasm in the first case and an urticarial rash in the second, are known to occur. In our report, both reactions responded to steroids and antihistaminics. The second patient had swollen joints indicating serum sickness-like reaction in a sensitized individual.\nWasp venom contains toxic melittin, apamine, phospholipases A1, hyaluronidase, acid phosphatase, histamine, and degranulating peptide mastoparan (4). These components have direct and indirect cytotoxic (hepatic, renal and myocyte membrane), hemolytic, neurotoxic and vasoactive properties, which can cause intravascular haemolysis and rhabdomyolysis (5, 6).\nWasp venom can cause ARF by several mechanisms, which include ATN, acute interstitial nephritis, pigment nephropathy resulting from rhabdomyolysis (myoglobinuria) or intravascular haemolysis (haemoglobinuria) and hypotension caused by an anaphylactic reaction (7, 8).\nPreviously rhabdomyolysis and renal ischemia were thought to be main causes of nephropathy. Sakhuja et al had postulated that direct toxic injury could be one of the possible mechanisms of ARF following wasp poisoning (9).\nMany cases of rhabdomyolysis-associated ARF have been published, but those due to wasp stings are rare. The wasp venom has deleterious effect on renal tubules and glomeruli (albuminuria, haematuria and ARF), red blood cells (haemolysis, reticulocytosis, unconjugated hyperbilirubinaemia), muscles (rhabdomyolysis, elevated creatinine phosphokinase and lactate dehydrogenase, myoglobinuria) and liver (elevated transaminases, hypoalbuminaemia and prolonged prothrombin time) (10). Kularatne et al had described similar multi-organ failure with high mortality following wasp poisoning owing to direct toxic effect (11).\nIn the first case we presented, the patient had myalgia, indicating muscle injury as evidenced by elevated CPK, LDH and AST and myoglobinuria (728 ng\/ml). He also had intravascular haemolysis and haemoglobinuria. Toxic pigments might have caused nephropathy resulting in ARF. The alternative mechanism of ARF postulated was direct nephrotoxicity by massive wasp venom.\nZhang R et al. (12) reported for the first time that acute tubulointerstitial nephritis could lead to ARF in wasp sting cases. In the second case we present, the patient had eosinophiluria, indicating interstitial nephritis. He recovered fully with the mentioned treatment. He did not report taking any medication which might have had nephrotoxic side-effects, and no other causes of ATN could be found. Ultrasound abdomen was unremarkable. Kidney biopsy revealed proximal peritubular necrosis and eosinophilic infiltration. Hence it can be hypothesized that the ATN was caused by a hypersensitivity reaction to the wasp venom.\nCONCLUSION\nWasp stings pose a great environmental hazard in Nepal and early recognition of anaphylactic shock, hepatic or renal dysfunction, rhabdomyolysis or haemolysis and rapid transport to hospital are essential steps of management to avoid fatalities. ARF due to toxic or pigment nephropathy and tubulointerstitial nephritis should be considered in any oliguric and azotemic patient following wasp attack.","keyphrases":["wasp envenomation","acute renal failure","rhabdomyolysis","interstitial nephritis","nepal"],"prmu":["P","P","P","P","P"]}
{"id":"Mol_Biochem_Parasitol-1-5-1885993","title":"Kinetoplastid PPEF phosphatases: Dual acylated proteins expressed in the endomembrane system of Leishmania\n","text":"Bioinformatic analyses have been used to identify potential downstream targets of the essential enzyme N-myristoyl transferase in the TriTryp species, Leishmania major, Trypanosoma brucei and Trypanosoma cruzi. These database searches predict \u223c60 putative N-myristoylated proteins with high confidence, including both previously characterised and novel molecules. One of the latter is an N-myristoylated protein phosphatase which has high sequence similarity to the Protein Phosphatase with EF-Hand (PPEF) proteins identified in sensory cells of higher eukaryotes. In L. major and T. brucei, the PPEF-like phosphatases are encoded by single-copy genes and are constitutively expressed in all parasite life cycle stages. The N-terminus of LmPPEF is a substrate for N-myristoyl transferase and is also palmitoylated in vivo. The wild type protein has been localised to the endocytic system by immunofluorescence. The catalytic and fused C-terminal domains of the kinetoplastid and other eukaryotic PPEFs share high sequence similarity, but unlike their higher eukaryotic relatives, the C-terminal parasite EF-hand domains are degenerate and do not bind calcium.\n1\nIntroduction\nProtein phosphorylation and dephosphorylation are critical processes in a variety of cellular mechanisms for the detection, transmission, and integration of intra- and extra-cellular signals. In eukaryotes, the extensively studied PPP family of serine\/threonine protein phosphatases function in cellular processes as diverse as regulation of the cell-cycle, RNA splicing and T cell activation [1]. PPPs have been divided into three subfamilies, commonly referred to as the \u2018Classical\u2019 PPP phosphatases; PP1, PP2A and PP2B [1]. Novel phosphatases that cannot be categorised into these subfamilies include the retinal degeneration C protein (RdgC) from Drosophila melanogaster\n[2]. RdgC homologues are found in a number of eukaryotic species, including Homo sapiens, Mus musculus, Gallus gallus and Caenorhabditis elegans\n[3,4] but have not been identified in fungi, yeast or plants to date. These novel phosphatases, distinguished by several putative EF-hand motifs within a fused C-terminal domain, have subsequently been termed PPEFs or Protein Phosphatases with EF-Hands [3].\nInterestingly, PPEFs exhibit a much narrower tissue distribution than classical PPPs, being restricted to the central nervous system or primary sensory structures in all metazoans studied to date. Thus, Dm RdgC has been principally localised to photoreceptors and the mushroom bodies of the central brain [2,5] while C. elegans CePPEF is highly enriched in primary sensory neurones [6]. In situ hybridisation and immunostaining have also localised mammalian PPEF isoforms to sensory structures such as the inner ear, dorsal root ganglia, embryonic brainstem nuclei, photoreceptors and pinealocytes [3,4]. These findings suggest that the PPEFs have conserved functions in diverse sensory systems and may have a role in development in mammals. The substrates of the RdgC\/PPEF phosphatases, however, remain elusive.\nThe domain architecture of RdgC\/PPEF homologues is highly conserved, consisting of three fused domains (Fig. 1A). The N-terminal regulatory domain often contains myristoyl\/palmitoyl acylation motifs and a downstream conserved IQ (isoleucine-glutamine) calmodulin binding domain. The catalytic domain contains several RdgC\/PPEF specific sequences\/motifs together with insertions of unknown function. The C-terminal domain contains at least two EF-hand motifs that have been shown to bind calcium in HsPPEF-1 and CePPEF [6,7] and one or more degenerate EF-hand like motifs.\nHere, we describe the characterisation of PPEF-like genes in the diverse lower eukaryotes Leishmania and Trypanosoma, the sole members of this phosphatase family in these parasites. The kinetoplastid PPEFs were identified following a genome-wide search for N-myristoylated proteins, carried out to identify downstream targets of the essential gene N-myristoyl transferase (NMT) in Leishmania major, Trypanosoma brucei and Trypanosoma cruzi. We show that LmPPEF and TbPPEF are substrates for NMT in vivo and that LmPPEF (and probably TbPPEF) can also be palmitoylated in vivo. Using immunofluorescence, LmPPEF has been localised to the endocytic system of Leishmania parasites, with some accumulation at the flagellar pocket. This location requires downstream regions of the protein in addition to the unique acylated N-terminus. Unlike other members of the RdgC\/PPEF family, the EF-hand domains within the C-terminus of LmPPEF are degenerate and do not bind calcium under the experimental conditions used here.\n2\nMaterials and methods\n2.1\nPCR amplification and sub cloning\nThe 2862-bp LmPPEF open reading frame (ORF) was amplified from cosmid 1567.3 (gift from Al Ivens) using Pfu DNA polymerase (Promega) at 64\u00a0\u00b0C annealing temperature and the primers LmPPEFFor (5\u2032-ATGGGGTGTGACTCATCCAAG-3\u2032) and LmPPEFRev (5\u2032-TTAGCGACTAGTGCCGAGGC-3\u2032). The amplified LmPPEF ORF was cloned into pPCR-Script AMP SK(+) (Stratagene) to generate pLmPPEF.\n236-bp and 1056-bp fragments from the 3\u2032 end of the LmPPEF ORF (nucleotides 2154\u20132862 and 1806\u20132862, respectively) were amplified at 60\u00a0\u00b0C annealing temperature using primers LmPPEF-Cterm1For (5\u2032-GACGATcatatgCGCATCTGGTAC-3\u2032) and LmPPEF-Cterm1Rev (-5\u2032-TGGCggatccTCTAGCCCTTA-3\u2032) or primers LmPPEF-Cterm2For (5\u2032-ATTAATTTcatatgCAGGTGGTGAGTCTA-3\u2032) and LmPPEF-Cterm2Rev (5\u2032-AATAggatccTTAGCGACTAGTGCC-3\u2032). Cloning sites are shown in lower case. The PCR fragments were digested with NdeI\/BamHI and cloned into pET-15b (Novagen) generating pLmPPEF-Cterm1 and pLmPPEF-Cterm2, respectively.\nThe 2775-bp TbPPEF ORF was amplified from T. brucei genomic DNA at 59\u00a0\u00b0C annealing temperature, using primers TbPPEFFor (5\u2032-CTTACGTTTccatggGTTGCTC-3\u2032) and TbPPEFRev (5\u2032-CCTCCcTcgagatCTCTCACAAA-3\u2032), digested with NcoI\/XhoI and cloned into pET-33b, generating pTbPPEF. The recombinant TbPPEF protein expressed from this plasmid has an N-terminal myristoylation motif (MGCSTSK).\n2.2\nParasite culture, membrane fractionation and nucleic acid analysis\nL. major Friedlin parasites (MHOM\/IL\/80\/Friedlin) were cultured, nucleic acids extracted and DNA\/RNA blotting and hybridisation carried out as previously described [8]. For membrane fractionation, mid-log phase parasites (5\u00a0\u00d7\u00a0107) were lysed by sonication on ice in either PBS alone, PBS plus 1\u00a0mM CaCl2 or PBS plus 1\u00a0mM EGTA. Undisrupted cells were removed by two centrifugation steps (500\u00a0\u00d7\u00a0g, 4\u00a0\u00b0C, 10\u00a0min). Cell lysates were separated into membrane and cytosolic fractions by ultra centrifugation (100,000\u00a0\u00d7\u00a0g, 4\u00a0\u00b0C, 1\u00a0h). Following separation, membrane fractions were washed twice in PBS and proteins from both fractions analysed by SDS-PAGE and immunoblotting as described [8].\n2.3\nAntibody production and immunoblotting\nExpression of N-terminally His-tagged recombinant LmPPEF-Cterm1 was induced by isopropyl-\u03b2-d-thiogalactopyranoside (IPTG) in Escherichia coli Rosetta (DE3) pLysS (Novagen). Cells were subsequently lysed in 6\u00a0M Gu-HCl prior to affinity chromatography using Talon Ni2+-nitrilotriacetic acid-agarose (Ni-NTA; BD Biosciences). Eluted protein was precipitated using 10% trichloroacetic acid, air dried and used for immunisation and generation of rabbit polyclonal antiserum (Eurogentech). Partial purification of LmPPEF-specific polyclonal antibodies was carried out using ammonium sulphate precipitation as described [9], followed by affinity purification against purified recombinant LmPPEF-Cterm1 as described [10].\nParasites were lysed in SDS-PAGE gel loading buffer, and immunoblotted as above with purified LmPPEF antiserum (abSK2031, 1:200 dilution), anti-NMT (abSK805, 1:2000 [8]), peroxidase anti-peroxidase (PAP) complex (P-2026, 1:2000, Sigma), or anti-GFP (ab290, 1:10,000, Abcam). Immune complexes were detected using an ECL kit (Amersham Biosciences).\n2.4\nL. major episomal expression constructs and parasite transfection\nA 111-bp fragment from the 5\u2032 end of the LmPPEF ORF (nucleotides1\u2013111) was amplified from pLmPPEF at 58\u00a0\u00b0C annealing temperature using primers Lm37WT-GFPFor (5\u2032-TAAAggatccATGGGGTGTGACTC-3\u2032) and Lm37WT-GFPRev (5\u2032-TTATAgatatcGCTACAAGTGCGTCG-3\u2032). The fragment was digested with BamHI\/EcoRV and cloned into pX-GFP [11], generating pLm37WT-GFP. Plasmids pLm37G2A-GFP, pLm37C3S-GFP and pLm37G\/A,C\/S-GFP were generated as above, using forward primers Lm37G2A-GFPFor (5\u2032-TAAAggatccATGGCGTGTGACTC-3\u2032), Lm37C3S-GFPFor (5\u2032-TAAAggatccATGGGGTCTGACTC-3\u2032) and Lm37G\/A,C\/S-GFPFor (5\u2032-TAAAggatccATGGCGTCTGACTC-3\u2032), respectively, and the reverse primer Lm37WT-GFPRev.\nThe 2862-bp LmPPEF ORF was amplified from pLmPPEF at 60\u00a0\u00b0C annealing temperature using primers LmPPEF-TAPFor (5\u2032-ATTAATTTcatatgGGGTGTGACTCAT-3\u2032) and LmPPEF-TAPRev (5\u2032-ATAtctagaCTTGCGGCTAGTGCC-3\u2032), digested with NdeI\/XbaI and cloned into the TAP vector pGL893 (gift from Sebastion Besteiro), generating pLmPPEF-TAP. The recombinant LmPPEF-TAP protein expressed from this plasmid has an N-myristoylation motif (MGCDSSK). All constructs used in this study are shown in Fig. 1B.\nMid-log phase L. major were electroporated with 20\u201350\u00a0\u03bcg of either pLm37WT-GFP, pLm37G2A-GFP, pLm37C3S-GFP, pLm37G\/A,C\/S-GFP or pLmPPEF-TAP as described [11] and cultures subsequently grown in media supplemented with 1\u00a0mg\/ml G418 (Life Technologies, Inc.).\n2.5\nMetabolic labelling and immunoprecipitation\nMid-log phase L. major promastigotes were metabolically labelled as previously described [11]. Cells were lysed for 1\u00a0h at 4\u00a0\u00b0C in lysis buffer (PBS containing 50\u00a0mM Tris, pH 7.5, 150\u00a0mM NaCl, 5\u00a0mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 100\u00a0\u03bcg\/ml leupeptin, 500\u00a0\u03bcg\/ml pepstatin, 198\u00a0\u03bcg\/ml 1,10 phenanthroline and 25\u00a0\u03bcg\/ml E64). The lysates were pre-cleared by incubation for 1\u00a0h at 4\u00a0\u00b0C with protein A-coupled Sepharose (Amersham Biosciences). Labelled proteins were then recovered from the supernatant by incubation with either anti-LmPPEF or anti-GFP antibodies overnight at 4\u00a0\u00b0C. After a second protein A-coupled Sepharose incubation, the beads were collected by centrifugation, washed twice in lysis buffer and proteins removed by boiling in SDS-PAGE gel loading buffer, prior to separation by SDS-PAGE. Detection of radiolabelling was improved using EN3HANCE\u2122 Autoradiography Enhancer (Kodak). DTT was omitted from the loading buffer for separation of [9,10-3H] palmitate-labelled proteins.\n2.6\nCalcium mobility shift assay\nThis assay was carried out as described [12]. In brief, proteins were lysed in SDS-PAGE gel loading buffer and separated by SDS-PAGE using either 5\u00a0mM CaCl2 or 5\u00a0mM EGTA in both the stacking and resolving gels. Separated proteins were Coomassie-stained or analysed by immunoblotting.\n2.7\nN-Myristoylation co-expression assay\nThis assay was performed as described [8,13]. In brief, E. coli BL21(DE3) pLysS cells were co-transformed with pTbPPEF and either pNMT [8] or pTbNMT [14]. Expression of recombinant TbPPEF and NMT protein was induced by IPTG in the presence of [3H]-myristate (Amersham Biosciences) and, following SDS-PAGE, radiolabelled proteins were detected by autoradiography.\n2.8\nFluorescence microscopy\nFor indirect immunofluorescence, L. major promastigotes were fixed and processed as previously described [15]. Cells were stained prior to fixation by incubation in 10\u00a0\u03bcM FM4-64 FX (Invitrogen) in serum-free medium at 26\u00a0\u00b0C for 10\u00a0min. For direct immunofluorescence, parasites were fixed in 4% (w\/v) paraformaldehyde at room temperature for 15\u00a0min, washed in PBS and immobilised on poly-l-Lysine coated coverslips (4\u00a0\u00d7\u00a0106 cells per cover slip). Slides were mounted using Vectashield plus 4\u2032,6\u2032-diamidino-2-phenylindole (DAPI; Vecta Laboratories).\n3\nResults\n3.1\nThe kinetoplastid N-myristoylomes\nN-Myristoylation describes the co-translational addition of the C14:0 fatty acid myristate to the amino-terminal glycine residue of a subset of eukaryotic and viral proteins. Catalysed by the enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT), N-myristoylation often plays a role in targeting proteins to membrane locations and can additionally be involved in mediating protein\u2013protein interactions and stabilising protein structure. We have previously shown that NMT is essential for viability in kinetoplastid parasites [8] while myristate analogues (non-specific inhibitors of NMT) are lethal to L. major promastigotes and bloodstream T. brucei\n[8,16,17]. A range of more specific NMT inhibitory compounds has recently been tested and several shown to inhibit T. brucei NMT activity in vitro, with toxicity to bloodstream T. brucei at low \u03bcM concentrations [18]. However, the cellular processes contributing to lethality in NMT-depleted cells are as yet unknown, although identification of the essential downstream targets of this enzyme may be crucial to the successful development of NMT as a potential drug target. Few kinetoplastid N-myristoylated proteins have been studied in depth to date\u2014the Leishmania HASPB and SMP-1 proteins and the T. brucei ARL1 protein are exceptions [11,14,19].\nWe used several strategies, all based on the well characterised eukaryotic N-myristoylation consensus motif, to search the L. major, T. brucei and T. cruzi genome datasets for putative N-myristoylated proteins. The first publicly available prediction algorithm for these proteins, the PDOC00008 PROSITE myristoylation signature [20], is known to generate high numbers of false positive as well as false negative predictions. The more recent NMT Predictor program was developed using a set of \u2018positive\u2019 amino acid sequences for profile training, substantially reducing the number of false results generated and extending the N-myristoylation motif from 6 to 17 amino acids [21,22]. Further refinement has come with the Myristoylator program, based on a neural network model and incorporating an additional \u2018negative\u2019 training set of amino acids [23]. We firstly used the \u2018relaxed\u2019 version of PROSITE PDOC00008 derived from the NMT Predictor, G-{EDRKHPFYW}-x(2)-[STAGCNDEF]-{P} (described in [21]), to probe the kinetoplastid genome datasets (http:\/\/www.genedb.org\/), thereby reducing the >8000 predicted ORFs to \u223c250\u2013300 potentially N-myristoylated proteins. The Myristoylator prediction algorithm was then applied to this refined data set, generating a final list of predicted N-myristoylated proteins within high confidence boundaries (scores: 0.85\u20131; Table 1; Tables S1\u2013S3, supplementary data). For the three genomes analysed, 62 N-myristoylated proteins were predicted in L. major, 62 in T. brucei and 123 in T. cruzi, with the latter higher figure a consequence of the presence of two different haplotypes in the T. cruzi CL Brener genome sequence [24]. Predicted N-myristoylated proteins contribute 0.5\u20130.8% of the proteomes of other eukaryotes studied to date [22,25], and the kinetoplastid data fall well within this range (Table 1). Further analysis of the N-termini of the high confidence kinetoplastid proteins revealed a strong preference (>70% of the total) for serine at position 6 in all three species, with threonine used in a further \u223c8% of proteins. In Saccharomyces cerevisiae, serine or threonine in these positions allows stabilisation of the peptide\u2013NMT complex through hydrogen bonding with Asp417, Gly418 and His221 in the ScNMT crystal structure [21].\nOf the putative N-myristoylated proteins identified with high confidence, 8 were specific to L. major, including the dual acylated HASPB [11] and the ADP-ribosylation factor LmARF3 [14], with a similar number of targets specific to T. brucei and T. cruzi (Tables S1\u2013S3, supplementary data). Other previously characterised N-myristoylated proteins included the calpain-like proteins, T. brucei CAP 5.5 [26] and L. major SMP-1 [19], while a number of putative signal transduction pathway proteins were also identified, including protein kinases and phosphatases (Table 2). However, the largest group of kinetoplastid N-myristoylated proteins was that currently characterised as of \u201cunknown function\u201d. A majority of the proteins within this group are conserved between the three Tri-Tryp species.\n3.2\nKinetoplastid PPEF-like protein phosphatases\nAnalyses of the kinetoplastid genomes have identified a range of kinases and protein phosphatases, some possessing novel motifs and insertions suggesting possible structural and functional differences from their mammalian homologues [24,27]. Of the phosphatases, only a few of the classical types have been characterised to date: protein phosphatase 2C (PP2C) from L. chagasi\n[28], PP1 from T. cruzi\n[29], PP1, PP2A and PP5 from T. brucei\n[30]. The N-myristoylome analysis described above identified a conserved serine\/threonine protein PPEF-like phosphatase (hereafter called PPEF) in all three kinetoplastid species: LmjF12.0660 in L. major, Tb927.1.4050 in T. brucei, Tc00.1047053506529.380\/Tc00.1047053510889.80 in T. cruzi. These kinetoplastid PPEFs are of similar size and composition: 954\u00a0aa, pI 8.0, 109.0\u00a0kDa in L. major; 925\u00a0aa, pI 7.7, 105.7\u00a0kDa in T. brucei; 923\u00a0aa, pI 7.7, 104.8\u00a0kDa in T. cruzi.\nThe phylogenetic relationship between these three proteins, the RdgC\/PP5 family and the classical phosphatase groups was investigated by multiple sequence alignment (using CLUSTAL W [31]) and generation of an unrooted tree (using the Tree View software, version 1.5.2 [32]). This analysis confirmed that the trypanosomatid PPEFs are more closely related to the RdgC\/PPEF subfamily than to the classical PPP subfamilies, PP1, PP2A or PP2B, and are also separate from the PP5\/PPT and PP7 subfamilies (Fig. 2A). Alignments revealed sequence similarity between the kinetoplastid PPEFs and other members of the RdgC\/PPEF subfamily (Fig. S1, supplementary data), with the overall percentage of amino acid identity between representative proteins of this group shown in Fig. 2B. While the kinetoplastid proteins share 60\u201370% identity with each other, they are only \u223c20% conserved when compared to their higher eukaryotic relatives. The similarity between these proteins is more apparent, however, when comparing their domain organisation, both at the primary sequence level (Fig. 1A; Fig. S2, supplementary data) and by homology modelling of the catalytic domain of LmPPEF with that of mammalian PP1 for which a high resolution structure is available [33] (Fig. S1, supplementary data). The central catalytic domains of the kinetoplastid and mammalian PPEFs, together with D. melanogaster RdgC, share significant sequence similarity (42%), including several RdgC\/PP5 specific catalytic motifs [1]. Kinetoplastid PPEFs also share RdgC\/PPEF specific mutations found in the conserved SAPNYC motif (common to all PPP phosphatases) that is found within the \u03b212\/\u03b213 loop of the catalytic domain (Fig. S2, supplementary data). The first of these, a Pro to Ser substitution at position 3, is found in all members of the Rdg\/PP5 family including the kinetoplastid proteins and at least two other protozoan phosphatases, PP1 from T. cruzi\n[29] and PfPPJ, a novel protein phosphatase from Plasmodium falciparum\n[34]. The second substitution within the SAPNYC motif, replacement of the Cys-6 residue by either Tyr, Asp or Asn, is restricted to the RdgC\/PPEF sub-family. Overall, the level of conservation within the catalytic domain of LmPPEF, including those residues acting as metal ligands, suggest that this protein and its kinetoplastid orthologues are functional phosphatases (Fig. S1, supplementary data).\nAt their C-termini, the kinetoplastid PPEFs are more closely related to the C-termini of the RdgC\/PPEFs than to any other calcium binding proteins or EF-hand domain-containing enzymes [35]. This observation supports the hypothesis that the catalytic domain of the ancestral form of RdgcC\/PPEF fused with an EF-hand Ca2+-binding protein prior to the acquisition of the N-terminal domain [1]. At their N-termini, there is little similarity between the kinetoplastid PPEFs and the RdgC\/PPEFs except for the presence of putative N-myristoylation and palmitoylation motifs, which are found in some but not all members of the RdgC\/PPEF phosphatase family.\n3.3\nGenomic organisation and expression of the L. major and T. brucei PPEFs\nThe Artemis annotation tool [36] was used to examine the level of gene synteny in the regions flanking the PPEF genes in the L. major and T. brucei genomes. LmPPEF is found on chromosome 12 in L. major while TbPPEF is found on chromosome 1 in T. brucei (http:\/\/www.genedb.org\/). The chromosomal regions surrounding these genes have been analysed previously as part of a wider study, revealing high conservation of gene order [37]. Thus the kinetoplastid PPEFs, including TcPPEF, can be classified as true orthologues.\nTo confirm the in silico gene analysis, genomic DNA blotting and hybridisation were used to show that both LmPPEF and TbPPEF are present as single copy genes in the L. major and T. brucei genomes, respectively (Fig. 3A and B). RNA expression from these genes in different parasite life cycle stages was initially demonstrated using RT-PCR (Fig. 3Ci and Di). RNA blotting and hybridisation were then used to confirm constitutive expression of single PPEF transcripts (of 4.3 and 4.8\u00a0kb, respectively) in insect and mammalian stages of L. major and T. brucei (Fig. 3Cii and Dii).\nExpression of the LmPPEF protein was analysed by immunoblotting, using affinity-purified antibodies raised against the C-terminal 236 residues of the recombinant protein (expressed from construct LmPPEF-Cterm1, Fig. 1B). These antibodies recognised a single polypeptide band of 109\u00a0kDa on immunoblots of whole L. major parasite lysates, correlating with the size of the deduced open reading frame of gene LmjF12.0660 (Fig. 4A). Whole cell lysates from L. major procyclics, metacyclics and amastigotes were then analysed and a single 109\u00a0kDa protein detected in all three life cycle stages (Fig. 4B). However, no signal was detected in T. brucei procyclic or bloodstream form parasite lysates using the same anti-LmPPEF (data not shown). This suggests that this C-terminal antibody does not cross react with TbPPEF (despite the 51% amino acid conservation in this domain) and\/or that TbPPEF is expressed at low levels during the parasite life cycle.\n3.4\nLmPPEF is not a calcium-binding protein\nGiven the degeneracy of EF-hand motifs in the C-terminal domains of the kinetoplastid PPEFs, and the absence of N-terminal IQ calmodulin binding motifs, we investigated whether LmPPEF can bind calcium, either as a recombinant or wild type protein, in a mobility shift assay. In this analysis, target and control proteins were separated by electrophoresis through denaturing gels in the presence of either Ca2+ or EGTA. Fig. 5A shows the positive (calreticulin) and negative (BSA) control proteins after separation: the migration of BSA was similar in both gel types whereas the migration of calreticulin was significantly altered on electrophoresis through Ca2+ as compared to EGTA, with >50% showing a mobility shift. To analyse the behaviour of LmPPEF in this assay, the C-terminal 351 residues, containing all three EF-hand like motifs, were expressed (from construct LmPPEF-Cterm 2) as a 40\u00a0kDa N-terminally His-tagged protein in L. major. Comparing the mobility of this protein and wild type LmPPEF in whole cell lysates, in the presence of Ca2+ or EGTA, revealed no significant differences in migration, suggesting that LmPPEF does not bind calcium (Fig. 5B).\nCell fractionation of total L. major proteins was also carried out, to analyse the relative distribution of LmPPEF between membrane and cytoplasm and to investigate whether this distribution was altered in the presence or absence of calcium. Wild type parasites were lysed in either PBS alone, PBS plus 1\u00a0mM CaCl2 or PBS plus 1\u00a0mM EGTA and separated by electrophoresis. Cell fractionation carried out in PBS alone revealed that LmPPEF is predominantly membrane-associated, with \u223c20% of the protein detected in the soluble or cytoplasmic fraction (Fig. 5B). The presence or absence of calcium did not alter this membrane versus cytoplasmic distribution, again suggesting that the degenerate EF-hands in LmPPEF are unlikely to bind calcium. A similar conclusion emerged from 45Ca overlay experiments with both wild type and recombinant protein (data not shown).\n3.5\nThe N-termini of recombinant and wild type kinetoplastid PPEFs are substrates for acylation in vivo\nGiven the presence of N-myristoylation motifs together with cysteine residues predicted to be palmitoylation sites at the N-termini of kinetoplastid PPEFs (Tables S1\u2013S3, supplementary data), we firstly confirmed that these proteins could act as templates for NMT in an E. coli co-expression assay [8]. In these experiments, plasmids expressing NMT and TbPPEF as substrate were co-expressed in the presence of [3H]-myristoyl CoA and incorporation of radioactivity into myristoylated product detected by autoradiography following SDS-PAGE (Fig. 6A). Using NMTs from both L. major and T. brucei with TbPPEF, these results demonstrate radiolabelling of a \u223c106\u00a0kDa product in the presence of each enzyme, indicative of the transfer of myristate to TbPPEF (as previously demonstrated for HASPA and TbARL1) [8,14]. While the amount of radiolabelled product appears greater with LmNMT rather than TbNMT in this experiment, this is likely due to an artefact of loading (as indicated by the higher amount of NMT-myristoyl CoA binary complex loaded on to the gel) rather than substrate specificity.\nTo demonstrate acylation in parasites in vivo, metabolic labelling experiments were carried out with L. major transgenic lines expressing the first 37 N-terminal residues of PPEF fused with GFP (Lm37WT-GFP, Fig. 1Bii) or a mutated version, in which the Gly in position 2, essential for N-myristoylation, is substituted with Ala (Lm37G2A-GFP, Fig. 1Bii). These parasites express equivalent amounts of the two fusion proteins, as indicated by immunoblotting with anti-GFP and anti-NMT (Fig. 6B). Following radiolabelling with [3H]-myristate or [3H]-palmitate, the fusion proteins were immunoprecipitated using anti-GFP and subjected to SDS-PAGE and autoradiography. This analysis detected a [3H]-myristate-labelled protein of 35\u00a0kDa, corresponding to the predicted molecular mass of the Lm37WT-GFP protein, which was absent from parasites expressing Lm37G2A-GFP lacking the residue required for N-myristoylation (Fig. 6C). Similarly, autoradiography of parasites labelled with [3H]-palmitate revealed a single radiolabelled protein of the same molecular mass, corresponding to [3H]-palmitate-labelled Lm37WT-GFP. This protein was again absent in parasites expressing Lm37G2A-GFP. These data correlate with similar experiments with the dual acylated HASPB protein, in which loss of the residue required for N-myristoylation also prevented palmitoylation, due to mislocalisation of the protein within the cell [11].\nThe experiments in Fig. 6A and C confirm that TbPPEF and LmPPEF can be acylated in vivo, both in a heterologous cell system and in live parasites. To confirm that endogenous LmPPEF is N-myristoylated in vivo, anti-LmPPEF was used in immunoprecipitations from parasite lysates following metabolic labelling with [3H]-myristate. As shown by autoradiography in Fig. 6D, a radiolabelled protein of \u223c109\u00a0kDa, corresponding to the predicted molecular mass of LmPPEF, was detected in the lysate from wild type L. major parasites, while no proteins were detected following precipitation with protein A beads alone. Radiolabelled parasites expressing LmPPEF-TAP (Fig. 1Bi) were also subjected to immunoprecipitation with anti-LmPPEF, generating two detectable [3H]-myristate-labelled proteins in the lysate: the smaller corresponding to the 109\u00a0kDa native LmPPEF and the larger weaker \u223c129\u00a0kDa band corresponding to the predicted size of the LmPPEF-TAP fusion protein.\n3.6\nLmPPEF is localised to the endomembrane system of L. major\nImmunofluorescence microscopy with the affinity-purified LmPPEF antibody was used to investigate the location of LmPPEF in procyclic L. major (Fig. 7A). Overlay of the fluorescent signal with DAPI (for identification of the nucleus and kinetoplast) indicated a punctate distribution of signal within the cytosol, reminiscent of the endocytic system, together with some concentration in the region of the flagellar pocket. These observations are consistent with the predominantly membrane association of LmPPEF revealed by cellular fractionation (Fig. 5). Counter-staining with anti-LCB2, which recognises an ER-resident sub-unit of serine palmitoyltransferase [38] showed no overlap with PPEF (Fig. 7B) while labelling with the endocytic lipophilic tracer, FM4-64, demonstrated some co-localisation with the flagellar pocket and endosomes (Fig. 7C). PPEF staining was excluded from the nucleus, plasma membrane and flagellum.\nWe attempted to express and analyse GFP-fusions with either full-length wild type LmPPEF or the N-terminal 473 residues alone (lacking the C-terminal EF-hands). However, these constructs did not express after transfection into L. major, probably due to the large sizes of the recombinant proteins generated. As an alternative approach, given that the N-terminus of LmPPEF is a target for acylation (Fig. 6), the N-terminal GFP transgenic parasite lines (see Fig. 1B) were used to investigate the role of these modifications in intracellular localisation of the fusion proteins. Counter-staining with FM4-64 was also utilized to detect the flagellar pocket in these analyses. Transfected parasites expressing the first 37 residues of LmPPEF fused to GFP (Lm37WT-GFP) targeted the fusion protein predominantly to the flagellum and the flagellar pocket, with weak staining only in the cell body (Fig. 7D). Loss of the N-myristoylation site by mutation of Gly to Ala (Lm37G2A-GFP) resulted in homogeneous distribution of cytosolic fluorescence throughout the parasite (Fig. 7E), in a pattern almost identical to the localisation of GFP alone (data not shown). Separate mutation of the putative palmitoylation site alone, by substituting Cys-3 with Ser (Lm37C3S-GFP), resulted in concentrated signal in a region adjacent to the flagellar pocket most likely to be the Golgi (Fig. 7F). Expression of the double mutant Lm37G2A, C3S-GFP, lacking both acylation sites, caused the fusion protein to remain localised within the cytosol (Fig. 7G). These observations correlate with those previously observed using similar N-terminal mutations of the L. major HASPB protein [11], suggesting that the wild type fusion protein (Lm37WT-GFP) requires N-myristoylation in the cytosol to reach the Golgi region, where palmitoylation further modifies the protein for trafficking to other locations, predominantly the flagellum. Clearly, the localisations of these GFP fusion proteins are distinct from the endocytic location of wild type LmPPEF, indicating that other regions and\/or signals within this large protein are important for subcellular localisation. More generally, these data suggest that N-terminal dual acylation might act as a primary signal in targeting proteins to the flagellum in Leishmania, as exemplified by HASPB, SMP-1 and PPEF [11,19 and this study].\n4\nDiscussion\nIn this paper, as part of a wider study of the downstream targets of NMT, we describe the PPEF-like protein phosphatases that are encoded by single copy genes in three kinetoplastid species. These phosphatases were identified following in silico genome analysis that predicted a subset of kinetoplastid proteins as \u201chigh confidence\u201d substrates for NMT. Within this subset were several proteins already confirmed as N-myristoylated in L. major or T. brucei, together with a number of species-specific molecules and a larger group of proteins found in all three species, many of no known function. We predict that all of these \u201chigh confidence\u201d putative substrates are acylated in vivo but do not exclude other \u201cmedium confidence\u201d proteins as candidates for NMT modification.\nLmPPEF and TbPPEF, studied in detail here, are constitutively expressed, membrane-associated acylated proteins. LmPPEF is found in the endocytic system and the flagellar pocket. Conservation of key features and residues within the catalytic domains, which have been modelled against the mammalian PP1 catalytic region, suggest that these proteins are active enzymes (although this has not yet been demonstrated biochemically). Unlike these conserved central regions, however, divergence in the N- and C-terminal domains has led to loss of the IQ calmodulin binding motif and degeneration of the EF-hands, features that characterise the higher eukaryotic PPEFs [39,40]. Thus, there is no evidence that the kinetoplastid proteins are regulated by cytoplasmic Ca2+ levels and\/or calmodulin, in contrast to the demonstrated interactions of both human PPEF and Drosophila RdgC with these regulators [39,40]. The analysis in Fig. 5 would support this conclusion.\nAlthough PPEF-like proteins have been characterised in a number of species, the functions of these unusual phosphatases are not well understood. The Drosophila RdgC protein is implicated in dephosphorylation of rhodopsin, a G protein-coupled receptor (GPCR) that initiates vertebrate and invertebrate phototransduction. However, mutant mice with targeted disruptions in each of their two PPEF genes show no retinal degeneration and normal rhodopsin dephosphorylation kinetics, suggesting interspecies functional differences despite high similarities in protein sequence [41].\nCompletion of the Tri-Tryp genome projects has confirmed earlier predictions that these parasites have \u2018stream-lined\u2019 signal transduction mechanisms as compared to their higher eukaryotic multicellular counterparts [42]. The lack of several classes of signalling molecules, including serpentine receptors, heterotrimeric G proteins and most classes of catalytic receptors, contrasts with the presence of a large and diverse family of kinase and phosphatases, suggesting complex cellular interactions [43]. While the conservation and expression of PPEF genes in the Kinetoplastida strongly suggest a functional role for these unusual phosphatases, it is clear that this is unlikely to be similar to that demonstrated for the RdgC protein. We have perturbed TbPPEF expression by inducible RNAi in both bloodstream and procyclic stages of T. brucei, which results in a partial growth defect under normal culture conditions (data not shown). However, it cannot be discounted in these experiments that a more extreme phenotype is masked by sufficient residual expression to support enzyme activity. Functional analysis of conditional gene deletion mutants will be required, together with substrate identification, to delineate roles for the kinetoplastid PPEFs in parasite viability.","keyphrases":["bioinformatics","n-myristoylation","protein phosphatases","palmitoylation","ppef, protein phosphatase with ef-hands","nmt, n-myristoyl transferase","bsf, bloodstream form","pcf, procyclic form"],"prmu":["P","P","P","P","R","R","M","M"]}
{"id":"Hum_Reprod-1-1-2387221","title":"A distinct cohort of the TGF\u03b2 superfamily members expressed in human endometrium regulate decidualization\n","text":"BACKGROUND Successful blastocyst implantation requires the differentiation of human endometrial stromal cells (HESC), a process known as decidualization. Activin A, a transforming growth factor \u03b2 (TGF\u03b2) superfamily member, enhances HESC decidualization and localizes to decidual cells in human endometrium. Other TGF\u03b2 superfamily members, including BMP2, BMP4, BMP7, GDF5, GDF8, GDF11, TGF\u03b2s and Nodal, may also play a role during decidualization. This study aimed to identify these TGF\u03b2 family members in human endometrium, and to determine whether they are involved in human decidualization.\nIntroductionThroughout each menstrual cycle the human endometrium undergoes morphological and physiological changes, in preparation for pregnancy should the cycle include conception (Loke et al., 1995). An absolute requirement for successful implantation is the differentiation and proliferation of endometrial stromal cells into enlarged, phenotypically different decidual cells, a process termed endometrial decidualization (Tang et al., 1994; Salamonsen et al., 2003). This process is initiated near the spiral arterioles (Bell, 1991), and occurs under the influence of progesterone (Psychoyos, 1973), although cAMP appears to be essential for priming the endometrial stromal cells to the actions of progesterone; hence both pathways are absolutely required for decidualization (Gellersen and Brosens, 2003). It is now clear that the decidualization response is mediated by a complex array of bioactive molecules, including IL-11, prostaglandin E2, relaxin and activins (Frank et al., 1994; Robb et al., 1998; Jones et al., 2002; Dimitriadis et al., 2005).The transforming growth factor \u03b2 (TGF\u03b2) superfamily is a large family of proteins that encompasses the sub-families TGF\u03b2s, activins, bone morphogenetic proteins (BMPs) and growth differentiation factors (GDFs). Overall, this superfamily of proteins exhibits functional diversity, with biological roles in cell differentiation, proliferation, apoptosis and tissue remodelling, all consistent with various reproductive processes (Jones et al., 2006).Activins consist of two \u03b2 subunits, \u03b2A and \u03b2B, that homo\/heterodimerize to form activin A, activin B and activin AB, respectively. The activin \u03b2 subunits are produced in the glandular epithelium during the proliferative and secretory phases, but in stromal cells only following decidualization during the mid-late secretory phase (Otani et al., 1998; Jones et al., 2000; Mylonas et al., 2004). High levels of dimeric activin A are secreted by cAMP-treated endometrial stromal cells. Addition of activin A to decidualizing cells in vitro significantly increased the secretion of prolactin (PRL) and IGFBP-1 (decidual cell markers), suggesting activin A drives decidualization (Jones et al., 2002; Tierney and Giudice, 2004). This decidualization response can be neutralized by follistatin, the naturally occurring activin antagonist (Jones et al., 2002; Tierney and Giudice, 2004). In addition to its tight regulation of activin (Shimonaka et al., 1991; Schneyer et al., 1994), follistatin can also bind and regulate other TGF\u03b2 members including the BMPs and GDFs (Table\u00a0I).Table I.Inhibitors of TGF\u03b2 superfamily: ligand\u2013receptor blocking.LigandFollistatinActivin-M108ASB431542Binds ligands and blocks association with type II receptorsActivin type II receptor (ActRIIA\/B) antagonistInhibitor of TGF\u03b2 and activin type I receptorsActivin A++++ (Schneyer et al., 2003)++ (Harrison et al., 2004)+++ (Inman et al., 2002)Activin B++ (Schneyer et al., 2003)+++a+++ (Inman et al., 2002)GDF 8++ (Amthor et al., 2004)+++a+++ (Inman et al., 2002)GDF11++ (Gamer et al., 1999)+++a+++ (Inman et al., 2002)BMP2+ (Iemura et al., 1998)\u2212\u2212BMP4+ (Iemura et al., 1998; Glister et al., 2004)\u2212\u2212BMP7+ (Iemura et al., 1998; Glister et al., 2004)(\u2212\/+)\u2212GDF5(+)\u2212\u2212Nodal\u2212(+++)+++ (Inman et al., 2002)TGF\u03b21\u2212\u2212+++ (Inman et al., 2002)TGF\u03b22\u2212\u2212+++ (Inman et al., 2002)TGF\u03b23\u2212\u2212+++ (Inman et al., 2002)aC.A. Harrison (unpublished observations); (), anticipated activity.BMPs have been identified in the rodent uterus (Cunningham et al., 1995; Ozkaynak et al., 1997; Fitzpatrick et al., 1998; Zhao et al., 1999; Ying and Zhao, 2000; Erickson et al., 2004; Lee et al., 2007; Li et al., 2007). In pregnant mice the temporospatial localization of BMPs 2, 4, 6 and 7 suggested roles during pregnancy (Ying and Zhao, 2000). BMP2 is present within the decidual area at implantation sites (Ying and Zhao, 2000; Li et al., 2007) and plays a role in decidualization in vitro (Li et al., 2007). Importantly, conditional ablation of uterine BMP2 in mice resulted in ineffective decidualization and disrupted pregnancy outcome (Lee et al., 2007). In a human model of endometrial stromal cell (HESC) decidualization in vitro, BMP2 mRNA increased during decidualization and addition of BMP2 to cultures accelerated PRL mRNA expression (a measure of the extent of decidualization). BMP2 protein has not been examined in vivo in human endometrial tissue. The only GDFs identified in the uterus are GDF9 and GDF10, detected by northern analysis in both human and mouse (Cunningham et al., 1995; Fitzpatrick et al., 1998; Zhao et al., 1999): no studies have shown GDF proteins.TGF\u03b2 isoforms (1\u20133), which cannot bind follistatin, have also been localized in human endometrial stroma (Jones et al., 2006), although it is not clear whether any isoform is specific to decidualized cells or increases as the cells decidualize. The TGF\u03b2s transmit signal through the TGF\u03b2 type I, II and III receptors, all of which are present on endometrial stromal cells (Chegini et al., 1994; Dumont et al., 1995). Microarray studies have demonstrated increases in both TGF\u03b21 and TGF\u03b22 mRNA with HESC decidualization in vitro (Popovici et al., 2000; Tierney et al., 2003), while in women treated with medroxyprogesterone acetate (MPA) for 10 days, stromal mRNA and protein increased for TGF\u03b23 but not TGF\u03b21 (Reis et al., 2002). Whether the TGF\u03b2s play a functional role during human decidualization remains unknown.Given that the TGF\u03b2 superfamily of proteins are such common and crucial differentiative and proliferative factors, it is surprising that very few members of the family, including the GDFs and BMPs, have been examined in the human endometrium, particularly in the mid-secretory phase when differentiation of stromal cells is initiated. The high level of redundancy between TGF\u03b2 family members and the promiscuity of receptor\u2013ligand interactions would suggest that it is likely that a number of other family members are involved in decidualization and in the other processes occurring in this highly dynamic tissue.In this study, the presence of BMPs (BMP2, BMP4, BMP7), GDFs (GDF5, GDF8\/Myostatin, GDF11), Nodal and TGF\u03b2s were examined in vivo by immunohistochemistry in secretory phase endometrial tissue, and in vitro for mRNA expression in both non-decidualized and decidualized HESC. Inhibitors with different specificities were administered to HESC to elucidate whether activin is the major family member driving decidualization or whether other family groupings (BMPs, GDFs, TGF\u03b2s) might equally contribute to the process. As these are secreted factors, individual ligands were measured in conditioned medium from non-decidualized and decidualized HESC to assess whether they increased with decidualization. The effect of the secreted ligands on decidualization was also examined. It is important to identify which TGF\u03b2 members are important during decidualization since this process is critical for implantation and the establishment of pregnancy.\nMaterials and MethodsTissue collectionsEndometrial biopsies (n = 18) were collected by dilatation and curettage from fertile women who were scheduled for tubal ligation or were undergoing testing for tubal patency. Tissues were assessed by a pathologist and had no obvious endometrial pathology. The women had no steroid treatment or other medication for at least 2\u20133 months before the collection of tissue. Written and informed consent was obtained from all women participating in the study, and the protocols were approved by Monash Medical Centre Human Ethics Committee.ImmunohistochemistryImmunohistochemical analysis was performed using a total of nine endometrial tissue biopsies from fertile women, confirmed by Noyes criteria (Noyes et al., 1975) as mid-late secretory phase tissue (POD 6\u201312). For each of the antibodies, activin \u03b2A and \u03b2B, BMP2, BMP4, BMP7, GDF5, GDF8, GDF11, Nodal and TGF\u03b21, n = 4\u20135 different tissue biopsies were used. For immunolocalization in first trimester placental tissue (kindly provided by Professor Euan Wallace, Obstetrics and Gynaecology, Monash University, Melbourne, Australia), n = 2 different biopsies were used per antibody. Briefly, 5 \u00b5m sections of formalin-fixed, paraffin-embedded tissues were dewaxed and rehydrated. For each antibody an antigen retrieval step was required, which involved microwave exposure or trypsin digestion (0.01% in CaCl2for 10 min at 37\u00b0C). Endogenous hydrogen peroxidase activity was quenched using 3% H2O2 in dH2O for 10 min at room temperature. Non-specific binding was prevented by pre-incubation of tissue sections with a non-immune block [5% fetal calf serum (FCS), 2% normal human serum in 0.1% Tween\/Tris-buffered saline (TBS) with addition of 10% normal horse serum for BMP2, BMP4, BMP7, GDF5, GDF8; 10% normal swine serum for Nodal; normal goat serum for activin \u03b2A and \u03b2B, GDF11, TGF\u03b21]. Primary antibodies were against activin \u03b2A and activin \u03b2B (400 and 600 \u00b5g\/ml, respectively; both gifts provided by W. Vale, Salk Institute, La Jolla, CA, USA); BMP2, BMP4, BMP7, GDF5, GDF8, Nodal (200 \u00b5g\/ml; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); GDF11 (1 mg\/ml; Orbigen, San Diego, CA, USA); TGF\u03b21 (\u03bcg\/ml; Abcam, Cambridge, UK). Specificity for these antibodies has been previously published (Nadiri et al., 2004; Kiyono and Shibuya, 2003; Mendler et al., 2000; McPherron et al., 1999; Garba and Relinger, 2001). For GDF5 and Nodal, the specificity has been shown by western blot analysis (unpublished observation). Antibodies were diluted to 2\u20134 \u00b5g\/ml in the non-immune block and applied overnight (18\u201320 h) at 4\u00b0C. A non-immune goat\/rabbit\/mouse IgG (R&D Systems Incorporated, Minneapolis, MN, USA) was used at a matching concentration to the primary antibody and was included for each tissue. After stringent washing with high salt TBS and 0.6% Tween 20 (BioRad Laboratories, Hercules, CA, USA), detection of positive binding was performed by the sequential application of the appropriate secondary antibody diluted in non-immune block and avidin\u2013biotin\u2013peroxidase conjugate (Dako, Glostrup, Denmark). This was followed by the substrate diaminobenzidine (Dako) for between 3 and 5 min. Sections were counterstained with Harris' hematoxylin (Sigma Chemical Company, St Louis, MO, USA), dehydrated, and mounted from Histosol with DPX mounting medium (BDH Laboratory Supplies, Poole, UK). Immunostaining was analysed by two independent observers for staining intensity and heterogeneity in each of the endometrial compartments (glandular and luminal epithelium; stroma, including decidualized stromal cells; vasculature).Stromal cell isolation and cultureTissue biopsies ranging from Days 10\u201321 were used for in vitro decidualization studies. HESC were isolated from tissue by enzymatic digestion and filtration as described previously (Dimitriadis et al., 2002). Briefly, tissue was finely minced with scissors and digested by bacterial collagenase type III (Worthington Biochemical Corporation, Freehold, NJ, USA) at a concentration of 45\u2003IU\/ml, in the presence of 3.5 \u00b5g\/ml deoxyribonuclease (DNase; Boehringer Mannheim Biochemica, Mannheim, Germany). After 30\u201345 min of agitation at 37\u00b0C, the digested tissue was filtered sequentially through 45 and 10 \u00b5m nylon filters to remove glands. Cell suspensions were centrifuged at 1500 rpm for 5 min. Cells were plated at a density of 5 \u00d7 106 in a 75 cm2 flask and grown to confluency in DMEM and Ham's F12 medium (1:1) (DMEM\/F12) (Trace Biosciences, Sydney, Australia), supplemented with 1% PSF (penicillin, streptomycin and fungizone) (Commonwealth Serum Laboratories, Melbourne, Australia) and 10% charcoal stripped FCS (csFCS) (Thermo Scientific, Maple Plain, USA). Approximately 30 min after plating, medium containing non-attached epithelial cells was removed and replaced with fresh medium. This results in a >97% pure stromal cell culture (Dimitriadis et al., 2002).In vitro decidualizationConfluent HESC were rinsed with phosphate-buffered saline, trypsinized and replated into 24-well plates using DMEM\/F12 and 10% csFCS. Once confluent, HESC were washed with DMEM\/F12 and the medium replaced with a serum-free medium containing DMEM\/F12 and a serum-free mix (SFM) including transferrin (10 \u00b5g\/ml; Sigma), sodium selenite (25 ng\/ml; Sigma), linoleic acid (10 nmol\/L; Sigma), bovine serum albumin (0.1%; Sigma) and insulin (5 \u00b5g\/ml; Actrapid, Novo-Nordisk Pharmaceuticals Pty Ltd, Sydney, Australia) for 48 h prior to treatment addition. HESC were decidualized by two distinct methods as previously described (Dimitriadis et al., 2005). For the inhibitor, mRNA expression and protein secretion studies, 0.5 mM cAMP (Sigma) was added to medium for 4\u20136 days. Alternatively, when exogenous proteins were added, cells were decidualized with E2 (10\u22128 M; Sigma) and MPA (10\u22127M; Sigma) for 8 days. All media were replaced every 48\u201372 h.RNA extraction and cDNA synthesisTotal RNA was extracted from non-decidualized and decidualized HESC from two separate biopsies at Day 4\u20136 using the RNeasy Minikit (Qiagen Sciences, Germantown, MD, USA), according to the manufacturer's instructions. RNA from positive control tissues was extracted by homogenization in Trizol reagent (Qiagen Sciences, Clifton Hill, Victoria, Australia), according to the manufacturer's instructions, with the exception of an additional chloroform extraction step to minimize carryover of phenol into the precipitate. The control tissues used were: term placenta (activin \u03b2A, \u03b2B, GDF11, TGF\u03b21, -2, -3); pregnant mouse endometrium (BMP2); mature rat ovary (BMP4, GDF5); cycling mouse endometrium (BMP7); mouse heart (GDF8); JEG3 cells (Nodal). All samples were treated with RNase-free DNase (Ambion, Austin, TX, USA) to remove the possibility of genomic DNA contamination. RNA samples were then analysed by spectrophotometry to determine RNA concentration, yield and purity. Total RNA (1 \u00b5g) was reverse transcribed at 46\u00b0C for 1.5 h in 20 \u00b5l reaction mixture using 100 ng random hexanucleotide primers and 6 IU AMV reverse transcriptase (Roche, Castle Hill, Australia) in the presence of cDNA synthesis buffer (Roche), 1 mmol\/l dNTPs (Roche), 10 mmol\/l dithiothreitol (Roche), 10 IU ribonuclease inhibitor (RNasin; Promega, Annandale, Australia). The resultant cDNA mixtures were heated at 95\u00b0C for 3 min before storage at \u221220\u00b0C. Negative controls were performed by omission of reverse transcriptase. Triplicate RNA samples were reverse transcribed in triplicate for each condition (non-decidualized and decidualized) with the successful conversion to cDNA monitored by 18S expression (data not shown).RT\u2013PCRMessenger RNA expression for all named TGF\u03b2 superfamily ligand members was determined using a conventional PCR block cycler (Hybaid, Middlesex, UK). All ligands (excluding Nodal), used a 1 \u00b5l aliquot of RT product, to be amplified in a total volume of 40 \u00b5l using 4 \u00b5l of RT single strength PCR buffer (10 mmol\/l Tris\u2013HCl, 1.5 mmol\/l MgCl2, 50 mmol\/l KCl, pH 8.3; Roche), 2.5 mmol\/l dNTPs (Gibco, Melbourne, Australia), 0.5 pmol\/\u00b5l sense and antisense primers (Sigma Genosys Australia Pty Ltd, Castle Hill, Australia) and 2.5 IU Taq DNA polymerase (Roche). For Nodal, 1 \u00b5l of RT was amplified in a total of 50 \u00b5l using the KOD-Taq PCR kit (Bioron, Germany), which included 10\u00d7 PCR KOD Hot Start buffer, 2 mM dNTPs, 0.5 pmol\/\u03bcl primers, 2 mM MgSO4 and 2.5 IU Taq DNA polymerase (Roche). For all ligands, the PCR was performed in three stages as follows: the first stage involved 94\u00b0C for 5 min, x C for 1 min, where x is the annealing temperature for the individual primer pairs (see Supplementary data) and 72\u00b0C for 3 min; the second stage involved 35\u201340 cycles of 94\u00b0C for 1 min, x C for 1 min, and 72\u00b0C for 1 min; and the final stage was 72\u00b0C for 7 min. PCR products including positive controls were analysed by electrophoresis on a 2% agarose gel (Roche) and stained with ethidium bromide. Bands of interest were excised from the gel, purified (DNA purification kit, Qiagen) and directly sequenced to confirm their identity.Inhibitor experimentsFor examination of the effect of inhibitors on decidualization, cells (from n = 10 biopsies) were plated at a density of 2.5 \u00d7 105\/well in 24-well plates until confluent, and then equilibrated in serum-free medium containing SFM for 48 h. This was designated Day 0 and first day of treatments. HESC were exposed to varying doses of either the inhibitor Activin-M108A (M108A) (0.39 1.56, 6.25 and 25 nM) (Harrison et al., 2004, 2006) or the inhibitor SB431542 (1.25, 2.5, 5 and 10 \u00b5M) (TOCRIS Bioscience, Northpoint, UK). Following 1 h incubation with either inhibitor, 0.5 mM cAMP (Sigma) was added as decidualizing stimulus. Each treatment was performed in triplicate wells of a 24-well plate and the experiments ran for 4\u20136 days with media collection and replenishment (including inhibitors) every 48\u201372 h. In each case, the final medium collection represented the final 48 h. Each experiment included a medium-only and a cAMP-only control. Cells were photographed for morphological analysis and cells from triplicate wells were pooled for trypan blue cell exclusion to check cell viability at the end of each experiment. Five separate cultures were conducted for each inhibitor.TGF\u03b2 superfamily assaysCulture medium from triplicate wells from three tissue biopsies (n = 3 separate decidualization experiments) were collected, pooled and concentrated 5-fold to measure BMP2 (ELISA; R&D Systems), BMP4, BMP7, TGF\u03b21 and TGF\u03b22 (ELISA; Ray BioTech., Norcross, GA, USA) according to the manufacturer's instructions. Mean sensitivities of the assays were: BMP4, 15 pg\/ml, BMP7, 10 pg\/ml, TGF\u03b21, 80 pg\/ml, TGF\u03b22, 15 pg\/ml, with intra-assay CV <10% and inter-assay CV <12% for each assay. Mean sensitivity for BMP2 was 11 pg\/ml and intra- and inter-assay variabilities were 2.6 and 6.3%, respectively. TGF\u03b21 and TGF\u03b22 ELISAs measured only the activated protein form and required activation steps prior to assay. Dimeric activin A secretion from stromal cells was measured by Activin A Immunofluorometric Assay (IFMA) (Harrison et al., 2006). The working range of the assay is 0.03\u20133 ng\/well, with a sensitivity of 0.03 ng\/well. All assays were read at 450 nm.Addition of exogenous BMP2 and TGF\u03b21 during decidualizationHESC from three separate cultures were decidualized using E2 and MPA as described above, in the presence of varying doses of either recombinant human (rh) BMP2 (5, 50, 500 ng\/ml; R&D Systems) or TGF\u03b21 0.5, 5, 50 ng\/ml; PeproTech Inc., NJ, USA) added every 48 h with media and decidualizing stimulus replenishment. At Day 8, cells were morphologically assessed and conditioned media were collected for PRL measurement.PRL and protein assaysPRL production by HESC was assayed in duplicate by ELISA (Bioclone Australia Pty Ltd, Sydney, Australia) to determine the extent of decidualization (Dimitriadis et al., 2002). Media collected at the end of the experiment were concentrated 5-fold for PRL measurement. A quality control sample (culture medium from a single endometrial cell culture) was included in every assay. The lower detection limit of the assay was 50 mIU\/l. The inter- and intra-assay variablilities were 5.3 and 3.0%, respectively. PRL concentrations (mIU\/l) were corrected for the amount of protein (\u03bcg\/\u03bcl) determined using the Bradford reagent.Statistical analysisData were expressed as mean \u00b1 SEM. Statistical significance for inhibitor studies was determined following confirmation of normal distribution, by one-way ANOVA followed by Dunnett's multiple comparisons test. Data from ELISA and recombinant protein studies were analysed by Student's t-test. A P-value of <0.05 was considered statistically significant.\nResultsLocalization of TGF\u03b2 superfamily ligands in human endometrium and first trimester deciduaAll TGF\u03b2 superfamily members studied, except Nodal, were detected in mid-late secretory endometrium and first trimester decidua. As previously described (Jones et al., 2000), activin A and activin B were localized to the morphologically distinct decidual cells (Fig.\u00a01A and B), glandular and luminal epithelial cells and some leukocytes (data not shown). Some glands had punctate staining for BMP2 (Fig.\u00a01C), but BMP2 was most strongly stained in decidual cells in mid-late secretory endometrium (Fig.\u00a01D). In first trimester decidua, BMP2 protein was detected in both the decidual cells (Fig.\u00a01E, inset) and glandular epithelium, while the vessels were devoid of stain (Fig.\u00a01E). GDF5 protein was observed in the decidualized stromal cells (Fig.\u00a01F and G) and was very low in the glands (Fig.\u00a01F) and luminal epithelium (data not shown). Similarly, GDF5 was very strongly expressed by the decidua of first trimester placenta, as well as the glandular epithelium (Fig.\u00a01H, inset). TGF\u03b21 was present in decidualized stromal cells (Fig.\u00a01I and J), with minimal staining evident in the glandular epithelium (Fig.\u00a01I). As previously described (Graham et al., 1992), TGF\u03b21 was detected in decidual cells and extravillous trophoblasts of first trimester placenta (Fig.\u00a01K). BMP4 was not detected in the glandular or luminal epithelium, but was present in the cytoplasm of both non-decidualized and decidualized stromal cells (Fig.\u00a01L and M). However, in first trimester placenta, BMP4 was reduced in the decidual cells, but strongly produced by the glandular epithelium (Fig.\u00a01N, inset). BMP7 was detected in the stroma and not in the glands in mid-late secretory endometrium (Fig.\u00a01O). Stromal staining for BMP7 did not appear to be decidual cell specific, however, in highly decidualized tissue stronger \u2018vesicle staining\u2019 was evident (Fig.\u00a01P). Similar but faint punctate staining for BMP7 was detected around spiral arterioles of first trimester placenta, but not specifically in decidual cells (Fig.\u00a01Q, inset). BMP7 was also strongly localized to glandular and luminal epithelium (Fig.\u00a01Q). GDF8 immunostaining in mid-late secretory endometrium showed minimal glandular and luminal epithelial staining, and intense stromal cell cytoplasmic staining, in both non-decidualized and decidualized cells (Fig.\u00a01R). GDF11 immunolocalized to glandular epithelium and endothelial cells, but was very low in the stroma (Fig.\u00a01S). GDF8 and GDF11 in first trimester placenta showed similar staining patterns to BMP4 (data not shown). No immunoreactive Nodal was detected in late secretory endometrium (Fig.\u00a01T), although the immunostaining protocol was verified by positive staining in term placenta (not shown).Figure\u00a01:Localization of TGF\u03b2 superfamily members during decidualization in mid-late secretory endometrium and first trimester decidua.Photomicrographs are representatives of immunostaining for activin A (A), activin B (B), BMP2 (C\u2013E), GDF5 (F\u2013H), TGF\u03b21 (I\u2013K), BMP4 (L\u2013N), BMP7 (O\u2013Q), GDF8 (R), GDF11 (S), Nodal (T). In mid-late secretory endometrium; scale bar = 100 \u00b5m (C, F, I, L, O, R, S); scale bar = 10 \u00b5m (D, G, J, M, P, T). In first trimester decidua; scale bar = 200 \u00b5m (E, H, K, N, Q); inserts show higher power images of same tissue (E, H, K, N, Q). Negative controls for each antibody are shown in inserts (C, F, I, L, O, R, S, T). Arrows highlight decidualized (D), non-decidualized (ND) stromal cells and glands (G).TGF\u03b2 superfamily mRNA expression in non-decidualized and decidualized HESCBy RT\u2013PCR, mRNA expression was evident for activin \u03b2A, activin \u03b2B, BMP2, BMP4, BMP7, GDF5, GDF8, GDF11, TGF\u03b21, TGF\u03b22 and TGF\u03b23 in both non-decidualized and decidualized HESC (Fig.\u00a02). Nodal mRNA was undetectable in both. As conventional RT\u2013PCR was used, only the presence\/absence of each gene was assessed. Positive control PCR products (Fig.\u00a02; see supplementary data) confirmed sequence identity and no signals were detected when reverse transcriptase was omitted (Fig.\u00a02).Figure\u00a02:mRNA expression for TGF\u03b2 superfamily members by decidualized and non-decidualized HESC.HESC were either non-decidualized or decidualized with cAMP for 7 days. Total RNA was extracted from cells for RT\u2013PCR. Representative products demonstrating Activin \u03b2A, Activin \u03b2B, BMP2, BMP4, BMP7, GDF5, GDF8, GDF11, TGF\u03b21, TGF\u03b22, TGF\u03b23 and Nodal mRNA are shown in non-decidualized (ND) and cAMP-decidualized (D) HESC; +ve=positive control; \u2212RT=negative control. Data shown is from a single experiment, which is representative of 2 independent culture experiments.Activin-M108A and SB431542 significantly decreases PRL secretion during HESC decidualizationTo establish whether HESC decidualization could be blocked or reduced, the newly developed activin type II receptor antagonist, Activin-M108A or the type I receptor kinase inhibitor, SB431542 were used in an in vitro decidualization model with PRL secretion, a marker of decidualization, as the end-point. These inhibitors affect activin and various other TGF\u03b2 family members (see Table\u00a0I). Cells maintained in medium alone (non-decidualized) showed non-detectable PRL levels over the final 48 h of culture (Fig.\u00a03). In contrast, cells treated with cAMP secreted PRL (mean 372.6 \u00b1 145.5 mIU\/l), and morphologically changed from elongated spindle-shaped cells to typical enlarged polygonal cells demonstrating successful decidualization, as previously described (Dimitriadis et al., 2002). Stromal cells cultured with cAMP and Activin-M108A showed a significant dose-dependent decrease in PRL secretion at 0.39, 1.56 and 6.25 nM with no further decrease at 25 nM (Fig.\u00a03A). At the 25 nM dose, a 60% reduction in PRL secretion was seen compared with cAMP-treated cells without inhibitor, which was similar to decidualization reduction seen with follistatin. Similarly cells co-cultured with cAMP and SB431542 showed a dose-dependent decrease in PRL secretion. Addition of SB431542 at a concentration of 1.25 \u00b5M significantly reduced PRL secretion compared with cAMP-only treated cells (Fig.\u00a03B). Further significant decreases were observed with doses ranging from 2.5\u201310 \u00b5M (Fig.\u00a03B). At 10 \u00b5M, SB431542 reduced PRL secretion by 77% compared with cAMP alone (Fig.\u00a03B). Higher doses were not tested. When either inhibitor (Activin-M108A or SB431542) was administered to cells in the absence of cAMP, PRL was not detected in culture medium. In addition, cell viability was tested using the trypan blue exclusion method at the end of the experiment and was found to not differ between treatments with medium-only, cAMP and either inhibitor. Microscopic visualization indicated cell morphology was not altered by inhibitor use, suggesting this effect was not due to toxicity to cells.Figure\u00a03:Activin-M108A and SB43 154 inhibitors significantly decrease HESC decidualization.Confluent stromal cells were cultured for 4\u20135 days with cAMP, with medium changes every 2\u20133 days. (A) M108A was added at doses 0.39, 1.5, 6.25 and 25 nM to cAMP-treated cultures. (B) SB431542 was added at doses 1.25, 2.5, 5 and 10 \u00b5M. Controls (not decidualized; ND) and cAMP (decidualized; D). PRL secretion from cells was used as a marker of decidualization, and values were corrected for total protein. All data (mean \u00b1 SEM) is expressed as % change from control (100%). Results are combined data from triplicate wells of five independent experiments for each inhibitor. *P < 0.05 and **P < 0.01 compared with cAMP alone.Secretion of activin A, TGF\u03b21, BMP2 and BMP4, but not TGF\u03b22 or BMP7 from non-decidualized and decidualized HESC in vitroTGF\u03b2 ligands are active as secreted proteins. Therefore we measured BMP2, BMP4, BMP7, TGF\u03b21, TGF\u03b22 from three different cultures by ELISA and activin A by IFMA (n = 1) in serum-free culture medium. There was a 4-fold increase in dimeric activin A secreted from decidualized (1.9 ng\/106cells) compared with non-decidualized cells (0.43 ng\/106cells), supporting previous published data from our laboratory (Jones et al., 2002). Non-decidualized and decidualized HESC secreted BMP2, TGF\u03b21 and BMP4, each in the range of 5\u201320 pg\/106 cells (Fig.\u00a04). BMP7 and TGF\u03b22 levels were below the sensitivity of the assays (data not shown). Decidualized stromal cells secreted significantly higher amounts of BMP2 and TGF\u03b21 compared with non-decidualized cells (TGF\u03b21 20.6 \u00b1 3.8 versus 14 \u00b1 3.1 pg\/106cells; BMP2 17.2 \u00b1 2.32 versus 8.44 \u00b1 1.5 pg\/106cells) (Fig.\u00a04). BMP4 secretion was increased slightly with decidualization (6.9 \u00b1 2.17 versus 10.1 \u00b1 3.45 pg\/106cells), but this was not significant (Fig.\u00a04). Western blot analysis was performed for GDF5, GDF8, GDF11 and TGF\u03b23 using 100\u2013150 \u00b5g of cell lysates from non-decidualized and decidualized HESC; however, these proteins were below the level of detection. Positive controls (GDF5\u2014rat heart and human term placenta; TGF\u03b23\u2014human term placenta; GDF8 and GDF11\u2014recombinant human myostatin\/GDF8 and recombinant human GDF11, respectively; data not shown) validated the technique.Figure\u00a04:BMP2, TGF\u03b21 and BMP4 protein secretion by HESC during decidualization.BMP2, BMP4 and TGF\u03b21 protein were measured in culture medium from non-decidualized (dark) and decidualized (grey) cells at Day 4 of culture. Results are combined data from n = 3 independent culture experiments and data (mean \u00b1 SEM) is represented as % difference from control (non-decidualized cells; defined as 100%). *P < 0.05 and **P < 0.01 compared to control.Exogenous BMP2 and TGF\u03b21 increases HESC decidualization in vitroTo assess whether addition of rhBMP2 and\/or rhTGF\u03b21 to HESC enhances decidualization in vitro, cells were decidualized with E2 and MPA under serum-free conditions for 8 days and PRL secretion measured. At Day 8, PRL secretion from non-decidualized cells was minimal but was increased when cells were decidualized (Fig.\u00a05). When rhBMP2 (5, 50 or 500 ng\/ml) was added to decidualizing HESC, PRL secretion was significantly increased at the 50 ng\/ml dose compared with the decidualized control (P < 0.05). The addition of rhTGF\u03b21 at 0.5, 5 or 50 ng\/ml to decidualizing HESC, however this was only significant at the 0.5 ng\/ml dose (P < 0.01) compared with decidualized cells alone.Due to the variability of endometrial biopsies only one dose for each BMP2 and TGF\u03b21 has resulted in a significant promotion of decidualisation. Also as the rhTGF\u03b21 is known to be quite potent, this result could be explained by the fact that at the higher doses it might be negatively affecting the response, for example receptor and downstream signalling disturbances.Figure\u00a05:Effect of exogenous BMP2 and TGF\u03b21 on PRL secretion from HESC.Cells were untreated or treated with E2 (10\u20138 M), MPA (10\u20137 M) alone or combined with recombinant human (rh) BMP2 (5\u2013500 ng\/ml) (A), rhTGF\u03b21 (0.5\u201350 ng\/ml) (B) for 8 day. PRL was measured in cultured medium from the last 48 h of treatment. Data are represented as percent fold change from control (decidualized; D) defined as 100%. ND, non-decidualized HESC. Results are combined data from triplicate wells of three independent cultures. *P < 0.05 and **P < 0.01 compared with D.\nDiscussionThis is the first study to identify BMP2, BMP4, BMP7, GDF5, GDF8 and GDF11 protein in secretory phase human endometrium and show the mRNA expression of these ligands in cultured HESC. In addition, the current study has immunolocalized BMP2, TGF\u03b21 and GDF5 protein to decidualized stromal cells, and provided further evidence for a role for secreted activin, BMP2 and TGF\u03b21 in decidualization through the use of novel inhibitors and a well-established ex vivo decidualization model.Decidualization of endometrial stromal cells is a pivotal event in the preparation for blastocyst implantation. Although this process is governed by hormonal regulation, it is becomingly increasingly evident that a multitude of factors, including cytokines and growth factors, are essential in ensuring this process occurs successfully such that endometrial stromal cells are differentiated into the implantation-favourable decidual cells. The identification of BMP2, TGF\u03b21 and GDF5 protein in mid-secretory human endometrial tissue, together with the increased secretion of both BMP2 and TGF\u03b21 from stromal cells following decidualization, suggests these TGF\u03b2 family members are hormonally regulated and important for decidualization.The finding that BMP2 secretion from HESC increases as they decidualize in vitro and that addition of rhBMP2 further drives decidualization as determined by secretion of PRL, extends the recent study (Li et al., 2007), which showed an increase of BMP2 mRNA with decidualization in a similar model and of PRL mRNA following treatment of HESC with rhBMP2. This is important given that translation and secretion do not necessarily follow increases in mRNA. The inhibitor M108A which acts primarily on those ligands acting through the activin type 11 receptor (Table\u00a0I) is not known to block the action of BMP2 which acts through the BMP type II receptor (BMPRII). Therefore, the inability of M108A to fully block the decidualization process suggests that factors other than activins and GDFs are likely to be important to this process: BMP2 is a likely candidate.Interestingly in mice, uterine-specific conditional ablation of BMP2 resulted in uterine stromal disruption and an inability to undergo normal decidual reactions and pregnancy progression (Lee et al., 2007). Our demonstration of BMP2 in human endometrium in vivo with the strongest immunostaining in decidualized cells in the mid-late secretory phase and in the decidua of early pregnancy, confirms the biological relevance in women. Given this data in conjunction with our functional studies of human decidualization, there appears to be little doubt that BMP2 is an important mediator of both mouse and human decidualization.This study also demonstrates, for the first time, the presence of GDF5 in human endometrium and first trimester placenta where it is intensely and specifically stained in the decidual cells. GDF5 is best known for its role in early chondrogenesis and joint formation, being involved in inducing cartilage differentiation, growth and maturation (Mikic, 2004), as well as bone formation and angiogenesis (Yamashita et al., 1997). Differentiation and angiogenesis are the main events of decidualization. Unfortunately, assays for GDF5 were not available to measure the secretion from decidualized cells. The decrease in decidualization seen following administration of M108A was not likely to be due to GDF5 blockade, as this ligand has a relatively low, if any, affinity for the activin type II receptor. Future studies should address whether GDF5 has a functional role in decidualization, whether it be in promoting differentiation or in the formation of associated blood vessels to support the blastocyst in early pregnancy and throughout gestation.Although the TGF\u03b2s have been previously detected in the human endometrium, the information regarding their role during decidualization is contentious, as studies report contrasting effects on PRL production by endometrial stroma and during decidualization in vitro (Kubota et al., 1997; Kim et al., 2005). We showed here that under serum-free conditions, TGF\u03b21 secretion increases during cAMP-induced decidualization in vitro. Microarray studies have likewise shown increased TGF\u03b21 and TGF\u03b22 mRNA when cAMP or progesterone is used as decidual stimuli (Popovici et al., 2000; Tierney et al., 2003). The protein for both isoforms localizes to stromal cells in human endometrium throughout the menstrual cycle (Gold et al., 1994; Godkin and Dore, 1998), although decidualized stromal cells were not shown. In contrast, the present study identified TGF\u03b21 protein in decidualized stromal cells in human endometrium, and its secretion increased in cAMP-decidualized HESC. Administration of SB431542, which effectively blocks the TGF\u03b2s as well as activins by blocking their structurally similar ALK5 and ALK4 receptors (Inman et al., 2002), resulted in a 77% reduction in decidualization, a greater decrease than that seen with M108A (this study) and follistatin (Tierney and Giudice, 2004). This, along with our demonstration that addition of TGF\u03b21 during progesterone-induced decidualization enhances decidualization, provides convincing evidence to suggest TGF\u03b21 is important during decidualization. The level of activated TGF\u03b22 was undetectable by ELISA in cAMP-decidualized endometrial stromal cells, and therefore this ligand is unlikely to contribute to the decidual response.The identification of BMP4, BMP7, GDF8 and GDF11 during the mid-secretory phase is also an important finding. In addition to decidualization, there are many remodelling processes occurring in the endometrium during this time. Both BMP4 and GDF8 protein showed intense immunolocalization to the cytoplasm of the stroma, irrespective of whether or not the cells were decidualized. This was verified for BMP4 by the minimal difference in secretion levels between non-decidualized and decidualized cells in vitro. BMP4 mRNA expression has been localized to the vascular endothelial cells in pregnant mice (Ying and Zhao, 2000), and in the non-pregnant rat uterus, where it is constitutively expressed throughout the cycle (Erickson et al., 2004). This differs to the localization of BMP4 protein shown here in human endometrium. It may be that the mRNA localized to blood vessels in the rodent studies is not translated into functional protein.GDF8 (more commonly referred to as Myostatin) is a negative regulator of skeletal muscle, but more recently has been shown to be involved in glucose metabolism (McPherron and Lee, 2002), particularly in placenta (Mitchell et al., 2006). This study verified the presence of GDF8 protein in first trimester placenta, and showed for the first time its presence in secretory phase endometrium and in HESC. Given the lack of specific decidual staining it is unlikely that the response from M108A inhibitor was due to blocking GDF8 in HESC, even though the predominant receptor for GDF8 is the activin type II receptor. The presence of GDF8 in non-muscular tissue and organs is rare and future experiments should determine whether it has a role in endometrial biology as it does throughout gestation in the placenta.Vesicular staining for BMP7 (or Osteogenic protein-1; OP-1) was evident in highly decidualized cells during the secretory phase of human endometrium with minimal staining in selected glandular and luminal epithelial cells. OP-1 has been identified in the uterine epithelium of non-pregnant mice and rat uterus (Ozkaynak et al., 1997; Erickson et al., 2004), with localization remaining unchanged across the cycle. In pregnant mice, however, BMP7 mRNA was detected in the decidualizing stromal cells surrounding the blastocyst with no staining in the epithelial cells (Ying and Zhao, 2000). In the present study, BMP7 secretion from HESC was undetectable by ELISA, making it difficult to assess whether it may have a functional role during decidualization.In conclusion, this study has identified that a wide range of TGF\u03b2 ligands are present in mid-late secretory human endometrium but of these, in addition to activins, only BMP2, GDF5 and TGF\u03b21 increase in stromal cells as they undergo decidualization. BMP2 and TGF\u03b21, along with activins, are important in driving the decidualization process. Functions for the remaining ligands remain to be determined but they are likely to participate in the extensive tissue remodelling that occurs in this highly dynamic tissue as it prepares for blastocyst implantation and pregnancy.\nSupplementary materialSupplementary material is available at HUMREP Journal online\nFundingThis work was funded by the National Health and Medical Research Council of Australia (#241000, #388901) and a Prince Henry's Institute Postgraduate Scholarship.\nSupplementary Material\n[Supplementary Data]","keyphrases":["human endometrium","decidualization","implantation","growth factors (activins, bmp, tgf\u03b2)"],"prmu":["P","P","P","R"]}
{"id":"Urol_Res-3-1-2082064","title":"The analgesic effect of inhalational Entonox for extracorporeal shock wave lithotripsy\n","text":"Extracorporeal shock wave lithotripsy (ESWL) is a non-invasive procedure that allows urinary stones to be fragmented using acoustic shock waves. The impact of the shock waves causes transient stinging pain at the entry site as well as deep visceral discomfort, requiring analgesia during the procedure. The objective of this study was to compare the clinical efficacy of Entonox and pethidine for pain relief during outpatient ESWL. We randomized 150 outpatients undergoing elective ESWL into three groups of 50 patients, each group receiving inhalational Entonox, intravenous pethidine, or inhalational compressed air during ESWL. Quantitative evaluation of pain was performed according to a visual analogue scale (VAS), before and after the intervention. Analysis of variance (ANOVA) and paired t tests were used to compare VAS scores in the three groups, before and after the intervention. Entonox and pethidine decreased the pain score significantly, while compressed air did not. There was no significant difference between pain relief by Entonox and pethidine. This study demonstrates for the first time that inhalational Entonox is an effective analgesic regimen for ESWL. Entonox can be regarded as an appropriate alternative to analgesics like opioids in relieving pain during ESWL.\nIntroduction\nSince 1980, extracorporeal shock wave lithotripsy (ESWL) has become the first-line treatment for most urinary stones in adults and children [1]. However, the vast majority of patients do not tolerate the procedure without analgesia or sedation. Several monitored anesthesia care techniques have been used to provide sedation and analgesia [2\u20135]. Some of the analgesic drugs administered for ESWL, however, carry the risk of respiratory depression, delayed discharge, and\/or unplanned hospital admission [6].\nEntonox is a mixture of 50% nitrous oxide and 50% oxygen. Inhalation produces analgesia without loss of consciousness [7]. Self-administration of Entonox as an analgesic has been widely used for many years, particularly in obstetric practice [8], and by paramedics for prehospital care [9]. Entonox has a good safety record, with no serious side effects recorded for intermittent use [7], and is rapidly cleared from the circulation by exhalation [10].\nThe aim of this study was to compare the clinical efficacy of inhalational Entonox and pethidine for pain relief during outpatient ESWL.\nMethods\nA total of 150 patients (97 men and 53 women) with stones located in the renal pelvicalyceal system were prospectively randomized to receive Entonox (prefixed equimolar nitrous oxide and oxygen mixture), pethidine, or compressed air for sedoanalgesia during lithotripsy with a third generation electromagnetic lithotripter (Dornier Compact Delta magneto lithotripter).\nAfter institutional review board approval, written informed consent was obtained during the anesthesia consultation, which was performed at least 48\u00a0h before ESWL. During this consultation, the method of pain assessment was explained to the patients. Patients were asked to rate their pain on a scale from 0 (no pain) to 10 (worst possible pain).\nPatients were included in the study if they were older than 15\u00a0years of age, had pelvicalyceal stones, and understood the pain scoring. Patients were excluded from the trial if they had any of the following: (1) serum creatinine\u00a0>\u00a0200\u00a0\u03bcmol\/l; (2) history of chronic use of analgesics and\/or sedatives; (3) allergy to any of the study medications; or (4) history of middle ear surgery within the previous month.\nJust before entering the lithotripsy room, the patients were randomly assigned to one of the three treatment groups (Entonox, pethidine, 1\u00a0mg\/kg intravenously over 10\u00a0min, or compressed air, each to be provided if the patient asked for pain relief during ESWL) by opening a sealed envelope. All patients were assessed by a single physician who was blinded to the patient group assignment. All patients received no premedication and they were told not to eat or drink for at least 4\u00a0h prior to the procedure.\nThe control group received compressed air, followed by Entonox gas if pain relief was considered inadequate by the patient.\nPain was assessed before and 2\u00a0min after receiving pain medication by using a visual analogue scale (VAS) with two anchor points, zero denoting no pain and ten for the worst pain the patient had ever experienced.\nStatistical analysis\nData are expressed as mean (\u00b1SD) and numbers (percentages). The paired t test was used to analyze VAS score before and after medication. Comparison of means (VAS decrease) in the three groups was performed using one-way ANOVA. Comparison of percentages was performed using Chi-square analysis.\nAll statistical analyses were performed using SPSS\u00ae software, version 9.0, for Windows. Results throughout the text and tables are presented as mean\u00a0\u00b1\u00a0SD unless otherwise specified, and statistical significance was defined as P\u00a0<\u00a00.05.\nResults\nThe three study groups were comparable with respect to demographic data, history of urinary calculi and ESWL, and location of calculi. There were no statistically significant differences between the three groups (Table\u00a01). All patients in the three groups required analgesia. Rescue medication in the form of pethidine was administered in only one case in the Entonox group after recording VAS scores because the analgesia was inadequate.\nTable\u00a01Demographic data and ESWL variables in three different groupsGroupsEntonoxPethidineControlAge (years)43.76\u00a0\u00b1\u00a011.8945\u00a0\u00b1\u00a013.4643.9\u00a0\u00b1\u00a014.21Weight (kg)75\u00a0\u00b1\u00a0278\u00a0\u00b1\u00a0379\u00a0\u00b1\u00a02Height (cm)165\u00a0\u00b1\u00a010167\u00a0\u00b1\u00a012164\u00a0\u00b1\u00a08Gender (M\/F) (n)30\/2034\/1633\/17Stone location (%)\u00a0\u00a0Renal807884\u00a0\u00a0Ureteral202015\u00a0\u00a0Both021Number of shocks delivered2,010\u00a0\u00b1\u00a03202,000\u00a0\u00b1\u00a05602,500\u00a0\u00b1\u00a0450Maximum voltage (kV) used4.04\u00a0\u00b1\u00a01.154.14\u00a0\u00b1\u00a00.754.51\u00a0\u00b1\u00a01.00Past history of urinary calculi (%)707276Past history of ESWL (%)424636Data are expressed as mean\u00a0\u00b1\u00a0SD, numbers (n), or percentages (%)\nNo ESWL procedure had to be prematurely terminated because of inadequate analgesia. A patient in the Entonox group had mild nausea that subsided spontaneously. The other patients did not develop any complications.\nMean\u00a0\u00b1\u00a0SD of VAS scores before and after intervention are shown in Table\u00a02. Statistical analysis showed significant decrease (Table\u00a02) in pain severity in patients receiving Entonox or pethidine. In contrast, patients receiving compressed air had no such change.\nTable\u00a02Mean\u00a0\u00b1\u00a0SD of VAS scores and P-value of comparing scores before and after Entonox, pethidine, and compressed air administration in different groupsVAS scoresEntonoxPethidineCompressed airEntonox in the compressed air groupBefore administration5.76\u00a0\u00b1\u00a01.926.04\u00a0\u00b1\u00a01.543.6\u00a0\u00b1\u00a01.14.96\u00a0\u00b1\u00a01.42After administration3.73\u00a0\u00b1\u00a02.134.11\u00a0\u00b1\u00a01.693.48\u00a0\u00b1\u00a01.33.8\u00a0\u00b1\u00a01.74P-value0.0010.0010.4260.001In control group, after testing with compressed air, we used Entonox. As can be seen in this group, like Entonox group, VAS score decreased significantly\nWhile VAS scores after receiving either Entonox or pethidine were significantly different from those in the control group (P\u00a0=\u00a00.001) after receiving compressed air, statistical comparison showed no significant difference between post-Entonox or post-pethidine VAS scores (P\u00a0=\u00a00.5), suggesting that inhalational Entonox may be as effective as intravenous pethidine in alleviating ESWL-associated pain.\nNo significant difference was found in VAS scores of male and female patients in each group (Fig.\u00a01).\nFig.\u00a01VAS scores in different groups before and after administration of Entonox, pethidine, and compressed air\nDiscussion\nExtracorporeal shock wave lithotripsy (ESWL) has become a valuable asset to the urologist and greatly benefits stone patients. In a short period of time, ESWL has completely changed the management of urinary stone disease and has almost entirely supplanted open surgical and most endourologic approaches [11]. Today, third-generation mobile electromagnetic lithotripters yield an average of 80% stone-free rate for kidney and ureteral calculi [12].\nAs ESWL is frequently carried out on an outpatient basis, it is crucial to provide adequate analgesia with minimal adverse effects [13].\nChaussy and Thuroff [14] demonstrated that analgesia requirements in ESWL depend on lithotripter, stone location, age, gender, and number of shock waves. The pathogenesis of pain in ESWL is still not clearly known. Whether it is due to cutaneous or deep visceral afferent stimulation is debatable [15]. The pain is presumably due to cavitation-mediated stimulation of nerve fibers. The intensity of pain perceived during ESWL depends on the energy level of shock waves passing through the tissues [16]. Although the development of new generation lithotripters has decreased pain during ESWL, some form of sedation and anesthesia may still be required to provide adequate patient comfort and effective treatment [15].\nAny technique for alleviating ESWL-associated discomfort\/anxiety should ideally be reliable in its effect, of rapid onset and short duration of action, free of adverse events, cost-effective, and easily administered [17].\nOpioids are the most commonly used analgesics during ESWL [18]. Despite their effectiveness, their use may be complicated by central nervous system (CNS) or respiratory depression, circulatory failure, or gastrointestinal problems [19].\nSeveral studies have compared different local and systemic analgesia regimens for pain relief during ESWL, but this, to our knowledge, is the first randomized clinical trial to evaluate the efficacy of Entonox in the treatment of ESWL-associated pain.\nNitrous oxide has been used for pain relief during childbirth since the 1930s and was initially delivered at a concentration of 50% in air, producing an effective analgesic mixture. In 1961, Mike Tunstall premixed 50% nitrous oxide with oxygen, and called it Entonox, which was stored as a compressed gas mixture in cylinders. Since then, the convenience of Entonox has made it a successful and popular analgesic for labour [20]. Entonox has also proved effective in providing pain relief in other specialties [21, 22].\nOur randomized clinical trial demonstrated that Entonox and pethidine were equally effective in providing analgesia during ESWL and that they were superior to compressed air. Entonox fulfills many of the criteria for an ideal analgesic during ESWL [17] and, compared to intravenously administered pethidine, provided comparable analgesia with less CNS, circulatory, and respiratory depression or nausea and vomiting.\nEntonox provides rapid and effective analgesia without heavy sedation and leads to adequate patient relaxation and cooperation. The effect of Entonox was of short duration, allowing the patients to leave the ESWL unit without the need for a long recovery period [23]. Entonox appeared to be associated with few, minor, and short-lived adverse effects such as nausea and vomiting, dizziness, dry mouth (breathing dry gas), buzzing in the ears, and rarely, pins and needles or numbness, dreams or drowsiness, ranging from 0 to 30% [24]. Nitrous oxide\u2013oxygen inhalation may thus provide a valuable alternative to conventional analgesia regimens during ESWL.\nIn addition to short-acting parenteral sedative narcotics, e.g., alfentanil, midazolam, and propofol, topical agents such as EMLA cream, lidocaine, prilocaine and piroxicam have been used to minimize pain during SWL [25]. Further studies are, therefore, warranted to compare the efficacy and safety of Entonox with those of other analgesic regimens.\nConclusions\nEntonox can provide easy, rapid, and adequate pain relief for patients undergoing ESWL, and is associated with few and minimal side effects.","keyphrases":["entonox","extracorporeal shock wave lithotripsy","pethidine","pain relief"],"prmu":["P","P","P","P"]}
{"id":"Immunome_Res-1-_-1312312","title":"IMGT, the international ImMunoGeneTics information system\u00ae: a standardized approach for immunogenetics and immunoinformatics\n","text":"IMGT, the international ImMunoGeneTics information system\u00ae, was created in 1989 by the Laboratoire d'ImmunoG\u00e9n\u00e9tique Mol\u00e9culaire (LIGM) (Universit\u00e9 Montpellier II and CNRS) at Montpellier, France. IMGT is a high quality integrated knowledge resource specialized in immunoglobulins (IG), T cell receptors (TR), major histocompatibility complex (MHC) of human and other vertebrates, and related proteins of the immune system (RPI) of any species which belong to the immunoglobulin superfamily (IgSF) and to the MHC superfamily (MhcSF). IMGT consists of five databases, ten on-line tools and more than 8,000 HTML pages of Web resources. IMGT provides a common access to standardized data from genome, genetics, proteome and three-dimensional structures. The accuracy and the consistency of IMGT data are based on IMGT-ONTOLOGY, a semantic specification of terms to be used in immunogenetics and immunoinformatics. IMGT-ONTOLOGY comprises six main concepts: IDENTIFICATION, CLASSIFICATION, DESCRIPTION, NUMEROTATION, ORIENTATION and OBTENTION. Based on these concepts, the controlled vocabulary and the annotation rules necessary for the immunogenetics data identification, classification, description and numbering and for the management of IMGT knowledge are defined in the IMGT Scientific chart. IMGT is the international reference in immunogenetics and immunoinformatics for medical research (repertoire analysis of the IG antibody sites and of the TR recognition sites in autoimmune and infectious diseases, AIDS, leukemias, lymphomas, myelomas), veterinary research (IG and TR repertoires in farm and wild life species), genome diversity and genome evolution studies of the adaptive immune responses, biotechnology related to antibody engineering (single chain Fragment variable (scFv), phage displays, combinatorial libraries, chimeric, humanized and human antibodies), diagnostics (detection and follow up of residual diseases) and therapeutical approaches (grafts, immunotherapy, vaccinology). IMGT is freely available at .\nIntroduction\nIMGT, the international ImMunoGeneTics information system\u00ae[1,2], was created in 1989, by Marie-Paule Lefranc, at the Laboratoire d'ImmunoG\u00e9n\u00e9tique Mol\u00e9culaire (LIGM) (Universit\u00e9 Montpellier II and CNRS) at Montpellier, France, in order to standardize and manage the complexity of the immunogenetics data. Fifteen years later, IMGT is the international reference in immunogenetics and immunoinformatics, and provides a high quality integrated knowledge resource, specialized in the immunoglobulins (IG) and T cell receptors (TR), major histocompatibility complex (MHC) of human and other vertebrates, and related proteins of the immune systems (RPI) of any species which belong to the immunoglobulin superfamily (IgSF) and to the MHC superfamily (MhcSF)[3-13]. The number of potential protein forms of the antigen receptors, IG and TR, is almost unlimited. The potential repertoire of each individual is estimated to comprise about 1012 different IG (or antibodies) and TR, and the limiting factor is only the number of B and T cells that an organism is genetically programmed to produce. This huge diversity is inherent to the particularly complex and unique molecular synthesis and genetics of the antigen receptor chains. This includes biological mechanisms such as DNA molecular rearrangements in multiple loci (three for IG and four for TR in humans) located on different chromosomes (four in humans), nucleotide deletions and insertions at the rearrangement junctions (or N-diversity), and somatic hypermutations in the IG loci (see FactsBooks[3,4] for review). Although IMGT was initially implemented for the IG, TR and MHC of human and other vertebrates [6], data and knowledge management standardization, based on the IMGT unique numbering [14-19], has now been extended to the IgSF [15-17,20-22] and MhcSF [18,23,24] of any species. Thus, standardization in IMGT contributed to data enhancement of the system and new expertised data concepts were readily incorporated.\nIMGT, the international ImMunoGeneTics information system\u00ae consists of five databases, ten on-line tools and Web resources [1,2]. Databases include sequence databases (IMGT\/LIGM-DB, IMGT\/PRIMER-DB and IMGT\/MHC-DB), one genome database (IMGT\/GENE-DB) and one three-dimensional (3D) structure database (IMGT\/3Dstructure-DB) [1,2] (Figure 1). Interactive tools are provided for sequence analysis (IMGT\/V-QUEST, IMGT\/JunctionAnalysis, IMGT\/Allele-Align, IMGT\/PhyloGene), genome analysis (IMGT\/LocusView, IMGT\/GeneView, IMGT\/GeneSearch, IMGT\/CloneSearch and IMGT\/GeneInfo) and 3D structure analysis (IMGT\/StructuralQuery) [1,2] (Figure 1). Web resources (\"IMGT Marie-Paule page\") comprise more than 8,000 HTML pages of synthesis [IMGT Repertoire (for IG and TR, MHC, RPI)], knowledge [IMGT Scientific chart, IMGT Education (IMGT Lexique, Aide-m\u00e9moire, Tutorials, Questions and answers), IMGT Medical page, IMGT Veterinary page, IMGT Biotechnology page, IMGT Index], and external links [IMGT Immunoinformatics page, IMGT Bloc-notes (Interesting links, etc.) and IMGT other accesses (SRS, BLAST, etc.)] [2]. Despite the heterogeneity of these different components, all data in the IMGT information system are expertly annotated. The accuracy, the consistency and the integration of the IMGT data, as well as the coherence between the different IMGT components (databases, tools and Web resources) are based on IMGT-ONTOLOGY[5], which provides a semantic specification of the terms to be used in immunogenetics and immunoinformatics. IMGT-ONTOLOGY, the first ontology in the domain, has allowed the management of knowledge in immunogenetics [2,25] and provided standardization for immunogenetics data from genome, genetics, proteome and 3D structures [3-13]. IMGT-ONTOLOGY concepts are available, for the biologists and IMGT users, in the IMGT Scientific chart[2], and for the computing scientists, in IMGT-ML which uses XML (eXtensible Markup Language) Schema [26].\nFigure 1\nIMGT, the international ImMunoGeneTics information system\u00ae . Databases and tools for sequences, genes and structures are in green, yellow and blue, respectively. The IMGT Repertoire and other Web resources are not shown. Interactions in the genetics, genomics and structural approaches are represented with dotted, continuous and broken lines, respectively.\nIMGT-ONTOLOGY concepts and IMGT Scientific chart rules\nThe IMGT Scientific chart[2] comprises the controlled vocabulary and the annotation rules necessary for the immunogenetics data identification, description, classification and numbering and for knowledge management in the IMGT information system. Standardized keywords, labels and annotation rules, standardized IG and TR gene nomenclature, the IMGT unique numbering, and standardized origin\/methodology were defined, respectively, based on the six main concepts of IMGT-ONTOLOGY: IDENTIFICATION, CLASSIFICATION, DESCRIPTION, NUMEROTATION, ORIENTATION and OBTENTION[2,5] (Table 1). The IMGT Scientific chart is available as a section of the IMGT Web resources (IMGT Marie-Paule page). Examples of IMGT expertised data concepts derived from the IMGT Scientific chart rules are shown in Table 1.\nTable 1\nIMGT-ONTOLOGY concepts, IMGT Scientific chart rules and examples of IMGT expertised data concepts.\nIMGT-ONTOLOGY main concepts 5\nIMGT Scientific chart rules [2]\nExamples of IMGT expertised data concepts [2]\nIDENTIFICATION\nStandardized keywords [5]\nSpecies, molecule type, receptor type, chain type, gene type, structure, functionality, specificity\nCLASSIFICATION\nReference sequencesStandardized IG and TR gene nomenclature (group, subgroup, gene, allele) [5]\nNomenclature of the human IG and TR genes (entry in 1999 in GDB, HGNC [27] and LocusLink at NCBI) [3, 4]Alignment of alleles [3, 4]Nomenclature of the IG and TR genes of all vertebrate species\nDESCRIPTION\nStandardized labels and annotations [5]\nCore (V-, D-, J-, C-REGION) Prototypes [5]Labels for sequencesLabels for 2D and 3D structures\nNUMEROTATION\nIMGT unique numbering [14-18] for: V- and V-LIKE-DOMAINs [16]C- and C-LIKE-DOMAINs [17]G- and G-LIKE-DOMAINs [18]\nProtein displaysIMGT Colliers de Perles [19]FR-IMGT and CDR-IMGT delimitations [16]Structural loops and beta strands delimitations [16, 17]\nORIENTATION\nOrientation of genomic instances relative to each other\nChromosome orientation\nLocus orientation\nGene orientation\nDNA strand orientation\nOBTENTION\nStandardized originStandardized methodology [2]\nThe IMGT Scientific chart rules, based on the IMGT-ONTOLOGY concepts [5], are used in the three major IMGT biological approaches, genomics, genetics and structural approaches [2], and corresponding data (Genes, Sequences, 3D structures) are available in the IMGT components (databases, tools and Web resources) [1,7-13].\nIMGT sequence databases, tools and Web resources\nIMGT sequence databases, tools and Web resources correspond to the IMGT genetics approach that refers to the study of genes in relation with their polymorphisms, mutations, expression, specificity and evolution (Table 2). The IMGT sequence knowledge management and the IMGT genetics approach heavily rely on the DESCRIPTION concept (and particularly on the V-REGION, D-REGION, J-REGION and C-REGION core concepts for the IG and TR), on the CLASSIFICATION concept (gene and allele concepts) and on the NUMEROTATION concept (IMGT unique numbering [14-18]).\nTable 2\nThe IMGT sequence databases, sequence analysis tools and Web resources\nIMGT sequence databases [1]\nIMGT sequence analysis tools [1]\nIMGT Repertoire\"Proteins and alleles\" section [2](2)\nIMGT\/LIGM-DB [7]IMGT\/PRIMER-DB [1]IMGT\/MHC-DB [28]\nIMGT\/V-QUEST [10]IMGT\/JunctionAnalysis [11]IMGT\/Allele-AlignIMGT\/PhyloGene [12]IMGT\/Automat [29, 30] (1)\nAlignments of alleles IG and TR [3, 4]Alignments of alleles RPI [22]Protein displays IG and TR [3, 4, 16, 17]Protein displays MHC [18]Protein displays RPI [16-18, 21]Tables of alleles IG and TRTables of alleles RPI [22, 24]Allotypes Isotypes, etc.\n(1) IMGT\/Automat [29, 30] is an integrated internal IMGT Java tool which automatically performs the annotation of rearranged cDNA sequences that represent the half of the IMGT\/LIGM-DB content. So far 7,418 human and mouse IG and TR cDNA sequences have been automatically annotated by the IMGT\/Automat tool, with annotations being as reliable and accurate as those provided by a human annotator.\n(2) IMGT publications from the IMGT Repertoire \"Proteins and alleles\" section are available as pdf in IMGT Locus in Focus , in IMGT Index (see also [2]).\nIMGT sequence databases\nIMGT\/LIGM-DB\nIMGT\/LIGM-DB is the comprehensive IMGT database of IG and TR nucleotide sequences from human and other vertebrate species, with translation for fully annotated sequences [7]. It was created in 1989 by LIGM (Montpellier, France), and is on the Web since July 1995 [6]. In August 2005, IMGT\/LIGM-DB contained more than 96,500 sequences of 150 vertebrate species [7]. The unique source of data for IMGT\/LIGM-DB is EMBL, which shares data with the other two generalist databases GenBank and DNA DataBank of Japan (DDBJ). Based on expert analysis, specific detailed annotations are added to IMGT flat files. The annotation procedure includes the IDENTIFICATION of the sequences, the CLASSIFICATION of the IG and TR genes and alleles, and the DESCRIPTION of all IG and TR specific and constitutive motifs within the nucleotide sequences. The Web interface allows searches according to immunogenetic specific criteria and is easy to use without any knowledge in a computing language. Selection is displayed at the top of the resulting sequences pages, so the users can check their own queries. Users have the possibility to modify their request or consult the results with a choice of nine possibilities. The IMGT\/LIGM-DB annotations (gene and allele name assignment, labels) allow data retrieval not only from IMGT\/LIGM-DB, but also from other IMGT databases. Thus, the IMGT\/LIGM-DB accession numbers of the cDNA expressed sequences for each human and mouse IG and TR gene are available, with direct links to IMGT\/LIGM-DB, in the IMGT\/GENE-DB entries. IMGT\/LIGM-DB data are also distributed by anonymous FTP servers at CINES  and EBI  and from many Sequence Retrieval System (SRS) sites . IMGT\/LIGM-DB can be searched by BLAST or FASTA on different servers (EBI, IGH, INFOBIOGEN, Institut Pasteur, etc.).\nIMGT\/PRIMER-DB\nIMGT\/PRIMER-DB[1] is the IMGT oligonucleotide primer database for IG and TR, created by LIGM, Montpellier in collaboration with EUROGENTEC S.A., Belgium, on the Web since February 2002. In August 2005, IMGT\/PRIMER-DB contained 1,827 entries. IMGT\/PRIMER-DB provides standardized information on oligonucleotides (or Primers) and combinations of primers (Sets, Couples) for IG and TR. These primers are useful for combinatorial library constructions, scFv, phage display or microarray technologies. The IMGT Primer cards are linked to the IMGT\/LIGM-DB flat files, IMGT Colliers de Perles and IMGT Alignments of alleles (IMGT Repertoire) of the IMGT\/LIGM-DB reference sequence used for the primer description.\nIMGT\/MHC-DB\nIMGT\/MHC-DB[28] comprises databases hosted at EBI and includes a database of human MHC allele sequences or IMGT\/MHC-HLA, developed by Cancer Research UK and maintained by ANRI, London, UK, on the Web since December 1998, and a database of MHC sequences from non human primates IMGT\/MHC-NHP, curated by BPRC, The Netherlands, on the Web since April 2002.\nIMGT sequence analysis tools\nThe IMGT sequence analysis tools comprise IMGT\/V-QUEST[10], for the identification of the V, D and J genes and of their mutations, IMGT\/JunctionAnalysis[11] for the analysis of the V-J and V-D-J junctions which confer the antigen receptor specificity, IMGT\/Allele-Align for the detection of polymorphisms, and IMGT\/PhyloGene[12] for gene evolution analyses.\nIMGT\/V-QUEST\nIMGT\/V-QUEST (V-QUEry and STandardization) is an integrated software for IG and TR [10]. This tool, easy to use, analyses an input IG or TR germline or rearranged variable nucleotide sequence. The IMGT\/V-QUEST results comprise the identification of the V, D and J genes and alleles and the nucleotide alignments by comparison with sequences from the IMGT reference directory, the FR-IMGT and CDR-IMGT delimitations based on the IMGT unique numbering, the translation of the input sequence, the display of nucleotide and amino acid mutations compared to the closest IMGT reference sequence, the identification of the JUNCTION and results from IMGT\/JunctionAnalysis (default option), and the two-dimensional (2D) IMGT Collier de Perles representation of the V-REGION [10] (\"IMGT\/V-QUEST output\" in IMGT\/V-QUEST Documentation).\nIMGT\/JunctionAnalysis\nIMGT\/JunctionAnalysis[11] is a tool, complementary to IMGT\/V-QUEST, which provides a thorough analysis of the V-J and V-D-J junction of IG and TR rearranged genes. IMGT\/JunctionAnalysis identifies the D-GENEs and alleles involved in the IGH, TRB and TRD V-D-J rearrangements by comparison with the IMGT reference directory, and delimits precisely the P, N and D regions [11] (\"IMGT\/JunctionAnalysis output results\" in IMGT\/JunctionAnalysis Documentation). Several hundreds of junction sequences can be analysed simultaneously.\nIMGT\/Allele-Align\nIMGT\/Allele-Align is used for the detection of polymorphisms. It allows the comparison of two alleles highlighting the nucleotide and amino acid differences.\nIMGT\/PhyloGene\nIMGT\/PhyloGene[12] is an easy to use tool for phylogenetic analysis of variable region (V-REGION) and constant domain (C-DOMAIN) sequences. This tool is particularly useful in developmental and comparative immunology. The users can analyse their own sequences by comparing with the IMGT standardized reference sequences for human and mouse IG and TR [12] (IMGT\/PhyloGene Documentation).\nIMGT sequence Web resources\nThe IMGT sequence Web resources are compiled in the IMGT Repertoire \"Proteins and alleles\" section that include Alignments of alleles, Proteins displays, Tables of alleles, Allotypes, Isotypes, etc. (Table 2). Standardized IMGT criteria for amino acid sequence analysis are described in [31].\nIMGT gene databases, tools and Web resources\nIMGT gene databases, tools and Web resources correspond to the IMGT genomics approach that refers to the studies of the genes within their loci and on their chromosome [2] (Table 3).\nTable 3\nThe IMGT gene database, genome analysis tools and Web resources\nIMGT genome database [1]\nIMGT genome analysis tools [1]\nIMGT Repertoire\"Locus and genes\" section [2] (1)\nIMGT\/GENE-DB [8]\nIMGT\/LocusViewIMGT\/GeneViewIMGT\/GeneSearchIMGT\/CloneSearchIMGT\/GeneInfo [13]\nChromosomal localizations [3, 4]Locus representations [3, 4]Locus descriptionGene exon\/intron organizationGene exon\/intron splicing sitesGene tablesPotential germline repertoiresLists of genesCorrespondence between nomenclatures [3, 4]\n(1) IMGT Web resources (IMGT Marie-Paule page) also include IMGT Index, IMGT Education (IMGT Lexique, Aide-m\u00e9moire, Tutorials, Questions and answers), The IMGT Medical page, The IMGT Veterinary page, The IMGT Biotechnology page, The IMGT Immunoinformatics page, IMGT Bloc-notes (Interesting links, etc.) [2] which are not detailed in this paper.\n(2) IMGT publications from the IMGT Repertoire \"Locus and genes\" section are available as pdf in IMGT Locus in Focus , in IMGT Index (see also [2]).\nIMGT\/GENE-DB, the IMGT gene database\nGenomic data are managed in IMGT\/GENE-DB, which is the comprehensive IMGT genome database [8]. IMGT\/GENE-DB, created by LIGM (Montpellier, France) is on the Web since January 2003. In August 2005, IMGT\/GENE-DB contained 1,377 genes and 2,207 alleles (673 IG and TR genes and 1,209 alleles from Homo sapiens, and 704 IG and TR genes and 998 alleles from Mus musculus, Mus cookii, Mus pahari, Mus spretus, Mus saxicola, Mus minuto\u00efdes). All the human and mouse IG and TR genes are available in IMGT\/GENE-DB. Based on the IMGT CLASSIFICATION concept, all the human IMGT gene names [3,4] were approved by the Human Genome Organisation (HUGO) Nomenclature Committee HGNC in 1999 [27], and entered in IMGT\/GENE-DB [8], Genome DataBase GDB (Canada) [32], LocusLink and Entrez Gene at NCBI (USA) [33], and GeneCards [34]. Reciprocal links exist between IMGT\/GENE-DB, and the generalist nomenclature (HGNC Genew) and genome databases (GDB, LocusLink and Entrez at NCBI, and GeneCards). All the mouse IG and TR gene names with IMGT reference sequences were provided by IMGT to HGNC and to the Mouse Genome Database (MGD) [35] in July 2002. Queries in IMGT\/GENE-DB can be performed according to IG and TR gene classification criteria and IMGT reference sequences have been defined for each allele of each gene based on one or, whenever possible, several of the following criteria: germline sequence, first sequence published, longest sequence, mapped sequence [2]. IMGT\/GENE-DB interacts dynamically with IMGT\/LIGM-DB [7] to download and display gene-related sequence data. As an example ans as mentioned earlier, the IMGT\/GENE-DB entries provide the IMGT\/LIGM-DB accession numbers of the IG and TR cDNA sequences which contain a given V, D, J or C gene. This is the first example of an interaction between IMGT databases using the CLASSIFICATION concept.\nIMGT gene analysis tools\nThe IMGT gene analysis tools comprise IMGT\/LocusView, IMGT\/GeneView, IMGT\/GeneSearch, IMGT\/CloneSearch and IMGT\/GeneInfo. IMGT\/LocusView and IMGT\/GeneView manage the locus organization and the gene location and provide the display of physical maps for the human IG, TR and MHC loci and for the mouse TRA\/TRD locus. IMGT\/LocusView allows to view genes in a locus and to zoom on a given area. IMGT\/GeneView allows to view a given gene in a locus. IMGT\/GeneSearch allows to search for genes in a locus based on IMGT gene names, functionality or localization on the chromosome. IMGT\/CloneSearch provides information on the clones that were used to build the locus contigs displayed in IMGT\/LocusView (accession numbers are from IMGT\/LIGM-DB, gene names from IMGT\/GENE-DB, and clone position and orientation, and overlapping clones from IMGT\/LocusView). IMGT\/GeneInfo[13] provides and displays information on the potential TR rearrangements in human and mouse.\nIMGT gene Web resources\nThe IMGT gene Web resources are compiled in the IMGT Repertoire \"Locus and genes\" section that includes Chromosomal localizations, Locus representations, Locus description, Gene exon\/intron organization, Gene exon\/intron splicing sites, Gene tables, Potential germline repertoires, the complete lists of human and mouse IG and TR genes, and the correspondences between nomenclatures [3,4] (Table 3). The IMGT Repertoire \"Probes and RFLP\" section provides additional data on gene insertion\/deletion.\nIMGT structure database, tool and Web resources\nThe IMGT structural approach refers to the study of the 2D and 3D structures of the IG, TR, MHC and RPI, and to the antigen or ligand binding characteristics in relation with the protein functions, polymorphisms and evolution (Table 4). The structural approach relies on the CLASSIFICATION concept (IMGT gene and allele names), DESCRIPTION concept (receptor and chain description, domain delimitations), and NUMEROTATION concept (amino acid positions according to the IMGT unique numbering [14-18]).\nTable 4\nIMGT structure database, analysis tool and Web resources\nIMGT structural database [1]\nIMGT structural analysis tool [1]\nIMGT Repertoire\"2D and 3D structures\" section [2]\nIMGT\/3D structure-DB [15]\nIMGT\/StructuralQuery [15]\n2D Colliers de Perles IG and TR [3, 4, 16, 17, 19] (1)2D Colliers de Perles MHC [18, 36]2D Colliers de Perles RPI [16-18, 21, 22, 24, 37]IMGT classes for amino acid characteristics [31]IMGT Colliers de Perles reference profiles [31]3D representations (1)\n(1) Cover of the Nucleic Acids Research 1999 database issue \nStructural and functional domains of the IG and TR chains comprise the variable domain or V-DOMAIN (9-strand beta-sandwich) which corresponds to the V-J-REGION or V-D-J-REGION and is encoded by two or three genes [3,4], the constant domain or C-DOMAIN (7-strand beta-sandwich), and, for the MHC chains, the groove domain or G-DOMAIN (4 beta-strand and one alpha-helix). A uniform numbering system for IG and TR V-DOMAINs of all vertebrate species has been established to facilitate sequence comparison and cross-referencing between experiments from different laboratories whatever the antigen receptor (IG or TR), the chain type, or the species [14-16]. In the IMGT unique numbering, conserved amino acids from frameworks always have the same number whatever the IG or TR variable sequence, and whatever the species they come from. As examples: Cysteine 23 (in FR1-IMGT), Tryptophan 41 (in FR2-IMGT), hydrophobic amino acid 89 and Cysteine 104 (in FR3-IMGT) (Figure 2). This numbering has been applied with success to all the sequences belonging to the V-set of the IgSF [20], including non-rearranging sequences in vertebrates (human CD4, Xenopus CTXg1, etc.) and in invertebrates (drosophila amalgam, drosophila fasciclin II, etc.) [15,16,21]. The IMGT unique numbering, initially defined for the V-DOMAINs of the IG and TR and for the V-LIKE-DOMAINs of IgSF proteins other than IG and TR, has been extended to the C-DOMAINs of the IG and TR (Figure 2B), and to the C-LIKE-DOMAINs of IgSF proteins other than IG and TR [17]. An IMGT unique numbering has also been implemented for the groove domain (G-DOMAIN) of the MHC class I and II chains (Figure 3), and for the G-LIKE-DOMAINs of MhcSF proteins other than MHC [18].\nFigure 2\nIMGT Colliers de Perles of a V-DOMAIN (A) and of a C-DOMAIN (B) (code PDB 1mcd in IMGT\/3Dstructure-DB [9]). IMGT Colliers de Perles are shown on one layer (on the left hand side) and on two layers with hydrogen bonds (on the right hand side). (A) The IMGT Collier de Perles of a V-DOMAIN is based on the IMGT unique numbering for V-DOMAIN and V-LIKE-DOMAIN [16]. The CDR-IMGT are limited by amino acids shown in squares, which belong to the neighbouring FR-IMGT. The CDR3-IMGT extends from position 105 to position 117. CDR-IMGT regions are colored as follows on the IMGT site: CDR1-IMGT (blue), CDR2-IMGT (bright green), CDR3-IMGT (dark green) and hydrogen bonds are shown as green lines. (B) The IMGT Collier de Perles of a C-DOMAIN is based on the IMGT unique numbering for C-DOMAIN and C-LIKE-DOMAIN [17]. Amino acids are shown in the one-letter abbreviation. Arrows indicate the direction of the beta strands that form the two beta sheets of the immunoglobulin fold [3, 4]. Hatched circles correspond to missing positions according to the IMGT unique numbering [16, 17]. In the IMGT Collier de Perles on the IMGT Web site  hydrophobic amino acids (hydropathy index with positive value) and Tryptophan (W) found at a given position in more than 50 % of analysed IG and TR sequences are shown in blue, and all Proline (P) are shown in yellow.\nFigure 3\nIMGT Colliers de Perles of the two G-DOMAINs of MHC class I (A) and of MHC class II (B) proteins (codes PDB 1bd2 and 1aqd, respectively, in IMGT\/3Dstructure-DB [9]). The IMGT Collier de Perles of a G-DOMAIN is based on the IMGT unique numbering for G-DOMAIN and G-LIKE-DOMAIN [18]. (A) The two MHC-I G-DOMAINs, G-ALPHA1 (top) and G-ALPHA2 (bottom), form the groove of the MHC class I chain (I-ALPHA). (B) The two MHC-II G-DOMAINs, G-ALPHA (top) of the MHC class II alpha chain (II-ALPHA) and G-BETA (bottom) of the MHC class II beta chain (II-BETA), form the groove of the MHC class II protein [36]. Amino acids are shown in the one-letter abbreviation. Hatched circles correspond to missing positions according to the IMGT unique numbering [18]. Positions in colour correspond to the IMGT contact sites provided, for each peptide\/MHC 3D structure, in IMGT\/3Dstructure-DB [36].\nIMGT\/3Dstructure-DB, the IMGT 3D structure database\nIMGT\/3Dstructure-DB is the IMGT 3D structure database, created by LIGM, and on the Web since November 2001 [9]. In August 2005, IMGT\/3Dstructure-DB contained 946 atomic coordinate files. IMGT\/3Dstructure-DB comprises IG, TR, MHC and RPI with known 3D structures [9,36,37]. Coordinate files extracted from the Protein Data Bank (PDB) [38] are renumbered according to the standardized IMGT unique numbering [16-18]. The IMGT\/3Dstructure-DB card provides, on-line, the complete information for each IMGT\/3Dstructure-DB entry. The IMGT\/3Dstructure-DB card shows a summary table and a menu that gives access to five sections: \"Chain details\", \"Contact analysis\", \"Visualization with Jmol\", \"Renumbered file\" and \"References and links\". The \"Chain details\" section provides chain description, IMGT gene and allele names, IMGT chain and domain labels, domain delimitations, amino acid positions according to the IMGT unique numbering, IMGT Colliers de Perles [16-19]. The \"Contact analysis\" section provides contact types and categories between domains (in IMGT\/3Dstructure-DB Domain contacts) and atom contacts at the residue and position level (in IMGT\/3Dstructure-DB Residue@Position contacts) [37]. (IMGT\/3Dstructure-DB Documentation). The \"Renumbered file\" section downloadable provides renumbered IMGT\/3Dstructure-DB flat files.\nIMGT\/StructuralQuery tool\nThe IMGT\/StructuralQuery tool [9] analyses the interactions of the residues of the antigen receptors IG and TR, MHC, RPI, antigens and ligands. The contacts are described per domain (intra- and inter-domain contacts) and annotated in term of IMGT labels (chains, domain), positions (IMGT unique numbering), backbone or side-chain implication [37]. IMGT\/StructuralQuery allows to retrieve the IMGT\/3Dstructure-DB entries, based on specific structural characteristics: phi and psi angles, accessible surface area (ASA), amino acid type, distance in angstrom between amino acids, CDR-IMGT lengths.\nIMGT structure Web resources\nThe IMGT stucture Web resources are compiled in the IMGT Repertoire \"2D and 3D structures\" section which includes 2D representations or IMGT Colliers de Perles [16-19], 3D representations, FR-IMGT and CDR-IMGT lengths [16], amino acid chemical characteristics profiles [31], etc. In order to appropriately analyse the amino acid resemblances and differences between IG, TR, MHC and RPI chains, eleven IMGT classes were defined for the 'chemical characteristics' amino acid properties and used to set up IMGT Colliers de Perles reference profiles [31]. The IMGT Colliers de Perles reference profiles allow to easily compare amino acid properties at each position whatever the domain, the chain, the receptor or the species. The IG and TR variable and constant domains represent a privileged situation for the analysis of amino acid properties in relation with 3D structures, by the conservation of their 3D structure despite divergent amino acid sequences, and by the considerable amount of genomic (IMGT Repertoire), structural (IMGT\/3Dstructure-DB) and functional data available. These data are not only useful to study mutations and allele polymorphisms, but are also needed to establish correlations between amino acids in the protein sequences and 3D structures and to determine amino acids potentially involved in the immunogenicity.\nConclusion\nIn order to allow any IMGT component to be automatically queried and to achieve a higher level of interoperability inside the IMGT information system and with other information systems, our current objectives include the modelling of the three major IMGT biological approaches, genomics, genetics and structural approaches, the analysis of the IMGT components (databases, tools and Web resources) in relation with the concepts, and the development of Web services [2]. They are the first steps towards the implementation of IMGT-Choreography [2], which corresponds to the process of complex immunogenetics knowledge [25] and to the connection of treatments performed by the IMGT component Web services. IMGT-Choreography has for goal to combine and join the IMGT database queries and analysis tools. In order to keep only significant approaches, a rigorous analysis of the scientific standards [3,4], of the biologist requests and of the clinician needs [39-42] has been undertaken in the three main biological approaches: genomics, genetics and structural approaches. The design of IMGT-Choreography and the creation of dynamic interactions between the IMGT databases and tools, using the Web services and IMGT-ML, represent novel and major developments of IMGT, the international reference in immunogenetics and immunoinformatics. IMGT-Choreography enhances the dynamic interactions between the IMGT components to answer complex biological and clinical requests.\nSince July 1995, IMGT has been available on the Web at . IMGT has an exceptional response with more than 140,000 requests a month. The information is of much value to clinicians and biological scientists in general. IMGT databases, tools and Web resources are extensively queried and used by scientists from both academic and industrial laboratories, from very diverse research domains: (i) fundamental and medical research (repertoire analysis of the IG antibody sites and of the TR recognition sites in normal and pathological situations such as autoimmune diseases, infectious diseases, AIDS, leukemias, lymphomas, myelomas), (ii) veterinary research (IG and TR repertoires in farm and wild life species), (iii) genome diversity and genome evolution studies of the adaptive immune responses, (iv) structural evolution of the IgSF and MhcSF proteins, (v) biotechnology related to antibody engineering (single chain Fragment variable (scFv), phage displays, combinatorial libraries, chimeric, humanized and human antibodies), (vi) diagnostics (clonalities, detection and follow up of residual diseases) and (vii) therapeutical approaches (grafts, immunotherapy, vaccinology).\nCiting IMGT\nIf you use IMGT databases, tools and\/or Web resources, please cite [1] and this paper as references, and quote the IMGT Home page URL address, .","keyphrases":["imgt","immunogenetics","information system","immunoinformatics","knowledge resource","immunoglobulin","t cell receptor","mhc","superfamily","database","three-dimensional","ontology","annotation","antibody","3d structure","collier de perles","polymorphism","hla"],"prmu":["P","P","P","P","P","P","P","P","P","P","P","P","P","P","P","P","P","U"]}
{"id":"Qual_Life_Res-3-1-1915610","title":"Evaluating the discriminatory power of EQ-5D, HUI2 and HUI3 in a US general population survey using Shannon\u2019s indices\n","text":"Objectives To compare quantitatively the discriminatory power of the EQ-5D, HUI2 and HUI3 in terms of absolute and relative informativity, using Shannon\u2019s indices.\nIntroduction\nThe need for assessing health-related quality of life (HRQL) has brought forth hundreds of HRQL instruments, both generic and disease-specific [1, 2]. Generic instruments fall into two main categories: (1) preference-based health classification systems, and (2) non-preference based measures, sometimes referred to as health profile or psychometric measures [1, 3, 4]. Preference-based classification systems, also referred to as multi-attribute utility instruments (MAUIs) are standardized health state classifications that can be used to obtain a single summary index (utility score) or so-called preference weight for different health states. At the core of any MAUI is a classification system consisting of multiple attributes (dimensions) with ordered levels for each dimension. Most MAUIs are generic and aim to cover the full spectrum of disease and disability. MAUIs are widely used as measures of health outcome and are applied in clinical and economic evaluation (to calculate QALYs) and in population health surveys. Three widely used MAUIs are the EQ-5D, the Health Utilities Index Mark 2 (HUI2) and the Health Utilities Index Mark 3 (HUI3) [5\u20137]. All three instruments have shown acceptable psychometric properties as established by conventional measures [8, 9].\nFeasibility, reliability, validity and responsiveness are important measurement properties in MAUIs, just as they are in non-preference based HRQL and health status measures such as the SF-36. However, these properties may be operationalized differently in MAUIs compared to non-preference based measures [4, 10, 11]. An underlying property to the concepts of reliability, validity and responsiveness is the ability of an instrument to discriminate between (\u2018true\u2019) different levels of health. This requires a MAUI to define the full range of potential health states, and to be sensitive over this range. A necessary measurement property for any health status measure (including MAUIs) is the ability to discriminate among people at a single point in time. This property is sometimes referred to as sensitivity or, more accurately: \u201cdiscriminatory power\u201d [12\u201314].\nGuyatt et\u00a0al. (1992) proposed a reliability coefficient as a suitable statistic to express discriminatory power [15]. Reliability essentially reflects two different concepts: (1) consistency, e.g. between raters (inter-rater reliability) or over time (test-retest reliability), and (2) discriminatory power: the ability of an instrument to discriminate among people [16]. We propose Shannon\u2019s indices of informativity as suitable measures that solely reflect discriminatory power [17].\nDiscriminatory power of MAUIs is usually investigated in an informal and partial manner by examining the frequency distributions, e.g. for floor or ceiling effects [12, 13, 18\u201320]. Shannon\u2019s indices are suitable to assess discriminatory power in MAUIs for two reasons: first, they are theoretically based and second, they incorporate the frequency distribution across all categories of a MAUI\u2019s health status classification system (not just the highest and lowest categories, as is the case with ceiling and floor effects).\nOur aim is to investigate the discriminatory power of the EQ-5D, HUI2 and HUI3 in a general population sample, as expressed by Shannon\u2019s indices. Informativity was assessed separately by dimension and by MAUI as a whole.\nMethods\nData\nA publicly available dataset was used (at http:\/\/www.ahrq.gov\/rice\/), resulting from the US EQ-5D valuation study [21, 22]. Collected data consisted of self-completed EQ-5D and HUI2\/3 data from a sample of the general adult US population, with an over-sampling of Hispanics and non-Hispanic Blacks. The HUI2\/3 data were collected using a standardized 15-item questionnaire, from which HUI2 and HUI3 health profiles were extracted using available recoding algorithms [23]. Only the responses of 3,691 respondents who had no missing data on any of the three instruments were included in this study (91.2% of the total number of respondents).\nInstruments\nThe EQ-5D descriptive system consists of 5 dimensions (items) with 3 levels each, logically defining 243 unique health states (permutations). The HUI2 was originally developed to assess outcomes in survivors of cancer in childhood and contains 6 dimensions (excluding the original HUI2 dimension of fertility) with 4\u20135 levels per dimension. The HUI3, originally developed for a general population health survey in Canada, has 8 dimensions with 5\u20136 levels per dimension. The HUI2 and HUI3 descriptive systems define 8,000 and 972,000 unique health states, respectively [6]. Table\u00a01 compares the 5 dimensions common to at least two of the classification systems: Mobility\/Ambulation; Anxiety\/Depression\/Emotion; Pain\/Discomfort (EQ-5D; HUI2; HUI3); Self-Care (EQ-5D; HUI2); and Cognition (HUI2; HUI3).Table\u00a01Level descriptions for common dimensions between EQ-5D, HUI2 and HUI3EQ-5DHUI2HUI3MobilityMobilityAmbulationNo problems in walking aboutAble to walk, bend, lift, jump, and run normally for ageAble to walk around the neighbourhood without difficulty, and without walking equipmentSome problems in walking aboutWalks, bends, lifts, jumps, or runs with some limitations but does not require helpAble to walk around the neighbourhood with difficulty; but does not require walking equipment or the help of another personConfined to bedRequires mechanical equipment (such as canes, crutches, braces, or wheelchair) to walk or get around independentlyAble to walk around the neighbourhood with walking equipment, but without the help of another personRequires the help of another person to walk or get around and requires mechanical equipment as wellAble to walk only short distances with walking equipment, and requires a wheelchair to get around the neighbourhoodUnable to control or use arms and legsUnable to walk alone, even with walking equipment. Able to walk short distances with the help of another person, and requires a wheelchair to get around the neighbourhoodCannot walk at allSelf-careSelf-careNo problems with self-careEats, bathes, dresses, and uses the toilet normally for ageSome problems washing or dressing selfEats, bathes, dresses, or uses the toilet independently with difficultyUnable to wash or dress selfRequires mechanical equipment to eat, bathe, dress, or use the toilet independentlyRequires the help of another person to eat, bathe, dress, or use the toiletPain\/DiscomfortPainPainNo pain or discomfortFree of pain and discomfortFree of pain and discomfortModerate pain or discomfortOccasional pain. Discomfort relieved by non-prescription drugs or self-control activity without disruption of normal activitiesMild to moderate pain that prevents no activitiesExtreme pain or discomfortFrequent pain. Discomfort relieved by oral medicines with occasional disruption of normal activitiesModerate pain that prevents a few activitiesFrequent pain; frequent disruption of normalactivities. Discomfort requires prescription narcotics for reliefModerate to severe pain that prevents some activitiesSevere pain. Pain not relieved by drugs and constantly disrupts normal activitiesSevere pain that prevents most activitiesAnxiety\/DepressionEmotionEmotionNot anxious or depressedGenerally happy and free from worryHappy and interested in lifeModerately anxious or depressedOccasionally fretful, angry, irritable, anxious, depressed, or suffering \"night terrors\"Somewhat happyExtremely anxious or depressedOften fretful, angry, irritable, anxious, depressed, or suffering \"night terrors\"Somewhat unhappyAlmost always fretful, angry, irritable, anxious, depressedVery unhappyExtremely fretful, angry, irritable, anxious, or depressed usually requiring hospitalization or psychiatric institutional careSo unhappy that life is not worthwhile\u00a0CognitionCognitionLearns and remembers school work normally for ageAble to remember most things, think clearly and solve day to day problemsLearns and remembers school work more slowly than classmates as judged by parents and\/or teachersAble to remember most things, but have a little difficulty when trying to think and solve day to day problemsLearns and remembers very slowly and usually requires special educational assistanceSomewhat forgetful, but able to think clearly and solve day to day problemsUnable to learn and rememberSomewhat forgetful, and have a little difficulty when trying to think or solve day to day problemsVery forgetful, and have great difficulty when trying to think or solve day to day problemsUnable to remember anything at all, and unable to think or solve day to day problems\nShannon\u2019s indices: background and properties\nThe Shannon index, named after Claude Shannon who is considered to be the founder of information theory, was initially developed to separate noise from information carrying signals in telecommunication systems [17]. The Shannon index is also known as the Shannon\u2013Weaver index because of Warren Weaver\u2019s contribution to Shannon\u2019s original paper, and as the Shannon\u2013Wiener index named after Norbert Wiener who independently developed a concept similar to Shannon\u2019s [24, 25]. The Shannon index has been applied in a variety of fields, ranging from ecology (as a measure of biodiversity) to psychology, record linkage and molecular biology (genetic diversity) [26\u201330].\nIn information theory, the information of a signal is distinguished from the meaning or the semantic content of a signal. Rather, the information is quantified and is identified with uncertainty. Informativity is dependent on the number of classes (e.g. bits or response options) and the distribution of the observations (the \u2018signal\u2019) among classes. For classifications, this implies that if one would want to develop a useful (informative) distinction between, say, European countries, distinguishing between Scandinavian and non-Scandinavian countries would be far less informative than distinguishing between Northern, Western, Eastern and Southern European countries. Note that the latter classification not only contains more categories but the countries are also more evenly distributed among categories.\nThe Shannon index is defined as:\nwhere H\u2032 represents the absolute amount of informativity captured, C is the number of possible categories (levels or permutations in this study), and pi\u00a0=\u00a0ni\/N, the proportion of observations in the ith category (i\u00a0=\u00a01,...,C), where ni is the observed number of scores (responses) in category i and N is the total sample size [17]. Any log base can be used, as long as one is consistent. Using log base 2, as did Shannon, allows the interpretation of the resulting units as bits per individual. The higher the index H\u2032 is, the more information is captured by the system. In case of a homogeneous (rectangular) distribution, i.e. ratings are evenly distributed among categories (pi\u00a0=\u00a0p* for all i), the optimal amount of information is captured and H\u2032 has reached its maximum (H\u2032max) which equals log2C. If the number of categories (C) is increased, H\u2032max increases accordingly but H\u2032 will only increase if the newly added categories are actually used. The variance of the Shannon index is defined as [31]:\nAccordingly, standard errors and 95% confidence intervals can be calculated.\nThe Shannon index combines the absolute information content as expressed by the number of categories with the extent to which the information is evenly spread over these categories. Shannon\u2019s Evenness index (J\u2032) exclusively reflects the latter component, i.e. the rectangularity of a distribution. This measure was first proposed by Lloyd and Ghelardi [32]; Shannon already referred to it as relative entropy and Pielou termed the concept \u2018evenness\u2019 [17, 33]. Shannon\u2019s Evenness index (J\u2032) is defined as: J\u2032\u00a0=\u00a0H\u2032\/H\u2032max, which expresses the use of the system (H\u2032) given its potential (H\u2032max). Shannon\u2019s index H\u2032 can be considered as an expression of the absolute informativity of a system whereas Shannon\u2019s Evenness index J\u2032 expresses the relative informativity of a system or \u2018evenness\u2019 of a distribution, regardless the number of categories.\nTwo alternative measures of (bio)diversity are the Simpson and the Brillouin index. We used the Shannon index, since the Brillouin index is dependent on sample size and the Simpson index gives very little weight to categories that are rarely occupied [26, 34, 35].\nShannon indices applied to MAUIs\nThe basic characteristics of Shannon\u2019s indices which make them suitable to reflect discriminatory power have been documented and are explained as follows. In an item where a response option has a very high (or low) endorsement, e.g. p is over 0.95 (or under 0.05), one learns very little because one can predict with more than 95% certainty what the answer will be. In other words, there is very little information being transmitted. Conversely, the maximum amount of information (uncertainty) is being transmitted when, in an item with two response options, p is 0.50 for each response option. As described above, this characteristic of an even distribution underlies the Shannon indices. In case of an even distribution, the item (dimension) is being most efficiently used, which means that the discriminant ability of the level descriptors is maximal.\nThe Shannon indices can be calculated by dimension separately or by MAUI as a whole. To calculate Shannon\u2019s indices by dimension, levels are treated as categories, so C represents the number of levels (L), pi is the proportion of responses of the ith level, and H\u2032max equals log2L. Suppose the EQ-5D Mobility dimension is scored by 10 respondents: no problems (n\u00a0=\u00a06), some problems (n\u00a0=\u00a03) and confined to bed (n\u00a0=\u00a01). Shannon\u2019s index for Mobility is calculated as H\u2032\u00a0=\u00a0\u2013((0.6 log2 0.6)\u00a0+\u00a0(0.3 log2 0.3)\u00a0+\u00a0(0.1 log2 0.1))\u00a0=\u00a01.30 and H\u2032max\u00a0=\u00a0log23\u00a0\u00a0=\u00a0\u00a01.58, so J\u2032\u00a0=\u00a01.30\/1.58\u00a0=\u00a00.82.\nFigure\u00a01 illustrates the difference between absolute and relative informativity (H\u2032, evenness J\u2032) relative to the number of levels (L) in a series of hypothetical health classification systems designed to describe the same underlying dimension. For illustrative purposes we consider only one dimension. Figure\u00a01a shows two distributions of responses corresponding to two different classification systems, both of which have 3 levels; one system results in a skewed distribution while the other results in a rectangular distribution. Assuming these responses are obtained within the same population, the system that yields the rectangular distribution is superior in discriminating between patients and the Shannon indices have both reached their maximum values. Figure\u00a01b illustrates the concept of relative informativity. The left panel shows the same skewed distribution as depicted in Figure\u00a01a, the right panel shows the same distribution of responses but now as it results from a 5 level classification system in which levels 2 and 4 are unused. Absolute informativity (Shannon\u2019s H\u2032) remains unchanged but J\u2032 decreases, expressing lower relative informativity. Clearly, adding 2 extra levels that do not represent anyone in the population (no individual shifts from a current level to any of the new levels) does not lead to a gain in absolute informativity (H\u2032) while the potential of a 5 level system is underutilized, compared to a 3 level system, which is expressed by a lower J\u2032. So why not use just the Shannon Evenness index? Figure\u00a01c shows the added value of absolute informativity (the H\u2032 index). If the 3 and 5 level systems both yield rectangular distributions, evenness J\u2032 will be the same but obviously H\u2032 increases since the 5 level system is much more refined in discriminating between patients.Fig.\u00a01Examples of Absolute Informativity (H\u2032) and Relative Informativity (J\u2032) with Skewed and Rectangular Distributions in a 3 Level System and 5 Level System\nTo calculate Shannon\u2019s indices by instrument as a whole, permutations are treated as unique categories (e.g. 243 categories for EQ-5D), so C is the number of permutations (Pmax), pi is the proportion of the ith permutation, and H\u2032max now equals log2Pmax.\nSince the number of observations in our study (N\u00a0=\u00a03,691) is lower than the number of theoretically possible permutations in HUI2 (8,000) and HUI3 (972,000), maximum informativity (H\u2032max) in HUI2 and HUI3, and consequently maximum relative informativity J\u2032 cannot be reached a priori. Therefore, Shannon\u2019s indices by MAUI as a whole were calculated using an estimation approach. Assuming that the current sample is representative, subsamples of the original set of observed health states were drawn in order to estimate the number of different health states in hypothetical populations of 1, 10, and 100 million respondents, by means of extrapolation. This procedure was repeated for different proportions of the population in relation to the number of health states (e.g. 11 different EQ-5D health states accounted for a 90% proportion of the respondents), in order to estimate the shape of the frequency distribution in the hypothetical populations of 1, 10, and 100 million respondents. Finally, Shannon\u2019s H\u2032 and J\u2032 could be calculated (details can be obtained from the authors).\nResults\nThe mean age of the respondents was 42.9\u00a0years (range: 18.0\u201399.3\u00a0years), with 42.2% of the respondents being male. White (non-Hispanic) respondents were 1,435 (38.9%), non-Hispanic blacks were 1,018 (27.6%) and Hispanic were 1,100 (29.8%).\nTable\u00a02 shows the frequencies of responses to the EQ-5D, HUI2 and HUI3 dimensions. The dominant response was \u2018no problems\u2019 (level 1) for all dimensions in all instruments, with a proportion larger than 90% for 1 out of 5 dimensions in EQ-5D (Self-Care), 1 out of 6 in HUI2 (Self-Care) and 3 out of 8 in HUI3 (Hearing, Speech, Dexterity). In all EQ-5D and HUI2 dimensions, frequencies decreased with increasing level severity. In the HUI3 Cognition dimension however, more respondents reported problems at level 3 (17.9%) and level 4 (7.4%) than at level 2 (4.1%). Although small, these differences also occurred in the HUI3 Vision and HUI3 Hearing dimensions.Table\u00a02Frequency distribution (%) of responses to the EQ-5D, HUI2 and HUI3 instruments (N\u00a0\u00a0=\u00a0\u00a03,691)\u00a0Level 1Level 2Level 3Level 4Level 5Level 6EQ-5DMobility82.1717.530.30\u2013\u2013\u2013Self care95.584.010.41\u2013\u2013\u2013Usual activities84.8813.571.54\u2013\u2013\u2013Pain\/Discomfort61.2834.714.01\u2013\u2013\u2013Anxiety\/Depression73.8623.572.57\u2013\u2013\u2013HUI2Sensation44.5443.5410.761.16\u2013\u2013Mobility87.248.483.600.680.00\u2013Emotion69.2027.851.820.650.49\u2013Cognition68.3629.941.630.08\u2013\u2013Self-care96.642.950.190.22\u2013\u2013Pain48.1740.947.102.980.81\u2013HUI3Vision48.5047.871.002.470.030.14Hearing94.580.921.521.650.301.03Speech92.684.822.030.430.03\u2013Ambulation87.248.482.551.060.510.16Dexterity92.445.820.790.700.140.11Emotion72.5022.323.741.160.27\u2013Cognition68.364.1517.857.372.190.08Pain49.3433.7311.464.011.46\u2013\nFigure\u00a02 shows absolute informativity (Shannon\u2019s H\u2032) and relative informativity (Shannon\u2019s Evenness J\u2032) of the common dimensions among the three instruments. Absolute informativity (H\u2032) was highest for HUI3 in all common dimensions, with largest differences between HUI3 and the other two instruments in the dimensions Pain\/Discomfort (0.52 compared to EQ-5D; 0.15 compared to HUI2) and Cognition (0.41 compared to HUI2).Fig.\u00a02The Shannon Index (H\u2032) and the Shannon Evenness Index  for the common dimensions between EQ-5D, HUI2 and HUI3: comparison by dimension. NA\u00a0=\u00a0not available;  Confidence intervals had an average range of 0.0012 (H\u2032) and of 0.00081 (J\u2032)\nRelative informativity (J\u2032) was highest for EQ-5D in all common dimensions, with largest differences with the other two instruments in the dimensions Mobility\/Ambulation (0.14 compared to HUI2; 0.16 compared to HUI3) and Anxiety\/Depression\/Emotion (0.14 compared to HUI2; 0.13 compared to HUI3).\nTable\u00a03 shows Shannon\u2019s indices by classification system as a whole. The EQ-5D, HUI2 and HUI3 descriptive systems distinguished 91, 322, and 694 observed different unique health states, accounting for 37.4%, 4.0%, and 0.07% of all possible permutations, respectively. The estimation procedure indicated that absolute informativity was highest for HUI3 (range 10.96\u201313.36), followed by HUI2 (range 8.57\u20139.48), and lowest for EQ-5D (range 6.24\u20136.41). Relative informativity was highest in EQ-5D (range 0.79\u20130.81), followed by HUI2 (range 0.66\u20130.73), and lowest for HUI3 (range 0.55\u20130.67).Table\u00a03Shannon\u2019s index (H\u2032) and Shannon\u2019s evenness index (J\u2032) for EQ-5D, HUI2, and HUI3: Comparison by instrument\u00a0EQ-5DHUI2HUI3Pmax (permutations)2438000972,000Observed health states91322694H\u2032max7.9212.9719.89EstimationH\u2032J\u2032H\u2032J\u2032H\u2032J\u2032N\u00a0=\u00a01,000,0006.240.798.570.6610.960.55N\u00a0=\u00a010,000,0006.370.809.120.7012.290.62N\u00a0=\u00a0100,000,0006.410.819.480.7313.360.67\nDiscussion\nWe compared the discriminatory power of the EQ-5D, HUI2 and HUI3 in the general population, using Shannon\u2019s indices of absolute and relative informativity, for each dimension separately and by MAUI as a whole.\nAs might be expected in a general population sample, most respondents reported no problems on all dimensions and there were fewer responses with increasing level severity. An exception is HUI3 Cognition, where respondents reported more problems on levels 3 and 4 than on level 2. This is probably due to the fact that this dimension is not unidimensional, and levels 2 and 3 are conceptualized parallel rather than ordinal. That is, HUI3 Cognition level 2 focuses on problems in thinking and problem solving, level 3 addresses problems in remembering, whereas level 4 combines the problems mentioned in levels 2 and 3.\nAbsolute informativity by dimension was highest for the HUI3 descriptive system. EQ-5D appears to underperform in the Pain\/Discomfort dimension. Moreover, EQ-5D appears to miss a considerable \u2018amount\u2019 of disability: 61.3% of the population indicated to have no problems on EQ-5D, against 48.2% on HUI2 and 49.3% on HUI3 (Table\u00a02). Shannon\u2019s H\u2032 \u2018translated\u2019 this difference adequately (Figure\u00a02). Apparently, for this population, the EQ-5D would benefit from more levels on the Pain\/Discomfort dimension. Regarding the Cognition dimension, the difference in absolute informativity between HUI2 and HUI3 might be explained by the 2 extra levels in HUI3, but the higher J\u2032 value in HUI3 suggests an alternative contributive factor. One explanation may be that HUI3 Cognition is not unidimensional and more sensitive to mild problems (levels 2\u20134) than HUI2 Cognition (level 2). Another explanation could be that the difference is due to currently suboptimal recoding algorithms.\nFor relative informativity by dimension, the EQ-5D descriptive system showed superior results in Mobility\/Ambulation, Self-Care and Anxiety\/Depression\/Emotion. The large differences in Mobility\/Ambulation could be due to a relatively large leap in the grading of the level descriptions in HUI3 Ambulation, where the difference between level 1 (\u2018without difficulty\u2019) and level 2 (\u2018with difficulty\u2019) can be considered disproportionately large in a 6 level dimension. The same leap from level 1 (\u2018normal\u2019) to level 2 (\u2018with difficulty\u2019) occurs in HUI2 Self-Care. We found that the 3 level EQ-5D Self Care outperformed the 4 level HUI2 Self-Care in both absolute and relative informativity (Fig.\u00a02), which is probably due to the severe grading of level 2 in HUI2. The difference in relative informativity between EQ-5D and the HUI instruments in Anxiety\/Depression\/Emotion is probably due to the 2 extra levels in HUI2 and HUI3 that are rarely endorsed.\nOverall, performance in terms of informativity of EQ-5D, HUI2 and HUI3 of the common dimensions varies over dimensions. The Pain\/Discomfort dimension of EQ-5D, but perhaps also other dimensions, might benefit from an extension to 4 or 5 levels. HUI2 and HUI3 might benefit from more sensitive grading terms in their level descriptions, especially the \u2018threshold\u2019 level 2, in Ambulation (HUI3) and Self-Care (HUI2).\nWhen assessing informativity by instrument, HUI3 shows the best results on absolute informativity but the lowest on relative informativity while EQ-5D shows highest relative informativity and lowest absolute informativity. HUI2 seems to be the optimal compromise. The importance of differences in the Shannon indices ultimately requires empirical evidence over a wider range of populations, conditions and instruments, including evidence on discriminant validity.\nAs Shannon\u2019s indices are new in the field of health status measurement, some methodological issues need to be addressed, taking into account that their principal focus is on classifications with mutually exclusive categories, rather than conventional (health status) measures which by design contain multiple partially overlapping items.\nThe Shannon indices share some properties with reliability coefficients. Like reliability indices, they express discriminatory power. Furthermore, they are also non-dimensional, i.e. they have no relation to the content, meaning or clinical relevance of what the instrument aims to measure, which make them suitable for comparability, between instruments as well as between populations. However, reliability reflects two different concepts: discriminatory power as such, and consistency, e.g. consistency between raters (inter-rater reliability) or consistency over time (test-retest reliability). This requires a repeated measurement (repetition \u2018over raters\u2019 or over time) which introduces an error component in case of a difference among the repeated measurements. Shannon\u2019s indices solely reflect discriminatory power, and need only a single measurement. Furthermore, the Shannon indices are non-parametric measures and therefore highly suitable for nominal or ordinal measurement scales.\nSince Shannon\u2019s indices have no dimension and are independent of any external standard, a rectangular distribution is always the ideal from the perspective of informativity. When comparing the discriminatory power of similar dimensions of different MAUIs, rectangularity is always optimal as it reflects which MAUI is the most sensitive in discriminating between different persons in that particular population. This implies that one MAUI cannot be superior in varying populations (e.g. a general population and a diseased population sample). Furthermore, MAUIs are bound to score rather low on discriminatory power in a general population sample, as the extreme categories, which have to be included for coverage of the full spectrum of diseases, will not be endorsed frequently.\nPreviously, the common approach to investigate discriminatory power was examining the frequency distributions of responses, e.g. for ceiling or floor effects. A comprehensive, formal measure to express discriminatory power such as Shannon\u2019s indices seems clearly superior to such a \u2018face-value\u2019 method. Furthermore, when the number of categories is large (e.g. when comparing MAUIs as a whole), it becomes very difficult to make a sound comparison by just looking at the distributions.\nWe have demonstrated the use of the Shannon indices to compare the discriminatory power of different MAUIs, to show which instrument is more sensitive in differentiating between levels of health in the population at hand. But they may also be used to guide the development of new, or optimization of existing MAUIs, by helping determine how many levels are efficient for each dimension. This is a particularly relevant consideration for MAUIs, since adding extra levels in a descriptive system makes it increasingly complex, and the derivation of a robust set of preference weights more challenging.\nApart from MAUIs, the Shannon indices can also be used in a wide range of other classifications in the medical domain (e.g. the Karnofsky scale, the Spitzer QL index) and in the clinical domain (e.g. the APGAR score, the Child-Pugh classification).\nA practical weakness of the Shannon approach is that when the sample size is exceeded by the total number of health states described by all permutations across all dimensions of a MAUI, informativity (for the instrument as a whole) has to be estimated. This implies that using the Shannon Evenness index by instrument is not very practical when a health classification system has a large number of permutations as was the case in HUI3 (972,000 permutations). This however is not a disadvantage of the Shannon methodology per se, but also a matter of classification design (overload of dimensions with detailed response options producing an excessive amount of \u2018empty\u2019 permutations), or a practical problem (excessive data collection).\nFrom a clinical or psychometric perspective it may seem tempting to extend any MAUI with extra levels or dimensions as it provides more clinically relevant detail generally and improves reliability. But Shannon\u2019s indices reveal that this may not always be a prudent approach. Increasing the number of levels per dimension (or permutations in the entire system) will probably result in higher H\u2032 values but J\u2032 values are likely to drop, as in fact our results for HUI3 indicate. This raises the question where the balance between H\u2032 and J\u2032 is optimal as more categories require more extensive subsequent studies to derive utility functions for the associated classification system.\nHow the Shannon indices will behave in a different population, such as patient populations, remains to be investigated. So far, Shannon\u2019s indices proved to be useful in showing weaknesses of level gradings used in EQ-5D, HUI2 and HUI3, and offers leads for improvement, establishing their practical psychometric value.","keyphrases":["psychometrics","population health","health status","methodology","health-related quality-of-life"],"prmu":["P","P","P","P","M"]}
{"id":"Arch_Orthop_Trauma_Surg-3-1-1914284","title":"Poor accuracy of freehand cup positioning during total hip arthroplasty\n","text":"Several studies have demonstrated a correlation between the acetabular cup position and the risk of dislocation, wear and range of motion after total hip arthroplasty. The present study was designed to evaluate the accuracy of the surgeon\u2019s estimated position of the cup after freehand placement in total hip replacement. Peroperative estimated abduction and anteversion of 200 acetabular components (placed by three orthopaedic surgeons and nine residents) were compared with measured outcomes (according to Pradhan) on postoperative radiographs. Cups were placed in 49.7\u00b0 (SD 6.7) of abduction and 16.0\u00b0 (SD 8.1) of anteversion. Estimation of placement was 46.3\u00b0 (SD 4.3) of abduction and 14.6\u00b0 (SD 5.9) of anteversion. Of more interest is the fact that for the orthopaedic surgeons the mean inaccuracy of estimation was 4.1\u00b0 (SD 3.9) for abduction and 5.2\u00b0 (SD 4.5) for anteversion and for their residents this was respectively, 6.3\u00b0 (SD 4.6) and 5.7\u00b0 (SD 5.0). Significant differences were found between orthopaedic surgeons and residents for inaccuracy of estimation for abduction, not for anteversion. Body mass index, sex, (un)cemented fixation and surgical approach (anterolateral or posterolateral) were not significant factors. Based upon the inaccuracy of estimation, the group\u2019s chance on future cup placement within Lewinnek\u2019s safe zone (5\u201325\u00b0 anteversion and 30\u201350\u00b0 abduction) is 82.7 and 85.2% for anteversion and abduction separately. When both parameters are combined, the chance of accurate placement is only 70.5%. The chance of placement of the acetabular component within 5\u00b0 of an intended position, for both abduction and anteversion is 21.5% this percentage decreases to just 2.9% when the tolerated error is 1\u00b0. There is a tendency to underestimate both abduction and anteversion. Orthopaedic surgeons are superior to their residents in estimating abduction of the acetabular component. The results of this study indicate that freehand placement of the acetabular component is not a reliable method.\nIntroduction\nAcetabular cup position after total hip arthroplasty is correlated with the risk of dislocation, wear and range of motion [1\u20136]. Lewinnek et\u00a0al. [7] described a safe range (5\u201325\u00b0 anteversion and 30\u201350\u00b0 abduction) to position the cup. Within this range the dislocation rate was 1.5% and outside this range 6.1%. Although the position of the cup is important for the prognosis and function of the hip, most surgeons place the cup without any specific guidance devices. During surgery the surgeon estimates the position of the acetabular component and decides if it meets the desired orientation before securing it.\nIn this study the accuracy of the surgeons peroperative estimation of the position of the cup is evaluated.\nMaterials and methods\nAll patients undergoing primary total hip arthroplasty were enrolled in the study. Patient characteristics as name, sex, age, operated side and the body mass index were recorded. All cooperating surgeons (three orthopaedic surgeons and nine residents) received a list on which the following data concerning the operation were recorded: fixation (cemented or uncemented), cup model, surgical approach, complications and the peroperative estimated anteversion and abduction of the acetabular component.\nTwo days after the operation two standardised X-rays were made. One anteroposterior X-ray was taken with the beam centered over the hip, the second was a plain AP radiograph centred on the symphisis, showing both hips. Radiographic cup anteversion was measured on the first radiograph, according to Pradhan [8], and cup abduction was assessed on the second by measuring the angle between the teardrop line and the line bisecting the opening of the acetabular cup. The combination of both radiographs was used to determine if a cup was in the ante- or retroverted position, as described by Flabeck et\u00a0al. [9].\nFor both anteversion and abduction, the inaccuracy of estimation was determined by calculating the difference between peroperative estimation and postoperative X-ray measurements. Mean values and standard deviations were calculated for measured cup position, estimated cup position and inaccuracy of estimation. A comparison of these values was assessed between orthopaedic surgeons and residents, using student\u2019s t test (P\u00a0<\u00a00.05 assumed as significant) and a multivariate linear regression analysis was applied on the data in order to investigate which factors influenced the inaccuracy of estimation.\nResults\nTwo hundred cups in 194 patients, placed between June 2003 and May 2005, were included in the study. There were 55 males and 139 females. At time of operation the mean age of the patients was 72.4\u00a0years (34\u201392) with a mean body mass index of 27.5 (16.6\u201338.1). The cups were placed by 12 different surgeons. Eighty-five cups (42.5%) were placed by three orthopaedic surgeons and 115 (57.5%) by 9 of their residents, always under supervision of one of the surgeons.\nOne hundred and fifty-seven (78.5%) cups had a cemented and 43 (21.5%) an uncemented fixation; 89 (44.5%) cups were placed in the patients\u2019 right hip and 111 (55.5%) in the left. Two surgical approaches were used. The anterolateral approach according to Mallory was used in 57 cups (28.5%), supervised by one orthopaedic surgeon and the postero-lateral approach by the other two surgeons in 143 cups (71.5%). The residents used the approach of their supervising surgeon. For the anterolateral approach according to Mallory, mean placement of the cup was in 11.8\u00b0 anteversion and 47.1\u00b0 abduction. Positioning of the cup differed significantly when a posterolateral approach was applied: 17.7\u00b0 anteversion and 50.7\u00b0 abduction (both P\u00a0<\u00a00.001). Comparing both anterolateral and posterolateral approaches, no significant differences were found for inaccuracy of estimation for either anteversion (4.9\u00b0 vs. 5.5\u00b0, respectively) or abduction (5.3\u00b0 vs. 6.1\u00b0).\nTable\u00a01 shows relevant data of estimations and the measurements of the acetabular component by both orthopaedic surgeons and residents. None of the cups was placed in retroversion.\nTable\u00a01Mean estimated and measured anteversion and abductionCups NAnteversion (degrees)DifferenceMean inaccuracy of estimationEstimatedMeasuredIa\u00a0Orthopedic surgeon8514.514.7\u22120.25.2 (SD 4.5)\u00a0Residents11514.716.9\u22122.25.7 (SD 5.0)\u00a0Total20014.6 (SD 5.9)16.0 (SD 8.1)\u22121.45.5\u00a0Significance\u2013\u2013\u2013\u2013Cups NAbductionDifferenceMean inaccuracy of estimationEstimatedMeasuredIb\u00a0Orthopedic surgeon8547.248.6\u22121.44.09 (SD 3.9)\u00a0Residents11545.650.5\u22124.96.28 (SD 4.6)\u00a0Total20046.3 (SD 4.3)49.7 (SD 6.7)\u22123.45.4\u00a0Significance0.0010.0430.0000.001\nFrom the data from which Fig.\u00a01 was constructed, it was derived that 129 cups (64.5%) were placed within 5\u00b0 of the estimated abduction, for anteversion this was the case in 122 cups (61%). There is a tendency to underestimate both abduction and anteversion.\nFig.\u00a01Results of the distribution of the difference between the estimated and measured values (degrees) for abduction (a) and anteversion (b) of the acetabular components (N)\nTable\u00a02 demonstrates the results of the chance for cup placement according to Lewinnek (5\u201325\u00b0 anteversion and 30\u201350\u00b0 abduction), based upon inaccuracy of estimation for the orthopaedic surgeons, their residents and the group in total. Virtual intended cup placement was set at 15\u00b0 anteversion and 40\u00b0 abduction. Only 56.5% of the cups were placed in this safe zone according to their measured abduction and anteversion. Table\u00a03 shows the percentage for the group and orthopaedic surgeons and residents separately, for cup placement within different ranges (20, 10, 5, 2.5 and 1\u00b0) of an intended cup position X.\nTable\u00a02Results of the chance for cup placement within the limits according to Lewinnek (5\u201325\u00b0 anteversion and 30\u201350\u00b0 abduction) for the orthopedic surgeons, their residents and the entire group. Intended cup placement was virtually set on 15\u00b0 anteversion and 40\u00b0 abductionAnteversion (%)Abduction (%)Placement according to Lewinnek (%)Orthopedic surgeons85.793.780.3Residents80.578.963.5Group82.785.270.5Table\u00a03Deviation in % of cup placement in between 20\u00b0, 10\u00b0, 5\u00b0, 2.5\u00b0 and 1\u00b0 of position X for the entire group (orthopaedic surgeons and residents): (a), the orthopaedic surgeons: (b) and the residents: (c)X\u00a0\u00b1\u00a020 (%)X\u00a0\u00b1\u00a010 (%)X\u00a0\u00b1\u00a05 (%)X\u00a0\u00b1\u00a02.5 (%)X\u00a0\u00b1\u00a01 (%)(a) Group\u00a0Abduction10085.246.82616.4\u00a0Anteversion99.982.74626.717.5\u00a0Combined99.970.521.56.92.9(b) Surgeon\u00a0Abduction10093.759.33421.2\u00a0Anteversion99.985.748.327.517.6\u00a0Combined99.980.328.69.43.7(c) Residents\u00a0Abduction99.878.93920.712.7\u00a0Anteversion99.880.544.526.217.4\u00a0Combined99.663.517.45.42.2\nA multivariate regression analysis was applied in order to identify any factors that might be responsible for the inaccuracy of the estimation. A significant result was found for abduction and anteversion, concerning age of the patient and if the patient was operated by an orthopaedic surgeon or resident. Other factors like body mass index, sex, operated side, (un)cemented fixation of the acetabular component, model of the cup and the surgical approach did not reveal any significant differences.\nDiscussion\nMalposition of the acetabular cup is probably the most important factor for dislocation of a total hip prosthesis. Therefore it is essential that the surgeon has maximum control over the position of the socket during the operation. Free hand positioning with the patient in a standardized position and a cup positioner with the patient and floor as reference is the routine method. Specific mechanical alignment guides were designed to add precision, as do navigation systems in the concept of CT-guided computer-assisted surgery. Surprisingly, there are very limited data on the precision rate of free hand positioning of the acetabular shell. The aim of our prospective study was to determine the accuracy of the free hand technique. Only then the presently introduced systems can be tested against this standard. The term \u201cfree hand\u201d is not used uniformly in the literature. Either it is referred to pure manual positioning or with the aid of the acetabular cup impactor-positioner provided with the implant. In the present study, the latter definition was applied.\nOne comparable study is that of Saxler et\u00a0al. [10] who showed, in a retrospective CT-controlled design, that 27 out of their 105 cups (25.7%) were placed within the safe zone of Lewinnek. A second study by DiGioa et\u00a0al. [11] with a specific mechanical acetabular alignment guide (with A-frame) where the results were controlled peroperatively with a hip navigation system in 74 hips aimed at 45\u00b0 of abduction and 20\u00b0 of flexion. With their specific guide only 22% of the cups were placed within Lewinneks safe zone. Compared with these two studies our prospective data demonstrate an accuracy within the safe zone of 70.5%.\nThere is an elegant in vitro study by Jolles et\u00a0al. [12] comparing free hand (without the help of any guide), mechanical alignment guide and computer-assisted cup placement. Onehunderd and fifty acetabular implants were placed in ten identical models of the pelvis (covered with artificial soft tissue of soft cast and foam) by ten surgeons. The mean accuracy for anteversion was 8\u00b0 (5.0\u201310.5) for free hand with cup positioner and 4\u00b0 (3.0\u20135.5) for abduction; with computer navigation this was 1.5\u00b0 (1.0\u20132.0) and 2.5\u00b0 (2.0\u20133.5), respectively. In the present in vivo study the data were 5.5\u00b0 for anteversion and 5.4\u00b0 for abduction with 200 acetabular shells. Jolles et\u00a0al. state that their in vitro conditions of ideal approach and anatomic relations and perfect placement of the patient would favour the free hand positioning as computer-assisted surgery devices are only slightly sensitive to modifications of these parameters and inaccuracy of the freehand will be enhanced greatly in vivo. Our study proves this statement not to be the case as results are quite comparable.\nWithout doubt it is essential to actually place what is aimed for. Our present study shows that the 70.5% positioned within the safe zone is remarkably good for free hand placement compared with literature. But when the target is reset from the wide safe zone of Lewinnek to for instance within 5\u00b0 or even 1\u00b0 of error from the judged position for both abduction and anteversion, only 21.5 and 2.9% of our sockets are placed within this narrow definition of safe zone. We believe that the power of our study comes from the distinctive set up where the recorded data are a reflection of the surgeon\u2019s perception of cup placement. To our opinion this is superior then to allow for a wide range or set a predefined target. At the actual point of cup placement a surgeon is also committed to or governed by the patient\u2019s anatomy, which will influence the actual position. For instance when less anteversion is accepted in the socket; the stem will be given some additional anteversion.\nAnother important item as stipulated by DiGioia et\u00a0al. [11] is the actual position of the patient in the lateral decubitus position. They demonstrated that the mean difference of pelvic orientation on the operating table with the desired position during acetabular alignment was 18\u00b0 in version and 3\u00b0 in abduction. This effect is not completely eliminated with the use of computer navigation as these systems are influenced by pelvic tilt [13].\nConsidering the estimation of abduction, significant results were found in favour of the orthopaedic surgeons compared to their residents. A learning curve as a result of experience is probably responsible for this difference. As to surgical approach, acetabular components placed posterolaterally were measured to have a significantly higher-degree of anteversion and abduction. This is obviously related with the intention to minimize the change of a dislocation for both approaches. Although both approaches take a different anatomical route to the acetabulum, visualization and presentation of the acetabulum do not seem to be important factors. The conclusion is that an anterolateral or posterolateral approach is not a distinguishing factor in the accuracy of cup placement in our series.\nAn important potential flaw in this investigation is the use of planar radiographs for the evaluation of true abduction and especially anteversion. Variations in pelvic flexion-extension during imaging are responsible of variations in flexion up to \u221226\u00b0 to +10\u00b0 [14]. This study concludes that radiographic measurements are not a reliable method to evaluate cup orientation, especially flexion or anteversion alignment [14]. Pradhan, however, stated that his method, as used in our study, proved to be reliable in an in vitro model in which the calculated anteversion on the X-ray was compared with the true (known) cup anteversion. In our study special attention was given for optimal positioning of the patients pelvis to minimize rotation and tilt before the X-ray was taken. From a practical point of view it is preferable to determine the actual cup position with planar radiographs rather then with CT as in most clinics the availability and the costs of a CT scan combined with the high radiation dose for the patient are reasons for not using this instrument as a routine practice.\nFrom this prospective study it is concluded that an accuracy of 70.5% for placement of the socket with an acetabular shell imactor-positioner within Lewinnek\u2019s safe zone and of only 21.5% within an error of 5\u00b0 for both abduction and anteversion is unacceptable.\nThe strength of our research is that literature is extremely scarce on studies as our prospective study on free hand positioning without special aiming devices. The results of the present study appear convincing but limitations are the high-number of participating surgeons, different surgical approaches (anterolateral and posterolateral) and two methods of cup fixation (uncemented and cemented). Multivariate analysis on these factors revealed only differences between the achievements of surgeons and residents.\nThe consequence of our conclusion that free hand cup positioning is an unreliable method is that attention now should be focussed on the results of a randomized trial comparing the different techniques of cup positioning.","keyphrases":["accuracy","freehand cup positioning","total hip arthroplasty"],"prmu":["P","P","P"]}
{"id":"Exp_Brain_Res-4-1-2335293","title":"The latency for correcting a movement depends on the visual attribute that defines the target\n","text":"Neurons in different cortical visual areas respond to different visual attributes with different latencies. How does this affect the on-line control of our actions? We studied hand movements directed toward targets that could be distinguished from other objects by luminance, size, orientation, color, shape or texture. In some trials, the target changed places with one of the other objects at the onset of the hand\u2019s movement. We determined the latency for correcting the movement of the hand in the direction of the new target location. We show that subjects can correct their movements at short latency for all attributes, but that responses for the attributes color, form and texture (that are relevant for recognizing the object) are 50 ms slower than for the attributes luminance, orientation and size. This dichotomy corresponds to both to the distinction between magno-cellular and parvo-cellular pathways and to a dorsal\u2013ventral distinction. The latency also differed systematically between subjects, independent of their reaction time.\nIntroduction\nNeurons in different cortical visual areas respond to different visual attributes with different latencies (Livingstone and Hubel 1987, 1988; Schmolesky et al. 1998; Bullier 2001). How do such differences affect the way in which visual information guides our movements? Two main streams of visual processing have been identified within the brain; each specialized for processing a different kind of information (Trevarthen 1968; Mishkin et al. 1983; Goodale and Milner 1992; Ungerleider and Haxby 1994). A ventral stream in which information travels from V1 to the inferotemporal cortex is crucial for the identification of objects, whereas a dorsal stream passing through the posterior parietal region plays a major role in spatial vision. A widely accepted explanation for this distinction is that the dorsal stream is specialized for quickly processing spatial information to control action, whereas the slower, detailed visual analysis of other attributes that are important for recognizing objects takes place in the ventral pathway (DeYoe and Van Essen 1988; Goodale and Milner 1992; Tanne et al. 1995; Desmurget et al. 1999; Rossetti et al. 2000; Lee and van Donkelaar 2002). This view implies that not all information can be used to quickly adjust hand movements. We tested this prediction.\nOne of the problems with comparing experiments that investigate whether various kinds of visual information can contribute to fast control of actions is that investigators use different experimental paradigms for different visual attributes, which themselves may give different results for a single attribute (e.g., compare Pisella et al. 1998; Schmidt 2002; Brenner and Smeets 2004; Cressman et al. 2006; White et al. 2006 for the role of color in guiding hand movements). It would therefore be very useful to systematically investigate the latency of responses to various visual attributes with a single paradigm. We therefore compared responses to changes in target location for hand movements directed toward targets defined by attributes that are normally relevant for goal-directed actions (luminance-contrast, size and orientation) with ones toward targets defined by attributes that are normally more relevant for object recognition (color, shape and texture). In all cases, there were three possible target positions. The target was at one of them. Other objects (references) occupied the other two positions. In half of the trials, the target switched locations with one of the references just after the hand started moving (Fig.\u00a01a, b).\nFig.\u00a01Schematic overview of the experiment. a The participant holds a pen at the starting position when a target (dark square in this example) and two other objects (references, the brighter squares in this example) appeared. b In half of the trials, the target switches to one of the other locations just after movement onset. c Ten paths of one participant\u2019s hand (P6) for a target that remained at the leftmost position in the \u201cluminance (390%)\u201d condition. d All ten paths of the same participant\u2019s hand in the condition in which the target jumped from the leftmost to the rightmost position at movement onset\nAn issue that cannot be ignored is how conspicuous the distinction between target and reference is within each attribute; i.e., how easily the target can be distinguished from the references. When comparing responses to targets defined by the same attribute, the response becomes faster if the relevant contrast is increased (Brenner and Smeets 2003). We therefore have to show that any differences in latency that we find cannot be simply caused by differences in conspicuousness. The question is how to evaluate conspicuousness independently from the response latency.\nIn previous studies, this issue was resolved by equating a critical stimulus parameter (e.g. cone contrast, White et al. 2006), or by equating the stimuli in terms of an independent perceptual judgment (e.g., how conspicuous the targets are judged to be, Brenner and Smeets 2003). The first approach is not feasible in our current experiment because it is not at all clear how stimulus parameters for different attributes could be matched. The second approach is also problematic, because the visual processing for the perceptual judgment task might differ from that for the real experiment. We will therefore develop a new method to deal with differences in conspicuousness. This method, which will be developed in the results section, is based on examining responses to various targets that are all distinguished on the basis of the same attribute, but that differ in conspicuousness. We will show that beside leading to shorter latencies, more conspicuous targets also lead to shorter reaction times, higher velocities, and a steeper slope of the responses. We will then compare latencies for targets defined by different attributes when they give rise to comparable reaction times, velocities, and response slopes. Differences in latency after such matching cannot be caused by differences in conspicuousness.\nMaterials and methods\nParticipants\nTwelve subjects took part in this study. Three of them were the authors and the others were colleagues and friends who were unaware of the hypothesis that was being tested. All of them had normal or corrected to normal vision. Two of the participants were left-handed. Each participant made 120 pointing movements in each of 13 different sessions. Six of the participants also made 120 pointing movements in each of 7 additional sessions in which various luminance contrasts were used. Each participant performed the sessions in a different random order. The ethics committee of the Faculty of Human Movement Sciences approved the study.\nApparatus and stimuli\nParticipants sat behind an A2-sized graphic tablet (Digitizer II, Wacom Ltd, Tokyo, Japan) and viewed a projection surface via a semitransparent mirror that was placed above this graphic tablet. The images were back-projected from above the projection surface. The distance between the mirror and the projection surface was identical to that between the mirror and the surface of the tablet, so that the projected image appeared to be at the surface of the tablet. Lamps underneath the mirror ensured that the participants could see their hands. The resolution of the projected image was 1,024 by 768\u00a0pixels, with 1\u00a0pixel corresponding to about 0.5\u00a0mm. The position of the pen was determined every 5\u00a0ms (200\u00a0Hz).\nAt the near edge of the screen was a starting point (a black, 1\u00a0cm diameter disk with a luminance of 2.3\u00a0cd\/m2 as measured with a Minolta LS-110 luminance meter). At a distance of 25\u00a0cm from this point in the sagittal direction, three objects were projected next to each other on a white background (35\u00a0\u00d7\u00a045\u00a0cm; 28.0\u00a0cd\/m2). The distance between the centers of the objects was 3\u00a0cm (Fig.\u00a01a). One of the three objects served as the target; this object differed from the other two objects (the references).\nWe varied the attribute in which the target differed from the references (and the magnitude of this difference) between conditions (Fig.\u00a02). Each condition was tested in a separate session. In the control condition, the reference objects were equal to the background so they were invisible: only the dark gray target square (1\u00a0cm sides, 5.1\u00a0cd\/m2) was visible.\nFig.\u00a02Schematic representations of the stimuli in the 20 conditions (depicted with the target on the left). The luminance of both the target and the references is 5.1\u00a0cd\/m\u00b2 and they are both 1\u00a0cm2 gray squares unless otherwise mentioned (see \u201crelevant details\u201d). a The 13 conditions that were performed by all participants. Red lettering indicates that the responses depend on attributes that are normally needed to recognize objects, and blue lettering indicates that all items change. b The seven additional luminance conditions that were performed by six participants (numbers between brackets are the percentage that the references are brighter than the target)\nIn the luminance (390%) condition, the target differed from the references in luminance only (for further details about this and subsequent conditions see Fig.\u00a02). There were seven additional conditions with different luminance contrasts between the target and the references. These were used to determine how responses depend on how conspicuous the distinction is between the target and the references.\nIn the three orientation conditions, the target and the references were rectangles (0.67\u00a0\u00d7\u00a01.50\u00a0cm, 5.1\u00a0cd\/m2). The target rectangle was orientated at an angle of 45\u00b0, 60\u00b0 or 90\u00b0 with respect to the parallel references. In the three size conditions the target squares had different sizes. The reference squares had surface areas of 1\u00a0cm2, as did all the objects in the other conditions. In the shape session the target was a circle, and the references were squares. In the color condition, the target was a green square (5.1\u00a0cd\/m2) and the references were two red squares (5.1\u00a0cd\/m2). In the texture condition, the target square was filled with ten 1-mm wide stripes, alternating between dark (2.3\u00a0cd\/m2) and bright (7.9\u00a0cd\/m2) ones. The average luminance of the stripes was equal to the luminance of the references (5.1\u00a0cd\/m2), to ensure that participants had to analyze the texture to recognize the target.\nDuring pilot-experiments, some of the participants had the impression (especially in the orientation and the shape conditions) that they were not responding to a difference between the target and the references, but to the change at the new target position. To investigate whether this was really an issue we performed two control conditions in which we masked such effects by also changing aspects of the references when the target changed its position. In a masked orientation condition, the references both changed their orientation by 90\u00b0 when the target jumped to a new location. In a masked shape condition, the reference squares increased in size at the same moment that the target circle changed position.\nProcedure\nParticipants started each trial by placing the pen on the starting point. After a random time (about 2\u00a0s), the target and references appeared (Fig.\u00a01a). The task was to reach the target with the pen and stop there as soon as possible. The sum of the movement time and the reaction time was to be minimized. Participants were shown which object was the target (and which the references) before they started each session.\nWithin each session of 120 trials, the target appeared 40 times at each of the 3 positions. In half of the trials the target and the references remained at their initial position (unperturbed trials, Fig.\u00a01c). In the other half of the trials the target changed its position to one of the two reference positions about 62\u00a0ms after the subject\u2019s finger reached a velocity threshold of 0.02\u00a0m\/s (perturbed trials, Fig.\u00a01b). At the same moment the reference (that had been at that position) changed its position to where the target had been. Whenever the target changed position participants had to adjust their hand movement to reach the new target location (Fig.\u00a01d). If a movement ended on the target within 1.2\u00a0s of the target first appearing, the participant was rewarded with auditory feedback. The 120 trials (three initial positions, change in position or not, two positions to which to change, ten replications) were presented in random order. Since different positions to which to change are irrelevant when there is no change, there were actually 20 trials in which the target remained at each initial position.\nAnalysis\nVelocities were calculated for the interval between every two measurements by dividing the displacement of the tip of the pen by the 5\u00a0ms between the measurements. The beginning of the movement was defined as the first position after the tangential velocity reached 0.02\u00a0m\/s. The end of the movement was defined as the first position after the tangential velocity fell below 0.02\u00a0m\/s. To evaluate the corrections, we only used the lateral component of the velocity (parallel to the displacement of the target).\nIn order to isolate the responses to changes in target position, we first synchronized all the measurements relative to the moment that the target changed\u2014or would have changed\u2014position. We then separately averaged the lateral velocity for each combination of initial and final target position. We defined the lateral velocity in the direction of the position change as being positive; when the position did not change, we defined the direction in which it would have changed as being positive. We characterize the response to a change in target position by the additional lateral velocity: the difference in the lateral component of the velocity between the trials in which the target changed from a certain initial position to a certain final position and the ones in which it remained at the same initial position. To obtain a single response per attribute for each participant we averaged the additional lateral velocity across the six combinations of initial and final target positions. We used these curves to determine the latencies of the responses.\nTo estimate the latencies of the responses we approximated the initial responses by straight lines (dotted lines in Fig.\u00a03). We used a robust method (one that is not too sensitive to the exact shape of the response near its onset) to approximate the initial response: we determined the amplitude of the peak in the average additional lateral velocity and then determined the points at which the additional lateral velocity reached 25 and 75% of this value. We consider a line through these two points as a good and robust approximation of the initial response. We considered the intersection of this line with a line at an additional lateral velocity of zero to give a reliable estimate of the latency of the reaction to the change in target position. We evaluated the influence of the conditions on the slopes of the above-mentioned lines and on their intersection points with the zero-velocity line (across and participants) using paired t-tests.\nFig.\u00a03Mean additional lateral velocity for each attribute as a function of the time after the perturbation. The mean additional lateral velocity is the mean difference between the lateral velocity on the trials in which the target did and did not change position. To estimate the latency of the response, a line through the points at 25 and 75% of the peak velocity (see dotted lines) was extrapolated to intersect the line representing an additional velocity of 0 (black horizontal line). The time at the point of intersection was considered to be the response latency. a Averages of six participants for the luminance contrast conditions. b Averages of 12 participants for the conditions in which they all participated\nThe slope of the line through the 25 and 75% points of the average additional lateral velocity does not only depend on the intensity of the response on individual trials (lower acceleration results in a shallower slope). If the response does not always occur at the same time, averaging will result in an average response that has a lower peak velocity, shallower slope and longer duration than the responses in individual trials. To evaluate the shape of the response curve without the influence of variability in timing, we used a second way of synchronizing trials before determining the additional lateral velocity.\nTo estimate the intensity of individual responses for each condition and participant, we synchronized the lateral velocity curves of all perturbed trials at the peak lateral velocity in the direction of the new target position (irrespective of when the target position changed), and produced an additional lateral velocity curve for each subject by averaging across replications and perturbation directions. We averaged these synchronized additional lateral velocity curves across positions and across participants to investigate whether there are systematic differences between the intensity of the responses for different attributes. We averaged the curves across conditions rather than participants to investigate whether there are systematic differences between the intensity of different participants\u2019 responses. In order to determine whether the response was proportional to the size of the perturbation, we also averaged the curves separately for all pairs of positions in which the target jumped 3\u00a0cm, and all pairs of positions in which the target jumped 6\u00a0cm.\nResults\nOverview of responses\nOn average, it took participants 365\u00a0ms to react to the appearance of the target (and the two references). The average movement time (time until participants stopped their movement on the target) was another 275\u00a0ms. Figure\u00a01 shows one participant\u2019s paths in one condition with the target initially positioned on the left. When the target stayed at the initial target position (Fig.\u00a01c), the hand moved to the target along a slightly curved path (note that the lateral deviations are exaggerated due to the unequal scaling of the figure). When the target jumped at the onset of the movement (Fig.\u00a01d), participants obviously initially moved along similar paths to those seen in Fig.\u00a01c, but approximately half way to the target the path curved toward the new target position.\nFigure\u00a03 shows the additional lateral velocity for the various conditions (summarized in Fig.\u00a02), averaged over all subjects. Each curve represents the difference in lateral velocity between trials in which the target did and did not change position (see \u201cmaterials and methods\u201d for details). The dotted lines are drawn through the points at which the mean additional lateral velocity curves reached 25 and 75% of their peak values. We consider the points at which these lines intersected the line at an additional lateral velocity of zero as the latencies of the responses. We used targets that only differed in luminance from the references to evaluate effects of conspicuousness (for half of our subjects). We found that lower luminance contrast (a less conspicuous target) results in both a longer latency and a shallower slope (Fig.\u00a03a). The conclusion that we can draw from the main conditions depicted in Fig.\u00a03b, is that both the timing and the slopes of the curves differ between conditions. Figure\u00a03 shows an analysis over responses averaged over subjects. The rest of the analysis is done on the basis of determining slopes and latencies for each subject (and condition) separately.\nDealing with conspicuousness\nIt is clear from Fig.\u00a03a that the latencies are larger for less conspicuous targets. In order to compare the latencies of responses to targets defined by different attributes, we have to take into account differences in conspicuousness. Figure\u00a04a, b and c shows how not only the latency of the response varies with conspicuousness, but also the reaction time of the initial movements toward less conspicuous targets are longer (Fig.\u00a04a), the slope of the response to a perturbation is shallower (Fig.\u00a04b), and both the peak velocity of the initial movement toward the target and that of the correction are slower (Fig.\u00a04c). All three plots yield significant correlations (P\u00a0<\u00a00.05), and the data are distributed along the fitted line with deviations that can be expected on the basis of the error bars.\nFig.\u00a04Comparing the responses across conditions and subjects. a\u2013c Relationship between various movement parameters for the luminance conditions. The latency and slope refer to the correction (Fig.\u00a03). Points are averages across subjects with standard errors. Dashed lines are linear fits through these points. d\u2013f Similar graphs for each of the conditions in which all subjects participated. Symbols in red are the conditions based on attributes for object recognition; symbols in blue are for conditions in which the change is masked by changing all items. g\u2013i Data for individual subjects, averaged across conditions. The brightness of the symbol indicates the average response latency of the subject (bright is short latency)\nSince several measures co-vary with the conspicuousness (i.e. the luminance contrast) of the target, we can use these measures to evaluate whether latency differences can be due to differences in conspicuousness. Except for the latency (the variable we are interested in), the slope is the variable that varies most clearly with conspicuousness. This is therefore the best measure to use to evaluate whether effects of condition on latency are caused by a difference in conspicuousness, followed by the reaction time (Fig.\u00a04a) and response velocity (Fig.\u00a04c). The correlation of the conspicuousness with the response velocity (Fig.\u00a04c) is less clear than its correlation with the slope (Fig.\u00a04b). This suggests that a shallower slope of the average response is not only caused by the response being less vigorous, but is also largely caused by more variability in the latency. To verify the effect of variability in latency on the slope of the response, we removed the effect of variability in the latency by averaging the responses after synchronizing them at the moment of peak lateral velocity. The responses resulting from this averaging method have almost the same slope for all conditions: 282\u00a0m\/s2 with a coefficient of variation of 0.13 (see Fig.\u00a05a). The slopes of the mean additional lateral velocity curves (Fig.\u00a03) are not only much shallower (on average 187\u00a0m\/s2), but are also much more variable between conditions (coefficient of variation 0.28). We conclude that the differences between the slopes of the mean additional lateral velocity curves are mainly caused by differences in the variability in timing between trials (rather than by differences in the intensity of the response). Thus a more conspicuous target results in less variable (Fig.\u00a04b) as well as more vigorous (Fig.\u00a04c) responses.\nFig.\u00a05Magnitudes of the responses as determined by averaging all trials after synchronizing at their peak lateral velocity. a Each curve indicates a condition; color coding as in Fig.\u00a04. The similarity between the curves indicates that the intensity of the response did not differ between conditions. b Responses of subjects. The brightness of the curve indicates the subject\u2019s average latency (bright is short latency). c Comparing the responses for the two jump amplitudes. The additional lateral velocity for all trials in which the target jumped 6\u00a0cm was twice as large as that for the trials in which the target jumped 3\u00a0cm\nDifferent attributes, different latencies?\nFigure\u00a04d and e shows the same relationships as Fig.\u00a04a, b for the main experimental conditions (Fig.\u00a02a). The dashed lines reproduce the relationship that we found for the luminance conditions (Fig.\u00a04a, b). Participants responded to the various size and orientation conditions with a relationship between latency and conspicuousness that was similar to that for the responses to luminance-defined targets. The targets defined by the attributes color, shape and texture were not less conspicuous than the other targets (reaction times and slopes are similar), but subjects take exceptionally long to react to changes in their position (t-test, P\u00a0<\u00a00.05). These responses were about 50\u00a0ms longer than the ones indicated by the dashed lines. The movements toward the targets defined by color, shape, or texture had similar peak velocities, both of the movements themselves and of the corrections, as the movements toward other targets (Fig.\u00a04f).\nFor two attributes (orientation and shape) we constructed a \u201cmasked\u201d condition, in which not only the target changed location, but also the references changed. This prevents subjects from using the change itself as an indicator of the new target location. The latency for these two conditions is 210\u00a0ms, which is longer than for the two corresponding original conditions (paired t-test, P\u00a0<\u00a00.05). The masking decreased the velocity of the movements and of the corrections (Fig.\u00a04f), but did not change the conspicuousness of the targets, either in terms of the reaction time (Fig.\u00a04d) or of the slope of the average response (Fig.\u00a04e). So the increase in latency due to the masking is not caused by masking making the targets less conspicuous, but by requiring additional processing, presumably for a renewed selection of the target. Due to the blocked design, subjects could counteract the effects of their later responses by slowing down their movements (low values for the masked targets in Fig.\u00a04f).\nSubjects and amplitudes\nBefore discussing how to interpret the results, we want to answer two additional questions. The first is, how does the variability between participants contribute to the results? To find this out, we plotted the data averaged over the conditions for each of the participants separately (Figs.\u00a04g\u2013i, 5b). We coded subjects according to the latency of their adjustments: bright curves and symbols correspond to subjects with short latencies. The intensity of the response differed much more between subjects (SD\u00a0=\u00a091\u00a0m\/s2, Fig.\u00a05b) than between conditions (SD\u00a0=\u00a037\u00a0m\/s2, Fig.\u00a05a), with no apparent correlation between the peak velocity and latency (Fig.\u00a04i: brighter symbols are not concentrated in one region of the figure). All subjects show more or less the same pattern of latencies as we found for the average data (not shown). This was so for the different attributes as well as for the different levels of conspicuity. There is quite some variability between the subjects, but subjects that are slow in one condition are generally slow in the other conditions as well. The result is that subjects differ systematically in their latency, without any correlation with their reaction time (Fig.\u00a04g) or with the slope of their response (Fig.\u00a04h). This performance difference cannot easily be explained by factors such as age, visual acuity or body size.\nThe second question is whether the similarity of the curves in Fig.\u00a05a means that subjects have a pre-programmed response to a perturbation that is used irrespective of the precise visual information. To answer this question, we compared the responses for the two amplitudes of the perturbation. When the target jumped 6\u00a0cm from its initial location (from one extreme position to the other), participants responded about twice as strongly as when the target jumped 3\u00a0cm (Fig.\u00a05c). Thus, the response is proportional to the position change, and therefore clearly tailored to the visual information rather than that pre-programmed and triggered by the change.\nDiscussion\nBy considering the relationship between response latency and measures of conspicuousness (reaction time and variability in the latency), we can conclude that participants reacted with a similar latency to differences in orientation and size than to corresponding differences in luminance. The attributes color, shape and texture gave rise to approximately 50\u00a0ms longer latencies than did differences in luminance, orientation and size that were equally conspicuous. Note that these latencies are still 150\u00a0ms faster than the original reaction times. We found a similar order of fast and more slowly processed attributes across participants, which supports our idea that we can use this method to measure systematic differences between attributes in terms of the time that it takes to process them, even if participants respond differently (see different velocity profiles in Fig.\u00a05b).\nAn important question in this kind of research is whether the apparent response to a particular attribute could actually be a response to a small difference in another attribute. For instance, in the color condition, participants could respond to small differences in luminance instead of to differences in color between the target and the references, since the luminance of the target and the references was not equated for individual participants. They clearly do not, because if they had done so, the response would be similar to that for one of the small luminance contrast conditions, which is clearly not the case (the slope of the response is much steeper for color).\nAs mentioned earlier, it takes about as long to respond to changes in position defined by target size as to those defined by target luminance. We cannot reject the possibility that the reactions to the difference in surface area between the target and the references in the size condition were actually responses to the average luminance within an area larger than that of the target itself. To be sure that this was not the case we would have had to vary the luminance of the targets and references, which we did not do in the present study.\nDesmurget et al. (1999), showed that applying transcranial magnetic stimulation over the left posterior parietal cortex disrupted corrections of reaching movements of the right hand after a perturbation in target location in four of their five subjects, thereby demonstrating that the dorsal pathway is necessary for fast corrections of the hand. The fact that participants are able to respond very quickly to changes in target location when the target is defined by the attributes orientation, size and luminance (but not color, texture or shape) is therefore an indication for dorsal processing of orientation, size and luminance (but not of color, texture and shape). This division of attributes is consistent with the distinction between pathways for \u201cwhat\u201d and \u201cwhere\u201d (Mishkin et al. 1983; Ungerleider and Haxby 1994). The fact that subjects were still able to correct their movements to targets defined by color, texture or shape (albeit at a longer delay) implies that these attributes also reach the parietal cortex, although at a longer latency. This is in line with studies using electrophysiology (Murata et al. 2000; Sakata et al. 2005), positron emission tomography (Faillenot et al. 1997; Vidnyanszky et al. 2000) and functional magnetic resonance imaging (Oliver and Thompson-Schill 2003) that all suggest that the parietal cortex is involved in at least some aspects of discriminating shape. These studies were, however, not concerned with the exact timing of the activity.\nWhy are responses for targets defined by shape relatively slow? Assuming that all the responses that we measured are indeed mediated by the posterior parietal cortex, the dichotomy in latencies could be explained in two ways. It could be that the parietal cortex mediates the responses to some targets after initial processing of the attributes that are relevant for the \u201cwhat\u201d of the object (like color, shape and texture (Mishkin et al. 1983) in the ventral pathway. This indirect pathway is likely to result in longer processing times than the direct dorsal pathway for the \u201cwhere\u201d attributes.\nAlternatively, the dichotomy may arise earlier, with the distinction between the magnocellular retinogeniculate pathway that is faster and more sensitive to luminance contrast, but is color-blind and has a low spatial resolution, and the parvocellular pathway that is color selective and has a higher spatial resolution, but is slower and less sensitive to luminance contrast (Livingstone and Hubel 1987, 1988). This distinction corresponds nicely with the dichotomy in the latencies that we found. Assuming that both the magnocellular and the parvocellular pathways provide input to the dorsal pathway could therefore account for the different latencies that we found for different attributes (Brenner and Smeets 2003), without requiring input from the ventral pathway.\nAlthough we cannot decide between these two lines of explanation, the two masked conditions indicate what is essential for the very fast responses. Being able to identify the target on the basis of \u201cwhere\u201d attributes (size, orientation, luminance) is necessary for fast responses. However this is not sufficient, because the latency of responses to the change in location of a target defined by orientation increases by more than 50\u00a0ms if the references change their orientation. Masking changes prevents responses to the location of the change. Presumably, the target has to be found (identified) again, which requires ventral processing (according to the first explanation).\nThe conclusion that identifying targets by their color takes 50\u00a0ms longer than doing so on the basis of luminance seems to be in conflict with earlier results where we reported that subjects could respond within 120\u00a0ms to color information (Brenner and Smeets 2004). In that study, we compared two conditions: one with one reference object of a different color and one with no reference object (similar to the control condition in the present experiment). The main difference between the responses in these conditions was the larger variation in latency for the condition with one reference object (a more shallow slope). It seems as if the distribution of responses consisted of a small peak at short latency, and a larger peak 50\u00a0ms later (Fig.\u00a03 in Brenner and Smeets 2004). Perhaps, the few very fast responses in the condition with the reference object were related to the very simple design of that experiment: the position to which the target could change was known in advance, so detecting the change in color at the original target location was enough to start an adequate correction. In the present experiment, the location of the new target position had to be determined on the basis of color information. That directly responding to a transient can lead to exceptional fast responses is evident from the fact that masking the jump of the oriented target by orientation changes of the references caused an increase in latency of 50\u00a0ms.\nA last issue to discuss is the variability between subjects. Some subjects responded consistently later to the perturbations than others (Fig.\u00a04g, h). The difference between the slowest and fastest subject was 70\u00a0ms; larger than the difference between the responses to targets defined by color and luminance. Such a large variation is in line with the remarkable finding that stimulating the posterior parietal cortex directly after movement onset disrupts adjustments in some subjects, but not in others (Desmurget et al. 1999). We can draw two conclusions from these variations in latency. The first is that since none of the subjects had particular complaints of clumsiness, the exact value of the latency is apparently not critical for performing every day tasks. The second is that in situations in which short latencies are important, such as when playing table tennis or boxing, some participants may perform better than others could ever hope to, due to differences between their minimal visuomotor latencies. This latter implication (and in particular whether a shorter latency can be achieved by training) is open for experimental verification.","keyphrases":["hand movement","color","shape","visual processing","dorsal stream","target perturbation","double step"],"prmu":["P","P","P","P","P","R","U"]}
{"id":"J_Struct_Funct_Genomics-3-1-2140095","title":"The scientific impact of the Structural Genomics Consortium: a protein family and ligand-centered approach to medically-relevant human proteins\n","text":"As many of the structural genomics centers have ended their first phase of operation, it is a good point to evaluate the scientific impact of this endeavour. The Structural Genomics Consortium (SGC), operating from three centers across the Atlantic, investigates human proteins involved in disease processes and proteins from Plasmodium falciparum and related organisms. We present here some of the scientific output of the Oxford node of the SGC, where the target areas include protein kinases, phosphatases, oxidoreductases and other metabolic enzymes, as well as signal transduction proteins. The SGC has aimed to achieve extensive coverage of human gene families with a focus on protein\u2013ligand interactions. The methods employed for effective protein expression, crystallization and structure determination by X-ray crystallography are summarized. In addition to the cumulative impact of accelerated delivery of protein structures, we demonstrate how family coverage, generic screening methodology, and the availability of abundant purified protein samples, allow a level of discovery that is difficult to achieve otherwise. The contribution of NMR to structure determination and protein characterization is discussed. To make this information available to a wide scientific audience, a new tool for disseminating annotated structural information was created that also represents an interactive platform allowing for a continuous update of the annotation by the scientific community.\nIntroduction\nThe long-term goal of structural genomics (SG) has been ambitiously defined as \u201cto make three-dimensional atomic level structures of most proteins easily obtainable from knowledge of their corresponding DNA sequences\u201d (http:\/\/www.nigms.nih.gov\/Initiatives\/PSI.htm). Long before this goal is achieved, the multiple specialized SG projects are expected to have a significant impact on many aspects of the biological sciences.\nThe most readily apparent contribution of SG is the rapid expansion in the number of available protein structures, derived at a reduced cost because of the efficiency of specialized centers. Proper target selection is critical to ensure that the structures solved by SG centers are indeed valuable to the research and industrial community, either because of the intrinsic interest of the proteins investigated, or because of the improved mapping of the protein structure universe, providing homologous structural models.\nA second important contribution of SG projects for the scientific community is the development of methods for efficient protein production and structure determination, which could be adopted in smaller research laboratories to improve productivity.\nOther scientific deliverables of structural genomics derive from the scale and nature of the operations, and include comparative studies on members of protein families, identifying determinants of specificity, deriving general rules, and improving the capability to predict protein structure and function from gene sequences.\nThe Structural Genomics Consortium (SGC), operating in the Universities of Oxford and Toronto and the Karolinska Institute, was initiated in 2003 to address needs of industrial and academic pharmaceutical research. The SGC investigates human and apicomplexan proteins; the targets are selected based on their potential as drug targets or involvement in disease processes. Technologically, the SGC focuses on interaction of proteins with small molecules (ligands, inhibitors, substrates and co-factors), and on coverage of protein families. This report provides several examples of the impact of research undertaken at the Oxford node of the SGC, including methodology for high-throughput structure determination, generic means for ligand screening, selected examples of insight from specific structures, insights from family coverage, and the possibilities resulting from the availability of large numbers of purified protein samples. The other SGC nodes share the core technologies but investigate non-overlapping target areas.\nFinally, the scientific impact depends on dissemination of structural data. We describe a new platform for distribution of annotated protein structures, which aims at making this data more meaningful to an audience beyond the usual users of the PDB.\nMethodology\nProtein production\nMethod adaptation and development for structural genomics involved a change of mindset, no less than developments in instrumentation, chemistry and computer software. Industrialization of protein production\u2013\u2013applied to a huge variety of proteins with very divergent chemical properties\u2013\u2013is not straightforward. Yet, extensive work in several SG centres have led to a convergence to core procedures, which are widely applicable, and often sufficient to generate purified proteins, crystals and structures (Table\u00a01). Where the core protocol fails, additional steps (e.g., further purification, crystal optimization), or alternative methods (e.g., different cloning vectors) are applied.\nTable\u00a01Core protocols employed at the SGC1. Source of DNA1. Sequence-verified cDNA clone collections.2. Synthetic DNA.3. RT-PCR, site-directed mutagenesis.4. Genomic (microbial).2. CloningLigation-independent cloning.Recombinase-based cloning (e.g., Gateway, InFusion).3. Expression vectors and hostsT7 promoters, controlled by Lac repressor.Bacterial vectorsN-terminal hexahistidine tag, cleavable by specific proteases (TEV, Thrombin, C3).Host strains based on BL21(DE3), often expressing rare-codon tRNAs or chaperone proteins.4. Eukaryotic expressionBacoluvirus-infected insect cells.5. Protein expressionRich media, grow at 37\u00b0C to mid-log, then induce at low temperature with IPTG.OR: Similar protocol using minimal medium for Selenomethionine or isotopic labelling.6. PurificationTwo-step purification: Affinity chromatography, Gel filtration, all in high-salt buffers (0.5\u00a0M NaCl). Optional: tag cleavage and re-purification.7. Ligand and buffer screeningThermal denaturation assays are used to screen purified proteins against 1\u2013103 small molecules and several buffer compositions, to identify stabilizing conditions and potential ligands.8. CrystalliationInitial coarse screens (2\u20134\u00a0\u00d7\u00a096 conditions; 3 protein concentrations each). Vapour diffusion, sitting drops, imaged by robots but scoring done by humans.Include ligands identified from screening or biochemical knowledge to promote crystallization.Follow-up screens and crystal optimization.9. Data collection and structure determinationManual or robotic screening of crystals for diffraction properties; data collection in rotating anode or synchrotron sources.Phasing: Molecular replacement (95%), experimental phasing using SeMet derivatives, and MIR.\nSeveral features of this protocol have been optimized to capture a large portion of target proteins. Gene clones have been predominantly obtained from public and commercial cDNA libraries. However, gene synthesis may become the method of choice, allowing to optimize codon frequency, restriction sites, and mRNA structure and to introduce site-directed mutations. Ligation-independent cloning is a generic, high-throughput process that can be uniformly applied regardless of the target gene or the cloning vector. Short N-terminal fusion tags, including a hexahistidine sequence and a specific protease cleavage site, are almost universally used. It has been widely documented, that larger fusion tags (e.g., GST, thioredoxin, MBP) can enhance solubility of proteins that are not soluble when expressed with a short peptide tag. However, such fusion proteins have not been widely used in the SGC, since removal of the tag often leads to loss of solubility.\nThe standard purification protocol is designed to be widely applicable, and experience has shown that it results in effective purification of a large fraction of proteins solubly expressed in E.\u00a0coli. A protein presented for crystallization must be homogeneous in composition, post-translational modification and oligomeric state; the presence of protein aggregates may be especially detrimental to subsequent crystallization. Affinity purification of highly-expressed proteins eliminates most other proteins, while gel filtration effectively separates different oligomeric forms of the protein and removes protein aggregates, which may otherwise promote irreversible aggregation of the protein preparation. The use of high salt concentration (typically, 0.5\u00a0M NaCl) throughout the purification process seems to reduce protein aggregation and non-specific binding of protein contaminants. Tag cleavage followed by another passage through the affinity column provides a further generic and highly effective purification step, which removes other proteins that bind adventitiously to the first affinity column. The generic purification procedure has provided in the majority of cases protein of sufficient purity to achieve crystallization. In most other cases, the generic procedure could be followed by polishing and protein modification steps to achieve homogeneous preparations.\nThe greatest barrier to production of human proteins in bacteria is recovery of soluble protein. Less than 15% of protein targets yielded detectable levels of soluble protein when tested as full-length constructs in the SGC, while more than 80% were expressed as insoluble aggregates. The key to achieving higher success rates has been the parallel production of large numbers of truncated constructs, often containing a compact protein domain. Construct design is initially based on domain boundary analysis, using a number of bioinformatic tools; 3\u20134 endpoints are designated around each of the predicted termini of the domain, resulting in 9\u201316 constructs. We have consistently found that this approach results in a 4-fold increase in the number of targets that can be produced as soluble proteins; a similar impact has been seen on the production of diffracting crystals, which can be dramatically affected by minute changes in protein termini. Although not rigorously tested, it is presumed that a protein construct that is inherently well-behaved (little tendency to aggregate or denature) will be less dependent on specialized conditions for expression and purification, and may crystallize in a wider range of conditions.\nCrystallization, crystal screening and data collection\nFor successful crystallization of a given target, the SGC\u2019s phase I operation appears to have confirmed that the most important driver for success is to explore protein diversity at the crystallization stage. One major form of variation was discussed above, namely testing multiple constructs of the target. Equally effective has been setting up co-crystallization with multiple ligands, along with varying protein concentration in the primary crystallization screens.\nAt the same time, it appears not to be vital to explore chemical space extensively for any given protein preparation; instead, the primary goal of the initial (coarse) screen can be to identify which preparations are \u201ccrystallizable\u201d, and a limited set of coarse screen conditions (\u223c200) generally seems sufficient. Practically, this requires only two 96-well crystallization plates, and by setting up three drops per condition, at different protein-well ratios (in Greiner 3-drop plates), the protein concentration is simultaneously varied. The conditions themselves are derived from those found to be most successful in other high-throughput initiatives [1\u20133], although according to this \u201ccrystallizability\u201d philosophy, the exact composition is probably not important. Naturally, coarse screens do not always yield high-quality crystals that can produce a dataset; however, the SGC operation does not rely on these crystals showing up in coarse screens, and a good optimization infrastructure is in place.\nIn practice, this diversity exploration leads to large numbers of parallel crystallization experiments, presenting a logistical challenge which, at this scale, can only be met with an efficient robotics and IT infrastructure. For the automation, the SGC has been able to exploit the devices developed on the back of the first wave of structural genomics initiatives, and our investment has been less in developing the machines, than in integrating them and implementing experimental best practices. Particular examples: by minimizing sample requirements with nanolitre crystallization, the available protein can be used in more experiments. The large numbers of drops thereby produced (1.5 million\/year) would be practically impossible to view by eye under the microscope, whereas automatic drop imaging on a fixed schedule allows images to be reviewed at leisure at the desk.\nAutomation has also played an important role in crystal characterization. An automatic sample changer has been used for initial characterization of diffraction quality of a vast number of crystals. This allows to rank the crystals for more careful data collection, especially at the synchrotron, and to direct further efforts at crystal optimization.\nA significant saver of upstream efforts has been to exploit each crystal\u2019s diffraction as efficiently as possible, even those traditionally considered to be marginal or problematic. Marginal diffractors would include crystals that are \u201cvery small\u201d (<40\u00a0\u03bcm in longest dimension), twinned, or have streaky or anisotropic diffraction. The latter cases generally require the undivided attention of experienced crystallographers.\nSmall crystals require an excellent X-ray beam: the PXII beamline of the Swiss Light Source synchrotron provides a beam which is reliably small but also well-aligned and very stable. Most efficient use of the beamline relied on pre-screening all crystals at the laboratory source for thorough work prioritization; real-time data processing during data collection; and close attention to radiation damage of crystals. It has been crucial to have experienced crystallographers on site. Adherence to these good practices has been highly productive: of datasets collected on 24-hour trips to SLS, 66% were used for final structures, while 90% of all depositions relied on synchrotron data. The ability to extract useful data from marginal crystals has been especially productive in combination with the protein\/ligand diversity approach of the SGC, as a significant fraction of structures (>50%) could be derived from crystals emerging from the primary screens, saving the need for further optimization.\nPhasing and structure solution\nDue to the family-based approach, for most SGC targets a homologous structure is already known, and most structures (>95%) can be phased by molecular replacement (MR). While this saves significant experimental efforts upstream compared to experimental phasing, by eliminating the need for selenomethionine-derived protein or heavy atom soaks, we find this does not actually save time overall, because starting phases from MR are heavily phase biased. Removing the bias has required many iterations of careful and incremental model building and refinement by experienced crystallographers who can see the danger signs of a poorly-refined model, and know how to deal with it [4, 5].\nThe final step, namely finalizing and depositing the model, is in fact a frequent stalling point, not only in high-throughput contexts. The reason is that the final model is not merely a result that can be trivially read off a few measurements, but instead is an interpretation of often rather noisy data, with a lot of detail that is easy to miss, where individual errors influence the clarity in all areas. Moreover, poor model definition affects biologically interesting parts of a structure, and interpreting it becomes a matter of judgment and using in orthogonal information. Indeed, the \u201cfinal\u201d model is as much scientific hypothesis as result, and depositing the model means signing off on the hypothesis\u2013\u2013which is why it has traditionally been a bottleneck in structural genomics efforts.\nThe SGC has used a peer proofreading system combined with strict timelines to counteract the problem: before deposition, the structure is reviewed by another crystallographer for errors or alternative interpretations, and comments passed back to the original refiner. The intention is threefold: First, to introduce quality control on the final output. Second, the refiner does not feel compelled to spend excessive time on the model to flush out the final errors, since she knows it will be checked. Third, by mixing up refiners and proofreaders, over time this should lead to common interpretations of marginal modeling decisions. The timelines depend on situation and difficulty, but typically allow two weeks for refinement, a day for proofreading, and two further days for deposition.\nThis approach has made it possible to deposit novel structures at a considerable rate (6 each month from a team of 6 dedicated and 4\u20135 occasional crystallographers) without compromising quality.\nInformation infrastructure\nAn efficient laboratory information management system (LIMS) has been vital to manage not only target tracking, but also capturing and integrating where possible information generated from robotics, as well as capturing human assessments of experimental outcomes, where these could be entered via a client (e.g., scoring of crystallization images).\nFortuitously, the solution we settled on, BeeHive from Molsoft (http:\/\/www.molsoft.com\/beehive.html), is in essence an extremely intuitive database query tool that enables even inexperienced users to extract information relevant to their current work\u2013\u2013including the simplification of data entry. This is a weak point of many LIMS solutions, whose focus often evolves around data entry but have very inflexible retrieval mechanisms. This has proved to be a powerful means of communication between all persons involved in a project, allowing immediate and error-free retrieval of \u201chard\u201d information (e.g., protein sequence, ligand and buffer conditions and project history), as well as evaluation and prioritization of crystals and of concurrent projects.\nProtein characterization and ligand screening\nOne of the major challenges in structural genomics is identifying the function and evaluating the functional integrity of the proteins. Examining the physical state of a protein\u2013\u2013by methods such as analytical ultracentrifugation, chromatography or dynamic light scattering\u2013\u2013is valuable in assessing the prospects for crystallization. In contrast, specific activity assays need to be tailored for each protein class, and may be impractical or impossible when the activity of the protein is not known. We have implemented a generic screen, based on the increase in thermal stability of a protein upon ligand binding. The fluorescent readout is based on monitoring of protein unfolding using a hydrophobicity-sensing dye. Differential Scanning Fluorimetry (DSF) assays [6\u20139] are ideal for screening a large number of compounds for binding to each target protein. Significantly, the shift in Tm (the unfolding transition midpoint) measured by this method is comparable to measurements obtained by differential scanning calorimetry (DSC), the well-established standard method for thermal shift measurements. In selected cases, a direct correlation between Tm shift and binding constants has been observed [8, 10].\nSeveral advantages have been derived from this capability: First, the identification of relatively strong interacting molecules out of several hundreds of candidates. As detailed below, the compounds discovered in this manner are then included in crystallization experiments; in many cases, only protein\u2013ligand complexes yielded diffracting crystals. Secondly, the reactivity profiles provide data on binding selectivity of the protein active site, which is the most crucial information for drug design; we have often followed up the results from ligand screens by analyzing the structures of several protein\u2013ligand complexes. In parallel, the properties of the protein\u2013ligand interactions are studied by biophysical methods and by enzyme inhibition studies. Third, such screens have allowed us to identify ligands or substrates of proteins with unknown function (sometimes termed \u201cde-orphanizing\u201d). Finally, DSF-based screens can be expanded to explore other conditions, such as buffer composition that enhance the stability of a protein. These conditions may then be introduced to improve the outcome of protein purification and crystallization [8].\nThe limited scale of protein production and other limitations on resources do not allow a full-scale screen as done in the pharmaceutical industry (105 compounds). Rather, we have assembled smaller family-specific compound libraries (10\u2013103 compounds each), which can reasonably be tested against available amounts of protein (\u223c200\u00a0\u03bcg for 100 assays). The compound libraries are based on the scientific and patent literature; the chemical structure of prospective compounds is used to search an in-house compilation of vendor databases to identify potential sources. Acquisition of desired compounds is not trivial: not all published compounds, even those appearing in vendor catalogues, are actually available when required; alternative vendors, or collaborative sources may then be accessed. With continuous updating based on current literature and our own experimental results, these libraries have allowed to derive binding profiles and new insights on ligand specificity.\nSGC target and biology area selection: relevance for the treatment of human diseases\nFor any structural genomic organisation target selection is an important consideration as it can have a major impact on the procedures that are implemented during the process of structure determination. There are a number of approaches applied by different structural genomics projects to select targets for structural analysis such as blanket coverage of an organism\u2019s genome, targets with potential novel folds, percentage cut off based on sequence identity or total coverage of selected protein families. The SGC has opted for the family-based approach with an emphasis on protein families whose members are important in human health, disease and are potentially druggable. From our point of view, the main advantages of this approach are 2-fold. Firstly, the methods and procedures identified for one family member can be applied to another family member improving everything from expression, solubility, stability, and purification, to crystallisation and structure determination. Secondly, analysis of the structures from all family members can reveal additional significant information such as ligand binding site specificity, conformational dynamics, understanding of aberrant behaviour of specific family members or the converse revealing common structural properties within all family members.\nThe availability of high resolution structures constitutes the foundation for structure-guided drug discovery projects. In recent years SG has significantly increased the number of human protein structures available for structure-based design projects [11]. In particular, protein family focused efforts originating from high-throughput structural biology projects have contributed to the structural description of a number of members from human protein families and thus provided valuable structural and chemical information for the design of bioactive compounds. In addition, established expression and crystallization conditions have been used to generate essential reagents, methodologies and technologies which have facilitated research projects in academia and drug discovery programs in industry.\nThe SGC has focused on providing protein structures to support drug development and understanding of the structural determinants for human disease. Of 160 unique targets deposited by the SGC (in phase 1), clear disease relevance has been established for 70% and a further 18% are likely to be involved in at least one disease. This pattern holds true for all the human protein families the SGC is working on. The following sections provide an overview of the three distinct biological areas selected at the Oxford site of the SGC.\nBiology area I: Structural Genomics of human metabolic enzymes\nSelection of metabolic enzymes as biological target area at the SGC was based on two distinct features: they are fundamentally involved in a multitude of human diseases, including cardiovascular, metabolic diseases or cancer, and in addition several enzymes constitute possible drug targets. Emphasis has been given to certain metabolic enzyme families such as oxidoreductases (mostly short-chain dehydrogenases\/reductases (SDR), medium-chain dehydrogenases\/reductases (MDR), long-chain dehydrogenases\/reductases, aldehyde dehydrogenases (ALDH), aldo keto reductases (AKR) and 2\u2032oxoglutarate dependent oxygenases (2OGs). In addition, pathways of importance, e.g., in lipid or amino acid metabolism were selected with a distribution of about 1:1 between oxidoreductases and other metabolic enzymes. The target list comprises about 300 metabolic enzymes, and after three years of operation, >60 unique novel structures have been solved. Three points of importance are highlighted in this review: structural characterization of enzymes shown to be causative of metabolic inherited diseases, structure determination of drug discovery targets in metabolic diseases such as metabolic syndrome or osteoporosis, and structure-guided \u201cde-orphanization\u201d of insufficiently characterized human gene products or even entire pathways.\nStructural basis of inherited metabolic diseases\nGenetic defects in enzymes involved in metabolic pathways such as amino acid or lipid catabolism are causative of a whole spectrum of symptoms, including dysmorphologies, mental retardation, neuropathies or life threatening situations like fasting induced hypoglycemia [12, 13]. Understanding of molecular causes and possible interventions of inherited metabolic diseases requires besides biochemical and clinical management a structural template for explanation of mutational effects.\nThus far the focus has been to a large extent on oxidoreductases in the area of metabolic diseases. Associated disorders comprise electron transfer reactions for energy production (e.g., mitochondrial myopathies), oxidative and reductive roles in the metabolism of amino acids (e.g., hyperprolinemia or branched-chain hydroxyacyl CoA dehydrogenase defects), fatty acids (e.g., inborn errors in \u03b1- and \u03b2-oxidation of short-, medium- or long-chain fatty acid metabolites), cofactors (e.g., phenylketonuria type 2), hormones (e.g., male pseudohermaphroditism or adrenal hyperplasia), mediators (e.g., congestive heart failure) and lipids (e.g., inborn errors in cholesterol synthesis, CHILD syndrome, Smitz-Opitz Laemmli syndrome as examples). The impact of the structural approach is illustrated by the successful structure determination of phytanoyl-CoA hydroxylase [14], the major molecular cause of Refsum disease, a peroxisomal disorder with severe neurological symptoms. The structure provides a framework to interpret the majority of the disease causing polymorphic alleles, and we were able to map those to changes in the active site, around the Fe2+ and 2-oxoglutarate binding sites in this 2OG enzyme [14].\nMetabolic enzymes as drug targets\nOxidoreductions at specific positions of lipid hormones such as steroids selectively alter nuclear receptor binding properties. Therefore, inhibition of dehydrogenases\/reductases carrying out these reactions selectively influences cellular hormone levels and transcriptional responses. This concept has recently found great attention with the development of specific inhibitors against 11\u03b2-hydroxysteroid dehydrogenase type 1 (11\u03b2-HSD1) as a novel drug target in diabetes and obesity [15\u201318]. Similar drug development efforts are underway regulating androgen or estrogen levels through specific modulation of distinct hydroxysteroid dehydrogenases (17\u03b2- and 3\u03b1-HSDs in cancer, inflammation, osteoporosis, ageing, and autoimmune diseases). We determined the structure of human 11\u03b2-HSD1 in complex with a clinically relevant inhibitor, carbenoxolone (Wu et\u00a0al., unpublished) and have provided a platform for drug development efforts. Other hydroxysteroid dehydrogenase structures comprise 17\u03b2-HSDs such as types 4, 8, 10, 11 and a novel type 14 (see below), necessary for determination of off-target activities of compounds directed against type 1 and 3 17\u03b2-HSDs. Other targets of pharmaceutical relevance successfully pursued are farnesyl diphosphate synthase (FDPS) and geranylgeranyl diphosphate synthase (GGPS), which are critical in synthesis of isoprenoids necessary for covalent modification of GTPases involved in cell signalling and survival. Crystal structures of FDPS complexed with nitrogen-containing bisphosphonates currently used for osteoporosis therapy allowed a molecular mechanism of action to be postulated for these drugs [19] (Fig.\u00a01). Furthermore, several prokaryotic and parasitic dehydrogenases have been identified as novel targets for antibiotic and antiparasite drug development, and thus allow synchronization with the SGC Toronto efforts, where an apicomplexan\/protozoan SG program has been established. Thus, structure determination of related human enzymes will facilitate structure aided drug design and allow virtual and focused screening efforts in this emerging disease area.\nFig.\u00a01Bisphosphonate binding to human farnesyl diphosphate synthase. Electron density is shown in green around the clinically used inhibitor risedronate\nDeorphanization of metabolic enzymes and pathways\nA significant proportion of the metabolic enzymes targeted were at the time of structure determination devoid of assigned activity or function. High throughput protein production, structure determination and functional characterization allowed \u201cdeorphanization\u201d of unknown enzymes. We employed ligand screening, enzyme activity assays, expression and subcellular localization data, as well as structure determination combined with docking analysis to describe novel human enzymes. In the absence of co-crystal structures, interpretation of results from biochemical assays and compound screening was rationalized by in silico docking of potential ligands into the active site of the orphan structures. Analysis of the different docking poses was correlated with experimental results, allowing direct visualization of the putative protein\u2013ligand complex. In this manner we determined a novel 17\u03b2-HSD14 [20], possibly involved in cancer, and a novel type-2 R-hydroxybutyrate dehydrogenase, involved in ketone body utilization [21]. Further emphasis was given on novel pathways such as mitochondrial fatty acid synthesis. This recently discovered pathway is important in the synthesis of lipoic acid, essential for mitochondrial function. Thus far we have determined three distinct enzymes of this metabolic route, namely the malonyl transferase (2c2n), ketoacyl synthase (2c9h) and the enoyl-ACP reductase (1zsy). These structures represent the only higher eukaryotic structures thus far available for this pathway. The data will be instrumental to compare to the multidomain type I fatty acid synthase, where we recently solved the structure of the malonyl\/acyl transferase domain (2jfk, 2jfd). This cytosolic enzyme is involved in production of endogenous fatty acids and lipids, and is discussed as potential target in metabolic diseases and cancer.\nBiology area II: Structural Genomics of transmembrane receptor signalling pathways\nComplete coverage of the14-3-3 protein family\nA human protein family that the SGC has completed the structure determination of all members is the 14-3-3 family. This family consists of seven members (\u03b2, \u03b5, \u03b7, \u03b3, \u03c3, \u03c4, and \u03b6) of which \u03c3 [22, 23], \u03c4 [24] and \u03b6 [25] structures were previously determined. This protein family plays a central role in many fundamental cellular roles such as cell cycle control, apotosis, protein trafficking, signal transduction and stress response [26\u201328].\nBefore the structural completion of the 14-3-3 family most of the structural studies utilised 14-3-3\u03b6 which provided details of the conserved peptide binding site [25], the primary peptide interaction [29, 30] and secondary target domain interactions [31]. As all of these structures displayed similar overall conformations, structurally it was proposed that 14-3-3s behaved as \u201cmolecular anvils\u201d in that their overall structure remained unchanged whether in the apo-form or bound to their target protein [32]. The structure determination of the remaining members allowed for a family-wide comparative study that revealed another story with a major emphasis on the flexibility of 14-3-3s [33]. This was most obviously with the apo-form of 14-3-3\u03b2 in which one of the subunits was in a similar conformation to all other 14-3-3 structure while the opposing monomer displayed a more open conformation for the peptide binding groove (Fig.\u00a02).\nFig.\u00a02The flexibility of the 14-3-3 is illustrated by the superimposition of 14-3-3\u03b2 (blue) with 14-3-3\u03b7 (orange). The monomer conformations of both isoforms are essentially identical on the left hand side. However, the beta monomer on the right side has a more open peptide binding groove and flexibility at the dimeric interface\nAdditional flexibility of 14-3-3 proteins was observed when all of the family members were superimposed against one subunit. It became instantly clear that the position of the second subunit varied between the different 14-3-3 isoforms [33]. This is achieved through the N-terminal helices that make up the dimeric interface sliding over one another (Fig.\u00a02). The significance of the interface flexibility is that it allows for the widening or shortening of the distance between the two peptide binding grooves hence allowing a 14-3-3 to accommodate structures of varying shapes and sizes. As 14-3-3 are known to have bind hundreds of partners [34\u201336] this interface flexibility would provide the necessary structural adaptability to accommodate the wide structural range of target proteins.\nAs all of the human 14-3-3 structures are now known they allow for a detailed bioinformatic analysis of the 14-3-3 family. This approach identified common protein\u2013protein interaction patches at the subunit interfaces plus two additional non-specific protein interaction sites that would attract and bind the globular structured regions of the target protein thus providing a mechanism by which the 14-3-3s can initially attract and then bind a wide range of structurally diverse target proteins [33]. Another more numerous protein\u2013protein interaction family that was targeted by the SGC are the PDZ domains which have been implicated in the regulation of drug transporters [37] and involved in the clustering, targeting and localisation of the target proteins [38]. These domains bind mostly to C-terminal peptides that fall into two classes: class I peptides are \u2013(Ser\/Thr)\u2013X\u2013\u03a6\u2013COO\u2212 while class II peptides are \u2013\u03a6\u2013X\u2013\u03a6\u2013COO\u2212 where X represents any amino acid and \u03a6 represents any hydrophobic residue [39, 40].\nPDZ domains\nInitial attempts at structure determination of 18 unique human PDZ domains resulted in a successful outcome for only 3 of these targets. To improve our success rate we took advantage of the family based approach and generated new expression clones of the remaining 16 targets with generic class I and II PDZ binding peptides attached to the C-terminus of each domain. The idea was for these peptides to bind adjacent PDZ domains initiating protein\u2013protein interactions and thus crystal nucleation. As such the linker between the predicted end of the PDZ domain and the C-terminal peptide was varied from 2 to 6 amino acids allowing for flexibility but restraining the distance between adjacent domains [41]. Using this approach we have now solved 11 of the remaining 15 targets many of which have thrown up new details regarding peptide selectivity and structural adaptability of the PDZ domain when bound with a peptide.\nAs expected for most of these domains the peptide interaction was similar to the standard configuration [42, 43] in that the side-chain of the C-terminal hydrophobic residue (position 0) was bound in a conserved hydrophobic pocket and that the peptide\u2019s -2 position Ser\/Thr coordinates the His side chain from the \u03b1B helix. However, there were a number of surprises of which the biggest was for MPDZ@3 in which a class II mode of binding was observed for a class I peptide which involved a translation of the \u03b1B helix (Fig.\u00a06a of [41]).\nBiology area III: Structural Genomics of human protein kinases\nKinases play an essential role in most (if not all) signalling pathways and dysregulation has often been linked to disease. Several successful inhibitors developed to target kinases have shown that members of this large protein family are excellent targets for the development of drugs. Currently protein kinases constitute about 25% of presently pursued drug targets in industry [44\u201347].\nThere are 518 identified human protein kinases constituting 1.7% of all human genes, which have been grouped into 10 families [48]. Despite the large number of members and their involvement in large variety of pathways, evidence points to a common single ancestral protein. As a result, the structural features as well as key regulatory elements and catalytic mechanism of phosphate transfer are all well conserved. High resolution structures are therefore essential for the rational design of potent and selective inhibitors. Before the contribution of SG efforts, the progression of publicly available kinase structures was linear with only 38 human kinase structures publicly available in 2004 [46]. Currently, 21 novel human kinases structures have been released by the SGC (19 from Oxford), which started to target this protein class in 2004. This increased the number of unique human kinase catalytic domain structures available in the pdb (http:\/\/www.pdb.org\/pdb\/home\/home.do) to 93 by the end of 2006.\nMany structures, released by SG, were only distantly related catalytic domain structures previously known and in some cases provided the first structural information for a subfamily. Thus, these structures significantly enriched the coverage of the three dimensional structure description of the kinome. Among the structures where the SGC determined the first representative structure of a family were: the NEK (\u201cnever in mitosis\u201d\/NIMA) family member NEK2, the CDC2 like kinases family member CLK1 and CLK3 as well as the first structure of a NAK (Numb-associated kinases) kinase MPSK1. These kinases are quite diverse in terms of primary structure and it is therefore not surprising that many novel structural features have been discovered. For instance, a novel activation loop architecture characterized by a large helical insert has been discovered in the structure of MPSK1, the structures of CLK1 and CLK3 revealed a family conserved antiparallel beta sheet flanking the kinase hinge region, and the structure of NEK2 identified a short helix following the activation segment DFG motif that may be explored for the development of specific inhibitors [49].\nKinases are extremely flexible proteins that may adopt a number of distinct catalytically active or inactive conformations during their catalytic cycle, upon activation by phosphorylation, or by binding of a regulatory protein, and consequently a number of clinically successful inhibitors have been developed to target specifically the inactive state of kinases [50]. For example the anti-leukaemia drug Imatinib binds selectively to the inactive state cABL characterized by an outward conformation of the DFG motif, a conserved tripeptide motif that ligates Mg2+ ions [51, 52]. It is not clear to date how many kinases are able to adopt this conformation, which makes development of these so-called type II inhibitors possible. In general, these are characterized by largely improved specificity.\nFor the development of conventional inhibitors that target the active state of kinases information about the plasticity of the catalytic domain greatly facilitates the rational design of inhibitors. Consequently it is desirable that several structures of the same target in complex with different ligands are available. Also here the structural information content regarding ligand binding was significantly increased during the last three years by SG. In 2004, only 38 human kinases had a structure available in the public domain and only 12 publicly available structures contained non-adenosine chemotypes [46]. From the 19 structures of kinase catalytic domains released by our laboratory, 16 were determined in the presence of an non-adenosine kinase inhibitor and several structures were determined in complex with more than one inhibitor scaffold (Table\u00a02, and Fig.\u00a03, showing PAK5 apo\/inhibitor).\nTable\u00a02Protein kinase structures determined by SGCNamePDB IDResolution [\u00c5]Inhibitor nameDisease linkFamilyCLK11Z571.70HymenialdisinePot. GeneticeCMGCCLK3c2EU91.53nonePot. GeneticeCMGCCK1\u03b312CMW1.75Compound 52CK1CK1\u03b322C472.405-IodotubercidinGeneticCK1CK1\u03b33d2CHL1.95Triazolodiamine 1CancerCK1ERK32I6L2.25noneCancerCMGCASK12CLQ2.30StaurosporineInflammation, CVfSTENEK22JAV2.10SU11652CancerOther-NEKPAK4a2CDZ2.40Cdk1 InhibitorCancerSTEPAK52F571.80Cdk1 InhibitorPot. CancerSTEPAK62C301.60noneCancerSTEPIM1b1XWS1.80BIM I, HB1Cancer, InflammationCAMKPIM22IWI2.80HB1Cancer, InflammationCAMKSLKc2J512.10Triazolodiamine 1Pot. CancereSTEMPSK12BUJ2.60StaurosporinePot. CancereOther-NAKSTK102J7T2.0SU11274Not knownSTEDAPK32J902.0Pyridone 6Cancer, InflammationCAMKCAMK1G2JAM1.7SU11652Not knownCAMKCAMK1D2JC62.5GSK inhibitor XIIIGeneticCAMKaPAK4 also deposited as an apo-structure in two different spacegroups: 2BVA, 2J01bStructures with different inhibitors and substrate: PIM1: 2BIK, 2BZH,2BZI, 2BZJ, 2BZK, 2C3I, 2BIL, 2J2I; SLK: 2JA0cCLK3 also deposited as phosphorylated protein: pdb-code: 2EXE. Detailed description of structures of targets solved in Oxford is available in form of \u201ciSee\u201d datapacks freely downloadable on: http:\/\/www.sgc.ox.ac.uk\/structures\/KIN.htmldCK1\u03b33 with different inhibitors: 2CHL, 2IZR, 2IZS, 2IZU, 2IZTeA formal link to the disease has not been established so far but is likelyfCardiovascular diseaseFig.\u00a03Superimposition of apo-PAK5 (cyan) and the PAK5 purine complex (orange), highlighting the decomposed movements of the glycine-rich loop (flapping) and the \u03b1C helix (swinging) [53]\nIn addition, the SGC has supported development of entirely new inhibitor classes exemplified by co-crystal structures with Ruthenium-half sandwich complexes. These stable organometallic compounds are extremely potent inhibitors for PIM1 kinases [54]. The co-crystal structure of three inhibitors of this class showed that the inert metal centre in this scaffold functions as a hypervalent carbon, allowing it to occupy the binding pocket efficiently with excellent shape complementarity.\nContributions of NMR to Structural Genomics\nNMR as a complementary method to crystallography for protein structure determination\nThe NMR spectroscopy can play an important role in structural genomics, providing complementary information to that obtained from X-ray crystallography. Importantly for large-scale structural genomics projects, NMR provides an alternative route to solving the high resolution, three-dimensional structures of proteins that prove refractory to crystallization. We were able to use NMR to solve the structures of a number of relatively small protein domains (\u223c20\u00a0kDa) in which the domain contained at least one flexible region. The RGS domains from the regulator of G-protein signalling proteins, RGS3, RGS10, RGS14, RGS18, RGS20 were all very good examples of this. Multiple constructs of these were designed, which expressed to high yield in stable, highly soluble form yet did not yield high quality crystals despite many months of concerted effort. The domains were therefore expressed as uniformly 15N-labelled proteins using standard growth methods in E.\u00a0coli, and their 15N-HSQC spectra were recorded to assess the feasibility of structure determination by NMR. In all cases, excellent spectral dispersion was observed and we were able to obtain almost complete assignment of the protein resonances. We have since deposited the high resolution NMR structures of three RGS domains in the PDB and the resonance assignments of four RGS domains in the BioMagResBank (BMRB). The structures and assignments of two further non-crystallizing domains (Spred2 EVH1 domain and JARID1CA Bright\/ARID domain) have also been deposited, and those of several other non-crystallizing domains \u2018rescued\u2019 by NMR are currently underway (Table\u00a03).\nTable\u00a03Deposited NMR structures and assignmentsGenePDB depositionResonance assignment depositionRGS3\u2013BMRB-15178RGS102I59BMRB-7272RGS142JNUBMRB-15128RGS182OWIBMRB-7106SPRED22JP2BMRB-5939JARID1CA2JRZBMRB-15348\nNMR as an assessment tool for the feasibility of structure determination\nFurther examples where NMR has proven useful as a rescue strategy include particular families of signalling domains which have a known tendency to be partially unfolded in their unliganded states. Some examples include certain WW domain [55, 56]. We successfully identified peptide binding partners for a WW-tandem construct using the SPOTs screening technique [57, 58] following which the most strongly binding peptides were synthesized on a large scale for NMR measurements. Although the 15N-HSQC spectra of this pair of tandem domains in isolation were very unhopeful, the spectra of their complexes showed significant improvements in signal dispersion, indicating that in the complexed form, the protein was better folded. At this point, the protein entered our NMR structure determination pipeline. The recording of a quick 15N-HSQC spectrum has in several cases allowed us to rescue protein constructs with promising but borderline behaviour, for example, proteins showing good signal dispersion but low-medium levels of aggregation. Far from abandoning these constructs, we took these constructs \u2018back to the drawing board\u2019 and made rational construct improvements with the help of bio-informatic tools. Successfully re-designed constructs were then re-screened for fold quality by 15N-HSQC. After 2\u20133 iterations of this procedure, it was often possible to refine promising constructs sufficiently for structure determination. For example, Fig.\u00a04 shows the stepwise improvement in the spectral properties of a hopeful, though initially problematic DNA-recognition domain from the oxygenase protein, JARID1CA. The NMR structure is now deposited (PDB code: 2JRZ). In all of the above cases, a quick 15N-HSQC showed immediately whether the structure determination of a protein, having to failed to crystallize, should be pursued or abandoned, hence reducing unnecessary attrition in the structure determination pipeline.\nFig.\u00a04Visible improvement in quality of 15N-HSQC spectra over two rounds of iterative construct re-design for the JARID1CA Bright\/ARID domain. The leftmost (initial) construct shows potential. The structure of the final construct on the far right was determined by NMR (PDB code: 2JRZ)\nThe study of protein dynamics by NMR\nThe use of NMR to study the rotational correlation times and internal dynamics of the proteins offers good explanations as to why crystallization sometimes fails even for well-folded proteins. In all of the proteins we rescued by NMR, 15N heteronuclear NOE and 15N T1, T2 relaxation data revealed regions of internal mobility within the proteins, which would have hindered long-range order and impaired or prevented efficient crystal packing. A striking example was the case of the RGS domain from RGS10, in which NMR relaxation data confirmed true local mobility in a region of the domain which not only lacked in NMR restraints, but also showed no electron density in the crystal structure of the complex of RGS10 with G-alpha-i3 (PDB 2IHB). Comparison of mobility in RGS domains from different branches of the phylogenetic tree leads to clues about their specificity and helps to guide further investigations. In some cases, the 15N T1 and T2 data have also identified partial dimerization in proteins that fail to crystallize, thus explaining the latter. NMR relaxation data were in each case confirmed by analytical ultracentrifugation (AUC). The combined information allowed us to decide whether these proteins should be highlighted as candidates for structure determination by NMR and to judge the best conditions under which they should be studied.\nFuture and outlook\nThe future role that NMR will play in structural genomics will depend heavily on the continued development and implementation of new, faster methods of data acquisition, processing, resonance- and NOE-assignment and structure determination and refinement. These topics have been covered extensively in other reviews; for a concise summary see [59] and references therein. The potential time gains that could be gained from these methods make high throughput structure determination by NMR a realistic possibility for the future.\nStructural bioinformatics and rationalisation of experimental results\nA crystal structure of a protein in absence of ligand or substrate may not always provide insight on reaction mechinasms or specificity. Ideally, such information can be derived from additional structures with bound ligands. In the absence of such co-crystals, interpretation of results from biochemical assays and compound screening is more speculative. However, these results can be rationalised with in\u00a0silico docking of potential ligands into the active site of unliganded protein structures. An example illustrating this point is the analysis of the DHRS10 structure [20]. Analysis of the different docking poses can be correlated with experimental results, allowing direct visualisation of the putative protein\u2013ligand complex. With these results, further modifications of the enzyme can be suggested more reliably, allowing a faster progress towards the complete elucidation of the mechanistics.\nDissemination of structural genomics data and knowledge\nStructural genomics produces a wealth of information of different types: DNA and protein seqeuences, biochemical information, coordinates of crystal structures, and structural annotation. This information is deposited in one or more public databases, predominantly the PDB, in addition to publication in journals. This form of data distribution does not adequately disseminate the full information to a wide scientific audience. The first issue is the fragmentation of data between different formats. A user may have to read text information in a journal paper, which may include a few two-dimensional Figures; then download a PDB structure file and image with a separate application; and then perform analysis and alignment of data from, say, SNP database using alignment software. The second issue is that non-structural biologists do not routinely access PDB files, especially of structures that were not published in pubmed-indexed journals.\nWe have approached this challenge by developing a new intuitive dissemination concept in conjunction with Molsoft LLC (San Diego, CA) [60]. This concept, (which we denoted iSee) integrates all the information associated with any given target solved by SGC into a small, self-contained file, annotated by the authors (Fig.\u00a05). The file not only allows the direct visualisation of text information, but also offers an interactive visualisation feature fully integrated to the structural data being presented. At any stage, the annotation written by the expert can be coupled with an interactive molecular graphics scene. Transition between each anotated viewpoint is fully animated on-the-fly, to convey a sense of three-dimensionality which is vital for the user to grasp the spatial relationship between different features on a structure.\nFig.\u00a05Screenshot of iSee datapack. The annotation text (top left panel) includes links (blue text), which lead to structural images focused at areas of interest, simultaneously accessing other types of information (sequence alignment, small molecule formulae, etc.)\nEach of these files (called an iSee datapack), as well as the software needed to visualise them (ICM-Browser) are available for free download from our website (http:\/\/www.sgc.ox.ac.uk\/iSee).\nWe also maintain and curate each of these files by revising each datapack quarterly to ensure that all the recently disclosed information is added (either by ourselves through follow-up experiments or by external collaborators working on the same targets). Each of the datapacks has a built-in automated updating function that can be executed on user\u2019s request.","keyphrases":["protein kinase","reductase","high-throughput","dehydrogenase","14-3-3","pdz","binding specificity","protein crystallography"],"prmu":["P","P","P","P","P","P","R","R"]}
{"id":"Matern_Child_Health_J-2-2-1592250","title":"Strategies to Reduce Alcohol-Exposed Pregnancies\n","text":"Introduction\nPrenatal alcohol exposure remains a leading preventable cause of birth defects and developmental disabilities in the United States, with the prevalence of alcohol consumption by women in childbearing age remaining high and unchanged over time (Fig. 1). This paper provides a summary of current knowledge and information on recognition and prevention of an alcohol-exposed pregnancy, including overall alcohol exposure burden among women of child-bearing age, diagnostic criteria for fetal alcohol syndrome (FAS), and recommendations for screening, assessment and interventions to reduce alcohol exposed pregnancies.\nBurden of alcohol exposure in pregnancy\nMost women reduce their alcohol use substantially when they realize they are pregnant, but significant numbers continue to drink at levels that can be hazardous to the fetus [1, 2]. Of particular concern are the group of sexually active women who are not planning to become pregnant (about half of all pregnancies in the US), but do so and continue alcohol use during the early stages of embryonic and fetal development [1]. For many women, pregnancy recognition does not occur until the 6th week of gestation [3]. According to national survey data, in 2002 about 8% of women aged 18 to 44 years were sexually active, fertile, not using any form of birth control, and at risk of becoming pregnant [1]. This group of women also report high rates of binge drinking, with 1 in 5 in the age category of 18 to 24 years reporting multiple episodes of binge drinking, and are therefore at risk for an alcohol-exposed pregnancy.\nFig. 1Weighted percentage of alcohol consumption for non-pregnant women aged 18 to 44 years during the previous 30 days, BRFSS, United States. Data were not collected in 1994, 1996, 1998, and 2000. Frequent refers to = 7 drinks\/week or binge. Binge drinking refers to = 5 drinks on one occasion\nAdverse fetal outcomes\nPrenatal alcohol exposure can result in a wide range of adverse outcomes including fetal alcohol syndrome (FAS), although not all infants exposed to alcohol in utero develop the same severity of effects as do others. The term fetal alcohol spectrum disorder (FASD) has been introduced to describe the range of physical, mental and behavioral effects that can occur in an individual exposed to alcohol in utero. Because diagnostic criteria are not available for all conditions along the spectrum, there are no prevalence rates available for the full spectrum. Prevalence rates of FAS range from 0.3 to 2.0 cases per 1,000 live births [4] depending on the methodology used, and the sub-populations assessed. Reported rates are higher among infants born to women who are American Indian\/Alaska Native or African American, unmarried, smokers, have low incomes, and have a history of previous drug use or mental health conditions [5, 6]. In an effort to promote the more complete recognition of FAS, guidelines for identifying and referring persons with FAS have been collaboratively developed and disseminated recently [4] (www.cdc.gov\/ncbddd\/fas\/documents\/fas_guidelines_accessible.pdf).\nApproaches to reducing alcohol-exposed pregnancies\nA 1996 report from the Institute of Medicine addressing prevention of FAS recommended implementation of preventative actions at multiple levels including individual, group, and universal levels [7]. To date, most of the effective prevention strategies identified have focused on the individual level. In 2004 the U.S. Preventive Services Task Force (USPSTF) released a report recommending screening and behavioral interventions to reduce alcohol misuse in adults in primary care settings [8], www.preventiveservices.ahrq.gov.\nThe report concluded that effective interventions include brief counseling comprised of feedback regarding screening and assessment information, advice, goal-settings and follow-up assistance. The report further cited complementary practices of motivational interviewing, assessing readiness to change, and use of the 5 A\u2019s behavioral counseling framework of assess, advice, agree, assist and arrange. Validated screening instruments are available for screening pregnant and non-pregnant women of reproductive age including the T-ACE, TWEAK, and AUDIT [9]. More information on these instruments is available at the following website: www.nih.gov\/publications\/Assessing\/Alcohol\/Index.htm.\nPrimary healthcare providers can play a pivotal role in identifying women of reproductive age (during pregnancy and before pregnancy occurs) who are at high risk for an alcohol-exposed pregnancy and providing them with advice, counseling, and referral as appropriate. This strategy has widespread support among professional organizations including the American College of Obstetricians and Gynecologist (ACOG), and the American Academy of Pediatrics (AAP), as well the U.S. Office of the Surgeon General, and the U.S. Department of Health and Human Services.\nA useful example of an intervention targeting groups of women using many of the components described in the recommendation of the USPSTF is found in a CDC sponsored study, Project CHOICES, a feasibility pilot study that targeted non-pregnant women at high risk for an alcohol-exposed pregnancy [10]. Project CHOICES was conducted in diverse sub-populations of women determined to have higher proportions of individuals at risk for an alcohol-exposed pregnancy. Compared to the overall estimated 2% of childbearing women at risk for an alcohol-exposed pregnancy, the Project CHOICES intervention settings (e.g. a jail, alcohol and drug treatment facilities, and primary care clinics) had an overall rate of 12.5% of women at risk for an alcohol-exposed pregnancy. A unique aspect of the intervention is that it focused not only on reducing risk drinking, but also addressed pregnancy postponement as a route for avoiding an alcohol-exposed pregnancy. The intervention consisted of 4 brief motivational interventions sessions and 1 consultation visit to a family planning provider. All participants were at risk for an alcohol-exposed pregnancy in that they were sexually active, fertile, risky drinkers and not taking effective measures to avoid pregnancy. At the 6 month follow-up assessment, 68.5% were at reduced risk because they had either changed their risk drinking, instituted effective contraception, or both. Subsequently, the Project CHOICES Research Group completed a randomized controlled trail to test the efficacy of the intervention with a report of the study findings currently underway.\nUniversal level interventions have not received recent attention with the exceptions of the 2005 release of U.S. Surgeon General\u2019s Advisory on Alcohol Use and Pregnancy, http:\/\/www.hhs.gov\/surgeongeneral\/pressrelease\/sg0222205.html. This advisory drew attention to the continuing problem of FASD and the continuing high rates of alcohol use, including binge drinking, among childbearing aged women in the U. S. The impact of the advisory has provided support for those seeking to inform and educate the public healthcare system overall to this important public health concern.\nSummary\nIn addition to the adverse effects of alcohol on the fetus, alcohol use can leads to other adverse effects on the health of women including reproductive health conditions that are not addressed in this brief. Though the approaches mentioned here are primarily aimed at reduction of prenatal alcohol-related pregnancies, any reduction in hazardous alcohol consumption among women will add to improvements in the general health of women. Multiple federal and non-fedral health agencies and organizations recommend that pregnant women and those planning a pregnancy should abstain from alcohol use. Although assessment and interventions are valuable tools to recognize and address alcohol use and secondary pregnancy outcomes, they are underutilized in primary care settings. To reduce the burden of alcohol exposed pregnancies and alcohol\u2019s impact on the health of families, it is critical for physicians and health care providers to consistently screen childbearing-aged women for alcohol use with validated screening tools which can be embedded in the patient screening protocol. Tools are now available for pediatricians and child health care providers to enhance early recognition of FASD and reduce secondary conditions that often accompany physical maturation [4]. Brief clinician-delivered behavioral interventions to women and their partners, counseling regarding effective contraceptive options when not planning a pregnancy, and improving access to such services for those who are unable reduce their alcohol intake can help more women reduce their risk for an alcohol exposed pregnancy [11]. Referral to needed social services should complement health services in order to achieve maximum benefits of primary care-based attempts to reduce alcohol-exposed pregnancies. Given the levels of alcohol use among childbearing aged women, primary care-based individual level assessment and intervention continues to remain a critical prevention strategy for reducing alcohol-exposed pregnancies.","keyphrases":["alcohol","pregnancy","preconception care"],"prmu":["P","P","M"]}
{"id":"Skeletal_Radiol-4-1-2226078","title":"Radiofrequency ablation of osseous metastases for the palliation of pain\n","text":"A number of different methods have been proposed for pain relief in cancer patients with bone metastases, each with different indications, contraindications and complications (systemic analgesics, bisphosphonates, antitumor chemotherapy, radiotherapy, systemic radio-isotopes, local surgery and vertebroplasty). The ideal treatment has to be fast, safe, effective and tolerable for the patient. CT-guided radiofrequency (RF) ablation may fulfill these criteria. Our experience in the treatment of 30 patients (34 lesions) with painful bone metastases using RF ablation was assessed. There was a significant decrease in the mean past-24-h Brief Pain Inventory (BPI) score for worst pain, for average pain and for pain interference during daily life (4.7, 4.8 and 5.3 units respectively) 4 and 8 weeks after treatment. There was a marked decrease (3 out of 30 patients 4 and 8 weeks after treatment) in the use of analgesics. CT-guided RF ablation appears to be effective for treatment of painful bone metastases.\nIntroduction\nPainful bone metastases are a common cause of morbidity in patients with metastatic cancer, especially when combined with possible neural compression and pathologic fractures. Several solid cancers are associated with bone involvement, most often, prostate and breast. Thirty to seventy percent of cancer patients develop bone metastases [1]. They indicate widespread disease. Treatment of local disease may reduce the pain of these patients who, in most cases, have a life expectancy of months. Such treatment must be fast, safe, effective and tolerable.\nA number of treatment methods are available that have variable success and complications. Radiation therapy is the preferred treatment in this setting, but other modalities such as chemotherapy, hormonal therapy, radiopharmaceutical therapy and surgery\u2014alone or in combination with non-steroid anti-inflammatory drugs (NSAIDs), opioids and adjuvant drugs\u2014are used for pain palliation [1\u20133].\nRadiofrequency (RF) ablation is a relatively new method for the treatment of painful bone metastases. Previously, tumour ablation was performed with percutaneous ethanol injection under CT guidance [4]. Administration of 95% ethanol was described in 25 terminally ill cancer patients with 27 bone lesions who had been unsuccessfully treated by radiation therapy and\/or chemotherapy.\nRadiofrequency ablation has been employed for the treatment of hepatocellular carcinoma (HCC), liver metastases, renal and lung tumours, as well as for the treatment of osteoid osteoma, for which it has become the treatment of choice [1\u20133]. Competing methods include chemical ablation (with ethanol or acetic acid) and thermal therapies, such as with laser, microwave, ultrasound and cryoablation [5]. The aim of this study was to demonstrate the effectiveness of RF ablation of bone metastases using CT guidance.\nMaterials and methods\nThirty patients were retrospectively identified. There were 19 men and 11 women. Their ages were between 47 and 91\u00a0years (mean \u00b1 standard deviation [SD]: 66.53\u2009\u00b1\u200910.56\u00a0years). The patients had bone metastases, which were treated with RF ablation under CT guidance, at our hospital, over a period of 4\u00a0years. All treated lesions were osteolytic with a combination of bone destruction and a soft tissue mass. In 26 there was a solitary lesion, and in 4 patients there were two such lesions, resulting in a total number of 34 metastases. Bone metastases were diagnosed by bone scintigraphy and spiral CT. The diagnosis was confirmed with a core biopsy obtained at the beginning of the procedure. Their topographical distribution and the originating primary malignancies are presented in Table\u00a01. In our study the most common treated metastases originated from colon cancer, which was probably related to the patient population treated at the oncology department of our hospital.\nTable\u00a01Bone metastasis classification, with regard to the primary malignant lesion and the site of the skeleton involvedSite of primary neoplasm originnSite of metastasisnColon13Pelvis15Breast7Ribs6Prostate2Sacrum5Lung4Femur3Renal2Spine3Thyroid1Scapula2Skin melanoma1Tibia 1Total30Total34\nLesion diameter was between 1 and 14\u00a0cm (mean\u2009\u00b1\u2009SD: 3.9\u2009\u00b1\u20092.6\u00a0cm). For sizes over 3\u00a0cm, two or more electrode placements were needed (with a maximum of five). Previously obtained imaging examinations were evaluated for lesion characteristics and feasibility of electrode positioning and ablation. Lesions located in proximity to the spinal cord and major nerves (less than 1\u00a0cm) were excluded from RF treatment. Patient selection criteria are summarised in Table\u00a02. The study was in accordance with the ethical principles of the Helsinki Declaration and informed consent was obtained in each case.\nTable\u00a02Criteria for the selection of patients to undergo radiofrequency ablationPatient selection criteriaBrief Pain Inventory (BPI) score above 4Lesions not responding to chemotherapy and\/or radiation therapy (completion of therapy at least 3 weeks before the radiofrequency ablation session)Chemotherapy-associated complications that halted this treatmentLesions adjacent to structures sensitive to irradiationPatients with life expectancy greater than 2\u00a0months who were not eligible for surgical treatment Patients who preferred this treatment over the other alternatives\nPhysical examination was performed by the oncologist and in collaboration with the radiologist performing the ablation. Pain was assessed with the Brief Pain Inventory (BPI) The use of analgesics was recorded the day before the procedure.\nBefore the procedure blood cell count and blood clotting analysis were performed. Minimal requirements were: platelet (PLT) count >50,000\/ml (normal range, 150,000\u2013350,000\/ml); prothrombin time (PT), international normalised ratio (INR) <1.3 (normal range, 0.8\u20131.2); and partial thromboplastin time (PTT) <34\u00a0s (normal range, 25\u201334\u00a0s). The procedure was performed under conscious sedation (administration of 3\u00a0mg of bromazepam PO and 50\u00a0mg of pethidine hydrochloric acid intramurally, 45\u00a0min prior to the procedure) and was trained in regular breathing and breath-holding (suspended respiration) before the procedure. He\/she was placed in the appropriate position (prone, supine, or lateral, depending on the site of the lesion) and a scan of the desired area with a 5-mm slice thickness was performed, using a Picker 5000\u00ae (Philips Medical Systems, Amsterdam, The Netherlands).\nAt least one of two staff radiologists with extensive experience in biopsies and tumour ablations was involved in all ablations.\nThe lesion\u2019s exact location and depth, in relation to the overlying skin, was determined on CT. The skin was then prepared with povidone iodine (10%) solution. Local anaesthesia (15\u00a0ml of 2% lidocaine hydrochloride solution) was administered.\nRadiofrequency ablation was performed with a RITA Model 1500\u00ae electrosurgical generator (RITA Medical Systems, Mountain View, CA, USA) and a seven-array, 2- to 3-cm multitined electrode for lesions smaller than 3\u00a0cm (20 out of 34), or a nine-array multitined electrode for larger lesions (14 out of 34). The electrode tip was inserted to approximately 1\u00a0cm from the centre of the target. The electrodes were then deployed slowly, taking into account the need to ablate the lesion\u2013bone interface. The net ablation time was ~15\u00a0min at an energy level of 90\u2013110\u00a0W, with the target goal temperature set to 80\u2013110\u00b0C. During the procedure the infusion port of the electrode was flushed with a 2% lidocaine hydrochloride solution in order to reduce patient discomfort and to decrease tissue overheating and vaporisation. The number of electrode placements, individual (per electrode) and total ablation times, the total energy delivered to the target and the lesion temperatures achieved were recorded.\nAfter each session a dual-phase spiral CT examination with intravenous contrast medium was performed in order to assess response, as confirmed by low lesion attenuation values and lack of contrast enhancement.\nPatients were hospitalised and observed for 24\u00a0h. Analgesics were administered if required. Before patient discharge the pain was re-evaluated with the BPI score. Post-ablation assessment was completed with telephone interview after one, four and eight week. The BPI score and the use of analgesics were recorded again.\nResults\nFor lesions smaller than 3\u00a0cm (20 out of 34), one placement was adequate, while for the remaining 14 out of 34 cases of lesions that were larger than 3\u00a0cm, two or more placements were required; one lesion sized 14\u00a0cm required five placements, accomplished in two sessions (since more deployments are required in larger lesions). The total procedure time ranged between 33 and 65\u00a0min (mean \u00b1 SD: 42\u2009\u00b1\u200911\u00a0min; Table\u00a03). There were no complications and post-treatment CT revealed a good response, as confirmed by low lesion attenuation values and a lack of contrast medium enhancement, consistent with necrosis. Post-procedural CT did not demonstrate any major complications (such as haemorrhage, thrombosis of neighbouring veins, or skin burns).\nTable\u00a03Lesion characteristics andtreatmentNumber of lesionsSize of lesion (cm)Number of electrode placementsTime of radiofrequency energy deposition (min)15<31553157427352816391841019410114515Total 34\nEleven patients reported no pain reduction during the first 24\u00a0h after the procedure and were treated with analgesics (opioids or an opioid\/NSAID combination). Nineteen of the 30 patients reported early pain reduction. In none of the patients was increased pain reported. Prior to the procedure, the mean past-24-h BPI score for worst pain was 8.3 (on a numerical rating scale where 0 indicates no pain, and 10 indicates worst pain imaginable), mean pain was 6.8, and mean pain interference with daily life 7.5. These scores were reduced to 7.4, 4.7 and 6.5 24\u00a0h after the procedure, dropped to 4.9, 3.2 and 4.0 after 1\u00a0week, to 3.6, 2.00 and 2.2 after 4\u00a0weeks, and to 2.1, 1.4 and 1.7 after 8\u00a0weeks respectively. These results revealed a marked decrease in pain with subsequent improvement in the life quality for all participants since the first week post-treatment that lasted throughout the 8-week follow-up (Fig.\u00a01). For all time points, the mean past-24-hour BPI score for worst pain, for average pain and for pain interference in daily life improved in comparison to preprocedural symptoms (p\u2009<\u20090.001, paired t test; Figs.\u00a02, 3, 4).\nFig.\u00a01Mean Brief Pain Inventory (BPI) scores over time for patients treated with radiofrequency ablation. a Worst pain. b Average pain. c Interference of pain in everyday lifeFig.\u00a02Computed tomography scan a during and b after the radiofrequency session with the patient in a supine position. The electrode is deployed inside the metastatic lesion of the left acetabulum (metastasis from breast cancer). There is no enhancement after intravenous contrast media administration. Before the radiofrequency session, the average pain score was 7. During the first 24\u00a0h after radiofrequency, it was 4 and 1, 4 and 8\u00a0weeks later the average pain scores were 3, 2 and 0 respectivelyFig.\u00a03Computed tomography scan images at the level of the sacrum show a, b two different electrode placements within a soft tissue mass involving the sacrum and right iliac bone (metastasis from thyroid cancer). Before the radiofrequency session, the average pain score was 7. During the first 24\u00a0h after radiofrequency, it was 4 and 1, 4, and 8\u00a0weeks later the average pain scores were 3, 2 and 1 respectivelyFig.\u00a04Computed tomography scan image during the electrode placement inside a a metastatic lesion involving a rib (the primary neoplasm originates in the lung). Immediately after the radiofrequency session the contrast enhancement CT scan revealed a hypodense area (necrosis) inside the lesion. Before the radiofrequency session, the average pain score was 6. During the first 24\u00a0h after RF, it was 4, and 1, 4 and 8\u00a0weeks later the average pain scores were 2, 2 and 1 respectively\nPrior to RF ablation 27 out of 30 patients received opioids or an opioid\/NSAID combination. The remaining 3 patients received NSAIDs. One week after treatment 5 out of 30 patients were treated with a combination of NSAID\/low-dose opioids. Six out of thirty used NSAIDs. After 4 and 8\u00a0weeks only 3 out of 30 patients received any medication NSAIDs. One patient died during the 8-week follow-up for reasons not related to RF ablation.\nDiscussion\nIn patients with cancer, pain originating from bone metastases can be difficult to treat. A number of treatment options are available, including NSAIDs, opioids, and adjuvant drugs medications, radiation therapy, chemotherapy, hormonal therapy, radiopharmaceutical therapy, surgery and vertebroplasty.\nMedication represents the first line of treatment. NSAIDs and adjuvant drugs represent basic medication, potentially followed by NSAID\/low-dose opioid combinations, and finally increasing the opioid dose.\nRadiation therapy is another treatment option that may also be employed in pathologic or impending fractures [6]. Approximately 70% of patients undergoing radiation therapy will experience pain relief after between 2\u20133\u00a0days and up to 4\u00a0weeks after treatment. However, radiation therapy may also cause complications, mostly from damage of adjacent soft tissues [6].\nChemotherapy and radiopharmaceutical therapy are the only systemic methods of treatment that deal with even small foci of metastatic cells. However, not all metastases are sensitive to chemotherapeutic agents. Chemotherapy is often not well tolerated and is associated with complications. Radiopharmaceutical therapy can be more useful in treating patients with multifocal bone metastases. It has been reported that radiopharmaceuticals proved efficient in pain palliation mostly in bone metastases from breast, prostate and perhaps small cell lung cancer. As in the case of chemotherapy, all agents have advantages and possible side effects. Radiopharmaceutical agents vary with regard to the analgesic efficacy, duration of pain palliation, ability to repeat treatments, toxicity and expense [7].\nThe term \u201cablation\u201d refers to the local destruction of the tumour by the means of application of either chemical agents (ethanol, acetic acid), or local deposition of some form of energy (radiofrequency, laser, microwave, ultrasound and cryoablation). Image-guided RF ablation is currently used for the treatment of various tumours with good results. According to preliminary results by Dupuy et al. [8], RF ablation can provide palliative treatment for patients with painful osseous metastases. Later on, Callstrom et al. [9] reported results after treating 12 patients and concluded that this modality provides an effective and safe alternative method of pain palliation in patients with osteolytic metastases. A multicentre study involving 43 patients with painful osseous metastases was carried out by Goetz et al. [10] and showed again significant reduction of pain and decrease in the use of opioids, with only minor complications.\nThe proposed mechanisms by which RF ablation decreases pain may involve: pain transmission inhibition by destroying sensory nerve fibres in the periosteum and bone cortex; reduction of lesion volume with decreased stimulation of sensory nerve fibres; destruction of tumour cells that are producing nerve-stimulating cytokines (tumour necrosis factor-alpha [TNF-\u03b1], interleukins, etc.) and inhibition of osteoclast activity [11, 12].\nIn our patients, we observed a considerable reduction of pain and improvement of the quality of life, as measured by the BPI score.\nA decrease in the use of analgesic medications was notable in our series and possibly greater than others reported in previous studies [9, 10].\nThe reduction of the procedural time is limited by both the time needed to achieve the optimal target temperature, and the size of the lesion, because more than one deployment is required in larger bone metastases. Although a few patients reported mild discomfort during the ablation, none of the sessions was forced to stop owing to considerable patient distress. There were none of the possible adverse effects, including infection, haemorrhage, neurological complications, skin burns, or the so-called post-ablation syndrome (low-grade fevers \u2264100\u00b0F [37.8\u00b0C]), myalgias, and malaise for up to 1\u00a0week after the procedure). Pain reduction was fast and occurred within the first 24\u00a0h for some and during the first week in the majority of the patients. This appears to be a fairly well-tolerated procedure and the combination of conscious sedation and local anaesthesia is adequate for its needs.\nIn the spine, RF ablation may be contraindicated due to the close relationship with the spinal cord and nerve root. Vertebroplasty may be used for pain relief and stabilisation of osteolytic lesions. Pain relief occurs within hours or days (mean 24\u00a0h) of the procedure, sometimes after a transient worsening of pain [13]. Mechanical or thermal damage to the adjacent soft tissue from needle positioning or cement leakage are the potential complications.\nIn our study, there were no lesions threatening the stability of the spine.\nThere are two conflicting studies in the literature concerning the use of RF ablation in spinal metastases. One of them has demonstrated that the presence of cancellous or cortical bone between the lesion and the spinal canal can provide adequate safety for the procedure [14, 15]. Another study in an in vivo animal model, with the use of magnetic resonance imaging (MRI) and pathological evaluation, has demonstrated that the placement of the electrode against the posterior vertebral body wall resulted in damage of the spinal cord [16]. In most series, lesions within 1\u00a0cm of the spinal cord, lesions involving the posterior wall and lesions with cortical bone destruction with involvement of soft tissue were considered to represent contraindications to treatment [17]. All spinal metastases treated in our series involved the anterior part of the vertebral body.\nThe follow-up period for this study was 8\u00a0weeks, a period that we believed was sufficient to demonstrate that RFA provides effective palliation. There is, however, a need for randomised prospective studies, to evaluate the method and to compare it with other treatment modalities, such as radiation therapy. Continued follow-up is warranted to determine the long-term efficacy of this interventional approach.\nIn conclusion, imaged-guided RF ablation of painful bone metastases is promising. It appears to be effective, safe and well tolerated by patients.","keyphrases":["radiofrequency ablation","osseous metastases","pain","minimally invasive treatment"],"prmu":["P","P","P","M"]}
{"id":"Int_J_Hematol-4-1-2276240","title":"Treatment of invasive fungal infections in clinical practice: a multi-centre survey on customary dosing, treatment indications, efficacy and safety of voriconazole\n","text":"Invasive fungal infections are frequent and often deadly complications in patients with malignant hematological diseases. Voriconazole is a third generation triazole antifungal with broad activity against most clinically relevant fungal pathogens. Clinical practice often deviates from insights gained from controlled randomized trials. We conducted a multi-centre survey to evaluate efficacy, safety, treatment indications and dosing of voriconazole outside clinical trials. Patients receiving voriconazole were documented via electronic data capturing. An analysis was conducted after submission of 100 episodes from September 2004 to November 2005. Voriconazole was administered for suspected or proven invasive fungal infection (IFI) (57%), as empirical treatment in patients with fever of unknown origin (21%) and secondary (19%) as well as primary (3%) prophylaxis of IFI. Investigators\u2019 assessment of fungal infection often diverted from EORTC\/MSG 2002 criteria. A favorable response was reported in 61.4% for suspected or proven IFI and 52.4% for empirical treatment. Mortality was 15%, 26.7% of which was attributable to IFI. Breakthrough fungal infections occurred in four (21.1%) patients with voriconazole as secondary prophylaxis. Toxicity and adverse events comprised elevated liver enzymes and visual disturbances. Although indications frequently deviated from clinical evidence and legal approval, voriconazole showed efficacy and safety, comparable to major controlled clinical trials. Data from this survey demonstrate the difficulty of putting drugs to their approved use in IFI.\nIntroduction\nPatients with malignant hematological diseases receiving remission-induction chemotherapy are at a high risk of contracting severe and sometimes lethal invasive fungal infections, especially invasive pulmonary aspergillosis (IPA) [1\u20134].\nVoriconazole follows itraconazole as the second triazole-antifungal agent with an enhanced spectrum of activity against numerous clinically important fungi. This activity is achieved by inhibition of fungal cytochrome P450-mediated 14\u03b1-lanosterol demethylase, a key enzyme in ergosterol biosynthesis. Subsequent loss of ergosterol in the fungal cell wall and accumulation of 14\u03b1-methyl sterols are the primary mode of action of voriconazole.\nAmong the properties of voriconazole are in vitro fungistatic activity against most Candida spp., including those resistant to fluconazole [5\u20137], fungicidal activity against several Aspergillus spp. [8\u201310] as well as profound activity against Fusarium spp. [8\u201313], Scedosporium spp. [14], Cryptococcus spp. [7] and other rare yeasts, molds and dermatophytes, such as Trichosporon spp., Blastomyces spp., Histoplasma capsulatum and Coccidioides immitis [14, 15]. In a controlled clinical trial, voriconazole proved superior efficacy and improved survival when compared with amphotericin B deoxycholate in the treatment of invasive aspergillosis [16]. Voriconazole has been approved by the US Food and Drug Administration (FDA) and European Medicines Agency (EMEA) for treatment of invasive aspergillosis, invasive candidiasis in non-neutropenic patients, esophageal candidiasis as well as serious infections caused by Scedosporium apiospermum and Fusarium spp. in cases refractory to or intolerant of first line treatment [17]. Availability of oral (tablets, suspension) and intravenous preparations along with a favorable safety profile make voriconazole a popular drug among clinicians in cases of suspected or proven invasive fungal infection alike. Voriconazole has become a well-accepted treatment option in probable and proven IFI [18].\nControlled clinical trials are an imperative tool for testing drug efficacy and safety in an idealized study population. To allow comparison between clinical trials, the European Organisation for Research and Treatment of Cancer (EORTC) and the Mycoses Study Group (MSG) have established criteria for the diagnosis of IFIs [21]. These criteria classify IFIs as possible, probable, or proven according to diagnostic test results. Basically, these criteria demand major clinical signs in combination with host factors for a possible diagnosis. For a probable diagnosis, certain microbiological criteria (e.g. serum galactomannan) must be fulfilled, while a proven diagnosis demands histopathological findings or positive culture from a primary sterile site. However, trial results do not always translate into everyday clinical practice. We therefore conducted a multi-centre survey to evaluate efficacy and safety as well as common treatment indications and prescribed dosing of voriconazole outside clinical trials.\nMethods\nParticipating investigators were asked to document all patients receiving voriconazole via online electronic data capture. Data were collected retrospectively after treatment completion. Collected data comprised demographic information, underlying disease, indication for treatment, earlier antifungal treatment, risk factors for invasive fungal infection, clinical outcome (including results of diagnostic imaging, microbiology, lab results, vital signs and survival), occurrence of adverse events, concurrent medication, and evaluation of response. Treatment outcome was evaluated by the investigator. Data were monitored electronically and manually for plausibility and completeness and queries were raised to the investigators in cases that were unclear or incomplete.\nAn analysis was conducted after submission of 100 episodes. The objective of the analysis was the evaluation of drug safety and efficacy as well as dosing habits and indications for voriconazole therapy by means of descriptive analysis. All episodes were registered in the period from September 2004 to November 2005. Drug toxicities were evaluated applying common toxicity criteria (CTC) [19].\nResults\nOne-hundred patient courses from the hematological units of five different centers (43 from Frankfurt am Main, 42 from Oldenburg, 10 from Cologne, 4 from Frankfurt an der Oder and 2 from Mainz) were successfully enrolled in the survey. All relevant data concerning the survey objectives were present. The most frequent underlying disease was hematological malignancy (94%). A total of 15 patients underwent stem cell transplantation, 12 of these were allogeneic and 8 showed signs of acute or chronic graft-versus-host disease (GvHD). The five patients with other hematological diseases were two patients with aplastic anemia, two with myelodysplastic syndrome and one with osteomyelofibrosis. Almost half of the patients (N = 48) were isolated by means of reverse isolation, 27 patients had air conditioning with high-efficiency particulate air filters (HEPA). Only 14 patients were accommodated with no isolation at all (Table 1).\nTable 1Patient characteristics at baseline and subsequent voriconazole exposure (N = 100)Age (years)a\n59 (24\u201384)Female37 (37%)Mean weight in kg (\u00b1SD)76.2 (\u00b114.7)Underlying diseaseb\nAcute myeloid leukemia72 Acute lymphoblastic leukemia9 Low grade non-Hodgkin lymphoma4 Other hematological malignancy9 Other non-malignant hematological disease5 Solid tumors3Stem cell transplantation15 Unrelated donor8 Sibling donor4 Autologous3Graft-versus-host disease8Isolation Reverse isolation48 HEPAc\n27 No isolation14 LAF9 HEPAc + reverse isolation2Treatment indication Primary prophylaxis3 Empirical therapy21 Suspected or proven IFI57 Secondary prophylaxis19Days on voriconazole (\u00b1SD)26.7 (\u00b143.3)\naMedian\nbTwo patients with multiple cancers\ncHigh efficiency particulate air filter\nThe most common indication for voriconazole treatment was suspected or proven IFI by assessment of the investigator (N = 57), 21 patients received voriconazole as empirical treatment for an episode of fever of unknown origin (FUO) while considered at risk for an IFI. Other treatment indications were prophylaxis of IFI (N = 22), 19 of which were considered as secondary prophylaxis after an earlier episode of IFI. On average, treatment duration was 27 days (Table 1). Fifteen patients died during the observation period. Causes of death were severe sepsis with multi-organ failure (N = 6), progression of the underlying disease (N = 4), progression of IFI (N = 4) and severe arrhythmia with cardiac arrest (N = 1). The investigator attributed the lethal arrhythmia to hypokalemia several days after switching antifungal therapy from voriconazole to liposomal amphotericin B.\nFor the 57 patients receiving voriconazole for treatment of suspected or proven IFI, the lung was the most common site of infection (N = 55). Microbiological evidence was obtained in two cases of candidemia, one invasive pulmonary aspergillosis and one Geotrichum capitatum fungemia. All other patients had suspected invasive pulmonary aspergillosis by the investigator\u2019s assessment. Frequent risk factors were exposures to dust due to nearby construction sites (N = 47), leukopenia (N = 43), indwelling central venous catheters (N = 39), immunosuppressive therapy (N = 35) and mucositis (N = 25). The initial daily voriconazole dose averaged 6.8 mg\/kg. Almost half of the patients were treated orally from the beginning (N = 27). In 21 of the remaining 30 patients, therapy was later switched to oral administration. Treatment success was rated by the investigators as complete or partial response in 35 (61.4%) patients. Disease remained stable in another 11 (19.3%) patients. Progressive disease was diagnosed in the remaining 11 patients (19.3%). Fifteen (26.3%) patients were switched to a different antifungal class (Table 2).\nTable 2Characteristics of patients with suspected or proven IFI by assessment of the investigator (N = 57)Site of infectiona\nLung55 (96.5%) Blood (fungemia)2 (3.5%) Sinus2 (3.5%) CNS1 (1.8%) Liver1 (1.8%)Risk factors Diabetes mellitus4 (7%) HIV1 (1.8%) Leukopeniab\n43 (75.4%) Mucositis25 (43.9%) Prior IFI7 (12.28%) Central venous catheter39 (68.4%) Dust exposure47 (82.5%) Surgery1 (1.8%) Cytarabine14 (24.6%) Purine analogues7 (12.3%) Steroids10 (17.5%) Other immunosuppressant4 (7%)Days with fever (\u00b1SD)7.6 (\u00b16.54)Total days on antibiotic treatment (\u00b1SD)29.3 (\u00b118.30)Days on voriconazole (\u00b1SD)26.6 (\u00b126.76)Average initial voriconazole mg\/kg (range)6.8 (3.125 \u2013 13.559)Route of administration Oral only27 (47.4%) Switch to oral21 (36.8%) Switch to intravenous4 (7.0%)Outcomec\nComplete response19 (33.3%) Partial response16 (28.1%) Stable disease11 (19.3%) Progressive disease11 (19.3%)Switch to other antifungal15 (26.3%) Liposomal amphotericin B8 (14.0%) Caspofungin5 (8.8%) Other2 (3.5%)\naSuper additive because of patients with multiple infection sites\nbAs defined as less than 1,000 leukocytes\/\u03bcl\ncAs assessed by investigator\nAn evaluation of the investigators\u2019 adherence to EORTC\/MSG criteria showed a discrepancy between the softer criteria used in recent clinical trials [16, 20] and the official 2002 criteria [21]. While recent trials allowed the investigator to rate evidence as probable without microbiological findings, the official criteria demand at least one microbiological criterion. Thus, 35 of the 57 cases with suspected or proven IFI had probable IFI according to the softer criteria, but only four patients had probable IFI when applying the official EORTC\/MSG criteria. Independent of the applied edition of the EORTC\/MSG criteria, 19 patients did not meet the requirements for classification of evidence even as possible (Table 3).\nTable 3Evidence rating for patients with IFI suspected or proven by the investigator (N = 57)Investigator assessmentAccording to criteria from recent clinical trials [16, 20]According to EORTC\/MSG 2002 [21]Proven3\/57 (5.3)2\/57 (3.5)2\/57 (3.5)Probable26\/57 (45.6)35\/57 (61.4)4\/57 (7.0)Possible14\/57 (24.6)1\/57 (1.8)32\/57 (56.1)Not defined14\/57 (24.6)19\/57 (33.3)19\/57 (33.3)Values in parenthesis are in percentage\nIn the 21 patients, where voriconazole was given as empirical therapy, oral administration was preferred (N = 16). Average duration of fever was higher than in those with suspected or proven IFI, averaging 11.6 days. Treatment duration averaged 9.7 days, markedly shorter than the other subgroups. Eleven patients improved under empirical treatment, six were stable while four showed signs of progressive infection. There was a frequent switch of therapy to antifungals of different classes, i.e. liposomal amphotericin B (N = 3), caspofungin (N = 4) or their combination (N = 1) (Table 4).\nTable 4Voriconazole dosing and treatment outcomeEmpirical treatment (N = 21)Secondary prophylaxis (N = 19)Average initial voriconazole dose in mg\/kg (range)5.7 (2.469\u201310.870)5.7 (3.3\u20137.5)Route of administration Oral only16 (76.2%)18 (94.7%) Switch to oral2 (9.5%)0 Switch to intravenous\u20131 (5.3%)Days with fever (\u00b1SD)11.6 (\u00b111.17)n. a.Days on antibiotic treatment (\u00b1SD)29.8 (\u00b113.55)n. a.Days on voriconazole (\u00b1SD)9.7 (\u00b16.94)48.8 (\u00b184.31)Breakthrough IFIn. a.4 (21.1%)Outcomea\nProgressive disease4 (19.1%)n. a. Stable disease6 (28.6%)n. a. Partial response1 (4.8%)n. a. Complete response10 (47.6%)n. a.Switch to other antifungal8 (38.1%)7 (36.8%) Liposomal amphotericin B3 (14.3%)2 (10.1%) Caspofungin4 (19.1%)5 (26.3%) Liposomal amphotericin B + caspofungin1 (4.8%)0\nAlmost all patients on voriconazole as secondary prophylaxis (N = 19) received their treatment orally (N = 18). Mean treatment duration was 48.8 days. Four breakthrough IFIs were reported. Therapy was switched to other antifungals in seven cases, most often to caspofungin (N = 5) (Table 5).\nTable 5Other adverse events (N = 100)Visual disturbance6Nausea3Rash3Hallucination2Vomiting2Diarrhea1Drug fever1Edema1Adverse events at least possibly related to voriconazole by judgment of the investigator\nThe most frequent adverse events at least possibly related to voriconazole treatment by the investigators were visual disturbances (N = 6), nausea (N = 3) and rash (N = 3) (Table 6). At least one liver function test showed grade 3\u20134 toxicity, applying the common toxicity criteria in 15 cases, seven of which were attributed to voriconazole by the investigators. In this survey, gamma glutamyl transpeptidase was the most sensitive marker for voriconazole toxicity, showing a marked elevation in 12 cases of investigator attributed toxicity. However, no definite preference could be detected for any of the liver parameters assessed. None of the four patients with grade 3\u20134 nephrotoxicity were attributed to voriconazole (Table 6).\nTable 6Highest grade of treatment-emergent renal and hepatic adverse eventsCTC-toxicity [19]Grade-1 (%)Grade-2 (%)Grade-3 (%)Grade-4 (%)\nN evaluableLiver GOT (AST)29 (31.5)5 (5.4)2 (2.2)2 (2.2)926 (6.5)a\n1 (1.1)a\n1 (1.1)a\n1 (1.1)a\nGPT (ALT)20 (20.2)4 (4)1 (1)1 (1)993 (3)a\n3 (3)a\n0 (0)a\n1 (1)a\nGGT14 (15.7)14 (15.7)11 (12.4)0 (0)893 (3.4)a\n4 (4.5)a\n5 (5.6)a\n0 (0)a\nAP17 (19.5)4 (4.6)2 (2.3)0 (0)877 (8)a\n1 (1.2)a\n1 (1.2)a\n0 (0)a\nBilirubin10 (11)5 (5.5)4 (4.4)2 (2.2)913 (3.3)a\n3 (3.3)a\n3 (3.3)a\n1 (1.1)a\nKidney Creatinine11 (11)6 (6)2 (2)2 (2)1002 (2)a\n1 (1)a\n0 (0)a\n0 (0)a\nValues given in parenthesis are in percentage\naToxicity judged at least possibly attributable to voriconazole treatment by the investigator\nDiscussion\nIn our survey, voriconazole was efficacious in 61.4% of patients treated for suspected or proven IFI (by assessment of the investigator). Favorable response was documented for 52.4% of patients receiving voriconazole as empirical therapy. This observation is comparable to prior results. For the treatment of suspected or proven IFI, especially IPA, investigator assessed favorable results defined as complete or partial responses were obtained in 61.4% as compared to 52.8% in Herbrecht\u2019s trial [16]. In the empirical setting, favorable response was reported in 52.4%, while only a 26% overall response was reported previously [22]. However, the outcome evaluation was supposedly far more rigid in the clinical trials than the self-assessment of the registry contributors.\nWhen compared to earlier prospective controlled clinical trials [16, 22], adverse events were less common. Visual disturbances were only reported in 6% and hallucinations in 2% as compared to 44.8 and 6.6%, respectively, in aspergillosis [16] and to 21.9 and 4.3%, respectively, in febrile neutropenia [22]. However, adverse event reporting is naturally less stringent in a survey than in controlled clinical trials.\nAnalysis of survey data did not reveal yet unknown toxicities of voriconazole. Treatment-related grade 3 or 4 hepatotoxicity was observed in 15 patients. Further frequent adverse events were visual disturbances (6%), rash (3%) and nausea (3%).\nSignificant elevations of serum alkaline-phosphatase activities (>3\u00d7 baseline value) occurred in 2.9% compared to 2.3% of grade 3\u20134 CTC toxicity in our study, aspartate aminotransferase (>3\u00d7 baseline value) in 8.9 versus 4.4%, alanine aminotransferase (>3\u00d7 baseline value) in 7.2 versus 2% and bilirubin (\u22651.5\u00d7 baseline value) in 27.2 versus 6.6% [22].\nOur survey demonstrates that clinical reality regularly strays from approved indications and evidence based choice of treatment. A total of 43% of the reported patients received voriconazole prophylactically or empirically. One third of the 57 patients with voriconazole for treatment of actual IFI lacked sufficient evidence of IFI according to EORTC\/MSG criteria [21]. Thus, a rate of as much as 62% may be considered as off-label use. Furthermore, all randomized controlled clinical trials on voriconazole used the intravenous formulation as initial treatment. Despite this, 39.5% of patients with at least possible IFI were started on oral voriconazole. Looking at these figures, one could claim that only 23% of the patients reported were treated as approved by the German health administration and according to best scientific evidence. But does this mean patients were not treated in their best interest?\nThe difficulty of proving IFI remains a dilemma in choosing adequate treatment. There is still no sensitive and specific non-invasive method for detection of invasive pulmonary aspergillosis (IPA) [21, 23\u201325]. Controlled clinical trials on antifungal agents normally use the current EORTC\/MSG criteria with or without modifications, while the consensus committee clearly discourages from clinical decision-making based on their guideline [21]. Additionally, especially in palliative situations, long-term inpatient treatment with intravenous antifungals is often undesirable. Finally, no antifungal has yet been approved for secondary prophylaxis of IPA.\nIn the above clinical situations, voriconazole offers a flexible and tolerable treatment option. Still, the many difficulties in the diagnosis and treatment of invasive fungal infections should not be used as a carte blanche in clinical decision making. Physicians should strive towards establishing the most accurate diagnosis possible and then treat according to current evidence.","keyphrases":["fungal infection","voriconazole","antifungals","prophylaxis","aspergillosis","candidiasis","drug therapy"],"prmu":["P","P","P","P","P","P","R"]}
{"id":"Pediatr_Nephrol-3-1-1805045","title":"Recurrence of nephrotic syndrome\/focal segmental glomerulosclerosis following renal transplantation in children\n","text":"The incidence of recurrence of nephrotic syndrome\/focal segmental glomerulosclerosis (NS\/FSGS) is variable (~30%) The incidence of recurrence is less in African-Americans than in whites and Hispanics Graft survival rates are decreased in recipients with FSGS, especially if remission of the NS is not achieved in those with recurrence Although controversial, the use of living donor (LD) transplants are not contraindicated; however, obligatory heterozygote parental grafts with a podocin mutation should be used with caution Optimal treatment to induce a remission post-transplant has not been delineated Pre-transplant and\/or prophylactic post-transplant pre-operative plasmapheresis (PP) for high-risk patients\u2014especially those with recurrence in a previous graft\u2014may be promising An international multicenter controlled study is required to delineate the optimal approach to prevent and\/or treat the recurrence of NS\/FSGS\nIntroduction\nFocal segmental glomerulosclerosis (FSGS) is a histologic (pathologic) diagnosis that encompasses numerous specific clinical entities with potential varied etiologies. Steroid-resistant nephrotic syndrome (SRNS) is a specific clinical phenomenon that may vary histologically from minimal change nephrotic syndrome (MCNS) to FSGS, but it also may be associated with other specific histological diagnoses.\nThe most frequently acquired disease resulting in end-stage renal disease (ESRD) in children is FSGS, accounting for 8.3% of the chronic renal insufficiency (CRI) patients, 14.3% of the dialysis patients, and 11.4% of the transplant recipients included in the North American Pediatric Renal Transplant Cooperative Study (NAPRTCS) registry [1].\nIn 1972, Hoyer et al. [2] from Minnesota described the recurrence of NS in 3\/4 pediatric renal allograft recipients whose original disease was FSGS. At post-mortem examination, the lesion of FSGS was present in the grafts of two recipients [2]. An editorial accompanying the publication of this article in Lancet stated that \u201crenal transplantation may be contraindicated (in patients with FSGS) leaving dialysis as probably the only means of prolonging life\u201d [3].\nThe purpose of this teaching review article will be to address the factors that influence the incidence of recurrence of NS\/FSGS in renal allografts, establish the known risk factors for recurrence, and delineate the current preventive and therapeutic approaches to the recurrence of NS\/FSGS in pediatric renal allograft recipients.\nWhat is the incidence of recurrence of post-transplant NS\/FSGS?\nThe general consensus is that 30\u201340% of recipients whose original disease was NS\/FSGS will clinically manifest recurrence [4]. In most instances, recurrence is clinically apparent in the immediate post-transplant period, frequently with substantive proteinuria in the initial 24\u00a0hours following transplantation. However, the onset of clinical recurrence has been reported to occur at more than one year following transplantation. The delay in initial clinical manifestations may be coincident to the occurrence of acute tubular necrosis (ATN), which occurs more frequently in live donor (LD) and deceased donor (DD) recipients with FSGS [5]. It has been proposed that the \u201cimmediate fulminate recurrence of NS\/FSGS post-transplant likely predisposes such grafts to ATN and explains the increased incidence of ATN in this patient population [5].\nWhat is the incidence of subsequent recurrence in a patient who manifested clinical recurrence in a previous graft?\nRe-transplantation of a recipient with recurrence of NS\/FSGS in a previous graft has been associated with a ~80% recurrence rate [4]. Recently, a 100% recurrence rate has been reported in ten recipients of a second (6), third (3), and fourth (1) transplant [6]. Therefore, proceeding with a subsequent transplant in a patient who manifested previous recurrence has been discouraged. Recently, pre-operative plasmapheresis (PP) was successful in preventing clinical recurrence in 3\/6 patients whose initial graft was lost in association with clinical recurrence [7].\nWhat is the incidence of recurrence in recipients with mutations of genes that encode for podocin?\nThe association of mutations impacting on the structure of the foot processes in the region of the slit diaphragm of the glomerular basement membrane in patients with sporadic and familial NS has potentially delineated the pathophysiology of the NS in such patients [8]. Initial publications indicated that the incidence of recurrence of NS\/FSGS in patients with podocin mutations was similar to that reported for all patients with NS\/FSGS [9]. However, a recent analysis of 73 patients with either homozygous\/compound heterozygous (65) or heterozygous (8) mutations revealed a 7.7% incidence in the former and a 62.5% incidence in the latter recipients [10]. If these data are confirmed, it would seem advantageous to test all potential recipients with ESRD secondary to NS\/FSGS for such mutations in order to delineate an optimal transplant strategy.\nDo patients with the glomerular tip lesion who receive a renal allograft manifest recurrence of the NS?\nHowie et al. [11] reported that 12 of 14 recipients whose native kidney manifested the glomerular tip lesion had clinical recurrence of the NS post-transplant. In all 12 recipients, the glomerular tip lesion was the only histologic abnormality on graft biopsy. These data support the concept that the glomerular tip lesion may be the earliest histologic abnormality in the NS\/FSGS spectrum of disease. Precise incidence and outcome data await subsequent reports.\nDoes race impact on the recurrence of NS\/FSGS?\nAlthough the disease process appears more virulent in African-American (AA) children with NS\/FSGS [12], it is interesting that the incidence of recurrence following transplantation in this ethnic population is decreased. At least three studies [5, 13, 14] have shown that the incidence of recurrence in the AA recipient was <50% of that in the Caucasian recipient. Hispanic recipients had an incidence similar to that of Caucasians. The precise reason for this disparity in incidence has not been delineated.\nHas the incidence of recurrence changed with the introduction of new immunosuppressive regimens?\nSince the initial publication in 1972 of Hoyer et al. [2], there has been a significant change in the available immunosuppressive agents and regimens to both prevent and treat rejections. Despite these regimens, which have substantially reduced the incidence of acute rejection episodes and markedly improved short-term allograft survival rates, the incidence of recurrent NS\/FSGS has remained unaffected [14]. Bartosh et al. [14] reviewed the NAPRTCS database for the period 1998\u20132003 and found the incidence of recurrence was 31%, which was similar to the incidence in the initial NAPRTCS report for the period 1987\u20131990 [4].\nWhat are the risk factors for the recurrence of NS\/FSGS?\nThree factors identified in the 1970s to be associated as risk factors for recurrence were: (1) the rapid progression to ESRD in a period of <3\u00a0years; (2) the presence of mesangial hypercellularity on biopsy; and (3) the age at onset of the clinical NS of >6\u00a0years. More recently, the presence of a circulating permeability factor [15], the use of induction therapy in the immunosuppressive regimen [16], and native kidney nephrectomy either prior to or at the time of transplantation [17] have been reported to be associated with an increased risk of recurrence.\nDoes the presence of an identified circulating factor predispose to the recurrence of NS\/FSGS?\nSavin et al. [15] showed that the presence of a permeability factor (Palb) with an activity of \u22650.50 was associated with a recurrence rate of 86% (6\/7), whereas an activity of <0.50 was associated with a recurrence rate of 17% (4\/23). Plasmapheresis was associated with both a reduction in the Palb activity and in urinary protein excretion.\nIs induction therapy a risk factor for the recurrence of NS\/FSGS?\nRaafat et al. [16] noted a recurrence rate of NS\/FSGS of 43% between 1968 and 1997. However, it was only 11% (1\/9) in those who did not receive induction, whereas it was 53% (15\/28) in those receiving some form of antilymphocyte serum (ALS). Conversely, Gagnodoux [17] reported an incidence of recurrence of 31% prior to 1985 when no induction therapy with antilymphocyte globulin\/antithymocyte globulin (ALG\/ATG) was used compared to 30% following the introduction of induction therapy. Both Sheth et al. [18] and Hubsch et al. [19] noted a significant increase in the incidence of recurrence (17.6% vs 37.5% and 38% vs 83%, respectively) with the use of anti-IL2 receptor antibody as induction therapy. In the most recent analysis of NAPRTCS for the period 1998\u20132003, Bartosh et al. [14] noted an overall incidence of recurrence of 31% (51\/165), which was similar to no induction (30%) or the use of polyclonal antibody (27%) or monoclonal antibody (33%). Therefore, advocating the restriction of induction therapy to reduce the incidence of recurrence awaits the performance of a multicenter prospective randomized controlled study.\nIs native nephrectomy a risk factor for the recurrence of NS\/FSGS?\nAnalysis of the recent NAPRTCS database [14] indicated a significant (p\u2009<\u20090.001) difference in the incidence of recurrence in recipients who had undergone prior native kidney nephrectomy (50%) compared to those whose native kidneys were not removed prior to or at the time of transplantation (22%). A possible explanation for this phenomenon was that the native kidneys provided a \u201csponge\u201d for any circulating putative antibody and that native nephrectomy facilitated a sufficient level of circulation of such antibodies which were deposited in the graft precipitating recurrence. Other reports [20] have come to the same conclusion.\nDoes the recurrence of NS\/FSGS impact on the allograft survival rate?\nBaum et al. [21] in 2002 and Bartosh et al. [14] in 2004 reviewed the NAPRTCS database, which indicated inferior long-term graft survival rates in both recipients of LD and DD grafts whose original disease was FSGS. Analysis of the UNOS database demonstrated a similar disadvantageous graft survival rate for recipients whose original disease was FSGS compared to recipients with other primary renal diseases [22].\nA consensus is that 50% of the grafts with the recurrence of NS\/FSGS are lost, presumably as a result of the recurrent pathologic process. However, data from Schachter and Harmon [23] noted that long-term survival rates significantly (p\u2009=\u20090.002) improved in those recipients with recurrence who achieved remission with some therapeutic intervention compared to those who failed to go into remission and had persistent NS.\nDoes age at the time of transplantation impact on graft survival in recipients with FSGS?\nAdolescent recipients (13\u201317\u00a0years of age) with FSGS have a significantly poorer graft survival rate with both LR and DD grafts compared to younger recipients (0\u201312\u00a0years of age) [21]. In actuality, the 0\u201312\u00a0year-old recipient DD graft survival rate was similar in the FSGS and no FSGS groups. The specific reason for this phenomenon is not known. However, Scientific Registry of Transplant Recipients (SRTR) data have consistently demonstrated that adolescent recipients have substantially poorer long-term graft survival rates, which approach that of recipients >65\u00a0years of age at transplantation [24].\nShould an LD graft be utilized in children with ESRD secondary to FSGS?\nAt least five reports from 1989 to 1999 [4, 25\u201328] concluded that recipients with FSGS had a greater risk of allograft failure from an LD graft than from a DD graft. A precise reason for this phenomenon was not delineated.\nIn 2001, Baum et al. [5] reviewed the NAPRTCS database and showed that the 5-year LD graft survival rate was 69% for recipients with FSGS compared to 82% with no FSGS (p\u2009<\u20090.001), whereas it was 60% and 67%, respectively, in the DD groups. The authors concluded that the long-term advantage of LD grafts was lost in recipients with FSGS.\nConversely, Huang et al. [22] proffered that, although the LD advantage was not observed in AA pediatric recipients, the authors recommend that LD transplantation be considered for all racial groups with FSGS.\nWith this potential excellent outcome with newer preventative and therapeutic regimens for recurrence to be detailed subsequently, the previous concerns regarding the use of an LD graft may not be appropriate. However, the recurrence of NS\/FSGS in two recipients with a homozygous podocin mutation who received an LD graft from their obligatory heterozygote mother indicates that the obligatory heterozygote parental donor may not be ideal [29, 30]. Similarly, the development of FSGS and ESRD in three donors [28, 31] at some point following donation also indicates that appropriate informed consent should be obtained prior to utilizing an LD graft in recipients with FSGS.\nWhat are the therapeutic options available to produce a remission following the recurrence of NS in recipients with FSGS?\nIn 1988, Laufer et al. [32] reported the remission of NS in two pediatric recipients following nine sessions of PP at 3 and 6\u00a0months post-transplant. Both recipients manifested prolonged ATN. This led to the general use of PP in patients with recurrence. However, no randomized control trial was ever performed and the rate of remission has been variable.\nIngulli et al. [33] in 1990 reported the success in inducing a remission with high-dose cyclosporine in three pediatric recipients. The rationale for the increased dosage was that hyperlipidemia bound the cyclosporine and higher doses were required to achieve adequate bioavailability. The precise dose and length of treatment with the \u201chigh-dose\u201d regimen were never delineated in a controlled trial. However, this report led to the general approach of using a higher maintenance dosage of cyclosporine once a remission was obtained.\nRecently, Raafat et al. [34] reported a recurrence rate of 67% (16\/24) in recipients with FSGS between 1991 and 2003. The oral dose of cyclosporine was increased to 24\u00a0mg\/kg\/day until either remission was achieved or renal toxicity was evident. Complete remission was achieved in 69% (11\/16) within 14\u00a0days to 19\u00a0months (10\/11 within 3\u00a0months) and 12% (2\/16) had a partial remission. Concomitant PP was administered to 7\/16 recipients with recurrence.\nCochat et al. [35] in 1993 introduced a regimen of PP and cyclophosphamide in three patients who remitted within 12\u201324\u00a0days. This regimen has been adopted by some centers, especially for patients who failed to remit with PP alone. However, again, no controlled trial has been performed.\nPlasma protein absorption was introduced by Dantal et al. [36] in 1994 for recipients with an identified circulating factor. Although proteineuria was reduced in the eight patients treated, it was permanent in only one patient following discontinuation of the procedure. Subsequent reports utilizing this technique in pediatric recipients are limited.\nRecently, Salomon et al. [37] reported an 82% (14\/17) remission rate utilizing IV Cyclosporine at 3\u00a0mg\/kg\/day to reach a cyclosporine level of 250\u2013350\u00a0mg\/dl. These data add to the potential utility of high-dose cyclosporine in inducing a remission following the recurrence of NS\/FSGS.\nWith the use of angiotensin-converting enzyme\/angiotensin receptor blocker (ACE\/ARB) therapy to reduce proteinuria in patients with various forms of renal disease, it would seem logical to extend the use of these agents to recipients with the recurrence of NS\/FSGS. However, there has been no controlled study of the use of these drugs either alone or as adjunctive to other therapeutic regimens.\nCan pre-operative PP reduce the risk of recurrence of NS\/FSGS?\nOhta et al. [38] in 2001 reported the recurrence of NS\/FSGS in 9\/21 grafts. Pre-operative PP was performed in 15 and recurrence developed in five, whereas 4\/6 without pre-operative PP developed recurrence. The authors concluded that pre-operative PP was effective in preventing the recurrence of NS\/FSGS.\nAt the International Pediatric Transplant Association (IPTA) Congress in August 2005, Rainthavorn et al. [39] from the UCLA reported a recurrence rate of 100% in three recipients of DD grafts who received one session of pre-operative PP, whereas it was 57% (4\/7) in recipients of LD grafts who received <5 pre-operative PP and 0% in three recipients who received >5 pre-transplant PP sessions. The authors concluded that >5 pre-operative PP sessions prevented the recurrence of NS\/FSGS.\nKawamura et al. [40], at the same IPTA congress, reported a recurrence rate of 15.4% (2\/13) in LD recipients who received 1\u20132 pre-operative PP sessions, as well as native kidney nephrectomy. Recurrence was evident at 6\u00a0days and 8\u00a0months post-transplant, respectively, and remitted with post-transplant PP.\nA recent publication involving primarily adult recipients (9\/10 were adult) utilized a combination of pre- and post-operative PP in high-risk patients\u2014six with recurrence in a prior graft and four with rapid progression to ESRD (<3\u00a0years) [7]. The LD recipients received eight courses of PP over a two-week period\u2014one week prior to and one week following transplantation. The DD recipients initiated PP within 24\u00a0hours of transplantation and continued for two\u00a0weeks post-transplant. There was no recurrence in the four recipients with rapid progression to ESRD and 3\/6 recipients with a recurrence in a prior graft had no recurrence.\nCertainly, the utilization of pre-operative and prophylactic post-operative PP appears promising in high-risk patients. Controlled multicenter trials are warranted to delineate the optimal preventative and therapeutic approaches.\nConclusions\nThe incidence of recurrence of nephrotic syndrome\/focal segmental glomerulosclerosis (NS\/FSGS) has remained at ~30% for the past four decades, despite the introduction of newer and more potent immunosuppressive regimens. Multiple risk factors have been identified to predispose or precipitate clinical recurrence. However, no prospective multicenter studies have validated the relationships. Mutations of the gene encoding for podocin may identify a population of patients with NS\/FSGS who are at a lower risk for clinical recurrence. Because of the potential for an increased incidence of recurrence and\/or graft loss, living donor (LD) grafts should be used with constraint. Graft survival rates in recipients with NS\/FSGS are decreased. However, if a remission can be induced, the outlook for long-term graft survival is substantially improved.\nVarious therapeutic regimens, including pre-operative plasmapheresis (PP), high-dose cyclosporine, IV cyclosporine, and adjunctive cyclophosphamide, have proven to be effective. However, there have been no multicenter controlled studies to delineate the optimal therapeutic approach. Recent use of pre-operative and\/or post-operative prophylactic PP to prevent the recurrence of NS\/FSGS is exciting. Controlled trials are required to validate efficacy and delineate a precise preventative regimen.\nQuestions (Answers appear following the references)The incidence of recurrence of FSGS is: \nComparable in whites and African-AmericansLess in whites than HispanicsLess with re-transplants than with primary graftsNot influenced by pre-transplant plasmapheresisBetween 30% and 40%The use of a deceased donor is mandatory in a patient with FSGS when: \nA living donor graft is not availableThe initial graft was lost as a result of recurrenceThe parental donor is not an obligatory podocin heterozygoteThe potential live-donor is a haplotype mismatchThe potential live-donor is a B-antigen mismatchGraft survival following the recurrence of NS in a patient with FSGS is dependent upon: \nA reduction in the magnitude of proteinuria to 3.0\u00a0g\/kg\/dayThe lack of histologic evidence of FSGS during the initial post-transplant monthInducing a complete remission of the NSThe immediate use of post-transplant plasmapheresisThe use of a one haplotype-matched live-related donorThe optimal approach to prevent the recurrence of NS in patients with FSGS: \nIncludes cyclophosphamide and plasmapheresisIncludes one\u00a0week of pre-transplant plasmapheresisIncludes post-operative tacrolimus and plasmapheresisIncludes post-operative sirolimus and plasmapheresisHas not been delineatedThe recurrence of NS in patients with congenital nephrotic syndrome: \nHas not been reported with the \u201cFinnish\u201d typeIs >80% in recipients with diffuse mesangial sclerosisIs uniform with Fin-major\/Fin-minor genotypeIs associated with the development of antinephrin antibodiesIs associated with anti-podocin antibodiesWhich of the following does not impact on the recurrence of NS in patients with FSGS: \nRaceNative kidney nephrectomyRecurrence in a prior graftPodocin mutationNone of the above","keyphrases":["plasmapheresis","nephrectomy","recurrent focal segmental glomerulosclerosis (fsgs)","immunosuppressive medications","long-term outcome"],"prmu":["P","P","R","M","R"]}
{"id":"Childs_Nerv_Syst-4-1-2367395","title":"Microsurgical third ventriculocisternostomy as an alternative to ETV: report of two cases\n","text":"Objective To describe a microsurgical alternative to endoscopic third ventriculocisternostomy.\nIntroduction\nIn the last two decades, endoscopic third ventriculocisternostomy (ETV) via a precoronal burr hole and transfrontal approach became a good therapeutic alternative to shunt placing and in non-communicating hydrocephalus, ETV has become the first choice of treatment [29]. Also, ETV is more and more offered to patients with shunt at the time of shunt malfunction or in case of slit-ventricle syndrome (SVS) as a means to become shunt independent. However, in a subpopulation of patients, ETV is technically not feasible due to small ventricular system or slit ventricles. Patients with such small ventricles and multiple shunt malfunctions possess a highly therapeutic challenge.\nOther means of internal shunting, as an alternative to ventriculoperitoneal or ventriculoatrial shunting, have been described in the decades before ETV became a routine procedure. Third ventriculocisternostomy (TVS) has been performed by subtemporal craniotomy [5], anterior subfrontal approach [6, 7, 24], Torkildsen ventriculocisternostomy [26, 27], microsurgical ventriculocisternostomy [17, 20], stereotactic ventriculocisternostomy [4, 13, 19], fluoroscopic ventriculocisternostomy [10\u201312], microsurgical opening of the lamina terminalis [21], reconstruction of or stent placement in the aqueduct [15], and endoscopic aqueductoplasty [22].\nTVS by lamina terminalis fenestration is still a routine procedure in aneurysm surgery for subarachnoid hemorrhage, especially in case of anterior communicating artery aneurysms. However, the possibility of a microsurgical TVS (MTV) for obstructive hydrocephalus seems to be lost to oblivion by the widespread use of ETV.\nIn this report, we describe two patients in whom an ETV could not be performed because of slit ventricles and who became shunt independent after MTV by a minimally invasive supraorbital approach.\nCase reports\nCase 1\nThis eight-year-old boy was born with an occipital meningocele and a thoracolumbar myelomeningocele. Both of these congenital abnormalities were operated upon 1\u00a0day after birth. Within several days, the patient developed a hydrocephalus which was treated with a ventriculoperitoneal shunt. One month later, a Chiari type II malformation was treated by a suboccipital craniotomy and a duraplasty. Since then, the patient experienced multiple drain infections and both proximal and distal drain dysfunctions. Ventriculoperitoneal shunting and ventriculoatrial shunting with different types of valves were implanted but none of them led to problem-free shunting, largely due to stiff slit ventricles. In March 2003, the patient experienced another drain dysfunction with an intracranial pressure (ICP) of 50\u00a0cm H2O while maintaining slit ventricles followed by infection necessitating external ventricular drainage and antibiotics treatment. In search for a more definitive treatment of his recurrent drain dysfunctions, we considered to perform an ETV. However, the very narrow slit-like lateral ventricles and third ventricle did not allow a safe execution of this procedure (Fig.\u00a01a\u2013c). As an alternative, a microsurgical fenestration of the lamina terminalis and Liliequist\u2019s membrane was performed via a right-sided eyebrow incision and a small supraorbital craniotomy (Fig.\u00a02). The postoperative course was uneventful. The patient did not experience any symptoms suspect for hydrocephalus or raised intracranial pressure during a follow-up of 3\u00a0years. MRI control examination did not reveal a significant increase in the size of the ventricular system (Fig.\u00a01d\u2013f).\nFig.\u00a01Case report 1. a, b Axial T1 MRI depicting very small slit-like lateral and third ventricles, c sagittal T2 MRI showing small third ventricle and lamina terminalis, d, e postoperative axial T1 MRI showing minimal increase in size of the lateral ventricles; f postoperative sagittal T2 MRI shows a minimal increase of the third ventricle, but no flow void phenomenon at the lamina terminalisFig.\u00a02Postoperative photograph of case 1 showing the hardly visible scar of the eyebrow incision\nCase 2\nThis 16-year-old boy, born with a lumbosacral meningocele and shunt dependent from birth on, developed with 13\u00a0years of age an increasing overdrainage syndrome with positional headaches. In another hospital, many valve revisions were performed in order to solve this problem and to rule out underdrainage. The original differential pressure valve was exchanged over a period of 3\u00a0years for several different Delta valves and a Codman Medos programmable valve, with which practical all settings were tried. Also, a shunt explantation was tried but after initial improvement, this led to a secondary increase of headaches and papilledema within several weeks, after which again, a Codman Medos valve was inserted. From this procedure, we obtained the security that the patient was still shunt dependant at that age. A new ICP monitoring was performed in the other hospital which showed negative intracranial pressures in upright sitting position and an ICP of 5\u201310\u00a0cm H2O in the supine position. The headache continued, even seemed to increase and became also chronic with positional increase of headaches in the upright position. The largest problem, however, became the frequent absenteeism from school because of these headaches. Chronic consultation of a pediatric neurologist and conservative treatment measures did not alter the symptoms either.\nAt this time, he was referred to our department. As a first measure, a PaediGav 9\/29 gravitational valve was applied because we had gained good experience with this type of valve but without any positive effect on the headaches. Although the patient did not improve after many shunt upgrading, it was felt that his headaches most likely were to be attributed to chronic overdrainage. Therefore, we sought for a means to have the patient shunt independent (after proven shunt dependency at the time). Because an ETV was considered to be impossible, a right supraorbital craniotomy with a lamina terminalis fenestration and fenestration of Liliequist\u2019s membrane was executed and the shunt was completely removed, except for the ventricular catheter that was fixated and therefore left behind. Again, no significant change of the headache could be observed. A 24-h continuous ICP monitoring, performed after 6\u00a0weeks because of unchanged symptoms, revealed a normal ICP in supine and upright positions with a maximum nightly increase to 13\u00a0mm Hg. Two years after surgery, the patient is still shunt independent with chronic headaches, no papilledema (contrary to the first shunt explantation before MTV), and in a rehabilitation program. A computed tomography (CT) scan 1.5\u00a0years after surgery shows a minimal increase in the size of the ventricles (Fig. 3).\nFig.\u00a03Case report 2. a, b Preoperative CT scan depicting slit-like ventricles; c, d postoperative CT scan with minimal increase of ventricle size and a residual ventricular catheter that could not be removed\nDiscussion\nThere are many complications related to shunt dependency. In 10.9\u201329% of cases complications (other than mechanical obstruction) occur, the majority of which is a shunt infection [9, 14, 18]. Mechanical complications in shunts including late shunt malfunction may even occur in up to 45.9% in the first postoperative year and up to 81% of patients at 12\u00a0years follow-up [8, 9, 18]. Also, the long-term shunt-related mortality rate of the shunt-treated patient, despite many improvements in shunt technique, diagnostics, and follow-up, remains high (2.9% to 12.4% at 10\u00a0years follow-up) [10, 16, 30]. Therefore, the ultimate goal in hydrocephalus therapy is to get the patient shunt free.\nSince the first experience by Guiot [10], transcortical or transventricular endoscopic third ventriculocisternostomy has proved to be a valid alternative in the treatment of obstructive hydrocephalus. Also, several studies have shown that ETV is an effective treatment in patients with obstructive hydrocephalus who had undergone previous shunting procedures [1, 23, 25]. It can provide a definitive cure for patients with difficult-to-manage shunt dysfunction.\nThe potential advantages of ETV over conventional shunting are clear. However, ETV requires a certain degree of lateral and third ventricle dilatation to be performed safely. In case of small ventricles, the potential risks of this procedure (injuring fornix, thalamostriate or internal cerebral veins, hypothalamus) outweigh the advantages. One might consider to enlarge the ventricles and, thus, to facilitate an ETV. This has been safely and successfully performed by Butler and Khan [2] with an external ventricular drain and by Chernov et al. [3] by using a programmable valve of which the pressure was increased in a stepwise fashion. In our department, we have practised both techniques for ventricular dilatation successfully, but we also encountered serious complications from this strategy.\nIn both cases described here, we already knew from previous shunt malfunctions that the ventricles hardly dilate with increased intracranial pressure. A shunt revision in case 1 for proximal catheter obstruction only several months before the MTV revealed an intracranial pressure of 50\u00a0cm H2O with only minimal ventricular dilatation. Therefore, in our two patients, we opted to perform an open microsurgical TVS by opening the lamina terminalis via a small supraorbital craniotomy and did not consider achieving ventricular expansion. Both patients became shunt independent.\nOpen surgical TVS once was a popular therapeutic technique for the treatment of obstructive hydrocephalus before the advent of VP shunt systems. The idea of third ventricular communication into the chiasmatic cisterns was initially proposed by Dandy [7] in 1922 as a surgical procedure to alleviate obstructive hydrocephalus. It was first performed by Dandy via a subfrontal approach and frequently required the sacrifice of one optic nerve and elevating the chiasm. Later, Dandy [5] described the alternative subtemporal approach.\nIn 1936, Stookey and Scarff [24] described a subfrontal anterior approach for TVS through the lamina terminalis and the hypothalamus to the lumen of the third ventricle. Scarff [21] published a review of 527 patients subjected to this operation by both the subfrontal and subtemporal procedures, as reported by many authors. The over-all operative mortality was 15% and the permanent arrest of the hydrocephalus was obtained in 70% of the survivors.\nOpen surgical TVS for obstructive hydrocephalus was then abandoned due to better and reliable shunting systems and because the advent of endoscopic techniques of this procedure is not reported to be in widespread use. Nevertheless, it remained a routine procedure during aneurysm surgery in order to get more slack ventricles and to prevent, or decrease the number of, posthemorrhagic hydrocephalus. To the best of our knowledge, MTV, by opening the lamina terminalis for the treatment of hydrocephalus, was last reported in 1988. In this report from Reddy et al. [17], five shunted patients with aqueduct stenosis and slit ventricle syndrome described are treated successfully with MTV via opening of the lamina terminalis. The lamina terminalis was approached in all patients via a classic pterional craniotomy. Since then, reports on ETV dominate the literature.\nOperative technique\nMany neurosurgeons consider craniotomy and MTV too large and risky a procedure. However, the procedure described in this report is a truly minimally invasive procedure. The operation time is about 1\u00a0h, the wound and scar are minimal, recuperation is very fast, and the potential risks are very low. One might even suggest that this procedure is safer than an ETV because it has an extracerebral approach, in contrast to ETV, and that surgery is better controlled with more opportunities for hemostasis.\nThe supraorbital craniotomy with eyebrow incision has been described previously [28]. The Sylvian fissure needs not to be dissected. The ipsilateral optic nerve and the optic chiasm, the ICA and the ipsilateral ACA as well as the ACoA are exposed after which the lamina terminalis is reached. Lamina terminals fenestration is a standard procedure easily performed with the bipolar forceps or an arachnoid diamond knife. This, however, is not sufficient. It is essential to open Liliequist\u2019s membrane to allow free and sufficient CSF communication between the posterior fossa and the suprasellar and frontobasal cisterns.\nRecovery of this operative procedure is fast and without major side-effects. Usually, hospitalization time is 2 to 3\u00a0days.\nConclusion\nIn conclusion, open microsurgical third ventriculocisternostomy by opening the lamina terminalis should be kept in mind as an alternative technique for treating patients with multiple shunt failures and small ventricles not accessible for an endoscopic approach in an effort to make these patients shunt independent.","keyphrases":["third ventriculocisternostomy","lamina terminalis","minimally invasive","supraorbital approach","microsurgery","neuroendoscopy"],"prmu":["P","P","P","P","U","U"]}
{"id":"Eur_J_Pediatr-3-1-1829442","title":"Effect of renal Doppler ultrasound on the detection of nutcracker syndrome in children with hematuria\n","text":"To assess the detection rate of nutcracker syndrome in children with isolated hematuria, renal Doppler ultrasound examinations were routinely performed on 216 consecutive children (176 microscopic hematuria and 40 gross hematuria). Renal Doppler ultrasound was also performed on 32 healthy normal children. The peak velocity (PV) was measured at the hilar portion of the left renal vein (LRV) and at the LRV between the aorta and the superior mesenteric artery. The PV at the aortomesenteric portion (P=0.003) and the PV ratios of the LRV (P=0.003) were significantly higher in children with hematuria than in normal children, while the PV at the hilar portion was not different. If a PV ratio of the LRV of at least 4.1 (the cut-off level set at the mean \u00b12 SD of the value for the normal children) was defined as abnormal, 72 cases (33.3%) in children with hematuria and no cases in normal children were diagnosed as having nutcracker syndrome. The prevalence of nutcracker syndrome is relatively high in children with isolated hematuria, and the inclusion of renal Doppler ultrasound as a screening examination has a substantial effect on the detection of nutcracker syndrome.\nIntroduction\nHematuria is a common urinary abnormality with a prevalence of 0.5\u20132.0% among school-aged children [5]. In South Korea, mass urinary screening was started in school-aged children in 1998, and therefore the incidence of patients with asymptomatic microscopic hematuria has been increasing. When routine diagnostic tests for hematuria are used, approximately 40% of children with isolated hematuria are classified as being idiopathic [14, 17].\nThe prevalence of nutcracker syndrome in children with hematuria is unknown, because evaluation of nutcracker syndrome has not been routinely performed in children with isolated hematuria. This might be due to the absence of both reliable and easily applicable diagnostic methods in diagnosing nutcracker syndrome in children. Recently, however, renal Doppler ultrasound has been developed to diagnose nutcracker syndrome, and it is now possible to apply this diagnostic method to children [10\u201312].\nTherefore, we carried out a prospective study of routine renal Doppler ultrasound in children presenting with isolated hematuria at our Nephrology Unit. The main aim was to assess the detection rate of nutcracker syndrome by renal Doppler ultrasound as a screening examination.\nMethods\nPatients\nAll consecutive 221 children with isolated hematuria (gross and microscopic) who visited our Nephrology Unit between 1 January 2002 and 31 December 2004 were routinely offered a renal Doppler ultrasound to detect cases of nutcracker syndrome in addition to a complete blood cell count, electrolytes, biochemistry, coagulation profile, serology for hepatitis B, antinuclear antibody, anti-streptococcal O titer, complement C3 and C4, urine culture, urinary calcium\/creatinine, two-dimensional ultrasonography and excretory urography.\nHematuria was defined as the presence of at least five red blood cells (RBCs) in a centrifuged specimen. A urine sample from each parent and sibling was examined for hematuria, and a family history of urolithiasis was considered positive if a sibling, parent, grandparent, or parental sibling had a history of renal-stone disease. We excluded proteinuria by urinalysis in most patients, but also collected 24-h urine collections if proteinuria was suspected by urinalysis in some patients with gross hematuria. We collected 24-h calcium and creatinine if the urinary calcium\/creatinine ratio was more than 0.2. Hypercalciuria was defined when 24-h calcium excretion was more than 4\u00a0mg\/kg. Children with a documented urinary tract infection (n=2), Henoch-Schoenlein purpura (n=1), and systemic lupus erythematosus (n=2) were excluded, because the causes of hematuria were evident in these patients.\nTherefore, 216 patients were included in this study: 176 showed microscopic hematuria on several examinations (170 through mass urinary screening and 6 through routine urinalysis during admission due to other diseases) and 40 gross hematuria. The hematuria was intermittent in 19 patients and permanent in 197. Blood pressure was normal in all patients. Also, 32 age- and sex-matched normal healthy children with no evidence of renal disease or other chronic diseases were selected for comparison. This study was approved by the institutional review board and the research ethics committee of Yonsei Severance Hospital.\nRenal Doppler ultrasound\nRenal Doppler ultrasound was performed at the first visit by one experienced radiologist, not knowing whether the subjects had hematuria or were control subjects. A HDI 5000 sonography system with 5- to 8- and 4- to 6-MHz convex transducers (Philips, Ultrasound, Bothell, Wash.) was used, and the Doppler spectrum could be successfully obtained in most cases, except in one with a retroaortic left renal vein.\nAfter the patients had fasted for 6 to 8\u00a0h, renal Doppler ultrasound was performed with the patients in the supine position. Peak velocity (PV) was measured in the transverse plane at two points in the LRV, one at the lateral portion of the LRV near the hilum (PV1) and the other where the LRV courses between the aorta and the superior mesenteric artery (aortomesenteric portion, PV2).\nDoppler spectra of the LRV at the hilum were obtained with the transducer placed on the middle of the upper abdomen. Doppler spectra of the LRV at the aortomesenteric angle were obtained with the transducer placed on the right or left subcostal area for keeping the Doppler angle of the LRV less than 60\u00b0. It was relatively easy to obtain the PV at the hilar portion of the LRV with the Doppler angle of less than 60\u00b0, but there were some patients in whom the measurements of the PV at or beyond the aortomesenteric portion of the LRV were somewhat difficult with the Doppler angle of 60\u00b0. The median Doppler angle was 60\u00b0 (range 32\u201366\u00b0) at the hilar portion and 60\u00b0 (range 56\u201370\u00b0) at the aortomesenteric portion of the LRV. Ratios of the PV of the LRV between the two portions (Aortomesenteric PV\/Hilar PV, PV2\/PV1) were calculated.\nStatistical analysis\nStatistical analysis was performed with Student\u2019s t-test and chi-square test, using SPSS for Windows (version 11.0). The mean \u00b12 SD of PV2\/PV1 ratios was calculated for normal controls and was used as the cut-off value for diagnosing nutcracker syndrome. A P value of 0.05 or less was defined as significant.\nResults\nFigure\u00a01 shows Doppler images of a patient with nutcracker syndrome (a, b) and a normal child (c, d). Color Doppler imaging of all LRVs showed blood flow from the renal hilum to the aortomesenteric portion.\nFig.\u00a01Renal Doppler ultrasound of the left renal vein in a patient with nutcracker syndrome (a, b) and a normal child (c, d). (a) The peak velocity was 194.8\u00a0cm\/s in the aortomesenteric entrapped portion. (b) The peak velocity was 21.3\u00a0cm\/s in the hilar portion (c). The peak velocity was 49.3\u00a0cm\/s in the aortomesenteric portion. (d) The peak velocity was 23.8\u00a0cm\/s in the hilar portion\nDoppler velocimetric findings in children with hematuria and the control subjects are summarized in Table\u00a01. In children with hematuria, the median PV2 and PV1 were 75.4 (range 23.9\u2013288.0) cm\/s and 23.8 (range 7.3\u201340.7) cm\/s, respectively. In normal children, the median PV2 and PV1 were 60.2 (range 20\u2013133.7) cm\/s and 23.9 (range 10.7\u201336.8) cm\/s, respectively. There were no differences in the PV1, PV2, and PV2\/PV1 ratios between children with gross hematuria and those with microscopic hematuria. The median PV2\/PV1 ratio was 3.14 (range 1.18\u201316.44) in children with hematuria and 2.56 (range 1.21\u20133.87) in normal children. The PV2 (P=0.003) and PV2\/PV1 ratios (P=0.003) were significantly higher and had a wider distribution (Fig.\u00a02) in children with hematuria than in normal children. On the basis of these data, we set the cut-off levels of the PV2\/PV1 ratios for diagnosing nutcracker syndrome at the mean \u00b12 SD of the value for the normal children. The calculated cut-off levels were 4.1. When these Doppler sonographic diagnostic criteria were applied, 72 cases (33.3%) in children with hematuria and no cases in normal children were diagnosed as having nutcracker syndrome. In the 72 children with nutcracker syndrome, the PV2\/PV1 ratios were 6.82\u00b12.51 (median 6.46, range 4.1\u201316.44) as compared with 2.57\u00b10.74 (median 2.56, range 1.21\u20133.87) in normal children (P<0.0001) (Tables\u00a01, 2). Collateral vessels around the LRV were observed in two patients with nutcracker syndrome. The body mass index was significantly lower in the nutcracker group than in the non-nutcracker group (P=0.006) (Table\u00a02). \nTable\u00a01Renal Doppler findings and anthropometric parameters (median values and ranges)\u00a0Gross hematuria (n=40)Microhematuria (n=176)Total hematuria (n=216)Control (n=32)P-valueDoppler findings\u00a0PV at AM portion (PV2, cm\/s)72.2 (23.9\u2013207)76.2 (30.1\u2013288)75.4 (23.9\u2013288)60.2 (20\u2013133.7)0.003\u00a0PV at hilar portion (PV1, cm\/s)22.6 (11.5\u201340.7)24.1 (7.3\u201338.4)23.8 (7.3\u201340.7)23.9 (10.7\u201336.8)NS\u00a0PV2\/PV1 ratio3.06 (1.18\u201310.34)3.19 (1.2\u201316.44)3.14 (1.18\u201316.44)2.56 (1.21\u20133.87)0.003\u00a0PV ratio >4.1*9 (22.5%)63 (35.8%)72 (33%)0 (0%)<0.0001Anthropometric findings\u00a0Height (cm)132 (72\u2013183)134 (88\u2013171)133 (72\u2013183)133 (109\u2013164)NS\u00a0Weight (kg)31.5 (13.5\u201371)30 (12\u201374)30 (12\u201374)30 (18\u201356)NS\u00a0BSA (m2)1.09 (0.53\u20131.78)1.06 (0.54\u20131.86)1.06 (0.53\u20131.86)1.05 (0.74\u20131.6)NS\u00a0BMI (kg\/m2)17.24 (12.98\u201327.23)16.88 (13.13\u201325.97)17.05 (12.98\u201327.73)17.09 (15.15\u201320.82)NS*Data are number (%)PV= peak velocity, AM= aortomesenteric, BSA= body surface area, BMI= body mass index, and NS= not significant (P>0.05)There were no differences between gross and microscopic hematuriaFig.\u00a02The peak velocity ratios of the left renal vein in children with gross (GH) or microscopic hematuria (MH) and normal controlsTable\u00a02Renal Doppler findings and anthropometric parameters in 216 children with and without nutcracker syndrome\u00a0Nutcracker group (PV ratio >4.1: n=72)Non-nutcracker group (PV ratio <4.1: n=144)P-valueDoppler findings\u00a0PV at AM portion, PV2 (cm\/s)129 (69.8\u2013288)*63.9 (23.9\u2013113)<0.0001\u00a0PV at hilar portion, PV1 (cm\/s)20.1 (7.3\u201330.1)26.3 (14.2\u201340.7)<0.0001\u00a0PV2\/PV1 ratio6.46 (4.1\u201316.44)2.45 (1.18\u20134.0)<0.0001Anthropometric findings\u00a0Height (cm)137 (72\u2013183)132 (90\u2013171)0.031\u00a0Weight (kg)32 (12\u201367)30 (13\u201374)NS\u00a0BSA (m2)1.1 (0.53\u20131.78)1.05 (0.58\u20131.86)NS\u00a0BMI (kg\/m2)16.12 (13.42\u201327.01)17.24 (12.98\u201327.73)0.006*Median and rangesPV= peak velocity, AM= aortomesenteric, BSA= body surface area, BMI= body mass index, and NS= not significant (P>0.05)\nClinical characteristics and diagnoses of 216 children with hematuria with or without nutcracker syndrome are listed in Table\u00a03. The median age and gender did not differ between the two groups. The incidence of gross hematuria, abdominal or flank pain and a family history of urolithiasis were also similar in both groups. Nutcracker syndrome was present in 60 of the 149 children (40%) in whom no other explanation for hematuria was ascertained, and it was also combined with other causes of hematuria in 12 patients. Among the children with nutcracker syndrome, renal biopsy findings were normal in three, and one had IgA nephropathy. \nTable\u00a03Clinical characteristics and diagnoses of 216 children with hematuria with and without nutcracker syndrome\u00a0PV ratios of the LRVNutcracker group (PV ratio >4.1: n=72)Non-nutcracker group (PV ratio <4.1: n=144)Clinical characteristics*\u00a0Age (years)9 (2\u201316)9 (3\u201315)\u00a0Sex (M\/F)37:3576:68\u00a0Gross hematuria**931\u00a0Abdominal or flank pain1117\u00a0Familial history of urolithiasis410Clinical diagnoses\u2020\u00a0IgA nephropathy1 (1)1 (1)\u00a0Thin GBM disease5 (5)\u00a0Hereditary nephritis with deafness2\u00a0Proliferative glomerulonephritis1 (1)\u00a0Postinfectious glomerulonephritis4\u00a0Familial hematuria320\u00a0Cystitis3\u00a0Trauma1\u00a0Hypercalciuria818\u00a0Hematuria unexplained60 (3)\u202189 (5)\u2021*There were no differences between the two groups (**P=0.107)\u2020Numbers in parenthesis indicate the number of diagnoses confirmed by renal biopsy\u2021Results of renal biopsies were normal\nThe results of the diagnostic studies are included in Table\u00a04. Complete blood count, electrolytes, biochemistry, and the coagulation profile were normal in all patients. Positive antinuclear antibody was detected in 13 patients (1:40 weak positive), but repeat testing and anti-ds DNA showed no evidence of autoimmune diseases. Serum C3 concentrations were low (more than 10% below the lower limit of normal) in four patients. Hypercalciuria was found in 26 patients (12%). Ninety-five percent of the two-dimensional ultrasonography and 97% of the excretory urography showed no abnormal findings. Of the 11 abnormal ultrasounds (2 increased echogenecity, 3 thickened bladder wall, 1 mild dilatation of the renal pelvis, and 5 urolithiasis) and 7 urographic examinations (2 duplications of the ureter, 1 aberrant small right upper calyx, and 4 bifid collecting systems), no finding was clinically significant. The detection rate of nutcracker syndrome by renal Doppler ultrasound was highest among all diagnostic tests for hematuria. \nTable\u00a04Diagnostic studiesDiagnostic studyNormal result (no. of patients)Abnormal result (no. of patients)Positive rate (%)Complete blood count21600Electrolytes and biochemistry21600Coagulation profile21600C321241.9C421420.9Anti-streptococcal O titer1793717Antinuclear antibody203136Hepatitis B surface antigen21510.5Urine culture21600Urinary calcium\/creatinine1902612Renal\/bladder ultrasonography205115Excretory urography20973Renal Doppler ultrasound1447233\nDiscussion\nAccording to Stapleton et al. [14], the cause of isolated hematuria was defined in only 27% of their patients when routine diagnostic tests for hematuria were performed. However, they found that idiopathic hypercalciuria was the most common cause of hematuria with a prevalence of 26% in children with isolated hematuria by the routine evaluation of urinary calcium excretion. Nevertheless, the cause of hematuria in the remaining 47% of the patients could not be explained.\nIn the present study, we could not identify the cause of hematuria in 149 (69%) of the 216 patients when renal Doppler ultrasound was not performed, but 40 percent was found to have nutcracker syndrome by renal Doppler ultrasound. Thus, nutcracker syndrome was the most common cause of isolated hematuria in children without urinary tract infection or proteinuria in our study, and we speculate that childhood nutcracker syndrome might have been underestimated in the past.\nCompression of the LRV between the aorta and the SMA by a mechanism similar to that of a nutcracker has been recognized by anatomists for about 70\u00a0years [6, 8]. However, the prevalence of this syndrome is unknown in both children and adults with hematuria. Even current textbooks of pediatrics rarely describe this syndrome [1, 5]. It is known that nutcracker syndrome is an uncommon cause of gross or microscopic hematuria from non-glomerular origin and may cause orthostatic or variable degrees of proteinuria [4, 9, 12]. Hematuria is believed to be caused by LRV hypertension, which may result in minute rupture of thin-walled collateral veins into the calyceal fornix [9, 10].\nLeft renal venography with measurement of the pressure gradient between the inferior vena cava and LRV has been used to confirm nutcracker syndrome in selected cases [10]. However, this procedure is invasive, time-consuming, and therefore not indicated if there are no severe symptoms. In addition, there were no reliable and easily-applicable non-invasive diagnostic methods in diagnosing childhood nutcracker syndrome in the past. Consequently, it has been thought that screening nutcracker syndrome in all children with hematuria is unnecessary and irrational, and therefore, the prevalence of nutcracker syndrome has not been evaluated. Recently, however, the introduction of renal Doppler ultrasound with color flow has improved functional diagnostic capabilities by showing the blood flow velocity of the LRV [10\u201312]. Compression of the LRV between the aorta and the SMA may cause an increase in flow velocity in the LRV at the aortomesenteric angle, and this flow can be measured noninvasively by using Doppler ultrasound. Although anatomical definition of nutcracker syndrome is the compression between the aorta and the SMA, the changes (narrowing at the aortomesenteric portion and dilatation at the hilar portion) in diameters of LRV are variable according to the stage of nutcracker syndrome. This would be the reason that Doppler ultrasound had been developed to detect nutcracker syndrome in both adults and children.\nKim et al. [10] first developed and applied this Doppler technique to adults with nutcracker syndrome confirmed by venography, because the measurements of diameters of the LRV had not been satisfactory [3], and it had been suggested that dilatation in the proximal LRV shown at either 2-dimensional ultrasonography or computed tomography might be a normal variant in both children and adults [3, 18]. Takebayashi et al. also found that nutcracker syndrome can exist in either nondistended or distended LRV, and normal flow also can exist in distended LRV [15]. However, a combination of different parameters might improve the correlation with hematuria [10]. According to the Kim et al.'s study [10], Doppler sonographic findings were comparable to venographic findings, and the sensitivity and specificity of the PV2\/PV1 ratios were higher than those of the ratio of LRV diameters (sensitivity 80% vs. 69%; specificity 94% vs. 89%) in adults with nutcracker syndrome. Park et al. [12] had also reported that the mean PV2\/PV1 ratios in 26 normal children aged 6\u201316\u00a0years were 2.57\u00b10.70 (range 1.3\u20133.9), which were similar to those (2.57\u00b10.74, range 1.21\u20133.87) in our control subjects (4.8\u201314.5\u00a0years). Thus, the Doppler sonographic cut-off value for the PV2\/PV1 ratios in our study (4.1) was similar to that of Park et al. [12] (4.0), but lower than that in adults [10] (5.0). Kim et al. [10] reported the PV at the hilar portion in hematuric adults with nutcracker syndrome was significantly lower than that in normal adults, suggesting compression of the LRV at the aortomesenteric portion could change the flow pattern in the hilar portion of the LRV. Similarly, our study also demonstrated that the PV at the hilar portion in the nutcracker group is significantly lower than that of the non-nutcracker group, suggesting the entrapment of the LRV might also influence the PV at the hilar portion.\nWe agree with the previous reports describing the necessity of limited evaluations in children with microscopic hematuria, because the extensive work-up resulted in a low rate of positive findings [7]. Nevertheless, our data suggest that the detection rate of nutcracker syndrome by renal Doppler ultrasound was highest among many other diagnostic tests. Regarding treatment of nutcracker syndrome, surgery should be considered if the patients have severe symptoms [2], but it has been suggested that childhood nutcracker syndrome might be a transient phenomenon resolving spontaneously with time by physical development [13, 16]. Therefore, renal biopsy should be delayed in children with asymptomatic microscopic hematuria if nutcracker syndrome is detected. However, if hematuria persists despite the improvement of Doppler findings, renal biopsy might be necessary to detect other causes of hematuria.\nHowever, our study has some limitations: (1) We did not perform left renal venography because most of our patients had mild renal symptoms, such as asymptomatic microscopic hematuria, detected through mass urinary screening. It was felt to be unethical to perform such an invasive procedure in these patients. (2) Renal Doppler ultrasound was performed by one pediatric radiologist, and we could not show inter-observer reproducibility. (3) We did not perform the urinary RBC morphology as a routine examination. However, a current textbook of pediatrics [5] describes: \u201cAlthough urinary RBC morphology may be normal with non-glomerular bleeding and dysmorphic from glomerular bleeding, cell morphology does not reliably correlate with the site of hematuria.\u201d Kim et al. demonstrated the percentage of isomorphic RBC was considerably variable (range 2\u201390%) in patients with nutcracker syndrome confirmed by venography [10]. Despite these limitations, our data suggested that nutcracker syndrome might be one of the leading causes of isolated hematuria during childhood.","keyphrases":["renal doppler ultrasound","nutcracker syndrome","hematuria","screening examination"],"prmu":["P","P","P","P"]}
{"id":"Sleep_Breath-4-1-2211364","title":"Position paper on the use of mandibular advancement devices in adults with sleep-related breathing disorders\n","text":"Custom-made mandibular advancement devices are an effective treatment option for snoring, upper airway resistance syndrome, and obstructive sleep apnea (OSA). Evidence-based data indicates their efficacy, and international sleep societies recommend oral appliance (OA) therapy for patients with sleep-related breathing disorders. The following position paper by the German Society of Dental Sleep Medicine (DGZS) is to guide the interdisciplinary team (sleep physician and sleep disorder dentist) in detail when to prescribe oral appliances. This position paper supports the responsible use of OA as an effective treatment option for patients with sleep-related breathing disorders. The paper advises of proper indication regarding OSA severity, body mass index (BMI), and dentition. It emphasizes the interdisciplinary approach of oral appliance therapy and suggests treatment under the guidance of dentists trained in dental sleep medicine.\nIntroduction\nVarious options are available in the treatment of obstructive sleep-related breathing disorders (SRBD):\nContinuous or bi-level positive airway pressure (nCPAP\/biPAP)Mandibular advancement devices (MADs)Surgical proceduresOther procedures\nThe use of oral appliances in treating SRBD was first described in 1982 [3]. The first findings, which document the therapeutic effect of MADs, were presented in 1984 [8]. Of the many oral appliances designed for use in the treatment of SRBD, mandibular advancement devices have been the most intensely researched. Progress in the areas of dentistry and appliance technology has lead to fundamental modifications in the design of mandibular advancement devices.\nThe clinical effectiveness of this treatment option has been documented with varying degrees of evidence in numerous studies. The clinical importance of this form of therapy is constantly being reinforced by the gathering of improved data on the validity of this form of treatment. The effectiveness of mandibular advancement devices in the treatment of SRBD has been documented by the Cochrane Collaboration as the highest level of scientific evidence [5]. The clinical analysis has been reviewed and revised twice since Cochrane\u2019s first evaluation in 2002 [6, 7]. The German Society of Dental Sleep Medicine (DGZS) takes this opportunity to issue its position with regard to this form of therapy and to make recommendations.\nThe definition of mandibular advancement devices in the treatment of SRBD\nA system of laboratory fabricated custom-made adjustable splints manufactured for the individual patient and adjusted to fit the upper and lower jaws. These splints position the lower jaw, tongue, and additional structures more anteriorly, and increase the occlusal vertical dimension, resulting in an opening of the pharynx lumen, which reduces resistance in the airway and mechanically keeps the airway open during sleep.\nGuidelines and indication recommendations from professional sleep-medicine societies\nAs early as 1995, the American Academy of Sleep Medicine (AASM) published recommendations on the indication and application of oral appliances in the treatment of SRBD [1]. These were updated and stated more precisely in 2005 [2]. In 2001, the German Society of Pneumology (DGP) in co-operation with the German Sleep Society (DGSM) published the \u201cS2\u201d guidelines on the diagnosis and treatment of SRBD. These guidelines envisaged the use of mandibular advancement devices as a treatment option [4]. A new description of the significance of mandibular advancement devices was formulated by the \u201cTask Force Apnea of the DGSM\u201d in the year 2006 [9].\nBased on scientific appraisals and international guidelines, the DGZS recommends the use of mandibular advancement devices in the treatment of SRBD:\nAs an initial treatment for\nPrimary snoringUpper airway resistance syndromeMild to moderate obstructive sleep apnea [apnea hypopnea index (AHI) up to about 25\/h] with minor clinical symptoms with a sufficient number of retaining teeth and a body mass index (BMI) of up to 30\u00a0kg\/m2In cases of SRBD with an approximate AHI of over 25\/h, MADs can be used as an alternative after a previous therapeutic trial with nCPAP.Regardless of SRBD severity in patients with CPAP failure and CPAP noncompliance.A special dental, oral, and functional diagnosis is necessary to check the dental requirements for long-term use before prescribing MADs.The procedure should take into account the various interdisciplinary and intradisciplinary treatment flows, particularly the preliminary medical diagnosis and the subsequent medical follow-ups.This service should be delivered by dentists who have specialized training in the field of sleep medicine.\nThe DGZS supports the specialized training of dentists and physicians in the field of dental sleep medicine. Patients and referring physicians can find the addresses of certified practitioners on the DGZS website at http:\/\/www.dgzs.de.","keyphrases":["position paper","mandibular advancement device","snoring","upper airway resistance syndrome","obstructive sleep apnea","oral appliance","german society of dental sleep medicine","dental sleep medicine","dgzs"],"prmu":["P","P","P","P","P","P","P","P","P"]}
{"id":"Eur_J_Pediatr-3-1-2042510","title":"Risk factors for severe RSV-induced lower respiratory tract infection over four consecutive epidemics\n","text":"Variability in severity among different respiratory syncytial virus (RSV) seasons may influence hospital admission rates for RSV-induced lower respiratory tract infection (LRTI) in young children. The aim of the present study was to identify through logistic regression analysis, risk factors associated with higher likelihood to acquire RSV-induced LRTI, in children with symptoms severe enough to lead to hospital admission. Over four consecutive RSV seasons (2000\u20132004), records from children <4 years of age admitted for RSV-induced LRTI (\u201ccases\u201d) were compared with those from children with LRTI not due to RSV and not requiring hospitalization (\u201ccontrols\u201d). 145 \u201ccase-patients\u201d and 295 \u201ccontrol-patients\u201d were evaluated. Independent from the severity of the four epidemic seasons, seven predictors for hospitalization for RSV infection were found in the bivariate analysis: number of children in the family, chronological age at the onset of RSV season, birth weight and gestational age, birth order, daycare attendance, previous RSV infections. In the logistic regression analysis, only three predictors were detected: chronological age at the beginning of RSV season [aOR = 8.46; 95% CI:3.09\u201323.18]; birth weight category [aOR = 7.70; 95% CI:1.29\u201345.91]; birth order (aOR = 1.92; 95% CI:1.21\u20133.06). Conclusions: Independent from the RSV seasonality, specific host\/environmental factors can be used to identify children at greatest risk for hospitalization for RSV infection.\nIntroduction\nLower respiratory tract infection (LRTI) due to respiratory syncytial virus (RSV) is a major cause of morbidity in infants and young children [21]. Although certain risk factors have been traditionally associated with a higher likelihood to develop severe RSV-induced LRTI, the vast majority of children hospitalized for this condition are previously normal babies [3, 21]. Solid epidemiologic data indicate that prematurity is a consistently proven risk factor for severe LTRI induced by RSV [3, 21]. However, there are other independent risk factors, which show a significant variability among studies performed in different countries and even among different RSV seasons within the same country with alternation of minor and major epidemics [1, 3, 4, 21]. This was recently demonstrated in the \u201cOsservatorio study\u201d, which was designed to collect epidemiological data on RSV infection in two consecutive winter epidemics (2000\u20132002) in Italy [16]. This study showed that, because of differences in severity of the two RSV seasons, some of the risk factors were significant in one but not in the subsequent epidemics [15]. To further expand the RSV surveillance, the \u201cOsservatorio study\u201d was extended for two additional winter epidemics (2002\u20132004). The aim of the present study was to use the data collected to identify, by means of logistic regression analysis, risk factors associated with higher likelihood to acquire RSV-induced LRTI in children with symptoms severe enough to lead to hospital admission.\nMaterials and methods\nStudy design\nA case-control study was used to evaluate the risk factors associated with higher likelihood to acquire RSV-induced LRTI, severe enough to lead to hospital admission, over four consecutive winter epidemics in Italy (2000\u20132004). The pediatric centres involved (scattered over the Italian national territory), the study population, the surveillance, and the laboratory methods have been described elsewhere [13]. Briefly, all consecutive children, aged \u22644\u00a0years, referred to emergency departments for acute respiratory infection on one day of the week (Tuesday), between 8 a.m. and 2 p.m., during the \u201cexpected\u201d epidemiological seasons (October \u2013 April) were enrolled and tested for RSV determination. Children who had received immunoprophylaxis with palivizumab (\u22647, each year) were excluded. We expanded the age limit of the children to be enrolled over the \u201cclassic\u201d 24\u00a0months, to also evaluate possible predictors of RSV-induced LRTI in an older age group. Each study site determined the date of onset and the end of the local RSV season according to a definition previously developed by the Pediatric Investigators Collaborative Network on Infections in Canada (PICNIC) group [20]. The study protocol was approved by the local ethics board of each hospital and written informed consent was sought from parents or guardians of all children evaluated. Medical history and physical examination were recorded and evaluated and clinical assessment, including nasal secretion specimens for RSV detection, were obtained according to the usual protocol at each hospital. Nasal secretion samples were sent to the microbiology laboratory of each hospital to check for RSV by an immunoenzymatic test (TestPack RSV, Abbott, Italy) within 24\u00a0hours. Other respiratory viruses were not tested. All children were treated according to standardized protocols and the decision to admit a child to hospital for a respiratory illness was made by local non-study physicians responsible for each child\u2019s care and was not influenced by the child\u2019s participation in the study.\nCase definition and control selection\nDuring a given study year, children were considered eligible for the case-control study on the base of his\/her first LRTI during that year. \u201cCase of disease\u201d was defined as LRTI due to RSV, occurring in a child \u22644\u00a0years of age and requiring hospital admission. From the cases we excluded patients with LRTI induced by agents other than RSV or RSV-positive but not severe enough to require hospitalization. \u201cControls\u201d were selected from children who had LRTI which was with certainty not due to RSV (negativity on immunoenzymatic test performed on nasal secretions) and who did not require hospitalization. This control population was selected to determine both predictors of acquiring RSV infection and developing severe disease. All controls should have come from the same residence areas as case-patients. Since case patients ranged from 0 to 30\u00a0months of age, control patients aged \u226531\u00a0months were excluded from the total control population (N\u2009=\u20091064). Within the remaining group of controls (N\u2009=\u2009729), a sample (N\u2009=\u2009295) was randomly selected (simple random selection procedure via SPSS software) and included in the study to obtain a case-control ratio of 1:2.\nFor both case patients and controls evaluated in the four epidemic seasons, data related to the characteristics of the family (e.g. mother\u2019s age, number of children in the family, father\u2019s and mother\u2019s educational level and social condition score) and of the children (e.g. chronological age of the child at the beginning of RSV season, birth weight, birth order, gestational age, length of breast feeding) were categorized according to cut-offs partially derived from literature [5, 10, 12] and partially imposed from the original categorization recorded in the \u201cOsservatorio\u201d database. In addition, children were considered to be small for gestational age (SGA) if their weight at birth fell below the 10th percentile based on gestational age and sex according to Oken\u2019s method [15] and using the United States of America national reference.\nStatistical analysis and data definitions\nDescriptive statistics of the characteristics of the patients were performed and reported in terms of mean and standard deviation (SD) for the quantitative variables (chronological age, birth weight, gestational age, etc.) and in terms of absolute frequencies and percentages for the qualitative variables. Firstly, a bivariate analysis was performed and the comparison of quantitative variables between two groups of subjects (i.e. cases vs controls) was made calculating the likelihood ratio test (LR test), and reporting the bivariate odds ratio (OR) and 95% confidence interval (CI). Comparison of frequency data was performed by the chi-square test or by the Fisher\u2019s exact test in case of expected frequencies less than five.\nIn order to evaluate the role of different predictors in the association with the outcome (hospitalization for RSV infection), multiple logistic regression analysis (step-down strategy) was performed. Variables that were statistically significant in the bivariate analysis or that were considered a priori important for the outcome were entered in the model. The following predictors were considered: epidemic season, father\u2019s educational level, chronological age category at the beginning of RSV season, birth order, birth weight category, small for gestational age, daycare attendance, and previous RSV infections. The effect was expressed in terms of adjusted (a)OR and 95% CI and the LR test was performed. The models\u2019 predictive ability has been investigated by calculating the area under the ROC curve of the model (AUC). All tests were two-sided and a P value less than 0.05 was considered statistically significant. The statistical packages used were the \u201cStatistica release 6\u201d (StatSoft Corp., Tulsa, OK, USA) for bivariate analyses and the \u201cStata release 7\u201d (Stata Corporation, College Station, TX, USA) for multivariate analyses.\nResults\nPatient populations\nThere were 145 case-patients included in the study (plus three cases not evaluated because of missing values). There were 61 males (42.1%) and 84 females (57.9%), with a median age of 3.5\u00a0months and a median birth weight of 3,050 grams. Seventeen of the 145 (11.7%) were born preterm (at less than 36\u00a0weeks of gestational age), two had congenital heart conditions and five had a previous history of chronic lung disease.\nThere were 292 control-patients (plus three cases not evaluated because of missing values). There were 124 males (42.5%) and 168 females (57.5%), with a median age of 5\u00a0months. Eighteen were preterm infants (6.2%) and their birth weight was greater but not significantly different (mean\u2009=\u20092,310; median\u2009=\u20092,175 grams) from the preterm case-patients\u2019 birth weight (mean\u2009=\u20091,993; median\u2009=\u20091,817.5) (P\u2009=\u20090.21).\nRisk factors associated with a higher likelihood to acquire RSV-induced LRTI, severe enough to lead to hospital admission\nIn the bivariate analysis we considered several risk factors that could be associated with a higher likelihood to acquire RSV-induced LRTI, severe enough to lead to hospital admission.\nIn Table\u00a01 only the statistically significant risk factors associated with a higher likelihood to acquire RSV-induced LRTI, severe enough to lead to hospital admission, are reported. The bivariate analysis showed that, although the severity of RSV-induced LRTI with hospital admission was different during the four epidemics, a variety of other risk factors could be detected. The only demographic-social characteristic associated with severe RSV-induced LRTI requiring hospitalization was the number of children in the family \u22652. Geographical area, social condition level and mother\u2019s age category were not significant risk factors (not shown). The major predictors were the characteristics of the child itself as follows: low chronological age at the beginning of RSV season, being at least the second baby in the family, having a low birth weight (1,500\u20132,499 g), and a low gestational age (<33\u00a0weeks). In addition, the absence of daycare attendance was also a risk factor and higher risks could be seen in children without previous exposure to RSV infections. Having a birth weight small for the gestational age was also a risk factor but the role of the birth weight uncorrected for gestational age was a stronger predictor. Breast feeding had no significant role due to the small number of data available (n\u2009=\u2009138). Mother\u2019s educational level was available only in a very small subset of patients (n\u2009=\u200980) (not shown) and because of the strong association with father\u2019s educational level (not shown) (P\u2009<\u20090.0001) only this latter variable was considered in subsequent analysis. \nTable\u00a01Distribution of risk factors associated with a higher likelihood to acquire RSV-induced LRTI, severe enough to lead to hospital admission\u00a0Cases (N\u2009=\u2009145) N (%)Controls (N\u2009=\u2009292) N (%)P valueOR (95%CI)Epidemic season\u00a02003\u2013200429 (20.0%)56 (19.2%)Reference\u00a02002\u2013200350 (34.5%)68 (23.3%)1.42 (0.80\u20132.53)\u00a02001\u2013200241 (28.3%)122 (41.8%)0.65 (0.37\u20131\u201315)\u00a02000\u2013200125 (17.2%)46 (15.7%)P\u2009=\u20090.0241.05 (0.54\u20132.03)Epidemic period (October to April): Yes137 (94.6%)247 (84.6%)P\u2009=\u20090.0033.12 (1.43\u20136.81)Sex: Female84 (57.9%)168 (57.5%)P\u2009=\u20090.941.02 (0.68\u20131.52)Exposure to cigarette smoking: Yes (N\u2009=\u2009432)74 (51.7%)165 (57.1%)P\u2009=\u20090.290.81 (0.54\u20131.21)Family history of allergy: Yes (N\u2009=\u2009333)50 (44.6%)75 (33.9%)P\u2009=\u20090.061.57 (0.99\u20132.50)No. of children in the family: \u22652 (N\u2009=\u2009393)97 (72.9%)155 (59.6%)P\u2009=\u20090.0091.83 (1.16\u20132.88)Chronological age at the beginning of RSV season\u00a0\u226512\u00a0months6 (4.1%)48 (16.4%)P\u2009<\u20090.0001Reference\u00a06\u201311\u00a0months31 (21.4%)98 (33.6%)2.53 (0.99\u20136.48)\u00a03\u20135\u00a0months48 (33.1%)85 (29.1%)4.52 (1.80\u201311.33)\u00a0<3\u00a0months60 (41.4%)61 (20.9%)7.87 (3.13\u201319.75) Birth order: \u2265 2nd (N\u2009=\u2009420)95 (69.3%)151 (53.4%)P\u2009=\u20090.0021.98 (1.28\u20133.05)Birth weight category (N\u2009=\u2009430)\u00a0\u22652500 grams113 (80.1%)259 (92.2%)P=\u20090.013aReference\u00a01500\u20132499 grams24 (17.0%)28 (7.2%)1.96 (1.09\u20133.54)\u00a0<1500 grams4 (2.8%)2 (0.6%)4.58 (0.83\u201325.39)Gestational age category (N\u2009=\u2009430)\u00a0\u226536\u00a0weeks126 (88.1%)269 (93.7%)P\u2009=\u20090.023aReference\u00a033\u201335\u00a0weeks8 (5.6%)14 (4.9%)1.22 (0.50\u20132.98)\u00a0<33\u00a0weeks9 (6.3%)4 (1.4%)5.35 (1.45\u201315.89)SGA: Yes (N\u2009=\u2009426)36 (25.7%)63 (22.0%)P\u2009=\u20090.401.23 (0.76\u20131.96)Breast feeding (N\u2009=\u2009138)\u00a0None or <3\u00a0months25 (71.4%)68 (66.0%)P\u2009=\u20090.14aReference\u00a03\u20136\u00a0months9 (25.7%)20 (19.4%)1.22 (0.49\u20133.04)\u00a0\u22657\u00a0months1 (2.9%)15 (14.6%)0.18 (0.02\u20131.44)Daycare attendance: No (N\u2009=\u2009430)138 (97.9%)265 (78.0%)P\u2009=\u20090.0134.17 (1.23\u201314.08)Previous RSV infections: No (N\u2009=\u2009426)126 (88.7%)230 (81.0%)P\u2009=\u20090.0421.85 (1.02\u20133.36)P values in the table refer to chi-square test, unless otherwise specifieda Fisher\u2019s exact test\nIn the logistic regression analysis variables that were statistically significant in the bivariate analysis or that were considered a priori important for the outcome were entered in the model (see Table\u00a01). As reported in Table\u00a02 only three independent predictors were associated with severe RSV infection with hospitalization: a lower age category of the child, a lower birth weight category (<1500 grams), and birth order (being at least the second child in the family). The number of children in the family and gestational age were not entered in the logistic model because of the strong autocorrelation, respectively, with the variables birth order and birth weight. Indeed, the vast majority of children (84.6%) belonging to the lowest gestational age category (<33\u00a0weeks) and more than 60% of those belonging to the intermediate gestational age category (33\u201335\u00a0weeks) had a birth weight <2,500 grams. \nTable\u00a02Best fitting logistic regression model for independent predictors of severe RSV infection with hospitalization (N\u2009=\u2009419)Explanatory variables\u00a0aOR95% CIP valueaChronological age at the beginning of RSV season  (reference category: \u226512\u00a0months)6\u201311\u00a0months2.4670.879\u20136.925P<\u20090.00013\u20135\u00a0months4.1531.506\u201311.451<3\u00a0months8.4623.088\u201323.185Birth weight category (reference category: \u22652,500 grams)1,500\u20132,499 grams2.4331.289\u20134.595P\u2009=\u20090.0023<1,500 grams7.7011.292\u201345.907Birth order (reference category: first)\u2265 second1.9241.210\u20133.058P\u2009=\u20090.0049a Likelihood ratio testThe area under the ROC curve of the model was 0.712\nDiscussion\nEvaluating data collected over four consecutive years in Italy, we found that, in addition to the severity of the RSV epidemics in a specific year, a variety of risk factors were associated with a higher likelihood to acquire RSV-induced LRTI, severe enough to lead to hospital admission. The only condition related to demographic and social characteristics of the family was the number of children in the family (being at least the second baby was a \u201crisk-factor\u201d). Among factors related to the characteristics of the children, major predictors were chronological age of the child at the onset of RSV season, birth weight and gestational age category, birth order, daycare attendance and previous episodes of RSV infections. However, in the logistic regression analysis, only three predictors turned out to be significant: chronological age at the beginning of RSV season, birth weight category, and birth order.\nThese results only partially confirm previous observations regarding risk factors for severe RSV infection [1, 2, 8, 11, 14, 17].\nChronological age of the child at the beginning of RSV season is a known factor for severe RSV-induced LRTI [1, 10, 17\u201319]. Indeed, in our study population only six children (4.1%) with a chronological age at the beginning of the season >12\u00a0months (and none between 2 to 4\u00a0years) had RSV-induced LRTI severe enough to lead to hospital admission. Aside from the incomplete development of the lung structure and the small size of the conducting airways related to the young age, data also suggest that such infants are less likely to have RSV-neutralizing maternal antibody as compared with infants born after the peak of the RSV season [6].\nBirth weight was also an important risk factor in our study population. Low birth weight is often linked to low gestational age and there is no question that premature birth is, in general, associated with acute and chronic pulmonary morbidity [3, 5, 7\u201310, 14, 22]. Prematurity, in RSV-induced LRTI, can lead to increased need for and lengthened duration of hospital admission, intensive care, mechanical ventilation and supplemental oxygen, as well as increased mortality [1, 21]. Increased risk to develop severe RSV-induced LRTI may also be observed in the \u201cless premature\u201d infants, i.e. those with a gestational age of 32\u00a0weeks or greater, in the presence of other concomitant host\/environmental unfavourable conditions, such as day-care attendance, low chronological age at the onset of RSV season, presence of preschool age sibling(s), and low birth weight [3, 5, 10, 21].\nIn the present study, gestational age, a strong risk factor in the bivariate analysis, was not included in the logistic model because of the strong autocorrelation with birth weight. Indeed, a great proportion of children belonging to two lower gestational age categories (<33\u00a0weeks and 33\u201335\u00a0weeks) had a birth weight <2,500 grams.\nSGA has been only recently examined as an independent risk factor for hospitalization for RSV infection and, in agreement with the result of the present study, was found to be significant in a population of infants born at 33 through 35\u00a0weeks of gestation [10]. An interesting finding of the present report is that a low birth weight, corrected or uncorrected for gestational age, was a strong predictor for severe RSV-induced LRTI.\nThe third important predictor in our study population was birth order, i.e. being at least the second child in the family was found to be a risk factor for severe RSV-induced LRTI. A variety of studies demonstrated a trend toward higher numbers of RSV hospitalization rates in infants with any older siblings, including preschool age siblings (younger than 6\u00a0years old) who attend daycare and school age siblings (older than 3\u00a0years) [1, 3, 5\u20137, 10, 18, 19, 21].\nIn agreement with the majority of reports, other conditions related to demographic and social characteristics of the family (the number of children in the family) or of the environment (daycare attendance) were found in the bivariate analysis to be able to increase the likelihood to be hospitalized for RSV-induced LRTI [1, 3, 5\u20137, 10, 18, 19, 21]. While the number of children in the family was not entered in the logistic model because of the strong autocorrelation with the variable birth order, daycare attendance did not reach a significant value in the logistic regression analysis, probably because of the small number of children attending daycare in our study population [3 (2.1%) in the \u201ccase\u201d group and 24 (22.0%) in the \u201ccontrol\u201d group]. Despite other conditions, such as father\u2019s and mother\u2019s educational level, mother\u2019s age category, social conditions, breast feeding, family history of allergy and gender and exposure to cigarette smoking, that were not statistically relevant in our study population, we cannot exclude that other socio-economical and environmental factors might be important [1, 3, 5\u20137, 10, 18, 19, 21]. As shown for other Mediterranean countries [5], exposure to tobacco smoke at home has decreased during recent years in Italy as a result of anti-smoking campaigns and better health education, and this may explain our surprising results.\nDue to the low number of children with specific characteristics, other risk factors such as presence of chronic lung diseases or of congenital heart diseases, immunodeficiency, haematological malignancies, bone-marrow or organ transplants, and cystic fibrosis were not analysed. Moreover, since only in the second, third and fourth RSV season a limited number of children had received immunoprophylaxis with palivizumab, its protective effect could not be evaluated.\nIn summary, evaluating four RSV seasons, characterized by inter-annual alternation of minor and major epidemics, we found, in addition to prematurity, a limited number of other conditions which appear to influence the severity of RSV-induced LTRI in infants and young children. However, before translating to clinical practice these indications, other similar studies performed in different countries are required, including a broader number of children and covering other RSV epidemic seasons. This information should be valuable to better determine hospital resource management and individualize RSV prophylaxis programs in preterm and term infants.","keyphrases":["infants","respiratory syncytial virus infections","emergency rooms"],"prmu":["P","R","M"]}
{"id":"Purinergic_Signal-3-4-2072917","title":"MPP+-induced cytotoxicity in neuroblastoma cells: Antagonism and reversal by guanosine\n","text":"Guanosine exerts neuroprotective effects in the central nervous system. Apoptosis, a morphological form of programmed cell death, is implicated in the pathophysiology of Parkinson\u2019s disease (PD). MPP+, a dopaminergic neurotoxin, produces in vivo and in vitro cellular changes characteristic of PD, such as cytotoxicity, resulting in apoptosis. Undifferentiated human SH-SY5Y neuroblastoma cells had been used as an in vitro model of Parkinson\u2019s disease. We investigated if extracellular guanosine affected MPP+-induced cytotoxicity and examined the molecular mechanisms mediating its effects. Exposure of neuroblastoma cells to MPP+ (10 \u03bcM\u20135 mM for 24\u201372 h) induced DNA fragmentation in a time-dependent manner (p < 0.05). Administration of guanosine (100 \u03bcM) before, concomitantly with or, importantly, after the addition of MPP+ abolished MPP+-induced DNA fragmentation. Addition of MPP+ (500 \u03bcM) to cells increased caspase-3 activity over 72 h (p < 0.05), and this was abolished by pre- or co-treatment with guanosine. Exposure of cells to pertussis toxin prior to MPP+ eliminated the anti-apoptotic effect of guanosine, indicating that this effect is dependent on a Gi protein-coupled receptor, most likely the putative guanosine receptor. The protection by guanosine was also abolished by the selective inhibitor of the enzyme PI-3-K\/Akt\/PKB (LY294002), confirming that this pathway plays a decisive role in this effect of guanosine. Neither MPP+ nor guanosine had any significant effect on \u03b1-synuclein expression. Thus, guanosine antagonizes and reverses MPP+-induced cytotoxicity of neuroblastoma cells via activation of the cell survival pathway, PI-3-K\/Akt\/PKB. Our results suggest that guanosine may be an effective pharmacological intervention in PD.\nIntroduction\nParkinson\u2019s disease (PD) is a progressive neurodegenerative disorder caused primarily by selective degeneration of dopaminergic neurons in the substantia nigra pars compacta [1, 2]. Apoptosis, a morphological form of programmed cell death [3], has been implicated in the pathophysiology of PD [4\u20136]. The biochemical pathways that mediate apoptosis may be initiated by extrinsic (e.g. activation of the death receptors) or intrinsic factors (e.g. mitochondrial insult) [7] and result in the activation of a set of cysteine-dependent aspartyl proteases (caspases) [8]. Both apoptotic pathways stimulate the effector enzyme, caspase-3 [7, 9, 10], which in turn activates a specific nuclear DNAse that degrades DNA into 200 base pair oligonucleosomal fragments, a hallmark of apoptosis [11, 12]. Apoptosis-related alterations have been reported in the dopaminergic brain regions in post-mortem tissues of PD patients, including increased caspase-3 activity in the substantia nigra [13, 14], and elevated immunoreactivity of the pro-apoptotic protein, Bax [15], indicating that apoptosis may play a role in the pathophysiology of PD.\nThe neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been used extensively to study PD [16, 17]. MPTP selectively targets and damages the dopaminergic neurons causing parkinsonism in humans and other primates [18, 19]. MPTP readily crosses the blood-brain barrier and is converted into the active metabolite, 1-methyl-4-phenyl pyridinium (MPP+) [17]. MPP+ is taken up by the dopaminergic neurons and accumulates in the mitochondria, where it inhibits complex I of the electron transport chain and ultimately causes neuronal cell death [5]. The mechanisms of MPP+-induced cytotoxicity most likely involve oxidative stress [20]. Several groups reported that treatment of mice with MPTP induces a Parkinson-like syndrome [16], accompanied by apoptosis of the dopaminergic neurons in the substantia nigra [21] and up-regulation of \u03b1-synuclein, a pre-synaptic protein, which plays an important role in PD pathogenesis [22]. In vitro studies using human SH-SY5Y neuroblastoma cells have produced comparable results [23, 24]. Exposure of these cells to MPP+ led to fragmentation of nuclear DNA [25], activation of caspase-3 [23, 26] and to up-regulation of \u03b1-synuclein [27\u201329]. In cultured SH-SY5Y cells dopamine-dependent cytotoxicity was enhanced by the accumulation of \u03b1-synuclein [30]. Therefore, targeting the molecular pathways activated during apoptosis may lead to novel treatment strategies for PD [5, 31].\nGuanosine and other non-adenine-based purines exert neuroprotective effects in the central nervous system [32]. In vitro guanosine also protects rat astrocytes from staurosporine-induced apoptosis [33] and SH-SY5Y cells from \u03b2-amyloid-induced apoptosis [34]. In both cases, the anti-apoptotic effect of guanosine was mediated by stimulation of the phosphatidylinositol-3-kinase (PI-3-K)\/Akt\/protein kinase B (PKB) and mitogen-activated protein kinase (MAPK) cell survival pathways [33, 34]. These pathways were activated via the pertussis toxin-sensitive Gi protein-coupled putative guanosine receptor that we have recently characterized pharmacologically [35\u201337].\nIn PD, symptoms appear only after the majority of the dopaminergic neurons in the substantia nigra pars compacta have degenerated, so putative neuroprotective drugs for PD should be tested in models in which the neurodegenerative process is already underway [38]. Therefore, we examined the effects of guanosine on MPP+-induced caspase-3 activation and DNA fragmentation in SH-SY5Y human neuroblastoma cells not only when it was added prior to, or at the same time as MPP+, but also when it was added to the cultures up to 48 h after MPP+ treatment, when the pro-apoptotic pathways were already activated. We also studied the molecular mechanisms that mediate the neuroprotective effects of guanosine.\nMaterials and methods\nTissue culture and treatment of human SH-SY5Y neuroblastoma cells\nHuman SH-SY5Y neuroblastoma cells (ATCC, Manassas, VA, USA) were cultured using 45% minimum essential medium, 45% F12 Hamilton\u2019s medium supplemented with 10% fetal bovine serum (FBS), 100\u00a0U\/ml of penicillin and 100\u00a0U\/ml of streptomycin at a pH of 7.4 (all from Invitrogen, Burlington, ON, Canada). Culture medium was changed every 3\u20134\u00a0days, and cells were maintained in a humidified 5% CO2 atmosphere at 37\u00b0C and subcultured at a ratio of 1:20 every 7\u201310\u00a0days. Culture medium was changed to 1% FBS for 24\u00a0h before the start of each experiments. All experiments were performed using 70\u201380% confluent cultures.\nApoptosis was induced by adding MPP+ (Sigma-Aldrich) to SH-SY5Y neuroblastoma cells, using a fresh stock solution of MPP+ (10\u00a0mM in reverse osmosis purified H2O). In all experiments, guanosine (Sigma-Aldrich) was dissolved in 0.1\u00a0M NaOH and added to the culture medium at a final concentration of 0.001\u00a0M NaOH. Some cells (control) were exposed to 0.001\u00a0M NaOH. In experiments in which the protective effect of guanosine was tested, guanosine (100\u00a0\u03bcM) was added to SH-SY5Y neuroblastoma cells either: 1\u00a0h prior to the administration of MPP+ (pre-treatment), or at the same time as MPP+ (co-treatment) or 24 or 48\u00a0h after the addition of MPP+(post-treatment at 24\u00a0h, or post-treatment at 48\u00a0h, respectively; Fig.\u00a01). Guanosine and MPP+ remained in the culture medium for the duration of the experiment (24, 48 or 72\u00a0h). In the first post-treatment experimental group at 24\u00a0h, cells were exposed to MPP+ for 24\u00a0h, followed by MPP+ plus guanosine for an additional 24\u00a0h (Fig.\u00a01). In the second post-treatment experimental group at 48\u00a0h, cells were treated with MPP+ for 48\u00a0h, followed by MPP+ plus guanosine for an additional 24\u00a0h (Fig.\u00a01).\nFig.\u00a01Experimental design for guanosine post-treatment of SH-SY5Y neuroblastoma cells added at 24\u00a0or 48\u00a0h after MPP+. SH-SY5Y neuroblastoma cells were exposed to either vehicle, or guanosine (100\u00a0\u03bcM) or MPP+ (500\u00a0\u03bcM) for various times. For post-treatment experiments with guanosine at 24\u00a0h, cells were exposed to MPP+ for 24\u00a0h, followed by MPP+ plus guanosine for an additional 24\u00a0h (total MPP+ exposure = 48\u00a0h). For post-treatment experiments with guanosine at 48\u00a0h, cells were exposed to MPP+ for 48\u00a0h followed by MPP+ plus guanosine for a further 24\u00a0h (total MPP+ exposure = 72\u00a0h). Once added, guanosine and MPP+ remained in the culture medium for the duration of the experiment. DNA fragmentation, or caspase-3 activity, was determined at 24, 48 or 72\u00a0h as described in the \u201cMaterials and methods\u201d section\nIn experiments in which enzyme inhibitors were tested, SH-SY5Y cells were pre-treated with various agents for 20\u00a0min prior to the addition of guanosine. These treatments included: the potent and selective inhibitor of PI-3-K, [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-1-hydrochloride] (LY294002; IC50\u2009=\u20091.4\u00a0\u03bcM; Sigma), or the selective inhibitor of MAP kinase kinase (MEK), [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-1] (PD98059; IC50\u2009=\u20092\u00a0\u03bcM; Sigma). In experiments using the ADP-ribosylating factor of the inhibitory guanosine nucleotide binding protein (Gi), pertussis toxin [PTX, 200\u00a0ng\/ml; in 50% glycerol, 0.5\u00a0M NaCl, and 0.05\u00a0M Tris-glycine (pH 7.5); Sigma] was added overnight (16\u00a0h). LY294002 and PD98059 were dissolved in and added to the culture medium at a final concentration of 0.01% dimethyl sulfoxide (DMSO).\nEvaluation of cell death and DNA fragmentation\nIn initial experiments, cell death was determined using acridine orange-ethidium bromide (AO-EB, Sigma-Aldrich) staining. Cells were seeded onto 6-well plates (diameter 10\u00a0mm; 2.5\u2009\u00d7\u2009104 cells\/well) and exposed to MPP+. After 24, 48 or 72\u00a0h, cells were washed three times with phosphate-buffered saline (PBS) and AO-EB solution (6\u00a0\u03bcM\/ml) was added to the wells. Cells were examined using a fluorescence microscope (Nikon, Tokyo, Japan). The number of fragmented nuclei and\/or condensed chromatin was determined by counting >200 cells. The percentage of apoptotic cells is defined as: (total number of cells with apoptotic nuclei\/total number of cells) \u00d7 100.\nIn subsequent experiments, DNA fragmentation was evaluated by the oligonucleosomal ELISA (Cell Death Detection ELISAPLUS; Roche Diagnostics, Laval, QC, Canada) and carried out according to the manufacturer\u2019s instructions. This assay measures the amount of oligonucleosomal fragments and is a marker of apoptosis [39]. Cells were exposed to MPP+ alone, or in combination with guanosine or pre-treated with the various inhibitors as described above. Following various treatments, cells were isolated for analysis of DNA fragmentation as described by Pettifer et al. [34]. For the oligonucleosomal ELISA, 10,000 viable cells\/treatment were lysed and centrifuged (200\u00a0g for 10\u00a0min) to isolate fragmented oligonucleosomal DNA. The cytosolic fractions of cell lysates were transferred into streptavidin-coated microplate wells, and a mixture of biotin-linked anti-histone antibody and peroxidase-linked anti-DNA antibody was added and incubated for 2\u00a0h at room temperature. Plates were washed with incubation buffer to remove the unfixed anti-DNA antibody and the peroxidase activity was determined spectrophotometrically with 2,2\u2032-azino-bis[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS) as the substrate (absorbance of 405\u00a0nm). The amount of DNA fragmentation is expressed as a percentage of the positive control provided with the kit.\nDetermination of caspase-3 activity\nCaspase-3 activity was determined using the caspase-3 colourimetric assay (Sigma-Aldrich). Cells were exposed to different treatments as described above; then cells were isolated, re-suspended in 100\u00a0\u03bcl\/107 cells of lysis buffer (50\u00a0mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4, 5\u00a0mM 3[(3-cholamidopropyl) dimethylammonio]-propanesulfonic acid (CHAPS), 5\u00a0mM dithiothreitol (DTT)) and incubated on ice for 20\u00a0min. Cell lysates were centrifuged (18,000\u00a0g for 10\u00a0min at 4\u00b0C) and the supernatants (5\u00a0\u03bcl\/well) were transferred to 96-well plates. A selective caspase-3 inhibitor (N-acetyl-Asp-Glu-Val-Asp-CHO; 10\u00a0\u03bcl\/well at 200\u00a0\u03bcM) was added to supernatants to control for the nonspecific hydrolysis of the substrate. Purified caspase-3 human recombinant protein, provided with the assay, was used (5\u00a0\u03bcl\/well at 5\u00a0\u03bcg\/ml) as a positive control for caspase-3 activity. Cell lysates were incubated in an assay buffer [40\u00a0mM HEPES, pH 7.4, 1% CHAPS, 10\u00a0mM DTT, 4\u00a0mM ethylenediaminetetraacetic acid (EDTA)], containing N-acetyl-Asp-Glu-Val-Asp p-nitroaniline (10\u00a0\u03bcl\/well at 2\u00a0mM) overnight at 37\u00b0C and the formation of p-nitroaniline was measured at 405\u00a0nm, using a microtiter plate reader. The activity of caspase-3 was calculated from the absorbance values using a calibration curve, and it is expressed as \u03bcg of substrate cleaved\/min per ml. As the number of cells in 1\u00a0ml of cell lysate is adjusted to 1\u2009\u00d7\u2009108 cells, this caspase-3 activity is generated by 1\u2009\u00d7\u2009108 cells.\nMeasurement of \u03b1-synuclein protein concentration by ELISA\nThe concentration of \u03b1-synuclein protein in SH-SY5Y neuroblastoma cells was determined by an ELISA (BioSource, San Diego, CA, USA). Cells were exposed to various treatments as described above and subsequently lysed using a cell extraction buffer (10\u00a0mM Tris, pH 7.4, 100\u00a0mM NaCl, 1\u00a0mM EDTA, 1\u00a0mM NaF, 20\u00a0mM Na4P2O7, 2\u00a0mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% sodium dodecyl sulfate, 0.5% deoxycholate; BioSource, San Diego, CA, USA), containing protease inhibitors [1\u00a0mM phenylmethanesulfonyl fluoride, 2\u00a0mM 4-(2-aminoethyl) benzenesulfonyl fluoride, 130\u00a0\u03bcM bestatin, 14\u00a0\u03bcM E-64, 1\u00a0\u03bcM leupeptin, 0.3\u00a0\u03bcM aprotinin; Sigma]. Cells were incubated on ice in cell extraction buffer for 30\u00a0min and centrifuged (14,000\u00a0g for 10\u00a0min at 4\u00b0C). Aliquots of supernatants (25\u00a0\u03bcl) were removed for the determination of the total protein concentration using the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA). We measured \u03b1-synuclein protein concentration using an ELISA kit according to the manufacturer\u2019s instructions. Briefly, cell lysates (diluted 1:10) were added to \u03b1-synuclein antibody-coated wells in duplicate, allowed to bind and then were treated with the anti-\u03b1-synuclein antibody. This was followed by the addition of an anti-rabbit IgG-horse radish peroxidase-linked antibody, and by the addition of a stabilized chromogen (tetramethylbenzidine). The reaction was terminated by adding 1\u00a0M HCl (stop solution) provided with the kit, and the absorbance of each well was read at 450\u00a0nm. Standard curves were prepared using purified recombinant \u03b1-synuclein protein (100\u00a0ng\/ml) provided with the kit. The concentration of the \u03b1-synuclein protein in each sample was determined from a nonlinear regression, and it is expressed as a ratio of \u03b1-synuclein protein (in ng\/ml) to total protein concentration (in ng\/ml).\nStatistical analysis\nData obtained in experiments testing the concentration dependence of the protective effect of guanosine on MPP+-induced DNA fragmentation (Fig.\u00a03) and the effect of PTX, and inhibitors of the PI-3-K, and the MAPK pathways on DNA fragmentation (Fig.\u00a06 and Table\u00a01) were analyzed by a one-way analysis of variance (ANOVA) followed by a Student-Newman-Keul post-hoc test. Two-way ANOVA, followed by a Bonferroni post-hoc test, was used to analyze the data obtained from experiments evaluating the effects of guanosine, administered at different time points on MPP+-induced DNA fragmentation (Fig.\u00a04) and on MPP+-induced caspase-3 activation (Fig.\u00a05). A p value of 0.05 or less was considered statistically significant. Results represent the mean \u00b1 SEM of a minimum of three independent experiments.\nTable\u00a01Effect of selected inhibitors of cell survival pathways on the anti-apoptotic effect of guanosine in SH-SY5Y cellsTreatmentDNA fragmentation (% of positive control)No inhibitorLY294002 10\u00a0\u03bcMPD98059 10\u00a0\u03bcMControl10.1\u2009\u00b1\u20091.316.3\u2009\u00b1\u20093.124.0\u2009\u00b1\u20094.7MPP+ (500\u00a0\u03bcM)34.5\u2009\u00b1\u20094.3a\u2013\u2013Guanosine pre-treatment15.9\u2009\u00b1\u20094.838.6\u2009\u00b1\u200910.3b38.3\u2009\u00b1\u20099.1bGuanosine co-treatment11.0\u2009\u00b1\u20090.732.9\u2009\u00b1\u20095.7b28.7\u2009\u00b1\u200910.4n.s.Guanosine post-treatment12.4\u2009\u00b1\u20091.848.2\u2009\u00b1\u20094.3c28.4\u2009\u00b1\u20094.8n.s.SH-SY5Y cells were exposed to MPP+ (500\u00a0\u03bcM) for 48\u00a0h to induce DNA fragmentation. Some cells were treated with guanosine (100\u00a0\u03bcM) either for 1\u00a0h prior to MPP+ (pre-treatment), or at the same time as MPP+ (co-treatment) or 24\u00a0h after MPP+ (post-treatment, see Fig.\u00a01). Some cultures were exposed to LY294002 (inhibitor of PI-3-K) or to PD98059 (inhibitor of MAP kinase kinase, MEK) for 20\u00a0min prior to the addition of guanosine. DNA fragmentation was determined at 48\u00a0h (n\u2009=\u20093) and is expressed as a percentage of the positive control. LY294002 abolished the protective effect of guanosine in cells pre-treated, co-treated or post-treated with this nucleoside. PD98059 also abolished the protective effect of guanosine in cells pre-treated with this nucleoside, but had no significant effect in cells co-treated or post-treated with guanosineap\u2009<\u20090.001 compared to controlbp\u2009<\u20090.01 compared to same treatment without inhibitorcp\u2009<\u20090.05 compared to same treatment without inhibitorn.s.\u2009not significant\nResults\nMPP+ induces DNA fragmentation in SH-SY5Y neuroblastoma cells\nSH-SY5Y neuroblastoma cells were exposed to different concentrations of MPP+ (10\u2013500\u00a0\u03bcM) for various times (0\u201372\u00a0h) to optimize the experimental conditions and to confirm that MPP+-induced DNA fragmentation was concentration and time dependent (Fig.\u00a02). DNA fragmentation in untreated control cells was around 10% and remained unchanged for up to 72\u00a0h (Fig.\u00a02). Treatment of cells with 500\u00a0\u03bcM MPP+ increased DNA fragmentation significantly after 48\u00a0h (p\u2009<\u20090.01) and 72\u00a0h (p\u2009<\u20090.001; Fig.\u00a02) compared to untreated controls. Since exposure of cells to 500\u00a0\u03bcM MPP+ for 48\u00a0h induced robust DNA fragmentation, these conditions were used in the following studies.\nFig.\u00a02MPP+ induces DNA fragmentation in SH-SY5Y cells. MPP+ (10, 100 or 500\u00a0\u03bcM) was added to SH-SY5Y cells and DNA fragmentation determined after 24, 48 or 72\u00a0h by oligonucleosomal ELISA as described in the \u201cMaterials and methods\u201d section. Data are presented as the percentage of DNA fragmentation relative to a positive control (n\u2009>\u20093). MPP+ (500\u00a0\u03bcM) increased DNA fragmentation significantly at 48 h (*p\u2009<\u20090.01) and at 72\u00a0h (**p\u2009<\u20090.001) compared to that of untreated cells at zero time\nPre-treatment of SH-SY5Y neuroblastoma cells with guanosine attenuates MPP+-induced DNA fragmentation\nSH-SY5Y cells were exposed to various concentrations of guanosine (1\u2013300\u00a0\u03bcM) for 1\u00a0h, followed by the addition of 500\u00a0\u03bcM MPP+ for a further 48\u00a0h, and DNA fragmentation was determined at 49\u00a0h by oligonucleosomal ELISA. As we observed in the previous experiment the addition of MPP+ (500\u00a0\u03bcM) led to significantly increased DNA fragmentation compared to controls (p\u2009<\u20090.001; Fig.\u00a03). Pre-treatment of cells for 1\u00a0h with various concentrations of guanosine (10\u2013300\u00a0\u03bcM) significantly reduced MPP+-induced DNA fragmentation (p\u2009<\u20090.05) for all four concentrations of guanosine compared to cells treated with MPP+ alone (Fig.\u00a03). Based on these results, we used 100\u00a0\u03bcM guanosine in all subsequent experiments.\nFig.\u00a03Pre-treatment of SH-SY5Y cells with guanosine decreases MPP+-induced DNA fragmentation. SH-SY5Y cells were exposed to MPP+ (500\u00a0\u03bcM) for 48\u00a0h to induce DNA fragmentation. Some cells were pre-treated with guanosine (1\u2013300\u00a0\u03bcM) for 1\u00a0h prior to MPP+. Guanosine and MPP+ remained in the cultures for the duration of the experiment. DNA fragmentation was determined after 48\u00a0h by oligonucleosomal ELISA as described in the \u201cMaterials and methods\u201d section and is expressed as the percentage of the positive control (n\u2009>\u20093). MPP+ increased DNA fragmentation significantly at 48 h compared to that of untreated controls (*p\u2009<\u20090.001). Pre-treatment with guanosine (10\u2013300\u00a0\u03bcM) reduced significantly the MPP+-induced DNA fragmentation compared to that in cells treated with MPP+ for the same time (#p\u2009<\u20090.05, at all four concentrations)\nPost-treatment with guanosine protects SH-SY5Y neuroblastoma cells against MPP+-induced DNA fragmentation\nWe next examined whether guanosine exerted any effect on MPP+-induced DNA fragmentation when it was added to neuroblastoma cells after apoptosis was already in progress. Cells were pre-treated with 100\u00a0\u03bcM guanosine prior to MPP+ or co-treated with guanosine plus MPP+ or, as described in Fig.\u00a01, guanosine was added as post-treatment. DNA fragmentation was determined in cells exposed to various treatments after 24, 48 and 72\u00a0h of MPP+ administration (Fig.\u00a04). The addition of MPP+ led to a significant increase in DNA fragmentation compared to controls at 48\u00a0h (p\u2009<\u20090.01) and 72\u00a0h (p\u2009<\u20090.001) (Fig.\u00a04). Guanosine alone had no significant effect on DNA fragmentation (Fig.\u00a04). Pre-treatment of cells with guanosine significantly reduced DNA fragmentation after 48\u00a0and 72\u00a0h compared to MPP+ treatment alone, at the respective time points (p\u2009<\u20090.05, at both time points; Fig.\u00a04). Similarly, co-treatment of cells with guanosine plus MPP+ significantly reduced DNA fragmentation after 48\u00a0and 72\u00a0h compared to cells treated with MPP+ alone at these times (p\u2009<\u20090.05, at both time points; Fig.\u00a04). Addition of guanosine 24\u00a0h after MPP+ (post-treatment at 24\u00a0h) or 48\u00a0h after MPP+ (post-treatment at 48\u00a0h) reduced DNA fragmentation significantly compared to cells treated with MPP+ alone for 48\u00a0or 72\u00a0h, respectively (p\u2009<\u20090.05, for both treatments; Fig.\u00a04). These results demonstrate that pre-treatment or co-treatment of cells with guanosine and MPP+ attenuates MPP+-induced DNA fragmentation, and guanosine added 24 or 48\u00a0h after the start of the DNA fragmentation (post-treatment) can actually reverse this process.\nFig.\u00a04Guanosine antagonizes and reverses MPP+-induced DNA fragmentation in SH-SY5Y cells. SH-SY5Y cells were exposed to MPP+ (500\u00a0\u03bcM) for 48\u00a0h to induce DNA fragmentation. Some cells were treated with guanosine (100\u00a0\u03bcM) either for 1\u00a0h prior to MPP+ (pre-treatment), or at the same time as MPP+ (co-treatment) or 24\u00a0or 48\u00a0h after MPP+ (post-treatment, see Fig. 1). DNA fragmentation was determined after 24, 48 or 72\u00a0h of MPP+ addition by oligonucleosomal ELISA as described in the \u201cMaterials and methods\u201d section and it is expressed as a percentage of the positive control (n\u2009>\u20093). MPP+ induced a significant increase in DNA fragmentation at 48\u00a0(*p\u2009<\u20090.01) and at 72\u00a0h (**p\u2009<\u20090.001) compared to that of untreated control cells at the same time. Pre-treatment or co-treatment of cells with guanosine significantly reduced MPP+-induced DNA fragmentation at 48 and 72\u00a0h compared to that in cells treated with MPP+ alone at the same time (#p\u2009<\u20090.05 at both time points, for both treatments). Post-treatment with guanosine at 24\u00a0or 48\u00a0h also reduced MPP+-induced DNA fragmentation at 48\u00a0and 72\u00a0h compared to that in cells treated with MPP+ alone at the same time (#p\u2009<\u20090.05, at both time points)\nGuanosine inhibits MPP+-induced caspase-3 activity\nExposure of SH-SY5Y cells to MPP+ leads to apoptosis via caspase-3 activation [23, 26]. We therefore examined if under our experimental conditions treatment of neuroblastoma cells with MPP+ activated caspase-3 and whether the addition of guanosine to MPP+-treated cells at various times (pre-treatment, co-treatment or post-treatment) had any effect on the activity of this enzyme. In untreated control cells caspase-3 activity was low and remained unchanged for up to 72\u00a0h (Fig.\u00a05). Addition of guanosine to cells had no significant effect on caspase-3 activity (Fig.\u00a05). In MPP+-treated cells caspase-3 activity increased significantly from 24 to 72\u00a0h (p\u2009<\u20090.001, at all time points; Fig.\u00a05). Pre-treatment of cells with guanosine prior to MPP+ addition significantly reduced caspase-3 activity after 48 or 72\u00a0h compared to that in cells treated with MPP+ alone for the same time (p\u2009<\u20090.01, at both time points; Fig.\u00a05). Similar results were also obtained when cells were co-treated with guanosine and MPP+ for 48 and 72\u00a0h (p\u2009<\u20090.01, at both time points; Fig.\u00a05). Post-treatment with guanosine at 24\u00a0h (see Fig.\u00a01) also reduced caspase-3 activity significantly at 48\u00a0h compared to that in cells treated with MPP+ alone at this time point (p\u2009<\u20090.01; Fig.\u00a05). When guanosine was added to cells 48\u00a0h after the addition of MPP+ (post-treatment at 48\u00a0h), caspase-3 activity determined after 72\u00a0h was unaffected compared to MPP+ treatment alone at this time point (Fig.\u00a05). These results thus demonstrate that pre-treatment, co-treatment or post-treatment at 24\u00a0h of neuroblastoma cells with guanosine prevents MPP+-induced activation of caspase-3. Administration of guanosine to cells 48\u00a0h after MPP+ however has no effect on caspase-3 activity.\nFig.\u00a05Guanosine inhibits MPP+-induced caspase-3 activation in SH-SY5Y cells. SH-SY5Y cells were exposed to MPP+ (500\u00a0\u03bcM) for 48\u00a0h to induce to induce caspase-3 activation. Some cells were treated with guanosine (100\u00a0\u03bcM) either for 1\u00a0h prior to MPP+ (pre-treatment), or at the same time as MPP+ (co-treatment) or 24\u00a0or 48\u00a0h after MPP+ (post-treatment, see Fig. 1). Caspase-3 activity was determined as described in the \u201cMaterials and methods\u201d section and is expressed as the amount of N-acetyl-Asp-Glu-Val-Asp p-nitroaniline cleaved by 1\u2009\u00d7\u2009108 cells, in \u03bcg\/min per ml (n\u2009>\u20093). MPP+ significantly increased caspase-3 activity from 24\u00a0to 72\u00a0h (*p\u2009<\u20090.001, at all three time points) compared to that of control cells at the same time. Pre-treatment or co-treatment of cells with guanosine significantly reduced MPP+-induced caspase-3 activity, at 48 and 72\u00a0h, compared to that in cells treated with MPP+ alone at the same time (#p\u2009<\u20090.01, at both time points, for both treatments). Post-treatment with guanosine at 24 h reduced MPP+-induced caspase-3 activity at 48\u00a0h compared to that in cells treated with MPP+ alone at the same time (#p\u2009<\u20090.01). Guanosine added to cultures 48\u00a0h after MPP+ had no effect on caspase-3 activity\nGuanosine has no effect on \u03b1-synuclein protein expression in MPP+-treated neuroblastoma cells\nSeveral authors reported that addition of MPP+ to SH-SY5Y cells increased \u03b1-synuclein protein expression [27, 29]. We therefore determined \u03b1-synuclein protein concentration in neuroblastoma cells under our experimental conditions (500\u00a0\u03bcM MPP+ for 48\u00a0h) and evaluated the effects of guanosine on the expression of this protein in MPP+-treated cells. The ratio of \u03b1-synuclein protein\/total protein in untreated control cells after 48\u00a0h was very low and exposure of neuroblastoma cells to MPP+ for 48\u00a0h had no significant effect on the expression of this protein (data not shown). Treatment of cells with guanosine prior to the addition of MPP+, or at the same time as MPP+ or 24\u00a0h after MPP+ had no significant effect on of \u03b1-synuclein protein expression (data not shown). These results indicate that under the present experimental conditions neither MPP+ nor a combination of guanosine plus MPP+ has any effect on \u03b1-synuclein protein expression.\nPertussis toxin abolishes the anti-apoptotic effect of guanosine\nWe reported recently that specific binding sites exist for guanosine in rat brain membranes [35], in cultured rat astrocytes [36] and in SH-SY5Y neuroblastoma cells (Traversa and Caciagli, 2005, personal communications). We have further shown that guanosine binding in these cells is sensitive to treatment with PTX [37, 40], indicating that the putative guanosine receptor is coupled to Gi protein. We therefore tested whether the anti-apoptotic effect of guanosine in the MPP+-treated cells is mediated via this putative guanosine receptor. Cells were exposed to PTX (200\u00a0ng\/ml for 16\u00a0h) prior to the various treatments with guanosine and MPP+ (pre-treatment, co-treatment or post-treatment at 24\u00a0h) and DNA fragmentation was determined at 48\u00a0h. As in previous experiments, MPP+ induced a significant increase in DNA fragmentation and the addition of guanosine alone had no significant effect on this process (Fig.\u00a06). Exposure of neuroblastoma cells to PTX alone increased DNA fragmentation slightly under basal conditions, but this was not statistically significant. Pre-treatment, co-treatment or post-treatment at 24\u00a0h of cells with guanosine and MPP+ significantly reduced DNA fragmentation compared to cells treated with MPP+ only (p\u2009<\u20090.01, for all three treatments; Fig.\u00a06). Addition of PTX to neuroblastoma cells prior to any of the three guanosine plus MPP+ treatments reversed the anti-apoptotic effect of guanosine (Fig.\u00a06). These results demonstrate that regardless of the time of its addition to MPP+-treated cells, the protective effect of guanosine is abolished by PTX. These findings are consistent with the interpretation that these effects are mediated by a Gi protein-coupled receptor, most likely the putative guanosine receptor.\nFig.\u00a06Pertussis toxin abolishes the anti-apoptotic effect of guanosine in SH-SY5Y cells. SH-SY5Y cells were exposed to MPP+ (500\u00a0\u03bcM) for 48\u00a0h to induce DNA fragmentation. Some cells were treated with guanosine (100\u00a0\u03bcM) either for 1\u00a0h prior to MPP+ (pre-treatment), or at the same time as MPP+ (co-treatment) or 24\u00a0h after MPP+ (post-treatment, see Fig. 1). Pertussis toxin (PTX, 200\u00a0ng\/ml for 16\u00a0h) was added to some cultures prior to pre-treatment or co-treatment or post-treatment with guanosine at 24\u00a0h. DNA fragmentation was determined at 48\u00a0h and is expressed as a percentage of the positive control (n\u2009>\u20093). MPP+ increased DNA fragmentation significantly at 48\u00a0h (*p\u2009<\u20090.001) compared to that of untreated control cells at the same time. Pre-treatment, or co-treatment or post-treatment of cells with guanosine at 24\u00a0h reduced significantly MPP+-induced DNA fragmentation compared to that in cells treated with MPP+ alone at the same time (\u00a7p\u2009<\u20090.01). PTX reversed the protective effect of guanosine. DNA fragmentation in cells exposed to PTX plus pre-treatment, or co-treatment or post-treatment with guanosine was significantly higher compared to that of cells without PTX (#p\u2009<\u20090.05, for all three treatments). These values were comparable to that obtained in cells treated with MPP+ alone\nGuanosine protects against MPP+-induced apoptosis by activating the cell survival pathways\nWe have shown earlier that guanosine activates the PI-3-K\/Akt\/PKB and MAPK pathways in neuroblastoma cells [34]. We therefore evaluated if the protective effects of guanosine against MPP+-induced DNA fragmentation were also mediated via these signaling pathways. Cells were exposed to either LY294002, or to PD98059, selective inhibitors of PI-3-K or MEK, respectively, for 20\u00a0min prior to various treatments with guanosine and MPP+ (pre-treatment, co-treatment or post-treatment at 24\u00a0h). Exposure of neuroblastoma cells to LY294002 alone had no significant effect on DNA fragmentation under basal conditions. Although in cells exposed to PD98059 alone DNA fragmentation increased slightly, this was not statistically significant (Table\u00a01). As in previous experiments, the addition of MPP+ induced significant DNA fragmentation and treatment of cells with guanosine under various conditions significantly reduced DNA fragmentation compared to cells treated with guanosine (Table\u00a01).\nExposure of cells to LY294002 prior to pre-treatment, or co-treatment or post-treatment at 24\u00a0h with guanosine and MPP+ reversed the protective effect of guanosine. DNA fragmentation in cells exposed to LY294002 plus guanosine plus MPP+ was significantly reduced compared to cells treated with guanosine plus MPP+ without the inhibitor (p\u2009<\u20090.05, for pre-treatment and co-treatment; p\u2009<\u20090.01, for post-treatment at 24\u00a0h; Table\u00a01). Addition of PD98059 to cells before pre-treatment with guanosine and MPP+ also abolished the protective effect of guanosine (p\u2009<\u20090.05; Table\u00a01). But this inhibitor had no significant effect on MPP+-induced DNA fragmentation in cells co-treated or post-treated at 24 with guanosine and MPP+ (Table\u00a01). These results demonstrate that the protective effects of guanosine are abolished by LY294002 in MPP+-treated cells regardless of the time of its administration and are consistent with a role of the PI-3-K\/Akt\/PKB cell survival pathway in guanosine-mediated reduction of DNA fragmentation. In contrast, the effect of PD98059 is limited to pre-treatment of cells with guanosine prior to MPP+, thus indicating that the MAPK pathway contributes to the protective effects of guanosine only when it is added before MPP+.\nDiscussion\nWe have shown earlier that pre-treatment with guanosine protects different cell types against apoptosis [33, 34]. Here, we report that guanosine not only protects human SH-SY5Y neuroblastoma cells against MPP+-induced apoptosis, but more importantly, it can reverse MPP+-induced cytotoxicity after the pro-apoptotic pathways have already been activated. Thus, these findings are novel and demonstrate for the first time that guanosine can in fact \u2018rescue\u2019 neuroblastoma cells from apoptosis when it is added to cells under experimental conditions that are \u2018clinically relevant\u2019 to the treatment of PD.\nUndifferentiated dopaminergic human SH-SY5Y neuroblastoma cells have been used widely to study the molecular pathways that mediate MPP+-induced apoptosis [23\u201329]. MPTP, after conversion to MPP+, activates the same intracellular pathways in vivo and in vitro as in idiopathic PD [2]. Thus, MPP+ is taken up by dopaminergic neurons, where it inhibits complex I of the mitochondrial electron transport chain. This leads to enhanced production of reactive oxygen species (ROS) and decreased synthesis of ATP [2]. Prolonged exposure to MPP+ results in the up-regulation of the pro-apoptotic protein Bax, and following its translocation to the mitochondria it promotes the release of mitochondrial cytochrome c. This may be initiated by the DNA damage caused by MPP+. Cytochrome c, together with pro-caspase-9, is incorporated into the complex, the apoptosome, which activates caspase-9, and this enzyme in turn activates caspase-3 [2, 41]. At the same time, the anti-apoptotic protein, Bcl-2, is down-regulated [42]. Taken together these data demonstrate that exposure of dopaminergic cells to MPP+ leads to cytotoxicity and cell death by apoptosis [2, 17]. Our findings are consistent with previous reports that MPP+ induces DNA fragmentation and activation of caspase-3 in SH-SY5Y cells [23, 25]. Interestingly, we detected no significant change in \u03b1-synuclein protein expression in MPP+-treated cells (data not shown). This may be the result of the relatively low MPP+ concentration (500\u00a0\u03bcM) and short duration of our experiments (48\u00a0h). Others reported that high concentrations of MPP+ (5\u00a0mM for 12\u00a0h [29]) or prolonged exposure of neuroblastoma cells to lower concentrations of MPP+ (5\u00a0\u03bcM for 4\u00a0days [28] or 1\u00a0mM for 3\u00a0days [27]) actually increased \u03b1-synuclein protein concentration. Mutations of this protein have been associated with a familial form of PD [43] and the wild-type protein is a major constituent of Lewy bodies [44]. In MPTP-treated mice \u03b1-synuclein expression is up-regulated [45], and in SH-SY5Y cells accumulation of this protein contributes to dopamine-dependent apoptosis [30]. Under our experimental conditions that were optimized to detect DNA fragmentation, we observed no change in \u03b1-synuclein protein expression.\nThe molecular mechanisms by which guanosine exerts its protective effects are complex. Results from the present study show that the effects of both pre-treatment and co-treatment with guanosine are pertussis toxin sensitive, and these may depend on the putative plasma membrane localized guanosine receptor [35\u201337] that is coupled to Gi proteins. There may be other possible explanations, which account for the effects of pertussis toxin on MPP+-induced DNA fragmentation. Since pertussis toxin enters cells and ADP-ribosylates the \u03b1-subunit of Gi proteins [46, 47], it may also attenuate signaling by other Gi protein-coupled receptors, resulting in the elevation of intracellular cAMP [48, 49] and this in turn may activate cell survival pathways, such as the MAPK [56]. Guanosine may also enter neuroblastoma cells via the equilibrative or the concentrative nucleoside transporters [50], cause an alteration in intracellular concentrations of guanine nucleotides [40] and this in turn may affect G protein activity.\nHowever, as we have shown before, guanosine binding to its cognate receptor leads to rapid activation of the cell survival pathways, PI-3-K\/Akt\/PKB and MAPK [33, 34], and these play a critical role in protecting neurons against cell death [51\u201356]. Although these signaling pathways are most often triggered by growth factor binding to their cognate receptors, G protein-coupled receptors have also been shown to activate them [57\u201360]. Thus, binding of guanosine to its Gi protein-coupled putative receptor may activate the class IB PI-3-K (PI-3-K\u03b3), via the dissociated \u03b2,\u03b3-subunits of Gi proteins, which promote the synthesis of phosphatidylinositol (3,4,5) trisphosphate at the plasma membrane and recruit the protein kinase, Akt\/PKB [60, 61]. Activation of Akt\/PKB by phosphorylation at Thr 308 and Ser 473 regulates cell survival and apoptosis by both transcription-dependent and transcription-independent pathways [7, 54, 61]. Thus, Akt\/PKB may suppress apoptosis by phosphorylating and inactivating the pro-apoptotic protein Bad [62], inhibiting the release of mitochondrial cytochrome c [63] and inhibiting the activation of caspase-9 and caspase-3 [64]. Expression of cell survival genes may also be triggered by the PI-3-K\/Akt\/PKB pathway, via activation of the transcription factors CREB and NF-\u03baB, and inhibition of Forkhead transcription factors of the FOXO family [54].\nSimilarly, activation of the MAPK pathway, specifically the extracellular signal-regulated kinases 1 and 2 (ERK1\/2), also prevent apoptosis and promote cell survival by both transcription-dependent and transcription-independent pathways [55, 56, 65, 66]. The specific downstream targets of the ERK1\/2 pathway include inhibition of caspase-9 by phosphorylation [67], suppression of the pro-apoptotic protein Bad by phosphorylation and activation of the transcription factor CREB [55]. Guanosine binding to its putative receptor may also stimulate this pathway via the dissociated \u03b2,\u03b3-subunits of Gi proteins [59]. Reports suggest that the PI-3-K\/Akt\/PKB and MAPK pathways do not function independently, but there is significant cross-talk between them [57, 68]. It has been suggested that whereas the PI-3-K\/Akt\/PKB pathway is the main signaling pathway for maintaining trophic support by pro-survival factors, ERK1\/2 mediates protection against damage-induced cell death [56]. Our results are thus consistent with the interpretation that protection of cells from MPP+-induced DNA fragmentation by guanosine pre-treatment is dependent on the activation of both these pathways. Following co-treatment with guanosine and MPP+, however, it is the PI-3-K\/Akt\/PKB pathway, which plays the essential role.\nIn the present study, we also examined the effect of guanosine on MPP+-induced DNA fragmentation by exposing cells to guanosine after caspase-3 activity was elevated and DNA fragmentation was already underway (post-treatment). This strategy is novel and is based on a recent proposal by Meissner et al. [38], who advocate that potential neuroprotective agents for the treatment of PD should be tested under conditions that are relevant to the clinical situation. Because clinical symptoms of PD are apparent only after the majority of nigrostriatal neurons have died, patients do not present for treatment until after the process of cell death is well established [38]. Most in vitro studies have been conducted by adding potential neuroprotective agents to cells prior to MPP+-triggered apoptosis [69\u201371] or at the same time as MPP+ [27, 72, 73], making these results irrelevant to the treatment of PD, where the process of neurodegeneration has already begun.\nAddition of guanosine to cells after MPP+-induced DNA fragmentation is already in progress may involve additional protective mechanisms. As DNA fragmentation is a late event in apoptosis [11, 12], some cells with damaged DNA may actually undergo DNA repair and survive [74, 75]. The preferred survival strategy of a cell is to attempt to restore any DNA damage by using a complex set of DNA repair pathways [74]. Only when the damage to the DNA becomes excessive and the repair capacity of the cell is overwhelmed will it undergo apoptosis. Therefore, adding guanosine 24 or 48\u00a0h after DNA fragmentation has been initiated may activate some of the DNA repair mechanisms, antagonizing and reversing the MPP+-induced DNA fragmentation. The protective effects of guanosine in these cells thus may include activation of both cell survival and DNA repair pathways.\nIn contrast, post-treatment with guanosine had no significant effect on MPP+-induced activation of caspase-3. As activation of this enzyme requires a cleavage of the interdomain region of the inactive dimer of caspase-3, once this process is initiated it cannot be reversed [8\u201310]. So addition of guanosine to cells after proteolytic cleavage of this enzyme will not alter caspase-3 activity.\nRecently administration of MPTP to mice has been reported to promote caspase-independent cell death of dopaminergic neurons [76]. This \u201capoptosis-like\u201d programmed cell death [77] is mediated by the release of the apoptosis-inducing factor (AIF) from the mitochondria and its subsequent translocation to the nucleus, where it induces DNA fragmentation [78\u201380]. If this process plays a significant role in MPP+-induced cell death of neuroblastoma cells, DNA fragmentation will still be reversed by the protective action of guanosine, but caspase-3 activation will not be affected.\nSeveral authors suggested recently that targeting programmed cell death in neurodegenerative diseases may lead to successful therapy [5, 31]. Drugs that will be effective in PD should protect the dopaminergic cells from apoptotic insult by either interrupting the signaling pathways that mediate cell death or activating the cell survival pathways. Our present results show that the non-adenine-based purine nucleoside guanosine, when added to MPP+-treated neuroblastoma cells, promotes their survival by inhibiting caspase-3 activation and DNA fragmentation via activation of the PI-3-K\/Akt\/PKB pathway. Furthermore, guanosine exerts these effects not only when it is pre- or co-administered with MPP+, but even when it is added to cells after caspase-3 is activated and DNA fragmentation is already in progress. Thus, these findings reveal a unique neuroprotective effect of guanosine with a potential for effective pharmacological intervention in PD.","keyphrases":["cytotoxicity","guanosine","apoptosis","parkinson\u2019s disease","dna fragmentation","caspase-3","cell survival","sh-sy5y human neuroblastoma cells","1-methyl\u20134-phenyl-pyridinium (mpp+)"],"prmu":["P","P","P","P","P","P","P","P","M"]}
{"id":"Naturwissenschaften-3-1-1829419","title":"Behavioral genomics of honeybee foraging and nest defense\n","text":"The honeybee has been the most important insect species for study of social behavior. The recently released draft genomic sequence for the bee will accelerate honeybee behavioral genetics. Although we lack sufficient tools to manipulate this genome easily, quantitative trait loci (QTLs) that influence natural variation in behavior have been identified and tested for their effects on correlated behavioral traits. We review what is known about the genetics and physiology of two behavioral traits in honeybees, foraging specialization (pollen versus nectar), and defensive behavior, and present evidence that map-based cloning of genes is more feasible in the bee than in other metazoans. We also present bioinformatic analyses of candidate genes within QTL confidence intervals (CIs). The high recombination rate of the bee made it possible to narrow the search to regions containing only 17\u201361 predicted peptides for each QTL, although CIs covered large genetic distances. Knowledge of correlated behavioral traits, comparative bioinformatics, and expression assays facilitated evaluation of candidate genes. An overrepresentation of genes involved in ovarian development and insulin-like signaling components within pollen foraging QTL regions suggests that an ancestral reproductive gene network was co-opted during the evolution of foraging specialization. The major QTL influencing defensive\/aggressive behavior contains orthologs of genes involved in central nervous system activity and neurogenesis. Candidates at the other two defensive-behavior QTLs include modulators of sensory signaling (Am5HT7 serotonin receptor, AmArr4 arrestin, and GABA-B-R1 receptor). These studies are the first step in linking natural variation in honeybee social behavior to the identification of underlying genes.\nIntroduction\nMapping genes for natural variation in behavior\nThe need to understand within- and between-population variation in behavior, especially social behavior and its evolution, remains a central theme in biology. Honeybee societies have facultatively sterile female workers and a specialized workforce. Honeybee social behavior is dramatically demonstrated by the efforts of worker bees collecting and hoarding pollen and nectar, and mass stinging responses in defense of the nest. Division of labor within a honeybee nest is a consequence of age-related changes in physiology plus genetic variation within the colony for tendency to perform specific tasks (Page and Robinson 1991). A number of studies have demonstrated genetic variation within and between honeybee populations in specialization for water, nectar, and pollen collecting (reviewed in Page et al. 2000) and variation in defensive responses (reviewed in Breed et al. 2004). The genetic architectures of these two task sets are more thoroughly characterized than other behavioral traits in the honeybee. For these reasons, we sought to determine whether it is possible to define, within a manageable number of candidate genes, regions influencing these behaviors.\nQuantitative trait locus (QTL)-mapping studies provide information about the genetic architecture of a trait that cannot be determined by other methods. This information includes estimates for the number and location of loci influencing population variation in the trait, the mode of inheritance of these loci (dominance, epistasis, or imprinting effects), and amount of phenotypic variance each locus accounts for (Lander and Botstein 1989). But QTL studies do not have a good track record for isolation of causal genes. For example, genes for only 20 of 2,000-rodent QTLs have been cloned (Flint et al. 2005). Identifying genes responsible for naturally occurring phenotypic variation is especially challenging for behavioral traits where complex interactions of genes and the environment are expected (Flint 2003; Plomin and McGuffin 2003; Arnholdt and Mackay 2004; Goldman et al. 2005).\nThe endgame in determining nucleotide sequence variation responsible for natural variation in honeybee behavior will require identification of QTLs, confirmation in independent crosses, fine-scale mapping, expression assays, and finally, experimental modulation of gene expression or complementation tests (Mackay 2004). The biggest problem in taking a map-based approach to isolation of genes is that confidence intervals (CIs) for QTL location are usually quite large. The size of the CI primarily depends on the number of individuals in the mapping population, the number of genetic markers scored, the magnitude of effect that the QTL has on the phenotype, and the recombination rate (numbers of crossovers per unit of chromosomal distance). The first two factors, sample size and marker density, are important for reducing CIs, but they are subject to diminishing returns as more individuals or more markers are added to the experiment. The effect of the QTL is a major concern because QTLs often account for only a small proportion (often <5%) of phenotypic variance. The QTL effect can only be increased up to a certain level by improving the phenotypic assay and using the most appropriate cross (Darvasi 1998; Arnholdt and Mackay 2004). On the other hand, recombination rate can have a large effect on the size of chromosomal regions covered by CIs because the physical size scales inversely with recombination rate. For this reason, fine-scale mapping studies are often designed to effectively increase recombination by taking advantage of historical recombination events through linkage-association studies in populations or by developing multigeneration, recombinant inbred lines (Darvasi 1998). In the honeybee, map resolution is enhanced by a meiotic recombination rate that currently ranks highest among metazoans (Hunt and Page 1995; Solignac et al. 2004), so that large genetic distances correspond to relatively small physical regions containing few genes.\nIn this study, we review what is currently known about the genetics of honeybee foraging and defensive behaviors, and how a map-based approach leads us to a manageable number of candidate genes that seem to fit what is known of the behavioral patterns. Information from the draft honeybee genome sequence (Honeybee Genome Sequencing Consortium, HGSC 2006) was used to delimit sets of candidate genes flanked by marker sequences that were identified in prior QTL studies, and expression data from candidate genes provided additional information on likely candidates.\nBehavioral genetics of foraging\nForaging specialization involves two components: the onset of foraging that establishes division of labor between foragers and bees that perform tasks inside the hive and the subsequent bias in foraging for either pollen or nectar. These components are related. Bees from lines selected for storing more surplus pollen (hoarding) are more likely to initiate foraging earlier in life and to specialize in pollen collection than bees from lines selected for low pollen hoarding. These associations are also linked to sensory response in that bees selected for higher levels of pollen hoarding are more responsive to low concentrations of sugar (less discriminatory) when tested with the proboscis extension assay (Fig.\u00a01 and Page et al. 1998). This link between sucrose responsiveness and the task of pollen foraging is also present in \u201cwild-type\u201d bees that have not been selected for pollen hoarding (Scheiner et al. 2004).\nFig.\u00a01Genetic and phenotypic associations involved in foraging division of labor. Arrows indicate significant correlations between phenotypic traits at the levels of behavior, hormonal signaling, and development and associations between traits and genotypes at specific QTLs (pln 1\u20134). Colored lines indicate relative titers of specific hormones. The picture illustrates a method for determining the threshold concentration of sugar that a bee will respond to by extending its proboscis or tongue\nQTLs were mapped based on whole-colony behavioral traits, and QTL effects were subsequently confirmed based on the behavior of individual bees. Three \u201cpollen\u201d QTLs, designated as pln-1, pln-2, and pln-3, were detected (Hunt et al. 1995; Page et al. 2000) based on the quantity of pollen in colonies from a backcross population derived from high and low pollen-hoarding strains. Association of marker alleles near the QTLs with individual foraging traits within single backcross families of bees confirmed the effects of pln-1, pln-2, and pln-3 on behavior. Pln-1 and pln2 were associated with the size of the pollen loads collected by workers (Fig.\u00a02a and b). Pln-2 and pln-3 were shown to influence the discrimination for the sugar concentration of the nectar collected (Hunt et al. 1995; Page et al. 2000; Fig.\u00a02b,c). Subsequent association studies using a candidate gene AmFor as a marker have mapped and confirmed an additional QTL region designated as pln-4 (Rueppell et al. 2004). This QTL maps to about 50\u00a0cM from pln-1. Allelic variation and pleiotropic effects of these QTLs have been associated with sucrose responsiveness and age at onset of foraging (Fig.\u00a01 and Rueppell et al. 2004, 2006).\nFig.\u00a02QTLs were mapped based on the amount of pollen stored in combs of colonies and confirmed based on individual behavior. Solid bars represent linkage groups with markers. Markers used in confirmation studies are shown. Orthologs of fly genes are indicated with arrows. Dashed lines indicate 97% CIs. Sequenced markers are underlined. No figure is shown for pln-4 because it was mapped by association to one marker (AmFOR) rather than by interval mapping. aPln-1 Map on the left is the localization of the QTL based on colony pollen stores. Map on the right is based on response thresholds to sucrose of individual worker bees. bPln-2QTL map on the left is based on colony pollen stores in a cross between European strains and the map on the right is based on a European by African strain cross. cPln-3QTL map is based on colony pollen stores\nPreferential foraging for either a nectar or protein source such as pollen are sequential parts of the gonotrophic cycle of many insect females. When nonreproductive, females tend to forage for nectar as a carbohydrate source for maintenance. When reproductively active, insects such as solitary bees and mosquitoes seek protein that is incorporated into eggs (Amdam et al. 2004). Although worker honeybees are facultatively sterile, they can produce eggs if their ovaries develop sufficiently as happens in the absence of a queen. Amdam et al. (2004, 2006) hypothesized that remnants of the ancestral gonotrophic cycle and the correlated foraging behavior remain and \u201cdrive\u201d foraging behavior (Fig.\u00a01). In support of this hypothesis, it was established that workers from the high pollen-hoarding strain are characterized by elevated titers of the conserved yolk precursor vitellogenin and have larger and more active ovaries than low strain bees. It was also found that bees that were unselected for pollen hoarding but had enlarged ovaries foraged earlier in life showed a preference for pollen foraging and collected nectar of lower concentration than bees with fewer ovarioles. Thereby, \u201cwild type\u201d bees show the same correlated phenotypes that differ between high and low pollen-hoarding strains (Amdam et al. 2006).\nIn honeybees, ovariole number is determined during the third larval instar through a nutrient-dependent endocrine signaling cascade. The endocrine factors, juvenile hormone and ecdysteroids, are involved in the initiation of vitellogenin expression at adult emergence, and vitellogenin and juvenile hormone interact during adult life to affect sensory responsiveness and onset of foraging behavior (Guidugli et al. 2005). In solitary insects, endocrine cascades involving juvenile hormone and ecdysteroids have pleiotropic effects on sensory tuning, yolk protein production, ovarian physiology, and life span (Amdam et al. 2004; Flatt et al. 2005; Guidugli et al. 2005). This hormonal pleiotropy appears to be regulated by upstream signaling through the insulin\/insulin-like signaling (IIS) pathway (Claeys et al. 2001; Flatt et al. 2005; Tu et al. 2005). The association between traits in high and low pollen-hoarding bees, therefore, suggests that honeybee foraging division of labor has evolved from an ancestral reproductive regulatory network involving IIS. With knowledge of the association between components of physiology and foraging behavior, we expected an overrepresentation of genes involved in IIS and ovarian development within the CIs for \u201cpollen\u201d QTLs.\nBehavioral genetics of defensive behavior\nHoneybee defensive behavior is not as thoroughly characterized as foraging behavior in terms of correlated physiological and sensory traits. Honeybees exhibit defensive behavior near the nest, but highly defensive bees may pursue for considerable distances away from the nest. Defensive behavior involves at least two tasks: guarding behavior at the hive entrance and flying out and stinging. Guards specialize in exploratory behavior in the nest entrance. They learn to recognize the hydrocarbon blend in the cuticles of their nestmates by olfaction, and they reject non-nestmates by biting or stinging. Only 10\u201315% of workers have been observed to guard the entrance during their lifetime (Moore et al. 1987). Both the number of days that individuals in a colony persist at guarding and the number of bees guarding the nest entrance correlate with the intensity of the stinging response (Arechavaleta-Velasco and Hunt 2003; Breed et al. 2004).\nMultiple sensory modalities influence stinging behavior. A moving visual stimulus usually is necessary to release stinging behavior (Free 1961). Substrate vibrations also increase the chance of a mass stinging response. Alarm pheromone also is an important component of colony defense. This pheromone blend is released from the sting apparatus during the act of stinging and as guards extrude their stings at the colony entrance in response to relevant stimuli. A transient increase in metabolic rate occurs after exposure of bees to alarm pheromone, and this increased rate genetically correlates with the defensiveness of colonies (Southwick and Moritz 1985; Moritz and Southwick 1987; Andere et al. 2002). Although the alarm pheromone components vary with strains of bees, QTLs influencing this variation were distinct from QTLs influencing stinging behavior (Hunt et al. 1999, 2003). Defensive strains of bees respond more quickly to all of these stimuli.\nCrosses involving highly defensive African-derived honeybees and low-defensive European races were used to map putative \u201csting\u201d QTLs based on colony-level stinging assays at hive entrances (Hunt et al. 1998). Subsequent crosses with stocks unrelated to the first studies confirmed that three \u201csting\u201d QTLs affect individual guarding behavior because guards from a backcross family were more likely to have the allele from the defensive parent of the F1 queen mother than were sisters chosen at random (Fig.\u00a03; Arechavaleta-Velasco et al. 2003). These sting QTLs also were associated with higher activity levels of colonies, which was assessed as their tendency to fly up or to sting when colonies were opened (Hunt et al. 1998). The QTL that had the largest effect on the phenotypic variance of colony stinging responses, sting-1, was shown to influence individual stinging behavior in two independent studies (Guzm\u00e1n-Novoa et al. 2002; Arechavaleta-Velasco et al. 2003). Higher activity levels, faster stinging responses, and greater sensitivity to stimuli exhibited by high-defensive strains suggest that sensory signaling pathways and heightened neuronal activity in the central nervous system (CNS) are involved in the defensive response, so we searched the QTL CIs for conserved genes with neuronal functions.\nFig.\u00a03Genetic and phenotypic correlations in defensive responses and specific QTLs. Arrows indicate significant associations between QTL genotypes and behavioral traits and between individual guarding behavior and colony stinging response\nMaterials and methods\nQTL mapping and confirmation\nQTLs\u2019 influencing traits related to foraging behavior or defensive behavior were mapped and confirmed previously (Hunt et al. 1995, 1998; Page et al. 2000; Rueppell et al. 2004, 2006), but new analyses were performed on the data to include additional markers, and in addition, \u201csting\u201d QTLs were reanalyzed by combining two traits as stated below. Maps were based on crosses that are appropriate for a haplodiploid, colonial species. For QTL detection, haploid drone progeny of an F1 queen were each backcrossed to sister queens (that were daughters of a single haploid drone). Colony phenotypes were correlated with inheritance of paternal marker alleles. Whole-colony phenotypes resulted from the behavior of many individuals, such as the number of stings in a leather patch in 1\u00a0min or the area of wax combs containing stored pollen. Confirmation studies all involved the use of F1 queens backcrossed to single drones and analyses of worker progeny for individual behavior and genotypes. Linkage maps that were used to identify candidate genes were constructed with JoinMap (3.0) and MapManager QTX software using the Kosambi mapping function (Van Ooijen and Voorrips 2001; Manly et al. 2001), and interval mapping was performed with MapQTL (v. 4.0; Van Ooijen et al. 2002). Interval mapping was performed as previously described, except that new analyses were used to map defensive-behavior QTL that combined two sets of phenotypic data. Individual z scores for the two correlated traits, the number of stings in 1\u00a0min, and ratings for the degree to which bees flew up at the beekeeper during colony manipulations were averaged to produce composite z scores used for interval mapping. The z scores were calculated using the formula z\u2009=\u2009(y\u2212\u03bc)\/\u03b4, in which y is the phenotypic value, \u03bc is the mean, and \u03b4 is the standard deviation. This provides a new trait value with a mean of zero and standard deviation of one. The colonies were rated on a relative scale of one to five based on the researchers\u2019 experience for the tendency of bees to fly up during colony manipulations. Ratings from two observation periods were averaged. This analysis resulted in somewhat reduced CIs and higher LOD scores for the three QTLs (3.84, 2.25, and 2.39, respectively for sting1, sting2, and sting3). A map with 1,154 markers was used to locate candidate genes for \u201csting\u201d QTLs, and two maps, each with about 400 markers, were used to locate \u201cpln\u201d QTLs. Markers in common between the dense map and other maps were used to interpolate the location of pln QTL and align QTL CIs with the physical map because more cloned markers were available from the dense-map population.\nSimple interval mapping was used rather than multiple factor interval mapping to determine CIs to have a more conservative (inclusive) search for candidate genes. The genomic region within 1.5 LOD value of the LOD-score peak was used to define each CI for QTL location. This corresponds to an approximate 97% CI for QTL location in a dense-marker map (Dupuis and Siegmund 1999). The LOD score is a log-likelihood estimator, for the probability that a QTL influencing the trait is present at a given map position (Lander and Botstein 1989). The honeybee genome assembly (v. 3.0) and positions of sequenced markers were used to identify predicted peptides that were evaluated for likely gene function.\nCloning and sequencing marker fragments\nSequences derived from DNA markers were obtained to integrate physical and genetic maps. More than 300 marker fragments not only from (primarily) amplified fragment length polymorphic markers (AFLPs), but also from random amplified polymorphic DNAs (RAPDs) and microsatellites linked to behavioral QTLs and throughout the genome, were cloned and sequenced. The first step in cloning was the excision of fragments from gels. For AFLPs from polyacrylamide gels, products were re-amplified and resolved on agarose gels to verify correct size before cloning with the TOPO-TA cloning kit and the pCR4-TOPO vector (Invitrogen, Carlsbad CA). RAPD marker fragments were excised from agarose gels and cloned into the same vector. Multiple sequences were obtained from each clone, and the consensus sequence was aligned with the genomic sequence scaffolds (HGSC 2006) using the nucleotide\u2013nucleotide basic local alignment search tool (blastn) algorithm with Pymood BLAST software (Allometra, Davis CA).\nRACE and cDNA cloning\nBefore expression analyses by quantitative real-time polymerase chain reaction (qRT-PCR), cDNA cloning was performed to confirm sequences and the gene prediction (location of introns and exons) and to provide information on sequence variation within some of the candidate genes. In the case of the serotonin receptor, this process provided complete sequence of the gene by making primers based on sequence of a putative G protein-coupled receptor (GPCR). Rapid amplification of cDNA ends (RACE) and cloning was performed using kits and manufacturers\u2019 instructions. Total RNA was extracted from individual bees using the RNAqueous kit (Ambion, Austin TX). The cDNA was synthesized using the SMART PCR cDNA Synthesis kit (SMART, simple modular architecture research tool, BD Biosciences, Palo Alto CA). The cDNA clones were obtained using the TOPO-TA kit and the pCR4-TOPO vector (Invitrogen). Clones were sequenced from multiple worker bees. Several sequence reads were obtained from each clone.\nComparative bioinformatics\nThis study is based on predicted peptides from the draft sequence (HGSC 2006). However, gene ontology (GO) terms and functional annotation of many of the homologous genes are incomplete. For this reason, further analyses were performed. Predicted peptides from the HGSC \u201cGlean3\u201d dataset that were found within QTL CIs by first using blastn to determine which sequence scaffolds from the genome assembly contained sequences corresponding to markers within the CI. Then, scaffolds were searched for presence of predicted peptides using protein\u2013nucleotide 6-frame translation (tblastn). Predicted peptides from the QTL CIs were used to search the nonredundant database (using protein\u2013protein BLAST [blastp]) at the National Center for Biotechnology Information website (http:\/\/www.ncbi.nlm.nih.gov). Protein domain information, gene similarities, and GO terms of homologs or orthologs were recorded. Literature and website searches were performed to assign putative functions based on reports from homologous genes of various species.\nQuantitative real-time PCR\nSome candidate genes for defensive behavior QTLs were tested for differential expression to better evaluate their potential for influencing behavior. These were chosen from a list of genes we were initially interested in based on functional studies in other species. Two families of worker bees were used: a low-defensive source and high-defensive source, each having a queen naturally mated to about 12\u201317 drones. The source families differed in the number of stings per\u00a0minute deposited in a leather target (0 for low line and more than 100 for the high-defensive source) using a standard assay (Hunt et al. 1998). Although it is not known at which life stage a gene might exert its influence on defensive behavior, bees that are 10- to 20-days old are much more likely to sting than younger bees. Workers were collected within 12\u00a0h of emergence from brood combs placed in an incubator or marked and co-fostered in an unrelated hive and then collected 20\u00a0days later. Co-fostering was performed to eliminate potential environmental effects between hives. All bees were frozen in liquid nitrogen and kept at \u221280\u00b0C before RNA extraction. Heads of eight to nine bees from each of the two families were removed, and RNA was extracted using the RNAqueous\u00ae kit (Ambion). RNA yield was quantified using Ribogreen\u2122 (Molecular Probes) dye on a fluorometer (Turner Biosystems, Sunnyvale CA). An aliquot of RNA was treated with DNAse (DNAfree\u00ae kit from Ambion) to remove any genomic DNA contamination before cDNA synthesis.\nThe cDNA template for qRT-PCR was generated according to Puthoff et al. (2005). For first-strand cDNA synthesis, the SuperScript First Strand cDNA Synthesis kit (Invitrogen) was used as per manufacturers protocol. The cDNA synthesis was monitored in a parallel tracer reaction as follows. A 5-\u03bcl aliquot was removed from each sample and mixed with 1\u00a0\u03bcl of a 1:5 dilution of 32P\u2013dCTP (dCTP, 2\u2032-deoxycytidine 5\u2032-triphosphate, Amersham, Piscataway, NJ) in water. The remaining 15\u00a0\u03bcl of each reverse transcriptase reaction and the corresponding 5-\u03bcl tracer reaction were incubated at 42\u00b0C for 2\u00a0h. Reactions were terminated at 70\u00b0C for 15\u00a0min then chilled on ice. The 32P-tracer reactions were used to quantify the amount of cDNA synthesized in the larger experimental samples. The 32P-tracer reactions were spotted onto DE-81 filters (Fisher, Fairlawn, NJ), dried for 10\u00a0min, and then washed four times for 4\u00a0min each in 0.5\u00a0M sodium phosphate buffer (1\u00a0M of monobasic and 1\u00a0M of dibasic in 4\u00a0l of water). After two 1-min rinses in water, filters were washed in 95% EtOH and allowed to dry. Each filter was placed in a scintillation vial containing 5\u00a0ml of ScintiVerse (Fisher), and radiation from the newly synthesized cDNA was quantified in a scintillation counter. Resulting counts were used to normalize the cDNA from the corresponding reverse transcriptase reactions to a final concentration of 10\u00a0ng per \u03bcl of sample.\nqRT-PCR was conducted on an ABI 7000 using the following mixture: 2\u00a0\u03bcl of normalized cDNA, 10\u00a0\u03bcl of 2X SYBR Green Mix (ABI, Foster City CA), and 0.25\u00a0\u03bcM of each primer in a 20-\u03bcl reaction. Reactions were carried out using the following cycling parameters: 50\u00b0C for 2\u00a0min, 95\u00b0C for 10\u00a0min, and 40 cycles of 95\u00b0C for 10\u00a0s and 60\u00b0C for 1\u00a0min. At the end of each run, a melt curve analysis was conducted to ensure primer specificity and purity of the PCR product. Relative mRNA levels were calculated by the standard curve method (User Bulletin 2: ABI PRISM 7700 Sequence Detection System) as described here. An aliquot was taken from each cDNA to construct a pooled sample. This pooled sample was serially diluted and subjected to qRT-PCR. The threshold cycle (Ct) for each dilution was plotted against its cDNA concentration (with an arbitrary starting quantity for the undiluted pooled sample assigned the value of 1) and used as the standard curve regression equation to generate the arbitrary expression values (AEVs). A standard curve was generated for each target gene on the same PCR plate that held the experimental samples. Linear standard curves, with a slope between \u22123.5 and \u22123.2 and R2 value of at least 0.98, were required for all primers used in this study. The AEVs were then normalized to the expression values of the eukaryotic initiation factor EIF-S8 for each bee. Two technical replicates for each bee were used, and results averaged before obtaining the family average. Normalized, average AEVs for each bee were analyzed by two-factor analysis of variance (ANOVA) without replication to compare transcript levels of the high-defensive family to the low-defensive family.\nResults and discussion\nBioinformatic analyses of putative gene functions and results of qRT-PCR allowed us to form hypotheses concerning gene networks involved in either foraging or defensive behaviors. Results of analyses and hypotheses concerning genes with potential to influence behavior are presented in the following two sections.\nCandidate genes for honeybee foraging behavior\nInspection of the predicted peptides (HGSC 2006) in genome sequence surrounding the mapped QTLs lends support to the hypothesis of the involvement of the IIS pathway in pollen foraging (Table\u00a01; Fig.\u00a04). Within, the pln-1 CI is the bee ortholog of the Drosophila gene bazooka, a gene involved in oocyte fate determination and influencing IIS through modulation of PI3K activity, as discussed below. Closer to the center of the CI is the honeybee ortholog of the fly gene, midway, which is involved in lipid metabolism and oocyte development. This gene is particularly interesting because it encodes diacylglycerol acyltransferase (DGAT) and changes in DGAT activity have been shown to alter sensitivity to IIS (Yu and Ginsberg 2004). Near the center of the CI of pln-1 is a gene encoding a protein with homology to class W phosphoinositolglycan-peptide (PIG-P), involved in the production of glycosylphosphatidylinositol (GPI) anchors to attach receptors to plasma membrane in various species. Lipids\/GPI anchors have insulin mimetic properties in some systems, where they modulate the IIS pathway by stimulating phosphoinositide-3-kinase (PI3K) activity (M\u00fcller and Frick 1999; M\u00fcller et al. 2002). \nTable\u00a01Annotation of peptides within 97% confidence intervals for foraging-behavior QTLsFig.\u00a04Hypothetical regulatory network influencing honeybee pollen foraging behavior modulated by insulin-like signaling and its effects on ovarian development. Inhibitory blue arrows bridging IIS with the reproductive physiology and hormonal dynamics of the honeybee denote the unique and mutually suppressive feedback interaction between vitellogenin and JII. This interaction is mediated via the allatoregulatory system (Guidugli et al. 2005, and references therein), which includes the IIS pathway (Flatt et al. 2005). ILPs Insulin-like peptides; PI phosphoinositol; PIP phosphoinositol phosphate; IRS insulin receptor substrate gene; PI3K phosphoinositide-3 kinase (class I or II); PIP5K 1-phosphatydylinositol-4-phosphate 5-kinase; PIG-P phosphatidylinositolglycan-peptide; PDK1 3-phosphoinositide-dependent kinase 1; PKB protein kinase B; HR46 honeybee ortholog of Dmel\/HR46; PTEN phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase; JH juvenile hormone\nThe pln-2 region contains two LOD-score peaks that were resolved into two CIs, suggesting separate but linked QTLs (Fig.\u00a02b). The CI with the higher LOD score contains a bee ortholog of a nuclear hormone receptor (Dmel\/HR46). Nuclear hormone receptors bind ligands (such as ecdysteroids) and enter the nucleus as transcription factors (Simonet et al. 2004). The bee ortholog of HR46 was differentially regulated in microarray experiments that compared bees with and without application of queen mandibular pheromone treatment, which inhibits worker ovarian development and delays onset of foraging behavior (Grozinger et al. 2003). The pln-2 region also contains the tyramine receptor AmTyr1, which has a higher expression level in young bees selected for high levels of pollen collection and storage (M. H. Humphries, unpublished data). A Drosophila mutant for the tyramine receptor (hono) is deficient in its behavioral response to olfactory stimuli, an observation that is relevant because honeybee foragers respond to floral scents and brood pheromone. In addition, tyramine increases responsiveness to sucrose and is found at elevated levels in the brains of egg-laying worker bees, a pattern that fits the behavioral and reproductive state of pollen foragers (Scheiner et al. 2004). The second CI associated with pln-2 contains an ortholog of Dmel\/skittles, encoding 1-phosphatydylinositol-4-phosphate 5-kinase (PIP5K), one of two phospholipid kinases known to produce phosphoinositol 4,5 phosphate (PI4,5P2), which is a key metabolite involved in IIS and the substrate of class I PI3K activity (Fig.\u00a04; Carricaburu et al. 2003). The Drosophila skittles mutant is deficient in oocyte polarity and nurse cell development, and the gene is essential for germ line development (Hassan et al. 1998; Table\u00a01).\nAt the most likely position for pln-3 is the honeybee ortholog of the fly gene for a class II PI3K (PI3K 68D). Class II PI3Ks have been shown to respond to insulin signals in mammals and use phosphoinositol to produce phosphoinositol phosphate, which influences glucose transport, a common effect of IIS (MacDougall et al. 2004; Shepherd 2005). In addition, three predicted peptides with glucose transport domains were found in the pln-3 CI. It is unknown whether class II PI3Ks can act directly on PI4,5P2 to produce PI3,4,5P3, thereby stimulating the primary downstream kinase in the IIS pathway 3-phosphoinositide-dependent kinase 1 (PDK1). However, the honeybee gene encoding PDK1 also lies within the pln-3 CI. PDK1 is a positive regulator of cell growth and size through its action on downstream protein kinase B (PKB; Fig.\u00a04; Rintelen et al. 2001), which, in Drosophila, is required for egg chamber development and influences egg follicle cell size (Cavaleire et al. 2005). The presence of these genes at pln QTLs suggests a possible network of genes influencing ovarian development and foraging (Fig.\u00a04). Linkage of a PI3K and PDK1 at pln-3 is intriguing, especially given the presence of PIG-P and the bazooka ortholog at pln-1 and the PIP5K ortholog near pln-2. PIG-P has the potential to activate PI3K, and thus PDK1, whereas bazooka has been shown to bind the protein tyrosine phosphatase PTEN. This phosphatase is a negative regulator of PI3K because it dephosphorylates PI3,4,5P3, and binding of dPTEN by the product of the bazooka modulates this process (Von Stein et al. 2005). The convergence of these pathways also suggests the potential for interaction between pln-3 and both the pln-1 and pln-2 regions, which has been observed experimentally (Rueppell et al. 2004, 2006).\nA search of the 10\u00a0cM window surrounding AmFOR (pln-4) revealed only three predicted peptides, one of which was the insulin receptor substrate (IRS). Although AmFOR was chosen as a candidate because of its influence on foraging behavior in Drosophila and association with foraging-related behavioral states in bees (Ben-Shahar 2005), the IRS could be partly or wholly responsible for the behavioral effects of this QTL. In Drosophila, ovarian expression of this gene is necessary for vitellogenesis, independent of the action of juvenile hormone and ecdysteroids (Richard et al. 2006).\nWhen the phenotypic architecture of foraging behavior is taken into consideration (Fig.\u00a01), the identification of genes encoding class II PI3K and PDK1 at pln-3 and other key components of IIS at pln-1, pln-2, and pln-4, supports the hypothesis that IIS is the upstream mediator of foraging division of labor (Fig.\u00a04). These IIS components do not constitute random distributions of genes. We obtained a rough estimate of the likelihood that a gene lies within the CIs for foraging behavior QTLs by comparing the genetic size of these four QTL regions relative to the genome (about 145:4600\u00a0cM, or 0.03). There are 12 Drosophila genes in Flybase with the GO term for insulin receptor signaling pathway (GO: 0008286), so the expected number of these genes within the CIs is 0.36. However, bee orthologs for two of these genes occur within foraging behavior QTL CIs, at least 5.5 times the expected number assuming independence of gene distributions. In addition, orthologs of four of these 12 genes were not in genome sequence assigned to chromosomes and so could not be sampled in our analyses. We also found at least four other genes known to interact with IIS, but not annotated as such with GO terms, and one nuclear hormone receptor within the QTL regions, which are characterized by epistatic interactions suggesting the presence of components of a common pathway. In contrast, we could not find any genes influencing IIS within CIs for QTLs that influence defensive behavior, although they represent a region of comparable size.\nCandidate genes for defensive behavior\nSting-1 had the highest LOD score for colony stinging response and was also the only QTL associated with initiation of stinging at the individual-bee level (Guzm\u00e1n-Novoa et al. 2002; Arechavaleta-Velasco et al. 2003; Fig.\u00a05a). Among the 50 predicted peptides in this 1.2-Mb region, at least nine are orthologs or homologs of genes reportedly involved in neuronal development and CNS activity (Table\u00a02). The interval includes the gene encoding 14-3-3 epsilon, a protein abundantly expressed in the CNS that modulates the activity of a number of kinases and ion channels (Berg et al. 2003). A Drosophila mutant for the ortholog (FBgn0020238) shows a failure to habituate to stimuli during nonassociative (unrewarded) learning trials (Skoulakis and Acevedo 2003), which is the type of learning that guard bees engage in when distinguishing nestmates from non-nestmates. The sting-1 CI contains six orthologs of Drosophila genes involved in CNS or antennal development. Of particular interest is the ortholog of the Dmel\/tango gene, which is an aryl hydrocarbon receptor nuclear transporter (ARNT)-like transcription factor. Tango is a basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) transcription factor that responds to hypoxia and is critical for the development of the fly neural midline and antennae. Other bHLH-PAS transcription factors act as heterodimers to sense light, temperature, oxygen, or endogenous hormones, and some have roles in regulating circadian rhythm (Roenneberg and Merrow 2003). The honeybee ortholog shares just 56% amino acid identity with tango and is diverged in the region important for activation of target genes, making it impossible to infer function (Sonnenfeld et al. 2005). The region also contains the gene for Huntingtin protein (htt), a large and unique protein with a complex structure that is conserved among metazoans. Expansions in htt cause Huntington\u2019s chorea. Htt interacts with many proteins and has roles in modulating neuronal transcription, intracellular neuronal transport, synaptic transmission, and morphology of dendrites (Harjes and Wanker 2003; Li and Li 2004). Finally, mRNA for a carboxylesterase of unknown function was more abundant in high-defensive bees compared to low-defensive bees (see Table\u00a03), and RACE sequences from seven workers revealed six amino acid substitutions and five alleles. Allelic variation for this gene was found in the population used to map sting-1, which is a necessary condition for a gene conferring variability in the behavior.\nFig.\u00a05Defensive-behavior QTLs were mapped based on the stinging response of colonies derived from crosses involving haploid drones of a F1 queen (European\u00d7African) each backcrossed to a European queen. Markers used for confirmation studies are indicated. Letters and numbers next to vertical bar represent linked markers. Sequenced markers are underlined. Dashed lines indicate approximate 97% CIs. Approximate positions of honeybee orthologs to Drosophila genes are indicated. a Sting-1. b Sting-2. c Sting-3Table\u00a02Annotation of peptides within 97% confidence intervals for defensive-behavior QTLsTable\u00a03Expression of candidate genes in high-defensive bees relative to low-defensive worker bees\nThe primary stimuli that elicit stinging behavior are moving visual targets and alarm pheromone. The sting-2 region contains two obvious candidates for modulation of response to these stimuli. At the most-likely position of the QTL is the bee ortholog of Drosophila arr1 (AmArr4, Fig.\u00a05b), an arrestin that binds metarhodopsin, the light-activated form of rhodopsin in the eye. Arrestins are involved in the desensitization of specific GPCRs and their recycling through clathrin-mediated endocytosis. But the so-called visual arrestins are also expressed in the antennae of Drosophila and are involved in olfaction. Fly arr1 mutants are insensitive to classes I and II odorants (Merrill et al. 2005). Near the edge of the CI is an ortholog of the fly gene encoding the metabotropic gamma-aminobutyric acid receptor (GABA-B-R1). GABA serves as the primary inhibitor of neuronal excitability in the CNS of both insects and mammals (Bettler et al. 2004).\nThe sting-3 CI contains only 17 predicted peptides in 0.96\u00a0Mb of DNA. Like sting-2, it has genes with the potential to modulate sensitivity to visual and olfactory stimuli. Results of cDNA sequencing of a putative GPCR revealed a 5-HT7 serotonin receptor (Am5HT7, Fig.\u00a05c). Serotonin influences associative learning and circadian rhythm in mollusks and insects, and both serotonin and GABA influence mood disorders in mammals (Hayley et al. 2005). Application of serotonin to the optic lobe of the bee brain reduced behavioral and neural responses to moving visual stimuli (Erber and Kloppenburg 1995). However, pharmacological experiments likely target all types of serotonergic neurons. The 5-HT7 receptor is just one of four serotonin receptor types known in insects and is unique in that it activates adenylate cyclase, resulting in increased levels of cyclic adenosine monophosphate (AMP) and activation of PKA. Therefore, activation of 5-HT7 receptors would likely cause a stimulatory response. Total serotonin levels have sometimes been reported to influence mammalian, insect, and crustacean aggressive interactions (Nelson and Chiavegatto 2001; Panksepp et al. 2003). Recent evidence suggests that 5-HT7 receptors modulate exploratory behavior and anxiety in mice (Takeda et al. 2005). The sting-3 region also contains one of the three catalytic subunits of cyclic AMP-dependent PKA, AmPKA-C1, a gene known to affect behavior in flies and mammals, including behavioral responses to alcohol, learning, and locomotor rhythm (Eisenhardt et al. 2001). Finally, the ortholog of homer, a dendritic gene involved in calcium signaling and synaptic plasticity, is closely linked to AmPKA-C1 (Diagana et al. 2002; Szumlinski et al. 2004).\nMeasurement of gene expression by qRT-PCR for defensive-behavior candidate genes showed several interesting trends. There were few statistically significant differences, because in this first screen, families were chosen that had divergent behavior but were genotypically diverse. Variation in expression between individuals was high, but numerical differences indicated a trend towards higher gene expression in older defensive bees. Only 14-3-3 Epsilon showed significantly higher transcript levels in the defensive family of bees. In contrast, the CG8165 ortholog, a putative jumonji-domain transcription factor, had numerically higher levels in newly emerged defensive adults, yet significantly lower levels in older defensive bees (Table\u00a03). This suggests an earlier peak in expression of this transcription factor in defensive bees. It was interesting that the mRNA levels of the GABA-B-R1 receptor, part of the major inhibitory pathway of neural signaling, were significantly lower in newly emerged defensive bees. Transcript levels of seven other genes showed trends for higher expression in defensive bees at the >1.3-fold level. At sting1, an unknown carboxylesterase showed the greatest numerical expression difference of all, but its levels also showed the highest interindividual variability, resulting in no significant difference between families. As previously stated, this gene also showed the highest allelic variability in cDNA sequence. At sting2, both the ortholog of Dmel\/discs lost and AmArr4 (arrestin) trended towards higher expression in defensive bees. Three other genes showed high numerical but not significantly different levels in defensive bees: oxysterol binding protein, the Dmel\/tango ortholog, and the Am5HT7 serotonin receptor (1.7-fold higher). Eight of the 19 genes tested appear to be more highly expressed in older defensive bees at 1.3-fold or higher although only one is significantly so. The fact that so many of these linked genes trended towards higher mRNA levels in defensive bees may mean that they are regionally regulated. More sampling of other bees and genomic regions is necessary. Some genes may be expressed at higher levels in high-defensive bees because of elevated metabolic rates (Harrison et al. 2005). Presumably, genes that influence behavior will show differences in expression levels of protein at some stage between low- and high-defensive alleles. However, demonstration of differences in transcript levels does not prove a causal connection to the phenotype. Conversely, failure to find a difference in transcript level does not disprove a causal connection to behavior and could be a consequence of not sampling the most relevant developmental stage, or that small differences in expression are still relevant to the phenotype.\nThe advantage of high recombination rates\nBy use of genome sequence and linkage maps, we reduced the list of candidate genes for honeybee foraging and defensive behavior from 10,157 (the current number of predicted peptides) to just 17 to 61 per QTL. As a consequence of high recombination rates, this level of resolution within 97% CIs was achieved in relatively large genetic distances, averaging 40\u00a0cM per CI. Table\u00a04 shows a comparison of our results to a study in which QTLs for ovariole number were mapped in Drosophila. Because of the low recombination rate in Drosophila, CIs totaling 158\u00a0cM represented half the genome in this cross and contained 9,100 genes (Orgogozo et al. 2006). But regions of similar size influencing foraging and defensive specialization in the bee contained only 113 and 128 genes, respectively. Other model organisms would provide less drastic comparisons. For example, a 40-cM window in mouse would be expected to contain about ten times as many genes compared to the bee, or about 510 genes (assuming a total of 20,000 genes evenly distributed over 2800\u00a0Mb and a recombination rate of 0.56\u00a0cm\/Mb). Of course, results will depend on local gene distributions and recombination rates. In practice, it is usually necessary to map a QTL to within 1\u00a0cM in mammalian species to reduce the list to five to ten candidate genes (e.g. Talbot et al. 2003; Flint et al. 2005). The genetic size of our QTL intervals compared to the size of the bee linkage map suggests that we should find 320 and 236 genes for the pln QTL and sting QTL, respectively, but instead, we found less than half this number (Table\u00a04). The expected number of genes based on physical distance is closer to the observed value. The discrepancy between these predictions can be explained by higher than average recombination rates in the QTL regions. The average recombination rates for the pln and sting QTL intervals are 28.2 and 32.8\u00a0cM\/Mb, respectively, but the genome average is 19\u00a0cM\/Mb. Consequently, there is a little less than one gene per centimorgan in the QTL regions. Our analyses do involve some degree of uncertainty. First of all, the estimate of recombination rate in QTL regions does not take into account sequence groups that have not yet been assigned to chromosomes (21% of the genome; HGSC 2006) that may lie between assigned groups. Therefore, recombination rates actually may be somewhat higher in QTL regions than we estimated. Sequence groups missing within CIs also may contain additional genes. In addition, the annotated set of 10,157 genes is a high-confidence set and eventually, may increase by several thousands as genes that are more novel in sequence are added (e.g., Drosophila has a gene count of about 13,000). Finally, our analyses focus on conserved genes of known function. A previous study of 81\u00a0Kb of sequence linked to sting2 revealed 13 expressed transcripts, none of which showed homology to known genes (Lobo et al. 2003). It cannot be ruled out that these behaviors may be at least partly influenced by completely novel genes.\nTable\u00a04Comparison of number of candidate genes in QTL confidence intervals from Apis and DrosophilaaInterval Size (cM)bGenusTraitNumber of QTLPredicted no. of genes based on genetic or physical sizecActual no. of genes145ApisPollen foraging4320222113107ApisDefensive behavior3236145128158DrosophilaOvariole number56,800\u20139,100aData for Drosophila are derived from a study of QTL for ovariole number in an interspecific cross between D. simulans and D. sechellia (Orgogozo et al. 2006). Data for Apis are from this report.bCumulative size of all confidence intervals.cCalculation of number of genes expected based on genetic distance was done by multiplying the proportion of total genetic distance that an interval contains by the number of predicted genes. An estimate based on physical distance in the honeybee is based on the current amount of genome sequence assembly (235\u00a0Mb).\nConclusions\nOur findings lead us to propose that foraging division of labor (Fig.\u00a01) is influenced by a gene network involving IIS (Fig.\u00a04). This is just a hypothesis, but a testable one. We also suggest that the genetically variable defensive responses of bees may be explained by allelic differences in neuronal transcription factors and genes involved in G protein-coupled signaling pathways. The potential involvement of the Am5HT7 serotonin receptor in defensive\/aggressive behavior implies that the bee may be used to elucidate a role of serotonin in novelty-seeking (guarding) behavior and that this behavior could be modified by specific agonists and antagonists of this receptor subtype. Linkage mapping at a finer scale using many single nucleotide polymorphisms combined with genome-wide expression assays could be the next step in finding the sequences responsible for behavioral variation. The bee is likely to become an important species in this process and may become the first invertebrate model for understanding how gene-regulation of life histories are remodeled by social evolution.","keyphrases":["behavior genetics","candidate genes","recombination rate","insulin-like signaling","aggressive behavior","foraging behavior","apis mellifera"],"prmu":["P","P","P","P","P","P","M"]}
{"id":"Eur_Radiol-3-1-1779628","title":"Local staging of rectal cancer: the current role of MRI\n","text":"With the advent of powerful gradient coil systems and high-resolution surface coils, magnetic resonance imaging (MRI) has recently extended its role in the staging of rectal cancer. MRI is superior to endorectal ultrasound, the most widely used staging modality in patients with rectal tumors, in that it visualizes not only the intestinal wall but also the surrounding pelvic anatomy. The crucial advantage of MRI is not that it enables exact T-staging but precise evaluation of the topographic relationship of a tumor to the mesorectal fascia. This fascia is the most important anatomic landmark for the feasibility of total mesorectal excision, which has evolved into the standard operative procedure for the resection of cancer located in the middle or lower third of the rectum. MRI is currently the only imaging modality that is highly accurate in predicting whether or not it is likely that a tumor-free margin can be achieved and thus provides important information for planning of an effective therapeutic strategy, especially in patients with advanced rectal cancer.\nIntroduction\nColorectal cancer is the third most common cancer worldwide [1, 2]. In the United States, about 145,000 new cases and 56,000 deaths were estimated for 2005 [1]. In recent years, mortality rates have decreased due to major changes in therapeutic management, in particular the standardization of the operative procedure and the introduction of adjuvant and neoadjuvant therapy [1].\nColorectal cancer primarily develops from adenomatous polyps over a period of 10\u201315 years, known as the adenoma-carcinoma sequence [3]. The incidence of polyps increases with age and the risk of malignant transformation of a polyp markedly increases with its diameter. The rate of malignant transformation is about 1% for polyps less than 1\u00a0cm in diameter, but 10% for larger ones [4, 5]. Around 40\u201350% of colorectal cancers are located in the rectum.\nRectal cancer is defined as a tumor whose aboral margin measured with the rigid rectoscope is 16\u00a0cm or less from the anocutaneous line. This distance serves to classify rectal cancer into tumors of the upper third (12\u201316\u00a0cm), the middle third (6\u201312\u00a0cm), and the lower third (<6\u00a0cm) [6] according to the UICC.\nThe mesorectal fascia is an important anatomic landmark for the diagnostic evaluation of local tumor extent [7] (Fig.\u00a01b). The fascia is a connective tissue sheath that encloses the rectum and the perirectal fatty tissue, including lymph nodes and lymphatic vessels down to the pelvic floor and acts as a natural barrier for tumor spread. The ability to visualize the mesorectal fascia on CT images has been described more than 20\u00a0years ago [8]. MRI currently is the most advanced staging modality able to depict the fascia and its relation to the tumor margins precisely. The following article will give an overview of the staging modalities currently used in rectal cancer staging, with an emphasis on the role of MRI and its significance for planning an effective therapeutical strategy for the individual patient.\nFig.\u00a01a Axial T2-weighted FSE (TSE) sequence of the pelvis depicting the layers of the rectal wall. The mucosa and submucosa can be visualized as a relatively hyperintense band (arrows). The hypointense line (arrowheads) represents the muscularis propria. b Axial T2-weighted FSE (TSE) sequence. The mesorectal fascia can be visualized as a thin line (arrowheads), enveloping the mesorectal compartment, containing the rectum, mesorectal fat, blood vessels, lymphatic vessels and nodes\nTherapeutic options\nSurgery\nThe anatomic position of the rectum in the true pelvis and its vicinity to adjacent anatomy, in particular the sphincter muscles, poses a challenge for the surgeon regardless of the surgical technique used. Surgical treatment of rectal cancer is a difficult balancing act between minimizing the risk of local recurrence and the preservation of anorectal and genitourinary function.\nTotal mesorectal excision (TME)\nThe introduction of standardized TME [9] has considerably improved prognosis in patients with cancer located in the middle or lower third of the rectum. Using this operative technique, the rectum is resected together with all surrounding lymphatic pathways, lymph nodes, mesorectal fatty tissue, and the mesorectal fascia while the parietal pelvis fascia and the pelvic splanchnic nerves (nervi erigentes) are spared. The widespread introduction of TME has markedly reduced the rate of non-continence-preserving abdominoperineal operations for rectal cancer.\nLocal excision is an option in patients with very small, well to moderately differentiated tumors that are confined to the mucosa and submucosa [10]. Techniques used for local excision are transanal surgical tumor removal and endoscopic microsurgical tumor ablation. Only a few patients are candidates for local excision (about 5%) and these must be selected with great care.\nCircumferential resection margin (CRM) and local recurrence\nThe local recurrence rate after surgery performed with curative intent ranges between 3% and 32% [11]. For TME, some studies report local recurrence rates that are markedly below 10% [12\u201314]. Lateral circumferential tumor extent is a much more important prognostic factor for local recurrence than longitudinal tumor extent. Incomplete resection of the lateral tumor margins is now considered the most important cause of local recurrence [15\u201317]. In a study by Quirke et al. [15], 83% of the patients with a positive CRM had local tumor recurrence. Hence, the topographic relationship of the tumor to the mesorectal fascia that serves as a natural barrier and anatomic landmark for TME is the most important criterion in local tumor staging for therapeutic decision making.\nAdjuvant\/neoadjuvant therapy\nThe aims of adjuvant or neoadjuvant therapy are to enable or facilitate total tumor resection even in advanced disease, to prevent local tumor recurrence, and to minimize the risk of distant metastases. Adjuvant or neoadjuvant therapy leads to downstaging of the tumor [18, 19] in terms of its T and N stages, and 20% of patients even show complete tumor regression (sterilization) [20].\nThe timing of adjuvant or neoadjuvant therapy is still a matter of debate. Based on the results of two large studies [21, 22], preoperative radiotherapy alone or combined radiochemotherapy is the preferred option for tumors of the middle or lower rectum in Europe. One of these studies, performed in Scandinavia [21], showed that a short cycle of preoperative radiotherapy reduces the local recurrence rate from 27% to 11%. The second study showed that even patients who underwent TME, which already has a lower recurrence rate than other operative approaches, benefit from preoperative radiotherapy [22]. Preoperative irradiation significantly reduced local recurrence compared to the group treated by TME only. In the United States, adjuvant therapy consisting of combined postoperative radiochemotherapy is favored for patients with T3 and\/or N1 tumors. [23].\nLocal tumor staging\nTumor staging is crucial for the prognosis and planning of therapy in the individual patient and aims at precisely determining the extent of the tumor as a basis for deciding whether surgery alone or surgery in combination with neoadjuvant therapy is the most suitable strategy. Of course, it is of great importance to avoid overtreatment or undertreatment of the patient. To reach a high level of accuracy in rectal cancer staging and to develop an adequate individual strategy for therapy, it is indispensable to establish a multidisciplinary team [24]. Rectal cancer staging is now mostly based on the TNM and UICC staging systems [6] (Tables\u00a01, 2), which have largely replaced the older Dukes classification. The most important anatomic structure on which staging is based using these staging systems is the lamina muscularis propria. While T1 rectal carcinomas are confined to the mucosa and submucosa, T2 tumors invade the muscularis propria (Figs.\u00a02a\u2013c and 3). A T3 cancer is defined as a tumor extending beyond the lamina muscularis propria (Figs.\u00a04, 5). However, none of the staging systems takes into account the fact that the T3 tumors are a very heterogeneous group, comprising tumors that just barely extend beyond the lamina muscularis propria as well as tumors that extend to or invade the mesorectal fascia (Figs.\u00a04, 5) without further subclassification. The therapeutically important topographic relationship of the lateral tumor margins to the mesorectal fascia is not taken into consideration. An adequate, state-of-the-art staging classification should be able to precisely determine this relationship and to predict whether a tumor-free CRM is likely to be achieved or not. In this way one would be able to differentiate patients with minimal mesorectal infiltration in whom neoadjuvant therapy is not mandatory from patients who would definitely benefit from neoadjuvant therapy because the mesorectal fascia is infiltrated or at risk. T4 rectal cancers are defined as tumors, that reach the peritoneal surface or adjacent organs (Figs.\u00a06a,b, 7, 8). \nTable\u00a01TNM classification for colorectal cancerTypeDescriptionT1Tumor involves submucosaT2Tumor involves muscularis propriaT3Tumor beyond muscularis propriaT4Tumor reaches peritoneal surface or invades adjacent organN0No involved nodesN1Up to three perirectal\/colic nodesN2Four or more perirectal\/colic nodesTable\u00a02UICC staging of rectal carcinomaStageDescriptionStage 0TisN0M0Stage IT1N0M0T2N0M0Stage IIAT3N0M0\u00a0BT4N0M0Stage IIIAT1, T2N1M0\u00a0BT3, T4N1M0\u00a0CEvery TN2M0Stage IVEvery TEvery NM1Fig.\u00a02a Paraxial T2-weighted FSE (TSE) sequence. T1\/2 rectal cancer. The relatively hyperintense intraluminal tumor (arrowhead) is confined to the rectal wall. Tumor invasion of the mesorectum is not visible. b Paraxial 3D-MPR and c intraluminal (virtual endoscopy) CT reconstuctions after rectal insufflation of CO2 showing the same tumor as aFig.\u00a03Paraxial T2-weighted FSE (TSE) sequence. Tumor of the rectal wall. Fibrous strands into the mesorectum represent desmoplastic reaction (arrow). A differention between desmoplastic reaction and tumor infiltration of the mesorectum can be difficultFig.\u00a04Paraxial T2 weighted FSE (TSE) sequence. A T3 rectal cancer breached through the muscularis propria (arrow) and invades the mesorectum. The tumor does not reach the mesorectal fascia (arrowheads). A tumor-free CRM can be expectedFig.\u00a05Paraxial T2-weighted FSE (TSE) sequence. A T3 rectal cancer widely invades the mesorectum. A tumor deposit (arrow) is located directly adjacent to the mesorectal fascia (arrowhead). A tumor-free resection margin cannot be predictedFig.\u00a06a Paraxial T2-weighted FSE (TSE) sequence and b sagittal T2-weighted FSE (TSE) sequence of a T4 cancer located in the upper third of the rectum invading the uterus (arrows)Fig.\u00a07Paraxial T2-weighted FSE (TSE) sequence of a low T4 rectal cancer with infiltration of the levator ani muscle (arrow)Fig.\u00a08Recurrent rectal cancer. Paraxial T1-weighted SE sequence with fat suppression after i.v. apllication of gadopentetate-dimelglumine at a dosage of 0.2\u00a0mmol\/kg body weight. The large extraluminal tumor shows central necrosis (arrowhead) and reaches the right pelvic wall\nStaging modalities\nEndorectal ultrasound (EUS)\nEUS is the oldest and most widely used imaging technique for evaluating the local extent of rectal cancer. EUS depicts the anatomic layers of the rectal wall with a high degree of accuracy and thus enables precise determination of the tumor extent in relation to the different wall layers. Reported accuracy rates of transrectal ultrasound in assessing the T stage are in the range of 69\u201397% [25\u201335]. EUS is most suitable for evaluating early rectal cancer while it is limited in assessing more advanced tumors. Although EUS allows the identification of transmural tumor growth, exact determination of the circumferential tumor spread and\u2014even more important\u2014depiction of the relation between the edges of the tumor and the mesorectal fascia is often not possible due to the limited scan depth caused by the high frequencies used. Moreover, the accuracy varies widely with the examiner\u2019s experience [28, 36].\nComputed tomography (CT)\nMost older studies report rather low accuracy rates of only 52\u201370% [32, 37\u201341] for T-staging by CT. It is remarkable that accuracy levels reported in studies including less advanced tumors were considerably lower compared with those including only advanced tumor stages. The poor accuracy of CT in the staging of superficial tumors is mainly attributable to the fact that these studies used conventional CT protocols with low spatial and contrast resolution. The accuracy has since been improved by the advent of the multislice technique (MSCT). In a study of 92 patients by Kulinna et al. [42], T-staging using MSCT was found to have an accuracy of 86%, while Filippone et al. [43] found an accuracy of 83% in a study of 41 patients. If one takes into account that four-row CT scanners were used in these studies, it is evident that further improvement is to be expected from state-of-the-art CT scanners with up to 64 detector rows that are already in use today. Hence, the role of MSCT in the local staging of rectal cancer remains to be defined. CT is superior to both EUS and MRI in that the scan typically covers the entire abdomen and pelvis and thus also allows evaluation of the liver, the most important target organ of hematogenic metastatic spread of rectal cancer.\nMRI\nIt is undisputed that MRI is the imaging modality with the highest soft-tissue contrast. This is why MRI is also used for staging rectal cancer. However, initial results with MRI were disappointing, with accuracies in T-staging reported in older studies ranging between 58 and 74% [39, 44\u201346]. These rather poor results are primarily due to the poor spatial resolution achieved with the whole-body coil systems used in these studies. When endorectal coils are used, MRI has similar accuracies as EUS [31, 47\u201349]. MRI using endorectal coil systems is comparable to EUS in that it allows highly accurate differentiation of the layers of the intestinal wall. However, endorectal coils also have a number of disadvantages. As with EUS, the field of view (FOV) is rather small and only allows adequate evaluation of early stages of rectal cancer because the evaluation of surrounding pelvic anatomy is limited. In patients with advanced tumors, insertion of the coil system may be impossible or is very painful. Another disadvantage is the high cost of endorectal coils, which are usually disposable.\nThe advent of powerful gradient systems and, above all, the development of high-resolution phased array surface coil systems in recent years brought the breakthrough in the staging of rectal cancer by MRI. The use of these phased-array surface coils combines a very high spatial resolution with a large FOV that allows not only detailed evaluation of the intestinal wall but also depicts surrounding anatomy including the mesorectal fascia.\nImaging technique\nRectal cancer staging by MRI is rather fast and straightforward. No special patient preparation is required. Some authors recommend administration of a positive or negative enteral contrast medium, but this seems not to be necessary as suggested by current data in the literature. A study published only recently even indicated that rectal distension significantly reduces the distance between the rectal wall and the mesorectal fascia and that this might impact on the ability of MRI to predict accurately the distance between the tumor and the potential resection margin [50].\nAt our department, we administer a spasmolytic agent (butylscopolamine) at a dose of 20\u201340\u00a0mg to prevent artifacts caused by peristalsis of the small intestine and to distend the sigmoid and rectum. The agent has a short half-life and is therefore injected intramuscularly immediately before MRI.\nFor efficient planning of the pulse sequences to be employed, the radiologist performing the examination should beforehand obtain information about the approximate tumor localization (distance from anocutaneous line in cm) from the referring surgeon and ask the patient about any previous surgery or diseases of the pelvic organs.\nThe patient is positioned comfortably on the back and a phased-array surface coil is placed on the pelvis in such a way that the lower edge of the coil comes to lie well below the pubic bone. The coil is kept in place with belts and the patient is then advanced head-first into the bore of the magnet.\nFollowing the usual localizer scans, a sagittal T2-weighted half-Fourier single shot turbo spin-echo (SSFSE, HASTE) sequence with a large field of view (FOV) should be acquired to obtain an overview and for planning of the subsequent sequences (e.g. TR \u221e, TE 62\u00a0ms, slice thickness 5\u00a0mm, FOV 255\u00d7340\u00a0mm, matrix size 116\u00d7256, voxel size 2.2\u00d71.3\u00d75\u00a0mm). Precise tumor localization is then achieved with an axial T2-weighted fast spin-echo (FSE) or turbo spin-echo (TSE) sequence with a large FOV and a slice thickness of 5\u00a0mm (e.g. TR 4,170\u00a0ms, TE 98\u00a0ms, FOV 300\u00d7220\u00a0mm, matrix 282\u00d7512, voxel size 0.8\u00d70.6\u00d75\u00a0mm). At the core of the examination is a high-resolution T2-weighted TSE sequence with a small FOV and a slice thickness of 3\u00a0mm (e.g. TR 3,570\u00a0ms, TE 68\u00a0ms, FOV 180\u00d7180\u00a0mm, matrix 179\u00d7256, voxel size 1.0\u00d70.7\u00d73\u00a0mm). It is mandatory to place the slices perpendicular to the longitudinal axis of the tumor or the intestinal lumen in the vicinity of the tumor. With this sequence, it is possible to precisely evaluate the tumor and its relationship to the intestinal wall, mesorectal fascia, the pelvic organs, and possibly also to the peritoneal fold. Moreover, mesorectal lymph nodes in the immediate vicinity of the tumor can be evaluated. For visualization of more distant lymph nodes in our institution a T1 to proton-density-weighted two-dimensional (2D) TSE sequence with a short echo train length (e.g. 3 or 5) in axial orientation (e.g. TR 1,980\u00a0ms, TE 10\u00a0ms, slice thickness 5\u00a0mm, FOV 300\u00d7225\u00a0mm, matrix 219\u00d7512, voxel size 1\u00d7 0.6\u00d75\u00a0mm), which covers the entire area up to the aortic bifurcation is used. Alternatively, a T1-weighted 3D gradient-echo sequence can be used for this purpose, allowing for the reconstruction of thinner slices. Possible infiltration of the anal sphincter muscles in patients with low tumors is evaluated using a coronal T2-weighted FSE (TSE) sequence (e.g. TR 3,570\u00a0ms, TE 68\u00a0ms, FOV 180\u00d7180, matrix 179\u00d7256, voxel size 1.0\u00d70.7\u00d73\u00a0mm) positioned parallel to the longitudinal axis of the anal canal. Current data in the literature suggests that intravenous contrast medium administration does not improve staging of rectal tumors by MRI [51, 52].\nSince differentiation with the T2-weighted sequences is based on the contrast between the high-signal-intensity mesorectal fatty tissue and the rather low signal intensity of the tumor, spectral fat suppression techniques are not needed. The duration of the MRI protocol as just outlined is about 25\u201330 min, including planning.\nT-staging\nAlthough the introduction of phased-array coil systems has improved the accuracy of MRI in staging rectal cancer, even more recent studies report accuracies of only 67\u201386 % for T-staging [53\u201356]. These disappointing results are primarily due to the poor differentiation of T1\/2 cancer from so-called borderline T3 cancer, where it is often not possible to distinguish true mesorectal tumor invasion from desmoplastic reactions (Fig.\u00a03) [49, 54, 57]. Desmoplastic reactions are reactive tissue alterations which often occur in the immediate surrounding of tumors, most frequently resulting in fibrotic extensions that may contain tumor cells or not. The failure to differentiate between desmoplastic reactions and tumor growth is not specific to MRI but is also a well-known problem in rectal cancer staging with EUS [27]. Clinically and therapeutically, however, this differentiation is of minor importance. As already mentioned, it is much more important to precisely describe the relationship of the tumor to the mesorectal fascia, representing the anticipated resection plane for TME in order to assess the likelihood of a tumor-free CRM. Several recent studies have confirmed that MRI is highly suited to provide this information [54, 57\u201360]. In a study of 43 patients, Bissett et al. [59] found good agreement between preoperative MRI and histopathology with regard to the demonstration of tumor penetration through the mesorectal fascia (accuracy: 95%). These results are underlined by the studies of Beets-Tan et al. [54, 61], who investigated 76 patients and likewise found preoperative MRI to be highly accurate in assessment of the CRM. The agreement was 100% in T4 tumors, and 97% and 93% for both readers in tumors with a histologically determined tumor-free CRM >10\u00a0mm. Regression analysis for histologically determined margins of 1\u201310\u00a0mm demonstrated that a tumor-free resection margin of 2\u00a0mm was predicted with an accuracy of 97% if the distance between tumor and mesorectal fascia measured by MRI was at least 6\u00a0mm. It is noteworthy that this study likewise showed only moderate results with regard to T-staging (accuracy of 83% and 67% for the two readers) [54, 61]. In a study of 98 patients published by Brown and co-workers in 2003, the agreement between MRI and histology in assessment of the CRM was 92% [60]. These figures indicate that MRI allows accurate prediction of the CRM status after resection. The expected CRM can be described as involved if tumor invasion of the mesorectal fascia is visible or the tumor has a proximity of 1\u00a0mm or less to the mesorectal fascia. A tumor-free CRM can be assumed with a high degree of accuracy if the shortest distance from the maximum tumor extension, a mesorectal tumor deposit or a suspect lymph node in the mesorectum is more than 6\u00a0mm [54]. The role of tumors that extend towards the mesorectal fascia to a distance of less than 5\u00a0mm on MR images remains controversial.\nThe study by Brown et al. [60] also suggests that other important prognostic factors besides the CRM are the infiltration of extramural veins and possible infiltration of the peritoneal fold and that these can also be identified by preoperative MRI.\nA study by Oberholzer and co-workers published in 2005 has shown that parallel imaging techniques do not compromise diagnostic accuracy with regard to the assessment of the CRM, but can considerable shorten the examination [62].\nN-staging\nIdentification of metastatic lymph nodes is the greatest challenge in preoperative staging of rectal cancer, regardless of the modality used (Figs.\u00a09, 10, 11). Exact staging is important because the number of metastatic nodes has been shown to affect the prognosis [63]. Involvement of lymph nodes in the vicinity of the mesorectal fascia is associated with a higher risk of local recurrence [16]. In patients with metastatic nodes outside the mesorectal fascia, extended lymph node resection with additional removal of the internal iliac nodes becomes necessary [64]. This lymph node group is not removed when regular TME is performed. A special problem associated with identifying lymphatic involvement in rectal cancer is that lymph node size is not a reliable criterion for metastatic involvement because micrometastasis in normal-sized lymph nodes is common [65, 66].\nFig.\u00a09Sagittal T2-weighted FSE (TSE) sequence. The enlarged mesorectal lymph node (arrow) shows heterogenous signal intensity indicating tumor invasionFig.\u00a010Axial PD-weighted FSE (TSE) sequence. A large lymph node metastasis (asterisk) located below the aortic bifurcation in a patient with rectal cancerFig.\u00a011Axial T2-weighted FSE (TSE) sequence. Inguinal lymph node metastases (arrows) in a patient with low rectal cancer\nThe accuracy rates reported in the literature for N-staging by the different imaging modalities vary widely (EUS: 61\u201380% [10, 25, 26, 30\u201332, 34, 35, 37, 67], CT: 56\u201379% [32, 39, 68, 69], MRI: 57\u201385% [32, 39, 47, 53, 55, 60]. In a current meta-analysis including 84 studies, Lahaye et al. [70] found EUS to be slightly superior in assessing nodal status, but there were altogether no significant differences between the three staging modalities investigated. In summary, these results suggest that none of the imaging procedures currently in use enables reliable detection of metastatic lymph nodes.\nIn their study of MRI with histologic correlation, Brown et al. [71] identified an irregular contour and inhomogeneous signal to be the most reliable MRI criteria for lymph node metastasis (Fig.\u00a09).\nFuture perspectives\nUSPIO\nA new promising approach to detect metastatic lymph nodes by MRI is imaging in combination with ultrasmall superparamagnetic iron oxide particles (USPIO) as a contrast medium for systemic MR lymphography (Fig.\u00a012a,b). Following intravenous administration, the particles are phagocytozed by nodal macrophages and, due to susceptibility effects, cause a signal decrease in normal or reactively changed lymph nodes on T2- and T2*-weighted images, which are usually acquired 24\u00a0h after administration of USPIO [72]. USPIO agents are currently under clinical evaluation and are not yet clinically available. Initial results of a study investigating this new approach in mesorectal lymph nodes are promising [73]. Further studies are needed to show whether USPIO can significantly improve lymph node staging by MRI.\nFig.\u00a012a Axial PD-weighted sequence of the pelvis. Two small lymph nodes are visible adjacent to the iliac vessels (arrows). b Axial T2*-weighted gradient echo sequence acquired 24 hours after i.v. infusion of USPIO (Sinerem). The two lymph nodes (arrows) show homogenous signal decrease indicating normal lymphatic tissue. As USPIO agents are currently under clinical evaluation and are not yet clinically available, this image was acquired during a clinical trial\nWhole-body MRI\nThe recent introduction of powerful whole-body MRI systems enables imaging of the whole body in a single session through repeated table movement. Several studies have already demonstrated the benefit of this approach for a variety of diagnostic queries in oncologic patients [74\u201377]. This technique may also be used for rectal cancer staging in the future and allow local staging and whole-body staging in a single session. In this way it would become possible to also evaluate the liver as the primary target organ of hematogenic spread of rectal cancer. The potential of parallel imaging to shorten the examination that has already been mentioned would be of particular significance in this respect [62].\nDiffusion\/perfusion-weighted MRI and PET\nIt has long been known that the pathophysiology and aggressiveness of a tumor are determined not only by the macroscopic tumor extent but also by other factors such as tumor microcirculation and angiogenesis. Several studies have shown the potential of diffusion- or perfusion-weighted imaging to indirectly determine these factors and to thus predict the response to adjuvant or neoadjuvant therapy [78\u201381]. However, extensive further research is necessary before the routine clinical use of these new techniques.\nRecent studies have indicated that PET is able to predict response to neoadjuvant treatment of locally advanced rectal cancer with a high degree of accuracy [82].\nConclusion\nThe advances that have been made in the treatment of rectal cancer in recent years and that have considerably improved the prognosis of affected patients rely on differentiated pretherapeutic tumor staging. Despite its known limitations in T-staging, MRI is currently the only imaging modality that enables highly accurate evaluation of the topographic relationship between lateral tumor extent and the mesorectal fascia and to thus make a prediction about the CRM. In this way it is possible to carefully select those patients who will benefit from neoadjuvant therapy and to avoid overtreatment or undertreatment.","keyphrases":["staging","rectal cancer","mri","rectal carcinoma"],"prmu":["P","P","P","P"]}
{"id":"Pharm_World_Sci-3-1-2039831","title":"Is melatonin helpful in stopping the long-term use of hypnotics? A discontinuation trial\n","text":"Objective To find out if administration of melatonin facilitates discontinuation of benzodiazepine (BD) therapy in patients with insomnia.\nImpact of findings on practice\nstopping long-term use of benzodiazepine hypnotics is difficult\nusers of a high daily dose might be more responsive to the effect of melatonin on the discontinuation of benzodiazepines\ninvestigators should take into account the difficulty of recruiting patients for studies on discontinuing the use of benzodiazepines\nIntroduction\nThe point prevalence of the use of sleeping medication, often benzodiazepines (BD), in countries such as the Netherlands is approximately 6% [1], with over one-third being long-term users. This does not meet the standard of the Dutch Society of General Practitioners, which advises that hypnotics should not be prescribed longer than for a period of 10\u00a0days [2]. After a minimal intervention strategy such as sending an educational letter, one in every four users stops [3\u20135]. As an aid to discontinuation, several treatments have been used, e.g., certain drugs [6] and cognitive therapy [7], without added value.\nThe circadian rhythm in humans is controlled by the endogenous biological clock, located in the suprachiasmatic nuclei of the hypothalamus and influenced by light and darkness to the body [8]. Suprachiasmatic projections regulate the pineal gland and its production of melatonin. The primary physiological function of melatonin is to convey information about the daily cycle of light and darkness to the body\u2019s physiology. Melatonin production changes with age and is lower in the elderly. Its secretion is high during the night and has an effect on falling asleep. There is evidence that low doses of melatonin improve initial sleep quality in selected elderly insomniacs [9]. Some BDs have shown to have a negative effect on melatonin production in the pineal gland [8].\nGarfinkel et\u00a0al. [10] investigated stopping the chronic use of BD sleeping medication with the help of melatonin (5\u00a0mg). In their randomized discontinuation trial melatonin appeared efficacious: 14 of 18 (78%) patients on melatonin stopped their BD compared to 4 out of 16 (25%) in a placebo control group.\nA study in elderly patients of Cardinali et\u00a0al. [11] does not support efficacy of melatonin to reduce the use of benzodiazepines in low doses. Forty-five patients were randomized to receive either melatonin (3\u00a0mg) or placebo for six\u00a0weeks. In two steps BD was tapered off and stopped after four\u00a0weeks. Several sleep parameters were assessed and found not to be different for both groups.\nThe administration of melatonin as a sleeping medication is a contradictory tool to help people discontinue use of BDs.\nAim of the study. The main research question in our trial was whether melatonin is helpful in discontinuing the use of BD sleeping medication, looking at the stopping rate. Secondary objectives are finding the possible influence of other variables on stopping, such as age, gender, period of BD use, and dependence on BD. Besides, we investigated a putative shift of addiction, e.g., to alcohol or smoking.\nMethod\nOur study was designed as a randomized placebo-controlled discontinuation trial. We gathered data in nine general practices (2001\u20132004). The patients, their GPs and the principal investigator were blinded for the study medication. All practices were located in Maastricht, in the south of the Netherlands.\nIncluded are adult patients who used BD as a sleeping medication for more than three\u00a0months (defined as long-term use) with a minimum use of three\u00a0days per week. Patients were selected via their GPs, in six practices through the desk prescriptions, in two on the basis of a pharmacy print out, and in one through the problem list in the medical file on the ICPC-code P17 (drug addiction) [12]. Exclusion criteria were the use of more than one BD at the same time, use of another type of sleep medication, use of stimulants and, according to their GP, alcohol misuse, serious mental\/somatic disease or unfit to participate.\nAll patients who were selected received an invitation letter from their GP that also informed them of the disadvantages of long-term use of BD. A short questionnaire was added to check the inclusion criteria. All respondents meeting the inclusion criteria received by mail a questionnaire (T0): about the use of sleeping medication, sleep complaints (SWEL) [13], sleep quality [14], general health experience (RAND-36) [15], BD dependence (Bendep-SRQ) [16] and habit formation (amount of alcoholic consumptions and\/or cigarettes\/cigars per day). The sleep wake experience list (SWEL) is a validated instrument to study chronic sleep complaints (insomnia, hypersomnia or the combination of those two). The benzodiazepine dependence self-report questionnaire (Bendep-SRQ) is a validated instrument to measure BD dependency, scoring in subscales the degree of awareness of problematic BD use, degree of preoccupation with respect to the availability of BDs, degree of lack of compliance with the therapeutic BD regimen, and the degree of unambiguity of experienced BD withdrawal (Bendep-SRQ, scale 4)\nAt the end of the questionnaire, we asked specifically whether the patient was willing to discontinue use of BDs. Participants had to send back an informed consent form. All participants were checked for liver and kidney function and their urine was tested for BD with a fluorescence polarization immunoassay (FPIA) [17].\nAfter returning the informed consent, patients were included for random allocation.\nThe use of sleeping medication at the start of the study was compared with the defined daily dose (DDD) of the patient\u2019s hypnotic. It was expressed as a quotient of prescribed daily dose (PDD) relative to the defined daily dose, i.e., PDD\/DDD. The PDD was based on the completed questionnaire (T0). We created three categories using this quotient: low (quotient\u00a0\u2264\u00a00.5), average (0.5\u00a0<\u00a0quotient\u00a0<\u00a01.0) and high use (quotient\u00a0\u2265\u00a01.0).\nAll participants received a stopping scheme of their BD via their own pharmacy. Their BD was converted to an equivalent dose of diazepam [18] that was stabilized for two\u00a0weeks and then further converted every two\u00a0weeks to 75%, 50%, 25%, 12.5% and 0% of the original dose. We added 5\u00a0mg melatonin or placebo which had to be taken 4\u00a0h before patients went to bed. After stopping BD we continued the use of melatonin or placebo for six\u00a0more weeks. Furthermore a questionnaire was sent at 18\u00a0weeks (T1), 26\u00a0weeks (T2) and 52\u00a0weeks (T3) after the beginning of the discontinuation. All questionnaires contained the same set of instruments. Also urine samples for BD determination were required at T1 and T3.\nResults\nThe GPs selected 503 patients for our trial, 138 males and 365 females. Of these, 80% responded to our invitation letter (Fig.\u00a01). Six patients reported to have stopped their use of sleeping medication already. Sixteen patients used two or more BDs, and were excluded from the study. Of all responders, 124 appeared to be willing to participate.\nFig.\u00a01Selection of patients and response\/non-response; participants\nReceiving the first questionnaire of these 124 patients, 22 did not respond, 56 decided not to participate and eight patients did not meet the inclusion criteria (six of whom had already stopped the BD use). At the end, only 38 participants (16 males and 22 females) were indicated to take part in the study. They were randomly allocated to melatonin or placebo.\nThe participants compared to the non-participants were more often males (40% vs. 25%), and had a lower age (<50\u00a0years, 16% vs. 5%) and less elderly (>80\u00a0years, 5% vs. 18%). Of the total group, 76% were 60 years or older.\nThe characteristics of all participants are listed in Table\u00a01. No differences were found between the melatonin and the placebo group. The sleep quality was rated bad in 58% of the cases. According to the SWEL 10 of the 38 participants reported that they did not have any sleeping complaints (any more) Of the other 28, 18 complained of insomnia, three of hypersomnia, and seven of a combination of both. Most participants (76%) used a low dose of BD (PDD\/DDD\u00a0<\u00a01.0). According to the score on the Bendep-SRQ list, 61% of the participants were aware that they had problematic BD use.\nTable\u00a01Characteristics of the 38 participants, and for melatonin and placebo separatelyMelatonin group n\u00a0=\u00a020 numbers (%)Placebo group n\u00a0=\u00a018 numbers (%)Total participants n\u00a0=\u00a038 numbers (%)GenderMale6 (30)10 (56)16 (42)Female14 (70)8 (44)22 (58)Age in years<503 (15)3 (17)6 (16)50\u2013593 (15)3 (17)6 (16)60\u2013696 (30)7 (39)13 (34)70\u2212797 (35)4 (22)11 (29)\u2265801 (5)1 (5)2 (5)Health insuranceNational health insurance16 (80)18 (100)34 (90)Private insurance4 (20)\u20134 (10)Sleep qualityGood7 (35)5 (28)12 (32)Bad11 (55)11 (61)22 (58)2 missing2 missing4 missingPeriod of use of BD<1\u00a0year\u2013\u2013\u20131\u22125\u00a0years7 (35)7 (39)14 (37)6\u22129\u00a0years4 (20)4 (22)8 (21)\u226510\u00a0years9 (45)6 (34)15 (39)1 missing1 missingDaily use of BD (PDD\/DDD)\u22640.5 (low)11 (55)14 (78)25 (66)0.5\u20131.0 (moderate)4 (20)\u20134 (10)\u22651.0 (high)5 (25)4 (22)9 (24)SmokingYes9 (45)7 (39)16 (42)No10 (50)11 (61)21 (55)1 missing1 missingAlcohol useYes15 (75)15 (83)30 (79)No5 (25)3 (17)8 (21)Body mass index (BMI)Normal8 (40)10 (56)18 (47)BMI\u00a0>\u00a02512 (60)8 (44)20 (53)Problematic BD use (subscale of Bendep-SRQ)Low6 (30)2 (11)8 (21)Average3 (15)2 (11)5 (13)High11 (55)13 (72)23 (61)1 missing1 missingNone of the differences were statically significant at the 5% level (\u03c72-test)\nTwenty-two participants had tried to stop BD use in the past, and 12 of them experienced a high level of withdrawal symptoms.\nIn total 21 patients discontinued the use of BD after the taper off: 12 in the melatonin group and nine in the placebo group (Table\u00a02). After one\u00a0year 15 patients remained (40%) without BD sleeping medication (eight out of 20 in the melatonin group and seven out of 18 in the placebo group). This difference was not significant.\nTable\u00a02Follow up after one\u00a0year. Use of BD sleeping medication after the taper off and during follow up. Separate data for the melatonin and placebo groupStopped on T1Did not resume on T2Definitely stopped on T3Melatonin *** on T3Melatonin group (n\u00a0=\u00a020)12108*2Placebo group (n\u00a0=\u00a018)977* (1**)1Total2117153T1\u00a0=\u00a0at the time of stopping the use of melatonin or placebo (six\u00a0weeks after the taper off of BD)T2\u00a0=\u00a0after six\u00a0monthsT3\u00a0=\u00a0after one\u00a0year*\u00a0One person in the placebo group and one in the melatonin group stopped participation in the trial after the taper off. According to their GPs, they no longer used any BD at T3**\u00a0One person had resumed using BD at T2, but stopped again at T3. He is not counted as a definite stopper***\u00a0Three persons received melatonin from their GP after discontinuing the use of melatonin in the trial\nThe urine analysis showed that two patients in the placebo group, who had reported that they had stopped using BD, were still positive. They had received new prescriptions of BD at one and three\u00a0months after T3, although at a lower dose.\nAt the end of the trial three persons used melatonin, two in the melatonin group and one in the placebo group. Of the patients who had stopped, four had switched to a herb product (valerian, Valdispert\u00ae) or homeopathic product (Nervogin\u00ae, passiflora).\nThe patients who had totally discontinued BD use were compared with the non-stoppers in terms of gender, age, weight, period of use, PDD, sleeping quality, general health experience and awareness of problematic use. We found small differences, which were not statistically significant at the \u03b1\u00a0=\u00a00.05 level (Table\u00a03). Of the patients who had already used BD for five\u00a0years or more, 35% stopped compared to 43% of the patients with a shorter period of use. For high daily use (PDD\/DDD\u00a0\u2265\u00a01), this was 33% compared to 41% for daily use <1. Of the patients who were aware of their problematic use, 54% stopped compared to 29% among the others.\nTable\u00a03Putative indicators of discontinuation of benzodiazepines (numbers and percentages)Definite stoppersNon-stoppersTotalMale6 (38%)10 (62%)16Female9 (41%)13 (59%)22Age <656 (33%)12 (67%)18Age 65+9 (45%)11 (55%)20Body mass index <256 (33%)12 (67%)18Body mass index \u2265259 (45%)11 (55%)20Period of use <5 year6 (43%)8 (57%)14Period of use \u22655\u00a0year8 (35%)15 (65%)23 (1 missing)PDD\/DDD\u00a0<\u00a01.012 (41%)17 (59%)29PDD\/DDD\u00a0\u2265\u00a01.03 (33%)6 (67%)9Awareness of problematic use, low7 (29%)17 (71%)24Awareness of problematic use, high7 (54%)6 (46%)13 (1 missing)General health, low6 (40%)9 (60%)15General health, high9 (41%)13 (59%)22 (1 missing)Sleeping quality, bad8 (36%)14 (64%)22Sleeping quality, good6 (50%)6 (50%)12 (4 missing)None of the differences were statistically significant at the 5% level (\u03c72 test)\nFour of the 19 patients who had stopped at T1 reported a high level of withdrawal symptoms during the taper off. We have no data for two people because they did not complete the questionnaire.\nTo measure the effects of discontinuation we compared the scores of T0 (initial measurement) with those at T3 (final measurement). In terms of health experience, there were no differences between the stoppers and non stoppers according to the scores on nine scales of the RAND questionnaire, sleeping quality, alcohol consumption, or smoking.\nDiscussion\nIn our trial, we could not confirm the positive outcome of Garfinkel et\u00a0al. [10]. Our findings are in agreement with those of Cardinali et\u00a0al. [11].\nThe success rate of our study was 40%, higher than of that of spontaneous stopping (10\u201315%) [19, 20] and stopping after an informative letter or minimal intervention strategy (25%) [3\u20135]. This may be caused by the use of our taper-off program, the selection by some GPs and the selective response, including the awareness of problematic use (in two-thirds of the participants). In general, readiness to stop using BD sleeping medication was low. In our trial we fell well short of the discontinuation rate in the melatonin group of the trial of Garfinkel et\u00a0al. (14 out of 18).\nAfter a minimum intervention, it appears that of the stoppers after two\u00a0years only half remain without this medication [21]. In our trial, 55% of the participants had stopped shortly after the taper off. However, after a year this dropped to 40%. We omitted any supportive measures during follow up as much as possible in order to measure the pure effect of melatonin.\nThe gender difference in our selected population (n\u00a0=\u00a0503) is comparable to figures in the literature [1]. BD use among Garfinkel et\u00a0al.\u2019s patients was higher than ours (only nine of our patients had PPD\/DDD\u00a0\u2265\u00a01.0). It has been shown that patients on a rather low dose of BD discontinue their use more easily and resume their BD use less rapidly [21]. For patients with a high daily use melatonin may still be valuable. If there will be future discontinuation trials there is reason to especially include patients who use a high dose of BD.\nIn our trial, in contrast to that of Garfinkel et\u00a0al., at the end only 9% still used melatonin. Four participants changed their use to a homeopathic or phytotherapeutic medication, which indicates a basic need to use something for their sleeping problem.\nHalf of the patients who tried to stop by themselves before participating in our study, without taper off, suffered from withdrawal symptoms. In our trial this was 25%, showing the advantage of gradual tapering. We did not find a shift of addiction behavior, sleep quality or general health among stoppers of BD.\nConclusions\nOur trial does not provide conclusive evidence that melatonin is helpful for BD discontinuation.\nThe overall question about the effectiveness of intervention remains. Another trial, in contrast to the first trial, shows a negative result. One should consider that the results of all three trials are influenced by a possible selection bias or lack of power, due to the low number of participants.\nFurther investigation is necessary, with special attention to the effect of the daily dose on stopping the use of BD. Finally one should take into account the difficulty of recruiting BD users for stop studies.","keyphrases":["melatonin","hypnotics","discontinuation","the netherlands","long-term benzodiazepine use","primary care"],"prmu":["P","P","P","P","R","M"]}
{"id":"J_Struct_Biol-1-5-2048824","title":"Reticulon 4a\/NogoA locates to regions of high membrane curvature and may have a role in nuclear envelope growth\n","text":"Reticulon 4a (Rtn4a) is a membrane protein that shapes tubules of the endoplasmic reticulum (ER). The ER is attached to the nuclear envelope (NE) during interphase and has a role in post mitotic\/meiotic NE reassembly. We speculated that Rtn4a has a role in NE dynamics. Using immuno-electron microscopy we found that Rtn4a is located at junctions between membranes in the cytoplasm, and between cytoplasmic membranes and the outer nuclear membrane in growing Xenopus oocyte nuclei. We found that during NE assembly in Xenopus egg extracts, Rtn4a localises to the edges of membranes that are flattening onto the chromatin. These results demonstrate that Rtn4a locates to regions of high membrane curvature in the ER and the assembling NE. Previously it was shown that incubation of egg extracts with antibodies against Rtn4a caused ER to form into large vesicles instead of tubules. To test whether Rtn4a contributes to NE assembly, we added the same Rtn4a antibody to nuclear assembly reactions. Chromatin was enclosed by membranes containing nuclear pore complexes, but nuclei did not grow. Instead large sacs of ER membranes attached to, but did not integrate into the NE. It is possible therefore that Rtn4a may have a role in NE assembly.\n1\nIntroduction\nThe nuclear envelope (NE) encloses the nucleus (Hetzer et al., 2005; Goldberg, 2004) and consists of two parallel sheets of membrane connected at nuclear pore complexes (NPCs). The outer membrane is continuous with the rough endoplasmic reticulum (ER). During mitosis and meiosis in higher eukaryotes the NE is dismantled. The lamina and NPCs are solubilised and the membranes disperse into the cytoplasm by retraction into the ER (Ellenberg et al., 1997; Yang et al., 1997) or by vesiculation (Vigers and Lohka, 1991).\nDuring telophase the NE is reassembled around chromosomes in a multistage process, involving accumulation of different membrane populations and NE and NPC proteins. Evidence from Xenopus egg extracts suggest that there are least two vesicle populations (Vigers and Lohka, 1991; Macaulay and Forbes, 1996; Drummond et al., 1999) which fuse to form tubules and sheets (Wiese et al., 1997; Goldberg et al., 1992; Hetzer et al., 2001) which enclose the chromatin. Fusion of nuclear membranes requires hydrolysis of GTP (Macaulay and Forbes, 1996) by Ran (Hetzer et al., 2000; Zhang and Clarke, 2000; Zhang et al., 2002) and also the p97\u2013UFD1\u2013NPL4 complex (Hetzer et al., 2001). The mechanisms of targeting and fusion are unknown. NPCs are apparent after a few minutes in egg extracts (Goldberg et al., 1992), but NPC proteins accumulate in a temporal order in culture cells (Bodoor et al., 1999) as do structural intermediates in the NPC assembly process (Goldberg et al., 1997; Kiseleva et al., 2001).\nDuring S-phase the NE grows as the DNA content in the nucleus is increased (Winey et al., 1997). The NE also grows during oogenesis. Although interphase NE growth is often considered separately from telophase, there are common features. Conceptually, once the chromatin is enclosed in telophase, there is no obvious difference between the subsequent growth phase and interphase growth. Like enclosure, growth requires the AAA-ATPase p97, but instead of UFD1 and NPL4 it is complexed with p47 (Hetzer et al., 2001), suggesting the mechanisms are related but distinct.\nRtn4a\/NogoA (hereafter referred to as Rtn4a) is a member of reticulon family and is one of three splice variants of the RTN4 gene (Oertle and Schwab, 2003). It has attracted much interest recently because of its inhibitory role in neurite outgrowth (Prinjha et al., 2000; Yan et al., 2006). Rtn4a has also been shown to localise to the ER (van de Velde et al., 1994) but is restricted to the tubular network and excluded from the peripheral ER sheets and NE (Voeltz et al., 2006). Rtn4a was shown to be required for formation of ER tubules from sheets and vesicles (Voeltz et al., 2006) and it was suggested that reticulons could induce and stabilise the high curvature of the membrane required to maintain tubules. It is possible that the unusual topography of the reticulons, with their long putative transmembrane domains, could induce curvature when clustered.\nRtn4a does not appear to be concentrated at the NE during interphase (Voeltz et al., 2006) possibly because the NE membrane bilayers consist of flat sheets with low membrane curvature. However, nuclear assembly involves considerable reorganisation of membrane topology. It is thought that ER tubules, shaped by Rtn4a (Voeltz et al., 2006), feed into the NE (Ellenberg et al., 1997) and vesicles may also contribute (Vigers and Lohka, 1991; Liu et al., 2003; Prunuske et al., 2005). These vesicles and tubules have to be converted to a large flattened double sheet during NE assembly. Recently, it was shown that Rtn4a may have an essential role in NE disassembly in C. elegans (Audhya et al., 2007). Therefore, we decided to test if Rtn4a could have a role in NE formation, both during interphase and telophase. We used a high resolution surface imaging technique, field emission in-lens scanning electron microscopy (feiSEM1) to look at the structure of interphase growing NEs during Xenopus oogenesis and in telophase in vitro. We show that highly curved membrane regions of the forming or growing NEs preferentially contain Rtn4a. Such regions include the junctions between apparently fusing vesicles and the edges of flattening membranes. An antibody against Rtn4a was also shown to affect NE assembly. We therefore suggest that Rtn4a could have a role in NE formation and growth.\n2\nResults\nThe ER connects to the NE in interphase and has been implicated in the post mitotic assembly of the NE (Ellenberg et al., 1997; Yang et al., 1997; Mattaj, 2004). Much of the interphase ER forms a tubular network which appears to feed into the NE during reassembly (Ellenberg et al., 1997; Hetzer et al., 2001). Rtn4a has been found to be involved in shaping the ER into tubules in specific regions (Voeltz et al., 2006). Therefore, we speculated that if the ER needs to be in a tubular form to contribute to NE growth and assembly then Rtn4a may have a role. First we asked whether Rtn4a is associated with membranes that are contributing to NE growth, and then we investigated whether Rtn4a might be required for NE assembly.\n2.1\nRtn4a is located at inter-membrane junctions between cytoplasmic vesicles near the NE\nDuring oogenesis in Xenopus, oocytes are arrested in pre-prophase when the nucleus grows to over 100\u00a0\u03bcM diameter. The NE must expand by the addition of membranes and assembly of lamina and NPCs. We previously observed, both in thin section TEM of whole oocytes and isolated nuclei and in feiSEM of isolated NEs, that growing stage III oocytes have more extraneous ER-like membranes associated with the NE than mature stage VI (Morozova and Kiseleva, 2006).\nFeiSEM analysis of the extraneous membranes localised near the NE showed that many were present as structures that look like long lines of inter-connected vesicles (Fig. 1a). We are not certain of the origin of these structures but their surface has an ER-like appearance, with ribosome-like particles on the surface (Fig. 1b and d, arrowheads). The vesicle-like structures are joined together by a short thin tubular connection of \u223c20\u00a0nm diameter (Fig. 1b, white arrows). The same structures were also observed in TEM thin sections of whole oocytes, showing that they are not artifacts of NE isolation or feiSEM specimen preparation (Fig. 1c). We have also used different fixation methods (see Section 4.1). In TEM sections (Fig. 1c) the 20\u00a0nm diameter inter-connecting tubes are continuous with the vesicle membranes and therefore appear to be membrane bridges between vesicle-like structures.\nAnti-Rtn4a immuno-gold labelling with a previously characterised affinity purified anti Xenopus Rtn4a antibody (Voeltz et al., 2006) showed that Rtn4a was present on the surface of vesicles (Fig. 1f). In the inter-connected vesicle structures, Rtn4a labelling was concentrated at the junctions between connected membrane structures where the membrane bridge is located (Fig. 1d and e). Inter-connected larger membrane structures were also observed (Fig. 1g and h) where Rtn4a was located at the junction between them. Immuno-gold labelling of TEM sections of whole oocytes also shows localisation of Rtn4a at the contact point of adjacent vesicles. (Fig. 1i). Rtn4a therefore appears to locate to the inter-connections between membrane structures (Fig. 1j). These results show that Rtn4a locates to specific regions on NE associated cytoplasmic membranes.\n2.2\nRtn4a is present on membranes attached to growing NEs\nWe isolated NEs from stage III oocytes and immuno-gold labelled them for Rtn4a. There was some labelling of the ONM (Fig. 2a) but membrane structures associated with the NE were heavily labelled and in some cases, in a specific pattern. Fig. 2a is an image of the surface of a stage III NE showing NPCs, rough (ribosome-containing) vesicles (RV), smooth (ribosome-free) vesicles (SV), and rough membranes that appear to be flattening onto the ONM (FM). We see that there is labelling of the ONM and vesicles are labelled to a varying degree. Smooth vesicles are not labelled, whereas rough ER type vesicles are. This shows that Rtn4a is associated with some but not all membranes that are associated with the ONM of a growing NE.\n2.3\nRtn4a localises to the junction between the ONM and membranes attached to it\nMembrane structures could be seen attached to the ONM. Rtn4a appeared to be located near the point of contact between the membrane structures and the ONM (Fig. 2b\u2013d). Therefore, it appears that Rtn4a accumulates both at junctions between ER-like membranes and between NE associated membranes that are attached to the ONM. NEs labelled with an antibody (CEL5C) to the ER protein ribophorin (Drummond et al., 1999) showed a more even distribution over flattened membranes, some vesicles and the outer nuclear membrane (Fig. 2e).\n2.4\nRtn4a localises to the edges of flattened membranes\nSome of the observed NE associated membranes had the appearance of collapsed spheres (Fig. 3a) whereas many are flattened (Fig. 3b\u2013f). It is not always clear from feiSEM images whether such membranes are fused to the ONM or simply lying on top, so thin section TEM was carried out and showed continuity between the ONM and overlying flattened membrane structures (Fig. 4, black arrow). Points of contact between the ONM and cytoplasmic membrane structures were also observed (Fig. 4, white arrows). Potentially corresponding images were seen by feiSEM, in which the flattened membrane structures appear continuous with the NPC-containing ONM (Fig. 3b, arrows).\nUsing immuno-gold labelling, we observed that Rtn4a appeared to locate preferentially around the edges of the flattened membranes (Fig. 3d\u2013f). This was quantified by counting the number of gold labels that were within 30\u00a0nm of the edge compared to greater than 30\u00a0nm from the edge (Fig. 5), which gave a ratio of approximately 2:1, for edge compared to the interior. This was in contrast to the ER protein, ribophorin, which was distributed more away from the edge (Fig. 5). The preferred edge location suggests that Rtn4a tends to locate or accumulate at regions with the highest curvature. We conclude that Rtn4a marks the highly curved edges of flattened sheets of presumed ER membrane attached to the ONM.\n2.5\nRtn4a is present at the edges of membranes that are flattening onto the chromatin\nDuring telophase, membranes also flatten onto chromatin (Goldberg et al., 1992; Macaulay and Forbes, 1996). We wanted to test if the edge location of Rtn4a that we observed in growing oocyte NEs also occurred in chromatin bound flattening membranes. To do this, we used a cell free system from Xenopus eggs which can be used to assemble nuclei in vitro (Goldberg and Allen, 1993; Lohka and Masui, 1984). Nuclei were isolated from assembly reactions, fixed and immuno-gold labelled for Rtn4a. At early stages (2\u20135\u00a0min) vesicles bind to the chromatin and flatten (Goldberg et al., 1992; Wiese et al., 1997). In such vesicles, Rtn4a localises preferentially around the edges (Fig. 6a and b), compared to a general ER protein, ribophorin (antibody CEL5C\u2014Drummond et al., 1999), which was more randomly distributed (Fig. 6d). To show this formally we counted the distribution of gold particles located within 30\u00a0nm of the vesicle edge, or further away in 30\u00a0nm increments, using images from three separate experiments (Fig. 6e).This shows that Rtn4a, compared to ribophorin, is preferentially located near the membrane edge. Therefore, as in growing oocytes, Rtn4a preferentially locates to the edges of flattening membranes. At later stages of assembly when the chromatin is enclosed, and there are no longer any highly curved membrane edges (except at the nuclear pores), the Rtn4a distribution on the NE appeared random and low level (Fig. 6c). This is consistent with Rtn4a\u2019s preference for curved membranes (Voeltz et al., 2006). Although there are highly curved membranes in the nuclear pores we see no Rtn4a labelling there. The high membrane curvature at the NPC might be maintained by nucleoporins which could exclude the accumulation of Rtn4a, or it is possible that Rtn4a is present but not detected by the antibody due to epitope masking.\n2.6\nDisrupting Rtn4a prevents NE growth\nAlthough Rtn4a is not preferentially located to the NE during interphase (Voeltz et al., 2006), our results in stage III oocytes show that it does locate to membranes that are attached to the interphase NE and suggested the possibility that it may be involved in NE assembly or growth. Therefore, we wanted to investigate if Rtn4a might be required for NE assembly. We incubated egg extracts with a previously characterised anti Xenopus Rtn4a antibody directed against the cytoplasmic facing N-terminus (Voeltz et al., 2006) which is specific to Rtn4a and not present in other Rtn4 spliced variants or other reticulon proteins.\nThis antibody inhibits the formation of ER tubules in similar egg extracts (Voeltz et al., 2006) and likewise we found that it inhibited ER tubule formation. Instead, ER membranes assembled into large vesicular structures (Fig. 7b) rather than tubules as seen in the no-antibody control reactions (Fig. 7a). These large membrane structures are ER-derived because they have ribosomes on their surface. This shows that the anti-Rtn4a antibody had a dominant effect on the formation of ER tubules.\nTo determine if the Rtn4a antibody affected NE assembly, the same egg extracts were incubated with the antibody (see Section 4.1) or with buffer or with an irrelevant antibody, before adding sperm chromatin to initiate nuclear assembly. Controls showed rapid binding, fusion and flattening of vesicles onto chromatin, following by chromatin decondensation, enclosure and NE growth to form large roughly spherical nuclei >10 microns in diameter, as expected (Goldberg et al., 1997; Wiese et al., 1997 and Fig. 7c). In the presence of the Rtn4a antibody we found that nuclei did assemble with an apparently completely enclosed unbroken NE (Fig. 7d\u2013g) that had apparently normal NPCs (Fig. 7i, black arrows). However, the chromatin failed to decondense and remained as a dense sperm-shaped object surrounded by NE (Fig. 7d\u2013g). To quantify this apparent NE growth defect, we measured the two-dimensional area occupied by each nucleus (excluding attached membranes, see below) as viewed from above by feiSEM (Fig. 7h). Nuclei in Rtn4a antibody-inhibited reactions had \u223c80% reduced area compared to controls, confirming the growth defect.\nThere were in addition, large (several microns) membrane structures extending from the nucleus (Fig. 7d\u2013g, arrows). The extensions are clearly rough ER-like and contain ribosomes (Fig. 7i, arrowheads). There is a sharp demarcation between the chromatin attached membrane which contains NPCs and the membrane extensions which contain ribosomes but not NPCs (Fig. 7i, white arrows). The extensions are clearly continuous with the NPC-containing NE. Therefore, it appears that perturbing Rtn4a does not prevent enclosure of the chromatin by NE. However, the chromatin and NE do not expand despite the attachment of large ER-like sacs to the ONM. These sacs are similar to the large ER vesicle formed in the cytosol in the presence of the antibody, except they are attached to the NE.\nAntibodies and reagents against other NE and ER proteins do not have such an affect on nuclear assembly or ER tubulation. Addition of wheat germ agglutinin, which binds to certain nucleoporins (Hanover et al., 1987), to extracts results in nuclei without NPCs but it has no effect on ER tubulation and does not result in membrane extensions (Goldberg et al., 1997). Addition of anti-nucleoporin antibodies (Mab414 and QE5) (unpublished results) or depletion of extracts with antibodies to specific nucleoporins, Nup214 (Walther et al., 2002) or Nup153 (Walther et al., 2001), also affected NPC structure but again does not affect the ER or NE membranes. Depletion of lamin B3 resulted in small spherical nuclei with normal NPCs but had no obvious effect on ER tubules or membrane extensions (Goldberg et al., 1995). The CEL5C antibody against the ER protein ribophorin (Drummond et al., 1999) also did not affect tubulation or membrane extensions when added to extracts (unpublished result). Similar experiments were also done (Voeltz et al., 2006) using antibodies to IP3R and TRAP\u03b1 which also did not affect ER tubule formation. Therefore, we believe that the growth defects and extensions are a specific effect of the anti-Rtn4a antibody used in this study.\n3\nDiscussion\nWe have found that Rtn4a is localised to junctions between membrane structures and at the edges of flattened membranes associated with growing NEs both in oocytes and in nuclei assembling in vitro. This is the first direct evidence to support the proposal that Rtn4a locates to regions of high curvature (Voeltz et al., 2006). It is a unique localisation that has not been seen for other NE or ER proteins. We believe this localisation may be driven by Rtn4a itself rather than by interacting with other proteins because high level over-expression of Rtn4a in COS cells (Voeltz et al., 2006) and the related Rtn1 in yeast (De Craene et al., 2006) does not change the proteins\u2019 localisations and therefore is unlikely to rely on other titratable factors. The location of Rtn4a at the junction between cytoplasmic membranes and NE membranes suggested to us the possibility that Rtn4a may also have a role in NE assembly.\nAddition of the Rtn4a antibody to nuclear assembly reactions allowed NE formation in egg extracts but chromatin remained condensed and the NE did not expand. Therefore, the antibody has a dominant effect on the growth phase of NE assembly but not on the initial enclosure and NPC assembly. As Rtn4a is an integral membrane protein that appears to be involved in shaping membranes (Voeltz et al., 2006) we suggest that the effect of the antibody is to perturb nuclear membrane dynamics and assembly, as shown previously for the ER (Voeltz et al., 2006).\nOne possible speculation for the localisation of Rtn4a at membrane\u2013membrane junctions in oocytes is that it may take part in fusion or stabilisation of membrane curvature during fusion. Inter-membrane fusion involves transitory extremes of curvature involving membrane stalks between vesicles (Smeijers et al., 2006; Yang and Huang, 2002). Rtn4a could be involved in stalk formation or stabilisation in certain NE membranes. We do not know if these membranes in oocytes are actively fusing membranes or if they are more stable or intermediate structures. Rtn4a could be involved in stabilising the conformation of these junctions by maintaining the high membrane curvature to facilitate membrane flow into the NE.\nOur in vitro antibody inhibition experiments suggest that ER-like membranes can attach to the NE when Rtn4a is perturbed but the NE fails to grow. When Rtn4a is perturbed the ER membranes are converted to large sacs, which can attach to the NE but fail to contribute to NE expansion. This suggests the possibility that the organisation of the interface between the NE and ER, as observed in oocytes, may be important for NE growth. Therefore we speculate that a possible function of localising Rtn4a to the inter-membrane junctions between ONM and ER is to maintain a particular interfacial organisation which contributes to the movement of membrane into the NE. Our results argue against a model in which membranes simply diffuse into the NE during assembly and growth (Ellenberg et al., 1997) because the NE fails to grow when Rtn4a is perturbed despite the attachment of ER membranes to the NE.\nRtn4a is not only located at the inter-membrane junctions but also throughout the tubular ER, where it appears to be involved in maintaining the tubularity (Voeltz et al. 2006; Fig. 7). Therefore, the tubular nature of the ER may be essential for its function in providing membrane for NE growth, at least in egg extracts.\nThe Rtn4a antibody does not inhibit the initial formation of NE around chromatin. This NE appears normal: it is flattened, fused and contains NPCs. Because it contains NPCs we can conclude that the contributing membranes carried the integral membrane nucleoporins such as POM121 and gp210 enabling NPC formation. This suggests that Rtn4a may only be required during the growth phase and that membranes that contribute to initial enclosure and NPC formation (Yang et al., 1997; Drummond et al., 1999) are not perturbed by the antibody.\nWe have also found that Rtn4a locates specifically to the edges of flattening membranes in growing and assembling NE in both in vivo and in vitro experiments. As Rtn4a has been implicated in the formation of highly curved membrane regions, it is possible to speculate that Rtn4a is important for formation of flattened sheets, by stabilisation of the high curvature at the edge regions. However, this may be a non essential function in the initial stages of NE assembly, which occur in the presence of the antibody.\n4\nConclusions\nWe have shown for the first time that Rtn4a partitions, within a single membrane structure, to the region of highest curvature, supporting the model that reticulons preferentially locate to curved membranes (Voeltz et al., 2006) and have a function related to membrane curvature. We have also observed that it locates to membrane junctions and other highly curved regions of membranes that may be involved in NE growth. Concordantly, an Rtn4a antibody perturbs NE growth. We therefore hypothesise that Rtn4a may have a role in maintaining functional ER\u2013NE junctions during NE growth and\/or the ER must be tubular to contribute to NE growth.\n4.1\nMaterials and methods\n4.1.1\nIsolation and fixation Xenopus oocyte for feiSEM\nStage III and VI NEs were isolated in 5:1 buffer, spread, fixed in 2% glutaraldehyde, 0.2% tannic acid, 10\u00a0mM Tris\u2013HCl (pH 7.4) and 0.3\u00a0mM MgCl2 and processed for feiSEM as described previously (Kiseleva et al. 2004). In some experiments tannic acid, which helps preserve protein filament (Maupin and Pollard, 1983) was omitted, as it can effect membrane preservation (MWG unpublished), but the same membrane structures were observed. Samples were coated with chromium using a Cressington 308R with additional cryo-pump or an Edwards Auto306 with cryo-pump to a nominal thickness of 2\u00a0nm. They were viewed using a Hitachi S-5200 feiSEM at 10\u00a0kV accelerating voltage.\n4.1.2\nFixation Xenopus oocyte for TEM\nOocytes were fixed (2.5% glutaraldehyde, 0,1M Hepes) for 1\u00a0h, then washed twice in 0.1\u00a0M Hepes buffer and postfixed 1\u00a0h in 1% OsO4 in ddH2O at 4\u00a0\u00b0C, stained 2\u00a0h in 1% aqueous uranyl acetate, washed in water, dehydrated through ethanol series and embedded in Agar-100 (Agar Scientific, UK). Sections were stained with Lead citrate and viewed with Leo 910 (Germany) TEM at 80\u00a0kV.\n4.1.3\nImmuno-TEM\nStage III oocytes were fixed in 4% formaldehyde (TAAB Labs) in Ringers (111\u00a0mM Nacl, 1.9\u00a0mM KCl, 1.1\u00a0mM CaCl2, 2.4\u00a0mM NaHCO3, pH 7.0) overnight at 4\u00a0\u00b0C, washed three times in Ringers, stain in 2% uranyl acetate in Ringers for 2\u00a0h at 4\u00a0\u00b0C, then washed in water twice, dehydrated in 30% ethanol, then further dehydrated by freeze substitution as follows through a series of ethanol (30% at 4\u00a0\u00b0C for 1\u00a0h, 50% at \u221220\u00a0\u00b0C for 1\u00a0h, 70% at \u221220\u00a0\u00b0C for 1\u00a0h, 95% at \u221220\u00a0\u00b0C for 2\u00a0h), embedded to LR Gold (Agar) at \u221220\u00a0\u00b0C and polymerised in gelatin capsules under UV light at \u221215\u00a0\u00b0C for 48\u00a0h. Ultrathin sections were cut and attached to nickel grids then incubated with 1% BSA in PBS for 30\u00a0min, washed in PBS, incubated with 1:100 dilution anti-Rtn4a rabbit antibody for 1\u00a0h, washed in PBS and incubated with a goat anti-rabbit secondary antibody conjugated to 10\u00a0nm colloidal gold (Amersham) for 1\u00a0h and washed with PBS. Sections were stained with lead citrate for 2\u00a0min.\n4.1.4\nImmuno-gold labelling and feiSEM imaging\nAffinity purified polyclonal antibody (40\u00a0M stock) against N-terminal domain of Xenopus Rtn4a (Voeltz et al., 2006) was diluted 1:100 with PBS. Nuclei were isolated and fixed for 20\u00a0min in 3.7% formaldehyde, 5:1 buffer, washed three times with PBS, incubated in PBS, 1% BSA 30\u00a0min, washed in PBS, and incubated 1\u20133\u00a0h primary antibody, PBS. Samples were washed three times in PBS, and incubated 1\u00a0h with 10\u00a0nm gold-conjugated secondary goat anti-rabbit antibody (Amersham Corp.). As negative controls, we used gold-conjugated secondary antibody diluted 1:100 in PBS. All samples were then washed three times in PBS then in 10\u00a0mM Tris\u2013HCl and processed for feiSEM as above. Both secondary electron images (for structure) and backscatter electron images (for gold label position) were collected simultaneously. Using Adobe Photoshop, the backscatter image was superimposed onto the secondary image as a separate layer. The positions of the gold particles were marked with a yellow dot and then the backscatter image removed.\n4.1.5\nIn vitro nuclear assembly\nEgg extracts were prepared as described previously (Goldberg et al., 1997). To perturb Rtn4a, extract was incubated with 4\u00a0\u03bcM affinity purified Xenopus Rtn4a antibody (Voeltz et al., 2006) on ice for 20\u00a0min then 1000 sperm chromatin per microlitre was added and the extract warmed to room temperature for 2\u201360\u00a0min for assembly. In controls an equal volume of either buffer or irrelevant antibody was added, which had no detectable effect on nuclear assembly. Extracts were resuspended in Membrane Wash Buffer (MWB: 250\u00a0mM sucrose, 50\u00a0mM KCl, 2.5\u00a0mM MgCl2, 10\u00a0mM Hepes, pH 7.4) and centrifuged at 1000g at 4\u00a0\u00b0C for 10\u00a0min onto silicon chips (Agar Scientific Ltd) and immersed in Membrane Fix (150\u00a0mM sucrose, 1\u00a0mM MgCl2, 80\u00a0mM Pipes\u2013KOH, pH 6.8, 2% paraformaldehyde, 0.25% glutaraldehyde) for 10\u00a0min and processed for feiSEM as previously described (Goldberg et al., 1997). Samples were imaged at 3\u00a0kV accelerating voltage.\n4.1.6\nAntibody labelling of in vitro nuclei\nExtract (5\u00a0\u03bcl) was resuspended in MWB and centrifuged at 1000g at 4\u00a0\u00b0C for 10\u00a0min onto silicon chips (Agar Scientific, UK). Chips were immersed in Membrane Fix without glutaraldehyde for 10\u00a0min, washed in PBS, immersed in PBS +100\u00a0mM glycine 10\u00a0min, washed in PBS, immersed in 1% fish skin gelatine (Sigma) for 1\u00a0h, then 1\u00a0\u03bcM primary antibody for 1\u00a0h, washed 3 times 5\u00a0min in PBS, incubated with 1:50 dilution anti-rabbit secondary antibody conjugated to 10\u00a0nm gold (Amersham) for 1\u00a0h, washed 3 times 5\u00a0min and 1 times 15\u00a0min in PBS and fixed in Membrane fix with 1% glutaraldehyde. Samples were then processed for feiSEM as previously described (Goldberg et al., 1997).","keyphrases":["reticulon 4a","scanning electron microscopy","nogoa nuclear envelope"],"prmu":["P","P","R"]}
{"id":"Dev_Genes_Evol-4-1-2362136","title":"Muscle precursor cells in the developing limbs of two isopods (Crustacea, Peracarida): an immunohistochemical study using a novel monoclonal antibody against myosin heavy chain\n","text":"In the hot debate on arthropod relationships, Crustaceans and the morphology of their appendages play a pivotal role. To gain new insights into how arthropod appendages evolved, developmental biologists recently have begun to examine the expression and function of Drosophila appendage genes in Crustaceans. However, cellular aspects of Crustacean limb development such as myogenesis are poorly understood in Crustaceans so that the interpretative context in which to analyse gene functions is still fragmentary. The goal of the present project was to analyse muscle development in Crustacean appendages, and to that end, monoclonal antibodies against arthropod muscle proteins were generated. One of these antibodies recognises certain isoforms of myosin heavy chain and strongly binds to muscle precursor cells in malacostracan Crustacea. We used this antibody to study myogenesis in two isopods, Porcellio scaber and Idotea balthica (Crustacea, Malacostraca, Peracarida), by immunohistochemistry. In these animals, muscles in the limbs originate from single muscle precursor cells, which subsequently grow to form multinucleated muscle precursors. The pattern of primordial muscles in the thoracic limbs was mapped, and results compared to muscle development in other Crustaceans and in insects.\nIntroduction\nAmongst all arthropods, Crustaceans display the greatest variety of different limb types, and the morphology of their appendages has played an outstanding role in the raging debate on arthropod phylogeny (reviews, e.g. Williams and Nagy 1996; Boxshall 1997; Kukalova-Peck 1997; Browne and Patel 2000; Bitsch 2001; Klass and Kristensen 2001; Schram and Koenemann 2001; Williams and Nagy 2001; Wolf and Harzsch 2002; Waloszek 2003; Williams 2004). The recent move towards integrating the fields of evolutionary and developmental biology (e.g. Averof and Akam 1995; Gilbert et al. 1996; Akam 1998a; Hughes and Kaufman 2000; Harzsch and Hafner 2006; Harzsch 2007) has fostered a surge of studies on Crustacean limb development which examined the expression and function of genes such as Distal-less (Panganiban et al. 1995; Popadic et al. 1996, 1998; Scholtz et al. 1998; Williams 1998, 2008; Williams et al. 2002), Ultrabithorax and AbdominalA (Averof and Akam 1995; Averof and Patel 1997), Extradenticle (Gonz\u00e1les-Crespo and Morata 1996; Abzhanov and Kaufmann 2000), Pdm and Apterous (Averof and Cohen 1997), Sex combs reduced (Abzhanov and Kaufman 1999), and Wingless (Nulsen and Nagy 1999) in various Crustacean taxa with uniramous, biramous or phyllopodous branched limbs. Interestingly, some of these studies failed to establish homologies between the function of these genes during development of the complex Crustacean limbs as compared to the uniramous limbs of Insecta (Williams and Nagy 1995, 1996; Averof and Patel 1997; Williams et al. 2002; Williams 2004) but instead established new hypotheses on the evolution of hox gene function (Averof et al. 1996; Akam 1998b). The emerging picture is that limb patterning genes seem to act differently in the insect with uniramous limbs and those Crustaceans with phyllopodous limbs, and therefore, a greater knowledge of the cellular foundations of limb development in Crustaceans is essential to establish an interpretative context in which to analyse gene functions. However, few papers have recently dealt with cellular aspects of Crustacean limb development other than gene expression (e.g. Williams and M\u00fcller 1996; Ungerer and Wolff 2005; Kiernan and Herzler 2006).\nConcerning the neuromuscular innervation, there is evidence for close similarities between Hexapoda and malacostracan Crustacea. In these animals, each thoracic walking leg is supplied by a set of exactly three inhibitory motoneurons in addition to its excitatory innervation. Wiens and Wolf (1993) have shown that the inhibitory limb innervation in a crayfish displays striking similarities to that in Hexapoda down to the level of single identified cells. The sets of inhibitors in these taxa share a number of morphological, physiological and biochemical characteristics which suggest homology, as discussed in greater detail by Harzsch (2007). Furthermore, the innervation pattern of particular excitatory motoneurons in crayfish and locusts provides new insights into the alignment of malacostracan Crustacean and insect trunk limbs (Wiens and Wolf 1993). These authors suggest a homology of the extensor muscles located within the second podomeres of insect and malacostracan limbs (merus and femur) and therefore support a close correspondence of limb segmentation in Malacostraca and Hexapoda (discussed in more detail by Wolf and Harzsch 2002; Harzsch 2007). Because information on inhibitory and excitatory leg motoneurons so far is only available for malacostracan Crustacea but not for the other Crustacean taxa, these comparisons so far are only of a limited phylogenetic value. However, these studies signify that comparative analyses of the neuromuscular system have a significant potential to contribute new insights into the evolution of arthropod appendages.\nThe cellular basis of embryonic muscle formation in Crustaceans is poorly understood, although ontogenetic aspects of the neuromuscular system (reviewed in Govind 1982; Govind and Walrond 1989; Govind 1995) and moult-induced muscle atrophy and regeneration (reviewed in El Haj 1999; Mellon 1999; Mykles 1999; Govind 2002) have been studied in some detail. The goal of the present project was to analyse muscle development in Crustacean appendages, and therefore, we generated monoclonal antibodies against arthropod muscle proteins. One of these antibodies, 016C6, strongly labelled muscle precursor cells in malacostracan Crustacea and on Western blots was shown to recognise several isoforms of myosin heavy chain in rabbit, grasshopper and Crustaceans. We used this antibody to study muscle formation in two isopods, the terrestrial Porcellio scaber Latreille, 1804 (Crustacea, Malacostraca, Peracarida, Isopoda, Oniscidea; Fig.\u00a01) and the marine Idotea balthica Pallas 1772 (Valvifera) by immunohistochemistry. In these animals with uniramous walking legs (Fig.\u00a01), muscles of the body wall and the limbs were shown to originate from single muscle precursor cells, which subsequently grow to form multinucleated muscle precursors. The pattern of primordial muscles in the thoracic limbs was mapped, and results compared to muscle development in insects (reviewed in Campos-Ortega and Hartenstein 1997; Abmayr and Keller 1998; Baylies et al. 1998; Paululat et al. 1999a, b; Roy and VijayRaghavan 1999).\nFig.\u00a01The walking limbs of adult isopods: A ventral view of an adult male of I. balthica. The labels Th2 to Th8 identify the thoracomeres. B Adult specimen of P. scaber, ventral view (medial is towards the right) of thoracomeres three to eight (Th3\u2013Th8) with the walking limbs attached. C Higher magnification of the walking limb from thoracomere three (P. scaber) to show podomeres (medial is towards the left). Abbreviations: letters in the upper right corners identify the species\nMaterials and methods\nAnimals\nI. balthica Pallas 1772 (Crustacea, Malacostraca, Peracarida, Isopoda, Valvifera; Fig.\u00a01A) were obtained from the Biologische Anstalt Helgoland and kept in artificial seawater at 16\u00b0C at the University of Konstanz (compare Kreissl et al. 1999). Eriphia spinifrons (Crustacea, Malacostraca, Decapoda, Brachyura) and Idotea emarginata (Crustacea, Malacostraca, Peracarida, Isopoda, Valvifera) were obtained from the Stazione Zoologica Anton Dhorn in Naples. P. scaber Latreille, 1804 (Crustacea, Malacostraca, Peracarida, Isopoda, Oniscidea; Fig.\u00a01B, C) were collected on the campus surrounding the University of Konstanz. For the experiments, embryos were gently removed from the brood pouches of ovigerous females.\nGeneration and characterisation of the antibody 016C6\nA crude myosin extract (d\u2019Albis et al. 1979) of meso- and metathoracic locust muscles was used as the antigen to immunise mice. Two mice (female Balb\/c, 6\u00a0weeks of age) received 0.1\u00a0ml of myosin extract emulsified in a 1:1 relation in RIBI adjuvant system, (MPL+TDM emulsion, R-700; RIBI Immunochem Research Inc.) at each of two subcutaneous sites on day 0 and day 28 and three final immunisations intraperitoneally with 50\u00a0\u03bcl myosin extract after 15\u00a0weeks. The mice sera were tested for antibodies on enzyme-linked immunosorbent assay (ELISA) coated with crude myosin extract. Both mice showed strong immune responses. Three days later, splenectomy was performed, and the splenocytes were fused with a mouse myeloma cell line (P3X63Ag8.653, ATCC CRL-1580) by PEG-1500. The successfully fused hybridomas were selected in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with HAT (10\u00a0mM hypoxanthine, 40\u00a0\u03bcM aminopterin, 1.6\u00a0mM thymidine; GIBCO BRL, Carlsbad, CA, USA), 10% foetal bovine serum, 24\u00a0\u03bcM \u03b2-mercaptoethanol and Nutridoma-CS (Roche, Indianapolis, IN, USA). The culture supernatants were collected and screened by ELISA. The reactions were confirmed with Western blot analysis and immunostaining on cryosections of native and paraformaldehyde fixed locust muscles using secondary antibody conjugated to Cy3 or Cy2 (Jackson Labs). Positive clones were selected by at least three sets of the limiting dilution technique. The antibodies generated against locust muscle proteins were also screened for their binding affinity to muscle tissue of malacostracan Crustaceans and of rabbits. For the identification of the antigen in Western blots, the crude myosin extracts (d\u2019Albis et al. 1979) of locust muscles, of abdominal muscles of the two Crustacean species E. spinifrons and I. emarginata and of adult White New Zealand rabbit muscles (psoas, soleus and diaphragm) were separated by sodium dodecyl sulfate\u2013polyacrylamide gel electrophoresis (SDS-PAGE) on 7.5% linear gels according to standard techniques. The proteins were transferred to cellulose nitrate membranes (Protran, Schleicher & Schuell GmbH, Dassel, Germany), and selective binding of monoclonal IgG1\u03ba 016C6 to proteins was detected with the IgG-ABC-ELITE-POD kit (Vector Labs) using ECL (Pierce) as a substrate. Molecular weight markers were biotinylated, allowing direct detection by the ABC reagent in the Western blots.\nWhole mount immunohistochemistry\nEmbryos were fixed for 4\u00a0h in 4% paraformaldehyde in 0.1\u00a0M phosphate buffer (pH\u00a07.4) at room temperature. Whole mounts of the embryos were incubated in 1\u00a0mg\/ml collagenase\/dispase (Sigma) for 20\u00a0min at room temperature or pretreated with short ultrasonic pulses to increase penetration of the antibodies then washed in several changes of 0.1\u00a0M phosphate-buffered saline (PBS) for 4\u00a0h and afterwards pre-incubated in PBS containing 1% normal goat serum and 0.3% Triton X-100 (PBS-TX) for 2\u00a0h at room temperature. Specimens were then incubated overnight at 4\u00b0C in supernatants of the monoclonal antibody 016C6 diluted 1:10 in PBS-TX. The omission of the primary antibody resulted in a complete absence of specific labelling. The embryos were then incubated in a biotinylated secondary antibody for 3\u00a0h (Jackson) and subsequently for another 3\u00a0h in peroxidase-conjugated streptavidin (Dianova). After washing for 4\u00a0h in PBS, the tissues were reacted with 0.013% diaminobenzidine and a reagent containing hydrogen peroxide, cobalt chloride and nickel chloride (Amersham, RPN 20) for 7\u20139\u00a0min to reveal the peroxidase label. Finally, whole-mount preparations were dehydrated and mounted in Eukitt (Riedel-de Haen). Preparations were observed with a Zeiss Axioskop and labelled structures drawn using a camera-lucida apparatus. The slides were also photographed on 35-mm colour slide film, the images transferred onto Kodak Photo CD and processed in Picture Publisher. Alternatively, specimens were photographed with the Polaroid DMC10 digital camera.\nHOECHST stain\nTo reveal the morphology of the early embryos, specimens were dissected out of the chorion and the yolk was removed. Specimens were then fixed in 4% paraformaldehyde in 0.1\u00a0M phosphate buffer (1\u00a0h, room temperature) and stained with the nuclear dye bisbenzimide (0.1%, 15\u00a0min at room temperature; Hoechst H 33258), washed in buffer overnight and mounted in Flouromount (Sigma). Specimens were viewed with a fluorescent microscope (Axioskop) and documented as described above.\nResults\nCharacterisation of monoclonal antibody 016C6\nScreening the supernatants of a number of monoclonal hybridoma cell lines producing antibodies against locust muscle proteins for their binding affinity to muscle tissue of other species revealed that monoclonal antibody (MAB) 016C6 shows a particularly strong affinity for muscle proteins of malacostracan Crustaceans. For the identification of the 016C6 antigen, muscle proteins of Locusta migratoria, I. emarginata, E. spinifrons and of adult White New Zealand rabbits were separated by SDS-PAGE and analysed in Western blots. Polyacrylamide gel analysis of proteins of the muscle homogenates shows prominent bands with apparent molecular weights of 180 to 200\u00a0kDA, indicating the presence of myosin heavy chains in all tissues examined (Fig.\u00a02A). In the four tested species, MAB 016C6 binds to muscle proteins with an apparent molecular weight of 180\u00a0kDA (Fig.\u00a02B).\nFig.\u00a02Characterization of monoclonal antibody 016C6: 016C6 specifically binds to myosin heavy chains, which co-migrates with a slow myosin heavy chain isoform of rabbit muscles. A Separation of muscle proteins by SDS-PAGE. Numbers at the left indicate molecular weight markers (MW), lanes 2\u20134 were loaded with muscle homogenates of the two malacostracan species E. spinifrons (Decapoda, Brachyura) and I. emarginata (Peracarida, Isopoda) and the hexapod L. migratoria. Lanes 5\u20137 were loaded with homogenates of the fast type psoas muscle, the mixed type diaphragm and the slow type soleus muscle of rabbits. Myosin heavy chains are prominent at MW of 180 to 200\u00a0kDA. B Identification of the 016C6 antigen by Western blot analysis reveals myosin heavy chains in arthropod muscles which co-migrate with a 180\u00a0kDA myosin heavy chain isoform of rabbit slow and mixed muscles\nIn mammals, myosin heavy chains (MHC) exist as isoforms of polypeptides with a molecular mass of about 180\u2013200\u00a0kDa. The homogenate of the rabbit psoas (fast twitch muscle) contains predominantly the MHC-IId isoform. The homogenate of the diaphragm contains MHC I, MHC IIa and MHC IId, the slow twitch and two fast twitch isoforms, respectively. The rabbit soleus (slow twitch muscle) contains predominantly myosin heavy chain MHC-I (Aigner et al. 1993). MAB 016C6 exhibits a considerably higher affinity to MHCs of the three arthropod species and to the slow MHC-I isorm of rabbit muscles as compared to the fast rabbit MHCs (Fig.\u00a02). We used MAB 016C6 as a general marker for differentiating muscle cells already containing myosin heavy chains in the present study.\nDevelopment of the embryos\nThe embryonic development of two species of the genus Idotea has been described by Str\u00f6mberg (1965), and the development of P. scaber was recently reviewed by Whitington et al. (1993) and Abzhanov and Kaufman (1999; see also Brena et al. 2005). Hejnol et al. (2006) explored germ band formation in this organism. For P. scaber, Whitington et al. (1993) established a percentage staging system based on developmental time. Under the rearing conditions described above, I. balthica embryos hatched after 27\u00a0days. However, a period of rapid organogenesis occurs during the last 6 or 7\u00a0days of embryogenesis (Fig.\u00a03) so that a direct comparison of the ontogeny of I. balthica and P. scaber based on a percentage scale was not possible. Therefore, we subdivided the embryonic period that we studied into five distinct stages, which are comparable between the two species:\nStage 1(I. balthica E70%, P. scaber E40%; the percentage values indicate embryonic development in a percentage staging system based on developmental time): The germ band is not completely elongated, and mitotic cells in the posterior growth zone are still visible; in the more anterior thoracomeres, distinct limb anlagen can be distinguished.Stage 2(I. balthica E80%, P. scaber E60%): All segments are formed, the embryo is not closed dorsally, and the limb anlagen are subdivided into distinct podomeres.Stage 3(I. balthica E85%, P. scaber E80%): Red pigments is visible throughout the entire embryo, the first ommatidia can be distinguished, and single muscle precursor cells can be labelled immunohistochemically in the limb anlagen.Stage 4(I. balthica E90%, P. scaber E90%): The growing tergites have dorsally enclosed the yolk.Stage 5(I. balthica E100%, P. scaber E100%): hatchingFig.\u00a03Embryonic staging system: A\u2013C embryos of I. balthica at A 70% of embryonic development (E70%; stage 1), B E85% (stage 3) and C E95% (stage 4\u20135). D Embryo of P. scaber at E60% (stage 2). E, F embryos of P. scaber at E E40% (stage 1) and F E60% (stage 2), HOECHST nuclear stain, ventral views. At stage 1, the germ band is not completely elongated, and limb anlagen are visible in the anterior thoracic segments. At stage 2, all segments are formed, and the thoracic limb anlagen are subdivided into distinct podomeres. Abbreviations: letters in the upper right corners identify the species and larval stages; A1-2 antenna 1 and 2, GZ posterior growth zone, LB labrum, MD mandible, MX1-2 maxilla 1 and 2, T1-8 thoracomeres 1 to 8, P1-5 pereomeres 1 to 5, the last pleomeres denoted. Scale bars A\u2013F, 100\u00a0\u03bcm\nDevelopment of the thoracic limbs\nA stage 1, embryo of P. scaber labelled with the HOECHST stain is shown in Fig.\u00a03E. Proliferating cells in the posterior growth zone have just generated the first pleomere, and limb buds are beginning to form in the thoracic segments at that stage. The thoracic limb anlagen subsequently enlarge, and bilobed anlagen of the pleopods appear (Fig.\u00a04A, late stage 1). In stage 2 embryos, the developing thoracopods begin to subdivide into distinct subunits, the podomeres (Fig.\u00a03E, F4B), and the adult subdivision of the thoracopods into seven subunits is established: coxa, basis and the five endopodal podomeres ischium, merus, carpus, propodus, dactylus (Fig.\u00a04B). At this stage, the limb anlagen are well developed in all pleonic and thoracic segments except thoracomere 8. The eighth thoracopods are not present in embryos (Fig.\u00a04A,B) but develop postembryonically (Fig.\u00a01; see Abzhanov and Kaufman 1999).\nFig.\u00a04Development of the walking limbs: A late stage 1 embryo (P. scaber), limb anlagen are present in the thoracic and pleonic segments except thoracomere 8; Normarsky interference contrast, ventral view, anterior is towards the right in all images of this figure. B stage 2 embryo (P. scaber), the thoracic limbs are subdivided into characteristic podomeres, thoracopod 8 is still undeveloped, lateral view, HOECHST nuclear stain. C\u2013E Immunohistochemistry with monoclonal antibody 016C6 (I. balthica). Muscle precursor cells in thoracic limbs 5 and 6 (C), antenna 2 of stage 3 embryos (D) and matured muscle in thoracopod 5 of a stage 5 embryo (E). Abbreviations: letters in the upper right corners identify the species and larval stages; Ba basis, Ca carpus, CX2-7 coxae of thoracopods 2 to 7, Da dactylus, Is ischium, Me merus, P1-5 pleomeres 1 to 5, Pr propodus, T8 thoracomere 8. Scale bars: A 30\u00a0\u03bcm, B 50\u00a0\u03bcm, C\u2013E 30\u00a0\u03bcm\nImmunohistochemistry with monoclonal antibody 016C6 revealed that muscle mononucleate precursor cells that express myosin heavy chain can be labelled for the first time in stage 3 embryos of I. balthica and P. scaber (Fig.\u00a04A,C). Sets of muscle precursors are arranged in a repetitive pattern within each podomere of the thoracic limbs and also the cephalic appendages, e.g. the second antennae (Fig.\u00a04C,D). During subsequent development, these precursors enlarge to form a complex pattern of muscle precursors in the thoracopods (Figs.\u00a04E, 5, 6 and 7).\nFig.\u00a05Myogenesis in the limb anlagen: Immunohistochemistry with monoclonal antibody 016C6 in the propodus of the thoracic limbs. Muscles propodus one and two (Pr1, Pr 2) originated from single, mononucleate cells, the muscle precursors or muscle founder cells, both in stage 3 embryos of P. scaber (A) and I. balthica (B). During subsequent development, the precursor cell of Pr1 enlarged (C) and eventually developed into a binucleate, syncytial muscle precursor (D, I. balthica). This muscle precursor further enlarged in size (E) and became a multinucleate muscle precursor (F, G, I. balthica). In stage 4 embryos, both muscles, Pr1 and the antagonistic Pr2, had developed into substantial primordial muscles (H). In stage 5 embryos, before hatching, Pr1 and Pr2 had subdivided into two or three distinct subunits (I, I. balthica). J: Development of muscles Pr1 and Pr2 in the propodus of I. balthica, schematic representation. Abbreviations: letters in the upper right corners identify the species and larval stages; Ca carpus, Da dactylus, Pr propodus. Scale bars: A\u2013I 5\u00a0\u03bcm (scale bar in B valid also for C\u2013F)Fig.\u00a06Muscles in the whole mount of a pre-hatching embryo: Immunohistochemistry with monoclonal antibody 016C6, stage 5 (pre-hatching) embryo of I. balthica. Abbreviations: A1, 2 antenna 1 and 2, MD mandible, MX1, 2 maxilla 1 and 2, OP operculum, P1-3 pleopods 1 to 4, T1-7 thoracic limbs 1 to 7 (T1 is a maxilliped), T8 thoracomere 8. Abbreviations: letters in the upper right corners identify the species and larval stages. Scale bar 100\u00a0\u03bcmFig.\u00a07Map of muscle precursor in a walking limb: A Immunohistochemistry with monoclonal antibody 016C6, thoracic limb 5, stage 5 (pre-hatching) embryo of I. balthica, frontal view, medial is towards the left. B Schematic drawing of the muscles in thoracic limb 5 of a stage 5 (pre-hatching) embryo of I. balthica (frontal view as in A). Anteriorly arranged muscles are drawn in light grey, posteriorly arranged muscles are drawn in dark gray. The color scheme of the podomeres is according to Walossek (1999) and Waloszek (2003); see also Wolf and Harzsch (2002): coxa\u2014red, basis\u2014yellow, endopodite\u2014green. C Schematic representations of the directions of movement of the joints between the podomeres. Abbreviations: letters in the upper right corners identify the species and larval stages; Ba basis, Ca carpus, Da dactylus, Is ischium, Me merus, Pr propodus; for the terminology to label the muscles see text. Scale bar 25\u00a0\u03bcm\nFormation of a single identified muscle in the propodus of the thoracic limbs\nMyogenesis will be exemplified by the formation of the identified, antagonistic muscles propodus 1 and 2 (Pr1, Pr2; Fig.\u00a05), both of which are located in the propodus of the thoracic limbs and which move the dactylus in the adult (see below). Pr1 originates from single, mononucleate muscle precursors or muscle founder cells, which express myosin heavy chain both in stage 3 embryos of P. scaber (Fig.\u00a05A,J; the mononucleate precursor cell of muscle Pr2 is also shown) and I. balthica (Fig.\u00a05B). During subsequent ontogeny, this mononucleate precursor cell enlarge (Fig.\u00a05C) and eventually develop into a binucleate, syncytial muscle precursor (Fig.\u00a05D,J). This muscle precursor further enlarges in size (Fig.\u00a05E) and becomes a multinucleate muscle precursor (Fig.\u00a05F,G,J). In stage 4 embryos, both muscles, Pr1 and the antagonistic Pr2, have developed into substantial primordial muscles (Fig.\u00a05H). In stage 5 embryos, before hatching, Pr1 and Pr2 have subdivided into two or three distinct subunits (Fig.\u00a05I,J).\nMuscle pattern in the thoracic limbs of pre-hatching embryos\nImmunohistochemistry with 016C6 enabled us to map the primordial muscles in the thoracic appendages of I. balthica embryos before hatching (Figs.\u00a06 and 7). As the adult pattern of muscles has not been mapped in the isopods which we examined, we labelled the primordial muscles with two letters to indicate the podomere in which they were located plus a number counting up the muscles from anterior to posterior (Fig.\u00a07B). Me2, for example, designates muscle number 2 in the merus. Most embryonic muscles are arranged in antagonistic groups and are restricted to one podomere (Fig.\u00a07B). Exceptions are Ba\/Is1 and Ba\/Is2, which span across the basis and the ischium (Fig.\u00a07B). In the coxae, a complex system of muscles is present, which we failed to map.\nTo determine the function that the embryonic muscles will exert in the adult limbs, we examined the articulations and directions of movement of the successive podomeres in adult limbs of P. scaber (Fig.\u00a01B,C) and I. balthica (Fig.\u00a01A). These functions can be tentatively summarised as follows (Fig.\u00a07B,C, Table\u00a01): Ba7 serves as abductor of the ischium while Ba1, 2, 3 and Ba4, 5 serve as adductors of the ischium that swings in a medial to lateral plane. The merus also moves in a medial to lateral plane and is abducted by Is1 and Ba\/Is1 and adducted by Ba\/Is2 and Is 2, 3. The carpus swings in an anterior to posterior plane perpendicular to the merus. It is retracted by Me2 and protracted by Me1. The carpus also seems to have a limited freedom of movement in the medial to lateral. The propodus once more is displaced exclusively in a medial to lateral plane with Ca3 serving as the abductor and Ca1 and Ca2 as adductors. The dactylus swings in the same plane and is adducted by Pr1 and abducted by Pr2 and Pr3. The coax\u2013basis joint was not examined in detail but seemed to allow displacement of the basis into several directions (Alexander 1972).\nTable\u00a01Muscles in the walking limbs of crayfish and other Decapoda (Evoy and Ayers 1982; Atwood and Govind 1982; \u201ccommon\u201d names are also indicated), Isopoda (present report and Alexander 1972), Amphipoda (Brusca 1981)Muscles located in this podomereCrayfish and other DecapodaIdotea (Isopoda)Cystisoma (Amphipoda)ThoraxNot shownNot shownNot shownCoxa2 Depressors of basis2 Extensor of basis2 Retractors of basis2 Levators of basis2 Flexors of basis2 Protractors of basis2 Rotators of basisadductor of basisBasisFlexor of ischium2 Flexors of ischiumBasis and ischium are fused in adult Decapoda and contain the reductor of merus3 Lateral extensors of ischium2 Extensors of ischium2 Lateral extensors of ischiumIschium2 Flexors of merusFlexor of merus2 Lateral extensors of merus2 Extensors of merus2 medial extensors of merusMerusAbductor of carpus (extensor)Abductor of carpus3 Abductors of carpusAdductor of carpus (flexor)Adductor of carpus2 Adductors of carpusAccessory adductor of carpusCarpusReductor of propodus (stretcher)Flexor of propodus2 Flexors of propodusProductor of propodus (bender)Lateral extensor of propodus2 Extensors of propodusAdductor of propodus (rotator)Medial extensor of propodusPropodusAbductor of dactylus (opener)Flexor of dactylus3 Flexors of dactylusAdductor of dactylus (closer)Lateral extensor of dactylus1 Extensor of dactylusmedial extensor of dactylusDactylusNo musclesNo musclesNo musclesMuscles located within the thorax are not shown.\nDevelopment of muscles in the body wall\nAlthough muscle precursor cells are not present in the anlagen of the thoracic limbs in late stage 1 embryos (Fig.\u00a04A), there is already a distinct pattern of muscle precursors in the body wall [Electronic supplementary material (ESM) Fig.\u00a01]. A bilateral band of ventral longitudinal (VL) muscle precursors is arranged parallel to the midline (ESM Fig.\u00a01A). This band displays an anterior\u2013posterior of maturation so that several steps of muscle formation can be observed in a single specimen (ESM Fig.\u00a01B,C). As in the limbs, the ventral longitudinal muscles also originate from single muscle mononucleate precursor cells (inset ESM Fig.\u00a01A), which span the length of one segment. These precursors subsequently develop into bi- (ESM Fig.\u00a01B) and multinucleated muscle precursors (ESM Fig.\u00a01C). In late stage 1 embryos, a band of dorsal longitudinal (DL) muscles is arranged in parallel to the ventral longitudinal muscles (ESM Fig.\u00a01D,E). The ventral and dorsal bands of muscles are connected by single, segmentally iterated transverse muscle precursor cells (arrows in ESM Fig.\u00a01E). In later stages, 016C6 also labelled a dense network of muscles surrounding the hindgut (ESM Fig.\u00a01F).\nDuring subsequent ontogeny, the muscle precursors of the longitudinal muscles mature into a more complex system of dorsal (extensors, ESM Fig.\u00a02A) and ventral (flexors, ESM Fig.\u00a02C) body wall musculature. A comparison of muscles in the leg-bearing thoracic segment and the leg-less eighth thoracomere enabled us to distinguish between muscles of the body wall and those that are associated with the coxa (Fig.\u00a06, ESM Fig.\u00a02B). In addition to the ventral and dorsal longitudinal muscles, distinct intersegmental extensor muscles are present in the eighth and the more anterior thoracomeres. These muscles transverse the border between two successive thoracomeres (Fig.\u00a06, ESM Fig.\u00a02A,B). Furthermore, two smaller transverse muscles are found in each thoracomere (ESM Fig.\u00a02B).\nDiscussion\nMyogenesis in Crustacea\nIn recent years, cell lineages analyses in malacostracan embryos have set out to explore the earliest stages of mesoderm formation (Gerberding et al. 2002; Hertzler 2002, 2005), and the molecular mechanisms that underlie mesoderm formation have been explored in the emerging Crustacean model system, the amphipod Parhyale hawaiensis (Price and Patel 2008). Furthermore, in decapod Crustaceans, considerable effort has been directed towards understanding aspects of the ontogeny of the neuromuscular system such as outgrowth of motoraxons, development of the innervation patterns of muscles and synapse formation. These processes have been examined, e.g. in the body wall of the pleon (Cole and Lang 1980; Stephens and Govind 1981; Govind et al. 1985), the pleopods (Davis and Davis 1973; Kirk and Govind 1992) and the thoracic limbs (Costello et al. 1981; Govind and Pearce 1981, 1982, 1989; Govind et al. 1982; Govind and Derosa 1983; Pearce et al. 1985; Lnenicka et al. 1991; Arcaro and Lnenicka 1995). Myogenesis has been studied in the pleon of embryonic American lobsters on the electron-microscopic level (review Govind 1982, 1995). According to these accounts, the first signs of muscle formation are the appearance of small localised patches of myofilaments in multinucleated structures, the presumptive myotubes. Surrounding undifferentiated cells have enlarged nuclei with diffuse chromatin, Golgi apparatus, ribosomes and mitochondria and seem to resemble premyoblast cells (Govind 1982, 1995). The myofilaments then become organised in longitudinal arrays, and later, distinct sarcomeres with A, Z and I bands appear. The muscle fibres grow in length either by the elongation of individual sarcomeres or the serial addition of sarcomeres (Govind 1982, 1995). Muscle development has also been studied in the embryos and nauplii of the dendrobranchiate shrimp Sicyonia ingentis using fluorescent phallotoxins to label F-actin (Kiernan and Hertzler 2006). In these animals, phalloidin labelling identifies muscle precursor that in the beginning are not striated and during mid-embryogenesis stretch along the entire length of the naupliar appendages (antenna one, antenna two and mandible). In this study, the analysis of myogenesis was restricted to these naupliar appendages and was followed up to the nauplius V stage when trunk appendages are still not developed (Kiernan and Hertzler 2006). Contrary to the dendrobranchiate S. ingentis which develops via nauplius larvae, the isopod species studied in the present study are direct developers so that a meaningful comparison of myogenesis in the trunk limbs is as yet not possible.\nHowever, we can compare myogenesis in the pair of second antennae of this dendrobranchiate shrimp to the Isopoda. One major difference is that in Dendrobranchiata, the muscle precursors in antenna 2 span across the entire length of these appendages (Kiernan and Hertzler 2006), whereas in Isopoda, sets of individual muscle precursors are arranged in an iterated pattern within each podomere of the second antennae. Studies on appendage development in embryos of the American lobster Homarus americanus (Malacostraca, Homarida) using monoclonal antibody 016C6 (Harzsch and Kreissl, unpublished data) revealed that the pattern of myogenesis in this organism is different from that in Isopoda but rather resembles that in Dendrobranchiata. In lobster embryos, syncytial muscle precursor cells establish the muscles in the endopodites of the thoracic appendages and also in the second antennae. As in Dendrobranchiata, these muscle precursors initially stretch along the entire length of the appendages. During subsequent embryogenesis, the muscle precursors subdivide into several distinct units, thereby giving rise to iterated pairs of antagonistic primordial muscles in each of the successive podomeres (Harzsch and Kreissl, unpublished data), a layout which more and more resembles the arrangement in the adult thoracopods (see below and Table\u00a01). We would predict that also in Dendrobranchiata, such an ontogenetic subdivision of the muscle precursors into individual iterated muscle primordia may take place, a question that should be explored by analysing later larval stages than have been studied so far. Considering that Kiernan and Hertzler (2006) reported striking similarities between the pattern of myogenesis between the dendrobranchiate S. ingentis and the brine shrimp Artemia salina (Crustacea, Branchiopoda) and considering that myogenesis in the American lobster H. americanus matches the Sicyonia\/Artemia pattern (Harzsch and Kreissl, unpublished data), we conclude that these organisms most likely represent the ancestral Crustacean mode of myogenesis and that the slightly different mode that we observed in the present study for the Isopoda is derived from this ancestral pattern.\nComparison of myogenesis in Crustacea and Insecta\nIn the developing body wall of grasshopper embryos, Ho et al. (1983) were the first to describe large mononucleate mesoderm cells that arise early in development to erect a scaffold for later developing muscles, and they termed these cells muscle pioneers. The development of muscle pioneers in the body wall subsequently was also examined with antibodies different from that which Ho et al. (1983) used (Xie et al. 1992, 1994; Steffens et al. 1995). Furthermore, the role of muscle pioneers was extensively studied in the developing limbs of the grasshopper (Ball and Goodman 1985a, b; Ball et al. 1985) and a moth (Consoulas and Levine 1997; Consoulas et al. 1997). Since the discovery of muscle pioneers in the grasshopper, detailed analyses of myogenesis in the fruit fly has led to the establishment of the founder cell model for muscle patterning (reviewed in Campos-Ortega and Hartenstein 1997; Abmayr and Keller 1998; Baylies et al. 1998; Paululat et al. 1999a, b; Roy and VijayRaghavan 1999). According to this model, mesodermal progenitor cells in insects undergo mitosis to produce mononucleate muscle pioneers (termed founder cells in the fruit fly). These pioneers then differentiate into bi-, tri- or multinucleate syncytial muscle precursors by fusion with surrounding undifferentiated myoblasts.\nOur present report applying a monoclonal antibody against myosin heavy chain extends the ultrastructural findings of Govind (1982, 1995) on muscle formation in malacostracan Crustaceans and suggests that in Isopoda, similar mechanisms of myogenesis as in insects may be present. This antibody labelled mononucleate myosin-expressing cells with morphological characteristics of insect muscle pioneers. Despite the differences between myogenesis in isopods and the American lobster noted above, the latter organisms nevertheless also seem to employ mononucleate myosin-expressing cells to found their limb muscles (Harzsch and Kreissl, unpublished data). Similar to insects, the muscle precursors in the limb anlagen of the isopod Crustaceans we examined were individually identifiable and established the primordial adult muscle pattern. Furthermore, these cells developed into bi- and multinucleate syncyctia similar to insect muscle precursors. However, our methods did not enable us to determine whether this process is achieved via fusion with surrounding myoblasts as it is in insects. We conclude that in the moment, our knowledge on Crustacean myogenesis is too limited and too few species studied as to suggest a homology of the mononucleate Crustacean muscle precursors and insect muscle pioneer cells. Although the musculature in the limbs of non-malacostracan Crustaceans has been examined in depth, e.g. in Copepoda (Boxshall 1985, 1990, 1997), Cephalocarida (Hessler 1964), Branchiopoda (Benesch 1969; Fryers 1988) and Cirripedia (Wallay 1969), myogenesis has only been studied in two representative of the Branchiopoda (Williams and M\u00fcller 1996; Kiernan and Hertzler 2006) and a cirripede (Semmler et al. 2006). However, these studies were not conducted at the cellular level that would allow for a meaningful comparison with our data. Therefore, it remains unclear at this point whether mononucleate muscle precursor cells are part of the Crustacean ground pattern. Similarly, as the mechanisms of muscle formation are not known in any representatives of the Chilopoda and Progoneata (Myriapoda), we cannot answer the question whether mononucleate muscle pioneers are a class of muscle founder cells that represent a synapomorphy of Hexapoda and Malacostraca or if they were already present in the ground pattern of Euarthropoda.\nMuscle arrangement in the adult limbs of Isopoda and Decapoda\nThe structural organisation of the neuromuscular system in adult Crustacean limbs is particularly well understood in Decapoda (reviewed in Wiersma 1961; Evoy and Ayers 1982; Govind and Atwood 1982; Rathmayer and Maier 1986; Wiens 1989; Rathmayer 1990, 2002; Cattaert and Le Ray 2001; Clarac 2002). In this taxon, muscles in the limbs exert their force on a system of levers formed by apodemes and joints, most of which operate in a single plane. Usually, the movements of an individual joint are brought about by a pair of antagonistic muscles located in the podomere proximal to the podomere which they displace. Successive joints along a particular appendage operate at an angle of approximately 90\u00b0 to one another, and most movements result from combinations of displacements at several joints. Three main joints are involved in locomotion: the thoraco-coxopodite joint allows forward and backward movements of the leg; the coxo-basipodite joint is responsible for upward and downward movements; the mero-carpopodite joint is responsible for extension and flexion of the leg (reviewed in Wiersma 1961; Evoy and Ayers 1982; Govind and Atwood 1982; Cattaert and Le Ray 2001; Clarac 2002).\nIn adult Isopoda, muscles have been extensively mapped in the cephalic appendages (Schmalfuss 1974) and the pleopods (Erhard 1997), but little information is available about the musculature of the thoracic limbs in the isopod taxa Oniscoidea and Valvifera. Von Haffner (1937) and Gruner (1954) described the morphology of the limbs in several isopod taxa but failed to individually identify the muscles, while Alexander (1972) only described the muscles located in the coxa. However, the limb musculature was mapped in several species of the amphipod genus Cystisoma Gu\u00e9rin-M\u00e9neville, 1842 (Hyperiidea; Brusca 1981). Table\u00a01 summarises the muscle arrangement in adult Decapoda (Eucarida) and compares it with Amphipoda (Peracarida; Brusca 1981) and the isopod pre-hatching embryos (Peracarida) we studied. In all three species, the principle of paired antagonistic muscles (or muscle groups) is realised. However, while in Decapoda, in most cases, pairs of single antagonistic muscles are present, pairs of functional groups composed of two to five distinct muscles are found in Peracarida (von Haffner 1937; Brusca 1981). Furthermore, all successive joints in Decapoda operate at an angle of approximately 90\u00b0 (Evoy and Ayers 1982), while in Peracarida, only the planes of the merus\u2013carpus joint and the carpus\u2013propodus joint are twisted against each other (Fig.\u00a07; and von Haffner 1937; Brusca 1981). Another difference is that in adult Decapoda, basis and ischium are fused and bear only one muscle, the reductor of the merus. These two podomeres are unfused in Peracarida and therefore contain sets of flexors and extensors (Table\u00a01; and von Haffner 1937; Brusca 1981).\nElectronic Supplementary Material\nBelow is the link to the electronic supplementary material.\nSupplementary Fig.\u00a01\nMyogenesis in the body wall: Immunohistochemistry with monoclonal antibody 016C6, muscle precursors in the body wall of late stage 1 embryos of P. scaber, dorsal views, anterior is towards the top. A bilateral bands of VL muscle precursors are arranged parallel to the midline. B, C Higher magnification of A, several steps of muscle formation from single muscle precursor cells (B and inset in A) to bi- and multinucleated muscle precursors (C) could be observed in this specimen. D, D\u2032 A dense network of muscles surrounding the hindgut was labelled in later embryonic stages. E, F late stage 1 embryo, a band of DL muscles was arranged in parallel to the VL muscles. The ventral and dorsal bands of muscles were connected by single, segmentally iterated transverse muscle precursor cells (arrows, E). Abbreviation: T 1-7 thoracomeres 1 to 7. Scale bars: A, B 40\u00a0\u03bcm, C 20\u00a0\u03bcm, D 40\u00a0\u03bcm, D\u2032 10\u00a0\u03bcm, E 5\u00a0\u03bcm (F same magnification as E). (GIF 693\u00a0kb)\nHigh resolution image file (TIF 15.0 MB)\nSupplementary Fig.\u00a02\nBody wall muscles in a pre-hatching embryo: Immunohistochemistry with monoclonal antibody 016C6, mature system of muscles in the body wall of late stage 4 embryos of I. balthica, lateral views (A, C). The segmentally iterated dorsal longitudinal muscles (A) and ventral longitudinal muscles are visible (C), and the muscles that move the coxa can be discerned (asterisks). B A comparison of muscles in the leg-bearing thoracomere 7 segment and the leg-less eighth thoracomere enabled us to distinguish between muscles of the body wall (IE intersegmental extensors, TM transverse muscles; compare A) and those that are associated with the coxa (asterisks). Abbreviations: CX coxa, DL dorsal longitudinal muscles, extensors, IE intersegmental extensors, T7 thoracic leg 7, T8 thoracomere 8, VL ventral longitudinal muscles, flexors. Scale bars: A\u2013C 30\u00a0\u03bcm. (GIF 650\u00a0kb)\nHigh resolution image file (TIF 14.8 MB)","keyphrases":["muscle precursor","crustacea","appendage","evolution","isopoda"],"prmu":["P","P","P","P","P"]}
{"id":"Histochem_Cell_Biol-4-1-2323030","title":"Endocytic downregulation of ErbB receptors: mechanisms and relevance in cancer\n","text":"ErbB receptors (EGFR (ErbB1), ErbB2, ErbB3, and ErbB4) are important regulators of normal growth and differentiation, and they are involved in the pathogenesis of cancer. Following ligand binding and receptor activation, EGFR is endocytosed and transported to lysosomes where the receptor is degraded. This downregulation of EGFR is a complex and tightly regulated process. The functions of ErbB2, ErbB3, and ErbB4 are also regulated by endocytosis to some extent, although the current knowledge of these processes is sparse. Impaired endocytic downregulation of signaling receptors is frequently associated with cancer, since it can lead to increased and uncontrolled receptor signaling. In this review we describe the current knowledge of ErbB receptor endocytic downregulation. In addition, we outline how ErbB receptors can escape endocytic downregulation in cancer, and we discuss how targeted anti-cancer therapy may induce endocytic downregulation of ErbB receptors.\nIntroduction\nProper endocytic uptake and endosomal sorting of signaling receptors are crucial mechanisms for the regulation of signaling activity involved in cellular growth, development, and differentiation (Crosetto et al. 2005; Fischer et al. 2006; Giebel and Wodarz 2006; Le Borgne 2006; Polo and Di Fiore 2006; Sorkin and von Zastrow 2002). This is not least true for the epidermal growth factor receptor (EGFR), a member of the ErbB family of receptor tyrosine kinases which contains four members: EGFR (ErbB1), ErbB2, ErbB3 and ErbB4. In addition to playing an important role in normal cellular functions, these receptors are involved in the pathogenesis of human cancers and receive much attention as targets for development of new anti-cancer drugs (Hynes and Lane 2005). There are several mechanisms whereby cancer cells can obtain uncontrolled ErbB receptor signaling, including increased receptor expression, activating mutations, and escape of endocytic receptor downregulation (Bache et al. 2004; Citri and Yarden 2006; Normanno et al. 2005; Polo et al. 2004; Warren and Landgraf 2006).\nOur current understanding of endocytic downregulation of ErbB receptors is largely based on knowledge of EGFR behavior upon binding of epidermal growth factor (EGF) (Bache et al. 2004; Dikic 2003; Gruenberg and Stenmark 2004). EGFR is among the most well studied receptors, and it is regarded as a prototype of a signaling receptor that is internalized and degraded following ligand binding. In contrast, surprisingly little is known about endocytosis of ErbB2-4 as well as about EGFR endocytosis following binding of ligands other than EGF. However, it is becoming increasingly clear that the other ErbB receptors do not behave like EGFR regarding endocytosis. In this review we outline the molecular mechanisms of EGF-induced endocytic downregulation of EGFR. In addition, we describe the current knowledge of internalization and downregulation of the other ErbB receptors and discuss how the various ErbB ligands differ in their potential to induce receptor downregulation. Finally, we focus on internalization of these receptors as a promising target in anti-cancer therapy.\nThe ErbB receptor system\nA thorough description of the structure and biological functions of the four ErbB receptors and their ligands is beyond the scope of this review and can be found elsewhere (Citri and Yarden 2006; Hynes and Lane 2005; Jones et al. 2006). In brief, the ErbB receptors are transmembrane receptor tyrosine kinases consisting of a glycosylated, extracellular N-terminal part containing a ligand binding site and a dimerization arm, a transmembrane segment, and an intracellular part containing a tyrosine kinase domain and a C-terminal tail with several phosphorylation sites (Fig.\u00a01) (Normanno et al. 2005). A large number of ErbB ligands have been identified, some of which can bind to more than one of the ErbB receptors with high affinity (Table\u00a01) (Breuleux 2007; Harris et al. 2003). The active, ligand-bound ErbB receptors function as homo- or heterodimers, which can activate a multitude of signaling pathways involved in proliferation, differentiation, cell survival, and migration (Normanno et al. 2005).\nFig.\u00a01Structure of EGFR. In the extracellular part of the receptor, EGFR harbours two domains (L1 and L2) that upon folding form the ligand-binding pocket. Between L1 and L2 is another domain (S1) that includes the dimerization arm. Intracellularly, EGFR has a kinase domain and a C-terminal tail with several amino acid residues that can be phosphorylated. The tyrosine residues (Y) that are involved in Cbl binding are shown. The parts of wild-type EGFR that are deleted in EGFRvIII, EGFRvIV, and EGFRvV are indicatedTable\u00a01ErbB receptors and their ligandsReceptorRemarksLigandsEGFR (ErbB1)Epidermal growth factor (EGF) Transforming growth factor \u03b1 (TGF\u03b1)Heparin-binding EGF (HB-EGF)\u03b2-CellulinAmphiregulinEpiregulinEpigenErbB2Constitutively exposed dimerization armErbB3Kinase deadHeregulin-1\/Neuregulin-1Heregulin-2\/Neuregulin-2ErbB4Heregulin-1\/Neuregulin-1Heregulin-2\/Neuregulin-2Heregulin-3\/Neuregulin-3Heregulin-4\/Neuregulin-4Heparin-binding EGF (HB-EGF)\u03b2-CellulinEpiregulin\nErbB receptor activation\nThe initial steps of ligand-mediated ErbB receptor activation have been well described upon solving the crystal structure of the EGFR extracellular domain bound to EGF (Garrett et al. 2002; Ogiso et al. 2002). EGF binding to EGFR has been proposed to induce a conformational change exposing the dimerization arm in the receptor extracellular domain (Burgess et al. 2003; Ferguson et al. 2003; Ogiso et al. 2002). Upon dimerization, the receptor\u2019s intrinsic kinase activity cross-phosphorylates specific residues in the C-terminal tail of the partnering receptor, allowing for the recruitment of phosphotyrosine-binding proteins to EGFR. ErbB3 and ErbB4 dimerize in response to ligand binding in a manner similar to EGFR, whereas ErbB2 differs from the other ErbB receptors by not binding to any ligand with high affinity (Bouyain et al. 2005; Burgess et al. 2003; Kani et al. 2005). Instead, ErbB2 is in a constitutively active conformation with an exposed dimerization arm even in the absence of ligand (Burgess et al. 2003). Its ability to homodimerize is limited due to the electronegativity of its extracellular part, but ErbB2 is the preferred heterodimerization partner for the other ErbB receptors (Garrett et al. 2003; Graus-Porta et al. 1997; Tzahar et al. 1996).\nThe signal transduction pathways activated by ErbB receptors include well-known signaling cascades such as the Ras-Erk1\/2 pathway, the phospholipase C\u03b3-protein kinase C pathway, the phosphatidyl inositol 3 (PI 3)-kinase-Akt pathway, and STAT signaling.\nEndocytic downregulation of signaling receptors has for many years been regarded solely as a means of attenuating receptor signaling. However, during the last decade an increasing amount of evidence has suggested that some receptors, including EGFR, may continue their signaling activity from endocytic compartments and that the signaling occurring from here is qualitatively different from the signaling taking place at the plasma membrane (Miaczynska et al. 2004). Thus, ErbB receptor endocytosis is not merely a means of turning off activated receptors; it may also be a regulatory mechanism altering the signaling outcome.\nEndocytic downregulation of EGFR\nRegarding ligand-induced endocytic receptor downregulation, EGFR has been the most popular model system for many years, and the mechanisms of EGFR endocytosis and intracellular trafficking are therefore relatively well understood.\nEGFR is activated upon EGF binding, leading to signaling and relocation to invaginating clathrin-coated pits (CCPs) on the plasma membrane. These pits give rise to clathrin-coated endocytic vesicles, and after the coat has been released from the membrane, the vesicles fuse with early endosomes and thereby deliver the receptor to this compartment. Here the receptor is sorted for further transport, either back to the cell surface by recycling, or to intraluminal vesicles (ILVs), a pathway that eventually leads to delivery of EGFR to lysosomes for degradation (Fig.\u00a02). Some of these steps are now well established, but others remain elusive. Below we describe both what is known and what challenges we are facing in understanding the crucial pathway of clathrin-mediated EGFR endocytosis and endosomal sorting.\nFig.\u00a02Internalization and endosomal sorting of EGFR. Main figure: upon activation, EGFR (green) is translocated to clathrin coated pits (CCP) on the plasma membrane and internalized (grey arrows). After transport to early endosomes (EE), EGFR is either recycled back to the plasma membrane (white arrows) or taken up into intraluminal vesicles (ILVs). EE will mature to late endosomes (LE), and EGFR in ILVs will eventually be degraded in lysosomes (Lys) (black arrows). Upper insert at the plasma membrane, EGF-activated EGFR dimerize and the kinase activity of the receptors phosphorylates tyrosine residues (P) in EGFR. This creates docking sites for intracellular proteins such as Grb2. Grb2 mediates binding of the ubiquitin ligase Cbl that adds mono- or polyubiquitins (Ub) to EGFR. Activated EGFR is transported to clathrin coated pits that in addition to clathrin also consists of Eps15 and other proteins. Lower insert At the EE vacuolar membrane, EGFR destined for degradation still binds Cbl and is continuously phosphorylated and ubiquitinated. The EGFR ubiquitins are bound by Hrs that resides in at Hrs\/STAM\/clathrin coat, and this is followed by binding of ESCRT complexes to the ubiquitinated EGFR leading to uptake into ILVs\nSorting of EGFR to clathrin coated pits: ubiquitination or not?\nOne of the first steps in clathrin-mediated endocytic downregulation is the transport of cargo into CCPs. Despite the importance of regulating whether EGFR is endocytosed, the molecular machinery controlling this is poorly understood.\nThe constitutively endocytosed receptors, the transferrin receptor and the low-density lipoprotein receptor, are sorted into CCPs by interaction with the clathrin-binding AP-2 complex through conserved tyrosine-based motifs in the receptor\u2019s intracellular tail. EGFR also interacts with AP-2, indicating a similar role of AP-2 in EGFR endocytosis (Sorkin et al. 1995; Sorkin and Carpenter 1993). However, EGFR is not sorted into CCPs in its resting state, and the direct interaction of AP-2 with EGFR does not seem to be necessary for EGFR internalization (Nesterov et al. 1995). Thus, additional interaction partners in the CCPs are required for EGFR to be sorted into these plasma membrane microdomains. Since the kinase activity of EGFR has been shown to be important for this sorting (Lamaze and Schmid 1995), such interaction partners should bind modifications of EGFR that are only present in the activated state; e.g. phosphorylated residues and\/or ubiquitinated residues.\nUbiquitination (Text box 1) has been known to mediate endocytosis of membrane receptors for vacuolar degradation in yeast (Hicke and Riezman 1996; Kolling and Hollenberg 1994), and ubiquitination has also been found to be involved in endocytic downregulation of mammalian receptors such as EGFR (reviewed in (Hicke 1999)). As a result, ubiquitination has long been considered a likely candidate as a CCP targeting signal at the plasma membrane.Text box\u00a01Ubiquitination\nUpon activation of EGFR, it is heavily ubiquitinated by the attachment of both monoubiquitin and polyubiquitin (Text box 1). As will be discussed below, this ubiquitination is a prerequisite for endosomal sorting of EGFR to ILVs in multivesicular bodies (MVBs), but its importance in the initial sorting to CCPs and internalization from the plasma membrane has been the subject of controversy for some time. The ubiquitin ligase responsible for EGFR ubiquitination is Cbl, a ring-finger domain E3 ubiquitin ligase (Levkowitz et al. 1999). Cbl is indeed necessary for EGFR endocytosis (Jiang and Sorkin 2003). Cbl can bind EGFR either directly by binding to phosphorylated Y1045, or indirectly via the adaptor protein Grb2, which binds to phosphorylated Y1068 and Y1086 (Fig.\u00a01) (Levkowitz et al. 1999; Waterman et al. 2002). Binding of Cbl via phosphorylated Y1045 is not necessary for EGFR endocytosis, since the Y1045F EGFR mutant that cannot bind Cbl directly is internalized almost as efficiently as wild-type EGFR (Gr\u00f8vdal et al. 2004; Jiang et al. 2003). In contrast, Cbl binding to EGFR via Grb2 is necessary for receptor internalization (Huang and Sorkin 2005). Thus, Grb2 knockdown inhibits EGFR endocytosis, but a chimeric protein consisting of the Y1068\/Y1086-binding domain of Grb2 fused to Cbl can rescue EGFR internalization in Grb2 depleted cells, showing that the prime role of Grb2 in EGFR internalization is recruitment of Cbl (Huang and Sorkin 2005).\nSince functional Cbl is a prerequisite for EGFR internalization (Jiang and Sorkin 2003), and Cbl ubiquitinates EGFR (Levkowitz et al. 1998), it has long been assumed that Cbl-mediated ubiquitination of EGFR is the prime signal for EGFR translocation to CCPs. In accordance with this, overexpression of the Cbl mutant 70Z-Cbl with impaired ubiquitin ligase activity inhibits EGFR internalization (Jiang and Sorkin 2003). To further investigate the role of EGFR ubiquitination in internalization, an EGFR mutated on 15 lysine residues was recently constructed. This mutant had an ubiquitination level corresponding to 1% of that seen for wild-type EGFR after EGF stimulation. Interestingly, the mutated EGFR still retained full kinase activity and displayed normal internalization (Huang et al. 2007). This could imply that EGFR ubiquitination is not a signal for internalization. Alternatively, the endocytic machinery recognizing ubiquitinated EGFR is highly sensitive, so that even a 1% residual ubiquitination is sufficient for proper targeting of EGFR to CCPs.\nImportantly, Cbl may serve other functions in addition to ubiquitin ligase activity. It has been reported that Cbl binds endophilin via CIN85 (Soubeyran et al. 2002). Endophilin is a known regulator of clathrin-mediated endocytosis (Reutens and Begley 2002), and this recruitment of CIN85 and endophilin to EGFR by Cbl has been shown to be important for EGFR internalization (Soubeyran et al. 2002).\nAlthough the role of EGFR ubiquitination as an internalization signal is controversial, several candidates, including Eps15, have been suggested as adaptors for sorting ubiquitinated EGFR into CCPs. Eps15 is found in CCPs and has an ubiquitin-binding domain (Hawryluk et al. 2006; Stang et al. 2004). Furthermore, Eps15 is recruited to the plasma membrane upon EGF stimulation of EGFR (Stang et al. 2004; Torrisi et al. 1999), and expression of dominant-negative Eps15 as well as siRNA-mediated knockdown of Eps15 has been shown to inhibit EGFR endocytosis (Bakowska et al. 2007; Benmerah et al. 1998; Fallon et al. 2006). Phosphorylation of Eps15 has also been shown to be necessary for EGFR internalization (Confalonieri et al. 2000). However, a direct evidence of Eps15 as an adaptor protein recognizing ubiquitinated EGFR via interactions with EGFR-attached ubiquitin has so far not been presented.\nAt present, it remains obscure which mechanisms are responsible for sorting of the activated EGFR to coated pits, although it is clear that Cbl and Grb2 are central, and Eps15 may have important functions. One possible explanation for the apparent contradictory results regarding ubiquitin as an endocytic signal for EGFR is that it is not ubiquitination of EGFR itself but rather ubiquitination of accessory proteins by Cbl that is necessary for EGFR internalization.\nEndosomal sorting of EGFR: a key role of ubiquitination\nUpon internalization by clathrin-mediated endocytosis, EGFR is transported to early endosomes (Fig.\u00a02). Both cargo destined for lysosomal degradation and for recycling back to the plasma membrane is delivered here, and the compartment is sometimes seen in the electron microscope as a vacuole with tubular elongations. The tubular elongated parts of the early endosomes are believed to be involved in recycling, whereas sorting for degradation initiates at the vacuolar membrane of the endosomes by inward budding, giving rise to ILVs containing membrane proteins destined for lysosomal degradation (Fig.\u00a02). The internalized EGFR can be sorted both for recycling and for lysosomal degradation. Whereas recycling seems to be the default pathway from early endosomes, sorting for lysosomal degradation is mediated by Hrs\/STAM and by the endosomal sorting complex required for transport (ESCRT) complexes (ESCRT-I to -III) that are believed to mediate receptor translocation into ILVs of MVBs (for recent review see (Williams and Urbe 2007)).\nThe EGF-activated EGFR is also associated with Cbl in early endosomes, resulting in continued ubiquitination of EGFR (Duan et al. 2003; Longva et al. 2002). Contrary to its uncertain role in receptor internalization (see above), ubiquitination has been convincingly demonstrated to be important for endosomal sorting of EGFR to lysosomes. EGFR mutated at intracellular lysine residues to give a receptor with decreased ubiquitination is severely degradation impaired following EGF stimulation (Huang 2006, 2007). Likewise, the Y1045F EGFR mutant that does not bind directly to Cbl and displays reduced ubiquitination is degradation impaired (Gr\u00f8vdal et al. 2004; Jiang and Sorkin 2003; Levkowitz et al. 1999). This mutant does not translocate to ILVs (Gr\u00f8vdal et al. 2004), giving further support to a model where ubiquitin is the signal for EGFR sorting for degradation.\nUbiquitin chains are recognized by ESCRT complexes, which retain ubiquitinated cargo in the membrane of early endosomes thereby hindering receptor recycling (Fig.\u00a02) (Raiborg and Stenmark 2002). The first described point of recognition is Hrs and STAM, also known as the ESCRT-0 complex. On early endosomes, Hrs and clathrin form dynamic microdomains, which can be seen by EM as flat electron-dense areas on the limiting membrane (Raiborg et al. 2001; Sachse et al. 2002). Hrs and STAM interact with ubiquitinated cargo via their ubiquitin-interacting motif (UIM) domains. The proteins preferentially bind polyubiquitin (Polo et al. 2002), and the UIM of Hrs has recently been shown to bind two ubiquitin molecules simultaneously (Hirano et al. 2006). Conceivably, cargo must be either polyubiquitinated by K63-linked polyubiquitin chains or multiply monoubiquitinated for efficient sorting towards the lysosome for degradation (Barriere et al. 2007). This is in concordance with more than 50% of EGFR ubiquitination being in the form of poly-ubiquitin chains, primarily via K63 (Text box 1) (Huang et al. 2006).\nAfter Hrs\/STAM has retained ubiquitinated cargo within microdomains in the vacuolar membrane of the early endosome, ESCRT complexes (ESCRT-I\u2013ESCRT-III) are sequentially recruited, eventually leading to sorting of EGFR into ILVs (reviewed in (Williams and Urbe 2007)). Recently, siRNA studies showed that knock-down of Hrs or ESCRT-I components caused increased recycling of EGFR. In contrast, knockdown of ESCRT-II and ESCRT-III components did not result in increased recycling of EGFR, but did impair degradation (Raiborg et al. 2007). This indicates that the fate of EGFR (recycling versus degradation) is determined upstream of ESCRT-II. ESCRT-II appears to be necessary for sorting of EGFR to ILVs. In contrast, the exact function of the ESCRT-III complex in EGFR sorting seems to be more complex. The original model was that ESCRT-III is important for formation of ILVs (Williams and Urbe 2007). However, it was recently reported that upon depletion of the ESCRT-III component VPS24, EGFR is still sorted into ILVs in MVBs and silenced (Bache et al. 2006). Thus the ESCRT-III complex might function downstream of internalization to ILVs, perhaps as late as in MVB fusion with lysosomes (Bache et al. 2006).\nDifferential effects of EGFR ligands on EGFR degradation\nThe model for EGFR sorting to lysosomes described above has been developed based on studies of EGF-stimulated cells. Six other ligands for EGFR have been described (Table\u00a01), all of which play important roles both during embryogenesis and in adult life. In addition, EGFR ligands have been found in high concentrations in human cancers (Normanno et al. 2001; Normanno et al. 2005; Revillion et al. 2008). It is therefore important to establish whether the additional ligands have the same effects on EGFR downregulation as EGF. The only other EGFR ligand that has been well investigated with respect to EGFR trafficking is transforming growth factor \u03b1 (TGF\u03b1). Whereas EGF targets EGFR to the degradative pathway, TGF\u03b1 allows receptor recycling (Decker 1990). The differential intracellular sorting of EGFR after stimulation with TGF\u03b1 compared to EGF is caused by differences in the pH-dependence of receptor-ligand binding. Whereas EGF binding to EGFR is relatively stable at the lower pH in endosomes, TGF\u03b1 dissociates from the receptor at endosomal pH (Ebner and Derynck 1991). It is believed that continuous ligand binding is necessary for continuous ubiquitination and thereby for translocation of EGFR to the ILVs. In case of TGF\u03b1, the ligand rapidly dissociates from the receptor in endosomes, leading to receptor dephosphorylation, de-ubiquitination, and recycling to the cell surface (Longva et al. 2002). Interestingly, this difference in their potential to induce EGFR degradation is thought to be the main reason why TGF\u03b1 is a stronger mitogen than EGF (Waterman et al. 1998).\nAt present, the effects of heparin-binding EGF (HB-EGF), \u03b2-cellulin, Epiregulin, and Epigen on EGFR trafficking has not been investigated. A single study has investigated the effect of Amphiregulin on endocytic EGFR downregulation, and found that Amphiregulin failed to induce EGFR degradation. This was the case even in cells overexpressing Cbl, although in these cells EGFR was highly ubiquitinated following Amphiregulin stimulation (Stern et al. 2007). Interestingly, this suggests that pronounced receptor ubiquitination is not sufficient to target receptors for lysosomal degradation. More studies are warranted to uncover how the remaining EGFR ligands affect receptor ubiquitination, endocytosis, and lysosomal degradation.\nClathrin-independent endocytic mechanisms involved in EGFR uptake\nAlthough clathrin-dependent endocytosis is generally considered to be the major mechanism for internalization and downregulation of EGFR, other endocytic pathways have also been proposed during recent years, especially following stimulation with high concentrations of EGF (Sigismund et al. 2005). The concentration of EGF varies greatly throughout the human body. Thus, while the EGF concentration in tissue fluid is about 1\u20132\u00a0ng\/ml, it is much higher, up to 100\u00a0ng\/ml or more, in tubular duct lumens of e.g., the kidney, salivary glands, and the mammary gland (Carpenter and Cohen 1979; Hayashi and Sakamoto 1988). Normally, EGFR is not reached by the high luminal concentrations of EGF in these systems, since the receptor is present at the basolateral site of the epithelial cells. However, during wound healing or malignant transformation the tight junctions disappear and allow the high concentrations of EGF to get access to the receptor (Mullin 2004). Very high EGF concentration can also be found in solid tumors (Salomon et al. 1995). It is therefore relevant to study whether the mechanisms of endocytic downregulation of EGFR are affected by the concentration of stimulating ligand.\nSigismund et al. (2005) have reported a role of caveolae in EGFR uptake. They found that at low concentrations of EGF (1\u20132\u00a0ng\/ml), EGFR was not ubiquitinated and became internalized by clathrin-dependent endocytosis. In contrast, at high concentrations of EGF (20\u00a0ng\/ml) the receptor became ubiquitinated and was to a high degree internalized by caveolae. This conclusion was based upon immunogold labeling electron microscopy showing that at 20\u00a0ng\/ml of EGF, about half of the receptors were localized to caveolae and to caveolae-like structures or \u201ccaveosomes\u201d apparently not connected to the plasma membrane (Sigismund et al. 2005). However, it is likely that such caveolae-like structures that appear to be freely located in the cytoplasm are actually surface-connected in another plane of sectioning (Sandvig et al. 2008). In another study, Orlichenko et al. (2006) showed that incubation of epithelial cells with 30\u00a0ng\/ml of EGF for 5\u201320\u00a0min resulted in an eight to tenfold increase in the number of plasma membrane caveolae due to EGF-induced tyrosine phosphorylation of caveolin-1. Moreover, live cell imaging revealed increased dynamics of green fluorescent protein (GFP)-tagged caveolin upon stimulation of cells with 30\u00a0ng\/ml EGF. After 15\u201320\u00a0min of treatment, fluorescent caveolae were seen to move from the plasma membrane deeper into the cytoplasm (Orlichenko et al. 2006). Thus, some studies suggest a role of caveolae in EGFR endocytosis.\nCaveolae are quite immobile under normal, nonstimulated conditions as revealed by FRAP (Fluorescence Recovery After Photobleaching) analysis of cells transfected with GFP-tagged caveolin and by other approaches (Hommelgaard et al. 2005; Kirkham et al. 2005; Thomsen et al. 2002). If caveolae were involved in EGFR-internalization stimulated by high concentrations of EGF to any significant degree, two possible mechanisms can be envisioned: either the EGFR-containing caveolae become internalized in a single-wave process, leading to depletion of the plasma membrane for caveolae as has been reported for SV40 virus (Pelkmans et al. 2001, 2002), or activation of EGFR stimulates an increased mobility or turnover of caveolae so that internalized caveolae become replaced by new caveolae at the plasma membrane. These scenarios have been tested by live cell imaging of cells expressing GFP- tagged caveolin (Kazazic et al. 2006). It was shown that even 100\u00a0ng\/ml of EGF did not cause any wave of internalized caveolae from the plasma membrane, or any increased turnover of caveolae at the plasma membrane. Moreover, it was found that inhibition of clathrin-mediated endocytosis by siRNA against clathrin heavy chain strongly inhibited internalization of EGF both at 1 and 60\u00a0ng\/ml EGF. It was therefore concluded that clathrin-dependent endocytosis is the major pathway for EGFR uptake also at high EGF concentrations, and that caveolae do not contribute significantly to endocytosis of EGFR at any EGF concentrations (Kazazic et al. 2006).\nAt high concentrations of EGF (100\u00a0ng\/ml), induction of ruffling and macropinocytosis can be observed in some cells (Fig.\u00a03, K. Roepstorff, unpublished observations). However, whether ligand and receptor internalization in this way leads to lysosomal degradation is uncertain. Furthermore, Orth et al. (2006) found that EGF (30\u00a0ng\/ml) stimulated the formation of dorsal, circular waves which progressed inward and resulted in sequestration of about 50% of the activated EGFR from the plasma membrane. The process resulted in the formation of EGFR-positive tubular structures, and it was dependent on actin reorganization, receptor phosphorylation, PI 3-kinase activity, and dynamin 2. The functional consequences of the process remain to be determined, but the dorsal wave formation seems to be less frequent in tumor cells than in normal cells (Orth et al. 2006).\nFig.\u00a03EGF induces membrane ruffling and macropinocytosis. HEp2 cells expressing GPI-GFP as a marker of the plasma membrane were followed by 3D confocal microscopy over time. The upper panel shows two cells that were imaged before and 5\u00a0min after stimulation with 100\u00a0ng\/ml EGF. Arrows indicate membrane ruffles formed after EGF stimulation. The lower panel shows that ruffling leads to formation of macropinosomes (arrows). The large image to the left shows a cell 7\u00a0min after stimulation with 100\u00a0ng\/ml EGF, and the small images show a time sequence during which macropinosomes are formed. Bars, 20\u00a0\u03bcm\nIn conclusion, although alternative clathrin-independent mechanisms of EGFR endocytosis have been suggested, clathrin-mediated endocytosis still appear to be by far the most important mechanism for EGFR downregulation.\nEndocytic downregulation of ErbB2\nInterestingly, the behaviour of ErbB2 regarding endocytic downregulation differs significantly from that of EGFR, and it is generally accepted that ErbB2 avoids efficient endocytic downregulation (Austin et al. 2004; Baulida et al. 1996; Hommelgaard et al. 2004; Longva et al. 2005; Sorkin and Carpenter 1993; Wang et al. 1999).\nSeveral studies have investigated why ErbB2 is less susceptible to endocytic downregulation than EGFR after activation, and two fundamentally different cellular mechanisms could explain this: inefficient internalization (Baulida et al. 1996; Hommelgaard et al. 2004; Lerdrup et al. 2006; Longva et al. 2005; Sorkin and Carpenter 1993; Wang et al. 1999), or efficient recycling of endocytosed ErbB2 back to the plasma membrane (Austin et al. 2004; Citri et al. 2003; Hendriks et al. 2003a, 2003b; Klapper et al. 2000; Lenferink et al. 1998; Yarden 2001). Using antibodies labeled with fluorophores or gold-particles to monitor ErbB2 trafficking, Austin and co-workers found that ErbB2 is constantly internalized and recycled (Austin et al. 2004). In contrast, two studies found no observable intracellular ErbB2 in unstimulated cells and that treatment with monensin, a compound known to inhibit recycling, led to very little intracellular accumulation of ErbB2 within two hours (Lerdrup et al. 2006; Longva et al. 2005). Importantly, immunogold labeling electron microscopy has demonstrated that ErbB2 is very infrequently found in clathrin coated pits: as little as 1 of 10,000 labeled ErbB2 molecules localize to these structures in unstimulated cells or in cells treated with ErbB ligands (Fig.\u00a04a) (Hommelgaard et al. 2004). This suggests that the inefficient downregulation of ErbB2 is due to a low basal rate of internalization, although it is possible that ErbB2 could be taken up by clathrin independent endocytosis.\nFig.\u00a04Crosslinking of ErbB2 induces receptor internalization. a Fixed SK-BR-3 breast cancer cells have been incubated with a mouse monoclonal antibody against the extracellular N-terminal part of ErbB2, and subsequently with a gold-conjugated anti-mouse antibody. Thereafter the cells were embedded in Epon, sectioned and examined in the electron microscope. It is seen that ErbB2 is mainly associated with membrane protrusions. An empty clathrin-coated pit (CCP) is also seen. b, c SK-BR-3 cells have been incubated for 1\u00a0h at 37\u00b0C with the anti-ErbB2 antibody followed by the gold-conjugated antibody before fixation and embedding. Note how the antibody crosslinking drives ErbB2 down from the protrusions to the bulk membrane, where it is seen in clathrin-coated pits (CCP). Labeling of a multivesicular body (MVB) is also seen in c, showing that crosslinked ErbB2 is internalized. For details see (Hommelgaard et al. 2004). Bar 0.5\u00a0\u03bcm\nA chimeric EGFR where the C-terminal tail has been replaced by that of ErbB2 is as endocytosis impaired as a chimeric EGFR where the entire intracellular part has been replaced by that of ErbB2 (Sorkin and Carpenter 1993). This suggests that the C-terminal tail of ErbB2 is responsible for its endocytic impairment, and an interesting hypothesis is that this is caused by an inhibitory signal rather than a lack of endocytic stimuli (Sorkin and Carpenter 1993). This is supported by the lower stability and increased endocytic downregulation of ErbB2 deletion mutants, which lack the C-terminal tail, compared to full-length ErbB2 (Lerdrup et al. 2007; Tikhomirov and Carpenter 2003). As mentioned previously, phosphorylation of EGFR on Y1045 is important for recruitment of Cbl and endocytic downregulation of EGFR. A Cbl docking site is also present in ErbB2 (Klapper et al. 2000; Levkowitz et al. 1999), and Cbl can be recruited to ErbB2 (Klapper et al. 2000; Levkowitz et al. 2000). However, the actual extent of Cbl binding is debated, and inefficient binding of Cbl could also explain the low lysosomal degradation of ErbB2 (Chan et al. 2004; Dankort et al. 1997; Levkowitz et al. 1996, 2000; Olayioye et al. 2000).\nSeveral groups have reported that ErbB2 can transfer its endocytic reluctance to EGFR, since ErbB2 overexpression protects EGFR from endocytic downregulation and can increase EGFR levels (Haslekas et al. 2005; Muthuswamy et al. 1999; Wang et al. 1999; Worthylake et al. 1999). However, the studies do not reach similar conclusions regarding the underlying mechanism, and different models have been presented including lack of Cbl recruitment to activated EGFR (Muthuswamy et al. 1999), inhibited formation of clathrin coated pits after EGFR stimulation (Haslekas et al. 2005), or reduced targeting of internalized EGFR to lysosomes (Worthylake et al. 1999).\nAlthough it is well established that ErbB2 is severely endocytosis impaired compared to EGFR, there is currently no consensus model of how this is achieved. Taking into account the major focus that ErbB2 receive as an oncogene and as a drug target, a better understanding of the mechanisms hindering ErbB2 endocytosis and lysosomal degradation is warranted.\nEndocytic downregulation of ErbB3 and ErbB4\nAt present, remarkably little is known about endocytosis of ErbB3 and ErbB4. In the first studies of their endocytosis, ErbB3 and ErbB4 were reported to be internalization impaired to the same extent as ErbB2 (Baulida et al. 1996). In accordance with this, the ErbB3- and ErbB4-specific ligand Heregulin1\u03b2 was found to be internalized less efficient and much slower than the EGFR-specific ligand EGF, supporting that ErbB3 and ErbB4 are not endocytosed as efficiently as EGFR (Baulida and Carpenter 1997; Waterman et al. 1998). Interestingly, the endocytic impairment of ErbB3 could be transferred to EGFR by replacing the EGFR C-terminal tail with the ErbB3 C-terminal tail, suggesting that there are regions in the C-terminus of ErbB3 that protect the receptor against internalization (Waterman et al. 1999).\nWhereas the abovementioned studies suggest that ErbB3 and ErbB4 are endocytosis impaired, both of the receptors can be ubiquitinated (Cao et al. 2007; Omerovic et al. 2007; Qiu and Goldberg 2002). In case of ErbB3, heregulin1\u03b2 stimulation leads to ubiquitination of the receptor by the ubiquitin ligase Nrdp1. Knockdown of Nrdp1 increases the steady-state levels of ErbB3, suggesting that Nrdp1-mediated ubiquitination may target ErbB3 to degradation (Qiu and Goldberg 2002). ErbB4 is ubiquitinated by the ubiquitin ligase Itch, which induces increased degradation of ErbB4 when overexpressed (Omerovic et al. 2007). The ubiquitination-stimulated degradation of both ErbB3 and ErbB4 depends upon proteasomal activity (Cao et al. 2007; Omerovic et al. 2007; Qiu and Goldberg 2002). Interestingly, the degradation appears to involve lysosomes as well, since inhibition of lysosomal function retards the degradation of both ErbB3 and ErbB4 (Cao et al. 2007; Omerovic et al. 2007). This apparent need for both lysosomes and proteasomes for receptor degradation may appear as a paradox, but it has been shown that proteasomal activity is required for lysosomal degradation of EGFR as well (Alwan et al. 2003; Longva et al. 2002). Thus, if this is also the case for ErbB3 and ErbB4, the block of receptor degradation seen after proteasomal inhibition could be due to an effect on lysosomal function like the one observed for EGFR. Obviously, this is in discrepancy with the studies reporting that neither ErbB3 nor ErbB4 becomes efficiently endocytosed, and more studies are therefore warranted addressing the role of endocytosis and lysosomal degradation of ErbB3 and ErbB4, not least in cancer cells.\nEscape of endocytic downregulation in cancer\nSeveral EGFR mutants have been found in tumors indicating an oncogenic role of these mutants. The mutations can be in the form of small or large deletions, point mutations, or amplifications and these alterations can be found both in the intra- and extracellular parts of EGFR (Fig.\u00a01). Whether the mutations are the cause of or a result of transformation is often not clear, but some EGFR mutants show increased signaling, which will contribute to oncogenesis. The increased signaling can be the result of constitutive activation and\/or impaired downregulation. In this review we will focus on examples of EGFR mutants with impaired downregulation.\nProper association of the activated receptor with the downregulation machinery is essential for stopping the signaling of EGFR. As described above, binding of the ubiquitin ligase Cbl to EGFR is central in the downregulation. EGFRvIV and EGFRvV are both found in glioblastomas (Frederick et al. 2000; Peschard and Park 2003; Zandi et al. 2007). Due to intracellular deletions, these mutants lack the direct binding site of Cbl and in addition EGFRvV also lacks the indirect Grb2-mediated binding sites (Frederick et al. 2000; Peschard and Park 2003; Zandi et al. 2007). The viral oncogene v-ErbB resembles EGFR, but lacks certain parts including the direct binding site of Cbl (Peschard and Park 2003; Zandi et al. 2007). The downregulation of EGFRvIV, EGFRvV and v-ErbB has not been investigated, but they would be expected to have an impaired downregulation.\nThe best characterized EGFR mutant with impaired downregulation is EGFRvIII. Initially found in glioblastomas (Sugawa et al. 1990), EGFRvIII has later been found in a range of other types of tumors (Garcia de Palazzo et al. 1993; Ge et al. 2002; Moscatello et al. 1995; Okamoto et al. 2003; Olapade-Olaopa et al. 2000). In concordance with this, EGFRvIII can transform fibroblasts and the receptor enhances proliferation and\/or tumorigenicity of cells both in vivo and in vitro (Batra et al. 1995; Moscatello et al. 1995; Nagane et al. 1996; Nishikawa et al. 1994; Pedersen et al. 2005a, 2005b; Tang et al. 2000). EGFRvIII is characterized by a deletion of amino acid residues 6\u2013273 found in the extracellular part of EGFR. This gives a truncated 145\u00a0kDa receptor with a non-functional ligand binding pocket and no dimerization arm. Thus, EGFRvIII is incapable of binding any ligands; the receptor is however constitutively active (Kuan et al. 2001), and it activates Ras-Erk1\/2 and PI 3-kinase-Akt (Antonyak et al. 1998; Chu et al. 1997; Montgomery et al. 1995; Moscatello et al. 1996, 1998). The constitutive activity may be sufficient for tumorigenicity, but impaired downregulation would enhance the effect. EGFRvIII can associate with overexpressed Cbl and this leads to downregulation (Davies et al. 2006). However, two recent reports show that EGFRvIII is not degraded in cells with endogenous levels of Cbl, and that this can be due to virtually no phosphorylation of Y1045 (Grandal et al. 2007; Han et al. 2006). In concordance with the hypophosphorylation at the direct binding site Y1045, Cbl binds primarily through the adaptor protein Grb2 (Grandal et al. 2007). This gives no detectable ubiquitination, and instead of being degraded, internalized EGFRvIII is recycled back to the plasma membrane (Grandal et al. 2007). Thus, as seen for the Y1045F EGFR mutant, direct binding of Cbl to the receptor appears to be necessary for efficient degradation.\nIn conclusion, endocytic impairment may be a returning theme of oncogenic EGFR mutants. ErbB signaling can also be sustained if the molecular machinery normally involved in receptor downregulation does not function optimally. Indeed, several mutations of such proteins have been found in tumors, including Cbl, TSG101 (an ESCRT-I subunit), and VPS25 (an ESCRT-II subunit) (recently reviewed in (Haglund et al. 2007)).\nEndocytic downregulation of ErbB receptors as a therapeutic target in cancer treatment\nSince lack of endocytic downregulation is an emerging theme in ErbB cancer biology, it is evident that stimulation of ErbB endocytosis and lysosomal degradation is an attractive means to inhibit tumor growth.\nAntibody-mediated crosslinking can induce internalization of ErbB receptors\nFrom a therapeutic perspective, an appealing way to induce endocytic downregulation of ErbB receptors is by antibody-mediated crosslinking. Indeed, an efficient way to induce endocytosis of both EGFR and ErbB2 is crosslinking with polyvalent antibody conjugates (Fig.\u00a04 b, c) (Friedman et al. 2005; Hommelgaard et al. 2004). The most studied ErbB2 antibody with an anti-tumor potential is Trastuzumab, which is currently used in the treatment of breast cancer (Hudis 2007). Although the dominating opinion has been that Trastuzumab causes endocytic downregulation of ErbB2 (Menard et al. 2003; Rubin and Yarden 2001; Yarden 2001), several recent studies suggest that Trastuzumab does not induce endocytosis of ErbB2 to a significant degree (Austin et al. 2004; Hommelgaard et al. 2004; Longva et al. 2005).\nCetuximab is an antibody targeting EGFR that is currently used in treatment of colorectal cancer and head and neck cancer (Harari et al. 2007). Several studies have shown that Cetuximab induces internalization of EGFR (Jaramillo et al. 2006; Sunada et al. 1986). In contrast to ligand-induced endocytosis, Cetuximab-induced EGFR internalization is independent of receptor tyrosine kinase activity, and it is both slower and less efficient in terms of receptor downregulation than the ligand-induced endocytosis (Jaramillo et al. 2006).\nAt present, the knowledge of mechanisms underlying antibody-mediated endocytic downregulation is relatively sparse. It is clear that the endocytic potential of antibodies vary, but the reason for this remains elusive. Furthermore, it is debated whether the endocytic potential of antibodies is correlated to their anti-tumor potential (Hurwitz et al. 1995; Klapper et al. 1997; Park et al. 1999). Indeed, in cases where the anti-tumor effect of therapeutic antibodies is in part due to antibody-dependent activation of an immune response, internalization of bound antibodies will be a therapeutic disadvantage, since it will clear the surface for antibodies that will otherwise be recognized by immune cells such as natural killer cells. However, antibody-mediated receptor endocytosis followed by degradation may be advantageous in the treatment of tumors that rely heavily upon ErbB receptor signaling as a growth-promoting signal.\nA useful observation is that extensive antibody-based crosslinking of ErbB receptors is far more efficient at inducing ErbB endocytic downregulation than single antibodies are (Friedman et al. 2005; Hommelgaard et al. 2004). Crosslinking can either be done using antibodies that forms multivalent aggregates via secondary antibodies or gold particles (Hommelgaard et al. 2004) or by a more clinically relevant approach using combinations of monoclonal antibodies against distinct epitopes in an ErbB receptor (Friedman et al. 2005). Thus, whereas administration of Trastuzumab alone did not induce significant ErbB2 endocytosis, the combination of Trastuzumab with another monoclonal antibody to ErbB2 was very efficient at downregulating ErbB2. In addition, the combination of two antibodies was much more efficient at inhibiting tumor growth in a mouse model compared to Trastuzumab administered alone (Friedman et al. 2005).\nHsp90 inhibition stimulates ErbB2 cleavage and internalization\nAnother efficient way to induce endocytosis and lysosomal degradation of ErbB2 is inhibition of the chaperone HSP90. HSP90 has gained a lot of attention due to the involvement of many of its clients in cancers, and several inhibitors are being tested in clinical trials, including the Geldanamycin derivative 17-AAG (Goetz et al. 2003; Solit and Chiosis 2008; Vastag 2006; Whitesell and Lindquist 2005). ErbB2 is one of the most prominent client proteins of the chaperone HSP90 (Citri and Yarden 2006; Linggi and Carpenter 2006), and this interaction is considered as a potentially valuable pharmacological target (Citri et al. 2004; Marmor et al. 2004; Neckers 2002; Neckers and Ivy 2003). ErbB2 overexpressing cancer cells often binds 17-AAG with an even higher affinity than other cancers cells, suggesting that ErbB2 overexpressing cells are highly dependent on HSP90 activity (Kamal et al. 2003). Inhibition of HSP90, e.g. by using Geldanamycin, leads to recruitment of the cochaperone and ubiquitin ligase CHIP to ErbB2 and ubiquitination of the receptor (Xu et al. 2002; Zhou et al. 2003).\nSeveral degradation mechanisms are involved in ErbB2 downregulation following its ubiquitination. Mimnaugh and coworkers (Mimnaugh et al. 1996) found that HSP90-inhibition led to degradation of ErbB2 in a proteasome-dependent manner, and this has been interpreted as if degradation of the transmembrane ErbB2 was carried out by the proteasome (Citri et al. 2002; Hong et al. 1999; Way et al. 2004; Xu et al. 2001; Zheng et al. 2000). However, later studies found that ErbB2 is endocytosed and degraded in lysosomes after Geldanamycin stimulation (Fig.\u00a05 a) (Austin et al. 2004; Lerdrup et al. 2006; Longva et al. 2005; Tikhomirov and Carpenter 2000), and that the proteasomal activity is actually needed for endocytosis and lysosomal degradation of ErbB2 after Geldanamycin stimulation (Lerdrup et al. 2006). Finally, Tikhomirov and Carpenter have demonstrated that the C-terminus of ErbB2 is cleaved by endoproteases into a transmembrane p135 and several cytosolic fragments in response to Geldanamycin-treatment (Tikhomirov and Carpenter 2000, 2001, 2003). Recent work from our group showed that this cleavage also promotes endocytosis and lysosomal degradation of ErbB2 (Lerdrup et al. 2007). Using a doubly fluorescent ErbB2 tagged with yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) in each terminus, we found that ErbB2 was cleaved at the plasma membrane after Geldanamycin stimulation. C-terminally cleaved ErbB2 as well as an ErbB2 deletion mutant lacking its C-terminal tail were endocytosed and degraded in lysosomes far more efficiently than full length ErbB2 (Fig.\u00a05b) (Lerdrup et al. 2007).\nFig.\u00a05HSP90 inhibition stimulates ErbB2 cleavage and internalization. a ErbB2 is internalized after 2\u00a0h of Geldanamycin-stimulation (3\u00a0\u03bcM) of SK-BR-3 breast cancer cells. Sequential immunocytochemistry was used to distinguish between internalized and surface associated ErbB2. Fixed cells were stained with antibodies against ErbB2 before permeabilization (green). After permeabilization, cells were once again stained with antibodies against ErbB2 (red). Only the latter antibody staining (red) gain access to intracellular ErbB2, which therefore appears red whereas surface ErbB2 is stained both red and green and therefore appears yellow. For details see (Lerdrup et al. 2006). b YFP-ErbB2-CFP is cleaved after 2\u00a0h of Geldanamycin-stimulation of SK-BR-3 cells. A construct with YFP fused to the extracellular N-terminus and CFP fused to the intracellular C-terminus of ErbB2 was expressed in SK-BR-3 cells for 48\u00a0h and followed by incubation with 0.3\u00a0\u03bcM Geldanamycin for 2\u00a0h as indicated. YFP-ErbB2-CFP where the C-terminal tail has been cleaved off appears green. Note the increased amount of such cleavage in vesicles compared to the plasma membrane after Geldanamycin stimulation. For details see (Lerdrup et al. 2007). Bars 20\u00a0\u03bcm\nAs opposed to ErbB2, wild-type EGFR is relatively insensitive to Hsp90 inhibition. However, the Geldanamycin derivative 17-AAG stimulates degradation of constitutively active EGFR mutants commonly found in lung cancer (Sawai et al. 2008). Whether 17-AAG induces degradation of mutant EGFRs by stimulating endocytosis and lysosomal degradation remains to be determined.\nPerspectives\nIn spite of its crucial importance both in health and disease, the ErbB receptor system still has hidden secrets regarding its regulation. It is evident that we need to know much more about the molecular mechanisms underlying endocytic downregulation of the otherwise heavily investigated ErbB receptors. One important area that deserves future attention is further mapping of the mechanisms responsible for recruitment of EGFR to CCPs. Moreover, EGFR endocytosis does not seem to be representative of the other ErbB receptor family members, which all appear to be endocytosis impaired to some extent compared to EGFR. It is especially of interest to decipher how formation of heterodimers between the different ErbB receptors affects their endocytic downregulation. In addition, the large number of ErbB ligands adds further complexity to the system, since they may vary in the capability to stimulate endocytosis and lysosomal degradation of their receptors. Interestingly, the few ligands that have been investigated so far differ significantly in their ability to induce endocytic downregulation of ErbB receptors.\nMore insight into endocytic downregulation of ErbB receptors is indeed relevant for understanding the role of these receptors in cancer, and it could lead to identification of novel targets for cancer therapy directed at downregulating signaling receptors. Although still in the making, the clinical as well as preclinical results show that targeted therapy stimulating endocytic downregulating of ErbB receptors is a promising tool in cancer treatment.","keyphrases":["erbb2","endocytosis","endosomal sorting","epidermal growth factor receptor","ubiquitin","lysosomal degradation"],"prmu":["P","P","P","P","P","P"]}
{"id":"Neuroradiology-3-1-2039855","title":"Superior cerebellar artery aneurysms: incidence, clinical presentation and midterm outcome of endovascular treatment\n","text":"Introduction The aim of this retrospective study was to determine the incidence, clinical presentation and midterm clinical and imaging outcome of endovascular treatment of 34 superior cerebellar artery (SCA) aneurysms in 33 patients.\nIntroduction\nAneurysms located on the superior cerebellar artery (SCA) are uncommon and their presentation, natural history and clinical management are poorly understood [1, 2]. Reports about the endovascular or surgical management of SCA aneurysms are rare and are usually incorporated in clinical series of basilar artery or posterior circulation aneurysms [3\u201318].\nThe aim of this retrospective study was to determine the incidence, clinical presentation and midterm clinical and imaging results of endovascular treatment of 35 patients with 36 SCA aneurysms.\nMethods\nPatients\nBetween January 1995 and January 2007, 2,112 aneurysms were treated in our institution. Of the 2,112 treated aneurysms, 324 (15%) were located in the posterior circulation and 1,788 (85%) in the anterior circulation. Surgery was performed in 970 aneurysms and endovascular treatment in 1,142 aneurysms. Of the 2,112 treated aneurysms, 36 aneurysms in 35 patients were located on the SCA, resulting in an incidence of 1.7% of all treated intracranial aneurysms and 11.1% of treated posterior circulation aneurysms. We excluded one patient with a peripheral SCA flow aneurysm associated with an arteriovenous malformation and one peripheral partially thrombosed SCA aneurysm presenting with trochlear nerve palsy. In one patient with four aneurysms, one aneurysm was located on the proximal right SCA and one aneurysm on the distal left SCA. All 33 proximal SCA aneurysms were primarily based on the basilar artery at the branching point of the SCA with or without incorporation of the SCA origin in the base. All 34 SCA aneurysms in 33 patients were treated by endovascular techniques. There were 6 men and 27 women with a mean age of 50.7\u00a0years (median 49\u00a0years, range 29\u201372\u00a0years). Of 34 aneurysms, 22 (65%) had ruptured, 7 (20%) were additional to another ruptured aneurysm, 4 (12%) presented with symptoms of a mass effect (trigeminal neuralgia 2, oculomotor palsy 2) and one (3%) was an incidental finding (Fig.\u00a01). The mean size of the 34 SCA aneurysms was 7.3\u00a0mm (median 6\u00a0mm, range 2\u201327\u00a0mm).\nFig.\u00a01Incidentally found SCA aneurysm in a 39-year-old man with vertebrobasilar embolic infarcts. a T1-weighted MR image shows a hyperintense lesion adjacent to the brainstem. b 3-D vertebral angiogram reveals a small SCA aneurysm\nThe clinical condition of the 22 patients with a ruptured SCA aneurysm at the time of treatment was HH I\u2013II in 16, HH III in 3 and HH IV\u2013V in 3. Seven SCA aneurysms in six patients were additional to another ruptured aneurysm and were coiled in the same session as the ruptured aneurysm. The clinical condition of these six patients was HH I\u2013II in five and HH IV\u2013V in one.\nOf the 33 patients, 14 (42%) had multiple aneurysms (Fig.\u00a02) for a total of 72 aneurysms: 5 patients had 2 aneurysms, 3 patients had 3 aneurysms, 3 patients had 4 aneurysms, 1 patient had 5 aneurysms, 1 patient had 7 aneurysms and 1 patient had 8 aneurysms. Of the 72 aneurysms in 35 patients, 53 were coiled, 13 were clipped, and 6 were small (<2\u00a0mm) and left untreated.\nFig.\u00a02Pre- and posttreatment images of two patients with multiple posterior circulation aneurysms. a A 44-year-old man with a ruptured middle cerebral artery aneurysm and four additional aneurysms. 3-D vertebral angiogram reveals right SCA aneurysm (short single arrow), left distal SCA aneurysm (long single arrow) and basilar tip aneurysm (pair of arrows). b Complete occlusion after coiling. c A 61-year-old woman with a ruptured middle cerebral artery aneurysm and additional aneurysms on the left SCA and basilar tip. d Adequate occlusion of basilar tip and SCA aneurysms\nCoiling procedure\nCoiling of aneurysms was performed on a biplane angiographic unit (Integris BN 3000 Neuro, Philips Medical Systems, Best, The Netherlands) with the patient under general anaesthesia and systemic heparinization. Heparin was continued intravenously or subcutaneously for 48\u00a0h after the procedure, followed by low-dose aspirin for 3\u00a0months orally. Coiling was performed with Guglielmi detachable coils (GDC, Boston Scientific, Fremont, Calif.) or TruFill DCS coils (Cordis, Miami, Fl.). The aim of coiling was to pack the aneurysm as densely as possible, until not a single additional coil could be placed. Four wide-necked aneurysms (12%) were coiled with the aid of a supporting balloon in the basilar artery. One aneurysm was located distal on the SCA and was selectively occluded with coils sparing the parent SCA. Complications of coiling were recorded.\nInitial angiographic results of coiling were classified as complete occlusion (100%), near complete occlusion (90\u2013100%) and incomplete occlusion (<90%).\nClinical and angiographic follow-up\nPatients who survived the hospital admission period were scheduled for a follow-up visit in the outpatient clinic 6\u00a0weeks after treatment and for follow-up angiography after 6 and 18\u00a0months. Neurological status according to the Glasgow Outcome Scale (GOS) was evaluated at every outpatient clinic visit and at every admission for follow-up angiography. The results of follow-up angiography were classified in the same way as for initial angiographic results.\nResults\nInitial angiographic results and complications\nInitial angiographic occlusion was complete or near complete (90\u2013100% occlusion) in 32 aneurysms and incomplete in 2 aneurysms (both 80% occlusion). Complications leading to permanent morbidity or death occurred in two patients. The first patient was a 42-year-old woman who presented with a ruptured 2-mm wide-necked SCA aneurysm in HH I. During insertion of a coil with the aid of a supporting balloon, a rupture occurred and the patient died 12 h later (mortality 1 of 33, 3.0%, 95% CI 0.01\u201316.65%). The second patient was a 71-year-old woman admitted with a ruptured 10-mm wide-necked SCA aneurysm (Fig.\u00a03). The SCA itself originated from the sac. The aneurysm was coiled with the aid of a supporting balloon with adequate occlusion and preserved flow in the SCA. However, the patient did not awake from general anaesthesia. Immediate control angiography revealed that the SCA had become occluded. MRI several days later showed infarctions in the vascular territory of the SCA in the brainstem and cerebellum. The patient gradually recovered but remained dependent. At the time of this report almost 4\u00a0years later she was still in a nursing home (procedural morbidity 1 of 33, 3.0%, 95% CI 0.01\u201316.65%).\nFig.\u00a03Ischemic complication of coiling in a 71-year-old woman with a ruptured SCA aneurysm. a Vertebral angiogram demonstrates a wide-necked right SCA aneurysm with the SCA arising from the sac. b, c Balloon-assisted coiling with adequate occlusion and preserved flow in the SCA. The patient did not awake from general anaesthesia. Immediate control angiography revealed that the SCA had become occluded (not shown). d MRI several days later showing brainstem and cerebellum infarctions in the SCA territory\nClinical follow-up\nClinical follow-up was available for all 33 patients. One patient died of a procedural rupture and another patient died of diffuse vasospasm after subarachnoid haemorrhage (SAH). The remaining 31 patients had clinical follow-up of mean 45\u00a0months (median 44.5\u00a0months, range 4\u2013103\u00a0months, 118 patient-years). Outcome at 6\u00a0months follow up of these 31 patients was GOS 5 in 26 (84%), GOS 4 in 4 (13%) and GOS 3 in 1 patient (3%). Symptoms of mass effect on cranial nerves in four patients were cured in all. During extended follow up, two patients died of unrelated disease 3 and 6\u00a0years after coiling of a SCA aneurysm: pulmonary embolism in one patient and disseminated bronchial carcinoma in the other patient. There were no episodes of (re)bleeding during the 118 patient-years of follow-up.\nAngiographic follow-up\nOf the 31 surviving patients, 4 refused follow-up angiography. The remaining 27 patients (with 28 SCA aneurysms) all had 6\u00a0months follow-up angiography and 19 had extended angiographic follow-up of a mean of 31\u00a0months (median 22\u00a0months, range\u00a018\u201384\u00a0months). Stable complete or near-complete occlusion was apparent in 25 of 28 aneurysms. In one patient, a 66-year-old man with a 27-mm partially thrombosed SCA aneurysm presenting with trigeminal neuralgia, the coils had migrated into the intraluminal thrombus at the 6-month follow-up angiography. However, the trigeminal neuralgia was cured and the aneurysm was not retreated. The patient died 6\u00a0years later of disseminated bronchial carcinoma. Two aneurysms in two patients that were initially incompletely occluded (both 80% occlusion) remained so at extended angiographic follow-up. Additional treatment was judged impossible in both patients.\nDiscussion\nIn this study, we found that SCA aneurysms are rare with an incidence of 1.7% of all treated intracranial aneurysms and 11.1% of treated posterior circulation aneurysms at our institution. The majority of patients presented with SAH and 42% of patients also had aneurysms at other locations. This frequent association with multiple aneurysms has not been established before. Endovascular occlusion with detachable coils is feasible with good angiographic and clinical results and with low morbidity [3].\nThe proximal SCA has an intimate relation with cranial nerves III, IV and V [19]. Aneurysms on the SCA may therefore present with palsies of these nerves, as was the case in four of our patients [10, 11]. After coiling, symptoms of nerve compression regressed in all four patients.\nOne of 34 SCA aneurysms was located distally on the artery. It was possible to occlude this aneurysm with sparing of the parent SCA. When selective occlusion of such an aneurysm is not possible, simultaneous occlusion of the aneurysm together with the parent artery can be performed. Collateral blood flow may limit ischemic infarctions and outcome is usually good [4, 12\u201315].\nAlthough surgical treatment of SCA aneurysms in experienced hands is associated with good results, access may be challenging needing destructive approaches [20\u201322]. The presence of important perforating arteries and adjacent cranial nerves makes clipping of SCA aneurysms difficult and complications may occur. Because SCA aneurysms are rare, not many surgeons will gain experience in the treatment of these aneurysms. In addition, many patients are in bad clinical condition and are not good surgical candidates in the acute phase.\nWhile surgery for SCA aneurysms is often difficult, limited to good-grade patients and associated with substantial morbidity, coiling is technically easy and can also be performed in the acute phase of haemorrhage in bad-grade patients. This makes coiling the method of choice to treat SCA aneurysms.\nConclusion\nSCA aneurysms are rare with an incidence of 1.7% of treated aneurysms at our institution. There is a frequent association with aneurysms at other locations. Most patients present with SAH. Since the SCA is closely related to cranial nerves III, IV and V, some aneurysms present with palsies of these nerves. Endovascular occlusion with detachable coils is feasible with good angiographic and clinical results.","keyphrases":["superior cerebellar artery","aneurysm","outcome","coiling"],"prmu":["P","P","P","P"]}
{"id":"J_Abnorm_Child_Psychol-3-1-1915626","title":"Being Admired or Being Liked: Classroom Social Status and Depressive Problems in Early Adolescent Girls and Boys\n","text":"This study investigates associations between depressive problems and classroom social status in a large population cohort of Dutch early adolescents (N = 1046, age 13.52 \u00b1 0.51, 52.4% girls). Depressive problems were assessed by parent and self-reports and classroom status by peer nominations. We assessed peer status with respect to both achievement-related (being a good learner, being good at sports, being good-looking) and affection-related (being liked, being disliked, being best friend) areas. In boys, depressive problems were most strongly associated with not being good at sports, while in girls the association was strongest for not being liked. The risk of a low status in one area could largely be compensated by a high status in another area.\n\u00a0\nDepressive problems and low social status have been associated since ancient times. In fact, the word depression is derived from the Latin term deprimere, which means being brought down in status or fortune (Jackson, 1986). The association between depression and social status has been confirmed empirically in numerous studies, in both animals and humans (e.g., Blanchard & Blanchard, 1990; Eaton & Harrison, 2000; Gilbert, Allan, & Trent, 1995; Hecht, Inderbitzen, & Bukowski, 1998).\nSocial status can be defined in at least two ways: in terms of achievements (being admired) and in terms of affection (being liked). These two definitions are related, but by no means interchangeable, and could be differentially linked to depressive problems. The former contains an element of competition (being better than others), while the latter relates to being accepted or rejected by the social group. We will compare the relative importance of both of them with respect to depressive problems in early adolescent girls and boys.\nAdolescence is a developmental context in which peer status is particularly salient (Connell & Dishion, 2006). Adolescents spend considerably more time in peer interactions than younger children (Larson & Richards, 1991), and use peers as primary sources for social comparison and self-appraisal (Prinstein & Aikins, 2004). Adolescence is also a highly interesting developmental stage for studying depressive problems, because the prevalence of these problems increases substantially in this phase of life, especially in girls (e.g., Hankin et\u00a0al., 1998; Oldehinkel, Wittchen, & Schuster, 1999).\nAchievement- and affection-related social status\nAchievement-related operationalizations of social status are usually based on Price\u2019s Social Ranking Theory (Gilbert & Allan, 1998; Price, 1972). Briefly, this theory postulates that in pursuing scarce resources (e.g., territories, sexual opportunities), animals will have to fight others aiming at the same resources. To prevent harm, animals who are likely to lose these conflicts need inhibitory mechanisms that force them to submit (withdraw, run away) and downgrade their aspirations so that they do not keep engaging in fights they cannot win. These inhibitory strategies may result in behavioral and physiological phenomena that closely resemble depressive symptoms in humans (e.g., Blanchard & Blanchard, 1990; Sapolsky, 1990). Hence, according to this theory, depression is an involuntary strategy to accept defeat in (ritual) agonistic encounters and to accommodate what would otherwise be unacceptably low social rank (Price, Sloman, Gardiner, Gilbert, & Rohde, 1994).\nHuman social hierarchy does not need actual (ritual) fighting to be established, but can also be formed by imagining what would happen in case of such an encounter. Just knowing the strength of your opponent may be enough to experience a sense of inferiority (Rohde, 2001), which in turn can boost depressive problems (Brown, Harris, & Hepworth, 1995; Gilbert & Allan, 1998). Internal hierarchical positions are also fed by the amount of social attention directed by others (Gilbert, 1989). As opposed to most other species, humans base (internal) hierarchies not just on physical strength, but on other capacities related to the availability of resources, such as intelligence and physical attractiveness (e.g., Anderson, John, Keltner, & Kring, 2001; Nettle, 2003), as well.\nWhereas Price\u2019s Social Ranking Theory is achievement-oriented and implies (possibly internal) social comparison and grading, peer status can also be defined in terms of affection, that is, being liked or disliked (rejected). While achievement-related status mainly refers to the hierarchical position within the group, affection-related status may also have implications for group membership in itself. Throughout human history, being banished from the group has been similar to a death sentence, so it is not surprising that being accepted by other people represents a core motive (e.g., Baumeister & Leary, 1995; Fiske, 2004). Hence, social rejection is a significant stressor (Coie, 1990), which can bring about feelings of loneliness (Asher & Wheeler, 1985) and low self-esteem (O\u2019Brien & Bierman, 1988), and may contribute to the development of depressive problems (Boivin, Poulin, & Vitaro, 1994; Monroe & Hadjiyannakis, 2002).\nSocial ranking and social rejection theories offer sound theoretical frameworks to predict depressive problems in those with a low status, but are less explicit regarding consequences of having a high status. Although there is a social gradient of (mental) ill-health, that is, health improves with each step one moves up the socioeconomic ladder (e.g., Siegrist & Marmot, 2004), this may not be translated directly to the association between peer status and depressive problems in adolescence. In fact, animal research suggest that both high- and low-status individuals can suffer from distress, among other things depending on their personality and the stability of the hierarchy (Sapolsky, 2005). Hence, high and low peer status may not be two sides of the same coin, and better be investigated separately.\nThough adolescents\u2019 social status is multidimensional (Rohde, 2001), the achievement-related and the affection-related approaches to social status have evolved independently from each other and have, to the best of our knowledge, never been studied in concert. Hence, it is not known how the two are interrelated and how they jointly affect depressive problems. Multiple hierarchies exist within each of the two status domains as well: achievement includes specific areas such as sports and school performance; and affection may relate to being disliked or being someone\u2019s best friend. This raises the question if and how a low status in one of the areas can be compensated by a high status in another area, or vice versa. Being caught in a (degrading) situation where no escape seems possible is a particularly potent trigger of depressive problems (Brown et\u00a0al., 1995). A position at the bottom of the status hierarchy may be experienced as such, but this is considerably less likely in adolescents who are highly regarded in other respects, since people tend to value most the hierarchy in which they rank highest (Sapolsky, 2005). We therefore expected that the effects of low status could be compensated by high status in another area.\nGender differences\nUntil now, only a limited number of studies have focused on gender differences in associations between social status in particular domains and depressive problems. With respect to achievement-related status, this is at least partly due to the fact that there are several well-documented strategies to induce achievement-related social stress in males (e.g., defeat in aggressive encounters), but hardly any for females, among other things because it is hard to obtain strong dominance relationships in female animals (Haller, Fuchs, Hal\u00e1sz, & Makara, 1998). Rather than winning or losing in competitive encounters, females seem to be sensitive to situations which disturb their relationships with other animals in the group, such as social isolation (Holston, Scallet, Ali, & Turner, 1991) and instability (Haller et\u00a0al., 1998). There is some evidence for gender differences in humans as well. In a study where men and women were subjected to a series of different stress conditions, men appeared physiologically more reactive to achievement challenges, and women to social rejection challenges (Stroud, Salovey, & Epel, 2002). Consistent with this, work problems and downward social mobility (i.e., lower achievement-related status) have been found to be associated with men\u2019s mental health in particular, while women are more sensitive to problems in getting along with dear ones and peer rejection (e.g., Brendgen, Wanner, Morin, & Vitaro, 2005; Kendler, Thornton, & Prescott, 2001; Tiffin, Pearce, & Parker, 2005). Taken together, these results suggest gender differences in the salience of specific domains of social status. We propose that autonomy and dominance are more important sources of well-being for males than for females, so that boys will be especially sensitive to achievement-related life events and status loss (Price et\u00a0al., 1994; Rose & Rudolph, 2006). Girls\u2019 depressive problems, on the other hand, are expected to be more strongly associated to affection-related peer nominations. Compared to boys, adolescent girls exhibit greater affiliative needs, increasing their sensitivity to conflict and rejection within interpersonal relationships (Cyranowski, Frank, Young, & Shear, 2000; Prinstein & Aikins, 2004).\nPresent study\nWe have studied the relationship between early adolescents\u2019 depressive problems and social status in peers comprising an everyday social network. We chose to study classroom social status as measured by peer nominations, as this is a social environment in which adolescents spend a large part of the day, and are engaged in comparable activities. The fact that school attendance is compulsory in early adolescence, hence the situation is inescapable, is also relevant in this respect, because entrapment has been found to be a particularly potent elicitor of depressive(-like) emotions and behaviors (Brown et\u00a0al., 1995; Gilbert & Allan, 1998). Peer status was assessed by sociometric nominations, which are generally considered the most reliable and valid indices of peer status (Coie & Dodge, 1983). Because the association between depression and peer status might be confounded by comorbid conduct problems (Connell & Dishion, 2006; Kiesner, 2002), we adjusted for comorbidity in our analyses.\nWe hypothesized that low peer status in the achievement-related domain would be most relevant for depressive problems in boys, whereas girls\u2019 depressive problems would be more strongly associated with low affection-related peer status. A further hypothesis was that high peer status in a particular area, although not necessarily associated with the probability of depressive problems in itself, would reduce the association between depressive problems and low status in another status area.\nMethod\nSample\nThe TRacking Adolescents\u2019 Individual Lives Survey (TRAILS) is a prospective cohort study of Dutch (pre)adolescents, with the aim to chart and explain the development of mental health from preadolescence into adulthood, which started in 2001.\nSample selection involved two steps. First, five municipalities in the North of the Netherlands, including both urban and rural areas, were requested to give names and addresses of all inhabitants born between 10\u201201\u20121989 and 09\u201230\u20121990 (first two municipalities) or 10\u201201\u20121990 and 09\u201230\u20121991 (last three municipalities), yielding 3483 names. Simultaneously, primary schools (including schools for special education) within these municipalities were approached with the request to participate in TRAILS. School participation was a prerequisite for eligible children and their parents to be approached by the TRAILS staff. Of the 135 primary schools within the municipalities, 122 (90.4% of the schools accommodating 90.3% of the children) agreed to participate in the study.\nIf schools agreed to participate, parents (or guardians) and children received brochures with information about the study; and a TRAILS staff member visited the school to inform eligible children about the study. Shortly thereafter a TRAILS interviewer contacted parents by telephone to ask whether they and their child were willing to participate in the study. Respondents with an unlisted telephone number were requested by mail to pass on their number. If they reacted neither to that letter, nor to a reminder letter sent a few weeks later, staff members paid personal visits to their house. Parents who refused to participate were asked for permission to call back in about 2 months. If both parents and children agreed to participate, parental written informed consent was obtained after the procedures had been fully explained. Children were excluded from the study if they were incapable of participating due to mental retardation or a serious physical illness or handicap, or if no Dutch-speaking parent or parent surrogate was available and it was not feasible to administer any of the measurements in the parent\u2019s language. Of all children approached for enrollment in the study (N\u00a0=\u00a03145), 6.7% were excluded because of mental or physical incapability or language problems. Of the remaining 2935 children, 76.0% (N\u00a0=\u00a02230, mean age\u00a0=\u00a011.09, SD\u00a0=\u00a00.56, 50.8% girls) were enrolled in the study (i.e., both child and parent agreed to participate). Responders and non-responders did not differ with respect to the prevalence of teacher-rated problem behavior, nor regarding associations between sociodemographic variables and mental health outcomes (De Winter et\u00a0al., 2005).\nThe present study is based on data from the second assessment wave of TRAILS (T2), which was held 2 to 3 years after baseline assessment (mean number of months 29.44, SD\u00a0=\u00a05.37, range 16.69\u201348.06). During this wave, questionnaires were filled out by the adolescents, their parents, and their teachers. The adolescents filled out their questionnaires at school, in the classroom, under the supervision of one or more TRAILS assistants. In addition to the regular questionnaires, which were filled out by TRAILS participants only, the T2 assessment wave also included peer nominations, which were collected in both TRAILS participants and their classmates, at a separate occasion. Of the 2230 baseline participants, 96.4% (N\u00a0=\u00a02149, 51.0% girls) participated in the first follow-up assessment (T2). Mean age at T2 was 13.56, SD\u00a0=\u00a00.53.\nSubsample with peer information\nPeer nominations were assessed in classes with at least three regular TRAILS participants. In total, 150 school classes within 34 schools met this criterion and agreed to participate. Two schools dropped out because of planning difficulties, while one school did not give permission to assess peer nominations within the class. At this school, we collected nominations from TRAILS participants only, providing there were more than 10 participants per class. Schools provided the names of classmates of TRAILS participants. Subsequently, all eligible students received an information letter for themselves as well as their parents in which they were asked to participate in this part of the data collection. If students or their parents refrained from participation they were requested to send a reply card within 10 days. Passive informed consent was adequate for non-TRAILS participants because they were used as informants rather than being study subjects themselves. A total number of 98 students, 3 of whom were regular TRAILS participants, refused to participate. Peer nominations were assessed approximately 2 weeks after the information letter had been sent. The assessment of the peer nominations lasted about 15\u00a0min and took place during the regular lessons. Two participating classes were excluded due to too many recent changes in the composition of the class. In total, 3334 students, 1012 of whom were regular TRAILS participants, filled out the questionnaire and nominated their classmates (outdegree), whereas 3798 students, 1078 of whom TRAILS respondents, were nominated by others (indegree). These 1078 adolescents did not differ from the rest of the TRAILS participants regarding gender, \u03c72 (1, N\u00a0=\u00a02149)\u00a0=\u00a01.17, p\u00a0=\u00a0.28, or depressive problems, t(2125)\u00a0=\u00a0\u22121.15, p\u00a0=\u00a0.25, but were slightly younger (mean age 13.52, SD\u00a0=\u00a00.51, versus 13.60, SD\u00a0=\u00a00.54), t(2085)\u00a0=\u00a03.49, p\u00a0<\u00a0.001. Persons with missing or unreliable data on depressive problems were excluded, leaving 1046 cases for analysis.\nMeasures\nAffection- and achievement related status\nRespondents could nominate any of their classmates on a total of 18 questions, covering a wide range of issues and behaviors (e.g., gossiping, helping others, substance abuse). For the purpose of this study, we selected six questions, three of which referred to affection-related status (Which classmates do you like? Which classmates do you totally dislike? Which classmates are your best friends?), and three to achievement-related status (Who are good at sports? Who are good learners? Who are good-looking?). For each of these questions, we used the proportion of nominations (indegree) as a measure of peer status. Despite the fact that being good-looking is usually more due to inherited features than to personal accomplishments, it was classified as achievement-related, because it is a salient social-ranking dimension in adolescence, which has been found to contribute to a higher social ranking and influence through halo effects (beautiful is good) (Anderson et\u00a0al., 2001; Eagly, Ashmore, Makhijani, & Longo, 1991; Feingold, 1992).\nDepressive problems\nInternalizing and externalizing problem behaviors were assessed by the Child Behavior Checklist (CBCL), one of the most commonly used questionnaires in current child and adolescent psychiatric research (Achenbach, 1991a; Verhulst & Achenbach, 1995). It contains a list of 120 behavioral and emotional problems, which parents can rate as 0 = not true, 1 = somewhat or sometimes true, or 2 = very or often true in the past 6 months. In addition to the CBCL, we administered the self-report version of this questionnaire, the Youth Self-Report (YSR, Achenbach, 1991b). The original, empirically derived, CBCL and YSR scales did not distinguish between anxiety and depressive problems. In order to improve the correspondence with clinical diagnostic categories, Achenbach, Dumenci, and Rescorla (2003) constructed DSM-IV scales for CBCL\/YSR problem behaviors, based on international experts\u2019 ratings. The resulting CBCL\/YSR Depressive Problems scale consists of 13 items (Cronbach\u2019s \u03b1 CBCL\u00a0=\u00a00.73, YSR\u00a0=\u00a00.77) covering depressed mood, anhedonia, loss of energy, feelings of worthlessness and guilt, suicidal ideation, sleep problems, and eating problems. The scale has been found to correspond more closely to DSM-IV Major Depressive Disorder than the original CBCL\/YSR scales (Van Lang, Ferdinand, Oldehinkel, Ormel, & Verhulst, 2005). Test-retest reliabilities of the DSM-IV scales are good, CBCL: r\u00a0=\u00a00.88; YSR: r\u00a0=\u00a00.79 (Achenbach et\u00a0al., 2003). The mean item score of the CBCL-scale was 0.15 (SD\u00a0=\u00a00.19, range 0\u20131.15) and the mean item score of the YSR-scale 0.27 (SD\u00a0=\u00a00.26, range 0\u20131.72), while 6.1% (CBCL) and 16.9% (YSR) of the scores were greater than 0.5 (which implies that more than half of the symptoms were rated as at least somewhat true and\/or more than 25% of the symptoms as very true).\nThe agreement between parent-reported and adolescent-reported depressive problems was moderate, r\u00a0=\u00a0.41. Because depressive problems rated as present by both parent and adolescent are assumed to be more severe (more generalized) than problems rated by only one informant, we used the mean of the standardized parent and adolescent scores as a measure of depressive symptoms in this study. When data of one informant were missing or unreliable (YSRs: n\u00a0=\u00a010, CBCLs: n\u00a0=\u00a081), the composite score was based on only one informant.\nDisruptive behavior\nDisruptive behavior was included to adjust for comorbidity. Disruptive behavior was assessed with the CBCL and the YSR, by combining the items for Oppositional Defiant Disorder and Conduct Disorder, which resulted in a scale of 22 (CBCL, Cronbach\u2019s \u03b1\u00a0=\u00a00.86) or 20 (YSR, Cronbach\u2019s \u03b1\u00a0=\u00a00.79) items.\nStatistical analysis\nFirst, means of and correlations between the variables used in the study were calculated and gender differences tested by means of, respectively, t-tests and z-tests.\nSubsequently, depressive problems were predicted by peer-status variables in a linear regression model. Standard errors were adjusted for possibly dependent observations within classes, using the cluster option available in the statistical package of STATA (StataCorp., 2003). We adjusted for disruptive behavior by including it as an independent variable in all regression analyses. Continuous variables were standardized to mean 0 and standard deviation 1 to ease interpretation of the regression coefficients. In order to be able to distinguish between associations with low status and associations with high status, each of the peer-status variables were categorized into low status (below the 20th percentile), high status (above the 80th percentile), and intermediate status (between the 20th and 80th percentile). For being disliked, high status referred to the lowest 20% (few nominations) and low status to the highest 20% (many nominations). Status was included in the analyses as dummy variables, with intermediate status as the reference category. We analyzed both the effects of each status area individually, and the effects of all areas adjusted for each other. The analyses were stratified by gender. To establish if gender differences were statistically significant, we performed a two-step regression analysis in the total sample. In the first step, main effects of all peer-status variables were entered in the model, as well as gender and disruptive problems. Interactions between gender and each of the peer-status variables, if any, were entered in a second step, by forward stepwise selection with a criterion for entry of p\u00a0<\u00a0.10.\nFinally, we examined whether low status in (at least) one area could be compensated by high status in one or more other areas. Peer-status variables that were significantly related to depressive problems (as established by correlations with the effects of gender and disruptive behavior partialled out) were used to create four combined status groups: 1 = low & high, referring to adolescents with a low status (i.e., below the 20th percentile) in at least one area and a high status (above the 80th percentile) in at least one area; 2 = only low, designating adolescents with a low status in at least one area and no high status; 3 = only high, representing adolescents with a high status in at least one area and no low status, and 4 = intermediate, that is, adolescents with an intermediate status in all areas. Depressive problems in the only-low, only-high, and intermediate group were compared to those in the low & high group (the reference category) by linear regression analysis, adjusting for disruptive behaviors and for dependent observations within classes. Gender differences in each of the associations were tested by interaction effects. A lower depression score in the low & high group than in the only-low group suggests that compensation of a low status by a high status in another area is possible; a lower depression score in the only-high group than in the low & high group suggests that a low status counteracts the benefits of a high status in another area. Comparison of the intermediate group with the low & high group provides insight in the differences between being extraordinary (in any direction) and being average. To take into account that the specific areas may represent the two broad domains of achievement-related and affection-related status disproportionately, this procedure was repeated for the strongest predictor of each.\nFor all analyses, a p-value smaller than .05 was considered statistically significant. Because we performed multiple statistical tests, the results may suffer from capitalization on chance: one would expect some 5% of the associations examined to be significant merely on the basis of chance, and the probability to find at least one significant effect increases with every additional statistical test. We therefore recommend to consider the general pattern of associations, rather than interpreting each individual effect as if it were the only one tested.\nResults\nDescriptive statistics\nTable 1 presents descriptive statistics of the variables used in this study. Girls had more nominations for being good learners and good-looking, while boys were nominated more often as being good at sports. Affection-related status domains showed no significant gender differences. Girls had more depressive problems, and boys more disruptive behavior problems.Table 1Mean (standard deviation) of peer status and mental health, by genderMean (SD)Girls (N\u00a0=\u00a0549)Boys (N\u00a0=\u00a0497)Difference t-testbEffect sizecProportion nominations for beingLiked0.56 (0.21)0.55 (0.20)t = \u22120.26, p = .800.05Disliked0.10 (0.13)0.12 (0.15)t = 1.61, p = .11\u22120.14Best friend0.20 (0.12)0.21 (0.13)t = 1.05, p = .30\u22120.04Good at sports0.22 (0.21)0.40 (0.29)t = 11.11, p < .001\u22120.71A good learner0.33 (0.26)0.28 (0.25)t = \u22122.60, p = .010.20Good-looking0.27 (0.22)0.12 (0.13)t = \u221213.87, p < .0010.83Mental healthDepressive Problemsa0.24 (0.21)0.19 (0.18)t = \u22123.57, p <.0010.26Disruptive Behaviora0.21 (0.15)0.25 (0.18)t = 3.26, p = .001\u22120.24aFor descriptive purposes, the means are based on mean unstandardized scores of parent and adolescent reports. The tests are based on mean standardized scores.bEffects are adjusted for possible dependence of observations within classes.cCohen\u2019s d\u00a0=\u00a0(M1\u2212M2)\/\u03c3pooled, where M1 is the mean of the first group, M2 is the mean of the second group, and \u03c3pooled is the square root of the mean variance of the two groups ().\nBivariate associations\nCorrelations between the peer nominations were generally moderate to high (Table 2). Being a good learner was not significantly related to being good-looking, and in girls neither to being liked and being someone\u2019s best friend. Strongest associations were found among the affection-related variables (being liked, being disliked, and being someone\u2019s best friend). In girls, depressive problems were most strongly associated with being disliked, in boys the correlation was highest for being good at sports.Table 2Correlations between peer status and mental health, in girls (above the diagonal) and boys (below the diagonal)DepressiveDisruptiveLikedDislikedBest friendGood at sportsGood learnerGood-lookingproblemsbehaviorLiked\u2212.62\u2007\u2007.4 9\u2007\u2007.34\u2007\u2007.06\u2007\u2007.55\u2212.17\u2212.07Disliked\u2212.68\u2212.34\u2212.23\u2212.18\u2212.36\u2007\u2007.21\u2007\u2007.14Best Friend\u2007\u2007.49\u2212.36\u2007\u2007.34\u2007\u2007.08\u2007\u2007.46\u2212.06\u2007\u2007.11Good at sports\u2007\u2007.38\u2212.25\u2007\u2007.34\u2007\u2007.21\u2007\u2007.44\u2212.15\u2212.04A good learner\u2007\u2007.14\u2212.16\u2007\u2007.15\u2007\u2007.15\u2007\u2007.07\u2212.14\u2212.21Good-looking\u2007\u2007.42\u2212.28\u2007\u2007.30\u2007\u2007.51\u2007\u2007.09\u2212.10\u2007\u2007.01Depressive Problemsa\u2212.16\u2007\u2007.16\u2212.11\u2212.19\u2212.09\u2212.12\u2007\u2007.52Disruptive Behaviora\u2212.09\u2007\u2007.20\u2212.02\u2007\u2007.03\u2212.20\u2212.04\u2007\u2007.48Note. Bold: p\u00a0<\u00a0.05, italics: significant gender difference.aStandardized scores, based on mean standardized scores of parent and adolescent reports..\nHierarchy of status areas\nTo distinguish between associations with low and high status, each of the peer-status variables was categorized into low status (below the 20th percentile), high status (above the 80th percentile), and intermediate status (between the 20th and 80th percentile). For being disliked, high status referred to the lowest 20% (few nominations) and low status to the highest 20% (many nominations). The distribution of the peer nominations precluded that the high-status group of being disliked contained approximately 20% of the cases: 32.0% of the adolescents were nominated by no one and hence classified as having high disliked status.\nTable 3 shows gender-specific regression coefficients for each of the low- and high-status groups, vis-\u00e0-vis the intermediate group, both without and with adjustment for the other peer-status dimensions. All effects were adjusted for disruptive behavior. Together, the peer-status variables explained 6.4% of the variance in boys, and 7.7% in girls.Table 3Prediction of depressive problems by low and high peer-status in several areas, adjusted for disruptive behavior, stratified by genderB (p)Unadjusted for other areasAdjusted for other areasStatus areaStatusGirlsBoysGirlsBoysAffectionLikedLow\u2007\u20070.41 (<.001)\u2007\u20070.16 (.05)\u2007\u20070.33 (.02)\u2007\u20070.02 (.85)High\u22120.07 (.48)\u22120.21 (.008)\u22120.03 (.80)\u22120.10 (.28)DislikedLowa\u2007\u20070.21 (.08)\u2007\u20070.00 (.95)\u2007\u20070.05 (.72)\u22120.11 (.33)Higha\u22120.13 (.11)\u22120.22 (.002)\u22120.06 (.47)\u22120.14 (.05)Best friendLow\u2007\u20070.24 (.02)\u2007\u20070.22 (.02)\u2007\u20070.08 (.47)\u2007\u20070.13 (.19)High\u22120.14 (.15)\u22120.15 (.046)\u22120.07 (.47)\u22120.05 (.49)AchievementGood at sportsLow\u2007\u20070.27 (.004)\u2007\u20070.44 (<.001)\u2007\u20070.19 (.08)\u2007\u20070.38 (.005)High\u22120.12 (.34)\u22120.17 (.02)\u22120.07 (.57)\u22120.10 (.17)Good learnerLow\u2007\u20070.12 (.35)\u2007\u20070.08 (.39)\u2007\u20070.05 (.68)\u2007\u20070.02 (.79)High\u22120.02 (.83)\u22120.01 (.91)\u2007\u20070.01 (.88)\u2007\u20070.04 (.68)Good-lookingLow\u2007\u20070.12 (.40)\u2007\u20070.17 (.04)\u22120.10 (.52)\u2007\u20070.01 (.92)High\u22120.11 (.13)\u22120.13 (.19)\u2007\u20070.06 (.45)\u22120.04 (.67)Note. Effects of low and high status are with regard to intermediate status. Depressive problems were standardized to mean 0 and standard deviation 1. Effects are adjusted for possible dependence of observations within classes.aAs opposed to other status areas, a low status at being disliked reflects many nominations, and a high status few.Bold: p\u00a0<\u00a0.05.\nIt is interesting to note that in girls, only low status appeared associated with depressive problems, while in boys, high status tended to be relevant as well, especially in the affection domain. Both without and with adjustment for other status areas, girls\u2019 regression coefficients were highest for a low status in being liked, and boys\u2019 coefficients for a low status in sports, indicating that girls and boys with few nominations regarding these areas had the highest number of estimated depressive problems. When all status areas were analyzed jointly, these two associations were the only significant ones. Without adjustment for disruptive problems, the effects remained largely the same, except that the effect of a high status for being disliked (reflecting no nominations) became significant in boys, B\u00a0=\u00a0\u22120.20, p\u00a0=\u00a0.02.1\nGender differences in the associations between peer status and depressive problems were tested by interaction effects of gender and each of the peer-status variables in the total sample, using a forward stepwise selection procedure (with criterion for entry p\u00a0<\u00a0.10) after all main effects had been entered in the model. Gender interactions with a low status on being liked, B\u00a0=\u00a00.32, p\u00a0=\u00a0.04, and a low status on being good at sports, B\u00a0=\u00a0\u22120.26, p\u00a0=\u00a0.09, were both selected by this procedure, but the latter only showed a trend and did not reach the significance criterion of p\u00a0<\u00a0.05. Other status variables did not the meet the entry criterion.\nAcross-area compensation\nWith the effects of gender and disruptive behavior partialled out, all peer-status variables were significantly associated with depressive problems, except being a good learner, r\u00a0=\u00a0\u22120.02, p\u00a0=\u00a0.60. This variable was excluded from further analyses, hence the index describing the combined statuses was based on five areas. Of the total sample, 17.5% fell in the low & high group, 32.0% in the only-low group, 40.8% in the only-high group, and 9.7% in the intermediate group. This distribution was approximately equal for girls and boys, \u03c72(3, N\u00a0=\u00a01046)\u00a0=\u00a02.23, p\u00a0=\u00a0.53. The regression coefficients for the only-low, only-high and intermediate group, in relation to the high & low group, are presented in Table 4.Table 4Prediction of depressive problems by cross-area peer status groups, adjusted for disruptive behaviorStatus groupnB (p)Low & high (reference category)183\u2013Only low335\u2007\u20070.30 (.001)Only high427\u22120.11 (0.12)Intermediate101\u2007\u20070.02 (0.87)Note. Low & High: low status in at least one area and high status in at least one area; Only Low: low status in at least one area and no high status; Only High: high status in at least one area and no low status, Intermediate: intermediate status in all areas. Effects are adjusted for possible dependence of observations within classes.Bold: p\u00a0<\u00a0.05.\nThe coefficients indicate that the only-low group had more depressive problems than the low & high group, while the only-high and intermediate group did not differ significantly from the low & high group. Thus, these results suggest that the risk associated with low status in one area can be compensated by a high status in another area, while the opposite (i.e., counteraction of the beneficial effects of a high status by a low status in another area) is not true, at least not to the same degree. None of the associations showed gender differences (all p-values >\u00a0.42).\nTo test the robustness of these findings, we also constructed a combined status index (with the categories only low, only high, low & high, and intermediate as defined in the method section) based on only the strongest predictors from each of the two broad domains of achievement-related and affection-related status, that is, being liked and being good at sports respectively. The use of this alternative status measure yielded similar results: more depressive problems in the only-low group than in the low & high group, B\u00a0=\u00a00.33, p\u00a0=\u00a0.004; no significant differences between the low & high group and, respectively, the only-high group, B\u00a0=\u00a0\u22120.10, p\u00a0=\u00a0.33, and the intermediate group, B\u00a0=\u00a00.07, p\u00a0=\u00a0.55; and no gender differences (all p-values >\u00a0.31).\nDiscussion\nStriving for status has been proposed as a universal human motive, and a wealth of evidence has indicated that persons\u2019 status within their group influences their psychological and physiological well-being (e.g., Anderson et\u00a0al., 2001). For adolescents, one of the most salient social groups is made up by their classmates. In this study on associations between classroom peer status and depressive problems in young adolescents from the general population, the peer status variables explained about 7% of the variance in depressive problems (unadjusted for measurement error, hence underestimating the true variance), which is a modest, yet noteworthy amount.\nWe hypothesized that peer status in the achievement-related domain would be most relevant for depressive problems in boys, whereas girls\u2019 depressive problems would be more strongly associated to their affection-related peer nominations. A further hypothesis was that high alternative hierarchical positions would (wholly or partly) counteract the association between depressive problems and low status in a particular status area. These hypotheses were largely supported by the data, although it should be noted that the expected patterns were not found in all status areas investigated. Compared with other status areas, not being good at sports, hence a low achievement-related status, was most strongly associated with depressive problems in boys; while not being liked, hence a low status on an affection-related area, had the strongest association with depressive problems in girls. However, our data suggest that not being good at sports may affect girls\u2019 well-being as well, and the gender difference regarding this status domain, although showing a trend, was not statistically significant. Not being liked, on the other hand, appeared to be related to depressive problems in girls only. Hence, girls seem to be more sensitive than boys to affection-related status, particularly to a lack of peers that like them.\nLow status versus high status\nIn general, the risk associated with a low group status was stronger than the resilience associated with a high status, especially in girls, and when the effects of all status areas were adjusted for each other, the only variables that remained significantly associated with depressive problems reflected a low status. Low peer status may lead to depressive problems through a variety of pathways, such as a reduced opportunity to make friends, controlling or dominating behavior by others, and expressions of disapproval or contempt (Deater-Deckard, 2001). All of these may undermine self-esteem and invoke feelings of loneliness (Asher & Wheeler, 1985; O\u2019Brien & Bierman, 1988), which are known risk factors for depressive problems (e.g., Monroe & Hadjiyannakis, 2002; Pelkonen, Marttunen, & Aro, 2003).\nAlthough a high peer status does not seem to be very effective in preventing depressive problems in itself, this may be different for those with a low status in another area: a high status appeared to have the potential to compensate the risk associated with a low status. Adolescents with a low status in at least one area and no high status to counterbalance this had most depressive problems, which supports the idea that depression is particularly likely when no escape from the degrading situation seems possible (Brown et\u00a0al., 1995), and underscores once more the importance of emphasizing and stimulating adolescents\u2019 strengths and talents, particularly for adolescents with weaknesses in other areas.\nGender differences\nThe gender differences found in this study, notably the fact that a direct association between affection-related status and depressive problems was found only in girls, add evidence to the notion that interpersonal stressors are more depressogenic for adolescent girls than for boys (e.g., Brendgen et\u00a0al., 2005; Shih, Eberhart, Hammen, & Brennan, 2006). Associations with depressive problems were adjusted for disruptive behaviors to take into account comorbidity of the two. Without adjustment for disruptive behaviors, being disliked was significantly related to boys\u2019 depressive problems, which may indicate that disliked boys tend to show disruptive behavior rather than (solely) depressive problems. Achievement-related status was most strongly related to depressive problems in boys. Compared to girls, boys focus more on agentic goals, including their own dominance on the peer group (e.g., Rose & Rudolph, 2006), which apparently makes them more vulnerable to low status in relevant areas. However, we found some indications that sports-related achievements may be relevant for girls too. This finding could hint at a dwindling of traditional socio-cultural gender differences; but it could as well be due to the fact that not being good at sports reflects not only a low probability of winning in ritual fights, but also lack of physical energy and activity, which is likely to occur in both depressed boys and depressed girls. The issue of reverse causality is discussed more extensively hereafter.\nDifferences between specific status areas\nWithin the achievement-related domain, sporting performance was more strongly associated with depressive problems than being a good learner and being good-looking. Sports, more than theoretical subjects and outward appearances, reflect ritual agonistic behavior, with the end being signified by the loser yielding and signals of superiority by the winner (Rohde, 2001). In other words, sports are much more closely related to social defeat than the other two achievement domains. But other factors may account for the relatively exceptional position of sports as well. To start with, adolescents who are good at sports may do relatively more physical exercise, which can help to reduce depressive problems (e.g., Nabkasorn et\u00a0al., 2006), so there may be a direct effect of sports on mental health, on top of the effect through peer status. Furthermore, being good at sports increases the likelihood of team membership, which in turn may result into admiration and new friendships. The latter could be especially relevant for boys, who tend to have multiple activity-related friendships rather than a few close friends (e.g., Moller, Hymel, & Rubin, 1992). The association between being a good learner and having a high peer status is probably more equivocal than that between being good at sports and peer popularity. Although some studies found positive correlations between grade point average and popularity among peers (e.g., Cauce, 1987; Green, Forehand, Beck, & Vosk, 1980), others suggested that intelligence is not associated with peer status (Luthar & McMahon, 1996), and that overachievers do not have a secure position in their peer group (Hartup, 1970). In fact, Bukowski, Sippola, and Newcomb (2000) showed that good classroom behavior starts to loose its attraction for peers during early adolescence, in favor of more aggressive behaviors. As concerning the role of being considered good-looking, our results seem to contradict earlier findings that physically unattractive adolescents are likely to receive more negative feedback (Burns & Farina, 1992; Cash, 1995), and that body dissatisfaction is an independent risk factor for (girls\u2019) depressive problems in adolescence (e.g., Stice, Hayward, Cameron, Killen, & Taylor, 2000). That peer nominations for being good-looking did not independently contribute to adolescents\u2019 (lack of) depressive problems in our study could be due to the fact that peer judgments have little to do with one\u2019s own body image (Rosenblum & Lewis, 1999). Furthermore, not being good-looking is not necessarily equivalent to being ugly; in other words, the question \u2018Who is unattractive?\u2019 might have been a stronger correlate of depressive problems than the question \u2018Who is good-looking?.\u2019\nWithin the affection-related domain, which appeared to be relevant to girls in particular, not being liked was the most salient factor in relation to depressive problems. In other words, it does not seem to matter much whether or not some people dislike you, as long as there are still others that like you. A lack of sympathizers is a threat to mental health. From an evolutionary point of view, this makes good sense, because being liked by others can offer social support and protection against being expelled from the group, and the \u2018need to belong\u2019 is a fundamental motivation (Baumeister & Leary, 1995; Fiske, 2004).\nLimitations, strengths, and recommendations for future research\nOur study has some limitations. To start with, the CBCL\/YSR Depressive Problems scale was not developed to assess depressive problems according to DSM-IV criteria, but constructed on the basis of expert ratings of the original, empirically derived, CBCL and YSR scale items. Consequently, the items do not represent one-to-one counterparts of all DSM-IV criteria, and inferences about associations between peer status and DSM-IV Major Depression are putative. Furthermore, we performed multiple statistical tests, which increases the probability of chance findings and warrants replications in other samples. A final limitation of the study is the cross-sectional and non-experimental nature of our data, which does not allow causal interpretation of the associations found (Kraemer et\u00a0al., 1997). Most likely, however, the association between peer status and depressive problems is bidirectional: not only does low peer status induce depressive problems, depressed children and adolescents tend to display maladaptive interpersonal behavior as well (e.g., Altman & Gotlib, 1988; Baker, Milich, & Manolis, 1996). A bidirectional association is especially plausible with respect to sporting performance. Being looked down on because you are bad at sports may reduce self confidence and through this incite the development of depressive problems; but it is just as likely that depressive problems, which often include lack of energy and motivation, have a negative impact on adolescents\u2019 sporting achievements. Similarly, one could argue that the association between depressive problems and not being liked is a two-way one. Having no allies in a peer group can be devastating for your self-worth and hence trigger depression, but depressed behavior may also fend off others (e.g., Prinstein, Borelli, Cheah, Simon, & Aikins, 2005). It seems plausible, however, that if depression were a cause of not being liked, we would expect that to happen in both boys and girls and not in girls only. We thus assume that not being liked is actually a risk factor for depression, at least in girls.\nCompared to the vast literature on peer processes predicting conduct problems, research on peer processes in depression has been relatively rare (Connell & Dishion, 2006; Deater-Deckard, 2001). To the best of our knowledge, our study is one of the first to examine the association between status-related peer nominations and depressive problems in a large population cohort of early adolescents. In addition to the size and representativeness of our sample, important assets of this study relate to the use of multiple informants, multiple aspects of peer status, and multiple outcomes, which allowed adjusting for comorbid disruptive behavior. Hence, we feel that it is an excellent starting point for further research. We propose three lines of extension. Firstly, the cross-sectional associations should be extended to (bidirectional) patterns of associations across time, to be better able to distinguish between concomitants and risk factors (Kraemer et\u00a0al., 1997). Secondly, the study could be extended to include other outcomes, not only within the domain of psychopathology, but also regarding somatic health. Thirdly, it is highly relevant from a public-health point of view to (further) explore to what extent and how peer processes can be manipulated in order to prevent aversive outcomes.","keyphrases":["adolescent","depressive symptoms","social hierarchy","gender"],"prmu":["P","P","P","P"]}
{"id":"Diabetologia-4-1-2292425","title":"Association analysis of the IGF1 gene with childhood growth, IGF-1 concentrations and type 1 diabetes\n","text":"Aims\/hypothesis Insulin-like growth factor-1 is a major childhood growth factor and promotes pancreatic islet cell survival and growth in vitro. We hypothesised that genetic variation in IGF1 might be associated with childhood growth, glucose metabolism and type 1 diabetes risk. We therefore examined the association between common genetic variation in IGF1 and predisposition to type 1 diabetes, childhood growth and metabolism.\nIntroduction\nType 1 diabetes is a common autoimmune disorder that arises by an interaction between genes and the environment. To date, ten loci have been identified including the HLA class II genes, the cytotoxic T-lymphocyte associated protein 4 (CTLA4) locus, protein tyrosine phosphatase, non-receptor type 22 (lymphoid) (PTPN22), IL-2 receptor, alpha (IL2RA), interferon induced with helicase C domain 1 (IFIH1) and four novel loci identified by a genome-wide association study [1].\nThe protein products of these genes play important roles in antigen presentation and the cellular immune response, highlighting the importance of the immune system in the pathogenesis of type 1 diabetes. However, not all patients with evidence of islet autoimmunity develop complete beta cell failure [2]. Given the evidence to suggest that early weight gain and growth in childhood are associated with type 1 diabetes [3], it is possible that factors influencing insulin action (body weight and fat mass) and islet function may predispose to or influence the presentation of type 1 diabetes.\nLower IGF-1 concentrations predict the development of glucose intolerance in adults [4]. George et al. reported that islet overexpression of IGF1 in diabetic mice enabled islet regeneration and gradual correction of hyperglycaemia and hypoinsulinaemia [5]. A microsatellite in the region 5\u2032 to IGF1 has been associated with adult height, cardiovascular risk, osteoporosis and type 2 diabetes in some [6] but not all [7] studies.\nSince common variation in IGF1 could alter circulating IGF-1 concentrations and therefore body habitus, insulin action and beta cell secretion, we sought to determine the associations, if any, between IGF1 and circulating IGF-1 concentrations, size at birth, both fasting and postprandial insulin concentrations, and type 1 diabetes.\nMaterials and methods\nPolymorphism identification\nDNA samples from 32 randomly selected type 1 diabetic probands were amplified using specifically designed forward and reverse primers. This provided 88% probability of detecting single nucleotide polymorphisms (SNPs) with minor allele frequencies (MAF) of 0.033, 96% probability for MAF 0.05 and 99.8% for MAF 0.10 [8]. Resequencing included the 3\u00a0kb region 5\u2032 to the gene, all exons and exon\u2013intron boundaries and 3\u2032 untranslated region. Four 1\u00a0kb segments in the second intron, ~10\u00a0kb intervals apart, were also resequenced.\nTag-SNP selection\nThe resequencing genotype data were used to select SNP subsets that predicted the genotypes of the remainder, using a coefficient of determination, R2, which measures the ability to predict each known SNP genotype by linear regression on the tag-SNP genotypes [9]. We considered only SNPs with a MAF\u2009\u2265\u20090.10 in the type 1 diabetes collection and \u22650.05 in the Avon Longitudinal Study of Parents And Children (ALSPAC) cohort (for details see below and Electronic supplementary material [ESM]), using a minimum R2 of 0.8.\nGenotyping\nAll genotyping data were double-scored to minimise error. All genotypes were in Hardy\u2013Weinberg equilibrium (p\u2009>\u20090.05).\nPopulations studied\nAll DNA samples were collected after ethics approval and informed consent had been obtained.\nType 1 diabetes family collection Type 1 diabetes families were of white European descent, with two parents and at least one affected child. The populations studied have been described previously [8].\nALSPAC Details of this birth cohort are available on the ALSPAC website (www.alspac.bris.ac.uk). The children in this study are from a 10% \u2018Children in Focus\u2019 sub-cohort (1,335 full-term singleton infants) randomly selected from the last 6\u00a0months of recruitment for more detailed measurements of growth. Birthweight was noted from hospital records, and length and head circumference were measured after birth. At age 7\u00a0years (mean age 7.5\u2009\u00b1\u20090.1\u00a0years), body weight and height were measured. Body composition was assessed at age 9\u00a0years by whole-body dual-energy X-ray absorptiometry. Internal SD scores were calculated for all parameters of growth to adjust for age and sex. IGF-1 concentrations were measured in cord blood samples (birth) and in venous blood (7 or 8\u00a0years of age). Fasting insulin sensitivity was assessed by the homeostatic model assessment index (www.dtu.ox.ac.uk\/homa\/) and insulin secretion at 30\u00a0min post oral glucose by the insulinogenic index at age 8\u00a0years.\nStatistical analysis\nAll statistical analyses to test the association between IGF1 and type 1 diabetes were performed in either Stata (www.stata.com\/) or R (www.r-project.org\/) statistical systems. Additional routines may be downloaded (www-gene.cimr.cam.ac.uk\/clayton\/software\/). Missing tag-SNP genotypes were imputed under the null hypothesis and were analysed using a multilocus test [9]. The microsatellite, 5\u2032 IGF1 CA repeat, was analysed using TRANSMIT [10]. The global effect of the IGF1 tag-SNP set on each outcome variable was entered into a multi-locus regression model. R2 change was taken as the contribution to the total variation in each outcome. Associations of IGF1 SNPs with growth and metabolic phenotypes were analysed using univariate ANOVA (general linear models). The association of the 5\u2032 IGF1 CA repeat was analysed by comparing the wild-type allele (192\u00a0bp) to all other alleles [6, 7].\nFor further details on Materials and methods section, see ESM.\nResults\nGenetic variation in IGF1\nResequencing identified 27 novel polymorphisms consisting of 24 SNPs, one rare non-synonymous SNP in exon 3 (Ala to Thr) and two deletion\/insertion polymorphisms. Eleven SNPs had a MAF\u2009>\u20090.05. Four tag-SNPs (Table\u00a01) were selected and genotyped in the type 1 diabetes families studied. In ALSPAC, seven tag-SNPs were selected (ESM Table\u00a01).\nTable\u00a01A summary of IGF1 tag-SNPs genotyped in 2,396 familiesParameterTag-SNPrs35140968ars6214rs3730220s6219SequenceAAATAACT[-\/AT]CTCAAATAGACTTAAC[A\/G]TGTTTTCTCAGGTTGG[A\/T]CTCAAACTAACCTCAA[A\/G]CTGTCTACAllele coding, 1 (minor)\/2Ins\/DelA\/GT\/AA\/GMAF in parents0.2770.4430.4930.0769Minor allele transmissionsTransmitted8151309915446Untransmittedb7901386942446Genotype 2\/2Transmitted1,150 (53.1)947 (33.3)798 (33.0)2,501 (84.8)Untransmittedb3,478 (53.6)2,724 (31.9)2,352 (32.47,489 (84.6)Genotype 2\/1Transmitted849 (39.2)1,382 (48.5)885 (36.6)423 (14.3)Untransmittedb2,541 (39.1)4,226 (49.5)2,685 (37.0)1,297 (14.7)Genotype 1\/1Transmitted166 (7.7)519 (18.2)737 (30.5)27 (0.91)Untransmittedb476 (7.3)1,594 (18.7)2,223 (30.6)67 (0.8)OR for minor allele (95% CI)1.03 (0.93\u20131.14)0.94 (0.87\u20131.01)0.97 (0.89\u20131.06)1.00 (0.88\u20131.14)aDeletion\/insertion polymorphismbUntransmitted (pseudocontrol) genotypes are estimated as previously described [8]Multilocus test p\u2009=\u20090.425\nIGF1 and type 1 diabetes\nThe selected tag-SNPs were genotyped in 2,439 families (3,121 parent\u2013child trios). The multilocus test of the IGF1 tag-SNPs provided no evidence of an association with type 1 diabetes (\u03c742\u2009=\u20094.4; p\u2009=\u20090.356; Table\u00a01). The microsatellite, 5\u2032 IGF1 CA repeat, was genotyped in 2,109 families and analysed using TRANSMIT [10]. There was no evidence of association (p\u2009=\u20090.358) (ESM Table\u00a02).\nWellcome Trust Case Control Consortium data\nWe used data from the Wellcome Trust Case Control Consortium (WTCCC) genome-wide association study [1] to test association of the extended IGF1 region with type 1 diabetes. The region contained 22 SNPs. When the WTCCC data were analysed using a logistic regression model adjusted for variation in allele frequencies across Great Britain, no evidence of association of these SNPs within the region was found (ESM Table\u00a03).\nAssociation with IGF-1 concentrations\nIn the ALSPAC children, multilocus regression models showed that the IGF1 tag-SNP set was associated with IGF-1 protein levels at birth (R2\u2009=\u20090.063; p\u2009=\u20090.029) and weakly associated at age 7 to 8\u00a0years (R2\u2009=\u20090.030; p\u2009=\u20090.055; ESM Table\u00a04). Results of IGF-1 protein level associations with individual SNPs are shown in Table\u00a02. However, the multilocus IGF1 tag-SNP set did not associate with any other childhood growth or metabolic phenotype (ESM Table\u00a04). No associations were observed between the IGF1 CA repeat and IGF-1 concentrations or any growth or metabolic phenotype (ESM Table\u00a05).\nTable\u00a02IGF-1 protein levels (ng\/ml) at birth and at age 7 or 8\u00a0years by genotypes of seven IGF1 tag-SNPs in a representative birth cohortParameternGenotype (%)p valuers35140968\u00a0I\/D (58)I\/I (35)D\/D (7)\u00a0\u00a0At birth31588.1 (2.8)91.1 (3.6)105.0 (8.9)0.179\u00a07 or 8\u00a0years547147.8 (2.9)147.3 (3.9)157.4 (9.1)0.581rs6214\u00a0G\/G (34)G\/A (50)A\/A (16)\u00a0\u00a0At birth30588.6 (3.6)87.7 (3.2)102.43 (5.30)0.048\u00a07 or 8\u00a0years556144.1 (4.0)148.9 (3.1)155.83 (5.70)0.239rs3730220\u00a0A\/A (38)A\/T (54)T\/T (9)\u00a0\u00a0Birth29994.1 (3.8)88.6 (2.9)89.6 (7.4)0.509\u00a07 or 8\u00a0years548152.0 (3.8)145.7 (3.0)141.8 (8.1)0.331rs6219\u00a0G\/G (84)*\/A (16)\u00a0\u00a0Birth30091.0 (2.4)85.0 (5.7)0.329\u00a07 or 8\u00a0years547150.1 (2.5)138.8 (5.8)0.072rs2946831\u00a0T\/T (89)*\/G (11)\u00a0\u00a0Birth29490.0 (2.3)95.2 (7.2)0.501\u00a07 or 8\u00a0years541148.0 (2.4)153.9 (7.1)0.432rs3730204\u00a0T\/T (97)*\/C (3)\u00a0\u00a0Birth294111.3 (13.5)90.3 (2.3)0.126\u00a07 or 8\u00a0years548173.8 (12.9)147.5 (2.3)0.046rs12579108\u00a0C\/C (97)*\/A (3)\u00a0\u00a0Birth30190.0 (2.2)101.4 (12.1)0.358\u00a07 or 8\u00a0years554147.6 (2.3)176.8 (15.9)0.017Values are mean (SE) for IGF-1 concentration and per cent for genotype*Additive models, adjusted for age and sex\nDiscussion\nWe used a UK birth cohort, ALSPAC, and a large family collection of type 1 diabetes to explore association with a subset of SNPs generated by in-depth resequencing of the IGF1 locus. A subset of SNPs that effectively predicted the genotype of the remainder was subsequently genotyped in these cohorts [9]. We report no evidence to support the hypothesis that genetic variation in IGF1 is associated with major susceptibility to type 1 diabetes. However, genetic variation in this locus may modestly influence circulating IGF-1 concentrations, at least at birth and during childhood.\nPrior studies examining the role of genetic variation in IGF1 in predisposition to common human disease have focused on a microsatellite in the region 5\u2032 to IGF1. This variant has been inconsistently associated with adult height and type 2 diabetes [6, 7]. Perplexingly, the same allele has been associated with high [6] and low [7] IGF-1 concentrations. It is possible that other common genetic variants, in different degrees of linkage disequilibrium with this variant, alter IGF-1 concentrations and explain the discrepancy in the published studies.\nWe found no association between this microsatellite and circulating IGF-1 concentrations, growth or type 1 diabetes. To exclude the possibility that other polymorphisms in the gene predispose to disease, we undertook a systematic analysis of IGF1 gene variation using tagging SNPs [8]. Our relatively large white type 1 diabetes family collection of European ancestry provided ~84% power to detect a causal allele for type 1 diabetes predisposition with MAF\u2009=\u20090.1 and OR of 1.2 at the 5% significance level. The study was therefore adequately powered to detect weak predisposition to type 1 diabetes conferred by IGF1. We did not directly examine whether these variants alter the age at diagnosis of type 1 diabetes, since there was no primary evidence of association.\nThe possibility remains that variation in IGF1 could alter insulin secretion or action as well as childhood growth. Therefore, to examine these associations, we adopted the same strategy in an established birth cohort. While we demonstrate that common genetic variation in IGF1 modestly influences circulating IGF-1 concentrations at birth and during childhood, we did not find a major contribution of IGF1 to birthweight, growth or response to oral glucose. It remains possible that maternal IGF1 genotypes that influence maternal metabolism might alter birth size, but they are unlikely to significantly impact on childhood growth.\nIn summary, this large systematic study shows that common IGF1 variants modestly influence circulating IGF-1 concentrations at birth and during childhood. However, these variants are not associated with birthweight, childhood growth and insulin secretion or action. Moreover they do not alter type 1 diabetes risk.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nESM Table\u00a01\nIGF1 SNPs genotyped in ALSPAC children (PDF 16.2 KB)\nESM Table\u00a02\nTRANSMIT analysis for association of the microsatellite in the 5\u2032 region of IGF1 with type 1 diabetes (PDF 62.5 KB)\nESM Table\u00a03\nSummary of 23 WTCCC SNPs in the extended IGF1 region (PDF 28.6 KB)\nESM Table\u00a04\nMultilocus test for associations with IGF-1 protein levels, childhood growth and glucose metabolism in a representative birth cohort (PDF 17.1 KB)\nESM Table\u00a05\nAssociation testing for the 5\u2032 IGF1 CA repeat with childhood growth and glucose metabolism in a representative birth cohort (PDF 72.3 KB)\nESM Table\u00a06\nESM Text (PDF 81.3 KB)","keyphrases":["growth","igf-1","type 1 diabetes","insulin-like growth factor-1","genetic variation","microsatellite","single nucleotide polymorphism","snp","tag-snps","children"],"prmu":["P","P","P","P","P","P","P","P","P","P"]}
{"id":"Anal_Bioanal_Chem-3-1-1914274","title":"An on-line post-column detection system for the detection of reactive-oxygen-species-producing compounds and antioxidants in mixtures\n","text":"Reactive oxygen species (ROS) can damage proteins, cause lipid peroxidation, and react with DNA, ultimately resulting in harmful effects. Antioxidants constitute one of the defense systems used to neutralize pro-oxidants. Since pro-oxidants and antioxidants are found ubiquitously in nature, pro-and antioxidant effects of individual compounds and of mixtures receive much attention in scientific research. A major bottleneck in these studies, however, is the identification of the individual pro-oxidants and antioxidants in mixtures. Here, we describe the development and validation of an on-line post-column biochemical detection system for ROS-producing compounds and antioxidants in mixtures. Inclusion of cytochrome P450s and cytochrome P450 reductase also permitted the screening of compounds that need bioactivation to exert their ROS-producing properties. This pro-oxidant and antioxidant detection system was integrated on-line with gradient HPLC. The resulting high-resolution screening technology was able to separate mixtures of ROS-producing compounds and antioxidants, allowing each species to be characterized rapidly and sensitively.\nIntroduction\nThe potentially toxic and beneficial properties of pro-oxidants and antioxidants have made them the focus of many studies. Pro-oxidants may represent a threat to health, whereas antioxidants may counteract these effects by scavenging pro-oxidants [1, 2]. Antioxidants are very important in industrial processes as well as in biological systems. They are known to possess anti-inflammatory [3], anti-cardiovascular disease [4], antineurogenerative [5], and anticancer properties [6]. Imbalances between pro-oxidants and antioxidants in favor of the pro-oxidants may result in oxidative stress, which in turn may result in oxidative damage [7] of cellular components in the form of lipid peroxidation, protein denaturation or DNA conjugation [8]. Oxidative stress has been associated with many diseases like cancer [6], post-ischemic and neural degradation [2], Parkinson\u2019s and Alzheimer disease [5], AIDS [9], and aging and cardiovascular diseases [4]. The metabolism of pro-oxidants by cytochrome P450s is another important process that can result in the formation of reactive oxygen species (ROS) [10, 11].\nBecause antioxidants may neutralize the potentially harmful effects of direct pro-oxidants or pro-oxidants formed upon bioactivation, much of the work done in this field has been directed at synthesizing antioxidants, in some cases organ- or tissue-specific ones [12\u201314]. In addition, the food industry pays a great deal of attention to antioxidants in foods. Phenols in tea [15], fish oils [16], curcumin in curry [17] and flavanoids in plants [18] are only a few examples of the large number of natural compounds that have been and are being studied for their positive, antioxidant-based, effects. When the pro-oxidant or antioxidant effects of natural extracts or synthetic compounds are investigated, batch assay formats are normally utilized [19\u201321]. On-line assays that measure the pro-oxidant or antioxidant effects of compounds are also described [22\u201324]. However, when individual compounds in mixtures need be analyzed for their pro-oxidant or antioxidant properties using these techniques, cumbersome gradient HPLC separations are required before the purified compounds can be characterized. Moreover, care must be taken to ensure that the purified compounds are not oxidized or degraded in air before analysis. One strategy that could be employed to circumvent the need for this cumbersome purification and subsequent screening process is high-resolution screening (HRS), which stands for \u201con-line post-column biodetection after HPLC separation.\u201d HRS methodologies, which screen individual compounds in mixtures for affinity, have been developed for receptors (e.g., the estrogen receptor [25]), enzymes (e.g., cathepsin B [26], cytochrome P450s [27]), and antibodies (e.g., digoxin antibodies [28]). These HRS strategies have proven to be very useful for the rapid profiling and identifying of individual ligands in active mixtures, especially when HRS systems are run simultaneously with MS [29]. Moreover, the use of these fast HRS strategies can result in a reduced risk of the oxidation or degradation of sample components before analysis.\nThis paper presents the development and validation of a HRS-based on-line post-column detection system for the detection of ROS-producing compounds as well as antioxidants in mixtures. This so-called pro-oxidant and antioxidant detection (PAD) system is based on the oxidation of 4-hydroxyphenylacetic acid (4HPAA) by H2O2 in the presence of horseradish peroxidase (HRP) to a highly fluorescent dimer [30, 31]. Scheme\u00a01 shows the general principles of the assay. H2O2 may be present as such or it may be formed from superoxide anion radicals (resulting from ROS-producing compounds) in the presence of superoxide dismutase (SOD). After optimization, the on-line detection system was validated with the well-known ROS-producing compounds paraquat, menadione and duroquinone, and the antioxidants L-ascorbic acid and glutathione, in flow injection analysis (FIA) mode. Finally, the on-line PAD system was coupled to gradient HPLC and thus used in HRS mode to screen individual compounds in mixtures for their ROS-producing and\/or antioxidant properties.\nScheme\u00a01Schematic representation of the principles of the biochemical assay\nExperimental section\nMaterials\nL-ascorbic acid, L-glutathione (reduced; GSH) and 4-hydroxyphenylacetic acid (4-HPAA) were purchased from Aldrich (Zwijndrecht, The Netherlands). Tween 20, menadione, polyethyleneglycol 6000 (PEG6000), polyethyleneglycol 3350 (PEG3350), methylviologen (paraquat dichloride), peroxidase (horseradish, type I; HRP), and superoxide dismutase (from bovine erythrocytes; SOD) were purchased from Sigma (Zwijndrecht, The Netherlands). \u03b2-Nicotinamide adenine dinucleotide phosphate tetra sodium salt (NADPH) was from Applichem (Lokeren, Belgium). Methanol (MeOH) and isopropanol (IPA) were purchased from Riedel de Ha\u00ebn (Seelze, Germany). Acetonitrile (ACN) was from Baker (Deventer, The Netherlands). The MeOH, ACN and IPA were of HPLC reagent grade. Rat liver microsomes (\u03b2-NF induced) were prepared as described elsewhere [32]. In short: livers from \u03b2-NF induced rats were homogenized at 4\u00a0\u00b0C in two volumes of 50\u00a0mM potassium phosphate buffer (pH 7.4) with 0.9% sodium chloride using a Potter-Elvehjem (Sigma) homogenizer. The homogenate was centrifuged for 20\u00a0min at 12.000\u00d7g, and the supernatant obtained was further centrifuged for 60\u00a0min at 100.000\u00d7g. The resulting pellet was washed twice and subsequently resuspended in 50\u00a0mM potassium phosphate buffer (pH 7.4), 0.9% sodium chloride, and 25% glycerol, and stored at \u221280\u00a0\u00b0C. The protein concentration in the microsomes was determined as 13.1\u00a0mg\/ml. Protein concentrations were determined with the standard Bio-Rad (Hercules, CA, USA) protein assay based on the method of Bradford.\nInstrumentation\nA Gilson 234 auto injector (Villiers-le-Bel, France) equipped with a Rheodyne (Bensheim, Germany) six-port injection valve (injection loop, 50\u00a0\u03bcl) was used for sample injections. A Knauer K-500 HPLC pump (Berlin, Germany) was used to deliver the injected samples into the on-line PAD system in FIA mode. Two Knauer K-500 HPLC pumps were used to deliver the cofactors, substrate and enzymes by means of superloops (SL-A and SL-B) (50\u00a0ml, Pharmacia, Peapack, NJ, USA), which were kept on ice, into the PAD system. Prior to detection via an Agilent 1100 (Waldbronn, Germany) series fluorescence detector (\u03bbex 320\u00a0nm; \u03bbem 409\u00a0nm), a knitted reaction coil (0.25\u00a0mm i.d.; 1.59\u00a0mm o.d.; internal volume of 75\u00a0\u03bcl) positioned in a Shimadzu CTO-10AC column oven (Duisburg, Germany), was used to perform the enzymatic reaction on-line. To reduce pump pulsing, flow restrictors were inserted between the pumps and the superloops. The flow restrictors were made in a similar way to the ones used by Kool et al. [27].\nPro-oxidant and antioxidant assay optimization\nThe initial optimization of the biochemical assay for the PAD system was performed off-line on a Shimadzu RF-1501 spectrofluorometer (\u03bbex 320\u00a0nm; \u03bbem 409\u00a0nm), before the biochemical assay was transferred to the on-line format for further optimization. All measurements were performed in triplicate at 37\u00a0\u00b0C in quartz cuvettes with total volumes of 2\u00a0ml. Different concentrations of enzymes (rat liver microsomal cytochrome P450s and cytochrome P450 reductase, SOD and HRP), cofactor NADPH, and 4-HPAA were tested. Blocking reagents and detergents (that can improve the resolution of the PAD system) [27] and organic modifiers (that are necessary when the PAD system is operated in on-line gradient HPLC mode) were investigated as well. Potassium phosphate buffer (50\u00a0mM; pH 7.8) was used in all experiments. Initial conditions were rat liver microsomes (50\u00a0\u03bcg\/ml), NADPH (40\u00a0\u03bcM), 4-HPAA (1\u00a0mM), SOD (10\u00a0U\/ml), and HRP (10\u00a0U\/ml). Reactions were started after 5\u00a0min of pre-incubation with the addition of paraquat (70\u00a0\u03bcM). The different parameters were optimized in the abovementioned order. When the optimal (or best compromise) concentration of every parameter was subsequently used in the optimization of the next parameter. After the optimization process, the optimized conditions were used in the PAD system in FIA and HPLC mode.\nPro-oxidant and antioxidant detection system in flow-injection analysis mode\nA schematic view of a similar on-line detection system to the PAD system used in FIA mode is shown and described elsewhere [27]. The main difference between the PAD system used in FIA mode and that used in HPLC mode is the replacement of a carrier solution used in FIA mode with a gradient reversed-phase HPLC system. The general scheme for a PAD system coupled on-line to gradient reversed-phase HPLC (described in the next paragraph) is shown in Fig.\u00a01, which also shows the schematics of the PAD system in general. The continuous mixing of enzymes (cytochrome P450s and cytochrome P450 reductase, HRP and SOD) from SL-A and cofactors\/substrate (NADPH\/4-HPAA) from SL-B with a carrier solution in a reaction coil is the basic principle of the PAD system (when used in FIA mode). After mixing in a knitted reaction coil, ROS formed from pro-oxidants (after cytochrome P450\/cytochrome P450 reductase-mediated bioactivation) are converted by SOD to (relatively stable) H2O2. The subsequent H2O2-dependent conversion of non-fluorescent 4-HPAA to its fluorescent dimer by HRP yields a spectroscopic handle for the efficient measurement of ROS formation. Thus, the ROS formed by eluting compounds give rise to a temporary increase in the formation of fluorescent product, which is seen in the PAD system as a peak. When SL-B also contains a ROS-producing compound, continuous oxidation of the 4-HPAA results in an elevated fluorescent baseline. In this situation, injection of antioxidants causes a temporary decrease in 4-HPAA oxidation, resulting in a negative peak in the PAD system. Thus, the system is sensitive to both ROS-producing compounds and antioxidants.\nFig.\u00a01Schematic view of the PAD system used in HPLC mode. Superloop A (SL-A) and superloop B (SL-B) are used to deliver enzymes and substrates to the reaction coil, respectively. ROS-producing pro-oxidants and antioxidants are introduced into the system by a gradient reversed-phase HPLC system. Antioxidants and ROS-producing compounds temporarily alter the amount of fluorescent product formed, and this change is detected with a fluorescence (FLD) detector. After HPLC, the make-up pumps produce a counteracting gradient, resulting in a PAD-compatible constant organic modifier concentration. The effluent is then split 1:9 (90% to UV detection and 10% to CYP EAD). AS, autosampler\nLiquid chromatography coupled to the pro-oxidant and antioxidant detection system\nA general scheme for the PAD system coupled on-line to gradient reversed-phase HPLC is shown in Fig.\u00a01. Gradient HPLC separations were performed using a 30\u00a0mm length \u00d7 2\u00a0mm i.d. stainless steel column (Luna 3\u00a0\u03bc C18(2), Phenomenex, Torrance, CA, USA). When the PAD system was operated in HPLC mode, two pumps were used to control the LC gradient and two pumps were used directly after the HPLC column to compensate for the increased concentration of organic modifier (during the gradient) before the delivery of the effluent to the PAD system. The following gradient was used for the HPLC separations: an initial flow rate of 300\u00a0\u03bcl\/min for 3\u00a0min at H2O:MeOH (95:5); a decreasie in the flow rate gradient for 6\u00a0min to 150\u00a0\u03bcl\/min H2O:MeOH (5:95); 14\u00a0min with H2O:MeOH (5:95) at a flow rate of 150\u00a0\u03bcl\/min. Thereafter, the column was re-equilibrated to the starting conditions in 5\u00a0min. To maintain a constant concentration of MeOH after the HPLC column, a second gradient with an increasing flow-rate was included in the system, after HPLC separation, with an initial flow-rate of 700\u00a0\u03bcl\/min H2O:MeOH (4:1) for 4.5\u00a0min. An increase in the flow-rate gradient for 6\u00a0min to 850\u00a0\u03bcl\/min H2O:MeOH (100:0) was then followed by a post-gradient flow-rate of 850\u00a0\u03bcl\/min H2O:MeOH (100:0) for 14\u00a0min. Finally, re-equilibration to starting conditions was performed in 5\u00a0min. The H2O and MeOH of the increasing flow-rate gradient contained 100\u00a0mg\/L Tween 20. The final constant flow-rate was 1000\u00a0\u03bcl\/min, with a MeOH concentration of 15% in H2O. This flow was connected to a T-piece and split 1\/9 with a flow splitter. 90% was directed to the UV detector, while10% was pumped into the PAD system. For HPLC analysis, all tested compounds were dissolved in 30% MeOH in water.\nResults and discussion\nThe primary aim of this study was to develop and validate a HRS-based on-line post-column detection system for the detection of ROS-producing compounds and antioxidants in mixtures. The biochemical assay is based on the oxidation of 4-hydroxyphenylacetic acid (4HPAA) to a highly fluorescent dimer (see Scheme\u00a01). After optimization, the on-line detection system was validated using the well-known ROS-producing compounds paraquat, menadione, and duroquinone, as well as the antioxidants L-ascorbic acid and glutathione, in flow injection analysis (FIA) mode. The on-line PAD system was then coupled to gradient HPLC and this was used in HRS mode to screen for individual compounds in mixtures based on their ROS-producing and\/or antioxidant properties.\nOptimization of pro-oxidant and antioxidant detection assay\nThe optimization of the biochemical assay for the PAD system was conducted first in an off-line batch format before the optimized biochemical assay was transferred to the PAD system in FIA and HPLC modes. Paraquat was used as a model ROS-producing compound. The following parameters were optimized: enzyme concentrations (rat liver microsomes, SOD, and HRP), cofactor NADPH and substrate 4-HPAA, blocking reagents PEG3350 and PEG6000, the detergent Tween 20 (which can improve the resolution of the on-line PAD system) and organic modifiers (that are necessary when the on-line PAD system is operated on-line in gradient HPLC mode). All optimizations were performed without the presence of paraquat as the continuous ROS-producing compound in SL-B. However, paraquat (0.036\u00a0mM) was added to the SL-B in the final optimized system in order to permit measurements of both pro-oxidants and antioxidants using the on-line PAD system.\nFirst, the concentration of cytochrome P450s\/cytochrome P450 reductase-containing rat liver microsomes was evaluated. Insertion of this important, mainly hepatic, biotransformation system permits the reductive bioactivation of compounds that need to be bioactivated before they can exhibit their ROS-producing effects (e.g., paraquat [33]). For the liver microsomes, it was found that higher concentrations resulted in an increase in fluorescence signals up to at least 150\u00a0\u03bcg\/ml (microsomal protein concentration). This is due to increased cytochrome P450s\/cytochrome P450 reductase-mediated redox cycling [33]. Since the use of high concentrations of rat liver microsomes increases the risk that on-line biochemical detection systems will become clogged [27], a concentration of 50\u00a0\u03bcg\/ml rat liver microsomes was used in subsequent experiments. For SOD, concentrations higher than 14\u00a0U\/ml did not increase the fluorescence signal significantly. Therefore, this SOD concentration was used in subsequent experiments. For HRP, the same effect was observed at a concentration of 18\u00a0U\/ml, and so this concentration was employed from then on. Increasing the concentration of 4-HPAA led to the increased formation of fluorescent dimer, up to a concentration of 1.2\u00a0mM, after which the additional increase in sensitivity was counteracted by an increase in noise. A 4-HPAA concentration of 1.2\u00a0mM was therefore used in later experiments. An NADPH concentration of greater than 44\u00a0\u03bcM did not increase the assay performance significantly and so this concentration was adopted. For the polymers PEG3350 and PEG6000, concentrations of up to 5\u00a0mg\/ml did not produce significant differences in assay performance. Since a PEG6000 concentration of 1\u00a0mg\/ml is sufficient to reduce possible peak broadening in on-line biodetection systems [27], this concentration was in subsequent experiments. Detergents, such as Tween 20, can be used to prevent (membrane-bound) enzymes from adhering to reaction coils in on-line biodetection systems, thereby resulting in reduced peak broadening [27]. Solubilization of membrane-bound enzymes, such as cytochrome P450s, can occur at high detergent concentrations and will inactivate the microsomes under the present conditions. A Tween 20 concentration of 100\u2013200\u00a0mg\/l showed minimal (5\u201320%) enzyme denaturation. These Tween 20 concentrations are known to efficiently reduce peak broadening when used in cytochrome-P450-containing on-line biodetection systems [27]. A Tween 20 concentration of 100\u00a0mg\/l was therefore used from then on. Organic modifiers may be useful for preventing enzymes and lipophilic compounds from adhering to the walls of reaction coils, thus preventing peak broadening [27]. Moreover, when on-line biodetection systems are operated in the HPLC mode, organic modifiers are automatically introduced via the HPLC gradient. When testing MeOH, ACN, and IPA in the off-line biochemical batch assay format, they were tolerated up to concentrations of 10%, resulting in approximately half the fluorescence signal compared to that obtained when organic modifiers were not used.\nOptimized conditions were derived from the abovementioned experiments performed in the off-line batch assay format and subsequently translated to the on-line PAD system in FIA mode. These final conditions were: a carrier solution consisting of 10% MeOH and 100\u00a0mg\/l Tween 20 at a flow rate of 100\u00a0\u03bcl\/min; SL-A containing potassium phosphate buffer (50\u00a0mM; pH 7.8), rat liver microsomes (50\u00a0\u03bcg\/ml), HRP (18\u00a0U\/ml), and SOD (14\u00a0U\/ml), and SL-B with the same buffer containing PEG6000 (1\u00a0mg\/ml), NADPH (44\u00a0\u03bcM), and 4-HPAA (1.2\u00a0mM). For continuous ROS production (resulting in a stable fluorescent baseline), paraquat was present in the optimized system in SL-B at a concentration of 0.036\u00a0mM. Both superloops had a flow-rate of 100\u00a0\u03bcl\/min.\nPAD system in flow-injection analysis mode\nBefore coupling the on-line PAD system to gradient HPLC, it was first evaluated and validated in FIA mode. Evaluation of the PAD system was done with three well-known model ROS-producing compounds (i.e., paraquat, menadione, and duroquinone), and two well-known model antioxidants (L-ascorbic acid and glutathione). First, paraquat was injected in triplicate at different concentrations (1.0, 0.25, 0.06, 0.015, 0.004, 0.001, 0\u00a0mM) into the PAD system in FIA mode. Figure\u00a02a shows the resulting signals. The highest concentration of paraquat resulted in fluorescence quenching. An analogous FIA trace for the redox cycling compound menadione is depicted in Fig.\u00a02b.\nFig.\u00a02Triplicate injections of a series of dilutions (dilution factor of 4) of different compounds into the PAD system used in FIA mode. a The pro-oxidant paraquat (starting with 1.0\u00a0mM). b The pro-oxidant menadione (starting with 0.15\u00a0mM). c The antioxidant ascorbic acid (starting with 1.0\u00a0mM). d The antioxidant glutathione (starting with 25\u00a0mM)\nFigure\u00a02c and d show the resulting PAD traces when L-ascorbic acid and glutathione (well-known antioxidants) were injected in triplicate in a dose-response manner, respectively. It was shown that ascorbic acid acted as a pro-oxidant compound at low concentrations (Fig.\u00a02c). Pro-oxidant effects of ascorbic acid at low concentrations were previously demonstrated by Abudu et al. [34], who stated that ascorbic acid can undergo one-electron reduction to form an ascorbyl radical. Ascorbic acid can also switch from being an antioxidant to being a pro-oxidant in the presence of transition metals [35].\nThe direct antioxidant effect of L-ascorbic was also shown using the PAD system (without paraquat in SL-B) by first injecting paraquat and then injecting a mixture of paraquat and L-ascorbic acid. Figure\u00a03 shows that the addition of L-ascorbic acid neutralizes the ROS formed from paraquat. The same effect is shown for menadione (Fig.\u00a03).\nFig.\u00a03Injections (triplicates) of different compounds into the PAD system in FIA mode (without the continuous addition of a pro-oxidant): 1) paraquat (0.05\u00a0mM); 2) paraquat (0.05\u00a0mM) and ascorbic acid (0.1\u00a0mM); 3) menadione (0.03\u00a0mM); 4) menadione (0.03\u00a0mM) and ascorbic acid (0.1\u00a0mM)\nTo determine the effects of the test compounds paraquat, menadione, L-ascorbic acid, and glutathione on the fluorescence baseline, injections of different concentrations of the test compounds into the PAD system were performed without HRP and SOD in SL-A. Menadione and duroquinone caused fluorescence signals when injected in concentrations larger then 0.8 and 0.4\u00a0mM, respectively. This implies that these compounds can only be measured efficiently at these or higher concentrations if a parallel signal measuring their auto-fluorescence is employed, so that this can be subtracted from the PAD signal. In order to determine the relative pro-oxidant or antioxidant effects of the compounds, and to compare the off-line batch assay and the on-line assay (in FIA and HPLC mode) with each other, the fluorescence signals per mole of the test compound were determined from the data obtained in the off-line batch assay and the PAD system in FIA and HPLC modes (Table\u00a01). When comparing the relative fluorescence signals per mole of test compound obtained using the off-line batch and both of the on-line systems, the relative fluorescence signals appeared to differ significantly for some compounds. This difference might be explained by the pre-incubation step, which was only performed for the off-line batch assays and not for the on-line PAD measurements. In contrast, for the off-line batch assay and both of the on-line PAD system formats, the measured pro-oxidant or antioxidant effects were comparable for all test compounds in terms of determinations of their individual pro-oxidant or antioxidant effects. Although the results obtained using the off-line batch format and both on-line formats were not similar, the comparable results still allowed the off-line batch assay to be transformed into both on-line assay formats. We can expect these relatively complex assay systems to give slightly different assay characteristics when performed under different assay conditions, like off-line batch or on-line assay conditions. Therefore, we can conclude that the off-line batch assay format can be transferred to the present PAD system used to screen for antioxidants and ROS-producing compounds.\nTable\u00a01Initial relative increases or decreases (in fluorescence units, FU) of different ROS-producing pro-oxidant compounds and antioxidants compared to paraquat for the PAD system used in FIA mode, in gradient HPLC mode and for a traditional batch assayPro-oxidant\/AntioxidantFIA PAD system (FU\/mol\u00b1SEM)Detection limit for the FIA PAD system (nmol)HPLC PAD system (FU\/mol\u00b1SEM)Detection limit for the HPLC PAD system (nmol)Batch assay set-up (FU\/mol\u00b1SEM)Paraquat1.00\u2009\u00b1\u20090.080.071.00\u2009\u00b1\u20090.330.91.00\u2009\u00b1\u20090.05Menadione1.55\u2009\u00b1\u20090.200.010.56\u2009\u00b1\u20090.010.41.60\u2009\u00b1\u20090.33Duroquinone0.13\u2009\u00b1\u20090.020.040.36\u2009\u00b1\u20090.191.30.16\u2009\u00b1\u20090.07Glutathione-2.20\u2009\u00b1\u20090.031.9-0.67\u2009\u00b1\u20090.188.0-1.01\u2009\u00b1\u20090.04Ascorbic acid-4.58\u2009\u00b1\u20090.220.1-1.35\u2009\u00b1\u20090.130.2-1.22\u2009\u00b1\u20090.12The detection limits of the PAD system used in the FIA and HPLC modes are also given\nInterday and intra-day variabilities were determined in the on-line PAD system in FIA mode as follows: intraday variability was determined by injecting paraquat (0.2\u00a0mM) in triplicate at 3.5-hour time intervals into the PAD system. The intraday variability was determined without changing the contents of the superloops. For the interday variability, paraquat (0.2\u00a0mM) was injected daily in triplicate for three days with fresh solutions in the superloops each day. Intraday variability was 3.3%\u00b11.1% and interday variability 4.1%\u00b10.6%, which are both within the ranges exhibited by bioanalytical screening methods [27, 29]. Detection limits (Table\u00a01) were determined by triplicate injections of a series of dilutions of every test compound. The detection limit was defined as the concentration of test compound that gave an average signal of three\u00a0times the noise (S\/N ratio\u2009=\u20093).\nThe sensitivities obtained for the different compounds, which are also intrinsically determined by their pro-oxidant or antioxidant potencies, are also indicated in Table\u00a01. These sensitivities were of the same order as those obtained with the off-line batch assay format (data not shown). Thus, the present PAD system provided a useful novel rapid screening tool for pro-oxidant and antioxidant activities of individual compounds in mixtures in this respect too.\nOn-line coupling of the PAD system to gradient HPLC\nThe PAD system in gradient HPLC mode was evaluated by analyzing the five test compounds after HPLC separation with a decreasing flow-rate gradient. The advantage of this decreasing flow-rate gradient lies with the initially high flow-rate through the column at low concentrations of organic modifier, which results in better eluting compound resolution at the start of the gradient. At higher concentrations of organic modifier, the flow rates are obviously gradually decreased (and the post-column counteracting flow rates are gradually increased) in order to obtain a continuous flow rate (1\u00a0ml\/min) and concentration of organic modifier (of 10%) after mixing in the post-column counteracting gradient. This results in a constant flow rate and organic modifier concentration (after the 1:9 split) when entering the on-line PAD. The added value of this approach is that alterations in the chromatographic method can be made without much effect on the on-line PAD assay.\nFirst, the individual test compounds were analyzed. The test compounds were injected (in triplicate) in five different concentrations prepared by serial dilution of 100\u00a0\u03bcl of stock solution with 300\u00a0\u03bcl MeOH (30% v\/v). Typical superimposed chromatograms of menadione and L-ascorbic acid are shown in Fig.\u00a04a and b, respectively. At high concentrations of L-ascorbic acid, two peaks were seen. This was the result of overloading the analytical column, as additional mass spectrometry data showed that both peaks were from L-ascorbic acid (data not shown). The relative pro-oxidant or antioxidant response of every test compound in the PAD system used in gradient HPLC mode is shown in Table\u00a01. The sensitivities obtained for the test compounds are also depicted in Table\u00a01. When comparing the PAD system used in HPLC mode with the PAD system used in FIA mode and the off-line batch assays, it is seen that all three assay formats allow the detection of both pro-oxidants and antioxidants. The differences between the three assay formats probably result from different factors, such as the pre-incubation step applied in the off-line batch assay format and the peak broadening of compounds during the chromatographic separation. Another possible cause derives from minor pro-oxidant or antioxidant impurities in the test compounds. As these impurities would be separated from the test compounds in the on-line PAD system used in HPLC mode, they would only influence the results obtained in the off-line batch format and the on-line PAD system used in FIA mode. However, comparable results are still obtained (no more than threefold differences in measured pro-oxidant or antioxidant effects were obtained per mole of test compound) in the different experimental set-ups. Therefore, it can be concluded that the PAD system used in gradient HPLC mode can be easily applied to and is a sensitive method for the screening of individual compounds for their ROS-producing capacity and antioxidant potentials.\nFig.\u00a04a Superimposed PAD traces of menadione injected in different amounts into the PAD system in HPLC mode (12.5, 50 and 200\u00a0\u03bcM from bottom to top chromatogram, respectively). b Superimposed PAD traces of ascorbic acid injected in different amounts into the PAD system in HPLC mode (50, 200 and 800\u00a0\u03bcM from top to bottom chromatogram, respectively)\nThe PAD system used in HPLC mode was also applied to the detection of individual antioxidants and ROS-producing compounds in mixtures. Typical PAD traces of two different mixtures that were injected and separated on HPLC are shown in Fig.\u00a05a and b, respectively. Figure\u00a05a shows that all three ROS-producing compounds\u2014paraquat, menadione and duroquinone\u2014were individually identified as oxidant species. The compounds in the second mixture, which contained two antioxidants and two ROS-producing compounds, were individually identified as antioxidants (L-ascorbic acid and glutathione) and pro-oxidants (menadione and duroquinone). Thus, mixtures in which ROS-producing compounds are present together with antioxidants could effectively be measured individually with the present PAD system in gradient HPLC mode. When analyzing such mixtures with traditional off-line batch assay formats, antioxidants can counteract the effects of pro-oxidants thereby reducing or even totally removing ability to detect the pro-oxidants. The relatively low resolutions obtained in the PAD assay directly reflect the resolutions resulting from the chromatographic part of the total system. Since this study was a proof-of-principle study, we did not perform a thorough optimization of the chromatographic part of the system. The resolutions that can be obtained with the PAD part of the system (used in FIA mode) are shown in Fig.\u00a02 and reflect the performance of the assay. Connecting the PAD part to a chromatographic separation system with higher resolution will obviously result in higher resolution, which may be needed when screening real life samples. The present PAD system in HPLC mode therefore opens up new avenues to the efficient and rapid screening of complex mixtures for individual pro-oxidant and antioxidant components.\nFig.\u00a05a PAD trace for a mixture of three pro-oxidants injected into the PAD system in HPLC mode. Injected compounds are: paraquat (0.12\u00a0mM; 3.5\u00a0min), menadion (0.05\u00a0mM; 17.5\u00a0min), and duroquinone (0.17\u00a0mM; 18.5\u00a0min). b PAD trace of a mixture of two pro-oxidants and two antioxidants injected into the PAD system in HPLC mode. Iinjected compounds are: ascorbic acid (0.14\u00a0mM; 5\u00a0min), glutathione (0.4\u00a0mM; 8\u00a0min), menadion (0.05\u00a0mM; 17.5\u00a0min), and duroquinone (0.17\u00a0mM; 18.5\u00a0min)\nConclusion\nThis paper presents the development and validation of a HRS-based on-line post-column detection system for the detection of ROS-producing compounds as well as antioxidants in mixtures. Different parameters, such as substrate (4-HPAA) and enzyme concentrations, reaction time, temperature, additives, and organic modifier concentrations were first optimized for the PAD system used in FIA mode. Several ROS-producing compounds as well as antioxidants were successfully measured with the optimized system. The intraday and interday variabilities of the PAD system used in FIA mode were determined and found to be lower than 5%. Good sensitivities, at least comparable with similar off-line batch assay formats for individual compounds, were obtained. On-line coupling of the novel PAD system to gradient HPLC permitted the screening of individual compounds in mixtures for ROS-producing and antioxidant properties. It should be noted, however, that compounds that show fluorescence quenching or intrinsic fluorescence may interfere with the methodology. However, it may be possible to adjust the system so that another split directs some of the flow to a second on-line assay that measures these artifacts with a \u201cnegative control PAD system\u201d in order to detect such interferences. This PAD system used in gradient HPLC mode is potentially of great value to drug discovery and toxicology and food research.","keyphrases":["on-line","ros","antioxidant","pro-oxidant","biochemical detection","bioactivation"],"prmu":["P","P","P","P","P","P"]}
{"id":"AIDS_Behav-3-1-1847541","title":"Acceptability of Male Circumcision for Prevention of HIV\/AIDS in Sub-Saharan Africa: A Review\n","text":"Based on epidemiological, clinical and experimental evidence, male circumcision (MC) could have a significant impact on the HIV epidemic in selected areas. We reviewed studies of the acceptability of MC in sub-Saharan Africa to assess factors that will influence uptake of circumcision in traditionally non-circumcising populations. Thirteen studies from nine countries were identified. Across studies, the median proportion of uncircumcised men willing to become circumcised was 65% (range 29\u201387%). Sixty nine percent (47\u201379%) of women favored circumcision for their partners, and 71% (50\u201390%) of men and 81% (70\u201390%) of women were willing to circumcise their sons. Because the level of acceptability across the nine countries was quite consistent, additional acceptability studies that pose hypothetical questions to participants are unnecessary. We recommend pilot interventions making safe circumcision services available in conjunction with current HIV prevention strategies and evaluating the safety and acceptability of circumcision.\nIntroduction\nNumerous observational studies have reported a significant protective effect of male circumcision (MC) against HIV and other sexually transmitted infections (STIs) in men (Bailey, Plummer, & Moses, 2001; Cameron et\u00a0al., 1989; Gray et\u00a0al., 2000; Lavreys et\u00a0al., 1999; Siegfried et\u00a0al., 2003; Urassa, Todd, Boerma, Hayes, & Isingo, 1997; Weiss, Quigley, & Hayes, 2000). Recently, a randomized controlled trial (RCT) of MC to reduce HIV incidence in Orange Farm, South Africa was stopped prematurely due to an observed protective effect of MC of 60% in intention to treat analysis and 76% in a per protocol analysis. This effect was consistent with the protective effect found in cohort studies (Auvert et\u00a0al., 2005).\nEcological studies have shown that the countries in sub-Saharan Africa with the highest HIV prevalence are those in which MC is little practiced (Halperin & Bailey, 1999; Moses et\u00a0al., 1990). Based on the epidemiological and experimental evidence to date, MC could have a significant impact on the HIV epidemic in these most highly affected countries. However, the effectiveness of the intervention will depend on many factors, not the least of which is the extent to which MC is accepted and taken up by males in these populations. If sufficient numbers of males are circumcised, there could be an effect similar to herd immunity since preventing men from becoming infected will also protect their sex partners. At more moderate levels of uptake, the effect is less clear.\nIn addition to the proportion of males who will become circumcised, the age at circumcision will also be a determinant of how rapidly the intervention results in reduction of HIV prevalence in the population. If infant circumcision is preferred over, say, pubertal circumcision, then the time lag from introduction of a large scale intervention until observable reductions in HIV prevalence could be decades. Because acceptance of MC by men and by parents of males in traditionally non-circumcising communities will be crucial to the success of a MC intervention for reducing HIV prevalence, we provide a review of the extant literature on acceptability of MC in sub-Saharan Africa.\nStudy Collection and Search Strategies\nCriteria for inclusion in this review were established before the literature searches were carried out and included studies researching acceptability of MC as an HIV prevention method formally or as a part of a larger study, conducted in sub-Saharan Africa and published in a peer-reviewed journal or presented at an international conference. Electronic searches were conducted in MEDLINE using the following strategy: term \u201ccircumcision\u201d in the title, abstract or keywords was combined with \u201cacceptability\u201d, \u201cattitudes\u201d or \u201cbeliefs\u201d in the title, abstract or keywords generating 920 articles, subset by \u201cHIV\u201d or \u201cSTIs\/STD\u201d in the title, abstract or keywords producing 244 articles, and finally limited to English language articles published from 1980 through 2006 resulting in 229 publications. Electronic search conducted in Google Scholar using phrase \u201cacceptability of male circumcision in Africa as HIV prevention\u201d resulted in 142 publications. Nine articles were directly related to the acceptability of circumcision in sub-Saharan Africa. Four additional studies were identified through personal communication with authors. A map of study sites in nine countries is presented in Fig.\u00a01. Key characteristics of the 13 studies included in this review are shown in Table\u00a01.\nFig.\u00a01Locations (by level 3 administrative unit) where male circumcision (MC) acceptability studies were conductedTable\u00a01Characteristics of male circumcision (MC) acceptability studies (N\u00a0=\u00a013), 1999\u20132006Country\/Authors\/YearTime of the studyStudy populationEthnic compositionCircumcision status of participantsData collection methodsBotswana\/Kebaabetswe et\u00a0al. (2003)2001316 Male and 289 female participants, age 18\u201374, in urban and rural settingsEthnically heterogeneous (over 15 ethnicities)Both circumcised and uncircumcised men Interviews, pre- and post-educational sessionKenya\/Bailey et al. (2002)1998Residents of Nyanza Province, age 16\u201380, men and women, 30 focus groups, each 6\u201314 people, urban and rural population, farmers, business people, teachers, sex workers, barmaids, and touts.Ethnically homogenous (Luo)Not recorded; nearly all likely uncircumcisedFocus groups; interviews with healthcare providersKenya\/Bailey (Unpublished report to AIDSMARK, 2002)199932 Clinicians were interviewed to assess their knowledge and practice of MC, records of MC performed in the area were reviewed, 7 circumcised men and their wives were interviewedEthnically homogenous (Luo)Both circumcised and uncircumcisedInterviews, KAB questionnaires, record reviewKenya\/Mattson et al. (2005)1999107 Men and 110 women, 16\u00a0years of age and older of Luo ethnicity, in urban and rural settingsEthnically homogenous (Luo)Both circumcised and uncircumcised menStructured interviewsMalawi\/Ngalande et al. (2006)2003318 Participants, 32 focus groups with men and women 16\u201380\u00a0years oldEthnically diverse (Chewa, Tonga, Yao, Ngoni, Lomwe, and Nyanja) Both circumcised and uncircumcised menFocus groupsSouth Africa\/Lagarde et al. (2003)2001482 Men aged 19\u201329\u00a0years and 302 women aged 14\u201325\u00a0yearsEthically heterogeneous (Sotho, Tswana, Xhosa and other ethnicities)22% of men 19\u201329\u00a0years old were circumcisedInterviews using standardized questionnaireSouth Africa\/Scott et al. (2005)2002100 Adult men and 44 adult women in rural Zulu land and 4 service providersEthnically homogenous (Zulu)Both circumcised and uncircumcised menInterviews, focus groupsSouth Africa\/Rain-Taljaard et\u00a0al. (2003)1999\u20132000Sample of 606 13\u201359\u00a0year old males interviewed in August 2000 and 723 14\u201324\u00a0year old males interviewed in August 1999Ethnically diverse (Sotho, Xhosa, Zulu, Tswana, Shangaan, and Venda)36% of men 25\u201359\u00a0years old were circumcisedInterviews and focus groupsSwaziland\/Tsela and Halperin (2006)2006409 Men aged 15\u201349 were interviewed in urban and rural settingNot reported, but likely majority were Swazi14% of men were circumcisedInterviewsTanzania\/Nnko et al. (2001)1991\u20131997998 Sukuma men from a cohort of factory workers in Mwanza town, 13 focus groups from mostly rural area, and population based surveysEthnically homogenous (Sukuma)21% of men in the sample were circumcisedInterviews and cohort data analysisUganda\/Bailey et al. (1999)1997188 Circumcised and 177 uncircumcised men 18 to 67\u00a0years old from the Industrial Borough, Mbale. Ethnically diverse (17 tribal groups, including Gisu)52% of men were circumcisedStructured interviewsZambia\/Lukobo and Bailey (submitted)2003160 Men and 162 women in the 34 focus groups in rural and urban settingsEthnically diverse (Lunda, Luvale, Chewa, Tonga)Both circumcised and uncircumcised menFocus groupsZimbabwe\/Halperin et al. (2005)2000200 Men attending beer halls in HarareNot reported, but likely majority were ShonaBoth circumcised and uncircumcised menInterviews, focus group\nDiversity of the Study Sample\nAll studies employed some variation of a convenience sample. Out of 13 studies reviewed, eight were designed specifically to study acceptability of MC (Bailey, Muga, Poulussen, & Abicht, 2002; Kebaabetswe et\u00a0al., 2003; Lagarde, Dirk, Puren, Reathe, & Bertran, 2003; Lukobo & Bailey, Submitted; Mattson, Bailey, Muga, Poulussen, & Onyango, 2005; Ngalande, Levy, Kapondo, & Bailey, 2006; Scott, Weiss, & Viljoen, 2005; Tsela & Halperin, 2006), two included questions on MC acceptability in the context of a larger study (Bailey, Neema, & Othieno, 1999; Halperin, Fritz, McFarland, & Woelk, 2005), and three included formal MC acceptability data collection as well as previously collected data as part of a larger scope of research (Bailey, Unpublished report to AIDSMARK, 2002; Nnko, Washija, Urassa, & Boerma, 2001; Rain-Taljaard et\u00a0al., 2003). Seven of the studies were performed in largely ethnically homogenous populations (Bailey, Unpublished report to AIDSMARK, 2002; Bailey et\u00a0al., 2002; Halperin et\u00a0al., 2005; Mattson et\u00a0al., 2005; Nnko et\u00a0al., 2001; Scott et\u00a0al., 2005; Tsela & Halperin, 2006), while the remaining studies implemented specific strategies to ensure an ethnically mixed sample (Bailey et\u00a0al., 1999; Kebaabetswe et\u00a0al., 2003; Lagarde et\u00a0al., 2003; Lukobo & Bailey, Submitted; Ngalande et\u00a0al., 2006; Rain-Taljaard et\u00a0al., 2003). Ten of 13 studies included both male and female participants (Bailey, Unpublished report to AIDSMARK, 2002; Bailey et\u00a0al., 2002; Kebaabetswe et\u00a0al., 2003; Lagarde et\u00a0al., 2003; Lukobo & Bailey, Submitted; Mattson et\u00a0al., 2005; Ngalande et\u00a0al., 2006; Nnko et\u00a0al., 2001; Rain-Taljaard et\u00a0al., 2003; Scott et\u00a0al., 2005). The remaining three studies were restricted to males (Bailey et\u00a0al., 1999; Halperin et\u00a0al., 2005; Tsela & Halperin, 2006). Only two studies addressed acceptability in adolescent populations separately from adults (Nnko et\u00a0al., 2001; Rain-Taljaard et\u00a0al., 2003). Three studies purposely included the participation of female sex workers (Bailey et\u00a0al., 2002; Lukobo & Bailey, Submitted; Ngalande et\u00a0al., 2006), and four studies included the opinions of MC providers in the assessment of circumcision acceptability\/promotion (Bailey, Unpublished report to AIDSMARK, 2002; Bailey et\u00a0al., 2002; Rain-Taljaard et\u00a0al., 2003; Scott et\u00a0al., 2005). Nine studies included both rural and urban populations (Bailey, Unpublished report to AIDSMARK, 2002; Bailey et\u00a0al., 2002; Kebaabetswe et\u00a0al., 2003; Lagarde et\u00a0al., 2003; Lukobo & Bailey, Submitted; Mattson et\u00a0al., 2005; Ngalande et\u00a0al., 2006; Nnko et\u00a0al., 2001; Tsela & Halperin, 2006); one study limited participation to rural groups only (Scott et\u00a0al., 2005); and three studies were restricted to urban groups (Bailey et\u00a0al., 1999; Halperin et\u00a0al., 2005; Rain-Taljaard et\u00a0al., 2003). All the studies were conducted in areas where circumcision is not traditionally practiced. Two purposely also included at least one area where most men are circumcised (Lukobo & Bailey, Submitted; Ngalande et\u00a0al., 2006). Ten studies assessed the circumcision status of male participants (Bailey, Unpublished report to AIDSMARK, 2002; Bailey et\u00a0al., 1999; Halperin et\u00a0al., 2005; Kebaabetswe et\u00a0al., 2003; Lagarde et\u00a0al., 2003; Mattson et\u00a0al., 2005; Nnko et\u00a0al., 2001; Rain-Taljaard et\u00a0al., 2003; Scott et\u00a0al., 2005; Tsela & Halperin, 2006) and all studies allowed participation regardless of circumcision status.\nSummary of Quantitative Results of Acceptability\nEight of the 13 studies reviewed included quantitative assessments of the acceptability of MC in six countries using interview questionnaires. Results are summarized in Fig.\u00a02 and Table\u00a02. Four of the eight studies included women respondents. Willingness of uncircumcised men to become circumcised varied from 29% in Uganda to 87% in Swaziland. The variation depended in part on how the question was posed and the context of the study. For example, one of the highest acceptability levels (81%) was recorded in Botswana after an informational session in which participants were told about the health benefits and risks associated with the procedure (Kebaabetswe et\u00a0al., 2003). In some studies, adults were asked if they would be circumcised or prefer their partner to be circumcised \u201cif MC were proven to be protective against HIV and STIs\u201d (Halperin et\u00a0al., 2005; Lagarde et\u00a0al., 2003; Rain-Taljaard et\u00a0al., 2003; Tsela & Halperin, 2006). In others, participants were asked if they would accept MC \u201cif it were safe and affordable\u201d (Bailey et\u00a0al., 1999; Kebaabetswe et\u00a0al., 2003; Mattson et\u00a0al., 2005; Scott et\u00a0al., 2005).\nFig.\u00a02Levels of male circumcision (MC) acceptability from eight quantitative studies in six sub-Saharan African countriesTable\u00a02Circumcision preference and conditions for acceptability reported in eight studies from six sub-Saharan African countriesAuthors\/year\/country% Of uncircumcised men willing to be circumcised% Of women favoring circumcision of their partners% Of men willing to circumcise their sons% Of women willing to circumcise their sonsKebaabetswe et\u00a0al. (2003)\/Botswana61% Before and 81% after information session, if procedure is done in safe hospital settings and is free50% Before and 79% after information session67% Before and 90% after information session, if procedure is done in safe hospital settings and is free 62% Before and 90% after information session, if procedure is done in safe hospital settings and is freeMattson et\u00a0al. (2005)\/Kenya70%, If procedure involved minimal cost and little pain69% 74%, If procedure was safe and affordable (Bailey, Muga, & Poulussen, 2000)89%, If little pain was involvedLagarde et\u00a0al. (2003)\/South Africa73%, If MC protected from STIs\/HIV47% Thought most women preferred circumcised men71% Of non-circumcised men and 82% of circumcised men, if MC protected from STIs\/HIV70%, If MC protected from STIs\/HIVScott et\u00a0al. (2005)\/South Africa51%, If performed safely and at low cost68% 50% 73% Rain-Taljaard et\u00a0al. (2003)\/South Africa59%, If MC reduced chances of STIs and HIVN\/aN\/aN\/aTsela and Halperin (2006)\/Swaziland54%; 87%, If MC protected against HIV\/STIsN\/a71% N\/aBailey et\u00a0al. (1999)\/Uganda29%, If cost was minimalN\/aN\/aN\/aHalperin et\u00a0al. (2005)\/Zimbabwe45% If MC protected against HIV\/STIs, and was safe and affordableN\/aN\/aN\/a\nIn general, approximately the same proportion of women would prefer circumcision for their partners or their sons as men would prefer circumcision for themselves or their sons. In Botswana, Kenya, South Africa and Swaziland, where men or women were asked about circumcision for their sons, more adults would agree to the procedure for their child than for their spouse or themselves. Approximately 75% of parents would seek circumcision for their son if it was safe, affordable and shown to be protective against HIV and STIs.\nAcross studies, the median proportion of uncircumcised men willing to become circumcised was 65% (range 29\u201387%). Sixty nine percent (range 47\u201379%) of women favored circumcision for their partners, and 71% (50\u201390%) of men and 81% (70\u201390%) of women were willing to circumcise their sons. The study restricted to rural population found that 51% of men were willing to become circumcised, while median proportion in the same category was 45% (range 29\u201359%) in three urban studies and 77% (70\u201387%) in studies that included both rural and urban population.\nBarriers to the Acceptability of MC\nPain\nApprehension about pain during and after the procedure was reported to be the major barrier to MC acceptability in most studies (Bailey et\u00a0al., 2002; Kebaabetswe et\u00a0al., 2003; Lukobo & Bailey, Submitted; Mattson et\u00a0al., 2005; Ngalande et\u00a0al., 2006; Scott et\u00a0al., 2005). Participants belonging to non-circumcising ethnic groups were familiar with the circumcision practices in neighboring circumcising tribes where pain was a key characteristic of the procedure. As a rite of passage to becoming a man, the endurance of the pain from circumcision is often an integral aspect of the ceremony. For example, of 108 circumcised participants in South Africa, 42.6% described the traditional procedure as \u201cvery painful\u201d, 34.4% as \u201cmildly painful\u201d, and 18.5% as \u201cnot painful\u201d (Lagarde et\u00a0al., 2003).\nCulture and Religion\nLack of circumcision was mentioned as an element of the ethnic identity of those who do not circumcise traditionally. However, remaining with one\u2019s foreskin is not considered crucial to one\u2019s own ethnic identity. It serves as an ethnic marker primarily used by others. In both Botswana and Swaziland studies, only 2% of participants, for example, felt that circumcision would lead to disapproval by their community (Kebaabetswe et\u00a0al., 2003; Tsela & Halperin, 2006), although in Botswana 22% cited \u201ccultural reasons\u201d as a factor in their decision not to circumcise their male child (Kebaabetswe et\u00a0al., 2003). It is fundamentally different from belonging to an ethnic group that does practice traditional circumcision. For the Yao in Malawi, for example, or the Lunda and Luvale tribes in Zambia, or the Bagisu in Uganda (Bailey et\u00a0al., 1999; Lukobo & Bailey, Submitted; Nnko et\u00a0al., 2001), it is unacceptable to remain uncircumcised, to the extent that forced circumcisions of older boys are not uncommon.\nIn some ethnic groups in which circumcision is not commonly practiced, disapproval of circumcision is evident in the existence of a derogatory term for a circumcised man or a man with a congenitally shortened prepuce. These terms include \u201crayuom\u201d in DhoLuo (Bailey et\u00a0al., 2002) and \u201cnjilwa\u201d in the Sukuma language (Nnko et\u00a0al., 2001). In ethnically homogenous areas, circumcision could lead to rejection by local women and serve as a barrier to marriage (Bailey et\u00a0al., 2002; Lukobo & Bailey, Submitted). In more ethnically diverse areas, however, circumcision among traditionally non-circumcising peoples could be held as a positive, increasing a man\u2019s chances of being accepted by the women of the surrounding circumcising groups.\nReligion is a major determinant of circumcision acceptability. MC is universally associated with Islam. It is also considered fundamental to some minority Christian and animist sects. There was no clear consensus on compatibility of MC with Christian beliefs (Bailey et\u00a0al., 1999; Lukobo & Bailey, Submitted; Ngalande et\u00a0al., 2006; Nnko et\u00a0al., 2001; Rain-Taljaard et\u00a0al., 2003; Scott et\u00a0al., 2005). Great variability in perceptions of Christian churches\u2019 positions on MC was described by different study populations, ranging from condemning MC as a pagan practice (Rain-Taljaard et\u00a0al., 2003) to viewing MC as consistent with Christian tradition according to the Bible and Jesus\u2019 circumcision status (Lukobo & Bailey, Submitted). In South Africa 38% of circumcised and 32% of uncircumcised study participants described circumcision as \u201cforbidden\u201d by their religion (Lagarde et\u00a0al., 2003). Sukuma study participants in Tanzania felt that the Christian religion did not theologically promote MC, while circumcision services were known to be available in church-run hospitals (Nnko et\u00a0al., 2001). Lukobo and Bailey describe the prevalent Zambian perception of circumcision being linked with Muslim or animist Chawa heritage, with several participants also reporting the belief that Christians should practice MC since Jesus was circumcised and the Bible teaches the practice (Lukobo & Bailey, Submitted). Similar findings were reported by Ngalande et\u00a0al. in Malawi (Ngalande et\u00a0al., 2006). In Kenya the Nomiya Church and a few other small Christian sects require circumcision for church membership (Mattson et\u00a0al., 2005).\nRain-Taljaard and colleagues report the South African belief that circumcision is fundamentally an African tradition and that Western ideas concerning the practice should not be taken seriously. Further, participants stated that many Christian churches opposed circumcision as a pagan tradition (Rain-Taljaard et\u00a0al., 2003). However, it was unclear whether this opposition was directed at circumcision itself or at rites and ceremonies with which it was associated.\nBefore MC is promoted in a country, it would be prudent to consult and collaborate with religious leaders to learn the stance of the various churches regarding MC. In many cases, churches can act as helpful advocates or obstructive opponents and may have significant influence on acceptability of MC.\nCost\nThe cost of the procedure was a significant barrier to MC acceptability by participants in many studies (Bailey, Unpublished report to AIDSMARK, 2002; Bailey et\u00a0al., 2002; Lagarde et\u00a0al., 2003; Lukobo & Bailey, Submitted; Mattson et\u00a0al., 2005). Some participants expressed the opinion that if circumcision were promoted by the government, it should be provided at health clinics and hospitals for free or at reduced cost (Bailey et\u00a0al., 2002; Lukobo & Bailey, Submitted; Ngalande et\u00a0al., 2006). Others recognized the need to pay for services because a free circumcision was viewed as being of potentially poor quality (Ngalande et\u00a0al., 2006). Male and female participants in Zambia believed that, if the MC procedure were free or extremely inexpensive, more men would be willing to get circumcised (Lukobo & Bailey, Submitted). In one study as many as 34% of participants who initially stated that their preference was to remain uncircumcised changed their minds when the proposed cost of the procedure was set at US$3.00 (Mattson et\u00a0al., 2005). Cost of traditional circumcision was considered to be high in many areas and there is a gradual shift from traditional to medical circumcision in part for this reason (Bailey & Egesah, 2006; Lukobo & Bailey, Submitted; Ngalande et\u00a0al., 2006; Rain-Taljaard et\u00a0al., 2003). Traditional circumcision is often expensive due to the costs of food, drink, special clothing and other items required during a sometimes prolonged celebration.\nComplications and Adverse Effects\nIf men and parents believe that circumcision leads to high rates of complications, then uptake of MC is likely to be slow. Concerns for safety were universal in the studies examined. Mothers were vocal in their concerns, especially in cases of infant and early childhood circumcision. Excessive bleeding was a major concern and this fear was heightened if the procedure was to be performed by a traditional circumciser outside the hospital setting (Bailey et\u00a0al., 2002; Lukobo & Bailey, Submitted; Ngalande et\u00a0al., 2006). Infection and difficulty in healing were expressed as concerns as well, but were generally believed to be minimized in clinical settings (Bailey et\u00a0al., 2002; Lukobo & Bailey, Submitted; Ngalande et\u00a0al., 2006). Using the same knife for several boys was believed to be common in traditional settings and a source of infections, including HIV (Bailey et\u00a0al., 2002; Halperin et\u00a0al., 2005; Lagarde et\u00a0al., 2003; Ngalande et\u00a0al., 2006; Rain-Taljaard et\u00a0al., 2003). Women were especially opposed to circumcision at the traditional initiation schools, as they feared that their children may be injured or die during the process (Rain-Taljaard et\u00a0al., 2003).\nOverall, there seemed to be a great deal of trust in medical practitioners and a strong preference for circumcision services to be made available in public health facilities by trained health professionals.\nPotential for Behavioral Disinhibition\nIf men and their partners believe that circumcision offers protection from HIV infection, they may be less inhibited (\u201cdisinhibited\u201d) in their sexual activities and engage in higher HIV risk behaviors, thereby mitigating a partially protective effect of MC. Fortunately, the perception that MC provides full protection against HIV and STIs was found to be generally rare, but it was expressed by a few study participants in South Africa and in Nyanza Province, Kenya (Bailey et\u00a0al., 2002; Rain-Taljaard et\u00a0al., 2003). In focus groups in Kenya, Malawi and Zambia a concern about the possibility of behavioral disinhibition was inevitably expressed. Most participants did seem to appreciate the concept of risk reduction opposed to risk elimination (Bailey et\u00a0al., 2002; Lukobo & Bailey, Submitted; Ngalande et\u00a0al., 2006). Similarly in Swaziland 87% of study participants advocated having only one partner and 94% promoted condom use for circumcised men (Tsela & Halperin, 2006).\nThere is some evidence of behavioral disinhibition among circumcised men. A study in South Africa found a significant association between circumcision status and the higher reported number of non-spousal lifetime partners (Lagarde et\u00a0al., 2003). Circumcised men in Uganda were found to engage in more HIV risk behaviors than uncircumcised men (Bailey et\u00a0al., 1999). In addition to reporting more extramarital partners in the previous year (1.13 vs. 0.62, P\u00a0<\u00a00.01), circumcised men had an overall higher \u201crisk profile\u201d. A few respondents in another South African study expressed the belief that MC potentially encouraged adultery as newly circumcised men were curious to test the new shape of the penis (Rain-Taljaard et\u00a0al., 2003).\nOne study in Botswana found that participants felt that circumcision before the age of six years may help to avoid a change in sexual behavior associated with sense of increased protection due to circumcision (Kebaabetswe et\u00a0al., 2003). Men attending beer halls in Harare, Zimbabwe were aware of the partial protection against HIV provided by MC, and had a good understanding of the limitations and the concept of risk reduction (Halperin et\u00a0al., 2005).\nOther Reasons Not to Circumcise\nOther barriers to circumcision, mentioned by participants, were lack of access to health care, required time away from work, the loss of penile sensitivity, reduction in penis size, decreased ability to satisfy women, excessive sexual desire, increased promiscuity (Bailey et\u00a0al., 2002; Rain-Taljaard et\u00a0al., 2003), and the perception of circumcision as old-fashioned (Lagarde et\u00a0al., 2003; Rain-Taljaard et\u00a0al., 2003).\nFacilitators of MC Acceptability\nHygiene\nPenile hygiene was universally recognized as being extremely important and was viewed as a major benefit of circumcision (Bailey et\u00a0al., 2002; Halperin et\u00a0al., 2005; Kebaabetswe et\u00a0al., 2003; Lukobo & Bailey, Submitted; Mattson et\u00a0al., 2005; Ngalande et\u00a0al., 2006; Nnko et\u00a0al., 2001). A great majority of participants, both male and female from multiple studies, agreed that it was much easier for a circumcised man to maintain cleanliness (Bailey et\u00a0al., 2002; Lukobo & Bailey, Submitted; Mattson et\u00a0al., 2005; Ngalande et\u00a0al., 2006; Nnko et\u00a0al., 2001; Rain-Taljaard et\u00a0al., 2003).\nThe majority of participants, including women, believed that it was worrisome that men do not maintain proper hygiene. Because women were the primary providers of water, poor penile hygiene was often seen as a woman\u2019s failing (Bailey et\u00a0al., 2002; Lukobo & Bailey, Submitted). In both Zambia and Malawi women were considered responsible for cleaning their partners\u2019 penises after sexual intercourse. Additionally, women in these populations linked their own risk of STIs to their partners\u2019 genital hygiene (Lukobo & Bailey, Submitted; Ngalande et\u00a0al., 2006). Ease of maintaining proper penile hygiene proved a major factor in women\u2019s acceptability of circumcision (Bailey et\u00a0al., 2002; Lukobo & Bailey, Submitted; Mattson et\u00a0al., 2005; Ngalande et\u00a0al., 2006).\nProtection from STIs and HIV\nHygiene as a mechanism of protection from STIs was mentioned by a great number of participants (Bailey et\u00a0al., 2002; Lukobo & Bailey, Submitted; Ngalande et\u00a0al., 2006). It was held that germs, dirt, bacteria, and viruses had a greater opportunity to proliferate in the warm moist environment beneath the foreskin (Bailey et\u00a0al., 2002; Lukobo & Bailey, Submitted; Ngalande et\u00a0al., 2006; Nnko et\u00a0al., 2001; Rain-Taljaard et\u00a0al., 2003). Participants also expressed a belief that it would be easier to detect rashes and\/or ulcerations with the foreskin removed allowing for earlier treatment (Bailey et\u00a0al., 2002; Ngalande et\u00a0al., 2006). The foreskin was also perceived as a portal of entry for sexually transmitted infection as the tissue is considered prone to traumatic injury during sexual intercourse (Bailey et\u00a0al., 2002; Lukobo & Bailey, Submitted; Ngalande et\u00a0al., 2006; Rain-Taljaard et\u00a0al., 2003). MC was recognized as a medical procedure to reduce or eliminate penile ulcerations and diseases of the penis (Bailey et\u00a0al., 2002; Lukobo & Bailey, Submitted; Ngalande et\u00a0al., 2006; Nnko et\u00a0al., 2001). Conversely, a minority of respondents in Zambia reported that the circumcised penis was \u201calways dry\u201d, \u201csusceptible to cracking\u201d, and that this state provided a portal of entry for bacteria and viruses (Lukobo & Bailey, Submitted).\nSeventy percent of Botswana study participants willing to circumcise their male child listed protection from STIs or HIV among their reasons for doing so (Kebaabetswe et\u00a0al., 2003). In Nyanza Province, Kenya, 79% of uncircumcised men and 81% of women believed that it was easier for uncircumcised men to acquire STIs compared to circumcised men. This belief dropped to 43% and 60%, respectively, concerning the acquisition of AIDS (Mattson et\u00a0al., 2005). In Swaziland, 81% of participants stated that MC reduced risk of STIs and 18% believed that MC reduced risk of HIV (Tsela & Halperin, 2006). In Tanzania STIs were considered more severe and more infective in uncircumcised men, with ulcers healing faster in those who are circumcised (Nnko et\u00a0al., 2001). Nearly all commercial sex workers believed that there exists a strong association between lack of circumcision and STIs, including HIV (Ngalande et\u00a0al., 2006). In South Africa (Scott et\u00a0al., 2005), no association was found between willingness to be circumcised and perceived health benefits. It was belief about sexual pleasure that was the strongest predictor of being willing to undergo circumcision.\nAcceptability by Other Ethnic Groups\nCommon reasons given for favoring MC were the social, political, and sexual benefits that could accrue when interacting with those in predominantly circumcising groups (Bailey et\u00a0al., 2002; Lukobo & Bailey, Submitted; Ngalande et\u00a0al., 2006). The Luo believed that they were often discriminated against by other Kenyans due to their circumcision status which led to political exclusion and even security concerns in times of social upheaval (Bailey et\u00a0al., 2002). Many younger men from traditionally non-circumcising groups cited being accepted as a sexual or marriage partner by women from other ethnic groups as an important reason to be circumcised (Bailey et\u00a0al., 2002; Lukobo & Bailey, Submitted; Ngalande et\u00a0al., 2006; Nnko et\u00a0al., 2001).\nSexual Pleasure Among Circumcised versus Uncircumcised\nHow circumcision is perceived to influence sexual drive, sexual performance, and sexual pleasure for the man himself or for his partner is likely to influence decision making around MC. Participants in many studies believed that circumcision enhances sexual pleasure (Bailey, Unpublished; Bailey et\u00a0al., 2002; Lagarde et\u00a0al., 2003; Lukobo & Bailey, Submitted; Mattson et\u00a0al., 2005; Ngalande et\u00a0al., 2006; Nnko et\u00a0al., 2001; Rain-Taljaard et\u00a0al., 2003).\nMost studies assessed three factors associated with sexual activity based on circumcision status: sexual performance, sexual pleasure for men, and sexual pleasure for women. Fifty percent of circumcised and 30% of uncircumcised participants in South Africa believed that MC increased sexual performance, while only 21% and 14%, respectively, believed that MC decreased sexual pleasure (Lagarde et\u00a0al., 2003). Other studies found that a high proportion of men and a majority of women believed that circumcised men enjoyed sex more than uncircumcised men (Mattson et\u00a0al., 2005; Rain-Taljaard et\u00a0al., 2003). About half of female participants reported preference for circumcised men (Lagarde et\u00a0al., 2003; Mattson et\u00a0al., 2005). Many had no preference. A study in South Africa found that men were 8 times more likely to prefer circumcision if they believed that circumcised men enjoyed sex more, and 6 times more likely to prefer circumcision if they believed that women enjoy sex more with circumcised men (Scott et\u00a0al., 2005). Other studies did not find a consensus about circumcision status and sexual pleasure on the part of the man or the woman (Bailey et\u00a0al., 2002; Lukobo & Bailey, Submitted; Ngalande et\u00a0al., 2006). For some, circumcision was irrelevant to pleasure, as pleasure was more related to emotional attachment and past sexual experience (Lukobo & Bailey, Submitted; Ngalande et\u00a0al., 2006). Attitudes about circumcision and pleasure may be different in areas where dry sex is practiced (Lukobo & Bailey, Submitted).\nOther Reasons to Circumcise\nOther reasons to be circumcised reported by participants included the belief that it was easier for circumcised men to use condoms (Bailey, Unpublished report to AIDSMARK, 2002; Bailey et\u00a0al., 2002; Kebaabetswe et\u00a0al., 2003), that MC proved manhood, that aim during urination was improved, and that not being circumcised brought bad luck (Rain-Taljaard et\u00a0al., 2003).\nTime and Setting of Circumcision Procedure\nPreferred Age at Circumcision\nThe ages at which males become circumcised will have an effect on how rapidly MC interventions may impact the HIV epidemic in any given area. Preferred age at circumcision varied both between and within studies. There appeared to be two leading directions exhibited by many studies: either circumcise males as babies due to a simpler procedure, less fear, easier care, and faster healing, or circumcise males around puberty and adolescence when boys can decide and take care of the wound for themselves (Bailey et\u00a0al., 2002; Lukobo & Bailey, Submitted; Ngalande et\u00a0al., 2006; Rain-Taljaard et\u00a0al., 2003; Scott et\u00a0al., 2005).\nAmong the nine countries where acceptability studies have been undertaken, only in Botswana were most participants in favor of circumcision in infancy and early childhood. Fifty-five percent of respondents were in favor of circumcising children under 6\u00a0years old with half of those preferring neonatal circumcision (Kebaabetswe et\u00a0al., 2003). In all other areas a significant minority were in favor of infant or early childhood MC, but most favored circumcision between ages 8\u201316\u00a0years with very few saying that over 18\u00a0years was best. Those who advocated for infant circumcision did so for reasons relating to decreased pain during the procedure and faster healing times (Bailey et\u00a0al., 2002; Lukobo & Bailey, Submitted), although babies under 1\u00a0year of age were thought to experience excessive pain, leading to crying and fevers (Lukobo & Bailey, Submitted). Participants from Malawi viewed infants especially vulnerable to potential complications of MC due to \u201clack of maturity\u201d and difficulty of timely detection of bleeding due to babies being carried on the mothers\u2019 backs (Ngalande et\u00a0al., 2006).\nMany studies reported strong beliefs among participants that circumcision should take place before the onset of sexual activity (Lukobo & Bailey, Submitted; Ngalande et\u00a0al., 2006; Rain-Taljaard et\u00a0al., 2003). Ages 7\u201313\u00a0years were thought to be best since the boy could make the decision for himself, understand the significance of the event, take care of the wound himself, heal faster than if done post-pubertally, and has likely not begun sexual activity (Bailey et\u00a0al., 2002; Lukobo & Bailey, Submitted; Ngalande et\u00a0al., 2006; Rain-Taljaard et\u00a0al., 2003).\nCircumcision as an adult or post-pubertally was reported by many to be undesirable due to higher risk of complications, pain during the procedure (Ngalande et\u00a0al., 2006; Rain-Taljaard et\u00a0al., 2003), and painful erections after MC, leading to complications and delays in healing (Bailey et\u00a0al., 2002; Lukobo & Bailey, Submitted; Ngalande et\u00a0al., 2006).\nMany people from traditionally non-circumcising communities felt that they had insufficient knowledge to make a decision about when best to circumcise. They preferred to consult clinical professionals to get their advice (Bailey et\u00a0al., 2002; Ngalande et\u00a0al., 2006). Practitioners interviewed in Kenya and Malawi preferred not to perform neonatal circumcision due to the small size of the penis and foreskin, potentially leading to higher rates of errors and complications. These providers preferred to perform the operation at ages 8\u201312\u00a0years (Bailey et\u00a0al., 2002).\nPreferred Circumcisers\nIn areas where traditional circumcision is uncommon, the preference is overwhelmingly for a medical practitioner to be the provider. All studies reported fear of infection, bleeding, excessive pain, and possible mutilation at the hands of traditional circumcisers (Kebaabetswe et\u00a0al., 2003; Lagarde et\u00a0al., 2003; Lukobo & Bailey, Submitted). In Zambia (Lukobo & Bailey, Submitted), even in the traditionally circumcising area of Zambezi District, the majority believed medical doctors to be experienced, more apt to use sterile equipment, able to minimize pain through anesthesia, and capable of dealing with complications. The few participants who preferred traditional surgeons viewed these practitioners as more experienced and more willing to maintain confidentiality (Lukobo & Bailey, Submitted).\nScott et\u00a0al. found that 77% of male Zulu preferred MC by a doctor or medical surgeon, 8% by a nurse, 11% by traditional circumciser, and 3% by other providers (Scott et\u00a0al., 2005). Another study based in South Africa observed that MC was commonly performed in both \u201cinitiation schools\u201d and by clinical providers. The more common circumcision was in an ethnic group, the less likely it was done in medical settings (Rain-Taljaard et\u00a0al., 2003).\nAcceptability in Certain Populations\nWomen\u2019s Beliefs and their Influence\nThe influence of women on the decision to circumcise is likely to be highly variable across cultures and across families within communities. However, in many settings, women, as mothers and as partners, are likely to have considerable influence, even if it is not overt. Any effort to promote MC will be more successful if it appeals to women as well as men.\nBailey et\u00a0al. (2002) found that women\u2019s beliefs may have a strong influence on male acceptability of circumcision in western Kenya. This influence may stem from women\u2019s strong emphasis on penile hygiene for their partners, and the wish to protect their young sons from acquisition of infections as they become sexually active. Scott et\u00a0al. (2005), on the other hand, suggested that in South Africa women are likely to have only an indirect influence through the male perception that women enjoy sex more with circumcised men. A different study from South Africa found that women had a strong influence on men\u2019s decision to circumcise, often scheduling the appointment for their boyfriends or husbands. Single mothers, however, were believed to have no influence over their teenage sons\u2019 decisions to circumcise (Rain-Taljaard et\u00a0al., 2003). Thirteen percent of circumcised participants in yet another South African study (Lagarde et\u00a0al., 2003) reported undergoing circumcision because their partner expressly requested it.\nAcceptability in Youth\nTwo out of thirteen studies assessed acceptability of MC among adolescents. In Tanzania, school aged boys and girls believed that it was easier for an uncircumcised man to acquire STIs, that it was easier for a circumcised man to maintain proper genital hygiene, and that circumcision enhanced sexual pleasure for both partners (Nnko et\u00a0al., 2001). As in most areas, adolescent boys linked circumcision with modernity and good hygiene. Overall, adolescent males and females proved to be knowledgeable about potential benefits of MC (Nnko et\u00a0al., 2001; Rain-Taljaard et\u00a0al., 2003). Nnko et\u00a0al. (2001) observed that knowledge and a positive attitude about MC became most obvious in secondary schools due to the effects of increased ethnic mixing.\nMany studies found that younger participants were more likely to view circumcision favorably than their elders (Lukobo & Bailey, Submitted; Mattson et\u00a0al., 2005; Ngalande et\u00a0al., 2006; Nnko et\u00a0al., 2001). In Botswana, only 43% of men ages 45\u201359\u00a0years were willing to be circumcised, compared to 65% of 25\u201334\u00a0year-olds (Kebaabetswe et\u00a0al., 2003). In Kenya, younger men were more likely to accept circumcision. Among those 16\u201321\u00a0years old, 71% said that they would prefer to be circumcised; whereas only 56% of those over 21\u00a0years preferred to be circumcised (Bailey, 2001). Results from more qualitative studies entailing focus group discussions were consistent with these quantitative results. Younger men in Zambia, Malawi and Tanzania were more likely to express a desire to be circumcised (Lukobo & Bailey, Submitted; Ngalande et\u00a0al., 2006; Nnko et\u00a0al., 2001).\nHypothetical versus Actual Acceptability\nAsking people whether they might prefer to be circumcised under various hypothetical scenarios (e.g., if it is found to reduce risk of HIV acquisition; or if it is at minimal cost and safe) is one means of assessing acceptability. A more realistic means is to discover where MC services are available and see who takes advantage of the services. Alternatively, one can offer the services in non-circumcising communities and see the response. This approach permits assessment of not just numbers seeking the services, but also the ages and population segments that respond as well as factors that inhibit or facilitate uptake of the services.\nA trial intervention in Siaya District, Kenya\u2013an area where circumcision is not traditionally practiced\u2014was introduced in 1999 (Bailey, Unpublished report to AIDSMARK, 2002). During a 25\u00a0month period, 433 circumcisions were performed in health facilities where only 6 procedures had been done in the previous year. In a comparison district, where no intervention was available, just 24 circumcisions were preformed over the same period. Demand for MC services was judged to be high but was highly dependent on cost. When the price charged for a circumcision was reduced from $3.62US to $1.45US, demand surged, and 50% of all circumcisions occurred during the 2\u00a0months when the price was reduced. The median age of those circumcised was 18\u00a0years; 25% were below age 12\u00a0years, and an estimated 35% were circumcised before their sexual debut. The researchers felt that a greater number of younger males would have been circumcised had parental permission not been required for those under age 18\u00a0years and if the cost were reduced permanently, since older males tended to have more financial support (Bailey, Unpublished report to AIDSMARK, 2002). The results from this trial intervention are consistent with results from studies of hypothetical acceptability indicating that cost is consistently found to be a major barrier to uptake of circumcision in traditionally non-circumcising communities.\nFurther evidence of acceptability comes from the one RCT completed in Orange Farm, South Africa and the two ongoing trials in Kisumu, Kenya and Rakai, Uganda, both of which have completed enrollment. Because every participant in these three trials stands a 50% chance of being circumcised immediately upon randomization, all of them must prefer to be circumcised in order to enroll in the study. The Orange Farm trial screened 3,483 young men, ages 18\u201324\u00a0years. We do not know what proportion of the total population of 18\u201324\u00a0year olds these men represent. However, that all but 203 (5.8%) of the men consented to enroll indicates that acceptability was high (Auvert et\u00a0al., 2005). In Kisumu, Kenya, a community in which 90% of adult men are uncircumcised, 6,686 of the 34,200 (19.5%) uncircumcised men in the population ages 18\u201324\u00a0years came to the study clinic seeking to enroll in the study. Of these, 4,489 (67.1%) were eligible to enroll, and of those eligible, 68.5% accepted to be randomized (Bailey, 2006). This acceptability rate agrees very closely with the 70% figure found in the sample by Mattson et\u00a0al. (2005) from the same area. In Rakai, Uganda, a rural community in which 83% of adult men are uncircumcised, approximately 45% of all eligible HIV uninfected men in the community enrolled in the trial before enrollment was closed (R. Gray, personal communication). That such large numbers of men are willing to join these trials suggests that circumcision acceptability is high and that uptake of MC in these communities could be rapid, if sufficient resources are available to accommodate large numbers of procedures.\nDiscussion\nThrough searching electronic databases and contacting authors, we identified 13 studies from nine countries that include investigation of the acceptability of MC in traditionally non-circumcising regions in sub-Saharan Africa. We found one additional report of a pilot intervention introducing MC services into health facilities where circumcision was little practiced. The level of acceptability across the nine countries appears greater than might be expected, considering that all thirteen communities where the studies were performed were all traditionally non-circumcising. The lowest level of acceptability by uncircumcised men (29%) was reported from eastern Uganda in a study conducted in 1997, before MC became well recognized as possibly being associated with STIs and HIV (Bailey et\u00a0al., 1999). More than half of men in the regions studied appear to be receptive, if not eager, to become circumcised.\nCost, fear of pain, and concern for safety were the three most consistent barriers to acceptability of MC. In communities where circumcision is the norm families expect to incur the obligatory circumcision expenses negating the importance of cost. In non-circumcising communities circumcision is regarded as a voluntary procedure that may be unlikely to take precedence over competing needs. Cost is viewed as including not only the payment for the procedure, but also the opportunity costs of time away from work and other income generating activities. Cost as a primary consideration was shown dramatically by the pilot intervention in Siaya, Kenya, where men came in large numbers when the charges were lowered to $1.45US (Bailey, Unpublished report to AIDSMARK, 2002). These results indicate that the true cost of the procedure will have to be supplemented to achieve significant uptake of MC.\nThe concerns for safety and pain are based partially on the perception of circumcision as a surgical procedure with inherent risks and partially on the occasional press releases publicizing mutilations and deaths. Personal knowledge of neighboring communities where traditional initiates withstand excruciating pain also likely plays a role. Sustained uptake of MC will require performance of the procedure with minimal adverse events. This can be achieved through proper training and supervision of practitioners, proper instrumentation and sterilization, complete instructions to patients, follow-up with patients, and over all attention to quality control (Krieger et\u00a0al., 2005).\nThe studies we reviewed revealed that it is virtually universal that Africans equate circumcision with improved hygiene. Also widespread is the belief that circumcision leads to reduced incidence of STIs achieved through improved hygiene, reduction in the number and severity of scratches, tears and abrasions to which the foreskin is susceptible and through earlier detection of ulcers, leading to earlier treatment. Although not as frequent, a significant proportion of participants in the studies also saw circumcision leading to reduced risk of HIV acquisition through the same route. If MC is proven in the remaining two clinical trials to reduce incidence of HIV and some STIs (e.g., HPV, HSV-2, chancroid and gonorrhea), this information will be consistent with the already existing beliefs of most sub-Saharan Africans.\nCultural norms, ethnic identity, and religious affiliation were viewed as central factors in acceptability of circumcision. Circumcision was associated with specific traditionally circumcising communities and with Muslims and members of a few minority Christian and animist sects. It will likely be important that confidentiality is maintained by circumcision practitioners, since stigmatization for being circumcised is a possibility in non-circumcising communities. An important conclusion reached by several studies was that circumcision was increasingly an issue of personal choice rather than ethnic identity (Rain-Taljaard et\u00a0al., 2003; Scott et\u00a0al., 2005). Urbanization, ethnic mixing, and exposure to other cultures and religions are conducive to higher acceptability of circumcision in traditionally non-circumcising ethnic groups.\nIn East and Southern Africa most MCs are done between ages 8 and 21 and the preferences for age at circumcision found in studies are consistent with these practices. However, a large enough proportion of people, especially mothers, preferred infant circumcision to consider making infant circumcision an available option. This should be an important consideration in designing MC interventions.\nInformation campaigns may be effective in increasing acceptability of MC. This was found to be true in Botswana and South Africa (Kebaabetswe et\u00a0al., 2003; Scott et\u00a0al., 2005). However, many studies demonstrated that both knowledge and acceptability of MC varies considerably by region within the same country. Therefore, informational campaigns may be more effective if targeted to particular communities.\nJust as the international health community is concerned about the possibility that promotion of circumcision could lead to increases in risky sexual behavior (World Health Organization, 2005), participants in many of the studies reviewed were similarly concerned. Higher risk behaviors have been found to be associated with circumcision status previously in Uganda, Rwanda and Kenya (Bailey et\u00a0al., 1999; Seed et\u00a0al., 1995; Tyndall et\u00a0al., 1996), as well as in the Orange Farm RCT (Auvert et\u00a0al., 2005). This underlines the importance of the counseling and education that must be provided to men who undergo circumcision, reinforcing the idea of MC reducing, not eliminating, the risk of HIV and other STIs.\nThere are several limitations to the studies that we reviewed. All used convenience sampling to recruit participants. The results could be biased if recruits were more likely to participate if they had a favorable view of MC. This may not be a concern, since most studies had nearly 100% participation by those who were asked to participate. Only two studies verified the circumcision status of the participants (Lagarde et\u00a0al., 2003; Nnko et\u00a0al., 2001) and none of the studies verified MC status of partners of interviewed women. The direction in which this may have biased results is not clear. There were differences across studies in design: some were more qualitative with open ended questions asked in a group discussion setting, others were more quantitative using closed-ended questions during a one-on-one interview. There was variation in the wording of questions to participants about the conditions under which they would accept circumcision. Some studies were geographically restricted and, as a result, may have limited generalizability and lack of representiveness of populations. Geographical coverage was spotty within study countries, and some high HIV prevalence countries where MC is little practiced (e.g., Mozambique, Lesotho, Namibia) were not included. Lastly, there was variation in the time when the studies were conducted (range 1991\u20132006). Attitudes toward circumcision assessed by early studies (Bailey et\u00a0al., 1999; Nnko et\u00a0al., 2001) may have changed since the time of the study.\nAll studies attempted to assess peoples\u2019 beliefs and attitudes toward circumcision and their willingness to be circumcised under some hypothetical conditions sometime in the future. We cannot know from these studies what the actual uptake of circumcision would be if it were found to be protective in three clinical trials and was actively promoted. We have only one example of an introduction of MC services in a traditionally non-circumcising community (Bailey, Unpublished report to AIDSMARK, 2002), and this was at a time when circumcision could not be actively promoted, but could only be made available. Results from that intervention were instructive in that demand for safe circumcision was robust, but depended very much upon price.\nThe results from the thirteen available studies of acceptability of MC in nine countries in sub-Saharan Africa where circumcision is little practiced are very consistent. Acceptability of MC is likely to be high enough to have a significant impact on HIV prevalence in these communities, if MC is proven to have a protective effect similar to that found in observational studies and in the Orange Farm RCT. It is doubtful, given the consistency of results to date, that we will learn a great deal more by additional acceptability studies that pose hypothetical questions to participants. Instead, we recommend pilot interventions making circumcision services available in health facilities after training of clinicians and provision of proper instruments and supplies. There are reports that demand for MC services is already high in many traditionally non-circumcising communities in East and southern Africa (Bangre, 2006; Nnko et\u00a0al., 2001; PlusNews, 2006; Timberg, 2005). There is a danger that this increasing demand will be filled by unqualified practitioners causing unnecessary adverse events. Pilot interventions will serve simultaneously to test whether there truly is a growing niche and, if so, to gain experience in filling the niche with safe, affordable services. At the same time, much will be learned about the operational requirements for training, instrumentation, safety, counseling and follow-up of patients, supervision of staff, monitoring of behavioral disinhibition, and about how MC services can be integrated with HIV\/STIs prevention services, including VCT, STIs diagnosis and treatment, behavioral counseling, condom promotion and anti-retroviral therapies.","keyphrases":["acceptability","male circumcision","africa","hiv-1"],"prmu":["P","P","P","U"]}
{"id":"Int_J_Cardiovasc_Imaging-3-1-2121119","title":"An integrated approach to determine left atrial volume, mass and function in hypertrophic cardiomyopathy by two-dimensional echocardiography\n","text":"Methods The study included 25 hypertrophic cardiomyopathy (HCM) patients (15 non-obstructive and 10 obstructive) and 25 controls for assessment of left atrial (LA) volume, mass and function by two-dimensional echocardiography. Measurement included mean LA diameter (LAD), LA mass = {(mean LAD + anterior LA wall + posterior LA wall)3 \u2212 mean LAD3} \u00d7 0.8 + 0.6, LA volume = [(8\/3 \u03c0 L \u00b7 A1 \u00b7 A2), where L is LA length, A1 and A2 are LA area in 4-chambers and 2-chambers, respectively] including maximum (Vmax), minimum (Vmin), and pre-atrial contraction (Vpre-A), total atrial stroke volume (TA-SV), TA emptying fraction (TA-EF), active atrial SV (AA-SV), AA-EF, passive atrial SV (PA-SV), PA-EF, atrial expansion index (AEI), and LA kinetic energy (LA-KE) = \u00bd \u00d7 AA-SV \u00d7 P \u00d7 V2.\nIntroduction\nHypertrophic cardiomyopathy (HCM) is a relatively common form of genetic heart disease affecting approximately 1 in 500 in the general population [1, 2]. The pathophysiologic appearance of HCM is complex and includes dynamic left ventricular outflow tract (LVOT) obstruction, mitral regurgitation, diastolic dysfunction, myocardial ischemia and cardiac arrhythmia [3]. Diastolic dysfunction is more common than systolic dysfunction in HCM due to marked left ventricular hypertrophy, interstitial fibrosis and myocardial ischemia. These factors contribute to elevated left atrial (LA) and pulmonary vascular pressures [4]. LA modulates left ventricular filling through three components, a phase of reservoir component or expansion during systole, a conduit phase during diastole and an active contractile component during late diastole [5]. The active LA contraction has an important role in patients with reduced left ventricular compliance as it is compensating the decreased early filling [6, 7]. In HCM, stiffness of LA increases and this affects its reservoir function and may in turn affects the cardiac output [8]. LA enlargement is a common findings in HCM and associated with increased morbidity and mortality [9, 10]. The present study aimed to assess LA size, mass and function in HCM patients compared with normal subjects.\nPatients and methods\nA retrospective study included 25 patients (80% males, mean age 38\u00a0\u00b1\u00a015\u00a0years) with an established diagnosis of HCM [11] and good two-dimensional echocardiography (2DE) image quality. According to the type of HCM, patients were classified into two groups the non-obstructive group included 15 patients with resting LVOT gradient <30\u00a0mm\u00a0Hg, and the obstructive group included 10 patients with resting LVOT gradient \u226530\u00a0mm\u00a0Hg. A group of 25 normal age-matched adults (mean age 35\u00a0\u00b1\u00a016\u00a0years) served as control subjects. None of them had evidence of cardiovascular disease by clinical examination, resting ECG and echocardiographic examination.\n2DE was undertaken for HCM patients and control subjects with lying in the left lateral decubitus using both apical and parasternal views. 2DE studies were performed using a 3.5\u00a0MHz transducer and a commercially available ultrasound system (Philips Sonos 7500, Best, The Netherlands). The following measures were obtained.\nLeft ventricular (LV) function\nLV fractional shortening and ejection fraction were selected as a marker for systolic function. LV end-diastolic (LV-EDD) and end-systolic (LV-ESD) dimensions were measured using M-mode from parasternal long axis view and thus LV fractional shortening were calculated by the traditional formula: Fractional shortening (%)\u00a0=\u00a0[LV-EDD\u00a0\u2212\u00a0LV-ESD]\/LV-EDD%. LV end-diastolic (LV-EDV) and end-systolic (LV-ESV) volumes were measured by 2D biplane modified Simpson\u2019s method and then ejection fraction was calculated by the formula: Ejection fraction\u00a0=\u00a0[LV-EDV\u2212LV-ESV]\/LV-EDV. Transmitral E\/A ratio was defined by pulsed wave Doppler and used as a marker of LV diastolic function.\nMitral regurgitation\nMitral regurgitation was defined by colour Doppler and graded according to the maximum regurgitant jet area as mild (jet area <4\u00a0cm2), moderate (jet area 4\u20138\u00a0cm2), and severe (jet area >8\u00a0cm2) [12]\nLA diameter (LAD)\nMaximum LAD was measured at three planes: antero-posterior from parasternal long axis view, medial-lateral and superior-inferior from apical four-chamber view. Then mean LAD (mean LAD) was defined as the average of the three LAD (See Fig.\u00a01)\nFig.\u00a01Measurement of maximum left atrial diameter (LAD) at three planes: (A) antero-posterior LAD from parasternal long axis view, (B) medial-lateral LAD and (c) superior-inferior LAD from apical four-chamber view\nLA mass\nLA mass was calculated with the same formula applied for left ventricular mass [13] {(mean LAD\u00a0+\u00a0anterior LA wall\u00a0+\u00a0posterior LA wall)3\u00a0\u2212\u00a0mean LAD3}\u00a0\u00d7\u00a00.8\u00a0+\u00a00.6. Thickness of anterior and posterior LA walls was measured from parasternal long axis view. Zooming was used to discriminate between posterior LA wall and pericardium (See Fig.\u00a02)\nFig.\u00a02M-mode obtained from parasternal long axis view (A). Zooming was used to measure anterior left atrial wall (B) and posterior left atrial\nLA volume (LAV)\nLAV was assessed according to the formula [6] 8\/3\u00a0\u03c0\u00a0L\u00a0\u00d7\u00a0A1\u00a0\u00d7\u00a0A2 where (L) is the LA longitudinal axis and (A1) is LA area at apical four-chamber and (A2) at apical two-chamber views. (L) was defined as the perpendicular line from mid point of the mitral valve plane to the tip of LA apex (See Fig.\u00a03). LA area was obtained by manual tracing of LA endocardial border excluding LA appendage and the pulmonary veins when visualized. The superior border of atrial outline was a straight line connecting both sides of the leaflet base attachment points. LAV was calculated at three phases of ventricular cardiac cycle: maximum LAV (Vmax) at the end-systole just before mitral valve opening, minimum LAV (Vmin) at end-diastole just before mitral valve closure, and LAV before atrial contraction (VPre-A): the last frame before mitral valve reopening. From the three LAV, the following measurements were selected as indices of LA function and calculated according to previous studies [14, 15]:\nFig.\u00a03Calculation of LAV using 2DE by manual tracing of LA endocardial border at apical four-chamber (A1) and apical two chamber views (A2), L is the long axis, then apply the formula.: 8\/3\u00a0\u03c0\u00a0L\u00a0\u00d7\u00a0A1\u00a0\u00d7\u00a0A2\n(1) Total Atrial Stroke Volume (TA-SV) defined as Vmax\u00a0\u2212\u00a0Vmin, 2) Total Atrial Emptying Fraction (TA-EF) defined as TA-SV\/Vmax\u00a0\u00d7\u00a0100%, 3) Active Atrial Stroke Volume (AA-SV) defined as VPre\u00a0A\u00a0\u2212\u00a0Vmin, 4) Active Atrial Emptying Fraction (AA-EF) defined as AA-SV\/VPre A\u00a0\u00d7\u00a0100%, 5) Atrial Expansion Index (AEI) defined as TA-SV\/Vmin\u00a0\u00d7\u00a0100%, 6) Passive Atrial Stroke Volume (PA-SV) defined as Vmax\u00a0\u2212\u00a0VPre A, and 7) Passive Atrial Emptying Fraction (PA-EF) defined as (Vmax\u00a0\u2212\u00a0VPre A)\/Vmax\u00a0\u00d7\u00a0100%.\nLA kinetic energy (LA-KE)\nLA-KE [16] was calculated according to the formula \u00bd\u00a0\u00d7\u00a0AA-SV\u00a0\u00d7\u00a0P\u00a0\u00d7\u00a0V2, where P\u00a0=\u00a01.06\u00a0g\u00a0cm\u22123 (blood density), and (V) is the peak velocity of transmitral A wave was measured by pulsed wave Doppler.\nTo characterize the three phases of LA activity, PA-SV and PA-EF were defined as indices for LA conduit function, AA-SV, AA-EF, and LA-KE for LA pump function, and AEI for LA reservoir function.\nStatistical analysis\nAll values were expressed as mean\u00a0\u00b1\u00a0SD. Paired t-test was used for comparison between all HCM patients and controls. Independent sample t-test was used for comparison between both HCM subgroups and between each HCM subgroup and controls. The difference in the values between groups was considered significant if P value\u00a0<\u00a00.05. The statistical package used was SPSS version 12.1.\nResults\nBaseline criteria (See Table\u00a01)\nBoth HCM patient group and controls were comparable with respect to age and sex distribution. All patients and controls were in sinus rhythm (mean heart rate 72\u00a0\u00b1\u00a013 beat per minute) and had normal LV systolic function. All HCM patients had type I diastolic dysfunction (increased A velocity, with an E\/A ratio <1) [17]. Twenty patients (80%) were under medications (10 patients under Verapamil, six under \u03b2-blockers, four under Amiodarone). LV-ESD and LV-ESV were significantly smaller in HCM patients than controls, while LV-EDD and LV-EDV showed no difference. LV-FS and LV-EF were significantly higher in HCM patients than controls (P\u00a0=\u00a00.001). HCM patients had a higher mean transmitral peak A velocity (61.5\u00a0\u00b1\u00a020.3 vs. 39.7\u00a0\u00b1\u00a09.9\u00a0cm\/s, P\u00a0<\u00a00.01). The prevalence and severity of mitral regurgitation were significantly higher in HCM patients compared to controls as 80% of HCM patients had mitral regurgitation (60% mild and 20% moderate to severe), while 20% of controls had mild mitral regurgitation (P\u00a0<\u00a00.0001). Both HCM patient subgroups (obstructive and non-obstructive) showed no significant differences in LV dimensions, volumes and function. Also, no significant difference in the prevalence and severity of mitral regurgitation was present.\nTable\u00a01Baseline clinical and echocardiographic data of HCM patients and controlsHCM patients N\u00a0=\u00a025Normal control n\u00a0=\u00a025P valueAge (yr)38\u00a0\u00b1\u00a015 35\u00a0\u00b1\u00a015 Male gender (%)24 (80%)18 (60%)Left ventricular FS (%)38.4\u00a0\u00b1\u00a09.630.3\u00a0\u00b1\u00a05.40.03Left ventricular EF (%)71.1\u00a0\u00b1\u00a017.757.4\u00a0\u00b1\u00a01.00.001Peak A velocity (cm\/s)61.5\u00a0\u00b1\u00a020.339.7\u00a0\u00b1\u00a09.90.01Mitral regurgitation\u00a0\u00a0\u00a0\u00a0No5 (20%)24 (80 %)0.0001\u00a0\u00a0\u00a0\u00a0Mild15 (60%)6 (20%)\u00a0\u00a0\u00a0\u00a0Moderate-severe5 (20%)0 (0%)Antero-posterior LAD (mm)42.8\u00a0\u00b1\u00a05.834.1\u00a0\u00b1\u00a04.20.0001Medial-lateral LAD (mm)45.6\u00a0\u00b1\u00a09.9 36.2\u00a0\u00b1\u00a03.80.001Superior-inferior LAD (mm)60.8\u00a0\u00b1\u00a09.044.8\u00a0\u00b1\u00a06.70.0001Mean LAD (mm)49.7\u00a0\u00b1\u00a07.238.4\u00a0\u00b1\u00a04.00.0001Anterior left atrial wall (mm)3.6\u00a0\u00b1\u00a01.03.5\u00a0\u00b1\u00a01.0NSPosterior left atrial wall (mm)3.5\u00a0\u00b1\u00a01.23.1\u00a0\u00b1\u00a01.1NSLeft atrial mass (g)89.8\u00a0\u00b1\u00a037.247.8\u00a0\u00b1\u00a018.10.0001Abbreviations: FS fractional shortening, EF ejection fraction, and LAD left atrial diameter\nLA size and mass (Table\u00a01)\nThe maximum LAD at the three planes and the mean LAD were significantly larger in HCM patients than controls (P\u00a0<\u00a00.001). Thickness of anterior and posterior LA walls showed no significant differences between HCM patients and controls. LA mass was significantly higher in HCM patients than controls (89.8\u00a0\u00b1\u00a037.2 vs. 32.3\u00a0\u00b1\u00a012.0\u00a0g, P\u00a0<\u00a00.0001). No significant differences were found between HCM patient subgroups in the mean LAD, wall thickness and LA mass.\nLAV and total LA function (Table\u00a02)\nLAV at the three phases of cardiac cycle (Vmax, Vmin, and VPre-A) was significantly higher in both HCM patient subgroups than control group. TA-SV, TA-EF showed no significant differences between both HCM patient subgroups and control group. Vmax was well correlated with the mean LAD (r\u00a0=\u00a00.89, P\u00a0<\u00a00.0001).\nTable\u00a02Left atrial mass and function in non-obstructive HCM and obstructive HCM patientsNormal control n\u00a0=\u00a025 Non-obstructive HCM n\u00a0=\u00a015Obstructive HCM n\u00a0=\u00a010 P value***Mean LAD (mm)38.4\u00a0\u00b1\u00a04.049.6\u00a0\u00b1\u00a07.649.9\u00a0\u00b1\u00a07.00.00010.0001LA mass (g)47.8\u00a0\u00b1\u00a018.186.8\u00a0\u00b1\u00a041.193.9\u00a0\u00b1\u00a032.50.0020.001V max (ml)38.2\u00a0\u00b1\u00a010.764.7\u00a0\u00b1\u00a026.765.9\u00a0\u00b1\u00a028.10.0020.009V min (ml)17.4\u00a0\u00b1\u00a07.632.7\u00a0\u00b1\u00a022.137.1\u00a0\u00b1\u00a019.70.020.008V pre A (ml)24.9\u00a0\u00b1\u00a07.744.7\u00a0\u00b1\u00a024.748.5\u00a0\u00b1\u00a021.50.0080.005TA-SV (ml)20.5\u00a0\u00b1\u00a06.932.0\u00a0\u00b1\u00a08.928.8\u00a0\u00b1\u00a013.1NSNSTA-EF (%)54.7\u00a0\u00b1\u00a014.253.6\u00a0\u00b1\u00a013.845.4\u00a0\u00b1\u00a012.8NSNSAA-SV (ml)7.3\u00a0\u00b1\u00a04.012.0\u00a0\u00b1\u00a06.211.4\u00a0\u00b1\u00a06.50.0010.001AA-EF (%)31.4\u00a0\u00b1\u00a017.829.7\u00a0\u00b1\u00a013.525.4\u00a0\u00b1\u00a012.8NSNSLA-KE (kdynes.cm)11.9\u00a0\u00b1\u00a07.418.3\u00a0\u00b1\u00a012.532.5\u00a0\u00b1\u00a023.3a0.030.001PA-SV (ml)13.4\u00a0\u00b1\u00a05.720.0\u00a0\u00b1\u00a08.817.5\u00a0\u00b1\u00a09.80.02NSPA-EF (%)33.9\u00a0\u00b1\u00a010.134.0\u00a0\u00b1\u00a014.827.1\u00a0\u00b1\u00a07.0NS0.03AEI (%)141.7\u00a0\u00b1\u00a074.0134.8\u00a0\u00b1\u00a074.491.1\u00a0\u00b1\u00a039.7NS0.02Abbreviations: see text*P value between non-obstructive HCM patients and controls**P value between obstructive HCM patients and controlsaP value\u00a0<\u00a00.001 between obstructive and non-obstructive HCM patients\nLA pump function\nAA-EF showed no significant differences between both HCM patient subgroups and control group, while AA-SV was significantly higher in both HCM patient subgroups than control group (12.0\u00a0\u00b1\u00a06.2\u00a0ml, 11.4\u00a0\u00b1\u00a06.5\u00a0ml vs. 7.3\u00a0\u00b1\u00a04.0\u00a0ml, P\u00a0<\u00a00.001). No significant differences were found between HCM patient subgroups in AA-EF and AA-SV. LA-KE was significantly higher in HCM patients than controls (24.3\u00a0\u00b1\u00a018.9 vs. 11.9\u00a0\u00b1\u00a07.4\u00a0kdynes\u00a0cm, P\u00a0=\u00a00.002). LA-KE was significantly higher in obstructive HCM patients than non-obstructive patients (32.5\u00a0\u00b1\u00a023.3 vs. 18.3\u00a0\u00b1\u00a012.5\u00a0kdynes\u00a0cm, P\u00a0<\u00a00.001).\nLA conduit function\nPA-SV was significantly increased in HCM patients than controls (18.9\u00a0\u00b1\u00a09.2 vs. 13.4\u00a0\u00b1\u00a05.7\u00a0ml, P\u00a0=\u00a00.01). PA-SV was significantly higher in non-obstructive HCM patients than controls (20.0\u00a0\u00b1\u00a08.8 vs. 13.4\u00a0\u00b1\u00a05.7\u00a0ml, P\u00a0=\u00a00.02), while in obstructive HCM patients; it was comparable to controls (17.5\u00a0\u00b1\u00a09.8 vs.13.4\u00a0\u00b1\u00a05.7\u00a0ml). PA-EF showed no significant difference between HCM patients and controls. PA-EF was significantly lower in obstructive HCM patients than controls (27.1\u00a0\u00b1\u00a07.0 vs. 33.9\u00a0\u00b1\u00a010.1, P\u00a0=\u00a00.02), while in non-obstructive HCM patients; it was comparable to controls (34.0\u00a0\u00b1\u00a014.8 vs. 33.9\u00a0\u00b1\u00a010.1).\nLA reservoir function\nAEI showed no significant difference between HCM patients and controls. AEI was comparable in non-obstructive HCM patients and controls (134.8\u00a0\u00b1\u00a074.4% vs. 141.7\u00a0\u00b1\u00a074.0%, P=NS) but it was significantly lower in obstructive HCM patients than controls (91.1\u00a0\u00b1\u00a039.7% vs. 141.7\u00a0\u00b1\u00a074.0%, P\u00a0=\u00a00.02).\nDiscussion\nIn the present study, LA size was increased in HCM patients as assessed by LA dimension at three directions (antero-posterior, medial-lateral and superior-inferior) and by LAV at three phases of cardiac cycle. Thus, LA mass was increased in HCM patients compared to normal subjects. According to Frank\u2013Starling mechanism, LA pump function in HCM was increased to help ventricular filling as shown by increased AA-SV and LA-KE. LA reservoir and conduit functions were decreased in obstructive HCM patients as shown by decreased AEI and PA-EF. The clinical importance of this study is the close relationship of LA dimension and function with the development of serious complications as atrial fibrillation, cerebral infarction, and progressive heart failure in HCM as shown in previous studies [18\u201320] as well as adverse outcome after myectomy [21]\nThe previous studies describing LA changes in HCM identified increased LAV [22, 23]. LA responds to the increased LAV by linear increase of LAD as shown in the present study. The nonlinear correlation between the increased LAD and increased LAV in previous studies [24] may be due to unidimensional M-mode assessment of LA size while, in the present study, the LA size was relied on 2D multidimensional assessment and the mean LA dimension was comparable to previous studies [10, 25].\nThe increased left ventricular wall thickness and elevated filling pressure in HCM frequently lead to development of abnormal diastolic function. In response to this overload, LA contractility increases to maintain adequate left ventricular filling. In accordance with previous studies [26, 27], AA-SV and LA-KE was significantly increased in our HCM patients than controls. Other studies reported no increase in LA contractility [28, 29]. The discrepancy between studies describing LA contractility in HCM is due to difference in hemodynamic conditions, atrio-ventricular interaction and left ventricular systolic function.\nIn HCM patients, the rate of left ventricular relaxation deteriorates due to markedly thickened and non-compliant left ventricle, which led to deterioration of LA conduit function. This was expressed by reduction in PA-EF in our HCM patients compared to controls.\nLA reservoir function is determined by LA relaxation and mitral annulus displacement during left ventricular contraction [30]. In accordance with previous studies [8], AEI was significantly reduced in obstructive HCM patients, which indicates impaired LA relaxation. AEI showed no difference between our non-obstructive HCM patients and controls indicating preserved LA reservoir function.\nStudy limitation\nThe study had some limitations. The sample size of HCM patients (25 patients) is small but due to low prevalence of HCM (0.2%). The assessment of LA mass is a new idea not discussed before by any imaging modalities. Of course it needs validation by autopsy or magnetic resonance imaging but at least it can pave a way for thinking about in further studies either by the same or another formula. Assessment of diastolic dysfunction was relied on transmitral flow by pulsed wave Doppler and other parameters by tissue Doppler imaging were not available. There are no data about prognostic value of atrial remodelling as a predictor for development of atrial fibrillation because it need long time follow up for large sample of patients. However data from previous studies could be relied on [18, 19].\nConclusion\nHypertrophic cardiomyopathy is associated with dilated LA and increased LAV that reflects the severity of LA overload. Higher AA-SV and LA-KE in HCM patients especially obstructive type indicates augmented LA pump function to overcome the high left ventricular filling pressure caused by severe left ventricular hypertrophy. Lower PA-EF and AEI in obstructive HCM patients indicate impaired LA reservoir and conduit functions. These findings may have clinical implication for the follow up of HCM patients","keyphrases":["left atrial volume","hypertrophic cardiomyopathy","left atrial function","left atrial mass"],"prmu":["P","P","R","R"]}
{"id":"Psychopharmacologia-2-2-1705494","title":"Impaired performance of alpha7 nicotinic receptor knockout mice in the five-choice serial reaction time task\n","text":"Rationale Nicotinic receptors have been implicated in attentional performance. Nicotine can improve attention in animals and humans, but knowledge about relevant receptor subtypes is very limited.\nIntroduction\nNicotinic acetylcholine receptors are pentameric cation channels, so-called because of the actions of nicotine in binding to these receptors and bringing about channel opening. Twelve subunits of neuronal nicotinic receptors have been identified, \u03b12-10 and \u03b22-4. From these subunits the two most common nicotinic receptors formed in the brain are the heteromeric \u03b14\u03b22 and the homomeric \u03b17 nicotinic receptors, at approximately 85 and 10% of total nicotinic receptors, respectively. The \u03b17 nicotinic receptors are especially highly expressed in the hippocampus where, as well as mediating cholinergic neurotransmission, their high Ca++ permeability (Seguela et al. 1993) engenders the capacity to modulate glutamatergic and GABAergic neurotransmission both pre- and post-synaptically (Berg and Conroy 2002). This study aims to shed further light on the role of \u03b17 receptors in behaviour and in the behavioural effects of nicotine.\nNicotine is the second most commonly abused drug in the world after alcohol; cigarette smoking, with its well-established risks of chronic disease, constitutes a great burden on societies worldwide. Considerable efforts have been expended in investigating the function of nicotinic receptors in nicotine addiction and in other neuropsychiatric conditions, and in mediating the cognitive effects of nicotine. For example, cigarette smoking is approximately twice as common in patients with schizophrenia, as it is within the general population. The \u03b17 nicotinic receptor has been implicated in the pathogenesis of schizophrenia and more specifically in the P50 sensory gating deficit measurable in approximately 90% of schizophrenics, about 50% of their relatives and about 10% of control subjects (Waldo et al. 1991). The sensory gating deficit in schizophrenia is thought to reflect an input dysfunction that allows greater passage of information through the hippocampus for processing by other brain areas (Freedman et al. 2002). The density of \u03b17 nicotinic receptors in hippocampus and cingulate cortex from schizophrenics is considerably reduced (Freedman et al. 1995; Marutle et al. 2001). It might, therefore, be expected that performance of tasks requiring sustained attention would require \u03b17 nicotinic receptors.\nAttempts to investigate the functional role of \u03b17 nicotinic receptors at the behavioural level have most often involved the use of selective agonists and antagonists to these receptors. Another avenue of research that has recently become possible uses genetically modified mice with specific deletions of genes for neuronal nicotinic receptors. Mice with targeted deletion of the gene for the \u03b17 nicotinic receptor lack \u03b1-bungarotoxin binding sites in the hippocampus and fast, rapidly desensitizing nicotinic currents in hippocampal neurons (Orr-Urtreger et al. 1997). Viability and brain morphology appear normal in these mice; of special interest is the lack of any impairment of sensorimotor gating as measured with both auditory and tactile stimuli in the prepulse inhibition paradigm (Paylor et al. 1998). This result is consistent with the lack of improvement in prepulse inhibition with nicotinic \u03b17 agonists (Olivier et al. 2001; Schreiber et al. 2002), although such agonists are able to improve auditory gating in animal models of sensory gating deficit (O\u2019Neill et al. 2003).\nIn other behavioural studies, \u03b17\u2212\/\u2212 mice have not proved to be considerably different in behaviour from wild-type littermates (Paylor et al. 1998). In locomotion, \u03b17\u2212\/\u2212 mice had a tendency towards less horizontal and vertical activity and spent significantly more time in the centre of the open field than the wild-type mice, but in the light\u2013dark test of anxiety there were no significant differences between the two groups (Paylor et al. 1998). In the delayed matching-to-place task in the Morris water maze, \u03b17\u2212\/\u2212 mice were slightly impaired in comparison with wild-types, as shown by a longer time to find the hidden platform (Fernandes et al. 2005). Nicotine-induced locomotor depression, hypothermia, seizures, drug discrimination and tolerance were unchanged in \u03b17\u2212\/\u2212 mice (Franceschini et al. 2002; Tritto et al. 2004; Stolerman et al. 2004; Naylor et al. 2005). However, the \u03b17\u2212\/\u2212 mice were less sensitive to impairments in contextual learning brought about by ethanol (Wehner et al. 2004).\nThe series of experiments presented in this paper use the five-choice serial reaction time task (5-CSRTT), a commonly used model of attentional performance that is considered analogous to the continuous performance test used in human research. The test requires responses to visual stimuli presented in one of five locations in one wall of an operant conditioning chamber. In a previous study Young et al. (2004) found that \u03b17\u2212\/\u2212 mice took longer to acquire the task than wild-type C57 Bl\/6J mice and made more omission errors, but they were not different with respect to accuracy or the latencies of correct responses. Young et al. (2004) also reported significant improvements in accuracy and reductions in omission errors at several doses of nicotine in C57 Bl\/6J mice, including a reduction in the latency of correct responses with a 0.3-mg\/kg dose of nicotine. In this paper, the first published report of nicotine improving the performance of mice in the 5-CSRTT and the effects seen were not dose-related, but the findings were important and need replication and investigation under a wider range of conditions.\nSeveral studies have reported that nicotine enhances the attentional performance of rats in the 5-CSRTT (Hahn and Stolerman 2002; Hahn et al. 2002, 2003a), although the use of selective nicotinic agonists and antagonists has provided no evidence for involvement of the \u03b17 receptor. The relatively specific \u03b17 agonist AR-R17779 had no effect upon task performance in an experimental procedure in which administration of nicotine, epibatidine and isoarecolone all improved performance (Hahn et al. 2003a). These agonists all have much higher relative affinities than AR-R17779 for heteromeric nicotinic receptors. The \u03b14\u03b22 nicotinic agonist SIB1765F has also been shown to improve 5-CSRTT performance in rats (Grottick and Higgins 2000). The \u03b17 antagonist methyllycaconitine did not block the effects of nicotine on response latency and anticipatory responding, whereas dihydro-\u03b2-erythroidine, an antagonist at heteromeric receptors, did block the effect of nicotine on these measures (Blondel et al. 2000; Grottick and Higgins 2000). However, the \u03b17 agonist GTS-21 has been reported to improve performance in various tests of attention and memory in healthy human volunteers when given in repeated doses (Kitagawa et al. 2003) and in a test of memory in monkeys 24\u00a0h after administration (Briggs et al. 1997). The small number of drugs investigated and their limited selectivity indicates a need for investigations on the role of \u03b17 receptors in attention by another approach, such as the \u03b17\u2212\/\u2212 mouse.\nThe aims of the present study were, therefore, to investigate the attentional performance of \u03b17\u2212\/\u2212 mice using the 5-CSRTT and to determine whether the gene deletion influenced any effects of nicotine on performance. If the \u03b17 nicotinic receptor is important in attentional mechanisms then it should be possible to confirm the attentional deficits reported by Young et al. (2004) in \u03b17\u2212\/\u2212 mice. If nicotine improves attention, at least in part, through these receptors then it should also be possible to show that nicotine-induced attentional enhancement is diminished in such animals. The procedures used in the initial stages of training were based on those of Patel et al. (2006) because they were close to those used in studies of rats where nicotine improved performance (Hahn et al. 2002; Hahn and Stolerman 2002). Mice were also trained with both fixed and variable inter-trial intervals (ITI) so that \u03b17\u2212\/\u2212 and wild-type animals could be compared for their abilities in coping with the temporally unpredictable stimuli in the variable ITI condition. In the later stages of the study, some parameters were modified to make the training procedure more similar to that of Young et al. (2004) so as to facilitate comparisons and to clarify reasons for certain differences in the results obtained.\nMaterials and methods\nAnimals\nThe animals used were descendants of \u03b17\u2212\/\u2212 mice produced by Orr-Urtreger et al. (1997), bred from heterozygotes purchased from the Jackson Laboratory, USA (B6.129S7-Chrna7tm1 Bay, stock no. 003232). The mice were at backcross generation N8, the background strain was C57BL\/6, donor strain 129S7 via ABI 2.1 ES cell line; subsequent breeding was always carried out using heterozygous pairs. Twenty-four male \u03b17\u2212\/\u2212 mice and 24 male wild-type littermates were used in initial training. Genotypes were determined with the methods used by Stolerman et al. (2004), and all mice were injected with TROVAN sterile transponder chips (Mid Fingerprint, Dorset UK) at age 10\u00a0weeks. These groups were divided into sub-groups of 12 mice each trained with fixed and variable ITI. One wild-type mouse was eliminated from the study at an early stage due to an unresolvable eye infection. Table\u00a01 lists the subsequent parameter changes that took place as training proceeded. \nTable\u00a01Parameters for all experimentsStudyStimulus durationLimited holdFixed ITIVariable ITITime-outAnticipatory time-outSessions to criteria1a60022 (0.25\u20133.75)00760044 (0.5\u20137.5)20210044 (0.5\u20137.5)2025144 (0.5\u20137.5)2042444 (0.5\u20137.5)2071544 (0.5\u20137.5)20281b154NA20NA2a12NA4 (0.5\u20137.5)22152b12NA4 (0.5\u20137.5)22NAStudies 1a and 2a refer to experiments looking at baseline (undrugged) performance at initial and final parameters, whereas studies 1b and 2b refer to the first and second of the experiments with nicotine. For experiment 1a, where ITI values are shown for both fixed and variable ITI mode, these refer to settings for different groups of mice. All times stated are in seconds. For variable ITI the mean, smallest and largest values are shown.NA Not applicable\nFood restriction was started at age 11\u201312\u00a0weeks and behavioural training began a week later. The mice were kept at 80% of their free-feeding weights as calculated from a previously obtained growth curve for animals from the same colony. The animals were weighed daily and fed accordingly. All animals had access to water while in their home cages and were individually housed in a room at 21\u00b0C and on a 12-h light\/dark cycle, with lights coming on at 0700 hours.\nThe studies complied with local ethical requirements and were carried out in accordance with the Animals (Experimental Procedures) Act, 1986.\nApparatus\nSound-insulated and ventilated enclosures containing aluminium operant chambers were used (CENES, Cambridge, UK). The chambers measured 14\u00a0cm across \u00d7 12\u00a0cm high \u00d7 11\u201313\u00a0cm deep. The back wall of the chamber was curved so that each nose-poke hole was equidistant from the feeder tray at the centre of the front of the chamber. There were five nose-poke holes 1\u00a0cm in diameter, 2\u00a0cm above the floor, 1.5\u00a0cm apart and 1.5\u00a0cm deep. Each hole had a green-light-emitting diode at the end and a photoelectric cell at the opening. The two house lights were situated 10\u00a0cm up the sides of the chamber. An Acorn computer running Arachnid software (Paul Fray, Cambridge, UK) under RISC OS collected the data.\nTraining procedure\nThe mice were trained for 5\u00a0days each week, Monday to Friday. The training procedure was based on that of Patel et al. (2006) and was modified from that of Humby et al. (1999) so as to resemble more closely the procedures in previous work carried out with the 5-CSRTT in rats (Bizarro et al. 2004). After the mice were habituated to handling, training began with four sessions of 5, 10, 15 and 15\u00a0min for the mice to habituate to the test chambers. The house lights were not illuminated at this stage. Before the next two sessions that were 15\u00a0min each in duration, 15\u201320 food pellets were placed in feeder trays before the start of each session. This was followed by two sessions of 15- and 20-min duration, respectively, in which food pellets were presented according to fixed-time schedules of 15 and 30\u00a0s. Nose-poke training then started with four 30-min sessions of non-spatial training with the stimulus duration set at 60\u00a0s and with an ITI of 2\u00a0s. As noted above, subgroups of mice were trained with ITI of fixed duration, whereas for other groups, individual ITI varied around the mean value (for details, see Table\u00a01). In non-spatial training sessions, all five holes were illuminated simultaneously and a nose-poke in any hole was reinforced by delivery of a food pellet. For the first of these sessions, 25\u00a0mg food pellets (PJPPP-0020, Sandown Chemical, UK) were placed at the front of each of the nose-poke holes to encourage the mice to nose-poke.\nFor spatial training, the house lights were on at all times except during time-outs. Each session lasted for 30\u00a0min and there was no limit to the number of trials in a session. A stimulus was randomly presented in one of the five holes and a response in the correct hole during the stimulus duration or the limited hold (LH) period after the stimulus was reinforced and followed by a 10-s allowance for the time taken to eat the food pellet. This was sufficient for mice to consume the food and return to attending to the task; the next ITI began at the end of the 10-s allowance for eating. In sessions in which there was a time-out, this was initiated by a nose-poke response into an incorrect hole during the stimulus duration or LH. Premature responses during the ITI in experiment 1 were recorded but had no programmed consequences. Time-outs involved turning off the lights for 2\u00a0s and were followed immediately by the start of the next ITI.\nExperiment 1a: initial acquisition of attentional task\nTable\u00a01 describes the stages of training, with criteria for progression to the next stage being that the mean percentage of correct responses for each group was greater than 70% and the mean response latency for each group was less than the stimulus duration for two consecutive days. The training parameters were not advanced if the mice had not been trained the day before. Acquisition was considered to be complete when the mean percentages of correct responses was stable for two consecutive weeks.\nExperiment 1b: first set of nicotine tests\nFor testing the effects of nicotine, a reduced number of 15 wild-type and 15 \u03b17\u2212\/\u2212 mice were used because it was known from previous studies that this was a sufficiently large group size for assessing drug effects (Humby et al. 1999; Hahn and Stolerman 2002). The selected animals were those that had all been trained at a similar time of day previously and no other selection criterion was used. Up to this stage, some mice had been trained with fixed and some with variable ITI. All selected mice were switched to fixed ITI mode before drug testing started to reduce the number of independent variables; all other parameters remained unchanged. These mice were given saline injections subcutaneously in the flank after training on each of the 3\u00a0days before the first nicotine test day to allow habituation to the procedure to take place. There were four test days on consecutive Tuesdays and Fridays. Mice were allocated to a dosing schedule for injection of saline or 0.001, 0.01 or 0.1\u00a0mg\/kg nicotine 10\u00a0min before the start of each test session; each mouse was tested once at each dose by the end of the study and dosing sequences were random. Young et al. (2004) found that these doses of nicotine significantly improved the task performance of nicotine-na\u00efve mice.\nExperiment 2a: retraining after changing parameters\nSeven weeks after the end of experiment 1b, retraining of the same 30 mice commenced, followed by alterations of experimental parameters to make them resemble more closely those used by Young et al. (2004). The changes entailed reducing the LH from 5 to 2\u00a0s, using variable ITI mode only, and including a 2-s time-out for anticipatory responses. House lights were extinguished during the time-outs during which no stimuli were presented, and the presentation of the next stimulus was delayed by the duration of the time-out. Although mice originally trained with fixed ITI mode were at this time trained exclusively in variable ITI mode, there were no differences in performance as a function of this variation in training history. Table\u00a01 lists the details of all the changes in task parameters. The animals were trained under the altered parameters for 7\u00a0weeks, by which time performance was relatively stable. One \u03b17\u2212\/\u2212 mouse developed an unusual behavioural phenotype. This mouse was markedly hyperactive in its home cage, required approximately twice as much food as the other animals to maintain its weight and made anticipatory responses at a rate 5\u201310 times greater than the next most impulsive mouse. No data from this mouse are presented.\nExperiment 2b: second set of nicotine tests\nA second experiment with nicotine began immediately after the end of experiment 2a. The mice were injected with saline for 3\u00a0days before the start of the test sessions so that they might habituate to the injection procedure. There were eight test sessions on consecutive Tuesdays and Fridays. Mice were allocated to a dosing schedule for injection of saline or 0.001, 0.003, 0.01, 0.03, 0.1, 0.3 or 1\u00a0mg\/kg nicotine 10\u00a0min before the start of each test session. Each mouse was tested once at each dose by the end of the study and dosing sequences were random.\nData analyses\nThe six behavioural measures selected for analysis were as follows: \nAccuracy (percentage of correct responses): 100 \u00d7 correct responses\/(correct + incorrect responses)Omissions (percentage of omission errors): 100 \u00d7 omission errors\/stimuli presentedLatency of correct responses: the mean time between stimulus onset and a nose-poke in the correct holeLatency of incorrect responses: the mean time between stimulus onset and a nose-poke in any incorrect holeReinforcers earned: equal to the number of correct responsesAnticipatory response rate: the mean number of responses per second during the ITI calculated by using the following formula: total number of responses in all ITI of a session\/number of trials\/ITI length in seconds.\nThe accuracy measure was not calculated when the sum of correct and incorrect responses was 15 or less. For analysis of variance (ANOVA), percentage data for accuracy and omissions were arc-sine transformed, latencies were log-transformed and anticipatory response data were subject to square root transformation, as in previous studies (Hahn et al. 2002, 2003a; Patel et al. 2006). For post hoc tests, the Bonferroni correction for multiple comparisons was applied to t tests. Graphs show untransformed data. Results for each measure were analysed by ANOVA for repeated measures using Unistat 5.0 for Excel, three-factor ANOVA was used to examine data from experiment 1a and two-factor ANOVA was used for experiments 1b, 2a and 2b. Further details of the factors are given below, at relevant places in the Results section.\nDrugs\nNicotine hydrogen tartrate (Sigma, UK) was dissolved in isotonic saline with pH adjusted to 7 with NaOH solution. All injections were given s.c. in the flank in a volume of 1\u00a0ml\/100\u00a0g and all doses were those of the nicotine base.\nResults\nExperiment 1a: initial acquisition of attentional task\nThere were no significant differences between any of the groups with respect to any parameter of performance at the first stage of training with the stimulus duration set at 60\u00a0s and the ITI at 2\u00a0s. Figure\u00a01a) shows the similarity in the changes in accuracy for \u03b17\u2212\/\u2212 and wild-type mice over these first seven sessions. It can be seen that the data show signs that the mice began to learn the task from the very earliest stage of training. Results for other variables are not shown because they were similarly negative with respect to genotype.\nFig.\u00a01Experiment 1a: accuracy of 5-CSRTT performance for wild-type (\u25cf) and \u03b17\u2212\/\u2212 mice (\u25cb) during the first seven sessions of training in the 5-CSRTT. Results show main effects of genotype and training day as means\u00b1SEM for groups of 23 mice. Subgroups of mice with each genotype were trained with fixed and variable inter-trial intervals without effect on performance (data not shown)\nDuring subsequent stages of training there were progressively greater demands made upon the animals\u2019 ability by altering task parameters, such as reducing the stimulus duration (Table\u00a01). Differences between the genotypes then emerged, including changes in accuracy, response latencies and anticipatory responding. The data for intermediate stages of training are not shown for brevity; they largely replicated the observations made when the final set of task parameters was in operation. The performance of the animals was relatively stable during the last 10\u00a0days of training under the final parameters and these data were selected for analysis and are presented next.\nData for the final 10\u00a0days of training were examined by three-factor analysis of variance, the factors being genotype, ITI mode, and days. This analysis yielded main effects of genotype on accuracy [F(1, 42)=8.67, p<0.01], reinforcers earned [F(1, 42)=12.3, p<0.01], anticipatory response rate [F(1, 42)=4.50, p<0.05] and arguably, on correct response latency [F(1, 42)=3.96, p=0.053]. Figure\u00a02a shows that \u03b17\u2212\/\u2212 mice were much less accurate than wild-types, were slightly slower to make a correct response, earned fewer reinforcers and responded more frequently during the ITI. Neither the three-way (group \u00d7 ITI mode \u00d7 day) nor any two-way interaction was significant for any of the parameters measured. For changes across the ten successive days of training, omission errors [F(9, 378)=4.99, p<0.0001], reinforcers earned [F(9, 378)=3.28, p<0.001] and anticipatory response rate [F(9, 378)=2.11, p<0.05] showed significant effects. Most of these effects did not show consistent patterns over days (Fig.\u00a02) and are not considered further.\nFig.\u00a02Experiment 1a: main effects of genotype and day of training on performance of wild-type (\u25cf) and \u03b17\u2212\/\u2212 mice (\u25cb) during the last 10\u00a0days before drug testing began (n=23). Subgroups of mice of each genotype were trained in fixed (F) and variable (V) inter-trial interval modes; inset bar graphs illustrate the significant main effects of this variable. All data shown are means\u00b1SEM (n=23)\nFor ITI mode, there were significant main effects for two variables, correct response latency [F(1, 42)=9.27, p<0.01] and reinforcers earned [F(1, 42)=6.25, p<0.02], with those mice trained on variable ITI mode being slower to make correct responses and obtaining fewer reinforcers. The inset bar graphs in Fig.\u00a02c and e illustrate the significant main effects of ITI mode.\nExperiment 1b: first set of nicotine tests\nData were examined with two-factor analysis of variance, the factors being genotype and drug (nicotine) dose. There were no significant effects of nicotine (0.001\u20130.1\u00a0mg\/kg) upon any measure of performance [largest F(3, 78)=2.32, p=0.082, for correct response latency]. The main effect of genotype was significant for accuracy [F(1, 26)=4.42, p<0.05] and reinforcers earned [F(1, 26)=6.55, p<0.02]. Table\u00a02 shows means\u00b1SEM. for both wild-type and \u03b17\u2212\/\u2212 mice, for each attentional parameter, over the 30-min sessions. As in experiment 1a, \u03b17\u2212\/\u2212 mice were less accurate than wild-types and earned fewer reinforcers. Genotypes did not differ significantly for other variables, although inspection of Table\u00a02 reveals trends for omission errors, correct response latencies and anticipatory response rate that were in the same direction as the effects seen in experiment 1a. There were no significant interactions between genotype and nicotine. \nTable\u00a02Mean\u00b1SEM effects of nicotine (0.001\u20130.1\u00a0mg\/kg) on attentional parameters in the 5-CSRTT in \u03b17 knockout (KO) mice and their wild-type (WT) littermatesAttentional parametersNicotine (mg\/kg)Accuracy (percent correct)Omission errors (percent omissions)Correct response latency (s)WTKOWTKOWTKO0.088.9\u00b11.784.7\u00b12.327.4\u00b12.830.5\u00b11.80.79\u00b10.030.85\u00b10.030.00189.7\u00b11.384.2\u00b12.725.7\u00b12.230.3\u00b11.70.78\u00b10.020.84\u00b10.040.0188.0\u00b12.181.7\u00b12.527.4\u00b12.331.5\u00b12.40.80\u00b10.020.82\u00b10.020.1088.8\u00b12.083.2\u00b11.826.1\u00b12.529.6\u00b12.40.81\u00b10.020.89\u00b10.03Incorrect response latency (s)Reinforcers earned (30\u00a0min)Anticipatory responses\/sWTKOWTKOWTKO0.02.27\u00b10.192.25\u00b10.2289.0\u00b12.982.7\u00b12.50.042\u00b10.0100.052\u00b10.0100.0012.05\u00b10.242.45\u00b10.1190.8\u00b12.582.9\u00b13.00.038\u00b10.0080.059\u00b10.0100.012.32\u00b10.162.16\u00b10.1488.1\u00b12.080.3\u00b12.70.045\u00b10.0130.061\u00b10.0130.102.32\u00b10.202.41\u00b10.1590.1\u00b12.982.6\u00b12.30.041\u00b10.0110.063\u00b10.013\nExperiment 2a: retraining after changing parameters\nAfter the parameter changes listed in Table\u00a01, animals were retrained on the task for several weeks and the relatively stable performance during the last ten sessions of training before tests with nicotine was selected for analysis. Data were examined by means of two-factor analysis of variance, the factors being genotype and day of training. The analysis yielded significant effects of genotype on omission errors [F(1, 29)=20.4, p<0.001] and reinforcers earned [F(1, 29)=19.7, p<0.001], but not on accuracy, correct or incorrect response latency or anticipatory response rate [maximum F(1, 29)=1.73]. The \u03b17\u2212\/\u2212 mice omitted responses to a greater percentage of stimuli and earned fewer reinforcers than wild-types; these effects were clearly manifested during each of the 10\u00a0days of training (Fig.\u00a03b,e).\nFig.\u00a03Experiment 2a: main effects of genotype and training day on performance of wild-type (\u25cf) and \u03b17\u2212\/\u2212 mice (\u25cb) after reduction of limited hold from 5 to 2\u00a0s and introduction of 2-s time-out for anticipatory responding (all mice trained in variable ITI mode). Results shown are for the last 10\u00a0days of training with the preceding altered parameters before the second set of tests with nicotine. Data shown are means\u00b1SEM (n=14\u201315)\nThere were also small but significant differences between correct response latencies and anticipatory responding over days. Anticipatory responding occurred at a lower rate in the later stages of training [F(9, 241)=7.07, p<0.0001]; the latency of correct responses also varied over days, but no clear pattern was evident [F(9, 241)=2.12, p<0.05]. The day of training had no effect on any other parameter. There were no significant genotype \u00d7 day interactions.\nExperiment 2b: second set of nicotine tests\nThese data were examined by two-factor analysis of variance, the factors being genotype and drug (dose of nicotine). There were no effects of genotype [F(1, 27)=2.79] or drug [F(7, 179)=1.44] on the percentage of correct responses; nor was there a significant group \u00d7 genotype interaction [F(7, 179)=0.97]. For omission errors there was a significant effect of genotype [F(1, 27)= 16.7, p<0.0001] and of drug [F(7, 189)=82.4, p<0.0001] and additionally, a drug \u00d7 group interaction was observed [F(7, 189)=6.40, p<0.0001]. From Fig.\u00a04b it can be seen that the effect of genotype arises from the slightly larger overall number of omission errors made by the \u03b17\u2212\/\u2212 mice. In view of the significant interaction, this difference was examined separately at each dose of nicotine by means of t tests with Bonferroni corrections for multiple comparisons. These analyses yielded significant differences between the genotypes at nicotine doses of 0.003, 0.01, 0.1 and 1.0\u00a0mg\/kg; in all cases the percentage of omission errors was greater in the \u03b17\u2212\/\u2212 than in the wild-type animals. Nicotine increased omission errors markedly at the largest dose tested, and this effect was greater in the \u03b17\u2212\/\u2212 than in the wild-type mice.\nFig.\u00a04Experiment 2b: main effects of genotype and nicotine dose on performance of wild-type (\u25cf) and \u03b17\u2212\/\u2212 mice (\u25cb) after reduction of limited hold from 5 to 2\u00a0s and introduction of 2-s time-out for anticipatory responding (all mice trained in variable ITI mode). Data shown are means\u00b1SEM (n=14\u201315). Asterisks represent differences between genotypes at each dose of nicotine determined by t tests with Bonferroni correction; *p<0.05, **p<0.01)\nThere was no main effect of genotype on correct latency [F(1, 27)=1.47]. The main effect of drug on this measure was significant [F(7, 183)=22.4, p<0.0001] and this appeared to be entirely due to an increase in latency at the largest dose of nicotine (Fig.\u00a04c). The interaction was not significant [F(7, 183)=1.63]. There was also a significant effect of drug on incorrect latencies [F(7, 166)=2.23, p<0.05), although its origin is not obvious from Fig.\u00a04d and there was no dose\u2013response relationship.\nWith respect to reinforcers earned, there were significant effects of genotype [F(1, 27)=20.7, p<0.0001], drug [F(7, 189)=82.0, p<0.0001] and a group \u00d7 drug interaction [F(7, 189)= 8.71, p<0.0001]. The overall number of reinforcers earned was slightly greater in wild-type than in \u03b17\u2212\/\u2212 mice, and Fig.\u00a04e shows that this difference was significant at four of the eight dose levels of nicotine that were tested. Nicotine itself decreased the number of reinforcers at the largest dose tested and this effect was greater in \u03b17\u2212\/\u2212 than in wild-type mice. Finally, there was a significant effect of nicotine [F(7, 189)=15.8, p<0.0001] but not genotype [F(1, 27)=1.10] on anticipatory responding. The effect of drug may be attributed to a decrease in the rate of responding at the 1.0\u00a0mg\/kg dose of nicotine (Fig.\u00a04f). A significant drug \u00d7 genotype interaction was seen [F(7, 189)=2.39, p<0.05], but post hoc tests did not identify a significant difference between the genotypes at any dose level of nicotine.\nDiscussion\nThe five-choice serial reaction time task revealed impairments in the performance of the \u03b17 nicotinic receptor knockout mice on a variety of measures, thus supporting the view that these receptors play a role in attentional performance. Analysis through variation in task parameters suggested that the primary deficit was an increase in omission errors; a general slowing in performance would have been reflected in corresponding increases in response latencies and decreases in anticipatory responding. Thus, the \u03b17\u2212\/\u2212 mice failed to detect significantly more stimuli than the wild-types, but when they did detect a stimulus they responded to it quickly and correctly. These findings confirm and extend the key observations of Young et al. (2004) and support the emerging consensus that although \u03b17\u2212\/\u2212 mice show deficits in the performance of several cognitive tasks, the differences from controls are modest and are manifest only with specific tasks used under precisely defined conditions (Paylor et al. 1998; Wehner et al. 2004; Fernandes et al. 2005). We now discuss in detail some differences between our results and those of Young et al. (2004) and indicate how they can be reconciled by considering parametric variations in the procedures used.\nIn the present study, \u03b17\u2212\/\u2212 mice did not differ from wild types until the later stages of training, where the more stringent task parameters placed greater demands upon their cognitive ability (Fig.\u00a02). These differences in the baseline (undrugged) performance of the \u03b17\u2212\/\u2212 mice and wild-type mice were largely replicated in our first study on the response to nicotine (Fig.\u00a03). The impaired accuracy and increased response latency suggested an attentional impairment, whereas the increase in anticipatory responding suggested greater impulsivity. Keller et al. (2005) also found that the amount of anticipatory responding was increased in \u03b17\u2212\/\u2212 mice, although they used a different behavioural procedure.\nThere is often an inverse relationship between accuracy and impulsivity in the 5-CSRTT, and from a comparison of inbred mouse strains it was argued that a decrease in accuracy could be secondary to increased impulsivity (Patel et al. 2006). The observed change in impulsivity may have contributed to the decrease in accuracy in experiment 1a, which should, therefore, be interpreted cautiously. We did not observe slow acquisition of task performance like that seen by Young et al. (2004); a primary effect on anticipatory responding may have contributed to their finding, too, rather than a deficit in learning ability. This interpretation is supported by the absence of impairments in \u03b17\u2212\/\u2212 mice in a variety of other learning tasks (Paylor et al. 1998) and is especially likely with the definition of accuracy used by Young et al. (2004), which included the number of anticipatory responses in the denominator for calculation of the proportion of correct responses.\nDuring experiment 1a we did not observe any difference between \u03b17\u2212\/\u2212 and wild-type mice with respect to omission errors. Interestingly, Young et al. (2004) reported an increase in omission errors in the \u03b17\u2212\/\u2212 mice. The difference in durations of the limited holds (LH) used in the two studies may explain this apparent disagreement. The LH, the period in which responses are recorded as being correct or incorrect following stimulus termination, was 2\u00a0s in Young et al. (2004) and 5\u00a0s in the final stage of experiment 1a. When, with a long LH, a stimulus is not detected, \u2018impulsive\u2019 responses in the period of the LH are more likely to be recorded as incorrect than as correct responses, as there are four times more incorrect response options than there are correct options. The increased impulsivity observed in \u03b17\u2212\/\u2212 mice may, therefore, have contributed to the observed reduction in accuracy.\nAt the same time, an increase in omission errors such as that reported by Young et al. (2004) may have been masked, as training with a long limited hold gives the animals more time to make an impulsive response. The trend (p<0.06) towards longer correct response latencies in \u03b17\u2212\/\u2212 mice in experiment 1a might also be explained by late \u2018correct\u2019 impulsive responses rather than slower reactions. Both groups had short response latencies and the differences between them were eliminated when impulsive responses were punished and the LH was reduced in experiment 2a. Additional and more detailed data on the temporal and spatial patterning of errors and systematic studies of the effects of punishment and of varying LH is needed to establish the validity of this interpretation.\nIn an attempt to understand the origin of the differences between the present results and those of Young et al. (2004), we retrained the mice with the LH reduced from 5 to 2\u00a0s, used only variable ITI and introduced a 2-s time-out for anticipatory responses; these task parameters resembled closely those of Young et al. (2004). In experiment 2a, carried out under these altered conditions, there was a clear increase in omission errors in the \u03b17\u2212\/\u2212 mice. In contrast, the percentage of correct responses was similar in wild-type and \u03b17\u2212\/\u2212 animals and the previously observed differences in the latencies of correct responses and in anticipatory response rates were no longer seen. This pattern of deficits in \u03b17\u2212\/\u2212 mice was indistinguishable from that observed by Young et al. (2004).\nThe absence of a difference in anticipatory responding between genotypes may have been associated with the reduction in its rate as a consequence of the time-out contingency in experiment 2a; the use of variable ITI in the retraining phase may also have been important because the initial difference between the wild-type and \u03b17\u2212\/\u2212 mice in anticipatory responding was greater in animals trained with fixed interval than with variable interval (Fig.\u00a02). This observation might reflect impaired utilisation of temporally predictable stimuli in the \u03b17\u2212\/\u2212 mice, rather than differences in impulsivity per se. Nevertheless, caution is needed because the altered performance after parametric changes may also have been associated with the longer training and greater age of the animals at this stage. \nIn experiment 1b, the absence of any significant effect of nicotine even in wild-type mice could have been attributed to the parameters of the schedule maintaining the behaviour. Nevertheless, the doses of nicotine used were within the range reported to improve accuracy and reduce omission errors in mice (Young et al. 2004). It, therefore, seemed possible that a nicotine effect would emerge when the training parameters were adjusted to resemble those used by Young et al. (2004). However, this was not the case and in experiment 2b; no dose of nicotine improved performance on any variable. The largest dose of nicotine impaired performance; omission errors and the latencies of correct responses increased, whereas reinforcers earned and anticipatory responses decreased (Fig.\u00a04f). This pattern of results can be attributed to a general deficit in operant response rates produced by the very large (1\u00a0mg\/kg) dose of nicotine. The lack of change in accuracy is compatible with explanations in terms of motor impairment rather than an attentional deficit. The data in Fig.\u00a04a indicate that these effects of nicotine were also present in the \u03b17\u2212\/\u2212 mice and, in some cases, their magnitude was increased (e.g. omission errors). The reason for this unexpected observation is unclear; it may represent a little more than a minor enhancement at certain doses of nicotine of the significant main effect of genotype on omission errors.\nWhy was there no improvement in attentional performance by any dose of nicotine? A ceiling effect associated with the very high baseline accuracy offers only a partial explanation because the baseline rate of omission errors was high (30%), but there was still no nicotine-induced improvement on either measure. Prior exposure to nicotine might be relevant, but Young et al. (2004) obtained positive effects of nicotine at 0.003 and 0.3\u00a0mg\/kg in mice previously exposed to the drug and in rats, nicotine-induced attentional enhancement did not show tolerance during 6\u00a0weeks of daily exposure to the drug (Hahn and Stolerman 2002). The parameters for training and testing during experiment 2b were also very similar to those used by Young et al. (2004). On the basis of the available information it appears that nicotine-induced improvement of performance in wild-type mice with a C57BL\/6 background is not a robust phenomenon.\nThis aspect of the study contrasts with the good agreement of the present results with those of Young et al. (2004) with respect to the performance of \u03b17\u2212\/\u2212 and wild-type mice in experiments 1a and 2a. Comparisons between C57BL\/6 and DBA\/2 mice in the 5-CSRTT have also been carried out in two different laboratories, and there was excellent agreement between the results; in each case, the DBA\/2 mice were the poorer performers, exhibiting increased impulsivity and lower accuracy (Greco et al. 2005; Patel et al. 2006). DBA\/2 mice had reduced levels of \u03b17 expression and impaired sensory gating that was corrected by \u03b17 agonists (Stevens et al. 1998).\nThe present work did not investigate the mechanisms underlying the impaired performance of \u03b17\u2212\/\u2212 mice, but some suggestions can be made. Investigations of brain areas important for attentional performance using localised lesions and drug administration have demonstrated the involvement of the medial prefrontal cortex and D1 dopamine receptor activation within this area (Muir et al. 1996; Granon et al. 2000; Hahn et al. 2003b). Dopamine utilisation in the frontal cortex also appears to be reduced in individual rats that perform badly in the task (Puumala and Sirvio 1998), whereas the \u03b17 agonist GTS-21 elevated dopamine and noradrenaline concentrations in the fronto-parietal cortex, which is implicated in attentional function (Summers et al. 1997); it is possible that a reduction or lack of \u03b17 receptors might lead to a deficit in dopamine levels in the fronto-parietal cortex, although changes in neurotransmitter systems other than dopamine may also contribute.\nIt can be suggested, speculatively, that the \u03b17\u2212\/\u2212 mouse may have some value as a model for very specific aspects of the cognitive dysfunctions in schizophrenia. Diminished frontal dopamine has been hypothesised to be important in schizophrenia (Davids et al. 2003; Laruelle et al. 2003), and post-mortem studies have shown diminished numbers of \u03b17 receptors in brains of schizophrenics. The \u03b17\u2212\/\u2212 mouse has not been tested for an auditory sensory gating deficit, the endophenotype of schizophrenia in which the \u03b17 nicotinic receptor has been strongly implicated (Freedman et al. 2002) and which may offer a cognitive explanation for the attentional deficits observed.\nIn conclusion, this study confirms that \u03b17\u2212\/\u2212 mice exhibit a weakened ability to detect visual stimuli in a test of sustained attention, suggesting that the presence of \u03b17 receptors is necessary for normal task performance. Manipulating task parameters can alter the apparent nature of the deficit, suggesting a need to compare different strains of mice under more than one set of conditions to characterise them adequately. While this observation encourages further investigation of the value of \u03b17 receptor agonists in neuropsychiatric states such as dementia (Kitagawa et al. 2003) and schizophrenia (Martin et al. 2004), the lack of effect with nicotine in the present experiments is a major concern. Identification of a protocol for assessing attention-enhancing effects of nicotine in mice may require further modifications of protocols or the use of different strains of animal.","keyphrases":["nicotinic receptor","nicotine","knockout","mice","attention","addiction","cognition","schizophrenia","drug abuse","behavior"],"prmu":["P","P","P","P","P","P","P","P","R","U"]}
{"id":"Eur_Child_Adolesc_Psychiatry-2-2-1705537","title":"Impaired working speed and executive functions as frontal lobe dysfunctions in young first-degree relatives of schizophrenic patients\n","text":"The aim of the investigation was to detect neuropsychological markers, such as sustained and selective attention and executive functions, which contribute to the vulnerability to schizophrenia especially in young persons. Performance was assessed in 32 siblings and children of schizophrenic patients and 32 matched controls using Wisconsin Card Sorting Test, Colour-Word-Interference-Test, Trail Making Test, and d2-Concentration-Test. The first-degree relatives showed certain impairments on all four tests, in particular, slower times on all time-limited tests. These results suggest the need for more time when completing neuropsychological tasks involving selected and focused attention, as well as cognitive flexibility, as a possible indicator of genetic vulnerability to schizophrenia.\nIntroduction\nSchizophrenic disorder is characterized by clinical heterogeneity and certain neurobiological alterations. The world-wide constant prevalence of schizophrenia disease suggests an genetic influence. Especially high-risk-studies have shown a broad genetic component in the traits of schizophrenia [20, 23, 39, 43]. However, little is currently known about the nature of the transmission of inheritance. Furthermore, genetic predisposition may be clinically unexpressed. Thus, it is necessary to examine ill, stabilized or recovered patients as well as unaffected individuals who are at an increased genetic risk for schizophrenia disease. Cognitive processes are a directly expression of brain functions and especially their interactions. Thus, failured, prolonged, complicated or rigided ways of thinking in persons with a genetic risk reflect disturbed neuronal connections. Studying cognitive processes of unaffected first-degree relatives with a genetic risk for schizophrenia in comparison with healthy controls allows estimating the genetic impact of the disease on infomation-processing only if both groups were matched. Then they only differ in their genetic risk and we can lead back the differences to the impact of the genes. Various vulnerability models [41, 58] and certain neuropsychological indicators of attentional and cognitive abilities such as selective attention, cognitive flexibility, inhibitory control and naming skills were examined in detail among schizophrenic patients and risk persons [11, 14, 40]. Therefore, the idea of cognitive endophenotypes is an important approach in the research of genetic risk of schizophrenia since years [24, 52]. In research the Stroop Test (FWIT), the Trail Making Test (TMT), and the Wisconsin Card Sorting Test (WCST), have been used especially to assess executive or higher-order functions such as working memory, cognitive adjustment and maintenance of set. However, only few studies have applied the d2-Concentration-Test (d2) or the above-mentioned tests to a young population with a genetic risk.\nSome studies have determined lower WCST-scores among schizophrenic patients. These have achieved a lower percentage of correct trials, fewer completed categories, and have made more preservative errors [16, 47]. Egan et al. [10] also determined impairments using the WCST and other tests among 193 unaffected siblings and even worse scores on these and other tests among 147 of their relatives who were patients. Further studies [12, 37, 38, 47, 48, 56] also indicate a genetic component regarding impairments among relatives of schizophrenic patients as compared to relatives of other patients with affective disorders and as compared to normal controls. Stratta et al. [53] showed lower WCST-scores among 92 schizophrenic patients, but not among 25 first-degree relatives. Other studies were also not able to detect any change in WCST-parameters among schizotypic persons or subjects with a genetic risk [28, 36]. Laurent et al. [33] showed deficits only in subgroups of relatives with higher rates of physical anhedonia and negative schizotypal symptoms. Therefore, the authors suggest that performance on the WCST is more likely a feature inherent to the disease process rather than an index of genetic susceptibility to the illness. WCST-deficits in schizotypic individuals [18, 42] support the hypothesis that executive function deficits may precede the onset of schizophrenia or indicate cognitive impairments in schizophrenic-spectrum syndromes.\nPerformance on the Stroop was also analyzed in detail among schizophrenic patients using various test versions. Impairments could be found repeatedly in patient groups. Nevertheless, some studies were not able to replicate these findings [33, 47]. In a review of 32 studies [19], inconsistent findings and the contribution of thought disorders and special components of the attention process to the Stroop-effect were emphasized. In genetic risk studies there are contradictory findings of poor performance among children, siblings and parents of schizophrenics [4, 9, 33, 47].\nImpairments on the TMT, especially in the second part, could also often be determined in schizophrenic patients [33, 47, 57]. However, only a few studies were able to demonstrate lower scores among those at genetic risk [10, 33].\nSome investigations showed impaired performance among schizophrenic patients or relatives on all of these tests: the Stroop, the TMT, and the WCST [13, 21, 27]. Only the investigations by Schreiber et al. [49] are known to have used the d2-Concentration-Test on schizophrenic patients and risk groups. On this test, patients, but not unaffected risks, performed more poorly (working speed and error rate) than did the controls. Furthermore, other tests have also shown reduced psychomotor speed and reaction time among schizophrenics [2, 49].\nThe findings described above suggest a subtle deficit in maintained and focused attention together with a subsequent impairment of stimulus discrimination among schizophrenics and schizotypical subjects. Due to these results, we assume these impairments principally to be an expression of the disease process, personality traits or symptomatology, such as schizoid, paranoid, extraversion, or anhedonia. Among subjects with a genetic risk for schizophrenia, cognitive dysfunctions have also been repeatedly determined [5, 32]. However, scores are inconsistent and display different impairment profiles [54]. Thus, it may be concluded that subgroups with various neurobiological and genetic profiles as well as certain symptomatology during the process of the disease can be distinguished and the vulnerable cognitive domains revealed.\nThe aim of this study (as part of an ongoing project called \u201cNeurobiological and neuropsychological risk factors for schizophrenia\u201d; [30]) is to consider the special cognitive abilities of working speed, inhibitory control, cognitive flexibility and executive functions, such as working memory, maintenance of set and naming skills using various tests among juvenile unaffected first-degree relatives of schizophrenic patients as compared to controls. Our hypothesis is that the genetic risk group of unaffected children and siblings of schizophrenics will score lower on the tests than will healthy controls. Based on frequently used tests and the results presented above, we selected the WCST, the TMT and the FWIT (Farbe-Wort-Interferenz-Test) as the German Version of the Stroop-Paradigm. A number of important studies [5, 11, 14, 32] refer to these test when assessing cognitive functioning and neuropsychological risk indicators in schizophrenic patients and persons at high risk, because these tests make demands on cognitive processes based in the frontal lobes, a brain area vulnerable to potential antecendents of schizophrenic disorder. All of these tests are supposed to have high effect sizes for test variation with a medium level of difficulty. Additionally, we used the d2-Test because it, in particular, makes demands on concentration and discrimination between similar stimuli under high time pressure in routine tasks.\nMethods\nSubjects\nA total of 32 children and siblings of schizophrenic patients without any clinical manifestation of schizophrenic symptoms (mean age in years: 16.0; males\/females: 13\/19) and 32 controls (mean age in years: 16.2; males\/females: 13\/19)\u2014carefully matched for age (range from 12 to 21\u00a0years), sex and educational level\u2014were examined. Additionally, in order to screen for the performance comparability of groups with respect to personal background data, the intellectual quotient was assessed using the Standard Progressive Matrices (SPM; [26] according to [37]). We assessed the educational level of probands and controls and also of parents of probands and controls as an indicator of their socio-economic status. Both groups did not differ significantly (Table\u00a01). Demographic characteristics are summarized in Table\u00a02. The high-risk subjects were drawn from a sample of schizophrenic in and outpatients of the Department of Psychiatry and Child- and Adolescent Psychiatry of the University of Jena and of two additional departments of psychiatry in the county of Jena. First-degree relatives of in and outpatients were asked for participation. The controls were recruited from all school types within the German School System. We also placed an ad in the local newspaper. People were asked to participate in a high-risk study conducted by the University of Jena as part of a neuropsychological study. Since recruitment was difficult we included both children and siblings of schizophrenic patients. Although siblings are a little more likely to be taken ill at schizophrenia, the genetic risk on average is nearly the same for children and siblings [51]. We made up only one group for relatives, because the risk of schizophrenia is almost equal for both. The study was approved by the ethical commission of the University of Jena. All tests took place in the Department of Child and Adolescent Psychiatry in Jena. After a full explanation of the procedures to the subjects, written informed consent was obtained from the young persons and their parents.\nTable\u00a01Education level of probands and controlsProbands with genetic riskControlsSchool typeLerning-Disabled School 11Secondary School1514Grammer School\/Advanced  Technical College\/University10\/313\/2Technical College\/Vocational School32Education level of the parents (always the highest qualification)Secondary School31Technical College\/Vocational School1717Advanced Technical College\/University1214Groups did not differ significantly Table\u00a02Demographic characteristics of probands Demographic dataSubjectsProbands with genetic riskControlsAge (years)16.0\u00a0\u00b1\u00a02.416.2\u00a0\u00b1\u00a02.0Gender (male\/female)13\/1913\/19Intelligence (SPM-IQ) Non verbal IQ101.6\u00a0\u00b1\u00a013.8100.5\u00a0\u00b1\u00a09.7Data of age and intelligence as mean values\u00a0\u00b1\u00a0standard deviationGroups did not differ significantly\nDiagnostic screening and evaluation\nPsychiatric status of both groups was determined according to the Schedule for Affective Disorders and Schizophrenia (SADS) or the Kiddie-SADS [8] by a board-certified psychiatrist or psychologist. First- or second-degree relatives of controls showing any schizophrenic symptoms were excluded, as were all probands suffering from internal or neurological disorders, schizotypical personality, affective or eating disorders, or diseases with a possible neurobiological basis, such as attentional deficit disorder or dyslexia. One subject in each group had an adjustment disorder, one had a minor depressive episode, two had social behavioral disorders without any symptoms of attentional deficit disorder, and one an enuresis. First-degree relatives showed no scores on SADS or were below threshold for diagnostic relevance. Inclusion criteria were an IQ of 70 or above according to SPM [31, 44] and an age between 12 and 21\u00a0years.\nNeuropsychological evaluation\nWe used the (WCST; [22]) in order to examine the probands\u2019 executive functions such as working memory and maintenance of set as well as cognitive flexibility. The probands were asked to successively place 128 response cards, showing symbols drawn in different combinations of color, form, and number, under one of four stimulus cards. After ten consecutive correct trials, the criteria according to which the response cards were selected changed. The examiner provided information only on correct or incorrect choices and the probands had to find out by themselves according to what criteria the response cards were to be placed. We evaluated the percentage of correct trials (WCST\/PCT), the percentage of perseverative errors (WCST\/PPE), and the number of completed categories (WCST\/NC).\nThe FWIT [1] as a version of the Stroop-Test was used to determine general alertness, naming skills and selectivity. At first the probands were asked to read aloud a list of color names printed in black, second to name the color of strokes, and third to name the color of different colored words. This run was repeated two times and the time required for reading was assessed. Finally, we calculated time needed (FWIT\/A), naming skills (FWIT\/N), selectivity of relevant information (FWIT\/S), and number of errors on the interference task (FWIT\/E).\nThe (TMT; [46]) provides information about psychomotoric speed, cognitive flexibility and working memory. Part A of the TMT requires the subjects to connect numbers in rising sequence. For Part B of the TMT, a line is drawn to connect alternating numbers and letters, starting with the number one, and then to the letter A, also in rising sequence. We determined time required for TMT\/A and TMT\/B as an indicator for working speed as well as cognitive flexibility using a ratio of TMT\/A and TMT\/B.\nThe d2-Concentration-Test (d2; [3]) provides information about the ability to discriminate between similar stimuli, measured by speed and accuracy of performance. The probands have to mark all \u201cd\u201ds that have two strokes within an array of 14 lines, with 20\u00a0s allowed per line. The probands must also discriminate between similar stimuli, because there are \u201cd\u201ds with one or more than two strokes, and \u201cp\u201ds with strokes. We counted the number of all scanned characters measuring the working speed (d2\/GZ as the German expression for number of scanned characters). Furthermore, the ratio of incorrectly marked characters (d2\/F as the German expression for error rate) to all scanned characters (d2\/GZ) as an indicator of accuracy was assessed.\nResults \nThe primary aim of the data analysis was to evaluate group differences for the parameters described above and to examine connections between the variables. Therefore, we tested our hypothesis that the genetic risk group of unaffected children and siblings of schizophrenics would perform more poorly on the tests than would healthy controls. Furthermore, we assumed that relatives of schizophrenics would show a wider pattern of impairments than would healthy controls. All statistical analysis were done using SPSS software (version 10.1).\nThe multivariate analysis of variance (MANOVA) showed significant differences between the group of children and siblings of schizophrenic patients and the controls for certain variables of performance, as well as a connection to age, intelligence and gender. In order to determine the normal distribution of parametric statistics for each variable, the score distributions of each group were examined using the Kolmogorov\u2013Smirnov-test. Homogeneity of variance of the groups was tested using the Levene Test. If there was no normal distribution, as was the case for the variables WCST\/NC and TMT\/F, then the non-parametric Mann\u2013Whitney-U-test was used to evaluate group differences. Otherwise comparisons were made using the t-test. Mean scores (\u00b1standard deviation) are shown in Table\u00a03. Correlations were determined using the non-parametric Spearman\u2013Rank-test, with selected values shown in Table\u00a04. Inhomogeneous variances arose in the variables WCST\/NC, WCST\/PCT, and FWIT\/F. The level of significance was fixed at 0.05 for all tests.\nTable\u00a03Comparison of neuropsychological data between high risk and control probandsNeuropsychological variableSubjectsComparisonsRisk probands (n\u00a0=\u00a032)Controls (n\u00a0=\u00a032)p-valuesWCSTPCT, Percentage of correct trials77.7\u00a0\u00b1\u00a010.584.0\u00a0\u00b1\u00a06.10.002+PPE, Percentage of perseverative errors9.8\u00a0\u00b1\u00a03.57.9\u00a0\u00b1\u00a02.80.010NC, Number of complete categories5.7\u00a0\u00b1\u00a00.86.0\u00a0\u00b1\u00a00.20.042*FWITA, Working speed 47.6\u00a0\u00b1\u00a08.052.2\u00a0\u00b1\u00a06.30.012N, Naming skill49.5\u00a0\u00b1\u00a07.049.0\u00a0\u00b1\u00a08.3nsS, Selectivity skill54.7\u00a0\u00b1\u00a07.155.5\u00a0\u00b1\u00a08.4nsE, Number of errors during the interference task14.9\u00a0\u00b1\u00a06.39.8\u00a0\u00b1\u00a05.10.000*TMTA, Time to execute TMT part A35.3\u00a0\u00b1\u00a010.627.8\u00a0\u00b1\u00a09.90.002B, Time to execute TMT part B83.0\u00a0\u00b1\u00a032.663.0\u00a0\u00b1\u00a018.60.002E, Number of errors0.47\u00a0\u00b1\u00a01.020.25\u00a0\u00b1\u00a00.57ns*A\/B, Ratio of times of part A to B0.462\u00a0\u00b1\u00a00.1670.452\u00a0\u00b1\u00a00.121nsd2GZ, Number of treated signs396.7\u00a0\u00b1\u00a097.7448.9\u00a0\u00b1\u00a092.10.016FGZ, Rate of errors to number of treated signs3.6\u00a0\u00b1\u00a02.13.1\u00a0\u00b1\u00a02.9nsns, Non-siginficant differencesStatistical analysis by t-test, not Bonferoni-corrected*Non-parametric test (Mann\u2013Whitney-U-test)+p-values for inhomogeneous variances Table\u00a04Selected correlations between the test variables for all probands (n\u00a0=\u00a064), shown as rho of Spearman-rank correlationWCSTFWITTMTD2PCTErrorsAction AGZWCSTPCT, Percentage of correct trials\u22120.354**0.316*\u22120.357**\u22120.387**FWITErrors\u22120.354**\u22120.471**0.347**\u22120.331**Action, Speed of a general alertness0.316*\u22120.471**\u22120.416**0.627**TMTA, Time to execute TMT part A as an inverse indicator of working speed\u22120.357**0.347**\u22120.416**\u22120.463**d2GZ, Number of treated signs as an indicator of working speed0.387**\u22120.331**0.627**\u22120.463***Correlation two-sited significant at the level of 0.05 **Correlation two-sited significant at the level of 0.01 \nGroup differences\nCompared to the controls, first-degree relatives of schizophrenic patients were significantly impaired in their performance on certain parts of all tests. For the WCST variables \u201cpercentage of correct trials\u201d (PCT), \u201cpercentage of perseverative errors\u201d (PPE), and \u201cnumber of complete categories\u201d (NCC), the analysis showed significantly worse results in the index group. The second finding of the three tests with time-limited demands, the FWIT, TMT, and d2-test, was that subjects with a genetic risk for schizophrenia worked at a significant slower rate as compared to the controls. Even though the variance of the significant results is greater in the high-risk group, these differences are not caused by extreme values. Statistical differences between the groups could not be detected for error rate on the TMT or on the d2, for cognitive control and flexibility or for naming skills on the FWIT (FWIT\/N). However, first-degree relatives of schizophrenic patients produced significantly more mistakes on the interference task of the FWIT as compared to their controls.\nEffects of age, intelligence and gender\nThese effects were taken into account in the analysis. As generally known, intelligence and speed on the FWIT, TMT, and d2, correct categorization on the WCST, as well as naming skills, improve with age for all probands. Intelligence correlated significantly with all measured performance except for selectivity and naming on the FWIT and for accuracy on the d2. With regard to gender effects (for a review, see [17, 55]) for all probands, females showed a significantly higher cognitive flexibility on the TMT\/B. Among all probands and within the risk group, males achieved significantly higher accuracy when demands were placed on cognitive adjustment during the interference task of the FWIT. They scored significantly lower on naming skills than did the females. No gender effects were found in the control group.\nRelationships between variables \nThe correlation between working speed and all time-limited tests (FWIT, TMT, d2) was highly significant, as was the correlation between speed and the PCT during the WCST. There is a positive connection between the accuracy of the d2 and the TMT. However, only the accuracy of the interference task on the FWIT correlated to parts of the WCST scores and the working speeds on the FWIT, TMT, and d2. While speed on the TMT is lower in the index group, there was no difference between the two groups regarding the ratio of the times required to complete the TMT\/A and the TMT\/B. Variable naming correlated only with general alertness on the FWIT. And selectivity on the FWIT did not correlate with any other performance variable.\nAdditionally, we did some factor analyses to see if different cognitive test variables could reflect the same cognitive function or a general vulnerability for schizophrenia. Surprisingly, after orthogonal rotation we found three factors explaining almost 60% of variance. But the other 4 of 7 components had \u201cEigenwerte\u201d between 0.55 and 0.97, so that we found almost as many factors as variables. After graphic representation, we are very cautious with an interpretation of the results of this factor analyses.\nDiscussion\nThis study examined the hypothesis that children and siblings of patients with schizophrenia, without a schizophrenic-spectrum disorder, show a pattern of decreased attentional and cognitive performance when compared to controls. Several findings of this study can be emphasized.\nFirst, subjects at risk for schizophrenia were slower on all three time-limited tests; the FWIT, TMT, and d2. These results suggest that young persons with a genetic risk need much more time to achieve the same accuracy as the controls during routine work on the d2 and the TMT\/A. The correlations among working speeds for all time-limited tests indicate that these tests assess the same components of an attentional process and that this may reflect a general vulnerability. On the other hand, on tasks with no time structure or constraints high risks showed a lower ability to establish, maintain or adjust a required conceptual level during the WCST. This finding suggests impaired executive functions, working memory, vigilance and cognitive flexibility, which cannot be compensated by using more time, as compared to the controls. Furthermore, it is fair to assume that parameters of the prolonged and more complex tasks of the WCST measure different cognitive processes than do those of the time-limited tests. Nevertheless, there are correlations between WCST-scores and other scores, especially regarding working speed.\nSecond, gender effects are greater within the risk group. In this group, females showed a worse selectivity, but only by making more mistakes. In contrast, females performed significantly better than males on naming skills on the FWIT. These findings support the assumption of gender-specific vulnerability markers, in particular that males with a genetic risk show vulnerability in respect to naming skills [50].\nThird, our findings regarding cognitive flexibility and naming skills on the FWIT are not reflected in the working speed for all time-limited tasks or in the intelligence of the probands. There is no correlation between these variables. However, there are significant correlations between the accuracy of the interference task on the FWIT with both the working speed for all time-limited tasks, as well as with WCST performance. Thus, both groups needed the same time to perform the interference task on the FWIT, but the high-risk probands made more mistakes. This is an indicator of impaired selectivity in distinguishing relevant from irrelevant stimuli. The results of the TMT test can be attributed to an impaired action speed but not to a lower ability of cognitive adjustment. Thus, the results do not allow a clear comparison of selectivity and naming performance in both groups. Probably the FWIT subtests, with their demands on naming and cognitive adjustment, reflect other aspects of the attentional process as compared to other parts of the tests used.\nThese results in first-degree relatives of schizophrenic patients without schizotypical or affective symptoms only partly confirm prior studies. Some studies show no differences on the Stroop and the WCST [28, 33]. Only the scores on the WCST variables, \u201ccompleted categories,\u201d \u201cpercentage of perceptual level response,\u201d and \u201cperseverative errors,\u201d are repeatedly lower among the genetic risks as compared to the healthy controls, especially among high-risk probands affected by symptoms such as anhedonia or those that are schizotypical [33, 56]. TMT\/B impairments could also be detected in groups with a genetic risk for schizophrenia [10, 33] suggesting a lower cognitive flexibility, again, particularly for subjects affected by schizoid or paranoid symptoms, extraversion or anhedonia [15, 34]. In contrast, we found lower working speeds on the TMT\/A in the risk group, but not decreased selectivity on the ratios of the TMT subtests. However, we were able to confirm lower scores on the WCST among the risks without these special symptoms.\nSome methodical issues must be discussed with respect to these findings. The sample size and power, the survey, the type of relatives and of control subjects may also account for the differences and the different results as compared to other studies. One factor relates to the motivation of the probands. Since they know themselves to be members of the index group it is possible that siblings or children of patients lack confidence in their own cognitive abilities and are, thus, less motivated than those in the control group. Nevertheless, abstract-logical intelligence is equal in both groups, although these scores are not influenced by time constraints. No ceiling or floor effects could be found so it may be assumed that all tests were sufficiently difficult. Furthermore, it is especially important to note that schizophrenia is etiologically heterogeneous. The higher standard deviation on almost all test variables relates to an inhomogeneous feature in the index group. Although subjects with a clinical diagnosis of a schizotypical personality disorder (SPD), affective or personality disorder were excluded, links to personality traits or other symptoms as described for our probands could also account for the lower scores in the risk group. We examined relatively small sample sizes. Therefore, an analysis of subgroups would not have been effective. In particular, a possible impact of the SPD dimensions could affect cognitive abilities, as suggested by the available studies of schizotypical persons. One could examine such effect by including a third group, namely patients with schizophrenic disorder. We took in consideration to include also patients into our statistical analysis in order to look for a continuum in behavioral symptoms and cognitive performances. In our case, the patients group was too heterogenous. We did not succeed in matching three parallel groups of first-degree relatives, healthy controls and patients. Therefore, our focus lies on cognitive impairments of relatives of schizophrenic patients. Especially the recruitment of adolescent patients and much more of their relatives is a fussy issue in the research of neuropsychological indicators of cognitive abilities. Nevertheless, there is a need for further neuropsychological research to evaluate the impact of subtypes of schizophrenia on cognitive abilities. The discussion concerning the variability of risk groups is also reflected in our results with respect to the contrast of trait markers versus prodromali, the sensitivity of test versions and the specificity to schizophrenic disease [6, 7, 25, 29]. Furthermore, comparisons between the patients and their symptomatology during the course of the disease, and between subjects with genetic or symptomatical risks and controls may provide more insight into possible traits signaling vulnerability to schizophrenia. It is important to identify risk factors for schizophrenia as early as possible because of the poor prognoses for early-onset schizophrenia [35, 45]. The design of the present study did not allow the analysis of such a complex issue. Effects are currently being evaluated in order to determine patterns of cognitive vulnerability of risk subgroups. However, the results from our ongoing project did confirm a link to genetic influences, because both groups were matched for age, gender and intelligence. So the differences between probands and healthy controls are due to their relationship with schizophrenic patients, respectively to the genetic vulnerability of first-degree relatives. Our results demonstrate that attentional impairments as shown by the data of the FWIT, TMT, and d2 may be a trait indicator of the disease and so, too, may deficits of cognitive adjustment and executive functions as determined by the WCST. We were able to show that starting at a young age persons at risk work more slowly in general and not only on specific interference tasks. This suggests a more easily distracted working memory even among these young subjects. Therefore, our findings support the need for future studies based on the same and similar multiple tasks in order to gather more evidence in the search to identify subgroups of vulnerable subjects.","keyphrases":["attention","schizophrenia","genetic vulnerability","cognitive functions"],"prmu":["P","P","P","P"]}
{"id":"J_Magn_Reson-2-1-2323944","title":"A comparison between equations describing in vivo MT: The effects of noise and sequence parameters\n","text":"Quantitative models of magnetization transfer (MT) allow the estimation of physical properties of tissue which are thought to reflect myelination, and are therefore likely to be useful for clinical application. Although a model describing a two-pool system under continuous wave-saturation has been available for two decades, generalizing such a model to pulsed MT, and therefore to in vivo applications, is not straightforward, and only recently have a range of equations predicting the outcome of pulsed MT experiments been proposed. These solutions of the 2-pool model are based on differing assumptions and involve differing degrees of complexity, so their individual advantages and limitations are not always obvious. This paper is concerned with the comparison of three differing signal equations. After reviewing the theory behind each of them, their accuracy and precision is investigated using numerical simulations under variable experimental conditions such as degree of T1-weighting of the acquisition sequence and SNR, and the consistency of numerical results is tested using in vivo data. We show that while in conditions of minimal T1-weighting, high SNR, and large duty cycle the solutions of the three equations are consistent, they have a different tolerance to deviations from the basic assumptions behind their development, which should be taken into account when designing a quantitative MT protocol.\n1\nIntroduction\nThe Magnetization Transfer (MT) effect is based on the exchange of magnetization occurring between groups of spins characterized by different molecular environments. In biological tissues, two or more \u201cpools\u201d of protons can be identified: those in free water (the free, or liquid, pool) and those bound to large molecules (referred to as restricted, semisolid, or macromolecular, pool). The latter protons are characterized by a very short transverse relaxation time (T2) and therefore do not directly contribute to signal intensity in conventional magnetic resonance (MR) images. Nevertheless, it is possible to sensitise an MR experiment to the magnetic resonance characteristics of macromolecular protons by exposing the sample to radio-frequency (RF) energy several kilohertz away from the Larmor frequency. Protons in free water are relatively insensitive to such irradiation, but it can cause saturation of protons in the semisolid pool which, due to their short T2 and correspondingly large line width, are responsive to irradiation at these frequencies. In these conditions, any exchange of magnetization between pools results in a decreased intensity of the observed MR signal.\nFrom a quantitative MT model based on the exchange between two pools Henkelman et al. [1] derived a signal equation for the continuous wave (CW) case, in which RF irradiation of particular (constant) amplitude and several seconds duration is used to saturate the macromolecular pool. The parameters characterizing the two pools in the model are potentially interesting to measure, and they can be estimated by fitting Henkelman\u2019s equation to a set of MR measurements obtained in the presence of MT pulses with a suitable set of amplitudes \u03c91 and offset frequencies \u0394f.\nAs CW irradiation is impractical and generally not available for in vivo imaging experiments, in vivo MT-weighted MRI is generally obtained using the so-called pulsed MT acquisition, in which the long period of saturation is replaced by a much shorter irradiation pulse (typically applied just before each excitation pulse) along with intervals without irradiation (during which data is collected). For data from this type of acquisition, Henkelman\u2019s equation must be modified to allow for the short duration of the saturation pulses relative to T1\n[2]. A number of such modified signal equations for pulsed MT, all based on the same original two-pool model, have been developed [3\u20135]. While the numerical results published so far suggest reasonable consistency across the solutions predicted by these equations, no direct comparison is available, and the differing conventions and symbols used mean that evaluating discrepancies and similarities between them is not straightforward.\nThis paper is concerned with the comparison of three of these signal equations\u2014two derived by Sled and Pike [3,6], plus that of Ramani et al. [5]. Ramani et al. used a CW power equivalent approximation (CWPE) [5] where the pulse is simply replaced by a CW irradiation with the mean square amplitude that would give the same power over the interval between MT pulses. By means of the CWPE approximation, Henkelman\u2019s steady state model can be straightforwardly applied to the in vivo MRI case, neglecting the imaging elements of the pulse sequence. Due its steady state nature, the implicit assumption within the equation is that the relative signal intensity in data obtained with different MT-weightings only depends on the characteristics of the MT pulse, and that T1 and T2 relaxations equally affect all measurements.\nAs Ramani\u2019s equation does not explicitly model the effects of the excitation pulses and TR, its description of the MT-weighted signal is valid only when the degree of T1-weighting in the acquisition sequence is minimal. As it effectively assumes that the MT pulse is applied continuously, another parameter likely to affect the accuracy of Ramani\u2019s equation is the duty cycle, i.e. the duration of the MT pulse relative to the repetition period, whose effect has never formally been investigated. Sled and Pike [6] propose an alternative equation which can be fitted directly to the measured signal. Their solution is derived by approximating the pulse sequence as a series of periods of free precession, CW irradiation and instantaneous saturation of the free pool. It has the advantage of incorporating the effect of the excitation RF pulses, and also makes it possible to account for saturation effects of the excitation. Together with this solution, the authors propose also a simpler variant which neglects free precession, thus assuming a succession of instantaneous saturation of the free pool and CW irradiation of the macromolecular pool for the total duration of the interval between pulses. Both equations presented by Sled and Pike for in vivo applications require the numerical evaluation of ordinary differential equations (when modeling the rate of saturation of the macromolecular pool with a super-Lorentzian, see next section), at least for the estimation of the effect of the MT pulse on the free pool, and they are therefore computationally intensive. Ramani\u2019s solution has the advantage of being simpler, at the price of its inability to account for the effects of the excitation pulses. A recent paper presented an evaluation of these signal equation, validated using animal data [7]. Here we first review the theory behind them and then use numerical simulations to extend the range of experimental conditions under which each can be tested (investigating how duty cycle, saturation effects of the excitation and noise affect the MT parameters fitted by each of them). We also perform a statistical comparison between MT parameters estimated using each signal equation in healthy brain tissue from in vivo data.\n2\nTheory\n2.1\nCoupled Bloch equations\nAssuming that the MT effect can be modeled using a liquid pool (A) and a macromolecular pool (B), the magnetization of either pool can be described by its longitudinal component  and its transverse components . The exchange between pools associated with the transverse components of magnetization can be considered negligible due to the extremely short T2 associated with the macromolecular pool [2,6]. The coupled Bloch equations for the system can thus be written as follows:\nIn Eqs. (1)\u2013(4),  represents the transverse relaxation time of the liquid pool,  and  are the fully relaxed values of magnetization associated with the two pools (assumed dimensionless), RA and RB are their longitudinal relaxation rates, and R is the exchange rate constant. \u0394f represents the frequency offset of the pulse, while \u03c91(t) is the time dependent amplitude of the pulse expressed in rad\u00a0s\u22121 (i.e. the angular frequency of precession induced by the pulse). RRFB(\u0394f, \u03c91(t)) is the rate of saturation of longitudinal magnetization in pool B due to the irradiation by the amplitude defined by \u0394f and \u03c91(t), and depends on the transverse relaxation time of the macromolecular pool, . Li et al. [8] show that, in brain tissue, the specra associated with macromolecular pool are better modeled by a super-Lorentzian, (with  being the average power of the MT pulse), yielding:and we adopt this model here.\nWe remark here that this notation is not universal. Some authors label the A and B pools as \u2018F\u2019 and \u2018R\u2019, respectively [6], or \u2018f\u2019 and \u2018m\u2019 [9,10] and use the symbol W instead of RRFB\n[6]. The pseudo first-order exchange rates,  and , are often referred to as kf (or simply k) and kr.\n2.2\nSled and Pike\u2019s RP signal equation\nAssuming that the pulse sequence consists of an MT pulse followed by an excitation pulse and by a period of recovery, Sled and Pike [6] decompose it into a series of periods where Eqs. (1)\u2013(4) have exact or approximate solutions. These solutions can then be concatenated by imposing the appropriate initial conditions, leading to an expression for the measured signal which is less expensive to compute than numerically integrating the full set of differential equations. The effect of an MT pulse on the macromolecular pool is modeled as a rectangular pulse whose width is equal to the full width at half maximum (\u03c4RP) of the curve obtained by squaring the instantaneous amplitude of the MT pulse throughout its duration, and whose amplitude is such that the pulses have equivalent average power (rectangular pulse, or RP, approximation). The effect of the pulse on the liquid pool is modeled as an instantaneous fractional saturation of the longitudinal magnetization. Such fractional saturation (S1A) is estimated by solving (numerically) the system of Eqs. (1), (3) and (4) when R and RA are set to 0.\nIn matrix form [6], considering the longitudinal components of magnetization onlyInstantaneous saturation of the free pool, caused by both MT and excitation pulses, is simply described by multiplying Mz by the matrix S (where \u03b8 is the excitation flip angle)The state of the magnetization after a period t1 (assuming starting time\u00a0=\u00a0t0) is given by the solution to the system of Eqs. (1) and (2) for either free precession [FP] or CW:with\nAccording to Sled and Pike\u2019s RP approximation, over the time interval T between application of MT pulses (typically the time required to excite and collect data for a single k-space line of a single image slice), Mz undergoes instantaneous saturation, CW irradiation for a period \u03c4RP\/2, FP for a period (T\u00a0\u2212\u00a0\u03c4RP), and CW for another \u03c4RP\/2. After including all thee steps, we can impose the equalityand solve for Mz yielding an equation for the longitudinal components of magnetization. Recalling that the signal observed at readout is(where the repetition time TR may be \u2a7eT depending of the details of the image acquisition) it is thus possible to model the MT-weighed signal. The solution to Eq. (10) can be easily computed in matrix form, and we adopt this procedure for all the following experiments.\n2.3\nSled and Pike\u2019s CW signal equation\nA simpler expression is presented in the same paper [6], where the effect of the MT pulse on the macromolecular pool is described by a CW irradiation of duration T. In this case, over the same period T, Mz undergoes instantaneous saturation, and CW irradiation (of the restricted pool only) for a period T. Following the same procedure as described above, a more manageable analytic expression can be derived in this case:Here  is the steady state solution obtained by Henkelman et al. [1] for CW irradiation of the restricted pool of duration T:which is equivalent to the first element of the vector . In Eq. (12),\nWe note here that for the specific case of the 3D spoilt gradient echo acquisition described in this paper (see Section 3) T\u00a0=\u00a0TR.\nSled and Pike also introduce, as a useful index which is believed to correlate with myelin content, the relative size of the macromolecular pool [6]\nF, defined asF can be fitted directly, by substituting  in Eq. (13) and previous. More details on these signal equations can be found in [3,6,11].\n2.4\nRamani\u2019s signal equation\nHenkelman\u2019s solution [1] for the CW case is obtained by solving Eqs. (1)\u2013(4) in the steady state, i.e. setting the derivatives on the left hand side to zero. Ramani et al. [5] adopt the same approach in the pulsed MT case, simply replacing the MT pulse with a CW irradiation with the same mean square amplitudewhere PSAT is the mean square saturating field.\nIn order to ease the comparison between the three signal equations, we break with the terminology of the original paper [5], where the macromolecular fraction f (with f\u00a0=\u00a0F\/(F\u00a0+\u00a01)) was used, and instead rewrite the Henkelman\u2013Ramani expression using F and  to obtainwhere M0 is the signal with no MT-weighting (again assuming a constant of proportionality of c\u00a0=\u00a01). Note that while in the equations presented in the previous section, M0 is the equilibrium magnetization, in Eq. (17) it simply represents the partially recovered magnetization available prior to the application of an MT pulse.\n2.5\nFitting\nAll the equations are written in terms of seven parameters:  and , but these cannot be uniquely determined [1,12]. Constraints are imposed by measuring the observed longitudinal relaxation rate of the sample, RAobs(=1\/T1obs) independently, linked to RA by Henkelman et al. [1]\nA further issue is that dependence of S(\u03c91, \u0394f) on RB is weak, making fitting of this parameter unstable. Since the estimates of the other parameters are largely insensitive to its value, RB is usually kept fixed at 1\u00a0s\u22121\n[1,5,6]. This reduces the number of free parameters to 5, which can be estimated by fitting the equation to five or more measurements with different combinations of \u03c91(t) and \u0394f.\n3\nMaterials and methods\n3.1\nNumerical simulations\nIn order to compare the three signal equations, and to highlight their shortcomings, we need to test their performance against data corresponding to a known set of parameters. The easiest way to obtain such data is to synthetically produce them, using numerical simulations.\nWe consider here the case of an MT-weighted spoiled gradient echo acquisition, where off-resonance saturation is achieved using Gaussian pulses (of duration \u03c4SAT) applied once every TR (just prior to RF excitation), while on-resonance excitation is obtained using short 5-lobe sinc pulses (in the presence of a \u2018slab selection\u2019 gradient).\nEqs. (1)\u2013(4) can be solved numerically to predict the longitudinal magnetization at the end of the MT pulse (i.e. just before the excitation pulse), . Since the measured signal intensity is proportional to the transverse magnetization at readout (if we neglect  decay):The MT pulse is characterized by its maximum amplitude, B1SAT, MAX, by its duration, \u03c4SAT, and by the standard deviation of the Gaussian envelope, \u03c3. The excitation pulse is characterized by its maximum amplitude, B1EXC, MAX, by its duration, \u03c4EXC, and by its bandwidth, BW. Both pulses can be described by their equivalent on-resonance flip angle, given by the integral over pulse duration of \u03c91(t). Additionally, we assume the excitation pulse to have no effect on pool B. The measured signal intensity can be estimated by calculating the solution of Eq. (19) as Mz tends towards a steady state, i.e. after solving Eqs. (1)\u2013(4) for several TRs, until the difference between Mz(TRn\u00a0+\u00a0\u03c4SAT) and Mz(TRn+1\u00a0+\u00a0\u03c4SAT) is less than 0.01% of Mz(TRn\u00a0+\u00a0\u03c4SAT).\nWe simulate four experiments to probe the effects of different parameters on the fits. Firstly we aim to identify any systematic biases in the three signal equations with respect to variable experimental conditions, by fitting them to noise-free simulated data (Experiments 1\u20133); next we investigate their robustness in the presence of noise, by using a Monte Carlo approach (Experiment 4). The simulated signal is computed by using a Runge\u2013Kutta ordinary differential equation (ODE) integrator with adaptive step-size control [13]. Spoiling of the transverse magnetization is simulated by setting the transverse components of magnetization equal to zero before the occurrence of every MT pulse. All experiments use the same set of MT parameters , chosen to be similar to values measured previously in white matter [14,15] and shown in Table 1 as \u2018test set\u2019. All also use the same excitation pulse parameters (\u03c4EXC\u00a0=\u00a03.2\u00a0ms, and BW\u00a0=\u00a02.5\u00a0kHz), but other values differ:\nExperiment 1: In the first simulation, we investigate the accuracy of Ramani\u2019s, Sled and Pike\u2019s CW and Sled and Pike\u2019s RP signal equations as a function of duty cycle, by simulating 5 MT experiments using an excitation flip angle of 5\u00b0, TR\u00a0=\u00a030\u00a0ms, and varying \u03c4SAT between 5 and 25\u00a0ms, in steps of 5\u00a0ms. The full width at half maximum (FWHM) of the pulses varies accordingly between 2.29 (\u03c3\u00a0=\u00a00.97) and 11.45\u00a0ms (\u03c3\u00a0=\u00a04.86\u00a0ms), in steps of 2.29\u00a0ms. Each simulated set consists of 60 points, generated using only two fixed values of \u03c91CWPE (250.2 and 850.7\u00a0rad\u00a0s\u22121) (and thus different MT flip angles for each value of \u03c4SAT), following Sled and Pike [6,15], and 30 values of \u0394f per flip angle. The offset frequency ranges from 400 to 30,000\u00a0Hz, sampled at regular interval on a logarithmic scale. The three equations are fitted to the synthetic datasets using the Levenberg\u2013Marquardt method, as implemented in Numerical Recipes [13], to yield the estimated parameters. Numerical derivatives are computed where an analytical expression is unavailable. The test set of parameters (see Table 1) are used to provide the initial parameter estimates, and RAobs is obtained by solving Eq. (18) with respect to this quantity. The same ODE integrator used to compute the simulations is used to estimate S1A at every step for Eqs. (7), (11) and (12).\nExperiment 2: Secondly, we explore the effects of saturation of the excitation on the estimated parameters, simulating the outcome of six MT experiments using regularly spaced excitation flip angles ranging from 5\u00b0 to 20\u00b0 and TR\u00a0=\u00a030\u00a0ms. We fix \u03c4SAT\u00a0=\u00a015\u00a0ms and keep the other parameters as in the first experiment. With the exception of the excitation flip angle, the input parameters are identical for all six cases.\nIn order to check that our results are not specific to the choice of the sampling scheme, we repeat Experiments 1 and 2 with an alternative sampling scheme generated using four fixed values of \u03c91CWPE (250.2, 450.4, 650.5 and 850.7 rad\u00a0s\u22121) and 15 values of \u0394f per flip angle.\nExperiment 3: As Sled and Pike used two sequences with two different MT pulse durations and two TRs to constrain the estimate of RM0B, we create a synthetic dataset formed by two \u201csequences\u201d (40 MT points with TR\u00a0=\u00a030\u00a0ms, \u03c4SAT\u00a0=\u00a015\u00a0ms, \u03b8\u00a0=\u00a05\u00b0, 2 MT flip angles equal to 250\u00b0 and 850\u00b0, respectively; and 20 with TR\u00a0=\u00a045\u00a0ms, \u03c4SAT\u00a0=\u00a020\u00a0ms, \u03b8\u00a0=\u00a06\u00b0, 2 MT flip angles equal to 353\u00b0 and 1202\u00b0, respectively). We then compare the accuracy of the MT parameters estimated fitting the three signal equations to such a dataset and to the \u201csingle sequence\u201d dataset obtained in Experiments 2 when fixing \u03c4SAT\u00a0=\u00a015\u00a0ms and \u03b8\u00a0=\u00a05\u00b0.\nExperiment 4: Finally, in order to investigate the sensitivity to noise, we add complex noise with zero mean Gaussian real and imaginary parts to the dataset obtained in Experiment 2 for \u03c4SAT\u00a0=\u00a015\u00a0ms and \u03b8\u00a0=\u00a05\u00b0. We then take the modulus to obtain a noisy data sample. The standard deviation of the Gaussian noise is set to be M0\/\u03a3, where \u03a3 is the desired SNR in the unweighted image, which we vary over the interval [20,\u00a0300]. (The SNR values typically observed in 3D spoiled gradient echo scans from our system, with acquisition parameters similar to those detailed below (see Section 3.2), typically range between 40 and 100 depending on the coil used, resolution, use of parallel imaging, etc.) For each level of noise, we generate 10,000 sets of noisy independent samples and fit the three equations to each set. Look-up tables are used for the super-Lorentzian lineshape, the fractional saturation S1A, and their derivatives, in order to speed up the computation.\n3.2\nIn vivo data\nA single subject (female, 34 years old) was scanned twice on a 1.5\u00a0T system (SIGNA Horizon Echospeed, General Electrics, Milwaukee, WI, USA) using a 3D MT-weighted fast spoiled gradient recalled-echo (SPGR) sequence [15] (TR\/TE\u00a0=\u00a028\/5.1\u00a0ms, Gaussian MT pulses, duration\u00a0=\u00a014.6\u00a0ms, standard deviation\u00a0=\u00a02.84\u00a0ms, bandwidth\u00a0=\u00a0125\u00a0Hz, matrix\u00a0=\u00a0256\u00a0\u00d7\u00a096\u00a0\u00d7\u00a032, FOV\u00a0=\u00a0240\u00a0\u00d7\u00a0180\u00a0\u00d7\u00a0160\u00a0mm3, to reconstruct twenty-eight 256 by 256 voxel slices). The excitation flip angle was 5\u00b0 on the first session and 15\u00b0 on the second one, the interval between scans was 7 days. On each occasion a dataset of 20 MT points was obtained, using 2 MT pulse flip angles (220\u00b0 and 820\u00b0, corresponding to a CWPE amplitude of 251.1 and 861.2\u00a0rad\u00a0s\u22121), and 10 values of \u0394f per flip angle. \u0394f ranged between 400 and 20,000\u00a0Hz, and was stepped using a constant logarithmic interval. In addition to the MT data, three 3D SPGRs (TR\u00a0=\u00a013.1\u00a0ms, TE\u00a0=\u00a04.2\u00a0ms, same FOV and resolution as the MT sequence) were also obtained on each occasion, with three different excitation flip angles (\u03b8\u00a0=\u00a025\u00b0, 15\u00b0, 5\u00b0) in order to independently estimate the longitudinal relaxation rate of the system, RAobs. The body coil was used for signal transmission and the manufacturer\u2019s eight-channel head coil was used for reception. The total scan time was about 45\u00a0min.\nThe study was approved by the Joint Research Ethics Committee of The National Hospital for Neurology and Neurosurgery and the Institute of Neurology, UCL, and the subject gave written informed consent before taking part.\n3.3\nImage analysis\nThe two datasets (one from each MRI session) were processed on a Unix workstation (Sun Microsystems, Mountain View, CA, USA), as described elsewhere [15]. Briefly: the 20 MT-weighted volumes obtained with the MT-weighted SPGR sequence and the three volumes obtained with the non-MT-weighted SPGR sequence were co-registered to the first MT-weighted volume using a modified [16] version of Automated Image Registration (AIR, available at http:\/\/air.bmap.ucla.edu:16080\/AIR) [17]. RAobs was estimated on a pixel-by-pixel basis by fitting the theoretical SPGR signal equation to the signal in the non-MT-weighted SPGR images, as a function of the flip angle [18]. The 3 MT signal equations were fitted to the remainder of the images (as described for the synthetic data) yielding estimates of the MT parameters. Six bilateral regions of interest (ROIs), three located in white matter (frontal, temporal and internal capsule) and three in gray matter (thalamus, putamen, caudate nucleus) were outlined on the T1-weighted images obtained from the non-MT-weighted SPGR scan (flip angle\u00a0=\u00a025\u00b0). The 12 ROI outlines were then superimposed on the MT parameter maps, yielding 6 (3 signal equations times 2 flip angles) estimates for each of the following: , and . Paired sample T-tests were used to compare the mean estimated parameters between equation solutions, and two-sample T-tests were used to test inequalities between flip angles, considering statistically significant two-tailed p values lower than 0.01.\n4\nResults\nTo provide a qualitative description of the accuracy of the three signal equations, we show in Fig. 1 the MT spectra simulated using Eq. (18) and the test set in Table 1, together with those simulated using each of the three equations and the same test set, for two of the cases explored in Experiment 1 (A: duty cycle\u00a0=\u00a050%, flip angle\u00a0=\u00a05\u00b0; B: duty cycle\u00a0=\u00a017%, flip angle\u00a0=\u00a05\u00b0). All curves are normalized to the maximum intensity. As expected, the largest deviations between signal equations are observed at high power, for small offset frequencies, with Ramani\u2019s equation providing the least accurate description. The deviation between Ramani\u2019s predictions and the others are more pronounced for lower duty cycles (Fig. 1B).\n4.1\nDuty cycle effect\nThe estimates of , and  against duty cycle are shown in Fig. 2. Results for  are omitted as the estimates from all signal equations converged to the test set irrespective of duty cycle. Both of Sled and Pike\u2019s equations are less sensitive to changes in the duty cycle than Ramani\u2019s equation (with the exception of ), providing very consistent estimates, except for , for which the CW variant yields a value closer to that used to create the simulation. The dependence of Ramani\u2019s on the duty cycle is non-linear, with estimated values tending towards a plateau for duty cycles \u2a7e50%. For Ramani\u2019s equation,  and  are the parameters most sensitive to duty cycle changes. Although more stable, the estimates obtained at larger duty cycles are not necessarily more accurate than those obtained at the lowest duty cycle simulated (17%).\nThe same experiment repeated with a different sampling scheme yielded almost identical results for duty cycles >17%. For duty cycle\u00a0=\u00a017%, Sled and Pike\u2019s CW equation converged to  and , and the RP variant converged to  and , while Ramani\u2019s equation produced results very similar to those obtained with the two-power scheme.\n4.2\nSaturation effect of the excitation\nThe MT parameter estimates obtained from noise-free simulated data at various flip angles are reported in Fig. 3, again with the exception of  which is accurately determined by fitting all signal equations irrespective of the excitation flip angle, and therefore is not shown. As the flip angle (and thus the amount of saturation) increases, the estimates of the MT parameters based on Ramani\u2019s signal equation increasingly deviate from the true values. Additionally, unlike the other parameters, the dependency of the estimated  on the amount of saturation does not appear to be monotonic. Sled and Pike\u2019s RP approximation provides the most consistent estimates across flip angles. The CW variant behaves similarly, at least for flip angles lower or equal to 15\u00b0, with increasingly biased results at higher flip angles.\nWe report in Table 1 the estimated MT parameters obtained by fitting the three equations to the simulated noise-free 60 point dataset with excitation flip angle of 5\u00b0 (minimum saturation effect from the excitation pulse) and duty cycle \u224850% (at which point duty cycle effects have reached a plateau, and the three signal equations seem to provide consistent estimates). All equations give similar results (very close to the \u2018test set\u2019 of parameters used in creating the simulation (see Table 1)). The two variants of Sled and Pike\u2019s solution provide more accurate estimates than Ramani\u2019s equation for all parameters, with the CW approximation yielding a more accurate value for , and RP approximation yielding a slightly more accurate value for F (which Ramani\u2019s equation tends to underestimate). For all the signal equations, the largest error is in the estimation of  (7.6% of true value for Sled and Pike\u2019s RP equation, 10.2% of true value for Sled and Pike\u2019s CW equation, 21.2% of true value for Ramani\u2019s equation). The results of Experiment 3 (fitting the signal equations to a combined dataset obtained with differing \u201csequences\u201d, i.e. different TR, \u03c4SAT, and flip angle) are also shown in Table 1. The estimates of  appear highly sensitive to the combination of acquisition parameters, while all other parameters are not. Interestingly,  is underestimated using both of Sled and Pike\u2019s equations when using 2 sequences (and the error becomes larger), while it is generally overestimated when using a single one.\nWhen using the four-power scheme the main difference compared to the two-power scheme was in the estimates of , which were slightly lower for the two variants of Sled and Pike\u2019s solution (between 2.98 and 3.1 for the CW and between 3.3 and 3.4 for the RP approximation).\n4.3\nSensitivity to noise\nFig. 4 compares the estimates of the MT parameters using the three signal equations in the presence of noise. Overall, the estimates obtained from the 60 point noisy dataset using any of the three signal equations are characterized by similar precision, although Ramani\u2019s solution seems to provide slightly more robust results than Sled and Pike\u2019s at SNR lower than 120 for  and . The two equations proposed by Sled and Pike\u2019s provide very similar estimates (with similar precision at all SNR levels) for  and . The CW variant provides more accurate values for . The parameter whose estimate deviates most from its true value is (for all signal equations) , with Ramani\u2019s estimates deviating more than the others. The standard deviation associated with this parameter, however, is smaller when using Ramani\u2019s equation, at low SNR. The opposite is true (with the two variants of Sled and Pike\u2019s formulation giving very similar performances) at high SNR.\n4.4\nIn vivo results\nThe MT parametric maps obtained by fitting all three equations to the 5\u00b0 dataset obtained in vivo are characterized by similar quality (examples of F maps are shown in Fig. 5). Fitting the two equations proposed by Sled and Pike to the 15\u00b0 dataset was more problematic; in some voxels (most commonly in gray matter, and at the boundary between tissues) the equations gave physically meaningless parameter estimates, particularly for  and . It is possible that this is the result of T1-contrast between tissues and CSF increasing the sensitivity of partial volume effect to motion. Fitting of Eq. (17) appeared to be more robust.\nTable 2 shows the values (mean and standard deviation) obtained for each white and gray matter ROI using each signal equation and both flip angles.\n4.4.1\n5\u00b0 data (between equations)\nAlthough the estimates of  and  obtained with Ramani\u2019s equation were statistically different (p\u00a0<\u00a00.001, providing higher estimates  and lower estimates of F and ) than those obtained with either variant of Sled and Pike\u2019s equations, for all parameters the absolute difference between Ramani\u2019s and Sled and Pike\u2019s estimates was always lower or comparable to the between-voxels standard deviation (within each ROI) (see Table 2). When comparing the two solutions proposed by Sled and Pike, none of the variables differed (p values of 0.02 for , 0.6 for F, 0.32 for  and 0.47 for ).\n4.4.2\n15\u00b0 data (between equations)\nConversely, when comparing the parameters obtained by fitting different equations to the 15\u00b0 dataset, the mean values were all significantly different (p\u00a0<\u00a00.001), with the exception of  obtained using the two variants of Sled and Pike\u2019s solution (p\u00a0=\u00a00.09). The largest differences were in the estimates of F and  obtained using Ramani\u2019s equation with respect to both Sled and Pike\u2019s equations. The absolute difference between estimates, in this case, were approximately one order of magnitude larger than the between-voxel standard deviation (within each ROI) of the same parameters.\n4.4.3\n15\u00b0 data vs. 5\u00b0 data\nThe estimates of  obtained fitting any of the three equations to the 5\u00b0 dataset were significantly different from those obtained by fitting the same equation to the 15\u00b0 dataset (p\u00a0<\u00a00.001). For Ramani\u2019s equation, the estimates of F and  obtained from the 5\u00b0 dataset were also significantly different from those obtained from the 15\u00b0 dataset (p\u00a0<\u00a00.001).\n5\nDiscussion\nWe have shown that the estimates of , and  obtained by fitting different equations describing the behavior of the two-pool model under conditions of pulsed MT to proton density-weighted noise-free simulated data with an MT duty cycle of approximately 50% (typical of most in vivo applications) are in good agreement and deviate only slightly from the values used to create the simulations. There are some systematic differences, however, with Sled and Pike\u2019s equations typically providing more accurate results. Overall, these findings are in line with those reported by Portnoy and Stanisz [7].  is substantially overestimated when using all signal equations, albeit less so when using the Sled and Pike\u2019s RP approximation. The discrepancy between the estimate of this parameter and the observed T2 of the system has been observed before [11]. As white matter is known to have multiple free water T2 components, one of the explanations provided by researchers is that the T2 measured by spin echo experiments and  measured by MT experiments represent different weighted averages of multiple water components. This explanation, however, does not apply to analogous results obtained in gels [1,11]. Similarly, the results of our simulations suggest that such a difference could merely result from the inadequacy of the model to estimate this parameter. It is unsurprising that, for both simulated and in vivo data, the largest quantitative difference between the results obtained when fitting different equations to the same (low flip angle) data is in the estimation of this parameter. When the effects of saturation from the excitation pulse are minimized, in fact, the main difference between Ramani\u2019s and Sled and Pike\u2019s equations is in the way the MT pulse effect on the liquid pool is modeled. It is interesting to notice that others have often reported this parameter to be underestimated\n[11], and our own in vivo results are consistent with such observations (Table 2). However, when fitting the MT signal equations to simulated data, we observed the opposite trend. A possible explanation for this inconsistency is the sensitivity of Levenberg\u2013Marquardt fitting to the values used as starting points: while for synthetic data we can use the real (test set) numbers, when fitting in vivo data we can only provide a \u2018best guess\u2019. In order to compensate for this effect we repeated Experiments 1 and 2 running the fitting procedure 10 times, each time perturbing the initial guesses (independently for each parameter) by a random factor from a Gaussian distribution with zero mean and standard deviation equal to 10% of the test parameter [19]. We retained and compared the set of parameters which gave the best fit out of the 10 trials in each case. This did not affect the estimates obtained using Sled and Pike\u2019s equations.  obtained using Ramani\u2019s equation was even larger in this case, with all other parameters almost unaffected (data not shown).\nAn alternative explanation is that we used a larger number of points for simulations than for the human brain data. The range and spacing of the sampling points is likely to affect the results of the fitting [20], and it is interesting to observe that even in the simulated data,  is underestimated when using two \u201csequences\u201d (as recommended by Sled and Pike [6,11]). Contrary to their observations, however, the estimates of  were virtually unaffected by this experimental parameter, and therefore our simulation experiments do not support the need for this type of acquisition. It is not clear why our result in this case should differ from that of Sled and Pike, but it is possible that this is due to our use of a different fitting routine. Because of our simulation results, we chose not to adopt the two-sequence approach for our in vivo comparison, as this would have required all the MT-weighted acquisitions to be normalized to the same maximum value, and also might introduce variable degrees of T1-weightings, which would further violate the assumptions underlying Ramani\u2019s equation.\nAlthough the reason why  is so strongly affected by the use of a dual TR protocol is unclear, it has been reported by others that even when using more accurate formulations, the estimates of R and  are highly sensitive to the choice of data points [7], and this has been explained as a result of the poor sensitivity of the equations to these parameters, or to systematic errors between the model and the data, which can vary with the sampling points.\nThe poor sensitivity of the two-pool model to changes in  partially explains the robustness of the other parameters against its misestimation. As T2 can be determined by the use of alternative techniques, the remaining parameters are generally of greater interest. We would like to stress, however, that we are not suggesting the two-pool model is an exact representation of the distribution of macromolecular pools or of their exchange. We simply report that all of these relatively simple signal equations appear to be able to provide consistent and practical information which might be useful in a clinical\/clinical research context. Although we did not directly compare the sensitivity of different signal equations to changes similar to those caused by pathology, we have attempted to provide useful guidelines to the choice of the most appropriate one for a particular experimental setting.\nAlthough suggesting optimal sampling schemes is beyond the scope of this paper, it is clear from Fig. 1 that low offset frequencies (<1\u00a0kHz) should be avoided when using Ramani\u2019s equation. This is consistent with the set of points used by Ramani et al. in their original paper [5], and with the findings of Portnoy and Stanisz [7], and confirms that in pulsed MT experiments it is difficult to model the behavior of magnetization at low off-resonance frequencies. Our results also confirm the observation of Portnoy and Stanisz [7] that Ramani\u2019s model tends to underestimate the signal especially close to the Larmor frequency. This deviation at low frequency offsets is likely to be a consequence of the poor ability of the CWPE approximation to characterize the liquid pool. As noted by Portnoy and Stanisz [7], the cut-off of 1\u00a0kHz they empirically determined might vary with field strength and MT pulse amplitude and bandwidth.\nWe should also note that it is apparent from Figs. 2\u20134 that F and RA are strongly inversely correlated. This is a trivial consequence of the typical values of F in the human brain (of the order of 10\u22121), for which the denominator on the right hand side of Eq. (18) is approximately equal to , effectively coupling RA and F. This corresponds to conditions of rapid exchange [9].\nThe use of numerical simulations allows the investigation of conditions for which it is impractical to acquire in vivo data, for example very narrow or very long MT pulses. As expected, and shown by others [7], even in noise-free condition, the estimates of the MT parameters provided by Ramani\u2019s signal equation depend on the duty cycle (in a non-linear fashion), suggesting the need for pulse sequences with a duty cycle of at least 50%. The estimates obtained using Sled and Pike\u2019s equations, on the other hand are, with the exception of , less sensitive to this parameter.\nWe also showed that the degree of T1-weighting (i.e. of saturation of the excitation) has a large effect on the MT parameters estimated by fitting Ramani\u2019s equation. This is a direct consequence of the assumptions underlying it. We therefore strongly recommend avoiding the use of Eq. (17) to fit data which deviate substantially from those assumptions. The two equations proposed by Sled and Pike, on the other hand, by removing the \u201csteady state\u201d assumption, appear to be less sensitive to this problem. The RP approximation gives estimates that are substantially insensitive to changes in T1-weighting, while the CW approximation becomes slightly affected by it for excitation flip angles larger than 14\u00b0 (assuming a TR\u00a0=\u00a030\u00a0ms).\nIn this respect, the results obtained from synthetic data are in keeping with in vivo measurements. Ramani\u2019s equation\u2019s estimates of F obtained from the more heavily T1-weighted data (Table 2, flip angle\u00a0=\u00a015\u00b0) are substantially lower than those obtained with less heavily weighted data, while estimates of  are larger. Conversely, when fitting Sled and Pike\u2019s equations to the 15\u00b0 dataset, the estimates of F are slightly larger compared to those from the 5\u00b0 dataset, although the difference is not statistically significant. A surprising finding from in vivo data was that estimates of  obtained from all the equations increased slightly with the excitation flip angle, while this was not observed with simulated data. This discrepancy between simulated and real data may be explained by the presence of noise, and by the use of a smaller number of points for the real data. Noise in the raw data critically affects the estimation of MT parameters, as shown by our fourth simulation (Fig. 3). Monte Carlo simulations also suggest that at high SNR (\u2a7e150) Sled and Pike\u2019s estimates (at least of some parameters) are at least as precise as (and generally more precise than) Ramani\u2019s estimates, but that Ramani\u2019s equation provides more precise answers at lower SNR. We also note that at low SNR the uncertainty associated with the estimated parameters is much larger than the systematic difference between the mean parameters estimated by each signal equation. For comparison of the in vivo results with simulations, an estimate of the in vivo SNR is needed. The typical SNR for our system, measured in white matter on 5\u00b0-excitation minimally MT-weighted 3D SPGR images acquired using a 8-channel head coil and the parameters described in the paper, ranges (due to non-uniformity of the receive coil) between 60 and 100 [20]; other systems are likely to be similar. It would be interesting to compare the standard deviations obtained by Monte Carlo iterations to the variance lower bound predicted by Cramer\u2013Rao theory [21], as well as to the standard deviation obtained from real data using the bootstrap method [22].\nA further limitation to this analysis of sensitivity to noise is in the use of suboptimal schemes for both synthetic and real data. For all quantitative techniques based on model fitting, the precision and accuracy of the parameter estimates depend on the choice of the sampling points, and we have previously shown [20] that the error in parameter estimates can be reduced by factors around 2 or 3 by using optimized sampling schemes. The precision of the estimates obtained from all three equations could therefore dramatically improve by using a more suitable set of MT points; we recommend such optimization for practical applications, but this was not possible here as we needed identical schemes to allow direct comparison of the three equations. Nevertheless, since the schemes here used were likely to be equally suboptimal for all signal equations (and are similar to those used by the authors of the original papers), we believe that our analysis should not have been unduly \u201cunfair\u201d to any of the solutions tested. It should also be noted, though, that the acquisition protocol reported by Sled and Pike [6,11] is based on a 60-point scheme to sample a single 7-mm thick slice. Here, despite the consequences on both scan time and SNR, we use only 20 points (for in vivo experiments) and smaller voxels as, for most clinical applications, such whole brain coverage and thinner slices are likely to be essential. Despite reducing the number of points by two thirds, a scan time of 35\u00a0min (required by the protocol we used in vivo) is still too long to be feasible in a clinical setting. We also estimated five parameters ( and M0) directly from the fitting, while the original paper by Sled and Pike [11] recommended the use of an independent estimate of the relative proton density, which allows the MT curves to be normalized to one, reducing to four the number of parameters to be extracted. While this clearly would improve the precision in the estimated quantities, it also lengthens the scan time, making it again less attractive for clinical applications, where the number of acquisitions is typically restricted by time constraints. A better fit could be obtained also by iteratively repeating the fitting using the estimate of M0 obtained from the current iteration as the starting point for the next. Preliminary results suggest that two iterations should suffice. Furthermore, simulated data suggest that when high SNR and a large number of MT points are available, fitting Sled and Pike\u2019s signal equations provides more accurate results, and therefore is preferable. Between the two variants, the main differences seems to be in sensitivity to the degree of T1-weighting in the acquisition sequence (as a consequence of saturation from the excitation pulse), suggesting that, in the absence of this confounding factor, the CW variant can be used without major disadvantages, given its reduced complexity. It should be noted, however, that we used a single set of MT parameters to create the simulations, without any attempt to explore the consistency of these results for a different type of tissue (e.g. gray matter). Our results, therefore, are limited to this specific case until confirmed by further experiments.\nRegarding the in vivo results, it should be noted that we made no attempt to correct in vivo data for B1 inhomogeneities (although the use of the body coil for transmission should provide a fairly uniform B1 distribution at 1.5\u00a0T). Both the measurements of RAobs, and the MT fitting are affected by deviations from the nominal flip angle [6,18]. However, we expect the error introduced by B1 inhomogeneity to equally affect the three solutions, and therefore not to have major consequences on the conclusions drawn from our experiments.\nIn the present work we have restricted our analysis to the comparison of three signal equations derived from the two-pool model to predict signal intensity in pulsed MT experiments, without any attempt to modify them, or compensate for their limitations. Several aspects of MT modeling such as the quantification of the effects of the excitation pulses on the macromolecular pool, which is typically considered negligible [2,6], need to be addressed. It also would be interesting to investigate the dependency of these 3 equations on other sequence parameters such as TR, as this quantity controls the efficiency of magnetization transfer [23]. This analysis would be complementary to the investigation of duty cycle effects we performed, and may confirm whether the relative insensitivity of Sled and Pike\u2019s CW approximation to duty cycle is maintained for all pulse sequences.\nFurthermore, it would be interesting to explore possible modifications of Ramani\u2019s equation to account for the imaging parameters of the pulse sequence (for example by incorporating an additional contribution of [1-cos(alpha)]\/TR to the CW saturation rate acting on the free pool). Finally, providing reliable information about the optimal number of sampling points and their distribution would yield an additional element towards the choice of the most appropriate equation for a given application. All these areas deserve further investigation, which we hope to pursue in the future.\n6\nConclusion\nWe have shown that (1) Sled and Pike\u2019s CW signal equation provides the most accurate estimates of MT parameters in the absence of noise; (2) Ramani\u2019s signal equation is sensitive to changes in the MT duty cycle, although this effect becomes stable for duty cycles \u2a7e50%; (3) Ramani\u2019s equation is (as expected) not suitable for fitting T1-weighted data, and doing so leads to underestimates F and  when the amount of T1-weighting is high. An error, albeit of smaller magnitude, is also introduced into Sled and Pike\u2019s CW approximation estimates when the amount of T1-weighting is high. The RP variant is, on the other hand, extremely robust to this effect; (4) in data with SNR typical of in vivo protocols, although the estimates of , and  obtained from the fit of all three equations show some differences, the magnitude of the difference is smaller than the between-voxels within-ROI standard deviation, provided that T1-weighting of the imaging sequence is minimal; (5) Sled and Pike\u2019s equations are slightly less robust than Ramani\u2019s one in conditions of low SNR.","keyphrases":["magnetization transfer","relaxation","mri","two-pool model","human brain"],"prmu":["P","P","P","P","P"]}
{"id":"Calcif_Tissue_Int-3-1-2039811","title":"Effect of Raloxifene Treatment on Osteocyte Apoptosis in Postmenopausal Women\n","text":"Increased osteocyte apoptosis, as the result of estrogen deficiency, could play a role in the decrease of bone mass and bone strength seen in postmenopausal osteoporosis. We investigated whether treatment with raloxifene of postmenopausal women with osteoporosis affects osteocyte apoptosis. Transiliac bone biopsies were obtained from 26 osteoporotic women at baseline and after 2 years of treatment with placebo or raloxifene. Immunohistochemical detection of cleaved caspase-3 was performed on sections from nondecalcified bone biopsies to visualize apoptosis. In the trabecular bone total osteocytes, positively stained osteocytes and empty lacunae were counted and percent positive cells and percent empty lacunae determined. Statistical evaluation was performed by Wilcoxon\u2019s paired t-test and Spearman\u2019s rank correlations. There was no significant difference in percentage positive osteocytes between baseline and follow-up biopsies in both the placebo and the raloxifene groups. The percentage empty lacunae increased significantly in the placebo group (11.20 \u00b1 1.43 vs. 9.00 \u00b1 2.25, P = 0.014) but not in the raloxifene group. At baseline in both groups combined, there was a negative correlation between indices of bone remodeling and the percentage positive osteocytes (bone formation rate\/bone volume r = \u22120.67, P = 0.001). We found no direct evidence for an effect of raloxifene treatment on osteocyte apoptosis, but small effects of raloxifene treatment cannot be excluded. The percent of apoptotic osteocytes was dependent on the level of bone remodeling in an individual.\nOsteocytes play a key role in the maintenance of bone mass and structure. The main function of osteocytes is to sense mechanical stress in the bone [1]. Osteocytes respond to this with the production of nitric oxide, prostaglandins, and other factors which are believed to restrain osteoclastic bone resorption or promote bone formation [2, 3]. A putative second role of osteocytes is to direct bone remodeling to foci of microdamage. Osteocyte apoptosis around the site of microdamage attracts bone remodeling cells, which resorb the damaged bone and replace it with new mechanically competent bone [4, 5].\nPostmenopausal osteoporosis generally results in a decrease in bone mineral density (BMD) and a higher susceptibility for osteoporotic fractures. It is characterized by high bone remodeling, with bone resorption exceeding bone formation. Estrogen deficiency results in an increase in the recruitment and activity of both osteoblasts and osteoclasts. It also leads to increased osteoclast survival, while the life span of the osteoblast is decreased [6, 7]. Recently it has been shown both in humans and in rats that estrogen deficiency also leads to increased osteocyte apoptosis [8, 9]. The resulting decrease in osteocyte number could, in time, impair the response of bone to mechanical stress and lead to an accumulation of microdamage. Therefore, it would be of interest to know whether therapies aimed at reducing postmenopausal bone loss affect the survival of osteocytes.\nRaloxifene is a selective estrogen receptor modulator (SERM) that can bind to the estrogen receptors ER\u03b1 and ER\u03b2. It has been shown to increase BMD and reduce vertebral fracture risk in women with postmenopausal osteoporosis [10, 11]. In vitro studies suggest that raloxifene exerts its effect, like estrogen, through modulation of the number and activity of both osteoclasts and osteoblasts [12]. Kousteni et al. [13] have recently shown, using in vitro experiments, that raloxifene does not inhibit etoposide-induced apoptosis of rat calvarial osteoblasts; but no clinical studies have examined the effect of raloxifene on osteocyte apoptosis in postmenopausal women.\nIn this study, we investigated whether treatment of postmenopausal women with raloxifene for 2 years would change osteocyte survival in trabecular bone and whether the level of osteocyte apoptosis would be associated with the level of bone remodeling.\nMaterials and Methods\nStudy Outline\nAll women in the study were participants in the Multiple Outcomes of Raloxifene Evaluation (MORE) trial. This was a placebo-controlled, double-blind, multicenter trial to test the efficacy of raloxifene. Details of this study have already been published elsewhere [10]. Briefly, 7,705 women were at least 2 years postmenopausal and had osteoporosis as defined by a BMD of at least 2.5 standard deviations (SDs) below the young adult mean and\/or one or more vertebral fractures. They were randomly assigned to one of the following three treatment groups: placebo, 60 mg\/day raloxifene, and 120 mg\/day raloxifene. Additionally, all women received daily vitamin D (400\u2013600 IU) and calcium (500 mg). Among the exclusion criteria for this study were the use of androgen, calcitonin, or bisphosphonates within the previous 6 months; oral estrogen within the previous 2 months; fluoride therapy for more than 3 months during the previous 2 years; or systemic glucocorticoid therapy for more than 1 month within the past year. During the study the women received no therapy with respect to their osteoporosis other than the study drugs. The use of other prescription drugs and over-the-counter drugs such as sedatives, antibiotics, and paracetamol was equally distributed in the placebo and raloxifene groups.\nIn this study, bone biopsies were obtained at baseline and after 2 years from 26 women who were enrolled in the European centers of the MORE trial. These women were part of the bone histomorphometry substudy of the MORE trial that included 65 women from two centers in the United States and two centers in Europe. Bone biopsies from the other 39 women were not available for sectioning. All women had given their informed consent, and the study was approved by the institutional ethics review boards.\nMarkers of bone turnover that were measured were serum osteocalcin, bone-specific alkaline phosphatase (BSAP), and urinary type 1 collagen C-telopeptide corrected for creatinine (CTX-I). Data are from the baseline and 24-month evaluations.\nBone Biopsies\nThe women received two doses of tetracycline with a 12-day interval. Transverse biopsy specimens were taken from the anterior iliac crest, the 2-year biopsy being on the opposite side from the baseline biopsy. The bone biopsies were immediately fixed in cold 4% phosphate-buffered formaldehyde, dehydrated in graded ethanol, and embedded in methylmethacrylate (MMA; BDH Chemicals, Poole, England) supplemented with 20% plastoid-N (R\u00f6hm und Haas, Darmstadt, Germany), 2.0 g\/L benzoylperoxide (Merck, Darmstadt, Germany), and N,N-dimethylaniline (Merck) [14]. Sections of 5\u00a0\u03bcm were cut with a Jung (Nussloch, Germany) K Polycut microtome. Sections were stained with Goldner\u2019s trichrome method. Static and dynamic histomorphometric measurements were performed as previously reported [15, 16]. Histomorphometric indices used for this study included the percentage of bone surface covered by osteoid (osteoid surface, OS\/BS) and the amount of mineralized bone formed per year on a given bone area (bone formation rate\/bone volume, BFR\/BV) as bone formation indices and the percentage of bone surface covered by osteoclasts or appearing eroded (eroded surface, ES\/BS) and the number of osteoclasts per bone area (osteoclast number, Ocl.N\/B.Ar) as bone resorption indices [17, 18]. Histomorphometric data and immunohistochemical data were obtained from the same biopsies.\nImmunohistochemistry\nApoptotic cells were visualized by immunohistochemical detection of activated caspase-3. The antibody against cleaved caspase-3 specifically stains apoptotic cells. Unlike the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) method, there is no staining of necrotic cells or cells with DNA damage. For optimal accuracy of the method, immunohistochemistry was performed on four 5 \u03bcm sections of each biopsy, which were obtained with an interval of 30 \u03bcm. Sections were cut and transferred to poly-l-lysine-coated slides. After deplastification and rehydration, sections were decalcified for 10 minutes with 1% acetic acid. Antigen retrieval was performed by 30-minute incubation with 0.5% saponin (Sigma, St. Louis, MO) in phosphate-buffered saline (PBS) and 10-minute incubation with 3.5 \u03bcg\/mL DNAse II (Sigma) in 25 mM Tris + 10 mM MgSO4. Sections were incubated with 3% H2O2 in methanol to block endogenous peroxidase and with 5% normal goat serum in PBS + 0.05% Tween to block nonspecific binding sites. Incubation with primary antibody was performed overnight at 4\u00b0C with 1\/100 rabbit-anticleaved caspase-3 antibody (Cell Signaling Technology, Beverly, MA) in PBS + 0.05% Tween. Sections were then incubated for 1 hour with 1\/100 biotin-labeled goat-anti-rabbit immunoglobulin G (Vector Labs, Burlingame, CA) in PBS + 0.05%Tween. The sections were incubated for 30 minutes with the ABC kit (Vector Labs) and developed for 10 minutes with 3,3\u2032-diaminobenzidine with nickel enhancement. Sections were counterstained with 0.025% toluidine blue in H2O, dehydrated, and sealed in DEPEX mounting medium (BDH). Random quality-control sections were measured in each assay. Sections of human ileum were tested as a positive control for apoptotic cells.\nCell Counting\nThe identity of the sections was blinded, and they were randomly numbered. In each section, the total trabecular bone area was measured using Osteomeasure software (Osteometrics, Atlanta, GA). In the entire trabecular bone area, the total number of osteocytes, the number of cleaved caspase-3-positive osteocytes, and the number of empty lacunae were counted with x200 magnification. Artefacts, such as areas where bone marrow obscured the trabeculae or where trabeculae were crossed, were avoided. All sections were analyzed by the same investigator.\nFrom these data we calculated the following parameters:\npercentage of positive osteocytes per total osteocytes (Pos.Ot.N\/Tt.Ot.N)percentage of empty lacunae per total lacunae (EL.N\/Tt.L.N)empty lacunae per bone area (EL.N\/B.Ar)total lacunae per bone area (Tt.L.N\/B.Ar)Calculations from the four sections per biopsy were averaged. The variation in the method was assessed by calculating the average standard deviation from all the quadruplicates according to the following formula:in which n is number of subjects, k is number of measurements per subject, nj is number of measurements in subject j, Xij is score measurement i in subject j, and \nis mean score in subject j.\nStatistics\nAll values are expressed as mean\u00a0\u00b1\u00a0SD. Differences in the calculated parameters between the 2-year biopsies and the baseline biopsies within groups were tested using a nonparametric paired t-test (Wilcoxon signed rank test). Differences between the treatment groups in the percent change after 2 years of treatment were tested with a nonparametric t-test (Mann-Whitney signed rank test). Correlations between the calculated parameters and histomorphometric indices or biochemical markers were assessed with Spearman\u2019s rank correlations. Statistical analysis was performed using SPSS (Chicago, IL) 12.0 software.\nResults\nThe data presented here are derived from 26 patients who attended the European centers of the MORE trial. Therefore, this study covers only part of the group of patients on which histomorphometry data were published earlier [16]. Of the 26 women in this study, 11 received placebo, ten received 60 mg\/day raloxifene, and five received 120 mg\/day raloxifene. Because the number of women in the 120 mg\/day raloxifene group was small and no difference in results between the two raloxifene treatment groups was detected, as tested using the Mann-Whitney signed rank test, data from the two raloxifene groups were combined for further analysis. The average age of the women in the placebo group was 67.9\u00a0\u00b1\u00a06.1 years; in the raloxifene group the average age was 67.1\u00a0\u00b1\u00a06.7 years.\nStaining for cleaved caspase-3 clearly identified apoptotic osteocytes (Fig.\u00a01a); in the negative control no cells showed any staining. Figure\u00a01b shows an example of an empty lacuna. In the human ileum sections, clear positive staining of intestinal epithelial cells was seen at the luminal surface of the villi while the negative control showed no staining (Fig.\u00a01c, d). Table\u00a01 shows the baseline and follow-up results of the calculated parameters in the placebo group and the raloxifene group and the average standard deviation of the parameters. Direct comparison of the follow-up biopsy with the baseline biopsy revealed no difference in the percentage of positive osteocytes (Pos.Ot.N\/Tl.Ot.N) in either the placebo group or the raloxifene group.\nFig.\u00a01Immunohistochemical staining of bone biopsy sections and human ileum sections for cleaved caspase-3. Human bone biopsy sections stained with cleaved caspase-3 (a, b). Arrows indicate apoptotic osteocyte (a) or empty lacuna (b). Human ileum sections stained with cleaved caspase-3 (c) or no first antibody (d). Arrows indicate apoptotic cellsTable\u00a01Results of baseline and follow-up biopsies for the calculated parametersParameter (average standard deviation)aTime pointPlacebo (n\u00a0=\u00a011, mean\u00a0\u00b1\u00a0SD)Raloxifeneb (n\u00a0=\u00a015, mean\u00a0\u00b1\u00a0SD)Pos.Ot.N\/Tt.Ot.N (%) (1.28)Baseline6.66\u00a0\u00b1\u00a05.285.30\u00a0\u00b1\u00a03.51Follow-up6.82\u00a0\u00b1\u00a05.274.99\u00a0\u00b1\u00a02.29EL.N\/Tt.L.N (%) (2.43)Baseline9.00\u00a0\u00b1\u00a02.259.11\u00a0\u00b1\u00a02.88Follow-up11.20\u00a0\u00b1\u00a01.43c9.74\u00a0\u00b1\u00a01.91EL.N\/B.Ar (n\/mm2) (6.29)Baseline21.2\u00a0\u00b1\u00a06.1321.0\u00a0\u00b1\u00a06.49Follow-up24.5\u00a0\u00b1\u00a03.6822.5\u00a0\u00b1\u00a04.67Tt.L.N\/B.Ar. (n\/mm2) (22.34)Baseline233.9\u00a0\u00b1\u00a028.2231.5\u00a0\u00b1\u00a022.9Follow-up219.5\u00a0\u00b1\u00a027.3231.1\u00a0\u00b1\u00a019.6a Average standard deviation calculated from all quadruplicatesb The 60 mg\/day and 120 mg\/day raloxifene groups were combinedc Statistically significant difference (P\u00a0=\u00a00.014) from baseline value\nAs empty lacunae are the result of osteocyte apoptosis and are only removed by bone remodeling, changes in the number of empty lacunae could be indicative of changes in osteocyte apoptosis. In the placebo group, the percentage of empty lacunae (EL.N\/Tt.L.N) increased significantly after 2 years (also shown in Fig.\u00a02). The empty lacunar density (EL.N\/B.Ar) showed a parallel change, but this was not significant. In the raloxifene group, the percentage of empty lacunae and the empty lacunar density did not increase significantly. The average standard deviation, calculated from the quadruplicate analysis, showed that the variation in the method was comparable to the variation between individuals, except for the measurement of percent positive cells, where the variation between the individuals was much higher.\nFig.\u00a02Change in percentage empty lacunae after treatment for 2 years with either placebo (A) or raloxifene (B). Empty lacunae and total lacunae were counted in each biopsy. The increase in the placebo group was significant (P\u00a0=\u00a00.014)\nAssociations of bone remodeling parameters with osteocyte apoptosis and empty lacunae are shown in Table\u00a02. At baseline, in the placebo group and the raloxifene group combined, there was a negative correlation of the histomorphometric indices (BFR\/BV, OS\/BS, ES\/BS, and Ocl.N\/B.Ar) with the percent positive osteocytes (Pos.Ot.N\/Tt.Ot.N). Histomorphometric indices were not correlated with empty lacunae. The regression lines for BFR\/BV with percent apoptotic osteocytes and percent empty lacunae are shown in Figure\u00a03. Correlations between biochemical indices (BSAP, osteocalcin, and CTX-1) and percent apoptotic osteocytes or percent empty lacunae were not found.\nTable\u00a02Association of histomorphometric indices and biochemical indices of bone remodeling with osteocyte apoptosis (Pos.Ot.N\/Tt.Ot.N) and empty lacunae (E.L.N\/Tt.L.N)Histomorphometric indicesOS\/BSBFR\/BVES\/BSOcl.N\/B.Ar% Positive osteocytes (Pos.Ot.N\/Tt.Ot.N)r\u00a0=\u00a0\u22120.64r\u00a0=\u00a0\u22120.67r\u00a0=\u00a0\u22120.48r\u00a0=\u00a0\u22120.43P\u00a0=\u00a00.0004aP\u00a0=\u00a00.001aP\u00a0=\u00a00.01aP\u00a0=\u00a00.03a% Empty lacunae (E.L.N\/Tt.L.N)r\u00a0=\u00a00.03r\u00a0=\u00a00.10r\u00a0=\u00a00.05r\u00a0=\u00a00.48P\u00a0=\u00a00.87P\u00a0=\u00a00.67P\u00a0=\u00a00.80P\u00a0=\u00a00.02aBiochemical indicesOsteocalcinBSAPCTX% Positive osteocytes (Pos.Ot.N\/Tt.Ot.N)r\u00a0=\u00a0\u22120.35r\u00a0=\u00a0\u22120.43r\u00a0=\u00a0\u22120.40P\u00a0=\u00a00.13P\u00a0=\u00a00.05P\u00a0=\u00a00.09% Empty lacunae (E.L.N\/Tt.L.N)r\u00a0=\u00a00.10r\u00a0=\u00a00.13r\u00a0=\u00a00.31P\u00a0=\u00a00.66P\u00a0=\u00a00.56P\u00a0=\u00a00.19aStatistically significant (P\u00a0<\u00a00.05)Fig.\u00a03Relationship between BFR\/BV and osteocyte apoptosis at baseline in the placebo and raloxifene groups combined. (A) Association between BFR\/BV and percent positive osteocytes. Correlation is significant for placebo and raloxifene groups combined (P\u00a0=\u00a00.001). (B) Association between BFR\/BV and percentage empty lacunae. No significant correlation (P\u00a0=\u00a00.67). \u25cf, placebo group; \u25b4, raloxifene group\nNo changes in the histomorphometric indices between baseline and follow-up were found, nor were there any differences between the placebo and raloxifene groups. All three biochemical markers showed a significant decrease at follow-up compared to baseline in the raloxifene group but not in the placebo group (Table\u00a03).\nTable\u00a03Results of baseline and follow-up biopsies for histomorphometric indices and biochemical markers of bone remodelingParameterTime point % changePlacebo (n\u00a0=\u00a011, mean\u00a0\u00b1\u00a0SD)Raloxifenea (n\u00a0=\u00a015, mean\u00a0\u00b1\u00a0SD)OS\/BS (%)Baseline10.2\u00a0\u00b1\u00a06.89.9\u00a0\u00b1\u00a05.4Follow-up7.5\u00a0\u00b1\u00a03.211.6\u00a0\u00b1\u00a04.6BFR\/BV (%\/year)Baseline28.8\u00a0\u00b1\u00a020.335.3\u00a0\u00b1\u00a018.6Follow-up19.8\u00a0\u00b1\u00a08.723.3\u00a0\u00b1\u00a012.6ES\/BS (%)Baseline5.3\u00a0\u00b1\u00a02.66.3\u00a0\u00b1\u00a02.9Follow-up5.3\u00a0\u00b1\u00a02.46.3\u00a0\u00b1\u00a02.4Ocl.N.\/B.Ar (n\/mm2)Baseline0.6\u00a0\u00b1\u00a00.30.8\u00a0\u00b1\u00a00.4Follow-up0.6\u00a0\u00b1\u00a00.30.6\u00a0\u00b1\u00a00.3BSAP (\u03bcg\/L)Baseline14.9\u00a0\u00b1\u00a07.314.4\u00a0\u00b1\u00a04.6Follow-up12.2\u00a0\u00b1\u00a05.610.0\u00a0\u00b1\u00a02.5bOsteocalcin (ng\/mL)Baseline22.9\u00a0\u00b1\u00a010.625.5\u00a0\u00b1\u00a09.1Follow-up18.2\u00a0\u00b1\u00a03.816.1\u00a0\u00b1\u00a03.1bCTX-1 (\u03bcg\/mmol creatinine)Baseline283.9\u00a0\u00b1\u00a0267.0277.4\u00a0\u00b1\u00a0198.2Follow-up137.7\u00a0\u00b1\u00a063.8132.3\u00a0\u00b1\u00a053.0baThe 60 mg\/day and 120 mg\/day groups were combinedbSignificantly different from baseline (P\u00a0<\u00a00.05)\nDiscussion\nWe investigated the effect of treatment with raloxifene for 2 years of postmenopausal osteoporotic women on osteocyte apoptosis as measured by activated caspase-3 immunohistochemistry in iliac crest bone biopsies. Direct comparison of follow-up with baseline in the placebo and raloxifene groups did not show differences in the percent positive osteocytes. This suggests that raloxifene has little or no influence on osteocyte apoptosis. We did find a significant increase in the percentage of empty lacunae at 2 years in the placebo group, while there was no change in the raloxifene group. This lack of accumulation of empty lacunae in the raloxifene group could be the consequence of an inhibitory effect of raloxifene on osteocyte apoptosis. At baseline, histomorphometric indices of bone remodeling were inversely correlated with apoptotic osteocytes but not with empty lacunae. Biochemical markers of bone remodeling were not correlated with apoptotic osteocytes or empty lacunae.\nThis is the first study to investigate the effect of antiresorptive treatment on osteocyte apoptosis in human bone biopsies. So far, the effect of raloxifene on osteoblast or osteocyte apoptosis has only been studied in in vitro studies. Kousteni et al. [13] studied the effect of raloxifene on etoposide-induced apoptosis of rat calvarial osteoblasts and did not find protection against apoptosis. On the other hand, Olivier et al. [19] found that raloxifene protected the osteoblast-like cell line MC3T3-E1 against apoptosis induced by a high concentration of nitric oxide. Comparable clinical studies with hormone replacement therapy or bisphosphonates have not been published, but in vitro studies [20, 21] and studies with mice [22, 23] have shown that both these treatments have an inhibiting effect on osteocyte apoptosis induced by glucocorticoids or etoposide.\nThe difference between the raloxifene group and the placebo group in percent empty lacunae provides some evidence for an inhibitory effect of raloxifene on osteocyte apoptosis, although this effect was not reflected in the empty lacunar density. The fact that apoptotic osteocytes and empty lacunae change differently in response to raloxifene treatment could be explained by the comparatively short time that apoptosis can be detected. It has been shown that nonviable osteocytes are detectable for up to 16 weeks [24], although remnants of apoptotic cells, such as apoptotic bodies, might exist a little longer. Therefore, the cleaved caspase-3-positive osteocytes that were detected are cells that became apoptotic in the last 16 weeks of the treatment period. Moderate changes due to treatment with raloxifene are probably not detectable in such a short period. Empty lacunae remain in bone until bone remodeling will remove them, and this period is longer then the 16 weeks that an apoptotic osteocyte is detectable.\nChanges in bone remodeling could have influenced the percentage of apoptotic osteocytes and empty lacunae that we found. The inverse correlation between bone remodeling indices and osteocyte apoptosis at baseline indicates that the level of osteocyte apoptosis is indeed associated with the level of bone remodeling. In our opinion, the explanation for this association is that with high bone remodeling the chance that apoptotic osteocytes are removed and replaced by new osteocytes is also high and, therefore, the percentage of detected apoptotic osteocytes is low. With low bone remodeling the chance that apoptotic osteocytes are removed and replaced by new osteocytes is also low and, therefore, the resulting percentage of observed apoptotic osteocytes is high. In this way, bone remodeling partly defines the percentage of apoptotic osteocytes, a mechanism which has already been suggested by Dunstan et al. [25]. This dependence of the level of osteocyte apoptosis on the level of bone remodeling complicates the detection of an effect of a treatment on osteocyte apoptosis if that treatment also has an effect on bone remodeling. In this study, there were no significant effects of raloxifene on bone remodeling parameters both in the placebo group and in the raloxifene group; however, changes in individual women over the 2-year treatment period could have influenced the level of osteocyte apoptosis found at follow-up. This could have obscured detection of a possible effect of raloxifene on osteocyte apoptosis.\nThe inverse correlation between osteocyte apoptosis and bone remodeling seems to be in contrast with the hypothesis put forward by several investigators [5, 26] that viable osteocytes inhibit bone remodeling and that lack of viable osteocytes, e.g., near sites of microdamage, attracts bone remodeling. We believe that our results do not contradict this hypothesis. Attraction of osteoclasts by dead osteocytes or by lack of osteocytes is probably a local process. If the occurrence of dead osteocytes and empty lacunae stimulates bone remodeling, the lacunae are more quickly removed and replaced with viable osteocytes.\nThe percent empty lacunae was not correlated to histomorphometric indices of bone remodeling. This suggests that the empty lacunae for a large part exist and increase in number in bone that is not participating in remodeling. In two studies, Qiu et al. [27, 28] have made the distinction between superficial bone and deep bone, i.e., bone at the surface of trabeculae and bone in the center of trabeculae. They postulated that deep bone is remodeled much more slowly than superficial bone. In this study, we did not make a distinction between superficial bone and deep bone, but it is possible that the increase in empty lacunae that we found primarily occurred in deep bone.\nBiochemical markers of bone formation and resorption were not correlated with osteocyte apoptosis or with empty lacunae. Biochemical markers reflect bone remodeling in the whole skeleton, both in trabecular bone and in cortical bone. Correlations between such general markers and parameters measured locally in trabecular bone of the iliac crest are possibly more difficult to find.\nThe major limitation of our study is the small number of patients. The percentage of apoptotic osteocytes varied considerably between individuals, and this makes it difficult to find significant differences between such small groups. The variation was not caused by variation in the method, as shown by the low average standard deviation, indicating that the percentage of apoptotic osteocytes has to be an individual characteristic.\nDetection of cleaved caspase-3, a key protease in the apoptotic process, is an established method for measuring apoptosis [29, 30]; and it has recently been used on bone tissue [31]. In the latter study, comparison between the TUNEL method and the cleaved caspase-3 method showed no significant differences. We confirmed the specificity of the detection of cleaved caspase-3 in sections of human ileum. In these sections, only the cells which are expected to be apoptotic [32] were stained by our method. Necrotic death of osteocytes might also occur in bone; however, we expect that necrotic cell death is low and not responsive to interventions such as estrogen, glucocorticoids, or mechanical loading. The average percentage apoptotic osteocytes we found was somewhat lower than what Tomkinson et al. [8] found in their study in young women who had received gonadotropin-releasing hormone analogue therapy. This could reflect differences in age and in treatment between the two studies.\nThe number of empty lacunae that we observed could be an overestimation caused by sectioning artefacts or the decalcification step in the immunohistochemistry method. We do not know to what extent this occurs, but this would be equal in all biopsies. Goldner-stained sections showed more sectioning artefacts, and therefore, empty lacunae in these sections were not counted. Inconsistency between the change in percent empty lacunae and empty lacunar density in the placebo group is probably also related to the small number of patients studied.\nIn this study, we did not find clear evidence that treatment with raloxifene influences osteocyte apoptosis. No change in the percentage of apoptotic osteocytes was detected, while the changes in the empty lacunae were inconclusive and at best indirect evidence that osteocyte apoptosis had been changed by raloxifene. Compared to estrogen, raloxifene treatment shows a similar reduction of vertebral fractures (but not nonvertebral fractures) [10, 33] but has less potent positive effects on bone quality assessed by BMD measurement and bone histomorphometry [34, 35]. It is conceivable that the weaker effect of raloxifene on bone is related to only a weak antiapoptotic effect or lack of antiapoptotic action. This is, however, difficult to measure in a study with such a small number of subjects. It would be of interest to compare the effects of different antiresorptive treatments on osteocyte apoptosis in larger clinical studies and to compare the mechanisms by which they exert their effect.\nIn conclusion, we did not find evidence for an effect of raloxifene treatment on osteocyte apoptosis in postmenopausal women, but small effects of raloxifene treatment on osteocyte apoptosis cannot be excluded. The percent of apoptotic osteocytes was dependent on the level of bone remodeling in an individual.","keyphrases":["raloxifene","osteocyte","apoptosis","postmenopausal osteoporosis","histomorphometry"],"prmu":["P","P","P","P","P"]}
{"id":"Sci_Eng_Ethics-4-1-2413105","title":"How Should We Foster the Professional Integrity of Engineers in Japan? A Pride-Based Approach\n","text":"I discuss the predicament that engineering-ethics education in Japan now faces and propose a solution to this. The predicament is professional motivation, i.e., the problem of how to motivate engineering students to maintain their professional integrity. The special professional responsibilities of engineers are often explained either as an implicit social contract between the profession and society (the \u201csocial-contract\u201d view), or as requirements for membership in the profession (the \u201cmembership-requirement\u201d view). However, there are empirical data that suggest that such views will not do in Japan, and this is the predicament that confronts us. In this country, the profession of engineering did not exist 10 years ago and is still quite underdeveloped. Engineers in this country do not have privileges, high income, or high social status. Under such conditions, neither the social-contract view nor the membership-requirement view is convincing. As an alternative approach that might work in Japan, I propose a pride-based view. The notion of pride has been analyzed in the virtue-ethics literature, but the full potential of this notion has not been explored. Unlike other kinds of pride, professional pride can directly benefit the general public by motivating engineers to do excellent work even without social rewards, since being proud of themselves is already a reward. My proposal is to foster a particular kind of professional pride associated with the importance of professional services in society, as the motivational basis for professional integrity. There is evidence to suggest that this model works.\nThere is an on-going shift in emphasis in the professional-ethics literature from misconduct to integrity [1, 2]. Integrity within this context generally means maintaining high moral standards and doing superior work in a professional capacity, as opposed to merely avoiding misconduct. The profession of engineering is of course no exception. However, even though the importance of professional integrity in society is obvious, the question of motivating students to maintain this integrity is not as straightforward a matter. Engineering-ethics courses are usually expected to do the job. Ethics teachers do not have to spend a great deal of time to explain why professionals should avoid misconduct, as long as students have already acquired everyday morality. Professional integrity and other special responsibilities by professionals, on the other hand, go beyond everyday morality, and need a different basis to convince students. How then can engineers (and other professionals) be motivated to provide efficient services to society? How can we foster professional integrity in engineers? These issues, which I will henceforth refer to as \u201cthe problem of professional motivation,\u201d have been discussed by many authors, and many different solutions have been proposed.\nTo clarify the issue, we need to distinguish the motivation to obey the codes of ethics and the justification of such codes. Professional codes of ethics have clear societal virtues, and often essential for the normal functioning of the society. In this sense, the existence of the code is morally desirable and justified. However, there are obligations in codes of ethics that seem to go beyond common sense obligations, such as \u201cengineers shall be guided in all their relations by the highest standards of honesty and integrity,\u201d and \u201cengineers shall at all times strive to serve the public interest,\u201d both listed as \u201cprofessional obligations\u201d in the Code of Ethics for Engineers by the NSPE (National Society for the Professional Engineers). To use a deontological term, these acts are usually regarded as supererogatory acts for ordinary people, and without special reasons, this should not be different for engineers. Thus, there is a gap between what society needs and what the society can require of engineers as ordinary obligations. The gap can be filled either by justifying the special obligations in a way engineers themselves feel happy with them, or by motivating engineers in other ways to accept these obligations. The problem of professional motivation can be solved and professional integrity will be fostered in either way, but the motivational aspect becomes more crucial when we have to take the second strategy.\nSince the effectiveness of a solution to the problem is partly dependent on the circumstances engineers are placed in, the most effective solution may differ from one situation to another. One of the main conclusions I intend to draw in this paper is that Japanese ethics teachers face a special predicament that teachers in many other countries (including the U.S.) might not. The quandary, if it is real, calls for a different solution to the problem of professional motivation. I will also provide a solution to the problem based on the notion of \u201cpride\u201d, which I take to be at least more promising in the current Japanese circumstances than the alternatives.\nGrounds for Professional Integrity of Engineers\nLet us first examine some common solutions to the problem of professional motivation. This issue is often discussed within the context of the nature of codes of ethics. Although the scope of professional integrity (which is my main concern here) often goes beyond written texts of codes of ethics, for now let us ignore this difference; for if we fail to persuade students to obey the codes of ethics of relevant professional societies, how can we persuade them to have professional integrity?\nProbably the most prevalent view is the so-called \u201csocial contract\u201d view (or, more accurately, the \u201ccontract-with-society\u201d view). This view sees an implicit contract between society and the professions and is based on particular notions of what professions are. According to this notion, professions have some characteristic features, such as autonomous professional societies, ethics codes, licenses, and privileges [3, 4]. These features are the basis of the contract, where society gives professions prestige, autonomy, educational opportunity, and other support for them to function; in exchange, professions maintain high moral standards and return to society high-quality services.\nIf students accept this scheme exists, it is rather easy to persuade them to participate in the contract, i.e., the contract is advantageous for them. This account of professional ethics is widely accepted (e.g., Harris et\u00a0al. [5] discusses this view).1\nAnother model, which I call the \u201cmembership-requirement\u201d view, has some strong supporters such as Davis [6, 7]. According to this model, engineers have special responsibilities simply because they have decided to take part in the profession, and all members are required to follow special standards (and the standards in turn are beneficial for the profession as a whole). According to this interpretation, professional responsibilities are basically derived from the principle of fairness (\u201cdo not cheat\u201d), rather than the assumed implicit contract with society.2\nBoth views have some advantages and disadvantages, but I will not discuss them here. What I would like to ask is a rather different question: can these views justify special obligations of engineers and motivate engineers to have a sense of professional integrity, especially in Japan? There are some reasons for believing that the situation is apparently different in this eastern country, and this is what I will try to demonstrate in the following.\nJapanese Peculiarities\nThe peculiarities of the Japanese situation I have in mind involve the environment Japanese engineers are placed in. Both the \u201csocial-contract\u201d view and the \u201cmembership-requirement\u201d view presuppose the existence of the profession of engineering as a matter of fact (rather than as an ideal), and the former view further presupposes the existence of an implicit contract between the profession and society (again, as a matter of fact). However, neither of these conditions is actually met in Japan.\nUnderdevelopment in Profession of Engineering\nFirst, until quite recently, engineers in Japan had never characterized themselves as professionals in a relevant sense. As a matter of fact, the very notion of profession was foreign to them. Engineering societies in Japan are not professional organizations in the proper sense, but academic societies; their main purpose is to organize academic meetings, and they rarely play a role in defending the interests of engineers. In fact, the lack of the notion of profession in engineering is frequently mentioned in the engineering-ethics literature in Japan as the main obstacle to the development of professional engineering ethics [8, 9]. Since they were (and still largely are) academic societies, most of the societies never had an ethics code (until recently). There was no accreditation system for engineering education, and professional engineer (PE) licenses did not exist.\nThis was the situation in Japan 10\u00a0years ago. Now, there is an on-going change toward the professionalization of engineering in Japan. Engineering societies have started to establish codes of ethics. An organization for accreditation, the Japan Accreditation Board of Engineering Education (JABEE), was established in 1999 and some engineering departments have already been accredited. To meet the accreditation criteria, engineering schools started to offer engineering-ethics courses, one of which I am teaching myself. The government amended the licenses for Consulting Engineers into those for Professional Engineers in 2000, and introduced some new features, such as the Continual Professional Development (CPD) requirement, to the licenses.3 Since licensing and accreditation have been used by various professions to attain privilege and autonomy, these changes seem to be steps toward the professionalization of engineering in Japan.\nHowever, the flip side of these positive developments is that those who are actively involved in these changes are still in the minority. Such changes are mainly caused by external factors irrelevant to most engineers. An effort was made in 1997 to make Japanese engineers internationally competitive.4 To achieve this purpose, the authorities had to establish an accreditation system compatible with ABET (Accreditation Board for Engineering and Technology) of the United States and other Washington Accord countries, but one conspicuously missing element in the Japanese engineering-education system was ethics education. They studied engineering-ethics education systems in other countries (especially in the U.S.), and noticed that such systems are firmly based on the notion of profession. They thus started the series of changes in professionalization.\nInternational competitiveness and the establishment of an accreditation system are not problems of their own making for most individual engineers in Japan. Even though codes of ethics as written texts have been established, what gives life to such texts is the members of the societies who consciously observe them, and in this sense the codes do not yet have real lives. It will take a long time for Japanese engineers to become aware of the importance of codes of ethics and regard engineering as a profession in the proper sense.\nEven after the recent amendments, there are still almost no privileges associated with PE licenses, and most engineers can still conduct their business without any license whatsoever.5 Many engineering schools (including most of the best engineering programs in the country) are not yet accredited, but their graduates do not seem to have any trouble finding jobs. Thus, the change toward professional privilege and autonomy is still nominal.\nIn summary, no autonomous professional groups existed for engineering students to take part in 10\u00a0years ago, and even now these are quite underdeveloped. Anyway, there are no advantages gained by becoming a member. Given these considerations, the \u201cmembership-requirement\u201d view is hopeless as a clue to motivating engineering students, and therefore does not seem to solve the problem of professional motivation, at least for the time being, in Japan. This also means that an important part of the supposed community deal in the \u201csocial-contract\u201d view is also missing. Do we have to wait until the profession is firmly established before teaching professional ethics?\nLack of Implicit Social Contract in Japan\nEven if engineering is not a profession in the proper sense, if there is an implicit social contract between engineers and society as a matter of fact, the \u201csocial-contract\u201d view will do. Privilege and autonomy are not the only things given to engineers by society. For example, if their social status is sufficiently high, that can be an implicit term in the social contract. However, there is evidence that suggests that the social status of engineers is not as high as they might wish. In the following, let us look at a couple of suggestive surveys in this regard.\nThere was an interesting survey conducted in 1998 by Matsushige et\u00a0al. [10] that showed that Japanese engineers do not earn that much (at least not enough to make the \u201csocial-contract\u201d view persuasive).6 Osaka University is one of seven major national (former Imperial) universities in Japan. They were classified by the degree they earned, and four of the categories were: Bachelor of Engineering, Master of Engineering, Bachelor of Social Science, and Bachelor of Humanities. They were further divided into age groups and the average salaries were calculated for all categories. The results for male graduates are summarized in Table\u00a01.7Table\u00a01Average salary in 1998 of Osaka University male graduates (reproduced from [10], p. 5)22\u201329\u00a0years old30\u201339\u00a0years old40\u201349\u00a0years old50\u201360\u00a0years oldBachelor of Social Sciences\u00a54,626,000 ($38,600)\u00a59,133,000 ($76,100)\u00a513,067,000 ($108,800)\u00a515,682,000 ($130,700)Bachelor of Engineering\u00a55,088,000 ($42,400)\u00a57,036,000 ($58,600)\u00a510,988,000 ($91,500)\u00a514,566,000 ($121,400)Master of Engineering and higher\u00a54,370,000 ($36,400)\u00a57,392,000 ($61,600)\u00a511,457,000 ($95,500)\u00a514,004,000 ($116,700)Bachelor of Humanities\u00a54,844,000 ($40,300)\u00a57,365,000 ($61,300)\u00a511,792,000 ($98,300)\u00a512,329,000 ($102,700)Dollar amounts are an approximation with $1\u00a0=\u00a0\u00a5120 at the current rate\nThere are several things to note here. First, social-science-major graduates are consistently better paid than other majors after they reach their thirties. Up to their forties, humanities majors are paid as much as engineering majors. In their fifties, the average salary of humanities graduates becomes lower than that of engineering graduates. However, according to the analysis done by Matsushige et\u00a0al., this difference is not caused by recognizing the status of engineers, but by the tendency for engineers to stay at a single company, while humanities majors leave their original company at around this age.8 Given that engineering majors spend longer in doing course work than social science or humanities majors, there is no point in majoring in engineering if one is interested in earning a good salary. There seems to be no recognition that the status of engineers is higher than that of the others in terms of salary.\nOf course, the data should be compared with similar data from other countries to analyze it objectively, especially that from the U.S., but so far I have found no comparable surveys.9 We also should note that as Osaka University is by no means an average university, it is possible that these results will not apply to less prestigious universities. Still this is a very suggestive case study.\nEven if they do not earn much, it is still possible for engineers to be highly regarded in society. However, another social survey in 1995 (part of a large survey called Social Stratification and Mobility) suggested that the social status of engineers is not as high as that of other professions [11]. Four-thousand ordinary people were asked to rank the social status of 56 occupations, and \u201cautomobile design engineers\u201d was one of them. This occupation was ranked 14th in the 56 occupations (Table\u00a02). This may not sound that serious, but other professions, such as doctors, judges, architects and accountants ranked much higher than engineers.10Table\u00a02Occupational Prestige Scores in 1995 \u201cStratification and Social Mobility\u201d Survey in Japan (reconstructed from [11]; the original result has 56 jobs listed)Doctor 90.1 (highest)Nurse 59.7CEO of large company 87.3Police officer 57.9Lawyer 86.9Municipal officer 56.8University professor 84.3Carpenter 53.1Congressman 74.9Restaurant cook 51.6Architect 71.9Mechanical assembly worker 51.1Accountant 70.8Farmer 45.6Manager of small company 68.9Insurance sales person 44.2Automotive design engineer 66.3Waitress 38.0School teacher 63.5Coal miner 36.6 (lowest)\nA similar poll on occupational prestige is conducted in the U.S. every year by Harris Interactive, and engineers in the 2006 poll had higher prestige than professions such as architects, athletes, lawyers, and accountants, even though firefighters, doctors, and nurses had yet higher prestige (Table\u00a03).11 This high status for engineers has been fairly stable since this poll began in 1977. Since the questions in the Japanese and American surveys were different, we cannot simply compare the two surveys; but the difference in the trend is remarkable.\nTable\u00a03The percentage of people who think that the occupation has \u201cvery great prestige\u201d. Based on survey conducted by Harris Interactive in 2006Firefighter 63%Athlete 23%Doctor 58%Lawyer 21%Nurse 55%Entertainer 18%Scientist 54%Accountant 17%Teacher 52%Banker 17%Military officer 51%Journalist 16%Police officer 43%Union Leader 12%Priest\/Minister\/Clergyman 40%Actor 12%Farmer 36%Business executive 11%Engineer 34%Stockbroker 11%Member of Congress 28%Real estate agent\/broker 6%Architect 27%\nTo sum up, most elements of the \u201csocial-contract\u201d view, i.e., the engineering profession, codes of ethics, privilege, high income, and prestige, are missing or underdeveloped in Japan. How can there be a social contract under such circumstances? Thus, this view does not seem to solve the problem of professional motivation in Japan either.\nPride-based Approach\nOutline of Pride-based Approach\nWhat I have argued so far is that both the \u201csocial-contract\u201d view and the \u201cmembership-requirement\u201d view lack a factual basis in Japanese society. This also means that they are hard to use in engineering-ethics education in Japan. What then are the alternatives available in Japan, especially in teaching professional integrity?\nAn appeal to common morality may do for many items in codes of ethics. For example, the avoidance of harm to the public can be derived from the harm principle. However, as I pointed out at the beginning of this paper, there are other obligations in codes of ethics that seem to go beyond common sense obligations, and the \u201csocial contract\u201d and \u201cmembership requirement\u201d are invoked because common morality do not seem sufficient for engineers.\nThe model of ethics education I will outline in the following is based on the notion of pride. The basic idea is simple. One can have pride as a professional even where the profession as an autonomous body is absent. Such pride will help engineers to adhere to high moral standards, even without being rewarded by society. We can expect to promote moral behavior in engineers (including engineering students) by developing their professional pride in engineering-ethics education.12 In terms of justification, this model does not try to justify special obligations of engineers; rather, the model tries to motivate students to accept supererogatory acts as their own obligations.\nConcept of Professional Pride\nTo explicate this approach, I need to say more about the very notion of pride in general, and professional pride in particular. First, let us examine the notion of pride itself. Pride is a positive feeling toward oneself and things associated with oneself. Let us call these things the \u201cobject\u201d of pride, following David Hume, whose analysis of this notion is one of the earliest [13].13 Groundless pride might be psychologically possible, but pride usually involves some beneficial properties about the object of pride as grounds (again, following Hume, let us call this the \u201ccause\u201d of pride). Since Hume, several moral philosophers have focused on this notion, especially in terms of virtue ethics. Some authors have considered pride as a vice (i.e., as contrary to the virtue of modesty), and others as a virtue [14, 15].14 When virtue ethicists (including Hume) talk about pride as a virtue, their reason for the positive evaluation of pride is based on its effect on oneself; pride is, according to these authors, a virtue because it promotes self-esteem and personal happiness.\nOf course, pride as a virtue is being considered within the present context. Just like other virtues in virtue ethics, such as courage, a misplaced pride and an excessive pride can be a vice rather than a virtue. Thus, just like a courage shown in a robbery does not count as a virtue, a pride as a robber does not count as a virtue. It is hard to make precise the distinction between misplaced and appropriate prides. This is a problem with the virtue ethics approach in general.15\nThis may be true of pride in general, but professional pride in particular can have a different other-regarding moral function. Professionals in various professions seem to have pride in their trade. What kind of pride is it? The object of professional pride may be the professional him or her as a professional, the profession as a collective body, or other professionals in the same profession. The cause of professional pride may be excellence in professional skill, the importance of the service they have done to society, or possibly high moral standards. To be justly proud of these things, professionals have to maintain these qualities in their professional work. This will of course benefit society. Here, we can see the possibility for professional pride to be an other-regarding virtue. The beautiful part of this scheme is that society does not necessarily have to reward good work; the feeling of pride itself functions as a reward. Of course, such feelings will be reinforced by the recognition of excellence by others.16\nWhen we think of professional integrity, professional pride has another virtue. Since pride is a positive feeling, it can motivate professionals to overachieve, i.e., to do things that are not required or expected (they overachieve simply because it feels good). This is in strong contrast to negative feelings, such as fear of punishment and feelings of guilt, which do not motivate professionals to achieve more than required. Given these limitations of negative feelings, some sort of positive feelings will be necessary as motivation in the transition from minimal professional responsibility to professional integrity, and professional pride is a good candidate for this.\nLet us now examine whether this will solve the problem of professional motivation in Japan. Given that the concept of the engineering profession as an autonomous body is absent in Japan, the profession itself may not be suitable as an object of pride. However, we do have individual engineers, and of course they can be the objects of their own professional pride. Some possible causes of professional pride (such as the high moral standards of the profession as a whole) are missing in Japan, but other important ones, such as their excellence as engineers and the importance of services they give to society, do exist. Rewards from society, which will reinforce this pride, can hardly be expected in Japan, but, as I said, pride is self-rewarding. Thus, the pride-based approach seems (at least) a possible solution to the problem.\nProspects for Pride-based Education\nThe pride-based approach is yet to be developed. There are many problems to be solved and many questions to be answered before the model outlined here can be implemented in engineering-ethics education. First, does real-world professional pride have desirable characteristics? It is possible for professionals to feel pride because of something other than their skills or services. For example, the sole cause of their pride may be their high income and prestige. If this is the case, the pride-based model does not work where the \u201csocial-contract\u201d view does not. This is a psychological question that calls for an empirical investigation. Second, can professional pride become strong enough to support professional integrity? The \u201csocial-contract\u201d view seems to provide a fairly strong motivational basis for professional ethics, where it works. Can pride be as strong as such motivation? This is another psychological issue, but harder to answer, given the speculative nature of the question. Third, is it possible to teach pride in the first place? Is pride not something we acquire by ourselves, rather than something taught in school? This is a pedagogical inquiry, which calls for classroom experiments.\nThere is an interesting survey that may illuminate some of these issues. Okamoto et\u00a0al. surveyed firefighters in Japan on their occupational self-esteem [17]. The results revealed that there were two independent factors in the sense of occupational self-esteem firefighters had (they conducted factor analysis on the replies to their questionnaire). The first was the esteem based on the service they gave, which Okamoto et\u00a0al. called \u201cself-esteem from occupational tasks\u201d, and the second was the esteem stemming from the very nature of the occupation (e.g., intense training and dangerous work), which they called \u201cself-esteem from occupational capacity\u201d (pp. 37\u201338). They further found that the self-esteem from occupational tasks was negatively correlated with delinquency, while the correlation between the self-esteem from occupational capacity and delinquency was not statistically significant (pp. 51\u201353).\nSince there are many differences between firefighting (which is not even a profession in the strictest sense) and engineering, we need to use a great deal of caution in applying the results of Okamoto et\u00a0al.\u2019s study. However, their results constitute positive empirical evidence that occupational self-esteem (which is often associated with, or even equated with, pride) can be caused by the importance of the service being provided, and that that kind of self-esteem helps the person to maintain high moral standards. If this is true, there are practical implications for engineering-ethics education under the pride-based approach. The best way to foster the desirable kind of professional pride in engineering students in the classroom is to stress the importance of the service provided by engineers. Professional pride acquired in this way will be conducive to professional integrity. Placing emphasis on professional capacity (e.g., higher education and skill) may also foster professional pride, but we cannot expect this kind of pride to be related to professional integrity.\nEven though I limited my discussion to Japan, it is plausible that many aspects of my discussion apply to other countries. Professionals play similar roles in other countries, and it is only natural that they have the similar motivational structure. Thus, in countries where the social contract model does not work, the pride-based approach may be a solution. In countries where we can assume the social contract between the society and engineering profession, we may not need the pride-based model, but still it may help in motivating engineering students.\nTo summarize, the notion of pride is a practical one, and engineering-ethics education based on this concept seems promising. Although we have to accumulate more empirical data and undertake more conceptual analyses before drawing conclusions on the effectiveness of such approaches, this approach is worth trying in a country where the profession of engineering and its implicit social contract with society are almost absent.","keyphrases":["professional integrity","japan","pride","engineering ethics"],"prmu":["P","P","P","P"]}
{"id":"Eur_Arch_Otorhinolaryngol-4-1-2358940","title":"Usefulness of three-dimensional computed tomography of the larynx for evaluation of unilateral vocal fold paralysis before and after treatment: technique and clinical applications\n","text":"Laryngoplasty is well-known technique for unilateral vocal fold paralysis (UVFP). However, operation result are sometimes not as good as expected before surgery. Three-dimensional Computed tomography (3DCT) is useful for visualizing complicated intralaryngeal structures. Moreover, 3DCT is suited for analyzing the movement of the vocal fold and arytenoid cartilage because the technique is based on actual data from live patients. We have been used 3DCT of the Larynx for evaluation of UVFP before and after treatment. We uncovered some new findings about UVFP and reasons of unsatisfactory outcomes after operation. Technique and clinical applications of 3DCT for UVFP are outlined in this paper.\nIntroduction\nThe movement of the arytenoid cartilage is complicated [1, 2]. Three-dimensional computed tomography (3DCT) is useful for visualizing the location of the arytenoid cartilage. However, complete understanding of the laryngeal anatomy is necessarily to reconstruct the cartilages in three dimensions. In our institution, an otolaryngologist who specializes in the larynx utilizes 3DCT reconstruction. The use of Windows\u00ae XP-compatible image-processing software enables various analyses in unilateral vocal fold paralysis. This study reports the clinical application of 3DCT in the laryngeal region using the image-processing software Mimics (Materialise, Yokohama, Japan).\nProduction of 3DCT\nComputed tomography was performed in the horizontal plane using a CT scanner (Sensation Cardiac 64; Siemens, Munich, Germany) with a 1\u00a0mm slice width, a helical pitch of 1, and an image production interval of 0.5\u00a0mm. Several other studies have used slice widths of around 1\u00a0mm [3\u20136].\nScans were made when the glottis was closed (with phonation) and open. Each patient was scanned for about 2\u00a0s. The speed of the scan enables scanning of patients with unilateral vocal fold paralysis during a brief phonation and minimizes motion artifact. Before scanning, doctors (the authors) were consulting the patient about the timing between phonation and the scan.\nThe data were transferred using the Dicom system. Mimics ver. 9.01 (Materialise) was used to create 3DCT images through its surface-rendering protocols. Using Windows\u00ae XP (Microsoft\u00ae, Redmond, WA, USA), 3DCT images can be shown in axial, coronal, sagittal, and 3D display windows (Fig.\u00a01).\nFig.\u00a01Actual view seen with the Mimics image-processing software. Using Windows\u00ae XP (Microsoft\u00ae), 3DCT images can be created with axial, coronal, sagittal, and 3D display windows. The use of Windows XP-compatible image-processing software enables various analyses in unilateral vocal fold paralysis\nThe threshold was set from 69 to 85\u00a0HU for the thyroid and cricoid cartilages and 52 to 69\u00a0HU for the arytenoid cartilage. Visualization of the vocal process is usually limited to its base because of its elastic cartilage. In addition, the laryngeal lumen was visualized. The threshold was set between \u2212600 and \u2212100\u00a0HU [7].\nStereoscopic location of the arytenoid cartilage in unilateral vocal fold paralysis\nFirst, we present a case of severe unilateral vocal fold paralysis with a mean flow rate (MFR) of 1,200\u00a0ml\/s (Fig.\u00a02). The patient was an 80-year-old man suffering from paralysis following aortic aneurysm surgery.\nFig.\u00a02The locations of the arytenoids in severe unilateral vocal fold paralysis. During inspiration, the healthy side (yellow) was located in the lateral part of the cricoarytenoid joint, while the paralyzed side (red) was located in the dorsocranial part of the cricoarytenoid joint. During phonation, the healthy side rocked and moved to the ventromediocaudal part of the cricoarytenoid joint, while the paralyzed side glided dorsocranially. This movement is a passive movement\nDuring inspiration, the arytenoid cartilage on the healthy side was located in the lateral part of the cricoarytenoid joint. Simultaneously, the vocal process was located outside and above the joint. In comparison, the arytenoid cartilage on the paralyzed side was located in the dorsocranial part of the cricoarytenoid joint, while the paralyzed vocal process was located inferiorly and the arytenoid cartilage dropped forward. This position on the paralyzed side was the result of posterior cricoarytenoid muscle paralysis.\nDuring phonation, the healthy side rocked and moved to the ventromediocaudal part of the cricoarytenoid joint to adduct the vocal cord, and the vocal process moved inward and downward. Conversely, the paralyzed side glided dorsocranially in comparison with its position during inspiration. Therefore, the arytenoid cartilage showed a rocking movement on the healthy side, and a gliding movement on the paralyzed side. This gliding movement on the paralyzed side was a passive movement. the paralyzed arytenoid underwent passive movement caused by contact with the mobile side during phonation. The vocal fold blown up may affect this passive movement. We call such arytenoids \u201cflaccid arytenoids.\u201d The position of the paralyzed arytenoid depends on the severity of paralysis.\nSurgical simulation\nThis section describes type I thyroplasty. It is important to determine the level of the vocal folds in this procedure. Note that the arytenoid cartilage on the paralyzed side glides dorsocranially, as mentioned above. The paralyzed vocal fold becomes higher than the vocal fold on the healthy side during phonation. The type I thyroplasty window should be based on the vocal cord level on the healthy side during phonation. Therefore, the vocal fold level of the healthy side must be projected onto the thyroid cartilage of the paralyzed side.\nFigure\u00a03 compares the upper surface of the vocal fold between our 3DCT simulation and the original type I procedure reported by Isshiki et al. [8]. The figure shows that the upper surface level of the vocal fold in the original procedure is consistent with the vocal fold level on the paralyzed side detected by 3DCT. This might have arisen because the upper surface level of the vocal cord in the original procedure was based on cadaveric studies of the larynx. However, the vocal process on the healthy side during phonation is lower than that on the paralyzed side. Therefore, the design should be made consistent with the healthy side. Individualized simulation for each case is needed because of the shape difference between left and right laminas of the thyroid cartilage (especially the laryngeal deviation) [9, 10]. The program Mimics allows distances to be measured on a personal computer; for example, the distances from the inferior notch or inferior nodule to the surface of the vocal fold may be measured. The 3DCT could contribute to a better oriented and designed thyroplasty window.\nFig.\u00a03A comparison of the upper surface of the vocal fold between the 3DCT simulation (red dotted line) and the original type I procedure reported by Isshiki (black dotted line). The figure shows a 3D image processed to make the thyroid cartilage more radiolucent, allowing the healthy vocal fold to be visualized. The upper surface level of the vocal fold with the original procedure is consistent with the vocal fold level of the paralyzed side detected by 3DCT. However, the vocal process of the healthy side during phonation is located below than that of the paralyzed side\nPostoperative evaluation\nCase 1\nA 69-year-old woman underwent arytenoid adduction for right recurrent nerve paralysis after thyroid carcinoma surgery. According to the postoperative 3DCT images (Fig.\u00a04), the vocal process adducted to the appropriate position. However, a large glottal chink was observed during phonation. In this case, the cricothyroid joint was released, and a thread placed on the muscular process was fixed to the anterior part of the thyroid cartilage. Since the cricothyroid joint was released, the cricoid cartilage (arytenoid cartilage) and thyroid cartilage were close together, which shortened the anteroposterior diameter of the vocal fold. The vocal fold relaxed and lost its tension.\nFig.\u00a04Case 1: A 69-year-old woman underwent arytenoid adduction for right recurrent nerve paralysis after thyroid carcinoma surgery. a In the postoperative 3DCT images, the vocal process appears to adduct to the appropriate position. b A large glottal chink is observed during phonation. c Since the cricothyroid joint is released, the cricoid (arytenoid cartilage) and thyroid cartilages are close together. d The anteroposterior diameter of the vocal fold (double-headed arrow) is shortened. The vocal fold relaxes and loses its tension\nCase 2\nA 64-year-old man underwent type I thyroplasty for left recurrent nerve paralysis after esophageal cancer surgery (Fig.\u00a05). This patient had relatively severe paralysis, but still underwent a type I thyroplasty only. The postoperative 3DCT image shows that the Gore-Tex\u00ae is inserted at the proper level of the vocal fold. However, the posterior parts of the vocal fold were at different levels because the flaccid paralyzed arytenoid cartilage was blown toward the dorsocranial part of the cricoarytenoid joint, as mentioned above. The MFR was improved from 770\u00a0ml\/s preoperatively to 290\u00a0ml\/s postoperatively. If the posterior support of the vocal fold had been fixed, the vocal fold would have developed tension against the flow of expired air during phonation, further improving the voice.\nFig.\u00a05Case 2: A 64-year-old man underwent type I thyroplasty for left recurrent nerve paralysis after esophageal cancer surgery. a The postoperative 3DCT image shows that the Gore-Tex\u00ae was inserted at the proper level of the vocal fold. b A level difference is seen in the posterior part of the vocal fold because the flaccid paralyzed arytenoid cartilage was blown toward the dorsocranial part of the cricoarytenoid joint during phonation\nCase 3\nA 70-year-old man was given a silicon injection 20\u00a0years earlier to treat left recurrent nerve paralysis after thoracic aortic aneurysm surgery (Fig.\u00a06). The MFR was 650\u00a0ml\/s, which improved insufficiently. The 3DCT showed that the silicon was injected widely from the anterior to posterior parts of the vocal fold. Silicon is also injected near the arytenoid cartilage. It was thought that the original surgeon had intended to adduct the arytenoid cartilage. However, the arytenoid cartilage glides dorsocranially, increasing the vertical difference in the level of the posterior part of the vocal fold. The vocal process was not adducted and the upper part of the arytenoid cartilage moved medially in an unnatural manner. As described earlier, during phonation, the arytenoid cartilage on the healthy side moves by rocking to the ventromediocaudal part of the cricoarytenoid joint, and the vocal process is moved inward. Arytenoid adduction [10, 11] is a procedure that resolves the level difference between the two vocal fold by reproducing the rocking motion of the paralyzed arytenoid cartilage. In contrast, injection and type I surgery are techniques to medialize the vocal fold by increasing its volume. An injection near the arytenoid cartilage moves the arytenoids to a dorsocranial position, and they never adduct naturally. If arytenoid adduction is scheduled after these techniques, the injected materials could interfere with the second procedure. The effect of the injection on posterior vocal fold closure arises mainly from the increased volume in the posterior part of the vocal fold.\nFig.\u00a06Case 3: A 70-year-old man underwent a silicon injection 20\u00a0years earlier to treat left recurrent nerve paralysis after thoracic aortic aneurysm surgery. a The 3DCT shows that the silicon is injected widely from the anterior to posterior parts of the vocal fold. b Silicon is also injected near the arytenoid cartilage. The arytenoid cartilage glides dorsocranially; the vocal process is not adducted and the upper part of the arytenoid cartilage is unnaturally shifted medially. The vertical difference in the level of the posterior part of the vocal fold increased. c The effect of the injection on posterior vocal fold closure is thought to be due mainly to the volume increase in the posterior part of the vocal fold\nCase 4\nA 77-year-old man received a fat injection for left recurrent nerve paralysis after esophageal cancer surgery. Three years after the fat injection, the maximum phonation time improved from 3\u00a0s before the injection to 7\u00a0s after the injection. The 3DCT image indicates that the vocal cord closure was sufficient (Fig.\u00a07a). In the lateral view, remaining fat is seen 3\u00a0years after the injection and it appears to be pushing the entire vocal fold (Fig.\u00a07b). However, the coronal section CT image revealed that the level difference of the two vocal folds had not improved. The vocal folds overlapped so that the incomplete closure was resolved (Fig.\u00a07c). Fat, unlike silicon, may not interfere with the vocal fold vibration of the healthy side, even if the vocal cords overlap, because fat is soft.\nFig.\u00a07Case 4: A 77-year-old man underwent fat injection for left recurrent nerve paralysis after esophageal cancer surgery. a The 3DCT (CT endoscopic image) suggests that the vocal cord closure is sufficient. b The lateral view shows fat remaining 3\u00a0years after the injection, which seems to be pushing the entire vocal fold. c The coronal view revealed that the difference in the levels of the vocal fold had not improved\nConclusions\nLaryngeal 3DCT is useful for determining the stereoscopic configuration of the arytenoid cartilage. In addition, 3DCT enables observation of the laryngeal framework from every angle, and is also useful for surgical simulation and feedback after surgery.","keyphrases":["three-dimensional computed tomography","unilateral vocal fold paralysis","thyroplasty"],"prmu":["P","P","P"]}
{"id":"J_Insect_Sci-1-_-355886","title":"Storage hexamer utilization in two lepidopterans: differences correlated with the timing of egg formation\n","text":"Most insects produce two or more storage hexamers whose constituents and developmental profiles are sufficiently different to suggest specialization in the ways that they support metamorphosis and reproduction. Hexamerin specializations are compared here in the Cecropia moth (Hyalophora cecropia), which produces eggs during the pupal-adult molt, and the Monarch butterfly (Danaus plexippus), which produces eggs under long-day conditions after adult eclosion. In both sexes of both species, reserves of arylphorin (ArH) were exhausted by the end of metamorphosis. In Cecropia, the same was true for the high-methionine hexamerins, V-MtH and M-MtH. But in short day Monarch females 20\u201330% of the pupal reserves of V-MtH and M-MtH survived metamorphosis, persisting until long-day conditions were imposed to stimulate egg formation. Differences in storage sites have been documented in other lepidopterans, with MtH reserves being found primarily in fat body protein granules and the ArH reserve being found primarily in the hemolymph. Similar differences could explain how a fraction of the MtH's, but not of ArH, escapes utilization during metamorphosis in a species with post-eclosion egg formation. No differences in utilization schedules were detected between V- and M-MtH, despite divergent compositions and antigenic reactivity.\nIntroduction\nInsects prepare for the synthetic demands of molting, metamorphosis and reproduction by accumulating hexamerins in their hemolymph and fat body. First to be described were two soluble storage hexamerins isolated from larvae of Calliphora erythrocephala (Munn and Greville, 1969). Two or more hexamerins were later reported in the hemolymph and fat body of many other insects, with some lepidopterans having as many as four. Hexamerins occurring within a single species can differ in amino acid composition, stage of synthesis, distribution between hemolymph and fat body, timing of clearance from the hemolymph, and antigenic reactivity (reviewed by Wyatt and Pan, 1978; Riddiford and Law, 1983; Kanost et al., 1990; Telfer and Kunkle, 1991).\nThe first storage proteins to be described in lepidopterans were two methionine-rich hexamerins from Hyalophora cecropia (Tojo et al., 1978). They differed in electrophoretic mobility in native PAGE and also in amino acid composition, methionine contents being 7.0% for the more slowly migrating and 4.9% for the faster. (We adopt here descriptive acronyms, V-MtH and M-MtH, for very and moderately methionine-rich hexamerins.) The two appear in the hemolymph late in the last larval instar, rise to maximum concentrations early in the larval-pupal molt, and are then largely endocytosed by the cells of the fat body, which store them in crystalline form until they are utilized during adult development. MtH's have been identified in a variety of moths, either as isolated proteins or from cDNA sequencing (Tojo et al., 1978; 1980; 1985; Ryan et al., 1985; Bean and Silhacek, 1988; Jones et al., 1990; Jones et al., 1993; Memmel et al., 1994). They are identified here in a butterfly by antigenic cross-reactions. They tend to be more abundant in female pupae than in males, and this has led to a prediction that they play special roles in egg formation.\nA third lepidopteran hexamerin, first described in Manduca sexta (Kramer et al., 1980), has a high aromatic amino acid content, resembling in this regard one of the prototypical hexamerins of Calliphora. The importance of aromatic amino acids in sclerotization led to the suggestion that hexamerins in this class, now known as arylphorins (ArH) (Telfer et al., 1983), are adapted to support cuticle deposition. While many lepidopterans produce just these three, some also contain a riboflavin-binding hexamerin, RbH (Telfer and Massey, 1987; Miller and Silhacek, 1992; Magee et al., 1994). Like ArH, RbH is a major component of pupal hemolymph that disappears during adult development (Pan and Telfer, 1999).\nLabeling experiments in saturniid moths failed to confirm the targeting of hexamerins for special developmental processes. In Actias luna M-MtH and ArH proved to be equivalent sources of labeled amino acids for the production of eggs and cuticle, as well as other adult tissues (Pan and Telfer, 1996), and RbH and ArH were found to be similarly equivalent in H. cecropia (Pan and Telfer, 1999). These tests were performed in species that produce eggs at the same time that they form the soma of moths. They thus left open the question of which of the hexamerins remain available to support egg formation when the latter is delayed until after adult eclosion.\nTo investigate this we compared the timing of hexamerin depletion in two lepidopterans that form eggs at different times relative to eclosion. The Cecropia silkmoth, Hyalophora cecropia, ecloses with a full complement of eggs that are ready to be fertilized and laid. The Monarch butterfly, Danaus plexippus, delays egg formation until it experiences a long-day photoperiod after eclosion (reviewed by Ackery and Vane-Wright, 1984). The strategy we used was to time in each species the disappearance of V-MtH, M-MtH and ArH during metamorphosis and egg formation. The accumulation of vitellogenin (Vg) was simultaneously measured, since synthesis of this abundant product by the adult fat body can be correlated with hexamerin depletion (Wheeler and Buck, 1996; Wheeler et al., 1999). RbH was not included because we could not detect it with rabbit antibodies in Monarch larvae or pupae (Pan and Telfer, 1999).\nMaterials and Methods\n1. Experimental Insects\nDiapausing pupae of field-reared Hyalophora cecropia were stored at 6\u00b0C for at least five months and then allowed to initiate adult development by transfer to 25\u00b0C. At this temperature development from apolyis to eclosion requires 23 days in females and 21 days in males.\nMigrating butterflies of Danaus plexippus were captured during mid-October in eastern Tennessee. Most of the catches were sufficiently fresh to suggest recent emergence in nearby regions. They were kept in cages at outdoor temperature and day length, and fed once per day with a 30% honey solution. After two to three weeks under these conditions the ovaries contained only small, translucent follicles, indicating an absence of yolk formation. Their fat bodies, relative to those of freshly caught butterflies, were greatly enlarged and full of lipid droplets.\nTo obtain pupae and second generation adults, some captured Monarchs were kept at 30\u00b0C under long day conditions, and fed twice daily with the honey solution. They terminated reproductive diapause and began mating in 1 to 2 weeks. Potted milkweed, Asclepias curasavica, was provided for egg laying. Hatched caterpillars were left on the plants until they reached the 3rd instar, and were then transferred to field-collected cuttings of Asclepias syriaca. Additional eggs and caterpillars were generously provided by Mr. Paul deMarrais of Afton, Tennessee.\n2. Antibodies\nV-MtH, M-MtH, ArH and Vg were measured in both species with rabbit antibodies against the corresponding proteins of H. cecropia. The reactions with Monarch proteins, though weaker than those with Cecropia, were still strong enough to be useful. This relationship is shown in Figure 1 for antibodies to Cecropia V-MtH.\nArH, Vg and what we identify below as M-MtH have been isolated from the hemolymph of Cecropia pupae by chromatographic methods, and the reactions of the antisera employed to measure them have already been described (Telfer et al., 1983; Telfer and Pan, 1988; Pan and Telfer, 1992). Each of the three antisera reacted with its homologous isolate to produce a single zone of heavy precipitation in immunodiffusion tests and showed no additonal reactions when combined with unfractionated hemolymph, whole-body extracts, or isolates other than the one used for immunization.\nThe antiserum against V-MtH has not been described before. We used native PAGE to isolate this hexamerin from pupal male hemolymph. Slab gels (0.3 \u00d7 12 \u00d7 17 cm) containing 4% acrylamide and lacking lane dividers were overlain with 40 \u00b5l of pupal male hemolymph diluted with twice its volume of native PAGE sample buffer. After electrophoresis a vertical strip from the center of the gel was stained with Coomassie Blue (Figure 2, lane 1). Horizontal strips corresponding to the positions of the two MtH's were cut from the unstained remainder of the gel and stored by freezing.\nThe two MtH's were identified by N-terminal amino acid microsequencing at a facility of the University of Pennsylvania School of Veterinary Medicine. For the faster of the two in native PAGE, the N-terminal sequence was RPDNDDVNFVVSM and for the slower band was SVVNDANYSF (40% of the fast component lacked the N-terminal RP but was otherwise identical to the other 60%.) These sequences matched N-terminal sequences deduced from cDNA clones of, respectively, M-MtH and V-MtH after signal polypeptide deletion (Massey, 1995; accession number AF032398 for M-MtH and AF032399 for V-MtH). The faster band corresponded to a 738 amino acid polypeptide with a methionine content of 4.76%, while the slower corresponded to a 737 amino acid polypeptide with a methionine content of 8.54%. Protein extracted from the slower band, thus identified as V-MtH, was used to immunize a rabbit (Harlow and Lane, 1988). In a variety of immunodiffusion tests with pupal hemolymph and extracts from H. cecropia, anti-V-MtH produced a single band of precipitation (Figure 1).\n3. Tissue Preparation\nHemolymph was drained through a dorsal slit into a pre-chilled, graduated test tube containing 0.2 ml of a buffer solution\u2014either, pH 7.2 phosphate-buffered saline (PBS) for Cecropia or 0.15 M Tris-citrate, pH 8.2, for the Monarch. (The Tris-citrate buffer used for the Monarch extraction was 6X that of the running buffer strength employed for Rocket Immunoelectrophoresis (Pan and Telfer, 1990). It was used in case the smaller amount of tissues collected from this species should prove to require a more sensitive technique for protein quantification, but this proved eventually not to be the case.) Both extraction media contained in addition a protease inhibitor cocktail, Control\u00ae (Boehringer Mannheim Biochemical), and 5mM phenylthiourea (PTU). The midgut and, when present, the bursa copulatrix were discarded and the rest of the soft tissues were scraped out of the abdominal cuticle and added to the collected blood. Additional buffer with the protease inhibitor and PTU was added to bring the suspended tissues to a volume that would yield antigen concentrations convenient to measure by the method described below. For Cecropia the final volume was 6 ml per pupa, and 3 or 1.5 ml per adult female and male, respectively. For Monarch pupae the final volume was 3 ml and for adults 0.75 ml. Comparisons between stages were made possible by normalizing to the pupal volume; e. g., concentrations for adult Cecropia females were divided by two and for males by four in order to compare them with the contents of pupal extracts.\nThe collected samples were stored frozen. For extraction, the tissue mixtures were thawed and transferred to a glass homogenizer. When chorionated eggs were present, they were crushed separately in a ceramic mortar and then combined with the rest of the tissues in the homogenizer.\n4. Measurement of Antigen Concentrations\nAn immunodiffusion method was used to measure changes in extractable stores of the four proteins. Antigen concentrations were measured by layering the extracts over antisera that had been solidified in 3 mm (i. d.) glass tubes with 0.3% agarose, PBS and 0.05% sodium azide. The rate of penetration of an antigen-antibody precipitation front through the agarose is a function of the rate of diffusion of the antigen into the agarose layer and this in turn is a measure of antigen concentration in the upper reservoir. Procedures for setting up the tubes, for measuring rates of advance of precipitation fronts, and for constructing standard curves have already been described (Telfer et al., 1983; Telfer and Pan, 1988). The method was developed for studies on human serum proteins (Oudin, 1948), and has been used many times to measure antigen concentrations in insect hemolymph and egg extracts. It requires larger volumes and higher antigen concentrations than rocket immunoelectrophoresis (Pan and Telfer, 1990, 1992), but when these conditions are satisfied, as they proved to be for hexamerin measurements in Cecropia and Monarchs, it is convenient and reliable.\nThe measurements are of relative concentrations and are expressed as percentages of the concentration in a standard solution. For the hexamerins, extracts of abdomens from female pupae of each species were used as the standards; for VG, extracts of females containing chorionated eggs were used. The latter included newly eclosed adults in Cecropia and day 7 adults in long day Monarchs.\n5. SDS-PAGE Electrophoresis\nInsoluble residues that would escape detection by immunodiffusion were monitored by SDS-PAGE. For this purpose 0.1 ml of each of the homogenized samples from a chosen stage were vortexed and pooled. The pooled sample was centrifuged in a microfuge (Beckman\u00ae) for 4 min. The lipid cap was removed, and the supernatant decanted. Pellet and supernatant were then separately dissolved in SDS-PAGE sample buffer so that equal volumes contained equal fractions of, respectively, the total pellet and supernatant material in the uncentrifuged pool. The samples were compared in neighboring lanes of pre-cast 4\u201315% gradient mini gels from Bio-Rad\u00ae. Electrophoresis was conducted at constant voltage (100 v), and terminated after 1 hr and 12 min. Coomassie Brilliant Blue staining was used to reveal the banding patterns.\nResults\n1. Vitellogenin\nCecropia produced Vg earlier than the Monarch (Figure 3). Like many other saturniids, Cecropia synthesizes part of its Vg, in this case about 30 mg per 6 g female, during the larval-pupal molt, and stores it in the hemolymph during the months of diapause (Telfer, 1954). Beginning 10 days before eclosion, when yolk formation begins, there is a second period of Vg synthesis (Pan, 1971) (Figure 3) that draws on precursors released from the hexamerins present at that time (Pan and Telfer, 1996, 1999). In Monarchs, Vg was not detected in either pupae or short day adults. In females raised under long day conditions it was first detected 2\u20133 days after eclosion (Figure 3) and it continued to accumulate until day 7 when eggs began to be laid.\nIn SDS-PAGE, Coomassie-stained Vg subunits were identified at 180 and 40 kDa by their presence in females and absence from males (Figure 4, Vg). They were found in pupal and adult extracts of Cecropia and in extracts of long day Monarch adults, the same samples in which Vg had been detected by immunodiffusion.\nVg is stored as a solute in the hemolymph of female Cecropia pupae and accordingly centrifugal pellets of extracts at that stage did not produce detectable 180 and 40 kDa bands. In adults most Vg has been transferred to the yolk; pellets at this stage produced weak Vg bands due, presumably, to small yolk spheres that are not readily disrupted by homogenization.\n2. Hexamerin utilization in females\nCecropia\nAll three Cecropia hexamerins were consumed during the first 13 days of somatic tissue metamorphosis, 55% of the soluble MtH's and 33% of soluble ArH disappeared before VG began its second rise (Figure 5A). The last eight days of adult development encompass late somatic tissue development as well as the second rise in Vg content, and during this period the three hexamerins fell to less than 2% of pupal levels.\nCoomassie staining of SDS-PAGE gels of pupal extracts was concentrated in a cluster of circa 80 kDa bands (Figure 4A, asterisks). Mini gels resolved the hexamerin subunit bands in the clusters poorly, but for present purposes that was not a problem. Low 80 kDa staining in centrifugal pellets of adult Cecropia extracts (Figure 4A, lanes A1 and A2) showed that PBS-insoluble reserves, like the soluble reserves assayed by immunodiffusion, were essentially negligible after eclosion.\nMonarch\nNearly all of the soluble ArH stored in Monarch pupae was consumed during metamorphosis, but substantial MtH reserves remained. This was first seen in individuals that had been collected in the field in October and then fed as captives for four weeks under short day conditions (Figure 6A). At the end of the four weeks, short-day females still contained about 20% as much M-MtH and 30% as much V-MtH as female pupae. When captive females were held for two weeks under short day conditions and then for two additional weeks in the long days that promote egg formation, these figures were much lower\u20141.5% for M-MtH (p < 0.01) and 0.7% for V-MtH (p < 0.02) (Figure 6A). These results were confirmed by SDS-PAGE, which detected a strong 80 kDa band in short day adults (Figure 4B, lanes Ast) and very little staining in long-day adults (lanes Alg). The time-course of the decrease was examined in a set of females that had been raised from eggs in the laboratory under long day conditions (Figure 7). In this case 32% of extractable pupal M-MtH and 47% of V-MtH remained at the time of eclosion. During one week of feeding as adults under continuing long day conditions three quarters of the reserves present at eclosion were consumed.\n3. Sexual Differences.\nWhile males contained roughly as much soluble ArH as females in Cecropia pupae, their contents of V-MtH and M-MtH were significantly lower (Figure 5B). A sexual difference was also seen during adult development: from days 13 to 21, when vitellogenesis and chorion deposition occur, utilization of V-MtH and M-MtH was more rapid in females than in males (Figure 5A and B). In males, as described above for females, immunodiffusion failed to detect more than 1\u20132% of the pupal levels of any of the three hexamerins at the time of eclosion (Figure 5B, Adult). In confirmation of this, SDS-PAGE revealed only traces of 80 kDa material in centrifugal pellets (Figure 4A, stages A1 and A2, lanes P).\nMonarchs differed from the lepidopteran norm in showing no sexual differences in the amounts of soluble V- and M-MtH stored in pupae (Figure 6B, green bars). In four week old adults raised under short day conditions, however, females retained larger reserves than males (Figure 6A and B, red bars), but had consumed more of these two proteins under conditions permitting egg formation (Figure 6A and B. blue bars).\nDiscussion\n1. Egg formation versus metamorphosis\nThe results are clear in showing that methionine-rich hexamerins in the absence of ArH support egg formation in Monarchs. Short-day adults, whose ovaries do not produce eggs, retained 20\u201330% of their pupal reserves of the MtH's, but only 0.3% of ArH (Figure 6A). Reduction of V-and M-MtH to only 1\u20132% of pupal levels in long-day females that had begun to lay eggs confirms that adult MtH's are in fact consumed during egg formation. The Monarch results also show that V- and M-MtH support somatic tissue development, as 50% of pupal V-MtH and 70% of M-MtH disappeared during adult development (Figure 7). The depletion of pupal V- and M-MtH in males (Figure 6B) also indicates functions other than the support of egg formation.\nArH, too, is multi-functional. It is known to support larval molting in the absence of the MtH's in Manduca sexta (Kramer et al., 1980; Riddiford and Hice, 1985; Webb and Riddiford, 1988) and Cecropia (Telfer et al., 1983). This situation differs in the dictyopteran, Blatta orientalis, whose two hexamerins are both present in larval hemolymph and cycle together during molting (Duhamel and Kunkel, 1978). ArH can also support reproductive functions, for it was as effective as M-MtH in providing precursors for the synthesis of Vg and chorions in pharate adult Luna moths (Pan and Telfer, 1996).\nSelectivity in the consumption of the multiple hexamerins within a lepidopteran species is shown by these results to be related to availability: the MtH's are not used in the larval molts because they are not synthesized until late in the last instar (Tojo et al., 1978; Riddiford and Hice, 1985; Jones et al, 1993; Memmel et al, 1994); and pupal reserves of ArH do not support egg formation in adult Monarchs because they are fully consumed during metamorphosis. When both are at hand, however, both contribute to whatever metamorphic and reproductive functions are under way.\nTwo kinds of processes are apparent that affect availability. Most obvious is the early synthesis of ArH that makes it uniquely available for the support of larval molting. The second involves differences in mode of storage. In insects generally, storage hexamers are secreted into larval hemolymph, where they can attain extraordinary concentrations. When metamorphosis approaches they are reclaimed to varying degrees by the fat body, and packaged in cytoplasmic storage granules. In an earlier study over 99% of injected M-MtH was cleared from the hemolymph of pharate pupae, compared with only 35% of ArH (Pan and Telfer, 1992). A consequence of this in both Luna and Cecropia pupae is that the MtH's are the most prominent proteins of fat body extracts while ArH is the most prominent protein of hemolymph (Figure 2 in Pan and Telfer, 1996, and Figure 17 in Tojo et al, 1978). There can also be differences in the disposition of proteins within the fat body, for in Cecropia the hexamerin storage granules contain protein crystals embedded in an amorphous material (Tojo et al., 1978). Differences in accessibility provide a plausible speculation on how V- and M-MtH, but not ArH, might survive metamorphosis in sufficient amounts to support post-eclosion egg formation.\n2. How hexamerins support egg formation\nHexamerins presumably support egg formation primarily by providing precursors for protein synthesis, for the synthesis of yolk proteins by fat body and of chorion proteins by the follicle cells both entail transcription and the incorporation of free amino acids (Wyatt, 1991; Paul and Kafatos, 1975; Nadel and Kafatos, 1980). Cases of intact hexamerin incorporation by ovaries have been described, but these are idiosyncratic rather than general. Antigenic epitopes of all four lepidopteran hexamerins are present among yolk proteins of saturniid moths, but they are only 0.2% as concentrated as those of Vg (Telfer and Pan, 1988). In Hyphantria cunea antigenic activity of an M-MtH-like protein was detected in intercellular spaces and yolk during vitellogenesis, but was absent from mature eggs (Seo et al., 1998). The authors speculated that this protein provides an internal source of amino acids for protein synthesis in the maturing egg. A more striking exception occurs in Riptortus clavatus, a hemipteran whose yolk contains approximately equal amounts of Vg and a 500 kDa, biliverdin-binding hexamerin (Chinzei et al., 1990; Miura et al., 1998). The yolk form of this otherwise conventional storage protein is secreted by the fat body of adult females in synchrony with Vg and selectively endocytosed by the vitellogenic oocyte. In another unusual case, the principal hemolymph storage proteins of last instar larvae and pupae of Bombyx mori are not hexamerins but a family of 30 kDa proteins that persist into the adult stage and are accumulated during vitellogenesis as major constituents of yolk (Zhu et al., 1986; Chen and Yamashita, 1990). But immunodiffusion and SDS-PAGE revealed nothing comparable to either the Riptortus or Bombyx storage proteins in egg-filled abdomens of long-day Monarchs.\n3. Hexamerin synthesis in adults?\nThere are other cases of the resumption of hexamerin synthesis in eclosed adults (e.g., Kunkel and Pan, 1976; Wyatt et al., 1992), but these occur in males as well as females and are thus not necessarily related to egg formation. Post-eclosion synthesis is not ruled out as a source of V- and M-MtH in adult Monarchs, but if it occurs it is not sufficient to prevent the depletion that accompanies egg formation. It would not, in any case, affect the argument that the consumption of residual pupal V- and M-MtH, rather than ArH, is correlated with post-ecdysial egg formation.\n4. V-MtH versus M-MtH\nThe two methionine-rich hexamerins are electrophoretically distinct and show no detectable similarities in precipitation by polyclonal rabbit antibodies. Yet they behaved here identically in the timing of their disappearance during adult development and in the fraction of their pupal reserves that survived metamorphosis. Cladistic analyses of amino acid sequences deduced from cDNA clones suggest that they are descended from products of a gene duplication occurring early in lepidopteran evolution (Burmester et al., 1998). But we are as yet left with no clue as to why natural selection has favored a pair of diverging MtH's over a single protein that might have averaged their amino acid compositions.","keyphrases":["no keywords available"],"prmu":["M"]}
{"id":"Clin_Auton_Res-3-1-1797062","title":"The autonomic nervous system at high altitude\n","text":"The effects of hypobaric hypoxia in visitors depend not only on the actual elevation but also on the rate of ascent. Sympathetic activity increases and there are increases in blood pressure and heart rate. Pulmonary vasoconstriction leads to pulmonary hypertension, particularly during exercise. The sympathetic excitation results from hypoxia, partly through chemoreceptor reflexes and partly through altered baroreceptor function. High pulmonary arterial pressures may also cause reflex systemic vasoconstriction. Most permanent high altitude dwellers show excellent adaptation although there are differences between populations in the extent of the ventilatory drive and the erythropoiesis. Some altitude dwellers, particularly Andeans, may develop chronic mountain sickness, the most prominent characteristic of which being excessive polycythaemia. Excessive hypoxia due to peripheral chemoreceptor dysfunction has been suggested as a cause. The hyperviscous blood leads to pulmonary hypertension, symptoms of cerebral hypoperfusion, and eventually right heart failure and death.\nIntroduction\nHigh altitude places are amongst the most inhospitable on earth. According to WHO [70] in 1966 there were approximately 140 million people living at altitudes over 2,500\u00a0m and there are several areas of permanent habitation at over 4,000\u00a0m. These are in three main regions of the world: the Andes of South America, the highlands of Eastern Africa, and the Himalayas of South-Central Asia. This review is concerned with the effects of the altitude on visitors and the ways by which the permanent high altitude dwellers have adapted to their environment.\nThe two main challenges to life at high altitude come from hypobaric hypoxia and the low ambient temperatures. Temperature decreases about 1\u00b0C for each 150\u00a0m elevation, so that at 4,500\u00a0m temperature is roughly 30\u00b0C lower than at sea level. Barometric pressure falls progressively with increasing altitude. Up to about 2,500\u00a0m there are few if any effects of hypoxia. Above 3,000\u00a0m some effects of hypoxia are likely to be experienced and above 4,000\u00a0m adverse effects would be experienced by most unacclimatized visitors. However, many people live and work at altitude with no apparent adverse effects. One such example is Cerro de Pasco a busy mining town of 72,000 population at 4,300\u00a0m in the Peruvian Andes, where much high altitude research has been undertaken. At this altitude barometric pressure is 450\u00a0mmHg and without hyperventilation alveolar oxygen tension would be only 34\u00a0mmHg.\nAutonomic control in visitors\nMost adaptive changes occur in the first days and weeks following arrival at altitude, and this is the period when acute mountain sickness with cerebral and\/or pulmonary oedema may occur. Recent studies in animals and man have highlighted the role of the autonomic nervous system in adaptation and in particular the importance of sympathetic activation following high altitude exposure.\nCardiovascular effects\nAcute hypobaric hypoxia results in an increase in resting heart rate and blood pressure and this is seen both during altitude exposure [5] and during simulated exposure using a hypobaric chamber [68]. Vogel et al. [68, 69] demonstrated that the rate of ascent influenced the magnitude of the tachycardia. Gradual increases in altitude over two weeks resulted in larger heart rate changes compared with an abrupt ascent. Later, as subjects acclimatise at altitudes up to about 4,500\u00a0m, much of the increase in heart rate is lost and resting heart rates return towards their sea level values.\nAcute hypoxia also causes an increase in cardiac output both at rest and for given levels of exercise. This was seen both when breathing hypoxic gas at sea level [34, 68, 69] and on acute exposure to high altitude [21]. As subjects acclimatise to the altitude cardiac output decreases although the heart rate can remain high with a low stroke volume. This may be due to a loss of plasma volume [23, 54].\nThe effect of hypoxia on the pulmonary circulation is rapid, resulting in an increase in pulmonary vascular resistance and pulmonary hypertension [49]. The maximum response occurs within 5\u00a0min [65]. Breathing 11% oxygen for 30\u00a0min increases pulmonary artery pressure from 16 to 25\u00a0mmHg [72]. The effect of hypoxia on the pulmonary circulation is even more pronounced during exercise, as demonstrated in the Operation Everest II studies [24] where pulmonary artery pressure increased during near-maximal exercise at 8840\u00a0m to 54\u00a0mmHg.\nThe mechanism of pulmonary artery vasoconstriction initially involves inhibition of O2 sensitive K+ channels leading to depolarization of pulmonary artery smooth muscle cells and activation of voltage gated Ca2+ channels causing Ca2+ influx and vasoconstriction [48]. This process is immediately reversed by breathing oxygen. However, lowlanders exposed to high altitude for 2\u20133 weeks develop pulmonary hypertension that is not completely reversed by oxygen breathing [24] suggesting remodelling of pulmonary arterioles. Remodelling involves proliferation of smooth muscle cells and thickening of the artery wall [57].\nSympathetic activity\nAcute hypoxia is a potent activator of sympathetic activity [39, 56]. Studies in several species, including dogs, rats and rabbits, showed that hypoxia stimulated the sympathoadrenal system. Acute hypoxia in spontaneously breathing anaesthetized animals causes increases in sympathetic nerve activity, increased release of catecholamines, increases in heart rate and regional vasoconstriction [28, 60]. However the effects of hypoxia on the human sympathetic nervous system are more difficult to determine and often indirect methods of assessment have been employed.\nOne method of assessment of sympathetic activity in humans is from blood or urine levels of catecholamines. However, catecholamine levels are the net resultant of secretion, spill-over, reuptake and excretion [59, 18] and results must be interpreted with caution. Mazzeo et al. [40] measured arterial noradrenaline and adrenaline concentrations in subjects at sea level, then after 4\u00a0h then 21\u00a0days at altitude (4,300\u00a0m). They reported an initial decrease in noradrenaline but by day 21 it had increased to 52% above sea level values. Arterial adrenaline values doubled following acute altitude exposure then declined to only 26% above sea-level by day 21. In a later study, however, the same authors [41] measured 24\u00a0h urinary noradrenaline and adrenaline excretion and venous plasma catecholames in women at sea level and during 12\u00a0days of exposure to 4,300\u00a0m and reported increases in both urinary noradrenaline and adrenaline excretion after only one day at altitude and increases in plasma catecholamines on day 4 at altitude. During the 12\u00a0day period noradrenaline continued to increase as assessed both from urinary excretion and in the plasma samples. Adrenaline values however fell back to those recorded at sea level. Rostrup [59] reported initial decreases in both plasma noradrenaline and adrenaline which subsequently recovered. Results from simulated hypoxia are also conflicting with an increase in urinary adrenaline but no change in noradrenaline [29] or no change in either [71]. These results illustrate the difficulty in assessing autonomic activity from blood or urine catecholamines, but they do suggest that in the early stages of exposure to altitude there is an increase mainly in adrenaline, but that later it is noradrenaline that predominates.\nThe changes in catecholamines are more consistent during exposure to chronic hypoxia. Calbet [10] measured systemic and skeletal muscle noradrenaline and adrenaline spillover in lowlanders after exposure to 5,260\u00a0m. After 9\u00a0weeks plasma noradrenaline and adrenaline concentrations were approximately 4 and 2 fold higher than the sea level values. These values were similar to those in patients with compensated chronic heart failure [2].\nThe heart rate at maximal exercise is reduced at altitude. In Operation Everest II maximal heart rates decreased from 160 at sea level to 118 at 8,848\u00a0m [54]. Given the evidence for elevated catecholamines at altitude, at least during chronic exposure, this suggests a down regulation of the cardiac \u03b2 -adrenergic receptors. Studies in rats following prolonged exposure to hypobaric hypoxia have shown a decreased \u03b2-adrenergic receptor density [30, 67]. Short exposures to hypoxia (1\u201315\u00a0days) did not affect \u03b2-adrenergic receptor density. However by 21\u00a0days there was a 24% reduction. Leon-Velarde et al [35] exposed rats to a simulated altitude of 5,500\u00a0m for 21\u00a0days and also found a 24% reduction in \u03b2-adrenergic receptor density in both left and right ventricles. They also reported an increase in \u03b11-adrenergic receptor density in the left ventricle, although Morel et al [48], who exposed rats to 5,500\u00a0m for 15\u00a0days, found no change in \u03b11-adrenergic receptor density in either left or right ventricle. Density of adenosine receptors has also been shown to be decreased by 46% following 30 day exposure to 5,500\u00a0m simulated altitude in the rat while muscarinic receptor density increased by 49% [31].\nChanges in receptor density have also been estimated indirectly in man by determining the rate of isoprenaline infusion required to increase heart rate by 25\u00a0beats\/min. The required rate increased with increasing exposure to altitude and this was attributed to a down regulation of the \u03b2-adrenergic receptors [56]. Platelet \u03b12-adrenergic receptor density decreased after 4\u00a0weeks exposure to 5,050\u00a0m [19]. Changes in \u03b12-adrenergic receptor density on platelets may indicate a similar change in the central nervous system [53]. In the central nervous system \u03b12-adrenergic receptors are known to play an important role in cardiovascular regulation [22]. Stimulation of these receptors in the ventrolateral medulla has been shown to reduce sympathetic and increase parasympathetic outflow [55]. If a change in the density of these receptors occurred it may explain many of the effects of altitude on the autonomic system.\nThe effect on the parasympathetic system has been assessed in humans from the responses to muscarinic blockade. Following short exposures to hypoxia the changes in heart rate following muscarinic blockade became smaller [11]. However, Boushel et al. [8] exposed subjects to an altitude of 5,260\u00a0m for 9\u00a0weeks and then studied the effects of muscarinic blockade both at rest and during exercise, and suggested that there was an enhanced parasympathetic activity and that this was responsible for the reduction in heart rate seen during chronic adaptation to altitude.\nMuscle sympathetic nerve activity\nSeveral studies have examined the effects of acute hypoxia on muscle sympathetic nerve activity and reported increases in discharge [17, 27]. Chronic hypoxia also enhances sympathetic activity and Hanson and Sander [27] reported that, following exposure to an altitude of 5,260\u00a0m for 4\u00a0weeks, activity remained elevated 3\u00a0days following descent.\nHeart rate variability\nCardiac autonomic nerve activity is often assessed noninvasively from spontaneous changes in heart rate. This is influenced by respiration [9] and this complicates many studies of hypoxia due to the concomitant respiratory stimulation. High frequency R\u2013R interval power is considered to be associated with cardiac parasympathetic activity whereas the low frequency components are associated with both parasympathetic and sympathetic activity. The ratio of low to high frequency power is said to be an index of the \u201csympathovagal balance\u201d. Power in both high and low frequency bands were found to decrease at altitude although the ratio of low to high frequency power increased [32, 16, 17]. The increase in the ratio is believed to imply that the sympathetic activity is dominant compared to parasympathetic. These findings imply that acute exposure to hypoxia causes decreased parasympathetic and increased sympathetic tone and during acclimatization there is a progressive shift toward still higher sympathetic tone.\nArterial baroreflex\nStudies in humans of baroreflex control at altitude or simulated altitude have yielded contradictory results. Sagawa et al. [61], using the neck chamber method, reported that acute exposure to simulated altitude of 4,300\u00a0m had no effect on carotid baroreflex set point for heart rate control but there was a 50% reduction in the gain of the reflex. Halliwill and Minson [25, 26], on the other hand, using nitroprusside and phenylephrine, found that hypoxic breathing increased set point but did not change gain. Studies of \u201cspontaneous\u201d baroreflex gain have indicated that it decreases at altitude or simulated altitude [5, 6, 7, 63]. Interpretation of these findings is complicated by the differences in baseline blood pressures and heart rate. Recently, Cooper et al. [15] reported that although acute hypoxia did not change baroreceptor control of heart rate it did decrease the gain of the vascular resistance response without changing \u201cset point\u201d.\nMechanisms for sympathetic activation at high altitude\nThe increase in sympathetic activity at altitude is caused by both the direct and indirect effects of hypoxia. The role of chemoreceptors is reviewed by Marshall [39]. Hypoxia acts directly on vascular smooth muscle in the systemic circulation causing relaxation and therefore hypotension. This, in turn, would lead to baroreceptor-mediated sympathetic excitation. Alterations in baroreflex function: an increase in \u201cset point\u201d and possibly a decrease in gain, are likely to contribute. An additional mechanism for exciting sympathetic activity may also arise through stimulation of pulmonary arterial baroreceptors. Hypoxia induces pulmonary hypertension and we have recently reported that, in the anaesthetized dog, increases in pulmonary artery pressure increase systemic vascular resistance [38, 46].\nAutonomic function in high altitude residents\nHealthy highlanders\nHealthy high altitude dwellers show excellent adaptation to their environment. These adaptations are likely to be associated with altered gene expression as the expression of genes associated with vascular control and reactions to hypoxia have been found to be high in altitude dwellers [1]. Different communities, however, seem to adopt different adaptation strategies [4]. For example Andeans hyperventilate to decrease end-tidal and arterial CO2 levels to as low as 25\u00a0mmHg and have haemoglobin levels well above those at sea-level. Ethiopian highlanders, on the other hand, have CO2 and haemoglobins levels similar to sea-level dwellers [13, 33].\nDespite the successful adaptation of permanent residents, some differences do occur. The carotid body shows age-related hyperplasia but blunted responses to hypoxia [62]. High altitude dwellers have been reported to show earlier cardiovascular degenerative changes with aging. Arterial wall stiffness is greater and blood pressures are higher [51, 52]. High altitude residents, both healthy subjects and patients with chronic mountain sickness (see below) have higher catecholamine levels when measured at altitude than following descent to sea level [20]. Baroreceptors, however, appear to function normally at altitude [5]. We [45] examined cardiac and vascular responses to neck chamber pressures and found that responses were unaffected by descent to sea-level. Moore et al. [47] also studied baroreceptor function in anaesthetized dogs, which allows better control of relevant variables. There was no difference in the gain of the baroreflex control of vascular resistance between lowland animals and animals reared at altitude (4,330\u00a0m). Set point, however, was lower in high altitude animals which seems surprising in view of the known hypertensive effect of hypoxia.\nBlood volumes are larger in high altitude dwellers. In Andeans this is due to a large packed cell volume whereas in Ethiopians plasma volume was large [12, 13]. Probably as the result of the large blood volumes, tolerance to orthostatic stress was greater than that in sea-level residents [12, 14].\nPatients with chronic mountain sickness\nChronic mountain sickness (CMS, Monge\u2019s disease; [43]) is a condition frequently found in long-term residents of high altitudes particularly in the Andes where it is a major public health problem. It also occurs in residents on the Tibetan plateau, although not in Ethiopians [13].\nPatients with CMS develop excessive polycythemia and various clinical features including dyspnoea, palpitations, insomnia, dizziness, headache, confusion, loss of appetite, lack of mental concentration and memory alterations. Patients may also complain of decreased exercise tolerance, bone pain, acral paraesthesia and occasionally haemoptysis. The impairment of mental function may be reversed by phlebotomy [42]. Physical examination reveals cyanosis, due to the combination of polycythemia and low oxygen saturation, and a marked pigmentation of the skin exposed to the sun. Hyperaemia of conjunctivae is characteristic and the retinal vessels are also dilated and engorged. The second cardiac sound is frequently accentuated and there is an increased cardiac size, mainly due to right ventricular hypertrophy. As the condition progresses, overt congestive heart failure becomes evident, characterized by dyspnoea at rest and during mild effort, peripheral oedema, distension of superficial veins, and progressive cardiac dilation. A clinical score was devised in the attempt to assess the severity of the syndrome and to compare CMS cases within and between different countries in the world [37].\nCMS is most likely the result of several influences acting on subjects living at high altitudes. Hypoventilation associated with aging has been proposed as one of the main underlying mechanisms [64]. Many of the clinical features may be attributed to the excessive polycythemia which leads to hyperviscosity of the blood and consequently impaired blood flow and impaired oxygen delivery to several organs including the brain [44].\nSime et al. [64] reported a blunted ventilatory response to hypoxia (VRH) in subjects with CMS and suggested this as the basic underlying cause. However, the same effect may also occur in some subjects without CMS [3, 62]. The function of the peripheral chemoreceptors has been shown to be abnormal [36].\nStudies have been carried out to examine the plasticity of chemoreflexes to both long and short term changes in blood gas tensions of chronically hypoxic high-altitude natives with blunted respiratory responses to hypoxia. It was found that natives who had migrated to live at sea level had ventilatory responses to acute hypoxia (few minutes) which had become similar to those of sea-level controls [21]. However, responses to sustained hypoxia (20\u00a0min) remained markedly blunted. These results may explain the apparent discrepancy in previous studies.\nRecent studies have examined cardiovascular and cerebrovascular control in subjects with CMS. Bernardi et al. [6] assessed baroreflex function and the chemo- and baroreflex interactions in Andean subjects with and without CMS. They found that subjects with CMS showed a reduction in the responses to stimulation of peripheral chemoreflexes. In addition, these subjects also showed a reduction in the baroreflex control of heart rate and blood pressure. The reduction in the arterial baroreflex correlated with an increase in CMS score, and with an increase of haemoglobin levels. They interpreted these findings as suggestive of a functional, reversible central depression rather than of the presence of an organic dysautonomia in CMS. They further suggested that the observed baroreflex alteration might be involved in the causation of some of the symptoms of CMS. In another study on subjects with CMS, performed at high altitude, the same authors showed that increasing oxygen saturation for an hour by a slow breathing pattern or by oxygen administration was associated with increased arterial baroreflex sensitivity [33].\nThe major mechanism for the control of blood pressure is through regulation of peripheral vascular resistance, but most studies have examined only the control of heart rate. We have recently studied the responses of forearm vascular resistance to carotid baroreceptor stimulation in high altitude residents with and without CMS, both at their resident altitude and shortly after descent to sea level. Results showed that baroreflex \u201cset point\u201d was higher in CMS, but only at altitude. At sea level, values were similar [45].\nCerebrovascular control Cerebral blood flow is known to be less in CMS patients than in healthy controls and this is attributed to the high viscosity of the blood. Villafuerte et al. [66] estimated that the ideal haemoglobin level for maximal oxygen delivery is 14.7\u00a0g\/dl, a value similar to that of sea-level dwellers. The cerebral circulation normally shows an efficient autoregulation whereby changes in cerebral perfusion pressure have minimal effect on flow. Claydon et al. [14] assessed autoregulation from the correlation between flow and pressure during orthostatic stress, where a significant correlation with a high coefficient indicates poor autoregulation. Results of this study showed that cerebrovascular autoregulation was impaired in CMS patients. These findings are compatible with those of Roach et al. [58] who reported reduced cerebrovascular sensitivity to carbon dioxide in the presence of hypobaric hypoxia in subjects with CMS.\nNorcliffe et al. [50] determined the cerebrovascular responses to hypoxia and hypercapnia, separately and together, in CMS patients and normal high altitude dwellers. CMS patients did not respond differently from the normals, but in both groups at altitude the sensitivity of the cerebral circulation to hypoxia was less than that in sea level residents. Shortly after descent to sea level, however, sensitivity increased. Sensitivity to hypercapnia during hypoxia decreased after descent.","keyphrases":["autonomic nervous system","altitude","hypoxia","chemoreceptors","baroreceptors","mountain sickness","respiration","polycythemia"],"prmu":["P","P","P","P","P","P","P","P"]}
{"id":"J_Neurol-2-2-1705488","title":"First aid in acute stroke\n","text":"Objective First aid training is well established to teach the public how to recognize a medical emergency and take appropriate action. Though it is now handled as a high priority emergency stroke is not among the main topics of first aid. We investigated if first aid training may be useful for enhancing stroke awareness.\nIntroduction\nFirst aid is a concept of first hands-on measures performed in a medical emergency by laypersons. The goal is to teach people how to recognize a medical emergency and start qualified action by providing measures of basic life support such as performing cardiopulmonary resuscitation. Other topics are injuries and burns, shock, unconsciousness or heart attack.\nIn Germany first aid training is mainly offered by charity organizations like the German Red Cross, or the St. John Ambulance. There are various training programmess differing in length and depth of information. First aid training was effectively used to teach the public in new methods of life-saving therapy like automated external defibrillation [1].\nStroke or brain attack is not or only marginally included in first aid training. This is in contrast to the fact that management of acute stroke changed as it is now handled as a high priority emergency [2]. Limited knowledge of signs, symptoms and risk factors for stroke was noted as an important factor associated with delay of admission [3\u20135]. To enhance awareness on stroke, various educational programmess have been initiated [3, 6\u20138\n]. The American Stroke Association\u2019s Operation Stroke, a huge awareness campaign on stroke, the Coordinated Stroke Programme of Southwestern Ontario and King County\u2019s Medic One at seattle are examples of continuous health education programmes using internet, designated courses and mass media. Regular first aid training was never used in a stroke information campaign though it is widely available and well accepted in many countries.\nIn a prospective study we tried to test the hypothesis that stroke information integrated in first aid training will improve knowledge of laypersons. Additionally a concept of first action will increase motivation and acceptance will be quite high with participants.\nMethods\nEducational procedure\nA teaching sequence was created by stroke neurologists of the University of Erlangen together with senior instructors of the St. John Ambulance (Johanniter-Unfall-Hilfe) in Bavaria. Educational objectives of the session were clearly defined. The sequence was designed for a 15 to 20\u00a0minutes audiovisual presentation and provided information basically what stroke means and about stroke risk factors, warning signs and symptoms. Warning signs presented included sudden uni- or bilateral weakness or numbness, facial paresis, speech disturbance, blurred vision, trouble in walking, vertigo\/ dizziness, diplopia. Then a concept of first action to be taken in case of stroke was presented (Figure\u00a01 shows a one-slide overview). including check of vital signs and immediate activation of EMS.\nFig.\u00a01Concept of first action presented in the stroke lesson (originally presented in German)\nThe teaching session was presented by first aid instructors integrated in regular first aid training programmes of 8 to 16\u00a0hours duration of the St. John Ambulance all over Bavaria. A small booklet was made for the first aid instructors to provide more detailed information about stroke.\nData collection and analysis\nTo investigate the effect of the teaching, all participants of designated courses were asked to fill out a questionnaire before the stroke lesson. At the end of the training program a similar questionnaire was presented slightly altered in structure and some questions. The overall length of the training courses differed as some courses were presented on a whole day schedule within one weekend while others offered one 90-minute teaching unit once weekly and were therefore scattered over a period of up to 6\u00a0weeks. Accordingly the time delay between the stroke lesson and the post-teaching questionnaire varied as well ranging from 1 to 28\u00a0days (mean 8.6\u00a0d).\nBefore teaching participants were asked to note in free text and explain what they think that a stroke is, to name signs and symptoms of stroke and to describe how they would act in case of witnessing stroke. Additionally sex, age and profession of participants was recorded as well as their previous information about stroke After the lesson we asked again for a definition of stroke and to list symptoms of stroke. Furthermore we asked how urgent they think that a stroke needs to get therapy. After all subjects were asked for their satisfaction with the training session, how they would rate their own interest in stroke and how they would rate the importance of stroke as a topic in first aid. Therefore a rating scale from 1 to 6 was given with 1 being the best, 6 the worst.\nParticipants with any profession within health care were excluded from the analysis. The questionnaire was not offered in trainings for special groups such as in schools or nursing homes.\nMean values were compared using paired t-tests while \u03c7\u00b2- statistics were used for proportions of nominal data.\nResults\nIn 38 first aid training courses 532 participants (86.6% of 614 subjects attending the training) completed the questionnaire before and after training, 53.6% of those were male, mean age was 28.6\u00a0years (+\/\u2212 11.1\u00a0y, range 15\u201361\u00a0y).\nBefore training 28.4% of all participants stated a correct description of stroke, another 32.7% gave at least the brain as the location of the disease. The mean number of stroke symptoms named was 1.52 (\u00b1 1.1), descriptions listed were mainly \u201cweakness\u201d, \u201cmotor problems\u201d \u201cdisturbance of consciousness\u201d or \u201cspeech problems\u201d, 12.8% of all respondents did not list at least one correct stroke symptom or warning sign, 40.7% listed symptoms that are not typically suggestive of stroke (coded as incorrect). The mean number of first aid measures named was 1.22 (\u00b1 1.0). 78.7% of respondents stated that they had any kind of information about stroke before the training.\nAfter the stroke lesson 69.9% of the participants stated a correct description of stroke, and 20.3% gave at least the brain as the location of the disease. Now the mean number of stroke symptoms named was 3.35 (\u00b1 1.4). 58.5 % declared that in case of stroke urgent treatment is needed . See table\u00a01 for an overview of key results.\nTable\u00a01Key results from the questionnaire before and after receiving the stroke lessonn = 532before stroke lessonafter stroke lessonmean numberno. of stroke signs1.523.35p < 0.001no. of first measures1.22not asked% of participantscorrect explanation what stroke means 28.469.9p < 0.001brain affected organ32.720.3p < 0.01no\/incorrect explanation38.9 9.8p < 0.01> 4 symptoms listed 2.628.4p < 0.001 no symptom listed12.8 3.6p < 0.001\u201cimmediate medical help required\u201dnot asked58.5prior information about stroke78.7\nMost participants were satisfied with the stroke lesson as the mean value in the score (1 = best to 6 = worst) was 1.76 (\u00b1 0.6), interest in topics of stroke was rated high (mean 1.56, \u00b1 0,8). Stroke was regarded as an important topic for first aid training. (mean 1.57, \u00b1 0,9). The difference in mean number of symptoms named before and after the lesson was highly significant (p < 0.001), as well as the difference in percentage of participants giving a correct definition of stroke (p < 0.001) and those naming more than 4 symptoms. In contrast the number of participants not listing at least one symptom and giving none or an incorrect definition of stroke was significantly reduced after the training.\nDiscussion\nTo our knowledge this is the first study published reporting about first aid training for public education on stroke. A teaching session was integrated in regular first aid training offered to the public by the St John Ambulance.\nFrom data of this pilot study the teaching session was very effective: Mean number of stroke symptoms and number of participants giving a correct description of stroke more than doubled.\nThe difference in number of symptoms and portion of participants describing stroke symptoms correctly was highly significant. Nevertheless with a mean delay between teaching and retention test below 10\u00a0days these results show only the short-term effect and durability of the information remains unclear. From other studies in teaching medical skills to laypeople we know that knowledge is reduced by about 25% over the first three months and may remain stable thereafter [9]. One year may be critical period for repetition of training [10]. However, participants showed also a high grade of satisfaction with the lesson and its presentation, and ranked importance of stroke as a topic quite high. As a consequence of these results the lesson is now integrated in every first aid training of the St. John Ambulance in Bavaria.\nBefore this study there was no clear cut concept existing for first action in acute stroke. Despite the fact that calling EMS accelerates admission [11] the effect of all these actions performed by medical laypersons on stroke progression or outcome is not yet proven.\nHowever, it takes about 15\u00a0minutes from starting the call to arrival of EMS on scene [12]. During this time a bystander should be able to offer help to the patient. By presenting simple hands-on measures it is also emphasized that something can and must be done in case of acute stroke.\nThe effect of various educational interventions was already demonstrated in previous studies [3, 6\u20138].\nNone of these studies handled information on stroke integrated in general health education campaigns such as routine first aid training. From our viewpoint integrating a stroke lesson in first aid training is not only effective but provides a number of advantages. First an integration makes it clear that stroke or brain attack is an important medical emergency as important as heart attack or trauma.\nConnecting brain attack with other medical emergencies also facilitates the demonstration of measures as they could be used for other situations as well and it might help to create a better understanding to stroke and its differences from other related diseases. We linked stroke to other related topics (e.g. \u201chead trauma\u201d) and formed a cluster of measures for \u201call problems in the head\u201d as an effort towards integrative problem-based teaching which was shown to be superior to conventional lessons also in medical education [13].\nSecond, by using first aid training information on stroke this will reach large numbers of subjects not particularly interested in stroke or sometimes health education at all. According to their annual review the St. John Ambulance, England, issued a first aid training certificate to more than 500000 people in the year 2003 [14], while the American Red Cross trained more than 11\u00a0million people in first aid [15]. In Germany within one year a driving license is issued to more than 800000 people mainly in the age of 16 to 21. By legal regulations every one of them must have completed at least an 8 hour first aid training. So first aid training may especially reach younger people that are not interested in stroke awareness campaigns. Furthermore, given the large numbers of people reached, this method of education is extremely cheap.\nTo summarize we found that a 15\u00a0minute presentation on stroke as a medical emergency improves stroke knowledge of participants in first aid training. Furthermore participants seem to appreciate getting information about stroke. Accordingly information on stroke or brain attack should be included in every first aid training thus reaching large numbers of subjects in many countries all over the world. Means of information may differ in their effectiveness [8, 16] but the question is not whether education integrated in training programs may be more effective than mass media campaigns. Both methods of information will rather be complementary as they use different approaches and may reach different target groups. The goal is not to miss any single opportunity to send out the message about stroke as a medical emergency that needs immediate reaction by anyone who is next.","keyphrases":["first aid","acute stroke","knowledge","emergency medical service","healtheducation"],"prmu":["P","P","P","M","U"]}
{"id":"Eur_Radiol-3-1-2039819","title":"Coronary fly-through or virtual angioscopy using dual-source MDCT data\n","text":"Coronary fly-through or virtual angioscopy (VA) has been studied ever since its invention in 2000. However, application was limited because it requires an optimal computed tomography (CT) scan and time-consuming post-processing. Recent advances in post-processing software facilitate easy construction of VA, but until now image quality was insufficient in most patients. The introduction of dual-source multidetector CT (MDCT) could enable VA in all patients. Twenty patients were scanned using a dual-source MDCT (Definition, Siemens, Forchheim, Germany) using a standard coronary artery protocol. Post-processing was performed on an Aquarius Workstation (TeraRecon, San Mateo, Calif.). Length travelled per major branch was recorded in millimetres, together with the time required in minutes. VA could be performed in every patient for each of the major coronary arteries. The mean (range) length of the automated fly-through was 80 (32\u2013107) mm for the left anterior descending (LAD), 75 (21\u2013116) mm for the left circumflex artery (LCx), and 109 (21\u2013190) mm for the right coronary artery (RCA). Calcifications and stenoses were visualised, as well as most side branches. The mean time required was 3 min for LAD, 2.5 min for LCx, and 2 min for the RCA. Dual-source MDCT allows for high quality visualisation of the coronary arteries in every patient because scanning with this machine is independent of the heart rate. This is clearly shown by the successful VA in all patients. Potential clinical value of VA should be determined in the near future.\nIntroduction\nCoronary fly-through or virtual angioscopy (VA) using various generations of computed tomography (CT) devices has been reported in a number of publications since its first mention in 2000 [1\u20133]. Main reason for the minimal research on this topic was the limited application because of the requirements for an optimal CT scan and time consuming post-processing. Recent advances in post-processing software facilitate easy construction of VA, but until now image quality of coronary CT was insufficient in most patients. The introduction of dual-source multidetector CT (MDCT) has shown a more stable quality of data in all patients because of higher temporal resolutions, which could prove to enable VA in all patients and allow investigation of the clinical value of this visualisation technique [4\u20136].\nMaterials and methods\nTwenty patients (13 male, mean age 60\u2009\u00b1\u200913\u00a0years old) were selected at random from patients scanned for coronary artery disease using a standard coronary artery protocol on a dual source MDCT (Definition, Siemens, Forchheim, Germany).\nPost-processing was performed on an Aquarius Workstation (TeraRecon, San Mateo, Calif.). The software allows an automatic vessel fly-through, which provides inside front and rear views, together with a view of the path and location on the surface of the heart (Fig.\u00a01). Besides this, three multiplanar reformation planes (sagittal, coronal and axial) are shown for the location of the camera. Length travelled per major branch was recorded in millimetres, together with the time required in minutes to perform the evaluation per major branch. Ostia of side branches were visualised, but no fly-through of those side branches was performed.\nFig.\u00a01Large trajectory visualisation of the right coronary artery (RCA)\nResults\nVA could be performed semi-automatically with minor user interaction in every patient for each of the major coronary arteries using dual-source CT (DSCT).\nThe mean (range) length of the automated fly-through was 80 (32\u2013107) mm for the left anterior descending (LAD), 75 (21\u2013116) mm for the left circumflex artery (LCx), and 109 (21\u2013190) mm for the RCA.\nCalcifications were clearly visualised as white structures floating inside the lumen. Stenoses were visualised when present by indentations of the lumen wall (Fig.\u00a02). Visibility of the actual size of the stent lumen was still limited, but fly-through was possible in the stents we found (Fig.\u00a03).\nFig.\u00a02Visibility of a stenotic lesionFig.\u00a03Stent visibility\nThe major side branches of the main coronary arteries could be detected during the fly-through (Fig.\u00a04). No fly-through was performed through those side branches.\nFig.\u00a04Visibility of major side branches\nThe mean time required was 3\u00a0min for LAD, 2.5\u00a0min for LCx, and 2\u00a0min for the RCA.\nDiscussion\nThe introduction of DSCT enables 100% visualisation of all major arteries and thus demonstrates a major gain in image quality compared with electron beam CT (EBCT) and MDCT, with only 76% and 84% assessable, respectively [3].\nFurthermore, the time required for processing the data decreased to about 2.5\u00a0min compared with the 6-13\u00a0min required in 2002 (Table\u00a01). Notice that the times shown in the table for EBCT and MDCT do not include the actual computing time, which could be hours with the hard- and software at that time. Currently, all computation and visualisation is in real time and thus included in the 2.5\u00a0min required for a coronary fly-through of a major branch using DSCT data. \nTable\u00a01Results of an earlier study using EBCT and four-slice MDCT (4-MDCT) [3] in comparison with the DSCT results of the current study\u00a0EBCT (2002)4-MDCT (2002)DSCT (2006)LAD\u00a0Percentage assessable14\/15 (93%)14\/15 (93%)20\/20 (100%)\u00a0Mean preparation time (min)12133\u00a0Mean number of key frames1316n.a.LC\u00a0Percentage assessable11\/15 (73%)10\/15 (67%)20\/20 (100%)\u00a0Mean preparation time (min)7102.5\u00a0Mean number of key frames913*n.a.RCA\u00a0Percentage assessable9\/15 (60%)14\/15 (95%)20\/20 (100%)\u00a0Mean preparation time (min)6102\u00a0Mean number of key frames814n.a.\u00a0Total percentage assessable34\/45 (76%)38\/45 (84%)60\/60 (100%)The number of key frames is no longer applicable (n.a.) for the current software tools that can perform the coronary fly-through in real time\nDifferent artefacts still hamper the fly-through. First, non-optimal reconstruction will present itself as a sudden change in the form of the vessel wall. The effects are shown in Fig.\u00a05, and it will be clear that these artefacts can be easily recognised because of the non-natural shape of the wall. A more difficult artefact is cause by motion. Here, the artefact can be easily appreciated on the three-dimensional view, but in the fly-through it presents as a apparent stenosis (Fig.\u00a06).\nFig.\u00a05Problems with artefacts in non-optimal reconstructed datasetsFig.\u00a06Problems with motion artefacts causing apparent stenoses\nFinally, surgical clips in bypasses still pose a problem as already shown an earlier publication using EBCT [1] (Fig.\u00a07).\nFig.\u00a07Visualisation is hampered by presence of surgical clips in a bypass graft\nAll three articles that (to our best knowledge) are available on this topic conclude with similar remarks [1\u20133]. They all state that the coronary fly-through or virtual coronary angioscopy (VCA) is a promising technique that, however, requires higher quality of the datasets and better image processing software.\nA shortcoming of our study was the lack of a \u201cgold standard\u201d to confirm the findings of the coronary fly-through.\nConclusion\nDual-source MDCT provides a high quality visualisation of the coronary artery tree, independently of the heart rate. This is clearly shown by the successful VCA in all 20 patients in this study. With recent advancements of image processing software and CT acquisition techniques, the conditions have been met for providing the technical basis for the clinical validation of VCA and the potential clinical value of VCA should be determined in the near future.","keyphrases":["coronary fly-through","virtual angioscopy","post-processing","coronary arteries","dual-source ct"],"prmu":["P","P","P","P","P"]}
{"id":"Health_Care_Anal-4-1-2362137","title":"Participatory Workshops are Not Enough to Prevent Policy Implementation Failures: An Example of a Policy Development Process Concerning the Drug Interferon-beta for Multiple Sclerosis\n","text":"A possible explanation for policy implementation failure is that the views of the policy\u2019s target groups are insufficiently taken into account during policy development. It has been argued that involving these groups in an interactive process of policy development could improve this. We analysed a project in which several target populations participated in workshops aimed to optimise the utilisation of an expensive novel drug (interferon beta) for patients with Multiple Sclerosis. All participants seemed to agree on the appropriateness of establishing a central registry of Multiple Sclerosis patients and developing guidelines. Nevertheless, these policy measures were not implemented. Possible explanations include (1) the subject no longer had high priority when the costs appeared lower than expected, (2) the organisers had paid insufficient attention to the perceived problems of parties involved, and (3) changes within the socio-political context. The workshops in which representatives of the policy\u2019s target populations participated did not provide enough interactivity to prevent policy implementation failure.\nIntroduction\nThere are numerous examples of health policy measures that have failed in their implementation. A review from the Netherlands Court of Audit indicated that no evidence of a full policy implementation can be found in previous audits [20]. For example, the applied measures for reducing greenhouse gas emissions have not met pre-determined targets, the legal rules for maintaining food safety have not been able to prevent targeted risks, and policy measures for cost control of medicines have not resulted in more structural cost control [19].\nThe theory of argumentative policy analysis offers a possible explanation for such policy implementation failures [8, 15]. The basic idea of this theory is that actors\u2019 behaviours can be explained by different views on a problem and the argumentation behind these views. According to the argumentative approach to policy analysis [7, 8], action is driven by processes of problem setting [27] in which actors define coherent sets of problems and solutions that correspond to these actors\u2019 normative and empirical background theories. The way in which problems are defined depends on the assumptions the actors make about the situation and their beliefs regarding what is good practice (normative values).\nCombining these findings with those of classical implementation theory, Grin and Van de Graaf [10] have argued that a policy will only be effective if both implementers and target populations consider the proposed policy measure meaningful. (Target populations consist of persons who will experience the consequences of a policy when implemented.) This means that the proposed policy should (a) make sense in the light of problems perceived by the target populations and (b) be consistent with their normative and empirical background theories.\nHowever, the fact that policy problems and associated solutions tend to shift over time, different actors with different background theories being involved successively, renders this rather complicated. In other words, a policy measure is not invented at a specific moment in time, but develops over time. Nevertheless, the challenge is to identify policy measures that cohere with the views of all actors involved.\nIf the argumentative policy theory is correct, then it is advisable to identify the policy\u2019s target populations and involve them in the process of policy development. An interactive process of policy making could thus ensure that policy coheres with and prevent that it diverges from the views of target groups.\nThe objective of this paper is to analyse a specific instance where target populations were involved in the process of policy development. The policy institution in this example was the Health Care Insurance Board in the Netherlands (HCIB). It is an advisory board to the Ministry of Health, particularly with respect to coverage and reimbursement issues. The Department of Policy Analysis of Medicines (PAM) is responsible for identifying developments that may jeopardise optimal medical care, analysing the nature and size of such threats and conducting further research that may provide a basis for policy decisions. The annual work programme in which topics are prioritised is submitted to the Ministry of Health for approval.\nThe policy objective discussed in this paper was to promote the appropriate use of a recently introduced drug for patients with multiple sclerosis. Multiple Sclerosis (MS) is a neurodegenerative disease characterised by neurological dysfunction. The drug interferon-beta (IFN\u03b2) appeared to be a promising treatment. However, its costs were high (\u20ac 12,000 per patient annually) and the evidence of its long-term effectiveness was limited. Therefore, policy measures to guide the prescription of this medicine were considered necessary. Representatives of prescribing physicians (neurologists), health insurance companies, and patients were invited to participate in two workshops. During these workshops, participants discussed policy measures that could promote the appropriate use of the new drug. Although two concrete policy measures had been proposed, these have never been implemented.\nIn this paper we evaluated whether the theory of the argumentative policy analysis could explain the proceedings in this case study. It offers a description of the process of the policy development that included the two workshops, as well as an analysis of the views of policy makers and target populations in order to assess whether the proposed policy measures fitted the perceived problems and underlying background theories.\nMethods\nRelevant documents were analysed and semi-structured interviews were held with the various stakeholders. These documents included correspondence, reports from the Board, internal memos on this subject, a report from Health Council on interferon-beta, reports from meetings, research proposals, and research reports. Interviews were held with three HCIB employees, two employees from the Ministry of Health, the organisers of the workshops, two neurologists, a patient, and a medical advisor from a health insurance company. Participants of the workshops were contacted and interviewed to reconstruct the proceedings of the workshops. MS patients were contacted via a Dutch MS patient organisation. Representatives of target populations were interviewed to assess their problem definitions and underlying argumentation.\nInterviews were transcribed verbatim. A summary from the interview and a concept report were sent to respondents for verification and literature was used to check the findings from interviews or documents. In line with the theory of argumentative policy analysis, we used the method of reconstructing interpretative frames to analyse target populations\u2019 argumentation [10, 11, 18]. The idea is to reconstruct (1) how problems are defined, (2) how solutions or policy measures are judged, (3) what theoretical and normative assumptions shaped them, and (4) what normative preferences underlie this all. Together, these four \u2018layers\u2019 of evaluation entail an individual\u2019s interpretative frame.\nResults\nProceedings of Project\nThe content of the workshops was, to a large extent, determined by two reports concerning IFN\u03b2. These had been issued by the HCIB and by the Health Council, respectively. In July, 1995, the Ministry of Health asked both institutions to provide advice regarding the introduction of IFN\u03b2 on the Dutch market. The Ministry asked for recommendations with respect to whether or not restrictions could be imposed on the reimbursement of the drug.\nIn May 1996, a committee from the Health Council reported to the Ministry of Health [9]. This committee was comprised of three neurologists, a chemical technologist, and a HCIB staff member. It concluded that IFN\u03b2 could be a promising new drug, but also cautioned against unrealistic expectations. Clinical trials had shown that IFN\u03b2 could decrease the rate and severity of exacerbations, but there was no evidence of IFN\u03b2 preventing the onset of disability. The Health Council emphasised that the drug should be prescribed only to patients who meet eligibility criteria for the trials. These criteria were: (a) clinically definite Relapsing Remitting MS; (b) at least two exacerbations in the two previous years; (c) mild to moderate disability; and (d) age of 18\u00a0years or older. The Council also proposed the following policy measures: (1) develop a guideline for treatment of MS patients with IFN\u03b2; (2) ensure that the drug is prescribed by neurologists who have sufficient experience with the diagnosis and treatment of MS patients; (3) properly instruct and guide MS patients; (4) conduct a systematic follow up of patients using IFN\u03b2 in order to evaluate side effects; (5) provide clear indications on which patients should be treated with IFN\u03b2; (6) establish a national registry of MS patients in order to conduct further research on the effectiveness of IFN\u03b2; and (7) conduct a re-appraisal after a number of years.\nJune 1996, the HCIB issued recommendations that corresponded partially with the Health Council\u2019s report [30]. The HCIB recommended that: (a) patients fulfil the criteria described in the Health Council\u2019s report; (b) a treatment protocol be developed; (c) health insurers approve reimbursement requests before starting treatment; (d) a prospective registration be established; and (e) the use of IFN\u03b2 be re-assessed after 3\u00a0years. Restricting prescription to a limited number of experienced neurologists was considered impossible, because IFN\u03b2 had already been included in health care packages.\nIn 1999, the department of Policy Analysis of Medicines (PAM) started a project on IFN\u03b2. Initially it they considered evaluating other, less expensive interventions that could be effective, but are of no interest to the industry. Indications had been received from the field that a much cheaper drug, namely methotrexate (used in oncology and rheumatoid arthritis), could be as effective as interferon. Furthermore, PAM considered developing a national database of MS patients. PAM later decided that conducting a clinical trial was not its responsibility. PAM staff, in collaboration with neurologists, decided to initiate the development of a treatment protocol and a national registry of MS patients. They proposed the organisation of two workshops.\nWorkshops\nIn 2000, these workshops were organised by an external institute for policy research. The first workshop aimed to provide an overview of the criteria that are used for prescribing IFN\u03b2. This workshop was preceded by an inventory study amongst health insurance companies into current reimbursement practices with respect to IFN\u03b2. This inventory study was performed by a HCIB department. The results from the inventory study on current reimbursement practices indicated that health insurance companies did not assess reimbursement requests against medical content. Assessments were limited to an administrative review of the completeness of data [4]. Nevertheless, health insurance companies considered the pre-utilisation approval effective, because it functioned as an administrative barrier. The authors of this report recommended the following: (a) assess whether a pre-utilisation approval by a central committee could improve the assessment of reimbursement requests; (b) develop a clear protocol and design application forms.\nNeurologists, medical advisors from health insurance companies, policy makers, and a representative from a patient organisation participated in the workshop. During the workshop, vignettes with patient descriptions were used to discuss which patients should be treated with IFN\u03b2. The organisers concluded that only a few problems were perceived with respect to criteria for IFN\u03b2 use. Health insurers questioned whether the situation was indeed problematic given the small size of the patient population and IFN\u03b2\u2019s status as an essential medicine. Neurologists argued that problems arose incidentally. Most often, these problems were related to reimbursement requests for continued use of IFN\u03b2. All participants agreed that subjectivity in decisions on prescription should be minimised. The participants claimed that clear distinctions should be made between the criteria for initialising treatment and the criteria for continuing treatment. The development of a guideline by neurologists was considered relevant. According to the participants, small adjustments of the current criteria would be sufficient [2].\nThe second workshop aimed to obtain advice on how existing databases could be improved so that the effects of IFN\u03b2 or other new drugs for MS patients could be evaluated. In preparation for this workshop, a neurologist was asked to make an inventory of available databases that contained data on IFN\u03b2 users. This inventory showed that data on MS patients were no longer being collected systematically in the Netherlands [2]. Former local databases were no longer up-to-date. The participants in the workshops agreed that a national database could be relevant and could serve the following goals: (a) policy making (such as financial surveys, planning health care capacity); (b) research (effects of treatment; monitoring for side effects); and (c) clinical practice support for neurologists (reflection on clinical practice, improvement of expertise). Participants agreed on the inclusion of general static data in the database, such as demographic details, diagnosis (type MS), the criteria used to make the diagnosis, and the initial treatment. Participants disagreed on the kinds of dynamic data, such as treatment details and information on physical functioning. A database including a broad range of data could be useful for research, but this was considered expensive. A small database that includes static data only could be used as a sample frame for selecting potential participants in further studies. In their report, the organisers concluded that additional research was needed to ascertain which variables should be included in the database.\nProposed Policy Measures\nBased on the organisers\u2019 report, the HCIB proposed the following policy measures to the Ministry of Health [5]:Have neurologists develop an evidence-based guideline for the treatment of patients with multiple sclerosis.Have reimbursement requests for IFN\u03b2 appraised on medical grounds. Possibly, implement a central pre-utilisation approval by experts (neurologists).Define clear criteria for (dis)continuing IFN\u03b2 reimbursement.Establish a national registry of MS patients. The database should enable scientific research, the improvement of treatments, monitoring, and the evaluation of efficiency and therapeutic value of new interventions.\nIn 2003, a medical advisor from a health insurance company mentioned that some small changes had been made to the procedures established for judging reimbursement requests (requests for continued use). A neurologist mentioned working on a guideline for diagnosis and treatment of MS patients. According to a PAM staff member, neither a central pre-utilisation approval nor a national database to prospectively register patient data had been established.\nReconstructed Interpretative Frames\nIn June 2003, we conducted interviews to reconstruct interpretative frames of policy makers and policy\u2019s target populations. The actors\u2019 views are summarised in Table\u00a01 (IFN\u03b2 guideline) and Table\u00a02 (national database).\nTable\u00a01Views towards guideline concerning IFN\u03b2 useActorJudgement of solutionaProblem definitionBackground theoriesNormative valuesMinistry of health (1996)+Clear criteria for reimbursement IFN\u03b2 neededIFN\u03b2 is an expensive medicine; uncertainty on meaning of effects Possibly inappropriate use because high expectationsOnly if evidence of effectiveness is available, a medicine can be at the expense of the community Cost control EfficiencyPAM staff+ A central pre-utilisation approval for IFN\u03b2 use A clear treatment protocol on IFN\u03b2 should be availableHealth insurance companies judged reimbursement requests differently Individual health insurer lack expertise to judge reimbursement requestsDifferences in judgement result in differences in care Situation IFN\u03b2 resembles situation growth hormones, for which a central pre-utilisation approval in combination with central registration was set up Central pre-utilisation approval prevents inappropriate useCost control; efficiency Equal accessibilityPAM staff (2003)\u2212 Central pre-utilisation approval no longer neededSituation concerning IFN\u03b2 was relatively stable; no longer problems concerning reimbursement requests Costs committee higher than possible savings IFN\u03b2 useCriteria for central pre-utilisation approval: \u2013 medicine is expensive \u2013 inappropriate use expected \u2013 decisions on treatment are complexCost control EfficiencyMedical advisor health insurance company+ Guidelines on IFN\u03b2 needed for decisions reimbursementUnclear when IFN\u03b2 is (still) meaningful Pharmaceutical companies benefit from extending criteria Expensive drugs are financed from public funds, on a solidarity baseCost control Neurologist+ Not guideline on IFN\u03b2 solely but guideline on diagnosis & treatment MSIncrease possibilities for diagnosis and therapy; variance in MS careAs in other fields increasing attention to evidence based medicine Provide clarity for neurologists on how to care patientsPatient+ National guideline might be usefulDifferences between hospitals care (IFN\u03b2 not offered in every hospital; contradictory advises on lifestyle)No common knowledge on MS Prognosis differs between individualsImprove or maintain quality of lifea\u2018+\u2019 actor defines the intervention meaningful; \u2018\u2212\u2019 intervention is not meaningfulTable\u00a02Views towards a national registry of MS patients or IFN\u03b2 usersActorJudgement of solutionaProblem definitionBackground theoriesNormative valuesMinistry of health (1996)+ For evaluation long-term effectsUncertainty on (meaning of) effects of IFN\u03b2Only medicines of which evidence of effectiveness is available at the expense of the community If no evidence on effectiveness, use is inappropriateEfficiencyPAM Staff + Database with only limited patient data to take samples for research or Database with all relevant data for researchFor each new medicine a study on effectiveness is inefficient Health insurance companies judged reimbursement requests differently, leading to difference in health careAim of national database to optimise IFN\u03b2 prescription: to monitor use of IFN\u03b2 in practice (which patients (indications), volume, costs) and to evaluate effectiveness IFN\u03b2 Efficiency PAM Staff (2003)\u2212 Registration without central judgement committee difficultCentral committee no longer relevant No problems perceivedCentral pre-utilisation judgement provides a minimal registration of patient data Efficiency Medical advisor of health insurance company+ Prospective database only way to assess long-term effectivenessJudgement of relative value as compared to alternative interventions not systematically but only in the neurologists headExpensive drugs are financed from public funds Pharmaceutical industry\u2019s power is underestimatedClear vision from the ministry of health on pharmacy Quality of careNeurologist+ Meaningful for evaluating effectiveness of medicines and knowledge on MS National coverage might be difficultClinical trials have limited follow up period Distrust amongst neurologistsMS is a chronic disease, invalidating on long term MS neurologists have their own \u201ckingdoms\u201dQuality of care Increase knowledge on disease and treatmenta\u2018+\u2019 actor defines intervention as meaningful; \u2018\u2212\u2019 intervention is not meaningful\nMinistry of Health\nInitially, the Ministry contended that the long-term effectiveness of the drug was not established sufficiently, while the acquisition costs were high. Trials had shown that IFN\u03b2 decreased the number of exacerbations, but it was unknown whether IFN\u03b2 would prevent disability. The potential target population, namely patients with MS, is large and the expectations of both patients and physicians may also have been high. As a result, the risk that the drug could be used inaccurately was considered to be high. Consequently, additional policy measures to control IFN\u03b2 use in clinical practice were requested.\nAccording to employees from the Ministry of Health, the HCIB report [5] had revealed that only health insurers perceived few problems with respect to judging reimbursement requests. The costs of a committee for a central pre-utilisation approval were considered relatively high, while the costs of the interferon-beta prescription were less than expected, namely \u20ac18\u00a0million (in 1999) versus the \u20ac90-180\u00a0million estimated by the Ministry. Therefore, both interventions were considered to be no longer relevant.\nPAM Staff\nAccording to the PAM staff, the main problem was that the use of IFN\u03b2 was expected to extend to other subgroups of patients. A central pre-utilisation approval in combination with a national registry of MS patients could provide a solution. This contention was made because the situation concerning IFN\u03b2 resembled the situation concerning other drugs for which a central pre-utilisation approval had already been established. In that case, requests for reimbursement of drug use were judged by a central committee of medical experts. For this purpose, a clear protocol including criteria for IFN\u03b2 use is needed. Simultaneously, patient data were recorded in a national registry.\nAt the time that PAM staff started their project, it was undesirable to evaluate why the proposed policy measures had not been implemented until then. Reason was a change in the relationship between the HCIB and the Ministry of Health. In 1999, the HCIB, an independent advisory board, was established as the successor to the Sickness Funds, which was a politically involved advisory board comprised of actors from the field.\nAfter completion of the project, the PAM staff considered the option of having reimbursement requests judged by a central committee no longer meaningful. They considered the implementation of policy measures difficult because the drug had already been introduced several years earlier. Limiting or discontinuing the reimbursement of IFN\u03b2 would have been practically impossible. Furthermore, the scale of problems relating to the prescription and\/or reimbursement of IFN\u03b2 and the costs related to IFN\u03b2 use turned out to be much smaller than expected. PAM considered the initiation of a national MS patient registry without the central pre-utilisation judgment to be unfeasible.\nMedical Advisor Health Insurance Company\nAccording to a medical advisor from a health insurance company, the problem was attributable to a lack of clarity on exactly what kind of patients benefit from IFN\u03b2. IFN\u03b2 is an expensive drug and the costs are either carried by the community (public health insurance) or reimbursed on an individual basis (private health insurance). Preferably, physicians should develop a guideline on the relative position of IFN\u03b2 in relation to alternative interventions. At the time of IFN\u03b2\u2019s introduction, a central pre-utilisation approval could have been relevant. However, time had passed and the prescription of IFN\u03b2 had become common practice. Unfortunately, new drugs are often introduced and included in the health care package long before all medical specialists agree on criteria for treatment. Professionals cannot develop guidelines quickly. In contrast, the time that passes between a drug being introduced into the market and its inclusion in the health care package is regulated by law and, in most cases, quite short. As a temporary solution, the drug could be included into the health care package under certain conditions, such as the registration of patient data.\nNeurologist\nA neurologist mentioned working on a guideline for diagnosing and treating MS patients as part of an initiative from the Dutch Society for Neurology. A guideline for diagnosis and treatment was considered relevant as it could support evidence-based practice. As in other medical fields, knowledge and treatment options have increased. The development of a guideline is a time-consuming endeavour as, often, it has to be done alongside the professional\u2019s usual activities. A central pre-utilisation approval was no longer relevant. Prescribing IFN\u03b2 was considered common practice, also in small general hospitals. Most neurologists were already familiar with the indications for treatment. IFN\u03b2 was proven to be effective in one type of MS that is characterised by invalidating exacerbations (relapsing remitting MS) through clinical trials that demonstrated IFN\u03b2\u2019s ability to decrease the severity and frequency of these exacerbations. A national database could still be useful. However, its purpose must be clear. An appropriate goal could be to evaluate the long-term effectiveness of IFN\u03b2. The neurologist questioned whether participants would have come to agreement on specific recommendations, such as which data should be collected and who should become the owner of the database. Most neurologists do have \u201ctheir own kingdoms\u201d and do not want to share these with others.\nPatient\nThe patient claimed that agreement amongst neurologists about MS treatment could be useful. For him, the main problem was that MS care differs between hospitals. He conveyed his experiences with receiving contradictory answers to questions from numerous health professionals and the option of using IFN\u03b2 had not been discussed in the hospital where he was initially treated. For this patient, the exacerbations are highly invalidating. Obviously, his preference is to lead a normal life, in so far as that is possible. MS has a high impact on his life.\nDiscussion\nAlthough results from the workshops showed that all respondents agreed that developing a guideline and a national database could be meaningful, these policy measures have never been implemented. A number of factors can provide an explanation for the proceedings in this project.\nFirstly, the costs of IFN\u03b2 appeared to be lower than expected. Perhaps the information campaign on IFN\u03b2 had had this effect [3]. It may also have been that the calculation by the Ministry of Health, which differed from calculations made by the Health Council and rested on the assumption that all MS patients are treated with IFN\u03b2, were unrealistic. In any case, the subject of IFN\u03b2 no longer had high priority. From the point of view of policy makers, the expected high costs of IFN\u03b2 in combination with its uncertain effectiveness were the main problems behind the proposed policy measures. Apparently, however, the problem of the high costs had still higher priority than the limited knowledge on IFN\u03b2\u2019s effectiveness. As a result, policy makers left it to the medical profession to implement policy measures. Neurologists, however, considered cost-containment not their problem and not their responsibility.\nThis also explains why the initial questions about the long-term effectiveness remained unanswered, without this being perceived as a problem. The effectiveness of IFN\u03b2 and the possible role of the industry was an issue of international debate. Clinical trials have shown that IFN\u03b2 decreased the number and severity of exacerbations in patients with relapsing-remitting MS [16, 23]. Probably, this is enough reason for neurologists to claim the right to prescribe IFN\u03b2. That evidence of long-term effectiveness, in terms of preventing disability, and safety was still limited [26] is not of great concern to them. Neither is the possible influence of the industry on prescription practices through the financing of clinical trials that had been discussed [13, 21, 22, 28]. Some authors have even challenged the hypothesis of MS as an inflammatory auto-immune disease [1]. A national registry could have been relevant from this point of view. In the UK, the Department of Health initially refused to reimburse IFN\u03b2 use for reasons of costs [17]. In 2002, however, agreements were made with the industry concerning the funding of IFN\u03b2. The Department of Health announced that it would reimburse IFN\u03b2 for MS patients who agreed to participate in a monitoring program on the effectiveness of IFN\u03b2.\nSecondly, the policy development process from the beginning centred around two specific policy measures, whereas it could have started with a broader scope, actors involved first eliciting the problems they perceive and adequately structuring the problem. Now, the proposed measures appeared not to be the most optimal solutions for the problems as perceived by neurologists, and therefore represented right solutions to the wrong problems. This typically concerns what Hischemuller and Dunn have coined an \u2018error of the third kind\u2019 [6, 14]. We have found that on a first order level, all actors agreed that a national guideline and a registration could be meaningful. But as it was, on a second order level they disagreed as to what goals such policy measures should serve. For example, neurologists considered a national guideline relevant, as long it was a broad guideline on the diagnosis and treatment of MS in general. All actors agreed that a national registry could be relevant, but defined different goals. Each goal came with different variables to be included in the registry.\nThirdly, during the workshops, the proposed policy measures remained rather vague and were not elaborated in detail. As a result, participants could easily consent without violating their background theories and preferences. They had not felt the urge to (re)consider these theories and preferences. This, however, was necessary, as is clearly illustrated by the respondents who questioned whether participants would have come to agreement on specific recommendations, such as which data should be collected and who should become the owner of the registry. Such an agreement requires that policy measures are made sufficiently substantive for every actor to understand what are the consequences. Moreover, it requires that background theories and normative preferences are elicited and scrutinised.\nFourthly, changes in the socio-political context affected policy development. Opinions with respect to which kind of policy measures are the responsibility of the HCIB had changed over time. The PAM staff considered conducting a clinical trial to obtain relevant information that would not be provided by the industry. Simultaneously, their position towards another Dutch institute involved with subsidising clinical research changed. Putting out clinical trials was then no longer their responsibility. Furthermore, the relationship between the HCIB and the Ministry of Health changed over the course of this project. These changes strongly affected ideas with respect to the Board\u2019s responsibility on initiating a national registry of patient data. On this basis we conclude that the workshops failed to meet the objectives set for interactive processes in policy development, namely to prevent policy implementation problems as a result of diverging views amongst target groups. Moreover, we concede that they could not have met those objectives. From the case of IFN\u03b2, we infer that an interactive process should meet the following criteria:The interaction should cover the whole process from problem structuring to policy implementation, in order to be able to deal with problem shifts and changes in the socio-political context. It is not enough to reduce interactivity to workshops at one or two moments in time.Actors involved should resist the temptation to think that policy problems can be understood at a first order level. That is, background theories and normative preferences should be explicated in the problem definition phase. Only if problem structuring is taken seriously in this sense can one think of developing solutions that could meet with the approval of all target groups.Developing widely endorsed solutions requires that actors involved are willing to learn from one another and adapt their views if necessary. A process of interactive policy development should include room for such learning processes [12, 24, 25, 29]. Grin and Van de Graaf [10] have argued that learning is likely to occur only if external events urge a revision of background theories and preferences, or if repeated failures show actors that their background theories are not functional.Proposed policy measures should be sufficiently elaborated, as to enable target groups to assess their consequences and constructively engage in the interactive process. As the case of IFN\u03b2 shows, actors involved are not willing to reconsider their background theories and preferences, should proposed policy measures not be sufficiently elaborated and actors involved not understand what is at stake.\nIn sum, this study has shown that the organisation of workshops in which target populations participate does not qualify as an appropriate process of interactive policy development. From the beginning, emphasis had been put on a limited number of interventions aimed to control the expected increase in treatment costs. Although target populations participated in policy development, perceived problems and which interventions could provide a solution had been discussed insufficiently. For policy development to be successful, interactive methods are needed, in which problem definitions and assumptions are explicated and discussed, providing an opportunity for mutual learning between actors involved.","keyphrases":["participatory workshops","interferon-beta","multiple sclerosis","argumentative policy analysis","interactive policy development"],"prmu":["P","P","P","P","P"]}
{"id":"Surg_Endosc-4-1-2422865","title":"Laparoscopic versus small-incision cholecystectomy: Health status in a blind randomised trial\n","text":"Background Gallstones are a major cause of morbidity, and cholecystectomy is a commonly performed procedure. Minimal invasive procedures, laparoscopic cholecystectomy (LC) and small-incision cholecystectomy (SIC), have replaced the classical open cholecystectomy. No differences have been found in primary outcome measures between LC and SIC, therefore secondary outcome measures have to be considered to determine preferences. The aim of our study was to examine health status applying evidence-based guidelines in LC and SIC in a randomised trial.\nCholecystectomy is a commonly performed procedure in patients with symptomatic cholecystolithiasis. With an estimated incidence up to 2.17 per thousand inhabitants [1, 2], and 500,000 cholecystectomies performed annually in the USA [3] and 21,000 in The Netherlands (an incidence of 1.31 per thousand inhabitants) [4, 5], gallstones are a major cause of morbidity in the Western world. During the 1980s, the preferred surgical technique for cholecystectomy changed from the classical open procedure to a smaller incision approach [6, 7] and eventually to laparoscopic cholecystectomy. Although evidence of superiority was never delivered, the laparoscopic technique was accepted as the gold-standard procedure by consensus [3].\nMultiple randomised trials comparing laparoscopic (LC) and small-incision cholecystectomy (SIC) have been performed and results are inconsistent. Some favour the SIC technique, others favour the LC technique, and many take a neutral position. All these randomised trials are included in our Cochrane review. Our review showed no differences in primary outcome measures between LC and SIC [8].\nIn comparing (surgical) treatments, primary outcome measures (mortality and severe complications) have to be considered prior to secondary outcome measures. As no significant differences between LC and SIC in primary outcome measures were found [8], it is justified to consider health status, an important secondary outcome measure. Frequently, quality of life is confused with health status. Quality of life measures the subjective judgment of patients about their condition, while health status refers to the impact of disease on patients\u2019 lives in the physical, psychological and social domains.\nQuestionnaires, both generic and condition-specific, have been shown to be useful in measuring changes in health status after cholecystectomy [9\u201311]. Several studies showed that health status was improved, both after LC and open cholecystectomy in patients suffering socially disabling uncomplicated symptomatic cholecystolithiasis [12\u201314]. Differences between the open and laparoscopic technique are not clear [15], although some studies found superior results using the laparoscopic technique [16, 17].\nTo date, differences in health status between LC and SIC are not very well examined [18\u201320]. Moreover, as the previous studies did not use the appropriate questionnaires as advised by evidence-based guidelines, there had been no possibility to correctly find differences in health status between both operating techniques.\nThe gastrointestinal quality of life index (GIQLI) and the short form (SF-36) are frequently used and validated questionnaires (disease-specific and generic, respectively) and are most suitable for evaluating patients\u2019 functional recovery after cholecystectomy [21].\nObjective\nThe aim of our study was to examine differences in health status in patients with symptomatic cholecystolithiasis before and after LC and SIC in a blinded randomised clinical trial. We used the GIQLI and the SF-36 questionnaires, as recommended by evidence-based guidelines [21].\nMethods\nAll patients with symptomatic cholecystolithiasis visiting the outpatients clinic of the St. Elisabeth hospital in Tilburg were considered for inclusion in a blind randomised trial comparing laparoscopic and small-incision cholecystectomy. Verbal and written informed consent was obtained from each patient, and patients were consecutively listed for elective cholecystectomy. Health status was a secondary outcome measure as part of the randomised clinical trial.\nSample size\nNo differences in primary outcome measures (mortality and complications) were expected between LC and SIC [8]. Consequently, a secondary outcome measure should be used to decide on preferences between both techniques. We decided to focus on costs between both techniques as the most important secondary outcome measure. Based on an anticipated difference of 10% in direct costs 120 patients had to be included in each group. However, multiple outcome measures including health status were evaluated in this randomised trial.\nBased on a previous study [18], it was calculated that 128 patients were needed in each group to detect a difference of 5 points (assuming a standard deviation of 20) in the gastrointestinal quality-of-life index (GIQLI) questionnaire with a type I error of 0.05 and a power of 0.8.\nRandomisation\nAs randomised trials with high bias risk may overestimate intervention effects [22], results of randomised trials with low bias risk are considered more reliable. Therefore, attention is warranted for correct generation of the allocation sequence, allocation concealment, blinding, and follow-up.\nA random-number table was used for the generation of the allocation sequence and allocation concealment was guaranteed by using sealed envelopes. To eliminate bias caused by preoperative expectations, patients were randomised in the operation theatre after induction of anaesthesia. A telephone call to the secretary office was made and an employee opened an envelope. All patient data were recorded in a case record form, with the procedure reported as \u2018trial cholecystectomy\u2019. Wounds and port sites were dressed with identical opaque dressings, stained using iodine, regardless of the surgical procedure performed, to allow blinding for patient, nurses, and physicians during the postoperative period. The type of operation was revealed just before discharge.\nNo patients were lost to follow-up. Operative procedures were standardised apart from using a laparoscopic or small-incision technique. Anaesthesia, postoperative care and analgesic use were also standardised.\nInclusion and exclusion criteria\nInclusion criteria were: male or female patients with symptomatic cholecystolithiasis, age 18 years or older at recruitment, with reasonable to good health according to American Society of Anaesthesiologists (ASA) classification (ASA I or II) [23], no known relevant allergies and a signed letter of informed consent.\nExclusion criteria were: age younger than 18 years, choledocholithiasis (icterus, acholic faeces and\/or bilirubine twice normal range), cholangitis, known pregnancy, moderate to severe systemic disease (ASA III and higher), known cirrhosis of the liver, history of abdominal malignancy, previous upper abdominal surgery (precluding laparoscopic approach), psychiatric disease, or another reason (e.g. lack of knowledge of the Dutch language) for making follow-up or completion of questionnaires unreliable.\nObesity was indexed but not considered an exclusion criterion [24]. Recovery after successful endoscopic treatment of choledocholithiasis was not a contraindication. Acute cholecystitis is a different disease with other complication rates, morbidity, and conversion rates, and patients suffering acute cholecystitis were, therefore, not included.\nSurgical procedures\nThe policy in our hospital was not to perform operative cholangiography in any patient in elective cholecystectomy. All patients had nasogastric intubations during the operation that were removed immediately afterwards. Bladder drainage was not performed. Abdominal wall and skin closure were standardised. In case of technical difficulties or for safety reasons, both laparoscopic and small-incision cholecystectomies were converted to open cholecystectomy by a subcostal incision (>8 cm). Reasons for conversion were registered. The wounds were covered with standard wound dressings as described by Majeed [24] to blind patient and ward personnel postoperatively. We did not use any local anaesthetic technique into the wounds nor intercostal nerve blocks.\nLaparoscopic cholecystectomy\nOpen introduction of trocars was performed in all patients, regardless of previous abdominal surgery. Pneumoperitoneum was created using the subumbilical trocar with an intra-abdominal pressure up to 12 mmHg. Three trocars for instruments were inserted. The dissection of the cystic artery and cystic duct, identifying Calot\u2019s triangle, was performed using a three-point \u2018flag\u2019 technique [25]. The cystic duct and artery were clipped and transsected. After complete dissection of the gallbladder, it was removed either through the subumbilical or the subxyphoidal trocar. Fascia defects as a result of the insertion of 10mm trocar and the open introduction of the subumbilical trocar were closed with UR6 vicryl 1.0\/2.0\u00ae sutures. All instruments, except for the subumbilical trocar, were reusable. No suction drains were left in the subhepatic space at the end of the procedure.\nSmall-incision cholecystectomy\nIn the literature most authors used 8 cm (or less) as a cut-off point to differentiate between small-incision and open cholecystectomy [24, 26\u201332]. Therefore, we performed small-incision cholecystectomy principally through an incision of 6 cm, maximally extended to 8 cm. As part of a separate research question, all patients had a preoperative ultrasound scan and the location of the fundus of the gallbladder was marked on the skin. We used the craniocaudal position of the mark for incision. The mediolateral position of the mark was not used, because in the pilot phase we found that the incision would be too lateral for adequate view of the hilus. The incision was placed over the musculus rectus abdominis. Only standard instruments were used and no special equipment. Access to the peritoneum was obtained by a muscle splitting (and not transsection) technique of the musculus rectus abdominis (like in an open appendectomy). The gallbladder was dissected by a fundus-first technique. If necessary the gallbladder was punctured to remove its liquid contents. The cystic duct and artery were ligated and the gallbladder was removed. No suction drains were left in the subhepatic space at the end of the procedure. Posterior and anterior fascias were closed separately with PDS 3.0\u00ae running suture. After wound closure, the length of the incision was measured. When the length exceeded 8 cm, the operation was considered to be a conversion to open cholecystectomy.\nPostoperative protocol\nEarly oral intake and mobilization were encouraged. Patients left the hospital as soon as they felt capable. As patients were admitted at the day of operation, hospital stay was defined as the number of nights (postoperative) in hospital. Shortly before discharge, wound dressings were removed for wound inspection. For logistic reasons, we were not able to blind the surgeon at the patients\u2019 follow-up. Follow-up took place according to a standardised scheme after 2 weeks, 6 weeks, and 3 months. Patients were encouraged to resume work and normal daily activity as soon as they felt capable to do so.\nMeasurements\nIn accordance with evidence-based guidelines [21], we decided to use the generic short form (SF-36) and the disease-specific gastrointestinal quality-of-life index (GIQLI) questionnaires. These questionnaires were completed preoperatively, on the first day postoperative and at each follow-up visit after 2 and 6 weeks and after 3 months. In addition, the body image questionnaire (BIQ) was completed preoperatively and at 6 weeks postoperatively in order to estimate differences in the patients\u2019 perception of their body image and cosmetics [33].\nThe SF-36 is a generic health status questionnaire that has 36 questions to assess eight domains (physical functioning, role physical, bodily pain, general health, vitality, social functioning, role emotional and mental health) [34]. Internal consistency measured by Cronbach\u2019s alpha was shown to be high (above 0.80 in all subscales) [34]. The Dutch version has been validated [35].\nThe GIQLI is a disease-specific health status measure. It includes both specific questions on gastrointestinal symptoms, for both the upper and the lower gastrointestinal tract, as well as questions on physical, emotional and social capabilities [36]. It is a mixed questionnaire that includes both generic and specific questions. Based on face validity, five subscales are distinguished in addition to a total score. Internal consistency measured by Cronbach\u2019s alpha was shown to be high (above 0.90 in all subscales) [36]. The Dutch version has been validated [37].\nThe body image questionnaire (BIQ) consists of nine questions evaluating three subscales: body image, cosmetic, and self-confidence. The BIQ has shown to consist of two factors, a body image and a cosmetic factor [33]. The body image scale measures patients\u2019 perception of and satisfaction with their own body and explores patients\u2019 attitudes toward their bodily appearance. The cosmetic scale assesses the degree of satisfaction of patients with respect to the physical appearance of the scar. Additionally, a question is added to assess patients\u2019 self-confidence before and after surgery. Internal validity (measured by Crohnbach\u2019s alpha) reliability coefficients were shown to be high for both the body image (0.80) and cosmetic scales (0.83) [33].\nStatistical analysis\nAnalyses were performed according to the type of operative procedure used, based on the intention to treat principle. Apart from this main analysis, one subgroup analysis was performed: converted procedures (LC and SIC) were compared with minimal invasive procedures (LC and SIC). This subgroup analysis was performed in order to illustrate the sensitivity of the questionnaires. Calculations were made using SPSS version 11.0\u00ae.\nRepeated measures analysis of variance (ANOVA) was used to evaluate health status differences over time between the two operative techniques.\nAdditional independent t-tests were performed to test for time-specific differences in scores at the preoperative measurements between two groups in order to check for a correct randomisation procedure. If appropriate, additional independent t-tests were performed to test for other time-specific differences in measurements.\nResults\nAll trial patients were included and operated between January 2001 and March 2004. Leaving unwilling and excluded patients out of consideration, 366 patients initially fulfilled the inclusion criteria and were initially included in the trial. A total of 102 patients were not randomised for a variety of reasons (Fig.\u00a01). After randomizing 264 patients, another seven patients were excluded (after their cholecystectomy) for the following reasons: unwillingness for further participation in the trial (2), intraoperative suspicion of malignancy (2), transfer to another ward not participating in the trial (1), participation in two trials (not in line with the Helsinki declaration) (1), and insufficient knowledge of the Dutch language (1). Excluding the data of these seven patients from our analyses did not affect the results of our questionnaires in any way. A total of 257 patients were left for analysis (LC:120 and SIC:137).\nFig.\u00a01Revised consort statement diagram showing the flow of participants through each stage of the randomised trial [38]\nBaseline characteristics and operative results\nThe groups (LC and SIC) did not differ regarding age, sex, body mass index (BMI) and ASA classification (Table\u00a01). The classical diagnostic symptoms of cholecystolithiasis as well as the duration of these symptoms were also equally distributed in both groups. In addition, the number of patients presenting with complicated gallstone disease who had received treatment by endoscopic retrograde cholangiopancreaticography (ERCP) (and papillotomy) were equally distributed and operated on in a later stage (Table\u00a02).\nTable\u00a01Patient characteristicsLaparoscopic cholecystectomy (n = 120)Small-incision cholecystectomy (n = 137)P valueMale31 (25.8%)30 (21.9%)0.459Female89 (74.2%)107 (78.1%)Age (years)\u00a0\u00a0Mean (SD)48.4 (14.1)48.5 (14.0)0.974\u00a0\u00a0Median (range)49 (17\u201377)48 (18\u201380)BMI\u00a0\u00a0Mean (SD)27.5 (4.8)27.9 (4.6)0.500\u00a0\u00a0Median (range)26.8 (18.5\u201345.9)27,2 (18.0\u201343.3)ASA stage\u00a0\u00a0I81 (67.5%)91 (66.4%)0.855\u00a0\u00a0II39 (32.5%)46 (33.6%)BMI body mass index; ASA american society of anaesthesiologists classificationTable\u00a02Operative features and difficulties of laparoscopic and small-incision cholecystectomyLaparoscopic cholecystectomy (n = 120)Small-incision cholecystectomy (n = 137)P valuePatients with complicated gallstone diseases before cholecystectomy18180.668Endoscopic retrograde cholangiopancreaticography12130.890Duration of symptoms (weeks)n = 107 (89.2%)n = 130 (94.9%)0.443\u00a0\u00a0mean (SD)61.1 (108.8)70.3 (147.2)\u00a0\u00a0median (range)26 (2\u2013884)17.5 (1\u20131040)Incision length (mm) #n = 95 (12 conversions)n = 134 (20 conversions)0.196\u00a0\u00a0mean (SD)76.1 (33.8)76.0 (24.0)\u00a0\u00a0median (range)65 (40\u2013200)66 (49\u2013165)Inflammation21250.876Operative team:\u00a0\u00a0surgeon\u2013resident15 (12.5%)21 (15.3%)0.515\u00a0\u00a0resident\u2013surgeon84 (70.0%)100 (73.0%)0.596\u00a0\u00a0resident\u2013resident21 (17.5%)18 (13.1%)0.331Hospital stay *\u00a0\u00a0mean (SD)2.4 (4.6)3.1 (12.4)0.560\u00a0\u00a0median (range)1 (1\u201336)2 (1\u2013144)Hospital stay * (without one extreme value)\u00a0\u00a0mean (SD)2.1 (3.38)2.04 (2.42)0.877Employed (n)5051Return to work (weeks)\u00a0\u00a0mean (SD)4.1 (2.3)3.7 (2.0)0.298\u00a0\u00a0median (range)4 (1\u201312)3 (0.5\u201312)# conversions were included in incision length measurements* hospital stay in postoperative nights\nThere was no mortality. There were five intraoperative complications in the LC group compared with three in the SIC group. There were 16 postoperative complications in the LC group and 13 in the SIC group. There were 21 and 16 total complications (intra- and postoperative) in the LC and SIC group, respectively. Of these, 11 and 7 complications were serious in the LC and the SIC group, respectively (Table\u00a03). We did not find a difference in the number or severity of the complications.\nTable\u00a03Serious complications in laparoscopic and small-incision cholecystectomy (intraoperative and postoperative)Laparoscopic cholecystectomy11Small-incision cholecystectomy7Intraoperative complicationsAsystole 1Cardiac ischemia, no elevated enzymes 1Common bile duct (CBD) injury, eventually hepatico-jejunostomy, complicated prolonged ICU stay, stenosis bile duct.1CBD injury, conversion, T-drain, ERCP and papillotomy for CBD stone1Bleeding requiring conversion (and transfusion)1Hepatic parenchyma rupture, conservative treatment (transfusion) 1Bowel injury at introduction (sutured)1Cerebrovascular accident at recovery1Total intraoperative5Total intraoperative3Postoperative complicationsPneumonia1Cystic duct leakage (ERCP + stent)1Cerebrovascular accident (6 weeks postoperative)1CBD injury, multiple relaparotomies and ICU stay 1Intra-abdominal fluid collection (haematoma); icterus (ERCP: no stones, complicated by bleeding)1CBD stone (ERCP) and abscess intra-abdominal (ultrasound drainage)1Pancreatitis (conservative treatment) 1CBD stone with pancreatitis (ERCP)1Intra-abdominal abscess (re-laparoscopy)1Epididimitis (operation by urologist)1Total postoperative6Total postoperative4CBD common bile duct; ICU intensive care unit; ERCP endoscopic retrograde cholangiopancreaticography\nOperative time was shorter for SIC compared to LC (60 versus 72 min, respectively; U = 6013.0, p < 0.001). Conversion rates were similar (p = 0.312), with similar reasons for conversion. The follow-up rate between the groups was not statistically different. Follow-up was 91.4\u201396.3% at six weeks, 82.2\u201382.8% at three months and 100% at either six weeks or three months. Complaints at follow-up were comparable.\nThere were no differences in the preoperative measurements of the SF-36 subscales, all the GIQLI subscales, the total GIQLI score and the BIQ subscales.\nHealth status\nThe questionnaires were obtained with a response rate varying from 87.5% preoperatively to 77.4% three months postoperatively. The nonresponders did not differ from those who remained in the study with regard to complications (16%), operative time (65 minutes), hospital stay (1.5 days), return to work (3.2 weeks) or baseline scores.\nWhen comparing LC with SIC (intention-to-treat), we found no differences in all SF-36 subscales, except for \u2018perceived health change\u2019. There were significant differences favouring the laparoscopic technique (F = 16.054, df = 1; p < 0.001) (Table\u00a04). Performing time-specific analyses, differences were identified at two weeks (p = 0.029) and six weeks (p < 0.001) postoperatively. There were no differences between LC and SIC with regard to the four GIQLI subscales, the total GIQLI score, and the body image subscales.\nTable\u00a04Comparison of GIQLI, SF-36 and BIQ scores in laparoscopic (LC) and small-incision cholecystectomy (SIC) according to intention-to-treat (mean scores and SD)PreoperativePostoperativeP valueday 12 weeks6 weeks12 weeksSF-36PhysicalLC77.0 (23.0)57.1 (29.5)67.5 (23.2)83.2 (21.2)87.8 (17.4)0.413SIC83.0 (18.3)39.9 (29.8)63.0 (22.8)83.1 (20.8)87.5 (19.3)SocialLC43.4 (14.2)77.8 (19.9)70.7 (23.9)86.5 (19.3)91.9 (15.9)0.260SIC42.7 (17.1)74.3 (22.6)66.3 (25.8)82.3 (22.9)90.4 (19.0)Role physicalLC56.4 (43.4)52.6 (43.2)26.7 (36.4)67.4 (40.1)81.1 (34.9)0.667SIC60.8 (44.4)53.0 (45.7)29.5 (49.4)54.8 (42.6)79.2 (35.8)Role emotionLC73.7 (39.4)70.5 (38.8)68.3 (41.7)82.7 (33.6)88.6 (27.8)0.797SIC74.3 (39.7)70.3 (40.7)66.4 (54.9)80.7 (36.5)88.7 (29.4)MentalLC61.7 (11.8)75.0 (17.1)77.3 (18.3)83.3 (16.2)85.1 (16.6)0.558SIC62.0 (10.9)72.1 (18.9)74.5 (18.7)81.2 (18.4)83.4 (17.4)VitalityLC54.1 (11.0)59.6 (22.4)52.1 (21.1)67.8 (20.1)73.5 (20.4)0.767SIC53.8 (12.2)58.4 (22.9)51.8 (21.6)66.9 (22.6)72.7 (21.7)PainLC56.5 (19.5)55.6 (22.5)52.2 (21.7)74.7 (20.2)82.4 (21.5)0.429SIC54.6 (17.1)55.9 (24.6)46.4 (21.9)69.3 (23.8)83.1 (21.4)General healthLC56.1 (11.7)69.7 (17.3)71.8 (19.3)74.9 (22.0)76.3 (21.2)0.457SIC57.4 (11.3)65.1 (19.2)70.1 (20.1)72.5 (21.5)76.4 (19.2)Health changeLC57.7 (21.2)57.7 (21.2)62.0 (26.9)76.7 (23.9)77.1 (24.4)<0.001*SIC55.4 (20.4)55.4 (20.4)53.5 (27.5)64.6 (25.1)71.5 (27.2)GIQLIPhysicalLC2.79 (0.76)2.82 (0.73)2.78 (0.75)3.20 (0.62)3.31 (0.59)0.790SIC2.95 (0.74)2.67 (0.83)2.69 (0.75)3.14 (0.73)3.30 (0.63)GastrointestinalLC3.01 (0.59)3.01 (0.54)3.22 (0.48)3.46 (0.41)3.50 (0.42)0.247SIC3.12 (0.58)3.13 (0.55)3.22 (0.45)3.46 (0.46)3.52 (0.40)SocialLC2.89 (0.48)2.82 (0.42)2.81 (0.52)2.93 (0.37)2.97 (0.29)0.056SIC2.90 (0.42)2.82 (0.43)2.76 (0.56)2.85 (0.50)2.85 (0.38)MentalLC2.55 (0.55)2.59 (0.45)2.88 (0.41)3.04 (0.35)3.07 (0.37)0.561SIC2.65 (0.49)2.58 (0.55)2.74 (0.45)2.99 (0.47)3.04 (0.44)TotalLC102.4 (17.0)102.6 (14.8)108.5 (15.0)116.4 (11.9)118.3 (11.7)0.607SIC106.7 (14.9)104.5 (16.0)107.4 (14.0)116.7 (13.2)118.0 (11.1)BIQBody imageLC6.42 (1.98)\u2013\u20136.03 (1.90)\u20130.530SIC6.26 (1.89)\u2013\u20135.85 (1.35)\u2013CosmeticLC\u2013\u2013\u201318.38 (3.88)\u20130.100SIC\u2013\u2013\u201317.52 (3.55)\u2013Self-confidenceLC6.95 (1.27)\u2013\u20137.68 (1.21)\u20130.647SIC7.02 (1.28)\u2013\u20137.49 (1.15)\u2013* significant difference\nSubgroup analysis\nIn checking for differences in preoperative data in the minimal invasive procedures versus conversions comparison, we only found a significant difference in the self-confidence subscale of the body image questionnaire (t = 2.821, df = 207, p = 0.005) with higher self-confidence scores in the minimal invasive operated group (7.08 versus 6.31). No other differences were found in preoperative data.\nIn order to assess differences between minimal invasive procedures (both laparoscopic and small-incision) and procedures converted to the classical open cholecystectomy, we examined patients\u2019 scores across the follow-up period (Table\u00a05).\nTable\u00a05Comparison of GIQLI, SF-36 and BIQ scores in minimal invasive laparoscopic (LC) and small-incision cholecystectomy (SIC) procedures versus converted (LC and SIC) procedures (mean scores and SD)PreoperativePostoperativeP valueday 12 weeks6 weeks12 weeksSF-36Physicalmin-inv79.9 (21.3)50.1 (30.9)67.3 (22.1)83.5 (21.1)87.6 (18.9)0.046*conv82.3 (17.5)31.6 (24.9)45.5 (22.6)79.6 (19.3)87.8 (14.6)Socialmin-inv43.2 (15.5)76.7 (21.3)69.9 (24.1)85.1 (20.9)90.9 (18.1)0.214conv41.7 (18.3)70.1 (21.9)55.1 (29.0)76.1 (24.7)93.2 (13.8)Role physicalmin-inv60.2 (43.4)54.0 (44.5)27.9 (36.3)61.3 (41.7)79.9 (35.2)0.415conv46.9 (46.8)43.1 (43.9)31.0 (87.6)55.0 (44.1)81.8 (37.1)Role emotionmin-inv74.9 (39.0)72.1 (38.8)67.4 (41.5)82.1 (34.6)89.3 (28.1)0.373conv66.7 (42.8)56.5 (45.4)66.7 (93.7)76.7 (40.6)83.3 (32.1)Mentalmin-inv61.8 (11.6)73.8 (17.9)76.6 (17.5)82.7 (16.4)84.9 (15.9)0.413conv62.6 (9.1)70.8 (19.5)69.0 (25.5)77.1 (24.4)78.6 (23.8)Vitalitymin-inv54.0 (11.9)59.5 (22.8)53.1 (21.1)67.8 (21.4)73.3 (20.6)0.180conv53.2 (8.8)55.0 (21.2)41.6 (21.1)63.3 (21.6)71.1 (24.9)Painmin-inv55.6 (18.4)56.5 (23.7)50.4 (22.1)72.5 (22.3)82.9 (21.7)0.038*conv55.2 (17.7)49.6 (22.2)37.1 (17.4)65.4 (22.4)81.9 (18.5)General healthmin-inv56.8 (11.4)68.2 (18.1)72.0 (19.4)74.2 (21.3)76.9 (19.7)0.136conv56.3 (12.4)59.7 (19.6)61.0 (20.2)67.8 (25.1)72.2 (23.3)Health changemin-inv56.8 (20.8)56.8 (20.8)58.8 (27.5)71.7 (24.9)74.9 (26.3)0.066conv54.2 (20.4)54.2 (20.4)46.3 (24.7)56.3 (24.2)67.1 (23.6)GIQLIPhysicalmin-inv2.90 (0.72)2.78 (0.76)2.79 (0.71)3.19 (0.65)3.33 (0.60)0.007*conv2.67 (0.96)2.40 (0.96)2.20 (0.87)2.96 (0.91)3.10 (0.71)Gastrointestinalmin-inv3.09 (0.57)3.10 (0.54)3.23 (0.47)3.47 (0.43)3.52 (0.39)0.052conv2.89 (0.68)2.89 (0.56)3.08 (0.37)3.42 (0.50)3.46 (0.50)Socialmin-inv2.89 (0.46)2.83 (0.42)2.83 (0.51)2.90 (0.43)2.92 (0.35)0.003*conv2.90 (0.35)2.75 (0.45)2.35 (0.62)2.74 (0.58)2.85 (0.35)Mentalmin-inv2.60 (0.53)2.58 (0.50)2.84 (0.41)3.04 (0.38)3.06 (0.38)0.031*conv2.63 (0.45)2.64 (0.53)2.48 (0.57)2.81 (0.70)2.96 (0.63)Totalmin-inv104.9 (16.0)104.3 (15.0)108.9 (14.3)117.1 (11.5)118.4 (11.0)0.020*conv101.4 (17.0)97.8 (17.8)98.5 (13.3)111.2 (19.5)115.8 (14.0)BIQBody imagemin-inv6.31 (1.80)--5.75 (1.27)-<0.001*conv6.60 (2.78)--7.55 (3.04)-Cosmeticmin-inv---18.27 (3.54)-<0.001*conv---14.86 (3.97)-Self-confidencemin-inv7.08 (1.16)--7.60 (1.13)-0.064conv6.31 (1.83)--7.38 (1.60)-* significant difference; min-inv: minimal invasive procedures (LC and SIC); conv: converted procedures (LC and SIC)\nThere were significant differences in the SF-36 subscales \u2018physical functioning\u2019 (F = 4.057, df = 1; p = 0.046) and \u2018pain\u2019 (F = 4.391, df = 1; p = 0.038). In the GIQLI questionnaire, there were significant differences in the total score (F = 5.593, df = 1; p = 0.020), and in the \u2018physical\u2019 (p = 0.007), \u2018social\u2019 (p = 0.003), and \u2018mental\u2019 (p = 0.004) subscales. Also, in the BIQ there were significant differences in the \u2018body image\u2019 and \u2018cosmetic\u2019 subscales between both operative groups, favouring the minimal invasive procedures (F = 13.939, df = 1; p < 0.001). No other differences were found.\nDiscussion\nWe have used both generic and disease-specific health status questionnaires and a body image questionnaire to evaluate the effect of LC versus SIC in patients having cholecystectomy for symptomatic cholecystolithiasis. No differences were found between laparoscopic and small-incision cholecystectomies (applying intention-to-treat). However, with regard to minimal invasive or converted procedures, we found significant differences in the \u2018physical\u2019 subscales in both SF-36 and GIQLI as well as differences in body image in favour of minimal invasive procedures. The fact that significant differences were found in the \u2018physical\u2019 subscales in both questionnaires illustrates construct validity between both health status instruments.\nLiterature\nA few other studies have compared health status after LC and SIC [18\u201320]. Two studies found that the laparoscopic technique was associated with a more rapid improvement in health status after cholecystectomy compared with the small-incision technique [18, 19]. One study found no differences at all between both techniques [20]. However, it is difficult to draw conclusions from three studies that used different questionnaires and suffer several methodological flaws. None of the mentioned studies combined the SF-36 and GIQLI as advised by evidence-based guidelines [21].\nBarkun studied 35 and 23 patients in the LC and SIC groups, respectively, and used the same GIQLI as we did in addition to the Nottingham health profile (NHP) and a visual analogue scale (VAS) for health [18]. Allocation concealment was unclear, no blinding was used, and eight dropouts occurred in their rather small, preliminary stopped trial. They used cumulative totals of both GIQLI and NHP data instead of using subscales. Changes in one dimension might be offset by changes in other dimensions. Both questionnaires have more than one dimension (the cumulative total); subscales indeed provide the advantage of additional information on several dimensions. As a rather small number of patients were included (the trial was stopped preliminary), no subscales were assessed, and no considerations were given to the construct or divergent validity of both questionnaires, their conclusion that LC was associated with a significantly quicker return to \u2018good health\u2019 seems inappropriate based on their results.\nMcMahon compared health status in 151 and 148 laparoscopic and small-incision cholecystectomy patients respectively using the SF-36 health survey questionnaire and the hospital anxiety and depression scale (HADS) [19]. Generation of the allocation sequence in their trial was unclear and no blinding was used. They found that patients recovering from LC enjoyed significantly better health 1 and 4 weeks after the operation compared with those recovering from SIC, but no significant difference was found at 12 weeks. The absence of preoperatively baseline measurements and the absence of considerations on the construct or divergent validity of the questionnaires make conclusions about postoperative data uncertain. Differences in SF-36 and HADS correlated with differences in return to domestic and leisure activities, but were not translated in differences in paid activity.\nSquirrell used the NHP in 100 patients (50 in each group) preoperatively, and 3 weeks and 6 months postoperatively [20]. This was the only study that used blinding in their methods. Generation of the allocation sequence in their trial was unclear. At no time there was a significant difference between the two groups. The study used a rather small sample size, and unfortunately they did not use a disease-specific questionnaire, but only one generic questionnaire. They concluded that it is necessary to take a broader view of health and not concentrate simply on pain when assessing postoperative recovery.\nIn our study, no significant differences were found between LC and SIC using both generic and disease-specific health status as well as body image with response in approximately 80% of patients. The response rate of 77.4% at 3 months follow-up may represent a possible source of bias. However, the nonresponders were comparable to those who remained in the study with regard to complications, operative time, hospital stay, return to work, and baseline scores of questionnaires. Moreover, our response rate is in line with the response rates in the studies of Barkun et al. (58%) and McMahon et al. (78%).\nWe conclude that there are no differences between both operative techniques regarding health status. The only exception is that in the SF-36 subscale perceived health change we found a difference between LC and SIC, which appeared to be caused by the scores at 2 and 6 weeks postoperatively and disappeared at 3 months follow-up. LC patients reported a larger health change. However, in the evaluation of 17 aspects of health status, only one difference was found. Moreover, this difference in perceived health change was not reflected in an earlier return to work in LC. In contrast, SIC patients returned to work quicker than LC patients, although this different was not significant. Therefore, our overall interpretation is that there are no differences between LC and SIC.\nThe comparable \u2018physical\u2019 subscales in SF-36 and GIQLI, which are supposed to measure the same effect, are both significantly different in the minimal invasive versus conversions comparison illustrating construct validity of both questionnaires. Subscales on different subjects in the questionnaires illustrate divergent validity. Significant differences between minimal invasive and converted procedures illustrate that the questionnaires used are able to measure what they are intended to do.\nConclusion\nIn our randomised trial with adequate generation of the allocation sequence, concealment of allocation, blinding, and follow-up we used both a generic and a disease-specific questionnaire in addition to a body image questionnaire. There is no significant difference in health status measured with SF-36, GIQLI, and BIQ between laparoscopic and small-incision cholecystectomy (applying the intention-to-treat principle). Additional calculations showed a significant difference between minimal invasive LC or SIC procedures and procedures converted to the classical open cholecystectomy.","keyphrases":["cholecystectomy","health status","surgery","surgery","laparoscopy","laparotomy"],"prmu":["P","P","P","P","U","U"]}
{"id":"Purinergic_Signal-4-2-2377324","title":"Purinergic inhibition of Na+,K+,Cl\u2212 cotransport in C11-MDCK cells: Role of stress-activated protein kinases\n","text":"Previously, we observed that sustained activation of P2Y1 leads to inhibition of Na+,K+,Cl\u2212 cotransport (NKCC) in C11 cells resembling intercalated cells from collecting ducts of the Madin-Darby canine kidney. This study examined the role of stress-activated protein kinases (SAPK) in NKCC inhibition triggered by purinergic receptors. Treatment of C11 cells with ATP led to sustained phosphorylation of SAPK such as JNK and p38. Activation of these kinases also occurred in anisomycin-treated cells. Surprisingly, we observed that compounds SP600125 and SB202190, known as potent inhibitors of JNK and p38 in cell-free systems, activated rather than inhibited phosphorylation of the kinases in C11 cells. Importantly, similarly to ATP, all the above-listed activators of JNK and p38 phosphorylation inhibited NKCC. Thus, our results suggest that activation of JNK and\/or p38 contributes to NKCC suppression detected in intercalated-like cells from distal tubules after their exposure to P2Y1 agonists.\nNa+,K+,Cl\u2212 cotransporter (NKCC), providing electroneutral symport of monovalent ions in the stoichiometry of 1Na+:1K+:2Cl\u2212 and selectively inhibited by bumetanide and other high-ceiling diuretics, belongs to the superfamily of Cl\u2212-coupled monovalent cation cotransporters. Two isoforms of this carrier have been cloned from vertebrate cDNA libraries. A ubiquitous NKCC1 isoform is expressed in all types of cells studied so far [1], including the basolateral membrane of epithelial cells derived from collecting ducts of the Madin-Darby canine kidney (MDCK) [2]. This isoform contributes to cell volume regulation, adjustment of [Cl\u2212]i above the values predicted by Nernst equilibrium potential and transcellular movement of salt and  in the secretory epithelium [1, 3]. Unlike NKCC1, three alternatively spliced variants of the NKCC2 isoform were found exclusively on the apical membrane of renal epithelial cells from the macula densa and thick ascending limb of Henle\u2019s loop. In the thick ascending limb, NKCC2 plays a key role in bulk salt reabsorption [3], whereas in the macula densa this carrier is involved in sensing extracellular Cl\u2212 concentration and regulating renal function via tubuloglomerular feedback [4].\nBecause of compensatory reactions occurring in proximal segments via tubuloglomerular feedback, collecting ducts are considered a major target for regulation of water\/salt and acid-base homeostasis by hormones and neurotransmitters, including vasopressin, bradykinin, atrial natriuretic peptide, prostanoids, mineralocorticoids, and catecholamines [5]. Under certain circumstances, extracellular nucleotides, such as ATP, UTP, and ADP, can also regulate renal epithelium function by activating two receptor subtypes. P2Y receptors are coupled to heterotrimeric G proteins and expressed in all types of renal cells studied so far, whereas P2X receptors correspond to ligand-gated cation channels, and their expression in the kidney is mainly limited to mesangial and vascular smooth muscle cells [6\u201310].\nThe renal ion transport systems affected by P2Y receptors have been explored mainly in MDCK cells where extracellular ATP leads to transient Cl\u2212 secretion [11]. Two populations of cells have been isolated from commercially available stocks of MDCK cells: C7- and C11-MDCK cells [12]. C7 cells have high transepithelial electrical resistance (Rte), are peanut lectin negative, maintain pHi at 7.39, and have large K+ conductance, thus resembling principal cells from the collecting ducts. C11 cells, on the other hand, resemble intercalated cells; they have low Rte, are peanut lectin positive, maintain pHi at 7.16, and have large Cl\u2212 and H+ conductances [12]. We were the first to report that transient activation of basolateral NKCC in ATP-treated C7 cells is evoked by elevation of [Ca2+]i [13] and contributes to transepithelial Cl\u2212 secretion [14]. We also noted that in contrast to C7 and other cells studied so far, sustained application of ATP to C11 cells resulted in sharp inhibition of this carrier [15] that was absent under the addition of other modulators of transepithelial ion fluxes [16].\nSeveral hypotheses might be proposed to explain the cell type-specific impact of extracellular nucleotides on NKCC activity. First, NKCC inhibition is mediated by the P2 receptor isoform whose expression is limited to intercalated cells. However, in a recent study, we established that NKCC inhibition in ATP-treated C11 cells is caused by activation of P2Y1 receptors [13] whose expression has been detected in most of the cell types studied so far, including C7 cells [17]. Second, in intercalated cells, sustained activation of P2Y1 receptors leads to inhibition of the cell type-specific NKCC isoform. This hypothesis contradicts data showing that prolonged exposure of C11 cell monolayers to ATP abolished the increment of bumetanide-sensitive 86Rb uptake across the basolateral membrane by transfection with human NKCC1 [18]. Third, delayed NKCC1 inhibition seen in ATP-treated C11 cells is mediated by the de novo expression of intercalated cell-specific inhibitor(s) of this carrier. However, the inhibitory action of ATP on NKCC activity in C11 monolayers was preserved in the presence of inhibitors of RNA and protein synthesis [18]. Fourth, NKCC1 inhibition is caused by the intercalated cell-specific signaling cascade that couples P2Y1 activation with NKCC1 inhibition.\nIn renal epithelial cells, P2Y receptors trigger diverse signaling, including transient elevation of [Ca2+]i, cAMP production, activation of phospholipase C (PLC), phospholipase A2 (PLA2), prostaglandin synthase, protein kinase C (PKC), and protein kinase A (PKA) [19, 20]. We did not observe any effect of extracellular Ca2+ and intracellular Ca2+ chelator on NKCC inhibition in C11 cells [15, 21]. The inhibitory action of ATP is also preserved in the presence of inhibitors of PLC, PLA2, and prostaglandin synthase (U73122, AACOCF3, and indomethacin, respectively) as well as under activation of adenylate cyclase with forskolin and isoproterenol and inhibition of PKA with H-89 [15, 21]. In C11 cells, NKCC inhibition was also revealed in the presence of the PKC activator 4\u03b2-phorbol-12-myristate-13-acetate (PMA). However, the same action of PMA was also documented in C7 cells [15]. Moreover, suppression of NKCC in ATP-treated cells was not affected in the presence of PKC inhibitors (staurosporine and calphostin C) and after downregulation of this enzyme by chronic exposure to PMA [15, 21]. Viewed collectively, these data strongly suggest that none of the above-listed intermediates of the intracellular signaling cascade is involved in the purinergic inhibition of NKCC [15, 21].\nIn contrast to the above-listed signals documented in both principal- and intercalated-like cells, activation of P2Y receptors resulted in phosphorylation of members of the superfamily of mitogen-activated protein kinases (MAPK), such as the extracellular signal-activated protein kinases Erk1 and Erk2 and the Jun N-terminal kinase JNK1. Compound PD98059, a potent Erk inhibitor, strongly suppressed Erk1 and Erk2 phosphorylation in ATP-treated C11 cells, but did not affect the inhibitory action of ATP on NKCC activity [15, 21]. Considering these results, we designed the present investigation to examine the involvement of JNK and p38, a member of the stress-activated protein kinases (SAPK), in P2Y-induced NKCC inhibition.\nMethods\nCell culture C11-MDCK cells were obtained from Dr. M. Gekle (University of W\u00fcrzburg, Germany) and cultured in DMEM supplemented with 2.5\u00a0g\/l sodium bicarbonate, 2\u00a0g\/l hydroxyethylpiperazine ethanesulfonic acid (HEPES), 100\u00a0U\/ml penicillin, 100\u00a0\u03bcg\/ml streptomycin, and 10% fetal bovine serum (Gibco Laboratories, Burlington, ON, Canada). The cells were passaged upon reaching subconfluent density by treatment in Ca2+- and Mg2+-free Dulbecco\u2019s phosphate-buffered saline (PBS) with 0.1% trypsin from Sigma (St. Louis, MO, USA), then scraped from the flasks with a rubber policeman. Dispersed cells were counted and inoculated at 1.25\u2009\u00d7\u2009103 cells\/cm2. Before experimentation, the cells were subjected to 24-h serum deprivation in the presence of 0.1% bovine serum albumin (BSA).\nNKCC measurement Serum-deprived cells seeded in 24-well plates were washed twice with 2\u00a0ml of PBS and incubated for 30\u00a0min at 37\u00b0C in 1\u00a0ml of Cl\u2212-depleted medium A containing 140\u00a0mM Na gluconate, 5\u00a0mM K gluconate, 1\u00a0mM MgSO4, 1\u00a0mM CaCl2, 5\u00a0mM D-glucose, and 20\u00a0mM HEPES-Tris (pH 7.4). After 30\u00a0min of Cl\u2212 depletion, ATP and other test compounds were added at concentrations indicated in the figure legends. In a major part of the experiments, preincubation with ATP was limited to 30\u00a0min. Then, Cl\u2212-depleted medium was replaced with 0.25\u00a0ml of medium B containing 140\u00a0mM NaCl, 5\u00a0mM KCl, 1\u00a0mM MgCl2, 1\u00a0mM CaCl2, 5\u00a0mM D-glucose, 20\u00a0mM HEPES-Tris (pH 7.4), ~1\u00a0\u03bcCi\/ml 86Rb, and 50\u00a0\u03bcM ouabain\u2009\u00b1\u200910\u00a0\u03bcM bumetanide. In 5\u00a0min, 86Rb uptake was terminated by the addition of 2\u00a0ml of ice-cold medium C containing 100\u00a0mM MgCl2 and 10\u00a0mM HEPES-Tris buffer (pH 7.4). The cells were then transferred on ice, washed 4 times with 2\u00a0ml of ice-cold medium C, and lysed with 1\u00a0ml of a 1% SDS\/4\u00a0mM ethylenediaminetetraacetate (EDTA) mixture. The radioactivity of the cell lysate was measured with a liquid scintillation analyzer. The rate of 86Rb (K+) influx was calculated as V\u2009=\u2009A\/amt where A was the radioactivity in the sample (cpm), a was the specific radioactivity of 86Rb (K+) (cpm\/nmol) in the incubation medium, m was the protein content in the sample measured by modified Lowry\u2019s method (mg), and t was the incubation time (min). NKCC activity was estimated as the rate of ouabain-resistant, bumetanide-sensitive 86Rb influx.\nWestern blotting C11 cells grown in 6-well plates were incubated for 24\u00a0h in DMEM containing 0.1% BSA and stimulated with ATP in Cl\u2212-depleted medium A. Then, the cells were washed twice with ice-cold PBS and lysed in 200\u00a0\u03bcl of lysis buffer containing 20\u00a0mM Tris-HCl (pH 7.5), 150\u00a0mM NaCl, 10% glycerol, 1% Triton X-100, 0.1% SDS, 2\u00a0mM EDTA, 2\u00a0mM ethyleneglycoltetraacetic acid (EGTA), 0.25% deoxycholate, 1\u00a0mM phenylmethylsulfonyl fluoride, 1\u00a0\u03bcg\/ml leupeptin, 1\u00a0\u03bcg\/ml aprotinin, 200\u00a0\u03bcM Na-orthovanadate, and 1\u00a0mM NaF. The lysed cells were scraped and centrifuged at 13,000\u00a0rpm for 5\u00a0min, and an equal volume of clear lysates containing 20\u00a0\u03bcg of protein was treated for 5\u00a0min at 95\u00b0C and applied on 10% polyacrylamide gel, followed by electrophoresis and transfer to Immobilon-P membranes (Millipore Corp., Bedford, MA, USA). The membranes were washed with PBS containing 0.05% Tween 20 (PBS-Tween) and 0.5% skim milk, and incubated overnight at 4\u00b0C with antibodies. After incubation, the membranes were washed 3 times with PBS-Tween, incubated for 1\u00a0h with horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), washed with PBS-Tween, and the protein bands were visualized with an enhanced chemiluminescence detection kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA) before exposure to X-ray film. Relative protein content was quantified by the NIH image program.\nChemicals ATP, ouabain, bumetanide, forskolin, and PMA were obtained from Sigma (St. Louis, MO, USA); anisomycin, cell-permeable inhibitors of JNK and p38 kinases (compounds SP600125 and SB202190, respectively) and their negative controls [N1-methyl-1,9-pyrazoloanthrone (MPA) and compound SB202474, respectively] were purchased from Calbiochem (La Jolla, CA, USA), and 86RbCl from Dupont (Boston, MA, USA). Anti-phospho-SAPK\/JNK (Thr183\/Tyr185) and anti-phospho-p38 (Thr180\/tyr182) antibodies were provided by Cell Signaling Technology Inc. (Hornby, ON, Canada). Salts, D-glucose, and buffers were obtained from Sigma (St. Louis, MO, USA) and Anachemia (Montreal, QC, Canada).\nStatistics The data were analyzed by Student\u2019s t-test or the t-test for dependent samples, as appropriate. Significance was defined as p\u2009<\u20090.05.\nResults\nIn a previous study, we observed that 1-h preincubation of C11 cells in Cl\u2212-depleted medium abolished the transient activation of NKCC by ATP but sharply increased baseline NKCC activity, allowing more precise estimation of the inhibitory action of P2Y-induced signaling [13]. Considering these results, Cl\u2212-depleted cells were used in the present experiments. Figure\u00a01 shows that NKCC inhibition in Cl\u2212-depleted cells occurred after a 10-min lag phase with maximal attenuation of the carrier\u2019s activity in 30\u201340\u00a0min of ATP addition.\nFig.\u00a01Time dependence of NKCC inhibition in C11 cells triggered by the addition of 100\u00a0\u03bcM ATP\nIncubation for 2\u00a0min with ATP was sufficient to induce full-scale phosphorylation of JNK1 (p46) and p38 kinases (Fig.\u00a02). Phosphorylation of JNK2 in ATP-treated cells was observed after a 5-min lag phase, and its time course was negatively correlated with NKCC inhibition. In contrast to sustained JNK phosphorylation, p38 phosphorylation was partially normalized in 40\u00a0min of ATP addition. Activation of adenylate cyclase with forskolin did not affect SAPK phosphorylation. Very modest p38 and JNK phosphorylation was detected in 20\u00a0min of PMA addition. These results show that neither PKA nor PKC is involved in SAPK phosphorylation by ATP, which is consistent with the lack of involvement of these serine-threonine kinases in inhibition of NKCC in ATP-treated C11 cells [15, 16, 21].\nFig.\u00a02Representative blots showing the time-dependent action of 100\u00a0\u03bcM ATP, 10\u00a0\u03bcM forskolin, and 0.1\u00a0\u03bcM PMA on SAPK phosphorylation in C11 cells\nConsistent with numerous previous observations, anisomycin sharply elevated SAPK phosphorylation (Fig.\u00a03). Figure\u00a04a reveals that the action of anisomycin on JNK phosphorylation was lower than that of ATP; in contrast, anisomycin was a more potent activator of p38 phosphorylation than ATP. However, these differences were not statistically significant. We observed that 40-min exposure to anisomycin resulted in the inhibition of NKCC to about the same level as was detected in ATP-treated cells (Fig.\u00a04b).\nFig.\u00a03Representative blots revealing the effects of SP600125 (a), MPA (b), SB202190 (c), and SB202474 (d) on JNK and p38 phosphorylation in C11 cells under control conditions and in the presence of 100\u00a0\u03bcM ATP or 0.1\u00a0\u03bcM anisomycin. ATP and anisomycin were added during the last 40\u00a0min of preincubation of cells in Cl\u2212-depleted medium. Compounds SP600125, MPA, SB202190, and SB202474 were added at the indicated concentrations 30\u00a0min before ATP or anisomycinFig.\u00a04Effect of ATP and anisomycin on JNK and p38 phosphorylation (a) and NKCC (b) in C11 cells. 100\u00a0\u03bcM ATP and 0.1\u00a0\u03bcM anisomycin were added during the last 40\u00a0min of preincubation of cells in Cl\u2212-depleted medium. Phosphoprotein content and NKCC activity in the absence of ATP and anisomycin were taken as 1.0 and 100%, respectively. Mean values from three (a) or four (b) independent experiments are shown\nWith cell-free systems, it was demonstrated that several newly synthesized cell-permeable compounds inhibited purified or recombinant JNK and p38 without significant action on other protein kinases. Thus, compound SP600125 inhibited recombinant JNK1-3 with an IC50 of 0.05\u20130.20\u00a0\u03bcM, whereas half-maximal inhibition of other members of the MAPK superfamily, such as Erk and p38 as well as PKA and cyclin-dependent kinases, was observed at concentrations higher than 5\u00a0\u03bcM [22, 23]. It should be underlined that SP600125 was a much less potent inhibitor of JNK in intact cells. Indeed, inhibition of JNK in the anisomycin-treated KB-3 carcinoma cell line was detected after 1-h preincubation with this compound at concentrations higher than 3\u00a0\u03bcM [23]. To decrease JNK phosphorylation by two- to threefold in PMA-treated Jurkat cells, SP600125 should be added at a concentration of 50\u00a0\u03bcM [22]. Surprisingly, we observed that at a concentration of 20\u00a0\u03bcM SP600125 and SB202190, i.e., potent inhibitors of p38 SAPK in cell-free systems, increased JNK and p38 phosphorylation to the level comparable with their phosphorylation in the presence of ATP and anisomycin (Fig.\u00a03a and c). Moreover, like other potent activators of JNK and p38 phosphorylation, such as ATP and anisomycin, both SP600125 and SB202190 sharply decreased NKCC activity in C11 cells (Fig.\u00a05). In contrast, neither JNK\/p38 phosphorylation (Fig.\u00a03b and d) nor NKCC activity in the presence and absence of ATP (data not shown) was affected by the inactive structural analogues of SP600125 and SB202190, compounds MPA and SB202474, respectively.\nFig.\u00a05Effect of ATP, SP600125, and SB202190 on NKCC activity in C11 cells; 100\u00a0\u03bcM ATP was added during the last 40\u00a0min of preincubation of cells in Cl\u2212-depleted medium. Compounds SP600125 and SB202190 were added at 20\u00a0\u03bcM concentration 30\u00a0min before ATP. NKCC activity in control cells was taken as 100%. Mean\u2009\u00b1\u2009SE values from experiments performed in quadruplicate are shown\nIncreased phospho-JNK and phospho-p38 levels in SP600125- and SB202190-treated C11 cells, revealed in our study, are in contrast with the inhibitory action on these SAPK in cell-free systems [22, 23]. Indeed, the Calbiochem catalogue notes that SP600125 and SB202190 inhibit purified JNK and p38 with IC50 of 50 and 16\u00a0nM, respectively. It should be underlined that both compounds are ATP-competitive kinase inhibitors, and the data reported were obtained at an ATP concentration of 5\u00a0\u03bcM, i.e., 1,000-fold lower than the intracellular ATP concentration. For this reason, in experiments with intact cells, these inhibitors are used at much higher concentrations where diverse side effects cannot be excluded. For more details, see [24].\nDiscussion\nThe data obtained in our study show that NKCC inhibition in ATP-treated C11 cells is accompanied by activation of JNK and\/or p38. We also report here that activation of these SAPK with three distinct stimuli, such as actinomycin, SP600125, and SB202190, results in NKCC inhibition. Additional experiments should be performed to establish the causal relationship between SAPK activation and NKCC inhibition. It should also be noted that the modest inhibitory action of ATP on NKCC was preserved in the presence of the above-listed activators of SAPK. This observation suggests that the signaling cascade is not limited to the activation of these kinases. Recent studies have demonstrated that the with no lysine (K) kinase member WNK4 downregulates NKCC1 via the STE20-related kinase PASK interacting with the conserved domain in the NKCC1 N-terminus [25\u201327]. The role of WNK kinases in ATP-induced NKCC1 inhibition limited to intercalated cells deserves further investigation.\nTwo questions should be answered to evaluate the role of purinergic-dependent ion transporters in the regulation of renal function in vivo.  First, are concentrations of ATP and other extracellular nucleosides sufficient for activation of P2Y receptors on the apical and basolateral surfaces of collecting duct epithelia?  Second, what is the physiological consequence of purinergic regulation of NKCC and other ion transporters in principal and intercalated cells comprising collecting ducts?\nIn peripheral blood, ectonucleotidases maintain circulating levels of ATP\u2009<\u200910\u00a0nM [28], a concentration at which renal P2 receptors with ID50(ATP)\u2009>\u20091\u00a0\u03bcM [29] cannot be activated. However, nucleosides can act in paracrine and autocrine ways, reaching high extracellular concentrations after sympathetic [30] and cholinergic [31] stimulation or exposure to shear [32, 33], osmotic [34\u201336], and ischemic stresses [33, 37\u201339]. Sympathetic innervation is probably the major source of nucleosides for basolateral purinergic receptors [38] whereas shear and osmotic stresses, occurring in proximal tubules and the juxtaglomerular apparatus, contribute to ATP release through the apical membrane in tubular fluid [20, 40]. Using chimeric Staphylococcus aureus protein A-luciferase bound to endogenous antigens on the human airway epithelium, it was shown that hypotonic shock leads to elevation of ATP concentration in surface fluid from ~0.001 to 1\u00a0\u03bcM [41]. Released ATP is rapidly metabolized by ectoenzymes to adenosine. Importantly, modest elevation of UTP [33] and adenosine [42] sharply increased ATP release from MDCK cells, suggesting a positive feedback loop. It is well documented that osmolality of tubular fluid in collecting ducts varies from ~400 to 1,500\u00a0mOsm [43]. Baseline concentrations of ATP and other P2Y agonists in collecting duct tubular fluid and their modulation by osmotic perturbations remain unknown.\nBoth C7 and C11 cells, resembling principal and intercalated cells, are highly abundant with P2Y1 and P2Y2 receptors [13, 17]. In these cells, P2Y1 and P2Y2 receptors have been shown to mainly reside on the basolateral and apical membranes, respectively [44]. Using monolayers of C7 cells, it was noted that basolateral P2Y1 receptors activate Cl\u2212 secretion via PLA2-PKA-mediated activation of Cl\u2212 channels [17]. Our studies demonstrated that P2Y2 receptors contribute to Cl\u2212 secretion via Ca2+-calmodulin-mediated activation of NKCC1 [14] (Fig.\u00a06a). Glanville and coworkers reported that in mouse collecting ducts, basolateral NKCC possesses the same affinity for K+ and  (K0.5 ~1.5\u00a0mM) [45]. Because bumetanide markedly decreased the acidification rate in NH4Cl-treated cells, they proposed a key role for NKCC1 in H+ secretion and  handling by intercalated cells. With intercalated-like C11 cells, we observed that apical P2Y2 receptors transiently activated NKCC1 via Ca2+-calmodulin-mediated signaling, whereas sustained activation of basolateral P2Y1 receptors inhibited this carrier [13]. The present data suggest that NKCC1 inhibition in intercalated cells evoked by P2Y1 agonists is caused by activation of SAPK (Fig.\u00a06b). Further studies should be performed to examine the relative contribution of purinergic signaling in the final adjustment of salt reabsorption and acid-base balance by principal and intercalated cells in vivo as well as the pathophysiological implications of these regulatory pathways.\nFig.\u00a06Possible mechanisms of purinergic signaling in the regulation of Cl\u2212 secretion and acid-base homeostasis by principal (a) and intercalated (b) cells from collecting ducts in vivo. 1 Cl\u2212 channels, 2 H+-ATPase, 3 anion exchange, PLC phospholipase C, IP3 inositol 1,4,5-triphosphate, COX cyclooxygenase, PGE1 prostaglandin E1, CAMK (Ca2++CaM)-dependent protein kinase, CA carbonic anhydrase, a.m. and b.m. apical and basolateral membranes, respectively. For other abbreviations and more details, see text","keyphrases":["k+","stress-activated protein kinases","intercalated cells","purinergic receptors","cl\u2212 cotransport","na+"],"prmu":["P","P","P","P","R","U"]}
{"id":"Diabetologia-3-1-1794136","title":"Does self-monitoring of blood glucose improve outcome in type 2 diabetes? The Fremantle Diabetes Study\n","text":"Aims\/hypothesis To assess whether self-monitoring of blood glucose (SMBG) is an independent predictor of improved outcome in a community-based cohort of type 2 diabetic patients.\nIntroduction\nIntensive glycaemic control is a cost-effective way of reducing the complications associated with type 2 diabetes [1]. Whether self-monitoring of blood glucose (SMBG) can improve glycaemia is subject to debate [2, 3]. The Retrolective Study \u2018Self-monitoring of Blood Glucose and Outcome in Patients with Type 2 Diabetes\u2019 (ROSSO) investigators recently reported that SMBG was associated with decreased diabetes-related morbidity and all-cause mortality in type 2 diabetes, despite the SMBG group having a higher mean baseline fasting plasma glucose than the non-SMBG group [4]. In an Australian community-based cohort of type 2 patients we found that SMBG was not a determinant of glycaemic control [5], and now investigate whether, as in ROSSO, those who performed SMBG had better outcomes than those who did not.\nSubjects and methods\nSubjects The Fremantle Diabetes Study (FDS) was a longitudinal observational study in a community of 120,097 people in the state of Western Australia. We identified 2,258 subjects between 1993 and 1996, using all available sources, and recruited 1,426 (63%) to attend annual assessments, of whom 1,294 (91%) had type 2 diabetes [6]. The FDS protocol was approved by the Fremantle Hospital Human Rights Committee and all subjects gave informed consent. The present study included (1) 1,280 type 2 patients (mean age 64.1\u2009\u00b1\u200911.3\u00a0years, 48.8% men) with complete diabetes treatment and mortality data who reported SMBG status at FDS entry, and (2) a subset of 531 patients (mean age 62.4\u2009\u00b1\u20099.4\u00a0years, 54.2% men) who attended six or more consecutive annual assessments.\nClinical assessment At baseline and annual reviews, a comprehensive history was taken and a physical examination was performed. Complications were identified using standard criteria [5, 7]. Microalbuminuria was defined as an urinary albumin:creatinine ratio (ACR) \u22653.0\u00a0mg\/mmol on a first morning sample, neuropathy as a score >2\/8 on the clinical portion of the Michigan Neuropathy Screening Instrument, and retinopathy as any grade in one\/both eyes on direct and\/or indirect ophthalmoscopy and\/or detailed specialist assessment. Self-report and hospitalisations were used to identify cerebrovascular disease (CVD; stroke, transient ischaemic attack) and CHD (myocardial infarction, angina, coronary revascularisation). Peripheral arterial disease (PAD) was defined as an ankle:brachial index \u22640.9 or diabetes-related amputation. Blindness in one or both eyes, foot amputation and end-stage renal disease were also defined to allow comparison with the ROSSO findings [4].\nHospital morbidity and mortality Western Australian government registers record details of all deaths and hospital admissions, and make up part of the Western Australian Data Linkage System (WADLS) [8], which provided endpoint data to the end of June 2006. The Confidentiality of Health Information Committee approved linkage with the FDS database. All hospitalisations for endpoints were identified using the International Classification of Diseases (ICD)-9-CM and ICD-10-AM diagnosis\/procedure codes. Causes of death were classified independently by two authors (D.G. Bruce, T.M.E. Davis) as \u2018cardiac\u2019 or \u2018other\u2019 [7].\nStatistical analysis Data were analysed using SPSS for Windows (version 11.5) and are presented as proportions or means\u00b1SD. Comparison of two independent proportions was by Fisher\u2019s exact test. Freedom from non-fatal endpoints, all-cause mortality or cardiac death was analysed by baseline SMBG status using the Kaplan\u2013Meier method and compared with the log-rank test. For outcomes with sufficient events, Cox proportional hazards modelling (forward conditional variable entry and removal with p\u2009<\u20090.05 and p\u2009>\u20090.05, respectively), was used to determine: (1) independent predictors of first ever occurrence of endpoints, with all clinically plausible univariate variables with a p value of less than 0.20 considered for entry; and (2) whether, after adjusting for these variables, either SMBG at baseline or the time-dependent covariate SMBG was independently associated with outcome. A p value of less than 0.05 was considered significant.\nResults\nAt baseline, 898 type 2 patients (70.2%) performed SMBG. During 12,491 patient-years of follow-up (mean 9.8\u2009\u00b1\u20093.5\u00a0years), 486 (38.0%) died, of which 196 (15.3%) deaths were from cardiac causes. SMBG was significantly less prevalent in those who died during follow-up than in those who were alive at the end of June 2006 (65.4 vs 73.0%, p\u2009=\u20090.005).\nSMBG and mortality In unadjusted survival analysis (Model 1), SMBG was associated with a significant 24% reduction in all-cause mortality (Table\u00a01). After adjusting for age, sex and diabetes duration (Model 2), this became a non-significant 11% increased risk. Additional adjustment for independent risk factors for all-cause mortality (Model 3) did not alter this finding. In patients on diet\u00b1oral hypoglycaemic agents (OHAs) and in those on insulin, baseline SMBG was associated with significant unadjusted 24% and 54% reductions in the risk of death, respectively, which became non-significant after full adjustment. \nTable\u00a01Hazard ratios for all-cause and cardiac mortality in 1,280 type 2 diabetes patients by SMBG status for: (1) all diabetes treatment types; (2) those who were treated by diet with or without OHAs; and (3) those taking insulin with or without OHAs\u00a0No SMBGSMBG\u00a0\u00a0Number of patientsNumber of eventsAbsolute riskaNumber of patientsNumber of eventsAbsolute riskaLog-rank p valueHR (95%CI)All patients\u00a0All-cause mortality\u00a0\u00a0Model 1 (unadjusted)38216846.789831835.80.0040.76 (0.63\u20130.92)\u00a0\u00a0Model 2 (adjusted)b1.11 (0.92\u20131.35)\u00a0\u00a0Model 3 (adjusted)c1.15 (0.93\u20131.44)\u00a0Cardiac mortality\u00a0\u00a0Model 1 (unadjusted)3826117.089813515.20.460.89 (0.66\u20131.21)\u00a0\u00a0Model 2 (adjusted)b1.35 (0.99\u20131.86)\u00a0\u00a0Model 3 (adjusted)d1.55 (1.07\u20132.24)Diet \u00b1 OHAs\u00a0All-cause mortality\u00a0\u00a0Model 1 (unadjusted)35414542.677325232.50.0080.76 (0.62\u20130.93)\u00a0\u00a0Model 2 (adjusted)b1.15 (0.93\u20131.43)\u00a0\u00a0Model 3 (adjusted)e1.20 (0.94\u20131.52)\u00a0Cardiac mortality\u00a0\u00a0Model 1 (unadjusted)3545115.077310813.90.650.93 (0.66\u20131.29)\u00a0\u00a0Model 2 (adjusted)b1.51 (1.06\u20132.14)\u00a0\u00a0Model 3 (adjusted)f1.79 (1.19\u20132.69)Insulin \u00b1 OHAs\u00a0All-cause mortality\u00a0\u00a0Model 1 (unadjusted)2823119.31256658.40.0010.46 (0.29\u20130.75)\u00a0\u00a0Model 2 (adjusted)b0.68 (0.41\u20131.14)\u00a0\u00a0Model 3 (adjusted)g0.73 (0.43\u20131.26)\u00a0Cardiac mortality\u00a0\u00a0Model 1 (unadjusted)281051.91252723.90.0260.45 (0.22\u20130.93)\u00a0\u00a0Model 2 (adjusted)b0.58 (0.27\u20131.27)\u00a0\u00a0Model 3 (adjusted)h0.52 (0.22\u20131.19)aEvents per 1,000 person\u2013yearsbModel 2 includes age, sex and duration of diabetes in each case. Model 3 includes all variables in Model 2, plus:cprior CHD, CVD, PAD, neuropathy, retinopathy, ln[ACR], any exercise in past 2\u00a0weeks (negative), abdominal obesity (negative), on lipid-lowering medications (negative), Australian Aboriginal, Asian (negative), current smokerdprior CHD, CVD, PAD, neuropathy, retinopathy, ln[ACR], systolic BP (negative), total serum cholesterol, Australian Aboriginal, current smokereprior CHD, CVD, PAD, neuropathy, retinopathy, ln[ACR], abdominal obesity (negative), on lipid-lowering medication (negative), Australian Aboriginal, current smokerfprior CHD, PAD, neuropathy, retinopathy, ln[ACR], systolic BP (negative), total serum cholesterol, current smokergprior CHD, diabetes education (ever; negative), HbA1c, Australian Aboriginalhprior CHD, retinopathy, HbA1c, Australian AboriginalIn unadjusted models for cardiac death, there was a significant 55% risk reduction in insulin-treated patients (Table\u00a01); after full adjustment, this became non-significant. SMBG was associated with a 55% increased risk of cardiac death in Model 3 as a result of a significant 79% increased risk in patients not treated with insulin.\nFive-year cohort Consistent with intensification of diabetes therapy, and as reported previously [5], the proportion of the 5-year longitudinal cohort using SMBG increased over a mean of 5.4\u2009\u00b1\u20090.5\u00a0years, from 75.2% at entry to 85.5% at third review, with little change thereafter.We ascertained the incidence of first ever occurrence of micro- and macrovascular complications by baseline SMBG status (Table\u00a02). Unadjusted survival analysis showed no significant benefit of baseline SMBG (p\u2009\u2265\u20090.11) for any endpoint, single or combination. Neither adjustment for age, sex and diabetes duration (Model 2), nor relevant additional variables independently associated with the outcomes in Cox proportional hazards modelling (Model 3), improved this result. \nTable\u00a02Hazard ratios of first-ever non-fatal complications in the 531 type 2 diabetes patients in the longitudinal arm for single and combined micro- and macrovascular events, and the ROSSO Study non-fatal endpoint, by SMBG status at: (1) baseline; and (2) each annual assessment (time-dependent covariate)\u00a0No SMBG at baselineSMBG at baseline\u00a0\u00a0Time-dependent SMBG useNo. of patientsNo. of eventsAbsolute riskaNo. of patientsNo. of eventsAbsolute riskaLog-rank p valueHR (95% CI)HR (95% CI)RetinopathyModel 1 (unadjusted)1164790.034911268.40.110.76 (0.54\u20131.07)0.51 (0.36\u20130.72)Model 2 (adjusted)b0.80 (0.57\u20131.13)0.52 (0.37\u20130.74)Model 3 (adjusted)c0.82 (0.58\u20131.16)0.52 (0.37\u20130.73)NeuropathyModel 1 (unadjusted)8462199.3256186203.00.761.05 (0.78\u20131.40)0.77 (0.57\u20131.03)Model 2 (adjusted)b1.16 (0.87\u20131.55)0.79 (0.59\u20131.07)Model 3 (adjusted)d1.30 (0.97\u20131.74)0.89 (0.66\u20131.20)Microalbuminuria Model 1 (unadjusted)8237104.125810390.60.380.85 (0.58\u20131.23)0.77 (0.52\u20131.14)Model 2 (adjusted)b0.88 (0.60\u20131.28)0.78 (0.53\u20131.15)Model 3 (adjusted)e0.91 (0.62\u20131.33)0.74 (0.50\u20131.10)Any microangiopathy Model 1 (unadjusted)5850280.5172153302.60.591.09 (0.79\u20131.51)0.99 (0.71\u20131.38)Model 2 (adjusted)b1.22 (0.89\u20131.68)0.96 (0.69\u20131.33)Model 3 (adjusted)f1.34 (0.97\u20131.86)0.98 (0.71\u20131.37)Myocardial infarction Model 1 (unadjusted)13168.6386188.80.971.02 (0.41\u20132.57)0.58 (0.24\u20131.41)Model 2 (adjusted)b1.13 (0.44\u20132.89)0.63 (0.26\u20131.54)Stroke Model 1 (unadjusted)13322.739820.90.280.35 (0.05\u20132.52)0.19 (0.03\u20131.36)Model 2 (adjusted)b0.82 (0.10\u20136.60)0.30 (0.04\u20132.28)Peripheral arterial diseaseModel 1 (unadjusted)10151119.330713499.30.290.84 (0.61\u20131.16)0.74 (0.53\u20131.04)Model 2 (adjusted)b0.97 (0.70\u20131.36)0.80 (0.57\u20131.13)Model 3 (adjusted)g1.13 (0.79\u20131.62)0.89 (0.62\u20131.28)Any macroangiopathyModel 1 (unadjusted)9954133.7297133103.80.110.77 (0.56\u20131.06)0.67 (0.48\u20130.93)Model 2 (adjusted)b0.88 (0.63\u20131.21)0.71 (0.51\u20130.99)Model 3 (adjusted)h0.88 (0.63\u20131.22)0.74 (0.52\u20131.04)All vascular diseaseModel 1 (unadjusted)4441222.0141128230.10.950.99 (0.69\u20131.41)1.38 (0.89\u20132.15)Model 2 (adjusted)b1.15 (0.80\u20131.64)1.43 (0.91\u20132.23)Model 3 (adjusted)i\u2013\u2013ROSSO endpointModel 1 (unadjusted)110813.83282615.30.761.13 (0.51\u20132.50)0.54 (0.26\u20131.14) Model 2 (adjusted)b1.29 (0.58\u20132.87)0.58 (0.27\u20131.23)aEvents per 1,000 person\u2013yearsbModel 2 includes age, sex and duration of diabetes in each case. Model 3 includes all variables in Model 2, plus:cfasting plasma glucosedAsian (negative), other European, mixed\/other ethnicity (negative)eBMI, ln[ACR], aspirin-use, any exercise in past 2 weeks (negative), married (negative)fln[ACR]gsystolic BP, aspirin-use, diabetes education (ever; negative), smoker, \u221adaily alcohol consumptionhsystolic BP, aspirin-use, smoker, \u221adaily alcohol consumptioninone after adjustment forbTime-dependent Cox models were included to allow for commencement (or discontinuation) of SMBG during follow-up. In both unadjusted and adjusted models, SMBG during follow-up was associated with a reduction in the risk of retinopathy of approximately 50%. In unadjusted analysis, the risk of the development of any macrovascular disease was reduced by 33% in patients who used SMBG, but this became non-significant after full adjustment.SMBG was not associated with the first occurrence of the composite ROSSO endpoint (which occurred infrequently), either at baseline or as a time-dependent covariate.\nDiscussion\nThe present study represents the second observational assessment of the relationship between SMBG and outcome in type 2 diabetes. With regard to all-cause mortality in the full FDS sample, our unadjusted data showed that baseline SMBG was associated with a significant risk reduction, a relationship observed in both non-insulin-treated and insulin-treated subgroups. After adjustment for other potential explanatory variables, this apparent benefit was lost in both subgroups. For both combined micro- and macroangiopathy outcomes, SMBG was not associated with a first ever event in the 5-year longitudinal cohort in either unadjusted or adjusted models, with or without time-dependent analyses. These findings are inconsistent with those of the ROSSO study [4], in which all-cause mortality and diabetes-related morbidity were reduced by 32 and 51%, respectively, in patients employing SMBG.\nWhat is the explanation for these discrepancies? ROSSO had a larger number of patients than the FDS (3,268 vs 1,280), followed for a similar period. However, while the FDS involved a prevalent, prospectively-studied, community-based cohort and all patients had access to subsidised SMBG [5], ROSSO was \u2018retrolective\u2019\u2014newly-diagnosed patients aged \u226545\u00a0years were selected\u2014and only those receiving insulin were reimbursed for SMBG [4]. Whereas there was a low rate of migration of the FDS sample out of Western Australia [9] (96% had been captured on the WADLS by the end of June 2006 [10]), the retention rate of the ROSSO primary care sample is unknown. The range of potential explanatory variables (including diabetes education, marital status, education, alcohol consumption, exercise and urinary ACR) and the completeness of data collection (e.g. HbA1c and serum HDL-cholesterol results from a single laboratory were available for \u226598.8% of FDS patients compared with only 45.5 and 30.2%, respectively, from multiple laboratories in ROSSO [4]) were both much greater in the FDS. The present analyses are therefore likely to have assessed the independent contribution of SMBG to diabetes-associated morbidity and mortality with greater validity. A healthy survivor effect may also have confounded the relationship between SMBG and non-fatal outcome in ROSSO, while these analyses were undertaken in FDS participants alive at the end of 5\u00a0years of follow-up.\nAfter adjustment, SMBG was associated with an increased risk of cardiac death in patients not treated with insulin. Although the ROSSO investigators suggest that, based on apparent benefits, SMBG may be a surrogate for greater patient empowerment and physician interest, with consequently improved compliance [4], it is equally plausible that SMBG may represent a belated attempt to improve glycaemic control by mostly non-insulin-treated patients who become aware of symptoms of coronary insufficiency and heart failure. Retinopathy was the only non-fatal complication with which SMBG was independently associated, with a significantly lower relative risk in our 5-year cohort in both unadjusted and adjusted time-dependent models. This could be a chance finding, but could also reflect the effect of other variables not measured in the FDS. It is not due to better glycaemic control in patients who perform SMBG, since SMBG use did not improve glycaemia [5]. The ROSSO study report does not contain a similar disaggregated analysis [4].\nThe results of the present study do not support a relationship between SMBG and improved survival in a well-characterised community-based sample of type 2 diabetic patients. We found evidence that SMBG was associated with an increased risk of cardiac death and a reduced risk of retinopathy. These conflicting results might reflect complex interactions between patient, physician and disease factors in particular circumstances, but may also represent the effects of confounding, incomplete covariate adjustment or chance.","keyphrases":["self-monitoring of blood glucose","outcomes","type 2 diabetes","cohort study","epidemiology"],"prmu":["P","P","P","R","U"]}
{"id":"J_Biomol_NMR-3-1-2039844","title":"Evidence of molecular alignment fluctuations in aqueous dilute liquid crystalline media\n","text":"Protein dynamics can be studied by NMR measurements of aqueous dilute liquid crystalline samples. However, the measured residual dipolar couplings are sensitive not only to internal fluctuations but to all changes in internuclear vectors relative to the laboratory frame. We show that side-chain fluctuations and bond librations in the ps\u2013ns time scale perturb the molecular shape and charge distribution of a small globular protein sufficiently to cause a noticeable variation in the molecular alignment. The alignment variation disperses the bond vectors of a conformational ensemble even further from the dispersion already caused by internal fluctuations of a protein. Consequently RDC-probed order parameters are lower than those obtained by laboratory frame relaxation measurements.\nIntroduction\nNative proteins are customarily pictured as highly structured entities; a view that is supported by many lines of evidence. Nonetheless, all proteins exhibit internal motions on various time scales and amplitudes. Fast internal motions, i.e. bond vibrations and librations as well as peptide plane movements, take place in picoseconds to nanoseconds (Bouvignies et\u00a0al. 2005; Kay et\u00a0 al. 1989; Lakomek et\u00a0al. 2006). Slow internal motions, i.e. secondary structure and protein domain motions, occur at a sedate pace in microseconds to milliseconds. NMR coherence decays have revealed that bond librations and side-chain reorientations amount only to a small reduction in structural order. The associated Lipari\u2013Szabo order parameter (SLS2) (Lipari and Szabo 1982a, b) in well-structured parts of a protein is typically above 0.85 for amide moieties (Kay et\u00a0al. 1989), 0.9 for backbone bonds (Ferrage et\u00a0al. 2006) and 0.2\u20130.9 for side-chain methyl groups depending on the length of the side-chain (Millet et\u00a0al. 2002; Skrynnikov et\u00a0al. 2002). On slower time scales, in the\u00a0\u03bcs-regime and above, relaxation dispersion and exchange rate measurements (Palmer 2004) have revealed larger but infrequent excursions away from the most populated states. Recently, residual dipolar coupling (RDC) data has revealed (Lakomek et\u00a0al. 2005; Meiler et\u00a0al. 2001; Tolman et\u00a0al. 2001) in the intervening time-scale, below\u00a0\u03bcs and above \u03c4c, anisotropic and correlated motions, that have been associated with order parameters in the range 0.6\u20131.0.\nRDCs (Tjandra and Bax 1997) can be regarded as ideal motional probes as they respond explicitly to all motions of internuclear vectors. Non-zero RDCs arise first when molecular tumbling in solution is slightly anisotropic. To that end a dilute liquid crystalline (LC) medium is used to impose a subtle alignment on a biological macro-molecule solute via steric and electrostatic interactions (Prestegard et\u00a0al. 2004). RDC observables are, of course, averages over a dynamic conformational ensemble (Bax et\u00a0al. 2001; Louhivuori et\u00a0al. 2006). The high structural similarity among the most populated states prompts to think that on average all conformations would, to an excellent approximation, adopt a similar alignment. This is the implicit assumption underlying the many structural and dynamic data analyses of weak alignment NMR spectroscopy\u2014a method that is widely used today in structural biology (Bax and Grishaev 2005; Blackledge 2005). Indeed it seems reasonable to imagine that the dispersion of molecular alignments among the conformations is small, perhaps negligible, in particular as no apparent inconsistencies have been noticed in protein structure determination. Furthermore it may also seem reasonable to use in the analysis of dynamics an effective alignment frame because usually the conformations are in a fast exchange compared to molecular reorientation.\nIn this study we inspect critically the concept of a common alignment frame for a protein structure on the basis of experimental data. We find that a variation in molecular alignment due to the ps\u2013ns fluctuations can contribute significantly to the RDC-probed order parameter in the case of a small globular protein.\nTheory\nProtein motions captured by RDCs\nA residual dipolar coupling reports essentially on the average direction of an internuclear vector, e.g. a chemical bond, relative to the main magnetic field of a NMR spectrometer. Both fast and slow internal motions perturb the directions of a chemical bond and since RDCs are measured as an average over a longer time period this reflects directly into the observed RDC. Besides internal motions, the molecule experiences also rotational and translational diffusion in the liquid sample. Anisotropic orientational sampling in a dilute liquid crystalline medium is the basis for observing a residual of a dipolar coupling (Tjandra and Bax 1997). In practice, it may not be possible to de-convolute the observed data to distinct motional modes, but their identification will clarify the analysis.\nFrom a molecular perspective, i.e. from the perspective of an observer on a single molecule, external motions are incidental and of no particular interest\u2014at least if we assume that molecule\u2013medium interactions are negligible. It would therefore be sufficient to consider any disorder present in molecular structures only in terms of internal motions (Fig.\u00a01). The averaging due to the fast ps\u2013ns bond fluctuations when inspected in the local sub-molecular frame is given by the aforementioned  values. The recently proposed (Bernad\u00f3 and Blackledge 2004; Lakomek et\u00a0al. 2006; Meiler et\u00a0al. 2003; Peti et\u00a0al. 2002; Tolman 2002) substantial and frequent fluctuation of secondary structures, i.e. \u03b1-helices or \u03b2-sheets, relative to a molecular frame will cause additional down-scaling of the order parameter. We denote the corresponding order parameter as  and note that it has been reported to range from 0.6 to 1.0.Fig.\u00a01In the molecular perspective internal motions like (a) bond fluctuations, (b) peptide plane motions and (c) secondary structure movements all contribute to disperse a chemical bond away from its average direction. (d) In the ensemble perspective both internal and external motions contribute to fill the available state space. In a RDC measurement the available state space is reduced to effective alignment tensors of the constituent conformations\nFrom an ensemble perspective, i.e. from the perspective of an observer regarding every molecule in the sample, both internal and external motions are manifestations of the state space of the system (Louhivuori et\u00a0al. 2006). Internal motions cause the conformational diversity and external motions are responsible for the various orientations and positions the molecules inhabit. A conformational change is, from the ensemble perspective, tied to a change in the effective alignment of that molecule. This connection between the alignment and the conformation seems like non-sense from the molecular perspective since a conformational change does not instantly change the orientation of the molecule. Since RDCs are a time and ensemble average the ensemble perspective is an appropriate choice. In the vicinity of a liquid crystalline particle conformations are not energetically equivalent (Louhivuori et\u00a0al. 2006) and therefore e.g. some side-chain conformations are not in a fast exchange. This means that conformational changes due to internal motions couple to the alignment and appear as fluctuations in the effective alignment frame of a molecule. We mark the corresponding order parameter as  It should be emphasised that the alignment is not a property of a free molecule, i.e. a molecule in void, but rather a property of the whole sample including the LC-particles.  does not therefore correspond to any intrinsic molecular motion, even though it might appear as one if the molecular perspective is chosen for RDC data interpretation.\nFinally, we remind that molecular tumbling in solution is the most effective averaging process with respect to the laboratory frame. The associated order parameter S\u03c42 is on the order of 10\u22126 in a dilute LC-medium (Bax et\u00a0al. 2001; Tjandra and Bax 1997). The order parameters are usually normalized relative to S\u03c42.\nDynamics modulated alignment\nWe reason that the variation in molecular alignment originates from the dynamics-induced modulation of van der Waals and Coulomb interactions between the protein conformations and the nematogens (Louhivuori et\u00a0al. 2006). It may appear paradoxical that a transient conformation could align as the internal ps\u2013ns fluctuations are much faster than the molecular translational and rotational diffusion. This quandary is resolved as explained above in what is formally referred to as Boltzmann\u2019s ergodic hypothesis of the equivalence between time and ensemble averages. The suppression of some of the molecules internal fluctuations by the presence of the LC-particle imposes an unique alignment on each transient conformation. In other words, a transient conformation does not align per se but rather may only exist if it is energetically allowed. It is thus mistaken to justify the use of a single, effective alignment frame solely on the fact that internal fluctuations are much faster than translational or rotational diffusion.\nIt is clear then that dynamics modulated alignment (DMA) is a conceivable phenomenon and could very well be expressed in experimental data. We will therefore proceed to search for experimental evidence of it. Our analysis will be restricted to the ps\u2013ns protein dynamics that is well characterized by NMR relaxation measurements. On such short time scales primarily bonds fluctuate and side-chains reorientate while motions of secondary structure elements are negligible (Palmer 2004).\nSignature of dynamics modulated alignment\nOf the many motions captured by residual dipolar couplings, bond fluctuations and the overall molecular tumbling are well understood. These two contributions should be removed from the total order to expose any additional motion attributable to either DMA or additional intrinsic motion, e.g. in the form of secondary structure fluctuations, or to both. If the additional motion is an exclusive property of the protein, i.e. independent of the LC-medium that was used in measurements, then it is likely evidence of the reported sub-\u03bcs intrinsic secondary structure fluctuations. On the other hand, if a change in LC-media makes a difference then the excess motion may just as well result from the alignment frame dispersion in DMA. Obviously neither secondary structure nor alignment fluctuations are mutually exclusive and indeed it is quite conceivable that both contribute to the observed additional motions. The presence of DMA does not therefore disprove for example the recent findings (Lakomek et\u00a0al. 2005) of correlated backbone motions.\nAll order examined by RDCs is captured by an overall molecular measure called the general degree of order (GDO) (Tolman et\u00a0al. 2001). We employ its squared value,  The components \u03d1ij are obtained from the measured RDCs, e.g. by singular value decomposition (SVD) (Losonczi et\u00a0al. 1999), using a set of equationswhere the refined structural model is given by cicj, i.e. internuclear vectors in the Cartesian {x, y, z} frame. The constants for the two nuclei A and B are the gyromagnetic ratios \u03b3A and \u03b3B, the internuclear distance rAB and as usual Planck\u2019s constant h and the permeability of vacuum\u00a0\u03bco.\nWe aim to determine the ps\u2013ns fluctuations, i.e. the SLS2 contribution to \u03d12, on the basis of the intrinsic difference between amide (NH) and backbone (C\u03b1CO) bond fluctuations. This difference is not large, but it is significant (Ferrage et\u00a0al. 2006). We find from molecular dynamics (MD) simulations of ubiquitin and the B3 domain of protein G that the NH order parameter squared is on the average  and the corresponding C\u03b1CO value  when flexible tails and loops are excluded from the evaluation. These values are consistent with the average model-free order parameter SLS2 for NH obtained from laboratory frame relaxation measurements (Hall and Fushman 2003; Tjandra et\u00a0al. 1995). The fluctuations of C\u03b1CO bonds are being investigated by cross correlated relaxation measurements (Chang and Tjandra 2005; Ferrage et\u00a0al. 2006; Wang et\u00a0al. 2003).\nIt follows that when \u03d12 is determined using only the NH couplings the result should be slightly smaller than when only the C\u03b1CO couplings are used, provided that no other motions play a role. Conversely, any additional motion will partially mask the intrinsic bond fluctuations and perturb the difference  Asymmetric motions, i.e. motions that affect the two bonds unequally, will either increase or decrease this difference, or even keep it invariant, whereas symmetric motions will only decrease it. Any change in  that is due to the intrinsic secondary structure fluctuations, i.e. S\u03b1\u03b22, will be independent of the alignment medium. Any medium-dependent variation in  cannot arise from medium-independent intrinsic molecular motions and must be attributed either to DMA or to some unknown, novel medium-induced motion.\nIn order to remove contributions from S\u03c42 and such superfluous factors as LC-medium density and sample temperature that are present equally in \u03d1NH2 and  we normalize  by Furthermore we should note that the sample conditions of the data sets used in this study to illustrate DMA are so similar (cf. Clore and Schwieters 2004a, b; Cornilescu et\u00a0al. 1998; Tossavainen et\u00a0al. 2003; Ulmer et\u00a0al. 2003) that we cannot see how they could have any significant effect on the results to be presented. Alignment tensors are determined from RDC data e.g. by using SVD (Losonczi et\u00a0al. 1999). The \u03b4 parameter and its error are then obtained using Eq.\u00a02.\nAmount of intrinsic bond fluctuations\nBased on the MD simulation derived  and  values, the normalized difference between the average NH and C\u03b1CO bond fluctuations is without any additional motion  If the measured  differs substantially from the simulated value of 0.955, this value should be adjusted accordingly. It should also be noted that if the internal bond fluctuations were highly molecule dependent, it might be more sensible to consider \u03b4o separately for each molecule rather than the general \u03b4o shown here. Nevertheless, a \u03b4o parameter derived from a system outside the influence of any alignment medium serves to establish a numeric value that includes all intrinsic motions and nothing else. Any deviation from this baseline is then a sign of additional motions.\nLocal alignment frames\nIt may appear strange to use the general degree of order, a molecular quantity, as a motional probe because local order parameters are more informative. A locus-specific measure of RDC-probed dynamics is obtained by combining, via transformations, a non-redundant data set of at least five alignments (Tolman et\u00a0al. 2001). The obtained local GDOs provide a clear measure of residue-specific dynamics, but unfortunately the procedure would also mix up the eventual fluctuations in alignments\u2014the very effect we aim to address. To differentiate between internal and alignment fluctuations it only matters to observe whether \u03b4 varies from one medium to another. To this end \u03d12 is a robust measure that samples various bond directions. By taking care to account for alignment frame fluctuations and population differences between the conformations (Louhivuori et\u00a0al. 2006), one could arrive at more precise local order parameters.\nMethods\nThe conformational ensembles of GB1 (850 members), GB3 (150 members) and ubiquitin (2,400 members) were generated by molecular dynamics using the CHARMM22 parameter set (MacKerell et\u00a0al. 1998) with a modified backbone potential (MacKerell et\u00a0al. 2004) and generalized Born model (Im et\u00a0al. 2003). The proteins were first heated to 300\u00a0K during 200\u00a0ps and then equilibrated for 2\u00a0ns at this temperature before sampling for 48, 34 and 3\u00a0ns for ubiquitin, GB1 and GB3, respectively. Subsequently the alignment of each conformation was simulated using the PALES program (Zweckstetter 2006; Zweckstetter and Bax 2000; Zweckstetter et\u00a0al. 2004) in order to achieve the alignment dispersion present in a real sample. The alignments were predicted in the presence of a neutral planar obstacle as well as using positively and negatively charged bacteriophages carrying a surface charge density 0.047\u00a0e\/nm2 and 10\u00a0mM ionic strength of monovalent salt in pH\u00a06.5 with a LC concentration of 1\u00a0mg\/ml.\nThe refined structures 1P7F (Clore and Schwieters 2004a; Ulmer et\u00a0al. 2003), 1D3Z (Clore and Schwieters 2004b; Cornilescu et\u00a0al. 1998) and 1NYA (Tossavainen et\u00a0al. 2003) of the B3 domain of protein G, ubiquitin and calerythrin were used to analyse experimental RDC data available in various LC media (Table\u00a01). The temperature dependence of the experimental parameters were reported (Cornilescu et\u00a0al. 1998; Ulmer et\u00a0al. 2003) to be minimal in the temperature range used in the experiments.Table\u00a01\u03d12 and \u03b4 of GB3, ubiquitin and calerythrin in various alignment media. \u03d12 are shown in units of 10\u22126ProteinMediumT (K)GB3 (1P7F)aCTAB2.0732.3490.117 \u00b1\u00a00.006302PEG0.6140.7500.181 \u00b1 0.009304PAG+1.0791.2420.131 \u00b1 0.007298PAG\u22121.1131.3200.158 \u00b1 0.006298Pf11.3771.7310.205 \u00b1 0.005302UBI (1D3Z)bBicelles0.6950.7880.118 \u00b1 0.005304CTAB2.2692.6850.155 \u00b1 0.003304CAL (1NYA)cPf14.4514.7280.059 \u00b1 0.001318Experimental temperature is shown for an easy comparison with \u03b4 variationapH\u00a06.5, \u025bNH =\u00a0 0.26\u00a0Hz,  Pf1 11\u00a0mg\/ml, 100\u00a0mM NaClb\u025bNH =\u00a0 0.15\u00a0HzcpH\u00a06.0; Pf1 30\u00a0mg\/ml\nAn effective alignment tensor for each ensemble was calculated using SVD (Losonczi et\u00a0al. 1999). Either the experimental or average PALES predicted RDCs were used. Besides the refined structures (Table\u00a01) experimental RDCs were used also with the MD simulated\u2014PALES aligned structures in what we call the hybrid model (Table\u00a05). PALES predicted average RDCs were used in conjunction with the MD simulated\u2013PALES aligned structures (Table\u00a02).Table\u00a02 and \u03b4 parameters for simulated conformational ensembles of the B1 and B3 domain of protein G and human ubiquitin based on PALES-predicted average RDCsProteinMedium (\u00b0) (\u00b0)\u03b4direct\u03b4SVDGB1Negative31.7 \u00b1 0.231.1 \u00b1 0.20.172\u00a0\u00b1 0.0070.109 \u00b1 0.008Neutral11.82 \u00b1 0.047.38 \u00b1 0.020.114 \u00b1\u00a00.0010.116 \u00b1 0.008Positive40.36 \u00b1 0.0536.23 \u00b1 0.030.385 \u00b1\u00a00.0040.208 \u00b1 0.002GB3Negative12.60 \u00b1 0.109.54 \u00b1 0.060.082 \u00b1\u00a00.0030.097 \u00b1 0.009Neutral11.82 \u00b1 0.097.75 \u00b1 0.050.096 \u00b1\u00a00.0030.100 \u00b1 0.009Positive23.0 \u00b1 0.420.6 \u00b1 0.40.119 \u00b1\u00a00.0100.107 \u00b1 0.007UBINegative13.21 \u00b1 0.049.87 \u00b1 0.020.090 \u00b1\u00a00.0010.106 \u00b1 0.003Neutral16.95 \u00b1 0.0913.16 \u00b1 0.080.080 \u00b1\u00a00.0020.085 \u00b1 0.005Positive15.84 \u00b1 0.0612.86 \u00b1 0.050.092 \u00b1\u00a00.0020.104 \u00b1 0.007\nResults and discussion\nAnalysis of experimental data\nThe wealth of high-quality residual dipolar coupling data of the B3 domain of protein G (GB3) and ubiquitin (UBI) and their refined structures (1P7F and 1D3Z, respectively) allow us to determine GDOs and to calculate \u03b4 values for several cases (Table\u00a01). The data for calerythrin (1NYA), a significantly larger protein (\u223c20\u00a0kDa v. less than 10\u00a0kDa), is also available (Tossavainen et\u00a0al. 2003). The quality of the refined structures in terms of an RMSD measure, referred to as the quality factor (Clore and Garrett 1999), is in all these cases good. Accordingly, in the evaluation of \u03b4 we employ only the NH and C\u03b1CO couplings that were used to refine the GB3, ubiquitin and calerythrin structures. The GDO values were calculated from the experimental dipolar coupling data using a SVD algorithm against the average structures of the refined ensembles.\nThe two most important observations are that  and that \u03b4 varies from one medium to another. Therefore it seems that there are motions in addition to bond fluctuations and we attribute them, owing to their medium-dependency, at least partly to dynamics modulated alignment.\nThe results are subject to the following uncertainties. The errors in \u03b4 were obtained by a Monte-Carlo (MC) method from the experimental errors in RDCs. The largest ambiguity originates from the ratio between the NH and C\u03b1CO bond length. We used rNH\u00a0=\u00a01.02\u00a0\u00c5 (Jeffrey 1992, p. 270) and  (Engh and Huber 1991; Lehmann et\u00a0al. 1972). An eventual error in the bond length ratio is systematic and affects the absolute \u03b4 value, but it does not abolish the variation in \u03b4 from one medium to another. It should be noted that customarily when a structure is refined against RDCs rNH is set to 1.04\u00a0\u00c5 (Ottiger and Bax 1998) to obtain the average directions of the more mobile amides consistent with less mobile bonds, e.g. those of the C\u03b1CO vectors. In other words, it is desirable to obtain the same magnitude of alignment tensor irrespective of the couplings employed. Further uncertainty stems from the discrete sampling of directions by the non-uniform and dissimilar distributions of NH and C\u03b1CO bonds, but as will be shown later on it does not seem to matter that much. Furthermore, as mentioned above, no motional effects including the consequences of discrete non-uniform sampling can be seen in \u03b4 when the excess fluctuations are small compared to SLS2. Large alignment fluctuations on the other hand lead categorically to nearly uniform sampling of directions.\nSimple model of wobble\nA simple approximation of internal bond motions considers fluctuations about the mean direction only as axial, i.e. including just the zero-order spherical harmonics Y00.Assuming that the internal bond (\u03b8) and alignment () fluctuations are independent and on different timescales, they can be separated and treated independently (Brainard and Szabo 1981; Petersen and Chan 1977). If an isotropic sampling of bond directions is also assumed, then the combined effect of the fluctuations will be  It would then be clear from Eq.\u00a02 that \u03b4 values remain invariant even in the presence of alignment fluctuations.\nHowever, it is evident from Table\u00a01 that the \u03b4 parameter varies from one medium to another implying a coupling between the alignment fluctuations and \u03b4. It is therefore reasoned that  cannot be factorised. This is not a contradiction since neither of the previously mentioned assumptions are entirely valid in this case. (i) The two fluctuations are not completely independent since there is a slight coupling between the alignment and the ps\u2013ns bond motions (Louhivuori et\u00a0al. 2006). (ii) Since alignment fluctuations are not a real motion, but rather a manifestation of the state space available in the system, alignment fluctuations can be said to affect all time-scales.\nTherefore we propose the following simple ad hoc model to describe the combined effect of internal bond motions and alignment frame fluctuations.Essentially this means that we are considering a model where a molecule\u2019s wobble about an average orientation amplifies the effect of internal bond motions. As a consequence the effective opening angle of internal bond motions is increased from the initial value of \u03b8o by \nIn the absence of alignment fluctuations () \u03b8o can be calculated using Eq.\u00a05 from the GDO values determined for the MD simulated ensembles.Accordingly, \u03b8o for NH and C\u03b1CO were calculated from the previously mentioned MD-derived  and  and found to be 11.82\u00b0 and 9.99\u00b0, respectively. Then  and  were calculated using Eq.\u00a04 and plotted as functions of increasing  (Fig.\u00a0 2). Finally, \u03b4 versus  was calculated from  and  using Eq.\u00a02.Fig.\u00a02Calculated effective order parameters  for the NH (dotted) and C\u03b1CO (dashed) bonds and the \u03b4 parameter calculated from them as the molecule is subject to a fluctuating alignment of increasing angle  in addition to internal bond fluctuations\nWhen the internal bond and alignment fluctuations combined reach the magic angle the alignment is lost all together. The normalized difference in the NH and C\u03b1CO probed order, denoted by \u03b4, is available from measurements and shown in Fig.\u00a02 for the B3 domain of protein G in CTAB doped bicelles, positively and negatively charged stretched polyacrylamide gels, polyethylene-glycol lamella and filamentous phages, and for ubiquitin in bicelles and CTAB doped bicelles. The \u03b4 value of calerythrin was smaller than the proposed general minimum of 0.081 and is therefore omitted. The symbols in the figure are larger than the errors obtained by MC analysis. It needs to be stressed that  should not be interpreted as a real measure of molecular motion since alignment fluctuations are only an ensemble property affecting RDC data.\nWhen the experimental \u03b4 values are placed on the (\u03b4, \u03a9)-curve we see that for both GB3 and ubiquitin in bicelle medium the effect of DMA is small enough to be comparable to that of bond fluctuations. Non-axial bond fluctuations will naturally complicate the situation and might invalidate such a comparison. The precision in \u03b4 is modest owing to the functional form of the -curve. In other media, particularly in those carrying surface charges on the nematogen, the alignments fluctuate noticeably. For example in filamentous phages the alignment of GB3 fluctuates even more than 13\u00b0. Thus \u03d12 may fall even below 0.7. However, this does not mean that the molecule is somehow reorienting itself with such large-amplitude motions. It only tells that quite likely there are more than one major alignment modes separated from each other sufficiently to give the impression of large-scale motions if the alignment fluctuations are interpreted simply as real, molecular fluctuations around an average direction as was done in Eq.\u00a04. Nonetheless, the medium-dependent variation in \u03b4 shows that DMA contributes to RDC-probed dynamics.\nAnalysis of simulation data\nThe sources of the medium-dependent variation in \u03b4 found from the experimental data can be many. Fortunately, they can be assessed via computer simulations. We consider the variation in the protein shape and charge distribution due to the ps\u2013ns dynamics. These fluctuations give rise to a family of conformations whose members do not all align in exactly the same way. Simultaneous variation in the shape and net electric dipole moment may lead to a substantial alignment fluctuation. However, it is also conceivable that dynamics of the nematogen contributes to the protein alignment dispersion. Other microscopic heterogeneity in obstructing nematogens may also add to the variation of protein alignments but we are unequipped to examine these effects. Neither are the possible effects of hydration fully explored in this study.\nThe average NH and C\u03b1CO RDCs of the simulated conformational ensembles were calculated and the GDO values were determined using SVD against the average structure of the ensemble. The average of the cosine matrix cicj of Eq.\u00a01 was taken to present the best possible average structure. In this way we aimed to mimic the experimental procedure where GDOs were determined from the measured couplings against the refined structure. Alternatively, the knowledge of all NH and C\u03b1CO vectors of the dispersed ensembles allowed us to calculate directly  of Eq. 3 and hence also the order parameters and \u03b4 for the various cases. The two methods, direct and SVD, give similar \u03b4 values, with only GB1 in positive alignment medium showing a larger discrepancy (Table\u00a02). Since both exhibit similar overall tendencies the methods can be assumed to be qualitatively consistent.\nThe alignment simulations in the neutral, positively and negatively charged media were not intended to match specifically any of the experimental conditions, but nevertheless the values are somewhat comparable. The simulated \u03b4 values are more uniform, but comparable in size (Table\u00a02). Only the B1 domain of protein G seems to be significantly susceptible to DMA. The lack of dispersion in \u03b4 was unexpected in light of the clear dispersion found from experimental data (Table\u00a01). Because of this and since the limited sampling of GB3 leaves a shadow of doubt on the exact values reported in Table\u00a02, further work was done to understand the source for the lack of dispersion in \u03b4. First, non-uniform sampling of bond directions was addressed. Second, the possible increase in PALES-predicted alignment dispersion due to hydration was considered. Third, a hybrid model was used to circumvent any shortcomings in PALES-predicted RDCs.\nNon-uniform bond direction sampling\nThe finite sampling of bond directions may influence the \u03b4 parameter. Therefore, we calculated the fraction of vectors oriented along the three principal directions as suggested by Fushman et\u00a0al. (2000). The fractions fi are a convenient measure of the uniformity of the bond direction sampling. In the case of uniform sampling  whereas a significant deviation from 1\/3 is a sign of non-uniform sampling. The Fushman-fractions calculated from the MD ensembles show gratifyingly that the sampling is rather uniform (Table\u00a03).Table\u00a03The fraction of vectors oriented along the three principal axis calculated for the MD simulated ensembles of GB1, GB3 and ubiquitinProteinBondfzfyfxGB1NH0.540.260.20C\u03b1CO0.450.290.26GB3NH0.550.260.19C\u03b1CO0.450.290.26UBINH0.490.310.21C\u03b1CO0.370.340.29\nNevertheless, the possible effects of discrete, non-uniform sampling of bond directions was tested using the MD ensembles. The C\u03b1CO bonds of every conformation were first rotated one residue at a time to point, on average, at the same direction as the NH bonds did, on average. A new set of RDCs was then calculated using the twisted structures and the original Saupe matrices. Singular value decomposition was then used to obtain an effective GDO value using the twisted structures and the new average RDCs. From the NH and C\u03b1CO GDO values the \u03b4 parameter was calculated using Eq.\u00a02. As can be seen from Table\u00a0 4, \u03b4 parameters changed, but mostly within the margins of error. Both GB1 in positively charged medium and ubiquitin in neutral medium experience a sharp drop in \u03b4. It seems that this is due to the fact that the C\u03b1CO bonds rotated pre-dominantly closer to the magic angle diminishing C\u03b1CO couplings sufficiently to render them much more labile in the face of the applied MC error. However, non-uniform bond direction sampling does not seem to influence \u03b4 significantly in most cases.Table\u00a04Alignment fluctuations in the MD ensemble before (\u03b4SVD) and after (\u03b4\u2032SVD) C\u03b1CO bonds were superimposed on NH bondsProteinMedium\u03b4SVD\u03b4\u2032SVDGB1Negative0.1090.113 \u00b1 0.008Neutral0.1160.116 \u00b1 0.007Positive0.2080.130 \u00b1 0.008GB3Negative0.0970.116 \u00b1 0.006Neutral0.1000.106 \u00b1 0.006Positive0.1070.113 \u00b1 0.006UBINegative0.1060.109 \u00b1 0.003Neutral0.0850.018 \u00b1 0.008Positive0.1040.092 \u00b1 0.007Table\u00a05Hybrid modelProteinMedium\u03b4\u03b4hybridGB3CTAB0.1170.133 \u00b1 0.005Pf10.2050.265 \u00b1 0.004UBIBicelles0.1180.093 \u00b1 0.008CTAB0.1550.139 \u00b1 0.006Alignment fluctuations in simulated conformational ensembles of the B3 domain of protein G and human ubiquitin based on experimental RDCs (\u03b4hybrid) compared to those in the refined, experimental structures (\u03b4)\nHydration shell\nSince the MD simulated conformational ensembles seemed to be less susceptible to DMA than NMR-based ensembles, it was plausible that PALES failed to impose a realistic dispersion of alignments. We hypothesised that the addition of a hydration shell to the molecules would make them more globular and therefore more susceptible to alignment dispersion due to steric interactions. To test whether this hypothesis could account for the observed lack of dispersion, a hydration layer needed to be added to the molecules. This was done with a rough model of the hydration shell in the form of increased atomic radii in PALES. Atomic radii were increased by 1, 2, 3, and 4\u00a0\u00c5, but no increase in DMA was observed neither for GB1 nor for ubiquitin. The lack of a hydration shell does not therefore seem to be the cause for the difference in DMA between simulations and experiments.\nHybrid model\nAnother possibility was that PALES dispersed the alignments sufficiently, but that the predicted RDCs had some issues that were reflected in the effective alignment and ultimately in \u03b4. To overcome this, we calculated the effective alignments of the MD ensembles using experimental RDCs instead of PALES-predicted ones. This was done for GB3 in Pf1 and CTAB-doped bicelle media and for ubiquitin in bicelle media with and without CTAB. As can be seen from Table\u00a05 and Fig.\u00a03 this seems to be the major cause for the lack of \u03b4-dispersion. What this suggests is that when experimental RDCs are available DMA can be estimated consistently from a MD-simulated conformational ensemble even without structures refined against experimental RDCs.Fig.\u00a03Hybrid model. Calculated effective order parameters  for the NH (dotted) and C\u03b1CO (dashed) bonds and the \u03b4 parameter calculated from them as the MD simulated conformations are subject to a fluctuating alignment of increasing angle  in addition to internal bond fluctuations\nCompetition of interactions\nIn the neutral medium only the molecular shape fluctuations contribute and DMA is comparable to the internal bond vector fluctuations. Neither in the simulation nor in the experimental data is there a lot of difference between the smaller molecules GB3 and ubiquitin. Calerythrin resembles more an oblate ellipsoid (Fig.\u00a04) and is expected to fluctuate less than the smaller, globular proteins in the absence of electrostatic interactions.Fig.\u00a04Causes of molecular alignment fluctuations for (a) GB1, (b) GB3, (c) ubiquitin and (d) calerythrin illustrated for example conformations. The variation in the molecular shape and charge distribution are simplified by showing the molecular principle axes (grey) and net electric dipole moments (orange)\nIt is more intricate to rationalize the reduced order parameters in a charged media as both molecular shape and charge distribution fluctuations are present. It may not be possible to simplify the dynamics of a complex set of interactions to provide lucid understanding to the causes of DMA. To begin with, GB1, GB3, ubiquitin and calerythrin carry under experimental conditions a net charge of \u22121.22, \u22123.09, +1.39 and \u22128.45, respectively. In the repulsive electrostatic potential the alignment is, to a crude approximation, governed by the direction and size of the net electric dipole moment in relation to the molecular shape tensor.\nA competition between the steric and electrostatic alignment arises when the dipole moment is mostly along the long axis of the molecule and comparatively small. GB1 is a good example of this (Fig.\u00a04). In GB1 dipole moments are pointing roughly to the middle of the long and short axis in two slightly separated groups. The situation is similar in GB3, but with the dipole moments concentrated in a single, tight group. Furthermore, the dipole moments in GB3 are larger than in GB1 and dominate the alignment. In the globular ubiquitin the dipole moments are directed along the shortest of the molecular axes. Thus both the shape and electrostatics act in synergy to narrow the cone of alignments. In spite of this the globular shape of ubiquitin ensures that modest alignment fluctuations remain in charged media. In the refined structures of calerythrin the dipole moments are spread out slightly more than in the MD simulated ensemble of GB1 and directed similarly roughly to the middle of the long and short axis. Alignment fluctuations in calerythrin are small (Table\u00a01), even though the combination of an oblate ellipsoidal shape with dipole moments directed away from the short axis suggest a competition between the steric and electrostatic alignments. Calerythrin contains a flexible hinge-region that has a slightly ambiguous structure in 1NYA (Tossavainen et\u00a0al. 2003). It is thus possible that the lack of restraints in the structural refinement has led to somewhat more compact structures with slightly distorted net dipole moments. Analysis of an attractive potential is more involved and not easily reduced to the analysis of the net electric dipole moment relative to the shape tensor. The variation in the position of the net charge influences the alignment that can fluctuate substantially as exemplified by the aforementioned \u03b4 values for GB3.\nIn general a small globular protein that carries many charges and yet only a small net electric dipole moment is the most vulnerable to molecular alignment fluctuations. The shape as well as the charge distribution is perturbed by dynamics of long side-chains at the molecular surface. For large proteins the effects are expected to have less impact on the overall molecular alignment.\nConclusions\nInternal motions are, at least to a good approximation, medium-independent. Therefore one would expect that e.g. relative differences between NH and C\u03b1CO bond fluctuations, denoted by the \u03b4 parameter, would remain the same from one medium to another. However, we find that this is not the case for protein G and ubiquitin. The changes in \u03b4 from one medium to another imply the presence of an additional medium-dependent ensemble property involved in the alignment phenomenon.\nThe simulations suggest that the effective alignment of a molecule fluctuates as a response to the variation in molecular shape and charge distribution caused by protein dynamics. This alignment modulation is at its highest when steric and electrostatic interactions compete for the dominance in a small globular protein. Also flexible loops and termini may cause a fluctuating alignment. The alignment fluctuation as a phenomenon is known and e.g. used to point out domain\u2013domain motions in modular proteins (Bewley and Clore 2000; Fischer et\u00a0al. 1999) and flexibility of polypeptides (Chou et\u00a0al. 2001; Goto et\u00a0al. 2001).\nHere we were able to consider only alignment effects due to protein dynamics that occur in the ps\u2013ns range owing to the limitations of molecular dynamics simulations. Other states accessible via slower motions are of course also expected to contribute to the variation in molecular alignment depending on their population, shapes and charge distributions. We also expect that dynamics of nematogens and other variation in the media contribute as well to the bond vector dispersion underlying the observed RDC signals.\nIt is perhaps perplexing that molecular alignment fluctuations do not show up as clear inconsistencies in refinement of protein structures in particular when a number of RDCs obtained from measurements in various media are used simultaneously. It should be emphasized that the dynamics modulated alignment is a process that is nearly uncorrelated from the intrinsic vector fluctuations. Consequently it will, to an excellent approximation, only scale RDCs but leave their directional information content, used in the structure determination, intact. Thus the averaging due to DMA appears much as the averaging due to the molecular tumbling. Even a substantial wobble in the alignment can be compensated by increasing the strength of LC-medium to acquire large enough couplings for quantitative analyses. It should be stressed that the presence of DMA does not, for example, invalidate the recent finding (Bernad\u00f3 et\u00a0al. 2005) of long-range interactions in \u03b1-Synuclein nor the previously mentioned supra-\u03c4c backbone motions, but gives a rationale for the low S2 values. Perhaps indicative of fluctuating alignments are the challenges in predicting precisely RDC-data on the basis of a single structure.\nIn studies of dynamics where the dispersion of a vector direction rather than its average direction is of interest we expect that the alignment fluctuations are worth to consider as they will inevitably incorporate into the RDC-based order parameter. In this study the evidence of molecular alignment fluctuation was established using the average molecular parameter, general degree of order. Consequently the site-specific variation in the order and its correlation to the secondary structure that have been reported in the RDC-probed dynamics studies was not addressed. It is conceivable that an alignment fluctuation could result in some site-specific variation in \u03d12 depending on the direction of the bonds, but unlikely in any correlated variations.","keyphrases":["protein dynamics","residual dipolar couplings","liquid crystal nmr spectroscopy"],"prmu":["P","P","M"]}
{"id":"J_Urban_Health-2-2-1705515","title":"Variance Estimation, Design Effects, and Sample Size Calculations for Respondent-Driven Sampling\n","text":"Hidden populations, such as injection drug users and sex workers, are central to a number of public health problems. However, because of the nature of these groups, it is difficult to collect accurate information about them, and this difficulty complicates disease prevention efforts. A recently developed statistical approach called respondent-driven sampling improves our ability to study hidden populations by allowing researchers to make unbiased estimates of the prevalence of certain traits in these populations. Yet, not enough is known about the sample-to-sample variability of these prevalence estimates. In this paper, we present a bootstrap method for constructing confidence intervals around respondent-driven sampling estimates and demonstrate in simulations that it outperforms the naive method currently in use. We also use simulations and real data to estimate the design effects for respondent-driven sampling in a number of situations. We conclude with practical advice about the power calculations that are needed to determine the appropriate sample size for a study using respondent-driven sampling. In general, we recommend a sample size twice as large as would be needed under simple random sampling.\nIntroduction\nTo understand and control the spread of HIV, it is important to have accurate information about hidden populations such as injection drug users and sex workers.1 However, these populations are difficult to study with standard sampling methods because sampling frames do not exist. The need to gather information about such hidden populations is not limited to public health. Social scientists and policy-makers are interested in many other hidden populations such as undocumented immigrants, artists, and members of some social movements.\nIn response to the problem of studying hidden populations, a new statistical approach called respondent-driven sampling has been developed.2\u20134 Respondent-driven sampling data are collected via a link-tracing (snowball) design, where current sample members recruit future sample members. For many years, researchers thought it was impossible to make unbiased estimates from this type of sample. However, it was recently shown that if certain conditions are met and if the appropriate procedures are used, then the prevalence estimates from respondent-driven sampling are asymptotically unbiased.4 For example, respondent-driven sampling can be used to estimate the prevalence of HIV among drug injectors in New York City.\nDespite the progress that has been made in making prevalence estimates, less is known about the sample-to-sample variability of these estimates. This gap in knowledge can lead researchers to construct inaccurate confidence intervals around estimates and to undertake studies with sample sizes that are too small to meet study goals. Filling this important gap in the respondent-driven sampling literature, this paper explores issues related to the sample-to-sample variability of estimates. The paper consists of four main parts. First, we briefly review the existing respondent-driven sampling methodology. Next, we develop and evaluate a bootstrap procedure for constructing confidence intervals around respondent-driven sampling estimates. Then, we estimate the design effect of the prevalence estimates in a number of simulated and real populations. The paper concludes with advice about the sample sizes needed for studies using respondent-driven sampling. In general, we recommend a sample size twice as large as would be needed under simple random sampling.\nReview of Respondent-driven Sampling\nA respondent-driven sample is collected with a link-tracing design, similar to a snowball sample.5\u20137 The sampling process begins with the selection of a set people in the target population who serve as seeds. After participating in the study, these seeds are each provided with a fixed number of unique recruitment coupons, which they use to recruit other people they know in the target population. After participating in the study, these new sample members are also provided with recruitment coupons, which they then use to recruit others. The sampling continues in this way, with subjects recruiting more subjects, until the desired sample size is reached.2\u20134 Experience has shown that this sample selection method is practical and it has already been used to study a number of different hidden populations, including jazz musicians,8 drug injectors,2 Latino gay men,9 and MDMA\/Ecstasy users.10\nIn addition to collecting information that addresses the substantive focus of the study, a researcher using respondent-driven sampling must also collect two other pieces of information: the recruiter of each sample member and the number of relationships that each sample member has in the population of interest (sometime called degree of each person). This additional information is necessary because the estimation procedure is a variation of the traditional sampling and estimation process. Ordinarily, researchers take a sample and then use that sample to make inferences about a population (Figure\u00a01a). However, when the data are collected with respondent-driven sampling, it is very difficult (and maybe even impossible) to make inferences from the sample directly to the population. Fortunately, such data can be used to make inferences about the social network connecting the hidden population, and this information about the social network can be used to estimate the prevalence a specific trait (Figure\u00a01b).3 Within this new framework, unbiased estimation becomes possible.1 A more detail description of the estimation procedure and the conditions under which it is unbiased is available in the literature.4Figure\u00a01Schematic of both traditional sampling and estimation and respondent-driven sampling. By not attempting to estimate directly from the sample to the population, respondent-driven sampling avoids many of the well-known problems with estimation from snowball samples.\nWhile the ability to make unbiased prevalence estimates represented a step forward for the study of hidden populations, it was an incomplete one. In order for respondent-driven sampling to be practical as a methodology, a procedure is needed in order to put confidence intervals around these prevalence estimates.\nConfidence Intervals\nBefore introducing the confidence interval procedure, we first need to introduce some language with which to describe the hidden population. In this paper we will consider the situation of a hidden population that is made up of two mutually exclusive and collectively exhaustive groups that, for the sake of generality, we will call group A and group B. The groups could be, for example, people with and without HIV. The proportion of the population in group A will be called PA. A point estimate of this prevalence is useful, but it is difficult to interpret without some measure of the precision of the estimate. One common way of describing this precision is with a confidence interval that provides a range within which the researcher expects to find the true population value with some level of certainty. Procedures to generate confidence intervals are well developed in the case of simple random sampling,12,13 but researchers using a complex sample design, where not all units have the same probability of selection, are often left without guidance. Despite numerous warnings,7,14 researchers often ignore the fact that their data were collected with a complex sample design and construct confidence intervals as if they had a random sample. This approach of ignoring the sampling design, which we will call the naive method, will generally cause researchers using respondent-driven sampling to produce confidence intervals that are too small. These incorrect confidence intervals are not just a technical concern; incorrect confidence intervals can lead to incorrect substantive conclusions.\nIn order to produce better confidence intervals, we will develop and evaluate a bootstrap method specifically designed for respondent-driven sampling.2 Although an analytic approach would be preferable,3 bootstrap methods are commonly used for variance estimation from complex sample designs because analytic solutions are often not possible.16,17 In the next sections, we will describe our proposed bootstrap procedure and then evaluate its performance using computer simulations.\nProposed Bootstrap Procedure\nThe general idea of the bootstrap procedure is to use the observed sample to generate a set of replicate samples. Then this set of replicate samples is used to produce a set of replicate estimates. By examining the variation in these replicate estimates, one can construct a confidence interval around the original point estimate. This three-step process can be seen in Figure\u00a02.17Figure\u00a02Schematic of the procedure for producing confidence intervals.\nThe first step in our procedure is the resampling step. In traditional bootstrapping, this resampling is done by randomly sampling with replacement from the original sample until the replicate sample is the same size as the original sample. This resampling procedure is well grounded theoretically for the case where the original sample is collected via simple random sampling.17 However, as described previously, in respondent-driven sampling there are dependencies in the sample selection process, and so we must use a modified resampling procedure which mimics these features. The modification of the resampling step is the main way that this approach deviates from traditional bootstrapping techniques.\nUnder our proposed procedure we divide the sample members into two sets based on how they were recruited: people recruited by someone in group A (which we will call Arec) and people recruited by someone in group B (which we will call Brec). For example, Arec could be the set of all sample members who were recruited by someone with HIV. Note that this set could include both people with and without HIV. In order to mimic the actual sampling process, the resampling begins when a seed is chosen with uniform probability from the entire sample. Then, based on the group membership of the seed, we draw with replacement from either Arec or Brec. For example, if the seed chosen for the replicate sample was a sample member with HIV, we draw from the set of sample members who were recruited by someone with HIV. Next, we examine the group membership of this newly chosen person and then draw again with replacement from either Arec or Brec.4 This process continues until the bootstrap sample is the same size as the original sample. Overall, this resampling scheme preserves some, but not all, of the dependencies that exist in the respondent-driven sampling data collection.5\nOnce the bootstrap samples are selected, we move to step 2 in Figure\u00a02: the estimation step. Here we use the normal respondent-driven sampling estimation procedure on each of the R replicate samples to produce a set of R replicate estimates. Finally, in step 3 of the bootstrap procedure, the R replicate estimates are converted into a confidence interval. One way to do this would be to construct a 90% confidence interval based on the normal approximation,\nwhere the estimated standard error, , is the standard deviation of the replicate estimates. While this approach is reasonable, it has two main disadvantages. First, it forces the confidence intervals to be symmetric, which can reduce accuracy, and second, it can produce intervals with endpoints outside of the range [0, 1].\nFortunately, there are several improvements over this standard error method, and in this paper we will use the percentile method.6 When using the percentile method, we define the endpoints of the 90% confidence interval to be the two replicate estimates, such that 5% of the replicate estimates fall below the interval, and 5% of the replicate estimates fall above the interval. For example, if a researcher generated 2,000 bootstrap replicates, a 90% confidence interval would be defined by the 100 and 1,900 ordered replicate estimates. As we shall see in the next section, the proposed resampling scheme combined with the percentile method produces confidence intervals that are generally good in an absolute sense and better than the naive method.7\nComparing the Naive and Bootstrap Methods\nThe quality of a confidence interval procedure can be measured by calculating \u03c6, the percentage of proposed intervals that contain the true population value. For example, if we took 1,000 samples from the population and produced a 90% confidence interval from each of these samples, then 900 out of 1,000 of these confidence intervals should include the true population prevalence.8 Unfortunately, due to resource constraints, we cannot repeatedly sample from real hidden populations. However, using computer simulations, we can construct hypothetical hidden populations and then repeatedly sample from them to evaluate the coverage properties of the different confidence interval procedures. Further, in these computer simulations we can systematically vary the characteristics of the hidden population in order to understand the effects of population and network characteristics on the quality of the proposed confidence intervals.\nFor example, to explore how network structure affects the quality of the confidence intervals, we constructed a series of hypothetical populations that were identical except for the amount of interconnectedness between the two groups. More specifically, we varied the ratio of the actual number of cross-group relationships to the number of possible cross-group relationship, and thus, our measure of interconnectedness, I, can vary from 0 (no connections between the groups) to 1 (maximal interconnection). All populations were constructed with 10,000 people, 30% of which were assigned a specific trait, for example HIV. Next, we began to construct the social network in the population by giving each person a number of relationships with other people in the population. The number of relationships that an individual has is called her degree. When assigning an individual\u2019s degree we wanted to roughly match data collected in studies of drug injectors in Connecticut,2 so each person with HIV was assigned a degree drawn randomly from an exponential distribution with mean 20, and those without HIV were assigned a degree drawn from an exponential distribution with mean 10; later in this paper we will explore other degree distributions. Once the degrees were assigned, we insured that the population had the appropriate amount of interconnection between the groups.9\nAfter each population was constructed, we took 1,000 samples of size 500, and for each of these 1,000 samples we constructed a confidence interval using both the naive method (i.e., ignoring the complex sample design and pretending to have a simple random sample) and the proposed bootstrap method. By seeing if each of these confidence intervals included the true population prevalence, we calculated  and . The results of these simulations are presented in Figure\u00a03 and reveal two important features. First, the figure shows that, for the populations used in these simulations, the proposed bootstrap procedure outperforms the naive procedure. Second, it shows that the bootstrap procedure also performs well in an absolute sense, meaning .\nFigure\u00a03Coverage probabilities of the naive and bootstrap procedure. Results indicate that the proposed bootstrap procedure outperforms the naive procedure and performs well in an absolute sense.\nTo test the robustness of these findings, we explored the coverage properties in a larger portion of the possible parameter space by varying the sample size, the proportion of the population in the groups, and the average degree of the groups (results not shown). To summarize these findings, in a few unusual portions of the parameter space, the proposed bootstrap procedure did not perform well in an absolute sense, but in most portions of the parameter space, the proposed procedure performed well.10 Additionally, in all cases the proposed bootstrap procedure outperformed the naive procedure. To conclude, in the situations that we have examined, the proposed bootstrap procedure works well in an absolute sense and better than the naive procedure. Further, these results seem robust. Therefore, until some superior procedure is developed, we recommend this bootstrap procedure for future researchers who wish to construct confidence intervals around prevalence estimates from respondent-driven sampling.\nDesign Effects\nEven though respondent-driven sampling produces unbiased prevalence estimates and allows researchers to produce approximate confidence intervals, respondent-driven sampling may not be appropriate for all situations. The unbiasedness of the estimates does not ensure that any particular estimate will be equal to the true population value. Rather, just like all other unbiased estimators, sometimes the estimate will be too low or too high; only on average will the estimate equal the true population value. If the respondent-driven sampling estimates are too variable, then, even if they are unbiased, they might not be useful in practice. For example, we constructed a population with 30% prevalence of a specific trait, for example HIV, and repeatedly sampled from that population using respondent-driven sampling. To provide a benchmark for interpreting the sample-to-sample variability, we also repeatedly sampled from the population using simple random sampling (even though this would not be possible in a real hidden population). Figure\u00a04 shows the distribution of prevalence estimates from these two methods. Both distributions center around 0.30, the true value, so both sampling methods produced unbiased estimates. But, the distribution of respondent-driven sampling estimates was more variable than the distribution of estimates under simple random sampling. A common way to quantify this difference is the design effect,12 which measures the increased variation of the estimates under respondent-driven sampling.11 That is,\nwhere  is the variance of under respondent-driven sampling and  is the variance of under simple random sampling. Because respondent-driven sampling generally provides less information, we expect that the design effect will generally, but not always, be greater than 1. For example, the results in Figure\u00a04 show a design effect of 2.8. In the next section we will explore the design effects for a range of different network structures to better understand the situations in which respondent-driven sampling is likely to be precise enough to be useful.\nFigure\u00a04Distribution of estimates from respondent-driven sampling and simple random sampling. The respondent-driven sampling estimates center around the true population value, 0.30, but they are more variable than the estimates from simple random sampling. This extra variation corresponds to a design effect of 2.8.\nSimulation Results on Design Effects\nEstimating the design effect requires comparing the variance of the prevalence estimate under different sampling methods. While the variability of the prevalence estimate under simple random sampling can be derived from basic probability theory,12 we must use simulations to discover the variability under respondent-driven sampling. Thus, as when we evaluated the confidence interval procedure, we constructed a range of populations and simulated repeated sampling from them. We observed several general patterns that seem to occur in all portions of the parameter space. First, generally, but not always, the design effects from respondent-driven sampling were greater than 1, which indicates that respondent-driven sampling estimates were less precise than estimates from simple random sampling. This finding is consistent with the literature on complex sampling designs, which generally finds that departures from simple random sampling lead to increased variability of estimates. Second, as the interconnectedness, I, increased, that is, as the two groups became more closely connected, the design effect decreased (see Figure\u00a05). Third, the minimum design effect for a given interconnectedness occurred not when the two groups had the same average degree (DA = DB), but when the two groups had the same total degree, (PADA = PBDB) (see Figure\u00a06). Fourth, the design effects were sensitive to the degree distribution assumed in the simulations. Previously in this paper we assumed an exponential degree distribution, but for specific subpopulations, such as drug injectors, the true functional form of the degree distribution is unknown. When we assigned a Poisson degree distribution for both groups, we observed much lower design effects, including some design effects below 1 (Figure\u00a07); the reason for this change is currently unknown.12 Overall, these observations should be viewed with some caution because they have not been verified analytically due to the previously mentioned inability to develop closed-form expressions for the variance of the prevalence estimate under respondent-driven sampling.\nFigure\u00a05Design effect as a function of interconnectedness I. In general, as the interconnectedness increases the design effect decreases. Results are based on 10,000 replicate samples.Figure\u00a06Design effect as a function of DA for different DB. In general, the minimum design effect, for a given interconnectedness, occurs when the two groups have the same total degree (PADA = PBDB). So if PA = 0.3 and PB = 0.7, then when DB = 10 the minimum design effect occurs when DA \u2248 23 and when DB = 20 the minimum occurs when DA \u2248 46. Results are based on 10,000 replicate samples.Figure\u00a07Design effect as a function of interconnectedness for different degree distributions. In general, the design effects are smaller when the degree distribution of the groups is Poisson rather than exponential. Results are based on 10,000 replicate samples.\nTaken together, these simulation results suggest that the design effect is a complex function of the network structure in the population.13 The simulation results also suggest that in some cases respondent-driven sampling can be quite blunt, with design effects as large as 10, but that in other cases it can be extremely precise, sometimes even more precise than simple random sampling.\nEstimated Design Effects in Real Studies\nThe simulation results indicate that a range of design effects are possible. Therefore, an important question becomes: What are the design effects in populations that people actually study? Our best attempt to answer that question is presented in Table\u00a01, where we report the estimated design effects from all studies that are currently available.14 To produce the estimated design effects we took the published estimates of PA and used them to estimate the variability of the prevalence estimates (). This variability is then compared to the published estimates of the variability under respondent-driven sampling ().15 We report only one design effect because, due to the symmetry of the two-group system, .\nTable\u00a01Estimated design effects from real respondent-driven sampling data.8\u201310 Sample sizes vary within the same study due to missing dataStudy descriptionStudy resultsPopulationLocationnTraitLatino gay menChicago69HIV+0.170.00240.00211.1Latino gay menSan Francisco72HIV+0.490.00410.00351.2MDMA\/Ecstasy usersOhio374Male0.580.00120.00071.7Jazz musiciansNew York City263Male0.760.00160.00072.3Jazz musiciansNew York City261Union member0.250.00100.00071.4Jazz musiciansNew York City253Received airplay0.750.00170.00072.4\nOverall, Table\u00a01 shows that the prevalence estimates from existing studies had design effects around 2, suggesting that respondent-driven sampling is reasonably precise in the situations in which it has been used so far.16 Based on this crude analysis of existing respondent-driven sampling data, we recommend that when planning a study using respondent-driven sampling researchers should assume a design effect of 2. This guideline should only be considered a preliminary rule-of-thumb and should be adjusted, if necessary, depending on pre-existing knowledge of the study population.\nSample Size Calculation\nInformation on design effects should be used when planning the sample size of a study using respondent-driven sampling, or else the sample size will not meet the goals of the study. Fortunately, once the researcher has an estimated design effect, it is rather straightforward to adjust the required sample size; the researcher need only to multiply the sample size needed under simple random sampling by the assumed design effect. Thus, for studies using respondent-driven sampling we recommend a sample size twice as large as would be needed under simple random sampling. However, calculating the appropriate sample size under simple random sampling is often difficult due to the overly general nature of the power analysis literature.20,21 Therefore, we will review the sample size calculations for two specific cases of most interest to researchers using respondent-driven sampling: estimating the prevalence of a trait with a given precision and detecting a change in prevalence over time.17\nOne common goal of studies is to estimate the prevalence of a characteristic with some pre-specified precision, for example, to estimate the proportion of sex workers in New York City that are HIV-positive with a standard error of no greater than 0.03. Since it is the case that,\nwe can solve for the required sample size, n, in terms of the desired standard error, which yields,\nTherefore, if based on pre-existing knowledge we suspect that 20% of the sex workers have HIV and that the design effect is 2, we would need a sample size of at least 356 sex workers to estimate the HIV prevalence with a standard error no greater than 0.03. Notice that this calculation depends on our initial guess of the prevalence. If researchers do not have enough information to make such a guess, they should assume a value of 0.5 which is maximally conservative.\nA second problem of interest to many researchers is comparing the prevalence of some behavior at two time points. For example, a researcher might want to test whether an outreach program was successful at getting drug injectors to stop sharing injection equipment. Assume that the researcher suspects that before the intervention 40% of drug injectors share injection equipment and that the researcher would like to choose the appropriate sample size to be able to detect a drop to 25% such that there is an 80% probability that a 95% confidence interval for the estimated difference will not include 0. Further, assume that the researcher suspects that each prevalence estimate will have a design effect of 2. Based on a derivation available in the literature,23 we can calculate that the required sample size is,\nMore generally, the required sample size for comparing prevalence in two populations is,\nwhere Z1- and Z1\u2212\u03b2 are the appropriate values from the standard normal distribution and deff is the design effect.18\nThese sample size calculations are based on assumptions about the prevalence of the characteristics and the design effect. Therefore, the sample sizes produced by Eqs. 4 and 6 should be considered approximate.\nConclusions\nThis paper makes two main contributions to the literature on respondent-driven sampling. First, we introduce a bootstrap confidence interval procedure that in simulations outperforms the naive method currently in practice. Therefore, we recommend this bootstrap procedure be used in future analysis of respondent-driven sampling data. The procedure requires some custom computer programming to implement, but, fortunately, it is already included in RDSAT, a software package for organizing and analyzing respondent-driven sampling data.19\nThe second major contribution of this paper is the information on design effects. The simulation results suggest that the design effects can range from as high as 10 to less than 1. These findings imply that, because of the possibility of high design effects, respondent-driven sampling is not appropriate in all cases. In some extreme network structures, the prevalence estimates could be so variable that, even though they are unbiased, they might not be very useful. Fortunately, data from existing studies suggest that, so far, respondent-driven sampling has been used in situations where it is reasonably precise, yielding estimated design effects around 2 (see Table\u00a01). Based on these data, we suggest that when using respondent-driven sampling, researchers collect a sample twice as large as would be needed under simple random sampling.\nThe sensitivity of the design effect to the functional form of the degree distribution further emphasizes the need for more research on methods to accurately measure the degree of each respondent. Currently, the estimated average degree depends on subjects' self-reported degree, and these reports may be inaccurate.26,27 In almost all cases, inaccuracy in the self-reported degree will introduce bias into the prevalence estimates.4 As far as we know, the best methods for estimating an individual\u2019s degree are scale-up method and summation method.28 However, it is not clear that either of these approaches, which were designed for the general population, is appropriate for studying hidden populations.\nTaken together, the results about the sample-to-sample variability presented in this paper add to the growing literature on respondent-driven sampling. By allowing researchers to obtain better information about key hidden populations, this research should allow public health professionals to monitor population dynamics more accurately, target resources more carefully, and intervene to slow the spread of disease more effectively.","keyphrases":["variance estimation","design effects","sample size","respondent-driven sampling","hidden populations","snowball sampling","power analysis"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Pediatr_Radiol-2-2-1764599","title":"Pediatric DXA: technique and interpretation\n","text":"This article reviews dual X-ray absorptiometry (DXA) technique and interpretation with emphasis on the considerations unique to pediatrics. Specifically, the use of DXA in children requires the radiologist to be a \u201cclinical pathologist\u201d monitoring the technical aspects of the DXA acquisition, a \u201cstatistician\u201d knowledgeable in the concepts of Z-scores and least significant changes, and a \u201cbone specialist\u201d providing the referring clinician a meaningful context for the numeric result generated by DXA. The patient factors that most significantly influence bone mineral density are discussed and are reviewed with respect to available normative databases. The effects the growing skeleton has on the DXA result are also presented. Most important, the need for the radiologist to be actively involved in the technical and interpretive aspects of DXA is stressed. Finally, the diagnosis of osteoporosis should not be made on DXA results alone but should take into account other patient factors.\nIntroduction\nAfter its commercial introduction in 1987, dual energy X-ray absorptiometry (DXA) has become a widely available and clinically useful tool in the evaluation and management of adult bone diseases. More recently, its utilization in the pediatric population has rapidly increased. DXA presents unique challenges to pediatric radiologists, and the aim of this article is to review the technique and interpretation of DXA. Special focus is on the issues unique to pediatric DXA emphasizing the need for an expanded role for the pediatric radiologist in the performance and interpretation of DXA examinations.\nIs the result of a DXA examination a number or a diagnosis? From the radiologist\u2019s perspective, it is both, making it different from all other imaging studies. Like many laboratory tests, the DXA examination generates a numerical result. The radiologist, like the clinical pathologist, must be knowledgeable about the processes by which that number is obtained and must ensure that meticulous technique is used [1]. Additionally, the radiologist must be knowledgeable about the statistical limitations and uses of a numerical result. For example, he or she must be familiar with the precision and accuracy of the technique and be able to consult regarding the least significant change and be able to suggest appropriate follow-up intervals [2]. The radiologist must also interpret the study in terms of the relevant patient factors that influence the numeric result. These factors include gender, ethnicity, height, weight, body composition, and physiologic maturity, and they might be incorporated in the interpretation of the study. Therefore, the result of a DXA examination is both a number and a diagnosis, and radiologists should be expert in understanding and reporting both aspects in the DXA report. The diagnosis of osteoporosis should not be made on DXA results alone but should take into account other patient factors. Additionally, the radiologist needs to be aware of the limitations of DXA in children [3]: there are difficulties in scan interpretation because DXA is an areal rather than a volumetric density measurement; the growing skeleton has an impact on follow-up measurements; there is a lack of consensus regarding the patient demographic and physiologic factors that should be incorporated into normative databases; and there is yet to be determined a prognostic value of pediatric DXA with regard to fracture risk or peak bone mineral density (BMD) [4].\nAreal vs. volumetric bone mineral density\nAs in all radiologic studies, DXA relies on the differential absorption of X-rays to differentiate tissues of different radiographic density. In addition, DXA can quantify (in grams) the bone mineral content (BMC) at various body sites. By selecting regions of interest, a bone area (BA) is selected with units of centimeters squared. (The abbreviation BA for bone area should not be mistaken for bone age.) BMD is measured directly for each pixel in the region of interest (ROI) by comparing the X-ray attenuation of that pixel to a reference standard. It has units of grams per centimeter squared. This value is multiplied by the pixel\u2019s area to derive the BMC of that pixel with units of grams. The areas of all the pixels in the ROI are summed to give BA. Thus, BMD = BMC\/BA.\nUnlike other density measurements, the DXA-derived BMD is based on the two-dimensional projected area of a three-dimensional structure. The third dimension, depth, cannot be accounted for directly because it is in the same direction as the X-ray beam. Therefore, DXA-derived BMD is an areal (aBMD) rather than a true volumetric (vBMD) density. Because the third dimension is unaccounted for, problems with DXA-derived BMD can arise [5]. Specifically, smaller bones will be found to have lower aBMD than larger bones, even when the vBMD is the same (Fig.\u00a01). For material of the same true density, a larger cube will be stronger than a smaller cube. Thus, in giving a lower aBMD for smaller bones when compared to larger bones with the same vBMD, an aBMD might give a more accurate indication of bone strength relative to size than a vBMD. Additionally, unlike adult patients in whom the bone volume does not change over time, a child\u2019s bones grow over time and the growth of individual bones is not uniform in three dimensions. Thus, errors resulting from areal measurements of BMD might be introduced with DXA and can make comparison of follow-up and baseline studies more challenging to interpret in pediatric patients [4].\nFig.\u00a01For two bones of known BMD=1g\/cm2, the DXA-derived areal BMD will be higher in the larger bone because of the lack of accounting for the true volume of the measured bone. It should be noted that the larger cube will be stronger than the smaller cube (adapted from Carter et al. [5], with permission)\nTechnical aspects of DXA performance\nPatient positioning and ROI selection as performed by the technologist require precision [6] and evaluation by the radiologist for each study. The lumbar spine should be straight and centered in the image, with visualization of the last rib pair and the upper sacrum. Artifacts, including enteric tubes, orthopedic hardware, and jewelry, should be excluded from the image, if possible. Vertebral segments with overlying artifacts should be excluded from BMD analysis. The ROIs are generated automatically using edge-detection software and are selected for the L1 to L4 vertebral bodies. These regions should include the entire vertebral body with a minimum of adjacent soft tissue (Fig.\u00a02). For evaluation of the hip, the femoral shaft should be parallel to the long axis of the image with only a small amount of the lesser trochanter visualized. When using the manufacturer\u2019s provided positioning device, there should be no overlap of the trochanters and the femoral neck or acetabulum. The ROIs include the femoral neck, trochanter, intertrochanteric, and total hip, (Fig.\u00a02). The region of lowest femoral neck BMD, known as Ward\u2019s triangle, is not used in pediatric DXA analysis, and, often, use of the total hip measurement is favored over the other subgroups in clinical practice because of its higher precision than the subgroups. Whole-body scanning provides total body BMC and BMD but also allows for subregions with appropriately drawn ROIs (Fig.\u00a02). Because of the normally low BMD in young children, software analysis is modified for children to improve edge detection of lower-density bone. Such algorithms have been validated in healthy, obese, and chronically ill children for the assessment of lumbar BMD [7]. The adult algorithm significantly overestimates lumbar BMD when compared to the pediatric low-density algorithm (Table\u00a01), because lower-density bone pixels will be excluded using the adult algorithm [8, 9]. It is suggested that normative pediatric data be collected using the low-density analysis routinely in children, thus allowing meaningful comparisons of patient data to normal data, and that the DXA report indicate which algorithm (adult or pediatric) was used [3].\nFig.\u00a02DXA images. a AP image of the lumbar spine shows regions of interest from L1 to L4. The bone area and mineral content are used to derive the bone mineral density at each level. The areal density is based on the bone area; the depth dimension is not directly assessed with DXA. Note transitional lumbosacral vertebral body. b AP image of the lumbar spine shows regions of interest from L1 to L4. AP DXA image of the left hip shows regions of interest of the femoral neck, greater trochanter, and total hip. c Total body scan with sub-regions of interests for trunk, extremities, and headTable\u00a01Effect of pediatric vs. adult software analysis on bone area and BMC results. Total body DXA from a 13-year-old patient processed using pediatric and adult software. Note decreased BA (1315 cm2) and BMC (1149\u00a0g) but increased BMD (0.874 g\/cm2) with the adult technique. Low-density portions of the bone are included using the pediatric technique and thus a larger BA (1810 cm2) with a greater BMC (1375\u00a0g) are obtained but the BMD (0.759 g\/cm2) is lower because of the inclusion of low-density bone pixelsRegionPediatricAdultBA (cm2)BMC (g)BMD (g\/cm2)BA (cm2)BMC (g)BMD (g\/cm2)Left arm201930.465114710.622Right arm196970.497116760.655Left ribs79400.50775390.511Right ribs98510.52592480.525Thoracic spine85480.56983480.524Lumbar spine49280.5033200.599Pelvis1791360.760115950.829Left leg3532690.762281990.873Right leg3382450.7242261870.829Subtotal157710080.63910817820.723Head2333671.5722333671.572Total181013750.759131511490.874\nThe sites selected for BMD analysis should provide a robust evaluation of the patient bone density status. If technically feasible, the pediatric DXA examination should measure lumbar spine and total body BMD [3]. The manufacturers\u2019 normative data for the hip in preadolescents are limited, and evaluation of the hip is not recommended in these young children. There are normative DXA data for the adolescent hip, especially in girls [10], and for these patients this site might be a useful addition to the standard examination. When evaluating group data of normal children, there is usually close correlation of BMD between the lumbar spine and hip [11]. For individuals, when the DXA results deviate from normal, there is less concordance between these two sites. For example, if the hip Z- score is between +1 and \u22121, the lumbar Z-score will vary an average of less than 0.5 SD from the hip score. As the hip Z-score decreases to \u22123, the lumbar score varies on average 1.7 SD from the hip Z-score. For example, immobilized or paraplegic children might have preserved lumbar BMD but low hip BMD. If evaluation of the spine and hip is not feasible because of extensive orthopedic hardware or patient positioning issues, DXA of the forearm can be performed. However, there are few normative data for pediatric forearm studies, and the value obtained might be useful only when compared to subsequent studies. Because of its reproducibility and lack of areal density-related errors, the total body BMC is preferred for the assessment of bone status by some clinicians and researchers [12].\nThe DXA lumbar ROI will not include the thoracic spine. However, the identification of a thoracic body compression deformity in patients with osteoporosis has significant prognostic value, indicating a higher risk of subsequent vertebral compression fractures. Evaluation of thoracic and lumbar vertebral bodies can be achieved with modern DXA scanners (Fig.\u00a03) and can identify thoracic compression fractures that would have been otherwise undiagnosed. Makitie et al. [13] found compressive thoracic deformities in 11 of 32 children suspected of having secondary osteoporosis who were studied with DXA. Of these 11 children, 8 had normal lumbar DXA, and the demonstration of a thoracic compressive deformity was thought to be an important diagnostic and prognostic finding. The addition of vertebral morphologic assessment might be an important adjunct in the diagnosis of pediatric osteoporosis.\nFig.\u00a03Lateral thoracic and lumbar spine image from DXA study for vertebral morphology. Note compressive deformities at T-7 and T-9, and a Schmorl\u2019s node at L1, with otherwise normal vertebral morphology of the lumbar spine\nDXA requires remarkably low radiation doses. Using manufacturers\u2019 data, the effective dose for lumbar spine and whole-body DXA is reported to be 1\u22125\u00a0\u03bcSv [14]. This is less than the dose of a standard PA chest radiograph. Scanning times are now less than 2\u00a0min for the spine, hip or forearm, and less than 3\u00a0min for the total body.\nAccuracy, precision, and least significant change\nDXA provides both accurate and precise bone mineral data. Accuracy refers to how close a measured value is to the true value as determined by a gold-standard technique. For BMC, the gold standard is the laboratory assessment of ashed bones, and DXA measurements of BMC are within 7\u20139% of ashed bone measurements [15, 16]. Precision is a measure of reproducibility of a measurement and can be expressed in terms of coefficients of variation (%CV). Both short- and long-term precision are important in performing DXA examinations. Short-term precision reflects both the imprecision of the equipment (manufacturers report this to be less than 1%) and the imprecision resulting from variation in patient positioning and motion effects (typical values are less than 2\u20133% for the spine, up to 5% for the hip, and 1\u20132% for the whole body) [14, 17, 18]. This component varies with each technologist and should be calculated with a repeated measures procedure [2]. Long-term precision is a measure of machine drift. It is normally well below 1%, and the radiologist should review the daily quality control scans of phantoms graphed over a period of weeks in order to detect this [15].\nBecause the DXA result is a number, the radiologist must be aware that the magnitude of change required to be statistically significant varies with the precision of the measurement technique. This is expressed in terms of the least significant change (LSC), and is equal to 2.8\u00d7%CV for the 95% confidence limit [2]. If the %CV was 1.5, then a change from the baseline measurement of 4.2% would be required for it to be considered statistically significant. The LSC can also be used to suggest the timing of follow-up measurements, i.e. if the LSC is 4.2% and the expected annual rate of change in the BMC or BMD is 2%, a follow-up study before 2\u00a0years would likely result in a value not statistically significantly different from the baseline. The annual rates of change in BMC and BMD vary considerably during childhood, with dramatic acceleration of bone accrual during the early pubertal years [19\u201321], especially in females [22]. The annual rates of change for early-stage and late-stage adolescents are approximately 25% and 10% for BMC and 10% and 3% for BMD [23]. For most pediatric conditions, follow-up examinations are obtained between 6 and 12\u00a0months.\nDXA in newborns\nDXA has been used to evaluate bone mineralization in newborns and young infants since its inception as a clinical tool, and numerous investigators have established its precision and accuracy [24\u201328]. The technique has a high degree of accuracy, even in preterm low-birth-weight neonates, with a slight underestimation of BMC of approximately 7% [26, 28]. The high precision of DXA has been shown in neonates with %CV of 2.4% [25] for measurements of the lumbar spine and of the total body BMC when assessed in piglets [26]. The absorbed dose is extremely low, and most neonates and infants can be imaged with gentle immobilization without the need for sedation.\nNormative data for neonates indicate a very close relationship of total body BMC and BA with weight; height is also strongly correlated with BMC in the newborn [25]. Koo et al. [29] found weight and, to a lesser extent, body length but not gender or race to predict total body BMC, BA and BMD in newborns. Additionally, body mass was more closely correlated with BMC and BA than BMD. This may be because of DXA-derived BMD is areal rather than volumetric. Last, the proportional contribution to total body BMC from each subregion (head, trunk, and upper and lower extremities) is maintained across gestational ages (27\u201342\u00a0weeks) and birth weights (1\u20134\u00a0kg) with only slight variation [29].\nIndications for pediatric DXA\nDespite many potential uses of DXA, there has been relatively little written regarding the indications for DXA in children. The International Society for Clinical Densitometry has suggested that any child being treated or considered for treatment of osteoporosis should undergo a DXA examination [3]. The National Osteoporosis Foundation lists the following indications for DXA in children: systemic long-term steroids, chronic inflammatory conditions, hypogonadism, prolonged immobilization, osteogenesis imperfecta, idiopathic juvenile osteoporosis, recurrent low trauma fractures, and apparent osteopenia on radiographs [14]. It is important to note that DXA would be inappropriate for skeletal pain, chronic disease and traumatic fractures without any of the additional risk factors listed above [14]. The National Institutes of Health recommend a baseline DXA examination for patients for whom systemic corticosteroids will be used for more than 2\u00a0months or who are at significant risk of osteoporotic fracture [30]. These guidelines are also suggested by the American College of Rheumatology [31]. The American College of Radiology has listed DXA of the lumbar spine and hip as highly appropriate for pediatric patients with a significant risk factor for osteoporosis [32]. Recommendations from other subspecialty societies have been slow in coming. Neither the Society for Pediatric Radiology nor the American Academy of Pediatrics has made recommendations regarding pediatric DXA.\nPediatric DXA interpretation\nAs with other laboratory tests, the numeric value reported is meaningless without comparison to the appropriate normal controls. After comparison is made, the reported value is given as a percentile or a standard deviation score, the Z-score. A Z-score of zero is equivalent to the mean, and Z-scores of \u22121 and +1.5 are equivalent to values one standard deviation below and 1.5 standard deviations above the mean, respectively. The T-score (comparison of the current Z-score with peak adult BMD) is used in adult interpretation of DXA but should not be included in the pediatric DXA report [3]. Because the T-score is a measure of bone density loss since early adulthood, its use in children whose BMD has yet to peak will always yield a low result. Because the World Health Organization\u2019s DXA-based definitions of osteopenia and osteoporosis are in terms of T-scores, T<\u22121.0 and T<\u22122.5, respectively [3], a different terminology is needed for pediatric patients. It is recommended that the phrase \u201clow bone density\u201d be used in DXA reports [3]. Some clinicians and researchers use the terms osteopenia and osteoporosis in children when Z-scores are less than \u22121.0 and \u22122.5, respectively. It is important to note that the diagnosis of osteoporosis should not be made on DXA results alone but should take into account other patient factors.\nMuch of the research in pediatric DXA has focused on determining which factors most influence BMD and should be accounted for in the development of normative pediatric datasets. The factors age, gender, ethnicity, and physiologic maturity level have been extensively studied and are included in most current normative datasets provided by the major DXA manufacturers. Some of the earliest reports indicate the influence of age on BMD values, and several authors have presented normal data accounting for age [10, 19\u201321, 33]. The effect that age has on BMD is largely related to the increase in frame size that occurs with increasing chronologic age. Increasing height and weight strongly correlate with increasing BMC and BMD. The changes in height and weight are most pronounced during the pubertal growth spurt. BMD increases rapidly during early puberty [21, 34], but because the age of pubertal onset is quite variable, physiologic maturity has a stronger influence on BMD than age. Several authors have included Tanner stage or gynecologic age as a primary factor in their normative datasets [21, 34\u201337]. Weight is an important factor influencing BMD for multiple reasons [4, 19, 20, 32, 38, 39] and has been included in normal datasets [10, 34]. Ethnicity has been determined to be important in the analysis of BMD results, with black children showing significantly higher BMD values than non-black children [10, 20, 36, 39\u201341]. Multiple factors are thought to account for this, including increased cross-sectional area in the axial skeleton and thicker trabecula in cancellous bone [36]. Lifestyle and anthropometric factors might also play a role [42].\nThere are numerous published pediatric normative datasets, many of which are summarized in Table\u00a02. These datasets have been developed using a variety of scanners and processing software and are based on various combinations of demographic and physiologic patient variables. Rather than simplifying pediatric DXA interpretation, the sheer number of available normal databases has made DXA interpretation complex, confusing, and at times erroneous [43]. To report the numeric result generated from the manufacturer\u2019s automated processing without consideration of factors specific to the patient being studied is unacceptable. This often will lead to misdiagnoses and can result in inappropriate therapy [44]. In fact, the diagnosis of osteoporosis in a child based on a DXA result is often a misinterpretation of the scan data. Gafni and Baron [45] found this to be the case in more than half of the pediatric patients referred to them with the diagnosis of osteoporosis. The most common causes for misdiagnosis were the use of T-scores, inappropriate normative datasets, inadequate ROIs, and inattention to short stature. \nTable\u00a02Normative pediatric DXA databases (C\/B\/H\/A\/O Caucasian\/black\/Hispanic\/Asian\/other, GA gestational age, (L) longitudinal study, SA surface area)ReferenceYearScannerNo. of patients (M\/F)Age rangeEthnicity (C\/B\/H\/A\/O)InputOutput251992Hologic 1000\u00a0PB29\/28NewbornGA + weight + height + SALumbar BMD and BMC251992Hologic 1000\u00a0PB22 total1\u201324\u00a0monthsGA + weight + height + SALumbar BMD and BMC291996Hologic 1000\u00a0PB82\/68GA 27\u201342\u00a0weeksWeightTotal BMD, BMC and BA351991Hologic 1000\u00a0PB84\/1342\u201317\u00a0years162\/56\/0\/0\/0Weight + Tanner stageLumbar BMD561990Lunar DP-3184 total5\u201311\u00a0yearsWeightLumbar BMD571993Norland XR-2686\/685\u201318\u00a0yearsGender + Tanner stageTotal BMC and % fat461997Hologic 1000\u00a0PB142\/2014\u201319\u00a0years (L)343\/0\/0\/0\/0Gender + Tanner stageTotal BMD, BMC and BA58, 592002Lunar DPXL\/PED188\/2564\u201320\u00a0years (L)444\/0\/0\/0\/0Gender + ageLumbar BMD and apparent BMD or total BMC, % fat and lean body massGender + Tanner stageLumbar BMD and apparent BMD or total BMD and BMC622002Hologic 4500\u00a0FB107\/1245\u201322\u00a0years226\/0\/0\/3\/2Gender + ageTotal BMC and BAGender + heightTotal BAGender + total BMCTotal BMD612005Hologic 4500\u00a0FBup to 19483\u201320\u00a0yearsGender + age Lumbar, total hip and total BMD211991Hologic 1000\u00a0PB109\/989\u201318\u00a0years207\/0\/0\/0\/0Gender + Tanner stage or age Lumbar BMC, BA and BMD, femoral neck BMD22, 331996Hologic 2000\u00a0FB110\/1248\u201317\u00a0years (L)220\/0\/0\/0\/0Gender + ageLumbar and total BMC and BMDFemoral neck BMC and BMD40a1999Hologic 1000\u00a0PB193\/2309\u201325\u00a0years (L)103\/114\/103\/103Gender + age + ethnicityLumbar, femoral neck, and total hip BMD and BMADTotal BMD and BMC\/Ht102004Hologic 4500\u00a0FB0\/42212\u201318\u00a0years153\/264\/0\/0\/5Age + weight + ethnicity Lumbar and femoral neck BMD, femoral neck apparent BMD60b2001Hologic 2000\u00a0PB0\/1519\u201314\u00a0years (L)151\/0\/0\/0\/0Breast stage + age Lumbar, femoral neck, trochanter and forearm BMC and BMD542003Lunar DPX PB210\/2493\u201330\u00a0years459\/0\/0\/0\/0Gender + height or ageTotal lean body mass and total BMC\/lean body massaFurther data available at http:\/\/www.stat-class.stanford.edu\/pediatric-bonesbFurther data available at http:\/\/www.bcm.edu\/bodycomplab\nAs with any other radiologic study, a methodical evaluation of the results should be undertaken in order to minimize the risk of misdiagnosis. The radiologist needs to review all input data, including patient age, gender, ethnicity, weight, height, and Tanner stage (if provided). Patient positioning should be evaluated, and the ROIs need to be analyzed for artifact and appropriateness. Comparison should be made with previous studies to ensure consistency of positioning and ROI selection. In addition, changes in patient height, weight, and Tanner stage should be noted. After these steps have been taken, interpretation of the numeric result is performed. An appropriate database for comparison purposes is selected. Ideally, this is based on data generated locally using the same equipment and technologists, but this is rarely possible. Normative data provided by the DXA manufacturers can be used, but historically these datasets do not include the parameters currently thought to be most important for interpretation. At a minimum, patient body size (height and weight) and physiologic maturity (Tanner stage, gynecologic or bone age) should be factors included in the normative dataset. Ethnicity and gender are also frequently included in the generation of normative data and are generally thought to affect BMD significantly. Table\u00a02 summarizes a large number of normal databases that can be used to best match the patient scan to be analyzed.\nMore complex and scientifically rigorous analyses of the BMD result have been suggested. Molgaard et al. [46] described a three-step analysis of BMD. Bone length is categorized as short or long by assessing the patient\u2019s height for age. Height is highly correlated with BMC [12] and thus needs to be accounted for, especially when BMD Z-score is abnormal (Z=\u00b12). Bone width is categorized as thin or thick by assessing BMC for height. Last, bone mineralization is categorized with assessment of BMC for BA. The first step takes into account height because of its profound effect on BMC; taller children will have a higher bone content. However, bone width is also important in determining bone content, thus the second step accounts for this. The last step is often the only step performed by many radiologists with reporting of BMC for BA which, by definition, is BMD. Height for age can be assessed using standard growth charts corrected for age, gender, and ethnicity. Percentile rankings can be easily converted to a Z-score [47]. Bone area for height tables are available for select groups, but normative data for all pediatric patients need to be developed. Using this three-step analysis, Molgaard and Michaelsen [48] found that the causes for low BMC might be various combinations of factors such as short stature and thin bones, as in children with cystic fibrosis, or short stature and reduced mineralization, as in children with milk allergy. BMC might be normal despite short stature in the presence of wide bones, as in children with previously treated leukemia.\nIn summary, an abnormal BMD Z-score should lead to evaluation of confounding patient factors that influence BMD, including height, weight, and physiologic maturity, before a diagnosis of low bone density is made. Until the manufacturers\u2019 databases sufficiently account for the physiologic factors that most impact BMD results, normative data derived locally or from the medical literature should be used in pediatric DXA interpretation. The report should include the DXA equipment and software algorithm used (pediatric or adult, low bone density or standard), the source of the normative reference data, the Z-score (not the T-score), and an impression giving a clinical context for the result. The diagnosis of \u201clow bone density\u201d does not rest solely on the DXA numeric result, and the report should indicate which patient factors were incorporated into the final impression. A specimen DXA report and the examination protocol from Columbus Children\u2019s Hospital are given in Appendix.\nBone growth over time and changes in BMD\nAs mentioned previously, bone size affects the DXA aBMD result. Changes in bone size over time, as occur in normal pediatric development, confound DXA interpretation. This is one of the major limitations in the use of pediatric DXA. On both follow-up studies, when the child\u2019s data are compared to prior results, as well as when the child\u2019s DXA values are compared to normative values, the effect of bone size needs to be accounted for. These problems could be avoided, at least in part, if a true volumetric BMD were available with DXA. As this is not possible with current DXA technology, investigators have attempted to account for the effect of bone volume on the DXA result and minimize the effect the growing skeleton has on the BMD value [49]. Katzman et al. [50] estimated the volume of the lumbar vertebral bodies, assuming it to be a cube, using this formula: apparent lumbar BMD (BMAD) = BMC\/BA1.5. Kroger et al. [51] suggested a volumetric correction based on the assumption that the vertebral body is a cylinder using this formula: BMAD=(BMC)(4\/[\u03c0{bone width}]). This correction can also be applied to the femoral shaft and neck [52]. These corrections do not fully account for changes over time in lumbar BMD but do eliminate age and height dependence of BMD at the hip. A different approach has been to not evaluate BMD at all, but to measure BMC only, because it is determined with greater accuracy and precision than BMD [53]. In an effort to incorporate the fundamental relationship between the mechanical stress a bone experiences through muscle action and its mineral content, Hogler et al. [54] and Crabtree et al. [55] used DXA to assess BMC and proposed algorithms that focus on the evaluation of lean total body mass (LTM) with respect to height and BMC. They found LTM explains greater than 95% of the variation in total body BMC in boys and girls. By incorporating LTM\/height and height\/age into the DXA interpretation, patients could be grouped as being normal or having a primary, secondary or mixed bone defect. For example, the diagnosis of low BMD for age might be a result of smaller bones and not osteopenia. Hogler et al. [54] found children with growth hormone deficiency to be short for their age (low height\/age ratio) but to have a normal amount of bone adaptation (BMC) for the muscle force (LTM). The BMC\/LTM ratio for these children is normal. In contrast, children with anorexia nervosa with normal height had low LTM and also low BMC\/LTM ratios. Their low BMD for age was a result of decreased muscle loading on bone (a secondary bone defect with low LTM\/height) and decreased mineralization for the level of muscle loading present (a primary defect with low BMC\/LTM). This indicated that a mixed defect caused their low BMD for their age.\nIn summary, smaller bones will have lower aBMD than larger bones as measured with DXA, even when the vBMD is the same because of limitations of the areal bone density technique. Attempts to correct for or to circumvent this limitation have been proposed and show promise in improving DXA interpretation and our understanding of bone physiology.\nClinical case studies\nThe following case studies are examples of common clinical indications for DXA in pediatrics and illustrate the principles used at Columbus Children\u2019s Hospital in DXA interpretation.\nPatient 1 was a 4-year-old white female with nutritional rickets and multiple fractures following minimal trauma. She presented for a baseline study and was Tanner stage 1 and weighed 19\u00a0kg. Her lumbar BMD was 0.399\u00a0g\/cm2. Using our normal database that corrects for Tanner stage and weight [35], the patient\u2019s lumbar BMD was far below the first percentile (Z-score was much less than \u22122.3). The manufacturer\u2019s database (which is based in part on the data from reference 35) yielded a Z-score of \u22122.1. The total body BMC was 543\u00a0g with a Z-score of \u22122.3 [43]. The patient was reported as having markedly reduced lumbar bone density and reduced total body BMC.\nPatient 2 was a 13-year-old black female with lupus. On baseline DXA the lumbar BMD was 0.719\u00a0g\/cm2, equivalent to a Z-score of 1.6 using our local database and was considered to be a high normal value for a 33-kg and Tanner stage 1 patient. The total body BMC was 1280\u00a0g, equivalent to a Z-score of 1.5 [43], and was also thought to be a high normal value. During the next 15\u00a0months, she was treated with corticosteroids and gained 15\u00a0kg and progressed to Tanner stage 2. On follow-up DXA the lumbar BMD was 0.723\u00a0g\/cm2. This value corresponds to the 25th percentile and a Z-score of \u22120.67 [35]. Thus, she had a slight decrease in her lumbar BMD value at a time when rapid bone mineral accrual would be expected [21]. This was made evident by the rather marked decrease in her Z-score, from 1.5 to \u22120.67. Her total body BMC was unchanged, but the Z-scores also decreased substantially.\nPatient 3 was a 16-year-old white female with Turner syndrome and of small stature (150\u00a0cm, first percentile, Z-score \u22122.3). Her lumbar BMD was 0.641\u00a0g\/cm2, equivalent to the eighth percentile (Z-score \u22121.3) for Tanner stage 3 and weight 50\u00a0kg [43]. It should be noted that the manufacturer\u2019s normal database yielded a lumbar BMD Z-score of \u22123.0, but it did not take into account height or weight. The low lumbar BMD Z-score was likely a result of the patient\u2019s short stature (low height Z-score). The weight correction in our normal database partially accounted for her short stature and gave a more meaningful interpretation of the numeric DXA result. This patient\u2019s bone age was 13\u00a0years (Z-score \u22122.3), and using this rather than her chronologic age (16\u00a0years) with the manufacturer\u2019s normal database would result in a Z-score of \u22121.0. This value would better reflect the patient\u2019s actual bone status. Because our database corrects for weight and Tanner stage, each patient is weighed and has a Tanner stage determination made by the referring clinician prior to the DXA study. Thus, we do not use the patient\u2019s bone age in DXA interpretation.\nFinal recommendations\nPediatric radiologists need to assume a more prominent role in DXA performance, interpretation and research. They require expertise with DXA as a laboratory procedure, a numeric result, and a clinical diagnostic examination. The radiologist must be a \u201cclinical pathologist\u201d closely supervising the acquisition of the DXA study with assessment of quality control data as well as the clinical images. The radiologist must be a \u201cstatistician\u201d understanding the principles of least significant change and the relationship of standard deviation scores and percentile rankings. The radiologist must also be a \u201cbone specialist\u201d providing a meaningful context for DXA, translating the numeric value to a clinically useful result. As always, pediatric radiologists must be children\u2019s health advocates by ensuring the appropriate clinical use of DXA as well as actively participating in research efforts.","keyphrases":["pediatric dxa","osteoporosis","technique interpretation"],"prmu":["P","P","R"]}
{"id":"Ann_Surg_Oncol-4-1-2234450","title":"The Lymphatic Anatomy of the Breast and its Implications for Sentinel Lymph Node Biopsy: A Human Cadaver Study\n","text":"Background Current understanding of the lymphatic system of the breast is derived mainly from the work of the anatomist Sappey in the 1850s, with many observations made during the development and introduction of breast lymphatic mapping and sentinel node biopsy contributing to our knowledge.\nManagement of the lymph nodes has been an important part of breast cancer treatment since the time of Halsted\u2019s radical mastectomy.1 Until the 1970s it was based on the perceived need to remove cancer cells before they spread via the lymphatics beyond the reach of the surgeon. The national surgical adjuvant breast and bowel project B04 study questioned the therapeutic benefit of axillary clearance, however it remained part of the treatment of invasive breast cancer because of the prognostic significance of the axillary lymph nodes status.\nSentinel node biopsy (SNB) has replaced axillary dissection in clinically node negative patients,2\u20134 with trials confirming a false negative rate of 5\u201310%. While it is agreed that SNB is appropriate, the optimal injection sites of dye and\/or colloid has not been defined.5\u20137 There are advocates of peritumoral,8\u20139 dermal and subdermal10\u201312 or subareolar and periareolar injection13\u201314 of the tracers. These recommendations regarding injection sites are derived from clinical experience rather than from anatomical studies.\nThe anatomical basis of axillary clearance was derived from the publications of Sappey in the 1870s15 who showed the breast lymphatics as separate from those of the underlying torso, with a subareolar plexus of lymphatics, and a small number of large lymphatic vessels draining into axillary lymph nodes (Fig.\u00a01). Despite the revolution in the surgical approach, there has been no advance in the underlying anatomical knowledge of the breast lymphatics.\nFig.\u00a01.Sappey\u2019s drawing of the superficial lymphatics of the upper torso (left) and female breast (right) in 1874.15\nThe lymphatics start as an avalvular lymph capillary network. In the skin they commence in the superficial dermis with diameters of 20\u201370 \u03bcm.16 They drain to precollectors that are up to 300 \u03bcm diameter, contain valves, have some smooth muscle cells in their wall,17 and drain this network to subcutaneous lymph collecting vessels (Fig.\u00a02). The lymph collecting vessels (lymph collectors) have a smooth muscle layer18 and are the vessels commonly identified as lymphatics. The breast develops as a modified skin gland appendage and expands into the subcutaneous tissues.19Fig.\u00a02.Schematic diagram of the relationship between the lymph capillaries, precollectors, and lymph collecting vessels.\nProgress in microsurgery has enabled new methods for investigating the lymphatic system. We developed a protocol for injecting radio-opaque contrast medium into lymph vessels of <0.3\u00a0mm diameter in cadavers. The contrast agent fills the draining lymph vessels, which are then radiographed.20,21We have previously published detailed investigations of the lymphatics of the upper limb.22\u201324\nThis study addresses the lymphatic anatomy of the breast and anterior upper torso. Our findings may explain the clinical experience in lymphatic mapping and sentinel node biopsy, and also the persistence of a false-negative rate of 5\u201310% irrespective of the experience of the surgeon.\nMaterials And Methods\nBilateral anterior upper torso specimens, which included both breasts, were harvested from 10 cadavers (4 male and 6 female) with incisions across the root of the neck, down the posterior axillary line and across the abdominal wall, just above the umbilicus. Another four unilateral studies were obtained from separate cadavers (1 male and 3 females) by a midline incision of the sternum, thus resulting in a total of 24 sides. Protocol refinements at the beginning of the study limited early results and there were always time limits to complete the work, especially on the contralateral side of bilateral subjects, before the specimen became putrefied. The injection and dissection for each side of a specimen took 4\u20135\u00a0weeks. The specimens were stored at \u221220\u00b0C prior to dissection and at 4\u00b0C during the dissection.\nThe lymphatic mapping technique has been described previously.20\u201321 Briefly 6% hydrogen peroxide was injected into the tissue being examined. Bubbles appear in the tissue and the lymphatic collectors and an operating microscope (Stemi 2000, Carl Zeiss Pty. Ltd. Germany) is used to identify and cannulate the lymphatic vessels with a 30-gauge needle (Precision glide needle: Becton Dickinson & Co., USA.) or an extruded glass cannula for very small vessels. Radio-opaque contrast medium containing lead oxide (P3O4 Red lead, AJAX Chemicals, Australia) was injected antegrade with a micromanipulator (UM-3C, Narishige Co., Japan). The contrast stopped after a variable distance because of coagulation requiring further cannulation and injection. This was repeated until the first lymph node for each collector was reached (sentinel node).\nThe procedure was performed: around the entire periphery of the specimens in the subcutaneous plane; around the internal thoracic artery on the inner aspect of the chest; and in the vicinity of the perforating branches of the internal thoracic artery to identify superficial lymphatics, internal mammary lymphatics, and perforating lymphatics respectively.\nIndocyanine Green dye (Pulsion Co., Germany) was injected into the internal mammary artery to demonstrate the artery and its perforating branches, which supply blood to the medial aspect of the breast, thus facilitating the identification of associated deep perforating lymphatics.\nA mixture of blue dye (Ultamarine, Educational Colours Pty Ltd., Melbourne) and hydrogen peroxide was used to stain the lymph collecting vessels originating from the nipple and areolar region. In previous studies of the upper limb we have first injected this blue dye into the dermis before injecting hydrogen peroxide into the subcutaneous tissues to help identify the collecting lymphatics.22 This was helpful in identifying lymphatics in the fingertips, where they exist at high concentration. This dye was injected also into the dermis over the breast mound but was only helpful in the nipple areolar area where again the lymphatics were concentrated.\nThe specimen was radiographed after completion of all injections to give the two-dimensional views of the lymph vessel anatomy. To provide the three-dimensional views, the female specimens were sliced parallel to the lymph vessels as they passed towards the axilla. These slices were then placed on their sides and radiographed to show the position of the lymphatic collectors with respect to the skin and the breast tissue.\nRadiographs were photographed (Nikon D100, Nikon Co., Japan) and transferred to the computer, then each lymphatic collector was traced (Adobe Photoshop CS, Adobe System Inc.) to its first-tier node and color coded. All collectors draining to the same first-tier node were assigned the same color after tracing them retrogradely from the sentinel nodes.\nResults\nSuperficial Lymphatic System\nLymphatic collectors were identified in the subcutaneous tissues near the peripheral cut edges of the specimens and the lateral border of the sternum. The collectors often branched in the peripheral region then combined to form larger collectors that remained approximately uniform in diameter until they reached the first lymph node (Figs.\u00a03 and 4). Some collecting lymph vessels joined with others within the same sentinel node territory, rarely with others, before reaching the lymph node. All superficial lymph vessels in these dissections entered a lymph node in the axilla, which was always close to the lateral edge of the pectoralis minor muscle. The findings were similar in both sexes (Fig.\u00a04). In all of the specimens we examined, many of the lymphatic collectors that passed over or through the breast ended by draining into the same first-tier lymph node. In some, almost the entire breast drained to one sentinel node. In others however, there was at least one other node that was the first-tier node for a collecting lymphatic that passed through part of the breast.\nFig.\u00a03.Radiographs in antero-posterior views of a male (left) and female (right) specimen after completing injections with the lead oxide mixture. Note that the torso lymph vessels radiate centripetally towards the axilla.Fig.\u00a04.Tracing distally of lymphatics of both hemi upper torsos (male: A and C, female: B and D) from each first-tier lymph node colour coded; pectoral node (green, orange, black and yellow), subclavicular node (light blue), and internal mammary node (red). Note (i) that the lymph collecting vessels from the nipple and areolar region on each specimen drain into the green-colored lymph node; (ii) the similar pattern of chest and breast drainage between the male and female studies; (iii) that the breast lies in the pathway of collecting lymphatics that start peripherally and (iv) that, although the majority of the breast drains to one sentinel node in D, every breast area is drained by more than one first-tier node in each study.\nThe lymphatics deep to the nipple and areola area were different from those of the other areas we examined. Microscope-assisted dissection of the areolar region revealed a dense network of lymph capillaries and precollectors in the dermis. This structure is presumably the subareolar plexus identified by Sappey. In most areas, dye injected subcutaneously over the breast mound was not taken up by the lymphatics. Dye injected in the nipple and areolar region was taken up by 2 or 3 superficial lymph collecting vessels via lymph capillaries and precollectors (Fig.\u00a05) each side of the areola and these drained into a first tier node in the pectoral group of axillary nodes (shown green in Fig.\u00a04).\nFig.\u00a05.Photograph of dissection in the areolar region after injecting the mixture of dye and hydrogen peroxide into the nipple. Lymph capillaries and precollectors (black arrows), and lymph collecting vessels (white arrow) were stained with the blue dye.\nThe radiograph of the cross-sectioned female specimen showed that the lymphatics have a \u201cwavy\u201d path through the superficial part of the subcutaneous tissue of the chest (Fig.\u00a06). In the breast region most of the lymph collectors ran between the dermis and the breast tissue. However, some passed through the breast tissue itself. After traversing the breast all of the collectors passed deeply to reach axillary lymph nodes.\nFig.\u00a06.The diagram (above) shows how the breast of Fig. 4B was sectioned to provide cross-section radiographs (middle of the breast and its lymphatic drainage (below). Note that some lymph collectors originating from the lower torso run through the breast tissue in both A and B sections.\nInternal Mammary Lymphatic System\nThe internal mammary lymphatic vessels were identified alongside the internal mammary artery and vein, deep to the parietal pleura, with lymph nodes present in the intercostals spaces. Collecting lymphatics were identified beside the perforating branches of the internal mammary arteries that were found in the deep medial aspect of the breast tissue (Fig.\u00a07). A single collector passed though the intercostal fascia and muscle beside each perforating artery to join the internal mammary lymphatic system.\nFig.\u00a07.The appearance of the perforating lymphatics (orange) along the branch from the internal mammary artery (green) in the right lateral sternal region of the female specimen.\nThere was no apparent connection between the collecting lymphatics accompanying the branches of the internal mammary artery and the superficial collecting lymphatics. Contrast injected into the superficial collecting lymphatics never flowed into the perforating collecting lymphatics. Even in the subcutaneous tissues overlying the lateral edge of the sternum, the superficial collecting lymphatics passed laterally towards the axilla. We found no evidence of direct anastomosis between the superficial collecting lymphatics and the collectors associated with perforating arterial branches, at the size of the collecting vessels. However, this does not exclude a connection at either the precollector or lymphatic capillary network as we were unable to demonstrate them radiologically.\nDiscussion\nCruikshank was the first to report the lymphatics of the breast in his book published in 1787.25 He used the cadaver of a pregnant woman injecting mercury into the mammary duct from the nipple. He reported seeing by chance mercury-filled lymphatics but did not make any diagrams of his findings.\nIn 1874, Sappey applied the same method,15 injecting mercury into the dermis of a semiputrefied cadaver to identify the superficial lymph vessels. A very thin adult cadaver was used and he described the superficial lymphatic drainage of the torso, dividing it into four territories by a sagittal midline and a horizontal line at the L2 level. The upper torso lymphatics always drained into the ipsilateral axilla (Fig.\u00a01). Sappey reported that the lymphatics of the breast collected in a subareolar plexus and then drained towards the axilla via lymph collecting vessels. Sappey\u2019s description of the breast lymphatics was adopted by anatomists and became the theoretical basis for the subareolar injection of dye and\/or isotope for lymphatic mapping as part of the sentinel node biopsy for breast cancer more than one century later.13\u201314\nIn 1903, Poirer and Cuneo summarized Sappey\u2019s results.26 They added their results from foetal studies using with Gerota\u2019s method27 using oil painting dye to stain the lymphatics, they reviewed other people\u2019s studies and published a comprehensive anatomy book of the lymphatic system. Poirer and Cuneo\u2019s famous picture of the lymphatics of the breast (Fig.\u00a08) was redrawn and is still being used in Gray\u2019s Anatomy.28Fig.\u00a08.Poirier and Cuneo\u2019s summary diagram of the breast lymph drainage.26 This diagram was composed based on the anatomical and clinical findings of several people, including Sappey.\nIn 1959, Turner-Warwick performed photographic and radiographic studies of the collecting lymphatics injecting them with iron Prussian blue or radioisotope gold (Au198) during surgery in breast cancer patients.29 He demonstrated lymphatic pathways that passed direct from the tumor injection site in the breast to the axillary lymph nodes that bypassed the subareolar plexus. He found that all quadrants of the breast drained to the axilla and either the internal mammary or posterior intercostal nodes. He suggested that Sappey had mistaken the mammary duct for a lymphatic vessel, thereby overemphasizing the importance of the subareolar plexus.\nThe more recent users of lymphoscintigraphy examinations of the breast are also skeptics of the centripetal lymphatic route towards the subareolar plexus. Tanis30 and Uren (Uren RF: personal communication, May 2007) stated that there is no constant route via the subareolar plexus.\nOur current anatomical knowledge of the breast lymphatics still depends on Sappey and Poierer and Cuneo\u2019s diagrams. Since Sappey used thin adult cadavers and Poirer and Cuneo used infant cadavers, the relationship between the superficial lymphatics and the adult breast tissue, and also the relationship of the lymphatic drainage of the breast tissue with that of the surrounding superficial tissues, has not been adequately described. Controversy still exists over the role of the subareolar plexus in the lymphatic drainage of the breast. There has been no concrete evidence of a centripetal anatomical lymphatic pathway that drains the breast tissue towards the subareolar plexus and then, via this plexus, towards the sentinel node.\nOur direct injection technique, however, gives a comprehensive image of the lymphatic system in individual specimens. Each lymph vessel that enters a lymph node can be traced retrogradely to provide an accurate map of the tissue for which that lymph node is \u201csentinel.\u201d We have photographed, radiographed, and recorded actual lymphatic pathways. In addition, cross-sectional studies have been performed to obtain three-dimensional images of these lymph collecting vessels.\nWe have shown that some of the torso vessels pass from the periphery through the breast tissue on their way towards the axilla. This is discordant with the conventional understanding that they run just underneath the skin.\nWe compared the torso lymphatic vessel pathways and breast tissue lymphatic vessel pathways in adult female cadavers. Although our results do not show conclusively that the lymph drainage of the breast tissue is via the collecting lymphatics that pass through or immediately superficial to the breast substance, this is likely to be the case since (i) the lymph collecting vessel has a microporous nature and drains surrounding tissue fluid along its entire course31 and (ii) lymphoscintigraphy in breast cancer patients after peritumoral injection, coupled with similar studies with radioactive gold by Turner-Warwick, usually reveals a direct pathway from the injection site to the axillary lymph node,29\u201330 not via a subareolar plexus. Therefore we conclude that the lymph collecting vessels that pass through the breast contribute to breast lymph drainage and must be the same vessels shown in lymphoscintigraphy examinations.\nEmbryologically, the development of the human peripheral lymphatics is poorly understood. However, the lymphatics of the foetus represent similar arrangements to those of adults.32\u201333 We assume that some of the lymph vessels are trapped inside the mammary gland as the breast develops around preexisting superficial lymph collectors. We have color coded a diagram of our dissection to simulate various injection sites in lymphatic mapping and sentinel node biopsy for breast cancer. Figure\u00a04 shows that, if the tracer is injected deep around the purple-colored vessel (lower outer quadrant), it reaches both the green and orange lymph node in the pectoral group. However, if the tracer is injected into the subareolar region or into the lower outer quadrant intradermaly near the tumor, it reaches only the green node. This anatomical analysis suggests a mechanism for false negative sentinel node biopsy since more than one sentinel node drains the breast in this example.\nFalse-negative results following sentinel node biopsy are of the order of 5\u201310% in different series.34\u201337 Reasons given to this have been technical; either related to:The surgeons\u2019 experience with the technique orThe size of the radioactive tracer which may not reach the lymph node, especially if sited in a peritumoral position.\nOur results now offer an anatomical explanation for these false negative results as a distinct third possibility.\nPerforating lymphatics that pierce the deep fascia are critical when discussing breast lymph drainage. The lymphatic system is classified conventionally into the superficial system and the deep system because of their relationship to the deep fascia. However, we subdivided the system draining the tissues above the deep fascia into the superficial system and the perforating system (Fig.\u00a09). The perforating system is connected to the deep lymphatic system and these collecting vessels have the same appearance as the superficial lymphatics as they course with the internal thoracic blood vessels. Variations of the blood supply to the breast have been reported.38\u201342 Therefore, we hypothesize that the perforating lymphatics may have the same variations. This helps explain the fact that in all quadrants of the breast, cancer has the potential to metastasize via the internal mammary lymphatics especially if the tumor is medial or deep in the breast parenchyma.43\u201344 It is accepted that intradermal injection of tracer rarely demonstrates the internal mammary lymphatics.10\u201312 The variable contribution of perforating lymphatics along the branches of the internal mammary artery to lymphatic drainage of the breast cannot be predicted clinically and therefore the likelihood of sentinel nodes being located along the internal mammary artery is also unpredictable. Our anatomical analysis suggests that accurate lymphatic mapping requires peritumoral injection.\nFig.\u00a09.Our concept of the breast lymph drainage, drained by both the perforating lymphatic system and the conventional horizontal superficial lymphatic system with their relationship to the lymphatic system beneath the deep fascia.\nConclusions\nCurrent anatomical knowledge of the breast lymphatics is derived from the work of Sappey, Poirier, and Cuneo. Our anatomical cadaver study of the relationship of the lymphatics of the torso and the adult breast goes a step further and provides additional information which has special significance for sentinel lymph node biopsy. We have used a refined protocol to accurately record the anterior upper torso lymphatics in adult male and female cadavers. The patterns of the superficial lymphatics were no different between sexes and frequently more than one sentinel node drained the breast. The cross-sectional studies of the female breast showed some lymph vessels of the torso coursing through the breast tissue. We found also perforating lymphatics coursing with similar branches of the internal mammary artery and vein.\nThis anatomical analysis suggests that (i) peritumoral injection is preferable for identification of the sentinel lymph node for breast cancer treatment and (ii) may help explain the incidence of negative sentinel node studies where only the subareolar plexus is injected with the radioisotope tracer.","keyphrases":["sentinel lymph node biopsy","cadaver study","subareolar injection","lymphoscintigraphy","peritumoral injection","dermal injection"],"prmu":["P","P","P","P","P","R"]}
{"id":"J_Gastrointest_Surg-3-1-1852394","title":"Hemangiopericytoma of the Greater Omentum\n","text":"A 41-year-old Chinese woman was admitted to our hospital with epigastric pain. Computed tomography detected a heterogeneous enhancement tumor fed by the left gastroepiploic artery in the left lower quadrant and cholelithiasis. Excision of the tumor in the greater omentum and cholecystectomy were performed laparoscopically. Histological findings confirmed a diagnosis of hemangiopericytoma with low-grade malignancy. To our knowledge, hemangiopericytoma of the greater omentum is very rare, and only 12 cases were reported in English literature. We report a case of hemangiopericytoma arising in the greater omentum and review the literature.\nIntroduction\nHemangiopericytoma is a rare tumor of the Zimmermann\u2019s pericyte, which was first described by Murray and Stout1 in 1942. Pericytes are rudimentary cells that have contractile properties and regulate the blood flow through capillaries. Although hemangiopericytoma may arise anywhere, the musculature of the lower extremities, the pelvic fossa, and the retroperitoneum are the predominant sites of origin2. The development of hemangiopericytoma in the greater omentum is rare; to our knowledge, only 12 cases were reported in the English literature until the end of 20033\u201311. We report a patient with hemangiopericytoma originating in the greater omentum.\nCase Report\nA 41-year-old Chinese woman was admitted to our hospital with epigastric pain of 6-months in duration. On physical examination, the abdomen was flat and no tumor was palpable. Enhanced computed tomography detected a well-defined tumor with heterogeneous contrast enhancement and no calcifications in the left lower quadrant whose arterial blood supply came from the left gastroepiploic artery (Fig.\u00a01). Cholecystolithiasis was an incidental finding. With a preoperative diagnosis of abdominal stromal tumor of the greater omentum, laparoscopic surgery was performed; a solitary tumor arose with a vascular pedicle originating from the greater omentum, which was free from adjacent organs and structures (Fig.\u00a02). There was no evidence of peritoneal or liver metastases. The tumor was excised with 10\u00a0cm of the vascular pedicle to secure sufficient surgical margin, and cholecystectomy was also performed. The resected tumor was a solid tumor with the largest diameter of 55\u00a0mm, measured 55\u2009\u00d7\u200945\u2009\u00d7\u200940\u00a0mm, weighed 68.5\u00a0g, and was encapsulated without central necrosis or hemorrhage (Fig.\u00a03). On histological examination, hematoxylin\u2013eosin staining demonstrated that spindle cells grew around the vascular endothelial cells, and no mitoses were found in high power fields. Immunohistochemical examination exhibited that the tumor was positive for CD34, factor-XIIIa, and HLA-DR. These findings confirmed a diagnosis of hemangiopericytoma with low-grade malignancy, and the resection margin was clear. The resected gallbladder demonstrated chronic cholecystitis with gallstones. The patient made a satisfactory recovery and was discharged on the fifth postoperative day. Histological findings and absence of mitoses suggests hemangiopericytoma with low-grade malignancy. Therefore, adjuvant chemotherapy was not given. She remains well with no evidence of tumor recurrence 6\u00a0months after resection.\nFigure\u00a01Enhanced computed tomography exhibited a well-defined heterogeneous tumor with contrast enhancement in the left lower quadrant of the abdomen, and demonstrated that the left gastroepiploic artery (arrow) was feeding the tumor.Figure\u00a02A solitary tumor arose in the greater omentum and was connected with the greater omentum by a vascular pedicle.Figure\u00a03The resected tumor measured 55\u2009\u00d7\u200945\u2009\u00d7\u200940\u00a0mm, weighed 68.5\u00a0g, and was solid and encapsulated without central necrosis or hemorrhage.\nDiscussion\nHemangiopericytoma arising in the greater omentum is extremely rare and only 12 cases were reported in the English literature3\u201311. A review of the reported cases revealed that three patients died of recurrence. Therefore, evaluation of the malignant potential seems important. Recent reports proposed that malignant hemangiopericytoma is suspected for tumor size of more than 5\u00a0cm, a high mitotic index with more than four mitoses per ten high power fields, and necrosis and hemorrhage within the tumor12. According to the 13 reported cases3\u201311, tumor size and mitotic index related to tumor recurrence after resection.\nBecause most recurrences developed at distant sites, i.e., the liver, lung, and peritoneum, systemic chemotherapy may be an additional treatment for hemangiopericytoma with high malignant potential after resection and for recurrence. However, effective chemotherapeutic regimens and molecular targeting therapy have not been established to date. Because three of the four patients who underwent omentectomy in the literature had peritoneal recurrences, the significance of omentectomy is questionable, especially for these with low-grade malignancy like in our patient. Therefore, surgical resection provides the only opportunity of cure for patients with hemangiopericytoma arising in the greater omentum. For pedunculated tumors like in our patient, laparoscopic excision is feasible.","keyphrases":["hemangiopericytoma","greater omentum","laparoscopic surgery"],"prmu":["P","P","P"]}
{"id":"Immunogenetics-3-1-1914303","title":"Lung response to Bordetella pertussis infection in mice identified by gene-expression profiling\n","text":"Host genetics determines the course of Bordetella pertussis infection in mice. Previously, we found four loci, Tlr4 and three novel loci, designated Bps 1\u20133, that are involved in the control of B. pertussis infection. The purpose of the present study was to identify candidate genes that could explain genetic differences in the course of B. pertussis infection, assuming that such genes are differentially regulated upon infection. We, therefore, studied the course of mRNA expression in the lungs after B. pertussis infection. Of the 22,000 genes investigated, 1,841 were significantly differentially expressed with 1,182 genes upregulated and 659 genes downregulated. Upregulated genes were involved in immune-related processes, such as the acute-phase response, antigen presentation, cytokine production, inflammation, and apoptosis, while downregulated genes were mainly involved in nonimmune processes, such as development and muscle contraction. Pathway analysis revealed the involvement of granulocyte function, toll-like receptor signaling pathway, and apoptosis. Nine of the differentially expressed genes were located in Bps-1, 13 were located in Bps-2, and 62 were located in Bps-3. We conclude that B. pertussis infection induces a wide and complex response, which appears to be partly specific for B. pertussis and partly nonspecific. We envisage that these data will be helpful in identifying polymorphic genes that affect the susceptibility and course of B. pertussis infection in humans.\nIntroduction\nThe airway pathogen Bordetella pertussis can cause the disease known as whooping cough. Despite high vaccination coverage, pertussis is still an endemic disease with peaks in incidence every 2\u20133\u00a0years during the last decade (Crowcroft and Pebody 2006; de Greeff et al. 2003, 2005). Susceptibility to and severity of B. pertussis infection in infants and children vary widely (Kimman 2001). The spectrum of clinical symptoms ranges from subclinical infection to mild disease, severe whooping cough, and death. In previous studies, we examined genetic susceptibility of mice to B. pertussis and identified loci in the mouse genome that are involved in restriction of colonization and\/or clearance of B. pertussis. We and others have provided indications for the role of several polymorphic host genes in the course of B. pertussis infection, including the interferon gamma receptor (Mahon et al. 1997), toll-like receptor 4 (Tlr4; Higgins et al. 2003; Mann et al. 2004, 2005), and three putative disease loci, B. pertussis susceptibility locus 1, 2, and 3 (Bps-1, 2, and 3; Banus et al. 2005). Tlr4 is a major host factor involved in the course of B. pertussis infection. C3H\/HeJ mice, which carry a nonfunctional Tlr4 gene, exhibit an aberrant course of infection. Functional Tlr4 is essential for an efficient IL1-\u03b2, TNF-\u03b1, and IFN-\u03b3 response, efficient clearance of bacteria from the lung, and therewith reduced lung pathology (Banus et al. 2006). The Bps-1 locus is located on chromosome 12, spanning a region of 185 genes and has a dominant positive effect on the clearance of B. pertussis from the lung. The function of most genes in this locus is unknown. Two other loci, Bps-2 and Bps-3, showed genetic interaction and are located on chromosomes 5 and 11, respectively (Banus et al. 2005).\nThe highest linkage between the number of colony-forming units (CFU) and the Bps-1 locus that we observed showed a logarithm of the odds (LOD) score of 4.6. This LOD score and the corresponding P value (P\u2009=\u20090.000025) are statistically significant also after correction for multiple comparisons (P\u2009=\u20090.0019). We interpreted these data as suggestive for linkage warranting further investigation (Banus et al. 2005). In the present study, we aimed to enhance the insight in the host response and pathogenesis of B. pertussis infection by identifying genes that are differentially expressed upon infection. In particular, we were interested in identifying candidate genes that could explain genetic differences in the course of B. pertussis infection, assuming that such genes are differentially regulated upon infection. For this purpose, we infected mice and analyzed gene-expression profiles in the lungs on days\u00a01, 3, and 5 after inoculation using microarrays. We were especially interested if genes, located in the Bps-1, 2, and 3 loci, were differentially expressed after B. pertussis infection or that the susceptibility of these loci was regulated alternately.\nMaterials and methods\nExperimental design\nFemale C3H\/DISnA mice (eight mice per group, total of 48) were intranasally inoculated with 2\u2009\u00d7\u2009107 CFU of the streptomycin-resistant Tohama strain of B. pertussis (B213) in 40\u00a0\u03bcl of Verwey medium (The Netherlands Vaccine institute, Bilthoven, The Netherlands) or as a control with Verwey Medium only. One, 3, and 5\u00a0days after inoculation, mice were euthanized. To remove blood cells from the lungs, mice were perfused with phosphate-buffered saline, and the lungs and trachea were collected (King et al. 2001; Willems et al. 1998). For the extraction of RNA, the right lung was collected in the RNA stabilization reagent (RNAlater, Qiagen, Venlo, The Netherlands). The left lung was fixed intratracheally using 4% formalin for histological examination.\nThe number of viable B. pertussis bacteria was determined in the trachea to confirm a proper infection (Verwey et al. 1949). Therefore, approximately 1\u00a0cm of the trachea was collected in 500\u00a0\u03bcl of Verwey medium. Bacterial suspensions were diluted in Verwey medium, and the number of CFU was determined by plating on Bordet Gengou agar supplemented with 15% sheep blood and 30\u00a0\u03bcg\/ml streptomycin (Tritium Microbiology, Veldhoven, The Netherlands). Plates were incubated for 4\u00a0days at 35\u00b0C before counting the number of CFUs using a ProtoCOL Colony counter (Synbiosis, Cambridge, UK).\nAnimals\nFemale C3H\/DISnA mice were kindly supplied by Dr. Peter Demant (Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY). Mice were bred to the appropriate numbers at our animal testing facility in Bilthoven, The Netherlands. Mice were acclimatized for at least 1\u00a0week before the start of the experiments. Mice received standard laboratory food (SRM-A, Hope Farms, Woerden, The Netherlands) and tap water ad libitum. All animal experiments were approved by the Institute\u2019s Animal Ethics Committee and were performed according to the National Institutes of Health (1996) guidelines.\nClinical and pathological examinations\nMice were weighed before inoculation and subsequently every day after inoculation to determine the relative change in weight. Lung weights were determined postmortem as a parameter for lung inflammation. Formalin-fixed lungs were embedded in paraplast (Monoject, St Louis, MO). Sections (5\u00a0\u03bcm) were stained with hematoxylin eosin. Lung lesions were examined for infiltration of inflammatory cells in the peribronchiolar space (peribronchiolitis), infiltration of inflammatory cells in the alveoli (alveolitis), infiltration of inflammatory cells in the perivascular space (perivasculitis), hypertrophy of mucus-producing glands, free protein (exudate), and eosinophilia. Lung lesions were scored semiquantitatively as absent, minimal, slight, moderate, marked, or strong as previously described (Barends et al. 2004).\nTranscription profiling\nLungs were incubated in RNAlater (Qiagen) at 4\u00b0C for 1\u00a0day, after which, the tissues were transferred to a fresh tube and stored at \u221280\u00b0C. RNA was extracted by using the midi-RNA isolation kit (Qiagen). RNA concentrations were measured using a NanoDrop Spectrophotometer (NanoDrop Technologies, Wilmington, DE). RNA quality was determined using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and was expressed as the RNA Integrity Number (RIN) as defined by the manufacturer. Only samples with a RIN score of at least eight (on a 1\u201310 scale) were used for expression profiling.\nMicroarray slides were spotted at the Microarray Department of the University of Amsterdam. The slides contain 21,997 65-mer oligo nucleotides from the Sigma-Compugen Mouse oligonucleotide library, 192 additional 65-mer oligo nucleotides of genes, of which 185 were located in the Bps-1 region spotted in duplicate (Isogen Life science, IJsselstein, The Netherlands), as well as appropriate controls and blank spots.\nBased on RNA quality and yield, RNA isolated from six mice per group was selected for amplification and labeling. RNA amplification and labeling were carried out using the Amino Allyl MessageAmp II aRNA kit (Ambion, Austin, TX) using 1\u00a0\u03bcg of total RNA as starting material. RNA samples from individual mice were labeled with Cy3. A common reference containing a RNA pool of all samples isolated was labeled with Cy5. After hybridization and washing, the arrays were scanned using a ScanArray 4000XL microarray scanner (Perkin-Elmer, Wellesley, MA).\nMedian Cy3 and Cy5 spot signals were determined using ArrayVision (Imaging Research, St. Catharines, ON, Canada). Quality control was performed by means of visual inspection of the scanned images, raw data scatter, and MA plots (M is a mnemonic for the log-ratios of expression, A is a mnemonic for the log-intensity of each spot; Smyth and Speed 2003), as well as a normal probability plot to assess signal distribution (Pennings and Heisterkamp 2004). If more than 10% of the spots were flagged as missing data, slides were excluded from data analysis. At least five slides per group were used for further analysis.\nRaw microarray signal data were normalized in the R statistical software environment (R Development Core Team 2005) using a four-step approach of (1) natural log transformation, (2) quantile normalization of all scans, (3) correcting for differences in the reference signal, and (4) averaging replicate spot data.\nSignificance of differences in gene expression between the experimental groups was calculated in R with analysis of variance. The false discovery rate (FDR) was calculated according to Benjamini and Hochberg (1995). The FDR is the expected proportion of false positives among the genes considered significantly differentially expressed (Benjamini et al. 2001; Benjamini and Hochberg 1995). Gene-expression data were visualized by hierarchical clustering (using Euclidian distance and Ward linkage) using GeneMaths (Applied Maths, St-Martens-Latem, Belgium). For this purpose, gene-expression values were corrected for the overall average to allow a neutral comparison between all B. pertussis- and mock-inoculated groups. Gene categories were defined by Gene Ontology (GO, http:\/\/www.geneontology.org). Classification and enrichment according to GO categories were determined using DAVID\/EASE (Dennis et al. 2003; Hosack et al. 2003), using EASE scores as P values for enrichment. Additional pathway analysis was performed using MetaCore\u2122 (GeneGo, http:\/\/www.genego.com).\nResults\nBacteriologic and pathologic findings\nAll B. pertussis-inoculated mice had between 102 and 105 CFUs in the trachea during the first 5\u00a0days after inoculation thereby confirming a proper infection. A statistically significant decrease in the number of bacteria in the trachea was observed over time during the first 5\u00a0days postinoculation (P\u2009=\u20090.022, linear regression, SPSS, data not shown).\nInfected mice lost up to 4% of their body weight during the first 5\u00a0days postinoculation, while all mock-inoculated mice gained weight up to 4% during these 5\u00a0days. This difference in relative gain of weight between mock- and B. pertussis-inoculated mice is significant from day\u00a01 to 5 postinoculation (Supplementary data Figure\u00a02).\nWe determined lung weights in relation to body weight as a marker for inflammation. All mock-inoculated mice had relative lung weights of approximately 1% during the first 5\u00a0days after inoculation, while B. pertussis-infected mice had relative lung weights of up to 3% of their total body weight. This difference in relative lung weights between mock- and B. pertussis-inoculated mice is significant from day\u00a01 to 5 postinoculation (Supplementary data Figure\u00a03).\nA summary of histopathological findings is presented in Table\u00a01. Intranasal inoculation of B. pertussis causes an acute inflammatory response that is characterized by influx of polymorphonuclear leukocytes (PMNs) and macrophages starting in perivascular and peribronchiolar areas on day\u00a01 and extending to alveolar walls and lumina on days\u00a03 and 5.\nTable\u00a01Histological evaluation of lung changes after B. pertussis inoculationParameter and gradeNo. of animalsaMockB. pertussisDay\u00a01Day\u00a03Day\u00a05Day\u00a01Day\u00a03Day\u00a05PeribrochiolitisbMinimal23422Slight125Moderate3Totalc(25%)(0%)(38%)(63%)(88%)(88%)PerivasculitisMinimal22221Slight433Moderate35Marked1Total(25%)(25%)(25%)(75%)(100%)(100%)HypertrophyMinimal1112Slight1Moderate31Marked24Strong1Total(13%)(0%)(13%)(13%)(88%)(88%)AlveolitisMinimal21Slight42Moderate31Marked13Strong31Total(0%)(0%)(0%)(75%)(100%)(88%)ExudateMinimal22Slight22Moderate11Marked1Strong1Total(0%)(0%)(0%)(0%)(100%)(63%)aEight mice per group were inoculated with medium (mock) or B. pertussis.bHematoxylin\/eosin-stained slides were examined for peribronchiolitis, alveolitis, perivasculitis, hypertrophy of mucus-producing glands, and free protein (exudate). Lung lesions were scored semiquantitatively as absent (not included in table), minimal, slight, moderate, marked, or strong.cTotal percentage of animals with at least minimal scoring in the specified category\nExpression profiling\nGene-expression profiles were determined in at least five animals per group. The three mock-inoculated groups, euthanized at days\u00a01, 3, and 5, were statistically indistinguishable (i.e., there were no genes with FDR\u2009<\u20090.6). Of the approximately 22,000 genes tested, 1,841 genes were significantly differentially expressed in B. pertussis-inoculated lungs (FDR\u2009<\u20090.05, corresponding to P\u2009<\u20090.0042). Expression levels of all 1,841 genes as well as comparisons to other studies are presented in the supplementary data Table\u00a05.\nHierarchical clustering showed time-dependent changes in gene expression with 1,182 upregulated genes and 659 downregulated genes. Of these genes, 439 were more than twofold upregulated, and 134 were more than twofold repressed. All genes followed a similar trend in time. One day postinoculation differential gene expression was already observed, but the effect was larger on days\u00a03 and 5 postinoculation (Fig.\u00a01). There was no identifiable subset of genes that displayed a different kinetic trend in gene expression.\nFig.\u00a01Presentation of microarray data from mock- and B. pertussis-inoculated mice. Analysis was performed on 1,841 genes whose expression was found to be significant at an FDR\u2009<\u20090.05. a Hierarchical clustering shows the segmentation of the genes clustered by gene expression. Relatively increased gene expression is presented in red, while relatively decreased gene expression is presented in green. The color scale shows log(2) ratios of gene expression levels compared to the global average. b Course of B. pertussis-regulated gene expression; diamonds represent the average of 1,182 upregulated genes, and squares represent the average of 659 downregulated genes. Error bars show the standard deviation (SD) of the average gene expression\nTo assign functions to differentially expressed genes, we classified them according to GO categories. In addition, we determined enrichment for GO categories to identify pathophysiological processes involved in response to B. pertussis. The most important GO categories, i.e., with the highest percentage differentially expressed genes, are summarized in Table\u00a02. Most upregulated genes are involved in immune response-related processes, such as the acute phase response, antigen presentation, cytokine activity, inflammation, and apoptosis; whereas downregulated genes are involved in nonimmune processes, such as development, muscle contraction, cytoskeleton, and general processes like metabolism.\nTable\u00a02Classification of genes induced upon B. pertussis infectiona\u00a0UpDownbTotal on array P valuecAcute-phase responsed12(55%)0(0%)222.4\u2009\u00d7\u200910\u22127 (up)Antigen presentation21(66%)0(0%)327.7\u2009\u00d7\u200910\u221215 (up)Apoptosis47(15%)6(2%)3248.0\u2009\u00d7\u200910\u22125 (up)Cell cycle43(8%)22(4%)5104.3\u2009\u00d7\u200910\u22121 (up)Chemokine activity21(58%)0(0%)361.2\u2009\u00d7\u200910\u221213 (up)Complement activation8(26%)1(3%)319.7\u2009\u00d7\u200910\u22123 (up)Cytokine activity48(25%)6(3%)1927.8\u2009\u00d7\u200910\u221213 (up)Cytoskeleton31(6%)40(8%)5312.0\u2009\u00d7\u200910\u22123 (down)Development96(7%)94(7%)1,4339.8\u2009\u00d7\u200910\u22121 (down)Immune response162(40%)3(1%)4086.3\u2009\u00d7\u200910\u221275 (up)Inflammatory response46(38%)2(2%)1211.1\u2009\u00d7\u200910\u221219 (up)Metabolism353(7%)220(5%)4,7711.8\u2009\u00d7\u200910\u22121 (down)Muscle contraction0(0%)12(24%)496.7\u2009\u00d7\u200910\u22126 (down)Listed in any of the above576(9%)318(5%)6,574Other328(6%)204(4%)5,639Unannotated278(3%)137(1%)9,744Total1,182(5%)659(3%)21,957aClassification according to gene-ontology (GO) categoriesbAnalysis was performed on 1,841 regulated genes.cEase score for GO category enrichment in the specified set of genesdSome genes are classified into multiple categories.\nTo reveal intergene relationships, we performed MetaCore\u2122 analysis on the 1,841 regulated genes in addition to the GO-based analysis presented above. The most significant pathways are presented in Table\u00a03. This analysis revealed the significance of several pathways, such as regulation of granulocyte function, the toll-like receptor (TLR) signaling pathway, the classic complement pathway, and apoptosis.\nTable\u00a03Pathway analysis of genes induced upon B. pertussis infectionName of Pathway involvedaMain processP valueNo. of genes in pathwayExpressedTotalTranscription regulation of granulocyte developmentImmune response0.00000192517(38%)45TLR ligands and common TLR signalling pathwayImmune response0.0000181416(34%)47ECM remodelingMetabolism0.0000390618(30%)60Role of IAP proteins in apoptosisApoptosis0.0000545813(36%)36HETE and HPETE diosynthesis and metabolismMetabolism0.000324913(31%)42TPO signaling via JAK-STAT pathwayCell cycle0.00038939(39%)23Leukotriene 4 biosynthesis and metabolismMetabolism0.000465516(27%)60RhoA regulation pathway (extension, GEFs\/GAPs)Immune response0.00241210(29%)34MIF in innate immunity responseImmune response0.00243514(25%)57Retinol metabolismMetabolism0.00284212(26%)46Classic complement pathwayComplement activation0.00284212(26%)46Putative erythropoietin signaling pathway (part 1)Immune response0.00290513(25%)52MAPK cascadeCell cycle0.00497512(24%)49Ceramide-dependent NO antiapoptotic actionApoptosis0.00673813(23%)57IL9 signaling pathwayCytokine activity0.0068099(27%)33Ephrins signalingOther0.00784713(22%)58Polyamine metabolismMetabolism0.0087317(30%)23Integrin outside-in signalingCell cycle0.00944416(20%)79Chemokines and adhesionChemokine activity0.00965734(16%)209Analysis was performed on 1,841 genes whose expression was found to be significant at an FDR\u2009<\u20090.05. The list is presented by descending P values. P values represent the probability of particular mapping arising by chance given the numbers of genes in all Metacore pathways, genes involved in the specified pathway, and genes regulated upon B. pertussis inoculation.ECM Extracellular matrix, GAP GTPase-accelerating protein, GEF guanine nucleotide exchange factor, HPETE hydroperoxyeicosatetraenoic acid, HETE hydroxyeicosatetraenoic acid, IAP inhibitor of apoptosis, IL9 interleukin 9, JAK Janus tyrosine kinase, MIF macrophage migration inhibitory factor, MAPK mitogen-activated protein kinase, NO nitric oxide, RhoA ras homolog gene family member A, STAT signal transducers and activators of transcriptionaClassification according to main gene-ontology (GO) categories\nGenes located in Bps-1 that were differentially regulated upon B. pertussis inoculation and their function according to the GO database are presented in Table\u00a04. Bps-1 contains 185 genes, of which nine were differentially expressed. The strongest upregulated genes in this locus were tryptophanyl-tRNA synthetase (Wars, fourfold) and tumor necrosis factor alpha-induced protein 2 (Tnfaip2, 5.4-fold). Zinc finger FYVE domain containing 21 (Zfyve21, 1.5-fold) and kinesin family member 26A (Kif26a, 1.7-fold) were the strongest downregulated genes.\nTable\u00a04Genes located in Bps-1 that are differentially regulated upon B. pertussis inoculationGenBank accessionGene symbolLocationaFold ratio changeP valueDescriptionPathwaybDay\u00a01Day\u00a03Day\u00a05NM_011705Vrk11061.291.521.410.00003Vaccinia-related kinase 1Protein kinase activityNM_011710Wars1091.953.584.010.00000Tryptophanyl-tRNA synthetaseAminoacyl-tRNA ligase activityNM_010480Hspca111\u22121.141.401.270.00064Heat shock protein 1, alphaProtein foldingNM_009396Tnfaip21121.434.485.400.00000Tumor necrosis factor, alpha-induced protein 2AngiogenesisXM_138272Gm266112\u22121.08\u22121.25\u22121.350.00110Gene model 266, (NCBI)G-protein coupled receptor activityNM_026752Zfyve21112\u22121.33\u22121.41\u22121.500.00001Zinc finger, FYVE domain containing 21Zinc ion bindingXM_138275Kif26a113\u22121.32\u22121.68\u22121.730.00030Kinesin family member 26AMicrotubule motor activityNM_178911Pld41131.211.822.520.00000Phospholipase D family, member 4Catalytic activityXM_619046LOC4341661131.131.421.420.00141Similar to hypothetical protein MGC37588UnknownaBps-1 is located on chromosome 12; location is presented as distance from centromere (Mb).bClassification according to gene-ontology (GO) categories\nGenes located in Bps-2 and -3 that were differentially regulated upon B. pertussis inoculation are presented in Supplementary data Table\u00a06. Bps-2 is located on chromosome 5 and contains 262 genes, of which 13 were differentially expressed. Interleukin-6 (IL-6) was the strongest upregulated gene (4.7-fold) in this locus, and general transcription factor IIIC, polypeptide 2, beta was the strongest downregulated gene (twofold) in this locus. Bps-3 is located on chromosome 11 and contains 922 genes, of which 62 were differentially expressed. Chemokine (C\u2013C motif) ligand 8 was the strongest upregulated gene (15.3-fold) in this locus, and RIKEN cDNA 9530033F24 gene was the strongest downregulated gene (2.3-fold) in this locus.\nDiscussion\nWe have recently shown that genetic differences of the host play an important role in the restriction of colonization and\/or clearance of B. pertussis from the lungs of mice (Banus et al. 2005, 2006). In the present study, we wanted to extend the understanding of the host response to B. pertussis and identify genes and pathways that play a role in the pathophysiological response to B. pertussis infection. In particular, we were interested in identifying candidate genes that could explain genetic differences in the course of B. pertussis infection, assuming that such genes are differentially regulated upon infection. For these purposes, we studied the time course of mRNA-expression in the lungs of mice after a B. pertussis infection by microarray analysis. We were especially interested if genes, located in the Bps-1, 2, and 3 loci, were differentially expressed after B. pertussis infection or that the susceptibility conferred by these loci was regulated alternately.\nOf the approximately 22,000 genes investigated, we found 1,841 (8.4%) genes to be significantly differentially expressed. Differential gene expression was already observed on day\u00a01 postinoculation, but the maximum effect was observed on day\u00a03 postinoculation. This effect remained constant till at least day\u00a05 postinoculation. The observed course in gene expression after B. pertussis inoculation was similar to that induced by another gram-negative lung pathogen, Francisella tularensis (Andersson et al. 2006).In a study of expression profiles in the lungs of F. tularensis-infected C57BL\/6 mice, 424 (2.3%) genes out of the 18,500 genes investigated were upregulated. Genes involved in immune responses were activated strongly on day\u00a04 postinoculation but hardly or not at all on days\u00a01 and 2 (Andersson et al. 2006).\nAs expected, most upregulated genes are involved in immune and inflammation-related processes, such as antigen presentation, complement, cytokine, and chemokine activity. Other processes involved include the acute phase response and metabolism. Most downregulated genes are involved in nonimmune processes, such as metabolism, development, cell cycle, or muscle contraction. We speculate that by downregulating these genes, the energy balance is shifted in favor of the immune response. Evidently, cellular influx in the lungs is likely to influence the gene-expression profile.\nOur data provide more detailed information on the central role of PMNs in the pathogenesis of B. pertussis infection. Pathogenesis of B. pertussis infection is characterized by colonization and proliferation of the ciliated mucosal cells resulting in damage of the respiratory epithelium and an acute increase in the levels of inflammatory cytokines resulting in cellular infiltrate in the alveolar spaces (Mattoo and Cherry 2005; Mills 2001; Preston 2005). Circulating PMNs are rapidly recruited to the lungs to bind and ingest B. pertussis, subsequently killing the bacteria by a combination of reactive oxygen and granule components. Finally, the PMNs undergo apoptosis (Kobayashi et al. 2005a). TLR ligands such as lipopolysaccharides (LPS) are critical components for the recruitment and priming of PMNs. Serum antibody-mediated clearance of B. pertussis also requires a TLR-induced early recruitment of PMNs. However, pertussis toxin limits this rapid serum antibody-mediated clearance by inhibiting PMN recruitment (Kirimanjeswara et al. 2005). As determined by pathway analysis in this study, the most significant pathways involved in the response to B. pertussis infection are regulation of PMN function, TLR signaling pathway, extracellular matrix remodeling, and apoptosis. It has been described that apoptosis of PMNs, in which TLR2 plays an important role (Jablonska et al. 2006), is accelerated after phagocytosis of bacteria (Kobayashi et al. 2005a, b). Therefore, the involvement of three out of four pathways may, at least partly, be explained by the influx of PMNs. Our data thus further illustrate the central role of PMNs in the course of B. pertussis infection and the involved mechanisms in PMN function.\nWe found the response to B. pertussis to be wide and complex. Some remarkable findings were the involvement of mucin genes, intracellular pathogen resistance gene, and the excessive upregulation of serum amyloid A (SAA) genes. We found the mucin (Muc) genes Muc4 and Muc5b to be significantly upregulated. Expression of mucin in the respiratory tract is one important innate host defense mechanism that respiratory pathogens must overcome (Belcher et al. 2000). Muc4, which encodes a respiratory tract mucin glycoprotein, was expressed 3.3-fold at day\u00a05 upon B. pertussis inoculation. Muc5b, which encodes mucin 5, subtype B protein, in tracheobronchial epithelial cells, was expressed 1.8-fold 3\u00a0days upon inoculation. Belcher et al. (2000) described that B. pertussis induces mucin gene transcription by BEAS-2B cells. Evidently, gene expression studies do not allow to define the functional role of regulated genes. Further studies should define whether the mucin response is favorable to the host, the pathogen, or both.\nIn our study, we found a threefold upregulation of the intracellular pathogen resistance gene 1 (Ipr1). Ipr1 mediates innate immunity to mycobacterium tuberculosis (Pan et al. 2005). Pan et al. (2005) speculated that the Ipr1 gene product might function in integrating signals generated by intracellular pathogens with mechanisms controlling innate immunity, cell death, and pathogenesis. Our data therefore suggest that Ipr1 may be involved in intracellular presence of B. pertussis. Interestingly, B. pertussis was shown to survive intracellularly in macrophages, PMNs and bronchoalveolar lavage (BAL) cells of mice (Hellwig et al. 1999; Mills 2001).\nAmong the upregulated genes upon B. pertussis inoculation, SAA genes were upregulated to a remarkably high extent. SAA is an acute phase protein whose expression is markedly upregulated during inflammation and infection (Uhlar and Whitehead 1999). Among other functions, this protein plays an important role in high density lipoprotein cholesterol metabolism by promoting cellular cholesterol efflux through a number of different efflux pathways (van der Westhuyzen et al. 2005). In our study, SAA1, 2, and 3 were induced 14-, 54-, and 44-fold, respectively, after B. pertussis infection, again underlining the important role for the acute phase response after B. pertussis infection. Although strongly upregulated, it is clear that the acute-phase response is not specific for B. pertussis infection.\nIn a previous study, we observed a 1.5-fold upregulation of the membrane-receptor Tlr4 and a fivefold upregulation of the downstream cytokine gene Tnf-\u03b1 3\u00a0days post-B. pertussis inoculation by means of quantitative real-time polymerase chain reaction (PCR). Thus, a slight upregulation (1.5-fold) of this receptor induces a more pronounced (fivefold) induction of this downstream proinflammatory cytokine (Banus et al. 2006). In the present study, we obtained similar results by using microarray analysis. Tlr4 expression was increased 1.6-fold, while the expression of Tnfaip was increased 4.5-fold 3\u00a0days postinoculation. The results of the present study hereby confirm our previous observations. In a recent study, a high correlation between gene-expression values determined by quantitative real-time PCR and microarray was observed (Canales et al. 2006). Given these results and our observations, we conclude that the results, at least for the two given genes, are similar for both techniques.\nIt is very likely that the expression profile to B. pertussis is partly unique to B. pertussis and partly nonspecific. To examine the specificity of B. pertussis-regulated gene expression, we compared our data to other studies. In a recent review by Jenner and Young (2005), a comparison was made between published transcriptional profiling data from 32 studies that involved 77 different host\u2013pathogen interactions. A set of 417 genes that comprise a common host transcriptional response was defined. We found that 122 genes affected by B. pertussis infection were listed in this common host\u2013pathogen response, further illustrating that many genes regulated by B. pertussis are indeed not specific for B. pertussis infection but are involved in a common response to pathogens. One explanation for the broad response in expression profiles is the complexity of the bacterium. It is, for instance, known that LPS is a very potent immune stimulus, which induces a strong inflammatory response (Kawai et al. 2001; Qin et al. 2005; Wells et al. 2003). Transcriptional profiling of LPS-induced acute lung injury in mice resulted in 71 immune-related upregulated genes (Jeyaseelan et al. 2004). Of these 71 genes, 43 were also regulated upon B. pertussis inoculation in this study.\nPreviously, we have demonstrated that the Bps-1 locus has a dominant positive effect on the clearance of B. pertussis from the lung. Of the 185 genes in this locus, nine were differentially expressed. Some of these genes could explain the linkage of this locus with susceptibility to B. pertussis infection. Tumor necrosis factor, alpha-induced protein 2 (Tnfaip2), has been described as a tumor necrosis factor-alpha-inducible primary response gene in endothelial cells (Sarma et al. 1992). Aminoacyl-tRNA synthetases catalyze the aminoacylation of tRNA with their cognate amino acid. Tryptophanyl-tRNA synthetase (Wars) catalyzes the aminoacylation of tRNA(trp) with tryptophan and is induced by interferon (Ibba and Soll 2000).The role of the nine genes in the B. pertussis infection process is unknown and will be investigated in future experiments. Thus, by using a microarray approach, we selected nine possible candidate genes from the 185 present in Bps-1. However, by this approach, we focused on transcriptionally regulated genes, and the possibility remains that susceptibility to B. pertussis is regulated in a different way, such as phosphorylation or protein binding.\nIn comparison to the Bps-1 region, the Bps-2 and -3 regions contain considerably more genes, namely, 262 and 922 genes, respectively, of which 13 and 62 were differentially expressed post-B. pertussis inoculation. Among these differentially expressed genes are several interesting candidate genes that may explain the linkage of these loci with susceptibility to B. pertussis infection. IL-6 was the most strongly induced gene in the Bps-2 region (4.7-fold). IL-6 is a proinflammatory cytokine that can be secreted by macrophages after Tlr4 activation (Akira and Takeda 2004). Among the genes differentially expressed in the Bps-3 region was a large group of chemokines, of which Chemokine (C\u2013C motif) ligand 8 was the strongest upregulated gene (15.3-fold). Upon B. pertussis infection, this large group of chemokines is induced to promote immune cells to migrate to the site of infection. The role of the other B. pertussis-induced genes in these loci is still unknown and will be investigated in future experiments.\nIn conclusion, we found 1,841 genes in the lungs of mice that are differentially expressed after B. pertussis inoculation. Most upregulated genes are involved in immune and inflammation-related processes or in nonspecific processes. Most downregulated genes are involved in nonimmune processes. A substantial number of genes and pathways provide more detailed information on the central role of PMNs in the pathogenesis of B. pertussis. In particular, the transcriptional profiles indicate the significance of genes involved in TLR activation, the recruitment and activation of PMNs, and apoptosis.\nElectronic supplementary material\nRaw data as well as the detailed description of the experiment will be uploaded to the freely accessible online database ArrayExpress (\nhttp:\/\/www.ebi.ac.uk\/arrayexpress\/\n) upon acceptance of the manuscript. Expression levels of all 1,841 genes as well as comparisons to other studies are presented in the supplementary data Table\u00a0\n5\n.\nSupplementary data Fig.\u00a02\nGain of body weight after infection. Relative gain of body weight at different days postinoculation with medium (mock) or B. pertussis. Boxes represent the mean of the relative weights of B. pertussis infected mice; diamonds represent the mean of the relative weights of mock inoculated mice (GIF 10\u00a0kb).\nHigh resolution image file (EPS 493\u00a0kb)\nSupplementary data Fig. 3\nLung weight after infection. Relative lung weight of mice after infection with B. pertussis at several days postinoculation. Solid bars represent the mean relative weight of B. pertussis infected mice; grey bars represent the mean relative lung weight of mock inoculated mice (GIF 13\u00a0kb).\nHigh resolution image file (EPS 680\u00a0kb).\nSupplementary data Table\u00a05\nAll B. pertussis induced genes and overlapped with other studies. a1,841 genes induced upon B. pertussis inoculation whose expression was found to be significant at an FDR\u2009<\u20090.05, of which 1,395 had an FR higher then 1.5; bJenner and Young (2005); cPalmer et al. (2006) BMC Genomics; dB cells and following columns are subsets described by Palmer et al. (2006) (XLS 665\u00a0kb)\nSupplementary data Table\u00a06\nGenes located in Bps-1, -2, and -3 that are differentially regulated upon B. pertussis inoculation (XLS 25\u00a0kb)","keyphrases":["bordetella pertussis","expression profiles","immunity","pathway analysis","gene expression"],"prmu":["P","P","P","P","P"]}
{"id":"Neurogenetics-3-1-1820748","title":"Functional analysis of a novel potassium channel (KCNA1) mutation in hereditary myokymia\n","text":"Myokymia is characterized by spontaneous, involuntary muscle fiber group contraction visible as vermiform movement of the overlying skin. Myokymia with episodic ataxia is a rare, autosomal dominant trait caused by mutations in KCNA1, encoding a voltage-gated potassium channel. In the present study, we report a family with four members affected with myokymia. Additional clinical features included motor delay initially diagnosed as cerebral palsy, worsening with febrile illness, persistent extensor plantar reflex, and absence of epilepsy or episodic ataxia. Mutation analysis revealed a novel c.676C>A substitution in the potassium channel gene KCNA1, resulting in a T226K nonconservative missense mutation in the Kv1.1 subunit in all affected individuals. Electrophysiological studies of the mutant channel expressed in Xenopus oocytes indicated a loss of function. Co-expression of WT and mutant cRNAs significantly reduced whole-oocyte current compared to expression of WT Kv1.1 alone.\nIntroduction\nMyokymia is defined as spontaneous involuntary muscle fiber group contraction, is visible as vermiform movement of the overlying skin, and may be hereditary or acquired. Electromyography (EMG) shows continuous motor unit activity with spontaneous discharges of high intraburst frequency. Myokymia occurs in autoimmune disease with antibodies to voltage-gated potassium channel subunits in Isaacs\u2013Merton syndrome and Morvan fibrillary chorea [1]. Hereditary myokymia with episodic ataxia type 1 (OMIM #160120) arises from mutations in the voltage-gated potassium channel KCNA1 [2]. The ataxia is characterized by profound attacks of cerebellar ataxia lasting seconds to minutes. Partial epilepsy has been reported in some families with KCNA1-associated disease [3, 4]. Another rare cause of myokymia with neonatal epilepsy but lacking episodic ataxia results from a KCNQ2 potassium channel mutation [5]. Reduced frequency and severity of myokymia and episodic ataxia have been reported after treatment with acetazolamide or carbamazepine [3, 6].\nApproximately a dozen missense mutations and one truncating mutation in KCNA1 have been reported [3, 4, 7\u201310]. The functional consequences of human KCNA1 mutations causing typical myokymia and EA1 have been reported [11], much less is known about the functional consequences of mutations causing unusual KCNA1-associated phenotypes.\nIn the present study, we describe the clinical and genetic features of a new family with hereditary myokymia lacking epilepsy or episodic ataxia. Molecular studies of KCNA1 in the proband revealed a c.676C>A transversion resulting in the substitution of Lys for Thr at codon 226 (T226K) and a c.1355A>C transversion resulting in Y452S. Further genetic studies showed that all affected family members carried the T226K mutation, which was absent in unaffected family members and ethnically matched normal controls, whereas the Y452S substitution was present in both affected and unaffected family members and normal controls. In transfected mammalian cells, electrophysiologic studies demonstrated functional effects of the c.676C>A transversion with no currents above background levels recorded from oocytes injected with the mutant subunit. Co-injection of the wild-type and mutant cRNAs produced significantly smaller whole-oocyte currents than injection with the WT cRNA alone.\nMaterials and methods\nClinical data\nThe family pedigree is shown in Fig.\u00a01. Six family members (two unaffected and four affected) were available for clinical and genetic studies approved by the Yale Human Investigations Committee.\nFig.\u00a01Family pedigree. Squares indicate males, circles indicate females. Blackened symbols denote individuals with myokymia. Alleles at codon 226 are indicated (Thr = wild-type, Lys = mutation)\nSubject II-1\nThe proband, a 13-year-old boy (II-1), was the product of an unremarkable pregnancy and delivery. He was diagnosed with cerebral palsy because of leg stiffness and delayed walking at 18\u00a0months old. Cognitive development was normal; his medical history was significant for esotropia. At 4\u00a0years old, he was hospitalized with increasing leg pain, stiffness, and inability to walk during the course of a flu-like illness. Creatine kinase (CK) was elevated at 520\u00a0U\/l acutely (normal <195\u00a0U\/l), but was normal when repeated interictally. Examination showed periorbital myokymia, mild abdominal and leg muscle hypertrophy, leg stiffness, spastic gait, hyperreflexia, and bilateral Babinski sign. Magnetic resonance imaging (MRI) of the brain and spine were normal. Metabolic studies including electrolytes, amino and organic acids, carnitine, ammonia, lactate, and thyroid functions were normal. Routine studies of cerebral spinal fluid and an electroencephalogram were normal. Needle EMG of deltoid and iliopsoas muscles showed irregular, polyphasic continuous motor unit discharges with normal interference pattern.\nAdditional family members\nSix family members were evaluated. Four affected members (mother and three sons) had myokymia on clinical examination, EMG examination, or both. Nerve conduction velocities were normal. There was no history of seizures or episodic ataxia in any of them. The mother (I-2, Fig.\u00a01) is a 40-year-old woman who had had muscle twitching since childhood, hyperreflexia, bilateral Babinski sign, and tendency to run on her toes. Her husband is unaffected and had a normal neurological exam. A 12-year-old son (II-2) also had delayed walking, leg stiffness, and was diagnosed with cerebral palsy. At age 3\u00a0years, he had an episode of prolonged generalized paralysis with no muscle response to tetanic stimulation after general anesthesia for esotropia surgery. He recovered within 1\u00a0day. MRI of the brain and spine were normal. An 8-year-old daughter (II-3) is unaffected. A 5-year-old son (II-4) had symptoms identical to his brothers and was hospitalized twice for episodes of increased muscle stiffness with presumed viral gastroenteritis, elevated CK during both episodes (606\u00a0U\/l and 997\u00a0U\/l) but had normal CKs measured while well. Carbamazepine treatment resulted in marked improvement of muscle symptoms in three of the brothers who remain asymptomatic. The mother, with milder symptoms, declined treatment. Three family members with myokymia had esotropia; one patient required three eye surgeries.\nMolecular studies\nGenomic DNA was extracted from blood samples or buccal swabs. The coding region of the single-exon 1,448-bp gene KCNA1 (GenBank sequence NM_000217) was amplified. Polymerase chain reactions (PCRs) were performed in a 25-\u03bcl reaction volume containing 50\u00a0ng genomic DNA, 50\u00a0ng of each primer, 1.5\u00a0mM MgCl2, 200\u00a0\u03bcM dNTPs, 1\u00d7 PCR buffer (Finnzyme), and 2.5\u00a0U of Finnzyme using a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA). Cycling parameters consisted of a modified touchdown (stepdown) protocol as follows: an initial hot start denaturation at 94\u00b0C for 5\u00a0min (hot start); 94\u00b0C for 45\u00a0s, annealing temperature (three cycles each at 70, 67, 64, 61,and 58\u00b0C) for 45\u00a0s, and extension at 72\u00b0C for 2\u00a0min for a total of 15 cycles followed by 25 cycles of 94\u00b0C for 45\u00a0s, 55\u00b0C for 45\u00a0s, 72\u00b0C for 1\u00a0min; a final extension step of 72\u00b0C for 10\u00a0min followed by a 4\u00b0C hold. PCR products were separated by electrophoresis on a 1% agarose gel and visualized by ethidium bromide staining. PCR products were purified by spin column (Qiagen, Valencia, CA) or by enzymatic method (ExoSAP-IT, USB, Cleveland, OH) and analyzed for mutations by automated sequencing (Applied Biosystems, Foster City, CA). The c.1355A>C transversion resulted in the creation of a DdeI restriction site, so the presence of the sequence variant was tested by sequencing and confirmed with restriction digest in all subjects. Restriction digests were performed with DdeI (New England Biolabs, Beverly, MA) at 37\u00b0C for 2\u00a0h.\nOocyte expression and electrophysiological studies\nHuman Kv1.1 cDNA was subcloned into Kpn1 and HindIII sites of psGEM. The Kv1.1-T226K mutation was introduced by site-directed mutagenesis and confirmed by automated sequencing. cRNAs were synthesized using an mMessage mMachine kit (Ambion, Austin, TX) and quantified by spectroscopy.\nOocytes were isolated from Xenopus laevis and defolliculated by collagenase treatment. Each oocyte was injected with either 5\u00a0ng of human Kv1.1-WT, 5\u00a0ng Kv1.1-T226K, or 2.5\u00a0ng WT plus 2.5\u00a0ng T226K cRNA. Whole-oocyte currents were measured with two electrode voltage clamp techniques (Oocyte Clamp, Warner Instruments, Hamden, CT) with constant perfusion (1\u00a0ml\/min, solution exchange\\3\u00a0s) after 16\u00a0h. Data were sampled at 1\u00a0kHz and filtered at 0.25\u00a0kHz. Standard bath solution was ND-96 (in mM): 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, and 5 HEPES\/NaOH, pH\u00a07.5.\nResults\nMutation analysis of KCNA1\nMutation analysis of KCNA1 identified two sequence variants: a c.676C>A transversion resulting in the substitution of Lys for Thr at codon 226 (T226K) and a c.1355A>C transversion resulting in Y452S and creating a DdeI restriction site. Either a Tyr (NM_000217) or a Ser (AAI01734) had been reported at codon 452, but the population frequency and association with disease were unknown. Therefore, we tested all six family members (four affected, two unaffected) and 10 normal controls for the SNP and found that all 16 subjects were homozygous for the Y452S substitution, compatible with a common polymorphism. In contrast, all (4\/4) affected subjects were heterozygous for the previously unreported T226K missense mutation, which was absent in both unaffected family members and absent in 190 chromosomes from ethnically matched normal controls (Fig.\u00a02).\nFig.\u00a02Sequence chromatograms demonstrating c.676C>A resulting in T226K substitution in KCNA1. A DNA sequence from an affected patient (I-2) heterozygous for the mutation (top) and from a normal control (bottom) are presented\nElectrophysiological studies of the human Kv1.1 mutant The genetic studies of the segregation of the T226K mutation favored it as a pathological mutation, but given that two missense mutations were identified, we sought additional supporting evidence. We tested whether the missense mutation alters human Kv1.1 channel function by expressing Kv1.1-T226K in Xenopus oocytes. No currents above background levels were recorded from Kv1.1-T226K cRNA-injected oocytes, whereas Kv1.1 wild-type channels produced typical delayed rectifier currents (Fig.\u00a03). We then mimicked the heterozygous situation by co-injecting equal amounts of Kv1.1 WT and T226K mutant cRNAs. Co-injection produced significantly smaller whole-oocyte currents than injection with Kv1.1 WT cRNA alone (Fig.\u00a03c and d). These results indicate that the T226K mutation significantly reduced efflux of potassium ions during depolarization and is therefore likely to result in increased muscle cell excitability.\nFig.\u00a03Missense mutation T226K induces loss of function of human Kv1.1 channels expressed in Xenopus oocytes. a\u2013c Representative family of whole-oocyte currents recorded from oocytes expressing human Kv1.1-WT (a), Kv1.1-T226K (b), and both Kv1.1 WT and T226K (c). Currents were evoked by a series of 350-ms test pulses from \u221280 to +60\u00a0mV in 10-mV increments from a hold potential of \u221290\u00a0mV. Tail currents were recoded at \u221240\u00a0mV. Scale bar, 100\u00a0ms and 1\u00a0\u03bcA. d I\u2013V curves for three experimental conditions in a\u2013c. Data were collected for 10\u201315 oocytes in each group. The peak currents in a and c are significantly different at test voltages between 0 and +60\u00a0mV (P\u2009<\u20090.05)\nDiscussion\nMutations in KCNA1 are typically associated with episodic ataxia type 1 (EA1), characterized by profound cerebellar ataxia lasting seconds to minutes and interictal myokymia [6]. The expanded phenotypic spectrum associated with proven KCNA1 mutation includes a family with prolonged episodes of episodic ataxia and epilepsy, but without myokymia [12]. Other families have been reported with myokymia and adducted thumbs, congenital contractures, kyphoscoliosis, or epilepsy with myokymia [4, 7\u201310]. We report a family with autosomal dominant myokymia lacking either ataxia or epilepsy. The mother had self-described lifelong \u201cmuscle twitchiness\u201d but never sought medical attention for her condition until the hospitalizations of her sons. Myokymia in the periorbital region and on EMG were the features that led to genetic studies of KCNA1.\nAlthough the stiffness, toe walking, and esotropia could be caused by continuous muscle contraction, the extensor plantar reflex [observed on multiple exams by experienced two neurologists (LRM and FMH)] is a new finding for KCNA1-associated disease and suggests involvement of the corticospinal tract. The KCNA1 knockout mouse has been reported to exhibit altered inhibition of layer V pyramidal neocortical neurons [13].\nKCNA1 on chromosome 12p13 encodes Kv1.1, a potassium channel subunit. Delayed-rectifier voltage-gated-type potassium channels are important in the rapid neuronal repolarization after an action potential and are multimeric proteins containing four subunits around a central pore. The Kv1.1 potassium channel subunit is expressed during development in neuronal and glial cells in the central and peripheral nervous systems [14]. Kv1.1 plays an important role in regulating neuronal excitability in the central nervous system, as shown by studies of the KCNA1 knockout mouse with an increased susceptibility to seizures [15].\nThe present family has generalized myokymia due to a T226K mutation in the voltage-gated potassium channel KCNA1. Unusual clinical features include pyramidal dysfunction and worsening with febrile illness or anaesthesia. One previously reported 3-year-old boy presented in a similar fashion to our patients with a 12-h episode of inability to walk, increased tone, and elevated CK during a febrile illness [3]. No anesthesia exposure was reported. That child had a P244H mutation in the intracellular loop between transmembrane segments 2 and 3. The whole-cell current amplitudes elicited by injection of P244H did not differ from wild-type amplitudes. Co-injection of P244H plus an equal amount of wild-type cRNA yielded a peak current amplitude that was 200% of wild-type alone.\nWe identified a novel, nonconservative T226K missense mutation that is located in the second transmembrane domain of Kv1.1 and is invariant from Drosophila melanogaster to mice and humans [6]. In contrast to the functional effects reported for the P244H mutation, heterologous expression of the T226K mutation identified in the present family showed that the mutant potassium channel expressed in oocytes demonstrated no current above background levels, and its co-expression with the wild-type channel significantly reduced whole-oocyte current, compatible with a severe effect of the mutation. The T226K mutation is likely to reduce efflux of potassium ions during depolarization, thereby increasing cellular excitability. The phenotypic similarity between the P244H and the T226K mutations contrasts with their biophysical effects. Therefore, additional factors must contribute to the functional effects of these mutations. Kv1.1 forms heteromeric channels with other subunits and interacts with other accessory subunits [16]. In addition to their well-known role as ion channels that regulate neuronal firing, potassium channels have also been shown to activate enzymes linked to intracellular signaling pathways [16, 17]. One of these novel interactions may contribute to differences in the manifestation of disease-causing mutations.","keyphrases":["potassium channel","mutation","myokymia"],"prmu":["P","P","P"]}
{"id":"Crit_Care-9-1-1065098","title":"Bench-to-bedside review: Ethical challenges for those in directing roles in critical care units\n","text":"Though much attention in the medical literature has focused on the ethics of critical care, it seems to be disproportionately weighted toward clinical issues. On the presumption that the operational management of an intensive care unit (ICU) also requires ethical considerations, it would be useful to know what these are. This review undertook to identify what literature exists with regard to the non-clinical issues of ethical importance in the ICU as encountered by clinician\u2013managers. We found that in addition to issues of resource allocation, there exist many areas of ethical importance to clinician\u2013managers in the ICU that have been described only superficially. We argue that a renewed focus on ICU ethics is merited to shed light on these other, non-clinical, issues.\nIntroduction\nAs a specialized field of philosophy, ethics has demanded that more institutions self-assess their actions so as to implement and maintain ethical practice (see the Additional file for definitions of 'ethical practice' and 'ethics'). In healthcare, technological and bureaucratic complexities have created dilemmas never before encountered, at least on the scale in which they now occur. Nowhere are these two issues, a push to self-analyze critically and an increase in novel dilemmas, more present than in the intensive care unit (ICU). The ICU is a place both where patients are exposed to modern advances in health technology and where some of the most challenging questions for bioethicists occur. Because of the pervasiveness of ethical considerations, it is logical to assume that the ethics knowledge base would be well documented in the ICU. In fact, a cursory search of the phrase 'critical care ethics' in PubMed between 1966 and 2004 cited an impressive 1090 articles. Because a more focused search on 'end of life ethics' returned 986 articles, it seems that much of the published literature has a particular focus.\nThe assumption that end-of-life issues represent the only ethical issue in the ICU was challenged by DeVita and colleagues' review [1] of all of the ethics manuscripts published in Critical Care Medicine. Although they identified a spectrum of ICU ethical issues in addition to end-of-life care such as futility, research, resource distribution, informed consent, and resuscitation, three issues are apparent to us. First, the number of manuscripts that they classified as 'end-of-life' far exceeded all other ethical topics (45 more than the second most common topic). Second, most of the additional ethical topics identified are issues that occur in the context of patient\u2013physician or patient\u2013nurse interactions. Third, on the presumption that the process of managing an ICU requires difficult moral decisions to be made, there seems to be a gap in the published literature with regard to the process of non-clinical decisions of ethical importance in the ICU.\nICU directors and nurse managers are required to make difficult decisions with respect to protocols, staffing, and administration of the ICU. Arguably, these have equal, maybe even greater, ethical importance than decisions made at the bedside, because management decisions can affect multiple patients, in a less direct, and transparent, manner. Perhaps the bedside can be described as a simpler ethical environment in that it involves fewer external factors and agents to consider, and the consequences of actions are immediately apparent to all.\nIf the dual roles that ICU physician directors and nurse managers occupy create unique ethical challenges that cannot be adequately captured by either the traditional principalism of medical bioethics because bioethics does not take into account the fundamentals of business, or the existing models of business ethics because these fail to account for the values of medicine, it is possible they should be addressed as a separate entity. The aim of this paper is to review briefly what ethical issues faced by ICU 'clinician\u2013managers' have been described, and to understand the context in which they are addressed.\nMethod\nTo identify publications that focused on ethical issues faced by professionals who occupy both clinical and administrative roles in ICUs, abstract and title searches were performed in Medline\/PubMed and CINAHL databases using combinations of the following keywords: Ethics, Clinician-Manager, Critical Care, Intensive Care Unit (ICU), Management, Leadership, Decision-making, Roles, Administration, Medical Directors, and Policy. Our search included primary literature, review, and opinion articles and the inclusion criteria were: 1966 to July 2004, English language, mention of critical or intensive care, direct mention of ethics OR discussed an ethical issue. An article was considered to have discussed an ethical issue if there was recognition of uncertainty about the correct choice of action in a given situation. Thus, any article that asserted one practice to be superior to another, whether anecdotally or as demonstrated by some research, was not considered to recognize an ethical concern. Articles were excluded if managed care was the source of the ethical dilemma and\/or the ethical issue was strictly clinical in nature, not including the use of treatment policies because clinician\u2013managers were considered to have a special interest in policy; that is, any ICU physician dealing with the issue was qualified to decide on the appropriate course of action.\nIn addition, references from captured articles were searched to identify additional literature that might not have been captured in the initial search. We also hand-searched the following journals: Bioethics (1997 to present), Critical Care, Critical Care Clinics, Critical Care Medicine (1985 to present), American Journal of Respiratory and Critical Care Medicine (1994 to present), and Intensive Care Medicine (1993 to present). Finally, the following key authors, identified by their previous ethics-related literature, were contacted to identify any articles that our search did not capture: Martin Strosberg, Kurt Darr, Dr G Rubenfeld, Dr C Sprung, and Dr J Luce.\nResults\nOf the roughly 1500 articles identified in our search, only 55 met the screening criteria that identified them as concerning an ethical challenge for clinician\u2013managers in critical care. A distribution across time of these articles can be seen in Fig. 1. After an initial review of all the articles that met the screening criteria, broad categories were arbitrarily chosen to sort the results as follows: resource allocation; organizational ethics (namely, how intensive care ought to be organized); policies and protocols (formulation and implementation); professional roles (namely, what the role of 'directors' should be); ethics and law; general ICU ethics (ICU ethics in the broadest sense); and other (Table 1). PubMed categorized 23 (42%) of the 55 articles that met all screening criteria as either review articles or editorials; these review articles and editorials were evenly distributed between our categories.\nArticles classified as 'resource allocation' papers mostly included discussions on which principles (such as justice, reasonableness as fairness) ought to apply when distributing scarce resources in the ICU. Some articles were more detailed, and described dilemmas such as age-based rationing [2] while still framing the question in terms of specific ethical principles and how they each affect the decision. One article sought to use cases to identify the ICU physicians' ethical role in distributing scarce resources [3].\nBy focusing attention on the physicians' role and not the ethical principles themselves, a whole new series of questions arose. It is noteworthy that the American Thoracic Society addresses the issue in a consensus statement outlining ethical guidelines for fair resource allocation [4], and the American College of Chest Physicians and the Society of Critical Care Medicine similarly discuss the ethics of resource allocation in their moral guidelines regarding the withdrawal of intensive care [5]. Articles classified as 'organizational' had a scope that ranged from what makes an ICU safe, to what should be taught to students in the ICU, to the ethical issues surrounding the use of restraints. Nelson describes an organization issue by illustrating how ethics is an everyday concern, with regard to issues like collaboration, staff conflict, and moral burnout [6]. This view is in contrast to the assumption that bioethics matters only when there is a specific dilemma. Additional articles on the use of restraints were classified as 'protocols and policies' because their focus was primarily on the policies themselves. Also in 'protocols and policies' was a discussion on the value of having a family presence protocol for life-saving procedures [7]; that is, is allowing families to witness resuscitation the right thing to do? The two articles under the heading 'professional roles' discussed the changing roles of ICU physicians, especially with regard to a transition to managerial duties; this issue is discussed below. 'General ethics' articles included all papers that discussed ICU ethics in the broadest sense with less attention to the details; many of these articles were brief reviews of the numerous ethical considerations in an ICU.\nDiscussion\nEthical issues are usually expressed as a conflict of ideas, values, and\/or norms that are often role dependent. It should therefore be expected that ICU physician directors and nurse managers, who have both clinical and non-clinical duties, should face some of the more difficult moral conflicts in the ICU. In one respect, these professionals follow a patient-centered code of conduct, either the Hippocratic oath or the Nursing Professional Code, which in part defines them. At the same time, they are also agents of the hospital as 'a business', and implicitly society as a whole. Although some clinicians completely relinquish their clinical duties on transition to management, most do not; the professional nature of medicine therefore gives the clinician a patient-centered outlook that is not as easily set aside: once a doctor or nurse, always a doctor or nurse. In summary, the physician director and nurse manager will always be in the unique position of having two separate professional standpoints from which to assess situations, which can therefore lead to unique ethical challenges.\nWe have begun to characterize the scope and uniqueness of ethical issues that are raised by the dual roles of clinician\u2013managers in the ICU. In reviewing the health literature, we found that almost half of the articles identified that discussed ethical concerns for ICU clinician\u2013managers were concerned with resource allocation. This is probably not surprising given that ICU clinicians are increasingly adopting the role of economic rationalist [8]. Perhaps the real surprise of these results is how many articles concerned issues other than resource allocation. Although DeVita and colleagues pointed out that 'end-of-life' was not the only ethical concern in the ICU [1], which they took to be a common assumption, we argue that there is more to the ethics of directing roles in the ICU than issues of resource allocation.\nIn this initial survey of the ethical issues experienced by those in dual management\u2013clinician roles in the ICU, important to our conclusion was the development of a categorization scheme. In the absence of any unique approach, we were arbitrary in our definition of categories. Although some of the articles we identified could have been placed in more than one category, or in categories not used, we believe our approach to be valid for the modest purposes of this survey. Additionally, some articles that discussed issues of clinical ethics in the ICU might have contained less prominent opinions or notes relevant to clinician\u2013managers and therefore might have been missed by our search. In spite of these potential issues, the articles selected were distributed to give both a clear and defined picture of what currently exists with regard to ICU ethics for those in both clinical and directing roles at the same time. We also believe it is likely that there were many articles that discussed ethical issues relevant to our review, yet failed to recognize the issues as being 'ethical' in nature. For example, many articles described the nuances of resource allocation in the ICU (see the Introduction), yet neither mentioned ethics or recognized any uncertainty with regard to the 'right' thing to do. The fact that many articles fail to address their ethical components might indicate a lack of awareness of what constitutes an ethical dilemma, but even if this is not so, the goal of better recognition and acknowledgement of the ethical issues that suffuse the operational management of the ICU is desirable.\nResource allocation is a well-defined topic of ethical interest that has stimulated much discussion. However, it is important not to perceive resource allocation as the beginning and end of the ethics discussion for clinician\u2013managers in critical care. Perhaps it is also time to move beyond the commentary on resource allocation and devote more research initiatives toward this topic (for example by studying the different approaches to resource allocation).\nThe term 'organizational ethics' is used to denote how a business or institution ought to be organized in any number of ways, including management functions, working environments, and its infrastructure. It should not come as a surprise that organizational ethics should constitute a concern for either an ICU director or a nurse manager, yet over the past 20 years only a handful of articles have been written about the organizational ethics of intensive care and have recognized them as such. Although policies and protocols for an ICU could also fall under the heading of 'organizational ethics', we believe that determining and implementing policies might require ethical concerns that merit special attention. The use of any policy that deals with either patients or staff is to apply one rule to many different people, and necessarily ignores factors that make individual cases unique. Because policies tend to generalize in this way, they create unique ethical challenges. Little reflection is required to determine that both of these issues, organizational ethics and the ethics of policy, constitute ethical concerns for directors in the ICU in which further study is merited.\nThe role of the ICU physician director, or nurse director\/ manager, is an ethical issue itself. Although two articles were identified that addressed this issue, we believe that the paucity of articles found indicate a need for greater consciousness of the ethical factors that influence and are influenced by clinical leaders in the dual roles of 'clinician' and 'manager'. Although healthcare leaders are familiar with the importance of ethics, they may be unaccustomed to thinking of their own role in terms of ethics [9]. Because the ICU director, or nurse manager, may engage in the decision making process from multiple professional standpoints (as both clinician and manager), the likelihood of conflicting rational and justifiable solutions, leading to ethical dilemmas, increases. The clinician\u2013manager role, then, may require a higher level of ethical proficiency, or perhaps expertise.\nConclusion\nIn summary, we believe it is important that future study be directed toward understanding ethical issues surrounding the dual roles of clinician\u2013managers in the ICU. Although it has been acknowledged that hospitals should pay as much attention to managerial ethics as to clinical ethics [10], it is not yet clear that this in fact occurs. One step in a research agenda would be to undertake a survey to determine if the published literature, identified by this review, is in fact an accurate representation of what is experienced. Understanding the scope of ethical issues experienced by clinician\u2013managers in the ICU will foster a more complete dialogue. Further, although there are studies that describe the process of ethical reasoning in nurses and other clinicians [11-13], similar studies have not been performed with clinician\u2013directors, be they nurses or physicians. These investigative steps toward a better understanding of the issues, and how they are understood and dealt with, ought to include direct input from ICU clinician\u2013managers. Input from ICU clinician\u2013managers with a background in, or special knowledge of, ethics would be particularly beneficial, because they would possess the language to describe what most feel by intuition. This experiential knowledge would best be conveyed in an open-interview or narrative format in which actual dilemmas can be discussed. Finally, the resulting data will require detailed qualitative analysis and might benefit from the support of a medical sociologist.\nFor all critical care leaders, there is now an opportunity to promote a better understanding of the complexity of the ICU environment and to prompt further learning.\nAbbreviations\nICU, intensive care unit.\nCompeting interests\nThe author(s) declare that they have no competing interests.\nSupplementary Material\nAdditional File 1\nA complete list of categorized references for all articles captured in this review can be found here.\nclick here for file","keyphrases":["ethics","critical care","management","clinician\u2013manager"],"prmu":["P","P","P","P"]}
{"id":"Breast_Cancer_Res_Treat-3-1-2001223","title":"Letrozole in the neoadjuvant setting: the P024 trial\n","text":"Neoadjuvant chemotherapy trials have consistently reported lower response rates in hormone receptor-positive (HR+) breast cancer when compared with HR\u2212 cases. Preoperative endocrine therapy has therefore become a logical alternative and has gained considerable momentum from the finding that aromatase inhibitors (AIs) are more effective than tamoxifen for HR+ breast cancer in both the neoadjuvant and adjuvant settings. The most convincing neoadjuvant trial to demonstrate the superiority of an AI versus tamoxifen was the P024 study, a large multinational double-blind trial in postmenopausal women with HR+ breast cancer ineligible for breast-conserving surgery. The overall response rate (ORR) was 55% for letrozole and 36% for tamoxifen (P < 0.001). Significantly more letrozole-treated patients underwent breast-conserving surgery (45 vs. 35%, respectively; P = 0.022). In addition, ORR was significantly higher with letrozole than tamoxifen in the human epidermal growth factor receptor HER1\/HER2+ subgroup (P = 0.0004). The clinical efficacy of letrozole in HER2+ breast cancer was confirmed by fluorescent in situ hybridization analysis and was found to be comparable to that of HER2\u2212 cases (ORR 71% in both subsets). Biomarker studies confirmed the superiority of letrozole in centrally assessed estrogen receptor-positive (ER+) tumors and found a strong relationship with the degree of ER positivity for both agents. Interestingly, letrozole was effective even in marginally ER+ tumors and, unlike tamoxifen, consistently reduced the expression from estrogen-regulated genes (progesterone receptor and trefoil factor 1). Furthermore, when analyzed by Ki67 immunohistochemistry, letrozole was significantly more effective than tamoxifen in reducing tumor proliferation (P = 0.0009). Thus, neoadjuvant letrozole is safe and superior to tamoxifen in the treatment of postmenopausal women with HR+ locally advanced breast cancer.\nIntroduction and rationale\nEarly-stage breast cancer is traditionally treated with an initial surgery such as lumpectomy or mastectomy followed by subsequent adjuvant therapy, including radiotherapy, chemotherapy, and hormone therapy [1, 2]. Numerous studies have investigated the use of preoperative (neoadjuvant) hormonal therapy or chemotherapy prior to surgical intervention, with the goals to improve surgical outcome and obtain long-term disease-free survival (DFS) (see Table\u00a01). The National Surgical Adjuvant Breast and Bowel Project B-18 trial demonstrated that doxorubicin and cyclophosphamide administered as neoadjuvant or adjuvant therapy had equivalent outcomes in terms of both DFS and overall survival (OS) [3]. Adding a taxane in the B-27 trial significantly reduced the local recurrence rate but did not significantly increase DFS or OS [4]. Importantly, neoadjuvant chemotherapy has been shown to increase the rate of breast-conserving surgery (BCS) without adversely affecting DFS or OS [3, 5\u20137]. Therefore, neoadjuvant chemotherapy has become the standard treatment approach for locally advanced breast cancer and an accepted option for patients with primary operable disease [8].\nTable\u00a01Aims of neoadjuvant therapy in different breast cancer populationsPopulationAimsTreatment optionLocally advanced breast cancerPrimary: to improve surgical optionsFit and healthy patients: chemotherapySecondary: to obtain freedom from disease, to gain information on tumor responseUnfit patients with hormone-sensitive disease: endocrine treatmentOperable breast cancer and candidates for adjuvant chemotherapyPrimary: to obtain freedom from diseaseChemotherapy\u00a0\u00b1\u00a0OFS and\/or AIsSecondary: to improve surgical options, to gain information on tumor responseSequence versus combinationLonger versus shorterOperable breast cancer and candidates for adjuvant endocrine treatment alonePrimary: to improve surgical optionsEndocrine treatment longer versus shorterSecondary: to gain information on tumor responseTamoxifen versus AIsReprinted from [8] with permission from the American Society of Clinical OncologyAIs aromatase inhibitors, NST neoadjuvant systemic therapy, OFS ovarian function suppression\nSignificant tumor reduction from neoadjuvant chemotherapy occurs in only subsets of breast cancer, however. For example, patients with estrogen receptor-negative (ER\u2212), high-grade, and highly proliferative tumors appear to benefit the most from neoadjuvant chemotherapy [9\u201311]. In contrast, significantly lower response rates have been reported in patients with ER+ tumors in neoadjuvant chemotherapy trials [12\u201315]. The German Breast Group demonstrated that the pathologic complete response (pCR) rates were 6.2 versus 22.8% for ER+ and ER\u2212 tumors, respectively (odds ratio 3.23, 95% confidence intervals [CIs] 1.91, 5.46; P\u00a0=\u00a00.0001) [12]. Recent studies indicate that the sensitivity to chemotherapy is likely determined by the underlying gene expression pattern and the molecular subtype of the tumor [16, 17]. The luminal subtype categorized by gene expression profiling, which encompasses most of the ER+ tumors, is less responsive to preoperative chemotherapy than the basal-like and human epidermal growth factor receptor 2-positive (HER2+) cancer. In addition, neoadjuvant chemotherapy is associated with substantial toxicity that may limit its clinical benefits and acceptability, especially in the elderly patient population [13, 18\u201320]. Thus, there is great need for effective alternatives to cytotoxic chemotherapy in hormone-responsive locally advanced breast cancer.\nEndocrine treatment is an attractive alternative to chemotherapy as neoadjuvant or primary systemic therapy for women with hormone-responsive primary breast tumors [21\u201323]. A study comparing neoadjuvant endocrine therapy with exemestane versus chemotherapy in 152 patients with ER+\/progesterone receptor-positive (PgR+) breast cancer found that while efficacy outcomes were comparable in the two groups, chemotherapy treatment was significantly more toxic, confirming that endocrine therapy could be used as an alternative to chemotherapy in older women [24]. Early studies of primary tamoxifen as an alternative to surgery were conducted in older women with hormone-responsive breast cancer who were unfit for chemotherapy [23, 25\u201327]. While these studies demonstrated a reduction in tumor size with primary tamoxifen, long-term local disease control was found to be poor. The omission of primary surgery resulted in an increased rate of progression, therapeutic intervention, and mortality [28]. Thus, primary tamoxifen treatment is indicated for only the most frail, medically ill, or noncompliant patients [29]. Although primary therapy with tamoxifen was found to be well-tolerated, adverse effects were reported including hot flushes, skin rash, vaginal discharge, breast pain, sleepiness, headache, vertigo, itching, hair loss, cystitis, acute thrombophlebitis, nausea, and indigestion [29]. Furthermore, more serious adverse effects, such as an increased risk of endometrial cancer and thromboembolic events, have been reported in large trials of adjuvant tamoxifen [30].\nIn view of the limitations of tamoxifen as a single modality treatment, attention has switched to the neoadjuvant use of endocrine therapy to increase the rate of breast conservation [21]. Data from preclinical models [31] and clinical studies in advanced breast cancer [32] predict that aromatase inhibitors (AIs) may be more effective than tamoxifen in the neoadjuvant setting. Furthermore, preclinical and clinical evidence suggests that letrozole may be the most effective AI in this setting [33\u201335]. In postmenopausal women with endocrine-responsive locally advanced or metastatic breast cancer, first-line treatment with letrozole was shown to be significantly more effective than tamoxifen in terms of response rate (overall response rate [ORR], 30 vs. 20%, P\u00a0=\u00a00.0006) and time to disease progression (41 vs. 26\u00a0weeks) [32]. A phase 1\u20132 pilot study showed a clinical response rate of 88% (21\/24 patients) in postmenopausal women with ER+ locally advanced breast cancer treated with letrozole for 3\u00a0months prior to surgery [36]. All patients in the study were eligible for breast conserving surgery following neoadjuvant letrozole.\nA large multinational trial, P024, was designed to assess the efficacy of neoadjuvant letrozole compared with tamoxifen in postmenopausal women with hormone receptor-positive (HR+) breast cancer who were ineligible for breast-conserving surgery [21]. The clinical objectives of the trial were to compare response rates and surgical outcomes between the two treatment arms. In addition, the trial provided the opportunity to conduct prospective biomarker studies to explore the biologic basis for response to neoadjuvant endocrine therapy [37]. This review describes the results of the P024 study and the follow-up biomarker studies, focusing on the treatment implications in the neoadjuvant setting.\nTrial design and patients\nPO24 was a multinational, randomized, double-blind controlled trial comparing letrozole and tamoxifen in postmenopausal women with hormone-responsive primary invasive breast cancer who were not eligible for breast-conserving surgery [21]. The trial was conducted in 55 centers in 16 countries between March 1998 and August 1999. Local ethics review boards approved the protocol and all patients gave written informed consent before study enrollment.\nRandomized clinical trial design\nPatients were randomly assigned to receive letrozole 2.5\u00a0mg or tamoxifen 20\u00a0mg administered orally once daily for 4\u00a0months prior to scheduled surgery [21]. Patients were considered to have completed the study when they had received 4\u00a0months of treatment and had been assessed for surgery. Following surgery, patients were treated at the investigator\u2019s discretion and were followed for 5\u00a0years for local recurrence, distant metastasis, and survival.\nPatient population\nA total of 337 postmenopausal women were enrolled into the trial, and 324 were included in the intent-to-treat population (see Fig.\u00a01). Eligible patients were postmenopausal women with untreated, primary HR+ (\u226510% nuclear staining for ER or PgR) invasive breast tumors (stages T2\u20134a\u2013c, N0\u20132, M0). The minimum tumor size was \u22653\u00a0cm, and all patients were considered inoperable or ineligible for breast-conserving surgery, mostly because of a non-favorable ratio tumor size\/breast size. Exclusion criteria included previous exposure to AIs, uncontrolled endocrine or cardiac disease, bilateral or inflammatory breast cancer, distant metastasis, and other malignant disease. In addition, administration of other cancer treatment or hormone replacement therapy was not allowed during study participation.\nFig.\u00a01Patient disposition [21] \nTrial end points\nThe primary end point was ORR, defined as the percentage of patients in each treatment arm with a complete response (CR) or a partial response (PR) as determined by breast palpation [21]. Response categories were CR, PR, no change, progressive disease, or not assessable\/not evaluable. The secondary end points were the percentage of patients who underwent breast-conserving surgery and the response rate (CR\u00a0+\u00a0PR) determined at 4\u00a0months by mammography and by ultrasound [21]. Safety was assessed and adverse events graded according to the National Cancer Institute Common Toxicity Criteria.\nCorrelative science studies\nIn parallel, studies were conducted in which tumor biopsies and blood samples were taken from all patients prior to the initiation of therapy and at the end of treatment to generate a database from which changes in molecular markers could be assessed [37\u201341]. These studies were prospectively designed to assess the biological basis for letrozole efficacy. The specific objectives of the biomarker sub-studies were as follows:To confirm ER and PgR status in a central laboratory. ER and PgR were determined using immunohistochemistry (IHC) performed on pretreatment biopsies; the criterion for minimal ER positivity was 10% positive cells [37]. The Allred histopathological score was applied to further assess the level of ER and PgR expression in both the baseline and surgical specimens [42].To explore relationships between ER and PgR expression levels and response to treatment [37].To examine the relationship between the expression of HER1 and HER2 and the likelihood of primary response [37]. Previous studies have shown that HER2 overexpression may be a predictor of tamoxifen resistance [43, 44], and HER1 has also been linked with endocrine therapy resistance [45]. HER2 IHC was initially scored as 0\/+ (negative) or ++\/+++ (positive or overexpressed) [46]. Since fluorescent in\u00a0situ hybridization (FISH) testing has replaced IHC as the gold standard for HER2 assessment, the P024 tumor bank was reassessed using HER1 and HER2 FISH probes, and these data on HER2 gene amplification status were supplemented with 106 tumor samples [40]. Pre- and post-treatment tumor biopsy samples were also analyzed for ER and several indices of ER function, including PgR and trefoil factor 1 (TFF1), HER1 and HER2, and the proliferation marker Ki67 [38].To assess gene expression profiling as a means to further investigate the transcriptional programs that underlie resistance and sensitivity to estrogen deprivation [39]. Gene expression (mRNA) profiles were collected from the tumor biopsies collected prior to surgery, at 1\u00a0month of treatment, and at surgery. The analysis was done using an Affymetrix U 133 subA Gene Chip.To measure aromatase expression in tumors before and after 4\u00a0months of letrozole or tamoxifen treatment. Aromatase is the key enzyme responsible for estrogen biosynthesis and is present in about 70% of tumors. IHC was performed with a monoclonal aromatase antibody (677) on trial samples prior to and following letrozole or tamoxifen treatment (n\u00a0=\u00a0185); scoring was measured as a proportion of immuno-positive cells and their intensity of reactivity in malignant epithelial, stromal, adipose, and normal compartments [41].\nEfficacy\nIn the intent-to-treat population, 154 patients received letrozole and 170 received tamoxifen. Similar proportions of patients in the letrozole and tamoxifen groups had inoperable tumors (13 and 14%, respectively). Other baseline characteristics were also well-balanced for age, race, HR status, and tumor\/nodal stage of disease. More patients in the tamoxifen group (n\u00a0=\u00a041) discontinued treatment than in the letrozole arm (n\u00a0=\u00a023). The main reason for premature discontinuation was disease progression.\nClinical response and breast-conserving surgery\nLetrozole was consistently superior to tamoxifen for primary and secondary efficacy end points [21]. The ORR was 55% for letrozole versus 36% for tamoxifen (P\u00a0<\u00a00.001). Median time to response was 66\u00a0days in the letrozole group and 70\u00a0days in the tamoxifen group. The odds ratio for achieving CR\u00a0+\u00a0PR was more than doubled with letrozole (2.23, 95% CI 1.43, 3.50; P\u00a0=\u00a00.0005). In terms of clinical progression, 12% of patients on letrozole and 17% on tamoxifen progressed, while 24% of letrozole- and 35% of tamoxifen-treated patients had stable disease. Letrozole was also shown to be significantly more effective than tamoxifen when response rates were assessed by mammography (34 vs. 16%, respectively; P\u00a0<\u00a00.001) and ultrasound (35 vs. 25%, respectively; P\u00a0=\u00a00.042).\nThe proportion of patients able to undergo breast-conserving surgery was significantly higher in the letrozole group than in the tamoxifen group (45 vs. 35%, respectively; P\u00a0=\u00a00.022). Of note, the odds ratio for breast-conserving surgery was 4.56 (P\u00a0=\u00a00.0001) for patients presenting with T2 tumors compared with all other T stages. The only other factor that increased the odds of undergoing breast-conserving surgery was treatment with letrozole (odds ratio 1.71, P\u00a0=\u00a00.03).\nER and PgR\nA supportive analysis of clinical efficacy data conducted in patients with centrally confirmed ER+ or PgR+ tumors also showed that outcomes were significantly improved with letrozole (n\u00a0=\u00a0124) compared with tamoxifen (n\u00a0=\u00a0126) [37]. In this subgroup, the ORR was 60% for letrozole versus 41% for tamoxifen (P\u00a0=\u00a00.004), and breast-conserving surgery was performed in 48 versus 36%, respectively (P\u00a0=\u00a00.036).\nThere was a linear relationship between ER Allred expression scores and response rates to both letrozole and tamoxifen (see Fig.\u00a02). Tumors with low ER expression were still responsive to letrozole but not to tamoxifen [37]. Of note, letrozole response rates were numerically superior to tamoxifen response rates in every ER Allred category from three to eight, indicating that letrozole is more effective than tamoxifen regardless of the level of ER expression [37]. This finding is important, because of all the AIs, only letrozole has demonstrated clear ORR superiority over tamoxifen in ER-poor tumors. When the effects of letrozole and anastrozole on tumors with low ER values (Allred scores 2\u20135) were compared in the neoadjuvant setting, only letrozole achieved a significant reduction in cell proliferation in ER-poor tumors [35].\nFig.\u00a02Clinical response rate versus estrogen receptor (ER) Allred score for letrozole and tamoxifen. The P value for a linear logistic model was 0.0013 for letrozole and 0.0061 for tamoxifen according the Wald test. In this analysis, ER\u2212, PgR+ cases (determined by conventional cut points) were excluded. Reprinted from [37] with permission from the American Society of Clinical Oncology\nLetrozole, but not tamoxifen, significantly reduced the expression of estrogen-regulated proteins PgR and TFF1 [38]. Average tumor PgR expression decreased dramatically on letrozole treatment (P\u00a0=\u00a00.0001), and only 4.4% of surgical specimens exhibited an Allred score of between six and eight for PgR expression. The significant decrease in PgR expression with letrozole remained significant in the ER+, HER2+ subpopulation. In contrast, changes in PgR expression with tamoxifen therapy were not consistent, with both increases and decreases in expression frequently observed in the overall and ER+, HER2+ subpopulations. Analysis of changes in TFF1 produced similar conclusions, with letrozole markedly reducing expression (P\u00a0=\u00a00.0001) and tamoxifen producing no overall trend in either direction.\nHER1\/HER2 and response\nApproximately 15% of tumors were ER+ and overexpressed both HER1 and HER2. The response rate in this patient subgroup was significantly higher with letrozole than with tamoxifen (88 vs. 21%) [37]. The odds ratio for response to letrozole versus tamoxifen was 28 (95% CI 4.5, 177; P\u00a0=\u00a00.0004). Letrozole was equally effective for HER1\/HER2+ and HER1\/HER2\u2212 tumors, whereas tamoxifen was significantly less effective in HER2+ compared with HER2\u2212 tumors (P\u00a0=\u00a00.045). These data suggest that although HER1 and HER2 status might not be the only explanation for the superiority of letrozole over tamoxifen, overcoming resistance pathways associated with HER1 and HER2 expression is a significant component of the improvement in outcomes associated with letrozole treatment observed in this clinical trial. FISH analysis of tumor samples confirmed the clinical efficacy of letrozole in breast cancers with or without HER2 amplification (ORR 71% in both subsets; P\u00a0=\u00a00.98). In contrast, tamoxifen-treated tumors with HER2 gene amplification had lower clinical response rates than tamoxifen-treated HER2\u2212 tumors (33 vs. 49%, P\u00a0=\u00a00.49) (see Table\u00a02) [40].\nTable\u00a02Analysis of clinical, ultrasound, and mammogram response data according to HER2 FISH status in letrozole-treated patients and tamoxifen-treated patientsResponse CategoryNo. responsesTotal No. (%)No. responsesTotal No. (%)HER2 FISH-positive Tamoxifen-treated patientsHER2 FISH-negative Tamoxifen-treated patientsP-valueaClinical39 (33)4490 (49)0.49Ultrasound 39 (33)2674 (35)0.99Mammography 19 (11)2290 (24)0.68HER2 FISH-positive Letrozole-treated patientsHER2 FISH-negative Letrozole-treated patientsP-valuebClinical1217 (71)131185 (71)0.98Ultrasound817 (47)91170 (54)0.61Mammography716 (44)84178 (47)0.79Reprinted from [40] with permission from the American Society of Clinical OncologyHER2 human epidermal growth factor receptor 2, FISH fluorescence in\u00a0situ hybridizationaFisher test P-valuebMantel\u2013Haenzel P-value\nBiomarkers of tumor proliferation\nLetrozole inhibited tumor proliferation, measured by the biomarker Ki67, to a greater extent than tamoxifen (reduction in geometric mean Ki67 level 87 vs. 75%, respectively; P\u00a0=\u00a00.0009). The differences in Ki67 reduction were also observed in ER+, HER1 and\/or HER2 overexpressing tumors (88% for letrozole vs. 45% for tamoxifen, respectively; P\u00a0=\u00a00.0018) [38]. Changes in the percentage of Ki67-positive cells in HER1\/2+ tumors treated with letrozole or tamoxifen are shown in Fig.\u00a03.\nFig.\u00a03A box plot of before and after treatment Ki67 values in the estrogen-receptor-positive, human epidermal growth factor receptor (HER) 1\/2+ subset. With letrozole (n\u00a0=\u00a015), 11 showed a decrease, one exhibited no change, and three showed an increase, of which only one was >2-fold (0.1\u20130.3%). With tamoxifen (n\u00a0=\u00a017), ten showed a decrease and seven an increase, of which three were relatively dramatic (9.5\u201322.7, 20.9\u201340.7, and 0.1\u201317.3%). Reprinted from [38] with permission from the American Association for Cancer Research\nMore recently, it was found that HER2 FISH-positive tumors showed higher histologic grade (P\u00a0=\u00a00.009), higher pretreatment Ki67 (P\u00a0=\u00a00.005), and less Ki67 suppression after letrozole when compared with HER2 FISH-negative tumors (P\u00a0=\u00a00.0001) [40]. Letrozole significantly decreased the geometric mean Ki67 level in HER2 FISH-negative tumors (from 6.25 [95%\u00a0CI\u00a05.16, 7.58%] to 0.68% [95%\u00a0CI 0.53, 0.87%]; P\u00a0=\u00a00.0001), but the decrease in HER2 FISH-positive tumors was blunted (from 14.73 [95%\u00a0CI 9.67, 22.44%] to 8.1% [95%\u00a0CI 4.16, 15.75%]; not significant). A similar observation for Ki67 was made in a smaller cohort of tamoxifen-treated tumors [40].\nThe relationship between cell cycle CR, defined as \u22641% of post-treatment Ki67 staining in the infiltrating component of the tumor, and HER2 status of tumors treated with letrozole was also analyzed [40]. Significantly more HER2 FISH-negative than FISH-positive tumors met the definition of a cell cycle CR at the time of surgery (60 vs. 12%; P\u00a0=\u00a00.0001). There was a high level of correlation between lack of cell cycle CR and the presence of a positive HER2 FISH test (P\u00a0=\u00a00.0001) for letrozole- and tamoxifen-treated tumors, which is consistent with the conclusion that HER2 gene amplification generates resistance at the level of cell cycle progression regardless of which endocrine therapy is used [40].\nThese biomarker findings are discordant with clinical observations that tumor regression is unaffected by HER2 amplification status in patients treated with neoadjuvant letrozole and may imply therapeutic resistance that could manifest later in the clinical course of the disease. Consistent with this finding, Miller and colleagues also reported that neoadjuvant letrozole produces rapid and profound decreases in expression of Ki67 and PgR that do not always correlate with clinical and pathological responses [47].\nGene expression profiling\nPreliminary gene expression profiling analysis of biopsies taken pretreatment and 1\u00a0month post letrozole treatment showed down-regulation of genes involved in DNA replication and synthesis, cell cycle progression, apoptosis suppression, and tissue invasion [39]. These results illustrate the molecular basis for estrogen-deprivation letrozole therapy which may be useful in the development of predictive models of ER+ breast cancer.\nAromatase\nSufficient pre- and post-treatment tumor material was available from 171 cases (81 on letrozole and 90 on tamoxifen) from the P024 trial for immunohistochemistry analysis of aromatase protein expression [41]. Aromatase was detected in all tumor compartments, with the strongest staining observed in malignant epithelial cells. Median aromatase values did not change significantly with letrozole or tamoxifen treatment; however, changes in score did occur in individual cases, with more noticeable effects observed in letrozole-treated patients [41]. A positive correlation existed between baseline ER and aromatase staining in cancer cells, while a negative correlation was observed between baseline Ki67 and aromatase expression in cancer plus stroma. Baseline aromatase expression did not predict response to letrozole or tamoxifen, or changes in Ki67 induced by treatment. However, negative staining in both stroma and cancer after treatment was strongly associated with fewer cell cycle CR and smaller Ki67 declines with letrozole (but not tamoxifen) treatment [48].\nSafety and duration of therapy\nP024 demonstrated that letrozole is well-tolerated in the neoadjuvant setting [21]. There were no major tolerability differences between letrozole and tamoxifen, and adverse effects of a similar nature were seen in 57% of patients in each arm [21]. The most common treatment-related adverse event was hot flushes, occurring in 20% of patients in the letrozole group and 24% of patients in the tamoxifen group.\nThe excellent tolerability, predictable pharmacokinetics, and minimal drug\u2013drug interactions [49, 50] make letrozole a particularly suitable option for older women unable to tolerate or unwilling to accept neoadjuvant chemotherapy and in whom the presence of comorbidities and use of concomitant therapies complicate treatment selection [20]. The median age of patients in P024 treated with letrozole was 68\u00a0years, and 46% of patients were at least 70\u00a0years old. The feasibility and safety of letrozole was also reported recently from another trial using letrozole as primary systemic therapy in elderly patients (median age 79\u00a0years) with breast cancer [51]. In addition, letrozole has been successfully administered to elderly patients (median age of the elderly subgroup 75\u00a0years; range 70\u201396\u00a0years) in the advanced breast cancer setting [52].\nThe favorable safety profile of letrozole also allows for the extension of the neoadjuvant treatment beyond the 4\u00a0months used in the P024 trial. In a recent study, 33 postmenopausal women with HR+ breast cancer ineligible for breast-conserving surgery were treated with letrozole for 4\u00a0months. Continued administration of letrozole for a further 4\u00a0months in responders and patients with stable disease resulted in a statistically significant improvement in tumor size reduction (P\u00a0=\u00a00.039); ORR was 90% in patients receiving preoperative treatment for longer than 4\u00a0months compared with 57% in patients receiving treatment up to 4\u00a0months [53].\nIn another study, 42 patients who were unsuitable for breast-conserving surgery or had refused surgery after responding to initial neoadjuvant therapy with letrozole for 3\u00a0months benefited from continuing tumor volume reduction during further letrozole treatment administered for up to 12\u00a0months [54]. The median reductions in tumor volume were 52% (95%\u00a0CI 37, 62) from 0 to 3\u00a0months, 57% (95%\u00a0CI 26, 100) from 3 to 6\u00a0months, and 66% (95%\u00a0CI 22, 100) from 6 to 12\u00a0months. Extending the duration of letrozole also improved the CR rate, which increased from 4\/42 patients (9.5%) at 3\u00a0months to 12\/42 (29%) by 6\u00a0months and 8\/22 (36%) by 12\u00a0months.\nConclusions\nNeoadjuvant therapy with AIs is a safe and effective treatment option for postmenopausal women who are unwilling or unable to undergo surgery or preoperative chemotherapy [21, 22, 55\u201357]. Letrozole is significantly more effective than tamoxifen in the neoadjuvant setting in terms of ORR and increased rate of breast-conserving surgery [21, 55].\nOne of the advantages of using a neoadjuvant strategy is the opportunity to gain information on tumor response early in the course of treatment. Detailed studies correlating clinical response to neoadjuvant therapy with changes in tumor biomarkers and gene expression may ultimately prove useful to tailor therapy for individual patients and to gain a better understanding of the biology of HR+ breast cancer. Correlative studies conducted using tumor samples from P024 have revealed important information about how breast tumors respond to letrozole [37\u201340]. Letrozole was shown to be significantly more effective than tamoxifen in the inhibition of ER+ tumor proliferation, since letrozole produced a greater reduction in levels of the proliferation biomarker Ki67 [38]. It has been suggested that a greater suppression in proliferation could lead to greater long-term survival in the adjuvant setting. Preliminary data from the Immediate Preoperative Anastrozole Tamoxifen or Combined with Tamoxifen trial have indicated that short-term changes in Ki67 levels, after 2 and 12\u00a0weeks, may be a useful predictive marker for relapse-free survival in patients treated with neoadjuvant AI therapy [58]. It has also been suggested that changes in proliferation and concurrent changes in apoptosis may be expected to be more predictive of adjuvant benefit from endocrine therapy than clinical response [59].\nCorrelative studies have also highlighted the complexity of breast cancer biology and revealed discordance between clinical and biomarker responses [40]. Amplification of HER2 was shown to be associated with a more aggressive breast cancer phenotype and greater resistance to tamoxifen [37, 40]. Clinical response data from the P024 trial have shown that letrozole is equally effective in HER2+ and HER2\u2212 tumors, whereas tamoxifen is less effective in HER2+ tumors [37]. These data suggest that letrozole could be a superior option to tamoxifen for postmenopausal women with HER2+, HR+ tumors [37]. However, analysis of proliferation markers has provided evidence of estrogen-independent proliferation of ER+, HER2+ breast cancer despite neoadjuvant letrozole [40]. It appears that cell-cycle regulation is partially or completely estrogen-independent in the majority of primary tumors showing HER2 gene amplification, and patients with such tumors may eventually develop resistance to adjuvant AI therapy. Novel strategies to delay or overcome hormone resistance are described elsewhere in this supplement, in the article, \u201cFemara and the future.\u201d\nGene expression profiling has demonstrated that letrozole targets genes responsible for DNA replication and synthesis, cell cycle progression, apoptosis, and tissue invasion [39]. Research into genetic profiling is continuing, with the aim of developing clinically relevant predictive models that can accurately classify ER+ disease according to likely response to specific neoadjuvant therapies. Predictive models will improve treatment individualization and help to avoid unnecessary treatment-related toxicity in patients unlikely to benefit from systemic therapies [17, 60].\nThe P024 trial has clearly demonstrated the therapeutic superiority of letrozole over tamoxifen for the neoadjuvant management of primary breast cancer. The trial has also provided the oncology community with a validated research setting within which to gain valuable insights into the molecular features of ER+ breast cancer and its treatment that will help shape new therapies in the years to come.","keyphrases":["letrozole","breast cancer","endocrine therapy","aromatase inhibitor","tamoxifen","postmenopausal","neoadjuvant therapy"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Naunyn_Schmiedebergs_Arch_Pharmacol-2-2-1705472","title":"Why are mineralocorticoid receptor antagonists cardioprotective?\n","text":"Two clinical trials, the Randomized ALdosterone Evaluation Study (RALES) and the EPlerenone HEart failure and SUrvival Study (EPHESUS), have recently shown that mineralocorticoid receptor (MR) antagonists reduce mortality in patients with heart failure on top of ACE inhibition. This effect could not be attributed solely to blockade of the renal MR-mediated effects on blood pressure, and it has therefore been proposed that aldosterone, the endogenous MR agonist, also acts extrarenally, in particular in the heart. Indeed, MR are present in cardiac tissue, and possibly aldosterone synthesis occurs in the heart. This review critically addresses the following questions: (1) is aldosterone synthesized at cardiac tissue sites, (2) what agonist stimulates cardiac MR normally, and (3) what effects are mediated by aldosterone\/MR in the heart that could explain the beneficial effects of MR blockade in heart failure? Conclusions are that most, if not all, of cardiac aldosterone originates in the circulation (i.e., is of adrenal origin), and that glucocorticoids, in addition to aldosterone, may serve as the endogenous agonist of cardiac MR. MR-mediated effects in the heart include effects on endothelial function, cardiac fibrosis and hypertrophy, oxidative stress, cardiac inotropy, coronary flow, and arrhythmias. Some of these effects occur via or in synergy with angiotensin II, and involve a non-MR-mediated mechanism. This raises the possibility that aldosterone synthase inhibitors might exert beneficial effects on top of MR blockade.\nIntroduction\nThe renin-angiotensin-aldosterone system (RAAS) has been viewed conventionally as a circulating system, involved in the regulation of salt, fluid homeostasis and blood pressure. Kidney-derived renin cleaves liver-derived angiotensinogen to form angiotensin (Ang) I in circulating blood (Fig.\u00a01). Angiotensin-converting enzyme (ACE), located at the luminal side of the endothelium, subsequently converts Ang I to Ang II. Ang II exerts its effects via stimulation of Ang II type 1 (AT1) and type 2 (AT2) receptors. Besides acting as a vasoconstrictor via AT1 receptors, Ang II also stimulates the formation of the sodium-retaining hormone aldosterone. Aldosterone mediates its cellular effects by binding to the mineralocorticoid receptor (MR), a member of the steroid\/thyroid\/retinoid\/orphan receptor family of transcription factors.\nFig.\u00a01The renin-angiotensin-aldosterone system. Angiotensin II activates angiotensin II type 1 and type 2 (AT1 and AT2) receptors. AT1 receptor activation in the adrenal results in the synthesis and release of aldosterone, which subsequently exerts \u2018genomic\u2019 (after hours, involving protein synthesis) or \u2018non-genomic\u2019 (within minutes, not involving protein synthesis) effects through either mineralocorticoid receptors (MR) or other, as yet unidentified, receptors. MR may occur both intracellular and on the cell membrane. Aldosterone upregulates both AT1 and AT2 receptors, and AT1 receptor activation also results in MR stimulation in an aldosterone-independent manner\nThis classic concept has been updated in the past two decades. It is now believed that some or all of the components of the RAAS are synthesized locally in tissues such as the heart and vessel wall (Danser 2003; Tom et al. 2003). For instance, in the heart, Ang II is synthesized locally by cardiac ACE following uptake of renin and angiotensinogen from the circulation (Danser et al. 1994; van Kats et al. 1998). This Ang II subsequently stimulates cardiac AT1 and AT2 receptors (Batenburg et al. 2004; van Kats et al. 2000). Local synthesis of Ang II at cardiac tissue sites is in agreement with the observation that the beneficial effects of ACE inhibitors in heart failure are, at least in part, independent of their effect on blood pressure (Yusuf et al. 2000).\nTraditionally, treatment of heart failure and hypertension has been performed mainly on the basis of the renin-angiotensin system (RAS) rather than the RAAS, with the assumption that aldosterone will be suppressed once Ang II formation is blocked. However, aldosterone formation does not stay suppressed during prolonged RAS blocking therapy. After 3\u00a0months of therapy, aldosterone levels start to rise again and they continue to do so due to \u201cAng II reactivation\u201d or \u201caldosterone escape\u201d (Farquharson and Struthers 2002; Jorde et al. 2002).\nTwo clinical trials, the Randomized ALdosterone Evaluation Study (RALES) (Pitt et al. 1999) and the EPlerenone HEart failure and SUrvival Study (EPHESUS) (Pitt et al. 2003), have convincingly indicated that MR antagonists reduce mortality in patients with heart failure and systolic left ventricular dysfunction post-myocardial infarction on top of ACE inhibition. In particular, a reduction in the rate of sudden death was observed. These results draw attention to the importance of aldosterone as an independent risk factor in the pathophysiology of cardiovascular disease. The benefit of MR antagonists during RAS blockade is not yet fully understood. Their effects cannot be attributed solely to blockade of the renal MR-mediated effects on blood pressure (Pitt et al. 1999, 2003), and it is now generally assumed that aldosterone also acts extrarenally, in agreement with the concept of local RAAS.\nIndeed, MR have been demonstrated in the heart, both at the mRNA and protein level (Lomb\u00e8s et al. 1995). Importantly, the enzymes required for the synthesis of aldosterone appear to be expressed in the human heart as well (Young et al. 2001). Together with the fact that Ang II is capable of increasing the aldosterone levels in isolated rat hearts and blood vessels (Silvestre et al. 1998; Takeda et al. 1997), these data suggest that aldosterone, like Ang II, may be formed and act locally in the heart. This review addresses the following questions: (1) is aldosterone really synthesized at cardiac tissue sites, (2) what agonist stimulates cardiac MR, and (3) what effects are mediated by aldosterone\/MR in the heart that could explain the beneficial effects of MR blockade in heart failure?\nAldosterone synthesis at cardiac tissue sites?\nAldosterone, a steroid that was originally discovered in 1953, is secreted by the zona glomerulosa cells of the adrenal cortex. The kidney is the major target for adrenal aldosterone to increase sodium (and consequently water) reabsorption and potassium excretion. The production of aldosterone is regulated at two critical enzyme steps: (1) the formation of pregnenolone from cholesterol by the mitochondrial enzyme P450scc (side-chain cleavage), and (2) the conversion of corticosterone to aldosterone by cytochrome P450 11\u03b2-hydroxylase 2 (CYP11B2, \u2018aldosterone synthase\u2019). Aldosterone synthesis in the adrenal cortex is regulated by Ang II, potassium and, more weakly, sodium and adrenocorticotrophic hormone.\nExtra-adrenal aldosterone synthesis has been proposed in heart (Silvestre et al. 1998), brain (Gomez-Sanchez et al. 1997) and vessel wall (Takeda et al. 1995, 1997). The rat heart expresses the steroidogenic acute regulatory (StAR) protein and aldosterone synthase, although at 100-fold lower levels than the adrenal (Casal et al. 2003; Silvestre et al. 1998). The StAR protein facilitates intramitochondrial cholesterol transfer, the rate-limiting step of steroidogenesis. Aldosterone synthase expression has also been proposed in the human heart (Tsybouleva et al. 2004; Young et al. 2001). In support of the functional importance of such expression, net release of aldosterone was observed across the human coronary vascular bed (Nakamura et al. 2004). However, other studies demonstrated the opposite (i.e., cardiac aldosterone extraction) (Hayashi et al. 2003), whereas it was also noted that, in humans, cardiac aldosterone correlated closely with the cardiac levels of renin. Since the latter is exclusively of renal origin (i.e., blood-derived) (Danser et al. 1994, 1997; Saris et al. 2001), its correlation with aldosterone argues against independent aldosterone synthesis at cardiac tissue sites. Furthermore, recent careful studies in rats, paying great attention to the measurement of aldosterone in cardiac tissue, did not confirm the idea of local synthesis of aldosterone in the rat heart (Fiebeler et al. 2005; Gomez-Sanchez et al. 2004). In these studies, the cardiac aldosterone levels were much lower than previously reported by others (Silvestre et al. 1998). Furthermore, the cardiac levels correlated closely with the plasma levels of aldosterone, and they decreased to levels at or below the detection limit after adrenalectomy.\nSubsequent perfusion studies with aldosterone in the isolated Langendorff heart (Chai et al. 2006) showed that the steroid rapidly accumulated in cardiac tissue, not only in extracellular (interstitial) fluid but also in a second, as yet unidentified, compartment. At steady state, the aldosterone tissue levels (expressed per g wet weight) were higher than its levels in coronary effluent (expressed per ml). Washout from the second compartment occurred relatively rapid (half life\u2009<10\u00a0min), suggesting that it represented cell surface-bound rather than internalized aldosterone (Fig.\u00a02). This pattern resembles that of cardiac renin, which also accumulates in extracellular fluid and binds to membrane receptors (Danser et al. 1994; de Lannoy et al. 1997; Saris et al. 2001). After prolonged washout, cardiac aldosterone became undetectable. Thus, on the one hand, the heart displays a large capacity to accumulate aldosterone. This explains why the levels of cardiac aldosterone in rats can be up to 10-fold higher than in serum (Fiebeler et al. 2005). On the other hand, cardiac aldosterone disappears rapidly during perfusion with aldosterone-free buffer. This provides an explanation for the \u2018release\u2019 of aldosterone across the coronary vascular bed in humans and rats (Takeda et al. 2000). The majority of cardiac aldosterone, if not all, is however derived from the circulation, i.e., is not synthesized locally, both under normal and pathological conditions (Chai et al. 2006; Fiebeler et al. 2005; Gomez-Sanchez et al. 2004).\nFig.\u00a02Washout of aldosterone from the isolated perfused rat Langendorff heart after its exposure to 10\u00a0nmol\/l aldosterone for 30\u00a0min. Data are modified from Chai et al. (2006)\nActivation of cardiac mineralocorticoid receptors by aldosterone?\nMRs occur both in Na+-transporting epithelia (e.g., kidney, colon) and non-epithelial tissues such as brain (de Kloet et al. 2000), heart (cardiomyocytes) (Lomb\u00e8s et al. 1995), and blood vessels (endothelial and smooth muscle cells) (Lomb\u00e8s et al. 1992; Oberleithner 2005; Oberleithner et al. 2004). The presence of MR in the cardiovascular system has been confirmed both at the mRNA and protein level in animals as well as in humans (Lomb\u00e8s et al. 1992).\nMRs bind mineralocorticoids and glucocorticoids with equal affinity (Kd\u2009\u2248\u20090.5\u20132\u00a0nM) (Arriza et al. 1987; Lomb\u00e8s et al. 1994). Yet, the circulating concentrations of glucocorticoids are several orders of magnitude higher than those of aldosterone (Fig.\u00a03). Selectivity of aldosterone binding to MR is achieved by co-expression of 11\u03b2-hydroxysteroid dehydrogenase type 2 (11\u03b2HSD2) (Alzamora et al. 2000). This enzyme converts cortisol (the endogenous glucocorticoid in humans) and corticosterone (the endogenous glucocorticoid in rats) to their non-MR-binding metabolites cortisone and 11-dehydrocorticosterone (Fig.\u00a03). In addition, the off-rate of aldosterone from the MR is five times lower than that of glucocorticoids (Lomb\u00e8s et al. 1994), and thus it is possible that MR also discriminate aldosterone from glucocorticoids, at least in part, independently of 11\u03b2HSD2.\nFig.\u00a03Diagram illustrating activation of glucocorticoid and mineralocorticoid receptors (GR, MR) by cortisol (top) or aldosterone (bottom). Selectivity of aldosterone binding to MR is achieved by co-expression of 11\u03b2-hydroxysteroid dehydrogenase type 2 (11\u03b2HSD2). 11\u03b2-hydroxysteroid dehydrogenase type 1 (11\u03b2HSD1), which acts as a reductase in vivo, reactivates cortisone to cortisol. The affinity constants refer to aldosterone-MR and cortisol-GR binding (Arriza et al. 1987; Hagendorf et al. 2005; Lomb\u00e8s et al. 1994). Note that, in the absence of 11\u03b2HSD2, MR bind cortisol with an affinity equal to that for aldosterone\nIn the kidney, 11\u03b2HSD2 expression is high enough to allow selective MR stimulation by aldosterone. In contrast, in the heart, the 11\u03b2HSD2 levels are almost negligible (Nagata et al. 2006), and it has therefore been proposed that cardiac MR are occupied by cortisol\/corticosterone rather than aldosterone (Funder 2005b; Gomez-Sanchez et al. 2004; Nagata et al. 2006). In fact, the endogenous glucocorticoid levels are high enough to keep all cardiac MR permanently occupied (Funder 2005b). Such permanent occupation does not apply to glucocorticoid receptors, since they have a \u224830-fold lower affinity for cortisol\/corticosterone than MR (Fig.\u00a03). Glucocorticoids are assumed to act as antagonists of MR (i.e., they exert no effect following binding to MR) in kidney (Good et al. 2002) and heart (Qin et al. 2003), whereas in the vessel wall, during 11\u03b2HSD2 blockade with carbenoxolone, they act as agonists (Alzamora et al. 2000). The latter could relate to the observation that glucocorticoid-MR complexes become activated as a result of the generation of reactive oxygen species (ROS). Under such circumstances, MR antagonists may exert effects by blocking the consequences of glucocorticoid-MR complex activation rather than aldosterone-MR activation (Funder 2005b; Nagata et al. 2006). The increased expression in the failing rat heart of the enzyme 11\u03b2-hydroxysteroid dehydrogenase type 1 (11\u03b2HSD1), which reactivates 11-dehydrocorticosterone to corticosterone, further supports this concept (Nagata et al. 2006).\nGenomic versus non-genomic effects\nThe classical MR-mediated effects of aldosterone are referred to as \u2018genomic\u2019 effects. These effects involve binding of aldosterone to intracellular MRs, and the translocation of the steroid-MR complex to the nucleus, where it acts as a transcriptional regulator, inducing protein synthesis (in particular synthesis of the epithelial Na+ channel) after several hours. Genomic effects can be inhibited by agents that block either transcription (e.g., actinomycin D) or translation (e.g., cycloheximide).\nIn addition to its genomic effects, which occur after hours, aldosterone also exerts rapid effects (within minutes) in various tissues, e.g., heart and vasculature. These effects are usually described as \u2018non-genomic\u2019, since they do not involve DNA-directed, RNA-mediated protein synthesis. For instance, aldosterone rapidly affects cardiac inotropy and facilitates both vasodilation and vasoconstriction (Barbato et al. 2002; Liu et al. 2003; Mazak et al. 2004; Michea et al. 2005; Mihailidou et al. 2004; Schmidt et al. 2003). In many (but not all) cases, these effects could not be blocked by the MR antagonist spironolactone, and therefore the existence of a novel (membrane-associated?) aldosterone receptor has been proposed (Funder 2005a; Wehling et al. 1995). However, despite numerous efforts in the past decade, no convincing data toward the characterization of a membrane receptor for aldosterone have been put forward (Funder 2005a; Wehling 2005). Consequently, it is not unlikely that these effects are after all also being mediated via the classical (intracellular) MR (Funder 2005a). If so, an explanation must be provided for the lack of effect of spironolactone toward the rapid aldosterone-induced actions in vitro. Possibly, via modification or dimerization, classical MR can attain an atypical pharmacology, thereby no longer allowing the binding of MR antagonists such as spironolactone. In addition, spironolactone exerts MR-independent effects of its own, i.e., it blocks human Ether-a-Go-Go-Related gene K+ channels (Caballero et al. 2003) and inhibits calcium entry (Cargnelli et al. 2001). Thus, to solve this issue, future studies should make use of alternative MR antagonists such as eplerenone. Eplerenone displays increased selectivity for the MR over other steroid receptors, although its affinity for the MR in vitro is 10- to 20-fold lower than that of spironolactone.\nThe rapid, non-genomic actions involve activation of the phospholipase C-protein kinase C (PKC)-inositol 1,4,5-trisphosphate (IP3)-1,2 diacylglycerol (DAG) pathway, which leads to an increase in intracellular Ca2+ and stimulation of the Na+\/H+ exchanger (Barbato et al. 2004b; Funder 2005a; Liu et al. 2003; L\u00f6sel et al. 2002; Mihailidou et al. 2004; Sato et al. 1997). The latter causes a rise in intracellular Na+, which subsequently activates Na+\/K+-ATPase. Interestingly, however, when the increase in intracellular Na+ is prevented, aldosterone decreases Na+\/K+-ATPase activity in a PKC-dependent manner (Mihailidou et al. 2004). Thus, aldosterone may exert both positive and negative inotropic effects. Other second messenger pathways that have been linked to the rapid effects of aldosterone include mitogen-activated protein (MAP) kinases, ROS and the epidermal growth factor receptor (Jaffe and Mendelsohn 2005; Mazak et al. 2004).\nEffects of aldosterone in the cardiovascular system\nSubstantial evidence has emerged showing that aldosterone induces adverse effects in the cardiovascular system. The co-expression of 11\u03b2HSD2 and MR in human heart and blood vessels (Glorioso et al. 2005; Jaffe and Mendelsohn 2005; Lomb\u00e8s et al. 1995), albeit at low levels, supports the concept that these organs possess the cellular machinery required for direct aldosterone action, irrespective of the source of aldosterone.\nEndothelial dysfunction\nAldosterone increases the volume and stiffness of endothelial cells and induces gap formation, allowing irregular diffusion pathways for large particles (Oberleithner 2005; Oberleithner et al. 2004). This mechanism could contribute to endothelial dysfunction observed in hyperaldosteronism. The normalization of endothelial function by spironolactone in patients with heart failure supports this concept (Abiose et al. 2004; Macdonald et al. 2004).\nOxidative stress, inflammation, fibrosis and atherosclerosis\nA growing number of studies support a specific role of the MR as a mediator of oxidative stress and subsequent inflammation, fibrosis and atherosclerosis. Elevations in circulating aldosterone are accompanied by a pro-inflammatory\/fibrogenic vascular phenotype (Ahokas et al. 2005; Blasi et al. 2003; Sun et al. 2002), and since this phenomenon can be blocked by both spironolactone and anti-oxidants (Sun et al. 2002), it appears that aldosterone, via MR, induces oxidative stress. Indeed, aldosterone upregulates various subunits of NADPH oxidase and induces ROS generation in mononuclear and vascular smooth muscle cells (Ahokas et al. 2005; Calo et al. 2004; Mazak et al. 2004; Sun et al. 2002). In addition, aldosterone stimulates collagen synthesis in cardiac fibroblasts (Brilla et al. 1994). Consequently, aldosterone-induced cardiac fibrosis may be due to both direct effects in the heart (mediated via fibroblasts) and indirect peripheral effects (mediated via oxidative stress-activated mononuclear cells) (Ahokas et al. 2005). The MR antagonist eplerenone inhibited atherosclerosis both in monkeys (Takai et al. 2005) and apolipoprotein-E deficient mice (Suzuki et al. 2006) fed a high-cholesterol diet, most likely by attenuating oxidative stress and inflammation.\nCardiac inotropy and coronary flow\nIn the isolated perfused rat Langendorff heart, aldosterone, like Ang II, rapidly increased left ventricular pressure and decreased coronary flow (Chai et al. 2005a; Moreau et al. 1996). Barbato et al. (2002) observed an increase in cardiac contractility in combination with an increase in coronary flow, but this may relate to the fact that the much larger positive inotropic effects in their study had favoured coronary vasodilation. Spironolactone and eplerenone did not block the inotropic and vasoconstrictor\/dilator effects of aldosterone in the rat heart (Barbato et al. 2002; Chai et al. 2005a). In fact, spironolactone exerted similar inotropic effects on top of aldosterone (Barbato et al. 2002; Chai et al. 2005a), and thus it appears that the effects of aldosterone on inotropy and flow occur in a non-MR-mediated manner.\nIn human myocardial trabeculae, aldosterone induced a negative inotropic response (Fig.\u00a04), in contrast to the well-known positive inotropic effects of Ang II in this preparation (Chai et al. 2005b). The PKC inhibitor chelerythrine chloride, but not spironolactone or eplerenone, blocked this negative inotropic effect, suggesting that it is mediated via a non-MR in a PKC-dependent manner. The aldosterone concentrations required to induce this effect were in the high nanomolar range, i.e. a range that occurred in failing hearts only (Chai et al. 2005b). Hydrocortisone, but not 17\u03b2-estradiol, mimicked the effects of aldosterone, although at lower potency (Fig.\u00a04). The contrast between the effects of aldosterone in the rat heart (positive inotropy) and the human heart (negative inotropy) may relate to species differences. However, there are alternative explanations. First, inotropic effects in isolated trabeculae do not necessarily parallel inotropic effects in intact hearts, since the latter also reflect responses on coronary flow (Barbato et al. 2002). Second, similar diametrically differing effects of aldosterone have been observed on flow, either because such effects involve different cells, or because different second messengers are activated depending on the experimental circumstances (Barbato et al. 2004b; Chai et al. 2005a; Mihailidou et al. 2004; Schmidt et al. 2003). Finally, the consequences of PKC-induced regulation of Na+-K+ pump activity are tissue-specific, and range from stimulation to inhibition, as described above (Mihailidou et al. 2004; Therien and Blostein 2000).\nFig.\u00a04Inotropic effects of aldosterone, hydrocortisone and 17\u03b2-estradiol in human atrial trabeculae. Left panel original tracing from an experiment with aldosterone (numbers represent -log[aldosterone] in mol\/L). Right panel % change from baseline contractile force. Data have been obtained from Chai et al. (2005b)\nIn human coronary arteries, aldosterone exerted no constrictor or dilator effect by itself. However, prior exposure to 1\u00a0\u03bcmol\/L aldosterone greatly enhanced the constrictor response to Ang II (Chai et al. 2005b). At the second messenger level, this was reflected by an increase in the level of phosphorylated p42\/p44 MAP kinase. Hydrocortisone and 17\u03b2-estradiol induced similar potentiating effects, but only in the case of aldosterone did these effects occur at the subnanomolar level, i.e., in a physiological range. Future investigations should now address to what degree this potentiation concerns aldosterone-induced endothelial dysfunction (Oberleithner 2005; Oberleithner et al. 2004), and\/or an interaction with Ang II at the level of smooth muscle cells, involving some or all of the mediators that have recently been coupled to aldosterone, e.g., the PKC-IP3-DAG pathway, Na+\/H+ exchange, Na+\/K+-ATPase, p38 MAP kinase, ROS and\/or the epidermal growth factor receptor (Jaffe and Mendelsohn 2005; Liu et al. 2003; Mazak et al. 2004). Finally, the possibility of aldosterone-induced, endothelium-dependent, NO-mediated vasodilation, as proposed by several investigators (Liu et al. 2003; Schmidt et al. 2003), needs to be addressed.\nArrhythmias\nMR blockade, in addition to standard therapy, reduced sudden death in RALES and EPHESUS (Pitt et al. 1999, 2003). The mechanism responsible for this favorable effect may rely on both renal changes in electrolyte excretion and myocardial fibrosis inhibition. In addition, conditional MR overexpression in the mouse heart, in the absence of aldosteronemia, has been reported to result in severe ventricular arrhythmias (Ouvrard-Pascaud et al. 2005). Apparently, cardiac MR trigger arrhythmias directly, thus providing an additional mechanism through which MR antagonists reduce sudden death in patients. In support of this possibility, spironolactone improved electrophysiological parameters such as QT interval dispersion (Yee et al. 2001), and, in combination with the ACE inhibitor fosinopril, reduced the arrhythmic score post-myocardial infarction (Beck et al. 2001).\nFurthermore, both spironolactone and eplerenone improved the condition of the isolated perfused rat Langendorff heart following ischemia and reperfusion, as evidenced by a decrease in infarct size, a decrease in arrhythmia incidence, and an increase in left ventricular pressure recovery (Chai et al. 2005a, 2006) (Fig.\u00a05). Given the virtual lack of aldosterone in the isolated perfused rat heart, it is unlikely that these effects are due to blockade of endogenous aldosterone. In fact, concomitant exposure to 100\u00a0nmol\/l aldosterone did not further deteriorate the condition of the heart during ischaemia and reperfusion (Chai et al. 2006). A more likely explanation of these findings is therefore that spironolactone and eplerenone had blocked MR activation by endogenous glucocorticoids. Given the 1,000-fold higher levels of corticosterone in the rat heart (Gomez-Sanchez et al. 2004), and assuming that the washout of glucocorticoids resembles that of aldosterone, it can be calculated that, at the time of ischaemia, sufficient glucocorticoid levels are indeed present to allow cardiac MR activation. Such activation might occur particularly under conditions which facilitate ROS generation, such as ischaemia and reperfusion (Funder 2005b; Nagata et al. 2006). Interestingly in this regard, epidemiological observations have recently shown that high-dose corticosteroids increase the risk of developing atrial fibrillation (van der Hooft et al. 2006). The cardioprotective effect of MR antagonism in the Langendorff heart during ischemia and reperfusion cannot be explained on the basis of the vasoconstrictor effect of aldosterone, as proposed by Fujita et al. (2005), since neither spironolactone nor eplerenone are capable of blocking aldosterone-induced vasoconstriction in vitro (Chai et al. 2005a, 2006).\nFig.\u00a05Infarct size (left panel), recovery of left ventricular pressure (LVP) (middle panel), and incidence of arrhythmias (right panel) in rat hearts that were subjected to 45\u00a0min left anterior descending coronary artery occlusion, followed by 3\u00a0h of reperfusion, after either no pretreatment (C, control) or a 15-min exposure to 100\u00a0nmol\/l aldosterone (A), 1\u00a0\u03bcmol\/l spironolactone (S), 1\u00a0\u03bcmol\/l eplerenone (E) or 100\u00a0nmol\/l aldosterone\u2009+\u20091\u00a0\u03bcmol\/L eplerenone (A\u2009+\u2009E). Data are from Chai et al. (2005a, 2006). *P\u2009<\u20090.05 versus control\nCardiac hypertrophy\nSerum aldosterone levels associate with the variability of left ventricular mass (LVM) in both healthy controls and subjects with hypertension (Schunkert et al. 1997). The CYP11B2 C-344T polymorphism associates with circulating aldosterone levels, subjects with the T allele having higher aldosterone levels than those with the CC genotype (Barbato et al. 2004a; Brand et al. 1998; Hautanena et al. 1998; Schunkert et al. 1999; Stella et al. 2004). Given the association between circulating aldosterone and LVM, it is not surprising that the T allele also associates with LVM, both in subjects with hypertension (Stella et al. 2004) and in patients with hypertrophic cardiomyopathy (Chai et al. 2005c). Interestingly, urinary 11\u03b2-HSD2 activity correlated directly with LVM in essential hypertension (Glorioso et al. 2005). This suggests that glucocorticoids also take part in the regulation of LVM. Furthermore, two independent investigations have shown that the CYP11B2 C-344T polymorphism is in strong linkage disequilibrium with polymorphisms of the nearby CYP11B1 gene (Ganapathipillai et al. 2005; Keavney et al. 2005). Since CYP11B1 (11\u03b2-hydroxylase) is the enzyme catalyzing the final step in the biosynthesis of cortisol, the association with the C-344T polymorphism might not only relate to increased aldosterone levels, but also to reduced 11\u03b2-hydroxylase activity (Hilgers and Schmidt 2005). Future studies, involving aldosterone, 11-deoxycortisol and cortisol measurements in serum and\/or urine (White and Rainey 2005), should address this possibility.\nInteraction with angiotensin II\nAng II stimulates the synthesis and release of aldosterone in the adrenal, and thus MR blockade and\/or aldosterone synthase inhibition will exert beneficial effects in Ang II-dependent models (Fiebeler et al. 2005; Virdis et al. 2002). Remarkably, however, aldosterone also appears to exert its effects, at least in part, via Ang II (or its receptors), and both agonists, when applied together, act synergistically (Gonzalez et al. 2005; Mazak et al. 2004; Xiao et al. 2004) (Fig.\u00a01). For instance, in cultured rat aortic smooth muscle cells, nanomolar concentrations of aldosterone enhanced the effect of Ang II on DNA synthesis (Chai et al. 2005a). Unexpectedly, higher (micromolar) aldosterone concentrations reduced DNA synthesis, both in smooth muscle cells and in cardiac myocytes. Such high aldosterone concentrations also reduced collagen synthesis in cardiac fibroblasts. These latter inhibitory effects most likely reflect glucocorticoid receptor activation by aldosterone (Arriza et al. 1987; Sato and Funder 1996). Furthermore, in human coronary artery smooth muscle cells, Ang II stimulated MR-mediated gene expression in an aldosterone-independent manner, suggesting direct MR activation by post-translational modifications such as phosphorylation (Jaffe and Mendelsohn 2005).\nClinical perspective: why are MR antagonists cardioprotective?\nThe beneficial effects of MR antagonists in heart failure cannot be explained on the basis of their renal and\/or blood pressure-lowering effects. Most likely they relate, at least in part, to blockade of a wide range of MR-mediated effects in the heart, including endothelial dysfunction, a decrease in cardiac inotropy and coronary flow, and the induction of fibrosis, hypertrophy, oxidative stress and arrhythmias. Some of these effects occur via or in synergy with Ang II. Given the virtual absence of aldosterone production in the heart, and in view of the low cardiac levels of the cortisol-inactivating enzyme 11\u03b2-HSD2, it is unlikely that, under all conditions, aldosterone is the endogenous agonist of cardiac MR. Thus, some of the MR-mediated effects in the heart may be due to MR activation by glucocorticoids, particularly when ROS levels are high. The aldosterone levels in the failing human heart, but not those in the healthy heart, are high enough to be of functional importance. Whether some of the above (acute, \u2018non-genomic\u2019) in vitro effects of aldosterone are mediated via a non-MR-mediated mechanism remains to be proven, in particular because no (membrane receptor) candidate has been identified so far that induces these effects. If true, however, aldosterone synthase inhibitors might be expected to exert beneficial effects on top of MR blockade.","keyphrases":["mineralocorticoid receptor","aldosterone","inotropy","arrhythmia","non-genomic","cortisol"],"prmu":["P","P","P","P","P","P"]}
{"id":"Am_J_Community_Psychol-2-2-1705531","title":"Being Silenced: The Impact of Negative Social Reactions on the Disclosure of Rape\n","text":"Rape survivors who speak out about their assault experiences are often punished for doing so when they are subjected to negative reactions from support providers. These negative reactions may thereby serve a silencing function, leading some rape survivors to stop talking about their experiences to anyone at all. The current study sought to examine this worst case scenario. Focusing on the qualitative narratives of eight rape survivors who initially disclosed the assault but then stopped disclosing for a significant period of time, this study sought to provide an in-depth description of how negative reactions silenced these survivors. Three routes to silence were identified: 1) negative reactions from professionals led survivors to question whether future disclosures would be effective; 2) negative reactions from friends and family reinforced feelings of self-blame; and 3) negative reactions from either source reinforced uncertainty about whether their experiences qualified as rape. Implications for future research and practice are discussed.\nFeminist activists and scholars have long been interested in the dynamics that keep women from speaking about their experiences. These analyses emphasize the sociopolitical nature of voice and silence. In this tradition, feminist sociologist Shulamit Reinharz describes voice as \u201chaving the ability, the means, and the right to express oneself, one's mind, and one's will. If an individual does not have these abilities, means, or rights, he or she is silent\u201d (Reinharz, 1994, p. 180). This conceptualization highlights social power structures that privilege some voices while excluding others. As metaphors for privilege and oppression, to speak and be heard is to have power over one's life. To be silenced is to have that power denied.\nSilence is thus emblematic of powerlessness in our society. It is therefore not surprising that rape survivors often remain silent about their experiences (George, Winfeld, & Blazer, 1992; Koss, 1985; McAuslan, 1998). Feminist scholars have long argued that rape serves an active function of reinforcing women's powerlessness and \u201ckeeping women in their place\u201d (e.g., Brownmiller, 1975; MacKinnon, 1987). How, then, can we expect women to break the silence about the very experience used to reinforce powerlessness?\nAmazingly enough, many women do find the strength to break this silence and speak out. Nearly two-thirds of all rape survivors disclose the assault to at least one person (Golding, Siegel, Sorenson, Burnam, & Stein, 1989; Fisher, Daigle, Cullen, & Turner, 2003; Ullman & Felipas, 2001; Ullman, 1996a, 1996b, 1996c). But, the consequences of speaking out are not always positive. Numerous studies have documented negative social reactions from significant others and community systems. Negative social reactions from informal support providers encompass both overtly negative reactions such as blaming or doubting victims (Davis, Brickman, & Baker, 1991; Golding et\u00a0al., 1989; Ullman, 2000) as well as well-intentioned support efforts that are nonetheless experienced as negative (e.g., encouraging secrecy, patronizing behavior)(Herbert & Dunkel-Schetter, 1992; Sudderth, 1998). Anywhere from one-quarter to three-quarters of survivors receive negative social reactions from at least one member of their informal support network (Campbell, Ahrens, Wasco, Sefl, & Barnes, 2001; Golding et\u00a0al., 1989; McAuslan, 1998; Filipas & Ullman, 2001).\nSurvivors are also likely to receive negative reactions from formal support providers. Negative reactions from professional sources may be particularly harmful for survivors. When \u201cexperts\u201d doubt survivors, hold them responsible for the assault, or refuse to provide assistance, survivors may question both the effectiveness of such services and the usefulness of reaching out for help to anyone at all. Unfortunately, negative reactions from community system personnel appear to be all too common. Rape victims frequently report receiving negative or unhelpful reactions from legal and medical personnel (Campbell, Sefl, Barnes, Ahrens, Wasco, & Zaragoza-Diesfeld, 1999; Campbell, Wasco, Ahrens, Sefl, & Barnes, 2001; Golding et\u00a0al., 1989; Filipas & Ullman, 2001).\nWhen rape survivors are exposed to victim-blaming behaviors or attitudes, the experience may feel like a \u201csecond assault\u201d or a \u201csecond rape\u201d, a phenomenon known as \u201csecondary victimization\u201d (Campbell, 1998; Madigan & Gamble, 1991; Martin & Powell, 1994; Williams, 1984). In many instances, these behaviors are overt as system personnel explicitly hold survivors responsible for the assault, doubt the veracity of survivors\u2019 stories, or minimize the seriousness of the crime (Madigan & Gamble, 1991). In other instances, this revictimization occurs when rape survivors are denied needed or desired services (Campbell & Bybee, 1997; Campbell et\u00a0al., 2001; National Victim Center, 1992).\nSpeaking out about the assault may therefore have detrimental consequences for rape survivors as they are subjected to further trauma at the hands of the very people they turn to for help. Negative reactions can thereby serve a silencing function. Women who initially break the silence and speak out about the assault may quickly reconsider this decision and opt to stop speaking. Negative reactions such as being blamed, being denied help, or being told to stop talking about the assault may effectively quash rape survivors\u2019 voices, rendering them silent and powerless.\nThe experiences of survivors who have been silenced by such reactions, however, has remained largely unexamined. As an initial examination of this unstudied area, the current study identified a select sample of rape survivors who had been so traumatized by negative social reactions that they stopped speaking about the assault altogether. This sample was selected from a larger project on the impact of community services on rape survivors\u2019 well-being (Campbell et\u00a0al., 1999). The author of the current study was the Interview Coordinator for this larger project. While conducting interviews, she became interested in survivors who ceased talking to anyone about the assault for significant periods of time. This interest led to the current project which sought to obtain an in-depth understanding of how the negative reactions rape survivors received led to their decision to stop speaking about the assault. Qualitative analysis is particularly suited to this type of inquiry. Engaging in an in-depth, context-laden analysis of a smaller number of cases enables us to gain a fuller understanding of how the phenomenon in question is experienced by our participants (Guba & Lincoln, 1994). Creating such a thick description (Geertz, 1973) of how negative reactions can silence some rape survivors is the goal of the current study.\nMethod\nRecruitment procedures\nRecruitment procedures for the larger study were modeled after techniques of adaptive sampling (Thompson & Seber, 1996) whereby researchers systematically sample from locations that are frequented by the population of interest (see also Campbell et\u00a0al., 1999 for a detailed review of this recruitment strategy). We sought to ensure both breadth of coverage by systematically recruiting from the 69 zip codes in Chicago and depth of coverage by targeting locations frequented by women during their daily lives (e.g., laundromats, bookstores, churches). Posters, fliers, and in-person presentations inviting rape survivors to call for more information were distributed in these locations. These recruitment efforts were systematically plotted and tracked to ensure breadth of coverage with intensive efforts in zip codes with high concentrations of traditionally overlooked populations (e.g., women of color, lower socioeconomic neighborhoods). In-person interviews were then scheduled with women who were at least 18 years old and had been raped when they were 16 years old or older.\nRecruitment was conducted between September 1997 and April 1998 and resulted in 102 interviews with adult rape survivors. A smaller subset of survivors who had been silenced by negative reactions was then selected for the current study. Specifically, survivors who initially disclosed the assault within 3 days, received at least one negative reaction during those initial disclosures, and then ceased disclosing altogether for 9 months or more were included in this sample. These inclusion criteria were purposefully stringent. As the first exploratory study of this phenomenon, an extreme sample was purposefully selected to begin to shed light on the relationship between negative social reactions and silence. The final sample of eight rape survivors was thus intentionally small in order to highlight the worst case scenario. The use of such small samples in critical case and narrative analyses has been deemed more than adequate in the qualitative literature (Sandelowski, 1995) and thus was deemed sufficient for the purposes of the current study. More subtle manifestations of the relationship between negative social reactions and silence can be examined in future studies.\nParticipant characteristics\nFive of the eight survivors were African American while the remaining three were White. None of these survivors were currently married and five did have children. Five of these survivors had at least a high school degree. Five survivors were also currently employed. Half of the women were raped by someone known to them. Their average age at the time of the assault was 23.88 (SD=7.32). Half of these assaults involved a weapon, five resulted in injuries, and two involved alcohol. The only significant difference between this smaller subset of survivors and the larger pool of 102 survivors was time since the assault. The average time since the assault was greater for the survivors in this smaller sample (M=16.10, SD=11.59).\nMeasures\nQualitative data was obtained from an in-person interview developed for the larger study. The interviews for the current sample lasted an average of 2.20\u00a0hr (SD=55.29\u00a0min) and were conducted by one of 13 ethnically diverse graduate and undergraduate interviewers who had received extensive training on sexual assault and interviewing skills. Great care was taken to provide a safe and sensitive interview environment, resulting in positive feedback from participants at the end of the interview (see Campbell et\u00a0al., 2004 for a lengthier description of steps taken to ensure participant well-being).\nThe semi-structured interview was designed to obtain a comprehensive picture of rape survivors\u2019 post-assault experiences. This interview consisted of 20 main content areas that covered the assault itself, initial disclosures, interactions with five key community systems (e.g., legal, medical, mental health, rape crisis centers, and religious community), reasons for non-disclosure, social reactions, the impact on survivors\u2019 social and sexual activities, psychological and physical health symptoms, and changes in survivors\u2019 worldviews. Survivors received $30 and a community referral booklet for their participation.\nAlthough information about survivors\u2019 assault and disclosure experiences emerged throughout this interview, several questions were more useful in generating data related to the aims of the current study. Each of these questions is described below.\nThe assault. Survivors were asked to describe the assault in their own words: \u201cCould you tell me about the assault? What happened? Would you tell me your story?\u201d\nFirst disclosures. Five questions were used to elicit information on survivors\u2019 first disclosures: 1) \u201cWho was the first person you told about the assault?\u201d; 2) \u201cWhy was __ the first person you told about the assault?\u201d; 3) \u201cHow did __ react?\u201d; 4) \u201cHow did __ reaction affect you?\u201d; and 5) \u201cLooking back over it now, how do you feel about telling __? Was this a good choice? a bad choice? Why?\u201d\nDisclosure to formal support providers. Separate sections focused on survivors\u2019 interactions with legal, medical, mental health, rape crisis, and religious communities. In each section, survivors were asked: 1) \u201cHow did you decide to contact the [community system] about the assault?\u201d; 2) \u201cWhat was it like for you [receiving each service]?\u201d; and 3) \u201cHow did [community system] treat you [while receiving each service]?\u201d Participants were also read a list of negative reactions (e.g., blamed, doubted, denied services) and were asked to indicate which of these reactions they experienced.\nDisclosure to informal support providers. Descriptions of survivors\u2019 interactions with family and friends emerged primarily during their responses to Ullman's (2000) Social Reactions Questionnaire. Survivors were read a list of positive and negative social reactions and were asked: 1) whether they received that reaction; 2) who reacted that way; and 3) how that reaction made them feel. While the SRQ is traditionally administered in written form, the oral presentation of these items allowed survivors to explain these interactions more fully.\nReasons for non-disclosure. Several questions assessed rape survivors\u2019 reasons for not telling other people about the assault. These questions allowed rape survivors to explain their reasons for non-disclosure in their own words. A series of four questions explicitly asked survivors about their reasons for not disclosing to specific community systems. These questions focused on survivors\u2019 reasons for not seeking out disclosure opportunities: 1) \u201cSo, you did not have any contact with the [specific community system], why was that?\u201d; 2) \u201cWere there things that prevented you from seeking assistance from [specific community system]? If so, what were they?\u201d; 3) \u201cIs there anything that could have been done to make it more likely that you would have turned to [specific community system] for help?\u201d; and 4) \u201cLooking back over it now, do you think it was a good choice, or bad choice, to not seek help from [specific community system]? Why is that?\u201d\nInformation about survivors\u2019 reasons for not telling friends and family about the assault emerged in three specific sections of the interview. Survivors discussed their reasons for not telling additional friends and family when discussing their first disclosure experiences (described above), their interactions with family and friends (described above), and when asked \u201chow did this experience change your understanding of \u2026 a) your family; and b) your friends?\u201d In these sections, survivors discussed reasons for not telling specific friends and family as well as reasons for not wanting to tell informal support providers in general.\nAnalysis procedures\nThis project used a qualitative approach to data analysis. In particular, narrative analysis, based on the recommendations of Miles and Huberman (1994), was used to help identify themes in survivors\u2019 narratives. The first step of this process was to reduce and organize transcribed data. The lead investigator first identified transcript segments that pertained to assault and disclosure experiences and noted key concepts in both the margins and on separate index cards. This process resulted in hundreds of index cards referring to dozens of concepts (similar to the labeling stage of open-coding advocated by Strauss & Corbin, 1990). Undergraduate research assistants who had been recruited from the lead investigator's research methods class were then trained to conceptually group these concepts into larger categories (similar to the discovering categories stage of open-coding advocated by Strauss & Corbin, 1990) by sorting the index cards into conceptually similar and dissimilar piles. A consensus model was used throughout this process whereby disagreements were discussed until agreement on the proper placement of the cards was reached. Each pile was then reviewed one more time and a consensus approach was used to create a name and definition for each concept being described. The resulting names and definitions were then used to create a codebook.\nThis codebook was then used to code the transcripts. To train the research assistants, the first transcript was coded together as a group. Each research assistant was then given her own transcript which she coded independently while the lead investigator simultaneously coded all eight transcripts. Appropriate code(s) were written in the margin next to the corresponding text segment. Codes were then compared, interrater reliability was computed, and final decisions about appropriate codes were made using a consensus approach. Two types of discrepancies were noted: disagreements and omissions. Disagreements occurred when a research assistant assigned one code while the author assigned a different code. Overall, there were few outright disagreements. The kappa coefficient for coding disagreements was .94 indicating excellent interrater reliability (Fleiss, 1971 as cited in Pett, 1997). More commonly, coding discrepancies involved omissions where either the research assistant or the author applied a code that the other coder overlooked. This occurred exclusively in cases where multiple codes were warranted. When omissions were included in the calculation of interrater reliability, the kappa coefficient was reduced to .63 indicating \u201cgood\u201d agreement (Fleiss, 1971 as cited in Pett, 1997).\nResults\nTo contextualize survivors\u2019 decisions to cease disclosing, vignettes summarizing their assault and disclosure experiences are presented first. Pseudonyms are used throughout.\nVignettes of survivors\u2019 experiences\nNatalie. Natalie was abducted from a bus stop by three men and taken to an abandoned building where she was assaulted both vaginally and anally. The ordeal lasted five hours and ended when they set the building on fire. After escaping, Natalie staggered home and passed out, injured and hemorrhaging. When she woke up, she was in the hospital and the police were there. The police took her back to the scene, but the woman who answered the door said she didn't know anything and that no-one else was there. The police did not investigate any further.\nNatalie felt that the police didn't care and weren't going to do anything to help:They wasn't going to do shit, wasn't nothing going to be done.\nShe was also distressed by their lack of sensitivity when she was recounting her experience:I remember one of the police officer laughed.\nNatalie also felt that the police doubted her story and held her accountable for the rape. These negative experiences with the police made her reluctant to have any further contact with them:The way they responded to me, I didn't want anything else to do with them.\nNatalie's experiences with the police served to silence her and she didn't speak of the assault again for a year, in part due to a perceived lack of options and in part due to fear that others would react as badly. She finally began speaking about the assault again when she entered a drug treatment program and began working with a counselor who was also a rape survivor.\nKaren. Karen was abandoned by her boyfriend at a coffee shop during a cross-country road trip. The manager offered her a room at the adjacent motel, but when the night watchman brought her food, he raped her. When Karen tried to tell her sister about the assault, her sister didn't seem to identify the experience as rape:Her comment was \u2026 you should never have sex with anybody you don't want to. I\u2019m like, duh. Like I had a choice, you know?\nThis unsupportive interchange caused Karen to question the efficacy of disclosure:It's just that it didn't do any good, it just made me angry.\nHer sister's response was so unsympathetic that she never spoke to her about the assault again. Believing that there was no-one in her life who would support her, Karen did not disclose again for 19 years. In effect, her sister's reaction confirmed her own doubts and fears about whether her experience qualified as rape:I never used the word rape \u2018til like a year ago honestly.\nThe fact that she was unsure of whether the experience qualified as rape also affected Karen's perception of options for disclosure. For example, she described never even considering reporting the assault to the police, going to the emergency room, seeking mental health services, or contacting a rape crisis center.I just felt \u2026 that anybody would say, well, ah, you know, it was your own fault or you were vulnerable or, you know, not that you asked for it, but you were in that position, what did you expect, or something like that, you know. There was like, it's not going to be any help. And, well, you just laid there, you know.\nAfter 19 years of silence, Karen began disclosing again after she ran into the ex-boyfriend who had abandoned her. After telling him, Karen was able to start telling other people.\nShawna. Shawna was a self-identified drug addict who prostituted when necessary to support her addiction. The assailant was a fellow drug user who assaulted her with a weapon one night when she was walking down the street. The first person Shawna told about the assault was her cousin who told her that she should have known better:She thought I knew about his, um, past. He was known to do that, take advantage of women.\nThis response reinforced Shawna's feelings of self-blame and fears of being blamed by others. Fears of being blamed were particularly salient because of her lifestyle, leading Shawna to believe that there was no-one out there that she could turn to:Being a drug, intravenous drug user, then you\u2019re like, that's your fault. That's\u2014what you coming here for? I mean, you keep using and you keep going on the streets, that's going to happen.\nFeelings of self-blame led Shawna to cease disclosing altogether for three years, a decision that was reinforced by her distrust of the police and fears of retaliation:Then they kill you, beat you up, lay in the garbage, you won't hear nothing about it.\nShawna began disclosing again when she entered a drug rehab center.\nMarie. Marie was assaulted by two strangers her boyfriend arranged to give her a ride home. They drove to a wooded area where they held a gun to her infant son's head and raped her. When she got home, she told her mother who told her to keep the rape a secret:And I went home, and it was my fault. Shut up and don't you tell anybody what you did.\nMarie also told two friends, but they blamed her and told her to try and forget that it happened:Forget it, it's over, it was your fault, leave it alone.\nShe then turned to her priest and told him about the assault in confession. But, he blamed her for the assault and told her that God was punishing her:He was in confessional. And just, you know, I\u2019m separated, I had no right dating.\nMarie internalized the shame and blame communicated by both her mother and the priest:I felt really, really, really bad. Feeling very bad. I couldn't talk, look at your face. I would, I would look down \u2018cause I\u2019d think you\u2019d look and I\u2019d be filthy, dirty whore \u2026 feel less than a whore, dirtiest thing in God's earth.\nHaving exhausted all of the options she felt were available to her, Marie stopped disclosing for several years. Although Marie did begin to seek counseling several years later and has found some support among new friends, she relies mainly on herself and God for support.\nLinda. After a party, Linda decided to spend the night on the sofa rather than take the bus home alone at night. She woke up to the host raping her anally. The next day, she told a counselor who had been helpful in the past. But, he was very judgmental and blaming:When I went to see him, he said, well, what do you expect? If you stay over at somebody's house like that that you barely know, that's an open invitation.\nThis experience was so traumatizing that Linda decided to stop disclosing altogether:After that, the red flag went up and I just said no, I\u2019m not speaking to anybody about this.\nHer inability to identify other support providers and her fears of similar negative reactions led her to stop disclosing for 13 years:Well, I figured they would do the same thing that this counselor did. They would just blame me and they would discount it.\nThese fears were reinforced by her own uncertainty about how to define her experience:I referred to it as an unfortunate incident. I \u2026 had a narrow view of what rape was.\nLinda broke her silence when a therapist was able to validate her experience for her. She now runs support groups for sexual assault survivors.\nRita. Rita's ex-boyfriend invited her out on his boat where he and one of his friends assaulted her at gunpoint. She remained in a state of shock for three days and then contacted her church's prayer line who told her she must have wanted it to happen:Well, they told me that\u2026that situation could not have occurred unless I\u2019d attracted it by thinking about it \u2026 they said, probably, it must be in your subconscious.\nRita then turned to friends who told her she should have known what would happen and she shouldn't report the assault because she knew the assailant. Rita decided to file a report anyway, but the police dismissed her claims because she knew the assailants:It was as if because I knew the men that suddenly, then, somehow that was not a legitimate complaint or something.\nRita considered contacting a counselor, but the negative reactions she received from the police and her church led her to question the efficacy of such services:I just wasn't in a place where I wanted to invest my money in that. Particularly in as much as everybody along the way that I had sought help from, I mean, always blowing me off. So, I\u2019m not going to pay money to have someone blow me off.\nSince Rita was unaware that rape crisis centers existed and really didn't have any close friends or family that she trusted to have a good reaction, she felt she was out of options for support and justice. This led her to stop disclosing altogether for the next 4 years. Although she has disclosed the assault to researchers and has written a magazine article in the hopes of helping other women, she continues to mainly rely on herself and her spirituality for healing.\nVanessa. Vanessa was assaulted when her ex-boyfriend offered to help her see her sisters whom she had been separated from when she was placed in the foster care system. When she arrived at his home, her sisters were not there and he raped her while threatening her with weights. After the assault, she ran to the nearest phone and called 911. The police took both the assailant and Vanessa to the police station for questioning. Vanessa was then taken to the hospital for the rape exam, a traumatizing experience for her:Well, for me, I was already feeling nasty and dirty and there was semen. Um. It was just like another assault. Cold and impersonal.\nShe was then taken back to the police station where a sergeant accused her of lying:He told me that he know my kind and I was messin up this boy future for college. And I couldn\u2019t-I know I heard what he said, but I couldn't understand. You know, like hey, I\u2019m the one that's the victim, you know. And he was all, pulled out some papers and threw them down and say, you\u2019re a repeated run-away, you in foster care. And all the time, I did not know what to say. I was just looking at him, like, why are you do this to me? He said, that boy have a future in there and you destroying it. You stay away from him.\nThe police refused to file rape charges but did file simple assault charges. When Vanessa arrived at the courthouse, however, one of the assailant's brothers pulled her aside:[He] said if I do not drop the charges, he will burn my mother house down and rape my little sisters.\nCombined with Vanessa's negative experiences with the police, this threat of retaliation served to silence her:I came to them in my most vulnerable state. I\u2019d just been victimized and I walked into a place that was male dominant and what they did or did not do for me hurt me. Emotionally, mentally, very bad. They formed an opinion that will probably take a lifetime to undo.\nAlthough she considered disclosing to friends and family, she was afraid of being punished for skipping school. She did not reach out to the mental health system because she didn't know that the rape would continue to affect her for so long. As the aftermath of the rape became clear to her, however, she still did not contact a therapist because she wasn't sure that such services were appropriate for her:[I didn't know] that it was OK for African Americans to go to a mental place. Back then, not too many people I knew who were my color was going to therapists.\nRape crisis centers were also not an option for Vanessa, primarily because she was unaware that they existed:I didn't even know what that is. And still don\u2019t.\nHaving nowhere else to turn, Vanessa stopped disclosing for 10 years. The current interview was the first time she told her story since her interactions with the legal system.\nTherese. Therese was assaulted by a stranger when she went to the apartment of a man who claimed to be an old acquaintance. She tried to leave when she realized that she had never met him before, but he grabbed her and proceeded to rape her. She remained in shock for three days, feeling unable to talk to anyone. When she contacted her best friend, her friend was sympathetic, but extremely upset:She took it almost as bad as I did.\nThis reaction was difficult for Therese to deal with because she felt guilty for upsetting her friend and felt she had to comfort her:It kind of made me feel like I had to comfort her because she was taking it so hard.\nEven though Therese's friend was trying to be supportive, she was more concerned about herself than Therese. Therese's own feelings of self-blame and shame were also too strong to be overcome. Unable to identify support providers she thought would react well, these high levels of self-blame were ultimately the reason Therese did not report the assault or contact any professional services:Cause I felt like it was my fault. And, ah, I really couldn\u2019t. If I felt like it was my fault, I knew everybody else would be looking at me like, well, it's your fault anyway.\nThese feelings of self-blame and fears of being blamed by others led Therese to cease disclosing for nine months. Unfortunately, her initial effort to break her silence by disclosing to a boyfriend was met by a blaming response. Nonetheless, she decided to participate in the current interview as a first step toward receiving needed services.\nCross-case analysis: Common experiences and themes\nThe preceding vignettes provide rich, contextualized descriptions of each survivor's experiences with disclosure and silence. In-depth analysis of these survivors\u2019 narratives revealed four general types of negative reactions experienced by these survivors: 1) being blamed; 2) receiving insensitive reactions; 3) experiencing ineffective disclosures; and 4) receiving inappropriate support.\nAll of the survivors described being blamed for the assault. These survivors were blamed for putting themselves in vulnerable positions and were frequently told that they should have known better. Such responses were particularly common from community system personnel, especially the legal system. Interactions with the legal system were characterized by questions about whether the assault qualified as rape, their role in the assault, and whether they deserved the assistance the legal system could provide.\nAll of the survivors also received insensitive reactions. These reactions included having a support provider question, doubt, or minimize their experience. Insensitive reactions also occurred when support providers showed no sympathy for her distress or didn't seem to consider what the survivor needed. Legal, medical, mental health, and religious system personnel were all described as reacting insensitively by at least half of the survivors who turned to them. Frequently, these insensitive reactions occurred in conjunction with blaming and doubting responses, reinforcing survivors\u2019 perceptions that community systems didn't care and would not provide any help.\nIneffective disclosures were also quite common. Ineffective disclosures were characterized by a lack of help\/support resulting from disclosure. This code was applied when support providers refused to help. In several cases, help-seeking attempts to counselors, church, or friends\/family did not result in support. In other cases, the police refused to take a report or charge the assailant with rape. Indeed, all three cases that were reported to the police were dropped and none resulted in prosecution of the offender for rape.\nAll but one of the survivors also described inappropriate support, mainly from friends, family, and religious personnel. Inappropriate support referred to support attempts that may have been well-intentioned, but were nevertheless perceived as inappropriate or unhelpful. Being told to keep silent or not report the assault, being treated as though they couldn't take care of themselves, or having to comfort their support providers ultimately interfered with these survivors\u2019 ability to receive support and comfort.\nThese negative reactions then affected survivors\u2019 decisions to cease disclosing the assault. In-depth analysis of these survivors\u2019 narratives revealed five common reasons for ceasing to disclose: 1) lack of options; 2) fears of negative reactions or consequences; 3) ineffectiveness of support; 4) self-blame or embarrassment; and 5) didn't qualify for support.\nAll of the survivors discussed a lack of options as a reason for not continuing to disclose the assault. Many of these survivors were unaware of services available in the community (e.g., rape crisis centers) and all of them felt that there were no additional sources of support available to them. Having evaluated the likelihood of receiving support from others and determined that such support was unlikely, these survivors described having nowhere else to turn.\nEven when survivors could identify providers, they all expressed extreme distrust and fears of negative reactions. Fears of being blamed, doubted, and treated insensitively were pervasive. For many, these fears were directly related to their previous negative disclosure experiences, leading them to conclude that additional disclosures would be harmful.\nSix of the survivors also feared that additional disclosures would be ineffective. These survivors believed that continued disclosure would be unhelpful or would not result in the type of support they needed. They didn't see the use of continued disclosure and felt that further discussion of the assault would be pointless. Combined with fears of negative reactions, their decision to cease disclosing was a form of self-protection.\nSix of these survivors also cited self-blame or embarrassment as reasons for ceasing to disclose. They felt that it was embarrassing to talk about rape and didn't want to put themselves through additional disclosures. The fact that they had been blamed for the assault previously served to heighten feelings of self-blame. These survivors described feeling somewhat responsible for the assault prior to disclosing, a view that was enhanced when they were blamed by others.\nFinally, two survivors described questioning whether their experience qualified as rape. Because these assaults were not particularly violent, these survivors were unsure whether their experience counted as rape even before they disclosed. The fact that their initial disclosure recipients also appeared to question whether their experiences were rape served to reinforce their own doubts.\nThe relationship between negative social reactions and reasons for ceasing to disclose\nAs the above analyses suggest, the survivors in this sample spontaneously referenced negative experiences from initial disclosure attempts when describing their reasons for ceasing to disclose (even though this question was never explicitly asked, suggesting that this was an extremely salient reason for ceasing to disclose). This suggests that these survivors were using their past experiences with disclosure to evaluate future disclosure opportunities. Receiving negative responses to their initial disclosures, these survivors became more cautious and critical of future disclosure opportunities and ultimately opted to remain silent rather than risk further harm.\nThe manner in which negative reactions led to being silenced, but, varied across survivors. Further examination of survivors\u2019 narratives revealed three primary routes to silence. First, three of these survivors described negative reactions that made them question whether future disclosures would be effective. Each of these survivors had disclosed to formal support providers and felt that these disclosures had been ineffective. For example, Natalie, the woman raped by a group of men in an abandoned building, attributed her decision to stop disclosing to the negative reactions she received from the police and medical system. She felt that talking to others would be ineffective so she tried to forget about the assault and move on with her life. Similarly, Rita, the woman assaulted on a boat, described initially seeking help from multiple sources. She ultimately decided that help would not be forthcoming and turned inward to heal herself. Finally, Vanessa, the woman assaulted at her ex-boyfriend's home, also attributed her decision to stop disclosing to her experience with the legal and medical systems. The ineffectiveness of her contact with the legal system was particularly poignant in Vanessa's case and reinforced her fears that the legal system could do nothing to protect her from retaliation by the assailant and his brothers.\nSecond, two survivors described negative reactions that reinforced their own doubts about whether their experience qualified as rape. Both Karen, the woman assaulted by the night watchman, and Linda, the woman assaulted by the host of a party, questioned whether the assault qualified as rape. Their own doubts about whether their assault qualified as rape and the reactions of others combined to make them reluctant to risk further disclosures.\nFinally, three survivors described disclosures which reinforced feelings of self-blame. These survivors\u2019 initial disclosures were either inadequate for overcoming their sense of self-blame or actively reinforced feelings of self-blame. For example, both Shawna, the woman assaulted by a fellow drug addict, and Therese, the woman assaulted at a stranger's home, turned to informal support providers who provided inappropriate support that was inadequate for countering their fears and self-blame. For Marie, the woman who was raped in a car, the negative reactions she received from her mother, friends, and priest reinforced her feelings of self-blame, making her too ashamed to talk about the assault with anyone else.\nDiscussion\nUnlike other crimes such as burglary and assault, rape survivors must prove not only that the crime did in fact occur, but that they had no role in its occurrence (Burt, 1980; Pollard, 1992; Ward, 1995). But, for most survivors, no matter what they did or how they behave, they are likely to be blamed for the assault. For some survivors, this blame may be so traumatizing that they are effectively silenced by the negative reactions they receive. Sadly, when rape survivors are silenced by negative reactions, their experiences and perspectives are concealed and our ability to identify the causes and consequences of rape are obscured. Such silences thereby obstruct our ability to engage in social change. A first step toward unearthing these untold stories may be to understand how and why rape survivors are silenced.\nResults suggested that rape survivors are silenced by a range of negative reactions including blaming, ineffective, insensitive, and inappropriate responses. Specific reactions appeared to be more common from different support providers, however. For example, being blamed, receiving insensitive reactions, and experiencing ineffective disclosures were particularly common among survivors who turned to formal community systems (especially the legal and medical systems). These experiences are consistent with previous research which has documented high levels of victim blame, doubt, insensitivity, and refusal of services by community system personnel (Campbell et\u00a0al., 1999; Madigan & Gamble, 1991; Filipas & Ullman, 2001). Such reactions may stem from organizational features of these systems. In an examination of unresponsive treatment of rape survivors by both legal and medical personnel, Martin and Powell (1994) argue that the organizational frameworks guiding these systems\u2019 activities are oriented towards the needs of the organization rather than the needs of survivors. They suggest that the needs of the systems and rape survivors may run counter to one another. Whereas the survivor needs to be believed and supported, the legal system needs to win cases and the emergency room needs to treat emergent patients. These conflicting needs often result in insensitive and unresponsive treatment of rape survivors (Martin & Powell, 1994). In the current study, survivors described police, medical staff, counselors, and pastors who laughed at their account of the assault, were cold and unsympathetic, and overtly blamed them for the assault. For three of the survivors in the current study, being silenced was a direct result of the accumulation of blaming, insensitive, and ineffective reactions from community system personnel which led them to question the effectiveness of disclosure.\nInappropriate support attempts, on the other hand, were more common from informal support providers such as family and friends. Inappropriate support attempts included suggestions or behaviors that may have been intended to be helpful but were experienced by survivors as hurtful or simply not what they needed. Herbert and Dunkel-Schetter (1992) first noted the distinction between intentionally negative reactions (e.g., blame) and unintentional negative reactions resulting from altruistically motivated, but ineffective support attempts. This distinction was further validated by Ullman (Ullman, 1996a, 1996b, 1996c) in her survey of rape survivors. In the current study, several of the survivors described interactions with informal support providers that were inadequate for overcoming their own feelings of self-blame. In essence, these survivors internalized many of the cultural narratives about rape that emphasize victim culpability. When support providers were unable to counter these messages, the victims engaged in self-silencing, choosing to censor themselves and remain silent about an experience they considered shameful and stigmatizing (Jack, 1991). These survivors did not receive any benefits from disclosure and often felt worse after speaking about the assault. Having lost faith in the efficacy of disclosure, these survivors opted to heal themselves.\nFinally, the current findings suggest that some rape survivors are silenced when disclosure recipients fail to confirm their victim status. Several of the survivors in the current study described knowing that something unwelcome had occurred, but they did not know whether the experience qualified as rape. This finding is consistent with previous research which suggests that rape survivors do not always identify as rape victims, particularly when their experiences do not conform to stereotypical rape myths (Allison & Wrightsman, 1993; Kahn, Jackson, Kully, Badger, & Halvorsen, 2003; Koss, Dinero, Seibel, & Cox, 1988; Layman, Gidycz, & Lynn, 1996). The results from the current study expand on this research, however, by suggesting that disclosure recipients play a role in how rape survivors interpret their experiences. For two of the rape survivors in this sample, initial disclosures were partially motivated by the desire to have others confirm or deny their victim status. Unfortunately, in these cases, the disclosure recipients failed to confirm their victim status and these survivors stopped disclosing as a result.\nThere are several limitations to this current research that are important to note. First, any effort to study silence is limited by the fact that, by definition, the population of interest is not disclosing the assault to anyone. As researchers, the best we can do is work to create recruitment strategies and interview environments that facilitate disclosure for previously silenced populations. In the current study, we went to great lengths to employ strategies that communicated support, understanding, and acceptance (see Campbell et\u00a0al., 2004 for a complete description of these recruitment strategies). These strategies may have enabled previously silenced survivors to come forward and speak about their assaults. Indeed, many of these survivors mentioned the fact that this interview appeared to be a safe place to talk about their experiences. For one survivor, this interview was the first time she had spoken about the assault since being silenced by negative reactions. For others, they only began disclosing once supportive providers were found (most commonly, counselors). Thus, supportive recruitment strategies and interview environments may facilitate disclosure for some survivors. But, it is likely that many others continue to remain silent about their experiences. Important differences may exist between survivors who do and do not choose to disclose to researchers.\nIt should also be noted that these results are not intended to reflect the experiences of all rape survivors. To the contrary, this study sought to examine the experiences of a previously hidden subpopulation of rape survivors\u2014those who have been silenced by negative disclosure experiences. This subpopulation has remained hidden due to both survivors\u2019 own silence about their experiences as well as researchers\u2019 overly simplistic conceptualizations of disclosure. To date, researchers have defined disclosure as a discrete yes\/no event\u2014a survivor disclosed or she didn't (Bachman, 1993, 1998; Binder, 1981; Feldman-Summers & Norris, 1984; Golding et\u00a0al., 1989; McAuslan, 1998; Neville & Pugh, 1997; Ullman, 1996c; Ullman & Filipas, 2001; Washington, 2001). This conceptualization has obscured the experiences of survivors who initially disclose but are then silenced by negative social reactions. As a result, almost nothing is known about how negative reactions can silence rape survivors. While there may only be a small population that is ultimately silenced by negative disclosure experiences, the fact that their voices have been silenced and their stories hidden is problematic. This study was intended as a first step toward unearthing such stories to add to our knowledge of the devastating impact that negative social reactions can have on rape survivors. As is true of most qualitative research, the goal was not to generalize to the entire population of rape survivors. Rather, the goal was to gain an in-depth understanding of the phenomenon in question (in this case, being silenced) and the context in which the phenomenon occurred (Goetz & LeCompte, 1984; Guba & Lincoln, 1982; Miles & Huberman, 1994). Future research can examine the findings in different populations of rape survivors, particularly among other populations that are likely to experience high levels of silence such as child sexual abuse and incest survivors.\nIn spite of these limitations, this exploratory study offers several avenues for further research. First, future research could benefit from larger samples in order to examine the impact of race, socioeconomic status, lifestyle characteristics, and assault characteristics. Increasingly, social identity theorists have argued that social group memberships are interactive such that one's race may affect how social class is experienced and vice versa (Frable, 1997; Weber, 1998). In the current context, it is possible that race, class, lifestyle, and assault characteristics may interact in varying combinations to lead to different outcomes. Unfortunately, the current study did not include a large enough sample to examine all of these combinations. This makes it difficult to interpret some of the racial, class, lifestyle, and assault characteristics that emerged in the current study. For example, all of the survivors who contacted the police were African American\u2014but they also experienced the most brutal assaults. All of the survivors who had difficulty defining their experiences were White\u2014but these assaults also involved less resistance by the survivor. Without comparison groups of White survivors who reported to the police or African Americans whose assaults were not as severe, it is not possible to determine the impact of race and type of assault on disclosure choices or outcomes. Similarly, there is some evidence that race and social class may have interacted in the current study: the police were far more blaming toward Vanessa, who was a young African American girl in the foster care system, than toward Rita, who was a middle-class African American professional. A larger sample would help confirm this possibility.\nFuture research could also further examine the role of support provider gender on the types of reactions provided and the impact these reactions have on survivors. In the current study, gender of support provider was not specifically assessed, leading to two cases where it was not clear whether the hospital personnel and police were comprised entirely of men or were a mixture of men and women. Although this prohibited a direct examination of support provider gender, the information that was available suggests that the vast majority of professionals who provided negative reactions were men. This is in line with previous research that suggests that men tend to respond more negatively than women (Ahrens & Campbell, 2000; Davis & Brickman, 1996). In contrast to previous research, however, the vast majority of informal support providers who provided negative reactions were women. Future research is needed to examine this distinction more methodically.\nFuture research should also compare the impact of positive and negative reactions. The current study focused on the impact of negative social reactions on rape survivors\u2019 decision to stop disclosing. This was, in part, because previous research has suggested that negative reactions are far more impactful than positive reactions (Campbell et\u00a0al., 2001; Davis et\u00a0al., 1991; Ullman, 1996b). It was therefore hypothesized that negative reactions would play an important role in rape survivors\u2019 decisions to stop speaking about the assault, a proposition that had yet to be examined in the literature. Future research is needed to determine how positive reactions affect this relationship, however. If victims receive both positive and negative reactions, how do they weigh these reactions when evaluating subsequent disclosure opportunities? Is there a threshold of positive disclosure experiences that serve to negate negative experiences? Is there a threshold of negative disclosure experiences that negate positive experiences? Or is a single positive or negative experience enough to determine whether victims will disclose? Future research on the impact of positive and negative social reactions on disclosure is needed to begin to answer these questions.\nFinally, the impact of such extensive periods of silence on survivors\u2019 recovery remains unknown. Predictions from Pennebaker's Psychoimmunology Theory of Disclosure would predict that the inhibition of emotional expression that accompanies such lengthy periods of silence would adversely affect survivors\u2019 psychological and physical health (Pennebaker, 1988, 1989; Pennebaker & Susman, 1988). On the other hand, these survivors chose to stop disclosing in order to avoid negative reactions that have been found to relate to higher psychological and physical health symptoms (Campbell et\u00a0al., 2001; Davis et\u00a0al., 1991; Ullman, 1996b; Ullman & Filipas, 2001). Ceasing to disclose may therefore have a positive impact on survivors\u2019 recovery by helping them avoid such negative reactions. Additional research is needed to understand the impact that their decision to stop disclosing has on their recovery.\nOn a more practical level, the results of the current study suggest that negative reactions are particularly detrimental as survivors use these reactions as a gauge for how others are likely to respond. These results attest to the importance of continued efforts to reduce rape myth acceptance and train support providers on how to effectively support rape victims. Combating rape myths, educating the public about sexual assault, and training potential support providers to avoid negative reactions may help reduce the trauma of the assault and increase the likelihood that victims receive the support they are seeking when they turn to others for help. Such efforts may be further enhanced by changes in the organizational context of formal community systems. Until the institutional orientation of the legal system is changed to reward prosecution of all rape cases rather than only those cases that are considered convictable, the legal system will likely continue to blame rape victims who come to their attention. Until the medical system comes to view providing support as part of their role, victims will likely continue to be treated insensitively. Thus, educational and training efforts should be used in conjunction with efforts to help shift the organizational orientation of formal community systems.","keyphrases":["disclosure","rape","secondary victimization","social relations"],"prmu":["P","P","P","R"]}
{"id":"Bioprocess_Biosyst_Eng-2-2-1705514","title":"Improving the batch-to-batch reproducibility in microbial cultures during recombinant protein production by guiding the process along a predefined total biomass profile\n","text":"In industry Escherichia coli is the preferred host system for the heterologous biosynthesis of therapeutic proteins that do not need posttranslational modifications. In this report, the development of a robust high-cell-density fed-batch procedure for the efficient production of a therapeutic hormone is described. The strategy is to guide the process along a predefined profile of the total biomass that was derived from a given specific growth rate profile. This profile might have been built upon experience or derived from numerical process optimization. A surprisingly simple adaptive procedure correcting for deviations from the desired path was developed. In this way the batch-to-batch reproducibility can be drastically improved as compared to the process control strategies typically applied in industry. This applies not only to the biomass but, as the results clearly show, to the product titer also.\nIntroduction\nBiologics are known to be rather complex products. Apparently small changes in the manufacturing processes can cause significant differences in their clinical properties. Hence, production processes for biologics are approved by authorities only with clearly defined constraints on their manufacturing procedures. Consequently, reproducibility is of utmost importance. Additionally, reproducibility is very important as it affects the downstream processing and thus quality of the final product.\nFrom the engineering point of view there are two challenges in guaranteeing batch-to-batch reproducibility. First of all, within the given constraints, the operational procedure, most robust with respect to typically appearing process fluctuations, must be found. And, secondly, while running the process along this robust path, the remaining randomly appearing disturbances must be eliminated by means of feedback control.\nWith respect to batch-to-batch reproducibility, production processes for recombinant proteins are lagging far behind most other industrial processes. Figure\u00a01 shows a typical example of the repeatability of biomass concentration profiles in a recombinant protein production process. The variability is quite high.\nFig.\u00a01Biomass (open circles) and target protein (open triangles) concentration profiles from 13 fed-batch fermentations for the production of a recombinant protein. Typically the batch-to-batch reproducibility of these production processes is rather low\nThis fact was recognized by the FDA. The agency responded with a couple of measures. One essential reaction is FDA\u2019s PAT initiative [2]. With PAT, improvements in pharmaceutical and biologics production processes with respect to real time automated process monitoring and control are demanded. A rigorous science-based approach to manufacturing is demanded, as better understanding is thought to lead to more efficient process control, lower process variability, thus high product quality and finally patient safety. For biologics it is particularly important to keep the processes under control early in the product synthesis process (e.g., [1]).\nIn bioprocess engineering, process monitoring and control is being developed since many years. A review was given by Lee et al. [8]. The objective was to keep the process on trajectories that finally lead to a high value of the desired mass mP of the product. Within a more or less well-defined production time tP, this can be related to biomass by the following expression:  where \u03c0 is the specific product formation rate and x the total biomass. Both arguments of the integral are functions of time, but more importantly, both are primarily dependent on the specific biomass growth rate \u03bc in most industrial production systems. The growth rate that a specific medium supports determines the physiological state of the cells, and particularly the cell\u2019s protein-synthesizing machinery that is important to recombinant protein production is also under growth rate control [12]. Hence, the variable that rules the final outcome of the process is the specific biomass growth rate \u03bc. Thus, design of effective cultivation processes should be based on an optimal or at least a quasi-optimal profile of the specific growth rate. This can be obtained by means of numerical optimization procedures (e.g., [10]) or simply by deriving a profile from data records and experience with the production system under consideration. In order to make sure that the process follows this profile it is straightforward to control \u03bc in the engineering sense (e.g., [5\u20137, 11, 13, 14, 16\u201318]).\nDirect control of \u03bc works perfectly as long as there are no severe disturbances in the process. When, however, some disturbances lead to a significant deviation of the biomass from its desired path, one must correct it before one can proceed with the desired optimal or quasi-optimal \u03bc profile. Otherwise reproducible process trajectories cannot be obtained.\nThe decisive innovation in this paper is to show that the batch-to-batch reproducibility of the production processes can be significantly enhanced when the process is controlled to a predefined profile of the biomass x. This does not mean to stay away from keeping the \u03bc profiles found to be optimal from the physiological point of view. The x-setpoint profiles are simply derived from predefined \u03bc profiles. Using start biomass and desired specific biomass growth rate profile it is easy to estimate total biomass profile during the cultivation: \nA given biomass profile xset(t) is then in close relationship with the specific biomass growth rate \u03bc. Hence, controlling the process to an x profile should satisfy the corresponding specific growth rate profile \u03bcset(t) as well. In this case the cultivation process is more robust because the deviations in biomass concentration can be eliminated directly by controlling the integral variable x.\nAs x cannot be measured directly with sensors that work reliably at a production fermenter and provide biomass values representative for the culture, it should be measured indirectly. This is a further advantage as compared to the \u03bc-control because we are able to estimate x much more reliably than the specific growth rate \u03bc [4]. In production environments, where sufficiently many data records are available from the process under consideration, artificial neural networks (ANNs) yield very accurate estimates. Hence we use ANNs to estimate biomass x. The ANN was trained on 26 data sets measured during a process development project with the strain used in this work.\nMaterials and methods\nExperiments were performed with Escherichia coli BL21(DE3) as the host cell. The recombinant target protein was coded on the plasmid pET 28a and expressed under the control of the T7 promoter after induction with isopropyl-thiogalactopyranosid (IPTG). The strain was resistant to kanamycin. The product appears as inclusion body within the cytoplasm. The particular strain used did not produce measurable amounts of acetate under the cultivation conditions adjusted in the experiments reported. This was tested in the beginning by means of appropriate test kits.\nThe main substrate was glucose. It was fed at a concentration of 600\u00a0g\/kg. The other components are compiled in Table\u00a01.\nTable\u00a01Composition of the mineral mediumMineral salt solutionTrace element solutionComponentConcentration (g\/kg)ComponentConcentration (g\/kg)K2HPO414.60Na2-EDTA20.10NaH2PO4\u00b7H2O3.60FeCl3\u00b76H2O16.70(NH4)2SO42.46CaCl2\u00b72H2O0.74Na2SO42.00CoCl2\u00b76H2O0.21MgSO4\u00b77H2O1.20ZnSO4\u00b77H2O0.18(NH4)2-H-citrate1.00CuSO4\u00b75H2O0.10NH4Cl0.50MnSO4\u00b7H2O0.10Kanamycin0.10Thiamin0.10Trace element solution2\u00a0mL\/kg\nAll experiments were performed within BBI Sartorius System\u2019s BIOSTAT\u00ae ED 15\u00a0L bioreactor with 8\u00a0L working volume (Fig.\u00a02). The fermenter was equipped with standard 6-blade Rushton turbines that could be operated at up to 1,400\u00a0rpm. The aeration rate could be increased up to 24\u00a0L\/min. Aeration rate and then stirrer speed were increased one after the other in order to keep the dissolved oxygen concentration at 25% saturation. In order to suppress foam formation, increase oxygen solubility and reduce the risk of contamination, the fermenter head pressure was kept at 0.5\u00a0bar above the ambient pressure in the laboratory.\nFig.\u00a02Experimental setup of the cultivation equipment\nThe fermentations were operated in the fed-batch mode from the early beginning with an initial volume of 5\u00a0L at pH 7 and a temperature of 35\u00b0C. All fermentations were started in the night by automatic transfer of the inoculation biomass from a refrigerator into the reactor. Substrate feeding starts immediately after inoculation with a fixed exponential profile. According to Jenzsch et al. [3], the specific growth rate setpoint was chosen as 0.5\u00a0h\u22121. Thus the glucose concentration appeared to be at a very low value just from the beginning on. Its value is in the order of the Ks value and thus below the values that can be measured during the fermentation. After the biomass concentration reached values of about 35\u00a0g\/kg, the culture was induced with 1\u00a0mM IPTG. From the seventh fermentation hour on, the growth rate was kept under feedback control along an x profile that was derived from a predefined profile of the specific growth rate. The corresponding profile of the substrate feed rate was determined from the profiles of \u03bc and x. This was taken as reference feeding profile Fref for all the fermentations described.\nTemperature was measured with a Pt-100, pH with an Ingold-pH-probe, pO2 with an Ingold pO2-Clark-electrode, CO2 in the vent line with MAIHAK\u00ae\u2019s Unor 610 and O2 there with MAIHAK\u00ae\u2019s Oxor 610. Further, the total ammonia consumption during pH control was recorded by means of a balance beneath the base reservoir. All these quantities were measured online. Additionally, enhanced foam levels could be detected with a foam sensor and, if the critical level was reached, a silicone antifoaming emulsion (ROTH\u00ae) was added.\nBiomass concentrations were measured offline (via the optical density at 600\u00a0nm) with a Shimadzu\u00ae photo-spectrometer (UV-2102PC). In preceding experiments a correlation was established between these values and the biomass dry weights, which were determined with the standard drying and weighting technique. Glucose was determined enzymatically with a YSI 2700 Select Bioanalyzer. The product was measured with SDS-PAGE after separation of the inclusion bodies and their solubilization.\nResults\nPreliminary simulations and experiments showed that the total biomass x(t) is better suited as the controlled variable than the biomass concentration X(t) itself. The control can then be performed with a simple adaptive control algorithm comprising the following steps:Estimate the total biomass xest at the current time instant t using the artificial neural network trained before on the available data records [4]. A simple feedforward artificial neural network with a single hidden layer was used which makes use of the online measured signals OUR, CPR, total base consumption resulting from pH control. With an additional bias node in the input layer the network has four input nodes. Five hidden layer nodes were used where the nonlinear response function of these nodes was chosen to be tangens hyperbolicus (tanh) functions. A single output, namely the biomass x, was taken, where the weighted sum of its input signals was directly used as the output signal.Compute the deviation of estimated biomass xest from its setpoint xset: The deviation \u0394x in x was used to correct the growth yield value Yxs in the feed function, which itself was limited to the interval given in order to avoid too big variations in cases where the measurements are disturbed in an unusual way: The concrete limits to F were chosen by experience in control practice.In order to determine the controller variable \u03b1 by which the growth yield is adapted to the current state of the process we found that it is better to make use not only of the deviation \u0394x but also the smoothing action of an integral correction term to the deviation: Again, the correction was limited. Here the limitation was chosen by experience in such a way that the resulting yield values are kept within reasonable limits. The parameters k1 and k2 were initially determined in simulation studies. Later, during preliminary control experiments, they were slightly adapted. Their final values are k1\u00a0=\u00a00.1\u00a0(kg(S))\u22121 and k2\u00a0=\u00a00.02\u00a0(kg(S))\u22121\u00a0h\u22121.\nThe controlled biomass profiles depicted in Fig.\u00a03 show that this simple control approach leads to a very good reproducibility of the total biomass profiles.\nFig.\u00a03Total biomass signals from five fermentations performed sequentially using the same setpoint profile\nAs one is usually not interested in the total biomass, the corresponding biomass concentrations are depicted in Fig.\u00a04. As can easily be seen from both plots, the batch-to-batch variability of the trajectories is rather small, i.e., the process total biomass control works well.\nFig.\u00a04Measurements of the biomass concentration during five experiments in which the total biomass was controlled. The induction time was tind\u00a0=\u00a011\u00a0h in all cases\nIn order to get a better impression of the controller action, the deviations from the desired profile, i.e., the relative deviations between the total biomass and its setpoint, are plotted in Fig.\u00a05. In all the experiments, the controller was switched on at t\u00a0=\u00a07\u00a0h. In the first 4\u00a0h thereafter, the relative deviations remain within a 5% interval. The controller action then improves so that finally the relative deviation of the total biomass remains within a 2% interval around the mean. The feed rate profiles F(t) applied in the experiments are shown in Fig.\u00a06.\nFig.\u00a05Relative deviations of the total biomass from the mean. The controller was switched on 7\u00a0h after the cultivation was inoculatedFig.\u00a06Substrate feed rate profiles for the six x-controlled fed-batch experiments\nThere is one exception. In the last experiment (S330) feed pump was switched off from 3 to 5\u00a0h in order to test the controller performance under process conditions with an extremely hard disturbance. The controller appeared to be robust enough to cope with this disturbance. Again in the end the relative deviation from the mean remained in the 2% interval.\nThe next question is what does this mean for the profiles of the specific biomass growth rate, which was initially chosen to determine the setpoint profiles for the total biomass. This comparison essentially should demonstrate data consistency. The specific growth rate \u03bc was determined from a Luedeking\u2013Piret type relationship between the biomass concentration, carbon dioxide production or oxygen uptake rate and the specific growth rate: \nBoth equations can be resolved for \u03bc. The parameters were already known from a fit of the models to the fermentation data.\nHence, with the biomass concentration profiles estimated and the measured OUR(t) and CPR(t) signals, \u03bc can easily be computed. The result is depicted in Fig.\u00a07. In the decisive phase of the process, the product formation phase, the resulting trajectories of the specific biomass formation rate \u03bc(t) resemble quite well the original setpoint profiles. It should be recalled that rather high noise is to be expected when computing \u03bc(t). However, the results depicted in Fig.\u00a07 nevertheless clearly show that the data are consistent.\nFig.\u00a07Specific biomass growth rate profiles determined from the measurement profiles of OUR, CPR as well as from the estimates for biomass concentration X(t). Note that the study 330 was the test case where the substrate supply was interrupted for some period during the biomass growth phase\nFinally the question arises what does a high batch-to-batch reproducibility in the biomass profiles mean for the variance in the product formation profiles. Results corresponding to the data shown before are depicted in Fig.\u00a08.\nFig.\u00a08Product concentration profiles for the cultivation processes already mentioned. All protein data stay within the error bar ranges representing the confidential interval of the protein analysis method\nThe corresponding protein concentration profiles are rather close together saying that the improved reproducibility in biomass profiles by means of x control also leads to an improved batch-to-batch reproducibility in the product concentration profiles. All profiles stay within the confidence interval of the analytical protein detection method. The relative error of \u00b115% of target protein analysis by SDS-PAGE was estimated from a fivefold analysis using the same fermentation samples.\nDiscussion\nPrevious work on controlling the fermentation processes performed to manufacture recombinant proteins focused on keeping the cultures on tracks that guaranteed a high productivity or yield. The obviously first quantity influencing the amount of protein that is produced in a cultivation run is the biomass employed, hence high-cell-density cultures are required (e.g., [9, 15]). As the cells\u2019 protein-synthesizing machinery that is particularly important to recombinant protein production is under growth rate control [12], the performance of the cells is ruled by the specific biomass growth rate \u03bc. Hence, much work has been put into closed loop control of fermentation processes along appropriate profiles \u03bcset(t) of the specific biomass growth rate [5\u20137, 11, 13, 16\u201318]. While advantageous from the cell physiological point of view, controlling the specific growth rate directly has the disadvantage of leading to a relatively low reproducibility of the fermentations [6]. This is a significant disadvantage from the process quality point of view.\nWe wished to extend the work on fermentation control towards quality assurance of process and thus product formation. This first of all requires improving the batch-to-batch reproducibility of the processes. There are two motivations for this. First, the product quality in recombinant protein manufacturing processes can be affected by changes in the fermentation operational procedure, hence the authorities link process approval with tight constraints on the process trajectories. Thus, good reproducibility increases product quality. Secondly the downstream processing can work much more efficiently when the cultivation results are highly reproducible. Therefore, having the same culture each time should be beneficial to the overall product yield as well.\nThe results of the new control procedure discussed below show that the batch-to-batch reproducibility is significantly improved. The procedure controls the biomass along a profile that is directly derived from the specific growth rate profile that was found to be desirable. The first decisive advantage of this approach is that the control procedure is robust to distortions usually appearing in practice. This is opposite to control along a predefined profile of the specific biomass growth rate. Practically all distortions lead to changes in the biomass and this is the controlled variable. A second advantage is that the actual biomass values can be estimated with higher accuracy than the specific growth rate from the online measurement signals available at most fermenters. As shown previously [4] x can be representatively estimated with a root mean square error of about 0.5\u00a0g\/kg by means of simple artificial neural networks.\nThe reason for the robustness of the x control is easy to understand. The x-setpoint profile for the control was determined from a \u03bc profile. This \u03bc profile leads to a feed rate profile F(t) and a biomass profile x(t). The F(t) profile is taken as the feed forward component of the process and programmed in the programmable controller at the fermenter. If due to distortion the biomass becomes smaller as expected, the cells will see more substrate than expected and respond temporarily by increasing their growth rate. If, on the other hand, the distortion leads to a higher biomass concentration, the cells see less substrate than expected and reduce their growth rate, thus correcting for their deviation in the total biomass. The feedback component of the process has thus only to correct for larger deviations.\nControl of the specific biomass growth rate or qualitatively similar control technique that uses the substrate feed rate F as the manipulated variable is currently not generally used in production practice. Its adoption is now supported by recent initiatives of the FDA in its PAT initiative [2], but will critically depend on the ease of the control algorithm. The algorithm used in this work is an extremely simple adaptive control approach which only corrects the biomass-on-substrate yield in the basic feed rate expression. The control algorithm proved to be stable in many fermentation runs with many different organisms and several different fermenter scales up to the large production scale.\nIt is very important to note again that the new x control approach does not require refraining from taking the \u03bc profile of the process as the basic process control variable. This basic concept can be maintained and it is easy to derive the x profile from a given \u03bc profile. As clearly shown in the examples, not only does this x-based control keep biomass and specific growth rate in tight limit, but also the batch-to-batch reproducibility with respect to the desired product concentration is high.","keyphrases":["reproducibility","recombinant proteins","pat","e. coli fed batch"],"prmu":["P","P","P","M"]}
{"id":"Clin_Oral_Investig-2-2-1705496","title":"Neurosensory disturbances after immediate loading of implants in the anterior mandible: an initial questionnaire approach followed by a psychophysical assessment\n","text":"The aim of the study was to assess past and present neurosensory disturbances using a questionnaire and a psychophysical approach in patients treated with immediate loaded implants in the edentulous anterior mandible. A group of 65 patients (age range 30\u201384 years, mean 58 years, 30 women) was enrolled. All were treated by means of three immediately loaded implants (Branemark Novum System\u00ae). A self-designed questionnaire was used for data collection. The response rate was 89%. Of the 58 responders, 33% (n=19) reported neurosensory disturbances after implant surgery. Nine of these patients (mean age 56 years, seven women) participated in an objective evaluation and were subjected to a psychological and several psychophysical tests. At the moment of the evaluation none of the nine patients still had clinical complaints. Psychological testing revealed no statistical differences between the patients, who had previously experienced subjective complaints, and the control group. Two-point discrimination and thermal sensation tests revealed no sensory lesions. The light touch sensation test at the lower lip indicated a more frequent reduction of tactility for the test group (p\u22640.03). Neurosensory disturbances can occur in the anterior region of the mandible after implant surgery.\nIntroduction\nNerve injury and specifically trigeminal nerve injury is known as a potential risk of many surgical procedures in the oral cavity in general [17, 23, 30].\nUsually after oral implant rehabilitation, the patient expects and experiences significant improvements, not only regarding jaw function, but also in relation to dental, facial, and even overall body image [22]. One can perfectly understand that the patient does not accept neural side effects, which might compromise his well-being.\nFollowing the definitions of the Subcommittee on Taxonomy of the International Association for the Study of Pain 1986, the types of sensorial disturbances are principally anesthesia, paresthesia, or dysesthesia. These changes can be persistent according to the degree of damage of the nerve [31].\nSensory disturbances in the maxillofacial region could be associated with different surgical procedures, like placement of endosseous implants [3, 36, 37]. With regard to immediately loaded implants, the presence of postoperative sensory disturbances was not documented.\nBranemark et al. [5] developed a new approach for immediate loading using fixed prosthesis of prefabricated standard components in the edentulous mandible. This technique involves a flattening of the jaw crest, followed by the placement of the implants by means of prefabricated and thus not individualized surgical guides. Thus the distances (but not the depth of drilling) between the implants were always the same, considerably reducing the variation of the surgical procedure. Sometimes the distal implants were positioned near the mental foramen [5].\nDamage to the mental nerve may result in loss of tactility and thus biting on the tongue or lip, drooling, painful sensations, and also interference with several jaw functions such as mastication, speech, hygiene maintenance, and social or psychosexual well-being [15].\nDifferent methods were used to evaluate such sensory disturbances after the placement of dental implants. Ellies [11] in 1992 and Ellies and Hawker [12] in 1993 published two retrospectives studies based on the analysis of questionnaires.\nWismeijer et al. [36] applied in a prospective study a self-administrated questionnaire and the somatic questionnaire, the Hopkins Symptoms Checklist (HSCL). The latter is a questionnaire routinely used in psychology to estimate a patient\u2019s psychoneurological and\/or psychosomatic discomfort. The somatic score of the HSCL was oriented on physical complaints and shows the level of a patient\u2019s perception of his\/her physical state. The higher the somatic score (i.e., 25% in the study of Wismeijer et al. [36]), the more the patient tends to exaggerate physical complaints, but the risk of a sensory disturbance of the lower lip is a possible complication after implant surgery [38].\nBartling et al. [3] analyzed the neurosensory disturbances in a population of patients after oral implant placement, using a combination of psychophysical methods like soft brush, two-point discrimination, pain perception, and temperature sensitivity. A small number (8\/94) of patients experience altered sensation after the placement of mandibular endosseous implants, but no permanent alteration was found.\nWalton [37] published a prospective study of 75 subjects using one objective (the light touch sensation) test, associated with a subjective analysis (questionnaire); both methods were used before and after placement of two implants in the anterior mandible. In this study, 24% of subjects reported neurosensory disturbances in the short term after implant surgery in the anterior mandible, but the problem appears to be a transient one with only about 1% experiencing sensation changes 1\u00a0year after implant surgery [37].\nAfter reviewing the related literature, it is clear that the proportion of patients with sensory disturbances varies among publications.\nIt is therefore essential to assess if neurosensory changes take place after the immediate loading of oral osseointegrated implants in the edentulous mandible in patients who received a fixed prosthetic construction on the day of implant insertion or the next day.\nDistinguishing between dysesthesia, an unpleasant abnormal tactile sensation, and paresthesia, an abnormal (not painful) and often decreased sensation, is the first step in defining the character of the neurosensory disturbance reported by a patient. Further differentiation between paresthesia and hypoesthesia, which is a reduction in the level of sensation, and finally anesthesia, the complete absence of any sensation, is important from all viewpoints. In the presence of dysesthesia, the differentiation must be established between spontaneous and a stimulus-induced unpleasant sensation. The clinical approach and sometimes legal implications of these different conditions are evident [6].\nBesides clinical evaluation, it must be understood that evaluation of nerve injuries such as demyelinization due to compression (neuropraxia), distal Wallerian degeneration with intact cell tubes (axonotmesis), or proximal and distal Wallerian degeneration with disparate Schwann cell tubes (neurotmesis) is an impossible mission for the clinician. Nevertheless, if no spontaneous return of tactile sensibility is noted within 3\u20136\u00a0months, the permanent loss of continuity of some or all the elements of the nerve trunk should be expected [11, 27]. Some observations indicate that the return to normal tactile sensibility may even occur after 2 to 3\u00a0years [12]. The differences between laboratory and clinical results are obvious.\nIn some studies, questionnaires were used to evaluate the presence of sensory disturbances. Such methodology is clinically helpful and is a good basis for more detailed and objective evaluations.\nImmediate loading of oral implants was proposed as an alternative protocol in the rehabilitation of partially or fully edentulous patients [8]. Surprisingly enough, no study has yet referred to the possible different neural sensibility when loading is imposed immediately after or together with the placement of the implants. This tactile sense aspect is relevant especially if we consider that the prosthetic rehabilitation (implying by example a full occlusal contact) is functional within one or a few hours after the implant placement. Thus, the patient\u2019s awareness of the load imposed on the implants can be a key issue to avoid undue load transfer on the implant\u2013bone interface.\nThe overall aim of the present research is to objectively evaluate the neurosensory disturbances and\/or function occurring after placement of oral implants in the anterior region of the mandible.\nMaterials and methods\nThis study comprised a total of 65 patients (age range 30\u201384\u00a0years, mean age 58\u00a0years; 30 women). All patients were treated by means of immediately loaded implants in the anterior mandible with the Br\u00e5nemark Novum\u00ae system approach. Surgery took place at the Department of Periodontology, Catholic University Leuven (55 patients, 3 surgeons), and at the Department of Oral and Maxillofacial Surgery, Erasmus Hospital, Free University of Brussels (10 patients, 2 surgeons).\nSelection criteria included (1) placement of implants in the anterior mandible using Br\u00e5nemark Novum System\u00ae (NobelBiocare AB, Gothenburg, Sweden); and (2) no history of neurological disorder.\nAn ad hoc multiple choice questionnaire (13 questions) was designed to record past and present neurosensory disturbances in these patients. The clinical history of the patient was used, and according to the selection criteria, a self-administered questionnaire was sent out to the 65 patients. The questionnaire was sent by mail to the patients with the request to complete it and return it to the clinic. The questionnaire was designed by experienced periodontologists, prosthodontists, and one psychologist (see Appendix for the questionnaire). The answers to the questions were analyzed and data were collected by this same team.\nAn objective evaluation took place after analysis of the questionnaire. During this evaluation psychological and psychophysical tests took place. The clinical evaluation of trigeminal nerve injuries suggested by Zuniga and Essick [39] in 1992 was used as basis in the test and control population.\nA psychological test: the SCL-90-R\u00ae (Symptom Checklist-90-Revised) was applied. The SCL-90-R\u00ae measures the psychoneurotic-somatic nonwellness (Psychoneuroticism) of patients [10]. The SCL-90-R\u00ae is different from the HSCL because it considers a broader area of psychopathology. The scale of psychoneuroticism in patients is assessed. It reveals the global level of recent psychological and also physical dysfunction. In the present study, the intensity of somatic complaints was considered important. The SCL-90-R\u00ae scale reveals complaints, loaded with a general feeling of physical dysfunction, as a result from functional problems.\nAfter completion of the questionnaire, each patient was interviewed using a standardized series of \u201ckey questions\u201d on altered feeling in the chin, lips, tongue, and cheek. Each time a distinction was made between the right and left sides of the face. Typical questions were as follows: \nHow would you describe the altered feeling?Do you notice the altered feeling constantly or only when touching the area, or chewing, or talking?Is it painful? Where? Transient or constant?Does it start spontaneously or is it evoked by touching, chewing, or speaking?What exacerbates the pain?What relieves the pain?\nAfter thorough questioning about the patient\u2019s general and oral medical history, an extra- and intraoral clinical examination took place, including palpation and\/or percussion, to detect eventual provoked pain at the site of injury. Finally, inspection of the oral cavity was performed to find eventual evidence of nerve injury, self-induced trauma, etc.\nIn the present study three psychophysical tests were selected: two-point perception at the lower lip, both left and right side (Fig.\u00a01); thermal sensitivity at the lower lip and gingiva of the anterior lower jaw, both left and right side (Fig.\u00a02); and light touch sensation at the lower lip and gingiva of lower jaw, both left and right side (Fig.\u00a03).\nFig.\u00a01Two-point discrimination instrumentFig.\u00a02Thermal sensitivity instrumentFig.\u00a03Light touch sensation (Semmes-Weinstein Aesthesiometer\u00ae) instrument\nPsychophysical tests demand a thorough consciousness and active participation of the patients and a quiet environment [20]. A combination of both psychophysical tests with testing tools adapted to the intraoral and perioral sites were used. The light touch sensation on the other hand was tested using the original Semmes-Weinstein Aesthesiometer\u00ae (Stoeling Company, Wood Dale, USA) device. To determine the threshold level the staircase method was applied. In the light touch sensation and static two-point discrimination, eight maximum and eight minimum values were recorded. Finally, in the thermal sensitivity test, all subjects were tested with ten trials. For a more detailed methodology of the procedures for sensory testing, see Jacobs et al. [21].\nThe collected data were statistically analyzed using the Statistica for Windows 5.1\u00ae (Stat Soft, Tulsa, OK, USA). A Mann\u2013Whitney U test was applied to the threshold levels in the test and the control groups.\nResults\nFifty-eight of the 65 patients (89%) completed the questionnaire and returned it to the hospital. Systemic diseases like cardiac, respiratory, endocrine, and renal diseases, allergic reactions, and psychological (depression) problems were detected in 46% (n=30) of the patients.\nThe mean time between the placement of the implants and the reception of the questionnaire was 20\u00a0months (range 8\u201340\u00a0months).\nThe analysis of the questionnaire showed that 33% (n=19) of patients reported a kind of neurosensory disturbance after the placement of the implants (range 8\u201324\u00a0months). The age range of this subpopulation was between 30 and 71\u00a0years (mean age 56, 13 women). This data are not different from the remaining group.\nThe duration of this postsurgical neurosensory disturbance after the implant surgery was less than 3\u00a0months in 58% (n=11) of the patients. However, eight patients were still suffering from a disturbance. For them the problem persisted for a period between 8 and 21\u00a0months.\nThe most commonly affected sites in the 19 patients were the gingiva only (6 patients), the inferior lip only (4 patients), and the chin (4 patients). One of the patients did not remember or could not determine the affected zone.\nSpeaking and drinking (five patients) were the oral activities most commonly impaired by the altered sensations. An important part of the affected population group (12 patients) didn\u2019t complain about problems with oral function or daily activity.\nThe most common reported sensation was numbness (nine patients), followed by cutting, beating, and itching reported by two patients.\nOnly one patient considered that the benefits of a fixed prosthesis did not outweigh the disadvantages that she had experienced as a result of disturbances in sensation of the lower jaw. She considered that she would not have done this surgery again, if she had been informed beforehand about the potential sensory changes in the orofacial region.\nFrom the 19 patients, 9 volunteered to participate in the objective evaluation (test group). The test group was 30\u201371\u00a0years old (mean age 56\u00a0years, seven women).\nA control group of volunteers (N=9) was also enrolled. Their age ranged from 49 to 71\u00a0years (mean age 63\u00a0years, all women). The mean observation time in this group since the surgery (two implants placed in the anterior mandible after a two-stage protocol) was 18\u00a0months (range 11\u201322\u00a0months).\nAs psychological test, the SCL-90-R\u00ae test was applied for symptom measurement of patient treatment outcomes and their degree of somatization of the symptoms. The psychophysical tests included three neurosensory evaluations: two-point discrimination, thermal sensitivity, and light touch sensation. The staircase method of limits was applied to determine the tactile threshold. An age- and gender-matched group of patients who had undergone similar surgery (two implants in the symphyseal region) in the same area, but without reporting sensory disturbances, served as control.\nResults of the psychological and psychophysical tests\nAs mentioned above, each of the 19 patients of the self-declared affected population was invited for a clinical evaluation and 9 of the 19 patients accepted to participate. The time span between surgery and psychophysical evaluation was on average 29\u00a0months (range 19\u201349\u00a0months). During the clinical interview, before the actual objective testing took place, none of the nine patients had remaining complaints or clinical symptoms such as drooling or tongue bite wounds that could indicate a sensory disturbance.\nHowever, at the reception of the questionnaire, which had to be mailed after the examination session, five of the nine patients with self-declared neurosensory disturbances still reported having them. In the remaining four patients the subjective neurosensory disturbances were completely resolved.\nNo major complaints were recorded after the evaluation of the SCL-90-R\u00ae. The scores of the SCL-90-R\u00ae of the test (N=9) and control group (N=10) revealed no statistic differences, neither on the global scale of neuroticism, nor on the dimension of somatic complaints. This means that the level of complaints of both groups was similar.\nAfter clinical examination and considering the negative interview for unpleasant dysesthesia and pain, it was concluded that only potential signs of paresthesia were currently present in the affected patients. Two-point discrimination, thermal sensation, and light touch sensation at the gingival level show no significant difference between test and control groups (Table\u00a01) [14, 16, 26, 28]. \nTable\u00a01Overview of psychophysical tests scores between test group, control group, and reference valuesType test\/RegionTest groupControl groupReference valuesReference numberT\u00b0S\/LLLS0.8 (CR)0.9 (CR)0.8 (CR)18T\u00b0S\/LLRS0.9 (CR)0.9 (CR)0.8 (CR)18T\u00b0S\/LGLS0.8 (CR)0.8 (CR)0.8 (CR)18T\u00b0S\/LGRS0.8 (CR)0.8 (CR)0.8 (CR)182PD\/LLLS3.4\u00a0mm4.5\u00a0mm6.1\u00b13.1\u00a0mm193.3\u00b11.6\u00a0mm202\u00b14\u00a0mm21, 222PD\/LLRS3.4\u00a0mm4.8\u00a0mm6.1\u00b13.1\u00a0mm193.3\u00b11.6\u00a0mm202\u00b14\u00a0mm21,22LTS\/LGLS7 NFa6 NFa4 (2.83) NF 19LTS\/LGRS7 NFa7 NFa4 (2.83) NF19T\u00b0S Thermal sensation, LLLS lower lip left side, LLRS lower lip right side, LGRS lower gingiva right side, LGLS lower gingiva left side, CR correct ratio, 2PD two-point discrimination, mm millimeters, LTS light touch sensation, and NF the number of the filamentaMean value of von Frey hair\nThe light touch sensation of the lower lip was significantly impaired in the test group (Mann\u2013Whitney U test). There was a statistical difference for both sides (p\u22640.03) between test and control groups.\nSystemic diseases have a significant effect on the outcome of the light touch testing at the lower lip. The light touch sensation of the lower lip revealed a statistical difference (p\u22640.04) for both sides.\nDiscussion\nThe anterior region of the mandible was always considered as a \u201csafe zone\u201d for the use of oral implants. This is particularly true concerning the high success rate of oral osseointegrated implants [25]. Nevertheless, there is an important difference between the reported implant success rate with specific surgical technique and the postsurgical changes to function, sensory mechanisms, and the physiological integration in the human body. Anatomical considerations in the anterior region of the mandible and the skills and experience of the surgeon are also important [24].\nIt is surprising to note that the present methodology allowed us to observe a very high percentage of subjective postoperative complaints. These results are in agreement with others, i.e, Ellies [11] with 37% and Ellies and Hawker [12] with 36%. This high degree of concordance for the percentages of incidence is interesting, considering the different surgical techniques used and cultural and sometimes ethnic differences between the three involved populations (Canada, Australia, and Belgium).\nIt was not possible, based on the subjective data, to establish any correlation between the systemic diseases presented in the study population and the reported sensory disturbances (i.e., impairment of perception in a patient with diabetes) [2, 4].\nIt is interesting to know that eight patients had the impression to be affected by some kind of persistent neurosensory disturbance, most probably paresthesia or hypoesthesia, more than 12\u00a0months after the surgery. Nevertheless, besides these results, a majority of these affected patients considered that the benefits outweigh this kind of transient or permanent disadvantages, and consider that they would accepted an implant surgery again even if they knew about the present complication.\nNormal somatic sensation reflects a continuous day and night monitoring process. Little of this activity reaches consciousness under ordinary conditions. Disordered sensation is alarming and dominates the sufferer\u2019s attention. As expected from the neuroanatomical knowledge recently collected, neurosensory disturbances regularly occur in the anterior region of the mandible after surgery. Anatomical factors like the presence of an anterior loop, handling of the mental nerve during surgery, or the perforation of the incisive nerve canal can all provoke such disturbances.\nAs mentioned above, the Br\u00e5nemark Novum\u00ae technique involves a flattening of the jawbone. This procedure can be considered rather invasive and can damage, in severely resorbed jaws, the incisive canal or the mental nerve emerging at the crestal level. The relationship between a reduction of the crestal jawbone and neurosensory disturbances could not be traced in the literature. However, bone chin grafting procedures present some similarities. von Arx et al. [35] recently reported 8.1% of neurosensory disturbances after 6\u00a0months in patients who underwent such procedures.\nFor some systemic conditions, it was reported that persons can be more prone to sensory disturbances. Peripheral neuropathy in people with scleroderma is thought to be rare, however, nerve conduction studies showed abnormalities in patients with a mean disease duration of 10\u00a0years or longer [29]. Polyneuropathies in mild or severe diabetic patients often cause subclinical damage of the trigeminal nerve. Moreover, their sensory complaints in the perioral area often remain unnoticed and the dysfunction undiagnosed [9]. Age by itself also has an influence, especially when repair is concerned. The younger the subject, the greater the degenerative response but the quicker and more complete the overall recovery [7].\nTactile threshold assessment reveals that detection of monofilaments up to 0.023\u00a0g (monofilament number 2) can be considered normal in the orofacial region [14, 37].\nNeurophysiological recordings of the masseter reflex, the mental nerve blink reflex, or evoked potentials are all useful in evaluating trigeminal nerve damage. This was specifically shown for damage of the inferior alveolar nerve [19, 32, 33]. However, evaluation of nerve damage in the symphyseal region was not reported in the literature. The number of nerves innervating this region, the vicinity of interfering structures (e.g., tongue, saliva, and labial muscles), and anatomical variations make this a difficult but challenging task.\nIn humans, several reports show that during the stimulation of peripheral sensory limb nerve examined after surgical repair (time span between 5 and 20\u00a0years), the sensory function remained deficient and often included abnormal sensory disorders [1]. It must also be considered that the loss of tactile sensitivity after surgery is not always reflected in abnormal psychophysical test results [13]. Furthermore, it is also possible, as shown by the collected data in this study, that an abnormal test result, particularly the light touch sensation test, does not reflect clinical reality. In other words, the patient does not always detect the loss of sensitivity.\nIt should be stressed that the selection of a control group of patients treated by means of two implants in the symphyseal region was principally motivated by the intent to have an age- and gender-matched group. The selection of a control group among patients treated with the Novum\u00ae system but without neurosensory complaints would not have allowed this.\nIt must also be considered that sometimes thresholds of the psychophysical test are not easy to reach in the oral cavity; the devices were not originally designed for this region, particularly light touch sensation [33].\nNot less important is the fact that 95% (n=17) of the affected population considers that the treatment benefits outweigh the transient disadvantages, and that 18\/19 of the patients consider that they would follow an implant surgery again if they knew beforehand the changes in sensation after the surgery.\nThere are no data in literature to compare neurosensory disturbances after immediate loading with those after the two-stage protocol. It is clear that it is difficult for the patient to distinguish between postoperative inconveniences, early functional adaptation, and real neurosensory dysfunction. Only a differential objective diagnosis can do so.\nA meticulous preoperative planning of the surgery, even in an improperly so-called safe region of the jaws, like the symphyseal region, might avoid many neural disturbances [18].\nIn this perspective, the use of cross-sectional images and the transfer of the planning to the operative field may be considered [34].\nConclusions\nThe use of a questionnaire to determine the presence or absence of a problem after a medical procedure is easy and inexpensive; but to clarify the type, magnitude, extension, and eventual persistence of the neurosensory disturbance, the use of objective methods (i.e., psychophysical methods) in the evaluation of a population affected by any sensory disturbance, when complaints are detected, is highly recommended.\nThe objective follow-up revealed that patients are often not impaired by, and even not aware of, neurosensory dysfunctions after implant surgery in the anterior mandible. Objective tests indicate however that tactile threshold levels may be elevated after such surgery. None of the patients suffering from this impaired tactile function seems to have functional deficits resulting from it.\nBased on these data, proper preoperative planning using cross-sectional imaging can be advised even for surgical procedures in the symphyseal region.","keyphrases":["neurosensory disturbances","psychophysical tests","oral implants","mental nerve","symphysis"],"prmu":["P","P","P","P","U"]}
{"id":"Med_Biol_Eng_Comput-3-1-1820753","title":"Algorithm for the classification of multi-modulating signals on the electrocardiogram\n","text":"This article discusses the algorithm to measure electrocardiogram (ECG) and respiration simultaneously and to have the diagnostic potentiality for sleep apnoea from ECG recordings. The algorithm is composed by the combination with the three particular scale transform of aj(t), uj(t), oj(aj) and the statistical Fourier transform (SFT). Time and magnitude scale transforms of aj(t), uj(t) change the source into the periodic signal and \u03c4j = oj(aj) confines its harmonics into a few instantaneous components at \u03c4j being a common instant on two scales between t and \u03c4j. As a result, the multi-modulating source is decomposed by the SFT and is reconstructed into ECG, respiration and the other signals by inverse transform. The algorithm is expected to get the partial ventilation and the heart rate variability from scale transforms among aj(t), aj+1(t) and uj+1(t) joining with each modulation. The algorithm has a high potentiality of the clinical checkup for the diagnosis of sleep apnoea from ECG recordings.\nIntroduction\nThe electrocardiogram (ECG) contains well-known PQRST(U) pattern. Its morphology has been extensively studied since the technique of recording was introduced in the beginning of the twentieth century. The use of computers for ECG recording enabled studies of dynamic properties of the ECG. In particular, two important characteristics have been revealed: amplitude and frequency modulation.\nThe modulation of the cardiac cycle is extensively studied based on the signal that is generated from intervals between consecutives R-peaks, known as heart rate variability (HRV). Numerous approaches have been proposed and the signal have been decomposed in various ways\u2014analyzing their spectral properties by frequency and time-frequency methods such as Fourier transform [2], autoregressive spectral estimation [11], wavelet transform [9, 17], or their scaling properties [6].\nThe amplitude characteristics and amplitude modulation received less attention, especially because the notion of the ECG amplitude is ambiguous: the heart as a source of the signal is moving and the signal is collected from the surface of the body that is moving as well. In addition, it is usually recorded as a 2-D projection. However, it has long been recognized that one of the sources of the amplitude modulation\u2014the respiratory related movement of the thorax can be usefully used to extract the information about the respiration.\nThe influence of respiration on recording cardiac potentials was reported as early as in 1967; Flaherty et al. [5] and Moody et al. [13] were probably the first to discuss the possibility to derive the respiratory signal from the ECG. Since then several algorithms were proposed to reconstruct respiration by demodulating the ECG signal (e.g. [1, 4, 8, 10]).\nThe ECG-derived respiration signal either based on changes in heart rate or based on direct effect of respiration on the ECG waveform has been particularly used in studies of obstructive sleep apnoea [3, 16].\nThe problem to reconstruct waveform from the discrete Fourier analysis is that the biological signal has complicated properties; being non-stationary wave, including random noise and having interactive components. However, though people rarely notice, the essential problem is that there is no way to gather impartial and statistical samples. That is why samples from an unique sampling frequency are biased for spectral analysis even if fine sampling.\nThe purpose of this study is to introduce an algorithm to classify the multi-modulating waveforms on ECG signals. The algorithm is composed by the three particular scale transforms and the statistical Fourier transform (SFT) with impartial and statistical samples [12]. As a result, the multi-modulating source is decomposed by the SFT and is reconstructed into ECG, respiration and the other signals simultaneously by particular transformations.\nAlgorithm\nThe principle of this algorithm is the selection of three particular scale transforms. Two scales convert modulating waveforms into the periodic signal and the other converts the periodic components into a few terms. The role of the SFT is to hold sample information for any transforms.\nScale transforms aj(t), uj(t) and oj(t) on modulating signal\nSince a source sample at any instant tk has the mutual phase relationship of instantaneous components on the SFT, any scale transform including permutation expresses the same sample as own instantaneous components by each scale (Appendix 1).\nThree scale transforms aj(t), uj(t), oj(t) are adopted to the periodicity and to the reduction of Fourier components.\naj\u00a0=\u00a0aj(t) is a scale to match time intervals in the standard\u2019s intervals and is called adaptive scale and uj\u00a0=\u00a0uj(\u03c4j) is a scale to match the magnitude spans in the standard\u2019s magnitudes. Those scales convert the source x\u2032(t) into the standard xj(t) as xj(t)\u00a0=\u00a0x\u2032(aj)\/uj(aj). aj(t) and uj(aj), then, are defined as a join of each akj(t) and ukj(akj) (k\u00a0=\u00a01,..., kj\u00b7) when the standard repeats kj times in a window Tj.\noj\u00a0=\u00a0oj(t), called inherent scale, is a timescale to convert the standard xj(t) into the Fourier series of rj components passing through 2rj poles of xj(t). The scale transform \u03c4j(t) is defined as the composite transform as follows (Appendix 2): \nThe transformed signal (x\u2032(\u03c4j)\u00a0\u2212\u00a0\u03b5j(\u03c4j))\/uj(\u03c4j) occupies rj instantaneous components of the skipped ikjth term in a window Tjincluding kj modulations. While, instantaneous components of the \u03b5j(\u03c4j)\/uj(\u03c4j) scatters into broad components from the 1st to the nth terms. As a result, (x\u2032(\u03c4j)\u00a0\u2212\u00a0\u03b5j(\u03c4j))\/uj(\u03c4j) fits the scales \u03c4j, uj and \u03b5j(\u03c4j)\/uj(\u03c4j) unfits scales \u03c4j, uj, and then, the source x\u2032(t) is decomposed into two class modulations of x\u2032(\u03c4j)\u00a0\u2212\u00a0\u03b5j(\u03c4j) and \u03b5j(\u03c4j) (Appendix 3).\nThat is, x\u2032(\u03c4j)\u00a0\u2212\u00a0\u03b5j(\u03c4j) is extracted by vj(\u03c4j)\u00a0=\u00a0\u03a3uj(\u03c4j)hij(\u03c4j) from the ikjth (i\u00a0=\u00a01,.., rj) components and \u03b5j(\u03c4j) is extracted by the other \u03b5j(\u03c4j)\u00a0=\u00a0\u03a3uj(\u03c4j)hij(\u03c4j) except the ikjth components when n instantaneous components of x\u2032(\u03c4j)\/uj(\u03c4j) are defined as hij(\u03c4j). The source x\u2032(t), therefore, is decomposed into x\u2032(t)\u00a0\u2212\u00a0\u03b5j(t) and \u03b5j(t) by inverse scale transform t\u00a0=\u00a0t(\u03c4j) under an adequate uj(\u03c4j).\nThe magnitude scale ukj(\u03c4j) is defined as an expression of the magnitude pattern in each window Tkj when the signals vj(\u03c4j)(=x\u2032(\u03c4j)\u00a0\u2212\u00a0\u03b5j(\u03c4j)), \u03b5j(\u03c4j) and uj(\u03c4j) are expressed by {vkj(\u03c4j)}k, {\u03b5kj(\u03c4j)}k and {ukj(\u03c4j)}k (k\u00a0=\u00a01,.., kj), and each changes its waveform independently. Especially, vj(\u03c4j) is expressed by a constant value uj(\u03c4j)\u00a0=\u00a0ukj in each window Tkj when its magnitude modulation takes a similar figure with the standard (Appendix 4).\nIterative scale classification of modulating signals\nThe timescale transform of the residual \u03b5j(t) is defined as \u03c4j+1(t)\u00a0=\u00a0oj+1(aj+1(t)) when several waves of {\u03b5kj(t)\/ukj+1(t)}k in \u03b5j(t)\/uj+1(t) reappears within a window Tj+1(Tj+1\u00a0\u2264\u00a0Tj). \u03b5j(t) is analyzed as the j\u00a0+\u00a01th modulation with the iterative algorithm as \u03b5j(t)\u00a0\u2212\u00a0\u03b5j+1(t) and \u03b5j+1(t) under aj+1(t), oj+1(aj+1(t)), uj+1(t) and xj+1(t) (Appendix 2).\nWhen x\u2032(t) is m classes of Fourier series with terms of r1, r2,..., rm (n\u00a0=\u00a0r1\u00a0+\u00a0r2\u00a0+\u00a0\n\u00a0+\u00a0rm), it expresses Eq. 2. \nIn Eq. 2, t\u00a0=\u00a0t(\u03c4j) is the inverse scale transform of the jth scale \u03c4j to scale t, vj(t) is a Fourier series of the jth class modulation, and \u03b5m(t) is the mth residual signal. Equation\u00a02 shows that x\u2032(t) is reconstructed completely by periodic Fourier series vj(\u03c4j) on scale \u03c4j (j\u00a0=\u00a01,2,..., m).\nQuantity of information on the modulating signal\nThe relation between the quantity of information (3) and the modulation (4) is derived from formula (2).\nSince the probability of components on the jth class modulation is to be pj\u00a0=\u00a0rj\/n, the probability to encounter x\u2032(t) with m modulations is p(r1,r2,..., rm)\u00a0=\u00a0p1r1, p2r2,..., pmrm which relates to the geometrical mean \n. Equation is as follows (Appendix 5). \nIt clearly shows that the quantity of information on multi-modulating signal is m itself.\nWhen hij(\u03c4j) is the ith instantaneous Fourier component of standard xj(\u03c4j) in scale \u03c4j and hij(t) is its modulating component in scale t, the morphological relation is given by the following Formula (4): \nIn Eq. 4, \u2202\u03c4j\/\u2202t is the Jacobian. Therefore, formula (4) shows that the modulation of every component at scale t is expressed by scale\u2019s modulations on its magnitude uj(\u03c4j) and its time \u03c4j(t)\u00a0=\u00a0oj(a(t)). Especially, the adaptive scale aj(t) describes composite transform aj(t)\u00a0=\u00a0c1(c2(\ncn(t))) and that of exchanged orders aj(t)\u00a0=\u00a0cn(\nc2 (c1 (t))), so the sth scale cs(t) will express its specific modulation from the standard such as modulations of PQ, QRS, QT and RR.\nMaterials and methods\nThe analysis of scale classification of the three modulating signals on V4 lead was carried out by using the statistical Fourier analysis with maximum terms of 256 with A\/D converted 16-bit samples of the sampling frequency 20\u00a0kHz over successive 50\u00a0s. To gather independent samples with 256 terms, samples needed to be accumulated by the high speed 20\u00a0kHz of A\/D converter with a large dynamic range of 16\u00a0bit to avoid the deform of a tiny respiratory signal (Fig.\u00a01).\nFig.\u00a01Synchronous frequency samplings on scale t and \u03c4\nThe ECG standard xj(t) was selected from the source x\u2032(t) without breathing. On the other hand, x\u2032(t) with natural breathing and the respiratory flow  are recorded simultaneously by using Number 3 Fleisch pneumotachograph. Both the ECG and the respiratory scale transforms were the same as in Fig.\u00a02a, b of Appendix 2. The ECG x\u2032(t)\u00a0\u2212\u00a0\u03b5j(t) and its baseline wander \u03b5j(t) were analyzed when uj(\u03c4j)\u00a0=\u00a01 and the respiratory standard xj+1(t) was selected from stationary sequences of \u03b5j(t) in 16 respiratory cycle. (\u03b5j(\u03c4j+1)\u00a0\u2212\u00a0\u03b5j+1(\u03c4j+1))\/uj+1(\u03c4j+1) and \u03b5j+1(\u03c4j+1)\/uj+1(\u03c4j+1) were analyzed when uj+1(\u03c4j) of the formula (5) in Appendix 4 and lung ventilation Vj+1(t) and its residual \u03b5j+1(t) were determined.\nFig.\u00a02ECG and respiratory standard waves and specific points on scale t. a Cardiac standard, b respiratory standard, and c detecting dots on a part of the source\nResults\nSignals corresponding to every process on the scale classification of m\u00a0=\u00a02 under constant magnitude scale of uj\u00a0=\u00a01 are illustrated in Fig.\u00a03 with five cardiac periods. That is, intervals among specific points in source x\u2032(t) in Fig.\u00a03a are transformed into the same intervals of the standard signal with the adaptive scale aj(t) to x\u2032(aj(t)) of Fig.\u00a03b and with the inherent scale \u03c4j(t) to x\u2032(oj(aj(t))) of Fig.\u00a03c. The transformed signal x\u2032(\u03c4j) is decomposed into statistical Fourier components on a fundamental period of 53 cardiac intervals. The transformed signal xj\u2032(\u03c4j) in Fig.\u00a03d is a reconstructed signal from the 53rd, 106th, 159th and 212th components, and its residual \u03b5j(\u03c4j) in Fig.\u00a03e from all other components. The extracted ECG xj\u2032(t) in Fig.\u00a03f and the base line wander \u03b5j(t) in Fig.\u00a03g are the inverse scale-transformed signals from scale \u03c4j to scale t. When tiny-leak signals on the \u03b5j(t) synchronized with intervals of QT are replaced with linear trends, the figure of pattern \u03b5j(t) takes that of the lung ventilation in Fig.\u00a03i. The smoothing signal, Vj(t) in Fig.\u00a03h is a filtered signal of \u03b5j(t) with low-pass filter (LPF) under 30 terms (0.6\u00a0Hz). Vj(t) is illustrated with enlarged four times. The signal V(t) in Fig.\u00a03i is the lung ventilation curve which is an integral signal of the direct measurement of the respiratory flow Fig.\u00a03Classified process of modulating signal of the algorithm\nAny differences of P\u00a0\u223c\u00a0U waveforms between the source x\u2032(t) and reconstructed ECG x\u2032j(t) bear getting information, and also the differences between ventilation V(t) and baseline wander Vj(t) are small when frequencies lower than breathing are neglected. x\u2032j(t) provides time-interval information on Minnesota codes better than that of x\u2032(t) clearly in visual.\nCorrelation coefficients \u03c1 between the signal Vj(t) and V(t) are calculated by each ventilation with scatter plots in Fig.\u00a04a, and are classified by P\u00a0\u223c\u00a0U waves of 53 cardiac signals in Table\u00a01a and by respiratory phases of 16 ventilations in Table\u00a01b. Total \u03c1 takes even values (\u03c1\u00a0\u2248\u00a00.82) and has less interference between P\u00a0\u223c\u00a0U waves in cardiac cycles. However, it takes low values (\u03c1\u00a0\u2264\u00a00.5) at the transient regions of I, III between the inspiration and the expiration corresponding to loops in Fig.\u00a04a. The inspiration and the expiration except transient regions are statistically dominant at higher value (\u03c1\u00a0\u2248\u00a00.95). It is clear that the scale classification extracts the electrical activity of the heart with constant magnitude scale of uj(\u03c4j)\u00a0=\u00a01 in hole window of Tj. The extracted baseline wander is constructed with lung ventilation and other biological information.\nFig.\u00a04X\u2013Y loop among modulating Vj(t), Vj+1(t) and V(t)Table\u00a01Correlation coefficients \u03c1i among Vj(t), Vj+1(t) and V(t) on cardiac and respiratory stagesCardiac stagesPQQRSST-TBase lineTotal(a) Correlation coefficients \u03c1i between Vj(t) and V(t) on ECG stages\u00a0\u03c1i0.83280.82430.81490.81510.8244\u00a0Number of samples13,56612,69033,01843,553102,827Respiratory stagesI II III VI V Total(b) Correlation coefficients \u03c1i between Vj(t) and V(t) on respiratory stages\u00a0\u03c1i0.39900.97880.57720.99700.95460.7273\u00a0Number of samples25,74725,21025,92814,57411,558103,017(c) Correlation coefficients \u03c1i between Vj+1(t) and V(t) on respiratory stages\u00a0\u03c1i0.99230.65890.99430.80320.73050.91648\u00a0Number of samples25,28622,81621,16315,15518,077102,497I inspirations before midpoints, II inspirations after midpoints, III expirations before midpoints, VI expirations after midpoints, V expirations after inflection points\nThe magnitude scale transform is applied to the scale classification of the base line wander Vj(t) with a respiratory standard in Fig.\u00a02b. Scale classification of m\u00a0=\u00a04 is illustrated in Fig.\u00a05, the source x\u2032(t) in Fig.\u00a05a, the extracted ECG xj(t) in Fig.\u00a05b, the extracted respiratory signal Vj+1(t) in Fig.\u00a05c, the residual \u03b5j+1(t) in Fig.\u00a05d, the leak ECG \u03b5(t) over 0.6\u00a0Hz of \u03b5j(t) in Fig.\u00a05e and the ECG of xj(t)\u00a0+\u00a0\u03b5(t) in Fig.\u00a05f. The signal V(t) in Fig.\u00a05g is 16 measured respiratory ventilations. The magnitude scale \u0394uj+1(t) is illustrated in Fig.\u00a05h.Vj+1(t) and \u03b5j+1(t) are displayed in the reverse because the extracted respiratory waveform is a visually reversed V(t). \u03b5j+1(t) is similar to base-line trends of V(t) which relate to the thoracic gas volume (TGV).\nFig.\u00a05Scale classification of the four modulating signals in an electro-cardiac signal\nThe scatter plots of 15 lung ventilations between Vj+1(t) and V(t) take the similar pattern as illustrated in Fig.\u00a04b. Correlation coefficients \u03c1 in Table\u00a01c takes higher value than the others though transient regions change I, III into II, IV.\nThe frequency distributions of the amplitude components on |V(f)|, |Vj+1(f)|, |\u03b5j+1(f)| and that of phase shifts \u0394\u03c6(f) between V(f) and Vj+1(f) are illustrated in Fig.\u00a06. On frequency ranges from 0.2 to 0.5\u00a0Hz, amplitude distribution in |V(f)| and |Vj+1(f)| takes similar pattern and those of phase shifts are invariant near 0\u00b0. Amplitude distribution in |\u03b5j+1(f)| has not only the lower frequency components under 0.06\u00a0Hz corresponding to the trends of TGV but also the leak components of the lung ventilation from 0.25 to 0.5\u00a0Hz. The components of |\u03b5j+1(f)|, moreover, has the harmonics among 0.04, 0.1, 0.18 and 0.28\u00a0Hz though those harmonics disappear in |V(f)| and |Vj+1(f)| at least.\nFig.\u00a06Frequency distributions of the measured V(f) and the extracted Vj+1(f), \u03b5j+1(f)\nAs a result, the time and magnitude scale is effective to classify modulating signals from the higher frequency Vj+1(t) to the lower frequency \u03b5j+1(t) because a residual \u03b5j+1(t) has less interference of the higher Vj+1(t) and successive sequential \u03b5j+1(t) will supply the signal for detecting other lower modulation.\nDiscussion and conclusion\nA discrepancy between time domain and frequency domain analysis happens commonly by using biased samplings for discrete signal processing in various way such as Hilbert transform [2] and wavelet analysis [9] relating with an unique sampling frequency. Moreover, the mathematical concepts of one-to-one correspondence, n unknown amplitudes and phases are derived from 2n samples, hinders to orientate the statistics in the signal processing.\nThe SFT, however, has no contradiction between Fourier analysis and the statistics which resolute components with the same signal-to-nose ratio (SNR) for any signal decreasing amplitude components with order 1\/f such as ECG signal, which is unsatisfied in the autoregressive spectral estimation [11]. The SFT component, moreover, is given independently by a time trace of instantaneous components at each instant as shown in Appendix 1. That is, its amplitude and phase are not calculated by the inner products between the source and trigonometric bases but by the extracted traces of instantaneous sin-wave themselves [12]. The concepts of signal arrangements at series {tk} is another thing on the SFT because the replacement between ti and tj needs the replacements of all the synchronous frequency samplings between ti and tjto hold the same instantaneous components. The summation of replacements, then, is about \n. In other words, the SFT supplies a signal ensemble arranging samples of the series {tk}. In this study, discrete samples from A\/D converter of 20\u00a0kHz satisfy the independency as shown in Appendix 6. As a result, the random noise including a signal is not analyzed as a white noise but as inherent components characterizing arrangements. Therefore, the reconstructed waveform of a baseline wander holds a prototype even if it includes many random noises.\nThe necessary condition of the spectral analysis known as Nyquist\u2019s condition [14, 15] becomes precise on the SFT as shown in Appendix 3, which confirms its condition statistically. The potentiality to scatter the aliased components impartially discriminates the waveforms under the unsatisfied Nyquist\u2019s condition. In this algorithm, while the inherent scale transform converges to the standard xj(\u03c4j) with four skipping components, others \u03b5j(\u03c4j), either continuous or random, disperse components into 256 terms.\nSince the scale-transformed samples on the SFT are gathered from a whole window at any instant, those samples reflect its scale transform better than any local one. The random and leak signal on the extracted ECG xj\u2032(t) are reduced into the tiny level under a stable behavior of the random noise. The 256 terms is enough to analyze scale-transformed signal in a window under 53 cardiac cycles because transformed components with the ranges, 8\u00a0\u2264\u00a0kj\u00a0\u2264\u00a053, takes almost the same figures. The high-resolution analysis, therefore, is accomplished by the relative high SNR from those instantaneous components.\nThe problem on the scale transforms is to be adequate scales, not to be expressing the true biological modulation. aj+1(t) is, however, selected a traditional scale for non-stationary sequences in each respiratory cycle which carry information stored in Fourier phases as already indicated in [6] and aj(t) is selected from cardiac cycles without breathing in addition to the non-stationary sequences. As a result, oj(aj) and oj+1(aj+1) make those mutual relationship of phases more simple relations. Taking own standard characterized with number of poles is also reasonable to determine the waveform reconstruction and to discriminate the own HRV. Moreover, the lost waveform is scattered into the other components without losing the morphologic information such as the leak ECG waveform.\nIt is well known the volume conductivity of the thorax takes inequality and changes the electro cardiac potentials on the thorax\u2019s surface with respiratory movements [1, 5].\nIn the scale classification of m\u00a0=\u00a04, it indicates that a constant magnitude scale ukj\u00a0=\u00a01 produces unchangeable heart-activity xj\u2032(t) so that the signal \u03b5j(t) appears in respiratory modulations from their changes of the conductivity. It suggests that the standard without breathing follows the formula (2) of the signal classification and the source follows the formula (4) which expresses the modulating standard with Jacobian, \u2202\u03c4j\/\u2202t. A composite scale cs(t), therefore, is expected to express the inherent modulation from the standard such as PQ, QT and RR. That is, the spectral analysis of cs(t) on RR provides the distribution of the HRV without a tachogram which is expressed in other ways by others [2, 9, 11]. The extracted ECG has the potentiality to detect R\u2013R modulations of the heart itself.\nThe algorithm extracts the lung ventilation curves {Vj+1(t)}k from V4 lead as changing the thoracic gas volume [1, 4]. The difference of amplitude distribution between V(t) and Vj+1(t) from spectral analysis takes the same pattern around 0.3\u00a0Hz [8]. The phase difference between V(t) and Vj+1(t), however, occurs in the transient regions between the inspiration and the expiration which deduce from inadequate respiratory standards, false respiratory peaks and the gas compression in the thorax [7]. It is effective to determine specific components, such as harmonics of \u03b5j+1(t) around (0.04, 0.1, 0.18 and 0.28\u00a0Hz) in cardiovascular responses [2, 17].\nInformation of the partial ventilation is also expected from other 12 leads [8]. The respiratory Jacobian \u2202\u03c4j+1\/\u2202t, like that of ECG, has the potentiality to detect respiratory modulation itself as a part of the HRV and uj+1(t) is expected to detect the amplitude modulation such as sighs distribution. The residual \u03b5j+1(t) supplies information of waveforms not only of the thoracic gas volume but also of the blood flow relating to cardiovascular responses [2, 11, 17].\nIt can be concluded that this algorithm reveals the simultaneous measurements of ECG and respiration. The comparison with the frequency distribution and the waveform evaluates new modulations clearly, such as ukj+1 for detecting changes of the tidal volume (TV) and for finding TGV and cardiovascular responses. This contribution of the algorithm indicates diagnostic potentials on the sleep apnoea from ECG recordings [16].","keyphrases":["electrocardiogram","scale transform","fourier transform","heart rate variability","respiratory waveform"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_Spine_J-2-2-1602203","title":"Thoracic arachnoiditis, arachnoid cyst and syrinx formation secondary to myelography with Myodil, 30 years previously\n","text":"Spinal arachnoiditis can rarely occur following irritation from foreign body substances, including certain oil based contrast agents used for myelography. We describe a patient with thoracic arachnoiditis, arachnoid cyst and syringomyelia, 30 years following a myelogram with Myodil. A 62-year-old female presented with chronic thoraco-lumbar back pain, a spastic paraparesis and sphincter disturbance. She had undergone a myelogram with Myodil, 30 years previously for investigation of back pain. A MRI scan revealed evidence of arachnoiditis, thoracic syringomyelia (T6\u2013T8) and an anteriorly placed, extramedullary, arachnoid cyst at T10\u2013T12, compressing the cord. At surgery, T7\u2013T10 thoracic laminectomies were carried out and syringo- and cysto-subarachnoid shunts were inserted. At 12 months follow-up, the sphincter disturbance, lower limb weakness and mobility problems had almost resolved. Although, the use of oil based contrast agents such as Myodil has been discontinued, the present case illustrates some of the rare sequelae of its use, manifesting decades later. Aggressive surgical intervention produced symptomatic benefit.\nIntroduction\nCauses of spinal arachnoiditis include infection, trauma, tumours, surgery and irritation from foreign body substances [3, 4, 9]. The latter include older radiographic contrast agents such as isophendylate (Myodil and pantopaque), which was widely used for myelography in Europe and North America until the 1980s [3, 4, 6]. We describe a 62-year-old lady who presented with thoracic arachnoiditis, arachnoid cyst and syringomyelia, 30\u00a0years following myelography-using Myodil.\nCase report\nA 62-year-old female presented with a 2\u00a0year history of chronic thoraco-lumbar back pain, and a progressive and bilateral lower limb weakness. This was associated with spasms affecting her legs and abnormal sensation on the soles of the feet. There was a 3\u00a0month history of urinary and faecal urgency. She was asymptomatic in the upper limbs. She had undergone a Myodil myelogram; 30\u00a0years previously as part of investigations for lumbosacral back pain. The myelogram was normal and her symptoms resolved one year later with conservative measures.\nOn examination, there was spastic paraparesis, with an up going plantar response on the right. There was bilateral weakness of hip flexion (MRC 4, motor power) and she had difficulty walking independently. Light touch, pinprick and temperature sensations were preserved, but proprioception was reduced at the toes bilaterally. Cranial nerves and upper limbs were neurologically intact.\nA MRI scan of the thoraco-lumbar region revealed an intramedullary lesion extending from T6\u2013T8 and an anteriorly placed extramedullary, intradural lesion at T10\u2013T12, compressing the thoracic cord (Fig.\u00a01). The abnormal areas were non-enhancing, low signal on T1 weighted images and high signal on T2 weighted images, in keeping with a thoracic syrinx and an arachnoid cyst, respectively (Fig.\u00a01). Axial MRI scans at the lumbar levels revealed clumping of the nerve roots, suggestive of arachnoidal adhesions, although Myodil droplets were not seen (Fig.\u00a02). Plain X-rays of the thoraco-lumbar spine, full blood count and serum inflammatory markers were normal.\nFig.\u00a01Sagittal T1 weighted (a) and T2 weighted (b, c) spin echo MRI scans of the thoracic region, demonstrate an intramedullary lesion from T6\u2013T8 and an anterior extramedullary, intradural lesion at T10\u2013T12. The abnormal areas were low signal on T1 weighted images and high signal on T2 weighted images, in keeping with a syrinx and an arachnoid cyst, respectivelyFig.\u00a02Axial T2 weighted spin echo MRI scans at the L2 (a) and L4 (b) levels reveal adhesions and clumping of the lumbar roots\nAs she was symptomatically deteriorating, surgical exploration was undertaken in the form of a T7\u2013T10 thoracic laminectomies. On opening the dura, dense arachnoidal adhesions were apparent. These were partially released to enter the anteriorly placed arachnoid cyst at T10 (Fig.\u00a03a) and the syrinx cavity at T8 level (Fig.\u00a03b). Fluid similar to cerebro-spinal fluid (CSF) was released under pressure from both the arachnoid cyst and the syrinx. Syringo- and cysto-subarachnoid shunts were inserted. Post-operative recovery was uncomplicated and at 12\u00a0months follow-up, the sphincter disturbance, lower limb weakness and mobility problems had almost resolved.\nFig.\u00a03Intra-operative view of the thoracic spinal cord under the operating microscope showing, dense arachnoidal adhesions (grey arrows), the anteriorly placed arachnoid cyst at T10 level (a, black arrow) and the syrinx cavity at T8 (b, black arrow)\nDiscussion\nThe present case is a rare report of widespread arachnoiditis, with the formation of an arachnoid cyst and syrinx in the thoracic cord (radiologically evident at operation), secondary to the use of Myodil, 30\u00a0years previously. Although, we did not observe Myodil droplets on the MRI scans, in the absence of other risk factors, the history of Myodil usage seems the likely contributory agent for the pathological changes noted [3, 4, 6]. Aggressive surgical intervention led to symptomatic benefit.\nSpinal arachnoiditis following myelography with certain agents such as isophendylate (Myodil and pantopaque) is a rare (approximately 1%) but a recognised complication [3, 4, 6]. This risk appears to be greater with oil based contrast agents such as isophendylate, than other modern water based agents such as metrizamide (amipaque) and iohexol (omnipaque) [3, 4]. Furthermore, the risk of arachnoiditis is augmented by previous spinal surgery and existing spinal canal stenosis at adjacent levels [3, 4]. However, this was not applicable to our patient.\nIn the present case there was radiological and operative evidence of not only arachnoiditis, but also an extramedullary arachnoid cyst and intramedullary syrinx formation. There have been rare reports of spinal arachnoiditis, arachnoid cyst and syrinx formation, but this typically followed spinal surgery or trauma [1, 2, 5, 8]. The present case is unusual in that our patient developed the same sequelae, but after myelography with Myodil.\nWhile the precise pathophysiology of these abnormalities is unclear, they may be interrelated. Thus, the arachnoidal adhesions may interfere with CSF flow pathways around the thoracic cord, causing trapping of CSF and the formation of the arachnoid cyst [3]. Moreover, the altered dynamics of CSF flow may also cause a pressure differential, promoting the formation of the syrinx [2, 5]. Although, only a partial release of the dense arachnoidal adhesions was possible in our patient, drainage of the cyst and the syrinx produced symptomatic relief [7].\nIn the modern era of computed tomography and magnetic resonance imaging, the need for myelography has diminished and the use of isophendylate has been discontinued. Nevertheless, the present case illustrates some of the rare sequelae of its use in the past, manifesting decades later.","keyphrases":["arachnoid cyst","myodil","syringomyelia","thoracic spine"],"prmu":["P","P","P","R"]}
{"id":"Breast_Cancer_Res_Treat-4-1-2244694","title":"Lifetime cumulative number of menstrual cycles and serum sex hormone levels in postmenopausal women\n","text":"Objective Lifetime cumulative number of menstrual cycles is related to breast cancer risk. The aim of this study is to investigate the relation between this index and serum sex hormone levels in postmenopausal women.\nIntroduction\nBreast cancer is strongly related to various hormonal factors. We know that different hormones play a key role in the pathogenesis of invasive breast cancer in both, premenopausal and postmenopausal women [1\u20134], likely representing a direct effect of circulating hormones [5\u20137].\nThe patterns of risk associated with reproductive history suggest that a prolonged exposure to ovarian hormones increases breast cancer occurrence [8]. It has been well established that early age at menarche, late age at menopause, null parity and late age at first full term pregnancy (FFTP) are important risk factors for breast cancer [9\u201311]. These characteristics may have long lasting effects on hormone metabolism, although data regarding reproductive characteristics and hormone levels are still inconclusive [12\u201316]\nIn an effort to measure the lifetime exposure to endogenous hormones, the concept of the \u201clifetime cumulative number of menstrual cycles\u201d was introduced as an index that could reflect total ovarian exposure [17, 18]. The \u201clifetime cumulative number of cycles\u201d summarizes information such as menarche, parity, menopause, regularity pattern, breast feeding and oral contraceptive (OC) use. In previous studies we, but also others, related the cumulative numbers of menstrual cycles to breast cancer risk [17, 18]. Ovarian function, and therefore, \u201clifetime number of menstrual cycles\u201d is defined by age at menopause, and age at menopause is related to postmenopausal estrogen levels [19]; with this rationale we studied the association between the \u201clifetime number of menstrual cycles\u201d and hormone levels after the menopause. The hypothesis fits in, where a common factor at hypothalamic level may affect the speed by which oocytes are being used, increasing postmenopausal gonoadothopin levels and maybe, stimulating ovarian testosterone release from the remaining theca cells, as well as non ovarian estrogens from adrenal origin.\nThe aim of this study was to evaluate the relation between the \u201clifetime cumulative number of menstrual cycles\u201d and the reproductive characteristics that contribute to this estimate on one side, and plasma concentrations of sex hormones on the other side, in order to elucidate the mechanism by which reproductive risk factors could influence hormone-related cancers.\nStudy design and methods\nThe Prospect-EPIC study is one of the two Dutch cohorts participating in the European Prospective Investigation into Cancer and Nutrition; a multi-center prospective study carried out in 23\u00a0centers from 10\u00a0European countries [20].\nA detailed description of the study population and data collection of the Prospect-EPIC study has been published elsewhere [21]. Briefly, 17,357\u00a0women aged 49\u201370, residing in Utrecht and its surroundings were recruited through a regional, population-based, breast cancer screening program between 1993 and 1997. Base-line characteristics were collected on the basis of two self-administered questionnaires (regarding lifestyle and nutrition). The lifestyle questionnaire contains information on demographic characteristics, family history, past and current morbidity, reproductive history (gynecologic and obstetric), smoking habits, and physical activity. In addition, anthropometric measurements (height, weight, and waist and hip circumference) were taken as well as a 30\u00a0ml blood sample. Within 24\u00a0h, samples of 4\u00a0ml serum, 9\u00a0ml citrate plasma, 2\u00a0ml white blood cells, and 2\u00a0ml red blood cells were fractionated into 0.5\u00a0ml aliquots and stored at \u2212196\u00b0C. Blood samples were successfully drawn from 97.5% of the participants [21]. All women signed a written informed consent and the study was approved by the Institutional Review Board of the University Medical Center Utrecht.\nOf all Prospect-EPIC participants a 10% random sample (n\u00a0=\u00a01736) was taken and hormone measurements were done. For the present study, participants who used hormone replacement therapy (HRT) or OC in the previous twelve months were excluded (n\u00a0=\u00a0311), as well as those women with history any cancer other than non-melanoma cancer (n\u00a0=\u00a025). From the remaining 1,400 participants only those who underwent natural menopause were eligible (n\u00a0=\u00a0860) because only in them we had reliable information about ovarian activity not masked by the absence of menstrual cycles due to hysterectomy. Natural menopause was defined as the complete cessation of menstrual bleeding for 12\u00a0months preceding enrollment due to natural causes (according to self-report).\nWe computed the \u201clifetime cumulative number of menstrual cycles\u201d of the women in this study. For parous women, the number of menstrual cycles before a FFTP was also estimated as we previously described [17]. Briefly, the \u201clifetime cumulative number of menstrual cycles\u201d was computed from age at first menstrual period to age at menopause. Ages at menarche, at menopause and at FFTP were recorded as continuous variables. Age at regularization of cycles was computed according to the time between menarche and the establishment of regular periods (immediately after menarche, after 1, 2, 3, 5, or 10\u00a0years, after FFTP or never regular). The regularity pattern was defined categorically as 24, 26, 28, 30 and 32\u00a0day cycles, or always irregular. Information about OC use included: age at start, number of years of use and age at last use. Data about number of and age at each pregnancy, stillbirth, miscarriage and abortion, if any, were available, as well as the number of weeks of breastfeeding for each pregnancy.\nWe took all the previously mentioned variables into account when computing the \u201clifetime cumulative number of menstrual cycles\u201d, considering that each pregnancy, stillbirth, miscarriage or abortion, resulted in an absence of cycles for a period of 36, 28 and 12\u00a0weeks respectively. We assumed that lactation results in a 6-week absence of cycles [22]. Menstrual cycles due to OC use were computed as 28\u00a0days duration (see Appendix). Considering that irregular cycles are a combination of long and short menstrual periods, the number of irregular cycles was computed according to each participant\u2019s regularity pattern; the total number of irregular years was recorded in order to make further adjustments. The number of menstrual cycles was impossible to compute in the participants who reported to be always irregular or never having regular periods, therefore we added the \u201cirregular\u201d category as such in the analysis (See appendix).\nBounded levels of estrogens and androgens were measured, levels of estrone (E1), estradiol (E2), androstenedione, testosterone, sex hormone binding globulin (SHBG) and dehydroepiandrostenedione sulfate (DHEAS) were measured in plasma using commercially available double-antibody radioimmunoassay (RIA) kits (Diagnostic System Laboratories Inc., Webster, TX, USA). The following kits were used: E1: DSL-8700, E2: DSL-39100, androstenedione: DSL-4200, TST: DSL-4100 SHBG: DSL-6300 and DHEAS: DSL-2700. The intra-assay coefficients of variation were 5.6%, 3.9%, 4.3%, 7.7%, 3.0% and 5.2% respectively. The inter-assay coefficients of variation were 11.1%, 4.1%, 6.3%, 8.1%, 4.0% and 5.3% respectively. All hormone assays were performed by the laboratory of the Hormones and Cancer group at the International Agency for Research on Caner (IARC). Although technically SHBG is not a hormone, for reasons of convenience it will be referred as such. We calculated the free-androgen index (FAI) dividing testosterone by SHBG and multiplying by 100, the SHGB\/E2 index was calculated dividing SHBG by E2.\nData analysis\nMeans with their standard deviation (SD) values, or frequencies of baseline characteristics were calculated. Concentrations of all hormones were logarithmically transformed to produce approximately normal distributions. To make the results easily interpretable, we transformed the mean and its 95% confidence interval (CI) back to their original scale, resulting in geometric means and 95% CIs. Geometric mean levels and 95% CIs were calculated for each hormone (SHBG, E1, E2, androstenedione, testosterone, DHEAS), SHBG\/estradiol index (SHBG\/E2) and free androgen index (FAI).\nUsing analysis of covariance models, geometric mean hormone levels were calculated for the different categories of cumulative number of menstrual cycles (lifetime and before a FFTP). The same analysis was also used for categories of age at menarche, age at menopause, parity, number of children and age of FFTP. In order to build the adjusted model we classified confounders in three categories as follows: Group A contained characteristics clearly related to hormone levels such as age, years since menopause and body mass index (BMI); Group B contained factors possibly related to hormone levels like OC and HRT use, smoking habits, physical activity and alcohol consumption; group C contained characteristics unlikely related to hormone levels such as marital status, education level and family history of breast cancer. We first adjusted for all confounders of type A, B, and C, then we built models by deleting B and C types of characteristics, without affecting validity but increasing precision. In the final model we included confounders in group A and B, therefore we adjusted for age at intake as a continuous variable, number of years from menopause to inclusion in the study, BMI at examination, use of HRT ever (Y\/N), use of OC ever (Y\/N), smoking habits, alcohol consumption and physical activity. Information on current alcohol use was obtained from the food frequency questionnaire, total alcohol consumption in grams per day was calculated from questions in the number of glasses of wine, beer, fortified wines, and liquor\/spirits per day, week, month or year. One glass of any alcoholic beverage was assumed to contain 10g of alcohol. With the daily physical activity data from the general questionnaire, the Voorrips score was calculated (and was entered in the model as a continuous variable), this score has been developed and validated especially for women in this age group [23]. For analysis of the \u201clifetime cumulative number of menstrual cycles\u201d and the \u201cnumber of cycles before a FFTP\u201d variables, we also adjusted for more than one year of irregular cycles (Y\/N). Given that age at menopause can be determined using age at intake in combination of years since menopause; when analyzing age at menopause, the covariate years since menopause was not entered in the model.\nTo test the significance of the models\u2019 linearity, median values in each category for every variable were computed accordingly. Subsequently these values were included as a continuous variable in a linear regression model to test for linear trends. Because there is no clear natural hierarchy, the \u201calways irregular\u201d category was not included when testing for trend for the \u201clifetime number of menstrual cycles\u201d and the \u201cnumber of menstrual cycles before a FFTP\u201d variables. Additional mutual adjustments for reproductive characteristics did not show any difference in the observed trends. The Statistical Package for Social Sciences for WINDOWS, version 14.0 (Chicago, IL), was used for all statistical analysis, and all tests were two sided.\nResults\nGeneral and reproductive characteristics of the Prospect-EPIC women that were included in this study are shown on Table\u00a0 1. On average, participants were 60.2\u00a0years old at enrollment; had a BMI of 26.2\u00a0kg\/m2 and the majority of them (69.4%) had at least low technical-secondary education. 113 (13.1 %) were nulliparous, and for the parous, the mean age for a FFTP was 25.5\u00a0years old. Mean age at menarche and menopause were 13.6 and 49.6\u00a0years old respectively, the time from menopause to inclusion, was on average 10.6\u00a0years. In 786 participants (91.4%) the lifetime cumulative number of cycles was calculated, as we had incomplete data regarding reproductive history in 74\u00a0women. The median number of lifetime menstrual cycles was 451.3, corresponding to 34.7\u00a0years of menstrual activity if considering an average of 13\u00a0cycles per year. From the 747\u00a0parous participants, 64 had incomplete data to compute the number of menstrual cycles before a FFT, in the remaining 683 participants (91.4%), the median number of menstrual cycles before a FFTP was 146.7 (approximately 11.3\u00a0years of menstrual activity). These results are consistent with the previous data on such estimates for a larger sample of the Prospect-EPIC participants as we previously published [17]. Table\u00a02 shows the geometric mean hormone levels for all participants with 95% CIs of SHBG, E1, E2, androstenedione, testosterone, DHEAS, SHBG\/E2 and FAI.Table\u00a01General and reproductive characteristics of the study participants (n\u00a0=\u00a0860)\u00a0Mean (SD)General characteristicsAge at enrollment60.2 (5.5)BMI26.2 (4.2)Waist Hip ratio0.797 (0.59)Length (cm)163.7 (6.1)Weight (Kg)70.1 (11.9)Educational level (%)- Primary education263 (30.6)- Low technical, secondary384 (44.6)- High technical, secondary98 (11.4)- Academic115 (13.4)Ever married (%)805 (93.6)Cigarette smoking habits (%)- Never smoked402 (46.7)- Past smoker275 (32)- Current smoker183 (21.3)Mother or sister with BC (%)123 (14.3)Reproduction characteristicsAge at menarche13.6 (1.6)Nulliparous (%)113 (13.1)Age at FFTPa25.46 (3.8)Number live born childrena2.9 (1.4)Age at menopause49.6 (4.0)Years since menopause10.6 (6.0)Oral contraceptive use (%)384 (44.6)Years of OC useb6HRT use (%)66 (7.7)Years of HRT usec1Lifetime number of menstrual cycles(n\u00a0=\u00a0786)165 (21.0)\u2003 \u2264415177 (22.5)\u2003 416\u2013453171 (21.8)\u2003 454\u2013490161 (20.5)\u2003 \u2265491112 (14.2)\u2003 Always irregulardNumber of menstrual cycles beforeFFTP (n\u00a0=\u00a0683)\u2003 \u2264118167 (25.5)\u2003 119\u2013147160 (23.4)\u2003 148\u2013185118 (17.3)\u2003 \u2265186143 (20.9)\u2003 Always irregulard95 (13.9)a\u00a0Among parous womenb\u00a0Median, Among OC usersc Median, Among HRT usersd Participants in the always irregular category, reported to be never regularTable\u00a02Geometric mean serum sex hormone and SHBG levelsHormoneMean, (95% CI)SHBG\u00a0\u03bcg\/ml18.1 (17.2\u201319.2)Estrone pg\/ml15.2 (14.48\u201316.0)Estradiol pg\/ml8.5 (8.2\u20138.8)Androstenedione ng\/ml0.47 (0.45\u20130.49)Testosterone total ng\/ml0.26 (0.25\u20130.27)DHEAS ng\/ml455.1 (432.9\u2013478.4)SHBG- Estradiol index (\u03bcg\/pg)0.47 (0.44\u20130.50)Free androgen index [(Testosterone \/SHBG*100 (ng\/\u03bcg*100)]4.6 (4.4\u20135.0)\nTable\u00a03 shows the unadjusted geometric mean hormone levels and 95% CI for each hormone according to some general and reproductive characteristics. When testing for trend participants in the lower quartile of \u201clifetime cumulative of menstrual cycles\u201d had higher levels of SHBG, androstenedione, testosterone and DHEAS than the participants with the highest quartile (P trend\u00a0=\u00a00.015, 0.003, 0.052 and 0.035 respectively), and lower levels of SHBG\/E2 (P trend\u00a0=\u00a00.031).Table\u00a03Hormones unadjusted means (95%CI) according to patient characteristics\u00a0SHBG (\u03bcg\/ml)Estrone (pg\/ml)Estroidal (pg\/ml)Androstenedione (ng\/ml)Testosterone (ng\/ml)DHEAS (ng\/ml)SHBG\/E2-indexFA-indexLifetime number of menstrual cycles\u00a0\u00a0\u00a0\u00a0<=41519.8 (17.3\u201322.5)15.6 (13.9\u201317.4)8.5 (7.8\u20139.2)0.56 (0.50\u20130.64)0.29 (0.26\u20130.31)515.3 (458.6\u2013579.1)0.431 (0.366\u20130.509)4.7 (4.05\u20135.46)\u00a0\u00a0\u00a0\u00a0416\u201345319.5 (17.1\u201322.1)13.5 (12.1\u201315.0)8.5 (7.8\u20139.2)0.45 (0.39\u20130.50)0.25 (0.23\u20130.27)448.1 (400.3\u2013501.5)0.435 (0.371-0.511)4.3 (3.73\u20134.97)\u00a0\u00a0\u00a0\u00a0454\u201349017.4 (15.3\u201319.8)16.1 (14.4\u201317.9)8.8 (8.1\u20139.5)0.43 (0.39\u20130.49)0.25 (0.23\u20130.27)436.4 (389.1\u2013489.3)0.504 (0.428\u20130.593)4.7 (4.09\u20135.49)\u00a0\u00a0\u00a0\u00a0\u00a0>= 49115.9 (13.9\u201318.2)15.9 (14.2\u201317.9)8.7 (7.9\u20139.4)0.44 (0.39\u20130.50)0.25 (0.23\u20130.27)434.8 (386.4\u2013489.3)0.542 (0.458\u20130.641)5.1 (4.36\u20135.89)\u00a0\u00a0\u00a0\u00a0Always irregular \u00a716.6 (14.1\u201319.5)14.9 (13.04\u201317.1)7.9 (7.1\u20138.7)0.49 (0.40\u20130.54)0.26 (0.23\u20130.28)425.4 (69.2\u2013490.2)0.476 (0.389\u20130.582)4.9 (4.07\u20135.85)\u00a0\u00a0\u00a0\u00a0P-trend0.150.4550.6480.0030.0520.0350.0310.406Number of menstrual cycles before a FFTP*\u00a0\u00a0\u00a0\u00a0<= 11819.5 (17.1\u201322.2)14.8 (13.2\u201316.6)8.3 (7.6\u20138.9)0.48 (0.42\u20130.54)0.27 (0.24\u20130.24)472.1 (420.1\u2013530.6).426 (.362-.500)4.5 (3.86\u20135.15)\u00a0\u00a0\u00a0\u00a0119\u201314718.2 (15.9\u201320.8)14.9 (13.2\u201316.7)8.6 (7.9\u20139.3)0.48 (0.42\u20130.54)0.26 (0.23\u20130.23)488.8 (433.8\u2013550.7).471 (.399-.555)4.6 (3.96\u20135.32)\u00a0\u00a0\u00a0\u00a0148\u201318517.3 (14.8\u201320.1)15.9 (13.9\u201318.2)9.2 (8.3\u201310.0)0.43 (0.37\u20130.50)0.26 (0.23\u20130.23)425.1 (369.9\u2013488.4).529 (.437-.641)4.9 (4.17\u20135.89)\u00a0\u00a0\u00a0\u00a0>= 18618.8 (16.3\u201321.6)14.5 (12.8\u201316.4)8.3 (7.6\u20138.9)0.45 (0.39\u20130.51)0.25 (0.22\u20130.22)419.4 (369.6\u2013475.7)0.440 (0.370\u20130.524)4.4 (3.76\u20135.15)\u00a0\u00a0\u00a0\u00a0Always irregular \u00a715.5 (13.1\u201318.4)14.8 (12.7\u201317.2)8.0 (7.2\u20138.8)0.48 (0.40\u20130.56)0.27 (0.24\u20130.24)440.3 (377.1\u2013513.9)0.515 (0.416-.638)5.5 (4.51\u20136.62)\u00a0\u00a0\u00a0\u00a0P-trend0.5740.9920.9330.3490.3330.0870.5650.999Age at intake\u00a0\u00a0\u00a0\u00a0<=\u00a05418.8 (16.5\u201321.5)15.9 (14.3\u201317.8)9.4 (8.7\u201310.2)0.53 (0.47\u20130.60)0.28 (0.26\u20130.31)611.5 (547.5\u2013682.9)0.499 (0.424\u20130.587)4.9 (4.19\u20135.62)\u00a0\u00a0\u00a0\u00a055\u20135918.9 (16.9\u201321.2)15.45 (14.1\u201316.9)8.6 (8.0\u20139.2)0.46 (0.42\u20130.51)0.25 (0.23\u20130.27)493.4 (448.6\u2013542.7)0.453 (0.394\u20130.522)4.5 (3.92\u20135.04)\u00a0\u00a0\u00a0\u00a060\u20136417.3 (15.6\u201319.3)14.3 (13.0\u201315.6)8.3 (7.8\u20138.8)0.47 (0.43\u20130.52)0.26 (0.24\u20130.28)424.3 (387.5\u2013464.5)0.480 (0.419\u20130.548)4.8 (4.26\u20135.42)\u00a0\u00a0\u00a0\u00a0>= 6517.7 (15.8\u201319.8)15.4 (14.0\u201316.9)7.9 (7.4\u20138.5)0.44 (0.39\u20130.49)0.25 (0.23\u20130.27)363.9 (330.9\u2013400.4)0.450 (0.391\u20130.518)4.6 (4.04\u20135.20)\u00a0\u00a0\u00a0\u00a0P-trend0.3030.4490.0020.0410.106<0.00010.4990.834Age at menarche\u00a0\u00a0\u00a0\u00a0<= 1118.9 (15.3\u201323.3)15.2 (12.7\u201318.2)8.0 (7.0\u20139.1)0.47 (0.39\u20130.57)0.28 (0.24\u20130.33)437.1 (363.9\u2013525.2)0.423 (0.325\u20130.550)4.9 (3.93\u20136.29)\u00a0\u00a0\u00a0\u00a01215.9 (14.1\u201318.1)14.5 (13.0\u201316.1)8.8 (8.1\u20139.4)0.47 (0.42\u20130.52)0.26 (0.24\u20130.28)439.4 (393.6\u2013490.5)0.549 (0.469\u20130.643)4.2 (4.51\u20135.99)\u00a0\u00a0\u00a0\u00a01317.5 (15.6\u201319.7)15.9 (14.4\u201317.5)8.3 (7.7\u20138.8)0.47 (0.42\u20130.52)0.26 (0.24\u20130.28)461.7 (417.2\u2013510.9)0.472 (0.408\u20130.546)4.7 (4.10\u20135.32)\u00a0\u00a0\u00a0\u00a01418.8 (16.6\u201321.3)15.1 (13.5\u201316.7)8.6 (8.0\u20139.3)0.44 (0.38\u20130.48)0.24 (0.22\u20130.26)447.9 (401.7\u2013499.4)0.458 (0.392\u20130.536)4.2 (3.69\u20134.88)\u00a0\u00a0\u00a0\u00a01520.8 (17.7\u201324.5)15.9 (13.9\u201318.2)8.5 (7.7\u20139.3)0.49 (0.42\u20130.57)0.28 (0.25\u20130.31)490.6 (426.2\u2013564.7)0.408 (0.334\u20130.499)4.4 (3.70\u20135.32)\u00a0\u00a0\u00a0\u00a0>=\u00a01618.8 (16.1\u201321.9)14.6 (12.7\u201316.6)8.5 (7.7\u20139.3)0.53 (0.46\u20130.61)0.27 (0.24\u20130.29)457.1 (399.0\u2013523.5)0.452 (0.372\u20130.549)4.6 (3.86\u20135.47)\u00a0\u00a0\u00a0\u00a0P-trend0.0690.9430.8050.2620.9060.4140.1820.147Reproductive status\u00a0\u00a0\u00a0\u00a0Parous17.5 (14.9\u201320.4)16.9 (14.8\u201319.4)8.8 (8.02\u20139.70)0.52 (0.45\u20130.60)0.26 (0.23\u20130.29)461.5 (402.0\u2013529.7)0.506 (0.415\u20130.616)4.8 (4.0\u20135.7)\u00a0\u00a0\u00a0\u00a0Nulliparous18.2 (17.2\u201319.414.9 (14.2\u201315.7)8.5 (8.13 8.76)0.47 (0.44\u20130.49)0.26 (0.25\u20130.27)454.1 (430.4\u2013479.1)0.463 (0.429\u20130.500)4.7 (4.3\u20134.9)\u00a0\u00a0\u00a0\u00a0P-equality of means0.6080.0830.4070.1680.8530.8310.4180.770Age at FFTP*\u00a0\u00a0\u00a0\u00a0<= 2119.5 (16.5\u201322.9)15.7 (13.6\u201318.1)8.5 (7.6\u20139.3)0.48 (0.41\u20130.56)0.26 (0.23\u20130.29)481.3 (416.4\u2013556.4)0.434 (0.355\u20130.531)4.4 (3.63\u20135.23)\u00a0\u00a0\u00a0\u00a022\u20132416.2 (14.3\u201318.2)14.7 (13.3\u201316.3)8.8 (8.2\u20139.4)0.47 (0.42\u20130.52)0.27 (0.25\u20130.29)468.1 (421.1\u2013520.3)0.544 (0.470\u20130.631)5.2 (4.57\u20135.96)\u00a0\u00a0\u00a0\u00a025\u20132718.5 (16.5\u201320.3)15.2 (13.8\u201316.6)8.2 (7.6\u20138.7)0.47 (0.42\u20130.52)0.26 (0.24\u20130.27)454.9 (414.8\u2013498.8)0.445 (0.391\u20130.506)4.6 (4.12\u20135.19)\u00a0\u00a0\u00a0\u00a0>= 2819.7 (17.4\u201322.2)14.5 (13.06\u201316.6)8.5 (7.8\u20139.1)0.45 (0.40\u20130.50)0.25 (0.23\u20130.27)425.6 (382.4\u2013473.6)0.429 (0.370\u20130.498)4.3 (3.75\u20134.90)\u00a0\u00a0\u00a0\u00a0P-trend0.2960.4970.5450.4140.3380.1380.2580.337Number of liveborn\u00a0\u00a0\u00a0\u00a0017.4 (14.9\u201320.4)16.9 (14.8\u201319.3)8.8 (8.0\u20139.7)0.52 (0.45\u20130.60)0.26 (0.23\u20130.29)461.5 (401.9\u2013529.7)0.506 (0.415\u20130.616)4.8 (4.00\u20135.71)\u00a0\u00a0\u00a0\u00a0117.5 (14.1\u201321.8)15.7 (13.1\u201318.9)7.9 (7.0\u20139.1)0.41 (0.33\u20130.50)0.24 (0.21\u20130.28)424.9 (351.1\u2013514.3).456 (.346-.600)4.5 (3.48\u20135.69)\u00a0\u00a0\u00a0\u00a02185 (16.7\u201320.3)14.8 (13.6\u201316.1)8.8 (8.2\u20139.2)0.48 (0.43\u20130.52)0.26 (0.24\u20130.28)474.9 (436.4\u2013516.8)0.473 (0.419\u20130.534)4.7 (4.21\u20135.23)\u00a0\u00a0\u00a0\u00a0318.2 (16.2\u201320.5)14.9 (13.4\u201316.4)8.4 (7.7\u20138.9)0.45 (0.40\u20130.50)0.26 (0.24\u20130.28)459.1 (414.4\u2013508.6)0.458 (0.395\u20130.531)4.6 (4.00\u20135.21)\u00a0\u00a0\u00a0\u00a0>= 418.1 (15.9\u201320.4)15.0 (13.5\u201316.6)8.2 (7.6\u20138.8)0.48 (0.43\u20130.54)0.27 (0.24\u20130.29)425.7 (381.8\u2013474.6)0.455 (0.389\u20130.532)4.7 (4.10\u20135.43)\u00a0\u00a0\u00a0\u00a0P-trend0.7700.1740.2890.6240.6680.4320.4370.946BMI\u00a0\u00a0\u00a0\u00a0<=\u00a024.925.8 (23.7\u201328.2)13.3 (12.3\u201314.4)7.5 (7.1\u20137.9)0.45 (0.41\u20130.49)0.23 (0.22\u20130.25)459.5 (459.5\u2013498.5)0.291 (0.262\u20130.323)3.1 (2.81\u20133.41)\u00a0\u00a0\u00a0\u00a025\u201329.916.8 (15.5\u201318.2)15.7 (14.6\u201316.8)8.6 (8.1\u20139.0)0.47 (0.44\u20130.51)0.27 (0.25\u20130.28)449.1 (449.1\u2013484.2)0.509 (0.461-0.561)5.1 (4.70\u20135.63)\u00a0\u00a0\u00a0\u00a030\u201334.910.5 (9.1\u201312.1)17.7 (15.5\u201320.1)10.0 (9.1\u201310.9)0.51 (0.44\u20130.58)0.31 (0.28\u20130.34)467.4 (467.4\u2013535.0)0.957 (0.803\u20131.14)8.5 (7.24\u20139.98)\u00a0\u00a0\u00a0\u00a0>=\u00a0359.9 (7.6\u201312.7)21.5 (17.0\u201327.0)13.5 (11.6\u201315.8)0.56 (0.43\u20130.72)0.31 (0.26\u20130.37)439.8 (439.8\u2013559.9)1.37 (1.00\u20131.87)9.0 (6.77\u201312.03)\u00a0\u00a0\u00a0\u00a0P-trend<0.0001<0.0001<0.00010.052<0.00010.959<0.0001<0.0001OC use ever\u00a0\u00a0\u00a0\u00a0yes17.6 (16.2\u201319.2)15.5 (14.4\u201316.6)8.4 (8.0\u20138.9)0.47 (0.43\u20130.51)0.26 (0.25\u20130.28)494.7 (459.4\u2013532.7)0.480 (0.431\u20130.533)4.9 (4.4\u20135.3)\u00a0\u00a0\u00a0\u00a0no18.6 (17.2\u201320.1)14.9 (14.1\u201316.0)8.5 (8.1\u20138.9)0.47 (0.44\u20130.51)0.26 (0.24\u20130.27)424.8 (397.1\u2013454.3)0.460 (0.417\u20130.507)4.5 (4.1\u20134.9)\u00a0\u00a0\u00a0\u00a0P equality of means0.3540.5530.7270.9380.6250.0030.5640.258HRT use ever\u00a0\u00a0\u00a0\u00a0yes17.9 (14.5\u201321.9)18.5 (15.5\u201322.0)11.9 (10.5\u201313.4)0.52 (0.4\u20130.6)0.26 (0.2\u20130.3)488.8 (408.1\u2013585.5)0.667 (0.515\u20130.863)4.6 (3.6\u20135.8)\u00a0\u00a0\u00a0\u00a0no18.2 (17.1\u201319.3)14.9 (14.2\u201315.7)8.3 (7.9\u20138.5)0.47 (0.4\u20130.5)0.26 (0.2\u20130.3)452.4 (429.4\u2013476.5).455 (.422-.490)4.7 (4.4- 4.9)\u00a0\u00a0\u00a0\u00a0P-equality of means0.8750.021\u00a0<0.00050.2990.8480.4180.0050.870Age at menopause\u00a0\u00a0\u00a0\u00a0<= 4718.4 (16.33\u201320.6)14.7 (13.3\u201316.2)8.5 (7.9\u20139.1)0.51 (0.46\u20130.57)0.28 (0.26\u20130.30)471.3 (425.7\u2013521.9)0.465 (0.402\u20130.539)4.9 (4.3\u20135.6)\u00a0\u00a0\u00a0\u00a048\u20135019.8 (17.94\u201321.8)15.3 (14.1\u201316.6)8.6 (8.1\u20139.1)0.49 (0.44\u20130.53)0.26 (0.24\u20130.27)485.1 (445.4\u2013528.4)0.433 (0.383\u20130.490)4.3 (3.8\u20134.8)\u00a0\u00a0\u00a0\u00a051\u20135217.5 (15.49\u201319.8)16.1 (14.5\u201317.9)8.3 (7.7\u20138.9)0.47 (0.42\u20130.52)0.26 (0.24\u20130.28)460.4 (413.4\u2013512.7)0.476 (0.408\u20130.556)4.8 (4.2\u20135.5)\u00a0\u00a0\u00a0\u00a0>= 5316.0 (14.15\u201318.2) 0.15914.6 (13.1\u201316.2)8.5 (7.8\u20139.1)0.41 (0.37\u20130.46)0.24 (0.22\u20130.26)389.2 (348.9\u2013434.1)0.527 (0.450-0.616)4.9 (4.3\u20135.7)\u00a0\u00a0\u00a0\u00a0P-trend0.7020.7390.0140.0170.0390.3350.950Time since menopause (years)\u00a0\u00a0\u00a0\u00a0<618.7 (16.7\u201320.9)16.0 (14.5\u201317.6)9.2 (8.6\u20139.9)0.51 (0.46\u20130.57)0.27 (0.25\u20130.29)567.5 (514.6\u2013625.9)0.493 (0.427\u20130.568)4.7 (4.14\u20135.35)\u00a0\u00a0\u00a0\u00a010-Jun17.9 (15.9\u201320.0)14.4 (13.1\u201315.9)8.6 (8.1\u20139.3)0.46 (0.42\u20130.51)0.25 (0.23\u20130.27)450.6 (408.0\u2013497.7)0.484 (0.419\u20130.559)4.6 (3.99\u20135.19)\u00a0\u00a0\u00a0\u00a015-Nov17.9 (15.9\u201320.1)15.5 (13.9\u201317.1)8.2 (7.6\u20138.8)0.45 (0.40\u20130.50)0.27 (0.24\u20130.29)432.7 (390.7\u2013479.2)0.457 (0.394\u20130.530)4.7 (4.13\u20135.39)\u00a0\u00a0\u00a0\u00a0>= 1618.1 (16.2\u201320.2)14.9 (13.6\u201316.4)8.1 (7.5\u20138.6)0.47 (0.42\u20130.52)0.26 (0.24\u20130.28)390.1 (354.9\u2013428.9)0.443 (0.386\u20130.509)4.7 (4.13\u20135.29)\u00a0\u00a0\u00a0\u00a0P-trend0.7250.4890.0030.2330.828<0.00010.2460.960* Among parous participants\u00a7 The always irregular category was not included when testing for trend\nIn table\u00a04 adjusted geometric means for each hormone according to the reproductive characteristics are shown. Participants in the lowest quartile of cumulative number of menstrual cycles tended to have higher SHBG (P trend\u00a0=\u00a00.013) and lower SHBG\/E2 (P trend\u00a0=\u00a00.050) levels. Androstenedione and DHEAS levels increased with increasing age at menarche (P trend\u00a0=\u00a00.027 and 0.022 respectively), a similar tendency was observed for E2, although the P for trend did not reach statistical significance (P trend\u00a0=\u00a0.058). Women with more children, tended to have higher SBHG levels (P trend\u00a0=\u00a00.030), number of children was also related to lower SHBG\/E2 (p trend\u00a0=\u00a00.010) levels. No association was found between number of cycles before a FFTP, parity, age at FFTP and any of the hormones. Age at menopause related inversely with androstenedione and testosterone; women with older age at menopause tend to have lower levels of such hormones (p trend\u00a0=\u00a00.024 and 0.015 respectively).Table\u00a04Hormones adjusted means\u2020 (95%CI) for serum sex hormones and SHBG levels according to reproductive characteristics\u00a0SHBG (\u03bcg\/ml)Estrone (pg\/ml)Estradiol (pg\/ml)Androstenedione (ng\/ml)Testosterone (ng\/ml)DHEAS (ng\/ml)SBHG\/E2 -INDEXFA-INDEXLifetime number of menstrual cycles\u00a0\u00a0\u00a0\u00a0<= 41519.50 (16.86\u201322.46)15.75 (13.83\u201317.94)8.62 (7.89\u20139.43)0.571 (0.495\u20130.659)0.285 (0.256\u20130.317)519.57(454.41\u2013594.67)0.444 (0.373\u20130.528)4.76 (4.06\u20135.59)\u00a0\u00a0\u00a0\u00a0416\u201345319.79 (17.51\u201322.35)13.28 (11.88\u201314.83)8.39 (7.78\u20139.04)0.448 (0.396\u20130.506)0.253 (0.231\u20130.278)436.16 (388.77\u2013488.82)0.424 (0.365\u20130.492)4.27 (3.72\u20134.89)\u00a0\u00a0\u00a0\u00a0454\u201349016.99 (14.98\u201319.26)16.26 (14.51\u201318.23)8.72 (8.06\u20139.41)0.434 (0.383\u20130.493)0.251 (0.228\u20130.276)427.96 (380.31\u2013481.06)0.513 (0.440\u20130.597)4.76 (4.14\u20135.48)\u00a0\u00a0\u00a0\u00a0>= 49115.94 (13.82\u201318.39)16.02 (14.07\u201318.24)8.53 (7.81\u20139.32)0.443 (0.384\u20130.511)0.257 (0.231\u20130.287)447.64 (391.51\u2013511.83)0.535 (0.450\u20130.636)5.15 (4.39\u20136.04)\u00a0\u00a0\u00a0\u00a0Always irregular \u00a716.48 (13.87\u201319.57)15.21 (13.01\u201317.78)8.04 (7.24\u20138.94)0.462 (0.389\u20130.548)0.255 (0.224\u20130.290)427.09 (363.58\u2013501.69)0.488 (0.397\u20130.601)4.93 (4.07\u20135.96)\u00a0\u00a0\u00a0\u00a0P-trend0.0130.4740.8420.0890.4460.3490.0500.192Number of menstrual cycles before a FFTP*\u00a0\u00a0\u00a0\u00a0<= 11819.83 (17.51\u201322.47)15.15 (13.49\u201317.01)8.22 (7.61\u20138.87)0.474 (0.416\u20130.540)0.265 (0.241\u20130.292)468.25 (415.30\u2013527.42)0.414 (0.356\u20130.482)4.37 (3.81\u20135.02)\u00a0\u00a0\u00a0\u00a0119\u201314718.41 (16.18\u201320.95)14.47 (12.83\u201316.33)8.38 (7.75\u20139.07)0.480 (0.419\u20130.550)0.253 (0.229\u20130.280)475.80 (420.31\u2013538.61)0.455 (0.389\u20130.533)4.53 (3.92\u20135.22)\u00a0\u00a0\u00a0\u00a0148\u201318516.09 (13.81\u201318.76)15.66 (13.57\u201318.06)8.85 (8.05\u20139.72)0.420 (0.358\u20130.493)0.258 (0.229\u20130.290)402.22 (347.23\u2013465.91)0.549 (0.456\u20130.662)5.17 (4.36\u20136.13)\u00a0\u00a0\u00a0\u00a0>= 18618.43 (16.13\u201321.05)14.86 (13.13\u201316.83)8.54 (7.88\u20139.27)0.461 (0.401\u20130.529)0.253 (0.228\u20130.280)445.86 (392.29\u2013506.74)0.463 (0.394\u20130.545)4.54 (3.92\u20135.27)\u00a0\u00a0\u00a0\u00a0Always irregular \u00a715.52 (12.92\u201318.65)14.81 (12.49\u201317.57)8.04 (7.19\u20138.99)0.467 (0.386\u20130.566)0.267 (0.232\u20130.307)438.34 (367.97\u2013522.69)0.518 (0.415\u20130.648)5.39 (4.397\u20136.60)\u00a0\u00a0\u00a0\u00a0P-trend0.2820.9290.4160.6050.5730.4230.1960.522Age at menarche\u00a0\u00a0\u00a0\u00a0<= 1119.26 (15.72\u201323.67)14.67 (12.23\u201317.58)7.56 (6.67\u20138.55)0.440 (0.360\u20130.538)0.272 (0.234\u20130.316)424.54 (352.13\u2013511.83)0.392 (0.307\u20130.502)4.71 (3.75\u20135.90)\u00a0\u00a0\u00a0\u00a01218.10 (16.04\u201320.43)13.98 (12.55\u201315.58)8.53 (7.93\u20139.19)0.454 (0.403\u20130.512)0.249 (0.228\u20130.272)423.27 (378.42\u2013472.95)0.471 (0.407\u20130.546)4.51 (3.94\u20135.16)\u00a0\u00a0\u00a0\u00a01316.35 (14.58\u201318.32)15.52 (14.01\u201317.18)8.17 (7.63\u20138.77)0.458 (0.409\u20130.513)0.254 (0.233\u20130.276)444.52 (400.21\u2013493.74)0.500 (0.436\u20130.574)4.87 (4.29\u20135.53)\u00a0\u00a0\u00a0\u00a01417.79 (15.78\u201320.06)15.41 (13.85\u201317.15)8.58 (7.98\u20139.23)0.440 (0.391\u20130.496)0.248 (0.227\u20130.271)448.99 (402.22\u2013501.69)0.482 (0.417\u20130.558)4.58 (4.01\u20135.23)\u00a0\u00a0\u00a0\u00a01519.73 (16.91\u201323.01)17.06 (14.86\u201319.57)9.02 (8.21\u20139.90)0.518 (0.444\u20130.603)0.286 (0.255\u20130.320)516.98 (448.54\u2013595.26)0.457 (0.379\u20130.550)4.78 (4.03\u20135.67)\u00a0\u00a0\u00a0\u00a0>=\u00a01618.58 (15.99\u201321.58)15.06 (13.16\u201317.22)8.78 (8.02\u20139.63)0.553 (0.476\u20130.641)0.276 (0.247\u20130.308)494.23 (430.09\u2013567.36)0.473 (0.395\u20130.567)4.79 (4.05\u20135.66)\u00a0\u00a0\u00a0\u00a0P-trend0.5770.1810.0580.0270.1940.0220.6210.739Ever pregnant\u00a0\u00a0\u00a0\u00a0yes18.17 (17.13\u201319.28)14.94 (14.17\u201315.75)8.41 (8.12\u20138.72)0.463 (0.437\u20130.491)0.258 (0.247\u20130.269)452.59 (428.80\u2013478.18)0.463 (431\u2013497)4.62 (4.33\u20134.94)\u00a0\u00a0\u00a0\u00a0no16.56 (14.19\u201319.34)17.12 (4.91\u201319.65)8.88 (8.08\u20139.76)0.528 (0.453\u20130.616)0.273 (0.244\u20130.306)466.85 (405.05\u2013538.08)0.536 (0.445\u20130.646)5.21 (4.39\u20136.18)\u00a0\u00a0\u00a0\u00a0P-equality of means0.2730.0720.2990.1190.8020.6930.1520.204Age at FFTP*\u00a0\u00a0\u00a0\u00a0<= 2119.89 (16.98\u201323.29)15.53 (13.45\u201317.94)8.26 (7.50\u20139.08)0.454 (0.386\u20130.534)0.248 (0.220\u20130.280)451.24 (388.77\u2013523.74)4.05 (3.39\u20134.82)0.415 (0.343\u20130.503)\u00a0\u00a0\u00a0\u00a022\u20132417.46 (15.52\u201319.63)14.41 (12.96\u201316.04)8.46 (7.88\u20139.07)0.466 (0.413\u20130.526)0.264 (0.242\u20130.289)464.05 (415.71\u2013518.53)4.84 (4.26\u20135.52)0.484 (0.420\u20130.558)\u00a0\u00a0\u00a0\u00a025\u20132717.32 (15.67\u201319.16)15.01 (13.69\u201316.44)8.06 (7.58\u20138.57)0.463 (0.417\u20130.513)0.257 (0.238\u20130.277)445.41 (405.05\u2013489.80)4.84 (4.33\u20135.41)0.465 (0.412\u20130.526)\u00a0\u00a0\u00a0\u00a0>= 2818.67 (16.63\u201320.99)15.07 (13.56\u201316.76)8.84 (8.24\u20139.48)0.466 (0.414\u20130.525)0.258 (0.237\u20130.282)452.59 (405.45\u2013504.72)4.59 (4.04\u20135.22)0.473 (0.411\u20130.545)\u00a0\u00a0\u00a0\u00a0P-trend0.7410.9880.3890.8580.8230.8500.4830.444Number of liveborn\u00a0\u00a0\u00a0\u00a0016.56 (14.18\u201319.32)17.12 (14.91\u201319.67)8.87 (8.08\u20139.76)0.527 (0.452\u20130.615)0.273 (0.244\u20130.306)466.85 (404.64\u2013538.08)0.536 (0.445\u20130.646)5.21 (4.39\u20136.18)\u00a0\u00a0\u00a0\u00a0116.14 (13.05\u201319.94)16.39 (13.56\u201319.81)8.33 (7.32\u20139.49)0.411 (0.333\u20130.508)0.237 (0.203\u20130.277)438.78 (361.04\u2013533.79)0.517 (0.400\u20130.667)4.69 (3.71\u20135.95)\u00a0\u00a0\u00a0\u00a0217.57 (15.99\u201319.32)14.88 (13.68\u201316.20)8.71 (8.22\u20139.23)0.471 (0.429\u20130.517)0.257 (0.240\u20130.276)456.69 (418.63\u2013498.19)0.496 (0.443\u20130.555)4.85 (4.37\u20135.39)\u00a0\u00a0\u00a0\u00a0318.05 (16.13\u201320.19)14.73 (13.33\u201316.29)8.28 (7.73\u20138.86)0.448 (0.401\u20130.502)0.259 (0.239\u20130.282)453.05 (408.71\u2013502.70)0.459 (0.401\u20130.525)4.62 (4.07\u20135.23)\u00a0\u00a0\u00a0\u00a0>= 420.18 (17.85\u201322.83)14.83 (13.28\u201316.56)8.12 (7.53\u20138.76)0.486 (0.430\u20130.550)0.263 (0.240\u20130.288)449.89 (401.82\u2013504.21)0.402 (0.346\u20130.467)4.23 (3.69- 4.85)\u00a0\u00a0\u00a0\u00a0P-trend0.0300.0800.1170.5780.9330.7800.0100.064Age at FFTP*\u00a0\u00a0\u00a0\u00a0<= 2119.89 (16.98\u201323.29)15.53 (13.45\u201317.94)8.26 (7.50\u20139.08)0.454 (0.386\u20130.534)0.248 (0.220\u20130.280)451.24 (388.77\u2013523.74)0.405 (0.339\u20130.482)4.15 (3.43\u20135.03)\u00a0\u00a0\u00a0\u00a022\u20132417.46 (15.52\u201319.63)14.41 (12.96\u201316.04)8.46 (7.88\u20139.07)0.466 (0.413\u20130.526)0.264 (0.242\u20130.289)464.05 (415.71\u2013518.53)0.484 (0.426\u20130.552)4.84 (4.20\u20135.58)\u00a0\u00a0\u00a0\u00a025\u20132717.32 (15.67\u201319.16)15.01 (13.69\u201316.44)8.06 (7.58\u20138.57)463 (0.417\u20130.513)0.257 (0.238\u20130.277)445.41 (405.05\u2013489.80)0.484 (0.433\u20130.541)4.65 (4.12\u20135.26)\u00a0\u00a0\u00a0\u00a0>= 2818.67 (16.63\u201320.99)15.07 (13.56\u201316.76)8.84 (8.24\u20139.48)0.466 (0.414\u20130.525)0.258 (0.237\u20130.282)452.59 (405.45\u2013504.72)0.459 (0.404\u20130.522)4.73 (4.11\u20135.45)\u00a0\u00a0\u00a0\u00a0P-trend0.7410.9880.3890.8580.8230.8500.4830.444Age at menopause\u221e\u00a0\u00a0\u00a0\u00a0<= 4718.16 (16.20- 20.37)14.74 (13.30\u201316.33)8.38 (7.81\u20138.98)0.511 (0.456\u20130.572)0.281 (0.258\u20130.306)445.41 (401.01\u2013494.72)0.461(0.402\u20130.530)4.94 (4.35\u20135.61)\u00a0\u00a0\u00a0\u00a048\u20135019.26 (17.53\u201321.16)15.41 (14.17\u201316.76)8.61 (8.12\u20139.15)0.484 (0.441\u20130.532)0.259 (0.241\u20130.277)481.06 (441.42\u2013524.79)0.447 (0.399\u20130.501)4.38 (3.95\u20134.86)\u00a0\u00a0\u00a0\u00a051\u20135216.76 (14.86\u20138.89)16.23 (14.57\u201318.06)8.36 (7.77\u20138.99)0.472 (0.419\u20130.532)0.261 (0.238\u20130.285)462.20 (413.64\u2013516.46)0.499 (0.431\u20130.577)5.01 (4.38\u20135.72)\u00a0\u00a0\u00a0\u00a0>= 5316.96 (15.01\u201319.18)14.47 (12.96\u201316.13)8.50 (7.88\u20139.15)0.411 (0.364\u20130.464)0.238 (0.217\u20130.261)416.55 (372.41\u2013466.38)0.501 (0.432\u20130.580)4.65 (4.06\u20135.33)\u00a0\u00a0\u00a0\u00a0P-trend0.3360.7700.8540.0240.0150.6450.3740.690\u2020 Adjusted for age at intake, years since menopause, body mass index (BMI) at examination, use of hormone replacement therapy (HRT) ever (Y\/N), use of oral contraceptive (OC) ever (Y\/N), smoking habits, total alcohol consumption (g\/day as a continuous variable) and physical activity (Voorrips total score as a continuous variable). For analysis of the \u201clifetime cumulative number of menstrual cycles\u201d and \u201cnumber of cycles before a FFTP\u201d variables, we also adjusted for more than one year of irregular cycles (Y\/N) \u00a7 The always irregular category was not included when testing for trend * Among parous participants \u221e Number of years since menopause was not included in the model\nDiscussion\nThe results of the present study suggest that the \u201clifetime cumulative number of cycles\u201d is related to SHBG and that some reproductive characteristics are also associated to postmenopausal serum sex hormone levels. We noted significant associations after adjusting for important confounders, the \u201clifetime cumulative number of menstrual cycles\u201d related inversely with levels of SHBG and positively with SHBG\/E2. Age at menarche directly relates to both DHEAS and androstenedione. Number of children had a significant relation with levels of SHBG and SHBG\/E2 and age at menopause was indirectly associated to androstenedione and testosterone.\nSeveral studies have investigated the relation between reproductive risk factors and endogenous sex hormone levels in postmenopausal women, but with conflicting results [6, 13, 15, 24\u201327]. The patterns of risk associated with reproductive history suggest that prolonged exposure to ovarian hormones increases breast cancer occurrence [8, 28, 29]. The cumulative number of menstrual cycles has been used as a proxy for ovarian activity, women in the highest quartile of the \u201clifetime cumulative number of menstrual cycles\u201d estimate, have an increased breast cancer risk when compared with women in the lowest quartile [17, 18]. A significant inverse relation between number of menstrual cycles and SHBG was observed in our study. It has been previously described that SHBG decrease the proportion of E2 that is able to leave the circulation and enter the cells, and an inverse relation between concentration of SHBG and breast cancer has been observed in epidemiological studies [1, 10]. Our findings suggest therefore, that postmenopausal free levels of estrogens or androgens may be related to the \u201clifetime cumulative number of menstrual cycles\u201d reflecting lifetime exposure to ovarian hormones.\nEarly age at menarche and late age at menopause have long being recognized as risk factors for breast cancer. A 1-year delay in the onset of menarche is associated with a 5% reduction in risk for developing breast cancer in later life [30]. We found that age at menarche has a direct relation with serum levels of DHEAS and androstenedione measured after the menopause. Androgens have been associated with breast cancer risk, women with breast cancer tend to have higher levels of DHEAS and androstenedione [2, 4, 31, 32]. It has been proposed that in postmenopausal women DHEAS might act as a moderate estrogen agonist, through competitive binding of its metabolite 5-androstene-3B,17B-diol [33]. It is also well known that the peripheral conversion of adrenal androstenedione is the principal source of E1 in postmenopausal women [34], therefore we would expect an inverse association between both DHEAS and androstenedione and age at menarche. Madigan et\u00a0al. showed, after adjusting for BMI and years since menopause, that young age at menarche, was associated with higher levels of androstenedione, but also of E1 and E2 [15]. Our results are in conflict with such findings, however several studies have found no association between age at menarche and SHBG, estrogens or androgens [14, 16, 35, 36].\nChildbearing and a young age at FFTP protect against breast cancer and additional pregnancies further reduce the risk [7, 10, 37, 38]. We found that parous women tend to have lower levels of E1, however this difference was not statistically significant (P\u00a0=\u00a0.072). Lamar et\u00a0al. [14] found that nulliparous women had higher testosterone levels, a finding that could contribute to the increased risk of breast cancer in nulliparous women. Our findings also showed that the number of live births is directly related to SHBG, and indirectly to SHBG\/E2 and FAI (P for trend for FAI did not reach statistical significance, P\u00a0=\u00a00.064). This observation is in conflict with the known association between low SBGH levels and breast cancer risk, and the protective effect that the number of pregnancies has on breast cancer [28, 29, 38, 39]. It is important to mention that a woman\u2019s genetic and environmental profile may influence both, her ability to have children and her postmenopausal sex hormone concentrations, making these observations difficult to interpret. It has been hypothesized that the overall effect of parity is due to a protective effect of a young age at FFTP [40], another proposed mechanism is through lactation, as breastfeeding induces final differentiation of the terminal duct epithelium making it relatively insensitive to hormonal stimulation [41, 42]. Despite these observations we found no relation between age at FFTP and any of the hormones. No relation between parity, number of pregnancies or age at FFTP and serum hormone levels was reported by several studies. [12, 14\u201316, 43, 44].\nWe observed an indirect association between age at menopause and both, androstenedione and testosterone. Several studies have failed in finding an association between age at menopause and sex hormones [12, 14, 16], however a direct association with menopause and androstenedione has been reported [15], as well as a relation between age at menopause and E2 in a group of 173 postmenopausal overweight women [13].\nTo appreciate the findings of our study, some strengths and limitations need to be mentioned. The main advantage is our large sample size, to the best of our knowledge this is the largest study assessing the relationship between reproductive risk factors and serum sex hormone levels in naturally menopausal women and the first one evaluating the relationship between sex hormones and an index that can be used as a proxy of ovarian exposure. Another advantage is that we attempted to adjust for all relevant covariates, in order to obtain fully adjusted estimates. The participants in this cross-sectional study originated from a random sample of a large cohort, and blood samples where drawn from 97.5% of the 17,357 participants, favoring the generalization of our results. Given the prospective nature of the Prospect-EPIC study, and because participants were recruited through an existing population-based breast cancer screening program, we included only disease-free participants at baseline blood collection.\nA limitation of this study is that only a single blood sample was assayed per participant, however it has been shown that a single measure provides substantial information in postmenopausal women for E1, E2 and SHBG [45]. Serial measurements of serum hormones in postmenopausal women suggest that a single measurement can reliably categorize women, at least over the short term. Intraclass correlation coefficients of hormone measurements in serum collected over a 3-year period were reported to be between 0.68 and 0.78 for the estrogens, 0.66 and 0.88 for the androgens, and 0.92 for SHBG [46]. We did not have information regarding premenopausal sex hormone concentrations in the participants of this study; it remains unknown whether some underlying hormonal profile could have influenced both, reproductive capabilities and postmenopausal sex hormone concentrations. It is also possible that premenopausal hormone levels are more related to reproductive factors than postmenopausal levels, and that is as a proxy for premenopausal levels that postmenopausal levels relate to reproductive characteristics. We should consider that the data used regarding reproductive characteristics are prone to recall errors. However, it is unlikely that recall errors are related to endogenous hormone levels themselves, if any misclassification occurred, it would have been non-differential.\nIn summary, in this group of naturally postmenopausal women, the \u201clifetime cumulative number menstrual cycles\u201d was related only to SHBG, suggesting that free levels of estrogens or androgens may be related to this index, reflecting lifetime exposure to ovarian hormones. Our findings show that some reproductive characteristics are related to postmenopausal serum sex hormone levels, however we do not find clear evidence to explain the mechanism by which reproductive risk factors influence hormone-related cancers in naturally postmenopausal women. It seems that reproductive and menstrual characteristics do not affect postmenopausal breast cancer risk via a long-term effect on sex hormone levels. The association of reproductive risk factors and breast cancer could be a result of prolonged exposure to ovarian hormones, and not only the product of a direct, long-lasting effect of sex hormone serum concentrations. Further research is needed in order to unravel the biological mechanisms through which reproductive characteristics relate to breast cancer risk.","keyphrases":["cumulative number of menstrual cycles","sex hormones","breast cancer","risk factors","reproductive characteristics","steroids"],"prmu":["P","P","P","P","P","U"]}
{"id":"Eur_Radiol-3-1-1914219","title":"Influence of convolution filtering on coronary plaque attenuation values: observations in an ex vivo model of multislice computed tomography coronary angiography\n","text":"Attenuation variability (measured in Hounsfield Units, HU) of human coronary plaques using multislice computed tomography (MSCT) was evaluated in an ex vivo model with increasing convolution kernels. MSCT was performed in seven ex vivo left coronary arteries sunk into oil followingthe instillation of saline (1\/\u221e) and a 1\/50 solution of contrast material (400 mgI\/ml iomeprol). Scan parameters were: slices\/collimation, 16\/0.75 mm; rotation time, 375 ms. Four convolution kernels were used: b30f-smooth, b36f-medium smooth, b46f-medium and b60f-sharp. An experienced radiologist scored for the presence of plaques and measured the attenuation in lumen, calcified and noncalcified plaques and the surrounding oil. The results were compared by the ANOVA test and correlated with Pearson\u2019s test. The signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) were calculated. The mean attenuation values were significantly different between the four filters (p < 0.0001) in each structure with both solutions. After clustering for the filter, all of the noncalcified plaque values (20.8 \u00b1 39.1, 14.2 \u00b1 35.8, 14.0 \u00b1 32.0, 3.2 \u00b1 32.4 HU with saline; 74.7 \u00b1 66.6, 68.2 \u00b1 63.3, 66.3 \u00b1 66.5, 48.5 \u00b1 60.0 HU in contrast solution) were significantly different, with the exception of the pair b36f\u2013b46f, for which a moderate-high correlation was generally found. Improved SNRs and CNRs were achieved by b30f and b46f. The use of different convolution filters significantly modifief the attenuation values, while sharper filtering increased the calcified plaque attenuation and reduced the noncalcified plaque attenuation.\nIntroduction\nMultislice computed tomography (MSCT) of the coronary arteries is becoming an established method to exclude the presence of significant stenosis because of the high negative predictive value [1\u20136]. Stenosis, however, occurs in a later stage of coronary artery disease (CAD); consequently, the atherosclerotic plaque composition and morphology may represent a better predictor of plaque stability [7\u20139]. Until recently, only invasive techniques, such as intracoronary ultrasound (ICUS), have been able to provide consistently reproducible quantitative and qualitative information on plaque composition [10]. ICUS, however, cannot be used for routine evaluation of plaque characteristics because of its invasiveness and related increased risk, additional time and cost.\nMagnetic resonance imaging may be used to characterize in vivo the atherosclerotic plaque morphology and composition in carotid [11] and coronary arteries [12, 13].\nSeveral recent in vivo studies have stressed that MSCT can be used to characterize coronary atherosclerotic plaque and has the potential to become the noninvasive modality of choice for plaque imaging [14\u201317]. This technique is able to provide information on noncalcified coronary plaques, which may be useful to identify a high-risk plaque [18\u201320].\nMSCT plaque assessment is based upon the variable X-ray attenuation of the tissue components. However, several parameters, such as lumen attenuation, convolution filtering, body mass index of the patient and contrast-to-noise ratio (CNR) of the images, are able to modify the attenuation values that are being used to define the composition of coronary plaques.\nThe aim of the present study was to investigate the effect of increasing convolution filtering on plaque components with varying coronary attenuation in an ex vivo coronary model.\nMaterial and methods\nSpecimens\nSeven ex vivo left anterior descending (LAD) coronary arteries were obtained at autopsy (Fig.\u00a01) from seven deceased patients: four male and three female, with a mean age of 68\u2009\u00b1\u200910\u00a0years: five (four males and one female with an age between 62 and 71\u00a0years) jad died of noncardiovascular diseases, and two (one male and one female with an age of 65 and 73\u00a0years, respectively) had died of ischemic heart disease. The Institutional Review Board approved the study protocol.\nFig.\u00a01Example of proximal left coronary artery specimen. CX Left circumflex, LAD left anterior descending artery, LM left main coronary artery\nThe specimens were prepared and examined separately. Each coronary artery was fitted with two cannulas fixed with surgical thread in the proximal (in the left main) and distal ends (in the LAD). The circumflex artery had been closed earlier at its end with thread. Only the LAD was used because of the major length (segments considered with a mean length of 7.57\u00a0cm) and higher prevalence of atherosclerotic disease and for technical reasons (ability to fix a surgical thread in the left main, without hampering the evaluation of the proximal LAD).\nContrast material\nTwo solutions were used: a saline and a 1\/50 dilution of contrast material (400\u00a0mgI\/ml Iomeprol; Bracco, Italy). The attenuation values (Hounsfield Units, HU) of the two solutions, measured in a 10-ml syringe after dilution, were 15.9\u2009\u00b1\u20091.8\u00a0HU (1\/\u221e; defined as saline; no contrast material was diluted) and 414.8\u2009\u00b1\u20095.6\u00a0HU (1\/50; defined as contrast solution). We preferred to use a 1\/50 dilution to achieve a mean coronary in-lumen density >300\u00a0HU \u2013 which is not typically achieved in in vivo cardiac computed tomography angiography (CTA) studies \u2013 in order to emphasize the difference with the saline solution. Our study benefits from two consecutive scans of the same vessel (e.g. with and without contrast media) for a comparative evaluation of plaques, which is not truly feasible during in vivo studies due to high radiation exposure.\nExperimental settings\nA box was filled with olive oil. Prior to positioning the specimen in the oil, saline was injected through the sheaths to wash out the air bubbles in the lumen as much as possible.\nOnce the specimen had sunk into the oil, which simulated epicardial fat, the solution was instilled through the sheaths using a 10-ml syringe from the proximal end of the specimen. The injection was finished when the solution was observed leaking out of the distal end of specimen. The leaking solution was removed from the specimen using a syringe. The same procedure was used to fill the LAD with both solutions. The specimen was kept for all investigations in the same longitudinal position (head-LM-to feet-distal end of LAD ) to obtain direct cross-section images.\nScan parameters\nA MSCT scan (Somatom Sensation 16; Siemens, Germany) was performed following the intra-coronary injection of two solutions.\nScans were performed at the following parameters: slices\/collimation, 16\/0.75\u00a0mm; rotation time, 375\u00a0ms; feed per rotation 3.0\u00a0mm (pitch: 0.25\u00a0mm); 120\u00a0kv; 400\u00a0mAs; effective slice thickness, 1\u00a0mm; reconstruction increment, 0.5\u00a0mm; field of view (FOV), 100\u00a0mm. Four convolution filters were used: b30f (smooth), b36f (medium smooth), b46f (medium) and b60f (sharp). Based on our clinical experience in routine evaluation, b30f was used instead of b20f because of a better delineation of the coronary arteries and atherosclerotic lesions.\nThe scan geometry was based on a retrospective electrocardiogram (ECG)-gated protocol (the same used for the in vivo examination). This protocol is based on a low pitch that allows a retrospective reconstruction of multiple phases within the cardiac cycle. A demo-ECG was switched on and a heart rate of 71 occurred during the scan. The half-rotation reconstruction algorithm brought the effective temporal resolution down to 187\u00a0ms.\nData collection and analysis\nA radiologist with a level 3 expertise in cardiac CT [21] loaded the data sets into a dedicated workstation (Leonardo; Siemens, Germany) and performed orthogonal views of all specimens for each convolution filter with two solutions.\nOne operator performed all of the measurements. Each specimen was evaluated for the presence of atherosclerotic plaque. The coronary wall thickening of plaque (minimum size observed: 0.3\u00a0mm2) was clearly visible from the surrounding oil with low attenuation and from the lumen after injection of contrast material. Plaques were targeted regardless of their size. The operator loaded the two data sets for each solution of the same specimen into a workstation screen, scrolling the data sets in parallel with standard soft-tissue window settings (window width:700\u00a0HU; window center:140\u00a0HU).\nOnce a plaque was detected in the two solutions by means of a comparative evaluation, the operator drew four regions of interest (ROIs) for the contrast material: in the lumen of the vessel (defined as lumen), the soft tissue of the coronary plaque (defined as noncalcified plaque), the calcification within the coronary artery wall (defined as calcified plaque) and the oil surrounding the plaque (defined as surrounding). The ROIs (minimum size: 0.1\u00a0mm2) were drawn as large as possible while avoiding the borders of each structure in order to limit the partial volume effect. Once the four ROIs were drawn, the operator could copy and paste these into other stacks of images. The two data sets for each solution were then evaluated using four convolution filters (b30f, b36f, b46f and b60f). For each ROI the mean and the standard deviation of the attenuation value were collected. Overall, 3200 attenuation values were obtained.\nStatistical analysis\nThe attenuation values are presented as means and standard deviations. The signal-to-noise ratio (SNR) was calculated as the mean attenuation value of the ROI\/standard deviation of the surrounding oil attenuation value. The standard deviation HU value of the surrounding air is often used for the estimation of noise. However, we did not include the air in our images. The standard deviation of the surrounding oil can estimate the noise because of its reliable signal. SNRs are presented as means for each kernel.\nThe contrast-to-noise ratio (CNR) was calculated as (mean attenuation A \u2013 mean attenuation B)\/image noise (defined as the standard deviation of the surrounding oil). CNRs of (lumen \u2013 noncalcified plaque), (noncalcified plaque \u2013 surrounding), (calcified plaque \u2013 noncalcified plaque) and (calcified plaque \u2013 lumen) were calculated and presented as average for each kernel and solution.\nStatistical evaluation was performed with dedicated software (SPSS ver. 10.1; SPSS, Chicago, Ill.). A one-way ANOVA test was performed to analyze the statistical difference between kernels in each structure for two solutions. A p\u2009<\u20090.0001 was considered for statistical significance. The Tukey HSD test evaluated the statistical absolute difference between any two groups of values. The Pearson\u2019s correlation was also tested between all the different convolution filters.\nAfter clustering for kernel and structure, the attenuation values obtained with the two solutions were compared with Student\u2019s t-test and correlated with Pearson\u2019s test.\nResults\nA total of 100 sections containing mixed plaques (n\u2009=\u200920) were evaluated in the seven coronary specimens. Two solutions and four convolution kernels were available (800 slices) at each level, and four ROIs (lumen, surrounding, calcified plaque, noncalcified plaque) were sampled in each slice. A total of 3200 measurements were performed. The results are summarized in Table\u00a01. The attenuation values were all significantly different between the convolution filters in each structure with both solutions (p\u2009<\u20090.0001). The results of the Tukey HSD to test the difference between pairs of four filters showed that the noncalcified plaque attenuation values were all significantly different using both solutions (p\u2009<\u20090.01), with the exception of pair b36f\u2013b46f. The attenuation values between b30f and b46f were significantly different at a high concentration in calcified plaques, whereas for the same pair of filters the attenuation values of the lumen and the surrounding were not significantly different. After clustering the paired attenuation values of the four structures for the convolution filter, a moderate-high correlation was generally found, with the exception of pairs b30f\u2013b46f in surrounding (r\u2009=\u20090.150), b30f\u2013b60f in lumen (r\u2009=\u20090.206) and surrounding (r\u2009=\u20090.311), b36f\u2013b60f in lumen (r\u2009=\u20090.132) and b46f-b60f (r\u2009=\u20090.201) in lumen in the saline solution. \nTable\u00a01Summary of the attenuation values measured in each structure in the two solutions (e.g. saline and contrast solutions) with increasing convolution filter\u00a0b30fb36fb46fb60fMeanSDSNRMeanSDSNRMeanSDSNRMeanSDSNRLumenSaline58.140.429.952.140.59.448.236.019.123.56.01.8Contrast329.193.7209.8312.685.544.0334.191.7169.7331.3138.317.8Noncalcified PlaqueSaline20.839.18.014.235.82.414.032.04.03.232.40.3Contrast74.766.639.368.263.39.266.366.525.948.560.02.4Calcified Plaque Saline740.5392.0327.0758.5360.3133.6785.7388.5294.91145.8517.487.8Contrast795.4333.8498.0838.7364.2116.0885.6382.6488.71194.8520.761.0SurroundingSaline127.12.7\u221254.7\u2212128.03.1\u221222.6\u2212128.72.7\u221247.6\u2212134.811.3\u221210.5Contrast\u2212128.22.6\u221284.5\u2212126.84.3\u221217.6\u2212128.52.4\u22127.2\u2212123.710.0\u22126.4\nWhereas the convolution filter did not significantly affect the attenuation values of the lumen and the surrounding, sharper filters decreased the attenuation of the plaque and, conversely, increased the attenuation of the calcification with both solutions (Fig.\u00a02).\nFig.\u00a02Mean attenuation of the four structures in the two solutions (e.g. saline and contrast) with increasing convolution filters. Whereas the convolution filter does not significantly affect the attenuation values of the lumen and the surrounding, sharper filters decrease the attenuation of the noncalcified plaque and, conversely, increase the attenuation of the calcified plaque with both solutions\nThe SNR values appear to have a common trend with increasing convolution filtering and both solutions. However, the best SNR was achieved with the b30f and b46f filters. Plaque values follow the lumen values, while the surrounding and calcification patterns are similar (Fig.\u00a03).\nFig.\u00a03The SNR values appear to have a common trend with increasing convolution filtering and both solutions (e. g. saline and contrast). However, the best SNR is achieved with b30 and b46 filters. Noncalcified plaque values follow the lumen values, while the surrounding and the calcified plaque patterns are similar\nThe CNR values were calculated between structures with increasing convolution filters and different solutions (Table\u00a02). Improved CNRs were achieved with the b30f and b46f filters. The lumen-noncalcified plaque CNR was particularly improved with the b30f and b46f filters using a high solution of contrast media. The noncalcified plaque-surrounding CNR was improved with the b30f and b46f filters in contrast solution. Calcified plaque-noncalcified plaque CNR increased with the contrast solution with the b30f and b46f filters, while it decreased using the b36f and b60f filters. The b46f filter was found to be ideal for calcified plaque-lumen contrast since it improved the CNR at contrast solution. \nTable\u00a02Summary of CNR values calculated between structures with increasing convolution filters and different solutions\u00a0b30fb36fb46fb60fSalineLumen \u2013 noncalcified plaque21.87.115.11.5Non \u2013 calcified plaque-surrounding62.725.051.610.8Calcified plaque \u2013 noncalcified plaque318.9131.3290.887.4Calcified plaque \u2013 lumen297.1124.2275.785.9Contrast solutionLumen \u2013 noncalcified plaque170.634.8143.815.4Noncalcified plaque \u2013 surrounding123.726.997.18.8Calcified plaque \u2013 noncalcified plaque458.7106.7462.858.5Calcified plaque \u2013 lumen288.271.9319.043.1\nAfter clustering the paired attenuation values obtained for the four structures in order to test the statistical difference between saline and contrast solution for each convolution filter, values were all significantly different (p\u2009<\u20090.05) apart from surrounding with b46f and calcification with b60f. Pearson\u2019s test has shown good correlation only between the attenuation values of calcification (r\u2009=\u20090.738, r\u2009=\u20090.826, r=\u20090.759, r\u2009=\u20090.723 with b30f, b36f, b46f, b60f, respectively).\nDiscussion\nThe evaluation of the coronary artery wall is a data set that has become available since the introduction of coronary MSCT. Several studies have reported the ability of MSCT to detect coronary plaques and subsequently characterize their tissue composition (i.e. calcified, mixed, noncalcified) [14, 20]. In an in vivo study, Kopp et al. showed an excellent correspondence between ICUS criteria (soft, intermediate and calcified plaques) and MSCT attenuation values [14]. Schroeder et al. subsequently found a strong correlation between tissue density measurements within the plaque and the qualitative ICUS criteria. The CT attenuation results were 14\u2009\u00b1\u200926\u00a0HU for plaques classified by ICUS as soft (i.e. predominantly lipid) plaques, 91\u2009\u00b1\u200921\u00a0HU for plaques classified by ICUS as intermediate (i.e. predominantly fibrous) plaques and 419\u2009\u00b1\u2009194\u00a0HU for plaques classified by ICUS as calcified. These researchers reported no overlap in the mean attenuation among the three groups [15]. Leber et al. showed that lesion echogenity correlated well with MSCT attenuation measurements in coronary plaque, with MSCT correctly classifying 78% of sections containing hypoechoic plaque areas (soft plaques), 78% of sections containing hyperechoic plaque areas (fibrous plaques) and 95% of sections containing calcified plaque tissue. The MSCT density measurements within coronary lesions revealed significantly different values for hypoechoic, hyperechoic, and calcified plaques [17]. The close correlation between ICUS and MSCT in terms of the detection of calcified and noncalcified coronary plaques was also shown by Achenbach et al., although this group found a sensitivity of only 53% for exclusively noncalcified plaque [16].\nBecker et al. compared the atherosclerotic lesions of 11 human cadaver heart specimens detected by MSCT with the histopathological macroscopic findings according to American Heart Association criteria. They concluded that MSCT is a promising tool for the characterization of atherosclerotic coronary plaques [22].\nSuch information is of great value since the risk of acute coronary syndromes caused by plaque disruption and thrombosis depends on plaque composition (i.e. noncalcified, predominantly lipid plaques with positive remodeling) rather than stenosis severity [18\u201320]. Reliable and reproducible non-invasive methods for the assessment of plaque constitution could be important in risk stratification of patients with CAD.\nNevertheless, several aspects (i.e. the impact of lumen attenuation, convolution filters, body mass index of the patient, CNR of the images and coronary calcification) of the methodology need to be better addressed in order to validate the accuracy of attenuation values measured within the coronary plaques using MSCT.\nIt is controversial if the absolute HU values are valid measurements for determining the plaque composition and identifying the corresponding tissue. A phantom study revealed that intracoronary attenuation can significantly affect the measured plaque attenuation so that accurate plaque tissue differentiation may be influenced [23]. Furthermore, Cademartiri et al. recently reported that the intravascular attenuation modifies significantly the attenuation of the coronary atherosclerotic plaques in an ex vivo coronary specimen [24]. Therefore, the characterization of the plaque on the basis of absolute attenuation values should be reported with caution, and the intraluminal attenuation should also be reported. The calibration of tissue attenuation values with contrast in-lumen values may improve accurate tissue component identification.\nNew possibilities for coronary artery plaque imaging may be offered by dual-source CT, which provides robust diagnostic image quality independent of the heart rate. Potential applications of dual-energy CT include tissue characterization and Ca quantification of atherosclerotic plaques [25, 26].\nIn our experimental study we addressed the issue of the variability of coronary plaque attenuation by varying the convolution filters (Fig.\u00a04).\nFig.\u00a04Example of plaque with increasing filtering using a saline and contrast solution\nThe direct back projection of CT attenuation profiles results in unsharp images. To prevent unsharp and approximate images, each projection has to be convolved with a predetermined mathematical function, the convolution kernel; this kernel represents a filtering procedure that is able to enhance or reduce object boundaries. Convolution influences image characteristics by the choice and design of the kernel (i.e. smooth, sharp). A smooth filter reduces spatial resolution as well as image noise; a sharp filter increases spatial resolution as well as image noise. The noise is caused by the fluctuations in the number of X-ray quanta registered by the detector and is affected by convolution and dose. The SNR evaluates the ratio between the intensity of the signal and its statistical fluctuation. Suzuki et al. have demonstrated that the accuracy of a vascular diameter measurement with an automated software is affected by the choice of convolution filtering [27]. Seifarth et al. recently compared the effect of different convolution filtering on the visualization of coronary artery stents. Using the edge-enhancing b46f, they observed a significant improvement in lumen assessment compared to the conventional b30f [28].\nOur study shows how the use of different convolution filters significantly affects the measured coronary plaque attenuation (Fig.\u00a03). The use of sharper convolution filters produces an increase in the mean attenuation value of the calcified component of plaque. Increasingly sharper filtering produces a reduction in the mean attenuation values of the noncalcified plaque component. On the basis of our results, it is still challenging to rely on absolute HU values since the protocol used in coronary MSCT has not been standardized. Furthermore, absolute plaque attenuation values determined in the various studies are not comparable. It is reasonable to use a proper convolution filtering with improved SNR and CNR to better focus on plaque components. The b36f intermediate filter obtained a slight decrease of noncalcified plaque attenuation and an increase of calcified plaque attenuation. However, improved SNRs and CNRs were also achieved by the b30f and b46f filters. The b36f underestimated the noncalcified plaque attenuation and overestimated the calcified plaque attenuation in the likely attempt to weigh both components.\nThere are several limitations to our study. The first one is inherent to the lack of an evaluation of intra- and inter-observer variability. A second limitation is related to the fact that the coronary specimens were collected from elderly patients with a high prevalence of calcification. The blooming artifacts of calcification may impair coronary noncalcified plaque visualization and characterization based on HU values [29]. Furthermore, we did not investigate the influence of mAs and voltage (kV) on the blooming effect. Nevertheless, it has been reported that the voltage setting employed (i.e.120\u00a0kV) might be reasonably chosen during in vivo studies [29]. In vivo studies should also better address the issue of body mass, which affects coronary plaque measurements [30]. Moreover, according to data mined from the literature, it is reasonable to expect that plaque size (especially in calcified plaques) and lumen area would be influenced by the use of different convolution filters due to variable partial volume effect. Edge-enhancing sharper reconstruction filters are reported to increase the visible in-stent lumen diameter [28, 31].\nThe pathological correlation has not been provided since the aim of our study was not to compare pathology with MSCT, but to assess the influence of convolution filtering on plaque attenuation measurement.\nIn conclusion, the use of different convolution filtering significantly modified plaque attenuation values. Therefore, convolution filtering should be reported when attenuation values are shown in order to provide a standardization of the methodology. Sharper convolution kernels increased the attenuation of the calcium within the coronary plaques and reduced the attenuation of soft plaque tissues. Improved SNR and CNR were achieved by using the b30f and b46f filters. A smooth filter (e.g. b30f) may be used for clinical routine evaluation, while a medium filter (e.g. b46f) may be considered the best choice for the assessment of highly calcified or stented vessels. The use of a proper filtering according to plaque type could give a more reliable assessment of plaque attenuation values in terms of HU.","keyphrases":["coronary plaque","multislice computed tomography","coronary angiography","convolutions kernels"],"prmu":["P","P","P","P"]}
{"id":"Anal_Bioanal_Chem-4-1-2287204","title":"Development, standardization and refinement of procedures for evaluating effects of endocrine active compounds on development and sexual differentiation of Xenopus laevis\n","text":"Xenopus laevis has been introduced as a model to study effects of endocrine-active compounds (EAC) on development and sexual differentiation. However, variable and inconsistent data have raised questions about the reliability of the test methods applied. The current study was conducted in two laboratories to develop, refine, and standardize procedures and protocols. Larvae were exposed in flow-through systems to 17\u03b2-estradiol (E2), at concentrations from 0.2 to 6.0 \u03bcg E2 L\u22121 in Experiment 1A, and 0.015 to 2.0 \u03bcg E2 L\u22121 in Experiment 1B. In both studies survival (92%, 99%) and percentage of animals that completed metamorphosis (97%, 99%) indicated reproducible biological performance. Furthermore, minor variations in husbandry led to significant differences in snout-to-vent length, weight, and gonad size. In Experiment 1A, almost complete feminization occurred in all E2 treatment groups whereas a concentration response was observed in Experiment 1B resulting in an EC50 of 0.12 \u03bcg E2 L\u22121. The final verified protocol is suitable for determining effects of EAC on development and sexual differentiation in X. laevis.\nIntroduction\nDuring the last ten years, the number of chemicals evaluated for endocrine-active effects has increased. Potential exposure of wildlife populations, including fish, amphibians, birds, and mammals, to environmental estrogens has attracted much attention due to the feasible impact of these substances on survival and population sustainability [1\u20136]. Laboratory studies using a range of wildlife species have demonstrated that physiological processes including development and reproduction, are sensitive to environmental estrogens [7, 8]. Due to the diversity of wildlife, certain species have been used as representatives of the various taxa that are considered in eco-toxicological hazard assessment. One example of a well established standardized animal model is the frog embryo teratogenesis assay (FETAX) [9] using Xenopus laevis embryos to determine impacts of chemicals on early development. This species has been investigated focusing on larval stages for studying the hormonal regulation of metamorphosis, development, sexual differentiation, and the impact of endocrine active compounds (EAC) [10\u201314]. However, the results in the literature for basic biological parameters such as mortality, growth, development, and sexual differentiation vary considerably among laboratories [15\u201317] making interpretation difficult. For example, it has been reported that the herbicide atrazine feminizes and demasculinizes amphibians [18\u201321] but other investigators have not been able to replicate these findings [22, 23].\nThe published data on potential effects of atrazine on gonadal development in laboratory studies and reproductive effects in wildlife populations were evaluated by a United States Environmental Protection Agency (EPA) Scientific Advisory Panel (SAP) in 2003. The SAP identified a number of factors that adversely affected the quality and comparability of data in the open literature on atrazine. These factors included variability in study design, differences in animal husbandry conditions (e.g., feeding rates), excess biomass in treatment chambers, and the effects of these factors on water quality. Other factors that contributed to difficulty in interpreting the results were variability in the response to the positive control substance and the negative control, as well as differences in the use of gross and microscopic pathology terminology, limited statistical power and lack of compliance with Good Laboratory Practice standards. In consequence, the SAP published a White Paper [24] that highlighted inconsistencies and variability among studies and by so doing provided guidance for the development of a standardized study design to evaluate potential effects of EAC on development and sexual differentiation of X. laevis.\nThe purpose of this study was to develop, standardize, and refine a protocol meeting specifications of the 2003 SAP that could be used to assess estrogenic effects on development and sexual differentiation in X. laevis. A team of scientists representing endocrinology, toxicology, chemistry, pathology, and statistics was involved in the creation of the study design, selection of endpoints, and choice of data analysis tools. The team concluded that it was necessary to conduct two independent studies consecutively in two different locations to develop, refine and validate the study design by assessing effects of E2 on gonadal development of X. laevis exposed from NF (Nieuwkoop and Faber) stages 46\u201348 through stage 66 in a flow-through exposure system. Experiment 1A was conducted at Wildlife International Ltd, MD, USA (WLI) in order to develop and standardize conditions for rearing and handling X. laevis larvae, operation of a flow-through system and maintaining water quality and environmental parameters. A concentration range for E2 of 0.2, 1.5 and 6.0\u00a0\u03bcg L\u22121 was chosen to calculate the effective concentration needed to achieve a 50% effect (EC50) for gonadal feminization and to evaluate abnormal development of gonads. In addition, this preliminary study was used to establish the terminology for classifying gross and histological gonadal abnormalities, to cross-train laboratory personnel, and to develop the appropriate statistical analysis methodology. The procedures developed in Experiment 1A were refined and, implemented in Experiment 1B conducted at the Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Germany (IGB).\nMaterial and methods\nTest organism\nTadpoles of X. laevis were obtained from the commercial supplier, Xenopus I, (Dexter, MI, USA). These larvae (approx. 1200 for each study) originated from ten pairs of adult breeders for each study. Xenopus I reared the eggs until day post fertilization (dpf) 3 and then shipped them to each laboratory.\nRearing\nUpon arrival on dpf 4, tadpoles were acclimated to water and temperature conditions (21\u2009\u00b1\u20092\u00a0\u00b0C) over a six-hour time period. During the acclimation period larvae were maintained in two 40-L glass aquaria containing 30 L well water (ca. 600 larvae\/tank, Experiment 1A) and three 40-L glass aquaria containing 30 L dilution water (ca. 400 larvae\/tank, Experiment 1B) derived from the flow-through system. At the initiation of treatment, twenty-five larvae were randomly assigned to each glass aquarium (n\u2009=\u2009200 larvae per treatment group) so that the biological loading rate was always less than 1g L\u22121 day\u22121. In Experiment 1A exposures were initiated at dpf 6 NF stage 45\u201346 and in Experiment 1B at dpf 8 when tadpoles had reached NF stage 47\u201348 as identified by visible hind limb buds [25]. Exposures continued until completion of metamorphosis or dpf 82, whichever came first. Throughout the acclimation period, water temperature was recorded twice daily whereas dissolved oxygen (DO) concentration and pH were measured daily. Experiment 1B was conducted in compliance with the local animal protection committee (G0048\/05).\nFeeding\nAnimals were fed Sera Micron (Lot No: 9698; Sera, Heinsberg, Germany), a commercially available food which was reported by the manufacturer to consist of 50.2% protein, 8.1% fat, 4.2% fibre, and 11.0% ash. A sample of Sera Micron was analysed by Lancaster Laboratories (Lancaster, PA, USA) for potential contaminants (metals and pesticides), and all tested contaminants were below their limits of quantification for analysis at that facility. A mixture of diet and dilution water was tested for estrogenicity and androgenicity using YES (yeast estrogen screen) [26] and YAS (yeast androgen screen) assays [27]. Based on these analyses no estrogenic or androgenic activity was detected.\nDuring acclimation of larvae in Experiment 1A, 1 g Sera Micron was added to each tank daily. This amount of food was found to be excessive. In experiment 1B, after temperature acclimation, larvae were transferred to the acclimation tanks where they promptly attached to the glass aquaria walls. Therefore, no food was added on dpf 4. The following day some of the tadpoles were free swimming and 150\u00a0mg Sera Micron was added that evening by rinsing through a sieve. The amount of feed was chosen based on the intake of food by the larvae, and care was taken to ensure that feeding did not adversely affect water quality. From dpf 6 to 8 the free swimming tadpoles were fed 200\u00a0mg Sera Micron in each tank twice a day. Throughout the remaining course of both studies, tadpoles were fed a suspension of Sera Micron (77\u00a0mg L\u22121) three times daily. The total quantity of food added per tank each day increased from 300\u00a0mg\/tank on dpf 8 to 900\u00a0mg\/tank on dpf 33. The quantity of food added to each tank was adjusted periodically in Experiment 1A and on a daily basis in Experiment 1B to account for mortality and removal of tadpoles as they completed metamorphosis.\nStudy design\nBecause an almost completely feminized phenotype was observed in Experiment 1A in all treatment groups, the nominal E2 concentrations in Experiment 1B were adjusted to 0.015, 0.2, and 1.5\u00a0\u03bcg E2 L\u22121, respectively. Mid and high concentrations were selected to correspond to experiment 1A and allow comparison of results from the two laboratories.\nReplicates\nEach study consisted of at least one negative control group plus three E2-treated groups, each comprising eight replicated glass aquaria\/treatment groups. Eight glass aquaria were arranged in two clusters of four tanks each. The clusters of negative control tanks and E2 treated tanks were randomly distributed within the environmental chamber in Experiment 1A and within water baths in Experiment 1B. Each cluster was supplied water via the same water flow meter and mixing chamber. Experiment 1A included 16 control tanks that were initially divided into two 8-tank groups designated as negative control and reference control, respectively. The animals from all treatment groups were handled in a blinded fashion so that the biologists and histologists involved in the experiments did not know the treatment applied to frogs being observed. An additional non-blind reference control group was included to provide reference information on rates of spontaneous incidence of gonadal abnormalities for blind histological assessment of gonads from the E2 treatment groups. Histological analysis was conducted on all frogs of Experiment 1A. At the end of Experiment 1A, it was discovered that slightly elevated temperature (1\u00a0\u00b0C) in one cluster of four negative control tanks had resulted in accelerated growth of the frogs in the tanks. Therefore, measurements of weight, length, gonad image area, and age at metamorphosis from those tanks were dropped from analysis, and the four remaining negative control tanks were combined with the reference control group to provide these measurements.\nFlow-through system\nBoth studies were conducted using continuous flow-through systems operating at a flow rate of at least 50\u00a0L dilution water per tank per day. This was equivalent to a water exchange rate of approximately seven tank volumes per day. Test tanks consisted of 9-L glass aquaria (30\u2009\u00d7\u200920\u2009\u00d7\u200914.5\u00a0cm) containing 7 L water. Dilution water for Experiment 1A was obtained from a well of approximately 40\u00a0m depth located on the WLI site. In Experiment 1B the dilution water used was non-chlorinated municipal water. Dilution water samples from Experiment 1A were analysed for possible metal or pesticides by Lancaster Laboratories and water samples of Experiment 1B by Environmental Chemistry and Pharmanalytics (RCC Ltd., Itingen, Switzerland).\nThe flow-through systems in the two laboratories were essentially identical. Prior to use, the water was passed through several filters (5, 1, 0.5, and 0.45\u00a0\u03bcm) to remove particulate material, a UV-sterilizer, and a final particle filter (0.45\u00a0\u03bcm). After being filtered and temperature conditioned, the dilution water was divided into 16 streams. Each stream passed through a flow-control assembly, consisting of a rotameter flow gauge and a needle valve, to a mixing chamber. The flow from each mixing chamber was then split to supply a cluster of four tanks. Each tank received 140\u00a0mL min\u22121 dilution water (\u00b15%).\nPrimary E2 solutions\nThe primary E2 stock solutions were continuously delivered to each mixing chamber (0.140\u00a0mL min\u22121), where it was vigorously mixed with the dilution water. Primary E2 stock solutions were metered into the mixing chambers by an eight-channel peristaltic pump (MasterflexL\/S, Model 752455, Cole\u2013Parmer, IL, USA). The flow-through system (temperature, water, filter pressure, flow-meter), and peristaltic pumps were calibrated four weeks prior to study initiation, and inspected visually twice daily throughout the study.\nE2 solutions\nOnce every seven days a primary E2 stock solution was prepared in amber bottles by dissolving 6\u00a0mg E2 (Lot No: 11121AB, CAS: 50\u201328\u20132, purity 99.9%, Sigma Aldrich; WLI, Allentown, PA, USA; IGB: Taufkirchen, Germany) in alkaline solution (0.01 mol L\u22121 NaOH) applying moderate heating and constant mixing of the solution overnight similar to procedures employed by the Duluth Laboratory of the USEPA, Office of Research and Development (personal communication by J. Tietge). In Experiment 1A, the working stock solutions were prepared at nominal concentrations of 200, 1500, and 6000\u00a0\u03bcg E2 L\u22121, and in Experiment 1B at 15, 200, and 1500\u00a0\u03bcg E2 L\u22121. The quantity of NaOH in E2 test solutions delivered to each tank was negligible, and therefore no NaOH was added to the negative control.\nAnalysis of E2 concentration\nSampling\nIn Experiment 1A, water samples were collected from each tank in all treatment groups to confirm the operation of the diluter two days prior to study initiation. In Experiment 1B, E2 concentrations were monitored twice weekly for a two-week period prior to study initiation. After study initiation, water sampling was carried out twice weekly from alternating tanks in two tanks per cluster until study termination. Throughout the studies water samples were collected routinely two days after the freshly prepared E2 solution was connected to the flow-through system and a second time in the same week. At mid-depth from each test tank 190\u00a0mL water was collected using a glass pipette and 10\u00a0mL methanol (MeOH) was added. Of this solution 20\u00a0mL was decanted into a 20\u00a0mL amber scintillation vial for E2-enzyme-linked immunosorbent assay (ELISA) analysis. The remaining volume of 180\u00a0mL was stored as backup at 4\u00a0\u00b0C until E2 analysis was completed. Water samples from Experiment 1B were shipped to WLI for E2 analysis. The stability of E2 was confirmed by shipping samples of E2 (0.1\u00a0\u03bcg E2 L\u22121) that were prepared and analysed in parallel with the water samples.\nSample analysis\nRapid analysis was performed using ELISA kits (Ecologiena, Abraxis LLC, Warminster, PA, USA) to ensure the absence of the test substance in the negative control tanks and to verify the proper E2 concentration in each E2 tank. The ELISA method was based upon the E2 kit manual provided by the manufacturer. The method detection range of this assay was 0.05\u20130.5\u00a0\u03bcg E2 L\u22121. Parallel analyses of water samples for E2 concentration were performed by direct aqueous injection using an Agilent Series 1100 High-Performance Liquid Chromatograph coupled with a MDS Sciex API 3000 tandem mass spectrometer (LC\u2013MS\u2013MS) (Applied Biosystems, Foster City, CA, USA) and MDS Sciex API heated nebulizer ion source (Applied Biosystems) operated in the multi-reaction mode (MRM). Chromatographic separations were achieved using a Keystone Betasil C-18 column (50\u00a0mm\u2009\u00d7\u20092\u00a0mm, 3\u00a0\u03bcm particle size) (Thermo Hypersil, Madison, WI, USA). The method limit of quantitation (LOQ) for HPLC\u2013MS\u2013MS estradiol analyses was defined as 0.005\u00a0\u03bcg L\u22121.\nEnvironmental conditions\nTemperature\nIn Experiment 1A, all glass aquaria were situated in a walk-in environmental chamber (21\u2009\u00b1\u20092\u00a0\u00b0C). In Experiment 1B, glass aquaria were placed in temperature controlled water baths (22\u2009\u00b1\u20091\u00a0\u00b0C). In both experiments water temperatures were continuously recorded, and temperatures in all glass aquaria were measured weekly using a liquid-in-glass thermometer.\nLight\nThe target light intensity at tank level was 100\u2013500 lux and was measured weekly using a Sper Scientific (AZ, USA) light meter. A photoperiod of 16\u00a0h of light and 8\u00a0h of dark was used in Experiment 1A, and was adjusted to a 12\u00a0h light, 12\u00a0h dark cycle in Experiment 1B. A 30-min low light (around 30% intensity of full light) transition period was installed in both laboratories.\nWater quality\nWater quality parameters were assessed according to ASTM (2003) [28] and US EPA guidelines. Water hardness, pH, conductivity, and the concentrations of nitrate, and ammonia were measured weekly in one tank from each cluster throughout the experiments. Hardness and alkalinity were measured by titration based on procedures described in \u201cStandard Methods for the Examination of Water and Wastewater\u201d [29]. Nitrate concentrations were analysed with a Hach DR\/700 colorimeter (Hach Company, Colorado, USA) and specific conductance was detected using a YSI conductivity meter (WLI: Yellow Springs Instruments Model 33, IGB: 3200, Yellow Springs, OH, USA). pH and ammonia concentrations were measured with an Orion pH\/ISE Meter 720Aplus (Thermo Electron, USA). DO concentrations were measured in alternate tanks per treatment three times a week with an Orion Model 850plus dissolved oxygen meter (Thermo Electron). Commencing at dpf 43 in Experiment 1A, and at dpf 36 in Experiment 1B, gentle external aeration was applied by a glass pipette in each test tank to ensure maintenance of an adequate DO concentration.\nMaintaining water quality\nThe tanks required daily cleaning to minimize microbial growth and to maintain adequate water quality. Bio-film that accumulated on the inner walls and bottom of the tanks was scraped off daily, and detritus was siphoned from the tanks. Daily cleaning proved inadequate to maintain appropriate water quality throughout the entire exposure period. Hence, in Experiment 1A all tanks were replaced with clean tanks weekly and in Experiment 1B twice throughout the experiment when siphoning did not lead to satisfactory results.\nIn-life assessment of tadpoles\nTadpoles were monitored for changes in general health, swimming behaviour and morphological appearance daily. The numbers of dead or moribund tadpoles and tadpoles completing metamorphosis were recorded daily for each tank.\nSnout-to-vent length and weight\nEach tadpole that had either completed metamorphosis by NF stage 66, or had failed to achieve metamorphosis by dpf 82, was removed from the test tank and euthanized by immersion in MS 222 (tricaine methanesulfonate) (WIL: Sigma Aldrich, Allentown, PA, USA; IGB: Sigma\u2013Aldrich, Taufkirchen, Germany) (2 g L\u22121 buffered to pH 7.5 with NaHCO3). Immediately thereafter, the snout-to-vent length was measured to the nearest millimetre. Each frog was blotted dry and then weighed to the nearest milligram. A unique identifier was assigned to each frog that included: study number, in-life laboratory ID, colour code, replicate, and animal number.\nGross pathology\nThe pleuroperitoneal cavity was opened to expose the visceral organs. The liver, stomach, and intestine were examined using a dissecting stereo microscope (WIL: Olympus SZ61-TR, Olympus America, Melville, NY, USA; IGB: Olympus, SZX7, Olympus, Hamburg, Germany), and unusual variations in size, colour, and\/or abnormal structure were recorded. The gastrointestinal tract was excised to expose the ventral surfaces of the kidney and gonads, which were similarly assessed for gross abnormalities. Tadpoles that did not complete metamorphosis at study termination (Experiment 1A: 25, Experiment 1B: 7) were staged according to NF and, the gonad morphology was assessed (if possible), and the carcasses were archived in fixative. Data derived from such animals were excluded from further analysis.\nAssessment of gonad morphology\nUsing a dissecting stereo microscope, gross evaluations of gonad morphology were performed on all animals that reached NF stage 66. To enhance visualization of the gonads, which presented as thin, pale-tan strips of tissue on the ventromedial margin of the kidney, several drops of Bouin\u2019s solution (WIL: Sigma Aldrich, Allentown, PA, USA; IGB: Sigma\u2013Aldrich, Taufkirchen, Germany) were applied to partially fix these organs. Based on gross observation, the gonads of each animal were identified as testes, ovaries, or malformed. Examples of observed malformations included mixed sex, intersex, pearling and segmental aplasia. Mixed sex was defined as the co-occurrence of both ovarian and testicular tissue in a single gonad. Intersex was assessed as ovarian and testicular tissue in the same individual as separate gonads (left\/right). The term pearling was characterized by the presence of multiple, prominent segmental enlargements and\/or attenuations along the length of one or both gonads, whereas gonads that presented as nodular islands of testicular or ovarian tissue, with either intervening membranous connections or no connections at all, were classified as segmental aplasia. Gross findings for the gonads of each frog were recorded separately for the left and right gonads. The gonadal findings for each frog were verified independently by a second biologist. If the two biologists differed in their interpretations, the disparate opinions were discussed and a consensus finding was recorded.\nPrior to the initiation of the first E2 experiment a list of standardized terms for changes of potential morphological gonadal observations was developed. Subsequent to the completion of both experiments the applied terminology was refined in order to better characterize and group different types of gonad changes observed and which in turn could be analysed. For example, the subcategories narrow, slightly narrow, truncated, slightly truncated, and margin entire were integrated into the main category hypoplasia. This term describes all gonads which appear smaller than those characterised as \u201ctypical\/normal\u201d. Since the term \u201ctypical\/normal\u201d is rather general, further sub-categories allow the biological variability of gonadal morphology to be described. However, during the investigations it was revealed that too few observations in each sub-category ruled out reasonable statistical analyses. Therefore, a refined list of qualitative descriptive terminology for gonadal features was created as presented in Table\u00a01.\nTable\u00a01Glossary of terms used to describe features observed during gross morphological examination of gonads in X. laevis NF stage 66\u00a0FeatureDefinition1.Adhesion (other tissue)Gonads joined to other abdominal tissue2.Aplasia (agenesis)Complete lack of gonad development3.Segmental AplasiaGonad is longitudinally discontinuous\u00a03-A. Tissue separationOne or more areas along the length of the gonad is poorly developed, attenuated with an essentially complete lack of gonadal tissue\u00a03-B. Extraneous gonadal tissueA small disjunct cluster of gonad-like tissue4.BifurcationDivision of the gonad oriented longitudinally, along the cranial\u2013caudal axis\u00a04-A. Protuberance(s)Small projection(s) of the gonad along the margins\u00a04-B. SymmetricA \u201cY\u201d -shaped single gonad with relatively equal division5.Angular deformityOne or more bends in the gonad to an excessive degree\u00a05-A. Gonad foldedThe gonad is bent over or doubled up so that one part lies on another6.DisplacedThe gonad, or a section of it, is not located in the typical location\u00a06-A. Displaced laterallyThe gonad, or a section of it, is not located in the typical location. Instead it is located further away from the frog\u2019s midline\u00a06-B. Displaced mediallyThe gonad, or a section of it, is not located in the typical location. Instead it is located closer to or crosses over the frog\u2019s midline\u00a06-C. Displaced craniallyThe gonad is not located in the typical location. Instead it is located more towards the anterior\u00a06-D. Displaced caudallyThe gonad is not located in the typical location. Instead it is located more towards the posterior7.FusedLeft and right gonads joined to varying degrees at one or more locations along cranial\u2013caudal axis8.HypertrophySize of the gonad is larger than typical\u00a08-A. WideGonad appears broad. Lateral width is large relative to the length\u00a08-B. ElongatedGonad appears long (cranial\u2013caudal). Length is large relative to the length of the kidneys and the torso of the frog\u00a08-C. ThickGonad relatively increased in the distance between the dorsal and ventral surfaces\u00a08-D. EnlargedA generalized increase in the size of the gonad along all axes9.Segmental hypertrophyA gonad where one or more areas are excessively large\u00a09-A. Enlargement (mass)A section of the gonad substantially bigger in width and\/or thickness than the remaining tissue\u00a09-B. PearlingPronounced multiple segmental enlargement of the gonad\u00a09-C. Partly thickA section or sections of the gonad is relatively increased in the distance between the dorsal and ventral surfaces10.HypoplasiaSize of the gonad is decreased\u00a010-A. NarrowGonad appears strap-like. Lateral width is markedly reduced relative to the length\u00a010-B. Slightly narrowLateral width is somewhat reduced relative to the length\u00a010-C. TruncatedGonad appears short (cranial\u2013caudal). Length is small relative to the length of the kidneys and the torso of the frog\u00a010-D. Slightly truncatedGonad appears somewhat short (rostal\u2013caudal). Length is somewhat reduced relative to the length of the kidneys and the torso of the frog\u00a010-E. ThinGonad relatively reduced in distance between the dorsal and ventral surfaces\u00a010-F. Margin entireAll of the ovary or indeterminate gonad has a smooth edge; no scalloping or lobes11.Segmental hypoplasiaA gonad where one or more areas are excessively reduced, attenuated, or poorly developed but not separated\u00a011-A. Partly narrowA section or sections of the gonad has a small lateral width relative to the length and the width of the other sections\u00a011-B. Partly thinA section or sections of the gonad is relatively reduced in distance between the dorsal and ventral surfaces\u00a011-C. Margin slightly sinuateGonad has only shallow waved or scalloped edge (typically associated with a narrow or partly narrow ovary)\u00a011-D. Margin partially entireA section or sections of the gonad has a smooth edge (typically associated with a narrow or partly narrow ovary)\u00a011-E. PearlingPronounced multiple segmental attenuation or narrowing of the gonad12.IntersexOvarian and testicular tissue present as separate structures (i.e. not contained in the same gonad) (left\/right)13.Mixed sexOvarian and testicular tissue present in the same gonad14.TranslucentGonad appears not so dense, light able to pass through diffusely\u00a014-A. Tissue slightly translucentTo a small extent the gonad appears not so dense, a small amount of light able to pass through diffusely15.Segmental translucenceA section or sections of the gonad appears not so dense, light able to pass through diffusely16.Melanophores decreasedApparently fewer pigment-containing cells than typically seen. Applies only to females, mixed sex frogs, and frogs of undetermined sex\u00a016-A. ApigmentationNo melanophores in the internal tissue. Applies only to females, mixed sex frogs, and frogs of undetermined sex17.Internal melanophoresPigment-containing cells within the gonadal tissue. Applies only to males, mixed sex frogs, and frogs of undetermined sex18.OtherAny finding not listed. Describe as needed. For any \u201cother\u201d finding that study personnel consider not incidental, notify the Study Director and other laboratories as soon as possible via email including a description and photograph\nPhotography\nAfter gross gonad inspection was complete, each gonad was photographed in situ using a digital camera (WIL: Olympus DP12\u20132, Olympus America, Melville, NY, USA; IGB: Olympus DT5, Hamburg, Germany) attached to the stereo microscope. Each digitalized image included a millimetre measurement scale placed adjacent to the gonads to permit gonad size to be determined from the photograph. Following photography, each frog was placed in a labelled individual container of 30\u00a0mL of Bouin\u2019s solution for approximately 48\u00a0h. At the end of the fixation period, each carcass was rinsed several times in 70% ethanol and was placed in 30\u00a0mL 10% neutral buffered formalin (WIL: VWR, Westchester, PA, USA; IGB: Histofix, Roth, Karlsruhe, Germany). A complete gonadal histological evaluation was performed on each frog of experiment 1A and results are presented in a separate paper (Wolf et al., in preparation). In some cases the growth and histological findings differ and a better understanding of results is gained by considering both types of observation.\nGonad measurement\nThe biological variability of morphological features of the gonads and the subjective nature of the assessment prompted us to develop a more quantitative metric of gonad size. Thus, individual measurements of gonad image area were derived from photographs of the gonads. Gonad image areas were obtained using image-processing software (Image Pro Plus, Version 5.1, Media Cybernetics, Silver Spring, MD, USA) that calculated the combined area of the left and right gonad whose outline had been manually traced around the digital image. The program was calibrated by the millimetre measurement scale prior to measurement for the gonad of each tadpole.\nStatistical analysis\nFor both experiments, observations were collected for each animal. However, consistent with the experimental design, the tank is considered the primary experimental unit. When tank differences are present, statistical analysis should accommodate this experimental structure directly using nested \u201crandom effects\u201d analyses or indirectly by analysing tank means or tank percentages. Neither approach is completely satisfactory for all endpoints. Nested models have specific distributional requirements and, in the case of incidence data, become unstable when frequencies are at or near zero. Analyses of tank means can also be problematic for these experiments. Estradiol-induced feminization causes the number of males per tank to be smaller for treated groups than for the control group. This, in turn, results in greater variation between estradiol-treated tanks than between control tanks. Most parametric and non-parametric statistical procedures, however, require equal variability between tanks within every group. When tank differences are small or absent, however, the individual data from all tanks within the same treatment group can be pooled and analysed more simply using the frog as the basic experimental unit.\nFor Experiments 1A and 1B, endpoints were tested for differences between tanks using a test of homogeneity. A chi-square homogeneity test was used for frequency endpoints (males, females, mixed sex) and a Kruskal\u2013Wallis test was used for measurement endpoints (snout-to-vent length, body weight, age at completion of metamorphosis, gonad image area). This homogeneity test was calculated for each experimental group and the p-values combined using Tippett\u2019s minimum-p method. Tank differences were found for several frequency endpoints in Experiment 1A. In addition, there was evidence that the presence of a humidifier caused a temperature-induced impact on measurement endpoints for two clusters of tanks. In contrast, Experiment 1B did not reveal any statistically significant (p < 0.05) tank effects. Consequently, all subsequent statistical analyses for Experiment 1B can be conducted on individual animals with all tanks pooled. Thus, analyses of Experiment 1A are quite complex and varied while Experiment 1B results can be analysed rather simply. In order to summarize the results consistently to allow comparisons of both experiments, only animal-level means and standard deviations are presented. The means and standard deviations given in this report for both experiments were calculated from individual animals using Excel Version 2000. For Experiment 1B these simple statistics are identical with those obtained from the statistical analyses described below. For Experiment 1A these means (and standard deviations) should be considered only approximate. They may differ slightly from means obtained using more sophisticated analyses that incorporate a tank structure.\nIn Experiment 1B, the estradiol-treated groups were compared with controls in a step-wise manner that preserved power and protected against excessive false positives. An overall test of group differences and a test for trend with dose were both conducted. If either of these two overall tests showed statistical significance at the 5% level (i.e., p < 0.05) then comparisons of each dose group to the control were made. For frequency endpoints a chi-square homogeneity test and a Cochran\u2013Armitage test for trend were used for the overall homogeneity and trend tests, respectively. Comparisons of dose groups to control were conducted using Fisher\u2019s exact test. For measurement data essentially all endpoints showed statistically significant deviations from normality (p < 0.01, Shapiro\u2013Wilk test). Consequently, a Kruskal\u2013Wallis nonparametric test was used for group homogeneity and a Jonckheere\u2013Terpstra test was used for trend. Comparisons of dose groups to control were conducted using the nonparametric Wilcoxon\u2013Mann\u2013Whitney U-test. All statistical analyses of Experiment 1B were calculated using Release 9.1 of the SAS statistical package (2004).\nResults\nIt is emphasized that Experiment 1A was performed first in order to standardize conditions for rearing X. laevis and for operation of the flow-through system. Improvements of these procedures were implemented in Experiment 1B resulting in optimised growth and development conditions for X. laevis tadpoles. Those parameters that were successfully achieved during Experiment 1B are summarised in Table\u00a02 and may be used as guidance for similar experiments. The following sections focus on results obtained in Experiment 1B, presenting results from Experiment 1A, as needed, to allow comparison of the two experiments.\nTable\u00a02Final study design specifications. Brief summary of the most important parameters for exposing X. laevis tadpoles in a flow-through exposure systemParameterCharacteristicWater-quality parameters\u00a0\u00a0Supply water: filtered, contaminant freeFiltered and UV sterilized; tested for chemical contaminants\u00a0\u00a0Dissolved oxygen\u226560% of saturation\u00a0\u00a0pH7.9 to 8.3\u00a0\u00a0Ammonia0.0 to 0.35\u00a0mg L\u22121\u00a0\u00a0Nitrate0.04 to 2.29\u00a0mg L\u22121\u00a0\u00a0Hardness90 to 130\u00a0mg CaCO3 L\u22121\u00a0\u00a0Alkalinity133 to 172\u00a0mg CaCO3 L\u22121\u00a0\u00a0Specific conductance726 to 817\u00a0\u03bcg S cm\u22121\u00a0\u00a0Supplemental aerationInitiated not later than dpf 36Animal husbandry\u00a0\u00a0Animal supplyXenopus, Dexter, MI, USA\u00a0\u00a0Shipped from SupplierDay 3 post fertilization (dpf)\u00a0\u00a0Acclimationdpf 4\u20137\u00a0\u00a0Treatment perioddpf 8\u201382\u00a0\u00a0Feed: Sera MicronSera Micron (contaminants and estrogenic potential evaluated) Contents: 50% protein, 8% fat; 4% fibre; 11% ash.\u00a0\u00a0Feeding rate\u2014acclimationdpf 4\u20135; per 300 larvae: 200\u00a0mg; if free swimming, dpf 6\u20138: 200\u00a0mg twice daily (based on the intake of food by the larvae)\u00a0\u00a0Feeding rate\u2014NF stages 46\u20136677\u00a0mg L\u22121: fed three times daily, 300\u00a0mg\/tank from dpf 8, increasing to 900\u00a0mg\/tank at dpf 33, then adjusted during experiment as animals were removed (dead, froglets)\u00a0\u00a0Exposure chamber cleaningDaily\u00a0\u00a0Exposure chamber replacementAs needed\u00a0\u00a0Water-quality parametersHardness, ammonia, nitrate, pH, specific conductivity twice a week; DO three times a week\u00a0\u00a0Temperature22\u2009\u00b1\u20091\u00b0C (verified twice daily, measured once a week)\u00a0\u00a0Light intensity100\u2013500 lux (measured once weekly, at level of surface of water)\u00a0\u00a0Light:dark cycle12:12; 30-min transition (30% of full light)Experimental design\u00a0\u00a0Performed in compliance with Good Laboratory Practice StandardYes\u00a0\u00a0Colour coded (blind) treatmentYes\u00a0\u00a0Randomization of tadpolesTadpoles distributed in rotation one or two at a time to transport vessels to contain 25, then vessels were randomly assigned to test tanks\u00a0\u00a0Randomization of tank clustersCompletely randomized placement; one cluster consists of four tanks\u00a0\u00a0Negative controlWater (eight replicates, two clusters)\u00a0\u00a0Treatment 17\u03b2-estradiol concentrations0.015, 0.2, 1.5, 6.0\u00a0\u03bcg E2 L\u22121; eight replicates; two clusters for each group\u00a0\u00a0Tank volume9 L (water volume 7 L), tanks covered\u00a0\u00a0Tank flow rate50 L day\u22121\u00a0\u00a0Number of animals200 larvae per treatment group\u00a0\u00a0Animal load densityLess than 1 g L\u22121 water; 25 larvae\/tank\u00a0\u00a0HPLC\u2013MS\u2013MS verificationdpf 8 all tanks; dpf 16\u201381 weekly (alternating tanks)\nEnvironmental conditions\nIn both experiments experimental conditions generally met the criteria listed in Table\u00a02. In Experiment 1B the mean and SD for hardness and alkalinity were 103\u2009\u00b1\u20095.8\u00a0mg and 162\u2009\u00b1\u20096.8\u00a0mg CaCO3 L\u22121, respectively. Specific conductance was 794\u2009\u00b1\u200918.0\u00a0\u03bcS cm\u22121, pH 8.1\u2009\u00b1\u20090.09, nitrate 1.6\u2009\u00b1\u20090.5\u00a0mg L\u22121 and ammonia 0.08\u2009\u00b1\u20090.09\u00a0mg L\u22121. The measurements from negative control tanks were not significantly different from those from the E2 tanks. Technical problems in Experiment 1B prevented accurate determination of DO concentration, however, based on subsequent studies using the same procedures, DO was consistently above 60% saturation.\nE2 exposure verification\nIn each experiment, E2 was not present in any of the negative control tanks. Measured concentrations of E2 stock solutions ranged from 96.6 to 106% in Experiment 1A and between 90.5 and 108% in Experiment 1B. In Experiment 1A, the mean concentration of E2 for three treatment groups began to decline after experiment initiation and fell below 80% of nominal E2 concentration by dpf 29. Between exposure dpf 43 and 50 mean concentration of E2 decreased to minima of 0.4, 43, and 38% of nominal concentrations of E2 in the 0.2, 1.5, and 6.0\u00a0\u03bcg E2 L\u22121 treatment groups, respectively. After dpf 50, concentrations of E2 began to increase in all treatment groups, and exceeded 60% of nominal concentration on dpf 64 (Fig.\u00a01a). In Experiment 1B, concentrations of E2 declined after study initiation and dropped below 60% of nominal E2 concentration by dpf 23. On dpf 37 concentrations of E2 fell to 47, 46, and 50% in the 0.015, 0.2, and 1.5\u00a0\u03bcg E2 L\u22121 treatment groups, respectively. The concentration of E2 increased to 60% of nominal concentration on dpf 65 (except the 1.5\u00a0\u03bcg E2 L\u22121 with 44.4%) (Fig.\u00a01b).\nFig.\u00a01Comparison of measured mean E2 concentrations in water samples of E2 treatments in the flow-through system. Mean values represent the concentrations of E2 of weekly sampling intervals from dpf 6 or 8 through the last sampling interval on termination of the experiments on dpf 82. a, E2 concentrations of Experiment 1A; b, E2 concentrations of Experiment 1B. The limit of quantification was 0.00500\u00a0\u03bcg E2 L\u22121, calculated as the product of the concentration of the lowest calibration standard (0.100\u00a0\u03bcg E2 L\u22121) and the dilution factor of the matrix blank samples (0.0500\u00a0\u03bcg E2 L\u22121)\nIn-life assessment of tadpoles\nAppearance and behaviour\nNo effects of E2 treatment on tadpoles condition, behaviour, or external appearance was observed in any of the treatment groups at any time throughout the entire course of Experiment 1A or Experiment 1B.\nSurvival\nExcellent survival was achieved in both experiments in controls as well as in E2 treated groups. The total survival was 92% in Experiment 1A and 99% in Experiment B. In Experiment 1A, survival in the combined negative control, 0.2, 1.5, and 6.0\u00a0\u03bcg E2 L\u22121 treatment groups was 90.8, 94.5, 95.0, and 88.0%, respectively. In Experiment 1B survival rates were 100% in the negative control and 97.5, 97.5, and 99.5% in the 0.015, 0.2, and 1.5\u00a0\u03bcg E2 L\u22121 exposure groups, respectively.\nSnout-to-vent length and weight\nIn Experiment 1A, for females the average length values were, 22.2\u2009\u00b1\u20090.1, 22.1\u2009\u00b1\u20090.1, and 22.8\u2009\u00b1\u20090.1\u00a0mm in the 0.2, 1.5, and 6.0\u00a0\u03bcg E2 L\u22121 treatment group compared with 22.0\u2009\u00b1\u20090.2\u00a0mm in the combined control group. Males treated with E2 had an average snout-to-vent length of 21.8\u2009\u00b1\u20090.1, 22.1\u2009\u00b1\u20090.6, and 23.0\u2009\u00b1\u20090.1\u00a0mm in the combined control, 1.5, and 6.0\u00a0\u03bcg E2 L\u22121 treatment group, respectively. The increase in the mean weight of males was more pronounced than the increase in the mean weight of females (Fig.\u00a02).\nFig.\u00a02Body weight (means\u2009\u00b1\u2009SD) of a, females, and b, males, after exposure to E2 from dpf 6 or 8 in a flow-through system until completion of metamorphosis or day 82 post-fertilization. 0.015\u00a0\u03bcg E2 L\u22121 was not used in Experiment 1A and 6.0\u00a0\u03bcg E2 L\u22121 was not used in Experiment 1B. Neg Ctrl, negative control group\nIn Experiment 1B no statistically significant differences between the snout-to-vent length for females and males were observed. The snout-to-vent length for females and males in the negative control group was 17.2\u2009\u00b1\u20091.5 and 17.0\u2009\u00b1\u20091.6\u00a0mm, respectively. For females and males treated with 1.5\u00a0\u03bcg E2 L\u22121 snout-to-vent length was 17.5\u2009\u00b1\u20091.5 and 16.3\u2009\u00b1\u20091.3\u00a0mm, respectively. No statistically significant treatment-related effect was observed for body weight either in females or in males. At completion of metamorphosis, frogs from Experiment 1A weighed more than twice as much as frogs from Experiment 1B, regardless of the gender (Fig.\u00a02).\nTime course of metamorphosis\nOn average, the process of metamorphosis for tadpoles took longer in Experiment 1A than in Experiment 1B. In Experiment 1A, the age at which the first frog in the experiment completed metamorphosis was delayed by 6 days (dpf 44) in each E2 treatment group compared with animals in Experiment 1B (dpf 38) (Fig.\u00a03). In Experiment 1A exposure of females to 0.2, 1.5, and 6.0\u00a0\u03bcg E2 L\u22121 resulted in a slight delay of metamorphosis. In Experiment 1B, the time course of metamorphosis for females in the 0.015\u00a0\u03bcg E2 L\u22121 treatment group was similar to that for females in the negative control group but exposure to 0.2 and 1.5\u00a0\u03bcg E2 L\u22121 caused a delay of completion of metamorphosis (Fig.\u00a03a,b). A comparable picture for the cumulative time course of metamorphosis of males is shown in Fig.\u00a03c,d. In Experiment 1A, males treated with 0.2 and 1.5\u00a0\u03bcg E2 L\u22121 had a delay in completing metamorphosis of approximately ten days compared with males in the negative control group. In Experiment 1B metamorphosis of male frogs in the 0.2\u00a0\u03bcg E2 L\u22121 treatment group was slightly postponed whereas for males treated with 1.5\u00a0\u03bcg E2 L\u22121 completion of metamorphosis was delayed by three days. The lowest E2 concentration used obviously did not affect the mean time to complete metamorphosis.\nFig.\u00a03Cumulative portion of X. laevis frogs completing metamorphosis after exposure to different concentrations of E2 from day 6 or 8 post-fertilization in a flow-through system until completion of metamorphosis or day 82 post-fertilization. The number of individuals per treatment group (n) is given in the legend. a, portion of females in Experiment 1A; b, portion of females in Experiment 1B; c, portion of males in Experiment 1A (just one male was observed in the 6.0\u00a0\u03bcg E2 L\u22121 group); d, portion of males in Experiment 1B. Neg Ctrl, negative control group\nExposure to E2 had no effect upon the percentage of surviving tadpoles that completed metamorphosis on dpf 82 (97.4% in Experiment 1A, 99.1% in Experiment 1B). By dpf 82, 25 (out of 920) and 7 (out of 789) individuals failed to complete metamorphosis in Experiment 1A and Experiment 1B, respectively, and no treatment-related effect was observed. In Experiment 1B, 1, 2, and 4 individuals failed to complete metamorphosis by dpf 82 in the negative control, 0.2, and, 1.5\u00a0\u03bcg E2 L\u22121 treatment groups, respectively.\nMean age at completion of metamorphosis\nThe differences in the time course of metamorphosis described in the previous section are reflected in the mean ages of frogs at completion of metamorphosis in each of the treatment groups. In Experiment 1A, the mean ages at completion of metamorphosis of females treated with 0.2, 1.5, and 6.0\u00a0\u03bcg E2 L\u22121 were dpf 64.2\u2009\u00b1\u20097.6, 62.9\u2009\u00b1\u20097.9, and 65.1\u2009\u00b1\u20099.2, respectively, and substantially greater than the mean age in the negative control group (dpf 61.4\u2009\u00b1\u20098.2) (Fig.\u00a04). In contrast, in Experiment 1B females treated with the different E2 concentrations showed a dose response with a significant increase noted in the 0.2\u00a0\u03bcg E2 L\u22121 group (dpf 50.7\u2009\u00b1\u20097.5) and a greater effect (dpf 51.5\u2009\u00b1\u20096.6) in the 1.5\u00a0\u03bcg E2 L\u22121 treatment group compared with dpf 47.8\u2009\u00b1\u20095.6 for females in the negative control group. For males in Experiment 1A, the mean age at completion of metamorphosis was increased from dpf 60.6\u2009\u00b1\u20099.4 in the negative control to dpf 70.9\u2009\u00b1\u20099.3 and dpf 72.3\u2009\u00b1\u20099.0 in the 0.2 and 1.5\u00a0\u03bcg E2 L\u22121 treatment groups, respectively. In Experiment 1B, the mean age at completion of metamorphosis of males was statistically significant greater in the 0.2\u00a0\u03bcg E2 L\u22121 (dpf 51.2\u2009\u00b1\u20096.6) and 1.5\u00a0\u03bcg E2 L\u22121 (dpf 58.0\u2009\u00b1\u20098.3) treatment groups than in the negative control group (dpf 48.5\u2009\u00b1\u20096.8) (Fig.\u00a04).\nFig.\u00a04Mean age at completion of metamorphosis (mean\u2009\u00b1\u2009SD) by treatment group for female, a, and male, b, frogs at completion of metamorphosis or on day 82 post-fertilization. 0.015\u00a0\u03bcg E2 L\u22121 was not used in Experiment 1A and 6.0\u00a0\u03bcg E2 L\u22121 was not used in Experiment 1B. Only one male frog was observed in the 6.0\u00a0\u03bcg E2 L\u22121 group in Experiment 1A. Neg Ctrl, negative control group. For Experiment 1B significant differences are marked by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001; two-sided Wilcoxon\u2013Mann\u2013Whitney test of equality with negative control group). A Kruskal\u2013Wallis test of overall group differences was significant (females p < 0.0001, males p\u2009=\u20090.0009) and a Jonckheere\u2013Terpstra test of trend was significant (females p < 0.0001, males p\u2009=\u20090.0062)\nAppearance and examination of non-gonadal organs\nNo externally visible morphologic abnormalities were observed in each treatment group in either experiment. Likewise, inspection of the liver, stomach, intestine, kidney for tumours, lesions or any other remarkable features had no effect.\nDetermination of sex, percent female, male, and mixed sex by gross examination\nThe percentages of male, female, and mixed-sexed animals were calculated based on frogs that completed metamorphosis (NF stage 66) among all animals. In Experiment 1A, the percentage of female phenotype was increased in all E2 treated groups. In the 0.2, 1.5, and 6.0\u00a0\u03bcg E2 L\u22121 treatment group 88.4, 95.8, and 98.3% of frogs showed female phenotype. In Experiment 1B, the incidence of female phenotype in the E2 treated groups displayed a statistically significant distinct dose-responsive increase with a high percentage of females observed for the 0.2 (70.5%) and 1.5\u00a0\u03bcg E2 L\u22121 (92.3%) treatment groups, respectively (Fig.\u00a05). The E2 concentration that results in an increase of female phenotype to 75% is defined as the effective concentration (EC50). Based on the feminization effect observed in Experiment 1B the EC50 was calculated to be 0.12\u00a0\u03bcg E2.\nFig.\u00a05Individual bars represent frequencies of sex of X. laevis frogs among all animals according to treatment group based on gross morphological evaluation; the total number of frogs (n) that completed metamorphosis, is given on top of the bars. Tadpoles were exposed to E2, in a flow-through system, from day 6 or 8 post-fertilization through completion of metamorphosis or day 82 post-fertilization. The EC50 was calculated from the results of Experiment 1B and was 0.12\u00a0\u03bcg E2. The numbers of frogs in the negative control in Experiment 1A was higher for evaluation of gender because the animals which were excluded from calculation of weight and length were included in calculation of the portion of males, females, and mixed sex. Neg Ctrl, negative control group. For Experiment 1B asterisks denote significant differences (**p < 0.01, ***p < 0.001; two-sided Fisher\u2019s exact test for comparison to negative control). Both an overall exact chi-square test of equality of all experimental groups and a Cochran\u2013Armitage trend test were statistically significant (p < 0.05) in every case\nIn Experiment 1A, the percentage of mixed sex individuals were 6.9% (13\/189), 2.1% (4\/191) and 1.1% (2\/176) in the 0.2, 1.5, and 6.0\u00a0\u03bcg E2 L\u22121 treatment groups, respectively. In Experiment 1B, the incidences of mixed-sex animals were statistically significant with 6.2% (12\/193) and 4.1% (8\/195) in the 0.2 and 1.5\u00a0\u03bcg E2 L\u22121 treatment groups, respectively (Fig.\u00a05). In both experiments the percentage of frogs displaying mixed sex gonads was higher in E2-treated groups than in the negative control groups. When results from the two experiments are compared, the percentage of mixed sex animals was seen to be similar in equal E2 concentrations.\nSegmental aplasia was detected in Experiment 1A in one of nine male individuals out of the 0.2\u00a0\u03bcg E2 L\u22121 treatment group. In Experiment 1B, segmental aplasia was observed in four of 101 (4.0%), nine of 92 (9.8%), eighteen of 45 (40.0%) and four of seven (57.1%) male frogs in the 0.015, 0.2, and 1.5\u00a0\u03bcg L\u22121 treatment groups, respectively. Thus, the frequency of segmental aplasia in the 0.2 and 1.5\u00a0\u03bcg E2 L\u22121 groups was significantly elevated. Pearling was observed in two of nine and one of five male frogs the 0.2, and 1.5\u00a0\u03bcg E2 L\u22121 groups, respectively, in Experiment 1A. In Experiment 1B, the occurrence of pearling was determined in two frogs out of 45 within the 0.2\u00a0\u03bcg E2 L\u22121 treatment group. Intersex was not found in any of the 1593 Xenopus evaluated for gonad morphology in both studies. Figure 6a,b demonstrate ordinary developed ovarian and testes tissue that was assumed to be \u201cnormal gonad morphology\u201d for gross morphological evaluation. Figure 6c to e illustrate gonads showing mixed sex, pearling, and segmental aplasia and clarify the terminology which was applied in these experiments to describe morphological changes in gonadal appearance of X. laevis due to E2 treatment. To allow the variability of the morphological appearance of gonads to be described, an extensive terminology to include the biological variability was developed and used in the two experiments (Table\u00a01). In Experiment 1A there were apparent differences in the occurrence of ovaries that were truncated, narrow entire margin, or had enlarged masses. However, no differences in morphological characteristics were observed between E2-treated and control frogs in Experiment 1B.\nFig.\u00a06a. Ovary of X. laevis revealing segmented, lobular structure and clearly visible internal melanocytes in the tissue; the length of the ovary is slightly shorter than the length of the kidney. b. In contrast, a testis is shorter relative to the length of the kidney (half of the kidney), straight shaped, and does not contain internal melanocytes. In general testis tissue appears much denser than ovary tissue. c. Mixed sex gonads were defined as the co-occurrence of both ovarian and testicular tissue in a single gonad. The ovary tissue in the presented gonad shows a reduced lobular structure, just a few melanophores, whereas the testis tissue appears bulbous. Arrows indicate the different gonad structures, f, ovary-like tissue, and m, male like tissue. d. Pearling (p) was characterized by the presence of multiple, prominent segmental enlargements and\/or attenuations along the length of one or both gonads. e. Segmental aplasia (segA) was classified as nodular islands of testicular or ovarian tissue, with either intervening membranous connections or no connections. The kidney and the attached gonad tissue were fixed with Bouin\u2019s solution\nGonadal measurement\nBecause of the subjective nature of gross gonad morphological assessment, quantitative measurements of gonad area were performed on digital images.\nIn Experiment 1A, mean gonad areas of female and male frogs were approximately twice as large as in animals of the corresponding gender in Experiment 1B. The mean gonad image areas for females in Experiment 1A were 2.20\u2009\u00b1\u20090.82\u00a0mm2, 2.21\u2009\u00b1\u20090.89\u00a0mm2, 2.09\u2009\u00b1\u20090.92\u00a0mm2, and 2.23\u2009\u00b1\u20090.92\u00a0mm2 in the combined negative control, 0.2, 1.5, and 6.0\u00a0\u03bcg E2 L\u22121 treatment groups, respectively. In Experiment 1B, for females treated with 0.015, 0.2, and 1.5\u00a0\u03bcg E2 L\u22121 mean gonad image areas were 1.21\u2009\u00b1\u20090.41\u00a0mm2, 1.27\u2009\u00b1\u20090.44\u00a0mm2, and 1.32\u2009\u00b1\u20090.41\u00a0mm2, respectively, compared with 1.23\u2009\u00b1\u20090.42\u00a0mm2 in the negative control group. Mean gonad areas in females treated with 1.5\u00a0\u03bcg E2 L\u22121 were significantly increased (p < 0.05). In Experiment 1A, the measured mean gonad areas of males in the combined control, 0.2 and 1.5 E2 L\u22121 were 1.28\u2009\u00b1\u20090.2\u00a0mm2, 1.39\u2009\u00b1\u20090.2\u00a0mm2, and 1.70\u2009\u00b1\u20090.3\u00a0mm2, respectively. In Experiment 1B, males treated with 0.015, 0.2, and 1.5\u00a0\u03bcg E2 L\u22121 had a slight increase of mean gonad areas 0.82\u2009\u00b1\u20090.20\u00a0mm2, 0.85\u2009\u00b1\u20090.19\u00a0mm2, and 0.89\u2009\u00b1\u20090.14\u00a0mm2, respectively, compared with the negative control (0.77\u2009\u00b1\u20090.22\u00a0mm2). A Kruskal\u2013Wallis test of overall group differences was marginally non-significant (p\u2009=\u20090.079) but a Jonckheere\u2013Terpstra test of trend was significant (p\u2009=\u20090.0092).\nDiscussion\nGonadal development in anuran amphibians has been proposed as a valuable model to evaluate the physiological consequences of exposure to EAC with (anti)estrogenic and\/or (anti)androgenic activity [30]. It has long been known that treatment of tadpoles with gonadal steroids can disrupt normal gonadal development in several anuran species and might even lead to complete sex reversal [31, 32]. However, studies on the potential effects of EAC on anuran gonadal development have produced variable and, in some cases, inconsistent results. From analysis of published studies it appears that some of the variability between EAC studies may be because of differences between the experimental conditions employed. One example to illustrate this situation is the outcome of three different exposure studies assessing the effects of bisphenol A (BPA) on gonadal development in X. laevis [15\u201317]. By using a semi-static exposure system, feminization of tadpoles due to BPA treatment was reported by Kloas [15] and Levy [17]. Attempts to replicate these findings in a flow-through exposure system by Pickford [16] were not successful. A more recent example includes inconsistent results observed in different studies on the putative effects of atrazine on survival, growth, metamorphosis, and sexual development of X. laevis [20, 21, 23]. The failure to reproduce the original findings leaves regulatory authorities with many uncertainties and in consequence, may prevent sound assessment of potential risks associated with the use of the chemicals of concern.\nContradictory results from several studies of atrazine, and the criticism associated with each of these studies, made it impossible for the US EPA to perform an unambiguous evaluation of the actual impact of atrazine on development and sexual differentiation in X. laevis. The US EPA therefore requested further independent studies to be conducted using a standardized test protocol including validated positive control treatments and an optimized study design [33]. To address uncertainties resulting from inconsistencies of previously reported data, the current multi-site study was undertaken in order to develop and refine an optimized testing protocol for the use in developmental studies with X. laevis tadpoles. The current paper presents the results of the two independent exposure experiments that were performed by two different laboratories. A first experiment (Experiment 1A) served as a pilot study to identify appropriate testing conditions for the development of a standardized testing protocol. Accordingly, the test protocol used in the follow-up experiment (Experiment 1B) included some modifications reflecting the experience gained in Experiment 1A. In combination, the current multi-site study aimed at a characterization of the general performance of the tadpoles using the optimized testing protocol and a detailed evaluation of concentration-dependent effects of E2 on tadpole growth, larval development and sexual differentiation.\nThe testing protocol under investigation specified the use of a flow-through exposure system to achieve E2 exposure of tadpoles. The main advantages of flow-through systems versus static renewal systems are the maintenance of constant testing conditions (e.g., water quality) and the continuous delivery of the test substance to the exposure vessels to achieve stable exposure of the test species to the test substance. In the two exposure experiments with E2, however, difficulties were encountered with regard to the maintenance of the target test concentration of E2 even under flow-through conditions. Although E2 concentrations in the stock solutions were within acceptable ranges in both exposure studies, and the quantity of E2 metered into the tanks was correct, actual E2 concentrations in the exposure tanks decreased to levels well below nominal test concentrations during the entire course of the experiments (Fig.\u00a01). These observations clearly indicate that in future studies care must be taken to maintain constant nominal concentrations of the test compound throughout exposure. It is recommended the flow rate of the stock solutions be adjusted on a daily basis according to the actual test substance concentration present in the tanks.\nComparison of the time-course of E2 concentration changes with estimates of the biomass present in E2 treatment tanks at different times during Experiments 1A and 1B suggests that E2 concentrations were inversely related to changes in total biomass. Aqueous E2 concentrations decreased concurrent with the increase in body size and body weight of tadpoles during premetamorphosis (NF stages up to 52), prometamorphosis (NF stages 53 to 57), and climax (58 to 66). Concentrations of E2 began to increase as tadpoles that completed metamorphosis were removed from the test tanks, and as remaining tadpoles began to lose weight during tail resorption. Figure 7 illustrates that E2 concentrations were inversely related to changes in total biomass of the tadpoles. Furthermore, it should be noted that the biomass was greater in Experiment 1A than in Experiment 1B and the decline in aqueous E2 concentration was more marked in Experiment 1A. These observations, combined with the relatively high octanol\u2013water partition coefficient of E2 (log Kow 3.94) suggest that additional measures (e.g., gradual increase of amounts of E2 delivered to the test tanks) are required to maintain a constant exposure to E2 in terms of actual aqueous E2 concentrations. In semi-static systems E2 disappears quickly, which suggests the animal may absorb the E2. Only few studies are available that have addressed this aspect. Results from Hayes and Licht [34] suggest that exogenous E2 is readily metabolized by X. laevis and other anuran species, and there was no evidence of bioaccumulation of E2 in anuran tadpoles. However, metabolic enzymes and the sites of E2 metabolism in anuran tadpoles are largely unknown. Possible candidate metabolites might include various E2 conjugates, but neither the test solutions nor the test animals have been examined analytically for the presence of such metabolites. To the best of our knowledge, there are also no data available regarding possible developmental profiles of E2-metabolising capacities that might explain the transient reduction of aqueous E2 concentrations in the present studies. It is, further, not known whether any of the putative metabolites still possess estrogenic or other hormonal activity. Finally, it is possible that factors other than changes in tadpole biomass may have contributed to the observed changes of E2 concentrations. For example, a small fraction of E2 may have been absorbed either in the biofilm or directly on the surface of the glass tanks. Another possibility could be that E2 partition from water to fat in the administered Sera Micron feed (~8% fat) occurred. Notably, daily food amounts administered to each tank were adjusted to total larvae biomass. Therefore, it is an interesting possibility that at least a portion of the E2 dose may reach biological targets in the larvae as a result of oral ingestion of the feed.\nFig.\u00a07Correlation of changes in E2 concentration and the biomass of growing tadpoles during the exposure period in Experiments 1A and 1B\nAn important objective underlying the development of a standardized testing protocol for exposure of X. laevis tadpoles was to maintain appropriate water quality throughout the exposure experiments. Thus special care was taken to standardize and refine water quality parameters to ensure optimal growth and development of X. laevis tadpoles. Except for a transient decline of DO concentration below desirable levels, all water-quality parameters were within acceptable limits. The decrease of DO concentrations through dpf 43 in Experiment 1A and inability to maintain the DO concentration above 60%, may have been due to insufficient cleaning. Based on this observation, the cleaning procedure was enhanced, and additional aeration was provided by gently bubbling air through glass pipettes in each tank.\nAn interesting finding in these two experiments was the effects on growth and development of X. laevis tadpoles that occurred due to slight differences in rearing procedures between Experiment 1A and Experiment 1B. Tadpoles in Experiment 1A required more time to complete metamorphosis and the metamorphic froglets (NF stage 66) were larger and heavier than those in Experiment 1B. Whereas the relationship between age and size is in accordance with established amphibian life-history models [25], the factors that are responsible for the inter-laboratory differences are not entirely clear in this case. In Experiment 1A, the photoperiod was 16 hours light:8 hours dark compared with 12 hours light:12 hours dark in Experiment 1B. The mean temperature was approximately 1\u00a0\u00b0C lower and more variable (\u00b11\u00a0\u00b0C) in Experiment 1A. The differences in photoperiod and water temperature are certainly prime candidates to explain the slightly slower development in Experiment 1A. On average, tadpoles in Experiment 1A consumed food 13 days longer than those in Experiment 1B. Subsequently, the longer food consumption of tadpoles in Experiment 1A led to greater weight compared with that of the froglets in Experiment 1B. Based on the results achieved in experiments 1A and 1B, and to consider the reported differences on growth and development of X. laevis in the literature, it is obviously important to standardize the test systems.\nResults from Experiment 1B indicated that most of the tadpoles (92%) from the negative control group completed metamorphosis within a relatively short time (18 days), whereas metamorphosis was delayed (26 days) in a few tadpoles only (7.5%; 15\/199). In contrast, time to metamorphosis in Experiment 1A, was much more variable, because 78.5% (216\/275) of the tadpoles completed metamorphosis within twenty-two days and 21.5% (59\/275) in thirteen days. Furthermore, tadpoles in Experiment 1A completed metamorphosis later than in Experiment 1B (Fig.\u00a08). Thus, the use of the improved testing protocol in Experiment 1B was associated with a reduction in the time needed for completion of metamorphosis, which resulted in much more homogenous development of the test organisms. The latter finding is particularly important given the fact that still only few data are available upon which one could build a set of performance criteria to assess the validity of exposure experiments with X. laevis tadpoles. Homogenous growth and development in the negative control group is certainly one important aspect that deserves special attention. Generally, it can be proposed that homogenous rates of tadpole growth and development will reflect appropriate culture and testing conditions. Furthermore, any reduction in the inter-individual variability of growth and development will enable more sensitive assessment of the possible effects of a test compound on the development of tadpoles. To this end, the robust demonstration of altered rates of development due to test compound exposure will also provide additional information for interpretation of the effects of the test compound on gonadal development (e.g., identification of systemic toxicity).\nFig.\u00a08Proportion of male and females frogs (n) of the negative control groups completing metamorphosis during the course of the studies, based on the total number of animals surviving in Experiments 1A and 1B\nIn contrast, it is still difficult to assess the validity of an exposure experiment based on the time required for completion of metamorphosis. According to Nieuwkoop and Faber [25] the average time to reach stage 66 is dpf 58. In Experiment 1B, the average time to stage 66 was still shorter, being dpf 47.8\u2009\u00b1\u20095.6 and 48.5\u2009\u00b1\u20096.8 for female and male frogs, respectively. This reduction of the time needed for tadpoles to complete metamorphosis is likely a result of the various efforts to optimize an array of biotic and abiotic factors known to affect tadpole growth and development.\nAn important element of experimental approaches used to assess the estrogenic activity of chemicals is the choice of an appropriate positive control treatment to confirm the validity of the experimental system. Therefore, a major focus of the current multi-site study was to establish a concentration\u2013response relationship for E2 treatment on sexual differentiation of X. laevis. The results from Experiment 1A were not appropriate to demonstrate this relationship because all E2 concentrations (0.2, 1.5, and 6.0\u00a0\u03bcg E2 L\u22121) employed in Experiment 1A caused a very high percentage of phenotypic females. Based on these observations, the E2 test concentrations were modified in Experiment 1B, and the use of E2 concentrations of 0.015, 0.2 and 1.5\u00a0\u03bcg E2 L\u22121 revealed a clear concentration-dependent increase in the number of phenotypic females. The data from Experiment 1B allowed the calculation of an EC50 of 0.12\u00a0\u03bcg E2 L\u22121 resulting in 75% gonadal female phenotype.\nLiterature information about E2 concentration-dependent effects on growth, development, and sexual differentiation in X. laevis is rare. In most studies single E2 concentrations ranging from 0.3 to 100\u00a0\u03bcg L\u22121 were used to study effects on gonadal development [15\u201317, 22, 31, 35, 36] and resulted in percentage of female phenotype between 60, and 100%. Hu et al. [14] applied 1, 10, and 100\u00a0\u03bcg E2 L\u22121 and reported an increase of female phenotype to 66, 99, and 100%, respectively. Rearing X. laevis larvae under optimized conditions in Experiment 1B demonstrated a distinctive sensitivity for gonadal differentiation. This is demonstrated by a clear concentration-responsive effect, and a distinct effective concentration of 0.2\u00a0\u03bcg E2 L\u22121, as shown by the frequency of female\/male individuals and the occurrence of substantial mixed sex and other abnormalities. Comparing the results of Experiments 1A and 1B regarding gonadal effects of 0.2 and 1.5\u00a0\u03bcg E2 L\u22121, a similar effect was observed, indicating high reproducibility of the testing protocol. Based on the endogenous E2 concentrations measured in adult X. laevis being approximately 28\u00a0ng mL\u22121 for females and 3\u00a0ng mL\u22121 for males, [37] and in froglets approximately 0.002\u00a0\u03bcg E2\/fresh body weight [38] the calculated EC50 appears to represent a physiological E2 concentration in X. laevis.\nAnother question of high interest is how estrogenic EAC might impact gonad gross morphology. While determination of phenotypic sex was a core endpoint in the assessment of the effects of E2 on sexual differentiation, a number of other gross abnormalities were also evaluated. To that end, a glossary of gonadal gross morphological terminology and criteria was included in the study protocol (Table\u00a01). The importance of this glossary cannot be understated, as a substantial degree of diagnostic variability, within and among studies, can be attributed to differences in terminology [39]. For example the word intersex is characterized differently by several authors and is often used interchangeably with the term mixed sex, whereas in the glossary, these terms have different meanings. Therefore, it is difficult to describe morphological changes triggered by EAC in a way that is clear and generally accepted. Furthermore, assessments of gonadal morphology involve subjective judgement, which make studies difficult to interpret and compare. In the current multi-site study the uncertainty arising from the subjective judgement that was necessarily involved in evaluation of the gross morphological assessment each gonad was reduced by having each finding verified by a second biologist. Additionally, digital photographs of each gonad were taken to allow later verification of assessments, and to allow measurement of gonadal image area using image-processing software. Measurement of gonad image area enables objective verification of some of the subjective size-related observations made during the gross assessments. Trend analysis revealed for both sexes in Experiment 1B an increase of mean gonadal area that coincides with an increased age at completion of metamorphosis.\nIn both experiments, gross morphological evaluation of the gonads revealed the presence of mixed sex, segmental aplasia, and pearling at 0.2\u00a0\u03bcg E2 L\u22121. It is noteworthy that intersex, characterized as ovarian and testicular tissue in the same individual as separate gonads (left\/right), was not found in any of the 1593 frogs evaluated in these two experiments. Interestingly, in both experiments the highest incidence of these testicular abnormalities was observed in tadpoles exposed to 0.2\u00a0\u03bcg E2 L\u22121. This was most likely due to the greater degree of complete sex reversal at the two higher E2 concentrations, as suggested by the significantly increased proportion of phenotypic females compared with phenotypic males in the 1.5 and 6.0\u00a0\u03bcg E2 L\u22121 treatment groups. Based on gross morphological evaluation no differences for the terms of main-categories such as hyper- or hypoplasia were observed compared with individuals in the negative control in Experiment 1B.\nIn conclusion, this multi-site study has developed and established an optimized study design and a standardized test protocol to determine optimal growth and development of X. laevis larvae. High survival rates, homogenous rates of development, relatively short times to completion of metamorphosis of control animals, and concentration-dependent effects of E2 treatment on gonadal development were achieved using appropriate testing conditions. Based upon these results, it is concluded that the protocol provides a basis for evaluation on potential effects of chronic EAC exposure on gonadal development in X. laevis using a flow-through system. Furthermore, it is our opinion that the protocol could also be successfully applied to investigate the effects of other types of EAC, such as (anti)thyroidal chemicals, on the development of X. laevis.","keyphrases":["development","sexual differentiation","xenopus laevis","17\u03b2-estradiol","flow-through exposure system","standardized protocol"],"prmu":["P","P","P","P","P","R"]}
{"id":"Diabetologia-3-1-1975734","title":"Long-term correction of diabetes in rats after lentiviral hepatic insulin gene therapy\n","text":"Aims\/hypothesis Type 1 diabetes results from the autoimmune destruction of pancreatic beta cells. Exogenous insulin therapy cannot achieve precise physiological control of blood glucose concentrations, and debilitating complications develop. Lentiviral vectors are promising tools for liver-directed gene therapy. However, to date, transduction rates in vivo remain low in hepatocytes, without the induction of cell cycling. We investigated long-term transgene expression in quiescent hepatocytes in vitro and determined whether the lentiviral delivery of furin-cleavable insulin to the liver could reverse diabetes in rats.\nIntroduction\nType 1 diabetes mellitus is caused by autoimmune destruction of pancreatic beta cells [1]. Treatment requires daily injections of insulin to normalise blood glucose levels. Tight glucose control delays, but does not prevent, the onset of complications, which increase morbidity and mortality. Transplantation therapy is limited by the scarcity of donors and chronic immunosuppressive regimens [2]. Physiological glucose control could be achieved by genetically engineering surrogate beta cells that are capable of synthesising, storing and secreting insulin in response to metabolic signals.\nOne approach to liver-directed gene therapy of diabetes is the transcriptional control of insulin expression without the induction of storage [3, 4], which is nonresponsive to minute-to-minute fluctuations in blood glucose. Another strategy is the delivery of beta cell transcription factors to the liver to induce pancreatic transdifferentiation. Studies have employed transiently expressing adenoviral vectors to deliver pancreatic and duodenal homeobox gene 1 (Pdx1) and neurogenic differentiation gene 1 (Neurod1)\/betacellulin gene (Btc) [5\u20138] to streptozotocin (STZ)-induced diabetic mice and isolated human liver cells, which have resulted in liver-to-pancreas transdifferentiation and reversal of diabetes. However, conversion of significant portions of the liver to \u2018true\u2019 pancreatic tissue may lead to an increased risk of autoimmune attack and development of hepatitis, due to the exocrine differentiating capacity of PDX1 [7].\nMost studies on the delivery of genes to the liver have employed adenoviral vectors [5\u20138] due to their ability to efficiently transduce non-dividing cells. However, the therapeutic gene remains episomal, often with consequent short-term gene expression and detrimental host immune responses [9]. Short-term euglycaemia has been achieved following delivery of insulin via an adeno-associated vector, with dose-dependent efficacy [10]. Lentiviral vectors (LV) are capable of stably integrating genes into the chromosomes of dividing and non-dividing cells [9, 11\u201313]. However, their ability to transduce quiescent hepatocytes in vivo remains low, as efficient transduction generally requires induction of cell cycling [14], which can be achieved by partial hepatectomy [15] or delivery of hepatotoxins [16, 17].\nWe have developed an improved method for high efficiency transduction of rat hepatocytes in vitro and describe a modified surgical technique for efficient delivery of LV to rat livers resulting in efficient transduction, without the requirement for cell division. We have used this technique to deliver lentivector-encoded furin-cleavable insulin (INS-FUR) [18], which enables cells to cleave proinsulin to mature insulin, in the livers of diabetic rats. This has resulted in permanent reversal of diabetes, insulin storage and restoration of glucose tolerance.\nMaterials and methods\nVector production INS-FUR cDNA [18] (proins.IfurIIfur.B10D; INS-FUR; gift from Genentech, San Francisco, CA, USA) was cloned into the multi-cloning site of the LV HIV\/MSCV (HMD) [19] (a gift from J. Choi, Department of Pathology and Laboratory Medicine, University of Pennsylvania, PA, USA) at the EcoRI site to produce the HMD\/INS-FUR construct. This LV vector has an HIV\/murine stem cell virus hybrid long terminal repeat as the promoter and an internal ribosomal entry site, allowing bicistronic expression of the genes encoding insulin and enhanced green fluorescent protein (EGFP) simultaneously. The vectors were produced by calcium phosphate precipitation cotransfection of three plasmids (10\u00a0\u03bcg HMD\/INS-FUR or 10\u00a0\u03bcg pHR\u2019CMVGFP [20] expressing EGFP alone, plus 20\u00a0\u03bcg pCMV\u0394R8.2 and 10\u00a0\u03bcg of the heterologous VSV-G envelope construct pCVSV-G; all other vector constructs were gifts from I. Verma, Salk Institute for Biological Sciences, CA, USA) into 293T cells [9]. Conditioned medium was collected 48\u00a0h after transfection, filtered and pelleted (50,000\u00a0g). Virus titre was determined by transducing 293T cells (5\u2009\u00d7\u2009105) with serially diluted vector stocks and quantifying numbers of EGFP-positive cells by flow cytometry as described [21]. Viral replication-competency was also assessed.\nTransduction of hepatocytes in vitro Maintenance and experimental manipulation of male Wistar rats (250\u2013300\u00a0g; Gore Hill Research Laboratories, Sydney, NSW, Australia) were performed in accordance with the NIH principles of laboratory care and regulations of the Australian Research Council and were approved by the University of Technology Sydney. Hepatocytes were isolated using a two-step EDTA\/collagenase digestion. The portal vein (PV) and the abdominal aorta were cannulated and the liver was flushed in situ with warmed (40\u00b0C) Hanks\u2019 buffered salt solution without Ca2+ and Mg2+ (Sigma-Aldrich, Castle Hill, NSW, Australia), plus 1\u00a0mmol\/l EDTA for 5\u20137\u00a0min. This was followed by perfusion with 0.05% collagenase (C5138; Sigma-Aldrich) in Hanks\u2019 buffered salt solution with Ca2+ and Mg2+ (Sigma-Aldrich) for 3\u20135\u00a0min. The hepatocytes were collected, filtered through sterile 250 and 100\u00a0\u03bcm mesh and washed three times in DMEM plus 10% FCS. Preparations containing greater than 85% viable cells were plated onto Matrigel (gift from C. Liddle, Westmead Hospital, Sydney, NSW, Australia) and cultured in DMEM supplemented with NaHCO3 (44\u00a0mmol\/l), insulin (100\u00a0mU\/l), glutamic acid (2.5\u00a0mmol\/l), proline (1\u00a0mmol\/l), ascorbic acid (56\u00a0mg\/l), penicillin (100\u00a0mg\/ml), nicotinamide (10\u00a0mmol\/l), epidermal growth factor (20\u00a0ng\/ml), selenium salts (0.1\u00a0\u03bcg\/ml) and hydrocortisone (1\u00a0nmol\/l). After 5\u00a0days, 1.5% DMSO together with CuSO4 (2.5\u00a0\u03bcg\/l), FeSO4.7H2O (834\u00a0\u03bcg\/l) and ZnSO4 (1.47\u00a0mg\/l) was also added to the medium. Although Matrigel inhibits cell division, hepatocytes were also irradiated (caesium-137 source; total doses of 0, 10 or 20\u00a0Gy) 24\u00a0h after seeding and DNA synthesis was assessed by [3H]thymidine incorporation [22]. The positive control was hepatocytes plated on rat-tail collagen 1 (Collaborative Biomedical Products, Bedford, MA, USA).At 24\u00a0h after cell attachment, hepatocytes (5\u00d7105 per well) were transduced with pHR\u2019CMVGFP LV (2.5\u2009\u00d7\u2009107 transduction units [TDU]) for 16\u00a0h in the presence of 8\u00a0\u03bcg\/ml polybrene. Transduction efficiency was assessed at 2, 7, 14, 21 and 28\u00a0days by fluorescence microscopy and flow cytometry. EGFP-expressing cells were also stained for the liver-specific marker glucose-6-phosphatase.\nTransduction of hepatocytes in vivo Rats were divided into four groups of 16 animals. For Group 1, the right adrenal vein was ligated and successively the infrahepatic vena cava (IVC), hepatic artery, PV and suprahepatic vena cava were clamped before pHR\u2019CMVGFP or HMD LV (5\u2009\u00d7\u2009108 TDU) was infused through the PV. During this procedure, blood supply to the liver was stopped for 5\u00a0min and then recommenced for 2\u00a0min. This procedure of intervallic infusion in full flow occlusion (FFO) was repeated three times. Animals in Group 2 were treated similarly; except 0.9% saline was infused. A 70% partial hepatectomy was performed on animals in Group 3 by resecting the left lateral and median liver lobes. The right adrenal vein was ligated and LV was infused 24\u00a0h later by the procedure detailed for Group 1. A fourth group of animals was treated the same as Group 3, except 0.9% saline was infused. Livers were removed at 1, 2, 4 and 8\u00a0weeks. Transduction efficiency was assessed by flow cytometric quantification of EGFP-positive cells.To assess cell proliferation, 5-bromo-2\u2032-deoxyuridine (100\u00a0mg\/kg; Sigma) was injected intraperitoneally at 18, 28 and 48\u00a0h after surgery (partial hepatectomy or sham-operated) and into untreated animals (three animals per time-point). After 2\u00a0h, livers were excised and processed by conventional histological techniques. Incorporation of 5-bromo-2\u2032-deoxyuridine was determined by immunohistochemistry.Diabetes was induced by an intraperitoneal injection of STZ (75\u00a0mg\/kg body weight; Sapphire Science, Crows Nest, NSW, Australia). Animals received HMD\/INS-FUR or empty HMD vector administered by FFO without partial hepatectomy. After treatment, animals were monitored for body weight and blood glucose. Animals were killed at 60 or 500\u00a0days and body tissues were sampled. To determine the proportion of EGFP-expressing cells that were hepatocytes, liver cells were isolated and stained with anti-cytokeratin 18 (CK18) antibody (Chemicon, Temecula, CA, USA) and R-phycoerythrin-conjugated secondary antibody (Chemicon). Numbers of insulin-positive cells were determined using an anti-insulin antibody (Sigma-Aldrich) and aminomethylcoumarin-conjugated secondary antibody followed by flow cytometric analysis.\nMicroscopic analyses Livers and pancreases were collected 60 and 500\u00a0days after vector infusion. Islet hormones were identified by peroxidase immunohistochemistry using diaminobenzidine as substrate. Primary antibodies against insulin (BioGenex, San Ramon, CA, USA), glucagon (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and somatostatin (Santa Cruz Biotechnology) were used. All cells in ten random fields were scored. Data were expressed as the number of positive cells per mm2 of tissue.For electron microscopy, tissue was fixed and processed using uranyl acetate block-staining and lead citrate counter-staining of ultra-thin sections (80\u00a0nm). For insulin immunoelectronmicroscopy, a post-embedding immunogold procedure was used. The beta cell line Nit-1 was the positive control. Tissues and cells were embedded in LR white and labelling procedures were performed as previously described [23].\nFunctional studies Intravenous glucose tolerance tests were performed on fasted (6\u20138\u00a0h) animals. After infusion of glucose (0.5\u00a0g\/kg body weight), blood samples were collected from the tail vein. Insulin content of pancreas and liver was determined after acid\/ethanol extraction [23].Insulin and proinsulin in acid\/ethanol extractions were separated on a Sephadex G50 superfine column (Sigma-Aldrich) [24]. Insulin in serum samples and column fractions was measured using an RIA for human insulin (Linco Research, St Charles, MO, USA), which has less than 1 and 6% cross-reactivity with rodent insulin and human proinsulin, respectively. Commercial RIAs (Linco Research) were used to measure human and rat C-peptide [25].To assess liver function, aspartate transaminase (AST) and alanine aminotransferase (ALT) were measured daily over the first 10\u00a0days and then monthly using commercial kits (Roche Diagnostics, Castle Hill, NSW, Australia).\nPCR analyses For RT-PCR, liver, pancreas, spleen, kidney and lung were collected at 60 and 500\u00a0days after vector infusion and snap-frozen in liquid nitrogen. Total RNA was extracted using Trizol (Invitrogen Australia, Mt Waverly, VIC, Australia). Samples were treated with DNase I (Invitrogen). Real-time quantitative PCR was conducted using SYBR green I and a sequence detection system (ABI 7500 HT; Applied Biosystems, Scoresby, VIC, Australia). Optimal primer and cDNA template concentrations were determined by titration. Amplification efficiencies for each primer set were determined to be similar. Data were represented as differences between threshold cycle values (\u0394CT) for the transcripts of interest and the internal standard, \u03b2-actin. The expression of the genes encoding human insulin and EGFP was examined in normal and HMD\/INS-FUR-transduced liver cells using semi-quantitative RT-PCR. Oligonucleotide sequences are available on request.\nQualitative western blot analyses Nuclear and cytoplasmic extracts of liver and pancreas were prepared using a nuclear extraction Kit (Chemicon). Samples (1\u00a0\u03bcg protein) were separated by SDS-PAGE and transferred to nitrocellulose. Primary antibodies against PDX1 and NEUROD1 (Santa Cruz) were used.\nStatistical analyses Differences between groups were determined by Student\u2019s paired t test or, if there were more than two groups, by one-way analysis of variance after log transformation of data. Data are expressed as means \u00b1 standard errors (SEM).\nResults\nEfficient transduction of hepatocytes Isolation of hepatocytes by dual perfusion of the PV and abdominal aorta resulted in increased cell viability as compared with perfusion of the PV alone (85.7\u2009\u00b1\u20096.2% vs 67.5\u2009\u00b1\u20097.8%, respectively, p\u2009=\u20090.03). The cells were morphologically normal, as assessed by light and electron microscopy, with maintenance of gap junctions between cells and limited vacuolation. Hepatocytes grown on Matrigel in the optimised medium, with or without irradiation, did not undergo cell division, as assessed by [3H]thymidine incorporation. Cells grown on collagen, which enhances cell growth, reached a maximum growth rate at day 2 and this plateaued between days 5 and 6 (data not shown). Expression of EGFP was seen 48\u00a0h post-transduction and persisted for over 1\u00a0month (Fig.\u00a01a). Up to 87\u2009\u00b1\u20091.2% of the irradiated cells expressed EGFP (Fig.\u00a01b). Most EGFP-positive cells (80.2\u2009\u00b1\u200910.7%) were also positive for glucose-6-phosphatase, which suggested a hepatocyte lineage (data not shown). Growth of hepatocytes on collagen did not increase transduction efficiency. Delivery of HMD\/INS-FUR to hepatocyte cultures resulted in low constitutive insulin expression (0.2\u00a0pmol per 3\u2009\u00d7\u2009105 cells) and no insulin storage; therefore transplantation of hepatocytes transduced in vitro was not performed.\nFig.\u00a01EGFP expression in hepatocytes in vitro and in vivo. a EGFP expression in primary hepatocytes in vitro at indicated times post transduction with pHR\u2019CMVGFP. Original magnification: 400\u00d7. The insets show untransduced hepatocytes. b Flow cytometric analysis of irradiated (10\u00a0Gy), non-dividing primary hepatocytes 14\u00a0days post-transduction with pHR\u2019CMVGFP. Negative control cells show background fluorescence only. EGFP-positive cells constituted 87\u2009\u00b1\u20091.2% of all cells following transduction. Graphs representative of three independent experiments. c Flow cytometric analysis of liver cells isolated from a normal control rat and a rat transduced with the HMD LV vector 1\u00a0month previously. EGFP-positive cells constituted 60\u2009\u00b1\u20093.2% of all liver cells. The results are representative of three experiments. d Representative flow cytometry dot-plot showing the proportion of EGFP-expressing cells that were hepatocytes (CK-18-positive: 58\u2009\u00b1\u20093.1%) from an animal that had been transduced with the HMD\/INS-FUR LV 2\u00a0months previously and e the proportion of insulin-positive (57\u2009\u00b1\u20092.8%) hepatocytes. Graphs are representative of three experimentsWe delivered pHR\u2019CMVGFP to the livers of diabetic rats by FFO and compared the transduction efficiency in non-hepatectomised and partially-hepatectomised animals. The total wet weights of liver 48\u00a0h after partial hepatectomy were similar to those of non-hepatectomised rats (14.3\u2009\u00b1\u20090.4 and 14.7\u2009\u00b1\u20090.8\u00a0g, respectively). We also determined whether FFO stimulated cell division. In control rats, 0.1\u2009\u00b1\u20090.06% of hepatocytes were proliferating at 18\u00a0h and this rate did not significantly alter at any time point. At 18\u00a0h in animals that had undergone FFO, 0.3\u2009\u00b1\u20090.2% of hepatocytes were proliferating, compared with 0.4\u2009\u00b1\u20090.3% at 28\u00a0h and 0.1\u2009\u00b1\u20090.04% at 48\u00a0h. These values were not significantly different to those recorded for control animals. By comparison, in animals that had undergone a partial hepatectomy, 12.8\u2009\u00b1\u20092.4% of hepatocytes were proliferating at 18\u00a0h. This value increased to 21.4\u2009\u00b1\u20094.3% at 28\u00a0h and then decreased to 7.4\u2009\u00b1\u20091.2% at 48\u00a0h.To determine transduction efficiency, liver cells were isolated after transduction with pHR\u2019CMVGFP and EGFP-positive cells were assessed by flow cytometry. The maximal number of EGFP-expressing cells was detected 2\u00a0weeks after transduction (57.0\u2009\u00b1\u20092%) and was not significantly different after 2\u00a0months (58.2\u2009\u00b1\u20093.5%). In animals that were partially hepatectomised prior to transduction, there was no significant difference in numbers of EGFP-expressing hepatocytes (59.2\u2009\u00b1\u20094%) after 2\u00a0months compared with non-hepatectomised animals. Use of the HMD vector also resulted in high levels of transduction (Fig.\u00a01c). Cells expressing EGFP\/INS-FUR were almost exclusively hepatocytes, as verified by CK18 and insulin labelling (Fig.\u00a01d,e).\nTransduction with HMD\/INS-FUR reversed STZ-diabetes  Blood glucose levels of STZ-diabetic rats decreased to subnormal levels immediately after transduction with HMD\/INS-FUR vector (Fig.\u00a02a). By day 5, blood glucose levels of insulin-transduced animals were not different to non-diabetic controls and reversal of diabetes was maintained for 500\u00a0days (experimental endpoint). STZ-diabetic rats treated with HMD alone remained hyperglycaemic until day 60, when they were killed. The body weights of rats treated with HMD\/INS-FUR were not significantly different to those of non-diabetic controls until day 125, lower between days 135 and 275, becoming not significantly different again after day 300 (Fig.\u00a02b). The body weights of rats treated with the empty vector alone continued to fall from day 50 to 60. Throughout the experimental period, there was no elevation of liver enzymes in animals treated with HMD\/INS-FUR (AST: 38.4\u2009\u00b1\u20096, ALT: 26.9\u2009\u00b1\u20095\u00a0U\/l) compared with control animals (AST: 44.2\u2009\u00b1\u200916, ALT: 19.1\u2009\u00b1\u20097).\nFig.\u00a02Stable expression of INS-FUR ameliorated STZ-induced hyperglycaemia. Blood glucose concentrations (a) and body weights (b) of non-diabetic control rats (black circles) and STZ-diabetic rats treated with HMD\/INS-FUR (white squares) or HMD alone (black triangles). Values are means\u00b1SEM (n\u2009=\u20095)\nHMD\/INS-FUR transduction stimulated expression of pancreatic hormones We examined pancreatic hormone-producing cells in the livers of rats transduced with LV after 2\u00a0months. Figure\u00a03a shows extensive staining for insulin in normal pancreatic islets. By comparison, pancreatic beta cells were rarely observed in STZ-diabetic animals (1.5\u2009\u00b1\u20091.4 cells) (Fig.\u00a03b). Normal liver was negative for insulin (Fig.\u00a03c), glucagon and somatostatin (not shown). No pancreatic hormones were detected in livers of animals treated with empty vector. Following transduction with HMD\/INS-FUR, significant numbers of hepatocytes were converted into insulin-positive cells (Fig.\u00a03d). There was also evidence of some pancreatic transdifferentiation, as glucagon- and somatostatin-positive cells were detected (Fig.\u00a03e,f). Cells positive for pancreatic hormones were distributed throughout the livers of animals transduced with HMD\/INS-FUR and not restricted to regions adjacent to the PV.\nFig.\u00a03Expression of pancreatic hormones following reversal of STZ-induced hyperglycaemia. Photomicrographs of anti-insulin staining of (a) normal pancreas, (b) STZ-diabetic HMD\/INS-FUR vector-treated pancreas, (c) normal liver and (d) STZ-diabetic HMD\/INS-FUR vector-treated liver at 2\u00a0months. Photomicrographs of anti-glucagon (e) and anti-somatostatin (f) staining in STZ-diabetic HMD\/INS-FUR vector-treated liver at 2\u00a0months. Positive cells appear brown. Original magnification: 200\u00d7. In a total of 1,200\u2009\u00b1\u200961 cells the number of insulin-positive cells (585.6\u2009\u00b1\u200973.8 cells\/mm2) was fivefold higher than the number of glucagon-positive cells (109.9\u2009\u00b1\u200937.1 cells\/mm2) and 11-fold higher than the number of somatostatin-positive cells (52.2\u2009\u00b1\u20098.1 cells\/mm2). The number of EGFP-positive cells was 854\u2009\u00b1\u200980.9 (data not shown). Therefore, some EGFP-positive cells did not store pancreatic hormones. No pancreatic hormones were detected in liver from animals treated with empty vector or from control animals. g Transmission electron micrograph showing secretory vesicles with dense granules surrounded by a pale halo (bar: 500\u00a0nm). Inset, vesicle 320\u00a0nm in diameter. h Immuno-electron micrographs showing localisation of insulin in liver 2\u00a0months after the transduction of an STZ-diabetic rat with HMD\/INS-FUR and (i) insulin-secreting granules in Nit-1 beta cells (positive control); bar: 400\u00a0nmElectron microscopy revealed cytoplasmic granules (270\u2013330\u00a0nm in diameter) in liver tissue from animals treated with HMD\/INS-FUR (Fig.\u00a03g). Immunoelectron microscopy of insulin-producing cells in the liver of STZ-diabetic rats transduced with HMD\/INS-FUR was comparable to those observed in Nit-1 beta cells (Fig.\u00a03h,i). Inflammatory cell infiltration and necrosis within the livers of transduced animals were not observed. Insulin-containing granules were absent in rats treated with empty vector and in untreated animals.\nHMD\/INS-FUR transduction induced hepatic insulin production Insulin storage was 4,165\u2009\u00b1\u2009180\u00a0pmol per liver in HMD\/INS-FUR-treated animals, compared with 10,418\u2009\u00b1\u2009210 pmol in normal rat pancreas (n\u2009=\u20093). STZ-treated rat pancreas only contained 255\u00b1110\u00a0pmol insulin. Separation of insulin from proinsulin in liver extracts from animals treated with HMD\/INS-FUR yielded two distinct peaks. The first represented proinsulin and partially processed material and the second larger peak corresponded to fully processed insulin (Fig.\u00a04).\nFig.\u00a04Stable expression of INS-FUR resulted in production of mature human insulin. Extracts of rat livers were separated by gel permeation chromatography and assayed for insulin. Arrows indicate mobility of mature human insulin (I) (5,800\u00a0Da) and proinsulin (pI) (9,400\u00a0Da). Results are representative of three independent experiments\nHMD\/INS-FUR transduced animals displayed regulated insulin secretion There was no significant difference between the ability of STZ-diabetic animals transduced with the HMD\/INS-FUR vector and untreated animals to normalise blood glucose levels after delivery of a bolus of intravenous glucose (Fig.\u00a05a). Levels of human C-peptide and human insulin in serum samples from HMD\/INS-FUR-treated animals (Fig.\u00a05b,c) were comparable to levels of rat C-peptide and insulin recorded for non-diabetic animals. Negligible levels of rat C-peptide were detected in serum from HMD\/INS-FUR-treated STZ-diabetic animals (Fig.\u00a05c).\nFig.\u00a05Plasma glucose (a), insulin (b) and C-peptide (c) levels after IVGTT. IVGTTs remained normal for animals maintained for 500\u00a0days (data not shown). White squares, HMD\/INS-FUR-treated animals (60\u00a0days) expressing human insulin and human C-peptide; black diamonds, normal control animals expressing rat insulin and rat C-peptide; black triangles, rat C-peptide in HMD\/INS-FUR-treated animals. Values are means\u00b1SEM (n\u2009=\u20095)\nHMD\/INS-FUR transduction stimulated expression of beta cell transcription factors EGFP expression was only detected in liver tissue of pHR\u2019CMVGFP- (Fig.\u00a06a) and HMD-treated (data not shown) rats. Similarly, INS-FUR expression was restricted to the livers of HMD\/INS-FUR-treated rats (Fig.\u00a06a). Insulin was not detected in livers from diabetic control animals and diabetic animals injected with HMD alone (data not shown). Mean ratios of insulin : \u03b2-actin (1.000\u2009\u00b1\u20090.069) and EGFP : \u03b2-actin (1.088\u2009\u00b1\u20090.062) in HMD\/INS-FUR liver, determined by semi-quantitative PCR, were not significantly different. Transduction of STZ-diabetic rats with the HMD\/INS-FUR vector induced sustained expression of several pancreatic transcription factors (Fig.\u00a06b). Normal liver and untreated diabetic liver (not shown) were negative for all pancreatic transcription factors, while normal pancreas was positive.\nFig.\u00a06Beta cell transcription factors and pancreatic hormones expressed in transduced rat livers. a RT-PCR analysis of transcription of the genes encoding EGFP or INS-FUR in animals transduced with pHR\u2019CMVGFP or HMD\/INS-FUR LV, respectively: liver (lane 1), pancreas (lane 2), spleen (lane 3), kidney (lane 4) and brain (lane 5) at 60\u00a0days. Results for HMD\/INS-FUR transduced animals at 500\u00a0days were identical (not shown). b RT-PCR analysis of transcription of the genes for: (1) beta cell transcription factors (Pdx1, Neurod1, Neurog3, Nkx2-2, Nkx6-1, Pax4, Pax6); (2) the pancreatic endocrine hormones somatostatin (Sst), glucagon (Gcg) and pancreatic polypeptide (Ppy); (3) GLUT2 (Slc2a2) and glucokinase (Gck); (4) rat insulin 1 and 2 (Ins1, Ins2); (5) insulin proconvertase PC2 (Pc2); (6) the exocrine marker p48 (P48); and (7) \u03b2-actin (Actb) in normal liver (lane 1), liver transduced with HMD alone at 60\u00a0days (lane 2), liver transduced with HMD\/INS-FUR LV at 60\u00a0days (lane 3) and 500\u00a0days (lane 4), and normal pancreas (lane 5). c Western blot analysis for PDX1 and NEUROD1 in normal pancreas (lane 1), liver transduced with HMD\/INS-FUR LV at 60\u00a0days (lane 2), liver transduced with HMD alone at 60\u00a0days (lane 3) and normal liver (lane 4). PDX1 and NEUROD1 generated bands at 43 and 50\u00a0kDa, respectivelyUnexpectedly, both Pdx1 and Neurod1 expression were detected by RT-PCR in empty vector-transduced livers; however, protein production was not detected (Fig.\u00a06c). HMD\/INS-FUR-transduced livers also expressed the genes for neurogenin 3 (Neurog3) and Nkx2-2 (Nkx2-2). Glucagon and somatostatin were present in liver transduced with HMD\/INS-FUR (confirmed by immunohistochemistry; Fig.\u00a03e,f). Pancreatic polypeptide was not expressed. The transduction of diabetic animals with HMD\/INS-FUR did not result in complete pancreatic transdifferentation of liver tissue since expression of the rat insulin genes (Ins1, Ins2) and the genes encoding the insulin prohormone convertase 2 (Pc2, also known as Pcsk2) and the exocrine marker p48 (Ptf1a, also known as P48) were not detected (Fig.\u00a06b). Quantitative real time PCR analysis of the expression levels of beta cell transcription factors Pdx1 (4.3\u2009\u00b1\u20090.9), Neurod1 (5.7\u2009\u00b1\u20091.1) and Neurog3 (7.4\u2009\u00b1\u20091.8) in HMD\/INS-FUR-treated livers at 500\u00a0days indicated that they were lower (p\u2009<\u20090.001) than levels expressed in normal pancreatic tissue (Pdx1: 12.5\u2009\u00b1\u20092.3, NeuroD1: 16.8\u2009\u00b1\u20091.4, Neurog3: 18.5\u2009\u00b1\u20092.2).\nDiscussion\nThis is the first report of permanent amelioration of STZ-induced hyperglycaemia using LV delivery of INS-FUR. Transduced animals processed, stored and secreted insulin in a glucose-regulated manner. Remission of diabetes, due to the secretion of insulin from residual beta cells, was discounted as insulin-positive pancreatic beta cells were rarely observed and levels of rat C-peptide were negligible. This is also the first report showing that transduction of INS-FUR alone can induce expression of several beta cell transcription factors resulting in hepatocyte differentiation along a pancreatic lineage.\nAll previous reports of liver\u2013pancreas transdifferentiation have been described in cells that endogenously express beta cell transcription factors [23, 26] or have been engineered to express these factors, leading to varying degrees of transdifferentiation [5\u20138, 27, 28]. Following immediate reduction in blood glucose due to transgene expression, induction of long-term euglycaemia in the present study was probably related to partial hepatic-to-pancreas transdifferentiation triggered by the lentiviral intrahepatic delivery method of INS-FUR or from expression of stably integrated transgene in the host genome. The HMD\/INS-FUR transgene alone, which constitutively expresses insulin, could not mimic the glucose-sensitive kinetics demonstrated by IVGTT, nor could it have solely induced insulin storage. Evidently, the hepatic delivery of the LV was a key factor, for expression of HMD alone triggered expression of Pdx1 and Neurod1, if only at the mRNA level. It is possible that the use of B10-insulin may have been another factor. This insulin is superior to normal insulin in biological activity [29] and has some mitogenic properties [30\u201332]. However, there was no evidence of excessive mitogenic activity or tumour development, while liver function tests remained normal throughout, with no lymphocytic infiltrates. Parallels can be drawn to the partial pancreatic transdifferentiation observed in Huh7ins cells, which endogenously produce NEUROD1 [23] and PDX1 (C. Tao, University of Technology Sydney; unpublished results). This cell line was transfected with normal human insulin under the control of a constitutive promoter. After this, secretory granules developed, which stored di-arginyl insulin and secreted it in a glucose-responsive manner, correcting diabetes. However, in Huh7ins cells the expression of fully processed native insulin was absent. Similarly, in the present study, pancreatic transdifferentiation was incomplete, as genes encoding transcription factors such as NKX6-1 and PAX6, which occur late in the hierarchy; were not expressed, nor were the rodent insulin genes or the genes for PC2 pancreatic polypeptide or exocrine markers. Therefore, the problems encountered with non-selective pancreatic transdifferentiation, produced after expression of PDX1 alone [5\u20137, 33, 34], were not encountered.\nHow the hepatic delivery of INS-FUR induced the expression of Pdx1 and other beta cell transcription factors requires further investigation. Liver and pancreas derive from common progenitor cells. Transdifferentiation between liver and pancreas is controlled by only a few transcription factors [35, 36]. Pdx1 is one such master switch gene. In a recent study, liver cells producing pancreatic markers (insulin and PDX1) were induced and localised to areas adjacent to or within injured tissue areas [37]. Lentiviral transduction may represent a cellular insult, making progenitor cells permissive to a pancreatic developmental shift.\nThe mechanisms that prevented both complete pancreatic transdifferentiation and expression of endogenous rat insulin are yet to be elucidated. Studies on the development of the rat pancreas indicate that chronologically glucagon and somatostatin appear prior to insulin [38]. The production of these pancreatic hormones may have prevented hypoglycaemia by providing counter-regulatory mechanisms. Complete transdifferentiation into true beta cells may require additional external factors, such as prolonged hyperglycaemia [39, 40]. If this is true, then the rapid reversion to euglycaemia following delivery of the insulin gene in our study may have prohibited complete liver-to-pancreas transdifferentiation. Additionally, the expression of human insulin may have exerted a negative feedback mechanism to inhibit endogenous insulin expression.\nUnlike other studies that have engineered beta cell neogenesis from liver cells [5\u20137], the insulin-containing cells were not isolated to the area surrounding the portal circulation. Insulin storage was 40% of the insulin content of a rat pancreas, satisfying clinical requirements for insulin independence [41]. Corroborating previous studies using INS-FUR, granules predominately stored fully processed insulin [18, 42, 43]. EGFP and insulin were produced at equivalent levels in liver tissue transduced with HMD\/INS-FUR. The majority of cells that expressed EGFP and insulin were also CK18-positive, indicative of a hepatocyte lineage for transduced cells.\nDespite the lack of the central polypurine tract, LV pHR\u2019CMVGFP efficiently transduced non-dividing hepatocytes in vitro, in the absence of cell division. We obtained an improvement in transduction efficiency of up to 87% in vitro, compared with 68% obtained by Zahler et al. [44]. Increased transduction efficiency may be attributable to several factors, including: (1) the isolation of hepatocytes by perfusion of both the PV and abdominal aorta as opposed to the PV alone, the most widely employed procedure [45]; (2) the optimisation of growth medium; (3) the use of LV at high multiplicity of infection (50:1); and\/or (4) the use of the R8.2 packaging plasmid, which encodes accessory HIV proteins. The use of the attenuated plasmid R8.91, used by Zahler et al., resulted in a 5% reduction in transduction efficiency (B. Ren, unpublished observations). Similarly, delivery of INS-FUR in the HMD hybrid vector, which lacks the central polypurine tract [19], to the portal circulation of STZ-diabetic rats by FFO resulted in up to 60% transduction of liver cells, without inducing cell division and with transgene expression being confined to the liver. The FFO technique, which employs repeated vector infusion, is probably responsible for the increased transduction efficiency and is more clinically applicable than the widely used perfusion method, where the IVC is cannulated, thereby increasing the risk of IVC narrowing and pelvic limb thrombosis [46, 47].\nThis novel approach offers potential as a beta cell replacement strategy with diabetic patients serving as their own donors. Current studies in our laboratory using nonobese diabetic mice, indicate that this procedure was able to reverse autoimmune diabetes for 5\u00a0months with no evidence of autoimmune destruction of insulin-secreting hepatocytes (B. Ren, unpublished observations), giving hope that this procedure will be useful for reversal of autoimmune diabetes. There are distinct advantages to the use of LV for transduction of hepatocytes. LV stably integrates into the genome of non-dividing cells, including hepatocytes, and could provide long-lasting expression of therapeutic genes [48, 49]. Whilst LV integration was not specifically examined in this study, the longevity of INS-FUR and EGFP expression provides some evidence for this. Further, human hepatocytes may be more susceptible to LV transduction [50] than rodent cells. LV are probably superior to adenoviral vectors where problems of short-term expression of genes and immunological responses have been reported [9]. Adeno-associated vectors are extremely efficient at transducing mouse livers; however, a recent study using adeno-associated viral 8-mediated delivery of Pdx1 did not result in correction of STZ-diabetes in mice [28]. The unique results reported in this study point to possible benefits of gene therapy using LV.","keyphrases":["gene therapy","type 1 diabetes","lentiviral vector","hepatocytes","beta cell transcription factors","insulin storage","wistar rats"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Knee_Surg_Sports_Traumatol_Arthrosc-4-1-2413120","title":"Comparison of two arthroscopic pump systems based on image quality\n","text":"The effectiveness of arthroscopic pump systems has been investigated with either subjective measures or measures that were unrelated to the image quality. The goal of this study is to determine the performance of an automated pump in comparison to a gravity pump based on objective assessment of the quality of the arthroscopic view. Ten arthroscopic operations performed with a gravity pump and ten performed with an automated pump (FMS Duo system) were matched on duration of the surgery and shaver usage, type of operation, and surgical experience. Quality of the view was defined by means of the presence or absence of previously described definitions of disturbances (bleeding, turbidity, air bubbles, and loose fibrous tissue). The percentage of disturbances for all operations was assessed with a time-disturbance analysis of the recorded operations. The Mann\u2013Whitney U test shows a significant difference in favor of the automated pump for the presence of turbidity only (Exact Sig. [2*(1-tailed Sig.)] = 0.015). Otherwise, no differences were determined (Exact Sig. [2*(1-tailed Sig.)] > 0.436). A new objective method is successfully applied to assess efficiency of pump systems based on the quality of the arthroscopic view. Important disturbances (bleeding, air bubbles, and loose fibrous tissue) are not reduced by an automated pump used in combination with a tourniquet. The most frequent disturbance turbidity is reduced by around 50%. It is questionable if this result justifies the use of an automated pump for straightforward arthroscopic knee surgeries using a tourniquet.\nIntroduction\nDuring arthroscopic surgery, the joint is continuously irrigated with saline fluid which is pressurized to create joint distension. The saline fluid flow removes disturbances in the arthroscopic view such as bleeding, air bubbles or synovial fluid. This improves the visibility of the arthroscopic view, which is an essential condition to perform an operation safe and fast. In this light, the quality of arthroscopic view has been defined as good or optimal if no disturbances are present [11]. In clinical practice, disturbances cannot always be prevented and maintaining a clear view is sometimes difficult [7, 10]. In a recent study performed by the authors, post-procedure analysis of 20 routinely performed arthroscopic knee procedures showed that 6% (SD 4%) of the total operation time was solely dedicated to obtaining a clear view. Since the number of arthroscopic procedures is large and still growing (60,000 per year in the Netherlands [8], and 1.7 million meniscectomies alone per year worldwide [3]), reducing the operating time by optimizing the view would be desirable.\nThe quality of the arthroscopic view is dependent on a number of factors such as the type of pump system, instruments, and condition of the joint [11]. In this study, the focus is on the type of pump system. The classical gravity pump and (automated) volumetric pumps are available for irrigation [10]. The main differences between these pumps are that the gravity pump causes a pressure due to a height difference caused by an elevated fluid bag and is manually controlled, whereas the volumetric pumps cause a fluid flow and have some type of automated pressure control. Since the automated pumps are regulated, in theory they should perform better in irrigation in the sense that the percentage of disturbed view over time should be less. In literature, the effectiveness of automated pump systems has been investigated [2, 4\u20136]. However, only subjective measures were used, such as visual clarity on a three-point scale or measures that were unrelated to the image quality, such as the number of fluid bags. The goal of this study is to determine the performance of an automated pump in comparison to the gravity pump based on objective assessment of the quality of the arthroscopic view. Therefore, a quantitative time-disturbance analysis was performed with uniquely described definitions for the different types of disturbances [11].\nMethods\nOnly routinely performed procedures were included such as meniscectomy, cyst removal, debridement of a partial rupture of the anterior cruciate ligament, and diagnostic arthroscopy. Cruciate ligament reconstructions were not included. The patients were not notified of the recordings, because the operation was not affected and the patients\u2019 identities could not be traced from the videos. No power analysis could be performed for this study, because no information was available on the variation of the disturbances. In accordance with other studies, it was decided to use ten operations for each of the two pump systems (20 in total) [2, 4]. The matching criteria were the number of operations, the duration of the operations, the duration of shaver usage, the type of operation, and the working experience of the surgeons. Matching of the shaver duration was performed, because some disturbances were expected to occur more frequently when using the shaver. Besides, a shaver is often used to clarify the view by temporarily increasing the flow. All surgeons who performed the operations had the same education and comparable working experience.\nTen arthroscopic knee procedures for which the gravity pump was used were recorded at 6 days within a time frame of 2 months. From the operations that met the inclusion criteria, ten were selected with randomization tables. Ten arthroscopic knee procedures performed with an automated pump were matched postoperatively to eliminate factors other than the irrigation pump systems that influence the arthroscopic view (Table\u00a01).\nTable\u00a01Matching of the two pump groups was performed based on the number of operations, the total duration of all operations, and the share of shaver usageOperationTime (min:s:frames)Shaver time (s)Gravity pump\u00a0Cyst removal17:55:11114\u00a0Meniscectomy26:03:15363\u00a0Meniscectomy33:15:09288\u00a0Meniscectomy19:56:10416\u00a0Meniscectomy13:37:18313\u00a0Meniscectomy17:55:13508\u00a0Meniscectomy12:10:15153\u00a0Meniscectomy15:09:02150\u00a0Debridement partial rupture of anterior cruciate ligament16:30:03290\u00a0Diagnostic7:12:03\u00a0Total duration 179:45:2443:15:0\u00a0Shaver time as percentage of total duration (%)24.1Automated pump\u00a0Meniscectomy30:37:11410\u00a0Meniscectomy13:49:15120\u00a0Meniscectomy16:07:12122\u00a0Meniscectomy22:10:01404\u00a0Meniscectomy15:08:16237\u00a0Meniscectomy11:05:19290\u00a0Meniscectomy27:35:00325\u00a0Meniscectomy14:16:13149\u00a0Debridement partial rupture of anterior cruciate ligament17:41:10284\u00a0Diagnostic7:08:21\u00a0Total duration 175:40:1839:01:0\u00a0Shaver time as percentage of total duration (%)22.2\nThe recordings of operations with a gravity pump were performed in the day care centre of Academic Medical Center (Amsterdam, The Netherlands). The recordings of operations with an automated pump were performed in the day care centre of the Sint Maartenskliniek (Nijmegen, The Netherlands). The latter hospital utilizes the automated FMS Duo pump (FMSGroup, Nice, France). Table\u00a02 shows the details on the standard operation setting for both hospitals using the different pumps. Most disturbances are more or less independent of the quality of the camera, the light source, and the arthroscope. It was assumed that these devices were functioning properly at the time of recording, because all operations were executed in acknowledged hospitals, in which the technical service department routinely inspects the equipment. A tourniquet was routinely applied at the start of the arthroscopic knee operations with a vacuum level of 300 mmHg. Drills, vaporizing devices, microfracturing, arthroscopic drills or meniscal repair devices were not used.\nTable\u00a02Standard operation setting for the gravity and the automated pumpGravity pumpAutomated pumpPortalsAnteromedial and anterolateralAnteromedial, anterolateral, and superomedialPump settingsPreset pressure of 49\u00a0mmHg (elevated saline fluid bag at 0.66\u00a0m)Suction pressure of \u2212500\u00a0mmHgIndicated setting for knee arthroscopy is 50, which implies a preset pressure of 113\u00a0mmHg and a continuous flow of 90\u00a0ml\/minInstrument set\u00d8 4\u00a0mm arthroscope (Karl Storz, Gemany)Probe and punches (Dyonics, UK)\u00d8 4.5 or 5.5\u00a0mm full radius shaver (Dyonics, UK)\u00d8 4\u00a0mm arthroscope (Karl Storz, Gemany)Probe and punches (Dyonics, UK)\u00d8 4.5 or 5.5\u00a0mm full radius shaver (Dyonics, UK)\nThe comparison of the pumps was conducted by assessing the quality of the arthroscopic view per operation. Extending the definition of arthroscopic image quality by Tuijthof et al. [11] to the setting of irrigation performance of the pump systems, it is proposed that the pump for which the least percentage of disturbances occurs is the most effective. Seven disturbances of the arthroscopic view have been uniquely described and defined [11]. Not all of them can be prevented or removed by irrigation. Therefore, a subset of these disturbances was chosen for comparison of the pump systems: bleeding, turbidity (caused by synovial fluid and debris, Fig.\u00a01), air bubbles, and loose fibrous tissue. The same recording equipment and quantitative time-disturbance analysis was performed as described in Tuijthof et al. [11]. Therefore, the recordings of the operations were analyzed frame by frame to assess the presence of the four disturbances in each frame. In addition to the total operation time, each operation was divided into phases to assess dominant prevalence of disturbances in a particular phase: (1) creation of portals, (2) joint inspection with or without a probe, (3) cutting, and (4) shaving. Phase 1 was the time required to achieve access to the joint by means of the arthroscope; thus the period from the first frame of the recorded digital video until the frame where a clear sharp arthroscopic view is presented for the first time. Added with this period is the time required to create the second and third portal: the period from the frame where the tip of a needle is seen for the first time until the frame where the tip of an instrument occurs. Phases 3 and 4 were defined as the frames in which the specific instrument was present in the arthroscopic view. The remainder of the operation time was indicated as Phase 2 inspection.\nFig.\u00a01Three images of the disturbance turbidity, which consists of synovial fluid and small particles\nThe 20 digital videos were analyzed by two testers who each assessed an equal number of operations of each group. Their tester agreement was determined prior to the time-disturbance analyzes by means of the adjusted kappa [1, 9]. For each of the four disturbances, the adjusted kappa was larger than 0.80 indicating good agreement. Statistical analysis was performed with SPSS 12.0.2 (SPSS Inc., Chicago, IL, USA). The presence of a normal distribution for both datasets was determined with the Kolmogorov\u2013Smirnov and Shapiro\u2013Wilk tests. With the help of a one-way analysis of variance test (ANOVA) significant statistical differences were assessed in case the data were normally distributed. Otherwise, the nonparametric Mann\u2013Whitney U test (Wilcoxon rank sum test) was applied.\nResults\nAlmost 3 h of digital video material has been analyzed for both groups (Table\u00a01). The mean operation time is around 18\u00bd\u00a0min for the gravity pump, and 17\u00bd\u00a0min for the automated pump. In each pump group, the total time of shaver usage is around 23% (Table\u00a01). The Kolmogorov\u2013Smirnov and Shapiro\u2013Wilk tests show that the datasets are not distributed normally. As a consequence, the results of the percentages of each disturbance of the total operation time are presented with the median, minimum and maximum (Fig.\u00a02). The disturbances show a substantial variation per operation. Turbidity was the dominant disturbance in both pump groups (median of 18% of the total operation time for the gravity pump versus 8% for the automated pump). The Mann\u2013Whitney U test shows a significant difference for the presence of Turbidity (Exact Sig. [2*(1-tailed Sig.)]\u00a0=\u00a00.015) between both pump groups in favor of the automated pump. No significant differences are found for the other three disturbances (Exact Sig. [2*(1-tailed Sig.)]\u00a0>\u00a00.436) (Fig.\u00a02).\nFig.\u00a02Results of the share of disturbances in the arthroscopic view as percentage of the total operation time for each operation and each of the two groups. The results are presented with the median, minimum, and maximum numbers, because the datasets were not distributed normally. *The Mann\u2013Whitney U test shows a significant difference for Turbidity (Exact Sig. [2*(1-tailed Sig.)]\u00a0=\u00a00.015)\nNot all of the four operation phases were present in the 20 operations. Phases 3 and 4 were absent in both diagnostic arthroscopies. Additionally, punches were not used in five operations in the gravity pump group, and three in the automated pump group. As was expected the creation of portals was the shortest phase (10% for the gravity pump versus 5% for the automated pump), and inspection was the longest phase (59% for the gravity pump versus 51% for the automated pump). Punches were used more frequently in the automated group (7% for the gravity pump versus 21% for the automated pump). Analyzing each of the four operation phases separately shows a significant difference in favor of the automated pump for Turbidity in Phase 2 (Exact Sig. [2*(1-tailed Sig.)]\u00a0=\u00a00.019). For all other disturbances and operation phases no significant differences are determined (Exact Sig. [2*(1-tailed Sig.)]\u00a0>\u00a00.165). The Mann\u2013Whitney U test within each of the pump groups shows a significant difference for loose fibrous tissue (Fig.\u00a03). In detail, the gravity pump shows the following results: Phase 1 versus 3 (Exact Sig. [2*(1-tailed Sig.)]\u00a0=\u00a00.002), Phase 1 versus 4 (Exact Sig. [2*(1-tailed Sig.)]\u00a0=\u00a00.010), and Phase 2 versus 3 (Exact Sig. [2*(1-tailed Sig.)]\u00a0=\u00a00.002). The automated pump shows these results: Phase 1 versus 2 (Exact Sig. [2*(1-tailed Sig.)]\u00a0=\u00a00.017), Phase 1 versus 3 (Exact Sig. [2*(1-tailed Sig.)]\u00a0=\u00a00.001), and Phase 2 versus 3 (Exact Sig. [2*(1-tailed Sig.)]\u00a0=\u00a00.022).\nFig.\u00a03Results of the share of disturbances in the arthroscopic view as percentage of the phase time for each operation in the gravity pump group (a), and the automated pump group (b). The results are presented with the median, minimum, and maximum numbers, because the datasets were not distributed normally. *The Mann\u2013Whitney U test shows only a significant difference for loose fibrous tissue in both pump groups\nDiscussion\nThe objective of this study was to compare two types of irrigation pump systems (the classic gravity pump and a widely used automated pump) based on their effectiveness in maintaining a clear arthroscopic view. Comparison of the effectiveness of pump systems has been performed [2, 4\u20136]. However, none of these studies measured the arthroscopic image quality objectively in detail. Besides the objective comparison, the time-disturbance analysis gave quantitative values for the frequency of four disturbances.\nTurbidity was the dominant disturbance in both pump groups being present in a considerable percentage of the operation time (Fig.\u00a02). The results show that for routinely performed arthroscopic knee operations, the automated pump was significantly more effective in reducing the presence of turbidity with 50% on average. This finding can be explained by the continuous flow of 90 ml\/min that is activated by the automated pump as opposed to the lower flow cause by leakage along the portals when using the gravity pump and a two-portal technique. No difference was determined for the other three disturbances. The low percentage of bleeding can be attributed to the use of a tourniquet in all operations. In order to determine the effectiveness of pump systems for this disturbance, analysis of shoulder surgeries would be more convenient.\nThe found variation in the duration of disturbances per operation could have been caused by the condition of the knee joints, because this factor could not be completely eliminated with precise matching. With this knowledge, a future suggestion is to analyze a larger number of patients per pump group to determine possibly other significant differences in pump performance.\nComparison of the operation phases independently showed similar results for the inspection phase compared to the total operation time, probably because this was the longest phase. Since the creation of portals was a relatively short phase, and the use of punches was absent for almost half of the operations, no significant differences were found. However, analysis of both groups internally showed that the presence of loose fibrous tissue was generally significantly higher in when an instrument was used (Phases 3 and 4). This is in accordance with the general expectation and confirms the applicability of this method for quantification of disturbances in the view.\nConcluding, this study shows that important disturbances as bleeding, air bubbles, and loose fibrous tissue are not affected by a pump system when performing a routine knee arthroscopy with a tourniquet. The most frequent disturbance turbidity is reduced by around 50% when using an automated pump. It is questionable if this justifies the use of an automated pump for straightforward arthroscopic knee surgeries using a tourniquet, since the purchase of an automated pump is more costly.","keyphrases":["pump","image quality","irrigation","arthroscopy","knee joint","video analysis"],"prmu":["P","P","P","P","P","R"]}
{"id":"Qual_Life_Res-4-1-2238779","title":"Validity, reliability, and responsiveness of the Kansas City Cardiomyopathy Questionnaire in anemic heart failure patients\n","text":"Background While generic health status measures quantify the impact of all patients\u2019 diseases on their health-related quality of life, disease specific measures focus on only one of the many conditions that a patient may have. If a patient has two diseases with similar clinical manifestations, they may respond differently to a disease-specific instrument if one of their conditions improves while the other worsens or remains stable, thus undermining the instruments in that patient population. We sought empirical evidence of the reliability and validity (including responsiveness) of the Kansas City Cardiomyopathy Questionnaire (KCCQ), a disease-specific measure for heart failure (HF), among HF patients with and without anemia, a condition that has similar symptoms to HF.\nIntroduction\nThe quantification of patients\u2019 health status (their symptoms, function, and health-related quality of life) is becoming increasingly important as an endpoint in clinical trials [1, 2], as a means of monitoring patients over time [3], and even as a marker of health care quality [4\u20136]. Measuring patients\u2019 health status is most commonly accomplished by direct solicitation from patients themselves, using either generic or disease-specific health status questionnaires [7]. Whereas generic health status measures seek to quantify the health-related quality of life limitations of patients\u2019 \u2018overall health\u2019, including all combinations of diseases and ailments, disease-specific measures focus upon a specific disease process. Accordingly, an advantage of disease-specific questionnaires is that they can be both more sensitive to clinical change and more interpretable than general health status questionnaires. Because disease-specific instruments tend to be more actionable to clinicians, they can be useful in monitoring patients\u2019 clinical status over time [8]. An unresolved challenge in the use of disease-specific measures, however, is whether they perform well when patients have other conditions that may also impact their symptoms, function, and quality of life in ways that are similar to the specific disease of interest. While this is not a problem with generic health status measures, if a co-occurring disease manifests similar effects to a primary disease of interest, and if one of the conditions worsens while the other remains stable or improves, then it is unknown whether the disease-specific measure will accurately measure the status of the disease it is designed to quantify. This can undermine the original validity, reliability and responsiveness of the disease-specific measure. One potential setting in which this type of confounding may occur is in the setting of heart failure and anemia.\nHeart failure (HF) affects over 5\u00a0million Americans and is responsible for enormous health care costs and resource utilization [9]. While several disease-specific measures for patients with HF have been created to quantify HF patients\u2019 health status [10\u201313], the psychometric properties of these instruments have all been demonstrated in patients whose predominant medical condition was heart failure. Recently, however, anemia has emerged as an important co-occurring condition that is prognostically important in HF [14\u201321]. Importantly, the symptoms of anemia may mimic those of HF. Specifically, chronic anemia manifests itself in patients as \u201cfatigue, loss of stamina, breathlessness, and tachycardia, particularly with exertion\u201d [22], coupled with the development of \u201cgradual fatigue and a reduced exercise tolerance\u201d [23]. The symptoms of anemia [24] are therefore remarkably similar to those of HF, for which breathlessness, reduced exercise tolerance, and fatigue are hallmarks of the disease [22, 23]. Because of the overlap in symptomatic manifestations of both diseases, it is not clear whether heart failure-specific health status assessments, such as the Kansas City Cardiomyopathy Questionnaire (KCCQ) which captures patients\u2019 perspectives of their disease rather than physiologic or anatomic characteristics (e.g. their hematorit or left ventricular function), would perform similarly in patients with and without anemia. For example, if a patient\u2019s heart failure symptoms improve from an increased use of diuretics, would there be an improvement in their KCCQ scores if their anemia did not change? Consequently, it is unknown whether the psychometric properties of a disease-specific HF instrument would be similar in anemic and nonanemic HF patients, given the potential for both conditions to induce similar symptoms in patients. The purpose of this analysis was to provide empirical evidence supporting the reliability and validity (including responsiveness) of the KCCQ in anemic HF patients. We sought to compare the psychometric properties, including reliability (internal consistency and test\u2013retest) and validity (including responsiveness to clinical change), in anemic and nonanemic HF patients.\nMethods\nPatient population\nThe STudy of AneMia IN A Heart Failure Population (STAMINA-HFP) was a prospective registry of 1,090 HF patients who were initially enrolled in the outpatient clinics of 58 U.S. cardiology centers. The purpose of STAMINA-HFP was to estimate the incidence and prevalence of anemia in patients with HF and to describe the association of anemia with HF progression. As such, the protocol mandated an initial clinical history and physical exam (including assignment of the New York Heart Association [NYHA] classification), a measurement of patients\u2019 hemoglobin, and an assessment of their health status, including the KCCQ. Enrolled patients were contacted by telephone within 1\u20132\u00a0days after their initial enrollment and every 3\u00a0months for a period of 1\u00a0year to assess their health status. In addition, data from outpatient visits during the year of observation were abstracted at each visit. To be eligible for participation, patients had to be >18\u00a0years of age, diagnosed with a history of symptomatic HF and willing to provide informed consent. Patients who had a prior organ transplant, were planning a major surgery, or were participating in another interventional trial were excluded. Data capture was completed in the STAMINA-HFP registry in July 2004. Patients participating in this study (n\u00a0=\u00a0811) had to have complete baseline health status assessments and a measure of hemoglobin. They were slightly more likely to be white (75% vs. 68%, P\u00a0=\u00a00.03) and to not smoke (90% vs. 86%, P\u00a0=\u00a00.03) but had similar distributions of NYHA class, KCCQ scores, and hemoglobin as compared with the entire cohort of 1,090 patients. For the longitudinal assessments of test\u2013retest reliability and responsiveness, a 3-month health status assessment was also required from these patients (n\u00a0=\u00a0698). At the time of follow-up interviews, a patient global assessment of change question, asking patients to describe \u2018how [they] think [their] condition has changed compared to the start of the study,\u2019 was also asked at each follow-up interview. Responses were on a 7-point categorical response scale ranging from \u2018markedly improved\u2019 to \u2018markedly worse.\u2019 The initial 3-month assessment was used for this study because it was the closest assessment to the time of their anemia evaluation and because it had the shortest recall period with which to assess patients\u2019 perspectives of clinical change of any of the other assessments.\nThe KCCQ is a 23-item, disease-specific measure that quantifies four clinically relevant domains of patients\u2019 health status including physical limitations, symptoms (frequency, severity, and change over time), a heart failure-specific assessment of their quality of life and their perceived social limitations due to heart failure, and a self-efficacy domain (a measure of patients\u2019 knowledge of how to best manage their disease). The four health status scales can be combined into a single, overall summary score [10]. Scores range from 0\u2013100, where higher scores indicate better functioning, fewer symptoms, and better disease-specific quality of life. The KCCQs validity and reproducibility have been previously supported and recent studies have demonstrated that both cross-sectional variations [25, 26] and changes [27] in KCCQ scores are prognostic of subsequent mortality and HF hospitalizations. A mean difference between groups of patients and an intra-individual change over time of \u22655 points is considered clinically significant. This was established in two studies: a prospective study of 476 outpatients in which a mean change of five points in the KCCQ overall summary scale was observed in those patients who experienced a small, but clinically significant change in their heart failure [28] and in a prospective study of 1,358 HF patients in which a 5-point change was associated with an 11% change in the multivariable-adjusted hazard ratio of hospitalization and cardiovascular death [27].\nClassification of anemia\nPatients were classified as being anemic or not based upon their hemoglobin at the time of study enrollment. Thus, the patients\u2019 hemoglobin value at baseline determined their anemia status throughout the remainder of the study. The World Health Organization (WHO) definition of anemia was used (hemoglobin <13\u00a0g\/dl for men, <12\u00a0g\/dl for women).\nStatistical analysis\nThe patient population was divided into those with and without anemia, and baseline demographic and clinical characteristics were compared with chi-square tests for categorical variables and t tests for continuous variables.\nTo evaluate the construct validity of the KCCQ in anemic and nonanemic patients, comparisons across NYHA classifications from the baseline interviews were conducted. A two-way ANOVA with KCCQ overall summary score as the dependent variable and baseline anemia classification, NYHA, and an anemia-by-NYHA interaction term as independent variables was constructed to establish whether the association of NYHA and KCCQ was different in patients with and without anemia.\nBoth internal consistency and test\u2013retest reliability estimates were compared among anemic and nonanemic patients. Internal consistency reliability was estimated using Cronbach\u2019s alpha. This value was calculated independently for patients with and without anemia. Test\u2013retest reliability was performed using a t test, by comparing the 3-month change in KCCQ scores for patients who responded on the global change in health question that their condition was stable over the previous 3\u00a0months. Three months was selected as the interval of analysis to maximize the number of patients included in the analyses, to leverage the existing data in which a 3-month interval was selected, and to replicate the time frame of the original validation studies evaluating the KCCQ [10].\nTo compare the responsiveness of the KCCQ in patients with and without anemia, the association of patients\u2019 means change in KCCQ scores with their responses to the global health question were assessed using linear regression. In these linear regression analyses, the mean change in KCCQ scores was used as the dependent variable and baseline anemia status, global assessment of change, and an anemia-by-global assessment of change interaction term were independent variables. As a secondary analysis, patients\u2019 perspectives of their 3-month change in status was categorized into improved (mildly\u2013markedly), stable (no change), and worsened (mildly\u2013markedly), and mean change in KCCQ scores were compared for those with and without anemia by these patient-perceived categories of change in health status. All analyses were conducted in SAS version 9.1 (SAS Institute Inc., Cary, NC, USA). All statistical tests were two-sided and a P value <0.05 was used as the threshold to declare statistical significance.\nResults\nPatient characteristics\nOverall, 811 patients completed the baseline clinical assessments, including the KCCQ interview, and were used to assess baseline construct validity and internal consistency reliability. Only 4% of the population had KCCQ scores of 100, suggesting a minimal ceiling effect of the instrument in this population. Among the patients who completed the baseline assessments, 268 (33%) met the WHO criteria for anemia. Baseline characteristics of the anemic and nonanemic patients are provided in Table\u00a01. Compared to nonanemic patients, anemic patients were significantly more likely to be older than  (mean\u00a0\u00b1\u00a0SD\u00a0=\u00a067\u00a0\u00b1\u00a013 vs. 63\u00a0\u00b1\u00a014\u00a0years, P\u00a0<\u00a00.001), non-Caucasian (30% vs. 23%, P\u00a0=\u00a00.03), and to have an ischemic HF etiology (46% vs. 38%, P\u00a0=\u00a00.04), diabetes (47% vs. 34%, P\u00a0<\u00a00.001), lower glomerular filtration rate (GFR) (50.8\u00a0\u00b1\u00a024.6 vs. 63.9\u00a0\u00b1\u00a023.1\u00a0ml\/min\/m2, P\u00a0<\u00a00.001), chronic renal insufficiency (33% vs. 15%, P\u00a0<\u00a00.001), higher serum creatinine (1.7\u00a0\u00b1\u00a01.5 vs. 1.3\u00a0\u00b1\u00a01.2\u00a0mg\/dl, P\u00a0<\u00a00.001), worse NYHA class (44% vs. 32% class III or IV, P\u00a0<\u00a00.001), lower KCCQ scores (61\u00a0\u00b1\u00a023 vs. 65\u00a0\u00b1\u00a023, P\u00a0=\u00a00.009), and to be treated with diuretics (90% vs. 84%, P\u00a0=\u00a00.04).\nTable\u00a01Baseline clinical characteristics of the populationCharacteristicAnemic (n\u00a0=\u00a0268)Nonanemic (n\u00a0=\u00a0543)P valueAge (mean\u00a0+\/\u2212\u00a0SD)67.0\u00a0+\/\u2212\u00a013.162.9\u00a0+\/\u2212\u00a013.5<0.001Male (n, %)105 (39.2%)239 (44.0%)0.190Non-Caucasian (n, %)80 (29.9%)123 (22.7%)0.026No insurance (n, %)6 (2.2%)19 (3.5%)0.329Etiology of HF (n, %)0.038\u00a0\u00a0\u00a0\u00a0Hypertensive 49 (18.3%)92 (17.0%)\u00a0\u00a0\u00a0\u00a0Ischemic124 (46.3%)208 (38.4%)\u00a0\u00a0\u00a0\u00a0Other95 (35.4%)242 (44.6%)Mean left ventricular ejection fraction (mean\u00a0+\/\u2212\u00a0SD)36.4\u00a0+\/\u2212\u00a015.935.9\u00a0+\/\u2212\u00a016.20.742Diabetes (n, %)125 (46.8%)185 (34.1%)<0.001COPD (n, %)51 (19.1%)87 (16.0%)0.273Chronic renal insufficiency (n, %)89 (33.3%)82 (15.1%)<0.001Current smoker (n, %)17 (6.3%)61 (11.2%)0.026Body mass index (mean\u00a0+\/\u2212\u00a0SD)30.3\u00a0+\/\u2212\u00a07.331.1\u00a0+\/\u2212\u00a07.40.181NYHA (n, %)0.001 \u00a0\u00a0\u00a0\u00a0I35 (13.1%)99 (18.2%)\u00a0\u00a0\u00a0\u00a0II116 (43.3%)268 (49.4%)\u00a0\u00a0\u00a0\u00a0III105 (39.2%)169 (31.1%)\u00a0\u00a0\u00a0\u00a0IV12 (4.5%)7 (1.3%)Mean KCCQ overall summary score (mean\u00a0+\/\u2212\u00a0SD)60.8\u00a0+\/\u2212\u00a022.865.3\u00a0+\/\u2212\u00a023.20.009Current medications (n, %)\u00a0\u00a0\u00a0\u00a0ACE inhibitor\/ARB232 (86.6%)486 (89.5%)0.217\u00a0\u00a0\u00a0\u00a0\u00df-blocker222 (82.8%)446 (82.1%)0.806\u00a0\u00a0\u00a0\u00a0Diuretic240 (89.6%)458 (84.3%)0.044\u00a0\u00a0\u00a0\u00a0Spironolactone76 (28.4%)132 (24.3%)0.214\u00a0\u00a0\u00a0\u00a0Digoxin126 (47.0%)245 (45.1%)0.610Mean hemoglobin (mean\u00a0+\/\u2212\u00a0SD)11.4\u00a0+\/\u2212\u00a01.114.1\u00a0+\/\u2212\u00a01.3<0.001Mean creatinine (mean\u00a0+\/\u2212\u00a0SD)1.7\u00a0+\/\u2212\u00a01.51.3\u00a0+\/\u2212\u00a01.2<0.001Glomerular filtration rate (ml\/min\/m2, mean\u00a0+\/\u2212\u00a0SD)*50.8\u00a0+\/\u2212\u00a024.663.9\u00a0+\/\u2212\u00a023.1<0.001*\u00a0Modification of diet in renal disease (MDRD) abbreviated formula\nFor the longitudinal analyses of test\u2013retest reliability and responsiveness, 698 (86.1%) patients provided both baseline and 3-month assessments. No statistically significant differences in any of the patient characteristics listed in Table\u00a01 were observed between those who did and did not participate in the 3-month interviews. Notably, no difference in anemia classification (33.0% among participants vs. 33.6% in those without follow-up, P\u00a0=\u00a00.89) or mean hemoglobin (13.2\u00a0\u00b1\u00a01.8 vs. 13.2\u00a0\u00b1\u00a02.1, P\u00a0=\u00a00.94) was observed between those with and without follow-up.\nConstruct validity\nFigure\u00a01 illustrates the relationship between the overall KCCQ score by NYHA class in anemic and nonanemic patients. Mean KCCQ scores tracked strongly with NYHA class (mean summary score\u00a0\u00b1\u00a0SE for the entire population\u00a0=\u00a080.3\u00a0\u00b1\u00a01.6, 68.0\u00a0\u00b1\u00a01.0, 50.9\u00a0\u00b1\u00a01.2, and 45.6\u00a0\u00b1\u00a05.4 for NYHA classes I\u2013IV respectively, P\u00a0<\u00a00.0001). While the mean KCCQ scores for the patients with NYHA class IV differed substantially, this was likely due to the small numbers of patients in these subgroups, resulting in a large standard deviation of scores (KCCQ scores in anemic [n\u00a0=\u00a012] and nonanemic [n\u00a0=\u00a07] patients\u00a0=\u00a039.5\u00a0\u00b1\u00a020 vs. 56.2\u00a0\u00b1\u00a028). Furthermore, the relationship between KCCQ and NYHA did not differ significantly between anemic and nonanemic patients (P\u00a0=\u00a00.38 for interaction).\nFig.\u00a01KCCQ overall summary score by NYHA class in anemic and nonanemic patients\nReliability\nThe internal consistency reliability (Cronbach\u2019s alpha) of the KCCQ overall summary score for the entire population was 0.93. In patients who were anemic and nonanemic, the Cronbach alphas were 0.92 and 0.93, respectively.\nThe mean (\u00b1SE) KCCQ overall summary score change in patients who reported no change over 3\u00a0months in their clinical condition (n\u00a0=\u00a0257) was \u22121.2\u00a0\u00b1\u00a00.7. Among anemic patients (n\u00a0=\u00a073) the mean change was \u22122.8\u00a0\u00b1\u00a01.4, and for nonanemic patients (n\u00a0=\u00a0184) the mean change was \u22120.5\u00a0\u00b1\u00a00.8. These mean 3-month changes were similar between anemic and nonanemic patients (P\u00a0=\u00a00.14). Importantly, the change between and within both groups was less than five points over time and, thus, not considered clinically significant. The intraclass correlation coefficient for the KCCQ overall summary score was 0.885 in the overall cohort and did not differ among those with and without anemia (0.865 for anemic patients and 0.892 for nonanemic patients [P\u00a0=\u00a00.11]).\nResponsiveness\nTo compare the sensitivity of the KCCQ to patients\u2019 perceptions of change, the mean change in KCCQ scores across each category of change were compared in anemic and nonanemic patients. Figure\u00a02 describes the means and 95% confidence intervals of change in KCCQ scores across crude categories of improved (mild to marked), no change, and worsening (mild to marked) of their overall clinical status during the first 3\u00a0months of the study. No significant differences by anemia status were detected within any of the three categories of reported health change. In regression analysis, mean 3-month KCCQ change was found to be linearly related to reported health change, with a slope of \u22122.7 points (95% CI\u00a0=\u00a0[\u22123.6, \u22121.9]) per 1 step down on the Likert-scale response. Nonlinearity was tested using cubic splines and was found to be nonsignificant (P\u00a0=\u00a00.68). The association was consistent in both anemic and nonanemic patients (slope estimates\u00a0=\u00a0\u22123.4 [95% CI\u00a0=\u00a0(\u22125.0, \u22121.8)] vs. \u22122.5 [95% CI\u00a0=\u00a0(\u22123.4, \u22121.6)], P-value\u00a0=\u00a00.33).\nFig.\u00a02Three-month KCCQ change by reported health change and anemia status\nDiscussion\nWe report empirical evidence to support the reliability and validity (including responsiveness) of the KCCQ in HF patients with anemia. We found similar associations between the KCCQ overall summary score and NYHA classification, similar internal consistency and test\u2013retest reliability in stable patients, and similar responsiveness of the KCCQ to patients\u2019 perceptions of 3-month clinical change. In light of the potential overlap in symptoms between anemia and HF, we tested and rejected the hypothesis that the psychometric properties of the KCCQ might differ between HF patients with and without anemia. Thus, the KCCQ could be a valid, reliable, and responsive outcome for clinical trials of anemia treatment in HF.\nTo date, there has been a paucity of literature examining attribution of symptoms to one of a spectrum of potentially co-occurring diseases in patient-reported health status assessments. A common recommendation for the design of clinical trials is to include both disease-specific and generic measures of health status. These recommendations are predicated upon the desire to capture the impact of treatments on a specific disease of interest, as well as the overall impact of treatment on patients\u2019 health outside the condition of interest. The latter intended to capture side effects or other unanticipated complications of therapy.\nHowever, since patients are often only aware of the symptoms that they experience, and not the underlying pathophysiology that is responsible for those symptoms, it is unclear whether a distinct pathophysiology (e.g. anemia) could confound the psychometric properties of a disease-specific health status measure quantifying a disease with similar clinical manifestations (e.g. HF). While a review of the literature and an independent investigation of Rijken et al. demonstrated that co-occurring diseases are associated, as expected, with worse scores on general health status measures, they did not examine the influence of co-occurring diseases on disease-specific instruments [29]. To examine the relative performance of generic and disease-specific instruments, Ren and colleagues demonstrated, in both cross-sectional [30] and longitudinal [31] analyses, that disease-specific measures outperformed generic measures in terms of their content and discriminative validity among veterans with multiple comorbidities. However, these latter studies did not focus upon the ability of a disease-specific measure to be a psychometrically valid representation of the disease of interest when another co-occurring illness may manifest similar symptoms.\nOne research situation in which one disease has similar symptoms to another is depression and heart failure. Numerous studies have documented that depressed patients have worse health status than nondepressed patients [32\u201335], yet both depression and heart failure can manifest symptoms of fatigue. Interestingly, Rumsfeld et al. were able to demonstrate that depression was associated with worse KCCQ scores over time, regardless of baseline scores, and that among nondepressed patients, poorer health status was associated with a greater likelihood of becoming depressed [36]. These studies did not address whether the KCCQ was equally valid in both depressed and nondepressed patients, but did show important interrelationships between diseases that can have similar clinical manifestations.\nSeveral potential limitations of this article should be considered in interpreting our findings. First, validation is always difficult to establish given the absence of a criterion standard for a patient-centered outcome such as health status. We selected the NYHA classification because it is a commonly used metric with which to quantify HF patients\u2019 health status. Yet the reproducibility and interobserver variability of NYHA assessments is known to be lower than directly soliciting such data from patients themselves. [37]. A second concern is that our assessments of change in HF came from a global patient-reported estimate of change. At best this is a crude assessment and much more detailed examinations of clinical change in the KCCQ have been conducted [28]. Although the quantitative assessments of change in KCCQ scores may be different than in previous studies, there is no a priori reason to suspect that these patient-reported assessments of global change would differ by patients\u2019 anemia status. Therefore, finding similar associations between 3-month change in KCCQ scores and patients\u2019 global assessments of change between those with and without anemia supports the fact that the responsiveness of the KCCQ to clinical change is similar in both populations of patients. In addition, concluding that no differences exist in the performance characteristics of the KCCQ in anemic and nonanemic patients could be incorrect due to a type II error. However, the point estimates for all of our analyses were extremely similar and included\u00a0more than\u00a0800 patients, which is greater than six\u00a0times as large as the original study documenting the reliability and validity (including responsiveness) of the KCCQ [10]. Finally, 12% of patients were lost to follow-up and we cannot exclude the introduction of a potential bias from failing to include these patients. Supporting the generalizability of our study, however, is the similar degree of association between KCCQ scores and NYHA classification, the similar estimates of internal consistency and test\u2013retest reliability, and similar responsiveness observed in this population as seen in other studies testing the psychometric properties of the KCCQ [10, 28].\nIn summary, we provide empirical evidence to support the ability of a disease-specific measure of heart failure patients\u2019 health status, the KCCQ, regardless of the presence of anemia. As such, we believe that patient-reported health status outcomes are a valid, reliable, and sensitive means to quantify the impact of anemia treatment on HF-specific outcomes. Given that the improvement in patients\u2019 health status is a primary goal of treatment, being able to accurately capture HF patients\u2019 health status in trials of anemia therapy are important. The KCCQ should be a valid and sensitive measure for accomplishing these goals.","keyphrases":["heart failure","health status","anemia"],"prmu":["P","P","P"]}
{"id":"Matern_Child_Health_J-2-2-1592153","title":"Health Care Provider Knowledge and Practices Regarding Folic Acid, United States, 2002\u20132003\n","text":"Objective: To assess health care providers (HCP) knowledge and practices regarding folic acid (FA) use for neural tube defect (NTD) prevention. Methods: Two identical surveys were conducted among 611 obstetricians\/gynecologists (OB\/GYNs) and family\/general physicians (FAM\/GENs) (2002), and 500 physician assistants (PAs), nurse practitioners (NPs), certified nurse midwives (CNMs), and registered nurses (2003) to ascertain knowledge and practices regarding FA. For analysis, T-tests, univariate and multivariate logistic regression modeling were used. Results: Universally, providers knew that FA prevents birth defects. Over 88% knew when a woman should start taking folic acid for the prevention of NTDs; and over 85% knew FA supplementation beyond what is available in the diet is necessary. However, only half knew that 50% of all pregnancies in the United States are unplanned. Women heard information about multivitamins or FA most often during well woman visits in obstetrical\/gynecology (ob\/gyn) practice settings (65%), and about 50% of the time during well woman visits in family\/general (fam\/gen) practice settings and 50% of the time at gynecology visits (both settings). Among all providers, 42% did not know the correct FA dosage (400 \u03bcg daily). HCPs taking multivitamins were more than twice as likely to recommend multivitamins to their patients (Odds Ratio [OR] 2.27 95%, Confidence Interval [CI] 1.75\u20132.94). HCPs with lower income clients (OR 1.49, CI 1.22\u20131.81) and HCPs with practices having more than 10% minorities (OR 1.46, CI 1.11\u20131.92) were more likely to recommend supplements. NPs in ob\/gyn settings were most likely and FAM\/GENs were least likely to recommend supplements (OR 3.06, CL 1.36\u20136.90 and OR 0.64, CL 0.45\u20130.90 respectively). Conclusions: Knowledge about birth defects and the necessity of supplemental FA was high. Increasing knowledge about unintended pregnancy rates and correct dosages of FA is needed. The strongest predictor for recommending the use of FA supplements was whether the provider took a multivitamin.\nIntroduction\nBirth defects continue to be the leading cause of infant morta-lity in the United States [1], and those of the nervous system, including neural tube defects (NTDs), are the third leading type of birth defect [2]. NTDs are serious birth defects of the brain (anencephaly) and spine (spina bifida), affecting appro-ximately 3000 pregnancies each year in the United States [3]. Studies show that 50\u201370% of these birth defects can be prevented if a woman consumes adequate folic acid (FA) daily before conception and throughout the first trimester of pregnancy [4, 5]. The U.S. Public Health Service (USPHS) (1992) and Institute of Medicine (IOM) (1998) issued separate recommendations that all women of childbearing age consume 400\u00a0\u03bcg of FA daily for the prevention of NTDs [5, 6]. Women can obtain adequate amounts of FA by taking a folic acid supplement, taking a multivitamin containing folic acid, or by eating cereal grain products fortified with 100% of the RDA of folic acid.\nIn 1998, the U.S. Food and Drug Administration required mandatory fortification of enriched cereal grains with 140\u00a0\u03bcg of FA per 100\u00a0g of cereal grain product in an effort to reduce the occurrence of NTDs [7]. It was estimated that FA fortification at this level would provide about 0.1\u00a0mg of additional folic acid in the average diet. Recent evidence from the literature suggests that fortification has in fact doubled this initial projection [8]. Fortification of cereal grain products has resulted in a 27% reduction in NTDs in the United States [3], which is relatively consistent with the observed population\u2019s increase of folic acid by 0.2\u00a0mg\/d [9]. While fortification has made a significant contribution in preventing NTDs, most women in the U.S. still do not get enough FA from fortification alone. In a recent study examining serum folate levels of women of childbearing age post fortification, \u201cless than 10% of women achieved a red blood cell folate level that is associated with a significant decrease of NTDs\u201d through diet\u00a0alone [10]. Therefore, it is still necessary for women of childbearing age to consume vitamins containing FA to get the daily recommended amount of FA (400\u00a0\u03bcg).\nSince 1995, the March of Dimes (MoD) and the Gallup Organization have conducted an annual national survey among women of childbearing age regarding their awareness, knowledge, and consumption of FA. Early results indicated a lack of awareness about FA among women of childbearing age. In 1999, in response to these results, the MoD, the Centers for Disease Control and Prevention (CDC), and the National Council on Folic Acid (NCFA) began a concerted effort to increase FA awareness through education campaigns targeting women and health care providers (HCPs). Based on the MoD survey, FA awareness in women of childbearing age has risen from 52% in 1995 to 77% in 2004 [11]. Lagging behind the increase in awareness, daily consumption of a multivitamin containing FA increased only slightly from 32% in 1995 to 40% in 2004 [11, 12]. When asked what types of things would encourage them to take a multivitamin daily, unprompted, 42% of women who were aware of, but not consuming, FA said they would take a multivitamin if advised to do so by an HCP [11]. Additionally, in the 2004 HealthStyles Survey, 91% of women 18 through 45 years of age agreed or strongly agreed that they would take a multivitamin daily to prevent birth defects during future pregnancies if their doctor encouraged them to do so (unpublished data) [13].\nTwo recent studies examined HCP practices regarding FA in the United States [14, 15]. While providers were generally aware of folic acid\u2019s role in NTD prevention, only half of providers surveyed in both studies discussed folic acid on a regular basis with their patients of childbearing age.\nIn 2001, the MoD, in partnership with the CDC, initiated this project to examine HCP practices regarding FA. The objectives of this research are to:Assess HCP knowledge of FA recommendations, including dosage and timing for prevention of NTDs, among different provider types: obstetricians\/gynecologists (OB\/GYNs), family\/general physicians (FAM\/GENs), nurse practitioners (NPs), certified nurse midwives (CNMs), physician assistants (PAs), and registered nurses (RNs).Assess current practices among HCPs with respect to recommending FA to women of childbearing age.Identify the best means of supporting HCP efforts to promote FA consumption in female patients of childbearing age.\nMethods\nRandom sample telephone surveys lasting an average of 12\u201318\u00a0minutes in length, were conducted in 2002 and 2003 among HCPs working in either obstetrical\/gynecology (ob\/gyn) practice settings or family\/general (fam\/gen) practice settings. These practice settings were selected because they represent the settings where the highest proportion of women of childbearing age receive care on a regular basis.\nGeographic quotas were established to ensure a representation of respondents proportional to the actual distribution of office-based providers nationwide. Providers were identified through random sampling lists purchased from American Medical Information Inc., a division of InfoUSA. To identify sufficient representation of providers based in federally qualified health centers, a list of centers from the Health Resources and Services Administration\u2019s Bureau of Primary Health Care website (http:\/\/ask.hrsa.gov\/pc\/) was added to the sampling frame.\nThe identities of respondents were not revealed. This research met the requirements of Title 45 CFR part 46 for the protection of human subjects.\nTo encourage participation, respondents were offered a $50 honorarium. The identity of the sponsors (CDC and MoD) was not divulged to respondents. A cover letter from the research firm was faxed to participants who requested additional background information about the survey.\nHealth care providers were eligible to participate if they personally saw adult female patients for office or clinic visits, and worked in ob\/gyn, or fam\/gen medicine practices.\nThe surveys were presented as a series of true\/false, multiple choice, and open-ended questions. The survey sought to determine the level of HCP knowledge regarding the role of FA in the prevention of NTDs, when HCPs recommended FA, and how often HCPs recommended FA to women of childbearing age.\nData were analyzed using SPSS software (SPSS, 12.0, SPSS, Inc., 2003, Chicago, IL). T-tests, univariate comparisons and multivariate logistic regression modeling of the data were conducted. All analyses were tested for significance at the 95% confidence level (p-value 0.05).\nResults\nInvestigators surveyed 611 physician HCPs in 2002 and 500 nonphysician HCPs in 2003 (Table 1).\nTable 1Provider type and practice settingPractice settingObstetrics\/Family\/GeneralProvider typeaGynecologyMedicineOB\/GYN362\u20070FAM\/GEN\u20070249PAs1638NPs5546CNMs200\u20070RNs7763Total710396Note. 5 missing provider variables not categorized.aProvider type abbreviations: OB\/GYN\u2014obstetrician\/gynecologist; FAM\/GEN\u2014family or general practice physician; PA\u2014physician assistant; NP\u2014nurse practitioner; CNM\u2014certified nurse midwife; RN\u2014registered nurse.\nIn 2002, 2552 physicians were screened as potentially qualified respondents. Of these 2552 physicians, 1213 (48%) did not participate in surveys, 366 (14%) requested additional information but did not schedule an interview, 184 (7%) did not meet practice setting requirements, 142 (6%) had fax numbers as the primary contact, 19 (1%) were qualified refusals and 11 (0.4%) had appointments set but not kept. The response rate for eligible physicians was 24%.\nIn 2003, 650 nonphysician HCPs were screened as potentially qualified respondents. Of these 650 nonphysician HCPs, 73 (11%) did not participate in surveys and 75 (12%) were qualified refusals. The response rate for eligible nonphysician HCPs was 77%.\nThe majority of physicians were male (77%), younger than 55 years of age (69%), and in practice over 20 years (50%). Nonphysician HCPs were typically female (97%) and younger than 55 years of age (82%); 28% had been in practice over 20 years. Nonphysician HCPs were two times more likely than physician HCPs to work with underserved populations (Odds Ratio [OR] 1.97, Confidence Interval [CI] 1.54\u20132.53). Nonphysician HCPs saw more women of childbearing age in their practices (67%) than physicans (52%) (p-value <0.000).\nThe following results were obtained from a series of true\/false questions.\nIn general, knowledge that women need a supplemental source of FA was high (\u223c85%). Universally, HCPs in both practice settings (97%) knew correctly that FA can prevent some birth defects. However, only \u223c60% of all providers knew that most grain products in the United States are fortified with FA. In addition, there were significant differences in response to this question among provider types according to practice setting, ranging from 71.3% of RNs in ob\/gyn settings to 44.7% of PAs in fam\/gen settings (p-value 0.030).\nThe following data were obtained from multiple choice questions. Providers in ob\/gyn settings were not more knowledgeable than providers in fam\/gen settings about when a woman should start taking FA for the prevention of NTDs (at least one month prior to conception through the first trimester of pregnancy) (88% vs. 89%, respectively, p-value 0.094).\nBecause the majority of respondents knew the correct timing for a woman to start taking FA, providers were asked to identify what percentage of pregnancies they thought were unintended in the United States. Only about half of HCPs in both settings knew that approximately 50% of all pregnancies in the United States are unplanned [16]. The most knowledgeable group was NPs in ob\/gyn settings (58.9%), and the least knowledgeable group was RNs in ob\/gyn settings (35.0%) (p-value 0.002). Providers answering incorrectly were more likely to underestimate rather than overestimate the unintended pregnancy rate. Although a woman\u2019s receptiveness to the FA message might increase during a preconception care visit, HCPs providing prenatal care saw only a quarter of their pregnant patients for a preconception care visit.\nOver half (58%) of all providers were knowledgeable about the amount of FA (400\u00a0\u03bcg\u00a0) a woman needs daily for prevention of NTDs. Providers in ob\/gyn settings were more knowledgeable than providers in fam\/gen settings. The most knowledgeable group was CNMs, with 73.4% knowing the correct daily dosage of FA; the least knowledgeable group was FAM\/GEN physicians (41.9%) (p-value <0.000). When asked about the amount of FA a woman with a prior NTD affected pregnancy should take to prevent the recurrence of a NTD (4\u00a0mg), overall knowledge dropped dramatically, with only about 30% of all providers knowing the correct dose. Providers in ob\/gyn settings were more knowledgeable than providers in fam\/gen settings, The most knowledgeable group was OB\/GYNs (46%) and the least knowledgeable were RNs in fam\/gen settings (11.1%) (p-value <0.000).\nOf critical importance to increasing consumption of FA among women of childbearing age is whether FA is incorporated into preventive health care messages during provider\/patient encounters. Providers were asked if they always, usually, sometimes, or never addressed multivitamins or FA during well-woman examinations. More providers in ob\/gyn settings than in fam\/gen settings stated they regularly (always\/usually) addressed multivitamins or FA during well-woman visits (65% vs. 50%, respectively). Women were 1.8 times more likely to hear about multivitamins or FA during encounters in ob\/gyn settings than in fam\/gen settings (CI 1.42\u20132.34). The group most likely to talk to patients about multivitamins and FA was NPs in ob\/gyn settings (83.9%); the least likely were FAM\/GEN physicians (45.6%) (p-value <0.00). Fewer counseling opportunities were taken at other types of examinations. Women heard the FA message about half the time from providers in both settings at routine gynecology visits. As expected, the fewest counseling opportunities taken were at episodic visits in both settings.\nPredictors for recommending a multivitamin at a well-woman visit were determined. The type of provider and clinic setting variables were combined as the base variable, with OB\/GYNs as the referent group. The dependant variable was whether the HCP always or usually recommended a multivitamin during a well-woman visit. Variables examined were number of weekly visits, provider age, sex, personal vitamin use, provider\u2019s years in practice, ethnicity of patients, practice setting, socioeconomic status of patient base, patient age, clinic setting (urban, suburban, or rural) and region of practice. Clinic setting (urban, suburban, or rural), provider age, region of practice, provider\u2019s years in practice, number of weekly visits, and patient age variables were not statistically significant in univariate analysis.\nTable 2 represents the variables included in the logistic regression model. In comparison with OB\/GYNs, FAM\/GEN physicians were statistically less likely (OR 0.64, CI 0.45\u20130.90) to recommend multivitamins, while NPs in ob\/gyn settings were three times more likely (OR 3.06, CI 1.36\u20136.90) than their OB\/GYN physician counterparts to provide counsel on multivitamins. While not quite reaching significance, CNMs were 1.6 times more likely (OR 1.56, CI 0.96\u20132.53) than OB\/GYNs to provide counsel on multivitamins. All other groups were not significantly different from OB\/GYNs. Interestingly, whether a provider personally took a multivitamin was one of the strongest predictors of recommending multivitamin use to patients. Providers who regularly took a multivitamin were 2.3 times more likely (OR 2.27, CI 1.75\u20132.94) to recommend multivitamin use to their patients. Additional predictors of recommending multivitamin use were whether a provider stated that his or her practice\u2019s patient base was classified as poverty or low income, or if the practice consisted of at least 10% minorities. Being a female provider was also correlated with recommending multivitamin use to women of child-bearing age.\nTable 2Factors associated with recommending multivitamins at well-woman visits included in multivariate logistic regression modelVariablesOdds ratio95% Confidence intervalProvider Type\u2003OB\/GYNReferentReferent\u2003FAM\/GEN0.640.45\u20130.90*\u2003CNM1.560.96\u20132.53\u2003NP in ob\/gyn setting3.061.36\u20136.90*\u2003PA in ob\/gyn setting0.890.30\u20132.66\u2003RN in ob\/gyn setting0.800.45\u20131.43\u2003NP in fam prac setting0.900.45\u20131.79\u2003PA in fam prac setting0.630.30\u20131.34\u2003RN in fam prac setting0.920.48\u20131.77Gender\u2003MaleReferentReferent\u2003Female1.621.11\u20132.36*Provider takes a multivitamin\u2003NoReferentReferent\u2003Yes2.271.75\u20132.94*SES of patient base\u2003Middle or high incomeReferentReferent\u2003Poverty or low income1.491.22\u20131.81*Minorities\u2003<10% minoritiesReferentReferent\u2003>10% minorities1.461.11\u20131.92**Denotes statistical significance.\nApproximately three-quarters of all respondents had seen information about FA in the last year. Unprompted, both physician and nonphysician HCPs reported that the organization most likely to have provided that information was the MoD (25% and 46% respectively). Medical journals were identified most frequently as a way to effectively reach providers with information about FA (66% physicians, 62% nonphysicians). Nonphysician providers reported the Internet as the second most effective way (15%), and physicians reported either medical associations or conferences as the second most effective way to reach them with information about FA (12 and 11%, respectively). Providers identified up to two types of resources they would like to have to promote FA intake among patients. Printed materials were the most popular type of resource requested by HCPs. Sixty percent (60%) of physician and 69% of nonphysician providers requested brochures to distribute to their patients. Additional print resource requests included charts and articles. Free vitamins for patients were also requested (data not shown).\nDiscussion\nThese findings suggest that physician and nonphysician providers in both ob\/gyn and fam\/gen settings know that FA can prevent NTDs and that FA must be used from at least one month before pregnancy and continued through the first trimester. Consistent with other studies, provider knowledge of FA did not necessarily translate into counseling patients about its benefits [15 ,17].\nProviders in ob\/gyn settings were almost twice as likely as providers in fam\/gen settings to regularly address multivitamin or FA use during well-woman visits. Although NPs and CNMs in ob\/gyn settings were most likely to regularly address multivitamin or FA use with their patients, these providers might not have as many opportunities to counsel women who are not contemplating pregnancy. Because providers in fam\/gen settings provide routine care to large numbers of women of childbearing age, they play an important role in counseling women about FA consumption. Healthy People 2010 calls for an increase in the number of pregnancies begun with an optimum FA level. Providers in fam\/gen settings are critical participants in the process of making the goals of Healthy People 2010 a reality [18, 19]. Greater opportunities for promoting FA exist through changing provider practice behavior in fam\/gen settings. It also appears that well-woman examinations in fam\/gen settings and routine gynecology visits in both settings are important yet underutilized opportunities for HCPs to counsel women about multivitamin and FA use. The episodic visit was the least utilized, but with all the competing priorities at these types of encounters, counseling of multivitamins might not take precedence over other topics to be covered within limited timeframes.\nProviders in ob\/gyn settings were more likely than providers in fam\/gen settings to know the correct amount of FA needed for NTD prevention (400\u00a0\u03bcg daily). Less than a third of all providers knew the correct amount of FA needed for the prevention of recurrence (4\u00a0mg). Because women with a previous NTD-affected pregnancy are at an increased risk for another NTD-affected pregnancy, it is important for all providers to know the correct amount of FA needed for recurrence prevention.\nOnly about half of providers in both practice settings knew that approximately 50% of pregnancies in the United States are unplanned. Without screening their female patients about pregnancy intention, perhaps providers should be recommending FA to all women of childbearing age.\nOne of the most interesting findings was that the providers surveyed \u201cpractice what they preach.\u201d Providers who regularly took multivitamins were more likely to recommend multivitamins to their patients. Studies looking at other health behaviors have shown this as well [20, 21]. Thus, including personal health behavior change messages in HCP outreach about multivitamins and FA might be advantageous.\nIn this dataset, a practice that served predominantly poverty or low income (low socioeconomic status) clientele or a practice consisting of at least 10% minorities predicted recommending a multivitamin. Among minorities and women with low socio-economic status, critical gaps in knowledge and practice on the benefits of periconceptional folic acid have been documented, especially among Hispanics, who are most vulnerable for NTDs [11, 22\u201324]. The findings in this survey that minorities and women of lower socio-economic status do appear to be targeted is encouraging, and underscores the importance of preconception care education programs for healthcare providers serving high-risk populations.\nThis study has certain limitations. Physician HCPs had a low response rate compared to nonphysician HCPs. We cannot comment on the providers who did not respond to the survey. Providers answering the survey might differ in knowledge and practice than non-responding providers. Additionally, one provider group surveyed (PAs) had small numbers. Many of the variables examined were associated with one another. In the logistic regression modeling, female sex of the HCP was a significant factor in predicting recommending multivitamins to female patients of childbearing age. This is most likely because the majority of the providers surveyed in 2003 were women.\nConclusions\nWhile it would be most beneficial for women of childbearing age to get adequate folic acid from fortified foods alone, alternatives include efforts to educate all childbearing aged women. Several approaches to educating and promoting folic acid consumption are endorsed by the U.S. Public Health Service. These approaches include direct to consumer marketing, public health awareness activities, health care provider outreach, and policy strategies. This study examined just one aspect (HCPs) of the multifaceted approach needed to increase FA intake among women of childbearing age. Collaboration between public health and medical care services is critical in educating women about FA and multivitamin use as health care providers are trusted professionals. Mobilizing folic acid awareness efforts to target HCPs might assist providers with patient preconception care education. Training providers while in school or residency programs, or by offering continuing education courses that cover methods of applied behavior change and health education might be useful for increasing the numbers of women hearing about folic acid from their health care provider. Additionally, further research on what barriers HCPs experience when providing preventive care, such as provider time constraints, self-efficacy, and what their perceptions are on the benefits of preventive care, is needed. As many preventive behavior and wellness messages exist, providers are forced to prioritize according to the length of and reason for patient visits. Finding a creative way to make FA counseling a priority is critical to making the FA message a consistent part of the patient\/health care provider dialogue.","keyphrases":["health care provider","knowledge","folic acid","behavior"],"prmu":["P","P","P","P"]}
{"id":"AIDS_Behav-2-2-1544374","title":"How Uganda Reversed Its HIV Epidemic\n","text":"Uganda is one of only two countries in the world that has successfully reversed the course of its HIV epidemic. There remains much controversy about how Uganda's HIV prevalence declined in the 1990s. This article describes the prevention programs and activities that were implemented in Uganda during critical years in its HIV epidemic, 1987 to 1994. Multiple resources were aggregated to fuel HV prevention campaigns at multiple levels to a far greater degree than in neighboring countries. We conclude that the reversed direction of the HIV epidemic in Uganda was the direct result of these interventions and that other countries in the developing world could similarly prevent or reverse the escalation of HIV epidemics with greater availability of HIV prevention resources, and well designed programs that take efforts to a critical breadth and depth of effort.\nIntroduction\nSince the beginning of the World Health Organization's collaboration with developing countries, Uganda was for many years the only country in Africa and only one of two countries (the other Thailand) in the developing world to have clearly reversed its HIV epidemic (Asiimwe-Okiror et al., 1997; Mason, Markovitz, & Kitsiripornchai, 1995; UNAIDS, 1998; USAID, 2003; US House of Representatives, Hearing on the Global AIDS crisis and pandemic in Africa, 2000), although more recently, other reports indicate additional countries are showing possible reductions (Gregson, Garnett, Nyamukapa, & Hallet, 2006; Kibian, 2006; USAID, 2003). Uganda's changes remain the first, most extensive, and of largest scale of decline (USAID, 2003) but are still subject to controversy as to how these changes occurred (Parkhurst, 2002; Stoneburner & Low-Beer, 2004).\nWith regard to the changes in Uganda, much has been written describing which sexual behaviors changed in the Uganda population (Asiimwe-Okiror et al., 1997; Stoneburner & Carballo, 1997; Stoneburner, Low-beer, Barnett, & Whiteside, 2002; UNAIDS, 1998), but there has still been little written about the strategies, activities and program elements that took place that affected these changes. In other words, the changes in sexual behaviors in Uganda that occurred during that time period are known - and the effects on the HIV epidemic are known, yet the continuously missing part of the discussion has been \u201cwhy did these behavior changes occur?.\u201d Similarly why did these changes occur in Uganda and seemingly not in the neighboring countries at the time, or to that degree still anywhere in the developing world?\nThis article attempts to fill this gap. We offer our description of these activities as persons who were involved in Uganda's AIDS program's inception and the first seven years (1987\u20131994) of the program when its effects occurred. Simplistic explanations such as leadership, migration, or awareness of the deaths of others, seem unlikely to be responsible on their own since country leadership principally defines the will for programs and the willingness to ensure activities occur, but does not explain what actual activities did occurred. Regarding migration, changes in HIV prevalence occurred in at least seven and possibly nine towns and villages simultaneously in Uganda (Stoneburner et al., 2002), making migration an unlikely explanation. Also, since similarly high rates of infection (15%\u201330%) were seen in the major cities of at least four neighboring countries (i.e. Rwanda, Burundi, Zambia and Malawi) during the same years (Stoneburner & Carballo, 1997; USAID, 2003) and without the same declines, we must look at what was the Uganda program itself. As will be seen, the program itself differed, both in substance and in intensity, from all neighboring programs in Africa.\nThis article focuses on the years when the HIV\/AIDS program in Uganda was first formed through the time when serologically measured changes in HIV prevalence occurred. Our analysis is based on the country documents, national program plans and reports of that period, and our experiences as part of that program. An attempt is also made to provide comparisons with other national AIDS programs in Africa that occurred in the same years and to describe the major differences between Uganda's program and other programs in the region at the time. First we describe our sources of information followed by our description of the Uganda AIDS prevention activities and their implementation.\nInformation sources\nThis paper focuses principally on program strategies, processes and activities of the Uganda National AIDS Control Program (NACP) for the years 1987\u20131994. Although noted here, we do not provide an in depth analysis of the trends in sexual behavior or HIV changes themselves which have been analyzed elsewhere and repeatedly. This article draws on sources from Ugandan Ministry of Health records and reports, World Health Organization (WHO) records and reports on the country program and programs of neighboring countries, Uganda country assignment reports, the Uganda National Program Review, and plans and country assessments by WHO and USAID written at the time. We also rely on data from specialized studies performed at the time and reports of those of us who were engaged directly in the program reviews, planning and implementation of the Uganda AIDS program during those years, some of whom were also involved in implementation or assistance to implementation of neighboring African countries at the same time.\nBrief history of the Uganda National AIDS Control Program (NACP) 1986\u20131995\nAIDS cases were first recognized in Uganda in 1983, with the first 900 cases reported by 1986, rising to 6,000 cases by 1988 (Uganda AIDS Control Programme, 1989). By 1986\u20131987, 86% of sex workers and 33% of lorry drivers studied were HIV positive and 14% of blood donors and 15% of antenatal clinic attendees in major urban centers were also HIV positive. In 1986, in response to these findings, the national government of Uganda formed the National Committee for the Prevention of AIDS (NCPA) and the NACP. The WHO assisted the government of Uganda in both of these endeavors beginning in January of 1987, when the first \u201cemergency\u201d national plan was written. Subsequently a short term plan (STP), and the 3\u20135 year medium term plan (MTP) were written in 1988\u20131989. A program review was performed in December of 1988 (the first of its kind in Africa and in the world), and the next MTP was drawn up immediately following the findings and recommendations of the first program review. All of these rapid and intense WHO assisted steps were set in motion for all countries in Africa, and subsequently for almost all countries in the world during this time. During the years 1987\u20131990, 120 countries developed NAPs, 40 developed MTPs, and 15 had program reviews before WHO all but disbanded in its prior formulation, and became less involved with direct country support (SIDA\/AIDS WHO, 1992; Slutkin, 2000; Tarantola, 1996; US House of Representatives, 2000; WHO Global Programme on AIDS, 1987\u20131995).\nDuring the years 1987\u20131990, although almost all AIDS control programs, especially those in East and Central Africa, were progressing, Uganda. was the first country in which the WHO worked with the concept of a single national plan and budget, which all donors would agree to use and fund to focus their respective aid, and with a local multi-donor round table aimed at meeting the targeted budget. Uganda was the first country to progress through all of the STP, MTP, joint donor meeting, program review and reprogramming processes. Uganda was then set up to lead the way among all countries in the extent of programming, the amount of on the ground national staffing, WHO staffing support, and global financial support (Mann, 1991; WHO, 1997).\nEmergency assistance \u2013 first plan in Africa\nThe strategies of Uganda's first national AIDS control plan of 1987 were described as \u201cto mount an educational campaign to inform the public on the modes of transmission and ways to avoid infection,\u201d \u201cto reduce transmission through blood transfusion by setting up laboratories to test all blood before transfusion and to reduce transfusion to a minimum,\u201d \u201cto advocate careful sterilization of instruments and contaminated hospital areas to assure patient and health worker safety,\u201d and \u201cto measure the extent of the outbreak and possible co-factors by case surveillance, seroprevalence studies and operational research projects\u201d (Uganda, 1989). These were the usual strategies for all early AIDS programs (WHO Global Programme on AIDS, 1987\u20131995).\nThis initial plan was supported by a WHO grant of $400,000 made available within weeks of the signature of the STP, and then $1\u00a0million more was provided in emergency assistance. Blood was largely screened in the major urban areas, a health education program started, and the sentinel surveillance program initiated (Slutkin, Chin, Tarantola, & Mann, 1988; Uganda, 1989). For the later two strategies, activities moved far in advance of other country programs at the time. This plan also laid the groundwork for the five-year plan, which was to be developed in 1989. The program review of 1988 summarized the accomplishments of the NACP of Uganda in its first 2 and half years.\nThe first Uganda program review\nThe program review of December 1988 (Ministry of Health Uganda, 1989) was performed for the stated purpose of reviewing accomplishments, adequacy, relevance, progress and effectiveness of stated activities, and to make recommendations for the next phase, in this case for the five year plan that was to be written in the following month. It should be noted that WHO Global Program on AIDS (GPA) at this time and the Ugandan government were both in an emergency mode of accelerated program development, cooperation and assistance.\nThe main findings of the Uganda NACP program review led to a series of recommendations. Program strengths highlighted by the review included: \u201ccontinued and increased political commitment of the Government of Uganda and especially the President,\u201d \u201cvital contribution(s) of non governmental organizations,\u201d and a \u201cstrong leadership role by ACP and an emerging coordinating role by (the national) AIDS Control Program (NACP) widely accepted by Government, non government agencies, and donors.\u201d Over 33 critical recommendations were made, commenting on the first two years of activities that were to instruct the next five years of work. Of these 33 recommendations, the following were thought important enough to be highlighted in the executive summary: \u201cthe intensity of the AIDS control activities must be accelerated especially at the district and community level\u201d; \u201c(the national level) ACP should take on a stronger (coordinating) role\u2009\u2026\u2009,\u201d \u201cprevention should remain the primary objective\u2009\u2026\u2009,\u201d that \u201c(there is a)lack of adequate renumeration for national staff\u2009\u2026\u2009and that Uganda and WHO explore all possible ways of overcoming this serious problem\u2009\u2026\u2009,\u201d and \u201c(the) NACP\u2009\u2026\u2009office needs to have more expertise in planning, manpower development, research direction, coordination, and administration\u201d (Uganda, 1989).\nThe first 10 recommendations of the Uganda Program Review (Uganda, 1989) provided insight into what was in process and what was thought to be needed by the combined international and national expert review team. These recommendations were then, within weeks, integrated into the 1989\u20131990 plan. The main recommendations, quoted from the report, were the need for \u201cstepwise application of well-conceived and integrated KAP\/IEC strategies,\u201d \u201cmake these methods\u2009\u2026\u2009available to all groups,\u201d \u201cmore widespread dissemination of factual information,\u201d \u201cincreased emphasis for illiterate people,\u201d \u201cmaterials designed for groups at high risk, and for families with AIDS,\u201d \u201cinformation that can be used by resistance committees (village level political structure through which the Musevini government operationalized new policies) to raise local awareness,\u201d \u201ceffective channels for distributing written and pictorial information,\u201d \u201ctraining,\u201d \u201ccommunication skills,\u201d teaching \u201ctraining skills at district level,\u201d \u201ctraining packages,\u201d \u201cdistributing training packages,\u201d \u201cfinancial support\u2009\u2026\u2009to enable district, community, and other training groups to run training courses,\u201d and \u201chealth educators to (be) fully equipped and functional within weeks rather than\u2009\u2026\u2009several months (to perform this education in the districts).\u201d\nRegarding condoms, the NACP was advised by the co-sponsored review only to \u201creview the use of condoms as part of the strategy particularly where transmission rates are high.\u201d The Ministry of Health, the lead ministry for the Uganda program at the time and for the next several years, was not eager to embrace condoms, for a number of reasons that included availability (dependence), acceptability, and disposal, and the fact that putting trust in one measure was not strategic. These reasons were constantly cited by the program and President Musevini. The review repeatedly noted the need for an urgent push toward the rapid expansion of the information, education, communication and training.\nThe Uganda medium term plan (MTP) 1989\u201394\nThe 1989\u20131994 Five Year plan, and 1989\u20131990 First Year Project Plan and Budget directed the immediate acceleration of the program as outlined in the program review (Ministry of Health Uganda, 1989; Amana, 1990; Moodie et al., 1991; Uganda, 1993).\nThis plan's priority strategies and budget\u00a0allocations are shown in Table 1. The sources of funds for the year 1989 were as follows: WHO Trust Fund $10.6\u00a0million, Bilateral funds (e.g. from USAID, ODA, etc) $7.1\u00a0million, and national (Ugandan) $0.5\u00a0million. Therefore, the Uganda program grew from annual expenditures of $1.0\u00a0mil to $4.0\u00a0mil to $18\u00a0million in its first three years. Dozens of organizations were involved in the management, planning and implementation of the Five Year Plan including local and international non-governmental organizations (NGOs), and all segments of Ugandan society. The management concerns discussed in the program review were addressed in part by assuring that the Ugandan NACP \u201cremain the central coordinating body,\u201d and its staffing was enhanced to 45 Ministry of Health staff, supplemented by 6 WHO staff. Each of these levels of staffing was substantially larger than any other program on the continent. We do not recall any other country having a program of more than 10 local staff or 4 WHO staff at the time.\nTable 1Uganda national AIDS control program budget 1989ActivityTotal (\u00d7 1\u00a0mill. USD)Management1.6Health education7.2Surveillance and care4.3Lab support5.0Total18.1Source: Uganda Aids Control Program 1989\u201390; Ministry of Health, Government of Uganda; Entebbe, Uganda 89.\nAs can be seen in Table 1, the Ugandan health education (IEC) budget was greatly prioritized among all strategies implemented. The IEC program is discussed in more detail below as it is thought essential. As was the case at the time for all programs in central and east Africa, it was essentially demanded of WHO by the country's government that enough funds be made available to ensure a safe blood supply, and have the laboratory supplies and facilities to perform this function. The Ugandan program also became the first country to both field test HIV sentinel surveillance at antenatal clinics and other settings (Nsambya, Uganda being the first sentinel surveillance site in Africa and is still operational), and then the first country to expand this system initially to six sites, and then country wide (Slutkin et al., 1988; Slutkin, Chin, Tarantola, & Mann, 1990).\nHowever with this infrastructure in place, including management, lab support, blood screening, and HIV monitoring, it was then completely unique on the continent to have funds remaining for a national AIDS health education budget to be supported at the level noted. Full country AIDS program budgets for the six neighboring countries were all in the range of $1\u20134\u00a0million total (cf. $18\u00a0million for Uganda). As was to be seen, the Uganda program was then free to prioritize and implement throughout the country the most widespread, extensive and well thought out information, education, and communication program for AIDS prevention (and care) of any country in Africa. As can be also seen in Table 2 (shown for both 1989 and 1990), the Uganda Health Education budget itself was to specifically prioritize local and decentralized programming, local rather than international expenses, and to ensure local staff salaries and perdiems.\nTable 2Uganda AIDS health education budget 1989 and 1990 (\u00d7 1,000 USD)Category19891990Salaries164203Consultants241164Duty travel1214Supplies-field projects238209Equipment-field projects388120Local costs1,7241,539Program support costs360292TOTALS3,1282,541Source: Uganda Aids Control Program 1989\u201390; Ministry of Health, Government of Uganda; Entebbe, Uganda 89.\nIn 1989, the first year in which financial information is available, $3.1\u00a0million of the $7.2\u00a0million available for health education activities were made available from the WHO Trust Fund. The additional $4.1\u00a0million were made available from bilateral sources, e.g. UNICEF, USAID, U.S. Red Cross, MSF and U.S. and other University projects. Of the $3.1\u00a0million, $1.7\u00a0million was made available for local costs. Similarly $1.5\u00a0million of the $2.5\u00a0million spent from the WHO Trust fund IEC budget was used for local costs. The 1991 Evaluation of the Uganda IEC activities stated that the DAMP and the ACP Mass Media activities were \u201ctwo of the most visible of the NACP's projects and together utilize more than half of the IEC budget.\u201d\nThe 1989\u20131992 health education campaign\nThe Health Education (Information, education and communication; IEC) Campaign was to be the center of the Uganda program (Amana, 1990; Moodie et al., 1991; Uganda, 1989). The main goals of the Ugandan IEC program were three fold: 1) mobilization of all formal and informal sectors of Ugandan society; 2) providing IEC materials to all districts; and 3) district level, decentralized information and training. The principal activities were, as proposed by the program review and plan, the development of training packages and a \u201ctraining of trainers\u201d program for \u201call sectors,\u201d public education materials development and dissemination, and district level \u201cmass mobilization\u201d with \u201ccross fertilization\u201d from one district to another, training educators from one district to be used in the next district. This IEC campaign was led by the Ugandan Ministry of Health with technical and implementing assistance from WHO and had very active support and involvement from UNICEF and from several critically important NGOs. The specific program elements are described below.\nTable 3The 1982\u201392 AIDS information, education and communication (IEC) campaign sectors trained (partial listing)Resistance committeesAgricultural workersPoliceSocial mobilizationTraditional healersPrisonsSchoolsUniversitiesYouthMediaMidwivesArmyPerformance artistsWomen's groupsReligious organizationsFamily planning workersRefugeesSource: Uganda Aids Control Program 1989\u201390; Ministry of Health, Government of Uganda; Entebbe, Uganda 89.Fig. 1District mobilization implementation model (DAMP). Source: Moodie, et\u00a0al. ACP\/MOH Uganda and WHO\/GPA, 1991\nTraining of trainers\nA partial listing of the specific \u201csectors\u201d trained by the program are listed in Table 3. The \u201cmain focus\u201d points of this multi-sector training were: correct information on how HIV\/AIDs is and isn't transmitted; destigmatization; care and compassion; and condoms (Moodie et al., 1991; Uganda, 1989). Each of these sectors had specific training activities that began centrally with workshops for Ministries or other \u201ccentral level\u201d personnel, e.g. for permanent secretaries, ministries, parliamentarians, police, teachers, family planning association workers, resistance committee members, etc, and then proceeded with a \u201ctop \u2013 down\u201d or \u201ccenter \u2013 out\u201d cascade of information dissemination and training. These training activities were meant mostly to simply convey the correct information about how HIV\/AIDS is and is not transmitted, and that persons with AIDS were to be treated humanely.\nDistrict mobilization\nThe District AIDS Mobilization Project (DAMP) model implemented by the Ugandan IEC program is shown in Fig.\u00a01. Resources used for the DAMP included 18 paid health educators of the Ministry of Health, 6 local graphic designers, and 3 international consultants (including 1 from WHO, and 1 from UNICEF). Seven vehicles for the central pool, 34 vehicles for the district health educators, and 40 motorcycles were provided by the international community and by the government for this campaign. Two film vans for mass addresses and a number of hand held megaphones and other public address systems were also purchased or made available for this campaign.\nThe District course content as noted in a WHO funded special IEC review stressed 11 topics including scientific overview, basic prevention messages, common questions and misconceptions, communicating with high risk groups, cultural practices, special concerns of women, effective communication skills, care, and counseling. District level activities used to convey this content included public gatherings, plays, songs, contests, and the distribution and discussion of posters, leaflets, fliers, comic books, documentaries and videos. The basic content of these district, village and community sessions were of a type of allowing the correct information to be discussed, to give it time, to allow everyone to be involved and to disseminate accurate information for the population in order for their to be an adequate opportunity to mull over, discuss between each other, understand, and facilitate a critical mass of persons who knew the facts. Norm change was intended to follow facilitated group decision-making.\nIEC materials on AIDS such as posters, pamphlets, and booklets were developed and translated into local languages, and distributed to District Councilors, Resistance Councilors and to members of NGOs at training workshops. Three day seminars were held for health workers, followed by two day seminars for Resistance Councilors and district department heads, and sub-county chiefs. The general public was to get their materials through the Resistance Councilors system and other key people such as \u201cmass mobilizers\u201d and community development workers.\nResistance committees were political administrative units that graduated into each other from the Resistance Councilors that was made up of 9 households. Resistance committees were later to be called local councils or local area councils, but they maintained the same function. These training activities were followed by a one week health education campaign conducted by Resistance Councilors in collaboration with assistant health educators, other health educators, community development workers and NGOs. The DAMP activities and the training of trainers through multiple formal and informal natural and other infrastructures would serve to cross fertilize, in other words allow multiple messengers to come at individuals and groups through multiple channels, but with the same message.\nAlthough DAMP was the \u201cbig concept,\u201d UNICEF funded health education activities included a community Health Education Network (HEN) with community based health educators imbedded in the community, meant to sustain DAMP, and SHAPE (School Health Education Project). Both of these activities cross fertilized with and were thought to have augmented the DAMP. Parents and the community were to get information from HEN and their children got information from the schools \u2013 so there would be multiple channels alongside the district level work, and specific synergy at the household level.\nCountry wide messaging\nThe dominant message of the Ugandan program, as is well known, and which was well known at the time both throughout Uganda and throughout Africa (as the Uganda message) was \u201cZero Grazing,\u201d alluding to the traditional way cattle were fenced in, or tied to a stick to limit grazing outside their own pasture. This clearly meant \u201cstick to one partner\u201d which was what was frequently said following the message or by way of explanation. This was a message of \u201cfidelity\u201d although that word was rarely used at the time, as it not only pertained to during marriage, but to during dating as well. One study in1990 supported by WHO\/GPA reported higher village recall of the saying or having seen the posters \u201cLove Faithfully\u201d (30%), and \u201cLove Carefully\u201d (25%) than \u201cZero Grazing.\u201d Over 70% of those aware of \u201cLove faithfully\u201d interpreted this to mean \u201cstick to one partner\u201d; over 50% of those who heard or saw \u201cLove carefully\u201d understood it to meant \u201cchoose your partner carefully\u201d (which became a large confusion in Uganda), and over half of those who saw or heard \u201czero grazing\u201d thought it to mean \u201cstick to one partner.\u201d\nAll options including delaying sex, not engaging in sex, sticking to one partner, and to a lesser extent, using condoms were also described in the materials and discussions in trainings and in the district level courses and discussions. The posters at the time emphasized a cow in a pasture surrounded by a fence, and many \u201cThank God I said no\u201d posters, as well as messages of care and compassion. The word abstinence per se was rarely if ever heard in Ministry conversations, although delaying onset of sex was part of usual information. Although all means of transmission noted here were provided as information, we believe the actual \u201cABC phrase\u201d came out of the USAID in the mid or late 90\u00a0s, and was not seen in any of the country plans or program documents, or known at the time of the first 6\u20137 years of the Uganda program.\nMaterials development and dissemination\nThere was an enormous emphasis in Uganda on material development, production and reproduction, and active dissemination out of the capital to the districts. Local graphic artists held subsidized workshops with local residents to design materials and information packets for use in the trainings and DAMP activities. A full time printing press was paid for by WHO and put into the Ministry of Health along with other equipment and staffing support to intentionally \u201cenhance the capacity of the Ugandan Ministry of Health - Health Education Unit.\u201d A full time \u201cU.N. Volunteer\u201d (UNV), funded by WHO\/GPA, was additionally supported for the Uganda program to run the printing press. The instructions to the UNV were to continue to print, produce, and disseminate materials no matter what else was occurring regarding the national program. Posters, leaflets, fliers, and comic books were produced by this and other means. Materials for persons with poor literacy skills were also developed and actively disseminated.\nMass media\nRepresentatives from Ugandan radio and television were included in the program and planning process, and both radio (thought to reach over 87% of the population) and television were widely used. Short \u201cjingles\u201d were designed by NACP for radio to convey information on AIDS. The Ugandan daily paper MUNNO ran daily articles on AIDS. The weekly Topic and daily New Vision ran a combined 17 articles on AIDS in 1990. The New Vision ran the \u201cAIDS Corner\u201d on the last page of every issue that posed a question and provided an answer. This campaign ran daily for three consecutive years. The Q and A's were later compiled into booklets used in other countries. Ugandan TV (UTV) ran regular spots discussion programs and documentaries. Theater was used to convey messages and almost all theater groups developed plays incorporating HIV\/AIDS messages. President Musevini, Dr. Okware \u2013 then National AIDS Program Director - and other Ministry officials were regular spokespersons in the media, and the program appeared to speak with one voice so there was little opportunity for confusion.\nChurch leaders and imams were a major sources of information for about 12% of the population, but between 81% and 95% of three villages reported having received messages on AIDS from the church. Fidelity and monogamy were emphasized \u201cin addition to abstinence.\u201d Eighty-five percent of respondents in the 1991 NACP study felt that abstinence \u201cwas not a practical prevention strategy or behavioral option for any or all members in their community\u201d; 44% similarly felt that monogamy was not practical or possible at that time.\nChanges in social norms\nAt the time there were several anecdotal reports of villages changing norms of the acceptability of sexual contacts between teachers, police and the military in terms of their relations with young women. These decisions were arrived at by group decision making - and these changes in enforced norms toward total unacceptability were also supported by Presidential directive at the time. The results reported were that teachers or police who did engage in sexual relations with young women and girls were removed from their assignments, and likewise forced to leave the towns or villages. The full extent of these specific changes in norms and practices is unknown.\nChanges in sexual behavior\nBetween 1989 and 1995 changes occurred for the spectrum of relevant sexual behaviors (Asiimwe-Okiror et al., 1997; Atkinson, 1989; Singh, Darroch, & Bankole, 2003; Stoneburner & Carballo, 1997; 2003; USAID, 1998, 2003; US House of Representatives, Hearing on the Global AIDS crisis and pandemic in Africa, 2000), including toward delay in age of first intercourse (fewer initiated at young ages), lesser frequency of multiple partners, fewer non-regular partners, a narrowing of the age gap between girls and their male partners (USAID, 1998) and toward more condom use. The proportion of male and female youth aged 15\u201319 years old who reported that they have never had sex increased from 31% and 26% in 1989, to 56% and 46% in 1995 respectively (Assimwe-Okror, 1997). It is reported that there was no general pattern of increased abstinence among those who had ever had sex (Singh et al., 2003), however overall sexual activity decreased among 15\u201319 year old men and women, and for most ages.\nCondom use was unusual prior to 1989 and increased from <1% to over 15% among unmarried women and men who had sex in the prior 4 weeks from the 1989 to 1995 period\u2014with larger increases for younger ages; up to 50% in \u201clast intercourse\u201d for sexually active 15\u201317 year olds (Singh et al., 2003). In the NACP study between 30% and 60% of persons in the three districts studied reported having made some changes in their sexual behavior; the overwhelmingly greatest change made was in the reduction in the number of partners (Moodie et al., 1991). Which changes occurred may or may not correspond directly to what was promoted by the program.\nChanges in HIV prevalence\nUganda's sentinel surveillance system was the earliest and most elaborate system of HIV monitoring in Africa at the time, allowing for continuous monitoring of the epidemic and help in planning in order to ensure strong enough efforts at sites (Asiimwe-Okiror et al., 1997; Moodie et al., 1991; Slutkin et al., 1988; Slutkin et al., 1990; Stoneburner & Carballo, 1997, 2002; UNAIDS, 1998). These surveillance studies were supplemented by other studies. The effects of such an extensive and strong surveillance system itself is unknown, but its use by the program as a monitoring tool to strengthen and help target implementation was highly emphasized within the Uganda program. Changes in HIV seroprevalence occurred during the period between 1989 and 1992, with continued reductions in 1995 and 1997. An analysis past this period is beyond the scope of this article. The changes in sexual behavior and in HIV occurred during and within two to three years of Uganda's planned and extensively implemented AIDS education effort.\nU.S. Census and UNAIDS estimates are of a 67% drop in Ugandan national prevalence between 1991 and 2001 (Singh et al., 2003; US House of Representatives, Hearing on the Global AIDS crisis and pandemic in Africa; 2000). For six sentinel surveillance sites measuring changes among 15\u2013 24 year old antenatal women, reductions occurred from HIV prevalence rates of 15.0%\u201330.8% in 1989 to 7.4% to 14.7% in 1995 (see Table 4).\nTable 4HIV prevalence (%) changes in Uganda for 15\u201324 years old 1990\u20131993 and 1995\u20131996Antenatal sentinelsurveillance sites1990\u201319931995-1996PNsambya30.6 (25\u201336)14.7 (13\u201317)<.001Rubaga24.0 (18\u201330)17.5 (14\u201321)<.05Mbarara30.7 (25\u201336)14.0 (11\u201317)<.001Jinja17.7 (14\u201322)12.1 (9\u201315)<.05Mbale15.0 (12\u201318)7.4 (5\u201310)<.001Tororo15.5 (11\u201320)12.2 (9\u201315)NS\nSummary and conclusions\nUganda developed the first, and what was soon to become, the strongest planned and best supported National AIDS program, with the largest national and international staffing and most intensive, broadly inclusive, decentralized and community based public education program in Africa. When Uganda was receiving $18\u00a0million in support, other countries in the region were receiving between $1\u00a0million to $4\u00a0million. External and national financial support to the Uganda program was 4 to 20 times the amount provided to any other national program outside of the industrialized world at the time (Mann & Tarantola, 1991; UNAIDS, 1998).\nThe messaging emphasized faithfulness and fidelity, although the IEC program at both national and district level provided information on all forms of HIV transmission and all forms of protection. Information on delaying age of onset of sexual activity and the protective value of condoms were included in the program. Counseling and testing and condom promotion were very small elements in the early years in which the changes in seroprevalence were first seen (Moodie et al., 1991; Uganda, 1989, 1993). Although condom knowledge was high, and use increased substantially, overall condom distribution was of the order of 4\u20136\u00a0million condoms\/year; an amount of condoms that could not have influenced overall reductions in HIV prevalence as substantial as observed (Moodie et al., 1991; USAID, 2003). The ABC phrase was not known during these years, and it is estimated that it began to be used in the mid 90s\u00a0\u2013 after the changes in Uganda had already taken place (USAID, 2003; Hardee, 2004). This does not take away from the fact that the full range of information was provided. Openness about AIDS, destigmatization, and promotion of care and compassion were also important components of the Ugandan program.\nAbstinence itself was not specifically promoted on any posters, billboards, or leaflets of the national program as far as we remember, or as noted in any of the reports of the time. Delaying onset among young persons was promoted. It was already known to WHO at the time that providing full information about all means of transmission and ways of protection had been demonstrated to be the most effective approach to sexual behavior change, in particular for non-AIDS programs such as STD control, family planning and other programs, and therefore comprehensive education was the standard guidance of WHO\/GPA health education guiding materials at the time (Grunsheit, 1994; UNAIDS, 1998).\nProbably most importantly, the intensity of messaging of the Uganda program was far above any other program at the time. We recall returning from usual \u201cmission travel\u201d to multiple countries in the region including Rwanda, Burundi, Kenya, Tanzania, and sometimes Malawi or Zambia, and noting that there was no comparison between what was going on in Uganda and the other countries. This usually referred to the amount of visibility of the program by way of billboards and other public education materials, and the amount of decentralized program activity. Nevertheless it was not our impression at that time that this would necessarily translate into effective change. The thinking at the time was that there needed to be a still more professionally designed IEC program with more of a strategic basis, findings and criticisms that were noted in the 1988 program review and common among many of the review participants at the time, and a problem thought common to all programs in the region. It was a few years later that Hornik (2002) would reveal the close relationship between intensity of messaging and behavior change, and the large importance of intensity for several other public health\/behavior change issues as well.\nPresident Musevini's commitment and personal leadership and the involvement of the whole government was well known to all staff at WHO at the time and throughout the continent. This allowed open discussion throughout the country and even more so encouraged it. The President guided all ministers and other senior level government officials to mention and speak about AIDS at all public functions. As a matter of policy President Musevini's speeches were to end with a note on HIV\/AIDS. As far as we know, no single speech, local or international, ended without talking about HIV\/AIDS. A note was always left to himself to use his knowledge and imagination to convey the message.\nThere is nothing intrinsically different about the Ugandan people, problem or program, although the Resistance Council structure did facilitate some aspects of the decentralization performed, and the post-revolutionary nature of the society at the time probably helped ensure an appropriate amount of zeal for the effort. However the implementation of the program itself was notably different as compared to all of the neighboring countries at the time. We believe that the principal difference between the Uganda's HIV\/AIDS prevention and education campaign and that of neighboring countries relates mostly to the intensity, depth, breadth, and extensiveness of programming of its behavior change campaign, the level of involvement of all sectors, the pragmatism and extension of the district level work deep into communities, including the involvement of local churches and mosques, and the high level of financial support that allowed this to occur. Although there are several matters different about the HIV\/AIDS situation now, most notably the increased availability of drug treatment, it is still believed that given the full support, proper planning, and strength of implementation, serious HIV\/AIDS prevention and public education campaigns of this nature, still largely missing in Africa, could and should be implemented in almost any country with this degree of problem or risk of problem.\nFor the most part in the late 1980s and early 1990s, the other countries in Africa were thought to be simply \u201cbehind\u201d Uganda in planning, implementation and in their own movement toward enhanced support. The Uganda program was the first program, and for this reason as well as reasons of high donor concern, obtained very strong financial and other support. Uganda also had the commitment, and the ability to mobilize and decentralize the program, and to develop the management capacity to implement the IEC program at this broad and high level of effort.\nBeginning in the early and mid 1990s serious interruptions in programming in neighboring countries began to occur as the WHO\/GPA global effort began to be less globally coordinated, more bilaterally fragmented, and less prevention focused for various reasons partially explained elsewhere, but which include the change in leadership at WHO, the international preference for bilateral support, and a move toward UN\/WHO coordination and consolidation without sufficient attention at the time to ensuring preservation of all relevant functions and priority to prevention (Slutkin, 2000). Some barriers to implementation and support may still exist in some countries, but there is no technical or program reason that the authors see today as to why all, or most other countries could not perform similar reversals today with appropriate planning, implementation, and intensity of effort and support to reverse their HIV\/AIDS epidemics. With 5 million people being newly infected each year and treatment lagging further and further behind, programs with this leadership, programming, and intensity of effort need to be common rather than rare.\nAuthors Biography\nDr. Slutkin was the WHO\/SPA and then WHO\/GPA epidemiologist assigned to support the Uganda program from 1987\u201394. He also co-developed (along with Drs. Chin and Tarantola) the serosurveillance methods used to monitor trends in HIV within and among all countries (global), and supported the 12 other countries of Central and East Africa during the same years. Dr. Sam Okware was the director of the National AIDS Programme of Uganda. Dr. Warren Naamara was the epidemiologist responsible for the serosurveillance system of Uganda, and director of the Uganda ACP from 1990\u20131993. Dr. Don Sutherland was the WHO Team Leader and epidemiologist assigned to Uganda following the work of Dr. Ros Widi-Wirski, now deceased whose trip reports were consulted. Donna Flanagan was the health educator assigned to Uganda from 1990\u20131994. Dr. Erik Blas provided the logistic, managerial and financial back-up support to the Uganda program, as well as dozens of other programs around the world from WHO\/GPA. Dr. Paul Delay was the epidemiologist of the Malawi AIDS program and then the global USAID program director for AIDS, and subsequently Chief of Monitoring and Evalution for UNAIDS. Dr. Michel Carael worked in the social and behavioral research unit of WHO\/GPA. Dr. Daniel Tarantola the Chief, National Programme Support was responsible for all national programs globally for WHO\/GPA. During this time frame Dr. Tarantola supervised the development of over 120 national programs. The authors would also like to acknowledge Adjoa Amana, the driving force for the IEC training and messaging in the period 1987\u201390.","keyphrases":["uganda","epidemic","prevention","aids"],"prmu":["P","P","P","P"]}
{"id":"Int_Arch_Occup_Environ_Health-3-1-1915587","title":"Monitoring of platinum surface contamination in seven Dutch hospital pharmacies using inductively coupled plasma mass spectrometry\n","text":"Objective: To develop, validate, and apply a method for the determination of platinum contamination, originating from cisplatinum, oxaliplatinum, and carboplatinum.\nIntroduction\nCytotoxic drugs are widely used for the treatment of cancer. Occupational exposure to these drugs has been recognised as a potential health hazard since 1970s (Donner 1978; Falck et al. 1979). Because cytotoxic drugs can affect the DNA, RNA, or protein synthesis, many of these drugs are classified as being carcinogenic, mutagenic, or teratogenic to humans (International Agency for Research on Cancer (IARC) 1997). Skin contact with cytotoxic drugs, due to contamination of the work area or contamination of packaging material, seems to play an important role in the uptake of these drugs by hospital personnel (Fransman et al. 2004; Sessink et al. 1994). Therefore, strict health and safety rules have been established and applied for the handling of these agents. Evidently, the potential health risks for persons manipulating cytotoxic drugs, such as pharmacists, pharmacy technicians, nurses, and cleaners, however, still are a concern. This concern is consolidated by a number of recent publications demonstrating workplace contamination (Crauste-Manciet et al. 2005; Hedmer et al. 2005; Leboucher et al. 2002; Mason et al. 2005; Schmaus et al. 2002; Zeedijk et al. 2005; Ziegler et al. 2002) and contamination of packaging of cytotoxic drugs (Connor et al. 2005; Hedmer et al. 2005; Mason et al. 2003; Nygren et al. 2002). Moreover, detection of cytotoxic agents in urine (Ensslin et al. 1994a, b, 1997; Minoia et al. 1998; Pethran et al. 2003; Schreiber et al. 2003; Sessink et al. 1994; Turci et al. 2002) and blood (Nygren and Lundgren 1997) of personnel who were involved in preparation or administration has been reported with increasing frequency.\nThe relationship between prolonged exposure to small quantities of cytotoxic drugs and harmful effects is difficult to establish. Based on current scientific knowledge, it is impossible to set a level of exposure that, beyond doubt, will not cause adverse effects. Because no regulations on the maximal acceptable amount of contamination for these drugs have been set so far, hospitals should aim for the lowest contamination as is reasonably achievable. Monitoring of contamination, therefore, is essential. This can aid in the identification of the main exposure routes and in assessing the effectiveness of cleaning and working procedures. Evaluation of environmental contamination will, moreover, lead to an increase of the consciousness among personnel, concerning the handling of the chemotherapeutic agents. This can lead to an improvement of and the compliance to working and cleaning procedures. Wipe sampling is a common method to monitor surfaces for the presence of cytotoxic drugs. Hence, sensitive and validated methods are indispensable to be able to detect the relatively low quantities of drug present on surfaces.\nPlatinum coordination complexes, such as cisplatinum, oxaliplatinum, and carboplatinum play a major role in the treatment of a variety of tumours. As a result, large amounts of these agents are processed in hospital pharmacies. Several wipe sample methods for platinum containing drugs have been used in earlier studies and platinum was detected as a surface contaminant in many of the workplaces (Leboucher et al. 2002; Mason et al. 2005; Schmaus et al. 2002; Ziegler et al. 2002) or drug vials (Connor et al. 2005; Nygren et al. 2002) investigated. A description of the validation of the analytical methods, however, has been scarce. Validation results were mentioned briefly for the method of Ziegler et al. (2002), using electro thermal vaporisation coupled to inductively coupled plasma mass spectrometry (ICP-MS). Raghavan et al. (2000) described the validation of a high-performance liquid chromatography method for the determination of cisplatinum in cleaning validation samples. The limit of quantification of this method, was 500\u00a0ng\u00a0l\u22121, which is high compared to the limits achievable with for example ICP-MS or voltammetry. Schmaus et al. (2002) reported the validation of a voltammetric method with a limit of quantification of 40\u00a0pg of platinum per sample.\nIn the present study, we describe the development and validation of an ICP-MS method for the evaluation of surface contamination by platinum originating from cisplatinum, oxaliplatinum, and carboplatinum. ICP-MS assures an ultra high sensitivity and specificity and requires relatively simple sample pre-treatment procedures. The validated method has been applied to measure surface contamination in seven Dutch hospital pharmacies.\nExperimental\nChemicals\nCisplatinum and carboplatinum reference standards were purchased from Calbiochem (San Diego, CA, USA). Oxaliplatinum was obtained from Sigma-Aldrich (St Louis, MO, USA). Chloroplatinic acid, containing 1,000\u00a0mg\u00a0l\u22121 platinum in 3.3% HCl, used for preparation of calibration solutions, was obtained from Inorganic Ventures\/IV Labs (Lakewood, NJ, USA). Iridium chloride, containing 1,000\u00a0mg\u00a0l\u22121 iridium in 3.3% HCl, used for internal standardisation, was also purchased from Inorganic Ventures\/IV Labs. Nitric acid (HNO3) 70% and hydrochloric acid (HCl) 35% Ultrex II ultrapure reagents were obtained from Mallinckrodt Baker (Philipsburg, NJ, USA). Water used for the ICP-MS analysis was sterile water for irrigation (Aqua B. Braun Medical, Melsungen, Germany). Ethanol 80% was purchased from Fresenius Kabi (Den Bosch, The Netherlands). A multi-element solution containing 10\u00a0mg\u00a0l\u22121 of Ba, Be, Ce, Co, In, Mg, Pb, Th, Tl (VAR-TS-MS) was purchased from Inorganic Ventures\/IV Labs. Hoek Loos (Schiedam, The Netherlands) provided argon gas (4.6) with 99.996% purity.\nInstrumentation\nAnalyses were performed on an ICP-quadrupole-MS (Varian 810-MS) equipped with a 90\u00b0 reflecting ion mirror (Varian, Mulgrave, Victoria, Australia). The sample introduction system consisted of a Micromist glass low flow nebuliser (sample uptake 0.4\u00a0ml\u00a0min\u22121), a peltier-cooled (4\u00b0C) double pass glass spray chamber and a quartz torch. The spray chamber was cooled to reduce the vapour loading on the plasma, increasing the available energy for atomisation and ionisation of the elements of interest. Sample transport from the SPS-3 autosampler (Varian) to the nebuliser was performed using a peristaltic pump (Watson-Marlow Alitea, Stockholm, Sweden). The instrument was cooled by using a K\u00fchlmobil 142 VD (Van der Heijden, D\u00f6rentrup, Germany). Data were acquired and processed using the ICP-MS Expert Software version 1.1 b49 (Varian). Further data handling was performed using Excel 2000 (Microsoft, Redmond, WA, USA). All measurements were carried out in a dedicated temperature-controlled, positively pressurised environment in order to maintain optimum instrument performance and minimise exogenous contamination. All solutions were prepared using pipettes (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ, USA) and polypropylene tubes 10\u00a0ml (Plastiques-Gosselin, Hazebrouck Cedex, France) and 30\u00a0ml (Sarstedt AG&Co, N\u00fcmbrecht, Germany). Filters (Minisart) used for filtration of wipe samples were obtained from Sartorius (Hannover, Germany). Prior to method development, tubes were checked thoroughly for platinum, iridium, and hafnium contamination and appeared to be suitable for platinum analyses.\nDetermination of platinum by ICP-MS\nTo optimise the ICP-MS signal for the high masses and to reduce the formation of oxides, a solution containing 1,000\u00a0ng\u00a0l\u22121 of Th, In, Ce, Ba and Pt was used. Typically this 1,000\u00a0ng\u00a0l\u22121 solution gave readings of 115In, 7\u00a0\u00d7\u00a0105\u00a0c\u00a0s\u22121; 232Th, 1\u00a0\u00d7\u00a0106\u00a0c\u00a0s\u22121 and 194Pt, 2\u00a0\u00d7\u00a0105\u00a0c\u00a0s\u22121. The production of CeO+ was less than 1.0% of the total Ce+ counts. The formation of doubly charged Ba++ was less than 3%. Performance was checked daily.\nThe platinum isotope used for calculation of platinum concentrations was 194Pt. Iridium was used as internal standard. Detection of platinum can be subject to the interference of hafmium oxides (Lustig et al. 1997). Therefore, hafmium signals were monitored for all samples. The detection mode for all isotopes was based on peak jumping with peak dwell times of 50\u00a0ms, 25 scans per replicate, and three replicates per sample. Quantitation was based on the mean concentration of three replicates analysed against a calibration curve using weighted linear regression analysis.\nAssay development\nThe most suitable wipe material, desorption solvent, and wipe solvent were selected using one surface sampling and desorption procedure. This will be described below. Recovery data were assessed for the three most commonly used platinum agents; cisplatinum, oxaliplatinum, and carboplatinum. The different molecular structures and, as a result, the variable physical characteristics, might possibly lead to a variation in absorption and desorption characteristics. Therefore, we decided to evaluate all three compounds instead of choosing one reference compound.\nSurface sampling and desorption procedure\nEach wipe tissue was moistened with 500\u00a0\u03bcl wipe solvent. In general, sampling was performed by wiping a defined surface area of 10\u00a0cm\u00a0\u00d7\u00a010\u00a0cm. However, for surfaces for which it was not possible to take a 10\u00a0cm\u00a0\u00d7\u00a010\u00a0cm sample, the complete top of the device was sampled and the area was estimated. All wipe samples were collected with a uniform sampling procedure by wiping in three different directions (vertical, horizontal, and diagonal). Wipe samples were stored in 50\u00a0ml disposable polypropylene flasks (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ, USA) at \u221220\u00b0C until further processing. Prior to analysis, 10\u00a0ml of desorption solvent was added to the sample and flasks were kept in an ultrasonic bath for 60\u00a0min. Then, samples were filtered to remove particles which could possibly obstruct the ICP-MS nebuliser, or could interfere with the analysis. Two millilitres of sample were, after addition of iridium as internal standard, introduced directly into the ICP-MS. Samples of locations which were expected to be highly contaminated, were diluted prior to analysis to prevent contamination of the sample introduction system of the ICP-MS.\nWipe material\nA variety of wipe tissues are available for collecting samples of surface contaminants. These vary in type of material, surface area, and content of platinum contaminants. Three types of wipes were evaluated for this study; Kimtech Science precision wipes (Kimberley-Clark Professional, Irving, TX, USA), Whatman glass fibre filters (Schleicher&Schuell Microscience GmbH, Dassel, Germany), and Klinion non-woven gauzes (Medeco, Oud-Beijerland, The Netherlands). The tissues were checked for platinum contamination and for their ability to release platinum from stainless steel surfaces.\nDesorption solvent\nOne percent HNO3 (v\/v), 5% HNO3 (v\/v), and 1% HCl (v\/v) were evaluated as desorption solutions. Kimtech Science precision wipes were spiked with cisplatinum, oxaliplatinum, and carboplatinum and the platinum recovery was determined after desorption with 10\u00a0ml of each solvent.\nWipe solvent\nInitially, water, 1% HCl, and 80% ethanol were selected as wiping solutions. To investigate the capability of these solutions to effectively wipe surfaces, 100\u00a0cm2 stainless steel surfaces were spiked with cisplatinum, oxaliplatinum, and carboplatinum. These surfaces were subsequently wiped and samples were desorbed using a 1% HCl solution.\nValidation procedures\nLinearity\nFor calibration, the chloroplatinic acid reference solution containing 1,000\u00a0mg\u00a0l\u22121 platinum was diluted with 1% HCl to obtain working solutions with concentrations ranging from 50.0 to 5.00\u00a0\u00d7\u00a0103\u00a0ng\u00a0l\u22121 platinum. Working solutions were diluted with 1% HCl to obtain calibration standards, ranging from 0.500 to 100\u00a0ng\u00a0l\u22121 platinum. Before analysis, 15\u00a0\u03bcl of iridium internal standard solution was added to 1.5\u00a0ml of each calibration standard (final internal standard concentration 100\u00a0ng\u00a0l\u22121). The seven non-zero calibration standards were processed and analysed in singular in three separate analytical runs. The calibrations were back-calculated from the responses. Deviations from the nominal concentration were evaluated.\nRecovery and precision\nQuality control (QC) samples were prepared to obtain information on the recovery and precision of the desorption method and platinum analysis. These samples were analysed in the validation runs and subsequently also during the analysis of the wipe samples of each hospital pharmacy. Therefore, stock solutions of the platinum agents in water, each containing a concentration of drug equivalent to 400\u00a0mg\u00a0l\u22121 platinum, were prepared. These stock solutions were further diluted to obtain spiking solutions with concentrations ranging from 10.0 to 2.00\u00a0\u00d7\u00a0103\u00a0ng\u00a0l\u22121. Tissues were spiked with these solutions serving as QC samples at the following concentration levels; 5.00\u00a0\u00d7\u00a010\u22123, 2.5\u00a0\u00d7\u00a010\u22122, 0.100, and 1.00\u00a0ng platinum on the tissues, corresponding to 0.500, 2.50, 10.0, and 100\u00a0ng\u00a0l\u22121 platinum in the final solution. These tissues were processed as described earlier. Three replicates of each sample were analysed in three analytical runs. Recovery was expressed as a percentage of the nominal concentration. Within-run and between-run precisions were calculated by analysis of variances (ANOVA) for each test concentration using the analytical run as the grouping variable.\nTwo of the most important surfaces, stainless steel and linoleum, were used to obtain information on the recovery and within-run and between-run precisions of the complete sampling procedure, including both wiping and desorption. Therefore, 1.00\u00a0ng of cisplatinum, oxaliplatinum, or carboplatinum was pipetted in triplicate on a 100\u00a0cm2 stainless steel and linoleum surface. After drying overnight, surfaces were wiped following the previously described procedure and analysed using ICP-MS. Three replicates of each sample were analysed in three analytical runs for both surfaces. Recovery was expressed as a percentage of the nominal concentration. Within-run and between-run precision were calculated by ANOVA for each test concentration using the analytical run as the grouping variable.\nSensitivity\nThe lower limit of quantitation (LLOQ) was defined as the concentration at which the analyte response was at least five times the response of a blank wipe sample. Besides, the LLOQ, when spiked on blank tissues, had to be determined with a precision less than 20% and the mean value should not deviate more than 20% of the actual value.\nStability\nStability of cisplatinum, oxaliplatinum, and carboplatinum spiked to tissues, at two concentration levels, was evaluated at ambient temperatures for 1 week and under storage conditions (\u221220\u00b0C) for up to 3 weeks. From each storage condition two wipe samples were analysed. Samples were considered stable when 80\u2013120% of the initial concentration was recovered.\nPlatinum determination in pharmacy facilities where no cytotoxic agents are processed\nPlatinum is an element that not only appears in the environment due to contamination with platinum containing cytotoxic drugs, but also due to pollution by car exhaust catalysts. As a consequence, road dust also contains platinum (Barefoot 1997). Even though, in the pharmacy facilities, precautions (use of slippers\/clogs) are taken to reduce the chance of contamination of the facility, road dust contamination might occur. Because ICP-MS does not differentiate between the sources of elemental platinum, this should be taken into account when considering this technique for evaluation of environmental contamination by cytotoxic platinum agents. Therefore, two additional locations were included in this study to set a threshold below which it was not possible to address the source of the contamination. The first location was the laminar-airflow (LAF) hood in a clean room in which no platinum contamination was expected. The second location was a preparation unit with LAF hood of a public pharmacy.\nMonitoring of surface contamination in seven Dutch hospital pharmacies\nThe wipe samples were taken in seven hospital pharmacies in The Netherlands. Characteristics of the facilities are shown in Table\u00a01. The facilities were selected to provide a representation of the diversity in hospital pharmacies in The Netherlands in terms of size and amount of platinum compounds handled. The facilities of each hospital consisted of a preparation room with at least one LAF hood and a room for storage and checking of the prepared drugs and administration purposes. In each facility, samples were taken at locations that were prone to contamination. For good comparison of the results 15 standard locations (Fig.\u00a01) were selected: (1) the middle of the bench-top of the LAF hood, (2) front edge of LAF hood, (3) floor in front of LAF hood, (4) handle of service hatch, (5) door handle, (6) waste bin top, (7) bench-top on which materials are placed in storage\/checking room, (8) floor in front of (7), (9) mouse computer, (10) handle telephone, (11) storage shelve of cisplatinum, (12) storage shelve oxaliplatinum, (13) storage shelve carboplatinum, (14) transport box, (15) handle refrigerator. Locations 1, 2, and 3 were sampled in duplicate to get an impression of the overall contamination of these locations. A new pair of gloves was used for each wipe sample. For each facility three blank samples were prepared by moistening tissue with 500\u00a0\u03bcl water. All samples were stored and processed as described earlier. The storage time from sampling until work-up procedure was less than 2 weeks.\nTable\u00a01Amount of platinum agents processed and years that the facilities are in serviceSite1234567Cisplatinum use in 2005 (in g)52.36.8064.014716.829.9104Oxaliplatinum use in 2005 (in g)47.58.6544.110923.498.862.0Carboplatinum use in 2005 (in g)25648.212463556.5223217Total amount of platinum processed in 2005 (in g) 19234.012948352.1185212Number of years in service510182.51.51015Wipe sampling frequencyOnce per year2005 first timeSporadic: last in 2004Once per yearOnce per yearOnce per year Twice per yearFig.\u00a01Sample locations in pharmacy facilities\nWipe sampling was announced in each facility in advance and was performed after the daily cleaning procedure of the LAF hoods, but before the daily cleaning procedure of the rest of the facility. Wipe sampling in all the facilities was performed by the same person.\nResults\nAssay development\nWipe material\nAs a result of high platinum backgrounds (10\u201320\u00a0pg platinum per filter depending on the batch analysed), Whatman glass fibre filters were found to be not suitable for platinum wipe sampling. Kimtech Science precision wipes and Klinion non-woven gauzes did not show platinum contamination. However, Kimtech Science precision wipes showed better recoveries of platinum compared to Klinion non-woven gauzes and consequently Kimtech Science precision wipes appeared to be the best choice.\nDesorption solvent\nThe most effective desorption of platinum from the wipe materials was achieved by 1% HCl (94\u201399%). As a result 1% HCl was selected as the desorption solution of choice.\nWipe solvent\nRecoveries were inadequate for 80% ethanol (<40% for all three compounds) and acceptable for water (50\u201377%) and 1% HCl (63\u201378%). Because 1% HCl appeared to be corrosive for some types of stainless steel, water was selected as wipe solution.\nValidation procedures\nLinearity\nThe calibration curve was best described by linear regression, using 1\/(sd of triplicate sample reading) as weight-factor, to avoid bias in favour of samples with high standard deviations. Deviations from the nominal concentration were between \u221210.0 and 10.2 for all concentration levels. Relative standard deviations for the calibration samples were up to 7.84%. Correlation coefficients were higher than 0.99999.\nRecovery and precision\nWithin-run and between-run precision data for spiked tissues, which served as QCs are summarised in Table\u00a02. Precision data showed that, for all QC concentration levels, the reproducibility of the desorption procedure and platinum analysis was excellent. Recoveries for cisplatinum, oxaliplatinum, and carboplatinum were between 86.7 and 103% for all concentration levels. These results indicated sufficient recovery.\nTable\u00a02Within and between-run precision data for quality control samplesAmount of platinum spiked to tissue (in ng)Final platinum concentration (in ng\u00a0l\u22121)CisplatinumOxaliplatinumCarboplatinumWithin-runBetween-runWithin-runBetween-runWithin-runBetween-run5.00\u00a0\u00d7\u00a010\u221230.5007.75a8.53a8.018.752.50\u00a0\u00d7\u00a010\u221222.504.104.051.661.152.39a0.10010.01.357.441.751.272.862.501.001001.077.791.63a0.841.96aNo statistically significant additional value was observed as a result of performing the assay in different runs (mean square within runs is greater than mean square between runs)\nFor recovery and within-run and between-run precision data from the spiked stainless steel and linoleum surfaces see, respectively, Tables\u00a03\u00a0and\u00a04. Precision data showed that the reproducibility of the method, including the wiping procedure was good. Recoveries from the spiked stainless steel surface were 50.4% for cisplatinum, 73.8% for oxaliplatinum, and 77.2% for carboplatinum (Table\u00a03). Recoveries for the linoleum surface were, respectively, 76.8, 77.9, and 81.4% (Table\u00a04).\nTable\u00a03Recovery of 1.00\u00a0ng platinum from a stainless steel surfaceCisplatinumOxaliplatinumCarboplatinumMean recovery (%)50.473.877.2Within-run precision (in %)2.214.632.53Between-run precision (in %)3.36aaNumber of days333Number of samples per day333aNo statistically significant additional value was observed as a result of performing the assay in different runs (mean square within runs is greater than mean square between runs)Table\u00a04Recovery of 1.00\u00a0ng platinum from a linoneum surfaceCisplatinumOxaliplatinumCarboplatinumMean recovery (%)76.877.981.4Within-run precision (in %)3.622.123.35Between-run precision (in %)12.25.416.82Number of days333Number of samples per day333\nSensitivity\nThe LLOQ of the assay was set at a platinum concentration of 0.5\u00a0ng\u00a0l\u22121 in 1% HCl, corresponding to 5\u00a0pg per wipe sample or 0.05\u00a0pg\u00a0cm\u22122 taking into account a surface of 10\u00a0cm\u00a0\u00d7\u00a010\u00a0cm. Signal to noise ratios at the LLOQ level exceeded 5 during all the experiments, which was in accordance with the requirement. The acceptance criteria, that the LLOQ was determined with a precision less than 20% and that the mean value should deviate no more than 20% from the actual value, were met for all three compounds (Table\u00a02).\nStability\nStability has now been established up to 3 weeks, but further testing is still ongoing. Sample storage at room temperature for 1 week was not possible. Tissues which were spiked with cisplatinum showed a decrease in platinum levels of 30% after 1 week. Oxaliplatinum and carboplatinum spiked tissues did not reduce under these conditions. Sample storage at \u221220\u00b0C was possible for at least 3 weeks. Platinum concentrations of cisplatinum spiked tissues were decreasing more obvious with time than oxaliplatinum and carboplatinum spiked tissues. However, no decrease of more than 20% of the initial concentration was observed after 3 weeks at \u221220\u00b0C.\nPlatinum determination in pharmacy facilities where no cytotoxic agents are processed\nNo platinum was detected in wipe samples from the LAF hood of the public pharmacy. Platinum levels of the LAF hood in the clean room and the floor in the public pharmacy ranged between 0.430 and 0.922\u00a0ng\u00a0l\u22121 (or 0.0430\u20130.0922\u00a0pg\u00a0cm\u22122). Therefore, it was recommended to set a threshold of 1.00\u00a0ng\u00a0l\u22121 platinum (0.100\u00a0pg\u00a0cm\u22122 when wiping a surface of 100\u00a0cm2), below which it was not possible to address the source of the contamination. All surfaces in the preparation units with platinum levels above this threshold were considered as being contaminated by platinum containing drugs.\nMonitoring surface contamination in seven Dutch hospital pharmacies\nIn February 2006, wipe samples were collected from seven Dutch hospital pharmacies with centralised units dedicated to the preparation of intravenous mixtures of cytotoxic drugs. The amount of platinum which was processed in these facilities ranged from 34.0 to 483\u00a0g per year (Table\u00a01). Surface contamination of all sample locations is depicted in Table\u00a05 in pg\u00a0cm\u22122. It is important to consider that recoveries of the samples, as assessed in the validation study, deviate from 100% dependent on the type of surface sampled and on the type of compounds present on the surface. Therefore, results depicted in Table\u00a05 represent \u226550.4% of the actual contamination present on the surface.\nTable\u00a05Platinum contamination in seven Dutch hospital pharmaciesS.no.Sampled surfacePlatinum contamination (in pg\u00a0cm\u22122)Site12345671Middle of bench LAF hood0.221800.5432.70.3607.222.94Duplicate of 10.1891240.64518.70.3288.222.282Front edge of LAF hood\u2013a3563.3299.50.13328.25.12Duplicate of 2\u2013a2688.34180\u2013a37.05.193Floor in front of LAF hood3.141738241,1070.2282.4821.7Duplicate of 33.202327282,2110.1861.9112.54Handle of service hatch\u2013a22.7\u2013a2,055\u2013a1.9611.85Door handle\u2013a3.17\u2013a16.8\u2013a21.416.16Waste bin top\u2013a1.020.39210.1\u2013a7.380.098c7Bench-top on which materials are placed0.8290.9490.29890.60.2020.37563.48Floor in front of bench0.105c19.758.938.1\u2013a0.311c11.99Mouse computer0.2520.8161.3410.2\u2013b0.7585.4110Handle telephone\u2013a3.220.5912.1\u2013a3.065.1211Storage shelve cisplatinum0.176c1.140.157c4.760.5364.0433612Storage shelve oxaliplatinum0.3680.9160.141c4.1082.71.152.2113Storage shelve carboplatinum3.251.530.1473.130.1860.9895,76014Transport box74.5\u2013a0.2854.44\u2013a0.828\u2013b15Handle refrigerator0.45226.31.4636.00.9481.425.71aRecovered platinum concentrations were below the thresholdbDevice was not present of available for wipe samplingcHafnium oxide might have accounted for up to 20% of the platinum content\nPlatinum was detected in 94% of the wipe samples and 88% of the samples contained levels above the threshold set. Six of the 126 samples showed raised hafmium signals which, considering a maximum oxide formation of 1%, might have accounted for up to 20% of the platinum signals of these samples. None of blank samples prepared for each facility by moistening tissues with wipe solvent, contained levels of platinum exceeding 20% of the LLOQ standard. The variation in the level of contamination between pharmacies was high. Pharmacies of site 1 and 5 showed overall low platinum contaminations. For these sites, respectively 33 and 39% did not contain platinum levels above the threshold set. Platinum levels detected at pharmacy 3 were relatively low as well, although the wipes taken from the floor were high at this site. These high values were, most probably, the result of a calamity in 2005 with a cisplatinum infusion mixture, which was spilled on the floor. Most locations wiped at the hospital pharmacies of site 2, 4, 6, and 7 showed high contaminations. Only one sample from these sites did not contain any detectable platinum. The high contamination of site 2 seemed to run counter to the quantities of drugs handled, because in this pharmacy relatively low amounts of platinum were processed. This site, however, was occasionally used, for preparation of larger amounts of cytotoxic drugs to serve another hospital. Therefore, the amount of drugs processed in 2005, was not fully representative for the amount of drugs processed in the 10 years that this site was in use. Site 4 showed the highest contamination, which paralleled the relative amount of drug handled in this unit.\nAs expected, platinum was found in most wipe samples taken from the middle of the LAF hood bench. Notable was that, in general, wipe samples of the front edge of the LAF hood were more contaminated than samples taken from the bench-top of the LAF hood. Furthermore, floor samples usually contained the highest platinum levels. Other locations showing substantial contamination were storage shelves, door handles, and handles of service hatches. Duplicate samples of locations 1, 2, and 3 showed similar results, indicating a homogeneous distribution over de surface area.\nThe number of years that the seven units were in use, did not parallel contamination levels and the amount of drug handled in 2005, overall, did not predict the level of contamination either.\nDiscussion\nThe presence of cytotoxic drug contamination in hospital pharmacies is recognized as a potential health risk. Therefore, it is important to monitor this contamination. Because platinum coordination complexes belong to the most extensively used anticancer agents, it is relevant to focus on the occupational exposure of these drugs. The rationale for evaluation of platinum contamination is also illustrated by several studies showing increased levels of platinum in blood (Nygren and Lundgren 1997) and urine (Ensslin et al. 1994a, 1997; Nygren and Lundgren 1997; Pethran et al. 2003; Schreiber et al. 2003; Turci et al. 2002) of hospital personnel working with these agents.\nTo be able to accurately assess the platinum contamination originating from cisplatinum, oxaliplatinum, and carboplatinum at different locations, we developed and validated a wipe sampling method. ICP-MS was used for quantification of platinum, because this technique assures a high sensitivity and relative simple sample pre-treatment. The sensitivity of the method was excellent. The LLOQ was set at a platinum concentration of 0.5\u00a0ng\u00a0l\u22121, corresponding to 5\u00a0pg per sample or 0.05\u00a0pg cm\u22122 when wiping a surface of 100\u00a0cm2. To our best knowledge, the method described here is 2\u2013300 times more sensitive than other methods described for determination of platinum in wipe samples (Connor et al. 2005; Mason et al. 2003, 2005; Raghavan et al. 2000; Schmaus et al. 2002; Ziegler et al. 2002).\nSample pre-treatment only involved surface sampling, desorption, and filtration. After filtration, samples could be analysed immediately. During method development it was shown that, in addition to tissue material and desorption solvent, also the wipe solvent affected the recovery to a considerable extent. This was in contrast with results described by Turci et al. (2003), who mentioned that the type of wipe solvent would not influence the recovery, since contaminants would be wept away from the surfaces independent of the composition or the pH of the solution itself. Best recoveries were achieved by wiping with Kimtech Science precision wipes moistened with 500\u00a0\u03bcl water and subsequent desorption with 1% HCl.\nValidation of the method was performed for the three, in oncology most prominently used platinum agents, cisplatinum, oxaliplatinum, and carboplatinum. We decided not to choose one reference compound, because the different molecular structures of the platinum agents and, as a result, the variable physical characteristics, might possibly lead to a variation in absorption and desorption characteristics. Excellent reproducibility (imprecision up to 8.75%) and recoveries (86.7\u2013103%) were demonstrated with spiked tissues, for all concentration levels and compounds. Up to 13.3% of the initial amount of platinum added to the tissues was not recovered after desorption and analysis. This could be due to variation in analysis, as well as loss due to adsorption to the tissues. Recoveries from the spiked stainless steel surface were 50.4% for cisplatinum, 73.8% for oxaliplatinum, and 77.2% for carboplatinum. Recoveries, for the three compounds, from the linoleum surface were, respectively, 76.8, 77.9, and 81.4%. These results showed that for stainless steel, depending on the compound analysed, up to 49.6% of the initial amount of platinum spiked to the surface was lost. This was, for the greater part, caused by the inability of the wipe procedure to remove all the added platinum and, for a minor part, by the variation in analysis and loss due to adsorption to the tissues. The lower recovery that was observed for cisplatinum, is, most probably, a consequence of its superior reactivity compared to oxaliplatinum and carboplatinum. This might lead to a stronger binding affinity of cisplatinum to materials and surfaces. For linoleum up to 23.2% of the initial amount of platinum spiked to the surface was not recovered. For this surface, recoveries were similar for all compounds.\nTo evaluate the stability of spiked samples, recoveries were assessed after storage at room temperature and \u221220\u00b0C. Storage of spiked tissues at \u221220\u00b0C for at least 3 weeks was possible. However, storage of tissues spiked with cisplatinum for 1 week at room temperature led to considerable decrease in platinum levels. Even though platinum concentrations from oxaliplatinum and carboplatinum spiked tissues did not reduce under these conditions, storage at room temperature was not recommended, also because the source of elemental platinum is not known in wipe samples performed in pharmacies. Differences in recovery of the cisplatinum and the oxaliplatinum and carboplatinum spiked samples, again, could be explained by the higher reactivity of cisplatinum.\nFor a correct interpretation of surface sampling results, it is relevant to take into account that platinum is an element that not only appears in the environment due to contamination with platinum containing cytotoxic drugs, but also due to pollution by car exhaust catalysts. By wipe sampling two locations where no cytotoxic drugs were handled, we, therefore, determined that below a threshold of 1.00\u00a0ng\u00a0l\u22121 platinum (0.100\u00a0pg\u00a0cm\u22122 when wiping a surface of 100\u00a0cm2), it was not possible to address the source of contamination. All surfaces in the preparation units with platinum levels above the threshold were considered as being contaminated by platinum containing drugs.\nTaking this threshold into consideration, platinum contamination was reported in 88% of the samples taken in the seven Dutch hospital pharmacies. It is important to consider that recoveries of the samples, as assessed in the validation study, deviate from 100% dependent on the type of surface sampled and on the type of compounds present on the surface. Therefore, results depicted in Table\u00a05 represent \u226550.4 of the actual contamination present on the surface.\nThe results of this study indicate that there is substantial variation in surface contamination of the pharmacies tested and that the amount of platinum processed in the pharmacies did not always parallel the level of contamination. The number of preparations with platinum drugs was, however, not assessed and might also be related to surface contamination. This suggests that variation in the application of or compliance to cleaning and working procedures and the incidence of calamities, rather than the amount of platinum processed, caused variation in surface contamination.\nIn general, results reveal that the LAF hoods, the floor in front of the LAF hoods, door handles, and handles of service hatches were often contaminated. This demonstrates that contamination is often spread throughout the pharmacy. Notable was that wipe samples of the front edge of the LAF hood were more contaminated than samples from the bench-top of the LAF hood. This is thought to be due to incorrect application of working procedures or insufficient cleaning.\nBy taking duplicate wipe samples of the LAF hood, the front edge of the LAF hood, and the floor, we demonstrated that these locations were overall contaminated and that contamination did not appear to be spotty as was mentioned by Zeedijk et al. (2005).\nWe also investigated storage shelves and, in most pharmacies, considerable platinum contamination was found. These elevated levels could be a consequence of elevated levels of platinum on packaging material (Connor et al. 2005; Mason et al. 2003; Nygren et al. 2002). Contamination of packaging can lead to a spread of cytotoxic drugs to locations where the drugs are stored or processed. Therefore, it was not surprising that the storage shelves of platinum agents were contaminated. Furthermore, it was noticed that in at least one of the hospital pharmacies (at site 4), secondary packaging and caps of the vials were discarded onto the floor during preparation, most probably to prevent the packaging from interfering with preparation activities. This pharmacy indeed showed considerable contamination of the floor.\nIn our study, surfaces showed platinum contamination of up to 5,760\u00a0pg\u00a0cm\u22122. The results are in the same range as findings of some other studies describing surface contamination of platinum in hospital pharmacies (Leboucher et al. 2002; Schmaus et al. 2002). In the study of Leboucher et al. (2002), however, no platinum was found outside the LAF hood, which could be due to the high detection limit of the atomic absorption spectrometry method used (10\u00a0\u03bcg\u00a0l\u22121). Schmaus et al. (2002) performed a study in 14 hospital pharmacies and all samples tested positive for platinum, even though the LLOQ of their method was eight times higher than of the method described here. Yet, although in our study not all samples tested positive for platinum, the highest contamination found (5,760\u00a0pg\u00a0cm\u22122) was comparable to the highest contamination found by Schmaus et al. (2,700\u00a0pg\u00a0cm\u22122). Mason et al. (2005) showed lower contamination levels than found in our study, despite that the amount of drug handled in these pharmacies was higher than in the pharmacies in our study.\nWhen comparing the amounts of platinum detected on different locations in this study (up to 0.576\u00a0\u03bcg per wipe sample) with the platinum content of one vial (between 6.50 and 237\u00a0mg platinum), contamination seems to be relatively low. Furthermore, the extremely sensitive technique used in this study, leads to a high percentage of positive samples. Interpretation of these results is rather complicated. It is important to consider that the total area of the contamination is large and that pharmacy personnel are at risk to be exposed to the contamination daily. Hence, for safety precautions, it is recommended to attempt to achieve the lowest possible contamination. Environmental monitoring therefore, may be used to monitor and control contamination and thereby evaluate working and cleaning procedures, rather than to interpret potential health risks.\nIn general, when a minimal contamination level is desired, the results of this study demonstrate that cleaning and working procedures do not sufficiently prevent contamination in most hospitals. This could be due to an inadequate compliance of personnel to these procedures. Moreover, contamination can spread out unconsciously by hands or feet of the personnel. It is also likely that cleaning procedures as applied in the different pharmacies are not fully optimised and validated, leading to contamination due to sub-optimal cleaning. With respect to the physical properties of the different cytotoxic drugs, it is recommended to consider cleaning techniques appropriate for specific agents. As was shown in this study for platinum, for instance, 80% ethanol did not effectively remove platinum from a stainless steel surface. Although water gave better recoveries for platinum, it was not capable of removing all of the added platinum from the stainless steel surface. This illustrates the importance to evaluate several cleaning procedures for the different cytotoxic agents handled and to optimise a procedure which does remove all drugs with acceptable recoveries.\nIn conclusion we developed and validated an ultra sensitive and reliable ICP-MS method for the determination of platinum in surface samples. This method was successfully applied in the evaluation of platinum contamination in the preparation units of seven Dutch hospital pharmacies. It was demonstrated that pharmacy personnel is at risk to be exposed to platinum, despite the use of cleaning and safety procedures. As long as the consequences of long-term exposure are not known, the aim should be to achieve a contamination levels as low as possible. This study, therefore, highlights the need to further evaluate cleaning and safety procedures. Wipe sampling can be applied to quantify improvements made through changes in procedures.","keyphrases":["platinum","surface contamination","hospital pharmacies","validation","icp-ms"],"prmu":["P","P","P","P","P"]}
{"id":"Exp_Appl_Acarol-3-1-2039815","title":"Spider mite (Acari: Tetranychidae) mitochondrial COI phylogeny reviewed: host plant relationships, phylogeography, reproductive parasites and barcoding\n","text":"The past 15 years have witnessed a number of molecular studies that aimed to resolve issues of species delineation and phylogeny of mites in the family Tetranychidae. The central part of the mitochondrial COI region has frequently been used for investigating intra- and interspecific variation. All these studies combined yield an extensive database of sequence information of the family Tetranychidae. We assembled this information in a single alignment and performed an overall phylogenetic analysis. The resulting phylogeny shows that important patterns have been overlooked in previous studies, whereas others disappear. It also reveals that mistakes were made in submitting the data to GenBank, which further disturbed interpretation of the data. Our total analysis clearly shows three clades that most likely correspond to the species T. urticae, T. kanzawai and T. truncatus. Intraspecific variation is very high, possibly due to selective sweeps caused by reproductive parasites. We found no evidence for host plant associations and phylogeographic patterns in T. urticae are absent. Finally we evaluate the application of DNA barcoding.\nIntroduction\nSpecies identification is the basis for understanding species diversity, phylogenetic patterns, and evolutionary processes. Only correct identifications allow for comparisons between studies and the repetition or expansion of earlier experiments. In pest species, species identification is also important in the development of (biological) pest control strategies.\nIdentification and delineation of species within the Tetranychidae has been an issue of debate for the past few decades. Within the family about 1200 different species are described, many of which are of agronomical importance (Bolland et\u00a0al. 1998). The genus Tetranychus is well studied and includes two common major agricultural pest species with a worldwide distribution: Tetranychus urticae Koch, 1836 and Tetranychus kanzawai Kishida, 1927. Morphological identification of tetranychid species is difficult. The number of potential diagnostic characters is limited (partly due to the small size of the mites) and key traits often exhibit large phenotypic plasticity. As a result, many species cannot be distinguished on the basis of external morphology. For example, in Japan 10 Tetranychus species are recognized (Ehara 1999). However, morphological identification using adult females is possible for only two of the species. The remaining eight species can only be identified by microscopic examination of the shape of the aedeagus (part of the male genitalia). Another example that shows our inability to identify species on the basis of morphology is the well-studied two-spotted spider mite species T.\u00a0urticae. This species is considered a species complex (Navajas et\u00a0al. 1998) and as many as 44 synonymous names are known (Bolland et\u00a0al. 1998). The question whether red T.\u00a0urticae mites should be considered a separate species (T.\u00a0cinnabarinus) has occupied taxonomists for many years (Dupont 1979; Gotoh and Tokioka 1996; Zhang and Jacobson 2000). The fact that there are only few taxonomists specialized in morphological identification of mites and that their number is decreasing adds to the problem of spider mite identification.\nDNA sequences are currently an indispensable tool for delineating and identifying species. In this context it is important to distinguish between DNA taxonomy and DNA barcoding. DNA taxonomy concerns the circumscription and delineation of species using evolutionary species concepts (Vogler and Monaghan 2007). DNA barcoding aims at the identification of pre-defined species and does not address the issue of species delineation per se (Monaghan et\u00a0al. 2005). In DNA barcoding a short standardized DNA sequence, usually the 5\u2032 end part of the mitochondrial cytochrome c oxidase subunit I (COI) gene, is used to identify species. DNA barcoding can be used to (i) identify and assign unknown specimens to species that have been previously described and (ii) enhance the discovery of new species using a threshold of sequence divergence (Hebert et\u00a0al. 2003; Moritz and Cicero 2004). DNA taxonomy may be based on one or several mitochondrial as well as nuclear DNA regions and can serve as a database for DNA barcoding. DNA taxonomy is an offshoot of phylogenetics, in which the evolutionary relationships between taxa (e.g., species) are investigated. Throughout this paper we use the term \u2018species delineation\u2019 when it concerns DNA taxonomy and \u2018species identification\u2019 when it concerns DNA barcoding.\nThe usefulness of the COI region for delineating tetranychid species has been investigated in several studies (Hinomoto et\u00a0al. 2001; Hinomoto and Takafuji 2001; Lee et\u00a0al. 1999; Navajas et\u00a0al. 1994, 1996a, 1996b, 1998; Toda et\u00a0al. 2000; Xie et\u00a0al. 2006a). Recently, a DNA barcoding approach was used to identify tetranychid species (Hinomoto et\u00a0al. 2007). Each of these studies used a different or sometimes partially overlapping subset of tetranychid sequences. Many studies extended their dataset with one or several tetranychid sequences from the GenBank database, serving as a reference for phylogeny reconstruction or species identification. However, sequence diversity within T.\u00a0urticae is substantial (e.g., Navajas et\u00a0al. 1998) so that different T.\u00a0urticae sequences are available from GenBank, some of which bear an incorrect species name (due to misidentification). As a consequence, different T.\u00a0urticae reference sequences were used in above-mentioned studies, leading to the emergence of variable taxonomic groupings and phylogenetic patterns. This, combined with the analyses of restricted subsets in each study, gives an incomplete and fragmented view of species delineations and phylogenetic relationships within the family Tetranychidae.\nIn this study we create an extensive COI dataset of the family Tetranychidae, with a wide coverage of the species T.\u00a0kanzawai and T.\u00a0urticae (including T.\u00a0cinnabarinus, which is currently considered synonymous to T.\u00a0urticae). We have collected all currently available mitochondrial COI sequences from GenBank and added data on mites collected from Europe and North America. We critically evaluate the assembled data and perform an overall phylogenetic analysis. This approach reveals novel patterns on species delineation and phylogenetic relationships. We discuss the use of COI for DNA barcoding purposes by considering the intra- and interspecific variation. In addition, we discuss the observed variation in COI in relation to associated host plant, phylogeographic patterns and the presence of endosymbionts (e.g., Wolbachia, Cardinium). Finally, we provide guidelines for future phylogenetic studies on (tetranychid) mites.\nMaterial and methods\nAdditional tetranychid samples\nTetranychid mites were collected in Europe (six locations), in North America (one location), and from two cultures maintained in our lab for 10\u00a0years (Table\u00a01). Mites were not identified morphologically to the species level. DNA was extracted from single individuals using a modified CTAB extraction method (Doyle 1991). A single adult female was ground in 5\u00a0\u03bcl of proteinase K (20\u00a0mg\/ml) and 100\u00a0\u03bcl CTAB (2% CTAB w\/v in 100\u00a0mM Tris-HCl [pH8], 20\u00a0mM EDTA, and 1.42\u00a0M NaCl) buffer was added. After vortexing, samples were incubated at 55\u00b0C for 1\u00a0h. Next, 100\u00a0\u03bcl chloroform: isoamylalcohol (24:1) was added and contents were gently mixed for 2\u00a0min. Tubes were centrifuged for 10\u00a0min. at 15,800\u00a0g. After centrifugation, 80\u00a0\u03bcl of the supernatant was transferred to a clean tube and DNA was precipitated by adding 200\u00a0\u03bcl ice-cold 96% ethanol. Tubes were incubated at \u221220\u00b0C for at least 1\u00a0h prior to centrifugation at 15,800\u00a0g for 15\u00a0min at 4\u00b0C. The supernatant was removed and the DNA pellet was washed with 70% ethanol. Next, the DNA was air dried for at least 15\u00a0min, eluted in 30\u00a0\u03bcl sterile water, and stored at \u221220\u00b0C.\nTable\u00a01Overview of samples sequenced in this study and the primer sequences used for COI amplificationStrainCountryLocalityHost plantCollection date\u00a0PrimerbCommon nameScientific nameFRNL1NetherlandsCastricumEuropean SpindleEuonymus europaeusSep-0612NL2NetherlandsCastricumEuropean HoneysuckleLonicera periclymenumSep-0612F1FranceVireuxBlackthornPrunus spinosaJul-0612T1UnknownUnknownCucumberaCucumis sativusa12T2NetherlandsAalsmeer (greenhouse)RoseaRosa spec.a12US1United StatesTucson (AZ)UnknownUnknownMay-0534P1PortugalCaldas de MonchiqueCitrusCitrus spec.Feb-0534S1SpainMont-roig del campOrangeCitrus spec.Apr-0434PL1PolandRabkowaPlumbPrunus spec.Aug-0534aMaintained in the lab on bean (Phaseolus vulgaris) for over 10\u00a0yearsb1\u00a0=\u00a0GGAGGATTTGGAAATTGATTAGTTCC (Navajas and Boursot 2003);\u00a02\u00a0=\u00a0AAWCCTCTAAAAATRGCRAATACRGC (modified from Hinomoto and Takafuji (2001); 3\u00a0=\u00a0TGATTTTTTGGTCACCCAGAAG (Navajas et\u00a0al. 1994); 4\u00a0\u00a0=\u00a0TACAGCTCCTATAGATAAAAC (Navajas et\u00a0al. 1994)\nPart of the mitochondrial COI gene was amplified using various primer combinations (Table\u00a01). Depending on the primer combination, this yielded a fragment size of 410\u2013863 basepairs (bp), excluding the primer annealing sites (Fig.\u00a01). PCR was performed in a 25\u00a0\u03bcl reaction mix containing 2.5\u00a0\u03bcl 10X Super Taq buffer (HT BioTechnology, Cambridge, U.K.), 1.25\u00a0\u03bcl bovine serum albumin (10\u00a0mg\/ml), 1.25\u00a0\u03bcl MgCl2 (25\u00a0mM), 5\u00a0\u03bcl dNTP mix (1\u00a0mM of each nucleotide), 0.2\u00a0\u03bcl of each primer (20\u00a0\u03bcM each), 0.2\u00a0\u03bcl of super Taq (5\u00a0u\/ \u03bcl) (HT BioTechnology), 11.9\u00a0\u03bcl water and 2.5\u00a0\u03bcl of DNA extract. PCR cycling conditions were 4\u00a0min. at 94\u00b0C, followed by 35\u00a0cycles of 1 min at 94\u00b0C, 1\u00a0min at 48\u00b0C and 1\u00a0min at 72\u00b0C, and a final extension at 72\u00b0C for 4\u00a0min. Products (2\u00a0\u03bcl) were visualized on a 1% agarose gel stained with ethidium bromide in 0.5X TBE buffer (45\u00a0mM Tris base, 45\u00a0mM boric acid, and 1\u00a0mM EDTA, pH 8.0).\nFig.\u00a01Overview of the COI fragments sequenced in different studies and their relative position after alignment. The position of the fragment analyzed in this study and of the standard DNA barcoding fragment (Folmer fragment) on the total mitochondrial COI gene (position 1474\u20133009) are indicated on top. Base pair numbers correspond to the Drosophila melanogaster mitochondrial DNA sequence (GenBank accession nr. U37541). For each study, thick horizontal lines represent the fragment sequenced in all specimens and thin lines indicate the maximum sequence length. Number of sequences (between parentheses) and references are listed at the right. For references of unpublished studies, see Appendix\nPCR products were purified using a DNA extraction kit (Fermentas, St. Leon-Rot, Germany). The purified products were directly sequenced using the ABI PRISM BigDye Terminator Sequence Kit (Applied Biosystems, Nieuwerkerk a\/d IJssel, The Netherlands) according to the manufacturer\u2019s instructions but diluted 16\u00a0times. Both strands of the products were sequenced using the same primers as used in the PCR amplification. Sequences were run on an ABI 3700 automated DNA sequencer. Obtained sequences were aligned using ClustalX v 1.8.0 (Thompson et\u00a0al. 1997) and compared to the sequences obtained from GenBank (see below).\nDatabase compilation\nSequence collection\nA single database was constructed comprising all available tetranychid COI sequences from GenBank and the sequences obtained in this study. Sequences were collected from GenBank on September 8th, 2006 (keywords for search were \u2018cytochrome oxidase subunit I AND Tetranychidae\u2019). This yielded a total of 165 sequences (156 from GenBank, 9 from this study), of which 79 (48%) were published in peer reviewed journals. An overview of all sequences, their GenBank accession numbers, assigned species names, references, sample locations, and associated host plants (if known) is given in the Appendix. Sequences were aligned using ClustalX. Due to the use of different primer combinations in the various studies, the sequences differed in length and in position on the COI region (Fig.\u00a01). A central part of 390\u00a0bp was chosen for subsequent analysis (highlighted region in Fig.\u00a01). Considering this central part, 25 sequences were found more than once (see Appendix). Prior to phylogenetic analysis, identical sequences (except one) were removed from the dataset, resulting in 96 unique sequences. In addition, six sequences with accession numbers AF131105\u2013AF131110 (Lee et\u00a0al. 1999) were excluded from analysis because of too many missing data in the region of overlap (272\u00a0bp of the 390\u00a0bp part are missing; Fig.\u00a01). The dataset was further adjusted for wrongly submitted sequences (see next paragraph) leading to the addition of one corrected sequence, yielding a final number of 91 aligned COI sequences. Of these, 71 were published in peer reviewed journals and 68 belong to the genus Tetranychus according to the GenBank submission info. A Clustal alignment of the 91 unique sequences can be obtained from the corresponding author upon request.\nData validation: Incongruencies in the database\nWhen compiling the dataset, two discrepancies were encountered between sequence information submitted to GenBank and the description in the associated articles. The first one concerns accession X80860. Its sequence was wrongly submitted to GenBank. In GenBank, accessions X80859 and X80860 are listed as T.\u00a0neocaledonicus and T.\u00a0gloveri respectively. According to the associated article, these sequences should differ 10% (Navajas et\u00a0al. 1996b). However, accessions X80859 and X80860 show identical sequences, both concurring with the sequence T.\u00a0neocaledonicus from the article. The correct sequence of T.\u00a0gloveri was obtained from the original article and added to the dataset. The second discrepancy concerns accessions X99873, X99874 and X99875. According to the description in GenBank, accession X99873 was obtained from the Amphitetranychus quercivorus strain Sapporo and accession X99874 from the A.\u00a0quercivorus strain Tsukuba. Comparing this to sequences in Navajas et\u00a0al. (1997), the X99873 GenBank sequence concurs with A.\u00a0quercivorus strain Tsukuba in the article. GenBank sequence X99874 concurs with the sequence of A.\u00a0viennensis in the article (and therefore is identical to the GenBank sequence of X99875, previously named T.\u00a0viennensis). This means that the sequences of accessions X99873 and X99874 are different from the article sequences. We included accessions X99873 as A.\u00a0quercivorus and X99875 as A.\u00a0viennensis in the dataset. Accession X99874 was excluded from the dataset.\nPhylogenetic analysis\nPAUP* version 4.0b10 (Swofford 2002) and DAMBE version 4.1.15 (Xia and Xie 2001) were used to calculate numbers of variable sites, uncorrected pairwise divergences, nucleotide composition, and transition and transversion ratios. PAUP was used to perform a chi-square test of base frequency homogeneity across all taxa.\nPhylogenetic analyses were conducted in PAUP using Neighbour-Joining (NJ) algorithms (p-distance) and Maximum Likelihood (ML) algorithms (TBR heuristics, random addition sequence with five replicates, reconnection limit of 10). Both PAUP and Modeltest 3.6 (Posada and Crandall 1998) were used to select the optimal evolution model for the ML analysis. The selected model was further optimized by critically evaluating the selected parameters (Swofford and Sullivan 2003) using the Akaike Information Criterion (AIC; Akaike 1974). Because COI is a protein coding gene, we tested if the likelihood of models with the lowest likelihood score could be further improved by incorporating specific rates for each codon position (Shapiro et\u00a0al. 2006). Under the selected model, parameters and tree topology were optimized using the successive approximations approach (Sullivan et\u00a0al. 2005). For the NJ analyses robustness of nodes was assessed with 1,000 NJ-bootstrap replicates. For the ML analyses bootstrap support was assessed by performing a NJ bootstrap (1,000 replicates) with distances calculated according to the selected ML model (because of computational constraints).\nPhylogenetic analyses were performed for i) the family Tetranychidae and ii) the genus Tetranychus separately. The analysis of the family Tetranychidae included all species. As the species T.\u00a0kanzawai, T.\u00a0urticae and T.\u00a0truncatus are over-represented, eight strains were selected representing these three species (Fig.\u00a04). The dataset for this analysis included 37 sequences. The final tree was rooted using the species Petrobia harti and Bryobia kissophila. These two species belong to a separate subfamily (Bryobiinae) from all other species (subfamily Tetranychinae). The analysis of the genus Tetranychus included 68 sequences. For this analysis, two Panonychus sequences and one Petrobia and one Bryobia sequence were used as an outgroup.\nBecause saturation of the third codon position is frequently observed for COI (Gleeson et\u00a0al. 1998; S\u00f6ller et\u00a0al. 2001), an analysis excluding this position was performed to see if this improved the resolution of the phylogeny.\nResults\nData acquisition: New sequencing\nEach COI sequence that we obtained in this study was identical to several sequences already present in the GenBank dataset. Two samples, from the USA (Arizona) and Portugal, were identical to Eutetranychus banksi. The sample from citrus in Spain was identical to Panonychus citri from Japan and the sample from Poland (from Prunus spec.) to P.\u00a0ulmi from Japan. The remaining samples (the two lab strains and a sample from France) were identical to an Asian T.\u00a0urticae strain (sampled from Japan, Thailand, and Taiwan) (see Appendix).\nAlignment and analysis of patterns of molecular evolution\nAll sequences could be unambiguously aligned; no insertions or deletions were found. Translation of all sequences into amino acids revealed no stop codons. The total alignment of the 91 tetranychid sequences was 390\u00a0bp (minimum sequence length was 304\u00a0bp; Fig.\u00a01); 146 sites were phylogenetically informative, 31 sites were variable but uninformative, and 213 sites were constant. On average across all taxa, the AT content was 75% (32%A, 43%T, 11%C, and 14%G). This high AT content is a general feature of the COI region in arthropods, and is comparable to other studies on insect and mite taxa (Lunt et\u00a0al. 1996; Navajas et\u00a0al. 1996b). However, the distribution of bias in base composition was not uniform with respect to the three codon positions (Fig.\u00a02). First, second, and third codon positions showed AT biases of 69, 64, and 94% respectively. In some haplotypes, no C or G base was found at the third codon position. Nevertheless, a chi-square test of base frequency homogeneity revealed no significant differences across taxa for the overall data set or for the three base positions separately (Fig.\u00a02). Note that this test ignores correlation due to phylogeny and therefore tends to reject the null hypothesis too easily, so that failure to reject can safely be taken as evidence of homogeneity (Frati et\u00a0al. 1997).\nFig.\u00a02Base compositions for each codon position of the 390-bp aligned COI region, averaged over all tetranychid samples. Error bars depict minimum to maximum range. Results of the homogeneity test are given for each codon position\nThe extent of saturation was assessed by plotting the transition and transversion rates against uncorrected p-distance divergences (Fig.\u00a03). At the third codon position, transversions outnumber transitions and the number of transversions begins to plateau (Fig.\u00a03), indicating saturation and making this position unsuitable for resolving more basal branching patterns. However, removing the third codon position from the analysis did not result in a more resolved phylogeny (results not shown). This is probably due to a conserved amino acid sequence (limiting the amount of variation in first and second base pair positions as changes in these positions in most cases change the amino acid sequence).\nFig.\u00a03Saturation plots of transversion and transition rates against uncorrected p-distance at each codon position\nThe model selected by Modeltest for the tetranychid dataset was the General Time Reversible Model with invariable sites and a gamma distribution of rate heterogeneity (GTR+I+G). However, implementing the rate class \u2018a b a b e f\u2019 significantly improved the likelihood (AIC) and was therefore used for parameter and tree topology estimation. For the Tetranychus dataset the General Time Reversible Model (GTR) with site-specific rates for the three coding positions was further optimized by incorporating the following rate class: a b c d e a. This slightly simpler model significantly improved the likelihood (AIC) and was used for parameter and tree topology estimation.\nPhylogenetic relationships between tetranychid genera\nThe ML tree of the overall analysis is shown in Fig.\u00a04. The phylogenetic relationship among the taxa is not well resolved. This is probably due to the strongly biased nucleotide composition and the saturation at the third codon position. It shows that this portion of the COI gene is not suitable for resolving the branching order of the genera and the more distantly related species. P.\u00a0harti and B.\u00a0kissophila, both belonging to the subfamily Bryobiinae cluster together, and group outside the subfamily Tetranychinae. There is however no high support for monophyly of any of the genera. The NJ tree shows a similar non-resolved phylogeny (not shown) with the only difference that the genus Eotetranychus appears as a monophyletic group with high bootstrap support (78%). Differences between genera range from 8 to 22% and between species within genera from 1 to 13%.\nFig.\u00a04Maximum likelihood tree of the tetranychid dataset based upon COI sequences. GenBank accession numbers and associated species names are given. If a haplotype is found more than once, the accession number is followed by the haplotype number (see Appendix) and the number of times the haplotype is found between parentheses. Numbers on the branches indicate the percentage bootstrap values (>50) based on NJ bootstrapping with ML settings (1,000 replicates). Bar at the lower left corner depicts the branch length corresponding to 10% maximum likelihood distance\nThe genus Tetranychus\nJust over 50% of the sequences in GenBank are unpublished. Hong, Xie and colleagues have submitted 27 sequences (accessions DQ437542 through DQ437568, submitted March 7, 2006) as T.\u00a0cinnabarinus. It is unclear why all these accessions were named T.\u00a0cinnabarinus. The fact that these T.\u00a0cinnabarinus accessions are scattered all over the phylogeny shows that these sequences do not concern a single species. Besides, the species name T.\u00a0cinnabarinus is not generally accepted (Dupont 1979; Gotoh and Tokioka 1996), and is also not mentioned in the World Catalogue of the spider mite family (only as synonym of T.\u00a0urticae) (Bolland et\u00a0al. 1998). In the remainder of this paper we will not use the name T.\u00a0cinnabarinus.\nFor the genus Tetranychus, the ML tree is shown in Fig.\u00a05. The NJ tree shows a similar topology as the ML tree, bootstrap support values are slightly lower in the ML tree (NJ tree not shown). Several clades emerge, although the exact branching order remains unresolved (Fig.\u00a05). The species T.\u00a0kanzawai, T.\u00a0urticae and presumably T.\u00a0truncatus (see below) have been widely sampled and intraspecific variation is substantial. These species form a monophyletic group (bootstrap support 65%). The relationship between all other Tetranychus species remains unresolved, except that T.\u00a0pacificus and T.\u00a0mcdanieli cluster together (bootstrap support 83%). Two potentially new species are found (DQ437551 and DQ437566). Sequence divergence between these two accessions is 9.2%, which is of the same order as found between other species. Although these accessions are described in GenBank as T.\u00a0cinnabarinus, this seems incorrect (see above).\nFig.\u00a05Maximum likelihood tree of the genus Tetranychus based upon COI sequences. GenBank accession numbers and associated species name are given (except for T.\u00a0cinnabarinus, see text). If a haplotype is found more than once, the accession number is followed by the haplotype number (see Appendix) and the number of times the haplotype is found between parentheses. Accessions deposited on GenBank as T.\u00a0turkestani are marked in grey. Accessions followed by the letter A or B indicate samples belonging to clade A and B respectively, deduced from Navajas (1998) and Navajas et\u00a0al. (1998). Numbers on the branches indicate the percentage bootstrap values (>50) based on NJ bootstrapping with ML settings (1,000 replicates). Bar at the lower left corner depicts the branch length corresponding to 10% maximum likelihood distance\nTetranychus urticae, Tetranychus kanzawai and Tetranychus truncatus\nThe species T.\u00a0urticae and T.\u00a0kanzawai have been investigated in several different studies and were sampled from all over the world (see Appendix). The analysis of COI variation reveals the existence of very divergent lineages (Fig.\u00a05). Clade 1 contains all T.\u00a0kanzawai specimens (bootstrap value\u00a0=\u00a071%). This clade contains two subclades that were previously described by Hinomoto and Takafuji (2001). On the other hand, T.\u00a0urticae specimens form a highly diverse group in which several well-supported clades are recognized. One clade (clade 3 in Fig.\u00a05) comprises T.\u00a0urticae specimens all originating from China (bootstrap value\u00a0=\u00a0100%). Hinomoto et\u00a0al. (2007) renamed this clade T.\u00a0truncatus. All other T.\u00a0urticae specimens form a group of highly divergent lineages (clade 2), which fall into several more or less supported subclades. Moreover, within this group several specimens have been identified as T.\u00a0turkestani, but these do not form a monophyletic group.\nDiscussion\nThe phylogenetic analysis of all COI sequences available in GenBank revealed novel patterns, which alter current views on species delineation and phylogeographic patterns in spider mites. In addition, we found that a number of accessions are probably registered under a wrong species name. This may in the past have led to erroneous interpretations of phylogenetic patterns that included these GenBank accessions.\nOne application of phylogenetic analysis is the identification of natural groupings in phylogenetic trees that represent biological species (DNA taxonomy). Our most inclusive assemblage of data shows new, and previously unnoticed, groups that most likely concern different species (Fig.\u00a05). In particular, the phylogenetic patterns within T.\u00a0urticae differ from these of previous studies and provide new insights in the evolutionary history of this group. Up to now, two clades within T.\u00a0urticae were recognized, named clade A and B by Navajas et\u00a0al. (1998) and Hinomoto et\u00a0al. (2001) and lineage I and II by Xie et\u00a0al. (2006a). The latter concluded that lineage I and II were consistent with the two clades A and B. However, this conclusion is not supported by our analysis. Our clade 2 (Fig.\u00a05) contains specimens of lineage II (Xie et\u00a0al. 2006a) and clade A and B (Navajas et\u00a0al. 1998). Moreover, clade A and B disappear in our total analysis and new groupings emerge. Clade 3 contains specimens of lineage I. In fact, clade 3 is a well supported clade restricted to China that clusters outside the other T.\u00a0urticae samples. It is unclear from the study of Xie et\u00a0al. (2006a) whether mites from clade 3 were morphologically different from other T.\u00a0urticae samples. Clade 3 presumably represents T.\u00a0truncatus, as suggested by Hinomoto et\u00a0al. (2007), based on morphological identification of newly sampled Japanese mites with highly similar COI sequences.\nTetranychus urticae and Tetranychus turkestani\nGenBank specimens listed as T.\u00a0urticae and T.\u00a0turkestani do not form separate monophyletic clades (Fig.\u00a05). This is in agreement with a study by Navajas and Boursot (2003) that was based on a smaller dataset. Although Navajas and Boursot (2003) were able to separate the two species based on ITS2 sequence, this distinction was based on three diagnostic sites only. Moreover, intraspecific and intra-individual variation within ITS2 was found, which further questions the recognition of two different species. In addition, there are no discrete morphological differences between the two species. Taxonomic identification is based on continuous traits (e.g., the shape of the aedeagus of males) and there is no thorough study describing variation of these traits within and between these species. The current data do not support the maintenance of T.\u00a0turkestani as a separate species.\nHost plant relationships\nHost race formation is another evolutionary process studied in spider mites that may explain the diversity in this group of mites. Phylogenetics is one approach to assess spider mite\u2013\u2013host plant associations. Most Tetranychus species are reported from many different host plant species. For example, Bolland et\u00a0al. (1998) described 911 different host plant species for T.\u00a0urticae, belonging to 121 plant families. We found no correlation between COI divergence and associated host plant species (Fig.\u00a05 and Appendix), similar to what was found by Navajas (1998). Even strains with identical COI haplotypes can be found on very different host plant species. Also the other two relatively well sampled species T.\u00a0kanzawai and T.\u00a0truncatus do not show host plant associations.\nPhylogeographic patterns\nPhylogenetic analysis is also used for determining phylogeographic distribution patterns (Avise 2000). Phylogeographic information is important for assessing historic migration and colonization routes and can also be used for tracing the origin of accidental introductions. For T.\u00a0kanzawai, two main clades are distinguished originating from eastern Asia (Japan, Taiwan and China), except one sample that originates from Congo. This suggests that T.\u00a0kanzawai has a mainly eastern Asian distribution. However, Bolland et\u00a0al. (1998) reported T.\u00a0kanzawai from all over the world, but it is not known if these samples fall within the clades found so far. Clade 3 (T.\u00a0truncatus) appears restricted to China (Fig.\u00a05).\nWithin T.\u00a0urticae no phylogeographic pattern is apparent with respect to COI variation. Samples form Europe, Asia, and North and South America are scattered over the tree. The phylogeographic patterns previously described by Navajas et\u00a0al. (1998) completely disappeared. They found an entirely Mediterranean clade (clade A) and a clade of mixed origin (clade B). Because the Mediterranean clade had the highest diversity, they argued that this region served as a source from which other non-Mediterranean regions of the northern hemisphere were recently colonized by a subset of the Mediterranean clades (Navajas et\u00a0al. 1998; Hinomoto et\u00a0al. 2001). However, inclusion of all currently available sequences does not support their conclusion. Clade A and B fall apart and moreover, many more clades are found. There are several possible explanations for the absence of clear phylogeographic patterns. First, such patterns may simply not exist. Second, T.\u00a0urticae is a pest species on many crops and ornamentals and it is likely that the international trade in crops has influenced the distribution of the mites around the world. This will obscure any correlation between geographical location and phylogeny. Finally, selective sweeps can greatly influence phylogenetic patterns (Ballard and Rand 2005). Evidence is accumulating that selective sweeps are often associated with the presence of reproductive parasites such as Wolbachia and Cardinium (Hurst and Jiggins 2005).\nPhylogenetic inferences and DNA barcoding\nThe analyzed COI region shows considerable variation among the tetranychids examined. The diversity within species is especially high with a maximum of 7.2%. This is a mixed blessing: it makes COI suitable for investigating intraspecific variation, but its usefulness for resolving phylogenetic species relationships remains limited. The latter is due to a strongly biased nucleotide composition at the third codon position and consequently saturation at this position. Variation at first and second codon positions is very low. As a result, relationships between taxa are difficult to resolve, especially at the deeper nodes. An extremely high AT content and saturation at the third codon position was also encountered in other studies, for the same COI fragment as used in this study in parasitengona mites and for the adjacent COI region in velvet worms (Onychophora) (Gleeson et\u00a0al. 1998; S\u00f6ller et\u00a0al. 2001).\nThe COI fragment analyzed in this study is different from the usual DNA barcoding fragment, which is located at the 5\u2032 end side of our fragment (Fig.\u00a01). Because this barcoding fragment is amplified by primers developed by Folmer et\u00a0al. (1994) it is referred to as the Folmer fragment (Erpenbeck et\u00a0al. 2006). Substitution patterns may differ between gene partitions and may result in different phylogenetic signals for these partitions (Erpenbeck et\u00a0al. 2006). To determine whether our fragment had the same phylogenetic signal as the commonly used Folmer fragment we compared the substitution patterns of both fragments. We did not find different substitution patterns when investigating 294\u2013448\u00a0bp of the Folmer fragment for samples for which this fragment was available (results not shown). A dataset of 27 unique sequences (representing 46 samples) revealed a highly similar substitution pattern with transversions outnumbering transitions resembling the patterns in Fig.\u00a03. We therefore assume that analyzing the Folmer fragment for tetranychid mites will reveal similar patterns as found in this study.\nCurrently, the Folmer fragment is widely used as a gene partition for barcoding species (e.g., G\u00f3mez et\u00a0al. 2007; Hebert et\u00a0al. 2003, 2004), although other fragments have also been proposed (e.g., in plants; Kress et\u00a0al. 2005). DNA barcoding assumes that genetic distances between species are greater than within species. In that way, clusters of similar sequences represent species, clearly separated from other clusters (species). Hebert et\u00a0al. (2003) proposed the use of a standard threshold (divergence value) to identify species. Up to now, several studies have reported successful barcoding of species (e.g., Barret and Hebert 2005; G\u00f3mez et\u00a0al. 2007; Hebert et\u00a0al. 2003, 2004). However, often, intraspecific variation was not at all or not thoroughly investigated, because only one or two individuals per species were analyzed or geographic sampling was restricted (Dasmahapatra and Mallet 2006; Prendini 2005). This may result in significant underestimation of the amount of intraspecific variation. Additionally, interspecific variation might be overestimated if closely related sister taxa are not included in the analysis. Therefore, it is necessary to analyze samples from more than one geographic region and to include closely related sister species. Our study comprises an analysis of three closely related species that were widely sampled and showed extensive amounts of intraspecific variation. Moreover, in several cases intraspecific variation exceeded interspecific variation between the species, as is illustrated by overlapping frequency distributions of intra- and interspecific pairwise p-distances (Fig.\u00a06). For example, differences within T.\u00a0urticae reach up to 7.2%, exceeding the minimum 3.7% difference between T.\u00a0urticae and T.\u00a0kanzawai. Thus the general barcoding assumption that intraspecific variation is smaller than interspecific variation is violated in tetranychids, indicating that simply relying on genetic distances is not sufficient for species identification. This clearly illustrates the importance of including samples of various, geographically different populations for each species, and to include comparisons with sister species, when investigating the efficacy of barcoding. It also shows the need to include phylogenetic information to delineate species groupings, instead of simply relying on sequence divergences (Prendini 2005; Rubinoff et\u00a0al. 2006). A careful analysis of the DNA phylogeny, preferably in a multi disciplinary approach (including multiple gene data, morphological, ecological or other relevant data), can assist in defining or delimiting species, but the use of single sequences in combination with a threshold seems insufficient to simply identify species.\nFig.\u00a06Histogram of pairwise differences (p-distance) between 91 COI sequences within the family Tetranychidae. Pairwise differences are separated into three categories: 1. between individuals in the same species; 2. between individuals in the same genus (excluding intraspecific differences); 3. between individuals in the same family (excluding intraspecific and intrageneric differences). n\u00a0=\u00a0number of pairwise comparisons\nReproductive parasites and selective sweeps\nThere are a number of additional problems associated with the use of a single mitochondrial gene for barcoding. Hybridization can result in reticulate evolutionary relationships between species and disturb groupings into species based on mtDNA. Selective sweeps of mtDNA can both homogenize or increase mtDNA diversity. The widespread occurrence of reproductive parasites in arthropods can both influence the frequency of hybridization between host species and indirectly cause selective sweeps of mtDNA (Hurst and Jiggins 2005). These parasites can cause homogenization of biological species after hybridization followed by spreading of the intracellular reproductive parasite. The mitochondrial haplotype is dragged along with these parasites resulting in replacement of the original mitochondrial haplotype and reducing mitochondrial diversity. In a recent study, Whitworth et\u00a0al. (2007) found a lack of species monophyly in the blowfly genus Protocalliphora due to introgressive hybridization associated with Wolbachia infection. On the other hand, the presence of different reproductive parasites co-infecting the same host species may increase the levels of mitochondrial diversity within that host species if each parasite is tightly linked to a different haplotype (Schulenburg et\u00a0al. 2002). Infection with reproductive parasites may thus increase or decrease mitochondrial diversity and severely influence the patterns of mitochondrial DNA variation.\nIntracellular reproductive parasites such as Wolbachia, Cardinium, and Rickettsia, are widespread in tetranychid mites (Breeuwer and Jacobs 1996; Gotoh et\u00a0al. 2003; Hoy and Jeyaprakash 2005; Xie et\u00a0al. 2006b). They can cause cytoplasmic incompatibility (CI) and hybrid breakdown in spider mites (Breeuwer 1997; Gotoh et\u00a0al. 2003, 2006; Vala et\u00a0al. 2000). It is possible that the COI variation found within and between closely related mite species is a result of selective sweeps caused by infection with reproductive parasites. Variation within species is relatively high. It is not linked to geographical location nor associated with the host plant. An interesting next step would be to investigate the link between haplotype variation within COI and variation in reproductive parasites. We should be especially cautious with the use of mitochondrial genes for delineating and barcoding biological species in light of the presence of reproductive parasites.\nConclusions and recommendations\nAccurate species delineation and identification is important for our ability to understand and interpret evolutionary processes and ecological diversity in mites. It is also clear that mites are a difficult group to identify morphologically, as many key traits exhibit large phenotypic plasticity and lack suitable characters for identification. Many ecological, behavioral, genetic and pest-control studies have been conducted on various tetranychid species or strains, without the concurrent storage of voucher specimens, leading to subsequent uncertainty about the identity of the investigated specimens. In such cases, DNA barcoding can be an important and powerful tool to assist in species identification (Will et\u00a0al. 2005). However, the use of a single (mitochondrial) gene for DNA barcoding or DNA taxonomy seems inappropriate. An integrative approach is needed combining nuclear and mitochondrial genes, morphological characters, and ecological information (and if possible crossing experiments).\nA combined analysis of mitochondrial and nuclear markers is commonly used to avoid the problem that gene trees are not necessarily congruent with species trees and for the detection of hybridization. The challenge is to find suitable nuclear markers and robust geographic sampling designs that allow for the assessment of intra- and interspecific variation. Navajas and Fenton (2000) and Cruickshank (2002) have investigated the suitability of various molecular markers, but there is still a need for nuclear markers suitable for distinguishing closely related species. Recently, Sonnenberg et\u00a0al. (2007) suggested the D1-D2 region of the nuclear 28S rDNA gene as a taxonomic marker. It could complement DNA barcoding studies based on mitochondrial DNA sequences. In addition, molecular testing for reproductive parasites and crossing experiments using isofemale lines should be standard procedure to delineate biological species.","keyphrases":["spider mites","tetranychidae","coi","reproductive parasites","barcoding","tetranychus urticae"],"prmu":["P","P","P","P","P","P"]}
{"id":"Matern_Child_Health_J-2-2-1592146","title":"The Implications of Maternal Overweight and Obesity on the Course of Pregnancy and Birth Outcomes\n","text":"The increasing prevalence of overweight and obesity among women of childbearing age is a growing public health concern in the United States. The average body mass index (BMI) is increasing among all age categories and women enter pregnancy at higher weights. Women are also more likely to retain gestational weight with each pregnancy. Women who are overweight (BMI 25\u201330) and obese (BMI \u226530) are at greater risk of adverse reproductive health outcomes compared to women of normal weight status (BMI 19.8\u201325). This article provides an overview of the complications associated with maternal overweight and obesity including diabetes, pre-eclampsia, c-sections, and birth defects. We present updated information on the weight trends among women. Finally, we present an overview of the prevention studies aimed at adolescents and women prior to pregnancy.\nIntroduction\nThe effect of maternal nutritional status prior to pregnancy on birth outcomes is of great public health importance. Epidemiological studies have shown a clear association between maternal pregravid weight and birth outcomes. As a marker of nutritional status, a woman's pregravid body mass index (BMI = kg\/m2), if low (<19.8 BMI), may reflect chronic nutritional deficiency whereas a high BMI (>26.1 BMI) reflects an imbalance between energy intake and expenditure, and thus varying degrees of adiposity [1]. The effects of each on birth outcomes differ, with low BMI being associated with intrauterine growth retardation (IUGR), preterm birth, and iron deficiency anemia [2\u20135]. Whereas, BMI above the normal range of 19.8 to 26.1 (IOM 1990) is associated with a number of adverse reproductive health outcomes. For example, infertility [6] gestational diabetes [7], pregnancy induced hypertension and pre-eclampsia [8], birth defects [9], large for gestational age (LGA) or macrosomia (>4500\u00a0g) [10], cesarean sections [11\u201313], prolonged labor [14], and recently postpartum anemia [15\u201317] have all been associated with maternal overweight yet the exact mechanisms have not been identified. The purpose of this paper is to describe an overview of the complications associated with maternal overweight and obesity; present updated information on the weight trends among women; and review prevention studies aimed at adolescents and women prior to pregnancy.\nPrevalence of overweight and obesity among women of childbearing age\nUntil recently, most studies on the topic of maternal pregravid weight focused on the lower end of the weight spectrum; however, with the shift in body weight that has occurred globally, more recent research has examined the effect of overweight and obesity on birth outcomes. Obesity world wide (>30 BMI) now exists at a prevalence of 15\u201320% and accounts for 2\u20137% of the total health care costs [18]. In the US, the latest NHANES survey (1999\u20132002) indicate that 26% of non-pregnant women 20\u201339 years of age are overweight (25\u201329.9 BMI), and 29% are obese (>29.9 BMI) [19]. On average, obesity among all women appears to have peaked at 33% with no appreciable increase between 1999\u20132000 and 2003\u20132004 [20].\nDeterminants of overweight among women\nA higher proportion of women of child bearing age are overweight or obese compared to men, and women of younger or older ages. In addition, upwards of 80% of African American women are either overweight or obese [19]. Using 2003\u20132004 data from NHANES, non-Hispanic black women are twice as likely to be obese than their non-Hispanic white counterparts (OR 2.01, 95% CI, 1.76, 2.29) [20]. Among individuals with less than a high school education, the prevalence of obesity was roughly twice that of college graduates [21]. Paradoxically, low-income households appear to have a higher risk of obesity [22], although, at the same time they are more likely to be at risk of food insecurity. Household food insecurity has been associated with increased BMI [23] and an increased risk of overweight [24] in women, but not among men or children [25\u201327].\nInfluences of adolescent overweight on reproductive health\nMoreover, thirty percent of non-pregnant adolescent girls 12\u201319 years of age are considered overweight or at risk, based on a BMI for age at the 85th percentile or higher [19]. Excessive weight gain at younger ages is associated with earlier menarche, especially among black and Hispanic girls. Younger and more severely obese girls reaching reproductive capacity perpetuate obesity if left untreated, directly through the influence of maternal weight on fetal origins and indirectly through maternal to child social and developmental interactions [28].\nPregravid BMI is a predictor of gestational weight gain\nAmong women who become pregnant, the shift towards higher pregravid weight also appears evident [29]. One study using a perinatal data base to ascertain weight before pregnancy for a 19 year period showed that the mean maternal weight of women at their first prenatal visit increased by 20% between 1980 and 1999, and the percentage of women weighing >200 lbs at that visit increased from 7.3 to 24% and for those weighing >300 lbs the percentage increased from 2 to 11% [30]. Another study using a perinatal data system of all live births from 8 contiguous counties in New York, an 11% increase in pregravid overweight and an 8% increase in pregravid obesity between 1999 and 2003 [29]. Not only are more women beginning pregnancy at a higher BMI, but women are also gaining in excess of the 1990 Institute of Medicine recommendation for gestational weight gain [1]. Specifically, and overweight and obese women are more likely to gain excessive gestational weight and keep it on after delivery [31\u201333]. In affluent countries, women retain some weight with each successive pregnancy, gaining more weight than their non-pregnant counterparts [34, 35]. These observations beg the far larger and more important question of how and when to intervene in order to optimize reproductive and individual health?\nWeight intervention studies\nMany research studies of dietary, physical activity and weight interventions focus on individuals who are already obese or experiencing co-morbidities such as heart disease, diabetes or cancer. In contrast, only a limited number of interventions have focused on weight loss and\/or maintenance of optimal weight in the context of reproductive health. On one end of the spectrum, surgical interventions such as gastric bypass and lap-band surgeries have been successful among obese women of child-bearing age with a decrease of reported rates of gestational diabetes, macrosomia, cesarean section while achieving adequate weight gain [36, 37]. Some interventions have been targeted to address obesity during adolescents, pregnancy, and the postpartum or inter-conceptional periods. These commonly have been school- or clinic-based in nature. School-based randomized intervention trails such as the Trial of Activity in Adolescent Girls (TAAG) are presently underway to determine if increased opportunities for moderate and vigorous activity may decelerate the age related decline in physical activity [38].\nClinic-based interventions\nClinic-based intervention usually begin with obesity management training for physicians because of the existence of a combination of a paucity of nutrition, physical and weight educational materials, lack of knowledge of how to counsel women, and lack of resources and personnel to assist with obesity management. For example, results from a survey of pediatricians found that only 12% of pediatricians reported high self-efficacy in obesity management, although 39% stated that physicians were important health care providers that could be effective with obesity management. Lack of non-MD staff reimbursement, of an on-site dietitian, and of patient educational materials were each highly associated with low self-efficacy in obesity management [39].\nWeight loss interventions\nTsai and Wadden [40] conducted a systematic review of major commercial weight loss programs undertaken to provide physicians with information on components, cost, and efficacy of these programs. eDiets.com, Health Management Resources, Take off Pounds Sensibly, OPTIFAST, and Weight Watchers were represented in the academic literature. The review identified Weight Watchers as the least costly with maintenance of 3.2% loss of initial weight at two years. Medically supervised very-low-calorie diet programs had the greatest weight loss (approximately 15 to 25% of initial weight), but were associated with high costs and high attrition rates. Finally, Internet (i.e., eDiets.com) and organized self-help programs (i.e., Overeaters Anonymous) produced minimal weight loss. In a clinical intervention conducted among pregnant women, physicians were trained to chart and monitor gestational weight gain adequacy based on the Institute of Medicine weight gain recommendations. Women received five patient education newsletters by-mail with action-oriented messages of how to gain adequate weight in pregnancy. Findings indicated that low-income women who received the intervention were less likely to gain excessive gestational weight. Overweight low-income women were at a reduced risk to retain more than 2.7\u00a0kg of weight at one year [41]. A hospital based randomized control trail with an intensive stepped-care, behavioral intervention found that the intervention significantly decreased the percentage of normal weight women gaining in excess of the IOM recommendations [42].\nSignificance of weight status during postpartum period\nThe postpartum period is hypothesized to significantly contribute to overweight and obesity through pregnancy weight retention. Olson et\u00a0al. [31] found that over 25% of women participating in a cohort study experienced major weight gain, defined as 4.55\u00a0kg or more, at one year postpartum. Gestational weight gain, postpartum exercise frequency, and food intake were all significantly associated with weight change from early pregnancy to one year postpartum. Lower income women who gained more than the IOM recommendation were at increased risk for major weight gain at one year postpartum. In a randomized trial of 40 overweight postpartum women receiving either a 12 week structured or self-directed diet and physical activity intervention, the structured intervention group had significant weight loss (7.3\u00a0kg), decrease in percent body fat (6%) and no change in fat-free mass, whereas the self-directed group had no significant change at one year postpartum [43]. Randomized control trials are currently underway in low-income WIC populations receiving usual care or a multi-component intervention including home visits, group classes and monthly telephone counseling [44].\nConclusion\nGiven that the costs associated with treating obesity are so high and their limited success rates, the best strategy is to prevent obesity from occurring in the first place. However, prevention first requires a clear understanding of its etiology. Obesity is a chronic disease thought to develop from a complex interaction of genotype and environmental factors [45]. Our knowledge of how and why this disease develops is far from complete, but currently funded research projects are targeting the integration of social, behavioral, cultural, and biological factors. In addition, best practices at obesity prevention, treatment and optimal weight maintenance must be identified to provide practitioners with an array of strategies to help curb the ensuing epidemic.","keyphrases":["obesity","pregnancy","women","nutrition","postpartum"],"prmu":["P","P","P","P","P"]}
{"id":"Clin_Oral_Investig-4-1-2238785","title":"Basic Erosive Wear Examination (BEWE): a new scoring system for scientific and clinical needs\n","text":"A new scoring system, the Basic Erosive Wear Examination (BEWE), has been designed to provide a simple tool for use in general practice and to allow comparison to other more discriminative indices. The most severely affected surface in each sextant is recorded with a four level score and the cumulative score classified and matched to risk levels which guide the management of the condition. The BEWE allows re-analysis and integration of results from existing studies and, in time, should initiate a consensus within the scientific community and so avoid continued proliferation of indices. Finally, this process should lead to the development of an internationally accepted, standardised and validated index. The BEWE further aims to increase the awareness of tooth erosion amongst clinicians and general dental practitioners and to provide a guide as to its management.\nIntroduction\nThe objective of tooth wear indices is to classify and record the severity of tooth wear or dental erosion in prevalence and incidence studies. There are a myriad of indices [1] which vary in type of assessment, scale, choice of teeth and other styles, resulting in non-comparability [4]. The main aim of these indices has been in research and health service planning, but since so many indices have been published it has not been possible to compare the outcomes of different studies and so provide an international overview of the present status of this condition. The variation in these indices, and for some their complexity, means that they remain a research tool of limited relevance. Further, there are no convenient and simple methods for general dental practitioners (GDPs) to record the level of tooth wear and erosion apart from using subjective terms such as mild, moderate and severe. The very nature of these terms means that their interpretation varies considerably between clinicians.\nThese are valid reasons for the need to establish a simple, repeatable and convenient index that is a valid research tool for the dental academic community but also of use in day-to-day dental practice for screening. Ideally, an index would have a basic structure that would allow for more sophisticated categories to be developed for specific research purposes, which could then be broken down again to the simplified version for clinical needs or for screening procedures. An example is the FDI DDE index [8] for enamel defects that has a basic screening index and an expanded epidemiological index.\nThe Basic Periodontal Examination (BPE) [5] or the Periodontal Screening Index adopted from the Community Periodontal Index [14] was developed to allow a convenient, repeatable method of recording periodontal diseases. Uniquely, these indices have been accepted not only by academics but also as part of the routine clinical examinations by GDPs. Development of these indices allows dentists not only to screen for periodontal diseases in a simple and effective manner but also to improve awareness and understanding of the disease. The same objectives are now needed for erosive tooth wear. Unlike caries and periodontitis, however, there is uncertainty about the validity of the criteria [9, 12] to define and grade tooth wear, erosion, abrasion and attrition, and at present, there is no conclusive knowledge how much erosive wear is physiological or can be accepted in relation to age [2, 19].\nThe Basic Erosive Wear Examination (BEWE) has therefore been designed to provide a simple scoring system that can be used with the diagnostic criteria of all existing indices aiming to transfer their results into one unit which is the BEWE score sum. The aim of the BEWE is to be a simple, reproducible and transferable scoring system for recording clinical findings and for assisting in the decision-making process for the management of erosive tooth wear.\nBasic Erosive Wear Examination (BEWE)\nThe BEWE is a partial scoring system recording the most severely affected surface in a sextant and the cumulative score guides the management of the condition for the practitioner. The four level score (Table\u00a01) grades the appearance or severity of wear on the teeth from no surface loss (0), initial loss of enamel surface texture (1), distinct defect, hard tissue loss (dentine) less than 50% of the surface area (2) or hard tissue loss more than 50% of the surface area (3). The differentiation between lesions restricted to enamel and dentine can be difficult particularly in the cervical area [11, 12]. Buccal\/facial, occlusal, and lingual\/palatal surfaces are examined with the highest score recorded.\nTable\u00a01Criteria for grading erosive wearScore\u00a00No erosive tooth wear1Initial loss of surface texture2*Distinct defect, hard tissue loss <50% of the surface area3*Hard tissue loss \u226550% of the surface area*in scores 2 and 3 dentine often is involved\nThe examination is repeated for all teeth in a sextant but only the surface with the highest score is recorded for each sextant. Once all the sextants have been assessed, the sum of the scores is calculated as indicated on the grid that follows:\nBEWE scores\nHighest scoreHighest scoreHighest score\u00a01. Sextant (17\u201314)2. Sextant (13\u201323)3. Sextant (24\u201327)\u00a0Highest scoreHighest scoreHighest scoreScore sum4. Sextant (37\u201334)5. Sextant (33\u201343)6. Sextant (44\u201347)\u00a0\nThe result of the BEWE is not only a measure of the severity of the condition for scientific purposes but, when transferred into risk levels, also a possible guide towards management (Table\u00a02).\nTable\u00a02Risk levels as a guide to clinical managementRisk levelCumulative score of all sextantsManagementNoneLess than or equal to 2aRoutine maintenance and observationRepeat at 3-year intervalsLowBetween 3 and 8aOral hygiene and dietary assessment, and advice, routine maintenance and observationRepeat at 2-year intervalsMediumBetween 9 and 13aOral hygiene and dietary assessment, and advice, identify the main aetiological factor(s) for tissue loss and develop strategies to eliminate respective impactsConsider fluoridation measures or other strategies to increase the resistance of tooth surfacesIdeally, avoid the placement of restorations and monitor erosive wear with study casts, photographs, or silicone impressionsRepeat at 6\u201312-month intervalsHigh14 and overaOral hygiene and dietary assessment, and advice, identify the main aetiological factor(s) for tissue loss and develop strategies to eliminate respective impactsConsider fluoridation measures or other strategies to increase the resistance of tooth surfacesIdeally, avoid restorations and monitor tooth wear with study casts, photographs, or silicone impressionsEspecially in cases of severe progression consider special care that may involve restorationsRepeat at 6\u201312-month intervalsaThe cut-off values are based on experience and studies of one of the authors (A. L.) and have to be reconsidered.\nThe management would include identification and elimination of the main aetiological factor(s), prevention and monitoring, as well as symptomatic and operative intervention where appropriate. It does not provide guidance for the most appropriate prosthodontic or operative technique as there is too much variation at this level of decision making between clinicians. However, for the highest risk levels special care is suggested.\nThe repetition of the BEWE will vary according to the severity and the relative importance of aetiological and risk factors. For patients particularly exposed to intrinsic or extrinsic acids [17], the procedure should be repeated at 6-month intervals, but for most other cases, annually is acceptable.\nDiscussion\nThe need for a standardised and internationally accepted index is obvious, but there continues to be research needs which may not be fulfilled with a simplified index. Over the past 20 to 30\u00a0years, different researchers have developed indices which suit their own research needs but do not allow comparison to assess the prevalence of tooth wear between countries and regions. Therefore, this new scoring system has been designed to allow existing and hopefully future indices to be collapsed and re-analysed. It will be important that this proposed system is validated against existing data or in field trials. In time, it should initiate a consensus within the scientific community and so avoid continued proliferation of indices. Finally, this process should lead to the development of an internationally accepted, standardised and validated index.\nThe structure of the BEWE is designed to allow fulfilling of most formal requirements generally formulated for indices. The grading includes four levels which is neither too precise nor too crude, and the threshold values should be easy to learn and to calibrate. In addition by removing the clear distinction between \u201cenamel loss\u201d and \u201cdentine\u201d exposed, it will not only evade diagnostic uncertainties but will open a broad applicability beyond the clinical situation. It can be used with study models or photographs which appear suitable for erosive wear as a surface phenomenon. Erosion already has been documented or diagnosed on study models and on photographs [10, 13, 15], and this could be of particular value in cross-sectional and incidence studies as well as for the monitoring of individual cases.\nIt will further allow a more reliable estimation of the severity of tooth surface loss on an individual basis rather than the current way to give data as \u201cx% of the subjects had at least one tooth with grade x or grade y erosion\u201d, and so avoid an overestimate of the problem. The BEWE is ideal for screening studies, but a longer version is required for a more detailed investigation.\nAs to the field of dental care, the clinical value of the BEWE, as with the BPE, should be highly significant as a convenient diagnostic tool for GDPs and as a model to increase awareness assuming that this relatively new entity is not well recognised. Even in UK, where the prevalence and incidence of erosive wear is relatively high [7, 18], a questionnaire survey has shown that only one third of the practitioners noted erosion on a frequent basis and the majority underestimated the prevalence of the condition [6]. The benefit of a system that encourages the more careful examination of the dental hard tissues other than for caries is therefore obvious and is one further aim of the BEWE.\nIn addition to diagnosis and scoring, also a strategy for treatment is included in the BEWE. There are a number of reviews with respect to the prevention and therapy of erosive wear (e.g. [3, 16]), but it is not only the variety of recommendations which makes the management of the condition difficult for the GDP, but also the fact that these recommendations were not made with respect to the severity of tooth surface loss in an individual. The risk levels presented aim to guide the management of erosive wear and allow sufficient flexibility so that different countries can adapt the index without compromising their unique needs. The levels defined, however, are suggestions at the present stage and need an ongoing review process.\nConclusion\nThe BEWE is a basic structure to initiate the development of an internationally accepted, standardised and validated index. This is to provide on one hand a clear and defined structure for scientific and clinical use, but on the other to be amenable for further development. It will encourage clinicians, students and GDPs to pay more attention to erosive wear and hence will be beneficial for patient care.","keyphrases":["erosion","examination","index","tooth wear","epidemiology","diagnosis"],"prmu":["P","P","P","P","P","P"]}
{"id":"Sleep_Breath-3-1-1794625","title":"Efficacy and patient satisfaction with autoadjusting CPAP with variable expiratory pressure vs standard CPAP: a two-night randomized crossover trial\n","text":"Expiratory pressure relief (C-Flex) technology monitors the patient\u2019s airflow during expiration and reduces the pressure in response to the patient. Increased comfort levels associated with C-Flex therapy have potential to improve patient adherence to therapy. The purpose of this study was to assess the combination of autoadjusting CPAP (APAP) and C-Flex in terms of (1) treatment efficacy, and (2) patient preference when compared to standard CPAP. Fifteen patients who had previously undergone formal CPAP titration polysomnography were treated with either one night of the APAP with C-Flex or one night of conventional CPAP, in a crossover trial. Patient satisfaction levels were recorded using visual analog scales (VAS) on the morning after the study. Mean patient age was 50 \u00b1 12 years, body mass index (BMI) was 36 \u00b1 6 kg\/m2, baseline AHI was 53 \u00b1 31 events\/h, and CPAP Pressure was 11 \u00b1 2 cm\/H2O. APAP with C-Flex was as effective as CPAP, with no differences detected in sleep latency (17 \u00b1 5 vs 12.3 \u00b1 3 min, p = 0.4), or respiratory indices (AHI of 4.2 \u00b1 2 vs 2.4 \u00b1 0.7 events\/h, p = 0.1). VAS scores (scale 0\u201310) indicated a trend towards increased patient satisfaction while using APAP with C-Flex (7.9 vs 7.2, p = 0.07). 10 patients expressed a preference for APAP with C-Flex (VAS, 0 to10) over standard CPAP (total positive score of 68, mean score of 4.8 \u00b1 4.3). One patient expressed no preference. Four patients expressed a preference for CPAP (total positive score of 13, mean score of 0.9 \u00b1 1.9) (APAP with C-Flex vs standard CPAP, p < 0.01 paired t test). APAP with C-Flex eliminates sleep disordered breathing as effectively as standard CPAP. Patients indicated a preference for APAP with C-Flex suggesting a possible advantage in terms of patient adherence for this mode of treatment.\nIntroduction\nContinuous positive airway pressure (CPAP) is the most commonly prescribed treatment for patients with obstructive sleep apnea (OSA). CPAP is effective in reducing sleep fragmentation, resolving nocturnal desaturation, and improving daytime sleepiness [1]; moreover, CPAP is a cost-effective use of healthcare resources, and may reduce risks of motor vehicle crashes and the development of cardiovascular morbidity [2\u20135].\nThe efficacy of CPAP is compromised by relatively poor compliance. A variety of measures have been shown to improve compliance with CPAP; these include aggressive follow-up of patients prescribed CPAP, group education, heated humidification, and mask adjustments [6\u201311]. Modifications in the algorithm, by which positive pressure is delivered, have been attempted to improve comfort and compliance with therapy.\nA typical complaint by those using conventional CPAP is that it is difficult to exhale against positive pressure. Therefore, reducing pressure during exhalation may improve tolerance with therapy. C-Flex (Respironics, Murrysville, PA, USA) is a novel algorithm designed to provide pressure relief during expiration, while maintaining optimal pneumatic splinting for effective therapy.\nAnother modification is the development of autoadjusting (APAP) devices. In contrast to conventional CPAP, APAP devices modify applied pressure in response to patient needs. APAP devices, through varied mechanisms, are able to detect obstructive events and change pressure accordingly to relieve the obstruction. This results in a reduction in mean applied CPAP pressure [12], and may be useful in patients who have difficulty tolerating CPAP. Furthermore, APAP may be useful for ambulatory titration of CPAP pressure in patients in whom in-laboratory therapy titration is either delayed or impossible [13\u201315]. The newest iteration of the REMStar Auto employs both the REMStar Auto CPAP algorithm and the C-Flex dynamic expiratory pressure relief.\nThis is the first study to evaluate the REMStar Auto with C-Flex (RSA C-Flex) as a therapy for patients with OSA. The major purpose of the study was to assess the ability of RSA C-Flex to effectively treat the sleep apnea events. Satisfaction and preference from the patients\u2019 perspective, and the number of required interactions per night by technicians were also determined.\nMaterials and methods\nStudy subjects\nConsecutive, recently diagnosed, adult patients (\u226518\u00a0years of age) with moderate to severe OSA [apnea hypopnea index (AHI) >15\u00a0events\/h of sleep as determined by PSG] [16] were asked to participate in our study. All subjects had a titration PSG that determined the effective pressure (i.e., AHI <5\u00a0events\/h during all sleep stages and positions). All subjects were using conventional CPAP at home on a regular basis with an average nightly usage >4\u00a0h, confirmed by objective evaluation of CPAP machines. Exclusion criteria included patients who were medically unstable, using oxygen, had surgery of the upper airway or nose within the previous 90\u00a0days, or who could not provide informed consent.\nThe study was approved by the Ethics Board at the University of British Columbia.\nProcedures and techniques\nSleep laboratory rooms were configured with a device that could deliver either conventional CPAP or RSA C-Flex. The study occurred on two separate nights, within 2\u00a0weeks of each other. For the first night, patients were randomized to either conventional CPAP at the previously determined effective pressure or RSA C-Flex. The patient was not aware of which of the therapies they would be receiving. However, the technician, by necessity, was aware of the therapy choice. On the second experimental night, subjects received the alternative therapy (crossover design).\nFor each night, all subjects were instrumented for polysomnography and PAP titration according to practice parameters published by the American Sleep Disorders Association Standards of Practice Committee [17]. Chest and abdominal excursion was measured using respiratory impedence plethysmography (Respitrace; Ambulatory Monitoring Equipment, Ardsley, New York, USA). Oxygen saturation was recorded using a pulse oximeter (Model N-100; Nellcor, Hayward, California, USA). Airflow was measured directly from the CPAP machine. Respiratory events were defined by standard criteria [16]. Obstructive apneas were defined as absence of airflow for greater than 10\u00a0s. Obstructive hypopneas as a 50% decrease in airflow, or a clear but lesser decrease in airflow if coupled with either a desaturation of >3% or an arousal in the context of ongoing respiratory effort. Central apneas were defined as absence of airflow and respiratory effort for greater than 10\u00a0s. Central hypopneas as a 50% decrease in airflow with concomitant reduction in respiratory effort, or a clear but lesser decrease in airflow if coupled with either a desaturation of >3% or an arousal. Arousals were counted per hour of total sleep time. Sleep efficiency index was defined as total sleep time\/total time in bed.\nSatisfaction with devices was assessed on the morning after the study (see attached surveys, Appendix\u00a01). All subjects completed a visual analog satisfaction (VAS) questionnaire. VAS scores are useful in assessing subjective patient experience and have been used for this purpose in a previous crossover trial comparing conventional CPAP and APAP [18]. Patients scored satisfaction (0\u201310) with different aspects of their experience from the previous night. Questions included: (1) How well did you sleep last night? (2) How rested do you feel this morning? (3) How often did you wake during the night? (4) How uncomfortable was your CPAP pressure? (5) How difficult was it to get to sleep? (6) Overall, how satisfied were you with your quality of sleep last night? Mean score was calculated for all 15 patients on both nights.\nPreference for device was assessed on the morning after the second experimental night. Subjects were asked \u201cIn comparison to the first night of the study, please state a preference for the CPAP machine set-up\u201d. Subjects were asked to place a mark on a line to indicate the degree of preference for the first night (0\u201310) or the second night (0\u201310). Mean score was calculated for both nights, with a score of less than 0 indicating preference for the other night.\nThe technicians recorded any required interactions with the subject, during the night.\nConventional CPAP without C-Flex\nCPAP pressure was set to the therapy pressure determined during a previous titration study. A 20-min ramp was used with the initial pressure being set as 66% of the previously titrated pressure.\nRSA with C-Flex\nThe REMStar Auto algorithm (Respironics, Murrysville, PA, USA) is a proactive, flow-based algorithm. The basic algorithm evaluates inspiratory flow, and determines impending or actual flow limitation. This flow evaluation occurs in concert with a program of pressure adjustments designed to evaluate the critical pressures (at which airways are susceptible to collapse) and resolve therapy pressures slightly above these critical pressures. The patient is protected from \u201cbreak-through\u201d events with a full complement of intelligent responses to airflow events and snoring. C-Flex technology monitors the patient\u2019s airflow during expiration and reduces the pressure in response to the patient. The relief pressure may vary on a breath-to-breath basis, depending on the actual patient airflow (Fig.\u00a01). The device can be preset to deliver varying degrees of C-Flex pressure relief (settings of 1\u20132\u20133).\nFig.\u00a01Pressure relief is shown on a breath by breath basis for the three standard gain settings. Pressure relief is dependant on patient flow according to the formulae\u2014pdelivered\u2009=\u2009pbase\u2212prelief. prelief\u2009=\u2009(flow*gain)\nRSA C-Flex was set at a minimum pressure of 66% of previously titrated therapy pressure and a maximum pressure of 20\u00a0cm H2O. Patients were arbitrarily set to a C-Flex setting of 3 (maximum pressure relief). If the patient commented about any sensation of \u201cbreathlessness\u201d due to the pressure relief, comfort settings were adjusted to 2 or 1 by the night technician.\nStatistical analysis\nBaseline characteristics were described using means and standard deviations for continuous variables. Differences between the CPAP and RSA C-Flex arms were compared using paired t tests. Two-tailed tests were used to assess significance (at the 0.05 level).\nResults\nFifteen patients were included in the trial. Baseline characteristics included (mean\u00b1SD) age 50\u2009\u00b1\u200912\u00a0years, BMI 36\u2009\u00b1\u20096\u00a0kg\/m2. Overall, the patients had substantial sleep apnea with a mean baseline AHI of 53\u2009\u00b1\u200931 with a mean CPAP pressure of 11\u2009\u00b1\u20092\u00a0cm H20.\nResults for the RSA C-Flex vs standard CPAP are shown in Table\u00a01. Overall, RSA C-Flex was as effective as standard CPAP in relieving upper airway obstruction with a mean AHI of 4.2 vs 2.4\/h (p\u2009=\u20090.1, Fig.\u00a02). One patient had central apneas during the RSA C-Flex night (index 10.2\/h). When these central events were excluded, the mean AHI during the RSA C-Flex night decreased to 3.2\u00a0events\/h (Fig.\u00a02). Furthermore, 12 out of 15 patients had an AHI of less than five on the APAP night, which is similar to the proportion on standard CPAP therapy (13\/15). Measures of sleep efficiency were similar between the groups. Although patients had more slow-wave sleep during the RSAC night, this was not statistically significant. \nTable\u00a01Outcomes with RSA C-Flex vs standard CPAP\u00a0RSA C-FlexCPAPP valueAHI (events\/h)4.2\u2009\u00b1\u20092.4\u2009\u00b1\u20090.70.1Sleep latency (min)17.0\u2009\u00b1\u2009512.3\u2009\u00b1\u200930.4Sleep efficiency (%)82\u2009\u00b1\u2009283\u2009\u00b1\u200920.6WASO (min)67\u2009\u00b1\u20091465\u2009\u00b1\u2009120.9REM %25\u2009\u00b1\u2009224\u2009\u00b1\u200920.7Stage 3\/4 (%)4\u2009\u00b1\u20091.51.6\u2009\u00b1\u20090.50.13Mean oxyhemoglobin saturation (%)97.3\u2009\u00b1\u20090.397.2\u2009\u00b1\u20090.30.93PLMI (events\/h)2.8\u2009\u00b1\u20091.03.8\u2009\u00b1\u20091.50.59Values given as mean\u00b1SEMAHI Apnea hypopnea index, WASO wake after sleep onset, PLMI periodic limb movement indexFig.\u00a02Mean oxygen saturation and AHI were compared between the treatments. Mean AHI was 4.5 in the C-Flex arm, as compared to 2.5 in the standard CPAP arm (p\u2009=\u20090.1). When central events were excluded, this decreased to 3.5 in the C-Flex arm compared to 2.5 in the CPAP arm (p\u2009=\u20090.12)\nPatient satisfaction levels were measured using a visual analog scale (Fig.\u00a03). In general, VAS scores tended to be higher on the RSA C-Flex night, although none of the scales individually demonstrated a significant difference. However, when Mean VAS scores were calculated for individual patients and compared using a paired t test, a trend towards an increased score was noted in the C-Flex group (7.9 vs 7.2, p\u2009=\u20090.07, Fig.\u00a04a).\nFig.\u00a03Visual analog scales were used to determine patient comfort levels. Scales ranged from 0\u201310 and were administered on the morning post sleep study. Although VAS scores were higher on the C-Flex night, differences were not significant using a paired t test (p\u2009>\u20090.1 for all outcomes)Fig.\u00a04a Mean VAS scores were calculated for individual patients and compared using a paired t test. A trend towards increased score was noted in the C-Flex group (7.9 vs 7.2, p\u2009=\u20090.07). b Patient scored their preference using VAS scores (1\u201310). A significant preference was noted for C-Flex (p\u2009<\u20090.01)\nIn addition, patients were asked to rate their preference for the treatment mode. Ten patients expressed a preference for the RSAC night with the sum of positive scores favoring the RSAC of 68 (mean 4.8\u2009\u00b1\u20094.3). Four patients expressed a preference for CPAP with the sum of positive scores favoring CPAP of 13 (mean 0.9\u2009\u00b1\u20091.9). One patient expressed no preference. A significant difference was found in preference between these two arms (paired t test, p\u2009<\u20090.01) (Fig.\u00a04b).\nSleep technicians recorded the number of interactions with each patient during the night. The majority of these interactions were to adjust recording leads, and a minority of occasions to adjust mask position. Technician interactions did not differ between nights (1.4 vs 2.3 per night, p\u2009=\u20090.14).\nDiscussion\nIn our study, RSA C-Flex was as effective as standard CPAP in treating sleep-disordered breathing. There were no differences in sleep-related breathing or architecture variables between the arms. However, the RSA C-Flex arm was associated with a trend in improvement in patient satisfaction as assessed by VAS scores. Patients preferred RSA C-Flex over standard CPAP.\nTo our knowledge, only one other published study has looked at the use of C-Flex in OSA. In a nonrandomized study of 89 patients, Aloia et al. [19] examined treatment adherence in patients with OSA assigned to either standard CPAP or C-Flex therapy. Clinical outcomes were measured at 3\u00a0months and although functional outcomes associated with sleep did not differ, adherence was significantly greater in the C-Flex group (4.8 vs 3.1\u00a0h\/night, p\u2009<\u20090.01). The crossover design of our trial using random treatment order guaranteed patient matching while allowing us to assess patient preference. Our study is not directly comparable to that of Aloia et al. as they examined long-term compliance. However, our results are consistent in that our two-night study demonstrated a preference of the patient for RSA C-Flex.\nOur findings are consistent with other studies of autoadjusting CPAP therapy. A metaanalysis of nine randomized controlled trials demonstrated that APAP is as effective as standard CPAP in treating sleep apnea (in terms of reducing apnea hypopnea index) [12]. Although this metaanalysis did not demonstrate a significant improvement in compliance with APAP, other studies have suggested that APAP may be preferred by patients, especially if their baseline CPAP pressures are elevated [20]. To control this, we used 20-min ramps during CPAP nights in an attempt to match initial pressures with the RSA C-Flex night.\nIn this two-night study, we showed that RSA C-Flex is as effective as standard CPAP in reducing AHI. We noted that one patient with a treatment AHI of >20 had a high proportion of central sleep apnea. Review of this patient\u2019s diagnostic study revealed a very high baseline AHI (>100\/h) with the presence of central apneas (index>20\/h). As such we feel that residual central apneas in this patient reflect baseline disease rather than being caused by RSA C-Flex. However, the possibility that the RSA C-Flex may predispose to central events in a subgroup of patients cannot be excluded.\nPatients preferred RSA C-Flex over standard CPAP, and may have found it more comfortable. As such, we speculate that RSA C-Flex may improve compliance if used as chronic therapy. Furthermore, one of the major determinants of long-term compliance with CPAP is the early experience with the therapy. For many patients, in-laboratory titration represents the first exposure of any duration to CPAP therapy (Appendix 2). Any positive impact on this initial experience has the potential to improve compliance in the longer term. Use of RSA C-flex may thus be useful for initial titration of CPAP in the laboratory. For similar reasons, RSA C-flex may be useful in patients who are being titrated at home with APAP. The impact of RSA C-Flex on long-term compliance with chronic use, as \u201csalvage\u201d therapy of patients intolerant of standard CPAP, or its utility as part of a laboratory\/ambulatory CPAP titration need to be studied in larger clinical trials.\nWe acknowledge that there are a number of limitations to our study. First, although the patient was not told of the nightly treatment assignment, the technician was not blinded. For our study, it was not possible for the technician to be unaware of the treatment. Second, we have studied a relatively small number of patients. Third, we have only studied patients for two nights in the laboratory and as such, we cannot extrapolate our study to chronic treatment. We have not tested CPAP adherence in this study; our results merely suggest a reasonable hypothesis for future study\u2014that use of the RSA C-Flex may have a favorable effect on compliance. Finally, this study included patients with predominately severe disease who were compliant with CPAP. We may not be able to extrapolate our results to patients with less severe sleep apnea, or to patients with other concomitant respiratory diseases (such as COPD).\nConclusion\nIn this small short-term study, both RSA C-Flex and standard CPAP (with pressure defined by an in-laboratory CPAP titration) were effective in treating sleep apnea. Most patients preferred RSA C-flex. Future studies with this technology are warranted to assess its effects on long-term compliance and its potential as rescue therapy for those initially noncompliant with standard CPAP therapy.","keyphrases":["cpap","c-flex","patient preference","auto-cpap"],"prmu":["P","P","P","U"]}
{"id":"Eur_J_Health_Econ-_-_-1388080","title":"Does the English NHS have a \u2018Health Benefit Basket\u2019?\n","text":"A \u2018health benefit basket\u2019 is a range of publicly entitled health-related goods and services. Primary legislation ensures the provision of broad categories of healthcare, but this provision is subject to political discretion. Case law has established that healthcare organisations may not operate a \u2018blanket ban\u2019 for particular services. This means that the English health basket currently has very few specific services explicitly included or excluded. Regulation may, however, be important in determining citizens\u2019 rights. With reference to \u2018services of curative care\u2019, this paper explores whether the NHS is moving towards a more explicit definition of a health basket.\nThe English National Health Service (NHS) was established in 1948 to provide healthcare for all, free at the point of use and irrespective of ability to pay. Legislation outlines the broad categories of healthcare service that should or could be provided within the NHS. However, the legal duties and powers of provision are not absolute, but tempered by powers of discretion as to what is a \u2018reasonable requirement\u2019 and by the right to take into account NHS financial capacity. Strictly speaking, this means that patients have no entitlement to specific services; this partly explains the existence of variation in local provision known as the \u2018postcode lottery\u2019 [1]. Furthermore, the courts have established that NHS organisations may not operate a \u2018blanket ban\u2019 on the provision of particular services [2]. Consequently there are few services that are explicitly unavailable to all NHS patients.\nWithin a health service with no specific entitlements but few explicit exclusions, internal quality control mechanisms are important to ensure that citizens\u2019 rights to health care, established under international law, are honoured [3]. National standards, embodied in guidance from National Service Frameworks (NSFs), the National Institute for Health and Clinical Excellence (NICE) and waiting time guarantees, are regulated by the Healthcare Commission, which monitors NHS organisations\u2019 compliance. The regulatory framework contributes to what may be considered as \u2018reasonable requirements\u2019 for health care provision and helps to specify entitlement. Fixed charges or payments, whilst in no way guaranteeing provision, implicitly brand services with an \u2018NHS\u2019 label and may therefore also serve to signal the service is, or should, be in the health basket. Incentive payments for clinicians may be seen in a similar light.\nThis paper describes the statutory and regulatory frameworks and discusses how these may impact upon patient entitlement to NHS \u2018services of curative care\u2019, \u2018HC1\u2019 of the International Classification for Health Accounts (ICHA) taxonomy [4]. An overview of the legal and regulatory framework defining benefits for England is given in Table\u00a01.Table\u00a01 Documents defining the English Health Basket, 2005Catalogue: type of document, actors and contentsCriteria used for defining benefitsType of documentLegally bindingPositive\/negative definition of benefitsDegree of explicitnessaUpdatingNCECEBOtherActs of ParliamentYesP1Irregular, amended by further legislation+\u2013\u2013\u2013\u2013Political judgement \u2018necessary to meet all reasonable requirements\u2019Statutory instruments (SI)YesP or N1\u20133Irregular, amended by further legislation+\u2013+++SafetyDirectionsYesP2No+\u2013+\u2013+\u2013National Service FrameworksNoP2 or 3Unclear+\u2013+\u2013\u2013\u2013NICE technology appraisalsYesbP or N3Every 4\u00a0years\u2013+++\u2013\u2013NICE clinical guidelinesNoP2 or 3Every 4 to 6\u00a0years\u2013+++\u2013\u2013NICE interventional proceduresNoP or N3Unclear\u2013\u2013+\u2013\u2013SafetyContractsYesP1\u20133Infrequent-although small amendments more frequent+\u2013\u2013\u2013+\u2013Waiting time guaranteescNoP2Irregular+\u2013+\u2013\u2013\u2013HRG tariffsNoP2 or 3Still evolving\u2013+\u2013\u2013+\u2013Devices tariffNoP3Monthly\u2013+\u2013++Safety, quality, appropriatenessFee schedulesNoP3Annually (at least)+\u2013\u2013\u2013+\u2013N need, C costs, E effectiveness, CE cost-effectiveness,\nB budget (from [1, 2, 3], DH website (http:\/\/www.dh.gov.uk\/home\/fs\/en), HMSO website (http:\/\/www.hmso.gov.uk\/), expert advice (see Acknowledgements))\na \u201cExplicit is subdivided as 1: all necessary\u201d; 2: areas of care; 3: items.b The statutory duty is upon PCTs to ensure funding is available to facilitate implementation, not upon doctors to adopt the approved technology.c The dominant instrument for securing these are performance ratings prepared by the Healthcare Commission.\nEngland\u2019s \u2018Health Benefit Basket\u2019: the case of services of curative care\nServices of curative care are defined as those where the principal medical intent of care is to relieve symptoms or reduce severity or protect against exacerbation or complication of illness or injury. In addition to in-patient and day care, HC1 includes \u2018out-patient care\u2019, defined as basic medical and diagnostic services, out-patient dental care and other specialist health care provided to outpatients by physicians or paramedics, including services provided at home [4].\nThe legal framework\nPrimary legislation addresses the establishment and promotion of \u2018a comprehensive health service\u2019, requiring the Secretary of State for Health \u2018to provide or secure the effective provision of services\u2019. However, discretion is at the heart of these duties and politicians are entitled to take into account the resources available to them.\nUnder the 1977 National Health Service Act (chap.\u00a049), the Secretary of State has a duty to provide \u2018to such extent as he considers necessary to meet all reasonable requirements\u2019: (a) hospital accommodation [s. 3 (1(a))], including high security psychiatric services (s. 4); (b) other accommodation necessary for the purpose of any other services required by the Act [s. 3 (1(b))]; (c) medical, dental and nursing services [s. 3 (1(c))] (N.B.: \u2018medical\u2019 includes \u2018surgical\u2019); and (d) such other services as are required for the treatment of illness [s. 3 (1(f))] (N.B.: \u2018illness\u2019 includes a mental disorder and any injury or disability requiring medical or dental treatment or nursing). The Secretary of State for Health also has powers to provide \u2018as he considers appropriate\u2019 facilities for the care of persons suffering from illness [s. 3 (1(e))]. The responsibility for providing these services has passed to local health authorities, which in the current organisational structure are known as Primary Care Trusts (PCTs). The 1977 NHS Act also places a duty on PCTs to provide general medical services (s. 29), general dental services (s. 35), pharmaceutical services (s. 41) and general ophthalmic services (s. 38).\nIn the law courts \u201cR v NW Lancashire Health authority, ex p A, D and G\u201d examined the case of three applicants suffering from \u2018gender identity dysphoria\u2019 [2]. The Health Authority had identified this illness as amongst the bottom 10% in terms of need (together with cosmetic surgery, reversal of sterilisation, correction of myopia and most \u2018alternative\u2019 medicines) and therefore transsexual surgery would be provided only in cases of \u2018overriding clinical need\u2019. The court acknowledged the need for priority setting in which issues of effectiveness, the seriousness of the condition and cost were taken into account. However, the court found that the Health Authority had in practice adopted a \u2018blanket ban\u2019 and recommended the authorities introduced a fair and consistent policy for decision making that adequately assessed exceptional cases by considering each request for treatment on its individual merits. The case therefore made it illegal for health authorities to refuse to provide specific services, with the possible exception of a treatment where \u2018the clinical evidence of its inefficacy is overwhelming\u2019 [2]. The case implies that costs and benefits should be evaluated on an individual patient basis, rather basing entitlement on the typical or average case.\nPrimary legislation and case law have therefore not prescribed which services are to be included in or excluded from the English health basket. However, secondary legislation on professional contracts goes some way towards defining entitlement to specific services. For example, the 2004 National Health Service (General Medical Services Contracts) Regulations provide details of the terms of service for general practitioners (GPs) and state that \u2018essential services\u2019 must be provided, covering emergency treatment and treatment for patients with chronic, terminal and self-limiting disease [National Health Service, General Medical Services Contracts, regulation SI 2004\/291, reg. 15 (3:5,6:8), 2004; see Table\u00a02].Table\u00a02 Eligibility for primary care \u2018essential services\u2019 under the 2004 General Medical Services Contract (from [19])Type of essential serviceEligibility for treatmentImmediate and necessary emergency treatment\u2018Any person to whom the contractor has been requested to provide treatment... at any place in its practice area\u2019 [reg.15 (6)]Management of terminal illnessRegistered patients and temporary residentsTreatment of conditions from which recovery is generally expectedRegistered patients and temporary residentsTreatment of chronic diseaseRegistered patients and temporary residentsAdvice in connection with the patient\u2019s health, including relevant health promotion adviceRegistered patients and temporary residentsReferral of the patient for other services under the 1977 ActRegistered patients and temporary residentsHome visitsWhere contractor considers it inappropriate, because of a patient\u2019s medical condition, for the patient to attend the practice premisesAnnual health checksPatients aged over 75\u00a0yearsPatients not seen within 3\u00a0years; newly registered patients\nFor dental practitioners the National NHS (General Dental Services) Regulations 1992 make provision for the Secretary of State for Health to determine dentists\u2019 remuneration, including a \u2018scale of fees\u2019 for providing particular services [reg. 19 (1)]. Updated at least annually, the Statement of Dental Remuneration describes over 400 services covered by the fees, including clinical examinations and treatment planning, diagnostic procedures, such as radiographic examinations, preventative, periodontal, conservation and surgical treatments and the supply of prostheses; patients pay 80% of the charge up to a stated maximum, although some exemptions apply (Department of Health, Statement of Dental Remuneration, amendment no. 93, 2005). The Doctors\u2019 and Dentists\u2019 Review Body reviews existing fee scales and makes recommendations regarding uplift. The Department of Health then considers these recommendations and the Chief Dental Officer for England notifies Strategic Health authorities, PCTs, NHS Trusts (groups of NHS hospitals, each operating as a single legal entity) and all general dental practitioners of the updated fee scales (ibid.).\nSimilarly, the 1986 NHS (General Ophthalmic Services) Regulations make provision for opticians and ophthalmic medical practitioners to charge for sight tests and for optical appliances (National Health Service, General Ophthalmic Services, regulation SI 1986\/975, 1986). Charges are determined by the Secretary of State in consultation with professional bodies, updated at least annually and amended by secondary legislation. For certain patients vouchers are available that reduce or remove the charge incurred by patients. Eligibility for general ophthalmic services, outlined in section 13 of the Health and Medicines Act 1988, is determined by age, disease (or risk of disease) or income. Voucher values are updated at least annually by secondary legislation.\nQuasi-law\nIn addition to legislation, NHS provision is shaped by a considerable amount of \u2018quasi-law\u2019. Quasi-law is defined as \u2018rules which are not usually legally binding, although they may have some legal force, but which will in practice determine the way in which people act\u2019 [3]. Amongst the regulation helping to define patient entitlement to services are NSFs, NICE guidance, waiting time guarantees, fee schedules, and incentive schemes. The Healthcare Commission is the key regulator, assessing the performance of NHS organisations against national standards in its \u2018annual health check\u2019, a monitoring process that assesses both existing performance (\u2018core\u2019 standards) and capacity to improve (\u2018developmental\u2019 standards) [5].\nThe programme of NSFs, launched by the Department of Health in April 1998, usually produces one new framework a year. NSFs set national standards, identify key interventions for a defined service or care group that should be available, establish strategies to support implementation and outline ways to ensure progress within an agreed time scale [6]. Frameworks cover some services of curative care (Table\u00a03). Each NSF is developed with the assistance of an external reference group (ERG), which seeks to engage a range of views from health professionals, service users and carers, health service managers, partner agencies and other advocates. However, the economic input into NSFs is sometimes weak. The Department of Health supports the ERGs and manages the overall process. It is unclear how NSFs are to be updated to reflect changes in the evidence base that underpins them.Table\u00a03 Services of curative care covered by selected National Service Frameworks; ICHA category HC.1: services of curative careCategoryServicesChildrenStandard 7: Guidance on hospital-based services for childrenStandard 3: Guidance on community-based careCoronary heart diseaseStandard 7: NHS Trusts to provide appropriate investigations and treatments for patients with suspected or confirmed coronary heart diseaseDiabetesStandard 7: NHS to provide rapid and effective treatment for diabetic emergenciesMental healthIn-patient hospital beds for persons needing a short period of intensive intervention and observationElderlyStandard 4: Need for appropriate specialist careStandard 7: Effective diagnosis, treatment and support for those with mental health problemsRenal diseaseQuality requirement 2: Timely, appropriate and effective investigation, treatment and follow-up for those with chronic kidney diseaseStandard 5: All likely to benefit from a kidney transplant to receive a high quality service which supports them in managing their transplant(from: DH website, http:\/\/www.dh.gov.uk\/PolicyAndGuidance\/HealthAndSocialCareTopics\/HealthAndSocialCareArticle\/fs\/en?CONTENT_ID=4070951&chk=W3ar\/W, accessed 12 July 2005)\nThere is no statutory obligation on health care organisations to implement NSF standards. However, the Health And Social Care (Community Health And Standards) Act 2003 [Health and Social Care (Community Health and Standards) Act, Chap. 43, 2003) gave the Secretary of State powers to publish standards for health care [s. 46 (1)] that NHS bodies are bound to take into account [s. 46 (4)]; NSFs could inform these standards. Furthermore, the Healthcare Commission reviews health care organisations\u2019 implementation of NSFs as part of its annual health check [5], reinforcing the quasi-legislative nature of NSF guidance.\nNICE, the organisation responsible for assessing whether new or existing technologies should be available on the NHS, produces three types of guidance which help define the availability of NHS services of curative care. Firstly, technology appraisals give guidance on the use of new and existing treatments within the NHS. Of the 91 technology appraisals published to date (July 2005) some relate to in-patient care, such as Appraisal No. 11 (the use of implantable cardioverter defibrillators for arrhythmias); to day care, such as Appraisal No. 48 (home compared with hospital haemodialysis for patients with end-stage renal failure); and to out-patient care, such as Appraisal No. 24 (debriding agents for difficult to heal surgical wounds). Secondly, NICE clinical guidelines offer guidance on the appropriate treatment and care of people with specific diseases and conditions within the NHS. Clinical guidelines are based on the best available evidence and are intended to help health care professionals in their work but not to replace their knowledge and skills. Services of curative treatment are included amongst the 40 currently published guidelines. For example, the dyspepsia guideline (Clinical Guideline, CG, 18) recommends life-style advice and appropriate medication as secondary preventative measures and the guideline on head injury (CG4) specifies treatment pathways covering emergency, out-patient and in-patient care. Thirdly, NICE produces guidance on whether interventional procedures used for diagnosis or treatment are sufficiently safe and effective for routine use in the NHS. Of the 127 currently published topics most relate to hospital-based care, such as radiotherapy for age-related macular degeneration (Interventional Procedure Guidance, IPG, 048) and auditory brainstem implants (IPG108).\nThe three types of NICE guidance are developed and updated using distinct methodologies. Guidance on technology appraisals is formulated by the NICE Appraisal Committee, an independent advisory body with individuals from a range of professional backgrounds. Preliminary guidance is based on evidence from an academic assessment group and from company submissions. Following consultation, the Final Appraisal Determination is developed, approved by the NICE Guidance Executive and put out again for consultation. An appeal process may ensue, after which the guidance is published. All guidance is reviewed at regular intervals [7]. To develop a guideline, NICE commissions one of the National Collaborating Centres to establish a Guideline Development Group. The Group appraises clinical and cost-effectiveness evidence from systematic reviews of the research evidence. Views of clinicians, consumers and stakeholders also inform the guideline [8]. To produce guidance on interventional procedures, the Interventional Procedures Advisory Committee, an independent body of 24 members with a range of expertise, considers the safety and efficacy of procedures, but does not examine clinical or cost effectiveness [9]. The Committee produces a Consultation document and published guidance reflects comments received over the 4-week consultation period.\nNICE guidance generally acts as quasi-law, but one aspect of guidance on technology appraisals is supported by statute. If NICE guidance is that a new technology should be made available to certain NHS patients, the funding bodies (PCTs) are obliged by law to ensure there are adequate resources to facilitate the implementation of NICE guidance (Secretary of State for Health \u201cNational Health Service Act 1977: Directions to Health Authorities, Primary Care Trusts and NHS Trusts in England\u201d, 2001). However, the guidance is not binding on individual clinicians, who must assess whether the technology is appropriate for the patients they treat [10].\nAnother illustration of quality standards impacting upon patient rights is Public Service Agreements. Published annually, these specify national goals within the public sector, including waiting time targets (or \u2018guarantees\u2019) for the NHS [11]: (a) By the end of 2005, patients will wait a maximum of 6\u00a0months for in-patient admission and no more than 13\u00a0weeks for an out-patient appointment. (b) By the end of 2008 the maximum wait from GP referral to hospital treatment will be 18\u00a0weeks.\nTargets that should already be achieved, and henceforth maintained, include the 4-hour maximum wait for emergency care and the 24\/48 target for accessing primary care. (The 24\/48 target refers to patients being able to see a general practitioner within 2 working days or another primary care professional within 1 working day, whilst the 4-hour wait target is that patients should spend no more than 4\u00a0hours in an accident and emergency hospital department from arrival to admission, transfer or discharge.)\nWhere fee schedules exist for NHS care, patient \u2018entitlement\u2019 to services might be inferred. For example, the new national tariff system of payments for hospital services, whilst in no way guaranteeing provision, \u2018suggests\u2019 services that should be accessible on the NHS. The national price schedule for patient services is classified by Health Care Resource Group (HRG) codes. A range of clinical procedures, treatments and diagnoses is included in the current list of 550 HRG tariffs for elective in-patient care [12].\nThe Quality and Outcomes Framework, part of the GP contract, is a voluntary mechanism for encouraging primary care provision of some services of curative care, such as antiplatelets for patients with coronary heart disease [13]. With total payments amounting to 15\u201320% of available total practice remuneration [14], there is a clear incentive for GPs to make these services available to NHS patients.\nDiscussion\nThe NHS is a complex and heavily regulated health care system in which the roles of actors are in general clearly defined. As the primary emphasis has historically been upon local cost control rather than quality or access issues, geographical variations in quality and quantity of provision have emerged [1]. The lack of explicitness in the definition of the health basket has led to a great deal of uncertainty about entitlement.\nEven if there is a statutory duty governing provision, this does not necessarily guarantee access to NHS services. For example, the 1977 NHS Act obliges PCTs to provide, or to arrange for the provision of, general dental services (National Health Service Act, Chap. 49, 1977). However, a large proportion of the population is unable to access NHS dental services because of a shortage of dentists willing to provide these services for the NHS. The rapid growth of private practice since 1992 was apparently precipitated by a 7% cut in NHS fees, which was designed to redress perceived excessive income [15, 16]. Access problems to NHS dentistry have triggered an urgent review of the regulations governing the dentists\u2019 terms of service with a recent cash injection of over \u00a3350 (\u20ac504) million, aimed at increasing the number of dentists working for the NHS [17]. The British Dental Association in is negotiations with the Department of Health to develop a new contract which is expected in April 2006. This example illustrates the general principle that private practice flourishes where there are access or quality problems within the NHS.\nThe use of regulation to address quality and access issues has had mixed results. A national evaluation of compliance by NHS organisations with NICE guidance found variable implementation. Looking at rates of prescribing and use of procedures and medical devices, the time-series analysis found significantly increased prescribing of some taxanes for cancer and orlistat for obesity in line with guidance. However, prescribing practice frequently appeared to have little relation to detailed guidance [18]. NICE guidance specifies entitlement in terms of patient groups and PCTs are obliged to provide funding only for these patients. Whether this practice is equivalent to the PCT operating a \u2018blanket ban\u2019 for patients whose condition lies outside the specified guidance is debatable and has not been tested in the courts.\nOver the past decade the growth in \u2018quasi-law\u2019 suggests that the English system is heading towards a more formal statement of benefits and entitlements. NICE guidance on new and existing technologies is in effect establishing a \u2018positive list\u2019 of technologies that the NHS should fund; NSFs describe interventions that should be implemented to achieve standards of care. The Payment by Results system will be expanded to cover non-elective care and the Healthcare Commission will increasingly encourage NICE and NSF implementation, reinforcing standards through the \u2018annual health check\u2019 [5]. These factors together will take the NHS forward in defining a more explicit health basket for England. However, variations in local capacity to comply with national standards may result in a more explicit rationing of healthcare services at the local level.","keyphrases":["health services","national health programmes","united kingdom","health benefit plans","public policy"],"prmu":["P","R","U","R","R"]}
{"id":"Purinergic_Signal-3-4-2072919","title":"P2 receptor-mediated modulation of neurotransmitter release\u2014an update\n","text":"Presynaptic nerve terminals are equipped with a number of presynaptic auto- and heteroreceptors, including ionotropic P2X and metabotropic P2Y receptors. P2 receptors serve as modulation sites of transmitter release by ATP and other nucleotides released by neuronal activity and pathological signals. A wide variety of P2X and P2Y receptors expressed at pre- and postsynaptic sites as well as in glial cells are involved directly or indirectly in the modulation of neurotransmitter release. Nucleotides are released from synaptic and nonsynaptic sites throughout the nervous system and might reach concentrations high enough to activate these receptors. By providing a fine-tuning mechanism these receptors also offer attractive sites for pharmacotherapy in nervous system diseases. Here we review the rapidly emerging data on the modulation of transmitter release by facilitatory and inhibitory P2 receptors and the receptor subtypes involved in these interactions.\nIntroduction\nIonotropic P2X receptors and metabotropic P2Y receptors are the principal cell surface proteins, conveying the action of extracellular ATP, the ubiquitous signaling substance. P2X receptors are ligand-gated cation channels, composed of three individual subunits, whereas P2Y receptors belong to the superfamily of G protein-coupled receptors (GPCRs), with seven transmembrane domains. Various subtypes of P2X and P2Y receptor families are expressed throughout the brain and involved in a wide array of functions from fast synaptic transmission to long-term plasticity and trophic changes important for development, neuron-glia interactions, and neuroimmunomodulation. In addition, ATP modulates synaptic transmission pre- and postsynaptically, both in a positive and negative direction via activation of P2X and P2Y receptors, respectively.\nThe presynaptic nerve terminal is an important regulatory site, whereby the efficacy of synaptic transmission could be locally and efficiently controlled. Accordingly, axon terminals in the central nervous system and in the periphery are equipped with a wide variety of auto- and heteroreceptors [1\u20134]. Whereas presynaptic metabotropic receptors convey negative feedback regulation of transmitter release, presynaptic ionotropic receptors could amplify synaptic transmission. Moreover the activation of ligand-gated cation channels with high Ca2+ permeability could directly elicit transmitter release triggered by the Ca2+ influx through the receptor-ion channel complex [2, 4]. Coactivation of different presynaptic receptors provides a fine-tuning mechanism whereby different neurotransmitters and modulators can mutually influence the activity of each other. Presynaptic and extrasynaptic receptors controlling transmitter release also offer attractive target sites for existing and future pharmacotherapy, as they may modify the normal and pathological synaptic information processing without all-or-none actions [3].\nSince ATP and its related nucleotides are ubiquitous signaling molecules, it is not surprising that their receptors, i.e., ionotropic P2X and metabotropic P2Y receptors, participate both in the negative and positive feedback modulation of neurotransmitter release. Although the principal function proposed for ATP-sensitive P2 receptors was that they mediate the fast transmitter action of extracellular ATP in neuro-neuronal and neuro-effector synapses in the nervous system, it was already recognized in the early 1990s that they are also involved in the regulation of transmitter release [5, 6]. It was subsequently revealed that the release of the major neurotransmitters of the brain and the peripheral neurons [acetylcholine (ACh), noradrenaline (NA), dopamine (DA), serotonin, glutamate, \u03b3-aminobutyric acid (GABA)] are modulated by P2X and\/or P2Y receptors. In 2000, Cunha and Ribeiro reviewed the literature on the presynaptic modulator role of ATP and suggested that there is a mismatch between the abundance of P2 receptor expression, the robust release of ATP in almost all parts of the central nervous system (CNS) and peripheral nervous system (PNS), and the relative paucity of identified P2 receptor-mediated synapses, which implicates the major role of ATP as a neuromodulator, rather than a classic transmitter [7].\nThe focus of this mini-review is the facilitatory and inhibitory modulation of neurotransmitter release by different subtypes of P2X and P2Y receptors, irrespective of their localization, i.e., whether they are pre-, post-, or extrasynaptic.\nTherefore, in addition to a brief summary of the determining factors of ATP availability in synapses, the structure, pharmacology, signal transduction, and distribution of P2X and P2Y receptors in the nervous system, available information on the release-modulating P2 receptors, and the receptor subtypes involved in these interactions will be detailed and updated.\nDetermining factors of ATP availability in synapses\nThe participation of ATP and related nucleotides in the regulation of neurotransmitter release presumes their accumulation in the extracellular space upon ongoing neuronal activity. Extracellular purine availability in the nervous system is basically determined by the balance of release and removal by enzymatic degradation and uptake.\nSources and stimuli that trigger ATP release\nSince ATP is ubiquitous, all metabolically active cells of the nervous system provide a potential pool for its release. Therefore, besides the nerve terminals themselves, the cellular source of released purines participating in the modulation of neurotransmitter release could be any cell type located in contact with nerve terminals, i.e., astrocytes, microglia, and endothelia. A wide variety of stimuli are known to release ATP to the extracellular space, which could lead to purine levels sufficiently high to activate nucleotide receptors expressed on the surface of pre- and postsynaptic membranes [8, 9]. Although the stimulation-dependent release of ATP upon conventional [10, 11] and high-frequency (e.g., [12]) neuronal activity is well documented, these stimuli probably result in a spatially restricted, localized increase in extracellular purine levels, which serve the fast synaptic transmission and its modulation within the synaptic cleft. Furthermore, ATP-metabolizing ectoenzymes, present on the nerve terminal membrane, and glial cells [13], such as ectoNTPDases, and the CD39\/ecto-5\u2032nucleotidase [14], may strongly limit nucleotide availability under these conditions. On the other hand, pathological events are known also to stimulate purine release. These signals include mechanical [15\u201317], chemical [18], and hypotonic stimuli [19], hypoxia\/hypoglycemia\/ischemia and consequent energy deprivation [20\u201325], inflammatory signals, such as bacterial lipopolysaccharide (LPS) [26, 27], interleukin-1\u03b2 (IL-1\u03b2) [28], and cellular injury. The pathological ATP release might result in a purine-rich extracellular milieu leading to a more widespread activation of receptors reaching also the extrasynaptic receptors on the neighboring nerve terminals or distant cells such as astrocytes. Therefore, P2 receptors could play a role in the modulation of not only neuronal but also astrocytic transmitter release. Finally, nucleotides and nucleosides may promote further release of purines, by a homo- or heteroexchange mechanism, if they reach a relatively high concentration in the extracellular space [29].\nMechanisms of ATP release\nSince ATP is a highly polarized molecule, which cannot pass freely the cell membrane being released to the synaptic cleft, it could enter the extracellular space by the following mechanisms: (1) vesicular exocytosis, (2) carrier-mediated release, (3) release through channels and membrane pores, and (4) cytolytic release. (1) Vesicular exocytosis is a prototype mechanism for neurotransmitters and neuromodulators to enter the extracellular space, which is expected to be a [Ca2+]o-dependent process. Indeed, ATP is taken up and stored in synaptic vesicles of nerve terminals [8] and astrocytes [15] and [Ca2+]o-dependent ATP release in response to neuronal stimulation appears in many areas of the central and peripheral nervous system (for further references see [8, 9, 24, 30, 31]). Moreover, recent findings indicate that vesicular ATP could be released not only from nerve terminals but also from neuronal somata [32] and astrocytes [15]. (2) Although specific transporters capable of transmembrane movement of ATP are yet to be molecularly identified in the nervous system, ABC (ATP binding cassette) proteins have been implicated as ATP transporters [19, 33, 34] in non-neuronal cells. These transporters are also expressed in glial cells [35] and mediate ATP release upon hypo-osmotic challenge [36, 37]. (3) Channels and pores, such as connexin hemichannels [38], are also potential candidates to drive the transmembrane movement of ATP. They have been identified to mediate ATP release from astrocytes and other non-neuronal cells in response to mechanical stress [39, 40] and other stimuli [31, 41]. (4) Although only scarcely supported by direct experimental proof [42], the general assumption is that any kind of cellular injury could result in high local ATP concentrations in the extracellular space. In this case the millimolar cytoplasmic ATP is expected to leak out of the cell through the membrane damage.\nMetabolism of ATP in the extracellular space\nSeveral enzyme families are responsible for the extracellular degradation of ATP in the nervous system. The first step of the inactivation of ATP is mediated by the family of ectonucleoside triphosphate diphosphohydrolases (ENTPDases, EC 3.6.1.5, also known as ectoATPase or apyrase), which are able to hydrolyze ATP and adenosine diphosphate (ADP) to AMP [14]. Among these enzymes ENTPDase 1, 2, 3, and 8 are present in the brain [43, 44], having low micromolar Km for ATP and ADP giving rise to rapid and highly effective hydrolysis of ATP in almost all neuronal tissues. In addition to the ENTPDase family, ATP and other nucleotides could also be dephosphorylated by ectonucleotide pyrophosphatases (ENPPs) and by alkaline phosphatases, both having broader substrate specificity, but also widespread tissue distribution [14]. The final step of extracellular inactivation is the hydrolysis of AMP by the ecto-5\u2032-nucleotidase (EC 3.1.3.5) enzyme, which is the rate-limiting step giving rise to the formation of adenosine that acts on P1 receptors, which include A1, A2A, A2B, and A3 receptor subtypes. Thus, endogenous ATP is converted to adenosine to activate A1 adenosine receptors within a second in the hippocampus [45, 46], whereas the hydrolysis of ATP seems slower in other brain regions, such as the cerebral cortex [47]. Ectoenzymes therefore have an important role in the substrate delivery to different subtypes of P2X and P2Y receptors. In spite of its short half-life, effective concentrations of nucleotides can be reached in the synapse for the activation of ionotropic P2X receptors and metabotropic P2Y receptors.\nStructure, pharmacology, and signal transduction of P2X receptors\nIonotropic P2X receptors are nonselective cation channels consisting of at least three subunits. P2X receptor subunits are 379-595 amino acid long polypeptide chains, having two transmembrane domains (TM1 and TM2) and a large extracellular loop [48, 49]. Until now seven members of this receptor family have been identified molecularly, which are numbered from P2X1 to P2X7, and have individual kinetics and pharmacological phenotype [50]. These receptor proteins coassemble into various homo- or heterooligomeric assemblies to form functional receptors. Among possible combinations so far 16 variations have been proved to be functional [51]. These are all of the homooligomeric receptors, except P2X6, which does not function in homooligomeric form, and the rest are heterooligomers, formed from P2X1-P2X6 subunits. However, recently it has been reported that by N-glycosylation even the homomeric P2X6 receptor could be rendered functional [52]. On the other hand, the P2X7 receptor functions only in homooligomeric form and does not coassemble with other known P2X receptor subunits. P2X receptors are permeable to both monovalent (Na+, K+) and divalent (Ca2+) cations and the activation of the receptor generates an inward current leading to the local depolarization of the cell membrane; in addition, the Ca2+ influx through the receptor-ion channel complex could directly trigger transmitter release. Moreover, upon prolonged or repetitive agonist application certain P2X receptors, especially the P2X7 receptor, display pore dilation which makes the channel permeable to high molecular weight cations up to 800\u00a0Da.\nBasically P2X receptors are sensitive to ATP and to its various synthetic analogues but not to AMP and adenosine and the ligand binding profiles of homomeric P2X receptors are well established (for further information, see [50, 53]). On the other hand, less is known about the pharmacology of heteromeric receptors; among them, the pharmacological profile of P2X2\/3, P2X2\/6, P2X1\/2, P2X1\/4, P2X1\/5, and P2X4\/6 are described [54\u201359]. However, the expression pattern and the ligand binding profile of individual assemblies of P2X receptors are highly overlapping, often creating difficulties in the identification of the P2X receptor subunit composition of receptors expressed in native tissues. Moreover P2X receptor assemblies also share common ligand binding properties with certain members of the P2Y receptor family. Therefore, transgenic mice genetically deficient in individual P2X receptor subtypes are being increasingly used for P2X receptor identification.\nThe distribution of P2X receptors in neuronal structures\nIn situ hybridization studies with specific riboprobes, and immunocytochemical studies using antibodies raised against individual P2X receptor subunits, revealed that all seven P2X receptors are widely expressed in the nervous system. However, the expression of different receptor subunits show species-, region-, and cell type-specific distinct distribution [60]. Among the P2X receptors, P2X2, P2X4, and P2X6 seem to be most abundantly expressed in the brain, whereas other subunits show more restricted localization [60\u201363]. The typical localization of the P2X2 receptor is on nerve terminals of the brain and the periphery [61, 64, 65], although it also appears postsynaptically [62]. P2X1 receptors had initially been suggested to be exclusively expressed on smooth muscle membrane consistent with its role in mediating fast synaptic transmission at the autonomic neuroeffector junction [60]. However, recent studies with more sensitive probes revealed that its expression is more widespread, i.e., it is also present on central and peripheral neurons [63, 66]. The same holds true for P2X3 receptors, which are primarily associated with sensory pathways, but functional studies indicate that they are also expressed in other brain regions and autonomic pathways [67\u201369]. The P2X4 receptor shows substantial expression in several brain areas such as the cerebral cortex, hippocampus, thalamus, and brainstem [70] and is associated with postsynaptic specialization of synaptic contacts [62]. P2X5 subunits have the most restricted localization in the brain, although it shows strong representation in certain areas, e.g., the nucleus tractus solitarii (NTS) [71]. Finally, P2X7 receptors are also expressed in the brain, especially in reactive microglia and astroglia [72] and immunoelectron microscopic studies revealed a widespread presynaptic expression of P2X7 receptor immunoreactivity in a number of different brain areas, including the brainstem, hippocampus, cortex, spinal cord, and the skeletal neuromuscular junction [73\u201375]. However, two studies, using the same antibodies, demonstrated that P2X7 receptor immunoreactivity is still observable in the brain of P2X7 receptor knockout animals and thereby raised doubts on the validity of previous immunocytochemical observations [76, 77]. Therefore, available P2X7 receptor antibodies either recognize a site, which is not the P2X7 receptor, or, as a recent study indicates [78], a brain analogue of the P2X7 receptor, which shares its antibody binding domain with the cloned P2X7 receptor and partially retains its functionality in P2X7 receptor knockout animals.\nStructure, pharmacology, and signal transduction of P2Y receptors\nP2Y receptors all belong to G protein-coupled receptors, having seven hydrophobic transmembrane domains, and possess their ATP binding site on the external side of TM3 and TM7 domains [79\u201382]. The P2Y receptor family has eight individual members, numbered P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14. P2Y receptors are basically activated by adenine and uridine nucleotides, such as ATP, ADP, uridine diphosphate (UDP), and uridine triphosphate (UTP), but not by nucleosides and they are classified according to their sensitivity to purines and\/or pyrimidines: P2Y1, 12 and P2Y13 are adenine nucleotide-preferring receptors; P2Y6 is preferred by uridine nucleotides; P2Y2, 4 and P2Y11 are receptors with mixed selectivity; whereas P2Y14 is activated by UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, and UDP-glucuronic acid [83]. Although a minority of P2Y receptor subtypes are incompletely characterized and the pharmacological profiles of individual P2Y receptors are partially overlapping, ligands are available which display some selectivity to certain subtypes of the P2Y receptor family [for further information see 84, 85]. Nevertheless, identification of individual P2Y receptors requires careful pharmacological analysis and the use of receptor knockout animals, if available, and exclusion of the involvement of adenosine receptors in the effect of nucleotides used as P2Y agonists, because they may be metabolized to adenosine (see, e.g., [45, 47]). In addition, the potential heteromerization of P2Y receptor subtypes with each other and with A1 adenosine receptors [86] should also be taken into account when individual P2Y receptors are identified.\nWhereas P2X receptors convey rapid changes in the neuronal excitability on the millisecond timescale, P2Y receptors act on a longer, second timescale, appropriate for the fine-tuning of synaptic transmission. As for the signal transduction pathways activated by various subtypes of P2Y receptors, P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors are coupled via Gq\/11 proteins to stimulate phospholipase C, followed by increases in inositol phosphates and mobilization of Ca2+ from intracellular stores; in addition, the P2Y11 receptor mediates an increase in adenyl cyclase activity [84]. On the other hand, P2Y12, P2Y13, and P2Y14 receptors are coupled via Gi\/o proteins to inhibit adenyl cyclase activity followed by a decrease in intracellular cAMP levels [84]. The activation of Gi protein-coupled P2Y receptors leads to the voltage-dependent inhibition of N-type voltage-sensitive Ca2+ channels directly or indirectly and subsequent inhibition of neurotransmitter release [85]. The inhibition of voltage-sensitive Ca2+ currents has also been demonstrated for those P2Y receptors, which are coupled to the Gq\/11 proteins [85]; however, this inhibition is voltage independent [85]. In addition, the Gi\/o-coupled P2Y receptors are known to activate voltage-sensitive GIRK K+ channels via direct interaction of Kir3 channel protein [85], which hyperpolarizes the neuronal membrane.\nThe distribution of P2Y receptors in neuronal structures\nmRNA encoding all known P2Y receptors, i.e., P2Y1, P2Y2, P2Y4, P2Y6, P2Y12, P2Y13, and P2Y14 are present in the brain [81, 87\u201389]. Although our knowledge of their cell-specific localization at the protein level is still incomplete, it appears that a number of them, such as P2Y1, P2Y2, and P2Y6 receptors, are expressed both on neurons and astrocytes [90\u201396], whereas others are not exclusively, but predominantly localized to astrocytes (P2Y13: [97] P2Y14: [98]), oligodendrocytes (P2Y12: [99]], or microglia (P2Y12: [100]). P2Y receptor mRNA and protein can be detected in a number of different structures, including sympathetic and parasympathetic and sensory nerve terminals, basal ganglia, brainstem, cerebellum, cortex, hypothalamus, and hippocampus (for further references, see [85]). However, immunocytochemical data should be handled with caution due to the lack of verification of the specificity of many of the available antibodies. For detailed information on the distribution of individual P2Y receptor mRNAs and proteins, we refer to recent reviews on this particular topic [85, 101].\nModulation of neurotransmitter release by facilitatory P2 receptors\nACh\nPNS\nP2X receptors Since P2X receptors have relatively high Ca2+ permeability [102, 103], this property makes them capable of initiating neurotransmitter release by Ca2+ influx through the receptor-ion channel complex or facilitating Ca2+-dependent neurotransmitter release, provided that they are located nearby the release sites (Table\u00a01). The first report suggesting that P2 receptors are involved in the facilitatory modulation of neurotransmitter release stems from 1991 when we found that opposite to the well-known inhibitory action of adenosine, \u03b1,\u03b2-methylene ATP, a metabolically stable analogue of ATP, enhanced electrically evoked acetylcholine release from the myenteric plexus of guinea pig and facilitated the related contractile response [6]. This effect was not blocked by the antagonists of adenosine receptors, and therefore was proposed to be mediated by ATP-sensitive P2 receptors [6]. In the same time Fu and Poo observed that ATP potentiates the spontaneous secretion of acetylcholine from developing neuromuscular synapses in Xenopus cell culture by promoting Ca2+ influx through the plasma membrane. However, this effect was not recognized to be the result of P2 receptor activation [104]. Later on, P2 receptor-mediated facilitation of acetylcholine release was confirmed by electrophysiological recordings in chicken ciliary ganglion [105] and mouse motor nerve terminals [106]. Presynaptic P2X receptors, involved in the facilitation of acetylcholine release, have also been identified in developing and adult neuromuscular synapses of Xenopus [107] and rat [108]. Homomeric P2X7 receptors are inserted into the membrane of mouse motor nerve terminals and their activation elicits vesicular exocytosis [74, 109]. However, there is no report about the presence of other subunit compositions of P2X receptors at the neuromuscular junction and it is also unclear whether such facilitatory receptors also exist on the terminals of central cholinergic neurons.\nTable\u00a01Facilitatory modulation of neurotransmitter release in the PNS and CNSPreparationMeasured effectProposed receptorReferenceAcetylcholine\u00a0Guinea pig ileumNT quantificationP2[6]\u00a0Chicken ciliary ganglionPresynaptic currentP2X[105]\u00a0Xenopus NMJsEPP\/mEPPP2[104, 107]\u00a0Mouse NMJEPP\/mEPPP2[106]\u00a0Mouse NMJVesicular destaining, EPSCP2X7-like[74, 109]\u00a0Rat NMJNT quantificationP2X[108]Noradrenaline\u00a0Rabbit ear arteryEJPP2[110]\u00a0PC12 cellsNT quantificationP2[111]\u00a0Guinea pig ileumNT quantificationP2[6]\u00a0Rat vas deferensNT quantificationP2X1, P2X3, P2X2\/3[113]\u00a0Guinea pig atriumNT quantificationP2X3, P2X2\/3[114, 115, 118]\u00a0Human and porcine heartNT quantificationP2X[119]\u00a0Sympathetic neuronsNT quantificationP2X2[116]\u00a0LC neuronsAP dischargeP2X[120]\u00a0Rat hippocampusNT quantificationP2X1, P2X3[68]Serotonin\u00a0Rat hippocampusNT quantificationP2[121]Dopamine\u00a0Rat striatumNT quantificationP2Y[122, 123]\u00a0Rat nucleus accumbensNT quantificationP2[124\u2013128]Glutamate\u00a0Rat brainstemmEPSC\/NT quantificationP2X1[131, 157]\u00a0Rat NTSEPSCP2X3, P2X2\/3[129, 130, 132]\u00a0Rat hippocampusNT quantificationP2X1, P2X3, P2X2\/3[69]\u00a0Rat hippocampusNT quantificationP2X7[75]\u00a0Mouse hippocampusNT quantificationP2X7[137]\u00a0Rat hippocampusEPSCP2X2[133]\u00a0Rat hippocampusEPSCP2X7[134]\u00a0Rat hippocampal neuronsEPSCP2[135]\u00a0Rat cortical synaptosomesNT quantificationP2X7[136]\u00a0Cultured astrocytesNT quantificationP2X7[142]\u00a0Retinal M\u00fcller glial cellsNT uptakeP2X7[143]\u00a0Rat spinal cordEPSC\/mEPSCP2X3, P2X1\/5, P2X4\/6[144\u2013148]\u00a0Rat nucleus accumbensNT quantificationP2[149]\u00a0Rat medial habenulaEPSCP2Y4[150]\u00a0Cultured astrocytesNT quantificationP2Y1[151]\u00a0Cultured Schwann cellsNT quantificationP2[152]GABA\u00a0Midbrain synaptosomesNT quantificationP2X3, dinucleotide R[153]\u00a0Cultured dorsal horn neuronsIPSCP2X[154]\u00a0Cultured hippocampal cellsIPSCP2[156]\u00a0Cultured cortical cellsNT quantificationP2X7[155]\u00a0Rat brainstemIPSCP2X1[157]\u00a0Rat, mouse, and guinea pig hippocampusNT quantification\/IPSCP2X7 (indirect)[75, 137, 158]\u00a0Cultured astrocytesNT quantificationP2X7[159]\u00a0Rat hippocampusIPSCP2Y1[163, 206]Glycine\u00a0Rat dorsal hornIPSCP2X[161]\u00a0Rat trigeminal nucleussIPSCP2X[162]AP action potential, EJP excitatory junction potential, EPP end plate potential, EPSC excitatory postsynaptic current, IPSC inhibitory postsynaptic current, mEPP miniature EPP, mEPSC miniature EPSC, NMJ neuromuscular junction, NT neurotransmitter, sEPP spontaneous EPP, sIPSC spontaneous IPSC\nMonoamines (NA, serotonin, DA)\nPNS\nP2X receptors The presynaptic facilitatory action of ATP on noradrenergic transmission was described for the first time by Miyahara and Suzuki in rabbit ear artery [110]. It was followed by the demonstration of the facilitatory effect of ATP and its metabolically stable analogue \u03b1,\u03b2-methylene ATP on [3H]noradrenaline efflux in PC12 cells [111] and in the guinea pig ileum [6]. However, the knowledge on P2 receptors at that time did not allow the identification of P2 receptor subtypes involved in these effects. The issue has been reinvestigated and it was found that sympathetic nerve terminals are equipped with ionotropic P2X receptors, activation of which directly elicits or facilitates noradrenaline release elicited by nerve stimulation [1, 112\u2013115] via a direct Ca2+ influx through the receptor-ion channel complex. The pharmacological phenotype of these receptors varies between species, between transmission sites of the sympathetic nervous system, and even between the somata and nerve terminals of an individual neuron. Thus, in cultured sympathetic neurons of the rat an \u03b1,\u03b2-methylene ATP-insensitive P2X2-like receptor was identified [116], whereas in the guinea pig right atrium we found that \u03b1,\u03b2-methylene ATP stimulates noradrenaline outflow and the pharmacological profile of the underlying receptor was similar to that of P2X3 or P2X2\/P2X3 receptors, consistent with the expression of their mRNA in the sympathetic ganglia [115]. In another study the facilitatory P2X receptors involved in the modulation of noradrenaline outflow in the rat vas deferens were identified as P2X1, P2X3, or P2X2\/P2X3 receptors [113]. In contrast, cultured mouse sympathetic nerve terminals do not seem to express facilitatory nucleotide-sensitive receptors [117]. Importantly, these receptors seem to be endogenously activated by ATP released in response to ongoing neuronal activity [115, 116] and by myocardial ischemia in the guinea pig [118], porcine, and human heart [119] and could contribute to ischemia-induced arrhythmia and ischemic heart dysfunction.\nCNS\nP2X receptors In the central nervous system, locus coeruleus (LC) neurons of the rat are equipped with ATP-sensitive P2X-like receptors, which facilitate the discharge of spontaneous action potentials [120]. Facilitatory P2X receptors have also been described in the noradrenergic axon terminals innervating the hippocampus, and the homomeric P2X1 and P2X3 receptors were identified as the most likely subunits responsible for this action [68].\nP2Y receptors P2 receptors enhance the release of serotonin from the hippocampus [121] and that of dopamine from the striatum [122, 123], and the latter effects are thought to be mediated by P2Y receptors. However, the pre- or postsynaptic localization of receptors responsible for these effects were not clarified in these studies. The P2 receptor agonist 2-methyl-thio ATP releases dopamine from the nucleus accumbens through direct and indirect mechanisms [124\u2013126] in vivo. Interestingly, P2 receptor activation-evoked dopamine release seems to play a role in the modulation of feeding behavior as P2 receptor antagonists inhibit feeding-induced dopamine release and concomitant behavioral changes after food deprivation [127, 128].\nExcitatory amino acids (glutamate, aspartate)\nCNS\nP2X receptors In addition to ACh and monoamines, the release of excitatory amino acid transmitters is also modulated by presynaptic P2X receptors in the CNS, as demonstrated partly by neurochemical and partly by electrophysiological methods.Activation of P2X receptors elicits glutamate release in the brainstem [129\u2013132], hippocampus [69, 75, 133\u2013135], and cortical synaptosomes [136]. As for the underlying receptor subunits involved in these effects, P2X1 [69], P2X2 [133], P2X3, and P2X2\/3 receptors [69, 130] as well as P2X7 [75, 132, 134, 136, 137] were identified. The involvement of P2X2 receptors [133] and P2X7 receptors [137] has been confirmed by the use of transgenic mice deficient in P2X2 and P2X7 receptors, respectively. Moreover the activation of P2X7 receptors not only elicits glutamate release but also permits the activation of other ligand-gated ion channels on the nerve terminals, such as \u03b17 nicotinic receptors, as demonstrated recently in rat cortical synaptosomes [136]. The activation of a P2X7-like receptor promotes Ca2+ influx in cortical synaptosomes [138] and in isolated midbrain synaptic terminals [139] and activates p38MAP kinase enzyme in the hippocampus [140]. This latter effect seems to participate in the effect of ATP to elicit glutamate release as it was sensitive to the inhibition by the specific p38MAP kinase inhibitor, SB203580 [140]. Nevertheless, the exact mechanism whereby the P2X7 receptor and subsequent activation of p38MAP kinase enzyme leads to increased glutamate release awaits further investigation. In addition, it has been reported that the P2X7 receptor agonist BzATP depresses synaptic transmission at the mossy fiber-CA3 synapse [141] in a p38MAPK-dependent way. However, more recently the participation of P2X7 receptors in this latter effect has been disproved [76, 77]. In addition to nerve terminals, P2X7 receptor activation also elicits glutamate release from cultured astrocytes [142] and inhibits the uptake of glutamate in M\u00fcller glial cells of the retina [143].The activation of P2X receptors facilitates excitatory transmission in the spinal cord, releasing glutamate from primary afferent fibers terminating in lamina II [144\u2013148] and lamina V [144]; these actions are mediated by P2X3, P2X1\/5, and P2X4\/6 receptors, respectively. Finally, the ability of the P2 receptor ligand 2-methyl-thio ATP to release glutamate has also been demonstrated in vivo in the dopamine-depleted nucleus accumbens [149], although the underlying receptor subtype was not identified in this study.\nP2Y receptors Interestingly, the activation of P2Y receptors is also implicated to elicit and potentiate glutamate release in the central nervous system. In the medial habenula nucleus UDP and UTP increase presynaptic release probability and elicit a non-Hebbian-type long-term potentiation of excitatory transmission, an effect probably mediated by P2Y4 receptors [150]. In addition, the activation of P2Y1 receptors elicits vesicular glutamate release from astrocytes [151] and from cultured Schwann cells [152].\nInhibitory amino acids (GABA, glycine)\nCNS\nP2X receptors ATP or P1,P5-di(adenosine-5\u2032) pentaphosphate (Ap5A) elicits an increase in the intrasynaptosomal calcium and induces subsequent GABA release in midbrain GABAergic synaptosomes via activation of P2X3 and a dinucleotide receptor [153]. The regulation of GABA release by P2X receptors has also been reported in the spinal cord [154], cultured cortical [155] and hippocampal [156] cells, and the brainstem, where the excitatory and inhibitory synaptic transmission is facilitated via P2X3 and P2X1 receptors, respectively [157]. In addition to direct modulation of glutamate release, P2X7 receptor activation also releases GABA from the hippocampus through the activation on non-NMDA-type glutamate receptors [75]. This effect is absent in mice genetically deficient in P2X7 receptors [137] and mediated by the sodium-dependent reversal of the GABA transporter [75]. P2X receptor-mediated, TTX-sensitive GABA release has been implicated in the accelerated recovery of guinea pig hippocampal slices from a hypoxic\/hypoglycemic insult [158]. The activation of P2X7 receptors also releases GABA from cultured RBA astrocytes, however, with a different mechanism, by participation of the HCO3-\/Cl- exchanger [159]. On the other hand, no evidence was found for a direct facilitation of GABA release by P2 receptors in the hippocampal nerve terminal preparation [160]. Nevertheless, the release of another inhibitory transmitter, glycine, is augmented by P2X receptor activation in the dorsal horn [161] and in the brainstem trigeminal nucleus [162].\nP2Y receptors In addition to P2X receptors, activation of P2Y1 receptors leads to an increase of the inhibitory postsynaptic current (IPSC) frequency in an acute hippocampal slice in a manner dependent on action potential generation, indicating that this effect is related to the activation of receptors present on the somata\/dendrites of hippocampal interneurons [163].\nModulation of neurotransmitter release by inhibitory P2 receptors\nIn addition to facilitatory modulation, P2 receptors are also involved in the inhibitory modulation of the release of various transmitters and the metabotropic P2Y receptors are thought to play a major role in these actions (Table\u00a02).\nTable\u00a02Inhibitory modulation of neurotransmitter release in the PNS and CNSPreparationMeasured effectProposed receptorReferenceAcetylcholine\u00a0Frog ganglionEPSCP2[165]\u00a0Guinea pig ileumNT quantificationP2[164, 166, 167]\u00a0Ileal synaptosomesNT quantificationP1[168]\u00a0Guinea pig submucosal neuronsEJPP3[169]\u00a0Frog NMJEPPP2[170]\u00a0Rat submandibular ganglionEPSCP2[171]\u00a0Rabbit retinaNT quantificationP2[172]\u00a0Mouse NMJsEPPP2Y[173]Noradrenaline\u00a0Mouse vas deferensNT quantificationP2Y-like[5, 174, 181, 182]\u00a0Rat vas deferensNT quantificationP3[178]\u00a0Rat vas deferensNT quantificationP2Y[181]\u00a0Rat vas deferensNT quantificationP2Y12, P2Y13[113]\u00a0Guinea pig vas deferensNT quantificationP2Y-like[179]\u00a0Guinea pig saphenous arteryEJPP2[175]\u00a0Rat caudal arteryNT quantificationP3, P2Y[176, 177, 180]\u00a0Rat atriumNT quantificationP2Y-like183\u00a0Rat irisNT quantificationP2Y-like[184]\u00a0Rat kidneyNT quantificationP2[185]\u00a0Rat pancreasNT quantificationP2[186]\u00a0Chick sympathetic neuronsNT quantificationP2, P2Y12[187, 188, 189]\u00a0Bovine chromaffin cellsNT quantificationP2Y12[190, 194]\u00a0Rat cortexNT quantificationP2Y[195]\u00a0Rat hippocampusNT quantificationP2Y[196]Serotonin\u00a0Rat cortexNT quantificationP2[197]Dopamine\u00a0Rat striatumNT quantificationP2[198]Glutamate\u00a0Rat cortexEPSCP2[199]\u00a0Rat hippocampusEPSCP2Y[200, but see 201]\u00a0Rat spinal cordPolysynaptic EPSPP2Y[204]\u00a0Hippocampal slice cultureEPSCP2Y[205]EJP excitatory junction potential, EPP end plate potential, EPSC excitatory postsynaptic current, EPSP excitatory postsynaptic potential, NMJ neuromuscular junction, NT neurotransmitter, SEPP spontaneous EPP\nACh\nPNS\nIt has been known for a long time that ATP is involved in the inhibitory presynaptic modulation of cholinergic transmission [164]. However, it has been the subject of a long-standing debate whether ATP itself is responsible for this effect or its degradation product adenosine [165\u2013167], whereas an alternative was that ATP itself acts on adenosine receptors [168] or activates a putative P3 receptor bearing pharmacological features of both P1 and P2 receptors [169]. A more definitive proof for the involvement of P2 receptors in the inhibition of acetylcholine release was obtained later in the frog neuromuscular junction [170], rat submandibular ganglia [171], and rabbit retina [172]. Presynaptic P2Y receptors responsible for the inhibition of spontaneous acetylcholine release were recently identified at the mouse neuromuscular junction [173]. In this study the underlying subcellular mechanism of the inhibition of acetylcholine release was also explored: the activation of P2Y receptors is coupled to Gi\/o proteins and modulates presynaptic Ca2+ channels related to tonic secretion of acetylcholine [173].\nCNS\nInhibitory P2 receptors involved in the modulation of ACh release have been demonstrated in rat cerebral cortex [47]. By contrast, in the hippocampus, ATP primarily inhibits acetylcholine release through its breakdown to adenosine and subsequent action on A1 adenosine receptors [47].\nMonoamines (NA, serotonin, DA)\nPNS\nThe presence of nucleotide-sensitive inhibitory P2 receptors on postganglionic sympathetic neurons was recognized relatively early [5, 174\u2013179], although initially these receptors were qualified as P2Y-like [5] or as putative P3 receptors, which are \u201chybrid\u201d receptors between P1 and P2 receptors and sensitive to adenine nucleotides but also to theophylline derivatives [176\u2013178]. These receptors have been described and characterized in sympathetic nerves innervating the rat caudal artery [176, 177, 180], guinea pig saphenous artery [175], vas deferens [5, 178, 179, 181, 182], atrium [183], iris [184], kidney [185], and pancreas [186] as well as in cultured sympathetic neurons [187, 188]. As for subtype-specific identification, Queiroz et al. [113] identified presynaptic inhibitory nucleotide receptors on the noradrenergic axon terminals of the rat vas deferens as P2Y12 and\/or P2Y13 receptors, whereas on cultured sympathetic neurons [189] and bovine adrenal chromaffin cells [190] only P2Y12 receptors have been identified. Interestingly, it appears that mouse sympathetic neurons [191] and noradrenergic nerves innervating the rat adrenal cortex [192] do not express an inhibitory P2 receptor. The mechanism of P2Y receptor-mediated inhibition of noradrenaline release has also been explored in several studies: the activation of P2Y receptors inhibits voltage-dependent Ca2+ influx and thereby limits the Ca2+-dependent vesicular exocytosis and subsequent efflux of noradrenaline to the extracellular space [189, 193, 194].\nCNS\nSimilar inhibitory P2Y receptors have also been reported in the CNS in the rat brain cortex [195] and hippocampus [196]; however P2Y receptor subtypes involved were not identified in these early studies.\nIn the CNS, ATP inhibits the release of serotonin [197] and dopamine [198] via activation of metabotropic P2 receptors.\nExcitatory amino acids (glutamate, aspartate)\nCNS\nATP and its metabolically stable analogue ATP-\u03b3-S inhibits depolarization-evoked glutamate release from rat brain cortex slices [199] and inhibits glutamatergic EPSPs in hippocampal CA1 synapses [200]. Although the underlying receptor was sensitive to theophylline derivatives, the authors proposed that ATP acted through a putative pertussis toxin-sensitive P2Y receptor. However, this hypothesis was challenged by showing the rapid and highly effective hydrolysis of ATP in the hippocampal slices [45, 46] and by the demonstration of the complete absence of nucleotide-mediated modulation of excitatory synaptic transmission in the hippocampi of A1 receptor-\/- mice [201]. In a recent study Rodrigues et al. demonstrated that single hippocampal pyramidal neurons do express P2Y1, P2Y2, and P2Y4 receptors, and the release of glutamate, measured by a neurochemical technique, is inhibited by these receptors [69]. The discrepancy between the observations obtained in electrophysiological and neurochemical studies might be explained by the fact that in the former, individual synapses, whereas in the latter, glutamate release from all synapses of the hippocampal slice were simultaneously investigated. Nevertheless, the exact conditions under which the activation of P2Y receptors by endogenous ligands gain significance remain to be identified.\nFunctional data suggest that the release of glutamate in the spinal cord is modulated by inhibitory P2Y receptors. The activation of P2Y receptors causes blockade of the N-type calcium channels in dorsal root ganglion (DRG) cells [202], and this effect may decrease the release of glutamate from DRG terminals in the spinal cord and thereby partly counterbalance the algogenic effect of ATP [203, 204]. This assumption is supported by the findings that the P2Y1\/12\/13 receptor agonist ADP-\u03b2-S inhibits polysynaptic, but not monosynaptic excitatory postsynaptic potentials in the hemisected spinal cord and exhibits antinociceptive potential in the tail flick test [204].\nRecent studies revealed that modulators released from glial cells also regulate neurotransmitter release from nearby nerve terminals by the activation of P2 receptors. Hence mechanical stimulation of astrocytes in hippocampal cell culture leads to the generation of Ca2+ waves in astrocytes, which spread by the release of ATP and subsequent activation of P2 receptors and lead to the depression of excitatory synaptic transmission between neurons [205]. This glia-driven synaptic depression is partly mediated by ATP itself acting on P2Y receptors and partly by adenosine acting on A1 adenosine receptors [205]. A similar mechanism has also been demonstrated in intact hippocampal slices, where ATP released from neurons and astrocytes acts on P2Y1 receptors to excite interneurons, resulting in increased synaptic inhibition within intact hippocampal circuits [206]. On the other hand, to our knowledge there is no information regarding whether the release of GABA and other inhibitory amino acids is subject to modulation via inhibitory P2 receptors.\nPotential therapeutic utilization of P2 receptors involved in the regulation of neurotransmitter release\nP2X and P2Y receptors involved in the regulation of neurotransmitter release offer attractive, although not yet utilized sites for pharmacotherapy in nervous system diseases. For instance, facilitatory P2X receptors present on axon terminals could be activated not only during normal neuronal activity, but also during pathological situations, when cellular damage provides an ATP-rich extracellular milieu nearby the receptors. Thus, P2X receptors present on the sympathetic nerve terminals supplying the heart seem to be endogenously activated by ATP by myocardial ischemia [118, 119] and could contribute to ischemia-induced arrhythmia and ischemic heart dysfunction. Therefore, inhibition of these facilitatory P2X receptors might have therapeutic relevance in ischemic heart disease. The pathological activation of CNS P2X receptors, regulating the release of glutamate during ischemic-like conditions, was also recently described [207]. Increased activation of P2X receptors could contribute to ischemia-evoked glutamate release and thereby to glutamatergic excitotoxicity and resultant neuronal death; therefore, inhibition of these receptors could be a promising approach to treat ischemia-related neurodegenerative diseases. An analogous mechanism could play a role in the spinal cord during the sensitization process leading to various forms of sensory neuropathy; therefore, attenuation of increased glutamate release from the central terminals of primary sensory neurons by the inhibition of P2X receptors is a potential pathway which could be utilized in neuropathic pain. Since inhibitory P2Y receptors are frequently coexpressed on nerve terminals that are equipped with P2X receptors, activation of P2Y receptors could have a similar effect as the inhibition of P2X receptors. Therefore P2Y receptor agonists might also have therapeutic value in the areas described above. However, one should bear in mind that different subtypes of P2X and P2Y receptors affect various other aspects of physiological and pathological neuronal functions, which could also modify their potential.\nConclusion\nIn conclusion, substantial advances have been obtained in the identification and characterization of neurotransmitter release modulating P2 receptors in recent years. It appears that almost all major neurotransmitters of the nervous system are subject to neuromodulation by nucleotide-sensitive P2 receptors. Although there are exceptions to this rule, in general the release of different transmitters is subject to a dual modulation similar to modulation of other transmitters of the CNS and PNS: facilitatory modulation is conveyed by ionotropic P2X receptors, whereas inhibitory modulation is mediated by G protein-coupled metabotropic P2Y receptors. Amongst P2X receptors, P2X1, P2X2, P2X3, P2X2\/3, P2X1\/5, P2X4\/6, and P2X7 receptors were identified to be responsible for facilitatory modulation in different areas of the CNS and PNS. In addition, P2Y receptors (P2Y1, P2Y4) could also mediate facilitation of transmitter release in certain areas. Inhibitory modulation of neurotransmitter release is mediated by P2Y12 and P2Y13 receptors; however, individual P2Y receptor subtypes involved in these interactions are far from fully explored yet. It appears that not only neuronal, but also glia-derived ATP play a role in the modulation of neurotransmitter release. The intensity of P2 receptor-mediated modulation, the balance between the facilitatory and inhibitory modulation and the participating individual receptor subtypes, however, varies between individual transmission sites, depending on the expression pattern of P2 receptors and the factors determining the nucleotide levels in the vicinity of release of modulatory P2 receptors. Therefore, further progress is necessary in order to obtain a precise mapping of P2 receptor-mediated modulation of neurotransmitter release. The in vivo relevance of most of the in vitro observations on presynaptic P2 receptors awaits further investigation. Finally, physiological and pathological situations where presynaptic P2 receptors become endogenously activated by released nucleotides need to be identified.","keyphrases":["transmitter","release","presynaptic","p2y receptors","p2x receptor","neuromodulation"],"prmu":["P","P","P","P","P","P"]}
{"id":"Anal_Bioanal_Chem-3-1-1592253","title":"Optical molecular imaging for systems biology: from molecule to organism\n","text":"The development of highly efficient analytical methods capable of probing biological systems at system level is an important task that is required in order to meet the requirements of the emerging field of systems biology. Optical molecular imaging (OMI) is a very powerful tool for studying the temporal and spatial dynamics of specific biomolecules and their interactions in real time in vivo. In this article, recent advances in OMI are reviewed extensively, such as the development of molecular probes that make imaging brighter, more stable and more informative (e.g., FPs and semiconductor nanocrystals, also referred to as quantum dots), the development of imaging approaches that provide higher resolution and greater tissue penetration, and applications for measuring biological events from molecule to organism level, including gene expression, protein and subcellular compartment localization, protein activation and interaction, and low-mass molecule dynamics. These advances are of great significance in the field of biological science and could also be applied to disease diagnosis and pharmaceutical screening. Further developments in OMI for systems biology are also proposed.\nIntroduction\nDue to the rapid progress in the field of molecular biology that has occurred over the last century, we have come to see that life involves thousands of genes, proteins, metabolites, etc. However, life is not invoked by simply collecting these together; an integrated system formed from those components is required. It is vital to understand biological systems such as molecular networks, cells, tissues and even the organism itself at the system level. This shift in biological paradigm from reductionism to integration has made the field of systems biology [1\u20135] a new challenge that is currently a hot topic. Although attempts to systematically understand biological systems can be dated back to Wiener\u2019s time [6], research into systems biology represent the first attempts to achieve this based on detailed knowledge of molecules. It promises to reveal the relationships among elements of systems that may include just a few proteins that together perform a defined task or more complex molecular machines, cells and groups of cells, with the goal of understanding their emergent properties [3].\nIt is widely agreed that systems biology is a large scientific field that relies greatly on collaborations between disciplines such as the life sciences, information science, system engineering and analytical technology. One of the keys to successful research in this field is considered to be work being done in analytical chemistry [7, 8] to develop innovative analytical methods that meet the needs of systems biology research. For example, capillary electrophoresis has been recognized as being the \u201cgold standard\u201d in genomics for DNA sequencing. Similar \u201cgold standard\u201d methods are available in transcriptomics (DNA microarray), proteomics (multidimensional separation coupling with mass spectrometry) and metabolomics (microseparation coupled with mass spectrometry and nuclear magnetic resonance). However, more of these methods are needed. As well as revealing genes, proteins and metabolites from those omic investigations, which relate to the basic structures of systems, it is important to have methods capable of monitoring their localizations, connections, and in particular their dynamics over time under various physiological or pathological conditions.\nOptical molecular imaging (OMI) is a versatile technique that can be used to investigate the dynamics of biological events in molecules, cells, tissues and organisms in real time and in vivo (Fig.\u00a01) [9\u201311]. Compared with other imaging approaches like magnetic resonance imaging and positron emission tomography etc., OMI exhibits the great advantages of high temporal (picosecond) and spatial (submicron) resolutions, high sensitivity (single-molecule level) and minimal invasion, and shows high potential for systems biology. This review highlights recent advances in OMI, focusing especially on the development of fluorescent probes such as fluorescent proteins and semiconductor nanocrystals (also referred to as quantum dots, QDs), OMI instrumentation and approaches (techniques). The application of OMI to studies of localization, conformations and interactions of biomolecules in vivo are also highlighted. In particular, this review describes the application of OMI for tracing the dynamics of single molecules and whole-body living organisms, which strongly suggest that OMI is an informative systems biology method that may be used to uncover biological events at different system levels. The future development of OMI is also predicted.\nFig.\u00a01Utilization of OMI in life science research. OMI can investigate the dynamics of biological events in real time from molecules (left), cells, tissues and organisms (right) digitally and quantitatively. It can handle a wide range of intensities (about 12 orders of magnitude), times (femtoseconds to years) and spatial dimensions (nanometers to centimeters), and it gives high spatial and temporal resolution of the targeted cellular structures\u2014better than any other method\nAdvances in fluorescent probes\nAequorea fluorescent protein (AFP)-derived mutants\nLabeling of biomolecules with fluorescent probes or other dyes has facilitated in vitro or in vivo studies of biomolecular structures and dynamics, as well as their interactions, which is critical if we are to understand the biomolecular mechanisms of cellular function. However, traditional methods of chemical labeling based on fluorescent dyes are often inadequate for biomolecular labeling, repurification and reintroduction into cells by invasive methods like microinjection. These limitations have spawned efforts to noninvasively and site-specifically label protein in living cells or tissues by using green fluorescent protein (GFP) and its variants. The fluorescent protein presents a relatively small size and a compact, single-domain structure, which allow it to fuse to other targeted proteins with little or no interference in native protein.\nGFP was cloned from Aequorea victoria. Excitation of the wild-type GFP by light with a wavelength of 398\u00a0nm can induce the emission of bright green fluorescence, peaking at 508\u00a0nm. Through continuous efforts to perform protein mutation of GFP, scientists have developed various kinds of AFP with different excitation and emission wavelengths, enhanced brightness, and improved pH resistance compared to the original wide-type, such as enhanced GFP (EGFP), cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) (Fig.\u00a02a) [12]. The original YFP exhibited several drawbacks when imaging in vivo [13]. Later generations of YFP mutants, including \u201cCitrine\u201d [14] and \u201cVenus\u201d [15], exhibit low pH (5.7) resistance, halide insensitivity, greatly improved photostability, brighter fluorescence, and faster maturation. The original generation of CFP mutants also displayed several spectroscopic disadvantages. By using similar methods to YFP mutagenesis, a CFP mutant named \u201cCerulean\u201d [16] was obtained, with an improved quantum yield, a higher extinction coefficient and a longer fluorescence lifetime. Another new color GFP mutant, cyan-green fluorescent protein (CGFP) [17], whose excitation and emission wavelengths are intermediate between those of CFP and EGFP, was also found by using this tactic. In addition, \u201cPA-GFP,\u201d a GFP mutant, has 100 times greater fluorescence than the original and remains stable for days [18]. These fluorescent proteins were termed molecular or optical highlighters, and represent perhaps the most promising tools for investigating protein lifetimes, transport, turnover rates and so on.\nFig.\u00a02a\u2013cVarious FP mutants. a Introduction of the mutation of Thr203His in GFP results in significantly red-shifted maximum excitation and emission wavelengths; this mutant is named YFP. b By using mutagenesis, the original tetrameric DsRed is reconstructed into the monomeric DsRed variant. c Interchanging the amino and carboxyl portions of GFP and rejoining them with a short spacer generates cpGFP\nOther kinds of fluorescent protein\nLong-wavelength fluorescence can provide greater tissue penetration and better spectral separation from cellular autofluorescence. Therefore, red fluorescent proteins (RFP) are very useful in applications related to multicolor protein-tracking and the construction of sensors. The first RFP with a red emission wavelength was cloned from Discosoma genus, named \u201cDsRed\u201d [19]. Its structure is similar to GFP, but it is tetrameric. By using mutagenesis, different derivations exhibiting several advantages have been generated, such as the fluorescent timer of DsRed which has fluorescence that changes from green to red over time [20], the monomeric DsRed, named \u201cmRFP1\u201d [21] (Fig.\u00a02b), as well as DsRed2 [22], T1 [23], and mCherry [24], which have chromophores that are brighter, more soluble, and mature faster than the original generation. Another orange-emitting fluorescent protein, which has higher pH-resistance and is more effective for imaging, was discovered in Fungia concinn [25]. In addition, \u201cKaede\u201d [26], which is cloned from Trachyphyllia geoffroyi, can convert from green to red fluorescence and brightens 2,000-fold under UV illumination.\nNew rearrangement variants of fluorescent protein\nIn previous studies, fluorescent proteins were treated as an indivisible entity, usually appended to the amino or carboxyl terminus of the targeted protein. However, several rearrangements of GFPs in which the amino and carboxyl portions were interchanged and rejoined with a short spacer still remained fluorescent (Fig.\u00a02c). These circular permutations exhibit several advantages, such as altered pKa values, orientations of the chromophore with respect to a fusion partner, and greater absorbance of the exciting energy [27, 28].\nNongenetic fluorescent probes\nHowever, there are some disadvantages of using FPs to perform OMI of the whole-body. Given the available wavelengths of excitation and emission and the power of the FP, optical imaging with a depth of penetration of approximately 1\u20132\u00a0mm limits their use to C. elegans, Drosophila or surface structures in small animals. To solve these problems, applying QDs (quantum dots) to OMI of the whole-body could allow us to visualize biological events in deeper tissues with better image quality.\nCompared with conventional organic dyes and fluorescent proteins, semiconductor nanocrystals\u2014quantum dots\u2014exhibit a narrow, tunable, symmetric emission spectrum and better photochemical stability. Also, their wavelengths of maximum excitation and emission shift to shorter wavelengths with decreasing size [29, 30]. QDs present several great advantages for whole-body OMI, such as their very large molar extinction coefficients and their very bright emission, which mean that the emission can be visualized in big animals [31]; the ability to perform simultaneous multiple color imaging of targets of interest [32]; and long excited state lifetimes that provide a way to separate the QD fluorescence from background fluorescence [33]. Because QDs are capped with a monolayer of organic ligands and are hydrophobic, they cannot be used for visualization in vivo. Goldman et al. [34] reported that QDs capped with fusion proteins of a specific antibody, protein G and leucine zipper adaptor protein could be used to image the target protein in vivo (Fig.\u00a03). Since then, bioconjugated QDs have provided new approaches for the ultrasensitive and multiple-color imaging of targeted molecules in vivo. In addition, integrating QDs with paramagnetic substances results in new multimodality imaging probes that cuple the deep imaging capability of magnetic resonance imaging with ultrasensitive OMI [35]; these would be very powerful probes if used in clinical research. More recently, a new QD conjugate was found to emit long waves in the absence of external excitation light through bioluminous resonance energy transfer [36]. Compared with existing quantum dots, self-luminous quantum dots provide great sensitivity when imaging small animals, giving high signal-to-noise ratios.\nFig.\u00a03Method of conjugating QDs to target proteins. The pG-zb acts as a molecular adaptor, connecting the QDs with the target protein through interactions of its protein G portion with a specific antibody as well as interactions of its positively charged tail with QDs capped with a negatively charged dihydrolipoic acid surface\nOMI techniques\nGeneral imaging microscopy\nLaser scanning confocal microscopy (LSCM) (Fig.\u00a04a) and wide-field microscopy (WFM) are the tools most widely used for in vivo OMI. The major difference between them is that LSCM only collects the fluorescence emission of in-focus light, whereas WFM collects all signals, including out-of-focus light [37]. LSCM can produce superior images of multiple-cell samples, with photobleaching. However, LSCM cannot perform multiple-color imaging because it only observes in-focus light [38]. WFM is among the most sensitive of all such methods, permits minimal exposure of the sample, uniform illumination, an unlimited choice of excitation wavelengths, and is relatively simple [37]. One of the disadvantages of WFM is that disturbances are seen in observations of thicker specimens, where the out-of-focus signals become substantial [38].\nFig.\u00a04a\u2013fFluorescent OMI approaches. a LSCM only collects in-focus emitted light. b The principle of multiple photon excitation is based on the use of pulsed long excitation wavelengths to excite fluorescence. c FRET occurs between a donor and an acceptor that are in molecular proximity if the emission spectrum of the donor overlaps the excitation spectrum of the acceptor. d FRAP can reveal the mobility of FP-labeling proteins. These images illustrate the change in fluorescence of cells expressed with YFP-hGR before and after photobleaching. Reproduced from [51] with permission. e FLIM can measure the time-dependent emission intensity. The histogram represents the fluorescence lifetime distributions for the donor in the presence of interactions (red) or not (yellow). Reproduced from [57] with permission. f FCS can monitor the fluorescence signals emitted from the ROI. The cross-correlation curve (black) indicates a higher level of dimer or oligomer formation in the R1- and R5-expressing cells. Reproduced from [59] with permission\nFurthermore, multiple-photon laser scanning microscopy (MPLSM) has also been used in OMI [39]. The principle of MPLSM is that pulsed long excitation wavelength light, such as that provided by an infrared (IR) laser beam, is used to excite a molecule at the focal plane by multiple photons, causing fluorescence (Fig.\u00a04b). The advantages of MPLSM are high tissue penetration ability (>30\u00a0\u03bcm), low photobleaching or photodamage, and a high signal\/noise ratio (due to low autofluorescence) [40].\nSecond-harmonic imaging microscopy (SHIM)\nSHIM is based on a nonlinear optical effect called second harmonic generation (SHG), commonly called frequency doubling. This phenomenon requires that intense laser light passes through a highly polarizable material with noncentrosymmetric molecular organization [41]. Biological macromolecules often assemble into large, ordered noncentrosymmetric structures and have highly polarizability. Therefore, high-resolution SHIM imaging can visualize living cells or tissues. This method provides several benefits: the targeted molecules are not excited, photobleaching does not occur, and it can detect information related to pathology. In addition, a combination of SHIM and MPLSM could make a powerful optical imaging technique.\nOptical coherence tomography (OCT)\nThe principle of OCT imaging is analogous to that of ultrasound B-mode imaging, except that OCT uses light rather than acoustic waves [42]. OCT can provide cross-sectional imaging of structures below the tissue surface, analogous to histopathology. By using a state-of-the-art laser as the light source, ultrahigh-resolution imaging with axial resolutions as fine as 1\u20132\u00a0\u03bcm can be achieved [43]. However, the penetration depth of the technique in most tissues is limited to approximately 2\u20133\u00a0mm [44]. Several features of OCT imaging make it well-suited to imaging-based diagnostics and surgical guidance, such as its high resolution, its ability to quantitatively assess, and its ability to image some tissue functions.\nFluorescent molecular imaging approaches\nFluorescence resonance energy transfer (FRET)\nFRET is a quantum mechanical phenomenon that occurs between a fluorescence donor and a fluorescence acceptor with a favorable dipole\u2013dipole orientation that are in molecular proximity to each other, provided the emission spectrum of the donor overlaps the excitation spectrum of the acceptor (Fig.\u00a04c) [45]. Therefore, imaging based on FRET can determine the proximity (within the nanometer range) between labeled biomolecules in living cells [46, 47]. FRET-based microscopy includes intensity-based detection methods and fluorescence decay kinetics-based detection methods [46]. Similar to general fluorescence imaging microscopy, FRET also suffers from various drawbacks, like autofluorescence, detector noise, optical noise and photobleaching. In addition, spectral bleed-through (SBT), or cross-talk, is a major problem in FRET [48]. FRET is currently widely used in studies of protein colocalization, conformational changes, protein interactions and signal transduction [47, 49].\nFluorescence recovery after photobleaching (FRAP)\nFRAP involves observing the rate of recovery of fluorescence resulting from the movement of a fluorescent marker into an area which contains the same marker that has been rendered nonfluorescent via an intense photobleaching pulse of laser light (Fig.\u00a04d). FRAP has proved to be a powerful method for measuring the mobilities of target molecules in various membranes, cytoplasms and nuclei [50]. For example, the mobility of the hormone receptor in the nucleus of a living cell [51], the mobility in subcellular compartments and the dynamics of vesicles inside the synapses of cultured hippocampal neurons [52] have been studied using this approach.\nFluorescence lifetime imaging microscopy (FLIM)\nFLIM is a technique by which the mean fluorescence lifetime of a chromophore is measured at each spatially resolvable element of a microscope image [53]. The fluorescence lifetime is an inherent property of a chromophore that is sensitive to environmental and physical processes. Therefore, FLIM can detect interesting physical processes that can influence the excited state of a probe in vivo [54]. In addition, the mean lifetime of a donor is shifted to shorter lifetimes in the presence of an acceptor in FRET (Fig.\u00a04e), so FLIM can detect changes in FRET [55], although this may not be valid in complex cellular systems that consist of several different lifetime components [56]. For example, using a FRET\u2013FLIM method, the regulation of the activity of receptor protein tyrosine kinase (RPTK) through its dimerization was demonstrated in living cells [57]. The main drawbacks of FLIM are that it has relatively low sensitivity and requires expensive instrumentation.\nFluorescence correlation spectroscopy (FCS)\nFCS is an approach that has emerged only recently. It is a high-sensitivity photon-counting technique that permits fluctuations in the fluorescence signal due to changes in the fluorescence quantum yield to be measured (Fig.\u00a04f) [58]. Therefore, FCS can directly measure the mobilities of biomolecules, and it can monitor the average association and dissociation of target labeled with fluorescence probe\u2014which vary with interaction kinetics, complex composition and complex size\u2014in vivo without photobleaching. For example, FCS can be used to detect the clustering of somatostatin receptors [59] and the binding affinities of glucocorticoids (GC) in different subcellular compartments [60].\nApplications of OMI\nOMI for biomolecules\nOriginally, OMI of biomolecule function was performed on dead fixed cells or tissue sections in vitro. The first application of fluorescent detection in situ emerged in 1980, when RNA that was directly labeled with fluorophore was used as a probe for specific DNA sequences [61]. However, this method is not sensitive enough. Another indirect detection method allows signal to be significantly increased by binding secondary reporter to the hybridization probes that bind to targeted DNA [62] and mRNA [63]. Recently, Santangelo et al. [64] indicated that coinjection of two beacons based on FRET could be used to map the localization of a specific hybridization in living cells. Another application of FISH is immunocytochemistry (IC), which is used to detect target protein in specimens in vitro as antigen by means of antibodies labeled with fluorescence probe [65]. The technique of FISH is still being developed, with more specific fine-tuning of sensitivity and multiplicity necessary, after which its applications should gradually move from being in vitro to in vivo [66].\nIn addition, Fourier transform infrared (FT\u2013IR) spectroscopy allows the simultaneous detection of nearly all organic molecules in a single spectrum and hence is ideally suited for the investigation of complex metabolic pathways. However, this advantage is only useful when powerful mathematical methods are available for the analysis of the highly complex spectra produced. Glycolytic intermediates In yeast extract were identified with FT\u2013IR spectroscopy. The results were in good agreement with known phase relationships in oscillatory glycolysis [67]. In another experiment, glycogen, protein, lipid and nucleic acid concentrations were monitored in fish liver [68] using this method.\nSingle-molecule OMI is a very young field that holds great promise. It is not a technique, but a way of thinking that can allow us to detect individual molecular interactions, protein dynamics and signaling transduction pathways in living cells, which is difficult and sometimes impossible to achieve using conventional techniques [46]. For example, Sonnleitner et al. observed that the voltage-gated ion channel does not directly open or close under the conditions of single-molecular interaction using this approach [69]. Karymov et al. reported a method that could be used to directly follow junction branch migration in the holiday junction at the single-molecule level, and they detected that branch migration was a stepwise random process where the overall kinetics were dependent on the Mg2+ concentration [70].\nUsing OMI to monitor physiological processes in living cells\nMonitoring gene expression and RNA localization\nThe fusion of gene promoter and fluorescent protein cDNA can be used to noninvasively analyze the expression pattern of a target gene, which is a very common approach used to study gene expression in vivo and is widely used in life science research. To minimize the accumulation of the background fluorescence that is generated by leaky, undegraded level (i.e. from previously expressed fluorescent protein that has not yet degraded), several chimeric EGFPs with shorter fluorescence half-lives were constructed by fusing protein [71, 72]. However, this approach leads to lower sensitivity, and cannot reveal low levels of expression. To overcome these drawbacks, a new approach that is similar to the amplification of an enzymatic reporter was applied, which was then used in \u201cenhancer-trap\u201d strategies [73, 74]. The principle behind this is that the fluorescence of FP, revealing targeted gene expression in different tissues, is magnified through a transcript factor, which allows weak signals to be seen.\nIn addition, DNA and RNA sequences and other molecules could be visualized indirectly by labeling their binding proteins with FP in vivo. For example, by using RNA binding protein MS2 labeled with GFP, the mobility of targeted RNA could be visualized in vivo [75]. In another study, a similar method was used to observe gene expression correlated with a change in chromatin structure under the progress of transcription in real time [76]. Compared with FISH in vivo, the disadvantage of this method is the introduction of a protein complex of considerable size.\nMonitoring protein and subcellular organelle dynamics\nFP can also be used to monitor the behavior of the targeted protein, such as its appearance, degradation, localization, translocation and interaction in vivo. For example, by using a fusion protein of GR and FP, the dynamics of the exchange of GR with its binding sequence array can be monitored [77]. In addition, visualization of the dynamics of the GFP-GR fusion protein in vivo in more physiological conditions was achieved by using GFP-GR knock-in mice [78]. Another interesting application involves identifying the localization of an unknown protein on a large scale, a method termed the \u201cprotein trap,\u201d in which the imaging of cells containing the fusion protein of FP and a cDNA library was used to screen for target protein at the localization of interest [79].\nFusion protein that targets a given subcellular organelle is often used to study the dynamics of subcellular organelles. For example, Shaw et al. [80] revealed the dynamics of migration across the cell cortex of individual cortical microtubules in Arabidopsis by using tubulin fused to FP. In a similar study, this method was used to measure the rates of tubulin polymer growth, shortening and transition [81]. Furthermore, new subcellular structures can also be discovered by visualizing fusion FP, such as the novel discrete area in the nucleus, in which phytochrome species were induced to accumulated by light [82].\nMonitoring general protein\u2013protein interactions\nDetecting the proximity of two biomolecules based on FRET is an approach widely used to observe protein interactions in real time in vivo. Most commonly, CFP and YFP are respectively fused to each of their putative interaction proteins, which are coexpressed in one living cell so that FRET between two fused FPs of interaction can be detected (Fig.\u00a05a). A variety of protein interactions in different cells have been successfully visualized so far using intermolecular FRET, such as the interaction between phytochrome B and cryptochrome 2 [83], the rearrangement of G-protein subunits [84], and the oligomeric states of the ligands B7-1 and B7-2 [85]. Protein\u2013protein interactions can also be imaged via protein complementation assays. The putative interaction proteins are respectively fused to two complementary fragments of one fluorescent protein, and then interaction of the proteins can reinvoke the fluorescence [46, 86], in a technique which is analogous to yeast two-hybrid assays (Fig.\u00a06a). Using this method, Hu et al. simultaneously visualized several interactions among bZIP and Rel family transcription factors in one cell [87, 88].\nFig.\u00a05a\u2013dGeneral designs of FRET-based fluorescent probes. a An intermolecular probe consists of two interacting proteins that are labeled with CFP and YFP, respectively, which interact and result in FRET. b An intramolecular probe consists of CFP and YFP fused together with a cleavable linker or protein, which can be cleaved by proteolysis and disrupt FRET. c An intramolecular probe consists of sandwiching two domains between CFP and YFP, which can interact after phosphorylation or binding to calcium, resulting in a change in FRET. d An intramolecular probe consists of CFP, YFP and a protein\/domain, which permits conformational change by binding to another biomolecule, leading to a change in FRETFig.\u00a06a\u2013dSingle FP-based fluorescent probes. a The probe consists of the fusion of two interacting proteins to two complementary fragments of one FP, respectively, which can interact and reinvoke the fluorescence. b Insertion of a conformationlly responsive domain\/protein into cpYFP can lead to a change of fluorescence when its conformation is changed. c The probe consists of the fusion of two interacting proteins\/domains to the amino and carboxyl termini of cpGFP, which can interact and change the cpGFP fluorescence. d By using mutagenesis, AFP can be engineered to be directly sensitive to a small molecule, such as Cl\u2212, H+\nMonitoring protease and kinase activity\nThe first reporter of proteolysis consists of BFP and GFP fusing together with a protease-sensitive linker, which exhibits the FRET phenomenon. Proteolysis can disrupt the FRET by separating the donor and acceptor fluorescent proteins [89]. In recent studies, fusion proteins of CFP, YFP and a specific recognition sequence were used to measure the different caspase proteolytic activities during different apoptosis pathways (Fig.\u00a05b) [90\u201392]. In another study, YFP-Bid-CFP fusion protein was used to visualize the activation of Bid protein by proteolytic cleavage, and translocation of the cleaved Bid to mitochondria was observed directly [93].\nReporters for the activity of tyrosine kinases and serine\/threonine kinases have been made by sandwiching a substrate peptide for the kinase of interest and a phosphoaminoacid-binding domain, such as Src-homology-2 (SH2) or 14-3-3 protein, between two FPs (Fig.\u00a05c). Phosphorylation of the substrate peptide induces the formation of an intramolecular complex with the phosphoaminoacid-binding domain, which leads to a change in the FRET. Meanwhile, these indicators also report the opposite phosphatase activity. This generic concept has so far been adapted to create probes for the phosphorylation of the kinase of interest in different signal transduction pathways, including phosphorylation of protein kinase A (PKA) [94], activation of serine\/threonine kinase Akt [95], and activation of Src kinase on the cell membrane [96].\nMonitoring changes in calcium\nGenetically encoded fluorescent indicators for calcium without cofactors that can target to specific intracellular locations were first constructed by Miyawaki et al. [97]. This indicator was termed \u201ccameleon.\u201d The cameleon consists of the tandem fusion of CFP, calmodulin, the calmodulin-binding peptide M13 and YFP. Binding of Ca2+ allows calmodulin to wrap around the M13 domain, which increases the FRET between flanking FPs (Fig.\u00a05c). Replacing YFP with cpYFP [28], Venus [98] or EYFP [99] led to a new generation of cameleon that exhibited better spatial and temporal resolution as well as better environmental resistance. In order to measure high concentrations of Ca2+, the CaM\u2013M13 interface of cameleon was re-engineered to reduce its affinity to Ca2+, which improved the imaging of Ca2+ in the endoplasmic reticulum [100].\nOther indicators for calcium that are not based on FRET have also been reported. cpGFP or cpYFP can tolerate the insertion of another entire protein, and conformational change of the fusion cpFP can change its fluorescence. Therefore, the insertion of calmodulin into cpYFP (called \u201ccamgaroos\u201d) (Fig.\u00a06b) [14, 27] and the tandem fusion protein of M13, cpGFP and calmodulin (termed \u201cG-CaMP\u201d) (Fig.\u00a06c) [101] have both been used as calcium indicators because a conformational change occurs in the fusion cpFPs upon calcium binding.\nMonitoring changes in cyclic nucleotides\nThe first fluorescent sensor of cAMP consists of cAMP-dependent protein kinase (PKA) in which the catalytic subunit and the regulatory subunit are labeled with a fluorescein and a rhodamine, respectively. Binding of cAMP to the labeled regulatory subunit leads to the dissociation of the labeled catalytic subunit, which disrupts the FRET between the two fluorescent dyes [102]. Later, this system was developed into a genetic cAMP indicator with BFP and GFP replacing the fluorescent dyes [103]. The most recent cAMP indicator was constructed by sandwiching a full length Epac1 between CFP and YFP (Fig. 5d) [104].\nA genetically encoded indicator for another cyclic nucleotide, cGMP, has been reported by Sato et al. [105]. This indicator consists of a tandem fusion of BFP, nondimerizing mutants of cGMP-dependent protein kinase I\u03b1 (PKG I\u03b1) and GFP. An increase in FRET between the two FPs can be detected upon the cGMP-induced conformational change of PKG I\u03b1. Another sensor, termed \u201ccygnet-1\u201d [106], was constructed to have high selectivity for cGMP over cAMP. Here, the truncation of PKG I\u03b1 consisted of residues 1\u201377 rather than 1\u201347 reported in a previous study [105], and it exhibited decreased FRET fluorescence upon the binding of cGMP.\nMonitoring changes in transmembrane voltage\nMeasuring electrical activity in living cells with high spatial and temporal resolution is a fundamental problem in studies of excited cell information processing. To address this problem, Siegel and Isacoff [107] first constructed a novel fluorescent probe that could be used to measure transmembrane voltage in vivo, termed the fluorescent shaker or \u201cFlaSh\u201d, in which the modified GFP was inserted into a voltage-sensitive K+ channel so that voltage-dependent rearrangements in the K+ channel fusion protein could induce changes in the GFP fluorescence. A similar voltage sensor was generated by inserting GFP into another channel, the rat \u03bcI skeletal muscle voltage-gated Na+ channel, and was named \u201cSPARC\u201d [108]. In other studies, Sakai et al. [109] developed a new voltage-sensitive indicator based on FRET, in which changes in membrane voltage can lead to changes in FRET.\nMonitoring changes in pH in living cells\nThe chromophore of a FP is surrounded by a hydrogen-bonding network within the \u03b2-barrel, which means that the FP is pH-sensitive because of direct interactions between its hydrogen-bonding network and external protons [12]. The majority of GFP, YFP and their circularly permuted constructions described in previous parts of this review exhibit sensitivity to acid (Fig.\u00a06d), whereas the original AFP\u2014Renilla reniformis GFP\u2014and DsRed are insensitive to acid. Therefore, acid-sensitive variants can serve as ideal donors in pH indicators based on FRET, and DsRed is the most desirable acceptor [110]. Another pH sensor has been generated by using a new group of pH-sensitive GFP variants, termed \u201cdeGFPs,\u201d which exhibit rapid changes in emission from green to blue as the pH decreases, and these are suitable for ratiometric measurements in vivo [111, 112].\nMonitoring signal transduction from cell to cell\nScientists have also monitored intercellular changes in biomolecules and physiological events, which have been visualized in single cells in real time in a cell population. For example, Bedner et al. monitored the intercellular permeability to cAMP of six different gap junction channels by using sensors that are highly sensitive to cAMP concentration [113]. The change in calcium concentration from cell to cell was also monitored via calcium dyes [114]. In addition to these biomicromolecules, the propagation of intercellular apoptotic or survival events in monolayer cells mediated by the gap junction channel were also studied through OMI [115].\nUsing OMI to investigate tissue structure and function\nIt has recently been shown that SHIM can be used to directly detect several structural protein arrays in tissues, such as collagen arrays in mouse [116], neurons and muscular structures of the pharynx in C. elegans [117] without the need for fluorescent labeling. SHIM also readily retrieves more detailed molecular information than that obtained through the application of fluorescent labeling. Therefore, SHIM can be used to study or diagnose several diseases that are related to the assembly polarity of microtubule complexes in native brain tissue [118]. In addition, SHIM has proven to be crucial to neuroscience investigations in thick tissue preparations. For example, the fast neuronal membrane potential transient in mammalian brain slices labeled with FM4-64 dye was investigated by this method [119]. By labeling with another dye, the action potential was recorded with high temporal and spatial resolution on soma and neurite membranes [120].\nOCT also has the ability to perform in situ, real-time imaging of tissue pathology, and it can be used to guide excisional biopsy in order to reduce false negatives caused by sampling errors. In particular, OCT enables the internal architectural morphology of the retina to be visualized noninvasively, and it can be used to diagnose and monitor retinal diseases, which cannot be achieved through any other methods [121]. The development of high-speed OCT imaging combined with small fiber-optic probes has enabled in vivo endoscopic imaging, such as the visualization of the oral cavity [122], the larynx and the bladder [123]. In addition, OCT can perform functional imaging of tissues, such as brain activity [124], inflammatory\/neoplastic morphologic changes [125]. Raman spectroscopy (RS) is another powerful diagnostic tool that enables tissue identification and classification, as demonstrated by measurements of the brain tissue of a six-month-old pig by fiber-optic probes [126] and analysis of the molecular composition of human bronchial tissue structures [127]. Also, a combination of confocal RS and LSCM has been employed to obtain detailed information about the subsurface structures in the skin with high spatial resolution in a completely noninvasive manner [128].\nOMI of organisms\nEmploying fluorescent protein\nWhen performing whole-body OMI of organisms, the utilization of genetically encoded fluorescence probes has enabled scientists to decipher spatial and temporal changes in biological events inside complex organisms [129]. Over the past few decades, FPs have been used to generate various fluorescent probes that have been used to image physiological and biochemical events in the monolayer cells. Extracting information from whole-body imaging is more relevant to real physiological conditions, but up to now, only a few fluorescent indicators have been used in complex organisms to visualize physiological activity in vivo.\nTo study the feeding behavior of C. elegans, Kerr et al. [130] introduced a calcium indicator based on cameleon to C. elegans, which allowed them to measure the Ca2+ signals evoked in pharyngeal muscles and individual neurons under the stimulation of feeding in vivo (Fig.\u00a07a,b). Similar visualization of cameleon localized in other tissues was used to study the functions of serotonin and G proteins [131], the role of G proteins in signal transduction [132], and the ASH neuron response [133] in C. elegans. The spatial and temporal representation of odorant-evoked Ca2+ signals in the Drosophila brain was explored using cameleon [134] and G-CaMP (Fig.\u00a07c\u2013f) [135]. Calcium imaging was also used in other animals, such as zebrafish (Fig.\u00a07g\u2013i) [136] and mouse [137, 138]. In addition, an indicator with pH sensitivity was used to visualize spatial patterns of defined neuronal activity in the mouse [139]. More recently, the proteolytic activity of calpain was visualized by introducing fluorescent indicator into living mouse muscle, which gave the first 3-D imaging of FRET in vivo [140].\nFig.\u00a07a\u2013kWhole-body OMI using FPs and QDs. Imaging of Ca2+ signals in pharyngeal muscles under the conditions of noncontraction (a) and contraction (b) (red color indicates higher calcium) in transgenic C. elegans expressing cameleon. Reproduced from [130] with permission. c\u2013f Ca2+ signals evoked by different odors in Drosophila brain expressing G-CaMP. Reproduced from [135] with permission. Imaging of a three-day-old transgenic fish (g) carrying the cameleon and its RB neurons (h) by confocal optical section, and the change in the fluorescence ratio (i) (representing the calcium concentration) in an RB neuron under electrical stimulation of the skin. Reproduced from [136] with permission. j Simultaneous multicolor imaging in a mouse injected with QD-encoded microbeads; k QD imaging of a prostate tumor in the mouse. Reproduced from [33] with permission\nEmploying QDs\nDue to several advantages of QDs over FP, QDs have been used to image large animals in vivo, which holds great promise for clinical applications, especially in the imaging of tumors. Akerman et al. [141] report the application of QDs coated with specific targeting peptides to the imaging of different tissues in tumor in vitro, but this QD probe cannot be detected in living animals. Gao et al. [33] report on a new class of multifunctional QDs that were encapsulated with an ABC triblock copolymer and then linked with a tumor-targeting antibody. Using subcutaneous injection or systemic injection of these QDs into the mouse, simultaneous multicolor fluorescence imaging of the prostate tumor with efficient background removal and precise delineation of weak spectral signatures based on wavelength resolution was achieved (Fig.\u00a07j,k). More recent research showed that the utilization of QDs in combination with GFP labeling can differentiate tumor vessels from both perivascular cells and the matrix, and QDs linked to bone marrow-derived precursor cells can visualize tumor vasculature [142]. These OMI techniques, as well as their applications at different biological levels, are summarized in Table\u00a01. \nTable\u00a01OMI in systems biologyTechniquesPhysical processesResolutionSensitivityPenetrationObservation methodsProbesIndicator localizationReferencesWFM LSCM MPLSMRNA transcription and expressionHighHighHighDFI, FRETFPsIn vitro, cells, tissues, organism[63, 64, 71\u201376]Protein localization and dynamicsDFIFPsIn vitro, cells, tissues, organism[51, 52, 65, 77\u201379]Subcellular organelle dynamicsDFI, FRAPFPsCells[80\u201382]Protein interactionFRET, FLIM, FCSFPs, cpFPsCells, mouse[46, 47, 49, 57, 59, 60, 83\u201388, 140]Protease and kinase activityFRETFPs[89\u201396]CalciumDFI, FRETFPs, cpFPsCells, C. elegans, fish, mouse, Drosophila[14, 27, 28, 97\u2013101, 130\u2013138]cAMP, cGMPFRETFPsCells[102\u2013106]Electrical activityDFI, FRETFPsCells[107\u2013109]pHDFI, FRETFPsCells, mouse[110\u2013112, 139]TumorDFIQDsMouse[33, 141, 142]SHIMBiomacromoleculesLowHighLowNI\u2013Mouse, C. elegans, brain tissue[116\u2013118]Membrane potentialNIDyesCells, brain tissue[119, 120]OCTMorphology of tissueHighLowHighEI\u2013Human[122, 123]Activity and disease of organEI\u2013Human[124, 125]RSComposition of structureLowHighLowNI\u2013Brain tissues, bronchial tissue[126, 127]FT-IRKinetics of reaction intermediatesLowHighLowNI\u2013Yeast extract, fish liver[67, 68]Abbreviations: DFI, Direct fluorescent imaging; NI, nonlinear imaging; EI, light-echo imaging\nFuture development\nAs described above, OMI is a feasible analytical technique for monitoring the dynamics of biological events in vivo and in real time anywhere from single-molecule to whole-body level, which makes it well-suited to systems biology measurements. Its noninvasive, ultrasensitive, high-resolution and real time nature has resulted in the widespread use of OMI. However, its disadvantages are also very apparent, such as its high cost, its low throughput, and its limited detectable depth, which is a particular hindrance in whole-body analysis. It should be pointed out that OMI is still far from fulfilling all of the requirements of systems biology (as concluded in our previous paper [8])\u2014in terms of sensitivity, selectivity or specificity, linear range of quantitation, throughput, robustness, flexibility and cost\u2014that are definitely required in genomics, transcriptomics, proteomics, and metabolomic profiling and dynamics, although it it clear that OMI is a promising method for these types of bioanalysis.\nIn the near future, several routes to improving OMI for systems biology need to be followed. Firstly, it is crucial to develop better molecular probes for OMI; in other words probes that have improved biocompatibilities and high quantum yields, are easy-to-tag, display multiple colors and have better penetration capabilities. The importance of the molecular probe to OMI cannot be overstressed. Current endeavors in QD development are heading in a good direction but this field is still in its infancy [143]. However, this doesn\u2019t mean that the development of small organic dyestuffs and biogenetic FP is less important: on the contrary, applications of these two kinds of molecular probes will still constitute most OMI studies [144]. Secondly, the integration of several modalities or approaches should be emphasized, since it makes OMI much more informative. Thirdly, efforts to improve OMI instrumentation are also important, since they will result in better time and space resolution and better sensitivity. For example, the 4Pi technique has improved the spatial resolution of OMI down to the mid-nanometer level, which is over the diffraction limit. The rapid progress currently being achieved in nanotechnology and nano\/microelectromechanical systems looks set to lead to the manufacture of low-cost and high-efficiency OMI instruments [145]. Finally, the application of OMI to the life sciences is important; it is the reason behind the development of OMI. In particular, the application of OMI to systematic measurements of biological systems will be of great interest, which is also the aim of systems biology.","keyphrases":["optical molecular imaging","systems biology","review","quantum dots","fluorescent protein"],"prmu":["P","P","P","P","P"]}
{"id":"Neurochem_Res-4-1-2226019","title":"\u03b2-Amyloid 25-35 Peptide Reduces the Expression of Glutamine Transporter SAT1 in Cultured Cortical Neurons\n","text":"\u03b2-Amyloid (A\u03b2) peptides may cause malfunction and death of neurons in Alzheimer\u2019s disease. We investigated the effect of A\u03b2 on key transporters of amino acid neurotransmission in cells cultured from rat cerebral cortex. The cultures were treated with A\u03b2(25-35) at 3 and 10 \u03bcM for 12 and 24 h followed by quantitative analysis of immunofluorescence intensity. In mixed neuronal\u2013glial cell cultures (from P1 rats), A\u03b2 reduced the concentration of system A glutamine transporter 1 (SAT1), by up to 50% expressed relative to the neuronal marker microtubule-associated protein 2 (MAP2) in the same cell. No significant effects were detected on vesicular glutamate transporters VGLUT1 or VGLUT2 in neurons, or on glial system N glutamine transporter 1 (SN1). In neuronal cell cultures (from E18 rats), A\u03b2(25-35) did not reduce SAT1 immunoreactivity, suggesting that the observed effect depends on the presence of astroglia. The results indicate that A\u03b2 may impair neuronal function and transmitter synthesis, and perhaps reduce excitotoxicity, through a reduction in neuronal glutamine uptake.\nIntroduction\nAlzheimer\u2019s disease (AD) is a neurodegenerative disorder, leading to progressive loss of memory, impaired cognition and debilitation. It affects about 2% of the population in industrialized countries, the risk of having the disease increasing rapidly with age beyond 70, hence the prevalence of AD will increase several fold in the coming decades [1, 2]. AD is characterized by extracellular deposits of \u03b2-amyloid (A\u03b2), associated with dystrophic neuronal processes, degenerating and apoptotic neurons showing neurofibrillary tangles (i.e. aggregates of hyperphosphorylated tau protein filaments).\nThe neurotoxic effects of A\u03b2 and some of its fragments have been demonstrated in in vitro primary cultures from human and rat cortical neurons, in hippocampal slice cultures and following in vivo injection into various areas of the rat and monkey brain [3]. A\u03b2 peptides cause neuronal cell death and also increase the vulnerability of neurons to excitotoxicity [4]. A\u03b2(25-35) has been frequently used in investigations of A\u03b2 properties as a less expensive and more easily handled substitute for the native full-length peptide, A\u03b2(1-42), the latter being implicated in the pathogenesis of AD. Implicit in these experiments is the assumption that the mechanism of action of A\u03b2(25-35) is similar to that of A\u03b2(1-42). Indeed, A\u03b2(25-35) mimics the toxicological and aggregational properties of the full-length peptide, though these characteristics are increased; i.e., the shorter peptide is more toxic to cultured neurons, exhibits toxicity earlier, causes more membrane protein oxidation, and aggregates faster than the native A\u03b2(1-42) [5].\nThe amino acids glutamine and glutamate are essential for brain metabolism and function. Glutamate is the major excitatory transmitter of the brain [6], particularly in the cerebral cortex and hippocampus, and therefore has important roles in cognition, learning and memory. After synaptic release, it is taken up mainly into astroglia by glutamate transporters [7]. Perturbed glutamate uptake may cause excitotoxicity and is implicated in the pathogenesis of AD [7]. Once accumulated into astrocytes, glutamate is converted to the non-excitatory amino acid glutamine by the glial enzyme glutamine synthetase. Glutamine is then returned to neurons via glutamine transporters of system N (SNs) and system A (SATs) and used as a source for the synthesis of glutamate through its conversion by phosphate-activated glutaminase [8, 9]. Glutamate is packaged for exocytosis by vesicular glutamate transporters (VGLUTs) [10, 11], or used as precursor of GABA in GABAergic neurons.\nThe aim of this study was to explore the effects of A\u03b2(25-35) treatment on main transporters implicated in sustaining synaptic release of amino acids, i.e. the glutamine transporters SN1 and SAT1 and the vesicular glutamate transporters VGLUT1 and VGLUT2, in two different in vitro models: neuronal cell cultures and mixed cell cultures of rat cerebral cortex.\nExperimental procedures\nCell cultures\nNeuronal cultures were prepared from embryos of Wistar rats at day 18 of gestation (E18) using a modification of an earlier described method [12]. In brief, the neocortices of embryos were dissociated by treatment with 0.25% trypsin\/calcium\u2013magnesium-free buffer\/EDTA for 10\u00a0min at 37\u25cbC. The suspension was settled for 2\u00a0min at 2,000\u00a0\u00d7\u00a0g and then the cells were mixed in DMEM, triturated and passed through a cell strainer. The cells were cultured in DMEM supplemented with 2% B27 and 1% GlutaMAX I. The cells were plated on poly-l-lysine (50\u00a0\u03bcg\/ml) coated glass coverslips at a density 0.5\u00a0\u00d7\u00a0106\u00a0cells\/ml and the cultures were maintained in a humidified atmosphere of 5% CO2\/95% air at 37\u25cbC for 5\u00a0days in vitro before incubation with peptide.\nPrimary mixed cell cultures were prepared from the cerebral cortices of 1-day-old (P1) neonatal Wistar rats and plated on poly-L-lysine-treated coverslips (see above). The dissociated cortical cells were suspended in Neurobasal-A medium containing 2% B27 and 1% GlutaMAX I and maintained as described for E18 cells.\nRats were kept according to nationally and internationally approved conditions. Culture media and reagents were from Gibco, Invitrogen Corporation (Carlsbad, CA, USA).\nIncubation with A\u03b2 peptide\nNeuronal and mixed cell cultures were treated with A\u03b2(25-35) (Sigma-Aldrich, St.Louis, MO, USA) at final concentrations of 3 and 10\u00a0\u03bcM, for 12\u00a0or 24\u00a0h. In this study the B27-containing medium was removed from the cultures at day 6. Cells were washed twice with DMEM or Neurobasal-A medium and then incubated in medium for 12 and 24\u00a0h in the absence or presence of A\u03b2(25-35). Controls received no peptide.\nAntibodies\nAntibody to SAT1 was raised in rabbit to a fusion protein corresponding to the N-terminal amino acids 1-71 of SAT1 (aka GlnT, [13]; aka SA2, [14]), and affinity purified and characterized (T.T. Solbu & F.A. Chaudhry, to be published) as described for SN1 [15]. A GST-fusion peptide was made by cloning the first 213 base pairs from SAT1 into a pGex3X vector (Amersham Biosciences, GE Healthcare, Oslo, Norway). Using primers 5\u2032- cccgggatccttatgatgcatttcaaa-3\u2032 and 5\u2032-gcggaattcggttgttcctggaat-3\u2032 (Eurogentech, Herstal, Belgium), the 5\u2032 end of SAT1 was amplified by PCR and ligated into pGex3X using BamH1 and EcoR1 (New England Biolabs, Ipswich, MA, USA). GST-SAT1 was purified on Gluthatione Sepharose 4B as prescribed by the manufacturer (Amersham Biosciences; \u201cGST Gene Fusion System Handbook\u201d). Antibodies to other transporters were prepared and characterized as described (VGLUT1 and VGLUT2: [16]; SN1: [15]). Antibodies to marker proteins [glial fibrillary acidic protein (GFAP), anti-microtubule associated protein-2A&B (MAP2)], raised in mice, were from Sigma-Aldrich. Secondary antibodies were Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 555 goat anti-rabbit IgG from Molecular Probes (Eugene, Oregon, USA).\nImmunocytochemical staining\nFollowing exposure to peptide, cells attached to 14-mm glass coverslips were fixed with formaldehyde (4% paraformaldehyde, freshly depolymerized, in 0.1\u00a0M sodium phosphate buffer pH\u00a07.4) for 20\u00a0min at room temperature followed by three washes in phosphate buffered saline (10\u00a0mM phosphate buffer in 150\u00a0mM NaCl) and then blocked with blocking buffer [10% (v\/v) newborn calf serum (NCS), 3% (w\/v) bovine serum albumin (BSA), 0.5% (w\/v) Triton X-100 in Tris buffered saline (TBS) pH\u00a07.4] for 1\u00a0h at room temperature. After blocking, sections were incubated with the primary antibodies (anti-VGLUT1 1:3,000; anti-VGLUT2 1:3,000; anti-SAT1 2\u00a0mg\/ml; anti-SN1 0.3\u00a0mg\/ml; anti-MAP2 1:200; GFAP 1:500; dilutions optimized in pilot experiments) in primary antibody solution [3% (v\/v) NCS, 1% BSA, 0.5% Triton X-100, 0.05% NaN3 in TBS pH 7.4] overnight at 4\u00b0C, and then the cells were washed and incubated with the secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 555 for 1\u00a0h at room temperature. The coverslips were then washed three times in fresh antibody solution. Cells on coverslips were sealed to microscopic slides with Fluoromout G water based (Southern Biotechnology Associates, Birmingham, AL, USA).\nQuantitative analysis of immunoreactivities\nDual immunofluorescence for VGLUT1, VGLUT2, SAT1, SN1, GFAP and MAP2 was examined and quantified with a Zeiss Axioplan 2 microscope equipped with an LSM 5 PASCAL confocal unit, LSM software and a 40x\/1.3 oil PlanApo objective (Carl Zeiss, Heidelberg, Germany). We used standard FICT-rhodamine filter settings. Other settings were optimized initially, and held constant throughout the study so that all sections were digitized under the same conditions of illumination. Care was taken to use similar times for orientation (with conventional fluorescence) and adjustments in all preparations, in order to avoid differences in photobleaching. The cells were selected on the criterion that they showed representative morphology (Fig.\u00a01) and strong immunoreactivity for MAP2 (neurons) or for SN1 (in the case of astroglia). The cell body and the most proximal parts of the processes were outlined for digitalization of area and of fluorescence intensity for two immunoreactivities (MAP2 and a transporter). The analysis was performed without notice of the treatment group; the lack of difference in neuronal area between treatment groups suggests that sampling bias did not interfere with the results obtained. Fluorescence intensity is expressed in arbitrary units, without subtraction of background. For each glass coverslip, five fields were randomly sampled and two cells quantified per field, i.e. 10 cells, to obtain an average value for each immunoreactivity and experiment.\nFig.\u00a01Classification of cell types. (a, c, e) MAP2 positive (green) neuronal cells (arrowheads). (b) GFAP positive (red) astroglial cell (arrow, same field as a). (d, e) SN1 positive (red) astroglial cell (arrow, same field as c); SN1 (in contrast to GFAP) reveals the entire extent of the astrocyte. (e) MAP2 and SN1 immunoreacivities superimposed. (f) DIC image (same field as c\u2013e). Arrowheads and arrows indicate the positions of the nuclei of sample neurons and astrocytes, respectively. Note that all of the 10 cells situated on or next to the astrocyte in (f) are identified as MAP2 positive neurons in (c). Double immunofluorescence observed by confocal microscopy in mixed cell cultures from rat cortex at P1\nData analysis\nData are expressed as mean\u00a0\u00b1\u00a0standard error of the mean (SEM) of four different experiments (four different sets of cultures prepared at four different times) for P1 and E18 cultures. Advanced statistical analysis was performed according to a linear mixed model regression analysis, using the function lme of the nlme package [17] in the statistical software R (http:\/\/www.r-project.org\/). The fundamental assumptions of the model are that the observations are normally distributed with a variance varying only with protein type, and that they are uncorrelated when they come from different experiments. Log (for P1 data) or square root (for E18 data) transformations were performed as quantile\u2013quantile plots showed these transformations to give the best fits to the Gaussian distribution.\nResults\nCell types\nCell types were characterized in control cultures incubated without A\u03b2. In mixed neuronal\u2013glial cultures (from cortex of P1 rat pups), neurons and astroglia were clearly identified by immunoreactivity for the markers MAP2 and GFAP, respectively (Fig.\u00a01a, b). Double labelling for MAP2 and system N glutamine transporter SN1, localized in the membranes of astroglia [15], again showed the two immunoreactivities in separate cells (Fig.\u00a01c\u2013e), SN1 displaying the full extension of protoplasmatic-like astrocytes (i.e. beyond the parts pervaded by GFAP filaments, Fig.\u00a01a, b). The MAP2 expressing cells show the morphological characteristics of neurons and usually grow in close apposition to astroglia (Fig.\u00a01a\u2013f). In neuronal cultures (from E18 rats), MAP2 positive neurons had similar morphology, but no GFAP positive astroglial cells were found (not shown).\nDouble labelling for MAP2 and different transporter proteins showed the vesicular glutamate transporters VGLUT1 or VGLUT2 to be in neurons. A slight signal for SN1 in neurons was close to background levels. The system A glutamine transporter SAT1 was mainly localized in neurons but slight immunoreactivity was seen in some astroglial cells (Fig.\u00a02a, b). In the neurons, SAT1 staining was strong in central areas of the perikaryon and pervaded the processes.\nFig.\u00a02SAT1 (red) colocalized (yellow-orange) with MAP2 (green) in neurons in P1 cortical mixed cell cultures. Images show sample cells, insets display framed areas separately for SAT1 immunoreactivity. Cultures were exposed to control medium for 12\u00a0h (a) or 24\u00a0h (b), or to 10\u00a0\u03bcM of A\u03b2(25-35) for 12\u00a0h (c) or 24\u00a0h (d). SAT1 is expressed in the soma and processes of neurons, as revealed by double labelling with antibodies to the somatodendritic neuronal marker MAP2 shown superimposed on SAT1. Foci of strong perikaryal immunoreactivity suggest intracellular stores of SAT1. (Slight SAT1 immunoreactivity occurs also in glia-like perikarya (b, centre right, arrow), immunonegative for MAP2.) The immunoreactive cells were morphologically similar in the different conditions (a\u2013d). Scale bar\u00a0=\u00a020\u00a0\u03bcm\nSurvey of preparations double stained for MAP2 and either VGLUT1 or VGLUT2 indicated that a large proportion of the neurons were VGLUT immunoreactive. Similarly, double staining for MAP2 and SAT1 suggested that most of the neurons in the cultures contained SAT1.\nEffects of A\u03b2 in mixed cell cultures\nCortical mixed cell cultures (P1) were exposed to A\u03b2. After day 5 in vitro cell cultures were incubated with 3 or 10\u00a0\u03bcM of A\u03b2(25-35) for 12 or 24\u00a0h and then processed for immunohistochemistry and quantitative analyses. Neurons with typical morphology and strong MAP2 staining were seen also after exposure to A\u03b2 (Fig.\u00a02c, d). The cells appeared morphologically similar to those in control cultures (Figs.\u00a01, 2a, b). Transporter immunoreactivities were quantitatively analysed (Fig.\u00a03a, b) in neurons with typical morphology and clear MAP2 immunoreactivity. In addition, SN1 was quantified in cells with typical astroglial morphology according to SN1 immunofluorescence. All immunoreactivities showed a tendency for decline on exposure to A\u03b2(25-35), at 12\u00a0h as well as at 24\u00a0h, but this attained statistical significance only for SAT1. (The low SN1 level in neurons is close to background and may not be biologically significant.) The size of the central areas of the cells, over which the fluorescence intensities were recorded, was independent of peptide exposure in the neurons and only slightly reduced (by about 16%) in the glia (Fig.\u00a03c, d). This indicates that, in the neurons recorded, A\u03b2 treatment did not cause general impairment resulting in a reduction of cell size.\nFig.\u00a03A\u03b2 induced a reduction of SAT1 immunoreactivity in neurons in P1 mixed cortical cell cultures. Transporter immunoreactivities (arbitrary units of fluorescence intensity, FL) and areas (\u03bcm2) were recorded in individual cells in cultures incubated with 0, 3 or 10\u00a0\u03bcM A\u03b2(25-35) for 12\u00a0h (a, c, e) or 24\u00a0h (b, d, f). Areas (c, d) comprising the perikaryon and proximal neurites were outlinded for recording of an average fluorescence intensity in each cell. To cancel out random differences between cells, transporter immunoreactivity was also expressed relative to MAP2 immunoreactivity in the same cell (e, f). All values are for neurons, except for SN1, which was recorded also in astroglial cells (area and relative immunoreactivity for SN1 in glia were 40 times the displayed numbers). Background was not subtracted; the low values for SN1 in neurons are close to non-immunoreactive background. Columns show mean\u00a0+\u00a0SEM of measurements from four independent experiments (n\u00a0=\u00a04), in each experiment 10 cells were averaged. Asterisks indicate a statistically significant difference compared with control (*P\u00a0\u2264\u00a00.05, **P\u00a0\u2264\u00a00.01, ***P\u00a0\u2264\u00a00.001) in a linear mixed model regression analysis (statistical software R). Only SAT1 showed a robust effect of A\u03b2\nTo adjust for differences in the condition of the individual cell, transporter immunoreactivity was expressed relative to the simultaneously recorded MAP2 immunoreactivity of the same cell (Fig.\u00a03e, f). In this analysis, the effect of A\u03b2 on SAT1 stood out even more clearly, showing the largest percentage changes, whereas there were no statistically significant effects of A\u03b2 on VGLUT1, VGLUT2 or SN1 in neurons, neither on SN1 in astroglia. After both 12 and 24\u00a0h incubation, SAT1 was reduced to about two thirds of the control level by 3\u00a0\u03bcM of A\u03b2, and about one half by 10\u00a0\u03bcM of A\u03b2. As statistical tests showed similar effects at 12 and 24\u00a0h, the data were reanalysed statistically with a simplified linear mixed model assuming the same fixed effect at 12 and 24\u00a0h. This analysis gave higher statistical significance for the effect on SAT1 (P\u00a0=\u00a00.0009 at 3\u00a0\u03bcM, P\u00a0=\u00a00.0000 at 10\u00a0\u03bcM) than did the full model reported in Fig.\u00a03. It confirmed the lack of effect on the other transporters (P\u00a0>\u00a00.2), and the lack of effect on perikaryal size (P\u00a0>\u00a00.1), except for the size of SN1 positive glia (P\u00a0<\u00a00.03 for 3\u00a0\u03bcM, P\u00a0<\u00a00.01 for 10\u00a0\u03bcM).\nLack of effect of A\u03b2 in neuronal cell cultures\nCultured cortical neurons (E18) were exposed to A\u03b2(25-35) in the same way as for P1 mixed cell cultures. MAP2 positive neurons had a similar appearance as in P1 cultures, in controls as well as after exposure to A\u03b2 (Fig.\u00a04a\u2013d). Quantification of the fluorescence intensities (Fig.\u00a05) indicated slight effects of A\u03b2 on SAT1 and SN1 (Fig.\u00a05a, b), but not on the other transporters, or on cell area (Fig.\u00a05c, d). However, when the fluorescence intensity for transporter was normalized to that for MAP2 in the same cell, there were no significant effects of A\u03b2, except on SN1 (which in neurons is very low and of questionable biological significance). Statistical tests suggested no difference between 12 and 24\u00a0h or between 3 and 10\u00a0\u03bcM. When statistically analysed according to a simplified linear mixed model, assuming the same fixed effect for the different incubation times and concentrations of A\u03b2, there were no significant effects of A\u03b2 on any of the transporter immunoreactivities (P\u00a0\u2265\u00a00.07).\nFig.\u00a04SAT1 (red) colocalized (yellow-orange) with MAP2 (green) in neurons in E18 cortical neuronal cell cultures. Sample neurons are shown. The immunoreactive cells were morphologically similar to those in P1 cultures (Fig. 2). No glia-like cells were present. Cells were incubated in the absence (a, b) or presence of 10\u00a0\u03bcM A\u03b2(25-35) (c, d) for 12\u00a0h (a, c) or 24\u00a0h (b, d). Labelling was strong in the perinuclear region and processes of neurons. Scale bar\u00a0=\u00a020\u00a0\u03bcmFig.\u00a05Lack of effect of A\u03b2 on SAT1 immunoreactivity in E18 cortical neuronal cells (cultured without glia). Data were obtained and presented as in Fig. 3\nFinally, to test more directly whether the results differed between the two types of culture used, the MAP2 normalized data for P1 and E18 cultures were combined into one linear mixed model for regression analysis. The fixed effect was now defined as the expected difference in effect of A\u03b2 exposure between P1 and E18 cultures. Assuming the same fixed effect at 12 and 24\u00a0h, the effect on SAT1 was significantly different between neurons in P1 and E18 cultures (P\u00a0=\u00a00.016 for 3\u00a0\u03bcM, P\u00a0=\u00a00.010 for 10\u00a0\u03bcM of A\u03b2) but those on the other transporters were not (P\u00a0>\u00a00.3). Assuming similar effect at the two concentrations of A\u03b2, the significance level was even higher (SAT1 P\u00a0=\u00a00.004; other transporters P\u00a0>\u00a00.2).\nDiscussion\nThe main finding of the present study is that A\u03b2 causes a reduction in neuronal SAT1, a transporter thought to be essential for neuronal uptake of glutamine [9, 13, 14] to support neuronal function including the formation of neurotransmitter in glutamatergic as well as GABAergic neurons. The transmitter of the neurons loosing SAT1 was not directly identified, but as double labelling for MAP2 with VGLUTs and MAP2 with SAT1 both showed major proportions of double labelled cells, they are likely to comprise glutamatergic neurons.\nThe concentrations of A\u03b2 were selected on the basis of literature data to be effective but with low to moderate toxicity levels [12]. To further minimize confounding influence of indiscriminate toxic damage, the immunoreactivities were measured in individual cells that showed near normal morphology and near normal immunoreactivity for the neuronal marker MAP2. Therefore, unlike in biochemical studies of whole cultures, cells dying or dead due to toxic effects of A\u03b2 [5] were excluded from analysis. Further, random variation in the condition of cells was corrected for by expressing the transporter immunoreactivities relative to that of MAP2, measured in the same cell by double immunofluorescence. A\u03b2 peptides have been found to induce proteolysis of MAP2 in cultured mouse cortical neurons [18], but in the cells sampled in the present study such proteolysis must have been minor, as the relation between the values for the different transporters and conditions were similar whether expressed as fluorescence units or relative to MAP2 fluorescence units.\nThe effect of A\u03b2 on neuronal SAT1 was observed in cultures containing neurons in close apposition to astroglial cells, but not in neuronal cultures without glia. This suggests that A\u03b2 does not produce the effect by acting directly on the neurons, but via the glial cells, an idea gaining some support from the observed reduction in astroglial cell size. However, as the astrocytes had only slightly reduced size and normal levels of their marker protein SN1, the changes in neuronal SAT1 cannot be ascribed simply to toxic damage in the glia.\nThe findings are consistent with previous reports that the presence of astrocytes enhances A\u03b2 induced neurotoxicity in hippocampal cell cultures [19] and that the presence of reactive astrocytes is a characteristic feature of the lesions in AD brain [20]. Notwithstanding, the effect on neuronal SAT1 cannot be ascribed simply to an indiscriminate toxic influence on the neurons from the A\u03b2 exposed glia, because there was no similar effect on VGLUTs, and because the effect was observed in neurons with apparently normal morphology and normal expression of the neuronal marker MAP2. Could the reduced level of neuronal SAT1 contribute to the impaired resistance of neurons to A\u03b2 when cultured in the presence of glia? While this does not appear likely, the observed 50% reduction in glutamine transporter level might possibly be critical in certain conditions.\nThe mechanism by which astroglial cells cause changes in neuronal expression of SAT1 must be determined by future research. As free radicals and oxidative stress play important parts in AD [21], it is interesting that crotonaldehyde accumulates in reactive astrocytes in AD brain [22]. Astrocytes mediate A\u03b2 induced neuronal death through oxidative stress [23]. One possible mediator of the observed glia-neuron influence is BDNF signalling, which is impaired in cortical neuron cultures exposed to A\u03b2 [24, 25], although A\u03b2 may affect this signalling cascade without the participation of glia [24]. An attractive possibility is offered by the observation that A\u03b2(25-35) causes astrocytes to produce nerve growth factor (NGF), thereby inducing tau hyperphosphorylation and reduced survival in hippocampal neurons [26]. As the effect is exacerbated by NO, this mechanism may offer a link with the effects of free radicals in AD pathology. It fits with the recent observation that reduction of endogenous tau protects against A\u03b2 toxicity in a mouse model of AD [27].\nThe mechanism of vulnerability of neurons in AD appears to involve excitotoxicity [4]. The increase in glial glutamate transporters induced by A\u03b2 [28] may represent a measure to limit excitotoxic damage. Similarly, the reduced neuronal levels of SAT1 observed here after exposure to A\u03b2 may limit the production of glutamate, but also of GABA, for synaptic release. It might represent an adaptation to curb excitotoxicity in AD, but might concomitantly impair synaptic function.","keyphrases":["glutamine transporter","alzheimer\u2019s disease","cell culture","vesicular glutamate transporter","\u03b2-amyloid peptide (25-35)"],"prmu":["P","P","P","P","R"]}
{"id":"Eur_J_Pediatr-4-1-2292482","title":"Erythropoietic protoporphyria without skin symptoms-you do not always see what they feel\n","text":"Erythropoietic protoporphyria (EPP) is an inherited disorder of the porphyrin metabolism that often remains undiagnosed in children. We report on a 4-year-old girl who had been suffering for 1 year from recurrent painful crises affecting her hands, feet, and nose following sun exposure. Objective skin lesions were absent until the age of 6. Porphyrin analysis revealed elevated free erythrocyte protoporphyrin (FEP) levels confirming the diagnosis of EPP. This illustrates that skin lesions might be completely absent in children affected with EPP, a fact that has only been reported once previously. Because EPP can manifest with few and unspecific cutaneous symptoms or no skin lesions at all, like in this patient, the diagnosis of EPP might be delayed or missed. EPP should be excluded in all photosensitive children, especially when discomfort is disproportionate to the extent of the cutaneous lesions. The clinic, pathophysiology, diagnosis, complications, and therapy of EPP are discussed.\nIntroduction\nErythropoietic protoporphyria (EPP) (OMIM 177000) results from a partial deficiency of ferrochelatase (FECH), the last enzyme in heme biosynthesis that is located in the mitochondrium [10, 13]. FECH catalyzes the insertion of iron into protoporphyrin IX to form heme (Fig.\u00a01) [10]. Although overall EPP is a rare disease, it is the most common type of porphyria manifesting in childhood with an estimated prevalence of 1 in 130,000 [10, 13, 23].\nFig.\u00a01In the mitochondrium, ferrochelatase catalyzes the formation of heme from protoporphyrin IX. ALA: \u03b4-aminolevulinic acid\nThe disease has no racial or sex predilection and no precipitating factors have been described to date [10, 20]. The major clinical feature of EPP is cutaneous photosensitivity that usually commences early in infancy or childhood, affects the sun-exposed body sites, and worsens in spring and summer [10]. The symptoms include pain, burning, itching, erythema, and swelling, which can develop within minutes of sun exposure [4, 10, 13, 14]. Petechiae, purpura, and vesicles may be seen, but are uncommon [4]. Typically, children experience relief when cooling the skin with cold water or wet towels [11]. Chronic UV (ultraviolet) exposure can lead to lichenification, postinflammatory hyperpigmentation, and scarring, particularly over the knuckles and on the nose [13, 23]. Of note, the cutaneous symptoms can be discrete and barely notable in early childhood despite significant subjective discomfort including burning and stinging sensations in the skin.\nHere, we describe a girl with EPP with an unusual clinical course, demonstrating that parents and physicians cannot always recognize how much the patients suffer.\nCase report\nAn otherwise healthy, 4-year-old Dutch Caucasian girl presented with a 1-year history of suddenly occurring severe itching and pain in the hands, feet, and nose after sunlight exposure. These sensations lasted up to 3\u00a0days, and she reported on three such incidents during the last 12\u00a0months. The symptoms developed either immediately or up to 8 h after UV exposure. Erythema or other skin manifestations were not seen. The condition worsened in the summer months and during vacations in sunny climates. Itching commenced immediately after sunlight exposure and reached a maximum at night. After a few hours, the itching changed into pain. Application of wet towels or cold baths led to partial relief, and the symptoms usually vanished spontaneously after a couple of cloudy days without intense UV exposure. Looking back, her parents also remembered that at the age of 1 year, during a summer vacation, she had suffered from an unexplained acute swelling of the hands and feet. At that time no specific diagnosis was made, besides the suspicion of an allergy. During that vacation at the sea side, the overall family life was disturbed because she was continuously agitated, cried a lot without an apparent reason, and did not sleep well.\nUpon physical examination in our outpatient clinic at the age of 4, she showed no skin symptoms except for few minimal excoriations on the dorsal aspects of the feet. The family history revealed eczema in the mother and atopy in the paternal grandmother, but was unremarkable regarding increased photosensitivity. Eventually, a pediatrician suggested the differential diagnoses polymorphic light eruption, warmth intolerance, or hypochondria, and the girl was treated unsuccessfully with antihistamines.\nDue to continuation of her complaints, a pediatric dermatologist was consulted who suspected EPP despite the absence of cutaneous manifestations. Blood porphyrin analysis revealed elevated free erythrocyte protoporphyrin (FEP) levels of 9,821\u00a0\u03bcg\/l (normal 0\u2013300\u00a0\u03bcg\/l), confirming the diagnosis of EPP. Affirmative phototesting showed maximal cutaneous sensitivity at a wavelength of 400\u00a0nm (violet light). Full blood count and liver enzymes were normal.\nTherapeutically, strict UV light avoidance, protective sunscreens, and annual follow-up visits were advised. Still, her photosensitivity did not improve, and mild hyperpigmentation and sores at her nose and upper lip as well as pronounced knuckles and progressive lichenification developed at the age of 6. To date, she has not shown liver enzyme alterations.\nDiscussion\nHere we present a girl with EPP who suffered from a significant decrease of quality of life due to severe itching and pain of the skin. Clinically, EPP is characterized by cutaneous photosensitivity manifesting early in life. Acute photosensitivity episodes include burning, stinging, and pruritus in sun-exposed skin, particularly on the nose, cheeks, and dorsal aspects of the hands, followed by erythema, edema, and wax-like scarring. Skin symptoms can occur within minutes of sun exposure, often starting early in spring time, continuing through the summer, and diminishing in fall and winter.\nInterestingly though, our patient did not show any visible skin lesions until the age of 6. Over the last 30\u00a0years there have been ten reports on children with EPP who developed mild to severe cutaneous photosensitivity and specific skin manifestations [1, 2, 11, 15, 19\u201321, 23, 30, 31]. With mild or absent visible skin symptoms, however, diagnosis may be delayed for several years or is not made at all because the discomfort experienced by the young patients is disproportionate to the objective cutaneous manifestations [22, 23]. Thus, children with EPP are often labeled as hysterical, hypochondriac, or even malingering, a distressing situation for both the patients and their parents [2, 22]. To the best of our knowledge the complete absence of visible skin lesions in early childhood EPP has only been described once to date [12].\nThe symptoms in EPP are associated with an overproduction and accumulation of lipophilic protoporphyrin as a result of FECH deficiency [28]. Excess deposition of protoporphyrin in the skin leads to cutaneous photosensitivity upon UV light exposure with maximum susceptibility at a wavelength around 400\u00a0nm (Soret band) [22]. Upon excitation, protoporphyrin exerts its phototoxic effects through the generation of reactive oxygen species (ROS). ROS can induce lipid peroxidation, oxidation of amino acids, and mediator release from mast cells, contributing to the acute photosensitivity, and protein cross-linking leading to cell membrane damage and cell death [3, 7, 13, 17, 25].\nThe diagnosis of EPP is based on the clinical symptoms and verified by a significant increase of FEP (more than five times the normal level of 0 to 300\u00a0\u03bcg\/l ) in peripheral erythrocytes [15, 18, 28]. Since protoporphyrin is a hydrophobic metabolite, it is not renally excreted. Hence, urinary porphyrin values are usually normal [15, 23]. Another diagnostic modality includes fluorescence microscopy of erythrocytes [2, 17].\nHistological changes are predominantly seen in the upper dermis and include deposition of amorphous material containing immunoglobulins, complement components, glycoproteins, acid glycosaminoglycans, and lipids around blood vessels [5, 21, 24, 26, 27, 29]. However, these alterations are not specific. Regardless of the aforementioned finding, we emphasize that it is unnecessary to take a skin biopsy if one of the cutaneous porphyrias is suspected because of two reasons. Firstly, simple non-invasive biochemical laboratory techniques can easily prove or exclude the presumptive diagnosis of porphyria. Secondly, any kind of external trauma, such as a biopsy or excision, inevitably constitutes an unnecessary risk for delayed or disturbed wound healing in porphyria patients.\nThe differential diagnoses of EPP include solar dermatitis, solar urticaria, polymorphous light eruption, lipoid proteinosis, hydroa vacciniforme, and lupus erythematosus [2, 22]. Elevated FEP levels can also be found in lead poisoning, anemia, renal failure, cholestasis, and liver failure. However, these conditions are not associated with photosensitivity [13, 18, 23].\nBeside the cutaneous manifestations, the most important concern in EPP patients is the development of cholestasis with rapid accumulation of protoporphyrin in hepatobiliary structures resulting in severe liver damage and hepatic failure. Although rarely occurring, progressive liver failure is a well-recognized complication in EPP, and about one third of EPP patients show biochemical liver abnormalities [6, 13]. Therefore, liver function tests should be performed annually [13, 28]. In our patient, no liver dysfunction has been detected so far.\nAlthough in rare instances an autosomal recessive inheritance pattern has been described, EPP is a predominantly autosomal dominantly transmitted disorder with incomplete penetrance resulting from mutations in the FECH gene on chromosome 18q21.3 [31, 32]. To date, more than 65 different FECH mutations have been reported, reflecting the genetic heterogeneity encountered in EPP [13, 28]. Recently, the genetic mechanisms underlying the cutaneous symptoms in EPP have been uncovered. It is clear now that only those individuals will develop skin symptoms who not only inherit a heterozygous FECH gene mutation on one parental allele, but also a specific intronic FECH polymorphism on the other parental allele [8]. Thus, clinically overt EPP results from a marked deficiency of FECH activity below a certain threshold due to co-inheritance of a specific FECH gene mutation with a low-penetrance IVS3-48C allele. Recent studies indicate that the frequency of the IVS3-48C allele shows a high degree of variability in different ethnic groups [9]. The identification of the molecular mechanisms underlying the manifestation of photosensitivity in EPP certainly has to be considered a milestone in porphyria research. Still, the development of protoporphyrin-induced hepatic disease and the molecular mechanisms governing the phenotype with severe liver injury are not well understood. It seems that other as yet unidentified factors may contribute to the pathogenesis of severe liver failure in EPP.\nThe current treatment modalities for EPP are limited and not effective in all patients [13]. In an ideal scenario, the harmful effects of exposure to visible light should be prevented. This can partially be achieved with sunscreens containing titanium oxide or zinc oxide. Common sunscreens, even those with high UV absorption capacity, do not block visible light and, thus, have no protective effect [2, 28].Consequent avoidance of UV light exposure and sun protection by protective clothing remains the most important measure in preventing acute photosensitivity and was also advised to our patient [13, 14, 18, 28]. The effectiveness of other modalities such as antioxidants (e.g., beta-carotene), cysteine, narrow-band UVB-phototherapy, and oral antihistamines remains doubtful and is certainly not of benefit in all patients. In the future, gene therapy approaches might play a role in the treatment of EPP because, e.g., normal ferrochelatase activity could be restored in vitro by transferring a wild-type copy of the FECH gene into cultured fibroblasts of EPP patients [16].\nIn conclusion, the diagnosis of EPP is often delayed or missed. Therefore, EPP should always be considered in photosensitive children, especially when the subjective discomfort is disproportionate to the extent of visible skin manifestations because we obviously do not always see what these patients feel. If cutaneous symptoms are completely absent, an accurate diagnosis is difficult and requires all diagnostic abilities of the attending physicians.","keyphrases":["erythropoietic protoporphyria","porphyrias","photosensitivity","ferrochelatase"],"prmu":["P","P","P","P"]}
{"id":"World_J_Urol-4-1-2295251","title":"Antibody therapy in renal cell carcinoma\n","text":"The treatment of metastasized renal cell carcinoma (RCC) still represents a formidable challenge, despite the development of small molecule, tyrosine kinase inhibitors (TKI) that have made a major impact on the disease. Although the percentage of patients achieving a partial response or stabilization of disease has been impressive, these effects are mostly non-durable. Additionally, drug-related side effects can be quite severe. Alternative treatment modalities might be monoclonal antibodies (mAbs). mAbs against RCC-associated antigens have been developed and have shown promise. Additionally, current efforts focus on Bevacizumab that recognizes vascular endothelial growth factor (VEGF). VEGF overexpression in RCC provides the opportunity to inhibit this proangiogenic pathway. Also with Bevacizumab, promising results have been obtained, particularly in combination with other treatment modalities. It is likely that mAbs, either as single agents or in combination with other agents, may become useful additions to the armamentarium to diagnose and treat RCC.\nIntroduction\nThe hypothesis by Ehrlich in early 1900s that malignant cells express unique structures that can be used to guide cytotoxic therapy to tumors [1] followed by the development of the hybridoma technique by Kohler and Milstein almost 70\u00a0years later [2] has led to the development of anti-cancer reagents with unique characteristics. One of the most distinguishing factors is the possibility to select monoclonal antibodies (mAbs) recognizing target molecules with very restricted expression in normal tissues. To date, tumor-specific antigens (antigens expressed on all tumor cells of a particular tumor type not expressed by normal cells) have not been identified. The members of the so-called cancer-testis family do exhibit highly tissue-restricted expression, but are considered promising target molecules for cancer vaccines, less for antibody therapy, particularly in view of the extreme intra- and inter-tumor heterogeneity [3].\nSimilar to other malignancies, monoclonal antibodies (mAbs) targeting renal cell carcinoma (RCC) associated molecules were developed without understanding the molecular events underlying RCC [4\u20138]. The increased understanding of molecular events important in the carcinogenesis of RCC led to the recognition that these aberrations can be used to target RCC. Specifically, aberrant von Hippel-Lindau (VHL) gene expression has been identified as a general event in clear cell RCC (ccRCC) [9], which represents 80\u201385% of localized cases and 90\u201395% of metastatic RCC (mRCC). The loss of a functional VHL gene product leads to accumulation of the transcription factor HIF-1\u03b1 that is an obligatory element for the transcription of several genes. This includes vascular endothelial growth factor (VEGF) and carbonic anhydrase 9 (CA9), targets for which most clinical experience with mAbs in RCC has been generated (Bevacizumab and G250, respectively).\nThe rationale and effects of Bevacizumab and G250-directed therapy are fundamentally different: Bevacizumab treatment leads to VEGF-depletion and consequently to diminished neovascularization followed by tumor cell death, mainly due to loss of vascularization. In contrast, G250 treatment targets the cell surface of RCC cells where it must exert toxic effects. Both approaches have advantages and disadvantages. Bevacizumab treatment has the advantage that VEGF depletion can be achieved in the circulation, and homing to all tumor vessels is not necessary. However, other regulatory pathways can also lead to neovascularization and small, non-vascularized tumor loci will not be affected. G250 treatment has the advantage that RCC cells can be targeted, irrespective of tumor size. However, in view of the generally poor perfusion rate and high interstitial fluid pressure in RCC, deep penetration of tumors may be difficult. Also, since G250-binding alone does not confer a lytic signal to RCC cells, tumor cell kill requires effector cells or coupling of G250 to toxic agents.\nBevacizumab\nBevacizumab is a humanized mAb against VEGF that binds and neutralizes all of the major isoforms of VEGF [10]. This prevents VEGF from interacting with its receptors and activation of downstream signaling pathways. This mode of action is thought to lead to regression of existing microvasculature, normalization of mature vasculature, and inhibition of the production of new vasculature [11]. Whether all these effects are true for RCC is unclear at the moment.\nSignificant protein dose levels are needed to maintain sufficiently high Bevacizumab levels to trap VEFG, the target of Bevacizumab. The first Bevacizumab trial in metastatic RCC (mRCC) patients addressed whether Bevacizumab treatment could lengthen the time to progression of disease and the response rate [12]. Survival was a secondary end point. In this randomized phase II trial, 116 patients with metastatic, refractory clear cell RCC were randomized to placebo, low-dose (3\u00a0mg\/kg) Bevacizumab, or high-dose (10\u00a0mg\/kg) Bevacizumab given intravenously every 2\u00a0weeks. All patients had prior disease progression while on systemic treatment; the vast majority had received prior interleukine-2. Patients with disease progression on placebo crossed over to receive low-dose Bevacizumab. Bevacizumab treatment resulted in a significant prolongation of the time to progression of disease in the high-dose antibody group (4.8\u00a0months as compared with 2.5\u00a0months). Possibly, the low-protein dose was inadequate to sufficiently deplete circulating VEGF levels in-between injections, explaining the poor outcome in this group. The study was inadequately powered to show a significant difference in overall survival between groups. Based on this encouraging result, Bevacizumab has been combined with other treatment modalities to augment the therapeutic index.\nBevacizumab in combination\nThe AVOREN trial investigated the effects of standard therapy of interferon alfa-2a plus placebo or interferon alfa-2a plus Bevacizumab, administered every 2\u00a0weeks at a dose of 10\u00a0mg\/kg [13]. In this randomized, double blind phase III trial, 649 patients with first-line mRCC were enrolled. The primary analysis endpoint was assessment of improvement in progression-free survival (PFS), defined as the length of time the tumor did not grow or patient death did not occur. Other endpoints of the study included overall survival, time to progression, time to treatment failure, overall response rate, and safety profile. The addition of Bevacizumab to IFN-\u03b12a significantly increased PFS (10.2 vs. 5.4\u00a0months) and objective tumor response rate (30.6 vs. 12.4%; P\u00a0<\u00a00.0001). Additionally, the combination treatment showed a trend toward improved overall survival (P\u00a0=\u00a00.0670), which leads to the conclusion that the combination of Bevacizumab with IFN-\u03b12a is superior to either of the single treatment regimens in mRCC. The working mechanism explaining the superiority of this combination treatment has not been defined yet, but most likely the superiority is the net effect of the reduction of the immunosuppressive effects due to decreased VEGF levels combined with the immunomodulatory effects of IFN.\nConsidering that the epidermal growth factor receptor (EGFR) is also overexpressed in RCC, a multicenter, phase II study evaluated the addition of erlotinib (Tarceva), an EGFR inhibitor, to Bevacizumab in metastatic RCC patients [14]. Treatment consisted of 10\u00a0mg\/kg Bevacizumab given intravenously every 2\u00a0weeks and 150\u00a0mg erlotinib given orally each day. With 15 (25%) patients showing objective responses, and an additional 36 patients (61%) with stable disease after 8\u00a0weeks of treatment, a randomized phase II trial was performed evaluating Bevacizumab\u00a0+\u00a0placebo versus Bevacizumab\u00a0+\u00a0erlotinib. Disappointingly, identical response rates and PFS rates for the two arms were observed [15], and it is doubtful that EGFR-targeting is of any benefit.\nThe effect of Bevacizumab and low-dose interleukine-2 (IL-2) in mRCC was evaluated in a phase II trial in previously untreated, good and intermediate risk, mRCC patients. Patients received 8-week cycles of IL-2 (250,000\u00a0U\/kg per day s.c. Day 1\u20135 during week 1 and 125,000\u00a0U\/kg per day s.c. Day 1\u20135 during weeks 2\u20136, followed by a 2\u00a0week break), and Bevacizumab 10\u00a0mg\/kg was administered i.v. every 2\u00a0weeks starting on day-14. With 16 of the planned 35 patients enrolled, and 11 evaluable patients for response, 1 partial response (PR) and 3 stable disease (SD) lasting >3\u00a0months were observed [16]. All patients with SD demonstrated some degree of tumor shrinkage. Similar to the working mechanism of the Bevacizumab\/IFN combination, the anti-tumor effects are possibly the result of the reduction of the immunosuppressive effects due to decreased VEGF levels combined with the general immune activating effects of IL-2. Interestingly, treated patients demonstrated an increase in the number of regulatory T cells without effect on DC activation. Larger, randomized studies will be necessary to address the value of this combination treatment.\nIn a phase I trial, Bevacizumab has also been combined with sunitinib (Sutent\u00ae), a tyrosine kinase inhibitor, with the hypothesis that this combination may increase antitumor efficacy by maximizing inhibition of the VEGF pathway. The Bevacizumab dose was kept constant (10\u00a0mg\/kg) while the sunitinib dose was escalated starting at 25\u00a0mg (escalation with 12.5\u00a0mg increments). Of 13 patients evaluated for best response, 4 had partial responses, 7 had stable disease, and 2 had PD [17].\nSimilarly, the combination of Bevacizumab and the mTOR inhibitor CCI-779 (Temsirolimus\u00ae) has been investigated. Patients received 25\u00a0mg\/week Temsirolimus and 5 or 10\u00a0mg\/kg Bevacizumab. In 12 evaluable patients, 7 PR and 3 SD were observed [18]. The encouraging results certainly deserve further testing of these combinations in phase II trials.\nMonoclonal antibody G250\nG250 is a mAb against CA9, a molecule which is ubiquitously expressed in ccRCC [19]. CA9 expression in non-ccRCC has also been documented, and there, it is most likely a reflection of (sustained) hypoxia [20]. Clinical efforts with mAb G250 in RCC have focused on radioimmunotherapy and passive immunotherapy. This mAb was described as a mAb recognizing an RCC-associated antigen, absent in normal kidney and homogeneously expressed in most RCC [4], most notably clear cell RCC [19]. In 2000, the G250 antigen molecule was identified and shown to be CA9 [21]. The molecular characterization allowed transcriptional regulation studies that revealed a strict dependence of G250 expression on HIF-1\u03b1 [22]. Thus, the molecular mechanism responsible for CAIX expression in ccRCC is similar to VEGF, namely due to non-functional VHL protein leading to HIF-1\u03b1 accumulation and gene expression.\nThe first clinical trials with mAbG250 were already performed and published before the molecular characterization of G250 antigen was achieved. The combined data from the immunohistochemical tissue distribution, animal experiments and ex vivo perfusion of tumor bearing kidneys had provided sufficient evidence to initiate a biopsy-based phase I protein dose escalation trial with murine mAbG250. The rationale of G250-directed therapy obviously differs from Bevacizumab: Bevacizumab treatment leads to VEGF-depletion and consequently to diminished neovascularization whereas G250 treatment targets RCC cells directly. This first mAbG250 clinical trial demonstrated various pivotal aspects: most notably, virtually no uptake in other tissues resulting in excellent tumor visualization, and very high tumor uptake [23].\nThe G250 antibody uptake that was observed was up to 10-fold higher than any other mAb uptake in solid tumors, which led to the design of a phase I\/II radioimmunotherapy (RIT) trial with murine mAbG250. RIT led to stabilization of disease in 17 of 33 patients, with tumor shrinkage observed in two patients. Transient liver toxicity was observed, quite likely the result of mAbG250 liver uptake, although there was no correlation between the amount of 131I administered or hepatic absorbed radiation dose and the extent and nature of hepatic toxicity [24].\nBecause the murine G250 antibody was highly immunogenic, restricting multiple injections, mAbG250 was chimerized. The results of the phase I protein dose escalation trial with chimeric G250 (cG250) basically duplicated the results from the murine G250 trial: virtually no uptake in other tissues resulting in excellent tumor visualization, and very high tumor uptake. The half-life of the antibody was extended, as was to be expected, but, more importantly, the chimerized from of G250 was almost immunosilent [25]. Thus, multiple injections became possible. Various phase I and phase II trials have been performed with cG250 aimed at therapeutic intervention. Based on the very high uptake levels, several RIT trials were performed. In the first phase I trial with 131I-cG250, one patient showed a partial response (>9\u00a0months) [26] which set the stage for phase II RIT trials in metastatic RCC patients. RIT studies with single high dose 131I-G250, rapid fractionated dose 131I-G250 [27], and sequential high dose 131I-G250 [28] have resulted in only occasional therapeutic responses, although dosimetric analyses suggest that tumor-sterilizing levels can be reached. Even two sequential high-dose treatments with 131I-G250 did not result in objective responses, but in stabilization of previously progressive disease in a few patients. RIT with G250 has been accompanied by bone marrow toxicity similar to mAb RIT in other tumor types and considering the minimal benefit, 131I-based RIT with cG250 have been abandoned. Since RCC is a radiotherapy resistant tumor, possibly even higher radiation doses are necessary to achieve tumor-sterilizing levels. Current G250 RIT efforts are directed to 177-lutetium and 90-yttrium labeled G250. It is hypothesized that the use of more powerful radionuclides that are also better retained in the tumor cells may lead to clinical responses. Animal experiments have demonstrated the superiority of 177Lu- and 90Y-labeled G250 over 131I-G250 [29]. Importantly, stabilization of previously progressive disease has been observed in almost all 177Lu-G250 treated patients, although the maximum tolerable 177Lu dose has not been achieved. Dosimetric analyses of the first patients treated with 177Lu-G250 suggest that indeed tumor-sterilizing levels may be achieved. Figure\u00a01 illustrates targeting of 177Lu-G250 in a patient with metastatic renal cancer.\nFig.\u00a01Anterior (left) and posterior (right) whole body scans acquired 7\u00a0days post-injection of patient injected with177Lu-cG250. Please note high uptake in both pulmonary lesions and in contralateral kidney lesions. Uptake in liver is due to the conjugation methodology and is not related to G250 antigen expression\nIn view of the obvious tumor-specific accumulation of cG250, passive immunotherapy of RCC patients has also been studied extensively. In vitro mAbG250 can elicit antibody-dependent cellular cytotoxicity (ADCC), which can be enhanced by low dose IL-2 [30]. Various (non-randomized) clinical trials have now been completed with cG250 alone, and in combination with IL2 or interferon [31, 32]. Thus far, these treatments appear to lead to extended survival time. The apparent clinical benefit appears to be quite substantial with a documented median survival of 22\u00a0months in patients with metastatic RCC who have progressive disease at study entry. Nevertheless, it is difficult to judge the value of this treatment. Clearly, randomized trials are necessary to unequivocally demonstrate whether passive immunotherapy with cG250 is of benefit for metastatic RCC patients.\nThe largest trial, which is currently ongoing, is the adjuvant ARISER trial (adjuvant Rencarex immunotherapy phase III trial to study efficacy in nonmetastatic renal cell carcinoma). In this phase III randomized, double blind, placebo-controlled trial, patients with ECOG performance status of 0 with completely resected primary clear cell RCC and no evidence of remaining local or distant disease, are treated. The study is designed to detect a significant difference between the two treatment arms with respect to disease-free survival; patients will be followed-up long-term to determine overall survival statistics.\nRecently, the potential utility of mAbG250 as a diagnostic imaging agent was investigated [33]. The excellent imaging capability had been noted in almost all patients, but this line of research was not pursued mainly because detection of suspect renal masses and occult metastatic RCC was not deemed advantageous. Additionally, treatment modalities for metastasized RCC were poor, and, therefore, efforts focused on treatment. However, with a steady increase of incidentally discovered renal masses and new therapeutic modalities becoming available, imaging might become of importance to distinguish more potentially malignant tumours from less aggressive variants. In the first prospective clinical trial with 124I-labeled cG250, a very high specificity and sensitivity to identify ccRCC in patients with suspect renal masses was demonstrated, a clear indication of the potential clinical utility. Whether this imaging modality can be used to follow therapy effects remains to be determined.\nIn conclusion, it is reasonable to assume that Bevacizumab and G250 monoclonal antibodies either as single agents or in combination with other agents may become useful additions to the armamentarium to diagnose and treat (cc)RCC. Several trials evaluating the combination of G250 or Bevacizumab with registered RCC treatments are currently in progress and will further define the role of these mAbs in RCC.","keyphrases":["therapy","renal","rcc","monoclonal antibody","vegf","g250","caix"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Ann_Surg_Oncol-4-1-2190342","title":"A Highly Active and Tolerable Neoadjuvant Regimen Combining Paclitaxel, Carboplatin, 5-FU, and Radiation Therapy in Patients with Stage II and III Esophageal Cancer\n","text":"Background The present phase II study aimed to assess the feasibility and efficacy of a new paclitaxel-based neoadjuvant chemoradiation regimen followed by surgery in patients with stage II\u2013III esophageal cancer.\nThe prognosis of patients with esophageal cancer who undergo surgery with a curative intention is poor, with a 5-year survival rate of 5\u201320%.1 In recent decades, several efforts have been made to improve outcome. Improved and standardized surgical techniques and supportive care as well as concentration of care have contributed to a better outcome; however, further improvement of survival from a single modality approach seems unlikely.2,3\nNeoadjuvant chemoradiation has become the focus of interest to improve long-term survival and reduce recurrence rates. Despite a large number of phase II and several phase III trials, the role of neoadjuvant chemoradiation remains unclear. Until now, only one phase III trial demonstrated a significant survival advantage.4 All neoadjuvant regimens used in these phase III trials were cisplatin-based, which leads to frequent toxicity-related dose reductions and delays and relatively low pathologic complete response rates (10\u201328%).\nNew neoadjuvant chemoradiation regimens including drugs such as paclitaxel have shown encouraging complete response rates (29\u201339%) and R0 resection rates.5\u20137 Since pathologic complete response and R0 resection have been shown to be associated with improved survival,8,9 it can be expected that these new regimens lead to better survival rates. Therefore, the optimal neoadjuvant treatment regimen with high pCR rates and minimal toxicity has to be determined.\nPaclitaxel interferes with mitotic spindle function by enhancing the rate and yield of microtubule assembly and preventing microtubule depolymerization.10 Paclitaxel is a potent radiosensitizer because it synchronizes cells at G2\/M, the phase of the cell cycle that is particularly sensitive to radiotherapy.11,12 Furthermore, paclitaxel radiosensitization persists well after the period of G2\/M synchronization, suggesting that other factors may also relate to its ability to enhance radiotherapy.13 The p53 gene is required for initiation of apoptosis in response to most chemotherapeutic agents and radiotherapy. In vitro, paclitaxel-mediated blockade at the G2\/M has shown to activate cell-cycle control pathways that induce apoptosis independent of p53.14\nTherefore, paclitaxel-based neoadjuvant chemoradiation seems a rational treatment approach for esophageal cancer. A recent study by Meluch et al.6 has reported an encouraging complete response rate of 38% with a paclitaxel-based regimen. However, toxicity was severe as only 54% of the patients received a full dose of the neoadjuvant regimen, because of complicating esophagitis and leucopenia. The goal of this study was to assess the feasibility and efficacy of a new paclitaxel-based neoadjuvant chemoradiation protocol in which we applied a reduced chemotherapy dose compared with the protocol of Meluch et al.6\nPATIENTS AND METHODS\nPatients\nReferral to our institute was accomplished as a result of agreements between hospitals affiliated to the Comprehensive Cancer Centre South in the Netherlands to concentrate surgical care of patients with esophageal cancer. The protocol of this prospective clinical phase II trial was approved by the Medical Ethics Committee, and from all patients written informed consent was obtained. All consecutive patients with a potentially resectable stage II and III esophageal cancer who were referred to our Centre between January 2002 and November 2004 were found eligible.\nFurther eligibility criteria included: patient age between 18 and 75 years, WHO performance status \u22642, written informed consent, central venous catheter, and adequate hematological, renal, and hepatic functions defined as white blood cell count > 3.5\u00a0\u00d7\u00a0109\/L, platelet count > 100\u00a0\u00d7\u00a0109\/L, creatinine level < 120 \u03bcmol\/L and\/or creatinine clearance > 60 mL\/min, and normal hepatic enzyme levels. Patients with distant metastatic disease or tumor positive supraclavicular lymph nodes were excluded from entry into the protocol. However, M1a disease in the regional lymph nodes was not.\nPretreatment staging evaluation included clinical examination, upper gastrointestinal endoscopy with histological biopsies of the tumor, computed tomography (CT) scan of chest and upper abdomen, and ultrasound of the neck. Endoscopic ultrasound (EUS) was performed when a T1 tumor could not be excluded by conventional techniques. More recently, PET imaging was introduced to exclude distant metastases.\nNeoadjuvant Treatment\nThe neoadjuvant regimen consisted of paclitaxel 175 mg\/m2 intravenously and carboplatin AUC 5 intravenously (IV) on day 1 and 22, and 5-FU 200 mg\/m2 continuous infusion on day 1\u201342. All patients were treated prophylactic with dexamethasone 20 mg and a 5HT3 antagonist IV followed by ranitidine 50 mg IV and clemastine 2 mg IV.\nRadiotherapy was performed with a linear accelerator with a minimal photon energy of 6 MV. The total dose of 45 Gy was given in 25 fractions of 1.8 Gy once daily, starting on day 1. The radiation fields encompassed the primary tumor and enlarged lymph nodes, if any, defined by endoscopy, CT scan, and EUS, surrounded by 5-cm proximal and distal margins and a 2-cm radial margin. Neoadjuvant treatment was given on a fully outpatient base.\nDose modifications were made for a toxic reaction as defined according to the criteria of the World Health Organization (WHO).15\nSurgery\nSurgery was planned 6\u20138 weeks after completion of neoadjuvant treatment. For tumors in the distal third of the esophagus, a transhiatal approach was used whenever possible. A thransthoracic Ivor-Lewis technique was used for tumors in the proximal and middle third of the esophagus. Esophagogastric continuity was reestablished using the stomach with a cervical anastomosis in all patients. A feeding jejunostomy was placed, and enteral feeding was started 24 hours after surgery.\nData Collection\nThe following data were prospectively collected: age, sex, WHO performance status, location of the tumor, histology of the tumor, comorbidity, toxicity of neoadjuvant treatment, type of resection, and postoperative course and complications.\nHistological Examination\nAll pathology reports and specimens were centrally reviewed by one pathologist (GL). Pathologic assessment was performed after standard hematoxylin-eosin (H&E) staining. The assessment included a determination of the histological type of the tumor, the depth of invasion of the tumor, whether there was nodal involvement, and the status of circumferential, proximal, and distal resection margins. The tumor stage was defined according to the classification of the American Joint Committee on Cancer.16 Patients with no residual viable tumor cells in the surgical specimen were classified as having a pathologic complete response (pCR). In addition, response after chemoradiation of the primary tumor was classified according to the Tumor Regression Grade (TRG) as described by Mandard et al.17 Categories according to the TRG ranged from TRG1, complete regression, to TRG 5 with no regressive changes. TRG 1\u20133 scores were defined as a major response to chemoradiation.\nStatistical Analysis\nThe analyses were performed on the basis of intention-to-treat. Survival time was calculated as the duration from the day of start of chemoradiation therapy until death or last day of follow-up, and recurrence free interval was calculated from the day of surgery until the day of diagnosis of recurrence. Overall survival and disease-free survival were estimated using the Kaplan-Meier method. Median survival time was obtained from the time corresponding to 50% survival based on the Kaplan-Meier survival curve. The log-rank test was used to assess survival differences. Significance was defined at a confidence level of P\u00a0<\u00a0.05. Statistical analyses were performed with SPSS version 15.\nRESULTS\nPatient Characteristics\nFifty-two patients with stage II\u2013III esophageal carcinoma were treated in the period between January 2002 and November 2004. Two patients were found ineligible. One patient had a history of Hodgkin\u2019s disease and squamous cell carcinoma of the tongue. Previous treatment of this patient included external beam radiotherapy on the chest. One patient had intellectual disability (mental retardation) and was extremely anxious. Therefore, our study population consisted of 50 patients. All 50 patients signed the written informed consent and were included. Patient characteristics are detailed in Table\u00a01.\nTABLE\u00a01.Patient characteristicsNo. of patients n\u00a0=\u00a050Age (years)\u00a0\u00a0Median60\u00a0\u00a0Range34\u201375Sex\u00a0\u00a0Male44\u00a0\u00a0Female6WHO performance status\u00a0\u00a0Median1\u00a0\u00a0Range0\u20132ASA classification\u00a0\u00a0I24\u00a0\u00a0II24\u00a0\u00a0III2\u00a0\u00a0IV\u2013Tumor location\u00a0\u00a0Upper third2\u00a0\u00a0Middle third1\u00a0\u00a0Lower third47Histology\u00a0\u00a0Adenocarcinoma42\u00a0\u00a0Squamous cell carcinoma8\nToxicity Related to Neoadjuvant Treatment\nForty-two patients (84%) completed full neoadjuvant treatment. Three patients discontinued chemotherapy prematurely due to grade II hand-foot syndrome, four patients due to esophagitis, and one due to grade IV leucopenia. Forty-seven patients (94%) received at least 5 weeks chemotherapy; the remaining three patients discontinued chemotherapy in week 4. Full dose of radiotherapy was received by 49 patients (98%). Grade III leucopenia occurred in 23 patients (46%), without febrile episodes. No other hematologic toxicity was recorded. All 50 patients experienced esophagitis, usually mild (\u2264 grade 2). A total of 13 patients (26%) needed nasogastric enteral feeding. Infection of the indwelling central venous catheter occurred in two patients. There were no neoadjuvant treatment related deaths. However, one patient died of myocardial infarction 3 weeks after completing neoadjuvant treatment.\nPostoperative Complications\nSurgery was performed after a median of 8.1 weeks (range 6.0\u201317.9 weeks). No significant difference was observed between responders (TRG 1\u20133) and nonresponders (TRG 4\u20135) with respect to the time between the end of chemoradiation and surgery. Two patients showed metastatic disease at the time of surgery; hence, 47 patients underwent surgery with a curative intention. Transhiatal esophageal resection was performed in 44 patients; a transthoracic Ivor-Lewis resection was performed in three patients.\nThe postoperative course was uneventful in 23 patients (49%).There were four postoperative deaths (8.5%). Two patients died of necrosis of the gastric tube. One patient developed a fistula between the gastric conduit and the trachea and died of pulmonary sepsis. One patient died of a progressive chylothorax. Other postoperative complications included major anastomotic leakage (n\u00a0=\u00a05), pulmonary complications (n\u00a0=\u00a015), recurrent nerve palsy (temporary n\u00a0=\u00a04, permanent n\u00a0=\u00a01), and cardiac arrhythmias (n\u00a0=\u00a03). An overview is outlined in Table\u00a02.\nTABLE\u00a02.Postoperative complicationsType of complicationNo. of patients (%)Major anastomotic leakage5 (11)Pulmonary15 (32)\u00a0Pneumonia7\u00a0Emphysema4\u00a0ARDS1\u00a0Mediastinal infection1\u00a0Trachea-esoph fistula1\u00a0Chylothorax1Recurrent nerve palsy\u00a0\u00a0Permanent1 (2)\u00a0\u00a0Temporary4 (9)Cardiac arrhythmias3 (6)Wound infection2 (4)Diaphragmatic hernia1 (2)Hospital stay (days)\u00a0\u00a0Median15\u00a0\u00a0Range9\u201383ICU\/MCU stay (days)\u00a0\u00a0Median2\u00a0\u00a0Range2\u201318Ventilation time (days)\u00a0\u00a0Median1Range0\u20139\nPathologic Response to Neoadjuvant Treatment\nPathologic complete regression in the primary tumor was achieved in 18 of 47 operated patients (38.3%). However, in two patients the primary tumor showed complete response, but microscopic residual tumor was detected in locoregional lymph nodes. The ypTNM stages of the remaining patients were: pT1N0M0 in six patients (12.8%), pT2-3N0M0 in 12 patients (25.5%), pT1-2N1M0 in four patients (8.5%), and pT3N1M0\/pT4N0-1M0 in seven patients (14.9%). Subsequent postoperative tumor stages are outlined in Table\u00a03. Postoperative tumor regression grades (TRG) scored by an independent pathologist are outlined in Table\u00a04. Eighteen patients (38.3%) showed a complete tumor regression (TRG1), and 38 patients (80.9%) displayed a major response (TRG1-3) after neoadjuvant treatment. Two patients had positive margin involvement of the resected specimen; therefore, a R0 resection was achieved in 45 of 47 patients (95.7%).\nTABLE\u00a03.Pathological stage of patients treated with neoadjuvant chemoradiation and surgeryStage, ypTNMNo. of patients (%)018 (38.3)I6 (12.8)II a12 (25.5)II b4 (8.5)III7 (14.9)TABLE\u00a04.Tumor regression grade (TRG) of patients treated with neoadjuvant chemoradiation and surgeryTRGaNo. of patients (%)1\u2014Absence of histologically identifiable residual cancer and fibrosis extending through the different layers of the esophageal wall18 (38.3)2\u2014Presence of rare residual cancer cells scattered through the fibrosis11 (23.4)3\u2014Increase in the number of residual cancer cells, but fibrosis still predominated9 (19.1)4\u2014Residual cancer outgrowing fibrosis7 (14.9)5\u2014Absence of regressive changes2 (4.3)a TRG according to Mandard et al.17\nSurvival and Pattern of Failure\nAll 50 patients were included in the survival analysis. The median follow-up for surviving patients was 41.5 months (range 21\u201359 months). The median overall survival and disease-free survival were 44 and 50 months, respectively. The estimated 3- and 5-year overall survival rates were 55.6 and 47.8%, respectively, whereas the estimated 3- and 5-year disease-free survival rates were 52.2 and 41.8%, respectively. The Kaplan-Meier curves for overall and disease-free survival are shown in Fig.\u00a01 and 2.\nFIG.\u00a01.Kaplan-Meier curve of overall survival of all patients (n\u00a0=\u00a050).FIG.\u00a02.Kaplan-Meier curve of disease-free survival (n\u00a0=\u00a050).\nPatients with TRG 1\u20133 showed a significant improved overall and disease-free survival compared with patients with TRG 4\u20135. Median survival time in TRG 1\u20133 patients was not yet reached and 23 months in patients with TRG 4\u20135. The estimated 3- and 5-year overall survival rates were 61.0 and 55.9% for the patients with a TRG 1\u20133 and 33.3 and 16.7% for the patients with a TRG 4\u20135. Median disease-free survival in TRG 1\u20133 patients was not yet reached and 11 months in patients with TRG 4\u20135. The estimated 3-year disease-free survival rates for patients with TRG 1\u20133 was 58.6 vs 25.0% for patients with TRG 4\u20135. Comparing the TRG 1\u20133 patients with TRG 4\u20135 patients, the log-rank test of equality of the survival curves generated a P value of .03 for overall survival and .02 for disease-free survival time in favor of patients with TRG1\u20133. The Kaplan-Meier curve for disease-free survival according to tumor regression grade is shown in Fig.\u00a03.\nFIG.\u00a03.Kaplan-Meier curve of disease-free survival by tumor regression grade (TRG). TRG1\u20133 (n\u00a0=\u00a038): major response, TRG 4\u20135 (n\u00a0=\u00a09): minor or no response (P\u00a0=\u00a0.0183).\nRecurrent disease was found in 20 patients surviving postoperatively (20 of 43, 47%) Five of them were still alive at the time of the analysis. Recurrence was only locoregional in three patients. Distant metastases occurred in 16 patients, and in one patient recurrence presented with locoregional and pulmonary metastases.\nTo date 22 patients have died. Of these 22 patients, three died from nondisease-related causes and were presumed disease free. Of the 19 patients who had disease-related deaths, four patients died of postoperative complications, and three patients died of complications from local recurrence, whereas 12 patients died due to systemic failure that included metastases to lung, liver, brain, and bone.\nDISCUSSION\nThis study shows the results of a phase II efficacy study, with a modified chemoradiation regimen according to Meluch et al.6 A reduction of the dose of chemotherapy components of this regimen resulted in a decrease of toxicity with preservation of its high efficacy rate. The pathologic complete tumor regression rate of 38.3% and R0 resection rate of 95.7 % are among the highest described in the literature. Toxicity and morbidity of this regimen compared with the original scheme were mild and acceptable.\nNumerous phase II trials using neoadjuvant chemoradiation showed marked downstaging and suggested survival benefits. In most trials, chemotherapy regimens included cisplatin and 5-FU. However, randomized controlled trials (RCT) comparing chemoradiation followed by surgery with surgery alone have yielded inconclusive results.4,18\u201322 One of these trials showed significant improvement in survival in the patients who received combined-modality therapy.4 The relevance of this study was disputed because the 3-year survival rate of 6% in the control arm was exceptionally low. Two additional trials showed a possible benefit of neoadjuvant chemoradiation without reaching a statistical significance.19,22 Furthermore, the power of these trials to detect modest but clinically relevant differences was limited by small patient numbers. In a meta-analysis report by Fiorica et al.23 six RCTs were pooled comparing neoadjuvant chemoradiation and surgery versus surgery alone. A significant difference in 3-year mortality favoring neoadjuvant chemoradiation with surgery versus surgery alone was detected (OR 0.53; 95% CI 0.31\u20130.93; P\u00a0=\u00a0.03 NNT\u00a0=\u00a010). It was concluded that neoadjuvant chemoradiation and surgery significantly improved 3-year survival compared with surgery alone in patients with resectable esophageal cancer. However, a careful interpretation of these results is warranted, because this meta-analysis was based on summarized patient data.\nNeoadjuvant chemoradiation induces acute treatment-related toxicity. These effects include myelotoxicity, esophagitis, nausea, and vomiting. The regimen used in this phase II trial produced moderate toxicity and is comparable with reported literature on neoadjuvant chemoradiation regimens including paclitaxel.5\u20137,24,25 The highly effective regimen used by Meluch et al.6 was modified to reduce toxicity. The modification consisted of dose reduction of 5-FU 200 mg\/m2 instead of 225 mg\/m2, paclitaxel 175 mg\/m2 instead of 200 mg\/m2 and carboplatin AUC 5.0 instead of AUC 6.0. This modification resulted in a reduction of toxicity. Forty-two patients (84%) received full doses of the planned 6 weeks chemotherapy, whereas in the group of Meluch et al. only 54% of patients received full dose chemotherapy. A relative large proportion of the patients (26%) received preoperative nasogastric enteral feeding. This was partly due to our liberal use of nasogastric enteral feeding, since in our experience optimizing preoperative nutritional status in patients treated with a neoadjuvant chemoradiaton protocol is critical. The neoadjuvant treatment was delivered on a fully outpatient basis. Further improvement of our regimen may be obtained by replacing 5-FU for novel oral fluoropyrimidines. In colorectal cancer, the novel fluoropyrimidines such as capecitabine and UFT show equal if not superior efficacy and improved patient convenience.\nThe reported postoperative mortality in the literature for neoadjuvant chemoradiation followed by surgery ranges from 0 to 29 % (median 9%). A nationwide study in the United States demonstrated in-hospital mortality rates of high-volume versus low-volume centers of 8.4 vs 20.3%.2 A comparative Dutch study found in-hospital mortality rates of 4.9 vs 12.1%.26 Therefore, the in-hospital mortality rate of 8.5% in our trial is within these boundaries. Several studies reported an increase in postoperative mortality after neoadjuvant chemoradiation. The aforementioned recent meta-analysis of six randomized trials confirmed a significant effect of chemoradiation on postoperative mortality (OR 2.10 95% CI 1.18\u20133.73, P\u00a0<\u00a0.01, NNT\u00a0=\u00a025).23 A relatively short interval between completion of neoadjuvant therapy and surgery of 2\u20134 weeks is noted in these trials and might be of influence in the relative high mortality rates described in these trials.\nWe decided to perform surgery after at least 6 weeks to achieve an adequate downstaging and permit maximal recovery; in addition, surgery was postponed in some patients due to a slow recovery from the chemoradiation. We believe that the timing of surgery is important. Performing an esophagectomy too soon after neoadjuvant chemoradiation may lead to increased morbidity due to prolonged myelosuppression and hence an increased risk for developing postoperative complications.\nUntil recently, neoadjuvant chemoradiation studies have been performed using cisplatin\/5-FU based regimens with a variation in dose and a variation in radiotherapy schemes. Moderate complete response rates of 8\u201326 % and R0 resection rates of 55\u201384% have been described in these studies.4,19,20,22,27\u201329 With the implementation of regimens including paclitaxel, 5-FU, and cisplatin, complete response rates of 32\u201338% and R0 resection rates of 78\u201395% have been documented.6,7,25,30 Our study reports a complete pathologic response of 38.3%, which is high. Furthermore, a R0 resection rate of 95.7% is among the highest described in the literature. The dismal prognosis of esophageal cancer has discouraged many clinicians. For many patients, the prognosis was supposed to be dependent on the presence of indolent distant metastasis despite the type of operative treatment given. In a recent phase III trial, however, extended radical transthoracic esophagectomy showed a trend to a better survival when compared with a less radical transhiatal esophagectomy, indicating that an adequate locoregional treatment may have a beneficial effect on survival.3 However, in both groups a R0 resection rate of only 72% was noted. This observation is an important rationale for neoadjuvant combined multimodality treatment. One of the goals of neoadjuvant chemoradiation therapy is increasing curative R0 resection rates by downsizing the tumor and theoretically eradicating micrometastasis. Achieving R0 resection in locally advanced (stage II\u2013III) esophageal cancer has been identified as a prognostic factor.8,9 Furthermore, complete pathologic response has been associated with an improved survival and local control.30,31 In the present study a major response, tumor regression grade 1\u20133, resulted in a significant increase in overall and disease-free survival. Since the time between surgery and the end of the chemoradiation did not significantly differ between the responders and nonresponders, a difference in response could not be explained by a lag-time difference. Thus, a regimen with high complete pathologic response and R0-resection rates will be very likely to improve survival. However, with our relatively short follow-up we cannot report definitive long-term survival data yet.\nIn conclusion, although the discussion regarding the value of neoadjuvant therapy is ongoing, the neoadjuvant treatment regimens used for the last 20 years produced only modest benefit, at best. Improving chemoradiation regimens as in our study, by incorporating modern chemotherapeutics such as paclitaxel in combination with modern 3-D conformal radiotherapy seems to be promising. Further improvement of the systemic therapy seems to be critical as survival is mainly determined by recurrences at metastatic sites. Data of ongoing phase III trials including novel chemoradiation protocols have to be awaited in the near future.","keyphrases":["paclitaxel","esophageal cancer","neoadjuvant chemoradiation","pathologic complete response","tumor regression grade"],"prmu":["P","P","P","P","P"]}
{"id":"BMC_Genomics-5-_-375527","title":"Assessment of clusters of transcription factor binding sites in relationship to human promoter, CpG islands and gene expression\n","text":"Background Gene expression is regulated mainly by transcription factors (TFs) that interact with regulatory cis-elements on DNA sequences. To identify functional regulatory elements, computer searching can predict TF binding sites (TFBS) using position weight matrices (PWMs) that represent positional base frequencies of collected experimentally determined TFBS. A disadvantage of this approach is the large output of results for genomic DNA. One strategy to identify genuine TFBS is to utilize local concentrations of predicted TFBS. It is unclear whether there is a general tendency for TFBS to cluster at promoter regions, although this is the case for certain TFBS. Also unclear is the identification of TFs that have TFBS concentrated in promoters and to what level this occurs. This study hopes to answer some of these questions.\nBackground\nUnderstanding the regulation of gene expression is a crucial issue in molecular biology. Since gene expression is mainly regulated by transcription factors (TFs), the elucidation of relationships among TFs, their binding sites (TFBS) and their controlling genes, is of great importance.\nAlthough TFBS can be predicted by computer searches on DNA sequences, false positives (FP) are often produced. Several computer programs use position weight matrices (PWMs) [1] to predict TFBS in silico, including MatInspector [2], MATCH [3], and TFBS perl modules [4]. PWMs represent positional base preferences or frequencies constructed by a set of experimentally determined TFBS, and typically correspond to a single TF. The transcription factor database, known as TRANSFAC, is a widely used collection of PWMs [5]. TRANSFAC provides several PWMs for single TFs with different quality levels. Computer programs predict TFBS from DNA sequences, which are the same or similar to known TFBS. The low information contents in the matrices leads to many false positives, due to the weak preference or shortness of the site length (6\u201330 bp). Various strategies have been proposed to allow correct identification of true positives (TPs) from predicted TFBS. One approach is to employ information from conserved regions in DNA sequences between different species, known as phylogenetic footprinting. Bayes block aligner (BBA) is a tool used to extract conserved regions from an alignment of two DNA sequences [6]. It was demonstrated that it could identify binding sites of muscle-specific transcription factors [6]. Another approach is to identify multiple TFBS that form a structural cluster on a DNA sequence coordinate. This seems a reasonable technique because the density of predicted TFBS in promoter sequences is reported to be higher than non-promoter sequences, especially in the region 300 bp upstream from the transcription start site [7]. Genes are regulated by interactions with multiple functional TFs in metazoans [8]. Therefore, many promoter prediction programs, such as promoterscan [9], TSSG, and TSSW [10], have been developed based on the density of TFBS. The identification of genuine TFBS by searching clusters of predicted TFBS has been successful; however, these studies were evaluated with only specific genes and TF sets, such as those found in Yeast[11], Drosophila (early developmental enhancer) [12-14], liver [15], LSF and muscle specific regulatory regions [16,17]. It is unknown whether this method is applicable to other species, or genes. Although many vertebrate promoter sequences have CpG islands (CGI), the relationship between clusters of predicted TFBS and CGI is often underestimated [18]. Another strategy for the identification of putative TFBS includes a combinatorial approach that uses both phylogenetic footprinting and cluster analysis [12,15,19]. The program rVISTA utilizes information from conserved regions between human and mouse, in addition to clusters of TFBS predicted by the MATCH (BIOBASE) program [19]. This approach was evaluated using several known TFs (AP-1, NFAT, and GATA-3) and genes from the cytokine gene cluster. It remains unclear whether the properties used for clusters of TFBS are general and can be applied to other TFs or regulatory regions. Several reports have described methods for determining the statistical significance of predicted TFBS [11,12,17,20-22]. These studies assume the use of appropriate PWMs to identify clustered TFBS. To determine if a particular cluster is genuinely related to the promoter, it is important to assess clusters of predicted TFBS for each individual PWM. This is done using real non-promoter sequences for the appropriate selection of the PWM and for the interpretation of clusters of predicted TFBS. Most of these studies use specific sets of coregulated genes to identify common predicted TFBS clusters, and therefore cannot be applied directly to the study of general properties of promoters.\nIn this study, we developed a measure that evaluates the degree of concentration of predicted TFBS to clarify whether predicted TFBS have a tendency to cluster in human promoter sequences rather than in non-promoter sequences for each PWM. We identified some PWMs in which predicted TFBS clusters occur more significantly in promoter than non-promoter sequences and vice versa. Using partial correlations among three properties (promoters, CGI and clusters of predicted TFBS), we identified two PWM groups, (1) those in which TFBS cluster in promoters as a result of the presence of CpG islands, and (2) those in which TFBS cluster in promoters independent of CpG islands. We show that transcription factors corresponding to the latter PWM group tend to be tissue-specific. In summary, this analysis is useful for the interpretation of predicted TFBS in regulatory regions.\nResults\nDivergent preferences of TFBS for promoter sequences\nWe determined whether predicted TFBS formed clusters in human promoter sequences or in non-promoter sequences for each PWM using the cluster score described in the Method section. The higher the cluster score (derived from a logarithm of the p-value), the stronger the cluster of predicted TFBS is related to the promoter sequence. The threshold T, used to determine whether a cluster of predicted TFBS is found on a sequence, was calculated simultaneously. Since a prediction for the presence of a TFBS was performed for each PWM, an assessment for TFBS clusters was performed using the cluster score for each PWM. As a result, a number of PWMs do not tend to have clusters of TFBS in the promoter sequence. We observed a divergence of cluster scores. Of the 199 vertebrate PWMs in TRANSFAC, 94 (47%) PWMs had significantly high cluster scores, while 22 (11%) PWMs had significantly low cluster scores. The remaining 83 (42%) PWMs did not show significant cluster scores. A p-value of 1.0% was used to identify the above PWM set with Bonferroni correction for multiple testing ([23] Section 3.8). Figure 1 shows a histogram of cluster scores. Although these results were derived from genes on chromosome 20, the results from other chromosomes were similar as described in the following subsection. PWMs with high cluster scores are shown in Table 1. Some of the PWMs have thresholds T (of accumulated score C) equal to or less than 1.0. This indicates that the occurrence of single predicted TFBS is more discriminative than clusters. Sequence logo [24] of the top three PWMs are depicted in Fig. 2-(a). See additional file 1 'PWMs sorted by cluster score' for the entire PWM list.\nFigure 1\nA histogram of cluster scores for PWMs.Each number of X-axis indicates the maximum score of PWMs in the bin.\nTable 1\nTop 50 PWMs for chromosome 20 sorted by cluster score S in descending order. Each column represents rank number, accession number in TRANSFAC, identifier in TRANSFAC, cluster score, and threshold.\nRank\nACCESSION\nID\nS\nT\n1\nM00736\nE2F1DP1_01\n189.3\n2.75\n2\nM00332\nWHN_B\n176.0\n1.90\n3\nM00652\nNRF1_Q6\n122.0\n0.93\n4\nM00649\nMAZ_Q6\n117.2\n4.35\n5\nM00491\nMAZR_01\n111.4\n1.78\n6\nM00739\nE2F4DP2_01\n103.8\n0.93\n7\nM00737\nE2F1DP2_01\n103.6\n0.94\n8\nM00108\nNRF2_01\n81.4\n0.92\n9\nM00665\nSP3_Q3\n72.1\n2.39\n10\nM00706\nTFIII_Q6\n61.4\n4.23\n11\nM00740\nE2F1DP1RB_01\n58.4\n0.90\n12\nM00324\nMINI20_B\n58.2\n1.61\n13\nM00032\nCETS1P54_01\n57.3\n3.70\n14\nM00743\nCETS168_Q6\n51.1\n1.75\n15\nM00341\nGABP_B\n48.6\n0.88\n16\nM00055\nNMYC_01\n41.1\n0.90\n17\nM00329\nPAX9_B\n39.2\n0.73\n18\nM00243\nEGR1_01\n37.3\n0.87\n19\nM00072\nCP2_01\n36.5\n1.66\n20\nM00054\nNFKAPPAB_01\n35.5\n0.85\n21\nM00056\nMYOGNF1_01\n35.1\n1.34\n22\nM00694\nE4F1_Q6\n35.0\n0.86\n23\nM00738\nE2F4DP1_01\n34.9\n0.91\n24\nM00143\nPAX5_01\n34.7\n0.84\n25\nM00235\nAHRARNT_01\n34.6\n0.92\n26\nM00698\nHEB_Q6\n33.6\n0.91\n27\nM00039\nCREB_01\n33.6\n1.00\n28\nM00514\nATF4_Q2\n33.1\n1.71\n29\nM00650\nMTF1_Q4\n31.4\n0.88\n30\nM00194\nNFKB_Q6\n30.8\n0.82\n31\nM00007\nELK1_01\n30.0\n0.85\n32\nM00733\nSMAD4_Q6\n29.7\n0.81\n33\nM00261\nOLF1_01\n28.8\n0.84\n34\nM00017\nATF_01\n26.7\n0.98\n35\nM00053\nCREL_01\n25.6\n0.81\n36\nM00691\nATF1_Q6\n25.5\n0.89\n37\nM00244\nNGFIC_01\n25.2\n0.88\n38\nM00041\nCREBP1CJUN_01\n24.9\n1.00\n39\nM00086\nIK1_01\n24.2\n0.90\n40\nM00287\nNFY_01\n24.0\n1.95\n41\nM00466\nHIF1_Q5\n22.7\n0.90\n42\nM00634\nGCM_Q2\n22.6\n0.84\n43\nM00273\nR_01\n21.8\n0.85\n44\nM00373\nPAX4_01\n21.7\n2.57\n45\nM00097\nPAX6_01\n21.5\n1.15\n46\nM00134\nHNF4_01\n21.1\n0.64\n47\nM00670\nTCF1P_Q6\n21.1\n0.80\n48\nM00057\nCOMP1_01\n21.1\n0.59\n49\nM00035\nVMAF_01\n21.0\n1.32\n50\nM00222\nHAND1E47_01\n20.3\n0.81\nFigure 2\nSequence logos. (a) Top three PWMs from Table 1, (b) representative PWMs from Table 2, (c) representative PWMs from Table 3.\nCluster scores for different datasets\nTo assess the robustness of the cluster score we compared cluster scores for different datasets from chromosomes 20, 21 and 22, respectively. Fig. 3 shows the correlation of cluster scores between chromosomes 20 and 21 (a), and between chromosomes 20 and 22 (b). Some PWMs, the matches of which were detected on less than 50 subsequences, are not shown. The correlation coefficient points were 0.91 (a) and 0.93 (b).\nFigure 3\nTitle: Correlation of cluster scores (a) between chromosomes 20 and 21, (b) chromosomes 20 and 22. Each dot represents a distinct PWM (defined by the TRANSFAC matrix). The correlation coefficients were (a) 0.91 and (b) 0.93.\nCorrelations among promoter sequences, CpG islands, and clusters\nAbout half of the human coding genes have a compositional bias for CGI over transcription start sites [25]. It is possible that some of the predicted TFBS clusters might be the consequence of the existence of a CGI. To investigate this possibility, we computed partial correlation coefficients of three categories of promoters, CGI and predicted TFBS clusters. In general, a partial correlation coefficient measures the correlation between any pair of variables when other, specified variables, have been held constant. For example, a partial correlation coefficient rIC.P is the correlation between I and C while controlling for P, where I denotes CGI, C denotes accumulated score (strength of predicted TFBS clusters, see Methods) and P denotes promoters. If we calculate simple correlation coefficients, rPI = 0.69, rIC ranged from -0.25 to 0.57 for various PWMs, and rPC ranged from -0.24 to 0.53 for various PWMs. These correlation coefficients are apparent ones. The partial correlation coefficients provide essential information and pure correlations, without the effect of the third variable. Fig. 4 shows a plot of rIC.P against rPC.I for various PWMs. For most of the PWMs, rPC.I is positive, although not particularly high (<0.3). This implies a correlation between clusters of these PWM matches and promoter sequences, separate to the effect of CGI. For the PWMs in the right circle in Fig. 4, rPC.I is high and rIC.P is approximately zero, where the cluster is more correlated with the promoter than the CGI. Some PWMs have a negative rPC.I, implying the absence of promoter sequences for these PWM matches. For the PWMs in the top circle in Fig. 4, rIC.P is high and rPC.I is approximately zero, suggesting that the correlation between promoters and clusters for these PWM matches is attributable to the presence of the CGI. While these promoters and clusters do not correlate directly, they appear to correlate because both are associated with CGI. \nFigure 4\nTitle: Plot of rIC.P against rPC.I for various PWMs. The top circle is the area where rPC.I is around zero and rIC.P is high. The right circle is the area where rIC.P is around zero and rPC.I is high. The two circles were drawn manually. Ideal CGI-related and CGI-independent PWMs are to be plotted in the top and right circles, respectively.\nUsing these two values, we identified two PWM sets, (1) a CGI-related set (37 PWMs, Table 2) in which TFBS clusters are correlated with CGI (independent of promoter), and (2) a CGI-independent set (54 PWMs, Table 3) in which clusters of TFBS are correlated with promoters (independent of CGI). These sets were used for the following analysis.\nTable 2\nCGI-related PWMs in descending order of rIC.P = Y. The columns are: rank number, accession number, Identifier in TRANSFAC, rPC.I (=X), rIC.P (=Y), cluster score and threshold.\nRank\nACCESSION\nID\nX\nY\nS\nT\n1\nM00332\nWHN_B\n0.09\n0.43\n158.4\n1.9\n2\nM00736\nE2F1DP1_01\n0.06\n0.39\n151.8\n2.6\n3\nM00739\nE2F4DP2_01\n0.09\n0.29\n91.4\n0.9\n4\nM00737\nE2F1DP2_01\n0.06\n0.27\n81.9\n0.9\n5\nM00108\nNRF2_01\n0.09\n0.25\n72.6\n0.9\n6\nM00055\nNMYC_01\n0.05\n0.25\n34.6\n0.9\n7\nM00235\nAHRARNT_01\n0.02\n0.23\n26.8\n0.9\n8\nM00740\nE2F1DP1RB_01\n0.04\n0.23\n48.1\n0.9\n9\nM00652\nNRF1_Q6\n0.05\n0.22\n105.3\n0.9\n10\nM00466\nHIF1_Q5\n0.01\n0.22\n19.7\n0.9\n11\nM00341\nGABP_B\n0.1\n0.19\n46.6\n0.9\n12\nM00738\nE2F4DP1_01\n0.02\n0.19\n28.6\n0.9\n13\nM00538\nHTF_01\n0\n0.16\n9.7\n0.8\n14\nM00694\nE4F1_Q6\n0.03\n0.16\n23.6\n0.9\n15\nM00743\nCETS168_Q6\n0.13\n0.14\n47.1\n1\n16\nM00650\nMTF1_Q4\n0.04\n0.14\n22.6\n0.9\n17\nM00243\nEGR1_01\n0.07\n0.12\n32.4\n0.9\n18\nM00251\nXBP1_01\n0.01\n0.12\n7.8\n0.9\n19\nM00691\nATF1_Q6\n0.07\n0.12\n17.3\n0.9\n20\nM00236\nARNT_01\n0.02\n0.11\n6.5\n1\n21\nM00143\nPAX5_01\n0.09\n0.11\n25.7\n0.8\n22\nM00273\nR_01\n0.06\n0.11\n23.8\n0.8\n23\nM00244\nNGFIC_01\n0.06\n0.1\n23\n0.9\n24\nM00280\nRFX1_01\n0.06\n0.1\n11.1\n0.9\n25\nM00121\nUSF_01\n0.03\n0.1\n7.6\n1\n26\nM00287\nNFY_01\n0.04\n0.1\n21.3\n1.9\n27\nM00039\nCREB_01\n0.04\n0.09\n23.2\n1\n28\nM00309\nACAAT_B\n0.04\n0.09\n6.8\n0.9\n29\nM00651\nNFMUE1_Q6\n0.03\n0.09\n13\n1.8\n30\nM00017\nATF_01\n0.06\n0.08\n19.2\n1\n31\nM00481\nAR_01\n0.05\n0.08\n7.5\n0.8\n32\nM00041\nCREBP1CJUN_01\n0.04\n0.08\n20.4\n1\n33\nM00040\nCREBP1_01\n0.03\n0.08\n4.7\n0.9\n34\nM00114\nTAXCREB_01\n0.02\n0.06\n7.3\n0.9\n35\nM00279\nMIF1_01\n0.02\n0.06\n10.9\n1.8\n36\nM00246\nEGR2_01\n0.04\n0.06\n9.7\n0.9\n37\nM00085\nZID_01\n0.05\n0.06\n8\n0.8\nTable 3\nCGI-independent PWMs in descending order of rPC.I (=X). The columns are: rank number, accession number, Identifier in TRANSFAC, rPC.I (=X), rIC.P (=Y), cluster score and threshold.\nRank\nACCESSION\nID\nX\nY\nS\nT\n1\nM00491\nMAZR_01\n0.27\n0.15\n117.4\n1.8\n2\nM00706\nTFIII_Q6\n0.24\n0.06\n52.7\n3.5\n3\nM00324\nMINI20_B\n0.22\n0.1\n53.2\n0.8\n4\nM00056\nMYOGNF1_01\n0.22\n0\n31.6\n1.3\n5\nM00649\nMAZ_Q6\n0.21\n0.19\n114.4\n3.7\n6\nM00665\nSP3_Q3\n0.2\n0.14\n67.7\n1.7\n7\nM00032\nCETS1P54_01\n0.19\n0.1\n47.7\n1.8\n8\nM00053\nCREL_01\n0.19\n0.04\n26.9\n0.8\n9\nM00054\nNFKAPPAB_01\n0.19\n0.06\n33.5\n0.9\n10\nM00632\nGATA4_Q3\n0.19\n0.04\n25.1\n0.6\n11\nM00373\nPAX4_01\n0.19\n0.05\n26.1\n0.6\n12\nM00072\nCP2_01\n0.19\n0.08\n32\n0.9\n13\nM00733\nSMAD4_Q6\n0.18\n0.05\n26.3\n0.8\n14\nM00134\nHNF4_01\n0.18\n0.06\n25.7\n0.6\n15\nM00194\nNFKB_Q6\n0.18\n0.02\n28.5\n0.8\n16\nM00445\nXVENT1_01\n0.17\n0.01\n19.9\n0.7\n17\nM00057\nCOMP1_01\n0.17\n0.05\n24.1\n0.5\n18\nM00097\nPAX6_01\n0.17\n0.06\n24.1\n0.5\n19\nM00104\nCDPCR1_01\n0.17\n0.03\n21.3\n0.6\n20\nM00222\nHAND1E47_01\n0.17\n0.02\n20.4\n0.8\n21\nM00626\nEFC_Q6\n0.17\n0.05\n22.6\n0.6\n22\nM00745\nLEF1_Q6\n0.16\n-0.02\n15.9\n0.8\n23\nM00707\nTFIIA_Q6\n0.16\n0.03\n20.2\n0.7\n24\nM00086\nIK1_01\n0.16\n0.06\n24.1\n0.9\n25\nM00329\nPAX9_B\n0.16\n0.1\n33.7\n0.7\n26\nM00478\nCDC5_01\n0.15\n0.03\n19\n0.6\n27\nM00670\nTCF1P_Q6\n0.15\n0.06\n22.7\n0.8\n28\nM00257\nRREB1_01\n0.15\n-0.02\n15.8\n0.8\n29\nM00007\nELK1_01\n0.15\n0.08\n31\n0.8\n30\nM00698\nHEB_Q6\n0.15\n0.08\n28.7\n0.9\n31\nM00052\nNFKAPPAB65_01\n0.14\n-0.05\n9.4\n0.9\n32\nM00514\nATF4_Q2\n0.14\n0.05\n21.8\n1.7\n33\nM00191\nER_Q6\n0.14\n-0.03\n11\n0.8\n34\nM00003\nVMYB_01\n0.14\n0.05\n18\n0.8\n35\nM00261\nOLF1_01\n0.14\n0.07\n24.6\n0.8\n36\nM00490\nBACH2_01\n0.13\n-0.03\n9.3\n0.7\n37\nM00001\nMYOD_01\n0.13\n-0.03\n10.4\n0.9\n38\nM00634\nGCM_Q2\n0.12\n0.05\n19.8\n0.8\n39\nM00035\nVMAF_01\n0.12\n0.06\n17.5\n0.7\n40\nM00340\nETS2_B\n0.12\n-0.08\n5\n0.8\n41\nM00005\nAP4_01\n0.12\n0.01\n14.1\n0.8\n42\nM00701\nSMAD3_Q6\n0.11\n0.03\n11.4\n0.8\n43\nM00531\nNERF_Q2\n0.1\n-0.08\n4.8\n0.9\n44\nM00339\nETS1_B\n0.1\n-0.07\n5.7\n0.9\n45\nM00657\nPTF1BETA_Q6\n0.1\n0\n7.5\n0.9\n46\nM00254\nCAAT_01\n0.1\n-0.01\n6.6\n0.9\n47\nM00118\nMYCMAX_01\n0.09\n-0.02\n6.2\n0.9\n48\nM00693\nE12_Q6\n0.09\n-0.01\n6.5\n0.9\n49\nM00004\nCMYB_01\n0.08\n0\n7.1\n0.9\n50\nM00238\nBARBIE_01\n0.08\n0.02\n9.4\n0.9\n51\nM00648\nMAF_Q6\n0.07\n0.01\n5.8\n0.8\n52\nM00002\nE47_01\n0.06\n0.02\n5.3\n0.9\n53\nM00262\nSTAF_01\n0.05\n0\n9.2\n0.9\n54\nM00119\nMAX_01\n0.05\n0.03\n4.9\n1\nCorrelation between clusters of predicted TFBS and gene expression\nSince all widely expressed, or housekeeping, genes have CGI [25], it is possible that clusters of PWM matches for CGI-independent sets are associated with tissue specific promoters. For this reason we examined the relationship between clusters of PWM matches and the tissue specificity of the associated genes using published gene expression data ([26,27]). The two resources used for this analysis are not consistent. Some genes annotated as housekeeping genes in one resource are referred to as tissue specific in another resource. We refer to these genes as mixed annotated genes. Genes with associated expression data were analysed and of these 72 were identified among the gene set covering the three chromosomes used in this study. They included 12 housekeeping genes, 9 mixed annotated genes, and 51 tissue specific genes. With the CGI-independent PWM sets we detected promoters with clusters of PWM matches. These clusters have significantly high Z-scores (see Methods) based on the accumulated score C in randomly generated DNA sequences (as control) with the same dinucleotide frequency of each promoter sequence. Table 4 shows the 40 genes detected, the DCC score (described below), their tissue specificity and start_p score. These genes are sorted according to the DCC score indicating the extent of association with CGI-independent PWMs over CGI-related PWMs. Results show that tissue specific genes tend to have high DCC scores and that transcription factors corresponding to CGI-independent PWMs are related to tissue specific genes. If we extract 20 genes which have DCC scores equal to or higher than -0.03, 18 (90%) of these are tissue specific genes. The 72 genes with known gene expressions data included 51 tissue specific genes (71%). The p-value of the event of extraction was 0.04 under cumulative hypergeometric distribution. The p-value of the ranking of the two groups (11 housekeeping and 29 tissue-specific) in Table 4 was 0.01 by Wilcoxon rank test. Note that DCC is not correlated with the CGI score (start_p).\nTable 4\nThe gene list sorted by DCC score. The genes, in which clusters of TFBS are found on promoters using CpG-related\/independent PWMs, and tissue specificity, have been previously identified. HK denotes housekeeping. Tissue specific genes can be selected independent of CpG islands (start_p) using DCC score.\n1\nNM006272\n0.43\nbrain\n0\n2\nNM007341\n0.4\nmuscle\n0\n3\nNM002592\n0.37\nbrain\n0.86\n4\nNM001819\n0.27\nbrain\n0.68\n5\nNM004414\n0.23\nkidney\n0.89\n6\nNM002999\n0.19\nkidney\n0.73\n7\nNM003195\n0.16\nbrain\n0.73\n8\nNM002591\n0.14\nliver\n0\n9\nNM000454\n0.11\nHK.liver\n0.87\n10\nNM003312\n0.1\nliver\n0.72\n11\nNM004339\n0.09\nbrain\n0.9\n12\nNM020708\n0.08\nbrain\n0.64\n13\nNM006870\n0.05\nHK\n0.7\n14\nNM003277\n0.04\nlung\n0.74\n15\nNM005194\n0.04\nbrain\n0.86\n16\nNM003610\n0.01\nbrain\n0.76\n17\nNM000355\n-0.03\nkidney\n0\n18\nNM002430\n-0.03\nmuscle\n0.75\n19\nNM006767\n-0.03\nbrain\n0.74\n20\nNM005137\n-0.03\nmuscle\n0.76\n21\nNM003279\n-0.05\nmuscle\n0\n22\nNM004535\n-0.05\nbrain\n0\n23\nNM007019\n-0.05\nHK\n0.72\n24\nNM013236\n-0.07\nHK\n0.69\n25\nNM004175\n-0.07\nbrain\n0.72\n26\nNM001958\n-0.07\nmuscle\n0\n27\nNM001338\n-0.13\nvulva\n0.84\n28\nNM002676\n-0.14\nHK\n0.63\n29\nNM003098\n-0.16\nmuscle\n0.71\n30\nNM002854\n-0.17\nbrain\n0\n31\nNM002305\n-0.23\nHK\n0\n32\nNM005080\n-0.25\nHK\n0.84\n33\nNM001024\n-0.25\nHK\n0.76\n34\nNM021974\n-0.26\nHK\n0.63\n35\nNM014876\n-0.3\nHK\n0.95\n36\nNM001098\n-0.34\nmuscle\n0.65\n37\nNM000071\n-0.37\nliver\n0.8\n38\nNM006198\n-0.37\nbrain\n0\n39\nNM001675\n-0.39\nHK.muscle\n0.8\n40\nNM005423\n-0.68\nbrain\n0\nDiscussion\nClusters of TFBS are an important property of regulatory regions [7,8,19,28]. To determine if this is a general tendency for PWM matches and all protein coding genes, we have developed a measure that evaluates the correlation between predicted TFBS concentrations and promoter sequences. We then examined the correlation for individual PWMs using an unbiased sequence set. Our results show that not all TFBS are clustered in promoter sequences. We found that TFBS clusters corresponding to 47% of PWMs are positively correlated with promoter sequences, and that TFBS clusters corresponding to around 11% of PWMs are negatively correlated with promoter sequences. \nIt is important to ascertain the relationship between cluster scores of PWMs and CGI, because CGI are a prominent feature of promoter sequences. The consensus sequences of the top-ranked PWMs (Table 1) are, 'ANNGACGCTNN' (WHN_B), 'TTTCSCGC' (E2F1DP1_Q6), 'NSGGGGGGGGMCN' (MAZR_01), and 'GGGGAGGG' (MAZ_Q6), where S represents C or G, M represents A or C, and N represents any bases. The sequence logos of the PWMs are depicted in Fig. 2-(a). The G+C % of base composition of each matrix is 70%, 56%, 91%, and 86%, respectively. The sequences with high cluster scores appear to be GC-rich. Larsen et al found that 57% of human genes are associated with CGI, that all housekeeping genes have CGI covering transcription start sites (TSS), and that 40% of tissue specific genes have CGI [25]. Therefore, the association of PWM-PCP with CGI may be significant, and CGI-related PWMs may play important roles in housekeeping regulation. \nTo evaluate the relationship between PWM-PCP and CGI, we calculated the partial correlation coefficient for each PWM. In general, if a correlation coefficient rXY is not small and rXY.Z (defined in Methods) \u2248 0, the probable hypotheses concerning cause and effect will be either 1) the correlation of X and Y is a consequence of Z, or 2) Z intervenes between X and Y. For the PWMs in the top circle in Fig. 4, rIC.P is high and rPC.I is approximately zero. This suggests that the correlation between promoters and TFBS clusters is attributable to the presence of the CGI and that while they do not directly correlate they appear to because both independently correlate with CGI. The characteristic PWMs in Table 2 are NMYC_01 (M00055:0.25), AHRARNT_01 (M00235:0.23), and HIF1_Q5 (M00466:0.22), where parentheses include the accession number referred to in TRANSFAC and the recorded rIC.P (Y-value in Fig. 4). Sequence logos are depicted in Fig. 2-(b). The PWMs in the middle right circle in Fig. 4 have an rIC.P of approximately zero and a high rPC.I  rPC.I value showing that the cluster is correlated with promoters independent of CGI. The predicted TFBS clusters corresponding to these PWMs could not be explained by the presence of CGI. Some of these PWMs have thresholds T less than 1.0 indicating that even the single occurrence of a predicted TFBS is more discriminative than clusters. Particular examples with high recorded rPC.I values and values for rIC.P < 0.1 are TFIII_Q6 (M000706), MYOGNF1_01 (M00056) and CREL_01 (M00053). Sequence logos are depicted in Fig. 2-(c). TFIII_Q6 is a matrix associated with a general transcription factor II-I with the consensus sequence RGAGGKAGG, where the K represents G or T. The matrix TFIII_Q6 contains many 'G', and 'C' is allowed only the fourth position with low frequency. MYOGNF1_01 is a matrix associated with myogenin, nuclear factor 1 or related factors, and is therefore involved in the regulation of differentiation. CREL_01 is a matrix associated with the C-Rel proto-oncogene protein (C-Rel protein). An understanding of the function of these factors is important to this study. The PWM groups described above may be involved in tissue-specific gene regulation. If all housekeeping genes have CGI [25] then genes without CGI can be assumed to be tissue-specific or rarely expressed. Thus, genes with a cluster of predicted TFBS not associated with CGI might be associated with tissue-specific regulation. Further analysis of extractions of tissue specific genes, shown in Results, supports the hypothesis.\nResults from this analysis provide a solution to the promoter prediction problem. Hannenhalli et al. used additional information, including profiles of TF binding sites, for promoter prediction based on CGI [29], with no significant improvement to prediction performance. The report using 7 manually selected PWMs confirmed that CGI are the most dominant feature. Our results show that Sp1 and ATF have a strong correlation with CGI; a result consistent with their result that information including both PWM did not improve prediction accuracy. This observation is consistent with other PWMs. More stringent selection of PWMs is required for an improved accuracy of promoter prediction. One strategy is to utilise the CGI-independent PWMs identified in this study. Another problem is exemplified by the under-representation of Oct-1 (M00138) in the (-600:600) region of the human promoter and the absence of positional preferences [29]. This under-representation was not expected but is observed in 10% of known PWMs. OCT1_04 (M00138) is not in the high quality list of TRANSFAC, OCT1_01 (M00135) and OCT1_C (M00210) was found to have minus cluster scores (-0.63 and -2.89) in our table (additional file 1).\nIt is noteworthy that Fig. 5 shows TFBS (AP2_Q6) in non-promoters to occur randomly under a certain distribution. This distribution can be modelled by a binomial probability distribution. A model of Poisson distribution, which is an approximation of binomial probability distribution for a certain condition, was proposed in [11] as the probability distribution of TFBS density. Although we have not tested the goodness-of-fit, our observation does not contradict the Poisson distribution model. \nFigure 5\nTitle: Distribution of accumulated score C for promoters and non-promoters for AP2_Q6\nTo assess the robustness of the cluster score, we compared cluster scores for different datasets from chromosomes 20, 21 and 22 (Fig. 3). The correlation coefficients were 0.91 (a) and 0.93 (b), proving that the significance would be similar if we utilized the whole human genome dataset in the analysis. The scale of the figures between the Y-axis and X-axis are different because of the different number of sequences taken from each chromosome.\nConclusions\nWe have developed a measure that statistically evaluates the degree of concentration of predicted TFBS in promoter sequences. Using this strategy to analyse various PWMs we have determined that predicted TFBS tend to cluster in human promoter sequences rather than in non-promoter sequences. Our results show that local concentrations of predicted TFBS in human promoter sequences are not a general characteristic of PWMs. Only a portion of identified PWM matches corresponded to TFBS occurring in clusters in promoter sequences. By computing partial correlation coefficients, we identified PWM sets associated with CGI and others that are independent of CGI. Transcription factors and binding sites associated with CGI-independent PWMs are likely to be involved in tissue-specific gene regulation. Indeed, using the CGI-related\/dependent PWM sets, we extracted tissue-specific genes with high accuracy by detecting clusters of predicted TFBS. These results will be useful to interpret predicted transcription factor binding sites and to further understand the role of their formation into clusters. Ultimately, these findings will further elucidate the various functions of promoters, genes and transcription factors.\nMethods\nData\nDNA sequences from the fully sequenced chromosomes (chromosomes 20, 21 and 22) were taken from the November, 2002 GenBank freeze (build 31) and assembled by NCBI, in accordance with the annotation of the UCSC genome browser [30]. RefSeq [31] genes were used as they have been reviewed by NCBI staff, are well studied, and are unlikely to be spurious. Some genes in the human genome have alternative promoters [32], complicating our analysis. For this reason, overlapping genes identified using the UCSC annotation were discarded. This check of RefSeq genes reduced the number of genes in the analysis from 527 to 373 for chromosome 20, 224 to 142 for chromosome 21, and 449 to 294 for chromosome 22. The resultant gene set U consists of 809 genes. \nTo increase the accuracy of the annotation of transcriptional start sites, we modified the annotation of RefSeq according to DBTSS (version 2, Mar 2002) [33], a database of transcriptional start sites for 5' end mRNA sequences. Suzuki et al. reported that a certain portion of sequences in DBTSS were longer (extended) toward 5' end of mRNA sequences than those in RefSeq [33]. We describe how the modification improved the first gene set U. Fig. 6 shows the composition of different gene collections. The RefSeq database is updated daily by increasing the number of entries and correcting others. For illustration purpose, two versions of RefSeq are shown in Fig. 6. The old RefSeq is the version analysed by Suzuki et al. The new RefSeq is the current version used in this study. Of the 217,402 sequences contained in DBTSS 7,889 correspond to sequences in RefSeq and are referred to as cloned RefSeq sequences. The extension rate, defined as the rate of extension of mRNAs sequences from cloned RefSeq by DBTSS, was 0.34. Therefore, |{D,G}| (the number of gene set {D,G}) = 7,889 genes, |{Dex,Gex}| = 2,683 genes and |{Dex,Gex}|\/|{D,G}| = 0.34. The ftp site of DBTSS provides the set of extended mRNA sequences (Iftp = {Dex,Gex}). The gene set from chromosomes 20, 21 and 22 is a partial set U = {Cu, Eu, Fu, , } from New RefSeq. We can identify the genes in set  as the conjunction of Iftp and U. The number of  was counted (273 genes). If the extension rate of mRNAs sequences for {, } is also 0.34, then |{}|\/|{, }| = 0.34. Therefore, {|, }| is estimated to be 802.9 genes. As |U| = 809, the number of genes in V = {Cu,Eu,Fu} is estimated to be 6.1 genes. If all human genes are cloned by the cap-targeted selection method called oligo-capping, a greater number will have extended 5' ends. If this were the case then 34% (or 2.1 genes) of V would have extended 5' ends. Thus, any further correction of TSS for our gene set is expected to be quite small.\nFigure 6\nA Venn diagram of three gene sets (DBTSS, old RefSeq, and new RefSeq). Gene sets from A to G (Bold alphabet) consist of genes in the regions bounded by the thick lines. D consists of Dn (genes whose 5' end sequences were not extended from the old RefSeq sequences with DBTSS data) and Dex (genes whose 5' end sequences were extended). G consists of Gn (genes whose 5' end sequences were not extended from the old RefSeq sequences with DBTSS data) and Gex (genes whose 5' end sequences were extended). Namely D  = {Dn,Dex} and G = {Gn,Gex}. Genes in chromosomes 20, 21, 22 were denoted by U = {Cu, Eu, Fu, , }. Gene sets Cu, Eu,Fu,  and  are parts of C, E, F, Gn and Gex, respectively. Some of the numbers of the sets are given in [33], that is, |{D,G}| = 7889, |{Dex,Gex}| = 2683 and |{Dex,Gex}| \/ |{D,G}| = 0.34, where |{D,G}|  denotes the number of genes in set {D,G}.\nWe identified the conjunction set  (273 genes) of the above collected 809 RefSeq genes and 2,683 genes in DBTSS. The set  was examined to determine if extended sequences existed on human genome sequences and if they are registered in the new RefSeq. Of this set, the BLAT program[34] identified 30 genes in which the 5' end sequences could not be detected. Due to the uncertainty of TSS these genes were not used in this study. Forty-one DBTSS mRNA sequences from  were shorter than corresponding sequences in the new RefSeq with regard to 5' end sequences. It is assumed that these RefSeq sequences were corrected following the old RefSeq release independent of DBTSS and were used as they were. Finally, we modified the exon annotation of 202 genes according to DBTSS. \nWe extracted promoter sequences at relative positions (-600:-1) from the TSS, and intron subsequences 600 bp in length from genome sequences. Only intron sequences were used for the non-promoter sequence data sets as exon sequences are known to have preferences in their oligomer statistics, such as G+C % and codon bias [35]. The first intron was not included in the data set as although regulatory elements are rare in introns, intron 1 occasionally contains regulatory elements such as enhancers. We investigated the frequency of enhancers in human introns by searching NCBI PubMed [36] with the keywords 'human', 'first intron', and 'enhancer'. This search yielded 194 papers. Replacing the keyword 'first intron' with 'second', 'third', 'fourth intron', 'fifth' or 'last intron' yielded 40, 15, 1, 1 and 6 papers, respectively. Replacing 'enhancer' with 'silencer' resulted in 281, 6, 3, 0, 0 and 0 papers, respectively. Removal of intron 1 from the data set greatly reduces the overall occurrence of regulatory elements in human intron sequences and allows our statistical analysis to be performed without significant interference from intronic regulatory sequences. Intergenic sequences are left out of the non-promoter dataset due the unknown occurrence of regulatory sequences.\nPrediction of TFBS\nEach promoter or non-promoter sequence was scanned by the MATCH program using 423 matrices in TRNASFAC version 6.3 (a transcription factor database) with options including 'vertebrate', 'minimize false negatives' (in cut-off selection) and 'use high quality matrices only'. As Kel et al. described, the cut-off was determined so that the false negative rate is 10% [3]. The option 'use high quality matrices only' uses approximately 70% of matrice [3]. Any PWM in the 'high quality' PWMs meet the criteria; When the PWM is used with a cut-off value which allows a false negative rate of 50%, then the match rate dropped below 1 match\/kb in exon2 sequences [3]. If more than one matrix was matched to same transcription factor (prefix of \"Identifier\"), we selected a representative matrix with the highest quality and smallest suffix number according to the TRANSFAC definition. After scanning the sequences by MATCH, we set consecutive sampling windows (600 bp) in introns and promoter sequences, and then recorded corresponding TFBS predictions. To prevent double counting of palindromic binding sites, two matches for the same matrix at the same position was regarded as a single match and the match with the higher score was taken. Before MATCH ran, repeat sequences were masked to 'N' according to the annotation by RepeatMasker in the UCSC genome browser. From the above analysis we extracted 361, 129, and 278 promoter sequences from chromosomes 20, 21 and 22, respectively. The promoter sequences identified contained repeat sequences (e.g. ALU, L1) and simple repeats with low complexity, as observed in intron sequences. These sequences account for about 20% of all bases. To balance the rate of repeats between promoters and introns, we discarded intron sequences with high rates of repeats, so that the average rate of repeats in the intron samples was at the same level as in promoter sequences. The number of 600 bp intron sequences included in the analysis was 6,589 (chromosome 20), 4,324 (chromosome 21) and 4,531 (chromosome 22).\nAccumulated scores of TFBS\nWhen predicted TFBS occur many times in a sequence there is a high probability that it contains functional regulatory regions or promoters [7,8,19,28]. We tested this hypothesis for individual PWMs. The degree of concentration of predicted TFBS in a sequence was defined as the accumulated score C, which is a summation of the MATCH score for PWMs in the subsequence and is calculated for each PMW and corresponding sequence. C is assumed to be almost proportional to the frequency of predicted TFBS for the corresponding PWM. Many sequences generate different C values although some are identical. We then generated a series of Cj (j = 1 ... n) values for a PWM, where n is the number of different C values. Fig. 5 shows the histogram of C for promoters and non-promoters respectively, using the TRANSFAC matrix of identifier 'AP2_Q6' as an example. Since C reflects the number of predicted TFBS found in a sequence, the figure shows the density of predicted TFBS in a sequence. This result is similar to the density plot described by Pestridge and Burks [7], although our figure (Fig. 5) is not a plot of predicted TFBS density for mixed PWMs, but instead is a plot of predicted TFBS density for individual PWMs. Also, the X-axis in our plot does not indicate the number of predicted TFBS but instead indicates the accumulated score C. The Y-axis is smoothed by averaging for the width of 5 in C value.\nCluster score and statistical significance for a PWM\nSignificance values for an individual PWM from a series of Cj can be determined from a contingency table. Table 5 shows a contingency table for the number of promoters and non-promoters above and below the threshold Cj for a given PWM. From this table, \u03c72 value for a given Cj is defined as \nTable 5\nA contingency table when predicted TFBS and a threshold T are given.\nSequences where TFBS clusters found\nSequences where TFBS clusters not found\nSum\n# of promoter\nA\n1\nA\n2\nA\n# of non-promoter\nB\n1\nB\n2\nB\nwhere\ndescribed in [37]. From the \u03c72 value, we computed the probability P that the \u03c72 value or greater is obtained by chance. The probability P was calculated from the \u03c72. Since P is calculated for many PWMs, we must deal with the problem of multiple testing. Using the Bonferroni correction [23], Pn was calculated using the formula 1 - Pn = (1 - P)n, approximately Pn = P \u00d7 n for small P \u00d7 n. The n is the number of PWMs. When we determine the set of significant PWMs, Pn were compared with the significance level (i.e. 0.01). We also defined the statistical significance Qj as Qj = -log10(Pn) if Rprom >Rnonprom and Qj = + log10(Pn) otherwise, where Rprom = A1\/A (a rate of sequences in promoters where clusters found), Rnonprom = B1\/B (a rate of sequences in non-promoters where clusters found). Although the P is an indicator of the difference between the occurrence of promoters and non-promoters, the probability P itself does not represent the preferences of PWMs for promoters. To represent the preference of predicted TFBS for or against promoters, we add signs for statistical significance Qj. Positive Qj indicates that predicted TFBS tend to appear frequently in promoters, while negative Qj indicates that predicted TFBS tend to avoid promoters. \nWe studied how statistical significance Qj varies with the threshold of C j. Fig. 7 shows the presence of a peak of Qj when we change the threshold. We define the cluster score S of a PWM in such a way that the significance is the maximum, namely\nFigure 7\nTitle: significant score Qj of matrix AP2_Q6 for different thresholds.\nWe simultaneously define a unique threshold T of the PWM by\nFor the all-vertebrate TRANSFAC PWMs, we determined thresholds T and calculated significance scores (or cluster scores) S. The highest scoring PWMs are listed in Table 1.\nCorrelations among promoter sequences, CpG islands, and clusters\nFor every 600 bp sequence, three numerical features (promoter, CGI, and clusters) were annotated. CGI were identified using the CpGProD program [38] for original long sequences (not for short sequences of 600 bp). Regions larger than 500 bp with a G+C % equal to or greater than 50% and 'observed CpG \/ expected CpG' equal to or greater than 0.60 were classified as CGI [38,39]. The CpGProD program outputs 'start_p' scores for the predicted CGI. This score indicates the probability that the region is a CGI located over a transcription start site (start CGI). Short 600 bp sequences sampled from long sequences containing CGI were annotated as CGI if the overlapping CGI region was longer than 300 bp. The accumulated score C was used for cluster annotation. From sequences with feature annotation, the correlation coefficients between every two of the three features were computed for each PWM by the statistical language R [40]. We use P to denote whether the sequences is promoter or not, namely P = {1,0}, and I to the denote 'start_p' score for CGI calculated using the CpGProD program [38]. A partial correlation coefficient for each PWM was calculated using the subsequences. For example, a partial correlation coefficient rPC.I is the correlation between P and C while controlling for I, defined by \nwhere rPI is a correlation coefficient between variable P and I, rPC is a correlation coefficient between variable P and C and rCI is a correlation coefficient between variable C and I. A partial correlation coefficient differs from a correlation coefficient. If the correlation between P and C depends entirely on the common cause I, then when I is constant, the correlation between P and C should be zero. The partial correlation rPC.I expresses such a relationship. Even when I varies, rPC.I is expected to be zero in such a situation, while the correlation coefficient rPC may not be zero. See chapter 16.4 in [41] for details. \nFig. 4 shows a plot of rIC.P against rPC.I for various PWMs. Using these two values, we identified two PWM sets including, (1) a CGI-related set consisting of 37 PWMs in which the clusters are correlated with CGI independent of promoters, and (2) a CGI-independent set consisting of 54 PWMs, in which the clusters are correlated with promoters independent of CGI. The CGI-related set requires that rIC.P >rPC.I and that the partial correlation coefficient (PCC) rIC.P is significantly high (p < 0.01) under the hypothesis that rIC.P is zero (see below). The CGI-independent set requires that rIC.P <rPC.I and that rPC.I is likewise significantly high. To calculate the statistical significance of PCC, a PCC r was subjected to z-transformation defined as\nThe values of z are supposed to be normally distributed and the expected variance is \nwhere n is the sample size [41]. The CGI-related PWMs and CGI-independent PWMs are listed in Tables 2 and 3.\nGene expression data\nTo examine the relationship between clusters of predicted TFBS and the tissue specificity of the genes where clusters were found, we generated a list of genes with expression data. This list includes 535 housekeeping or maintenance genes expressed in 11 human adult and foetal tissues from [26], and 451 housekeeping or maintenance genes available at HugeIndex database [27]. We then identified 581 non-redundant housekeeping genes and 'tissue-selective' genes, which are predominantly, but not exclusively, expressed in one tissue type. Tissue-selective genes were expressed in brain (589 genes), kidney (129 genes), liver (271 genes), lung (68 genes), muscle (302 genes), prostate (45 genes) and vulva (95 genes) [27]. These genes corresponded to 2,069 RefSeq entries. Seventy-two of these genes were identified in our gene set covering chromosome 20, 21 and 22 and were used for further analysis.\nTissue specific gene detection based on clusters of predicted TFBS\nUsing the two PWM sets described above, we searched clusters of predicted TFBS in promoter sequences. We calculated C and statistical significance of C as follows. For each promoter sequence, 30 random 600 bp sequences were generated under the first order Markov model, which is based on dinucleotide frequency and can identify promoter CpG bias and G+C%. The MATCH program was run with given matrix thresholds. The accumulated score C of PWMs was computed in every random sequence. A mean  and a variance  of C for each PWM were estimated from the random sequences. Then, for the given promoter sequence Sp and the PWM (M), we can run MATCH and calculate C and its significance score (Z-score), namely \nIf the Z-score value was above three, the promoter sequence was taken and considered together with the PWM. Table 4 lists genes identified with clusters of predicted TFBS with significant C values for the CGI-related\/dependent PWM set. The computation of p-value of cumulative hypergeometric distribution was performed using the AS R77 algorithm [42].\nAuthors' contributions\nKM designed the study and carried out statistical analysis. TK participated in the design and carried out functional analysis. YS directed the study. All authors read and approved the final manuscript.\nSupplementary Material\nAdditional File 1\nThe list of PWM-PCP\/NCP sorted by cluster score. Each column represents rank number, accession number in TRANSFAC, identifier in TRANSFAC, cluster score, threshold.\nClick here for file","keyphrases":["promoter","tissue-specific gene expression","position weight matrix","regulatory motif"],"prmu":["P","R","R","M"]}
{"id":"Crit_Care-9-3-1175868","title":"Bench-to-bedside review: Inotropic drug therapy after adult cardiac surgery \u2013 a systematic literature review\n","text":"Many adult patients require temporary inotropic support after cardiac surgery. We reviewed the literature systematically to establish, present and classify the evidence regarding choice of inotropic drugs. The available evidence, while limited in quality and scope, supports the following observations; although all \u03b2-agonists can increase cardiac output, the best studied \u03b2-agonist and the one with the most favourable side-effect profile appears to be dobutamine. Dobutamine and phosphodiesterase inhibitors (PDIs) are efficacious inotropic drugs for management of the low cardiac output syndrome. Dobutamine is associated with a greater incidence of tachycardia and tachyarrhythmias, whereas PDIs often require the administration of vasoconstrictors. Other catecholamines have no clear advantages over dobutamine. PDIs increase the likelihood of successful weaning from cardiopulmonary bypass as compared with placebo. There is insufficient evidence that inotropic drugs should be selected for their effects on regional perfusion. PDIs also increase flow through arterial grafts, reduce mean pulmonary artery pressure and improve right heart performance in pulmonary hypertension. Insufficient data exist to allow selection of a specific inotropic agent in preference over another in adult cardiac surgery patients. Multicentre randomized controlled trials focusing on clinical rather than physiological outcomes are needed.\nIntroduction\nDespite improvements in surgical technique and myocardial protection, pharmacological support for low cardiac output is often required during and after weaning from cardiopulmonary bypass (CPB) [1]. This acute deterioration in ventricular function may continue into the postanaesthesia care unit or intensive care unit (ICU). Because cardiac surgery is conducted in an increasingly aged population, with coexisting pathology, these patients are at increased risk for developing a low cardiac output syndrome (LCOS) during the postoperative period.\nThere is no consensus definition of what constitutes LCOS, but it would be reasonable to define it as a low cardiac output (cardiac index [CI] < 2.4 l\/min per m2 is used as a criterion in some studies) with evidence of organ dysfunction, for example elevated lactate or urine output under 0.5 ml\/hour for more than 1 hour. Such LCOS can persist for several hours to days, despite optimization of volume status, temporary pacing, or exclusion of mechanical factors (e.g. cardiac tamponade and mechanical assistance with intra-aortic balloon counter-pulsation). Causes are multifactorial but include myocardial ischaemia during cross-clamping, reperfusion injury, cardioplegia-induced myocardial dysfunction, activation of inflammatory and coagulation cascades, and unreversed pre-existing cardiac disease. LCOS can result in reduced oxygen delivery to vital organs [2]. Organ dysfunction and multiple organ failure are among the main causes of prolonged hospital stay after cardiac surgery, and this increases resource use and health care costs as well as increasing morbidity and mortality. Optimization of cardiac output and oxygen delivery may decrease morbidity and reduce length of stay [3].\nDespite a wide range of available inotropic agents, no consensus exists regarding the treatment of LCOS post-CPB. This review examines the pharmacological options for providing inotropic support in the period after CPB and evaluates the literature systematically in order to establish, present and classify the available evidence regarding the use of inotropic drugs after cardiac surgery in adults. We do not discuss exclusive or mostly vasopressor drugs such as vasopressin and norepinephrine (noradrenaline).\nMethods\nWe conducted a systematic Medline and PubMed search, over the period 1982\u20132003, using the following keywords: cardiac surgery, cardiopulmonary bypass, coronary artery bypass grafting, inotropic support, epinephrine, dopamine, dopexamine, dobutamine, amrinone, enoximone, milrinone and levosimendan. Agents considered to be primarily vasopressors (e.g. norepinephrine, arginine vasopressin and phenylephrine) and mechanical support (e.g. intra-aortic balloon counter-pulsation and assist devices) are not considered. For reasons of space and the likelihood that they would behave like other agents in their class, the more obscure phosphodiesterase inhibitors (PDIs; e.g. olprinone) not in common usage in the UK and Australia are not considered.\nThe bibliographies of articles identified through this methodology were also studied for reports that might have been missed in our initial searching of electronic reference libraries. Non-English language papers, animal studies, paediatric studies and in vitro studies are not included. Using this search strategy we identified 210 papers. This selection was further refined to 142 reports in which the agent in question was used for support of cardiac function or vital organ perfusion in patients who had undergone cardiac surgery. All articles in question were obtained.\nPapers were selected and graded for quality of evidence according to the methodology of Cook and coworkers [4] (Table 1). Particular attention was given to the following issues regarding each agent in patients who have undergone cardiac surgery: what are the effects of each inotropic drug on systemic haemodynamics?; does the inotropic drug alter vital organ perfusion?; does the inotropic drug affect major clinical outcomes (e.g. time spent in hospital or ICU or requiring ventilation or artificial renal support) or survival?; and does the inotropic drug have any important side effects?\nData covering the application of each therapy were examined. Where possible, 'evidence-based' recommendations were developed.\nResults\nThe results of our literature search are considered by pharmacological groups and agent. A full pharmacological profile of each agent is beyond the scope of the present review, but the proposed cellular mechanisms of action and receptor activation for each agent are schematically summarized in Fig. 1.\nCatecholamines\nNatural and synthetic catecholamines have different haemodynamic effects because of their differential abilities to stimulate adrenergic receptors. Accordingly, each must be considered separately.\nEpinephrine\nEpinephrine (adrenaline) is a naturally occurring catecholamine that binds to both \u03b1- and \u03b2-receptor subgroups, with \u03b2 effects predominating at low doses and \u03b1 effects predominating at high dose. Fifteen reports relating to the use of epinephrine in cardiac surgical patients were retrieved using our search strategy. No study yielding 'level I' evidence (Table 1) was identified. Only one uncontrolled study, that by Gunnicker and coworkers [5], specifically investigated its effectiveness in LCOS.\nIn that report [5], epinephrine at a dose of 0.03 \u03bcg\/kg per min produced significant increases in CI and heart rate (HR) of 24.1% and 14.1%, respectively, compared with placebo. HR was minimally affected in all studies except that of Gunnicker and coworkers, in which it increased by 14%. All studies recorded significant increases in mean arterial pressure (MAP) [6]. A recent observational study on the effects of 5 \u03bcg boluses in patients undergoing cardiac surgery [7] revealed a biphasic effect on systemic vscular resistance (SVR), with an initial increase followed by reduction.\nEpinephrine has also been directly compared with amrinone, milrinone and dobutamine. Two small, randomized controlled trials [5,8] compared epinephrine with the PDI amrinone. In both studies, both drugs significantly increased CI from baseline. In the randomized, open-label trial conducted by Gunnicker and coworkers [5] in 20 patients with LCOS, epinephrine produced a significantly greater increase in CI, HR and MAP than did amrinone. However, this was accompanied by significantly greater increases in myocardial workload and oxygen consumption. Lobato and coworkers [9] conducted a prospective, randomized, blinded trial comparing the myocardial relaxation effects of epinephrine with those of milrinone in patients undergoing elective coronary artery bypass grafting (CABG). Epinephrine (0.03 \u03bcg\/kg per min) had no effect on left ventricular end-diastolic area, as measured using trans-oesophageal echocardiography, whereas milrinone significantly increased it by 15%. Two studies [10,11] compared epinephrine with dobutamine and found epinephrine to be less effective at increasing CI than dobutamine over what the authors considered a reasonable clinical range of doses (0.01\u20130.03 \u03bcg\/kg per min for epinephrine and 2.5\u20135 \u03bcg\/kg per min for dobutamine). However, dobutamine was associated with significantly more tachycardia for the same stroke volume index (SVI).\nNo studies were found regarding the effect of epinephrine on blood flow to vital organs. A prospective, randomized trial [12] showed that epinephrine produces lactic acidosis in some post-CPB patients.\nTwo randomized controlled trials, one of which used laser Doppler flowmetry, investigated the effect of epinephrine on internal mammary artery (IMA) graft flow [13,14]. Both of these studies found epinephrine to have no effect on IMA blood flow. An earlier crossover study of 28 patients [15], using an electromagnetic flowmeter, had shown that epinephrine significantly increased flow through IMA and saphenous vein grafts.\nNo studies were found regarding the effect of epinephrine on major clinical outcomes or survival.\nDopamine\nDopamine is a naturally occurring catecholamine that binds to both \u03b1- and \u03b2-receptor subgroups, with \u03b2 effects predominating at low dose and \u03b1 effects predominating at high dose. Doses of 2\u201310 \u03bcg\/kg per min are commonly used for inotropy, with doses of 1.5\u20133.0 \u03bcg\/kg per min still used by some for renal protection ('renal dose') because of the binding of the drug to specific dopaminergic receptors in the kidney. Our literature search identified a total of 21 papers relating to the use of dopamine in cardiac surgical patients, all of which were retrieved.\nNone of these papers specifically compared the haemodynamic effects of dopamine with those of placebo. When the effects of dopamine on CI were compared with baseline data, dopamine at a dose of between 2.5 and 5.0 \u03bcg\/kg per min produced significant increases in CI (range 16.3\u201357.9%). In all studies except one there was a significant rise in HR (range 4.5\u201345.7%).\nAt doses of up to 5 \u03bcg\/kg per min, significant decreases in SVR (range 13.1\u201346.1%) were recorded. However, in 1982 Saloman and coworkers [16] conducted a prospective, randomized, blinded trial of 20 patients and found that increasing dopamine from 5.0 to 7.5 \u03bcg\/kg per min caused significant increases in MAP and pulmonary vascular resistance (PVR) without increasing cardiac output. In a multicentre, prospective, blinded, randomized trial of 70 patients, Rosseel and coworkers [17] examined the use of dopamine in LCOS after cardiac surgery. The study compared dopamine with dopexamine in patients with CI below 2.2 l\/min per m2. Dopamine produced a 57.9% increase in CI compared with baseline. However, this was accompanied by a 25.5% increase in HR. Clinical efficacy (defined as CI > 2.5 l\/min per m2 and urine output > 0.5 ml\/kg per hour) was significantly greater in the dopexamine group at 1\u20132 hours after commencement of the infusion and approached significance at other time points. Moreover, 63% of patients in the dopamine group had an adverse cardiac event (defined as arrhythmias, ischaemia and hypertension), which was significantly greater than with dopexamine.\nTarr and coworkers [18] compared the efficacies of dopamine, dobutamine and enoximone for weaning from CPB in a randomized trial of 75 patients. Nine of the 25 patients randomly assigned to dopamine failed to respond adequately, and the remaining 16 recorded an increase in CI of 25.7% but this was accompanied by an increase in HR of 44.3%, with little change in SVI. The CI in the dopamine treated group was significantly lower than in patients treated with either dobutamine or enoximone.\nDopamine has been studied extensively with regard to regional perfusion of the gut and kidney. Other than a case series of 15 patients reported by Davis and coworkers in 1982 [19], which suggested that low-dose dopamine might increase postoperative urine output and serum creatinine in CPB patients, several level II studies [20-23] have failed to provide any evidence to support its use. Jakob and coworkers [24,25] and Thoren and colleagues [26] conducted observational studies on the effect of dopamine on splanchnic perfusion using indocyanine green (ICG) dye clearance and laser Doppler flowmetry, respectively. They observed significant increases in splanchnic blood flow in the order of 27\u201336%. Two level II studies [27,28] failed to demonstrate any effect of dopamine on gastric intramucosal pH (pHi). A significant worsening in pHi associated with low CPB flow rate and dopamine was observed by Schneider and coworkers [29] in a randomized, double-blind, placebo-controlled trial (n = 100) conducted in 1998.\nNo data were found regarding the effect of dopamine on major clinical outcomes or survival.\nDobutamine\nDobutamine is a synthetic catecholamine and is a derivative of isoprenaline. It has strong affinity for \u03b2-receptors with little affinity for \u03b1-receptors because of the configuration of the terminal amine. Twenty-six studies investigating the effects of dobutamine in cardiac surgical patients were identified and retrieved. These studies are summarized in Table 2.\nAdministration of dobutamine in cardiac surgery patients produces a dose-dependent rise in CI. In the study conducted by Ensinger and coworkers [31], in which they compared dobutamine at 6.0 \u03bcg\/kg per min with placebo, a significant increase in CI of 46% was recorded. Studies by Feneck and coworkers [2] and Tarr and colleagues [18], investigating the haemodynamic effects of dobutamine in LCOS, identified increases in HR in excess of 25%. Significant reductions in SVR (> 40% in the study by Tarr and coworkers) were also recorded.\nRomson and coworkers [32] conducted an observational study of 100 patients who had undergone cardiac surgery and were administered dobutamine at doses of 0\u201340 \u03bcg\/kg per min, where tolerated, and compared these with 10 control patients who received no dobutamine. Those investigators found that HR increased by an average of 1.45 beats\/min per \u03bcg\/kg per min in patients who were able to receive the full dose (66 out of 100 patients). Of the patients who were unable to receive the full dose, more than half (52%) developed tachycardia greater than 85% of predicted maximum HR by age. Romson and coworkers concluded that, in post-CPB patients, the dominant method of increasing CI was by increasing HR.\nThe Milrinone Multicentre Trial Group provided the most recent randomized controlled trial data concerning dobutamine [2]. That multicentre, randomized but not blinded study compared the haemodynamic effects of dobutamine with those of milrinone. A total of 120 patients with CI below 2.0 l\/min per m2 were studied and dobutamine was used at doses of 10\u201320 \u03bcg\/kg per min. Dobutamine increased CI by 55% versus 36% with milrinone at 1 hour, and this effect was accompanied by a 35% increase in HR (versus 10% with milrinone) and a 31% increase in MAP (versus 7% with milrinone). Dobutamine was also associated with significantly higher incidences of hypertension and new atrial fibrillation (18% versus 5%; P < 0.04).\nThe randomized trial of 75 patients conducted by Tarr and coworkers in 1993 [18] identified no statistically significant difference in CI between enoximone and dobutamine (both drugs effectively increased CI). However, dobutamine produced significantly more tachycardia, and enoximone produced significantly greater increases in SVI. A further five small randomized trials compared dobutamine with enoximone [33,41-44], but only one of these studies [44] demonstrated any difference between drugs, specifically a significantly greater increase in CI in the enoximone-treated group.\nTwo small randomized trials [34,39] compared dobutamine with amrinone and found no significant differences in haemodynamic effect. Dupuis and coworkers [39], however, did note an increase in incidence of arrhythmias in the dobutamine group, and 40% of patients treated with dobutamine suffered postoperative myocardial infarction versus none in the amrinone group (P = 0.017).\nRegarding comparisons with other catecholamines, MacGregor and coworkers [37] conducted a randomized, blinded comparison of dopexamine and dobutamine in 10 patients undergoing CABG. No significant differences in haemodynamic variables were found, but there was a significantly greater incidence of supraventricular tachycardias in the dopexamine group. As mentioned above, Butterworth and coworkers [10] found no significant differences between epinephrine and dobutamine other than a significantly greater HR in the dobutamine-treated group.\nSix studies investigated the effects of dobutamine on regional perfusion. The study by MacGregor and coworkers [37], outlined above, showed no difference in net sodium excretion or urinary output compared with dopexamine. The remaining studies investigated the effects of dobutamine on splanchnic blood flow. Four studies demonstrated significant increases in splanchnic blood flow as measured by ICG clearance [31,36,38] or laser Doppler flowmetry [26]. In studies in which pHi was measured, dobutamine had no effect [31,34] or decreased pHi [36].\nWe were unable to find any data relating to the effect of dobutamine on major clinical outcomes or survival.\nDopexamine\nDopexamine is a synthetic catecholamine with agonist activity at \u03b22-receptors and indirect action at \u03b21-receptorsby inhibiting the uptake of endogenous catecholamines [46]. This agent is not available in some developed countries. Our literature search identified 20 papers investigating the effects of dopexamine in patients who had undergone cardiac surgery, all of which were retrieved.\nTwo randomized controlled trials [47,48] compared dopexamine with placebo. Hurley and coworkers [47] reported a study of 23 low-risk post-CABG patients in 1995. In that study, dopexamine at a dose of 2.0 \u03bcg\/kg per min significantly increased CI by 41% and HR by 19%. SVR was also reduced by 45%. In their randomized, double-blind, placebo-controlled trial, Sherry and coworkers [48] similarly found significant increases in HR and CI over placebo (one patient was withdrawn from the study because of tachycardia).\nWe identified five studies investigating the effects of dopexamine in LCOS, the largest of which is the multicentre, randomized, blinded comparison of dopexamine with dopamine reported by Rosseel and coworkers [17]. In that study, the increased CI in the dopexamine group was accompanied by an increase in HR of 37%. As previously discussed, there was significantly greater efficacy and fewer adverse events in the dopexamine group (although 54% of the dopexamine group still suffered an adverse cardiac event in the form of arrhythmia or ischaemia).\nMcGregor and coworkers [37] conducted a prospective, randomized, blinded comparison of dopexamine with dobutamine (n = 10) in patients with LCOS after CABG. They found no difference between the agents other than the fact that tachycardia of greater than 120 beats\/min was more common in the dopexamine group.\nWe were unable to find any studies comparing dopexamine with PDIs. One study, reported by Honkonen and coworkers [49], compared dopexamine with iloprost (a prostacyclin analogue) in a randomized, double-blind, crossover trial of 20 patients with total proximal occlusion of the right coronary artery. Dopexamine increased right ventricular ejection fraction significantly more than did iloprost at a dose of 0.68 \u03bcg\/kg per min.\nEight studies investigated the effects of dopexamine on regional perfusion. A randomized, placebo-controlled trial of 44 patients undergoing CABG conducted by Berendes and coworkers [50] in 1997 found improvement in creatinine clearance in the dopexamine-treated groups. However, four subsequent small randomized trials [37,48,51,52] failed to provide any evidence that the use of dopexamine improves renal function or perfusion.\nBerendes and coworkers [50] also assessed the effects of dopexamine on splanchnic oxygenation in a randomized, placebo-controlled trial of 44 patients with normal left ventricular ejection fraction (LVEF; > 0.5) who received dopexamine at doses of 0.5, 1.0 and 2.0 \u03bcg\/kg per min. There was no difference in hepatic venous oxygenation, and pHi decreased during and after CPB in all patients. A further three randomized controlled trials [28,53,54] concluded that dopexamine had no influence on pHi compared with dopamine or placebo. Dopexamine has also been shown to increase jejunal perfusion (as measured by laser Doppler flowmetry) and ICG dye clearance [55].\nNo studies were found relating to the effect of dopexamine on major clinical outcomes or survival.\nPhosphodiesterase inhibitors\nThe cardiac effects of PDIs are characterized by positive inotropy and improved diastolic relaxation (lusitropy; Fig. 1). These agents also cause potent vasodilation, with reductions in preload, afterload and PVR. Acute tolerance is not a feature.\nAmrinone\nAmrinone (known as inamrinone in North America) is a bipyridine phosphodiesterase-III inhibitor. It is typically given as a loading dose of 0.75\u20131.5 mg\/kg, followed by an infusion of 10 \u03bcg\/kg per min. It has an elimination half-life of 3.5 hours in post-CPB patients [56]. Our literature search identified 27 papers, all of which were retrieved. One of these studies provided level I data regarding the use of amrinone in patients who have undergone cardiac surgery [57].\nLewis and coworkers [57] reported a prospective, randomized, placebo-controlled trial of 234 patients. In that study the amrinone group received a bolus of 1.5 mg\/kg followed by an infusion of 10 \u03bcg\/kg per min to wean from CPB. Phenylephrine or glyceryl trinitrate were also used to optimize perfusion pressure. Significantly fewer patients failed to wean in the amrinone group than in the control group (7% versus 21%; P = 0.002). Amrinone improved weaning success regardless of LVEF, although this benefit was only statistically significant in the group with a preoperative LVEF greater than 55%.\nAnother randomized controlled trial was undertaken by Ramsay and coworkers [58]. A total of 100 patients undergoing CABG were randomly assigned to receive a single bolus of 0.75 mg\/kg amrinone (with no subsequent infusion) or saline before separation from CPB. Haemodynamic measurements were similar between the two groups at all times, but the amrinone group received a higher dose of phenylephrine. The authors of that study conceded that an insufficient amrinone dose might explain the lack of haemodynamic effect.\nOf the remaining level II evidence available, Badner and coworkers [59] also conducted a randomized, blinded, placebo-controlled trial of 30 patients undergoing mitral valve replacement in which amrinone at 2.0 mg\/kg or placebo was given before weaning from CPB. The amrinone group had a significant increase in CI (52% versus 10%) and decreases in SVR index (47% versus 10%), but there was no statistically significant difference in requirement for other inotropes or vasopressors between groups. Kikura and Sato [60] conducted a randomized, blinded comparison of amrinone, milrinone and placebo in 45 patients for weaning from CPB. Compared with placebo, amrinone significantly improved CI and SVI, and reduced dopamine requirements.\nThe remaining studies largely echo these findings. In two of these studies [61,62], however, more than 50% of patients in the amrinone group required concomitant infusions of phenylephrine to maintain MAP.\nTwo studies compared amrinone with milrinone [60,62] and one compared amrinone with enoximone [63]. None of these studies found significant differences in haemodynamic profiles. A further two randomized trials [5,8] compared amrinone and epinephrine; in both these studies amrinone produced a similar increase in CI and SVI, with significantly greater reductions in SVR.\nJenkins and coworkers [34] conducted a randomized, double-blind comparison of amrinone with dobutamine in 20 patients with severe pulmonary hypertension undergoing mitral valve replacement. Amrinone was associated with a reduction in pulmonary artery pressures and an increase in CI and right ventricular ejection fraction compared with dobutamine. Six patients in the dobutamine group suffered postoperative myocardial infarctions, as opposed to none in the amrinone group \u2013 a similar finding to that reported by Dupuis and coworkers [39].\nOur literature search returned only one study relating to the effect of amrinone on vital organ perfusion. This was a prospective, randomized study of 29 patients, reported by Iribe and coworkers in 2000 [64]. That study compared the effects of amrinone, milrinone and olprinone on hepatic venous oxygen saturation. No significant change in hepatic venous oxygen saturation was demonstrated in the amrinone group (n = 8).\nAlthough no studies used major clinical outcomes as primary end-points, the study by Lewis and coworkers [57], the largest randomized controlled trial, did not detect any statistically significant difference in length of ICU or hospital stay and mortality. The study by Butterworth and colleagues [61] similarly found no difference in mortality between amrinone and placebo groups.\nAmrinone has been reported to impair coagulation because of a reduction in platelet count and function [65,66], and concerns over this have limited its use in some countries.\nEnoximone\nEnoximone is an imidazolone derivative phosphodiesterase-III inhibitor. It is typically used in doses of 0.5\u20131.5 mg\/kg followed by an infusion of 5\u201310 \u03bcg\/kg per min. It has a half-life of 2 hours in normal patients but this may be prolonged in patients with cardiac failure.\nOf the 24 papers identified in our literature search, 19 investigated the effects of enoximone on systemic haemodynamics in post-CPB patients. Of these 19 papers, two were prospective, randomized, placebo-controlled trials. Boldt and coworkers [67,68] conducted both of these studies. The most recent of these studies [67] was published in 2002 and is a prospective, randomized, blinded, placebo-controlled trial of 40 patients aged 80 years or older. The patients in that study received either enoximone (bolus dose of 0.5 mg\/kg followed by an infusion of 2.5 \u03bcg\/kg per min) or normal saline. Compared with placebo, enoximone-treated patients recorded a significant increase in CI (25.9%) and reduction in SVR (27.5%). No significant differences in HR and MAP were recorded. In 1992, Boldt and coworkers [68] conducted a further prospective, randomized, blinded, controlled study, again of 40 patients. Patients received either a single dose of enoximone 1.0 mg\/kg or served as controls. Enoximone produced significant increases in CI (50%) and SVI (28.8%), and decreases in SVR (45.3%) and PVR (30.4%). No significant changes in HR and MAP were recorded. Oxygen delivery and consumption were also significantly higher in the enoximone group. Another study conducted by Boldt and coworkers [69] compared 40 patients who had received a single dose of enoximone 1.0 mg\/kg with 40 historical controls. The enoximone-treated group required significantly less epinephrine, calcium and nitroglycerin than did the control group.\nSeveral level II studies compared enoximone with other inotropic agents, both catecholamines and other phosphodiesterase-III inhibitors. One small, prospective, randomized trial failed to show any significant haemodynamic differences between amrinone and enoximone [62]. In the prospective, randomized, blinded trial conducted by Tarr and coworkers in 75 patients [18], only in the enoximone group were all patients successfully weaned from CPB, whereas three patients from the dobutamine group and nine from the dopamine group were withdrawn from the study because of inadequate response. The enoximone group exhibited a significantly lesser increase in HR and a greater increase in stroke index than did either the dopamine or dobutamine group, and also exhibited significantly a greater increase in CI and decrease in SVR in comparison with dopamine. Birnbaum and coworkers [70] conducted an earlier, prospective, randomized, blinded comparison of enoximone (two boluses of 0.5 mg\/kg followed by an infusion of 5 \u03bcg\/kg per min) with dopamine (3.0\u20134.0 \u03bcg\/kg per min) in 20 patients and obtained similar results.\nAs previously mentioned, we were able to find a further five studies comparing enoximone with dobutamine. The largest of these studies is the previously cited study conducted by Zeplin and coworkers [44]. That study (n = 50) found that enoximone significantly increased CI in comparison with dobutamine.\nTwo small, randomized, controlled trials investigated the effect of enoximone on vital organ perfusion. These showed no effect on pHi and significant reductions in endotoxin release [71], interleukins and \u03b11-microglobulin in the enoximone-treated group [67]. Two randomized controlled trials (n = 80 and n = 36) [72,73] investigated the effects of enoximone on coagulation parameters and platelet count and function, and found no difference from control groups.\nFinally, in a prospective trial [74] 88 elective CABG patients were randomly pretreated with enoximone, clonidine, enalalapril, or placebo. The enoximone-treated group exhibited lower troponin T and creatine kinase-MB levels compared with clonidine or placebo [74].\nThere are no data regarding the effect of enoximone on major clinical outcomes or survival other than those from the study conducted by Boldt and coworkers in 2002 [67], which found that tracheal extubation was performed significantly earlier in the enoximone-treated group.\nMilrinone\nMilrinone is a bipyridine methyl carbo-nitryl phosphodiesterase-III inhibitor. Loading doses of 20\u201350 \u03bcg\/kg are typically given, followed by an infusion of 0.2\u20130.75 \u03bcg\/kg per min. It has a half-life of 30\u201360 min.\nOur literature search identified 29 papers relating to the use of milrinone in adults after cardiac surgical procedures. These papers are summarized in Table 3. Nineteen of the papers provided data on the haemodynamic effects of milrinone following cardiac surgery and 14 of the papers were prospective randomized trials.\nFour prospective randomized trials [60,75,83,85] demonstrated the effectiveness of milrinone compared with placebo for weaning from CPB. In the study by Doolan and coworkers [85], all patients in the milrinone group (n = 15) were successfully weaned from CPB, as compared with only five out of the 15 in the group randomly assigned to placebo. In their prospective, blinded, randomized controlled trial, Yamada and coworkers [80] compared two groups of 24 patients with low and normal pre-CPB CI. In both these groups, the patients randomly assigned to milrinone exhibited significantly higher CI (46% in the low pre-CPB CI group) and significantly lower SVR (52% in the low pre-CPB CI group) than controls. HR was not significantly affected, but six out of 12 patients with a low CI required norepinephrine to maintain adequate systemic blood pressure. Similarly, Lobato and coworkers [83] found that a single dose of milrinone 50 mg\/kg administered before separation from CPB significantly increased CI (43%) and decreased SVR and catecholamine requirement compared with placebo in 21 patients with pre-existing left ventricular dysfunction. Again, more patients in the milrinone group required vasopressor support. Kikura and Sato [60] obtained similar results with milrinone, and these effects were sustained into the first 24 hours after surgery.\nMilrinone has been compared with catecholamines for postoperative support in LCOS, notably by the European Milrinone Multicentre Trial Group, which published the results of a randomized, open label, multicentre study of 120 patients treated with milrinone or dobutamine for LCOS after cardiac surgery [2]. The significant findings of this study are described above in the section on dobutamine.\nAs previously mentioned, two studies [60,62] compared amrinone with milrinone. Neither of these studies found significant differences in haemodynamic profiles.\nSolina and coworkers [79] compared the use of milrinone with nitric oxide (NO) in cardiac surgery patients with pulmonary hypertension. Those investigators found that the effects of milrinone on right ventricular ejection fraction were comparable with NO at 20 ppm but significantly less effective than NO at 40 ppm.\nSmall randomized controlled trials [27,64,78,81] have examined the effects of milrinone on pHi, splanchnic blood flow and inflammatory markers. Two of these [78,81] suggested that the administration of milrinone may attenuate the fall in pHi associated with CPB and the increase in some markers of inflammation. The remaining studies showed no difference.\nIn a prospective, randomized study of 20 patients conducted by Lobato and coworkers [13], milrinone produced a 24% increase in grafted IMA flow, as measured by laser Doppler flowmetry. In an observational study of 25 patients [76] milrinone also increased cerebral blood flow, as measured by transcranial Doppler, after separation from CPB.\nWe were unable to find any data relating to the effect of milrinone on major clinical outcomes or survival in cardiac surgery patients.\nLevosimendan\nThere is little published work on the use of the novel calcium sensitizer levosimendan in cardiac surgical patients with LCOS. Levosimendan is a new inodilator that exerts its inotropic effect by interacting with troponin C (the binding protein for calcium) to enhance the calcium sensitivity of cardiac myocytes.\nIn a multicentre, randomized, double-blind trial of 203 patients [95], the efficacy and safety of levosimendan were compared with those of dobutamine in severe low-output heart failure (the LIDO study). Levosimendan improved haemodynamic performance more effectively than did dobutamine in patients with severe, low-output heart failure, and there was significantly lower mortality in the levosimendan group. However, only 2\u20134% of the study population had postoperative cardiac failure. A recent uncontrolled pilot study in cardiac surgery patients with LCOS found that levosimendan increased CI and stroke volume while lowering pulmonary artery occlusion pressure [96].\nConclusion\nIt is well recognized that myocardial dysfunction occurs after cardiac surgery. Because LCOS is common, contributes to morbidity and mortality, and increases length of ICU and hospital stay and costs, it is desirable to minimize its occurrence or attenuate its severity. A summary of the significant findings of our literature review are presented in Table 4. It is evident that there are two main classes of inotropic agents that should be used for support of cardiac output after cardiac surgery: catecholamines and PDIs (data for use of calcium sensitizers in this setting is scant). Moreover, all of these agents have been demonstrated to be effective at improving myocardial contractility or HR, or both. Although some reports in the literature suggest that catecholamines are more potent inotropic and chronotropic agents, serious drawbacks associated with their use include increased myocardial oxygen consumption, tachycardia, increased afterload and arrhythmias. \u03b2-Adrenergic receptors may also be downregulated in patients with pre-existing cardiac failure. This has led to interest in the use of phosphodiesterase-III inhibitors and, more recently, the calcium sensitizer levosimendan.\nStudies investigating the use of PDIs in cardiac surgery have shown them to be potent inotropes, but vasodilation is a prominent feature of their use, and so concomitant administration of a vasoconstrictor such as norepinephrine or phenylephrine is often required. Such vasoconstrictor agents may or may not have adverse effects of their own. The effects of PDIs on HR appear to be minimal, and there is evidence to suggest that diastolic relaxation and flow through arterial grafts is improved. However, because of their pharmacokinetic profile, the time of onset and offset are longer (a loading dose is required) and they have the potential to accumulate in renal failure. These features can render the PDIs clinically less practical.\nThe effect of using either catecholamines or PDIs on major clinical outcomes or survival is unknown. We conducted an extensive literature search for data reported during the past 20 years relating to the use of inotropic agents in adult patients who have undergone cardiac surgery. Perhaps the most important finding of this review is the lack of large, double-blind, randomized controlled trials focusing on important clinical outcomes for drugs that are probably given to 250,000\u2013500,000 people each year in western countries alone. Of course, there is overwhelming evidence that the agents considered in this review increase cardiac output, but the question of their comparative effects in the post-CPB heart and their effects on important clinical outcomes remains unclear. The available evidence is often not homogenous and is completely unsuitable for meta-analysis. Also, many of the data rely on physiological end-points, and there are clearly inherent pitfalls in this. Of 125 retrieved papers, only one 'level I' study was identified. The study by Feneck and coworkers [2] provides the only direct comparison between catecholamines and PDIs in patients with LCOS. A summary of haemodynamic changes between the milrinone group and the dobutamine group from the study is outlined in Fig. 2. Although that study included a reasonably large number of patients (n = 120), no convincing advantage was shown for either drug. Moreover, the observation period was only 4 hours and the outcomes were only physiological. This is disappointing because there are several theoretical advantages of PDIs over catecholamines: less tachycardia and myocardial oxygen consumption, improved diastolic relaxation, peripheral and pulmonary vasodilation, and increased IMA graft flow. On the other hand, such advantages are theoretically diminished by the need for vasopressor support.\nHoffman and coworkers recently reported the findings of the PRIMACORP study [97], a randomized, blinded, placebo-controlled trial that investigated the efficacy and safety of prophylactic milrinone in paediatric patients at risk for developing LCOS. Of the 239 patients investigated, high-dose milrinone (75 \u03bcg\/kg per min followed by an infusion of 0.75 \u03bcg\/kg per min) reduced the risk for LCOS by 48%. A randomized controlled study of suitable statistical power must be conducted to compare fully the benefits of PDIs with those of dobutamine in adult cardiac surgical patients, with the focus on clinical rather than just physiological outcomes.\nFollowing our systematic analysis of the literature, we believe that \u2013 despite the limitations of the data \u2013 some recommendations can be made, each with a particular level of evidence.\n\u2022 Recommendation 1 (level C). \u03b2-Agonists or PDIs are more efficacious at increasing cardiac output than placebo for the treatment of LCOS after cardiac surgery. Beta-agonists are associated with a greater incidence of tachycardia and tachyarrhythmia. Administration of a vasoconstrictor is often required with PDIs.\n\u2022 Recommendation 2 (level C). Catecholamines such as dopamine, epinephrine and dopexamine have no clear advantages over dobutamine and may be associated with a greater incidence of adverse effects. Epinephrine has been successfully used as salvage therapy.\n\u2022 Recommendation 3 (level C). Administration of PDIs before separation from CPB increases the likelihood of successful weaning compared with placebo, and decreases the use of catecholamines during the postoperative period. Concerns regarding amrinone and thrombocytopenia have limited its use.\n\u2022 Recommendation 4 (level C). There is no evidence that inotropes should be selected for their effects on regional perfusion.\n\u2022 Recommendation 5 (level C). Administration of milrinone increases flow through arterial grafts.\n\u2022 Recommendation 6 (level C). Milrinone and probably other PDIs reduce mean pulmonary artery pressure and improve right heart performance in pulmonary hypertension.\nWe believe that the field of clinical research into inotropic support for adult cardiac surgery has reasonably established the superiority of catecholamines and PDIs over placebo. However, insufficient evidence exists to guide the choice of one group of drugs versus the other. The role of the new calcium sensitizers remains unknown. It is biologically plausible that the use of catecholamines or PDIs may lead to different clinical outcomes and the clinical scenario of LCOS is relatively common, and so suitably powered, multicentre, randomized controlled trials should be a clinical research priority in adult cardiac surgery patients.\nAbbreviations\nCABG = coronary artery bypass grafting; CI = cardiac index; CPB = cardiopulmonary bypass; HR = heart rate; ICG = indocyanine green; ICU = intensive care unit; IMA = internal mammary artery; LCOS = low cardiac output syndrome; LVEF = left ventricular ejection fraction; MAP = mean arterial pressure; NO = nitric oxide; PDI = phosphodiesterase inhibitor; pHi = intramucosal pH; PVR = pulmonary vascular resistance; SVR = systemic vascular resistance; SVI = stroke volume index.\nCompeting interests\nThe author(s) declare that they have no competing interests.","keyphrases":["cardiovascular drugs","coronary artery bypass surgery","postoperative care"],"prmu":["M","R","R"]}
{"id":"Eur_J_Pediatr-3-1-2039787","title":"Outcome of paediatric intensive care survivors\n","text":"The development of paediatric intensive care has contributed to the improved survival of critically ill children. Physical and psychological sequelae and consequences for quality of life (QoL) in survivors might be significant, as has been determined in adult intensive care unit (ICU) survivors. Awareness of sequelae due to the original illness and its treatment may result in changes in treatment and support during and after the acute phase. To determine the current knowledge on physical and psychological sequelae and the quality of life in survivors of paediatric intensive care, we undertook a computerised comprehensive search of online databases for studies reporting sequelae in survivors of paediatric intensive care. Studies reporting sequelae in paediatric survivors of cardiothoracic surgery and trauma were excluded, as were studies reporting only mortality. All other studies reporting aspects of physical and psychological sequelae were analysed. Twenty-seven studies consisting of 3,444 survivors met the selection criteria. Distinct physical and psychological sequelae in patients have been determined and seemed to interfere with quality of life. Psychological sequelae in parents seem to be common. Small numbers, methodological limitations and quantitative and qualitative heterogeneity hamper the interpretation of data. We conclude that paediatric intensive care survivors and their parents have physical and psychological sequelae affecting quality of life. Further well-designed prospective studies evaluating sequelae of the original illness and its treatment are warranted.\nIntroduction\nThe development of paediatric intensive care has contributed to improved survival rates in children with critical illnesses [68, 69]. Consequently, new disease patterns have emerged due to long-term complications and effects of the original illness and its treatment. In addition to survival and morbidity, physical and psychological sequelae, as well as the quality of life (QoL) in survivors and in their families are important outcome measures.\nHistorically, outcome research in paediatrics is either based on an age-specific approach, such as follow-up studies of premature infants [41, 72, 73], or on a more disease-oriented approach, such as follow-up studies in survivors of cardiothoracic surgery or trauma [15, 55, 64, 70]. These studies have shown substantial physical, psychological and neuro-cognitive sequelae, interfering with daily life and normal development. In addition, effects on parents and siblings have been shown [26]. Evaluative research of adult intensive care survivors showed the effect of intensive care treatment per se. Irrespective of the underlying illnesses, sequelae on all domains with effects on QoL were found [2, 19, 58, 75]. In multi-disciplinary paediatric intensive care unit (PICU) populations, reports on outcome are scarce [24, 25].\nBased on these observations, we believe that follow-up research of paediatric intensive care survivors and their families is needed to evaluate: (1) physical sequelae and their impact during growth and development; (2) psychological sequelae in patients and their families and their impact on the QoL of patients and family members; and (3) the need for treatment and support after discharge.\nThe aim of this article is to provide an overview of the available literature concerning the different domains of QoL (i.e. physical, psychological and social functioning) in children surviving paediatric intensive care, including the effect on parents, and to suggest directions for future follow-up research.\nMethods\nTo identify studies eligible for this review, we searched Medline (1966\u20132006), EMBASE (1974\u20132006), CINAHL (1982\u20132006), pre-CINAHL and the Cochrane Library (2006) in March 2006. In the search strategy, all terms mapped to the appropriate MeSH\/EMTREE subject headings and \u201cexploded\u201d were used; among them were: paediatric intensive care unit (PICU), septic shock, respiratory insufficiency, meningococcal disease, central venous catheterisation, intubation, physical and psychological sequelae, post-traumatic stress disorder (PTSD), QoL, health status and long-term outcome.\nDefinitions\nFunctional health is defined as an individual\u2019s ability to perform normal daily activities, to fulfil usual roles and to maintain health and well-being.\nQoL is defined as an individual\u2019s perception of their position in life, in the context of the culture and value systems and in relation to their goals, expectations, standards and concerns [1].\nHealth-related QoL (HRQoL) is defined as QoL in which a dimension of personal judgement over one\u2019s health and disease is added [21].\nStudy selection\nStudies were selected for review if they met two inclusion criteria: (1) study of a representative population of PICU survivors (defined as a population consisting of medical and\/or surgical PICU patients <18\u00a0years old) and (2) evaluation of physical sequelae, measurement of QoL or functional health >30\u00a0days after PICU discharge. Because of the limited number of studies, the measurement tools did not need to be standardised. Studies with a retrospective and prospective design were included.\nExcluded were: (1) studies in homogeneous PICU populations (e.g. survivors of cardiothoracic surgery and trauma) reporting diagnosis-related outcome in particular but not intensive care treatment as such, and (2) studies evaluating mortality only.\nResults\nEligible studies and quality of the studies\nTwenty-seven studies were found in which one or more aspects of long-term sequelae in PICU survivors and\/or their families were described. The patient characteristics, populations, measurement tools and outcomes are described in Tables\u00a01 and 2. The quality criteria are described in Table\u00a03. None of the studies met all of the quality criteria. In studies describing the same outcome aspect, differences in study population, follow-up time and measurement tools make the comparison and synthesis of results difficult.\nTable\u00a01Patient characteristics, measurement tools, physical and functional health outcomeReferencePopulationa, nbAgec (yrs)Follow-up timed (yrs)Severity of illnesseLOSf (days)Measurement toolOutcome (n)gInterpretation of outcome23Meningococcal disease0.1\u201315.38\u201312GMSPSNANeurological examination1 spastic quadriplegiaMajority of children surviving meningococcal disease neurologically normal.n\u2009=\u2009115 (139)Median 5Cognitive tests5 hearing loss4 major impairments35Cardiopulmonary resuscitation (CPR)0\u2013161NANAPCPCh26 normal, 12 mild, 7 moderate, 3 severe disability60% of survivors of CPR neurological normal.n\u2009=\u200944 (48)37 same as prior to CPRLocation, underlying cause and duration of CPR determinants of outcome.43CPR0\u2013171NANAPCPCh54 normal or mild disability, 6 moderate, 5 severe disability80% of survivors of CPR neurological normal.n\u2009=\u200965 (94)POPCiLocation, underlying cause and duration of CPR not determinants of outcome.53Acquired brain injury<3>0.5GCS<9NAGOSjGOS: 23 good recovery, 7 moderate, 5 severe disabilityMajority of children with acquired brain injury dead or disabled.n\u2009=\u200938 (53)BSID-IIkBSID-II: 8 normal, 12 cerebral palsy, 11 cognitive delayNeuro-developmental examination59CPR0\u2013181NAPICUPCPCh5 normal, 1 mild, 4 moderate, 1 severe disability, 2 persistent vegetative state38% of survivors of CPR neurological normal.n\u2009=\u200913Median 5POPCiUnderlying cause and duration of CPR determinants of outcome.6ARDS2\u2013135.6\u00b14.3NANAChest radiography1 SaO2 94% during exerciseARDS survivors at risk for hypoxaemia during exercise.n\u2009=\u20097 (15)Pulmonary function1 reduced diffusion capacity14Meningococcal disease with ARDS0.3\u20133.70.5\u20132.1PRISM 12-53%NAPulmonary function1 wheezing for which salbutamolARDS survivors possibly at risk for mild obstructive lung disease.n\u2009=\u2009122 obstructive disease22ARDS4.6\u201315.90.9\u20134.2NANAPulmonary function3 restrictive or obstructive diseaseARDS survivors at risk for restrictive and obstructive lung disease.n\u2009=\u20099 (12)Electrocardiography EchocardiographyCardiac function normal in all30ARDS5\u2013144.4NANAPulmonary function1 limitations in activityARDS survivors at risk for restrictive and obstructive lung disease.n\u2009=\u200954 restrictive disease48Meningococcal septic shock1.6\u201315.42.2\u20134.9NANAPulmonary function18 normal lung functionMeningococcal septic shock survivors at risk for hypoxaemia during exercise.n\u2009=\u2009186 SaO2\u226495% during exercise74ARDS0.5\u2013160.3\u20135.5PRISM 18\u00b114%NAChest radiography7 (11) restrictive or obstructive diseaseARDS survivors at risk for restrictive and obstructive lung disease.n\u2009=\u200914 (20)Pulse oximetry4 (7) decreased diffusion capacityPulmonary functionElectrocardiographyEchocardiography62Meningococcal sepsis with renal replacement therapy0.5\u2013152.7\u20137.1NAPICUGlomerular filtration rate (GFR)2 decreased GFR, proteinuria, hypertensionChildren surviving acute renal failure due to septic shock at risk for long-term renal dysfunction.n\u2009=\u200912 (15)Median 12Serum creatinine1 parenchymal defectProtein excretion in urine1 proteinuriaDMSA scan9Near drowning admitted to PICU0.7\u201314\u22650.5PRISM 71%NAFunctional health:8 severe neurological impairmentMajority of near-drowning survivors lead a normal life.n\u2009=\u200925 (27)GCS\u22645Contact primary physician or examination by study facility17 normal or mildly impaired12PICU0\u2013182.5\u20133NAPICUFunctional health:7% moderate or severe handicapMajority of PICU survivors seem to lead a normal life.n\u2009=\u2009775Mean 2.8Written questionnaire or telephone contact with the specialist physician or parents12% mild handicap91% will lead independent life44Bacterial meningitis with respiratory insufficiency0\u2013120.6\u20136.4PRISMNAFunctional health:12 normalHalf of children surviving severe bacterial meningitis seem to lead a normal life.n\u2009=\u200921 (22)Mean 22%Telephone interview1 independentRange 1\u201347%2 partially dependent6 dependentaStudied population. bn\u2009=\u2009studied patients (eligible patients). cAge of patients at admission to PICU (mean\u00b1SD or range). dFollow-up time (mean\u00b1SD or range). eSeverity of illness (PRISM, Glasgow Coma Score (GCS), Glasgow Meningococcal Septicaemia Prognostic Score (GMSPS) and\/or parent rating). fLength of stay (LOS) in PICU and\/or hospital (mean\u00b1SD or range). gn=\u2009examined patients (eligible patients)hPCPC=Paediatric Cerebral Performance Category. iPOPC=Paediatric Overall Performance Category. jGOS=Glasgow Outcome Scale. kBSID-II=Bayley Scales of Infant Development-IITable\u00a02Patient characteristics, measurement tools and psychological and quality of life (QoL) outcomeReferencePopulationa, nbAgec (yrs)Follow-up timed (yrs)Severity of illnesseLOSf (days)Measurement toolOutcome (n)gInterpretation of outcome40PICU children and mothers2.1\u201315.90.3\u20131PRISM 0.4\u201376%PICU 1\u201330Psychological outcome:Children: Behaviour high 3 (8), SDQ high 3 (21), IES PTSD 3 (29)PICU survivors and their mothers at risk for psychological distress and PTSD.n\u2009=\u200929 (33)Child: Behaviour Check List, SDQh,IESiMothers: GHQ high 11 (26), IES high 13 (27)Mother: GHQj, IESi50PICU5\u201318Median 0.6Parent rating 10Hospital 4\u201314Psychological outcome:Children: PTSD 4 (19) PICU, 0 (27) ward IES high 4 (21) PICU, 2 (17) wardPICU survivors and their parents at risk for psychological distress and PTSD.n\u2009=\u200935 (46)Child: PTSDk, SDQh, IESi, depression, anxiety, CSIlParents: PTSD 9(33) PICU, 2 (29) wardGeneral wardParent: GHQj, IESi depressionn\u2009=\u200933 (41)Children and parents51PICU11.3\u00b13.20.5PRISM 25\u00b123%Hospital 13.0Psychological outcome:IES and CMFS dependent on invasive procedures, CMFS and CHLOC on ageStress symptoms in children possibly dependent on number of invasive procedures.n\u2009=\u200960 (69)Children: IESi, CMFSm, CHLOCnGeneral wardn\u2009=\u200960 (69)52PICUMean0.5PRISMNAPsychological outcome:IES higher in high risk, not decreasing over time, IES related with invasive proceduresStress symptoms possibly dependent on invasive procedures. Stress symptoms not decreasing over time.n\u2009=\u200960Low risk 11.5Low risk<34%Children: IESi, CMFSm, CHLOCnHigh risk 11.1High risk\u226534%61Meningococcal disease PICU and wardMedian 6.80.3GMSPS 6.9\u00b13.3PICU LOS 0\u201362Psychological outcome:Child: PTSD 4 (26)PICU survivors and their parents at risk for psychological distress and PTSD.Children and parentsParent rating median 7Hospital LOS 2\u201387Child: SDQh, IESiMothers: PTSD 22 (58)n\u2009=\u200978 (118)Parent: GHQj, IESiFathers: PTSD 8 (43)4PICU parents25% <1, 25% 1\u20134, 25% 5\u201311, 25% >110.2\u20130.9PRISM 0\u201326%PICU 1\u2013200Psychological outcome:ASD 87Parents of PICU survivors with ASD more at risk for PTSD.n\u2009=\u2009272 (291)Parent rating 1\u20139Parents: Acute Stress Disease symptomsPTSD 33PTSDk symptoms8PICU1.2\u00b11.3<0.5PRISM 12\u00b17%PICU 10.5\u00b111.5Psychological outcome mothers:Mothers PICU more stress. Stress decreases over time in all groupsMothers of PICU survivors at risk for psychological distress; families at risk for dysfunctioning.n\u2009=\u200931Parent rating 8.3\u00b11.9Parental Stress Scale SCL-90-RoAll families dysfunctioningGeneral wardFAM IIIpn\u2009=\u200932FILEqERn\u2009=\u200932 Mothers20Meningococcal Disease Parents1\u2013180.25\u20137NANAPsychological outcome parents:High psychological distress in mothers and fathers, not decreasing over timeMothers and fathers of PICU survivors at risk for psychological distress.102 mothers, 90 fathersGHQj27PICU4.61PRISMNAQoL: MAHSCr106 equal to before PICU50% of PICU survivors seem to have the same QoL as before admission; 10% normal QoL.n\u2009=\u2009226 (241)n\u2009=\u2009223\u2009<\u20095%58 improvedn\u2009=\u200919\u2009>\u200916%62 deteriorated26 normal38PICU5.7\u00b13.61PRISMPICU 5.7\u00b15.5QoL: MAHSCr52 improved50% of PICU survivors seem to have good QoL.n\u2009=\u2009138 (150)n\u2009=\u200979\u2009<\u20095%29 deterioratedn\u2009=\u20094\u2009>\u200916%65 normal after PICU47PICUMedian 2.30.3\u20132PRISMNAQoL: RAHCs measure of function256 normal QoL60% of PICU survivors seem to have normal QoL.n\u2009=\u2009432 (906)Mean 5.5%140 fair QoL9 poor QoL65PICU0\u201329.32.3\u20136PRISMPICU 0\u201357.4QoL: HSUIt GOSuHSUI (727): 608 normal, 29 (very) poor QoL70% of PICU survivors seem to have good QoL.n\u2009=\u2009868 (1265)n\u2009=\u2009554\u2009<\u20095%GOS (727): 515 normal, 137 mild disability, 75 moderate\/severe disability60% seem to have normal functional health.n\u2009=\u2009137\u2009>\u200916%aStudied population. bn\u2009=\u2009studied patients (eligible patients). cAge of patients at admission to PICU (mean\u00b1SD or range). dFollow-up time (mean\u00b1SD or range). eSeverity of illness (PRISM, Glasgow Coma Score, GMSPS and\/or parent rating). fLength of stay (LOS) in PICU and\/or hospital (mean\u00b1SD or range). gn\u2009=\u2009examined patients (eligible patients).hSDQ=Strength and Difficulties Questionnaire. iIES=Impact of Event Scale. jGHQ=General Health Questionnaire. kPTSD=Post-traumatic stress disorder. lCSI=Child Somatization Inventory. mCMFS=Child Medical Fears Scale. nCHLOC=Child Health Locus Control Scale.oSCL-90-R=Symptom Checklist-90 Revised. pFAM III=Family Assessment Measure III. qFILE=Family Inventory of Life Events and Change. rMAHSC=Multi-attribute health status classification. sRAHC=Royal Alexandra Hospital for Children. tHSUI=Health State Utility index. uGOS=Glasgow Outcome ScoreTable\u00a03Quality assessment of reviewed studiesReferenceSelection bias excludedaSelective loss to follow-up excludedbExposure clearly definedcOutcome clearly defineddControl group includede4yesyesyesyesno6nonoyesyesno8nonoyesyesyes9noyesyesnono12yesyesyesyesno14yesyesyesyesno20yesyesyesyesno22nononoyesno23nonoyesyesyes27yesnoyesyesno30nononoyesno35yesnoyesyesno38nonoyesyesno40noyesyesyesno43nonoyesyesno44yesyesyesyesno47yesnoyesyesno48nonoyesyesno50yesnoyesyesyes51noyesyesyesyes52noyesyesyesyes53yesyesyesyesno59yesnoyesyesno61noyesyesyesno62yesnoyesyesno65nonoyesyesno74yesyesyesyesnoaSelection bias excluded (i.e. exclusion of >10% of the studied population excluded). bSelective loss to follow-up excluded (i.e. description of patients lost to follow-up and comparison with those remaining in the study). cExposure clearly defined (i.e. clear definition of the studied population). dOutcome clearly defined (i.e. clear definition of outcome measures). eComparison with control group (i.e. children admitted to general ward)\nPhysical and neuro-cognitive sequelae (Table\u00a01)\nIn 12 studies that included in total 340 patients, aspects of physical and neuro-cognitive sequelae were evaluated.\nNeurological evaluation was conducted in five studies including 275 survivors. The majority of the children were neurologically normal. In the remaining children, disabilities such as hearing loss, coordination, cognition and developmental problems turned out to be severe [23, 35, 43, 53, 59].\nPulmonary evaluation was conducted in six studies including 65 patients [6, 14, 22, 30, 48, 74]. Restrictive and obstructive disease and hypoxaemia during exercise was found.\nCardiac evaluation was conducted in two studies including 23 survivors [22, 74]. No abnormalities were found, except for left ventricular hypertrophy in one child.\nRenal evaluation was conducted in one study including 12 survivors [62]. In two children, glomerular filtration was impaired, one had hypertension and one had proteinuria.\nPsychological sequelae (Table\u00a02)\nVarious questionnaires were used. Cut-off points for the diagnosis of PTSD differed between studies but all of them showed high scores for PTSD in children and parents.\nPsychological evaluation of children was conducted in five studies including 202 children [40, 50\u201352, 61]. Symptoms of PTSD were found in 11 of 74 evaluated children. In one study, a relation was found between invasive procedures and high scores [52].\nPsychological evaluation of parents was conducted in six studies including parents of 547 children [4, 8, 20, 40, 50, 61]. Symptoms of PTSD were found in 72 of 295 evaluated parents. In some studies, a relation was found between high scores and illness severity as perceived by parents [4, 50, 61]. In one study, these high scores decreased over time [8].\nFunctional health and QoL (Tables\u00a01 and 2)\nEvaluation of functional health was conducted in three studies including 821 children [9, 12, 44]. The majority of the children seemed to have normal functional health; the remainder was found to be seriously impaired.\nEvaluation of QoL was conducted in four studies including 1,664 children [27, 38, 47, 65]. QoL was evaluated using three different questionnaires. In the majority of children, the QoL was normal or equal to the QoL before PICU admission. In all studies, some of the children had poor QoL.\nDiscussion\nOnly 27 studies consisting of 3,444 PICU survivors met our inclusion criteria. The small numbers, heterogeneity of the studied populations and the used measurement tools, the frequent use of non-validated measurement tools and the various aspects of outcomes studied make aggregation of the data and, therefore, strong conclusive statements difficult.\nPhysical sequelae\nThe reviewed studies report distinct physical sequelae, including neurological abnormalities in PICU survivors. Standardised neurological examination of PICU survivors was validated in 1994 but very few studies have been carried out since [24, 25]. As neurological problems have a great impact on daily life, standardised evaluation and adequate support and rehabilitation seem to be relevant, similar to in NICU survivors [11, 46, 56].\nFollow-up studies evaluating lung function in children are hampered by the small incidence of severe respiratory insufficiency in children [49]. In adult respiratory distress syndrome (ARDS), the recovery of lung function is shown during the first year and physical limitations seem to be partly dependent on lung function [34, 58]. In infants and children, post-natal lung growth may contribute to the improvement of lung function after critical illness. In addition to lung function, the long-term effect of small airway disease should be evaluated, for instance, in children with respiratory syncitial virus infection.\nData on the structured evaluation of cardiac and renal function in paediatric and adult ICU survivors is not available. In young children, septic shock and the need for vasoactive support of the circulation may interact with the developing myocardium and may have persistent effects on cardiac growth and function [10, 67, 77].\nComplications of intensive care procedures per se, (e.g. vascular complications due to intra-vascular catheters and side-effects of ototoxic drugs and sedatives) are not evaluated [5, 18, 32, 33, 45, 54, 57, 63]. One can assume the exact incidence of physical sequelae to be higher than has been reported so far.\nPsychological sequelae and functional health and QoL\nIn the reviewed studies, psychological sequelae have been established in 10\u201314% of survivors and their parents. The comparison of findings is hampered due to different measurement tools and cut-off points for the diagnosis of PTSD and various follow-up intervals. Risk factors accounting for hampered psychological outcome could be diverse (severity of illness, being removed from one\u2019s child, having been witness to the accident, mental health, family functioning, social support, coping strategies and lack of information from the medical team) [17, 26, 29, 31]. Psychological support to improve coping strategies and prevent over-protection might improve psychological outcome in children and parents [3, 28]. Further research is essential to establish the appropriate time and extent of the psychological support needed.\nCognitive sequelae have rarely been studied in the reviewed studies. Adequate neuro-cognitive evaluation is both expensive and time-consuming. Studies in neonatal ICU survivors show substantial cognitive dysfunction with great impact on daily life [7]. Consequently, early intervention, education and rehabilitation are expected to improve daily life [11, 46].\nA majority of PICU survivors seem to have unchanged functional health and good QoL. In the reviewed studies, functional health is evaluated by telephone interviews [27, 38, 47, 65]. In most of these studies, the physician rather than the child or its parents evaluates functional health. Ideal (HR)QoL questionnaires should measure all aspects of QoL and preferably be filled in by the children themselves. Proxy investigation of functional health and (HR)QoL (in children <6\u20138\u00a0years of age) is second best [36, 37, 39, 66]. Besides, the pre-morbid state is probably an important factor which is difficult to assess [16].\nSuggestions for future follow-up research\nThe reviewed studies have a number of methodological limitations. Heterogeneity is the most important one. Consensus on all aspects of follow-up research is essential for well-founded conclusions. For example, structured and standardised evaluation of: (1) organ system function with a validated tool such as the Paediatric Logistic Organ Dysfunction (PELOD) score [13, 42, 60, 71]; (2) neuro-cognitive function; (3) complications of PICU treatment; and (4) (HR)QoL are warranted. Multi-centre studies as proposed by the Collaborative Pediatric Critical Care Research Network (CPCCRN) with a uniform approach will provide answers either in general PICU cohorts or in disease-oriented study groups [76].\nIn conclusion, this review indicates that PICU survivors and their parents may have substantial physical and psychological sequelae interacting with QoL. Because of longer life expectancy, longer follow-up time is warranted, emphasising the consequences for health care in children. We believe that paediatric intensivists and psychologists should be involved as core members of follow-up teams.","keyphrases":["quality of life","paediatric intensive care unit","post-traumatic stress disorder","health status","outcome assessment (health care)"],"prmu":["P","P","P","P","R"]}
{"id":"Qual_Life_Res-4-1-2238782","title":"Nonparametric IRT analysis of Quality-of-Life Scales and its application to the World Health Organization Quality-of-Life Scale (WHOQOL-Bref)\n","text":"Background This study investigates the usefulness of the nonparametric monotone homogeneity model for evaluating and constructing Health-Related Quality-of-Life Scales consisting of polytomous items, and compares it to the often-used parametric graded response model.\nIntroduction\nQuestionnaires for health-related quality-of-life (HRQoL) measurement are important for several reasons. First, they may be used to compare the mean level of different patient groups with respect to physical, mental and social health. A researcher may want to find out whether these patient groups have different needs with respect to, for example, therapy or medication or whether different adaptations of their environment are in order so as to improve their conditions of living. Second, HRQoL questionnaires are also important for the measurement of mean change\u2013\u2013either progress or deterioration\u2013\u2013of such groups due to, for example, therapy. The researcher\u2019s interest then lies in the effectiveness of therapy with respect to HRQoL. Third, the total score a patient obtains on an HRQoL questionnaire may be used to diagnose this patient\u2019s general level of physical health and psychological well-being, for example, so as to be able to estimate the budget needed for his\/her treatment during a particular period.\nTo effectively measure HRQoL, we argue that an instrument must meet two requirements. The first requirement is that it is clear what the instrument measures: one overall dimension of HRQoL or several dimensions reflecting different aspects of HRQoL. If the instrument measures one dimension, one can use the total score on all items to obtain an impression of the overall level of HRQoL. If the instrument measures multiple dimensions, it may be recommendable to determine total scores on subsets of items (e.g., domain scores), each reflecting a particular aspect of HRQoL (e.g., HRQoL with respect to physical, psychological, and social limitations) and then assess individuals or compare groups on a profile of scores. These two cases may be characterized as unidimensional and multidimensional measurement.\nThe second requirement is that the psychometric properties of the items are known and found sufficient. One important psychometric item property is the item\u2019s location on the scale that quantifies the HRQoL aspect of interest. For example, patients are likely to experience fewer problems when engaging in activities like bathing and dressing than in more demanding activities such as shopping and travelling. The items concerning bathing and dressing require a lower level of physical functioning than the other two items. Thus, bathing and dressing are located further to the left (at a lower level of the scale) than shopping and travelling. A good diagnostic HRQoL instrument contains items of which the locations are widely spread along the scale. Such a scale allows for measurement at varying levels of physical functioning and may be used, for example, for assessing mean differences between groups, mean change due to therapy, and individual patients\u2019 levels of physical functioning.\nAnother important psychometric item property is the item\u2019s discrimination power. This is the degree to which the item distinguishes patients with relatively low psychological well-being levels from patients with relatively high psychological well-being levels. The higher the discrimination power the higher the item\u2019s contribution to reliable measurement ([1], pp. 101\u2013124). A good diagnostic instrument has items with high discrimination power that each contributes effectively to reliable measurement of patients at different locations along the scale. Such an instrument picks up differences between groups, effects of therapy, and individual levels of activity limitation.\nItem response theory (IRT) models [1] are becoming more popular as statistical tools for scale construction in the HRQoL context. IRT can be used effectively to investigate the dimensionality of an instrument and the psychometric properties of its constituent items. The goal of this study is to discuss one particular class of IRT models known as nonparametric IRT models [2\u20135], and to argue that this class in particular provides a general and flexible data analysis framework for studying the dimensionality of a set of polytomously scored items (with dichotomously scored items as special cases) and ascertaining ordinal scales for the measurement of HRQoL aspects which contain items that have varying locations and sufficient discrimination power. Over the past few years, nonparametric IRT models already have been used occasionally for constructing HRQoL scales; see [6\u20139].\nOur point of view is that, given that the researcher has formulated desirable measurement properties, (s)he should construct his\/her scale by means of an IRT model that is as general as possible while satisfying the desired measurement properties. Examples of such properties are that the items measure the same dimension, that the measurement level is at least ordinal, and that measurement values are reliable. An HRQoL researcher who has constructed and pre-tested a questionnaire consisting of, say, 40 items is not served well when his\/her data are analyzed by means of an IRT model that is unnecessarily restrictive, the result of which is that, say, half of the items are discarded. We will argue that the most general IRT model that serves one\u2019s purposes well, often (but not always) is a nonparametric IRT model.\nMany questionnaires are used for assessing differences in HRQoL between groups, change due to therapy, and individual patients\u2019 scale levels, and a general nonparametric IRT model then is the perfect choice for analyzing one\u2019s data. Nonparametric IRT models have several advantages over more-restrictive parametric IRT models [1]: Nonparametric IRT models (1) are based on less-restrictive assumptions, thus they allow more items into the scale while maintaining desirable measurement properties; (2) offer diverse tools for HRQoL analysis that give ample information about the dimensionality of the data and the properties of the items; and (3) provide patient measurement values and item location and discrimination values, which have an interpretation that is close to intuition and therefore easy to interpret for users of HRQoL scales. For computerized adaptive HRQoL testing, more-restrictive parametric IRT models such as the Rasch [10] model (dichotomous items) and the generalized partial credit model [11] and the rating scale model [12] (polytomous items) are more appropriate than nonparametric models.\nParametric IRT models have been used more than nonparametric IRT models, especially in psychological and educational measurement, and also in HRQoL research (e.g., [13\u201315]). A reason for this may be that nonparametric IRT models were developed later than parametric IRT models. See [16\u201318] for reviews of nonparametric IRT.\nThis paper is organized as follows. First, we explain assumptions of IRT and compare parametric IRT and nonparametric IRT. Second, we discuss methods and software from nonparametric IRT that can be used for analyzing the polytomous item scores obtained from HRQoL questionnaires. Third, we use this software to analyze data from the World Health Organization Quality-of-Life Scale (WHOQOL-Bref) [19]. The results are compared to those obtained by means of a parametric IRT model. Finally, we provide recommendations for HRQoL researchers on how to use nonparametric IRT methods for analyzing their data.\nDefinitions and assumptions\nIRT models are suited for the analysis of multi-item questionnaire data that typically result from HRQoL questionnaires. The data are discrete scores representing the responses of N respondents to J items (items are indexed j; j\u00a0=\u00a01, ..., J). Many HRQoL questionnaires use items that have three or more ordered answer categories represented by three or more ordered scores, also called polytomous item scores. For simplicity, we assume that all items have the same number of ordered answer categories; this number is denoted by M\u00a0+\u00a01. Let Xj be the random variable representing the discrete score on item j, and let the items be scored Xj\u00a0=\u00a00,...,M. For example, for an item asking whether one is satisfied with one\u2019s sleep, score 0 may represent \u2018very much dissatisfied\u2019 and score M may represent \u2018very much satisfied\u2019, and the intermediate scores represent intermediate levels of satisfaction. With dichotomous item scoring, Xj\u00a0=\u00a00,1, and possible intermediate satisfaction levels are not quantified separately but collapsed into the score categories 0 and 1. Usually, researchers summarize the J item scores for each patient by the total score (sometimes referred to as the sum score), which is formally defined as \n. Total score X+ is an estimate of a patient\u2019s true score T; this is the expectation of X+ across independent replications of the measurement procedure ([20], pp. 29\u201330).\nIRT models distinguish observable or manifest variables such as item score Xj and total score X+ from latent variables. These latent variables play the role of summaries of the behavior that is described by the responses to the items. Sometimes, latent variables are interpreted as if they were causal agents driving responses to items and individual differences between patients. We will also use the distinction between latent and manifest variables in our examples. Thus, we assume that patients are characterized by either one latent HRQoL attribute (meaning that measurement is unidimensional) or different HRQoL attributes (meaning that measurement is multidimensional) which together represent the patient\u2019s latent physical, mental or social health. For example, assume that measurement is unidimensional and that the latent variable is mental health or psychological well-being. Psychological well-being then is an unobservable state in each patient, and inferences about it are made on the basis of the manifest responses reported by patients in reaction to the items in an HRQoL questionnaire. Latent variables are denoted by notation \u03b8. If measurement is unidimensional, the IRT model contains one latent variable \u03b8, and if it is multidimensional multiple latent variables are needed. We only consider unidimensional IRT models here; see [21] for a discussion of multidimensional IRT models.\nSeveral families of IRT models for polytomous item scores have been proposed (e.g., [22]). The family of graded response models (GRMs; [23]) is suitable for analyzing ordered item scores collected by means of polytomous response scales [22, 24, 25]. Suppose the item \u2018How much do you enjoy life?\u2019 has four ordered answer categories running from \u2018Not at all\u2019 (score 0) to \u2018Very much\u2019 (score 3); thus Xj\u00a0=\u00a00,...,3 and M\u00a0=\u00a03. For this item, GRMs conceptualize the response process by means of four conditional response probabilities, called item-step response functions (ISRFs) and denoted by P(Xj\u00a0\u2265\u00a0m|\u03b8)=Pjm(\u03b8) (m\u00a0=\u00a00,...,M). It may be noted that for m\u00a0=\u00a00 we have that Pj0(\u03b8)\u00a0=\u00a01 by definition, irrespective of the latent variable level; thus, this response probability is uninformative about the response process and may be ignored. The ISRF describes the relationship between expressing at least a particular minimum level of enjoying life (i.e., having at least a score of m on the example item) and the latent variable of psychological well-being (\u03b8).\nFigure\u00a01a shows an example of the ISRFs of two items having four answer categories each (i.e., M\u00a0=\u00a03). The solid lines denote the ISRFs for one item and the dashed lines denote the ISRFs for the other item. Two things are noteworthy. First, the ISRFs of different items may intersect but based on the cumulative character of the definition of the ISRFs, the ISRFs of the same item cannot intersect. Second, the ISRFs have been drawn as monotone curves with rather irregular shapes. Such shapes are typical of a nonparametric GRM [24] and found not to prevent a set of items from having favourable measurement properties, as we will see shortly. Figure\u00a02b also shows monotone ISRFs but now having smooth S-shapes, typical of a parametric GRM. This parametric GRM nearly has the same measurement properties as the nonparametric GRM, but because it assumes smooth ISRFs (Fig.\u00a01b) instead of irregular ISRFs (Fig.\u00a01a), it is more restrictive and often leads to the rejection of more items (and thus shorter scales) than its nonparametric counterpart.Fig.\u00a01Examples of item step response functions of (a) a nonparametric item response model and (b) a parametric item response modelFig.\u00a02Examples of (a) flat ISRFs and (b) non-monotone ISRFs\nGRMs have the next three assumptions as a point of departure. The first assumption is unidimensionality (UD); that is, each model assumes that one latent variable \u03b8 summarizes the variation in the J item scores in the questionnaire. Assumption UD implies that respondents can be ordered meaningfully by means of a single number. The second assumption is local independence (LI); that is, if we condition on \u03b8, the J item scores are statistically independent. An implication of LI is that in a subgroup of patients who have the same \u03b8 value, all covariances between item scores are 0. The third assumption is monotonicity (M); that is, the ISRFs are each assumed to be monotone increasing functions in \u03b8 (see Figs.\u00a01a and b). Applied to the latent variable of psychological well-being (\u03b8) and the items from the WHOQOL-Bref, assuming UD, LI and M means that we hypothesize that (1) only psychological well-being drives responses to items and has a systematic effect on individual differences in item scores and total scores (UD); (2) given a fixed level of psychological well-being, relationships between concrete aspects of psychological well-being such as represented by the items \u2018How much do you enjoy life?\u2019 and \u2018Are you able to concentrate?\u2019 are explained completely (i.e., the covariance between these items, conditional on \u03b8, equals 0) (LI); and (3) the higher the level of psychological well-being, the higher the probability that one enjoys life and is able to concentrate.\nParametric and nonparametric graded response models\nParametric Graded Response Model. The ISRFs of the parametric GRM [26] are defined by logistic functions that have the following parameters:\u03b4jm: the location parameter of the mth ISRF of item j (i.e., Pjm(\u03b8)) on the scale of \u03b8;\u03b1j: the slope parameter or \u2018discrimination power\u2019 of item j.\nThe meaning of these parameters is explained after the ISRF of the parametric GRM is introduced. This ISRF is defined as\nFigure\u00a01b shows the logistic ISRFs of two items (M\u00a0=\u00a03). The two items are denoted by j (solid ISRFs) and k (dashed ISRFs). For each item, the three location parameters are also shown. For item j, by definition we have that \u03b4j1\u00a0<\u00a0\u03b4j2\u00a0<\u00a0\u03b4j3, and for item k by definition \u03b4k1\u00a0<\u00a0\u03b4k2\u00a0<\u00a0\u03b4k3. An ISRF\u2019s location parameter is the value of \u03b8 for which the probability of having an item score of at least m equals .5: that is, Pjm(\u03b8)\u00a0=\u00a0Pkm(\u03b8)\u00a0=\u00a0.5, m\u00a0=\u00a01,..., M.\nFigure\u00a01b also shows that the slopes of the ISRFs of the same item are equal (mathematically, they must be equal or the ISRFs would intersect; this is impossible given the cumulative definition of the ISRFs), but also that the ISRFs of item j are steeper than the ISRFs of item k. Steepness of slopes is evaluated as follows. For ISRF m of item j, consider the point with coordinates (\u03b4jm, .5). This is the point in which the slope of a logistic ISRF is steepest, and this steepest slope is taken to be typical for the whole ISRF. Parameter \u03b1j expresses this maximum steepness (but is not exactly equal to it). In the example in Fig.\u00a01b, we have that \u03b1j\u00a0>\u00a0\u03b1k.\nNonparametric Graded Response Model. Instead of choosing a parametric function, nonparametric IRT models typically define order restrictions on the ISRFs. The nonparametric GRM, better known as the monotone homogeneity model (MHM) for polytomous items [27, 4], assumes UD, LI, and M: that is, for any pair of \u03b8s, say, \u03b8a and \u03b8b, the MHM assumes that\nThus, the ISRF is monotone non-decreasing in \u03b8; see Fig.\u00a01a for examples of ISRFs that are monotone but not logistic. This assumption says that a higher level of psychological well-being induces a higher probability of obtaining at least an item score of m (i.e., a higher item score). The ISRFs of different items can have any monotone form and be very different. Requiring monotone ISRFs only is less restrictive than requiring monotone logistic ISRFs; thus, the MHM is a more general model for describing the data than the GRM (henceforth, we call the nonparametric GRM by its better known name (in fact, acronym) MHM, and the parametric GRM simply the GRM).\nUnlike the GRM, the MHM does not provide numerical estimates of the latent variable \u03b8 . Instead, the MHM allows that total score X+ orders patients stochastically on latent variable \u03b8 in almost all practical measurement situations [28]. This means that, for two total scores X+ denoted v and w,\n[4]. This inequality says that as the total score increases the mean \u03b8 also increases (or stays the same). Thus, groups of patients that have higher total scores, on average also have higher latent variable values. This result may not seem spectacular at first sight, but it (1) ascertains an ordinal scale for patient measurement (2) using only observable total scores (without requiring the actual estimation of \u03b8). For the psychological well-being example, if the MHM fits the data, ordering patients by means of the total score by implication orders them on the latent variable \u03b8.\nAlso, the MHM does not provide numerical estimates of the item parameters \u03b4 and \u03b1. Instead, a distinction can be made between drawing information about item functioning from estimates of the complete ISRFs and item parameters typical of the MHM. Estimates of the complete ISRFs provide much information about the exact relationship between the item scores and the latent variable [16, 29]. ISRFs that are relatively flat or fail to be monotone can be studied in much detail so as to reveal why they dysfunction.\nFigure\u00a02a shows three relatively flat ISRFs of a hypothetical item (M\u00a0=\u00a03). This item does not distinguish low \u03b8 and high \u03b8 patients well. It may be noted that the ISRFs do not all need to be flat simultaneously, but given that they  cannot intersect if one ISRF is flat others are likely to be relatively flat as well and the item as a whole contributes little to the reliable ordering of patients on \u03b8.\nFigure\u00a02b shows three non-monotone ISRFs of another hypothetical item. Each shows relative good distinction between low \u03b8 values and between above-average \u03b8 values but bad distinction just below the middle of the scale. Again, non-intersection of the ISRFs of the same item implies that often several ISRFs simultaneously show such disturbing non-monotonicities. For the example item one may conclude that when the questionnaire contains few items, which are effective in the high \u03b8 area, this item may be retained to cover this area even though this would be at the expense of measurement quality just below the middle of the scale.\nFor each item, the MHM framework provides M location parameters and a scalability coefficient, which provides information about item discrimination. The location parameters are the proportions of the population of interest, which have at least a score m on item j, and which are denoted by \u03c0jm, m\u00a0=\u00a01, ..., M. For the same item, due to non-intersection of ISRFs we have that \u03c0j1\u00a0\u2265\u00a0 ...\u00a0\u2265\u00a0\u03c0jM, whereas in the GRM item location parameters are ordered oppositely, \u03b4j1\u00a0\u2264\u00a0...\u00a0\u2264\u00a0\u03b4jM. The item scalability coefficient Hj (e.g., [2], pp. 148\u2013153; [18], chap. 4) summarizes the discrimination power of an item across its M ISRFs. The numerical Hj value is determined by the interplay of the slope and the location of the ISRFs of all J items and the distribution of the latent variable \u03b8 [30], and it expresses how well item j separates patients given the ISRFs of item j relative to the other items\u2019 ISRFs and the distribution of \u03b8. Mathematically, holding constant this distribution and the location of all items\u2019 ISRFs, coefficient Hj is higher when the slopes of ISRFs of item j are steeper [18].\nNonparametric versus parametric graded response models\nRelationships Among Models. The MHM shares assumptions UD, LI and M with the GRM, but the MHM is less restrictive with respect to the shape of the ISRFs. Thus, the MHM is more general than the GRM or, equivalently, the GRM is a special case of the MHM. This hierarchy implies that, if the GRM fits the data, by implication the more general MHM also fits but if the MHM fits the data, this does not imply that the GRM also fits. Fit of the GRM then needs to be investigated separately. Because of the hierarchical relationship, for any data set the MHM fits as least as many items as the GRM (Table\u00a01).Table\u00a01Comparison of monotone homogeneity model (MHM) and graded response model (GRM)Nonparametric IRT (MHM)Parametric IRT (GRM)Restrictiveness of modelsLow; many items admitted to the scaleHigh; fewer items admitted to the scaleInterpretation of parametersIntuitively appealingMore-complicatedParameters(typical range)\u00a0\u00a0\u00a0\u00a0Person levelT, X+\u03b8(\u22123\u00a0\u2264\u00a0\u03b8\u00a0\u2264\u00a03)\u00a0\u00a0\u00a0\u00a0ISRF location\u03c0jm\u03b4jm(\u22123\u00a0\u2264\u00a0\u03b4jm\u00a0\u2264\u00a03)\u00a0\u00a0\u00a0\u00a0ISRF discriminationHj\u03b1j(0.5\u00a0\u2264\u00a0\u03b1j\u00a0\u2264\u00a02.5)Data analysisExploratory, data as point of departureConfirmatory, model as null hypothesisApplicationsComparing groupsMeasuring changeDiagnosing patientsComparing groupsMeasuring changeDiagnosing patientsConstructing item banksAdaptive testing\nPatient and Item Parameters. The MHM and the GRM provide the following patient and item parameters (also, see Table\u00a01):For patient measurement, the MHM uses total score X+ to order patients on latent variable \u03b8. Because total score X+ has an easy interpretation and, moreover, in many IRT models X+ and \u03b8 tend to correlate extremely high suggesting a strong linear relationship [31], total score X+ may be preferred in practice. Total score X+ is the sum of the rating scale scores on the J items, whereas estimates of \u03b8 are expressed on a logit scale, which does not have a straightforward interpretation for users of HRQoL scales. In general, the ordinal relationship of \u03b8 with total score X+ (which can be approximated well by a linear relation) enables users to switch between scales, and use the one that suits their goals best.Because item location \u03b4jm is expressed on the same scale as latent variable \u03b8, it also has an interpretation in logits. For many users, proportion \u03c0jm, the proportion of patients who have at least an item score of m, has an easier interpretation.Item discrimination \u03b1j gives the maximum slope of the logistic ISRF irrespective of the locations of the other ISRFs of item j and the other items in the questionnaire, and irrespective of the distribution of \u03b8. Thus, information on ISRF slopes is absolute in the sense that a particular \u03b1j value does not provide information on the item\u2019s suitability for measurement in a particular group (characterized by a particular distribution of \u03b8) by means of a set of J items (characterized by particular location and slope parameters). On the other hand, item scalability coefficient Hj depends explicitly on the interplay of the distribution of \u03b8, the spread of locations of the ISRFs, and the slopes of the ISRFs. In particular, keeping two of these factors fixed, Hj tends to increase in the third. This dependence on the distribution of \u03b8 and the item properties informs the researcher precisely how well item j separates patients with low and high \u03b8 values in the particular group of patients under consideration using the particular set of items. The difference between absolute slope information (\u03b1j) and relative slope information (Hj) is illustrated as follows.\nTwo data sets of size N\u00a0=\u00a05,000 and five items (J\u00a0=\u00a05) (M\u00a0=\u00a03 for each item) were generated using the item parameters in Table\u00a02. The first data set came from a hypothetical clinical population with \u03b8 \u223c N(\u22122,1) (i.e., low psychological well-being level), and the second data set came from a hypothetical healthy population with \u03b8\u00a0\u223c\u00a0N(1,1) (i.e., high psychological well-being level). It may be noted that for each of the five items, \u03b1j\u00a0=\u00a01.4 by definition, irrespective of ISRF location parameters and the \u03b8 distribution. The Hj values in the clinical group were computed and found to range from .36 to .39. In the healthy group, the Hj values were found to be smaller for items 1, 4, and 5 (H1\u00a0=\u00a0.26, H4\u00a0=\u00a0H5\u00a0=\u00a0.25). In the clinical group, the ISRFs of these three items were located more closely to the middle of the \u03b8 distribution (Fig.\u00a03 shows this for Item 4) such that higher Hj values resulted, but in the healthy group the ISRFs were located further in the lower tail of the \u03b8 distribution (Fig.\u00a03) resulting in lower Hj values (.3 is considered minimally acceptable; [2], chap. 5). Thus, the items 1, 4, and 5 are well suited for measurement in the clinical group but not in the healthy population, despite their overall discrimination power expressed by \u03b1j\u00a0=\u00a01.4, for J\u00a0=\u00a01, ..., 5. For item 3, H3 was a little higher in the healthy group because the second and third ISRFs discriminate particularly well at higher ranges of \u03b8. For item 2, the location parameters of the ISRFs were widely spread across the \u03b8 distribution, resulting in good discrimination both at lower and higher levels of \u03b8 in both distributions. We conclude that item scalability coefficient Hj has the advantage that it takes the item (and not the individual ISRF) as a unit and depends simultaneously on the \u03b8 distribution, the slopes of the ISRFs, and the spread of the locations of the ISRFs. Thus, Hj informs the researcher whether item j discriminates well in the group under consideration using the particular set of items, whereas item discrimination \u03b1j provides information about the discrimination power irrespective of the patient group under consideration and the item properties of item j and the other items in the scale.Table\u00a02Example of item parameters of the graded response model (GRM), and Item H Values in two different populations with normally distributed latent traitsItem jItem parameters GRMHj\u03b1 j\u03b4j1\u03b4j2\u03b4j3\u03b8\u223cN(\u20132,1)\u03b8\u223cN(1,1)11.4\u20132.4\u20132.2\u20131.00.360.2621.4\u20134.0\u20132.01.00.370.3931.4\u20131.02.02.50.390.4241.4\u20133.0\u20132.0\u20131.00.360.2551.4\u20132.5\u20132.0\u20131.50.360.25Note: Hj values are based on simulated data for sample size N\u00a0=\u00a05,000Fig.\u00a03Three ISRFs of the same item (Item 4) relative to two different distributions of the latent variable\nConfirmatory and exploratory data analysis. In general, before IRT models are accepted as reasonable descriptions of the data their goodness-of-fit to these data must be investigated and assessed. In general, goodness-of-fit research is different for parametric and nonparametric IRT (with the GRM and the MHM as special cases, respectively). In a parametric IRT analysis the model often serves as null-hypothesis and it is tested whether this null-hypothesis must be rejected or may be supported by the data. Nonparametric IRT analysis in general takes the data as point of departure and (1) instead of positing a unidimensional or multidimensional latent variable structure analyzes the data to find its true dimensionality, and (2) instead of positing a logistic or other functional shape estimates the ISRFs from the data so as to diagnose the items\u2019 functioning [16, 18]. This research strategy renders nonparametric IRT a more flexible data-analysis tool than parametric IRT. One could also characterize this distinction as confirmatory (parametric IRT) versus exploratory (nonparametric IRT) (Table\u00a01).\nApplication of parametric and nonparametric IRT. If a nonparametric IRT model such as the MHM fits the data, the result is a scale on which patients can be ordered by means of the total score X+. This total score has a strong linear correlation with latent variable \u03b8. Such a scale suffices in many applications. Examples are the comparison of groups, the measurement of change due to therapy, and the establishment of the patient\u2019s psychological well-being level as low, medium, or high (Table\u00a01). The practical advantage of nonparametric IRT models over parametric IRT models is that the scales they produce contain more items thus reducing the risk of wasting items that have non-logistic but monotone ISRFs that discriminate well in (part of) the group under consideration (e.g., Fig.\u00a02b). Such items contribute well to reliable measurement. In addition, rejection of such items may also harm the coverage of the latent attribute.\nIf a fitting parametric IRT model is obtained for a set or a subset of the items, one has a parsimonious description of the item characteristics, and one can use the estimated item parameters to scale the items, and the estimated \u03b8s as interval level measures to locate patients on this scale. If a large set of items, also known as an item bank [32], is available, and if a parametric IRT model fits the item bank, parametric IRT models have the advantage that the patient\u2019s \u03b8 can be assessed using different sets of items from the item bank. This may be useful for the measurement of change when change is so large that the set of items that was used initially no longer captures the higher or lower \u03b8 levels needed for the second measurement, thus necessitating the use of other items. Another application of parametric IRT is computerized adaptive testing (CAT), which selects items that match the patient\u2019s \u03b8 level well from a huge item bank so as to optimize accuracy of \u03b8 measurement. In principle, CAT requires different item sets for different \u03b8 values (Table\u00a01).\nNonparametric IRT analysis in practice \nSoftware for nonparametric IRT analysis. Several programs are available for data analysis using nonparametric IRT but not each can handle polytomous item scores. We briefly discuss the programs MSP ([33]; also see [4]) and TestGraf98 [34]. Both programs are used regularly and, together, they provide the researcher with a clear and informative picture of (1) the dimensionality of the data, (2) the (lack of) monotonicity of the ISRFs, and (3) estimates of item locations and item discrimination.\nProgram MSP uses the MHM as the main analysis model but another nonparametric model not discussed here is also included in the program. Basically the program consists of two parts. The first part of MSP is an algorithm for exploring the dimensionality of the data ([2], chap. 5; [4], chap. 5). The algorithm uses item scalability coefficient Hj to select items. Items that are related to the same latent variable \u03b8 are selected one by one on the basis of their Hj value. Suppose that one latent variable drives the responses to one subset of the items, another latent variable drives the responses to another subset of items, and so on. Then, the algorithm selects mutually exclusive clusters of items each of which is driven by a different latent variable.\nThe second part of MSP provides several statistical tools for exploring the shape of the ISRFs [29]. This is most useful after the dimensionality of the data has been ascertained. For example, due to their strong positive tendency the non-monotone ISRFs in Fig.\u00a02b may have an Hj value that is high enough for the items to be selected in a unidimensional cluster, but the non-monotonicity also may distort parts of the ordinal scale defined by the items in the cluster. MSP estimates ISRFs by means of a number of discrete points that are connected to form a jagged \u2018curve\u2019. Figure\u00a04a shows four discrete ISRFs of the same item (i.e., M\u00a0=\u00a04) each estimated by means of eleven points. The researcher can manipulate the number of estimated points. If more points are estimated from the same data more details of the ISRF become visible (i.e., bias is reduced) but because for each point fewer data are available, accuracy decreases. In statistics, this is known as the bias-accuracy trade-off, and it is advisable to try several options to reach a good decision. MSP tests observable deviations from monotonicity for significance. Figure\u00a04a also shows a \u2018mean\u2019 ISRF (dashed curve), which we may call the item score function (ISF) and which is not standard output of MSP. This function must be monotone nondecreasing.Fig.\u00a04(a) Discrete estimates of four ISRFs (solid curves; from MSP) of Item 16 (\u2018Satisfied doing daily activities?\u2019, from Physical Health and Well Being domain), and the corresponding discrete item-score function (dashed curve), (b) also estimated as a continuous curve (from Testgraf98)\nProgram TestGraf98 [34] can be used for studying the shape of the ISF. Unlike MSP, TestGraf98 produces continuous estimates of response functions ([35]; here, only the ISFs); and like MSP, TestGraf98 shows graphical displays of these estimates that can be manipulated with respect to bias and accuracy, and also here it is advisable to try several options. The quality of the decision can be improved by using the confidence envelopes for the estimated ISFs for statistical testing. Figure\u00a04b shows an example of an estimated ISF (solid curve) and its confidence envelopes (dashed curves), which were estimated by means of TestGraf98. \nResearch strategies for nonparametric IRT analysis. MSP provides a method for investigating the dimensionality of the data, and MSP and TestGraf98 both can be used to investigate assumption M. The investigation of dimensionality and monotonicity serves to identify the items that together constitute an ordinal patient scale for the same latent variable.\nFor investigating dimensionality, MSP offers the researcher the possibility to set a positive lower bound c on Hj. Under the MHM, the lowest admissible value is c\u00a0=\u00a0.0; MSP\u2019s default is c\u00a0=\u00a0.3 ([2], chap. 5; [4, 33]). This default value ascertains a lower bound on the overall discrimination power of the items (but researchers are free to choose a higher value) and, as a result, item clusters consist only of sufficiently discriminating items that measure the same latent variable. Thus, MSP aims to produce unidimensional scales that allow accurate patient measurement.\nTestGraf98 estimates the ISFs (e.g., Fig.\u00a04b) by means of the nonparametric regression method known as kernel smoothing (e.g., [36], chap. 2; [35]). The availability of confidence envelopes for the continuous ISF estimates provides detailed information of (lack of) monotonicity for each item. TestGraf98 provides these estimates irrespective of the dimensionality of the data. Thus, a good research strategy is to first investigate item-set dimensionality by means of MSP and then use MSP and TestGraf98 to study the ISRFs and the ISFs in dimensionally distinct clusters. See [37] for another method for assessing the shape of these curves.\nA real-data example: The World Health Organization Quality-of-Life Scale\nThe WHOQOL-Bref was developed for assessing individuals\u2019 perception and feelings of their daily life. The questionnaire starts with two items, which ask for global estimates of one\u2019s quality of life, and then continues with 24 items covering four domains: (a) physical health and well-being (seven items); (b) psychological health and well-being (six items); (c) social relations (three items); and (d) environment (eight items). The two general items were left out of the analysis. In agreement with their numbering in the WHOQOL-Bref, the other 24 items were numbered from 3 to 26. Examples of items are:Do you have enough energy for daily life? (physical domain)How much do you enjoy life? (psychological domain)How satisfied are you with your personal relationships? (social domain)How safe do you feel in your daily life? (environmental domain)\nEach item uses a five-point rating scale (i.e., Xj\u00a0=\u00a00, ..., 4 ); the higher the item score, the better one\u2019s quality of life on the specific domain covered by the item.\nThe data were collected by undergraduate psychology students of Tilburg University as part of a course Research Practical in the academic year 2005\u20132006. Students were instructed to strive for a sample of participants equally distributed across both sexes and the following age categories: 30\u201339, 40\u201349, 50\u201359, and more than 60\u00a0years. The final sample consisted of N\u00a0=\u00a0589 respondents from the Dutch population. Of these respondents, 55% were women, mean age was 55.2\u00a0years (SD\u00a0=\u00a014.6), 32% had completed community college or university, 36% had completed vocational school, 20% had high school at most, and 12% had only elementary school or less.\nN\u00a0=\u00a055 cases had missing item scores. Missing values were estimated using two-way imputation. Comparable to an analysis-of-variance layout, this method uses both a person effect and an item effect for estimating a missing score (for details, see [38]). MSP and TestGraf98 were used to analyze these data and construct one or more scales, thus illustrating the possibilities of the MHM. For the sake of comparison, we ran a principal component analysis and a GRM scale analysis on the data.\nResults\nSample statistics of item and scale scores \nTable\u00a03 shows that the mean item scores ranged from 2.58 (Item 20: \u2018Satisfied with sex life?\u2019) to 3.46 (Item 25: \u2018Moving around well?\u2019). The mean X+ scores were 21.04 (physical domain), 17.01 (psychological domain), 8.52 (social domain), and 24.27 (environmental domain). Correlations between the domain scores ranged from .37 (between physical and social domains) to .51 (between physical and environmental domains). Table\u00a03Results from MSP item selection procedure (Item clusters, item Hj values, and total H), and Item Hj values and total H for each content domainMSP item selection procedureHj per content domainc\u00a0=\u00a0.3c\u00a0=\u00a0.4jMean1212345Physical health and well-being3Distraction due to paina3.04.59.59.4010Experiencing energya2.98.43.53.4615Satisfied with sleep2.66.22\u2013\u2013\u2013\u2013\u2013\u2013.2825Moving around well3.46.36.43.4116Satisfied doing daily activities2.84.41.56.524Need medical treatment for daily functioninga3.17.59.59.4317Satisfied work capacity2.89.40.57.52Scale value21.04.43Psychological health and well-being5Enjoying life2.66.34.42.377Being able to concentrate2.80.32\u2013\u2013\u2013\u2013\u2013.2918Satisfied with yourself2.95.41.48.4511Acceptance physical appearance3.23.33\u2013\u2013\u2013\u2013\u2013.3526Experiencing negative feelingsa2.72.30\u2013\u2013\u2013\u2013\u2013.346Life meaningful2.66.30\u2013\u2013\u2013\u2013\u2013.37Scale value17.01.36Social relations19Satisfied relationship with other people3.06.34.50.5020Satisfied with sex life2.58.30.42.4221Satisfied support from others2.88.28\u2013.40.40Scale value8.52.44Environment8Feeling safe in daily life3.08.30\u2013\u2013\u2013\u2013\u2013.3322Satisfied living conditions3.13.43\u2013\u2013\u2013\u2013\u2013.4312Enough financial resources3.08.33.53.4223Satisfied getting adequate health care2.87.29\u2013.52.3613Availability information needed in daily life3.06.34.53.4014Opportunities leisure2.90.34.49.399Healthy environment2.88.29\u2013\u2013\u2013\u2013\u2013\u2013.3124Satisfied with transport in daily life3.28.34.52.40Scale value24.27.38aReversely scored items \nDimensionality analysis\nPrincipal components analysis. Dimensionality was explored by means of a principal components analysis using polychoric correlations. The ratio of the first to the second eigenvalue of the polychoric correlation matrix was 8.428\/1.986\u00a0=\u00a04.24. A ratio of 4:1 is taken as evidence of considerable strength of the first dimension (e.g., [39]). The first factor explained 32.4% of the variance. A confirmatory factor analysis of the four a priori domain scales of the WHOQOL-bref improved fit over the one-factor model (P\u00a0\u2264\u00a00.001). However, the factors correlated from .50 (physical and social domain) to .79 (psychological and social domain). The explorative factor analysis in conjunction with the confirmative factor analysis justifies the assumption of a general HRQoL dimension underlying each scale.\nMonotone homogeneity model analysis. Next, MSP was used treating all 24 items as a fixed scale. The MHM does not allow negatively correlating items in one scale. Item 4 (\u2018Need medical treatment for daily functioning?\u2019) and Item 20 (\u2018Satisfied with sex life?\u2019) correlated negatively but not significantly (P\u00a0>\u00a0.05); thus all 24 items were used for analysis. The item Hj values (not tabulated) ranged from .21 (Item 15: \u2018Satisfied with sleep?\u2019) to .40 (Item 22: \u2018Satisfied with living conditions?\u2019 and Item 10: \u2018Enough energy for everyday life?\u2019). The total-scale H coefficient was equal to .30. The results suggest that the items tend to cover one latent HRQoL aspect, which, however, induces only weak general association between the items. Thus, in addition to this common aspect it seems reasonable to also look for more-specific HRQoL aspects that are covered by subsets of items.\nDimensionality was investigated by means of the MSP search algorithm using several c values, starting with .3 (default) and then increasing c with steps of .05 in each next analysis round. We only report results for c\u00a0=\u00a0.3 and c\u00a0=\u00a0.4 (other values did not reveal interesting results). For c\u00a0=\u00a0.3, one scale consisting of 18 items (H\u00a0=\u00a0.35) and one scale consisting of 2 items (H\u00a0=\u00a0.59) were found (Table\u00a03). The four remaining items were not selected because their Hj values were under .3 (i.e., .22 and .28, .29, and .29). The 18-item scale had a rather heterogeneous content. The 2-item scale asked about distraction due to pain (Item 3) and the need for medical treatment for daily functioning? (Item 4). Thus, their high scalability may be explained by the use of palliative medicines. \nFor c\u00a0=\u00a0.4, five scales were found consisting of 6, 2, 3, 2, and 3 items, respectively (Table\u00a03). The first scale consisted of items from the physical domain and the psychological domain. The other scales consisted of items from one domain. Scale 2 again covered Item 3 and Item 4. Scales 3 and 4 covered environmental-domain aspects. Scale 5 contained all social-domain items.\nThus, for default c\u00a0=\u00a0.3, 18 of the 24 items were selected in one scale. The pattern of item selection for higher c values such as c\u00a0=\u00a0.4 showed that the item set progressively crumbled into many smaller scales while other items remained unselected. Sijtsma and Molenaar ([4], pp. 80\u201386; see also [7] [40]) argued that this typical pattern of results gives evidence that the 18-item set constitutes a unidimensional scale. The total-scale H equaled .35, giving evidence of weak scalability ([2], p. 185). Most of the item Hj values were between .3 and .4, also suggesting a weak relationship with the latent variable ([2], p. 185). \nFinally, it was investigated whether the four a priori identified item domains could be considered as separate scales. Table\u00a03 (last column) shows that the total-scale H values ranged from .36 (environmental domain) to .44 (social domain). Thus, based on Mokken\u2019s classification of scales [2] the four a priori item domains constituted weak to medium scales. Two items had H values just smaller than c\u00a0=\u00a0.3 (i.e., Item 15 (H15\u00a0=\u00a0.28): \u2018Satisfied with sleep?\u2019 and Item 18 (H18\u00a0=\u00a0.29): \u2018Satisfied with yourself?\u2019). The content domains may be considered as unidimensional clusters of items measuring distinct aspects of HRQoL, each of which are related to a more general underlying HRQoL construct. Because of their conceptual clarity, the remaining analyses were done on the a priori defined item domains.\nMonotonicity assessment in each item domain\nFor each item domain, MSP was used to assess the ISRFs\u2019 shapes. First, ISRFs were estimated accurately (i.e., many cases were used to estimate separate points of the ISRFs) but at the expense of possible bias (i.e., only few points were estimated). Second, ISRFs were estimated with little bias (i.e., many points were estimated) but at the expense of accuracy (i.e., few cases were used to estimate each point).\nFor the physical domain, the first analysis (high accuracy, more risk of bias) revealed four items of which one or more ISRFs showed minor violations of monotonicity, but none of these violations were significant (5% level, one-tailed test, because only sample decreases are tested as violations; increases support monotonicity). The second analysis (more inaccuracy, less bias) revealed that for all seven items one or more ISRFs showed one or more local decreases, but none them were significant. Figure\u00a05a shows the local, nonsignificant decreases in the ISRFs for Item 3 (\u2018Distraction due to pain?\u2019).Fig.\u00a05Four ISRFs of Item 3 (\u2018Distraction due to pain?\u2019, from Physical Health and Well Being domain) showing nonsignificant violations of assumption M and rejected by the GRM: (a) Results from MSP (including the ISF); (b) results from Testgraf98 (ISF and confidence envelopes); (c) and results from Multilog7.0 (GRM)\nFor the psychological domain, for both analyses (i.e., high accuracy versus little bias) two items were found which had ISRFs showing significant local decreases. For example, Fig.\u00a06a shows for Item 7 (\u2018Being able to concentrate?\u2019) that the estimate of ISRF P72(\u03b8)\u00a0=\u00a0P(X7\u00a0\u2265\u00a02|\u03b8) (the second curve from the top) has several local decreases, the largest of which was significant (5% significance level, P\u00a0=\u00a00.019). The estimate of ISRF P73(\u03b8)\u00a0=\u00a0P(X7\u00a0\u2265\u00a03|\u03b8) shows two small decreases; they were not significant. For the social domain and the environmental domain, no significant violations of the monotonicity assumption were found. It can be concluded that assumption M holds for each of the four scales.Fig.\u00a06Four ISRFs of Item 7 (\u2018Being able to concentrate?\u2019) showing significant violations of assumption M and rejected by the GRM: (a) Results from MSP (including the ISF); (b) results from Testgraf98 (ISF and confidence envelopes); and (c) results from Multilog7.0 (GRM)\nNext, TestGraf98 was used to investigate assumption M for the ISFs. Several sample sizes were used for estimating curve fragments of the ISFs and balancing the bias-accuracy trade-off. Figure\u00a05b shows the estimated ISF of Item 3 (\u2018Distraction due to pain?\u2019). The confidence envelopes show that the local decrease of the estimated ISF can be ignored safely. For the estimated ISF of Item 7 (\u2018Being able to concentrate?\u2019), Fig.\u00a06b suggests a violation of assumption M for high latent variable levels (i.e., \u03b8\u00a0>\u00a02). With MSP the user specifies the minimum number of observations used for estimating each point of an ISRF, but Testgraf98 controls the bias-accuracy trade-off by means of a bandwidth parameter, also to be specified by the user but without being able to control the number of observations for estimating separate curve fragments. The effect may be that, in particular at the lower end and higher end of the scale, the ISF is estimated very inaccurately. Combining the results from MSP (no significant decreases of the ISRFs at the higher end of the \u03b8 scale) and Testgraf98 (a smooth monotone increasing ISF in the middle of the \u03b8 scale), we may conclude that assumption M holds for item scores. Thus, the expected item score monotonically increases in the latent variable.\nComparison of the MHM with the GRM \nThe GRM was fitted using Multilog7.0 [41]. Estimation problems occurred for item scoring 0\u20134 because some score categories were (almost) empty. This was resolved by combining scores 0 and 1 into score 0, and re-scoring the items 0\u20133. Table\u00a04 provides the estimated slope parameters (\u03b1j) and the three location parameters (\u03b4jm, j\u00a0=\u00a01, 2, 3), and also the estimated item Hj coefficients (also in Table\u00a03, last column) and the nonparametric location parameters (\u03c0jm, j\u00a0=\u00a01, 2, 3). The location parameters indicate that the items are relatively popular (highly endorsed).Table\u00a04Results of monotone homogeneity model (MHM) scale analysis and estimated item parameters from the graded response model (GRM)MHMGRM bjHj\u03c0j1\u03c0j2\u03c0j3\u03c0j4\u03b1j\u03b4j1\u03b4j2\u03b4j3Physical health and well-being3Distraction due to painb.40.99.93.72.401.14\u22122.71\u22120.990.4910Experiencing energya.46.98.95.79.451.97\u22122.24\u22120.630.5615Satisfied with sleep.28.99.95.70.340.85\u22122.09\u22120.791.7825Moving around well.41.99.96.91.611.23\u22123.08\u22122.24\u22120.4116Satisfied doing daily activities.52.98.93.72.213.96\u22121.59\u22120.580.874Need medical treatment for daily functioninga.43.98.92.75.231.23\u22122.82\u22121.290.2417Satisfied work capacity.52.99.98.77.203.58\u22121.56\u22120.690.81Scale value.43Psychological health and well-being5Enjoying life.371.00.98.61.071.93\u22122.79\u22120.381.977Being able to concentrate.29.99.98.77.200.82\u22124.270.681.6918Satisfied with yourself.451.00.93.64.151.99\u22122.85\u22120.971.1211Acceptance physical appearance.35.99.98.80.451.08\u22123.93\u22121.560.2226Experiencing negative feelingsa.341.00.97.61.081.11\u22122.84\u22120.601.906Life meaningful.371.00.96.62.231.90\u22122.71\u22120.351.94Scale value.36Social relations19Satisfied relationship with other people.50.99.96.82.292.52\u22122.21\u22121.100.9720Satisfied with sex life.40.97.88.58.151.32\u22123.19\u22120.901.3821Satisfied support from others.42.99.97.72.201.38\u22121.88\u22120.341.62Scale value.44Environment8Feeling safe in daily life.331.00.98.78.321.12\u22124.12\u22121.370.8422Satisfied living conditions.43.98.90.68.341.96\u22122.71\u22121.270.6312Enough financial resources.42.99.95.70.441.84\u22122.22\u22120.730.2123Satisfied getting adequate health care.36.99.97.68.231.32\u22122.84\u22120.931.3313Availability information needed in daily life.40.99.95.72.211.64\u22123.21\u22120.980.6014Opportunities leisure.39.99.98.75.341.61\u22121.94\u22120.690.609Healthy environment.31.99.98.83.321.02\u22123.95\u22120.891.4324Satisfied with transport in daily life.40.99.97.86.451.66\u22122.77\u22121.550.17Scale value.38a\u00a0Reversely scored items; b\u00a0For the GRM analysis, items were recoded by collapsing item scores 0 and 1 into item score 1\nSeveral methods are available for assessing the fit of the GRM, but many are problematic and no generally accepted standard goodness-of-fit method for the GRM is presently available ([42] pp. 85\u201389). We investigated goodness-of-fit of the GRM by means of posterior predictive assessment [43], which can provide graphical and numerical evidence about model fit. Model fit was investigated at the level of items. The most interesting results were found for items from the physical and psychological domains. Results are only discussed for these domains. For the physical domain, Item 3 (\u2018Distraction due to pain?\u2019) and Item 7 (\u2018Being able to concentrate\u2019) showed significant misfit (P\u00a0<\u00a0.01). In Fig.\u00a05c, the curve made up by dots connected by straight line pieces represents the estimated nonparametric ISF of Item 3, the solid curve represents the expected ISF of Item 3 under the GRM, and the dotted curves represent the 95% confidence envelopes. The GRM rejects this item. Thus, modeling the jagged pattern of the estimated ISF by means of logistic ISRFs having the same slopes would do injustice to the data. However, it is noteworthy that Item 3 has good measurement properties (Table\u00a04: e.g., H3\u00a0=\u00a0.40) under the more general MHM, and from this model\u2019s perspective it might be retained in the scale.\nItem 7 (\u2018Being able to concentrate?\u2019) was a popular item (Table\u00a04; \u03c0j1\u00a0=\u00a0.99, \u03c0j2\u00a0=\u00a0.98; also, see Fig.\u00a06a, upper two ISRFs). As a result, the GRM could not be estimated accurately; item parameters were estimated very inaccurately (standard errors >.25). Figure\u00a06c shows evidence of misfit at \u03b8\u00a0>\u00a02, for which the observed ISF fell outside the 95% confidence interval. This means that the GRM gives biased results for \u03b8\u00a0>\u00a02. The nonparametric estimates of the ISRFs were monotone. This provides evidence that the MHM adequately fitted Item 7. However, H7\u00a0=\u00a0.29, which is rather low. A reason to keep this item in the scale is that it may help measuring differences at the lower and middle ranges of the \u03b8 scale, which are the most relevant ranges for measuring HRQoL. \nSummary of the scale properties\nThe WHOQOL-Bref is most often used in scientific research (e.g., epidemiological studies and clinical trails) and by health professionals (e.g., to assess treatment efficacy) [19]. The nonparametric MHM analyses revealed that the scales have adequate properties for comparing groups on the underlying HRQoL aspects. Each of the four domains of the WHOQOL-Bref constitutes a unidimensional scale, each scale measuring a different aspect of HRQoL in addition to a weak common HRQoL attribute. This justifies reporting both separate domain scores and possibly an overall HRQoL score. The scalability results showed that the domain scales are weak to moderate, with scalability coefficients H ranging from .36 to .44. The test-score reliabilities of the four domain scores were .82, .76, .66, and .81, respectively. The rank correlations between sum score X+ and the estimated \u03b8 from the GRM varied from .91 (\u2018physical health\u2019) to .96 (\u2018environment\u2019) (Pearson correlations ranged from .94 (\u2018physical health\u2019) to .99 (\u2018environment\u2019)). Thus, X+ and estimated \u03b8 carry nearly the same rank order (and numerical) information. This interesting result further justifies the use of the nonparametric MHM for scale analysis, and the use of X+ for (at least) ordinal measurement of persons. \nBecause the item-score distributions were severely skewed to the left, the lower response categories 0 and 1 were ineffective for HRQoL measurement in the general population. The locations of the ISRFs for the higher response categories 2, 3, and 4 were well spread along the \u03b8 scale. The ISRFs\u2019 discrimination power as reflected by the Hj values often was in the weak to medium range. Thus, the higher response categories are modestly informative across a wide range on the \u03b8 scale.\nThe relatively short WHOQOL-Bref may also be considered for use as a tool for assessing HRQoL at the individual level in clinical and medical settings. For example, the WHOQOL-Bref may be used to evaluate whether a patient\u2019s HRQoL has improved after taking medication. An interesting feature of a fitting IRT model is that psychometric properties can be evaluated conditionally on the latent variable. For example, the measurement error of X+ can be evaluated at different values of the latent variable. TestGraf98 provides graphical information about the standard error of measurement based on the MHM model. For example, for the physical-health domain Testgraf98 estimated a standard error of measurement ranging from 2.8 for X+\u00a0\u2264\u00a020 to 1.8 for X+\u00a0\u2265\u00a028. Thus, to be significant at the 5% significance level differences between two observed X+ scores have to be larger than  for X+\u00a0\u2264\u00a020, and larger than  for X+\u00a0\u2265\u00a028 (e.g., see [44], p. 209). This is a substantial standard error of measurement relative to the length of scale. This relatively large measurement error appears to be consistent with the observed Hj values, which indicate weak to moderate scalability. For the other three content domains, the standard error of measurement was also substantial. Thus, caution has to be exercised when drawing conclusions about differences and changes in individual levels of HRQoL based on observed X+ scores from the WHOQOL-Bref and any other HRQOL measure\u2014see [45].\nDiscussion\nThis study explained how the nonparametric monotone homogeneity model contributes to the construction of scales for the measurement of HRQoL. The MHM is more general than parametric IRT models [24], such as the much-used parametric graded response model [26] but also the partial credit model [13\u201315] and the generalized partial credit model [11]. Hemker et\u00a0al. [46] showed that all known parametric IRT models for polytomous items are special cases of the nonparametric MHM. This means that any item set satisfying the requirements of a parametric IRT model for polytomous items also satisfies the requirements of the nonparametric MHM. Given the greater generality and flexibility of the nonparametric MHM, which results in longer scales, and because X+ and estimated \u03b8 carry the same rank order information (based on the approximate stochastic ordering property of \u03b8 given X+), the nonparametric MHM is highly suited for person measurement.\nIn an HRQoL context, often little is known about the psychometric properties of new questionnaires. A typical nonparametric MHM analysis explores the dimensionality of the data by capitalizing on model assumptions such as monotonicity (MSP), and studies the shapes of the ISRFs and the ISFs in order to learn more about the (mal-)functioning of individual items (MSP and TestGraf98). This results in scales on which groups can be compared and changes monitored without making unduly restrictive assumptions about the data.\nThe properties of any IRT model only hold for the application at hand when the model fits the data. In case of misfit, the structure of the model does not match the structure of the data. One cause of misfit is that the data are multidimensional while the model assumes unidimensionality. Another cause of misfit is that the real ISRFs may not be monotone or that they are monotone but fail to have the logistic shape assumed by many parametric models. Other causes of misfit, such as a multiple-group structure as in differential item functioning (e.g., [47]) or person misfit [48] were not considered here.\nWhen the MHM fits the data, the researcher may decide to also investigate goodness of fit of the GRM or other parametric IRT models for polytomous items. The choice of a parametric model may be based on the flexibility of the model. For example, the partial credit model only has item location parameters but assumes the slopes of the response functions to be the same within and between items, whereas the generalized partial credit model also allows for varying slope parameters between items, just as the GRM. If one pursues a parametric IRT analysis, misfit may be a good reason to resort to a nonparametric IRT model and still have an ordinal patient scale. If CAT is pursued, one of the parametric models is a better option provided the model fits the data well. In an HRQoL context, CAT indeed could prove to be successful because patients have a definitive interest in providing truthful answers (in the educational context, in which CAT originated, CAT requires that items be kept secret. This requires item banks often containing hundreds of calibrated items). As a result, in HRQoL measurement CAT presently meets with a growing interest (e.g., [49\u201354]).","keyphrases":["nonparametric monotone homogeneity model","parametric graded response model","item response theory","health-related quality-of-life measurement"],"prmu":["P","P","P","R"]}
{"id":"Arch_Dermatol_Res-3-1-1950346","title":"Expression of the chemokine receptor CCR5 in psoriasis and results of a randomized placebo controlled trial with a CCR5 inhibitor\n","text":"Several reports have indicated that the chemokine receptor CCR5 and its ligands, especially CCL5 (formerly known as RANTES), may play a role in the pathogenesis of psoriasis. The purpose of this investigation was to examine the expression of CCR5 and its ligands in chronic plaque psoriasis and to evaluate the clinical and immunohistochemical effect of a CCR5 receptor inhibitor. Immunohistochemical analysis showed low but significant increased total numbers of CCR5 positive cells in epidermis and dermis of lesional skin in comparison to non-lesional skin. However, relative expression of CCR5 proportional to the cells observed revealed that the difference between lesional and non-lesional skin was only statistically significant in the epidermis for CD3 positive cells and in the dermis for CD68 positive cells. Quantification of mRNA by reverse transcriptase-polymerase chain reaction only showed an increased expression of CCL5 (RANTES) in lesional skin. A randomized placebo-controlled clinical trial in 32 psoriasis patients revealed no significant clinical effect and no changes at the immunohistochemical level comparing patients treated with placebo or a CCR5 inhibitor SCH351125. We conclude that although CCR5 expression is increased in psoriatic lesions, this receptor does not play a crucial role in the pathogenesis of psoriasis.\nIntroduction\nPsoriasis is a chronic skin disease affecting approximately 2\u20133% of the population worldwide. Despite its common occurrence, the exact pathogenesis of psoriasis remains unclear and adaptations to the pathogenesis of this inflammatory disease are continuing [4, 9, 31, 35, 40, 52]. Although the specific effector cells responsible for the inflammatory process in psoriasis are not known, reported beneficial effects of specific T cell targeted therapies, such as cyclosporine A, DAB389IL-2 and alefacept support a central role for T cells in the pathogenesis of psoriasis [3, 5, 10, 13, 15, 24, 25]. Further investigation on the immunophenotype and cytokine secretion patterns of T cells have indicated that specifically Th1-cells are involved in psoriasis [2, 32, 46].\nIn the many aspects encompassing T-cell homeostasis, the trafficking of T cells from blood to tissues is thought to be relevant in chronic inflammatory diseases such as psoriasis. Key factors in this migration are chemo-attractant cytokine molecules known as chemokines and their receptors [30, 33, 37, 38, 57, 61, 62]. The predominant chemokine receptors expressed on Th1-cells are CCR5 and CXCR3 [7, 9, 29, 30, 33, 34, 47, 50]. Besides its preferential expression on Th1 cells, CCR5 is also expressed on monocytes, macrophages, natural killer and dendritic cells: all thought to be significant elements in the pathogenesis of psoriasis [8, 11, 12, 18, 26, 27, 36, 39, 41, 43, 54].\nThe ligands of CCR5 [CCL3, CCL4 and CCL5 (formerly known as MIP1\u03b1, MIP1\u03b2 and RANTES, respectively)] are highly expressed by keratinocytes in psoriatic tissue [19, 22, 42, 48]. Furthermore, it has been demonstrated that the proinflammatory cytokines IFN-\u03b3 and TNF-\u03b1 can induce the expression of these chemokines [19, 22] and that treatment of psoriasis resulted in a significant decrease of CCL5, as well as a reduction of CCR5+ T cells in the skin [19, 20, 58].\nSeveral animal models resembling psoriasis have been developed, yet, none of these models imitates psoriasis completely, hence limiting their utility [51]. Investigations by Mack et al. [36] showed a different expression pattern of CCR5 in mice and humans. Additionally, different expression of a single amino acid in the CCR5 molecule between rhesus macaques and humans resulted in a different response to inhibitors of the receptor in the species [6]. Therefore, research on the expression of CCR5 in psoriasis, as well as clinical efficacy of a CCR5 inhibitor, is limited to humans. So far, the data available on the expression of CCR5 in psoriatic skin in humans are not univocal (varying from high to minimal) and were obtained with divergent methods in investigations in which CCR5 was never the main focus [20, 27, 49, 58, 59].\nThe primary purpose of this study was to determine the expression of CCR5 and its ligands in chronic plaque psoriasis in situ compared to non-lesional skin, through analysis by immunohistochemistry and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). In order to examine the possibility that CCR5 plays a functional role in the pathogenesis of psoriasis, we also analyzed clinical and immunohistochemical data obtained from lesional and non-lesional skin biopsies of psoriasis patients before and after treatment with a CCR5 inhibitor.\nMaterials and methods\nStudy design and patients\nLesional and non-lesional skin biopsies were obtained from nine patients with moderate to severe chronic plaque psoriasis, defined by the psoriasis area severity index (PASI)\u00a0\u2265\u00a08. These skin biopsies were analyzed by manual quantification of immunohistochemical double-staining and quantitative RT-PCR. In order to get insight in the possibility of a functional role of CCR5 in the pathogenesis of psoriasis, 34 patients, including the previous 9 patients, participated in an 8\u00a0week, randomized, placebo-controlled, parallel group, multi-centre, double-blind clinical trial in which patients received either 50\u00a0mg twice daily of the CCR5 inhibitor SCH351125 (23 patients) or matched placebo (11 patients) orally for 28\u00a0days, followed by a follow-up period of 4\u00a0weeks. During the follow-up period patients were only allowed to use emollients as treatment and on day 56 vital signs, PASI and blood were assessed. Patients were included at the dermatology outpatient departments of four academic hospitals from April 2004 to December 2004. At baseline and the last day of treatment (day\u00a028), lesional biopsies were taken to evaluate the immunohistochemical effect of the CCR5 inhibitor. For this immunohistochemical evaluation, single-stained sections were analyzed with digital image analysis, semi-quantitative analysis (SQA) and confocal scanning microscopy, and double-stained sections on baseline and day\u00a028 were analyzed by manual quantification. To evaluate the clinical effect of the CCR5 inhibitor the PASI was assessed at baseline, day\u00a028 and day\u00a056.\nIn all patients, psoriasis was diagnosed at least 12\u00a0months prior to enrolment and patients were not allowed to use systemic psoriasis treatment or phototherapy within 4\u00a0weeks of study entry. Only emollients were allowed as topical treatment. All other topical anti-psoriasis therapy (e.g. corticosteroids, vitamin D derivates, etc.) had to be stopped 2\u00a0weeks before study entry. The protocol was reviewed and approved by the medical ethical committees of all participating centres and all patients gave their written informed consent before enrolment. The study was conducted according to the Declaration of Helsinki principles and is registered at the ISRCT register (http:\/\/www.controlled-trials.com\/ISCRT14986467).\nBiopsies\nFour-millimeter biopsies were taken from the inside border of a target psoriatic plaque, preferentially from a non-sun-exposed area. Lesional biopsies from each patient were obtained from the same target lesion, separated by at least 1\u00a0cm. The biopsy samples were randomly coded, snap-frozen in Tissue-Tek OCT compound (Sakura Finetek Europe, Zoeterwoude, The Netherlands) by immersion in liquid nitrogen and stored at \u221280\u00b0C until processing. Five-micrometer cryostat sections were cut and mounted on glass slides (Star Frost adhesive slides, Knittelgl\u00e4ser, Braunschweig, Germany), before being stored at \u221280\u00b0C until immunohistochemical staining. For each staining three sections of each biopsy were analyzed to minimize random variation.\nImmunohistochemistry\nAfter fixation of the slides, the endogenous peroxide activity was inhibited with 0.1% sodium azide and 0.3% hydrogen peroxide in Tris-buffered saline (TBS), before incubating for 15\u00a0min with 10% normal goat serum in TBS. Next, the sections were incubated for 1\u00a0h at room temperature (overnight at 4\u00b0C in the case of CCR5) with the primary antibody in 1% bovine serum albumin (BSA; Sigma-Chemical Co, St. Louis, MO, USA) in TBS. The following mouse anti-human monoclonal antibodies were used: FITC-conjugated anti-CD3 (BD Pharmingen, San Jose, CA, USA), FITC-conjugated-anti CD68 (clone EBM11; Dako), anti-human neutrophil elastase (Dako, Glostrup, Denmark), anti-cytokeratin 8.12 (keratin 16; Sigma, Saint Louis, MO, USA), anti-CD161 (NK-T cells; BD Pharmingen) and anti-CCR5 (CD195; BD Pharmingen). After rinsing with TBS, sections were further incubated with biotin-conjugated goat anti-mouse antibody (Dako) or, in case of CD3 and CD68 staining, with rabbit anti-FITC (Dako) in 10% normal human serum (NHS) in TBS for 30\u00a0min. Following a wash step with TBS, sections were subsequently incubated with horseradish peroxidase (HPR)-conjugated streptavidin (Dako) or, in case of CD3 staining, with HRP-conjugated goat anti-rabbit antibody (Dako), in 1% BSA in TBS for 30\u00a0min. In case of CCR5 staining an amplification step was performed with the TSA biotin system (Perkin Elmer, Boston, MA, USA). Sections were counterstained with Mayer\u2019s haematoxylin (Merck, Darmstadt, Germany) and mounted in Kaiser\u2019s glycerol gelatine (Merck). Skin sections were double stained with anti-CCR5 together with anti-CD3 or macrophage marker anti-CD68. The double stained sections were manually counted by two independent observers blinded for order, patient and clinical data. Using a 0.5\u00a0\u00d7\u00a00.5\u00a0mm ocular grid and at 200\u00d7 magnification, single red (CCR5+), single blue (CD3+ or CD68+), and purple double-positive cells were counted in the entire section. The epidermal and dermal regions were separately counted. The results are expressed as the number of double-positive cells\/mm2.\nRNA analysis\nRNA was extracted from frozen skin biopsies using the RNeasy mini kit (Qiagen). RNA quantity was assessed by OD at 260\u00a0nm and RNA quality was analyzed by measuring the ratio of 28s and 18s rRNA using the Agilent 2100 bioanalyzer.\nQuantitative PCR\nTaqman primers and probes were designed with Primer Express software (ABI), and purchased from ABI. The sequences of the human primers and probes are available upon request. For the human skin tissue, quantitative PCR was carried out with an ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The PCR reactions were prepared using the components from the Invitrogen Platinum Quantitative RT-PCR One-Step kit and assembled according to the manufacturer\u2019s instructions (Invitrogen, Carlsbad, CA, USA). The final concentrations of the primers and probe in the PCR reactions were 200 and 100\u00a0nM, respectively. The RT-PCR reactions for each gene were performed in a single 384-well plate. Separate plates of the same RNAs were used to quantitate 18S RNA as an internal control for RNA quality, and a primer\/probe set for the CD4 promotor was used to check the RNAs for genomic contamination. The PCR data was quantitated based on a standard curve generated using fourfold serial dilutions of the target genes. The fourfold dilutions began at 0.25\u00a0ng, and eight dilutions were used to generate the standard curve. This procedure provides an absolute quantitation of the amount of CCR5 mRNA in a given tissue. Data were analyzed using Sequence Detection Systems software version 1.7 (Applied Biosystems, Foster City, CA, USA).\nDigital image analysis\nSingle stained sections were randomly coded and analyzed by computer-assisted image analysis as described previously in detail [23]. In short, images were acquired and analyzed using Syndia algorithm on a Qwin based analysis system (Leica, Cambridge, UK). Twenty high power fields per section were analyzed. Positive staining of cellular markers was expressed as positive cells\/mm2.\nSemi-quantitative analysis\nFor keratinocyte expression of K16 keratin, a semi-quantitative score was done by two independent observers, blinded for order, patient and clinical data, with a standard binocular light microscope (Olympus) at 200\u00d7 magnification. The semi-quantitative score ranged from 0 to 4+. A score of 0 represented no expression, while a score of 4 represented abundant expression in all layers of the epidermis.\nConfocal scanning microscope\nCryosections of 5\u00a0\u03bcm on silanized slides were fixed with acetone and dried at room temperature, and stored at \u221280\u00b0C. Sections were incubated in PBS-3% BSA for 30\u00a0min and washed with PBS before all steps. Next, monoclonal anti-human CCR-5 antibody (R&D Systems, MAB183, clone 45549.11), Texas red immunoglobulins diluted in PBS-3% BSA (1:100) was applied, followed by application of the primary monoclonal specific antibodies CD3 (clone SK7; BD Biosciences), CD4 (clone SK3; BD Biosciences), CD8 (clone DK25, DakoCytomation) or CD68 (clone KP1, DakoCytomation). Next, FITC-conjugated affinipure rabbit anti-mouse IgG (Jackson, A\u00a0=\u00a0492, E\u00a0=\u00a0520) diluted in PSB-3% BSA (1:200) was applied and sections were mounted in fluorescent mounting medium (DakoCytomation). Of each double stained slide three pictures were taken with an image definition of 1024\/1024 pixels at a magnification of 25\u00d7.\nSample size calculation\nThe randomized placebo controlled clinical trial was targeted to randomize a total of 30 subjects (20 on active treatment and 10 on placebo). With this sample size, the trials would be able to detect a difference of 38% in the response rate from the placebo group assuming a 0% response rate in the placebo group with 80% power at an alpha level of 0.05 (two-sided test).\nRandomization\nRandomization was stratified by sites. Each site was assigned a fixed number of subjects numbers; e.g. site\u00a01 would get numbers 1\u20139 and so on. Once the physician of the study site would enroll a subject, the subject would be assigned the next available subject number assigned to the site, starting with the bottom of the list; e.g. the first subject enrolled in site\u00a01 would get number 1, the second subject would get number 2, and so on. Treatments would be assigned in an active to placebo ratio of 2:1 according to a computer generated randomization schedule. No stratification based on age, sex or other characteristics was performed. Throughout the study both patient and treating physician were blinded to the group assignment.\nStatistical analysis\nWe used SPSS 12.0.1 for Windows (SPSS, Chicago, IL, USA) for the statistical analysis of clinical data. The Mann\u2013Whitney test was used to compare lesional and non-lesional skin at baseline and to compare skin biopsies before and after treatment. All statistical tests were two-sided: P values less than 0.05 were considered significant. The results are expressed as median\u00a0\u00b1\u00a0standard error of the mean. To evaluate the clinical effect of treatment with a CCR5 inhibitor, an intention to treat analysis was performed. Quantitative PCR data analysis was performed by two-sided t test as implemented by Graphpad Prizm (version 4.0 Graphpad Software, San Diego, CA, USA). A P value less than 0.05 was considered significant.\nResults\nComparison of CCR5 expression in lesional versus non-lesional psoriatic skin\nThe expression of CCR5 in T cells (CD3) and macrophages (CD68) in lesional and non-lesional skin of nine psoriasis patients was compared at baseline. We observed a clear expression of CCR5 which was primarily present in the dermis. In absolute numbers approximately half of the T cells and half of the macrophages co-expressed CCR5 (Fig.\u00a01). The CD3+CCR5+ and CD68+CCR5+ double positive cells showed a low but statistically significant increased expression of CCR5 in epidermis and dermis of lesional skin in comparison to non-lesional skin, as shown in Fig.\u00a01. Focussing on the expression of CCR5 as the percentage of all T cells or macrophages present in the sections, the difference between lesional and non-lesional skin was only statistically significant in the epidermis for CD3+ cells (P\u00a0<\u00a00.05) and in the dermis for CD68+ cells (P\u00a0<\u00a00,001).\nFig.\u00a01Immunohistochemical analysis of CCR5 in lesional versus non-lesional skin. Data are shown as mean\u00a0\u00b1\u00a0SD; ns non-significant, *\u00a0P\u00a0<\u00a00.05, **\u00a0P\u00a0<\u00a00.01, ***\u00a0P\u00a0<\u00a00.001\nQuantitative RT-PCR analysis indicated no increased expression of mRNA for CCR5 and CCR5-ligand CCL4 (MIP-1\u03b2) in lesional skin (Fig.\u00a02), only the expression of CCR5-ligand CCL5 (RANTES) and IL-8 was significantly increased in lesional skin (P\u00a0<\u00a00.0001 and P\u00a0<\u00a00.05). The well-known enhanced expression of IL-8 in psoriatic skin was included as positive control.\nFig.\u00a02mRNA analysis of CCR5 in lesional psoriatic skin in comparison with non-lesional skin. IL-8 is used as a control marker. *\u00a0P\u00a0<\u00a00.0001, **\u00a0P\u00a0<\u00a00.05\nLack of clinical efficacy of SCH351125: a CCR5 inhibitor\nIn total 34 patients were randomized, as shown in Fig.\u00a03. The demographical data of the patients are shown in Table\u00a01. With regards to co-morbidities, one patient had hypercholesterolemia and one had obesity in the placebo group. In the SCH351125 group one patient also had obesity and two had a history of hypertension. After treatment with the CCR5 inhibitor there was no change in mean PASI in the SCH351125 group (n\u00a0=\u00a023) [15.5\u00a0\u00b1\u00a03.8 at baseline, 15.4\u00a0\u00b1\u00a07.4 at day\u00a028 (Fig.\u00a04a)]. Three of the patients treated with SCH351125 (13%) attained an improvement of 50% or more compared to baseline (PASI 50 responders), showing improvements of 67, 77 and 69%. In the placebo group (n\u00a0=\u00a09) the mean PASI slightly decreased (14.2\u00a0\u00b1\u00a04.7 at baseline, 12.9\u00a0\u00b1\u00a03.7 at day\u00a028). None of the patients treated with placebo showed an improvement of more than 50%. All changes observed were not statistically significant. In the follow-up period no changes in mean PASI were seen in either treatment groups.\nFig.\u00a03Flow chart of randomized, placebo controlled clinical trial. SCH351125 CCR5 ligand inhibitor, SAE serious adverse event, AE adverse eventTable\u00a01Demographical data patientsRandomized clinical trialPlaceboSCH351125Number1123Male:female7:418:5Age (years)a41.8 (10.2)49.4 (14.3)Duration of skin disease (years)a20.6 (9.8)19.8 (11.6)Baseline PASIa14.9 (4.7)15.7 (4.3)aMean (\u00b1SD)SCH351125 CCR5 ligand inhibitor, PASI psoriasis area and severity index, BSA body surface areaFig.\u00a04Clinical and immunohistochemical respons after treatment with a CCR5 receptor inhibitor. In a randomized placebo controlled clinical trial 34 psoriasis patients were randomized for treatment with a CCR5 receptor inhibitor (SCH351125) or placebo for 28\u00a0days. Clinical efficacy was measured by psoriasis area and severity index (PASI) (a). Lesional skin biopsies were taken from all patients at baseline and day 28 to evaluate immunohistochemical effect. Immunohistochemical single staining of CCR5 of a patient treated with SCH351125 at baseline (b) and day 28 (c). Immunohistochemical markers in relation to clinical response (d). PASI 50 non-responder, improvement of PASI of less than 50%; PASI 50 responder, improvement of PASI of 50 or more percent; data are shown as mean\u00a0\u00b1\u00a0SD; ns non-significant; 1P\u00a0=\u00a00.05\nIn the treatment group four patients discontinued. One patient developed an erythrodermic eruption after 4\u00a0days of treatment, which was considered by the site physician as a serious adverse event (SAE). Two patients discontinued due to adverse events (AEs): one developed shingles in the n.trigeminus area of the right side of his face after 8\u00a0days of treatment and one patient discontinued due to hair loss after 21\u00a0days of treatment. One patient discontinued due to non-compliance. In the placebo group two patients discontinued due to AEs: both exacerbation of their psoriasis after 2\u00a0weeks of treatment.\nCCR5 expression before and after treatment with SCH351125\nImmunohistochemical analysis of lesional tissue samples from the SCH351125 group and the placebo group revealed no statistically different expression of CCR5 between baseline and day 28 in both treatment groups, as illustrated by Fig.\u00a04b, c. When focusing on the markers CD3, CD68, CD161, elastase and K16 in relation to the clinical response, no statistically significant difference after 28\u00a0days of treatment with either SCH351125 or placebo was found (Fig.\u00a04d), except for elastase and dermal CCR5+CD3+ cells, which were statistically significantly lowered in the three PASI 50 responders treated with SCH351125. Additional data obtained by confocal scanning microscopy corresponded with the digital image and SQA (data not shown).\nDiscussion\nThe primary objective of this study was to explore the possibility of involvement of CCR5 in the pathogenesis of chronic plaque psoriasis. Therefore we determined the expression of CCR5 in situ at the protein and mRNA level by immunohistochemical analysis and, quantitative RT-PCR, respectively. The total number of single positive (CCR5+) and double positive (CCR5+CD3+ and CCR5+CD68+) cells in lesional psoriatic skin significantly outnumbered those in non-lesional skin. However, when expressed as percentage of CD3 or CD68 cells, the difference between lesional versus non-lesional expression was less clear. With the latter approach we found that the proportion of CCR5 expression was significantly higher in the epidermal CD3+ cells and dermal CD68+ cells only, when comparing lesional skin to non-lesional skin.\nAnalysis of CCR5 mRNA expression demonstrated a slight, though not significant, increased expression of CCR5 in lesional psoriatic skin, perhaps due to the small numbers of patients. In line with earlier observations we found that the mRNA expression for RANTES [19, 48] and IL-8 [1, 17, 21, 45, 53] was significantly higher expressed in the lesional samples. However, in contrast to previous research [42] the expression of MIP1\u03b2 was not increased in lesional psoriatic skin.\nIn summary, our results do not provide a clear answer to our objective of determining whether the percentage of CCR5 expressing cells is similar in lesional and non-lesional skin, or if this percentage is increased in lesional skin.\nTo assess any possible functional participation of CCR5 in the development or maintenance of psoriatic plaques we investigated the clinical response to treatment with a CCR5 inhibitor as well as the effect of this drug on the inflamed skin in situ. The randomized placebo-controlled clinical trial revealed no significant clinical effect and changes at the immunohistochemical level between patients treated with placebo or the CCR5 inhibitor. The specific type and dose of the CCR5 inhibitor used in this clinical trial has proven its efficacy previously in vitro, in vivo and clinical studies with other diseases in which CCR5 is known to play a pivotal or significant role [14, 44, 55, 56, 60]. However, only three patients treated with the CCR5 inhibitor demonstrated a clinical improvement of 50% or more. It cannot be excluded that this low and not statistically significant number of patients is due to a spontaneous improvement, reflecting the unpredictability of psoriasis. Surprisingly, the immunohistochemical markers analyzed in the skin biopsies of the so-called PASI 50 responders, did only partially correspond with the clinical response. Of all immunohistochemical markers only elastase and dermal CCR5+CD3+ showed a significant decline after 28\u00a0days of treatment. Notably, the expression of CD3, a marker known to correspond well with the clinical severity as measured by PASI, was increased after treatment with SCH351125 in two out of the three responding patients (Fig.\u00a04d). In addition, their baseline expression of CD3 was lower in comparison with the other patients while their PASI was similar. This inconsistency could be due to the low number of patients or suggests an individual difference in CD3 kinetics.\nThe increased expression of CCR5 observed by immunohistochemistry and the increased mRNA expression of CCR5-ligand RANTES in lesional psoriatic skin may suggest an involvement of this receptor and ligand in psoriasis. However, our clinical trial with an effective CCR5 inhibitor unequivocally demonstrated that this is not the case. Previous research has shown increased expression of several chemokines and chemokine receptors in psoriasis, [16, 21, 27, 28, 49] indicating that multiple receptors may participate in regulating T cell recruitment to the inflamed skin. Furthermore, RANTES is known to also bind with CCR1 and CCR3, whereas MIP1\u03b2 is solely connected to CCR5. Given this complexity of interactions between chemokines and chemokine receptors, it is not unlikely that blocking a single chemokine receptor (i.e. CCR5) would have been insufficient in diminishing the inflammatory process.\nAccording to Homey [26], chemokine antagonistic approaches to impede with the inflammatory process may perhaps be preventive rather than therapeutic. Chemokines and their receptors play an essential role in the trafficking of T cells to all kinds of tissue, including the skin. Yet, once leucocytes have entered the target organ and underwent activation processes, impairment of recruitment of pathogenic T cells is likely to be less effective in reducing the clinical symptoms. When combined with a successful eradicative treatment of the T cells, chemokine antagonists could perhaps be promising candidates for prevention of acute flares, prolongation of lesion-free interval and therefore provide optimized long-term management of patients suffering from chronically relapsing inflammatory skin disease such as psoriasis. So, although CCR5 does not seem to be a key chemokine receptor in the pathogenesis of psoriasis, further efforts are needed to unravel the complete set of chemokines and chemokine receptors significant in the recruitment of inflammatory cells in psoriasis, and may help to identify crucial molecules, as demonstrated previously by TNF in various immune mediated inflammatory diseases.","keyphrases":["ccr5","psoriasis","chemokine inhibitor"],"prmu":["P","P","R"]}
{"id":"Clin_Oral_Investig-4-1-2238786","title":"Current erosion indices\u2014flawed or valid? Summary\n","text":"The problem of erosive tooth wear appears increasingly to be encountered by clinicians and researchers. An adequate way of defining and recording erosive tooth wear is essential in order to assess the extent of this clinical phenomenon, both on an individual level and in the population, and for the adequate provision of preventive and therapeutic measures. Well-established erosion indices have been used in most of these studies, although in many cases modifications have been made to suit the different research aims. This use of different indices is one reason why it still cannot be claimed that there is enough current knowledge on this clinical phenomenon. This article summarises the proceedings of a workshop to discuss the topic of dental erosion indices. The result of the workshop is the proposal for a new scoring system (Basic Erosive Wear Examination, BEWE) designed for use both within the research field and for dental clinicians, with the aims of standardising assessment of erosion for international comparisons, raising awareness and providing guidelines for treatment of erosive tooth wear in dental practice.\nIntroduction\nAt the 1995 International Life Sciences Institute\u2014Europe Workshop on Dental Erosion, amongst the recommendations made were the development of a clinical index for assessment of progression of dental erosion and the validation of a proposed erosion index for large-scale epidemiological surveys. One decade later, it appears that these recommendations are still relevant in that such tools have not been validated. Several different indices for the clinical diagnosis of erosive tooth wear are still in use by researchers and clinicians around the world and research on dental erosion carried out over the past decade indicates a lack of consensus. This problem applies both in relation to the prevalence, the distribution, as well as the progression of erosive tooth wear, its early detection and in the evaluation of the efficacy of preventive\/therapeutic measures. Although both patients and dental clinicians are becoming increasingly aware of the erosive component of tooth wear, there is still a need to raise diagnostic competence through the use of a simple, standardized index that properly reflects the nature, extent and progression of the defects.\nThis paper summarises the result of the workshop \u2018Current erosion indices\u2014flawed or valid?\u2019 held in Basle in 20071, the aim of which was to substantiate and critically discuss the current research tools in this field.\nAnthropological aspects of tooth wear\nIn contrast to the dental profession, anthropologists have considered tooth wear as a normal physiological phenomenon. Skeletal material from ancient remains, such as the pre-contemporary Australian Aboriginal populations, shows clear evidence of attrition and abrasion. The most common cause of these forms of wear is believed to be food mastication, that is to say hard, fibrous material typically consumed by these populations and tooth surface loss appears to be directly related to the age of the individual.\nAnthropologists describe a tooth wear model where genetic factors influence tooth morphology and occlusion, and, in turn, occlusion and food consistency influence the chewing pattern. The resulting gradual tooth wear is accompanied by physiological adaptation involving bone remodelling and compensatory tooth eruption. It is postulated that from an evolutionary perspective, tooth wear was one of the main selective factors leading to changes in both tooth morphology and properties of dental tissues. Masticatory efficiency is strongly related to tooth anatomy and wear characteristics as highlighted in comparative studies of dentitions of many different species including herbivores, carnivores, primates and humans.\nA basic understanding of the anthropological perspective of tooth wear is highly valuable both in the diagnosis and treatment planning of contemporary erosive tooth wear and particularly in relation to discussion about the relevance of erosive wear for oral health.\nDiagnosis and risk factors\nDental erosion or erosive tooth wear is a multi-factorial condition. Tooth wear mechanisms seldom happen in isolation, each occurring with different intensity and duration to produce a multitude of wear patterns. This interplay is probably the reason for confusion when attempting to use any tooth wear index based mainly on aetiology. Diagnosis requires a thorough knowledge of both morphological patterns typical of this type of wear and of the factors that are likely to contribute to the development of erosion.\nRisk factors for erosive tooth wear can be categorized into chemical, biological and behavioural factors. The two most often cited chemical parameters, pH and titratable acidity, can only partly explain the erosive potential of acidic food or drinks. Mineral content, especially the common ions (calcium and phosphate) and fluoride, as well as calcium-chelation properties that vary for different dietary acids, are also important. Saliva is considered to be one important biological factor in erosion protection, but also the acquired enamel pellicle, tooth structure and positioning in relation to soft tissues and tongue may be of particular relevance. Behavioural factors can play a role in dental erosion both during and after an acidic challenge. The manner by which dietary acids are introduced and kept in the mouth before swallowing, the timing of acidic consumption\/exposure and daily work\/pleasure\/sport activities can all have a significant effect on the development and location of erosive tooth wear.\nIndividuals suffering from anorexia and bulimia nervosa, rumination, chronic alcoholism or gastro-oesophageal reflux are at risk of erosive tooth wear caused by intrinsic acid and certain occupations are also conducive to erosive tooth wear such as professional wine tasting and industrial work involving exposure to acidic vapours or dust.\nCurrent tooth wear indices\nMeasurement of tooth wear has been performed over the years using a large variety of both quantitative and qualitative methods. A number of indices have been developed typically using grading or scoring designed to identify increasing severity or progression of the condition, but traditionally with a focus on only one aetiological factor. Many diagnostic indices however, do not properly reflect the morphological defects and furthermore there is little international standardisation.\nThe earliest published attempt to diagnose tooth wear dates to the late 1800s. This index did not presuppose aetiology and formed the foundation for the development of further indices. In the latter half of the 1900s, many other indices came about largely based on work by Eccles and Jenkins [1]. The Eccles index for dental erosion of non-industrial origin denotes three classes of lesion assigned to four tooth surfaces. A few years later, based on Eccles work, Smith and Knight [2] produced the Tooth Wear Index, a comprehensive system whereby all four visible surfaces (buccal, cervical, lingual and occlusal\/incisal) of all teeth present, are scored for wear. Further modifications of these indices have been published, designed for specific purposes by researchers in order to suit their own work. However, some problems have been encountered concerning the amount of data produced and inter- and intra-examiner reliability in large-scale epidemiological surveys, as well as threshold values in different population groups. The challenge still remains to develop a simple index that can be used clinically to assess progression of wear and in epidemiological prevalence studies.\nValidity of current diagnostic criteria and tools for grading\nThe diagnosis of tooth wear and of erosive tooth wear in particular, involves the process of identifying the condition by the lesion characteristics and by obtaining medical, nutritional, and occupational information about the patients. On an individual basis this process can be very specific compared with the case for epidemiological research where, for practical reasons, diagnosis is often restricted to lesion characteristics. Although there appears to be relatively widespread consensus as to the characteristic signs and symptoms of erosive tooth wear, dating back approximately 60\u00a0years, true validation of these clinical criteria is lacking. At that early stage, the presence of (amalgam) fillings appearing to project above the surface of the surrounding tooth was considered to be the most important clinical sign. However, hypersensitivity of the affected teeth, absence of stain and tooth wear defects with rounded margins were also mentioned. More clinically precise descriptions of the lesions have appeared in the literature over time, describing tissue loss at the gingival third of the facial surfaces that were shallow, disc-shaped, smooth, polished or with a scooped out appearance. It is currently accepted that loss of surface contour, shallow concavities on smooth surfaces, cupping and grooving on occlusal\/incisal surfaces and \u2018proud\u2019 restorations are characteristic signs of erosive tooth wear, but reviewing the literature, it turns out that these criteria derived from observations in small groups and individual experience rather than from systematic research.\nThe results of epidemiological studies aimed at relating the occurrence of erosion lesions with known aetiological factors, are somewhat unclear, and in some cases controversial. Most of these studies are performed on random cross-sectional or cluster samples of children or adolescents, with few addressing the adult and aged population. Whether the current diagnostic criteria are valid in reflecting the effect of chronic acid exposure remains to be tested.\nComparative studies on wear-lesion characteristics aimed at differentiating between abrasive and erosive wear by examining dietary and nutritional differences have concluded that occlusal\/incisal lesions need to be quantified by depth\/breadth ratios in order to obtain a correct diagnosis, but that the presence of shallow defects on smooth surfaces is almost certainly a more valid criterion than occlusal cupping and incisal grooving. Except in cases of occlusal cupping in young individuals, it is suggested that the presence of occlusal cupping and incisal grooving are at best uncertain diagnostic criteria for erosive tooth wear, but that shallow defects on smooth surfaces coronal to the CEJ may be pathognomonic of erosive tooth wear.\nGrading the degree of erosion in an individual is important as it reflects the net exposure to the erosive challenges and the opposing protective forces, and plays a part in the assessment of the problem and of the need for intervention for prevention of further tooth wear. The reliable diagnosis of both early enamel and dentine erosive tooth lesions is challenging and not very reproducible. In cases involving cupping of the molar cusp tips, determining whether dentine is exposed at the base of the cup can be very difficult. Histological studies would tend to indicate that even experienced clinicians underestimate dentine exposure.\nAssessing tooth wear in the primary dentition provides further challenges. Primary teeth do not appear to withstand wear forces to the same degree as permanent teeth and commonly show signs of wear. This fact affects the scoring of severity and makes determination of aetiology in many cases more difficult.\nPathological or physiological erosion: is there a relationship to age?\nIn order to be able to diagnose erosive tooth wear as unacceptable or even pathological, it is imperative that acceptable levels of tooth wear be established. Partly due to the lack of effective methods for evaluating the amount of tooth loss, these data do not exist apart from a few epidemiological surveys. These studies, combined with experienced clinical judgment currently form the basis for a diagnosis of severity.\nDefining pathological erosion has proved problematic. Authors addressing this concept have considered cases to be \u2018pathological\u2019 when excessive loss of tooth tissue has occurred affecting appearance or function, or when it causes pain. This is further complicated by attempting to predict if a tooth, at any age, would survive its current rate of wear, or, if and when interceptive treatment is required, and what level of wear is acceptable to the patient. However, an erosion lesion judged to be pathological at one age may revert to be within physiological boundaries at a later age if it remains quiescent and does not progress further during the intervening period. Thus, when defining pathology, the severity of loss, the current activity and the age of the patient must be considered.\nFurthermore, the concept of age-related pathological tooth wear becomes interesting when assessed from different perspectives. State health care authorities, private insurance companies, industry, dental clinicians and patients will all have differing interpretations of what constitutes \u2018pathological\u2019 tooth wear.\nRequirements and demands on an erosion index\nIndices have been formulated based on varying criteria, either to satisfy a particular purpose\/condition or to enhance reproducibility. Among the factors considered in the development of currently existing indices are clinical versus epidemiological purposes, children versus adults, permanent versus primary teeth, full versus partial mouth scoring, exclusion\/inclusion of dentine exposure and\/or occlusal cupping and incisal grooving, etc. None of these indices has been found to describe dental erosion in such a way that they can be regarded as the \u2018gold standard\u2019 for measurement of erosion.\nA simple and standardized index that is suitable for assessment of erosion should ideally be (1) easily applicable in general dental practice, (2) adaptable for epidemiological prevalence studies, (3) suitable for monitoring erosive lesion activities such as progression or arrestment of lesions, (4) easily reproducible under varying conditions for examination such as with\/without magnification devices, ambient light, and hydration state of the tooth surface (dry\/wet), (5) capable of reflecting net exposure of an affected individual to the erosive challenge, (6) capable of indicating the need for treatment, and (7) should serve for both children and adult as well as permanent and primary teeth.\nDevelopment of one erosion index capable of satisfying all the above conditions poses a lot of challenges and the implications of the exclusion or inclusion of certain factors and conditions may need thorough consideration. An index involving partial scoring of the dentition used for rapid screening would be more acceptable than one incorporating full mouth scoring, which is both time consuming and believed to lower the accuracy of the index. While limiting the scoring to anterior teeth (e.g. central incisors) may eliminate a considerable number of individuals with posterior located erosion, it is considered that the use of a sextants technique, which involves a full mouth examination but scoring the most severely worn surface on each sextant, would favour easy application and reproducibility. In summary, the application of a sextant method, assessing only wear defects on the coronal smooth surfaces, and estimating severity by the diameter and depth (regardless of dentine involvement) of wear, may be considered for the development of a simple and standardised index acceptable for clinical practice and epidemiological studies.\nMethodological considerations\nThe WHO Oral Health Program (2003) [3] focuses on oral health as an integral and essential component of general health and an important factor in quality of life. To date, only the DMFT index has been assessed and recorded on an international basis. No indicator of dental erosion is currently included in any EU or WHO oral health policy, most likely due to the lack of a standardized index for measurement of dental erosion. As part of a revision of the WHO Oral Health Surveys Basic Methods, there is now an emphasis on development of methodologies and approaches for evaluating newer disease entities such as dental erosion.\nImportant quality characteristics of epidemiological tools include validity and reliability and sensitivity and specificity. Content validity describes whether all aspects that are relevant for the \u2018construct\u2019 or content of the index are adequately considered. The content validity is based on the subjective meaning of established experts. When comparing results using a new index with results using an existing \u2018gold standard\u2019, it can be stated that the construct validity is high if results are well correlated with each other. When a gold standard is not available, approved hypotheses concerning the construct of interest can be confirmed empirically using the new instrument or index. Reliability of an erosion index indicates how precise that index is able to record dental erosion and encompasses both inter-examiner and intra-examiner reliability. The sensitivity of an erosion index indicates its ability to detect dental erosion (compared to a gold standard) and is calculated by dividing the frequency of correct positive results by the sum of correct positive and false negative results. The specificity of an index indicates how well the index can record erosion as absent when erosion is not present and is determined by dividing the frequency of correct negative results by the sum of correct negative and false positive results.\nValidation studies should be carried out on an international basis and should consider the mentioned quality criteria, most of which are relatively easy to prove. A challenge however is the question of content validity which requires research and final consensus on the construct of erosive wear.\nNew scoring system: the Basic Erosive Wear Examination (BEWE)\nAfter lengthy discussions flaws in current approaches were identified and it was agreed that there is need for a validated, standardised and internationally accepted index. To initiate the development of such a tool, a universal scoring system was suggested. The new BEWE proposed in this issue records at the sextant level, based on the model of the BPE (Basic Periodontal Examination). It is designed to be a simple, reproducible and transferable scoring system that can be used with the diagnostic criteria of all existing indices. In time, use of the BEWE will generate comparable prevalence data, thus allowing ongoing consensus processes in the scientific community. Researchers within this field should, in practice, be able to transfer previous results using other indices based on other diagnostic criteria over to the BEWE index thereby allowing for improved comparisons.\nFurthermore, the BEWE will help students, clinicians and general practitioners to screen for erosive tooth wear and to guide them in the decision-making process for the management of erosive tooth wear.\nFuture research perspectives\nAn international alliance between dentists, epidemiologists, statisticians and other scientists and politicians is recommended in order to develop an internationally agreed and accepted dental erosion index. Efforts should be made:\nto re-evaluate existing data with the BEWE system to gain comparable data on the relevance of erosive wear for oral health and to initiate the consensus processto prove the content validity of the construct \u2018dental erosion\u2019 and to define valid diagnostic criteriato use these criteria with the BEWE system thus creating a new indexto validate this index on an international basis with the perspective to implement erosive wear data in the WHO and EU oral health data bases and erosion indicators in indicator sets for oral healthto further develop the link between the sum score of the BEWE and recommendations for prevention and treatment in the individual patient","keyphrases":["erosive tooth wear","index","epidemiology","diagnosis","prevalence"],"prmu":["P","P","P","P","P"]}
{"id":"J_Hum_Genet-2-2-1705471","title":"Association and synergistic interaction between promoter variants of the DRD4 gene in Japanese schizophrenics\n","text":"Recent association studies suggest that polymorphisms in the promoter and exon 1 upstream region of the dopamine D4 receptor (DRD4) gene play a functional role in the development of common psychiatric illnesses, although there are also conflicting results. In this study, we re-sequenced this region to identify all genomic variants, and tested them for association with schizophrenia. A total of 570 Japanese schizophrenic cases with matched controls were studied by genotyping all identified\/validated common polymorphisms (\u22121106T>C, \u2212906T>C, \u2212809G>A, \u2212616G>C, \u2212521T>C, \u2212376C>T, \u2212291C>T and 12-bp repeat) and a known microsatellite (120-bp tandem duplication) in the upstream region. A single nucleotide polymorphism (SNP) \u2212809G>A in the promoter region was found to be significantly associated with disease (P=0.018 and 0.032 for allelic and genotypic comparisons, respectively), although not surviving after Bonferroni correction. Logistic regression analysis showed that a combination of the four polymorphisms, \u2212809G>A, \u2212616G>C, \u2212291C>T and the 12-bp repeat, conferred a susceptibility to schizophrenia. These results suggest that the upstream variants have a primary functional effect in the etiology of schizophrenia in the Japanese population.\nIntroduction\nDisturbances in the dopamine neurotransmitter system have long been suggested to play a crucial role in the pathogenesis of schizophrenia (Prasad et al. 2002). However, involvement of dopamine-related genes in the manifestation of this disorder has been difficult to determine. The dopamine D4 receptor (DRD4) gene, located at chromosome 11p15.5 (Gelernter et al. 1992; Petronis et al. 1993), has received considerable interest because clozapine, a neuroleptic which is often effective against treatment-resistant symptoms, has a high affinity for this receptor (Sanyal and Van Tol 1997; Van Tol et al. 1991). Also, DRD4 is up-regulated in postmortem brain tissue from schizophrenic patients (Murray et al. 1995; Seeman et al. 1993; Stefanis et al. 1998; Sumiyoshi et al. 1995).\nTo investigate its genetic association with schizophrenia, many groups have examined polymorphisms of the DRD4 gene. Early studies focused on a 48-bp variable number of tandem repeat (VNTR) in exon 3. This tandem repeat comprises 2 (2R) to 11 (11R) 48-bp repeat units and codes for the third intracellular loop of the receptor protein (Van Tol et al. 1992). There is growing genetic evidence linking the VNTR with psychiatric traits or illnesses, such as novelty-seeking (Benjamin et al. 1996; Ebstein et al. 1996), attention-deficit hyperactivity disorder (ADHD) (Faraone et al. 2001; LaHoste et al. 1996), and schizophrenia (Hwu et al. 1998; Jonsson et al. 1996; Kaiser et al. 2000; Sanak et al. 2005; Serretti et al. 2001), but the results are still variable.\nIn recent years, promoter region polymorphisms of the DRD4 have also received particular attention because of their possible role in the regulation of gene transcription. To date, two polymorphisms in the promoter, T allele of the single nucleotide polymorphism (SNP) \u2212521T>C and a 120-bp tandem duplication (1.2\u00a0kb upstream from the initiation codon), are reported to reduce transcriptional efficiency (D\u2019Souza et al. 2004; Okuyama et al. 1999). Numerous studies have examined the association of upstream (promoter region and exon 1) polymorphisms with schizophrenia and some have shown a positive association (Okuyama et al. 1999; Xing et al. 2003), although not all were in full agreement (Hong et al. 1998; Jonsson et al. 2001; Kohn et al. 1997; Lung et al. 2006; Mitsuyasu et al. 2001; Petronis et al. 1995).\nSince previous studies focused primarily on individual polymorphisms, allelic heterogeneity and\/or a relatively weak effect of the variants may partly explain these inconsistencies. When haplotype construction is difficult because of a weakness or absence of linkage disequilibrium (LD) in a genomic interval, as is the case for the promoter region of DRD4, it may be important to test for a synergistic interaction between genetic variants in addition to performing single SNP analysis. Haplotype analysis is a reasonable strategy, assuming that the causative allele arises from a specific ancestral haplotype. However, this method is inadequate when there is an accumulation of multiple causative variants in a restricted genomic stretch. Additionally, haplotypic analysis is further compromised in regions where LD is decayed, because of the low statistical power to detect association due to inflated degrees of freedom. Therefore, clusters of potentially functional variants in genomic region with tenuous LD are better analyzed using a logistic regression framework that provides a powerful test of SNP etiology.\nIn this study, we examined the 5\u2032 upstream region of DRD4, by first re-sequencing the region to identify all genomic variants. We then tested the association of all identified\/validated common variants individually, as well as performing synergistic interaction analyses between them and schizophrenia.\nMaterials and methods\nSubjects\nSamples from 570 unrelated cases of schizophrenia (285 men, 285 women; mean age 47.0\u00b111.4\u00a0years), and 570 age and sex matched controls (285 men, 285 women; mean age 46.7\u00b111.1\u00a0years) were analyzed. The diagnosis of schizophrenia was made by consultation according to DSM-IV criteria with consensus from at least two experienced psychiatrists. All available medical records were taken into consideration. Control subjects were recruited from hospital staff and volunteers who showed no evidence of present or past psychoses during brief interviews with psychiatrists. All subjects were from central Japan. The study was approved by the Ethics Committees of RIKEN, Hamamatsu University and Chiba University. All participants provided written informed consent.\nSequence analysis\nThe upstream region encompassing the promoter region and exon 1 of the DRD4 gene (from 1,216\u00a0bp upstream of \u201cA\u201d in the start codon to 293\u00a0bp downstream of this \u201cA\u201d) was analyzed by the direct sequencing of PCR amplification from the genomic DNA of 30 unrelated Japanese schizophrenics. Primer sequences and detailed information on the reaction conditions are available upon request. Sequencing was performed using the DYEnamic ET terminator cycle sequencing kit (Amersham Biosciences, Piscataway, N.J., USA) and the ABI PRISM 3730 Genetic Analyzer (Applied Biosystems, Foster City, Calif., USA). Polymorphisms were detected by the SEQUENCHER program (Gene Codes Corporation, Ann Arbor, Mich., USA).\nGenotyping\nSNPs were typed by the TaqMan system (Applied Biosystems). PCR was performed using an ABI 9700 thermocycler and fluorescent signals were analyzed by an ABI 7900 sequence detector single point measurement and SDS v2.0 software (Applied Biosystems). Conflicts or flagged alleles were resolved by re-genotyping. Two microsatellite marker loci, the 120-bp tandem duplication and the 12-bp repeat polymorphism (Fig.\u00a01), were amplified by PCR using fluorescently labeled primers. PCR fragments were analyzed on an ABI PRISM 3730 Genetic Analyzer (Applied Biosystems). Genotypes were determined using GeneScan 3.5.2 and Genotyper 3.6 software (Applied Biosystems).\nFig.\u00a01Genomic structure and location of polymorphic sites within DRD4. Exons are denoted by boxes, with untranslated regions in white and translated regions in black. The sizes of exons and a scale are also shown. SNPs and microsatellites are indicated by solid squares and triangles, respectively. Re-sequencing was performed on the upstream region (from 1.2\u00a0kb upstream of the initiation codon to the 3\u2032 end of exon 1)\nStatistical analyses\nThe allelic and genotypic distributions were tested for association by Fisher\u2019s exact test for biallelic markers (SNPs, the 120-bp tandem duplication and the 12-bp repeat polymorphism).\nHaplotypic association analysis was performed using COCAPHASE in the UNPHASED package (http:\/\/www.rfcgr.mrc.ac.uk\/\u223cfdudbrid\/software\/unphased\/) (Dudbridge 2003). We employed a 2- and 3-marker sliding window analysis using COCAPHASE. To estimate the degree of LD between pairs of loci, the standardized disequilibrium coefficient (D\u2032) was calculated and haplotype blocks were defined using the Haploview program (http:\/\/www.broad.mit.edu\/mpg\/haploview\/) (Barrett et al. 2005).\nLogistic regression analysis\nTo test the multiple polymorphisms in the upstream region for a synergistic effect, stepwise logistic regression analysis was performed using the SPSS Software (Release 11.0J) (SPSS Japan, Tokyo, Japan). We employed a procedure of backward selection in which we started with all the markers genotyped in the present study. According to the approach of Cordell and Clayton (2002), we set dummy variables, x1 and x2, taking values x1=\u22121, 0 and 1, x2=\u22120.5, 0.5 and \u22120.5, for genotypes w\/w, w\/m and m\/m, respectively (w: wild-type, m: mutant).\nResults\nWe re-sequenced the upstream region of the DRD4 gene, and identified a total of eleven SNPs: \u22121106T>C, \u2212930C>G, \u2212906T>C, \u2212873G>A, \u2212809G>A, \u2212616G>C, \u2212603G>T, \u2212595G>del, \u2212521T>C, \u2212376C>T, and \u2212291C>T, and a 12-bp repeat polymorphism (Fig.\u00a01). One novel variant, \u2212930C>G (deposited into GenBank as ss61570833), was detected in only 1 out of 58 chromosomes. The remaining variants were already present in the public databases and were validated in Japanese subjects during the course of this study.\nThe eight markers, \u22121106T>C, \u2212906T>C, \u2212809G>A, \u2212616G>C, \u2212521T>C, \u2212376C>T, \u2212291C>T, and the 12-bp repeat polymorphism in exon 1, were selected for genotyping using the criterion of a minor allele frequency >0.05 from our sequencing data. We also typed the size of the 120-bp tandem duplication (bi-allelic polymorphism with either one or two repeat alleles). The information on markers is shown in Table\u00a01. The 12-bp repeat polymorphism was mostly bi-allelic with the exception of four chromosomes (three from schizophrenics and one from controls) that contained a three-repeat allele.\nTable\u00a01Genotype distributions of bi-allelic polymorphisms from the upstream region of DRD4MarkerdbSNP IDPhysical positionCase or controlnGenotype frequenciesP value for allelic associationP value for genotypic association\u22121.2\u00a0kb120-bp repeat\u2013625826\u20136260651\/11\/22\/2Case5690.04 0.32 0.64 0.202 0.377 Control5700.06 0.34 0.61 \u22121106 T>Crs936460626199T\/TT\/CC\/CCase5690.79 0.20 0.02 0.522 0.644 Control5700.76 0.22 0.02 \u2212906 T>Crs3758653626399T\/TT\/CC\/CCase5690.66 0.31 0.03 0.116 0.223 Control5690.62 0.34 0.05 \u2212809 G>Ars936461626496A\/AA\/GG\/GCase5620.03 0.33 0.64 0.018 0.032 Control5640.06 0.35 0.59 \u2212616 G>Crs747302626689C\/CC\/GG\/GCase5650.09 0.44 0.47 0.769 0.257 Control5680.10 0.39 0.50 \u2212521 T>Crs1800955626784T\/TT\/CC\/CCase5660.34 0.48 0.19 0.371 0.377 Control5690.34 0.50 0.16 \u2212376 C>Trs916455626929T\/TT\/CC\/CCase5610.01 0.18 0.81 0.732 0.838 Control5620.01 0.19 0.80 \u2212291 C>Trs916457627014T\/TT\/CC\/CCase5650.01 0.21 0.79 0.518 0.080 Control5660.02 0.19 0.79 Exon 112-bp repeatrs4646983627392\u20136273911\/1a1\/2a2\/2aCase5680.02 0.25 0.73 0.512 0.248 Control5690.03 0.21 0.76 aAlleles are coded by the number of repeats. Four subjects (three from the schizophrenia and one from the control group) with three repeats are omitted from this table. Significant results are shown in bold type\nAllelic and genotypic distributions of the promoter SNP \u2212809G>A differed significantly between cases and controls (P=0.018 and 0.032 for allelic and genotypic comparisons, respectively). In the exploratory haplotype window analysis, the two-SNP haplotype [(\u22121106C)\u00a0\u2212\u00a0(\u2212906C)] tended to be over-represented in schizophrenia (frequencies in cases and in controls were 0.70 and 0.66, respectively, P=0.0656) (data not shown).\nIn LD analysis, D\u2032 values were close to 1 in the distal [(120-bp repeat)\u00a0\u2212\u00a0(\u22121106T>C)\u00a0\u2212\u00a0(\u2212906T>C)] and proximal [(\u2212376C>T)\u00a0\u2212\u00a0(\u2212291C>T)\u00a0\u2212\u00a0(\u221212-bp)] segments of the upstream region, with each defined as a haplotype block, but it did not hold for the region in between (Fig.\u00a02). Decay of LD in this short stretch (1.2\u00a0kb) may imply a high local recombination rate. Therefore, in addition to conventional haplotype-window analysis, we employed logistic regression analysis, which can test a combinatorial effect of multiple SNPs simultaneously. A combination of four polymorphisms (\u2212809G>A, \u2212616G>C, \u2212219C>T and 12-bp repeat) appeared to affect susceptibility to schizophrenia (Table\u00a02).\nFig.\u00a02Haplotype organization of DRD4 in Japanese. The haplotype block pattern was constructed by the Haploview program using the genotype data from both case and control samples (1,140 subjects). The number in each cell represents the LD parameter D\u2032 (\u00d7100), blank cells mean D\u2032=1. Each cell color is graduated relative to the strength of LD between markers, which is defined by both the D\u2032 value and confidence bounds on D\u2032. Strong LD was observed in the discrete distal [(120-bp repeat)\u00a0\u2212\u00a0(\u22121106T>C)\u00a0\u2212\u00a0(\u2212906T>C)] and proximal [(\u2212376C>T)\u00a0\u2212\u00a0(\u2212291C>T)\u00a0\u2212\u00a0(12-bp repeat)] segments, but not in the intervening regionTable\u00a02Logistic regression analysis in the upstream region of DRD4Marker\u03b2SEWald\u2019s \u03c72P valueOROR 95% CI\u2212809G>A0.346 0.127 7.384 0.007 1.414 1.10\u20131.82\u2212616G>C0.247 0.125 3.872 0.049 1.280 1.00\u20131.64\u2212291C>T1.031 0.400 6.649 0.010 2.804 1.28\u20136.2412-bp repeat\u22120.725 0.274 6.988 0.008 0.484 0.28\u20130.83\u03b2 Logistic regression coefficient in the model, SE standard error of the coefficient, Wald \u03c72 Wald statistic to test significance of the coefficient, OR the odds ratio, CI 95% confidence interval of the odds ratio\nDiscussion\nIn this study of the DRD4 gene, we focused on the upstream region, where associations with psychiatric phenotypes have been previously reported. However, genetic variants in this region do not show consistent association with schizophrenia. This may have been due to insufficient statistical power or inadequate interrogation of genomic variations in some studies. In addition, the differences in marker sets between studies increase the ambiguity when interpreting data. To minimize these problems, we adopted the thorough approach of re-sequencing and genotyping all validated polymorphisms.\nBoth upstream region variants, the single marker \u2212809G>A and a multi-marker combination, displayed association with schizophrenia in this study. The SNP \u2212521T>C, which was previously reported to show association with schizophrenia in an independent Japanese sample set (Okuyama et al. 1999), was not significant in our data set, as in other population data sets (Ambrosio et al. 2004; Jonsson et al. 2001; Segman et al. 2003). Since it is conceivable that particular combinations of multiple variants may confer an enhanced susceptibility to schizophrenia, we performed a logistic regression analysis to test all SNPs in the upstream region for association. This approach may be more appropriate than conventional haplotype analysis, when examining the upstream region of the DRD4 gene where there is evidence of LD decay in a relatively short genomic stretch and most markers do not correlate with each other. Results appear to indicate a synergistic interaction among the four promoter polymorphisms in the DRD4 gene. These results should be treated with caution since there are limitations to the present association study. Firstly, this is a case-control study, where false positive findings due to population stratification can occur. Analysis of this sample set using STRUCTURE software (Pritchard et al. 2000) detected no evidence of population stratification (Shimizu et al. 2006). To address this issue further, we also performed a transmission disequilibrium test. This testing failed to replicate the association of \u2212809G>A in a sample set of 80 complete schizophrenic trios (data not shown). This sample size would have only a limited statistical power to detect true association. Secondly, the significance of the \u2212809G>A polymorphism in the promoter region disappeared after Bonferroni correction for multiple testing. Thirdly, the sample size is obviously limited and so the insignificant result for \u2212521T>C needs to be interpreted cautiously. The sample size in this study had a power of 0.59 to detect a susceptibility variant with a relatively small effect (relative risk = 1.2 and 1.44 for heterozygotes and homozygotes, respectively), when the allele frequency is 0.41. This frequency equals that of SNP \u2212521C, whose association with schizophrenia has been previously reported (Purcell et al. 2003; Xing et al. 2003).\nIn conclusion, to our knowledge, this is the first study that highlights a possible combinatorial effect of promoter SNPs in the DRD4 gene. It will be interesting to determine whether this multi-marker association can be confirmed in independent data sets, where samples are well-defined in terms of clinical variables such as symptoms, age-at-onset, severity, medication type, and response to antipsychotic treatment. Furthermore, it will be important to examine the functional effects of variant combinations, to understand the mechanisms by which they increase or decrease susceptibility to schizophrenia.","keyphrases":["dopamine d4 receptor","re-sequencing","promoter region","logistic regression analysis","linkage disequilibrium","haplotype block"],"prmu":["P","P","P","P","P","P"]}
{"id":"Eur_J_Clin_Pharmacol-4-1-2329724","title":"Should medical students learn to develop a personal formulary?\n","text":"Objective This study was performed to determine whether students who are trained in developing a personal formulary become more competent in rational prescribing than students who have only learned to use existing formularies.\nIntroduction\nMany medical schools in developed as well as developing countries are changing their undergraduate education in pharmacotherapy from classic lecture-based learning to problem-based learning (PBL). They are moving away from the presentation of classic pharmacological knowledge on medicines towards promoting problem-solving and prescribing skills [1, 2]. The World Health Organisation (WHO) Guide to Good Prescribing and several training courses on problem-based pharmacotherapy teaching have contributed to this development [3, 4].\nThe WHO Guide to Good Prescribing gives medical students a normative model for therapeutic reasoning and prescribing and provides a six-step guide to the process of rational prescribing: (1) define the patient\u2019s problem, (2) specify the therapeutic objective, (3) choose a (drug) treatment, (4) write the prescription and start the treatment, (5) give patient information and warnings, and (6) monitor the treatment. One of the key principles of the WHO approach is the division of step 3 [choose a (drug) treatment] into two steps. Step 3a, consider the suitability of a standard (p-drug) treatment for the disease in general, and step 3b, verify its suitability for the particular patient and alter the (drug) treatment if necessary. Consequently, undergraduate students are taught how to define their own p-drugs and are encouraged during their medical education and clinical activities to develop a personal formulary [5].\nThe WHO method emphasises that future doctors should master both steps of the drug-selection process. Selecting a p-drug trains students in pharmacological, clinical and epidemiological principles and in collecting and comparing evidence. By considering various treatment alternatives when developing their personal formulary, students are able to choose an alternative drug treatment more rationally when the drug of first choice is not suitable for a particular patient; for example, because of contraindications or side effects. In addition, by considering treatment alternatives, students are able to evaluate the information provided about new drugs during their professional career more critically.\nApplication of the WHO method has improved the prescribing skills of undergraduate medical students [4]. However, it is not known to what extent the p-drug concept and developing a personal formulary has contributed to this improvement. Some have argued that existing formularies and standard treatment guidelines, developed by experts, would lead to better prescribing than would a personal formulary developed by students. Furthermore, doctors are usually expected to work within the limitations of existing hospital formularies or practice guidelines. Despite the fact that teaching the p-drug concept and developing a personal formulary is a challenging and time-consuming task, we hypothesise that training in developing a personal formulary would lead to increased prescribing skills compared with using an existing formulary.\nTherefore, the aim of this study was to determine whether there is a difference in prescribing skills between medical students who have been trained in creating and using a personal formulary and those who have been trained in using existing formularies only.\nMaterials and methods\nStudy design\nA randomised controlled pre and posttest design with three arms was implemented (Fig.\u00a01). All students participated in a pretest. After the pretest at each university, students were randomly divided into three equally sized groups: a personal formulary (PF) group, an existing formulary (EF) group and a control group. Students in the PF and EF groups received two different training programmes before taking the posttest. The control group received no additional training and was given only the pre- and posttests.\nFig.\u00a01Study design: PF personal formulary group, EF existing formulary group and C control group. Clinical subjects: A  essential hypertension, B  osteoarthritis, C  acute bronchitis and D  gastroenteritis. Complexity levels: 1  straightforward case of a middle-aged adult patient, 2  patient with a contraindication to a generally accepted drug of first choice, 3  severe case and  4 case without effect of previous treatment and side effects\nAfter the posttest, a structured questionnaire with 21 questions was administered to identify differences in the type of teaching students received prior to the study. The questions were about two major teaching issues: curriculum type; and teaching methods in pharmacology, clinical pharmacology and pharmacotherapy. Other questions were designed to verify whether the universities had carried out the study in full accordance with the standard protocol. The study was approved by the educational research committee of the VU University Medical Center (VUMC).\nStudy population\nSix hundred and nine undergraduate medical students from eight countries were selected on a voluntary basis (Vellore in India, Yogyakarta in Indonesia, Maastricht in the Netherlands, Kazan in the Russian Federation, Bratislava in Slovakia, Cape Town in South Africa, La Laguna in Spain and Aden\/Sana\u2019a (jointly) in Yemen). All students had passed their pharmacology examinations (basic pharmacology and clinical pharmacology) and were about to start their clinical internships. Participation in the study was in addition to their normal curriculum. All students were informed in advance about the general aim of the study and the method of testing. Cross-contamination of the students in the study groups and the control group was prevented by giving the students in the control group the opportunity to participate in a training programme after the study.\nMaterials\nFor the intervention and tests, 16 written patient cases were developed using four clinical subjects (A\u2013D), each with a different level of complexity (1\u20134) (Fig.\u00a01). All cases were formulated according to a standard design:\nGeneral patient information (e.g. age, gender, allergy, occupation and pregnancy)Summary of previous and current diseases and treatments (comorbidity and comedication)Extensive description of the present history and a physical examinationDiagnosis\nEach case differed in disease severity and complicating factors such as age, comorbidity and comedication, drug allergy, pregnancy and breast feeding. All three study groups received a copy of the British National Formulary (BNF) as a drug reference [6]. In addition, the PF group received the WHO Guide to Good Prescribing, whereas the EF group received an adapted version of the guide without the chapters about the p-drug concept. The EF group also received the Oxford Formulary, containing treatment advice for symptoms and diseases that occur frequently in general practice, including the four clinical subjects used in this study [7].\nIntervention\nThe training course for the PF and EF groups lasted for 5\u00a0weeks. The intervention for both groups consisted of an introductory session and four training sessions (Fig.\u00a01)\u2014one per week for about 2 h. The chosen teaching time was based on earlier experiences with teaching programmes using the PF concept [4, 8]. In the introductory session, the WHO step-wise approach to prescribing drugs, was explained. However, the p-drug concept was explained only to the PF group. In each training session, two written patient cases of one clinical subject were discussed. Students completed a treatment plan form, following the six steps of the WHO model. The students prepared for the sessions by studying the treatment of the clinical subject but in different ways. The PF group was asked to make an inventory of available treatments and select a p-drug from this group (on the basis of efficacy, safety, suitability and cost), whereas the EF group was asked to study the relevant treatment guidelines of the Oxford Formulary. One facilitator per university supervised the sessions of both PF and EF groups only by guiding the discussions and clarifying aspects of the case if necessary. Facilitators were specifically instructed not to provide solutions to the patient problems but only to advise students on how to solve these themselves. All facilitators had received specific training on this teaching methodology in a research meeting at the VUMC. The control group received no additional training between the pre- and posttest but proceeded only with their routine curriculum.\nTest and scoring\nThe pre- and posttests consisted of four written patient cases. Each case contained a different clinical problem of a different level of complexity (Fig.\u00a01). The combination between the clinical problem and level of complexity were randomly chosen. For each case, the students had to complete a treatment plan form. They were allowed to use any reference materials but not to consult one another. Directly after the posttest, the answer sheets for both pre- and posttests were blindly scored by the facilitator in a standardised manner using a 5-point scale (0 = no answer, 1 = poor answer, 2 = arguable answer, 3 = acceptable answer and 4 = good answer). Prior consensus about the scoring system had been reached between all facilitators during the research meeting at the VUMC. The scores were sent to the researchers at the VUMC for analysis. Individual student scores were not used by the participating universities for formal student assessments.\nStatistical analysis\nThe results were analysed in SPSS 13.0 for Windows. The scores on the six steps of the WHO model were clustered into four prescribing skills: treatment choice, prescription writing, patient information and treatment monitoring. The differences between groups were analysed by analysis of variance (ANOVA) with the university as a factor, as well as separately. For pair-wise differences between the three groups, a multiple comparison procedure was applied. Significance was accepted at a two-tailed p\u2009<\u20090.05. Only the results of students who finished both pre- and posttest were included in the analysis.\nResults\nA total of 609 students participated. Five hundred and eighty-three students (96%) completed the pre- and posttests:194\/203 (95.6%) students from the PF groups, 198\/203 (97.5%) from the EF groups and 191\/203 (94.1%) from the C groups. The three groups did not differ in their averages for previous examination results and pretest scores. The increases in the mean scores of the PF group (0.93, 23%) and the EF group (0.78, 19%) were both significantly higher than in the C group (0.23, 6%) (Table\u00a01). In addition, the increase in the PF group was significantly higher than in the EF group (Table\u00a01). Within this total, the differences were significant for three of the four prescribing skills (treatment choice, patient information and monitoring treatment but not prescription writing) and for three of the four complexity levels (straightforward case, cases with a contraindication to first-line drugs and severe cases but not cases with no effect of previous treatment and side effects present) (data not shown).\nTable\u00a01Pre- and posttest scores per university\u00a0NujmberPretestPosttestPFEFCPFEFCYemen730.410.350.332.98ab1.56b0.46Russia880.830.910.871.83ab1.67b1.03Indonesia751.121.201.342.45ab1.98b1.42Netherlands642.202.062.253.04b3.06b2.63cSlovakia722.272.132.123.203.122.90cIndia722.172.392.232.192.51b2.08Spain732.382.332.282.612.76b2.36South Africa662.542.482.383.022.972.81cAll Increase5831.711.621.692.64a,b 0.93 (23%)2.40b 0.78 (19%)1.92c 0.23 (6%)PF personal formulary group, EF existing formulary group, C control groupaPF > EF (p\u2009<\u20090.05), bPF, EF > C (p\u2009<\u20090.05), cC > pretest (p\u2009<\u20090.05)\nResults of medical students from the eight universities are listed separately (Table\u00a01). In Yemen, the Russian Federation and Indonesia, pretest scores were lower than in the other five universities. In addition, the increase in their scores was significantly higher in the PF groups than in the EF groups. The differences among the PF and EF groups were also significant for all levels of complexity and all four prescribing skills (Fig.\u00a02). In the remaining five schools, there was no significant difference between the PF and EF groups.\nFig.\u00a02Pre- and posttest scores for different prescribing skills of students from countries with a traditional curriculum (Yemen, the Russian Federation and Indonesia). PF personal formulary group (\u2013), EF existing formulary group (.....), C control group (- -). Posttest scores are annotated, # = PF > EF\nThe results of the questionnaire about the type of teaching that students received indicated that Yemen, the Russian Federation and Indonesia had a predominantly classic curriculum. On average, the participating students from these universities had received 90\u00a0h (range 52\u2013160 h) of pharmacology teaching by lectures, 23\u00a0h (range 3\u201350 h) of clinical pharmacology teaching also by lectures and no explicit pharmacotherapy teaching. These students were not familiar with PBL but were somewhat accustomed to discussion in small groups. The remaining five universities had a predominantly problem-based teaching curriculum. The participants had received an average of 43\u00a0h (range 6\u2013100 h) of pharmacology teaching by lectures, including small group teaching; 24\u00a0h (range 6\u201340 h) of clinical pharmacology teaching, mainly by small group teaching; and no explicit pharmacotherapy teaching. The students from these universities were all familiar with PBL and were all accustomed to discussion in small groups. Some of these students already had some clinical experience (Slovakia and Spain). Except for the presence (or absence) of PBL, no other differences in teaching could be identified between any of the eight universities. No specific differences between universities in the way the study was performed or indications for possible bias were identified.\nDiscussion\nThe results of this study show that developing a personal formulary and using an existing formulary both increase the competence of medical students in rational prescribing. However, the overall differences between the PF group and the EF group (23% vs. 19% increase in mean score, p\u2009<\u20090.05) should be interpreted with caution. It is tempting to conclude that teaching students to develop a personal formulary adds to their prescribing skills. However, this effect was only visible in the universities in Yemen, the Russian Federation, and Indonesia. No significant differences between PF and EF scores were found in the universities in the Netherlands, Slovakia, Spain, India or South Africa. A possible reason could be the high pretest scores in the last five universities, which reduced the likelihood of a significant increase after a relatively short intervention period of 5\u00a0weeks. A similar effect was seen in the first multicountry study where universities with a problem-based curriculum showed less benefit from a short intervention course than those without a problem-based curriculum [4].\nAn important question is why these five universities had such high initial scores? The questionnaire was specifically designed for the purpose of this study, and the only explanation that emerged was that students from these five universities had received problem-based teaching and were therefore familiar with the process of solving patient problems. In the universities with lower pretest scores, classic teaching was paramount and students had less experience with solving patient problems.\nThis finding is in accordance with the fact that successful clinical problem solvers possess comprehensive knowledge, but the way they organise and understand their knowledge is even more critical. Modern teaching is aimed at creating both a knowledge structure and the necessary \u201csearch-and-retrieve\u201d strategy for responding to complex diagnostic problems [9, 10]. Students who were already trained in this way via problem-based curricula seemed better able to transfer this ability to therapeutic problem solving and did not show an additional increase in such skills when they participated in our study. However, students from universities with a classic curriculum quickly assimilated the p-drug concept, and their results came very close to those of the other five universities, even after a short intervention of only 5\u00a0weeks.\nThe limitations of our study need to be addressed. The voluntary participation of students may have created a positive bias for the total group, but allocation to the intervention groups was randomised and therefore the comparison was unbiased. In addition, to ensure baseline comparability among the groups, previous results on university examinations were averaged for the three groups. The scoring by the medical schools was blinded, with the pre- and posttest forms of all three groups being mixed during the scoring. Cross-contamination between the study and control groups was prevented by explaining to the students the importance of not exchanging any information and by reassuring the control group that it would receive the training after finishing the study.\nTherefore, taking the above-mentioned limitations and considerations into account, it can be concluded that training in developing and using a personal formulary or in the use of an existing formulary may increase the rational prescribing of medical students. However, with regard to universities with a general problem-based curriculum, training in the development of a personal formulary is probably not worth the additional effort. In these circumstances, pharmacotherapy teaching can be based on existing formularies instead of a personal formulary.\nIn this international study, only the short-term effects on rational prescribing of developing a personal formulary or using an existing formulary have been studied. Despite the fact that the long-term effects are unknown, it can be hypothesised that after developing a personal formulary, students may judge new drugs more effectively and be more capable of deciding whether or not to include these drugs into their (personal) formulary. Furthermore, the results may diminish the concerns ventilated by some clinical pharmacology teachers regarding the changes in the style of undergraduate medical curricula from classic to PBL teaching [11, 12].\nIn practice, this implies that the decision to organise a teaching programme for medical students on how to develop and use a personal formulary needs to be based on an initial assessment of both the students and the curriculum. The same may be true for postgraduate and continuing education of doctors who have been trained in the classic manner [13, 14]. A screening test, such as the pretest in this study, could perhaps be used to identify universities, students or prescribers that would benefit most from this type of training.","keyphrases":["personal formulary","rational prescribing","prescribing","drug treatment","undergraduate medical education"],"prmu":["P","P","P","P","R"]}
{"id":"Apoptosis-4-1-2257993","title":"Rho-kinase-dependent F-actin rearrangement is involved in the inhibition of PI3-kinase\/Akt during ischemia\u2013reperfusion-induced endothelial cell apoptosis\n","text":"Activation of cytoskeleton regulator Rho-kinase during ischemia\u2013reperfusion (I\/R) plays a major role in I\/R injury and apoptosis. Since Rho-kinase is a negative regulator of the pro-survival phosphatidylinositol 3-kinase (PI3-kinase)\/Akt pathway, we hypothesized that inhibition of Rho-kinase can prevent I\/R-induced endothelial cell apoptosis by maintaining PI3-kinase\/Akt activity and that protective effects of Rho-kinase inhibition are facilitated by prevention of F-actin rearrangement. Human umbilical vein endothelial cells were subjected to 1 h of simulated ischemia and 1 or 24 h of simulated reperfusion after treatment with Rho-kinase inhibitor Y-27632, PI3-kinase inhibitor wortmannin, F-actin depolymerizers cytochalasinD and latrunculinA and F-actin stabilizer jasplakinolide. Intracellular ATP levels decreased following I\/R. Y-27632 treatment reduced I\/R-induced apoptosis by 31% (P < 0.01) and maintained Akt activity. Both effects were blocked by co-treatment with wortmannin. Y-27632 treatment prevented the formation of F-actin bundles during I\/R. Similar results were observed with cytochalasinD treatment. In contrast, latrunculinA and jasplakinolide treatment did not prevent the formation of F-actin bundles during I\/R and had no effect on I\/R-induced apoptosis. Apoptosis and Akt activity were inversely correlated (R2 = 0.68, P < 0.05). In conclusion, prevention of F-actin rearrangement by Rho-kinase inhibition or by cytochalasinD treatment attenuated I\/R-induced endothelial cell apoptosis by maintaining PI3-kinase and Akt activity.\nIntroduction \nExperimental ischemia and reperfusion (I\/R) induces endothelial cell apoptosis in several organs [1, 2]. We and others have previously shown that activation of RhoA and downstream effector Rho-kinase during experimental I\/R plays a major role in I\/R injury and associated apoptosis [3\u20137]. Indeed, Rho-kinase inhibition has beneficial effects on I\/R injury in several organs [4, 6\u201312]. Endothelial cells may be essential for mediating the beneficial effects of Rho-kinase inhibition on I\/R injury, because, for instance inhibition of endothelial nitric oxide synthase blocked the beneficial effects of Rho-kinase inhibition on rat myocardial infarct size [12].\nRho-kinase activation during the execution of apoptosis results in increased myosin activity, bundling of F-actin by activated myosins, actin\u2013myosin contractile force generation, cell contraction and ultimately membrane blebbing [13\u201315]. Recent in\u00a0vivo and in\u00a0vitro studies suggest that Rho-kinase and the effects of Rho-kinase on the actin\u2013myosin cytoskeleton may also be involved in the initiation of apoptosis [4, 12, 16\u201320]. Rho-kinase is a negative regulator of the pro-survival phosphatidylinositol 3-kinase (PI3-kinase)\/Akt pathway and it upregulates mitochondrial death pathway protein Bax [4, 12, 16, 19]. The effects of Rho-kinase on the cytoskeleton may therefore be important in linking Rho-kinase activation and PI3-kinase\/Akt inactivation.\nWe hypothesized that (1) inhibition of Rho-kinase can prevent I\/R-induced endothelial cell apoptosis by maintaining PI3-kinase and Akt activity and (2) prevention of F-actin rearrangement facilitates the protective effects of Rho-kinase inhibition (Fig.\u00a01). To test this hypothesis, we assessed whether specific inhibition of Rho-kinase with Y-27632 [21] attenuates apoptosis via phosphorylation of Akt in an in\u00a0vitro model of simulated I\/R. The involvement of the F-actin cytoskeleton was explored by the use of F-actin depolymerizers cytochalasinD and latrunculinA and F-actin stabilizer jasplakinolide [22].\nFig.\u00a01Proposed mechanism for modulation of I\/R-induced apoptosis involving RhoA\/Rho-kinase and PI3-kinase\/Akt. Simulated I\/R activates RhoA\/Rho-kinase [3\u20137], which leads to F-actin rearrangement. F-actin rearrangement facilitates reduction of PI3-kinase\/Akt activity and induction of apoptosis. This can be prevented by inhibition of Rho-kinase with Y-27632. The protective effects of Y-27632 can be blocked with PI3-kinase inhibitor wortmannin. Several other pathways for I\/R-induced apoptosis are represented by the dashed arrow [2, 19]\nMaterials and methods\nCell culture\nHuman Umbilical Vein Endothelial Cells (HUVECs) were isolated, cultured and characterized as previously described [23]. Briefly, HUVECs were cultured on gelatin-coated wells\/glass cover slips until confluent at 37\u00b0C under 5% CO2\/95% air atmosphere. Culture medium was renewed every other day (medium 199 supplemented with 10% heat-inactivated human serum, 10% heat-inactivated new born calf serum, 150\u00a0\u03bcg\/ml crude endothelial cell growth factor, 2\u00a0mM l-glutamine, 5\u00a0U\/ml heparin, 100\u00a0IU\/ml penicillin, and 100\u00a0\u03bcg\/ml streptomycin).\nSimulation of I\/R\nWe used an established in\u00a0vitro endothelial cell model for simulation of ischemia and reperfusion [24, 25]. Figure\u00a02 shows a schematic representation of the experimental protocol. Cells were washed twice with washing buffer (1.2\u00a0mM MgSO4\u00a0\u00b7\u00a07H2O, 116\u00a0mM NaCl, 5.3\u00a0mM KCl, 1.13\u00a0mM NaH2PO4\u00a0\u00b7\u00a0H2O, 1.8\u00a0mM CaCl2\u00a0\u00b7\u00a02H2O, 20\u00a0mM HEPES) and treated for 1\u00a0h with washing buffer with or without 10 \u03bcM Y-27632 (Tocris Cookson Ltd., Bristol, United Kingdom (UK)), 250\u00a0nM cytochalasinD (Sigma-Aldrich, Saint Louis, Missouri, United States of America (USA)), 150\u00a0nM latrunculinA (Calbiochem, Darmstadt, Germany), 20\u00a0nM jasplakinolide (Calbiochem) and 50\u00a0nM wortmannin (Sigma-Aldrich). This was followed by 1\u00a0h of simulated ischemia by covering cells with 1\u00a0ml mineral oil (nitrogen bubbled) and subsequently simulated reperfusion by replacement of culture medium for different time points: no reperfusion for adenosine tri-phosphate (ATP) measurement and F-actin staining, 1\u00a0h for ATP measurement, F-actin staining and western blotting to analyze phospho Akt (pAkt) levels and 24\u00a0h for quantification of apoptosis. Drugs were also present during simulated reperfusion. As controls, we used cells treated for 1\u00a0h with washing buffer followed by treatment with medium (control) or medium supplemented with 10% mineral oil (control oil) to assess any injurious effects of products within the oil.\nFig.\u00a02Schematic representation of the experimental protocol. Cells were pre-treated for 1\u00a0h with Y-27632, cytochalasinD, latrunculinA, jasplakinolide or wortmannin. One hour of simulated ischemia was followed by 1\u201324\u00a0h of simulated reperfusion. No reperfusion for ATP measurement and F-actin staining, 1\u00a0h for ATP measurement, F-actin staining and analysis of Akt activity by western blotting and 24\u00a0h for quantification of apoptosis. The drugs were also present during the reperfusion phase\nATP measurement\nWe measured ATP after ischemia and I\/R (1\u00a0h of reperfusion) in order to confirm ischemic substrate deprivation. Cells were grown in a gelatin-coated 12-well chamber and treated according to the simulated I\/R protocol. ATP was measured using the ENLITEN ATP Assay System Bioluminiscence Detection Kit for ATP (Promega Corporation, Madison, Wisconsin, USA). Proteins in lysate were measured with BCA Protein Assay Kit (Pierce, Rockford, Illinois, USA) in order to calculate nmol ATP\/\u03bcg protein.\nQuantification of apoptosis\nThe cells were grown in a 24-well chamber on gelatin-coated glass cover slips and treated according to the simulated I\/R protocol (24\u00a0h of reperfusion). Cells were fixed in 4% formaldehyde and permeabilized with 0.2% triton X-100 (Sigma-Aldrich). The nucleus was stained with 4\u2032,6-diamidino-2-phenylindole (DAPI) in Vectashield\u00ae Mounting medium (Vector Laboratories, Inc., Burlingame, California, USA). Apoptosis on each glass cover slip was quantified using a DeadEndTM Fluorometric TdT-mediated dUTP Nick End Labeling (TUNEL) System (Promega Corporation) with fluorescence microscopy using a MarianasTM digital imaging microscope and Slidebook 4.2 software (Intelligent Imaging Innovations, Inc., Denver, Colorado, USA). The number of apoptotic nuclei (TUNEL-positive) and the total number of nuclei (DAPI-positive) were counted in three non-overlapping microscope fields\/glass cover slip using a 10\u00d7 air lens (Carl Zeiss B.V., Sliedrecht, The Netherlands) and averaged. The number of apoptotic nuclei was expressed as percentage of the total number of nuclei.\nF-actin cytoskeleton staining\nThe cells were grown in a 24-well chamber on gelatin-coated glass cover slips and treated according to the simulated I\/R protocol (1\u00a0h of reperfusion). Cells were fixed in 4% formaldehyde and permeabilized with 0.2% triton X-100. F-actin was stained with rhodamin-phalloidin (1:100) (Molecular Probes, Inc., Eugene, Oregon, USA) and the nucleus with DAPI in Vectashield\u00ae Mounting medium. Cells were visualized with fluorescence microscopy and Slidebook software.\nAnalysis of Akt phosphorylation\nCells were grown in a gelatin coated 6-well chamber, treated according to the simulated I\/R protocol (1\u00a0h of reperfusion) and lysated with lysis buffer (20\u00a0mM Tris\/HCl pH 8.0, 150\u00a0mM NaCl, 90\u00a0mM KCl, 2\u00a0mM EDTA\/NaOH pH 8.0, igepal (1:200), triton X-100 (1:200), 1\u00a0mM Na3VO4, 10\u00a0mM NaF, protease inhibitors (1:100), phosphatase inhibitors (1:100)). Protein concentration was measured (BCA method) and equal amounts of protein from each sample were separated by SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, California, USA). Membranes were incubated with polyclonal antibodies against pAkt (serine-473, rabbit, 1:1,000) and total Akt (tAkt, rabbit, 1:1,000) (Cell Signaling Technology, Inc., Danvers, Massachusetts, USA). tAkt was used as loading control. Goat anti-rabbit immunoglobulins horseradish peroxidase (HRP) from DakoCytomation (Glostrup, Denmark) was used for the detection of the primary antibodies at 1:1,000. Detection of the HRP reaction was performed with ECL plus Western Blotting Detection System (Amersham Biosciences, Little Chalfont Buckinghamshire, UK). Imaging and analysis were performed with LAS-3000 (Fuji Photo Film Co., Ltd., Tokyo, Japan) and AIDA Image Analyzer (Raytest GmbH, Straubenhardt, Germany). Data were expressed as pAkt\/tAkt as percentage of control.\nStatistical analysis\nA one-way analysis of variance (ANOVA) was conducted to explore the effects of I\/R, Y-27632, cytochalasinD or wortmannin treatment, and their interaction on apoptosis. The t-test was used to further analyse the differences between the groups when ANOVA indicated statistical significance. We adjusted for multiple testing with help of the Bonferroni Holm method. The paired t-test was used for paired ATP data and logarithmic transformed absolute pAkt\/tAkt data. Absolute pAkt\/tAkt data were logarithmic transformed to obtain a Gaussian distribution. We adjusted for multiple testing with help of the Bonferroni Holm method. The Pearson coefficient of determination (R2) was used to express the relation between the percentage of apoptotic nuclei and the logarithmic transformed absolute pAkt\/tAkt data. Differences were considered significant at the P\u00a0<\u00a00.05 level. Data are presented as means\u00a0\u00b1\u00a0standard error of the mean (SEM).\nResults\nATP levels and apoptosis following simulated ischemia and I\/R\nIn order to confirm ischemic substrate deprivation in simulated I\/R, we measured intracellular ATP levels. Ischemia was simulated by covering the monolayer of cells with nitrogen bubbled mineral oil. Reperfusion was simulated by replacement of culture medium (Fig.\u00a02). Intracellular ATP levels decreased following simulated ischemia compared to the levels in cells treated with medium supplemented with 10% mineral oil (control oil) (0.15\u00a0\u00b1\u00a00.04\u00a0nmol\u00a0ATP\/\u03bcg protein versus 0.22\u00a0\u00b1\u00a00.04\u00a0nmol ATP\/\u03bcg protein, P\u00a0<\u00a00.05, n\u00a0=\u00a07). Intracellular ATP decreased even further during the reperfusion phase (0.10\u00a0\u00b1\u00a00.04\u00a0nmol ATP\/\u03bcg protein, P\u00a0<\u00a00.05 versus control oil, n\u00a0=\u00a07). ATP levels of control oil cells were comparable to those of control cells (0.20\u00a0\u00b1\u00a00.04\u00a0nmol ATP\/\u03bcg protein, n\u00a0=\u00a07). Simulated I\/R induced a significant percentage of apoptotic nuclei (ANOVA for the effect of I\/R on apoptosis, P\u00a0<\u00a00.0001), while hardly any apoptosis was observed in control and control oil cells (Fig.\u00a03).\nFig.\u00a03Rho-kinase inhibition reduces the degree of apoptosis during I\/R via PI3-kinase. Non-treated control (C, n\u00a0=\u00a011), control oil (C O, n\u00a0=\u00a02), Y-27632-treated control (C Y, n\u00a0=\u00a011) and wortmannin-treated control (C W, n\u00a0=\u00a05) showed hardly any apoptosis. Simulated I\/R induced a significant percentage of apoptotic nuclei (minimum P\u00a0<\u00a00.005 versus the controls, n\u00a0=\u00a012). Y-27632 treatment reduced the percentage of apoptotic nuclei during simulated I\/R (I\/R Y, *P\u00a0<\u00a00.01, n\u00a0=\u00a012). This protective effect was completely blocked by wortmannin (I\/R YW, #P\u00a0<\u00a00.05, n\u00a0=\u00a05). The number of apoptotic nuclei is expressed as percentage of total number of nuclei. Data are presented as means\u00a0\u00b1\u00a0SEM\nThe effect of Rho-kinase inhibition on apoptosis\nTo investigate the role of Rho-kinase during I\/R-induced apoptosis, HUVECs were treated with inhibitor Y-27632 during simulated I\/R (Fig.\u00a03). Y-27632 had no effect on basal apoptosis, but reduced the degree of apoptosis during I\/R (ANOVA for the effects of Y-27632 and the interaction between I\/R and Y-27632 on apoptosis, both P\u00a0<\u00a00.01). To determine whether the protective effects of Rho-kinase inhibition were mediated by PI3-kinase, cells were co-treated with PI3-kinase inhibitor wortmannin. Wortmannin had no effect on basal apoptosis, but blocked the protective effect of Rho-kinase inhibition during I\/R (ANOVA for the effect of wortmannin on apoptosis, P\u00a0<\u00a00.05).\nAkt activity in Y-27632-mediated protection against apoptosis\nTo investigate the effect of Y-27632 treatment during I\/R on Akt activity, Akt phosphorylation was analyzed by western blotting (Fig.\u00a04). Simulated I\/R reduced Akt activity compared to non-treated or Y-27632-treated control cells. Y-27632 treatment maintained Akt activity in I\/R cells. To determine whether this effect was mediated by PI3-kinase, cells were co-treated with wortmannin. Wortmannin blocked the Y-27632-induced maintenance of Akt activity. Akt activity in Y-27632- (Fig.\u00a04) or wortmannin-treated control cells (preliminary data) was comparable to the activity in non-treated control cells.\nFig.\u00a04Rho-kinase inhibition maintains Akt activity during I\/R. A representative western blot of pAkt and tAkt is shown in the upper panel and a graph of pAkt\/tAkt as percentage of control in the lower panel. Simulated I\/R (I\/R, n\u00a0=\u00a015) induced a decrease in pAkt compared to the control and the Y-27632-treated control (C Y, *P\u00a0<\u00a00.05, n\u00a0=\u00a015). Y-27632 treatment during simulated I\/R maintained pAkt levels (I\/R Y, **P\u00a0<\u00a00.05, n\u00a0=\u00a014). Wortmannin blocked the Y-27632-induced maintenance of Akt activity (I\/R YW, #P\u00a0<\u00a00.05, n\u00a0=\u00a012). Data are presented as means\u00a0\u00b1\u00a0SEM. Absolute pAkt\/tAkt data in arbitrary units (a.u.): control: 0.75\u00a0a.u.; C Y: 0.64\u00a0a.u.; I\/R: 0.32\u00a0a.u.; I\/R Y: 0.36\u00a0a.u.; I\/R YW: 0.04\u00a0a.u\nThe F-actin cytoskeleton in Y-27632-mediated protection against apoptosis\nTo investigate whether Y-27632-mediated protection against apoptosis paralleled the absence of Rho-kinase-mediated F-actin rearrangement, the F-actin cytoskeleton was visualized with rhodamin-phalloidin in non-treated or Y-27632-treated control, simulated ischemia and simulated I\/R cells (n\u00a0=\u00a09, representative pictures are shown in Fig.\u00a05). Y-27632-treated control cells showed a slight decrease in peripheral F-actin bundles compared non-treated control cells, indicating that in\u00a0vitro inhibition of Rho-kinase was able to reduce F-actin bundle formation. After simulated ischemia in non-treated cells, some F-actin bundles appeared in the cytoplasm. Y-27632-treated ischemic cells showed only F-actin clumps. After simulated reperfusion, even more F-actin bundles appeared in the periphery and the cytoplasm of the non-treated cells. In contrast, Y-27632-treated I\/R cells, showed no F-actin bundles.\nFig.\u00a05Y-27632 and cytochalasinD prevent F-actin bundle formation during I\/R. Shown is fluorescent imaging of rhodamin-phalloidin stained F-actin structures and DAPI stained nuclei in non-treated, Y-27632-, cytochalasinD-, latrunculinA- and jasplakinolide-treated control, simulated ischemia and simulated I\/R cells. The arrow and * indicate F-actin bundles and F-actin clumps, respectively. Control cells: In non-treated cells, most F-actin bundles were seen in the periphery of the cell. Y-27632-treated cells showed a slight decrease in peripheral F-actin bundles. CytochalasinD-treated cells showed only F-actin clumps. Cells treated with latrunculinA or jasplakinolide showed a slight decrease and increase in F-actin bundles, respectively. Ischemic cells: Non-treated cells showed F-actin bundles throughout the whole cell. Cells treated with Y-27632, cytochalasinD or latrunculinA showed only F-actin clumps. Jasplakinolide-treated cells showed F-actin bundles. I\/R cells: Non-treated cells showed F-actin bundles throughout the whole cell. In Y-27632-treated cells, no F-actin bundles were seen. CytochalasinD-treated cells showed also no F-actin bundles, only F-actin clumps were visible. LatrunculinA-treated cells showed some F-actin bundles. Jasplakinolide-treated cells showed as many F-actin bundles as non-treated I\/R cells\nTo further investigate the role of the F-actin cytoskeleton in I\/R-induced apoptosis, we used F-actin depolymerizers cytochalasinD and latrunculinA and F-actin stabilizer jasplakinolide (Fig.\u00a05). CytochalasinD-treated control cells showed no F-actin bundles. LatrunculinA-treated control cells showed a slight decrease and jasplakinolide-treated control cells showed a slight increase in F-actin bundles compared to non-treated control cells. CytochalasinD- and latrunculinA-treated ischemic cells showed no F-actin bundles, only F-actin clumps were visible. Jasplakinolide-treated ischemic cells showed F-actin bundles throughout the whole cell. In cytochalasinD-treated I\/R cells, no F-actin bundles were visible, only F-actin clumps could be seen. In contrast, latrunculinA-treated I\/R cells still showed some F-actin bundles and jasplakinolide-treated I\/R cells showed as many F-actin bundles as non-treated I\/R cells.\nSince cytochalasinD treatment prevented F-actin rearrangement similarly to Y-27632 treatment, we investigated whether cytochalasinD treatment could also prevent apoptosis during simulated I\/R (Fig.\u00a06). CytochalasinD had no effect on basal apoptosis, but reduced the degree of apoptosis during I\/R (ANOVA for the effects of cytochalasinD and the interaction between I\/R and cytochalasinD on apoptosis, both P\u00a0<\u00a00.05). Wortmannin blocked the protective effect of cytochalasinD in I\/R (ANOVA for the effect of wortmannin on apoptosis, P\u00a0<\u00a00.0001). In contrast to the protective effect of Y-27632 and cytochalasinD on apoptosis, latrunculinA and jasplakinolide did not affect I\/R-induced apoptosis, also not in co-treatment with wortmannin (data not shown). LatrunculinA and jasplakinolide had no effect on basal apoptosis (data not shown).\nFig.\u00a06CytochalasinD reduces the degree of apoptosis during I\/R via PI3-kinase. Non-treated control (C, n\u00a0=\u00a06) and cytochalasinD-treated control (C CytoD, n\u00a0=\u00a06) showed hardly any apoptosis. Simulated I\/R induced a significant percentage of apoptotic nuclei (P\u00a0<\u00a00.0001 versus the controls, n\u00a0=\u00a06). Treatment with cytochalasinD reduced the percentage of apoptotic nuclei in simulated I\/R (I\/R CytoD, *P\u00a0<\u00a00.05, n\u00a0=\u00a06). This protective effect was completely blocked by wortmannin (I\/R CytoDW, #P\u00a0<\u00a00.005, n\u00a0=\u00a06). The number of apoptotic nuclei is expressed as percentage of total number of nuclei. Data are presented as means\u00a0\u00b1\u00a0SEM\nFurthermore, cytochalasinD maintained Akt activity during simulated I\/R (Fig.\u00a07). Co-treatment with wortmannin completely blocked the cytochalasinD-induced maintenance of Akt activity. Akt activity in cytochalasinD-treated control cells was comparable to the activity in non-treated control cells.\nFig.\u00a07CytochalasinD treatment maintains Akt activity during I\/R. Simulated I\/R (I\/R, n\u00a0=\u00a011) induced a decrease in pAkt compared to the control and the cytochalasinD-treated control (C CytoD, *P\u00a0<\u00a00.01, n\u00a0=\u00a011). CytochalasinD treatment during simulated I\/R maintained pAkt levels (I\/R CytoD, **P\u00a0<\u00a00.05, n\u00a0=\u00a011). Wortmannin blocked the cytochalasinD-induced maintenance of Akt activity (I\/R CytoDW, #P\u00a0<\u00a00.01, n\u00a0=\u00a07). Data are presented as means\u00a0\u00b1\u00a0SEM. Absolute pAkt\/tAkt data in arbitrary units (a.u.): control: 0.61\u00a0a.u.; C CytoD: 0.84\u00a0a.u.; I\/R: 0.36\u00a0a.u.; I\/R CytoD: 0.54\u00a0a.u.; I\/R CytoDW: 0.05\u00a0a.u \nInterestingly, the percentage of apoptotic nuclei inversely correlated to Akt activity (Fig.\u00a08), indicating that a decrease in Akt activity correlated to an increase in apoptosis.\nFig.\u00a08The percentage of apoptotic nuclei inversely correlates to Akt activity (R2\u00a0=\u00a00.68, P\u00a0<\u00a00.05). Mean percentage of apoptotic nuclei and mean logarithmic transformed absolute pAkt\/tAkt levels in non-treated (C), Y-27632 (C Y)- and cytochalasinD (C CytoD)-treated control cells and in non-treated (I\/R), Y-27632 (I\/R Y)-, cytochalasinD (I\/R CytoD)- and wortmannin (I\/R YW and I\/R CytoDW)- treated I\/R cells are shown as *\nDiscussion\nThis study shows that (1) inhibition of Rho-kinase attenuates I\/R-induced endothelial cell apoptosis by maintaining PI3-kinase and Akt activity and (2) the effects of Rho-kinase inhibition on PI3-kinase\/Akt and apoptosis are facilitated by prevention of F-actin rearrangement.\nOur data point to a novel mechanistic link between activation of Rho-kinase during I\/R and negative regulation of PI3-kinase\/Akt, namely the effect of Rho-kinase on F-actin rearrangement (Fig.\u00a01). Prevention of F-actin rearrangement by Rho-kinase inhibition or by treatment with actin depolymerizer cytochalasinD, independent of Rho-kinase, both attenuated I\/R-induced endothelial cell apoptosis by maintaining PI3-kinase and Akt activity. Indeed, it has been reported previously that cytochalasins inhibited in\u00a0vivo and in\u00a0vitro apoptosis induced by a variety of stimuli, including I\/R [5, 13, 26]. This study is therefore an extension of and a link between reports on Rho-kinase as negative regulator of the PI3-kinase\/Akt pathway and reports on the involvement of Rho-kinase-dependent cytoskeletal rearrangement in the initiation of apoptosis [4, 12, 16\u201320].\nActin depolymerizer latrunculinA did not affect I\/R-induced endothelial cell apoptosis. This may be explained by the nature and degree of F-actin rearrangement during I\/R, since F-actin bundles were completely absent in Y-27632- and cytochalasinD-treated I\/R cells, while latrunculinA-treated cells still showed some bundles. Indeed, cytochalasinD and latrunculinA depolymerize F-actin by different mechanisms and induce different patterns in\u00a0vitro [22, 27]. CytochalasinD caps the barbed end of actin filaments, severs actin filaments, sequesters actin monomers or dimers, promotes nucleation and stimulates the ATPase activity of G-actin. In\u00a0vitro in hamster fibroblasts, it caused a complete collapse of the stress fibers at concentrations comparable to ours [22, 27]. LatrunculinA on the other hand binds to monomeric G-actin to form a non-polymerizable complex and caused concentration-dependent shortening and thickening of stress fibers in the fibroblasts at concentrations comparable to ours [22, 27]. Jasplakinolide, which promotes actin polymerization and stabilizes actin filaments by binding to F-actin [22], induced as many F-actin bundles in I\/R as non-treated I\/R cells and consequently did not affect I\/R-induced endothelial apoptosis. However, in\u00a0vivo it has been demonstrated that jasplakinolide prevented I\/R-induced apoptosis in the rat kidney, while latrunculinB increased I\/R-induced apoptosis in rat kidney [28, 29]. Furthermore, we observed hardly any apoptosis in our Y-27632-, cytochalasinD-, latrunculinA- and jasplakinolide-treated control cells, while some reports describe the induction of apoptosis in control cells by latrunculinA or jasplakinolide at much higher concentrations than ours [30, 31], by cytochalasinD in airway epithelial cells, but not in canine kidney epithelial cells [32] or by Y-27632 in airway epithelial cells [32]. Therefore, these discrepancies may be explained by the use of different concentrations, cell types or the use of an in\u00a0vivo\/in\u00a0vitro model.\nAkt activation after 1\u00a0h of reperfusion inversely correlated to the percentage of apoptosis after 24\u00a0h of reperfusion. Therefore, early reduced Akt activity may have a predictive value for the development of late I\/R-induced apoptosis. The preventive effect of Y-27632 and cytochalasinD treatment probably takes place early in the process of apoptosis, because the Y-27632-induced increase in Akt activity is rapid and transient with a maximum after 30\u00a0min [12, 16]. Indeed, we could neither detect increased Akt activity in our Y-27632-treated control cells after 3\u00a0h nor in our Y-27632-treated I\/R cells after 24\u00a0h of reperfusion (pilot study, data not shown), possibly because this was to late to detect the peak in Akt activity. I\/R-induced apoptosis was maximal as late as after 24\u201348\u00a0h of reperfusion [25]. Therefore, apoptosis was quantified after 24\u00a0h of reperfusion. Indeed, we did not see any TUNEL-positive nuclei after 1\u00a0h of reperfusion (pilot study, data not shown). Our percentage of apoptotic nuclei was comparable to the percentage reported by the developers of the simulated I\/R model [25]. However, less endothelial cell apoptosis was observed in a model of hypoxia\/reoxygenation [33], possibly because this induces less severe metabolic deprivation. In addition, PI3-kinase inhibition did not decrease basal Akt activity nor increased basal apoptosis, while it blocked the effects of Rho-kinase inhibition on I\/R-induced apoptosis and Akt activity. This suggests that the basal situation is independent of PI3-kinase, while the effects Rho-kinase inhibition during I\/R are dependent on PI3-kinase.\nAlthough it is unclear by which mechanism F-actin rearrangement facilitates the effects of Rho-kinase on PI3-kinase\/Akt and apoptosis, several mechanisms can be proposed. First, RhoA and Rho-kinase may regulate pAkt levels by regulating phosphatase and tensin homologue (PTEN) activity. PTEN is in inactive state in the cytosol and translocates to the membrane upon RhoA or Rho-kinase-induced activation, where it can inhibit the phosphorylation of Akt via PI3-kinase [34]. Disruption of the F-actin bundles by Rho-kinase inhibition or cytochalasinD treatment or the direct effects of Rho-kinase inhibition on PTEN may prevent the translocation and activation of PTEN, thereby preventing its inhibitory effect on Akt phosphorylation. Furthermore, there may be some PI3-kinase independent mechanisms involved in the Y-27632-induced protection against apoptosis. It is reported that F-actin polymerization and actin\u2013myosin contractility are necessary for membrane blebbing and chromatin condensation during execution of apoptosis induced by tumor necrosis factor-\u03b1 or overexpression of Rho-kinase [13, 26]. Moreover, the actin\u2013myosin cytoskeleton may be involved in facilitating the cellular localization of specific components of the apoptotic machinery and be required for caspase-3 activation [17, 18].\nOur study carries some limitations. First, we cannot conclude on the mechanisms by which F-actin rearrangement facilitates the effects of Rho-kinase on PI3-kinase\/Akt and apoptosis. Second, Rho-kinase inhibition and F-actin depolymerization could establish only a partial protection against apoptosis, in agreement with the literature [4, 5, 8, 12], possibly because other mechanisms are also involved and the I\/R-induced injury is too extensive. Third, we may have underestimated the number of apoptotic cells, because endothelial cells that detached from the basement membrane during simulated I\/R and did not reattach later were not counted in the quantification of apoptosis. Indeed, Prahalad et\u00a0al. [35] describe this reversible cell detachment during ATP depletion of Madin-Darby canine kidney cells.\nConclusion\nPrevention of F-actin rearrangement or F-actin bundle formation by Rho-kinase inhibition or by treatment with actin depolymerizator cytochalasinD, independent of Rho-kinase, attenuated I\/R-induced endothelial cell apoptosis by maintaining PI3-kinase and Akt activity. The present finding suggests that the changes in the F-actin cytoskeleton play a pivotal role in the negative regulation of PI3-kinase and Akt by Rho-kinase. Further studies are required to conclude on the mechanisms by which F-actin rearrangement facilitates the effects of Rho-kinase on PI3-kinase\/Akt and apoptosis.","keyphrases":["cytoskeleton","y-27632","cytochalasind","latrunculina","jasplakinolide","huvec"],"prmu":["P","P","P","P","P","P"]}
{"id":"Pediatr_Nephrol-4-1-2275772","title":"Therapeutic strategies to slow chronic kidney disease progression\n","text":"Childhood chronic kidney disease commonly progresses toward end-stage renal failure, largely independent of the underlying disorder, once a critical impairment of renal function has occurred. Hypertension and proteinuria are the most important independent risk factors for renal disease progression. Therefore, current therapeutic strategies to prevent progression aim at controlling blood pressure and reducing urinary protein excretion. Renin-angiotensin-system (RAS) antagonists preserve kidney function not only by lowering blood pressure but also by their antiproteinuric, antifibrotic, and anti-inflammatory properties. Intensified blood pressure control, probably aiming for a target blood pressure below the 75th percentile, may exert additional renoprotective effects. Other factors contributing in a multifactorial manner to renal disease progression include dyslipidemia, anemia, and disorders of mineral metabolism. Measures to preserve renal function should therefore also comprise the maintenance of hemoglobin, serum lipid, and calcium-phosphorus ion product levels in the normal range.\nNatural course of chronic kidney disease\nProgression of chronic kidney disease (CKD) toward end-stage renal failure is common in CKD patients, and once significant impairment of renal function has occurred, it tends to progress irrespectively of the underlying kidney disorder. However, information on the natural course of CKD progression in children is still limited. The prospective, population-based ItalKid registry, including almost 1,200 CKD children with various renal diseases over a 10-year period, reported a prevalence of 23% of patients suffering from severe kidney disease with chronic renal insufficiency (CRI). The incidence of renal replacement therapy was 7.3 per year per 100 patients with CRI, and the risk of developing end-stage renal disease (ESRD) by age 20 was 68% [1]. The decline of renal function was not linear but rather characterized by a sharp decline during puberty and at early postpubertal age. This finding supports the general clinical impression that in many children with renal hypodysplasia, kidney function deteriorates more rapidly around the time of puberty. This notion received further support by a recently published retrospective analysis of 176 children with renal hypodysplasia [2]. The authors postulated that the natural course of chronic renal failure in these patients can be divided into three time periods: an initial period, usually lasting the first 3 years of life characterized by an improving renal function, a subsequent period of stable renal function attained by 50% of patients for a mean of 8\u00a0years, and a phase with renal function gradually deteriorating toward ESRD. The latter period started just after infancy in 48% and around puberty in 23%. In 30% of patients, renal function remained stable even beyond puberty.\nFactors affecting renal disease progression\nThere is clear evidence from clinical studies that hypertension and proteinuria are key players in the pathophysiology of CKD progression in humans [3\u20135]. The renin-angiotensin system (RAS) is intrinsically involved in the process, and other potential contributors include genetic background, renal anemia, altered mineral homeostasis, dyslipidemia, chronic inflammation, and oxidative stress as well as general cardiovascular risk factors such as diabetes, smoking, and obesity. As a consequence of the mechanistic insights in renal disease progression obtained by experimental work, several principal renoprotective strategies have emerged in recent years (Fig.\u00a01). These are based mainly on clinical evidence established in adult patients, but growing evidence supports their efficacy also in children. Efficient control of blood pressure and minimization of proteinuria appear as the two most important measures to preserve residual kidney function. Other issues, such as prevention and treatment of renal anemia, uremic dyslipidemia, and disorders of mineral metabolism, have an experimental basis, although their clinical importance is less clear to date. In the following, we review current treatment strategies to slow renal disease progression in childhood CKD.\nFig.\u00a01Pathophysiology, consequences, and treatment options of hypertension and proteinuria in chronic kidney disease. EPO erythropoietin, P reduction  serum phosphate reduction, RAS renin angiotensin system, Non-DHP CCP non dihydropyridine calcium channel blockers, TGF\u03b2 transforming growth factor \u03b2, TIMP tissue inhibitors of metalloproteinases, ET1 endothelin 1, PAI plasminogen activator inhibitor, \u22a5 inhibitory effect\nHypertension\nHypertension is an independent risk factor of renal failure progression in adults [3\u20135]. Whereas the degree of hypertension correlates with the severity of the underlying renal disease, interventional studies have provided evidence that high blood pressure actively contributes to renal failure progression in human CKD. In pediatric nephropathies, renal hypertension is common, although typically less severe than in adult kidney disorders. Hypertension prevalence estimates in children with CKD range from 20% to 80% depending on the degree of renal dysfunction and underlying renal disease [6, 7]. However, even children with CKD stage 2 or renal hypodysplasia may present with significantly elevated blood pressure [8]. The European Study Group for Nutritional Treatment of Chronic Renal Failure in Childhood demonstrated that in CKD children, a systolic blood pressure greater than 120\u00a0mmHg was associated with a significantly faster glomerular filtration rate (GFR) decline [9].\nNumerous studies in adults have provided proof to the concept that consequent antihypertensive therapy slows down the rate of renal-failure progression [10]. A close linear relationship between the blood pressure level achieved by antihypertensive treatment and the rate of renal failure progression in adult CKD patients has been noted, which appears to persist well into the normal range of blood pressure [11, 12].\nThe firm evidence of a favorable effect of intensified blood pressure control in patients with CKD has resulted in generally lower target blood pressure recommendations in this patient group. In the most recent guidelines by the Joint National Committee in the US (JNC7) [13] and the Guidelines of the European Hypertension Society [14], 120\/80 mmHg has been defined as the upper limit of the \u2018optimal\u2019 blood pressure range, particularly when proteinuria is present, and any blood pressure > 130\/80 in CKD patients should be actively lowered by therapeutic intervention [15]. These blood pressure targets are equivalent to the 50th to 75th distribution percentile in the general young adult population. It is as yet unknown whether these blood pressure targets hold true for the pediatric population and whether glomerular damage in children correlates with absolute or age-specific relative blood pressure. The Kidney Disease Outcomes Quality Initiative (K\/DOQI) guidelines on blood pressure control in CKD children [15] adopted the recommendations of the task force that target blood pressure should be <90th percentile for normal values [16] adjusted for age, gender, and height percentile. Assuming that equivalent blood pressure percentiles should be targeted in children as in adults, the adult recommendations would, for instance, correspond to an acceptable upper blood pressure level of 106\/66\u00a0mmHg (75th percentile) in an 8-year old child with CKD. The final results of the Effect of Strict Blood Pressure Control and ACE Inhibition on Progression of Chronic Renal Failure in Pediatric Patients (ESCAPE) trial, to become available in mid-2008, will provide pediatric evidence as to whether intensified blood pressure control (targeting to below the 50th percentile of 24-h mean arterial pressure) will confer a renoprotective advantage over a more conventional target (50th to 95th percentile) [17]. The integrity of the normal diurnal blood pressure pattern may play a significant role in renal failure progression in addition to and independent of the absolute blood pressure level. Nondipping, a well known independent cardiovascular risk factor and common characteristic of renoparenchymal hypertension, is associated with more rapid progression of renal failure in adult CKD patients [18, 19], and nondipping is believed to reflect sympathetic hyperactivation in CKD.\nProteinuria\nPopulation-based studies in healthy individuals have demonstrated that proteinuria is a powerful independent risk factor for ESRD and overall mortality [20\u201322]. Proteinuria is certainly predictive of the renal prognosis in adults with diabetic and nondiabetic kidney disorders [23\u201325]. Urinary protein excretion was the only baseline variable correlated with GFR decline and progression to ESRD in the Ramipril Efficacy in Nephropathy (REIN) trial [26]. However, the spectrum of underlying renal disorders in children differs markedly from adults. In the pediatric CKD population, congenital renal hypodysplasia with or without urinary tract abnormalities is the leading underlying renal disorder, affecting more than 60% of children. The European Study Group for Nutritional Treatment of Chronic Renal Failure in Childhood first demonstrated in 200 children with CKD stage 3\u20134 that proteinuria and hypertension are major independent determinants of GFR decline in pediatric nephropathies [9]. The ItalKid Project confirmed that proteinuria predicts renal disease progression in children with renal hypodysplasia [27]. In addition, there is evidence from the ESCAPE trial that residual urinary protein excretion during angiotensin-converting enzyme (ACE) inhibition is quantitatively associated with renal failure progression [28]. Even in children with normal kidney function, persistent proteinuria in the nephrotic range is a risk factor for progressive renal injury, and early detection and treatment of proteinuria is essential [29]. In nonproteinuric children with CRI of nonglomerular origin, the level of protein excretion does not appear to play a major role in CKD progression, which seems to be best predicted by rapid somatic growth, age, and blood pressure [30].\nIn line with evidence from animal models, multiple clinical studies have confirmed that proteinuria is not only a marker but also an important mechanism of CKD progression. Reduction of proteinuria is associated with a slowing of GFR loss in the long term [25, 31\u201333]. In the Modification of Diet in Renal Disease (MDRD) trial, for each 1\u00a0g\/d reduction in proteinuria observed within 4\u00a0months of antiproteinuric treatment (i.e. blood pressure reduction and dietary interventions), the subsequent GFR decline was slowed by about 1\u00a0ml\/min per 1.73\u00a0m2 per year [25]. In the REIN study, reduction of proteinuria at 3\u00a0months of ACE inhibitor therapy by 1\u00a0g\/d resulted in slowing down GFR decline by 2\u00a0ml\/min per year [34]. This degree of proteinuria reduction appears to be associated with the maximal renoprotective effect [35, 36]. Hence, the goal of any antiproteinuric treatment is to reduce proteinuria as much as possible, ideally to <300\u00a0mg\/m2\/day.\nDyslipidemia\nEpidemiological studies suggest that dyslipidemia is an independent risk factor not only for cardiovascular disease but also for progressive chronic renal failure [37]. The dyslipidemic pattern differs between the major renal disease entities [38], and the degree of dyslipidemia parallels the degree of renal function impairment. In animal models, hypercholesterolemia clearly accelerates the rate of progression of kidney disease [39]. A high-fat diet causes macrophage infiltration and foam-cell formation in rats, leading to glomerulosclerosis [40]. Dyslipidemia may damage glomerular capillary endothelial and mesangial cells as well as podocytes. Macrophages are the major cell types expressing scavenger receptors; however, mesangial cells, as well, express receptors for low-density lipoprotein (LDL) and oxidized LDL, which upon activation induce mesangial cell proliferation, increase mesangial matrix deposition, and enhance production of chemokines, cytokines, or growth factors and increase oxidative stress.\nA relationship between serum cholesterol levels and GFR decline was shown in adult patients with type 1 diabetes and overt nephropathy [41]; patients with a total cholesterol level >7\u00a0mmol\/L showed an at least three times faster decline in GFR than subjects with lower cholesterol levels. For the general adult population, the Arteriosclerosis Risk in Communities (ARIC) study demonstrated that elevated triglycerides and low high-density lipoprotein (HDL) cholesterol but not LDL cholesterol were associated with an increased risk of renal dysfunction [37]. In a cohort of more than 11,000 middle-aged adults with normal kidney function, hypertriglyceridemia was associated with a 1.68 times increased risk of a 0.4\u00a0mg\/dl increase in serum creatinine within the 3-year observation period [37].\nThere are also observations that insulin resistance syndrome may underlie or mediate the association between lipids and loss of renal function. In humans, a strong relationship between metabolic syndrome and the risk for chronic renal disease and microalbuminuria was found in a large nondiabetic general population [42].\nAnemia\nThere is increasing evidence that anemia is an independent risk factor for progression of chronic renal failure. Anemia is a surrogate marker for tissue hypoxia that might perpetuate preexisting renal tissue damage. In patients with reduced nephron number, hypoxia of tubular cells is favored by an increase of oxygen consumption by tubular cells of the remaining nephrons, a decrease in the number of interstitial capillaries [43], and an accumulation of extracellular matrix between interstitial capillaries and tubular cells, which hampers oxygen diffusion. Hypoxia appears to have at least three consequences: It stimulates production of profibrotic molecules such as transforming growth factor (TGF)-\u03b2 or endothelin-1 by tubular cells, synthesis of extracellular matrix [44], and increased oxygen consumption, which also enhances production of reactive oxygen species (ROS) that may play an additional role in CKD progression.\nThe renoprotective effect of erythropoietin (EPO) in CKD might be partially related to an attenuation of interstitial fibrosis and tubuloepithelial cell loss by improved oxygen supply and reduced oxidative stress via correction of anemia. In addition, EPO might exert direct protective effects on tubular cells and might help maintain integrity of the interstitial capillary network and stimulate regenerative progenitor cells [45]. The combination of antiapoptotic effects of rhuEPO in renal tissue and stimulation of regenerative progenitor cells may play a role in organ protection.\nOxidative stress\nOxidative stress is defined as an imbalance between ROS and endogenous levels of antioxidant substances. High oxidative stress and low availability of the substrate of nitric oxide (NO) synthase, L-arginine, and an accumulation of endogenous NO inhibitors such as asymmetric dimethylarginine (ADMA) may induce endothelial dysfunction. Several studies reported on increased oxidative stress in CKD patients. This increase appears to correlate with the extent of deterioration of renal function [46]. Increased oxidative stress contributes to the release of proinflammatory and profibrotic molecules and thereby directly enhances the production of extracellular matrix by fibroblastic cells. This may lead to accelerated progression of CKD, hypertension, and cardiovascular complications, and it was suggested that increased oxidative stress in CKD patients may be both a cause and an effect of renal injury. Anemia, hypercholesterolemia, and chronic inflammation are conditions known to further promote oxidative stress.\nNutrition\nNutrition has been considered an important instrument for slowing down renal disease progression in individuals with impaired renal function. In animals, high protein diet results in renal scarring, whereas restriction of dietary protein diminishes or even prevents progressive renal damage [47]. In end-stage renal failure, uremic symptoms can be often diminished and renal replacement therapy postponed by the restriction of dietary protein intake. This led to the hypothesis that restriction of protein intake might also slow down the progression of chronic renal failure in patients with CKD stages 2\u20134.\nDisorders of calcium-phosphate metabolism\nDisorders of the calcium-phosphate metabolism are additional risk factors for renal disease progression. On one hand, renal insufficiency causes disturbances of the calcium-phosphate homoeostasis and alters serum lipid profiles. On the other hand, the resulting vasculopathy and hypertension promote progression of chronic renal failure toward end-stage renal disease. Several factors related to disturbed calcium-phosphorus metabolism, such as hyperphosphatemia, hyperparathyroidism, lack of active vitamin D, and possibly the phosphaturic hormone FGF23, may be considered to be\u2014at least to a minor extent\u2014involved in the progression of renal dysfunction [48].\nTreatment strategies and their impact on renal disease progression\nSeveral antihypertensive and antiproteinuric therapies have proven effective. Blood pressure control per se has a proteinuria-lowering effect, as demonstrated by three large trials: the MDRD study [25], the Appropriate Blood Pressure Control in Diabetes (ABCD) study [49], and the African American Study of Kidney Disease and Hypertension (AASK) [32]. A low blood pressure goal, i.e. <125\/75\u00a0mmHg in adults, either reduced proteinuria absolutely by 50% [25] or prevented the two- to threefold increase in proteinuria observed in patients with the more conventional blood pressure goal of 140\/90\u00a0mmHg [49]. A low blood pressure goal appears to be very well tolerated by the vast majority of patients and in terms of cardiovascular outcomes; the \u201cJ curve\u201d phenomenon (a slight increase of cardiovascular events in patients achieving a very low blood pressure level) seems to be confined to aged patients with advanced atherosclerosis.\nThe goal of any antiproteinuric treatment is to reduce proteinuria as much as possible, ideally to <300\u00a0mg\/m2\/day. This degree of proteinuria reduction appears to be associated with the maximal renoprotective effect [35, 36]. Whereas the different classes of antihypertensive agents are comparable with respect to their blood pressure-lowering efficacy, they differ markedly regarding their effects on proteinuria and CKD progression [32, 35, 50, 51].\nBlockade of the renin-angiotensin system\nAntagonists of the RAS, such as ACE inhibitors and, more recently, angiotensin II type I receptor blockers (ARB) have become pharmacotherapeutics of first choice in adults [15] as well as children with CKD by virtue of their pharmacological properties. They significantly reduce blood pressure as well as urinary protein excretion and have an excellent safety profile, which is almost indistinguishable from placebo. In adults with essential hypertension, treatment with RAS antagonists has been associated with the best quality of life among all antihypertensive agents.\nRAS antagonists suppress the local angiotensin II tone (ACE inhibitor) or action (ARB). This results in a reduction of intraglomerular pressure and proteinuria, diminished local release of cytokines and chemokines, and alleviated activation of inflammatory pathways, with consequently attenuated glomerular hypertrophy and sclerosis, tubulointerstitial inflammation, and fibrosis [8], as well as in a normalized central nervous sympathetic tone by reduced renal afferent nerve stimulation. In addition, oxidative stress is reduced independently of the blood-pressure-lowering effect [52].\nIn adults with diabetic or nondiabetic kidney disease, several randomized trials demonstrate a more effective reduction of proteinuria, usually by 30\u201340%, by ACE inhibitors compared with placebo and\/or other antihypertensive agents [35]. This is associated with a significantly reduced rate of renal failure progression in the long term [31, 35, 53\u201361].\nVery similar results were obtained in randomized studies comparing ARBs with placebo or conventional antihypertensive agents in diabetic nephropathy [51, 62, 63]. It has been reasoned that ACE inhibitors might have a specific renoprotective advantage by inducing accumulation of vasodilatory and antifibrotic bradykinins; however, the course of GFR was similar in two clinical trials comparing ACE inhibitors and ARB therapy [64, 65]. The size of the advantage of RAS antagonists over other antihypertensive agents is still under debate [66]. The risk of doubling serum creatinine or attaining ESRD is typically reduced by 30\u201340%, but the superiority of RAS antagonists is related to the prevailing degree of proteinuria [35, 36]. In adults, ACE inhibitors are believed to provide better renoprotection than other antihypertensive agents in patients with proteinuria exceeding 500\u00a0mg\/day.\nHowever, there is some evidence that previous studies may not have used sufficiently high ACE inhibitor doses to achieve effective RAS suppression at the kidney tissue level and obtain a maximal renoprotective effect. Furthermore, at least a subset of patients appears to develop partial secondary resistance to ACE inhibition (aldosterone escape by compensatory upregulation of ACE-independent angiotensin II production) [67\u201369]. It is currently an open issue whether such patients would benefit from the primary use of ARBs alone or in combination with ACE inhibitors.\nWhereas the maximal antiproteinuric and renoprotective effects of ACE inhibitors and ARBs seem to occur at doses that are supramaximal with respect to maximal antihypertensive action, regulatory authority approval is usually available only for the indication of hypertension in the respective dose range. Therefore, it is generally recommended to administer these drugs, after confirming tolerability in a short run-in period, at their highest approved doses [32, 70].\nLimited information is available regarding the efficacy of RAS antagonists for renoprotection in children with CKD. Small uncontrolled studies showed stable renal function in children with sequelae of hemolytic uremic syndrome during long-term ACE inhibitor treatment [71], stable GFR during 2.5\u00a0years of losartan treatment in children with proteinuric CKD [72], and attenuated histopathological progression in children with IgA nephropathy receiving combined RAS blockade [73]. Data from the ItalKid Study did not show a significant modification of CKD progression by ACE inhibitor treatment in children with hypodysplastic kidney disease [74] compared with matched untreated subjects. However, the overall CKD progression rate in the total cohort was very slow (< \u20132\u00a0ml\/min per 1.73\u00a0m2 per year), thereby making the detection of significant differences (ACE inhibitors -1.08 vs. non-ACE inhibitors 1.80; not significant) difficult. In addition, no information was available with respect to the types and dosages of ACE inhibitors used and the prevailing degree of proteinuria.\nThe ESCAPE trial demonstrated efficient blood pressure and proteinuria reduction by ramipril in almost 400 children with CKD [17]. However, an interim analysis of the 3-year results revealed a gradual rebound of proteinuria after the second treatment year. This effect was dissociated from a persistently good blood pressure control and may limit the long-term renoprotective efficacy of ACE inhibitor monotherapy in pediatric chronic kidney disorders [28].\nAldosterone antagonists also lower blood pressure by RAS suppression. Whereas the use of spironolactone is limited by endocrine side effects, the new aldosterone antagonist, eplerenone, has minimal affinity for progesterone and androgen receptors. Apart from the risk of hyperkalemia, reported side effects are similar to placebo [75]. Combined therapy of eplerenone and an ACE inhibitor increases patient survival in adults with congestive heart failure [76]. However, combination therapy appears limited in CKD patients due to the potentiated risk of hyperkalemia [77, 78].\nAliskiren, a renin-antagonist, blocking the conversion from angiotensinogen to angiotensin I, has been shown to effectively lower blood pressure in animals and humans. The effect on blood pressure is comparable with that of ARBs, and combination therapy of aliskiren and valsartan at maximum recommended doses provided significantly greater reductions in blood pressure than did monotherapy, with a tolerability profile similar to that of aliskiren or valsartan alone [79]. However, there are no data on the effect of aliskiren on renal disease progression in adults nor on its applicability in children available to date.\nCalcium-channel blockers\nCalcium-channel blockers (CCBs) are safe and can achieve blood pressure goals in patients with CKD. However, CCBs of the dihydropyridine type (amlodipine, nifedipine) fail to reduce progression of chronic renal failure and may even increase proteinuria and promote more rapid CKD progression [33]. Therefore, dihydropyridine CCBs may be acceptable as first-line antihypertensive monotherapy only in nonproteinuric patients and should be avoided unless in combination with RAS antagonists to improve blood pressure control in proteinuric patients [70]. In contrast, nondihydropyridine CCB (diltiazem, verapamil) may have some antiproteinuric effect and may be therefore renoprotective [33]. However, data are not conclusive. An antiproteinuric effect was not observed in type 2 diabetes [80], and amlodipine exerted a renoprotective effect comparable with ACE inhibitors in one study [81].\nBeta-blockers\nCKD is often a state of overactivation of the sympathetic nervous system, and antiadrenergic drugs play an important role in its management. Beta-blockers are effective in lowering blood pressure in CKD patients by blockade of postsynaptic beta receptors, resulting\u2014among others effects\u2014in a reduction of pulse rate, cardiac output, afterload, and renal renin release. Metoprolol and atenolol were the first antihypertensive agents for which beneficial effects on the decline of renal function in CKD patients were demonstrated [54]. In the AASK trial, the beta-blocker metoprolol had an antiproteinuric effect almost comparable with ramipril, in marked contrast to amlodipine [32]. The antiproteinuric action may be due to sympathicoplegic effects. Newer beta-blockers, such as carvedilol, have even improved antiproteinuric effects compared with atenolol [82, 83].\nCombination therapy\nBecause hypertension is a multifactorial disorder, monotherapy is often not effective in lowering blood pressure or reducing proteinuria to the target range. Treatment with a single antihypertensive agent usually controls blood pressure in less than half of the patients. According to the JNC7 guidelines, subjects with blood pressure >20\/10\u00a0mmHg above the normal range (i.e. >160\/100\u00a0mmHg in adults) should be started on combination drug therapy [13]. In CKD patients, RAS antagonists are most commonly combined with a diuretic or with a CCB, whereas their combination with a beta-blocker usually does not exert an additive effect on blood pressure control. Fixed-dose combination preparations are becoming increasingly popular in antihypertensive therapy and may help maximize treatment adherence and efficacy.\nCombined RAS blockade using ACE inhibitors and ARB concomitantly has only a minor effect on blood pressure (3\u20134\u00a0mmHg vs. monotherapy) but increases the antiproteinuric effect of ACE inhibitors or ARB monotherapy by 30\u201340% [64, 84\u201386]. The prospective randomized Combination Treatment of Angiotensin II Receptor Blocker and Angiotensin-Converting-Enzyme Inhibitor in Non-diabetic Renal Disease (COOPERATE) trial, performed in adults with nondiabetic nephropathies, suggested that combination therapy may also provide better long-term renoprotection [64]. However, in most ACE inhibitor\/ARB combination studies, it remained unclear whether maximally efficient single-drug doses were used, a formal prerequisite to demonstrate true synergism of the two drug classes. The few published studies assessed the effects of single-drug dose escalation followed by combined administration of an ACE inhibitor and an ARB at maximally effective single doses found synergistic antiproteinuric effects of combined treatment [87]. Otherwise, a recent study demonstrated additional proteinuria reduction by escalating candesartan exposure to an ultrahigh dose [88]. Notably, raising the dose from 16 to 32\u00a0mg daily had no effect on proteinuria, whereas a further increase from 32 to 64\u00a0mg was highly effective, suggesting that the dose\u2013response relationship may be nonlinear. Hence, the issue of whether ACE inhibitor and ARB combination therapies have a synergistic renoprotective potential remains an exciting field of clinical research.\nRestoration of blood pressure day\u2013night rhythm\nIn view of the fact that nondipping of nocturnal blood pressure is an independent risk factor for CKD progression, effects of the timing of application of antihypertensive drugs may be an issue of interest. Even using agents with a long half-time and recommended administration on a once-daily basis, evening administration lowers nighttime blood pressure more effectively, increasing the day\u2013night ratio and partially restoring the physiological nocturnal dipping pattern. However, these effects seem to differ for individual antihypertensive drug classes. Whereas bedtime administration of CCBs and ACE inhibitors tends to restore the dipping pattern, evening dosing of beta-blockers has no effect on the circadian blood pressure rhythm [89]. In a substudy of the Heart Outcomes Prevention Evaluation (HOPE) trial, adult patients were evaluated by ambulatory blood pressure monitoring (ABPM) after evening administration of the ACE inhibitor ramipril. A more marked blood pressure reduction during nighttime was observed, compatible with the notion that the beneficial effects of ramipril on cardiovascular morbidity and mortality in the HOPE study were related to the 8% increase in the day\u2013night ratio of blood pressure obtained with evening dosing [90]. Although this association appears firm, it is as yet unclear whether pharmacological restoration of the dipping pattern will result in any long-term clinical benefit for cardiovascular health in general and for renal function preservation in CKD. However, it is of note that the antiproteinuric efficacy of the ARB valsartan was found correlated with the increase in blood pressure day\u2013night ratio induced by evening dosing [91].\nTreatment of dyslipidemia\nGeneral measures to prevent dyslipidemia in CKD patients include prevention or treatment of malnutrition and correction of metabolic acidosis, hyperparathyroidism, and anemia, all of which may contribute to dyslipidemia [92\u201394]. In addition, referring to evidence from the general population, therapeutic life-style modification is recommended for adults and children with CKD-related dyslipidemia [95]. However, the lipid-lowering effect of life-style modifications in CKD patients is usually not impressive. Lipid-lowering medical treatment is commonly prescribed in adults with CKD based on the evident benefit of this approach for primary and secondary prevention of cardiovascular disease in the general adult population. Statin therapy is effective in reducing cardiovascular morbidity and mortality in adults with moderate to severe CKD although not in patients with ESRD [96, 97].\nWith respect to renoprotection, experimental evidence suggests that statins may retard renal disease progression not only by their lipid-lowering but also by lipid-independent pleiotropic effects. Statins inhibit signaling molecules at several points in inflammatory pathways. Anti-inflammatory effects, reduction of oxidative stress, and improved endothelial function are thought to be partially responsible both for CVD risk reduction and improved renal function [98]. Furthermore, there is also evidence for synergistic effects of statins and RAS inhibitors on the prevention of renal disease progression [99]. However, a recent meta-analysis of published clinical trials concluded that the intrinsic antiproteinuric and renoprotective effects of statins, albeit significant, are quantitatively small [100]. To date, no studies have evaluated the usefulness of statins in children with progressive nephropathies.\nErythropoietin treatment\nIn rats undergoing acute ischemic renal injury, pretreatment with recombinant human erythropoietin (rhuEPO) reduces renal dysfunction and morphological damage. This effect appears to be mainly mediated by a reduction of apoptotic cell death [101]. Darbepoetin, a long-acting EPO analog, ameliorated podocyte injury and decreased proteinuria by maintenance of the podocyte actin cytoskeleton and nephrin expression in puromycin-induced nephrotic rats [102]. Even more interesting than treatment of acute renal failure may be tissue protection in chronic renal failure. In a recently published clinical trial, early initiation of rhuEPO therapy in patients with CKD and mild to moderate anemia significantly slowed down the progression of renal disease and delayed the need for renal replacement therapy [103]. However, other data in patients with more advanced CKD and high-dose rhuEPO treatment revealed no beneficial effect on renal survival [104]. The role of EPO in pediatric CKD progression has not been defined yet.\nNutrition and vitamin D supplementation\nFor decades, low-protein diets have been prescribed for preventing CKD progression. However, the effects of these diets on CKD progression and delay of ESRD are still inconclusive. One of the largest trials, the MDRD trial, could not prove efficacy of a low-protein diet on progression in nondiabetic kidney disease [105], whereas a recent Cochrane Review [106] found a risk reduction of renal death in patients with protein restriction. Thus, the progression rate was not significantly influenced by protein restriction, whereas renal replacement therapy could be postponed. In children, reducing protein intake to the maximal acceptable lower limit was ineffective to slow down renal disease progression [9, 107]. Further reductions may be effective but not acceptable. Furthermore, therapeutic strategies of protein reduction in children may be conflicting, since a low-protein diet bears the risk of low calorie intake, whereas a high calorie intake is needed for optimal growth. Therefore, at present, it seems not to be justified to prescribe low-protein diets to children early in the course of chronic renal failure.\nSome studies in adult CKD patients suggest that dietary phosphorus restriction may stabilize kidney function [108]. However, conclusions in this regard could not be drawn from studies in children [109]. A high calcium\u2013phosphorus product may be detrimental to renal survival by aggravating intrarenal vasculopathy as well as by causing tubulointerstitial calcifications, which may stimulate tubulointerstitial inflammation and fibrosis. In view of these pathophysiological associations, it is currently discussed whether calcium-free phosphate binders may have some renoprotective potential in patients with CKD. Sevelamer may prove beneficial beyond phosphate lowering due to its pleiotropic effects, which include lipid-lowering and anti-inflammatory properties. Treatment with nonhypercalcemic doses of active vitamin D attenuates renal failure progression in chronically uremic rats. This effect may be brought about by the immune modulatory and antifibrotic properties of vitamin D. In addition, a negative endocrine regulation of the RAS through 1,25-Dihydroxyvitamin D3 has been reported [110]. In humans, an antiproteinuric effect of oral paricalcitol was demonstrated in adult CKD patients [111]. These exciting experimental and early clinical findings provide an additional rationale beyond mineral metabolism for close monitoring and early intervention to maintain mineral, vitamin D, and PTH homeostasis in CKD [109].\nConclusion\nIn conclusion, hypertension and proteinuria are key players in renal disease progression. Therapeutic strategies to prevent progression should comprise blood pressure control and lowering of proteinuria. RAS antagonists preserve kidney function, not only by lowering blood pressure but also through antiproteinuric and antifibrotic properties. Other factors contributing to renal disease progression in a multifactorial manner include anemia, dyslipidemia, and disorders of mineral metabolism, and measures to preserve renal function should therefore also comprise the maintenance of hemoglobin and serum lipid and calcium-phosphorus ion product levels in the normal range.\nQuestions\n(Answers appear following the reference list.)\nProgression of chronic renal failure toward ESRD is common in children with CKD and:\nDeclines linearlyNonlinearly and often characterized by a sharp decline in renal function during pubertyInevitablyStrongly, depending upon the underlying renal diseaseResults in ESRD in less than 50% of patients at age 20 years\nThe most important factors influencing renal disease progression are:\nAge at onset of chronic renal failure, gender, and underlying renal diseaseResidual renal function and blood pressureBlood pressure and proteinuriaRapid somatic growth during puberty, and age\nThe different antihypertensive drug classes are comparable regarding:\nAntihypertensive efficacyAntiproteinuric efficacySide effects and safety profileEffect on CKD progression\nThe antihypertensive and antiproteinuric effects of RAS antagonists are:\nStrictly dose dependentBasically mediated by reduction of systemic hypertensionMediated by bradykinin releaseAlso mediated by antifibrotic and vasodilatory effectsDue to restoration of the often disturbed day\u2013night blood pressure pattern (dipping) in CKD patients\nAngiotensin receptor antagonists in children:\nAre approved for the indication proteinuria and hypertensionDo not exert additional antiproteinuric effect when combined with ACE inhibitorsShould be given at the highest approved dose for maximal antiproteinuric effectShould not be combined with ACE inhibitorsHave the same side effect profile as ACE inhibitors\nDecline of GFR:\nIs independent of serum cholesterol levelCan be reversed by dietary protein restrictionCan be reversed by the use of statins in younger childrenMay be accelerated by prescription of erythropoietinMay be retarded by the lipid-independent pleiotropic anti-inflammatory effects of statins","keyphrases":["chronic kidney disease","progression","hypertension","proteinuria","prevention","children"],"prmu":["P","P","P","P","P","P"]}
{"id":"Matern_Child_Health_J-2-2-1592162","title":"Preconception Care and Women with or at Risk for Diabetes: Implications for Community Intervention\n","text":"Introduction\nDiabetes is a chronic and, often, disabling disease, which has reached epidemic proportions in America and worldwide. When a person has diabetes their body cannot produce or properly use insulin \u2013 a hormone needed to convert sugar, starches, and other foods into energy. This leads to high levels of sugar in the bloodstream, which can result in serious complications and premature death, if diabetes is not controlled. There are three main types of diabetes [1]. In type 1 diabetes, the body does not produce any insulin; daily injections of insulin are required for survival. Typically beginning in childhood or young adulthood, type 1 accounts for approximately 5\u201310% of all cases of diabetes. Autoimmune, genetic, and environmental factors influence type 1 diabetes risk [1].\nIn type 2 diabetes, which accounts for 90\u201395% of all diagnosed cases, the body\u2019s cells do not secrete or use insulin adequately. Risk factors for type 2 diabetes include obesity, physical inactivity, family history of diabetes, and history of gestational diabetes (GDM). GDM, defined as diabetes that develops or is first recognized during pregnancy, is the third type of diabetes. Risk factors for GDM include obesity, pregnancy weight gain, age, and family history of diabetes [1\u20135].\nIn the weeks after pregnancy, 5\u201310% of women who had GDM are diagnosed with type 2 diabetes [1]. Subsequently, women with a history of GDM have a 20\u201350% chance of developing type 2 diabetes five to 10 years after the index pregnancy, with a lifetime risk near 80% [1, 4\u20137]. Both type 2 diabetes and GDM are diagnosed more frequently in African Americans, Hispanic\/Latino Americans, and American Indians compared to non-Hispanic whites [1].\nNational Health and Nutrition Examination Survey III data for nonpregnant women aged 20\u201349 years indicate that during the period 1988\u20131994, 27.6% of Mexican American women and 22.4% of African American women of childbearing age had diabetes or impaired glucose tolerance, in comparison to 10.1% of non-Hispanic white women [8]. Approximately one third of women of childbearing age have undiagnosed diabetes [9]. Additionally, between 3 and 8% of pregnant women have gestational diabetes (GDM) [2, 3, 9, 10].\nA study of women with pregestational diabetes (type 1 and type 2) found that 60% of the women had suboptimal glucose control before conception [11]. Women who had a poor outcome in a previous pregnancy were more likely to enter a subsequent pregnancy with poor glucose control then were women with good outcomes [11].\nDiabetes during pregnancy is associated with increased risk for miscarriages, stillbirth, macrosomia and obstetric complications [12\u201316], intrauterine developmental and growth abnormalities, birth and neonatal complications, and later development of obesity and type 2 diabetes [3, 10\u201315, 17, 18]. Treatment to normalize maternal blood glucose prior to conception and throughout pregnancy is necessary to reduce the likelihood of maternal, obstetric, and infant complications [12\u201316]. While treatment and monitoring are common practice during prenatal care, many women and their families may not know about the importance or even the existence of preconception care interventions for women of childbearing age who have or are at risk for diabetes.\nGreater awareness of the potential contribution of preconception care to diabetes prevention and control may help reduce the devastating impact of diabetes and its complications on the lives of women and their families. The objectives of this paper are to: 1) review barriers that can impede a woman\u2019s ability to receive preconception care, and 2) recommend novel interventions to reach reproductive-aged women with or at risk for diabetes.\nPrevention trials have demonstrated that type 2 diabetes and its complications can be prevented or at least delayed through healthful dietary practices, regular moderate physical activity, weight loss, and medication use [19, 20]. The clinical practice guidelines of the American College of Obstetrics and Gynecologists [21] and the American Diabetes Association [15] suggest that preconception care is an ideal primary prevention opportunity during which modifiable risk factors can be identified and reduced.\nPreconception care may be defined as a window of opportunity for comprehensive health care to: 1) identify conditions that may have detrimental effects on the mother or fetus, and 2) recommend necessary medical, behavioral, and educational interventions for increasing the likelihood of achieving optimal pregnancy outcomes. A major goal of preconception care for women with diabetes is to reduce the risk of diabetes-related complications by obtaining the lowest possible glycated hemoglobin (HbA1C [a measure of glucose control]) without significant episodes of hypoglycemia [15].\nWomen with diabetes who receive preconception care obtain intensive treatment to assist them with developing diabetes self-management skills, and obtaining nutritional, physical activity, and medical support needed to promote optimal glucose control and health status before becoming pregnant. During interconception periods, diabetes education, postpartum glucose testing, and ongoing support to reduce postpartum weight retention and maintain a healthy weight and glucose control may also help reduce risk factors for subsequent morbidity [3, 19, 24].\nPrevious studies have found that women with diabetes who received preconception care demonstrated improved glucose control during pregnancy, their offspring had fewer congenital anomalies, and the women\u2019s hospital stays were shorter in comparison with women who did not receive preconception care [25\u201327]. Although these findings are very positive and they support the importance of preconception care for women with diabetes, other studies have indicated that many women who could benefit from preconception care are not receiving this intervention [25, 28].\nBarriers to receiving preconception care\nAt every health care encounter, a woman of childbearing age should be informed about the importance of preconception care and, if she has diabetes, the steps required to maintain appropriate blood glucose control [15, 28, 29]. Unfortunately, however, reaching women who may be in need of preconception care has proven to be difficult, with only a quarter to a third of women with diagnosed diabetes receiving this care [25, 28].\nThere are many barriers to providing and\/or receiving preconception care. Among them are: 1) Many women with diabetes do not know that they have the illness and, thus, they are undiagnosed [1], 2) Approximately 50% of all pregnancies are unintended [28, 30, 31], 3) Even among women planning to become pregnant, lack of health insurance or a regular care primary care or obstetric provider reduces contact with the health care system [32\u201334], 4) Many primary care practices do not have or use established guidelines for providing preconception care, or identifying women with risk factors [32, 34], 5) Some women and health care providers may not know about about the existence or the importance of preconception care or do not see it as a high priority [15, 32, 34, 35], and 6) Women with incomplete health care coverage, lack of child care or transportation, geographic isolation, distrust of health care providers or other social and economic challenges have additional barriers to receiving preconception care [15, 32, 34\u201336].\nWomen are more likely to receive preconception care if they are married or in a stable relationship, are comparatively older, are nonsmokers, are non-Hispanic whites, are more educated, have annual incomes above $20,000, have private medical insurance, and have a positive bond with their prepregnancy care provider [25, 26, 28, 33, 35]. Younger women with diabetes and those who are single, have low income, or are less educated may be particularly vulnerable to unplanned pregnancies, which greatly reduces the chances that they will receive any form of diabetes-related preconception counseling [16, 32, 36].\nInterventions to reach women of childbearing age with diabetes\nSince the establishment of preconception care programs, health care centers have employed numerous marketing approaches geared toward physicians, and for patients in need of preconception care intervention. Janz et\u00a0al. [28] identified marketing approaches which were employed in five Michigan medical centers to encourage the use of preconception care programs in treating high-risk women. These included journal advertisements, newsletters, brochures, flyers, posters, and patient education programs. However, the results of these outreach efforts were \u201cgenerally disappointing [28].\u201d\nIn a Maine study, a diabetes registry was developed to promote the availability of preconception care programs [27]. In addition, health care providers formed a network to enhance the care of pregnant women using locally developed patient care guidelines. From 1987\u20131990, there were a total of 185 pregnancies in women with diagnosed diabetes. Of the total pregnancies, only 34% of these women received preconception care. These findings support those of Janz et\u00a0al. [28], which demonstrate that very few women with diabetes are receiving preconception care. Additional innovative strategies are needed to increase awareness of the availability of preconception care.\nNovel interventions to reach reproductive-aged women with or at risk of diabetes\nThe National Public Health Initiative on Diabetes and Women\u2019s Health (cosponsored by the American Diabetes Association, the American Public Health Association, the Association of State and Territorial Health Officials, and the Centers for Disease Control and Prevention) is a partnership devoted to increasing public and provider awareness of the importance of interventions such as preconception care in the prevention and management of diabetes for women [37, 38]. Using a life stage approach, the Initiative\u2019s cosponsors and partners developed and disseminated a national action plan, The National Agenda for Public Health Action [39], to identify 10 priority action steps to improve the lives of women with or at risk of diabetes, from adolescence to the older years (see Table 1). The National Agenda serves as a blueprint to guide the nation in implementing strategies related to diabetes and women\u2019s health. Given the devastating effects that diabetes can have on women\u2019s health, the National Agenda recognizes and identifies the need for effective interventions, such as quality health care and programs, public health research and surveillance, and specific policies to address the burden of this chronic illness on the lives of women and their families [37, 38].Table 1The National Agenda\u2019s 10 priority recommendations for action1. Encourage and support diabetes prevention and control programs \u2003 in state health departments to develop prevention programs for all \u2003 women and establish efficient links for women at risk for type 2 \u2003 diabetes2. Expand community-based health promotion, education, activities, \u2003 and incentives for all ages in a wide variety of settings\u2014schools, \u2003 workplaces, senior centers, churches, and other locations where \u2003 women live, learn, work, and play3. Strengthen advocacy on behalf of women with or at risk for \u2003 diabetes4. Fortify community programs for women with sufficient training, \u2003 tools, and materials5. Expand population-based surveillance to monitor and understand:\u2003a. Variations in the distribution of diagnosed and undiagnosed \u2003 diabetes\u2003b. The factors\u2014cultural, racial, ethnic, geographic, demographic, \u2003 socioeconomic, and genetic factors\u2014that influence the risk for \u2003 diabetes and complications among women at all life stages6. Educate community leaders about diabetes and its management \u2003 and about the value of healthy environments7. Encourage healthcare providers to promote risk assessment, \u2003 quality care, and self-management for diabetes and it \u2003 complications in their practice settings8. Ensure access to trained healthcare providers who offer quality \u2003 services consistent with established healthcare guidelines9. Encourage healthcare coverage and incentives for recommended \u2003 diabetes prevention management practices by:\u2003a. Promoting partnerships between insurers and workplaces or \u2003 labor communities and encouraging employers and employees to \u2003 discuss needed diabetes benefits in offered health care packages\u2003b. Working with health insurers and policymakers to expand \u2003 coverage and reimbursement policies to include prevention \u2003 services for women throughout their lives10. Conduct public health research to further our knowledge about \u2003 the epidemiological, socioenvironmental, behavioral, \u2003 translational, and biomedical factors that influence diabetes and \u2003 women\u2019s health\nSeveral of the National Agenda\u2019s recommendations support the need for preconception care and follow-up care for women with or at risk for diabetes through community intervention efforts and policy development. These recommendations identify the need to: 1) expand our outreach to communities where women live, learn, work, and play by offering health promotion, education, activities and other incentives; 2) provide sufficient funding, tools, training, and materials to fortify community programs that focus on women\u2019s health needs; 3) educate community members and leaders about diabetes prevention and control and the roles that they can play in promoting healthy environments; 4) encourage healthcare providers to promote risk assessment and quality care for women, including preconception care; and 5) conduct public health research to better understand multiple factors that influence diabetes and women\u2019s health.\nCommunity intervention and the involvement of community health workers\nOne of the community-focused strategies from the National Agenda, is the need to \u201cidentify and develop links with community agencies that interface with women at greatest risk for diabetes, including organizations in the non-health related sectors [39].\u201d Given that preconception care programs have had difficulty reaching high-risk women, nontraditional approaches to outreach and preconception care should be explored [32, 36].\nCommunity health organizations and community health workers (CHWs) appreciate and understand the social, political, environmental, and cultural factors that affect individuals within their own neighborhoods and have the potential to influence the consumers\u2019 relationship with the health care system [33]. They serve as \u201cbridges\u201d\u2014connecting community members who have traditionally lacked access to the appropriate services needed [40, 41]. The Institute of Medicine (IOM) recommends supporting the use of CHWs. The IOM identifies CHWs as an integral component of the health care system that help to address racial and ethnic disparities in access to care [41, 42].\nMany community-based projects funded by the Centers for Disease Control and Prevention have included CHWs in community-based programs aimed at health intervention for people with diabetes [43]. Programs employing CHWs have reported that they improve access and health outcomes for persons with chronic diseases, including diabetes, particularly in minority and underserved populations [44\u201348]. Although, no empirical studies have been published that assess the effectiveness or impact of CHWs in providing preconception care services for women with diabetes, CHWs have also played an important role in maternal and child health care. Thus, studying the methods for, and outcomes of an expanded role for CHWs with preconception outreach and education for women with, or at risk for diabetes, is recommended.\nCommunity health workers could play a vital role in linking women to preconception care services in the following manner: 1) increasing women\u2019s awareness about the importance of preconception care programs, 2) providing culturally and linguistically appropriate diabetes-related health information and education, 3) reminding women about scheduled health provider visits, 4) providing a communication bridge linking women and providers in terms of patients\u2019 needs and providers\u2019 recommendations.\nThe prevalence of diabetes among women of childbearing age is rising, particularly in communities with ethnic minorities and low resources [4, 8]. There is a strong relationship between these conditions and adverse maternal, child and subsequent adult health outcomes. Assessing the processes and effects of preconception care interventions implemented by providers and CHWs, especially in those communities with varied ethnic and socioeconomic backgrounds, is a critical first step toward identifying and promoting effective preconception care [18, 49\u201355].","keyphrases":["preconception care","women","diabetes"],"prmu":["P","P","P"]}
{"id":"Behav_Genet-4-1-2226023","title":"Modeling Genetic and Environmental Factors to Increase Heritability and Ease the Identification of Candidate Genes for Birth Weight: A Twin Study\n","text":"Heritability estimates of birth weight have been inconsistent. Possible explanations are heritability changes during gestational age or the influence of covariates (e.g. chorionicity). The aim of this study was to model birth weights of twins across gestational age and to quantify the genetic and environmental components. We intended to reduce the common environmental variance to increase heritability and thereby the chance of identifying candidate genes influencing the genetic variance of birth weight. Perinatal data were obtained from 4232 live-born twin pairs from the East Flanders Prospective Twin Survey, Belgium. Heritability of birth weights across gestational ages was estimated using a non-linear multivariate Gaussian regression with covariates in the means model and in covariance structure. Maternal, twin-specific, and placental factors were considered as covariates. Heritability of birth weight decreased during gestation from 25 to 42 weeks. However, adjusting for covariates increased the heritability over this time period, with the highest heritability for first-born twins of multipara with separate placentas, who were staying alive (from 52% at 25 weeks to 30% at 42 weeks). Twin-specific factors revealed latent genetic components, whereas placental factors explained common and unique environmental factors. The number of placentas and site of the insertion of the umbilical cord masked the effect of chorionicity. Modeling genetic and environmental factors leads to a better estimate of their role in growth during gestation. For birth weight, mainly environmental factors were explained, resulting in an increase of the heritability and thereby the chance of finding genes influencing birth weight in linkage and association studies.\nIntroduction\nBirth weight is a complex trait in which both environmental and genetic factors are involved. Low birth weight is not only associated with higher neonatal morbidity and mortality (Kramer et\u00a0al. 2005), but also with post-natal psychopathology (Wichers et\u00a0al. 2002). It is even stated that birth weight is a causal risk factor for child problem behavior and that the effects may well extend into adulthood (van Os et\u00a0al. 2001).\nHeritability estimates for birth weight using twin pairs, based on structural equation modeling or study of the offspring of twins, have given inconsistent results ranging from 10 to 40% (Baird et\u00a0al. 2001; Clausson et\u00a0al. 2000; Hur et\u00a0al. 2005; Luciano et\u00a0al. 2004; Magnus et\u00a0al. 2001; van Baal and Boomsma 1998; van Dommelen et\u00a0al. 2004; Vlietinck et\u00a0al. 1989; Whitfield et\u00a0al. 2001). The relatively low heritability estimates may be explained by unequal sharing of nutrients or unequal placental blood supply to the twin pair (Machin 1996). Vlietinck et.al. (Vlietinck et\u00a0al. 1989) revealed that chorionicity explained 12% of the variance of birth weight with DC twins having a significantly higher similarity than MC twins. Random environment explained 23%, additive genetic factors 22.5%, and maternal age 0.7% of the total variance. But, the largest source of variation on birth weight in this study was gestational age (42%). Until now however, no study has focused on the change of variance of birth weight during gestation. We have shown that this variance increases during gestation (Gielen et\u00a0al. 2007). As a consequent, heritability of birth weight may change over gestational age or different covariates may explain different parts of the variance-covariance structure. For example, it is more likely that at 37\u00a0weeks of gestation, nutrient factors (environmental factor) will explain a larger part of the covariance matrix as compared to 13\u00a0weeks of gestation.\nThe advantage of twin studies, is that the total variance can be split up into genetic (A), shared or common environmental (C) and unique environmental (residual, E) components, enabling an accurate estimation of heritability (=\u00a0genetic variance\/total variance) (Boomsma et\u00a0al. 2002). The degree of heritability determines the power to localize and identify the genes involved (Snieder et\u00a0al. 2003). For linkage and association studies a high degree of heritability is preferable. Since heritability is the proportion of phenotypic variation of a trait that can be attributed to genetic variation (Boomsma et\u00a0al. 2002), reducing environmental and residual components will give an increase of the heritability. The latter can be achieved by adjusting for covariates that explain part of the environmental factors in the analysis. Additionally, a covariate may also explain part of the genetic variance and as a result this may provide an indication for finding genes for this covariate, but of course the drawback is that with this covariate in the model the success of finding the latent gene (s) will be reduced. Consequently, it is mandatory to determine how each covariate influences the genetic, common environmental and residual factors of the variance. Covariates that influence birth weight of twins are gestational age, maternal factors (parity and maternal age), twin specific factors (zygosity, sex, birth order), and placental factors (chorionicity, fusion of placentas and insertion of the umbilical cord) (Gielen et\u00a0al. 2007).\nThe aim of this twin study was to model birth weight across gestational ages and to quantify the genetic and environmental components in order to explain the effects of covariates on the variance of birth weight. We hypothesize that heritability changes during gestation and that covariates mainly explain the common environmental and residual factors of the variance. By entering these covariates in the means model and in the covariance matrix, heritability will increase and, as a consequence, the power to find genes by linkage and association studies will increase, although the effects of each covariate on the genetic and environmental component may be different.\nMaterials and methods\nSubjects\nThe study sample consisted of live born twin pairs selected from the East Flanders Prospective Twin Survey (EFPTS), Belgium (Loos et\u00a0al. 1998). Between July 1964 and the end of December 2002, the EFPTS registered 6315 twin pairs who met the World Health Organization criteria for live born infants (birth weight \u2265 500 grams or gestational age \u2265 22\u00a0weeks, if birth weight unknown). The vast majority consisted of twins of Caucasian origin. Methods of data collection have previously been described in detail (Gielen et\u00a0al. 2006; Gielen et\u00a0al. 2007; Loos et\u00a0al. 2005). Briefly, a defined set of obstetric and perinatal data were recorded (including gestational age, mode of conception, maternal age, birth order, parity, sex and neonatal survival) and placentas were examined within 24\u00a0hours after delivery according to a standardized protocol. Based on the site of the cord insertion two groups were distinguished: (1) central insertion (central and eccentric insertions), and (2) peripheral insertion. Gestational age was reported by the obstetrician, based on the last menstruation or a first trimester ultrasound investigation, and was calculated as the number of completed weeks of pregnancy. Zygosity was determined through sequential analysis based on sex, fetal membranes, umbilical cord blood groups (ABO, Rh, CcDEe, MNSs, Duffy, Kell), placental alkaline phosphatase (Decorte et\u00a0al. 1990; Vlietinck 1986), and, since 1982, DNA fingerprints. Zygosity and chorionicity were determined with an accuracy of 99%.\nTwin pairs of whom one or both children were stillborn (205 pairs) or suffered from major congenital malformation (120 pairs) were excluded (Loos et\u00a0al. 2001). Two MC pairs were excluded because they were recorded as being MC but at birth had two separate placentas. Twin pairs with missing values in one or more covariates for one or both twins (birth weight, gestational age, parity, chorionicity, mode of conception, unknown whether staying alive postnatally, maternal age, placental type and weight, site of insertion of the umbilical cord) were excluded (1756 pairs) (Gielen et\u00a0al. 2007). Finally, 8464 twins (4232 pairs) with complete datasets were analyzed.\nStatistical analysis\nThe birth weights of pregnancies of different gestational ages were analyzed using a non-linear multivariate Gaussian regression, MVN ((\u03bc, \u03a32) where \u03bc is the means model and \u03a32 is the covariance matrix.\nMeans model\nGrowth was best modeled by a logistic growth curve (non-linear multivariate Gaussian regression) in which the time of conception (gestational age minus two weeks) was chosen as time point zero ( where \u03b21 t\/m \u03b23 are the coefficients of the logistic growth curve and Time is time since conception; unadjusted model) (Neale and McArdle 2000). Furthermore, the two first weeks of pregnancy (the time between the last menstrual period and conception) were set to be zero. The logistic growth curve was modified to allow a change of growth rate ( where \u03b24 is the change in growth rate after the time point described by coefficient \u03b25). The following covariates were considered: sex of the individual and of the co-twin, chorionicity, number of placentas, site of umbilical cord insertion, birth order, maternal age, mode of conception, parity, and neonatal death (Gielen et\u00a0al. 2007). Because in 1964 all gestational ages were based on the last menstruation, while in 2002 almost all gestational ages were based on first trimester ultrasound and this might influence gestational age, we controlled for birth year. The covariates were consecutively added in the logistic growth curve,\nwhere X represents possible covariates and their interactions included in the model and \u03b2 represents the corresponding coefficients. Finally, members of different groups were allowed to have different growth rates after a certain point in time as shown in the following equation\nwhere Z is a binary covariate (indicator for belonging to group Z) and \u03b26 is the additional change in growth rate after the time point described by coefficient \u03b27 for members of group Z. (adjusted model). Examples of how to use the estimates can be found elsewhere (Gielen et\u00a0al. 2007).\nCovariance matrix\nThe twin model assumes that the total phenotypic variance can be decomposed into additive genetic effects (A), dominance genetic effects (D), effects of common environment (C) and effects of unique environment (E) (Neale and Maes 2002). The common environmental variance is assumed to be the same for both the MZ and DZ twin pairs and will account for the intra pair correlation between twin pair weights. MZ twins are assumed to share the same genetic variance (A and D), whereas DZ twins share half of the additive genetic variance and a quarter of the dominance genetic variance. In the case of a covariance matrix containing an additive genetic effect, common environment, and unique environment (ACE), the covariance matrix is respectively  and  for the MZ and DZ pairs. Because former analyses showed that a model in which the variance allowed to change linearly during gestational age fitted best (Gielen et\u00a0al. 2007), we allowed the variance and covariance to change over time (for example, A\u00a0=\u00a0e\u03b11\u00a0+\u00a0e\u03b12Time, C\u00a0=\u00a0e\u03b41\u00a0+\u00a0e\u03b42Time, and ln(E)\u00a0=\u00a0\u03c31\u00a0+\u00a0\u03c32Time, where Time is gestational age, not time since conception).\nFirst, models containing the ACE and ADE components were fitted separately. Both models were fitted with only gestational age in the mean regression (unadjusted model) and with covariates in the mean regression (adjusted model). The best fitting unadjusted and adjusted models were tested with the (co-)variance being allowed to change during gestation. Next, the covariates of the mean regression of the adjusted model were entered as moderators in the covariance matrix the same way the mean regression of the adjusted model was built. This model is called the full model (for example, ln(E)\u00a0=\u00a0\u03c31\u00a0+\u00a0\u03c32 Time\u00a0+\u00a0\u03c33X where \u03c33 is the coefficient of covariate X), which corresponds to the method described by Shaun Purcell (Purcell 2002). In this way, the covariance of the first-born twin was allowed to differ from the second born twin. The full model will show how heritability, A\/(A\u00a0+\u00a0C\u00a0+\u00a0E), depends on the representative state of the covariates in the model.\nFor example, let us assume a model in which genetic, common and unique environmental components are defined as \nA\u00a0=\u00a0e7.403\u00a0\u00d7\u00a0Time, where the intercept \u03b11 is set to zero\nC\u00a0=\u00a0e6.688\u00a0\u00d7\u00a0Time, where the intercept \u03b41 is set to zero\nIn(E)\u00a0=\u00a05.4054\u00a0+\u00a00.1435\u00a0\u00d7\u00a0Time\u00a0+\u00a00.4324\u00a0\u00d7\u00a0SB\u00a0+\u00a00.724\u00a0\u00d7\u00a0ND\u00a0\u2212\u00a00.163\u00a0\u00d7\u00a0MP\u00a0+\u00a00.2441\u00a0\u00d7\u00a0OPM, where SB is the second born, ND is neonatal death, MP is multiparity, and OPM is one placental mass. The heritability for a first-born twin with a separate placenta, staying alive after one week of birth, of a multiparous mother, in case of two separate placentas would be at 25\u00a0weeks\nand at 40\u00a0weeks of gestation\nModel selection\nModels were compared with the Akaike information criterion (AIC) (Akaike 1973; Lindsey and Jones 1998). Because the modeling process is exploratory, the inference criterion used for comparing the models under consideration is their ability to fit the observed data; i.e. models are compared directly through their minimized minus log likelihood. When the numbers of parameters in models differ, they are penalized by adding the number of estimated parameters. Smaller values indicate more preferable models. This criterion allows direct comparison among models, which are not required to be nested.\nEffect of each separate covariate on the genetic and environmental component\nTo explain the effect of each covariate on the (co-)variance of birth weight at different gestational ages, we removed a significant covariate completely from the full model (out of the mean regression and out of the covariance matrix) and compared the (co-)variances, so that the influence of that specific covariate became apparent.\nAll analyses were conducted with R (Ihaka and Gentleman 1996) version 2.0.1 using the twin library (Lindsey 2001).\nResults\nHeritability decreases during gestation\nFor both the unadjusted model with only gestational age in the mean regression, and the adjusted model with covariates in the mean regression, the ACE model with additive genetic (A), common environmental (C), and unique environmental (E) variances fitted best. Allowing the covariance to change over time improved the fit (Table\u00a01).Table\u00a01Model fit statistics of ACE and ADE models for the unadjusted model with only gestational age in the mean regression, the adjusted model with covariates in the mean regression, and the full model with covariates in the mean regression and in the covariance matrix. The best fitting model (lowest AIC) is presented in boldUnadjusted model Adjusted model\u2212Log likelihooddfAIC\u2212Log likelihoodDfAICACE61696.84845761703.8461350.49844161373.49ADE61727.45845761734.4561380.6844161403.6CE61711.88845861717.8861364.28844261386.28AE61727.45845861733.4561380.58844261402.58E62189.72845962194.7261826.93844361847.93ACE; A and C allowed to change over time61688.44845761695.4461343.61844161366.61Full modelACE; A and C allowed to change over time and covariates in covariance matrix61297.26843761324.26A: Additive genetic effects, D: Dominance genetic effects, C: Effects of common environment, E: Effects of unique environment\nFor both the unadjusted and adjusted ACE model, the common environmental variance was higher than the genetic variance. Although the variances of the adjusted model were lower, the proportions of the standardized variances of both models remained the same, and therefore heritability (standardized A-variance). Heritability decreased from 38% at 25\u00a0weeks to 15% at 42\u00a0weeks (Fig.\u00a01). Allowing the covariance to change during gestation increased the genetic and common environmental variances from 25 to 42\u00a0weeks. The common environmental variance was only a little bit higher than the genetic variance, thereby increasing heritability at the end of gestation (from 36% at 25\u00a0weeks to 18% at 42\u00a0weeks). The model without an intercept for the genetic (\u03b11) and common environmental variance (\u03b41) had a better fit, because it imposes these variance components to be zero at conception (Table\u00a02).Fig.\u00a01Genetic (A), common environmental (C) and unique environmental (E) variance and standardized variance of birth weight of the unadjusted (unadj) model and the adjusted model (adj). E-variance was allowed to change over time, but the A- and C-variance notTable\u00a02Regression coefficients of the covariance matrix of the unadjusted model, the adjusted model, and the full modelACLog(E)Log(estimate)SELog(estimate)SEestimateSEUnadjustedbIntercept10.3700 0.173510.5800 0.11546.00550.3462Gestational age\u2013\u20130.14050.0094UnadjustedaIntercept0.0000\u20130.0000\u20136.30610.3468Gestational age6.89300.16046.9420.12500.13170.0094AdjustedbIntercept10.28000.185710.48000.12226.14360.3495Gestational age\u2013\u20130.13470.0094AdjustedaIntercept0.0000\u20130.0000\u20136.45520.3489Gestational age6.78300.17376.86000.12970.12560.0094Full modelIntercept0.0000\u20130.0000\u20135.40540.3963Gestational age7.04300.14216.68800.15710.14350.0103Second born0.43240.0642Neonatal death0.72400.1981Multiparity\u22120.16300.0491One placental mass0.24410.0636Unadjusted:model with only gestational age in the mean regressionAdjusted:model with covariates in the mean regressionFull model:model with covariates in the mean regression and in the covariance matrixThe unadjusted and adjusted models are presented witha and withoutb A and C allowed changing during gestation  and  where Time is gestational age and X represent the covariates with the corresponding estimates \u03b2.\nEffect of covariates in the covariance matrix on heritability\nThe full model with also covariates in the covariance matrix was the best fitting model (Table\u00a01; for estimates of the means model see Table\u00a03). The genetic variance became larger than the common environmental variance (Fig.\u00a03; Table\u00a02). Not all covariates of the mean regression explained part of the covariance matrix (Table\u00a02 and Table\u00a03). The genetic and environmental variance were only influenced by gestation, the unique environmental variance by parity, birth order, neonatal survival and number of placentas (estimates in Table\u00a02, path diagram in Fig.\u00a02). Although these dichotomous covariates were only present in the unique environmental variance, heritability will nevertheless change, because heritability is the proportion of the total variance that can be explained by genetic variation.Table\u00a03Regression coefficients of the full model with covariates in the mean regression and in the covariance matrixEstimateSEMeans modelCoefficients of the logistic growth curveb1\u00a0=\u00a0saturation level at which growth stops3688.098084.9100b2\u00a0=\u00a0growth rate5.80610.1762b3\u00a0=\u00a0location parameter\u22120.18420.0068b4\u00a0=\u00a0decrease in weight after inflection point0.95630.3017b5\u00a0=\u00a0inflection pointa37.59340.3216b61\u00a0=\u00a0decrease in weight for second bornb0.10540.0021b71\u00a0=\u00a0inflection point for second borna30.72210.8399b62\u00a0=\u00a0decrease in weight for neonatal deathb0.57330.1846b72\u00a0=\u00a0inflection point for neonatal deatha29.00001.098b63= decrease in weight for MZ MCb1.14380.5059b73\u00a0=\u00a0inflection point for MZ MCa37.78690.4227CovariatesMultiparity0.11650.0148Male0.14050.0118Birth year (form 0 [1964] to 38 [2002])0.00180.0007One placental mass\u22120.05250.0127Peripheral umbilical cord insertion\u22120.05930.0227Maternal age0.00460.0014Interaction termsBirth yeara Peripheral umbilical cord insertion\u22120.00270.0009Multiparitya Second born0.03170.0131aWeeks since conceptionbAfter inflection pointMZ MC\u00a0=\u00a0monozygotic monochorionic. Inflection point\u00a0=\u00a0weeks of gestational age since conception (Time). Equation: b1 t\/m b7 coefficients, Z\u00a0=\u00a0second born, Neonatal death or MZ MC, X\u00a0=\u00a0covariates, and \u03b2\u00a0=\u00a0coefficient of covariatesFig.\u00a02Partial path diagram of the full model shown for one twin only. For the estimates and interactions, see table 1 the adjusted model. BWT1\u00a0= Birth weight twin 1, Circle\u00a0=\u00a0latent variables, square\u00a0=\u00a0measured variables, triangle\u00a0=\u00a0definition variables in the means model, MZ MC\u00a0=\u00a0monozygotic monochorionic, A\u00a0=\u00a0additive genetic effects, C\u00a0=\u00a0effects of common environment, E\u00a0=\u00a0effects of unique environment\nFirst-born twins of multipara with two separate placentas, who stay alive neonatally, have the lowest unique environmental variance (Table\u00a02, Fig.\u00a03: Lowest) and therefore the highest heritability (from 52% at 25\u00a0weeks to 30% at 42\u00a0weeks) (Fig.\u00a04: Lowest). Allowing a dichotomous variable to be fixed to another representative state showed that the unique environmental variance remained lower than in the adjusted model, except for twins who died neonatally (Fig.\u00a03). However, heritability was always higher in the unadjusted model than in the adjusted model (Fig.\u00a04). When the covariates were chosen to represent second born twins of primipara with one placental mass and who died neonatally, the unique environmental variance became extremely high (Fig.\u00a03: Highest) and heritability became lower than that of the adjusted model (Fig.\u00a04: Highest).\nFig.\u00a03Genetic (A), environmental (C) and residual (E) variance of birth weight of the adjusted (Adj) model and the full model (Full). Legend: Lowest: E-variance for a first-born twin of a multipara with two separate placentas who is staying alive. Primipara: E-variance for a first-born twin of a primipara with two separate placentas who is staying alive. One placenta: E-variance for a first-born twin of a multipara with one placental mass who is staying alive. Second born: E-variance for a second born twin of a multipara with two separate placentas who is staying alive. Neonatal death (ND): E-variance a first-born twin of a multipara with two separate placentas who died neonatally. Highest: E-variance for a second born twin of a primipara with one placental mass who died neonatallyFig.\u00a04Heritability (standardized A-variance) of birth weight of the adjusted (Adj) and full model (Full) of Fig.\u00a03. Legend: Lowest: Standardized E-variance for a first-born twin of a multipara with two separate placentas who is staying alive. Primipara: Standardized E-variance for a first-born twin of a primipara with two separate placentas who is staying alive. One placenta: Standardized E-variance for a first-born twin of a multipara with one placental mass who is staying alive. Second born: Standardized E-variance for a second born twin of a multipara with two separate placentas who is staying alive. Neonatal death (ND): Standardized E-variance a first-born twin of a multipara with two separate placentas who died neonatally. Highest: Standardized E-variance for a second born twin of a primipara with one placental mass who died neonatally\nEffect of each separate covariate on the genetic and environmental component\nComparing the variances of the full model with and without a specific covariate showed that maternal factors (age and parity) had minor influence on the genetic variance, but no influence on heritability. Removing sex, birth order and neonatal survival gave an increase in genetic variance. However, only for sex, heritability increased if removed from the model. Removing the placental factors from the full means model resulted in a decrease of the genetic variance and a decrease of heritability. Insertion of the umbilical cord (unique for each twin) explained mainly the unique environmental variance, whereas number of placentas also explained common environment.\nChorionicity or other placental factors\nSince chorionicity, number of placentas and insertion of the umbilical cord are related (MC twins always have one placenta and have more often a peripheral cord insertion), as an additional step we built a model in which we replaced cord insertion and number of placentas with chorionicity (chorionicity model). Although the AIC of the chorionicity model was higher, it roughly explained as much of the genetic and unique environmental variances but not of the common environmental variance (Fig.\u00a05). Removing of either the two placental factors or chorionicity resulted in a decrease of genetic variance and decrease of heritability. The variances of the DC twins were comparable to the variances of two separate placentas, whereas variances of the MC twins were comparable to the variances of one placental mass (Fig.\u00a05).\nFig.\u00a05Effect of either placental factors (site of insertion of umbilical cord and number of placentas), or chorionicity on the variance and heritability of birth weight The curves represent the differences between the full model and the model without the specific covariate. The presented curves are the curves for the first born twin of a primipara, who is staying alive\nDiscussion\nThe aim of this study was to model birth weight at different gestational ages and quantify the genetic and environmental components. To our knowledge we are the first to show that heritability of birth weight seems to decrease during gestation, in our case from 38% at 25\u00a0weeks to 15% at 42\u00a0weeks. Entering the covariates of the mean regression as moderators in the covariance matrix, explained more of the common environmental variance than of the genetic variance. However, the unique environmental variance depended upon the representative state of the covariates and was lowest for first-born twins of multipara with two separate placentas, who stayed alive neonatally. Therefore these twins have the highest heritability: from 52% at 25\u00a0weeks to 30% at 42\u00a0weeks.\nPart of the explanation for a decrease in heritability during gestation could be the increasing demand for nutrients and oxygen in twin gestation, especially in the third trimester (Loos 2001). After about 29\u00a0weeks of gestation, intrauterine growth of twins deviates from growth of singletons, (Loos et\u00a0al. 2005; Luke et\u00a0al. 1993; Soucie et\u00a0al. 2006), resulting in a lower birth weight and placental weight (per twin) (Gielen et\u00a0al. 2006; Loos et\u00a0al. 2005; Pinar et\u00a0al. 1996). In addition, placental factors can be involved. In twin pregnancies, a lower placental weight, monochorionicity, one placental mass (one placenta or two fused), and a peripheral cord insertion result in a lower birth weight (this study) (Gielen et\u00a0al. 2007; Loos et\u00a0al. 2001). Also, vascular anastomoses in MC twins result in higher discordancy rates (Machin et\u00a0al. 1996). These placental factors can partly determine the (unequal) blood supply to the fetus and therefore the amount of nutrients and oxygen for each fetus, resulting in a higher degree of discordancy (Machin 1996) and low heritability estimates in twins in late gestation. In this study, first-born twins of multipara with two separate placentas, who stay alive, were genetically the most homogeneous group. This group also had the highest birth weights and placental weights (Gielen et\u00a0al. 2006; Gielen et\u00a0al. 2007), suggesting that they may have minimal environmental restriction and therefore the highest heritability.\nModeling common and unique environmental factors can increase heritability and therefore optimize finding genes responsible for the genetic variance in combined linkage and association studies. By partitioning the association effects into between twin pair and within twin pair components, true from false associations can be distinguished in combined linkage and association studies (Fulker et\u00a0al. 1999; Posthuma et\u00a0al. 2004). As a rule, the power in statistical analysis will increase as the sample size increases, if a quantitative trait locus (QTL) explains a larger part of the variance it is easier to find, and if multivariate models are used (Peeters 2005). For example, if a QTL effect explains 0.05 of the variance (\u03b1\u00a0=\u00a00.05 \u03b2\u00a0=\u00a00.80), with genetic factors explaining 20%, and common and unique environmental factors each explaining 40% of the variance, 6422 sib-pairs are needed. If genetic factors explain a larger part of the variance e.g. 40%, and common and unique environmental factors each explaining 30% of the variance, only 3751 sib-pairs are needed. If in the latter case the QTL effect is 0.10 even less sib-pairs (n\u00a0=\u00a01080) are needed. Lack of statistical power may also be a reason for the inconsistent results of linkage studies to identify quantitative trait loci (QTLs) for birth weight. Only three genome wide linkage studies in different populations have identified QTLs on chromosome 6 (Arya et\u00a0al. 2006), 7 (Fradin et\u00a0al. 2006), and 11 (Lindsay et\u00a0al. 2002) (LOD\u00a0>\u00a03) with relative small sample sizes (less then 413 sibships (Fradin et\u00a0al. 2006), (Lindsay et\u00a0al. 2002) and 840 individuals from families (Arya et\u00a0al. 2006)) and only two covariates taken into account (sex and gestational age).\nBy building a means model and then entering the significant covariates in the covariance matrix, we controlled for environmental variables correlated with the genetic effects on the trait (gene environment correlation\u00a0=\u00a0rGE) (Purcell 2002). All the covariates with a unique environmental component, besides gestational age, must either explain differences between twins or describe gene environment interactions (Purcell 2002; van der Sluis et\u00a0al. 2006). Parity is common for both twins, but because of the interaction with birth order in the mean regression, parity explains part of the unique environmental variance.\nCovariates also contain useful information in order to identify genes, which may be associated with the trait. In this study, only sex contained latent genetic information. For all other covariates used in this study, either heritability did not change, or environmental factors were explained with heritability depending on the representative state of the covariate. Since sex explained part of heritability, entering sex in the means model will mask genes that act differently according to sex and it is better to have these genes in the model instead of sex.\nIn the past, Vlietinck et\u00a0al (1989) showed an effect of chorionicity, although fusion of the placentas and site of insertion of the umbilical cord was not taken into account. Our analyses showed that only by replacing the placental factors cord insertion and number of placentas with chorionicity, the effect of chorionicity became visible, although the model fitted worse. Chorionicity certainly plays a role in growth (in mean regression), but mainly after 40\u00a0weeks of gestation if fusion of the placentas and insertion of the umbilical cord are taken into account (Gielen et\u00a0al. 2007). Consequently, in order to achieve a higher heritability by explaining environmental factors, number of placentas and site of the insertion of the umbilical cord are preferable above chorionicity.\nThe strength of our study lies in the large sample size, accurate zygosity and chorionicity determination. This results in a decrease of the unique environmental variance and therefore a better estimation of heritability of birth weight. The large sample size further offers the opportunity to determine heritability at different gestational ages, although twins might not be representative for singletons due to differences in growth restriction. On the other hand, in studies without MZ and DZ twins, no difference can be made between genetic and common environmental factors, resulting in an overestimation of the genetic factors with, in case of birth weight, heritability estimates up to 80% (Arya et\u00a0al. 2006; Clausson et\u00a0al. 2000; Magnus et\u00a0al. 1984; Nance et\u00a0al. 1983).\nInformation on parental ethnicity was not always available. However, it has been shown that the genetic variance of birth weight does not differ between populations in which the majority (>95%) is Caucasian and Asian (Hur et\u00a0al. 2005), suggesting that the genetic variance might be equal for populations. Since a vast majority of the inhabitants of East Flanders are Caucasians, we assume that our results are not biased by ethnicity. Finally, parental weight and height were not available. We assume however no biased results, since both traits are highly heritable (Carmichael and McGue 1995; Preece 1996; Silventoinen et\u00a0al. 2001). Because the aim of linkage and association studies is to identify loci or alleles responsible for birth weight, adjusting for genetic factors will reduce the chance of finding them as stated above. But also, because we studied birth weight of twin pairs, rather than offspring of twins, the genes implicated in our study relate to fetal rather than maternal effects (Luciano et\u00a0al. 2004). Therefore, maternal genes will be seen as common environment (Luciano et\u00a0al. 2004).\nIn conclusion, modeling genetic and environmental factors gives a better insight in factors influencing growth during gestation, and therefore birth weight. Heritability of birth weight changed during gestation with a decrease from 25 to 42\u00a0weeks with highest heritability for twins with the least environmental restriction (first-born twins of multipara with two separate placentas who stayed alive neonatally). For this complex trait, modeling the covariance matrix with covariates, mainly environmental factors were explained, resulting in an increase of the heritability and subsequently the chance of finding genes by linkage and association studies.","keyphrases":["heritability","birth weight","twins","gestational age","variance.","growth curves"],"prmu":["P","P","P","P","P","P"]}
{"id":"Histochem_Cell_Biol-4-1-2413112","title":"Origin of renal myofibroblasts in the model of unilateral ureter obstruction in the rat\n","text":"Tubulo-interstitial fibrosis is a constant feature of chronic renal failure and it is suspected to contribute importantly to the deterioration of renal function. In the fibrotic kidney there exists, besides normal fibroblasts, a large population of myofibroblasts, which are supposedly responsible for the increased production of intercellular matrix. It has been proposed that myofibroblasts in chronic renal failure originate from the transformation of tubular cells via epithelial\u2013mesenchymal transition (EMT) or from infiltration by bone marrow-derived precursors. Little attention has been paid to the possibility of a transformation of resident fibroblasts into myofibroblasts in renal fibrosis. Therefore we examined the fate of resident fibroblasts in the initial phase of renal fibrosis in the classical model of unilateral ureter obstruction (UUO) in the rat. Rats were perfusion-fixed on days 1, 2, 3 and 4 after ligature of the right ureter. Starting from 1 day of UUO an increasing expression of alpha-smooth muscle actin (\u03b1SMA) in resident fibroblasts was revealed by immunofluorescence and confirmed by the observation of bundles of microfilaments and webs of intermediate filaments in the electron microscope. Inversely, there was a decreased expression of 5\u2032-nucleotidase (5\u2032NT), a marker of renal cortical fibroblasts. The RER became more voluminous, suggesting an increased synthesis of matrix. Intercellular junctions, a characteristic feature of myofibroblasts, became more frequent. The mitotic activity in fibroblasts was strongly increased. Renal tubules underwent severe regressive changes but the cells retained their epithelial characteristics and there was no sign of EMT. In conclusion, after ureter ligature, resident peritubular fibroblasts proliferated and they showed progressive alterations, suggesting a transformation in myofibroblasts. Thus the resident fibroblasts likely play a central role in fibrosis in that model.\nIntroduction\nTubulo-interstitial fibrosis is a landmark of chronic renal failure, regardless of the underlying disease. It has been proposed that fibrosis is not only a response to tubular injury but that it may be the main driving force of nephron loss (Remuzzi and Bertani 1998; Zeisberg et al. 2001).\nThe basic aspects of fibrosis are similar in various organs. The increased accumulation of matrix is associated with a change of the phenotype of interstitial cells. Myofibroblasts are found in addition to or in the place of the quiescent fibroblasts of healthy tissues (Liu 2004; Desmouliere et al. 2005; Mucsi and Rosivall 2007). Compared to quiescent fibroblasts myofibroblasts show, besides their increase of the production of matrix and of the synthesis of matrix-degrading enzymes, structural characteristics, such as frequent intercellular contacts and a unique set of cytoskeletal proteins, the most distinctive being smooth muscle \u03b1-actin (\u03b1SMA) (Desmouliere et al. 2005;Hinz 2007; Hinz et al. 2007;Essawy et al. 1997).\nAs discussed in recent reviews myofibroblasts may have different origins, possibly depending on the organ and on the pathology (Desmouliere et al. 2005; Hinz et al. 2007). They may originate from the differentiation of resident fibroblasts, from bone-marrow-derived precursors which infiltrate the injured tissue or from transformation of epithelial cells, the so-called epithelial\u2013mesenchymal transition (EMT).\nThe origin of myofibroblasts in the kidney has been the object of intensive research. There is evidence of a role of bone marrow-derived precursors in models of adriamycin nephropathy (Li et al. 2007) and of ischemia\/reperfusion injury (Broekema et al. 2007). In contrast no significant contribution of such precursors to fibrosis was found in a murine model of unilateral ureter obstruction (UUO) (Roufosse et al. 2006). Numerous research reports and review papers discuss the role of EMT in renal fibrosis (Lan 2003; Iwano and Neilson 2004; Li et al. 2007; Mucsi and Rosivall 2007). In contrast, the possibility of a transformation of resident fibroblasts into myofibroblasts in renal fibrosis has hardly been considered. There is little information on the behavior and fate of resident fibroblasts in fibrosis in the kidney, in particular whether they proliferate and differentiate into myofibroblasts.\nThe aim of our present investigation was to study the morphology of cortical peritubular interstitium, with a focus on the resident fibroblasts, during the first 4\u00a0days following ureteral ligature in rats. UUO is a commonly used experimental model for inducing renal fibrosis and is furthermore of outmost clinical relevance. We choose to study the early events after ureteral ligature because in early stages the identification of cell types and their putative phenotypical changes might be better traceable than in advanced stages of fibrosis. We include in our study also the early effects of UUO on tubular epithelia.\nWe performed our investigation by light and electron microscopy, as well as immunofluorescence, and we included morphometric quantification of tissue components and analysis of changes in the proliferation rates of interstitial cells.\nWe demonstrate that resident fibroblasts progressively gain the phenotype of myofibroblasts and massively increase their rate of cell division during the first days after ureteral ligature. Our data suggest that resident peritubular fibroblasts transform to myofibroblsts and participate importantly in the development of renal fibrosis.\nMethods\nExperimental design\nFor induction of UUO male 20 Sprague\u2013Dawley rats weighing 160\u00a0\u00b1\u00a03\u00a0g were anesthetized using isoflurane and the abdominal cavity was opened by a midline incision. The right ureter was tied with 4\/0 silk 3\u20135\u00a0mm below the renal hilum. The rats of the control group (four rats) were also laparatomized, the ureter exposed, but no ligature was made. After closure of the abdomen the animals were returned to the cage with free access to standard lab chow and water ad libitum.\nThe experimental procedure conformed to the Institutional Guidelines of Experimental Animal Care and Use and was approved by the Veterinary office of the Kanton Z\u00fcrich.\nTissue fixation\nAfter 1, 2, 3 and 4\u00a0days of the operation, five ureter-ligated rats were fixed by vascular perfusion through the abdominal aorta as described previously (Dawson et al. 1989). The control group was perfused on day 4. The occluded renal pelvis was cut open just before perfusion to allow outflow of the fluid from the kidney. The fixative solution was composed of 3% paraformaldehyde, 0.01% glutardialdehyde and 0.05% picric acid in a 6:4 mixture of 0.1\u00a0M cacodylate buffer (pH 7.4, adjusted to 300\u00a0mOsm with sucrose) and 10% hydroxyethyl starch in saline (HAES steril TM; Fresenius AG, Germany). Five minutes after onset of the fixation both kidneys were removed.\nLight and electron microscopy\nFrom the unligated left and from the ligated right kidney large tissue blocks extending from the renal capsule to at least the upper third of the inner zone were immersed for at least 24\u00a0h in the fixative solution described above to which 1% glutardialdehyde was added. This tissue was then embedded into epoxy resin and used for light and electron microscopy. Semithin (1\u00a0\u03bcm) sections and ultrathin (80\u00a0nm) sections were cut with an ultramicrotome. Semithin sections were stained with 1% methylene blue and 1% azure II and were studied with a Polyvar microscope (Reichert Jung, Vienna, Austria). Ultrathin sections were postfixed with osmium tetroxyde and contrasted with uranyl acetate and studied with a CM100 Philips electron microscope.\nImmunofluorescence microscopy\nCoronal slices across the right and left kidney, comprising the cortex and the outer medulla were frozen in liquid propane, cooled with liquid nitrogen. From these slices, 2\u20133\u00a0\u03bcm thick sections were cut in a cryostat (Microm, Walldorf, Germany). The sections were pre-treated with 5% normal goat serum diluted in PBS with 1%BSA, which was also the diluent for all other antibodies. They were incubated at 4\u00b0C overnight in a humidified chamber with primary antibodies described in Table\u00a01. Binding sites of the primary antibodies from rabbit origin were detected using Alexa 555-conjugated goat anti-rabbit (Invitrogen, Carlsbad, CA, USA) and the primary antibody from mouse origin was detected using Alexa 488-conjugated goat anti-mouse (Invitrogen). For chromatin staining 4,6-diamidino-2-phenylindole (DAPI; Sigma, St Louis, MO, USA) was added to the secondary antibodies. The slides were mounted in DAKO-glycergel (Dakopatts, Glostrup, Denmark) containing 2.5% of 1,4-diazabicyclo (2.2.2)octane (DABCO; Sigma, St Louis, MO, USA) as fading retardant.Table\u00a01List of primary antibodiesPrimary antibodiesHostSourceS100A4RabbitDako, Glostrup, DenmarkEcto-5\u2032nucleotidase (5\u2032NT)RabbitM. Le HirEcto-5\u2032nucleotidase (5\u2032NT)MouseBD Biosciences, Franklin Lakes, NJ, USAAlpha smooth muscle actin (\u03b1SMA)MouseDako, Glostrup, DenmarkAlpha smooth muscle actin (\u03b1SMA)RabbitAbcam, Cambridge, UKRat MHC class IIMouseClone OX 6Phospho-S6-kinaseRabbitCell Signaling, Danvers, MA, USACollagen Type IMouseMD Biosciences, Zurich, SwitzerlandVimentinMouseChemicon, Temecula, CA, USA\nSections were studied by epifluorescence with a Polyvar microscope (Reichert Jung, Vienna, Austria), and digital images were acquired with a CCD camera.\nQuantitative evaluation\nIn order to quantify changes in the structural composition of the renal cortex and to assess the mitotic rates of interstitial cells within this area, a morphometrical analysis was undertaken on sections of kidneys from three rats in each of the five experimental groups.\nIn 1\u00a0\u03bcm thick epon sections with a surface of approximately 2\u00a0\u00d7\u00a04\u00a0mm, a randomly selected cortical area of approximately 1\u00a0mm2 was photographed at 100\u00d7 magnification using a Leica DMRBE microscope (Leica Microsystems, Heerbrugg, Switzerland) connected to a Olympus SIS colorview camera (Olympus Soft imaging systems, M\u00fcnster, Germany). The individual micrographs were merged to one large image and the exact size of the total image area was subsequently determined by analySIS software (Olympus Soft imaging systems, M\u00fcnster, Germany).\nThe images were overlaid with a 15\u00a0\u00d7\u00a015 grid to assign the histological structures covered by each of the 225 intersections to one of five categories, namely tubules, capillaries, interstitium, renal corpuscles and large vessels. The fractional areas of these histological structures were defined as the ratio of the number of category-specific intersections to the total number of intersections (225).\nFurthermore, the total numbers of peritubular interstitial cells in the image were counted and the numbers of mitotic cells in the cortical interstitium were recorded. This allowed calculating the fraction of mitotic cells in relation to all interstitial cells. The identity of each mitotic cell in the cortical interstitium was attributed to one of the two categories \u201cfibroblasts\u201d or \u201cmononuclear cells\u201d (dendritic cells, infiltrating leucocytes) independently by two investigators. Mitotic cells that could not be assigned to one of these two categories or that were classified differently by the two investigators recorded as \u2018non-identified\u2019.\nStatistics\nNumerical data are expressed as mean values (n\u00a0=\u00a03)\u00a0\u00b1\u00a0standard deviation (SD).\nResults\nMacroscopy\nThe ligature of the right ureter resulted invariably in each rat in a large expansion of the renal pelvis within the renal sinus and of the ureter above the ligature. The enlargement appeared more prominent at the first and second day after the operation than after the third and fourth day. The fluid contained in the expanded renal pelvis was clear up to the second day and appeared trouble and viscous the third and fourth day. In sham operated rats no expansion of the renal pelvis was observed.\nMicroscopy\nTubular epithelia\nIn control and contra-lateral kidneys the tubules were patent with rather homogeneous luminal diameter (Figs.\u00a01a, 2a).Fig.\u00a01Rat renal cortex of contra lateral (a, e) and of ureter ligated (b\u2013d, f\u2013h) kidneys; 1\u00a0\u03bcm epon sections. Overviews (a\u2013d) show the dilatation of distal segments after 1\u00a0day, the collapse and shrinkage of predominantly proximal tubules after 2\u20134\u00a0days and the progressive enlargement of the interstitium. Higher magnifications (d\u2013h) reveal the increasing complexity of interstitial cells; asterisk fibroblasts; D distal segment, P proximal tubule (cut tangentially in H); arrow fibroblast in prophase of mitosis, arrowhead mononuclear cell in mitosis. Bara\u2013d ~100\u00a0\u03bcm, e\u2013h ~10\u00a0\u03bcmFig.\u00a02Proximal tubules in the cortex of a sham operated kidney (a) and of a ureter-ligated kidney on day 3 (b, c). 80\u00a0nm epon sections. In b and c the outer diameters of the tubules as well as the width of their lumina are decreased and the fractional area of nuclei in the epithelium is abnormally high. d Higher magnification of the area framed in c, revealing junctional complexes (arrowheads) and microvilli. Bars in a, b, c (same magnification): 10\u00a0\u03bcm; in d 1\u00a0\u03bcm\nStarting from day 1 the most striking feature in tubules of kidneys with occluded ureter was the heterogeneity of tubular diameter.\nProximal tubules\nAfter 1\u00a0day (Fig.\u00a01b) most proximal tubular profiles appeared similar as in controls, only a few proximal tubular profiles were markedly smaller in diameter than in controls. As soon as after 2\u00a0days (Fig.\u00a01c) the majority of proximal tubules displayed narrowed lumina and decreased outer diameters. That tubular shrinkage progressed through the 4\u00a0days of experiment (Figs.\u00a01d, 2). The density of nuclei was strikingly increased in shrunken tubules (Fig. 2), thus, the decreased epithelial volume reflected cellular atrophy rather than cell death. The brush border remained well visible even after nearly complete collapse of the lumen (Figs.\u00a01g, 2c, d). Although proximal and distal tubules in the cortex revealed severe regressive changes no disintegration of the epithelial layer was detected either by light or by electron microscopy. In all cases the proximal tubule were lined by a continuous epithelium showing ultra-structural evidence of polarity like microvilli at the luminal aspect and morphologically intact junctional complexes. We found no instance of disruption of the tubular basement membrane. The polarized expression of 5\u2032NT at the luminal pole of all proximal tubules further evidenced the preservation of epithelial architecture in proximal tubules. Tubular cell death was not conspicuous in the cortex. Mitotic cells were rather frequently observed in proximal tubules from day 2 on. At the same time epithelial cells, including mitotic cells, were occasionally seen in the lumen, indicating some degree of anoikosis. From day 3 on an increasing number of proximal tubular profiles bound anti collagen 1-antibodies. Even in those profiles the individual epithelial cells were still recognizable.\nDistal segments and collecting ducts\nAfter 1\u00a0day dilation of the luminal diameters of most distal segments was eye-catching (Fig.\u00a01b). Occasionally their lumen was filled with hyaline material. From day 2 on cellular hypotrophy associated with a collapsed lumen, as described above for proximal tubules, became apparent also in distal segments. At day 4 many distal profiles were massively shrunken and atrophic (Figs.\u00a01d, h, 3). However, some profiles remained histologically almost normal up to day 4. The diameters of collecting duct profiles were dilated from day 1 on and their largely open lumina were strikingly prominent among the shrunken tubular profiles in the cortex at day 3 and 4 (Fig.\u00a01c, d).Fig.\u00a03Thick ascending limb in the cortex of a sham operated kidney (a) and of a ureter ligated-kidney on day 2 (b); 80\u00a0nm epon sections. In b the outer diameters of the tubule as well as the width of the lumen is decreased. c Higher magnification of the area framed in b, revealing junctional complexes (arrowheads) and microvilli. Bars in a, b (same magnification): 5\u00a0\u03bcm, in d 1\u00a0\u03bcm\nQuantitative estimation of changes of cortical tubular volume and peritubular spaces\nThe fractional area of tubules, capillaries and the interstitium was assessed in 1\u00a0\u03bcm epon sections of perfusion fixed kidneys (Fig.\u00a04). The epithelial shrinkage of most proximal tubules and of some distal tubules was reflected in significantly decreased fractional volume of tubules from day 2 (Fig.4).Fig.\u00a04Quantitative analysis of the structural changes in rat renal cortex in response to UUO. The fractional area of the tubular system decreased while that of the interstitium increased continuously during the first 3\u00a0days after UUO. Tubular and interstitial areas differ significantly from control values from day 2 on. The fractional area of capillaries did not change significantly during the 4\u00a0days of UUO. Values are means\u00a0\u00b1\u00a0SD, n\u00a0=\u00a03\nThe peritubular interstitial space is defined as the space between the tubular, glomerular and capillary basement membranes. After ligature of the ureter its fractional volume increased significantly from about 11% in control kidneys (day 0) to more than twice this value on days 3 and 4 of UUO (Figs.\u00a01d, 4). The fractional volume of capillaries did not change significantly (Fig.\u00a04).\nCortical peritubular interstitium\nThe major cellular constituents of the peritubular interstitium are peritubular fibroblasts (Dawson et al. 1989; Gandhi et al. 1990; Kaissling and Le Hir 1994; Kaissling et al. 1996) and dendritic cells (Kaissling and Le Hir 1994; Dong et al. 2005; Soos et al. 2006). In addition a few lymphocytes and rare granulocytes may be encountered (Kaissling and Le Hir 1994). The precise delimitation of the individual interstitial cells within the narrow interstitial space is difficult in conventional light microscopic preparations. Fixation by vascular perfusion resulted in unfolded intercellular spaces and in thin sections (80\u00a0nm and 1\u00a0\u03bcm epon sections, 3\u00a0\u03bcm cryostat sections) delimitation of the cytoplasm of individual interstitial cells is possible.\nPhenotypical changes of peritubular fibroblasts\nIn control animals the morphology of fibroblasts (Fig.\u00a01e) and dendritic cells was as described previously (Kaissling et al. 1996). In fibroblasts the nucleus was generally angular and surrounded by a sparse cytoplasmic rim, most organelles being located in the extended cytoplasmic processes (Figs.\u00a01e; 5a). The latter showed connections with tubules and capillaries and to each other, as seen in electron microscopy (EM; Figs.\u00a05a, 6b, c). Thereby fibroblasts establish a continuous framework within the peritubular interstitium (Kaissling and Le Hir 1994; Kaissling et al. 1996).Fig.\u00a05Characteristic fibroblasts in a sham-operated kidney (a) and in a ureter-obstructed kidney on day 2 (b). In a the RER (arrow) is flat and concentrated in a cell process whereas the cytoplasmic rim around the angular nucleus is narrow and virtually organelle-free. The fibroblast displays the characteristic narrow association with a tubule and a blood capillary. Collagen fibrils are hardly detectable. The fibroblasts in b reveal inflated cisterns of RER (arrows) distributed all over the cytoplasm, including the direct surroundings of the oval nucleus. In B most collagen fibrils are cross-sectioned and appear as dots (arrowheads). They are abundant along the basement membranes of tubules (lower right and upper left corners). Bars in a and b (same magnification): 2\u00a0\u03bcmFig.\u00a06Ultrastructural details of fibroblasts in ureter-ligated kidney (a\u2013c: day 2). a A bundle of actin filaments displays dense bodies (arrows). b Adherent contact between two fibroblasts. c Intercellular contact between two fibroblasts that display different types of RER, inflated on the left, flat as in controls on the right. d Fibroblast in a ureter-ligated kidney (day 4). The profile of the cell reveals large irregularly formed cytoplasmic processes. Different types of RER are visible even within the same cell; in the pericaryon the cisterns are flat (arrowheads), in the large process they are inflated (arrows), masses of collagen fibrils are seen in the immediate vicinity of the process. Bars 0.5\u00a0\u03bcm and in d 5\u00a0\u03bcm\nThe most conspicuous organelles in the cytoplasm of fibroblasts were the large, mostly flattened, cisterns of rough endoplasmic reticulum (RER) (Fig.\u00a04a). Actin filaments were found exclusively in a narrow rim of cytoplasm underlying the plasma membrane, most frequently at sites of contact with tubules and vessels (Kaissling et al. 1996).\nStarting from day 1 (Fig.\u00a01f\u2013h) the appearance of fibroblasts became strikingly heterogeneous. Whereas a small fraction almost retained the morphology found in control kidneys, others displayed profound changes. In general the nucleus was rather rounded (Fig.\u00a05b), often with indentations, and it was surrounded with a broad cytoplasmic rim. The cytoplasmic processes were often extensively branched (Fig.\u00a01f\u2013h). The RER was more abundant than in controls. In a large fraction of fibroblasts the RER cisterns were inflated and filled with flocculent material (Figs.\u00a05b, 6c) consistent with enhanced synthesis of matrix. Indeed, massive accumulations of collagen fibrils were seen in the immediate vicinity of fibroblasts with such inflated cisterns of RER. In other cells the RER cisterns were flat (Fig.\u00a06c) as in controls. The two forms could be found in neighboring fibroblasts (Fig.\u00a06c) and even occasionally within a single cell (Fig.\u00a06d). Intermediate forms of the RER were also found suggesting a progressive activation of the fibroblasts. Bundles of actin filaments were observed in the cytoplasmic processes. The presence of dense bodies (Fig.\u00a06a) identified them as \u03b1SMA filaments. There was no obvious correlation between the abundance of bundles of actin filaments and the abundance or shape of the RER. In some fibroblasts of obstructed, but not of control kidneys intermediate filaments were seen (not shown). Adherent junctions between fibroblasts were sparse in controls but were common in obstructed kidneys (Fig.\u00a06b, c).They sometimes connected a fibroblast with a flattened RER with one with an inflated RER but never a fibroblast with a mononuclear cell or two mononuclear cells with each other.\nThe study of the peritubular interstitium in the renal cortex by immunofluorescence takes advantage of the fact that fibroblasts in healthy kidneys of rats and mice strongly express 5\u2032NT in their plasma membrane (Dawson et al. 1989; Kaissling and Le Hir 1994). Only a very small population of lymphocytes that are rarely encountered in the interstitium and easily distinguished from fibroblasts on account of their different shape may weakly express 5\u2032NT (=CD 73) (Kaissling and Le Hir 1994).\nAlpha SMA is the hallmark of myofibroblasts. In the kidney of adult healthy controls there is no trace of \u03b1SMA, as well as of vimentin (Essawy et al. 1997) in peritubular 5\u2019NT-positive fibroblasts (Marxer-Meier et al. 1998). Alpha SMA was apparent in the peritubular interstitium as soon as after one day of ureteral obstruction. Double labeling for 5\u2032NT and \u03b1SMA (Figs.\u00a07, 8) revealed that \u03b1SMA was localized consistently within 5\u2019NT-positive fibroblasts (Fig.\u00a07d\u2013f), in agreement with the observation of bundles of actin filaments and dense bodies in fibroblasts in the electron microscope. Alpha SMA was detectable in the majority of 5\u2032NT-positive fibroblasts on day one, though in highly variable intensity, and in virtually all of them on day 4 of UUO (Fig.\u00a08). The average intensity of immunolabeling for \u03b1SMA in individual fibroblasts progressively increased during the 4\u00a0days. The intensity of labeling of 5\u2032NT and of \u03b1SMA seemed to be reciprocal. In fibroblasts strongly positive for \u03b1SMA the 5\u2032NT labeling was weaker than in normal fibroblasts and the staining pattern for 5\u2032NT changed from a sharp outline of the plasma membrane to a slightly diffuse, granular pattern over the plasma membrane and cytoplasm (Fig.\u00a08, see also Fig.14). Large areas with strongly \u03b1SMA- and weakly 5\u2032NT-positive fibroblasts were found around shrunken tubules, whereas prominent 5\u2032NT and weakly \u03b1SMA-positive cells were found in areas where the tubules seemed to be rather intact (Fig.\u00a08).Fig.\u00a07Renal cortex in sham-operated (a) and in ureter-ligeted kidneys (b, c). 3\u00a0\u03bcm cryostat sections; immunofluorescence; ecto-5\u2032nucleotidase (5\u2032NT)\u2014red channel, alpha smooth muscle actin (\u03b1SMA)\u2014green channel); chromatin staining by DAPI\u2014blue channel. In controls (a) 5\u2032NT strongly labels cells in the interstitium and the brush border of proximal tubules, \u03b1SMA labels exclusively smooth muscle cells in arterial vessels (a). In the interstitium of ureter-ligated kidneys (b, c) 5\u2032NT staining decreases, whereas that of \u03b1SMA appears and becomes increasingly prominent. d\u2013f interstitial fibroblast in ureter-ligated kidney after 2\u00a0days. 5\u2032NT is distributed over the plasma membrane and cytoplasm in a granular manner, \u03b1SMA is apparent along the plasma membrane and in the cellular processes. Bara\u2013c ~100\u00a0\u03bcm, d\u2013f ~10\u00a0\u03bcmFig.\u00a08Renal cortex after 4\u00a0days of ureter ligation; 3\u00a0\u03bcm cryostat sections; immunofluorescence; ecto-5\u2032 nucleotidase (5\u2032NT)\u2014red channel, alpha smooth muscle actin (\u03b1SMA)\u2014green channel); chromatin staining by DAPI\u2014blue channel. a Merge of channels, b 5\u2032NT, c \u03b1SMA; the framed area 1 comprises interstitium with high 5\u2032NT and faint \u03b1SMA staining, adjacent to rather intact tubules, the framed area 2 comprises interstitium with strong \u03b1SMA and faint 5\u2032NT staining, adjacent to collapsed tubules. Bara\u2013c ~100\u00a0\u03bcm; 1, 2: 10\u00a0\u03bcmFig.\u00a09Renal cortex after ureter ligation; 3\u00a0\u03bcm cryostat section, immunofluorescence for \u03b1SMA\u2014green channel and collagen type I (Col I)\u2014red channel; chromatin staining by DAPI\u2014blue channel; a arteriole. a\u2013c Overview on the renal cortex after three days of ureter ligation; localization and intensity of \u03b1SMA and of Col I staining in the intertstitium coincide; heavy collagen deposits are seen around collapsed tubules (see also Fig.\u00a013 where the ring of collagen is shown by TEM). d\u2013f: a single fibroblast with distinct \u03b1SMA under the plasmalemm and immediately adjacent collagen deposits 2\u00a0days after ureter-ligature. Bar ~50\u00a0\u03bcmFig.\u00a010MHC II-positive interstitial cells in the renal cortex. a\u2013e 3\u00a0\u03bcm cryostat sections, immunofluorescence for MHC II, f 1\u00a0\u03bcm epon section. In controls faintly stained MHC II-positive cells are present throughout the interstitial spaces, a few are seen also in the glomerulus (G). The amount and size of MHC II-positive cells in the peritubular interstitium increases after 2 and even more after 4\u00a0days of ureter ligation, a few MHC II-positive cells are seen in the Glomerulus (G); d\u2013f characteristic morphology of dendritic cells (asterisks) with a smooth, rounded or ovoid nucleus and lace veil-shaped large processes; in (f) two mononuclear cells in mitosis are seen in left half. Barsa\u2013c ~100\u00a0\u03bcm; d\u2013f ~10\u00a0\u03bcmFig.\u00a011FSP1\/S100A4-positive cells in the renal cortex. 3\u00a0\u03bcm cryostat sections, immonofluorescence; 5\u2032NT\u2014red channel; FSP 1\/S100A4\u2014green channel. In controls a very few FSP1\/S100A4-positive cells are seen in the peritubular interstitium among the 5\u2019NT-positive interstitial cells; the abundance of FSP1\/S100A4 positive cells in the interstitial spaces increases at day 2 and 3 after ureteral obstruction; a few are found in glomeruli (G); arterioles (arrow) are weakly labeled by the antibody to FSP1\/S100A4, tubular epithelia are negative for FSP1\/S100A4. Bar ~100\u00a0\u03bcmFig.\u00a012FSP1\/S100A4 and \u03b1SMA in renal cortex after 4\u00a0days of ureteral obstruction. 3\u00a0\u03bcm cryostat sections, immonofluorescence; red channel\u2014FSP1\/S100A4, green channel\u2014\u03b1SMA; FSP1\/S100A4-positive cells in the interstitial space never stained for \u03b1SMA; tubular profiles never stained for FSP1\/S100A4 or \u03b1SMA; (G)\u2014glomerulus, insert: at higher magnification the shapes of FSP1\/S100A4-positive cells suggest that they are lymphocytes and not fibroblasts. Bar ~100\u00a0\u03bcm; insert ~10\u00a0\u03bcmFig.\u00a013Mitotic figures in ureter-ligated kidneys on day 2; 80\u00a0nm epon sections. The fibroblast in a (prophase) displays a rather normal morphology with its close association with a capillary (upper left corner); the RER appears abnormally abundant but the shape of the cisterns is not altered. The fibroblasts in b shows inflated cisterns of RER. Note the abundance of collagen fibrils along the basement membrane of the tubule (arrowheads, compare with Fig.\u00a09). The mononuclear cell in c cannot be precisely identified. d Proximal tubular cell. Bars 4\u00a0\u03bcm. Bars ~100\u00a0\u03bcm and 10\u00a0\u03bcmFig.\u00a014Mitotic cells in the peritubular interstitium in ureter-ligated kidneys. 3\u00a0\u03bcm cryostat sections, immonofluorescence; \u03b1SMA\u2014green channel; 5\u2032NT, or phopho-S6 kinase (p-S6\u00a0K)\u2014red channel, chromatin staining by DAPI\u2014blue channel. The mitotic cell in a heavily expresses 5\u2032NT and faintly \u03b1SMA, the adjacent cell shows strong \u03b1SMA and faint 5\u2032NT staining. The two mitotic cells in B display strong staining for \u03b1SMA along the plasma membrane and in the cell processes, 5\u2032NT staining appears granular and is seen over the cytoplasm. The mitotic cell in C shows strong \u03b1SMA along the plasma membrane, in the cell processes and in a reticulated manner also in the cytoplasm; p-S6\u00a0K is up regulated in the cytoplasm of mitotic cells, whereas in quiescent cells the nucleus shows weak staining. Bar ~10\u00a0\u03bcm\nAccumulation of collagen type I in the peritubular interstitium\nAccumulation of collagen I (Col I) in the interstitial space is a hallmark of renal fibrosis. Collagen type I is produced by (myo)fibroblasts. By immunofluorescence Col I was virtually absent in the cortical peritubular interstitial space of controls. A modest immunofluorescence was apparent along the tubular profiles.\nIn the obstructed kidneys increasing amounts of Col I was found beginning with day 1 after ligature. In the cortex of ureter-ligated kidneys immunolabeling revealed heterogeneity in the abundance of Col I and this correlated closely with the intensity of immunolabeling for \u03b1SMA (Fig.\u00a09). Thus interstitial Col I staining was the most abundant at sites with strongly \u03b1SMA-positive fibroblasts. At sites with moderate \u03b1SMA expression the collagen deposits could be attributed to single interstitial \u03b1SMA-positive cells (Fig.\u00a09d, e). Col I staining became particularly strong around the tubular basement membranes of shrunken tubules, in agreement with the accumulation of collagen fibrils along the tubular basement membranes, seen in EM (Figs.\u00a05b, 13b). Col I was more abundant in regions where the tubules were atrophic than in regions with apparently intact tubules. At these sites the tubular epithelium revealed some binding to antibodies to Col I.\nIncreases in mononuclear cells in the cortical interstitium\nMononuclear cells can be distinguished from fibroblasts on account of morphological criteria (Kaissling and Le Hir 1994; Kaissling et al. 1996) and in immunofluorescence by their absence of 5\u2032NT and presence of specific marker proteins. In the healthy kidney the majority of mononuclear cells in the interstitial space are dendritic cells. The dendritic cells in rats can be recognized by their high expression of MHC class II. Morphologically these cells are distinguished from fibroblasts by their usually ovoid nucleus and extensive formation of cell projections that appear lighter than those of fibroblasts and that reveal frequent holes like in a lace-veil (Fig.\u00a010) (Kaissling and Le Hir 1994; Kaissling et al. 1996). In sharp contrast to (myo)fibroblasts dendritic cells lack actin filaments and junctions with other cells.\nMassive invasion of mononuclear cells in the obstructed kidney was observed from day 2 after ureter ligature. In the light- and the electron microscope most infiltrating cells in the occluded kidneys displayed the morphology of dendritic cells, of lymphocytes, or of macrophages. Frequency and size of dendritic cells increased dramatically during the 4\u00a0days of ureteral obstruction (Fig.\u00a010).\nFSP1\/S100A4-positive cells (FSP1\/S100A4)\nFSP1\/S100A4 has been implicated in development of renal fibrosis. It had been suggested that proximal tubular cells up-regulate FSP1\/S100A4 and transform to myofibroblasts (Iwano et al. 2002). Immunostaining for FSP1\/S100A4 (Figs.\u00a011, 12) revealed cells in the interstitial space that displayed the smooth outlines of lymphocytes, as described previously (Le Hir et al. 2005). The abundance of these cells was low in control kidneys and increased sharply on day 2 after ligature and thereafter remained approximately stable (Fig.\u00a010b, c). Occasionally these cells were found also within the lumen of capillaries and venules, as well as within glomeruli. In no instance we observed FSP1\/S100A4 positive tubular cells or cells, double positive either for FSP1\/S100A4 and 5\u2019NT or for FSP1\/S100A4 and \u03b1SMA (Fig.\u00a012).\nProliferation of interstitial cells\nMitoses in interstitial cells are rarely encountered in control kidneys. Thus, the occurrence of numerous mitoses within the interstitial space after ureteral ligature was particularly striking. All stages of mitoses were found in the light- (Fig.\u00a01) and electron microscope (Fig.\u00a013) and by immunofluorescence (Fig.\u00a014) in all types of interstitial cells. Immunostaining for phospho-S-kinase, which is up regulated in the cytoplasm in all stages of mitosis (Fig.\u00a014c), was helpful for easy spotting of mitoses at low magnification (Schmidt et al. 2007).\nQuantitative analysis of mitotic interstitial cells in 1\u00a0\u03bcm epon sections showed a sharp increase of the mitotic index in the peritubular interstitium on day 2 in UUO (Fig.\u00a015a). Thereafter the mitotic index decreased but it remained significantly higher in the occluded kidney at days 3 and 4 than in controls. Even though up to 20% of mitotic cells could not be identified it was evident that fibroblasts and mononuclear cells contributed similarly to the increase. This was confirmed by electron microscopy (Fig.\u00a013), since of a total of 32 mitotic cells found on days 2, 3 and 4 in the interstitium, 17 were fibroblasts and 15 mononuclear cells.Fig.\u00a015Quantitative analysis of mitotic cells in the interstitium in response to UUO. a Overall mitotic rates in interstitial cells. Values on day 1, 2, 3 and 4 differ significantly from values of controls. b Relative contribution of fibroblasts and mononuclear cells to mitotic activity in the interstitium. Means\u00a0\u00b1\u00a0standard deviation; n\u00a0=\u00a03\nIn the electron microscope mitotic figures were seen in fibroblasts with normal RER as well as in fibroblasts with inflated RER (Fig.\u00a013). In fluorescence microscopy mitotic figures in fibroblasts were independent of the levels of expression of 5\u2019NT and \u03b1SMA (Fig.\u00a014). Thus, cell proliferation affected the various stages of transformation of fibroblasts in myofibroblasts.\nMitotic figures in tubules and in capillaries were rare in control kidneys. They were easily found in occluded kidneys on day 2, and less so on days 1, 3 and 4. Quantification was not carried out.\nDiscussion\nThe main purpose of the present study was to investigate the early response to ureter obstruction of resident fibroblasts in the renal cortex. Two major alterations were observed, starting from the first day after ligature of the ureter in the rat. Firstly, ultrastructural features and the expression of specific proteins suggested the progressive acquisition by virtually all fibroblasts of characteristic features of myofibroblasts. Because the transformation did not affect all fibroblasts simultaneously a large heterogeneity in the phenotypes of fibroblasts was present. Secondly, the incidence of mitosis of fibroblasts increased dramatically, affecting the various phenotypes of fibroblasts.\nTransformation of resident fibroblasts to the myofibroblast phenotype\nImmunofluorescence data as well as ultrastructural data showed the acquisition by resident fibroblasts of features of myofibroblasts. In immunofluorescence cortical interstitial fibroblasts were identified in the present study by their expression of 5\u2032NT (Kaissling and Le Hir 1994; Kaissling et al. 1996; Le Hir et al. 2005). Co expression of 5\u2019NT with \u03b1SMA was never observed in interstitial cells of control or contralateral kidneys but appeared already on day 1 after UUO, and became progressively more frequent up to day 4, where nearly all 5\u2019NT-positive fibroblasts expressed more or less strongly \u03b1SMA. Thus, it appears that during the first days of UUO virtually all resident fibroblasts acquired the most characteristic feature of myofibroblats.\nIn the electron microscope the increased size of cisterns of RER suggested an increased production of extracellular matrix proteins by fibroblasts of obstructed kidneys. This also is a feature of the transformation of fibroblasts into myofibroblasts (Desmouliere et al. 2005; Hinz 2007; Hinz et al. 2007). Accordingly, the incidence of collagen fibrils was markedly increased in the interstitium after day 3 and 4\u00a0days of UUO. The presence of intercellular junctions is considered characteristic for myofibroblasts (Powell et al. 1999). It must be stressed that previously junctions were found between interstitial fibroblasts of normal rat kidneys (Kaissling and Le Hir 1994; Kaissling et al. 1996). However, in the present study they were much more easily found in obstructed kidneys compared to control or contralateral kidneys. These collected data suggest that the different shapes and phenotypes of fibroblasts seen in obstructed kidneys are different functional stages of one cell type. The striking differences between neighboring fibroblasts pertaining to the general cell morphology, to the abundance and shape of cisterns of RER and to the expression of 5\u2032NT, \u03b1SMA and vimentin in obstructed kidneys suggest that alterations of fibroblasts following UUO are not attributable only to diffusible factors, for example secreted by infiltrating mononuclear cells. Forces exerted on individual cells might play a role as well. Indeed, a study of uranyl acetate-induced acute renal failure in rats (Fujigaki et al. 2005) suggested that forces exerted on fibroblasts are responsible for expression of \u03b1SMA in fibroblasts around dilated tubules and that under this condition myofibroblasts are derived from the progressive transformation of resident fibroblasts. Transformation of 5\u2032NT-positive fibroblasts to the myofibroblast phenotype with expression of \u03b1SMA and vimentin was observed also around dilated, damaged distal convoluted tubules after treatment with thiazides (Loffing et al. 1996; Le Hir et al. 2005). The role of the tension exerted on stress fibers in the differentiation of myofibroblasts has been discussed recently (Hinz 2007).\nControversial identity of FSP1\/S100A4-expressing cells\nAs found before (Anders et al. 2002; Iwano et al. 2002), in the present study UUO resulted in an increased incidence of FSP1\/S100A4-positive cells in the renal cortical interstitium. In previous studies those cells were considered as fibroblasts\/myofibroblasts (Anders et al. 2002; Iwano et al. 2002). That interpretation is not supported by our observations since we did not find cells double positive for FSP1 and \u03b1SMA or 5\u2032NT in UUO. Similarly, in polycystic kidneys in mice (Okada et al. 2000) and in TGF over-expression-driven fibrosis in mice (Chai et al. 2003) FSP1-positive cells were generally negative for \u03b1SMA. Furthermore in the present study the FSP1-positive cells were generally ovoid with one or two plump cell processes. Finally frequent FSP1-positive cells adhered at the luminal aspect of endothelia. Thus FSP1-positive cells are probably not myofibroblasts but rather infiltrating leukocytes as found in a previous study (Le Hir et al. 2005).\nProliferation of interstitial cells\nThe increased abundance of fibroblasts in the UUO kidneys may be explained by the proliferation of resident cells, with no need for the extra-renal source proposed by others (Broekema et al. 2007; Li et al. 2007). Indeed, a peak of mitotic activity was observed on day 2 in tubular, vascular and interstitial cells. This is in agreement with the time course described in the same model in a previous study (Pat et al. 2005). Duymelinck et al. (Duymelinck et al. 2000) observed a peak in Ki-67-positive cells in \u03b1SMA-positive myofibroblasts in rats at day 5 after UUO. Also the nuclear incorporation (Hughes et al. 1999) of bromo-2\u2032-deoxyuridine in \u03b1SMA-positive interstitial cells in a mouse model of UUO strongly suggested proliferation of myofibroblasts (Hughes et al. 1999). This is clearly confirmed by the detection of mitotic figures in the present study. Mitoses were found in cells with various levels of \u03b1SMA and 5\u2032NT. In the electron microscope some mitotic fibroblasts displayed a normal RER, others an inflated RER. Thus, proliferation was not restricted to a specific stage of differentiation of fibroblasts. The origin of mitotic factors might be infiltrating cells but also tubular cells. The latter produce M-CSF in the UUO model in rats (Isbel et al. 2001).\nChanges of tubular morphology\nA further observation was made, which is highly relevant to the discussion about the role of EMT in renal fibrosis: whereas distal and proximal tubules in the cortex displayed profound regressive and atrophic alterations after ureter ligature, they preserved their epithelial architecture. In other words there was no morphological evidence of EMT during the 4\u00a0days following UUO. The dilatation of distal tubules following UUO has been reported before (Kida and Sato 2007). We found that the major morphological alterations in the proximal tubule are cellular atrophy accompanied by decreased luminal and outer diameters, affecting a majority of, but not all nephrons. The role of atrophic tubules in development of interstitial fibrosis has been pointed out in a rat model for renal failure, induced by disturbance of microcirculation following microsphere injection (Suzuki et al. 2001) and in a model using administration of aristolochic acid (Pozdzik et al. 2008). The signals, implicated in activation, proliferation and transformation of resident fibroblasts to myofibroblasts are among others TGF\u03b2 and PDGF-D, produced and released by damaged tubules (Suzuki et al. 2001; Taneda et al. 2003; Forino et al. 2006; Pozdzik et al. 2008). Tubular shrinkage affected also the distal segments of some nephrons. Virtually identical tubular alterations occurred in nephrons in which urine flow was decreased as a consequence of obstruction of the urinary pole by crescents in models of glomerular diseases (Le Hir and Besse-Eschmann 2003; Kriz and LeHir 2005).\nAlso previous other studies showed that fibrosis developed and myofibroblasts appeared without contribution of EMT in nephritic syndrome in humans (Kuusniemi et al. 2005) and in laboratory animals (Duymelinck et al. 2000; Fujigaki et al. 2005; Pozdzik et al. 2008). A similar conclusion was drawn from a rat model of angiotensin II-induced renal fibrosis (Faulkner et al. 2005) and a murine model (Chai et al. 2003).\nIn other studies FSP1 was detected by immunofluorescence in tubules of obstructed kidneys in animal models of UUO (Iwano et al. 2002; Kida et al. 2007; Sakairi et al. 2007). This finding was interpreted as evidence of EMT. We never detected FSP1 in tubular cells in the present study. It must be noted that in the previous studies tubular immunoreactivity for FSP1 was shown only on day 7 after ureter ligature or later, whereas our study ended on day 4.\nIn conclusion, during the first days following ureter ligature the resident peritubular fibroblasts differentiate into myofibroblasts and they proliferate strongly. These events are likely essential to fibrosis in UUO.","keyphrases":["myofibroblast","fibroblast","fibrosis","kidney","morphology","s100a4","fsp1","epithelial mesenchymal transition (emt)"],"prmu":["P","P","P","P","P","P","P","M"]}
{"id":"Ann_Surg_Oncol-3-1-1914261","title":"Non-Radical Diagnostic Biopsies Do Not Negatively Influence Melanoma Patient Survival\n","text":"Background In fair-skinned Caucasian populations both the incidence and mortality rates of cutaneous melanoma have been increasing over the past decades. With adjuvant therapies still being under investigation, early detection is the only way to improve melanoma patient survival. The influence of incisional biopsies on melanoma patient survival has been discussed for many years. This study investigates both the influence of diagnostic biopsy type and the presence of residual tumor cells in the re-excision specimen on disease free and overall survival.\nIn fair-skinned Caucasian populations cutaneous melanoma is an important growing public health problem, causing a heavy burden on healthcare services. Both its incidence and mortality rates have been increasing in Europe over the past decades.1 The absolute total number of new cases of melanoma in the Netherlands is expected to be more than 4800 in 2015, compared with around 2400 in 2000.2 The Netherlands, as many other countries, has a two-tiered medical care system in which patients need to seek a medical opinion initially from a general practitioner (GP) before referral if necessary, to a specialist.\nThe mean number needed to treat (NNT), defined as the mean number of pigmented lesions needed to be excised to identify one melanoma, among 468 GPs in Perth, Australia, was 29 and ranged from 83 in the youngest patients (\u226419 years) to 11 in the oldest patients (\u226570 years).3 Assuming that for each new case of melanoma another 20\u201350 patients with pigmented skin lesions will visit the GP, the demand for detection will increase quite markedly. With adjuvant therapies still being under investigation, early detection is the only way to improve melanoma patient survival.4 Dutch guidelines recommend pigmented skin lesions suspect for melanoma to be removed through a diagnostic excision biopsy with a minimal lateral clearance of 2 mm.5 This recommendation is inline with the advice of the National Institutes of Health (NIH) to remove any suspicious lesion through excisional biopsy with a narrow margin of normal-appearing skin.6 The influence of an incisional biopsy on melanoma patient survival has been discussed for many years and different investigators have found contradicting results.7\u201315 Two recent publications concluding incisional biopsies not to interfere with melanoma patient survival were not able to end this discussion since patient groups were not fully comparable16 or follow up (FU) was short.17\nThis study investigated both the influence of diagnostic biopsy type and the presence of residual tumor cells in the re-excision specimen on melanoma patient disease free survival (DFS) and overall survival (OS), in 471 patients with a mean FU of more than 5 years. Survival analysis was done using Cox\u2019s proportional hazard model adjusted for; gender, age, site of primary melanoma, Breslow thickness, type of melanoma, ulceration, lymphatic invasion and sentinel node (SN) status. Both the diagnostic biopsy type and the presence of tumor cells in the re-excision specimen were found not to influence melanoma patient survival.\nMETHODS\nPatients\nBetween August 1993 and September 2004, 551 patients were diagnosed with clinical stage I\/II cutaneous melanoma according to criteria of the American Joint Committee on Cancer (AJCC) and underwent re-excision of the primary melanoma site and a SN biopsy. If patients were referred to us from another institution the pathologic characteristics of the primary melanoma were reviewed in our hospital before the SN procedure. All patients were treated according to the same protocol; re-excision margins were 1 cm for melanomas with a Breslow thickness of\u00a0\u2264\u00a02 mm and 2 cm for melanomas with a Breslow thickness of >2 mm. To identify and retrieve the SN, the triple technique was used as described previously.18\u201320 In short, the day before surgery patients underwent a dynamic and static lymphoscintigraphy to determine the lymphatic drainage pattern. Just prior to surgery, Patent Blue V (Laboratoire Guerbet, Aulnay-sous-Bois, France) was injected intradermally next to the initial site of the melanoma. During surgery, guided by a hand held gamma probe and the blue staining of the draining tissues, the SN was removed.\nTo investigate the influence on survival, patients were divided both according to their diagnostic biopsy type; wide excisional biopsy (lateral clearance\u00a0\u2265\u00a02 mm), narrow excisional biopsy (lateral clearance\u00a0<\u00a02 mm), excisional biopsy with positive margins and incisional biopsy (includes punch) and the presence of residual tumor cells in their re-excision specimen.\nStatistical Analysis\nData were processed with the Statistical Package for the Social Sciences software for Windows 2000 (SPSS 11.5, Chicago, IL). Cox\u2019s proportional hazard model was used for survival analysis. P values <0.05 were considered significant.\nRESULTS\nPatient Population\nBetween August 1993 and September 2004, 551 patients were diagnosed with clinical stage I\/II cutaneous melanoma, 257 male (46.6%) and 294 female (53.4%) with a mean age of 49.9 years (Table 1). Most primary melanomas were located on the trunk (43.7%) or on the lower extremities (36.7%). Breslow thickness was categorized into four groups (\u00a0\u2264\u00a01.00 mm; 1.01\u20132.00 mm; 2.01\u20134.00 mm; >4.01 mm), but due to spontaneous regression of the primary lesion remained unknown in 38 patients. The majority of patients had a superficial spreading melanoma (65.0%) or a nodular melanoma (26.7%). In 46 patients the type of melanoma was different or remained unknown (8.3%). Ulceration, defined as the absence of intact epidermis overlying the major portion of primary melanoma, was diagnosed in 80 patients (14.5%), unknown in 1 patient (0.2%) and absent in 470 patients (85.3%). Lymphatic invasion was present in 25 patients (4.5%), absent in 521 patients (94.6%) and remained unknown in 5 patients (0.9%). The SN was negative in 446 patients (80.9%) and positive in 94 patients (17.1%). In 11 patients the SN was not removed and the SN status remained unknown (2.0%). In total, there were 101 missing variables in 80 patients; all were excluded from the study.\nSN Identification\nIn 11 of the 551 patients the SN status remained unknown (2.0%), in 5 of these patients the SN was located in the deep lobe of the parotid gland and in one patient the SN was located high in the left axilla, in all cases the decision was made not to remove the SN to avoid potential morbidity associated with the intervention. The SN was not identified in 3 cases due to non-visualization by preoperative lymphoscintigraphy. In one patient the SN was located in the right axilla and could not be removed because the patient was suffering from frozen shoulder syndrome, the physical condition of another patient did not allow further treatment. Therefore, the success rate of SN identification was 98% (540 of 551 patients). Two of the patients with the SN located in the deep lobe of the parotid gland experienced metastasis of the parotid gland, one patient is still alive with disease and one patient is dead of disease. The patient whose physical condition did not allow further treatment, passed away soon after re-excision of the primary melanoma site, from massive hematogenic and lymphogenic metastasis. The 8 remaining patients have shown no evidence of disease.\nDiagnostic Biopsy Type and Survival\nThe influence of diagnostic biopsy type on DFS and OS was tested in 471 patients with a mean FU of more than 5 years; 279 patients (59.3%) underwent a wide excision biopsy, 109 patients (23.1%) a narrow excision biopsy, 52 patients (11.0%) an excision biopsy with positive margins and 31 patients (6.6%) an incision biopsy (Table\u00a02A).\nTABLE\u00a01.Patient characteristicsCharacteristicsPatients (n\u00a0=\u00a0551)Follow up (years)\u00a0\u00a0Mean (SD)5.1 (2.8)Gender\u00a0\u00a0Male257 (46.6%)\u00a0\u00a0Female294 (53.4%)Age (years)\u00a0\u00a0Mean (SD)49.9 (15.3)Site of primary melanoma\u00a0\u00a0Lower extremity202 (36.7%)\u00a0\u00a0Upper extremity 63 (11.4%)\u00a0\u00a0Head\/Neck45 (8.2%)\u00a0\u00a0Trunk241 (43.7%)Breslow thickness (mm)\u00a0\u00a00\u00a0<\u00a0x\u00a0\u2264\u00a01153 (27.8%)\u00a0\u00a01\u00a0<\u00a0x\u00a0\u2264\u00a02207 (37.6%)\u00a0\u00a02\u00a0<\u00a0x\u00a0\u2264\u00a04114 (20.7%)\u00a0\u00a0> 439 (7.1%)\u00a0\u00a0Unknown (regression)38 (6.9%)Type of melanoma\u00a0\u00a0Superficial spreading358 (65.0%)\u00a0\u00a0Nodular147 (26.7%)\u00a0\u00a0Other\/Unknown46 (8.3%)Ulceration\u00a0\u00a0No470 (85.3%)\u00a0\u00a0Yes80 (14.5%)\u00a0\u00a0Unknown1 (0.2%)Lymphatic invasion\u00a0\u00a0No521 (94.6%)\u00a0\u00a0Yes25 (4.5%)\u00a0\u00a0Unknown5 (0.9%)Sentinel node status\u00a0\u00a0Negative446 (80.9%)\u00a0\u00a0Positive94 (17.1%)\u00a0\u00a0Unknown11 (2.0%)TABLE\u00a02.Patient distribution according to (A) diagnostic biopsy type and (B) the presence of residual tumor cells in the re-excision specimenAPatients (n\u00a0=\u00a0471)Follow up (years; mean\u00a0\u00b1\u00a0SD)Diagnostic biopsy type\u00a0\u00a0Wide excision biopsy (\u00a0\u2265\u00a02 mm)279 (59.3%)5.0\u00a0\u00b1\u00a03.0\u00a0\u00a0Narrow excision biopsy (0<x<2 mm)109 (23.1%)6.0\u00a0\u00b1\u00a02.7\u00a0\u00a0Excision biopsy with positive margins52 (11.0%)5.8\u00a0\u00b1\u00a02.9\u00a0\u00a0Incision biopsy31 (6.6%)5.4\u00a0\u00b1\u00a02.4BPatients (n\u00a0=\u00a0441)Follow up (years; mean\u00a0\u00b1\u00a0SD)Re excision specimen\u00a0\u00a0No residual tumor cells400 (90.7%)5.1\u00a0\u00b1\u00a02.9\u00a0\u00a0Residual tumor cells41 (9.3%)5.8\u00a0\u00b1\u00a02.6\nIn 91\/471 patients (19.3%) the SN was positive, 58\/279 patients (20.8%) after a wide excision biopsy, 14\/109 patients (12.8%) after a narrow excision biopsy, 15\/52 patients (28.8%) after an excision biopsy with positive margins and 4\/31 patients (12.9%) after an incisional biopsy.\nIn 79\/471 patients (16.8%) a recurrence was found during FU, 45\/279 patients (16.1%) after a wide excision biopsy (21 locoregional skin, 8 SN basin and 16 systemic), 17\/109 patients (15.5%) after a narrow excision biopsy (7 locoregional skin, 2 SN basin and 8 systemic), 10\/52 patients (19.2%) after an excision biopsy with positive margins (5 locoregional skin, 2 SN basin and 3 systemic) and 7\/31 patients (22.6%) after an incision biopsy (5 locoregional skin, and 2 systemic). But confounding factors were not equally distributed between the diagnostic biopsy groups. Therefore, survival analysis was done using Cox\u2019s proportional hazard model adjusted for; gender, age, site of primary melanoma, Breslow thickness, type of melanoma, ulceration, lymphatic invasion and sentinel node (SN) status.\nIn univariate analysis; gender, age, Breslow thickness, type of melanoma, ulceration, lymphatic invasion and SN status were all significantly related to both DFS and OS (Table\u00a03). Site of primary melanoma on the head\/neck was in univariate analysis only significantly related to DFS (Table\u00a03). Diagnostic biopsy type did not have a significant relation with either DFS or OS (Table\u00a03).\nTABLE\u00a03.Univariate and multivariate Cox regression analysis of disease-free survival and overall survival according to diagnostic biopsy typeDisease Free SurvivalOverall SurvivalUnivariateMultivariateUnivariateMultivariateTested variablesPHR95% CIPPHR95% CIPDiagnostic biopsy type\u00a0\u00a0Wide excision biopsy0.6911.00\u20130.2040.5001.00\u20130.765\u00a0\u00a0Narrow excision biopsy0.3600.710.39\u20131.280.2550.3150.740.37\u20131.510.411\u00a0\u00a0Excision biopsy with positive margins0.8700.590.29\u20131.180.1320.7480.750.34\u20131.650.476\u00a0\u00a0Incision biopsy0.5810.470.19\u20131.160.1010.3870.740.29\u20131.890.530Gender0.0181.150.70\u20131.890.5730.0041.440.81\u20132.570.215Age (years)0.0041.021.00\u20131.040.0180.0021.031.01\u20131.050.007Site of primary melanoma\u00a0\u00a0Lower extremity0.0911.00\u20130.0230.153\u00a0\u00a0Upper extremity0.8250.910.41\u20132.010.8090.071\u00a0\u00a0Head\/Neck0.0143.401.45\u20137.970.0050.095\u00a0\u00a0Trunk0.7310.930.54\u20131.610.7900.063Breslow thickness (mm)\u00a0\u00a00\u00a0<\u00a0x\u00a0\u2264\u00a01<0.0011.00\u2013<0.001<0.0011.00\u20130.001\u00a0\u00a01\u00a0<\u00a0x\u00a0\u2264\u00a020.00313.191.77\u201398.410.0120.0168.151.07\u201362.190.043\u00a0\u00a02\u00a0<\u00a0x\u00a0\u2264\u00a04<0.00134.114.50\u2013258.600.001<0.00121.432.79\u2013164.840.003\u00a0\u00a0> 4<0.00115.731.86\u2013132.810.011<0.00111.851.35\u2013103.700.025\u00a0\u00a0Type of melanoma<0.0011.580.95\u20132.620.078<0.0011.570.88\u20132.790.128\u00a0\u00a0Ulceration<0.0011.831.09\u20133.070.023<0.0011.640.89\u20133.040.116\u00a0\u00a0Lymphatic invasion<0.0013.982.05\u20137.72<0.001<0.0012.191.07\u20134.480.032\u00a0\u00a0Sentinel node status<0.0013.872.23\u20136.70<0.001<0.0013.191.75\u20135.81<0.001\nEven though, diagnostic biopsy type did not have a significant relation with either DFS or OS in univariate Cox regression analysis it was also tested in multivariate Cox regression analysis together with all significant variables from univariate analysis, to investigate its influence on DFS and OS after correction for the confounding factors. In multivariate analysis, the only significant and independent predictors of both DFS and OS were; age, Breslow thickness, lymphatic invasion and SN status (Table\u00a03). Site of primary melanoma on the lower extremities or head\/neck and ulceration were in multivariate analysis only significant and independent predictors of DFS (Table\u00a03). Also in multivariate analysis, diagnostic biopsy type did not have a significant relation with either DFS or OS (Fig.\u00a01A, B).\nFIG.\u00a01.A Disease free survival rates and B overall survival rates according to the initial biopsy type.\nThe same analysis was done after combining the groups; the wide excision biopsy group was joined with the narrow excision biopsy group and compared to the excision biopsy group with positive margins joined with the incision biopsy group. Still, diagnostic biopsy type did not have a significant influence on either DFS or OS (data not shown).\nResidual Tumor Cells in the Re-Excision Specimen and Survival\n441\/471 Patients (93.6%) underwent re-excision of the primary melanoma site and in 30 patients (6.4%) the diagnostic biopsy was found to be sufficient. In 41\/441 patients (9.3%) residual tumor cells were found in the re-excision specimen (Table\u00a02B). All 41 patients with residual tumor cells in the re-excision specimen underwent either an excision biopsy with positive margins or an incision biopsy. In none of the patients with a wide or narrow excision biopsy residual tumor cells were found.\nIn univariate analysis; gender, age, Breslow thickness, type of melanoma, ulceration, lymphatic invasion and SN status were all significantly related to both DFS and OS (Table\u00a04). Site of primary melanoma on the lower extremity or head\/neck was in univariate analysis significantly related to DFS and site of primary melanoma on the head\/neck or trunk was significantly related to OS (Table\u00a04). Residual tumor cells in the re-excision specimen did not have a significant relation with either DFS or OS (Table\u00a04).\nTABLE\u00a04.Univariate and multivariate Cox regression analysis of disease-free survival and overall survival according to the presence of residual tumor cells in the re-excision specimenDisease Free SurvivalOverall SurvivalUnivariateMultivariateUnivariateMultivariateTested variablesPHR95% CIPPHR95% CIPResidual tumor cells0.0940.790.38\u20131.640.5320.2300.830.36\u20131.920.668Gender0.0251.140.68\u20131.920.6110.0091.220.65\u20132.280.531Age (years)0.0071.021.00\u20131.040.0380.0071.021.00\u20131.040.040Site of primary melanoma\u00a0\u00a0Lower extremity0.0321.00\u20130.0330.1051.00\u20130.123\u00a0\u00a0Upper extremity0.8300.950.41\u20132.200.9100.0692.210.85\u20135.710.102\u00a0\u00a0Head\/Neck0.0043.221.35\u20137.660.0080.0403.221.03\u201310.090.045\u00a0\u00a0Trunk0.6470.960.54\u20131.740.9030.0492.020.98\u20134.200.059Breslow thickness (mm)\u00a0\u00a00\u00a0<\u00a0x\u00a0\u2264\u00a01<0.0011.00\u2013<0.001<0.0011.00\u20130.004\u00a0\u00a01\u00a0<\u00a0x\u00a0\u2264\u00a020.00512.821.71\u201396.070.0130.0207.991.04\u201361.490.046\u00a0\u00a02\u00a0<\u00a0x\u00a0\u2264\u00a04<0.00130.163.97\u2013229.220.001<0.00120.132.59\u2013156.250.004\u00a0\u00a0> 4<0.00115.911.86\u2013136.250.012<0.00111.841.32\u2013106.540.028\u00a0\u00a0Type of melanoma<0.0011.610.95\u20132.730.076<0.0011.630.87\u20133.040.128\u00a0\u00a0Ulceration<0.0011.550.90\u20132.670.119<0.0011.610.85\u20133.050.141\u00a0\u00a0Lymphatic invasion<0.0014.162.13\u20138.14<0.001<0.0012.241.04\u20134.800.038\u00a0\u00a0Sentinel node status<0.0013.301.87\u20135.83<0.001<0.0013.051.56\u20135.960.001\nEven though, residual tumor cells in the re-excision specimen did not have a significant relation with either DFS or OS in univariate Cox regression analysis it was also tested in multivariate Cox regression analysis together with all significant variables from univariate analysis, to investigate its influence on DFS and OS after correction for the confounding factors. In multivariate analysis, the only significant and independent predictors of both DFS and OS were; age, site of primary melanoma on the head\/neck, Breslow thickness, lymphatic invasion and SN status (Table\u00a04). Also in multivariate analysis, residual tumor cells in the re-excision specimen did not have a significant relation with either DFS or OS (Fig.\u00a02A, B).\nFIG.\u00a02.A Disease free survival rates and B overall survival rates according to the presence of residual tumor cells in the re-excision specimen.\nConsistent Confounders of Melanoma Patient Survival\nAge, Breslow thickness, lymphatic invasion and SN status were the most consistent, independent and significant confounders of melanoma patient DFS and OS (Table\u00a03, 4). Site of the primary melanoma was not always an independent and significant confounder of melanoma patient survival, but location on the head\/neck region consistently carried the highest hazard ratio (HR) (Table\u00a03, 4). Surprisingly, ulceration was not an independent and significant confounder of OS (Table\u00a03, 4).\nDISCUSSION\nNumerous investigators have studied the influence of incisional biopsy on melanoma patient survival and found contradicting results. Fitzpatrick et al. found a five-year OS rate of 30% in the incisional biopsy group as compared to 48% in the excisional biopsy group but the different biopsy groups were not matched for important prognostic factors.8 Epstein et al. found a more favorable ten-year OS in the biopsy group (65%) as compared to the primary wide excision group (56%) but biopsy was loosely defined as less than optimal or complete surgical excision.9 Ironside et al. found a five-year OS rate of 66% in the excision- and 43% in the incision biopsy group but failed to describe the distribution of prognostic factors between both groups.10 Rampen et al. also found a worse prognosis for patients after an incision biopsy (14 patients) but the study was small and retrospective.11 Griffiths et al. found no difference in survival but 7\/19 incisional biopsy patients were excluded because of missing histopathological data.12 Survival rates were not significantly different between the incision- and excision biopsy groups of Lederman et al., but patient groups were matched for Breslow thickness only.13 Lees et al. also indicated no significant averse effect of incisional biopsy in 96 patients, but 40% of the histopathological data was not assessable.14 Austin et al did find a significantly reduced survival in the incisional biopsy group, but patients in the incisional biopsy group were also significantly older.15 The two most recent publications found no negative influence of incisional biopsies on melanoma patient survival but in Bong et al. patients groups were not fully matched and in Martin et al. median FU was only 28 months.16,17\nThis study not only investigated the influence of diagnostic biopsy type but also the presence of residual tumor cells in the re-excision specimen on melanoma patient survival in patient groups adjusted for 8 important confounders of survival with a mean FU\u00a0>\u00a05 years. Excision biopsies with positive margins and incisional biopsies were found not to influence melanoma patient survival. Interestingly, DFS and OS even seemed slightly better in the non-radical biopsy groups (Fig.\u00a01A, B). In line with this, patients with residual tumor cells in their re-excision specimen also had a slightly better survival as compared to patients without residual tumor cells in their re-excision specimen (Fig.\u00a02A, B). Melanoma is the most immunogenic tumor identified to date; melanoma specific T cells are detectable both in the blood and in tumor draining lymph nodes from melanoma patients and their frequency can be increased by specific vaccination.21\u201323 This intrinsic immunogenicity makes melanoma lesions particularly amenable to therapeutic approaches aimed at strengthening tumor immune surveillance. Did residual tumor cells combined with biopsy induced wound healing trigger the melanoma patient\u2019s immunesystem? In non-melanoma skin cancer (NMSC) biopsy induced tumor regression has previously been described, Swetter et al reported that 24% of NMSCs transected on the initial biopsy showed no residual tumor in the excision specimens and suggested biopsy induced wound healing to play an important role.24\nEven though non-radical diagnostic biopsies and residual tumor cells in the re-excision specimen do not have a negative influence on melanoma patient survival, the routine use of incision biopsies is not recommended. Incisional biopsies often consist only of a small percentage of the surface area of the pigmented skin lesion making it difficult to sample a representative area within the tumor. Somach et al found that 40% of the histopathological features were more pronounced in the re-excision specimen as compared to the incision biopsy and in 20% areas of invasive melanoma not detected in the incisional biopsy were observed in the re-excision specimen.25 Further more when melanoma is diagnosed, attempting to evaluate the depth of invasion in an incisional biopsy is treacherous and may lead to over- or underestimation of the invasion.26,27 Of course these problems are less prominent in excision biopsies with positive margins, here the majority of the lesion has been removed and only the outer borders are compromised making a sampling error highly unlikely.\nIn conclusion; age, Breslow thickness, lymphatic invasion and SN status were the most consistent, independent and significant confounders of melanoma patient DFS and OS. The site of primary melanoma and ulceration were also important confounders of survival. Both the diagnostic biopsy type and the presence of residual tumor cells in the re-excision specimen did not have a negative influence on melanoma patient DFS and OS. With melanoma incidence rates rising1 and early detection of melanoma still being the only way to improve melanoma patient survival,4 it is important for all physicians to feel confident about removing a pigmented skin lesion suspect for melanoma. Incisional biopsies are not recommended but there is no cause for concern when an excision biopsy turns out to have positive margins.","keyphrases":["diagnostic","melanoma","survival","initial","re-excision and biopsy"],"prmu":["P","P","P","P","R"]}
{"id":"Exp_Brain_Res-3-1-1914280","title":"Auditory grouping occurs prior to intersensory pairing: evidence from temporal ventriloquism\n","text":"The authors examined how principles of auditory grouping relate to intersensory pairing. Two sounds that normally enhance sensitivity on a visual temporal order judgement task (i.e. temporal ventriloquism) were embedded in a sequence of flanker sounds which either had the same or different frequency (Exp. 1), rhythm (Exp. 2), or location (Exp. 3). In all experiments, we found that temporal ventriloquism only occurred when the two capture sounds differed from the flankers, demonstrating that grouping of the sounds in the auditory stream took priority over intersensory pairing. By combining principles of auditory grouping with intersensory pairing, we demonstrate that capture sounds were, counter-intuitively, more effective when their locations differed from that of the lights rather than when they came from the same position as the lights.\nIntroduction\nSense organs like the ears and eyes are continuously bombarded with information. Yet, observers perceive distinct objects or events. The way information is assigned to objects has, for vision, been described with Gestalt principles like \u2018similarity\u2019, \u2018good continuation\u2019, or \u2018common fate\u2019, and similar principles have also been discovered for audition (Bregman 1990). It occurs, for instance, when a sequence of alternating high- and low-frequency tones is played at a certain rate. When the frequency difference between the tones is small, listeners group the tones into a single stream, but at bigger frequency differences, the sequence splits into two streams, one high and one low in pitch. Typically, these grouping principles apply within a single modality like vision or audition. However, sense organs not only work in isolation, they also have to cooperate to form a coherent multisensory representation of the environment. The notion on how information from different sense organs is assigned to a multisensory event is usually referred to as the \u2018assumption of unity\u2019. It states that as events from different modalities share more amodal properties, in particular space and time, it is more likely that they originate from a common object or source (e.g. Welch and Warren 1980; Bedford 1989; Stein and Meredith 1993; Radeau 1994; Bertelson 1999; Welch 1999). Following this notion, the assignment of information from different modalities to a single multisensory event will be reduced or absent when stimuli are too far apart in space or time, because in that case two objects or events will be perceived rather than a single multimodal one.\nHere, we explored how principles of auditory grouping relate to intersensory pairing. Previous work on this topic suggests that auditory grouping may take priority over intersensory pairing. For example, Vroomen and de Gelder (2000) used a task in which participants had to detect a visual target in a rapidly changing sequence of visual distracters. They observed that a high tone embedded in a sequence of low tones enhanced detectability of a synchronously presented visual target. There was no such intersensory enhancement when the tone was embedded in a sequence of tones with the same frequency or when the tone was part of a melody. The cross-modal enhancement thus only occurred when the sound segregated from the sound sequence in which it was embedded. Similar results were obtained by Watanabe and Shimojo (2001). They explored how the \u2018bounce illusion\u2019 is affected by contextual auditory information. The bounce illusion is a cross-modal phenomenon in which a \u2018collision\u2019 sound presented near the crossover of two moving balls enhances the perception of the balls \u2018bouncing\u2019, whereas the absence of the sound results in a \u2018streaming\u2019 percept. The authors showed a reduction of the bounce illusion when the sounds were embedded in a sequence of similar sounds, as opposed to when the sounds were flanked by sounds of a different frequency.\nHere we tested the generality of these findings by examining how auditory grouping affects auditory\u2013visual (AV) pairing in the temporal domain using the so-called temporal ventriloquist effect (Scheier et\u00a0al. 1999; Fendrich and Corballis 2001; Aschersleben and Bertelson 2003; Bertelson and Aschersleben 2003; Morein-Zamir et\u00a0al. 2003; Vroomen and de Gelder 2004; Stekelenburg and Vroomen 2005; Vroomen and Keetels 2006). Temporal ventriloquism refers to the phenomenon that when a sound and light are presented at slightly different onset times (usually in the order of \u223c100\u00a0ms), the sound will attract the temporal occurrence of the light. This phenomenon can be demonstrated in a visual temporal order judgment (TOJ) task in which participants are presented two lights at various stimulus onset asynchronies (SOAs) and judge, which came first. By presenting a sound before the first and after the second light, the just noticeable difference (JND) improves (i.e., participants become more sensitive), presumably because the two sounds attract the temporal occurrence of the two lights, and thus effectively pull the lights further apart in time (Scheier et\u00a0al. 1999; Morein-Zamir et\u00a0al. 2003; Vroomen and Keetels 2006). Judgments about which light came first are therefore more accurate if there is a \u223c100\u00a0ms interval between the sounds and lights rather than when the sounds are presented simultaneously with the lights.\nHere we asked what happens if the sounds that capture the onset of the lights are assigned to a stream of other sounds with which they form a well-formed sequence. If auditory grouping takes priority over intersensory pairing, one expects an improvement on the visual TOJ task only if the capture sounds segregate from the auditory stream. Alternatively, though, audio\u2013visual pairing might take priority over auditory grouping in which case observers should improve on the visual TOJ task no matter whether the sounds segregate or not.\nIn Experiment 1, these predictions were tested with two capture sounds that were embedded in a sequence of flanker sounds, which either had the same or a different frequency. When the frequency of the flanker and the capture sounds were the same, the sequence was heard as a single stream which, following previous findings (Vroomen and de Gelder 2000; Watanabe and Shimojo 2001), should prevent temporal ventriloquism to occur. When the flankers differed from the capture sounds, stream segregation was more likely to occur in which case the two sounds could possibly interact with the lights and thus improve performance on the visual TOJ task. Other stream segregation cues besides the frequency of the flanker and capture sounds were further explored in Experiment 2 (rhythm) and Experiment 3 (sound location).\nExperiment 1: Capture and flanker sounds with the same or different frequency\nParticipants performed a visual TOJ task in which they decided which of two lights appeared first. Two task-irrelevant high tones were presented either simultaneously with the lights (i.e., the \u223c0\u00a0ms AV interval), or the first tone was presented \u223c100\u00a0ms before the first light and the second tone \u223c100\u00a0ms after the second light (i.e., the \u223c100\u00a0ms AV interval). The two high tones were embedded in a sequence of other tones, which either had the same (high) or a different (low) frequency (see Fig.\u00a01a for a schematic overview of the conditions).\nFig.\u00a01a A schematic illustration of a trial. Lights were presented with a particular SOA ranging between \u221275 and 75\u00a0ms, with negative values indicating that the lower light was presented first. Two capture sounds were presented either simultaneously with the lights (\u223c0\u00a0ms AV-interval), or \u223c100\u00a0ms before the first and \u223c100\u00a0ms after the second light (\u223c100\u00a0ms AV-interval). The capture sounds were embedded in a sequence of flanker sounds which either had the same or a different frequency (Exp1), rhythm (Exp 2), or location (Exp 3). Perceptual grouping of the sounds is illustrated by the grey ovals. Temporal ventriloquism only occurred when the capture sounds were different from the flanker sounds at the \u223c100\u00a0ms interval, as illustrated on the second line. b Schematic set-up of Experiment 1, 2 and 3\nMethod\nParticipants Thirteen students from Tilburg University were given course credits for their participation. All reported normal hearing and normal or corrected-to-normal seeing. They were tested individually and were unaware of the purpose of the experiment. The experimental procedures were approved by the Institute and were in accordance with Declaration of Helsinki.\nStimuli Two auditory stimuli, a low (1,500\u00a0Hz) and a high (3,430\u00a0Hz) pure tone of 3\u00a0ms at 72\u00a0dB(A) were used that clearly differed in pitch. The sounds were presented via a hidden loudspeaker located at eye-level, at central location and at 90\u00a0cm distance. Visual stimuli were presented by two red LEDs (diameter of 1\u00a0cm, luminance of 40\u00a0cd\/m2), positioned 5\u00b0 below and above the central loudspeaker. A small green LED was placed at the center of the loudspeaker and served as a fixation point (see Fig.\u00a01b for a schematic set-up). Trials in the \u223c0\u00a0ms AV interval consisted of a sound sequence of 40 sounds in which the interval between the successive tones was equal to the SOA between the two lights. The lights were presented simultaneously with the 25th and 26th sound. Trials in the \u223c100\u00a0ms AV-interval consisted of a tone sequence of 15 sounds with the interval between the tones equal to the SOA of the lights plus 200\u00a0ms. The two lights were presented in the middle of the temporal gap, \u223c100\u00a0ms after the 10th and \u223c100\u00a0ms before the 11th sound.\nDesign The experiment had three within-subjects factors: Frequency of the flanker sounds (same or different frequency as the capture sounds), the AV-interval between the capture sounds and the lights (\u223c0 or \u223c100\u00a0ms), and the SOA between the two lights (\u221275, \u221260, \u221245, \u221230, \u221215, +15, +30, +45, +60, and +75\u00a0ms; with negative values indicating that the lower light was presented first). These factors yielded 40 equi-probable conditions, each presented 20 times for a total of 800 trials (10 blocks of 80 trials each).\nProcedure Participants sat at a table in a dimly lit and soundproof booth. The fixation light was illuminated at the beginning of the experiment, and participants were instructed to maintain fixation on this central green LED during testing. The participant\u2019s task was to judge whether the lower or the upper LED was presented first. Responses (unspeeded) were made by pressing one of two designated keys with the right thumb (lower light first) or right index (upper light first). Whenever a response was detected, both LEDs were turned off and the next trial started after 2,000\u00a0ms. A practice block was included consisting of 16 trials in which the four longest SOAs were presented once in each condition. During practice, participants received verbal feedback (\u201cCorrect\u201d or \u201cWrong\u201d).\nResults and discussion\nTrials of the practice session were excluded from analyses. The proportion of \u2018up-first\u2019 responses was calculated for each condition and converted into equivalent Z-scores assuming a cumulative normal distribution (cf. Finney 1964). For each of the four conditions, the best-fitting straight line was calculated over the ten SOAs. The lines\u2019 slopes and intercepts were used to determine the point of subjective simultaneity (PSS) and the just noticeable difference (JND\u00a0=\u00a00.675\/slope). The PSS represents the average interval by which the upper stimulus had to lead the lower one in order to be perceived as simultaneous. The JND represents the smallest interval between the onsets of the two lights needed for participants to correctly judge which stimulus had been presented first on 75% of the trials. Temporal ventriloquism was measured by subtracting the JND in the \u223c100\u00a0ms AV interval from the \u223c0\u00a0ms AV interval (see Table\u00a01 for the average JNDs).\nTable\u00a01Mean just noticeable differences (JND) in ms, and standard errors of the mean (in parentheses) of Experiment 1 and 2Flanker soundsSame as capture soundsDifferent from capture soundsExperiment AV-interval (ms)JNDTVEJNDTVEExp 1 (Frequency)021.0 (0.8)0.522.1 (1.1)3.8*10020.5 (1.1)18.3 (0.7)Exp 2 (Rhythm)029.1 (2.5)\u22123.723.9 (1.9)3.6*10032.8 (2.5)20.3 (0.8)Capture Sounds Presented at \u223c0 or \u223c100\u00a0ms audio\u2013visual intervals; flanker Sounds with the same or different frequency (Exp 1) or rhythm (Exp 2) as capture sounds. The temporal ventriloquist effect (TVE) is the improvement in JND between the \u223c0 and \u223c100\u00a0ms audio\u2013visual intervals*P\u00a0<\u00a00.05\nA 2\u00a0\u00d7\u00a02 ANOVA with as within-subjects factors the frequency of the flanker sounds (same or different frequency as the capturer sounds) and the AV-interval (\u223c0 and \u223c100\u00a0ms) was conducted on the JNDs and PSSs. In the ANOVA on the PSSs, no effect was significant (all P\u2019s\u00a0>\u00a00.30), which is in line with our expectations since no shift towards more \u2018up\u2019 or \u2018down\u2019 responses was expected. In the ANOVA on the JNDs, the important interaction between the AV-interval and the frequency of the flanker sounds was significant, F(1, 12)\u00a0=\u00a04.90, P\u00a0<\u00a00.05. Separate t tests comparing the \u223c0\u00a0ms AV interval with the \u223c100\u00a0ms AV interval showed that JNDs improved by 3.8\u00a0ms when the frequency of the capture and flanker sounds differed, t(12)\u00a0=\u00a03.06, P\u00a0<\u00a00.01, but no significant difference was obtained (0.5\u00a0ms) when the capture and flanker sounds were the same, t(12)\u00a0=\u00a00.71, P\u00a0=\u00a00.49. As predicted, temporal ventriloquism thus only occurred when the capture and flanker sounds differed. It seems therefore likely that segregation of the capture sounds from the flankers was necessary before the capture sounds could interact with the lights. When the capture and flanker sounds were the same, auditory grouping thus took priority over AV pairing.\nExperiment 2: Capture and flanker sounds with the same or different rhythm\nTo further explore the relation between auditory grouping and intersensory pairing, we presented the capture sounds in or out of rhythm with the flankers. Rhythm is, besides frequency, another important auditory segregation cue (Bregman 1990). It was expected that sounds presented out of rhythm would segregate from the sound sequence, thus enhancing performance on the visual TOJ task. Capture sounds presented in rhythm should not segregate from the auditory stream, and they should thus have no effect on the visual TOJ task.\nMethod\nParticipants Twenty new students participated.\nStimuli and design Stimuli and design were as in Experiment 1, except that the time interval between the capture and flanker sounds was varied rather than their frequency. The auditory stimuli consisted of 5\u00a0m\u00a0s sound bursts presented at 72\u00a0dB(A). When the capture sounds were presented in rhythm with the flankers, the same interval between consecutive sounds in the sequence was used as in Experiment 1 (i.e., SOA between the two lights\u00a0+\u00a02\u00a0\u00d7\u00a0AV-interval). When the capture sounds were presented out of rhythm, the interval between the capture and flanker sounds was increased, such that there was a short pause before the first and after the second capture sound. In the \u223c0\u00a0ms AV-interval condition, the two longer intervals were 8\u00a0\u00d7\u00a0SOA of the lights, in the \u223c100\u00a0ms AV-interval condition they were 17\u00a0\u00d7\u00a0SOA of the lights +200\u00a0ms.\nResults and discussion\nIn the 2 (Capture sounds in or out of rhythm)\u00a0\u00d7\u00a02 (AV-interval \u223c0 or \u223c100\u00a0ms) ANOVA on the PSSs, no effect was significant (all P\u2019s\u00a0>\u00a00.30). The same ANOVA on the JNDs showed that the important interaction between AV-interval and rhythm was significant, F(1, 19)\u00a0=\u00a015.35, P\u00a0<\u00a00.001. Separate t tests showed that the 3.6\u00a0ms temporal ventriloquist effect (lower JNDs in the \u223c100\u00a0ms AV interval rather than \u223c0\u00a0ms) of sounds presented out of rhythm was significant, t(19)\u00a0=\u00a02.37, P\u00a0<\u00a00.05. Performance got actually worse (\u22123.7\u00a0ms) when the capture sounds were presented in the same rhythm as the flanker sounds, t(19)\u00a0=\u00a0\u22122.15, P\u00a0<\u00a00.05. Capture sounds thus again only improved performance on the visual TOJ task when they segregated from the flanker sounds.\nExperiment 3: Capture and flanker sounds from the same or different location\nIt is known that auditory stream segregation may also occur when there is a difference in the location of consecutive sounds (Bregman 1990). In Experiment 3, we therefore varied the location of the capture and flanker sounds. The sounds could emanate either from a central loudspeaker near the two lights, or from a lateral loudspeaker on the far left or far right. If intersensory pairing only occurs when the capture sounds segregate from the flankers, then temporal ventriloquism should be obtained when the locations of the capture and flanker sound differ, but not when they are the same.\nThis set-up also allowed us to explore whether spatial disparity between the capture sounds and lights affects intersensory pairing. The common notion on intersensory pairing states that commonality in space between the auditory and visual signal matters. However, in contrast with this notion, it has recently been shown that temporal ventriloquism may not be affected by spatial discordance between the sounds and lights. In a study by Vroomen and Keetels (2006), it was shown that there were equal amounts of temporal ventriloquism when the two capture sounds came from the same or a different position as the lights, when the sounds were static or moved, or when the sounds and lights came from the same or opposite sides of fixation. Assuming that these results would be replicated in the present set-up as well, we expected sound location to be unimportant for intersensory pairing. Equal amounts of temporal ventriloquism were therefore expected from segregated sounds presented from the central location (near the lights) and the lateral location (far from the lights).\nThe notion that sound location matters for auditory grouping, but not for intersensory pairing also lead to a very counter-intuitive prediction. In case flanker sounds were presented near the central lights, there should more temporal ventriloquism by capture sounds presented from a lateral position than from central position, because only the lateral sounds segregate. With central flankers, there should thus be more temporal ventriloquism when the location of the sounds and lights differ, rather then when they are the same.\nMethod\nParticipants, stimuli and procedures were the same as in Experiment 1, except for the following changes. Eighteen new students from the same subject pool participated. The auditory stimuli consisted of 5\u00a0ms sound bursts presented at 72\u00a0dB(A), presented from one of two loudspeakers (see Fig.\u00a01b). One speaker was located at central location at eye-level and at 90\u00a0cm distance (as in Experiments 1 and 2), the other was located on either the far left or the far right (at 90\u00b0 azimuth). Four within-subjects factors were used: Location of the two capture sounds (central or lateral), Location of the flanker sounds (same or different position as the capture sounds), the AV-interval between the capture sounds and lights (\u223c0 or \u223c100\u00a0ms), and the SOA between the two lights (\u221275 to +75\u00a0ms). The 80 conditions were each presented 20 times in 10 blocks of 160 trials each. In half of the blocks, the lateral speaker was on the left, in the other half it was on the right.\nResults\nA 2\u00a0\u00d7\u00a02\u00a0\u00d7\u00a02 ANOVA with as within-subjects factors Location of the two capture sounds (central or lateral), location of the flanker sounds (same or different position as the capture sounds) and the AV interval (\u223c0 and \u223c100\u00a0ms) was conducted on the JNDs and PSSs. In the ANOVA on the PSS, there was an interaction between the location of the capture and flanker sounds, F(1, 17)\u00a0=\u00a06.31, P\u00a0<\u00a00.025, indicating that there were slightly more \u2018up\u2019 responses in trials in which the capture and flanker sounds were presented centrally (mean PSS\u00a0=\u00a0\u22121.84\u00a0ms) rather than in the other conditions (mean PSS\u00a0=\u00a02.61\u00a0ms), a finding for which there is no clear explanation.\nIn the ANOVA on the JNDs (see Table 2) there was no main effect of the location of the capture sounds, F(1, 17)\u00a0=\u00a01.67, P\u00a0=\u00a00.21, indicating that JNDs were unaffected by whether the capture sounds were presented centrally (near the two lights) or laterally. Most importantly, there was an interaction between the AV-interval and the location of the flanker sounds, F(1, 17)\u00a0=\u00a05.11, P\u00a0<\u00a00.05. Separate t tests confirmed that the temporal ventriloquist effect (better performance at \u223c100\u00a0ms rather than \u223c0\u00a0ms AV interval) was only significant when the flanker sounds came from a different position than the capture sounds. There was thus no temporal ventriloquism when the capture and flanker sounds came both from central or lateral positions (both P\u2019s\u00a0>\u00a00.6), while there was a 3.9\u00a0ms improvement for central capture sounds with lateral flankers, t(17)\u00a0=\u00a02.175, P\u00a0<\u00a00.05, and a 3.1\u00a0ms improvement for lateral capture sounds with central flankers, t(17)\u00a0=\u00a02.55, P\u00a0<\u00a00.025. These two improvements were not significantly different from each other, t(17)\u00a0=\u00a00.52, P\u00a0=\u00a00.60. Moreover, as predicted, with central flankers, temporal ventriloquism by lateral capture sounds was bigger than that from central ones, t(18)\u00a0=\u00a01.798, P\u00a0<\u00a00.05.Table\u00a02Mean just noticeable differences (JND) in ms, and standard errors of the mean (in parentheses) of Experiment 3Location of flanker soundsSame as capture soundsDifferent from capture soundsLocation of capture soundsAV-interval (ms)JNDTVEJNDTVECentral028.2 (2.1)\u22120.927.7 (2.0)3.9*10029.1 (2.3)23.8 (1.4)Lateral030.0 (2.2)1.729.0 (1.8)3.1*10028.3 (2.0)25.9 (1.8)Capture Sounds Presented at \u223c0 or \u223c100\u00a0ms audio\u2013visual intervals from central or lateral location; flanker sounds presented from the same or different location as the capture sounds. The temporal ventriloquist effect (TVE) is the improvement in JND between the \u223c0 and \u223c100\u00a0ms audio\u2013visual intervals*P\u00a0<\u00a00.05\nGeneral discussion\nThis study examined how principles of auditory grouping relate to intersensory pairing. Two capture sounds that normally enhance performance on a visual TOJ task (i.e. temporal ventriloquism) were embedded in a sequence of flanker sounds which could differ in frequency (Exp. 1), rhythm (Exp. 2), or location (Exp. 3). In all experiments, we found that temporal ventriloquism only occurred when the capture sounds differed from the flankers, and there was thus no effect when flanker and capture sounds were the same. Presumably, when the capture sounds differ, they segregate from the auditory stream, and only then they can be paired cross-modally with the lights. When the two capture sounds do not differ from the flankers, they are perceptually grouped in an auditory stream, in which case they lose their saliency and cannot interact cross-modally anymore.\nThese results are similar to previous findings, which have shown that intersensory interactions do not occur when sounds that normally enhance performance belong to another auditory group (Vroomen and de Gelder 2000; Watanabe and Shimojo 2001; See also Sanabria et\u00a0al. 2004a, b). The results also imply that a sound can only be assigned to a single event: it either belongs to the auditory stream, or it is paired with the lights, but it cannot be assigned to both simultaneously. In this respect, it is analogous to many of the well-known ambiguous figure-ground displays (e.g., the Face-Vase illusion or the Necker cube), where it is known that only one interpretation of the scene can be maintained.\nAnother important finding was that commonality in space between the capture sounds and lights did not affect temporal ventriloquism. The temporal ventriloquist effect was thus equally big for segregated capture sounds that were presented near the lights or far away from the lights. A few other studies have demonstrated before that spatial disparity between sound and vision may not affect intersensory interactions (Welch et\u00a0al. 1986; Bertelson et\u00a0al. 1994; Stein et\u00a0al. 1996; Colin et\u00a0al. 2001; Murray et\u00a0al. 2004; Vroomen and Keetels 2006). However, these studies always relied on null-effects, which entails the danger that they simply lacked the power to detect any effect of spatial disparity. Participants in previous studies might, for example, not have been able to perceive spatial disparity, or they might have learned to ignore it in the experimental task. Our findings, though, counter these arguments. By combining principles of auditory grouping with intersensory pairing, we were able to create a situation where the capture sounds were actually more effective when their locations differed from that of the lights rather than when they came from the same position as the lights. Within the same experimental situation, we thus demonstrated that sound location mattered for auditory grouping, but not for intersensory pairing. Such a finding makes it highly unlikely that sound location was not perceived or simply ignored. Rather, it becomes more likely that, at least in the temporal ventriloquist situation, commonality in space between sound and vision is not relevant for AV pairing.\nThis may, at first sight, seem unlikely, because after all, most natural multisensory events are spatially an temporally aligned, except for some minor variations in time or space that people are readily able to adjust (e.g. Vroomen et\u00a0al. 2004). However, a critical assumption that underlies the idea of spatial correspondence for intersensory pairing is that space has the same function in vision and audition. This notion, though, is arguable as it has been proposed that the role of space in hearing is only to steer vision (Heffner and Heffner 1992), while in vision it is an indispensable attribute (Kubovy and Van Valkenburg 2001). If one accepts that auditory spatial perception evolved for steering vision, but not for deciding whether sound and light belong together, there is no reason why intersensory interactions would require spatial co-localization. Our results therefore have also important implications for designing multimodal devices or creating virtual reality environments, as they show that the brain can, at least in some cases, ignore intersensory discordance in space.","keyphrases":["auditory grouping","intersensory pairing","temporal ventriloquism","temporal order judgment","multisensory perception"],"prmu":["P","P","P","P","R"]}
{"id":"J_Digit_Imaging-3-1-2039840","title":"Design and Implementation of an Open Source Indexing Solution for a Large Set of Radiological Reports and Images\n","text":"This paper hopes to share the insights we experienced during designing, building, and running an indexing solution for a large set of radiological reports and images in a production environment for more than 3 years. Several technical challenges were encountered and solved in the course of this project. One hundred four million words in 1.8 million radiological reports from 1989 to the present were indexed and became instantaneously searchable in a user-friendly fashion; the median query duration is only 31 ms. Currently, our highly tuned index holds 332,088 unique words in four languages. The indexing system is feature-rich and language-independent and allows for making complex queries. For research and training purposes it certainly is a valuable and convenient addition to our radiology informatics toolbox. Extended use of open-source technology dramatically reduced both implementation time and cost. All software we developed related to the indexing project has been made available to the open-source community covered by an unrestricted Berkeley Software Distribution-style license.\nBACKGROUND\nWhenever a person or an institution builds a large enough collection of anything, searching through it becomes tedious or even unfeasible. An obvious solution is indexing: the process of compiling a list of pointers to items in the collection based on alphabetical order or any other item attribute. Indexing can add useful metadata, which is data about data, to a collection. A well-known example is the Science Citation Index for papers published in academic journals as envisioned by Eugene Garfield in 1955.1\nHospitals typically generate a lot of data about patients, their examinations, and their treatments. Due to the patient-oriented workflow, hospital information systems have a strong patient-centric architecture; hence, problems tend to arise whenever information across patients is required. For purposes of business intelligence, data mining and data warehousing can provide an adequate solution. When cross-patient information is needed in a research or training context, collecting medical data will become labor-intensive or practically impossible if the relevant data only exist in a free textual form. A solution for this problem consists in building an index of all free-text words that allows for efficient queries.\nIn our institution, the university hospital closely associated with the largest Belgian university, close to two million radiological reports and corresponding images were made instantaneously searchable by implementing a language-independent, feature-rich indexing solution. This paper hopes to share the insights we experienced during designing, building, and running an indexing system in a production environment for more than 3\u00a0years. Extended use of open-source technology dramatically reduced both implementation time and cost.\nMETHODS\nTo achieve a complete indexing solution that is ready for a production environment, we designed a system consisting of five major sections: the report warehouse, the search engine, specific import filters, efficient indexing, and finally the user interface. These sections correspond to five discrete phases in the implementation scheme. An overview of the architecture is presented in Figure\u00a01.\nFig\u00a01.The architecture of the complete indexing system.\nReport Warehouse\nIndexing a large set of reports requires fast access to all text files to reduce the time needed to build such an index. Our radiology information system (RIS) did not comply with this rule because reports were stored in an Oracle database using a Lempel\u2013Ziv related compression scheme.2 To counter this problem we decided to set up an independent report warehouse that did not have an impact on RIS performance.\nWe started out by recycling two 5-year-old servers and fitting both of them with enough hard disk capacity (160 gigabytes) and a small uninterruptible power supply. We chose to run the open-source Free Berkeley Software Distribution (FreeBSD) operation system,3 which had the important ability to maintain disk integrity after a crash or power outage. This technique, called soft updates, works by properly ordering file system metadata-writes to guarantee consistency and was introduced by Marshall Kirk McKusick.4 Both servers were configured as a high-availability cluster.\nWe achieve synchronization with the RIS through two parallel processes. First, a structured query language (SQL) statement interrogates the RIS to find out which reports were cancelled since the last synchronization. Subsequently, these cancelled reports are removed from the report warehouse. The second process is similar to the first; a list of new reports and reports updated with an addendum is obtained. Next, by means of another SQL statement, the report binary large objects are retrieved from the RIS database and decompressed. Alternatively, we could have been using a standardized, but more complex, method of capturing outbound Health Level 75 observation results unsolicited messages originating from RIS. At the time of implementation this method had the major disadvantage of only supporting converted flat text representations of the reports; meanwhile, our RIS gained the possibility to send out rich text format (RTF), retaining report layout and style. In an ideal situation, indexing should be built right into RIS; this way both extracting and synchronizing reports and data become obsolete. Our report warehouse only contains validated reports by policy.\nSearch Engine\nWith the advent of the Internet and ever-increasing computing power, indexing software became widely available. Our report warehouse made it possible to plug in and evaluate several search engines with a limited set of reports. Due to our characteristic European setting, language independence was needed to support Dutch, French, English, and German reports. The ability to include RIS data in the index as report metadata was also an important factor; this allows searching for (or sorting on) specific authors and requesting physicians and departments and other administrative information. Four more features we required were phrase and wildcard searching, Boolean logic, and the possibility to exclude common stop words. The section on semantics in this paper contains a detailed motivation for why these last four features are essential in a medical context.\nBoth commercial and open-source indexing engines were tested and compared, but in-depth evaluation reports have not been included here because they have become seriously outdated after more than 3\u00a0years. Commercial engines were difficult or impossible to integrate due to their closed-source nature. Most engines could be discarded quickly because of a missing feature or a time-consuming indexing process. Note that the Google Search Appliance6 was not available in Europe at that time. We stopped looking for other candidates after we evaluated the highly recommended Swish-e indexing engine,7 which had all the desired features and a very efficient indexing process.\nImport Filters\nOur evaluation showed that the filters provided by most search engines, including Swish-e, had issues with importing our reports. Most problems were related to file formats; old formats were converted incorrectly, international characters often disappeared, and automatically generated text fragments (Microsoft Word AutoText) were not being filled in. One issue was observed across file formats: hyphenated words did not always make it into the index in one piece.\nSolving these problems required programming import filters for all historically and currently used file formats: both American Standard Code for Information Interchange flat text and flat text containing multiple international character sets, WordPerfect, Microsoft Word, and RTF. These in-house developed filters properly undo hyphenation and combine the imported report with corresponding RIS data into a single Extensible Mark-up Language (XML) file ready for indexing, an example of which is presented in Figure\u00a02. At our institution there is no mandatory structure for radiological reports. Additionally, we have put a quality assurance process in place; whenever word processing software or a report template is changed, the import results are tested on international characters, automatically generated fragments, and hyphenation.\nFig\u00a02Example of a XML file containing the imported report and its corresponding RIS metadata.\nEfficient Indexing\nIndexing without tuning becomes very computing-intensive as the collection of document content, and thus the number of word\/document combinations, grows. Fortunately, limiting the size of the index itself strongly decreases the computing power needed. In our situation, patient and study IDs in the radiological reports were safely excluded from the index without loss of information; using the metadata from the RIS yielded the same results and was more efficient.\nA second technique consists in excluding some, but certainly not all, of the very frequent words present in almost all documents. These common stop words were found by building an index and evaluating the significance of the thousand most frequent words. Amongst the 39 manually excluded stop words, good examples like \u201ca,\u201d \u201cthe,\u201d \u201cof,\u201d and closing statements like \u201cgreetings\u201d were found. The very frequent word \u201cno\u201d was not excluded because its meaning was highly significant in combination with other keywords.\nIn the section on semantics we will show that excluding common stop words leads to semantic advantages. We will also discuss building indexes in an incremental fashion in the section on scalability.\nUser Interface\nDue to our completely digital and highly optimized reporting workflow,8 the only good place to put the search interface was right at the radiologists\u2019 fingertips. To attain this, we created a full front-end integration with RIS and the picture archiving and communication system (PACS). By means of a generic search button, the interface can be launched from any RIS or PACS context. The integration also includes a security layer serving two distinct purposes. Firstly, our existing single-sign-on\/single-sign-off mechanism allows radiologists and researchers to provide their authentication credentials using an encrypted hardware key, defying the need to type in many usernames and passwords. The second function of the security layer consists of auditing the search queries and the displayed reports.\nBecause Internet technology was being used, it was straightforward to make a web-based interface that resembles well-known Internet search engines. Such simple and user-friendly interfaces lower the threshold for new radiologists. As shown in Figure\u00a03, users simply enter the desired keywords, possibly accompanied by parentheses and the binary operators \u201cand,\u201d \u201cor,\u201d and \u201cnot.\u201d Wildcards are denoted by an asterisk, and quotes can be used for phrase searching. Between keywords or phrases, an implicit \u201cand\u201d is assumed. The user can quickly pick a previous query from his complete query history. The results can be sorted on a calculated page rank (default), descending and ascending report time, and RIS metadata. Useful query feedback is shown: the number of results, the query duration, and the excluded stop words. Fast reviewing of reports is essential; to meet this end, the user can choose between displaying summarized or full reports\u2014in both cases, keywords are highlighted and the matching ranking score is displayed graphically. With a single click of the mouse, radiologists can open both images in PACS and the historical reports of the selected patient.\nFig\u00a03.The user interface for radiologists and researchers.\nAlas, trying to comprehend the ranking code of Swish-e is not for the faint of heart because of its inherent complexity and the speed optimizations used. The algorithms take into account the binary structure of the search query, details of keyword occurrences, and the relative frequency of words in the index. Luckily, unlike searching the Internet, queries in a radiological context tend to be very specific (see results), making fine-tuning of ranking unnecessary.\nRESULTS\nPreceding the go-live of our indexing solution, over one million reports and associated data were extracted from the RIS. Some filter fine-tuning was necessary to pass all reports through the layer of import filters. The initial import was spread over a 14-day period without performance penalties for production systems. At the time of writing, our report warehouse contained 1.8 million reports and corresponding administrative data, their XML versions and backup copies not included. The system holds 7.2 million files in total. The oldest report dates back to 1989, but not many reports of that era were available in digital form.\nEach night the index is rebuilt completely, which facilitates early detection of disc decay because all XML reports get reread in the course of this process. Alternatively, incremental reindexing could have been used. Building a full index is very efficient; it only takes 76\u00a0min worth of CPU time on 8-year-old hardware to process 104 million words. Currently, the index contains 332,088 unique words in four languages, including names of patients and requesting physicians. Alas, all spelling mistakes ever made by our typists and radiologists are also included.\nThe usefulness of the indexing solution is indicated by the fact that all resident radiologists make use of it despite the nonmandatory character. Figure\u00a04 illustrates the nearly linear increase in users since the initial deployment in May 2004.9Fig\u00a04.The increase in users over time.\nIndexing reports added useful metadata to our collection of radiological examinations. Early generations of the RIS did not track the supervisor of a radiologist in training; instead, the supervisor\u2019s name was added manually to the text of a report. Indexing made it possible to retrieve historical supervisor information.\nHaving an independent report warehouse available that contains all radiological reports avoids vendor lock-in. During PACS implementation we encountered a 6-month delay in the PACS\/RIS front-end integration due to a difficult multivendor situation. We successfully avoided this delay by making historical reports available at the radiologists\u2019 work stations through the report warehouse.\nQuery Analysis\nThe search engine was consulted 7,071 times by our radiologists mainly to find interesting cases for research or training purposes. Occasionally, a query is performed to find a recent clinical exam in a fast and convenient manner.\nTable\u00a01 presents the usage of search engine features. Apparently, 61% of the radiologists are aware of and use at least one of the features despite the lack of formal training; 39% of them have never used a search engine feature. Nine in ten queries consist of keywords only, which means that results are mostly found without having to resolve to more complicated queries. Users tend to start out with a simple query and quickly skim across the clearly presented and highlighted results. Binary operators turned out to be the most popular feature, especially the \u201cand\u201d operator, which is used in 4.6% of the queries to narrow the returned results. Phrase and wildcard searching each are used in 3% of the queries, whereas parentheses account for 0.6%.\nTable\u00a01.The Usage of Search Engine FeaturesFeatureQueries (n\u2009=\u20097,071)Users (n\u2009=\u200938)Keywords only6,344 (89.7%)15 (39%)Phrases215 (3.0%)14 (37%)Wildcards208 (2.9%)14 (37%)Binary operator \u201cand\u201d325 (4.6%)15 (39%)Binary operator \u201cnot\u201d117 (1.7%)9 (24%)Binary operator \u201cor\u201d83 (1.2%)8 (21%)Parentheses40 (0.6%)6 (16%)\nQuerying the index is very fast; the median query duration is 31\u00a0ms. The most complex query took 2.6\u00a0s: results for each keyword need to be binary combined and ranked; a processing delay is noticeable in the case of very frequent keywords or when broad wildcards are used. The users enter fairly specific queries; medially, only 36 results are returned. A statistical analysis of the query duration and the number of results can be found in Table\u00a02. The cumulative frequency distribution of the query duration, presented in Figure\u00a05, can give the reader an idea of the response rate of the search engine. Caching performed by the operating system has a strong influence on the query duration because 36% of the queries yield results within the 13-ms average access time of the disk drive used; Figure\u00a06 visualizes the relevant detail of the frequency distribution.\nTable\u00a02.Statistical Analysis of the Query Duration and the Number of Results\u00a0Query Duration (ms)Number of ResultsMedian3136Maximum2,598962,629Mean1295,878Standard deviation27459,286Skewness4.415Kurtosis24237Fig\u00a05.The cumulative frequency distribution of the query duration (ms).Fig\u00a06.Detail of the frequency distribution of the query duration (ms), showing the influence of caching performed by the operating system.\nOpen Source\nOur indexing solution is a prime example of an open-source success story; extended use of this technology dramatically reduced both implementation time and cost. Designing and building the system took a mere 6\u00a0weeks, which was less than the \u201cred tape\u201d time needed to approve a commercial offering. Apart from the implementation man-hours, the total cost of ownership consists of low-budget hardware and support costs. Support calls are rare due to the high-availability design of the system and the excellent stability of the Swish-e search engine. In fact, users almost never call; the interface is very user friendly and similar to well-known interfaces of Internet search engines. Furthermore, passwords cannot be forgotten because of the single-sign-on mechanism. About four times a year, manual intervention is required when the synchronization link with the RIS breaks down; in this case, the administrator is automatically notified by e-mail. There has only been one hardware failure during the system\u2019s lifetime, a broken power supply, but this did not cause any downtime.\nAll software we developed in the course of this project has been made available10 to the open-source community covered by an unrestricted BSD-style license. The source code included linking and synchronization with a RIS, five import filters, the security layer and audit logging, the complete user interface, and the integration code for both RIS and PACS.\nDISCUSSION\nAlthough indexing technology has undoubtedly been used in a clinical setting before, the authors believe they have followed a novel approach in designing and implementing a complete and scalable indexing solution. This high-availability and high-performance system has been running without issues for more than 3\u00a0years in a production environment. Furthermore, the independent and flexible design of the platform allows for hosting other applications; several research projects have already found a home under its wings. In this section, Internet indexing technology, scalability, and semantics are briefly discussed.\nInternet Indexing Technology\nOur decision to make use of Internet indexing technology in a medical context was straightforward because of its ubiquitous presence, the available software components, and the maturity this technology has gained over the years. Three generations of automated Internet indexing systems can be identified as to their methods of compiling their data sets, their search interfaces, and their associated etymological metaphors and mythologies.11 Firstly, the Archie search engine dating back to 1990 allowed for single keyword and regular expression searches of the file lists of anonymous File Transfer Protocol sites. Gopher, a distributed document network protocol preceding the World Wide Web,12 featured a second generation; the Veronica system allowed for Boolean queries of directory information and filenames. Thirdly, search engines that compile searchable databases of information accessible via the World Wide Web are in wide use today, such as the popular Google.13 Indexing technology slowly found its way into computer operating systems like Microsoft Corporation\u2019s Windows Vista (released in 2007) and Apple Corps\u2019 Mac OS X 10.4 (released in 2005).\nTwo aspects of Internet indexing technology could not be applied to our medical documents because of absent hyperlinking. The first was \u201cspidering\u201d: robots crawling the web to search for new and updated material by investigating hyperlinks. However, this did not pose a problem because the RIS kept track of new and updated reports. Secondly, page-ranking algorithms could not use rules based on the number of referrals by other documents.\nA nationwide study conducted by Vorbeck et al14 in Austria, apart from the Alps, a country similar to Belgium, showed that radiologists were already familiar with the Internet in 1999. Today, all radiologists at our institution have a broadband Internet connection at home, including secure access to the electronic medical records and the PACS.\nScalability\nGenerally, the duration of building an index should be less than the desired timeframe in which new or updated documents show up in the system. Build times can increase beyond this point when document access speeds decrease or when the total size of the document collection grows. To counter this limitation, indexes can be built incrementally; however, care must be taken because some indexers do not recalculate frequencies of very common words for automatic exclusion. Similarly, indexes can be built in parallel; both mechanisms make use of index merging. Judging by our current results and the obsolete hardware used, we estimate that our indexing system scales up by two orders of magnitude, the main bottleneck being the nondistributed index.\nTo scale a cross-patient search engine, hospital-wide privacy considerations have to be taken into account. The hospital\u2019s policy on patient privacy should be implemented in strict access rules based on document metadata.\nSemantics\nA disadvantage of keyword-based searching is the simple fact that radiologists or researchers want to find the meaningful concepts behind their keywords. To make word indexing more or less suitable for concept searching, four search engine features are necessary. Firstly, because medical terms mostly consist of more than one word, it has to be possible to search for phrases. Page-ranking algorithms can help by giving a higher score to documents with adjacent keywords, but in practice, the sole use of this technique alone is not specific enough. Secondly, very frequent words with low significance can hamper phrase searching and should not be indexed; when \u201cof\u201d and \u201cthe\u201d are excluded, the phrases \u201cMRI of the brain\u201d and \u201cMRI brain\u201d yield the same results. A third helpful feature is Boolean logic to facilitate binary operations like \u201cand,\u201d \u201cor,\u201d and \u201cnot\u201d in combination with parentheses. Finally, a word-stemming technique can be used to find all words with a common root. Although we did not implement word stemming because our multilanguage set-up required building a separate stemmed index for each language, the use of wildcards turned out to be an adequate alternative. Word stemming is part of the broader method of fuzzy indexing, which can be used to find words with similar pronunciations.\nTo find conceptual information, users build their queries in multiple steps; hence, the user interface should allow for fast reviewing of results. Table\u00a03 shows a typical user refining his query. Note that structured reporting, which uses standardized information concepts, does not have the semantic problems of free-text reporting.\nTable\u00a03.Refining of a Query by a Typical UserTimeQueryNumber of Results12:00cirrhose52812:02cirrhose ascites18312:02cirrhose ascites MR not angio*100\nA lot of research regarding medical semantics has been conducted, ranging from natural language processing techniques15\u201317 to building unified medical lexica18,19 for multiple languages.20 The authors hope to combine the indexing solution with some of these scientific methods and technology regarding the semantic web21 in the near future.\nCONCLUSION\nIn this digital age, special care should be taken that the ever-growing amount of on-line information does not deteriorate into an inaccessible swamp of mere bits and bytes. On should strive to provide fast, clear, and easy access to information sources and, hence, increase the total value of stored data.\nDesigning and implementing an indexing solution for a large set of radiological reports and images turned out to be a technically challenging but educational endeavor. For research and training purposes, it certainly is a valuable and convenient addition to our radiology informatics toolbox. The use of open-source technology is highly recommended to reduce both implementation time and cost.","keyphrases":["open source","radiology reporting","search engines","radiology information systems (ris)","pacs integration","medical informatics applications","document indexing"],"prmu":["P","P","P","P","R","R","R"]}
{"id":"Histochem_Cell_Biol-3-1-2137940","title":"Morphological and cytochemical determination of cell death by apoptosis\n","text":"Several modes of cell death are now recognized, including necrosis, apoptosis, and autophagy. Oftentimes the distinctions between these various modes may not be apparent, although the precise mode may be physiologically important. Accordingly, it is often desirable to be able to classify the mode of cell death. Apoptosis was originally defined by structural alterations in cells observable by transmitted light and electron microscopy. Today, a wide variety of imaging and cytochemical techniques are available for the investigation of apoptosis. This review will highlight many of these methods, and provide a critique on the advantages and disadvantages associated with them for the specific identification of apoptotic cells in culture and tissues.\nIntroduction\nThe mode of cell death referred to as apoptosis, and now defined by a variety of techniques, including biochemistry, flow cytometry, and imaging was originally defined based solely upon morphological criteria. In 1972, Kerr et al. (1972) introduced the neologism \u201capoptosis\u201d to describe a type of cell death which had previously been referred to as \u201cshrinkage necrosis\u201d. Recognizing the importance of its kinetic mechanism in controlled cell deletion, either occurring spontaneously or in response to a stimulus, they offered the term apoptosis (from Greek, meaning \u201cto fall away from\u201d, as in leaves from a tree; thus, the falling away of cells from a tissue). They observed apoptotic cells in a wide variety of tissues, including during development and neoplastic transformation. Although they could detect apoptotic cells in many instances by light microscopy, it was their observations by transmission electron microscopy that established the characteristic ultrastructural features now considered the hallmark of apoptosis. These features include (1) cytoplasmic and nuclear condensation (pyknosis); (2) nuclear fragmentation (karyorrhexis); (3) normal morphological appearance of cytoplasmic organelles; and (4) an intact plasma membrane (Kerr et al. 1972; Wyllie et al. 1980; Galluzzi et al. 2007). Frequently, the pyknotic nucleus assumes the appearance of a half-moon or crescent shape, a feature most indicative of an apoptotic cell (Fig.\u00a01). Following nuclear fragmentation, the cell disaggregates into a number of membrane-bound apoptotic bodies, which are engulfed via phagocytosis by neighboring epithelial cells or macrophages.\nFig.\u00a01Transmission electron microscopic image of an apoptotic cell in a human kidney biopsy. Note the pyknotic, shrunken nucleus and the very condensed cytoplasm\nIn more recent years, evaluation of cells and tissues for apoptosis has evolved towards staining for light microscopic and flow cytometric analysis. As the biochemical and cell signaling events involved in the apoptotic cascade have been revealed, new tools for the analysis of apoptosis have emerged. Many of these tools are in the form of antibodies raised against proteins specific for the apoptotic pathway, or against neoepitopes on proteins resulting from action of an activated enzyme. While these new probes may specifically target aspects of the apoptotic pathway, they do not address the ultrastructural changes upon which the term apoptosis was originally defined.\nThis review will focus on some morphological and cytochemical techniques used to demonstrate the presence of apoptotic activity in tissue and cultured cells. For further information on this topic, the reader is referred to the earlier excellent reviews by Willingham (1999), Barrett et al. (2001) and Watanbe et al. (2002). However, before we begin to describe these specific detection methods, we need to examine more closely the different classifications of cell death, and then delve into the mechanisms responsible for initiating the apoptotic cascade.\nClassification of cell death\nCell death is now known to be perpetrated through a variety of mechanisms. According to Galluzzi et al. (2007), cell death can be classified into four different types, based upon morphological characteristics: apoptosis (Type 1), autophagy (Type 2), necrosis (oncosis, Type 3), and mitotic catastrophe. The morphological changes accompanying apoptosis have been described in detail above. Whereas apoptosis is manifested by volume reduction of the nucleus and cytoplasm (cell shrinkage), necrosis (the mode of cell death with which apoptosis is most often confused) is evinced by cytoplasmic swelling, rupture of the plasma membrane, swelling of cytoplasmic organelles (particularly mitochondria), and some condensation of nuclear chromatin (Galluzzi et al. 2007). Autophagy is distinguished by the accumulation of cytoplasmic vacuoles and membranes, and mitotic catastrophe by multinucleation. Clearly, cellular morphological characteristics must be taken into consideration when determining the mode of cell death. This is of utmost importance when employing the TUNEL assay, since this technique stains both apoptotic and necrotic cells, which as just described display widely different morphological characteristics. This issue will be addressed in detail below.\nPathways of apoptosis\nCell death through apoptosis is known to occur through two primary pathways, an extrinsic pathway involving death receptors, and an intrinsic pathway via members of the Bcl-2 family (Adams and Cory 1998). The extrinsic pathway uses the classical death receptors such as Fas (CD95\/APO1), TNF Receptor1, and TRAIL Receptors. Engagement of these receptors triggers a now well-defined process of recruitment of proteases known as caspases (cysteine-dependent aspartate-cleaving proteases). In the case of Fas, oligomerization by its ligand causes recruitment of the adaptor protein FADD (Fas-associated via death domain) through interactions between the death domains (DD) found in each protein (Krueger et al. 2003). FADD then recruits caspase-8 through their common death effector domains (DED). Homodimerization of caspase-8 results in its activation through sequential cleavage at critical aspartate residues. Fully active caspase-8 then cleaves downstream targets such as the Bcl-2 family member Bid to form truncated Bid or tBid (Li et al. 1998). tBid localizes to the outer mitochondrial membrane where it promotes oligomerization of another Bcl-2 member, Bax, which results in breakdown of the mitochondria and release of cytochrome c and Smac\/Diablo (Verhagen et al. 2000). Cytochrome c engages a complex containing Apaf-1 and caspase-9 that in the presence of ATP forms a large molecular weight complex of active caspase-9 known as the apoptosome (Cain et al. 2000). The apoptosome can then activate the effector caspases, such as caspase-3 and caspase-6 that ultimately lead to breakdown of cellular components. Among these is cleavage of an inhibitor of caspase-activated DNAse (ICAD) that allows the DNAse to degrade DNA. This process is fundamental to various assays of DNA breaks that are described in this review.\nIn parallel with the extrinsic death pathway, the intrinsic pathway ultimately engages the same mitochondria-initiated downstream death pathways. In this case the cascade is initiated by any of a variety of sensors that detect cellular stress. The initiators in this case include a panel of Bcl-2 family members that contain only the BH3 domain (so-called BH3-only proteins) (Strasser 2001). These pro-apoptotic Bcl-2 family members are located throughout the cell somewhat as sentinels that detect cellular perturbations. Upon their activation they induce aggregation and activation of Bax or Bak, which then feeds into the mitochondrial death pathway described above. Activation of the intrinsic death pathway may occur in the form of DNA damage by UV irradiation that activates p53 and then two BH3-only proteins, Puma and Noxa. Glucocorticoids similarly activate Puma and Bim that are critical to induction of cell death by steroids. In fact most chemotherapeutic agents ultimately engage a BH3-only protein in the process of killing cells. As a consequence of these pathways, several death assays measure mitochondrial integrity or membrane potential as described in this review. Additional assays might assess cleavage of caspases or their substrates.\nGiven the localization of many Bcl-2 family members at the mitochondria, considerable interest is now developing over the concept that these proteins may function as well in some normal metabolic pathways. An early and somewhat overlooked study from the Korsmeyer laboratory showed that increased expression of Bcl-2 in T lymphocytes decreased their production of the growth cytokine IL-2 and resulted in somewhat reduced proliferative capacity following stimulation of the T cell antigen receptor (TCR) (Linette et al. 1996). Bcl-2-deficient T cells manifested the opposite phenotype. More recent studies suggest that this may function via regulation of calcium signaling (White et al. 2005). Other studies suggest that BAD may regulate glucokinase activity in mitochondria and that BAD-deficient \u03b2-islet cells are less responsive with insulin secretion to glucose levels (Wikstrom et al. 2007). In a similar manner, caspase-8 has been shown to be critical to growth of various cell types including T lymphocytes (Chun et al. 2002; Salmena et al. 2003). Thus, analysis of activation of \u201cdeath\u201d molecules will, in the future, need to consider these potential overlapping functions with normal cell metabolism.\nDetection of apoptosis by electron microscopy\nIt was the early electron microscopic investigations of Kerr et al. (1972) and Wyllie et al. (1980) that led to their formulation of the concept of apoptosis as a wide-spread form of cell death in a variety of tissues and physiological situations. Electron microscopy is still considered the \u201cgold standard\u201d for the identification of apoptotic cells. However, likely due to the costs associated with performing electron microscopy, the specialized equipment and technical expertise required, and the limited tissue area represented, this technique is no longer routinely used as an apoptosis assay. In many instances, the degree of apoptosis detected, for example following treatment with a specific reagent, is an experimental endpoint. This can easily be accomplished with light microscopy or flow cytometry-based methodologies, but is exceedingly laborious by transmission electron microscopy. However, in instances where quantitation is unimportant, and the mere presence of specifically identifiable apoptotic morphology is the goal, electron microscopy is indispensable. For instance, Abbate et al. (2007) recently employed this approach as a means to characterize the nature of cell death in the myocardium after an ischemic event. They investigated the ultrastructural appearance of cardiomyocytes by transmission electron microscopy in failing rabbit hearts 16\u00a0days following coronary artery ligation. They described cardiomyocytes with severe nuclear derangements, peripheral coarse chromatin condensation, and prevalent mitochondrial and myofibrillar alterations. Although these ultrastructural characteristics are not typical of apoptotic cells, the cells were deemed to be apoptotic based upon an intact cardiomyocyte plasma membrane. These results would suggest that the \u201ctypical\u201d ultrastructural features ascribed to apoptotic cells may in some instances be cell-type specific. However, further experimentation will be necessary to confirm this interpretation.\nScanning electron microscopy has also been used to examine apoptotic cells, with their characteristic rounded-up phenotype well represented by this imaging modality (Wyllie et al. 1980; Germain et al. 2007).\nDetection of apoptosis by cytochemical optical microscopy\nA wide variety of probes are commercially available today for the detection of apoptosis (see Table\u00a01). Many of these are in the form of antibodies raised against proteins specific for the apoptotic pathway, or against neoepitopes on proteins resulting from action of an activated enzyme. These will be discussed later, however, since we will begin with ostensibly the most common cytochemical method for the determination of apoptosis, the TUNEL assay.\nTable\u00a01Commercial sources for apoptosis reagentsReagentCommercial sourceTUNEL assay kitMBL International, Woburn MAMillipore Corporation, Billerica, MAInvitrogen Corporation, Carlsbad, CAPromega, Madison, WIRoche Applied Science, Indianapolis, INApo stain (F7-26) (anti-ssDNA)Axxora, LLC, San Diego, CAAnti-cleaved caspase 3 (ASP 175)Cell Signaling Technology, Inc., Danvers, MAAnti-cleaved cytokeratin 18Chemicon, Temecula, CAAnnexin V-FITCBD Biosciences Pharmingen, San Diego, CARoche Applied Science, Indianapolis, INInvitrogen Corporation, Carlsbad, CALectins specific for \u03b1-d-mannose and \u03b2-d-galactoseLectinotest Laboratory, Lviv, UkraineEY laboratories, San Mateo, CACalbiochem, San Diego, CASigma-Aldrich, St Louis, MOAnti-cytochrome c oxidase subunit 1Invitrogen Corporation, Carlsbad, CABD Biosciences Pharmingen, San Diego, CAAnti-apoptosis-inducing factorSanta Cruz Biotechnology, Santa Cruz, CASyntem, Nimes, FranceThis list is not meant to be exhaustive, but to serve as a starting point to search for reagents. Apologies to those companies not listed\nThe TUNEL assay\nThe presence of apoptotic cells has often been inferred from a positive reaction to the TUNEL assay [terminal deoxynucleotidyl transferase (TdT)-mediated dUTP in situ nick end labeling]. However, the lack of specificity of the TUNEL assay for apoptotic cells has been well documented (Sloop et al. 1999; Jung et al. 2000; Walker and Quirke 2001; Watanabe et al. 2002; Groos et al. 2003; Takemura and Fujiwara 2006). TUNEL will detect free 3\u2032-OH terminals of both single- and double-strand DNA breaks, thus potentially labeling both apoptotic and necrotic derived DNA strand breaks. Moreover, the TUNEL assay has also been reported to label mitotic cells (Ito et al. 2006), cells in the process of gene transcription (Kockx et al. 1998), cells undergoing DNA repair (Kanoh et al. 1999), and cells with DNA breaks caused by the microtomy sectioning procedure (Sloop et al. 1999). The effects of tissue fixation and processing may also alter TUNEL reactivity (Negoescu et al. 1996; Labat-Moleur et al. 1998; Tateyama et al. 1998). In a rat model, we found that exposure to asbestos and cigarette smoke resulted in strong TUNEL positivity in bronchiolar epithelial cells from paraffin-embedded sections stained by immunoperoxidase (Jung et al. 2000). However, when bronchioles from the same animals were examined by transmission electron microscopy, only necrotic epithelial cells were observed, indicating that the TUNEL-positive cells detected by light microscopy were likely necrotic, not apoptotic. The TUNEL-positive nuclei did not display the characteristic shrunken appearance expected for an apoptotic cell, again emphasizing the need to corroborate staining results with morphological observations. Similar conclusions were reached by Negoescu et al. (1996) and Labat-Moleur et al. (1998) in their efforts to improve tissue preparation and staining procedures for the TUNEL method.\nThe TUNEL assay maintains its popularity as a marker of apoptotic cells; it is available in a kit form, is easy to use, and has been available for a number of years, becoming entrenched in the literature. However, given the concerns raised above with its specificity for apoptotic cells, we are of the opinion that other methods should be employed concomitantly with this assay in order to make a determination that the mode of cell death observed is the result of an apoptotic mechanism.\nAnti-single-stranded DNA antibody (Apostain)\nFrankfurt et al. (1994, 1996) and Frankfurt and Krishan (2001) have pioneered the application of a monoclonal antibody raised against single-stranded DNA (ssDNA) as a more specific cytochemical probe for apoptotic cells than the TUNEL assay. They have shown that the combination of a commercially available ssDNA antibody with formamide-induced denaturation of condensed chromatin, results in specific labeling of apoptotic cells in culture and in formalin-fixed, paraffin embedded tissues (Frankfurt and Krishan 2001). Ito et al. (2006) expanded upon these observations by adapting the formamide DNA denaturation followed by anti-ssDNA antibody staining technique for the electron microscopic level. They show very convincingly by immunoelectron microscopy that the TUNEL assay results in staining of condensed chromatin in mitotic and necrotic cells in addition to apoptotic cells, whereas the formamide-ssDNA technique stained only the condensed chromatin from apoptotic cells.\nWe have also used this Apostain method for the specific detection of apoptotic pulmonary epithelial (Buder-Hofmann et al. 2001) or rat pleural mesothelial cells (Shukla et al. 2003) after exposure to crocidolite asbestos in culture, and in human lung adenocarcinoma cells (A-549) following treatment with the chemotherapeutic agents paclitaxel and vinorelbine (Jung et al. 2004). Moreover, we recently combined staining with the ssDNA antibody with a second specific marker of apoptosis in a novel dual staining method on tissue sections, as described below. In our hands, in agreement with studies described above, we find the formamide condensed DNA denaturation\/ssDNA technique to be a reliable indicator of apoptotic cells in a variety of cell cultures and tissues.\nApoptosis-related neoepitopes\nAntibodies raised against caspase-cleaved proteins are now commercially available. The enzymatic cleavage of proteins during the apoptotic cascade results in the formation of \u201cneoepitopes\u201d on these proteins which can be used as antigens for the production of caspase-mediated apoptotic reagents. Although several such antibodies are currently available, those raised against neoepitopes present on cleaved cytokeratin 18 and caspase 3 have been most frequently used for the detection of apoptosis. Willingham and colleagues (Barrett et al. 2001) demonstrated the utility of using an anti-cleaved cytokeratin 18 antibody for the specific detection of apoptotic cells in culture and in archived paraffin embedded tissues. Specifically stained cells showed the shrunken condensed morphology expected of an apoptotic cell. Naturally, this antibody can only be employed for cells and tissues that express cytokeratin 18.\nLikewise, Gown and Willingham (2002) used a commercially available antibody raised against cleaved caspase 3 to specifically label apoptotic cells in culture and paraffin embedded tissues. Given the ubiquitous presence of caspase 3 in most cells, and the formation of the neoepitope following activation of the apoptotic cascade, this reagent should prove to be a most useful and specific label for cells undergoing caspase-mediated apoptotic cell death.\nRecently, we combined immunostaining for anti-cleaved caspase 3 with the formamide denaturation\/ssDNA method for the detection of two specific apoptosis probes in the same tissue section using confocal scanning laser microscopy (Taatjes et al. 2007). In dual stained cells in paraffin sections from infarcted mouse ventricle, the anti-cleaved caspase 3 antibody labeled the cytoplasm, whereas the anti-ssDNA antibody stained the cell nucleus. Cells were observed stained singly with either of the two probes, suggesting that cells in different phases of the apoptotic cascade may be identified by their staining properties with these antibodies. We include sections from the thymus of dexamethasone-treated mice as a positive control (Figs.\u00a02, 3) for the apoptosis assays (Taatjes et al. 2007), since steroid hormone treatment has been shown by transmission electron microscopy to induce thymic apoptosis (Wyllie et al. 1980).\nFig.\u00a02Paraffin section of thymus from mouse treated with dexamethasone and stained with anti-cleaved caspase 3 antibody (green), and anti-ssDNA antibody (red). Confocal microscopy demonstrates doubly stained thymic cells (arrowheads). Note that the cleaved caspase 3 is localized to the cytoplasm, whereas the ssDNA is localized to the nucleus. Some cells only stained by cleaved caspase 3 antibody are indicated by solid arrows. The nucleus is labeled with DAPI (blue)Fig.\u00a03Paraffin section of thymus from mouse treated with dexamethasone and stained with anti-cleaved caspase 3 antibody (red). Confocal microscopy image shows intensely stained cells in the cortex, with neighboring unstained cells. Nuclei have been stained with DAPI (blue). This represents a good positive control for the anti-cleaved caspase 3 antibody technique\nAnnexin V\nSome markers for apoptotic cells require the use of live cells. For instance, early in the apoptotic cascade of events, phosphatidyl serine residues flip from the internal to external side of the cells\u2019 plasma membrane. Accordingly, annexin V, which binds specifically to phosphatidyl serine residues has been used in this manner as a specific probe for apoptosis (Koopman et al. 1994). However, as pointed out by Willingham (1999), this assay does not seem to label all apoptotic cells, and moreover will stain necrotic cells once the plasma membrane is compromised.\nOther probes for apoptosis\nBilyy et al. (2004, 2005) and Bilyy and Stoika (2003) have championed the examination of plasma membrane glycoconjugates as indicators of apoptosis. They have detected an augmentation in the expression of \u03b1-d-mannose- and \u03b2-d-galactose-containing glycoproteins in the plasma membrane in response to apoptotic signals. These glycoconjugates can be visualized using a battery of lectins (carbohydrate-binding proteins of nonimmune origin), in conjunction with fluorescence- or peroxidase-conjugated probes for light microscopy. Unlike most of the markers for apoptosis which bind to cytoplasmic or nuclear constituents, these plasma membrane-specific probes offer the means to assess activity occurring at the cell surface during the apoptotic cascade (similar to annexin V binding to phosphatidylserine). Given the enormous diversity in carbohydrate structure, and the role played by carbohydrates in a variety of cell\u2013cell interactions, it is feasible that they may offer more insight into cellular mechanisms of apoptosis in the future.\nAn early event in the apoptotic cascade is the release of cytochrome c from the outer mitochondrial membrane. Accordingly, the intracellular localization of cytochrome c could serve as an indicator for apoptosis. Jones and colleagues (Jiang et al. 1999) used this approach to show that in a control (untreated) osteosarcoma cell line, immunostaining for cytochrome c (as assessed by confocal microscopy) was observed in a punctuate cytoplasmic pattern, coincident with mitochondrial staining by MitoTracker Red. When cells were treated with staurosporine to induce apoptosis, the immunolabel for cytochrome c was found to be diffusely present throughout the cytoplasm, consistent with its release from the mitochondria. We have also used immunolabeling for cytochrome c as a marker for mitochondria in intact cells (Fung et al. 1998), and its further application as a potential marker for apoptotic cells dependent upon its intracellular staining pattern should be further explored.\nApoptosis-inducing factor (AIF) is a mitochondrial membrane protein involved in caspase-independent cell death (Daugas et al. 2000). Immunostaining for AIF revealing a translocation from the mitochondrion to the nucleus has been used as a marker for caspase-independent apoptosis (Daugas et al. 2000; Susin et al. 2000). However, Daugas et al. (2000) have shown that the nuclear translocation of AIF also occurs during a necrotic response, so other methods should be employed in conjunction with AIF immunostaining to differentiate between these two modes of cell death. Indeed, we have used immunostaining of AIF translocation from the cytoplasm (mitochondrion) to the nucleus, together with cell morphological assessment by phase contrast microscopy, as an indicator of apoptosis in lung C10 cells in response to asbestos exposure (Yuan et al. 2004).\nDetection of apoptosis by video optical microscopy\nMorphological assessment of cultured cells by video microscopy has been successfully used to follow the cellular changes induced by apoptosis. Cells treated with apoptosis-inducing agents are imaged with a light microscope, typically operating in phase contrast or differential interference contrast mode, and time-lapse recording is made with a ccd camera connected to a video recorder (Collins et al. 1997; Willingham 1999). A major advantage of this type of temporal microscopy is the ability to document cellular morphological changes associated with apoptosis with the time course of the event. Differences in cellular changes observed in necrosis versus apoptosis can be well documented, and the slow course of apoptosis is well suited to time-lapse recording (Collins et al. 1997). Using phase contrast microscopy in conjunction with video recording has produced dramatic footage of cells exposed to apoptotic agents rounding up and displaying intense surface blebbing (Collins et al. 1997). Of course, the full apoptotic cascade may require up to 24\u00a0h in cultured cells, so movies can also be made by merging files from single ccd camera images and exporting them in a movie-type format. Thorburn and colleagues (Morgan et al. 2002) used a combination of fluorescence and phase contrast microscopy to image apoptosis in live HeLa cells. A Hamamatsu digital camera was used to acquire combination images every 30\u00a0min over a 24\u00a0h period. The merged image series were exported in Quicktime format, and a representative movie sequence can be seen in the online supplemental material referenced in the manuscript (Morgan et al. 2002). This type of analysis is particularly useful for determining the temporal sequence of morphological events associated with the introduction of an apoptosis-inducing agent to cell culture media.\nDetection of apoptosis by laser scanning cytometry\nLaser scanning cytometry (LSC) is a method which combines the efficiency and precision of flow cytometry with the morphological characterization ability of a microscope-based imaging system (Kamentsky and Kamentsky 1991; Kamentsky et al. 1997; Taatjes et al. 2001; Pozarowski et al. 2006). The ability to examine the morphological appearance of cells following flow cytometry-like analysis overcomes the limitations posed by flow cytometry itself in the analysis of apoptosis; that is, apoptotic cells appear in the subdiploid peak of a flow cytometric histogram, together with apoptotic bodies and fluorescence debris, and cannot be directly visualized. Darzynkiewicz and colleagues have pioneered the use of LSC for analysis of apoptosis, and the reader is referred to their excellent review for further details (Bedner et al. 1999). In particular, they have used LSC in combination with a variety of apoptosis markers, including annexin-V, markers of mitochondrial transmembrane potential (rhodamine 123 and DiOC6), nuclear markers (DAPI, 7-ADD, PI), and markers of DNA strand breaks. Importantly, in all of these investigations, they emphasized the combination of fluorescence staining and morphological determination with LSC as being of utmost importance for the identification of apoptotic cells.\nWe have also used LSC for the cytometric and morphological characterization of apoptotic cultured mouse lung epithelial cells (C10 cell line) (Taatjes et al. 2001). Cells were exposed to hydrogen peroxide for 24\u00a0h to induce apoptosis, followed by staining with the anti-ssDNA antibody and an Oregon green-conjugated secondary antibody to stain apoptotic cells. All cells were then stained with propidium iodide and analyzed by LSC. Scattergrams from the analysis (Fig.\u00a04) showed that hydrogen peroxide induced an increase in apoptotic cells, evinced by an increase in the population of cells depicted in the subdiploid area. Cells located in this subdiploid portion were then visually confirmed through relocation using the automated stage and microscope of the LSC. These cells were found to be labeled with the anti-ssDNA antibody, and showed the condensed morphological characteristics of apoptotic cells (Fig.\u00a04).\nFig.\u00a04Laser scanning cytometry determination of apoptosis in lung epithelial cells using anti-ssDNA antibody. Upper panel scattergrams showing percentage of ssDNA-positive cells (green integral vs. orange\/red integral), the apoptotic fraction (orange\/red integral vs. orange\/red maximum pixel), and a DNA histogram for untreated C10 cells. Middle panel scattergrams showing percentage of ssDNA-positive cells (green integral vs. orange\/red integral), the apoptotic fraction (orange\/red integral vs. orange\/red maximum pixel), and a DNA histogram for C10 cells treated with 200\u00a0\u03bcM H2O2 for 24\u00a0h. Note the increased number of cells ssDNA-positive, and the enhanced subdiploid DNA fraction in cells treated with H2O2 as compared with sham controls. Bottom panel cell relocation feature of LSC demonstrated for eight cells. Cells within the subdiploid fraction (elliptical region highlighted on scattergram in middle panel) were relocated and visually confirmed as apoptotic by morphological appearance and positive staining with the ssDNA antibody. Reprinted with permission from BioTechniques (Taatjes et al. 2001)\nDetection of apoptosis by atomic force microscopy (AFM)\nThe atomic force microscope is a member of the scanning probe microscope family (Binnig et al. 1986). This instrument utilizes a tactile sensing system, in which a probe (typically composed of silicon nitride) is attached to the end of a cantilever and raster scanned in x- and y-directions across a specimen through the activity of a piezoelectric tube to which the cantilever assembly is mounted (Montigny et al. 2006). A laser beam is directed onto the surface of the cantilever, and the reflected beam is directed up to a four quadrant photodiode, which converts laser movement on the surface into a measurable electrical voltage change. This voltage change is then translated via computer interface into height and amplitude information about the specimen. The complexity of the system belies the simplicity of specimen preparation; fixed or unfixed cells grown on glass coverslips can be readily imaged by AFM without further processing, or importantly introduction of artifacts. Although AFM initially found its greatest utility in the imaging of isolated molecules and their dynamic interactions (Hansma and Ho 1994; Taatjes et al. 1997; Rand et al. 2003), more recently whole cell studies have become more prevalent (Madl et al. 2006). With this development, and since AFM typically images surface phenomena, it was a natural extension to use this technology to image cells undergoing apoptosis. Hessler et al. (2005) used AFM to image and measure the cellular volume decrease occurring during apoptosis. They treated the human epidermoid carcinoma cell line KB with staurosporine to induce apoptosis, and determined that after a 3-h exposure, the total cell height decreased 32%, the total cell volume was decreased by 50%, and the cell diameter changed by 15%. Given the temporal experimental capabilities of AFM, it seems as if this would be an ideal modality, in combination with other imaging methods, to investigate the cellular dynamics accompanying the apoptotic cascade, as elegantly shown by Hessler et al.\nAn example of the importance of delineating apoptosis: cell death in the heart\nIn this brief review, we have attempted to highlight some of the morphological and cytochemical techniques currently in use to classify cell death as an apoptotic response. There are a wide ranging variety of methods to choose from, rendering the choice a matter of the experimental system employed and personal preference. However, the determination of cell death as apoptotic, or through another mode should not be viewed simply as a matter of semantics. For instance, an example from clinical cardiovascular disease will illustrate the importance of the precise determination of the mode of cell death. For several decades the conceptual basis underlying treatment of acute coronary syndromes has been that limitation of myocardial oxygen requirements by diminution of afterload and heart rate coupled with augmentation of myocardial oxygen supply through restoration of myocardial perfusion are the pivotal components (Maroko et al. 1972; Shell and Sobel 1974; Sobel and Braunwald 1980). The potential value of \u201ctissue protective\u201d interventions including favorable modification of myocardial metabolism has long been recognized as well (Maroko et al. 1972).\nSeveral different types of cell death have been characterized in the heart including coagulation necrosis, the typical manifestation of ischemia sufficient to render myocardium nonviable, contraction band necrosis typical of ischemia followed by reperfusion and presumably mediated in part by calcium overload, autophagy typical of cells insulted by deprivation of nutrients that consequently consume their own components, and apoptosis. The extent to which this final modality, programmed cell death or apoptosis, contributes to overall cardiomyocyte cell death is far from a trivial matter (Sobel et al. 2007). If apoptosis plays a substantial role as a contributor to total cardiomyocyte cell death and if it can be modified or prevented despite the presence of ischemia, the potential, favorable impact of tissue protective interventions is likely to be considerable. Conversely, if apoptosis plays a small role in overall cardiomyocyte cell death consequent to ischemia, an anti-apoptotic strategy would likely be ineffective. Unfortunately, the implication of apoptosis in cardiomyocyte cell death in studies in laboratory animals has been based virtually exclusively on results with the TUNEL assay (see above for issues of nonspecificity of this assay). As noted by Takemura and Fujiwara (2006), there is no unequivocal evidence that apoptosis occurs in zones of infarction.\nAs described above, we have recently sought to quantify apoptosis in myocardium and have developed a novel dual staining method for confocal microscopic identification of apoptotic cells (Taatjes et al. 2007). Our initial findings indicate that despite massive necrosis induced by ischemia in a transgenic strain of mice deficient in plasminogen activator inhibitor type-1 (PAI-1) hardly any cells were apoptotic as judged from positive reactions to antibodies against both cleaved caspase 3 and ssDNA. Others have observed an absence of apoptosis as judged from electron microscopic criteria (Ohno et al. 1998) despite TUNEL positivity in hearts of animals subject to transitory ischemia followed by reperfusion for several hours.\nIn view of these considerations, it appears important to determine definitively whether the presence of ssDNA coupled with the presence of caspace 3 cleavage product does, indeed, map isomorphically with the cells that exhibit electron microscopic criteria of apoptosis. Furthermore, it will be important to determine the extent to which the positivity of each of these criteria of apoptosis occurs in hearts of experimental animals subjected to ischemia for selected intervals followed by reperfusion for selected intervals as well as in those from animals subjected to myocardial ischemia that is persistent and to define the time course of the evolution of apoptosis, if present, under both conditions. It is, of course, possible that apparently scanty apoptosis at any given time could be of considerable biological importance since apoptotic cells are rapidly destroyed or removed by macrophages. Thus, if a given percentage of cells, though a small one, were exhibiting apoptosis repetitively, the cumulative amount could be substantial. Accordingly, both the temporal and the spatial evolutions of apoptosis associated with myocardial ischemia and infarction require elucidation. This is particularly important because apoptosis in the heart may be secondary to necrosis rather than a primary mode of cell death, a distinction that needs to be clarified because of the disparate therapeutic implications of the two possibilities. Only when armed with information needed to characterize apoptosis in the heart more definitively will it be possible to delineate the potentially beneficial effects of tissue protective interventions designed to retard or prevent apoptotic cell death in cardiomyocytes subjected to ischemia.\nConcluding remarks\nDetermining the mechanisms of cell death is an area of intense research interest in a wide range of fields including cancer biology, pathology, and toxicology. Certainly, the role of apoptosis as a mode of \u201cprogrammed cell death\u201d during embryonic development has been unequivocally established (Penaloza et al. 2006). We hope that this brief review has highlighted a variety of imaging modalities suitable for differentiating amongst the different modes of cell death. What should be apparent is that rarely will one method be sufficient for such a determination. Each imaging method has its own strengths with respect to the morphological information it can provide. Such morphological information, combined with biochemical or immunohistochemical assessment can provide a strong indication of the specific mode of cell death observed. The key, in our estimation, is a multiparameter observational approach.\nAccordingly, the take-home message for any morphological or cytochemical investigation of apoptosis is to employ more than one detection method and run multiple controls.","keyphrases":["morphology","cell death","apoptosis","microscopy","immunostaining","cytochemistry"],"prmu":["P","P","P","P","P","U"]}
{"id":"Intensive_Care_Med-4-1-2271085","title":"Assessment of stroke volumeindex with three different bioimpedance algorithms: lack of agreement compared to thermodilution\n","text":"Objective The accuracy of bioimpedance stroke volumeindex (SVI) is questionable as studies report inconsistent results. It remains unclear whether the algorithms alone are responsible for these findings. We analyzed the raw impedance data with three algorithms and compared bioimpedance SVI to transpulmonary thermodilution (SVITD).\nIntroduction\nAdditional information about the cardiovascular status of critically ill patients can be obtained by measuring cardiac output (CO). Pulmonary artery thermodilution CO monitoring has remained the reference technique for three decades\u00a0[1] but is invasive and associated with specific complications\u00a0[2\u20134]. Thoracic bioimpedance cardiography, a\u00a0noninvasive CO monitoring technique, exhibits many qualities of the ideal CO monitor: it is operator independent, continuous, and cost-effective\u00a0[5]. Since the late 1960s a\u00a0number of bioimpedance devices have been developed with cylinder- or cone-based models of a\u00a0homogeneously with blood filled human thorax. Method comparison studies have demonstrated conflicting results with respect to validity and reliability\u00a0[6], varying from satisfactory correlations\u00a0[7\u20139] to poor correlations\u00a0[10,\u00a011]. Inaccuracies can result from irregular cardiac rhythms, abnormal ventilatory patterns, motion artifacts, valvular heart diseases, electrocautery, changes in hematocrit, excessive changes in body temperature, and an obese body habitus\u00a0[5]. Thereby, it remains unclear whether the methodology (i.e. detection of impedance signals from the thorax using a\u00a0small number of electrodes) per se or limitations of the underlying algorithms are responsible for these conflicting results. We hypothesized that bioimpedance SV measured with any of three well-established bioimpedance algorithms is valid and reliable. We compared bioimpedance stroke volume index (SVI) with transpulmonary thermodilution stroke volume index (SVITD) as a\u00a0reference of proven accuracy\u00a0[12].\nMaterials and methods\nAfter approval by the institutional review board and written informed consent, patients scheduled for elective coronary artery bypass graft (CABG) surgery with cardiopulmonary bypass were included. Exclusion criteria were: ejection fraction less than 40%, femoral arterialdisease, and valvular heart disease. A\u00a0total intravenous anesthesia technique was used during the operation. Normocapnia was maintained during mechanical ventilation (inspired fraction of oxygen 0.4, positive end-expiratory pressure 5\u202fcmH2O).\nA4-Fthermodilutioncatheter(PulsiocathPV2014\u202fL16) was introduced into the femoral artery and connected to a\u00a0commercially available CO device (PiCCO, Pulsion,Munich, Germany). Transpulmonary thermodilution cardiac output (TPCO) was measured by quadruple injections of 15\u202fml ice-cold saline into the right atrium and used for transpulmonary thermodilution stroke volume calculation.\nAfter rubbing and cleaning the skin with alcohol to achieve a\u00a0skin-to-electrode impedance as low as possible, two \u201ccurrent injecting\u201d electrodes were placed on the forehead and the left hip, and two voltage sensing electrodes were placed on the lateral side of the neck just above the left clavicle and in the left midaxillary line at the level of the sternal xiphoid. An alternating current of 0.3\u202fmA (64\u202fkHz) was applied. A\u00a0thoracic bioimpedance cardiograph (HL-4, Hemologic, Amersfoort, The Netherlands) was used for recording raw bioimpedance signals in the perioperative period. The first derivative of the thoracic impedance (dZ\/dt) and the electrocardiographic signal were displayed on the screen. Raw data were analyzed off-line over a\u00a020-s period (LabView, E-solutions, Arnhem, The Netherlands) and used for bioimpedance SV calculation using three distinct reconstruction algorithms: Kubicek et\u00a0al.\u00a0[13], Sramek\u2013Bernstein\u00a0[14], and Wang et\u202fal.\u00a0[15].\nData collected after induction before skin incision (t1), after weaning from cardiopulmonary bypass (t2), after sternal closure (t3), after admission at the intensive care (t4), after reaching normothermia (36.5\u202f\u00b0C) (t5), after extubation (t6), and before discharge to the ward (t7) were: heart rate, mean arterial pressure, central venous pressure, bioimpedance raw data and TPCO measurements. SVI was calculated by dividing stroke volume by body surface area.\nSample size calculation was performed to limit the width of a\u00a095% confidence interval for the mean error; based on a\u00a0mean CO of 5.0\u202fl\/min, a\u00a0correlation coefficient of 0.65, a\u00a0mean error of 30%\u00a0[16], and a\u00a0confidence interval of 95%, a\u00a0sample size of 20 patients was calculated.\nStatistical analysis was performed using PRISM 4.0 (GraphPad, San Diego, Calif., USA) and SPSS 12.0.2 (SPSS, Chicago, Ill., USA). If the analysis of variance revealed a\u00a0significant interaction, post-hoc analysis was performed using Student's t test with Bonferroni's correction. Validity and reproducibility between bioimpedance SVI and SVITD were tested according to Bland and Altman\u00a0[17]: bias, precision (=\u202fSD of bias), limits of agreement (LOA), and mean error\u00a0[15] for absolute SVI values and for relative changes in SVI (\u0394SVI). Mean error was calculated as 2\u202f\u00d7\u202fprecision divided by the mean SVITD. Pooled data and data per time moment were analyzed. A\u00a0p value less than 0.05 was considered to indicate statistical significance.\nResults\nThe population studied included 15 men and 5 women (age 64\u202f\u00b1\u202f10\u202fyears, weight 79\u202f\u00b1\u202f12\u202fkg, height 171\u202f\u00b1\u202f8\u202fcm; body surface area 1.64\u20132.20\u202fm2). Of 140 SVI series obtained 93 with each technique were available for statistical analysis. Forty seven series of SVI data could not be used for further analysis because of failure to obtain SVI due to insufficient raw bioimpedance signals. Time course of SVI and Bland\u2013Altman analysis for each method are shown in Fig.\u202f1. Bias, precision, LOA, and mean error between SVITD and SVIK were 1.0\u202f\u00b1\u202f10.8\u202fml\/m2, \u221220.2 to +22.1\u202fml\/m2, and 63%, respectively, while the results for SVITD and SVISB were 9.8\u202f\u00b1\u202f11.4\u202fml\/m2, \u201312.5 to +32.2\u202fml\/m2, and 67% and for SVITD and SVIW \u221215.7\u202f\u00b1\u202f8.2\u202fml\/m2, \u221231.6 to +0.3\u202fml\/m2, and 48,% respectively. Analysis of bioimpedance data for each algorithm at each time point are given in Table\u202f1.\nFig.\u00a01Time course of stroke volume index (SVI) and Bland\u2013Altman analysis obtained by each method. SVITD, Transpulmonary thermodilution stroke volume index; SVIK, stroke volume index according to Kubicek et\u202fal.\u00a0[13]; SVISB, stroke volume index according to Sramek\u2013Bernstein\u00a0[14]; SVIW, stroke volume index according to Wang et\u202fal.\u00a0[15]. *p\u202f<\u202f0.05 vs. t1Table\u00a01Bias, precision, limits of agreement (LOA), and mean error of stroke volume index calculated with three bioimpedance algorithms vs. transpulmonary thermodilution stroke volume index (SVITD) for each time moment (t1, after induction before skin incision; t2, after weaning from CPB; t3, after sternal closure; t4, after admission at the intensive care unit; t5, after reaching normothermia at 36.5\u202f\u00b0C; t6, after extubation; t7, before discharge to the ward; SVIK, stroke volume index according to Kubicek et\u202fal.\u00a0[13]; SVISB, stroke volume index according to Sramek\u2013Bernstein\u00a0[14]; SVIW, stroke volume index according to Wang et\u202fal.\u00a0[15])SVIKSVISBSVIWtime Bias (ml\/m\u20132) Precision (ml\/m\u20132) LOA (ml\/m\u20132) Mean error (%) Bias (ml\/m\u20132) Precision (ml\/m\u20132) LOA (ml\/m\u20132) Mean error (%) Bias (ml\/m\u20132) Precision (ml\/m\u20132) LOA (ml\/m\u20132) Mean error (%)t16.413.9\u201320.8, +33.67715.512.3\u20138.6, +39.668\u201314.79.8\u201333.9, +4.554t20.68.8\u201316.6, +17.85111.89.3\u20136.4, +30.054\u201313.04.8\u201322.5,\u2013 3.528t3\u20133.07.8\u201318.3, +12.4516.213.9\u201321.0, +33.490\u201314.28.1\u201330.2, +1.753t41.08.4\u201315.5, +17.6569.95.4\u20130.7, +20.436\u201313.85.8\u201325.1,\u2013 2.538t50.410.0\u201319.2, +20.05810.110.7\u201310.9, +31.162\u201316.38.6\u201333.1, +0.650t61.513.3\u201324.5, +27.5737.212.1\u201316.4, +30.966\u201317.89.5\u201336.4, +0.852t7\u20130.610.7\u201321.5, +20.4608.313.0\u201317.2, +33.872\u201318.08.1\u201334.0, \u20132.145\nDiscussion\nThis study compared three bioimpedance algorithms assessing bioimpedance SVI to SVITD during the perioperative period in CABG patients. However, significant deviations were found, and accurate clinical decision making was not possible based on absolute values or changes in bioimpedance SVI. No single algorithm was superior to another. Interestingly, application of the Wang algorithm produced consistent underestimation, whereas the two other algorithms overestimated SVI. Our study differed from previous studies in several important aspects. Raw voltage data were measured and used for off-line calculation of bioimpedance SVI on the basis of different bioimpedance algorithms commonly used in commercially available devices. Therefore data were obtained without using different bioimpedance devices and calculation was independent from built-in proprietary software algorithms. Measurements were performed not only in the operating room but also in the intensive care in ventilated as well as in spontaneously breathing patients.\nThe difference between bioimpedance SVI and SVITD for any of the three algorithms may be explained by the fact that the relationship between the signal on the voltage sensing electrodes and the resulting SVI is based on assumptions in relation to multiple effects. Whereas SV is equal to the change in the left ventricular volume during the systole, the voltage signal measured with bioimpedance is a\u00a0result of volume changes in different intrathoracic compartments during the cardiac cycle, such as the intracardiac cavities, aorta, superior and inferior vena cava, and pulmonary circulation on the \u201cinjected\u201d current\u00a0[18]. Vascular diseases (atherosclerosis) can affect the relative contribution of the aorta to the bioimpedance signal because the volume change in the aorta during the cardiac cycle depends on aortic compliance. Moreover, a\u00a0considerable anatomical variability exists between patients and within the cardiac cycle. The orientation of the central heart axis in relation to the thorax cavity varies considerably between patients but also during the cardiac cycle. Both influence the main current density field and hence the relative contribution of SV to the bioimpedance signal. It is questionable whether it is even possible to measure SVI reliably using thoracic bioimpedance with only one single voltage input stream given the fact that each of three distinctly different algorithms failed to produce satisfactory agreement with SVITD. Therefore an increase in the number of data input streams (i.e. electrodes) may improve the validity and reliability of the technique. Consequently suggestions have been made to optimize the measurement technique and the basic bioimpedance SV equation\u00a0[19].\nRecently Spiess et\u202fal.\u00a0[8] and Sageman et\u202fal.\u00a0[9] studied a\u00a0second-generation thoracic bioimpedance cardiograph (BioZ System 1.52, Cardiodynamics International, San Diego, Calif., USA) in CABG patients and found a\u00a0clinically acceptable correlation between pulmonary artery thermodilution and bioimpedance. However, mean error in the study by Spiess et\u202fal. was 26% after induction of anesthesia and exceeded the clinically acceptable 30% during the other measurements\u00a0[8]. In contrast, our study showed a\u00a0mean error exceeding 30% with the exception of t2 using the Wang algorithm.\nIn conclusion, common cylinder- and cone-based models for bioimpedance SVI calculation are not reliable compared to SVITD measurements in CABG patients. These models are oversimplifications of the complex electrical events occurring inside the thorax during the cardiac cycle. The problem of retrieving SV from voltage data may be considered as a\u00a0special case of the general inverse conductivity theory\u00a0[20]. There is need for a\u00a0more robust mathematical approach (see Electronic Supplementary Material), including an increase in the number of voltage measurement input streams, an accurate description of the physics of current density distributions and taking into account the full spectrum of all relevant patient anatomical variabilities.\nElectronic supplementary material\nElectronic Supplementary Material (DOC 54K)","keyphrases":["stroke volumeindex","bioimpedance","transpulmonary thermodilution","cardiac output","method comparison","coronary artery bypass graft"],"prmu":["P","P","P","P","P","P"]}
{"id":"Ann_Biomed_Eng-2-2-1705506","title":"Heart Valve Tissue Engineering: Concepts, Approaches, Progress, and Challenges\n","text":"Potential applications of tissue engineering in regenerative medicine range from structural tissues to organs with complex function. This review focuses on the engineering of heart valve tissue, a goal which involves a unique combination of biological, engineering, and technological hurdles. We emphasize basic concepts, approaches and methods, progress made, and remaining challenges. To provide a framework for understanding the enabling scientific principles, we first examine the elements and features of normal heart valve functional structure, biomechanics, development, maturation, remodeling, and response to injury. Following a discussion of the fundamental principles of tissue engineering applicable to heart valves, we examine three approaches to achieving the goal of an engineered tissue heart valve: (1) cell seeding of biodegradable synthetic scaffolds, (2) cell seeding of processed tissue scaffolds, and (3) in-vivo repopulation by circulating endogenous cells of implanted substrates without prior in-vitro cell seeding. Lastly, we analyze challenges to the field and suggest future directions for both preclinical and translational (clinical) studies that will be needed to address key regulatory issues for safety and efficacy of the application of tissue engineering and regenerative approaches to heart valves. Although modest progress has been made toward the goal of a clinically useful tissue engineered heart valve, further success and ultimate human benefit will be dependent upon advances in biodegradable polymers and other scaffolds, cellular manipulation, strategies for rebuilding the extracellular matrix, and techniques to characterize and potentially non-invasively assess the speed and quality of tissue healing and remodeling.\nINTRODUCTION\nPotential applications of tissue engineering in regenerative medicine range from structural tissues (e.g., skin, cartilage, bone) to complex organs (e.g., heart and other components of the cardiovascular system, liver, kidney, pancreas). In each case, there are limitations to conventional surgical approaches and existing prosthetic devices, serious complications associated with transplantation, and critical shortages of available donor tissues. Cardiovascular tissue engineering has primarily considered blood vessels,58,102,103,104,114,178 myocardium,27,36,74,80,123 and heart valves.14,119,122,176 This review focuses on the application of tissue engineering technology to heart valves.\nCurrently, adults who undergo replacement of diseased valves by either mechanical prosthetic or tissue valves (including bioprosthetic valves [porcine aortic valve or bovine pericardial xenograft], cadaveric allograft, or pulmonary-to-aortic autograft valves [Ross procedure]) generally have enhanced survival and quality of life.127 Nevertheless, each of these valve types has its limitations\u2014in particular, mechanical valves require anticoagulation to control thromboembolism, while bioprosthetic and allograft valves frequently undergo calcification and structural deterioration.50,64,146\nAdvantages of an engineered tissue heart valve would likely include nonthrombogenicity, infection resistance, and cellular viability. The design criteria and characteristics for conventional and tissue engineered replacement heart valves are summarized and compared in Table 1. The most immediate need for heart valve tissue engineering and regeneration technology is in the pediatric and young adult population in which the results of valve replacement are not as favorable as those in older adults.35,68 Most exciting is the possibility of growth, repair, and remodeling as a child recipient matures, thus eliminating the repetitive surgeries typically necessitated by the inability of a valve substitute to enlarge as an individual grows. Only autografts (such as Ross valves transplanted from the pulmonary-to-aortic position in an individual) presently are viable,120 but the Ross procedure is technically difficult, risky, only serves a small patient subset, and has controversial results, including uncertainty over whether the grafts will grow commensurate with recipient growth.67TABLE 1.Design objectives for and characteristics of replacement heart valves.Feature to optimizeConventional (Mechanical, bioprosthetic)Tissue engineeredClosure of leafletsRapid and completeRapid and completeSize of orifice areaLess than that of natural valvesBetterMechanical propertiesStableStableSurgical insertionEasy and permanentEasy and permanentRisk of thrombosisYes, especially mechanical valves, which require anticoagulation, causing vulnerability to hemorrhageNo; endothelial surface to inhibit thrombogenesisRisk of structural dysfunctionDegradation of synthetic materials rare with mechanical valvesResistant to degradation and calcificationTissue degradation and calcification of leaflets with bioprosthetic valvesRisk of InfectionEver presentResistant to infectionViabilityNoYes, able to repair injury, remodel, and potentially grow with patientFIGURE 1.Specialized ECM enables dynamic aortic valve function. (a) Photograph of the aortic valve in open and closed position (from the aorta). (b) Aortic valve histology emphasizing trilaminar structure and presence of valvular interstitial and endothelial cells. (c) Biomechanical cooperativity between elastin and collagen during valve motion. (d) Schematic depiction of layered aortic valve cuspal structure and configuration of collagen and elastin during systole and diastole. (a) and (b) reproduced by permission from Schoen FJ. \u201cValvular heart disease: General principles and stenosis,\u201d IN: Cardiovascular Pathology, 3rd Ed, Silver MD, Gotlieb AI, Schoen FJ (eds.), WB Saunders 2001, pp. 402\u2013442; (c) and (d) reproduced by permission from Schoen FJ. Aortic valve structure-function correlations: Role of elastic fibers no longer a stretch of the imagination. J Heart Valve Dis 6: 1\u20136, 1997.\nThe goal to engineer functional heart valve tissue presents a unique combination of challenges. Normal heart valves are vital and dynamic tissues composed of specialized cells and extracellular matrix (ECM) that respond and remodel in response to changes in local mechanical forces.121,143 Approximately 40 million times a year, opening and closing of the leaflets induces repetitive changes in the shape, dimensions, and stress of the leaflets and supporting valvular structures (Fig. 1).147 A successful tissue engineered valve and its components must not only accommodate those deformations but also have ongoing strength, flexibility, and durability, beginning at the instant of implantation and continuing indefinitely thereafter, possibly despite an evolving tissue architecture.\nThis review provides a contemporary analysis of heart valve tissue engineering and regeneration, emphasizing the evolving understanding of heart valve biology, the promise and difficulties demonstrated by in-vivo studies done to date, and the critical challenges that will be encountered in translating the potential of this exciting therapeutic modality from the laboratory to the clinical realm. To put the technology and the evolving enabling science into context, we begin by examining the complex and dynamic structural components that are needed to accomplish normal heart valve function, biomechanics, physiological tissue maintenance, homeostasis, and ongoing health. Our discussion of the basic principles of tissue engineering summarizes current principles of scaffolding and cell sourcing. Subsequently, we examine three potential approaches to the field of tissue-engineered heart valves: (1) cell seeding of biodegradable synthetic scaffolds, (2) cell seeding of processed tissue scaffolds, and (3) in-vivo repopulation by circulating endogenous cells of implanted substrates without prior in-vitro cell seeding. Finally, we analyze challenges to the field and suggest future directions for both preclinical and translational (clinical) studies that will be needed to address key regulatory issues for safety and efficacy of the application of tissue engineering and regenerative approaches to heart valves. Our primary goal is to stimulate thinking in the field by introducing concepts such as outcome criteria, biomarkers, molecular imaging, product release criteria, questions raised by the integration of engineered heart valves with the host tissues, and uncertainties engendered by patient-to-patient heterogeneity in key biological processes such as tissue remodeling capability. Our discussion in this paper underscores that the development and application of innovative approaches to repair and regenerate damaged or diseased heart valves requires the integration of numerous biological, engineering, and technological principles.\nTABLE 2.Key structural elements of heart valves.ElementSub-structureFunctionExtracellular matrixCollagenProvides strength and stiffness to maintain coaptation during diastole, when cusp has maximal areaElastinExtends in diastole; contracts in systole to minimize cusp areaGlycosaminoglycansAccomodates shear of cuspal layers, cushions shock during valve cycleCellsInterstitialSynthesize ECM; express MMPs and TIMPs that mediate matrix remodelingEndothelialMaintain nonthrombogenic blood-tissue interface; regulate immune and inflammatory reactionsBlood vesselsFew and focal; valve cusps and leaflets sufficiently thin to be nourished by diffusion from the heart\u2019s bloodNervesPresent, with uncertain functionOther principlesCorrugationsAccordian-like folds in cusps; allows cuspal shape and dimensions to vary with cardiac cycleCrimpMicroscopic collagen folding, allows lengthening at minimal stressAnisotropyPermits differences in radial and circumferential extensibilityCordsMacroscopic collagen alignment; transfers forces from cusps to aortic wall\nHEART VALVE FUNCTION AND STRUCTURE\nHealthy native heart valves maintain unidirectional blood flow via an extraordinarily dynamic functional structure with several key characteristics: viability, sufficient strength to withstand repetitive and substantial mechanical stress, and ability to adapt and repair injury by connective tissue remodeling. A rational approach to heart valve tissue engineering depends on a thorough understanding of the complex normal functional elements and their coordinated interactions (Table 2).185 For the trileaflet semilunar valves (aortic and pulmonary), the important structures are the cusps (which avoid prolapse by substantial coaptation), the commissures, and the supporting structures in the aortic and pulmonary roots. For the atrioventricular valves (mitral and tricuspid), the key components are the leaflets, commissures, annulus, chordae tendineae, papillary muscles, and atrial and ventricular myocardium. Valve leaflets and cusps have few and only focal blood vessels (vessels may be present in the proximal portion near the myocardium), likely because valves cusps and leaflets are sufficiently thin to be nourished predominantly by diffusion from the heart\u2019s blood. Valve leaflets and cusps also have nerves, but their significance is uncertain.87\nThe four cardiac valves have microstructural similarities; however, the aortic valve best illustrates the essential features and serves as a paradigm for microstructural and cellular adaptation to functional requirements. The aortic valve is the most frequently diseased and also commonly transplanted. The aortic valve cusps undergo substantial changes in shape and dimension during the cardiac cycle (see Fig. 1a). Microscopically, the aortic valve is composed of three distinct layers: (1) the ventricularis closest to the inflow surface, rich in elastin, (2) the fibrosa closest to the outflow surface, primarily composed of densely packed collagen, and (3) the centrally located spongiosa, largely composed of glycosoaminoglycans (GAGs). Together, collagen, elastin, and GAGs comprise the valvular ECM (see Fig. 1b). Studies of normal, pathological, and substitute valves have demonstrated that the principal determinant of valve durability is the valvular ECM, whose quantity and quality depend on the viability and function of valvular interstitial cells (VIC).145 Recent research suggests that cell-matrix interactions in heart valves are highly stress-dependent and likely reciprocal. In vivo, heart valve leaflets experience a dynamic and complex mechanical stress state during every cardiac cycle: sheer stress due to blood flow (open valve), flexure (opening and closing), and tension (closed valve).92,93 Forces acting on the valve at the macroscopic level (pressure, sheer stress, and tension) are translated into specific biomechanical responses at the tissue level (collagen uncrimping, reorientation, and fiber compaction) which are transduced into a VIC response at the cellular level (with intracellular signaling events leading to changes such as increased alpha smooth muscle actin (\u03b1-SMA) expression, increased VIC stiffness, and increased ECM biosynthesis). Indeed, the higher absolute pressure and transvalvular pressure gradients on the left side of the heart impose larger local tissue stress on the VICs than those on the right side, which leads to higher VIC stiffness (through \u03b1-SMA content) and collagen biosynthesis in the left-sided valves.94\nComplex microstructural rearrangements and several specializations of collagen accommodate the cyclical pressure fluctuations during the cardiac cycle.143 Collagen fibers in a planar orientation in the fibrosa comprise the strongest portion of the leaflet that is responsible for bearing diastolic stress. GAGs in the spongiosa probably serve predominantly as a shock and shear absorber. The large cuspal deformation during the cycle between systole and diastole is facilitated by biomechanical cooperativity between collagen and elastin (see Fig. 1c). Collagen fibrils are inelastic and incapable of supporting large strains; they therefore have adaptations (macroscopic corrugations and microscopic crimp) that permit collagen stretching at minimal stress by unfolding (Fig. 1d). During valve opening, elastin stretches during extension of collagen crimp and corrugations. When the valve is closed, the collagen is fully unfolded and the load is shifted from elastin to collagen; elastin restores the contracted configuration of the cusp during systole.\nTwo types of cells are present in the aortic valve: endothelial cells covering the surface and interstitial cells with variable properties of fibroblasts, smooth muscle cells, and myofibroblasts in the interior (see Fig. 1b).1,121,168,169 Like endothelial cells elsewhere in the circulation, valvular endothelial cells (VEC) maintain a nonthrombogenic blood-tissue interface and regulate immune and inflammatory reactions. Nevertheless, endothelial cells isolated from different vascular and valvular sources display differences in gene expression and other properties (endothelial cell heterogeneity).21 Indeed, heart valve endothelial cells are different from those in the aorta.16,17 For example, in response to mechanical stress, porcine aortic valve endothelial cells align perpendicular to flow whereas endothelial cells from the aorta align parallel to flow and their gene expression is different from that of aortic endothelial cells exposed to the same mechanical environment. These differences suggest a unique phenotype of VEC not mimicked by vascular cells and could have implications for cardiovascular cell biology and cell-source considerations for tissue-engineered heart valves. Moreover, different transcriptional profiles are expressed by the endothelium on the aortic side versus ventricular side of normal adult pig aortic valves.155 The reasons for these differences are not yet known.\nVIC, the most numerous valvular cell type, synthesize ECM and express matrix degrading enzymes, metalloproteinases (MMPs), and their inhibitors (TIMPs) that mediate matrix remodeling.1,169 Moreover, VIC comprise a dynamic population of resident cells of diverse and dynamic phenotypes, in a spectrum that ranges from fibroblast-like to myofibroblast-like; their phenotype is regulated by environmental conditions.1,25,121,168,169, We and others have evaluated VIC phenotypes in normal and diseased human valves. In adult heart valves in situ, VIC are quiescent and display a fibroblast-like phenotype, characterized by the presence of vimentin (intermediate filaments), and very low levels of \u03b1-SMA, MMP-13 (proteolytic enzymes), and SMemb (non-muscle myosin heavy chain). Indeed, only 2\u20135% of normal adult VIC express \u03b1-SMA, which is a marker of myofibroblast-like function.1,121,142,148 Myofibroblasts are activated fibroblasts that synthesize and remodel the specialized ECM, facilitate tissue remodeling and wound healing, and play a pathological role in fibrotic disease.99,148 In contrast, as demonstrated by previous in-vitro studies using isolated cells cultured from heart valves, 56\u201378% of cells are \u03b1-SMA-positive.169 This suggests that removal of cells from the environment of the intact valve (i.e., in culture) or their manipulation stimulates VIC. Moreover, treatment of isolated VICs with TGF-\u03b2 strongly activates interstitial cells to the myofibroblast phenotype.177\nBiomechanical and biochemical factors play an important and potentially synergistic role in determining the local homeostatic environment of the aortic VIC. As demonstrated in in-vitro studies, mechanical stimulation (tension) and cytokine stimulation (TGF-\u03b21) were found to synergistically alter the contractile (\u03b1-SMA) and biosynthetic (heat shock protein, Hsp 47) proteins of aortic VICs to a greater extent than each factor alone.93 The need for mechanical stimulation and\/or cytokines and potentially other soluble factors for the maintenance of appropriate cellular biosynthetic activity will be an important influence on tissue engineering efforts.\nHEART VALVE DEVELOPMENT, MATURATION, ADAPTATION, AND REPAIR\nA thorough understanding of developmental biology, physiology, and pathophysiology of heart valves will likely inform tissue engineering. Indeed, some of the processes and regulatory pathways active in valvular development and maturation may be recapitulated in tissue engineered valves. During embryological development, the three germ layers\u2013ectoderm, endoderm, and mesoderm\u2013give rise to cells that differentiate to form the body\u2019s tissues and complex organs. The heart develops primarily from the embryonic layer called mesoderm.160 The initial commitment of mesodermal precursor cells to a cardiac lineage depends on complex signaling pathways.106 Cardiac myocytes become organized into a linear heart tube that subsequently undergoes looping.174 Growth of the looped heart tube and development of septa leads to the multichambered heart.\nDuring cardiac development, the valve cusps and leaflets originate as outgrowths (known as endocardial cushions) from mesodermal derived connective tissue called mesenchyme.30 Endothelial cells lining the inside surface of the cushion forming area undergo an epithelial-to-mesenchymal transdifferentiation (EMT) and migrate from the blood-contacting internal heart surface deep into the connective tissue of the subendocardium (called cardiac jelly) to become precursors of mature VICs.5 Widespread in embryological development, epithelial mesenchymal transitions comprise a series of cell\u2013cell and cell-matrix interactions that release epithelial cells from a surface and confer the ability to move through three dimensional ECM and synthesize ECM.124 The newly formed mesenchymal cells remodel the cushions into leaflets and cusps. Evidence for EMT is provided by mesenchymal cellular expression of \u03b1-SMA, a marker that is not typically expressed by endothelial cells.31,110 Numerous signaling pathways, growth and transcriptional factors (including vascular endothelial growth factor [VEGF], nuclear factor in activated T cells [NFATc1], and Notch) regulate the process of heart valve formation.5,9,43,78\nWe studied the subsequent maturation and evolution of human semilunar valves in fetuses, neonates, children, and adults.1 We demonstrated that fetal valves contain immature activated cells and are dynamic and adaptable structures; the architecture, collagen content, and organization were immature compared with that of adult valves. During valve development and maturation, fetal VIC have a myofibroblast-like phenotype, characterized by expression of \u03b1-SMA, MMP-13, and SMemb, and continuously remodel the ECM. The cells become quiescent in the normal valve post-natally, suggesting progressive adaptation to the prevailing hemodynamic environment. Moreover, the cell density progressively decreases (by nearly 90%) throughout life.\nThe role of myofibroblasts in valvular wound healing, adaptation, and remodeling is best illustrated by comparisons of normal, diseased, autograft, and tissue engineered valves.121 In conditions of disease (e.g., myxomatous mitral valve),117 adaptation (early pulmonary-to-aortic autograft),120 or remodeling (tissue engineered valves in-vitro and in-vivo),118 VICs have an activated myofibroblast phenotype, similar to that of fetal valves. Normal and pathological cardiac valves respond to environmental conditions, such as mechanical loading, by cell activation and matrix remodeling. In contrast, following return of a stable equilibrium of mechanical state in development, adaptation, or remodeling, VIC return to their normal quiescent fibroblast phenotype, as exemplified by late pulmonary-to-aortic autografts (>3 years postoperative) and tissue engineered valves implanted in-vivo. Therefore, heart valves respond to environmental change via reversible phenotypic modulation of their resident VICs.\nSCAFFOLDS FOR TISSUE ENGINEERING: GENERAL CONCEPTS\nA widely accepted paradigm of tissue engineering comprises (1) a scaffold, that is pre-seeded with (2) cells, followed by (3) an in-vitro stage of tissue formation typically conducted in a bioreactor (that recapitulates a physiological metabolic and mechanical environment), and subsequently, following implantation of the construct, (4) an in-vivo stage of tissue growth and remodeling (Fig. 2, Pathway A).77 The key pathophysiological processes occurring during the in-vitro and in-vivo phases are cell proliferation and migration, ECM production and organization, scaffold degradation, and tissue remodeling. The in-vivo but not the in-vitro phase can involve recruitment of the recipient\u2019s inflammatory cells. The resulting tissue engineered construct will likely contain some combination of seeded and\/or recipient-derived new cells. An alternative pathway (Fig. 2, Pathway B) utilizes an unseeded scaffold that incorporates biological \u201cinformation\u201d designed to attract and direct the formation of circulating endogenous precursor cells (potentially both endothelial and mesenchymal) in-vivo. Both of these pathways are considered in detail in subsequent sections of this paper. Moreover, host inflammatory cells may play a role in the in-vivo phase of either approach. In the following sections we examine the characteristics of biodegradable synthetic scaffolds and natural allograft or xenograft scaffolds; a composite biodegradable polymer with collagen or other natural material may also be considered (Table 3).\nTABLE 3.Comparative analysis of scaffolds.Synthetic scaffoldsNatural scaffoldsAdvantages\u2003Control of material structure and properties (e.g. pore size, stability, degradation rate)Maintain architecture of the native tissue (potentially valve)\u2003Easily reproducedMaintain biological information (e.g., reactive sites, growth factors)\u2003ResorbablePotentially resorbableDisadvantages\u2003Difficulty in controlling cell adhesion and tissue reorganizationDecellularization may alter physical properties\u2003Inflammation due to incomplete polymer degradation or lack of biocompatibilityDifficulty of cell penetration into interior\u2003Space formerly occupied by polymer and its interstices is replaced by fibrosis\/scarMay induce immunologic reaction\u2003Limited perfusion to deep cellsPotential for calcificationFIGURE 2.Tissue engineering paradigms. (Pathway A) The conventional paradigm of tissue engineering comprises a scaffold that is seeded with cells, an in-vitro stage of tissue formation typically conducted in a bioreactor, and an in-vivo stage of tissue growth and remodeling. The key pathophysiological processes occurring during the in-vitro and in-vivo phases are cell proliferation, ECM production and organization, scaffold degradation, and tissue remodeling. The resulting tissue engineered construct will contain some combination of seeded and\/or new cells. A modified paradigm (Pathway B) might utilize an unseeded scaffold that is fabricated with biological \u201cinformation\u201d designed to attract and provide a suitable substrate for differentiation of circulating precursor cells in-vivo.\nSynthetic Scaffolds\nSynthetic polymeric scaffolds generally have well controlled and easily reproduced properties. Ideally, a tissue engineered scaffold should be biocompatible, resorbable, have a highly porous macrostructure (necessary for cell growth, nutrient supply and waste removal) and a surface conducive to cellular attachment and potentially migration, proliferation, differentiation, matrix formation and\/or remodeling. A bioresorbable scaffold serves as a temporary matrix until the seeded cells are capable of producing their own matrix proteins; the chemical and physical properties and the rate of degradation are generally tailored to the application and the rate of new tissue evolution.41,53,69,83,100\nPolyglycolic acid (PGA) and polylactic acid (PLA) and their copolymers are currently the synthetic, biodegradable polymers most widely considered for tissue engineering applications. PGA is a highly crystalline, linear, aliphatic polyester, with a high melting point and low solubility in organic solvents. PGA was used as the first totally synthetic resorbable suture, commercially available under the trade name \u201cDexon\u201d since 1970. PGA is rapidly resorbable; thus, Dexon sutures tend to lose their mechanical strength rapidly, usually within 2 to 4 weeks after implantation. Copolymers of the hydrophilic PGA with the more hydrophobic PLA increase the range of material properties and hence possible applications; for example, the addition of PLA limits the water uptake and reduces the rate of hydrolysis (the predominant model of degradation) as compared to PGA alone. Considered safe, nontoxic, and biocompatible, these polymers are used successfully in a number of approved medical implants.48\nThe use of a synthetic scaffold introduces challenges in the regulation of cell adhesion and three-dimensional tissue reorganization since such materials are usually isotropic and ECM proteins are not usual constituents of synthetic polymers. For example, PGA, PLA, and their copolymers are generally considered poor substrates for cell growth in-vitro.71 Therefore, bioactive synthetic biomaterials are being developed for use as three-dimensional microenvironments that mimic ECM function.84,85 Recent advances include nanofibrillar networks formed by self-assembly of small molecules and artificial ECM networks from protein polymers or synthetic polymers that present bioactive ligands and respond to cell-secreted signals to enable remodeling.115\nAn especially attractive concept is that of smart biomaterial scaffolds, which carry spatial and chemical information that affects cellular function and\/or responds to changes in the environment.76 Smart scaffolds are exemplified by biodegradable elastic shape-memory polymers that predictably alter their shape with changes in temperature79 and polymers that transition between hydrophobic and hydrophilic states in response to electric potential.2,75 Moreover, normal tissue culture dishes grafted with a temperature sensitive polymer PIPAAm (poly N-isopropylacrylamide) respond to temperature changes, which alter the adhesion properties of cells to this matrix. At elevated temperatures (above 37\u00b0C), the dish surfaces are relatively hydrophobic and cells attach, whereas at lower temperatures (20\u00b0C) the polymer surface becomes hydrophilic and swells, forming a hydration layer between the dish surface and the cultured cells. The cells along with their framework ECM spontaneously detach and may be harvested as intact sheets.186 As the technology develops, smart scaffolds may prove important to applications of stem cells in tissue engineering; for example, a new three dimensional material with the ability to direct the differentiation of neural progenitor cells into a specific lineage without the help of growth factors has been reported.154\nTissue engineered biomaterial constructs must also exhibit mechanical and structural properties comparable to the native tissue they replace, including dynamic anisotropic behaviors optimized for tissue specific function, from the time of implantation of a construct through the lifetime of the recipient. This implies central requirements of relatively constant mechanical properties despite potentially considerable tissue remodeling and scaffold resorption. Moreover, mechanical anisotropy (certainly a characteristic of the natural valves)92 may be desirable; towards this end, electrospun scaffolds were fabricated which exhibited highly anisotropic mechanical properties that resembled native pulmonary heart valve cusps.136\nPotential undesirable features of synthetic scaffolds include local tissue inflammation owing to the foreign body reaction and slow and\/or incomplete polymer degradation. As the scaffold degrades, the space formerly occupied by a polymer and its interstices is progressively filled by cells and ECM which may eventuate in fibrosis (scar) that poorly resembles specialized native tissue and may contract and distort during maturation. In some cases, cells on the scaffold periphery are healthy and resemble native differentiated parenchymal (i.e., function-specific) tissue whereas cells at the interior become necrotic due to restricted deep delivery of oxygen and nutrients, and removal of wastes.\nNatural Scaffolds\nAlternative to synthetic scaffolds, natural scaffolds usually comprise pure ECM components (such as collagen or fibrin) or decellularized but otherwise intact allograft or xenograft tissue (such as heart valve or small intestinal submucosa). In cardiovascular tissue engineering, multi-step static seeding methods have been most commonly used; alternatively, efficient single step seeding may be achieved via cellular encapsulation in collagen and fibrin gels. Collagen based constructs seeded with vascular smooth muscle and\/or endothelial cells have been utilized to generate tubular blood vessels,178 one-dimensional strings to serve as a component of composite aortic valve cusps150 or chordae tendineae151 and molds for heart valve leaflets.171 Cells entrapped in collagen gels contract and compact the gels in a process similar to the contraction of a wound during healing, thus increasing their density and enhancing their properties.170 Alternatively, cells can be encapsulated in an autologous fibrin gel which initially serves to obtain uniform cell distribution and to improve the seeding efficiency.66,97,134,187 With gel encapsulated cells, newly synthesized ECM accumulates in the immediate extracellular space, rather than diffusing into the surrounding medium.187 Reported limitations of the encapsulated cell approach to tissue engineering include excessive shrinkage of the gel\/cell construct,66 and the possibility that the entrapped cells become necrotic or apoptotic.166 In one in-vitro study, cells encapsulated in a fibrin gel were seeded onto a synthetic PGA scaffold to optimize tissue formation and organization. Following fibrin degradation, the underlying synthetic scaffold contributed to the structural integrity of the developing tissue.97 Additionally, hyaluronan, a viscoelastic and broadly biocompatible material that plays a role during embryonic cardiac development, is also being explored as a potential ECM scaffold material.88,129\nSome investigators favor enzymatically decellularized tissue as a natural scaffold. This treatment is done to decrease antigenicity and the risk of calcification (both of which are enhanced by cells and their debris). The hope with a decellularized scaffold is that preseeded or endogenous circulating cells can repopulate such a scaffold. A decellularized allograft or xenograft tissue scaffold may best serve as a template for cellular attachment and retain many of the mechanical and structural properties of native tissue such as tensile strength and unique ECM composition.54\nDecellularized porcine small intestinal submucosa (SIS) has been studied extensively as a natural resorbable scaffold material that does not require cell seeding.7 In both animal and human clinical studies, SIS was rapidly remodeled by the host tissue.133 Useful in implants ranging from particulate material related to the bladder,182 to sheets that might be used to repair the infarcted left ventricle,132 SIS has exhibited good vascularization and tissue growth without excessive inflammation and foreign body reaction. The success of SIS has been attributed to its intrinsic ECM proteins, GAGs, cytokines, and growth factors (VEGF and TGF-\u03b2).130\nCELLS FOR TISSUE ENGINEERING: GENERAL CONCEPTS\nWhile the ultimate goal of tissue engineered heart valves is to recapitulate the matrix and cells found in native tissue, there exists variability in potential strategies and sources of cells. The predominant paradigm provides cell-seeded scaffolds (biodegradable or natural) with the ingredients and environment to form tissue and mature in-vitro in a bioreactor, in order to generate a construct which is then implanted in-vivo in the desired anatomic location (recall Fig. 2, Pathway A). Potential cellular sources for seeding of scaffolds to fabricate heart valves include differentiated tissue-specific cells (such as endothelial and\/or smooth muscle cells91) and stem cells that may be autologous or allogenic. In a clinical study, an in-vitro endothelialization procedure, in which femoropopliteal arterial grafts composed of expanded polytetrafluoroethylene (ePTFE) were confluently lined with cultured autologous endothelial cells before implantation, was assessed for its ability to improve the long-term patency of these prosthetic bypass grafts. The results (regrettably not compared with concurrent unseeded control grafts) suggested that autologous endothelial cell lining improved the patency of these small-diameter vascular grafts.26 Another option is to harness the potential of endogenous cells by utilizing decellularized biological scaffolds that contain intact ECM and other chemical signals necessary to recruit the appropriate cell populations (recall Fig. 2, Pathway B and see below); unseeded scaffolds that could attract endogenous cells in-vivo to the site of implantation might permit bypassing the in-vitro stage of cell seeding, by facilitating repair by endogenous cells. Nevertheless, presently used tissue heart valve replacements do not endothelialize from circulating blood to a degree sufficient to provide functional benefit. This holds true for both glutaraldehyde-pretreated porcine valves,59 in which the barrier to endothelialization may result from toxicity induced by residual glutaraldehyde,37 and cryopreserved allograft heart valves.95\nThe unique properties of stem cells, such as multipotency and capacity for self-renewal, make them attractive cells for tissue engineering.22,46 Stem cells are found in the bone marrow of adults, including hematopoietic stem cells (which form the mature blood cells), endothelial stem cells (endothelial progenitor cells, which form components of the cardiovascular system), and mesenchymal stem cells (which form bone, cartilage, muscle, fat, and fibroblasts). Bone marrow-derived adult somatic stem cells are an attractive cell source because they are multipotential cells, in principle capable of differentiation, transformation, and regeneration. We will not discuss stem cells of embryonic origin owing to the complicated scientific ethical and moral issues surrounding their usage. Bone marrow comprises an ideal cell source for tissue engineering because its cells are easily accessible, its primary isolate is a cell suspension that is easier to process and less prone to contamination than solid tissue, and bone marrow will likely be the primary source of cells for endogenous repopulation.164 Throughout post-natal life, both bone marrow-derived and organ-resident adult stem cells continuously regenerate some tissues (such as skin epithelium, intestinal epithelium, blood cells, and liver when stimulated). Nevertheless, increasing evidence suggests that the heart and the brain can regenerate some of their mass, defying the conventional wisdom that these organs cannot replenish cells lost as a result of maturation, senescence, and injury.33,81\nEndothelial progenitor cells (EPCs) are bone marrow-derived hematopoietic stem cells capable of differentiating into the endothelial cells that line the blood vessels and cardiac valves.56,61,125,173 EPCs maintain vascular homeostasis by promoting reendothelialization after endothelial injury and neovascularization after tissue ischemia. Endothelial progenitor-derived cells obtained from peripheral blood have been expanded in-vitro and seeded on a three dimensional biodegradable PGA-PLLA scaffold. When seeded alone, they maintained an endothelial phenotype for the entire six-week duration of implantation and when co-seeded with smooth muscle cells, the endothelial progenitor derived endothelial cells formed microvessels on the scaffold.184 EPCs obtained from peripheral blood have also been used to line small diameter vascular grafts.70\nMesenchymal stem cells (MSCs), have the potential for differentiating into osteogenic, chondrogenic, adipogenic, and myogenic lineages.113,116 These can be isolated from adult bone marrow and represent another adult stem cell population that can be used as a cellular source in tissue engineering.86,90,112 The concept of continuous replacement of connective tissue with bone marrow mesenchymal stem cells parallels the known continuous replacement of blood by bone marrow hematopoietic cells. Cultured MSCs display the spindle shape morphology characteristic of myofibroblasts and express cell markers characteristic of VIC. Upon implantation, these cells retain profiles identical to those seen in-vitro. Following myocardial infarction in a mouse model, injected MSCs improve recovery of the infarcted tissue and were thought to differentiate into cardiac myocytes, endothelial cells, and vascular smooth muscle cells.107 Marrow progenitor cells or multipotent adult progenitor cells (MAPCs) have many attributes of MSCs but they are reported to expand indefinitely (compared to 1 million fold expansion of MSCs) and may have lineage potential that includes ectodermal and endothelial cell types, making them similar to embryonic stem cells.65\nBIOREACTORS FOR TISSUE ENGINEERING\nMechanical stimulation through the use of bioreactors during in-vitro tissue development is widely utilized in cardiovascular tissue engineering for improving tissue formation, organization, and function. The bioreactor exposes the developing tissue to mechanical conditioning, primarily through cyclical flow and pressure changes that mimic physiological conditions. Bioreactors have been developed that use flow52,101,158,181 and strain103,161 as their main mechanical cues to engineer blood vessels and heart valves. For the engineering of heart valves, a diastolic pulse duplicator bioreactor has been developed to mimic only the diastolic phase of the cardiac cycle, resulting in dynamic tissue straining.96 Additionally, electrical stimulation (designed to mimic native excitation-contraction coupling) has been used as a cue to enhance the structure and function of pulsatile myocardium in-vitro.123 The optimal conditioning protocol depends on numerous parameters such as the sensitivity of the cell phenotype to mechanical cues, the scaffold used, the transfer of the mechanical cues from the scaffold to the cells, and the magnitude and type of mechanical cues.\nKEY IN-VIVO STUDIES IN HEART VALVE TISSUE ENGINEERING\nMany published studies have used in-vitro methods to investigate critical variables and demonstrated \u201cproof-of-principle\u201d of important concepts in tissue engineering germane to heart valves. Only a few key studies have been conducted using synthetic and natural scaffolds in animal models (Table 4). Early studies focused on the design of individual valve leaflets188 whereas later studies emphasize design of complete valved conduits. In this section, we focus on studies in which scaffolds seeded in-vitro were implanted in-vivo. Following this section, we discuss the possibility that the recipient can provide all the cells needed to populate and\/or remodel a scaffold to yield a functional heart valve in-vivo.\nTABLE 4.Representative, animal, and clinical implant studies using seeded and non-seeded matrices.StudyScaffoldCellsSiteIn-vitro seeding\u2003(A) Shinoka (1995\u201396)Polyglycolic acid (PGA)Autologous ovine endothelial cells and fibroblastsReplacement of one pulmonary valve (PV) leaflet in sheep\u2003(B) Hoerstrup (2000)Poly-4-hydroxybutyrate (P4HB) coated PGAAutologous ovine endothelial cells and myofibroblastsReplacement of all three PV leaflets in sheep\u2003(C) Steinhoff (2000)Decellularized pulmonary sheep valvesAutologous ovine endothelial cells and myofibroblastsPV conduits implanted into sheep\u2003(D) Dohmen (2002)Decellularized cryopreserved pulmonary allograftAutologous human vascular endothelial cellsReconstruction of the right ventricular outflow tract (RVOT) in a human patient\u2003(E) Perry (2003)P4HB coated PGAAutologous ovine mesenchymal stem cellsIn-vitro only, no in vivo implantation\u2003(F) Iwai (2004)Poly(lactic-co-glycolic acid) (PLGA) compounded with collagen microspongeAutologous endothelial and smooth muscle cells; w\/ and w\/o in-vitro precellularizationPatch implant in canine pulmonary artery\u2003(G) Sutherland (2005)PGA and poly-L-lactic acid (PLLA)Autologous ovine mesenchymal stem cellsReplacement of all three PV leaflets in sheepIn-vivo only (No in-vitro seeding)\u2003(A) Matheny (2000)Porcine small intestinal submucosaN\/AReplacement of one PV leaflet in a pig\u2003(B) Elkins (2001)Decellularized (using SynerGraft treatment) human (CryoValve SG) and sheep pulmonary valvesN\/ASynerGraft-treated and cryopreserved sheep PVs implanted in RVOT in sheep; CryoValve SG human PVs implanted in patients\u2003(C) Simon (2003)Decellularized porcine Synergraft valveN\/AImplanted in RVOT in children\nImplant Studies Using Synthetic Scaffolds Seeded In-Vitro\nIn this approach a bioabsorbable polymer provides a temporary scaffold until cells seeded in-vitro produce their own matrix proteins (see Fig. 2, Pathway A). The biodegradable polymer PGA and related compounds have been used as biodegradable synthetic polymer scaffolds because this polymer (albeit with suboptimal mechanical properties) is well characterized and approved by the FDA in sutures and other devices for human implantation. In one of the earliest experiments in the field, an isolated tissue engineered heart valve leaflet was implanted in the pulmonary position of sheep using a PGA scaffold seeded with vascular wall cells.152,153 Histologic evaluation of the constructs showed development of an ECM, endothelialization of the surface, and scaffold remodeling. While this preliminary experiment showed that a tissue engineered valve leaflet constructed from its cellular components can function in the pulmonary valve position, the resultant engineered tissue heart valve cusps were thicker, stiffer, and less pliable than native valves.\nTo alleviate the problem of scaffold thickness and rigidity, Hoerstrup et\u00a0al.52 designed a scaffold composite that combined PGA with the strong, flexible poly-4-hydroxybutyrate (P4HB) formed in the configuration of a trileaflet heart valve. P4HB is added to prolong mechanical integrity because PGA degrades faster than P4HB (approximately 4 weeks versus 8 weeks respectively). This scaffold was seeded with differentiated autologous vessel-derived ovine endothelial cells and smooth muscle cells. To simulate a biomimetic environment during tissue formation, the constructs were grown for varying time points in an in-vitro pulse duplicator system under gradually increasing flow and pressure conditions while controls were grown in static nutrient medium. After 14 days of in-vitro culture, the constructs (valves) grown in the bioreactor showed significantly increased DNA content, higher formation of matrix proteins, a more organized histological structure, and more favorable mechanical properties than did constructs grown under static culture conditions. Based on these preliminary in-vitro studies, seeded constructs that had been matured in-vitro in the bioreactor for 14 days were implanted in-vivo as a pulmonary valve replacement in an ovine animal model. After 20 weeks in vivo, the polymer had been degraded and replaced by a partially endothelialized uniform, layered tissue with layer-specific ECM predominance similar to that of the native valve, including a layer containing elastin near the inflow surface, gylcosaminoglycans centrally, and a fibrous layer with abundant collagen near the outflow surface. Mechanical properties were comparable to those of native tissue at 20 weeks.52 Particularly exciting was the presence of a trilaminar structure resembling the native pulmonary valve, which indicates that dynamic growth, remodeling, and asymmetric structural differentiation had occurred in-vivo, probably regulated by the mechanical environment.\nBuilding on these principles, an autologous trileaflet heart valve was created from a biodegradable synthetic scaffold [PGA and poly-l-lactic acid (PLLA)] seeded with autologous bone marrow-derived MSCs. The cell-scaffold construct was cultured in-vitro then implanted into the pulmonary artery position of sheep for up to 8 months. The results showed that the construct remodeled in-vivo to a tissue with features that simulated those of native valves. In particular, the tissue-engineered valves displayed a trilaminar distribution of cells and ECM, analogous to those of native valves\u2013myofibroblasts immediately below the endothelium, fibroblasts expressing vimentin distributed throughout the remainder, and endothelialization. These findings support the potential use of bone marrow derived MSCs as a cell source for the fabrication of heart valves. The editorial accompanying this paper emphasized that, although proof-of-concept has been demonstrated based on remodeling of the construct to a trilaminar structure following implantation, there remain numerous challenges to overcome before such technology can be used in human patients.6\nImplant Studies Using Biologic Scaffolds Seeded In-Vitro\nAn especially attractive concept is that of a native valve biological scaffold, either decellularized xenograft28,72,141,163 or allograft, that has been seeded with autologous cells in-vitro.10,158 One study suggested that allogenic decellularized sheep matrix conduits seeded with autologous myofibroblast and endothelial cells may yield viable valves. Following almost complete removal of cells, control unseeded allogenic acellular valves implanted in sheep for up to 3 months showed partial degeneration and no interstitial tissue reconstitution whereas the counterparts seeded with autologous vascular wall cells were reported to show restitution of the endothelial cell surface, myofibroblasts, and matrix synthesis.162\nOwing to the antigenicity of xenograft ECM proteins, xenograft scaffolds likely provide a more inflammatory stimulus than allograft scaffolds, exemplified by the cryopreserved homografts presently used clinically.131 An inflammatory reaction could weaken or scar the heart valve scaffold, making it more susceptible to biomechanical damage. Therefore, human allograft heart valves, either decellularized or not, have been considered as an alternative scaffold. Presently used cryopreserved allograft valves are effectively decellularized following several months of in-vivo function, yet they do not grow, remodel, exhibit active metabolic functions, or recellularize, even following long-term function.95 Moreover, decellularization may alter the physical properties of native valves and newly seeded cells may initially have difficulty growing into a decellularized matrix.FIGURE 3.A representative hypothesis for the population of a tissue engineered heart valve by endogenous cells. Key processes include proliferation, differentiation, and mobilization of endothelial progenitor cells within the bone marrow, followed by recruitment in the blood and adhesion to the valve. Subsequently, recruited cells might undergo an epithelial to mesenchymal transdifferentiation within the valve (recapitulating development), followed by differentiation to interstitial cells that ultimately synthesize and remodel the ECM.\nHARNESSING THE REPARATIVE POTENTIAL OF CIRCULATING ENDOGENOUS CELLS: UNSEEDED SCAFFOLDS\nAs discussed in the previous section, the basic paradigm of tissue engineering uses a cell seeded scaffold, an in-vitro stage of tissue formation, and an in-vivo stage of tissue growth and remodeling (see Fig. 2, Pathway A). In this section, we examine an alternative pathway (see Fig. 2, Pathway B) in which an unseeded scaffold could have the potential for attracting circulating precursor cells (endothelial and mesenchymal) in-vivo (Fig. 3).\nAccumulating evidence suggests that circulating endogenous cells can be recruited in-vivo to adhere to intravascular sites via a pathway that likely mimics the adherence of inflammatory cells to the endothelium during physiological inflammation.73 EPCs promote endothelial regeneration in dog models by covering implanted Dacron grafts149 and in human studies by covering the blood-contacting surfaces of implanted ventricular assist devices,40,126 and homing to stents that have been coated with CD34 antibody (a marker found on EPCs).3 Recent studies have suggested therapeutic potential of EPCs in humans.125 Ischemic heart disease patients with naturally higher levels of EPCs had a reduced risk of death from cardiovascular causes.180 Following experimental myocardial infarction, bone marrow derived cells are recruited by selective homing to the area of injury.98 It has been suggested that intracoronary infusion of EPCs in patients with acute myocardial infarction might potentially contribute to restoring myocardial and endothelial function to the damaged area.109 Endogenous stimuli such as tissue ischemia and exogenous cytokines promote mobilization of EPCs.18 Patients with vascular trauma such as acute myocardial infarction display increased numbers of EPCs, which is positively correlated to elevated plasma VEGF.165 VEGF and other angiogenic growth factors such as angiopoietin-1, fibroblast growth factor and stromal cell-derived growth factor-1 promote EPC mobilization and recruitment.172 One potential strategy may be to coat a scaffold with appropriate cell-signaling molecules in an effort to encourage EPC adhesion and differentiation. An experiment utilizing decellularized porcine aortic valves containing fibronectin and hepatocyte growth factor suggested that the growth factor enhances early endothelial cell recruitment and coverage of the grafts.108\nRecent experimental evidence suggests that human bone marrow may be a source of progenitor cells contributing smooth muscle-like cells to adult human heart valves.23,157 Like endothelial cells, smooth muscle cells can also be recruited to sites of vascular injury.82,138 Experimental evidence in mouse models suggests that bone marrow derived smooth muscle cells may be implicated in degenerative aortic stenosis167 and atherosclerosis.135,137,138,139 Evidence of smooth muscle cell recruitment is exemplified by a prototype tissue-engineered vascular graft. In an experiment in which rat arteries were acellularized, recellularized with endothelial cells, and implanted as grafts in the femoral artery for four weeks, immunohistochemical staining of explanted grafts demonstrated a complete layer of endothelial cells on the luminal surface and smooth muscle cell repopulation. Since smooth muscle cells were not originally seeded onto the graft, the authors suggested that they were recruited to the graft from the bone marrow by a mechanism that involved endothelial cells.13\nUnseeded scaffolds have been examined experimentally, with the goal of achieving in-vivo recellularization by circulating endogenous cells. In one experiment, each of four pigs had one pulmonary valve leaflet excised and replaced with a leaflet constructed from porcine SIS. Histology indicated that the implanted matrix was progressively resorbed and replaced by fibrous connective tissue that had features of adult valve.89 Alternatively, a biodegradable graft containing collagen microsponge was fabricated and tested with and without preseeding.60 In both cases (SIS and collagen microsponge) there was no thrombus formation, the scaffold was absorbed, and there was endothelialization, parallel smooth muscle cell alignment, elastin, and collagen fibers. These results suggested that the patch promoted in-situ cellularization and regeneration of autologous tissue. However, an important limitation of these studies was that the implanted leaflets and patches were small; cellularization of a large implant may be less efficient.\nCLINICAL STUDIES USING ENGINEERED MATRICES AS HEART VALVES\nIn this section we examine the limited number of studies using engineered matrices in clinical settings. One study used a decellularized pulmonary allograft seeded with autologous endothelial cells and conditioned in bioreactor to reconstruct the right ventricular outflow tract of adults undergoing the Ross procedure.29 According to the investigators, based on a one-year follow-up, seeded endothelial cells remained on the construct and were fully functional and the construct mechanical strength was maintained. There was no calcification and\/or thrombogenesis. However, whether the seeded cells contributed to valve function is yet uncertain.\nDespite promising results in animal experiments using decellularized xenograft scaffolds, translation to humans has been difficult. Clinical applications of implanted decellularized xenograft tissue heart valves have been largely unsuccessful.156 Histological examination of decellularized porcine aortic SynerGraft valves (Cryolife Inc.) implanted for 6 months in sheep without in-vitro preseeding suggested some growth of host cells on intact leaflets and showed a lack of calcification.32 Simon et\u00a0al. used SynerGraft decellularized porcine heart valves as valve replacements in the right ventricular outflow tract during Ross procedures in children. The decellularized valves were not seeded or conditioned in a bioreactor before implant with the hope that the unseeded scaffold could attract endogenous cells. These valves had a high rate of failure; examination of failed valves revealed incomplete initial decellularization, lack of cell repopularization, lack of endothelialization, severe inflammation, fibrous sheath formation, calcification and severe degeneration of both leaflets and wall.156 A recent report showed a case with infiltration of a Synergraft valve by inflammatory cells (neutrophils and macrophages) at 5 weeks post implantation.140\nCHALLENGES FOR FUTURE TRANSLATION TO THE CLINIC\nHeart valve tissue engineering has exciting potential but many unanswered questions and challenges remain before human implantation can be considered. A successful tissue engineered valve must be vital, complex, dynamic, composed of specialized cells and ECM that remodel in response to changes in local mechanical forces, and have ongoing strength, flexibility, and durability, beginning at the instant of implantation and continuing indefinitely thereafter. A schema for the interrelationships among and challenges in tissue characterization for heart valve tissue engineering is summarized in Fig. 4. To provide an agenda for translating the notion of TEHVs from an extraordinarily interesting research curiosity to a clinically useful surgical tool, we discuss below both the major research goals\u2014i.e., understanding mechanisms, defining animal models, developing biomarkers, developing assays\/tools, defining surrogate and true endpoints\u2014and the major clinical goals\u2014i.e., characterizing and assuring quality tissue constructs, accommodating patient-to-patient heterogeneity in tissue remodeling, and predicting outcomes as early as possible.\nTABLE 5.Critical challenges to clinical translation of heart valve tissue engineering.ChallengesStrategy for translationTEHV components and function are complex, heterogeneous and dynamicDevelop guidelines for the pre-implantation characterization of TEHV structure, function and qualityTEHV function depends upon patient response to implantation and integration with the recipient\u2019s tissues more than conventional valve replacementIdentify\/validate biomarkers predictive of implant success\/failure and capable of non-invasive in-vivo monitoringIndividuals differ in the speed and effectiveness of their tissue remodelingAssess\/control patient variability in tissue remodeling capabilityOwing to the key role of patient response, animal models may not reliably predict human outcomesValidate suitable animal models that will test key biological processes and correlate with human outcomesRemodeling processes after implantation may release or change seeded cells and recruit host cellsDevelop tools to monitor the fate of transplanted and endogenous cells (location, function, viability, phenotype)FIGURE 4.Paradigm for translating research in heart valve tissue engineering from the laboratory to the clinic. Biomarkers for cell and tissue characterization in conjunction with structural, chemical and molecular information obtained via in-vitro and in-vivo models are necessary for understanding key biological processes in tissue engineering and regenerative medicine. These concepts and data can be used to predict and measure patient success and failure. Data from clinical experience further informs the development of appropriate biomarkers, which may result in reassessment of the appropriate characterization parameters.\nNumerous steps must be surmounted in the laboratory before heart valve tissue engineered constructs can be made clinically useful. Typical biomaterial-tissue interactions in medical devices, such as thrombosis, infection, and inflammatory interactions, will have to be acceptable. Another important consideration is whether calcification, the major pathologic process in bioprosthetic valve degeneration, will be problematic. Evidence suggests that calcification may not be a major problem as long as polymer or other scaffold is resorbed and\/or not intrinsically mineralizable, the interstitial cells are viable, and the ECM is capable of remodeling.\nWhile studies using animal models such as sheep are promising, further detailed studies will be needed in these models, other animal models, and in humans. There is considerable controversy over to what extent results from available animal models translate directly to humans and the most suitable animal model for testing tissue-engineered valves has not yet been determined. Sheep for example, produce an exuberant fibrotic response to cardiovascular implants; valves implanted in sheep generally overgrow more rapidly with fibrotic tissue than they do in humans, and likely overestimate tissue remodeling relative to humans.144 Owing to immunologic considerations, the choice of an animal model for preclinical testing for allogenic or xenogeneic cell-based therapies presents unique challenges.\nA key consideration is that currently available heart valve replacements have predictable behavior in many recipients whereas in-vivo remodeling of tissue engineered heart valves will likely display considerable variability among patients, owing to heterogeneity among individuals in physiological tissue remodeling potential. As the field of tissue engineering evolves it may become important to apply principles analogous to those of pharmacogenetics, a field which seeks to understand the role of genetics in inter-individual variation in drug metabolism.179 Simply stated, some patients might not appropriately remodel their tissue-engineered valves, and this could lead to failure (Fig. 5). This could be a result of mutations or polymorphisms in key proteins central to remodeling.11,24,38,91 Indeed, as implants have become more interactive and integrative with the host tissues, there has arisen a corresponding need to understand and potentially control human variation in different facets of biomaterial-tissue interaction and the healing process. To accommodate TEHVs, the usual mechanism for demonstrating pre-clinical safety and efficacy of medical devices and biologics may need to be altered due to unpredictability of the engineered tissue with the recipient\u2019s native tissue.FIGURE 5.A hypothesis for inter-individual variability in tissue remodeling. While most individuals will remodel tissue with a usual speed and quality of remodeling, some people will display slow and poor quality of remodeling while others will show fast and better quality of remodeling. Inadequate remodeling could lead to implant failure and its consequences for the patient. The threshold of properties needed for tissue engineered heart valves and the means of conducting post-implantation surveillance of the patient and graft need to consider this variability. Success or failure may be followed and predicted non-invasively.\nTo understand, monitor, and potentially control patient-to-patient differences in wound healing and tissue remodeling capability in-vivo, biomarkers that predict implant outcomes must be identified. Conventional and innovative invasive and\/or non-invasive anatomic and functional imaging modalities will certainly be important tools to assess success and failure. Specific molecular biomarkers may be identified and validated by assessing tissue healing and remodeling during in-vitro and in-vivo experiments; suitable biomarkers will need to be followed in-vivo, possibly via chemical assays in the serum or urine or via molecular imaging. Key targets for characterizing tissue-engineered constructs include tissue composition, cellular gene expression and phenotype, ECM, key effectors of tissue remodeling and tissue quality. For example, researchers are currently working to identify serum-specific biomarkers of ECM remodeling in disease such as MMPs in acute coronary syndromes,4,42 and urine-based biomarkers for cancers of the breast,39,136 bladder,49,175 and prostate.55 These biomarkers should correlate directly with success and failure in order to generate surrogate endpoints, namely outcome measurements (such as laboratory assays or imaging results) that substitute for but reflect the mechanism of a significant clinical event or characteristic (such as regurgitation, stenosis, thromboembolism, calcification, infection, or death). Validated surrogate end points could be assessed in an individual patient, in order to predict outcome as early as possible in the patient\u2019s course and influence necessary changes in management.\nThe potential for molecular imaging is particularly exciting in this regard; it requires the identification of a molecular target, selection of a ligand that binds the target, selection of an appropriate imaging system, and synthesis of a molecular imaging agent to detect the desired target.8,44,57,62,63In-vivo molecular imaging has been used to demonstrate key enzymatic and cellular events in atherosclerosis and thrombosis. For example, imaging studies on inflammatory markers such as proteases (cathepsins and MMPs), activated macrophages (expressing iron oxide), and activated endothelium (intercellular and vascular adhesion molecules) have been performed in atherosclerotic mice.19,24 Molecular imaging can probe polymorphisms of ECM gene expression in-vivo in models of cardiovascular disease,12,105 and can potentially be translated to perform real-time in-vivo characterization of scaffold matrices (either seeded or with the potential of attracting endogenous cells) implanted in animal models. Other imaging modalities such as optical coherence tomography (OCT)15,47 and intravascular ultrasound (IVUS)111 have been used to assess collagen content of coronary atherosclerotic plaque; allowing real-time in-vivo analysis without tissue sampling. Such imaging modalities may prove useful in assessing tissue remodeling for heart valve tissue engineering applications.\nAnother important laboratory consideration for seeded scaffolds is the origin of the cells seeded in-vitro and whether the seeded cells remain viable and attached to the scaffold following in-vivo implantation. In the absence of in-vitro cell labeling, it is not possible to ascertain the fate of preseeded cells and the precise origin of the cellular phenotypes observed in the explanted tissue engineered valves. Molecular imaging could be utilized to track the presence, migration, proliferation, and function of bone marrow derived progenitor cells used to seed scaffolds both in-vitro and in-vivo.62,63 Moreover, molecular resonance imaging (MRI) of magnetically labeled mesenchymal stem cells injected into porcine myocardium has been performed in-vivo,51 a technique which can potentially be expanded to study magnetically labeled endothelial and mesenchymal progenitor cells seeded on a scaffold and implanted into an animal or human model. In future experiments, endothelial progenitor and mesenchymal stem cells might be labeled during the in-vitro stage and then analyzed using molecular imaging to ensure that they differentiate into appropriate cell lineages and that they remain functional and attached to the scaffold over time.\nThere is a need to develop clinical guidelines that specify how to characterize the safety, efficacy, and quality of a tissue engineered product before it can be implanted in humans. Demonstration of long-term efficacy (implantability, functionality, long-term performance) and safety (biocompatibility, durability, modes of failure, ease of monitoring) of these valves in humans will be a particular challenge. Risk\/benefit relationships of engineered tissue may be less predictable than those of accepted technology. Since contemporary heart valve replacements have considerable success in most situations (not withstanding the limitations, and except in pediatrics), acceptance of tissue engineering by the surgical community may be slow. It has been suggested that surgeons will consider the use of a tissue-engineered valve in a patient beyond appropriately controlled clinical research only after the 15-year lifetime of conventional valve substitutes can be exceeded with a high degree of certainty.128\nAnother key need is the development of science-based approaches to the characterization of fabricated\/manufactured engineered tissue products in general and heart valves in particular. These will likely include measurement of mechanical properties of the scaffold and the tissue-scaffold complex, characterization of the dynamic cell phenotypes and ECM components, and the evolution of the final manufactured product, including shelf-life, stability, and shipping considerations.\nCONCLUSIONS\nThe goal of heart valve tissue engineering is to regenerate a functional structure containing endothelial and interstitial cells capable of continuously remodeling the ECM that functions structurally and biomechanically as a valve leaflet. Despite an exciting potential for tissue engineered heart valves, significant technical barriers must be overcome before widespread clinical application can be envisioned. Further success toward a clinically useful tissue engineered heart valve will be dependent upon additional advances in biodegradable polymers, stem cell manipulation, strategies for recreating the ECM, understanding how to harvest the potential of endogenous recruitment of cells, and techniques to non-invasively assess the speed and quality of tissue healing and remodeling. This need is likely to engender a host of novel testing strategies and methods, which will include in-vitro safety studies, ex-vivo performance characterization in functional testing devices akin to bioreactors, and in-vivo preclinical studies.\nNOTE ADDED IN PROOF\nSeveral studies relevant to and that became available during final production of this manuscript may interest readers.\nVisconti, R.P., Y. Ebihara, A.C. LaRue, P.A. Fleming, T.C. McQuinn, M. Masuya, H. Minamiguchi, R.R. Markwald, M. Ogawa, and C.J. Drake. An invivo analysis of hematopoietic stem cell potential: hematopoietic origin of cardiac valve interstitial cells. Circ. Res. 98:690\u2013696, 2006.\nTo test the hypothesis that hematopoietic stem cells (HSC) may be a source of adult valve interstitial cells, single lineage-negative (Lin-), c-kit(+), Sca-l(+), CD34- cells from the bone marrow of mice that ubiquitously express enhanced green fluorescent protein (EGFP) were transplanted into a lethally irradiated congenic non-EGFP mouse. Histological analyses of valve tissue from clonally engrafted recipient mice revealed the presence of numerous EGFP+ cells within host valves, some of which exhibited synthetic properties characteristic of fibroblasts (expression of mRNA for procollagen 1 alpha 1). The cells were shown to be the result of HSC-derived cell differentiation and not fusion with host somatic cells. Together, these findings demonstrate a contribution by HSCs to the adult valve interstitial cell population in mice.\nCao, F., S. Lin, X. Xie, P. Ray, M. Patel, X. Zhang, M. Drukker, S.J. Dylla, A.J. Connolly, X. Chen, I.L. Weissman, S.S. Gambhir, and J.C. Wu. In vivo visualization of embryonic stem cell survival, proliferation, and migration after cardiac delivery. Circulation 113:1005\u20131014, 2006.\nAs discussed in the body of the present manuscript, monitoring the trafficking and function of stem cells in vivo remains problematic owing to limitations of conventional histological assays and imaging modalities. A recent study demonstrated a method by which embryonic stem (ES) cells could be stably transduced with a lentiviral vector carrying a novel triple-fusion (TF) reporter gene, tracked by positron emission tomography, and monitored for survival, proliferation, and migration. This imaging platform should have broad applications for basic research and clinical studies on stem cell therapy.\nKiernan, T.J. Endothelial progenitor cells in 2006 \u2013 where are we now? Cardiovasc. Pathol. 15:236\u2013239, 2006.\nA recent brief review of the current status of endothelial progenitor cells (EPCs), including their role as biomarkers and potential therapeutic applications, may be useful to the reader of the present manuscript. The authors emphasize critical questions relating to the characterization of the biological phenotype of \u201ctrue\u201d EPCs and the mechanisms of interaction of EPCs with resident cells of the vascular wall.","keyphrases":["engineered tissue heart valves","decellularized scaffold","heart valve remodeling","biodegradable scaffold","circulating stem cells"],"prmu":["P","P","R","R","R"]}
{"id":"Purinergic_Signal-4-1-2246000","title":"Purinergic junctional transmission and propagation of calcium waves in cultured spinal cord microglial networks\n","text":"In order to elucidate the mechanisms of purinergic transmission of calcium (Ca2 + ) waves between microglial cells, we have employed micro-photolithographic methods to form discrete patterns of microglia that allow quantitative measurements of Ca2 + wave propagation. Microglia were confined to lanes 20\u2013100 wide and Ca2 + waves propagated from a point of mechanical stimulation, with a diminution in amplitude, for about 120 . The number of cells participating in propagation also decreased over this distance. Ca2 + waves could propagate across a cell-free lane from one microglia lane to another if this distance of separation was less than about 60 , indicating that propagation involved diffusion of a chemical transmitter. This transmitter was identified as ATP since all Ca2 + wave propagation was blocked by the purinoceptor antagonist suramin, which blocks P2Y2 and P2Y12 at relatively low concentrations. Antibodies to P2Y12 showed these at very high density compared with P2Y2, indicating a role for P2Y12 receptors. These observations were quantitatively accounted for by a model in which the main determinants are the diffusion of ATP released from a stimulated microglial cell and differences in the dissociation constant of the purinoceptors on the microglial cells.\nIntroduction\nMicroglia give a calcium (Ca2\u2009+\u2009) transient in mixed cultures of microglia and astrocytes, following mechanical stimulation of a single astrocyte [1, 2]. This transient in the microglia is dependent on the release of ATP by the astrocytes [1]. However, nothing is known about the mechanism of transmission of a Ca2\u2009+\u2009 signal between microglial cells that allows for propagation of a Ca2\u2009+\u2009 wave in populations of microglia [3]. In the absence of neurons, a Ca2\u2009+\u2009 wave in astrocytes propagates for hundreds of microns from a point of mechanical stimulation (e.g., [4, 5]). It remains to be seen whether a Ca2\u2009+\u2009 signal can be transmitted between microglia in such a way that there is propagation of a Ca2\u2009+\u2009 wave.\nThe Ca2\u2009+\u2009 waves in astrocytes, when initiated at a point in a culture, can jump cell-free gaps of different widths formed by scraping away cells [4, 6]. Such Ca2\u2009+\u2009 waves propagate across these gaps with a delay that increases with gap width until this width reaches about 150 , when such propagation fails. More recent research has used micropatterned arrays of astrocytes in which lanes of cells about 110  wide alternate with cell-free lanes about 40  wide [4, 5]. If a Ca2\u2009+\u2009 wave is initiated by mechanical stimulation in an astrocyte in just one lane it propagates both along the lane as well as transversely across the cell-free lanes into the adjacent astrocyte lanes with a delay of about 10 s. These observations support the idea that the transmission of Ca2\u2009+\u2009 waves between astrocytes involves the release of a diffusible substance. The question of whether this wave is transmitted between microglia by a diffusible substance has not been addressed. It is known that microglia collecting in the vicinity of a stab wound express connexin CX43 and that these cells are coupled by such connexins in vitro under the control of certain cytokines [7]. However, given the relative low density of resting microglia in the brain compared to that of astrocytes [8\u201310], it seems unlikely that connexins are used by microglia in normal circumstances to transmit Ca2\u2009+\u2009 waves. We have therefore used micropatterned arrays of microglia to ascertain if the Ca2\u2009+\u2009 wave can propagate across cell-free regions, thus indicating that a diffusible substance is involved in transmission. As shown below, this turns out to be the case.\nMicroglia possess P2X7, P2Y1, P2Y2\/4, P2Y6, P2Y12, P2Y13 and P2Y14 purinoceptors [11\u201314] with P2Y12 receptors and P2X7 receptors unique to microglia, at least in the hippocampus [15]. There is evidence that some microglia cells possess predominantly P2X receptors and others predominantly P2Y receptors [16]. P2X7 receptors on microglia are involved in apoptosis, transcription and microvesicle shedding [17]. Activation of P2X7 receptors can lead to the release of pro-inflammatory cytokines, such as TNF\u03b1 [18, 19]. P2Y1 and P2Y2\/4 receptors are involved in the release of the cytokine IL-10 that acts to markedly reduce the release of the pro-inflammatory cytokines [20]. Most (85%) resting microglia respond to ATP with a Ca2\u2009+\u2009 transient [21] as a consequence of an action of ATP on P2X and P2Y receptors [11, 14]. The activation of P2X receptors leads to an influx of calcium ions whereas the activation of P2Y receptors releases calcium from internal stores [21\u201326]. Microglia can also release ATP using in part ATP-binding cassette (ABC) proteins, such as P-glycoproteins (mdr 1a and mdr 1b) and multi-drug resistant associated proteins (mrp1 and mrp4; [27]). Taken together, these observations on the action of ATP on purinergic receptors possessed by resting microglia suggest the hypothesis that the transmission of Ca2\u2009+\u2009 waves between microglia is due to ATP, and this we have investigated.\nThe experimental work is supplemented by calculations using a theoretical model of extracellular communication in cellular networks, originally developed for astrocytes [28], and here modified for microglia.\nMethods\nThe experimental methods for immunohistochemistry, mechanical stimulation of leading to Ca2\u2009+\u2009 waves in cells, application of drugs, recording Ca2\u2009+\u2009 waves and seeding cells into lanes were the same as that previously described [5]. The purification of microglia started when plated mixed glia culture from Sprague-Dawley rat pup spinal cord formed a confluent monolayer (usually between 1 and 2 weeks). The culture was shaken at 200 rpm for 1 h at 37\u00b0C using rotating shaker (IKA-Vibrax-VXR). During shaking, the astrocytes remained adhered to the poly-D-lysine coating whereas the microglia and oligodendrocytes detached from the astrocyte monolayer. Immediately after shaking, the medium containing the detached cells was transferred to a 15-ml centrifuge tube and centrifuged for 5 min at 500 rpm. The supernatant was discarded and the pellet was resuspended in 1 ml DMEM and triturated. Cell density was adjusted by adding fresh DMEM (typically 1-2 ml) after cell trituration and 300 \u03bcL of the cell suspension was pipetted onto each coverslip containing previously prepared microchannels and then incubated for 15 min. Incubating DMEM was replaced with fresh DMEM containing 50% of spinal cord astrocytic conditioned medium (ACM). After 15 min, microglia selectively adhere to the poly-D-lysine coating whereas other cell types that may be present, such as any remaining astrocytes and oligodendrocytes, take a longer period of time to adhere [29]. The purity of the microglial cultures was greater than 98% according to live staining of the cells with the microglial marker FITC-IB4 (Invitrogen). Microglia-plated microchannels were incubated in ACM supplemented DMEM for 48 h before use in experiments (Fig.\u00a01). The medium was removed and replaced daily.\nFig.\u00a01The distribution of microglia, immunostained with anti-CSF-1R, in parallel lanes of width 20  and separation 45 ; the calibration bar is 45 \nFor Ca2\u2009+\u2009 recording, the relative fluorescence amplitude (\u0394F\/F), was calculated using the formula\nwhere F is the fluorescent intensity during the Ca2\u2009+\u2009 transient,  is the intensity averaged over the interval immediately before the calcium transient and  is the average fluorescence intensity measured in several cell-free areas. Ca2\u2009+\u2009 transients with a maximum \u0394F\/F value less than 0.3 (being 15% of the largest Ca2\u2009+\u2009 transient observed in a lane of microglia) were discounted as being too close to the noise level to be reliable. All experiments were repeated at least three times and values are presented as mean \u00b1 s.d. Statistical significance was determined with the use of unpaired t-tests and ANOVA, and P\u2009<\u20090.05 was considered significant. All P2Y receptor antagonists were obtained from TOCRIS.\nMathematical model\nA detailed description of a mathematical model of purinergic transmission in glial networks was given in [28] and subsequently applied to experimental results on such transmission between astrocytes [5]. This model has been adapted to the present case of microglial networks. The basic model is the same, so here we give only a brief summary of the main features, highlighting the changes that have been made.\nCommunication between the model microglia is mediated by ATP diffusing in the extracellular space. This ATP binds reversibly to metabotropic receptors (P2Y) on the surface of cells so that the ratio of bound to total receptors is given by\nwhere [ATP] is the extracellular ATP concentration and KR is the dissociation constant for ATP binding. The usual meaning of KR is the concentration of ATP at which half the total receptors are bound; however, in the present context KR, as well as being a measure of the affinity of receptor types, also reflects additional variables such as spatial variations in receptor density, since \u03c1 is a measure of the effective activity of ATP as a function of space and time. Thus KR is to be interpreted as an effective, rather than an actual, dissociation constant (see the section \u201cReceptors\u201d in [5]).\nEach microglial cell is represented by a cube of side 8.3 , and these cubes are arranged in 2D arrays with their centres 25  apart. As explained in [28], this simplified geometry does not model the spatial complexity of a real cell, but is a lumped approximation. The Ca2\u2009+\u2009 wave can be initiated either by increasing the IP3 concentration in a single cell, or by applying ATP extracellularly. In the present calculations, a fixed concentration of ATP (typically 20 \u03bcM) is applied for an extended time (typically 5 s) to the surface of one model microglial cell. The parameters used are those given in Table 1 of [28], except that the parameter governing the ATP release rate, VATP, has been reduced from 2\u00d710\u2009\u2212\u200911 to 5\u00d710\u2009\u2212\u200912\u03bcmol \u2009\u2212\u20092 s\u2009\u2212\u20091 in order to obtain agreement with the experimental observations.\nResults\nQuantitative characteristics of Ca2\u2009+\u2009 wave propagation\nThe amplitude and velocity of propagating Ca2\u2009+\u2009 waves in microglia at different positions along microglia lanes, from a point of mechanical initiation in a microglia, were determined. Figures\u00a02A and B show the extent of propagation from the point of stimulation in two lanes, ~80  and ~40  wide, respectively. The Ca2\u2009+\u2009 wave propagates with diminution in amplitude (Fig.\u00a02C), at a velocity of about 5\u00a0s\u2009\u2212\u20091, over a distance of at most 120  before becoming undetectable. This velocity is about one-quarter of that for Ca2\u2009+\u2009 wave propagation in astrocyte lanes [5]. Quantitation of these observations for four different sets of microglia lanes in four cultures is shown in Fig.\u00a03. Figure\u00a03a shows that there is a continual decrease in amplitude of the Ca2\u2009+\u2009 wave over 120\u00a0 at which distance the amplitude falls below 15% to 20% of the initial amplitude and could no longer be reliably detected. The percentage of microglia cells across a lane that gives a Ca2\u2009+\u2009 peak amplitude change that is greater than 15% of the peak amplitude at the site\u00a0of initiation remains high for about the first 80  and then declines rapidly over the succeeding 40  (Fig.\u00a03b). On the other hand, the peak Ca2\u2009+\u2009 amplitude increases rapidly with an increase in the local density of cells that give an observable Ca2\u2009+\u2009 transient response (Fig.\u00a03c). \nFig.\u00a02The Ca2\u2009+\u2009 wave propagates along a lane of microglia with decreasing amplitude from the point of initiation. A and B show results for two different lanes of microglia, of widths 82\u00b15  and 38\u00b14 , respectively. In each case, (a) shows the site of Ca2\u2009+\u2009 initiation by mechanical stimulation with a micropipette and (b) shows the microglia that responded with a Ca2\u2009+\u2009 transient (indicated by open circles) after stimulation at the site indicated by the arrow; the calibration bar is 45 . C shows the timecourse of the Ca2\u2009+\u2009 wave at different distances along a lane from the site of initiation, as indicated. The mechanical stimulus was applied for 1 s starting at time zeroFig.\u00a03Quantitative characteristics of the Ca2\u2009+\u2009 wave for a number of microglia lanes. a shows the average peak amplitude of Ca2\u2009+\u2009 deliveries along the lanes before becoming undetectable at about 120  (values normalized to the peak Ca2\u2009+\u2009 concentration at the stimulating electrode). b shows the number of microglia that give a Ca2\u2009+\u2009 response at different positions along the length of a lane, expressed as a percentage of the total number of Ca2\u2009+\u2009 indicator-labeled microglia at that position; this remains high for about 80  and then declines. c shows that the average amplitude of the peak Ca2\u2009+\u2009 wave in equal-width segments of a microglia lane increases with the number of microglia that propagate Ca2\u2009+\u2009 in the segment. Results in a, b and c are for three different lanes in three different cultures. In a and b the distance is from equal-width segments along a lane (for which the average peak Ca2\u2009+\u2009 was calculated for all microglia in the segment) to the site of mechanical initiation of the Ca2\u2009+\u2009 wave\nThe restricted local propagation of the Ca2\u2009+\u2009 wave from the point of initiation is emphasized by experiments in which different sites of initiation along a single lane of microglia are determined. As Fig.\u00a02 shows, in each case the Ca2\u2009+\u2009 wave propagation is restricted to regions of about 100  diameter around the site of initiation.\nEvidence for release and diffusion of ATP during Ca2\u2009+\u2009 wave propagation\nIn order to test for the possibility that mechanically stimulated microglial cells release a diffusible substance, parallel lanes of microglia were constructed, separated by cell-free lanes (see Fig.\u00a01). Tests were then made of the extent to which Ca2\u2009+\u2009 waves could propagate across these cell-free lanes of widths 70  or more, independent of the width of the microglia lanes (Figs.\u00a04A and B). No such propagation was observed in 20 experiments. On the other hand, if the cell-free lanes were less than ~60  wide there was always successful propagation of the Ca2\u2009+\u2009 wave across the cell-free lanes (Fig.\u00a04C). It appears then that a diffusible substance is released by the excited microglia and is able to initiate Ca2\u2009+\u2009 transients in them.\nFig.\u00a04Propagation of a Ca2\u2009+\u2009 wave occurs between microglial lanes if these are not separated by distances greater than about 60 . A and B show parallel lanes of microglia in which the lane widths are 75\u00b13  and 25\u00b13 , respectively, separated by cell-free lanes of 165\u00b13  and 70\u00b14 , respectively; the open circles indicate the microglia that gave a Ca2\u2009+\u2009 response following mechanical excitation of the microglial cell indicated by the arrow; there is no propagation of the Ca2\u2009+\u2009 wave across these lanes. C shows parallel lanes of microglia in which lane widths are 50\u00b16  and the cell-free lane 46\u00b110 ; the open circles indicate that a Ca2\u2009+\u2009 wave response was able to propagate across cell-free lanes as well as along the lanes. The calibration bar is 45  in A, B and C. The position of the micropipette in C(a) is not evident as it is out of focus\nSince astrocytes use ATP as a chemical transmitter, and it is known that microglia initiate Ca2\u2009+\u2009 transients in response to ATP, we determined if ATP was likely to be the diffusible substance released by microglia in order to promote Ca2\u2009+\u2009 wave propagation. First, Ca2\u2009+\u2009 wave propagation was blocked by the ATP-degrading enzyme apyrase (grade III, 60 units per m\u2113; Fig.\u00a05). Second, the effects of antagonists to the P2Y class of purinergic receptors on Ca2\u2009+\u2009 wave propagation were tested. It is known that the pharmacological profile of P2Y receptor activation on spinal cord microglia and the expression of their mRNA clearly favours P2Y12 receptors, followed by P2Y6 and P2Y1 according to [12] and P2Y2, P2Y6, P2Y12 and P2Y14 according to [14]. Suramin (100 \u03bcM) completely blocked all propagation of the Ca2\u2009+\u2009 wave along microglial lanes (compare Fig.\u00a06B with 6A), indicating that P2Y6 and P2Y14 are not involved, and this was confirmed for P2Y6 using the specific P2Y6 antagonist MRS 2578 (30 \u03bcM). The specific P2Y1 antagonist MRS 2500 (100 \u03bcM) did not block the Ca2\u2009+\u2009 wave. On the other hand, the P2Y12-specific antagonist 2-MeSAMP (300 \u03bcM) blocked Ca2\u2009+\u2009 wave propagation. Any contribution of P2X receptors known to be present on microglial cells, such as P2X4 and P2X7 [11], to Ca2\u2009+\u2009 wave propagation is likely to be minimal given the blocking effects of the P2Y12-specific antagonist 2-MeSAMP. We conclude that Ca2\u2009+\u2009 wave propagation between microglial cells involves the release of ATP onto at least P2Y12 receptors. Visentin et al. [14] have also placed emphasis on the role of P2Y12 receptors in calcium signalling.\nFig.\u00a05Propagation of Ca2\u2009+\u2009 waves is blocked by the ATP-degrading enzyme apyrase. A and B show the extent of propagation of Ca2\u2009+\u2009, from the point of mechanical stimulation of a microglial cell, to other microglial cells in a lane. In each case, the top panel (a) shows the position of the mechanically stimulating micropipette and the lower panel (b) shows the microglial cells that gave a Ca2\u2009+\u2009 signal (open circles) in response to mechanical stimulation at the arrow. A is the control and in B apyrase (60 units\/m\u2113; grade III, Sigma) was present with only the stimulated cell now giving a Ca2\u2009+\u2009 transient. The calibration bar is 45Fig.\u00a06Transmission of the Ca2\u2009+\u2009 wave between microglial cells is chemical due to the release of ATP. A and B show the extent of propagation of Ca2\u2009+\u2009 from the point of mechanical stimulation of a microglial cell to other microglial cells in a lane. In each case, the top panel ((a)) shows the position of the mechanically stimulating micropipette and the lower panel ((b)) the microglial cell(s) that gave a Ca2\u2009+\u2009 signal (open circles) in response to mechanical stimulation at the arrow. A is the control and in B suramin (100 \u03bcM) was present blocking P2Y receptors and only the stimulated cell responded with a Ca2\u2009+\u2009 transient. The calibration bar is 40 \nDensity of P2Y receptors on microglia\nThe theoretical requirement that KR take values from 25 to 45 \u03bcM may reflect different densities of P2Y receptors on the microglia, since in the present theory KR depends on this density as well as on the dissociation of ATP from the receptors (see Theory section above, and also the following section). Polyclonal antibody labelling of P2Y12 (Fig.\u00a07a) receptors indicated that these are localized in clusters of average diameter 0.45 , as are P2Y receptors on astrocytes [5] and smooth-muscle cells [30]. The density of P2Y12 receptors, measured over nine microglial cells, varied about four-fold (Fig.\u00a07b). We suggest that P2Y12 receptors mediate the Ca2\u2009+\u2009 wave propagation. Since in the present model KR values need to vary over at least a two-fold range, it may be that this variability is partly due to differences in P2Y12 receptor density.\nFig.\u00a07Density of P2Y receptors on microglia. a Distribution of anti-P2Y12 receptor immunofluorescence on single spinal-cord microglia in lanes; the shape of the cell is given by the borders of immunohisto chemical staining. b Histogram of the density of and anti-P2Y12 labelled receptors for different microglia. The error bars indicate \u00b1SEM. At least ten areas on each cell were used to determine the P2Y receptor density\nModelling the quantitative characteristics of Ca2\u2009+\u2009 wave propagation amongst microglia\nThe model of purinergic transmission of the Ca2\u2009+\u2009 wave given in the Methods was used to give a quantitative description for comparison with the experimental results. A lane of microglia five cells wide and 528  long was considered in which the centre-to-centre distance between the microglia was 25 . The whole lane of microglia was placed on a 2D surface 528  by 528\u00a0 (Fig.\u00a08). Each row of five cells possessed KR values assigned by random permutations of the values 25, 30, 35, 40 and 45 \u03bcM. Note that these are effective KR values that take into account other properties besides dissociation of ATP from P2Y receptors. Activation of a microglial cell in the centre of the lane, by increasing the ATP concentration about the cell to 20 \u03bcM for 5 s, generated a Ca2\u2009+\u2009 wave that propagated with diminution as shown in Fig.\u00a08. The Ca2\u2009+\u2009 wave varied in amplitude and velocity, both across the width of the lane as well as along its length (Fig.\u00a08).\nFig.\u00a08Diagrammatic representation of the theoretical spatial and temporal changes in a Ca2\u2009+\u2009 wave in a lane of microglia five cells wide following excitation of the central microglia, according to the mathematical model. The Ca2\u2009+\u2009 wave is initiated by a 5-s pulse of ATP of concen tration 20 \u03bcM. The vertical bars give Ca2\u2009+\u2009 in \u03bcM at times t = 7.5, 15, 22.5 and 30 s, as indicated. KR values range from 25 to 45 \u03bcM for different microglia across each lane\nA quantitative analysis of Ca2\u2009+\u2009 wave propagation in a lane, such as that shown in Fig.\u00a08, gives the results summarised in Fig.\u00a09. The peak amplitude of Ca2\u2009+\u2009 in each microglial cell of the lane varied significantly both along the length and across the width of the lane (Fig.\u00a09a). Normalizing the Ca2\u2009+\u2009 to the largest amplitude observed at the site of initiation shows that many of the cells give a Ca2\u2009+\u2009 amplitude that is less than 15% of the largest one (Fig.\u00a09a). Using this as a cut-off for the \u0394F\/F value that would be observed experimentally (see Methods) gives a rate of decline of Ca2\u2009+\u2009 with distance similar to that observed, from 100% to 20% over about 120  (compare Fig.\u00a09a with Fig.\u00a03a). The percentage of cells that gives a Ca2\u2009+\u2009 amplitude greater than 15% of the largest amplitude at the site of initiation, for different rows of five cells along the length of the lane, remains high for the first 75  and then declines to about 40% of maximum at 120 . This is a similar pattern of Ca2\u2009+\u2009 changes to that observed experimentally along a lane of microglia (compare Fig.\u00a09b with Fig.\u00a03b). The average amplitude of the peak Ca2\u2009+\u2009 across rows of cells in a lane increases with the number of cells that are activated in a row (Fig.\u00a09c). This is also observed experimentally (compare Fig.\u00a09c with Fig.\u00a03c).\nFig.\u00a09Theoretical characteristics of a Ca2\u2009+\u2009 wave in a microglia lane, according to the mathematical model as in Fig.\u00a08, for quantitative comparison with the observed characteristics (see Fig.\u00a03). a shows that the predicted peak amplitude of Ca2\u2009+\u2009 for all cells in a lane, normalized to that at the site of stimulation, declines with distance along the length of the lane until it becomes undetectable at about 120  (compare to Fig.\u00a03a); the horizontal line indicates the values of the Ca2\u2009+\u2009 amplitude below which experimental detection is in the noise level (set at 15%). b shows the predicted number of microglia that give a Ca2\u2009+\u2009 response greater than 15% of the maximum value at different positions along the length of a lane expressed as a percentage of the total number of cells at that position; this is maintained for about 70  and then declines rapidly (compare to Fig.\u00a03b). c shows that the predicted amplitude of the average peak Ca2\u2009+\u2009 in rows of a microglia lane increases with the number of microglia that propagate Ca2\u2009+\u2009 in the row (compare to Fig.\u00a03c)\nThese theoretical results highlight the fact that Ca2\u2009+\u2009 wave propagation amongst microglial cells is very limited compared with that amongst astrocytes [5]. Experimentally, this was highlighted by mechanical stimulation of microglial cells at different well-separated sites along a single microglial lane, showing that Ca2\u2009+\u2009 wave propagation was restricted to within about 90  of the stimulating micropipette (see Fig.\u00a010). Such restricted propagation was also observed along the model lane following stimulation at well-separated sites (compare Fig.\u00a011 with Fig.\u00a010). The clustering of activated cells near the site of initiation of the Ca2\u2009+\u2009 wave is, in the model, due to the large amount of ATP released in this region.\nFig.\u00a010Stimulation of microglia at different sites in a lane leads to Ca2\u2009+\u2009 wave propagation confined to the vicinity of the stimulating electrode. Shown in (a), (b) and (c) are three different sites, greater than 140  apart, of mechanical stimulation by a micropipette of a single cell in a single lane of microglia. In (d) is shown that the Ca2\u2009+\u2009 waves initiated in each case ((a) to (c)) are confined to a region within 90  of the stimulating micropipette, with each set of symbols indicating the extent of Ca2\u2009+\u2009 wave propagation. The calibration bar is 45 . The position of the micropipette in C(a) is not evident as it is out of focusFig.\u00a011Theoretical predictions of the distribution of microglia in a lane five cells wide that gave a Ca2\u2009+\u2009 response, with an amplitude greater than 15% of maximum, following excitation of a single microglial cell at three different sites along the length of the lane, indicated by filled circles (\u2219). The dots indicate the positions of microglia in the lane. The diamonds (\u22c4) indicate microglia that gave a response following stimulation of the microglia near the left-hand end of the lane (sixth cell from the left at \u2212\u2009250\u00a0), the open circles (\u2218) responses following stimulation of the microglia near the right-hand end of the lane (sixth cell from the right at 250\u00a0) and the plusses (+) microglia in the centre of the lane (at 0 ). (A longer lane (800 ) has been used for this calculation.) The KR values range from 25 to 45 \u03bcM for different microglia across each lane. Note that there is no overlap in the Ca2\u2009+\u2009 wave domains of each stimulated microglial cell, the diamonds, crosses and circles designating discrete regions\nThe model of purinergic transmission of Ca2\u2009+\u2009 waves was used to see if it could account for Ca2\u2009+\u2009 propagation along and between lanes of microglia, such as those shown in Fig.\u00a04. Figure\u00a012 shows the propagation of Ca2\u2009+\u2009 waves in three such parallel lanes of cells, separated by cell-free lanes 42  wide, following initiation of the Ca2\u2009+\u2009 wave in the middle row of the middle lane by applying 20 \u03bcM of ATP for 5 s at a central cell. There is propagation of the Ca2\u2009+\u2009 wave over about six cells of the middle lane before the side lanes are engaged, at about 7 s after application of the initiating stimulus (Fig.\u00a012). Both side lanes first generate a Ca2\u2009+\u2009 wave that is in cells in a row opposite or nearly opposite the row containing the initiating cell in the middle lane. By 13 s the crest of the Ca2\u2009+\u2009 wave has reached the limits of Ca2\u2009+\u2009 propagation at about 100  from the site of initiation, by which time it has travelled less than 75% of that distance along adjacent lanes (Fig.\u00a012). Very few cells are engaged in Ca2\u2009+\u2009 wave propagation in these adjacent lanes and propagation fails over distances of about 70  along the lane and 50  across it.\nFig.\u00a012A diagrammatic representation of the theoretical spatial and temporal changes in Ca2\u2009+\u2009 in three lanes of microglia each five cells wide, separated by cell-free lanes 42  wide, following excitation of a single microglial cell in the middle row of the middle lane. The Ca2\u2009+\u2009 wave is initiated by a 5-s pulse of ATP of concentration 20 \u03bcM on the central microglial cell at time t\u2009=\u20090. The vertical bars give Ca2\u2009+\u2009 in \u03bcM at times t = 7.5, 15, 22.5 and 30 s, as indicated. KR values range from 25 to 45 \u03bcM for different microglia across each lane. Note the limited propagation of the Ca2\u2009+\u2009 wave in both the middle and side lanes\nThe question arises as to whether regeneration of ATP in each cell is necessary. Repeating calculations with regenerative release switched off, and thus only pure diffusion of ATP from its initial release site, gave travel distances along lanes reduced by about 20%. We investigated whether this reduced distance could be compensated for by increasing the initial release of ATP, but this then recruited cells in adjacent lanes in a way not observed experimentally. Thus the theoretical calculations indicate a role for intracellular regeneration of ATP, but at a much lower rate than in astrocytes.\nDiscussion\nAlthough activation of P2X7 receptors by ATP leads to an influx of calcium ions in microglial cells [16, 31\u201333] it does not seem that these receptors mediate the Ca2\u2009+\u2009 wave propagation in these cells as this is blocked by suramin which does not block P2X7 receptors. It seems likely that P2X7 receptors are activated at higher concentrations of ATP than is required for P2Y receptor activation [33], suggesting that the concentration of ATP reached at the receptors after its release from microglia is insufficient to excite P2X7 receptors. ATP acts on microglial cells to both release calcium from internal stores and to evoke an influx of calcium [34, 35]. P2Y receptors mediate Ca2\u2009+\u2009 release from internal stores in microglial cells [14, 21, 32, 33, 36, 37], the extent of this release being under the modulatory control of P2X receptors [25, 32] and of toll-like receptors [38].\nATP released following stimulation of astrocytes can generate Ca2\u2009+\u2009 transients in nearby microglial cells [2]. Repeated stimulation of the astrocytes releases sufficient ATP to activate P2X7 receptors on the microglial cells, greatly increasing membrane permeability in the microglial cells [1] and leading to the release of inflammatory cytokines, such as IL-2, from the cells [39]. The present work suggests that in these experiments the ATP released in moderate amounts from singly stimulated astrocytes most likely acts on P2Y12 receptors on microglial cells. We could block the ATP-dependent Ca2\u2009+\u2009 propagation in these cells with suramin, which blocks P2Y1, P2Y2 and P2Y12 receptors, but could not be blocked by P2Y1 receptor antagonists. Given that the predominant receptor on microglial spinal cord cells shows the pharmacological profile of P2Y12 [12] (see also [14]), we conclude that this receptor, which we found in relatively high density, is most likely to be mediating the effect of ATP. Thus there is no evidence that P2X7 receptors are engaged in Ca2\u2009+\u2009 propagation in microglia. It is interesting to note in this regard that microglial cells rapidly re-orientate their processes towards a site of ATP release in vivo as a consequence of the action of the released ATP on P2Y receptors, an action that is blocked by apyrase [40] and that nucleotides acting on P2Y12 receptors of microglia exert a chemotactic effect [41].\nWe have used soft lithography techniques of micro-fabrication to allow controlled and discrete patterning of microglia so that quantitative investigations can be carried out [42]. Such an approach avoids the difficulties inherent in the random seeding of microglial cells on a homogeneous substrate for carrying out quantitative measurements of the properties of propagating calcium waves [43]. The technique allows lanes of microglial cells with controlled widths of from 15  to over 150 , separated by cell-free regions with the same range of widths [44]. It is unlikely that this fabrication method affects the seeded microglial cells, since when applied to astrocytes the rates of propagation of the Ca2\u2009+\u2009 waves remain about the same as determined in random seeded cultures [4, 5].\nThe amplitude of the Ca2\u2009+\u2009 wave, and the percentage of microglial cells excited to give a Ca2\u2009+\u2009 response, were correlated along the length of a microglial lane, both decreasing from near the site of initiation over a distance of about 120  before the wave ceased to be detectable. Decreases in amplitude of Ca2\u2009+\u2009 waves from a site of mechanical initiation have often been observed in randomly seeded astrocytes over a homogeneous substrate, but in this case over several hundred microns [45\u201347]. The purinergic gliotransmission model suggests that the decline in amplitude of the Ca2\u2009+\u2009 wave is due to the relatively high level of ATP released from the stimulated microglial cell, which then dominates the concentration profile of ATP within approximately 100 . The decline in amplitude of the wave approximately follows this concentration gradient of ATP. The decline in the percentage of microglial cells excited to give a Ca2\u2009+\u2009 response within this 100  range is then attributed to a failure of microglial cells possessing a relatively high KR to generate a Ca2\u2009+\u2009 response as the concentration of ATP declines over the same range. The more than two-fold range in the KR values used in our model is comparable to the range of P2Y12 receptor densities found in microglial cells using immunohistochemistry.\nOur work shows that there can be propagation of Ca2\u2009+\u2009 waves between parallel lanes of microglial cells when these are about 30  wide and separated by cell-free lanes about 40  wide, but this does not occur until many microglial cells in the initiating lane undergo a Ca2\u2009+\u2009 response. It seems likely that a certain minimum amount of ATP must be released from the initiating lane, involving a certain minimum number of microglial cells undergoing a Ca2\u2009+\u2009 response, before nearby microglial lanes are excited. Since ATP can diffuse across cell-free lanes as wide as 150  from astrocyte-seeded lanes [5], it seems likely that the ATP released from microglial cells is much less than that from astrocytes since the P2Y12 receptors in the model for microglia possessed lower KR values (25\u2009\u2212\u200945 \u03bcM) than in the model for astrocytes (25\u2009\u2212\u2009125 \u03bcM; [5]).\nThe purinergic transmission model can account for the observed extent of the Ca2\u2009+\u2009 wave provided pure diffusion of ATP from the stimulation site is supplemented by regenerative release of ATP from the microglia. Fluctuations in the density of excited microglia along a lane leads to local fluctuations in the amplitude of the Ca2\u2009+\u2009 wave due to changes in the local ATP concentration, consequential on the changes in the local number of microglial cells excited. This is modelled by assigning a range of KR values to the P2Y receptors on the microglia, and this also reflects the density of receptors on individual microglial cells.\nStimulation of three different microglial cells some hundreds of microns apart gave a Ca2\u2009+\u2009 response in microglia at highest density closest to the site of stimulation. Our model quantitatively explains these observations as arising from the large amount of ATP released by the stimulated microglial cell diffusing to activate microglia within about 100 .","keyphrases":["transmission","propagation","calcium waves","microglia","purines"],"prmu":["P","P","P","P","U"]}
{"id":"Eur_Arch_Otorhinolaryngol-2-2-1705529","title":"Canal wall reconstruction and mastoid obliteration with composite multi-fractured osteoperiosteal flap\n","text":"We used inferior pedicled composite multi-fractured osteoperiosteal flap (CMOF), our original and new surgical approach, to obliterate the mastoid cavity and reconstruct the external auditory canal (EAC) to prevent the open cavity problems. CMOF was used to obliterate the mastoid cavity and reconstruct the EAC in 24 patients (13 women, 11 men; age span 12\u201351 years) who underwent radical mastoidectomy to treat the chronic otitis media between 1998 and 2004. Small meatoplasty was done in all 24 patients to relive their aesthetical concerns. Temporal bone CT scanning was done to observe the neo-osteogenesis in the mastoidectomy cavity and the CMOF, and the EAC volume was measured postoperatively. All our patients were followed-up for 2 years. The epithelization of the new EAC in our patients was complete at the end of the second month. Cholesteatoma, granulation, and recurrence of osteitis did not occur in any of the patients. We saw the new bone formation filling the mastoid cavity in the postoperative temporal bone CT scanning images. The mean volume of the new EAC on the 24th month was 1.83 \u00b1 0.56 cm3. We had an almost natural EAC, which owed its existence to the neo-osteogenesis that grows behind the CMOF, which we use to obliterate the mastoid cavity and to reconstruct the EAC.\nIntroduction\nOpen cavity mastoidectomy techniques applied for chronic otitis media cause some cavity problems. These patients need life-long otologic care for the debris that accumulates due to the insufficient self-cleaning mechanism of mastoidectomy cavities. Sixteen percent of ears without cavities needs cleaning, whereas this ratio climbs to 42% for ears with cavities [11]. These patients complain of dizziness in cold weather and during swimming. Other problems are non-aesthetic meatoplasty and difficulties in the placement of hearing-aid devices [2, 7].\nVarious obliteration and external auditory canal (EAC) reconstruction techniques have been recommended to eliminate open cavity problems. Mosher was the first to use soft tissue flap in 1911 and has described the postaural subcutaneous flap technique for cavity obliteration [10]. In the following years soft tissue flaps, free bone, cartilage grafts, autograft and synthetic filling substances have been used to reduce mastoid cavities [1]. Mastoid cavity obliteration by neo-osteogenesis under the periosteal flap has been first shown by Kahramanyol in 1992 [8]. This result has been my start-point and I have used inferior pedicled CMOF, which is our modification for EAC reconstruction and to reduce the volume of radical mastoidectomy cavity. In addition, small meatoplasty was done to lessen aesthetic concerns.\nMaterials and methods\nTwenty-four patients (13 women and 11 men ranging in age from 12 to 51\u00a0years) were operated between 1998 and 2004. Various stages of cholesteatoma were detected during preoperative otoscopy and intraoperative observations in all those patients who had otorrhea and hearing loss for years. Four patients had complications such as facial paralysis, labyrinthine fistula and intracranial abscess. Ossicle chain injury of various severities was present in all these participants.\nAll patients were operated under general anaesthesia. Prior to operation, each patient had otoscopic examination, pure tone and speech audiometry and temporal bone CT scans. Approval for radical mastoidectomy indication was granted after simple mastoidectomy and atticotomy.\nOperative technique\nThe incision line that was 1.5\u20132\u00a0cm posterior to the postauricular sulcus was kept as a margin as the subcutaneous tissues were dissected into fascia. Dissection was advanced to the skin of the posterior meatal wall along the fascia. The skin of the posterior meatal wall was horizontally transected between 6 and 12 O\u2019\u00a0clock. Then, the periosteum was incised till the bone was reached about 1\u20132\u00a0mm anteriorly to the postauricular incision. The superior part of the incision runs parallel to the inferior border of the temporalis muscle. From the upper margin of the incision that extends to the bone, the periosteum was elevated to the inferior pedicle with a 7-mm chisel. During this procedure bone lamellae with utmost 1-mm thickness were raised as a part of a fractured bone panel attached to the periosteum. By this way the mastoid apex was reached inferiorly and EAC skin posteriorly. Periosteum was dissected from the EAC posterior wall skin. Thus, CMOF with an inferior base was formed. The bone lamellae under the periosteum were fractured into fragments as much as possible (Fig.\u00a01). The flap was given the functionality of a flexible metal watch belt to place it properly into the mastoid cavity and let it reconstruct the EAC. The flap was retracted inferiorly to be able to work on the mastoid cortex.\nFig.\u00a01Preparation of the composite multi-fractured osteoperiosteal flap; CMOF composite multi-fractured osteoperiosteal flap, MT musculus temporalis, BL bone lamellae, EAC external auditory canal, MC mastoid cortex, C chisel\nThe following rules were attended during radical mastoidectomy. (1) All bony buttresses were excised and lowered as much as possible. This is called saucerization or skeletonization. (2) The mastoid apex was excised. (3) All related anatomic structures and loci were visualized. (4) All diseased tissues were removed. (5) White cortical bony tissues were reached in all directions. Moreover, large defects at the dural plate and or the sigmoid sinus were reconstructed with modelled autologous bone chips obtained from the operative field.\nCleansing of the tympanic cavity was emphasized. Patients with bone conduction thresholds better than 35\u00a0dB and an open eustachian ostium and a healthy peri-eustachian mucosa were treated with tympanic reconstruction. The prepared CMOF was placed over the facial ridge and attached to the anterior attic wall in all cases. The flap could be elongated to the attic with the help of small incisions. The bone lamellae beneath the periosteum are supportive material to our flap and thus became a skeleton for the expected EAC. Absorbable gelatine sponge was laid over the mastoid bone behind the CMOF. The EAC skin was incised longitudinally at the 6 O\u2019\u00a0clock direction and the resultant skin flap having its nourishing root at the zygoma was laid posteriorly and inferiorly over the CMOF. We cared to keep the skin margins over the fascial flap. That is how the new EAC was composed. Then EAC was filled with absorbable gelatine sponge (Fig.\u00a02). Siebenmann technique was used to form a small meatoplasty. A \u2018\u2018Y\u201d incision was used with its concavity looking to the posterior to elevate a flap of choncal skin and a sufficient part of the choncal cartilage was excised. The triangular skin flaps were just laid down such that they could touch the CMOF. The EAC in expectation was filled with absorbable gelatine sponge and extraphore with antibiotic ointment. The subcutaneous and cutaneous postauricular incisions were closed (Fig.\u00a03).\nFig.\u00a02Transposition of the composite multi-fractured osteoperiosteal flap (above), and fixation of it (below); SF skin flap, MC mastoid cavity, CMOF composite multi-fractured osteoperiosteal flap, SS sigmoid sinus, BL bone lamellae, EAC external auditory canal, S spongeFig.\u00a03Shematic represantation of surgical procedure. Pr Periosteum, BL bone lamella, E extraphore, S sponge, FG fascia graft, SF skin flap, SS sigmoid sinus\nPostoperative antibiotics and anti-inflammatory medicines were continued for 1\u00a0week. Skin sutures were removed on the 7th postoperative day. The cavity dressings were removed on the 14th day. Antibiotic and steroid drops were used at the end of the second postoperative month. In all patients epithelization of the cavity completed at the end of the 2nd month.\nTemporal bone CT scanning was done to observe the neo-osteogenesis in the mastoidectomy cavity and the CMOF on the 1st, 12th and 24th months. The EAC volume was measured on the second, 6th, 12th and 24th months. The volume measurements were done with saline, warmed up to the body temperature that was filled into the EAC up to the level of the posterior line of the meatoplasty. The head position was horizontal during the measurement. Data of the study were analysed by the statistical package for social sciences (SPSS) for Windows 10.0.\nResults\nThis study was done between the years 1998 and 2004 in 24 patients. All patients were followed-up for at least 2\u00a0years. EACs were smooth and healthy. In all patients epithelization was complete after the 2nd month of the operation. None had recurrent osteitis, cholesteatoma or granulation. Otoscopic examination could reveal the entire cavity in all these cases and debris and cerumen cleaning was feasible.\nThe mean volume of the new EAC on the 2nd, 6th, 12th and 24th months were 0.93\u00a0\u00b1\u00a00.32, 1.43\u00a0\u00b1\u00a00.48, 1.66\u00a0\u00b1\u00a00.53, 1.83\u00a0\u00b1\u00a00.56\u00a0cm3, respectively. The canal volume measurements of the patients that we were able to follow after 24 months revealed no significant difference. The evaluations of the postoperative temporal bone CT scanning revealed no retraction or displacement around, but demonstrated new bone formation obliterating the mastoid cavity behind the CMOF that was used to build the posterior wall of the EAC (Fig.\u00a04). The CMOF never displaced toward the EAC or the sigmoid sinus, and it always remained where it was placed during the operation.\nFig.\u00a04Postoperative temporal bone CT images 1\u00a0month later (a), and 2\u00a0years later (b)\nNone of our patients returned with vertigo induced by cold air or swimming. No aesthetical disapproval occurred. Introducing the hearing aid placements into the EAC was easy and almost natural. The therapeutical results in four patients with preoperative complications were successful. In one patient conchal perichondritis emerged soon after the operation and was treated with medical therapy.\nDiscussion\nVarious treatments have been proposed for mastoid cavity obliteration and reconstruction [1, 10]. Extended meatoplasty has been used for auto-cleansing and sufficient aeration [4, 8, 13]. Some authors offered a small meatoplasty to avoid the inevitable aesthetic concerns stemming from a wider meatoplasty [6]. However, small meatoplasty has restricted the cavity drainage and aeration by increasing the cavity depth posteriorly. In our technique a small meatoplasty became a functional continium of the almost a normal EAC anatomy that was obtained by the placement of the CMOF to the posterior edge of the meatoplasty. This newly formed EAC has provided sufficient drainage and aeration, and a preferable aesthetic appearance (Fig.\u00a05).\nFig.\u00a05The external ear meatus in the second postoperative year\nThe bilateral perfusion obtained by the postauricular artery supplying the inferior pedicled CMOF and the branches of internal maxillary artery supplying the superior pedicled EAC flap have accelerated cavity healing and epithelization. The absorbable gelatine sponges placed into the mastoidectomy cavity and new EAC have helped the immobilization of the CMOF by supporting it placed at the facial ridge. Moreover, they have contributed to the formation of a homogenous haematoma under the CMOF. Many authors have detected that haematoma at the fracture site has aided in fracture healing [5, 12]. Numerous callus areas emerging in interlamellar fracture lines under the CMOF have supplied both skeletal support and obliteration of the mastoidectomy cavity by neo-osteogenesis.\nHistological investigations relating fracture-healing process have shown that intramembranous bone formation begins under the periosteum in a few days after fracture formation and bridging between fracture fragments begins in 4\u00a0weeks [3]. We observed calcification foci indicating osteoblastic activity in histological investigations of a cavity wall biopsy taken from a patient in 6\u00a0months after surgery (Fig.\u00a06). New bone formation filling the mastoidectomy cavity under the CMOF was detected in the postoperative temporal bone CT images taken in the 1st, 12th and 24th months.\nFig.\u00a06Calcification foci indicating osteblastic activity are seen in histologic investigations 6\u00a0months after surgery (HE \u00d7400)\nIn a study where Palva flap was used for mastoid obliteration, the ear canal was found enlarged at both the early and the late postoperative check-up compared to the preoperative values [11]. The results of the studies, reported by Kahramanyol et al. [8] in which mastoid cavities were obliterated with fascioperiosteal flap, have shown that the mastoid cavity volume was decreased by 39% due to neo-osteogenesis under fascioperiosteal flap, and that the obliteration has stayed in long term has shown radiologically [9]. In our study it has shown a nearly twofold increase in EAC volume and no significant difference after 24th months. Thus the difference in EAC volumes between the 2nd month and the 2nd year of the postoperative period was assessed as a result of shrinking of the tissues, composing the EAC, especially the periosteum.\nConclusion\nSome large meatoplasties performed for open cavity problems are not preferable due to non-aesthetic appearance. On the other hand, small meatoplasties that are preferred to overcome this concern cause aeration and drainage problems by increasing the depth of mastoidectomy cavity posteriorly. Therefore, in this study we both performed small meatoplasties to avoid the aesthetic concerns and at the same time filled the mastoid cavity up to the posterior edge of the meatoplasty to obtain efficient aeration and drainage.","keyphrases":["canal wall reconstruction","mastoid obliteration","osteoperiosteal flap","radical mastoidectomy","neo-osteogenesis"],"prmu":["P","P","P","P","P"]}
{"id":"Calcif_Tissue_Int-3-1-2151961","title":"Polymorphisms in the Low-Density Lipoprotein Receptor-Related Protein 5 (LRP5) Gene Are Associated with Peak Bone Mass in Non-sedentary Men: Results from the Odense Androgen Study\n","text":"Purpose To investigate the impact of the Ala1330Val (rs3736228, exon 18) and Val667Met (rs4988321, exon 9) polymorphisms of the low-density lipoprotein receptor-related protein 5 (LRP5) gene on peak bone mass in young men.\nFamily and twin studies have demonstrated that genetic factors account for 50\u201380% of the inter-individual variation in bone mineral density (BMD) in both women and men [1\u20139]. Polymorphisms in a number of genes affect the bone phenotype regarding BMD, bone size, or fracture risk [10]. Only a small part of the overall variation in BMD, however, has been explained by the polymorphisms identified so far.\nThe transmembrane proteins low-density lipoprotein receptor-related protein 5 (LRP5) and LRP6 are essential in Wnt signaling. They are expressed in many tissues including osteoblasts and have been implicated in the adaptive response of bone to mechanical load [11]. Located on chromosome 11q12\u201314, activating mutations in the LRP5 gene are responsible for rare conditions with a \u201chigh bone mass phenotype\u201d [12, 13] as well as autosomal dominant osteopetrosis type 1 [14], and transgenic mice carrying the LRP5 G171V mutation have increased bone mass and bone biomechanical properties [15]. Conversely, inactivating mutations are responsible for the osteoporosis pseudoglioma syndrome [16] and this syndrome is recapitulated by LRP5 inactivation in mice [17].\nIn addition to these mutations, a number of polymorphisms have been described in the LRP5 gene (http:\/\/www.ncbi.nlm.nih.gov\/SNP). One of these, the Ala1330Val (rs3736228, exon 18) polymorphism, has been associated with attenuated bone gain in prepubertal boys [18], decreased BMD in Japanese [19], American [20], Australian [21], and Dutch [22] women as well as in elderly Dutch men [22]. It has also been associated with an increased risk of fragility fractures in postmenopausal Australian women [21] and in elderly Dutch men [22]. No association, however, was found between this polymorphism and bone loss in elderly men and women [22]. Similarly, the Val667Met (rs4988321, exon 9) polymorphism was associated with attenuated bone gain in prepubertal boys but not in girls participating in a 1-year longitudinal Swiss study [18]. In the same study, this polymorphism was also associated with decreased lumbar spine bone mineral content and projected bone area and there was a trend toward lower BMD in the spine [18]. These associations were mainly driven by men [18] and in haplotype analysis including Ala1330Val, most of the effect was conferred by the Val667Met polymorphism. Moreover, in a case-control study by Ferrari et al. [23] on idiopathic osteoporosis in males, both LRP5 polymorphisms significantly increased the odds ratio for osteoporosis. Additionally, Val667Met was associated with low stature in adults of both sexes [18]. Thus, current evidence suggests that polymorphisms in the LRP5 gene may be involved in the control of peak bone mass and may increase the risk of osteoporosis in males [16,18, 23].\nIn the present study, we investigated the association between the Ala1330Val and Val667Met polymorphisms and peak bone mass in men in a population-based study.\nSubjects and Methods\nThe Odense Androgen Study (OAS) is a population-based, prospective, observational study on the interrelationship between endocrine status, body composition, muscle function, and bone metabolism in young men. Details on the design and inclusion of participants are reported elsewhere [24]. In brief, 3,000 men aged 20\u201330 years were randomly selected from the civil registration database in Fuenen County, Denmark, and invited by mail to participate in the study. These subjects also received a questionnaire regarding smoking, medication, chronic diseases, drug abuse, etc. A total of 2,042 questionnaires were returned, and from these 859 men expressed interest in participation in the clinical part of the study. Nineteen withdrew before an appointment for inclusion was made. Ten men did not meet with the inclusion criteria (Caucasian and age between 20\u201329 years), and another 3 men gave their notice after the inclusion had stopped. Aside from these latecomers none of the responders were excluded. Of the remaining 827 eligible subjects, 43 did not attend for their appointment and did not wish to reschedule, but informed consent was obtained from 784 men, with dropout from 1 subject. Thus 783 Caucasian men gave written informed consent and were included in the OAS. These participants had similar age, body mass index (BMI), lifestyle, socio-economic status, and educational level as the background population, as evaluated by questionnaires and demographic data (manuscript in preparation). The protocol stipulated that subjects with massive substance or alcohol abuse, malignant disease or severe chronic disease be excluded; however, none of the responders fulfilled any of these criteria. The study was approved by the local Ethics Committee of Fuenen and Vejle Counties (file number 20010198), conducted according to the Helsinki Declaration, and declared in ClinicalTrials.gov: NCT00150163, registered September 6, 2005.\nGenotyping\nDNA was isolated from whole blood samples using QIAamp DNA Blood Midi kits (Qiagen, Valencia, CA, USA).\nThe Ala1330Val polymorphism was analyzed using Taqman primers and probes that were designed using Primer Express software (Applied Biosystems):Sense: 5\u2032-GACGGCGAGGCAGACTGT-3\u2032Antisense: 5\u2032-GCAGGGCCAGGGTCTTG-3\u2032C probe: 5\u2032-FAM-TCAAAGTCCGCCTCGT-NFQ-3\u2032T probe: 5\u2032-VIC-CACAGTCCACCTCGTC-NFQ-3\u2032The real-time analysis was performed using the ABI PRISM 7700 Sequence Detection System. To validate the method, approximately 10% of the samples were repeated and in total 17 samples\u2014at least 5 of each genotype\u2014were sequenced.\nThe Val667Met polymorphism was analyzed using fluorescence polarization as described previously [25], using AcycloPrime-FP SNP Detection kits (Perkin Elmer Life Sciences, Boston, MA, USA). PCR fragments containing the polymorphisms were generated using standard conditions with the following PCR primers:Sense: 5-gccTTcTTggTcTTcAccAg-3\u2032Antisense: 5\u2032-cTTTgAggcAggAAcAgAgg-3\u2032Enzymatic clean-up and subsequent AcycloPrime-FP reaction with SNP primer (sense 5\u2032-ccTcgAgAccAATAAcAAcgAc-3\u2032) were performed according to the instructions of the kit manufacturer. Fluorescence polarization was read in a Wallac Victor multilabel plate reader (Perkin Elmer Life Sciences, Boston, MA, USA). Blank samples and samples with known genotype were included as negative and positive controls, respectively. A total of 10% of all samples were sequenced and genotyping confirmed in all cases.\nAnthropometrics and Lifestyle\nBody weight, body height, sitting height, and arm span were measured. Data regarding exercise habits were collected using a self-administered questionnaire. Non-sedentary lifestyle was defined as participation in any form of regular exercise.\nDual-Energy X-ray Absorptiometry (DXA)\nBMD of the lumbar spine, hip, and whole-body was measured using a Hologic 4500 DXA-scanner (Waltham, MA, USA). The coefficient of variation (CV) for BMD is 1.5%, 1.5%, and 0.7% in the lumbar spine, total hip, and whole body, respectively. Lean body mass (LBM) and fat mass was obtained from the whole-body scans.\nBiochemistry\nSerum samples were drawn after an overnight fast between 08.00 and 10.00 hours and stored for later analyses at \u221280\u00b0C. Serum concentrations of osteocalcin were measured by a luminoimmunoassay (BRAHMS Diagnostica, Berlin, Germany) with an intra-assay and inter-assay CV of 5% and 8%, respectively.\nSerum levels of type 1 collagen C-terminal telopeptide (1CTP) were measured using radioimmunoassay (RIA) (Orion Diagnostica, Espoo, Finland) with an intra-assay and inter-assay CV of 4% and 6%, respectively. Bone-specific alkaline phosphatase (bone-AP) concentration in serum was determined enzymatically after monoclonal antibody capture (Metra Biosystems, Mountain View, CA, USA). The intra-assay CV was 2% and the inter-assay CV 12% for this analysis. Serum levels of 25-OH-vitamin D were measured using a competitive radioimmunoassay (DiaSorin, Stillwater, MN, USA) with an intra-assay CV of 6% and an inter-assay CV of 15%. Serum IGF-I was determined in acid\u2013ethanol extracts as described previously [26]. Intra- and inter-assay variation averaged less than 5% and less than 10%, respectively.\nStatistics\nData are reported as the mean\u00a0\u00b1\u00a0SD, median (range), or median [95% CI], as appropriate. BMD values are shown in absolute values. Regarding the allele dose-analyses, BMD values were transformed to Z-scores using the SD observed in our population. Hardy-Weinberg equilibrium was checked using chi-square analysis. Linkage disequilibrium (D\u2019) between polymorphisms was evaluated by using GOLD software [27]. Haplotype analysis was undertaken using FAMHAP software for constructing haplotypes [28]. Comparisons between groups were performed using one-way ANOVA, unpaired t-test or similar nonparametric tests. Associations between parameters were analyzed using Pearson\u2019s regression analysis and multiple linear regression analysis. Dichotomies were tested using the chi-square test. A p value of less than 0.05 was considered significant. Statistical analyses were performed using SPSS 11.5 and SPSS 12.0.\nResults\nGenotype Frequencies\nFor the Ala1330Val polymorphism, the CC genotype was found in 589 (75.6%), the CT genotype in 170 (21.8%), and the TT genotype in 20 (2.6%), respectively (Table\u00a01) allele frequencies of 0.87 and 0.13 for the C- and T-alleles, respectively.\nTable\u00a01Covariates, BMD, and lifestyle (sedentary\/non-sedentary) by LRP5 genotypes (Ala1330Val and Val667Met polymorphisms)PolymorphismsAla1330ValVal667MetCCCTTTp values (ANOVA)GGGAAAp values (ANOVA)n58917020\u2013699764\u2013Age (years)25.5\u00a0\u00b1\u00a02.825.3\u00a0\u00b1\u00a02.825.9\u00a0\u00b1\u00a02.7NS25.5\u00a0\u00b1\u00a02.825.3\u00a0\u00b1\u00a02.627.0\u00a0\u00b1\u00a01.8NSBody height (cm)181.7\u00a0\u00b1\u00a06.8181.5\u00a0\u00b1\u00a06.1182.8\u00a0\u00b1\u00a07.7NS181.7\u00a0\u00b1\u00a06.8181.3\u00a0\u00b1\u00a05.6190.7\u00a0\u00b1\u00a06.30.02Arm span (cm)183.4\u00a0\u00b1\u00a08.2 183.1\u00a0\u00b1\u00a07.0185.4\u00a0\u00b1\u00a08.9NS183.3\u00a0\u00b1\u00a08.0182.6\u00a0\u00b1\u00a06.9193.5\u00a0\u00b1\u00a06.80.04Body weight (kg)81.9\u00a0\u00b1\u00a011.881.1\u00a0\u00b1\u00a012.485.9\u00a0\u00b1\u00a016.7NS81.7\u00a0\u00b1\u00a012.182.2\u00a0\u00b1\u00a012.990.4\u00a0\u00b1\u00a012.2NSBody mass index (kg\/m2)24.8\u00a0\u00b1\u00a03.324.6\u00a0\u00b1\u00a03.725.6\u00a0\u00b1\u00a04.1NS24.8\u00a0\u00b1\u00a03.325.1\u00a0\u00b1\u00a04.324.8\u00a0\u00b1\u00a01.8NSTotal fat mass (kg)14.9\u00a0\u00b1\u00a06.214.9\u00a0\u00b1\u00a07.317.7\u00a0\u00b1\u00a08.5NS15.3\u00a0\u00b1\u00a06.215.1\u00a0\u00b1\u00a06.717.9\u00a0\u00b1\u00a07.9NSLean body mass (kg)64.0\u00a0\u00b1\u00a07.363.3\u00a0\u00b1\u00a06.865.3\u00a0\u00b1\u00a09.0NS64.7\u00a0\u00b1\u00a06.9 64.4\u00a0\u00b1\u00a06.070.4\u00a0\u00b1\u00a07.0NSUnits alcohol\/week (n)11.4\u00a0\u00b1\u00a010.210.7\u00a0\u00b1\u00a08.612.3\u00a0\u00b1\u00a08.7NS11.0\u00a0\u00b1\u00a09.410.8\u00a0\u00b1\u00a08.16.5\u00a0\u00b1\u00a07.3NSCigarettes\/day (n)0 [0\u201345]0 [0\u201335]0 [0\u201325]NS0 [0\u201335]0 [0\u201330]0 [0\u20131]NSSerum 25-OH-D3 (nmol\/l)65.1\u00a0\u00b1\u00a027.964.2\u00a0\u00b1\u00a026.967.9\u00a0\u00b1\u00a028.6NS65.2\u00a0\u00b1\u00a027.563.0\u00a0\u00b1\u00a029.160.0\u00a0\u00b1\u00a025.4NSSerum IGF-I (\u03bcg\/l)199.9\u00a0\u00b1\u00a052.8202.4\u00a0\u00b1\u00a050.3191.0\u00a0\u00b1\u00a036.1NS27.6\u00a0\u00b1\u00a07.525.5\u00a0\u00b1\u00a06.627.3\u00a0\u00b1\u00a07.8NSSerum osteocalcin (mmol\/l)3.0\u00a0\u00b1\u00a01.13.1\u00a0\u00b1\u00a01.33.0\u00a0\u00b1\u00a01.0NS3.1\u00a0\u00b1\u00a01.22.8\u00a0\u00b1\u00a01.02.3\u00a0\u00b1\u00a00.5NSSerum 1CTP (\u03bcg\/l)5.0\u00a0\u00b1\u00a01.44.9\u00a0\u00b1\u00a01,55.1\u00a0\u00b1\u00a01.3NS5.0\u00a0\u00b1\u00a01.44.9\u00a0\u00b1\u00a01.44.9\u00a0\u00b1\u00a00.9NSBone-AP (U\/l)26. 6\u00a0\u00b1\u00a08.128.4\u00a0\u00b1\u00a010.026.7\u00a0\u00b1\u00a08.3NS26.7\u00a0\u00b1\u00a08.225.8\u00a0\u00b1\u00a07.033.4\u00a0\u00b1\u00a016.4NSBMDspine (g\/cm2)1.08\u00a0\u00b1\u00a00.121.07\u00a0\u00b1\u00a00.171.05\u00a0\u00b1\u00a00.13NS1.08\u00a0\u00b1\u00a00.121.07\u00a0\u00b1\u00a00.221.07\u00a0\u00b1\u00a00.15NSBMDhip (g\/cm2)0.95\u00a0\u00b1\u00a00.140.94\u00a0\u00b1\u00a00.140.94\u00a0\u00b1\u00a00.15NS0.95\u00a0\u00b1\u00a00.140.96\u00a0\u00b1\u00a00.150.84\u00a0\u00b1\u00a00.070.03BMDWB (g\/cm2)1.22\u00a0\u00b1\u00a00.10*1.21\u00a0\u00b1\u00a00.091.21\u00a0\u00b1\u00a00.11NS1.22\u00a0\u00b1\u00a00.101.21\u00a0\u00b1\u00a00.111.20\u00a0\u00b1\u00a00.08NSSedentary\/non-sedentary (n)150\/44037\/1334\/16NS171\/52818\/581\/3NSNo significant differences were found between the groups (ANOVA). This was true also if the CT and TT genotypes were pooled (t-test). Genotypes were in Hardy-Weinberg equilibrium (Ala1330Val: \u03c72\u00a0=\u00a03.2, p\u00a0=\u00a00.07; Val667Met: \u03c72\u00a0=\u00a01.48, p\u00a0=\u00a00.22)*p\u00a0=\u00a00.05 comparing the CC genotype with CT\u00a0+\u00a0TT pooled; NS, not significant\nSimilarly, the GG, GA, AA genotypes of the Val667Met polymorphism were found in 699 (89.7%), 76 (9.8%), and 4 (0.5%), respectively. This corresponds to allele frequencies of 0.95 and 0.05 for the G- and A-alleles, respectively.\nBoth allele distributions were compatible with Hardy-Weinberg equilibrium (\u03c72\u00a0=\u00a03.2, p\u00a0=\u00a00.07 and \u03c72\u00a0=\u00a01.48, p\u00a0=\u00a00.22). The two polymorphisms were not in complete linkage disequilibrium (D\u2019\u00a0=\u00a00.955, R\u00b2\u00a0=\u00a00.33).\nCovariates\nData on covariates in relation to genotypes are shown in Table\u00a01. No significant differences regarding covariates (age, body height, body weight, BMI, sitting height (not shown), total fat mass, lean body mass, alcohol consumption, smoking, serum 25-OH-D3, or serum IGF-I) were seen between the genotypes of the two polymorphisms. Regarding the Val667Met genotypes, however, subjects with the AA genotype had significantly increased body height (190.7\u00a0\u00b1\u00a06.6 vs. 181.7\u00a0\u00b1\u00a06.8 and 181.3\u00a0\u00b1\u00a05.6\u00a0cm, p\u00a0=\u00a00.02) and arm span (193.5\u00a0\u00b1\u00a06.8 vs. 183.3\u00a0\u00b1\u00a08.0 and 182.6\u00a0\u00b1\u00a06.9\u00a0cm, p\u00a0=\u00a00.04).\nBMD in Relation to Age\nIn the spine, no relationship between BMD and age could be detected. Maximum BMD in the total hip was observed around the age of 22 years. In subjects above this age BMD was significantly lower (ANOVA, p\u00a0<\u00a00.05). In subjects aged 30 years, BMD was 5.5% lower that the observed maximum.\nGenotypes in Relation to BMD\nRegarding the Ala1330Val polymorphism, no significant differences between BMD in the spine, hip, or whole body were found between subjects with the CC, CT, and TT genotypes. When the CT and TT genotypes were pooled, whole-body BMD was significantly lower in the CT\u00a0+\u00a0TT compared with the CC genotype (1.21\u00a0\u00b1\u00a00.10 vs. 1.22\u00a0\u00b1\u00a00.10, p\u00a0=\u00a00.05).\nRegarding the Val 667Met polymorphism, a significant difference in hip BMD was observed, since subjects with the AA genotype had lower BMD (0.84\u00a0\u00b1\u00a00.07 vs. 0.95\u00a0\u00b1\u00a00.14 and 0.96\u00a0\u00b1\u00a00.15, p\u00a0=\u00a00.03).\nTable\u00a01 also shows the distribution of genotypes in relation to sedentary\/non-sedentary lifestyle, but no significant difference in lifestyle was found between any of the genotypes.\nTable\u00a02 shows the gene-dose effect on BMD expressed as the change in Z-score of each copy of the T- or A-allele, respectively. The analysis was performed both with and without adjustment for BMI, smoking (0\/1), any continuous medication (0\/1), and serum 25-OH-D3 in the overall study population (multiple regression analysis). Regarding the Ala1330Val polymorphism, whole-body BMD tended to decrease with each copy of the T-allele (p\u00a0=\u00a00.07) in the overall study population (n\u00a0=\u00a0779). No significant association between number of T-alleles and BMD at the other skeletal sites was found. Regarding the Val667Met polymorphism, no significant gene-dose effect could be demonstrated in the overall population.\nTable\u00a02Multiple regression analysis determining the change in BMD Z-score for each copy of the T- or A-allelePolymorphismAla1330ValVal667MetZ-scoreUnadjustedAdjustedaUnadjustedAdjustedaTotal population (n\u00a0=\u00a0779)BMDspine\u22120.11 (\u22120.29; 0.06)NS\u22120.10 (\u22120.27; 0.06)NS\u22120.044 (\u22120.28; 0.19)NS\u22120.054 (\u22120.27; 0.16)NSBMDhip\u22120.03 (\u22120.20; 0.11)NS\u22120.02 (\u22120.17; 0.11)NS0.018 (\u22120.20; 0.23)NS0.002 (\u22120.22; 0.21)NSBMDWB\u22120.13 (\u22120.27; 0.11)p\u00a0=\u00a00.07\u22120.12 (\u22120.25; 0.01)p\u00a0=\u00a00.07\u22120.085 (\u22120.30; 0.13)NS\u22120.11 (\u22120.33; 0.10)NSNon-sedentary (n\u00a0=\u00a0589)BMDspine\u22120.21 (\u22120.40; \u22120.03)p\u00a0=\u00a00.02\u22120.20 (\u22120.37; \u22120.02)p\u00a0=\u00a00.01\u22120.26 (\u22120.51; \u22120.01)p\u00a0=\u00a00.04\u22120.23 (\u22120.46; \u22120.002)p\u00a0=\u00a00.048BMDhip\u22120.08 (\u22120.26; 0.09)NS\u22120.06 (\u22120.22; 0.10)NS\u22120.08 (\u22120.33; 017)NS\u22120.07 (\u22120.32; 0.19)NSBMDWB\u22120.17 (\u22120.33; \u22120.01)p\u00a0=\u00a00.04\u22120.15 (\u22120.30; 0.01)p\u00a0=\u00a00.07\u22120.17 (\u22120.41; 0.08)NS\u22120.18 (\u22120.44; 0.07)NSData are shown as mean effect and 95% confidence interval. Z-scores were derived from the study population itselfaAdjusted for BMI (ln-transformed), lean body mass (ln-transformed), smoking (0\/1), any continuous medication (0\/1), and serum 25-OH-D3\nWhen analysis was restricted to non-sedentary men (n\u00a0=\u00a0589), however, a significant correlation between the number of T-alleles at the Ala1330Val polymorphism and BMD in the spine was found in both the unadjusted (\u22120.21 [95% CI: \u22120.40; \u22120.03], p\u00a0=\u00a00.02) (Fig.\u00a01) and adjusted analysis (\u22120.20 [95% CI: \u22120.37; \u22120.02], p\u00a0=\u00a00.01) (Table\u00a02). Similarly, each copy of the A-allele in the Val667Met polymorphism decreased the lumbar spine BMD in unadjusted (\u22120.26 [95% CI: \u22120.51; \u22120.01], p\u00a0=\u00a00.04) and adjusted (\u22120.23 [95% CI: \u22120.46; \u22120.002], p\u00a0=\u00a00.048) analysis.\nFig.\u00a01BMD of the lumbar spine, total hip, and whole body in relation to the genotype regarding the Ala1330Val (upper panel) and the Val667Met (lower panel) polymorphisms in the LRP5 gene. Participants with a non-sedentary (left) and sedentary lifestyle (right) are displayed. Data are shown as the mean\u00a0\u00b1\u00a0SE. (*)p\u00a0<\u00a00.10, *p\u00a0<\u00a00.05\nGenotypes in Relation to Bone Markers\nNo significant differences were observed between genotypes of the Ala1330Val or Val667Met polymorphisms regarding bone markers (Table\u00a01).\nHaplotype Analysis\nThe effect of haplotypes of the Ala1330Val and Val667Met polymorphisms on BMD adjusted for BMI, lean body mass, smoking, drugs, and vitamin D are shown in Table\u00a03. No significant association between BMD and haplotypes was found. This was also the case when sedentary and non-sedentary men were analyzed separately (not shown).\nTable\u00a03The effect of haplotypes of Ala1330Val and Val667Met polymorphisms on BMD adjusted for BMI, lean body mass, smoking, drugs, and vitamin DHaplotypesAla1330ValCTVal667MetGAGApn1331312981BMDspine (g\/cm2)0.008\u00a0\u00b1\u00a00.96\u22120.42\u00a0\u00b1\u00a00.65\u22120.09\u00a0\u00b1\u00a00.90\u22120.04\u00a0\u00b1\u00a01.60NSBMDhip (g\/cm2)\u22120.002\u00a0\u00b1\u00a01.010.97\u00a0\u00b1\u00a01.48\u22120.0005\u00a0\u00b1\u00a00.95\u22120.04\u00a0\u00b1\u00a01.0NSBMDWB (g\/cm2)0.02\u00a0\u00b1\u00a01.010.70\u00a0\u00b1\u00a00.59\u22120.10\u00a0\u00b1\u00a00.87\u22120.14\u00a0\u00b1\u00a01.04NSData are shown as the mean\u00a0\u00b1\u00a0SD. No significant associations between BMD and haplotypes were found when sedentary and non-sedentary men were analyzed separately (not shown)\nDiscussion\nWe found a significant association between the Ala1330Val and the Val667Met polymorphisms in the LRP5 gene and BMD in young men, especially when physical activity was taken into account as confounder. This finding supports a number of recent papers reporting significant associations between polymorphisms in this gene and BMD [18, 19, 21, 22]. Some of these studies involved or were restricted to men [18, 22, 29, 30]. Moreover, Ferrari et al. [18] suggested that the polymorphisms in the LRP5 gene are associated with bone gain in prepubertal boys. Only one study [25] failed to demonstrate an association between variations in the LRP5 gene and BMD, but most likely this is due to their restricted sample size (n\u00a0=\u00a0219).\nMajor disruption of the LRP5 gene (homozygous nonsense or frame-shift mutations) resulting in loss-of-function has been demonstrated to be responsible for the osteoporosis pseudoglioma syndrome [16]. Conversely, a number of dominantly inherited gain-of-function mutations result in high bone mass phenotypes [12\u201314] and autosomal dominant osteopetrosis type 1 [14]. Minor changes in this gene, i.e., frequently occurring polymorphisms, may also be of importance in the regulation of bone mass as suggested by the association studies mentioned above. Further support comes from mapping a quantitative trait locus for BMD to chromosome 11q12\u201313 in some [31, 32] but not all studies [7, 33, 34]. In a case-control study comprising 78 men with idiopathic osteoporosis and 86 controls, Ferrari et al. [23] found that Ala1330Val and Val667Met conferred an odds ratio for osteoporosis of 2.98 (95% CI: 1.03\u20138.81) and 3.27 (95% CI: 1.43\u20137.44), respectively. Also, a single prospective study [21] comprising 1,301 elderly Australian women demonstrated that the c.3357A\u00a0>\u00a0G (rs556442) polymorphism was associated with decreased BMD and increased incidence of fractures. This polymorphism located in exon 15 is synonymous but was found to be in linkage disequilibrium with Ala1330Val [21]. Similarly, Mizuguchi et al. [19] found a linkage equilibrium block comprising exons 7 to 18. In our study, no significant effect of Ala1330Val or Val667Met on BMD was found apart from the ANOVA analysis which suggested that the AA genotype at Val667Met was associated with low BMD of the hip and increased body height. The number of participants with this genotype, however, was low (n\u00a0=\u00a04) and we found no significant gene-dose effect to back this finding. In non-sedentary subjects, however, we found a significant association between Ala1330Val genotype and the Val667Met and Z-score BMD in the spine both in unadjusted analysis and when adjusted for BMI, smoking, continuous medication, and serum levels of 25-OH-D3. Further, we observed an association between Ala1330Val and whole-body BMD in the unadjusted analysis. This interaction between lifestyle (sedentary\/non-sedentary), LRP5 genotype, and BMD has not previously been reported. Such an association is fully in line with the proposed role of LRP5 as a player in adapting bone to weight-bearing. LRP5 acts as a co-receptor for Wnt proteins and is expressed in the osteoblast and a number of other cell types. The Ala1330Val (exon 18) polymorphism results in a substitution of alanine with the chemically similar valine. No functional studies have been performed on this polymorphism; however, the polymorphism is located within the second LDL-repeat of LRP5 [22] and this region is involved in the ligand binding of the LDL-receptor [35]. Also, preliminary results have shown that drugs that inhibit Dkk binding to LRP5 increase bone formation in vitro [36] and LRP5 seems to be involved in the adaptive response of bone to mechanical load and may be partly responsible for the \u201cmechanostat\u201d [11]. It is presently unclear why association between the Ala1330Val and Val667Met genotypes and BMD was found only in the spine and not in the hip in the non-sedentary group. Differences between the skeletal sites regarding loading pattern (compression vs. bending) and bone architecture (trabecular vs. cortical) may be important.\nIn our study, the numbers of T-alleles and A-alleles of the Ala1330Val and Val667Met polymorphisms, respectively, were associated with a decrease in BMD of the lumbar spine of 0.2 SD in the non-sedentary participants (i.e., approximately 2.5%). Previous studies have assessed this impact to be from 1% [20] to 15% [18] in women and men and women, respectively. While a number of genes have been associated with bone mass in women (e.g., PTH [37], alpha-1 chain of type 1 collagen [10], vitamin D receptor [38], transforming growth factor beta-1 [39], interleukin-6 [40], and osteoprotegerin [41]), only a few genes have been shown to be associated with peak bone mass in men. We have previously reported that the MTHFR gene is associated with peak bone mass in the same series of men as reported here [42]. Each of these polymorphisms including the LRP5 gene only accounts for a fraction of the variability in BMD; however, the 2.5% impact suggested by our study is not trivial.\nWe found no association between either projected bone area at the spine or hip, or body weight and any of the polymorphisms; however, our data demonstrated a significant impact of the AA genotype of the Val667Met polymorphism and body height as previously reported by Ferrari et al. [18] and Koay et al. [29]. In our study, the number of subjects with the AA genotype was, however, very low (n\u00a0=\u00a04). Thus, this finding clearly needs confirmation in other study designs.\nThe allele frequencies found in our study corresponded closely to those reported in Dutch, Australian, Japanese, and Korean populations [19, 21, 22, 30].\nOur study has some limitations. We only tested two polymorphisms in the LRP5 gene. We cannot, therefore, rule out the possibility that the associations found in our study could be due to other polymorphisms in the LRP5 gene or even in neighboring genes in linkage with the polymorphisms tested. Indeed, Bollerslev et al. [21] found that several of the polymorphisms in the LRP5 gene were in linkage disequilibrium with each other in Australian women. In our study, haplotype analyses were negative. This might be due to the limited number of participants. Testing large arrays of polymorphisms or haplotypes in relation to many outcome variables (i.e., bone mass at several skeletal sites), however, carry an increased risk of detecting spurious associations. Also, our study had a limited size. Given the observed genotype frequency and BMD values, our study had the power to detect differences in BMDof the spine between CC and CT\u00a0+\u00a0TT of 0.037 g\/cm2 (alpha\u00a0=\u00a00.05 and beta\u00a0=\u00a00.80). Similarly, our study had the power to detect differences of 0.070 g\/cm2 between GG and GA\u00a0+\u00a0AA in the overall analysis.\nOur study also has several strengths. First, it was population-based. In contrast to most similar studies [18, 20] that recruited participants by advertisements, we recruited our participants by direct mailing on the basis of the National Personal Registry that includes all Danish citizens. This enabled us to account meticulously for the recruitment of our participants. Thus, our sample was not skewed with respect to key socio-economic parameters [24]. Second, our population had a relatively homogeneous genetic background both due to a small influx to our population and since participants with a first- or second-generation immigrant background were excluded on the basis on data from our national registry. Third, we selected young men aged 20\u201330 years since these were expected to have reached their peak bone mass. This was confirmed by our data demonstrating an infinitesimal decrease in BMD by age. Moreover, very few were afflicted with chronic diseases or had long-term exposure of environmental factors that may affect bone mass.\nWe conclude that the Ala1330Val (exon 18) and Val667Met (exon 9) polymorphisms of the LRP5 gene are significantly associated with lumbar spine peak bone mass in physically active men, potentially accounting for 2.5% of BMD in the spine. This gene-environment interaction provides support for LRP5 as a mediator of load-induced bone formation and suggests that this gene is involved in the pathogenesis of osteoporosis in men.","keyphrases":["polymorphisms","low-density lipoprotein receptor-related protein 5","association","men","bone mineral density"],"prmu":["P","P","P","P","P"]}
{"id":"Protein_Eng_Des_Sel-1-1-2427320","title":"Directed evolution of a biterminal bacterial display scaffold enhances the display of diverse peptides\n","text":"Bacterial cell-surface display systems coupled with quantitative screening methods offer the potential to expand protein engineering capabilities. To more fully exploit this potential, a unique bacterial surface display scaffold was engineered to display peptides more efficiently from the surface exposed C- and N-termini of a circularly permuted outer membrane protein. Using directed evolution, efficient membrane localization of a circularly permuted OmpX (CPX) display scaffold was rescued, thereby improving the presentation of diverse passenger peptides on the cell surface. Random and targeted mutagenesis directed towards linkers joining the native N- and C-termini of OmpX coupled with screening by FACS yielded an enhanced CPX (eCPX) variant which localized to the outer membrane as efficiently as the non-permuted parent. Interestingly, enhancing substitutions coincided with a C-terminal motif conserved in outer membrane proteins. Surface localization of various passenger peptides and mini-proteins was expedited using eCPX relative to that achieved with the parent scaffold. The new variant also permitted simultaneous display and labeling of distinct peptides on structurally adjacent C- and N-termini, thus enabling display level normalization during library screening and the display of bidentate or dimeric peptides. Consequently, the evolved scaffold, eCPX, expands the range of applications for bacterial display. Finally, this approach provides a route to improve the performance of cell-surface display vectors for protein engineering and design.\nIntroductionDisplay methodologies have proven invaluable to the discovery, production and optimization of proteins and peptides in a variety of biotechnological applications. Various approaches including phage display (Smith, 1985), mRNA (Wilson et al., 2001) and DNA display (Yonezawa et al., 2003), ribosome display (Mattheakis et al., 1994; Hanes and Pluckthun, 1997), eukaryotic virus display (Muller et al., 2003; Bupp and Roth, 2002), yeast display (Boder and Wittrup, 1997) and bacterial display (Lu et al., 1995) have been developed to screen diverse molecular repertoires for desired activities. In particular, bacterial display libraries have enabled antibody affinity maturation (Daugherty et al., 2000), the discovery of protein-binding peptides (Bessette et al., 2004), the isolation of cell-specific ligands (Nakajima et al., 2000; Dane et al., 2006) and the identification of optimal protease substrates (Boulware and Daugherty, 2006). One of the key advantages of bacterial surface display is the ability to use flow cytometry for quantitative screening of the libraries, allowing for real-time analysis of binding affinity and specificity to optimize the screening process (Wittrup, 2001). Additionally, ease of genetic manipulation, high transformation efficiency and rapid growth rate make Escherichia coli a well-suited host for display. A broad range of bacterial surface display systems have been developed allowing for insertional or terminally fused peptides and proteins to be displayed on the cell surface. Several outer membrane proteins and cellular appendage proteins have been used to present polypeptides as insertional fusions (Charbit et al., 1986; Taschner et al., 2002; Bessette et al., 2004). Ice nucleation protein (Jung et al., 1998), intimins (Christmann et al., 1999) and LppOmpA (Francisco et al., 1992) have been used to display proteins on the C-terminus of a transmembrane scaffold while N-terminal display has been accomplished using autotransporters IgA1 protease (Maurer et al., 1997) and EstA (Becker et al., 2005).Recently, a unique bacterial display scaffold was developed that allows for both N- and C-terminal display from a circularly permuted variant of outer membrane protein OmpX (CPX) (Rice et al., 2006). This scaffold enabled display of peptides on both termini, but with reduced efficiency when compared to that obtained using insertions into OmpX. Reduced membrane localization of CPX may result from slower folding rates and reduced stability that has been described previously for circularly permuted proteins (Heinemann and Hahn, 1995). Regardless, reduced display efficiency makes it necessary to employ longer induction times to achieve sufficient display for screening via fluorescence-activated cell sorting (FACS). Importantly, inefficient display can also create an undesired selection pressure resulting in growth biases, reduced viability or differing levels of passenger localization on the cell surface. As a result, library screening based on cell fluorescence can favor passengers most efficiently localized to the surface, rather than passengers improved for the properties of interest (e.g. binding affinity).A variety of proteins have been circularly permuted to create novel proteins of desired topologies. For instance, Guntas et al. coupled a circularly permuted \u03b2-lactamase with maltose binding protein to create a switch in which \u03b2-lactam hydrolysis activity is increased in the presence of maltose (Guntas et al., 2005). Also, fluorescent proteins have been circularly permuted to create molecular sensors that respond to Ca2+ (Baird et al., 1999). In these instances, the point of circular permutation can have important consequences for protein function. Random circular permutations often yield inactive or unfolded variants (Graf and Schachman, 1996), and those variants retaining activity often fold more slowly and are less stable than the non-permuted parent (Lindqvist and Schneider, 1997). With the aim of rescuing the reduced surface localization of CPX relative to the non-permuted OmpX, we used targeted combinatorial mutagenesis and directed evolution to identify CPX variants that would localize passenger peptides to the cell surface with an efficiency equivalent to that of native OmpX. This strategy yielded a more robust display scaffold to present diverse peptides as C- and N-terminal fusions on the surface of bacteria.\nResultsCircularly permuted OmpX librariesWe previously reported a new type of protein scaffold for polypeptide display that allows for both N- and C-termini to be exterior to the cell, using circularly permuted outer membrane protein OmpX (CPX) (Rice et al., 2006). The CPX protein scaffold consists of the native OmpX signal sequence, which is cleaved after translocation; a sequence with an embedded SfiI restriction site (GQSGQ) after which peptides may be inserted; a flexible linking sequence (GGQSGQ); amino acids S54\u2013F148 of the mature OmpX; a GGSG linker joining the native C- and N-termini; and finally, amino acids A1\u2013S53 of the mature OmpX. To assess the extent of display, a disulfide-constrained streptavidin-binding peptide (SApep) with the amino acid sequence AECHPQGPPCIEGRK (Giebel et al., 1995) was fused to the N-terminus of CPX, allowing cell labeling using a fluorescently conjugated streptavidin probe and measurement of display levels using cytometry. CPX yielded a reduced level of peptide display when compared to cells displaying the same peptide presented as an insertion within the corresponding region of OmpX (Fig.\u00a01). The reduced display ability requires longer induction periods to allow for sufficient fluorescence labeling and library screening, thereby causing cell stress that can result in growth biases during library screening (Daugherty et al., 1999).Fig.\u00a01Display of a streptavidin-binding peptide using OmpX, CPX and eCPX. Flow cytometric analysis of E. coli displaying a streptavidin-binding peptide (AECHPQGPPCIEGRK) (Giebel et al., 1995) as an insertion into OmpX, an N-terminal fusion to CPX or an N-terminal fusion to eCPX after varying durations of induction. The cells were induced at room temperature for time increments between 0 and 90 min then labeled with 100 nM SA\u2013PE.The CPX scaffold was constructed using an arbitrarily chosen flexible linker (GGSG) to join the native N- and C-termini. Thus, we reasoned that alternative linkers and point mutations within CPX could enhance the display of peptides on the cell surface. To improve the display characteristics of CPX, various regions of the transmembrane protein were targeted for mutagenesis. An optimized linker region joining the native N- and C-termini was identified by generating and screening four libraries allowing for three (3X), four (4X), five (5X) and six (6X) random residues to be inserted in place of the GGSG linker using the degenerate codon NNK. Each library was screened separately using FACS for clones exhibiting a high level of fluorescence after 50 min of induction, indicating increased display of SApep binding to streptavidin\u2013R-phycoerythrin (SA\u2013PE). Under these conditions, the parent CPX scaffold yielded display levels only slightly greater than background autofluorescence, making the selection of improved mutants efficient. After sorting, the display level of several clones was measured using cytometry, and their sequences were determined by DNA sequencing (Table\u00a0I). Isolated clones exhibited 3- to 15-fold improved display compared to CPX after only a 30 min induction period. The identified linker sequences exhibited a preference for basic residues, and glycine was present at the first position of the linker in 14 of 16 clones characterized. In addition, four of the clones had unintended mutations preceding the native C-terminus, two with the substitution A165V and two others with G166S. Interestingly, these four clones were among the most improved display scaffolds isolated. The average display level of the selected clones from each library increased with increasing linker length, and was highest for 5- and 6-mer linker clones.Table\u00a0IMutations improving CPX surface localizationClonePositionsLinkerRelative display level165166CPXAGGGSG2.3a\/1.5bThree residue linker library\u2003CPX-3X-1AGGRK8.9a\u2003CPX-3X-2AGGRK8.3a\u2003CPX-3X-3AGGTK7.1a\u2003CPX-3X-4AGGKK10aFour residue linker library\u2003CPX-4X-1AGGSKR18a\u2003CPX-4X-2AGGRQK14a\u2003CPX-4X-3AGSWPN15a\u2003CPX-4X-4VGPRKS22aFive residue linker library\u2003CPX-5X-1AGGRTRK24a\u2003CPX-5X-2AGGRKRN22a\u2003CPX-5X-3VGGATRR32a\u2003CPX-5X-4ASGSQSK36aSix residue linker library\u2003CPX-6X-1AGGTKRYH35a\u2003CPX-6X-2AGGRRHYK28a\u2003CPX-6X-3AGGNRRHR24a\u2003CPX-6X-4ASGSKQSK38aSecond generation library\u2003CPX-L2-1LSGSKSRR33b\u2003CPX-L2-2FSGRKNSH19b\u2003CPX-L2-3ISGTRGSQ29b\u2003CPX-L2-4LSGHRSHR27b\u2003CPX-L2-5ISGDRKRR28b\u2003CPX-L2-6VAGARGRH24b\u2003CPX-L2-7VSGTHNSQ26b\u2003CPX-L2-8VSGPNKSR17b\u2003CPX-L2-9ISGPHNSR23b\u2003CPX-L2-10ISHRGYHAQR33baFold fluorescence above background after 50 min of expression; bfold fluorescence above background after 25 min of expression.In an attempt to further enhance peptide display, the amino acid linker that joined the passenger peptide to the N-terminus of the display scaffold was also targeted for mutagenesis and screening for improved variants. A library was created in place of the original linking sequence GGQSGQ by randomizing these six residues. Screening yielded four clones exhibiting a 10-fold improved display when compared with CPX. Sequencing did not reveal a consensus within the target linker region. Instead, clones with enhanced display possessed a non-targeted substitution of either A165V or G166S. Since the randomly selected library members from the initial pool did not possess mutations outside of the intended region, these advantageous substitutions were rare and likely arose from PCR errors. In parallel, a library was generated with random residues at the surface exposed C-terminus of CPX, since native outer membrane proteins (Omps) possess a conserved C-terminal motif thought to aid in Omp membrane insertion or assembly (Bos and Tommassen, 2004). Four clones were isolated from this library that exhibited improved peptide display. Again, these variants did not share consensus in the randomized region, but each carried the spontaneous mutation G166S. These results suggest that the amino acid compositions of the new termini derived from circular permutation have little effect on the rate of assembly and display of CPX, whereas residues A165 and G166 play a key role in proper translocation and insertion of the protein.A final library was designed to combine the display-enhancing mutations identified within the most improved clones from the first generation libraries. A six residue linker library was chosen to identify optimal linkers to connect the native N- and C-termini, since longer linkers appeared to improve display (Table\u00a0I). After 50 min of induction, individual library members exhibited higher display levels on average when compared with clones with five amino acid linkers (Table\u00a0I). The first amino acid of the linker was fixed to glycine since it was highly conserved in the isolated clones. The third and sixth positions were restricted to R\/K\/S\/H\/Q\/N using the codon MRM, given the increased frequency of these residues at the proposed positions in clones with improved function, and the remaining three positions were fully randomized. Positions A165 and G166, where beneficial substitutions were observed, were also fully randomized. Improved variants were identified using two rounds of MACS followed by two rounds of FACS, sorting clones exhibiting the highest display of the SApep after 30 min of induction. Ten clones were isolated after the final FACS screening (Table\u00a0I). All variant scaffolds identified possessed a more bulky hydrophobic residue (I\/L\/V\/F) in place of alanine at position 165, a consensus for serine at position 166 and a high frequency of basic residues Arg and Lys within the linking region. The display enhancing substitutions A165V and G166S are located immediately upstream of the native C-terminus of OmpX (Fig.\u00a02).Fig.\u00a02Locations of enhancing mutations within eCPX. Image was created using Molecular Operating Environment using the reported structure of OmpX (Vogt and Schulz, 1999), joining the N- and C-termini with the six residue linker GSKSRR, and creating the new termini within the second extracellular loop. Enhancing substitutions A165L and G166S are shown as space filling residues.Expression characteristics of optimized CPXThe scaffold variant exhibiting the most improved display characteristics, CPX-L2-1, or enhanced CPX (eCPX), was then compared to parental OmpX and CPX scaffolds. The cell-surface display level of SApep was measured at incremental times after induction of expression for these three scaffolds. This peptide was displayed either at the N-terminus (CPX and eCPX) or as an insertional fusion within the second extracellular loop of OmpX. The level of display was measured before and after 30, 60 and 90 min of induction using flow cytometry (Fig.\u00a01). The display rate of eCPX was substantially improved relative to that of CPX, and even slightly higher than that of OmpX. After only 30 min of expression, the level of display of eCPX-nSApep was 50-fold above background autofluorescence. Introducing A165L and G166S into OmpX resulted in nearly identical display of SApep relative to that obtained with OmpX (data not shown).To determine whether the enhanced display using the eCPX scaffold was a general effect or specific to the streptavidin binding peptide SApep, several unrelated passenger peptides were fused to the N-terminus of CPX and eCPX and their display levels were compared (Fig.\u00a03). Surprisingly, a disulfide-constrained peptide binding to C-reactive protein (CRPpep) (EWACNDRGFNCQLQR) identified by bacterial display (Bessette et al., 2004) displayed with eCPX yielded nearly 50-fold higher florescence labeling than that for CPX, after only 30 min of expression. In fact, the fluorescence of cells displaying CRPpep from CPX could not be distinguished from background (Fig.\u00a03). Similarly, the T7\u2022tag epitope (T7pep) (MASMTGGQQMG) was displayed more efficiently by eCPX than by CPX. A disulfide-constrained 19-mer peptide binding to vascular endothelial growth factor (VEGF) identified previously using phage display (Fairbrother et al., 1998) was also displayed over 3-fold more efficiently within eCPX. Additionally, an IgG binding mini-protein [a minimized version of the Z-domain from protein A (Braisted and Wells, 1996)] composed of 33-amino acids that form two antiparallel \u03b1-helices exhibited a display level roughly 3-fold higher than that from CPX after a 2 h induction period. Finally, P2, a proline-rich peptide (PAPSIDRSTKPPL) known to bind to the C-terminal SH3 domain of Mona (Harkiolaki et al., 2003), was expressed as a C-terminal fusion using both CPX and eCPX. Similar to the improvements of display at the N-terminus, the display level after only 30 min of expression of P2 using eCPX was improved by 9-fold compared to display with CPX. Thus, for all peptides investigated, the eCPX scaffold increased display levels when compared to the parental CPX.Fig.\u00a03Display of various peptides and mini-proteins using eCPX (shaded) and CPX (white) as measured using flow cytometry. The X-axis indicates the fold fluorescence above background for each protein target in the corresponding fluorescent channel. P2 was labeled with Mona, which is fused to the fluorescent protein YPet. CRPpep and V114 were labeled with biotinylated CRP and VEGF, respectively, then labeled with SA\u2013PE. Mini-Z and T7pep were labeled with Alexa488 -conjugated human IgG and anti-T7\u2022tag monoclonal IgG, respectively. SApep was detected with SA\u2013PE.Since the increased expression of the scaffold could potentially result in a reduction in cell viability, the viability of E. coli populations displaying two different peptides was measured as a function of the duration of induction of expression. The IgG binding mini-Z helix was selected since it exhibited reduced display in the improved eCPX scaffold. Viability was measured at 30, 60 and 90 min after induction using CFU assays and compared to that for parental strain (MC1061\/pBAD33) lacking an expression cassette. Under ordinary display conditions (i.e. induction for less than 60 min), a measurable loss in viability could not be observed. After prolonged induction (90 min), cells expressing the IgG binding minihelix (miniZ) exhibited a 15% loss of viability (data not shown), while cells displaying SApep exhibited viability indistinguishable from that of cells that do not express the scaffold (MC1061\/pBAD33).Biterminal display using eCPXTwo distinct peptides were simultaneously displayed on the structurally adjacent N- and C-termini of eCPX. SApep was fused to the N-terminus, and the P2 peptide fused to the C-terminus (eCPX-nSApep-cP2). Labeling with fluorescent probes SA\u2013PE (red) and YPet-Mona (Nguyen and Daugherty, 2005) (green) enabled independent detection of each peptide using flow cytometry. To determine the ability of eCPX to simultaneously display these two peptides, cells expressing the biterminal display scaffold were labeled with SA\u2013PE only, YPet-Mona only or both probes concurrently. If the peptides bound to their respective receptors independently (i.e. without any steric clashes), there should be no difference between the extent of single color labeling (fluorescence intensity) of the sample labeled with one probe and that labeled with both fluorescent probes simultaneously. However, simultaneous labeling of cells expressing a fusion protein of the form eCPX-nSApep-cP2 with SA\u2013PE and YPet-Mona, or with each probe separately yielded differing extents of labeling, consistent with steric interference between these two large fluorescent probes (290 and 34 kDa, respectively). Specifically, the fluorescence of the cells when labeled with only one probe was always greater than the fluorescence in the corresponding channel of the cells when labeled with both probes simultaneously. In an attempt to reduce steric interference, a long flexible linker of the form (GGGS)5 was inserted between SApep and eCPX, resulting in a total linker length of 26 amino acids causing SApep to be further from the cell surface and thus increasing the distance between the two peptides. Using this long linker, independent labeling of each displayed peptide was improved (Fig.\u00a04). These results indicate that the use of a long, unstructured linker can increase the accessibility of large proteins to peptides simultaneously displayed at both termini of eCPX, without substantially reducing the level of display.Fig.\u00a04Flow cytometric measurement of simultaneous N- and C-terminal display (bi-terminal display). Overlays of two-parameter histograms resulting from clones displaying SApep and P2 on the N- and C-terminus, respectively, with a 6 (A) or a 26 (B) residue linker between SApep and the N-terminus of eCPX. In both (A) and (B) plots, the four distinct populations consist of non-displaying cells mock-labeled with SA\u2013PE and Ypet-Mona (bottom left population), cells that display SApep and P2 labeled with only SA\u2013PE (top left population), with SA\u2013PE and YPet-Mona (top right population), or with only YPet-Mona (bottom right population).\nDiscussionThe application of bacterial display technology to a broader range of protein engineering applications has been hindered by the absence of robust, validated display scaffolds. We recently reported a circularly permuted outer membrane protein X (CPX) having the unique characteristic that both C- and N-termini of the scaffold become localized on the bacterial cell surface (Rice et al., 2006). This \u2018biterminal\u2019 scaffold provides a means for expression normalization to improve affinity screening, analogous to yeast display (Boder and Wittrup, 1997), as well as a means to display dimeric peptides. However, the permuted scaffold exhibited reduced surface localization efficiency, precluding presentation of large peptides or the display of two unique peptides simultaneously from structurally adjacent termini. Here, semi-rational design and directed evolution were used to enhance the surface localization efficiency of CPX to improve the display of diverse peptides. To identify CPX scaffold variants with optimal linker sequences for joining the native C- and N-termini, four separate libraries with three, four, five or six random linker amino acids were screened. Improved scaffold variants revealed a preference for longer linkers of five to six residues, a strict consensus for glycine at the first position of the linker, and a high frequency of basic residues in the remaining positions. Unintended substitutions (A165V, G166S) near the native C-terminus of OmpX greatly increased the display level. These substitutions probably arose from rare errors introduced during PCR which were enriched from the large libraries (109). After directed evolution, the final variant with the most improved display (eCPX) carried substitutions A165L and G166S, with a linker sequence of GSKSRR.Two substitutions (A165L, G166S) identified to improve surface localization occur at the native C-terminus of OmpX and have a high degree of similarity to the C-termini of outer membrane proteins that lack periplasmic domains (Table\u00a0II), even though these Omps share little identity. These results support previous observations that the C-terminal sequence serves an important role in membrane assembly and\/or translocation (Struyve et al., 1991). The beneficial eCPX substitutions discovered could potentially enhance the ability of chaperones to recognize and assemble this topological variant. Interestingly, the extended C-terminal motif resulting from directed evolution (\u2212L\/VXYRFG) is conserved in OmpA homologs which utilize this motif in their last transmembrane \u03b2-strand that precedes a C-terminal periplasmic domain (Table\u00a0II). Our results suggest that glycine is required to allow the internal, as opposed to terminal, YRF motif to interact with a putative chaperone. The improved linkers also include an increased frequency of charged residues that could serve to increase solubility and decrease aggregation to allow more protein to insert into the membrane.Table\u00a0IIComparison of bacterial outer membrane proteins\u2019 C-terminal sequences with eCPXOmp IDC-terminal sequenceaeCPXDVGTWILSVGYRFGSKSRRAT\u2026NP_992891NIGTWVLGIGYRFYP_624929NPIVTFLSVGYRFYP_156535NPRTVSLSVGYRFYP_341424DPRTVSLTVGYRFZP_00792472TPVTFNLNVGYRFSequence of last membrane \u03b2-strand of OmpA homologsbEAQ20637LVSVGLIYRFGGKTQAPAEAP54071YWGLELSYRFGTPVAAAAAAN42586LLSLGVSYRFGQGEAAPVNP_992692LLSVGVSYRFGQEDAAAPZP_00826943MLSVGVSYRFGQDDVVAPaIdentities between eCPX (with substitutions A165L, G166S) and other bacterial outer membrane protein C-termini.bOmpA homologues with a periplasmic domain following the transmembrane strand exhibit a highly conserved glycine residue following the C-terminal YRF motif of transmembrane proteins.The evolution of an efficiently localized biterminal display scaffold (eCPX) provides several important new capabilities for peptide engineering. First, eCPX increased the display rate of various unrelated peptide fusions, on either the N- or C-terminus when compared to that achieved using the parental CPX. Increased display improves library screening efficiency using FACS and promotes the recovery of peptides that are otherwise difficult to display. For example, the display of CRPpep using CPX was only marginally detectable after 30 min of expression, but the same peptide displayed using eCPX provided a fluorescent signal 50-fold above background. If a peptide library displayed on CPX was screened for CRP binding peptides, this sequence and others like it may not have been isolated due to the difficulties in presenting this peptide using CPX. Though, it should be noted that excessive display levels, above that required for screening by FACS, should be avoided to minimize avidity interactions with target proteins. In fact, using eCPX, induction periods as short as 10 min can be sufficient for affinity screening of peptide libraries (unpublished observations). Importantly, quantifying the display level during library screening by labeling of a C-terminal peptide such as P2 allows fine affinity discrimination (VanAntwerp and Wittrup, 2000); that is, peptides with a high affinity but low display level can be differentiated from peptides with a high display but moderate affinity. Moreover, efficient biterminal display allows for the potential to generate peptide libraries on each terminus where both peptides can bind to distinct epitopes of a target protein, resulting in increased binding affinity and specificity through avidity. Finally, C-terminal display with eCPX should enable new applications such as screening for preferred PDZ C-terminal binding motifs (Harris and Lim, 2001).Engineering of the bacterial display scaffold eCPX yielded a circularly permuted transmembrane protein that has both termini presented on the exterior of the cell and inserts into the outer membrane as efficiently as the non-permuted parent. The amino acid sequence used to join the termini played a major role in the proper function of CPX, and two substitutions adjacent to the native C-terminus aided in display of peptides using eCPX. This novel protein scaffold substantially improves the robustness of bacterial surface display via CPX and, when combined with modern cell sorting instrumentation, creates new opportunities for peptide engineering. More generally, similar linker mutagenesis strategies coupled with efficient screening methods should prove useful for optimizing other display scaffolds and circularly permuted proteins.\nMaterials and methodsBacterial strains, reagents and plasmidsAll experiments were performed with E.coli strain MC1061 (F- araD139 \u0394(ara-leu)7696 galE15 galK16 \u0394(lac)X74 rpsL (StrR) hsdR2 (rK\u2212 mK+) mcrA mcrB1) (Casadaban and Cohen, 1980). All plasmid constructs utilize pBAD33 (Cmr) (Guzman et al., 1995), with the promoter araBAD operon and the p15A origin of replication (low-copy number). KOD Hot Start DNA Polymerase (Novagen) was used for PCRs. Primers were from Operon, restriction enzymes from New England BioLabs, SA\u2013PE from Molecular Probes and streptavidin-coated magnetic microbeads (MyOne\u2122 Streptavidin T1) were from Invitrogen. Qiagen mini-preps and gel extraction kits were used for DNA preparation. Ni-NTA agarose for protein purification was from Qiagen, and B-PER II bacterial protein extraction reagent was from Pierce Biotechnology.Vector and library constructionConstruction of circularly permutated OmpX (CPX) was described previously (Rice et al., 2006). To monitor the display level, a streptavidin binding peptide was fused to the N-terminus of CPX as described in earlier work; this plasmid was termed pB33CPX-nSApep. To generate the libraries that joined the original N- and C-termini of OmpX with three to six random residues, primers PD1237\u20131240 (see Supplementary data available at PEDS Online) were used (respectively) as the reverse primer and PD179 as the forward primer, with pB33CPX-nSApep as the template. Primers are listed in the supplementary material. The random positions were encoded using NNK codons allowing for all amino acids and the amber stop codon. The product of the PCR reaction was gel purified then used as a forward primer for the next reaction, using PD180 as a reverse primer, and again with pB33CPX-nSApep as the template. The product was then gel purified, digested with SfiI and gel purified again. The digested insert was ligated into the similarly digested vector pB33CPX-nSApep. Ligation products were desalted and electroporated into electro-competent MC1061 yielding 7.5 \u00d7 107, 7.5 \u00d7 107, 1.5 \u00d7 108, 5.0 \u00d7 108 transformants, respectively. To create the second generation library (CPX-directed), primers PD1282 and PD179 were used to randomize positions A165 and G166 using pB33CPX-nSApep as the template. The product was then used as a template for PCR with PD1281 and PD179, adding the second generation library residues. The primer encoded for G at the first position of the linker and used a restricted codon of MRM to encode for residues R, K, S, H, Q or N at positions 3 and 6 of the linker; the remaining positions used NNK. The product of the previous PCR reaction was then used as a forward primer for the next reaction, using PD180 as a reverse primer and pB33CPX-nSApep as the template. The product was then digested and ligated into the similarly digested vector pB33CPX-nSApep. Ligation products were desalted and electroporated into electro-competent MC1061 yielding 1.0 \u00d7 109 transformants.The various binding peptides were fused to the N-terminus of CPX and eCPX using a linker of GGQSGQS. PCR was used with pB33CPX-nSApep as the template, PD180 as the reverse primer and with forward primers PD1192\/PD1193 for the CRP binding peptide (EWACNDRGF.NCQLQR) or PD961\/PD962 for the VEGF binding peptide (VEPNCDIHVMWEWECFERL). The products were digested with SfiI and ligated into similarly digested vector and electroporated into MC1061. Primers PD1130\u20131133 were used in an assembly PCR to create the forward primer for the mini-Z-domain (FNMQQQRRFYEALHDPNLNEEQRNAKIKSIRDD). This primer with PD180 and template pB33CPX-nSApep was used in PCR. The product was digested with SfiI and ligated into similarly digested vector and electroporated into MC1061. The CPX-T7pep (MASMTGGQQMG) was created using overlap extension PCR with the products from the PCR reaction with primers PD179\/PD705 and PD180\/PD706. The products were digested with SfiI and ligated into similarly digested vector and electroporated into MC1061. To transfer these peptides to eCPX, the vectors containing the peptide CPX fusion was digested with PstI and KpnI, the smaller fragment was gel extracted and ligated to the similarly digested vector of eCPX, transferring the displayed peptide to the eCPX plasmid. To insert the P2 peptide (PAPSIDRSTKPPL) at the C-terminus of CPX and eCPX, PCR was used with PD179 as the forward primer and primers PD950\/PD951 as the reverse primers with pB33CPX-nSApep at template. The primers also encoded a linker of GGQSGQ preceding the P2 peptide. The products were digested with SfiI and ligated into similarly digested vector and electroporated into MC1061. The gene is eCPX-nSApep-cP2. The streptavidin binding peptide was removed using KpnI and HindIII and ligation with a similarly cut insert that contained no N-terminally fused peptide, creating CPX-cP2.To insert an extended linker of (GGGS)5 between the streptavidin binding peptide and eCPX-cP2, PCR was used with forward primer PD179 and reverse primers PD1429\/PD1430\/PD31 with pB33eCPX-nSApep-cP2 as template. The product was then gel extracted and used as the forward primer with PD180 as the reverse primer and pB33eCPX-nSApep-cP2 as template. The product was gel extracted and digested with SfiI and ligated to similarly digested vector. The portion after the OmpX signal sequence was now GQSGQ (encoding a SfiI site), AECHPQGPPCIEGRK (the streptavidin binding peptide), (GGGS)5 (the additional linker) and GGQSGQ (original linker) followed by the S54 of eCPX with the P2 peptide on the C-terminus; the gene construct is termed eCPX-nSApep-linker-cP2.Magnetic selection and screening by FACSMagnetic selections were performed for the first round of selection using the libraries CPX-5x, CPX-6x and CPX-directed. An overnight culture of cells corresponding to 5\u00d7 the library diversity was inoculated to LB medium containing 34 \u00b5g\/ml chloramphenicol (Cm) for a final cell concentration of 0.05 OD600, or 100 \u00b5l of overnight cultures into 5 ml LB Cm, which ever was greater. The cultures were then grown at 37\u00b0C to 0.5 OD600 with shaking (250 rpm), at which time the culture was moved to room temperature (22\u00b0C) to equilibrate and then induced with l-arabinose to a final concentration of 0.04% (w\/v). The cells were induced by shaking (250 rpm) for 50 min, at room temperature. A volume of cells corresponding to 5\u00d7 the library diversity was concentrated by centrifugation (3000 g, 4\u00b0C, 5 min) and resuspended in cold PBS to 10\u201330 OD600. Dynal MyOne SA beads were added to a ratio of approximately one bead per four cells. Magnetic separation was used to wash the beads four times with a volume of LB equivalent to the volume used in the initial labeling, and the beads plus bound cells were finally resuspended in LB with Cm and 0.2% glucose (w\/v) for overnight growth.For flow cytometric sorting, 50 \u00b5l of overnight cultures of the libraries were inoculated to 5 ml LB Cm. Cells were induced as described in the previous paragraph, in future rounds of sorting the induction time was decreased to 30 min. Ten microliter of cells were labeled with 100 \u00b5l of 100 nM SA\u2013PE in PBS on ice for 45 min, pelleted by centrifugation and the supernatant was removed. Cells were resuspended in ice-cold PBS at approximately 107 cells\/ml and immediately analyzed and sorted using a FACSAria cytometer with 488 nm excitation. Between 1 and 5% of the most labeled cells were collected and amplified for further rounds of analysis and\/or sorting by growing overnight in LB medium containing glucose and Cm. A subset of the sort was plated directly on agar for isolation of single clones. Typically 4\u201310 selected clones were assayed for antigen binding by flow cytometry, and the identity of each peptide insert was determined by DNA sequencing.Determination of peptide display levelsTo compare the display level of CPX and eCPX as a function of induction time, CPX and eCPX, cells were subcultured 1:50 from overnight stocks into 5 ml LB Cm and grown for 2 h with shaking (250 rpm) at 37\u00b0C. The cells were then moved to room temperature (22\u00b0C) to equilibrate and induced with 0.04% (w\/v) l-arabinose still shaking at 250 rpm. Samples (5 \u00b5l) were taken prior to induction, 30, 60 and 90 min after induction then added to 50 \u00b5l of 100 nM SA\u2013PE in PBS and incubated on ice for 45 min. The cells were then centrifuged (3000 g, 5 min), supernatant removed and resuspended in 500 \u00b5l ice cold PBS. Cells were immediately analyzed with a FACSAria using 488 nm excitation and fluorescence data collected at 576 nm.To compare the display of various peptides using CPX and eCPX, cultures were started using 50 \u00b5l of overnight culture in 5 ml LB Cm. Cultures expressing the mini-Z domain, SApep, P2 and T7pep were grown until an OD600 of 0.4 and moved to room temperature (22\u00b0) to equilibrate and then induced with l-arabinose to a final concentration of 0.04% (w\/v) for 30 min or 2 h for the mini-Z domain. Cultures expressing the CRPpep and V114 peptides were induced at 37\u00b0C at an OD600 of 0.4 for 30 min. Induced cells (5 \u00b5l) were added to 50 \u00b5l of PBS containing the respective antigens at the following concentrations: 50 nM YPet-Mona, 65 nM biotinylated VEGF, 100 nM biotinylated CRP, 300 nM Alexa labeled human IgG, 100 nM SA\u2013PE and 6.7 nM anti-T7\u2022tag monoclonal IgG. Samples were labeled on ice for 45 min. Biotinylated samples were spun down at 3000 g for 5 min and the supernatant removed. Cells were resuspended in 50 \u00b5l of PBS with 10 nM SA\u2013PE and put on ice for 45 min. Before cytometric analysis, samples were spun down at 3000 g for 5 min, supernatants removed and 500 \u00b5l of PBS added to resuspend the cells. Samples were excited at 488 nm; fluorescence data were collected at 576 nm for SA\u2013PE labeled samples and 530 nm for Alexa labeled samples and YPet conjugated samples.For the dual labeling experiments, cells were subcultured 1:50 from overnight stocks into 5 ml LB Cm and grown for 2 h shaking (250 rpm) at 37\u00b0C. The cells were then induced with 0.04% (w\/v) l-arabinose. Cells expressing eCPX-nSApep-cP2 were expressed for 25 min at 37\u00b0C, and cells expressing eCPX-nSApep-linker-cP2 were expressed for 45 min at 37\u00b0C. Cells (5 \u00b5l) were labeled with 50 \u00b5l of PBS with 100 nM SA\u2013PE only, 40 nM Ypet-Mona only or with both probes simultaneously. The negative controls were non-displaying cells labeled with SA\u2013PE and Ypet-Mona. The cells were incubated at room temperature for 45 min, centrifuged at 3000 g for 5 min and supernatant removed. The cells were left on ice before resuspension with 500 \u00b5l ice cold PBS and analyzed using cytometry with 488 nm excitation and measuring emission at 576 and 530 nm.\nSupplementary dataSupplementary data are available at PEDS online.\nFundingNational Science Foundation CAREER award (BES-0449399) to P.S.D., U.S. Army Research Office\u2014Institute for Collaborative Biotechnologies (DAAD19-03-D-0004).\nSupplementary Material\n[Supplementary Data]","keyphrases":["directed evolution","bacterial display","ompx","circular permutation","peptide display"],"prmu":["P","P","P","P","P"]}
{"id":"Diabetologia-4-1-2292421","title":"Fatty acid-induced mitochondrial uncoupling in adipocytes as a key protective factor against insulin resistance and beta cell dysfunction: do adipocytes consume sufficient amounts of oxygen to oxidise fatty acids?\n","text":"To the Editor: The phenotype of patients with various forms of congenital lipodystrophy has indicated that proper storage of triacylglycerol in peripheral adipocytes is essential to prevent formation of ectopic fat deposits and development of type 2 diabetes [1]. A similar situation with induced peripheral lipodystrophy and elevated risk for type 2 diabetes is seen in patients undergoing highly active anti-retroviral therapy (HAART). In these patients, there is a 30\u201350% decline in mitochondrial copy number as a result of HAART [2].Based on these and other observations, we have recently put forward the hypothesis that one of the functions of mitochondria in adipose tissue is to prevent leakage of fatty acids into the circulation [3]. This mitochondrial activity attenuates the development of ectopic triacylglycerol deposits in the liver and other tissues that would otherwise contribute to the development of whole body insulin resistance and pancreatic beta cell damage. Mitochondria contribute to efficient confinement of fatty acids within adipocytes by oxidative removal of fatty acids, liberated from the triacylglycerol pool, by uncoupled beta oxidation. In addition, mitochondria may provide glyceroneogenic substrates, which contribute to re-esterification of fatty acids. As a net result, there are less fatty acids available to the circulation for redistribution to other compartments of the body [3]. A recent study provided supporting evidence for this concept by showing that the mitochondrial DNA copy number in human adipocytes is positively associated with lipogenesis in adipocytes [4].Recently, Frayn et al. have argued against this biochemical mechanism [5]. They point out that fatty acid oxidation is not a major pathway in white adipocytes and that oxygen consumption by adipose tissue is insufficient to oxidise substantial amounts of fatty acids [6].We fully agree with the point made by the authors that fatty acid oxidation by adipocytes is not a major pathway compared with muscle. However, we hold a different view on the quantitative interpretation of these data: in our model there is no need for a high rate of fatty acid oxidation in adipose tissue per time unit, as fatty acid redistribution from peripheral tissue to the liver is a slow process, taking several years. For instance, in individuals in whom mitochondrial copy number is acutely reduced as result of starting HAART, it takes 18\u201324\u00a0months before redistribution of body fat becomes clinically manifest [7].Therefore, the amount of fatty acids that undergoes redistribution per time unit is very small. Thus, mitochondria in adipocytes only need to remove a small amount of fatty acids per time unit to protect against redistribution of body fat. By using the oxygen consumption data of Frayn et al. and others [5, 6] or using similar data from other studies [8, 9], one can calculate that oxygen consumption by adipose tissue is sufficient to prevent redistribution of >1.5\u00a0kg of fat out of 10\u00a0kg of peripheral adipose tissue to the liver within a 3\u00a0year period, a realistic clinical situation.Therefore, in our opinion, adipose tissue is able to oxidise substantial amounts of fatty acids over a long period by mitochondrial beta oxidation. This process may over time attenuate ectopic deposition of those fatty acids. Since mitochondria also contribute to formation of glycerol 3-phosphate, needed for re-esterification of fatty acids within adipocytes, mitochondria in adipocytes have the ability to protect the organism to a certain extent against leakage of fatty acids into the circulation and thus, redistribution of body fat. As Frayn et al. [5] have pointed out, brown adipose tissue is indeed more active in uncoupled oxidative disposal of fatty acids compared with white tissue. Recent evidence also suggests that brown adipose tissue is present in variable amounts and at multiple sites in humans, contrary to earlier reports [10]. This recently recognised situation further enhances the capacity of the adipose compartment to remove fatty acids through uncoupled beta oxidation.In order to understand the mechanism by which mitochondria in adipocytes contribute to the control of body fat distribution during the development of the metabolic syndrome, additional studies are needed, comprising quantification of fatty acid fluxes in relation to re-esterification and oxidation in adipose tissue. These studies should include study of adipose tissue from individuals with normal glucose tolerance and from those with developing glucose intolerance.","keyphrases":["fatty acids","adipocytes","haart","mitochondria","type 2 diabetes mellitus"],"prmu":["P","P","P","P","M"]}
{"id":"Eur_J_Nutr-3-1-1914246","title":"Safe addition of vitamins and minerals to foods: setting maximum levels for fortification in the Netherlands\n","text":"Background In 2004, the European Court of Justice decided that the prohibition of fortification with vitamin A, vitamin D, folic acid, selenium, copper, and zinc in the Netherlands conflicts with the principle of free movement of goods in the European Union. This decision led to a change in the Dutch policy, resulting in a more flexible handling of requests for exemption from this prohibition to fortify. Therefore, an investigation was proposed in which it would be determined whether a general exemption could be granted for food fortification with a certain maximum safe amount per micronutrient.\nIntroduction\nFoods are fortified with micronutrients and other bioactive compounds to prevent deficiencies or to provide additional health effects. Furthermore, the use of health claims on products after fortification is thought to result in better marketing. As fortification with micronutrients should be safe for the whole population, unacceptably large intakes of micronutrients from all sources should be prevented. For this purpose, tolerable upper intake levels (ULs) are established [28].\nBecause of the small range between recommended daily intakes and ULs and the lack of proof of a nutritional need, fortification with vitamin A, vitamin D, folic acid, selenium, copper, and zinc is prohibited in the Netherlands [1]. Addition of these six micronutrients was only allowed in the case of substitution (e.g., vitamins A and D in margarine as a substitute for butter) or restoration (for instance, because of losses during processing) [1]. In December 2004, the Court of Justice of the European Communities decided that the prohibition of fortification with these six micronutrients contradicted the free movement of goods, a fundamental principle in the Treaty Establishing the European Community [5]. This decision means that market introduction of fortified foods can only be prohibited if it can be proved that the degree of fortification (e.g., level of fortification or quantity of foods fortified) may harm public health. This decision led to a change of Dutch policy, resulting in a more flexible handling of requests for exemption from the Dutch Commodity Act that prohibits fortification with these six micronutrients [6]. Exemption will only be granted if there is no harm to public health, based on the results of the most recent international scientific research.\nBecause of this court decision, each request for individual exemption had to be evaluated for the risk of exceeding the UL. At first, requests for individual exemption were evaluated in order of receiving. To evaluate the risk of exceeding the UL, consumption of the new fortified product by the Dutch population was simulated by replacing the unfortified food products with the new fortified product within the Dutch National Food Consumption Survey (DNFCS) (e.g., the consumption of all kinds of breakfast cereals as monitored within the DNFCS was replaced by the new fortified breakfast cereals). The resulting habitual daily intake (P95) of the micronutrient was compared with the UL. If the UL was not exceeded, the exemption was granted. This approach resulted in the exemption of very few products and turned out to be an unrealistic worst-case approach. Furthermore, the procedure resulted in an administrative overload and long waiting periods for the industry. Therefore, and in anticipation of upcoming European legislation [11], an investigation was proposed in which it would be determined whether a general exemption could be granted for food fortification with a certain maximum safe amount per micronutrient. A model was needed to calculate the national maximum level of a specific micronutrient that can safely be added to foods. Several such models have recently been developed [11\u201314, 25]. First, an International Life Science Institute (ILSI) Europe model (2002) [14], which was followed by models by Domke et al. [12, 13] and Rasmussen et al. [25]. The latter is an adapted version of the ILSI Europe model [14]. These models were developed for the purpose of calculating the maximum amount of micronutrients that can be added to each 100\u00a0kcal of a given food [14, 25] or each food portion [12]. The intake of micronutrients from nonfortified foods and dietary supplements must be taken into account for this calculation. Domke et al. also estimate the maximum amount of micronutrients in dietary supplements by dividing the space between the UL and the level of intake from nonfortified foods. Most models are theoretical approaches, and data regarding their practical use in decision- or policy-making have not yet been published, although the published acceptable levels of addition per 100\u00a0kcal by Rasmussen et al. have been implemented as the Danish maximum levels for fortification (Rasmussen, personal communication, 2006). Besides these models that calculate a maximum fortification level, models that calculate the optimal intake for the population (taking into account both too low and too high intakes) are also being developed [15, 26]. In view of the present application, the derivation of maximum safe fortification levels (MSFLs), these models on optimal intake were not taken into account. The question how to create an optimal micronutrient intake is outside the scope of this paper.\nOn the basis of the existing models to calculate a maximum fortification level, a model adapted to the Dutch practical situation was developed for the Dutch authorities to derive for the fortification with micronutrients. This article first describes the model for the derivation of maximum amounts of micronutrient fortification in the Netherlands, and then it illustrates the use of the micronutrient specific data and assumptions in the calculation of the MSFLs for folic acid, vitamin A, and vitamin D. The article concludes with the considerations and the final allowed fortification level (AFL) of the risk management.\nMethods\nModels to calculate safe maximum levels of addition of micronutrients are published, among others, by ILSI, Rasmussen and Domke [12\u201314, 25]. We developed a model for the Dutch situation, based on (elements of) the models of ILSI, Rasmussen and Domke. Below, we will amplify on the main differences between the models and our filling-in for the Dutch model.\nThe ILSI Europe and Rasmussen et al. models differ in several aspects from Domke et al.\u2019s model. Instead of the calculation per portion size as applied by Domke et al., both ILSI Europe and Rasmussen et al. calculate the maximum fortification level per 100\u00a0kcal. For our Dutch model, we also calculate with 100\u00a0kcal portions because the variation in mean daily energy intake is low compared to for instance the variation in micronutrient or carbohydrate intake [29]. Moreover, there is no univocal definition of portion size in the Netherlands. Another important difference is that Domke et al. not only establish maximum fortification levels, but they also establish maximum levels for dietary supplements. Because maximum safe addition levels of vitamins and minerals in dietary supplements will be regulated via European Union legislation, we did not determine them in our model [11]. Similarly to ILSI Europe and Rasmussen et al., we estimated the current intake of micronutrients from dietary supplements and took them into account. Our Dutch model is principally based on the ILSI Europe model [14] and Rasmussen et al.\u2019s extended model [25], with adaptations to the Dutch practical situation. The formula is equal to the one used by Rasmussen et al., however some of the factors are differently derived (see below).\nOur model calculates the maximum safe level for fortification with a micronutrient per 100\u00a0kcal of the food (MSFL) for various age groups with the mathematical formula presented in Box 1. The lowest MSFL, i.e., the MSFL for the most sensitive group, is then advised to be the overall maximum acceptable fortification, as is common practice in toxicological risk assessment.\nTolerable upper intake level (UL)\nThe UL is the maximum level of total chronic daily intake of a nutrient that is unlikely to pose a risk of adverse health effects to humans. In general, the UL is related to total daily intake from all sources including nonfortified foods as well as fortified foods and dietary supplements. In Europe, the Scientific Committee on Food (SCF) and the Scientific Panel on Dietetic Products, Nutrition and Allergies (NDA) set age-specific ULs [28]. Due to lack of data, the ULs for children are usually extrapolated from the ULs for adults based on bodyweight. In anticipation of future European policy [11], these age-specific European ULs were used as model input rather than the national ULs, which is similar to the model of Rasmussen et al. [25]. ILSI Europe has also used European ULs, although only the values for men [14].\nBox 1. Formula for the calculation of the maximum safe fortification level with micronutrients per 100 kcal food (MSFL) [25].\nCurrent intake of energy and micronutrients from nonfortified foods (EI95 & CI95)\nMicronutrients are naturally present in the diet (further referred to as \u2018background diet\u2019). Because it is important to consider the intake from all possible sources to assess the risk, the intake from the background diet has to be included in the calculations. To estimate the intake of energy and micronutrients from the background diet, it is important to use the most up-to-date country- and age-specific data available. In view of the precautionary principle [3, 10], we calculated the age-specific habitual intake (also referred to as usual intake) at the 95th percentile of the distribution of energy and the specific micronutrients from the background diet on the basis of data from the most recent Dutch National Food Consumption Surveys (DNFCS): DNFCS-3 and DNFCS-2003. The DNFCS-3 was carried out in 1997\u20131998 for the whole population (1\u201397\u00a0years) and the DNFCS-2003 was carried out in 2003 for young adults (19\u201330\u00a0years) [16, 23]. In the DNFCS-3, a 2-day dietary record method was used to collect data on two consecutive days. In the DNFCS-2003, data were collected with a 2-day, 24-hour recall method using EPIC-SOFT, a computer-assisted interview method. We estimated the habitual intake of both micronutrients and energy with statistical correction for the within-person variation using the Iowa State University method (C-SIDE software) [18, 20].\nRasmussen et al.\u2019s model uses results from the Danish dietary survey in which a 7-day prospective food recall method was used to calculate the daily intake of micronutrients from nonfortified foods (95th percentile) and energy intake (95th percentile) [25]. ILSI Europe used pan-European estimates of the 95th percentile of intake of micronutrients based on seven national surveys. Some of these surveys already include voluntary fortification of foods with micronutrients and the intake of dietary supplements [14]. Furthermore, the pan-European 95th percentile of energy intake was estimated from five national surveys.\nCurrent intake of micronutrients from dietary supplements (SI)\nIn addition to the intake from the background diet, the micronutrient intake from dietary supplements (SI) has to be taken into account in order to get a good estimate of the total intake from all sources. In the Netherlands, similar to other Western countries, the use of dietary supplements has increased and takes a more important place in the total micronutrient intake [22]. Preferably, the habitual micronutrient intakes of nonfortified foods and dietary supplements are calculated together. This method takes into account the facts that not everybody uses dietary supplements, not all supplement users use the supplements daily, and supplement users and nonusers may have different eating habits [19]. However, due to lack of information about supplement use, a calculation of combined habitual intake is not possible at this time. Therefore, we used two realistic high-intake scenarios of dietary supplements based on the limited Dutch dietary supplement data available: one for adults (\u226518\u00a0years old) and one for children (1\u201317\u00a0years old). The scenario for adults was based on the P90-P95 of both the intake of micronutrients from dietary supplements in young adults (19\u201330\u00a0years) reported by the DNFCS-2003 [22] and the levels of micronutrients present in dietary supplements (for adults) available in the Netherlands [4]. The scenario for children was based on P90\u2013P95 of the infants\u2019 intake of dietary supplements [9] and the levels of micronutrients present in dietary supplements for children, that are available in the Netherlands [4].\nWe determined the SI differently than Rasmussen et al. and ILSI Europe did. Rasmussen et al. chose to set the SI at 100% of the reference values for the recommended daily intake of vitamins and minerals because this is the content of the most used multi-vitamin\u2013mineral supplement in Denmark [25]. The ILSI Europe model does not include the intake from dietary supplements because only a minority of the population is expected to use them [11, 14].\nFraction of energy intake that can and will be fortified (PFFn)\nNot all foods on the market can or will be fortified. The fraction of total energy intake that will finally be fortified consists of two parts: (1) the proportion of total energy intake that can be fortified; for example, it is unlikely that fresh products such as vegetables, fruits and meat will be fortified; and (2) the proportion of the energy intake of foods available for fortification that actually will be fortified. Due to practical constraints such as costs and processing, not all foods available for fortification will be fortified with a particular micronutrient.\nThe first part of the fraction was set at 30%, which is lower than the 50% set by both ILSI Europe and Rasmussen et al. Main argument for the lower percentage was that the large part of the energy intake in the Dutch population is delivered by the traditional dinner, which consists in general of fresh products like meat, vegetables, and potatoes. The second part of the fraction was equal to the one ILSI Europe and Rasmussen et al. set at 50% (worst case). This resulted in a fraction of the energy intake available for fortification (PFFn) of 15% (i.e., 30%\u00a0*\u00a050%).\nResults\nRisk assessment: deriving MSFLs\nWe illustrate the applicability of the Dutch model by deriving MSFLs of folic acid, vitamin A, and vitamin D. Tables\u00a01, 2, 3 present the factors used for the calculation of the MSFLs for folic acid, vitamin A, and vitamin D. The classification into age groups is based on the age categories used by NDA\/SCF to define the ULs [28] and is different for folic acid, vitamin A, and vitamin D. We calculated the 95th percentile of habitual energy intake for each age group. The habitual energy intake of children increases with age (Tables\u00a01\u20133). The 95th percentile of the habitual energy intake for men is greater than that for women; there is a small decline of habitual energy intake with age for both genders.\nTable\u00a01Maximum safe fortification levels and allowed fortification level of folic acid based on Dutch consumption data and European tolerable upper levels of intake for specific age groupsAge groupnUL (\u00b5g\/day)CI95a (\u00b5g\/day)SI (\u00b5g\/day)EI95 (SE) (kcal\/day)Max safe fortification level (MSFL) (\u00b5g\/100 Kcal)Allowed fortification level (AFL) (\u00b5g\/100 kcal)PFFn\u00a0=\u00a00.15PFFn\u00a0=\u00a00.10bChildren1\u20133c25420003001,890 (67)0Children4\u20136c27630003001,995 (56)0Children7\u201310c31240003002,456 (63)27Children11\u201314c33660003003,154 (117)63Adolescents15\u201317c28080003003,518 (150)95Men18\u201330c5251,00006004,010 (112)67100100Women18\u201330c5791,00006002,867 (62)93140Men19\u201330d3521,00006003,751 (130)71106100Women19\u201330d3981,00006002,581 (76)103155Men31\u201350c9501,00006003,529 (64)76113Women31\u201350c1,1001,00006002,721 (48)98147Men51\u201365c4201,00006003,427 (109)78117Women51\u201365c4791,00006002,647 (74)101151Men>65c2601,00006003,106 (119)86129Women>65c4101,00006002,432 (65)110164aDietary intake of folic acid only, folate is not taken into account because the UL is based on folic acid onlybAdditional calculation with exactly the same results as for PFFn\u00a0=\u00a00.15 in combination with a scenario of low dietary supplement intake (i.e., 400\u00a0\u00b5g), only calculated for adultscBased on consumption data from the DNFCS-3 (1997\u20131998) [2]dBased on consumption data from the DNFCS 2003 [17, 21]The most sensitive age groups are bolded; CI95, 95th percentile of habitual dietary folic acid intake; EI95, 95th percentile of habitual energy intake; SI, supplement intake; PFFn, proportion of energy intake that can and will be fortified; UL, tolerable upper intake levelTable\u00a02Maximum safe fortification levels of vitamin Aa based on Dutch consumption data and the European upper levels of intake for specific age groupsAge groupNUL (\u00b5g \/day)CI95 (SE) (\u00b5g \/day)SI (\u00b5g \/day)EI95 (SE) (kcal\/day)Max safe fortification level (MSFL) (\u00b5g \/100\u00a0kcal) PFFnb\u00a0=\u00a00.15Children1\u20133c2548001,057 (167)8001890 (67)0Children4\u20136c2761,1001,553 (262)8001995 (56)0Children7\u201310c3121,5001,055 (116)8002456 (63)0Children11\u201314c3362,0001,153 (131)8003154 (117)10Adolescents15\u201317c2802,6001,429 (205)8003518 (150)70Men19\u201330b3523,0001,886 (261)1,2003751 (130)0Women19\u201330b3983,0001,203 (162)1,2002581 (76)154Men\u226518c2,1553,0002,363 (132)1,2003642 (50)0Women18\u201350c1,6793,0001,350 (83)1,2002785 (39)108Women>50c8891,5001,441 (108)1,2002555 (53)0aNot including provitamin AbBased on consumption data the DNFCS 2003 [17, 21]cBased on consumption data from the DNFCS-3 (1997\u20131998) [2]The most sensitive age groups are bolded; CI95, 95th percentile of habitual dietary vitamin A intake; EI95, 95th percentile of habitual energy intake; SI, supplement intake; SE, standard error; PFFn, proportion of energy intake that can and will be fortified; UL, tolerable upper intake levelTable\u00a03Maximum fortification levels and allowed fortification level of vitamin D based on Dutch consumption data and European tolerable upper levels of intake for specific age groupsAge groupnUL (\u00b5g\/day)CI95 (SE) (\u00b5g\/day)SI (\u00b5g\/day)EI95 (SE) (kcal\/day) Max safe fortification level (MSFL) (\u00b5g\/100 Kcal)Allowed fortification level (AFL) (\u00b5g\/100 kcal)PFFn\u00a0=\u00a00.15PFFn\u00a0=\u00a00.10aChildren1\u20133b254254.8 (0.6)101,890 (67)3.65.4Children4\u201310b276254.4 (0.2)102,334 (50)3.04.54.5Children11\u201313b312506.5 (0.6)102,935 (100)7.611.4Adolescents14\u201318b336507.6 (0.5)103,509 (116)6.29.2Men19\u201330c352506.5 (0.4)103,751 (130)6.08.9Women19\u201330c398505.2 (0.4)102,581 (76)9.013.5Men31\u201350b950508.9 (0.4)103,529 (64)5.98.8Women31\u201350b1,100506.4 (0.3)102,721 (48)8.212.3Men51\u201360b3145011.3 (1.2)103,410 (128)5.68.4Women51\u201360b332506.7 (0.6)102,623 (90)8.512.7Men61\u201370b197509.8 (1.0)103,315 (124)6.19.1Women61\u201370b270506.6 (0.6)102,528 (87)8.813.2Men>70b1695012.2 (1.8)103,067 (141)6.09.1Women>70b287508.6 (0.8)102,487 (86)8.412.6aAdditional calculation with exactly the same results as for PFFn\u00a0=\u00a00.15 in combination with low scenario of dietary supplement intake (i.e., 5\u00a0\u00b5g)bBased on consumption data from the DNFCS-3 (1997\u20131998) [2]cBased on consumption data from the DNFCS 2003 [21]The most sensitive age groups are bolded; CI95, 95th percentile of habitual dietary vitamin D intake; EI95, 95th percentile of habitual energy intake; SI, supplement intake; SE, standard error; PFFn, proportion of energy intake that can and will be fortified; UL, tolerable upper intake level\nFolic acid\nFolic acid is the synthetic form of folate, a natural component in the diet. An UL has been established only for folic acid and not for folate. For this reason, the calculation does not consider the intake of folate from the background diet. At the time the food consumption surveys were conducted, fortification with folic acid was not permitted in the Netherlands, which results in an intake from the background diet (CI95) of 0\u00a0\u00b5g\/day. The SI of folic acid was estimated to be 300\u00a0\u00b5g\/day for children and adolescents and 600\u00a0\u00b5g\/day for adults. Filling in these factors in the formula of Box 1 resulted in a maximum fortification level for folic acid of 0\u00a0\u00b5g\/100\u00a0kcal for children 1\u20136\u00a0years old to 110\u00a0\u00b5g\/100\u00a0kcal for women older than 65\u00a0years (Table\u00a01). For children and adolescents, the MSFL for folic acid fortification increased with age because of an increase in UL over the age groups. For adults, men generally had lower MSFLs than women due to a higher energy intake. Based on the most sensitive age group (i.e., children 1\u20136\u00a0years) the MSFL is 0\u00a0\u00b5g\/100\u00a0kcal. For the most sensitive adult group (i.e., men 18\u201330\u00a0years old) the MSFL is 67\u201371\u00a0\u00b5g\/100\u00a0kcal. Our calculated MSFL for the most sensitive group is lower for folic acid than the acceptable level of addition of Rasmussen et al., 23\u00a0\u00b5g\/100\u00a0kcal for the most sensitive group (1\u20133\u00a0years old), while we calculated 0\u00a0\u00b5g\/100\u00a0kcal for children 1\u20136\u00a0years old. This difference is mainly caused by a lower estimation of the SI by Rasmussen et al., which was based on Danish legislation. In our calculations, the current intakes from dietary supplements were estimated based on the amount of folic acid available in dietary supplements on the Dutch market.\nVitamin A\nPart of the vitamin A is consumed as provitamin A (for instance, carotenoids). However, the UL is defined for the intake of vitamin A as pure retinol and retinyl esters only because it is thought that the intake of provitamin A will not significantly contribute to the toxicity of high intakes of vitamin A [28]. When we use the term \u2018vitamin A\u2019, we refer to pure retinol and retinyl esters. Due to the risk of osteoporosis, the UL for postmenopausal women is much lower than that for other women [28]. In our study population, the distinction between premenopausal and postmenopausal women could not be made exactly; therefore, it was assumed that women older than 50\u00a0years are representative for the group of postmenopausal women.\nWhen the food consumption surveys were carried out, vitamin A addition was only allowed for margarine and low-fat margarine as a substitute for butter. This substitution will continue in the Netherlands, and therefore the fortification of (low-fat) margarine with vitamin A was included in the calculation of the CI95. The SI was estimated to be 800\u00a0\u00b5g vitamin A\/day for children and adolescents and 1,200\u00a0\u00b5g vitamin A\/day for adults. The habitual dietary vitamin A intake varied from 1,055\u00a0\u00b5g\/day to 2,363\u00a0\u00b5g\/day. For children, the age group 4\u20136\u00a0years had the greatest vitamin A intake (1,553\u00a0\u00b5g\/day). Men had a larger intake than women. The vitamin A intake of premenopausal and postmenopausal women (i.e., older than 50\u00a0years) was comparable (Table\u00a02). Filling in these factors in the formula (Box 1) resulted in a MSFL for vitamin A of 0\u00a0\u00b5g vitamin A\/100\u00a0kcal for children 1\u201310\u00a0years old, men and post-menopausal women, 154\u00a0\u00b5g vitamin A\/100\u00a0kcal for women 19\u201330\u00a0years old and for older children and adolescents, the MSFL is 10\u201370\u00a0\u00b5g vitamin A\/day. In conclusion, the MSFL for vitamin A will be 0\u00a0\u00b5g vitamin A\/100 kcal on the basis of the most sensitive groups (i.e., children 1\u201310\u00a0years, men 18\u00a0years old or more, and postmenopausal women). Our results for vitamin A were the same as Rasmussen et al.\u2019s, i.e., 0\u00a0\u00b5g vitamin A\/100 kcal.\nVitamin D\nAt the time when the DNFCS-3 and the DNFCS-2003 were carried out, addition of vitamin D was only allowed for margarine and low-fat margarine as a substitute for butter. Because this substitution is expected to continue in the Netherlands, this fortification practice was included in the calculation of the CI95. The 95th percentile of habitual dietary vitamin D intake varied from 4.4\u00a0\u00b5g\/day to 12.2\u00a0\u00b5g\/day (CI95). Vitamin D intake increased with age for children, although children 1\u20133\u00a0years old had a slightly greater intake than children 4\u201310\u00a0years old. For men, the 95th percentile of habitual vitamin D intake lies between 6.5\u00a0\u00b5g\/day and 12.2\u00a0\u00b5g\/day and for women, between 5.2\u00a0\u00b5g\/day and 8.6\u00a0\u00b5g \/day (Table\u00a03). The SI was estimated to be 10\u00a0\u00b5g vitamin D\/day for children, adolescents, and adults.\nEntering these factors into the formula (Box 1) resulted in a MSFL for vitamin D of 3.0\u00a0\u00b5g\/100\u00a0kcal for children 4\u201310\u00a0years old to 9.0\u00a0\u00b5g\/100\u00a0kcal for women 19\u201330\u00a0years old. The MSFL increased with age for children, although children 4\u201310\u00a0years old had a slightly lower MSFL than children 1\u20133\u00a0years old. Due to a smaller habitual energy and vitamin D intake, the MSFL for women is greater than that for men. In conclusion, the MSFL of vitamin D is 3.0\u00a0\u00b5g\/100\u00a0kcal based on the most sensitive group (i.e., children 4\u201310\u00a0years). For the most sensitive adult group (men 51\u201360\u00a0years old), the MSFL is 5.6\u00a0\u00b5g\/100\u00a0kcal. Our MSFL for vitamin D (3.0\u00a0\u00b5g\/100\u00a0kcal for children 4\u201310\u00a0years old) is greater than Rasmussen et al.\u2019s acceptable level of addition: 1\u00a0\u00b5g\/100 kcal for children 7\u201310\u00a0years old. This is mainly due to the difference in PFFn. Rasmussen et al. used a more strict PFFn of 25% whereas we used a PFFn of 15%.\nRisk-management: setting AFLs for general exemption\nAfter the risk was assessed, a risk manager considered all the important issues and decided what maximum level of fortification will be allowed and included in a general exemption. In the Netherlands, proposed changes and additions to the Dutch Commodity Act are discussed with stakeholders (e.g., organizations of trade and industry, consumers organizations, authorities) in a Regular Consultation on the Commodity Act. Representatives of individual companies cannot participate in the Regular Consultation on the Commodity Act.\nSeveral consultations regarding the results (Tables\u00a01\u20133) of the risk assessment for fortification with folic acid, vitamin A, and vitamin D resulted in a general exemption for folic acid of 100\u00a0\u00b5g\/100\u00a0kcal and for vitamin D of 4.5\u00a0\u00b5g\/100\u00a0kcal. No general exemption was given for vitamin A. A main general consideration is that this national policy will be temporary, as European legislation is expected to come into effect within a few years [11].\nFolic acid\nThe UL of folic acid for adults is based on the risk of masking vitamin B12 deficiency, for children an UL is extrapolated from this adult value based on bodyweight. Because of an ongoing discussion about the value of the UL for children extrapolated from the UL for adults, the risk manager decided to focus on the results of the risk assessment for adults only. It was thought that such an extrapolation would be conservative because the risk of vitamin B12 deficiency for children was thought to be low. Furthermore, at that time, exemptions had been requested for only a few product types and some specific brands. Therefore, the risk manager expected that the chance of children consuming all available folic-acid-fortified products was small. In discussions with the stakeholders, which considered the temporary character of this national policy and the number of requests for exemption, the PFFn of 15% was judged to be rather conservative. Using the dietary intake at the 95th percentile of the distribution (CI95) and the 90\u201395th percentile of intake from dietary supplements (SI) was also judged to be quite conservative. Therefore, two additional calculations were done, one with a PFFn of 10% and the other with a low intake scenario of dietary supplements (i.e., 400\u00a0\u00b5g for adults). Both scenarios resulted in a maximum level for fortification in the most sensitive adult group (men aged 18\u201330\u00a0years) of 100\u2013106\u00a0\u00b5g\/100\u00a0kcal (Table\u00a01). For practical reasons, the risk manager rounded this value off to an AFL of 100 \u00b5g folic acid\/100\u00a0kcal. Because of this relatively liberal AFL and the fact that the value was based on the MLF of adults, it was decided to monitor the intake of folic acid from fortified foods. If the results of the monitoring show that the long-term intake exceeds the UL in specific population groups, appropriate measures can be taken.\nVitamin D\nFor vitamin D the derivation of the UL for children is based on critical effects observed in children, whereas for folic acid UL-values for children were established by extrapolation only [28]. Therefore, the maximum fortification levels for children from the risk assessment were considered. As for folic acid, this will be a temporary policy for only a couple of years and it was thought that the PFFn of 15% and the high-intake scenario from dietary supplements are rather conservative estimates. In addition, only one request for exemption was received at that time for vitamin D fortification, and the risk manager did not expect more exemption requests. Thus, similar additional calculations with a PFFn of 10% or a scenario of smaller intakes from dietary supplements (i.e., 5\u00a0\u00b5g) were done for vitamin D. The calculations resulted in a MSFL of 4.5\u00a0\u00b5g vitamin D\/100\u00a0kcal for the most sensitive group (children aged 4\u201310\u00a0years) (Table\u00a03). The risk manager adopted this MSFL and set the AFL at 4.5\u00a0\u00b5g\/100\u00a0kcal. Again, the risk manager desires monitoring of vitamin D and energy intake for insight into current intakes and proportions of energy intake fortified. If long-term intakes exceed the ULs, appropriate measures can be taken.\nVitamin A\nIn contrast to the liberalization for folic acid and vitamin D, the risk manager did not liberalize the results from the risk assessments for vitamin A. To the opinion of the risk manager, exceeding the UL of vitamin A has more serious consequences for public health than exceeding the ULs for folic acid and vitamin D. In addition, the 95th percentile of habitual dietary vitamin A intake of several age groups already exceeded the UL, even without considering the vitamin A intake from dietary supplements. Furthermore, at this time no request for exemption has been received, although one product for which an exemption for folic acid was requested also contained vitamin A. So the AFL was set at 0\u00a0\u00b5g\/100\u00a0kcal. The stakeholders in the consultation agreed with this decision.\nException for low-energy products\nAn exception was made for light variants of products (low-energy products). According to the model, products low in energy may be fortified with smaller amounts of the micronutrient than the more energy-dense counterparts. In view of the obesity trend and the healthy image of light products, this was considered undesirable. Therefore, light products may be fortified with amounts similar to the amounts approved for their more energy-dense counterparts.\nDiscussion\nWe presented a model for the derivation of maximum amounts of micronutrient fortification in the Netherlands. A novelty of this publication is that besides the theoretical model, the practical applicability for the Netherlands is shown including both the risk-assessment and risk-management aspects. The model has already contributed to new Dutch policy-making, as the allowed level for fortification for folic acid and vitamin D are applied as a general exemption in 2007 [8].\nApplicability of the model\nAlthough the model can already be applied with the available data in the Netherlands, it is recommended to improve the monitoring of consumption of fortified foods and dietary supplements within the Netherlands. Such data are very useful to check the model parameters and to keep them up-to date. If necessary, AFLs can be adjusted. It is recommended to monitor the intake of fortified foods and dietary supplements as part of the national food consumption survey [7, 27]. Food frequency questionnaires specifically for fortified functional foods and dietary supplements in a representative sample of the population would be a useful tool. In addition composition databases of fortified foods and dietary supplements are recommended [27].\nRisk management\nThe discussions about the risk-assessment results with the different stakeholders worked well for the risk manager. This resulted in broad insight into the various points of view of the stakeholders. Ultimately the risk manager took the final decision, and in all these cases the stakeholders agreed with the final outcome. The risk assessment was based on the precautionary principle. However, with a view to the fact that this will be a temporary policy, the outcome of the risk assessment was judged to be rather conservative for folic acid and vitamin D. In contrast, the market may change, the date the European legislation will come into effect can be later than expected, and people may consume more fortified foods than estimated. Therefore, monitoring the intake of micronutrients from fortified foods is a very important tool for warranting the safety of the population. If monitoring shows that some groups within the population have habitual intakes that exceed the ULs, adaptations should be made. Especially children may be vulnerable. A food consumption survey is currently being conducted among children in the Netherlands. These data will provide insight into the micronutrient intake from natural foods, fortified foods and dietary supplements during 2005\/2006.\nAdditional considerations\nSome factors that were not taken into account in MSFL risk assessment are worth considering, and they can be implemented in the model or taken into account in the risk management if necessary. First, we made an exception for the fortification level of \u2018light\u2019 products in our risk management. This exception was not considered in the calculation and can therefore theoretically lead to micronutrient intakes that are too large even if the energy intakes are not exceptionally high. Monitoring the use of fortified \u2018light\u2019 products will be necessary to avoid frequent users exceeding the UL. Second, some products are intended for a specific population or target group only. If there is little risk that the product will be used by nontarget groups, the risk manager can make an exception to the model. Instead of using the calculated MSFL for the most vulnerable group, the MSFL for the specific target group can be used as a maximum for this specific product. Again, monitoring of the use of these products is important, and mentioning the target groups, as well as nontarget groups, on the product package might be considered.\nThe vitamin levels in products may decrease over time. Therefore, it is possible that the amount of vitamin added by manufacturers is greater than the amount declared on the packages [24]. To be able to protect all consumers and avoid underestimation of the intake, this so-called overage should be integrated into the MSFL in the model. Rasmussen et al. also noted this problem. In Denmark the currently accepted practice is to add up to 150% of the declared amount. Our calculated MSFL includes overage.\nInternational applicability of the risk-assessment model\nCurrently, the European Commission is preparing to set maximum and minimum amounts for vitamins and minerals in both foods and dietary supplements [11]. The simultaneously setting of both the maximum levels for dietary supplements and for foodstuffs can result in a well based division of micronutrients over dietary supplements and fortified foods. Once the choice has been made which micronutrient is allowed to be added to dietary supplements and\/or fortified foods, models like the one presented can be helpful to set the MSFL and AFL for micronutrients in food products.\nThe applicability of the model for this purpose depends on the availability of the input data. When calculating MSFLs valid for Europe as a whole, the availability of European data is necessary. However, data from a food consumption survey that cover all Member States of the European Union representatively are not available. Such data are not even available for all Member States individually. Furthermore, consumption data available in various Member States are often collected with different methods, in a different time frame, and for different specific population groups and the fraction of energy intake that can be fortified may differ greatly from country to country because of the various traditional diets. This makes it difficult to estimate the total habitual intake for each micronutrient and energy type at a European level.\nTo overcome these problems, MSFLs in Europe can be set by using the available country-specific calculations and to select the most sensitive country (i.e., the country with the lowest MSFL). The maximum safe level for fortification for this country can then be applied in all Member States.\nConclusion\nAs this paper illustrates, our model for risk assessment can be used in the Netherlands to help risk managers to set maximum levels for safe addition of vitamins to foods. This has resulted in two general exemptions, one for folic acid at 100\u00a0\u00b5g\/100\u00a0kcal and one for vitamin D at 4.5\u00a0\u00b5g\/100\u00a0kcal. The model is flexible and can be adapted to new insights. For example, aspects concerning overage and \u2018light\u2019 products can be included with minor changes in the formula. Monitoring of the total micronutrient intake after applying a general exemption, as well as monitoring of the fortified fraction of the energy intake and the effect of the exception for light products, is recommended for evaluating the model parameters and AFLs and for making adjustments when needed.","keyphrases":["safe addition","vitamins","minerals","food","fortification","micronutrients","tolerable upper intake level","maximum fortification levels"],"prmu":["P","P","P","P","P","P","P","P"]}
{"id":"Calcif_Tissue_Int-3-1-1914289","title":"In Vivo Mechanical Loading Modulates Insulin-Like Growth Factor Binding Protein-2 Gene Expression in Rat Osteocytes\n","text":"Mechanical stimulation is essential for maintaining skeletal integrity. Mechanosensitive osteocytes are important during the osteogenic response. The growth hormone-insulin-like growth factor (GH-IGF) axis plays a key role during regulation of bone formation and remodeling. Insulin-like growth factor binding proteins (IGFBPs) are able to modulate IGF activity. The aim of this study was to characterize the role of IGFBP-2 in the translation of mechanical stimuli into bone formation locally in rat tibiae. Female Wistar rats were assigned to three groups (n = 5): load, sham, and control. The four-point bending model was used to induce a single period of mechanical loading on the tibial shaft. The effect on IGFBP-2 mRNA expression 6 hours after stimulation was determined with nonradioactive in situ hybridization on decalcified tibial sections. Endogenous IGFBP-2 mRNA was expressed in trabecular and cortical osteoblasts, some trabecular and subendocortical osteocytes, intracortical endothelial cells of blood vessels, and periosteum. Megakaryocytes, macrophages, and myeloid cells also expressed IGFBP-2 mRNA. Loading and sham loading did not affect IGFBP-2 mRNA expression in osteoblasts, bone marrow cells, and chondrocytes. An increase of IGFBP-2 mRNA-positive osteocytes was shown in loaded (1.68-fold) and sham-loaded (1.35-fold) endocortical tibial shaft. In conclusion, 6 hours after a single loading session, the number of IGFBP-2 mRNA-expressing osteocytes at the endosteal side of the shaft and inner lamellae was increased in squeezed and bended tibiae. Mechanical stimulation modulates IGFBP-2 mRNA expression in endocortical osteocytes. We suggest that IGFBP-2 plays a role in the lamellar bone formation process.\nMechanical stimulation is important for the maintenance of the skeleton of humans and other animals [1\u20139]. Bone serves as a structural support for the body and has the ability to modify its architecture throughout life. Mechanical stimulation regulates bone mass and architecture according to Frost\u2019s mechanostat hypothesis. Physical activity results in increased bone formation and decreased bone resorption, whereas inactivity has the opposite effect [10, 11].\nThe growth hormone-insulin-like growth factor (GH-IGF) axis plays an important role in the regulation of bone remodeling. Of the insulin-like growth factors (IGFs), IGF-I is predominantly expressed in rat bone and IGF-II is predominantly expressed in human bone [12\u201314]. IGFs are involved in bone formation after mechanical stimulation. Mechanical loading upregulates IGF-I mRNA expression in osteocytes, as shown in rat vertebra by Lean and colleagues using the caudal vertebra compression model [15]. Northern blot analysis also showed enhanced periosteal IGF-I mRNA expression after mechanical stimulation [16]. Besides an increase in IGF-I mRNA synthesis, Bravenboer and colleagues reported upregulation of IGF-I protein concentration in bone after mechanical stimulation using additional weight bearing in running rats (rat-with-backpack) [17].\nAs in other tissues, the autocrine and paracrine actions of IGFs in bone are modulated by a family of six structurally related binding proteins, the IGF binding proteins (IGFBPs). IGFBPs can be synthesized by osteoblasts [18, 19] and are capable of modulating the effects of IGFs by potentiating or inhibiting their action; some IGFBPs also have IGF-independent actions that can either inhibit or stimulate cellular function.\nSince rat osteoblasts do not express IGFBP-1 [18, 20, 21], it is most likely that IGFBP-1 does not play a significant role during bone formation after mechanical stimulation in rats. Although IGFBP-2 is the major IGFBP synthesized by rat osteoblastic cells [18], a role for IGFBP-2 during mechanical loading has not been established yet. IGFBP-3 has both inhibitory and stimulatory effects on bone cells. IGFBP-4, which is locally produced by osteoblasts, has an inhibitory effect on bone metabolism, whereas locally synthesized IGFBP-5 by osteoblasts has a stimulatory effect [18, 22, 23]. However, the effects of IGFBP-4 on osteoblasts are IGF-I-dependent, whereas IGFBP-5 probably acts as a store for IGFs in bone tissue [18, 22, 23]. IGFBP-6 is expressed in rat osteoblasts [24]. In conclusion, IGFBP-2 is the most abundant IGFBP in rat osteoblasts; however, the role of IGFBP-2 during bone formation after mechanical stimulation in vivo has not been determined yet. We hypothesized that IGFBP-2 mRNA expression level might be changed in bone cells during bone formation after mechanical stimulation.\nThe aim of this study, therefore, was to determine the localization of IGFBP-2 mRNA in the cortical tibial shaft after mechanical stimulation. To this end, we induced a single period of mechanical loading using the four-point bending model of Forwood and Turner [1, 2, 7]. This resulted in bone formation in the rat tibia 5\u20138 days after stimulation [25]. We developed an in situ hybridization method especially for bone tissue to detect the local osteogenic response at the cellular level 6 hours after a single period of dynamic loading.\nMaterials and Methods\nAnimals\nFifteen female 12-week-old Wistar rats (235 \u00b1 12 g; Harlan, Zeist, The Netherlands) were randomly assigned to three weight-matched groups (n = 5\/group): load, sham, and control. The animal experiment was in accordance with the governmental guidelines for the care and use of laboratory animals and approved by the Institutional Animal Care and Use Committee of the VU University Medical Center (Amsterdam, The Netherlands).\nIn vivo Mechanical Loading\nThe right tibiae underwent \u201cmediolateral\u201d loading (load), sham loading (sham, in which the opposed pads were placed at the inner position, 11 mm apart), or no loading (control) using the four-point bending system of Forwood and Turner [1, 7]. Since loading will result in bending and squeezing of the tibia and sham loading only in squeezing of the tibia, the sham group was used as a control for the load group. The left tibiae served as contralateral controls. The four-point bending model [25] was used to generate a single period of dynamic loading of the right tibia in rats in vivo in order to detect acute changes of IGFBP-2 mRNA locally in bone tissue after stimulation by mechanical stress. The rats were subjected to a single episode of loading comprising 300 cycles (2 Hz) using a peak magnitude of 60 N. Forwood and Turner showed that a single loading session resulted in bone formation in the rat tibia 5\u20138 days after stimulation [25]. Characterization of the in vivo strain using the four-point bending system was reported by Akhter and colleagues [26]. Using the four-point bending system at our laboratory, we demonstrated bone formation at the endosteal surface of the rat tibia 15 days after a single loading session with a frequency of 2 Hz during 300 cycles and an applied peak magnitude of 60 N [27]. The loading experiment was performed under general anesthesia (2% isoflurane in 1 L\/min O2 and 2 L\/min N2O). The rats were killed exactly 6 hours after loading. This time point was based on the literature [15], which was confirmed by a time-course pilot experiment at our laboratory using real-time reverse-transcription polymerase chain reaction analysis (H. W. van Essen, personal communication) showing the highest IGF-I mRNA expression 6 hours after loading. Since the actions of IGFs are influenced by their IGFBPs, we examined IGFBP-2 mRNA expression at 6 hours after loading. The tibiae were dissected and immediately fixed in 4% (w\/v) paraformaldehyde (buffered in phosphate-buffered saline [PBS], pH 7.4) at 4\u00b0C for 24 hours.\nTissue\nAfter fixation, the tibiae were decalcified in 10% ethylenediaminetetraacetic acid (EDTA) with 0.5% paraformaldehyde in PBS at 4\u00b0C for 4.5 weeks. Finally, the tibiae were washed in PBS and dehydrated through a series of ethanol and xylene at room temperature and embedded in paraffin.\nAs a positive control, brain tissue was used [28]. Brains were dissected rostrally to the cerebellum (interaural coordinate 0 mm) and the hippocampus (interaural coordinate 4 mm) in three coronal blocks and immediately fixed in 4% (w\/v) paraformaldehyde (buffered in PBS, pH 7.4) at 4\u00b0C for 24 hours, followed by washing in PBS, dehydration through a series of ethanol and xylene at room temperature, and embedding in paraffin.\nReagents\nAll restriction enzymes and modifying enzymes were purchased from Roche Molecular Biochemicals (Mannheim, Germany), as well as digoxigenin-uridine triphosphate (UTP), antidigoxigenin Fab fragments, nitroblue tetrazolium chloride (NBT), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), and blocking reagent. Nylon membranes were purchased from Qiagen (Hilden, Germany). Polyvinyl alcohol was obtained from Aldrich (Milwaukee, WI). Euparal mounting medium was purchased from Chroma Gesellschaft (Waldeck, Division Chroma, M\u00fcnster, Germany). Silane-coated glass slides were obtained from Sigma-Aldrich (St. Louis, MO).\nMouse IGFBP-2 cDNA was kindly provided by Dr. S. L. S. Drop and Dr. J. W. van Neck (Department of Pediatrics, Sophia Children\u2019s Hospital, Erasmus University, Rotterdam, The Netherlands) via Dr. S. C. van Buul-Offers (Department of Metabolic and Endocrine Diseases, University Medical Center, Utrecht, The Netherlands).\nSynthesis of Digoxigenin-Labeled Complementary RNA (cRNA) Probes\nStandard in vitro transcription reactions were carried out using T7- and Sp6-RNA polymerase with digoxigenin-UTP as a substrate [29]. cDNA encoding mouse IGFBP-2 cDNA, corresponding to amino acid position 98\u2013258 and showing 95% homology with the corresponding rat IGFBP-2 cDNA fragment, was used as a template for the synthesis of antisense and sense digoxigenin-labeled RNA probe. The probe was specific for the mRNAs analyzed. The IGBP-2 probe did not show cross-reactivity with different types of mouse tissues (spleen, thymus, and brain) [28, 30, 31].\nNonradioactive In Situ Hybridization\nSerial, longitudinal tibial sections (5 \u03bcm), which were cut in the posterior-anterior direction, and cross-sectional control brain sections (5 \u03bcm) were mounted onto RNase-free silane-coated glass slides and dried at 56\u00b0C for at least 3 days. Corresponding sections of the right and left tibia of one rat were mounted on the same glass slide. In situ hybridization was performed on every fiftieth section with a total of five slides per tibia (i.e., sections I, II, II, IV, and V). All sections were dewaxed, rehydrated, and rinsed in water. The sections were pretreated with 0.2 N HCl for 15 minutes at room temperature, permeabilized in proteinase K (15 \u03bcg\/mL) for 30 minutes at 37\u00b0C, and subjected to an acetylation treatment [32]. Sections were rinsed in 2\u00d7 SSC (0.3 M sodium chloride and 0.03 M sodium citrate) and kept in this solution until the start of hybridization.\nHybridization was performed in a solution containing 50% formamide, 2\u00d7 SSC, 1\u00d7 Denhardt\u2019s solution, 250 \u03bcg\/mL transfer RNA, 480 \u03bcg\/mL herring sperm RNA, 10% dextran-sulfate, and the mouse IGFBP-2 digoxigenin-labeled cRNA probe at concentrations of 500 pg\/\u03bcL (brain) and 1,000 pg\/\u03bcL (tibia). Sections were hybridized overnight at 53\u00b0C. After hybridization, sections were washed with 50% formamide in 2\u00d7 SSC at the hybridization temperature for 30 minutes and treated with RNase A (1 unit\/mL) for 30 minutes at 37\u00b0C. Subsequently, sections were rinsed in 2\u00d7 SSC, treated with 1% blocking reagent for 30 minutes, and incubated with sheep antidigoxigenin Fab fragments conjugated with alkaline phosphatase (1:1,500) overnight at 4\u00b0C.\nChromogenesis was performed in the dark with 0.38 mg\/mL NBT and 0.19 mg\/mL BCIP in the presence of 6% (w\/v) polyvinyl alcohol [33], resulting in a blue precipitate. Finally, sections were counterstained with nuclear fast red, dehydrated through a series of ethanol, and mounted with Euparal. Sense probes were used to investigate the level of nonspecific binding.\nQuantification and Statistics\nFirst, a semiquantitative screening of all animals per group was performed on the antisense and sense slides to collect the in situ hybridization data. Sections I-V of all tibiae were scored in a semiquantitative manner, which was defined, on a Nikon Eclipse E800 microscope (Uvikon, Bunnik, The Netherlands), as follows: no expression (0), low expression (1), average expression (2), and high expression (3).\nEffects of mechanical loading were expected in the shaft of the tibiae. Therefore, a second quantitative evaluation of the endocortical osteocytes expressing IGFBP-2 mRNA was performed with Lucia G Version 4.82 (Uvikon) using coded slides of sections II and III. The defined area of interest, which comprised the loading zone, started below the primary spongiosa at the endosteal side of the shaft following the total length of the shaft to the distal side (total length maximal 3,852 \u03bcm). At the endosteal side of the shaft, all osteocytes, which were positioned within 100 \u03bcm of the endosteal surface, were included in the area of interest. Within the area of interest, total osteocyte number and total IGFBP-2 mRNA-positive osteocyte number were measured.\nThe paired-samples Student\u2019s t-test and independent samples Student\u2019s t-test were used for statistical analysis using SPSS (Chicago, IL) version 9.0 for Windows. P < 0.05 was considered to reflect statistical significance.\nPhotography\nBrightfield photographs with different magnifications (objectives \u00d710, \u00d720, and \u00d740) were made using a Leica microscope (DM4000B) with a digital camera (Leica DC500) and Leica software IM50 (Leica Microsystems, Rijswijk, The Netherlands).\nResults\nEndogenous IGFBP-2 mRNA Expression in Tissue\nThe control brain showed IGFBP-2 mRNA expression in the choroid plexus (Fig.\u00a01A) and the neurons of the medulla oblongata (data not shown). In the internal control, i.e., the growth plate, IGFBP-2 mRNA was located in chondrocytes of the proliferative and hypertrophic zone (Fig.\u00a01C). In control tibiae, IGFBP-2 mRNA was expressed in osteoblasts, which were situated against the surface of trabecular bone (Fig.\u00a01C, E) and the endosteal side of the shaft (Fig.\u00a01G). IGFBP-2 mRNA expression was also observed in some trabecular osteocytes and in cortical osteocytes, which were situated within the first lamella at the endosteal side of the shaft (Fig.\u00a01G). The endocortical osteocytes, which were located within the deeper lamellae (Fig.\u00a01G), and the periosteal osteocytes (Fig.\u00a01D) did not express IGFBP-2 mRNA. IGFBP-2 mRNA was also expressed in the intracortical endothelial cells of blood vessels (Fig.\u00a01G) and in the periosteum of control tibiae (Fig.\u00a01D). Some cells of the bone marrow, i.e., megakaryocytes, macrophages, and myeloid cells, also expressed IGFBP-2 mRNA (Fig.\u00a01E). No differences of IGFBP-2 mRNA expression between the right and left tibiae of the control group were observed using semiquantitative analysis.\nFig.\u00a01Expression of IGFBP-2 mRNA in rat brain sections (A, B) and in longitudinal control (C-G), sham-loaded (H), and loaded (J) tibial sections of 12-week-old female Wistar rats. The RNA signal is shown as a blue precipitate. The sections were counterstained with nuclear fast red and visualized under brightfield illumination. The scale bars represent 100 \u03bcm (A, B), 50 \u03bcm (D, E, G-J), and 100 \u03bcm (C, F). (A) IGFBP-2 mRNA expression within the choroid plexus of a control rat brain; red blood cells are negative and do not express IGFBP-2 mRNA. (B) Hybridization with representative IGFBP-2 sense probe showed no signal in the choroid plexus. (C) IGFBP-2 mRNA expression in the chondrocytes of the growth plate and osteoblasts of the trabecular bone. (D) IGFBP-2 mRNA expression in the periosteum of a control tibia; the osteocytes in the periosteal side of the shaft do not express IGFBP-2 mRNA. (E) IGFBP-2 mRNA expression in osteoblasts situated against the surface of normal trabecular bone and IGFBP-2-positive bone marrow cells. (F) Hybridization with a corresponding IGFBP-2 sense probe showed no signal in the osteoblasts, chondrocytes, and bone marrow cells of a control tibia. (G) IGFBP-2 mRNA expression within the superficial endocortical osteocytes (indicated by arrowheads), osteoblasts, and intracortical endothelial cells of a blood vessel of a control tibia. (H) IGFBP-2 mRNA expression in the endocortical osteocytes within multiple layers (indicated by arrowheads) and osteoblasts of a sham-loaded tibia. (J) IGFBP-2 mRNA expression in the endocortical osteocytes within multiple layers (indicated by arrowheads) and osteoblasts of a loaded tibia.\nControl hybridizations with the corresponding sense RNA probe exhibited no signals in the brain (Fig.\u00a01B) and tibia (Fig.\u00a01F).\nEffect of Mechanical Loading on IGFBP-2 mRNA Expression in Osteocytes of the Tibial Shaft\nInduced IGFBP-2 mRNA expression was observed in osteocytes within the first lamella and within multiple layers at the endosteal side of the shaft of loaded tibia (1.68-fold increase, n = 5) (Fig.\u00a01J) and sham-loaded tibia (1.35-fold increase, n = 5) (Fig.\u00a01H) in contrast to the control tibia (n = 10), where IGFBP-2 mRNA expression was only seen within the first lamella of the endosteal side of the shaft (Fig.\u00a01G).\nThe proportion of IGFBP-2 mRNA-positive osteocytes was 25.5 \u00b1 12.6% (mean \u00b1 standard deviation [SD], n = 5) for loaded tibiae and 17.2 \u00b1 10.7% (mean \u00b1 SD, n = 5) for the contralateral control tibiae (Figs.\u00a01G, J and 2A). Sham-loaded tibiae and the contralateral control tibiae showed a proportion of IGFBP-2 mRNA-positive osteocytes of 20.5 \u00b1 7.5% (mean \u00b1 SD, n = 5) and 13.2 \u00b1 4.2% (mean \u00b1 SD, n = 5), respectively (Figs.\u00a01H and 2B). Mechanical loading significantly increased the number of IGFBP-2 mRNA-synthesizing osteocytes in loaded tibiae (P = 0.001) and in sham-loaded tibiae (P = 0.031) (Fig.\u00a02A, B). No statistical differences of IGFBP-2 mRNA-positive osteocytes were observed between the contralateral controls of the loaded and sham-loaded tibiae (P = 0.475) or between the loaded and sham-loaded tibiae (P = 0.468).\nFig.\u00a02Influence of mechanical loading on IGFBP-2 mRNA expression in osteocytes at the endosteal side of the shaft within individual rat tibiae. Values are expressed as (A) percentage IGFBP-2 mRNA-positive osteocytes in control versus contralateral loaded tibiae (**P = 0.001, n = 5) and (B) percentage IGFBP-2 mRNA positive osteocytes in control versus contralateral sham-loaded tibiae (*P = 0.031, n = 5). No statistical differences of IGFBP-2 mRNA-positive osteocytes were observed between the control load and control sham groups (P = 0.475) or between the load and sham groups (P = 0.468). The lines connect the left and right tibiae of the same rat. The mean values of each group are represented by a bar.\nEffects of Mechanical Loading on IGFBP-2 mRNA Expression in Osteoblasts, Chondrocytes, and Bone Marrow Cells\nNo differences in IGFBP-2 mRNA expression between loaded, sham-loaded, and control tibiae were observed in the osteoblasts, chondrocytes, and bone marrow cells by semiquantitative screening. No differences in morphology of the cells were observed between groups.\nDiscussion\nThis in vivo study showed an increase in the number of endocortical osteocytes expressing IGFBP-2 mRNA in loaded (1.68-fold) and sham-loaded (1.35-fold) tibiae 6 hours after a single period of mechanical loading with the four-point bending system. The upregulation of IGFBP-2 mRNA expression was restricted to osteocytes at the endosteal side of the shaft and multiple inner lamellae. No difference between IGFBP-2 mRNA expression in the left and right control tibia was observed. We conclude that bending and squeezing of rat tibiae result in an increase of locally synthesized IGFBP-2 mRNA by endocortical osteocytes. We suggest that IGFBP-2 plays an as yet unidentified role in the lamellar bone formation process.\nWe suggest that the IGFBP-2-producing osteocytes of the loaded tibiae are involved in the increased lamellar bone formation after mechanical loading. Forwood and colleagues reported that a single short period of loading using the four-point bending system resulted in an increased lamellar bone formation rate at the endosteal surface of rat tibia [25]. This has been confirmed in a validation study at our laboratory [27]. The lamellar bone formation is located at the endosteal side of the shaft, which is identical to the location of newly synthesized IGFBP-2 mRNA after mechanical loading, as demonstrated in this study. IGFBP-2 mRNA expression was restricted to this area and was not observed in the osteocytes at the periosteal side of the shaft. Mechanical loading with an external bending load of 60 N in vivo will result in an increased woven bone formation rate at the periosteal surface as a result of irritation of the periosteum and increased lamellar bone formation rate at the endosteal surface [1]. We suggest that IGFBP-2 mRNA may be less involved in woven bone formation because the osteocytes at the periosteal side of the shaft did not express IGFBP-2. This suggests that IGFBP-2 mRNA in the mechanosensitive osteocytes is specifically important for lamellar bone formation.\nIn addition, this study showed that the sham-loaded tibiae have increased IGFBP-2 mRNA production in endocortical osteocytes 6 hours after sham loading. Apparently, besides bending, squeezing alone is also responsible for the upregulation of IGFBP-2 mRNA in the endocortical osteocytes. However, squeezing did not induce lamellar bone formation.\nRemarkably, there is a large range in the data for the percentage of IGFBP-2 mRNA-positive osteocytes in the contralateral control tibiae of the load group in comparison to the data of the contralateral tibiae of the sham group. This could be caused by the biological variation between the individuals because the animals were randomly divided between the groups and there is no significant statistical difference between those tibiae.\nFor this study we used the four-point bending model of Forwood because this apparatus produces a controlled mechanical strain in the tibia of living rats. An advantage of this approach is that it does not require surgical intervention and allows normal physical activity after the loading session [1, 7]. The osteogenic response will occur at a distinct location in bone. Therefore, we used the in situ hybridization technique in order to detect the local osteogenic response at the cellular level. Nonradioactive in situ hybridization is a powerful and sensitive technique to localize gene expression within decalcified rat tibiae. It was demonstrated that the IGFBP-2 probe is specific because IGFBP-2 mRNA expression is present in the choroid plexus and the neurons of the medulla oblongata, as described earlier [28], and in the chondrocytes of the proliferative and hypertrophic zone of the growth plate [34].\nNevertheless, this study has some limitations. The applied load of 60 N is supraphysiological, and 6 hours after a single loading session is too early to demonstrate bone formation.\nIn summary, this study shows an increase of the number of osteocytes at the endosteal side of the shaft and inner lamellae expressing IGFBP-2 mRNA 6 hours after mechanical loading and sham loading in vivo. We conclude that these IGFBP-2-producing osteocytes in the endosteal shaft are mechanosensitive. We suggest that IGFBP-2 plays a role in the lamellar bone formation process.","keyphrases":["in vivo","mechanical loading","igfbp-2 mrna expression","nonradioactive in situ hybridization","four-point bending system"],"prmu":["P","P","P","P","P"]}
{"id":"Anal_Bioanal_Chem-4-1-2228378","title":"Formation of gutingimycin: analytical investigation of trioxacarcin A-mediated alkylation of dsDNA\n","text":"Formation and fragmentation of recognition complexes between trioxacarcin A and various DNA sequences were examined by temperature-dependent UV and CD spectroscopy, HPLC analysis, and ESI mass spectrometry with regard to reaction conditions, intermediates, products, mechanism, and sequence specificity. Cleavage of the trioxacarcin\u2013DNA complexes provided the natural product gutingimycin by guanine abstraction. The resulting DNA with an abasic site was further cleaved into a DNA fragment with a furanyl unit at the 3\u2032-end and an oligonucleotide with a phosphorylated 5\u2032-end.\nIntroduction\nTrioxacarcins are complex natural antitumor antibiotics with anti-malaria activity [1\u20133]. The first trioxacarcins were isolated from streptomycetes in 1981 by Tomita [1, 2] and their structure was elucidated by Shirahata [4]. Gutingimycin (1) was isolated in 2004 from a marine streptomycete together with further trioxacarcin derivatives and named according to the ancient name of G\u00f6ttingen [3, 5, 6]. It turned out to be an interesting natural product because of its mode of formation. The structure of gutingimycin (1), elucidated by X-ray analysis, suggested that gutingimycin (1) results from nucleophilic attack of the N7 atom of a guanine subunit in DNA at the epoxide of trioxacarin A (2) [5]. Natural antibiotics like hedamycin and altromycin B are able to intercalate into the dsDNA followed by alkylation of guanine at the N7 position because of their epoxide functionality [7\u20139]. Like hedamycin, altromycin B or kapurimycin A3 [10, 11], trioxacarcin A (2) contains an epoxide. Therefore, it has been proposed that gutingimycin (1) is not built up from low-molecular-weight precursors [5]. As for other cytotoxic natural products, it is likely that alkylation of trioxacarin A (2) is followed by depurination and DNA strand scission at the abasic site [7, 12\u201314]. Saito obtained derivatives structurally related to gutingimycin (1) by reaction of kapurimycin A3 with various oligonucleotides. At 0 \u00b0C a stable ionic intermediate is formed; on heating to 90 \u00b0C this dissociates into a depurinated oligonucleotide and a kapurimycin derivative [15, 16]. Hedamycin binds to dsDNA by intercalation and alkylation with a distinct preference for the central guanine in the sequence d(CGT) or d(CGG) [8, 17, 18]. The covalent hedamycin-DNA adduct was isolated by Sheil and characterized by ESI MS [19, 20].\nFrom previous studies it was known, that trioxacarcin A (2) also forms a stable complex with dsDNA, but did not react with deoxyguanosine even at higher temperatures [1]. The aim of this study was to investigate the conditions and mechanism of the trioxacarcin-mediated alkylation of dsDNA by applying high-performance liquid chromatography and high-resolution electrospray mass spectrometry together with temperature-dependent UV and CD spectroscopy. In particular, the influence of the DNA sequence on binding and adduct formation and the identification of intermediates and cleavage products like gutingimycin (1) and DNA fragments is described in detail.\nExperimental\nMethods and materials\nThe DNA oligomers d(AATTACGTAATT) (4a), d(CAATTATAATTG) (4b), d(AATTXGXAATT) (5a\u2013d), and the complementary oligonucleotides d(AATTGGGAATT) (7) and d(TTAACCCTTAA) (8) were purchased from IBA (G\u00f6ttingen, Germany). An oligomer length of eleven or twelve nucleotides was sufficient to provide stability at room temperature (Tm\u2009=\u200922\u201335 \u00b0C). Trioxacarcin A (2) and trioxacarcin B (3) were obtained by fermentation of Streptomyces sp. isolate B8652 [6]. The reactions were carried out in aqueous phosphate buffer (10\u00a0mmol L\u22121 Na2HPO4\/NaH2PO4, 0.1\u00a0mol L\u22121 NaCl, H2O, pH 7.0) with 5\u00a0\u03bcmol L\u22121 dsDNA and trioxacarcin A (2) in a 1:2 ratio.\nUV spectroscopy\nMelting curves were measured on a Perkin\u2013Elmer Lambda 10 UV\u2013visible spectrophotometer in connection with a Perkin\u2013Elmer PTP-1 Peltier system following the temperature program: 0 \u00b0C (15\u00a0min) \u2192 90 \u00b0C (180\u00a0min) \u2192 0 \u00b0C (180\u00a0min). dsDNA (30\u00a0nmol) was dissolved in 1.5\u00a0mL phosphate buffer (10\u00a0mmol L\u22121 Na2HPO4\/NaH2PO4, 0.1\u00a0mol L\u22121 NaCl, H2O, pH 7.0), trioxacarcin A (2) (60\u00a0nmol) in 1.5\u00a0mL phosphate buffer was added and the measurement was performed at a wavelength of \u03bb\u2009=\u2009260\u00a0nm in a quartz cuvette (1\u00a0cm).\nCD spectroscopy\nCD spectra were obtained on a Jasco J-810 spectropolarimeter at room temperature (data mode: CD, band width 1.0\u00a0nm, slit width: auto, sensitivity: 5\u00a0mdeg, time const.: 4.0\u00a0s; step resolution: 0.1\u00a0nm, scan speed: 50\u00a0nm min\u22121, five accumulations). dsDNA (30\u00a0nmol) was dissolved in 1.5\u00a0mL phosphate buffer (10\u00a0mmol L\u22121 Na2HPO4\/NaH2PO4, 0.1\u00a0mol L\u22121 NaCl, H2O, pH 7.0), trioxacarcin A (2) (60\u00a0nmol) in 1.5\u00a0mL phosphate buffer was added and the measurement was performed in a quartz cuvette (1\u00a0cm).\nKinetic measurements\nA solution of trioxacarcin\u2013DNA (30\u00a0nmol) adduct in phosphate buffer (600\u00a0\u03bcL, 10\u00a0mmol L\u22121 Na2HPO4\/NaH2PO4, 0.1\u00a0mol L\u22121 NaCl, H2O, pH 7.0) was heated to 80 \u00b0C for 4\u00a0h. Samples of 60\u00a0\u03bcL were taken after 0, 15, 30, 45, 60, 75, 90, 120, and 240\u00a0min and analyzed by HPLC. The area under the curve of the trioxacarcin\u2013DNA adduct peak was plotted versus time to determine the time constant as the slope of the regression line.\nHigh-performance liquid chromatography\nHPLC separations were performed with an \u00c4kta purifier from Amersham Pharmacia Biotech, a UV900\u00a0UV detector operated at 260\u00a0nm, a NewGuard RP-8 Brownlee Precolumn (15\u2009\u00d7\u20093.2\u00a0mm, 7\u00a0\u03bcm), and an Aquapore RP-300 Brownlee Column (220\u2009\u00d7\u20094.6\u00a0mm, 7\u00a0\u03bcm) from Perkin\u2013Elmer. Samples were eluted within 45 min with a flow rate of 1\u00a0mL min\u22121 by applying a two-stage gradient (5\u201320% B in 20\u00a0min and 20\u201345% B in 25\u00a0min). Eluent A: 0.1\u00a0mol L\u22121 triethylammonium acetate buffer (H2O, pH 7.0). Eluent B: 0.1\u00a0mol L\u22121 triethylammonium acetate buffer (80% acetonitrile and 20% water, pH 7.0).\nElectrospray mass spectrometry\nTo a solution of 120\u00a0nmol dsDNA in phosphate buffer (10\u00a0mmol L\u22121 Na2HPO4\/NaH2PO4, 0.1\u00a0mol L\u22121 NaCl, H2O, pH 7.0) a fivefold excess of trioxacarcin A (2) was added. The resulting mixture was incubated several hours at 0 \u00b0C and the trioxacarcin\u2013DNA adduct formed was isolated by HPLC. High-resolution mass spectrometry was performed using an Apex IV 7 T FTICR MS (Bruker Daltonics, Billerica, MA, USA) equipped with an Apollo electrospray ion source which was used in the negative ion mode (capillary voltage 4.2\u00a0kV, end plate voltage 3.8\u00a0kV). Analytes were dissolved in a water\u2013methanol (50:50) solution and introduced by a syringe pump at a flow rate of 2\u00a0\u03bcL min\u22121. Nitrogen was used as drying gas at 100 \u00b0C. Hexapole accumulation was set between 0.2 s to 3.0\u00a0s and mass spectra were acquired within the mass range m\/z 400 to 2000 with a resolution of about 100,000 at m\/z 600. The capillary exit voltage was \u221280\u00a0V, \u2212100\u00a0V, \u2212140\u00a0V, or \u2212170\u00a0V. Deconvolution and mass calculation for the corresponding ions were done using Xmass 6.1.2 from Bruker.\nResults and discussion\nIn order to investigate the sequence dependency of guanine abstraction from dsDNA by trioxacarcin A (2), the two self complementary DNA oligomers d(AATTACGTAATT) (4a) and d(CAATTATAATTG) (4b) were investigated with the reactive guanine located in the center or at the end of the double strand. The position of guanine was varied to determine the influence of the helix topology on alkylation. Four additional non-self complementary oligonucleotides with the general sequence d(AATTXGXAATT) (5a\u2013d) together with the corresponding complementary strands were examined with respect to sequence specificity. The sequences were limited to only one guanine in the double strand in order to limit alkylation to a single site. It was placed in the central position investigating all possible alterations of the neighboring nucleobases using adenine and thymine.\nFig.\u00a01Gutingimycin (1), trioxacarcin A (2), and trioxacarcin B (3)\nA first approximation of the interaction of trioxacarcin A (2) with double-stranded DNA can be obtained from temperature dependent UV-spectroscopic measurements of double-strand stabilities, because double-strand recognition, formation of a covalent adduct, nucleobase excision, or strand scission are expected to affect DNA stability. A mixture of double-stranded oligonucleotide d(AATTACGTAATT) (4a) with two equivalents trioxacarcin A (2) gave a stability of Tm\u2009=\u200941 \u00b0C which is significantly higher than the double strand transition of oligomer 4a without addition of the natural product (Tm\u2009=\u200934 \u00b0C) (Fig.\u00a02) indicating a DNA interaction with the trioxacarcin A (2). For this interaction intercalation and groove binding of the aglycon is more likely than covalent linkage of 2, because double-strand stability obtained for d(AATTACGTAATT) (4a) with trioxacarcin B (3) is similarly increased (Tm\u2009=\u200942 \u00b0C) even though trioxacarcin B (3) is not able to alkylate DNA. In the cooling cycle the UV melting curve of the 4a\/2 mixture provided only a stability of Tm\u2009=\u200928 \u00b0C. This destabilization is most likely because of nucleobase excision initiated at higher temperatures to yield gutingimycin (1) and a DNA double strand with apurine site which might further lead to DNA strand scission.\nFig.\u00a02UV-melting curve of d(AATTACGTAATT) (4a) with and without trioxacarcin A (2) (5\u00a0\u03bcmol L\u22121, 1:2)\nIn contrast, for the oligomer with the terminal guanine 4b the differences in double-strand stability were not significant comparing the self pairing (Tm\u2009=\u200931 \u00b0C) with the 1:2 mixture of oligomer 4b and natural product 2 in the heating (Tm\u2009=\u200929 \u00b0C) or cooling cycle (Tm\u2009=\u200929 \u00b0C) (Fig.\u00a03). Apparently, for DNA recognition of trioxacarcin A (2) the double helix topology is required which is not sufficiently assured at the terminus of the helix. Therefore, the guanine placed at the terminal position cannot act as a nucleophile without the help of helix recognition. This is in agreement with guanosine itself not being a nucleophile for trioxacarcin A (2) [1].\nFig.\u00a03UV-melting curve of d(CAATTATAATTG) (4b) with and without trioxacarcin A (2) (5\u00a0\u03bcmol L\u22121, 1:2)\nThe modification of DNA 4a by trioxacarcin A (2) was also indicated by CD spectroscopy (Fig.\u00a04). The spectra of 4a and the mixture of 4a with 2 measured at room temperature are quite similar, whereas the CD spectrum of the 4a\/2 mixture heated to 90 \u00b0C differed by a shift and the intensity of the Cotton effect around 270, 250, and 220\u00a0nm.\nFig.\u00a04CD-spectra of self pairing d(AATTACGTAATT) (4a) with (grey) and without (black) trioxacarcin A (2) (5\u00a0\u03bcmol L\u22121, 1:2). The dashed line indicates the spectrum obtained after interim heating to 90 \u00b0C\nFor a more detailed picture of trioxacarcin A (2) adduct formation with dsDNA, high-performance liquid chromatography was applied determining the components present after mixing at room temperature and after heating to 90 \u00b0C. The HPLC chromatogram of the mixture of oligonucleotide 4a with trioxacarcin A (2) before starting the heating cycle provided three peaks (Fig.\u00a05a). Next to DNA (4a) and trioxacarcin A (2), an additional compound was indicated which was assigned to the stable covalent complex between the oligonucleotide and the natural product trioxacarcin A (4a + 2). The compound corresponding to peak (4a + 2) was isolated and characterized by high-resolution ESI MS as the DNA\u2013trioxacarcin A adduct. After heating to 90 \u00b0C additional peaks appeared in the oligonucleotide area together with gutingimycin (1) and trioxacarcin B (3) generated by epoxide ring opening of trioxacarcin A (2) with water (Fig.\u00a05b). The assignment of all HPLC peaks was confirmed by co-injection and mass spectrometry.\nFig.\u00a05HPLC chromatogram of a mixture of oligonucleotide 4a and trioxacarcin A (2): a at room temperature; b after heating to 90 \u00b0C; next to DNA 4a further oligonucleotides and oligonucleotide fragments are eluting between 11 and 17\u00a0min\nExamining the trioxacarcin A (2) interaction with oligomer d(CAATTATAATTG) (4b) carrying the guanine at the terminal position, the HPLC chromatogram turned out to be quite similar to the HPLC chromatogram obtained after interaction with DNA 4a. Nevertheless, the yield of the respective DNA\u2013trioxacarcin adduct (4b + 2) was significantly lower and gutingimycin (1) was only obtained in traces after heating (Fig.\u00a06). As indicated in Table\u00a01, adduct formation of trioxacarcin A (2) with oligonucleotide 4a is about five times more efficient than binding to DNA 4b. Sterically, the nucleophilic attack should be easier at the end of the double helix, but the ease of adduct formation turned out to be higher in the central position of the double strand. Therefore, trioxacarcin A (2) seems to take advantage of dsDNA recognition by intercalation or groove binding prior to nucleophilic attack. This recognition behavior is also described for the natural products hedamycin and altromycin B of the pluramycin family [7, 8, 21, 22].\nFig.\u00a06HPLC chromatogram of a mixture of the oligonucleotide 4b and trioxacarcin A (2): a at room temperature; b after heating to 90 \u00b0CTable\u00a01Examined oligomers and adduct formationDNA oligomers\u00a0Adduct formula[Madduct \u2212 6H]6\u2212 a calcd. \/ found mass[Madduct + Na \u2212 7H]6\u2212 a calcd. \/ found massAdduct (%)bAATTACGTAATT4aC161H202N43O90P11752.32 \/ 752.33755.99 \/ 755.9953CAATTATAATTG4bC161H202N43O90P11752.32 \/ 752.39755.99 \/ 756.0010AATTAGTAATT5aC152H190N40O84P10704.15 \/ 704.16707.81 \/ 707.8313AATTTGAAATT5bC152H190N40O84P10704.15 \/ 704.16707.81 \/ 707.825AATTTGTAATT5cC152H191N37O86P10702.65 \/ 702.65706.31 \/ 706.3116AATTAGAAATT5dC152H189N43O82P10705.65 \/ 705.66709.31 \/ 709.321a Adduct ions up to the sevenfold charged ion were observed; the relative intensities varied from oligomer to oligomerb Conversion rate given by the trioxacarcin A to adduct proportion\nThe isolated adduct of oligonucleotide 4a with trioxacarcin A (2) is stable at room temperature for more than 24\u00a0h and at 56 \u00b0C for at least 4\u00a0h. At 80 \u00b0C within 4\u00a0h, decay to gutingimycin (1) was observed obeying first-order kinetics (Fig.\u00a07).\nFig.\u00a07Kinetic analysis based on a decay of the d(AATTACGTAATT)\u2013trioxacarcin A adduct (4a + 2) [23]\nIn order to get preliminary information about sequence selectivity in trioxacarcin A (2) recognition and alkylation, all adenine and thymine combinations were investigated as the neighboring nucleobases of a central guanine. The four oligonucleotides 5a\u2013d reacted with trioxacarcin A (2) to yield the corresponding adducts, which were characterized by high-resolution ESI MS. The corresponding HPLC profiles of oligomers 5a\u2013d with trioxacarcin A (2) before and after heating to 90 \u00b0C were almost similar, except for the peak height of the generated adducts. The conversion rate was calculated from the peak area proportion of trioxacarcin A (2) to the adduct. As indicated in Table\u00a01, the conversion rate from trioxacarcin A (2) to the corresponding DNA adducts varied from 1 to 16%. Based on non-self complementary double strands 5a\u2013d and results obtained by HPLC and ESI MS, adduct formation seems to be preferred when thymine is placed next to guanine with thymine neighboring the 3\u2032-site having a greater impact. It still needs to be investigated if this result holds only for thymine in comparison with adenine or if this is a more general preference for pyrimidines over purines neighboring the trioxacarcin-substituted guanine.\nThe alkylation of oligomers with more than one guanine was investigated with the double strand formed by the complementary strands d(AATTGGGAATT) (6) and d(TTAACCCTTAA) (7) which provide a quite stable double strand (Tm\u2009=\u200935 \u00b0C). In a mixture of 6 + 7 with six equivalents of trioxacarcin A (2) the double strand stability decreased to Tm\u2009=\u200929 \u00b0C (heating cycle) whereas double strand formation was no longer detected after heating to 90 \u00b0C. This leads to the conclusion that trioxacarcin A (2) addition and DNA degradation is accelerated with three neighboring guanines. The high tendency for adduct formation of trioxacarcin A (2) to dsDNA 6 + 7 at 0 \u00b0C was indicated by an HPLC signal at 26.0\u00a0min nearly the size of the remaining oligomer 7 (Fig.\u00a08a). Even after 24\u00a0h signals appeared at 30.8 and 31.6\u00a0min that were assigned to DNA 6 functionalized with two equivalents of trioxacarcin A (2). The amount of double substitution increased significantly within 72\u00a0h reaction time (Fig.\u00a08b).\nFig.\u00a08HPLC chromatograms of a mixture of the DNA double strand d(AATTGGGAATT) (6) + d(TTAACCCTTAA) (7) and trioxacarcin A (2): a 24\u00a0h at 0 \u00b0C and b 72\u00a0h at 0 \u00b0C\nFurther insight into the trioxacarcin A (2)-initiated substitution reaction and the DNA cleavage products were obtained by additional ESI MS experiments stepwise increasing the capillary-skimmer dissociation energy in the ion source [24\u201330]. Like increasing the temperature this also initiated fragmentation of alkylated DNA followed by strand cleavage. The adduct of trioxacarcin A (2) with oligomer 5d was unequivocally assigned by ESI MS with a low capillary exit voltage of \u221280\u00a0V providing exclusively ions of the DNA\u2013trioxacarcin complex 5d + 2 (Fig.\u00a09a): ([M \u2212 3H]3\u2212, [M \u2212 4H]4\u2212, [M \u2212 5H]5\u2212, and [M \u2212 6H]6\u2212). By applying capillary\u2013skimmer dissociation by raising the capillary exit voltage to \u2212140\u00a0V the signals for the DNA\u2013trioxacarcin A adduct were also found (Fig.\u00a09c). Nevertheless, the adduct ions were less intense. One of the peaks with the highest intensity appears at m\/z 1026.37; this is assigned to the single negatively charged ion of gutingimycin (1) comparable to the thermal cleavage product observed already by HPLC. Depurination is known to result in oligonucleotide strand scission [19, 20, 27]. Furthermore, it has been reported that collision-activated dissociation of negatively charged oligonucleotide ions starts with cleavage of a base followed by cleavage of the 3\u2032 C\u2013O bond of the sugar unit from which the base was lost [31]. In accordance, two ESI MS peaks were correlated to the doubly charged w5-fragment ion at m\/z 781.64 (calculated 781.63) and the doubly charged (a6-B)-type fragment ion at m\/z 821.66 (calculated 821.64) of the oligonucleotide 5d. The accompanying smaller peaks to the right of w52\u2212 and [a6-GT]2\u2212 were assigned to the corresponding [M + Na \u2212 3H]2\u2212 ions. These fragments might mechanistically be explained by the initiation of strand cleavage at C3\u2032 leading to a furanyl unit at the 3\u2032-end of d(AATTA) and a phosphoryl group at the 5\u2032-end of the second fragment d(AAATT) [31]. An additional small peak m\/z 801.90 [10 \u2212 4H]4\u2212 can be assigned to the depurinated oligonucleotide 10 (Scheme\u00a01, together with its sodium peak m\/z 807.40 [10 + Na \u2212 5H]4\u2212). Nevertheless, oligonucleotide 10 with a double bond between C1\u2032 and C2\u2032 (Scheme\u00a01) has a calculated mass [10 \u2212 4H]4\u2212 of m\/z 801.89 and [10 + Na \u2212 5H]4\u2212 of m\/z 807.38 corresponding to the mass of the intermediate fragment. This assignment is clearly supported by the corresponding deconvoluted mass spectra (Figs.\u00a09b and d).\nFig.\u00a09a ESI mass spectrum of trioxacarin-DNA d(AATTAGTAAATT) 5d + trioxacarcin (2) adduct, capillary exit potential \u221280\u00a0V; b Respective deconvoluted spectrum of trioxacarin-DNA 5d adduct; c ESI mass spectrum of trioxacarin-DNA d(AATTAGTAAATT) 5d + trioxacarcin (2) adduct, capillary exit potential \u2212140\u00a0V; d Respective deconvoluted spectrum of trioxacarin-DNA 5d adductScheme\u00a01Proposed cleavage reaction of DNA\u2013trioxacarcin A adduct\nThe fragments observed for depurination and DNA strand scission of trioxacarcin\u2013DNA adducts are in agreement with the results from Sheil reporting similar oligonucleotide fragmentation in ESI MS\u2013MS experiments for various hedamycin\u2013DNA adducts [19]. Based on the ESI MS fragments detected by cleavage of the trioxacarcin\u2013DNA adduct (5d + 2) the following products and intermediates for the fragmentation of the trioxacarcin\u2013DNA adduct are proposed (Scheme\u00a01): Depurination is generating gutingimycin (1) and the oligonucleotide with an abasic site 10 dissociating in fragment 8 with a furanyl unit at the 3\u2032-end (w5-type fragment) and fragment 9 with a phosphorylated 5\u2032-end (a6-B-type fragment). The phosphoester 9 seems to be a perfect leaving group for generation of a furan.\nThe results described for the trioxacarcin\u2013DNA 5d adduct were supported by the mass spectra of the trioxacarcin adducts with oligonucleotides 4a\u2013b and 5a\u2013c. The signals assigned to adducts, abasic sites, fragments, and gutingimycin were also observed for these oligomer adducts. With low dissociation energy in the ion source (capillary exit potential \u221280\u00a0V) the adduct peak was obtained for the self complementary double strand 4a with guanine in the central position almost exclusively, whereas fragmentation already occurred in the case of oligomer 4b with the terminal guanine obtaining the gutingimycin signal as the most prominent. Trioxacarcin A adducts with oligonucleotides 5a\u2013c always provided the strongest ESI MS signals for the adduct peaks using the lowest dissociation energy; nevertheless, the peak intensities corresponding to the other fragments varied. The trioxacarcin adducts with oligomers 5a and 5d were the most stable.\nConclusions\nThe formation of stable adducts of the natural product trioxacarcin A (2) with guanine-containing dsDNA was investigated by a combination of temperature-dependent UV and CD spectroscopy, high-performance liquid chromatography, and high-resolution mass spectrometry. Recognition and non-covalent binding of trioxacarcin A (2) to dsDNA seems to be a prerequisite for nucleophilic attack of the guanine to form a covalent complex. Preliminary evidence indicates that trioxacarcin A-DNA interaction has sequence specificity with greater reactivity observed for guanines 5\u2032 to thymidines. Cleavage of the trioxacarcin\u2013DNA adducts can be initiated at higher temperatures or by applying capillary-skimmer dissociation by raising the capillary exit potential in mass spectrometry experiments leading to the formation of the bioactive secondary metabolite gutingimycin and DNA strand scission. As DNA cleavage products, a fragment with a furanyl unit at the 3\u2032-end was identified next to an oligonucleotide with a phosphorylated 5\u2032-end.","keyphrases":["gutingimycin","trioxacarcin a","electrospray mass spectrometry","dna alkylation","dna strand cleavage"],"prmu":["P","P","P","R","R"]}
{"id":"Eur_Spine_J-2-2-1602183","title":"Pure cervical radiculopathy due to spontaneous spinal epidural haematoma (SSEH): report of a case solved conservatively\n","text":"Introduction: Spontaneous spinal epidural haematoma (SSEH) is widely recognised throughout the literature as a cause of myelopathy, radicular compression being very rarely reported. Surgical management is almost always recommended, especially in the cases of spinal cord compression. Conservative treatment is reported as a curiosity and only in the case of spontaneous improvement. This report presents the particular case of a 64-year-old patient undergoing anticoagulant therapy that had a cervical radiculopathy due to a SSEH confirmed by MRI. The patient improved spontaneously and symptoms were solved with unconventional conservative treatment and without stopping the anticoagulant therapy. Conclusions: Spontaneous epidural haematoma must be kept in mind when patients undergoing anticoagulant therapy have a sudden onset of cervicobrachialgia. Even though most spinal surgeons advocate surgical treatment, a conservative approach may lead to a complete recovery and may be considered as a good option in the case of radicular involvement. Discontinuation of the anticoagulant therapy may not always be needed, especially when the clinical syndrome improves spontaneously.\nIntroduction\nAlthough spontaneous spinal epidural haematoma (SSEH) is a low incidence condition, it is widely recognised throughout the literature as a cause of myelopathy [5, 15]. The relationship between SSEH and anticoagulant therapy is well known and the probable cause of bleeding is thought to be the rupture of the venous epidural plexus during a sudden elevation of thoracic or abdominal pressure [4, 6, 8, 10, 11, 13, 15].\nThe most common symptoms are cervical or thoracic pain followed by cord compression signs. Radicular involvement is very rare and, when it appears, it mostly affects the lumbar spine, producing ciatalgia [1, 3, 11]. Although the currently suggested approach is an urgent surgical decompression [9, 10], conservative treatment is recommended when there is an objective improvement of the neurological status [2, 11, 12]. In general, anticoagulation treatment must be suspended or adjusted before surgery.\nThe originality of this report arises in three points:The case involved the cervical spine producing simple radiculopathy.It was solved spontaneously.Anticoagulation therapy was not discontinued.\nCase report\nA 64-year-old white man undergoing anticoagulant therapy because of cardiac valve prosthesis arrived at the emergency room of our Hospital suffering from a sudden onset of cervical pain and a left C5 brachial pain and weakness. The physical exam revealed a 4\/5 deltoid weakness and abolition of the bicipital reflex. The patient showed no signs or symptoms of cord compression. Standard radiographs of the cervical spine were normal.\nBased on the suspicion that it was a case of disc herniation with left C5 root involvement, symptomatic treatment by means of non-steroid anti-inflammatory drugs and painkillers was indicated. A cervical MRI was scheduled and the patient was invited to undergo further examination at the Spinal Surgery Unit. Owing to the fact that the first physician was not a spinal surgeon and therefore not aware of the relationship between anticoagulation and SSEH, discontinuation of anticoagulant therapy was not indicated. Two days later, when the patient was examined by the Spinal Surgery Unit, the pain had been completely relieved and the weakness had also decreased. The MRI revealed a left posterolateral ovoid mass compatible with a haematoma extending from C4 to C5 (Fig.\u00a01). Symptomatic medical treatment was interrupted. Once the specialist knew the real cause of the radicular syndrome, the haematologist was consulted and they preferred not to suspend the anticoagulant therapy (despite an adequate level of anticoagulation, INR 2,7) in order to prevent thromboembolism.\nFig.\u00a01MRI 2\u00a0days after the onset of symptoms. Posterolateral placement of the haematoma at C4\u2013C5 level. a, c Isointense appearance of the haematoma on the MRI T1-weighted sagittal and axial images. b, d Hyperintense appearance of the haematoma on the MRI T2-weighted sagittal and axial images\nSeven days later, the patient was free of weakness and the MRI showed a decrease in the haematoma size (Fig.\u00a02). Successive MRI results were obtained 1\u00a0month (Fig.\u00a03) and 1\u00a0year later on (Fig.\u00a04) to document the resolution of the haematoma. One year on, the patient remains asymptomatic.Fig.\u00a02MRI performed 7\u00a0days later. The haematoma is smaller nevertheless it is located at the same C4\u2013C5 levelFig.\u00a03MRI 1\u00a0month later: disappearance of the haematomaFig.\u00a04MRI 1\u00a0year later: normal MRI\nDiscussion\nRadicular compression due to SSEH is far less frequent than myelopathy. Groen and Van Alphen [6] reported that 4.5% of a series of 320 SSEH cases treated surgically presented radiculopathy; all cases affecting the lumbar spine. Recently, Groen [7] reported that 9% of a series of 64 SSEH cases treated conservatively presented an isolated radicular compromise; only one of his cases was located on the cervico\u2013thoracic spine. Our case adds little in terms of number but the fact that it may be the only report of its kind on purely cervical radiculopathy due to SSEH, which may be considered anecdotic, as well as interesting.\nAlthough the spreading of the haematoma throughout the epidural space is the most likely hypothesis proposed for spontaneous recovery in case of neurological impairment [7, 14], our case does not support that theory because the haematoma did not spread throughout the epidural space. (Figs.\u00a01, 2)\nConservative management is currently indicated in uncommon situations or when neurological symptoms improve before medical evaluation. Geographic isolation, initial inaccurate diagnosis, neurological improvement pending an adequate coagulation level prior to surgery, high surgical risks and several other reasons had been referred to as the likely causes that led to the opportunity for spontaneous recovery [4, 7, 9, 11, 16]. Our case can be categorised as wrong diagnosis initially, but we have to admit that the first non-specialised consultation spared our patient from the operation. If the patient had first consulted a more skilled specialist, following the state of the art, he would have probably been operated on, thus obtaining a good result and a proud surgeon.\nFinally, the main interest of our case would be focused on the controlled maintenance of the anticoagulant therapy in patients with mild neurological compromise such as radiculopathy due to SSEH in order to avoid thromboembolic risk.","keyphrases":["conservative treatment","anticoagulant therapy","cervicobrachialgia","spontaneous spinal epidural hematoma"],"prmu":["P","P","P","M"]}
{"id":"Virchows_Arch-3-1-1888721","title":"Invasive carcinomas of the male breast: a morphologic study of the distribution of histologic subtypes and metastatic patterns in 778 cases\n","text":"The current investigation was conducted to evaluate the proportional distribution of the various histologic subtypes (including newly recognized variants) of male breast carcinomas, to determine whether any histologic subtypes occur with a frequency that is markedly discordant with the expected frequencies from published data on parallel female breast tumors. We also aimed to document the distribution of malignancies metastatic to the breast. Seven hundred fifty-nine archived cases of primary invasive carcinoma involving the male breast were retrieved and subcategorized into histologic subtypes according to contemporary criteria. Six hundred forty-three (84.7%) tumors were pure infiltrating ductal carcinoma (IDC) not otherwise specified. The most common of the remainder included papillary carcinoma with invasion in the form of IDC (n = 34), mixed IDC and mucinous carcinoma (n = 26), and pure mucinous carcinoma (n = 21). In 19 cases, metastases from other sites involved the breast, most commonly (58%) cutaneous melanoma. Invasive carcinoma of the male breast appears to display a morphologic spectrum and distribution of histologic subtypes that is comparable to those of the female breast, with some expected variation. Compared with published experience on their female counterparts, there is a two-fold increase in the frequency of invasive papillary carcinoma in the male breast. Finally, the most common tumor metastatic to the male breast in this series was cutaneous melanoma.\nIntroduction\nBreast cancer in males is relatively uncommon, with approximately 1,450 newly diagnosed cases annually in the United States, and an associated mortality rate that accounts for less than 0.2% of all cancer-related deaths in men [1, 4, 9, 15, 17, 20, 28]. However, recent epidemiologic studies suggest that the incidence of male breast cancer has been steadily increasing [7, 14]. Utilizing data from the National Cancer Institute\u2019s Surveillance, Epidemiology and End Results database for the 26-year period between 1973 and 1998, Giordano et al. [7] found that the incidence of male breast cancer increased from 0.86 to 1.08 per 100,000 population. This increasing incidence, in addition to the better elucidation of the role of BRCA2 mutations in male breast cancer, has renewed interest in specifically identifying the underlying pathogenesis of male breast cancer and clarifying the specific differences, if any, between male and female breast cancer.\nOn a broad demographical level, male and female breast cancers are fundamentally similar with the main differences lying in older age of occurrence and lower incidence of the former. However, significant differences have been noted between male and female breast cancers with respect to the expression of a variety of biologic factors, including hormone receptors such as estrogen receptor, progesterone receptor, c-erbB-2, estrogen-inducible proteins such as pS2, Cathepsin D, hsp27, proteins related to basement membrane and extracellular matrix degradation such as the urokinase system of plasminogen activation and their inhibitors, and protooncogenes such as bcl-2 [2, 6, 7, 18, 21, 22, 24, 27]. At the morphologic level, male ductal intraepithelial neoplasia (ductal carcinoma in situ), in contrast to similar lesions in females, displays a distinct histologic profile in which the majority of tumors are of the papillary type, with cribriform, micropapillary, and solid types being much less common [13]. In particular, the comedo-type of DIN (comedo DCIS) is notably rare in the male breast [13]. For invasive carcinomas, the female and male tumors are morphologically indistinguishable [26] and many published studies on male breast cancer frequently represent the major histologic subtypes of nonlobular breast carcinoma, such as ductal, medullary, mucinous, and papillary [3, 7, 8, 12, 19, 22, 25]. However, these studies generally do not have the distribution and relative frequencies of the various histologic subtypes as their main focus, and as such, pathologically important distinctions such as signet ring vs colloid carcinoma (both subsumed under mucin producing carcinoma), or metaplastic carcinoma vs infiltrating duct carcinoma not otherwise specified are not emphasized. Over- or underepresentation of a particular histologic subtype in male breast cancers may provide valuable insight into the etiopathogenetic differences between male and female breast cancers and may provide an important nidus for further studies.\nIn this study, we investigated the frequencies and distribution of the various histologic subtypes in a large data set of invasive carcinomas seen at a large tertiary center over a 40-year period. Our goals are to document these frequencies and distribution and to see whether any histologic subtypes are notably distinct in frequency of occurrence in the male breast, as compared with historical published data on similar lesions in the female breast. The frequencies and sites of origin of metastatic tumors to the male breast during the study period were also investigated.\nMaterials and methods\nAfter approval from our institutional research review committee, cases of invasive carcinoma involving the male breast diagnosed between 1957 and 1997 were retrieved from the archived institutional files of the Armed Forces Institute of Pathology (AFIP, Washington DC, USA). The available records and slides on each patient were reviewed. All cases with ambiguous gender (even if one among the multiple entries for gender was coded as female rather than male) were excluded (n\u2009=\u200938). Diagnostic designations were assigned according to contemporary WHO criteria [23]. All special types of carcinoma inclusive of mucinous, tubular, secretory, adenoid cystic, lobular, metaplastic, etc. (n\u2009=\u2009116) were reviewed extensively; many of these cases had been evaluated repeatedly as part of various previously published studies from the department and slides of good quality were available on these cases for review. Among the 643 cases of infiltrating duct carcinoma not otherwise specified, only cases with good quality and adequate representative slides (n\u2009=\u2009365) were graded, using the modified Scarf\u2013Bloom Richardson scoring system. On 78 cases, the slides were so old that we could only confirm the presence of a ductal type carcinoma. The other 200 cases did not have all of the patients\u2019 slides pertaining to the lesion available and the final interpretation was based on the descriptions of morphology in the AFIP files. Because cases sent to AFIP are mostly for confirmation of diagnosis, information about therapy beyond what was in the surgical pathology report or the accompanying cover letter are generally not available. Some patients had repeated material sent to AFIP as the lesion recurred, which is how follow-up information was collected in a small subset of cases.\nInformation about age, operative procedure, nodal excision, and distant metastases were retrieved from the patients\u2019 AFIP files. Diagnostic designations were assigned largely based on morphologic and not immunohistochemical features since in many cases there were no blocks or unstained slides available for additional studies. Thirty of these cases were the subject of a previous report [13], which focused on in situ carcinomas and were reported in 1998. Another 113 cases, 8 of which were ductal in situ and another 9 papillary carcinomas, were subject of another report in 1969 [19] Cases of nonhematopoietic or lymphoreticular malignancies metastatic to the male breast during this period were also catalogued.\nResults\nSeven hundred and seventy-eight cases of breast carcinoma were reported as involving the male breast during the study period. Seven hundred fifty-nine of these cases were considered primary to the breast and 19 as metastatic to it. The patients ranged in age from 9 to 94\u00a0years (mean 61\u00a0years). The age distribution available for 520 cases was as follow: 8 were \u226430\u00a0years, 22 were \u226440\u00a0years, 205 were 41 to 60\u00a0years, 251 were 61 to 80\u00a0years, and 34 were over 80\u00a0years of age. Among the 490 for whom the racial information was available, 401 were Caucasian, not of Hispanic origin, 82 were Black, not of Hispanic origin, 6 were Asians or Pacific Islander, 1 was Hispanic, and the race was unknown for 269. The carcinomas ranged from \u22641\u00a0mm (microinvasive) to 12\u00a0cm. Many of the cases with multifocal early invasion of 1 to 7\u00a0mm were associated with large solitary or multifocal papillary intraductal carcinomas. The various histologic subtypes and their proportional distribution are outlined in Table\u00a01, whereas representative images are illustrated in Fig.\u00a01. Pure infiltrating duct carcinoma not otherwise specified was the most common histologic subtype, representing 84.7% of all cases. Six hundred forty-three cases were in the latter group, including 34 cases associated with Paget\u2019s disease, 17 cases with prominent apocrine features in <50% of tumor volume (i.e., insufficient to qualify as apocrine carcinomas), 4 cases with prominent medullary features (tumor circumscription, cellular syncytia, sparse lymphocytic infiltrate), 2 cases of infiltrating duct carcinoma with signet ring cells (<5% of tumor volume), 3 cases of infiltrating duct carcinoma with focal clear cells (<5% of tumor volume), and 3 cases of carcinoma with osteoclast-like giant cells. Papillary intraductal carcinoma with invasion in the form of infiltrating duct carcinoma represented 4.5% (34\/759 each) of all cases. Other well-represented histologic subtypes or combinations included mixed mucinous and infiltrating duct carcinoma (n\u2009=\u200926, 3.4%), pure mucinous carcinoma (21, 2.8%), and adenoid cystic carcinoma (n\u2009=\u20098,1.05%). Other variants each constituted less than 1% of all cases (Table\u00a01). Notably, there were three cases of lobular carcinoma. As noted previously, these diagnoses were not based on immunohistochemical confirmation by immunostain for E-cadherin, but all three showed archetypal morphologic features of lobular carcinoma with loosely cohesive single cell files and rare intracytoplasmic lumens. Two of these cases had evidence of lobular development in the surrounding breast but the slides available for review did not show lobular intraepithelial neoplasia (LIN). There were also three cases of mixed lobular and ductal carcinomas, in which these areas were spatially separate. The youngest patient, a 9-year-old boy was the only one with pure secretory carcinoma. Five of 759 cases were microinvasive (invasive focus less than 1\u00a0mm in maximum size in a predominantly intraepithelial lesion); three of these five cases were multifocal, whereas the other two were unifocal. Of the 365 cases of infiltrating duct carcinoma not otherwise specified that were graded, 104 were grade 1, 137 were grade 2, and 124 were grade 3. Among the 93 with lymph nodes available for assessment, 61 (66%) cases had axillary metastases. Forty of the women with axillary node metastases also had distant metastases. The most common distant metastatic sites were lung, adrenal glands, and bone. In addition, there were 19 examples of metastases to the breast that were documented. Of these, 58% (11\/19) were cutaneous melanomas, and 21% (4\/19), 10% (2\/19), and 10% (2\/19) represented carcinomas from the prostate, lung, and larynx, respectively. \nTable\u00a01Distribution of the various histologic subtypes in 778 invasive carcinomas involving the male breastHistologic subtypeNumber of casesPercentageInfiltrating duct carcinoma not otherwise specified, includinga64384.7Carcinoma associated with Paget\u2019s disease34Carcinoma with prominent apocrine features17Carcinoma with medullary features4Carcinoma with osteoclast-like giant cells3Carcinoma with focal signet rings2Carcinoma with focal clear cells2Pleomorphic carcinoma2Carcinoma with no notable distinctive features579Papillary intraductal carcinoma with invasion in the form of infiltrating duct carcinoma344.48Mixed infiltrating duct and mucinous carcinoma263.4Pure mucinous carcinoma212.8Adenoid cystic carcinoma81.05Tubular carcinoma60.8Small cell carcinoma60.8Invasive lobular carcinoma30.39Solid neuroendocrine carcinoma30.39Mixed lobular and ductal carcinoma30.39Adenocarcinoma with spindle cell  metaplasia20.26Pure signet ring carcinoma (no in situ component)10.13Invasive cribriform carcinoma10.13Secretory carcinoma10.13Squamous cell carcinoma10.13Total759100Metastatic lesions19Final total778aSubsets included for information only (not WHO categories)Fig.\u00a01Representative pathologic images of the various histologic subtypes. a and b Gross and microscopic images of infiltrating duct carcinoma, the most frequently encountered histologic subtype (b original magnification \u00d7160). c A colloid (mucinous) carcinoma (right field) occurring in a gynecomastoid breast (left field) (original magnification \u00d7100). d The single case of a secretory carcinoma was in a 9-year-old boy, the youngest patient in this series (original magnification \u00d7140). e and f Examples of papillary intraductal carcinoma with invasion in the form of infiltrating duct carcinoma [original magnifications: \u00d7100 (e), \u00d7120 (f)]\nDiscussion\nThe frequency, distribution, and morphologic spectrum of the various histologic subtypes of male breast cancer as compared with its female counterpart may provide a valuable insight into the etiology and\/or pathogenesis of both. This was perhaps best illustrated in the study of ductal carcinoma in situ by Hittmair et al. [13]. The authors showed that in the male breast, papillary DCIS occurred with a much higher frequency (46% of pure lesions) than expected, while solid, micropapillary, and cribriform patterns occurred with a much lower frequency (22% of pure DCIS combined). Notably, only 5 of 114 cases were of comedo-type DCIS and only 7 of 114 cases were considered high grade. The authors concluded that predominance of papillary patterns among intraepithelial male breast lesions is a reflection of the relative abundance of ducts and the poorly developed terminal-ductal lobular units in male breast. Given the rarity of true comedo DIN in male breast, the authors speculated that the fully developed lobular environment and the female type hormonal milieu may be a requirement for development of comedo DIN [13].\nIn this study, we investigated in detail the frequencies and distribution of the various histologic subtypes in a large data set of invasive carcinomas seen at a large tertiary center over a 40-year period. Our findings suggest that invasive carcinoma of the male breast displays a morphologic spectrum and distribution of histologic subtypes that is comparable to invasive carcinoma of the female breast and that no specific histologic subtypes are notably over- or underrepresented with the exception of papillary and lobular variants.\nPapillary intraductal carcinomas basically assumed an infiltrating duct carcinoma pattern once they invaded the stroma (Fig.\u00a01e,f). A papillary component was present in 34 (4.5%) of 759 cases, representing the second most common histologic subtype. These papillary carcinomas appeared to have invaded either through massive expansion and total disruption of ductal confines with typical invasive patterns at their periphery or only focally disrupted the duct wall and invaded the surrounding stroma as an infiltrating duct carcinoma. The latter represents approximately 2% [5] of female breast invasive carcinomas and appears overrepresented in males. In a large population-based study, Giordano et al. [7] reported that 2.6% of invasive malignancies in males were papillary carcinomas, as compared with 0.6% in females (p\u2009<\u20090.0001). However, the differences between males and females with respect to the frequencies of the other histologic subtypes were also statistically significant. The significance of the papillary histologic subtype or papillary architecture in male ductal carcinoma requires further investigation but is most probably related to the architecture of the male mammary duct system with dominance of larger ducts.\nThe possibility of metastases to the breast should be considered when evaluating breast masses particularly if there is a history of cancer elsewhere. Nineteen cases of carcinoma metastatic to the male breast were seen over this study period, representing 2.4% (19\/778) of all nonhematopoietic or lymphoreticular malignancies. This is again a twofold increase comparable to the 1.2% reported in female breast malignancies over a 10-year period [11]. Of the 19 metastatic tumors, 58% (11\/19) were cutaneous melanomas, and 21% (4\/19), 10% (2\/19), and 10% (2\/19) represented carcinomas from the prostate, lung, and larynx, respectively. It is interesting to note that prostatic carcinoma, which has been the subject of most reports of carcinomas metastatic to the male breast [10], was not the most common metastatic tumor in this series. The number of metastatic tumors in this series is relatively small, which is a limitation in interpreting the distribution of originating sites. Nonetheless, in the investigation of metastatic tumors to the male breast of an unknown primary site, our findings provide additional data on some of the most likely sites.\nThis study also confirms the occurrence of lobular carcinoma in the male breast. Three cases of invasive lobular carcinoma, diagnosed purely on a morphologic basis, were present among 759 cases (0.4%). Three additional cases of mixed lobular and ductal carcinoma were also diagnosed over this period (0.4%). Joshi et al. [16] found one (4%) lobular carcinoma out of 27 cases, while Goss et al. [8] found 4 (1.9%) cases out of a total of 229. In the series of Giordano et al. [7], lobular carcinomas constituted 1.5% for their male breast cancers. It is noteworthy that all of these cases were interpreted as lobular based on morphology without E-cadherin immunostain confirmation.\nIn summary, invasive carcinoma of the male breast appears to display a morphologic spectrum and distribution of histologic subtypes, which is comparable to invasive carcinoma of the female breast with some variation. As expected, lobular carcinoma is exceedingly rare but was encountered in this series. There appears to be a twofold increase in the frequency of invasive papillary carcinoma and metastatic cancers to the male breast as compared to published data in the female breast. Finally, the most common tumor metastatic to the male breast in this series was cutaneous melanoma.","keyphrases":["carcinoma","male","breast","papillary","cancer"],"prmu":["P","P","P","P","P"]}
{"id":"Arch_Dermatol_Res-_-_-1461773","title":"The long-term efficacy and safety of new biological therapies for psoriasis\n","text":"Long-term therapy is often required for psoriasis. This article reviews the most recent long-term clinical data for biological agents that have been approved or for which late-stage development data have been released for the treatment of patients with moderate to severe plaque psoriasis. Efficacy data are available for up to five 12-week courses of alefacept (approximately 60 weeks of therapy), 36 months (144 weeks) of continuous efalizumab, 48 weeks of continuous etanercept, and 50 weeks of bimonthly infliximab. Data sources include publications, product labeling, and posters presented at recent international scientific meetings. Alefacept appears to continue to be efficacious over multiple treatment courses for some responsive patients. The efficacy of efalizumab achieved during the first 12\u201324 weeks of therapy appears to be maintained or improved through at least 60 weeks of continuous treatment. The efficacy of etanercept appears to be maintained through at least 48 weeks of continuous treatment. Infliximab demonstrates a high response rate soon after initiation, which appears to be maintained through 24 weeks but declines modestly with therapy out to 50 weeks. After 48 weeks, approximately 60% of efalizumab-treated and 45% of etanercept-treated patients remaining on therapy achieved \u226575% improvement from baseline in Psoriasis Area and Severity Index, as did 70.5% of infliximab patients who did not miss more than two infusions. Safety data suggest that these agents may be used for long-term administration. Long-term data from psoriasis trials continue to accumulate. Recent data suggest that biological therapies have efficacy and safety profiles suitable for the long-term treatment of patients with moderate to severe psoriasis.\nIntroduction\nPsoriasis is a chronic inflammatory, immune-mediated disease characterized by periods of spontaneous remission and relapse [22]. Conventional nonbiological therapies for moderate to severe psoriasis, such as cyclosporine, methotrexate, and phototherapy, have proved effective in suppressing symptoms; and many dermatologists have at least a decade of experience using these treatment modalities [21]. Because of the chronic nature of psoriasis, many patients require lifelong symptom management. Conventional nonbiological systemic therapies have been successfully used as continuous therapy in some patients for extended periods (e.g., methotrexate); however, for some patients, the long-term use of these agents may be limited by known toxicities, including nephrotoxicity (cyclosporine) [21], hepatotoxicity (methotrexate) [10], skin cancer (psoralen-ultraviolet A phototherapy, or PUVA) [23], and prolonged teratogenicity (oral retinoids) [5].\nCurrent treatment guidelines recommend limiting the administration of cyclosporine to short-term (3-month) intermittent therapy or to no longer than 1\u20132\u00a0years continuously [11], reducing the dosage and minimizing lifetime exposure to methotrexate and ultraviolet irradiation [12, 27], and avoiding treatment with oral retinoids and several other conventional antipsoriatic therapies in women of childbearing potential [4]. Available data suggest that, when used according to contemporary guidelines and with appropriate monitoring for toxicity, conventional nonbiological treatments can be reasonably safe and tolerable [29]. However, minimizing toxicity while still providing continuous control of psoriasis symptoms over a patient\u2019s lifetime represents a clinical challenge.\nAn improved understanding of the immunologic basis of psoriasis led to the development of several targeted biological therapies that attempt to address the challenge of providing long-term continuous disease control. The first biological agent approved for the treatment of adult patients with moderate to severe chronic plaque psoriasis, alefacept (Amevive\u00ae, Biogen Idec Inc.), is currently registered for use in several countries, including the United States but not the European Union (EU). Two others, efalizumab (Raptiva\u00ae, Genentech, Inc.; Serono International S.A.) and etanercept (Enbrel\u00ae, Amgen, Inc.; Wyeth Pharmaceuticals), are also registered for this indication in multiple countries, including the EU and the United States. Another biological, infliximab (Remicade\u00ae, Centocor, Inc.; Schering-Plough Corp.), has presented Phase III clinical trial data for the treatment of psoriasis and recently became registered for use in the EU. The clinical trials programs for these four agents have demonstrated their safety and efficacy in patients with psoriasis over an initial period of several months; longer-term results are now becoming available. One other biological agent, adalimumab (Humira\u00ae, Abbott Laboratories), is at an earlier stage of development for psoriasis and has completed Phase II clinical trials for the disease. This article reviews the latest clinical data available regarding the long-term efficacy of these biological agents for treatment of psoriasis.\nAlefacept\nAlefacept is a fusion protein between human leukocyte function-associated antigen-3 (LFA-3) and the human IgG1 antibody Fc region. By binding to the CD2 antigen on T cells, alefacept prevents T-cell activation and triggers apoptosis, preferentially targeting memory-effector T cells [3]. Alefacept has been evaluated as a weekly intravenous (IV) 7.5-mg administration and as a weekly intramuscular (IM) 15-mg injection (Amevive\u00ae [alefacept] prescribing information; Cambridge, Mass: Biogen, Inc.; August 2004); however, only the IM dose is currently available. The efficacy and safety of a 12-week course of weekly IM alefacept 15\u00a0mg were evaluated in a randomized, placebo-controlled Phase III trial of adult patients with chronic plaque psoriasis [minimum of 10% body surface area (BSA) affected by psoriasis] [15]. Two weeks after the treatment phase was completed (study week 14), the Psoriasis Area and Severity Index (PASI) improved by at least 75% from baseline (PASI-75) in 21% of the 166 patients who received alefacept 15\u00a0mg per week, with 42% achieving at least a 50% improvement from baseline (PASI-50) (Amevive\u00ae [alefacept] prescribing information). This compares with rates of 5 and 18%, respectively, for patients randomized to placebo (n=168; P<0.001 for both comparisons). Mean percentage PASI improvement from baseline for a course of alefacept 15-mg IM therapy is shown in Fig.\u00a0 1.\nFig.\u00a01Mean percentage psoriasis area and severity index (PASI) improvement from baseline in randomized placebo-controlled, Phase III studies of biological agents for the treatment of psoriasis. ETANERCEPT Results for etanercept 25 or 50\u00a0mg twice weekly for 24\u00a0weeks [18]. Sample size at each PASI assessment was not reported. EFALIZUMAB Results for the use of efalizumab 1\u00a0mg\/kg\/week for 12\u00a0weeks followed by a 12-week open-label extension phase [6, 19]. The efficacy-evaluable population for this study included all patients randomized to efalizumab treatment (n=369), which differs from other efalizumab trials (and those of the other agents discussed) that include only patients who received at least 1 dose of the study drug. Following are the sample sizes at each PASI assessment, represented by the data points. For efalizumab: week 0, 369; week 2, 357; week 4, 353; week 8, 352; week 12, 347; week 16, 322; week 20, 323; week 24, 312. For placebo: week 0, 187; week 2, 186; week 4, 184; week 8, 180; week 12, 175 [19]. ALEFACEPT Alefacept results for the available 15-mg intramuscular (IM) dose [15]. Drug was administered weekly for 12\u00a0weeks, followed by observation for 12\u00a0weeks. In this study, 166 patients were randomized to receive alefacept 15\u00a0mg IM and 168 patients to receive placebo. Sample sizes at each PASI assessment were not provided. Statistical comparisons were not reported. Note Data for infliximab were not available\nAlefacept has demonstrated the longest psoriasis remission times among biological agents for psoriasis. Patients who achieved PASI-75 after a 12-week course of alefacept 15\u00a0mg per week (n=54) maintained at least a PASI-50 response for a median period of approximately 7\u00a0months [15]. Thus, the long-term use of alefacept as a remittive therapy has been explored through extension studies of additional treatment courses for patients who were enrolled in alefacept IV or IM Phase III trials. Efficacy data have been presented for up to five 12-week courses of alefacept therapy (i.e., approximately 60\u00a0weeks of treatment), although PASI responses are available for only the IV formulation [IM results were provided using Physician\u2019s Global Assessment (PGA)] (A. Menter et al., unpublished data presented at the 63rd Annual Meeting of the American Academy of Dermatology, 2005). The analyses were performed on the as-treated patient population, and a patient was considered a responder if he or she achieved a response anytime during each 12-week treatment course. Using these criteria, PASI-75 response rates for IV alefacept increased from 29% during the first course of therapy (n=521) to 54% during course 5 (n=39). PGA response rates for the IM formulation increased from 21% of patients classified as \u201cclear\u201d or \u201calmost clear\u201d during course 1 (n=457) to 41% during course 4 (n=100), but the response rate decreased to less than 30% during course 5 (n=50).\nAlefacept therapy appears to be well tolerated, even with long-term use. The primary concern with alefacept is T-lymphocyte depletion. Alefacept should not be initiated in patients with CD4+ T-lymphocyte counts below normal, and biweekly CD4+ T-cell monitoring is recommended during each treatment course (Amevive\u00ae [alefacept] prescribing information). Alefacept should be withheld if T-cell counts fall below 250 cells\/\u03bcl and discontinued if they remain low for a month. Patients treated with alefacept during Phase III trials demonstrated a low incidence of malignancy, serious infections requiring hospitalization, and hypersensitivity reactions (Amevive\u00ae [alefacept] prescribing information). The integrated safety database for alefacept includes a total of 1,869 alefacept-treated psoriasis patients and includes patients who have received up to nine courses of alefacept therapy (n=8) over a 5-year period. Despite the paucity of data for multiple treatment courses, the incidence of common adverse events (e.g., headache, nasopharyngitis, influenza, upper respiratory tract infection, and pruritus), serious adverse events, infections, malignancies, and autoantibody production appeared to fluctuate little with additional treatment, although the safety of long-term alefacept therapy will need to continue to be assessed.\nEfalizumab\nEfalizumab is a recombinant humanized monoclonal IgG1 antibody against the \u03b1 subunit (CD11a) of LFA-1, which modulates several key steps in the immunopathogenesis of psoriasis, including initial T cell activation, trafficking from the circulation into psoriatic lesions, and reactivation therein [13]. Multiple Phase III clinical trials have demonstrated the efficacy, safety, and health-related quality-of-life (HRQOL) benefits of 12\u00a0weeks of subcutaneous (SC) efalizumab therapy in patients with moderate to severe chronic plaque psoriasis [6, 16, 20, 24, 28]. As will be discussed later, available clinical data support the efficacy of extending efalizumab treatment beyond 12\u00a0weeks, as reflected in the European guidelines (European Medicines Agency: Raptiva [efalizumab] summary of product characteristics; http:\/\/www.emea.eu.int\/humandocs\/Humans\/EPAR\/Raptiva\/raptiv\/htm; 2005).\nThe Phase III study that served as the basis for efalizumab labeling evaluated the efficacy and safety of efalizumab 1\u00a0mg\/kg weekly [6]. A total of 556 adult patients with moderate to severe chronic plaque psoriasis (baseline PASI \u226512.0 and \u226510% BSA affected) were randomized to receive efalizumab (n=369) or placebo (n=187) double-blind for 12\u00a0weeks; all patients were then eligible to receive extended efalizumab open-label treatment for an additional 12\u00a0weeks [19]. A significant efalizumab treatment effect relative to placebo was achieved at 12\u00a0weeks, and extended treatment conferred additional clinical benefit. Using an intent-to-treat (ITT) analysis with the last observation carried forward (LOCF) for patients with missing data, the percentage of efalizumab-treated patients who achieved PASI-75 increased from 27% at week 12 (vs. 4% with placebo, P<0.001) to 44% at week 24. Similarly, the percentage of efalizumab-treated patients who achieved PASI-50 and PASI-90 increased from 59 to 67% and from 5 to 15% at weeks 12 and 24, respectively. The mean percentage PASI improvement relative to baseline increased from 52% at week 12 to 67% at week 24 (Fig.\u00a01) [6, 19]. Mean percentage PASI improvement appeared to increase throughout the 24-week duration without any evidence of a plateau effect. Two additional Phase III studies with similar placebo-controlled, double-blind initial 12-week treatment periods support the efficacy and safety of efalizumab therapy over 24\u00a0weeks [16, 17].\nAn open-label Phase III study evaluated the efficacy, safety, and tolerability of up to 36\u00a0months of continuous efalizumab treatment. At the time of this writing, results through 60\u00a0weeks of the trial and through 27\u00a0months (108\u00a0weeks) of the trial have been published [7, 9] and data through 36\u00a0months (144\u00a0weeks) have been presented (A.B. Gottlieb et al., unpublished results reported at the 63rd annual meeting of the American Academy of Dermatology, 2005). A total of 339 patients with moderate to severe plaque psoriasis received weekly efalizumab 2\u00a0mg\/kg for 12\u00a0weeks; of the 308 patients who completed the initial treatment period, 290 patients (94%) achieved PASI-50 or a static Physician\u2019s Global Assessment (sPGA) of mild, minimal, or clear at week 12 and were allowed to receive maintenance treatment with efalizumab 1\u00a0mg\/kg per week for up to 33 additional months (132\u00a0weeks) [7, 9]. At completion of the first 12-week treatment period, 41% of patients achieved PASI-75. Preliminary results showed that, by week 60, PASI-75 responses were observed in 49% of the ITT population (n=339) and in 57% of patients (n=290, last observation carried forward) who were analyzed by means of a maintenance group analysis, which included only patients eligible to receive continued efalizumab. An as-treated analysis indicated that 64% of patients (n=228) achieved PASI-75 at week 60 (PASI-75 results through week 48 are shown in Fig.\u00a02) [7]. Results in this trial support the sustained efficacy of up to 36\u00a0months of continuous efalizumab treatment (A.B. Gottlieb et al., unpublished results reported at the 63rd annual meeting of the American Academy of Dermatology, 2005).\nFig.\u00a02Percentage of patients achieving 75% improvement from baseline psoriasis area and severity index (PASI). Solid lines indicate intention-to-treat analyses; broken lines indicate as-treated analyses (efalizumab) or modified ITT analysis (infliximab) ETANERCEPT Results shown are from an etanercept study in which patients received etanercept 25\u00a0mg (n=196), etanercept 50\u00a0mg (n=194), or placebo (n=193) twice weekly for 12\u00a0weeks followed by open-label etanercept 25\u00a0mg twice weekly for up to 36 additional weeks [25]. Efficacy was evaluated in the ITT population. Published data beyond 24\u00a0weeks are not available; however, the data presented at several international medical congresses [e.g., European Academy of Dermatology and Venereology (EADV), American Academy of Dermatology (AAD)] suggest that the additional efficacy initially achieved with etanercept 50\u00a0mg twice weekly beyond that obtained with the 50-mg-per-week dose might not be sustained in the long term following dose reduction (unpublished data presented at the 2004 EADV and 2005 summer AAD meetings). EFALIZUMAB Results are shown for efalizumab from an open-label Phase III study (n=339) of efalizumab 2\u00a0mg\/kg\/week for 12\u00a0weeks, followed by 1\u00a0mg\/kg\/week for up to 33 additional months in patients who achieved PASI-50 or a static Physician\u2019s Global Assessment (sPGA) of mild, minimal, or clear at week 12 [7]. The dosage received during the first 12\u00a0weeks was higher than that later approved for clinical use (1\u00a0mg\/kg\/week). Efficacy was evaluated in the intent-to-treat (ITT) population throughout the study and is presented through 48\u00a0weeks; as-treated analysis is shown after 12\u00a0weeks through week 48. Sample sizes for as-treated analysis are as follows: week 24, 290; week 36, 269; week 48, 247 [7]. The shaded portion specifies a response window framed by the most conservative measure of efficacy (ITT) and the response of patients that remain on therapy (as-treated). ALEFACEPT Results are shown for alefacept 7.5\u00a0mg administered intravenously (IV) (A. Menter et al., unpublished data presented at the 63rd annual meeting of the American Academy of Dermatology); long-term PASI-75 response rates for the alefacept intramuscular (IM) dosing regimen have not been reported. Each course represents 12\u00a0weekly alefacept infusions followed by a minimum of 12\u00a0weeks of treatment-free observation. The second course was initiated 12\u00a0weeks after the first course [14]; additional treatment courses were initiated when the patient was deemed by the investigator to require systemic therapy or phototherapy for psoriasis recurrence. Course 5 represents patients who may have received up to 60 total weeks of alefacept treatment. Sample sizes are as-treated numbers. INFLIXIMAB Results are shown for infliximab patients who were randomized to receive IV infliximab 5\u00a0mg\/kg or placebo at initiation, week 2, week 6, and then every 8\u00a0weeks through week 46 [26]. Analyses through week 10 were performed using the ITT population (infliximab, n=301; placebo, n=77); analyses through week 50 were based on a \u201cmodified ITT\u201d population (n=234) where missing data were considered as nonresponse for patients who withdrew from the study due to preselected reasons (mainly related to lack of efficacy; patients who withdrew for other reasons were omitted from the analysis)\nSome limitations of the 3-year study are noted. At initiation of the trial, the label-supported dosage of 1\u00a0mg\/kg had not been established; thus, a dosage of 2\u00a0mg\/kg was administered during the initial 3-month period. It was subsequently demonstrated that the safety and efficacy do not differ for the 1- and 2-mg\/kg doses [16, 17]. In addition, although relatively few patients were affected (\u22644%), a dose increase of up to 4\u00a0mg\/kg was allowed up through 15\u00a0months of the study. Use of the LOCF procedure during analysis of ITT data might introduce bias in the final result; depending on the response of the patient, the bias could overestimate or underestimate the final result.\nClinical trials have shown that efalizumab is generally well tolerated and has a favorable safety profile over an initial 12-week treatment period [6, 16, 24, 28]. Results of the extended studies described here support the continued safety and tolerability of efalizumab treatment periods longer than 12\u00a0weeks [7, 9, 16, 17, 19]. The most common adverse events associated with efalizumab administration are acute flulike symptoms (headache, chills, fever, myalgia, vomiting, and nausea) observed primarily following the first two doses. After the third and subsequent doses, the incidence of acute adverse events in efalizumab-treated patients is comparable to that observed in placebo recipients. The extended treatment studies have shown that, with the exception of an expected reduction in acute adverse events, the incidence and intensity of adverse events during the second and subsequent treatment periods are similar to those observed during the first 12\u00a0weeks. To date, no evidence of cumulative or end-organ toxicity has been found with efalizumab. Thrombocytopenia has been reported during efalizumab clinical trials (Raptiva\u00ae [efalizumab] package insert; South San Francisco, Calif: Genentech, Inc.; June 2005), and recently a statement about the development of hemolytic anemia has been added to the prescribing information (Raptiva\u00ae [efalizumab] package insert). Worsening of psoriasis and psoriasis variants has been observed in 3% of efalizumab patients during therapy (i.e., generalized inflammatory flare), and in 14% of patients following abrupt discontinuation of efalizumab (i.e., rebound) [1]. The likelihood of experiencing rebound was inversely related to the PASI response at week 12; 72% of patients experiencing rebound were nonresponders, and only 10% had achieved PASI-75 [1]. New-onset or worsening arthritis has been infrequently reported during clinical trials and postmarketing (Raptiva\u00ae [efalizumab] package insert). Based on available clinical data from up to 36\u00a0months of continuous treatment, efalizumab does not appear to be associated with an increased risk of malignancy or opportunistic infections observed in immunosuppressed hosts [7, 16, 17, 19, 28]. Because the numbers of patients evaluated in the long-term studies have been relatively small, the incidence of malignancy and infection will be assessed postmarketing (Raptiva\u00ae [efalizumab] package insert).\nEtanercept\nEtanercept is a recombinant fusion protein comprising domains of the 75-kDa human tumor necrosis factor (TNF) receptor and human IgG1, which inhibits the activity of TNF-\u03b1, a proinflammatory cytokine implicated in psoriasis. The efficacy, safety, and HRQOL benefits of 12 and 24\u00a0weeks of etanercept therapy in patients with moderate to severe chronic plaque psoriasis (baseline PASI \u226510 and \u226510% of BSA affected) have been demonstrated in Phase III clinical trials [18, 25]. There is also clinical experience with etanercept in other indications, such as rheumatoid arthritis (RA). Recommended dosage in the EU is 25\u00a0mg administered SC twice weekly for up to 24\u00a0weeks. Dosing at 50\u00a0mg twice weekly is also possible for the first 12\u00a0weeks followed by a \u201cstep-down\u201d to 25\u00a0mg twice weekly. Based on published clinical data, European guidelines indicate that nonresponders should discontinue etanercept after 12\u00a0weeks and that reinitiation of etanercept treatment is possible after discontinuation (European Medicines Agency. Enbrel [etanercept] summary of product characteristics; http:\/\/www.emea.eu.int\/humandocs\/Humans\/EPAR\/enbrel\/enbrel.htm; 2005). In the United States, a 50-mg prefilled syringe recently became available, and the recommended dosing of etanercept is 50\u00a0mg twice weekly for the first 12\u00a0weeks of treatment, followed by step-down to 50\u00a0mg once weekly (Enbrel\u00ae [etanercept] package insert; Thousand Oaks, Calif: Immunex Corporation; October 2005).\nA placebo-controlled, double-blind Phase III study demonstrated the safety and efficacy of 12 and 24\u00a0weeks of etanercept treatment [18]. A total of 672 patients were randomized 1:1:1:1 to receive placebo or etanercept 25\u00a0mg once weekly, 25\u00a0mg twice weekly, or 50\u00a0mg twice weekly via SC injection for 12\u00a0weeks. Etanercept recipients who completed the initial treatment period continued on their assigned dosage for an additional 12\u00a0weeks. Whereas efficacy was originally reported only in patients who received at least 1 dose of study drug (n=652) [18], some data were subsequently reanalyzed for all patients who were enrolled and randomized, irrespective of whether the study drug was administered (Enbrel\u00ae [etanercept] package insert). These data are discussed herein to allow for a more accurate comparison with ITT data from efalizumab clinical trials. This reanalysis showed that among patients randomized to receive etanercept 25 or 50\u00a0mg twice weekly (the dosages most similar to those now recommended in Europe and the United States), 32 and 47% of patients, respectively, achieved PASI-75 at week 12, increasing to 41 and 54% at week 24 (Enbrel\u00ae [etanercept] package insert). The mean percentage PASI improvement reported in patients who received at least 1 dose of etanercept 25 or 50\u00a0mg increased from 53 to 64%, respectively, at week 12 to 62 and 71% at week 24. However, it appears that the mean percentage PASI improvement began to plateau around week 20 (Fig.\u00a01).\nResults from the first 24\u00a0weeks of a 48-week continuous etanercept therapy global Phase III psoriasis study were recently published. In the initial 12-week, placebo-controlled, double-blind phase of the study, 611 patients were randomized 1:1:1 to receive placebo or etanercept 25 or 50\u00a0mg twice weekly; all patients were then eligible to receive open-label etanercept 25\u00a0mg twice weekly for up to 36 additional weeks. Results at 12 and 24\u00a0weeks were reported for all randomized patients who received at least 1 dose of study drug (n=583) using the LOCF procedure for analysis and for all randomized patients regardless of whether they received study drug (n=611), with the assumption that patients with missing data had not met the criteria for response at that end point [25]. At 12\u00a0weeks, 32 and 46% of all patients randomized to receive etanercept 25 or 50\u00a0mg twice weekly, respectively, achieved PASI-75 (vs. 3% for placebo, P<0.0001 both comparisons). The PASI-75 response rate was slightly higher among patients who received at least 1 dose of etanercept 25 or 50\u00a0mg (34 and 49%, respectively), increasing to 45 and 54% at week 24 [25]. Data presented for this study at scientific meetings [2004 meeting of the European Academy of Dermatology and Venereology [EADV] and 2005 meeting of the American Academy of Dermatology (AAD)] indicated that among patients continuing on etanercept 25\u00a0mg twice weekly, the PASI-75 response achieved at week 24 was maintained through week 48 of continuous treatment and suggested that the greater PASI-75 response rate initially observed after 24\u00a0weeks in the group initially randomized to etanercept 50\u00a0mg twice weekly decreased slightly after per-protocol dose reduction, with PASI-75 response rates reaching a similar level in both groups during the open-label treatment phase (W. Sterry, unpublished data).\nA randomized, placebo-controlled, multicenter Phase III study to evaluate the safety and efficacy of etanercept 50\u00a0mg twice weekly over the course of 2\u00a0years is currently ongoing. Preliminary results after 48\u00a0weeks of therapy were recently reported (S.K. Tyring et al., unpublished data presented at the summer meeting of the American Academy of Dermatology, 2005). In this trial, patients were randomized to receive either etanercept 50\u00a0mg (n=311) or placebo twice weekly for 12\u00a0weeks (n=307), followed by open-label etanercept 50\u00a0mg twice weekly. Etanercept demonstrated statistically significant improvements in PASI response rates compared with placebo by week 4 of therapy. Response rates continued to increase through 24\u00a0weeks of therapy, after which they appeared to plateau. Approximately 60% of patients achieved a PASI-75 response at week 24, with a similar proportion at week 48.\nClinical data support the safety and tolerability of 12\u201324\u00a0weeks of etanercept treatment in patients with psoriasis [18, 25]. Published results from psoriasis clinical trials showed that etanercept was generally well tolerated. Adverse events observed during the initial 12-week placebo-controlled period were typically mild to moderate in intensity and occurred with similar frequency in etanercept and placebo groups [18, 25]. No apparent change in the adverse events profile occurred during the second 12-week period of the trials [18, 25].\nSafety outcomes of the long-term Phase III psoriasis clinical trial, particularly for patients who received 50\u00a0mg twice weekly, have not yet been presented in detail; however, a preliminary analysis integrating data from the Phase II and 2 Phase III etanercept clinical trials, in which many patients received the 25-mg dosage, suggested that no new pattern of adverse events emerged during up to 60\u00a0weeks of treatment (A.B. Gottlieb et al., unpublished data presented at the 62nd annual meeting of the American Academy of Dermatology, 2004). Psoriasis clinical trials revealed no evidence of increased risk of opportunistic infections, tuberculosis, or skin cancers during up to 60\u00a0weeks of etanercept treatment (A.B. Gottlieb et al., unpublished data, 2004). Whereas long-term data from clinical trials in many patients receiving etanercept for other indications, such as RA, support the general safety and tolerability of the 25-mg dosage, there have been infrequent cases of tuberculosis and rare cases of demyelinating disorders, pancytopenia, and congestive heart failure (Enbrel\u00ae [etanercept] package insert, European Medicines Agency; Enbrel [etanercept] summary of product characteristics; http:\/\/www.emea.eu.int\/humandocs\/Humans\/EPAR\/enbrel\/enbrel.htm; 2005).\nInfliximab\nInfliximab, a chimeric monoclonal antibody that binds membrane-bound and soluble TNF-\u03b1, is still under evaluation for use in patients with psoriasis. Infliximab has demonstrated efficacy in psoriasis patients in randomized, placebo-controlled Phase II trials [2, 8]. However, the results of an international multicenter, randomized, placebo-controlled Phase III trial of adult patients with plaque psoriasis (baseline PASI \u226512 and \u226510% of BSA affected) were recently reported [26]. Patients were randomized to receive IV infliximab 5\u00a0mg\/kg (n=301) or placebo (n=77) at initiation, week 2, and week 6 and then every 8\u00a0weeks through week 46. The placebo group crossed over to infliximab treatment at week 24, but the study remained blinded throughout. Patients who discontinued the study early because of lack or loss of response or for whom insufficient data were available were treated as not achieving the end points for analysis of the ITT population. At week 10 of treatment, the PASI-75 response rate was 80.4% in the infliximab-treated group and 2.6% in the placebo-treated group. This level of response was sustained through week 24. At week 50, the proportion of the ITT population that achieved PASI-75 dropped to 60.5%. However, excluding patients who missed 2 infusions, the PASI-75 rate at week 50 was 70.5% (n=234). The reason for the moderate reduction in efficacy is unclear but may be due to the development of inhibitory antibodies in a percentage of the patients.\nThe safety profile for infliximab during this extended treatment Phase III trial appears to be comparable to those observed during earlier studies [26]. The incidence of adverse events and serious adverse events in infliximab-treated patients was slightly elevated relative to those in placebo recipients through the first 24\u00a0weeks of treatment, and one patient who received infliximab died of sepsis. Elevations in aminotransferases were also observed in some infliximab-treated patients. In this trial, infection rates were comparable between the treated and placebo groups; however, like etanercept, postmarketing data from infliximab-treated patients with RA, Crohn\u2019s disease, or other indications for which infliximab is approved suggest a potential increased risk for events such as opportunistic infections (e.g., tuberculosis), lymphoma, or congestive heart failure.\nAdalimumab\nAdalimumab is a fully human monoclonal antibody that binds TNF-\u03b1. It is currently being evaluated in Phase III clinical studies for the treatment of moderate to severe plaque psoriasis. Results from a Phase II randomized, double-blind, placebo-controlled, multicenter clinical trial have been presented (K.B. Gordon et al., unpublished data presented at the 62nd annual meeting of the American Academy of Dermatology, 2004). Patients were randomized to receive placebo (n=52) or adalimumab, either an 80-mg first dose followed by 40\u00a0mg every other week (EOW; n=46) or an 80-mg first and second dose followed by 40\u00a0mg every week (n=50), for 12\u00a0weeks. In the preliminary analysis and presentation, 53 and 80% of the patients receiving adalimumab EOW or every week, respectively, achieved a PASI-75 response, compared to 4% of the patients receiving placebo (n=52). However, a careful reanalysis of the data suggested that the true efficacy was several percentage points lower (R.G.B. Langley et al., unpublished data, presented at the Fall European Academy of Dermatology and Venereology, 2005). Adverse events were similar to placebo, with headache, injection site pain, nausea, elevated triglycerides, cough, sinus congestion, and fatigue most common. Injection site pain was more frequent in the adalimumab 40-mg\/week group (12%) than in the placebo group (6%).\nComparison of biological agents\nIn the absence of head-to-head clinical trials, it is difficult to accurately compare the efficacy of the biological agents for psoriasis. However, most of these agents use comparable evaluations of treatment efficacy. Alefacept is an exception, as it is used as remittive therapy; and alefacept data are presented using the as-treated population and \u201cresponse at any time\u201d analyses. Thus, although alefacept appears to maintain efficacy in responsive patients over multiple courses of treatment (Fig.\u00a02), its efficacy cannot be readily compared with efalizumab, etanercept, or infliximab.\nAvailable long-term data indicate that the improvements in psoriasis achieved during the first 12\u201324\u00a0weeks of efalizumab therapy are maintained or improved through at least 60\u00a0weeks of continuous treatment. Data support the efficacy of efalizumab through at least 36\u00a0months of continuous treatment. The efficacy of etanercept 25\u00a0mg administered twice weekly appears to be maintained through at least 48\u00a0weeks of continuous treatment. Preliminary findings of the PASI-75 response rate in patients who \u201cstep down\u201d from etanercept 50\u00a0mg twice weekly to 25\u00a0mg twice weekly suggest that the initial improvements achieved with etanercept 50\u00a0mg twice weekly beyond what is observed with the 25-mg twice-weekly dose are slightly reduced following the recommended dose reduction. For infliximab, the very high response rates obtained after 10\u00a0weeks of treatment appear to be sustained through 24\u00a0weeks but show a moderate reduction through 48\u00a0weeks with infusions every 8\u00a0weeks. Week 48 PASI-75 response rates were 62% for efalizumab-treated patients and approximately 45% for etanercept-treated patients (as-treated populations; Fig.\u00a02). The PASI-75 response rate at week 50 for infliximab was 70.5% in patients who did not miss more than 1 infusion (Fig.\u00a02).\nThe longer-term studies summarized here are not directly comparable because of differences in patient eligibility for initial enrollment and maintenance treatment and inclusion in efficacy analyses. For example, in contrast to the long-term etanercept study, patients in the efalizumab open-label trial were eligible for maintenance treatment only if they achieved a reasonable response during the first 12\u00a0weeks. This included the majority (94%) of patients completing the initial treatment phase. For the ITT efficacy analyses (presented through 12\u00a0weeks), all efalizumab-treated patients were included, even those who did not achieve PASI-50 or sPGA of mild, minimal, or clear at week 12 and were required to discontinue the trial. Direct data comparisons are further compromised by the available analyses. Efalizumab data are presented as both an ITT analysis for 48\u00a0weeks and an as-treated analysis for weeks 12 through 48. Etanercept data are presented as an ITT analysis through 48\u00a0weeks. Results from the long-term infliximab study are presented as an ITT analysis for the first 10\u00a0weeks of the trial and thereafter are presented as a \u201cmodified ITT\u201d population, where missing data were considered as nonresponsive for patients who withdrew from the study for preselected reasons.\nGiven that psoriasis is a chronic, incurable disease, the availability of long-term data is critical for dermatologists evaluating treatment options. There is a paucity of long-term data derived from robust clinical trials for conventional nonbiological psoriasis therapies; therefore, dermatologists generally rely on treatment guidelines and personal experience in making their decisions about long-term administration of therapies such as methotrexate and cyclosporine. Because biologicals represent a new approach to the management of psoriasis, long-term data are of particular importance for this therapeutic class. Alefacept appears to be an effective therapy for inducing extended remissions in a small population of patients, and it has a demonstrated safety profile for remittive use. The long-term data available to date for efalizumab indicate that initial improvements in psoriasis are sustained for up to 36\u00a0months. Data from multiple clinical trials indicate that 22\u201341% of patients achieved PASI-75 in the first 3\u00a0months of therapy [6, 7, 16, 17, 24]; in an extended trial of patients receiving continuous therapy up to 36\u00a0months, 45.4% of the patients achieved PASI-75 at the 36-month end point (C.L. Leonardi et al., manuscript in preparation). Studies evaluating the long-term use of etanercept are under way, but only limited data are currently available, as suggested by the current 24-week limit for etanercept therapy in Europe. At 12\u00a0weeks, 49% of patients who received etanercept 50\u00a0mg twice weekly achieved PASI-75 [25]. Available data suggest that improvements are maintained for 48\u00a0weeks, as 60% of patients receiving 50\u00a0mg twice weekly achieved PASI-75 (S.K. Tyring et al., unpublished data presented at the summer meeting of the American Academy of Dermatology, 2005). Importantly, the safety data reported to date indicate that the safety profiles of efalizumab and etanercept allow for continuous long-term administration. Infliximab has demonstrated excellent response rates with induction and extended therapy, with an every-8-week infusion schedule optimal.\nThe approval of biological therapies, that is, the T-cell-modulators alefacept and efalizumab and the TNF\u03b1 antagonist etanercept, along with the development of other TNF\u03b1 antagonists such as infliximab and adalimumab, represent significant advances for the management of psoriasis. Their efficacy and safety profiles suggest that they may be suitable for remittive or continuous long-term administration, providing dermatologists with new options for the long-term management of their patients with psoriasis. The safety and efficacy data from ongoing clinical trials and the experience of dermatologists are important to define further how these agents can best be integrated into the psoriasis armamentarium.","keyphrases":["biological","psoriasis","alefacept","efalizumab","etanercept","infliximab","long-term treatment"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Neuroradiology-3-1-2151779","title":"How long does it take to coil an intracranial aneurysm?\n","text":"Introduction The change in the treatment of choice for intracranial aneurysms from clipping to coiling has been associated with an important change in logistics. The time needed for coiling is variable and depends on many factors. In this study, we assessed the procedural time for the coiling of 642 aneurysms and tried to identify predictors of a long procedural time.\nIntroduction\nCoiling of intracranial aneurysms has become the treatment of choice and has partly replaced neurosurgical clipping in most neurosurgical centers [1\u20133]. This change in treatment modality has been associated with an important change in logistics: patients are no longer treated in the operating room but in the radiology department. Coiling of intracranial aneurysms has to fit into the program of the angiography suite and an anesthetic team has to leave the operating room complex to assist in coiling. Since many aneurysms are treated acutely after subarachnoid hemorrhage (SAH), planning may be difficult. The time slot needed for coiling is variable and depends on many factors such as complexity of the aneurysm, the number of aneurysms that should be treated, the experience and skills of the treating endovascular team and local preferences. In this study, we assessed the procedural time for coiling of 642 aneurysms and tried to identify predictors of a long procedural time.\nPatients and methods\nPatients\nFrom January 1998 to September 2005, 817 aneurysms were coiled. The starting point of the study was chosen because of installation of a new biplane angiographic unit (Integris 3000 BN Neuro, Philips Medical Systems, Best, The Netherlands) at the end of 1997.\nWe excluded 65 patients (with 146 aneurysms) with more than one aneurysm coiled in the same session. Of the remaining 671 aneurysms, 642 had complete imaging data available for review. The time of each angiographic run was printed on the images automatically. The procedural time for coiling was defined as the number of minutes between the first diagnostic angiographic run and the last angiographic run after embolization. Thus, induction of general anesthesia and catheterization of the first vessel were not included in the procedural time. In the vast majority of patients, 3-D rotational angiography (3DRA) of the vessel harboring the aneurysm was performed [4]. Many patients with acute SAH confirmed on CT scan were immediately transferred to the angiography suite and underwent complete diagnostic angiography and coiling of the aneurysm under general anesthesia. Only in patients with unruptured aneurysms, was diagnostic angiography usually available before coiling, and in these patients only 3DRA was performed during the process of coiling.\nOf the 642 patients, 178 were men and 464 were women with a mean age of 52.9\u00a0years (median 52\u00a0years, range 19\u201383\u00a0years). Of the 642 aneurysms, 523 had ruptured and 119 had not. Among the 523 patients with a ruptured aneurysm at the time of treatment, clinical condition was Hunt and Hess (HH) I\/II in 337, HH IV\/V in 68, and HH III in 118. The mean aneurysm size was 8.0\u00a0mm (range 2\u201355\u00a0mm). The aneurysm was located in the posterior circulation in 154, in the anterior cerebral artery in 246, in the middle cerebral artery in 46, and in the carotid artery in 196 patients. The type of coil used was mostly Guglielmi detachable coils (GDC; Boston Scientific, Fremont, Calif.), followed by TruFill\/Orbit coils (Cordis Neurovascular, Miami Lakes, Fl.) and straight coils 50\u00a0cm long (Cook Detach, Cook, Copenhagen, Denmark) for large and giant aneurysms [5]. A supportive device was used during coiling in 60 aneurysms, a supportive balloon in 55, a TriSpan in 4, and a stent in 1 aneurysm [6]. In 26 aneurysms a procedural rupture occurred. Complications leading to permanent neurological deficit or death occurred in 40 patients [7, 8].\nStatistical analysis\nTo assess possible predictors of a long procedural time, we defined a long procedural time as the upper quartile of the procedural times (70\u2013158\u00a0min) and performed logistic regression analysis for the following variables: male gender, age greater than the median age of 52\u00a0years, ruptured aneurysm, aneurysm size in the upper quartile (10\u201355\u00a0mm), occurrence of procedural rupture, HH grade III\u2013V, occurrence of procedural morbidity defined as complications leading to death or permanent neurological deficit [7, 8], use of a supportive device, location on the anterior cerebral artery, carotid artery or middle cerebral artery, and location in the posterior circulation. Subsequently, to identify independent predictors, multiple logistic regression analysis was performed for all significant predictors.\nTo assess whether recent technical developments had influenced procedural time, we compared the mean procedural time of the first 321 aneurysms with the mean procedural time of the last 321 aneurysms using a t-test. In addition, we assessed whether the implementation of 3DRA in October 2000 had influenced procedural time.\nResults\nThe mean procedural time was 57.3\u00a0min (median 52\u00a0min, range 15\u2013158\u00a0min). More than half of the coiling procedures lasted between 30 and 60\u00a0min (Fig.\u00a01), 49 procedures (7.6%) lasted less than 30\u00a0min, and 68 procedures (10.6%) lasted more than 90\u00a0min.\nFig.\u00a01Procedural times in minutes for 642 coiling procedures displayed as number of procedures in 15-min time periods\nMedian and mean procedural times with ranges and standard deviations for aneurysm coiling procedures in relation to patient and aneurysm characteristics are summarized in Table\u00a01. The predictors of a long procedural time are displayed in Table\u00a02. The use of a supportive device was the strongest predictor of a long procedural time (OR 5.4), followed by the occurrence of procedural morbidity (OR 4.5), a large aneurysm size (OR 3.0), and patient age (OR 1.5). A poor clinical condition of the patient, rupture status of the aneurysm, gender, occurrence of procedural rupture, aneurysm location, and implementation of 3DRA had no influence on procedural time. Multiple logistic regression analysis identified the use of a supportive device, occurrence of procedural morbidity, and large aneurysm size as independent predictors of a long procedural time.\nTable\u00a01Procedural times for all 642 aneurysms in relation to patient and aneurysm characteristicsVariableNo. of aneurysmsProcedural time (min)MedianMeanRangeStandard deviationAall aneurysms64257.352.015\u201315824.1Use of supportive device6080.580.235\u201315030.7Procedural morbidity4072.575.628\u201315830.7Aneurysm size in upper quartile (10\u201355\u00a0mm)1636470.525\u201315826.4Age more than median 52\u00a0years3215560.717\u201315825.0Hunt and Hess grade III\u2013V1865459.218\u201312222.6Ruptured aneurysm5235258.116\u201315824.6Male gender1825560.417\u201313123.8Procedural rupture265053.816\u201310721.3Aneurysm location\u00a0Middle cerebral artery465361.819\u201313126.8\u00a0Posterior circulation1545461.722\u201314627.1\u00a0Anterior cerebral artery24653.557.116\u201311221.2\u00a0Carotid artery1964853.115\u201315823.9Implementation of 3-D angiography\u00a0Before1675258.618\u201315825.5\u00a0After4755256.915\u201314623.7Table\u00a02Odds ratios with 95% confidence intervals for different variables for the upper quartile of procedural times (70\u2013158\u00a0min)VariableOdds ratio95% CIUse of supportive device5.403.11\u20139.34Procedural morbidity4.492.33\u20138.64Aneurysm size in upper quartile (10\u201355\u00a0mm)3.032.06\u20134.45Age more than median age1.471.02\u20132.10Hunt and Hess grade III\u2013V1.320.88\u20131.97Ruptured aneurysm1.410.87\u20132.30Male gender0.790.54\u20131.16Procedural rupture0.730.27\u20131.98Aneurysm location\u00a0Middle cerebral artery1.810.97\u20133.40\u00a0Posterior circulation1.300.87\u20131.95\u00a0Anterior cerebral artery0.930.65\u20131.35\u00a0Carotid artery0.680.46\u20131.02Implementation of 3D angiography1.110.74\u20131.66\nThe mean procedural time for the first 321 coil procedures was 56.5\u00a0min (median 51\u00a0min, range 18\u2013158\u00a0min) and for the last 321 this was 58.1\u00a0min (median 54\u00a0min, range 15\u2013124\u00a0min). This difference was not significant (t-test, P\u2009=\u20090.40)\nDiscussion\nIn this study, the mean procedural time for coiling was 57\u00a0min. Most procedures were performed within 1\u00a0h. Procedural time was defined as the time between the first and the last angiographic run, since this time period could easily be assessed from time prints on the images. The time for patient preparation, anesthetic care, insertion of angiographic sheet and catheterization of the first vessel were not included in the procedural time. In our department, this additional time is usually between 30 and 45\u00a0min. This means that the typical time needed for coiling of an intracranial aneurysm is around one and a half hours, varying from one to two hours.\nA long procedural time was associated with the use of a supportive device, occurrence of procedural morbidity and large aneurysm size. Although coiling of middle cerebral artery aneurysms tended to take more time and coiling of carotid artery aneurysms tended to take less time, these differences were just not statistically significant. The strongest predictor of a long procedural time was the use of a supportive device, mostly a supportive balloon [6]. When the use of a supportive device is not anticipated from the beginning of the procedure, an additional guiding catheter must be placed in the groin and navigated to the vessel harboring the aneurysm, or the guiding catheter has to be changed for a catheter with larger inner lumen. In addition, the balloon catheter has to be prepared and navigated across the neck of the aneurysm and this may be time consuming since 0.010-inch guidewires are usually difficult to steer. Another predictor of a long procedural time was large aneurysm size. In larger aneurysms a higher number of coils can usually be inserted and the accumulated electrolytic detachment time thus increases. The last predictor of a long procedural time was the occurrence of morbidity (not the occurrence of procedural rupture). Most morbidity is caused by thromboembolic complications. Several actions may be undertaken when such a complication occurs, such as mechanical thrombus fragmentation and selective injection of thrombolytic drugs, which both lead to a longer procedural time.\nIt is of note that no difference in procedural time was found between the earlier and later procedures, despite the technical advances in microcatheters and guidewires and increased experience. In both groups, there was no difference in mean aneurysm size (8.7 versus 7.2\u00a0mm), use of a supportive device (both 30) or occurrence of morbidity (both 20). We did not include the first 3\u00a0years of coiling in our hospital because the time was not printed on the images by the angiographic equipment used during that period. The mean procedural time may have been longer on the steep side of the learning curve during those first 3\u00a0years.\nIn our high-volume department, logistics have been optimized. In patients scheduled for coiling, an intravenous line and urinary catheter are placed on the ward, the treating endovascular team is experienced, a biplane angiographic unit with 3DRA is available, all catheters and devices needed are available in the angiography room, microcatheters and guidewires are prepared by technicians, and in recent years the puncture site is closed with a dedicated device. For coiling of large and giant aneurysms, we use for the most part mechanically detachable coils 50\u00a0cm long. All anesthetists involved in coil procedures were experienced, with no residents in training. Our time slot for anesthesia is in the afternoon from 1300 to 1600 hours with a \u201cdeadline\u201d at 16.30, after which time an anesthetist on call has to be notified to take over care. With these logistics we normally plan two patients, and regularly treat three patients within this time slot.\nBoth of the senior authors (M.S. and W.J.v.R) have performed many coil procedures in four other hospitals in The Netherlands. The procedural time may differ greatly between centers, depending on many factors such as availability of single- or biplane angiographic equipment and 3DRA, the experience of the operator, supporting technicians and anesthetic team, and local preferences such as liberal or restricted use of supportive devices.\nConclusion\nWith optimal logistics, coiling of most intracranial aneurysms can be performed in one to two hours, including patient handling before and after the actual coiling procedure.","keyphrases":["coiling","intracranial aneurysms","procedural time"],"prmu":["P","P","P"]}
{"id":"Eur_J_Clin_Pharmacol-3-1-2071964","title":"How useful are prescribing indicators based on the DU90% method to distinguish the quality of prescribing between pharmacotherapy audit meetings with different levels of functioning?\n","text":"Objectives The objective of the study was to assess the association between the quality of drug prescribing based on three indicator types derived from the DU90% method and different levels of functioning in pharmacotherapy audit meetings (PTAMs).\nIntroduction\nIn The Netherlands, pharmacotherapy audit meetings (PTAMs) are established since 1990. These are local meetings of GPs and community pharmacists to make agreements on pharmacotherapy based on national guidelines and with regard to the costs of medicines to improve prescribing and dispensing of drugs. Depending on the local character, these groups differ concerning participating number of GPs and pharmacists, frequency and duration of the meetings, choice and the way of discussing topics and the adherence to agreements made in the daily practice of prescribing. By this, the nowadays more than 800 PTAMs in the Netherlands differ in the quality of functioning. It is to be expected that PTAMs with strict rules, which meet more regularly and reach obligatory agreements, will prescribe and dispense drugs more efficiently with regards to quality and costs. Research of this association, however, is still scarce. In The Netherlands, there were two studies with different results. Muijrers et al. [1] did not find any association between the quality of prescribing and the quality of PTAMs. Florentinus et al. [2], however, showed that PTAMs functioning on a higher level of cooperation were less susceptible to marketing influences to prescribe newly introduced drugs, and therefore, showed a higher quality in prescribing. Regarding these conflicting results, we decided to investigate the association between the quality of PTAMs and the quality of drug prescribing, taking into account a number of lessons learnt from the earlier studies. First, the levels of PTAMs in one of the earlier studies had been judged by self-made questionnaires [3]. We made use of the routine assessment of the quality of functioning in PTAMs from 2004 performed annually by The Dutch Institute for the Proper use of Medicines (DGV) since 2000 on behalf of the Dutch Ministry of Health [4, 5]. Second, for the judgement of the quality of prescribing, we used the drug utilization 90% (DU90%) method [6]. In Sweden, this method has been introduced as an inexpensive, flexible and simple manner to assess the quality of drug prescribing which is recommended for that aim by the Swedish Medical Quality Council. The DU90% method focuses on 90% of the delivered drug volume and supplies three general quality indicators for prescribing [7]. Third, we applied the DU90% to each of the seven mostly prescribed groups of drugs where we expected the influence of PTAMs to become most obvious. Fourth, we restricted the DU90% method to first prescriptions, and by this, excluded repeat prescribing, as changes in prescription behaviour become better visible in new than in existing drug regimens [8]. Fifth, for the quality of prescribing, we made use of dispensing data of pharmacies collected by the Foundation of Pharmaceutical Statistics (SFK). Concerning drug exposure, pharmacy databases are superior to prescribing data reflecting the medication actually dispensed [9]. Dispensing data have proven to correlate well with patient questionnaires on actual drug intake for chronically used drugs [10]. In this cross-sectional population-based study, we investigated the association between the quality of functioning in PTAMs, categorised into four levels by annual quality assessments, and the quality of prescribing, measured by the DU90% method with first dispensing of participating GPs for three indicator types within the seven mostly prescribed groups of drugs based on dispensing data.\nMaterials and methods\nInclusion of PTAMs\nIn the end of 2005, 257 of the more than 800 PTAMs were asked to join the study. In stratified selection, 50 PTAMs for each of the four levels (based on the quality assessment of DGV in 2004) and 57 PTAMs with no level available were invited. As it is possible that more than one community pharmacy participates in a PTAM, a total of 387 pharmacies had to be asked for permission to use their data in this study. Pharmacists further had to identify the GPs participating in their PTAM in 2004 by the anonymous codes from their computer systems. For this purpose, an online accessible web form was created. A third precondition for PTAMs to join the study was that pharmacies had delivered complete dispensing data to SFK from the year 2004 and from the second half of 2003 to distinguish first prescriptions from repeat prescribing. The SFK routinely collects dispensing data from more than 90% of the total of about 1,800 community pharmacies in The Netherlands. These data include detailed information on drugs dispensed such as the codes from the anatomic therapeutic chemical (ATC) system [11] and cost price. Dispensings are linked to the patients having been prescribed the drug in question. Patients are identified by an anonymous code, gender and year of birth, being the only information of the patient himself. Furthermore, for each dispensing, prescribers can be identified by speciality, and GPs further have an anonymous code.\nLevels for the quality of functioning of PTAMs\nLevels of functioning were assigned by a standard questionnaire developed by DGV for annual quality assessments [4, 5]. Six objective criteria were judged by an expert panel to be most important for cooperation within PTAMs such as the frequency and duration of the meetings, a jointly preparation of GPs and pharmacists, coming to agreements and checking the implementation of agreements in prescribing practice (Table\u00a01). These questionnaires were sent to a GP and a pharmacist as the declared contacts of each PTAM. The results collected for six criteria in 2004 were used to scale all PTAMs into one of the four different levels of cooperation by an investigator of DGV (Table\u00a02).\nTable\u00a01Six domains for quality assessment of the PTAMsParametersNumber of FTO meetingsMedian duration of FTO meetingsJoined preparation of GP and pharmacist (yes or no)Use of feedback information by prescription data concerning prescribed volume, costs\/DDD and preferences in drug choice per GP (yes or no)Written down agreements on drug choices (yes or no)Routine check of adherence to the agreements made with prescription data (yes or no)Table\u00a02Four levels for the quality of functioning of PTAMsLevelDescriptionLevel 1No structured meetingsLevel 2Frequent meetings without concrete decisionsLevel 3Frequent meetings with concrete decisions, supported by use of feedback information with prescription data, but without evaluation of these decisionsLevel 4Frequent meetings with concrete decisions and evaluation of these decisions\nIndicators for the quality of prescribing based on an adjusted DU90% method\nThe DU90% method was applied to the seven groups of drugs that had more than 4 million prescriptions in 2004: HMGCoA reductase inhibitors, drugs for acid-related disorders, antibacterials for systemic use, antidepressants, benzodiazepine derivatives, drugs for obstructive airway diseases and oral blood-glucose-lowering drugs (Table\u00a03). For these groups, only prescriptions of the GPs participating in the included PTAMs were selected in 2004. From these, only first dispensings were derived from the SFK database. \u2018A first dispensing\u2019 was defined as the delivery of a drug to a certain patient who, in the preceding half year, had not had a similar prescription of the same substance (on fifth ATC level and thereby regardless of dosage, dosage form, pack size and brand) [11]. For each FTO group, the first dispensings of the participating GPs expressed as the number of defined daily doses (DDDs) per ATC-5 code were calculated separately within the seven medication groups. Within each of the seven groups of drugs and for each PTAM, the substances were ranked by descending volume of DDDs to estimate DU90% segments for drug utilisation as described earlier by Bergman et al. [7]. The DU90% method focuses on 90% of the delivered drug volume. The borderline of 90% is an arbitrary but useful choice to study the bulk of prescribing while still allowing some leeway for individual variation due to special patient conditions. The DU90% method supplies three general quality indicators for prescribing [7]. One indicator type addresses the number of drugs within the DU90% segment (defined as substance of the same ATC code, fifth level). The second indicator is based on the percentage of drugs according to accepted guidelines within the DU90% segment. The third indicator measures economic aspects by the relationship between the costs per DDD in the DU90% segment and the costs per DDD in the remaining segment.\nTable\u00a03Selection of drugs from the seven groups, classification of guideline drugs and non-guideline drugs mostly encounteredGroup of drugsATCGuideline drugsMostly encountered non-guideline drugs in our study populationHMG CoA reductase inhibitorsC10AAC10AA01, C10AA03Simvastatin, PravastatinFluvastatin, Rosuvastatin, AtorvastatinDrugs for acid-related diseasesaA02A02A, A02BAAntacids, H2-receptor antagonistsProton pump inhibitorsAntibacterials for systemic useJ01J01A, J01BTetracyclines, AmfenicolesQuinolone antibacterialsJ01C except J01CR02Beta-lactam antibacterials except amoxicilline and enzyme inhibitorAmoxiciline and enzyme inhibitorCefalosporinsJ01ESulfonamids and TrimethoprimJ01FMacrolides, lincosamides, streptograminsJ01XOther antibacterials (e.g. Fusidic acid)AntidepressantsN06AN06AA09, N06AA02Amitryptiline, ImipramineMaprotiline, Clomipramine, FluoxetineN06AA10, N06AB08Nortriptyline, FluvoxamineCitalopram, Trazodone, Venlafaxine, MirtazapinN06AB05, N06AB06Paroxetine, SertralineBenzodiazepine derivatives and related drugsbN05BAN05CD07, N05CF02Temazepam, ZolpidemTriazolam, Lormetazepam, Flunitrazepam, Loprazolam, Clobazam, Bromazepam, Lorazepam, Oxazepam, Chloordiazepoxide, Alprazolam, ZopicloneN05CDN05BA01DiazepamN05CFDrugs for obstructive airway diseasesR03R03ACSelective beta-2-adrenoreceptor agonistsAdrenergis for systemic use, Theophylline,MontelukastR03BA, R03BBGlucocorticoids, AnticholinergicsR03BC01Cromoglicic acidOral blood glucose lowering drugsA10BA10BBSulfonamides, urea derivativesRepaglinide, ThiazolidinedionesA10BA02, A10BF01Metformin, AcarboseaDrugs of first choice according to the corresponding NHG standard were only suitable for patients without use of NSAIDs. Therefore, for the index of adherence of the antacids, special DU90% segments had to be calculated for all PTAMs, with the additional postulation that only first deliveries of selected GPs were included for those patients who had not had a prescription of a NSAID in the preceding 4\u00a0months.bThe valid NHG standard for hypnotics dated from 1992 and was quite obsolete. To better meet the actual standards and recommendations, we labelled the guideline drugs, recommended by another commonly used standard [23].\nFrom this, we deduced the following three indicators to assess the quality of prescribing:\nPrescribing according to ATCPrescribing according to guidelinesCost-effective prescribingPrescribing according to ATCWithin the DU90% segments of all seven groups of drugs, it was determined how many substances (fifth ATC level) accounted for the DU90% segment. Physicians are expected to manage prescribing by restricting it to a limited number of drugs. High quality of prescribing is then associated with the use of a relatively limited number of pharmaceutical products within a drug group [6]. PTAMs are expected to come to agreements on the drugs preferred for treatment of certain diseases. As PTAMs functioning on a higher level should be more able to make specific choices and follow these, we expected the number of drugs within a certain group of drugs to decrease with higher levels of PTAMs.Prescribing according to guidelinesThe national practice guidelines for GPs provide evidence-based recommendations for drugs of first choice for average patients with the indication in question. For each group, drugs were classified as permitted or not recommended by the guidelines. Classification was done on the basis of the Dutch Practice Guidelines for GPs (NHG-standards) valid in 2004 (the Dutch Standards have been described elsewhere [12]). We made use of the online accessible updates on the web site of the Dutch organization of GPs [13\u201319]. The particular choices made are listed in Table\u00a03. Consecutively, the number of DDDs of the guideline drugs was related to the whole number of DDDs in the DU90% segment [6]. Better functioning PTAMs are more likely to come to binding agreements on using drugs recommended by the guidelines and not for instance advocated by industrial marketing. We expected the index of adherence for each group of drugs within the DU90% segment to increase with higher levels of PTAMs.Cost-effective prescribingFor each ATC code, the actual cost per DDD as cost price for the pharmacy were recorded at the moment of dispensing (without value-added tax and regardless of the amount of reimbursement per prescription or discount). Per PTAM, the mean cost\/DDD were estimated for each DU90% segment and for the remaining 10% segment of the seven drug groups. Consecutively, the relationship of the cost\/DDD in the remaining 10% segment and the DU90% was calculated (cost\/DDD of remaining 10% segment divided by cost\/DDD of DU90% segment) [6]. When coming to agreements of drugs to be used preferably, PTAMs are also expected to take costs into account. Comparison between the median costs\/DDD within the remaining segment of scarcely prescribed drugs and the DU90% segment for each of the seven drug groups is likely to elucidate in how far the drug choices were also related to costs. As PTAMs on a higher level were likely to succeed better in consistent cost-effective choices, we expected the indicator to be increased with higher levels of PTAMs.\nAnalyses\nWithin all three indicator types, mean results (plus standard deviation) for the seven groups of drugs were calculated for each PTAM. With univariate and multivariate analyses of variance (MANOVA), variation between the levels of PTAMs was checked for all indicators of the seven drug groups within all three types of indicators. With MANOVA, analyses could be performed for all seven drug groups within one type of indicator simultaneously, adjusting for the fact that within a type of indicator, the same PTAMs were repeatedly measured for outcome of the seven drug classes. Further, we adjusted for the number of GPs per PTAM in categories from 1 to 10\u00a0GPs, 11 to 20\u00a0GPs, 21 to 30\u00a0GPs and more than 30\u00a0GPs per PTAM. All analyses were done with SPSS 12.1 for Windows (SPSS, Chicago, IL, USA).\nResults\nParticipating PTAMs\nFrom the 387 pharmacies belonging to 257 PTAMs invited for participation in this study, 182 pharmacies (corresponding to 122 PTAMs) gave permission to use their data (47% response). One hundred twenty-two pharmacists belonging to totally 96 PTAMs identified their participating GPs (67% of responding pharmacists). Among these were 11 PTAMs which not had permitted the use of their data. One pharmacy had switched from computer system in 2005 so that the codes for the GPs when filling the web form did not correspond any longer with the data delivered in 2004. Therefore, the data of the corresponding PTAM could not be used. For our study, data of 102 pharmacies were available belonging to 84 PTAMs (26% of the contacted pharmacies and 33% of the invited PTAMs). The distribution of these PTAMs within the different levels is shown in Table\u00a04. The number of participating GPs did not differ significantly between these groups.\nTable\u00a04Numbers of PTAMs and GPs per level of PTAMLevel of PTAMsNumber of PTAMs (%)Number of GPs (SD)Not classified7 (8)6.6 (2.2)121 (25)10.4 (7.0)223 (27)8.1 (3.5)315 (18)9.3 (6.7)418 (27)10.9 (5.5)Total84 (100)\nResults of the three indicators\nPrescribing according to ATC\nThe mean number of drugs within the DU90% segment for the seven groups of drugs ranged from of 3.1 for the HMG CoA reductase inhibitors to 9.6 for the antibacterials for systemic use. Within a certain group of drugs, however, the number of drugs was quite similar for all levels of PTAMs (Fig.\u00a01). MANOVA could not detect any differences in the number of DU90% drugs for the levels of PTAMs (p\u2009=\u20090.144). The number of GPs had no significant impact on the number of DU90% drugs (p\u2009=\u20090.884).\nPrescribing according to guidelines\nThe index of adherence to guidelines for the seven groups of drugs on average ranged from 48% for the drugs for acid related disorders to 96% for the antibacterials for systemic use. Within a certain group of drugs, however, the index of adherence was quite similar for all levels of PTAMs (Fig.\u00a01). MANOVA could not detect any differences in the index of adherence for the four levels of PTAMs (p\u2009=\u20090.836). The number of GPs had no significant impact on the index of adherence (p\u2009=\u20090.963).\nCost-effective prescribing\nThe cost per DDD within the DU90% segment on average ranged from 0.14 \u20ac\/DDD for the benzodiazepine derivatives up to 0.99 \u20ac\/DDD for the antibacterials for systemic use. In the remaining segment, the cost \/DDD varied from 0.22 \u20ac\/DDD for the benzodiazepine derivatives up to 1.62 \u20ac\/DDD for the antibacterials for systemic use. The relationship between the cost\/DDD of the rest segment and the cost\/DDD of the DU90% segment was taken as indicator. A high result indicated a large difference in costs for the drugs seldom dispensed compared to those responsible for 90% of dispensed volume. Except for the group of the oral blood-glucose-lowering drugs (ratio 3.6), the groups had mean ratios between 0.9 and 1.8 for the costs\/DDD in the remaining segment divided by the costs\/DDD for the DU90% segment.\nMANOVA could not detect any differences in cost effectiveness for the four levels of PTAMs (p\u2009=\u20090.636; Fig.\u00a01). The number of GPs had no significant impact on the indicator for cost effectiveness (p\u2009=\u20090.744).\nFig.\u00a01\nPrescribing according to ATC, according to guidelines and cost-effective prescribing for different groups of drugs and all levels of PTAMs\nDiscussion\nIn this cross-sectional study, we found no association between the level of functioning of PTAMs and the quality of prescribing for any drug group within the three indicator types chosen. Although we made several improvements in the choice of the prescribing indicators and the quality assessment of PTAMs compared to earlier studies, our findings were similar to those of Muijrers et al. [1]. However, these results are counterintuitive to the prevailing and general opinion that it makes sense to have well-organised PTAMs. We therefore critically evaluated our methods and results to see if we could come up with a plausible explanation for our findings. First of all, we looked at the selected prescribing indicators. In Sweden, the selected DU90% method had been used successfully to assess and distinguish the quality between primary healthcare centres [7]. In our study, however, differences between the levels of PTAMs turned out to be less substantial than those between the Swedish healthcare centres [6]. Apparently, the quality of prescribing concerning a restricted number of drugs, a choice concerning the guidelines and the cost efficiency in The Netherlands, is generally high and may approach an optimum in daily practice. This is quite obvious for the index of adherence found for the oral blood-glucose-lowering drugs and the antibacterials for systemic use. In acid-related disorders, however, the guideline acceptance seemed to be generally low, possibly due to considerations in daily practice that overall do not match with the recommendation in the standards. For the prescribing according to ATC, there is no general agreement on the lowest number possible within one drug class still warranting a desired level of prescribing quality for the individual patient. However, the low numbers of drugs within HMGCoA inhibitors, oral blood-glucose-lowering agents and agents for acid-related disorders found in our study seem to represent an overall high quality of prescribing. The cost-effectiveness was also overall high for oral blood-glucose-lowering drugs which was the only drug class with substantial differences in costs\/DDDs. Possibly due to high quality of prescribing for these three aspects in general, our three indicator types were not suitable to detect differences in the quality of prescribing for different levels of functioning in PTAMs. To distinguish the quality of drug prescribing in our country, indicators should be more sophisticated and address specific aspects in the daily practice of good prescribing within each drug class [9]. For instance, with antidepressants, the suitable duration of treatment may be more relevant for good prescribing than using the first drug choice from the guidelines [20]. In diabetes, the supply of HMGCoA reductase inhibitors as co-medication may be more important than the number of different oral blood-glucose-lowering drugs [21]. For obstructive airway diseases, co-prescribing and concordance with corticosteroids is more decisive for patients prognosis than choices according to guidelines [22]. Second, in using the DU90% method, the estimation of segments may need further specification, especially for the aspect of cost effectiveness. As DDDs in general are not always equipotent and may not, in all cases, represent the prescribed daily practice, the costs of the DU90% may be misclassified. For atorvastatin for instance, normally two DDDs are used as daily dosage. Based on the DDD classification, the DU90% volume in our study overestimated the volume of HMGCoA reductase inhibitors and underestimated the costs per effective DDD.\nWe also looked at the tool for measuring the quality of PTAM. This tool was based on criteria that had been valued as crucial and suitable by an expert panel and had been proven to be applicable in practice [4]. However, other criteria than the selected ones might be more decisive for public health. An example of an additional criterion is the one Florentinus et al. [2] found to be related with the quality of prescribing, namely to agree on one representative within a PTAM to receive the visitors of the pharmaceutical industry. Evaluation of the classification of PTAM levels of 2004 showed that only half of the PTAMs, being able to make and to evaluate decisions, actually were classified as the highest level. The main reasons for a lower classification were due to the lack of meeting the criteria on the number and duration of meetings. However, PTAMs meeting less than five times per year and being in conference for less than 1.5\u00a0h might be quite able to make and fulfil agreements on high quality of prescribing. Thus, the functioning of PTAMs may have been underestimated in our study, which may explain that also PTAMs of lower levels were able to reach high scores in the quality of prescribing.\nAnother possible explanation could be selection bias caused by the voluntary participation in our study which was quite low. In 2004, there were in total 824 PTAMs in The Netherlands, of which, 14% were in level 1, 43% were in level 2, 20% were in level 3 and 23% were in the highest level 4. Due to our stratified invitation of 50 groups from each level and 57 groups of no classified level, we managed to include sufficient numbers within each level. On average, PTAMs in The Netherlands had 9.6\u00a0GPs, comparable to the number of participating PTAMs in our study. Only a quarter of the pharmacists corresponding with one third of the PTAMs invited finally succeeded in participation. Possibly, by this, we included primarily those PTAMs being well organised and with a high level of cooperation regardless of the quality level assessed. The classification into the different PTAM levels might not have created substantial differentiation between participating groups, and this could be an explanation for the fact that the results of our indicators were nearly the same for the different PTAM levels.\nA further limitation was our lack of information about the topics that have actually been discussed by the PTAMs in 2004 and earlier. It is possible that PTAMs made agreements on other aspects than the three indicator types chosen. Perhaps, agreements were made on the duration of treatment with antidepressants, on co-medication with HMGCoA inhibitors in patients with diabetes or on usage of new drugs, but as mentioned above, these were not measured by our three indicator types.\nIn conclusion, it is difficult to define indicators based on the DU90% method that can readily distinguish differences in the quality of drug prescribing between PTAMs with different levels of functioning. Indicators have to be more sophisticated and to meet relevant aspects of the pharmacotherapy within certain drug classes. Items for classification of PTAM levels may need some reconsideration.","keyphrases":["prescribing indicators","pharmacotherapy audit meetings","drug use 90% method"],"prmu":["P","P","R"]}
{"id":"Matern_Child_Health_J-2-2-1592150","title":"Human Immunodeficiency Virus-1 and Preconception Care\n","text":"Human immunodeficiency virus (HIV) is a condition that increasingly affects women, especially women of childbearing age. Early in the HIV epidemic, HIV infection and AIDS were diagnosed in relatively few women in the United States. Today, the HIV\/AIDS epidemic represents a growing and persistent health threat to women, especially young women and women of color. In 2001 and 2002, HIV infection was the leading cause of death for African American women aged 25\u201334 years and was among the four leading causes of death for African American women aged 20\u201324 and 35\u201344 years, as well as Hispanic women aged 35\u201344 years [1, 2].\nAlthough HIV is increasing among women, the incidence in children from perinatal transmission is decreasing. With recommendations for routine screening for HIV during pregnancy and the availability of effective interventions to dramatically reduce the risk of perinatal transmission, the number of annual perinatally acquired AIDS cases from 1985\u20131994 has declined approximately 95% [3], representing one of the most striking success stories in the 25-year effort to reduce the impact of HIV\/AIDS in the United States.\nHIV infection can be transmitted from mother to child in utero, during labor and delivery and through breastfeeding. In the absence of intervention and without breastfeeding, the risk of perinatal HIV transmission is approximately 25% (range 13\u201339%) [4]. Evidence suggests that in the absence of breastfeeding, 30% of transmission occurs before birth and 70% around the time of delivery [4].\nIn February 1994, the results of the Pediatric AIDS Clinical Trials Group (PACTG) Protocol 076 documented that zidovudine (ZDV) chemoprophylaxis, initiated at 14\u2013 34 weeks gestation and continued through pregnancy, followed by intravenous ZDV during labor and oral ZDV to the infant for the first 6 weeks of life, could reduce perinatal HIV transmission by nearly 70% [5]. A U. S. Public Health Service (USPHS) task force subsequently issued recommendations for the use of ZDV for reduction of perinatal HIV transmission [6] and universal prenatal HIV counseling and HIV testing with consent for all pregnant women in the United States [7]. Epidemiologic data have further confirmed the efficacy of ZDV for reduction of perinatal transmission, even among women with advanced disease, low CD4+ T-lymphocyte counts, and prior ZDV therapy [8\u201313]. In addition, substantial advances have taken place in the treatment and monitoring of persons with HIV disease, which have resulted in changes in standard antiretroviral therapy for HIV infected adults. More aggressive combination drug regimens are now recommended to maximally suppress viral replication. For pregnant women, however, the benefits of antiretroviral therapy must be weighed against the risk of adverse events to the woman, fetus, and newborn; however pregnancy is generally not a reason to defer standard therapy. It should be offered with the addition of ZDV for prevention of perinatal HIV transmission to HIV infected pregnant women [4, 14].\nFor women with HIV infection, preconception care must focus on recommendations for safe sexual practices that prevent HIV transmission to sexual partners and protect women from acquiring potentially harmful sexually transmitted diseases or even additional strains of HIV that may be more virulent or resistant to therapy. Women also need education about perinatal transmission risks and prevention strategies, expectations for the child's future, and where desired, effective contraception until the optimal maternal health status for pregnancy is achieved, including appropriate therapy to maximally reduce viral load and optimize immune function. For HIV-infected women desiring pregnancy, specific counseling regarding available reproductive options that both prevent HIV exposure to uninfected partners, as well as preventing superinfection with resistant or more virulent virus should be discussed. The USPHS recommends that the following be included in preconception counseling for HIV-infected women: 1) selection of effective and appropriate contraceptive methods to reduce the likelihood of unintended pregnancy; 2) education and counseling about perinatal transmission risks, strategies to reduce those risks, and potential effects of HIV or treatment on pregnancy course and outcomes; 3) initiation or modification of antiretroviral therapy avoiding agents with potential toxicity for the developing fetus (e.g., efavirenz, hydroxyurea), choosing agents effective in reducing perinatal HIV transmission, attaining a stable, maximally suppressed maternal viral load, evaluating and controlling for therapy associated side effects which may adversely impact maternal-fetal health outcomes (e.g., hyperglycemia, anemia, hepatic toxicity); 4) evaluation and appropriate prophylaxis for opportunistic infections and administration of immunizations (e.g., influenza, pneumococcal, or hepatitis B vaccines) as indicated; 5) optimization of maternal nutritional status; 6) institution of the standard measures for preconception evaluation and management (e.g., assessment of reproductive and familial genetic history, screening for infectious diseases\/sexually transmitted diseases, and initiation of folic acid supplementation); 7) screening for maternal psychological and substance abuse disorders, and 8) planning for perinatal consultation if desired or indicated [4]. The USPHS further notes that HIV infected women of childbearing potential receive primary healthcare services in various clinical settings, e.g., family planning, family medicine, internal medicine, obstetrics\/gynecology, and stress that primary health-care providers should consider the fundamental principles of preconception counseling an integral component of comprehensive primary health care for improving maternal and child health outcomes [4].\nThe Infectious Disease Society of America (IDSA) recommends in-depth discussions about childbearing early in the course of HIV care for women who express a desire for a future pregnancy, who are not trying to conceive but are not using appropriate contraception consistently or who express uncertainty about reproductive plans. The goal of these discussions is to ensure informed decisions about contraception and to offer preconception counseling if pregnancy is desired. IDSA recommends that providers regularly assess women's reproductive plans and their desire to have questions answered and, that providers question patients at each visit about interval menstrual history and sexual and contraceptive practices, offering pregnancy testing as indicated [15].\nThe American College of Obstetricians and Gynecologists (ACOG) and the American Academy of Pediatrics (AAP) recommend that screening for HIV infection be strongly recommended for women with unknown HIV status and who are considering pregnancy [16, 17]. The Centers for Disease Control and Prevention (CDC) is considering this recommendation for its revised recommendations for HIV testing in healthcare settings planned for 2006 [3]. In addition, these organizations as well as the Institute of Medicine and the U. S. Preventive Services Task Force recommend universal prenatal screening for HIV infection [3, 17\u201321], so that HIV-infected pregnant women can take full advantage of the available interventions to reduce perinatal transmission as well as to receive care for themselves. These organizations support an opt-out approach to HIV screening, (i.e., patients are notified that HIV testing will be done and consent is inferred unless testing is declined) [3] to normalize HIV testing in health care settings and to reduce the barriers clinicians experience with specific HIV test counseling and written informed consent.\nPre-test discussions regarding risk reduction for HIV infection are not a requirement for HIV testing and can be provided in the context of discussions of reproductive health and STD prevention. Routine HIV testing during preconception care can be integrated with other aspects of preconception care that require a blood sample and should not require substantial additional time commitments by providers. Improved implementation of preconception care will further extend the success in reducing perinatal HIV transmission in the U. S. as well as improve access to HIV testing and care to women. Because the issues accompanying HIV treatment during pregnancy and perinatal prophylaxis are rather complex, the case for preconception counseling and care for known HIV-infected women is clear.","keyphrases":["preconception care","hiv","perinatal","pregnancy"],"prmu":["P","P","P","P"]}
{"id":"Acta_Neuropathol_(Berl)-3-1-1868652","title":"Silver diagnosis in neuropathology: principles, practice and revised interpretation\n","text":"Silver-staining methods are helpful for histological identification of pathological deposits. In spite of some ambiguities regarding their mechanism and interpretation, they are widely used for histopathological diagnosis. In this review, four major silver-staining methods, modified Bielschowsky, Bodian, Gallyas (GAL) and Campbell\u2013Switzer (CS) methods, are outlined with respect to their principles, basic protocols and interpretations, thereby providing neuropathologists, technicians and neuroscientists with a common basis for comparing findings and identifying the issues that still need to be clarified. Some consider \u201cargyrophilia\u201d to be a homogeneous phenomenon irrespective of the lesion and the method. Thus, they seek to explain the differences among the methods by pointing to their different sensitivities in detecting lesions (quantitative difference). Comparative studies, however, have demonstrated that argyrophilia is heterogeneous and dependent not only on the method but also on the lesion (qualitative difference). Each staining method has its own lesion-dependent specificity and, within this specificity, its own sensitivity. This \u201cmethod- and lesion-dependent\u201d nature of argyrophilia enables operational sorting of disease-specific lesions based on their silver-staining profiles, which may potentially represent some disease-specific aspects. Furthermore, comparisons between immunohistochemical and biochemical data have revealed an empirical correlation between GAL+\/CS-deposits and 4-repeat (4R) tau (corticobasal degeneration, progressive supranuclear palsy and argyrophilic grains) and its complementary reversal between GAL-\/CS+deposits and 3-repeat (3R) tau (Pick bodies). Deposits containing both 3R and 4R tau (neurofibrillary tangles of Alzheimer type) are GAL+\/CS+. Although no molecular explanations, other than these empiric correlations, are currently available, these distinctive features, especially when combined with immunohistochemistry, are useful because silver-staining methods and immunoreactions are complementary to each other.\nIntroduction\nThe year 2006 was the centenary anniversary of the first clinical description of a presenile patient with progressive cognitive decline, later defined as Alzheimer\u2019s disease (AD) [7]. It was the same year (1906) when Santiago Ram\u00f3n y Cajal and Camillo Golgi were awarded the Nobel prize for their contributions on neuroanatomy achieved by using silver-staining methods, especially Golgi-silver technique. In the field of neuropathology as well, silver staining was the principal method of choice at that time, which visualized senile plaques (SPs), neurofibrillary tangles (NFTs) and Pick bodies [2]. More than 80\u00a0years have passed before biochemical compositions of these pathological structures were unveiled. During this period, technical trials to identify and localize these molecules on histological sections showed the effectiveness of immunohistochemistry and revolutionized not only research but also routine diagnosis in the field of neuropathology. Probably because of the apparent success in the molecular explanation of diseases, recent attention to silver staining has not been very enthusiastic. Although most silver-staining methods are still left with some ambiguities, neuropathologists have profited by their efficacies. Silver-staining methods have been steadily improved and one of the recent advances is undoubtedly Gallyas method [41]. In practice, it allows stable, reproducible and sensitive visualization with low background. However, it is unfortunate that their principles, technical details and interpretations, which are different according to each method, are sometimes not sufficiently shared among neuropathologists and technicians. It frequently happens that some time-honored protocols are left without possible explanations and their interpretation is often restricted to \u201cargyrophilic or not\u201d. This naive interpretation is based on the assumption that \u201cargyrophilia\u201d is a homogeneous phenomenon irrespective of the staining method and the lesion. Recent studies have demonstrated, on the contrary, that \u201cargyrophila\u201d is heterogeneous and dependent on the staining method and the lesion [85, 119\u2013122]. Here, established silver-staining methods in the field of neuropathology are reconsidered with the intension of improving their use and interpretation.\nThe first part of this review summarizes basic principles and outlines protocols about four principal silver-staining methods for formalin-fixed, paraffin-embedded samples from human autopsy brains mainly with degenerative diseases, currently in use in our laboratory and their modifications. Although other silver-staining methods have also been invented to visualize normal neuronal structures, astrocytes, microglia and experimental tissue samples, it is not possible to encompass the whole range of these techniques mainly because of the limitation of my own experience. The second part provides an overview of similarities and differences between silver-staining methods and immunohistochemistry, so that a comprehensive interpretation, including their pitfalls results. Nevertheless, this review is not intended to establish a relative grading of these methods by identifying the possible superiority of a method (for example, immunohistochemistry) over another (for example, a silver-staining method). Instead, it is designed to show how unique each staining method can be, by providing a distinct point of view, each independent of the other. If we are prepared to take these possible differences of staining methods into account, such an approach will provide a less biased, multi-faceted and fuller stereoscopic picture than can be achieved with immunohistochemistry alone.\nGeneral principle of silver staining\nThe coloration in conventional histology is based on the selective affinity of a dye to some tissue elements relative to surrounding elements and their permeability. This differential affinity to specific tissue elements is finally represented by the distinctive original color of the dye as seen with its solution. This is essentially a single step procedure, called dyeing, and does not involve chemical transformation of the dye. Subsequent washing step is also selective in dissolving out the attached dye, which is called differentiation [5]. Various combinations (for example, hematoxylin and eosin, the Kl\u00fcver-Barrera method, etc.) are currently in use for neuropathological examinations. The mechanism of silver staining is, however, completely different. Because the solutions containing coloring agents used for silver-staining methods are usually colorless, simple application of these solutions to tissue sections, by itself, does not allow visualization through light microscope even if the reagent has some affinity to the tissue elements. Subsequent chemical transformation is necessary for visualization. In silver solutions, most of the silver is present as ion or complex salt. The silver ions should be first attached to the target elements as silver ions or silver salts (step 1). This attachment, however, does not allow visualization by itself. The attached silver ions (Ag+) have to be reduced (=accept electrons) to yield, in situ, metallic silver particles (Ag) now visualized only if their amount and size are sufficient for microscopic detection (step 2). Therefore, not only the differential affinity of the silver ion to some tissue elements (step 1) but also its chemical transformation for visualization (step 2) is essential. Although the attachment of silver ions to tissue elements is based on the \u201cin situ\u201d formation of silver salt, these silver ions, either in the solution or as salts, are reducible. They undergo reduction to form metallic silver. Gallyas demonstrated that there is a time lag between the rapid attachment of silver ions onto the section (step 1, within 15\u00a0min) and delayed formation of metallic silver particles (step 2) there [42]. Because these two steps are independently affected by pH, temperature, the concentration of silver ion and coexisting molecules [42], these steps are independent even if both take place in a single manipulation. This dual function of silver, that enables \u201csite-directed chemical transformation\u201d, is fundamental to various silver-staining methods.\nBecause some tissue elements (glutathione, creatine, adrenaline etc.) exhibit an intrinsic reducing capacity, which may be strong enough to reduce silver ions into metallic silver particles in situ, these two steps (selective attachment and visualization after reduction of silver ions) may take place in a single manipulation. This is called silver reaction or \u201cargentaffin\u201d reaction [35, 102]. When the reducing capacity of the tissue elements is absent or not sufficient, it is necessary to provide reducing agents externally after silver ions have been attached to the tissue elements. It is also possible to facilitate or modify these steps by some reagents [40]. Anyway, this two-step procedure is called postblackening (Nachschw\u00e4rzen) or \u201cargentophilic\u201d reaction [35, 102] and most of the current silver-staining methods for neuropathology consist, in essence, of at least these two steps. If sufficient amount of silver ions have been attached to the tissue elements but not have been reduced spontaneously after step 1, it is possible to visualize them simply by applying an appropriate reducing agent externally. This is called chemical development, during which the amount of deposited silver in the tissue section remains unchanged. However, it is not easy to increase the amount of silver deposits during step 1. For example, increasing the concentration of silver reagents or prolongation of incubation time is frequently hampered by nonspecific staining. This trade off between sensitivity and specificity, principally encountered in all kinds of staining, is especially troublesome with silver-staining methods because reactions in the silver solutions and those in reducing agents are both capricious and difficult to standardize. This is one of the reasons why more sensitive, stable and reproducible silver solutions and staining methods have been a matter of considerable endeavor for a long period, as will be discussed later.\nParticle size and possible determinants of selective staining\nIt has been reported that the apparent color of silver-stained elements is correlated with the size of final silver particles. In the test tube, the smallest range in their diameter (10\u201320\u00a0nm) is correlated with yellow color. The largest range (>100\u00a0nm) is associated with black color. Intermediate ranges are correlated with red (>30\u00a0nm) or blue-purple (50\u201360\u00a0nm) [102, 103], as reported by Liesegang [70]. The emission spectra of fluorogenic semiconductor nanocrystals, recently introduced for fluorolabeling, are dependent not only on their materials but also on their particle size [20, 22]. Although the size range of semiconductor nanocrystals (2\u20136\u00a0nm) is much smaller than that of silver particles, it is interesting to note that these differences in their color spectra are dependent on the particle size in both silver particles and the nanocrystals. Others reported, however, that black color is correlated with a larger amount of deposits [35]. Although the deposition of silver particles is primed by an affinity of the silver ion to some tissue elements, is this affinity the only major determinant of the selective staining? According to Seki [102], some colloidal substance in the tissue may hinder formation and deposition of metallic silver particles (Schutzkolloid). In addition, packing density of tissue ultrastructures (for example, fiber density) may also be responsible, because an excessive looseness of the structure may not allow secure settlement of silver particles. It is also possible that its extreme tightness does not permit appropriate growth of silver particles. He then concluded that a small quantity of this intervening colloid (eine Armut an Schutzkolloid) and an appropriate density of the structure (eine passende Strukturdichte) of the target structure are also important for the selectivity of silver staining. This idea that some spatial constraints around silver particles, in addition to the primary affinity of the silver ion, are essential in determining the selectivity of silver staining may be related to the current concept of disease-specific \u201cconformational status\u201d of pathological deposits and their constituent molecules.\nThese explanations are, however, far from complete for the majority of silver-staining methods. Furthermore, because these experiments have been performed mainly in animal tissues, in which sampling and handling of tissue materials are optimal, it remains to be clarified whether similar explanations are applicable to human samples obtained for neuropathological examinations, often containing distinct pathological lesions and suffering from suboptimal handling such as postmortem delay, suboptimal fixation and embedding in paraffin wax. Even though various methods for celloidin- or paraffin-embedded autopsy samples have been invented. Table\u00a01 shows fundamental steps and contents of the solutions for the silver-staining methods currently in use in our laboratory.\nTable\u00a01Protocols for different silver-staining methodsBielschowsky method (original) for frozen sections [8]Modified Bielschowsky method for paraffin sections [131]Bodian method [9,10]Gallyas method [41]Campbell\u2013Switzer method [21]PretreatmentPyridine (24\u201348\u00a0h)\u2013\u20130.25% potassium permanganate (RT, 15\u00a0min), then2% oxalic acid (RT, 2\u00a0min) [114], then0.4% lanthanum nitrate\/2% sodium acetate (RT, 60\u00a0min) [47]\u2013WashingDW\u2013\u2013DW\u2013Silver solution I3% silver nitrate(RT, 24\u00a0h)20% silver nitrate(RT, 20\u00a0min)1% silver proteinate\u00a0+\u00a0metallic copperf (37\u00b0C, 24\u00a0h)Alkaline silver iodate (RT, 4\u00a0min)\u00a00.035% silver nitrate\u00a010% potassium iodide\u00a04% sodium hydroxidePyridine silver (RT, 40\u00a0min)\u00a00.52% silver nitrate\u00a08.7% (volume) pyridine\u00a00.39% potassium carbonateWashingDWaDWDW0.5% acetic acid 3\u00a0min\u00a0\u00d7\u00a03 times0.5% acetic acid 3\u00a0min\u00a0\u00d7\u00a03 timesSilver solution II4\u20135%b ammoniacal silver20%c ammoniacal silver(RT, 15\u00a0min in the dark)\u2013Physical developerg (RT, 5\u201310\u00a0min)\u00a00.1% silver nitrate\u00a00.5% tungsto-silicic acid\u00a00.1% ammonium nitrate\u00a02.5% sodium carbonate\u00a00.03\u20130.2% formalinPhysical developergWashingDWDW\u00a0+\u00a0ammoniad\u2013Chemical developer20% formalin in tap waterDevelopere in 20% ammoniacal silver 1% hydroquinone\/5% sodium sulfiteDW distilled water, RT room temperaturea20% formalin in tap water is optional [50]b5\u00a0ml of 20% silver nitrate\u00a0+\u00a0five drops of 40% sodium hydroxide, followed by titration with ammonia to dissolve the precipitates. DW (20\u00a0ml) is then added [8]cAmmonium hydroxide was added to 20% silver nitrate drop by drop until the precipitates turn clear. Additional two drops of ammonium hydroxide is recommended [131]dAdd three drops of ammonium hydroxide to DW [131].eAdd three drops of the developer (formalin 20\u00a0ml, DW 100\u00a0ml, concentrated nitric acid one drop, citric acid 0.5\u00a0g) to the 20% ammoniacal silver used as silver solution II [131].f4\u20136\u00a0g of metallic copper in 100\u00a0ml of the silver proteinate solution is optimal [9, 10]gThe physical developers reported for Gallyas and Campbell\u2013Switzer methods are essentially identical. In our laboratory, equal amount of stock solutions A (0.5\u00a0g sodium carbonate-anhydrous, 100\u00a0ml DW) and B (0.2\u00a0g silver nitrate, 1\u00a0g tungusto-silicic acid, 0.2\u00a0g ammonium nitrate, formalin 0.73\u00a0ml, 100\u00a0ml DW) are mixed immediately before use\nBielschowsky methods\nImprovements in silver-staining methods have often been derived from chemistry. The ammoniacal silver solution had been used for production of mirror, based on the nature of the complex \u201csilver diamine [Ag(NH3)2]+ \u201c in the solution that is liable to precipitate, after being reduced, at the interface between the glass surface and the solution. This is called the silver mirror reaction and it was Fajersztajn who initially applied this phenomenon to tissue staining [34]. Later, Max Bielschowsky [8] improved this method by incubating formalin-fixed frozen sections in the silver nitrate prior to the ammoniacal silver solution (Table\u00a01). Although the exact mechanism still remains to be clarified [100], it is plausible that this prior treatment with the silver nitrate provides active foci for subsequent silver deposition in the ammoniacal silver solution [50]. Anyway, it is reasonable to suppose that this stepwise impregnation (silver nitrate-ammoniacal silver) improved the staining probably by increasing the amount and the size of silver precipitates [66]. Early modifications of Bielschowsky method were summarized by Beech and Davenport [6]. Garven and Gairns [50] provided a comprehensive review on the possible mechanism of the so-called Bielschowsky-Gros method and its modifications. The presence of a huge number of modifications, however, indicates that this method is highly unstable and still awaits improvements. A modification for paraffin-embedded samples of normal neural tissues from various animal species was first proposed by Sevier and Munger [105]. Practically, it is probably Yamamoto\u2013Hirano\u2019s modification [131], which is currently accepted as the standard Bielschowsky method for neuropathological examination of paraffin-embedded autopsy samples (Table\u00a01). This Yamamoto\u2013Hirano\u2019s modification is different from other conventional modifications of Bielschowsky method because the usual silver nitrate\u2013ammoniacal silver sequence is followed by another ammoniacal silver solution containing developers.\nBodian method\nBodian established a new method, applicable to paraffin-embedded sections, for staining nerve fibers and nerve endings [9, 10]. This method is rather stable and reproducible and is currently in use for neuropathology. He used 1% silver proteinate (which usually contains 8% of silver) solution and placed metallic copper in it. Because the staining does not work well in the absence of metallic copper, he speculated that metallic copper might regulate the deposition of metallic silver by decreasing the concentration of silver ion [9, 10]. However, the mixture of silver nitrate and powdered egg albumin was reportedly successful even in the absence of metallic copper [92]. Nevertheless, the measured concentration of silver ion gradually decreases after addition of metallic copper, as demonstrated by two independent groups [64, 93]. Concomitant and gradual increase in the concentration of copper ion [64, 93] is accompanied by a decrease in pH [93]. These reciprocal changes between concentrations of silver and copper ions are mainly explained by a primary difference in ionization energy between copper and silver. In other words, copper is more liable to be ionized than silver. Because their concentrations are not influenced by the presence of sections in the solution, the amount of silver ion trapped in the section is practically negligible. While the addition of metallic copper up to 5\u00a0g\/100\u00a0ml progressively decreases the concentration of silver ion in dose-dependent fashion (from 1,000 to 250\u00a0\u03bcg\/ml), further increase in the amount of metallic copper up to 15\u00a0g\/100\u00a0ml does not lead to further increase in the copper ion nor further decrease in the silver ion in the solution [64]. Indeed, this amount of copper, 5\u00a0g\/100\u00a0ml, is in perfect agreement of the amount, 4\u20136\u00a0g\/100\u00a0ml, originally recommended by Bodian [9, 10] and the amount of metallic copper more or less than this range does not give optimal results. Interestingly, not only the amount of metallic copper but also the shape of metallic copper (foil, plate, chip or granules) influences the concentration of copper and silver ions and the results of staining. The metallic copper as \u201cfoil\u201d is correlated with the highest concentration of copper ion and the most intense staining [64, 93]. Moreover, these reactions in the silver proteinate solution occur slowly and the equilibrium is reached after 12\u201316\u00a0h. Figure\u00a01 illustrates putative chronological events during incubation in the silver proteinate solution and development. In the early phase of silver impregnation, the high concentration of silver ion facilitates its attachment to the section (step 1, Fig.\u00a01). This initial attachment is apparently not selective, because early interruption of incubation yields an overall nonselective nerve staining [93]. Gradual deposition of silver ion as metallic silver on the surface of metallic copper (step 2, Fig.\u00a01) leads to a gradual decrease in the concentration of silver ion and a concomitant increase in the concentration of copper ion in the solution. Continued incubation in the solution with a decreasing concentration of silver ion facilitates liberation of the silver ion, once trapped in the section, back into the solution (step 3, Fig.\u00a01). The selective staining of Bodian method is explained if this rate of liberation of silver ion back to the solution (step 3, Fig.\u00a01) is different according to the tissue elements. This chronological change may be necessary to yield the selective staining and explains why an equivalent concentration of copper ion provided as copper sulfate instead of metallic copper does not yield similar results [64]. Some copper ions are also deposited onto the section (step 4, Fig.\u00a01). Residual silver proteinate as colloidal fraction may release silver ions gradually throughout the incubation (step 5, Fig.\u00a01). For practice, (1) 5\u00a0g of metallic copper as foil in 100\u00a0ml of 1% silver proteinate solution and (2) incubation for 16\u201324\u00a0h are recommended as optimal for Bodian method [64].\nFig.\u00a01Mechanism of the Bodian method. Step 1: Attachment of silver ions to the section driven by high concentration of silver ions; single asterisk: a fraction of silver ions are already reduced to metallic silver even before chemical development. Step 2: Deposition of silver ions as metallic silver leads to a decrease in the concentration of silver ion and reciprocal liberation of copper ions. Step 3: Delayed liberation of silver ions from the section back to the solution with decreased concentration of silver ion after step 2. Step 4: Copper ions are now available in the solution after step 2 to be attached on the section. Step 5: Silver proteinate gradually releases silver ions during steps 1\u20134. Step 6: Not only silver but also copper ions\/salts on the section are reduced to metallic particles. Step 7: These metallic particles are replaced with metallic gold; double asterisk: retrieval of copper ions in the gold chloride solution indicates that metallic copper has been deposited on the section (steps 4 and 6). Symbols: circle: silver; square: copper; pentagon: gold; blank symbols: reducible (ion\/salts); filled symbols: metallic\nAfter being incubated in the silver proteinate solution, sections are treated with 1% hydroquinone with 5% sodium sulfite for reduction (step 6, Fig.\u00a01) [9, 10]. It has been reported that formaldehyde, suitable for reducing the silver diamine of Bielschowsky method, is not appropriate by itself for reducing the silver ions in Bodian method. In contrast, hydroquinone suitable to reduce the silver ions in Bodian method is not appropriate for reducing the silver diamine of Bielschowsky method [35]. However, some recommended formaldehyde in combination with hydroquinone for the reducing step of Bodian method [9, 10]. Because it is usually after the incubation in the developer when the sections appear contrasted for visualization, it seems reasonable to suppose that deposited silver ions are reduced to form metallic silver only after exposure to hydroquinone. If this is the case, treatment of the silver impregnated sections with sodium sulfite, which dissolves out silver salt but not metallic silver, will diminish the staining. On the contrary, Peters [93] demonstrated the presence of sodium sulfite-resistant metallic silver and suggested that at least some fraction of silver ions have already been reduced to metallic silver during incubation in the silver proteinate solution (asterisk, step 4, Fig.\u00a01). They may serve as nuclei that attract surrounding reducible silver ions\/salts to form larger metallic silver particles during subsequent development. Indeed, Gallyas demonstrated that radiolabeled silver ions in the silver proteinate solution are deposited to the section in the form of metallic silver even before the development. This is based on the finding that the radiolabeled silver on the section is resistant to the treatment with 1% silver nitrate\/10% acetic acid that washes out reducible silver ions but not metallic silver particles. Because the localization of this radiolabeled silver, now deposited as metallic silver, is in good agreement with the final image of the adjacent section stained by Bodian method, it is likely that these metallic silver deposits play a determinant role in defining final image obtained with Bodian method [44]. At any rate, it is possible to intensify the staining through subsequent gold toning by incubating in 1% gold chloride with glacial acetic acid (three drops for 100\u00a0ml). Although the mechanism of Bodian method, especially that of copper ion, is not yet completely clarified, not only silver but also copper ions are retrieved in the gold chloride solution for toning (double asterisk, step 7, Fig.\u00a01) [64]. This indicates that the ionized copper in the silver proteinate solution has been deposited on the tissue and that the deposited copper, in addition to silver, is replaced with gold [64]. This dual replacement may explain why this gold toning is necessary and particularly effective for Bodian method. After gold toning, it is now possible and necessary to treat the sections with 5% sodium thiosulfate to dissolve out silver ions that remain unreduced in the tissue. Otherwise, these remaining ions may be reduced after exposure to light to form undesirable precipitates. Because Bodian method was originally invented to stain normal axons and neurofibrils, these normal structures are always superimposed on pathological structures such as SPs and NFTs. These intervening normal structures are one of the major hindrances of Bodian method especially when examining silver-stainable deposits overlaid on normal structures.\nGallyas method\nAnother way of improvement had been achieved based on the chemical principle of photography, \u201cphysical development\u201d. As opposed to the chemical development, additional silver ions are provided with reducing agents to form silver particles around the reactive foci, which have been already activated after exposure to light in dose-dependent manner. This greatly increases the amount of metallic silver or oxidized silver that are now visible as photographic images. It was Liesegang who first introduced this idea to histological staining in an attempt to establish a more consistent and reproducible method of silver staining [70, 88]. The protocols with physical development were later customized successfully for neuropathology by Gallyas [40]. Because physical developer solutions contain, in general, silver ions and reducing agents that together react rapidly, protecting colloid (tungsto-silicic acid for Gallyas method) is included in order to control by retarding this reaction [43].\nAs with other silver-staining methods, the attachment of silver ion to specific targets determines final localization of silver particles. According to Gallyas, this specific attachment is mediated by some determinant substance, which binds to the target tissue elements or the tissue elements themselves, followed by their chemical modifications. These foci are enabled to attract silver ions to form silver salts. Otherwise, the determinant substance renders these foci capable of reducing silver ions to form submicroscopic metallic silver particles bound to the target elements. The chemical nature of the original determinant substance may determine which components (for example, fibrous astrocytes, microglia or oligodendrocytes) in the section are finally silver-stained [37\u201339]. For visualization of NFTs, sections are incubated with silver iodide in alkaline condition that exhibits affinity to NFTs [41] (Table\u00a01). Although Gallyas customized protocols according to various target structures other than NFTs [37\u201339], this silver iodide protocol for NFTs, currently in routine use for neuropathological diagnosis, is defined in this review as \u201cGallyas method\u201d for simplicity and convenience. If this silver solution is prepared with radiolabeled silver, it is possible to quantify the silver ions\/particles after each step of the staining protocol. Gallyas demonstrated that most of the silver attached to the section after incubation in this silver iodide solution is reducible silver salt but not metallic silver, because it is washed out after incubation in nonlabeled 1% silver nitrate\/10% acetic acid (Table\u00a01 of Ref. [44]), a treatment that expels reducible silver ions\/salts from the sections. Incubation in sodium thiosulfate or sodium cyanide, alternate ways to expel these reducible silvers, diminishes the final image, again compatible with the assumption that the attached silver after incubation in the silver iodide solution is silver ions\/salts but not metallic silver [41].\nIs a separate step necessary to transform these attached silver ions\/salts into metallic silver particles so that they may subsequently work as nuclei for subsequent growth of silver particles in the physical developer? Because the reducing agent in the physical developer is ready to reduce these attached silver ions\/salts into metallic silver nuclei, an isolated step for reduction is not necessary before the treatment in the physical developer. However, a brief exposure of the silver-impregnated sections to formalin prior to the physical development leads to an instantaneous completion of the physical development [41]. However, it is possible that silver ions contained in the physical developer potentially attaches to the tissue and are physically developed to form separate metallic silver particles. Because this may happen independently of the silver from the initial alkaline silver iodide solution [45], a prolonged incubation in the physical developer may be hampered by this unexpected staining. Therefore, pretreatments have been invented in order to minimize this background. A pretreatment initially proposed by Gallyas was 5% periodic acid for 30\u00a0min. He tried various pretreatments [46] and finally proposed an alternative pretreatment with 0.4% lanthanum nitrate\/2% sodium acetate in H2O2 for 1\u00a0h, which is more effective in eliminating the background [47]. An additional pretreatment with sodium permanganate followed by oxalic acid [114] (initially intended for melanin bleaching [71]) prior to this lanthanum nitrate\/sodium acetate treatment of Gallyas [47] was found highly effective in eliminating the background and is currently in use as the standard in our laboratory. The background staining is practically negligible because the expected target staining is usually obtained before long and this untoward background reaction is generally slow. Similar oxidative pretreatments are not beneficial for other silver-staining methods [68]. The subsequent gold toning is essentially similar to that for Bodian method.\nCompared with Bodian and Bielschowsky methods, Gallyas method is characterized by its stability, reproducibility and ability to visualize abundant deposits [15]. Moreover, normal structures are not stained in practice. This allows easy and reproducible examination especially when looking for pathological deposits such as NFTs or glial cytoplasmic inclusions (GCIs) of multiple system atrophy (MSA) [90, 119]. However, amyloid deposits are hardly visualized and it is usually neuritic components of SPs that are labeled by Gallyas method.\nCampbell\u2013Switzer method\nThe initial impregnation solution of Campbell\u2013Switzer method contains pyridine (Table\u00a01). Pyridine was initially used for tissue fixation. Bielschowsky [8] used pyridine for postfixation of formaldehyde-fixed frozen sections and found an enhanced selectivity. In order to avoid instability and risk of explosion of the ammoniacal silver solution, Hicks [57] added pyridine (4.4 vol%) to the silver nitrate solution (0.55%) buffered with potassium carbonate at alkaline range. Thereafter, Campbell et al. [21] combined this silver-pyridine (2.5 vol%)-carbonate solution with the physical developer invented by Gallyas and found that this staining is not only highly sensitive in detecting SPs and NFTs in AD brains but also as stable and reproducible as Gallyas method. The solution was later modified by increasing the concentration of pyridine up to 8.7% [13] and was found to be equally very powerful in detecting \u03b1-synuclein-related pathologies [101]. Gallyas demonstrated that the presence of pyridine markedly decreases the amount of silver, either metallic or reducible, attached to the section [42]. This is compatible with the observation that pyridine enhances the selectivity of Bielschowsky method [8] and that a higher concentration of pyridine is associated with reduced background [21]. Because the standard pretreatment (potassium permanganate\/oxalic acid followed by lanthanium nitrate) for Gallyas method does not affect the results obtained with Campbell\u2013Switzer method and there are essentially no differences between the physical developers used for Gallyas and Campbell\u2013Switzer methods, the only significant difference between them is the silver solutions [86]. The reason for these differences between Gallyas and Campbell\u2013Switzer methods remains unexplained.\nEnhancement of silver staining by methenamine\nMethenamine has been used for histological demonstration of glycogen and mucin [52]. Periodic acid-methenamine silver stain has been used for staining of the basement membrane of the glomeruli [61]. Dekura et al. [30] first described the use of the methenamine-silver solution for neurofibrils without prior periodic acid. Further modification of this methenamine-silver method was found highly sensitive in detecting SPs, which is equivalent to immunohistochemistry for amyloid \u03b2-protein (A\u03b2) [1, 53]. Interestingly, formic acid pretreatment, which enhances immunoreactivity for A\u03b2 of SPs, completely diminishes argyrophilia of SPs by the methenamine-silver method or congophilia, suggesting that the methenamine-silver method detects specific structures, such as \u03b2-pleated sheets of amyloid, which are destroyed by formic acid [130]. It also stains perivascular amyloid but fails to stain Kuru plaque of Gerstmann\u2013Str\u00e4ussler\u2013Scheinker disease [130] and renal deposits of amyloid [53]. This is in agreement with the early speculation by Seki [102] that factors other than direct affinity of silver to some tissue elements, such as the quantity of intervening colloid or packing density of tissue components are also responsible for final results of the silver staining. The paucity of normal neurofibrils with this method is helpful in delineating SPs on electron microscopy [76]. It is then possible to detect even sparse aggregations of amorphous, often ramified structures with fine granular silver deposits, suggesting that this highly sensitive method is capable of detecting the earliest stage of A\u03b2 deposition [58]. Although the mechanism of lesion-specific enhancement remains unknown, introduction of 5% of methenamine in the silver proteinate solution of Bodian method is similarly effective in enhancing the selective affinity to SPs and NFTs [65]. It is interesting that this methenamine-silver method exhibits more affinity to Pick bodies when they are extracellular [89], while intracellular counterparts are more readily stained by conventional Bodian method even with electron microscopy [91].\nPretreatment with microwave\nA microwave treatment of sections in silver solution was first reported by Brinn [19]. This treatment causes an increase in molecular movements across the tissue. This presumably facilitates quicker silver impregnation. At the same time, shortened incubation time for impregnation and activated molecular movements may minimize precipitate formation. Microwave activation of silver impregnation for SPs and NFTs was first introduced in order to apply King\u2019s method [63] (originally designed for frozen sections) to paraffin-embedded sections [74]. Microwave activation is effective with Bielschowsky [23] and the methenamine silver [36] methods in shortening the incubation time for silver impregnation and eliminating the background. Although the elevated temperature may partly explain the improvements of these silver-staining methods, it has also been reported that a lowered temperature at 5\u00b0C for initial impregnation in 20% silver nitrate for Yamamoto\u2013Hirano\u2019s modification of Bielschowsky method gives more reproducible results in visualizing a larger number of diffuse plaques [73].\nMolecular species detected by silver staining\nIt is interesting to identify isolated molecular species detected by silver-staining methods, but these attempts have been so far limited. Gambetti et al. used Bodian method and anti-neurofilament antibodies in parallel to stain an extract from rat spinal cord electrophoresed on the SDS-polyacrylamide gel. The typical triplet of 200, 145 and 68\u00a0kDa identified on the Bodian-stained gel is in agreement with the neurofilament triplet identified on the counterpart stained with anti-neurofilament antibodies [48]. They confirmed similar parallelism in other vertebrate and nonvertebrate species as well [94]. Additional less intense bands at a lower molecular weight range may correspond to tubulin or degradation products of neurofilament proteins [4]. Because these findings have been obtained with normal nerve tissue, further analyses based on brains containing pathological lesions are mandatory to identify components responsible for their argyrophilia on the pathological lesions. Iqbal et al. [60] used Gallyas method to stain the SDS-gel after electrophoresis of the paired helical filament (PHF) fraction extracted from AD brain. The electrophoretic mobilities of the Gallyas-positive bands correspond to those of PHF tau (intense), normal tau (less intense) and high molecular weight MAPs (weak). The absence of Gallyas-positive bands in the range of neurofilament proteins is in sharp contrast with the Bodian-positive triplet of neurofilament [48]. This difference may explain why Gallyas method is practically free from the staining of normal structures such as neuronal soma and neurites. This discrepancy suggests that molecular species detected by different silver-staining methods can be completely different, even though both of the methods label apparently identical structures such as NFTs on histological sections. Because several silver-staining methods have been modified to stain SDS gels [62, 78, 107], it may be possible to identify molecular species, either normal or specific to some pathological structures, which have a selective affinity to some silver-staining methods. Because extracted proteins electrophoresed on gel are necessarily modified or significantly degenerated, this approach may be fruitful only if the disease-specific nature, if detectable with silver-staining methods, is readily represented by a single isolated molecule even when it is modified for electrophoresis.\nQuantitative comparison of silver-staining methods and immunohistochemistry\nEven after the introduction of immunohistochemistry, modification of classic silver-staining methods or invention of new silver-staining methods have been attempted [11, 25, 26, 98]. Two major deposits targeted by these silver-staining methods are undoubtedly NFTs and SPs of AD [31, 32, 79, 82]. Different silver-staining methods [24, 27, 31, 54, 56, 67, 99, 124, 125, 127] have been compared quantitatively for their relative ability to detect NFTs or SPs, as represented in Fig.\u00a02 [56]. In respect to detection of NFTs, these comparative studies are in agreement that Gallyas method is most powerful, followed by one of the three methods: modified Bielschowsky, Bodian or Cross methods. The methenamine-silver method is rated as least powerful for NFTs. There are two studies, each claiming a superior or equivalent number of NFTs detected with Reusche method [99] or with the nickel peroxidase method [27] compared with Gallyas method. This rating of putative sensitivity, however, is not shared when SPs are examined. SPs are well stained by modified Bielschowsky [125, 131], the methenamine-silver [1], periodic acid methenamine silver [128] or thioflavin-S [124] to similar extent. Bodian method is less powerful in detecting SPs , especially diffuse deposits [54, 56, 67, 127, 128]. Gallyas, Cross [56, 67] and Garvey [27] methods are least powerful in detecting SPs.\nFig.\u00a02Density of NFTs (a: upper panel) and that of SPs (b: lower panel) as a function of intellectual status evaluated by the Blessed Test Score. Both are dependent on the staining methods. Reproduced with permission [56]\nIt is needless to say that a more sensitive detection is preferable in order to identify specific deposits in more detail with ease. However, if one deals with clinicopathological correlation of a disease such as AD, where symptoms and deposits are more or less quantifiable under the background of normal aging, it is possible to identify a threshold of the deposits with any of the methods that may correlate with a threshold of the cognitive decline [118]. For this purpose, stability and reproducibility of the method are preferable [31\u201333, 54, 79\u201382]. Reliable agreement between the conventional Braak staging of neurofibrillary pathology based on Gallyas method [17] and the revised protocol based on AT8 immunohistochemistry [18] is a good example. On the other hand, if one is looking for specific pathological deposits, such as Pick bodies, GCIs or astrocytic plaques not observed in normal aging [118], specificity is the primary concern for the staining method. Some of these comparative studies included immunohistochemistry for tau [31, 56, 99], A\u03b2 [27, 54, 56, 99, 124, 127], ubiquitin [56] or neurofilament [24] for comparison with silver-staining methods. Some studies with highly sensitive A\u03b2 immunohistochemistry demonstrated that it visualizes a larger number of deposits than with conventional silver-staining methods [1, 56]. This suggests that amorphous A\u03b2 deposition, not identified by conventional silver-staining methods, represents the earliest stage of A\u03b2 deposition [59, 95, 129]. On the other hand, it is noteworthy that none of the series found any quantitative superiority of immunohistochemistry in identifying NFTs over the best-possible silver-staining method. For example, it is rather frequent that pathological structures in the form of NFT do not exhibit tau-like immunoreactivity any more, while most of them are stained by some silver-staining methods or fluorochromes [116]. This is explained if some tau epitopes in NFTs are truncated out during their evolution from pretangle to extracellular NFTs [12, 116]. Furthermore, immunohistochemical detection is highly dependent on the antibody and experimental procedures. These technical uncertainties make it difficult to place immunohistochemical detection as a standard for neuropathological diagnosis, especially when the quantity of deposits is of primary importance as with the histological diagnostic criteria for AD [32, 79]. It is needless to say that the molecular specificity of immunohistochemistry is a potentially reliable and logical way in order to sort different diseases under the diagnostic flags of molecules. This highly specific detection by pinpointing a single target molecule is, however, \u201ca very restricted, monochromic view\u201d as pointed out by Switzer [108]\u201d. Identification of the disease-specific molecular species and their reliable probes applicable to histological sections are desirable. Even if this kind of probes are available, immunohistochemical procedures are also difficult to standardize and much more costly than most of silver-staining methods [67]. Immunohistochemistry is highly costly especially when staining large histological samples, for example, hemispheric sections of human brain. Moreover, penetration of antibodies is sometimes not sufficient when staining thick histological sections. Silver-staining methods are more readily applicable even to large and thick histological sections [13]. It is of note as well that archival materials preserved for a long period, especially in formaldehyde, sometimes fail to exhibit immunoreactivity. It is frequently possible to visualize the lesions with silver-staining methods even in these archival materials [13, 123].\nQualitative representation by silver-staining methods\nAs summarized in the previous section, differences in the quantity of argyrophilic AD lesions are dependent on the silver-staining method. As has been considered frequently, it may be \u201cthe sensitivity\u201d of a staining method that governs the quantity of the argyrophilic lesions. This is probably in agreement with our general impression that \u201cmodified Bielschowsky method is more powerful than Bodian method in detecting AD lesions.\u201d [131] This interpretation is based on the assumption that so-called \u201cargyrophilia\u201d is a homogeneous phenomenon regardless of the staining method and the nature of lesion, which is not the case. For example, Gallyas method is one of the most \u201csensitive\u201d methods in detecting NFTs and neuropil threads in AD, while it is far less \u201csensitive\u201d in detecting SPs. This empirical but evident discrepancy is not readily explained by a mere difference in the \u201csensitivity\u201d of the methods and rather indicates that argyrophilic properties are dependent not only on the staining methods but also on the target lesions. Careful neuropathologists have been aware of these \u201cmethod- and lesion-dependent natures\u201d of various argyrophilic deposits in neurodegenerative diseases [16, 85, 96]. Table\u00a02 provides a summary of the heterogeneity of argyrophilia according to the staining method and the lesion. Gallyas method is one of the most \u201csensitive\u201d silver-staining method that clearly labels NFTs of AD, as well as neuronal and glial lesions of corticobasal degeneration\/progressive supranuclear palsy (CBD\/PSP) [113, 120]. This \u201csensitive\u201d method, however, fails to stain Pick bodies, another distinct tau-positive deposit [16, 85, 96, 117, 118]. On the other hand, Campbell\u2013Switzer method labels Pick bodies but not CBD\/PSP-related lesions [120, 122]. This reversed and complementary profile with Gallyas and Campbell\u2013Switzer methods is hardly explained by a superior sensitivity of Gallyas method over Campbell\u2013Switzer method or vise versa. These discrepancies between Gallyas and Campbell\u2013Switzer methods are readily demonstrable if mirror section pairs are initially fluorolabeled and one of the section pair is subsequently stained with Gallyas and the counterpart with Campbell\u2013Switzer method, as shown in Fig.\u00a03. It is now known that molecular species of tau are different according to the disease-specific lesions. Namely, Pick bodies are usually composed of three-repeat (3R) tau, while CBD\/PSP-related tau pathologies are positive for four-repeat (4R) tau [28]. Because Pick bodies are usually positive with Campbell\u2013Switzer method but negative with Gallyas method (Fig.\u00a03a\u2013d), one of the possible interpretations is that 3R tau deposits, such as Pick bodies, exhibit argyrophilia with Campbell\u2013Switzer but not with Gallyas method. In contrast, the lesions of 4R tauopathy (CBD\/PSP and argyrophilic grain disease [110, 111]) are related to the argyrophilia with Gallyas but not with Campbell\u2013Switzer method (Fig.\u00a03i\u2013l) [120]. Indeed, NFTs of AD, Down syndrome, and diffuse neurofibrillary tangles with calcification, all containing both 3R and 4R isoforms, similarly exhibit argyrophilia with either method (Fig.\u00a03e\u2013h) [120\u2013122]. It is interesting that Gallyas-positive Pick-like bodies were found to be positive for 4R tau but sparsely positive for 3R tau [132], which corroborates the empirical correlation between 4R tau and Gallyas method. Moreover, in one of the largest studies on sporadic and familial frontotemporal degeneration with tau-positive deposits, \u201cPick bodies\u201d defined as \u201cinclusions stained by Bielschowsky but not by Gallyas\u201d were found in 17 cases. All exhibited the immunoreactivity with a 3R-tau specific antibody as well as 3R tau predominance in tau biochemistry except for a single case with E342V mutation in the tau gene [29]. This particular case is characterized by diffuse cytoplasmic tau staining in neurons and PHF-like electron microscopic features [72], both of which are not typical of Pick bodies. In another sporadic case, Bodian-positive inclusions positive for 4R but not 3R tau have electron microscopic features with twisted ribbon distinct from those of typical Pick bodies [83]. These apparent heterogeneities necessitate a revision of the classical definition of Pick bodies \u201cround argyrophilic, tau-positive inclusions in neuronal cytoplasm\u201d, because argyrophilic features and tau-species are both heterogeneous in this classical category. Among argryophilic cytoplasmic inclusions in neuron, it seems that those positive for 3R-tau [84] and those negative with Gallyas method [29] are correlated to form a cluster to be defined as Pick bodies. Argyrophilia with the Campbell\u2013Switzer method [122] is helpful for more precise and straightforward definition of Pick body. In contrast with Gallyas and Campbell\u2013Switzer methods, argryrophilia with Bielschowsky and Bodian methods are less dependent on the target lesions (Table\u00a02). It is noteworthy that tau-positive cortical lesions of PSP are silver-stained with Bodian method [55], while those of CBD frequently fail to exhibit argyrophilia with Bodian method [69, 109, 115] especially in the cerebral cortices. Although possible biochemical distinctions of tau between CBD and PSP have been described [3, 104], it remains to be clarified how these biochemical differences are related to these morphological differences between CBD and PSP. Another possible limitation is related to the observations that the expression of tau-isoforms is not homogeneous throughout the brain [51, 126]. Because most of the data on tau-positive deposits summarized in Table\u00a02 and Fig.\u00a03 have been obtained on cerebral cortices, it remains to be determined whether these findings are similarly applicable to extracortical areas such as the brainstem. Yoshida [132] demonstrated that 3R tau immunoreactivity is represented in the brainstem even in so-called 4R tauopathies such as CBD\/PSP. Because neurofibrillary pathologies related to AD or aging are sometimes found in the brainstem nuclei and usually positive for both 3R and 4R isoforms, it remains to be clarified whether the presence of 3R tau in brainstem nuclei in CBD\/PSP is ascribed to tauopathies independent of normal or accelerated aging.\nTable\u00a02Heterogeneity of argyrophilia according to disease and methodStaining methodsCampbell\u2013SwitzerBodianBielschowskyGallyasPick bodies [16, 85, 96, 117, 118]++++++\u2212AD-NFTs++++++++Down-NFTs++++++++DNTC-NFTs++++++++PSP\/CBD-neurons\u2212+a+b++PSP\/CBD-glia\u2212\u00b1c+b++Argyrophilic grains [14, 77, 112]-++++AD-SPs+++++++AD-diffuse deposits++-\u00b1-Lewy bodies [75, 119]+++++-GCIs [87, 90]++++++AD Alzheimer\u2019s disease, NFTs neurofibrillary tangles, DNTC diffuse NFTs with calcification, PSP progressive supranuclear palsy, CBD corticobasal degeneration, SP senile plaques, GCIs glial cytoplasmic inclusions, ++: easily recognizable, +: positive, \u00b1: questionably positive, \u2212: negativeaPositive in PSP cases [55] but less evident in CBD cases [67, 109, 115]bNot consistent[97], positive in some cases, not appropriate for histological examinationcThe argyrophilia (Bodian) is less evident in glia than in neurons, especially in CBD casesFig.\u00a03Silver-staining profiles of various deposits in neurodegenerative diseases compared with immunofluoresence images. (a\u2013d Pick bodies[122]; e\u2013h neurofibrillary tangles; i\u2013l argyrophilic grains [121]; m\u2013p glial cytoplasmic inclusions; q\u2013t Lewy bodies [119]). Mirror section pairs were initially multi-fluorolabeled for PHF-tau (AT8, green for b, c, f, g, j, k) or for \u03b1-synuclein (green for n, o, r, s), with thiazin red (red, fluorochrome that labels fibrillary structure such as neurofibrillary tangles for b, c, f, g, j, k, n, o, r, s) and for ubiquitin (blue for f, g, j, k, n, o, r, s). After recording the fluorescent images from the same area of the mirror section pairs, one of the section pair was stained with Campbell\u2013Switzer (CS: a, e, i, m, q) and the other with Gallyas (GAL: d, h, l, p, t) method. Fluorescent images and silver staining profiles of the same area from the mirror section pair were compared. Pick bodies (a\u2013d) in the pyramidal layer of hippocampus are stained with CS (a) but not with GAL (d) [122]. Neurofibrillary tangles (NFTs, e\u2013h) from the frontal cortex of diffuse neurofibrillary tangles with calcification are stained with both CS (e) and GAL (h) [121]. This staining profile is shared with NFTs of AD [122]. Argyrophilic grains (i\u2013l, in the square) and tau-positive neurons are present (j, k) in the parahippocampal gyrus. The tau-positive neuron (double asterisk) and neuropil thread (arrow) stained with both CS and GAL are similar to those observed in DNTC (e\u2013h), representing neurofibrillary pathology. Grains and pretangle neuron (single asterisk) are stained with GAL (l) but not with CS (i) in contrast. This staining profile is shared with cortical lesion of corticobasal degeneration\/progressive supranuclear palsy [120]. Glial cytoplasmic inclusions (m\u2013p) in the putamen, positive for \u03b1-synuclein (n, o), are stained with both CS (m) and GAL (p) [119]. Lewy bodies (arrowhead, q\u2013t) and Lewy neurites (arrow, q\u2013t) in the dorsal motor nucleus of vagus, positive for \u03b1-synuclein (r, s), are stained with CS (q) but not with GAL (t) [119]. The same blood vessel is indicated with asterisk (q\u2013t). Bar\u00a0\u00a050\u00a0\u03bcm (a\u2013d, m\u2013p, q\u2013t), bar\u00a0\u00a030\u00a0\u03bcm (e\u2013l). Reproduced with permission. a\u2013d [122]; e\u2013l [121]; m\u2013t [119]\nInterestingly, argyrophilic differentiation with Gallyas and Campbell\u2013Switzer methods is not limited to tau-positive deposits. Both of the two staining methods label GCIs of MSA (Fig.\u00a03m\u2013p) [119]. Although GCIs are positive for some restricted tau epitopes [106], their major constituent is now considered to be \u03b1-synuclein. Indeed, Campbell\u2013Switzer method clearly labels Lewy bodies and their neurites, another representative deposits of \u03b1-synuclein [13]. It is interesting that Gallyas method fails to label Lewy bodies and Lewy neurites (Fig.\u00a03q\u2013t). Therefore, these two silver-staining methods, when performed in parallel, provide staining profiles that may allow qualitative distinction between GCIs and Lewy bodies [119]. Because no biochemical markers that enable distinction between Lewy bodies and GCIs are not readily available, these differential profiles in silver staining provides an additional qualitative distinction possibly linked to underlying molecular differences.\nAt present, these empirical distinctions are useful for diagnostic differentiation but await further explanations preferably at molecular or ultrastructural level. These distinctive features of silver-staining profiles may be explained if (1) molecular species specific to each subcategory (for example, 3R vs. 4R tau or LBs vs. GCIs) has different affinity to each silver-staining method; (2) some disease-specific ultrastructures of pathological deposit are related to each staining profile; (3) other independent molecules in these deposits play some roles in characterizing the pathological deposits so that distinctive silver-staining profiles are engendered; or (4) some disease-specific conformational changes are related to a silver-staining profile specific to a disease. Because Campbell\u2013Switzer and Gallyas methods are useful in discriminating not only tau-positive deposits but also \u03b1-synuclein-positive deposits even with their molecular and ultrastructural diversities, it is plausible that factors such as (3) or (4) shared by tau-positive deposits and synuclein-positive deposits may play roles, at least partly, in these discriminating abilities with these silver-staining methods.\nConclusions and perspective\nEven with my limited experience on these silver-staining methods, it is apparent that the argyrophilic profiles are dependent not only on the silver-staining method but also on the target lesion. This indicates that \u201cargyrophilia\u201d is highly heterogeneous. However, this heterogeneity is not capricious but rather representative of some disease-specific aspects. The apparent empirical correlations between the argyrophilic profiles and the disease-specific pathological structures are useful in sorting these neurodegenerative disorders. Combined silver-staining profiles, for example with Gallyas and Campbell\u2013Switzer methods, are more convenient than modified Bielschowsky and Bodian method for sorting different disease-specific lesions. Although these staining profiles are discussed in this review as if disease-specific, more detailed comparison at cellular level demonstrated that they can be different from cell to cell even in the same section (intercellular differences) [117]. Furthermore, staining profiles are heterogeneous even in a single cell (intracellular differences) [49]. These silver-staining and immunohistochemical profiles are much more informative especially when combined together than when used in isolation because such combination is not a mere summation of the results obtained with different methods. It will rather provide a more stereoscopic and multi-faceted view, which may allow more precise morphological delineation of disease-specific deposits. In my view, comments such as \u201cSilver-staining methods are unreliable, nonreproducible, nonscientific and out-dated.\u201d are now out-dated. Awareness of their utility and reliability of silver-staining methods, different from immunohistochemistry, may facilitate diagnosis and accelerate research, which hopefully clarifies molecular basis of each silver-staining method and improves our understanding of diseases.","keyphrases":["gallyas","campbell\u2013switzer","argyrophilia","senile plaques","methenamine","tauopathies","synucleinopathies"],"prmu":["P","P","P","P","P","P","U"]}
{"id":"Osteoporos_Int-4-1-2358936","title":"Progressive vertebral deformities despite unchanged bone mineral density in patients with sarcoidosis: a 4-year follow-up study\n","text":"Summary To evaluate the incidence of new and\/or progressive vertebral deformities and changes in bone mineral density, we re-examined 66 patients with sarcoidosis after a follow-up period of four years. In 17 subjects (26%) new and\/or progressive vertebral deformities were found, though BMD did not change significantly.\nIntroduction\nSarcoidosis is a T-cell driven chronic inflammatory disease. Although chronic inflammation has been associated with decreased bone mineral density as a result of the effects of cytokines on bone metabolism [1\u20134], we and others could not demonstrate changes in BMD in subjects with this condition, even if treated with glucocorticoids (GCs). In a cross-sectional study of 124 subjects with sarcoidosis, BMD values similar to an age- and sex-matched reference population were found [5]. Comparable observations were made in three small studies in untreated patients [6\u20138]. These studies also found a normal BMD relative to age and sex-matched controls, except for a small group of postmenopausal women in which BMD was moderately decreased at the spine in longstanding sarcoidosis only [7].\nAlthough in our cross-sectional study normal BMD values were observed, increased levels of the bone resorption marker serum carboxy-terminal cross-linked telopeptide of type I collagen (ICTP) and the bone formation marker serum procollagen type I amino-terminal propeptide (PINP) suggestive of increased bone turnover were found [5]. ICTP levels correlated with markers of disease activity such as soluble IL-2 receptor (sIL2R) and angiotensin converting enzyme (ACE). In addition, vertebral deformities suggestive of fracture were demonstrated in 20% of the subjects studied in this series. This may imply that the fracture risk in sarcoidosis is increased due to an increased bone turnover with consequent changes in microarchitecture and decrease of bone strength which is not reflected by changes in BMD [9, 10].\nIf so, this may result in progressive vertebral deformities during the course of the disease. For this reason we re-examined individuals with sarcoidosis four years after the initial measurements to determine the incidence of new and\/or progressive vertebral deformities and their relation with changes in BMD.\nSubjects and methods\nSubjects\nSixty-six of the 124 subjects with sarcoidosis that were studied in 2002 [5] agreed to participate in the follow-up study performed in 2006. None of the 56 subjects who declined or were unable to participate had impaired mobility or a history of vertebral fractures. The mean age of this group was 45 years and did not differ with respect to gender or glucocorticoid (GC) use from the group of subjects that were re-examined in 2006.\nDemographic, clinical and treatment data of the subjects studied in 2002 and 2006 are summarized in Table\u00a01. The group consisted of 22 pre-menopausal women, 11 post-menopausal women, and 33 men; median age of the total group (all Caucasian) was 43 years (20\u201366\u00a0y). The clinical records of all patients were reviewed. In 2002 patients were evaluated according to a standard protocol that included questionnaires, measurement of height and weight, lung function, measurement of BMD, a single energy densitometry of the spine, and laboratory evaluation [5]. In 2006 the same protocol was repeated. Informed consent was obtained from all participants and the study was approved by the medical ethics committee of our institution.\nTable\u00a01Baseline and follow-up demographic, clinical, and treatment variables (n\u2009=\u200966)VariableBaseline (n=66)Follow-up (n=66)P*Demographic variablesFemale sex33 (50%)Postmenopausal11 (17%)14 (21%)nsAge, years43 (20\u201366)Body mass index, kg\/m226.9\u2009\u00b1\u20095.727.2\u2009\u00b1\u20095.3nsSmoking7 (11%)Daily dietary calcium intake, mg740 (110\u20132360)758 (150\u20131340)nsClinical variablesDisease duration, years3 (1\u201322)7 (5\u201326)Chest X-ray stage (0-I-II-III-IV)24\/11\/12\/16\/327\/5\/12\/15\/7nsFEV1,% of predicted87\u2009\u00b1\u20092891\u2009\u00b1\u200926nsDLCO,% of predicted87\u2009\u00b1\u20091692\u2009\u00b1\u200918nsPhysical activity8.6\u2009\u00b1\u20093.78.1\u2009\u00b1\u20093.7nsLaboratory values (in serum)Calcium, mmol\/l (2.1\u20132.6)2.4\u2009\u00b1\u20090.12.4\u2009\u00b1\u20090.08ns1,25(OH)2D3, nmol\/l (0.040\u20130.200)0.14\u2009\u00b1\u20090.03ACE, U\/l (9\u201325)22.5\u2009\u00b1\u20099.815.3\u2009\u00b1\u20097.90.001sIL-2R, kU\/l (241\u2013846)654 (188\u20134315)Hs-CRP, mg\/l (<10)3.2 (0.2\u2013191)2.0 (1\u201316)<0.05Z-score ICTP0.7\u2009\u00b1\u20091.4Z-score PINP\u22120.1\u2009\u00b1\u20090.9Treatment variablesGC use never31 (47)26 (39)nsGC use previous14 (21)25 (38)<0.01GC use current21 (32)15 (23)nsLifetime GC dose, mg9240 (200\u201348750)11187 (200\u201356700)<0.001Daily dose, mg12.4\u2009\u00b1\u20096.210.5\u2009\u00b1\u20093.3<0.05Started on bisphosphonates after baseline measurement6 (9)Clinical risk factors for osteoporosisFracture2 (2\/24\u2009=\u20098%)5 (5\/28\u2009=\u200918%)nsLow body weight (< 60\u00a0kg)8 (12)7 (11)nsSevere immobilization00nsLow physical activity index \u2264 518 (27)18 (27)nsMother with hip fracture6 (9)7 (11)nsData are given as mean \u00b1 SD, median (range) or number (%); *=\u2009p value between baseline and follow-up measurement. Reference parameters in bracketsAbbreviations: GC, glucocorticoid; FEV1, forced expiratory volume in one second; DLCO, diffusion capacity for carbon monoxide; 1,25(OH)2D3, 1,25 dihydroxyvitamin D; ACE, angiotensin converting enzyme; sIL-2R, soluble interleukin-2 receptor; Hs-CRP, high-sensitivity C-reactive protein; ICTP, carboxy-terminal cross-linked telopeptide of type I collagen; PINP, procollagen type I amino-terminal propeptide\nPulmonary evaluation\nLung function measurements, including forced expiratory volume in one second (FEV1) and forced vital capacity (FVC), were measured with a pneumotachograph. The diffusion capacity for carbon monoxide (DLCO) was measured using the single-breath method (both Masterlab, Jaeger, W\u00fcrzburg, Germany). Values were expressed as a percentage of those predicted [11].\nChest radiographs were graded according to the radiographic staging of DeRemee (0 to III), adding stage IV, the end stage of lung fibrosis [12, 13]. All interpretations were made by a radiologist who was blinded to the patient\u2019s history.\nLaboratory assays\nSerum 1,25-dihydroxyvitamin D concentration was determined by radioimmuno-assay using a commercially available kit [(IDS Ltd, Boldon, England, interassay coefficient of variation (IE-CV) 18%, intra-assay CV (IA-CV) 15%)]. High-sensitivity C-reactive protein (hs-CRP) was measured by particle-enhanced immunonephelometry on the BN Prospec (Dade Behring). The detection limit is 0.175\u00a0mg\/L and the measuring range is 0.175\u20131100\u00a0mg\/L. Soluble IL-2 receptor (sIL-2R) was determined on the IMMULITE automated analyzer, by means of a two-site chemiluminescent enzyme immunometric assay with a measuring range of 50\u20137500\u00a0kU\/L (Diagnostic Product Corporation, Los Angeles, CA, cat no LKIP1). Serum angiotensin converting enzyme (ACE) was measured using a colorimetric method. The precision of the ACE assay was < 5.6% and the reference interval for ACE was 9\u201325\u00a0U\/L.\nAs a marker for bone formation, serum procollagen type I amino-terminal propeptide (PINP) was measured. As a marker for bone resorption, serum carboxy-terminal cross-linked telopeptide of type I collagen (ICTP) was assessed. Both PINP (IE-CV 3.2%, IA-CV 2.5%, lowest detectable concentration 0.4\u00a0\u03bcg\/l) and ICTP (IE-CV 3.5%, IA-CV 2.3%, lowest detectable concentration <0.1 \u03bcg\/l) were measured using commercial RIA kits (Orion Diagnostica Oy, Espoo, Finland). To adjust for age and gender Z-scores for these bone markers were obtained using a Dutch reference group (300 women, 150 men), checked for normal BMD of the lumbar spine and femur and normal 25-hydroxyvitamin-D levels [14, 15].\nBone mineral density and vertebral morphometry\nBone mineral density (BMD) was measured by dual X-ray absorptiometry (DXA, Hologic QDR 4500). In 2002 only the BMD of the hip was measured. In 2006 the BMD of both the hip and of the lumbar spine were determined. As reference group for the hip the NHANES III database (sex- and age-matched) was used. A standard protocol as described previously was used for measurement of BMD. To adjust for age and gender, Z-scores were used. To examine changes in Z-scores between baseline and follow-up measurements a \u0394 Z-score was calculated reflecting the difference between the Z-score at follow-up and the Z-score at baseline.\nFurthermore, after bone density measurement a lateral single energy densitometry of the thoracic and lumbar spine for vertebral fracture assessment (VFA) was performed (also called Morphometric X-ray absorptiometry (MXA)) [16]. The scans obtained were analyzed twice by one trained operator (intra-observer correlation: 0.85), using the semi-quantitative method of Genant [17]. In addition we measured every vertebra quantitatively. The observer was blinded to the T-score values and to the values of the first set of measurements. After visual examination six points were placed on each vertebral body from T4 to L4. From these points three vertebral heights were measured anterior (Ha), mid (Hm) and posterior (Hp). On the basis of the average score of these morphometric measurements, ratios were calculated and a prevalent vertebral deformity was defined as a reduction of height of 20% or more (Ha\/Hp; Hm\/Hp and Hp\/Hp below). Severity of deformities was assessed using the scoring system of Genant [17]. A score of \u20180\u2019 was assigned to normal, non-fractured vertebra; \u20181\u2019 for a mild deformity (20\u201325% reduction in anterior, middle or posterior vertebral height); \u20182\u2019 for a moderate deformity (25\u201340% reduction) and \u20183\u2019 for a severe deformity (>40% reduction). A new vertebral deformity was scored if a normal vertebra (grade 0) became deformed (grade \u2265 1) and a progressive deformity if the grade increased [17].\nQuestionnaires\nCalcium intake of all patients was scored in 2002 as well as 2006 on the basis of a detailed dietary list. Known clinical risk factors for osteoporosis (weight below 60\u00a0kg, mother with hip fracture, history of fractures after age 50, menopausal status and severe immobilization) as well as daily activities and exercise were assessed by a validated questionnaire [18], in which sports, daily and work activities are scored with a minimum of zero and a maximum of eighteen. GC therapy was evaluated by means of a patient questionnaire and verified using all the records of the patient\u2019s pharmacist. It was scored as never, previous or current use and if subjects were currently using GCs, the daily dose was noted.\nStatistics\nStudent t-tests, chi-square tests, and one-way ANOVAs were used, depending on the variables and subgroups tested. Depending on the analysis, change scores or actual scores were used. Patients with new and\/or progressive vertebral deformity were clustered for the multivariate and the receiver-operating characteristics (ROC) analysis. Multivariate logistic regression analyses was performed to assess the strength of association between the incidence of new and\/or progressive vertebral deformities and gender, age, weight, clinical risk factors, GC use, lifetime GC dose, daily GC dose, disease activity, bone markers, calcium intake, physical activity and BMD measurements. The variables that were entered in the multivariable analysis were those variables that appeared related (p\u2009<\u20090.10) to this outcome measure in univariate analyses. Odds ratio (OR) and its 95% confidence intervals (CI) were calculated by using SPSS version 12.0. ROC analysis was used to assess the ability of various levels of the T-score femoral neck to predict the incidence of a new and\/or progressive vertebral deformity. The ROC curve indicates the probability of a true-positive result as a function of the probability of a false-positive result for all possible threshold values [19]. A p value < 0.05 was considered statistically significant.\nResults\nBone mineral density and bone turnover parameters\nThe results of BMD measurements are shown in Table\u00a02. The BMD of the total group remained unchanged after a median follow-up of 45 months (range 35\u201349 months). When stratifying patients according to GC use, no decrease in each of the subgroups was found. Patients that never used GCs showed a \u0394 Z-score of the femoral neck (FN) of 0.03\u2009\u00b1\u20090.36 and a \u0394 Z-score of the trochanter of \u22120.08\u2009\u00b1\u20090.37. In patients with previous use of GCs these \u0394 Z-scores were 0.10\u2009\u00b1\u20090.36 and 0.22\u2009\u00b1\u20090.43, respectively. Even the group currently on GCs revealed no decrease of Z-score (\u0394 Z-score FN: 0.06\u2009\u00b1\u20090.30 and \u0394 Z-score trochanter: 0.00\u2009\u00b1\u20090.18) and also the subgroup of postmenopausal women (n\u2009=\u200911) did not show significant bone loss (\u0394 Z-score FN: 0.06\u2009\u00b1\u20090.45 and \u0394 Z-score trochanter: \u22120.05\u2009\u00b1\u20090.52). In the total group, bone turnover parameters at baseline showed an increased Z-score of ICTP compared to norm scores (0.7, 95% confidence interval (CI):0.4\u20131.1; p\u2009<\u20090.001). on the other hand, the marker of bone formation (Z-score PINP) did not differ from the reference population.\nTable\u00a02BMD variables at baseline and follow-up for the total group (n\u2009=\u200966, median follow-up duration 45 months (range 35\u201349 months)VariableBaseline measurementFollow-up measurementP*BMD, mean \u00b1 SD gm\/cm2Femoral neck0.84\u2009\u00b1\u20090.120.83\u2009\u00b1\u20090.12nsTrochanter0.74\u2009\u00b1\u20090.130.74\u2009\u00b1\u20090.12nsTotal hip0.97\u2009\u00b1\u20090.130.98\u2009\u00b1\u20090.14nsLumbar spine1.04\u2009\u00b1\u20090.14Z-score, mean \u00b1 SDFemoral neck0.17\u2009\u00b1\u20091.00.23\u2009\u00b1\u20091.1nsTrochanter0.27\u2009\u00b1\u20091.10.32\u2009\u00b1\u20091.1nsTotal hip0.18\u2009\u00b1\u20091.00.32\u2009\u00b1\u20091.00.001Lumbar spine0.03\u2009\u00b1\u20091.4T-score, mean \u00b1 SDFemoral neck\u22120.42\u2009\u00b1\u20091.0\u22120.46\u2009\u00b1\u20091.0nsTrochanter\u22120.02\u2009\u00b1\u20091.1\u22120.03\u2009\u00b1\u20091.0nsTotal hip\u22120.14\u2009\u00b1\u20091.0\u22120.10\u2009\u00b1\u20091.0nsLumbar spine\u22120.50\u2009\u00b1\u20091.3Abbreviations: BMD, bone mineral densityP*\u2009=\u2009p value between baseline and follow-up measurement\nClinical fractures and vertebral deformities\nThree new non-vertebral fractures occurred during the follow-up period. These included a hip fracture (twice in the same patient), an ankle fracture and a fracture of the thumb. All these fractures were related to trauma and occurred in subjects older than 50 years.\nMorphometric data are summarized in Table\u00a03. In 2002 vertebral deformities (ratio of <0.80) were found in 19 vertebrae of 13 subjects. Seventeen of these were wedge and two biconcave deformities. No crush deformities were seen. The majority of these deformities were found in the low thoracic region. At follow-up a new vertebral deformity was scored if a normal vertebra (grade 0) became deformed (grade \u2265 1) and a progressive deformity if the grade increased [17]. With this method, 36 vertebral deformities were found in 21 subjects. In one subject a vertebral deformity (ratio 0.78 of T11) found in 2002 was not found at follow-up (ratio 0.81). So, in total nine new subjects revealed one or more vertebral deformities, which means an increase of vertebral deformities from 20 to 32% of the subjects studied (p\u2009<\u20090.05). From the 21 subjects with a vertebral deformity in 2006, 17 subjects (26% of total group) were diagnosed with one or more new or progressive vertebral deformities and in four subjects the deformity was unchanged compared to baseline. Data on number and severity of the deformities can be found in Table\u00a03. Six patients were started on a bisphosphonates after baseline measurement and from these six patients, two had a new or progressive vertebral deformity at follow-up.\nTable\u00a03Number and grade of deformitiesBaselineFollow-upNo. of subjects with deformity13 (20%)21 (32%)*No. of deformities Mild1728 Moderate28 Severe00Total1936*P\u2009<\u20090.05 between number of subjects with deformity at baseline and follow-up\nComparing the groups with and without new or progressive vertebral deformities at follow-up, no differences in \u0394 Z-scores of BMD of the trochanter or femoral neck (FN) were found (\u0394 Z-score trochanter \u22120.02\u2009\u00b1\u20090.41 and 0.08\u2009\u00b1\u20090.38 respectively and \u0394 Z-score FN 0.01\u2009\u00b1\u20090.32 and 0.08\u2009\u00b1\u20090.35). In addition no differences in baseline Z-scores of ICTP and PINP were seen between these groups. Multivariable logistic regression analysis, including factors that correlated in the univariate analysis, revealed that a T-score of the femoral neck at baseline (OR per 1 SD T-score reduction\u2009=\u20092.5 (CI: 1.0\u20135.9), p\u2009=\u20090.04), and a mother with a hip fracture (OR\u2009=\u200914.1 (CI:1.4\u2013142,6), p\u2009=\u20090.02) were determinants of a new and\/or progressive morphometric vertebral deformity at follow-up measurement. Factors such as age, gender, calcium in take, GC use, daily GC dose, lifetime GC dose, disease activity, bone markers, radiographic stage and disease duration at baseline did not predict new and\/or progressive vertebral deformities.\nThe threshold level of the T-score FN that maximized the combined specificity and sensitivity on the ROC curve (Fig.\u00a01) was < \u22120.45 for predicting a new and\/or progressive deformity (sensitivity 88%, specificity 51%).\nFig.\u00a01ROC curve using Femoral neck T-score to identify patients with new and\/or progressive vertebral deformity. Area under the curve (AUC): 0.72. Arrow: The threshold level of T-score FN that maximized combined specificity and sensitivity was < \u22120.45 (sensitivity 88%, specificity 51%)\nDiscussion\nIn this cohort of subjects with sarcoidosis, a high prevalence of morphometric vertebral deformities suggestive of fracture was found, as well as a substantial increase in vertebral deformities during a follow-up period of four years. In 2002 20% of subjects were diagnosed with vertebral deformities according to the criteria of Genant [17], which increased to 32% of all subjects in 2006. In parallel, the total number of deformities in these subjects almost doubled. However, BMD of the trochanter and femoral neck did not change over time and BMD of the lumbar spine at follow-up measurement did not differ from the reference population. These data are suggestive of an increased risk of progressive vertebral deformities in individuals with sarcoidosis despite preservation of BMD.\nAlthough data on prevalent or incident fractures in younger healthy populations are lacking, data from other studies suggest that the incidence and prevalence of vertebral deformities in this population are indeed high. Prevalence rates of 30% asymptomatic vertebral fractures are demonstrated in elderly post-menopausal women on chronic GC therapy using the same techniques [20]. In a previous study in 60 subjects (mean age 49\u2009\u00b1\u200913 years) with differentiated thyroid carcinoma we found vertebral deformities in 7% of patients [21]. Data from the European Vertebral Osteoporosis Study (EVOS), a very large cross-sectional population based study on European subjects aged 50 to 79 years, showed a prevalence of vertebral deformities of 12% (range 6\u201321%) in males and females [22]. In the Rotterdam study, in which 3469 men and women aged 55 years and older were studied, the prevalence of vertebral deformity suggestive of fracture was 6.9% in men and 7.5% in women [23]. The epidemiology of vertebral fractures in women aged 50\u201354 years turned out to vary in different countries from 4.7% \u2013 11.5% [24]. All these studies indicate that the fracture risk in subjects with sarcoidosis is substantial, regardless the differences in populations studied and differences in methodology.\nA new vertebral deformity was found in 15 subjects (23%). To identify incident deformities several approaches can be followed. Measurement of changes in vertebral heights of the same vertebral body from a baseline to a later radiograph in which a decrease in height of 15 or 20% or 4\u00a0mm is suggestive of fracture [25, 26], changes in indices of vertebral area [27] or changes in the number or presence of prevalent deformities [17, 28]. Black and coworkers evaluated these different approaches and concluded that none of these were consistently better than any other method [29]. As we aimed to assess the change of numbers of subjects with one or more vertebral deformities over time we used the last method [17], in which changes in number of prevalent deformities are scored. A comparable approach was followed in the European Prospective Osteoporosis Study (EPOS) [30], which revealed an incidence of new deformities of 3.4% after a similar follow-up period. As the mean age of subjects included in this study was substantially higher than that of our cohort, these data cannot be used as a reference, although it is likely that in younger age groups even lower incident deformities would be observed. The high prevalence of vertebral deformities at baseline, the significant increase of more than 50% of subjects after follow-up with one or more deformity and the increase of severity of prevalent deformities all imply that sarcoidosis is a relevant risk factor for vertebral deformity.\nWhat is the underlying mechanism of this predisposition to vertebral deformities in view of the lack of effects on BMD in sarcoidosis? The load bearing capacity of bone, also referred to as \u2018whole bone strength\u2019, depends on the amount of bone, the spatial distribution of the bone mass, and the intrinsic properties of the materials that comprise the bone. Thus, properties at the cellular, matrix, micro- and macro-architectural levels may all impact the mechanical properties of bone [31, 32]. Apparently, in sarcoidosis mechanisms are involved that influence bone strength without having a significant impact on bone mass. As we found in the total group an increased marker of bone resorption (Z-score ICTP) at baseline, one of the possible mechanisms could be increased bone remodelling with a negative effect on bone micro-architecture that is not reflected by a change in BMD. It is well known that chronic inflammatory diseases influence bone physiology by the production of cytokines stimulating bone turnover [1, 2, 33]. Increased bone remodelling is associated with an increased bone fragility and thus fracture risk [34, 35] and in postmenopausal women the level of bone turnover turned out to be an as strong and independent predictor of fractures as BMD [36, 37]. These data may support the hypothesis that the chronic inflammatory state in sarcoidosis results in increased bone remodelling with a negative effect on bone strength and thus an increased fracture risk.\nNo changes in BMD in the group currently treated with GCs were found. This is unexpected as GCs are known to effect BMD via several mechanisms with consequent decrease of BMD. It may well be that this is due to intermittent GC use, as most of our patients were on intermittent glucocorticoids. Other studies have demonstrated that intermittent GC use has no major effects on BMD [38]. A recent large retrospective cohort study on clinical fracture risk among patients from the UK General Practice Research Database showed that intermittent use of high dose of oral GCs was associated with only a small increase in the risk of osteoporotic fractures [39].\nDespite the on average normal BMD, we found T-score of the femoral neck and a family history of hip fractures to be predictors of a new and\/or progressive vertebral deformity. This suggests that the combination of a lower BMD in combination with the increased bone turnover in sarcoidosis predisposes to progressive vertebral deformity. If so, this would mean that in these high risk individuals preventive treatment should be considered to reduce fracture risk. Controlled trials are needed, however, to substantiate this suggestion.\nOne of the limitations of our study is the lack of an age and sex matched control population. Unfortunately data on vertebral deformities in younger populations are at present not available. The aim of this study was, however, not to compare sarcoidosis patients with healthy subjects, but to follow a cohort of these patients and to compare follow-up with baseline measurements. Another limitation is the use of morphometric X-ray absorptiometry (MXA) instead of spine radiographs. MXA is less reliable for the detection of deformities at the upper thoracic spine, where deformities are less frequent as compared to the lumbar and mid-thoracic spine. A recent study comparing MXA with lateral spine X-ray found that vertebral morphometry using MXA allowed diagnosis of vertebral fracture in the lumbar and mid thoracic spine, where vertebral fractures are most common [40]. The advantage of MXA is the low dose of radiation and the convenience of the technique for patients. The present quality of the images, with ongoing refinement of this technology, is considered sufficient to be used for the diagnosis of vertebral deformity consistent with fracture [41]. Furthermore there is a lack of a \u201cgold standard\u201d for VFA. We followed the method of Genant [17], which is based on a reduction of the ratios of anterior, middle or posterior heights and all measurements were performed twice to improve accuracy. This is the simplest and most practical method [42] and an association with future fracture risk is documented [43, 44]. The above mentioned EVOS study, however, applied the methodology described by McCloskey and Eastell and co-workers in which measurements are corrected for normal variations in vertebral shape [25]. Relative to the method of Genant, the method of Eastell [25] or McCloskey [28] may have resulted in lower prevalences of vertebral deformities. This does not, however, explain the differences in prevalence of vertebral deformities reported elsewhere and in this paper. The restrictions of the methodology are also the limited ability to provide a differential diagnosis for the detected deformities, a lower sensitivity for milder fractures and the inability to evaluate the uppermost thoracic levels. Other disorders that may cause changes in vertebral shape involve congenital abnormalities and conditions as severe osteoarthritis [45] and Scheuermann\u2019s disease. We have, however, no indications that these relatively rare conditions may have interfered with our observations.\nIn conclusion, we have shown that in subjects with sarcoidosis the number of vertebral deformities, diagnosed with morphometric assessment, increases during the course of this disease despite preservation of BMD. Although this is an uncontrolled study, it appears that subjects with sarcoidosis have an increased fracture risk, even if BMD is normal. High risk individuals can be identified by a low-normal BMD and by a family history of hip fractures. Probably these individuals will benefit from therapies that increase bone strength. A T-score FN below \u22120.45 may be used to identify these individuals with a high sensitivity and an acceptable specificity. Studies evaluating the effects of such therapies in individuals with sarcoidosis are however clearly needed.","keyphrases":["vertebral deformities","bone mineral density","sarcoidosis"],"prmu":["P","P","P"]}
{"id":"Mol_Genet_Genomics-4-1-2270915","title":"Characteristic expression of twelve rice PR1 family genes in response to pathogen infection, wounding, and defense-related signal compounds (121\/180)\n","text":"Pathogenesis-related (PR) proteins have been used as markers of plant defense responses, and are classified into 17 families. However, precise information on the majority members in specific PR families is still limited. We were interested in the individual characteristics of rice PR1 family genes, and selected 12 putatively active genes using rice genome databases for expressed genes. All were upregulated upon compatible and\/or incompatible rice-blast fungus interactions; three were upregulated in the early infection period and four in the late infection period. Upon compatible rice\u2013bacterial blight interaction, four genes were upregulated, six were not affected, and one was downregulated. These results are in striking contrast to those among 22 ArabidopsisPR1 genes where only one gene was pathogen-inducible. The responses of individual genes to salicylic acid, jasmonic acid, and ethylene induced defense signaling pathways in rice are likely to be different from those in dicot plants. Transcript levels in healthy leaves, roots, and flowers varied according to each gene. Analysis of the partially overlapping expression patterns of rice PR1 genes in healthy tissues and in response to pathogens and other stresses would be useful to understand their possible functions and for use as characteristic markers for defense-related studies in rice.\nIntroduction\nPathogenesis-related (PR) proteins, which are classified into 17 families, accumulate after pathogen infection or related situations in many plant species (van Loon et al. 2006). Among the PR gene family, PR1 genes have been frequently used as marker genes for systemic acquired resistance in many plant species. However, only a small proportion of PR1 genes have been studied among the many members in this family, and information on the other members is limited. For example, NtPR1a, b, and c genes for acidic proteins, and NtPRB1 (PR-1g) and NtPRB1b genes for basic proteins were reported in tobacco (Nicotiana tabacum) (van Loon and van Strien 1999), but little information is known on other PR1 members. To understand the characteristics and redundancy of the majority of PR1 family members, genome-based studies are necessary. For such studies, dicot Arabidopsis and monocot rice (Oryza sativa L.) plants have been used as the model plants.\nIn Arabidopsis, 22 genes are listed as predicted PR1 genes that encode homologous proteins to tobacco PR1a protein, which was first reported as an acidic protein in tobacco leaves infected with Tobacco mosaic virus (TMV) (van Loon et al. 2006). Among the 22 genes, only one PR1 gene (At2g14610), which encodes a basic protein, is known to be pathogen-responsive, and the other PR1 genes reportedly did not respond to either bacterial or fungal pathogens (van Loon et al. 2006). From these results, we tend to suppose that only one PR1 gene relates to pathogen resistance in Arabidopsis and the others contribute to other functions.\nIn rice, the induction of two PR1 genes, OsPR1a and 1b, by blast fungus infection was reported (Agrawal et al. 2001). They encode putative acidic and basic proteins, respectively, and also responded to environmental stresses and treatments with some chemicals (Agrawal et al. 2000a, b). However, for other rice PR1 gene family members, there is only limited information except for their presence and expression: (1) at least 4 signals for possible rice PR1 proteins responsive to anti-tobacco PR1a antibodies were found in an extract of blast fungus-infected rice leaves (Schweizer et al. 1997; Iwai et al. 2007), and (2) 32 predicted PR1 genes were proposed to be present in the rice genome (van Loon et al. 2006).\nTo study the response of individual rice PR1 genes to pathogens, we selected active rice PR1 genes from the rice genome databases for expressed genes, and studied their induced expression by real time RT-PCR (qPCR). In striking contrast with the result in Arabidopsis, all 12 rice genes selected here were upregulated by blast fungus infection. The levels of constitutive expression in organs and induced expression by different treatments varied according to the gene. In addition to the data on tissue-specific PR1 expression in transgenic rice with a OsPR1 promoter::\u03b2-glucuronidase (GUS) fusion gene, we list the characteristics of the 12 rice PR1 genes. This is the first example of a comparison of the expression of the majority of members of a monocot PR family to our knowledge. This information will be useful for further studies on PR genes and on resistance mechanism in rice plants.\nMaterials and methods\nPlant materials\nWild-type (WT) rice (Oryza sativa cv. Nipponbare) and the near isogenic line IL7 (Ise and Horisue 1988), which contains the R gene Pi-i against blast fungus (Magnaporthe grisea) race 003 (Yamada et al. 1976), were grown for 14\u201316\u00a0days in soil (Bonsol No. 1, Sumitomo Chemicals L., Japan) in a greenhouse at 28\u00b0C in the day time and 25\u00b0C at night. The fourth leaves of plants at the 4-leaf stage were mainly used as the experimental material. The seeds of transformants with OsPR1::GUS were germinated on agar medium containing 30\u00a0\u03bcg\u00a0ml\u22121 hygromycin, transferred to soil at 7\u00a0days after imbibition, and grown in the greenhouse. Five-day-old seedlings on agar medium and 2-month-old plants in the greenhouse were used for GUS-staining assays.\nInfection with pathogens\nMagneporthe grisea race 003 (isolate Kyu-89-241) was grown on oat-meal medium (Difco) for 2\u00a0weeks and conidia were induced under BLB light (FL20S BLB, Toshiba) for 2\u00a0days at 25\u00b0C. The rice seedlings of Nipponbare and IL7 plants at the 4-leaf stage were spray-inoculated with a conidia suspension (1\u00a0\u00d7\u00a0105 conidia ml\u22121) containing 0.05% Tween 20, and the inoculated plants were incubated under high humidity in the dark for 20\u00a0h, and then moved to the greenhouse. Under these conditions, about 100 local lesions were induced per leaf on Nipponbare and IL7. For bacterial blight infection, Nipponbare plants, which are compatible with Xanthomonas oryzae pv. oryzae (Xoo) strain T7174 (race I, MAFF 311018), were inoculated by cutting the leaf top with scissors that had been dipped in a suspension containing 108 cfu\/ml of Xoo, and incubated in the greenhouse.\nTreatment with chemicals\nPlastic pots (15\u00a0\u00d7\u00a05.5\u00a0\u00d7\u00a010\u00a0cm) with 12 rice seedlings at the 4-leaf stage, were dipped in 500\u00a0ml solutions containing 1\u00a0mM 1-aminocyclopropane-1-carboxylic acid (ACC) or 3\u00a0mM sodium salicylate (SA) solution, and incubated for 24\u00a0h in the greenhouse. For jasmonic acid (JA) treatment, pots with 12 seedlings each was put in an air-tight clear plastic box, and a cotton pad with volatile methyl jasmonate (MeJA) dissolved in EtOH was put at the corner of the box to give a final concentration of 100\u00a0\u03bcM, and incubated for 24\u00a0h. The fourth leaves were used for RNA extraction.\nQuantitative real-time RT-PCR\nTo analyze the response of OsPR1 genes to biotic and abiotic stresses, quantitative real-time RT-PCR (qPCR) was conducted using iQ SYBR Green Supermix (BioRad, Hercules, CA, USA) and an iCycler (BioRad) according to the manufacturers\u2019 instructions. At least three independent biological samples were used with specific primers for each individual gene (Table\u00a02). The data were normalized by the value of an actin gene (AK060893), and fold change in the expression level was calculated compared with that of healthy fourth leaves, and standard deviation (SD) values are shown.\nConstruction of plasmids for transformation of rice\nThe promoter region of the OsPR1.1 gene (OsPR1#074 in this work) was obtained through PCR amplification from the Nipponbare genome, using a GenomeWalkerTM kit (Invitrogen, CA, USA) in accordance with the manufacturer\u2019s instructions (Accession no. AP008213, Kawahigashi et al. 2007). The amplified fragment was digested by NotI and BamHI and inserted into the pTH2 vector to construct the PR1T::GUS plasmid (Fig.\u00a07a). The PR1T::GUS plasmid contained 1,919\u00a0bp of the promoter and the coding region for 16 amino acids at the N-terminus of the OsPR1 gene to express a fused GUS protein effectively (Kawahigashi et al. 2007).\nTransformation of rice\nAgrobacterium tumefaciens LB4404 was transformed with the constructed vector (Kawahigashi et al. 2007). The transformation of rice was performed by Agrobacterium infection as described by Toki et al. (2006).\nHistological GUS analysis\nHistological analysis for GUS activity was performed at 37\u00b0C essentially as described by Ohshima et al (1990) using a modified reaction mixture: 50\u00a0mM phosphate buffer (pH 7.0) containing 1\u00a0mM 5-bromo-4-chloro-3-indolyl glucuronide (X-gluc), 5% methanol, 10\u00a0\u03bcg\/ml cycloheximide, and 1\u00a0mM dithiothreitol. The reaction was stopped by the addition of ethanol.\nResults\nCharacterization of the rice PR1 gene family\nUsing the amino acid sequence of the mature tobacco acidic PR1a protein (NtPR1a; 138 amino acids, Ohshima et al. 1987) as the probe, we searched for expressed genes in rice (Oryza sativa cv. Nipponbare) encoding homologous proteins with a similar size range (within 150\u2013200 amino acids) using a full-length cDNA database (http:\/\/www.cdna01.dna.affrc.go.jp\/cDNA) and rice EST database (http:\/\/www.riceblast.dna.affrc.go.jp\/). The presence and location of these genes were confirmed in the genome database of rice (http:\/\/www.rapdb.dna.affrc.go.jp\/). After removing repetitive clones and confirmation of the sequences, finally 12 genes were selected as candidates for active rice PR1 genes, which encode proteins with sequences \u226541% identical to that of tobacco PR1a protein at least in the C-terminal 65 amino acids. Previously, two rice PR1 genes were reported to encode an acidic PR1a protein and a basic PR1b protein (Agrawal et al. 2000a), which were upregulated by blast fungus infection. In addition to the two, we found another 10 active genes on chromosomes 1, 2, 5, 7, 10, and 12 (Table\u00a01). Proposed names were given to distinguish each gene to avoid confusion in this study according to the location on the chromosomes. For example, OsPR1b (AK107926, Os01g28450), located on the 5\u2032 side of the gene for AK121108 (Os01g28500) on chromosome 1, was designated as OsPR1#011 and AK121108 was OsPR1#012. On chromosome 7, four PR1-like genes, OsPR1#071, #072, #073, and #074, were clustered in tandem from the 5\u2032 side to the 3\u2032 side in the sense orientation in this order. The predicted isoelectric point (pI) of each mature protein is shown in Table\u00a01 for reference. The amino acid sequences of the predicted mature proteins for the 12 OsPR1 genes were compared with those of representative acidic and basic PR1 proteins from Arabidopsis (AtPR1-like and AtPRB1) and tobacco (NtPR1a and NtPRB1b), respectively (Fig.\u00a01a). The sequences of the 12 OsPR1s, 2 AtPR1s, and 2 NtPR1s listed here are well conserved. As the N-termini of OsPR1#051, #052, and #121 are not conserved, these regions were eliminated for the alignment in Fig.\u00a01a. Ten amino acid residues (closed circles) are completely conserved among the 12 rice, 2 Arabidopsis, and 2 tobacco genes, indicating they are important for the predicted roles of PR1 proteins.\nTable\u00a01Classification of rice PR1 protein genesPlantAccession No. (Symbol name)Locus nameReferencesPIProposed symbolsRiceAK107926 (OsPR1b)Os01g28450Agrawal et al. (2000b)7.6OsPR1#011AK121108Os01g28500\u20138.7OsPR1#012AK107467Os02g54540\u20138.5OsPR1#021AK105575Os02g54560\u20135.9OsPR1#022AK071326Os05g51660\u20137.1OsPR1#051AK100748Os05g51680\u20135.5OsPR1#052AK060057Os07g03279\u20134.2OsPR1#071AK062949Os07g03580\u20134.7OsPR1#072AK063248Os07g03590\u20135.8OsPR1#073AU163470 (OsPR1a)Os07g03710Agrawal et al. (2000a)4.4OsPR1#074AU070895Os10g11500\u201310.7OsPR1#101AK100940Os12g43700\u20135.1OsPR1#121Arabidopsis(PR-1 like)At2g19990Metzler et al. (1991) 6.0(AtPRB1)At2g14580Santamaria et al. (2001)8.8TobaccoX06361 (NtPR1a) (X12737, X05959, X06930)Ohshima et al. (1987)4.4X66942 (NtPRB1b)Eyal et al. (1992)7.6Fig.\u00a01Characterization of the PR1 gene family in rice plants. a Amino acid sequence alignment of 12 putative PR1 proteins from rice. The predicted mature proteins encoded by these genes were aligned by GENETICS 9.0 (Software Development Co., Tokyo). Because the N-termini of OsPR1#052, #051, and #121 are considerably different from those of the other 9 OsPR1s, these regions were eliminated from the figure and the conserved C-termini among the 12 OsPR1s were aligned. The acidic and basic PR1 proteins from Arabidopsis (AtPR1-like and AtPRB1) and tobacco (NtPR1a and NtPRB1b) were used as representatives of acidic and basic PR1 proteins, respectively. The conserved amino acid residues among the 16 proteins are marked with black circles. Identical amino acids are boxed. b Phylogenetic analysis of rice PR1 proteins based on the mature proteins using UPGMA method. The predicted isoelectric point (pI) for each mature protein is shown on the right. Acidic, basic, and neutral proteins are shown in red, blue, and green, respectively. Refer to the description in a for details\nUsing the data of Fig.\u00a01a, a phylogenetic tree was constructed (Fig.\u00a01b). Among the 12 OsPR1 proteins, 7 acidic and 4 basic protein members were found (Fig.\u00a01b). The C-terminal region of OsPR1#051 was indicated to be neutral. The PR1 proteins from Arabidopsis and tobacco grouped in one clade (framed), and OsPR1 proteins belonged to different clades.\nResponses of OsPR1 genes to blast-fungus infection in young rice plants\nCompatible interaction\nThe response of individual OsPR1 genes to blast-fungus infection was analyzed by qPCR using a set of primers specific for each gene (Table\u00a02). A conidia suspension of M. grisea race 003 was sprayed on the seedlings of Nipponbare at the four-leaf stage. In this system, the fungus infection results in the formation of whitish expanding lesions at 3\u00a0days post inoculation (dpi), and increasing lesion size thereafter with conidia formation. The infected plants were severely wilted and died within 7\u20139\u00a0days indicating a typical compatible host\u2013parasite interaction (Sasaki et al. 2004; Iwai et al. 2006). Using triplicate cDNA samples originating from independently prepared RNA templates from inoculated fourth leaves, the expression levels of each gene were compared with that of an actin gene, which was selected as a control gene. Although expression levels of OsPR1 genes in healthy Nipponbare leaves varied according to each gene (0\u00a0dpi, white column in Fig.\u00a02a), all OsPR1 genes tested were up-regulated by blast-fungus inoculation at 3 and\/or 6\u00a0dpi (black column). When compared with mock-inoculation (gray column), the enhanced expression by the fungus was significant for each gene at 6 dpi, at which lesions were vigorously expanding. The transcripts of OsPR1#074, #011, and #012 had already accumulated to near maximal levels at 3\u00a0dpi maintaining almost the same levels at 6\u00a0dpi, indicating they are rapid response genes to blast fungus infection. However, the transcripts of OsPR1#073, #022, #101, #051, and #121 had not increased significantly at 3\u00a0dpi compared with mock-inoculated controls, indicating these are late response genes. Blast fungus infection induced around 1,000-fold transcript accumulation at 6\u00a0dpi compared with 0\u00a0dpi in 7 genes: OsPR1#074, #011, #071, #073, #052, #072, #101, and #121.\nTable\u00a02Primers for qPCR of OsPR1s and actin genesNameForward primerReverse primerOsPR1#074GTATGCTATGCTACGTGTTTATGCGCAAATACGGCTGACAGTACAGOsPR1#011ACGCCTTCACGGTCCATACAAACAGAAAGAAACAGAGGGAGTACOsPR1#071CTTTAACTATGTATGGAGTATGATATAAATGTGTTATTTTCTTCTTTTATTCGAACGACAACOsPR1#012CGCTGTGTGTTTGTGTTATGTCCGTGGTTTTGTCTTTATTTCAATCCOsPR1#073TTATATATGTATGTTCGTATGTATGTATGCTGATGTACTTATTCCATCCGACACOsPR1#021CGCAGCAACCAACCAATCTTGACAGTTGTAGTACTCTTGTAACATCATCOsPR1#022CCACAGAGTTTGTCAGGATTGTCCAGATTGCACACACCTGATTCCOsPR1#052AGCTACCTGTCATTTCTTCATTTCTGCTACTCCAGAAGGAAATTAAAAGOsPR1#072AATTAATACTGGAGTAGATGCATGTACACGAATAACGTACTGTATTCTGTATGOsPR1#121ACCATCGTCGTCGTCTCATCAGCCTCTAGGGCATATCACTAACOsPR1#101TCGCTGCCGCTAGTACATTTCATTAAGATCATTACATGCTTTATTGTTCACOsPR1#051CCTGCCTGCCTTCCTCATTCAGTGAAGATTTGGTTTCCATTGTATTGActinGAGTATGATGAGTCGGGTCCAGACACCAACAATCCCAAACAGAGFig.\u00a02Response of OsPR1 genes to pathogen infection. a Blast fungus infection. A conidia suspension of blast fungus race 003 was spray-inoculated onto Nipponbare (compatible, upper panel), and IL7 (incompatible, lower panel). The inoculated fourth leaves were harvested at 0, 3, and 6\u00a0days post inoculation (dpi) for RNA extraction. To semi-quantify the transcript level, RNA samples were subjected to qPCR, and the relative expression levels compared with that of a control actin gene are shown. The transcript levels at time 0, mock-inoculated, and blast-fungus inoculated leaves were shown as white, gray, and black columns, respectively. Bars indicate mean\u00a0\u00b1\u00a0SD. The up-regulated genes by blast fungus-infection are marked with arrowheads. Gray arrowheads in the lower panel indicate early responsive genes. b Bacterial blight infection. The fourth leaves of Nipponbare rice plants were inoculated with a suspension of Xoo for bacterial blight disease, and the leaves were harvested at 0, 4, and 8 dpi for RNA extraction. The transcript level was analyzed by qPCR. The relative expression levels compared with that of the control actin gene are shown. Transcript levels at time 0, mock-inoculated, and bacteria-blight inoculated leaves are shown as white, gray, and black columns, respectively. Xoo-upregulated and -downregulated genes are marked with forward and reverse arrowheads, respectively. Bars indicate mean\u00a0\u00b1\u00a0SD\nIncompatible interaction\nIL7 is a near isogenic line of Nipponbare, into which the resistance gene Pi-i for blast fungus race 003 has been introduced. In fungus-inoculated IL7, small HR lesions were induced at 2\u00a0dpi with no remarkable fungal development thereafter, indicating a typical incompatible interaction (Sasaki et al. 2004; Iwai et al. 2006). The transcript levels of OsPR1#074, #011, and #012 were maximized at 3\u00a0dpi, and slightly reduced at 6 dpi (Fig.\u00a02a, lower part, gray arrowheads), indicating they are early responsive genes to blast fungus infection. The 3 genes had been recognized as early responsive genes in compatible interaction. The levels of OsPR1#074 and #011 transcripts increased by about 1,000-fold at 3 dpi. The transcripts of OsPR1#071, #072, #073, and #101 were higher at at 6 dpi than at 3 dpi, indicating they are late responsive genes (black arrowheads). The level of OsPR1#121 was suppressed by mock-inoculation but the level was not changed by incompatible interaction. OsPR1#051, whose transcript increased in the compatible interaction at 6 dpi, was not clearly upregulated in the incompatible interaction.\nResponses of OsPR1 genes to bacterial blight infection in young rice plants\nTo study the response of the 12 OsPR1 genes to a bacterial pathogen, the fourth leaves of Nipponbare were inoculated with Xoo for leaf blight disease. In the compatible rice\u2013bacterium interaction, the infected region was visualized as wilted necrosis, which spread from the cross cut end to the leaf base with time, and the area of necrosis grew to 2\u20133\u00a0cm long at 8 dpi. At 4 and 8 dpi, 5\u00a0cm long leaf pieces from the inoculated point were used to prepare RNA samples. Transcripts of OsPR1#074, #011, and #012, which are early responsive genes to blast fungus (Fig.\u00a02a), accumulated remarkably at both 4 and 8 dpi compared with 0 dpi or a mock-inoculated control (Fig.\u00a02b), indicating they were responsive to Xoo infection (closed forward arrowheads). Expression of OsPR1#021 and #022 were slightly suppressed after the infection compared with mock-inoculated leaves (closed reverse arrowheads). Expression of OsPR1#074, #011, #012, and #101 were upregulated by Xoo infection but other OsPR1 genes did not respond or rather suppressed, while all OsPR1 genes were upregulated in compatible blast fungus interaction (Fig.\u00a02a).\nWound-induced expression of OsPR1 genes in young rice plants\nTo study the response of OsPR1 genes to wounding, the fourth leaves were wounded by cross cutting. Triplicate samples were extracted at 0, 1, 6, and 24\u00a0h after wounding, and used for qPCR. Among the 12 genes, OsPR1#074 was wound-inducible with peak accumulation of the transcript at 6\u00a0h after wounding, and OsPR1#051 was found to be an early wound-responsive gene (Fig.\u00a03, forward arrowheads). The OsPR1#121 and #052 transcripts decreased in response to wounding (reverse arrowheads) (Fig.\u00a03).\nFig.\u00a03Response of OsPR1 genes to wounding The fourth leaves were cut into 1\u00a0cm length sections for wounding, and floated on water for 0, 1, 6, and 24\u00a0h under light, and then the leaf pieces were homogenized for RNA extraction. Relative expression levels compared with that of the control actin gene are shown. The wound-upregulated and -downregulated genes are marked with forward or reverse arrowheads, respectively. Bars indicate mean\u00a0\u00b1\u00a0SDFig.\u00a04Organ-specific expression of OsPR1 genes Leaves and roots of the seedlings at the fourth leaf stage (left panel), and flowers of adult plants (right panel) were subjected to qPCR analysis. Relative expression levels compared with that of the control actin gene are shown. The genes that express at a high level in roots and flowers are marked with white and gray arrowheads, respectively. Bars indicate mean\u00a0\u00b1\u00a0SD\nOrgan specific expression of OsPR1 genes\nThe basal transcript levels in various organs varied according to each gene; in the roots of healthy young rice plants at the 4-leaf stage, the transcript levels of the 5 genes, OsPR1#074, #071, #073, #072, and #101, were clearly higher than in the fourth leaves (Fig. 4) (open arrowheads). In flowers, the basal transcript levels of five genes, OsPR1#074, #011, #012, #121, and #051, were at a high level (gray arrowheads). These results indicate that the organ-specific expression profiles of OsPR1 genes varied considerably. The OsPR1#074, #071, #073, and #072, which are located on chromosome 7, were highly expressed in healthy roots. In healthy flowers the expression levels of five genes, OsPR#074, #011, #012, #121, and #051, were also higher.\nResponses of OsPR1 genes to defense signal compounds in young rice plants\nThe responses of OsPR1 genes to defense signal compounds were studied. Intact young rice plants in soil pots were dipped in water, SA, or ACC solutions, or exposed to the vapor of MeJA. RNA from the fourth leaves was subjected to qPCR (Fig.\u00a05). OsPR1#074 and #101 were upregulated by all three signal compounds tested, while OsPR1#071, #073, #021 and #121 were upregulated by JA but not by ACC or SA. OsPR1#074, #011, and #012, which were early responsive genes to blast fungus (Fig.\u00a02), were SA-inducible. OsPR1#011 was upregulated by SA but suppressed by ACC. Among the six genes that were inducible by the wound signal compound JA, OsPR1#074 and #051 were also upregulated by wounding (Fig.\u00a03). OsPR1#022 and #052 were not particularly sensitive to any of these compounds. OsPR1#011 and #072 were downregulated by ACC and SA respectively, and OsPR1#071 was downregulated by both ACC and SA.\nFig.\u00a05Response of OsPR1 genes to defense-signal compounds ACC, SA, and JA. Expression levels of each OsPR1 gene in fourth leaves at 24\u00a0h after treatment with defense-signal compounds were analyzed by qPCR. Relative expression levels compared with that of the control actin gene are shown. The ACC-, SA-, and JA-inducible genes are marked with gray, dark gray, and black arrowheads, respectively. For ACC or SA treatment, plastic pots with rice seedlings were dipped in solutions containing 1\u00a0mM ACC or 3\u00a0mM SA solution, and incubated for 24\u00a0h in the greenhouse. C1 control for ACC- and SA-treatment (leaves of water treated plant); For JA treatment, pots with seedlings each was put in an air-tight clear plastic box, and a cotton pad with volatile methyl jasmonate (MeJA) dissolved in EtOH was put at the corner of the box to give a final concentration of 100\u00a0\u03bcM, and incubated for 24\u00a0h. C2 control for JA-treatment (leaves of plant treated with 0\u00a0\u03bcM of JA), bars indicate mean\u00a0\u00b1\u00a0SD\nComparison of the defense-signaling pathways conferred by ACC, SA, and JA in rice with those in tobacco and Arabidopsis\nUsing the 12 OsPR1 genes as probes, we analyzed the relationship of responsiveness to three defense signal compounds in rice. In tobacco, SA signaling was antagonistic to JA signaling on PR gene expression: Niki et al. (1998) showed that the expression of acidic PR1, 2, and 3 genes was upregulated by SA-treatment and it was suppressed in the presence of JA in a dose-dependent manner, while the expression of basic PR2, 5, and 6 genes was upregulated by JA and suppressed by SA. Thomma et al. (1998) reported that the JA-signaling pathway is different from the SA-signaling pathway in Arabidopsis. Glazebrook et al. (2003) reported that the expression patterns of some genes revealed mutual inhibition between SA- and JA-dependent signaling using global expression phenotyping analysis of mutant Arabidopsis plants. Synergistic induction by ET and JA was reported in basic tobacco PR genes (Xu et al. 1994). However, the expression patterns of OsPR1 genes in Fig.\u00a05 showed no clear mutual inhibition between SA- and JA-dependent signaling, except for OsPR1#071. We further analyzed the relationship by means of Pearson\u2019s correlation coefficient (Fig.\u00a06). Correlation coefficients for SA versus ACC, ACC versus JA, and SA versus JA were 0.75, 0.61, and 0.22, respectively. The linear correlation coefficient, r, represents the strength and the direction of the linear relationship between two variables. A correlation of +1 means an increasing linear relationship and complete correlation, while \u22121 means a decreasing linear relationship. Cohen (1988) proposed that |r| below 0.1 is regarded as insubstantial, 0.1\u00a0\u2264\u00a0|r|\u00a0<\u00a00.3 as weak, 0.3\u00a0\u2264\u00a0|r|\u00a0<\u00a00.5 as medium, and 0.5\u00a0\u2264\u00a0|r|\u00a0<\u00a01.0 as strong correlation. While, levels of statistical significance at 5 and 1% (n\u00a0=\u00a012) are 0.576 and 0.708, respectively. The r values in Fig.\u00a06 suggest synergism between ACC versus SA and ACC versus JA, and low or no correlation between SA versus JA rather than antagonism.\nFig.\u00a06Relationship of the three signaling pathways in OsPR1 genes. Based on the data in Fig.\u00a05, the response of young rice plants to ACC- , SA-, or JA-treatments was compared with other treatments, and the correlation coefficients between the responses to pairs of signals were calculated. See the text for details\nTissue specific expression of a representative OsPR1 gene in rice plants\nAnalysis of the tissue specific expression of rice PR1 gene by means of histological studies has not been performed previously. To study the tissue specific expression profiles of a representative OsPR1 gene, we selected OsPR1#074 because it clearly responded to both compatible and incompatible fungal interactions, bacterial bright infection, wounding and the 3 defense signal compounds. The promoter sequence of OsPR1#074 was obtained by PCR amplification from the Nipponbare genome. The promoter sequence, containing 1,919\u00a0bp, was inserted into the pTH2 vector (Kawahigashi et al. 2007) to construct the plasmid PR1T::GUS, which contains the promoter and the coding region for 16 amino acids of the N-terminus of the OsPR1 protein to express the GUS protein effectively as a translational fusion (Fig.\u00a07a). The OsPR1T::GUS gene was introduced into rice plants (Oryza sativa cv. Nipponbare). Among 30 hygromycin-resistant plants regenerated, three independent lines were selected as representatives exhibiting a negligible level of GUS activity in healthy leaves and more than 30-fold higher levels after wounding or inoculation with a conidia suspension of M. grisea. The OsPR1T::GUS plants of the second or third generation were subjected to histological GUS analysis using X-gluc as the substrate.\nFig.\u00a07Histological analysis of OsPR1#074 gene expression in transgenic rice plants. a Schematic diagram showing the construct of the introduced gene. The promoter sequence of OsPR1#074 was used. b Localized GUS staining in healthy 5\u00a0day-old transgenic rice seedlings (a\u2013e). Because similar expression profiles were obtained in three independent transgenic lines, the data from a representative line is shown. f unhulled rice. c GUS staining of 2\u00a0month-old transgenic rice plants. a Healthy roots, b roots at 2\u00a0days after wounding, c\u2013e leaves at 2\u00a0days after wounding. d GUS staining assay of 4th leaves at 5 (a, c, d) or 11 (b) dpi. Black arrows indicate stained mesophyll tissue. Brown arrows indicate the necrotic regions\nFigure\u00a07B shows the localization of GUS activity in healthy 5-day-old transgenic rice seedlings. A high level of GUS activity was detected in the primary root (arrows, Fig.\u00a07B a\u2013c) and the root tip (arrowheads), but it was absent or considerably reduced in the elongation zone (white arrowheads). In cross sections of the middle part of the root 80\u00a0\u03bcm in thickness (Fig.\u00a07B e), a strong GUS activity was detected in the vascular cylinder (vc) and sclerenchyma (sc), and a weak activity in the cortex (co). GUS staining was found in the slit surface of the coleoptile (yellow arrowheads, Fig.\u00a07B a, b), and on the surface of unhulled rice (white arrow, Fig.\u00a07B a, f). In the anther, a weak blue staining was found in the matrix of pollen loculus (data not shown). In the vector control plants, no GUS activity was detected in roots (Fig.\u00a07B d) or any other part analyzed under these in condition (data not shown).\nIn 2\u00a0month-old OsPR1T::GUS plants, no GUS activity was found in healthy roots (Fig.\u00a07C a), while high levels of GUS activity were found in healthy 5\u00a0day-old (Fig.\u00a07Ba, b, and c) and 2\u00a0week-old (data not shown) plants. Thus, OsPR1 expression in roots is likely to be regulated developmentally. Wounding induced a high level of GUS activity in the roots of 2-month-old plants. When the aerial parts were removed by cross cut from of the plants, a strong GUS activity was induced in the roots of cut plants at 2\u00a0days after cutting (Fig.\u00a07C b). In cross sections of the root in the middle region, GUS staining was found in all tissues, including the vascular cylinder, cortex, and sclerenchyma. No detectable GUS activity was found in healthy leaves of transgenic rice plants (data not shown); however, wounding induced a strong GUS activity in the neighbor region of the cut surface especially in the vascular systems including xylem parenchyma (xy) and phloem (ph) (Fig.\u00a07C c\u2013e).\nNext, GUS activity in rice leaves infected with blast fungus was analyzed. In the compatible cultivar (WT) lacking the Pi-iresistance gene, enlarged lesions were found 3\u20135 days after inoculation developing in size with time and with conidia formation on the infected leaf surface. After GUS staining, many blue spots were found at or around the lesions in the leaves 5 days after inoculation (Fig. 7D a). When the leaf of Fig. 7D a was cross cut at the marked line, and shown in Fig. 7D c, the blue staining was found to\nlocalize in the mesophyll cells (black arrows) around the necrotic regions (brown arrows, Fig. 7D d). At 11 days after inoculation, GUS activity was found around the necrotic lesions (Fig. 7D b).\nDiscussion\nWe studied here the response of 12 OsPR1 genes to pathogens, wounding, and defense signal compounds and their constitutive expression in organs. This is the first example of a comparative study on the characterization of a set of PR 1 family genes by qPCR, a high throughput method. The 12 genes include PR1a and b, which have been reported as pathogen-inducible genes by Agrawal et al. (2001), and ten of the 12 genes are contained in the 32 listed rice PR1-like genes including at least 3 pseudogenes whose expression profiles were not obvious (van Loon et al. 2006). We searched rice PR1-like genes from full-length cDNA (http:\/\/www.cdna01.dna.affrc.go.jp\/cDNA) and EST (http:\/\/www.riceblast.dna.affrc.go.jp\/). Because strongly expressed genes should be easily extracted from the databases for expressed genes, a majority of highly expressed rice PR1-like genes are thought to be contained in the 12 genes. The sequences of the 12 OsPR1 proteins were well conserved with complete matches in 10 amino acid residues in the C-terminal half (Fig.\u00a01a). Because the 10 residues were also very well conserved in all 36 PR1-type proteins selected from 14 different plant species including Brassica napus, Hordeum vulgare, Lycopersicon esculentum, Medicago trunculata, Triticum aestivum, and Zea mays (van Loon and van Strien 1999), these regions in the C-terminus should be important for the putative functions of PR1 proteins in plants.\nAll 12 OsPR1 genes selected here were clearly upregulated in a compatible rice-blast fungus interaction, indicating that they really are pathogen related. Notable is that the induction pattern of each OsPR1 gene by different pathogen\u2013host interactions was characteristic (Fig.\u00a02). Four genes, OsPR1#074, #011, #012, and #101, responded positively to blast fungus infection in both compatible and incompatible interactions, and also to Xoo infection. Among these, OsPR1#074, #011, and #012 were responsive to blast fungus in the early infection period in both interactions. Conversely, the expression of OsPR1#021 and #022 was reduced by Xoo infection compared with the control after mock-inoculation, which is a wound treatment. Assuming that Xoo infection may inhibit wound-induced expression of these genes, it is consistent that they are wound-inducible (Fig.\u00a03). The above result that \u201call 12 OsPR1s are characteristically pathogen inducible\u201d is apparently different from the data of Arabidopsis where only one PR1 gene was pathogen-inducible (AT2g14610) among 22 predicted ArabidopsisPR1 genes (van Loon et al. 2006). We studied this result using an AtGenExpress Visualization Tool from http:\/\/www.arabidopsis.org\/, and confirmed that only the PR1 gene was upregulated by infection with either Pseudomonas syringae pv. tomato DC3000 or Pytophythora infestans, and the other PR1 genes were not. The reason for different responses of PR1s to pathogen infection in rice and Arabidopsis is not clear, but we could speculate that OsPR1s have evolved in the rice genome to provide resistance to a broad range of pathogens, but a limited set of ArabidopsisPR1s has evolved to respond to pathogens.\nIn rice, the level of free SA in young healthy rice leaves was as high as about 10\u00a0\u03bcg per g fresh leaf (Silverman et al. 1995), but it was only about 20\u00a0ng (Seo et al. 2007) or 100 \u2013 200\u00a0ng (Silverman et al. 1995) in tobacco leaves, and about 30\u00a0ng (Park et al. 2007) or 150\u00a0ng (our unpublished data) in Arabidopsis leaves. Thus, the SA content in rice leaves corresponds to 50- to 500-fold of that in tobacco or Arabidopsis leaves, which is comparable to the induced SA level after HR lesion formation by TMV-infection. Such a high SA level in rice plants could affect OsPR1 expression after pathogen infection, treatment with SA, JA, or ACC, and wounding. Actually, the responses of the OsPR1 genes to defense signal compounds indicate that the signaling pathways in rice conferred by SA and JA were synergistic as well as that by ET and JA, and SA and ET (Fig.\u00a06). This finding is considerably different from the \u201cantagonistic relationship of SA and JA signaling\u201d found in tobacco (Niki et al. 1998) and Arabidopsis (Gupta et al. 2000). In tobacco, acidic PR1 proteins such as NtPR1a, b, and c were inducible by SA (Ohashi and Matsuoka 1987), which is a defense signal compound for systemic acquired resistance. The NtPRB1b (Eyal et al. 1992; Niki et al. 1998) and AtPRB1 (Santamaria et al. 2001) genes, which encode basic PR proteins in tobacco and Arabidopsis, respectively, were reported to respond positively to JA and ET, and negatively to SA. The ArabidopsisPR1 gene (At2g14610) encodes a basic protein that is inducible by SA.\nTo compare the natures of the OsPR1 genes, the results of the expression of 12 OsPR1 genes are summarized in Fig.\u00a08. It is again impressive that the expression upregulated by various treatments differed considerably depending on the gene, and the transcript levels constitutively found in leaf, root, and flower also varied according to the genes. When comparing the four genes OsPR1#071, #72, #073, and #074, which are clustered in a locus on chromosome 7, we found the following common characteristics: all four genes (1) encode acidic proteins, (2) are constitutively expressed in healthy roots at a high level, and (3) are upregulated by blast fungus-infection. In spite of these similarities, their expression profiles after certain treatments were significantly different from each other. For example, (1) the transcript of OsPR1#074 accumulated at an earlier time period than those of OsPR1#071, #072, or #073 in fungus-infected resistant cultivar IL7, (2) OsPR1#074 but not #071, #072, or #073 was upregulated by Xoo-infection, (3) OsPR1#074, but not OsPR1#071, #072, or #073, was wound-inducible, (4) OsPR1#074 was ACC-, SA-, and JA-inducible, OsPR1#071 was JA-inducible, OsPR1#072 was not inducible by any signal compounds used here, and OsPR1#073 appeared to be suppressed by ACC, (5) OsPR1#074 was expressed in flowers at a high level, but OsPR1#071, #072, and #073 were not.\nFig.\u00a08Comparison of expression characteristics of OsPR1 genes Induction levels of OsPR1 genes by pathogens, wounding, and defense signal compounds, and their constitutive expression levels in organs were compared. The transcript levels in healthy 4th leaves are shown in the right most column. ++ very highly inducible or expressed gene, + highly inducible or expressed gene, \u00b1 gene whose induction or expression was not clear, \u2212 not inducible or expressed gene\nFrom the expression characteristics of the 12 OsPR1 genes obtained here, the possible contribution of all OsPR1 genes for plant self-defense against pathogen attack was indicated. Although the functions of OsPR1 genes are not fully understood, PR1 proteins in some plant species have been reported to have antifungal activity including tobacco acidic PR1a overexpression that induced increased tolerance to two oomycete pathogens in tobacco (Alexander et al. 1993), and basic PR-1 proteins of tobacco and tomato, which have an antimicrobial activity against Phytophythora infestans (Niederman et al. 1995). Thus, the redundant expression of the 12 OsPR1 genes may effectively contribute to defend the attack of various pathogens.\nThe expression levels of the 12 genes in healthy tissues and wounded leaves were also very different according to each particular gene. The data in Fig.\u00a07 shows a representative example of tissue- and organ-specific expression of OsPR1. The level of GUS activity in transgenic rice plants carrying the OsPR1T::GUS gene was confirmed to be similar to the OsPR1#074 transcript level as shown in tobacco PR1a::GUS plants (Yamakawa et al. 1998), in which the GUS gene was expressed as a similar translational fusion gene described in Fig.\u00a07A. GUS analysis using independent transgenic lines showed OsPR1#074 expression was developmentally regulated in roots and constitutively expressed in young rice plants, but not in 2-month old plants, in which the expression was strongly upregulated by wounding (Fig.\u00a07A, B). The wound-induced OsPR1#074 expression in adult plants was localized mainly in the vascular system, which is important as the sensing tissue of the water pressure change after wounding and as the site of bacterial pathogen propagation.\nThe information on the 12 PR1 genes obtained here will be useful for further studies on self-defense mechanisms in rice plants, and for the usage of each PR1 gene as a characteristic marker gene.","keyphrases":["rice","bacterial blight","pathogen resistance","histological analysis","pr protein","defense gene expression"],"prmu":["P","P","P","P","P","R"]}
{"id":"Diabetologia-4-1-2362135","title":"Reconstituted HDL infusion restores endothelial function in patients with type 2 diabetes mellitus\n","text":"To the Editor: HDL-cholesterol is inversely correlated with cardiovascular events in all major epidemiological studies [1]. HDL-increasing strategies have demonstrated that HDL-cholesterol increase is associated with decreased cardiovascular risk in high-risk individuals such as patients with type 2 diabetes [2]. Endothelial dysfunction, a hallmark of type 2 diabetes patients, has been shown to predict future cardiovascular events [3]. Therefore, we investigated the effect of reconstituted HDL (rHDL) on endothelial function measured both acutely (4\u00a0h after infusion) and 7\u00a0days after infusion in type 2 diabetes patients. Control volunteers were measured only at baseline and 4\u00a0h after infusion.\nSeven non-smoking patients with type 2 diabetes (four men and three women, BMI 24.4\u2009\u00b1\u20091.6\u00a0kg\/m2) and seven matched control volunteers (four men and three women, BMI 22.9\u2009\u00b1\u20091.8\u00a0kg\/m2) were enrolled. Inclusion criteria for type 2 diabetes patients were: (1) fasting glucose >7.0\u00a0mmol\/l; (2) no insulin therapy; and (3) triacylglycerol and LDL-cholesterol levels <2.0 and <3.5\u00a0mmol\/l, respectively. Matched control individuals were volunteers who were recruited via advertisements. The presence of macrovascular disease (ECG abnormalities, abnormal ankle\u2013brachial index or a history of cardiovascular events) served as exclusion criteria. Female participants were postmenopausal and not using hormone replacement therapy. The study protocol was performed at least 4\u00a0weeks after discontinuation of vasoactive medication, including ACE inhibitors, angiotensin receptor blockers and non-steroidal anti-inflammatory drugs. None of the patients or control volunteers used lipid-lowering medication. The Internal Review Board of the Academic Medical Center approved the study and all individuals gave written informed consent.\nVascular function was assessed using venous occlusion strain-gauge plethysmography (EC-4; Hokanson, Washington, DC, USA) [4]. Forearm blood flow (FBF), expressed as ml min\u22121 100\u00a0ml\u22121 forearm tissue volume (FAV), was measured simultaneously in both arms. FBF responses to cumulative doses of the endothelium-dependent vasodilator serotonin (Sigma, Poole, UK; 0.6, 1.8 and 6\u00a0ng 100\u00a0ml\u22121 FAV min\u22121), the endothelium-independent vasodilator sodium nitroprusside (SNP; Spruyt Hillen, IJsselstein, the Netherlands; 6, 60, 180 and 600\u00a0ng 100\u00a0ml\u22121 FAV min\u22121), and the competitive inhibitor of endothelial nitric oxide (NO) synthase NG-monomethyl-l-arginine (l-NMMA; Kordia, Leiden, the Netherlands; 50, 100, 200 and 400\u00a0\u03bcg 100\u00a0ml\u22121 FAV min\u22121) were measured. Agents were administered intra-arterially for 6, 4 and 8\u00a0min at each dose, respectively. Average FBF values of the measurement (cannulated) and control arm were obtained from the last six measurements of each measurement period. The three different infusion blocks proceeded after a 15\u00a0min rest period or until FBF had returned to baseline. The ratio of flow in the infused measurement (M) and non-infused control (C) arm was calculated for each recording (M\/C ratio). The average value of the M\/C ratio was calculated from these four to six M\/C ratios, thus providing an internal control by excluding systemic factors from influencing the results [5]. Subsequently, a venous catheter was inserted in the contralateral arm for administration of rHDL (CSL-111; CSL Bioplasma, Parkville, VIC, Australia) at a dose of 80\u00a0mg\/kg body weight over a period of 4\u00a0h. Subsequently, the infusion blocks were repeated. Blood samples were drawn from the individuals after a 12\u00a0h overnight fast, and at 4\u00a0h and 7\u00a0days after rHDL infusion. Descriptive statistics between the two groups were compared by two-tailed independent Student\u2019s t tests or non-parametric tests, depending on skewedness of the data. Analysis of measurements for individuals between baseline and 4\u00a0h as well as baseline and 7\u00a0days after rHDL infusion was performed by two-way ANOVA for repeated measures with Bonferroni correction.\nInfusion of rHDL was well tolerated and no adverse events were recorded. Characteristics of type 2 diabetes patients and control volunteers during each measurement are listed in Table\u00a01. Baseline FBFs were not significantly different between type 2 diabetes patients and control individuals (Table\u00a01). Intra-arterial infusion of serotonin increased FBF in a dose-dependent manner in both groups (see Fig.\u00a01a). At baseline, the FBF response to serotonin was attenuated in type 2 diabetes compared with control volunteers (M\/C ratio in type 2 diabetes: 1.5\u2009\u00b1\u20090.2 vs controls: 2.5\u2009\u00b1\u20090.3; p\u2009<\u20090.05). Four hours after rHDL infusion in type 2 diabetes, FBF response to serotonin increased significantly (M\/C ratio to 1.9\u2009\u00b1\u20090.2; p\u2009<\u20090.05 compared with baseline). rHDL infusion had no significant effect on serotonin-induced vasodilation in control volunteers. In type 2 diabetes, 7\u00a0days after rHDL infusion serotonin responses had returned to baseline values (7\u00a0days, 1.5\u2009\u00b1\u20090.2). At baseline, the maximal vasoconstrictor response to L-NMMA was blunted in type 2 diabetes compared with control volunteers (M\/C ratio controls 0.6\u2009\u00b1\u20090.1 vs type 2 diabetes 1.0\u2009\u00b1\u20090.2; p\u2009<\u20090.05, Fig.\u00a01b). After rHDL infusion, the L-NMMA response improved in type 2 diabetes compared with baseline (0.7\u2009\u00b1\u20090.1; p\u2009<\u20090.05). Although not significant, 7\u00a0days after rHDL infusion there still was a tendency towards improvement in type 2 diabetes (M\/C ratio 0.8\u2009\u00b1\u20090.1 compared with baseline). rHDL infusion had no effect on L-NMMA response in control individuals. Finally, SNP responses were lower in type 2 diabetes vs control individuals at baseline (M\/C ratio in type 2 diabetes, 3.0\u2009\u00b1\u20090.6 vs control individuals, 5.3\u2009\u00b1\u20090.7; p\u2009<\u20090.05, Fig.\u00a01c) and rHDL infusion had no effect on SNP responses.\nFig.\u00a01Change in M\/C ratio after stimulation with the endothelium-dependent vasodilator serotonin (a), NO inhibitor L-NMMA (b) and the endothelium-independent vasodilator SNP (c) before (white circles) and 4\u00a0h (black circles) and 7\u00a0days (diamonds) after rHDL infusion in type 2 diabetes patients as well as in control volunteers (before, white triangle; after 4\u00a0h, black triangle). bp\u2009<\u20090.05 for type 2 diabetes patients compared with control individuals and ap\u2009<\u20090.05 for changes after rHDL within type 2 diabetes patients. Data are means\u2009\u00b1\u2009SEMTable\u00a01Clinical characteristics of type 2 diabetes patients and control volunteers\u00a0Type 2 diabetes patients (n\u2009=\u20097)Control volunteers (n\u2009=\u20097)Baseline4\u00a0h7\u00a0daysBaseline4\u00a0hWaist circumference (cm)101\u2009\u00b1\u20095\u00a0\u00a090\u2009\u00b1\u20096\u2020\u00a0Metabolic syndrome6\/7\u00a0\u00a00\/7\u00a0Systolic BP (mmHg)148\u2009\u00b1\u200912146\u2009\u00b1\u200911143\u2009\u00b1\u20097135\u2009\u00b1\u200916138\u2009\u00b1\u200912Diastolic BP (mmHg)78\u2009\u00b1\u20091379\u2009\u00b1\u20091382\u2009\u00b1\u2009883\u2009\u00b1\u2009985\u2009\u00b1\u20098Heart rate (beats per min)65\u2009\u00b1\u2009967\u2009\u00b1\u2009966\u2009\u00b1\u20091261\u2009\u00b1\u2009964\u2009\u00b1\u20098Total cholesterol (mmol\/l)5.6\u2009\u00b1\u20090.46.5\u2009\u00b1\u20091.45.4\u2009\u00b1\u20091.35.3\u2009\u00b1\u20090.46.7\u2009\u00b1\u20091.3LDL-cholesterol (mmol\/l)2.9\u2009\u00b1\u20090.63.3\u2009\u00b1\u20091.13.1\u2009\u00b1\u20091.03.0\u2009\u00b1\u20090.73.9\u2009\u00b1\u20091.2HDL-cholesterol (mmol\/l)1.1\u2009\u00b1\u20090.22.7\u2009\u00b1\u20090.7*1.6\u2009\u00b1\u20090.61.2\u2009\u00b1\u20090.32.5\u2009\u00b1\u20090.4*ApoA-I (g\/l)1.2\u2009\u00b1\u20090.12.8\u2009\u00b1\u20090.4*1.5\u2009\u00b1\u20090.31.2\u2009\u00b1\u20090.22.7\u2009\u00b1\u20090.4*Triacylglycerol (mmol\/l)1.5\u2009\u00b1\u20090.41.6\u2009\u00b1\u20090.61.6\u2009\u00b1\u20090.50.8\u2009\u00b1\u20090.3\u20201.8\u2009\u00b1\u20091.3*Glucose (mmol\/l)8.3\u2009\u00b1\u20091.26.8\u2009\u00b1\u20091.77.3\u2009\u00b1\u20091.35.2\u2009\u00b1\u20090.4\u20204.9\u2009\u00b1\u20090.2hsCRP (mg\/l)3.5\u2009\u00b1\u20091.64.4\u2009\u00b1\u20091.73.6\u2009\u00b1\u20090.81.0\u2009\u00b1\u20090.9\u20201.8\u2009\u00b1\u20091.1ASAT (U\/l)22.5\u2009\u00b1\u20092.520.4\u2009\u00b1\u20094.022.4\u2009\u00b1\u20094.320.2\u2009\u00b1\u20091.821.4\u2009\u00b1\u20092.7ALAT (U\/l)31.2\u2009\u00b1\u20096.926.0\u2009\u00b1\u20099.727.8\u2009\u00b1\u20098.714.9\u2009\u00b1\u20091.7\u202015.6\u2009\u00b1\u20093.2Basal FBF (ml 100\u00a0ml\u22121 FAV min\u22121)4.1\u2009\u00b1\u20092.03.7\u2009\u00b1\u20090.83.9\u2009\u00b1\u20091.32.6\u2009\u00b1\u20090.92.8\u2009\u00b1\u20090.7Values are means\u2009\u00b1\u2009SD*p\u2009<\u20090.05 compared with baseline within one group; \u2020p\u2009<\u20090.05 between type 2 diabetes and control volunteers ALT, alanine aminotransferase; ASAT, aspartate aminotransferase; hsCRP, high-sensitivity C-reactive protein\nIn conclusion, the present study confirms that basal and stimulated NO bio-availability is reduced in type 2 diabetes patients compared with control volunteers. Besides hyperglycaemia-induced reactive oxygen radical formation, other components of type 2 diabetes-associated dyslipidaemia such as small dense LDL are known to influence endothelial function in type 2 diabetes [3, 6]. Moreover, the high prevalence of metabolic syndrome in our patients is in line with previously published data [7]. rHDL resulted in a significant improvement of endothelial function within several hours in type 2 diabetes patients. More importantly, there was still a tendency towards improved NO availability 7\u00a0days after infusion, at a time when apolipoprotein A-I (ApoA-I) increase had largely disappeared. Acute HDL-increasing strategies are actively being pursued for further reducing cardiovascular burden [8], and thus far the lack of selective and potent HDL-increasing drugs has limited the success of the HDL-cholesterol increase concept. Our reported beneficial effects of ApoA-I infusion may lend further support to the development of ApoA-I-increasing strategies, also for patients with type 2 diabetes.","keyphrases":["type 2 diabetes mellitus","hdl-cholesterol","therapy","vascular function"],"prmu":["P","P","P","P"]}
{"id":"Psychopharmacologia-3-1-1915592","title":"Effects of the cannabinoid CB1 receptor antagonist rimonabant on distinct measures of impulsive behavior in rats\n","text":"Rationale Pathological impulsivity is a prominent feature in several psychiatric disorders, but detailed understanding of the specific neuronal processes underlying impulsive behavior is as yet lacking.\nIntroduction\nDespite its relative recent discovery, the central endocannabinoid system has been implicated in a variety of behaviors such as food intake (for review, see Di Marzo and Matias 2005) and nociception (for review, see Cravatt and Lichtman 2004), and in mediating the reinforcing properties of drugs of abuse (for review, see De Vries and Schoffelmeer 2005). In addition, the endocannabinoid system has been shown to play an important role in various cognitive processes. In this respect, memory encoding, retrieval, and extinction processes have received most interest (e.g., Marsicano et al. 2002; Takahashi et al. 2005; Varvel et al. 2005; for review, see Lichtman et al. 2002), presumably due to the high expression level of CB1 receptors in the hippocampal formation (Egertova and Elphick 2000; Tsou et al. 1998). High densities of CB1 receptors are also present in frontal cortical and striatal regions (Egertova and Elphick 2000; Tsou et al. 1998), suggesting involvement of the endocannabinoid system in executive functions that appear to be largely controlled by frontal corticostriatal systems (for review, see Miller and Cohen 2001). Indeed, several clinical and preclinical observations have demonstrated that \u03949-tetrahydrocannabinol (THC), the principle active cannabinoid of Cannabis sativa and other synthetic cannabimimetics impair selective attention (Arguello and Jentsch 2004; Solowij et al. 1995; Verrico et al. 2004) and behavioral flexibility (Egerton et al. 2005; Hill et al. 2006), alter time estimation (Han and Robinson 2001, McDonald et al. 2003), and impair working memory (Ilan et al. 2004; Jentsch et al. 1997). Nonetheless, to date, little is known about the involvement of the endocannabinoid system in other executive functions such as inhibitory control processes subserving impulsivity.\nPathological levels of impulsive behavior are important features in attention-deficit\/hyperactivity disorder, substance-related disorders, bipolar disorders and personality disorders (American Psychiatric Association 2000). Further elucidating the neurobiological basis of impulsivity may therefore enhance our understanding of these psychiatric disorders. It is becoming increasingly clear, however, that the concept impulsivity is multifaceted and covers various distinct and independent measures. These measures range from poor inhibitory control (impulsive action) to probability and delay aversion or impulsive choice (Barkley 1997; Evenden 1999; Moeller et al. 2001). Recent studies have implicated CB1 receptors in some of these measures of impulsivity. For instance, it has been shown that acute THC impairs response inhibition in healthy volunteers, whereas time estimation and impulsive choice were not affected (McDonald et al. 2003). On the other hand, it has been demonstrated more recently that marijuana acutely increases risk taking in volunteers (Lane et al. 2005). Collectively, these data suggest a role of the cannabinoid system in impulsivity, although its precise role therein is still unclear.\nThe present experiments were aimed at further elucidating the importance of cannabinoid CB1 receptor activation on distinct measures of impulsivity. To this end, we tested the effects of the potent and selective CB1 receptor antagonist rimonabant (SR141716A; Rinaldi-Carmona et al. 1994) and agonist WIN55,212-2 (D\u2019Ambra et al. 1992) on impulsive behavior in various operant paradigms measuring different and presumably independent aspects of impulsivity (for review, see Winstanley et al. 2006), namely, (1) the five-choice serial reaction time task to measure inhibitory control; (2) the delayed reward paradigm to measure impulsive choice, and (3) the stop-signal paradigm to measure response inhibition.\nMaterials and methods\nSubjects\nIn total, 48 male Wistar rats were obtained from Harlan CPB (Horst, The Netherlands). At the start of the experiments, animals were 12\u00a0weeks old, weighed approximately 250\u00a0g, and were housed in pairs in macrolon cages (42.5\u2009\u00d7\u200926.6\u2009\u00d7\u200918.5\u00a0cm; l\u2009\u00d7\u2009w\u2009\u00d7\u2009h) under a reversed 12\u00a0h light\/dark cycle (lights on at 7:00\u00a0p.m.) at controlled room temperature (21\u2009\u00b1\u20092\u00b0C) and relative humidity of 60\u2009\u00b1\u200915%. Animals were maintained at approximately 90% of their free-feeding weight, starting 1\u00a0week before the beginning of the experiments by restricting the amount of standard rodent food pellets (Harlan Teklad Global Diet, Blackthorn, UK). Water was available ad libitum throughout the entire experiment. All experiments were conducted with the approval of the animal ethical committee of the Vrije Universiteit, Amsterdam, The Netherlands.\nApparatus\nExperiments were conducted in 12 identical rat five-hole nose poke operant chambers with stainless steel grid floors (MED-NPW-5L, Med Associates, St. Albans, VT, USA) housed in sound-insulating and ventilated cubicles. Set in the curved wall of each box was an array of five circular holes, 2.54\u00a0cm in diameter, 2.2\u00a0cm deep, and 2.25\u00a0cm above floor level. Each hole was equipped with an infrared detector located across each nose poke unit 1.0\u00a0cm from the front, and a yellow LED stimulus light (6.4\u00a0mm in diameter). Rodent food pellets (45\u00a0mg, Formula P, Research Diets, New Brunswick, NJ, USA) could be delivered at the opposite wall via a dispenser. In addition, the chamber could be illuminated by a white houselight, and sound stimuli were generated using a programmable audio generator (ANL-926, Med Associates). A computer equipped with MED-PC version 1.17 (Med Associates) controlled experimental sessions and recorded data. Animals were tested once daily from Monday until Friday, during the dark phase of the light\/dark cycle.\nBehavioral procedures\nSeparate groups of n\u2009=\u200916 animals were trained for each different paradigm, and for all procedures, a similar habituation and magazine training protocol was used. This protocol consisted of a habituation exposure to the boxes for 20\u00a0min with the houselight on and the food cup containing three food pellets for two consecutive sessions. Subsequently, in the next two sessions, in total, 100 pellets were delivered with an average delay of 15\u00a0s, to allow the animals to associate the sound of pellet delivery with reward.\nFive-choice serial reaction time task\nA more detailed description of training in the 5-CSRTT in our laboratory has been reported previously (Van Gaalen et al. 2006a). In short, rats were trained 5\u00a0days per week to detect and respond to a brief visual stimulus in one of five holes to obtain a food reward. Each session terminated after 100 trials or 30\u00a0min, whichever occurred first. Initially the duration of this stimulus was 32\u00a0s and was gradually decreased to 1\u00a0s over sessions until animals reached stable baseline performance (accuracy >80% correct choice and <20% errors of omission). Responding during stimulus presentation or within the limited hold (LH) period of 2\u00a0s was counted as a correct response. Incorrect, premature responses and errors of omission (no responses or a response after the LH) did not lead to the delivery of a food reward and resulted in a 5-s time-out period during which the houselight was extinguished, whereas perseverative responses, i.e., repeated responding during the presentation of the stimulus, were measured but did not have any programmed consequences. Two different measures of inhibitory control were measured, namely, (1) the number of premature responses before the onset of the visual stimulus, reflecting aspects of loss of inhibitory control and (2) the number of perseverative responses into the stimulus hole after correct choice, presumably measuring aspects of compulsive behavior. In addition, the following other behavioral parameters were measured that reflect task performance, namely, (3) accurate choice, i.e., percentage correct responses calculated as [number correct trials\u2009\/\u2009(correct\u2009+\u2009incorrect trials)]\u2009\u00d7\u2009100; (4) latency to make a correct choice, i.e., the mean time between stimulus onset and nose poke in the illuminated hole; (5) omission errors, i.e., the number of omitted trials during a session; and (6) feeder latency, i.e., the latency to collect a pellet following correct choice.\nDelayed-reward paradigm\nIn addition to the 5-CSRTT, the delayed reward paradigm, as employed in our laboratory, has also been described more elaborately (Van Gaalen et al. 2006b). Briefly, rats were trained 5\u00a0days per week in this paradigm. In the final stages of training and during drug testing, a session was divided into 5 blocks of 12 trials, each block starting with 2 forced trials during which, after initiating the trial through a nose poke into the central hole, either the left hole or the right hole was illuminated in a counterbalanced fashion. In the next ten trials, the animals had a free choice, and both the left and right hole were illuminated. Poking into one position resulted in the immediate delivery of a small reinforcer (one food pellet), whereas a nose poke into the other position resulted in the delivery of a large, but delayed, reinforcer (four food pellets). If an animal did not make a response during this choice phase within 10\u00a0s, an intertrial interval was initiated, and the trial was counted as an omission. The position associated with the small and large reinforcer was always the same for each individual and counterbalanced for the group. The delay for the large reinforcer progressively increased within a session per block of 12 trials as follows: 0, 10, 20, 40, and 60\u00a0s. Responding into non-illuminated holes during the test was recorded, but had no programmed consequences. The behavioral measure to assess task performance, i.e., the percentage preference for the large reinforcer as a function of delay, was calculated as: the number of choices for the large reinforcer\u2009\/\u2009(number choices large\u2009+\u2009small reinforcers)\u2009\u00d7\u2009100. In addition, we calculated the total number of omitted choice trials within a session.\nStop-signal paradigm\nShaping During initial shaping for two consecutive sessions, both the middle nose poke hole and the hole immediately adjacent to the right or left were illuminated (counterbalanced for all subjects). A nose poke into either one of the two active holes extinguished the visual stimuli in both holes and resulted in delivery of a pellet. After an intertrial interval of 10\u00a0s, the next trial started. Nose poking within this intertrial interval period did not have any programmed consequences. A session ended when the rat had earned 100 pellets or after 30\u00a0min, whichever occurred first.\nShaping: go trials During the next phase, only the stimulus light in the middle nose poke hole was illuminated (start stimulus). A response into the active middle hole switched off the stimulus light and was followed by the illumination of the stimulus light (go stimulus) in the hole immediately adjacent to the left or right. A nose poke into this illuminated hole switched off the stimulus light and resulted in the delivery of a pellet. After an intertrial interval of 10\u00a0s, the next trial started. Responding in the start stimulus hole during presentation of the go stimulus was counted as perseverative start pokes, whereas prestimulus responses into the go stimulus hole resulted in a timeout period of 5\u00a0s. Subsequently, the response requirements into the start stimulus hole before onset of a go stimulus were varied into a variable ratio 2 schedule (VR2, i.e., either FR1, FR2, or FR3) to avoid the development of a prepotent response pattern from start stimulus to go stimulus hole and to ensure that animals waited until the appearance of a go stimulus. During this phase, rats were trained until they reliably completed 200 successful go trials. Following this phase, a LH period was introduced for the go stimulus and only during this period was the go stimulus present. Initially, the LH was set at 5\u00a0s, and in subsequent sessions, was individually titrated to meet performance criterion of 80% successful hits and <20% prestimulus responses. Omissions of a go stimulus response within the LH resulted in a 5-s time-out period, during which both the houselight and stimulus light were turned off. From initial shaping to criterion performance in this phase, 26 sessions were required.\nShaping: introduction stop signal During the final training phase, a stop signal was introduced in 25% of all trials. Initially, this stop signal (duration, 50\u00a0ms; frequency, 4,500\u00a0Hz; intensity 80\u00a0dB) was contingent with the appearance of the go signal. Responding during the onset of the stop signal or during the LH immediately extinguished the go stimulus and houselight, turned off the stop signal, and was followed by a 5-s time-out. In contrast, if the animal successfully refrained from responding during a stop trial, a pellet was delivered. Initially, the LH during stop and go trials were equal; however, when performance during stop trials was below 80% successfully inhibited stop trials, the LH during stop trials was lowered over sessions in steps of 100\u2013200\u00a0ms until animals improved performance. Subsequently, the LH was then gradually increased in these individuals over sessions until the LH during both go and stop trials were equal. As soon as animals reached the criterion of approximately 90% successfully inhibited stop trials, delays for the onset of the stop signal were introduced. The stop-signal delays (SSD) were presented in a pseudorandom order, and to compensate for differences between rats, SSDs were based on each individual rats\u2019 mean reaction time on go trials in the preceding drug-free training session. SSDs were calculated as follows: mRT minus either 25, 50, 100, 200, or 400\u00a0ms. In addition, an equal amount of zero delays were presented during sessions. Drug testing commenced upon stable baseline performance for at least five consecutive sessions, i.e., 80% accuracy during go trials and a significant SSD-dependent decrease in correctly inhibited stop trials. It took 30 sessions before animals reached stable baseline performance after the introduction of the stop signal; therefore, from initial shaping to stable baseline performance in the stop-signal paradigm, in total, 56 sessions were required.\nStop-signal paradigm: estimation stop-signal reaction time and correction for omissions during go trials\nCalculations to estimate the stop-signal reaction times (SSRT) and a correction for omission errors were adapted from Logan (1994) and Solanto et al. (2001). For estimating the SSRT, data of the three SSDs of 200, 100, and 50\u00a0ms were used, as the probability of correct inhibition on these intervals was within the range of 0.2\u2009<\u2009p\u2009<\u20090.8, and thus, most informative for estimating SSRT (Band et al. 2003). For each of the three intervals, the probability of responding was calculated including a correction for nonresponses based on the number of omissions during the go trials, the latter, as omissions cannot be distinguished from successful inhibitions during stop trials. The following formula, adapted from Solanto et al. (2001) was used for these calculations:\nwhere x is the number of stop-signal trials at each delay interval; correct inhibitions are the number of correctly inhibited trials, and y is the probability of omissions during the go trials within the entire session. To calculate SSRTs, reaction times on all go trials were rank ordered. From this list with RTs, the \u201cnth\u201d RT was taken, where \u201cn\u201c was obtained by multiplying the total number of go trials by the probability of responding for a particular SSD. This RT value approximates the latency between onset of the go stimulus and completion of the stopping process. The SSRT for each interval is then obtained by subtracting the SSD interval from this RT. The average estimated SSRT that is used for the analyses in the present study is calculated by taking the mean of each SSRT at the three SSDs (200, 100, and 50\u00a0ms).\nDrugs\nSR141716A was generated and kindly donated by Solvay Pharmaceuticals (Weesp, The Netherlands), whereas WIN55,212-2 was purchased from Tocris Biosciences (Bristol, United Kingdom). Both SR141716A and WIN55,212-2 were dissolved as described previously in a mixture of ethanol, Tween80, and sterile saline (ratio 1:1:18; cf. De Vries et al. 2001). In all experiments, SR141716A was injected 30\u00a0min before testing, whereas WIN55,212-2 was injected 20\u00a0min before testing. In all paradigms, the order of testing the drugs was (1) SR141716A and (2) WIN55,212-2. The drug combination was only performed in the five-choice serial reaction time task and followed the studies with SR141716A and WIN55,212-2. Drugs were freshly prepared each day before testing and intraperitoneally injected in a volume of 1\u00a0ml\/kg bodyweight according to a Latin square design for both the dose\u2013response studies and the drug combination on Tuesdays and Fridays, with baseline training sessions on the other weekdays.\nStatistical analyses\nData were subjected to repeated measures analysis of variance (ANOVA) with drug dose (all paradigms), delay to large reinforcer (delayed reward paradigm), and stop-signal delay (stop-signal paradigm) as within subjects variables using the Statistical Package for the Social Sciences version 11 (SPSS, Chicago, IL, USA).\nThe homogeneity of variance across groups was determined using Mauchly\u2019s tests for equal variances, and in case of violation of homogeneity, corrected, and therefore, more conservative Huynh\u2013Feldt probability values were used for subsequent analyses. In the stop-signal paradigm, some further exploratory analyses were performed between mean go reaction times and the limited hold period using a bivariate Pearson correlation. In addition, the estimated SSRT data were also subjected to a median split analysis to assess whether SR141716A or WIN55,212-2 had differential effects on response inhibition in individuals with relatively \u201cfast\u201d or \u201cslow\u201d stopping abilities. In case of statistically significant main effects, further post hoc comparisons were conducted using Student\u2013Newman\u2013Keuls Tests. The level of probability for statistically significant effects was set at 0.05.\nResults\nEffects of SR141716A and WIN55,212-2 on measures of inhibitory response control in the five-choice serial reaction time task\nThe number of premature responses, a measure of inhibitory control reflecting impulsive behavior, was dose dependently decreased by SR141716A [Fig.\u00a01a; F3,45\u2009=\u200912.24, p\u2009<\u20090.001], and further post hoc analyses revealed that all doses significantly lowered premature responding compared with vehicle, whereas the highest dose (3.0\u00a0mg\/kg) even further lowered the number of premature responses compared with 1.0\u00a0mg\/kg SR141716A. In contrast, perseverative responding after correct choice, a different measure of inhibitory control reflecting compulsive behavior, was not affected at any dose [Fig.\u00a01b; F3,45\u2009=\u20091.84, p\u2009=\u20090.15]. Attentional function was improved by SR141716A, and further analyses revealed that only 0.3\u00a0mg\/kg SR141716A significantly increased the percentageof accurate choice from approximately 77% under vehicle conditions to 82% as shown in Table\u00a01 [F3,45\u2009=\u20093.43, p\u2009=\u20090.025]. In addition, a marginal, but significant, increase in the latency to make a correct choice was also detected, and further comparisons showed that only the high dose of 3.0\u00a0mg\/kg significantly slowed correct response reaction time [Table\u00a01; F3,45\u2009=\u20094.53, p\u2009=\u20090.007]. The latency to collect a food reward and errors of omission, however, were not affected at any dose [Table\u00a01; feeder latency: F3,45\u2009=\u20090.39, p\u2009=\u20090.65 and omissions: F3,45\u2009=\u20091.30, p\u2009=\u20090.29].\nFig.\u00a01Effects of SR141716A (a, b) and WIN55,212-2 (c, d) on different measures of inhibitory control in the 5-CSRTT. Data depict mean (\u00b1SEM) numbers of premature responses (a, c) and perseverative responses after correct choice (b, d). *p\u2009<\u20090.05 and **p\u2009<\u20090.005 vs vehicleTable\u00a01Effects of SR141716A and WIN55,212-2 on measures of attentional function and motivation in the 5-CSRTT. Data depict mean\u2009\u00b1\u2009SEMSubstances\u00a0Accuracy (%)Response latency (ms)Omissions (%)Feeder latency (ms)SR141716AVehicle77.2\u2009\u00b1\u20092.4336\u2009\u00b1\u200995.4\u2009\u00b1\u20091.1954\u2009\u00b1\u2009480.3\u00a0mg\/kg82.0\u2009\u00b1\u20091.9*338\u2009\u00b1\u2009105.6\u2009\u00b1\u20091.0954\u2009\u00b1\u2009441.0\u00a0mg\/kg80.5\u2009\u00b1\u20092.5347\u2009\u00b1\u2009145.1\u2009\u00b1\u20090.7937\u2009\u00b1\u2009433.0\u00a0mg\/kg80.2\u2009\u00b1\u20092.3374\u2009\u00b1\u200914*7.3\u2009\u00b1\u20091.0939\u2009\u00b1\u200937WIN55,212-2Vehicle 77.7\u2009\u00b1\u20092.5324\u2009\u00b1\u200973.9\u2009\u00b1\u20090.7956\u2009\u00b1\u2009530.3\u00a0mg\/kg79.6\u2009\u00b1\u20092.3333\u2009\u00b1\u2009103.4\u2009\u00b1\u20090.7910\u2009\u00b1\u2009411.0\u00a0mg\/kg77.2\u2009\u00b1\u20092.3380\u2009\u00b1\u200923*14.4\u2009\u00b1\u20094.3*943\u2009\u00b1\u2009443.0\u00a0mg\/kg76.8\u2009\u00b1\u20092.7399\u2009\u00b1\u200917**20.2\u2009\u00b1\u20094.1**1212\u2009\u00b1\u2009138*p\u2009<\u20090.05**p\u2009<\u20090.005 vs vehicle\nWIN55,212-2 did neither change the number of premature responses nor the number of perseverative responses after correct choice [Fig.\u00a01c and d; premature responses: F3,45\u2009=\u20091.02, p\u2009=\u20090.39 and perseverative responses: F3,45\u2009=\u20091.64, p\u2009=\u20090.21; respectively]. Furthermore, as indicated in Table\u00a01, WIN55,212-2 did not change accurate choice or the latency to collect a pellet after correct choice [accurate choice: F3,45\u2009=\u20090.72, p\u2009=\u20090.54 and feeder latency: F3,45\u2009=\u20093.19, p\u2009=\u20090.052]. In contrast, errors of omission were increased, and latencies to make a correct choice were lengthened by WIN55,212-2 [omissions: F3,45\u2009=\u20097.44, p\u2009=\u20090.002 and correct response latency: F3,45\u2009=\u20096.59, p\u2009=\u20090.004].\nAs shown in Fig.\u00a02, the decrements in premature responding by 3.0\u00a0mg\/kg SR141716A were prevented in the presence of WIN55,212-2 at a dose of 1.0\u00a0mg\/kg [F3,45\u2009=\u20097.33, p\u2009<\u20090.001]. In addition, this selected dose of WIN55,212-2, by itself, did not affect inhibitory control as indicated by the absence of an effect of this dose on the number of premature responses.\nFig.\u00a02Coadministration of WIN55,212-2 at 1.0\u00a0mg\/kg (WIN1) prevents the effects of 3.0\u00a0mg\/kg SR14716A (SR3) on inhibitory control in the 5-CSRTT. All data are depicted as mean (\u00b1SEM). *p\u2009<\u20090.05 and **p\u2009<\u20090.005\nSR141716A and WIN55,212-2 do not affect decision making in the delayed reward paradigm\nStable baseline performance on the delayed reward paradigm occurred after approximately 30 training sessions on a full delay range (0, 10, 20, 40, and 60\u00a0s), and therefore, we commenced drug testing from session 35 onwards. A clear, highly significant delay-dependent decrement in the percentage preference for the large reinforcer was observed [Fig.\u00a03; delay: F4,60=270.22, p\u2009<\u20090.001]. Nonetheless, SR141716A did not shift the preference for a large reinforcer over delays at any of the tested doses [dose: F3,45\u2009=\u20091.56, p\u2009=\u20090.21 and dose\u2009\u00d7\u2009delay: F12,180\u2009=\u20091.24, p\u2009=\u20090.29]. In addition, SR141716A also did not change the total numbers of omitted choice trials, i.e., the failures to start a trial during the choice phase [dose: F3,45\u2009=\u20093.10, p\u2009=\u20090.052].\nFig.\u00a03Effects of SR141716A (a) and WIN55,212-2 (b) on the mean (\u00b1SEM) percentage preference for the large reinforcer in the delayed reward paradigm\nIn the WIN55,212-2 experiments, at the highest dose, in total, five animals omitted all choice trials of some delays, and therefore, were excluded from all analyses of the delay discounting data. Similar to SR141716A, increasing the delay highly significantly shifted the preference from large to the small reinforcer [Fig.\u00a04; delay: F4,40\u2009=\u2009179.06, p\u2009<\u20090.001]. Although there was no overall effect of WIN55,212-2 on decision making [dose: F3,30\u2009=\u20090.68, p\u2009=\u20090.57], there was a dose by delay interaction effect suggesting a shift in preference for the large reinforcer over delays [dose \u00d7 delay: F12,120\u2009=\u20092.61, p\u2009=\u20090.011]. Nonetheless, further post hoc comparisons revealed no differences between vehicle and any of the other doses. The number of omitted choice trials was not affected by WIN55,212-2 [dose: F3,30\u2009=\u20091.61, p\u2009=\u20090.21].\nFig.\u00a04Effects of SR141716A on response inhibition as measured in the stop-signal paradigm. Data are depicted as mean (\u00b1SEM) percentage of correctly inhibited stop trials with varying SSDs before the mean go RT (a), go reaction times (b) and estimated stop signal reaction times from SSDs 200, 100, and 50\u00a0ms (c). **p\u2009<\u20090.005 vs vehicle\nEffects of SR141716A and WIN55,212-2 on response inhibition in the stop-signal paradigm\nUnder baseline conditions in the stop-signal paradigm, there was stable individual variation in mean reaction times during go trials over consecutive sessions ranging from 290 to 470\u00a0ms (mean: 365\u00a0ms; standard deviation: 58\u00a0ms). Likewise, the limited hold periods during go and stop trials also stably varied and ranged from 400 to 1,100\u00a0ms (mean: 594\u00a0ms; SD: 167\u00a0ms). A highly significant positive correlation indicated that individuals with \u201cshorter\u201d limited hold periods also displayed shorter mean go reaction times compared to individuals with \u201clonger\u201d limited hold periods that were slower in go reaction speed [r\u2009=\u20090.85, p\u2009<\u20090.001].\nResponse inhibition, as displayed in the inhibition function curve in Fig.\u00a04a, was not affected by SR141716A at any of the tested doses [dose: F3,45\u2009=\u20091.28, p\u2009=\u20090.29 and dose\u2009\u00d7\u2009SSD: F12,180\u2009=\u20091.25, p\u2009=\u20090.26]; however, the percentage of correctly inhibited stop trials significantly declined with decreasing stop-signal delays [SSD: F4,60\u2009=\u200939.24, p\u2009<\u20090.001]. SR141716A did slow mean reaction times during go trials by approximately 30\u00a0ms [Fig.\u00a04b; dose: F3,45\u2009=\u20097.93, p\u2009=\u20090.001], and further analyses indicated that both the 1.0 and 3.0\u00a0mg\/kg dose significantly slowed reaction times compared with vehicle. Nonetheless, the number of omissions during go trials were not significantly affected [mean\u2009\u00b1\u2009SEM: vehicle\u2009=\u200916.2\u2009\u00b1\u20093.0; 0.3\u00a0mg\/kg\u2009=\u200918.1\u2009\u00b1\u20091.2; 1.0\u00a0mg\/kg\u2009=\u200917.7\u2009\u00b1\u20091.7 and 3.0\u00a0mg\/kg\u2009=\u200923.3\u2009\u00b1\u20092.6; dose: F3,45\u2009=\u20091.76, p\u2009=\u20090.17]. Lastly, the average estimated SSRT across three stop-signal delays, putatively reflecting response inhibition, was not significantly changed by SR141716A at any dose [Fig.\u00a04c; dose: F3,45\u2009=\u20091.47, p\u2009=\u20090.24]. Furthermore, a median split analysis (median SSRT: 238\u00a0ms) did not reveal differential effects of SR141716A on the estimated SSRT in individuals with relatively \u201cfast\u201d vs \u201cslow\u201d stopping abilities [slow vs fast stopper: F1,14\u2009=\u20099.54, p\u2009=\u20090.008; dose: F3,42\u2009=\u20091.04, p\u2009=\u20090.38 and dose\u2009\u00d7\u2009slow vs fast stopper: F3,42\u2009=\u20092.48, p\u2009=\u20090.074].\nIn the WIN55,212-2 experiments, at the highest dose, in total, four animals did not start any go trials and were therefore excluded from all stop-signal data analyses. Response inhibition, as displayed in the inhibition function curve in Fig.\u00a05a, was affected by WIN55,212-2 [SSD: F4,44\u2009=\u200923.51, p\u2009<\u20090.001; dose: F3,33\u2009=\u20095.12, p\u2009=\u20090.005 and dose\u2009\u00d7\u2009SSD: F12,132\u2009=\u20091.11, p\u2009=\u20090.36], and further comparisons revealed that 0.3\u00a0mg\/kg WIN55,212-2 significantly deteriorated the percentage of correct inhibition compared to vehicle. Mean reaction times during go trials were slowed by WIN55,212-2 [Fig.\u00a05b; dose: F3,33\u2009=\u200915.07, p\u2009<\u20090.001], and further analyses indicated that 1.0 and 3.0\u00a0mg\/kg WIN55,212-2 significantly slowed mean reaction times compared to vehicle. Nonetheless, the number of omissions during go trials were not significantly affected [mean\u2009\u00b1\u2009SEM: vehicle\u2009=\u200914.8\u2009\u00b1\u20091.9; 0.3\u00a0mg\/kg\u2009=\u200923.2\u2009\u00b1\u20094.8; 1.0\u00a0mg\/kg\u2009=\u200926.1\u2009\u00b1\u20095.4 and 3.0\u00a0mg\/kg\u2009=\u200934.7\u2009\u00b1\u20097.0; dose: F3,33\u2009=\u20093.06, p\u2009=\u20090.073]. Lastly, the average estimated SSRT across three stop-signal delays, putatively reflecting response inhibition, was not significantly changed by WIN55,212-2 at any dose [Fig.\u00a05c; dose: F3,33\u2009=\u20092.06, p\u2009=\u20090.13]. Furthermore, a median split analysis (median SSRT: 227\u00a0ms) did not reveal differential effects of WIN55,212-2 on the estimated SSRT in individuals with relatively \u201cfast\u201d vs \u201cslow\u201d stopping abilities [slow vs fast stopper: F1,10\u2009=\u20099.57, p\u2009=\u20090.011; dose: F3,30\u2009=\u20092.12, p\u2009=\u20090.12 and dose\u2009\u00d7\u2009slow vs fast stopper: F3,30\u2009=\u20091.30, p\u2009=\u20090.29].\nFig.\u00a05Effects of WIN55,212-2 on response inhibition as measured in the stop-signal paradigm. Data are depicted as mean (\u00b1SEM) percentage of correctly inhibited stop trials with varying SSDs before the mean go reaction times (a), go RTs (b) and estimated stop signal reaction times from SSDs 200, 100, and 50\u00a0ms (c). *p\u2009<\u20090.05 and **p\u2009<\u20090.005 vs vehicle\nDiscussion\nTo our knowledge, this is the first report demonstrating behavioral effects of a CB1 receptor agonist and antagonist on independent measures of impulsivity. Thus far, evidence pointing towards cannabinoid involvement in impulsivity mainly originates from studies in which effects of THC or marijuana were tested in human volunteers (Lane et al. 2005; McDonald et al. 2003). Our data obtained in the 5-CSRTT strongly suggest that inhibitory control is modulated by an endogenous cannabinoid tone, as premature responding was dose dependently decreased by SR141716A. This notion is further supported by the observation that in the presence of WIN55,212-2, the effects of 3.0\u00a0mg\/kg SR141716A on premature responding were no longer observed. Furthermore, the finding that the CB1 receptor agonist WIN55,212-2, by itself, did not impair inhibitory control by increasing the number of premature responses is in keeping with previous findings (Arguello and Jentsch 2004). It should be noted though, that in the study by Arguello and Jentsch (2004), SR141716A (dose range: 0.1\u20131.0\u00a0mg\/kg), by itself, did not change premature responding in a lateralized reaction time task, thereby, contrasting our findings. Nonetheless, differences in baseline performance may have contributed to the discrepancy in findings, as premature responding in well-trained rats in the task by Arguello and Jentsch (2004) was low (approximately 3 per session) compared to premature responding in well-trained rats in the present study (approximately 22 per session). Presumably, a floor effect may have masked the effects of SR141716A on premature responding in the lateralized reaction time task.\nTogether, our data indicate that performance in the 5-CSRTT is associated with profound occupation of CB1 receptors by endogenously released cannabinoids. As might then be expected, the endocannabinoid uptake inhibitor AM404 did not affect inhibitory response control in the 5-CSRTT (unpublished data). In contrast to premature responding, perseverative responding after correct choice, a different measure of inhibitory control that putatively reflects compulsive behavior (Robbins 2002), was neither altered upon activation nor blockade of CB1 receptors. This finding further underscores the dissociation between premature and perseverative responding in the 5-CSRTT that has been shown to have a neuroanatomical basis as well (Chudasama et al. 2003).\nThe endocannabinoid system interacts with many other central neurotransmitters systems including the cholinergic, GABAergic, glutamatergic, and opioid systems (Schlicker and Kathmann 2001; Schoffelmeer et al. 2006). Moreover, cannabimimetics have been shown to indirectly modulate the release of dopamine and glutamate in corticostriatal regions (e.g., Cheer et al. 2004; Szabo et al. 1999; Tanda et al. 1997; Xi et al. 2006), most notably in the nucleus accumbens, and particularly, these effects have been linked to the involvement of the cannabinoid system in addiction (for review, see De Vries and Schoffelmeer 2005). Although it is beyond the scope of the present study and additional experiments are required to substantiate this, one might speculate that the present findings are explained by these modulatory effects of the cannabinoid system on mesolimbic dopamine release, as inhibitory control processes in the 5-CSRTT have been shown to depend upon dopamine receptor activation within the nucleus accumbens (Cole and Robbins 1987; Pattij et al. 2007). On the other hand, explanations for the beneficial effects of SR141716A on inhibitory control may be due to the role of the endocannabinoid system in feeding behavior (for review, see Di Marzo and Matias 2005). Accordingly, it has been demonstrated that SR141716A reduces intake of normal and palatable food (e.g., Arnone et al. 1997; Freedland et al. 2000; Thornton-Jones et al. 2005) and the motivation to obtain food (Solinas and Goldberg 2005). Nonetheless, despite these reported effects of SR141716A on food intake, anorexic effects of this compound seem unlikely to explain the current data, as both primary indices of food-motivated behavior in the 5-CSRTT, namely, errors of omission and feeder response latencies, were not changed.\nIt is interesting to note that we also observed a moderate beneficial effect of SR141716A on visuospatial attention, as it increased the level of accurate choice at 0.3\u00a0mg\/kg. Likewise, it has been shown that SR141716A improves social recognition and spatial and aversive memory at comparable dose ranges between 0.3 and 3.0\u00a0mg\/kg (Lichtman 2000; Takahashi et al. 2005; Terranova et al. 1996; Wolff and Leander 2003). Although the mechanisms responsible for the beneficial effects of disrupting endocannabinoid signaling on mnemonic and attentional processes need to be elucidated further, it is interesting to note that SR141716A increases, among others, cholinergic neurotransmission in the medial prefrontal cortex in vivo (Tzavara et al. 2003). This finding may be particularly relevant for attentional function, as performance in the 5-CSRTT has been shown to be accompanied by increments in acetylcholine release in the medial prefrontal cortex (Passetti et al. 2000), whereas decrements in acetylcholine release in this brain region have been shown to correlate with poor visuospatial attention (McGaughy et al. 2002).\nA different measure of impulsivity we studied was impulsive choice in the delayed reward paradigm. Conceptually, impulsive choice differs from inhibitory control in the 5-CSRTT and rather reflects a cognitive decision-making process, as rats have to weigh the immediate vs delayed outcomes of their behavior. Impulsive choice then is reflected in insensitivity towards the delayed larger reward and a preference for the small immediate reward. In the present experiments, we observed that neither SR141716A nor WIN55,212-2 did have any effects on impulsive choice. In accordance with these findings, it has been shown recently that THC did not change delay and probability discounting in human volunteers (McDonald et al. 2003), and together, these data suggest that the (endo)cannabinoid system is not critically involved in impulsive choice. Our observations do contrast with previous findings indicating that marijuana elevates risk-taking behavior in volunteers (Lane et al. 2005). Risk taking, however, differs from delay and probability aversion in that it is most likely to occur when the behavioral options may result in losses or have aversive consequences (Rachlin et al. 1986). In the delayed reward paradigm and the paradigm used by McDonald et al. (2003), subjects could only win and not lose reward (food or money, respectively), whereas only the magnitude of the reward depended upon choice. It is therefore possible, that these procedural and conceptual differences explain the discrepancy in findings, and furthermore, that delay aversion and risk-taking processes are differentially regulated by the (endo)cannabinoid system.\nWhile response inhibition has been shown to be impaired in human volunteers after THC administration (McDonald et al. 2003), neither disruption of endocannabinoid signaling nor administration of a CB1 receptor agonist had clear observable behavioral effects on stop-signal task performance. In agreement with the assumption of a \u201crace model\u201d between \u201cgo\u201d and \u201cstop\u201d processes (Logan 1994) and previous stop-signal data in rats (Eagle and Robbins 2003a, b), the probability of successfully inhibiting a response during a stop trial decreased in rats when the onset of the stop signal was delayed in time. Although SR141716A did not shift this inhibition curve as a function of increasing stop-signal delays, the low dose WIN55,212-2 (0.3\u00a0mg\/kg) significantly deteriorated the ability to inhibit responding with increasing stop-signal delays. However, the primary parameter in this paradigm, the average estimated stop-signal reaction time, or simply put, speed of stopping, was not changed by any dose of SR141716A or WIN55,212-2. In addition, median split analyses revealed no differential drug effects in individuals with \u201cslow\u201d vs \u201cfast\u201d stopping abilities as has been reported previously for the effects of d-amphetamine on stopping speed in both rats and humans (De Wit et al. 2002; Feola et al. 2000). Collectively, our findings suggest that response inhibition processes, as measured in the stop-signal paradigm, are not under control of CB1 receptors. In the present study, the speed of stopping was estimated from separate SSRTs obtained from three stop-signal delays (200, 100, and 50\u00a0ms before mean go RT), and consistent with this estimation method, there was some variability in the SSRTs depending on the delay (Band et al. 2003; Logan 1994), with shorter stop-signal delays resulting in SSRTs of approximately 140\u00a0ms and longer delays resulting in SSRTs of approximately 300\u00a0ms. However, the variability in SSRTs over delays was similar across all doses of both drugs (data not shown), thus, ruling out the possibility that this variability may have masked effects of SR141716A or WIN55,212-2 on response inhibition. In contrast to the measures of response inhibition, reaction times during performance on go trials were slowed by both compounds in line with their effects in the 5-CSRTT. With regard to these effects of SR141716A, in the absence of changes on omission errors in both the 5-CSRTT and the stop signal paradigm, they cannot be solely interpreted in terms of motor effects. Rather, the minor but significant increase in response latencies of approximately 30\u00a0ms in both paradigms (Table\u00a01 and Fig.\u00a04b) may indicate changes in information processing speed induced by blockade of endocannabinoid signaling. However, this notion is not confirmed by previous sensorimotor gating data (Mansbach et al. 1996; Martin et al. 2003). In contrast, the effects of 1.0 and 3.0\u00a0mg\/kg WIN55,212-2 on reaction times in the stop-signal paradigm may in part be secondary to changes in locomotor activity, as these doses also increased the number of omissions in the 5-CSRTT.\nRemarkably, although both the stop-signal paradigm and 5-CSRTT measure aspects of impulsive action, only inhibitory control in the 5-CSRTT was improved by SR141716A. Impulsive action in the latter paradigm is mainly measured as the inability to inhibit inappropriate (premature) responses, whereas in the stop-signal paradigm impulsive action is reflected in the inability to inhibit ongoing behavior, i.e., the inability to stop a behavioral response that has just been initiated. Our observations support the notion of different and separable forms of inhibitory control, and moreover, suggest a differential role for the endocannabinoid system therein. Likewise, lesion studies have suggested that different brain regions are involved in inhibitory control measured in either the 5-CSRTT or stop-signal paradigm, as for instance, lesions of the nucleus accumbens or subregions of the medial prefrontal cortex have been shown to impair inhibitory control in the 5-CSRTT (Christakou et al. 2004; Chudasama et al. 2003; Muir et al. 1996) and not in the stop-signal paradigm (Eagle and Robbins 2003b).\nIn summary, the present study provides evidence for a differential involvement of the endocannabinoid system in independent measures of impulsivity, as SR141716A primarily affected inhibitory control, and neither impulsive choice nor response inhibition, whereas WIN55,212-2 only slightly affected response inhibition. In this regard, the present data add to existing clinical and preclinical evidence demonstrating that distinct measures of impulsivity can be dissociated at a pharmacological and neuroanatomical level (e.g., Chudasama et al. 2003; De Wit et al. 2002; McDonald et al. 2003; Winstanley et al. 2004). Our findings may be of particular interest with respect to the heterogeneity observed in attention-deficit\/hyperactivity disorder (Sonuga-Barke 2002) and suggest that possible novel pharmacotherapies targeted at the cannabinoid system may benefit the subtype resulting from poor inhibitory control, but not the motivational style, or delay aversion, subtype.","keyphrases":["cannabinoid","cognition","inhibitory control","impulsive choice","response inhibition","sr141716a","win55,212-2"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Neuroimage-2-1-2278232","title":"Phonological processing in deaf signers and the impact of age of first language acquisition\n","text":"Just as words can rhyme, the signs of a signed language can share structural properties, such as location. Linguistic description at this level is termed phonology. We report that a left-lateralised fronto-parietal network is engaged during phonological similarity judgements made in both English (rhyme) and British Sign Language (BSL; location). Since these languages operate in different modalities, these data suggest that the neural network supporting phonological processing is, to some extent, supramodal. Activation within this network was however modulated by language (BSL\/English), hearing status (deaf\/hearing), and age of BSL acquisition (native\/non-native). The influence of language and hearing status suggests an important role for the posterior portion of the left inferior frontal gyrus in speech-based phonological processing in deaf people. This, we suggest, is due to increased reliance on the articulatory component of speech when the auditory component is absent. With regard to age of first language acquisition, non-native signers activated the left inferior frontal gyrus more than native signers during the BSL task, and also during the task performed in English, which both groups acquired late. This is the first neuroimaging demonstration that age of first language acquisition has implications not only for the neural systems supporting the first language, but also for networks supporting languages learned subsequently.\nIntroduction\nPhonology describes the level of analysis at which meaningless, contrastive units of language combine to form meaningful units. In spoken languages these are auditory\/articulatory elements. Substitution of a single element creates a new lexical item, e.g., in English \/pin\/\u2013\/bin\/. The same level of analysis has been applied to signed languages, where phonology is visual, with handshapes, movements and locations combined to form signs (Stokoe, 1960; Brentari, 1999; Sandler and Lillo-Martin, 2006). As with words, the substitution of just one element can create a new sign. For example, the BSL sign NAME is located at the forehead while AFTERNOON differs only in that it is located at the chin (see Fig. 1).\nThe primary aim of the current study was to examine whether the application of the term \u2018phonology\u2019 to signed languages has neurological as well as linguistic and psycholinguistic validity. We address, for the first time, whether similar neural processing is involved in phonological analysis of both signed and spoken languages. We asked participants to judge whether spoken word labels for pictures rhymed or not. Of the phonological parameters of signs, location is one of the primary factors determining whether signs are judged to be similar (Hildebrandt and Corina, 2002). Therefore, we also asked deaf participants to judge if BSL signs for pictured items shared the same location.\nStudies of written rhyme judgement by hearing people report reliable activation of the posterior portion of the left inferior frontal gyrus (IFG) and the ventral premotor cortex (Broca's area; Sergent et al., 1992; Poldrack et al., 1999; Kareken et al., 2000; Lurito et al., 2000; Xu et al., 2001; Seghier et al., 2004; Burton et al., 2005). Temporary disruption of this region using transcranial magnetic stimulation (TMS) impairs phonological processing (Gough et al., 2005). Given the well-established role of the posterior IFG in speech production (Ojemann and Mateer, 1979), the contribution of the left posterior IFG and ventral premotor cortex to phonological processing is often attributed to articulatory processes or representations (D\u00e9monet et al., 1996). In addition to the left IFG, the left parietal lobe is also recruited during rhyme judgement tasks using written stimuli (e.g., Pugh et al., 1996; Lurito et al., 2000; Xu et al., 2001; Seghier et al., 2004). However, the precise role of this region in phonological processing remains unclear (see Eden et al., 2004) and will be addressed further in the Discussion section. If similar processing is required to make phonological similarity judgements about BSL and English, left inferior frontal and left parietal regions should be recruited during both tasks.\nAn important feature of the current study was the use of picture stimuli (see Fig. 2). This enabled the same stimuli and response requirements to be used in both the BSL and English tasks. This is not possible in studies contrasting comprehension (e.g., MacSweeney et al., 2002b) or production (e.g., Braun et al., 2001) of speech and sign. Picture stimuli also allowed us to tap individuals' own phonological representations of words and signs. Previous studies of rhyme judgement with hearing people have used auditory or written stimuli, which directly or indirectly provide the phonology of the item. To determine whether rhyme judgements in response to pictures elicit the pattern of activation reported in studies using written words, hearing participants were also tested. Including this group also allowed us to directly contrast activation patterns observed in deaf and hearing participants to determine the effect of hearing status on the neural systems supporting phonological processing.\nA second aim of this study was to determine if the neural systems supporting language are influenced by the age of first language acquisition. To address this, deaf native and non-native signers were contrasted. Approximately 5% of deaf people are born to deaf, signing parents (Mitchell and Karchmer, 2004). Typically, these children learn a signed language as their native language, reaching acquisition milestones along the same timescale as hearing children acquiring speech (Morgan and Woll, 2002). However, for nearly all of the 95% of deaf children who are born to hearing parents (non-native signers), exposure to a signed language is delayed.\nDeaf native and non-native signers should differ on sign-related tasks since this is the language acquired early or late. In addition, Mayberry et al. have shown that deaf native signers perform better than deaf non-native signers on grammaticality judgements of written English (Mayberry et al., 2002; Mayberry and Lock, 2003). In these studies, both groups encountered written English at the same age. However, what did differ between these groups was their early language experience. Native signers have a well-established first language which can facilitate the acquisition of a later learned language; non-native signers do not. For all deaf people, both native and non-native signers, exposure to spoken language is late and incomplete. This is because speechreading cannot provide full access to speech since many of the articulators of speech are invisible. From this perspective, Mayberry and Lock (2003) have argued that deaf non-native signers can be considered to have \u2018no early language\u2019. This is a very different situation to that of hearing people learning a signed language late since they already have a native spoken language. Studies contrasting early and late acquisition of signed language in hearing participants (cf., Newman et al., 2002) cannot therefore be generalised to the deaf population. We examined how the incomplete acquisition of a language early in life is reflected in the brain. Consistent with the behavioural findings of Mayberry et al., we predicted differences between deaf native and non-native signers, on both the BSL and English tasks, despite these groups' similar experience of English.\nIn summary, in the current study profoundly deaf and hearing adults made phonological similarity judgements in response to picture pairs (see Fig. 2). Participants judged if the spoken English labels rhymed or whether the BSL labels shared the same location (deaf group only). Activation during these experimental conditions was contrasted with a \u2018same picture?\u2019 control task. The following research questions were addressed: are the same neural networks recruited during phonological decisions based on signed and spoken language? What is the impact of age of signed language acquisition and hearing status on this network?\nMaterials and methods\nParticipants\nTwenty-three deaf adults and 24 hearing adults were scanned. All were right-handed and had normal or corrected to normal vision. Of the deaf participants, one was excluded because of excessive movement in the scanner and another two were excluded because they did not complete both the sign and speech tasks. Therefore, 20 deaf and 24 hearing participants, matched on age and non-verbal IQ (Block Design, WAIS-R: p > 0.1), were included in the analyses (see Table 1 for participant characteristics). The deaf participants included in the fMRI study were selected from a larger sample of volunteers. To enhance the likelihood that they would perform the phonological judgement tasks well, those selected were good readers and had performed well on a test of rhyme ability in a previous test session outside the scanner. Therefore, the deaf participants included in the fMRI study were good readers (mean reading age = 15years, 6months) in comparison to the population mean for deaf people, generally considered to be approximately 9 or 10years (see Conrad, 1979; Allen, 1986; Holt, 1993). The deaf participants were also skilled speechreaders, outperforming the hearing participants on the Test of Adult Speechreading (Mohammed et al., 2003; t = 5.1, (34), p < 0.0005). Nevertheless, the hearing group were significantly better readers (Vernon-Warden, 1996: t = \u2212 3.9, (42), p < 0.0005) and had a higher English vocabulary score (shortened version of the Boston Naming Test, Kaplan et al., 1983: t = \u2212 4.1, (42), p < 0.0005) than deaf participants. These differences are accounted for in the fMRI data analyses.\nAll deaf participants reported being born profoundly deaf and audiograms obtained at the time of testing confirmed that all had a mean hearing loss greater than 92dB in the better ear over four octaves, spanning 500\u20134000Hz. All deaf participants encountered written English upon entering primary school, aged 4\/5. Twelve of the deaf participants were native signers, having acquired BSL from their deaf, signing parents. The remaining eight deaf participants (non-native signers) had hearing parents. One native signer and one non-native signer reported attending schools which used a total communication approach, in which signs are used to support spoken English. The remaining 18 of the 20 deaf participants had attended \u2018oral\u2019 schools in which spoken English was the main form of communication. This educational approach was the norm for this generation of deaf adults in the UK, even for those who used BSL as their native language. Of the eight non-native signers, five learned BSL after leaving secondary school, aged 17 to 21. One participant learned BSL at their total communication primary school aged 4\/5. Two other participants who attended oral schools reported learning BSL at school; one aged 4\/5, the other aged 11. These participants will have been exposed to BSL by their deaf native signing classmates.\nThe deaf subgroups were well matched. There were no significant differences between deaf native and deaf non-native signers in age (p = 0.06), non-verbal IQ (p > 0.1), reading age (p > 0.1), English vocabulary (p > 0.1) or speechreading skill (p > 0.1). All participants gave informed, written consent to participate in the study, which was approved by the Institute of Psychiatry\/South London and Maudsley NHS Trust Research Ethics Committee.\nStimuli\nThe same pictures were presented in both the rhyme and location judgement tasks (n = 60). All pictures represented highly familiar\/frequent, monosyllabic English words. Fifty-eight of the pictures were black and white line drawings, taken predominantly from the Snodgrass and Vanderwart (1980) standardised picture set and other language assessments. Two colour pictures were also included, to represent \u2018red\u2019 and \u2018blue\u2019.\nRhyme task (shared English phonology pairs)\nThirty pictures were combined as 15 rhyming pairs. Orthography was inconsistent so that rhyme decisions could not be based on spelling, (e.g., chair\u2013bear, tail\u2013whale; see Fig. 2A).\nLocation task (shared BSL phonology pairs)\nThe remaining thirty pictures were combined as 15 pairs sharing the same location when signed in BSL, but differing in handshape and movement (e.g., pig\u2013witch; hat\u2013cow, see Fig. 2B). Only signs touching the body or occurring in close proximity to it were considered to have a defined location. Participants were told that any pairs articulated in neutral space in front of the signer's body (see Stokoe, 1960) should receive a \u2018no\u2019 response.\nThe two sets of experimental pictures (rhyme and location pairs) were matched on familiarity (Coltheart, 1981) (t = 0.64, 53, p > 0.1) and concreteness (Coltheart, 1981) (t = \u2212 1.1, 53, p > 0.1) and the English labels were matched on frequency (Kucera and Francis, 1967) (t = 0.92, 56, p > 0.1) and length (t = 1.5, 58, p > 0.1). The \u2018no\u2019 (non-shared phonology) trials were established by re-pairing the pictures from the complementary task. Rhyming pictures were re-paired such that there was no overlap in signed or spoken phonology to form the \u2018no\u2019 trials in the location task (e.g., chair\u2013whale). Likewise, location pairs were re-paired to form the \u2018no\u2019 trials in the rhyme task (e.g., hat\u2013pig). Thus, the same stimuli were presented in both tasks.\nThe stimuli used in the experimental conditions doubled as their own controls in the \u2018same picture?\u2019 control task. Fifteen of the pictures were presented as identical pairs (e.g., chair\u2013chair, see Fig. 2C). Another 30 pictures were re-paired to form different picture trials. The labels for these items did not share any phonological features in either English or BSL. Thus, of the 60 pictures seen in both experimental conditions, 45 were also presented in the \u2018same picture?\u2019 control condition. Whether an item was first seen in the experimental or control condition was counterbalanced such that any repetition effects were balanced across conditions.\nAll participants performed a picture naming pre-test before the scan session. If an unexpected label was generated, the desired English word or BSL sign (deaf participants only) was supplied. Correct naming of these items was checked again at the end of the pre-test session.\nDesign\nDeaf participants performed the rhyme and location similarity judgement tasks in separate, counterbalanced runs. Hearing participants performed only the rhyme task. Each run consisted of six 30-s blocks of the experimental task (rhyme or location), alternating with six 30-s blocks of the \u2018same picture?\u2019 control task. Each run lasted 6 min.\nIn the English phonology task, participants were required to decide whether the English labels for two pictures rhymed. Deaf participants had already been involved in a behavioural study of rhyme awareness as part of a wider project. They were reminded of the concept of rhyme and were given examples and practice trials prior to the start of the experiment in the scanner. In the sign phonology task, signing participants were required to decide if the BSL labels for the two pictures shared the same location. The control condition was interleaved between the same phonology? task blocks. This consisted of deciding if two pictures were the same. The trials in each condition were half \u2018yes\u2019 trials and half \u2018no\u2019 trials. Subjects indicated their response using a two-choice button box.\nA one-syllable task prompt appeared at the bottom of the screen, without a pair of pictures, for 2000ms at the beginning of each block (\u2018Rhyme?\u2019\u2014rhyme task; \u2018Place?\u2019\u2014location task; \u2018Same?\u2019\u2014picture matching task). The prompt remained on the screen throughout the block. Each pair of pictures was presented for 5s. This relatively long presentation duration was selected on the basis of pilot studies in which deaf people made self-paced rhyme decisions in response to pictures. The inter-stimulus interval was 500ms. Each 30-s block was a mixture of five \u2018yes\u2019 and \u2018no\u2019 trials (see Fig. 2).\nfMRI parameters\nGradient echo echoplanar MRI data were acquired using a 1.5-T GE NVi MR system (General Electric, Milwaukee, WI, USA) using a standard quadrature head coil. Head movement was minimised by positioning the participant's head between cushioned supports. One hundred and twenty T2\u204e-weighted images depicting BOLD contrast were acquired during one experimental session at each of 38 near-axial 3mm thick planes parallel to the intercommissural (AC\u2013PC) line: 0.3mm interslice gap; TR = 3s, TE = 40ms; flip angle = 90\u00b0). The field of view for the fMRI runs was 240mm, and the matrix size was 64 \u00d7 64, with a resultant in-plane voxel size of 3.75mm. An inversion recovery EPI dataset was also acquired to facilitate registration of individual fMRI datasets to Talairach space (Talairach and Tournoux, 1988). This comprised 43 near-axial 3mm slices (0.3mm interslice gap), which were acquired parallel to the AC\u2013PC line (TR = 16s; TE = 80ms; TI = 180ms; flip angle = 90\u00b0). The field of view for the EPI dataset was 240mm, and the matrix size was 128 \u00d7 128, with a resultant in-plane voxel size of 1.875mm.\nfMRI data analysis\nThe fMRI data were analysed using an in-house non-parametric software package (XBAM_v3.2) which uses standard preprocessing steps: realignment, normalisation, baseline correction, spatial smoothing, and GLM parameter estimation using a combination of gamma variate basis functions (for details see Brammer et al., 1997; Bullmore et al., 1999, 2001; Suckling and Bullmore, 2004). The data were realigned to minimise artefacts due to subject motion. First, a template was computed by averaging the image intensity over all time points at each voxel. The 3D volumes at each time point for each participant were then realigned to the template by computing the rigid body motion parameters (3 rotations, 3 translations) that maximised the correlation between each volume and the template. Normalisation was conducted using an affine transform and by computing the parameter set that maximised the correlation between the template image (in standard space\u2014Talairach and Tournoux) and the image to be normalised. The data were then smoothed using a Gaussian filter (FWHM 7.2mm). Experimental responses were then analysed by convolving the experimental design with two gamma variate functions (peak responses four and eight seconds) with delays chosen to span the likely range of BOLD delays and computing the least squares fit of the resulting convolution to the time series at each voxel. A goodness of fit statistic was derived by calculating the ratio between the sum of squares due to the model fit and the residual sum of squares (SSQ ratio). The value of this statistic was then tested for significance using the wavelet-based time series permutation method (Bullmore et al., 2001; Suckling and Bullmore, 2004).\nGroup analysis\nData were transformed into standard space (Talairach and Tournoux, 1988). Voxel size in standard space was 3.3 \u00d7 3.3 \u00d7 3.3mm. Significant activations were identified using data-driven significance testing of the median activations at each voxel over all members of the group (Brammer et al., 1997). Median statistics were used to minimise outlier effects in the group sizes normally used in fMRI studies. Analysis was extended to the cluster level with the clusterwise false positive threshold set to less than one across the whole brain (Bullmore et al., 1999). Since the XBAM analysis method takes into account first level as well as second level variance, it resembles what Thirion et al. (2007) have called a \u201cpseudo mixed effects analysis\u201d.\nGroup differences\nDifferences in activation between groups and conditions were assessed by fitting the following linear model to the data at each voxel, Y = a + bX + e, where Y is the vector of BOLD effect sizes for each individual, X is the contrast matrix for the particular inter-condition\/group contrasts required, a is the mean effect across all individuals in the various conditions\/groups, b is the computed group\/condition difference, and e is a vector of residual errors. The model was fitted by minimising the sum of absolute deviations to reduce outlier effects. The null distribution of b was computed by permuting data between conditions (assuming the null hypothesis of no effect of experimental condition) and refitting the above model. Group difference maps were computed as described above at voxel or cluster level by appropriate thresholding of the null distribution of b.\nConjunction analysis\nConjunction analyses were carried out to identify brain regions in which there was consistent activation across tasks. First, the minimum SSQ ratio (effect\/error) at each voxel across conditions was determined. This measure was then tested (at appropriate voxelwise and clusterwise p-values), as described above under Group analysis, to determine whether it was significantly different from zero. Brain areas showing significant levels of activation were considered to show significant conjunctions of brain activation.\nResults\nBehavioural data\nSee Table 2 for accuracy and reaction time data.\nDeaf participants only\nA mixed-model ANOVA was conducted on the accuracy data (Task (rhyme\/location\/control) \u00d7 Group (native\/non-native signers)). A main effect of Task indicated that the control task was performed better than both experimental tasks (F(2,36) = 25.5, p < 0.0005). There was no significant effect of Group and no interaction. Excluding the control task from the ANOVA yielded no significant main effects and no interaction. Thus, deaf native and non-native signers were equally accurate on both the rhyme and location tasks.\nThe same mixed-model ANOVAs were applied to the reaction time data. Deaf participants were faster on the control than experimental tasks (F(2,36) = 211.7, p < 0.0005). There were no further significant effects. When the control task was omitted from the model, a main effect of Task (F(1,18) = 9.7, p < 0.01) indicated faster reaction times to the rhyme than location task.\nAll deaf versus all hearing participants performing the rhyme task\nA mixed-model ANOVA was conducted on the accuracy data (Task (rhyme\/control) \u00d7 Group (deaf\/hearing)). A main effect of Task indicated better performance in the control than rhyme task (F(1,42) = 52.5, p < 0.0005). A main effect of Group indicated better performance by hearing than deaf participants (F(1,42) = 13.4, p < 0.002). This was qualified by a significant interaction (F(1,42) = 11.4, p < 0.005) indicating that the hearing group performed better than the deaf group on the rhyme task, with no difference on the control task.\nWith regard to the reaction time data, a main effect of Task (F(1,42) = 744.7, p < 0.0005) indicated faster responses on the control than rhyme task. A significant interaction (F(1,42) = 11.5, p < 0.005) indicated slower performance by deaf than hearing participants to the rhyme task, but no group difference on the control task.\nfMRI data\nRhyme and location similarity judgements in deaf participants only\nTo identify neural systems involved in phonological processing of sign and speech in the deaf group, data from all deaf participants were combined. Analysis of the rhyme (English) and location (BSL) tasks separately, relative to the control task (voxelwise p = 0.025; clusterwise p = 0.01), resulted in remarkably similar patterns of activation (see Figs. 3A and B\/Table 3).\nA conjunction analysis was performed to clarify the overlap in activation between the two phonological tasks (English rhyme and BSL location) in deaf participants (voxelwise p = 0.05; clusterwise p = 0.025). As in the individual task analyses, a network consisting of three regions was identified. The most extensive activation was in the left frontal cortex (19.41cm3 volume; X = \u2212 40, Y = 30, Z = 17; these Talairach and Tournoux coordinates, and those reported in the text to follow, represent local maxima). This extended from the insula, through the inferior and middle frontal gyri into the ventral precentral gyrus. The second significant activation extended from the superior portion of the supramarginal gyrus (SMG) into the superior parietal lobule (SPL) and the precuneus (11.28cm3 volume; X = \u221229, Y = \u221267, Z = 40). Finally, significant activation was identified in the medial portion of the superior frontal gyrus (SFG), incorporating the anterior cingulate (7.37cm3 volume; X = 0, Y = 1, Z = 50). These data suggest that a left-lateralised network of three regions is engaged by deaf participants performing a phonological similarity judgement task, regardless of whether the task is performed in English or BSL.\nDifferences between the rhyme and location tasks and the effect of age of BSL acquisition in deaf participants\nTo determine the differences between the networks recruited during the rhyme and location judgement tasks and to examine the effect of age of signed language acquisition, a mixed-model ANOVA was conducted. This included Task (rhyme\/location) as a within subjects factor and Age of BSL Acquisition (native\/non-native) as a between subjects factor (voxelwise p = 0.025; clusterwise p = 0.005). The main effect of Task showed that two regions were recruited to a greater extent for the rhyme than location task. These were the left dorsal IFG, extending into the precentral gyrus (4.13cm3 volume; X = \u2212 51, Y = \u2212 4, Z = 40; BA 6), and the medial portion of the SFG, at the junction with the anterior cingulate (2.30cm3 volume; X = 0, Y = \u2212 11, Z = 53; BA 6). In contrast, a region in the left parietal lobe was recruited to a greater extent for the location than rhyme task (see Fig. 4). This extended from the superior portion of the SMG, into the SPL and medially to include the precuneus (5.10cm3 volume; X = \u2212 4, Y = \u2212 70, Z = 43; BA 7).\nNo regions were recruited to a greater extent by native than non-native signers. In contrast, non-native signers recruited the left inferior frontal cortex to a greater extent than native signers (see Fig. 5A; 5.32cm3 volume; X = \u2212 40, Y = 19, Z = 30). This activation extended from the IFG (BA 44\/45), into the middle frontal gyrus and precentral gyrus. Follow-up analyses were conducted comparing native and non-native signers on the rhyme and location tasks separately (voxelwise p = 0.025; clusterwise p = 0.005). These analyses confirmed that non-native signers engaged the posterior IFG more than native signers, during both the location task (3.45cm3 volume; X = \u2212 40, Y = 15, Z = 30) and the rhyme task (3.74cm3 volume; X = \u2212 40, Y = 7, Z = 23).\nA significant Group \u00d7 Task interaction was also identified, the focus of which was at the junction of the left IFG (BA 44), the precentral gyrus (BA 6) and the middle frontal gyrus (BA 9; see Fig. 5B; 2.44cm3 volume; X = \u2212 43, Y = 19, Z = 30). Follow-up analyses (voxelwise p = 0.025; clusterwise p = 0.005) demonstrated that there was no significant difference in the extent to which non-native signers recruited this region during the rhyme and location tasks. In contrast, native signers engaged this region more during the rhyme task, performed in English which they learned late, than the location task, performed in their native language (2.87cm3 volume; X = \u221247, Y = 0, Z = 36, precentral gyrus (BA 6)). The medial portion of the SFG, at the junction with the anterior cingulate, also demonstrated the same effect (2.16cm3 volume; X = 0, Y = 11, Z = 53).\nRhyme similarity judgements in hearing participants (voxelwise p = 0.025; clusterwise p = 0.01)\nHearing participants performing the rhyme task engaged four regions (see Table 3): [1] the left prefrontal cortex extending from the insula, through inferior and middle frontal gyri and into the precentral gyrus, [2] the superior portion of the SMG, extending into the SPL and medially into the precuneus, [3] the anterior medial portion of the SFG and superior portions of the anterior cingulate, and [4] the right inferior occipital gyrus extending into the fusiform gyrus and incorporating superior parts of the cerebellum.\nThe pattern observed in hearing participants performing the rhyme task was similar to that observed in deaf people performing the rhyme and location tasks. To clarify the overlap in regions recruited during phonological similarity judgements a conjunction analysis was conducted on the data from deaf and hearing participants performing the rhyme task and deaf participants performing the location task (voxelwise p = 0.05; clusterwise p = 0.01). Not surprisingly, given the individual group patterns, this analysis identified three regions as being significantly activated across all tasks\/groups: the dorsal portion of the left IFG, extending into the middle frontal gyrus (14.81cm3 volume; X = \u221240, Y = 7, Z = 33 BA 44\/9); the left SPL (5.82cm3 volume; X = \u221229, Y = \u221263, Z = 46 BA 7) and the medial portion of the SFG (5.61cm3 volume; X = 0, Y = 11, Z = 50 BA 6). These regions appear to make up a core network involved in phonological processing of both signed and spoken language, recruited by both deaf and hearing participants.\nDeaf versus hearing participants performing rhyme task, matched for performance\nTo determine the effect of hearing status on the neural system supporting phonological processing, we contrasted activation patterns in deaf and hearing participants performing the rhyme task. Since group performance on the rhyme task was poorer in deaf than hearing participants, subsets of 12 participants were selected from each group who were matched on accuracy and reaction time on the rhyme task performed in the scanner. To further control for differences between deaf and hearing participants the subgroups were also matched for age, non-verbal IQ, reading age, and accuracy and reaction time on a more extensive test of rhyme awareness, run prior to the scan session. There were no significant differences between the two groups on any of these variables (all p-values > 0.1; see Table 4).\nA between subjects ANOVA (voxelwise p = 0.05; clusterwise p = 0.005) indicated no regions in which hearing participants showed greater activation than deaf participants. The deaf subgroup showed greater activation than the hearing subgroup in the left IFG, extending into the middle frontal and the precentral gyri (7.01cm3 volume; X = \u221240, Y = 0, Z = 33; BA 44\/6) and in a small portion of the SFG, at the junction with the anterior cingulate (1.29cm3 volume; X = \u2212 4, Y = 4, Z = 50; BA 6\/32). Further analyses confirmed that the same pattern was observed when deaf native signers and deaf non-native signers were compared separately to matched hearing participants. This suggests that combining native and non-native signers, in order to carefully match subgroups of deaf and hearing participants, did not influence the outcome of this analysis.\nDiscussion\nOur results demonstrate that a very similar neural network supports phonological similarity judgements made in both English and British Sign Language (BSL). Given that these languages operate in such different modalities, these data suggest that this phonological processing network is multimodal or possibly to some extent supramodal: that is, involving representations that in some way \u2018transcend\u2019 the sensory modalities (see Fowler, 2004 for discussion). This network, which was also recruited by hearing people making rhyme judgements, consists of the medial portion of the superior frontal gyrus (SFG), the left superior parietal lobule (SPL) incorporating the superior portion of the supramarginal gyrus (SMG), and, most extensively, the left posterior inferior frontal gyrus (IFG) extending into the ventral precentral gyrus. We do not argue that these regions are dedicated to phonological processing. Rather we argue that they act together as a network to support phonological similarity judgements and other linguistic and, it is likely, non-linguistic processes (see Corina and Knapp, 2006). Nevertheless, our data are consistent with prior demonstrations, concerning semantic and syntactic processing, that modality has relatively little influence on the neural systems that support language (Neville et al., 1998; Petitto et al., 2000; Braun et al., 2001; Emmorey et al., 2002; MacSweeney et al., 2002b; Corina et al., 2003; MacSweeney et al., 2006). Demonstrating this in the context of phonological processing is even more striking since awareness of phonology is more directly linked to sensory input (which differs for sign and speech) than either semantic or syntactic processing.\nAlthough the observed network is recruited by both signed and spoken language, we demonstrate that it does not perform identically across languages or groups. Recruitment of different parts of this network is modulated by age of first language acquisition, language modality and hearing status. The impact of age of first language acquisition was explored by comparing deaf native and non-native signers. Non-native signers engaged the left posterior inferior frontal cortex to a greater extent than native signers. This was the case not only during the task performed in BSL, of which both groups had different language experience, but also during the task performed in English, of which both groups had similar experience. The differential recruitment of the left posterior IFG is even more striking given that native and non-native signers were matched on non-verbal IQ, English vocabulary score and reading age and that there were no significant group differences in accuracy or reaction time on the two phonological tasks. These are the first neuroimaging data to demonstrate the impact of age of acquisition of a first language in the brain. Lack of exposure to a fully accessible language early in life has implications for the neural systems supporting not only that language, but also for languages learned subsequently, whether signed or spoken. In conjunction with the behavioural data of Mayberry and colleagues (Mayberry et al., 2002; Mayberry and Lock, 2003) these data highlight the importance of early exposure to an accessible language for those born profoundly deaf. Even when signed, early language leads to the normal establishment of language systems that may then be used to facilitate a later learned language.\nEnhanced recruitment of the left posterior IFG has previously been reported during grammaticality judgements performed by late in contrast to early learners of German (Wartenburger et al., 2003) and during semantic judgement tasks in low- in contrast to high-proficiency late language learners (Chee et al., 2001; Wartenburger et al., 2003). Regions within the left IFG are differentially modulated not only by age of language acquisition and proficiency level, but also by extent of language use (Perani et al., 2003), age at time of testing, and task demands (Tatsuno and Sakai, 2005). Thompson-Schill et al. (2005) argue that the left IFG is increasingly engaged as selection demands increase. In particular, it is argued that this region is involved in regulating the cognitive control necessary to resolve competition between incompatible responses (Snyder et al., 2007). It is possible that selection demands increase for bilinguals because responses from both first and second languages are available. This situation applies to native signers (first language: BSL; second language: English), but may apply to a greater extent to non-native signers. Deaf non-native signers have knowledge of both BSL and English; however, both languages are acquired late. Despite equivalent behavioural proficiency on our tasks, both languages are likely to be poorly established in non-native signers, leading to greater conflict between potential responses, possibly resulting in enhanced recruitment of the left IFG.\nAn alternative, phonology-specific argument can also be made for the role of the posterior IFG. It has been argued that different parts of the left IFG may show preferential engagement in different aspects of language processing: phonological (the dorsal region: BA 44\/6), syntactic (the more anterior region: BA 45), and semantic (the ventral portion: BA 47) (Fiez, 1997; Price et al., 1997, Poldrack et al., 1999; Bookheimer, 2002; Devlin et al., 2003). While the baseline task used in the current study did not require picture name retrieval, given the relatively long presentation time (5s) it is likely that participants did name these stimuli (cf. Meyer and Damian, 2007). Furthermore, the network we identify in the current study has been reported in numerous previous studies of phonological processing involving written words (e.g., Lurito et al., 2000; Xu et al., 2001; Seghier et al., 2004). The most parsimonious interpretation of the current data is thus in terms of phonological processing. With regard to the left frontal cortex, as in previous studies, it was specifically the posterior portion of the IFG, extending into the ventral premotor cortex within precentral gyrus, that was the focus of activation involved in the phonological similarity matching tasks reported here. Moreover, this region was engaged to a greater extent by deaf participants during the rhyme than location task and more by deaf than hearing participants performing the rhyme task (see also Aparicio et al., 2007), even when behavioural performance was taken into account. To account for these findings, we suggest that when the auditory component of speech is absent, the articulatory\/motoric component makes a greater contribution to speech-based phonological processes. A similar explanation may account for the observed increased involvement of the left IFG during reading in children with developmental dyslexia following phonological remediation (e.g., Temple et al., 2003). Further studies with deaf participants are underway to test this hypothesis. We also found that the posterior portion of the left IFG was engaged more by non-native than native signers during tasks performed in both languages. Broca's area, in left posterior IFG, is engaged in sign production, just as it is in speech production (Braun et al., 2001; Corina et al., 2003; Emmorey et al., 2007). One possibility that may account for the effect of age of first language acquisition in this region is that the articulatory component of both speech and sign is less established in deaf non-native than native signers, leading to enhanced recruitment of this region during both tasks.\nFrom our data, it is not possible to distinguish between phonology-specific and cognitive control\/selection demands accounts of the differential engagement of the left IFG by native and non-native signers. Indeed both accounts may apply since the area showing differential activation in all of the contrasts reported here involved both the posterior portion of the left IFG and the ventral premotor cortex, in the precentral gyrus. Snyder et al. (2007) propose that the left IFG is involved in cognitive control, while the ventral premotor cortex demonstrates phonology specific properties. The relative role of these regions in language processing, and phonological processing in particular, will be greatly informed by future studies examining different domains of language (phonology, syntax, semantics) within the same group of deaf late language learners, while manipulating age of acquisition and proficiency.\nThe left parietal lobe was also recruited during both the location task (deaf participants) and the rhyme task (deaf and hearing participants). In all groups and tasks this activation included the superior portion of the supramarginal gyrus (SMG), extending into the superior parietal lobule (SPL) and medially into the precuneus. Previous studies of rhyme judgement of written words by hearing adults also report activation of this area (Lurito et al., 2000; Xu et al., 2001; Seghier et al., 2004; Snyder et al., 2007). Nevertheless, the exact role of this region in phonological processing remains unclear. It has been proposed that this multimodal integration region may be recruited during mapping between orthographic and phonemic representations (Booth et al., 2002; Eden et al., 2004). The fact that the stimuli in the current study were pictures, not written words, does not necessarily pose a problem for this account (but see Katzir et al., 2005). Hearing adults have been shown to engage this region more than children during auditory rhyme decisions (Booth et al., 2004). In addition, deaf people are more accurate and faster to make rhyme judgements when the labels for picture stimuli share orthography, e.g., cat\u2013mat, than when they do not, e.g., chair\u2013bear (Sterne and Goswami, 2000). Both of these lines of evidence suggest that orthographic representations may be activated in both deaf and hearing participants when making spoken language phonological decisions in response to pictures. Further studies are needed to examine this hypothesis and the role of the left parietal lobe in phonological processing.\nWhatever the functional role of the left parietal lobe in spoken language phonological processing, a growing number of studies suggest that this area may play a particularly important role in signed language processing (see Corina and Knapp, 2006). Perception of BSL sentences that involve spatial description engage the left inferior parietal lobule (IPL) and SPL to a greater extent than \u2018non-spatial\u2019 sentences in deaf native signers (MacSweeney et al., 2002a). Direct stimulation of the IPL induces phonological errors during sign production (Corina et al., 1999). Emmorey et al. (2007) have reported that sign production engages the left IPL (X = \u221260, Y = \u221225, Z = 27) and the left SPL (X = \u221226, Y = \u221251, Z = 54) more than speech production. Emmorey et al. (2007) propose that the left IPL may be involved in phonological processing while the left SPL may be involved in proprioceptive monitoring of motor output. In the current study, the observed greater activation in deaf signers during the location task than the rhyme task was located between the two foci reported by Emmorey et al. (2007) and incorporated the superior portion of the SMG in the IPL, extending into the SPL (X = \u22124, Y = \u221270, Z = 43). Given the nature of the current task, a phonological account of this activation seems more likely than an account based on proprioceptive monitoring. Corina and Knapp (2006) have argued that the left parietal lobe plays a greater role in signed than spoken language processing because signed language can build on the \u201c...prior existence of a general human system for manual action observation and production\u201d (p. 537). Portions of the parietal lobe are particularly attuned to the location of the hands\/body in space (Gerardin et al., 2000; Hermsdorfer et al., 2001). Accordingly, this region may be particularly engaged in tasks that focus on these spatial relationships, including those involving sign phonology. Whether this activation is specifically related to linguistic processing or to more general processing of body-related information requires further investigation, using linguistic tasks exploring both comprehension and production, and non-linguistic tasks.\nExamining other parameters of sign phonology, e.g., movement and handshape, is also required. It is not possible to claim a straightforward parallel between rhyme in spoken language and any of the parameters of sign phonology. It has been suggested that when any sign parameter is shared, this is more analogous to alliteration than to rhyme (Sutton-Spence, 2004). Location was chosen in the current study because it has been demonstrated to be important in judging sign similarity (Hildebrandt and Corina, 2002) and because, among the phonological parameters of signed languages, it appears to have the smallest inventory, as do vowels, which are the nucleus of the syllable and crucial for rhyme in spoken languages. However, sign phonologists may argue that movement is more analogous to vowels with respect to syllable structure because a sign is ill-formed without movement, just as a syllable is ill-formed without a vowel (Brentari, 1999; Sandler and Lillo-Martin, 2006). Whether different patterns of activation are observed when different sign parameters are examined or whether any form of sublexical analysis of signs elicits activation in the network reported here remains to be seen.\nIn summary, these data suggest that phonological processing, at least in the context of a phonological similarity judgement task, is to some degree supramodal. We show that a similar neural network supports phonological processing of both signed and spoken language in signed language users born profoundly deaf. Furthermore, this network was also engaged by hearing non-signers performing the similarity judgement task in English. However, different parts of this network were differentially weighted depending upon language modality, hearing status and, most importantly, age of first language acquisition. These data highlight the importance of learning a language, whether signed or spoken, early in life. Early acquisition of a first language is critical not only for processing that language, but also appears to form a base on which subsequently learned languages can successfully build.","keyphrases":["phonology","deaf","rhyming","sign language","inferior frontal gyrus","pictures","age of acquisition","plasticity","phonological awareness"],"prmu":["P","P","P","P","P","P","P","U","R"]}
{"id":"Int_J_Parasitol-1-5-1906861","title":"Altered phenotype and gene transcription in endothelial cells, induced by Plasmodium falciparum-infected red blood cells: Pathogenic or protective?\n","text":"Severe malaria is associated with sequestration of Plasmodium falciparum-infected red blood cells (PRBC) in the microvasculature and elevation of intercellular adhesion molecule-1 (ICAM-1) and TNF. In vitro co-culture of human umbilical vein endothelial cells (HUVEC), with either PRBC or uninfected RBC, required the presence of low level TNF (5 pg\/ml) for significant up-regulation of ICAM-1, which may contribute to increased cytoadhesion in vivo. These effects were independent of P. falciparum erythrocyte membrane protein-1 (PfEMP-1)-mediated adhesion but critically dependent on cell\u2013cell contact. Further changes included increases in IL8 release and soluble TNF receptor shedding. Microarray analysis of HUVEC transcriptome following co-culture, using a human Affymetrix microarray chip, showed significant differential regulation of genes which defined gene ontologies such as cell communication, cell adhesion, signal transduction and immune response. Our data demonstrate that endothelial cells have the ability to mobilise immune and pro-adhesive responses when exposed to both PRBC and TNF. In addition, there is also a previously un-described positive regulation by RBC and TNF and a concurrent negative regulation of a range of genes involved in inflammation and cell-death, by PRBC and TNF. We propose that the balance between positive and negative regulation demonstrated in our study will determine endothelial pathology during a malaria infection.\n1\nIntroduction\nPathogenesis of severe malaria has stirred considerable interest over the years, however there are many questions at the mechanistic level that remain unanswered. Severe malaria is characterised, in part, by sequestration of Plasmodium falciparum-infected red blood cells (PRBC) at microvascular sites in the brain tissue, leading ultimately to coma and subsequent death. There is evidence that the sequestration of PRBC on the endothelial surface can result in loss of integrity, thus compromising blood\u2013brain barrier function either directly (Brown et al., 1999a,b, 2001a), by indirect mechanisms including binding via platelets (Wassmer et al., 2004; Combes et al., 2006) or by the production of microparticles from endothelial cells (EC) activated by adhering PRBC (Combes et al., 2005, 2006), and also cytokine-driven modulation of endothelial cell metabolism, but that this is usually a small, transient effect in vivo.\nOne question is the mechanism underpinning the accumulation of PRBC leading to micro-vascular occlusion as seen in post mortem cerebral malaria (CM) brain tissue, which is believed to be a progressive phenomenon of PRBC sequestration. We suggest that the initial sequestration of PRBC, if maintained for a prolonged period of time, has the ability to activate the endothelium to promote sequestration, leading to deleterious effects on the host. Our studies investigate the direct effects on the endothelium of PRBC retention for prolonged periods of time.\nFirst, sustained exposure of the endothelium to PRBC can result in modulation of the endothelium, thus increasing its responsiveness to low levels of TNF (TNFlow). This may contribute to promoting further cytoadhesion in microvasculature containing sequestered PRBC. This hypothesis is based on previous evidence of endothelial cell activation by abnormal red blood cells in various conditions, including sickle cell disease, diabetes and also malaria (Shiu and McIntire, 2003). Sickle cell RBC (sRBC), diabetic RBC (dRBC) and also PRBCs can all induce expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) and E-selectin (Brown et al., 2001b; Shiu and McIntire, 2003; Viebig et al., 2005; Tripathi et al., 2006). In addition, recent co-culture studies have demonstrated PRBC-induced increases in the activity of pro-apoptotic genes involved in the caspase pathway (Pino et al., 2003). Other changes induced on EC by sRBCs include induction of endothelin and prostacyclin production (Shiu et al., 2002) and by PRBC include increased expression of pro-inflammatory genes (Pino et al., 2003) and release of IL6 (Viebig et al., 2005).\nSerum levels of IL8 were elevated in severe malaria compared with healthy volunteers (Lyke et al., 2004), correlated with parasite count and severity of disease at the time of admission (Burgmann et al., 1995) and there were no apparent differences between febrile and a-febrile volunteers (Hermsen et al., 2003). IL8 is an endothelial-derived acute response activation marker. Prestored IL8 is exocytosed at a basal level from Wiebel palade bodies in ECs and is rapidly released in response to various stimuli (Wolff et al., 1998; Utgaard et al., 1998; Oynebraten et al., 2004).\nExposure of ECs to sRBCs in vitro resulted in increased sensitivity to the pro-inflammatory cytokine IL1\u03b2 (Shiu et al., 2000). We propose that local increases in TNF levels occur in the microvasculature in early stages of infection, during shizogony (Bisser et al., 2006). In a similar fashion to that observed with sRBCs, contact between PRBCs and the endothelium could potentially sensitise the ECs to TNF. There is evidence for an important role for TNF receptors (TNFR) in CM pathology, however, most of this comes from studies on animal models. Lucas et al. (1997a) showed a close correlation between absence of TNFR II expression and protection from CM-associated brain damage in TNFR II knock-out mice, while TNFR II was up-regulated on brain endothelium in CM-susceptible mice (Lucas et al., 1997b). Up-regulation of the soluble TNFR I, which is the predominant type in human serum, was reported in the serum of patients with acute P. falciparum infection (Wenisch et al., 1994). Interestingly, EC from CM-susceptible mice had a greater sensitivity to TNF than that of CM-resistant mice. TNF induced up-regulation of TNFR I and II together with an associated increase in IL6, ICAM-1 and VCAM-1 to a greater extent in CM-susceptible mice than in CM-resistant mice (Lou et al., 1998). In addition, there was an absence of ICAM-1 up-regulation in TNFR II knock-out mice, suggesting a link between TNFRs and ICAM-1 up-regulation during malaria infection (Lucas et al., 1997a).\nOur aim was to investigate the ability of PRBC to modulate the endothelium in the presence and absence of the inflammatory cytokine, TNF, in a co-culture system. Firstly, functional markers of EC modulation included endothelial expression of ICAM-1, which has been attributed a critical role in parasite adhesion, and the release of IL8. Changes in levels of TNFR I and II, were also investigated as a potential mechanism for any changes in the sensitivity of EC to TNF. Second, we sought to assess the global transcriptional changes in ECs and elucidate the regulation of cellular processes following co-culture under the same conditions, using a human genome Affymetrix (Affymetrix, Santa Clara, CA, USA; http:\/\/www.affymetrix.com) chip.\nOur results have led us to propose a novel mechanism for the modulation of the endothelium during malaria infection that is dependent on low level TNF and involves a pro-inflammatory component but also a concurrent down-modulation of RBC-induced inflammation due to the presence of the parasite within the infected cell.\n2\nMaterials and methods\n2.1\nMalarial parasites\nPlasmodium falciparum ItG strain was derived from the Brazilian line IT4\/25\/5 (Ockenhouse et al., 1992). This strain was used for the PRBCs in these studies. The ItG strain is a strong ICAM-1 binder and also binds to CD36 (Gray et al., 2003). The PRBCs were cultured in RPMI-1640 supplemented with 2\u00a0mM l-glutamine, 37.5\u00a0mM N-2-hydroxyehtylpiperazine-N\u2032-2-ethanesulfonic acid (Hepes), 10\u00a0mM glucose, 25\u00a0\u03bcg\/ml gentamicin and 10% human serum, at pH of 7.2 (Trager and Jensen, 1976). All reagents were obtained from Sigma, UK. Human serum was isolated from whole blood obtained from the Royal Liverpool Hospital and was approved by the Liverpool School of Tropical Medicine Ethical Committee.\n2.2\nEndothelial cells\nPooled human umbilical vein endothelial cells (HUVEC) were obtained from Promocell (Heidelberg, Germany), HUVEC from different batches were used for each experiment, at passages three to five. HUVEC were grown to confluence on 1% gelatin (Sigma, UK) coated flasks and plates. All co-culture experiments were performed in serum-depleted basal HUVEC medium (quiescing medium) which consisted of M199 (Invitrogen, UK) containing 1% FCS. These conditions were designed to increase the signal window, while maintaining the integrity of the HUVEC monolayer (i.e. there was no indication of cell apoptosis or necrosis during the experiments).\nFor all co-culture studies, ECs were co-cultured with PRBCs and uninfected RBCs in the absence and presence of TNFlow. The sub-optimal dose was verified in separate TNF dose response studies; 5\u00a0pg\/ml was 1\/100th of the dose of TNF used as standard for optimal induction of ICAM-1 on HUVEC in our laboratory (0.5\u00a0ng\/ml), and had no significant effect on ICAM-1 expression (data not shown). For the positive control a high dose of TNF (TNFhigh) was used (10\u00a0ng\/ml) to stimulate the HUVEC, while medium alone served as a negative control. All parasite and EC cultures were regularly monitored for mycoplasma using the Takara PCR mycoplasma detection kit (Cambrex Biosciences).\n2.3\nPRBC-EC co-culture conditions\nHUVEC grown to confluence in either 24-well plates or 25\u00a0cm2 flasks, for functional studies and microarray studies, respectively, were co-cultured with PRBCs and uninfected RBCs in the absence and presence of TNFlow at 37\u00a0\u00b0C. The RBC suspension was adjusted to 1% haematocrit. PRBC at the trophozoite stage (20\u201328\u00a0h post-invasion) were used in all co-culture studies; for the functional studies the parasites were synchronised using sorbitol, while for the microarray studies the trophozoites were additionally enriched using plasmagel flotation. In selected experiments, the parasites were retrieved after the 20-h, stained with giemsa and examined by light microscopy. There was no apparent rupture of RBC or change in the parasite stage over this period. For all functional studies the PRBCs were at a parasitaemia of 3% and co-cultured with HUVEC for 20\u00a0h. For microarray studies, the PRBCs were enriched on Plasmagel to parasitaemia ranging between 50% and 60% and co-cultured for 6\u00a0h. The uninfected RBCs were from the same batches used for parasite culture and were maintained under the same conditions as PRBCs, in separate flasks.\n2.4\nRNA expression: microarray analysis\nFollowing incubation, the supernatant was aspirated, HUVEC was washed with cold RPMI-1640 and then with 0.02\u00a0M EDTA to remove the adherent RBCs and the cells harvested using Trizol (Invitrogen, UK). RNA integrity was evaluated by electrophoresis on a 1% agarose gel and by spectrophotometry using the absorbance ratio at 260\/280\u00a0nm.\nFive micrograms of each complementary biotin-labelled RNA was hybridised on the Affymetrix GeneChip, Human Genome version 2, HGU133plus2.0 (Santa Clara, CA), according to the manufacturer\u2019s instructions. The signal intensity for each feature on the array was determined using the 70th percentile method provided by GCOS software (Affymetrix). Hybridisations were performed for four replicates of each of the seven conditions (control, RBC, PRBC, RBC\u00a0+\u00a0TNFlow, PRBC\u00a0+\u00a0TNFlow, TNFlow and TNFhigh).\nData analysis was performed using Bioconductor (Gentleman et al., 2004). The microarrays were pre-processed using a Robust Multiarray Averaging program (Irizarry et al., 2003). The expression values were calculated using the R statistical computing environment (http:\/\/www.r-project.org\/) with the \u201caffy\u201d package (Gautier et al., 2004). The differential expression was assessed and variability estimated by fitting a linear model to the data that fully models the systematic part of each gene, using the \u201climma\u201d package. From the \u201climma\u201d output, log-fold changes from one condition compared with another, P-values corrected for a 5% false discovery rate (Hochberg and Benjamini, 1990), together with B-statistic (log-odds that a gene is differentially expressed) can be obtained and used to determine up- and down-regulation of genes. Due to the complexity of the microarray design (i.e. four replicates, seven conditions); for non-hierarchical clustering, the 47,000 transcripts present on the human chip were filtered down to 8105, based on their moderated F-statistic at P-value <0.001. These were then clustered by similarity. The unsupervised clustering program enabled us to discriminate biological clusters (ArrayMiner, OptimalDesign). For each cluster Gene Ontology (GO; http:\/\/www.geneontology.org) assignments were extracted using the GOstats package (http:\/\/bioconductor.org\/packages\/1.9\/bioc\/html\/GOstats.html). The gene list of each cluster was used to identify all the unique GO terms in order to determine whether genes that comprise a given cluster have a common function, process or location in the cell. For each GO term, GOstats analysed whether that term was being significantly over-represented in the data, using a P-value cut-off of P\u00a0\u2a7d\u00a00.05.\n2.5\nICAM-1 protein expression\nHUVEC grown to confluence in 24-well plates were co-cultured with PRBC and uninfected RBC at a haematocrit of 1% and a parasitaemia of 3% and suspended in M199 supplemented with 1% FCS in the absence and presence of 5\u00a0pg\/ml TNF (TNFlow) for 20\u00a0h at 37\u00a0\u00b0C. Following the incubation period, the supernatant was removed, centrifuged at 300g for 3\u00a0min to remove any RBCs and stored at \u221280\u00a0\u00b0C.\nThe HUVECs were washed once with cold RPMI-1640 and then with 0.02\u00a0M EDTA to remove the adherent RBCs and subsequently harvested by trypsinisation for analysis by flow cytometry. FACS ICAM-1 expression on HUVEC was determined by staining the cells using a fluorescein isothiocyanate (FITC)-conjugated mouse anti-human ICAM-1 antibody (MCA1615F; Serotec) using standard staining protocols and the cells fixed in 2% paraformaldehyde and analysed by flow cytometry. ICAM-1 expression was expressed as geometric mean of the fluorescence intensity.\n2.6\nIL-8 and TNF receptor expression\nThe supernatants stored from the co-culture studies were analysed using a standard sandwich ELISA kit (IDS), using a horse-radish peroxidise based colorimetric detection system, to quantify IL8 released from ECs. IL8 production was expressed as a concentration in pg per ml.\nSimilarly, soluble TNFR I, sTNFR I (p55) and soluble TNFR II, sTNFR II (p75), were detected using sTNFR I (KAC1761) and sTNFR II (KAC1771) ELISA kits (Biosource). TNFR level was expressed as TNFR concentration in ng per ml.\nIn order to understand the kinetics of TNFR expression on the surface of ECs in response to co-culture with PRBCs, the ECs were co-cultured with PRBCs and uninfected RBCs for 0.5, 1, 2 and 3\u00a0h. Following the incubation period, HUVECs were harvested and dual stained for surface TNFRs with monoclonal anti-human RII-FITC (FAB226F) and monoclonal anti-human RI-PE (FAB226F) antibodies (R&D Systems Europe). The receptor expression was expressed as the geometric mean of the fluorescence intensity.\n2.7\nTrypsin digestion of RBC\nThe ability of PRBCs to induce changes in surface ICAM-1 levels following trypsinisation was determined. PRBCs and uninfected RBCs were washed twice with PBS and incubated with 0.1\u00a0mg\/ml trypsin in PBS for 15\u00a0min at room temperature with gentle mixing (modified from Chaiyaroj et al., 1994). After incubation the trypsin was inhibited with FCS at a final concentration of 10%. Conditions for trypsinisation were optimised to prevent cell lysis and the cells were stained with giemsa before and after trypsinisation to confirm RBC integrity was maintained. The RBCs were washed three times in PBS and resuspended in M199 supplemented with 1% FCS and co-cultured with HUVEC, as described.\nWe also analysed adhesion of PRBCs to microslides coated with 50\u00a0\u03bcg\/ml Fc-tagged recombinant ICAM-1, ICAM-1-Fc (Gray et al., 2003), under laminar flow conditions at shear stress of 0.05\u00a0Pa, before and after trypsinisation, in order to evaluate PfEMP-1 mediated cytoadhesion.\n2.8\nTranswell experiments\nConfluent HUVECs were co-cultured with PRBCs separated using a 0.4\u00a0\u03bcm transwell filter (Falcon), which prevents contact between HUVECs and RBCs but allows soluble factors to diffuse through. This set-up allowed us to determine whether contact between RBCs and ECs was necessary for induction of ICAM-1 expression on the ECs.\n3\nResults\n3.1\nUp-regulation of ICAM-1 expression following co-culture with infected and uninfected RBC\nICAM-1 is expressed constitutively at low levels on the surface of HUVEC in culture. Neither PRBCs nor uninfected RBCs was capable of inducing appreciable ICAM-1 expression over 20\u00a0h (Fig. 1). However, exposure to both PRBCs and TNFlow simultaneously, resulted in significant up-regulation of ICAM-1 expression (P\u00a0<\u00a00.05). TNFlow alone was incapable of inducing ICAM-1 expression (Fig. 1).\n3.2\nUp-regulation of IL-8 release following co-culture with infected and uninfected RBC\nFollowing incubation with either PRBCs or uninfected RBCs for 20\u00a0h, IL8 levels in the supernatant were increased over basal levels (P\u00a0<\u00a00.01) (Fig. 2). When the HUVECs were concurrently exposed to PRBCs and TNFlow, IL8 release was further amplified over basal levels (P\u00a0<\u00a00.0002). Uninfected RBCs in combination with TNFlow induced similar levels of IL8 release (P\u00a0<\u00a00.001) (Fig. 2).\n3.3\nUp-regulation of soluble TNF receptor release and down-regulation of surface expression following co-culture with infected and uninfected RBC\nSoluble forms of both TNFR I (p55) and TNFR II (p75) were significantly increased by 35% (P\u00a0<\u00a00.05) following treatment with either PRBCs or uninfected RBCs for 20\u00a0h. Thus, the phenomenon of soluble receptor release from the surface of ECs, when co-cultured with RBCs, appeared to be independent of the parasite (Fig. 3a and b).\nSurface levels of the TNFRs were monitored over 3\u00a0h to understand the dynamics of TNFR expression. Both TNFR I and TNFR II were significantly reduced in response to both PRBCs and uninfected RBCs by 70% (P\u00a0<\u00a00.05) and 50% (P\u00a0<\u00a00.01), respectively, within 30\u00a0min (Fig. 4a). Interestingly, the levels of the TNFR I, but not TNFR II, fell over time under basal conditions, albeit at a slower rate than co-culture with RBCs. This could be a result of serum depletion when complete medium was replaced with M199\/1% FCS for the duration of the co-culture experiment. In the case of both receptors, the observed down-regulation in response to RBCs was significantly greater than that seen under basal conditions.\n3.4\nLoss of ICAM-1-mediated adhesion phenotype following trypsinisation of PRBC\nFollowing trypsinisation, PRBCs did not lose the ability to up-regulate ICAM-1 expression on co-culture with ECs (Fig. 5a). As before, both PRBCs and uninfected RBCs induced an increase in surface levels of ICAM-1 in the presence of TNFlow. ICAM-1 mediated adhesion under laminar flow, however, was abolished following trypsin treatment of PRBCs (Fig. 5b). In additional experiments, when the PRBCs were separated from the ECs in 24-well plates using 0.4\u00a0\u03bcm transwell inserts, there was no stimulation of ICAM-1 expression (data not shown). Thus, the EC activation observed in our system was critically dependent on cell\u2013cell contact, although it did not involve specific PfEMP-1-mediated cytoadherence.\n3.5\nModulation of the endothelial cell transcriptome following co-culture with infected and uninfected RBC\nUsing ArrayMiner software (OptimalDesign), seven non-hierarchical clusters were identified (Fig. 6a). As illustrated on the resulting heatmap, there is minimal change in the gene expression profiles with either PRBCs or RBCs compared with controls (Fig. 6b). However, when HUVECs were exposed to either PRBCs or RBCs in combination with TNFlow, differential expression was visible in three out of the seven clusters, compared with control or TNFlow alone (Fig. 6a and b).\nA comprehensive analysis of the microarray data was performed and has been submitted to Array Express (Accession No. E-SGRP-3). Our analysis focuses on genes that were differentially and significantly expressed at P\u00a0<\u00a00.001, compared with control levels. We have concentrated on over-represented gene ontologies and cited genes that best exemplify these GO terms.\nIn cluster 3, both PRBCs and uninfected RBCs, in combination with TNFlow, produced over-expression (Fig. 6a and b). TNFlow alone, however, produced a significant under-expression. Genes in this cluster included the inflammatory protease, caspase 1, the acute response cytokine, IL8, inflammatory cytokines IFN-\u03b3 and lymphotoxin \u03b2, adhesion molecules ICAM-1, E-selectin and chemokines, CXCL2, CXCL3, CX3C1 and CXCL6.\nIn clusters 1 and 2, opposing effects were induced when HUVECs were exposed to either PRBCs or RBCs in combination with TNFlow. Over-expression of genes was observed in response to uninfected RBCs and TNFlow and conversely, PRBCs and TNFlow resulted in under-expression of genes in these clusters (Fig. 6a and b).\nIn cluster 1, TNFlow on its own had no effect compared with controls, while in cluster 2 TNFlow alone produced a small reduction in gene expression (Fig. 6b). Genes in cluster 1 included the protease calpain 13, toll-like receptor-6 and the junction protein desmocollin 2, while genes in cluster 2 included the junction protein decorin, MHC class II, transferrin, endothelin 3 and endothelin receptor type A. While it is difficult to extrapolate roles for these genes from this in vitro analysis of the transcriptome, we can make some speculations based on recent studies. For instance, transferrin induction may be a mechanism for iron homeostasis to prevent accumulation of iron in endothelial cells which can lead to oxidative damage of the cells (Nanami et al., 2005). Endothelin is a known vasoconstrictor, in vivo; down-regulation of endothelin and its receptor may be a mechanism for maintaining vascular tone and integrity during malaria infection (Basilico et al., 2004). Down-regulation of MHC class II may represent suppression of the host immune response by the parasite.\nThe genes in each cluster were further analysed and categorised by GO) terms using the GOstats package of R\/Bioconductor to understand their basic biological functions. The over-represented GO terms, defining genes in each cluster, in terms of biological process, cellular component and molecular function, showing \u2a7e2% of the total number of genes being annotated with that term, were evaluated. Fig. 7 illustrates distribution of the over-represented gene ontologies with respect to biological process and cellular component, in clusters 1 and 2, which reflect inflammatory and protective functions in the endothelial cells. The full analysis for all three clusters is detailed in Supplementary Fig. S1(see on-line supplementary data). Whilst some GO terms fell below the 2% cut-off, it is important to note that they were nonetheless significant and have been tabulated for further information (Supplementary Tables 1, 2 and 3; see on-line supplementary data). A wide range of GO terms were over-represented in each cluster within categories of biological process, cellular component and molecular function, with a number of common and parallel themes, including cell communication, signal transduction, cell adhesion, organismal physiological process, ion transport, response to external stimuli and immune response. Interestingly, the genes in each of these clusters were primarily cell membrane-associated, or related to the extracellular region and the extracellular matrix. The molecular functions of the genes in each cluster were wide ranging, although receptor activity, signal transducer activity, ion channel and protein binding activities were parallel themes in all three clusters.\nThe fold changes for the 8105 genes were calculated for each of the conditions for visualizing the changes in expression, compared with control levels. Expression of ICAM-1 and IL8 RNA was up-regulated, reaching 3.7-fold and 7-fold, respectively, over control levels (P\u00a0=\u00a00.001), when HUVECs were incubated with a combination of PRBCs and TNFlow. Interestingly, ICAM-1 transcription reached similar levels in the presence of a combination of uninfected RBCs and TNFlow. Table 1 illustrates the changes in ICAM-1 and IL8 expression under the different co-culture conditions.\nTable 2 shows the changes in expression of selected genes which illustrate specific biological processes of ECs that may have a role in mediating either pathology or protection, and their respective fold changes in the presence of PRBCs, uninfected RBCs and TNFlow compared with controls. Although the magnitudes of change are relatively small, they are highly reproducible and biologically significant at P\u00a0<\u00a00.001, over four separate replicates.\nThe chemokines, CXCL2 (Gro-\u03b2), CXCL3 (Gro-\u03b3), CXCL6 and E-selectin were all over-expressed in the presence of PRBCs and TNFlow, with variable increases in expression of CXCL2 and CXCL6 in the presence of uninfected RBCs and TNFlow (Table 2). Gro-\u03b2 has been shown to have a role in the adhesion of monocytes to ECs (Schwartz et al., 1994) while CXCL6 has similar properties to IL8 and can be co-induced with IL8 (Gijsbers et al., 2005). E-selectin was also over-expressed in response to PRBCs, uninfected RBCs and TNFlow, reaching up to 7-fold over controls with PRBCs and TNFlow.\nMolecules involved in cell adhesion such as ICAM-1 and CX3C1 were both over-expressed in PRBCs, uninfected RBCs, and TNFlow, with a greater effect in PRBCs (Table 2). CX3C1 (fractalkine) has recently been implicated in mediating adhesion of PRBCs to ECs (Hatabu et al., 2003). Other adhesion receptors such as desmocollin 2 and integrin \u03b21 were both under-expressed with PRBCs and TNF, and over-expressed with uninfected RBCs and TNF (Table 2).\nA number of inflammatory genes such as MHC class I, superoxide dismutase 2 (SOD 2) and lymphotoxin-\u03b2 were all over-expressed in the presence of PRBCs and TNFlow (Table 2). Whilst both MHC class I and SOD 2 were also over-expressed with uninfected RBCs and TNFlow, lymphotoxin \u03b2 was under-expressed.\nTable 2 illustrates differential expression of molecules such as TNFR-associated factor 1 (TRAF 1), Fas associated factor 1 (FAF-1), prostaglandin receptor 3 and TGF-\u03b23, which all have a role in signal transduction.\nA number of cell apoptosis-related genes were differentially regulated in our co-culture system (Table 3). Both caspase 3 and caspase 9, which are believed to be inducers of apoptosis (Ho and Hawkins, 2005), were under-expressed in the presence of PRBCs and TNFlow. In addition, caspase 3 expression was markedly increased in the presence of uninfected RBCs and TNF. Calpains, which are inducers of cell death via a caspase-independent pathway (Utz and Anderson, 2000; Perrin and Huttenlocher, 2002), were also regulated in our system. We looked at the expression of two calpains, 9 and 13. Calpain 9 was under-expressed and there was a small increase in calpain 13 expression in the presence of PRBCs and TNFlow (Fig. 8). Thus, our data does not suggest induction of apoptosis; in fact, there appears to be a trend towards protection from apoptosis and this was supported by our inability to detect any positive staining with annexin V and the absence of any morphological changes in the ECs after co-culture.\n4\nDiscussion\nSequestration of PRBCs at vascular sites is a critical event in the pathogenesis of severe malaria. Here we have investigated the effect of prolonged exposure to PRBCs on the endothelium. We have demonstrated a phenomenon whereby ECs are differentially modulated when co-cultured with PRBCs in the presence of TNFlow, which could occur at microvascular sites early in infection, for instance during schizogony. Unlike previously published work, in our system there was no apparent induction of ICAM-1 or modulation of RNA transcription by PRBCs alone. Significant de novo ICAM-1 induction and differential regulation of gene transcription were dependent on the presence of low levels of TNF. PRBCs alone did modulate certain EC functions such as IL8 release and TNFR shedding from the cell surface, but IL8 release was further enhanced in the presence of TNFlow.\nUp-regulation of ICAM-1 was maintained following removal of surface expressed PfEMP-1 by trypsin digestion, but was abolished when cell\u2013cell contact was prevented. This demonstrated that while cell-to-cell contact was crucial for modulating EC function, there was no requirement for adhesion via specific receptors in this model. Despite the differences in the reported effects of PRBCs on ECs, the unifying theme in all the co-culture studies is this critical requirement for close apposition of the two cell types in the co-culture system, in the induction of EC activation. The transwell system, whilst preventing cell-to-cell contact, allowed the movement of parasite-derived soluble factors. Therefore we could exclude the role of soluble parasite-derived factors in EC activation. In all cases, when the cell\u2013cell contact was compromised using a filter, EC activation was abolished (Randolph and Furie, 1996; Pino et al., 2003; Viebig et al., 2005). Thus the variable observations may reflect differences in the respective co-culture conditions, for instance, studies by Viebig et al. (2005) were performed in complete HUVEC culture medium and studies by Pino et al. (2003) were performed in parasite culture media, while our studies were performed in a serum-depleted basal HUVEC medium in order to enhance the signal window for the EC measurements, whilst maintaining EC integrity. It is clear from our work and that of others, that care will need to be taken in interpreting the results of model co-culture systems and that further work will be required to develop ex vivo models of severe disease. It is possible that no single model will be able to reflect the variable pathology represented in malaria infection. Our study is unique in the observation that concomitant presence of low level TNF is necessary for PRBCs to modulate the endothelium, namely HUVECs. Indeed, a recent study also suggested that PRBCs alone do not have the ability to induce ICAM-1 in HUVECs, and that this may be a phenomenon specific to brain-derived ECs (Tripathi et al., 2006).\nUsing HUVEC as an in vitro model for studying PRBC-EC interactions in vivo, we have demonstrated that close apposition or cell\u2013cell contact of the PRBCs with ECs is a critical factor in mediating this activation. Whilst uninfected RBCs also had an effect on ECs in the presence of low level TNF, it is noteworthy that in vivo, apposition of uninfected RBCs on ECs only occurs secondary to cytoadhesion or as a result of vessel occlusion. The ability of normal RBCs to stick to and modulate ECs in vitro, however, is not a novel observation. Indeed, normal RBCs can bind to ECs mediated by plasma factors such as fibrinogen and fibronectin (Wautier et al., 1983) and the binding can induce low-grade ICAM-1 expression in vitro (Brown et al., 2001b). RBCs express a wide range of molecules on their surface which have been implicated in mediating adhesion to endothelial cells under different conditions. These include: (i) VLA-4 (alpha4beta1) which can bind to endothelial VCAM-1 (Walmet et al., 2003); (ii) the blood group Lutheran molecule (LU) over-expressed on sRBCs can bind to laminin present on cells or in the intercellular space (Eyler and Telen, 2006); (iii) advanced glycation end products (AGEs) present on RBCs bind to their receptor (RAGE) on endothelium (Wautier and Schmidt, 2004), activating endothelial cells; and (iv) a molecule related to blood group Rhesus, ICAM-4 (Hermand et al., 2003) binds to integrins present on leukocytes (CD11\u2013CD18) and on platelets (alpha2beta4) offering a surface which can be involved in thrombosis (reviewed by Wautier and Wautier, 2004). Thus, although PfEMP-1 does not play a role in the induction of ICAM-1 expression seen by us, it would normally be required to bring the PRBCs and ECs together in vivo.\nContrary to our hypothesis that up-regulation of TNFRs in response to PRBCs contributed to the increased sensitivity to TNF, unexpectedly, we observed significant down-regulation of surface TNFR expression and increased receptor shedding. It is possible that the enhancement of the effects of RBCs is at the level of intracellular signal transduction. Rapid TNFR shedding by cleavage of the extracellular region of the receptors as observed in our studies is a common response to various stimuli (Aderka et al., 1998) and may be a mechanism for down-modulation of both TNF receptors. This rapid release of TNFRs in response to an insult to the endothelium or physical cell contact with the endothelium is believed to be a non-specific pro-inflammatory response and up-regulation of soluble TNFR I has been correlated with parasitaemia in the serum of patients with acute P. falciparum infection (Wenisch et al., 1994). The TNFR shedding seen in response to PRBCs or uninfected PRBCs alone, may be part of a non-specific inflammatory response of ECs to prolonged cell\u2013cell contact. Similarly, IL8 release in our system may also represent a non-specific inflammatory response to cell\u2013cell contact with RBCs, as was demonstrated in a monocyte\/EC co-culture model and was further shown to be independent of ICAM-1 mediated binding (Lukacs et al., 1995).\nSimilarly to our direct measurements of ICAM-1, there was no significant change in EC gene transcription in response to PRBCs or RBCs alone but in combination with TNFlow, significant regulation of the EC transcriptome was induced. Analysis of the transcriptome revealed a wide diversity of gene ontologies representing genes that were significantly regulated, however, care must be taken in interpreting the data since there is a high degree of redundancy across GO terms, which was not accounted for in this analysis. IL8, for instance, has diverse biological roles including cell communication, cell adhesion, signal transduction and immune response. In spite of such deficiencies, the GO terms that were over-represented, including cell communication, cell adhesion, signal transduction, ion transport and immune response, fit well with our hypothesis that the infected RBC is able to modulate the host endothelial response.\nFrom previous work, we had expected to see a pro-inflammatory response induced by PRBCs (cluster 3, Fig. 6b). Less predictable was the down-regulation of genes in the presence of PRBCs and TNFlow (clusters 1 and 2, Fig. 6b), which may represent a parasite-specific adaptive response that favours parasite survival and development by protecting the integrity of the host endothelium. Up-regulation of these genes under the same conditions may result in the induction of a pro-adhesive effect that can potentially feed back to reinforce pathological sequestration that is typically seen in the later stages of severe malaria. In addition, induction of an immune response, as in cluster 3, may confer a protective effect on the host. Thus, we propose that the balance between these converse effects of PRBCs and TNF on the endothelium may be the critical factor in determining the response by endothelium and the clinical outcome of a malaria infection in vivo.\nThe over-expression of genes in response to uninfected RBCs and TNFlow in two of the clusters, whilst unexpected, is a common phenomenon in static in vitro co-culture studies. For instance, normal RBCs can interact with ECs to induce the expression of a variety of pro-inflammatory molecules including ICAM-1 (Brown et al., 2001b), VCAM-1 (Brown et al., 2001b; Wautier et al., 2001) and activation of NADPH oxidase, which mediates the generation of reactive oxygen intermediates (Wautier et al., 2001). This is a potential mechanism to induce expression of genes to counteract the response to PRBCs and may reflect a partially protective host response during a malaria infection, balanced against inducing a pro-adhesive environment, particularly early in infection when systemic inflammation is unlikely to be a major contributor to pathogenesis.\nModulation of host cells has been demonstrated in other co-culture models, for instance tyrosine phosphorylation of scyncitiotrophoblasts in a model of placental malaria (Lucchi et al., 2006) and ecto-phosphorylation of CD36 following the initial PRBC attachment which is believed to further enhance CD36-mediated cytoadhesion (Yipp et al., 2003). While co-culture models give useful insights into mechanisms of action of PRBCs on host cells in malaria pathogenesis, care must be taken in the interpretation and extrapolation of this data to pathogenesis in vivo, since these mechanisms may not be reflected in the in vivo situation.\nIn summary, our co-culture model identifies a novel mechanism whereby PRBCs, whilst mobilizing a pro-inflammatory and a seemingly pro-adhesive effect on the host endothelium, have the additional ability to suppress gene expression pathways involved in signal transduction, apoptosis and immune response as part of a mechanism to support parasite survival through maintaining the overall integrity of the host EC. This fits well with histo-pathological observations in severe malaria where transient dysfunction of the blood\u2013brain barrier is observed (Brown et al., 1999a) but the overall health of the endothelium seems to be maintained despite the presence of many adherent PRBCs (Medana and Turner, 2006). Our suggestion is that the host response to sequestration is complex and involves a balance between pro- and anti-inflammatory pathways, both mediated by apposition of the PRBC, and interacting with other mechanisms of host response to malaria infection variably throughout an infection. This balance is critical to the health of an EC and the resulting pathology associated with severe malaria.","keyphrases":["severe malaria","icam-1","co-culture","microarray","vascular endothelium","tumour necrosis factor"],"prmu":["P","P","P","P","R","M"]}
{"id":"Mol_Syst_Biol-3-_-1800356","title":"Tissue-specific regulatory elements in mammalian promoters\n","text":"Transcription factor-binding sites and the cis-regulatory modules they compose are central determinants of gene expression. We previously showed that binding site motifs and modules in proximal promoters can be used to predict a significant portion of mammalian tissue-specific transcription. Here, we report on a systematic analysis of promoters controlling tissue-specific expression in heart, kidney, liver, pancreas, skeletal muscle, testis and CD4 T cells, for both human and mouse. We integrated multiple sources of expression data to compile sets of transcripts with strong evidence for tissue-specific regulation. The analysis of the promoters corresponding to these sets produced a catalog of predicted tissue-specific motifs and modules, and cis-regulatory elements. Predicted regulatory interactions are supported by statistical evidence, and provide a foundation for targeted experiments that will improve our understanding of tissue-specific regulatory networks. In a broader context, methods used to construct the catalog provide a model for the analysis of genomic regions that regulate differentially expressed genes.\nIntroduction\nReverse engineering mammalian transcriptional regulatory circuits can be achieved using systematic methodology that includes both computational and experimental techniques, working in tandem to generate, refine and verify hypotheses. Understanding tissue-specific transcription is a necessary step for extending regulatory circuit reverse-engineering efforts from single-cell eukaryotes to metazoans. We recently demonstrated that the information in proximal promoters can predict a significant portion of tissue-specific elevated or inhibited expression (Smith et al, 2006). Here, focusing on tissue-specific regulatory pattern identification and prediction accuracy instead of proof of existence, we use refined analysis and data curation methods to discover and catalog high-confidence regulatory interactions and sites. This catalog will assist experimental efforts to reverse engineer tissue-specific transcriptional regulatory networks from the bottom up.\nNumerous techniques for analysis of regulatory sequences have been proposed, and the problem of module identification is now receiving due attention (Zhou and Wong, 2004; Gupta and Liu, 2005; Zhu et al, 2005). Previously characterized binding site motifs have been used to infer transcription factor function in certain tissues (Nelander et al, 2005). Xie et al (2005) identified conserved motifs across ortholog promoters of four mammalian genomes. Robertson et al (2006) describe cisRed, a database that integrates genome annotation data, homology data and genome alignments to identify motifs with conserved sites across mammals. We analyzed proximal promoters with evidence for tissue-specific regulation in order to identify tissue-specific motifs, modules and their sites in proximal promoters. We developed a new technique for characterizing tissue-specific modules that ensures that each module component significantly improves tissue-specific module enrichment.\nWe integrated multiple sources of expression data to identify reliable sets of transcripts that are under tissue-specific regulation in human and mouse. Using transcription start site (TSS) annotation in Cold Spring Harbor Mammalian Promoter Database (CSHLmpd) (Xuan et al, 2005), we compiled sets of proximal promoters corresponding to transcripts with evidence for specific regulation in the selected tissues. Our analysis was based on motifs discovered de novo (called novel motifs) using DME (Smith et al, 2005a) and DME-B (Smith et al, 2006), as well as previously characterized vertebrate binding-site motifs (called known motifs) from TRANSFAC (Matys et al, 2003) and JASPAR (Sandelin et al, 2004). We evaluated motifs according to enrichment in tissue-specific promoters relative to other promoters from the same species. We showed that motifs associated with factors with known tissue-specific roles rank high for enrichment, that motif ranks are significantly correlated between human and mouse and that this same set of motifs and their corresponding cis-elements are unlikely to be identified using traditional, order-preserving alignments of ortholog promoter sequences. We constructed modules of interacting motifs (both novel and known motifs), ensuring that each component contributed significantly to the enrichment of the whole module. We annotated tissue-specific promoters with predicted tissue-specific regulatory elements and demonstrated that these sites are in excellent agreement with experimentally annotated liver-specific sites in the human albumin promoter and skeletal-muscle-specific sites in the human \u03b1-actin promoter. Both promoters are particularly well annotated with experimentally verified tissue-specific regulatory elements and permit an informative comparison. In other tissues, we gave predicted sites for tissue-specific motifs in representative promoters. The complete data and analysis are available in TCat: The Catalog of Tissue-Specific Regulatory Motifs (http:\/\/rulai.cshl.edu\/tcat).\nResults\nWe describe first steps toward cataloging high-confidence tissue-specific motifs, modules and their sites. We first collected and integrated expression and function data from various sources, and identified transcripts that are likely to be under tissue specific regulation. We demonstrated that transcripts with evidence for tissue-specific regulation from multiple expression sources in one species (human or mouse) are significantly more likely to have evidence for tissue-specificity in the other species. We analyzed and annotated proximal-promoter sets in seven representative tissues from both human and mouse, demonstrating that motifs and predicted binding sites are in agreement with experimentally verified data and that analyses in human and mouse are significantly correlated. We also showed that the top-scoring sites in orthologous tissue-specific promoters from human and mouse rarely have significant conservation of site order, suggesting that comparative genomics alone may not be sufficient to decode the regulatory signals in these proximal promoters.\nTranscripts under tissue-specific regulation\nFew transcripts have expression restricted to a single tissue, but many transcripts appear to be regulated in a tissue-specific manner (Su et al, 2004), and the corresponding promoters are likely to contain tissue-specific regulatory elements. To circumvent problems associated with individual sources of information, we used a voting system that combined information about expression, function and tissue specificity from different sources. Table I gives the number of transcripts with single and multiple sources of evidence (votes) for tissue-specific regulation in each tissue. Orthologs of transcripts with multiple votes for tissue-specific regulation were more likely to have evidence for specific regulation in that tissue, suggesting that the false-positive rate for calling a transcript tissue specific is lower when based on multiple votes. The number of ortholog transcript pairs with multiple votes for tissue-specific regulation in both species ranged from 1 in CD4 T cells to 69 in liver. Table II lists genes and orthologous transcripts with votes for skeletal-muscle-specific regulation in both human and mouse. Gene and transcript lists for other tissues are given in Supplementary Section 1.5.\nEnrichment of known tissue-specific motifs\nKnowledge of factors and corresponding binding sites that regulate tissue-specific transcription can be used to evaluate motif ranking. We measured motif enrichment in tissue-specific promoter sets using balanced error rates, evaluating motifs for their ability to distinguish tissue-specific sets (foreground sets) from background sets that are composed of non-tissue-specific promoter samples from CSHLmpd. Balanced error rates measure proportions of misclassified promoters after normalization of foreground and background sizes. We ranked motifs according to enrichment and determined whether the ranks assigned to binding-site motifs for factors with known tissue-specific roles are significantly elevated in the corresponding tissues. The results presented in Table III demonstrate that binding-site motifs for these factors ranked significantly high (P<0.01) according to a Wilcoxon signed-ranks test in almost all tissues tested. Excluding DBP in human liver and HNF-3 in mouse liver, these factors had evidence for expression in their respective tissues, and their binding-site motifs were highly enriched in our foreground sets. Results are summarized in Table IV, and the motifs with greatest enrichment in each tissue are given in TCat. Table IV also includes information for HNF-6 in liver. HNF-6 is a known liver regulator, but there is no evidence for its expression in liver based on our data, and its binding-site motif was not enriched in our liver foreground sets. In addition, C\/G- or A\/T-rich motifs are likely to be enriched in foreground sets that are C\/G or A\/T rich relative to promoter base composition. To eliminate this potential bias, we adjusted the GC content in background sets to match foreground sets. Some known tissue-specific motifs were identified as enriched only after GC content correction.\nNuclear receptor binding-site motifs and E-box motifs are among the top enriched motifs in 11 and 10 of the 14 human and mouse tissues, respectively. A Wilcoxon signed-ranks test showed that nuclear receptor and E-box motifs (represented by 54 and 39 TRANSFAC motifs, respectively) are enriched in the union of our foreground sets with P-values below 6.07E\u221214 and 2.02E\u221213 (nuclear-receptor motifs) and 2.22E\u221210 and 4.57E\u221203 (E-box) in human and mouse, respectively. These results suggest a diversity of tissue-specific roles for nuclear receptors and E-box binders, likely mediated by tissue-specific cofactors.\nTissue-specific cis-regulatory elements\nHighly enriched motifs, and the associated score thresholds identified by our methods, provide a starting point for targeted experimental annotation of tissue-specific promoters. Figure 1 shows known and predicted sites mapped on the \u2212500 to +100 region of the human albumin promoter and the \u2212250 to +50 region of the human skeletal muscle \u03b1-actin promoter. Human albumin has known functional binding sites for HNF-1, C\/EBP, AFP and NF-Y (Paonessa et al, 1988; Sawadaishi et al, 1988; Frain et al, 1990; Li et al, 1990), all of which were identified among the top predicted motifs or included in top modules for liver. Locations of known sites for C\/EBP (at \u2212437) and NF-Y (at \u2212125) do not perfectly align with the corresponding predicted sites (at \u2212462 and \u2212143, respectively), but current knowledge about binding sites for those factors raises the possibility that the predicted locations are more accurate. The only predicted binding site for these factors in the human albumin promoter that is not depicted in the figure is a C\/EBP site at \u2212956. Human skeletal muscle \u03b1-actin has known sites for SRF, TEF and a known TATA box (Boxer et al, 1989; MacLellan et al, 1994), all among top predicted motifs for human skeletal muscle (TATA box motifs have high similarity with MEF-2 motifs). Figure 2 gives predictions in a representative human promoter in each of the remaining tissues.\nComparison to previous results\nPrevious analysis of tissue-specific patterns in regulatory regions includes analysis based on cross-species conservation (Xie et al, 2005) and coexpression (Smith et al, 2006).\nXie et al (2005) identified conserved elements in orthologous promoters of four mammalian genomes. They found 59 experimentally validated motifs that are significantly conserved and enriched in at least one human tissue. These include E-box, ETS, MEF2, MEIS1 and NF-1 in skeletal muscle; Chx10 in kidney; NRF-1, ELK-1, GABP and E12 in CD4 T cells; AP-4 and MEF-2 in heart; and NRF-1 in testis. Our results agree with Xie et al (2005) on the enrichment of E-Box and MEF-2 in skeletal muscle, ETS in CD4 T-cells and E-box in pancreas.\nIn previous work (Smith et al, 2006), we tested the hypothesis that proximal promoters contain information that can be used to predict tissue-specific expression. We were not concerned with identifying the most significant tissue-specific motifs, modules and sites. Considering the difference between the goals of the two projects, it is not surprising that the predictive models described by Smith et al (2006) have little similarity to our top motifs and modules. The most significant similarities between our top tissue-specific patterns and the predictive models of Smith et al (2006) include the enrichment of ETS in CD4 T-cell-specific promoters and the enrichment of Smith et al (2006) motifs Novel3 and Novel6 in mouse testis and Novel1 in human testis. The three novel testis motifs are very similar to motifs that rank in the top 100 in our analysis, but the enrichment of these motifs was not sufficiently high for inclusion in TCat.\nCorrelation between human and mouse regulatory regions\nWe compared motif enrichment ranks in each human foreground set to ranks in the corresponding mouse foreground set using Spearman's rank correlation test, and found that enrichment ranks across species are highly correlated (P<0.001) for all but CD4 T cells (Supplementary Table 15). In CD4 T cells, motif enrichment ranks are similar only for few highly enriched motifs. Despite the motif-enrichment ranks correlation, the order of the top predicted binding sites is not usually conserved between orthologous promoters. Fewer than 10% of orthologous pairs showed significant (P<0.01) conservation of site order. Weak site-order conservation suggests that the top tissue-specific sites would be difficult to identify using traditional cross-species alignment alone, and methods that rely on co-linear promoter alignment may have high false-negative detection rates. This evidence is in agreement with Frith et al (2006), who found that homologous transcription start sites can be separated by more than 100 nucleotides. A list of the nine genes (out of 102 candidates) with significant conservation of site order is given in Supplementary Section 2.3.\nMaterials and methods\nThe steps used in creating the catalog include (1) identifying tissue-specific transcripts, (2) identifying factors that are expressed in each tissue, (3) obtaining promoter sequences for tissue-specific transcript, and (4) identifying individual motifs and modules (i.e. sets of interacting motifs) that characterize tissue-specific promoter sets.\nIdentifying tissue-specific transcripts\nTo identify motifs and modules that regulate tissue-specific transcription, we analyzed promoters of transcripts that appear to be regulated in a tissue-specific manner. If an information source indicated that a transcript has restricted expression, unusually high expression, or a specific function in the tissue, that source voted for tissue specificity of the transcript. For each tissue, we sorted the transcripts according to the number of votes received, retaining the top 100 with distinct TSS as tissue specific. Ties in the ranking were broken according to intensity values from the GNF SymAtlas expression data (discussed below), which we have found to be the most complete and the most reliable source of tissue-based expression information. We used the same number of transcripts for each tissue to facilitate comparison across tissues, and 100 sequences provided sufficient information for our analysis while allowing identification of well-known tissue-specific motifs.\nMicroarray data\nThe GNF SymAtlas microarray data were generated using Affymetrix HG-U133A array and the custom GNF1H and GNF1M Affymetrix arrays, and include expression profiles for 79 human and 61 mouse tissues (Su et al, 2004). Among these are the seven tissues we selected to include in the catalog. Tissues were selected with consideration to data availability in GNF and other sources and interest from Zhang lab members and collaborators. A transcript received a vote for tissue specificity from this information source if it was called present and its intensity exceeded its mean across all tissues by 3 standard deviations.\nThe Hughes Toronto microarray data (Zhang et al, 2004), which was generated using custom-built oligonucleotide arrays, provide mouse expression profiles for 55 tissues, including all of our tissues but CD4 T cells. A transcript received a vote for tissue specificity if it was called present in the tissue, and had intensity at least 10 standard deviations above its mean across all 55 tissues. This large number of standard deviations was required to limit the number of transcripts receiving positive votes.\nThe GeneNote expression profiles (Shmueli et al, 2003), which were generated using the Affymetrix GeneChip HG-U95, provide human expression data for 12 tissues, including all of our tissues but CD4 T cells and testis. The GeneNote data were used in the same way as the GNF SymAtlas data, with a transcript being called tissue specific if it is present in that tissue and has intensity at least 3 standard deviations above its mean across all 12 tissues.\nEST data\ndbEST is a database of expressed sequence tags (Boguski et al, 1993), and contains source information, such as the tissue of origin, for each EST. This information is used to annotate UniGene clusters with the source data, and a UniGene is said to have restricted expression in a tissue if more than half of the ESTs contributing to that UniGene have the same source tissue. A transcript received one vote for specificity in a particular tissue if the corresponding UniGene cluster is annotated as having expression restricted to that tissue.\nGO terms\nWe associated a set of GO Terms with each tissue. This was performed by compiling a set of keywords for each tissue (e.g. \u2018renal' was associated with kidney; \u2018sperm' was associated with testis), and searching GO Term names and definitions for those keywords. This produced, for each tissue, a set of GO Terms that were subsequently reviewed to ensure that the context of the keywords was appropriate. A transcript of a gene annotated with a GO Term that is associated with a tissue received a vote for specificity in that tissue.\nSelecting promoter sequences\nAlthough regulatory elements can exist almost anywhere in the genome, they are concentrated near the TSS (Cooper et al, 2006). We used the CSHLmpd to map transcripts to promoters, using experimentally confirmed promoters from EPD (Perier et al, 1998), DBTSS (Suzuki et al, 2002) and GenBank, as well as computationally predicted promoters. For each promoter, we used the proximal sequence region of \u22121000 to +100 relative to the TSS.\nEach part of our analysis is based on comparing the tissue-specific promoter sets to a background of random promoters from the same species. For each tissue, a background set was constructed by selecting 1000 transcripts uniformly at random from the set of RefSeqs for the corresponding species with TSS annotation in CSHLmpd. For each tissue, transcripts with at least one vote for specificity in that tissue were removed from consideration before selecting the background.\nBecause our analysis focused on proximal promoters, \u22121000 to +100 relative to the TSS, if the TSS annotation is off by several hundred base pairs, important promoter regions might be excluded. AKR1D1, identified as liver specific by our voting system, has two known TSSs within 500 bp of the first exon for the corresponding RefSeq (NM_005989) (Charbonneau and Luu-The, 1999). We used the TSS located upstream of the first exon, but could have chosen to use the other promoter, which was annotated by a generally more reliable source (DBTSS versus GenBank). Currently, in such situations, there is little information that identifies the promoter responsible for observed tissue-specific regulation, but comparative genomics and rapidly improving arrays promise better 5\u2032 end identification, thus improving proximal promoter annotation and association between transcripts and promoters (Kim et al, 2005; Carninci et al, 2006). Negative promoter sets can be used to cancel out patterns that are not related to tissue-specific transcription regulation. We use random negative promoter sets with and without GC-content correction; this correction cancels the inuence of genomic GC-content isochore variability.\nIdentifying and evaluating motifs\nGiven a motif M (represented as a position\u2013frequency matrix) and a sequence S, the max-score of M in S, max-score (M, S) is the score of the top scoring subsequence of S when aligned against the scoring matrix for M. Details on constructing and using scoring matrices can be found in Stormo (2000). For a fixed threshold \u03bb, the max-score classification method classifies S as belonging to the foreground if max-score (M,S)\u2a7e\u03bb, and the background otherwise. Given a set of foreground sequences FG (i.e. the tissue-specific promoters) and a set of background sequences BG, the sensitivity of M and \u03bb under max-score classification is\nand the specificity is\nThe balanced error rate for M and \u03bb under max-score classification is then\nThe quantity of interest in our analysis corresponds to the optimal value of \u03bb for M in distinguishing FG from BG:\nMany known motifs are similar to each other, usually owing to similar binding specificities for distinct factors or distinct origins for mofits associated with a single factor. We used MATCOMPARE (Schones et al, 2005) to eliminate redundancies in the sets of known and novel motifs.\nIdentifying and evaluating modules\nModules are used to classify sequences based on the max-score values of the motifs they contain. Let \u2133={M1, \u2026, Mk} be a module and \u039b={\u03bb1, \u2026, \u03bbk} be an associated set of thresholds. The max-score classification for modules assigns sequence S to the foreground if and only if max-score (Mi, S)\u2a7e\u03bbi holds for all i (1\u2a7di\u2a7dk). Given sets of sequences FG and BG, the sensitivity of \u2133 and \u039b under max-score classification is\nand the specificity is\nThe balanced-error rate for \u2133 and \u039b under the max-score classification is\nAs with mofits, we are interested in the optimal value of \u039b and define\nBecause modules are intended to describe synergistic function of a set of motifs, we are interested in modules whose performance is better than expected given the performance of the individual motifs composing the module. For a module \u2133 composed of k motifs, let \u2133\u2032\u2282\u2133 minimize B(\u2133\u2032, FG, BG) over all size k\u22121 modules built from motifs in \u2133, and let \u2133\u2032=\u2133\\\u2133\u2032. To assess whether \u2133, with balanced-error rate u, significantly improves over \u2133\u2032 and \u2133\u2032, we use the probability\nWe estimated this probability empirically, by sampling from the distribution of balanced-error rates resulting from intersections of sets with balanced-error rates B(\u2133\u2032, FG, BG) and B(\u2133\u2032, FG, BG).\nWe used MODULATOR, which is available in CREAD (Smith et al, 2005b), to construct modules. Given a set of motifs, a set of foreground sequences and a set of background sequences, MODULATOR identifies those modules composed of the given motifs that have the best balanced-error rates. A branch-and-bound algorithm is used to simultaneously optimize the score thresholds for the motifs in a module. Modules are constructed by adding motifs to existing modules until a user-specified module size is reached or until motif addition does not significantly improve enrichment. Each time a motif is added to a module, the resulting larger module is retained only if the balanced-error rate of the larger module is improved significantly above expectation. The initial modules of size two are obtained by combining pairs of motifs.\nFor modules that are entirely composed of known motifs, the top 100 motifs (before eliminating redundancies) were used. Modules were allowed to contain up to four motifs for reasons of computational feasibility, but many top modules are smaller. Novel modules, which must contain at least one novel motif, were constructed using the top 100 novel motifs and the top 100 known motifs. Redundancies were removed from the lists of top modules using a procedure described in Supplementary Section 2.\nMeasuring the significance of motifs and modules\nTo measure significance of enrichment for top known motifs we used known motifs to classify randomly assembled promoter sets. We constructed 1000 foreground\/background pairs for each species by selecting 100 sequences for each foreground and 1000 for each background uniformly at random from CSHLmpd. For each foreground\/background pair we calculated the balanced-error rate of each known motif. The best balanced-error rates overall obtained on random samples for human and mouse were 0.364 and 0.368, respectively. We used the distribution of these error rates to identify the q-value (Storey and Tibshirani, 2003) significance of the error rate of each motif. Tissues whose highest ranking motifs fail the q<0.05 test include CD4 T cells and heart in human, and CD4 T cells in mouse. TCat includes q-value annotation for each ranked known motif. The full set of motifs for which q<0.05 is estimated to include five false leads per 100 predictions. We did not obtain statistical significance measures for novel motifs, because this will require running DME and DME-B more times than is computationally feasible.\nModules were identified by combining motifs whose cooccurrence was enriched in the foreground sets. We measured enrichment of modules using the balanced-error rate (analogous to that of motifs), and we required that each motif in a module contributes significantly to the enrichment of the module as a whole. To test significance, we randomly selected 100 of the 1000 foreground\/background pairs used to evaluate individual known motifs, and performed the module identification procedure on each of the 100 selected pairs. The best balanced-error rates for modules that are entirely composed of known motifs (called known modules) in random human and mouse sets were 0.3145 and 0.304, respectively. We used these balanced-error rates as an estimate of the critical value for P<0.01. We opted for using P-value cutoffs instead of computing q-values because accurate q-value estimation for modules is computationally prohibitive. Top known modules in human kidney (0.2955), liver (0.3105), pancreas (0.3125) and testis (0.3) scored better than the cutoff, as did top known modules in mouse kidney (0.3005), liver (0.3025) and testis (0.2945).\nSupplementary Material\nSupplementary information","keyphrases":["tissue-specific regulation","cis-regulatory modules"],"prmu":["P","P"]}
{"id":"Dev_Genes_Evol-4-1-2292471","title":"Hedgehog signaling pathway function conserved in Tribolium segmentation\n","text":"In Drosophila, maintenance of parasegmental boundaries and formation of segmental grooves depend on interactions between segment polarity genes. Wingless and Engrailed appear to have similar roles in both short and long germ segmentation, but relatively little is known about the extent to which Hedgehog signaling is conserved. In a companion study to the Tribolium genome project, we analyzed the expression and function of hedgehog, smoothened, patched, and cubitus interruptus orthologs during segmentation in Tribolium. Their expression was largely conserved between Drosophila and Tribolium. Parental RNAi analysis of positive regulators of the pathway (Tc-hh, Tc-smo, or Tc-ci) resulted in small spherical cuticles with little or no evidence of segmental grooves. Segmental Engrailed expression in these embryos was initiated but not maintained. Wingless-independent Engrailed expression in the CNS was maintained and became highly compacted during germ band retraction, providing evidence that derivatives from every segment were present in these small spherical embryos. On the other hand, RNAi analysis of a negative regulator (Tc-ptc) resulted in embryos with ectopic segmental grooves visible during germband elongation but not discernible in the first instar larval cuticles. These transient grooves formed adjacent to Engrailed expressing cells that encircled wider than normal wg domains in the Tc-ptc RNAi embryos. These results suggest that the en\u2013wg\u2013hh gene circuit is functionally conserved in the maintenance of segmental boundaries during germ band retraction and groove formation in Tribolium and that the segment polarity genes form a robust genetic regulatory module in the segmentation of this short germ insect.\nIntroduction\nThe ontogenic stage at which the body plans of animals belonging to the same phylum reach maximum morphological similarity is called the phylotypic stage. In insects and other arthropods, the phylotypic stage is the elongated germband at which the three germ layers have formed and segments are morphologically evident along the entire anterior\u2013posterior axis (Sander 1997). In Drosophila, segment polarity genes, most of which are components of two major signal transduction pathways (the Wingless and Hedgehog signaling pathways), control the formation of grooves between segments and anterior\u2013posterior patterning within each segment. Initially, pair-rule genes activate engrailed (en) expression at the anterior boundary of each parasegment and wingless (wg) at the posterior boundary DiNardo and O'Farrell (1987; Howard et al. 1988; Ingham et al. 1988). Engrailed protein activates expression of hedgehog (hh), which encodes a secreted protein that signals to surrounding cells (Hidalgo and Ingham 1990; Ingham and Hidalgo 1993). Hh signaling leads to the continued activation of wg, whose secreted protein product is necessary for the continued activation of en. This positive feedback loop ensures the interdependence of these three genes for the maintenance of each other\u2019s expression until embryonic stage 9\u201310 (Forbes et al. 1993). At the end of stage 10, en expression becomes independent of wg, and it is around this stage that segmental boundaries are morphologically visible at the posterior edge of cells expressing en and hh.\nEvidence that this segment polarity network might be conserved is primarily based on the expression patterns of en, wg, and more recently, Hh pathway component genes in other insects as well as non-insect arthropods and annelids (Patel et al. 1989; Brown et al. 1994; Kraft and Jackle 1994; Nagy and Carroll 1994; Grbic et al. 1996; Peterson et al. 1998; Damen 2002; Dhawan and Gopinathan 2003; Simonnet et al. 2004). There is also limited data suggesting this network is functionally conserved among insects. Ectopic expression of Drosophila wg in Tribolium induces ectopic en in the anterior half of the parasegment suggesting functional conservation of the wg\u2013en interaction (Oppenheimer et al. 1999). wg and\/or en have been implicated by RNAi analyses in the proper formation of segmental boundaries in the milkweed bug Oncopeltus faciatus (Angelini and Kaufman 2005), the honey bee Apis mellifera (Beye et al. 2002), the blowfly Lucilia sericata (Mellenthin et al. 2006) and the beetle Tribolium (Ober and Jockusch 2006). While RNAi analysis of wg and hh in the cricket Gryllus (Miyawaki et al. 2004) failed to reveal significant effects on segmentation, RNAi analysis of armadillo, a wg pathway component, does implicate wg signaling in segmentation in this short germ insect. In the long germ embryo of Drosophila, where all segments initiate virtually simultaneously, loss of any one of these three genes destabilizes the expression of the others and they eventually fade, resulting in shorter embryos that, in addition to the loss of segmental grooves, also show misspecified epidermal cell fates (Ingham et al. 1991; Forbes et al. 1993). We have investigated genes that encode the Hh-signaling pathway components hedgehog (hh), patched (ptc), smoothened (smo), and cubitus interruptus (ci) to determine whether they function with en and wg in the formation of segmental boundaries in this short germ band insect.\nThe Hh-signaling pathway is well-conserved between insects and vertebrates (Huangfu and Anderson 2006) and is thus likely to be conserved in Tribolium. The main components were first elucidated in Drosophila, where Hh is secreted by cells in the posterior compartment of embryonic segments and larval imaginal discs. It diffuses to the anterior compartment (Lee et al. 1992; Tabata et al. 1992; Tashiro et al. 1993) where the signal is controlled by two membrane proteins: Patched (Ptc), a twelve pass transmembrane protein (Hooper and Scott 1989; Nakano et al. 1989) and Smoothened (Smo), a seven pass transmembrane protein (Alcedo et al. 1996; van den Heuvel and Ingham 1996). In the absence of Hh signal, Ptc represses Smo activity (Chen and Struhl 1996). Signaling is initiated by binding of Hh to its receptor Ptc, which relieves this repression and allows Smo to signal to a multimeric complex inside the cell. This complex is composed of the serine threonine kinase Fused (Alves et al. 1998), the kinesin related protein Costal-2 (Sisson et al. 1997), a novel cytoplasmic protein Suppressor of fused (Monnier et al. 1998) and a zinc finger transcription factor Cubitus Interruptus (Ci; Motzny and Holmgren 1995). In unstimulated cells, this complex sequesters ci, inhibits nuclear import of the full-length 155\u00a0kDa protein and promotes its cleavage to generate an N-terminal 75-kDa form containing the Zn finger DNA-binding domain, which can enter the nucleus and repress transcription of Hh target genes (Aza-Blanc et al. 1997). When Hh signal is transduced, activation of Smo inhibits ci cleavage and activates the full-length protein, which then translocates to the nucleus, resulting in the transcription of Hh-responsive target genes including wg, ptc, gooseberry, and decapentaplegic (Alexandre et al. 1996; Dominguez et al. 1996; Hepker et al. 1997; Ingham and McMahon 2001).\nConsistent with reports on other arthropods, we found that the expression patterns of hh, ci, smo, and ptc were largely conserved in Tribolium. Using RNAi to study the function of these genes during segmentation in Tribolium, we followed embryonic development in these embryos using En as a marker of segment development and integrity. When the Hh signal was depleted by RNAi, segments were specified normally in the posterior growth zone and the embryos elongated as fully as wild-type. En and wg expression in these embryos, although properly initiated, was not maintained and defects appeared during germ band retraction, resulting in tiny, sphere-shaped embryos lacking segmental grooves. On the other hand, overactivation of the pathway by ptc RNAi produced embryos with transient ectopic segmental grooves and embryonic cuticles with enlarged heads and thoracic appendages. All together, these results indicate that Hedgehog signaling is an essential component of the segment polarity network in Tribolium, which is necessary to maintain segmental integrity during germband retraction after the segments have been enumerated in the growth zone. The conserved function of an en\u2013wg\u2013hh gene circuit during segmentation suggests that the segment polarity genes constitute a robust gene regulatory module in this short germ insect.\nMaterials and methods\nBeetle husbandry\nTribolium castaneum strain GA-1 was reared in whole wheat flour supplemented with 5% dried yeast at 30\u00a0C (Beeman et al. 1989).\nIdentification of hedgehog pathway component genes in the Tribolium genome\nPartial cDNAs of tc-hh, tc-ci, tc-smo and tc-ptc, cloned into the pCR4TOPO vector (Invitrogen), were obtained from Y. Tomoyasu. Orthologs of each gene were identified in the annotation of the Tribolium genome (the Tribolium genome consortium, in review). The sequences of the partial cDNAs matched those deduced from the gene models with minor differences. hh, glean gene number tc01364 or NCBI mRNA accession number XM961615, is located on LG 2; smo, TC05545 or XM966834, is on LG 8; ci, TC03000 or XM965017, is on LG 3 and ptc, TC04745 or XM962700 is on LG 1\u2009=\u2009X.\nIn situ hybridization and immunostaining\nWhole mount in situ hybridizations were performed according to established protocols (Tautz and Pfeifle 1989). Expression of Engrailed in Tribolium embryos was determined using the \u03b1-Invected antibody, 4D9 which cross-reacts with Tc-En (Brown et al. 1994). Double staining for the different mRNAs in addition to En protein was performed simultaneously according to the protocol of Nagaso et al. (2001).\nRNA interference (RNAi)\nTemplates for dsRNA synthesis were amplified as described (Tomoyasu et al. 2007). Double stranded RNA was synthesized using the T7 MEGAscript kit (Ambion) and purified using the MEGAclear kit (Ambion). Different amounts of dsRNA (Table\u00a01) were mixed with injection buffer (5\u00a0mM KCl, 0.1\u00a0mM KPO4 pH 6.8) prior to injection. Parental RNAi was performed and affected embryos were analyzed as previously described (Bucher et al. 2002).\nMicroscopy and imaging\nStained embryos and larval cuticles were documented with a Nikon Digital DXM 1200F camera on an Olympus BX50 microscope using Nikon ACT-1 version 2.62 software. Brightness and contrast of all images were adjusted and some were placed on a white background using Adobe Photoshop 7.0.1 software.\nTable\u00a01\nSummary of RNAi effects\nGene\nDsRNA \u03bcg\/\u03bcl\nClass I (%)\nClass II (%)\nClass III (%)\nUndeveloped (%)\nWild type (%)\nTotals (n)\nTchh\n3.0\n0\n6\n83\n11\n0\n464\n1.5\n18\n73\n0\n9\n0\n477\nTcsmo\n3.0\n0\n0\n89\n11\n0\n579\n1.5\n0\n43\n43\n14\n0\n477\n0.75\n38\n44\n0\n20\n0\n230\nTcci\n3.0\n0\n33\n61\n5\n0\n694\n1.5\n0\n30\n63\n7\n0\n405\n0.75\n73\n19\n0\n8\n0\n293\nTcptc\n4.0\n0\n76\n\u2013\n24\n0\n271\n2.0\n13\n68\n\u2013\n19\n0\n189\nEmbryos were collected every 48\u00a0h for the first 2 weeks after injection. Phenotypic classes are unique for each gene. See text for details. Percentages have been rounded up.\nResults\nExpression patterns of Tc-hh, Tc-smo, and Tc-ci\nTc-hh transcripts are first detected in the presumptive head lobes on either side of the ventral mesoderm (arrowhead in Fig.\u00a01a) and at the posterior end of the embryo. As the embryonic rudiment condenses, faint stripes of Tc-hh expression appear immediately posterior to the intense stripes in the head lobes and in the presumptive mandibular segment (arrowheads in Fig.\u00a01b). Gnathal and trunk stripes appear in an anterior to posterior progression (Fig.\u00a01c). Double staining for En expression revealed that Tc-hh and Tc-En are coexpressed in cells of the posterior compartment in each segment (Fig.\u00a01d). During germband elongation, expression at the posterior end of the embryo resolves into spots on either side of the mesoderm and eventually into a ring surrounding the proctodeum (Fig.\u00a01b\u2013d and arrowhead in f). In the head, twin spots appear on either side of the presumptive stomodeum. As the head lobes mature, expression in the anterior region of the developing stomodeum increases (Fig.\u00a01c,d and black arrowhead in e), while the anterior-most stripes of Tc-hh expression resolve into spots in the brain that overlap with the Tc-En-expressing cells (Fig.\u00a01c,d and blue arrowhead in e). Expression at the posterior end of the embryo continues throughout germ band extension (Fig.\u00a01b,d and f) eventually surrounding the proctodeum as it invaginates (Fig.\u00a01f). As segments mature, Tc-hh expression at the ventral midline clears and Tc-hh is not expressed in the CNS. In total, there are three gnathal, three thoracic, and ten abdominal Tc-hh stripes with additional expression in the stomodeum, proctodeum, antennae, and brain (Fig.\u00a01d).\nFig.\u00a01Expression of Tc-hh, Tc-ci, and Tc-smo in Tribolium during segmentation. In these ventral views, anterior is to the left. a\u2013cTc-hh; d\u2013fTc-hh (purple) and Tc-En (gold); g\u2013hTc-smo; i\u2013kTc-ci; l, mTc-ci (purple) and Tc-En (gold); a Anterior stripes of Tc-hh (arrowhead) in the presumptive head lobes of a blastoderm embryo. Expression at the posterior end of the embryo is not in the plane of focus. b Weak stripes appear in the antennal and mandibular segments (arrowheads) posterior to the dark stripes in the head lobes. Twin spots of expression flank the mesoderm at the posterior end of the embryo and near the stomodeum. c In addition to expression in the head lobes and antennae, three gnathal stripes and one trunk stripes appear in an elongating germband embryo. d Coexpression of Tc-En (gold) and Tc-hh (purple) in the posterior compartment of each segment. Note Tc-En expression at the ventral midline in the absence of Tc-hh. e\u2013fTc-hh expression in the stomodeum and the proctodeum (arrowheads) in the absence of Tc-En. Expression of Tc-hh in the head lobes has resolved into spots that overlap Tc-En expression (blue arrowhead). g Ubiquitous expression of Tc-smo in an early germ band embryo. h In an elongating germ band embryo, Tc-smo expression appears ubiquitous with some segmental modulation (arrowheads). i Expression of Tc-ci at the anterior edge of the head lobes (arrowhead) of a young germband embryo. j Gnathal stripes appear first in a slightly older embryo. Expression throughout the posterior region of the embryo fades anteriorly. k Wide Tc-ci stripes in the head lobes and antennal segments as well as in the gnathal and thoracic segments of an elongating germ band embryo. l Tc-En (gold) expression does not overlap Tc-ci (purple) expression in cells of the anterior compartment of each segment. m Enlarged view of a few thoracic segments from the embryo shown in l reveals a gap of two or three rows of cells anterior to the Tc-En stripes that do not express Tc-ci (arrowhead)\nTc-smo is expressed ubiquitously in the germband (Fig.\u00a01g) in Tribolium. As the germ band elongates, expression appears to modulate slightly within each segment (arrowheads in Fig.\u00a01h). smo transcripts are expressed in a similar manner in Drosophila (Alcedo et al. 1996).\nTc-ci expression is first detected at the anterior edge of each head lobe in the early embryonic rudiment (arrowhead in Fig.\u00a01i). Dynamic expression of Tc-ci in the head lobes eventually resolves to a wedge of cells in the lateral eye field (Fig.\u00a01j). Expression is detected in the labrum (Fig.\u00a01l) and in a broad posterior region, which fades toward the anterior (Fig.\u00a01j). As the germ band elongates, Tc-ci is expressed in a broad stripe in every segment in an anterior to posterior progression (Fig.\u00a01i\u2013k). Double staining for Tc-En indicates that Tc-ci is expressed in the anterior compartment where the anterior-most Tc-ci expressing cells are immediately posterior to the Tc-En-expressing cells of the preceding segment (Fig.\u00a01m). Segmentally reiterated stripes of ci expression are also observed in the mulberry silkworm Bombyx mori (Dhawan and Gopinathan 2002), the spider Cupiennius salei (Damen 2002), and the millipede Glomeris (Janssen et al. 2004), suggesting that the segmental expression of ci in the anterior region of each segment is conserved among arthropods. Closer examination of Tc-ci expression revealed a gap of two or three rows of cells immediately anterior of the Tc-En-expressing cells that do not express Tc-ci (Fig. lm). In contrast, expression of ci throughout the anterior compartment of each segment in Drosophila is thought to be essential to the function of the Hh-signaling pathway in maintaining wg expression (Orenic et al. 1990; Hepker et al. 1997). Expression of ci immediately anterior to En-expressing cells is conserved in the spider (Damen 2002), but has not been reported for Bombyx or Glomeris. Thus, it is not clear whether this unusual expression pattern is unique to Tribolium.\nRNAi analysis of Tc-hh, Tc-smo, and Tc-ci\nThe conserved expression patterns of Tc-hh and Tc-smo are consistent with the hypothesis that the Hh function in segmentation is conserved in Tribolium, but the lack of Tc-ci gene expression in cells immediately anterior to Tc-hh is not. To determine whether the Hh-signaling pathway is required for proper segmentation, we performed parental RNAi analysis of these Hh pathway components. Three different amounts of Tc-hh and Tc-smo dsRNA were injected, which uncovered a range of hypomorphic phenotypes (Table\u00a01). Similar RNAi phenotypes were produced for both genes, and the RNAi cuticles were classified into three different categories (Class I, II, and III) based on severity as listed in Table\u00a01. Regardless of the severity, none of the RNAi embryos hatched and the cuticle had to be dissected out of the vitelline membranes. Class I cuticles display the weakest phenotypes, in which the head is severely reduced and contains only rudimentary limb structures. The legs are present but slightly warped, and segmental grooves in the abdomen are occasionally fused but all eight abdominal segments are present (Fig.\u00a02b (Tc-hh), f (Tc-smo)). Class II cuticles are small and spherical, with a large protuberance at the anterior end and three small warped pairs of limbs. The presence of the spiracle on the second thoracic segment (arrowhead in Fig.\u00a02c and g) allowed us to identify these structures as rudimentary legs. The small amount of cuticle posterior to these small warped legs is smooth and lacked any abdominal features (Fig.\u00a02c (Tc-hh), g (Tc-smo)). The most severely affected embryos, Class III, produced small spherical cuticles with a large protuberance at the anterior end and no obvious head structures or thoracic limbs (Fig.\u00a02d (Tc-hh), h (Tc-smo)). These cuticles are very smooth on all sides, with no sign of grooves and are quite small relative to the size of similarly aged wild-type cuticles (Fig.\u00a02a Tc-hh, e Tc-smo). The head and gnathal appendages appear to be more sensitive to the depletion of Tc-hh or Tc-smo than the legs, as even in the weakly affected individuals the head, including gnathal segments, failed to form properly (Fig.\u00a02b,f).\nFig.\u00a02Tc-hh, Tc-smo, and Tc-ci RNAi phenotypes in first instar larval cuticles. a\u2013dTc-hh RNAi; e\u2013hTc-smo RNAi; i\u2013lTc-ci RNAi. a Comparison of a severely affected small spherical Tc-hh RNAi cuticle and a wild-type cuticle at the same magnification. b Lateral view of a mildly affected Class I cuticle containing reduced gnathal appendages, normal legs and all abdominal segments. c Ventral view of a Class II cuticle that is similar in size and shape to more severely affected Class III cuticles but retains three pairs of thoracic appendages. Note spiracle on second thoracic segment (arrowhead). d Ventral view of a small spherical Class III cuticle with rudimentary head structures, no appendages and no segmental grooves. e Comparison of a severely affected small spherical Tc-smo RNAi cuticle and a wild-type cuticle at the same magnification. f Class I cuticle with highly reduced head structures and mouthparts but a full complement of body segments. g Class II cuticle with three pairs of legs and rudimentary head structures. Note spiracle on second thoracic segment (arrowhead). h A severely affected Class III Tc-smo RNAi cuticle phenotypically similar to the most severely affected Tc-hh RNAi cuticle. i Comparison of a severely affected small spherical Tc-ci RNAi cuticle and a wild-type cuticle at the same magnification. j Mildly affected Class I cuticle with reduced head structures, three pairs of legs and mildly fused abdominal segments. k Class II cuticles are slightly longer than intermediate Tc-hh or Tc-smo RNAi cuticles with highly fused abdominal segments. l A severely affected Class III Tc-ci RNAi spherical cuticle with three pairs of legs\nThe phenotypes of Tc-ci RNAi embryos are not as severe as those observed for Tc-hh or Tc-smo. The most weakly affected, Class I Tc-ci RNAi cuticles (Fig.\u00a02j), are shorter and fatter than wild-type with severely reduced heads, but contain all thoracic and abdominal segments. The Class II Tc-ci RNAi cuticles are also shorter than wild-type with a protuberance at the anterior end and fairly normal thoracic limbs, but little to no sign of segmental grooves in the abdomen (Fig.\u00a02k). Similar to Class III RNAi embryos of Tc-hh and Tc-smo, Class III Tc-ci RNAi embryos produced smooth unsegmented cuticles lacking heads and gnathal appendages (Fig.\u00a02l) that are considerably smaller than wild-type cuticles (Fig.\u00a02i). Unlike Tc-hh and Tc-smo Class III RNAi embryos, they still produced fairly normal legs (Fig.\u00a02l). Interestingly, loss of function mutants of ci in Drosophila also produce milder limb phenotypes than do hh mutants (Methot and Basler 1999).\nTo understand how the segmentation process is affected by loss of Hh signaling, we followed Tc-En expression during elongation and retraction in Class III RNAi embryos (Fig.\u00a03). In RNAi embryos for all three genes, segmental stripes of Tc-En initiate normally during germ band elongation (blue arrowheads in Fig.\u00a03a,d and g). Tc-En expression fades as the segments matured (black arrowheads Fig.\u00a03a,d and g), suggesting that Hh signaling is required for the maintenance of Tc-En. However, Tc-En expression in cells along the midline, presumably in the developing CNS (wild-type, Fig.\u00a03i), is maintained in the RNAi embryos, which allowed us to follow segmentation in RNAi embryos. Tc-hh, Tc-smo, and Tc-ci RNAi embryos completed elongation more or less normally (Fig.\u00a03a,d and g), but abnormalities became evident during retraction. The head lobes fail to mature, and there is no evidence of antennal Tc-En stripes. There is also a protuberance at the anterior end of the embryo that is likely to correspond to the anterior protuberance seen in the RNAi cuticles (Fig.\u00a02d,h and l, and arrow in 3 h). In embryos that completed retraction, the unsegmented germbands are highly compacted, and Tc-En expression pattern is very irregular (Fig.\u00a03c,e and h). Compared to wild-type germ bands at a similar stage, these germ bands occupy only a portion of the egg (Fig.\u00a03j,k). In addition, loss of early Hh signaling at the ventral midline affected cell fate, as indicated by the loss of Tc-En expression here (Fig.\u00a03b,e). Loss of Hh signal in Drosophila similarly affects En expression in midline cells (Bossing and Brand 2006). We also found that Tc-wg expression was initiated but not maintained in these embryos (data not shown), suggesting Tc-wg is a target of the Hh pathway.\nFig.\u00a03En staining in wild-type and severely affected Tc-hh, Tc-smo and Tc-ci RNAi embryos during elongation and retraction. a\u2013cTc-hh RNAi; d\u2013f, kTc-smo RNAi; g, hTc-ci RNAi; i, j wild-type. a Tc-En expression initiated normally in the posterior segments of an elongating Tc-hh RNAi germband embryo (blue arrowhead) but did not persist in older, more anterior segments (black arrowhead). b During germband retraction, patches of Tc-En expression are visible near the ventral midline, and laterally in the posterior segments. c In a highly compacted germ band with fused segments, Tc-En expression is disrupted along the ventral midline. d Expression of Tc-En in an elongating Tc-smo RNAi embryo initiated normally (blue arrowhead) but failed to persist in older, more anterior segments (black arrowhead). e During germband retraction, Tc-En is expressed in patches along the ventral midline, but has faded laterally. f In a highly compacted germ band, persists in a disrupted pattern along the ventral midline. g In an elongating Tc-ci RNAi embryo, similar to Tc-hh and Tc-smo RNAi, Tc-En initiated normally (blue arrowhead) but is not maintained (black arrowhead). h Tc-En expression at the ventral midline of a retracted germband embryo. Note the extended stomodeum (arrowhead) near the anterior end. i Tc-En expression in the CNS along the ventral midline and in the lateral ectoderm of each segment of a wild-type embryo during germband retraction. j Lateral view of a wild-type embryo inside the vitelline membrane at the end of germ band retraction. Tc-En expression persists laterally to the edge of the germband. k Ventral view of a severely affected Tc-smo RNAi embryo located near one end of the egg\nhedgehog was first isolated in a screen for mutations that disrupt the Drosophila larval cuticle pattern and identified as one of the segment polarity genes with a \u2018lawn of denticles\u2019 phenotype (Nusslein-Volhard and Wieschaus 1980). In segment polarity mutants (e.g., wg, hh, and smo), deletion of a portion of the larval epidermis in each segment is accompanied by a mirror image duplication of the remaining structures. As a result, they contain the normal number of segments, but are smaller than wild-type due to partial deletion of each segment. In our study, we found Tc-hh and Tc-smo RNAi embryos to be smaller in size than the wild-type Tribolium larva (Fig.\u00a02a,e), which may be due to cell death, fusion, or failure of cell division in segments during germ band retraction. The few random bristles produced in severely affected RNAi cuticles do not have any definable polarity suggesting that, unlike Drosophila, loss of function of these genes in Tribolium does not appear to produce mirror image duplications or affect polarity within the segments.\nAnalysis of Tc-ptc and overactivation of the Hh pathway\nTranscripts of Tc-ptc are first detected in a broad domain in the posterior regions of the head lobes in the embryonic rudiment encompassing the antennal segments and the stomodeum, and at the posterior end of the embryo (Fig.\u00a04a). Broad segmental stripes of Tc-ptc appear in an anterior to posterior progression during germband elongation (Fig.\u00a04b). Expression fades in the middle of each initial stripe resulting in two narrow Tc-ptc stripes per segment (Fig.\u00a04c). Double staining with Tc-En and Tc-ptc indicates that in each segment, one of the narrow Tc-ptc stripes marks the anterior boundary of a segment while the other is located immediately anterior to Tc-En-expressing cells (Fig.\u00a04c and arrowhead in inset). This pattern persists even after germ band retraction; the anterior stripe appears stronger than the posterior stripe in each segment. ptc expression during segmentation in Drosophila is similarly dynamic (Nakano et al. 1989; Hidalgo and Ingham 1990).\nFig.\u00a04Analysis of Tc-ptc in Tribolium.a\u2013cTc-ptc expression in wild-type embryos; d\u2013fTc-ptc RNAi cuticles; g wild-type; h\u2013mTc-ptc RNAi. a Expression of Tc-ptc in three gnathal stripes, in a broad posterior region of the head lobes and in the posterior region of an early germ band embryo. b Segmental stripes appear sequentially during elongation and resolve in double stripes. c Tc-En (gold) expression in the posterior compartment abuts, but does not overlap the double Tc-ptc stripes (extent denoted by black lines) in the anterior compartment of each segment. Inset: enlarged view of Tc-ptc stripes adjacent to a stripe of Tc-En (arrowhead). d A mildly affected Class I Tc-ptc RNAi cuticle with deformed legs and head appendages. e A more severely affected Class II cuticle with enlarged misshapen legs but a normal complement of abdominal segments. f Comparison of a Class II Tc-ptc RNAi cuticle and a wild-type cuticle at the same magnification. g Expression of Tc-En (gold) and Tc-wg (purple) in a wild-type embryo during elongation. h Segmental expression of Tc-En and Tc-wg initiated fairly normally in a Tc-ptc RNAi germband. Tc-wg expression domains in the head lobes and around the proctodeum are expanded. i Expanded expression of Tc-wg in a slightly older embryo. j Ectopic stripes of Tc-En expression (blue arrowhead) in a germband undergoing retraction. k Ectopic expression of Tc-En at the ventral midline (arrowhead) in a close up of the embryo in h. l Transient grooves (arrowheads) around expanded Tc-wg expression domains in a close up of the embryo in i. m Ectopic Tc-En stripes (blue arrowhead) between each set of normal Tc-En stripes (black arrowhead) in a close up of the embryo in j. T1, first thoracic segment\nAs discussed above, Ptc is a negative regulator of the Hh-signaling pathway in Drosophila. Depletion of a negative regulator of the pathway would ectopically activate the pathway. To understand what happens when the Hh pathway is overactivated in Tribolium, we performed parental RNAi using two different amounts of Tc-ptc dsRNA (Table\u00a01). The resulting cuticles (Fig.\u00a04d,e,f) are very different from the ones described above for the other three genes. In mildly affected embryos (Class I), the head appendages are misshapen, while the legs are relatively normal (Fig.\u00a04d). In the most severely affected embryos (Class II), all segments are present, but the head and thoracic appendages are enlarged and misshapen (Fig.\u00a04e,f).\nTo understand the phenotype of the Tc-ptc RNAi embryos, we examined the expression of Tc-wg and Tc-En. In Tc-ptc-depleted embryos, segmental expression of Tc-En and Tc-wg is initiated normally (Fig.\u00a04h), although the domains of Tc-wg expression in the head and at the posterior end of the embryo are expanded. Closer inspection revealed ectopic Tc-En expression at the ventral midline (Fig.\u00a04k). In slightly older embryos, Tc-wg is expressed more intensely than normal in enlarged domains (Fig.\u00a04i,l). Cells surrounding the expanded Tc-wg domains appear to be invaginating, as if beginning to form grooves. Closer inspection revealed a row of non-Tc-wg-expressing cells between the Tc-wg domain and the grooves (Fig.\u00a04l). Tc-En is expressed in ectopic stripes that, in combination with the normal Tc-En stripes, would surround the expanded Tc-wg domains (Fig.\u00a04j,m). In addition to the normal grooves that form posterior to the normal Tc-En stripe, ectopic grooves initiate anterior to the ectopic Tc-En stripes. Thus, overactivation of the Hh-signaling pathway in Tribolium leads to overexpression of Tc-wg, and ectopic expression of Tc-En, which results in ectopic groove formation. In Drosophila, binding of Hh to Ptc receptor relieves repression of wg and allows expression of target genes. In Drosophila ptc mutants, ectopic induction of wg and En result in the formation of extra grooves (Nakano et al. 1989), suggesting that the functional role of Ptc is highly conserved between these two species. However, unlike Drosophila, the ectopic grooves in Tribolium are transient and it appears that a late regulatory action restores the normal number of segments in this insect.\nDiscussion\nExpression patterns suggest that the function of the Hh-signaling pathway is conserved in several developmental pathways in Tribolium.\nIn Tribolium, the expression patterns of the four Hh-signaling pathway components we examined are highly similar to those of their Drosophila counterparts. In both Drosophila and Tribolium, the domains of Tc-hh are very similar to those of Tc-En. However, there are some significant differences in their expression dynamics. For example, the antennal Tc-En stripes appear after the three gnathal Tc-En stripes, whereas antennal Tc-hh stripes appear before the gnathal Tc-hh stripes. Each metameric stripe of Tc-hh is laterally continuous across the width of the germband after mesoderm invagination. Later, Tc-hh expression has disappeared at the ventral midline in anterior segments, while in posterior segments the stripes are still continuous. In contrast, Tc-En expression continues in the CNS after Tc-hh expression has faded there. Tc-hh expression in the stomodeum and at the posterior end of the blastoderm embryo in the absence of Tc-En suggests that, similar to hh in Drosophila (Lee et al. 1992; Mohler and Vani 1992; Tashiro et al. 1993), Tc-hh is involved in some En-independent processes in these regions.\nIn both Drosophila and Tribolium, expression of segment polarity genes is initiated by pair-rule genes (recently reviewed by Damen 2007). Soon thereafter, their expression is controlled by interactions between the segment polarity genes themselves. In Drosophila, Ptc is constitutively active unless or until Hh represses Ptc activity. In the absence of Hh, unbound Ptc keeps the pathway switched off. One of the targets of the Hh pathway is ptc itself. In cells where its activity is antagonized upon binding of Hh, ptc continues to be expressed, but ptc expression disappears in cells that do not receive the signal. Thus, an initially broad domain of ptc expression resolves into two narrow stripes flanking the En expression domain. Tc-ptc expression pattern is similar: each broad stripe splits into two, such that each En stripe is bracketed by two Tc-ptc stripes, suggesting that Tc-ptc itself might also be a target of the Hh pathway in Tribolium.\nThe expression patterns of Hh pathway component genes are highly conserved between Drosophila and Tribolium, except for that of Tc-ci (Fig.\u00a01l,m). In Drosophila, ci transcripts are initially expressed uniformly in the early cellular blastoderm and persist until the end of germ band elongation. At that point, ci expression is directly repressed by En in cells of the posterior compartment in each segment. In Tribolium, expression of Tc-ci is somewhat different in that there is a narrow region of two to three cells between the Tc-ci and En-expressing cells that does not express Tc-ci. In Tribolium, the absence of Tc-ci transcripts in cells just anterior to En-expressing cells might suggest the existence of an En-independent mechanism regulating Tc-ci expression. Alternatively, it is possible that Tc-ci transcripts turn over rapidly in these cells, but the expression pattern is conserved at the protein level. In Drosophila, ci is regulated post-transcriptionally. At stage 11, ci transcripts are localized throughout the anterior compartment of each segment whereas protein levels are lower at the center of each transcriptional stripe and higher in cells that bracket the En-expressing cells (Motzny and Holmgren 1995; Slusarski et al. 1995) much like what we describe here for ptc transcription in Tribolium and previously for Ptc protein levels in Drosophila. It will be interesting to see if Tc-ci is also post-transcriptionally regulated in Tribolium.\nFunctional analysis of Hh pathway component genes supports conserved roles in segment boundary formation.\nIn Drosophila, mutations in genes encoding positive regulators of the Hh pathway including hh, smo, and ci produce smaller than wild-type embryos with asegmental phenotypes (Nusslein-Volhard and Wieschaus 1980). In Tribolium, Tc-hh, and Tc-smo RNAi embryonic phenotypes are nearly identical; both produce highly compacted spherical cuticles with no evidence of appendages or segmental grooves. The most severe Tc-ci RNAi phenotypes are not as severe as those of Tc-hh or Tc-smo RNAi. Interestingly, the ci94 allele in Drosophila is a null allele (Slusarski et al. 1995; Methot and Basler 1999) and these mutant embryos differ considerably from hh mutants. hh mutants are much shorter in length and have a continuous \u2018lawn of denticles\u2019 phenotype whereas ci94 mutants are almost normal in size and have alternating naked cuticle and denticle belts on the ventral surface. Target genes of hh are partially derepressed in the absence of ci, producing a ci phenotype that is milder than that of hh (Methot and Basler 2001). An analogous situation has been described for the Wingless signaling pathway in Drosophila where derepression of target genes in the absence of pangolin results in a milder segment polarity phenotype compared to that of wg null mutants (Cavallo et al. 1998; Waltzer and Bienz 1998). In hh and smo mutants, only the repressor function of ci remains, producing the catastrophic phenotype. In contrast, loss of both the activating and repressor forms results in the milder phenotype seen in ci null mutants. In Tribolium, the most severe Tc-ci RNAi phenotype is milder than the most severe Tc-hh RNAi phenotype, suggesting similar regulation of the Hh pathway in the beetle.\nIn Drosophila, loss of Wg or Hh-signaling results in larvae that are smaller than wild-type due, at least in part, to epidermal cell death during and after germband retraction (Martinez-Arias and Lawrence 1985) or a combination of transformation and cell death (Klingensmith et al. 1989). In Tribolium, hh, smo, and ci RNAi individuals are greatly reduced in size compared to the wild-type (Fig.\u00a02). These embryos go through the events of early embryogenesis normally, producing the full complement of segments and initiating Tc-En and Tc-wg expression. Tc-En and Tc-wg expression fade and the segmental remnants become highly compacted along the anterior\u2013posterior axis during retraction. Tribolium embryos lacking Tc-wg also elongate normally but fail to maintain En expression and form shorter than wild-type embryos during retraction (Ober and Jockusch 2006). While it is likely that cell proliferation and programmed cell death both contribute to shaping the embryo in Tribolium, during normal development cells divide randomly throughout the elongating germ band (Brown et al. 1994); organized patterns of cell division or cell death have not been reported. Similarly, it is likely that excessive cell death or the lack of cell proliferation contributes to the severely compacted terminal phenotype of Tc-hh, Tc-smo or Tc-ci RNAi embryos, but closer examination will be required to determine if this is so.\nIn Drosophila ptc mutants, deletion of the midregion of each segment is accompanied by a mirror image duplication of the remaining denticles (Nusslein-Volhard and Wieschaus 1980). In contrast, Tribolium Tc-ptc RNAi embryos, which also display the correct number of segments, are characterized by distended gnathal and thoracic appendages. Although there are some random bristles, we could not identify any noteworthy difference in the polarity of these bristles that could be attributed to a characteristic loss of function phenotype for genes belonging to this class. This suggests that unlike Drosophila, the loss of segment polarity gene function in Tribolium does not result in any morphologically identifiable polarity defect in the cuticle.\nIn the absence of ptc in Drosophila (DiNardo et al. 1988) and Tribolium, En expression is established properly; but later, de novo En stripes appear between the normal stripes. Ectopic expression of En in Drosophila is not due to regulation of ptc by pair-rule factors (DiNardo et al. 1988). In the absence of ptc, the wg expression domain broadens anteriorly. Expanded wg expression induces ectopic En stripes, which cause ectopic groove formation, suggesting that the principal function of Ptc is to repress wg. Similar expression of Tc-ptc in Tribolium, considered with the expanded Tc-wg domains that are surrounded by ectopic Tc-En-expressing cells and the ectopic grooves transiently formed in Tc-ptc RNAi embryos suggest that the role of Tc-ptc is likely to be functionally conserved between Drosophila and Tribolium. However, in Tc-ptc RNAi embryos, ectopic Tc-En is also detected along the ventral midline, a phenotype that has not been described for Drosophila ptc mutants. hh RNAi has been attempted in the orthopteran Gryllus bimaculatus (Miyawaki et al. 2004). Unfortunately, this organism seems to be resistant to hh dsRNA. In the RNAi embryos, the level of hh is not reduced. The embryos develop normally and hatched larvae show no cuticular defects. Lack of ptc analysis in other insects makes it difficult to speculate as to whether this novel expression of Tc-En along the ventral midline is specific to Tribolium, or a general feature related to short germ development. Finally, although ectopic grooves appear to form around the expanded Tc-wg domains, they are not detected in the terminal cuticles, implying that events late in embryogenesis restore the normal number of segmental grooves.\nConservation of the hh\u2013wg\u2013en gene circuit in short germ segmentation\nSeveral lines of evidence suggest that the segment polarity gene circuit, in which the expression of the hh, wg, and en genes are dependent upon one another in the long germ mode of segmentation elucidated in Drosophila, is conserved in the short germ mode of segmentation found in Tribolium. As in Drosophila, in the absence of smo, hh, ci (this paper), or wg (Ober and Jockusch 2006) in Tribolium, En expression is not maintained. In addition, Tc-wg mRNA fails to persist in Tc-hh, Tc-smo and Tc-ci RNAi embryos (data not shown). Furthermore, expression patterns and RNAi phenotypes of the Hh pathway components we examined (Tc-hh, Tc-smo, Tc-ci, and Tc-ptc) suggest that the regulation of the Hh pathway is also conserved. Segment polarity genes, which function last in the segmentation gene hierarchy, are expressed in segmental fields that have been predefined by genes at higher levels (gap and pair rule). Conservation of the segment polarity gene circuit in Tribolium suggests that that segment polarity genes form a robust regulatory module in the short germ mode of segmentation in this beetle and their expression patterns in numerous insects and chilicerates suggest that this module is likely to be conserved among the Insecta, and perhaps the Arthropoda.\nWhile the expression patterns of segment polarity orthologs are highly conserved, the expression patterns of pair-rule gene orthologs vary greatly among insects and other arthropods (recently reviewed in Tautz 2004; Peel et al. 2005; Damen 2007). Functional analysis in Tribolium indicates that interactions among pair-rule gene homologs differ from those of their Drosophila counterparts (Choe et al. 2006) and that some secondary pair-rule genes function in opposite parasegmental registers in Drosophila and Tribolium (Choe and Brown 2006). These findings suggest that the inputs from pair-rule genes to the segment polarity gene module are likely to be quite different in each of these insects. Functional analysis of how pair-rule genes regulate the alternating expression of en and wg in Tribolium will provide insight into these differences, which will ultimately help us understand the evolution of genetic regulatory networks. Interestingly, the segment polarity gene module seems to be resilient enough to withstand such evolutionary changes in input from the pair-rule gene module.","keyphrases":["hedgehog","segmentation","segmental groove","smoothened","patched","cubitus interruptus"],"prmu":["P","P","P","P","P","P"]}
{"id":"Pediatr_Nephrol-3-1-1994209","title":"Genetic approaches to human renal agenesis\/hypoplasia and dysplasia\n","text":"Congenital abnormalities of the kidney and urinary tract are frequently observed in children and represent a significant cause of morbidity and mortality. These conditions are phenotypically variable, often affecting several segments of the urinary tract simultaneously, making clinical classification and diagnosis difficult. Renal agenesis\/hypoplasia and dysplasia account for a significant portion of these anomalies, and a genetic contribution to its cause is being increasingly recognized. Nevertheless, overlap between diseases and challenges in clinical diagnosis complicate studies attempting to discover new genes underlying this anomaly. Most of the insights in kidney development derive from studies in mouse models or from rare, syndromic forms of human developmental disorders of the kidney and urinary tract. The genes implicated have been shown to regulate the reciprocal induction between the ureteric bud and the metanephric mesenchyme. Strategies to find genes causing renal agenesis\/hypoplasia and dysplasia vary depending on the characteristics of the study population available. The approaches range from candidate gene association or resequencing studies to traditional linkage studies, using outbred pedigrees or genetic isolates, to search for structural variation in the genome. Each of these strategies has advantages and pitfalls and some have led to significant discoveries in human disease. However, renal agenesis\/hypoplasia and dysplasia still represents a challenge, both for the clinicians who attempt a precise diagnosis and for the geneticist who tries to unravel the genetic basis, and a better classification requires molecular definition to be retrospectively improved. The goal appears to be feasible with the large multicentric collaborative groups that share the same objectives and resources.\nIntroduction and definition\nCongenital abnormalities of the kidney and urinary tract are frequently observed in the first year of life, when they collectively represent a significant cause of morbidity [1] and mortality. Data from birth defects registries [Metropolitan Atlanta Congenital Defects Program (MACDP); California Birth Defects Monitoring Program (CBDMP) [2] indicate an overall frequency from three to six per 1,000 births, and the abnormalities seriously impact life expectancy (http:\/\/www.marchofdimes.com). Human urinary tract abnormalities are phenotypically variable and may affect several segments simultaneously, often aggregating to form complex phenotypes. Hence, clinical classification and diagnosis may be difficult. As a consequence of the overlap between anatomical defects, many investigators have opted to group renal and urologic malformations under the single label of Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) [3]. This broad classification is supported by the fact that a mutation in a single gene can have pleiotropic effects on the development of the urogenital tract. For example, mutations in the PAX2 gene cause the renal-coloboma syndrome, but the clinical features of the trait vary significantly between affected individuals, ranging from renal agenesis\/hypoplasia to vesicoureteral reflux (VUR) and secondary obstruction [4]. Conversely, mutations in different genes can result in similar renal phenotypes, e.g., EYA1 and PAX2 mutations both can cause the development of hypoplastic kidneys [5]. Hence, improved classification of urinary tract malformations may require understanding of primary molecular defects. A broad but clinically useful diagnostic scheme consists of classifying malformations depending on whether the kidney, the collecting system, or both are affected. This scheme stems from the fact that the upper tract (glomeruli and tubules) is derived from the metanephric mesenchyme (MM), and the lower urinary tract (collecting duct, renal pelvis, ureter) is derived from the ureteric bud [1]. Even if this is in contrast with more recent data about the reciprocal interaction between the ureteric bud and the MM (see below), this classification can be clinically useful to partition patients with different types of urinary tract abnormalities. In this review, we focus on the malformations that primarily involve a reduction of renal parenchyma in the form of renal agenesis and\/or hypoplasia\/dysplasia, occurring both as isolated forms or in association with other malformations of the lower urinary tract (see below).\nPrimary renal agenesis\nBilateral renal agenesis is a rare and fatal event, usually associated with severe oligohydramnios, which produces a characteristic clinical pattern with facial compression and pulmonary hypoplasia (Potter syndrome). An estimate of the incidence of bilateral agenesis is 0.1\/1000 births. Unilateral renal agenesis is more common, although the frequency is difficult to estimate, as it is usually clinically silent and is commonly detected as a chance observation by autopsy or by prenatal ultrasound [6].\nPrimary renal hypoplasia and dysplasia\nStrictly speaking, renal hypoplasia is defined as a small kidney, which contains intact nephrons that are reduced in number, whereas a dysplastic kidney contains disorganized elements and maldifferentiated tissue. Noninvasive imaging studies such as ultrasounds and dimercaptosuccinic acid (DMSA) scan offer limited information to help distinguish a hypoplastic kidney from a dysplastic one. Unequivocal distinction between these two entities therefore depends on histological examination of renal tissue obtained from kidney biopsy or surgical nephrectomy, which are rarely performed. A further confounding factor is the reduction of kidney size due to chronic injury and scarring from VUR. Most of the time, a DMSA scan helps differentiate primary hypoplasia or dysplasia from small kidneys secondary to VUR. However, a DMSA scan has a low negative predictive value in distinguishing primary hypoplasia or dysplasia from a secondary reduction in kidney size from VUR when scars or areas of negative isotope uptake are present. In practice, the diagnosis of primary renal hypoplasia is favored when the following criteria are satisfied: (a) a reduction of renal size by 2 standard deviations (SDS) from the mean size for the age, (b) exclusion of renal scarring by DMSA scan, and (c) a presence of compensatory hypertrophy of the contralateral kidney. In all cases, the exclusion of renal cysts by ultrasonography is mandatory to avoid confusion with primary renal hypoplasia associated with fibrosis and cysts, nephronophthisis being the most pertinent example. The presence of VUR and\/or ureteropelvic junction obstruction (UPJO) does not automatically exclude the diagnosis of hypoplasia, as both conditions are frequently associated with primary renal-size defects. It is clear that this problem is difficult to resolve if the ureteral defect presents ipsilateral to renal hypoplasia. For example, severe antenatal hydronephrosis due to UPJO can determine the involution of the renal parenchyma and lead to an erroneous diagnosis of primary renal agenesis after birth. In bilateral cases, syndromic traits as well as inherited disorders such as medullary cystic kidney disease\/nephronophthisis have to be excluded. Unequivocal exclusion of renal dysplasia is usually not feasible except in rare cases for which histology is available. It is possible that in the near future, molecular genetic advances could modify our present understanding and allow for a more direct separation of the two pathological entities based on laboratory tests.\nThese challenges in clinical diagnosis of renal hypoplasia complicate studies attempting to discover new genes underlying this anomaly. For research purposes, we utilize a tentative classification scheme for categorizing our subjects for genetic studies: (1) isolated bilateral hypoplasia\/dysplasia, (2) isolated unilateral hypoplasia\/dysplasia, and (3) hypoplasia\/dysplasia associated with lower tract abnormalities such as VUR or UPJO. Once the genetic basis of different subsets of urinary tract malformations is identified, the classification will likely be retrospectively changed and improved.\nKidney development and mouse models\nThe development of mammalian kidney derives from reciprocally inductive events between two tissue compartments of the embryonic metanephros: the ureteric bud (UB), an outgrowth of the nephric duct, and the MM. The ureteric bud invades the metanephric blastema at embryonic day 10.5\u201311 in the mouse and 35\u201337 in humans. The MM induces the ureteric bud to grow and branch while the ureteric bud induces the MM to transdifferentiate and form the nephrons\u2019 epithelia (see recent reviews in kidney development in human and mice [7, 8]).\nIn recent years, many factors, specific for either the UB or the MM, have been demonstrated to induce and regulate the epithelial conversion in the mesenchymal cells and the UB branching, leading to the development of the final structure and function of the kidney. Most data constituting the basis of our current knowledge on the topic are based on gene targeting studies in mice (Table\u00a01). A partial list of genes includes protooncogenes RET and Wingless-related 11 (WNT11) that are well recognized UB-specific molecules, whereas glial cell-line-derived neurotrophic factor (GDNF), Wilms tumor 1 (WT1), and Eyes absent 1 (EYA1) represent important examples of MM-specific factors. The paired-box gene 2 (PAX2) appears to be expressed in both structures during kidney development [7, 9]. It is noteworthy that almost half of the genes on the list are transcriptional factors or encode for proteins that are involved in the mesenchymal to epithelial conversion. GDNF signaling through the RET receptor is one of the best studied pathways, representing a critical step in the normal growth and branching of the UB during kidney development [10]. Perturbation of Gdnf\/Ret signaling has been shown to be the downstream mechanism underlying impaired nephrogenesis in many other mutant models (e.g., in Gdf11 and Six1 null mice). Numerous factors other than the Gdnf\/Ret pathway also participate in kidney and urologic development (e.g. Wnt signaling), as evidenced by the long list of mutant mice with malformations in the kidney and urologic tract (Table\u00a01).\nTable\u00a01Principal genes targeted in mice leading to renal agenesis, hypoplasia, dysplasiaGeneHuman homologKidney phenotypeReferenceFoxd1FOXD1Small, fused, undifferentiated kidneysHatini et al. [59]Eya1EYA1Absent kidneysJohnson et al. [60]Xu et al. [61]Emx2EMX2Absent kidneysMiyamoto et al. [62]Hoxa11\/Hoxd11HOXA11\/HOXD11Small or absent kidneysDavis et al. [63]Lhx1LHX1Absent kidneysShawlot and Behringer [64]Pax2PAX2Small or absent kidneysTorres et al. [65]Wt1WT1Absent kidneysKreidberg et al. [66]Agtr2AGTR2Multiple urinary tract malformationsNishimura et al. [67]Bmp4BMP4Altered ureteric bud (UB) branchingMiyazaki et al. [68]Bmp7BMP7Disrupted nephrogenesisDudley et al. [69]Wnt4WNT4Undifferentiated kidneysStark et al. [70]RetRETAbsent kidneys, severe dysgenesisSchuchardt et al. [71]GdnfGDNFAbsent kidneys, severe dysgenesisSanchez et al. [72]Moore et al. [73]Pichel et al. [74]Six1SIX1Absent kidneysXu et al. [75]Six2SIX2Small kidneysSelf et al. [76]Sall1SALL1Absent kidneysNishinakamura et al. [77]Fgfr1\/Fgfr2FGFR1\/FGFR2Absent kidneysPoladia et al. [78]Slit3SLIT3Small or absent kidneysLiu et al. [79]Pbx1PBX1Small or absent kidneysSchnabel et al. [80]Fgf8FGF8Small kidneysPerantoni et al. [81]Rara\/Rarb2RARA\/RARB2Small kidneysMendelsohn et al. [82]Lim1LIM1Absent kidneysKobayashi et al. [83]\nThe interdependence between developmental pathways explains why defects in different genes result in similar phenotypes and why morphologic classification of abnormalities alone cannot predict the location or nature of primary defects. Available data thus suggest a large list of candidate genes for human renal and urologic malformations, highlighting the potential for genetic heterogeneity of the trait.\nGenetic contribution to human renal agenesis\/hypoplasia and dysplasia\nA genetic contribution to the development of renal hypoplasia\/dysplasia has been recognized for many years. For the isolated, nonsyndromic renal agenesis\/hypoplasia and dysplasia, only segregation studies have been performed, and no loci and\/or genes have been mapped so far. Much more is known about rare syndromic forms, for which several genes have been already implicated.\nSyndromic forms\nSyndromic forms of renal hypoplasia\/dysplasia include rare disorders affecting extrarenal organs such as the eye, the central nervous system, the skin, the limbs, and others. The list of syndromes that include the renal agenesis\/hypoplasia\/dysplasia phenotype consists of at least 73 clinical conditions (for more details, see Limwongse and Cassidy [11]). Several genes underlying these defects having been identified (Table\u00a02). Renal-coloboma syndrome, orofaciodigital syndrome, branchiootorenal syndrome, renal cysts and diabetes syndrome, and Fraser syndrome are the most frequent syndromes associated with renal parenchymal defects. It seems clinically relevant that the renal abnormalities may represent the first manifestation of the disease, thus requiring a detailed evaluation of other organs. A list of extrarenal signs and symptoms that clinicians should look for to define these syndromes include retinal coloboma [4], deafness, external ear abnormalities including cysts and fistulas [12, 13], anus imperforates and limb and ear anomalies [14], diabetes and renal cystic dysplasia [15], and others. Finally, renal agenesis\/hypoplasia is frequently part of chromosomal disorders (Table\u00a03) that must be recognized for genetic counseling. Most common syndromes that should be considered in the initial differential diagnosis are listed in Tables\u00a02 and 3, and we suggest referring to popular Web sites for further details (links provided at the end).\nTable\u00a02List of human malformation syndromes with kidney hypoplasia\/dysplasiaGeneHuman syndromeKidney phenotypeOMIMJAG1, NOTCH2Alagille syndromeMCDK, kidney dysplasia, kidney mesangiolipidosis#118450#610205BBS1-BBS11Bardet-Biedl syndromeRenal dysplasia and calyceal malformations#209900EYA1, SIX1, SIX2Branchiootorenal syndromeRenal agenesis\/dysplasia#113650SOX9Campomelic dysplasiaDiverse renal malformations#114290CHD7CHARGE syndromeDiverse urinary tract malformations#214800Del. 22q11Di George syndromeRenal agenesis, dysplasia, VUR#188400GATA3Hypothyroidism, sensorial deafness, renal anomalies (HDR)Renal agenesis, dysplasia, VUR#146255DNA repairFanconi anemiaRenal agenesis#227650FRAS1, FREM2Fraser syndromeRenal agenesis, dysplasia#219000KALL1, FGFR1Kallman\u2019s syndromeRenal agenesis, dysplasia#308700, #147950PAX2Renal coloboma syndromeRenal hypoplasia, MCDK, VUR#120330TCF2Renal cysts and diabetes syndromeRenal dysplasia, cysts#137920GPC3Simpson-Golabi-Behmel syndromeRenal dysplasia, cysts#300209DHCR7Smith-Lemli-Opitz syndromeRenal dysplasia, cysts#270400SALL1Townes-Brocks syndromeRenal dysplasia, lower urinary tract malformations#107480LMX1BNail-patella syndromeGlomerulus malformation, renal agenesis#161200NIPBLCornelia de Lange syndromeRenal dysplasia#122470CREBBPRubinstein-Taybi syndromeRenal agenesis#180849WNT4Rokitansky syndromeRenal agenesis#277000PEX-familyZellweger syndromeRenal dysplasia, cysts#214100GLI3Pallister-Hall syndromeRenal agenesis, dysplasia#146510p57(KIP2)Beckwith-Wiedemann syndromeRenal dysplasia#130650SALL4Okihiro syndromeRenal ectopia with or without fusion, lower urinary tract malformations#607323TBX3Ulnar-Mammary syndromeRenal agenesis#181450MCDK multicystic dysplastic kidney, VUR vesicoureteral refluxTable\u00a03Common chromosomal disorders associated with urinary tract anomaliesChromosomal disordersRenal agenesisHypoplasiaOther associated anomaliesPatau syndrome (trisomy 13)+Holoprosencephaly, midline anomalies, cleft lip\/palateMiller-Dieker syndrome (17p13 deletion)+MR, lissencephaly, microgyria, agyria, typical facie, seizuresEdward syndrome (trisomy 18) 18q deletion+IUGR, CHD, clenched hands, rocker bottom feet SS, MR, microcephaly, narrow external ear canals, long handsDown syndrome (trisomy 21)+MR, hypotonia, CHD, typical face, clinodactylyCateye syndrome (tetrasomy 22p)+MR, CHD, colobomas, anal\/digital anomaliesVelocardiofacial syndrome (22q11 deletion)++Conotruncal CHD, thymic aplasia, typical face, cleft palateTurner syndrome (45,X or 46,X,i(Xq))++SS, amenorrhea, webbed neck, cubitus valgus, hypogonadismMR mental retardation, IUGR intrauterine growth retardation, CHD congenital heart disease, SS short stature\nNonsyndromic forms\nIt is well known that nonsyndromic renal malformations may occur as hereditary traits and can present with familial aggregation. Evidence in favor of a genetic determination of the disease is raised by an increased recurrence risk among first-degree relatives and by several reports of familial occurrence of multiple malformations, including renal agenesis\/hypoplasia and dysplasia. The relative recurrence risk of bilateral and unilateral agenesis has been estimated at 4\u20139% [6, 16, 17]. For familial cases, in most of the pedigrees, the suggested mode of inheritance was autosomal dominant with reduced penetrance, estimated to range between 50% and 90% [16]. For example, a large pedigree with an autosomal dominant mode form of nonsyndromic renal hypoplasia and dysplasia has recently been described [18]. However, a Somalian kindred in which the trait was segregating in an autosomal recessive fashion has been reported [19]. Nevertheless, until recently, no linkage studies in familial renal agenesis\/hypoplasia and dysplasia have been reported. Incomplete penetrance, variable expression and the fact that anatomical defects in many family members can be clinically silent, complicate recruitment of large pedigrees that would be suitable for linkage analysis.\nStrategies for gene discovery\nStrategies to find genes causing renal agenesis\/hypoplasia and dysplasia vary significantly depending on the characteristics of the study population available. Different data sets of patients have potential advantages and possible pitfalls.\nCandidate gene studies\nSo far, candidate gene studies have been the only alternative to linkage analysis to find genes underlying both Mendelian and complex traits. Such studies have identified many genes causing rare genetic diseases [20] (The Human Gene Mutation Database, http:\/\/www.hgmd.cf.ac.uk\/ac\/index.php) and most of the genes that are known contribute to susceptibility to common diseases [21, 22]. Large cohorts of sporadic cases or small pedigrees can be utilized in case-control association studies to find common disease associated alleles. Such cohorts can also be screened by resequencing of candidate genes to detect rare variants with large effects that account for disease in a small proportion of the patients. Selection of one approach over the other depends on the expected degree of genetic and allelic heterogeneity of the trait under investigation. Genetic heterogeneity refers to the situation where mutations in different genes account for disease in different affected individuals. Allelic heterogeneity refers to the presence of many independent mutations in a given gene. For a trait with high locus and allelic heterogeneity, the search for common disease-contributing alleles is problematic, and resources would be better directed toward comprehensive resequencing of candidate genes to discovery the rare disease-causing variants. In practice, the heterogeneity parameters are difficult to predict a priori. The resequencing approach has been successfully applied to find several genes causing kidney developmental disorders. As an example, mutations in the uroplakin III gene, which produce VUR in mice [23], explain a small fraction of human renal hypodysplasia [24\u201328]. Similarly, results from the ESCAPE study recently provided the first comprehensive analysis of renal developmental genes in children affected by nonsyndromic renal hypodysplasia, showing a fairly high prevalence of PAX2 and TCF2 mutations [5, 29]. Another success of the candidate gene approach is the latest discovery of mutations in genes of the renin-angiotensin system (RAS) in severe forms of renal tubular dysgenesis [30]. The search for common variants predisposing to nonsyndromic renal hypodysplasia has not been frequently applied. However, these common predisposing alleles may not be recognized until a comprehensive search is undertaken. As an example, a common noncoding variant in a RET enhancer has recently been shown to be a strong risk allele for Hirschsprung disease, explaining the paucity of coding mutations found in families showing linkage to the RET locus [31].\nTraditional linkage studies and genetic isolates\nThe genome-wide linkage analysis\/positional cloning approach is a time-tested method used to identify disease-causing mutations, and it has been extremely successful in the past few decades for mapping genes that underlie monogenic Mendelian diseases [32, 33]. This approach hinges on availability of single, uniquely large pedigrees that segregate genes with large effect or a large number of small-sized pedigrees. Mutations in genes underlying Mendelian forms of disease usually account for a fraction of sporadic forms (e.g. PAX2 and TCF2).\nFor renal agenesis\/hypoplasia and dysplasia, large pedigrees amenable for linkage analysis are very difficult to ascertain because these traits have incomplete penetrance (due to genetic and environmental modifiers). Moreover, many malformations, such as unilateral agenesis can be clinically silent and will not be detected without systematic screening of family members. As for candidate gene studies, locus heterogeneity is another potentially complicating factor that may dilute the power of linkage studies. Our previous data demonstrated that in the setting of reduced penetrance, variable expressivity, and very high genetic heterogeneity, approaches based on a limited number of uniquely large pedigrees or a very large number of medium-sized kindreds, are more likely to be successful to map a disease gene [34]. As a result of these difficulties, no linkage studies of renal agenesis\/hypoplasia have been published so far. These kinds of patient cohorts are very arduous to collect and require multicenter collaborative efforts. We have been able to collect seven multigenerational extended pedigrees segregating congenital anomalies of the kidney and urinary tract, including renal agenesis\/hypoplasia, as an autosomal dominant trait with reduced penetrance trait. These families allowed us to localize a gene for this trait to a ~7\u00a0Mb interval to chromosome 1p32\u201333 in a setting of genetic heterogeneity [35]. This work represents the first step toward the discovery of a new gene and, possibly, a new pathway, in kidney development.\nGenetic isolates represent a population structure that can greatly facilitate gene identification efforts. The genetic isolates are populations that are originated from a limited group of founders with little subsequent immigration into the population. Without an inflow of genes, a long period of time would be required for spontaneous mutations to rebuild genetic diversity. Therefore, genetic isolates are likely to harbor few disease-contributing alleles that have been inherited identical by descent from common ancestors [36\u201338]. These ancestral mutations can be detected by searching for a shared haplotype signature in affected individuals, representing a powerful shortcut to narrow down a linkage interval to a handful of genes. This strategy, called linkage disequilibrium (LD) mapping, has allowed the identification of several genes for Mendelian disorders [39\u201341]. Hence, the advantages of studying a genetic isolate rely on: (a) a higher prevalence of certain diseases, allowing traits with reduced penetrance to express and show their hereditary component, (b) a more uniform genetic background, thus reducing the genetic heterogeneity, (c) usually good genealogical records, (d) a more uniform environment, and (e) the possibility of speeding up gene discovery through linkage disequilibrium mapping. We have recently characterized a genetic isolate in an Italian valley, in which different glomerular diseases occurred at a much higher prevalence compared with the general population, in apparently unrelated individuals. The genealogical reconstruction allowed us to reconnect most of the patients to a few founders up to the sixteenth century [42]. This study is an example of how an isolate can allow traits that display reduced penetrance and variable expressivity to express their genetic component and represent a first step to find genes causing or predisposing to such diseases. Further investigation of recognized population isolates for developmental disorders, especially renal agenesis\/hypoplasia and dysplasia, might help to accelerate gene mapping.\nGenome-wide association studies\nThe genome-wide association study is an approach aimed at exhaustively covering the genome to look for causative variants. Similar to genome-wide linkage studies, no assumptions are made about either the location of the causative variant or the biological role of the disease gene. Therefore, this approach represents an unbiased method to find disease-causing genes, with also a very high probability of discovering new genes, thus unraveling new pathophysiological pathways. Genome-wide association studies were not feasible until now because of the lack of information about the variability in the human genome and lack of low-cost, high-throughput genotyping technology. This situation has changed in the past 2\u00a0years: dbSNP (http:\/\/www.ncbi.nlm.nih.gov\/projects\/SNP\/) now contains about 5 million SNPs, including most of the SNPs with a minor allele frequency higher than 1% estimated to exist in the human genome [43]. Moreover, the HapMap project [44] represents a fundamental advance to performing efficient and successful genome-wide studies through the determination of LD patterns and haplotype blocks across the genome. Another important step has been the tremendous improvement in genotyping technology, with the development of platforms for fast, high-throughput, low-cost SNPs genotyping. Such platforms allow the simultaneous genotyping of 100\u2013500,000 SNPs in a single assay, allowing a dense coverage of the human genome [45, 46]. Some examples of success of this approach have been recently published. For example, genome-wide association studies on patients affected by age-related macular degeneration allowed the individuation of a common variant in the complement factor H as a major risk-associated allele [47, 48]. Similarly, polymorphisms in the transcription factor TCF7L2 have been found to confer risk to type 2 diabetes in different populations [49, 50]. Whether genome-wide association studies will lead to significant discoveries in renal agenesis\/hypoplasia and dysplasia is still unclear, but certainly, this approach represents a very promising strategy to identify common variants conferring susceptibility to more frequent, complex traits.\nSearch for structural variations in the genome\nA number of urogenital malformations are associated with chromosomal abnormalities. For example, a deletion on chromosome 10q26 has been implicated in urogenital development [51]. Similarly, two distinct loci for renal malformations, including VUR, have been mapped to chromosome 13q by deletion mapping using microsatellites in a limited number of affected individuals [52, 53]. Advances in technology, mainly, genome-scanning array technologies and comparative DNA-sequence analyses, have identified a high prevalence of DNA variations that involve segments that are smaller than those recognized by standard cytogenetics techniques [54]. These structural variations are a common feature of our genomic landscape, encompassing deletions, duplications, inversions, and translocations, which range from a few bases up to hundreds of kilobases. These rearrangements comprise benign polymorphisms, as well as deleterious mutations that can disrupt gene structure or affect gene regulation. Newer techniques now allow for the identification of structural variation at the genome-wide level, enabling examination of single patients to rapidly define a chromosomal region (locus) of interest. Several studies have already reported structural variations associated to human disease, leading in some cases to a molecular definition of a disorder before a recognized clinical syndrome [55\u201357].\nThese technologies have also been already successfully applied to developmental disorders. A genome-wide search for structural variations using comparative genomic hybridization (CGH) array allowed the discovery of the gene CHD7 as a cause of CHARGE syndrome, a rare, complex disorder in which congenital anomalies affect in a nonrandom fashion several tissues, including the urinary tract [58]. Careful clinical selection of patients and application of genome-wide methods for searching structural variation in renal agenesis\/hypoplasia and dysplasia can help find new loci linked to the disease, confirm and narrow loci obtained by linkage analysis, and speed up the discovery of causative genes.\nConclusions\nRenal agenesis\/hypoplasia and dysplasia still represents a challenge for both the clinicians who attempt a precise diagnosis and for the geneticists who try to unravel the genetic basis. Genetic and clinical approaches are now converging toward a common goal, which is the discovery of genetic markers, to make the diagnosis of this trait easier. The final objective is to improve classification, to make a reliable prognosis, and to attempt prevention. Based on advances from the last few years, the goal appears to be more feasible with large multicentric collaborative groups that share the same objectives and resources.","keyphrases":["renal agenesis\/hypoplasia and dysplasia","linkage analysis","association studies","gene mapping","structural variants"],"prmu":["P","P","P","P","R"]}
{"id":"Mol_Biochem_Parasitol-1-5-1906845","title":"An approach to classifying sequence tags sampled from Plasmodium falciparum var genes\n","text":"Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) appears to play an key role as both a virulence factor and as a target of naturally acquired immunity [1,2]. This large family of molecules is encoded by the highly polymorphic superfamily of var genes of which there are 60 variants in every genome [3].\nA rapidly growing collection of var sequences is now available from clinical isolates around the world [4\u201311]. Despite immense diversity both in terms of overall organization and primary sequence, the majority of var genes contain a DBL1\u03b1 region [3]. The existence of short islands of homology within this region has enabled the design of primers that can be used to sample sequence from most var genes to create DBL1\u03b1 sequence tags [5]. A standard approach to classification of these sequence tags would enable direct comparisons to be made between different studies. However, the extreme diversity of var genes and the fact that they undergo intra-genic recombination [4,12,13], makes this difficult.\nDespite the high diversity there does appear to be underlying simplicity to the var genes that supports the use of information present in DBL1\u03b1 sequence tags in making comparisons between the expression levels in different isolates. Analysis of the fully sequenced genome of a single P. falciparum isolate 3D7 suggests that the genomic location of the 60 var genes promotes genetic structuring and the maintenance of genetically distinct sequence types [14\u201316]. In addition, structural features of the genes within the single genome of 3D7 closely mirrors the range of structural features among collections of DBL1\u03b1 sequence tags from clinical parasite isolates [9]. We previously used a small number of key sequence features in an algorithm to classify the DBL1\u03b1 sequence tags from a single geographical location in Kenya into six groups [9] (see Fig. 1A and below). This var tag grouping system, though it is based on portion of the DBL1\u03b1 domain (see Supplementary information), corresponded well with whole var gene classification based on the whole genome sequence of the parasite line 3d7 [9]. This grouping system appears to be biologically meaningful. Expression of group 2 sequences was strongly associated with the parasite rosetting phenotype in Kilifi whereas expression of group 1 sequences was negatively associated with the repertoire of antibodies to infected erythrocyte surface antigens carried by the patient at the time of disease [9]. Thus DBL1\u03b1 sequence tags appear to contain useful information about the genes to which they belong that is currently not directly accessible in field studies of clinical parasite isolates.\nWe have developed a rapid approach to performing the classification using text string analysis functions in Microsoft Excel and Perl (see Supplementary files). This classifies sequence tags directly without the need for prior alignment and can be performed on many sequences simultaneously. The approach is summarized in Fig. 1A. The classification is based around a count of the number of cysteine residues within the tag region and a set of sequence motifs at four positions of limited variability (PoLV 1\u20134) whose positions within the sequence are fixed in relation to four anchor points (a\u2013d, marked with arrows in Fig. 1A). Thus PoLV1 and PoLV4 are fixed in relation to the 5\u2032 and 3\u2032 ends of the sequence, respectively (anchor points a and d). PoLV2 and PoLV3 are fixed in relation to a \u201cWW\u201d motif (anchor point b). The definition of the groups defined by these features is summarized in the box in Fig. 1. Henceforth we will refer to these groupings as cyteine\/PoLV groups.\nThis text string analysis approach was tested on the original set of sequences from Kilifi, Kenya [9] and sequences from 9 other studies (see Fig. 1B\u2013E). The sequences were pre-screened to ensure that they contained a 5\u2032DIGDI and 3\u2032PQFLR consensus sequences. Overall 99.6% of sequences could be classified using this approach. This included 100% of sequences from Malawi (J. Montgomery unpublished), Papua New Guinea [7,17], Mali [10], Solomon Islands [7], and The Philippines [7] together with 100% of sequences from one dataset from Brazil [6]. A dataset from Venezuela (52 non-identical sequences [8]) carried two sequences that could not be classified. A dataset from Brazil (137 non-identical sequences, [18]) carried one sequence that could not be classified. The original dataset from Kilifi (878 non-identical sequences [9]) carried two sequences that could not be classified. All five of these sequences lacked WW or VW motifs required as anchor points within the sequence.\nPart of the rationale for this grouping system came from a search for PoLV motifs that were associated with sequences with distinct length distributions [9]. Two motifs were identified which were independently associated with short sequences. These are MFK* at PoLV1 and *REY at PoLV2 (an asterisk here denotes any amino acid). We hypothesised that if sequences of different length recombine with each other they will generate a wide range of sequences of different lengths whereas genetically isolated sequences, i.e., those that are not recombining with one another are able to maintain distinct distributions in their length. If these groupings are genuine the sequences classified into different groups should have similar lengths in different settings. As shown in Fig. 1B\u2013E, broadly similar distributions of sequence length are observed within the six different groups between three different continents, suggesting that sequences generated in these different studies shared the same set of structural features. Specifically, MFK* (carried at PoLV1 in group 1) and *REY (carried at PoLV2 in groups 2 and 5) are associated with short sequences in each geographical region. No examples of sequences with both MFK* and *REY motifs were found, suggesting that these motifs are mutually exclusive. In addition, though *REY motifs were found in sequences with 2 or 4 cysteine residues (cys2 or cys4), with the exception of a single cys4 (group 4) sequence from the Philippines, MFK* motifs were found exclusively in cys2 (group 1) sequences.\nFurther support for the cysteine\/PoLV groupings comes from recent publications. Trimnell et al. found a good correspondence between cysteine\/PoLV groupings of cys2 sequences and groups defined phylogenetically within a globally sampled subset of var genes with a specific upstream control region, upsA [11]. Also evident from sequences reported in that study is the fact that DBL1 from two other globally sampled subsets of var genes can be easily distinguished from DBL1 domains from other vars using unique PoLV motifs. var2csa vars have a unique PoLV2 motif \u201cEVIT\u201d, whereas Type3 vars have a unique PoLV4 motif \u201cPPVV\u201d (data not shown).\nKraemer et al. have recently performed an analysis and re-classification of whole var genes from 3D7, HB3 and IT4 [19]. Fig. 2A and B summarizes the relationship between the cysteine\/PoLV groupings and whole var gene classification. With the exception of group 6 sequences which were not found in HB3 var genes all sequence groups were represented. In all three genomes cysteine\/PoLV group 1 sequences are exclusively found in group A var gene and long genes with >5 domains whereas cysteine\/PoLV group 5 are found only in non-group A genes and those with 4\u20135 domains. Cys2 sequence tags (groups 1\u20133) were never found in group C var genes.\nKyriacou et al. used a phylogenetic approach to compare DBL1\u03b1 sequence tags from Mali [10]. Visual inspection of the layout of these sequences reveals three main groups and a minor group. There was good correspondence between these groups and the cysteine\/PoLV groupings (Fig. 2C [10]). This study showed that cys2 sequence tags were more frequent among parasite isolated from children with cerebral malaria than those from children with hyperparasitaemia. However, division of the sequences into cysteine\/PoLV groups suggests that the frequency of group 2 sequences is similar in parasites from these two groups of children (Fig. 2D [10]).\nAt a higher level of resolution, the distinct sequence identifier (DSID) (see Fig. 1A) is a potentially useful method of further classifying sequence tags. This consists of a string of sequence features in the form \u201cPoLV1-PoLV2-PoLV3-number of cysteines-PoLV4-sequence tag length\u201d. The DSID captures more of the overall sequence diversity than the previously described \u201csequence signature\u201d [9] whilst remaining robust to minor changes introduced by sequencing or PCR errors. Among the 1595 non-identical sequences identified in all the studies described here, there were 1111 DSIDs. Fig. 1F\u2013G illustrates the potential usefulness of this approach to classification. In Fig. 1F, 44 \u201ccommon\u201d sequences that were shared between more than one study were selected. Fishers exact test was used to determine whether these common sequences were shared between two studies more or less than would be expected by chance (+ or \u2212 symbols, respectively). Fig. 1G is the same except that the analysis was done at the level of 157 \u201ccommon\u201d DSIDs that were shared between more than one study. In contrast to Fig. 1F, there was a highly significant similarity between var genes from South American isolates in support a recent study of Amazonian isolates [18]. In contrast to the low overlap between DSIDs from Kilifi and from South America (Fig. 1G) there is considerable overlap in the constituent PoLV motifs themselves (see Supplementary information). This illustrates the potential for recombination to generate diversity from a limited number of sequence blocks [4,12,13].\nSince the cysteine\/PoLV system of classification is based on commonly occurring sequence features it is hoped that it will useful for initial analysis and annotation, comparison of different geographical regions over time and identification of unusual sequences.","keyphrases":["plasmodium falciparum","var","pfemp1","malaria","pfemp1, plasmodium falciparum erythrocyte membrane protein 1","dbl, duffy binding like","polv, position of limited variability","dsid, distinct sequence identifier"],"prmu":["P","P","P","P","R","M","R","R"]}
{"id":"Mol_Genet_Genomics-4-1-2329726","title":"Transcriptomics and adaptive genomics of the asymptomatic bacteriuria Escherichia coli strain 83972\n","text":"Escherichia coli strains are the major cause of urinary tract infections in humans. Such strains can be divided into virulent, UPEC strains causing symptomatic infections, and asymptomatic, commensal-like strains causing asymptomatic bacteriuria, ABU. The best-characterized ABU strain is strain 83972. Global gene expression profiling of strain 83972 has been carried out under seven different sets of environmental conditions ranging from laboratory minimal medium to human bladders. The data reveal highly specific gene expression responses to different conditions. A number of potential fitness factors for the human urinary tract could be identified. Also, presence\/absence data of the gene expression was used as an adaptive genomics tool to model the gene pool of 83972 using primarily UPEC strain CFT073 as a scaffold. In our analysis, 96% of the transcripts filtered present in strain 83972 can be found in CFT073, and genes on six of the seven pathogenicity islands were expressed in 83972. Despite the very different patient symptom profiles, the two strains seem to be very similar. Genes expressed in CFT073 but not in 83972 were identified and can be considered as virulence factor candidates. Strain 83972 is a deconstructed pathogen rather than a commensal strain that has acquired fitness properties.\nIntroduction\nUrinary tract infection (UTI) is one of the most common infectious diseases in humans and a major cause of morbidity. It is estimated that 40\u201350% of adult healthy women have experienced at least one UTI episode (Foxman 2002). UTI can be caused either by pathogenic strains leading to symptomatic UTI or by asymptomatic bacteriuria (ABU) strains resulting in a symptom-free carriage resembling commensalism. Escherichia coli is responsible for more than 80% of all UTIs. Acute pyelonephritis is a severe acute systemic infection caused by uropathogenic E. coli (UPEC) clones with virulence genes clustered on \u201cpathogenicity islands\u201d (PAIs) (Eden et al. 1976; Funfstuck et al. 1986; Stenqvist et al. 1987; Orskov et al. 1988; Johnson 1991; Welch et al. 2002). Paradoxically, a large proportion of UTIs are caused by ABU E. coli. Individuals infected with ABU-class E. coli may carry high urine titres of a single E. coli strain for months or years without provoking a host response.\nEscherichia coli 83972 is a prototype ABU strain and undoubtedly the best-characterised ABU-class E. coli to date. Strain 83972 was originally isolated in the 1970s from a young girl who had carried it for at least 3\u00a0years without symptoms (Lindberg et al. 1975; Andersson et al. 1991). The strain is well adapted for growth in the human urinary tract (UT) where it establishes long-term bacteriuria (Hull et al. 2000). It has been used for prophylactic purposes in numerous studies; as such it has been used as an alternative treatment in patients with recurrent UTI who are refractory to conventional therapy (Hull et al. 2000). An ongoing study on patients infected with strain 83972 has so far reported over 50 patient years with no serious side effects (Sund\u00e9n et al. 2006).\nABU patients may carry a single strain for months or years, creating a condition that resembles commensalism, but with a strain that may have evolved from a pathogenic ancestor. Several lines of evidence support the notion that the ancestor of strain 83972 was a pyelonephritic UPEC strain; it belongs to the B2 clonal group, a group associated with pyelonephritis and other extra-intestinal invasive clinical syndromes such as bacteremia, prostatitis and meningitis; the strain also contains gene clusters in various stages of erosion encoding the three UPEC-class fimbriae, i.e. the fim, pap and sfa\/foc clusters (Klemm et al. 2006; Roos et al. 2006a).\nSeveral studies have investigated the virulence characteristics of uropathogenic E. coli (UPEC) isolates and ABU isolates, in order to get better understanding of how some UTI strains can cause severe disease, while others can be used prophylactically to prevent the same (Blanco et al. 1996; Dobrindt et al. 2003; Vranes et al. 2003; Johnson et al. 2005; Marrs et al. 2005). ABU strains have been shown to lack many of the virulence-associated phenotypes; many of them are nonhaemolytic, nonadherent and lack haemagglutination ability (Vranes et al. 2003). Strain 83972 lacks many of the virulence-associated phenotypes but has been shown to carry many of the virulence-associated genes, such as kps, iutA, fyuA and malX (Dobrindt et al. 2003). However, apart from the fimbrial clusters, the strain has not been sequenced and it is not known which genes it shares with other E. coli isolates, which of the genes in the E. coli \u201ccore genome\u201d it carries and which genes it shares with other UTI isolates.\nThus far, although a number of UPEC isolates (i.e. CFT073, 536, UTI89 and F11) have been completely sequenced, no genomic sequencing of any ABU strain has been reported. Comparative genomics profiling using microarray chips designed to cover entire genomes is one strategy to obtain information about variability between different strains of the same species and indication of horizontal gene transfer (Willenbrock et al. 2006). DNA microarray-assisted functional genomics provides the global expression profile of a strain, revealing which genes are expressed under certain conditions. Global gene expression profiling of ABU strain 83972 employing the GeneChip E. coli Genome 2.0 Array (Affymetrix), containing four E. coli genomes including that of the UPEC isolate CFT073, will not only provide information regarding the up- and down-regulation of genes comparing different conditions, but will also reveal which genes are actually present (expressed) in the genome of 83972. Bacterial pathogens differ from commensals by expression of specific virulence factors such as those that mediate histological damage. Commensals, in contrast, have generally been regarded as bacteria lacking such virulence factors or other specific mechanisms for interaction with host tissues. Here we compare the global expression profiles of E. coli ABU strain 83972 grown under a number of different in vitro conditions and in three patients in order to get a representative picture of which genes are present\/expressed in the genome of this asymptomatic UTI strain.\nMaterials and methods\nBacterial strain\nEscherichia coli 83972 is a prototype ABU strain and lacks defined O and K surface antigens (Lindberg et al. 1975). It belongs to the ECOR group B2 together with many other UTI strains such as the well-characterized and virulent E. coli isolates CFT073, 536 and J96.\nGrowth conditions and stabilisation of RNA for microarray experiments of E. coli 83972 grown on urine agar plates\nHuman urine was collected from four healthy men and women volunteers who had no history of UTI or antibiotic use in the prior 2\u00a0months. The urine was pooled, filter sterilised, stored at 4\u00b0C, and used within the following day. E. coli 83972 was grown aerated in triplicates in 10\u00a0ml of human urine for 6\u00a0h. Thereafter, 100\u00a0\u03bcl of each culture was spread on urine plates (1:1 ratio of human urine and 0.9% NaCl) containing 1.5% agar. The plates were incubated at 37\u00b0C for 16\u00a0h. Subsequently, 600\u00a0\u03bcl of a 1:2 mixture of PBS and RNAprotect\u2122 Bacteria Reagent (QIAGEN AG) was poured on the plates, mixed with the lawn of cells and incubated for 5\u00a0min at room temperature to stabilise RNA. The stabilised mixture was then centrifuged and pellets were stored at \u221280\u00b0C. The samples from 83972, grown exponentially in MOPS and urine, in urine biofilms and in patients (>108\u00a0CFU\/ml) were all treated identically with RNAprotect Bacteria Reagent and have been described previously (Roos and Klemm 2006; Hancock and Klemm 2007).\nRNA isolation and microarray hybridisation\nTotal RNA was isolated using the RNeasy\u00ae Mini Kit (QIAGEN AG) and on-column DNase digestion was performed using RNase-Free DNase Set (QIAGEN AG). The quality of the total RNA was examined by agarose gel electrophoresis and by measuring the absorbance at 260 and 280\u00a0nm to ensure intact high-quality RNA. Purified RNA was precipitated with ethanol and stored at \u221280\u00b0C until further use. Conversion of RNA (10\u00a0\u03bcg per sample) to cDNA, labelling and microarray hybridisation were performed according to the GeneChip Expression Analysis Technical Manual 701023 Rev. 4 (Affymetrix, Inc., Santa Clara, CA). GeneChip E. coli Genome 2.0 Arrays (Affymetrix) were used for hybridisation of the labelled cDNA. The microarrays were scanned using the GeneChip Scanner 3000.\nData analysis\nThe raw intensities from the microarray experiments were background corrected and quantile-normalised. All microarray data in the study were obtained from mRNA being converted to cDNA, i.e. no genomic DNA was used for hybridisation. Probe intensities were summarised to yield expression values for each probe set or gene. These calculations were performed using the implementation of GCRMA (Wu et al. 2004) in Bioconductor (Gentleman et al. 2004) (http:\/\/www.bioconductor.org, http:\/\/www.r-project.org). In order to derive a cut-off expression value for making presence\/absence calls, we made use of intensities due to control probe sets with IDs beginning with AFFY. There were 96 such probes. The cut-off value was set so that only the top 1\/16th of these control probes would be flagged as present. As a result 4,109 genes in the array were marked as present; the remaining genes are referred to as \u201cabsent\u201d throughout this report, i.e. these genes could be truly absent, non-homologous or not expressed during any of the seven different growth conditions. Orthologs of all the genes in the array across E. coli K12 MG1655, E. coli O157:H7 Sakai, E. coli O157:H7 EDL933 and E. coli CFT073 were identified using bidirectional best hit BLAST.\nMicroarray data accession number\nThe supporting microarray data have been deposited in ArrayExpress (http:\/\/www.ebi.ac.uk\/arrayexpress) with accession numbers E-MEXP-584 (MOPS, urine and patient arrays), E-MEXP-926 (biofilm arrays) and E-MEXP-1453 (urine-agar plate arrays).\nResults\nGenes expressed in ABU E. coli 83972\nThe bacterial transcriptome is a dynamic entity that reflects the organism\u2019s immediate, ongoing response to its environment. DNA microarray-assisted functional genomics provides the global expression profile of the genome. The genomic expression profiles of the urinary tract infectious E. coli isolate 83972 were analysed under several different growth conditions and in different media using the GeneChip E. coli Genome 2.0 Array (Affymetrix). This array contains approximately 10,000 probe sets for all 20,366 genes present in E. coli strains MG1655 (K-12), CFT073 (UPEC), EDL933 (EHEC) and O157:H7-Sakai (EHEC). Due to the high degree of similarity between the E. coli strains, whenever possible, a single probe set is tiled to represent the equivalent ortholog in all the four strains.\nIn total, 21 microarrays were included in the study; arrays in triplicates were hybridised with RNA of the ABU strain 83972 cultured (1) aerobically to exponential phase in MOPS minimal medium, (2) aerobically to exponential phase in pooled human urine, (3) on urine agar plates, (4) statically in urine biofilm on Petri dishes and finally, (5\u20137) in three patients (Pat1, Pat2 and Pat3) in vivo. Figure\u00a01 shows the expression levels of all CFT073 genes in strain 83972 during growth in the different environments; many genes were similarly expressed during all seven conditions. However, some genes were expressed only during one or a few of the conditions. For example, the genes encoding yersiniabactin in the high pathogenicity island (HPI), i.e. PAI-asnT, were highly expressed in Pat2 (and in biofilm), but much lower during the other conditions. The c2557\u2013c2563 genes (around 2.4\u00a0M in Fig.\u00a01), involved in nucleotide sugar and mannose metabolism and encoding hypothetical proteins, were highly expressed in Pat3 but not under any other condition. Another example is the c1968\u2013c1971 genes (around 1.8\u00a0M), i.e. ydfI encoding a d-mannonate oxidoreductase, ydfJ encoding a metabolite transport protein and rspAB involved in the starvation response, which also were highly expressed only in Pat3.\nFig.\u00a01The expression levels of CFT073 genes in strain 83972 during seven different growth conditions. The outer blue circle shows the calculated absence (0.0) and presence (1.0) of CFT073 genes in ABU strain 83972. The seven PAIs of CFT073 are indicated in red\nIn total, there were 108 genes that were significantly changed in all six urine environments compared with MOPS. Twenty of these genes were up-regulated in all six urine conditions whereof half were related to different iron systems, i.e. iroN, fepA, fecI, iucBC, fhuA and exbD, as well as b3337 and b1995 involved in iron storage and encoding a putative haemin receptor, respectively. The other urine up-regulated genes were marA, a multiple antibiotic resistance gene, sodA, encoding superoxide dismutase, ahpC, encoding hydroperoxide reductase, b1452, c1220, c4210, lysA, rrsG, rrsH and yrbL. Most iron acquisition systems were expressed in all the six urine environments; the enterobactin, salmochelin, aerobactin, haem and sitABCD systems were all expressed in all the six urine conditions (although weaker in the urine plates). Interestingly, the fec system, which is a citrate-dependent iron uptake system found in K-12 but missing in CFT073 and other UPEC strains, was highly expressed in Pat3. Up-regulation of all these iron-uptake systems revealed that the strain has an impressive array of iron acquisition systems and all of these are active in the human bladder.\nNineteen of the top 31 highest expressed genes overall were genes involved in ribosomal synthesis. The high expression of ribosomal genes in E. coli 83972 suggests a rapid growth rate; the highest expression values were obtained in Pat1 followed by MOPS, urine and Pat2, indicating a growth rate just as fast in the patients in vivo as in exponential growth phase in a shake flask. This supports our hypothesis that the strain\u2019s optimized growth properties in human urine explain its ability to successfully colonize the human urinary tract in the absence of functional fimbriae (Roos et al. 2006b).\nFigure\u00a01 reveals that strain 83972 almost exclusively expresses the iron uptake and transport systems in the seven CFT073 PAIs, almost none of the other genes in these islands are expressed. There are only two exceptions; c0300, located in PAI-aspV encoding a hypothetical protein, and c3686\u20133690, located in PAI-pheV encoding YrbH and KpsEDC. The yrbH gene belongs to the 131 genes that were recently identified as UPEC specific and it was the second highest expressed UPEC-specific gene in mice (Lloyd et al. 2007); in our samples the highest expression was found in the three patients and in MOPS. Outside the PAIs there are a few genes\/gene clusters that are highly expressed in all urine samples or only in the patients. The enterobactin system was up-regulated during all urine conditions and the chu cluster (involved in haem uptake and transport) was highest up-regulated in the patients followed by in vitro urine growth. The ycdO and ycdB genes were highly expressed in the three patients; these have recently been identified to encode haemoproteins, probably involved in iron transport, induced at acidic conditions (Sturm et al. 2006).\nLooking at the significantly changed genes for all six urine conditions compared with MOPS (in total 1,897 genes) revealed that Pat2 and Pat3 shared the largest number of similarly changed genes; 75% of all changed genes in Pat2 are regulated in the same way (i.e. up or down-regulated) in Pat3 (Fig.\u00a02). Interestingly, Pat1 shared the largest number similarly regulated genes with the biofilm growth mode; also for Pat2 and Pat3, the biofilm growth mode showed a larger number of similarly regulated genes than Pat1 or any other condition. This could indicate that the expression profile of strain 83972 during in vivo growth is closer related to biofilm growth than to growth in shake flasks or plates.\nFig.\u00a02Number of significantly up- and down-regulated genes in strain 83972 during the different growth conditions (i.e. exponential growth in urine, on urine-agar plates, in urine biofilm, in vivo in three patients) compared with exponential growth in MOPS minimal lab medium. The diagonal boxes (dark blue colour) show the number of significantly changed genes during cultivation in that specific condition compared with MOPS (e.g. 664 genes were up- or down-regulated in urine compared with MOPS and 938 genes were changed in plates compared with MOPS) and the other boxes show the number of significantly changed genes shared between two conditions (e.g. 311 of the 664 and 938 significantly changed genes in urine and plates compared with MOPS were shared between these two conditions, i.e. up- or down-regulated in both urine and plates compared with MOPS). Stronger blue colour indicates larger number of significantly changed genes shared between two conditions\nCloseness to CFT073\nGiven the different growth conditions analysed, it is not unrealistic to assume that most genes present in strain 83972 would be expressed, to some extent, under at least one of these seven different conditions\/environments, i.e. growth in liquid and on solid media; during exponential phase, in biofilm and during colony-forming conditions; in different growth media (human urine and minimal lab medium); as well as in vivo in three different individuals.\nData analysis of the 21 microarrays revealed that of the 8,716 E. coli transcripts on the microarray (not including probes representing intergenic regions and controls), 4,109 transcripts (47%) showed expression levels above detection limit during at least one of the growth conditions investigated (referred to as \u201cpresent\u201d, see blue, outer circle in Fig.\u00a01). Figure\u00a03 shows the distribution among the four E. coli genomes represented on the microarray of these 4,109 transcripts expressed in E. coli 83972. Not surprisingly, the UTI strain 83972 shows highest similarity with the UPEC isolate CFT073 of the four genomes on the array; the large majority of the 4,109 transcripts found present (96.3%) can be found in CFT073, corresponding to 71% of the CFT073 genome. E. coli 83972 expressed 150 genes that do not exist in CFT073; 85 of these can be found in MG1655 and the remaining 65 genes can be found exclusively in one or both of the two EHEC strains present on the array (Fig.\u00a03). Thirty of the 65 genes homologous to EHEC genes are encoding proteins of cryptic prophages, whereas the large majority of the remaining 35 genes encode unknown or hypothetical proteins. The 85 genes that can be found in MG1655 but not in CFT073 includes the fec cluster encoding an iron citrate transport system (fecABCDEIR). In total, 3,959 CFT073 genes were expressed in strain 83972; this could be compared with 4,162 CFT073 genes present in the UPEC (cystitis) isolate F11 (Lloyd et al. 2007).\nFig.\u00a03Venn diagrams showing the distribution of the 4,109 genes filtered present in strain 83972. The percentages indicated below each strain show how large part of the genome of the corresponding strain was filtered present in strain 83972\nE. coli core genome\nThere is a large diversity in size of the chromosome of E. coli; in all 32 E. coli (and Shigella) genomes that have been fully sequenced, or at least with an expected coverage of greater than 99%, the size of the chromosome ranges from 4.5 to 5.6\u00a0Mbp. The genomes show a considerable amount of diversity, and the estimated size of the current pan-genome was estimated to contain 9,433 different genes (Willenbrock et al. 2008). Several studies have identified sets of \u201ccore genes\u201d found in most E. coli genomes. However, the number of these core genes tends to decrease as the full genomic sequences of new E. coli strains become available. The size of the E. coli core genome has recently been predicted to contain 1,563 genes for an infinite number of E. coli strains, and the number of new genes predicted from each new E. coli genome that is sequenced is \u223c79 (Willenbrock et al. 2008). In our analysis, 2,472 (60%) of the genes found present in strain 83972 were common in all the four E. coli genomes on the array (Fig.\u00a03), which is well above the estimated E. coli core genome and also above the 2,241 common genes conserved among the 32 sequenced E. coli strains (Willenbrock et al. 2008). Furthermore, considering the fact that the microarray contains only four E. coli genomes, the total number of genes detected present (4,109 genes) in 83972 seems reasonable comparing the size of other sequenced UTI E. coli genomes. The genome size of strain 83972 has been reported to be 4.9\u00b10.2\u00a0Mbp (Zdziarski et al. 2007), indicating that the strain contains roughly an additional 800 genes, not identified in the present analysis.\nOf the 2,734 transcripts on the chip that are present in all the four strains represented on the microarray, 393 transcripts were below detection limit on all 21 microarrays and filtered as \u201cabsent\u201d in strain 83972. These included 81 genes encoding hypothetical proteins. Several of the absent genes were found in clusters, many of which are involved in surface structure elements and chemotaxis. These included genes involved in flagellar biosynthesis (flgABCDEFGHIJKL, flhABE, fliACDEFGHIJKLNOPQRSTZ and motAB), curli production (csgABCEFG), colanic acid synthesis (wcaABCDEFGHI and wza) and chemotaxis (cheBRWYZ and tap). Other whole cluster of genes that were not expressed in the ABU strain but found in all the four E. coli present on the chip were hyaBCDEF (hydrogenase I), hycACD (hydrogenase 3), tauABCD (responsible for taurine uptake in E. coli) and b1500\u20131505 (containing the fimbrial-like genes ydeQRST), as well as the fimEAIC genes which previously have been shown to be absent in strain 83972 (Klemm et al. 2006).\nUPEC-associated genes present in strain 83972\nThe four UPEC isolates that have been sequenced, CFT073, UTI89, 536 and F11, contain 5,379, 5,154, 4,766 and 4,467 genes, respectively, on the chromosome. CFT073 and 536 are both O6 strains and yet show a large diversity; the genome of 536 is almost 300\u00a0kb smaller than that of CFT073 (Brzuszkiewicz et al. 2006). The genomic differences are mainly restricted to large pathogenicity islands, the additional DNA in CFT073 are genes of five cryptic prophages, which are absent in 536 (Brzuszkiewicz et al. 2006). The 427 genes that are present only in the strain 536, and the 432 genes present only in the two UPEC (compared with other sequenced E. coli) are scattered all over the genome (Brzuszkiewicz et al. 2006). Over 70% of the CFT073 transcripts were present in strain 83972 compared with 89% of the CFT073 transcripts found in strain 536. Figure\u00a04 shows the homology of 16 sequenced E. coli and Shigella isolates including the three sequenced UPEC strains (UTI89, 536 and F11) pasted on the CFT073 genome; the outer, red circle in the figure shows the results from the presence\/absence analysis on strain 83972. Many virulence-associated genes are located on the large pathogenicity islands (PAIs) found in different UPEC strains. The large pathogenicity island at pheV in CFT073 (also called PAI ICFT073) encodes haemolysin (hlyCABD), aerobactin biosynthesis proteins (iutA and iucABCD), antigen 43 (c3655) and the secreted autotransporter toxin (sat); these were all filtered present in our analysis, suggesting that strain 83972 harbours a similar island on its chromosome. Interestingly, the aerobactin system is missing in the other three UPEC isolates. Furthermore, this PAI contains genes encoding the uropathogenic-associated P fimbriae (papIBAHCDJKEFG). The pap gene cluster of 83972 has been sequenced (Klemm et al. 2006); the pap genes are all present and show 72\u2013100% sequence homology with the corresponding genes in CFT073. The results of the microarray analysis corresponded very well to the observed sequence homology of the different genes in the cluster (i.e. if a specific gene on the microarray is represented with probes that contain a non-homologous region compared with the corresponding gene in the hybridised sample, that gene will not hybridise and will be filtered absent); the six genes with highest sequence homology were filtered present (i.e. papHCDJKF with 98, 100, 100, 98, 99 and 95% homology, respectively) and the four with least sequence homology were filtered absent (i.e. papIAEG with 94, 83, 77 and 72% homology).\nFig.\u00a04BLAST atlas comparing the absent (0.0) and present (1.0) CFT073 genes in strain 83972 with other sequenced E. coli and Shigella strains, including the three sequenced UPEC isolates 536, UTI89 and F11. The UPEC CFT073 genome is used as reference. The outer blue circle represents the calculated absence\/presence in 83972 followed by the three UPEC isolates; the six inner circles represent Shigella strains. The seven PAIs of CFT073 are indicated in red. The blow-up shows the presence\/absence of the fim cluster (c5391\u20135400) in strain 83972\nThe employed microarray contains probes for all ten known and putative fimbriae-encoding gene clusters in CFT073. Together with the pap cluster, two other fimbrial clusters that have been associated with UPEC virulence are known to be present in strain 83972 and have been sequenced, i.e. the fim and sfa\/foc clusters. As for the pap cluster, the filtering of absent genes corresponded very well to the actual presence and sequence homology of the genes; strain 83972 contains a large deletion in the fim cluster but shows high sequence homology with the present genes, and all the genes in the deleted part of the cluster, i.e. fimEAIC, were filtered absent (see blow-up in Fig.\u00a04). Also, the sfa\/foc cluster in 83972 shows high homology with that in CFT073 (98\u2013100%), and eight of nine genes were filtered present; the putative regulatory gene, sfaC, was filtered absent. Regarding the other fimbrial clusters present on the microarray, none of the genes encoding F9 fimbriae, which appear to be common in UPEC and plays a role in biofilm formation (Ulett et al. 2007), and another putative fimbriae (yehABCD) were expressed and might be absent in strain 83972 (Table\u00a01).\nTable\u00a01Analysis of fimbriae-encoding genes in strain 83972Descriptionc numberGenesNo of genesNo (%) of absent genesAbsentPutative chaperone-usher fimbrial operonc0166\u20130172yadN-ecpD-htrE-yadMLKC73 (43)ecpD, yadMKF1Cac1237\u20131245sfaCB-focAICDFGH91 (11)sfaCF9c1931\u20131936c1936-34-ydeSRQ66 (100)AllPutative chaperone-usher fimbrial operonc2635\u20132638yehABCD44 (100)AllPutative chaperone-usher fimbrial operonc2878\u20132884yfcOPQRSUV75 (71)yfcQRSUVP fimbriaeac3583\u20133593papIBAHCDJKEFG114 (36)papIAEGbPutative chaperone-usher fimbrial operonc3791\u20133794ygiLGH-c379441 (25)ygiLAuf fimbriaec4207\u20134214aufABCDEFG87 (88)aufBCDEFGP fimbriae (2)ac5179\u20135189papIBAHCDJKEFG2 (papAD)1 (50)papA_2Type 1 fimbriaec5391\u20135399fimBEAICDFGH94 (44)fimEAICaFimbrial operons present on PAIs. The two pap clusters share the same probes on the array with exception for papA and papD, which are represented by two separate probe sets eachbNone of these pap genes are absent in strain 83972, but papIAEG were filtered absent in the microarray analysis due to non-homologous sequence regions compared with the CFT073 pap probes present on the array\nPresence of other pathogenicity islands in 83972\nStrain 83972 seems to carry most of the pathogenicity islands of CFT073 (or PAIs similar to the ones in CFT073) according to our present\/absent analysis (Table\u00a02). The only PAI of CFT073 in which most genes (i.e. 93%) were filtered absent in strain 83972 is PAI-pheU (PAI IICFT073), the island that contains a second pap cluster. The three genes filtered present in this PAI are present in several of the other sequenced E. coli and Shigella strains indicating that these three genes not are unique\/characteristic for this island wherefore this PAI is most probably absent in strain 83972.\nTable\u00a02Analysis of presence of pathogenicity islands in strain 83972Island nameCommon namec numberNo of genesaAbsent (%)Virulence-associated genesbPAI-CFT073-aspVPAI III CFT073c0253\u2013c03689640 (42)cdiA (c0345), picU (c0350)PAI-CFT073-serXc1165\u2013c12939233 (36)mchBCDEF (c1227, c1229\u20131232), sfa\/foc (c1237\u2013c1247), iroNEDCB (c1250\u2013c1254), ag43 (c1273)PAI-CFT073-icdAc1518\u2013c1601425 (12)sitDCBA (c1597\u20131600)PAI-CFT073-asnTHPI CFT073c2418\u2013c2437193 (16)fyuA (1246)PAI-CFT073-metVc3385\u2013c34102617 (65)hcp (c3391), clpB (c3392)PAI-CFT073-pheVPAI I CFT073c3556\u2013c369811951 (43)hlyA (c3570), pap (c3582\u2013c3593), iha (c3610), sat (c3619), iutA, iucDCBA (c3623\u20133628), ag43 (c3655), kpsTM (c3697\u2013c3698)PAI-CFT073-pheUPAI II CFT073c5143\u2013c52164643 (93)pap2 (c5179\u2013c5189)aNo of genes in the PAI that were present on the array with unique probes (i.e. genes that are not orthologues to any other E. coli transcripts present on the array)bBoldface indicates genes filtered present in strain 83972\nInsertion of the high pathogenicity island (HPI) of Yersinia pestis has been suggested to be one of the earliest events in the evolution of extraintestinal E. coli strains (Welch et al. 2002). The genes of HPI encoding yersiniabactin (Ybt) were all expressed in strain 83972. The HPI genes have been found up-regulated during urine biofilm growth of 83972 indicating that Ybt-mediated iron-uptake might play an important role in biofilm growth (Hancock and Klemm 2007) and a deletion mutant in the Ybt uptake receptor (FyuA) exhibits reduced biofilm formation (Hancock et al. 2008). The HPI genes have also been found up-regulated in vivo in two of the three patients (particularly in Pat2, see Fig.\u00a01) infected with this strain (Roos and Klemm 2006).\nThe pks island, a recently characterised and widely spread genomic island found in, for example, meningitis strains and the uropathogenic strain CFT073, encodes a machinery for the synthesis of peptide\u2013polyketides hybrid compounds (Nougayrede et al. 2006). The presence of the island is associated with the accumulation of double-strand DNA breaks in host cells and has genotoxic activity (Nougayrede et al. 2006). This island was expressed in strain 83972 and up-regulated in urine and in vivo (Table\u00a04; Fig.\u00a01). The pks island is widely distributed within E. coli phylogenetic group B2, and has been found in both pathogenic and commensal isolates; in commensal strains the cell-cycle-blocking activity might slow the turnover of the intestinal epithelium, and therefore prolong colonisation.\nPresence of positively selected UPEC genes\nA recent paper comparing the UPEC isolates CFT073 and UTI89 with six other finished E. coli genome sequences presented 29 genes that are under positive selection only in UPEC strains (Chen et al. 2006). These 29 genes are involved in various aspects of cell surface structure, DNA metabolism, nutrient acquisition and UTI. Of these 29 genes, 25 were filtered present in our ABU strain 83972; many of these genes are represented by more than one transcript on the array due to sequence differences among the four strains present on the array, in all cases the gene filtered present in 83972 corresponded to the CFT073 transcript. Four genes were filtered absent, agaI, yjiL, recC and yegO; they encode a putative galactosamine-6-phosphate isomerase, a hypothetical protein, exodeoxyribonuclease V gamma subunit and a hypothetical transport protein, respectively. The genes in the two COG categories that were significantly enriched in the two UPEC strains, i.e. \u201ccell wall\/membrane biogenesis\u201d (amiA, cutE, fepE, ompC, ompF and yfaL) and \u201csecondary metabolites biosynthesis, transport and metabolism\u201d (entD, entF and yojI) (Chen et al. 2006), were all present in strain 83972.\nFunctional analysis of MG1655 transcripts of ABU E. coli 83972\nTo gain more information concerning what type of genes were absent, the MG1655 genes were grouped into functional categories defined by the clusters of orthologous groups (COGs) of proteins (Tatusov et al. 1997). Previous studies have, in attempts to identify essential genes and the E. coli core genome, found that groups with genes involved in metabolism and various cellular processes (excluding cell motility) contain a substantially higher percentage of conserved and essential genes, while COGs with genes of unknown function and external origin as well as genes involved in signalling and motility contain fewer essential genes (Anjum et al. 2003; Gerdes et al. 2003). Classification of the absent genes of strain 83972 revealed that the groups \u201ccell motility\u201d, \u201cdefence mechanisms\u201d and \u201cnot in COGs\u201d had a significant overrepresentation of absent genes (Table\u00a03). A significantly lower proportion of absent genes were found in the groups: \u201ccell cycle control\u201d, \u201cposttranslational modification\u201d and \u201ctranslation\u201d. This is in agreement with a previously published study of pathogenic E. coli; Anjum et al. (2003) studied 26 strains of E. coli and found that the two groups with largest proportion of absent genes were \u201cnot in COGs\u201d and \u201ccell motility\u201d, while the six groups with the lowest proportion of absent genes were \u201ctranslation\u201d, \u201ccell division\u201d, \u201cposttranslational modification\u201d, \u201ccoenzyme metabolism\u201d, \u201cnucleotide transport and metabolism\u201d and \u201cenergy production and conversion\u201d, which all, with exception for the last group, contained significantly fewer absent genes in strain 83972 (Table\u00a03). This suggests that strain 83972 utilises a similar set of core genes as other E. coli strains.\nTable\u00a03Distribution of absent genes in functional categoriesFunctional categoryAbsentTotalZ testNo.%P valueAmino acid transport and metabolism9833.12960.636Carbohydrate transport and metabolism12642.32980.025Cell cycle control, cell division and chromosome partitioning516.1310.000Cell motility7784.6910.000Cell wall\/membrane\/envelope biogenesis8140.52000.087Coenzyme transport and metabolism2523.11080.001Defense mechanisms1748.6350.000Energy production and conversion8836.72400.563Function unknown6124.72470.003General function prediction only8330.92690.255Inorganic ion transport and metabolism5534.41600.921Intracellular trafficking, secretion and vesicular transport822.2360.000Lipid transport and metabolism2129.6710.129Nucleotide transport and metabolism1924.4780.002Posttranslational modification, protein turnover, chaperones2420.21190.000Replication, recombination and repair7042.41650.023Secondary metabolites biosynthesis, transport and catabolism2240.7540.075Signal transduction mechanisms3933.61160.748Transcription7833.22350.654Translation, ribosomal structure and biogenesis2113.51560.000Not in COGs57754.210650.0001,59539.24,070\nCFT073 genes absent in strain 83972\nThere were 1,636 CFT073 genes that could not be detected according to our expression profiling in ABU strain 83972; 961 of these genes are exclusively found in CFT073, i.e. not present in the other three strains represented on the array. The majority, 645 genes, corresponded to hypothetical, putative or unknown proteins. Considering the very different patient symptom profiles of strains CFT073 and 83972 (one being a true pathogen, while the latter is a commensal-like strain), genes that are present in UPEC isolate CFT073 but not expressed in ABU strain 83972 can be considered as virulence factor candidates. However, most genes associated with UPEC pathogenesis were expressed in strain 83972 and up-regulated during growth in urine, e.g. all iron-related genes encoding uptake and transport of aerobactin, salmochelin, yersiniabactin and haem\/haemoglobin (Table\u00a04). Two exceptions were the ireA gene encoding an iron-regulated outer-membrane protein that was filtered absent as well as the tsx gene encoding a nucleoside-binding outer-membrane protein. Although the tsx gene has not previously been associated with UPEC virulence, it has just recently been identified together with more well-known UPEC genes as involved in movement from the intestinal tract to the bladder and vagina (i.e. occurred significantly more often in multiple-site isolates than in rectal site-only isolates) (Xie et al. 2006); furthermore, Tsx was also recently identified together with 22 other outer-membrane proteins from CFT073 cells grown under conditions mimicking the urinary tract (Hagan and Mobley 2007).\nTable\u00a04Characteristics of ABU isolate 83972 compared with UPEC isolates CFT073, UTI89 and 536CharacteristicaCFT073UTI8953683972Expression in 83972bSerotypeO6O18O6O?cCapsuleK2K1K15K?cChu++++U, BF, PatEnt++++U, BF, PatFep++++Pl, U, BF, PatFeo++++BF, PatFhu++++Pl, U, BF, PatIro++++U, PatIuc+\u2212\u2212+Pl, U, BF, PatIutA+\u2212\u2212+U, BF, PatSit++++U, BF, PatFyuA++++U, BF, PatIha+\u2212\u2212+U, PatIreA+\u2212\u2212\u2212Pks island++++U, PatRfaH++++BFd-serine++++PatPap+++\u2212Fim+++\u2212Foc\/sfa+++\u2212Vat++++BF, PatSat+\u2212\u2212+UTsx+++\u2212Biofilm formation1.01.314.4aBoldface indicates genes that were filtered absent in strain 83972bUp-regulation in urine (U), biofilm (BF), plates (Pl) and patients (Pat) compared with MOPS minimal mediumcUndefined. Extensive electron microscopy analysis of the strain has never reported any capsule\nType IV fimbriae are assembled by the type II general secretory pathway. They occur in a wide range of species and frequently are associated with diseases. The ppdD and hofBC genes (b0106\u20130108), which encode type IV prepilin and are present in CFT073, EDL933 and MG1655, were filtered absent in strain 83972.\nCFT073 genes present in strain 83972 but not found in other UPEC strains\nThe majority of the genes that are absent in the other three UPEC isolates (i.e. 536, UTI89 and F11) were filtered absent in strain 83972 as well (gaps in Fig.\u00a04). However, there are a few exceptions where a gene that is not found in any of the other UPEC strains is filtered present in strain 83972. The aerobactin system belongs to one of the exceptions, indicating that strain 83972 is particularly well equipped with iron uptake systems. The other exceptions are all but one located on PAIs and they all encode hypothetical proteins: c1194\u2013c1204 (on PAI-serX), c1522\u2013c1528 (on PAI-icdA), c3394\u2013c3396 (on PAI-metV), c3681\u2013c3682 (on PAI-pheV where the aerobactin genes also are found) and c5372\u2013c5382. c3394\u2013c3396 and c5372\u2013c5382 are not present in any of the 16 sequenced E. coli and Shigella strains represented in Fig.\u00a04, indicating that some genes unique to CFT073 can be found in strain 83972 as well.\nDiscussion\nBacterial genomes are under constant change. New genes are acquired by horizontal transfer and old ones are lost by mutations. It is generally believed that commensal E. coli can become pathogenic through the acquisition of novel genes encoding virulence factors and niche-adaptation factors (Kaper et al. 2004). In contrast to organisms that have acquired genes for pathogenesis, E. coli 83972 is an example of an organism that has adapted to a commensal-like existence through gene deletions and point mutations. Using primarily the CFT073 as a scaffold, we used presence\/absence data from seven sets of different gene expression profiles (in total 21 microarrays) to model the gene pool of strain 83972. Given the limitations of the approach, i.e. genes not present on the employed chip have been ignored, a substantial body of information was gathered concerning the genomic content of the strain. As it turned out the strain was highly similar to CFT073; 96% (3,959) of the genes found to be expressed on the employed microarray by 83972 are also found in CFT073, and genes on six of the seven pathogenicity islands of CFT073 were expressed by 83972; furthermore, CFT073 genes not found in any other UPEC isolate were expressed by 83972. An estimated \u223c900 CFT073 genes are not expressed by 83972. Arguably, in the light of the difference in patient symptoms invoked by encounters with the two strains, this list represents virulence gene candidates.\nAlthough strain 83972 seems to be a deconstructed uropathogen and does not provoke symptoms in the human host it grows fast in urine and is an excellent colonizer of the human bladder (Roos and Klemm 2006; Roos et al. 2006b; Klemm et al. 2007). It can do so because it has kept a large assortment of fitness factors required for this particular ecological niche. Among the genes expressed under realistic environmental conditions such as in the human bladder are candidates for fitness factor genes, e.g. the many iron acquisitions systems expressed by the strain and many genes involved in sugar acid and amino acid metabolism. Interestingly, many of the known and putative virulence factors of the urinary tract are expressed by strain 83972 and might therefore be considered as fitness factors rather than virulence factors; these include 25 of 29 positively selected UPEC genes as well as the newly characterised pks island inducing breaks in double-stranded DNA in host cells. Also, virulence-associated genes such as cdiA, mchBCDEF, flu, hcp, rfaH, sat, picU and vat were all expressed by strain 83972. Very few of the known or putative virulence factors were absent in (or not expressed by) strain 83972. The pap, fim and foc\/sfa clusters encoding UPEC-class fimbriae are dysfunctional in strain 83972 and the clpB, ireA and tsx genes were not expressed in the ABU strain. These stand out as potential virulence candidates together with a number of uncharacterised genes encoding hypothetical proteins.\nThus from the analyses performed here we can make predictions about several gene categories such as potential virulence genes, fitness genes and \u201chousehold-class\u201d genes. It is also noteworthy that the information reported herein complements a potential genome sequence of strain 83972. Whole genome sequencing can identify the presence of genes but is unable to reveal if they are transcribed. Genes can be silenced not only due to lesions in the actual gene and its promoter but also due to mutations of genes encoding regulatory factors. The methodology employed in the present work reveals the active genome of strain 83972.\nABU strain 83972 is closely related to fully virulent uropathogenic strains. All evidences suggest that the strain is a deconstructed pathogen. This study dispels the commonly held idea that ABU strains are commensals that have picked up niche-adaptation genes by horizontal gene transfer. Rather, strain 83972 was originally a true pathogenic strain that has lost whole or part of operons that contribute to virulence.","keyphrases":["asymptomatic bacteriuria","urinary tract infections","global gene expression","virulence factors","microarray"],"prmu":["P","P","P","P","P"]}
{"id":"Plant_Mol_Biol-4-1-2268730","title":"Deoxymugineic acid increases Zn translocation in Zn-deficient rice plants\n","text":"Deoxymugineic acid (DMA) is a member of the mugineic acid family phytosiderophores (MAs), which are natural metal chelators produced by graminaceous plants. Rice secretes DMA in response to Fe deficiency to take up Fe in the form of Fe(III)\u2013MAs complex. In contrast with barley, the roots of which secrete MAs in response to Zn deficiency, the amount of DMA secreted by rice roots was slightly decreased under conditions of low Zn supply. There was a concomitant increase in endogenous DMA in rice shoots, suggesting that DMA plays a role in the translocation of Zn within Zn-deficient rice plants. The expression of OsNAS1 and OsNAS2 was not increased in Zn-deficient roots but that of OsNAS3 was increased in Zn-deficient roots and shoots. The expression of OsNAAT1 was also increased in Zn-deficient roots and dramatically increased in shoots; correspondingly, HPLC analysis was unable to detect nicotianamine in Zn-deficient shoots. The expression of OsDMAS1 was increased in Zn-deficient shoots. Analyses using the positron-emitting tracer imaging system (PETIS) showed that Zn-deficient rice roots absorbed less 62Zn-DMA than 62Zn2+. Importantly, supply of 62Zn-DMA rather than 62Zn2+ increased the translocation of 62Zn into the leaves of Zn-deficient plants. This was especially evident in the discrimination center (DC). These results suggest that DMA in Zn-deficient rice plants has an important role in the distribution of Zn within the plant rather than in the absorption of Zn from the soil.\nIntroduction\nZn, an essential element for plant growth, is absorbed from the rhizosphere by higher plants through specific transporters. The Zinc-regulated and Iron-regulated transporter Proteins (ZIP) have been isolated from both graminaceous (Ramesh et\u00a0al. 2003; Ishimaru et\u00a0al. 2005) and non-graminaceous plants (Grotz et\u00a0al. 1998; Lasat et\u00a0al. 2000; Moreau et\u00a0al. 2002). Several ZIP transporters have been shown to be transcriptionally upregulated in Zn-deficient roots, but their contribution to the absorption of Zn from the rhizosphere into roots has not yet been demonstrated. Zn is also absorbed from the rhizosphere as complexes of Zn(II)\u2013mugineic acid family phytosiderophores (MAs). The maize protein YS1 transports Fe(III)\u2013MAs complexes (Curie et\u00a0al. 2001), as well as other micronutrient-MAs complexes, including Zn(II)\u2013MAs (Schaaf et\u00a0al. 2004). von Wir\u00e9n et\u00a0al. (1996) reported that the ys1 mutant absorbed less 65Zn(II)-MAs than did the wild type, and Suzuki et\u00a0al. (2006) showed that Zn-deficient barley absorbed more 62Zn(II)-DMA than 62Zn2+. These two studies also showed that free Zn2+ ions are absorbed into roots to some extent, suggesting that graminaceous plant roots absorb both Zn-DMA and Zn2+.\nGraminaceous plants secrete MAs into the rhizosphere from their roots, although the amount differs among species in the order of: barley\u00a0>\u00a0wheat\u00a0=\u00a0rye\u00a0>\u00a0oat\u00a0>\u00a0maize\u00a0>\u00a0sorghum\u00a0>\u00a0rice (Takagi 1993). While the amount of MAs secreted is increased dramatically by Fe deficiency, it is not clear if Zn deficiency increases the secretion of MAs. Zn deficiency has been reported to increase (Cakmak et\u00a0al. 1994; Walter et\u00a0al. 1994; Zhang et\u00a0al. 1989) or have no effect on the secretion of MAs from wheat and barley roots into the rhizosphere (Gries et\u00a0al. 1995; Pedler et\u00a0al. 2000).\nThe biosynthesis of MAs and their corresponding genes have been characterized (Fig.\u00a03b) as being synthesized from methionine (Mori and Nishizawa 1987). Three molecules of S-adenosyl-l-methionine (SAM) are combined to form one molecule of nicotianamine (NA) by nicotianamine synthase (NAS). NA is then converted to the 3\u2032\u2032-keto acid by NA aminotransferase (NAAT), and 2\u2032-deoxymugineic acid (DMA) is synthesized by DMA synthase (DMAS). In some graminaceous species, including barley, DMA is further hydroxylated by two dioxygenases, IDS2 and IDS3 (Kobayashi et\u00a0al. 2001; Nakanishi et\u00a0al. 2000). The genes encoding the enzymes involved in DMA synthesis have been well-characterized in Fe-deficient rice and barley. The expression of HvNAS1, HvNAAT-A, HvNAAT-B, HvDMAS1, HvIDS2, and HvIDS3 is increased in Fe-deficient barley roots (Nakanishi et\u00a0al. 1993; Okumura et\u00a0al. 1994; Higuchi et\u00a0al. 1999; Takahashi et\u00a0al. 1999; Bashir et\u00a0al. 2006). In rice, the expression of OsNAS1, OsNAS2, OsNAAT1, and OsDMAS1 is increased in both roots and shoots by Fe deficiency (Inoue et\u00a0al. 2003; Bashir et\u00a0al. 2006). The expression of OsNAS3 is increased in Fe-deficient roots, but decreased in Fe-deficient shoots (Inoue et\u00a0al. 2003). Promoter-GUS analysis suggests that OsNAS1 and OsNAS2 are involved in DMA secretion, because those genes are expressed in all root cells. On the other hand, OsNAS3 may not be involved in DMA secretion because its expression is restricted to the pericycle and companion cells of the roots (Inoue et\u00a0al. 2003).\nRecently, we showed that the expression of NASHOR2, a NAS gene in barley (Herbik et\u00a0al. 1999), and HvNAAT-B was increased in Zn-deficient barley shoots, but that the expression of IDS2 and IDS3 was not detected in Zn-deficient shoots (Suzuki et\u00a0al. 2006). On the other hand, the expression of HvNAS1, HvNAAT-A, HvNAAT-B, IDS2, and IDS3 was increased in both Zn- and Fe-deficient barley roots, while the expression of these genes was not evident in Fe-deficient shoots (Suzuki et\u00a0al. 2006). This suggests that in barley Zn deficiency induces DMA synthesis in shoots, while both Zn and Fe deficiency induce MAs synthesis and secretion in roots.\nThe aims of the present study were to examine if the stimulation of MAs secretion by Zn deficiency is common in graminaceous plants and roles of MAs in Zn homeostasis of rice plants. In this report, we shows that DMA secretion is decreased in rice roots, while the concentration of endogenous DMA is increased in Zn-deficient rice shoots.\nMaterials and methods\nPlant material and growth conditions\nRice seeds (Oryza sativa L. cv. Nipponbare) were germinated for 7\u00a0days at room temperature on paper towels soaked with distilled water. After germination, the seedlings were transferred to a Saran net floating on nutrient solution (Higuchi et\u00a0al. 2001) and grown in a glasshouse. After 4\u00a0days, 270 seedlings were transferred to six 20-l plastic containers containing nutrient solution. After a further 5\u00a0days, 90 seedlings were transferred to two 20-l plastic containers containing nutrient solution without Zn followed 7\u00a0days later when a further 90 seedlings were transferred to two 20-l plastic containers containing nutrient solution without Fe. All samples were harvested 5\u00a0days later for analysis. Consequently, the Zn-deficient treatment was performed for 12\u00a0days, and Fe-deficient treatment for 5\u00a0days since Fe deficiency has a much more rapid deleterious effect. The nutrient solutions were renewed weekly with adjustment to pH 5.5 every 2\u00a0days with KOH.\nMeasurement of DMA secretion and endogenous levels of DMA and NA\nDMA secretion was measured as described by Suzuki et\u00a0al. (2006) and the concentrations of endogenous DMA and NA were measured as described by Higuchi et\u00a0al. (2001). Briefly, the roots of 12 plants were rinsed with deionized water and then placed in 600\u00a0ml of deionized water at sunrise. Root exudates were collected for two periods of 2 and 3\u00a0h. After collection, Micropur (Katadyn, Switzerland), an antimicrobial agent, was added to prevent microbial degradation of the MAs Both solutions containing root exudates were combined and filtered (Advantec 5C, Toyo Roshi Kaisha, Ltd., Japan).\nThe shoots of the plants from which root exudates were collected were ground in liquid nitrogen using a mortar and pestle. Approximately 1\u00a0g of material was suspended in 40\u00a0ml of distilled water at 80\u00b0C for 30\u00a0min and filtered. The cationic fraction of the samples was prepared as a 2-M NH4OH eluate from Amberlite IR(H+)120 (Rohm and Haas Co., USA). The condensed and microfiltered samples were subjected to HPLC analysis, as described previously (Mori et\u00a0al. 1987; Shojima et\u00a0al. 1989). To ensure better separation of DMA, the pH of 0.15-N Li-citrate buffer was decreased to 2.97 and the pH of 0.2-N Li-citrate buffer increased to 3.30 for better separation of NA.\nNorthern blot analysis\nThe samples for Northern blot analysis were harvested 3\u00a0h after sunrise. The roots and shoots were separated immediately and stored at \u221280\u00b0C. Total RNA was extracted from the roots and shoots using SDS-phenol. Ten micrograms of total RNA were electrophoresed in 1.2% (w\/v) agarose gels containing 0.66\u00a0M formaldehyde, then transferred to a Hybond-N+ membrane (Amersham, USA) and hybridized with probes at 65\u00b0C. The blots were analyzed using a Bio-imaging Analyzer System (BAS) 3000 (Fuji Film, Japan). The following specific primers were used to prepare specific probes: OsNAS1, 5\u2032-GTCTAACAGCCGGACGATCGAAAGG-3\u2032 and 5\u2032-TTTCTCACTGTCATACACAGATGGC-3\u2032; OsNAS2, 5\u2032-TGAGTGCGTGCATAGTAATCCTGGC-3\u2032 and 5\u2032-CAGACGGTCACAAACACCTCTTGC-3\u2032; OsNAS3, 5\u2032-GACTGCTTCCATCGCTTGCTACCTC-3\u2032 and 5\u2032-CGCAACAGAGACAATGGTTGATTGT-3\u2032; OsNAAT1, 5\u2032-TAAGAGGATAATTGATTTGCTTAC-3\u2032 and 5\u2032-CTGATCATTCCAATCCTAGTACAAT-3\u2032; and OsDMAS1, 5\u2032-GCCGGCATCCCGCAGCGGAAGATCA-3\u2032 and 5\u2032-CTCTCTCTCTCGCACCTGCTAGCGT-3\u2032. The accession numbers of the genes are AB021746 (OsNAS1), AB023818 (OsNAS2), AB023819 (OsNAS3), AB206814 (OsNAAT1), and AB269906 (OsDMAS1).\nPETIS analysis of 62Zn translocation\nPETIS experiment was established by Kume et\u00a0al. (1997). 62Zn (half life: 9.13\u00a0h) was produced by the method of Watanabe et\u00a0al. (2001). After adjustment of the 62Zn2+ solution to about pH 4 with 1\u00a0M KOH, 62Zn2+ was chelated with DMA in darkness for more than 3\u00a0h. Prior to the analysis, Zn-deficient rice plants were grown as described above but in a growth chamber (day: 30\u00b0C, 14\u00a0h of light at 320\u00a0\u03bcmol photons m\u22122\u00a0sec\u22121; night: 10\u00a0h at 25\u00b0C). The Zn-deficient rice plants were each supplied with 15\u00a0ml of culture solution lacking Zn in a polyethylene bag. The plants and bags were fixed between two acrylic boards and placed between a pair of PETIS detectors in a chamber set at 30\u00b0C, 65% humidity, and a light density of 320\u00a0\u03bcmol m\u22122\u00a0s\u22121. In the root absorption experiment, 62Zn (0.6\u00a0nmol) with DMA (79\u00a0nmol) was dissolved in 1\u00a0ml of distilled water and added to the culture solution. In the leaf absorption experiment, 62Zn (0.5\u00a0nmol) with DMA (33\u00a0nmol) was dissolved in 4.5\u00a0ml of nutrient solution containing cold Zn (2.5\u00a0\u03bcmol) and additional DMA (308\u00a0\u03bcmol), and supplied at the cut end of the second newest leaf. The individual leaves were cut to ensure a constant distance from discrimination center (DC) which is the junction of shoot and root (Kiyomiya et\u00a0al. 2001). The \u03b3-rays emitted from decaying positrons from 62Zn were counted over time using the coincident method with the paired detectors. The data were automatically corrected using 9.13\u00a0h as the half-life of 62Zn. The radioactivity of selected region was measured as region of interest (ROI) analysis. After PETIS analysis for 8\u00a0h, the plants were removed from the polyethylene bags, and the roots were gently washed for 1\u00a0min in 50\u00a0ml of 0.01\u00a0mM EDTA. Next, each plant was placed under a Bio-Imaging plate inside a cassette. The plate was scanned using an image analysis system (BAS-1500, Fuji Film, Japan). The plants were then cut into pieces, and the absolute amount of radioactivity was analyzed using \u03b3-ray spectrometry with an ORTEC HPGe detector (Seiko EG & G Co., Ltd., Japan). Each experiment was conducted in triplicate. Due to the very short half-life of 62Zn, the radioactivity was different in each experiment. Therefore, images of individual plants and PETIS results are representative of those obtained.\nResults\nPlant growth, DMA synthesis and gene expression\nIn contrast with the good growth of the control rice plants, Zn or Fe deficiency had a markedly detrimental effect when plants were grown for 12\u00a0d or 5\u00a0d in solutions containing no Zn or Fe, respectively. Shoot growth was reduced by 50% in the absence of Zn and by 10% without Fe (Fig.\u00a01). The corresponding figure for the Zn-deficient roots was a reduction of 20%, while the absence of Fe supply for 5\u00a0d had no significant effect on root growth. As previously established in our studies (Suzuki et\u00a0al. 2006), there was no measurable effect of no Zn supply during the first week of growth (data not shown), but thereafter plants developed symptoms of Zn deficiency and growth declined. Chlorotic symptoms of Fe deficiency developed after 5\u00a0days of treatment while the fresh weights of the shoots and roots were almost the same as in the control plants.\nFig.\u00a01Fresh weights of rice shoots and roots. 31-days old rice seedlings grown in hydroponically were harvested. Zn-deficient treatment (-Zn) was performed for 12\u00a0days, and Fe-deficient treatment (-Fe) was performed for 5\u00a0days. Values are mean \u00b1 SD (n = 4)\nThe amount of DMA secreted from the roots of Fe-deficient rice was much higher than that of the control plants (Fig.\u00a02). On the other hand, Zn deficiency slightly decreased the level of DMA secretion. In fact, the amount of DMA secreted per plant in Zn-deficient rice was significantly decreased compared to that in the control plants (P\u00a0<\u00a00.05).\nFig.\u00a02Amount of DMA secreted from rice roots. The growth condition and harvest time are the same in Fig. 1. Root exudates were collected for 5\u00a0h after sunrise. -Zn, zinc deficiency; -Fe, iron deficiency. Values are mean \u00b1 SD (n = 4)\nWe next analyzed the expression pattern of the genes participated in DMA synthesis in roots and shoots under Zn or Fe deficiency (Fig.\u00a03a, b). As reported previously, the transcription of OsNAS1, OsNAS2, OsNAS3, OsNAAT1, and OsDMAS1 in roots was increased by Fe deficiency (Inoue et\u00a0al. 2003, 2008; Bashir et\u00a0al. 2006). On the other hand, Zn deficiency enhanced the transcription of OsNAS3 and OsNAAT1, but not that of OsNAS1, OsNAS2, and OsDMAS1. The expression of OsNAS2 in roots was slightly decreased by Zn deficiency. The expression patterns of OsNAS1 and OsNAS2 were well correlated with the amount of DMA secreted from the roots of Fe-deficient or Zn-deficient rice plants (Figs.\u00a02, 3a).\nFig.\u00a03Expression patterns of genes involved in DMA synthesis (a) Northern blot analysis of each gene involved in DMA synthesis. Each lane contained 10\u00a0\u03bcg of total RNA. C, control; -Zn, zinc deficiency; -Fe, iron deficiency. (b) DMA synthesis from S-adenosyl-l-methionine (SAM)\nIn the shoots, Fe deficiency increased the expression of OsNAS1, OsNAS2, OsNAAT1, and OsDMAS1, but decreased that of OsNAS3. In comparison, Zn deficiency increased the expression of OsNAS3 and especially that of OsNAAT1. The expression of OsDMAS1 was slightly increased in Zn-deficient shoots. Transcripts of OsNAS1 and OsNAS2 were not detected in control or Zn-deficient shoots (Fig.\u00a03a).\nHiguchi et\u00a0al. (2001) reported that the concentration of endogenous DMA was increased in Fe-deficient rice shoots. We confirmed that the concentration of endogenous DMA in Fe-deficient shoots was about three times higher than that in control shoots (Fig.\u00a04a). The concentration of endogenous DMA in Zn-deficient shoots was about two times higher than that in control shoots (Fig.\u00a04b). Although the concentration of endogenous DMA in Zn-deficient shoots was lower than that in Fe-deficient shoots, the expression of OsNAAT1, which encodes the enzyme that converts nicotianamine to its 3\u2032\u2032-keto acid, in Zn-deficient shoots was much higher than that in Fe-deficient shoots (Fig.\u00a03a). Therefore, we measured the endogenous NA, which is an intermediate of DMA. Corresponding to the expression pattern of OsNAAT1, endogenous NA in Zn-deficient shoots was under the detection limit by HPLC, while in Fe-deficient shoots NA was lower than in the control shoots (Fig.\u00a04b).\nFig.\u00a04Concentration of endogenous DMA and NA in rice shoots (a) Concentration of endogenous DMA. (b) Concentration of endogenous NA. The plants were harvested after collection of root exudates (Fig. 2), then DMA or NA was measured using HPLC. -Zn, zinc deficiency; -Fe, iron deficiency; N.D., not detected. Values are mean \u00b1 SD (n = 4)\nContribution of DMA to Zn translocation in Zn-deficient rice\nTo investigate whether DMA is involved in Zn absorption and translocation, a PETIS experiment was performed using Zn-deficient rice (Fig.\u00a05; supplemental movie 1, supplemental Fig.\u00a01). 62Zn chelated with DMA (62Zn-DMA) or 62Zn without DMA (62Zn2+) was supplied to Zn-deficient rice roots. Images of the plants and the localization of 62Zn after 6\u00a0h of absorption are shown in Fig.\u00a05a. In both plants, most of the 62Zn absorbed into the plant was observed at the DC and in the bottom of the leaf sheath (Fig.\u00a05a). The radioactivity of the plant supplied with 62Zn2+ was higher than that of the plant supplied with 62Zn-DMA both in the roots and shoots (Fig.\u00a05b, c). This indicates that compared to Zn-DMA, Zn2+ is the preferred form for root absorption in Zn-deficient rice. Real-time imaging of the 62Zn absorption was observed by PETIS analysis (Fig.\u00a05d). More 62Zn was observed in the bottom of the leaf sheath supplied with 62Zn2+ than in that supplied with 62Zn-DMA. This observation was quantified by ROI analysis (Fig.\u00a05e) On the other hand, beginning at 90\u00a0min after the start of the experiment, more 62Zn was observed in the newest leaf of the plant supplied with 62Zn-DMA than in that supplied with 62Zn2+ (Fig.\u00a05d, f). This indicates that Zn-DMA is preferred over Zn2+ for translocation within Zn-deficient rice plants. Continuous PETIS imaging is shown in supplemental movie 1. We performed three experiments of 62Zn absorption. Due to the very short half-life of 62Zn, however, the radioactivity was different in each experiment. Therefore, we showed the relative radioactivity of each experiment in supplemental Fig.\u00a01. In each experiment, 62Zn2+ was more absorbed than 62Zn-DMA.\nFig.\u00a0562Zn movement from roots in Zn-deficient rice using PETIS (a) Plant image (left) and BAS image (right). (b) Radioactivity in roots after 6\u00a0h of absorption. (c) Radioactivity in shoots after 6\u00a0h of absorption. (d) Time course of the accumulation of radioactivity as determined by PETIS analysis. The data were scored every 3\u00a0min, and the images shown were taken at 90-min intervals for 6\u00a0h after supplying 62Zn through the roots. (e) Region of interest (ROI) analysis of 62Zn movement in the bottom of the sheath white boxed in (d). (f) ROI analysis in the newest leaf yellow boxed in (d)\nWe conducted an additional PETIS experiment in which Zn-DMA or Zn2+ was supplied to Zn-deficient rice at the cut second newest leaf (Fig.\u00a06; supplemental movie 2, supplemental Fig.\u00a02). Plant images and the localization of 62Zn after 6\u00a0h of absorption are shown in Fig.\u00a06a. Most of the 62Zn absorbed from the second newest leaf remained in that leaf, but some 62Zn was translocated to the other leaves and roots as well as to the DC and leaf sheath. The level of radioactivity in the entire plant, except for the second newest leaf, was higher in the plant supplied with 62Zn-DMA than in the plant supplied with 62Zn2+ (Fig.\u00a06b), while that of the second newest leaf was not higher in the plant supplied with 62Zn-DMA than in the plant supplied with 62Zn2+ (supplemental Fig.\u00a03). PETIS analysis showed that more 62Zn appeared continuously in the plant supplied with 62Zn-DMA than in the plant supplied with 62Zn2+. This suggests that 62Zn-DMA is translocated more rapidly than 62Zn2+ within the plant.\nFig.\u00a0662Zn movement from cut leaf in Zn-deficient rice using PETIS (a) Plant image (left) and BAS image (right). Arrows indicate the point where 62Zn was absorbed. (b) Radioactivity in the entire plant except for the second newest leaf after 8\u00a0h of absorption. (c) Time course of the accumulation of radioactivity as determined by PETIS analysis. The data were scored every 3\u00a0min, and the images shown were taken at 120-min intervals for 8\u00a0h after supplying 62Zn through the shoots\nDiscussion\nThe concentration of endogenous DMA is increased in Zn-deficient shoots, but DMA secretion from the roots is not increased\nPreviously we reported increased MAs secretion in Zn-deficient barley (Suzuki et\u00a0al. 2006); however, in rice, Zn deficiency did not increase the secretion of DMA, but rather slightly decreased it (Fig.\u00a02). Fe deficiency increased MAs secretion in both rice and barley (Suzuki et\u00a0al. 2006; Fig.\u00a02). In barley, expression of the genes encoding the MAs biosynthetic enzymes was increased in the roots of both Zn- and Fe-deficient plants (Suzuki et\u00a0al. 2006). On the other hand, in rice, the expression pattern of the genes involved in DMA synthesis in roots was increased in Fe-deficient roots, while the expression of OsNAS1, OsNAS2, and OsDMAS1 was not increased in Zn-deficient roots (Fig.\u00a03a). Inoue et\u00a0al. (2003) suggested that OsNAS1 and OsNAS2 are involved in DMA secretion. Supporting this idea, the expression pattern of OsNAS1 and OsNAS2 in roots was in good correlation with the amount of DMA secreted (Figs.\u00a02, 3a). On the other hand, OsNAS3 is thought not to be involved in DMA secretion, because the expression of OsNAS3 is restricted to the pericycle cells adjacent to the protoxylem and companion cells but not in other cortical cells (Inoue et\u00a0al. 2003). The expression of OsNAS3 is observed in whole leaf tissues in the control plants (Inoue et\u00a0al. 2003). The expression of OsNAS3 was increased in Zn-deficient roots and shoots (Fig.\u00a03a); therefore, OsNAS3 may function to synthesize NA or subsequent components, such as DMA, for the distribution of Zn within Zn-deficient plants. It is noteworthy that Zn deficiency enhanced the expression of OsNAS3 in shoots, while Fe deficiency enhanced the expression of OsNAS1 and OsNAS2. This suggests that NA and DMA are involved both in Fe and Zn homeostasis, but that their synthesis under conditions of Fe and Zn deficiency is catalyzed by different isozymes.\nThe concentration of endogenous DMA in the shoots was increased by Zn and Fe deficiencies (Fig.\u00a04a). Although the induction of OsNAAT1 in Zn-deficient shoots was much higher than that in Fe-deficient shoots, the concentration of DMA in Zn-deficient shoots was lower than that in Fe-deficient shoots. In accordance with the high level of OsNAAT1 expression in Zn-deficient shoots, the concentration of NA in Zn-deficient shoots was low (Fig.\u00a04b). This result led to us hypothesize that OsDMAS1 is a rate-limiting enzyme for DMA synthesis in Zn-deficient rice.\nZn-DMA is the preferred form for long-distance transport of Zn in Zn-deficient rice\nRecently, we showed that Zn-deficient barley absorbs more Zn-DMA than Zn2+ from its roots (Suzuki et\u00a0al. 2006). In the present paper, however, less Zn-DMA than Zn2+ was absorbed from the roots of Zn-deficient rice (Fig.\u00a05). These findings are correlated with the change in the amount of MAs secreted under Zn deficiency. In barley, MAs secretion is increased by Zn-deficiency, and this contributes to the absorption of Zn from the soil. On the other hand, in rice, the level of DMA secretion was slightly decreased (Fig.\u00a02). This suggested that rice may prefer to absorb Zn2+ rather than Zn-DMA.\nOur PETIS experiment clearly showed that more 62Zn was translocated to the newest leaf when 62Zn-DMA was supplied than when 62Zn2+ was supplied, while the opposite tendency was observed at the bottom of the leaf sheath (Fig.\u00a05, supplemental movie 1). This finding suggests that Zn-DMA is the preferred form for long-distance transport of Zn in Zn-deficient rice. The preferential translocation of 62Zn-DMA was also observed in the PETIS experiment in which 62Zn-DMA and 62Zn2+ were supplied from the Zn-deficient leaf (Fig.\u00a06, supplemental movie 2). These data also support the idea that Zn-DMA is the preferred form for long-distance transport of Zn in Zn-deficient rice.\nSince DMA synthesis might be promoted by Zn deficiency in barley shoots (Suzuki et\u00a0al. 2006), graminaceous plants have a common system for Zn translocation within the plant, while the contribution of MAs to Zn uptake from the soil differs among species. Further studies will enable us to understand the mechanism of Zn transport, to manipulate the distribution of Zn in order to produce crops that tolerate Zn-deficient stress, and to enhance the Zn content of crop seeds.\nElectronic supplementary material\n(EPS 348 kb)\n(EPS 362 kb)\n(EPS 353 kb)\n(MOV 2400 kb)\n(MOV 2257 kb)","keyphrases":["deoxymugineic acid","rice","nicotianamine","transport","zinc","iron"],"prmu":["P","P","P","P","P","P"]}
{"id":"Pediatr_Nephrol-4-1-2214826","title":"Rituximab therapy for juvenile-onset systemic lupus erythematosus\n","text":"Rituximab (RTX), an anti-CD20 monoclonal antibody, has been proposed for use in the therapy of systemic lupus erythematosus (SLE). We present the initial long-term experience of the safety and efficacy of rituximab for treatment of SLE in children. Eighteen patients (mean age 14 \u00b1 3 years) with severe SLE were treated with rituximab after demonstrating resistance or toxicity to conventional regimens. There was a predominance of female (16\/18) and ethnic African (13\/18) patients. All had lupus nephritis [World Health Organization (WHO) classes 3\u20135] and systemic manifestations of vasculitis. Clinical disease activity of the SLE was scored with the SLE-disease activity index 2K (SLEDAI-2K). Patients were followed-up for an average of 3.0 \u00b1 1.3 years (range 0.5 to 4.8 years). B-cell depletion occurred within 2 weeks in all patients and persisted for up to 1 year in some. Clinical activity scores, double-stranded DNA (dsDNA) antibodies, renal function and proteinuria [urine protein to creatinine ratio (Upr\/cr)] improved in 93% of the patients. Five patients required multiple courses of RTX for relapse, with B-cell repopulation. One died of infectious endocarditis related to severe immunosuppression. In conclusion, our data support the efficacy of rituximab as adjunctive treatment for SLE in children. Although rituximab was well tolerated by the majority of patients, randomized controlled trials are required to establish its long-term safety and efficacy.\nIntroduction\nSystemic lupus erythematosus (SLE) in children is known to carry a worse prognosis than in adults, especially in those of African and Hispanic ethnicity [1, 2]. Although treatment protocols for proliferative lupus nephritis have evolved during the past two decades, pediatric regimens have been center based and selectively derived from adult protocols [3]. These protocols primarily include cyclophosphamide (CYC), which carries long-term toxicities related to malignancy and gonadal dysfunction [4].\nMost recently, B-lymphocyte function has been recognized as a major component in the pathogenesis of autoimmune diseases [5]. Rituximab (RTX), a chimeric monoclonal antibody directed against CD20 lymphocytes (commonly called B-cells), was developed as a primary treatment against B-cell lymphoma [6] and is now approved for use in the treatment of rheumatoid arthritis [7]. Although clinical trials of the use of RTX in adults with SLE are substantial and ongoing [8], studies in children have been limited [9, 10]. A collaborative retrospective and uncontrolled multicenter experience from Europe reported mixed results [11]. Unfortunately, that study failed to use a single protocol, and many patients were treated concurrently with more toxic medications, which made safety analysis unreliable [11].\nThe purpose of our study was to determine the safety and efficacy of RTX therapy in the treatment of children with severe SLE. Over the course of 7\u00a0years, we retrospectively assessed the indications and responses to RTX therapy in a cohort of children with active SLE that was refractory to conventional therapies or who had clinical indications prohibiting the use of more toxic immunosuppressant drugs.\nPatients and methods\nA retrospective analysis was performed on a cohort of 51 patients diagnosed with SLE and lupus nephritis who received their care at Holtz Children\u2019s Hospital, University of Miami Miller School of Medicine, between January 1996 and June 2007. The study was approved by the institutional review board, with waiver of consent authorization, and all subjects were assured anonymity in compliance with the Health Insurance Portability and Accountability Act (HIPAA). The medical records were reviewed for patients\u2019 demographic characteristics, age at diagnosis, prior and current medical treatments, type of lupus nephritis and serologic and clinical response to therapy.\nChildren were considered eligible for inclusion in the analysis if they fulfilled the following criteria: (1) American College of Rheumatology (ACR) criteria for the diagnosis of SLE [12]; (2) age < 16\u00a0years at the time of diagnosis of SLE; (3) treatment with the anti-CD20 monoclonal antibody, rituximab, during the observation period from January 2000 through October 2007.\nClinical protocol\nRituximab (RTX) was administered weekly for two to four doses. The initial dose was 188\u00a0mg\/m2, infused over 4\u00a0h. Subsequent doses were 375\u00a0mg\/m2 per dose, infused over 6\u00a0h to 8\u00a0h. Premedication initially consisted of diphenhydramine and acetaminophen, administered 30\u00a0min before the rituximab infusion. However, mild infusion reactions, including pruritis, nausea and emesis, resulted in our adding hydrocortisone 50\u2013100\u00a0mg intravenously 30\u00a0min prior to each infusion. Lymphocyte subsets were assayed before and after each course of RTX therapy at 1- to 3-month intervals. This included B cells assayed as CD19+ lymphocytes and T-cells assayed as CD4 and CD8 lymphocytes as total and percent of the total lymphocyte cell count. The serum immunoglobulin levels were measured every 1 to 6\u00a0months prior to and after the course of RTX. Adverse events were recorded from the time of the first infusion until 1\u00a0year following the treatment course.\nLupus disease activity index and serology\nClinical and biological parameters were assessed prior to the RTX infusion course, at 1- to 3-month intervals thereafter. The clinical disease activity was scored with the SLE-disease activity index\u00a02K (SLEDAI-2K), which has a maximum activity score of 105 [13]. Laboratory evaluations included serum complement components (C3 and C4) and anti-double stranded DNA (Anti-dsDNA) antibody and anti-nuclear antibody (ANA).\nLupus nephritis and renal disease activity\nRenal biopsies were classified according to the new proposed modifications of the World Health Organization (WHO) classifications [14]. Three patients were treated at the time they were on hemodialysis for extra-renal manifestations of SLE. For those patients not on dialysis, degree of proteinuria and renal function were assessed in response to RTX therapy. Degree of proteinuria was determined by the random urine protein to creatinine ratio (Upr\/cr) in milligrams per milligram (mg\/mg), with normal <0.2 and nephrotic range >1.0 [15, 16]. Serum albumin (Salb) was assayed as an indirect measure of the nephrotic syndrome. Serum creatinine (Scr) was assayed, and an estimation of glomerular filtration rate (eGFR) was derived by the height index formula of Schwartz, expressed as milliliters per minute per 1.73 meters squared (ml\/min per 1.73 m2) [17]. Complete renal remission was defined as normalization of Upr\/cr and eGFR. Partial remission was defined as >\u00a050% decline in Upr\/cr. All serum and urine chemical analyses and immunoassays were performed in the central hospital clinical laboratory or the Quest referral laboratories.\nStatistical methods\nAll data sets were analyzed for Gaussian distribution using the D\u2019Agostino\u2013Pearson omnibus test for normality. The data were calculated into average values at baseline and at 2\u00a0months, 4\u00a0months, and 6\u00a0months after initiation of treatment. Variables at baseline, during and after the treatment were compared by the Wilcoxon matched-pairs signed-ranks test. Differences in parameters between the 2 independent groups were analyzed using Mann-Whitney and the Friedman test with Dunn\u2019s multiple comparisons post tests for repeated measures of nonparametric data. P values of less than 0.05 were considered significant. All results are expressed as mean \u00b1 standard deviation (SD).\nResults\nPatients\u2019 demographics\nDuring the period of observation 18 patients were treated with RTX for at least one course of two to four doses. There were 16 female patients and two male patients. Mean age at the time of diagnosis of SLE was 10.7\u2009\u00b1\u20092.5\u00a0years (range 7 to 14\u00a0years). Mean duration of disease prior to treatment with RTX was 3.1\u2009\u00b1\u20092.5\u00a0years (range 2\u00a0months to 8\u00a0years). Age at the time of first RTX treatment was 14.2\u2009\u00b1\u20093.3\u00a0years, and current age at the time of this report was 17\u2009\u00b1\u20095.0\u00a0years.\nAll patients had clinical evidence of lupus nephritis. Individual patient demographics, clinical manifestations of SLE and indications for rituximab therapy for patients not on hemodialysis are shown in Table\u00a01. All patients received concurrent therapy with low-dose corticosteroids (CSs) and hydroxychloroquine (HCQ). Sixteen patients had failed or had suffered toxic effects from multiple courses of intravenous therapy with cyclophosphamide (CYC) and corticosteroids. Treatment with RTX included concurrent or subsequent treatment with maintenance doses of mycophenolate mofetil (MMF) or azathioprine (AZT) similar to recently published adult protocols [18].\nTable\u00a01Patients\u2019 baseline characteristics, indications for rituximab treatment, and clinical course (F female, M male, H Hispanic, B Black, HT hypertension, CYC cyclophosphamide, CyA cyclosporine\u00a0A, AZT azathioprine, MMF mycophenolate mofetil, IVIG intravenous therapy with gamma globulin, CS corticosteroids, HXQ hydroxychloroquine, FR full remission, PR partial remission)PatientGenderAge at diagnosis (years)RaceIndication for rituximabNephritis classPrevious and\/or concurrent therapiesaUpr\/cr (mg\/mg)OutcomeBefore RTXAfter RTX1F12.0BNephritis, serositis, Coomb\u2019s (+) anemia, CYC-toxicityIVCYC, MMF13.00.1FR2M10.0WNephritis, serositis, CS toxicityIVCYC, MMF2.10.6PR3F11.0BNephritis, serositis, Coomb\u2019s(+) anemia, CYC- toxicity sepsisIVCYC, MMF4.01.7PR4F8.0HPulmonary HT, nephritis, Coomb\u2019s(+) anemiaIV-VCYC, MMF, IVIG2.60.2FR5F12.0BOphthalmic vasculitis, nephritisIV-VCYC, AZT0.40.1FR6F14.0BNephritis, Coomb\u2019s(+) anemia, CS toxicityNACYC, AZT, MMF, IVIG0.60.3PR7F12.0BNephritis, serositis, CYC-toxicity sepsisIV-VCYC, AZT, MMF, IVIG2.6NADied8F12.0BNephritis, serositis, Coomb\u2019s(+) anemiaIVCYC, AZT, IVIG3.61.9PR9F14.0BNephritis, serositis, ataxia, miliary tuberculosisIIIIVIG1.70.2FR10F7.0HNephritis, serositis, arthritis, CYC-toxicity sepsisIVCYC, MMF, IVIG3.00.7PR11F12.8HNephritis, serositis, arthritis, obesityVMMF9.70.2FR12F7.0BNephritis, thrombocytopenia, CYC toxicityIV-VCYC, AZT, MMF, IVIG, CyA5.40.2FR13F7.0BNephritis, Coomb\u2019s(+) anemia, skin vasculitisIVMMF1.50.2FR14M12.5HNephritis, serositis, arthritis, CYC-toxicity sepsisIIICYC, MMF, IVIG2.30.8PR15F13.0BNephritis, arthritis, skin vasculitis, Coomb\u2019s(+) anemiaIVCYC, MMF, IVIG6.80.9PRaAll patients were treated with CS and HXQ\nAll three patients on hemodialysis responded positively to the RTX therapy. Patient HD-1 had Raynaud\u2019s syndrome and finger necrosis; her symptoms resolved, and she recently received a transplant, 4\u00a0years after RTX treatment. Patient HD-2, who had been on ventilator support with pneumonitis, pleuritis and carditis for 1\u00a0month prior to receiving RTX, recovered and has been stable on hemodialysis as an outpatient for >4\u00a0years since RTX therapy. Patient HD-3, after 3\u00a0years on hemodialysis, developed debilitating chorea with increased lupus serology. She did not respond to anti-convulsant therapy. The chorea resolved within weeks of the RTX therapy. She remains on dialysis without recurrence of chorea.\nB-cell depletion and re-population\nFigure\u00a01 shows the average B-cell depletion relative to the beginning of the first dose of RTX. Patients received an average of 4.3\u2009\u00b1\u20091.6 doses overall. B-cell depletion was achieved within 2\u00a0weeks of the first dose of RTX and lasted an average of 3\u2009\u00b1\u20094\u00a0months (range 3 to 12\u00a0months). Five patients (28%) received repeated courses of RTX therapy ranging from two to four courses of one to four additional doses following re-population of the B-cell line in the setting of clinical relapse of SLE. Overall follow-up period from the initial dose of RTX to this report was 3.0\u2009\u00b1\u20091.3\u00a0years. It should be noted that those patients who did not have clinical relapse were not routinely assayed for lymphocyte subsets. Therefore, it is not known if or when those patients with persistent clinical remission had their B-lymphocyte lineage re-populated.\nFig.\u00a01Graph of B-cell depletion over time from before rituximab therapy to 12\u201318\u00a0months after rituximab therapy. Asterisks indicate significant difference from the post-rituximab values; P\u2009<\u20090.01\nTable\u00a02 describes the changes in the lymphocyte subsets from baseline to the nadir drop in the B-lymphocyte lineage. Total lymphocyte counts, as well as CD4 and CD8 T-cell counts, tended to be lower before RTX treatment and increased after RTX therapy. Similarly, the levels of serum immunoglobulins remained normal in comparison with the pre-RTX values.\nTable\u00a02Clinical parameters before and after rituximab therapy (NS not significant)ParameterBefore rituximab therapyAfter rituximab therapyPTotal lymphocyte count1044\u2009\u00b1\u20095961734\u2009\u00b1\u20091535NSCD4+ lymphocytes344\u2009\u00b1\u2009217556\u2009\u00b1\u2009264NSCD19+ lymphocytes243\u2009\u00b1\u200922374\u2009\u00b1\u2009710.005Immunoglobulin IgG1603\u2009\u00b1\u20098481596\u2009\u00b1\u20091055NSImmunoglobulin IgM273\u2009\u00b1\u2009514283\u2009\u00b1\u2009531NSUpr\/cr (mg\/mg)4.0\u2009\u00b1\u20093.50.6\u2009\u00b1\u20090.60.001Serum albumin (g\/dl)2.6\u2009\u00b1\u20090.73.5\u2009\u00b1\u20090.60.001Scr (mg\/dl)1.2\u2009\u00b1\u20090.40.6\u2009\u00b1\u20090.20.001eGFR (ml\/min\/1.73m2)86\u2009\u00b1\u200932144\u2009\u00b1\u2009370.0003Corticosteroid dose (mg\/m2 per day)79\u2009\u00b1\u20092613\u2009\u00b1\u200920<0.0001SLEDAI-2K score47\u2009\u00b1\u20091925\u2009\u00b1\u2009140.0004\nLupus activity and auto-antibody response\nTable\u00a02 and Fig.\u00a02 summarize the clinical response to RTX therapy. The average lupus activity score (SLEDAI) improved in all patients. Average dsDNA antibody decreased and serum C3 complement increased significantly after RTX therapy. Other serological parameters showed a trend towards improvement as well.\nFig.\u00a02Composite graphs depicting response to rituximab therapy on clinical parameters. Panela SLEDAI [13] scores before and after rituximab therapy. Panelb proteinuria (Upr\/cr) before and after rituximab. Panelc auto-antibody titers before and after rituximab. Paneld serum complements as C3 and C4 complements. Asterisks indicate significant differences from the post-rituximab values; P\u2009<\u20090.01\nAll 15 patients not on dialysis had active lupus nephritis with significant proteinuria. Table\u00a01 provides detailed information regarding the classification of the lupus nephritis for each patient, together with extra-renal manifestations and concurrent or previous therapies, as well as their final outcome. All parameters improved or stabilized in 14\/15 (93%) patients. Seven had total remission of proteinuria, while the other seven had partial remission, with significant improvement in proteinuria and renal function. Coincident with the improvement in renal disease, the requirement for maintenance corticosteroid therapy was reduced in most patients, with an average fall in corticosteroid dosing from 79\u2009\u00b1\u200929\u00a0mg\/m2 per day before RTX therapy to 13\u2009\u00b1\u200920\u00a0mg\/m2 per day after RTX therapy (P\u2009<\u20090.0001).\nAdverse events\nOne patient (#7) received three doses of RTX shortly after a cycle of intravenous cyclophosphamide (CYC) administration in an effort to control lupus serositis and nephritis. She developed Staphylococcus aureus endocarditis, with vegetations on the mitral and aortic valves. She died after open heart surgery. This was considered a death from overwhelming infection most likely related to excessive immune suppression. Her CD4 count prior to RTX was 220\/mm3, possibly related to prior CYC therapy. At the time of her death her serum immunoglobulin levels were normal.\nPatient #13 had systemic vasculitis and class\u00a0IV lupus nephritis. Since she was only 7\u00a0years of age, it was decided to treat her with intravenous administration of Solu-Medrol followed by weekly RTX for four doses and concurrent mycophenolate mofetil (MMF) as induction therapy. She responded well to RTX, with resolution of her proteinuria and clinical remission of the lupus nephritis. However, she developed symptoms of cerebral vasculitis, with seizures and a cerebral infarct, demonstrated by magnetic resonance imaging (MRI) after her fourth dose of RTX. She was treated with intravenously administered gamma globulin (IVIG) and two doses of cyclophosphamide. Her neurologic deficits resolved and her vasculitis reversed, as demonstrated by MRI. She remains in remission of the lupus nephritis and cerebritis 6\u00a0months after RTX therapy.\nMild adverse reactions occurred in approximately half the patients and included pruritis, nausea, or malaise associated with the infusions. These became less when hydrocortisone was added to the pre-medication regimen and the infusion rate was slowed to 6 to 8 hours.\nDiscussion\nTo date, this report has provided the largest long-term experience in children treated with targeted B-cell depletion using the monoclonal antibody, rituximab, for severe SLE. We showed a beneficial effect of the drug in over 90% of the children in this series, which is consistent with that of previous reports [8, 19\u201321]. Moreover, we were able to demonstrate efficacy in controlling proteinuria in conjunction with a fall in auto-antibodies in those patients with active lupus nephritis, which mirrored that in a series of adult patients [19].\nB-lymphocyte dysregulation is central in the pathogenesis of systemic lupus, with disruption of an individual\u2019s immune homeostasis. These mechanisms are multi-dimensional and are not limited to the single role of B cells in antibody production [22]. Hence, the elimination of the B-cell lineage, with potential re-population with a \u201chealthier\u201d line, is theoretically curative to the patient.\nThe safety of rituximab in young lupus patients remains unanswered. Most of the patients in our cohort tolerated the rituximab well. However, as in the European trial [11], our patients were on variable multi-drug regimens. The one patient who died did so from infectious complications in conjunction with severe immune suppression from multiple cytotoxic agents, including both rituximab and cyclophosphamide. Another young patient developed symptoms of cerebral vasculitis, with seizures and encephalopathy. The differential diagnosis included the dreaded progressive multifocal leukoencephalopathy (PML), recently reported to occur in patients treated with rituximab [23]. Although she showed clinical recovery after cyclophosphamide therapy, the case emphasizes the uncharted risks imposed by the use of drugs without well-studied indications.\nThe efficacy of rituximab may be short-lived in some patients. Resistance to RTX B-cell depletion may be related to antibodies against the human-mouse chimeric protein [8]. It may also be genetically influenced by polymorphisms of the FcRIIIa gene, responsible for the Fc-\u03b3 receptor required for RTX binding to phagocytes [24]. In our series, all patients had some degree of B-cell depletion after the initial course of RTX. However, five of the 18 patients (28%) developed clinical relapse in association with re-population of the B-cell lineage. All but one of these responded to additional courses of RTX. This is similar to recent long-term reports in a series of adults [25].\nTreatment regimens in children with SLE do not conform with those of adults developed by carefully designed randomized controlled trials. This may be partially attributable to the fact that children \u2264\u00a016\u00a0years of age are excluded from these trials. This has resulted in an ambiguous and often anecdotal approach to pediatric patients, especially those with early onset and severe disease. Although intravenous cyclophosphamide treatment was established as induction therapy for proliferative lupus nephritis over two decades ago [26], its short- and long-term toxicity delayed its incorporation into pediatric regimens [27]. Paradoxically, those with severe disease have received excessive courses of toxic medications in futile efforts to preserve renal function [27]. More recently, less toxic alternative treatment regimens, including mycophenolate mofetil for both induction and maintenance therapy, have resulted in equal or improved renal survival over conventional therapies [18, 28]. Since treatment protocols of children do not conform to conventional protocols for adults, attempts at reporting multicenter experiences are hampered by a lack of consistency in treatment regimens [11, 22]. Therefore, anecdotal center experiences such as our own are necessary in developing treatment protocols tailored towards efficacy while decreasing long-term risks on growth, fertility and malignancy.\nIn conclusion, RTX therapy offers a potential innovation in the current treatment regimens for children with aggressive SLE that includes both renal and extra-renal manifestations. The development of a randomized controlled treatment protocol for induction and maintenance therapy in pediatric patients is very much warranted.","keyphrases":["rituximab","systemic lupus erythematosus","children"],"prmu":["P","P","P"]}
{"id":"Matern_Child_Health_J-2-2-1592247","title":"A Perspective of Preconception Health Activities in the United States\n","text":"Objectives: Information regarding the type and scope of preconception care programs in the United States is scant. We evaluated State Title V measurement and indicator data and abstracts presented at the National Summit on Preconception Care (June 2005) in order to identify existing programs and innovative strategies for preconception health promotion.\nIntroduction\nThe June 2005 National Summit on Preconception Care, sponsored by the Centers for Disease Control and Prevention (CDC) and the March of Dimes, assembled an array of health care providers, public health practitioners, and researchers in an effort to promote the exchange of information and ideas related to preconception care at the local, state, and national levels. A key issue emerging from conference discussions was the perceived lack of information pertaining to the nature and scope of preconception health services currently being provided in the United States, whether explicitly defined or falling under another umbrella of services. Another issue was the recognition that a compilation of local, state, and national resources would provide important information related to the ability of current public health systems to address preconception care needs, as well as allow for the description of innovative models for the delivery of such services.\nAttended by more than 400 registered participants representing a myriad of agencies and organizations, the summit generated significant response within the public health and clinical communities and reflects a perspective that cannot be readily gleaned from literature reviews or the inspection of existing programmatic guidelines: that is, the topic of preconception health has garnered substantial interest and support and has already been incorporated into numerous existing programs, policies, and strategies in the United States. Unique to the summit was an opportunity to learn about the development and administration of preconception care programs within clinical and population-based settings as Medline, PubMed, and other Internet searches have revealed very little information regarding the development and implementation of preconception care programs within a local context. However, as evidenced by the numerous programmatic components featured in the conference abstracts, such programs are being introduced in many different formats, among various private and public organizations, and across the entire nation.\nAlthough little is now known at this writing about the scope and extent of U.S. programs and projects to promote preconception health, this article uses readily available sources (in the published professional literature and the gray literature of government and organization reports) to provide a snapshot of related activities in the United States. Thus, we are able to report the findings of a scan of State Title V Performance Measures and Priority Needs pertaining to preconception care and a detailed summary of the abstracts of the papers presented at the Summit.Fig. 1State Title V priority needs focused on preconception health and health care, U.S. 2005\nMethods\nThe Maternal and Child Health Bureau website (http:\/\/mchb.hrsa.gov\/) was used to ascertain the proportion of states including preconception health indicators in State Performance Measures and Priority Needs, as reported in the State Title V Maternal and Child Health Service (MCH) Block Grant FY 2004 Annual Report and FY 2006 Application, Form 14. The web-based Title V Information System (https:\/\/perfdata.hrsa.gov\/mchb\/mchreports\/Search\/core\/ measureindicatemenu.asp) was used to search State Title V Performance Measures and Priority Needs for indicators related to preconception health. Initial search parameters included all keywords and all populations and were further refined by selecting for birth outcomes, reproductive health, and women. Those indicators for which infants, newborns, children, men, or service providers represented the sole target population were excluded. The remaining measures were evaluated based on their relevancy to preconception health. Indicators using the term \u201cpreconception\u201d or \u201cpreconceptual\u201d and those that did not use such terminology but adequately captured the concept were considered to fall within the domain of preconception health and health care. States identifying preconception health Priority Needs were depicted on a national map. States reporting relevant Performance Measures were identified and selected measures were described in tabular format. Finally, a comprehensive list of State Title V Performance Measures and Priority Needs related to preconception health, that reflected the tone and language of multiple states was developed.\nAll abstracts submitted and accepted for presentation at the National Summit on Preconception Care (June 2005) were pooled and subjectively categorized into five broad topic areas: research pertaining to preconception risk assessment and outcome evaluation; preconception care programs and activities; tools intended for provider or patient preconception health education; clinical practice strategies; and public policy related to preconception care.\nResults\nState Title V Performance Measures and Priority Needs\nThe federal MCH Block Grant guidance requires states to report annually on National \u201cCore\u201d Performance Measures and to assess needs and set priorities every 5 years. In addition, each state must develop 7 to 10 additional Performance Measures relevant to its own priority needs and programs. Priority Needs reflect a state\u2019s focus for programmatic efforts throughout the next five years and can be adjusted during interim years in response to variations in needs. Performance Measures describe a specific maternal and child need that, when effectively addressed, can lead to improvements in health. In 2005, states completed the 5-year needs assessments and set out priorities and targets for 2010. A previous analysis of the 2000 priorities and measures found that some states, but not all, included measures for preconception care or visits, as did Healthy People 2000 National Health Promotion and Disease Prevention Objectives [1].\nTwenty-three states reported a Priority Need that focused on preconception health and health care (Fig. 1). \u201cPreconception\u201d or \u201cpreconceptual\u201d health was mentioned by 10 states as a Priority Need in maternal and child health. In other states, Priority Needs indirectly addressed preconception care via broad objectives such as improving the health of women of childbearing age, promoting reproductive health and sexual responsibility, and advancing a holistic care continuum for women\u2019s health.\nA total of 42 states and jurisdictions reported at least one Performance Measure related to preconception care, neural tube defects, birth spacing, family planning, unintended pregnancy, and healthy weight\/obesity (Table 1). Unintended pregnancy and healthy weight or obesity were the measures identified most frequently (43% and 19%, respectively). The majority of the measures pertaining to birth spacing or parity, or both, addressed repeat pregnancies among adolescent populations. A total of thirty states or territories reported measures on tobacco use during pregnancy; however, only one focused on the proportion of women who smoked during 3 months prior to pregnancy. Measures pertaining to smoking rates among women of childbearing age were reported by two states. Four states addressed mental health among women of childbearing age and postpartum depression was identified in three others. Two states included measures pertaining to preventive oral health services among women of childbearing age (data not shown).\nTable 2 provides examples of selected State Performance Measures and Priority Needs derived from 18 states. Variations in state-selected preconception health indicators are evident, with some states detailing various components of preconception care and others employing a broader focus on reproductive health.\nTable 1States or U.S. jurisdictions reporting Performance Measures related to selected preconception health topicsPerformance measureStatePreconception careNeural tube defects\/folic acidBirth spacing\/parityFamily planningUnintended pregnancyHealthy weight\/obesityArizona\u00d7Arkansas\u00d7California\u00d7Delaware\u00d7District of Columbia\u00d7a\u00d7Florida\u00d7aGeorgia\u00d7\u00d7aGuam\u00d7Hawaii\u00d7Illinois\u00d7Indiana\u00d7Kentucky\u00d7Louisiana\u00d7Maine\u00d7Marshall Islands\u00d7bMaryland\u00d7Massachusetts\u00d7\u00d7Michigan\u00d7Minnesota\u00d7Mississippi\u00d7aMontana\u00d7Nebraska\u00d7Nevada\u00d7New Jersey\u00d7aNew Mexico\u00d7\u00d7New York\u00d7North Carolina\u00d7\u00d7\u00d7North Dakota\u00d7Northern Mariana Islands\u00d7Ohio\u00d7Oklahoma\u00d7Puerto Rico\u00d7South Carolina\u00d7TennesseeTexas\u00d7South Dakota\u00d7Wyoming\u00d7cUtah\u00d7Vermont\u00d7Washington\u00d7bWisconsin\u00d7aMeasure references repeat births\/pregnancies among adolescents onlybMeasure references family planning counseling or postpartum birth control planning as addressed during routine prenatal carecMeasure references multivitamin use in month prior to conceptionTable 2Sample of selected state Performance Measures and Priority Needs related to preconception healthSelected performance measures and priority needsPreconception health and health care\u2003Performance measure\u2003\u2003\u2022Percentage of women of childbearing age who receive preconception health care in the local health department\u2003Priority need\u2003\u2003\u2022Enhance preconception care and work toward eliminating disparities in infant and maternal morbidity and mortality\u2003\u2003\u2022Improve women\u2019s preconception and interconception health\u2003\u2003\u2022Improve indicators of health in the preconception and perinatal periods, including but not limited to smoking, alcohol, folic acid use,\u2003\u2003\u2003family violence, intention of pregnancy, access to and use of health care\u2003\u2003\u2022Promote sexual responsibility and reproductive healthNeural tube defects\/folic acid\u2003Performance Measure\u2003\u2003\u2022Percent of women of reproductive age who consume at least 400 mcg of folic acid daily\u2003\u2003\u2022Percent of women of childbearing age taking folic acid regularlyBirth spacing and parity\u2003Performance Measure\u2003\u2003\u2022Percentage of live births to women who have another birth in less than 18 months\u2003\u2003\u2022Percentage of repeat pregnancies among adolescents 15\u201319 years of age\u2003Priority need\u2003\u2003\u2022Reduce repeat teenage pregnancies, unintended pregnancies and inadequate spacing of births through widely available, adequately\u2003\u2003\u2003 funded comprehensive, efficiently run family planning and support services\u2003\u2003\u2022Increase the proportion of births that are intended, including promotion of healthy interpregnancy spacingFamily planning\u2003Performance measure\u2003\u2003\u2022Proportion of low-income women who receive reproductive health\/family planning services\u2003Priority need\u2003\u2003\u2022To improve access to and utilization of contraceptive servicesUnintended pregnancy\u2003Performance measure\u2003\u2003\u2022Percentage of pregnancies (live births, fetal deaths, abortions) that are unintended\u2003\u2003\u2022Percentage of women responding to the Pregnancy Risk Assessment Monitoring System (PRAMS) survey that they wanted to be\u2003\u2003\u2003pregnant later or not then or at any time in the future\u2003Priority need\u2003\u2003\u2022Improve the health of children and families by increasing the percent of births that are intended\u2003\u2003\u2022Promote planned pregnancies and child spacingHealthy weight\/obesity\u2003Performance measure\u2003\u2003\u2022Percent of women (18\u201344) with healthy weight (BMI)\u2003\u2003\u2022To increase the percentage of Family Planning clients with BMI greater than the 85th percentile who receive educational materials in\u2003\u2003\u2003 the Family Planning Clinics, and are referred to community sources\u2003Priority need\u2003\u2003\u2022To reduce proportion of women of childbearing ages, pregnant women and children and youth with BMIs in the underweight,\u2003\u2003\u2003 overweight and obese categories\nAbstracts from the National Summit on Preconception Care\nA total of 59 abstracts accepted for presentation at the National Summit on Preconception Care were reviewed and categorized according to primary area of focus. Approximately 32% addressed preconception health research; 27% described preconception care programs and activities; 22% outlined tools for provider or patient education; 15% detailed clinical practice strategies; and 3% highlighted policy-based strategies for increasing access to preconception care services.\nAmong the research abstracts, the topics noted most frequently pertained to methods for preconception risk assessment, potential predictors of adverse pregnancy outcomes, and variations in preconception health indicators across high-risk populations. Three abstracts described the use of the Perinatal Periods of Risk (PPOR) model [2] for assessing risks among various populations. Other issues included folic acid awareness among women and health care providers, maternal nutrition, pregnancy planning and risk behaviors, preconception hepatitis B prevention, and workplace hazards.\nThe PPOR model facilitates the identification of four contributors to infant mortality: maternal health and prematurity, maternal care, newborn care, and infant health. Excess rates of fetal and infant mortality in any of the four components indicate a need for targeted interventions within that construct [2\u20134]. CityMatCH\u2019s 2000\u20132002 PPOR Practice Collaborative provided public health workers in 14 U.S. cities with the skills, knowledge, and support to implement the PPOR method in various urban communities [2]. The PPOR approach is also is being used statewide by Florida and Ohio and in selected areas across the country [2\u20134].\nThe Fountain Project in Kansas City, Missouri linked PPOR techniques with the Fetal Infant Mortality Review (FIMR) process in an effort to identify the causes of consistently higher rates of infant mortality among African-American residents compared with their non-Hispanic White counterparts. The findings of the analysis indicated an excess rate of maternal health or prematurity-related deaths among African-American infants. In light of the contributing social factors identified by the FIMR, the development of a Women\u2019s and Children\u2019s Wellness Center was proposed to provide social and clinical services for high-risk families (Cook BE, Guillory VJ, Cai J, Hoff GL, Manning J, unpublished data, 2005).\nIn an effort to facilitate the analysis of various perinatal health outcomes, a group of nine counties surrounding the San Francisco Bay (California) formed the Maternal, Child and Adolescent Health Bay Area Data Collaborative (BADC). The group used the PPOR model to evaluate recent county and regional fetal-infant mortality rates and reported substantial excess in the proportion of deaths among African-American infants, particularly in the prematurity and maternal health component of the model. Using a national comparison group, these investigators were able to determine that approximately 56% of the excess deaths among African-American infants could be prevented by targeting preconception and interconception risk factors (Stein EJ, Abramowitz A, Brown J, Chabra A, unpublished data, 2005).\nFederally funded Healthy Start projects were highly represented among the 16 abstracts detailing preconception care programs and activities presented at the Summit. Of these, seven abstracts described the modification or expansion of existing programs to include direct services or community-based interventions for high-risk postpartum mothers or all women of reproductive age. The Magnolia Project in Jacksonville, Florida is an example of a Healthy Start program in which a comprehensive array of health services was offered in an effort to improve birth outcomes. Services included, but were not limited to, case management, education and risk reduction, and well-woman care. The target population was high-risk African-American women aged 15\u201344 years who lived in five zip code areas of Jacksonville. The project operates as a collaborative between the Northeast Florida Healthy Start Coalition, the Duval County Health Department, and local community-based organizations. As reported, a 2004 assessment of project services conducted by the Health Resources and Services Administration (HRSA) Office of Performance Review found high rates of success (>70%) in the resolution of key risks (for example, lack of family planning, and rates of repeat sexually transmitted diseases) among the project participants (Brady, CM, unpublished data, 2005).\nThe Missouri Bootheel Healthy Start project, a provider of referral and education services in a five county area of southeast Missouri, uses community-based education and interventions to improve adverse perinatal outcomes. The project has been one of few in the area that has promoted a family-focused approach to health education through the inclusion of various services directed at men. The curriculum for fathers has addressed a variety of issues, such as communication with the mother, addressing stress in the relationship, and the impact of nutrition on birth outcomes (Dean CG, Campbell T, Frazier V, Washington J, unpublished data, 2005).\nTitle X family planning clinics were identified as providers of preconception primary care and folic acid awareness interventions in two programs and activities abstracts. The Women Enjoying Life Longer (WELL) Project was initiated by the Maryland Department of Health and Mental Hygiene following a community needs assessment. Comprehensive preventive women\u2019s health services were added to three Title X family planning clinics in Baltimore County, using Title X and Maternal and Child Health funding. Augmented services included nutrition and physical activity counseling, adult immunizations, smoking cessation interventions, and preconception counseling, as well as referrals for problems such as substance abuse, depression, domestic violence, and chronic disease. Early evaluation data indicated that patients and staff have responded positively to new services, patient knowledge of women\u2019s health has improved, and patient volume has increased 37% (Cheng D, unpublished data, 2005).\nThe Oklahoma Birth Defects Registry developed, implemented, and evaluated a preconception \u201cwomen\u2019s health appraisal\u201d project. The intervention was comprised of a 3-page health appraisal for women, with follow-up risk counseling, in selected family planning clinics (two rural and one urban). Pre-evaluation results indicated that 84% of nurses found the questionnaire helpful in assessing risk factors, 90% of nurses found it helpful as a guide to counseling and referrals, and 86% of patients increased their understanding of risk factors. Post-evaluation results found that 62% of patients modified one to three risk factors in a three-month period (Feuerborn VR, Pearson K, unpublished data, 2005).\nThe remaining programs were funded either through grants, private organizations, or state\/local health departments and comprised comprehensive health care programs for women with a history of preterm delivery or a low birth weight infant and broad women\u2019s wellness interventions aimed at improving women\u2019s health or folic acid utilization. For example, the Interpregnancy Care Program at Grady Memorial Hospital in Atlanta, Georgia employs a \u201cresource mother\u201d to coordinate the provision of primary health care and dental services, enhanced nurse case management, and other outreach services to African-American women who delivered a very low birth weight infant at the hospital and who qualified for indigent or charity care. For 24 months postpartum, the women were offered health care visits every 1\u20133 months in order to address risks known to be associated with the delivery of a low birth weight infant such as poorly controlled chronic disease, short intervals between pregnancies, reproductive tract infections, periodontal disease, nutritional deficits, substance abuse, and stress. Preliminary evaluation of the 22 women retained in the pilot program indicated that approximately one-quarter of them were affected by unrecognized or poorly managed chronic health problems and none of the participants wanted to become pregnant during the next 2 years (Dunlop A, unpublished data, 2005).\nThirteen abstracts (22%) described the creation, dissemination, and evaluation of tools for patient or provider education. Of those, eight described marketing campaigns and toolkits for patient education, three specifically referenced provider education, and three assessed the use of women\u2019s health appraisals by health care providers. For example, the California Preconception Care Initiative, Every Woman, Every Time, was created in 1989 through a partnership between Sutter Medical Center in Sacramento and the March of Dimes. The project conducted a metanalysis of the preconception care literature [5] and used this information in a consensus development process to produce a marketing packet for providers. Key components of the packet included the rationale for providing preconception care, a description of the essential elements of care, patient education materials, and information on billing methods. More than 9,000 packets were distributed statewide. An evaluation found that, among 187 providers responding, 75% indicated the information was very useful, 80% said they would distribute materials to patients, and 72% said they would use the billing codes provided. A further impact evaluation is being considered (Cullum AS, unpublished data, 2005).\nA research project conducted at a New York inner-city hospital served by Albert Einstein College of Medicine\/Montefiore Medical Center sought to evaluate the knowledge and awareness of providers regarding preconception care. A pre-intervention chart review and a provider survey were conducted to evaluate delivery or preconception care. The two-part intervention included: [1] a 1-h lecture for all providers, and [2] a standardized preconception care form inserted into all charts. A post-intervention chart review of a convenience sample and repeated provider survey were then conducted. The result was a significant improvement in documentation of the delivery of preconception care (p<0.05); however, provider knowledge and attitudes, as measured by the survey, did not change significantly (Bernstein P, unpublished data, 2005).\nClinical practice strategies were discussed in nine abstracts and included topics such as genomics, group care, screening for environmental exposures and maternal depression, smoking cessation, and provider knowledge of current practice guidelines. An example of a promising practice strategy presented at the Summit is the use of a group model of prenatal care to address topics relevant to preconception care. In 2002, the Comprehensive Family Care Center at Montefiore Medical Center implemented the group model of prenatal care developed by the Centering Pregnancy and Parenting Association. Traditional prenatal visits were replaced with group appointments lasting approximately 2 hours and attended by 10 to12 women. Typical clinical care services were provided during the visits and were supplemented with group discussions on pregnancy-related topics. Issues related to preconception health were covered during many of the meetings and included nutrition, substance abuse, contraception, and family planning. Since its inception, the group care model has been employed for 14 patient groups with high levels of patient and provider satisfaction reported, particularly related to enhanced opportunities for patient education (Bernstein P, Rising SS, Dolan S, Pardanani S, Merkatz IR, unpublished data, 2005).\nFinally, policy-based strategies for the funding of preconception care were described in a small number of abstracts. One pair of abstracts highlighted Illinois\u2019 efforts to promote preconception health using public policy and funding through Medicaid waivers, Title V, and other resources (Murphy AM, unpublished data, 2005; Saunders SE, unpublished data, 2005). Identified as a priority area, preconception care has been integrated into a number of programs, most notably the Illinois Healthy Women program. This Medicaid waiver program initiative extends coverage for family planning services to women who would otherwise lose their benefits after 60 days postpartum and to all women 19 through 44 years of age who were previously enrolled in Medicaid but who had lost their benefits (Murphy AM, unpublished data, 2005). Additional efforts have been made through the Illinois Family Planning Program. Preconception education is currently provided at all state-funded family planning clinics, with 16 state family planning agencies offering additional counseling and referral for high-risk clients [6]. Other strategies employed by the Illinois Department of Human Services include statewide genetic counseling programs and folic acid campaigns, screening mothers of children enrolled in the state\u2019s Medicaid program for perinatal depression, and programs aimed at increasing birth spacing and promoting the health of teen parents (Saunders SE, unpublished data, 2005).\nConclusions\nIn public health, effective programs are those developed in response to a well-defined problem analysis, are mindful of the local context, and apply proven interventions. Although the scientific evidence in support of some preconception interventions is relatively well described within the literature, topics discussed during the National Summit on Preconception Care reframed the issues in light of new findings regarding unmet needs, health disparities, service delivery models, and tools such as the PPOR to assess risks among populations. Variations in the type and scope of policies, programs, and strategies were evident both in the diversity of the summit abstracts and in the state Title V indicators. Clearly, a preconception focus is being woven into existing programs and guiding the development of new initiatives. Innovative strategies are being implemented that target a variety of populations including community groups, health care providers, medical students, adolescents, and fathers. Finally, many state indicators are adopting a broader language to encompass the health needs of women of childbearing age and support the notion of a lifespan approach to preconception health.\nIt should be noted that the findings reported in this brief are limited by a number of factors. First, state measures and priorities reported by the federal Maternal and Child Health Bureau might not reflect the programs and projects actually being implemented by states and communities; they are only an expression of interest and concern. Next, the content of the abstracts accepted for the National Summit on Preconception Care presented a selection of current research, policies, and programs and, therefore, does not fully represent the scope of national preconception care projects. Finally, the identification of preconception health state Title V indicators required a certain amount of subjective interpretation regarding the actual focus of the Priority Need or Performance Measure. Although the authors have considerable knowledge of the definition and content of preconception care, it is possible that certain indicators might have been incorrectly excluded or included due to misinterpretation.\nNotwithstanding these limitations, the results of the present analysis indicate a widespread recognition of the need for a continuum of care that starts well before birth, involves both men and women, and employs a lifespan approach to emotional and physical well-being. Because preconception interventions can take place at any time during a woman\u2019s reproductive life and across a variety of dimensions, countless opportunities exist for positively influencing the health of women, children, and families. The variations among domestic preconception health projects and related public health priorities are reflective of the true breadth of the topic. If a national agenda for preconception health is to be moved forward, it is imperative that these efforts continue to be fostered and expanded in all settings and across a multitude of disciplines.","keyphrases":["preconception care","reproductive health","pregnancy"],"prmu":["P","P","P"]}
{"id":"Anal_Bioanal_Chem-3-1-1764596","title":"Instrumental methods and challenges in quantifying polybrominated diphenyl ethers in environmental extracts: a review\n","text":"Increased interest in the fate, transport and toxicity of polybrominated diphenyl ethers (PBDEs) over the past few years has led to a variety of studies reporting different methods of analysis for these persistent organic pollutants. Because PBDEs encompass a range of vapor pressures, molecular weights and degrees of bromine substitution, various analytical methods can lead to discrimination of some PBDE congeners. Recent improvements in injection techniques and mass spectrometer ionization methods have led to a variety of options to determine PBDEs in environmental samples. The purpose of this paper is therefore to review the available literature describing the advantages and disadvantages in choosing an injection technique, gas chromatography column and detector. Additional discussion is given to the challenges in measuring PBDEs, including potential chromatographic interferences and the lack of commercial standards for higher brominated congeners, which provides difficulties in examining degradation and debromination of BDE congeners, particularly for BDE 209.\nIntroduction\nOver the past 5\u00a0years there has been a surge in the number of studies investigating polybrominated diphenyl ethers (PBDEs) in the environment. In 1981 the first report on PBDEs was made in biota samples from a Swedish river [1], which was followed by a handful of studies throughout the 1980s and 1990s. Then, in 2000 Noren and Meironyte [2] published a paper on organohalogen temporal trends in human breast milk from Sweden that generated rapid interest in the fate of PBDEs. This study demonstrated that PBDE levels appeared to be increasing at an exponential rate, and since then the number of studies investigating PBDEs has increased as well.\nPBDEs are used as brominated flame retardant additives which are incorporated into a number of polymers and resins found in a majority of consumer products. PBDEs are the most widely used additive flame retardant and, until recently, almost 70,000\u00a0t was produced every year, half of which was used in products sold in the USA and Canada. In 2004 the European Union phased out the use of two of the three PBDE commercial mixtures, PentaBDE and OctaBDE. Following this action, and owing to growing concerns in some US state governments, the sole US chemical manufacturer voluntarily agreed to stop manufacturing these two PBDE commercial mixtures beginning in January 2005 [3]. Presently, the only remaining unregulated PBDE mixture in production is DecaBDE, with an annual global market demand of 56,000\u00a0t (http:\/\/www.bsef.com). DecaBDE is composed almost exclusively (more than 97%) of decabromodiphenyl ether (BDE 209), a fully brominated BDE congener.\nMuch of the concern regarding PBDEs has been focused on the elevated levels measured in human tissues within the US population [4]. The BDE congeners typically measured in human tissues are associated primarily with the PentaBDE mixture, and to some extent with the OctaBDE mixtures. While production of PentaBDE and OctaBDE has been halted, there are still a number of products left on the market and in use today that contain these mixtures. On the basis of their persistence and current reservoirs, the fate of PBDEs will most likely need to be monitored for years to come.\nOne of the greatest challenges to measuring PBDEs in environmental samples has been developing methods to accurately quantify BDE 209. While analytical methods are readily available for quantifying tribrominated through heptabrominated congeners found in the PentaBDE and OctaBDE mixtures, the analysis of higher brominated compounds (i.e., congeners with eight or more bromines) has proven to be difficult. In 1999\u20132000, the first worldwide interlaboratory comparison on PBDE measurements was conducted. Laboratory agreements were acceptable for most of the lower brominated congeners; however, reported values for BDE 209 varied over almost 2 orders of magnitude [5]. Over the past few years, improved methods have been developed to measure BDE 209 with increased accuracy and precision, and with the use of these methods, more data on BDE 209 have been generated.\nA number of papers have described analytical methods for extracting and quantifying environmental samples for the determination of PBDEs [6\u201311]. In 2003 Covaci et al. [9] published an excellent review on extraction methods, cleanup techniques and analytical detection methods for brominated flame retardants. Since the publication of that review, several interesting papers have been published which highlight improvements in injection techniques, analyte resolution and selectivity, particularly for BDE 209. When choosing a method one must find the best compromise in considering cost, reproducibility, chromatographic and mass resolution, sensitivity, and analysis time. The aim of this paper is to review and disseminate the latest information available and discuss the advantages and disadvantages found when using different analytical techniques to determine PBDEs in environmental and human samples.\nThe physical-chemical properties of PBDEs are very similar to those of PCBs [12], and as such, PBDEs are typically extracted using nonpolar solvents (e.g., hexane, dichloromethane) in methods typically employed for PCB analysis. In this paper all PBDE congeners are labeled and numbered according to the same scheme developed by Ballschmitter and Zell [13], for PCB congeners. A variety of extraction and cleanup methods have been reported [6, 9, 14\u201316], but for the purpose of this paper, we will focus on instrumental methods of analysis for PBDE congeners.\nInjection techniques\nOwing to their vapor pressures and polarity, gas chromatography (GC) has become a standard analytical separation method employed for the analysis of PBDEs. The injection of samples into the GC system is an important and crucial step for the accurate and optimal determination of compounds with relatively high boiling points like PBDEs. Various injection methods have been reported which introduce sample analytes into the GC analytical column. The three most common injection techniques for PBDE analysis are split\/splitless injection, on-column injection and programmable temperature vaporization (PTV) injection. All three methods possess advantages and disadvantages that are dictated primarily by availability, price, acceptable detection limits and discrimination of congeners on the basis of molecular weight. Split\/splitless injection is the most routinely used, and is capable of analyzing dirty samples; however, this method can be limited by small injection volumes (typically 1\u20133\u00a0\u03bcl) and high injection temperatures (250\u2013300 \u00b0C). During split\/splitless injection the analytes are immediately vaporized in the inlet liner and are transferred to the column in the gas phase. In some cases, the high inlet temperature can lead to thermal degradation and discrimination of higher molecular weight PBDEs, particularly the fully brominated BDE 209. Evaporation of the sample can begin inside the injection needle or on the tip, and differences in evaporation rates can lead to discrimination of the higher molecular weight congeners. Inefficient transfer of the sample to the column can be difficult for analytes with high boiling points. Furthermore, active sites on dirty liners can result in thermal degradation; therefore, the temperature of the injection port and the time spent in the injection port prior to transfer are important mediating factors in the response of the PBDE congeners. A detailed study by Bj\u00f6rklund et al. [17] found that the optimal injector temperature and splitless time should be kept as high as possible, in their case, 325 \u00b0C and 4\u00a0min, respectively (Fig.\u00a01). Using these optimal setpoints, Bj\u00f6rklund et al. demonstrated that the BDE 209 response increased by almost 25% when compared with average settings used in an interlaboratory comparison [5]. However, they also noted a significant decrease in the precision of higher molecular weight congeners, particularly BDE 209, compared with the case for on-column techniques, which are less prone to discrimination effects.\nFig.\u00a01Detector response for BDE 209 as a function of splitless time and injector temperature, with pressure held constant at 2.3 bar. BDE brominated diphenyl ether. (Reprinted from Bj\u00f6rklund et al. [17] with permission)\nOn-column injection has become a popular method for introducing samples into the gas chromatograph for PBDE analysis [18\u201320] and is comparable in cost to split\/splitless injectors. The design is much simpler relative to a split\/splitless injection port and involves direct injection of the sample, dissolved in a carrier solvent, onto the head of the column. This reduces the potential for thermal degradation and discrimination experienced in split\/splitless injectors. In the same study mentioned previously, Bj\u00f6rklund et al. [17] observed the highest precision in PBDE measurements, particularly for BDE 209, when using on-column injection compared with split\/splitless and PTV injectors. However, when using on-column injection, care must be employed to ensure a clean sample, to prevent coextracted compounds from building up in the retention gap and column leading to increased noise, peak tailing, retention shifts and reduced lifetimes of the columns. On-column injections are also limited by injection volumes (typically 1\u20132\u00a0\u03bcl) to prevent excess solvent from overwhelming the detectors.\nPTV inlets have become a more popular choice for injection over the past 5\u00a0years, particularly for the analysis of PBDEs [21\u201323]. The primary advantage in using PTV injectors is an increase in injection volumes (up to 125\u00a0\u03bcl), which can drastically improve detection limits, and can be useful in measuring low-level samples such as human serum [14, 24\u201326]. With this type of injection, multiple injections are made into a liner while the solvent is vented off, trapping the analytes and increasing the mass of analytes present in the liner. Transfer of the analytes to the head of the column occurs after the solvent vent is closed, and rapid heating of the injection port (200\u2013700 \u00b0C\/min) aids the carrier gas in transferring the analytes to the column. Several studies have demonstrated that the PTV injectors must be optimized prior to use as they are more complicated than the conventional split\/splitless injector. Analyte discrimination can result if the following are not optimized: injection rate or flow, injection temperature, vent flow, solvent elimination time, injection volume, transfer time and transfer temperature.\nTollb\u00e4ck et al. [25] published a review on optimization parameters for PTV injection and their optimal values are reported in Table\u00a01. In general, it is important that the rate of injection is equivalent to the rate of solvent evaporation to avoid flooding the injector. Care must also be taken to avoid discriminating low molecular weight compounds through solvent vent losses. The rate of injection will influence the solvent introduction mode (i.e., drops versus spray), which can discriminate congeners of different masses on the basis of the point (vertical position) of solvent evaporation within the injection liner. BDE 209 responses can be particularly sensitive to injection flow rate. To balance the solvent removal from the injector, the temperature and vent flow must also be optimized. At elevated injector temperatures, low molecular weight compoundss can be lost through solvent venting and at high flow rates the risk of analyte loss increases. The type of liner used in the injector can also influence PBDE responses. Some liners contain activated sites, which can lead to irreversible adsorption or catalysis of thermal degradation of BDEs, particularly the high molecular weight congeners. Tollb\u00e4ck et al. [25] recommended adding 0.1\u20130.35% (v\/v) of dodecane with multibaffled liners to trap low molecular weight congeners and changing the liner every 200 injections. Transfer of analytes to the GC column is accomplished by rapid heating of the injector port after the solvent vent is closed. PBDE responses are not significantly affected by the analyte transfer temperature ramp; therefore, steep ramps (700 \u00b0C\/min) should be used to increase the rate of transfer to GC columns. A final injector temperature of 325 \u00b0C was found to be a good compromise to balance transfer efficiency of high molecular weight analytes relative to the risk of their thermal degradation in the injector. \nTable\u00a01Optimized injector parameters for polybrominated diphenyl ether (PBDE) analysis using programmable temperature vaporization as reported by Tollb\u00e4ck et al. [25]ParameterDomain Injection rate300\u00a0\u03bcl\/minInjection temperature80 \u00b0CVent flow150\u00a0ml\/minTemperature rate700 \u00b0C\/minTransfer temperature325 \u00b0CTransfer time0.3\u20130.6\u00a0minSolvent elimination time0.1\u00a0minCarrier gas pressure650\u00a0kPa\nWhile split\/splitless, on-column and PTV injections are the most common methods used in PBDE analysis, other injection techniques have been reported. Large volume injections up to 500\u00a0\u03bcl have been reported using both loop-type [26] and automated rotary valves [27]. Loop-type injectors can easily be constructed and used but require day-to-day checks for stability and reproducibility owing to problems with the injection pressure and temperature which can result in peak distortion. Bj\u00f6rklund et al. [27] used a stainless steel high-performance liquid chromatography (LC) automated rotary valve to inject PBDEs into a GC system. This technique reduces discrimination of higher molecular weight congeners and thermal degradation similar to on-column injection, but offers the advantage of injecting larger volumes (up to 50\u00a0\u03bcl). However, care must be taken to incorporate a solvent vent system for certain detector systems, particularly mass spectrometry (MS). Lastly, Sjodin [28] used a septum-equipped programmable injector (SPI), which resulted in reproducible results with minimal congener discrimination; however, these injectors are no longer produced by the manufacturer.\nGC column selection\nThe next crucial step in the method development is the selection of an appropriate column system. The optimal chromatographic column is one that optimizes resolution and discrimination of congeners with the greatest sensitivity in the shortest amount of time. Of the three primary injection methods described earlier, both on-column and PTV injections require the use of a retention gap or guard column to reduce column deterioration and to aid in focusing the initial band of analytes. Most guard columns are either composed of untreated fused silica with active silanol groups or deactivated fused silica that ranges in polarity. Bj\u00f6rklund et al. [17] recommends using a Siltek deactivated guard column, which the authors found had the best precision (measured by peak response) and displayed the least degradation of higher molecular weight congeners. In contrast, a nonpolar fused silica guard column resulted in a 50% reduction in sensitivity for the higher molecular weight PBDE congeners.\nA variety of different columns have been used for the determination of PBDEs in environmental samples. When choosing a column one must consider the column stationary phase (polarity), column length, film thickness and inner diameter, all of which will influence the response towards PBDEs. Generally speaking, the most sensitive method for measuring the entire range (low to high bromine substitution) of PBDE congeners is found when using short (10\u201315-m) nonpolar DB columns with thin (0.1-\u03bcm) stationary phases [17]. Most GC\/MS methods employed for PCB quantification use a 60-m column to increase the resolution power; however, longer columns are not well suited for PBDE analysis, particularly for the higher molecular weight congeners. Longer columns result in longer residence times for the analytes, initiating degradation of the higher brominated congeners (noticeably for heptaBDE, octaBDE, nonaBDE and decaBDE congeners) in the column. Bj\u00f6rklund et al. [17] determined that the DB-5MS column (Agilent Technologies, Palo Alto, CA, USA) produced the lowest discrimination and the highest precision compared with five other columns tested (Fig.\u00a02). Columns that contained similar stationary phases but that were produced by two different manufacturers also resulted in different column performance. An HP-1 column from Agilent Technologies significantly degraded nonaBDEs and BDE 209, whereas a comparable DB-1 column from J & W Scientific (Folsom, CA, USA) did not. Narrow-bore columns (inner diameters 0.1\u00a0mm or less) have been used recently in combination with PTV injectors, resulting in faster and more efficient separation of congeners [24, 25]. Narrow-bore columns produce extremely narrow peaks, with average peak widths at half peak height of less than 1\u00a0s, compared with almost 2\u00a0s using columns with 0.25-mm film thickness.\nFig.\u00a02The relative response of five selected BDE congeners using different gas chromatography capillary columns. The response for DB-5MS was set to 1. Mean values are plotted (n=5) and the error bars correspond to the standard deviation. (Reprinted from Bj\u00f6rklund et al. [17] with permission)\nAnother consideration in choosing a GC column is its resolving power. Koryt\u00e1r et al. [29] recently published a retention time database for 126 PBDE congeners using seven different capillary columns with varying film thicknesses (Table\u00a02). This database is useful for determining the most suitable column for quantitative, congener-specific PBDE analysis. Of the seven columns tested, the DB-XLB column (30\u00a0m \u00d7 0.25\u00a0mm \u00d7 0.25\u00a0\u03bcm, J & W Scientific) was found to have the fewest number of coelutions with other BDE congeners and with other brominated flame retardants, followed closely by the DB-1 column (30\u00a0m \u00d7 0.25\u00a0mm \u00d7 0.25\u00a0\u03bcm, J & W Scientific). However, the DB-XLB column demonstrated greater degradation of the higher molecular weight congeners relative to the DB-1 column. Koryt\u00e1r et al. [29] compared the relative retention times (RRTs) from their study to a GC-RRT model developed by Rayne and Ikonomou [30] which was based on the number of ortho-, meta- and para-bromine substituents, dipole moments and the molecular weight of the compounds. In general the RRTs of lower molecular weight PBDE congeners compared very well between the two studies. In contrast, the higher molecular weight congeners, noticeably the heptaBDE and octaBDE congeners were not tightly correlated, most likely owing to a lack of commercially available standards for heptaBDE and octaBDE congeners when the GC-RRT model was developed. Additional standards are now available and it will be useful to update these types of models in the future. \nTable\u00a02An assessment of PBDE coelution from seven gas chromatography columns as tested and reported by Koryt\u00e1r et al. [63]ColumnDB-1DB-5HT-5DB-17DB-XLBHT-8CP-Sil 19Dimension (m \u00d7 mm \u00d7 \u03bcm)30 \u00d7 0.25 \u00d7 0.2530 \u00d7 0.25 \u00d7 0.2530 \u00d7 0.25 \u00d7 0.1030 \u00d7 0.25 \u00d7 0.2530 \u00d7 0.25 \u00d7 0.2525 \u00d7 0.22 \u00d7 0.2517 \u00d7 0.15 \u00d7 0.30Number of coeluting BDEs62636667566272Number of coelutions with flame retardants24262730222629Co-elution with major BDE congenersMajor BDE\u00a02816, 3316, 3316, 33, 3816, 33, 3816, 33\u00a047\u00a04968, 8068686242, 48, 68, 716851, 75\u00a085155114\u00a099116127\u00a0100109101109, 120\u00a0138166HBCD166\u00a0153HBCD168\u00a0154MTBBP-A, BB-153MTBBP-A, BB 153105126BB 153\u00a0183BB 169BB 169MTBBP-A dimethylated tetrabromobisphenol-A, HBCD hexabromocyclododecane\nChromatographic interferences\nCoelution of compounds can be an important consideration when using GC\/electron capture detection (ECD) and GC\/electron capture negative ionization (ECNI) MS techniques to quantify environmental samples, especially as more PBDE congeners become available for analysis. The database of Koryt\u00e1r et al. [29] found that at a minimum, 56 of the 126 PBDE congeners tested were coeluted on the GC columns. The majority of these coelutions were observed for congeners not typically observed in environmental samples; however, a few major congeners (i.e., BDE 28, BDE 49 and BDE 154) can often be composed of different BDE congeners and other brominated flame retardants. Because ECD and ECNI-MS techniques are not as selective as electron ionization (EI) MS for the analysis of lower brominated congeners, coextracted halogenated compounds can sometimes be mistaken for PBDE congeners. Several papers have reported the coelution of 2,2\u2032,4,4\u2032,5,5\u2032-hexabromobiphenyl (BB 153) with BDE 154 and of tetrabromobisphenol-A with BDE 153 [6, 9, 29], typically on 15- and 30-m capillary columns. PCB congeners can also be coeluted if not specifically separated during the extraction process. Alaee et al. [31] reported ten possible interferences between PBDEs and organochlorines. Of particular notice was the interference of BDE 47 with CB-180, and that of BDE 99 with CB-205. The additional mass accuracy of high resolution MS (HRMS) may not be adequate to distinguish certain PCBs and BDEs. Alaee et al. [31] found that the isotopic cluster of [M-Cl2]+ from heptachlorinated biphenyls contains the same mass fragments found in tetrabrominated diphenyl ethers [M-Br2]+ and resolving powers of 25,000 (m\/\u0394m) were required for discriminating the two.\nNatural brominated compounds have recently been identified in some marine algae, mammals and birds [32\u201334]. Because GC\/ECNI-MS methods rely upon selective ion monitoring (SIM) of Br\u2212 ions [79Br and 81Br], other brominated compounds can produce the same fragment ion and confound analysis of PBDEs. Methoxylated PBDEs (MeO-BDEs) are often extracted with PBDEs in environmental samples and can cause interferences when using both GC\/EI-MS and GC\/ECNI-MS methods. As seen in Fig.\u00a03, the BDEs and MeO-BDEs are eluted very closely together using common techniques and differentiation must be based solely on retention time. Two MeO-BDEs, (6-methoxy-2,2\u2032,4,4\u2032-tetrabromodiphenyl ether and 2\u2032-methoxy-2,3\u2032,4,5\u2032-tetrabromodiphenyl ether) have been identified in several marine species [32, 34] and are eluted between BDE 47 and BDE 100 with identical Br\u2212 ion ratios. Other studies have also identified a brominated bipyrrole known as 1,1\u2032-dimethyl-tetrabromo-dichloro-2,2\u2032-bipyrrole (DBP-Br4Cl2) in marine species [35\u201337]. DBP-Br4Cl2 is also eluted very closely to BDEs and MeO-BDEs using common methods of analysis (Fig.\u00a04).\nFig.\u00a03Comparison of elution order for methoxylated BDEs (MeO-BDE) and BDE congeners on a 30-m DB-5MS capillary column using gas chromatography (GC)\/electron capture negative ionization (ECNI) mass spectrometry (MS). PDBE polyBDEFig.\u00a04GC\/ECNI-MS chromatogram of sea lion blubber containing both PBDEs and a marine natural product, DBP-Br4Cl2\nCalibration and quantification standards\nPBDE calibration standards are commercially available from a few suppliers, including Accustandard (New Haven, CT, USA), Cambridge Isotope Laboratories (Andover, MA, USA), Wellington Laboratories (Guelph, ON, Canada) and Chiron Co. (Trondheim, Norway). All of these suppliers provide the suite of major PBDE congeners typically identified in environmental samples. To the author\u2019s knowledge, about 160 of the 209 possible BDE congeners are currently available commercially. There are still many hexaBDE, heptaBDE and octaBDE congeners that are not available and it can sometimes be difficult to identify unknown peaks in chromatograms that may be those of BDE congeners. For example, octaBDE congeners have been identified as degradation products of BDE 209 through apparent debromination mechanisms [38\u201340]; however, the bromine substitution of these congeners is unclear owing to a lack of commercially available octaBDE congeners for comparison. Hopefully in the future, more standards will be available for comparison and will provide more information on the fate of higher brominated congeners.\nAnother facet to consider in measuring PBDEs is the choice of an appropriate internal quantification standard. Ideally, isotopically labeled (13C) PBDE standards would be the best choice for quantifying PBDEs. Unfortunately, for some detection methods (see later), 13C PBDEs cannot be distinguished from native PBDEs and cannot be used. Several studies have used alternative internal standards such as 13C-labeled bromobiphenyls and chlorinated diphenyl ethers [41], polychlorinated biphenyls [42], or unlabeled BDE congeners [5, 20] that are not typically identified in samples such as BDE 15 and BDE 75. Chiron Co. recently announced the availability of fluorinated BDEs for use as internal standards. For these standards one fluorine atom is substituted for hydrogen on a BDE congener, resulting in a slightly different vapor pressure. These fluorinated BDEs fragment in a manner similar to that of native BDEs, but have slightly different retention times from their nonfluorinated analogues (Fig.\u00a05), making them very useful as internal standards in GC\/ECNI-MS methods when 13C-labeled BDE congeners cannot be used.\nFig.\u00a05GC\/ECNI-MS chromatogram (a) illustrating the elution order of 6-fluro-BDE 47 relative to the PBDE congeners and its comparable ion fragmentation pattern (b)\nDetection techniques\nDetermination of PBDEs in environmental samples is sometimes conducted using ECD, but more commonly mass spectrometers are used, the latter being classified into low-resolution (LR) and HR instruments. ECD is advantageous because is relatively inexpensive, user-friendly and sensitive for measurements of halogenated organics; however, ECD is limited in selectivity because detection and identification is based solely on retention time and halogenated interferences can lead to misidentification [31]. Determination of PBDEs using LR instruments is typically made with the instruments operated in either EI-MS or ECNI-MS modes. In contrast, HR instruments are used almost exclusively in EI mode (EI-HRMS). HRMS offers the best selectivity for PBDE measurements, with a mass spectrometric resolution (m\/\u0394m) of approximately 10,000, resulting in fewer coeluting interferences and facilitating the use of isotopically labeled internal standards. However, HR instruments are more expensive relative to LR instruments. In addition, HRMS is labor-intensive, requiring trained personnel to maintain the instruments and keep them actively running. A brief review of LRMS and HRMS techniques are given next.\nA variety of papers have reported PBDE measurements in environmental samples using both GC\/LR-EI-MS [43\u201345] and GC\/LR-ECNI-MS [19, 46, 47]. In general, LR-EI-MS is more selective than LR-ECNI-MS methods because molecular fragments (typically [M-Br2]+) are monitored for each homologue group, in contrast to the bromide ions ([Br\u2212]\u2212, m\/z 79 and 81) monitored for all homologue groups in LR-ECNI-MS methods. However, LR-ECNI-MS is a much more sensitive method with lower limits of detection (LOD) relative to LR-EI-MS, even when using SIM mode. The LOD reported for LR-EI-MS range from 0.53 to 32.09\u00a0pg, whereas for LR-ECNI-MS the LOD is an order of magnitude lower, ranging from 30\u00a0fg to 1.72\u00a0pg [48], which is particularly useful for the analysis of low-concentration samples such as human serum and plasma [14, 33, 49]. Additionally, there is a large drop in sensitivity when measuring BDE congeners with more than six bromine atoms using quadrupoles in LR-EI-MS; therefore, one must prioritize selectivity versus sensitivity when choosing a reliable detection method. However, recent work conducted by Ackerman et al. [50] suggests that selectivity can be retained when using LR-ECNI-MS under optimized conditions. The authors reported that optimization of the electron energy, emission current, source temperature and system pressure increased the relative abundance of molecular fragment ions [M-xH-yBr]\u2212, which can be monitored for each homologue group in place of the nonspecific bromide ions. With this method one could use isotopically labeled BDE congeners as internal standards, which cannot be used when monitoring bromide ions (79Br and 81Br) in typical LR-ECNI-MS methods. The exception is BDE 209, in which 13C-labeled BDE 209 can be used in all LR-ECNI-MS applications, since fragmentation produces a higher abundance of the [C6Br5O]\u2212 ion relative to bromide [51].\nDifferent reagent gases have also been used to increase PBDE response in LR-ECNI-MS. Typical methods use methane gas as a buffering reagent in the ionization process, but ammonia and isobutane can also be used. Eljarrat et al. [48] found that ammonia and methane gas provided similar LOD for PBDEs, but required significantly different ion source temperatures and system pressure. Ackerman et al. [50] found that isobutane resulted in a 20% increase in the abundance of molecular fragments relative to methane; however, isobutane appeared to contaminate the ion source very quickly, resulting in decreased sensitivity after only 12\u00a0h of use and was therefore not recommended for use.\nHRMS offers both good sensitivity and optimum selectivity. Alaee et al. [52] described a GC\/HRMS method for the determination of PBDEs in fish. HRMS instruments have a dual magnetic and electrostatic sector, which increases sensitivity for higher molecular weight analytes such as the higher brominated congeners. This is particularly useful for quantifying heptaBDE through decaBDE congeners with greater confidence. Additionally, HRMS allows one to use isotope dilution with 13C-labeled BDE standards, which are ideal for accurate and precise measurements. In a comparison between GC\/HR-EI-MS and GC\/LR-ECNI-MS, Thomsen et al. [49] found that both methods provided similar LOD and repeatability. And, as stated previously, HRMS methods will limit the number of potential mass interferences. Therefore, if an instrument and funds are available, HRMS methods provide the best standards for selectivity and sensitivity.\nAdditional ionization methods and detectors have also been used to measure PBDEs in environmental samples. Ikonomou [53] used GC\/HRMS with metastable atom bombardment with nitrogen gas as an alternative to electron impact ionization. This method provided less fragmentation of the parent BDE congeners and was useful for identifying the degree of bromination on unknown BDE congeners relative to conventional GC\/HR-EI-MS methods. Tandem mass spectrometers using ion traps have also been reported for the analysis of PBDEs [16, 54]. Ion traps offer the advantage of increased selectivity at a low mass resolution because the analytes are fragmented twice. This type of detection minimizes the chance of isobaric interferences and significantly reduces background noise. Ion traps typically use a collision-induced dissociation (CID) cell to fragment a precursor ion, forming a secondary fragment ion. For PBDEs, the precursor ion is typically a molecular ion [M]+or [M-Br2]+ and the secondary fragment formed from the CID is a [M-COBr]+ ion. Wang et al. [54] optimized a GC\/ion trap method for 20 BDE congeners and noted that the relative abundance of the fragment ions was dependent upon the number of ortho-substituted bromines on the congener. They also pointed out that coelution could be a potential problem for higher brominated compounds that could produce similar precursor and fragment ions and contribute to additional signals to the MS\/MS channel monitored. More work is needed to determine if other brominated compounds would be isobaric using this method.\nHR time-of-flight (TOF) mass spectrometers have also been used to determine PBDEs in environmental samples [55, 56], with detection limits comparable to those of most other MS methods. TOF methods offer the advantage of acquiring spectral data across a wide mass range without compromising sensitivity, which is often the case with quadrupole mass filters. TOF-MS can be operated in both electron impact and ECNI modes, similar to quadrupole methods. Operation of a TOF mass spectrometer in ECNI mode results in the same type of fragmentation pattern observed in quadrupole MS, and therefore offers little improvement and\/or advantage in spectral data because the bromide ions, and not molecular fragments, are found in greatest abundance. HRTOF instruments offer increased mass selectivity and also permit the estimation of elemental composition; however, like most high mass resolution instruments, TOF-MS can be expensive and require high maintenance. Additionally, Cajka et al. [56] found that concentrated samples can result in inaccurate mass determination owing to saturation of the instrument\u2019s time-to-digital converter. Therefore, TOF mass spectrometers have a limited linear range, and dilution and reanalysis would be necessary to quantify concentrated samples.\nAlternate analytical techniques\nWhile a gas chromatograph connected to a mass spectrometer is generally the method of choice for PBDE measurements, alternate methods have been investigated. Despite the limited chromatographic resolving power of LC, methods employing LC\/MS and LC\/MS-MS offer promise for PBDE determination. Debrauwer et al. [57] recently investigated the use of atmospheric pressure photoionization (APPI) using LC\/MS-MS. Most traditional LC\/MS-MS methods use electrospray ionization (ESI) or atmospheric chemical ionization; however, PBDEs do not ionize well with either of these two techniques. Using APPI however, PBDEs will ionize in both negative and positive modes, depending on the degree of bromine substitution. Positive ion mode was more sensitive towards diBDE through pentaBDE, whereas negative ion mode was more sensitive towards pentaBDE through decaBDE. APPI appears to be a softer ionization technique relative to EI as M+ ions are the most intense ions produced by the interaction of PBDEs with charged dopants (charged by photons), compared with the [M-Br2]+ ions formed in EI. Multiple reaction monitoring in the MS-MS system follows the M+ to [M-Br2]+ transition.\nThe use of ESI may be limiting for PBDEs, but the analysis of PBDE metabolites can be conducted using ESI-LC\/MS methods. Hydroxylated BDEs (OH-BDEs) have been identified in environmental samples in several studies [58\u201360] and have been observed in serum samples of rats dosed with PBDEs [61], suggesting they are products of PBDE metabolism. A method for measuring OH-BDEs using LC\/MS-MS has recently been reported [62]. LC\/MS-MS holds much promise for investigating other potential metabolic and breakdown products of PBDEs and other brominated flame retardants in general.\nThe development of comprehensive two dimensional GC (GC \u00d7 GC) has improved the GC resolving power for organohalogen determination and GC \u00d7 GC can be used to sidestep some of the coelution problems encountered in standard GC\/MS methods. The use of two columns with different separation characteristics increases the chromatographic resolving power and has been proven successful in separating PBDEs from other halogenated compounds. Koryt\u00e1r et al. [63] used a DB-1 column in combination with 65% phenylmethylspolysiloxane (007-65HT) to efficiently separate PBDEs from polychlorinated alkanes. Focant et al. [55] used GC\u00d7GC coupled to a TOF mass spectrometer to resolve 58 different halogenated compounds, including PBDEs, PCBs, bromobiphenyls and organochlorine pesticides.\nChallenges in PBDE analyses\nIncrease in the fate and transport of PBDEs in the environment has led to the development of a number of different analytical methods for measuring this class of persistent halogenated contaminants. An interlaboratory comparison exercise for the determination of PBDEs in marine sediment conducted during 2004\u20132005 [64] found that laboratory agreement in measurements of PBDEs have improved since the first PBDE intercomparison exercise was conducted in 2001 [5]. Of particular note was the improvement of BDE 209 measurements among laboratories. The development of methods to limit degradation of BDE 209 in the injection port and column of GC systems has drastically improved the accuracy and precision of measurements. With this advance, an increasing number of studies are reporting BDE 209 measurements in environmental samples [65, 66], particularly in human samples [67, 68], suggesting that BDE 209 is more diffuse and ubiquitous in the environment than originally observed.\nTheoretically, there are 209 different BDE congeners that can potentially be found in the environment. As stated previously, commercial standards are not currently available for all 209 different congeners and thus one of the major challenges in PBDE analysis is the lack of data on the prevalence of higher brominated BDE congeners in the environment. Several laboratory studies have demonstrated that PBDEs, particularly BDE 209, may debrominate both through abiotic [69, 70] and biotic [38, 39, 71\u201373] pathways, resulting in the formation of pentaBDE through nonaBDE congeners. The lack of standards has made it difficult to determine the products and elucidate the mechanisms of selective bromine loss. Very little is known about the potential for debromination in the ambient environment and few laboratories routinely measure octaBDE and nonaBDE congeners. The identification of octaBDE and nonaBDE congeners in human samples [40, 74] has led to many questions about the origin of these congeners, such as if they are a result of selective uptake of impurities in the technical mixtures, or if they are a result of debromination. Studies have attempted to identify all major and minor BDE congeners present in the commercial mixtures [29, 75]; however, the lack of standards has made congener identification difficult.\nWith the implementation of the European Union\u2019s directive on waste electrical and electronic equipment and on the restriction of the use of certain hazardous substances (including PBDEs) in electrical and electronic equipment (RoHS; 2002\/95\/EC), fast and reliable methods for determining PBDE content in electrical equipment will be needed to ensure compliance. The use of X-ray fluorescence (XRF) portable analyzers is a promising method that would allow measurement of the total bromine content of a plastic component by scanning its surface with the handheld device. Obviously there will be difficulties in determining which type of brominated flame retardants are present in materials analyzed with this instrument. However, the XRF may be useful for determining if PBDE degradation occurs after exposure to light and it may be possible to determine the percentage of bromine lost through this pathway in an easy and fast method. Future directions in PBDE research may also wish to examine the fate of the bromine atoms that could be lost from PBDEs via the aforementioned degradation\/debromination processes.\nConclusions\nIn conclusion, a variety of methods are now available to accurately and precisely measure a suite of PBDE congeners in environmental samples. The choice of method selection will involve a compromise between cost, selectivity and sensitivity, particularly since different congeners require different instrumental optimization techniques, especially BDE 209. The Environmental Protection Agency has outlined a protocol for measuring PBDEs (EPA method 1614), but to the author\u2019s knowledge, this method is still in draft form.","keyphrases":["methods","polybrominated diphenyl ethers","review","analysis","brominated flame retardants"],"prmu":["P","P","P","P","P"]}
{"id":"Mol_Genet_Genomics-4-1-2413074","title":"Aspergillus niger genome-wide analysis reveals a large number of novel alpha-glucan acting enzymes with unexpected expression profiles\n","text":"The filamentous ascomycete Aspergillus niger is well known for its ability to produce a large variety of enzymes for the degradation of plant polysaccharide material. A major carbon and energy source for this soil fungus is starch, which can be degraded by the concerted action of \u03b1-amylase, glucoamylase and \u03b1-glucosidase enzymes, members of the glycoside hydrolase (GH) families 13, 15 and 31, respectively. In this study we have combined analysis of the genome sequence of A. niger CBS 513.88 with microarray experiments to identify novel enzymes from these families and to predict their physiological functions. We have identified 17 previously unknown family GH13, 15 and 31 enzymes in the A. niger genome, all of which have orthologues in other aspergilli. Only two of the newly identified enzymes, a putative \u03b1-glucosidase (AgdB) and an \u03b1-amylase (AmyC), were predicted to play a role in starch degradation. The expression of the majority of the genes identified was not induced by maltose as carbon source, and not dependent on the presence of AmyR, the transcriptional regulator for starch degrading enzymes. The possible physiological functions of the other predicted family GH13, GH15 and GH31 enzymes, including intracellular enzymes and cell wall associated proteins, in alternative \u03b1-glucan modifying processes are discussed.\nIntroduction\nAspergillus niger is a saprophytic fungus well known for its production and secretion of a variety of hydrolytic enzymes contributing to its ability to degrade plant polysaccharides such as cellulose, hemicellulose, pectin, starch and inulin (De Vries and Visser 2001; Tsukagoshi et al. 2001; Yuan et al. 2006). Starch is the most abundant storage carbohydrate in the plant kingdom and is present in tubers, seeds and roots of a variety of crop plants including cereals, potatoes and manioc (Peters 2006). Starch is composed of two different molecules: (1) amylose, an unbranched, single chain of \u03b1-(1,4)-linked glucose residues and (2) amylopectin, consisting of a \u03b1-(1,4)-linked glucose chain with \u03b1-(1,6)-branches on every 12\u201325 glucose residues along the \u03b1-(1,4)-linked backbone (Robyt 1998). The degradation of starch is performed by a variety of enzymes, which are divided over three Glycoside Hydrolase (GH) families based on their sequence similarity (http:\/\/www.cazy.org) (Coutinho and Henrissat 1999). The first step in starch degradation is the endo-hydrolysis of the long polysaccharide chains into shorter maltooligosaccharides and \u03b1-limit dextrins by \u03b1-amylases (EC 3.2.1.1). \u03b1-Amylases belong to family GH13, a large family containing various hydrolysing and transglycosylating enzymes, mostly acting on \u03b1-(1,4) or \u03b1-(1,6)-glycosidic bonds. Members of family GH13 have a (\u03b2\/\u03b1)8 barrel structure and can be recognized by four highly conserved amino acid regions containing three catalytic residues (MacGregor et al. 2001; Nakajima et al. 1986). After endo-hydrolysis, subsequent steps in starch degradation involve exo-acting enzymes releasing glucose. This reaction is performed by glucoamylase type enzymes of family GH15 (EC 3.2.1.3), a relatively confined family with regard to enzyme specificity, as all its studied members hydrolyse either \u03b1-(1,4) or \u03b1-(1,6)-bonds to release \u03b2-glucose from the non-reducing end of maltooligosaccharides. Most GH15 enzymes described thus far possess a starch-binding domain (SBD) (Sauer et al. 2000), a discrete C-terminal region of the protein that binds to starch and facilitates hydrolysis (Southall et al. 1999). Additionally, \u03b1-(1,4)-glucosidases of family GH31 may release \u03b1-glucose from the non-reducing end of starch (EC 3.2.1.20). This family also harbours other enzyme specificities such as \u03b1-xylosidase activity.\nSeveral A.\u00a0niger enzymes involved in starch degradation, and their corresponding genes, have been characterized and isolated. A.\u00a0niger glucoamylase GlaA (family GH15) is an important enzyme for the modification of starch in the food industry (Boel et al. 1984; van Dijck et al. 2003). Additionally, one GH31 \u03b1-glucosidase (AglA, renamed AgdA) (Nakamura et al. 1997) has been characterized previously, as well as three family GH13 \u03b1-amylases: acid amylase AamA, and the almost identical AmyA and AmyB (Boel et al. 1990; Korman et al. 1990). The transcriptional regulation of the genes encoding starch-degrading enzymes has been studied in several aspergilli (Nakamura et al. 2006). In general, their expression is high on starch and induced by the presence of (iso)maltose (Tsukagoshi et al. 2001; Kato et al. 2002a). The presence of the inducer activates the Zn(II)2Cys6 transcription factor AmyR which binds to CGGN8(C\/A)GG sequences in the promoter regions of AmyR target genes thereby activating their transcription (Petersen et al. 1999; Gomi et al. 2000; Tani et al. 2001; Ito et al. 2004).\nRecent studies have indicated that some GH13 enzymes in fungi may be involved in the synthesis or modification of \u03b1-glucan in the fungal cell wall, rather than in starch degradation. The cell wall of aspergilli contains four major classes of polysaccharides: chitin, \u03b1-glucan, \u03b2-(1,3)-glucan and galactomannan (Fontaine et al. 2000; Beauvais and Latg\u00e9 2001). The \u03b1-glucan fraction identified in A.\u00a0niger consists of two types of molecules: a linear polymer with alternating \u03b1-(1,3)\/(1,4)-glycosidic bonds called nigeran (Barker and Carrington 1953) and pseudonigeran, a linear \u03b1-(1,3)-glucan molecule with some (3\u201310%) \u03b1-(1,4)-linkages (Johnston 1965; Horisberger et al. 1972). Synthesis of \u03b1-glucan is thought to be carried out by \u03b1-glucan synthase enzymes encoded by ags genes. The first putative \u03b1-d-glucan synthase encoding gene (ags1) was identified in Schizosaccharomyces pombe (Hochstenbach et al. 1998). The ags1 gene encodes a large, three-domain protein. In addition to the multi-pass transmembrane domain in the C-terminal part of the protein, two predicted catalytic domains are present. The middle domain shows strong similarity to glycogen and starch synthases in Glycosyltransferase family (GT) 5 and is predicted to be involved in the synthesis of \u03b1-glucan. The N-terminal part of the protein is similar to \u03b1-amylases and belongs to family GH13. This part of the protein is predicted to be localized extracellularly and might be involved in connecting two \u03b1-(1,3)-glucan chains (Gr\u00fcn et al. 2005). Apart from the \u03b1-glucan synthases, two types of family GH13 enzymes were recently identified in fungi to play a role in fungal cell wall biosynthesis. Marion et al. (2006) provided evidence for the involvement of a putative \u03b1-amylase (Amy1p) in the formation of \u03b1-(1,3)-glucan in the cell wall of Histoplasma capsulatum. An AMY1 knockout was unable to produce \u03b1-(1,3) glucan and showed reduced virulence. In dimorphic fungi like H. capsulatum, cell wall \u03b1-glucan is a known virulence factor (Rappleye et al. 2004; Rappleye and Goldman 2006). The second \u03b1-amylase-like enzyme Aah3p was first studied in S. pombe (Morita et al. 2006). Disruption of aah3 encoding a GPI-anchored protein resulted in hypersensitivity towards cell wall-degrading enzymes and an aberrant cell shape, indicating that normal cell wall biosynthesis was affected. Disruption of a homologous gene (agtA) in A.\u00a0niger also affected cell wall stability (van der Kaaij et al. 2007a).\nWe previously surveyed the A.\u00a0niger genome sequence to identify all GH13, GH15 and GH31 family members present in this important industrial source for amylolytic enzymes (Pel et al. 2007). This resulted in identification of a surprisingly large number of previously unknown enzymes. In this study, we have analysed their phylogeny, the presence of specific protein features and synteny with other Aspergillus species, which allowed the division of the members of each GH family into several groups. Additionally, we studied the transcriptional regulation of the genes encoding these proteins in a wild type A.\u00a0niger strain, and in a derived amyR deletion strain, during their growth on xylose and maltose. Only few of the identified proteins were induced by maltose. Expression of many of the identified groups of enzymes, including the homologues of both S. pombe Aah3p and H. capsulatum Amy1p, was not induced by maltose and was not dependent on the presence of AmyR. The possible involvement of these enzymes in cell wall \u03b1-glucan synthesis and modification is discussed.\nMaterial and methods\nDatabase mining of A.\u00a0niger genome and analysis of predicted proteins\nThe full genome sequence of A.\u00a0niger strain CBS 513.88 has been deposited at the EMBL database with accession numbers AM270980\u2013AM270998 (Pel et al. 2007) and was used for database mining. The nucleotide accession numbers of A.\u00a0niger genes, as listed in Tables\u00a01 and 2, refer to this database. Hidden Markov Model (HMM) profiles were built with the HMMER package (Durbin and Eddy 1998) (http:\/\/hmmer.wustl.edu\/) based on the amino acid sequences of known members of GH13, GH15 and GH31. Proteins belonging to these families, originating from the different kingdoms of life, were retrieved from the CAZy website at http:\/\/www.cazy.org (Coutinho and Henrissat 1999), and the protein sequences were extracted from the GenBank\/GenPept database at http:\/\/www.ncbi.nlm.nih.gov\/entrez\/ and Swiss-Prot database at http:\/\/www.expasy.org\/sprot\/. The A.\u00a0niger genome was searched with the HMM profiles using the WISE2 package (Birney et al. 2004) (http:\/\/www.ebi.ac.uk\/Wise2\/).\nTable\u00a01All members of family GH13, GH15 and GH31 identified in the genome sequence of A.\u00a0niger CBS 513.88 using HMM profiles Accession no.GeneFamilyEnzyme activityFeaturesaAmyR binding motifbProposed biological functionRefcAn11g03340aamAGH13Acid \u03b1-amylaseSS Starch degradation1An12g06930amyAGH13\u03b1-AmylaseSS+970; +252Starch degradation2An05g02100amyBGH13\u03b1-AmylaseSS+252Starch degradation2An04g06930amyCGH13\u03b1-AmylaseSS+787; +664; \u2212531Starch degradation3An09g03100agtAGH13\u03b1-GlucanotransferaseSS, GPICell wall \u03b1-glucan synthesis4An12g02460agtBGH13\u03b1-GlucanotransferaseSS, GPI+810Cell wall \u03b1-glucan synthesis4An15g07800agtCGH13Putative \u03b1-glucanotransferaseSS, GPICell wall \u03b1-glucan synthesis4An02g13240agdCGH13Putative \u03b1-glucosidase+368UnknownAn13g03710agdDGH13Putative \u03b1-glucosidaseUnknownAn01g13610amyDGH13Putative \u03b1-amylase+504; \u221232Cell wall \u03b1-glucan synthesisAn09g03110amyEGH13Putative \u03b1-amylase\u221276Cell wall \u03b1-glucan synthesisAn01g06120gdbAGH13Glycogen debranching enzyme\u2212487; +393Glycogen metabolismAn14g04190gbeAGH13Glycogen branching enzymeGlycogen metabolismAn04g09890agsAGH13Putative \u03b1-glucan synthaseSSCell wall \u03b1-glucan synthesis5An15g07810agsBGH13Putative \u03b1-glucan synthaseSS+287Cell wall \u03b1-glucan synthesis5An12g02450agsCGH13Putative \u03b1-glucan synthaseSS\u2212973; +622, \u2212185Cell wall \u03b1-glucan synthesis5An02g03260agsDGH13Putative \u03b1-glucan synthaseSSCell wall \u03b1-glucan synthesis5An09g03070agsEGH13Putative \u03b1-glucan synthaseSSCell wall \u03b1-glucan synthesis5An03g06550glaAGH15GlucoamylaseSS, SBD\u2212792; \u2212669; +423; \u2212301Starch degradation6An12g03070glaBGH15Putative glucoamylase\u2212878UnknownAn04g06920agdAGH31\u03b1-GlucosidaseSS+574; +191, Starch degradation7An01g10930agdBGH31Putative \u03b1-glucosidaseSS+904; \u2212334Starch degradationAn09g05880agdEGH31Putative \u03b1-glucosidase IISSProtein glycosylationAn18g05620agdFGH31UnknownUnknownAn07g00350agdGGH31UnknownSS+402UnknownAn09g03300axlAGH31Putative \u03b1-xylosidaseSS+126Xyloglucan degradationAn01g04880axlBGH31Putative \u03b1-xylosidase+430; +138, Xyloglucan degradationThe newly identified proteins are indicated in boldaSS\u00a0predicted N-terminal Signal Sequence; GPI\u00a0\u00a0predicted Glycosylphosphatidylinositol anchor signal; SBD\u00a0\u00a0predicted starch-binding domainbThe presence of consensus AmyR binding motif (CGGN8(A\/C)GG) was analysed in the promoter region up to 1\u00a0kb upstream of the start codoncReferences: 1 (Boel et al. 1990); 2 (Korman et al. 1990); 3 (R. M. Van der Kaaij and X. L. Yuan, unpublished). ; 4 (van der Kaaij et al. 2007); 5 (Damveld et al. 2005); 6 (Boel et al. 1984); 7\u00a0(Nakamura et al. 1997)Table\u00a02Functionally described family GH13 and GH31 members from other organisms, used for the multiple sequence alignments in Fig.\u00a01Accession no.NameFamilyEnzyme activityFeaturesaBiological functionOrganismRefbBAA78714 AndGbe1GH13Glycogen branching enzymeGlycogen metabolismA.\u00a0nidulans1BAA34996ScGdb1GH13Glycogen debranching enzymeGlycogen metabolismS. cerevisiae2P19571BsAmyAGH13\u03b1-AmylaseSSStarch degradationBacillus sp.3CAA54266BsAglAGH13\u03b1-GlucosidaseStarch degradationBacillus sp.4CAA21237 SpAah1GH13UnknownSS, GPI\u03b1-Glucan biosynthesisS. pombe5CAA91249SpAah2GH13UnknownSS, GPI\u03b1-Glucan biosynthesisS. pombe5CAB40006SpAah3GH13UnknownSS, GPI\u03b1-Glucan biosynthesisS. pombe5CAA16864SpAah4GH13UnknownSS, GPI\u03b1-Glucan biosynthesisS. pombe5ABK62854HcAmy1GH13Unknown\u03b1-Glucan biosynthesisH. capsulatum6ABF50883AN7345.2GH31\u03b1\/\u03b2-GlucosidaseSSStarch\/cellulose degradationA.\u00a0nidulans7ABF50846AN7505.2GH31\u03b1-XylosidaseXylan degradationA.\u00a0nidulans7BAB39856AndAgdBGH31\u03b1-GlucosidaseSSStarch degradationA.\u00a0nidulans8AAU87580 TrAguIIGH31\u03b1-Glucosidase IISSProtein glycosylationT. reesei9A45249CAMAL2GH31MaltaseMaltose degradationC. albicans10For each A.\u00a0niger protein identified, a functionally or biochemically characterized protein with the highest similarity was used in the phylogenetic analysis aSS\u00a0\u00a0predicted N-terminal Signal Sequence; GPI\u00a0\u00a0predicted Glycosylphosphatidylinositol anchor signalbReferences: 1 (Sasangka et al. 2002); 2 (Teste et al. 2000); 3 (Tsukamoto et al. 1988); 4 (Nakao et al. 1994); 5 (Morita et al. 2006); 6 (Marion et al. 2006); 7 (Bauer et al. 2006); 8 (Kato et al. 2002b); 9 (Geysens et al. 2005); 10 (Geber et al. 1992)\nThe presence of signal peptidase cleavage sites, glycosylphosphatidylinositol (GPI-) attachment sites and SBD in the obtained sequences were predicted by Web-based tools at URL: http:\/\/www.cbs.dtu.dk\/services\/SignalP\/ (Bendtsen et al. 2004), URL: http:\/\/mendel.imp.ac.at\/sat\/gpi\/gpi_server (Eisenhaber et al. 2004), and URL: http:\/\/www.ncbi.nlm.nih.gov\/BLAST\/ (Marchler-Bauer and Bryant 2004), respectively.\nMultiple sequence alignments of GH13, 15 and 31 family members were performed using DNAMAN version 4.0 (Lynnon BioSoft, Canada). The alignments were based on the full length of the predicted proteins, except in case of predicted \u03b1-glucan synthases for which only the N-terminal part, encoding the family GH13 domain, was used for the alignment. The phylogenetic relationship was calculated by using Optimal Alignment (Thompson et al. 1994) with gap opening penalty and gap extension penalty of 10 and 0.05, respectively. A bootstrapped test of phylogeny was performed by the Neighbour-Joining method using 1,000 replicates. Wherever possible, one protein with described activity was included for each of the groups identified based on phylogenetic analysis.\nStrains and transformations\nThe wild type A.\u00a0niger strain used in this study is N402, a cpsA1 derivative of A.\u00a0niger van Tieghem (CBS 120.49, ATCC 9029) (Bos et al. 1988). Strain AB4.1 is a pyrG negative derivative of N402 (van Hartingsveldt et al. 1987) and was used to construct the amyR disruption strain. A.\u00a0niger strains were grown in Aspergillus minimal medium (MM) (Bennett and Lasure 1991), or Aspergillus complete medium (CM) consisting of MM with the addition of 0.5% (w\/v) yeast extract and 0.1% (w\/v) casamino acids. Growth medium was supplemented with 10\u00a0mM uridine (Serva, Germany) when required. Transformation of A.\u00a0niger AB4.1 was performed as described earlier (Punt and van den Hondel 1992) using lysing enzymes (L1412, Sigma, USA) for protoplastation. The bacterial strain used for transformation and amplification of recombinant DNA was Escherichia coli XL1-Blue (Stratagene, USA). Transformation of XL1-Blue was performed according to the heat shock protocol (Inoue et al. 1990).\nDisruption of the maltose utilization activator amyR in A.\u00a0niger\nPlasmid pJG01 containing the A.\u00a0nigeramyR gene as a 4.3\u00a0kb NsiI fragment in pGEM11 was kindly provided by P. van Kuyk (Wageningen University, the Netherlands) and was used to disrupt the amyR gene. The construction of the amyR deletion cassette was performed as follows. The BamHI\u2013EcoRI fragment and NsiI\u2013SalI fragment flanking the amyR ORF at the 5\u2032 and 3\u2032 region, respectively, were isolated from pJG01. The isolated NsiI\u2013SalI fragment was cloned into pUC19 to obtain plasmid pAmyRF3. Subsequently, a BamHI\u2013SalI fragment carrying the A. oryzaepyrG gene, obtained from plasmid pAO4-13 (de Ruiter-Jacobs et al. 1989) was inserted into pAmyRF3 which resulted in plasmid pAmyRF3-pyrG. The BamHI\u2013EcoRI fragment isolated from pJG10 was ligated into pAmyRF3-pyrG resulting in the amyR deletion plasmid (p\u0394amyR). Prior to transformation to AB4.1, p\u0394amyR was linearized with EcoRI. Uridine prototrophic transformants were selected by their ability to grow on MM without uridine. After two rounds of purification, transformants were tested for their ability to grow on starch. Approximately 10% of the pyrG+ transformants showed defective growth on MM agar plates containing starch as sole carbon source. Six independent putative amyR deletion strains (YvdM1.1-1.6) with identical phenotypes were obtained. Southern blot analysis confirmed proper deletion and a single integration of the amyR disruption cassette at the amyR locus. Strain YvdM1.1 was used for further analysis and we will refer to this strain as the \u0394amyR strain in the remaining of this paper.\nCulture conditions, RNA preparation, microarray experiments and data analysis\nRNA extracted from the A.\u00a0niger \u0394amyR strain and its parental strain (N402) grown on different carbon sources were used for microarray experiments using custom-made \u2018dsmM_ANIGERa_coll\u2019 Affymetrix GeneChip\u00ae Microarrays kindly provided by DSM Food Specialties (Delft, The Netherlands). All experiments for each growth condition (culturing the mycelia, RNA extractions and microarray hybridizations) were performed twice as independent biological experiments.\nAspergillus niger spores (2\u00a0\u00d7\u00a0106\u00a0spores\u00a0ml\u22121) were inoculated in 250\u00a0ml MM supplemented with 2% (w\/v) xylose (Sigma) and 0.1% (w\/v) casamino acids and grown for 18\u00a0h at 30\u00b0C on a rotary shaker at 300\u00a0rpm. The mycelium was harvested by suction over a nylon membrane and washed with MM without carbon source. Aliquots of 1.6\u00a0g wet weight of mycelium were transferred to 300\u00a0ml Erlenmeyer flasks containing 70\u00a0ml MM supplemented with 1% (w\/v) carbon source [maltose (Sigma) or xylose] and incubated at 30\u00b0C for a further 2 or 8\u00a0h. The pH of all cultures grown for 2\u00a0h was equal to the pH at the time of transfer (pH 6.2). Cultures grown for 8\u00a0h were buffered at pH 4 by the addition of 100\u00a0mM of citric acid\/sodium citrate to allow comparison between the N402 and the \u0394amyR strain at this time point. The mycelium was harvested over Miracloth filter, frozen in liquid nitrogen and stored at \u221280\u00b0C. Total RNA was isolated from mycelia using TRIzol reagent (Invitrogen) and RNA quality was verified by analyzing aliquots with glyoxal\/DMSO gel electrophoresis and Agilent Bioanalyzer \u201cLab on chip\u201d system (Agilent Technologies, USA). Processing, labeling and hybridization of cRNA to A.\u00a0niger Affymetrix GeneChips were performed according to the corresponding Affymetrix protocols for \u201cEukaryotic Target Preparation\u201d and \u201cEukaryotic Target hybridization\u201d. For probe array washing and staining, the protocol \u201cAntibody Amplification for Eukaryotic Targets\u201d was followed. Hybridized probe array slides were scanned with Agilent technologies G2500A Gene Array Scanner at a 3\u00a0\u03bcm resolution and a wavelength of 570\u00a0nm. Affymetrix Microarray Suite software MAS5.0 was used to calculate the signal and P-values and to set the algorithm\u2019s absolute call flag, which indicates the reliability of the data points according to P (present), M (marginal) and A (absent). The data on each chip were globally scaled to an arbitrary target gene intensity of 500. The complete microarray data were deposited into ArrayExpress with an accession E-TABM-324 at http:\/\/www.ebi.ac.uk\/miamexpress.\nThe pre-scaled data from each hybridization experiment was then normalized for statistical analysis using Genespring 7.0 software (Silicon Genetics, USA). The per chip normalization was performed to ensure that the overall characteristic of the expression distribution such as median should be the same for all the chips. For the genome-wide analysis, we focused on maltose-induced genes and therefore a pre-filtering of data was performed to select for genes whose detection calls are present in both maltose duplicate samples in the wild-type strain (N402). The selected dataset was used to perform a one-way ANOVA analysis under the test type of \u201cparametric test, don\u2019t assume variances equal\u201d. Fold changes in expression between two different conditions were then computed for genes with P\u00a0<\u00a00.08 based on one-way ANOVA analysis.\nResults\nIdentification of glycoside hydrolase family 13, 15 and 31 genes in the A.\u00a0niger CBS 513.88 genome sequence\n\u03b1-Amylases, glucoamylases and \u03b1-glucosidases, members of families GH13, 15 and 31, respectively, are the three main types of enzymes involved in breakdown of starch by aspergilli (Tsukagoshi et al. 2001). To identify all genes encoding enzymes that might play a role in starch utilization, or other \u03b1-glucan modifying processes in A.\u00a0niger, the genome of A.\u00a0niger CBS 513.88 was searched with HMM profiles based on known enzymes from families GH13, 15 and 31. This resulted in the retrieval of a total of 27 protein sequences including 17 previously unknown proteins (Pel et al. 2007), as listed in Table\u00a01. The predicted proteins were annotated based on their similarity to known enzymes. Gene names were assigned based on this annotation and do not necessarily match the activity of the enzymes, as this is often unknown.\nTwo approaches were combined to predict putative functions in cellular processes for this surprisingly large number of newly identified proteins. First, phylogenetic trees were constructed using the GH13, GH15 and GH31 family members identified in the A.\u00a0niger genome, as well as functionally characterized proteins from other organisms with similarity to the identified A.\u00a0niger proteins (Fig.\u00a01). Second, using DNA microarrays, the expression of all the A.\u00a0niger genes encoding GH13, GH15 and GH31 enzymes was examined in both the A.\u00a0niger wild type strain N402 and the \u0394amyR strain derived, after growth on different carbon sources (Fig.\u00a02; Table\u00a0S1). Both the N402 and the \u0394amyR strains were pregrown in xylose for 18\u00a0h, and mycelia were transferred to either xylose or maltose media and grown further for 2 or 8\u00a0h. Expression levels were determined based on geometric mean data of biological duplicate samples. We will discuss each enzyme family in detail and combine the findings in A.\u00a0niger with the predicted proteins present in the genomes of A.\u00a0fumigatus (Nierman et al. 2005), A.\u00a0nidulans (Galagan et al. 2005) and A. oryzae (Machida et al. 2005).\nFig.\u00a01Bootstrapped phylogenetic tree of A.\u00a0niger GH13 (a) and GH31 (b) enzymes and several closest homologues from other species. Newly identified proteins in the genome of A.\u00a0niger are shown in bold. A description of each protein is listed in Tables\u00a01 and 2. Bootstrap values are indicated on the node of each branch. The tree was created with DNAMAN 4.0 using gap and extension penalties of 10 and 0.5, respectively. The scale bar corresponds to a genetic distance of 0.05 substitution per positionFig.\u00a02Expression profiles of A.\u00a0niger family GH13, 15 and 31 enzymes. Accession numbers of the gene names are given in Tables\u00a01 and 2. Strain and time points after transfer from the preculture are indicated on the right-hand side. The numeric values and Present\/Absent calls from the expression data are provided as Supplementary Tables\u00a01, 2 \nIdentification and transcriptional regulation of GH13 family members\nThe HMMer search for family GH13 enzymes in the A.\u00a0niger genome resulted in the identification of 18 protein sequences of which 10 had not been identified previously (Table\u00a01). Table\u00a03 displays the four conserved regions typical for family GH13 proteins as identified in these enzymes. A phylogenetic tree was produced combining the A.\u00a0niger family GH13 enzymes with several functionally characterized GH13 family proteins from other organisms (Fig.\u00a01). The combination of this phylogenetic analysis with a functional annotation of the proteins revealed six recognizable subgroups.\nTable\u00a03Alignment of the four conserved regions of all family GH13 enzymes identified in A.\u00a0niger, as well as in four Aah proteins from S. pombe and Amy1p from H.\u00a0capsulatumThe seven residues generally conserved in family GH13 are indicated in bold and the three catalytic residues are additionally underlined. The group to which the proteins are assigned, as described in this paper, is indicated\nGroup I consists of four extracellular \u03b1-amylases. Three of these are the previously characterized extracellular \u03b1-amylases acid-amylase (AamA) and \u03b1-amylases AmyA and AmyB (Korman et al. 1990). One new extracellular \u03b1-amylase was identified and named AmyC. This protein displays high similarity with the known A.\u00a0niger \u03b1-amylases (74% identity to AmyA and AmyB, 65% identity to AamA). The amyC gene is located in a gene cluster also containing an \u03b1-glucosidase gene (agdA) and the amyR gene encoding the AmyR transcription factor. Expression of aamA in A.\u00a0niger N402 was not detectable in xylose media, but was strongly induced in the presence of maltose (Fig.\u00a02). The expression of aamA was reduced to a non-detectable level in the \u0394amyR strain. Expression of amyA and amyB was not detected in A.\u00a0niger N402 in any of the conditions tested (Fig.\u00a02) (see Discussion). The expression of the newly identified amyC gene was relatively low compared to the aamA gene. At 2\u00a0h after transfer from the preculture, the expression level of amyC was independent of the carbon source, while after 8\u00a0h the expression level was reduced 3-fold on xylose compared to maltose. Additionally, the expression on maltose was reduced 2- to 3-fold in the \u0394amyR strain (Table\u00a0S1). The presence of three putative AmyR-binding elements in the promoter region of amyC further suggests that its expression is controlled by AmyR (Table\u00a01).\nThe A.\u00a0niger acid amylase (encoded by aamA) in the CBS 513.88 strain does not contain a predicted SBD, whereas its homologues in A.\u00a0nidulans, A.\u00a0fumigatus and other aspergilli contain a full-length SBD (Table\u00a0S2). In addition, the presence of a functional SBD in the AamA protein of A.\u00a0niger N402 was suggested because of the purification of AamA via its SBD from culture fluid of a N402 \u0394glaA strain, and subsequent demonstration of the SBD with specific antibodies (M. F. Coeffet-Le Gal and D. Archer, personal communication, University of Nottingham, UK). We therefore PCR amplified the aamA gene and its 3\u2032 flanking regions using N402 genomic DNA as a template, determined its DNA sequence and compared it to the aamA gene and its 3\u2032 flanking region from CBS 513.88. The comparison revealed that the aamA gene of A. niger strain N402 does include an SBD and that the CBS513.88 strain harbours a deletion of 230 nucleotides right after the part encoding the (\u03b2\/\u03b1)8 barrel, causing a frame shift and the introduction of a stop codon resulting in a truncated protein (Fig.\u00a0S2). After the deletion, the DNA sequence continues by encoding part of a predicted SBD, but this part is not translated (Fig.\u00a0S2). The SBD in AamA found in the A. niger N402 strain is also predicted to be present in A. niger strain ATCC 1015 (Baker 2006).\nGroup II contains three putative GPI-anchored enzymes, recently identified as \u03b1-glucanotransferases, and named AgtA, AgtB and AgtC, respectively (van der Kaaij et al. 2007a). This subgroup of proteins is characterized by the presence of two hydrophobic signal sequences. The N-terminal signal sequence is predicted to serve for translocation to the endoplasmic reticulum (ER), whereas the C-terminal sequence is predicted to be replaced by a preassembled glycosylphosphatidylinositol (GPI) anchor in the ER (Orlean 1997). The three enzymes cluster together with the \u03b1-amylases in the phylogenetic tree (Fig.\u00a01a), but can be distinguished from the \u03b1-amylases by their catalytic domains which are clearly different from the consensus sequence for the \u03b1-amylase family. In all three proteins, one or two highly conserved histidines in conserved regions I and IV are replaced by other hydrophilic residues (Table\u00a03). Family GH13 members without these histidine residues, which are part of the active site, are very rare (Uitdehaag et al. 1999). Mutation of these residues generally results in reduced activity or altered reaction specificity of the enzymes (Chang et al. 2003; Leemhuis et al. 2004). Interestingly, both the conserved His-residues in Regions I and IV are also missing in all (putative) \u03b1-glucan synthases (Table\u00a03). Other aspergillus genomes harbour two or three Agt homologues (Fig.\u00a0S1A; Table\u00a0S2), all sharing the aberrant conserved regions and predicted GPI-anchoring. In the A.\u00a0niger genome, both agtB and agtC are located next to genes encoding putative \u03b1-glucan synthases, and this arrangement of genes is conserved in other aspergilli. The agtA gene is constitutively expressed in both the wild type strain N402 and the \u0394amyR strain under all growth conditions examined (Fig.\u00a02). Expression of agtB was only detected 8\u00a0h after transfer, regardless of the carbon source and independent of AmyR, while expression of agtC was not detected.\nGroup III consists of two putative, intracellular \u03b1-glucosidases, named AgdC (An02g13240) and AgdD (An13g03710). The protein sequences contained all residues commonly conserved in the \u03b1-amylase superfamily (Table\u00a03). The predicted intracellular proteins lack clear similarity to any previously characterized fungal protein, although similar enzymes are predicted in A. oryzae, A.\u00a0nidulans and A.\u00a0fumigatus (Fig.\u00a0S1A). Their most related functionally characterized homologue is an \u03b1-glucosidase from Bacillus sp. SAM1606 (Nakao et al. 1994). Expression of agdC was low and not induced on maltose, and expression of agdD was not observed (Fig.\u00a02).\nGroup IV contains two putative intracellular \u03b1-amylases, named AmyD (An01g13610) and AmyE (An09g03110), which share 55% identity. AmyD was recently characterized as an \u03b1-amylase with low-hydrolyzing activity on starch and related substrates (Van der Kaaij et al. 2007b). AmyD and AmyE are similar to a recently identified protein Amy1p from H. capsulatum, which was shown to be involved in cell wall \u03b1-glucan synthesis. The latter protein has not been characterized biochemically. Functionally characterized enzymes with similarity to this cluster therefore included only bacterial enzymes, of which maltohexaose-forming \u03b1-amylase of alkalophilic Bacillus sp. #707 (Tsukamoto et al. 1988) had the highest similarity. No significant expression of amyD or amyE was detected in our experiments (Fig.\u00a02). Predicted enzymes highly similar to A.\u00a0niger AmyD and AmyE were also present in other Aspergillus species (Fig.\u00a0S1A; Table\u00a0S2). Like the A.\u00a0niger amyE gene, the orthologues in A.\u00a0nidulans (AN3309.3) and in A. oryzae (AO003001497) are clustered in the genome with genes encoding \u03b1-glucanotransferases and \u03b1-glucan synthases (Table\u00a0S2).\nThe two A.\u00a0niger proteins in Group V could be reliably annotated as enzymes involved in glycogen metabolism: glycogen branching enzyme (GbeA) and glycogen debranching enzyme (GdbA). A homologue for each of these enzymes is present in the other Aspergillus genome sequences. Transcriptional analysis in A.\u00a0niger showed that both genes were expressed both on xylose and maltose, and that their expression was unaffected in the \u0394amyR strain (Fig.\u00a02).\nGroup VI contains the five predicted \u03b1-glucan synthase genes (Damveld et al. 2005). The derived proteins are highly similar (66\u201383%) to each other, and all contain the two catalytic domains (GT5 and GH13) characteristic for these proteins. Both agsB and agsC are clustered in the genome with genes encoding the aforementioned \u03b1-glucanotransferases agtC and agtB, respectively. In both cases, the direction of transcription of the pair of genes is such that they can be transcribed from their intergenic region. Expression analysis in our microarray data collection, which includes data from various time points on several carbon sources (glucose, maltose, xylose, inulin, sucrose) indicate that agsC and agtB are co-expressed at a later growth stage, independent of the carbon source (data not shown). Expression of both agsB and its neighbouring gene agtC was not detected in any of the growth conditions. The expression levels of agsA and agsD were very low, or not detectable. AgsE was highly expressed in all experiments independent of AmyR (Fig.\u00a02). As expected, several proteins with high similarity to the A.\u00a0niger \u03b1-glucan synthases are predicted from the other Aspergillus genomes, although the number of homologues present is highest in A.\u00a0niger (Fig.\u00a0S1A; Table\u00a0S2).\nIdentification and transcriptional regulation of GH15 family members\nAn HMMer search for GH15 family members in the A.\u00a0niger genome returned two predicted proteins: the previously described glucoamylase GlaA (Boel et al. 1984) and an unknown predicted protein named GlaB (An12g03070). The GlaB protein lacks both an N-terminal signal sequence and an SBD, and displays a low similarity of 26% with GlaA. These two types of family GH15 enzymes are also recognized in other aspergilli studied (Fig.\u00a0S1B). All predicted proteins similar to GlaB lack both a signal for secretion and an SBD, features typically present in the fungal GH15 enzymes described to date. A.\u00a0niger glaA was expressed on xylose and strongly induced on maltose, as described previously (Fowler et al. 1990). The induction of glaA was AmyR dependent. Expression of glaB was not detected in any of the conditions tested (Fig.\u00a02).\nIdentification and transcriptional regulation of GH31 family members\nHMMer searches in the genome of A.\u00a0niger revealed the presence of seven GH31 family members, of which only one was previously identified as an \u03b1-glucosidase (aglA) (Nakamura et al. 1997). We propose to name all (putative) \u03b1-glucosidases in A.\u00a0niger Agd enzymes, similar to the nomenclature in A.\u00a0nidulans, and to rename AglA as AgdA, to prevent confusion with \u03b1-galactosidases (den Herder et al. 1992). The phylogenetic tree (Fig.\u00a01b) shows the presence of (at least) four subgroups within family GH31. In the group of the \u03b1-glucosidases, AgdA clusters with AgdB, a predicted extracellular protein. AgdB has some similarity to two A.\u00a0nidulans enzymes: AN7345.2 (62% identity) with both \u03b1- and \u03b2-glucosidase activity (Bauer et al. 2006) and \u03b1-glucosidase B (AndAgdB\/ AN8953.3, 53% identity), with strong transglycosylation activity (Kato et al. 2002b). However, none of the true orthologues of AgdB has been characterized (Fig.\u00a0S1C). Transcript analysis revealed that agdB was regulated similar to agdA, as the strong induction of both genes in the presence of maltose was dependent on the presence of amyR. However, where most AmyR-regulated genes (aamA, glaA and agdA) reached their highest level of induction after 8\u00a0h growth on maltose, the expression level of agdB was decreased after 8\u00a0h compared to 2\u00a0h (Fig.\u00a02).\nFrom the remaining five GH31 family members, AgdE (An09g05880) shows similarity to Trichoderma reesei glucosidase II (TrAguII) (Geysens et al. 2005). This type of \u03b1-glucosidases is located in the ER where it is involved in the trimming of \u03b1-(1,3)-linked glucose residues from N-glycan core structure Glc3Man9GlcNAc2, which may be attached to proteins designated to be secreted (Geysens et al. 2005). AgdE contains a predicted signal sequence which might serve to direct the protein to the ER. The agdE gene was constitutively expressed in all conditions tested (Fig.\u00a02), consistent with its predicted function as an \u03b1-glucosidase II. Two additional GH31 family members, AgdF (An18g05620) and AgdG (An07g0350), lack similarity to any functionally described proteins. Expression of both genes was not detected in any of the tested growth conditions.\nThe two final family GH31 members, named AxlA and AxlB, are highly similar to AN7505.2 from A.\u00a0nidulans, which was recently characterized as an \u03b1-xylosidase (Bauer et al. 2006). Gene axlA was highly expressed in the presence of xylose, whereas no expression was detected in maltose grown cultures (Fig.\u00a02). The high expression of axlA on xylose further supports its putative function as a xylanolytic enzyme. The gene encoding the putatively intracellular AxlB was expressed at a very low level independent of the carbon source (Fig.\u00a02).\nA BLASTP search in the genomes of A.\u00a0fumigatus, A.\u00a0nidulans and A.\u00a0oryzae for family GH31 enzymes resulted in a similar collection of enzymes as identified in A.\u00a0niger (Fig.\u00a0S1C). Several clusters of orthologous proteins are distinguishable, but the assignment of an enzymatic activity to these clusters is not yet possible due to a lack of well studied homologues for these enzymes, thus requiring biochemical studies in our future work.\nGenome-wide analysis of AmyR dependent maltose induced genes using microarrays\nThe expression analysis of genes encoding family GH13, GH15 and GH31 enzymes in the A.\u00a0niger genome revealed that the expression of only a limited number of genes was induced by maltose in an AmyR dependent way. In fact, only four genes (aamA, glaA, agdA, and agdB) showed the predicted expression pattern for genes encoding enzymes involved in the breakdown of starch (Fig.\u00a02). To identify additional genes with a possible role in starch metabolism, the expression of all 14,509 predicted open reading frames on the Affymetrix microarray chips was analysed. Comparison of the transcriptome of the wild type strain (N402), grown for 2\u00a0h after transfer on either xylose or maltose, identified 634 genes that were significantly induced by maltose (One-way ANOVA analysis P\u00a0<\u00a00.08 and >2-fold change in expression level). A set of 12 genes was expressed >2-fold higher in N402 compared to the \u0394amyR strain (One-way ANOVA analysis P\u00a0<\u00a00.08) after the transfer of these strains from xylose to maltose. Combining the two gene sets resulted in a collection of six genes that were >2-fold induced on maltose in an AmyR-dependent manner (Fig.\u00a03, Tables\u00a04, 5). Five of these were assigned to the category of carbohydrate transport and metabolism according to FunCat (Ruepp et al. 2004), including the three genes encoding known extracellular starch degrading enzymes (aamA, glaA and agdA) and a putative \u03b1-glucosidase (agdB) (Table\u00a04). Gene An15g03940, encoding a protein with high similarity (68%) to a Candida intermedia glucose\/xylose symporter (Leandro et al. 2006), was 2.3-fold induced by maltose; this induction was not observed in the \u0394amyR strain. The sixth induced gene (An11g02550) encodes a protein highly similar (72%) to Kluyveromyces lactis phosphoenolpyruvate carboxykinase (Kitamoto et al. 1998) which functions in gluconeogenesis by catalyzing the conversion of oxaloacetate into phosphoenolpyruvate. It should be noted that the pH of the growth medium remained 6.2 during the 2\u00a0h growth period after transfer.\nFig.\u00a03Results of microarray analysis for maltose induced and AmyR dependent genes. Venn-diagram showing the number of genes induced on maltose compared to xylose in A.\u00a0niger N402, and the number of genes induced in N402 compared to the \u0394amyR deletion strain. The number of genes both induced by maltose and dependent on AmyR is indicated in bold. The maltose induced and AmyR dependent genes which are present in both 2 and 8\u00a0h after transfer from a preculture are shown in the boxTable\u00a04Overview of six maltose induced and AmyR dependent genes in A.\u00a0niger at 2\u00a0h (pH 6.2) after transfer from precultureORF no.aN402 maltosebCallcN402 xyloseCall\u0394amyR maltoseCallFold inductionP-valuePutative functionMaltose vs xyloseN402 vs \u0394amyRCarbohydrate transport and metabolismAn11g033403.15\u00a0\u00b1\u00a00.12P0.08\u00a0\u00b1\u00a00.04A0.13\u00a0\u00b1\u00a00.09A38.624.20.080Acid alpha-amylase AamAAn04g0692085.96\u00a0\u00b1\u00a022.54P6.24\u00a0\u00b1\u00a00.98P5.03\u00a0\u00b1\u00a00.03P13.817.10.064Extracellular alpha-glucosidase AgdAAn01g1093096.50\u00a0\u00b1\u00a031.50P10.47\u00a0\u00b1\u00a02.59P8.35\u00a0\u00b1\u00a02.13P9.211.50.062Extracellular alpha-glucosidase AgdBAn03g06550117.80\u00a0\u00b1\u00a043.16P13.78\u00a0\u00b1\u00a04.14P5.85\u00a0\u00b1\u00a00.47P8.520.10.072Glucoamylase GlaA An15g03940152.30\u00a0\u00b1\u00a019.00P65.74\u00a0\u00b1\u00a03.04P70.59\u00a0\u00b1\u00a02.88P2.32.20.074Putative monosaccharide transporterEnergyAn11g0255058.87\u00a0\u00b1\u00a03.86P10.13\u00a0\u00b1\u00a01.41P25.27\u00a0\u00b1\u00a05.35P5.82.30.033Putative phosphoenolpyruvate carboxykinase aGene name in bold indicates that the gene is >2-fold induced on maltose in an AmyR dependant way after both 2 and 8\u00a0hbThe expression level was based on the geometric mean value of the duplicate samples, and the deviation values between the duplicate samples are indicated. The P-value was based on 1-way ANOVA analysiscP, M or A represent detection calls for present, marginal or absent, respectively. Genes were divided over different functional groups according to FunCat (Ruepp et al. 2004)Table\u00a05Overview of the 18 maltose induced and AmyR dependent genes in A.\u00a0niger at 8\u00a0h (pH4.0) after transfer from precultureORF no.aN402 maltosebCallcN402 xyloseCall\u0394amyR maltoseCallFold inductionP-valuePutative functionMaltose vs xyloseN402 vs \u0394amyRCarbohydrate transport and metabolismAn11g0334065.26\u00a0\u00b1\u00a07.48P0.58\u00a0\u00b1\u00a00.57A0.08\u00a0\u00b1\u00a00.01A113.0808.40.004Acid alpha-amylase AamAAn01g1093049.46\u00a0\u00b1\u00a03.42P2.37\u00a0\u00b1\u00a00.08P6.84\u00a0\u00b1\u00a01.99P20.97.20.006Extracellular alpha-glucosidase AgdBAn04g06920179.10\u00a0\u00b1\u00a013.90P9.89\u00a0\u00b1\u00a03.63P3.36\u00a0\u00b1\u00a00.46P17.651.70.012Extracellular alpha-glucosidase AgdAAn02g0354062.79\u00a0\u00b1\u00a08.36P8.81\u00a0\u00b1\u00a00.61P0.96\u00a0\u00b1\u00a00.82P, A6.761.90.012Putative hexose transport protein MstCAn15g03940144.40\u00a0\u00b1\u00a06.80P35.93\u00a0\u00b1\u00a02.72P3.04\u00a0\u00b1\u00a00.03P, M4.047.60.003Putative monosaccharide transporterAn03g06550239.30\u00a0\u00b1\u00a02.05P83.03\u00a0\u00b1\u00a04.35P19.03\u00a0\u00b1\u00a01.75P2.912.60.012Glucoamylase GlaA An04g069304.36\u00a0\u00b1\u00a00.50P1.50\u00a0\u00b1\u00a00.16P1.40\u00a0\u00b1\u00a00.03P2.93.10.062Extracellular alpha-amylase AmyCAn09g0481018.47\u00a0\u00b1\u00a00.44P6.46\u00a0\u00b1\u00a00.35P2.52\u00a0\u00b1\u00a00.02P2.97.40.003Putative hexose transporterAn12g07450137.60\u00a0\u00b1\u00a013.00P51.57\u00a0\u00b1\u00a011.82P52.50\u00a0\u00b1\u00a06.31P2.72.60.064Sugar\/H+ symporter MstAEnergyAn03g062704.72\u00a0\u00b1\u00a00.29P1.59\u00a0\u00b1\u00a00.14A0.20\u00a0\u00b1\u00a00.25A3.325.60.035Putative isoamyl alcohol oxidase An16g060105.18\u00a0\u00b1\u00a01.55P1.80\u00a0\u00b1\u00a00.39P0.22\u00a0\u00b1\u00a00.02A2.923.80.032Putative phosphoglycerate mutaseAmino acid transport and metabolismAn03g002808.35\u00a0\u00b1\u00a00.90P3.57\u00a0\u00b1\u00a00.74P, A0.12\u00a0\u00b1\u00a00.06A2.368.00.050Similarity to tyrosinase protein Cell rescue, defence and virulence An14g057303.99\u00a0\u00b1\u00a00.59P0.23\u00a0\u00b1\u00a00.03A1.37\u00a0\u00b1\u00a00.73P, A18.83.20.037Similarity to integral membrane protein An03g002905.96\u00a0\u00b1\u00a00.89P1.68\u00a0\u00b1\u00a00.47P0.64\u00a0\u00b1\u00a00.21P3.59.30.068Similarity to integral membrane protein An06g004908.14\u00a0\u00b1\u00a00.89P3.81\u00a0\u00b1\u00a00.30P, A2.23\u00a0\u00b1\u00a00.44A2.13.70.066Similarity to integral membrane proteinProtein fateAn18g042605.44\u00a0\u00b1\u00a00.40P2.57\u00a0\u00b1\u00a00.07P1.77\u00a0\u00b1\u00a00.28M, A2.13.10.056Similarity to UDP-galactose transporterUnclassifiedAn16g0129015.04\u00a0\u00b1\u00a00.28P5.22\u00a0\u00b1\u00a01.69P, A1.97\u00a0\u00b1\u00a00.26A2.66.90.027Unknown An09g0613012.62\u00a0\u00b1\u00a02.51P5.41\u00a0\u00b1\u00a00.93P2.69\u00a0\u00b1\u00a00.30P, A2.34.70.059Unknowna,b,cSee footnotes of Table\u00a04\nA similar comparative analysis was performed for samples taken for cultures that had grown for 8\u00a0h after the transfer. To prevent differences in pH between the N402 and the \u0394amyR strain after 8\u00a0h of growth, the pH of the medium was buffered to 4.0 using 100\u00a0mM of citric acid\/sodium citrate to allow comparison between the N402 and the \u0394amyR strain at this time point, as the medium of the \u0394amyR strain did not acidify as quickly as the medium of the N402 strain. A total of 28 genes were significantly induced by maltose in comparison with xylose, and 161 genes were 2-fold higher expressed in N402 compared with the \u0394amyR strain (one-way ANOVA analysis P\u00a0<\u00a00.08). By combining the two sets, we identified 18 genes which were induced by maltose and whose induction was AmyR dependent (Fig.\u00a03, Table\u00a05). Nine of these genes encode proteins involved in carbohydrate transport and metabolism, from which five were also identified as differentially expressed after 2\u00a0h (aamA, glaA, agdA, agdB, and An15g03940). Additionally identified genes included amyC, encoding an extracellular \u03b1-amylase, three genes encoding putative sugar transporters, and several other genes belonging to various functional categories (Tables\u00a04, 5). At this stage we cannot exclude that additional overlapping genes have escaped our attention, because of the different pH values of the medium after transfer, which also could affect gene expression. The results from the microarray experiments were validated using Northern blot analysis for a selected number of amylolytic genes. These genes include glaA, aamA, agdB and amyC. As shown in supplementary Fig.\u00a03, the results of the Northern hybridizations are in good agreement with the microarray data (Supplementary Table\u00a01). The Northern analysis also revealed an additional mRNA of a larger size for the amyC gene, 8\u00a0h after transfer. The detection of two different-sized mRNAs suggests two different mRNA start sites or different polyadenylation sites, but the exact sequence of the different mRNAs and possible consequences for the gene model have not been addressed so far.\nInterestingly, the transcription factor gene amyR was also induced 2 and 8\u00a0h after transfer to maltose compared to the transfer to xylose (2.6-fold, P-value 0.014; 2.8-fold, P-value 0.027, respectively), indicating transcriptional regulation of the amyR gene itself. As the AmyR transcription factor is per se missing in the deletion strain, it is not appropriate to include the amyR gene in the group of maltose induced, AmyR dependent genes. However, the 1-kb promoter region of the amyR gene contains two AmyR binding motifs, allowing for the possibility that AmyR can induce its own expression.\nDiscussion\nThe full inventory of glycoside hydrolases belonging to families GH13, GH15 and GH31 in A. niger strain CBS 513.88 has recently been described (Pel et al. 2007). Members of these protein families in aspergilli, including A.\u00a0niger, have been studied extensively mainly because of their industrial relevance. Nevertheless, several groups of new alpha-glucan acting enzymes that had not been identified previously were identified. These novel enzymes are conserved among several Aspergillus species as well as other Ascomycetes, indicating that they may play an important role in fungal metabolism. In the present study, we have combined transcriptional analysis and more detailed phylogenetic analysis to further understand their function.\nThe GH13 family in A.\u00a0niger contains six separate groups of amylase-type enzymes (Fig.\u00a01), of which three groups had not been described thus far. The best-described group comprises the extracellular \u03b1-amylases, and is now extended with AmyC in addition to the three known \u03b1-amylases. Overexpression of the amyC gene in A.\u00a0niger resulted in increased levels of endo-\u03b1-amylase activity in the medium, indicating that this gene indeed encodes an extracellular \u03b1-amylase (R. M. Van der Kaaij and X. L. Yuan, unpublished results). The relatively low expression of amyC compared to other starch degrading enzymes may explain why this protein has not been identified previously. The localization of the amyC gene in the genome is noteworthy, as it is part of a small cluster of amylolytic genes (with agdA) and their transcriptional regulator gene amyR. A similar organization is observed in the genomes of other aspergilli such as A.\u00a0nidulans and A. oryzae RIB40 (Gomi et al. 2000). In A.\u00a0fumigatus the same cluster is extended with a glucoamylase (similar to GlaA). Gene clusters of transcriptionally co-regulated genes in filamentous fungi are often involved in the same process, e.g. secondary metabolite production (Woloshuk et al. 1995) or catabolism of amino acids (Hull et al. 1989). A possible function of AmyC might be to act as a scouting enzyme for the presence of starch, resulting in the subsequent activation of AmyR by starch-derived molecules (maltose or isomaltose). Alternatively, AmyC might be regulated by another system additional to AmyR, e.g. pH-regulated expression, or its expression might be upregulated locally, e.g. in hyphal tips.\nThe lack of detectable expression of amyA and amyB in A.\u00a0niger N402 is puzzling. Both genes were expressed in A.\u00a0niger strain CBS 513.88 in a fed-batch fermentation using glucose as a carbon source (Pel et al. 2007), but batch cultivation of A.\u00a0niger N402 on glucose did not result in detectable expression in microarray studies (E. Martens, H. Kools, P. Schaap, personal communication). These results indicate a difference between both strains with regard to either the presence of amyA\/B in A. niger strain N402 or in the transcriptional regulation of amyA\/B. In the recently released genome sequence of A. niger strain ATCC 1015 from the Joint Genome Institute (Baker, 2006) we could not identify orthologues of the AmyA\/AmyB proteins suggesting that these genes might be actually absent from the N402 strain. The absence, or lack of expression of amyA\/amyB, is consistent with the observation that the A.\u00a0niger MGG029\u0394aamA strain (N402 background) with gene disruptions for both aamA and glaA grows very poorly on starch (Weenink et al. 2006), confirming that AmyA and AmyB do not contribute significantly to the amylolytic potential of N402. The microarray analysis revealed that in addition to the amyA\/B genes, five more amylolytic genes (agtC, amyD, agdD, agdG and glaB) were not detected (Absent calls) in any of the conditions tested. However, orthologous genes were identified in the genome of A. niger strain ATCC1015, suggesting that these genes are not absent from the N402 genome, but rather not expressed under the tested conditions.\nIn this study we have identified three new groups within the GH13 family that had not been described before in aspergilli: \u03b1-amylase-like proteins predicted to be GPI-anchored enzymes (group II) (subfamily GH13_1, as defined by Stam et al. 2006), intracellular \u03b1-glucosidases (group III) (unknown subfamily) and intracellular \u03b1-amylases (group IV) (subfamily GH13_5). Detailed biochemical studies of two GPI-anchored proteins revealed that they are \u03b1-glucanotransferases, acting mainly on maltooligosaccharides (van der Kaaij et al. 2007a). The expression of these genes was not regulated in response to maltose metabolism or AmyR. The Agt enzymes are homologues of a group of four GPI-anchored amylase-like proteins recently identified in S. pombe (Aah1-4p), which have similarly aberrant conserved regions (Table\u00a03). A deletion of one of these homologues resulted in aberrant morphology of the cell and an increased sensitivity towards cell wall degrading enzymes (Morita et al. 2006). A similar phenotype was observed for an A.\u00a0niger knockout of agtA (van der Kaaij et al. 2007a). Such a phenotype can be explained if \u03b1-glucan synthesis is affected. The clustering of agt genes with genes encoding \u03b1-glucan synthases in all four Aspergillus species studied, and the expression pattern of the A.\u00a0niger agt genes both support the hypothesis that Agt enzymes play a role in \u03b1-glucan synthesis.\nAspergillus niger contains two putative intracellular \u03b1-amylases, AmyD and AmyE, that belong to the recently identified subfamily GH13_5 (Stam et al. 2006). A homologous gene in H. capsulatum, AMY1, was shown to be essential for the formation of \u03b1-(1,3)-glucan in the cell wall (Marion et al. 2006). The possibility of a role for subfamily GH13_5 enzymes in the cell wall of Aspergilli is strengthened by their location in the genome: A. niger, A.\u00a0nidulans and A. oryzae, all contain a cluster of genes, containing a homologue of AMY1 as well as ags and agt genes (Table\u00a0S2). Both the GPI-anchored \u03b1-glucanotransferases and the family GH13_5 \u03b1-amylase-like proteins are only present in fungi with cell wall \u03b1-glucan and not in the true yeasts, which lack this type of cell wall glucans. However, a study with heterologously expressed AmyD did not confirm a role for this enzyme in \u03b1-(1,3)-glucan formation, as the enzyme only showed a (low) hydrolyzing activity on substrates with \u03b1-(1,4) glycosidic bonds in vitro (van der Kaaij et al. 2007b). The expression levels of amyD and amyE were below detection level, and thus could not confirm nor refute the possible involvement of these proteins in cell wall \u03b1-glucan synthesis in A.\u00a0niger.\nAt this moment, no function can be assigned to the predicted intracellular \u03b1-glucosidases AgdC and AgdD, and their homologues identified in other fungi. The enzymes show similarity to a Bacillus \u03b1-glucosidase as well as to a maltase from C. albicans, and might therefore be involved in the intracellular hydrolysis of maltose. Alternatively, these enzymes could play a role in trehalose metabolism, as they show some similarity to bacterial trehalose-6-phosphate hydrolases. Trehalose is known to be a common reserve carbohydrate in fungi including A.\u00a0niger (Wolschek and Kubicek 1997; Arguelles 2000).\nA surprising finding from the genome mining of A.\u00a0niger was the high number of GH enzymes with a predicted intracellular location. This group included not only the starch-acting enzymes described here, but also other predicted intracellular glycoside hydrolases such as \u03b2-glucosidase, chitinase, \u03b2-mannosidase, \u03b2-xylosidase, rhamnosidase and invertase, present in various Aspergillus genomes (Pel et al. 2007; Goosen et al. 2007). Most of these proteins were predicted to be exo-acting, i.e. releasing terminal mono- or disaccharide residues from the non-reducing end of the substrate. In A.\u00a0niger, the genes encoding these proteins are expressed at a relatively low level compared to the extracellular starch degrading enzymes, sometimes even below the detection limit. In a recent publication, expression of intracellular chitinases in A.\u00a0nidulans was found to be induced during the autolysis phase of the culture (Yamazaki et al. 2007). A role for these and other predicted intracellular proteins during the autolysis phase would explain their low expression levels observed in A.\u00a0niger during vegetative growth, as well as their lack of secretion signals.\nThe genome-wide expression analysis identified only five genes that were >2-fold induced by maltose in an AmyR dependent way, 2 and 8\u00a0h after the transfer to maltose. Among them are genes encoding known starch degrading enzymes (aamA, glaA), two (putative) \u03b1-glucosidases (agdA, agdB), and a putative sugar transporter protein (An15g03940). As described in the Results section, the samples taken after 2 and 8\u00a0h after transfer were grown at different pH values (pH 6.2 and 4.0, respectively), which might influence our results and lead to an underestimation of the number of genes that are induced by maltose in an AmyR dependant way at both 2 and 8\u00a0h as additional pH-controlled gene expression might also occur. To fully explore this, both strains should be cultivated at controlled conditions at identical pH.\nThe specificity of the putative sugar transporter encoded by the An15g03940 gene has not been examined, but its expression profile suggests that it might function as a glucose or a maltose transporter. Three additional sugar transporters were expressed higher on maltose than on xylose after 8\u00a0h (Table\u00a05). One of them, An12g07450\/MstA has been functionally characterized as a high-affinity glucose transporter (Vankuyk et al. 2004) which is in-line with its expression profile. The two additional sugar transporters encoding genes (An02g03540\/mstC and An09g04810) showed an interesting expression pattern, with induced expression on maltose but also relatively high expression levels on xylose (Table\u00a05), suggesting that these transporters might have a broad sugar transporting activity. In the \u0394amyR strain both their expression levels were strongly reduced, also relative to the xylose conditions, indicating that the deletion of AmyR might have had an indirect effect. A possible explanation is that disruption of the AmyR transcription factor resulted in low levels of extracellular enzymes converting maltose to glucose, and consequently stress due to low glucose levels. The low availability of glucose might be a signal to downregulate mstC and An09g04810 if these genes encode low-affinity sugar transporters. To further address these observations additional array analysis should be performed using the \u0394amyR strain grown on xylose. Interestingly, the amyR gene itself was also induced by the presence of maltose suggesting that its regulation takes place, at least partly, at the transcriptional level. Furthermore, conserved AmyR binding motifs are present in the promoters of all co-regulated genes, including the 1-kb amyR promoter region, with the exception of aamA (Table\u00a01). Possibly the aamA promoter contains a currently unknown AmyR binding motif. Alternatively, an AmyR binding site might be present in the promoter region of aamA in the N402 background.\nThe results presented in this paper suggest that A.\u00a0niger can metabolize maltose by inducing the expression of a limited amount of enzymes and sugar transporters. Other members of the GH13, GH15 and GH31 protein families might function in starch metabolism in different conditions (solid state growth, or influenced by pH, nitrogen metabolism, etc.), or they may be involved in other processes such as the synthesis of cell wall \u03b1-glucan, or the glycosylation of proteins. To determine the exact function of these enzymes will require further biochemical characterization in combination with the detailed analysis of gene deletion mutants.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nSupplementary material (DOC 581 kb)","keyphrases":["aspergillus niger","alpha-glucan","starch","glucoamylase","maltose","amyr","cell wall","starch-binding domain","alpha-amylase","alpha-glucosidase"],"prmu":["P","P","P","P","P","P","P","P","P","P"]}
{"id":"Ann_Hematol-3-1-2082654","title":"The diagnosis of BCR\/ABL-negative chronic myeloproliferative diseases (CMPD): a comprehensive approach based on morphology, cytogenetics, and molecular markers\n","text":"Recent years showed significant progress in the molecular characterization of the chronic myeloproliferative disorders (CMPD) which are classified according to the WHO classification of 2001 as polycythemia vera (PV), chronic idiopathic myelofibrosis (CIMF), essential thrombocythemia (ET), CMPD\/unclassifiable (CMPD-U), chronic neutrophilic leukemia, and chronic eosinophilic leukemia (CEL)\/hypereosinophilic syndrome, all to be delineated from BCR\/ABL-positive chronic myeloid leukemia (CML). After 2001, the detection of the high frequency of the JAK2V617F mutation in PV, CIMF, and ET, and of the FIP1L1\u2013PDGFRA fusion gene in CEL further added important information in the diagnosis of CMPD. These findings also enhanced the importance of tyrosine kinase mutations in CMPD and paved the way to a more detailed classification and to an improved definition of prognosis using also novel minimal residual disease (MRD) markers. Simultaneously, the broadening of therapeutic strategies in the CMPD, e.g., due to reduced intensity conditioning in allogeneic hematopoietic stem cell transplantation and the introduction of tyrosine kinase inhibitors in CML, in CEL, and in other ABL and PDGRFB rearrangements, increased the demands to diagnostics. Therefore, today, a multimodal diagnostic approach combining cytomorphology, cytogenetics, and individual molecular methods is needed in BCR\/ABL-negative CMPD. A stringent diagnostic algorithm for characterization, choice of treatment, and monitoring of MRD will be proposed in this review.\nIntroduction\nChronic myeloproliferative disorders (CMPD) are clonal stem cell disorders encompassing a very heterogeneous complex of different entities which are defined by distinct clinical and cytomorphological phenotypes and, in some part, known genetic features. They are characterized by increased and effective proliferation of one to three hematopoietic cell lineages in the bone marrow associated to increased peripheral blood parameters. The recent detection of the high incidence of the JAK2 mutations in polycythemia vera (PV), chronic idiopathic myelofibrosis (CIMF), and essential thrombocythemia (ET) [1\u20135], the detection of the FIP1L1\u2013PDGFRA gene fusion in chronic eosinophlilic leukemia (CEL) [6, 7], and the introduction of tyrosine kinase inhibitors such as imatinib in chronic myeloid leukemia (CML) or hypereosinophilic syndrome (HES)\/CEL shed new interest on molecular diagnostics and detection of minimal residual disease (MRD) in CMPD.\nToday, CMPD are primarily separated in CML as defined by the Philadelphia translocation t(9;22)(q34;q11)\/BCR\u2013ABL and in all other so-called BCR\/ABL-negative CMPD. According to the WHO proposal of 2001 [8], the latter are subdivided into PV, CIMF, ET, and unclassified CMPD (CMPD-U). Some very rare disorders as chronic neutrophilic leukemia (CNL), HES, and CEL are additionally included. This broad spectrum becomes even more heterogeneous due to the continuous progress of stages, as all CMPD have the potential of clonal evolution and stepwise progression. They often terminate in bone marrow failure due to myelofibrosis or ineffective hematopoiesis or in acceleration and finally transformation to blast crisis. Differential diagnosis in CMPD is further hampered by the biologically given overlap of the diverse morphologic phenotypes and the sometimes close relationship to reactive conditions, and even show overlaps to myelodysplastic disorders. The WHO, thus, created a category of disorders combining myeloproliferative and myelodysplastic features in which chronic myelomonocytic leukemia (CMML), unclassified myelodysplastic\/myeloproliferative disorder (MDS\/MPD, U), and some very rare disorders as juvenile myelomonocytic leukemia (JMML) were incorporated [8, 9].\nIt is well known that PV has a median survival of 10\u00a0years, ET of 10\u201315\u00a0years, but CIMF of only 4\u00a0years. However, the clinical course in CMPD ranges from a few months with rapid leukemic transformation to several decades. These uncertainties in prognosis and the similarities in the clinical and morphological phenotypes at diagnosis plead for inclusion of other than clinical and morphologic parameters only into classification.\nAlthough chromosomal abnormalities are not specific for distinct CMPD, they contribute to the definition of the prognosis and to the classification of the CMPD. However, aberrant karyotypes are detectable in only 5\u201345% of all BCR\/ABL-negative CMPD, depending on the specific subtype.\nRoutinely applied methods further include by now polymerase chain reaction (PCR) screening for the V617F mutation in the JAK2 non-receptor tyrosine kinase [1\u20135] and will probably soon be included as diagnostic criteria in a revised WHO classification of the CMPD [10]. This novel marker is highly utile for the confirmation of a BCR\/ABL-negative CMPD and might contribute to the definition of the prognosis and even for MRD strategies in the future [11]. Molecular methods in the CMPD further encompass PCR analyses of some rare gene fusions, e.g., in HES\/CEL [6, 7] or in the 8p11 syndrome [12].\nAdaptation of these extensive diagnostic procedures in CMPD to the laboratories\u2019 resources becomes a major challenge. Major goals are standardization of diagnostic workflow, hierarchical order of methods, and combination of single results. This review intends to propose specific diagnostic algorithms for scenarios in BCR\/ABL-negative CMPD.\nPreanalytic conditions\nTo achieve optimal conditions in the diagnostic procedures, a standardized preparation of the samples and optimal conditions for transport are essential in the CMPD: Cytomorphology requires 3\u00a0ml bone marrow and 2\u00a0ml peripheral blood anticoagulated with ethylenediaminetetraacetic acid (EDTA), being aware that cytomorphology is hampered by heparine. Cytogenetics, in contrast, requires 5\u201310\u00a0ml heparinized bone marrow and 10\u201320\u00a0ml heparinized peripheral blood, as cultivation of metaphases is nearly inhibited by EDTA which induces apoptosis of cells. Multiparameter flow cytometry and all molecular genetic methods can be performed either on EDTA or heparinized material. Trephine biopsies should be performed for histomorphology and immunohistochemistry and allow cytomorphological evaluation by smears from the trephine cylinder in case of a dry tap. In the latter case, also for cytogenetics, a trephine cylinder can be transferred to isotone saline solution plus heparine, which, in many cases, makes metaphases after cultivation in cytogenetic medium possible.\nCytomorphology in CMPD\nDifferential diagnosis in the CMPD should always include investigation of peripheral blood smears, bone marrow aspirates, and trephine biopsies in parallel. Smears from peripheral blood and bone marrow are stained according to May Grunwald Giemsa. This may be completed by other stainings: Myeloperoxidase (MPO) reaction and non-specific-esterase (NSE) should be performed in blast crisis after CMPD and are warranted in cases of CMML. Iron staining may be performed additionally, but is of minor importance for the differential diagnostics, as in an early PV, iron will not always be absent. MPO and iron staining are further helpful in all cases with an overlap between the CMPD and MDS for detection of MPO deficiency and ringed sideroblasts. Cases with suspicious HES or CEL should, in addition, undergo toluidine blue staining for detection of mast cells which are frequently increased in CEL with the FIP1L1\u2013PDGFRA gene fusion.\nIn PV, cytomorphology shows increased cellularity with trilineage cell proliferation. Stainable iron is completely missing in many cases. PV is further characterized by elevated blood counts in either cell line (Hb\u2009>\u200918.5\u00a0g\/dl in men, >16.5\u00a0g\/dl in women, platelets \u2265400\u2009\u00d7\u2009109\/l, WBC \u226512\u2009\u00d7\u2009109\/l). In addition, other criteria as reduced serum erythropoietin levels below normal ranges [10, 13] or in vitro formation of endogenous erythroid colonies are included in the classification criteria [8]. In CIMF, bone marrow cytology is often hampered by myelofibrosis, and the peripheral blood shows the characteristical poikilocytosis and leukoerythroblastosis. However, these findings are not specific but occur as well in secondary myelofibrosis following other CMPD. ET is characterized by proliferation of the megakaryocytic lineage with clusters of enlarged mostly mature megakaryocytes in normocellular bone marrow. Granulopoiesis and erythropoiesis present as normal. According to the WHO [8], a diagnosis of ET requires a sustained peripheral platelet count \u2265600\u2009\u00d7\u2009109\/l, whereas reactive thrombocytosis must be excluded. Recently, an international expert panel recommended to lower the threshold for the diagnosis of ET to 450\u2009\u00d7\u2009109\/l [10]. All CMPD, but especially CML, may show the so-called pseudo-Gaucher cells in the bone marrow, which represent glycolipide storing histiocytes resulting from increased cell proliferation [14]. Another specific cytomorphological subentity is represented by refractory anemia with ringed sideroblasts associated with marked thrombocytosis >500\u2009\u00d7\u2009109\/l (RARS-T). This rare subtype may be included within the category of overlapping myelodysplastic and myeloproliferative diseases in the upcoming WHO classification [15\u201317].\nHistomorphology in CMPD\nBone marrow histology has a central role in the diagnosis of CMPD. Staining is performed according to Giemsa, PAS, and chloroacetate esterase. Gomori silver impregnation allows assessment of reticulin fibers to quantify bone marrow connective tissue.\nThus, a diagnosis of CIMF requires at least a bone marrow histology, as bone marrow aspirates are hampered in most cases. Histology shows variable reticulin or collagen fibrosis, osteosclerosis, and sometimes even decreased cellularity. Sinuses are dilated with intraluminal hematopoiesis and erythrocyte extravasates. Megakaryopoiesis is increased with signs of dysplasia [8, 18].\nPV demonstrates increased megakaryopoiesis, increased granulopoiesis, and erythropoiesis without storage iron, sinusoid hyperplasia, and variable myelofibrosis in combination with osteopenia. These characteristics allow discrimination from reactive erythrocytosis [19]. In more detail, differentiation from cases of secondary polycythemia includes the evaluation of megakaryopoiesis which shows clustering and pleomorphous appearance with very small and giant megakaryocytes with the characteristical staghorn-like nuclei neighboring each other [19, 20].\nET is characterized by clusters of enlarged mature megakaryocytes close to the sinusoids, whereas erythropoiesis and granulopoiesis are normal [10, 18]. A specific problem is provided by the differentiation between ET and prefibrotic CIMF, as both might be characterized by thrombocytosis in initial stages, and reticulin fibrosis might be minimal or absent in prefibrotic CIMF. However, prefibrotic CIMF is characterized by marked hypercellularity, left-shifted increased granulopoiesis, and a particular megakaryocyte morphology with nuclear features, whereas ET shows hyperlobulated and mature-appearing megakaryocytes [10, 21].\nCytogenetics in BCR\/ABL-negative CMPD\nThe frequency of clonal karyotype anomalies varies considerably between the different BCR\/ABL-negative CMPD entities. The presence of karyotype abnormalities at diagnosis per se seems to be prognostically negative [9]. CIMF has the highest karyotype aberration rate with 33\u201343% of all cases, followed by PV in 33\u201335%, whereas in ET, clonal abnormalities are extremely rare (<5%) [22\u201325]. In CMPD-U, aberrant karyotypes were reported in \u223c20% [22], but definition of the true incidence is difficult due to the heterogeneity of subtypes in this category.\nChromosomal changes in the CMPD are not specific, but their presence at least confirms the diagnosis of a malignant hematopoietic disorder and contributes additional aspects to differential diagnosis. This can be exemplified in the 9p-aberrations which are closely associated to PV and to CIMF. In addition, translocations involving ABL, PDGFRA, PDGFRB or other tyrosine kinases can be detected by chromosome banding analyses, allowing the identification of patients who probably benefit from treatment with tyrosine kinase inhibitors.\nThus, chromosome banding analyses contribute a lot at diagnosis in many in cases with a suspicious or proven CMPD. However, they do not lead to important information for clinically clearly proven cases of ET. Cytogenetics may also be needed for the follow-up of the CMPD, as leukemic transformation is characterized in many cases by clonal evolution to more complex karyotypes resulting in higher rates of chromosomal abnormalities of \u226590% [9, 23, 26].\nInterphase (IP-), metaphase (HMF-), and 24-color fluorescence in situ hybridization (FISH) may further confirm and clarify the results of the chromosome banding analyses. IP-FISH probes can be used for future MRD studies. Nearly all typically observed aberrations\u2014e.g., +8, +9, gain of 9p, or del(20q)\u2014can be monitored.\nTrisomy 8 is the most frequent aberration in the CMPD being detected in \u223c20% of all cytogenetic aberrant PV cases and in \u223c10% in chromosomally aberrant CIMF\u2014mostly as sole abnormality or in combination with +9. This is followed by trisomy 9 in \u223c10% of all cytogenetically aberrant cases. Partial trisomies of 9p are equally frequent with a special association to PV [27\u201330]. Other recurrent aberrations are deletions of 13q and 20q and partial trisomies of 1q [9, 22, 31], whereas +19, +21, \u22127, \u2212Y, del(12p), and i(17q) are less frequent.\nChromosomal changes show a characteristic distribution within the diverse CMPD. In detail, PV shows, as the most frequent changes, +9, followed by +8 and by del(20q) [23, 26]. CIMF has a more heterogeneous pattern with deletions of 13q and of 20q both in \u223c9% of all cases [9, 25], structural abnormalities of 1q and 5q, and chromosome 7 abnormalities [23, 31]. In ET, chromosomal abnormalities are found in <5% of cases only, mostly represented by numerical gain of chromosome 9. Table\u00a01 presents an overview on recurrent cytogenetic and molecular markers in the CMPD.\nTable\u00a01Chromosomal and molecular markers in CMPD [8, 9, 22, 23, 52, 69]\u00a0Karyotype abnormalitiesMolecular markersMolecular MRD markersCMLt(9;22)(q34;q11), in all cases BCR\u2013ABLBCR\/ABL+PVIn some cases: +8, +9, del(20q), del(13q), del(1p)JAK2V617F+JAK2 exon 12 in V617F-negative casesCIMFIn some cases: del(13q), del(20q), +8, +9, partial trisomy 1qJAK2V617F+MPLW515ETIn rare cases +8, +9, del(13q), JAK2V617F+MPLW515CMPD-UIn some cases +8, +9, del(20q)JAK2V617F+In rare cases 8p11 translocationsFGFR1 rearrangementsCEL\/HESin some cases: +8, i(17q),PDGFRA\/FIP1L1 in CEL+CNLin some cases: +8, +9, del(20q)\u2212\u2212CMML\u22127, +8, del(20q)NRAS in some cases+\nBalanced translocations as revealed by cytogenetics are rare in the CMPD. Many of these lead to the disruption of genes encoding tyrosine kinases. The breakpoints cluster in two regions at 5q31-33 and 8p11 which target the platelet-derived growth factor receptor beta [e.g., in the t(5;12)(q31q33;p12)\/ETV6-PDGFRB)] and the fibroblast growth factor receptor 1 kinase [e.g., in t(8;13)(p11;q12)\/FGFR1-ZNF198)]. Further, the ABL non-receptor tyrosine kinase might be involved in these rare rearrangements as in the t(9;12)(q34;p13)\/ETV6-ABL [32]. The 8p11 myeloproliferative syndrome shows a specific profile outlined by frequent association to Non-Hodgkin\u2019s lymphoma, high leukemic transformation rates, eosinophilia, and CML-like findings in bone marrow cytomorphology. It is most frequently caused by the t(8;13)(p11;q12)\/FGFR1-ZNF198, but many other variants all involving 8p11\/FGFR1 have been described. Bone marrow cytomorphology shows CML-like findings and eosinophilia [12]. As patients with PDFRB and ABL rearrangements are all candidates for tyrosine kinase inhibitor treatment, detection of these rare rearrangements by cytogenetics in combination with FISH and PCR is obligatory. For an overview on these reciprocal gene fusions, we refer to Cross and Reiter [32].\nMolecular mutations in BCR\/ABL-negative CMPD\nAs published in 2005 by several study groups, a high proportion of patients with BCR\/ABL-negative CMPD have a somatic point mutation in the JAK2 gene on 9p24 (V617F) which codes for the JAK2 kinase. In detail, the mutation was found in 80\u201397% of all patients with PV, in >50% of all patients with CIMF, and in 40\u201357% in ET [1\u20135]. Janus kinases are non-receptor TKs which regulate the phosphorylation of several signaling pathways, e.g., JAK\/STAT, whose activity is increased by the JAK2 mutation [3, 5]. Interestingly, JAK2V617F-positive ET cases were found to show considerable clinical similarities to PV. This pleads for common pathogenetic pathways in part of the ET cases and in PV [33]. It has to be expected that the JAK2V617F mutation will soon be included as a major criterion for PV diagnosis in a revised WHO classification, as nearly all cases were found to be positive [10]. According to this proposal, also in ET or in CIMF, the respective mutation will serve likewise to any other clonal marker as criterion for the diagnosis of ET or CIMF, which further emphasizes its value for diagnostics in the CMPD.\nA positive mutation status seemed correlated with further advanced stages. Homozygous JAK2 mutations are more frequent in PV and CIMF than in ET [26] and are associated with a longer history of disease than heterozygous mutations [34]. Thus, a homozygous mutation status correlates with a more aggressive course and might indicate an inferior outcome.\nDifferent assays were developed for JAK2 mutational analyses, e.g., allele specific PCR, real-time PCR, or pyrosequencing, which is able to convey information on the frequency of mutated alleles [35]. Kroger et al. [11] showed that quantitative assessment of the JAK2 mutation with real-time PCR after allogeneic hematopoietic stem cell transplantation was valid as minimal residual disease parameter allowing the adaptation of adoptive immunotherapy accordingly. Thus, determination of the JAK2V617F mutation status is highly valuable for all cases with a suspicious or proven BCR\/ABL-negative CMPD and even contributes to determination of the prognosis and to MRD strategies.\nThe role of the JAK2V617F mutation is not limited to the \u201cclassical\u201d CMPD but was detected in other myeloid malignancies as well, e.g., in 3\u201310% of all MDS cases [1, 36, 37] or in 50\u201390% in RARS-T representing an ambiguous subentity with overlapping myeloproliferative and myelodysplastic features [17, 38]. Interestingly, the JAK2V617F mutation was as well found in 20\u201330% of patients with abdominal vein thromboses and in 5% of patients with cerebral vein thromboses without signs of an overt hematologic disorder [39, 40].\nSince the detection of the JAK2617F, the panel of known activating mutations in the CMPD is continuously increasing: PV patients who were JAK2V617F-negative were detected to carry somatic gain-of-function mutations within exon 12 of the JAK2 gene in 40% of cases in a recent study. This mutation subtype was shown to stimulate erythroid proliferation in in vitro experiments [40, 41]. Further on, \u223c5% of CIMF and \u223c1% of all ET cases show somatic mutations in codon 515 within the transmembrane domain of the MPL gene which encodes the thrombopoietin receptor. The respective point mutations lead to single amino exchanges (W515L and W515K) and induce constitutive cytokine-independent activation of the JAK-STAT pathway as gain-of-function-mutations likewise to the V617F [42\u201344].\nChronic neutrophilic leukemia\nCNL is a very rare CMPD defined by persistent leukocytosis in pB \u226525\u2009\u00d7\u2009109\/l, segmented neutrophils and bands >80% of WBC, immature WBC <10%, and myeloblasts <1% [8]. Survival is extremely heterogeneous and was reported from a few months to 20\u00a0years. Cytogenetic aberrations are rare and include +8, +9, del(20q), and del(11q) [8, 45, 46]. The JAK2 mutation was described in few cases of CNL [4, 22, 37, 45\u201348], but determination of the true incidence is extremely difficult due to the rare occurrence. Some CNL cases were identified to bear rare BCR\u2013ABL fusion transcripts with a breakpoint between exons c3 and c4 of the BCR gene leading to a 230-kDa fusion protein [49, 50].\nHypereosinophilic syndrome\/chronic eosinophilic leukemia\nPersisting hypereosinophilia is, in most cases, reactive and is only rarely caused by a malignant disorder such as HES or CEL or other eosinophilia-associated CMPD [6, 7, 51\u201353]. HES is defined by persistent eosinophilia \u22651.5\u2009\u00d7\u2009109\/l in peripheral blood >6\u00a0months and an increased number of bone marrow eosinophils of unknown origin; this is accompanied by organ involvement and dysfunction. Classification as HES requires exclusion of all other causes and failure of detection of the underlying genetic defect by cytogenetic and\/or molecular screening.\nA diagnosis of CEL requires >2% blasts in peripheral blood and >5\u201319% bone marrow blasts or evidence of clonality [8, 54]. The most frequent aberration in CEL is the FIP1L1\u2013PDGFRA fusion which results from a cryptic interstitial deletion on chromosome 4q12. Whereas chromosome banding analyses fail to detect the respective cytogenetic correlate, it is revealed by IP-FISH with differently marked probes for CHIC2, FIP1L1, and PDGFRA, and with reverse transcription (RT)-PCR for FIP1L1\u2013PDGFRA [55, 56].\nSome more rare TK gene fusions were detected in eosinophilia-associated CMPD, which involve the tyrosine receptor kinases PDGFRA on 4q12, PDGFRB on 5q31, FGFR1 on 8p11, and the non-receptor kinase JAK2 on 9p24 [9, 57]. The beneficial response of patients with PDGFRA and PDGFRB rearrangements to imatinib makes the detection of these rearrangements obligatory [6, 7, 53, 58]. Therefore, a combination of cytomorphology, cytogenetics, IP-FISH, and RT-PCR based on the patient\u2019s history provides the basis for optimized diagnosis in eosinophilia-associated CMPD followed by targeted therapy [32, 59, 60].\nChronic myelomonocytic leukemia\nDue to its ambiguous character, CMML was incorporated by the WHO into a category which overlaps between myelodysplastic and myeloproliferative disorders [8] and was defined by peripheral monocytes >1\u2009\u00d7\u2009109\/l and by dysgranulopoiesis >10%. Blasts and promonocytes were defined by <20% of WBC and by <20% of all nucleated cells in bone marrow. NSE was strongly recommended for bone marrow examination. Clonal karyotype abnormalities occur in 25\u201335% of all cases. Most frequent are changes of chromosome 7, trisomy 8, and complex aberrant karyotype which is defined by three or more chromosomal aberrations [9, 22, 61]. Mutations of the NRAS protooncogene are the most frequent so far identified molecular markers with variable incidences of 10\u201366% in this entity. Although there was a wide range in these studies (probably due to the limited samples size in the different studies), this high incidences suggest parallels to MDS or AML [61\u201363]. The JAK2 mutation was detected in 3\u201313% of all cases with CMML [1, 4, 37], which illustrates the vicinity to the CMPD in another part of CMML cases. This molecular heterogeneity correlates with the clinical and morphological diversity of CMML and supports the ambiguous position as suggested in the WHO classification.\nRemission criteria in the CMPD\nNew therapeutic strategies including allogeneic stem cell transplantation as potentially curative option or targeted therapies in the CMPD implicate the need for more differentiated and sensitive criteria of remission. For CIMF, an international working group formulated new consensus criteria for response to treatment [64]. These criteria are based on a combination of clinical and laboratory parameters and include cyto- and histomorphological, cytogenetic, and molecular findings. Thus, highest so far applicable sensitivity is achieved. Clinical criteria include disappearance of clinical symptoms as palpable hepatosplenomegaly. The laboratory and cytomorphological criteria of complete remission imply normalization of trilineage peripheral blood count and the absence of blasts, immature progenitor cells, or nucleated erythroid precursors in peripheral blood. Bone marrow histologic remission criteria apply to cellularity, myeloblast percentage, and osteomyelofibrosis. Finally, cytogenetic response criteria discriminate major cytogenetic response, meaning absence of chromosomal abnormalities in cases with a preexisting aberration from minor cytogenetic response requiring a \u226550% reduction of abnormal metaphases. As the most sensitive criterion, major molecular response defines absence of a specific disease-associated mutation in previously positive cases [64].\nApproach to a diagnostic algorithm in the CMPD\nA diagnostic algorithm for all cases with diagnosis or suspicion of CMPD (Fig.\u00a01) should start with the cytomorphologic evaluation of peripheral blood and bone marrow. This allows, in many, cases a differentiation between CML and a BCR\/ABL-negative CMPD. In parallel, all cases should be evaluated by histomorphology.\nFig.\u00a01Proposal for a diagnostic algorithm in BCR\/ABL-negative CMPD. HES Idiopathic hypereosinophilic syndrome, CEL chronic eosinophilic leukemia, PV polycythemia vera, CIMF chronic idiopathic myelofibrosis, CMPD-U unclassifiable CMPD, ET essential thrombocytosis, RARS-T refractory anemia with ringed sideroblasts, CMML chronic myelomonocytic leukemia, MFC multiparameter flow cytometry, RT-PCR reverse transcription polymerase chain reaction. Dashed line may add information in difficult cases, but not obligatory\nChromosome banding analyses might gain important information in PV, CIMF, and also in CMPD-U, as chromosomal aberration rates of \u223c20\u201345% were reported [22]. According to the recent guidelines of a British Committee, cytogenetics are considered as \u201cstage 2 investigations\u201d in case of PV and erythrocytosis which should be performed in dependence on the results of clinical evaluation, blood count, and the JAK2 mutation as well as other laboratory parameters such as serum ferritin which are obligatory as \u201cstage 1 investigations\u201d [65]. In contrast, cytogenetics can be neglected in clinically clear cases of ET due to the extremely low incidence of aberrant karyotypes. In cases in which the discrimination of a CMPD from a reactive disorder is not possible according to clinical, laboratory, and cyto-\/histomorphological aspects, cytogenetics might contribute to differential diagnosis, as, in some cases, clonal abnormalities confirm the diagnosis of a hematologic malignancy. CMML should as well undergo cytogenetic analyses; in addition, molecular screening for NRAS mutations might be discussed due to their frequent occurrence and new drugs that may come up. This also includes the investigation of PDGF receptors expression.\nThe chromosomal aberrations as revealed by chromosome banding analyses\u2014e.g., +8, +9, del(20q)\u2014can be verified by FISH techniques. This further allows the selection of IP probes for distinct numerical and structural aberrations to provide parameters for MRD diagnostics.\nToday, the backbone in all cases of suspicious or proven CMPD should be screening for the JAK2V617F mutation status by PCR, e.g., by a melting curve light cycler assay [66]. According to the proposals for a revised WHO classification, evidence of the JAK2V617F mutation (localized in exon 14 of the JAK2 gene) or a functionally similar mutation will be required as major criterion for the diagnosis of PV. This might justify additional screening for JAK2 mutations in exon 12 in cases which are highly suspicious for PV from morphological aspects but are negative for the JAK2V617F [10].\nFinally, all cases with a suspected or proven CMPD should be evaluated by IP-FISH or PCR for BCR\u2013ABL to exclude a diagnosis of CML due to the therapeutic consequences. This is even more emphasized by single cases showing a coincidence of the BCR\u2013ABL fusion and the JAK2V617F mutation where the cytomorphological features of the CMPD can mask the CML aspect [67].\nIn cases with diagnosis or suspicion of HES\/CEL, cytogenetic analysis is informative only in very few cases and may be omitted, whereas molecular diagnostics in search of the FIP1L1\u2013PDGFRA gene fusion by IP-FISH and\/or RT-PCR is obligatory.\nConclusions\nFor many years, diagnostics in the BCR\/ABL-negative CMPD were mainly based on clinical symptoms, cytomorphology, and histomorphological findings. This has dramatically changed in recent years. The WHO (2001) [8] included cytogenetic aspects directly and indirectly in their classification system. This can be exemplified in ET which is excluded by evidence of a del(5q) or an inv(3)(q21q26), as both the 5q- syndrome in MDS or AML with the respective inversion 3 can show microkaryocytes and high thrombocytes. Clonal chromosomal abnormalities other than the Philadelphia translocation support the diagnosis of CMPD and allow to discriminate this from reactive conditions.\nAdditionally, the spectrum of molecular mutations considerably increased. Most molecular events target receptor TK (such as FGFR1, PDGFRA, and PDGFRA) and non-receptor-TK (such as ABL or JAK2). Especially, the detection of the JAK2 mutation in the majority of patients with a CMPD has revolutionized diagnostics in the CMPD [13] and allows, in many cases, now a rapid and clear discrimination of the CMPD from reactive conditions. It further may abolish the measurement of the PRV1 expression in cases with suspected PV. JAK2 mutated cases seem to be associated with higher complication rates and an increased need for therapeutic interventions when compared to wild-type patients [3]. This might play a role for the choice of therapy in the future. Finally, JAK2 might represent a target for specific compounds with anti-tyrosine kinase activity [2, 34]. The recent description of JAK2 mutations in exon 12 in V617F-negative PV cases [40, 41] and of the W515 mutations of the MPL gene in a low frequency in ET and CIMF [42\u201344] illustrate that we have to assume a complex network of activating mutations in the CMPD of which only parts are so far identified. Thus, a new classification of the CMPD according to the molecular substrate, e.g., the JAK2 mutation, likewise to the definition of CML, will be more appropriate [10, 68]. The inclusion of JAK2 mutation analysis as a major criterion for the PV diagnosis within the current and upcoming World Health Organization (WHO) diagnostic criteria was suggested [10].\nThe detection of the FIP1L1\u2013PDGFRA fusion in CEL\/HES in association to the good response to imatinib [6, 52] and the increasing detection of rare fusion transcript in the BCR\/ABL-negative CMPD [32] contributed as well significantly to an improved molecular classification in CMPD.\nMRD strategies are so far poorly established in the Philadelphia negative CMPD when compared to the acute leukemias or to CML. This might change in the near future, as it was already shown in a post-transplantation setting that quantitative assessment of the JAK2 mutation qualifies as MRD marker [11]. In CML, the definition of remission criteria allowed an international standardization in clinical studies and was helpful for clinical routine. This approach is increasingly important also for the BCR\/ABL-negative CMPD as realized by an international working group proposing cytogenetic and molecular response criteria in CIMF [64].\nIn conclusion, diagnostics in the BCR\/ABL-negative CMPD have abandoned the former perception which classified these complex disorders mainly on clinical and morphological aspects and are on the way to a comprehensive approach focussing increasingly on cyto- and molecular genetic aspects. Individual treatment is already available or will hopefully follow.","keyphrases":["cmpd","molecular marker","cytomorphology","bcr\/abl"],"prmu":["P","P","P","R"]}
{"id":"World_J_Urol-3-1-1913182","title":"Overdiagnosis and overtreatment of early detected prostate cancer\n","text":"Early detection of prostate cancer is associated with the diagnosis of a considerable proportion of cancers that are indolent, and that will hardly ever become symptomatic during lifetime. Such overdiagnosis should be avoided in all forms of screening because of potential adverse psychological and somatic side effects. The main threat of overdiagnosis is overtreatment of indolent disease. Men with prostate cancer that is likely to be indolent may be offered active surveillance. Evaluation of active surveillance studies and validation of new biological parameters for risk assessment are expected.\nIntroduction\nWhat is the rationale for screening?\nScreening for diseases, especially cancer, has become part of modern medicine. Screening for breast, cervical and colorectal cancer is already normal practice in some countries, and will probably become routine in other countries in the future. Screening for prostate, melanoma and lung cancer are subject to ongoing studies [1\u20135]. The rationale behind screening is simple: to detect cancers at an early stage, when they are still curable. Screening is currently performed using one of the three methods: mass screening (i.e. large scale screening of an entire population), selective screening (i.e. screening of high-risk populations) or opportunistic screening (e.g. incorporated as part of a medical consultation). Diagnostic testing differs from screening because it attempts to identify the disease in the presence of symptoms, while screening is offered to symptom-free individuals.\nIn any population screened for cancer, four basic groups of patients exist: those diagnosed with cancer who would not have developed cancer symptoms during their lifetime (overdiagnosis); those diagnosed with cancer at an early stage that might otherwise have led to symptoms and\/or the need for more aggressive curative treatment; those diagnosed with cancer at a curable stage with aggressive disease that might otherwise have progressed to metastatic disease at the time of diagnosis; and those whose cancer is diagnosed by screening at the same stage as it would have been diagnosed through clinical routines, and that involves cancers that are too late for curative therapy. Ideally, screening should reduce the number of patients in the fourth group (that cannot be cured), and increase those in the second and third group. The window of opportunity for decreasing cancer-mortality by screening for cancer lies with the second and third group. Randomized clinical trials, considered the gold standard for the evaluation of a screening test, have to show how sizeable the window of opportunity is. The difference between the first group and the second is however not always clear at the time of initial diagnosis. Any screening procedure carries a risk of overdiagnosis and overtreatment, which should be balanced against the benefits for those in which the cancers are diagnosed at a curative stage. Whether this balance is justifiable depends on more than mortality differences of randomized study groups only, but also on quality of life issues measured against the cultural background of the population studied.\nIncidence\nDoes screening influence prostate cancer incidence?\nSince the potential value of PSA for the early detection of prostate cancer was described in the early 1990s, both prostate cancer incidence and mortality rates have changed profoundly [6]. Between 1989 and 2003, for example, the age-standardized incidence rate of prostate cancer increased by 48.4% in The Netherlands (reaching an incidence of 93.2 cases per 100,000 men). Based on rates from 2001 to 2003, 17.1% of U.S. men born today will be diagnosed with cancer of the prostate at some time during their lifetime [http:\/\/www.cancer.gov]. It is now the most frequently diagnosed non-cutaneous cancer, with 225,000 new cases reported each year in Europe alone [7]. This increase of incidence suggests that this is due to the detection of cancers in the first three groups described above. This is supported by the reports on autopsy studies. These autopsy studies have revealed that histologic prostate cancer occurs in an even larger proportion of men compared to the screening incidence: up to 55% of men in their fifties, and 64% of men in their seventies have prostate cancer diagnosed at autopsy, while only 5\u201310% are detectable in a screening setting during life [8].\nA number of influences might have contributed to the increase of cancer diagnosis over the last decades, apart from the structured screening studies that have been initiated at various places. First, the PSA thresholds for biopsy appears to have been reduced gradually in some areas of the world due to the detection of significant cancers in the low PSA range [9]. Most guidelines, however, still mention the traditional cut-off of 4\u00a0ng\/ml as an indication for biopsy. Secondly, an increase in the number of core samples per biopsy have been advocated, based on the observation that more cancers are diagnosed when more biopsies are taken. Thirdly, awareness of prostate cancer within the general population has increased stimulated by the information obtained predominantly from the urologic profession [10]. If these current trends continue, the number of living men diagnosed with prostate cancer will increase even further [11].\nMortality\nWhat happens to prostate cancer mortality by screening and detecting indolent tumors?\nDespite this rising incidence, the age-standardized prostate cancer mortality rate has decreased in many countries around the world with or without early detection programs. In The Netherlands, for example, rates fell by 11% between 1989 and 2003, to 28.4 deaths per 100,000 men or 2,349 in total [http:\/\/www.cancer.gov]. It is however unclear whether the stage and grade migration observed in screening studies results in a reduction in the mortality, although case-control studies with conflicting results are available [12\u201314]. A decrease of mortality shown in randomized studies would form a strong argument in favor of population screening.\nScreening results in the more frequent detection of small volume, low grade and organ confined prostate cancers, which are diagnosed earlier in their course [6, 15]. Many of these tumors have the histological characteristic of autopsy tumors, that is, tumors that have not become symptomatic during life [16]. They have been called indolent or clinically insignificant cancers. Various definitions of clinically insignificant tumors have been designed based on the characteristics of the autopsy studies, of which the Epstein definition is widely known [17]. Detecting such tumors will increase the detection frequency of cancer, but it is unlikely that they will influence the prostate cancer specific mortality, as they do not alter the course of life.\nEarly diagnosis\nScreen detected tumors are diagnosed more early\nScreen detected tumors are not only diagnosed more often, but can be expected more early during their natural course. Lead-time is defined as the time period from detection by a screening procedure to the time of diagnosis in absence of screening due to symptoms. If the patient dies during the lead-time period of the tumor, the lead-time is indefinite and therefore equal to overdiagnosis.\nEarly detection by PSA advances prostate cancer diagnosis in time (i.e. lead time) [18]. For men aged 55\u201375\u00a0years lead time amounts to 12.3\u00a0years in a screening setting [19]. The lead-time is likely to be shorter for aggressive cancers and longer for indolent ones. Early detection also causes a significant stage shift towards more locally confined and less aggressive cancers [20]. The long lead-time, stage reduction and natural history data cited above indicate that for properly selected cases there is a long \u2018time-window\u2019 during which observation is safe without losing the opportunity for cure. This is supported by evidence from nomograms, prognostic tables, and by the only available randomized study of observation against radical prostatectomy [21].\nThe impact of overdiagnosis and unnecessary treatment and of its side effects on patient health is also unclear; however, application of a mathematical model (the Miscan model) on data from the ERSPC has shown that, in an annual screening program for men aged 55\u201367\u00a0years, 56% of diagnosed cases would constitute instances of overdiagnosis [22]. If this estimate proves to be realistic (as it appears to be the case), nationwide screening programs may not be acceptable using the present screening regimens, even if benefits in terms of mortality reduction were shown. Research aimed at the development of more selective screening tools is therefore very important.\nThe natural course of screen-detected cancers\nThe current knowledge of the natural history of prostate cancer is mainly based on clinically detected cases, diagnosed by disease-related symptoms, or coincidentally at the time of unrelated symptoms of the urinary tract. Natural history data on screen-detected cases of prostate cancer will be created as a result of the large ongoing screening studies worldwide in which tumors might have been treated without invasive therapy [1]. Most studies show histological and cytological grade to be the most important prognostic variable [23, 24]. There is a steep decline in the 5-, 10- and 15-year cancer-specific survival with increasing grade. Most prostate cancer cases diagnosed with present diagnostic techniques fall into the moderately differentiated group (grade 2, Gleason 6), of which the cancer-specific 10\u201315-year mortality is 18\u201330%. This is even lower in the group of prostate cancers diagnosed with grade 1 and 2 or Gleason 4\u20136, and there are subgroups of patients who are not at risk of dying from prostate cancer even within 15\u00a0years. Overall mortality is then determined by comorbidity. The majority of these men are currently treated for their prostatic disease with invasive procedures, which might not be needed. Adequate prediction of the outcome of comorbid diseases would be of great support in predicting the outcome of the prostate cancer patient.\nOverdiagnosis\nOverdiagnosis and overtreatment, what does it mean?\nDuring recent years, increased interest has risen to the possibility that increased detection of prostate cancer may lead to the diagnosis of cancers that rather should not have been diagnosed, and certainly should not have been treated, as their detection and subsequent treatment is unlikely to benefit patients, or even might harm them. Related to this, the terms \u2018overdiagnosis\u2019 and \u2018overtreatment\u2019 are being used. So, when is prostate cancer overdiagnosed?\nBy using the clinical definition of overdiagnosis, that is diagnosing tumors that would otherwise remain clinically unrecognized until the individual died from other causes, it is clear that this definition can only be applied in retrospect in the evaluation of studies. There are currently no clinical or biological parameters that can identify such tumors 100% adequately at the time of diagnosis. By studying the natural course of prostate cancer, and comparing autopsy results with findings from screened populations, clinical and histological parameters can be identified that predict indolent tumors best. Those indolent tumors are likely to be only a subset of the tumors that are overdiagnosed in retrospect.\nOverdiagnosis is predominantly being associated with early detection or screening programs. Overdiagnosis appears to be especially harmful when it results in invasive treatment of the tumors that would unlikely to be harmful. This is called overtreatment.\nOverdiagnosis occurs when screening detects small tumors that would otherwise remain clinically unrecognized until the individual dies from other causes. Such tumors are predominantly found in the low PSA ranges. Unfortunately, an unknown number of biologically more aggressive cancers may hide between the larger number of detectable tumors with favorable stages. Though some of the aggressive tumors can be diagnosed by adverse histological criteria such as high Gleason score in the biopsy, some of these features might be missed due to the heterogeneity of prostate cancers and their representation in the biopsy sampling. This might justify the amount of overtreatment that has been practiced in various areas of the world. Overtreatment is thus defined as unnecessary invasive treatment with respect to the outcome of the natural course of the tumor in combination with its host.\nOne can wonder what number needed-to-treat to prevent one prostate cancer death we are prepared to accept. Based on the Swedish randomized trial of radical prostatectomy versus watchful waiting [22], the Connecticut observation series [15], and the Toronto active surveillance experience [25], a number needed to treat analysis of the benefit of radical treatment of all newly diagnosed favorable-risk prostate cancer patients, compared with a strategy of active surveillance with selective delayed intervention, has been presented by Klotz [25]. This suggested that approximately 73 patients will require radical treatment for each prostate cancer death averted. This translates into a 3- to 4-week survival benefit, unadjusted for quality of life. If any, the number of life-years gained will be small, because of the fact that prostate cancer is a disease of old age. This should be contrasted to the side-effects of all applied treatments. As a minimum, the number of men needed to treat should be higher than the number of men dying from intervention related causes.\nInformed decision making, reduction of unnecessary prostate biopsies\nThe increasing number of diagnostic procedures and subsequently of prostate cancer diagnosis in various areas of the world is partly fed by an increased awareness of prostate cancer and the anxiety raised of suffering from a devastating disease at the end of life. Men should ask themselves if they are at specific risk for having prostate cancer, and if they want to follow the step wise procedure of PSA testing, urologic investigations, prostate biopsies, and potential treatment. Balanced information regarding this procedure and its consequences should be offered to every man considering prostate testing. Validated information (that is: well understandable text that delivers the information that is required) has been made available in several countries around the world, and it has been shown that such information reduces the number of men who initially wanted to be screened.\nBased on population data it is possible to provide risk assessments for every step of the screening procedure. Such assessments produce an individual risk calculation based on relevant risk parameters. This might support patients and doctors in their decision to follow or refrain from further steps, dependent on their interpretation of the risk calculated. At the time of cancer diagnosis, it may provide information on the risk of the presence of an indolent tumor, as discussed above.\nCurrent risk calculators have incorporated family history, age, micturition complaints, PSA, DRE, TRUS results, and histologic features of the prostate biopsies into their assessment. With an increasing number of relevant parameters, the level of predictive accuracy is enhanced. It is likely that new parameters might be added once tested in population-based biorepositories. Candidate parameters therefore obviously are serum and urine markers for the early diagnostic steps, and histologic markers at the time that biopsies have been taken.\nMuch interest has been given to the increase of specificity of the biopsy procedure in the general population with serum markers like PSA isoforms and kallekreins in order to diminish the number of false negative biopsies [26, 27]. Enhancing specificity always resulted in a reduction of sensitivity of prostate cancers. As we have seen based on autopsy incidence, the number of potentially detectable tumors is manifold the number of currently diagnosed cancers. It is therefore not the absolute number, but the number of clinically relevant cancers that is of interest [28]. The PSA isoforms and kallekreins should therefore ideally be related to the characteristics of the cancers detected.\nScreening efficiency\nA small number of studies already have provided evidence that supports strategies to reduce the number of screens in the general population. Such strategies will inevitably lead to the reduction of overdiagnosis of prostate cancer.\nInformation obtained from population based screening studies illustrates that the absolute value of serum PSA is related to the proportion of detectable carcinomas in subsequent screening rounds. Data from the ERSPC as well as from the PLCO [29, 30] showed that men with a PSA less than 1\u00a0ng\/ml did not develop invasive cancer over the time period of more than 5\u00a0years of repeated check-ups. In the Rotterdam site of the ERSPC, 1703 men with an initial PSA of less than 1\u00a0ng\/ml men were followed during two consecutive 4\u00a0year screening rounds. Eighty percent of men attended the second screening round, and 77% the third round. In total, only 8 cancers were found in 47 prostate biopsies on the indication of PSA of >3\u00a0ng\/ml. In the PLCO screening, every 5\u00a0years for baseline PSA less than 1\u00a0ng\/ml and every 2\u00a0years for PSA 1\u20132\u00a0ng\/ml could result in a 50% reduction in PSA tests and in less than 1.5% of men missing earlier positive screens.\nIn men who were enrolled onto a cardiovascular study in Sweden, 21,277 men aged <50\u00a0years old were assessed over a period of more than 20\u00a0years starting between 1974 and 1986. Two decades later, 498 (2.3%) were eventually diagnosed with prostate cancer (outside a structured screening procedure). In retrospect, the level of serum kallikreins (hK2, total PSA, and free PSA) at baseline and thereafter were strongly associated with emerging prostate cancer. This supports the idea of risk stratification for screening on prostate cancer in an early age, that is during the fourth decade of life. Men at low risk may refrain from frequent serum testing for long periods of time based on their individual risk assessment that incorporates the information obtained from currently available and newly validated parameters.\nOvertreatment\nSide effects of treatment are substantial\nTreatment for prostate cancer may involve surgery, external beam radiation therapy, brachytherapy, high intensity focused ultrasound (HIFU), watchful waiting, active surveillance, chemotherapy, cryosurgery, hormonal therapy, or combinations. The most frequently applied treatments for organ confined prostate cancer are radical prostatectomy, external beam radiotherapy and brachytherapy.\nAlthough, severe or life-threatening complications with radical prostatectomy are rare, the adverse effects of greatest concern are damage to the urinary sphincter and erectile nerves (nervi erigenti), resulting in urinary incontinence and impotence, respectively. Complete incontinence is uncommon after radical prostatectomy, although a significant number of men experience some degree of stress-urinary incontinence [31\u201333]. In the Prostate Cancer Outcomes Study, a population-based study of 1,291 men who underwent radical prostatectomy for localized prostate cancer and were followed for 2\u00a0years, 1.6% reported no urinary control at 24\u00a0months following surgery (compared with 0.7% at baseline prior to surgery), while 7 and 42% reported frequent and occasional leakage, respectively (compared with 2 and 9% at baseline) [32]. Age had an impact on the degree of incontinence; 14% of men aged between 75 and 79\u00a0years experienced the highest level of incontinence compared with 0.7\u20134% of younger men. In the Prostate Cancer Outcomes Study, 42% of men reported that sexual performance was a moderate to large problem at 24\u00a0months (compared with 18% at baseline); 60% were not able to have erections firm enough for sexual intercourse (compared with 16% at baseline) [32]. At 24\u00a0months postoperatively, men over the age of 60 were more likely to be impotent than younger men (78\u201385 vs. 61%, respectively).\nComplications after external beam radiotherapy include bladder irritation (urgency, pain, frequency) in up to 5% of men, and impotence in 40\u201350% [34]. In contrast to surgery, these complications tend to increase over time. The reported incidence of radiation proctitis ranges from 2 to 39%, depending upon the definition used, and the dose field, and technique of radiotherapy. Prostate inflammation and swelling can occur acutely following brachytherapy, suggesting that men with significant urinary symptoms or a large prostate are not good candidates. Urinary retention can be severe enough to require self-catheterization; transurethral resection to improve micturition is contraindicated until a substantial portion of the radioactivity (usually five half-lives) has dissipated because of the risk of incontinence and radiation risks to the surgeon and pathologist. Later complications include irritative voiding symptoms, urinary retention, rectal urgency, bowel frequency, rectal bleeding or ulceration, and prostatorectal fistulas [35\u201337]. The incidence of erectile dysfunction ranges from 14 to 52%, depending on whether it is physician- or patient-reported.\nIt is obvious that invasive treatment may influence the quality of life of men with prostate cancer and their families substantially. But so does a potential threat of prostate cancer that is not actively treated or not even diagnosed yet. It is unlikely that quality of life studies will be able to indicate the best balance between these points of view for management decisions on an individual patient level.\nActive surveillance as alternative to invasive treatment\nBecause not all cancers diagnosed require treatment, one of the major challenges for the future is to determine which diagnosed cancers should be treated, and which can be managed by active surveillance. Active Surveillance manages selected men with prostate cancer expectantly with curative intent. This means men are carefully selected and subsequently actively observed in order to have the possibility to offer them deferred curative treatment once the tumor seems to progress. Active surveillance should be clearly differentiated from watchful waiting. Watchful waiting entails a strategy for all men who are managed expectantly, whereas active surveillance focuses on men for whom therapy is delayed until the tumor becomes progressive and curative treatment can be offered. This offers an attitude of active control over the cancer diagnosed for patients and their doctors. The stage migration that screening provides has resulted in an over-representation of low-risk cancers. Therefore, studies which validate monitoring algorithms in active surveillance regimens are ongoing [38].\nRisk stratification for indolent disease\nOver the last decade, a number of nomograms have been composed to predict the presence of an indolent cancer [39, 40]. The identification of indolent cancers was strongly based on histologic information of prostate biopsies, and power of Gleason score as a predictive parameter for aggressiveness of prostate cancer was unsurpassed. As a single serum parameter at the time of diagnosis, the level of PSA contributed most to prognosis.\nThese nomograms were based on extensive clinical series. A new nomogram recently appeared based on information obtained from a screening series of the general population [41]. Screening series differ from clinical studies, as the incidence of indolent cancer is almost 50% compared to maximal 20% in multicentred clinical series. With the use of the nomogram, at a 70% probability cut-off, at least 69% of all indolent cancers would be diagnosed as such, and be treated with active surveillance.\nConclusions\nIt is still too early to say whether population-based prostate cancer screening is a useful tool with regard to cancer mortality. We must wait until the results of ongoing prostate cancer screening trials are available. Until then, opportunistic screening should not be encouraged and those men who do want a PSA test should participate in carefully designed, balanced information program. Even if PSA screening is found to reduce prostate-cancer-specific mortality, levels of overdiagnosis may remain unacceptable for population-based screening.\nTo reduce overdiagnosis in a screening setting, markers are needed that reduce the risk on a positive prostate biopsy, increasing the specificity of this procedure. Men from the age of forty, as well as their advising doctors, need instruments to reduce their doubts and anxiety of the potential presence of a prostate cancer. This, together with balanced information about the benefits and risks of the individual outcome of screening procedures, might induce a more selective and step-wise screening action. Risk assessment, incorporating the main determinants known for the presence of prostate cancer from the age of 50, such as age, family history, and micturition complaints, should form the base of an individual screening approach. Objective values of serum markers might enhance the accuracy of such of risk predictors.\nVarious efforts are performed to find new markers in the proteome and genome of blood and urine. Based on large and longitudinal serum collections of men diagnosed with prostate cancer in screening settings, the EC-sponsored P-MARK consortium evaluates candidate markers as prognostic tools [42].\nUntil alternative screening tools are found, PSA will continue to be used, and overdiagnosis will remain an unavoidable drawback of prostate cancer screening. The current challenge is to ensure that in the still growing numbers of men diagnosed with prostate cancer world-wide, overdiagnosis does not result in overtreatment. To this end, research efforts presently focus on clarifying which cancers can be managed through active surveillance.","keyphrases":["overdiagnosis","overtreatment","prostate cancer","active surveillance","rationale for screening"],"prmu":["P","P","P","P","P"]}
{"id":"J_Mol_Model-3-1-2039870","title":"\u03c0-\u03c0 stacking tackled with density functional theory\n","text":"Through comparison with ab initio reference data, we have evaluated the performance of various density functionals for describing \u03c0-\u03c0 interactions as a function of the geometry between two stacked benzenes or benzene analogs, between two stacked DNA bases, and between two stacked Watson\u2013Crick pairs. Our main purpose is to find a robust and computationally efficient density functional to be used specifically and only for describing \u03c0-\u03c0 stacking interactions in DNA and other biological molecules in the framework of our recently developed QM\/QM approach \"QUILD\". In line with previous studies, most standard density functionals recover, at best, only part of the favorable stacking interactions. An exception is the new KT1 functional, which correctly yields bound \u03c0-stacked structures. Surprisingly, a similarly good performance is achieved with the computationally very robust and efficient local density approximation (LDA). Furthermore, we show that classical electrostatic interactions determine the shape and depth of the \u03c0-\u03c0 stacking potential energy surface.\nIntroduction\nTwo major factors for the structure and stability of DNA are the hydrogen bonds between two adjacent bases from opposite strands (forming a Watson-Crick pair), and the \u03c0-\u03c0 stacking interactions between two bases within each of the two DNA strands. The hydrogen bonding interactions in DNA are well understood by high-level Density Functional Theory (DFT) calculations [1\u20133]. They were shown to be provided by nearly equally important contributions from electrostatic attraction and donor-acceptor orbital interactions (e.g., from an N lone pair to an N-H \u03c3* orbital), and to be sensitive to effects from the molecular environment (counter-ions, solvent) [1, 2]. A similar understanding of the \u03c0-stacking interactions is currently lacking due to several reasons: (1) \u03c0-\u03c0 stacking interactions have proven to be sensitive to the methodology used (see below); (2) \u03c0-\u03c0 stacking interactions have proven to be very dependent on the size of the basis set used in (ab initio) calculations, resulting in a large basis-set superposition error (BSSE); (3) high-level ab initio benchmark studies (CCSD(T) with at least triple-zeta basis sets) have only recently become available; and (4) the notorious problems of DFT to correctly describe dispersion interactions, [4\u201316] which constitute one of the components involved in \u03c0-stacking interactions (see below). The latter problems of DFT resulted in potential energy surfaces (PES) that, erroneously, lack any equilibrium configuration, i.e., the stacked systems were incorrectly described as being purely repulsive.\nBecause of the technical difficulties involved in obtaining accurate benchmarks (CCSD(T) using large basis sets), Sponer, Hobza and co-workers have studied in a ground-breaking series of papers [17\u201322] the stacking energies of DNA bases and DNA base analogs using a variety of ab initio methods and basis sets. Earlier studies showed that reasonable interaction energies could be obtained when employing the MP2\/6-31G*(0.25) method, which was confirmed by later and more accurate studies [17\u201322]; in the aforementioned 6-31G*(0.25) basis set, the standard d-polarization functions (with exponent of 0.80) were replaced by more diffuse ones (exponent of 0.25) with the aim to improve description of the dispersion attraction [22]. Thus, for the anti-parallel displaced pyrimidine dimer, the MP2\/6-31G*(0.25) energy is only 1.1\u00a0kcal mol\u22121 lower than the CCSD(T)\/6-31++G*(0.25,0.15) energy, for the anti-parallel cytosine dimer it is 0.6\u00a0kcal mol\u22121 higher than the CCSD(T)\/6-31++G**(0.25,0.15) energy, and for the twisted uracil dimer the MP2\/6-31G*(0.25) is equal to the CCSD(T)\/6-31++G**(0.25,0.15) energy. Although there seems to be no systematic shift in energy when comparing MP2\/6-31G*(0.25) with the more elaborate CCSD(T) stacking energies, the MP2 stacking energies are always larger in magnitude than the corresponding CCSD(T) ones when using the same basis set for both methods. Interestingly, in connection with the quest for reliable ab initio benchmarks, preliminary results [23\u201327] indicate that the recently developed spin-component scaled MP2 (SCS-MP2) yields systematically lower stacking energies than MP2. This suggests that SCS-MP2 is in good agreement with the CCSD(T) results at only a fraction of the computational cost.\nSeveral approaches have been proposed to remedy the \u201cdispersion problem\u201d of DFT, ranging from Time-Dependent DFT frameworks [28] to more ad-hoc procedures such as including (C6) van der Waals terms in the interaction energy (DFT-D) [29] or fitting (reparameterizing) the form of the density functional to some reference energies and\/or distances. A more elegant solution was recently proposed by Tozer and co-workers [30], who analyzed the DFT exchange-correlation (xc) potential in comparison with the near-exact multiplicative Zhang-Morrison-Parr (ZMP) potential from coupled cluster Brueckner doubles (BD) densities. Their resulting KT1, and the related KT2, functional [30] produces xc potentials that closely resemble those from ZMP. As a result, KT1 and KT2 produce NMR chemical shifts [4, 31] that in general show smaller deviations from experimental values than those from standard density functionals. Furthermore, the ZMP potential from Brueckner (BD(T)) coupled cluster densities gives dispersion forces in good agreement with near-exact dispersion forces [32]. It is therefore to be expected that the KT1 and KT2 functionals behave properly for dispersion forces as well. However, the application of these new functionals to \u03c0-stacking interactions has so far not appeared in the literature.\nHere, we report the results of a systematic investigation of \u03c0-stacking interaction energies, potential energy surfaces (PES) and stacking distances for dimers of, among others, the five DNA\/RNA bases. A long list of standard as well as recent density functionals (including the KT1 and KT2 functionals) has been considered in this study, ranging from the Local Density Approximation (LDA), via the Generalized Gradient Approximation (GGA), and meta-GGA, to hybrid functionals. We have also examined BHandH, which was recently [12] shown to give reasonable agreement with ab initio data. The DFT results are compared to benchmark data that were taken from the literature, which in some cases contained energy profiles as a function of the torsion associated with the rotation of one base with respect to the other.\nThe present investigation is part of a larger undertaking. We aim at developing an extended QM\/QM scheme, designated QUILD [33], which allows a (complex) molecular system to be decomposed into arbitrary, interpenetrating domains such that each type of interaction can be described with a different method, i.e., the density functional that performs best for that type of interaction or process, for example, electron-pair as well as hydrogen bonding (BP86: see [2, 3]), \u03c0-\u03c0 stacking (LDA, KT1, KT2: this work) or reaction barriers (OLYP, OPBE: see [34, 35]). This is a pragmatic (of course not a fundamental) approach to cope with the present-day shortcomings of DFT, which features density functionals that perform satisfactorily for a number but not all types of interactions and phenomena. The present study focuses on identifying which DFT approaches serve best to describe the \u03c0-\u03c0 stacking interactions in the above-mentioned QM\/QM scheme.\nFurthermore, we have analyzed the \u03c0-stacking interactions in more detail by decomposing the interaction energy into three physically meaningful components, namely, electrostatic energy, Pauli repulsion (i.e., the destabilizing interactions between occupied orbitals), and bonding orbital interactions [36]. Our analyses reveal, among others, that the electrostatic attraction between the stacked molecules is the most important component of the \u03c0-\u03c0 stacking interaction (see also [37]) which determines the shape and depth of the PES. We have also analyzed the \u03c0-\u03c0 interactions between two stacked Watson\u2013Crick base pairs with the purpose to differentiate between inter- and intra-strand stacking interactions.\nMethods\nComputational details\nAll calculations were performed with the Amsterdam Density Functional (ADF) program. [38\u201349] Molecular orbitals (MOs) were expanded using a large, uncontracted set of Slater-type orbitals: TZ2P [40]. The TZ2P basis is an all-electron basis of triple-\u03b6 quality, augmented by two sets of polarization functions (2p and 3d on H; d and f on heavy atoms). An auxiliary set of s, p, d, f, and g STOs was used to fit the molecular density and to represent the Coulomb and exchange potentials accurately in each SCF cycle.\nThe equilibrium structure of individual benzene analogs, DNA bases and Watson-Crick base pairs were optimized at the BP86 level of DFT, i.e., using the local density approximation (LDA; Slater exchange and VWN [42] correlation) with non-local corrections due to Becke [43] (exchange) and Perdew [44] (correlation) added self-consistently, which was previously shown to be one of the best DFT methods for the accuracy of geometries [41, 45] and hydrogen-bonding interactions [1\u20133]. The \u03c0-\u03c0 stacking interaction energies for all density functionals were evaluated post-SCF using LDA as SCF functional. This procedure was recently shown to introduce an error of only a few tenths of a kcal mol\u22121 [46].\nStacking distances (vertical separation) and orientations (twist angle) were explored with the various density functionals through scans of the potential energy surface (PES) in which the BP86 geometries of the monomers (e.g., DNA bases or Watson\u2013Crick base pairs) were kept frozen. PES scans as a function of the twist angle (see below) were done using steps of 30\u00b0 in case of homo-base stacks, 60\u00b0 in the case of hetero-base stacks and 36\u00b0 for stacks of Watson\u2013Crick base pairs. PES scans as a function of the vertical separation (see below) were done in steps of 0.1\u00a0\u00c5.\nThe interaction energy (see below) was studied as a function of the vertical separation between the two monomers in the \u03c0-stacked dimer, and as a function of the twist angle between these monomers. The latter is defined as the right-handed rotation of the upper base around the axis that runs through the center of mass of both the upper and lower base, and perpendicular to the plane of the base (see Scheme\u00a01).\nScheme\u00a01Definition of the twist angle (TW). The black dot is the center of mass\nIn the stacked DNA systems, the twist angle of 0\u00b0 is defined as that twist angle at which the respective \u201cglycosidic\u201d N-H bonds (more precisely, the N-H bonds that replace the glycosidic N-C bonds to the sugar in the backbone) are oriented in parallel.\nBond-energy decomposition\nThe overall \u03c0-stacking energy \u0394E is made up of two major components [36] (Equation\u00a01):\nIn this formula, the preparation energy \u0394Eprep is the energy needed to deform the separate molecular fragments from their equilibrium structure to the geometry that they attain in the overall molecular system. The interaction energy \u0394Eint is the energy released when the prepared fragments are brought together into the position they have in the overall molecule. It is analyzed for our model systems in the framework of the KS-MO model using a Morokuma-type decomposition [47] into electrostatic interaction, Pauli repulsion (or exchange repulsion), and (attractive) orbital interactions (Equation\u00a02).\nThe term \u0394Velstat corresponds to the classical electrostatic interaction between the unperturbed charge distributions of the prepared (i.e. deformed) fragments and is usually attractive. The Pauli-repulsion, \u0394EPauli, comprises the destabilizing interactions between occupied orbitals and is responsible for the steric repulsion. The orbital interaction \u0394Eoi in any MO model, and therefore also in Kohn-Sham theory, accounts for electron-pair bonding, charge transfer (i.e., donor-acceptor interactions between occupied orbitals on one fragment with unoccupied orbitals of the other, including the HOMO-LUMO interactions), and polarization (empty-occupied orbital mixing on one fragment due to the presence of another fragment). The orbital interaction energy can be further decomposed into the contributions from each irreducible representation \u2118 of the interacting system (Equation\u00a03) using the extended transition state (ETS) scheme developed by Ziegler and Rauk [48, 49].\nResults and discussion\nWe have analyzed \u03c0-\u03c0 stacking interactions using different systems that were taken mainly from reference data present in the literature. We start with the prototypical system for studying \u03c0-\u03c0 stacking, namely, the benzene dimer. The second set of reference data was taken from the supporting information of a paper by Mignon and co-workers [50], who studied hydrogen bonding and stacking in complexes of substituted benzene C6H5X with pyridine at MP2\/6-31G*(0.25) with substituents X = H, F, NH2, Cl, CH3, OH, CN, COOH, CHO and NO2. All substitutions of H by another X in benzene were done at the para-position with respect to the nitrogen of pyridine (see Scheme\u00a02). The third set of reference data was taken from a paper by Jurecka and co-workers [21], who investigated hydrogen bonding and stacking interactions in the cytosine dimer with MP2 and CCSD(T) methods, using several orientations (see Scheme\u00a03) to investigate the potential energy surfaces. The fourth set of reference data was taken from a paper by Wu and Yang [14] (based on previous work by Hobza, Sponer and co-workers [17, 19, 22]) who studied the dependence of the stacking energy of DNA bases on the twist angle (see Scheme\u00a04) and vertical separation using MP2\/6-31G*(0.25) calculations.\nScheme\u00a02Stack of substituted benzene and pyridineScheme\u00a03Orientations of the stacked cytosine dimer from Jurecka and co-workers ([21]). The upper base is drawn in bold, the center of mass is indicated by a black dot, and the carbonylic oxygen and the hydrogen replacing the glycosidic bond are indicated for clarityScheme\u00a04Geometries of stacked base dimers with a twist angle of 0 and 120 degrees. The upper base is drawn in bold, the center of mass is indicated by a black dot, and the hydrogens replacing the glycosidic bond are indicated for clarity. Note that for the homo dimers at zero twist angle the lower base is hidden behind the upper base\nAs mentioned already, the prototypical system for studying \u03c0-\u03c0 stacking interactions is posed by the (parallel sandwich) benzene dimer. Because of its high symmetry (D6h), CCSD(T) energies using large basis sets (aug-cc-pVQZ) are available [51] to compare with (see Table\u00a01). The minimum is found at 3.9\u00a0\u00c5 for this system with a stacking energy of \u22121.7\u00a0kcal mol\u22121. The standard density functionals are unable to give a proper description of this system, i.e., they either predict a purely repulsive energy surface (e.g., BP86; see Table\u00a01), or give a shallow well (e.g., OLYP). Interestingly, the KT1 functional predicts a minimum that is very close (3.8\u20133.9\u00a0\u00c5) to that of the CCSD(T) method, and has a well depth that is equally close (\u22121.6\u00a0kcal mol\u22121; see Table\u00a01). The related KT2 and, surprisingly, the LDA functionals have a minimum at the same distance as CCSD(T) and KT1, but with a somewhat smaller well depth (\u22121.3\u00a0kcal mol\u22121). Also, the BHandH functional performs reasonably well with a similar equilibrium distance, but a reduced well depth (\u22120.9\u00a0kcal mol\u22121; see Table\u00a01).\nTable\u00a01Stacking energy (in kcal mol\u22121) of the benzene-benzene complex as a function of the vertical separation Rvert (in \u00c5), computed with various density functionalsa and CCSD(T)\/aug-cc-pVQZ*bRvertCCSD(T)bLDAKT1KT2BHandHPW91BLYPBP86OLYPB3LYPX3LYP2.913.5314.5914.0817.0824.4131.6327.4638.6730.1328.923.08.609.299.0411.2717.9824.2020.8430.9322.7821.653.15.115.525.477.1313.1818.5315.8724.8517.2316.173.23.712.702.902.984.239.6014.2212.1420.0513.0412.063.31.681.061.101.292.246.9310.949.3416.229.898.983.40.30\u22120.01\u22120.090.170.924.958.457.2513.147.526.683.5\u22120.62\u22120.68\u22120.83\u22120.540.063.486.555.6810.655.734.963.6\u22121.19\u22121.07\u22121.27\u22120.96\u22120.452.405.104.508.634.403.693.7\u22121.51\u22121.27\u22121.50\u22121.17\u22120.741.593.993.626.983.392.743.8\u22121.66\u22121.33\u22121.58\u22121.25\u22120.861.003.152.945.622.632.043.9\u22121.70\u22121.31\u22121.56\u22121.25\u22120.890.572.502.434.512.061.524.0\u22121.67\u22121.24\u22121.48\u22121.18\u22120.850.252.002.033.601.631.144.1\u22121.58\u22121.13\u22121.37\u22121.09\u22120.780.021.611.722.851.300.854.2\u22121.46\u22121.02\u22121.24\u22120.98\u22120.68\u22120.151.311.472.241.050.644.3\u22120.90\u22121.11\u22120.86\u22120.58\u22120.271.071.271.750.860.494.4\u22120.78\u22120.97\u22120.75\u22120.48\u22120.360.881.111.350.710.374.5\u22121.08\u22120.67\u22120.85\u22120.64\u22120.39\u22120.420.730.971.030.590.285.0\u22120.58\u22120.28\u22120.39\u22120.26\u22120.06\u22120.450.300.520.200.260.095.5\u22120.27\u22120.09\u22120.15\u22120.070.08\u22120.310.130.280.000.140.046.0\u22120.11\u22120.01\u22120.050.000.12\u22120.160.050.15\u22120.030.080.036.5\u22120.040.020.000.030.12\u22120.060.020.08\u22120.030.050.037.00.030.020.040.11\u22120.010.000.05\u22120.010.040.037.50.030.020.040.090.020.000.03\u22120.010.030.038.00.030.020.090.080.01\u22120.010.020.000.020.02aPost-SCF using LDA\/TZ2P orbitals and densities, see Computational detailsbFrom supporting information of [51]\nReference data from Mignon and co-workers\nScheme\u00a02 shows the structure of the \u03c0-stacked complexes of pyridine with (substituted) benzene, i.e. with benzene, fluorobenzene, aminobenzene, chlorobenzene, toluene, phenol, cyanobenzene, benzoic acid, benzaldehyde and nitrobenzene. For all of these complexes the coordinates were taken from the supporting information of the original paper by Mignon and co-workers [50]. The geometry of each dimer had been fully optimized and therefore the vertical separation is different for each complex; it is found in the range of 3.2\u20133.4\u00a0\u00c5.\nThe interaction energies for several typical density functionals are compared to the original MP2 data in Table\u00a02. Consistent with previous reports, the mean absolute deviations (MAD) with respect to the reference MP2 data are much larger for density functionals containing the Becke88 [43] exchange functional (e.g., BP86, MAD value 7.68\u00a0kcal mol\u22121) than for functionals based on PW91 [52] or PBE [53] exchange (e.g., PW91, MAD value 5.33\u00a0kcal mol\u22121). However, these standard density functionals all show repulsive interactions, in contrast to the MP2 results that indicate bound systems (see Table\u00a02). Much better agreement with the MP2 data is observed for the KT1, KT2 and, surprisingly, LDA functionals, with MAD values of 0.25\u00a0kcal mol\u22121 (see Table\u00a02). The BHandH functional, recently [12] shown to give good agreement with MP2\/CCSD(T) energies, shows a larger MAD value of 0.4\u00a0kcal mol\u22121 (see Table\u00a02). Because of the uncertainty of ca. \u00b1 0.5\u00a0kcal mol\u22121 in the reference MP2 data with respect to the high-level CCSD(T) data (see above), it is uncertain which of the four density functionals (LDA, KT1 , KT2 or BHandH) performs best. However, the improvement over standard density functionals is obvious and promising.\nTable\u00a02Stacking energy (in kcal mol\u22121) of substituted benzene-pyridine complexes (see Scheme\u00a02), computed with various density functionalsa and MP2\/6-31G*(0.25)bX=MP2bLDAKT1KT2BHandHPW91BLYPBP86OLYPB3LYPX3LYPH\u22122.8\u22123.4\u22123.4\u22123.2\u22122.72.36.14.611.15.04.1F\u22122.9\u22123.4\u22123.3\u22123.2\u22122.82.56.44.911.55.14.2NH2\u22123.2\u22123.6\u22123.6\u22123.3\u22123.02.26.14.711.24.93.9Cl\u22123.4\u22123.4\u22123.4\u22123.2\u22122.82.66.75.111.95.44.4CH3\u22123.3\u22123.3\u22123.4\u22123.2\u22122.81.24.53.58.73.52.7OH\u22122.7\u22123.2\u22123.1\u22123.0\u22122.52.86.85.211.95.54.6CN\u22124.1\u22123.9\u22123.9\u22123.7\u22123.50.94.23.18.43.12.3COOH\u22123.5\u22123.6\u22123.6\u22123.4\u22123.02.46.44.811.55.14.2CHO\u22123.9\u22123.8\u22123.8\u22123.6\u22123.30.94.23.28.43.22.4NO2\u22123.8\u22123.8\u22123.7\u22123.6\u22123.32.05.84.310.84.63.7MADc0.260.250.240.395.339.077.6813.907.896.99aPost-SCF on LDA\/TZ2P orbitals and densities, see Computational detailsbFrom [50]cMean Absolute Deviation (in kcal mol\u22121) of DFT energies from reference MP2 data\nReference data from Jurecka and co-workers\nScheme\u00a03 shows the fourteen orientations of the stacked cytosine dimer that have been considered in the paper by Jurecka and co-workers [21] (with a vertical separation of 3.3\u00a0\u00c5 for the first orientation, and 3.4\u00a0\u00c5 for all others). The corresponding reference energies and our DFT interaction energies are collected in Table\u00a03. The latter also contains the MAD values with respect to two sets of reference energies, i.e. the usual MP2\/6-31G*(0.25) results (MAD1) as well as the MP2 energies obtained after complete-basis-set (CBS) extrapolation corrected for the difference between MP2 and CCSD(T) with the 6-31G*(0.25) basis set (MAD2). Similar to what was found for the benzene-pyridine systems (see above), the MAD values for standard density functionals are found between 5 and 14\u00a0kcal mol\u22121, resulting in many cases in erroneous repulsive stacking interactions between the cytosine fragments (see Table\u00a03). Improved results are again obtained with LDA, KT1, KT2, and to a lesser extent BHandH, which have MAD1 values (i.e. deviations with respect to MP2\/6-31G*(0.25)) of 0.9, 0.8, 0.6 and 1.5\u00a0kcal mol\u22121 respectively, and MAD2 values of 0.4, 0.5, 0.7 and 0.5\u00a0kcal mol\u22121 respectively with the CCSD(T) data (see Table\u00a03).\nTable\u00a03Stacking energies (in kcal mol\u22121) for several orientations of the cytosine dimer (see Scheme\u00a03), computed with various density functionals and MP2a,borientationMP2b,cMP2ccb,dLDAKT1KT2BHandHPW91BLYPBP86OLYPB3LYPX3LYP12.22.52.72.32.73.98.011.710.916.510.99.92\u22123.1\u22123.8\u22123.7\u22123.9\u22123.5\u22123.32.66.55.412.35.24.13\u22127.2\u22128.9\u22128.8\u22128.7\u22128.3\u22129.0\u22122.11.70.78.00.2\u22121.04\u22128.3\u22129.9\u22129.4\u22129.2\u22128.9\u221210.1\u22122.90.8\u22120.16.9\u22120.9\u22122.150.20.30.60.10.61.56.410.39.315.79.38.360.50.60.80.40.81.86.810.89.716.39.88.87\u22120.5\u22121.0\u22120.7\u22120.9\u22120.60.22.85.55.28.75.04.28\u22127.3\u22129.1\u22128.4\u22128.3\u22127.9\u22128.8\u22123.00.2\u22120.25.6\u22121.2\u22122.29\u22127.6\u22129.1\u22128.7\u22128.5\u22128.2\u22129.4\u22122.31.50.57.3\u22120.2\u22121.410\u22126.6\u22128.3\u22127.9\u22127.7\u22127.5\u22128.3\u22121.81.80.97.60.3\u22120.811\u22127.6\u22129.4\u22128.8\u22128.5\u22128.3\u22129.5\u22122.80.6\u22120.16.2\u22121.0\u22122.112\u22125.5\u22127.4\u22126.7\u22126.6\u22126.2\u22127.0\u22123.6\u22121.6\u22121.41.5\u22122.4\u22123.113\u22127.4\u22128.8\u22128.3\u22128.1\u22127.9\u22128.6\u22123.00.4\u22120.45.2\u22121.0\u22122.014\u22127.0\u22129.1\u22128.8\u22128.7\u22128.4\u22129.4\u22122.11.60.78.0\u22120.1\u22121.3MAD1e0.940.810.601.494.878.357.5813.637.066.03MAD2f0.380.470.720.526.049.528.7514.808.247.21aPost-SCF on LDA\/TZ2P orbitals and densities, see Computational detailsbFrom [21]cMP2\/6-31G*(0.25) resultsdMP2 with complete-basis-set (CBS) extrapolation, i.e. MP2\/CBS, corrected for \u0394[CCSD(T)\/6-31G*(0.25) - MP2\/6-31G*(0.25)]eMean Absolute Deviation (in kcal mol\u22121) of DFT energies from MP2\/6-31G*(0.25) datafMean Absolute Deviation (in kcal mol\u22121) of DFT energies from MP2\/CBS corrected for \u0394[CCSD(T)\/6-31G*(0.25) - MP2\/6-31G*(0.25)]\nReference data from Wu and Yang\nIn the paper by Wu and Yang, [14] the potential energy surface (PES) was calculated as a function of the twist angle between two stacked bases (see Scheme\u00a01 and methodological section for the definition of the twist angle) using MP2\/6-31G*(0.25), which was chosen as a reliable reference method based on the paper by Hobza and co-workers [22]. Although thymine was not included by Wu and Yang, we report here the energy profiles for all combinations of the five RNA\/DNA bases, i.e., thymine, adenine, guanine, cytosine, uracil (see Scheme\u00a04 for the geometries). The vertical separation is 3.4\u00a0\u00c5 for the C-C, G-C and G-G stacked base dimers, and 3.3\u00a0\u00c5 for all other systems. This difference in vertical separations was chosen based on earlier papers by Sponer and co-workers [19].\nFrom the tests with the benzene dimer (Table\u00a01), the Mignon (Table\u00a02) and Jurecka reference data (Table\u00a03), it is already evident that standard density functionals do not give reliable interaction energies, which is now shown to be true also for the energy profiles as function of the twist angle. Figure\u00a01 shows the PES for two stacked bases, namely C-C and G-U, which are representative for all possible base pair combinations (the complete figure for all combinations of stacked bases can be found in the Supporting Information). Interestingly, although the standard density functionals typically underestimate the interaction energy, the shape of the PES is in all cases highly similar to that based on the more accurate MP2 data. This suggests that, at least trends and qualitative features in the rotational energy profile can be correctly reproduced with DFT which otherwise shows a (functional and basis-set dependent) constant shift with respect to the more accurate MP2 rotational profile.\nFig.\u00a01Stacking interaction (in kcal mol\u22121) as function of the twist angle (in degree) for \u03c0-\u03c0 stacks of two bases, C-C and G-U, computed with various density functionals. The thick lineswithfilled circles, triangles, squaresand diamonds show the results obtained with LDA, PW91, BLYP, and BP86, respectively. The dashed lines with open squares, trianglesand diamonds show the results obtained with OLYP, B3LYP and X3LYP, respectively. The bold linewithcrosses represents the MP2\/6-31G*(0.25) reference data\nIt is, however, more instructive to look at functionals that by themselves already give accurate interaction energies, such as LDA, KT1, KT2 and BHandH. Figure\u00a02 shows the corresponding rotational-energy profiles, again for the stacked bases C-C and G-U. From Fig.\u00a02 it is evident that the LDA, KT1, KT2, and to a lesser extent BHandH are visually (and virtually) indistinguishable from the reference MP2 data.\nFig.\u00a02Stacking interaction (in kcal mol\u22121) as function of the twist angle (in degree) for \u03c0-\u03c0 stacks of two bases, C-C and G-U, computed with various density functionals. The red, blue and green lines show the results obtained with BHandH, KT1 and KT2, respectively. The bold line with crosses represents the MP2\/6-31G*(0.25) reference data\nFigure\u00a03 shows the interaction energy as a function of the vertical separation for both the standard (upper diagrams) and promising (lower diagrams) density functionals, together with MP2 reference curve in each of the four diagrams. Not surprisingly, the density functionals that gave larger errors for the rotational energy profile, also give larger errors for the vertical separation profiles: almost all standard density functionals show potential energy surfaces that are repulsive, or at best weakly attractive with a very shallow minimum at elongated distances. In contrast, the promising functionals (i.e., KT1, KT2, LDA and BHandH) do provide well-defined minima with equilibrium distances (3.2\u20133.3\u00a0\u00c5) close to the reference MP2 data. Similar to the uncertainty in the reference energy data of some 0.5\u20131.0\u00a0kcal mol\u22121 (see above), we may expect a similar uncertainty for the vertical separation, which we very roughly estimate at 0.1\u20130.2\u00a0\u00c5. The differences between the equilibrium distance of the promising density functionals and that of the reference MP2 falls well within this estimated uncertainty. Thus, KT1, KT2, LDA and BHandH perform well not only for rotational energy profiles but also for the PES as a function of the vertical separation.\nFig.\u00a03Stacking interaction (in kcal mol\u22121) as function of the vertical separation (in \u00c5) for \u03c0-\u03c0 stacks of two bases, A-A and G-G, computed with various density functionals. The thick lines with filled triangles, squares and diamonds in the upper diagrams show the results obtained with PW91, BLYP, and BP86, respectively. The dashed lines with open squares, triangles and diamonds in the upper diagrams show the results obtained with OLYP, B3LYP and X3LYP, respectively. The red, blue, green and orange lines in the lower diagrams that truly show well-defined minima are obtained with BHandH, KT1, KT2 and LDA respectively. The bold black line with crosses represents the MP2\/6-31G*(0.25) reference data\nDecomposition of interaction energy\nNow that we have established the reliability of the KT1, KT2, LDA and BHandH functionals for the interaction energy of stacked DNA bases, it is interesting to study the actual interaction into greater detail. This is done through a quantitative decomposition of the interaction energy \u0394Eint into electrostatic attraction \u0394Velstat, Pauli repulsion \u0394EPauli and orbital interactions \u0394Eoi (see Methods). Table\u00a04 provides the results of the energy decomposition at KT1\/TZ2P for the five \u03c0-stacked homo dimers, for each one at a twist angle of 0\u00b0 and 180\u00b0 (which is the lowest-energy conformation) and at two different vertical separations. Note that the lowest-energy conformation is always achieved at a twist angle of 180\u00b0 but that the optimal stacking distance (vertical separation) varies by 0.1 \u2013 0.2\u00a0\u00c5 between the different \u03c0-stacked homo dimers. The energy changes by \u22126.8 (A-A), \u221211.8 (C-C), \u221210.2 (G\u2013G), \u221211.2 (T-T) and \u22127.9 (U-U) kcal mol\u22121, respectively, when going from a twist angle of 0\u00b0 to 180\u00b0 (the lowest-energy conformation). This change in energy is almost completely resulting from a change in electrostatics that change by \u22125.4 (A-A), \u221213.1 (C-C), \u221210.3 (G-G), \u22126.6 (T-T) and \u22128.3 (U-U) kcal mol\u22121 respectively. The sum of Pauli repulsion and orbital interactions changes much less, by ca. 1.4\u00a0kcal mol\u22121 or less, except for T-T where the sum of Pauli repulsion and orbital interactions changes by 4.6\u00a0kcal mol\u22121 due to the influence of the peripheral methyl groups. But also the changes in each term individually, i.e., either \u0394EPauli or \u0394Eoi, are much smaller (typically 2 to 5 times, but in some cases even more) than the changes in \u0394Velstat (again, only in the case of T-T, the change in \u0394EPauli is nearly as large as that in \u0394Velstat). Here we note that the same energy decomposition analyses for the five \u03c0-stacked homo dimers at LDA\/TZ2P instead of KT1\/TZ2P yield values that differ by only 1 kcal mol\u22121 or less (not shown in Table\u00a04).\nTable\u00a04Decomposition of interaction energiesa (in kcal mol\u22121) between stacked DNA bases in different geometries, computed at KT1\/TZ2P\u00a0A-AC-CG-GT-TU-UTwist angle 0\u00b0(TW, Rvert)(0\u00b0, 3.3 \u00c5)(0\u00b0, 3.4 \u00c5)(0\u00b0, 3.4 \u00c5)(0\u00b0, 3.3 \u00c5)(0\u00b0, 3.3 \u00c5)\u0394EPauli5.352.432.1311.003.72\u0394Velstat\u22123.311.801.97\u22122.48\u22120.07\u0394Eoi\u22122.30\u22121.88\u22122.49\u22125.66\u22122.11\u0394Eint\u22120.262.351.612.861.54Lowest energy conformation(TW, Rvert)(180\u00b0, 3.2 \u00c5)(180\u00b0, 3.1 \u00c5)(180\u00b0, 3.2 \u00c5)(180\u00b0, 3.3 \u00c5)(180\u00b0, 3.1 \u00c5)\u0394EPauli5.867.014.524.286.13\u0394Velstat\u22128.72\u221211.32\u22128.33\u22129.04\u22128.38\u0394Eoi\u22124.17\u22125.12\u22124.80\u22123.58\u22124.12\u0394Eint\u22127.02\u22129.44\u22128.61\u22128.34\u22126.36aSee Computational details\nAdditivity of interaction energies of stacked base pair dimers\nWith the good performance of KT1 for stacking interactions kept in mind, we went one step further, and looked also at the interactions within stacked dimersof hydrogen-bonded base pairs (see Scheme\u00a05) [54]. The geometry was obtained by taking the geometry of one hydrogen-bonded base pair and putting another one at 3.4\u00a0\u00c5 on top of the former. The base pair dimer is stabilized by hydrogen bonding between the bases within one layer, and stacking interactions between the bases in different layers of the stack. In this case, because there are two bases per layer, there are also interactions present between bases that are, in a sense diagonally, on opposite sides and in different layers [55], the so-called cross terms. For example for the AT-AT system as shown in Scheme\u00a05, this refers to the interaction between the thymine of the top layer with the adenine of the bottom layer.\nScheme\u00a05Structure of stacked DNA base pair dimer AT-AT with twist angle of 36\u00b0. The dot represents the B-DNA helical axis of rotation.[54]\nIn order to investigate in more detail the importance of the various energy terms, in particular, the cross-terms, we approximate the total stacking interaction between the base pairs in the stacked base pair dimer by the sum of four pairwise interactions between two bases, i.e., we approximate the total (stacking) interaction energy \u0394Eint between the layers AB and CD in Scheme\u00a06 by the sum \u0394Eadd of interactions A-C, B-D, A-D and B-C as shown in Equations\u00a04a,b:\nScheme\u00a06Additivity approximation for the \u03c0-\u03c0 interaction between two stacked Watson-Crick base pairs in terms of pairwise interactions between individual bases\nThe remaining term \u0394Ecoop that, added to the approximate \u0394Eadd yields again the real stacking interaction \u0394Eint, consists of cooperativity effects between the different interactions.\nIn Table\u00a05, we report the energy values resulting from the additivity scheme for the stacked AT-AT, GC-GC and AU-AU base pair dimers at three different values of the twist angle (0, 36 and 180\u00b0). Importantly, the additive approximation \u0394Eadd of the real stacking interaction \u0394Eint largely accounts for the latter. \u0394Ecoop has in most cases a positive sign and is small compared to either \u0394Eadd or \u0394Eint. Furthermore, the dependence of the \u0394Ecoop term on the twist angle is much smaller than that of the interaction energy \u0394Eint. For example, \u0394Ecoop varies from 1.0 to 1.6\u00a0kcal mol\u22121 whereas \u0394Eint varies from \u22121.4 to \u221210.7\u00a0kcal mol\u22121 for the stacked AT-AT base pair dimer (see Table\u00a05). Larger cooperativity effects are found in the case of the stacked GC-GC base pair dimer, where \u0394Ecoop values are found between 2.0 and 4.4\u00a0kcal mol\u22121. Note however that the changes in \u0394Ecoop are again small compared to the changes in \u0394Eint.\nTable\u00a05Interaction energy (kcal mol\u22121) between stacked DNA base pairs in terms of the additivity scheme, computed at KT1\/TZ2Pa,bSystemTW\u0394EAC\u0394EBD\u0394EAD\u0394EBC\u0394Eadd\u0394Ecoop\u0394EintAT-AT0\u22121.23+1.26\u22121.24\u22121.24\u22122.45+1.04\u22121.41AT-AT36\u22125.27\u22123.93\u22121.11\u22120.93\u221211.24+1.11\u221210.13AT-AT180\u22121.84+0.39\u22125.44\u22125.44\u221212.33+1.62\u221210.71AU-AU0\u22121.23+0.94\u22121.37\u22121.37\u22123.03+1.17\u22121.86AU-AU36\u22125.27\u22123.00\u22121.20\u22121.08\u221210.55+1.20\u22129.35AU-AU180\u22121.84+0.28\u22124.67\u22124.67\u221210.90+1.73\u22129.17GC-GC0+2.15+2.83\u22124.90\u22124.90\u22124.82+4.42\u22120.40GC-GC36\u22122.08\u22121.32\u22123.44\u22124.71\u221211.55+3.92\u22127.63GC-GC180\u22127.59\u22122.60\u22121.52\u22121.52\u221213.23+1.98\u221211.25aSee Scheme\u00a06: bPost-SCF on LDA\/TZ2P orbitals \/ densities.\nThe cross-terms in the additivity scheme are as important as the non-cross terms, especially when rotating the base pair dimer from a twist angle of 0 degrees to finally a twist angle of 180 degrees. At the end point of 180\u00b0, the A and B base have switched, and the cross terms are no longer AD and BC, but AC and BD; this switch occurs after a twist angle of 90\u00b0. For instance for the AT-AT base pair dimer at 0\u00b0, the cross terms are stabilizing (\u22122.5\u00a0kcal mol\u22121) and dominate the \u0394Eadd of \u22122.2\u00a0kcal mol\u22121. At 180\u00b0, the non-cross terms dominate (\u221210.9\u00a0kcal mol\u22121) with still a substantial contribution from the cross terms (\u22121.4\u00a0kcal mol\u22121). Note also the dominance of the cross terms (\u221210.2\u00a0kcal mol\u22121) over the non-cross terms (\u22123.0\u00a0kcal mol\u22121) for the GC-GC base pair dimer at 180\u00b0. This is mainly due to favorable hydrogen-bonding interactions between the cross G-G pair.\nWhen rotating base pair dimers from a twist angle of 0\u00b0 to 36\u00b0, the cross terms in the additivity scheme reduce slightly. However, this is dominated by the increase of favorable interactions between the stacked bases. For instance for AT-AT, the cross terms drop from \u22122.5 to \u22122.0\u00a0kcal mol\u22121, but the non-cross terms increase from +0.0 to \u22129.2\u00a0kcal mol\u22121. The same trend is observed for the AU-AU and GC-GC base pair dimers.\nAgain we note that the same energy decomposition analyses for stacked DNA base pairs at LDA\/TZ2P instead of KT1\/TZ2P yield values that differ by only 1\u00a0kcal mol\u22121 or less (not shown in Table\u00a05).\nSignificance of the observed trends for the structure of DNA\nThe stabilizing interactions for the structure of DNA mainly originate from hydrogen-bonding interactions between bases in one plane and the \u03c0-\u03c0 stacking interactions between stacked bases. However, we have shown here that in addition to these terms there is also an important contribution of the cross terms that arise between bases that are neither hydrogen-bonded within one layer, nor stacked above each other. All these interaction energies can now be understood and quantified with density functional theory using appropriate functionals. For the hydrogen-bonding interactions, the BP86 and PW91 functionals seem to perform best, [3] while for \u03c0-stacking interactions KT1, KT2 and (surprisingly) LDA work well.\nThe experimentally observed value for the twist angle (36\u00b0) is retrieved in our study on base pair dimers as the orientation with optimal stacking interactions. We have shown that this is predominantly determined by the classical electrostatic component of the stacking energy. The rotation to a twist angle of 36\u00b0 destroys part of the favorable cross terms (see above), which is however completely overcome by the increase in favorable stacking interactions between the stacked bases. The twist angle and vertical separation that are experimentally observed are of course influenced by both the sugar-phosphate backbone and the presence of solvent and\/or counter ions. These factors will be tackled in forthcoming work.\nConclusions\nWe have analyzed \u03c0-\u03c0 stacking interactions between two benzenes or benzene analogs, between two DNA bases, and between two Watson-Crick base pairs using Density Functional Theory (DFT) in combination with large basis sets. The interaction energies for a large number of density functionals have been compared with ab initio reference data. In line with previous studies, most standard density functionals recover, at best, only part of the favorable stacking interactions.\nAn exception is the new KT1 functional, which has been constructed to closely resemble the near-exact Zhao\u2013Morrison\u2013Parr (ZMP) exchange-correlation potential. Thus, not unexpectedly, KT1 gives a good description for \u03c0-\u03c0 stacking energies and potential energy surfaces (PES) resulting in bound systems. Surprisingly, however, a similarly good performance is achieved with the Local Density Approximation (LDA). In view of the accurate prediction of PESes and the low computational cost, we recommend the use of either KT1 or LDA for treating the \u03c0-\u03c0 stacking interactions in larger systems, using a QM\/QM approach (e.g., our QUILD approach which is under development [33]) that allows this type of interaction to be separated from other types of interaction, such as hydrogen bonding but also regular covalent bonds.\nTo gain insight into the origin of \u03c0-\u03c0 stacking interactions, we have decomposed the interaction energies into the classical electrostatic attraction, Pauli repulsion and orbital interactions. Interestingly, the electrostatic interactions appear to be the most important factor that determines the shape and depth of the PES. In the case of two stacked Watson-Crick base pairs, this classical electrostatic attraction causes a minimum to occur along the energy profile at a twist angle of 36\u00b0. Furthermore, the stabilizing contributions to the stacking interaction between two Watson-Crick base pairs is shown to originate from the inter-strand stacking terms, that is, from the interaction between two bases that are in different Watson-Crick pairs and also not directly stacked on top of each other.\nThe above-mentioned insight that electrostatic attraction plays an essential role in \u03c0-\u03c0 stacking interactions nicely consolidates and extends the recent finding by Krapp, Bickelhaupt and Frenking [56] that classical electrostatic attraction is essential for understanding trends in bond strength of diatomic molecules. It is interesting to note that qualitative models of both \u201cregular\u201d chemical bonds and those of \u03c0-\u03c0 stacking interactions often completely ignore classical electrostatic attraction. These qualitative models attempt to rationalize trends in bonding entirely in terms of orbital interactions (in the case of regular bonds) and dispersion interactions (in the case of \u03c0-\u03c0 stacking).\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nSupporting Information\nEnergy profiles for various \u03c0-\u03c0 stacked systems as a function of the twist angle and the vertical separation computed with MP2 and various density functionals (PDF 1.90 mb)","keyphrases":["density functional theory","\u03c0-\u03c0 stacking interactions","hydrogen bonding","dna stability","dna structure"],"prmu":["P","P","P","R","R"]}
{"id":"J_Mol_Med-3-1-1820751","title":"An unexpected new role of mutant Ras: perturbation of human embryonic development\n","text":"The Ras signaling pathway controls important cellular responses to growth factors, and somatic mutations in RAS genes and other components of the Ras pathway, such as PTPN11 (encoding the protein-tyrosine phosphatase SHP-2) and BRAF, are found in human malignancies. Ras proteins are guanosine nucleotide-binding proteins that cycle between active guanosine triphosphate (GTP)-bound and inactive guanosine diphosphate (GDP)-bound conformations. Neoplasia-associated Ras mutations frequently affect amino acids G12, G13, or Q61 and decrease the intrinsic guanosine triphosphatase (GTPase) activity by ten- to twentyfold. The GTPase activity is crucial for Ras inactivation by hydrolysis and release of a phosphate group from Ras\u00b7GTP to produce Ras\u00b7GDP. We and others have recently discovered germline mutations in the KRAS gene in individuals diagnosed with Noonan and cardio\u2013facio\u2013cutaneous (CFC) syndrome, two clinically overlapping disorders characterized by short stature, distinct facial anomalies, heart defects, and other abnormalities. Noonan syndrome-associated mutations V14I and T58I K-Ras activate Ras but have milder biochemical effects than somatic mutations encountered in cancers, offering an explanation why these K-Ras lesions are tolerated during embryonic development. Together with recent findings of BRAF, MEK1, and MEK2 mutations in CFC syndrome and HRAS mutations in Costello syndrome, another clinically related disorder, it has now become clear that Noonan-like features (short stature, relative macrocephaly, facial anomalies, learning difficulties) that are found in these three related disorders are a result of constitutive activation of the Ras\u2013Raf\u2013extracellular signal-regulated and mitogen-activated protein kinase pathway.\nRAS genes\nThe three human RAS genes, KRAS (isoforms A and B), NRAS, and HRAS (isoforms 1 and 2), encode small, highly conserved guanosine triphosphatases (GTPases) that relay growth signals to a number of effector proteins to control fundamental cellular pathways (reviewed in [1]). Ras proteins act as molecular switches by cycling between an active guanosine triphosphate (GTP)-bound and an inactive guanosine diphosphate (GDP)-bound state. Stimulated growth factor receptors recruit a number of adaptor proteins that activate guanosine nucleotide exchange factors (GEFs) to remove guanine nucleotides from Ras. Ras is then activated by binding to GTP, which is present at a tenfold higher concentration than GDP. In the GTP-bound state, the two switch regions of Ras (switch I and II) change their conformation. This conformational switch allows Ras to bind and activate Ras effector proteins such as Raf-1. The \u201con\u201d position is turned \u201coff\u201d by an intrinsic GTPase activity, which hydrolyses and releases a phosphate group from Ras\u00b7GTP to produce Ras\u00b7GDP. The conformational transition of the switch I and II regions that is associated with this reaction disrupts the interaction between Ras and its effectors. The intrinsic GTPase activity of Ras is slow and accelerated about 105-fold by GTPase activating proteins (GAPs), such as neurofibromin or p120 GAP. These GAPs mediate Ras\u00b7GTP hydrolysis by inserting an arginine residue (arginine finger) into the phosphate-binding pocket of Ras (reviewed in [2, 3]; Fig.\u00a01).\nFig.\u00a01Ras cycles between an active GTP-bound and an inactive GDP-bound conformation. In the active state, the two switch regions, switch I and II, change their conformation allowing Ras to activate effector proteins. The intrinsic GTPase hydrolyzes a phosphate group to produce Ras\u00b7GDP. This reaction is accelerated by GTPase activating proteins (GAPs). A similar version of this figure has been previously published [61], republication with permission\nFor several decades, it has been well recognized that RAS genes are frequently mutated in human cancers (reviewed in [4]). These mutations predominantly lead to amino acid substitutions at residues G12, G13, or Q61 and lock Ras in the active GTP-bound state by diminishing the intrinsic Ras GTPase activity and\/or by causing resistance to GAPs. These mutations were long believed to occur only as somatic events, and in a mouse model system, widespread expression of endogenous K-RasG12D leads to embryonic death [5]. The highest incidence of RAS mutations are found in adenocarcinomas of the pancreas (90%), the colon (50%), and the lung (30%), in thyroid tumors (50%), and in myeloid leukemia (30%) [4].\nNoonan syndrome and related disorders\nNoonan syndrome (NS; MIM 163950) is an autosomal dominant disorder characterized by short stature, distinct facial anomalies (Fig.\u00a02), a typical spectrum of congenital heart defects, including pulmonic stenosis, hypertrophic cardiomyopathy, and septal defects, and developmental delays (reviewed [6]). The disorder occurs in approximately 1 out of 2,000 individuals and shares many features with the much less common disorders of Costello syndrome (CS), cardio\u2013facio\u2013cutaneous (CFC) syndrome, and lentigines, electrocardiographic conduction defects, ocular hypertelorism, pulmonary stenosis, abnormalities of the genitals; retarded growth resulting in short stature, and deafness (LEOPARD) syndrome (LS) [6]. Patients with these disorders have in common Noonan-like facial features, a similar spectrum of cardiac anomalies, delayed growth, and\u2014to a variable degree\u2014developmental retardation, which is stronger in patients with CS or CFC. In addition, each of these disorders is characterized by unique phenotypic patterns: (1) CS patients have nasal papillomata, loose skin, and a strong predisposition to tumors (mainly rhabdomyosarcoma) [7]; (2) CFC patients have ectodermal abnormalities with sparse curly hair, sparse, or absent eyelashes [8]; and (3) LS patients have multiple lentigines typically emerging during adolescence [6]. NS and LS may be familial with an autosomal dominant inheritance pattern (except for rare instances of NS where rare recessive inheritance has been suspected [9]), but most cases of these disorders and virtually all of the more severe conditions, CFC and CS, occur sporadically, suggesting dominant new mutations. Until recently, the genetic basis for these disorders was unknown. It was not clear whether they represented genetically distinct entities or if they were different (allelic) variants of a common disorder. By linkage analysis in families with NS, the disorder was mapped to chromosome 12q24 [10], and subsequently, it has been shown that approximately 50% of patients with NS carry germline mutations in PTPN11 [11]. The PTPN11 gene encodes for tyrosine-protein phosphatase (SHP-2), a phosphatase that relays growth signals from activated growth factor receptors to other signaling molecules, including Ras (reviewed in [12]). Most PTPN11 mutations are predicted to disrupt the auto-inhibition of the catalytic phosphatase domain (PTPase) by the N-terminal src-homology 2 (N-SH2) domain thereby promoting the active conformation of the protein [12]. PTPN11 mutations were not found in patients with CS or CFC syndromes (reviewed in [6]). However, specific mutations in the same gene were uncovered in patients with LS [13]. Surprisingly, in contrast to NS, LS mutants are catalytically defective and may act as dominant negative mutations [14, 15]. Molecular modeling and biochemical studies suggest that LS mutations disrupt the SHP-2 catalytic domain to result in open, inactive forms of SHP-2. Thus, the pathogenesis of LS and NS is distinct. It is unknown why the clinical phenotypes of LS and NS are similar although the underlying mutations have opposite biochemical effects. There are several possible explanations for this paradox. These are discussed in detail by Kontaridis et al. [14]: (1) The NS and LS phenotypes potentially result from differential effects of mutant SHP-2 on different receptor tyrosine kinase pathways at distinct developmental times. For example, recent work by others suggests that the main effect of NS mutants is to enhance epithelial\u2013mesenchymal transformation\/mesenchymal cell proliferation by increasing ErbB2\/3 (and\/or ErbB3\/4) signaling. By contrast, LS mutants antagonize HB-EGF\/ErbB1 signaling at later times [14]; (2) Other phenotypes common to NS and LS such as facial abnormalities and short stature might involve defective migration and\/or differentiation, which might result from increased or decreased signaling involving the same pathway; (3) Yet, undetermined or poorly understood functions of SHP-2 might underlie pathogenesis of one or both of these disorders [14].\nFig.\u00a02a Craniofacial phenotype of young children with NS, CFC, and CS aged between 10 and 18\u00a0months. Their genotypes are PTPN11 G503R, BRAF K499E, and HRAS G13C, respectively. Note the similarities of facial features, including hypertelorism, broad forehead, and low-set ears. Coarse facial features are particularly typical of CS. b The same disorders in older children and adolescents with the mutations PTPN11 N308D, MEK2 F57I, and HRAS G12S, respectively. Facial features become more distinct with age, although the similarities of the three syndromes are still evident. Courtesy of Prof. Rainer K\u00f6nig, Frankfurt (images of CFC patients) and Prof. Kerstin Kutsche, Hamburg (images of CS patients)\nThe Noonan gene PTPN11 also acts as oncogene\nOne of the myeloid malignancies that has been found to be particularly related to perturbed Ras signaling is juvenile myelomonocytic leukemia (JMML), a myeloproliferative disorder (MPD) of early childhood [16], in which mutations occur in NRAS or KRAS (\u223c25%) or in NF1 (clinical diagnosis of neurofibromatosis type 1 in \u223c11%) [17\u201319]. In a murine model, somatic activation of K-Ras in hematopoietic cells initiates a rapidly fatal MPD modeling JMML [20, 21]. Despite a low annual incidence estimated at 1\u20132 per million, JMML has attracted many clinical and basic researchers because of the severe and often lethal clinical course and the association with NS [22] and neurofibromatosis type 1 (NF1; MIM 162200; reviewed in [19]). Shortly after the discovery of PTPN11 mutations in individuals with NS, specific germline PTPN11 alterations were identified in young children with NS who developed a JMML-like disorder within the first few weeks of birth (NS\/JMML) [23, 24], and somatic PTPN11 mutations were subsequently uncovered in JMML cells from 35% of children with non-syndromic JMML [23\u201325]. Somatic PTPN11 mutations also occur in B-cell precursor acute lymphoblastic leukemia [26] and rarely other malignancies [27]. Somatic mutations observed in patients with JMML differ from those mutations found in patients with NS\/JMML and from those detected in patients with NS alone. In sporadic JMML, the most common mutation predicts an E76K substitution. This mutation has never been found in patients with NS (N308D most common) or NS\/JMML (T73I most common) [6, 25]. In elegant functional experiments, several groups have shown that somatic PTPN11 mutations associated with sporadic JMML exhibit stronger biochemical and biological effects than germline PTPN11 mutations, leading to the concept that only milder SHP-2 activation may be tolerated during embryonic development [28\u201330]. The situation is too complex however to be explained by a simple model of strong activating somatic vs less activating germline mutations alone. Enzymatic, structural, and mathematical modeling analyses show that these mutants can affect basal activation, SH2 domain-phosphopeptide affinity, and\/or substrate specificity to varying degrees, and there is no absolute correlation between the mutants\u2019 extents of basal activation and the diseases they induce [31]. A murine knock-in Ptpn11D61G\/+ model of NS has been constructed, revealing that endocardial cushions of these mice have increased activation of extracellular signal-regulated kinase [32]. This finding suggests that the phenotype exhibited by PTPN11 mutations is mediated through hyperactive Ras signaling.\nNeurofibromatosis type 1\nJMML is also associated with NF1 (MIM 162200), an autosomal dominant disorder that occurs in 1 of 4,000 births and is characterized by pigmentary anomalies (multiple caf\u00e9-au-lait spots) and a predisposition to benign and malignant tumors of mainly neurogenic origin. The disorder is caused by mutations in the NF1 tumor suppressor gene, which encodes the RasGAP neurofibromin. The incidence of JMML is increased approximately 200-fold in children with NF1 (reviewed in [33]). Loss of the normal NF1 allele (LOH, loss of heterozygosity) is common in JMML cells from children with NF1 [34, 35], and this results in severely deregulated Ras signaling and causes aberrant growth of hematopoietic progenitor colonies in vitro [36]. In addition, adoptive transfer of homozygous Nf1 mutant fetal liver cells or somatic inactivation of a conditional mutant Nf1 allele in hematopoietic cells induces a JMML-like MPD in mice [37, 38]. Remarkably, many NF1 patients have mild features reminiscent of NS [39], and some may even develop a mixed phenotype, which has prompted the definition of neurofibromatosis\u2013NS (NFNS; MIM 601321) as a separate entity. Most patients who are diagnosed with this condition harbor NF1 mutations without an obvious genotype\u2013phenotype correlation [40].\nAdditional genes mutated in Noonan syndrome\nTo date, the identification of additional NS genes was hampered by the fact that the vast majority of PTPN11 mutation-negative NS-patients represent sporadic cases [41], rendering genetic linkage studies impossible. However, recent findings by our group and others identified germline mutations in components of the Ras signaling pathway in individuals with NS or related syndromes [42\u201346]. We systematically screened patients with NS and CFC for RAS mutations after having discovered a novel de novo germline KRAS mutation in a patient with NS\/JMML [42]. The mutation c.173C>T (p.T58I) detected in the index patient affects a highly conserved amino acid residue of K-Ras flanking the switch II region (amino acids 59\u201367) of the protein. De novo KRAS mutations were identified in 4 of 174 sporadic cases with NS previously excluded for PTPN11 mutations. All germline KRAS mutations were novel and not known to occur in human cancer. NS-associated KRAS alleles included recurrent mutations, V14I and D153V. The latter was confirmed in patients with more severe phenotypes [44, 46]. Analogous to the dual role of PTPN11 as both oncogene and developmental gene, this discovery led to the hypothesis that activating KRAS mutations do not only act as oncogenes; aberrant K-Ras can also cause developmental disorders when mutations\u2014probably conferring relatively mild effects\u2014emerge in the germline. To prove our hypothesis, we studied the functional properties of V14I and T58I K-Ras [42] and found both NS-associated mutants V14I and T58I to have intermediate biochemical (e.g., ability to hydrolyze Ras\u00b7GTP) and biological properties (e.g., growth behavior in response to growth factors after retroviral transfection) when compared with wild-type K-Ras and the oncogenic mutant K-Ras G12D [42].\nThe two aforementioned mutations, V14I and T58I, are located adjacent to amino acid residues that are typically altered in cancer (G12, G13, or Q61). They locate to regions of Ras that are known to be involved in GTP binding. By contrast, in a subset of NS patients, KRAS mutations lead to substitutions in protein regions not obviously involved in GTP binding (e.g., D153V K-Ras), indicating the existence of previously unappreciated mechanisms of Ras activation. Carta et al. [46] performed a structural analysis on the two K-Ras mutants, V152G and D153V, that these investigators identified in two patients with severe NS. Their computer-based analysis indicated that both mutations disturb the conformation of the guanine ring-binding pocket favoring the active GTP-bound conformation by increasing the guanine nucleotide dissociation rate. Additionally, these residues are predicted to be important for binding the RasGEF son of sevenless (SOS) [47]. In the meantime, we have detected a number of additional KRAS mutations associated with NS (Q22R, P34L, P34Q, I36M), all of which affect highly conserved amino acid residues and are assumed to confer (mild) gain of function [48]. In this study on additional 236 PTPN11-negative NS patients, we detected KRAS mutations in seven individuals [48], thus confirming that KRAS accounts for less than 5% of NS cases.\nGenes mutated in cardio\u2013facio\u2013cutaneous syndrome\nIn our initial study, we also discovered a novel K-Ras mutation, K-Ras P34R, in 1 of 12 patients with CFC syndrome [42]. This mutation is now known to be located in another mutational hotspot associated with NS [48]. A P34R H-Ras lesion has been previously characterized by Stone et al. [49]. These investigators employed a mutagenesis strategy during which they found that H-Ras P34R binds to GTP in vivo. In vitro, H-Ras P34R is not stimulated by GAPs [49]. Considering the highly conserved G-domain structure of Ras proteins, the P34R K-Ras mutation presumably has very similar (if not identical) biochemical properties. The presence of K-Ras mutations in CFC syndrome was confirmed by Niihori et al. [44] who analyzed DNA specimens from 43 individuals with CFC syndrome and found two KRAS germline mutations (G60R and D153V) in three patients. Notably, the D153V K-Ras mutation also has been described in patients with NS [42, 46, 48]. In a more recent study, we identified additional K-Ras mutations in individuals with CFC or NS\/CFC including Q22E and F156I. The observations that mutations, such as K-Ras F156I, may be associated with an overlap of NS and CFC suggest that there is not a very strict genotype\u2013phenotype correlation [48]. Simultaneous to the finding of mutated K-Ras in CFC, Niihori et al. identified BRAF germline mutations in 16 of 43 CFC patients [36]. At the same time, another group found BRAF germline mutations in 18 of 23 CFC patients and MEK1 or MEK2 germline mutations in 3 of the remaining 5 CFC patients studied [45]. Like PTPN11 and RAS, BRAF is a known oncogene, and somatic mutations are frequently found in cancer [50].\nThe molecular basis of Costello syndrome\nShortly before germline KRAS mutations were described in patients with NS and CFC, HRAS germline mutations were reported to cause CS [43]. Later, other investigators also detected these mutations in the majority (\u223c90%) of CS patients studied [51\u201353]. Surprisingly, these germline lesions affect the same amino acid residues of H-Ras that are also mutated in cancer. The observation that activating oncogenic H-Ras mutations affecting codons G12 or G13 are tolerated in the germline, whereas oncogenic K-Ras mutations affecting these residues are not, underscores the notion that although H-Ras and K-Ras are structurally highly similar, they play different roles during embryonic development. Knockout studies have shown that only K-Ras is essential for embryonic development, whereas N-Ras and H-Ras are not [54]. Different phenotypes exhibited by mutations in various Ras isoforms may be due to heterogenous expression patterns of these proteins. Additionally, various Ras isoforms undergo different processing, e.g., de\/repalmitoylation kinetics that regulate subcellular localization and activity of Ras isoforms [55, 56]. Although the vast majority of patients with CS harbor HRAS mutations, very recent studies have indicated that a CS phenotype may occasionally be associated with specific mutations of BRAF [57] or KRAS [48]. It remains to be determined if these patients also have the increased risk of neoplasia, which is typical of CS. Therefore, we doubt it is useful to classify these patients with mutations in Ras pathway genes other that HRAS as CS.\nNeuro\u2013cardio\u2013facial\u2013cutaneous syndrome\nThe molecular explanation of how different germline K-Ras or H-Ras mutations cause a wide spectrum of different phenotypes is still largely unknown. Activating germline K-Ras mutations may be associated with the broadest spectrum of clinical manifestations [42, 44, 46]. However, these syndromes are not just a less or more severe expression of essentially the same disorder. Therefore, it is conceivable that the different RAS mutations associated with divergent phenotypes do not only vary quantitatively in their degree of constitutive activation. Unique lesions may also have different qualitative effects on downstream signaling pathways. Additionally, modifier loci may play a role. Recently, the term \u2018neuro\u2013cardio\u2013facial\u2013cutaneous\u2019 (NCFC) syndrome [58] was coined to illustrate that clinically overlapping disorders of the NS spectrum, including NF1, NS, CS, LS, and CFC syndrome, are caused by mutations in components of the Ras signaling pathway (Fig.\u00a03). We speculate that the phenotypic variability between these disorder results from (1) different expression patterns of affected genes\/isoforms and (2) variable mechanisms by which certain mutants interact with downstream effectors or regulatory proteins; these mutants therefore perturb Ras signaling in varying degrees. Further research is required to determine the basis of genotype\u2013phenotype correlations in NS and related disorders.\nFig.\u00a03The Ras signaling pathway relays growth signals from activated growth factor receptors to the nucleus. Somatic mutations in several molecules of the pathway have been implicated in cancer. It is now recognized that germline mutations of identical molecules may cause disorders of the Noonan spectrum. A similar version of this figure has been previously published [61], republication with permission\nNeurofibromatosis Noonan syndrome and NS-like features in neurofibromatosis\nThe rapidly increasing knowledge of the Ras signaling pathway and its relation to developmental disorders of the NS spectrum suggests that Noonan-like features are the clinical correlate of any genetic abnormality occurring in the germline and leading to a generalized mild deregulation of the Ras signaling cascade(s) during embryonic development. The new insights gained by recent research in this field may explain the phenotypic similarities between individuals with NF1 and NS. In addition to the well-known symptoms defining NF1, namely multiple caf\u00e9-au-lait spots and cutaneous neurofibromas, patients with NF1 are known to exhibit NS features, including relative short stature, relative macrocephaly, mild facial anomalies, thorax deformities, and learning difficulties. NFNS probably just represents the extreme end of a highly variable expression of these features [59]. Although the association of NF1 and NS was described in one family with independently segregating mutations in NF1 and PTPN11 [60], this double chance event is not the causative mechanism in many other patients with NF1 and NS-like features. The majority of patients with the NFNS phenotype have been found to harbor NF1 mutations, some of which also occur in patients with \u201cpure\u201d NF1 [40, 59]. We propose that the NS-like features that are often present in patients with NF1 are potentially due to NF1 haploinsufficiency leading to decreased inactivation of Ras\u00b7GTP. This might cause mild constitutional deregulation of Ras signaling, which may occasionally reach a level that leads to a NF1-NS phenotype. Frequently, patients with NF1 have mild features of NS. These may be explained by a mild activation of Ras, which is due to loss-of-function of one NF1 allele. Unknown genetic modifiers may play a role. Malignant cells arising in patients with NF1 somatically loose the wild-type NF1 allele. This second somatic hit ablates the GAP function of neurofibromin resulting in strongly enhanced Ras signaling (Fig.\u00a04).\nFig.\u00a04Model illustrating the molecular basis of how Ras signaling may be increased in patients with neurofibromatosis type 1 leading to Noonan-like feature in these patients. Although only one NF1 allele is inactive in the germline, tumors of patients with neurofibromatosis type 1 somatically lose the second, wild-type NF1 allele\nWork in progress\nAlthough the vast majority of cases with CFC and CS are explained by mutations in KRAS, BRAF, MEK1, MEK2, and HRAS, the underlying genetic defect in 50% of NS and 10\u201330% of CFC cases remains unknown. Based on the assumption that the NS phenotype results from hyperactive Ras signaling, we and others are currently screening DNA specimens from patients for mutations in genes encoding for other components, including negative regulators, of the Ras pathway. Analogous to PTPN11 and KRAS, currently unknown genes of the Ras pathway mutated in NS are presumably playing double roles in both development and oncogenesis. Mutations in BRAF and MEK1\/2 have been excluded as major NS genes (M.Z., unpublished data).\nIt will be of interest to study how the different expression patterns of various RAS genes and isoforms influence clinical phenotypes when these genes\/isoforms are mutated in the germline. It will be crucial to elucidate the structural mechanisms to understand how these new lesions perturb signaling on a molecular level. Characterization of the biological consequences of these mutations will be largely improved by the construction of murine knock-in models.\nNote added in proof\nAfter this paper was accepted we uncovered mutations in SOS1 encoding for the homonymous RasGEF in patients with NS. Meanwhile, however, two other groups have published elegant reports on activating SOS1 mutations in approximately 10% of cases with NS: Roberts AE, Araki T, Swanson KD, Montgomery KT, Schiripo TA, Joshi VA, Li L, Yassin Y, Tamburino AM, Neel BG, Kucherlapati RS. Germline gain-of-function mutations in SOS1 cause Noonan syndrome. Nat Genet. 2006 Dec 3; [Epub ahead of print] Tartaglia M, Pennacchio LA, Zhao C, Yadav KK, Fodale V, Sarkozy A, Pandit B, Oishi K, Martinelli S, Schackwitz W, Ustaszewska A, Martin J, Bristow J, Carta C, Lepri F, Neri C, Vasta I, Gibson K, Curry CJ, Siguero JP, Digilio MC, Zampino G, Dallapiccola B, Bar-Sagi D, Gelb BD. Gain-of-function SOS1 mutations cause a distinctive form of Noonan syndrome. Nat Genet. 2006 Dec 3; [Epub ahead of print]\u201d.","keyphrases":["ras signaling","noonan syndrome","costello syndrome","neurofibromatosis type 1","cardio\u2013facio\u2013cutaneous syndrome"],"prmu":["P","P","P","P","P"]}
{"id":"Dev_Biol-1-5-2082130","title":"Differential activities of the core planar polarity proteins during Drosophila wing patterning\n","text":"During planar polarity patterning of the Drosophila wing, a \u201ccore\u201d group of planar polarity genes has been identified which acts downstream of global polarity cues to locally coordinate cell polarity and specify trichome production at distal cell edges. These genes encode protein products that assemble into asymmetric apicolateral complexes that straddle the proximodistal junctional region between adjacent cells. We have carried out detailed genetic analysis experiments, analysing the requirements of each complex component for planar polarity patterning. We find that the three transmembrane proteins at the core of the complex, Frizzled, Strabismus and Flamingo, are required earliest in development and are the only components needed for intercellular polarity signalling. Notably, cells that lack both Frizzled and Strabismus are unable to signal, revealing an absolute requirement for both proteins in cell\u2013cell communication. In contrast the cytoplasmic components Dishevelled, Prickle and Diego are not needed for intercellular communication. These factors contribute to the cell\u2013cell propagation of polarity, most likely by promotion of intracellular asymmetry. Interestingly, both local polarity propagation and trichome placement occur normally in mutant backgrounds where asymmetry of polarity protein distribution is undetectable, suggesting such asymmetry is not an absolute requirement for any of the functions of the core complex.\nIntroduction\nThe term planar polarity was first used to describe the polarisation of structures within the plane of the insect cuticle (N\u00fcbler-Jung et al., 1987); however, the phenomenon is widespread in nature (reviewed in Klein and Mlodzik, 2005). Genetic analysis, particularly in Drosophila, has identified a planar polarity or PCP (planar cell polarity) pathway, dependent on the function of Frizzled (Fz) family receptors. Interestingly, not only are elements of this pathway conserved throughout the animal kingdom, but it is also required for developmental patterning processes that are distinct from planar polarity, such as polarised cell rearrangements during vertebrate gastrulation (Wallingford et al., 2002).\nTo date, planar polarity patterning has been best studied in the Drosophila wing, which provides a simple model in which each cell becomes coordinately polarised and produces a single distally pointing trichome (Fig. 1A). It is widely considered that this pattern is produced by three tiers of gene activity (Tree et al., 2002a; Klein and Mlodzik, 2005; Strutt and Strutt, 2005). At the top of the hierarchy the type II transmembrane protein Four-jointed (Fj) and the atypical cadherins Dachsous (Ds) and Fat (Ft) act (probably with other unidentified factors) to provide a long-range (or \u201cglobal\u201d) patterning cue across the axis of the tissue (Adler et al., 1998; Zeidler et al., 2000; Strutt and Strutt, 2002; Ma et al., 2003). In a manner which is not understood, but is possibly dependent on widerborst gene function (Hannus et al., 2002), this long-range cue is thought to be interpreted by the middle tier of genes which include fz and a number of other factors known as the \u201ccore\u201d polarity genes (Shulman et al., 1998). The final tier consists of tissue-specific effectors, which modulate cellular behaviours such as polarisation of the cytoskeleton and transcription, in response to activity of components of the core.\nThe definition of the \u201ccore\u201d polarity proteins is somewhat fluid, but was originally used to refer to factors that act together with Fz in all tissues examined in Drosophila. A notable property of Fz during planar polarity patterning is that it adopts an asymmetric subcellular localisation in polarising cells, for instance in the wing becoming localised to the junctional zone at the distal cell edge (Strutt, 2001). Five other proteins that act with Fz also adopt asymmetric localisations, either at the proximal or distal edges of wing cells, and loss of any one of these proteins prevents the distal localisation of Fz. As these proteins colocalise to junctions with Fz and are required for Fz localisation, it seems reasonable to regard them as the \u201ccore\u201d. They consist of the multidomain cytoplasmic protein Dishevelled (Dsh) and the ankyrin repeat protein Diego (Dgo) that localise distally with Fz (Axelrod, 2001; Das et al., 2004), the fourpass transmembrane protein Strabismus (Stbm, also known as Van Gogh [Vang]) and the LIM-domain protein Prickle that localise proximally (Bastock et al., 2003; Tree et al., 2002b), and the sevenpass transmembrane cadherin Flamingo (Fmi, also known as Starry Night [Stan]) that localises both proximally and distally (Chae et al., 1999; Usui et al., 1999) (Fig. 1B). We note, that by this definition, the G\u03b1o subunit encoded by the brokenheart gene may also be regarded as a component of the \u201ccore\u201d (Katanaev et al., 2005), but this requires further investigation.\nFz is thought to perform at least three functions in planar polarity patterning. The first is to receive long-range pattering information from upstream cues, for instance provided by the activities of Fj\/Ds\/Ft. Experiments analysing the temporal requirements of fz and ds suggest that such coupling may occur around 6 to 24\u00a0h of pupal life (Strutt and Strutt, 2002; Matakatsu and Blair, 2004). Recent models have suggested that this information could be provided either by generation of a gradient of Fz activity across the whole axis of the wing or alternatively via generation of a gradient of Fz activity across the axis of individual cells (Lawrence et al., 2004; Amonlirdviman et al., 2005). Notably, there is currently no evidence that other components of the core are involved in this coupling.\nSecond, Fz is involved in a process of cell\u2013cell communication that locally coordinates cell polarity (Adler et al., 2000; Ma et al., 2003; Lawrence et al., 2004) and also occurs after 6\u00a0h of pupal life (Strutt and Strutt, 2002). Historically, models to explain this coordination have invoked the production of a diffusible ligand for Fz (Park et al., 1994; Zheng et al., 1995; Adler et al., 1997). However, more recent models based on the observation of core polarity protein localisation to cell junctions have suggested that cell\u2013cell signalling is contact-dependent (Tree et al., 2002b; Lawrence et al., 2004; Amonlirdviman et al., 2005; Klein and Mlodzik, 2005; Le Garrec et al., 2006). Generally, it has been assumed that all components of the core act with Fz in local coordination of polarity, but the exact roles of each protein have not been defined.\nThe third function of Fz is to provide a subcellular cue for trichome growth, apparently via its localisation to the distal cell edge (Wong and Adler, 1993; Strutt, 2001). In the absence of Fz, or several other core components, trichomes form in the cell centre. Provision of Fz activity after 24\u00a0h of pupal life is sufficient to permit asymmetric localisation and polarised trichome growth (Strutt and Strutt, 2002); however, distal polarity is lost, presumably due to disruption of earlier fz functions. As all core components asymmetrically localise together with Fz prior to trichome formation, it is tempting to conclude that all are required for trichome placement, but this has not been definitively demonstrated.\nAsymmetric localisation of the core components only becomes clearly visible during pupal life by about 24\u00a0h of pupal life (but has also been observed earlier in development, see Classen et al., 2005), and hence it has been suggested that this probably follows the cell\u2013cell communication phase (Strutt and Strutt, 2002; Lawrence et al., 2004). However, other workers have argued that asymmetric complex formation may occur progressively over a longer period of pupal life, and be intrinsically required for cell\u2013cell communication and local coordination of polarity (Tree et al., 2002b; Amonlirdviman et al., 2005). In this context, it is important to consider that the spatial relationships observed during asymmetric complex formation (Fz, Dsh, Dgo and Fmi\/Stan colocalising at distal cell edges; Stbm\/Vang, Pk and Fmi\/Stan at proximal edges) may not necessarily reflect earlier functional relationships. Notably, associations have also been reported between Dsh and Pk (Tree et al., 2002b; Jenny et al., 2005), Dsh and Stbm\/Vang (Bastock et al., 2003; Jenny et al., 2005), Dgo and Pk (Das et al., 2004) and Dgo and Stbm\/Vang (Das et al., 2004).\nIn this manuscript, we address three key issues: First, which components of the core act together with Fz during the different planar polarity patterning processes? Second, are the spatial relationships seen during the later phase of asymmetric localisation also relevant during the phase of cell\u2013cell communication and local coordination of polarity? Third, is asymmetric core protein localisation absolutely required for planar polarity patterning?\nMaterials and methods\nFly strains and genetics\nAlleles and transgenes used are described in FlyBase, except where noted. Temporal rescue of polarity phenotypes in the wing and eye was carried out and analysed as described (Strutt and Strutt, 2002). Actin\u00a0\u226b\u00a0fz-EYFP and Actin\u00a0\u226b\u00a0stbm-EYFP have been described (Strutt, 2001; Strutt et al., 2002), Actin\u00a0\u226b\u00a0dsh-ECFP, Actin\u00a0\u226b\u00a0fmi-FLAG, Actin\u00a0\u226b\u00a0pkpk, sev-stbm and sev-pksple were constructed as previously (Strutt and Strutt, 2002). Note that the pk locus produces two protein isoforms, of which the Pk variant is sufficient for wing patterning and the Sple variant is sufficient for eye patterning (Gubb et al., 1999). For double mutant clones, rescue of fz activity on the X and 2R was provided by Arm-fz-EGFP transgenes (Strutt, 2001) and rescue of stbm\/Vang activity on the X was provided by an Actin-stbm-EYFP transgene. fz;stbm twinclones were generated by inducing clones of FRT42 stbm6\nArm-fz-EGFP in a fz background, resulting in cells homozygous for stbm6\nArm-fz-EGFP juxtaposed to twinspot cells lacking the transgene. Clones in the wing were generally induced using Ubx-FLP, kindly provided by J\u00fcrgen Knoblich.\nExact genotypes used are as follows:\nFigure 1\nTemporal rescue of stbm\/Vang in wing: w hsFLP1; stbm6\/stbmVang-A3; Act-FRT-polyA-FRT-stbm-EYFP\/+\nTemporal rescue of pkpk-sple in wing: w hsFLP1; pkpk-sple-13\/pkpk-sple-13; Act-FRT-polyA-FRT-pk\/+\nstbm\/Vang phenotype in eye: w; FRT42 stbm6\/FRT42 P[w+] stbmVang-A3\nRescue of stbm\/Vang phenotype in eye by sev-stbm: w; FRT42 stbm6\/FRT42 P[w+]stbmVang-A3; sevE-sevP-stbm7.1\/+\npkpk-sple-13 phenotype in eye: FRT42 pkpk-sple-13\ncn sp\/FRT42 pkpk-sple-13\ncn\nRescue of pkpk-sple phenotype in eye by sev-pksple: w; FRT42 pkpk-sple-13\ncn\/FRT42 pkpk-sple-13\ncn; sevE-sevP-sple2.2\/+\nGenotypes shown in graph in (S): w; stbm6\/FRT42 P[w+] stbmVang-A3\nw; FRT42 stbm6\/FRT42 P[w+] stbmVang-A3; sevE-sevP-stbm2.2\/+, w; FRT42 stbm6\/FRT42 P[w+] stbmVang-A3; sevE-sevP-stbm7.1\/+\nGenotypes shown in graph in (T): FRT42 pkpk-sple-13\ncn\/FRT42 pkpk-sple-13\ncn\nw; FRT42 pkpk-sple-13\ncn\/FRT42 pkpk-sple-13\ncn; sevE-sevP-pksple[2.2]\/+\nw; FRT42 pkpk-sple-13\ncn\/FRT42 pkpk-sple-13\ncn; sevE-sevP-pksple[14.2]\/+\nFigure 2\nfz clones using rescuing transgene on 2R: w; FRT42D\/FRT42D Arm-fz-EGFP, Arm-lacZ; fz15\/fz21, Ubx-FLP\nstbm\/Vang clones: y w Ubx-FLP\/+; FRT42D stbm6\/FRT42D Arm-lacZ\nstbm\/Vang; fz double clones: w; FRT42D stbm6\/FRT42D Arm-fz-EGFP; fz21\/fz21, Ubx-FLP\nstbm\/Vang and fz twin clones: w; FRT42D stbm6, Arm-fz-EGFP\/FRT42D Arm-lacZ; fz21\/fz21, Ubx-FLP\nfmi\/stan clones: y w Ubx-FLP\/+; FRT42D fmiE59\/FRT42D Arm-lacZ\nfmi\/stan; fz double clones: w; FRT42D fmiE59\/FRT42D Arm-fz-EGFP, Arm-lacZ; fz21\/fz21, Ubx-FLP\nstbm\/Vang fmi\/stan double clones: y w Ubx-FLP\/+; FRT42D stbm6\nfmiE59\/FRT42D Arm-lacZ\nFigure 3\ndsh3 clones: y w dsh3\nFRT18A\/w Arm-lacZ FRT18A; FLP38\/+dsh; fz double clones:\ny w dsh3\nf36a\nFRT19A\/w Arm-fz-EGFP FRT19A; fz21\/fz21, Ubx-FLP\ndsh; stbm\/Vang double clones: y w dsh3\nf36a\nFRT19A\/w Act-FRT-polyA-FRT-stbm-EYFP FRT19A; stbm6\/stbm6, Ubx-FLP\npkpk-sple clones: y w Ubx-FLP\/+; FRT42D pkpk-sple-13\/FRT42D Arm-lacZ\npkpk-sple; fz double clones: w; FRT42D pkpk-sple-13\/FRT42D Arm-fz-EGFP; fz21\/fz21, Ubx-FLP\npkpk-splestbm\/Vang double clones: y w Ubx-FLP\/+; FRT42D pkpk-sple-13\nstbm6\/FRT42D Arm-lacZ\nFigure 4\nstbm\/Vang dgo double clones: y w Ubx-FLP\/+; FRT42D stbm6\ndgo380\/FRT42D Arm-lacZ\npkpk-spledgo double clones: y w Ubx-FLP\/+; FRT42D pkpk-sple-13\ndgo380\/FRT42D Arm-lacZ\npkpk-spledgo; fz triple clones: w; FRT42D pkpk-sple-13\ndgo380\/FRT42D Arm-fz-EGFP; fz21\/fz21, Ubx-FLP\nFigure 5\nstbm\/Vang overexpression in fz background: y w hsFLP1\/+; Act-FRT-y+-FRT-GAL4, UAS-lacZ\/+; fz15, UAS-stbm\/Df(3L)fzD21\nfz-EGFP overexpression in stbm\/Vang background: w hsFLP1\/+; Act-FRT-y+-FRT-GAL4, stbm6\/stbm6, UAS-fz-EGFP\nfz-EGFP overexpression in pkpk-sple background: w hsFLP1\/+; Act-FRT-y+-FRT-GAL4, pkpk-sple-13\/pkpk-sple-13, UAS-fz-EGFP\nfz-EGFP overexpression in pkpk-sple\ndgo380 background: w hsFLP1\/+; FRT42D pkpk-sple-13\ndgo380\/FRT42D pkpk-sple-13\ndgo380; Act-FRT-CD2-FRT-GAL4, UAS-fz-EGFP\/+\nfz-EGFP overexpression in dsh1: w dsh1\/Y; FLP38\/Act-FRT-y+-FRT-GAL4, UAS-lacZ; UAS-fz\/+\nfz-EGFP overexpression under ptc-GAL4 control in wings containing dsh3 clones: y w dsh3\nf36a\nFRT19A\/y w w+FRT19A; ptc-GAL4\/+; UAS-fz-EGFP, Ubx-FLP\/+\nFigure 6\nAct-fz-EYFP expression in pkpk-sple background: w hsFLP1\/+; pkpk-sple-13, Act-FRT-polyA-FRT-fz-EYFP\/pkpk-sple-13\nAct-fz-EYFP expression in dsh1 background: w dsh1\/Y; Act-FRT-polyA-FRT-fz-EYFP\/FLP38\ndgo clones: y w Ubx-FLP\/+; FRT42D dgo380\/FRT42D Arm-lacZ\npkpk-sple clones: y w Ubx-FLP\/+; FRT42D pkpk-sple-13\/FRT42D Arm-lacZ\npkpk-spledgo double clones: y w Ubx-FLP\/+; FRT42D pkpk-sple-13\ndgo380\/FRT42D Arm-lacZ\ndsh3 clones: y w dsh3\nFRT18A\/w Arm-lacZ FRT18A; FLP38\/+\nSupplementary Figure 1\nTemporal rescue of fz: y w hsFLP1; Act-FRT-polyA-FRT-fz-EYFP\/+; fz21\nTemporal rescue of fmi\/stan: y w hsFLP1; fmiE45, GAL4-1407\/fmiE59; Act-FRT-polyA-FRT-fmi-FLAG\/UAS-fmi\nGAL4-1407 and UAS-fmi provide rescue of fmi activity in the embryonic nervous system (Usui et al., 1999)\nTemporal rescue of dsh: w dsh1\/Y; FLP38\/+; Act>FRT-poly-FRT-dsh-ECFP\/+\nSupplementary Figure 2\nw hsFLP1; stbm\n6\n\/stbm\nVang-A3\n; Act-FRT-polyA-FRT-stbm-EYFP\/+\ny w hsFLP1; fmiE45, GAL4-1407\/fmiE59; Act-FRT-polyA-FRT-fmi-FLAG\/UAS-fmi\nw dsh\n1\n\/Y; FLP38\/+; Act>FRT-poly-FRT-dsh-ECFP\/+\nw hsFLP1; pk\npk-sple-13\n\/pk\npk-sple-13\n; Act-FRT-polyA-FRT-pk\/+\nSupplementary Figure 3\nstbm\/Vang clones: w hsFLP1; FRT42 stbm6\/FRT42 P[w+]\nstbm\/Vang clones rescue by sev-stbm: w hsFLP1; FRT42 stbm6\/FRT42 P[w+]; sevE-sevP-stbm7.1\/+\nNote that fz21, stbm6, dsh3, fmiE59, pkpk-sple-13 and dgo380 have been molecularly characterised and are thought to be null alleles on the basis of being unable to give rise to functional proteins (Jones et al., 1996; Wolff and Rubin, 1998; Wehrli and Tomlinson, 1998; Usui et al., 1999; Gubb et al., 1999; Feiguin et al., 2001). fmiE45 contains a missense mutation that generates an amorphic mutation in the wing by genetic criteria (Usui et al., 1999). fz15 contains a nonsense mutation that gives rise to a truncated protein that has been characterised as amorphic in the wing (Jones et al., 1996). stbmVang-A3 has not been molecularly characterised, but has been defined by genetic criteria to be amorphic in the wing (Taylor et al., 1998). dsh1 contains a missense mutation in the DEP domain which has been reported to be a strong mutation for planar polarity functions of the gene (Perrimon and Mahowald, 1987; Axelrod et al., 1998; Boutros et al., 1998).\nHistology\nPupal wings were processed for immunofluorescence and imaged as previously (Strutt, 2001). Primary antibodies used for experiments or confirmation of genotypes were mouse monoclonal anti-\u03b2gal (Promega), rabbit anti-\u03b2gal (Cappel), rabbit anti-GFP (Abcam), mouse monoclonal anti-Fmi#74 (DSHB, Usui et al., 1999), rabbit anti-Pk (Tree et al., 2002b), rabbit anti-Stbm (Rawls and Wolff, 2003), rat anti-Dsh (Shimada et al., 2001) and rabbit anti-Dgo (Feiguin et al., 2001). Actin was visualised using Texas-Red-conjugated phalloidin (Molecular Probes). Adult wings were mounted in GMM and eye sections were prepared as described (Tomlinson and Ready, 1987).\nResults\nDiffering temporal requirements of the core polarity proteins during wing development\nWe previously analysed the temporal requirements of fz for planar polarity patterning in the wing, by rescuing the phenotype of fz mutant flies using an inducible fz-expressing transgene (Strutt and Strutt, 2002). Expression of the transgene is activated at different times during pupal development, by administration of a heat-shock, allowing determination of the latest timepoint that gene expression is sufficient to permit normal patterning. These studies found no requirement for fz function prior to 6\u00a0h after prepupa formation (APF). Progressively later heat-shocks up to 24\u00a0h APF produced stronger phenotypes that were qualitatively and quantitatively different from the reported fz loss-of-function phenotype. We classified this stronger phenotype as ds-like, as Fz protein was still localising at cell edges and specifying the site of trichome formation, but due to a loss of non-autonomous coordination of polarity Fz localisation was seen in a swirling pattern rather than uniformly at distal cell edges (Strutt and Strutt, 2002). Heat-shocks after 28\u00a0h APF resulted in the reported fz loss-of-function phenotype, consistent with this being produced by loss of the later autonomous function that places trichomes at the cell edge.\nAs the core polarity gene stbm\/Vang shows similar phenotypes to fz, exhibiting both strong domineering non-autonomous effects on trichome polarity and being required for trichome placement at the cell edge (Taylor et al., 1998), we considered it a good candidate for sharing common functions with fz. Using the same methodology, we analysed its timecourse of requirement in wing patterning (Figs. 1C\u2013I). In common with fz, stbm\/Vang is not required prior to 6\u00a0h APF, but then shows progressively stronger phenotypes when induced between 12 and 24\u00a0h APF, with induction at 30\u00a0h APF mimicking the normal loss-of-function phenotype (seen when no heat-shock is administered, Fig. 1I). For comparison, we repeated our analysis of the timecourse of fz-requirement (Supplementary Fig. 1A), but this time using the molecularly characterised fz21 null allele (Jones et al., 1996). This gave the same timecourse as observed for stbm\/Vang, although generally with slightly stronger phenotypes being observed.\nNext we analysed the temporal requirement of the core polarity gene pk, which produces a protein that colocalises with Stbm\/Vang at the proximal cell edge and which has been implicated in cell\u2013cell coordination of planar polarity (Tree et al., 2002b). Interestingly, induction of pk expression as late as 20\u00a0h APF resulted in only negligible polarity defects in the adult wing, with induction at 24\u00a0h and 28\u00a0h still providing partial rescue of pk function (Figs. 1J\u2013N).\nThese results indicate that whereas stbm\/Vang shares an early requirement with fz in the wing, pk has only a relatively late function. We further extended these results by investigating the requirements of the other two core components fmi\/stan and dsh (Supplementary Figs. 1B, C). To circumvent the embryonic lethality of dsh null alleles, we analysed rescue of the strong planar polarity phenotype of the viable dsh1 allele (Perrimon and Mahowald, 1987) (the core component dgo was not examined, as the adult wing phenotype is too subtle for this approach to be feasible, Feiguin et al., 2001).\nInduction of fmi\/stan expression between 12 and 24\u00a0h APF resulted in progressively stronger phenotypes that differ from the loss-of-function phenotype (Supplementary Fig. 1B), as observed for fz and stbm\/Vang. Conversely, induction of dsh at 16 to 20\u00a0h APF resulted in relatively minor defects, although later induction revealed a strong requirement for dsh function after 20\u00a0h APF (Supplementary Fig. 1C). Hence, fmi\/stan appears to share early requirements with fz and stbm\/Vang, whereas dsh exhibits later temporal requirements. However, we cannot rule out the possibility that the dsh1 allele exhibits residual activity in planar polarity, which might contribute to the apparently later requirement.\nFor all genotypes, early transgene induction can rescue, indicating that the transgenes provide appropriate levels of expression throughout the wing. Consistent with this, almost all cells express detectable protein after transgene induction (Supplementary Fig. 2). Furthermore, without induction, we observe the expected loss-of-function phenotype seen in the absence of the transgene, indicating that our results are unlikely to be due to \u201cleaky\u201d expression from the transgenes. We tested whether the differences might be due to transmembrane proteins taking longer to be synthesised and targeted to the appropriate subcellular sites; however, we found that after induction both Fz-EYFP and Dsh-ECFP show the appearance of junctional staining within 2\u20133\u00a0h (data not shown).\nDiffering temporal requirements of the core polarity proteins during eye development\nWe also find a common early requirement for fz and stbm\/Vang in the eye. We previously distinguished between early and late activities of fz in the eye, by expressing fz under control of the sevenless promoter which is not active until the time of photoreceptor differentiation (Strutt and Strutt, 2002). Providing fz activity only at the time of photoreceptor differentiation resulted in defects in the dorsoventral polarity of ommatidia, indicating that fz activity is specifically required prior to photoreceptor differentation for correct specification of dorsoventral polarity. However, lack of fz activity after photoreceptor differentiation results in randomisation of all aspects of ommatidial polarity including both dorsoventral and anteroposterior polarity and rotation. Hence, fz shows two phases of requirement during eye development, an early phase needed just for dorsoventral patterning and a later phase required for dorsoventral and anteroposterior polarity and rotation of ommatidia.\nIn contrast, we showed that ommatidial polarity and rotation defects within null mutant dsh3 tissue can be rescued completely by expression of sev-dsh, indicating that dsh does not share the early dorsoventral patterning function with fz, but nevertheless is required for the later phase of activity.\nWe have now extended this work to stbm\/Vang and pk. Rescuing the phenotype of stbm\/Vang in the eye by expression of sev-stbm reduces general polarity and rotation defects but reveals an underlying randomisation of dorsoventral polarity (Figs. 1O, P, S) indicating that stbm\/Vang shares with fz an early dorsoventral patterning function.\nConversely, the pk phenotype is almost completely rescued by a sev-pksple transgene (Figs. 1Q, R, T) which expresses the Sple isoform of the Pk protein which is specifically required for eye patterning (Gubb et al., 1999). Thus pk, like dsh, does not exhibit an early patterning function in eye development.\nInterestingly, while investigating the functions of stbm\/Vang in the eye, we found that stbm\/Vang clones also show equatorial non-autonomy of the polarity phenotype. In a number of cases we observed dorsoventral polarity inversions in ommatidia on the equatorial sides of clones, in which all 8 photoreceptors of the ommatidium retain stbm\/Vang activity (Supplementary Fig. 3). This is consistent with the observed non-autonomy of clones in the wing (Taylor et al., 1998). However, a previous analysis of over 169 misoriented ommatidia on the edges of clones found no significant evidence of non-autonomy of the polarity phenotype of stbm\/Vang (Wolff and Rubin, 1998). Re-examination of this original data set in the light of our results again failed to find evidence of non-autonomy (T. Wolff, personal communication). The reasons for this discrepancy are currently unclear (but see legend to Supplementary Fig. 3 for further discussion of this issue).\nMutual dependence of fz, stbm\/Vang and fmi\/stan for intercellular communication\nThe common early function of fz, stbm\/Vang and fmi\/stan is likely to be either receiving long-range patterning cues and\/or local coordination of polarity. Little is understood about how the long-range signal might be received, rendering this activity difficult to study. However, the effects of fz and stbm\/Vang on local coordination of polarity can be assayed, as groups of cells lacking the activity of either gene cause neighbouring cells to mispolarise: fz clones cause neighbouring cells to point their trichomes towards the clone (arrow Fig. 2A, Gubb and Garc\u00eda-Bellido, 1982; Vinson and Adler, 1987), whereas trichomes point away from stbm\/Vang clones (arrow Fig. 2B, Taylor et al., 1998).\nAs during later pupal life Fz and Stbm\/Vang are seen localised to adjacent cell boundaries (Strutt, 2001; Bastock et al., 2003), it has been proposed that polarity coordination requires signals to pass between Fz and Stbm\/Vang expressing cells. Some evidence for this has been presented in the abdomen (Lawrence et al., 2004), and recent models for planar polarity coordination in the wing are based on this hypothesis (Amonlirdviman et al., 2005; Klein and Mlodzik, 2005; Le Garrec et al., 2006). However, there is no rigorous experimental evidence for signals passing between Fz and Stbm\/Vang expressing cells in the wing. Furthermore, if such signalling does occur, it is not known whether signals might pass monodirectionally from Fz to Stbm\/Vang, monodirectionally from Stbm\/Vang to Fz or bidirectionally.\nTo address this issue, we generated clones of cells simultaneously mutant for both fz and stbm\/Vang. We reasoned that if signals pass strictly monodirectionally from Stbm\/Vang to Fz, then wild-type cells outside of a clone would receive the same aberrant polarity cue from a stbm\/Vang; fz double mutant clone as from a stbm\/Vang single mutant clone. Thus, stbm\/Vang; fz double mutant clones should show the same polarity phenotype as stbm\/Vang single mutant clones.\nConversely, if signals pass strictly from Fz to Stbm\/Vang, cells outside should polarise as if neighbouring a fz clone and not a stbm\/Vang clone. In this case, cells require Fz to send polarity cues and cells mutant for both fz and stbm\/Vang provide the same aberrant polarity cue as cells mutant for only fz.\nHowever, if there is a bidirectional interaction, such that cells expressing Fz need to contact cells expressing Stbm\/Vang and vice versa, then the result is harder to predict. In this case, signal receiving cells would require both Fz and Stbm\/Vang and similarly signal sending cells would require both Fz and Stbm\/Vang. Hence, one possibility is that clones of cells doubly mutant for fz and stbm\/Vang would send or receive no polarity signals, and thus might have no effect on the polarity of their neighbours. A precedent for this prediction has been provided by work in the abdomen, where experimental results suggest that cells that lack Fmi\/Stan are unable to send or receive polarity cues, and in this case the neighbours to exhibit normal polarity (Lawrence et al., 2004). However, it is also possible that a failure to send or receive cues might result in neighbouring cells adopting a randomised polarity.\nControl clones lacking only fz activity (marked by lack of lacZ expression) show trichomes pointing towards the clone (arrow Fig. 2A); whereas stbm\/Vang clones (marked by lack of lacZ) show trichomes pointing away (arrow Fig. 2B). We then generated double mutant stbm\/Vang; fz clones using null alleles of both fz and stbm\/Vang and an Arm-fz-EGFP transgene (which rescues fz activity, see Strutt, 2001) located on the same chromosome arm as stbm\/Vang. This resulted in genetically mosaic wings containing clones of cells of the genotype stbm6\/stbm6; fz21\/fz21 juxtaposed to twinspot tissue of the genotype Arm-fz-EGFP\/Arm-fz-EGFP; fz21\/fz21 or heterozygous tissue of the genotype Arm-fz-EGFP\/stbm6; fz21\/fz21 (see Materials and methods). Such clones of stbm\/Vang; fz cells (marked by lack of Fz-EGFP, green) show negligible effects on the polarity of trichomes in neighbouring cells (Fig. 2C, trichomes visualised by labelling for Actin, magenta). This result fits the hypothesis that bidirectional interactions occur between Fz and Stbm\/Vang expressing cells, and that lack of communication with cells within a clone leads to neighbouring cells adopting a wild-type polarity.\nInterestingly, within the double mutant clones, trichome polarity is also relatively unperturbed (Fig. 2C). This is in contrast to single mutant fz and stbm\/Vang clones, where trichomes of a sufficient age adopt polarities consistent with those shown by trichomes outside the clone (e.g. Fig. 2B). We do not fully understand this phenomenon; however, it is well-established that trichomes within fz and stbm\/Vang tissue largely emerge in the cell centre without obvious polarity (Wong and Adler, 1993; Taylor et al., 1998). We surmise that such \u201capolar\u201d trichomes subsequently align themselves with the strongly polarised trichomes in the wild-type tissue surrounding the clone- possibly as a result of cytoskeletal interactions between adjacent cells.\nWe also examined the distribution of the core polarity protein Fmi\/Stan on the boundaries of clones of cells singly or doubly mutant for fz and stbm\/Vang. It has previously been shown that Fmi\/Stan strongly localises to the boundaries between fz+ and fz\u2212 tissue and stbm\/Vang+ and stbm\/Vang\u2212 tissue (arrowheads Figs. 2A\u2032, B\u2032; Usui et al., 1999; Bastock et al., 2003). Although not formally proven, it is widely thought that such localised protein localisation might mediate cell\u2013cell communication (Amonlirdviman et al., 2005; Klein and Mlodzik, 2005; Le Garrec et al., 2006). Consistent with this view, there is no strong localisation of Fmi\/Stan on the boundaries of stbm\/Vang; fz double clones (Fig. 2C\u2032).\nSo far our results suggest that Fz in one cell and Stbm\/Vang in the adjacent cell is necessary for cell\u2013cell communication and polarisation of trichomes. We next investigated whether Fz and Stbm\/Vang in adjacent cells were sufficient for this process. To do this, we examined the effect of juxtaposing cells that lack fz activity to cells that lack stbm\/Vang activity. This was achieved by generating clones homozygous for the genotype FRT42D stbm6, Arm-Fz-EGFP; fz21 juxtaposed to twinspots of the genotype FRT42D Arm-lacZ; fz21 (see Materials and methods), such that cells lacking stbm\/Vang activity also lacked lacZ expression, whereas cells lacking both fz activity and the rescuing Arm-fz-EGFP transgene exhibited high levels of lacZ expression.\nOn the boundaries where stbm\/Vang tissue is juxtaposed to fz tissue, we observe strong Fmi\/Stan localisation (Fig. 2D, arrowheads in Fig. 2D\u2032) resembling that seen on the edges of fz or stbm\/Vang clones (Figs. 2A\u2032,B\u2032). Notably, at the edges of fz and stbm\/Vang clones, localised Fmi\/Stan is associated with production of polarised trichomes (Figs. 2A, B), apparently as a result of assembly of a polarised asymmetric core polarity protein complex with Fz on one side of the cell\u2013cell boundary and Stbm\/Vang on the other side (Fig. 1B). We also observe polarised trichomes produced at the site of Fmi\/Stan localisation on boundaries between stbm\/Vang and fz tissue, which point towards the fz tissue (Figs. 2D\u02ba,D\u2034). Taken together, the localisation of Fmi\/Stan and the production of polarised trichomes suggest that a functional core polarity protein complex assembles on the boundaries between stbm\/Vang and fz tissue and that this complex is sufficient to specify polarised trichome formation.\nWe note that within the stbm\/Vang and fz mutant tissue, there is no assembly of asymmetric complexes and trichome placement is unpredictable (Figs. 2D, D\u2032), as expected from previous work (Wong and Adler, 1993; Taylor et al., 1998).\nWe next analysed the role of Fmi\/Stan in cell\u2013cell communication of polarity cues in the wing. Unlike fz or stbm\/Vang clones, clones of cells lacking fmi\/stan activity do not strongly affect the polarity of neighbouring cells (Fig. 2E, Chae et al., 1999; Usui et al., 1999). This could be interpreted to suggest that fmi\/stan is not required for cell\u2013cell communication and coordination of cell polarity. However, the Fmi\/Stan protein is thought to act as a homophilic cell adhesion molecule (Usui et al., 1999) and so alternatively Fmi\/Stan may be required in both sending and receiving cells for coordination of cell polarity and loss of fmi\/stan blocks cell\u2013cell communication. Support for this second view comes from experiments in the abdomen, where cells overexpressing Fz or Stbm\/Vang are unable to repolarise their neighbours if they also lack fmi\/stan activity (Lawrence et al., 2004).\nWe generated clones of cells double mutant for either fz and fmi\/stan or for stbm\/Vang and fmi\/stan. Both showed a phenotype typical of single mutant fmi\/stan clones (Figs. 2F, G), indicating that Fmi\/Stan is required in both Fz and Stbm\/Vang expressing cells for cell\u2013cell communication, and thus by extension is required on both sides of the cell\u2013cell boundary.\nThus, for the fz-dependent process of cell\u2013cell communication that is thought to locally coordinate cell polarity, we have demonstrated that Fz and Stbm\/Vang are required in opposite cells and that Fmi\/Stan is required in both cells. This spatial arrangement is as seen in the asymmetric complex that assembles at the site of trichome initiation (Fig. 1B), and supports models in which a subset of this complex is also involved in intercellular communication.\ndsh and pk are not required for intercellular communication\nIn the late asymmetric complex, Dsh associates with Fz at distal cell edges whereas Pk localises with Stbm\/Vang at proximal cell edges. Hence, it is possible that the association of Dsh with Fz is essential for signalling to Stbm\/Vang in the adjacent cell, and similarly that Pk association with Stbm\/Vang is required for signalling to Fz in the adjacent cell. However, interactions have also been reported between Dsh and Pk and between Dsh and Stbm\/Vang (Tree et al., 2002b; Bastock et al., 2003; Jenny et al., 2005), and during an early phase of cell\u2013cell communication these interactions might also be important.\nClones of cell mutant for dsh alone do not strongly affect the polarity of neighbouring cells (Fig. 3A, Theisen et al., 1994), in this respect resembling fmi\/stan clones. Thus, like fmi\/stan, it is possible that dsh might be required on both sides of cell\u2013cell boundaries for communication to occur, consistent with its known physical associations with both Fz and Stbm\/Vang. However, we found that both dsh; fz and dsh; stbm\/Vang double mutant clones showed non-autonomous effects on the polarity of neighbouring cells, typical of either single mutant fz or stbm\/Vang clones respectively (arrows Figs. 3B, C), with normal accumulation of Fmi\/Stan at clone boundaries (arrowheads Fig. 3B\u2032). These results demonstrate that dsh activity is not required for Fz-Stbm\/Vang-dependent intercellular communication to occur, in either Fz-expressing or Stbm\/Vang-expressing cells.\nWe carried out similar experiments using a null allele of pkpk-sple. Single mutant clones of pk also do not significantly affect the polarity of neighbouring cells (Fig. 3D, Gubb et al., 1999). However, pk-sple; fz and pk-sple stbm\/Vang double mutant clones still show the typical non-autonomous effects of fz or stbm\/Vang clones respectively (arrows Figs. 3E, F) and accumulation of Fmi\/Stan at clone boundaries (arrowheads Fig. 3E\u2032). We conclude that pk activity is also not essentially required for Fz-Stbm\/Vang-dependent intercellular communication.\nThe range of non-autonomous alterations in cell polarity around fz and stbm\/Vang clones is generally up to 10 cell diameters; however, it varies with clone size, shape and position (Vinson and Adler, 1987; Taylor et al., 1998; Adler et al., 2000). We observed similar ranges of non-autonomy for the double mutant clones generated with null dsh and pk alleles, suggesting that the strength of intercellular signalling remained in the normal range.\nAnalysis of dgo function\nIt has been recently reported that during planar polarity patterning of the Drosophila eye disc, the core polarity gene dgo acts redundantly with stbm\/Vang and pk (Das et al., 2004). If such a situation also pertained in the wing, we reasoned that this might mask specific roles of either pk or dgo in intercellular signalling. However, we find that stbm\/Vang dgo clones still exhibit the proximal non-autonomy typical of stbm\/Vang single clones (arrow Fig. 4A) and pk dgo clones behave like pk single clones, showing no non-autonomous effect on trichome polarity (Fig. 4B). In an attempt to test the hypothesis of redundant functions of core polarity proteins as rigorously as possible, we generated clones of cells triply mutant for pk, dgo and fz. These also behaved like single mutant fz clones, showing typical non-autonomous effects on trichome polarity (arrow Fig. 4C).\nIn the eye, dgo has been particularly implicated in cooperating with pk and stbm\/Vang to maintain the junctional localisation of Fmi\/Stan (Das et al., 2004). Interestingly, in the pupal wing Fmi\/Stan remains junctional in stbm\/Vang dgo clones (Fig. 4D), although proximodistal asymmetric localisation is lost as previously reported for stbm\/Vang clones (Bastock et al., 2003). The same is true of Fmi\/Stan localisation in pk dgo clones (Fig. 4E). We also find that contrary to the reported situation in the eye, Stbm\/Vang apicolateral localisation is maintained in pk dgo clones (Fig. 4F).\nPolarity defects propagate through tissue mutant for pk, pk dgo and dsh to different degrees\nMosaic experiments so far described have analysed the ability of clones of cells lacking the function of one or more core polarity genes to influence the polarity of neighbouring cells via intercellular signalling. However, for polarity defects to propagate away from such clones, intracellular signalling is required across the axes of individual cells, in addition to intercellular signalling between cells. The nature of intracellular signalling is poorly understood. One recent model proposes that it depends upon detection of levels of intracellular fz activity by transmembrane receptors on different sides of the cell (Lawrence et al., 2004), whereas others suggest that it relies on asymmetric assembly of protein complexes on one cell edge in response to the presence of an asymmetric complex at the opposite cell edge (Amonlirdviman et al., 2005; Klein and Mlodzik, 2005; Le Garrec et al., 2006).\nAlthough dsh, pk and dgo do not have essential functions in intercellular signalling, they might nevertheless be required for intracellular communication. However, in this context, it is interesting to note that polarity defects can still propagate around fz clones in abdomens wholly mutant for a null allele of pk (Lawrence et al., 2004) and in wings mutant for the pk1 mutation which mutates one of the pk isoforms (Adler et al., 2000). Taken at face value, these data suggest that pk may not be essentially required for either intercellular or intracellular signalling.\nTo investigate this further, we generated clones of cells with altered fz or stbm\/Vang activity in wings wholly mutant for the function of other core polarity genes. As it simplified the generation of the appropriate fly strains, in these experiments we generated clones of cells with increased Fz activity (which cause neighbouring trichomes to point away from the clone, see e.g. Strutt, 2001) or Stbm\/Vang activity (which cause trichomes to point towards the clone, Amonlirdviman et al., 2005).\nFor control experiments, we analysed wings entirely mutant for fz or stbm\/Vang activity. These factors are required both for intercellular communication and trichome placement at the cell edge, so overexpression clones in these backgrounds are not expected to alter the polarity of neighbouring cells (e.g. Taylor et al., 1998). Consistent with this, clones of cells that overexpress Stbm\/Vang cannot alter trichome polarity in fz wings (Fig. 5A), and clones of cells overexpressing Fz cannot alter trichome polarity in stbm\/Vang wings (Fig. 5B).\nHowever, if Fz is overexpressed in clones of cells in wings wholly mutant for a null allele of pk, we observed a strong effect on trichome polarity in neighbouring cells (Fig. 5C), which extends 8\u20139 cells (average 8.6, n\u00a0=\u00a011) from the clone boundary, similar to the effect seen in wild-type wings and in agreement with previous observations (Adler et al., 2000; Lawrence et al., 2004). Interestingly, in wings double mutant for pk and dgo, clones of cells overexpressing Fz also affect the polarity of neighbouring cells (Fig. 5D), but the effect only extends for 5\u20136 cells (average 5.6, n\u00a0=\u00a08). Hence, although patterning is essentially normal in dgo wings, and polarity defects propagate a normal distance though pk tissue, when both factors are absent the propagation of polarity defects is substantially reduced, revealing an unexpected redundancy between these factors for this process.\nWe also investigated the effect of overexpressing Fz in clones of cells in wings mutant for dsh. To circumvent the lethality of dsh null mutations, we used the dsh1 point mutation which affects only planar polarity functions. We again observed an effect of the clones on the polarity of neighbouring cells (Fig. 5E); however, in this case, polarity defects only propagated at most 3\u20134 cells from the clone (average 3.7, n\u00a0=\u00a07). Finally, we tested propagation through cells mutant for a null allele of dsh. Non-autonomous polarity defects were induced by overexpressing Fz-GFP in the patched expression domain at the anteroposterior compartment boundary (Adler et al., 1997), in wings containing dsh3 clones (Fig. 5F). Within the clones, we observed trichomes emerging largely in the cell centre as previously reported (Wong and Adler, 1993), and were unable to detect propagation of polarity defects.\nAsymmetry of polarity protein activity exists in the absence of asymmetry in distribution\nRecent models have suggested that asymmetric localisation might be essential for polarity propagation (Amonlirdviman et al., 2005; Klein and Mlodzik, 2005; Le Garrec et al., 2006). This is at variance with observations of propagation of polarity defects in pk or dsh1 backgrounds (Figs. 5C, E and Adler et al., 2000; Lawrence et al., 2004), in which asymmetry is not observed (Figs. 6A, B and e.g. Axelrod, 2001; Strutt, 2001; Usui et al., 1999). However, it is possible that weak asymmetry exists in these backgrounds, which is difficult to detect. We investigated this more closely by generating clones of cells expressing Fz-EYFP. In general, a subtle increase or decrease in Fz-EYFP at the edge of one cell might be masked by Fz-EYFP localisation on the adjacent boundary of a neighbouring cell (as it is not possible to distinguish between localisation at adjacent cell boundaries by visible light microscopy). However, by looking at Fz-EYFP distribution on the edges of clones, it should be easier to discern subtle asymmetry of proximodistal localisation (see Strutt, 2001). Hence we examined the distribution of Fz-EYFP expressed in clones in null pk and dsh1 backgrounds, but still were unable to observe any evidence of asymmetric localisation (Figs. 6A\u2032,B\u2032).\nNotwithstanding our failure to find asymmetry of distribution of polarity proteins in pk and dsh1 wings, we nevertheless suppose that there must be asymmetry of activity in order for propagation of polarity and asymmetric trichome placement to occur. Interestingly, in clones of cells lacking dgo or pk activity, trichome formation occurs both at the same time as in adjacent non-mutant tissue and at the distal cell edge (Figs. 6C, D). Hence in these backgrounds there is clearly an asymmetric signal for trichome formation. In contrast, in clones of cells doubly mutant for dgo and pk, trichome formation in often seen to be delayed and when it does occur is often seen in the cell centre (Fig. 6E), indicative of loss of the asymmetric trichome placement cue. The phenotype in pk dgo clones is similar to that seen in tissue mutant for dsh, fz or stbm\/Vang (Fig. 6F, Wong and Adler, 1993; Taylor et al., 1998). Hence, consistent with the differing effects of dsh, pk and dgo on propagation of polarity defects, we see a similar progression of effects on the asymmetric placement of trichomes at the cell edge, suggesting that these two processes are linked.\nDiscussion\nAs described in the Introduction, it is possible to define a core group of polarity proteins, but this does not imply that all components of the core make equal contributions to planar polarity patterning. In this work, we have attempted to systematically analyse the contributions of the core proteins to the processes of coupling to the global cue, local coordination of polarity and asymmetric trichome placement.\nOur key findings are as follows:(i)The transmembrane proteins Fz, Stbm\/Vang and Fmi\/Stan have a common early function during planar polarity patterning in the wing and eye, with the cytoplasmic factors Dsh and Pk playing only later roles.(ii)The transmembrane core of Fz, Fmi\/Stan and Stbm\/Vang is absolutely required for intercellular communication. We demonstrate that the asymmetric relationship of these proteins seen at the time of trichome placement, with Fmi\/Stan in both communicating cells and Fz in one cell juxtaposed to Stbm\/Vang in the adjacent cell, is also necessary and sufficient for such intercellular communication. In addition we provide evidence that information passes both from Fz to Stbm\/Vang expressing cells and vice versa.(iii)Intercellular communication does not require Dsh or Pk in either Fz or Stbm\/Vang expressing cells and Dgo also does not play redundant roles with Pk in intercellular signalling.(iv)Pk and Dgo act redundantly in propagation of polarity from cell to cell, most likely by promoting intracellular communication. Dsh plays a prominent role in such propagation, greater than that of both Pk and Dgo. We speculate that the intracellular communication required for such polarity propagation is dependent on establishing intracellular asymmetries of protein activity.(v)Although even subtle asymmetry of Fz localisation is not apparent in pk tissue, not only does polarity propagate between cells, but trichome placement also occurs at the normal time and place. Hence, asymmetry of polarity protein activity exists in the absence of detectable asymmetry of localisation.(vi)Neither Pk nor Dgo are directly required for determining the site of trichome placement.\nCoupling to the global polarity cue\nAs noted in the Introduction, one of the putative functions of the core polarity proteins is to couple to long-range polarity patterning cues. It has been suggested that these cues are provided by fj\/ds\/ft (Ma et al., 2003; Yang et al., 2002), but little is understood regarding the molecular mechanism of any such coupling. We suppose that fz, stbm\/Vang and fmi\/stan are involved as they show the earliest requirement. Comparison with the temporal requirements of ds (Matakatsu and Blair, 2004), argues against a role for pk, and probably dsh (with the caveat that we were unable to analyse a null allele). Interestingly, it has been argued that during abdomen patterning, pk may play a particular role in \u201crectifying\u201d the global signal in different compartments (Lawrence et al., 2004). In the wing, such a function is not necessary, possibly explaining why we do not find a corresponding early role for pk.\nLocal coordination of polarity\nA better understood and major function of Fz and the core polarity proteins is the local coordination of cell polarity. All recent models for this coordination have proposed a role for cell\u2013cell contact mediated signalling, as opposed to schemes requiring the secretion of a diffusible ligand. A key feature of such models is that they require both intercellular communication to pass polarity cues between adjacent cells and intracellular communication to pass information across the axes of individual cells. Experimental support for cell\u2013cell contact mediated signalling has been provided by experiments in the abdomen, showing that the atypical cadherin Fmi\/Stan is required in both signal sending and receiving cells, suggesting that signals pass between Fmi\/Stan homodimers (Lawrence et al., 2004). Theoretical evidence has also been provided by a number of mathematical models that have confirmed the feasibility of locally coordinating polarity via assembly of asymmetric junctional complexes containing Fz in one cell and Stbm\/Vang in the adjacent cell (Amonlirdviman et al., 2005; Le Garrec et al., 2006).\nOur results rigorously demonstrate that, in the wing, intercellular signalling events that locally coordinate polarity require Fmi\/Stan in both communicating cells and Fz in one cell and Stbm\/Vang in the other. This supports models in which the asymmetric junctional distributions that are observed by immunofluorescence are required for intercellular signalling (Amonlirdviman et al., 2005; Klein and Mlodzik, 2005; Le Garrec et al., 2006). However, it should be noted that although signalling may require the assembly of complexes with Fz in one cell adjacent to Stbm\/Vang in the next, this does not necessarily imply that detectable asymmetric subcellular distribution of proteins within cells is necessary. Indeed the persistence of signalling in pk and dsh1 backgrounds where subcellullar asymmetry of the core polarity proteins is not observed argues against this being essential. In addition, our data raise the possibility of bidirectional cell\u2013cell communication via Fz-Stbm\/Vang, and are inconsistent with a monodirectional signal as proposed to occur in the abdomen (Lawrence et al., 2004).\nIt is also evident from our results that intercellular signalling does not require association of Dsh, Pk or Dgo with Fz or Stbm\/Vang. Indeed, fz or stbm\/Vang clones that also lack any of these factors are not obviously impaired in their ability to alter the polarity of neighbouring cells.\nWhat then are the roles of Dsh, Pk and Dgo? We show that propagation of polarity defects away from a clone is reduced in dsh and pk dgo tissue, indicating that they are required for local relay of polarity cues. Hence, a likely role would be in the intracellular signalling required to pass polarity cues across the axes of cells. Previous experiments in which polarity was seen to propagate normally in a pk background (Adler et al., 2000; Lawrence et al., 2004) argued against such a function for pk, which is only revealed when dgo function is also absent.\nOne proposed mechanism for intracellular signalling is that each cell acquires a particular level of Fz activity, which is communicated by intercellular signalling to all surrounding cells (Lawrence et al., 2004). In this case, roles for Dsh, Pk and Dgo in modulating intracellular levels of Fz activity could be envisaged. However, the majority of models suggest roles for Dsh and Pk in intracellular feedback loops that amplify differences in the asymmetric localisation of the core polarity protein complexes within cells (Amonlirdviman et al., 2005; Klein and Mlodzik, 2005; Le Garrec et al., 2006). For instance, it has been proposed that association of Dsh with Fz might be antagonised by high local concentration of Stbm\/Vang-Pk (Amonlirdviman et al., 2005), or alternatively that Fz-Dsh antagonise Stbm\/Vang-Pk interactions (Le Garrec et al., 2006). In either case, if asymmetric complexes containing Fz, Fmi\/Stan and Stbm\/Vang were somehow stabilised by addition of Dsh, Pk and\/or Dgo to the complex, then such antagonistic interactions would provide feedback that would amplify asymmetries of protein localisation across the axes of individual cells. Notably, our experimental results suggest that if such feedback is occurring, then the relative importance of the cytoplasmic factors in stabilising complex formation follows the hierarchy Dsh\u00a0>\u00a0Pk\u00a0>\u00a0Dgo. Such a scheme would also explain the redundant functions of pk and dgo, even though these factors act at opposite cell edges, as Pk on one side of a cell\u2013cell boundary could bind to and stabilise a complex that was also being stabilised by Dgo binding on the opposite side of the cell\u2013cell boundary. Simultaneous loss of both Pk and Dgo would have a greater destabilising effect than loss of either factor alone.\nHowever, models that depend on amplification of differences in asymmetric subcellular protein distribution have to be reconciled with the failure to observe protein asymmetries in pk or dsh1 tissue, through which polarity can still propagate. Possibly in these backgrounds there are subtle asymmetries which cannot be observed\u2014notably at least one recent model predicts such subtle asymmetry in pk clones (Amonlirdviman et al., 2005). But another explanation is that receptor proteins such as Fz may be uniformly distributed, but nevertheless exhibit differential signalling activity across the axes of cells.\nDespite the apparently non-essential role of protein asymmetry either in polarity propagation over short distances or in trichome placement, it nevertheless seems likely that it is an active mechanism in ensuring robust coordination of polarity and correct trichome placement over the whole wing (Ma et al., 2003; Amonlirdviman et al., 2005), as otherwise the failure of long-range coordination of polarity in pk wings cannot be explained. Interestingly, it has recently been reported that asymmetry is present from as early as the third instar stage of development, but is subsequently lost during junctional remodelling in pupal stages (Classen et al., 2005), suggesting that such asymmetry could be playing a role from much earlier in development than previously suspected.\nSpecification of the site of polarised trichome production\nThe precise mechanism by which asymmetric trichomes are generated remains unknown, although there appears to be a role for asymmetric subcellular activities of polarity effector proteins such as Inturned (Adler et al., 2004). As asymmetric trichomes can be generated in the absence of Pk or Dgo, it seems unlikely that either of these proteins interacts directly with the trichome placement machinery; however, all of the other core polarity proteins are candidates for such a role.\nConcluding remarks\nIn conclusion, we note that there has recently been great interest in attempting to mathematically model the processes underlying propagation of planar polarity between cells (e.g. Lawrence et al., 2004; Amonlirdviman et al., 2005; Klein and Mlodzik, 2005; Le Garrec et al., 2006). Although the presented models have been very successful at reproducing known phenomena, they have nevertheless been based on limited experimental data. This work both provides support for some of the assumptions of such models, for instance by directly testing the central role of core transmembrane proteins such as Fz, Fmi\/Stan and Stbm\/Vang in intercellullar signalling, but also provides challenges, for instance by demonstrating the propagation of polarity in the absence of visible protein asymmetry.","keyphrases":["planar polarity","drosophila","frizzled","strabismus","flamingo","dishevelled","prickle","diego","pcp"],"prmu":["P","P","P","P","P","P","P","P","P"]}
{"id":"Eur_Spine_J-2-2-1602194","title":"Textiloma: a case of foreign body mimicking a spinal mass\n","text":"Items such as cotton or gauze pads can be mistakenly left behind during operations. Such foreign materials (called textilomas or gossypibomas) cause foreign body reaction in the surrounding tissue. The complications caused by these foreign bodies are well known, but cases are rarely published because of medico-legal implications. Some textilomas cause infection or abscess formation in the early stage, whereas others remain clinically silent for many years. Here, we describe a case of textiloma in which the patient presented with low-back pain 4 years after lumbar discectomy. Imaging revealed an abcess-like mass in the lumbar epidural space.\nIntroduction\nTextiloma and gossypiboma are non-medical terms used to describe a mass of cotton matrix that is left behind in a body cavity during an operation [9, 18, 20]. Such foreign bodies can often mimic tumors or abscesses clinically or radiologically. Although these masses and their associated complications may occur, they are rarely reported due to medico-legal implications [7]. Most cases of textiloma in the literature have been connected with abdominal or thoracic surgery, very few have been linked with spinal surgery. From 1965 to 2006, only 32 cases of spinal or paraspinal textiloma have been documented in the literature [1, 3\u20137, 9, 10, 12, 14, 16, 19\u201321]. Here, we describe a case in which cotton, a foreign body, was left behind during an operation for lumbar disc herniation. The patient presented 4\u00a0years later with a symptomatic mass in the lumbar epidural space, and imaging indicated a possible abscess.\nCase\nA 62\u00a0year old woman presented a complaint of low-back pain. She had had this problem for 1\u00a0year, and the pain had been radiating to her left calf for 1\u00a0month. The patient had undergone surgery for lumbar disc herniation 4\u00a0years prior to presentation. Physical examination indicated good health status. There was no tenderness, swelling or erythema at the incision site. The straight leg-raising test was positive at 60\u00b0 on the left. The patient\u2019s left hamstring muscle strength was 4\/5, and she had diminished patellar deep tendon reflex on that side. Routine laboratory testing (complete blood count, erythrocyte sedimentation rate, blood biochemistry panel) revealed nothing abnormal. Magnetic resonance imaging (MRI) of the lumbosacral spine showed the laminectomy defect at L3 and a mass lesion in the posterior paravertebral region at this level. The mass appeared hypointense to spinal cord tissue on T1-weighted images and hyperintense on T2-weighted images. Injection of contrast medium revealed an enhanced hyperintense rim around a hypointense center (Figs.\u00a01, 2). The lesion was interpreted as a possible epidural abscess. At surgery, a foreign body composed of cotton was found and was completely removed (Fig.\u00a03). No infection or abscess was detected. Histological examination of the tissues around the material revealed only chronic inflammatory infiltration and granuloma formation. One month after the operation, there were no abnormal findings either on physical examination or on MRI of the lumbosacral spine.\nFig.\u00a01MRI of the lumbosacral spine showing the mass lesion in the posterior paravertebral regionFig.\u00a02The sagittal section of MRI showing the left laminectomy at L3 vertebra and the mass lesionFig.\u00a03Cotton materials extracted from the patient during the operation\nDiscussion\nCotton pads, towels and sponges are used to achieve hemostasis during surgical procedures, including dissection for intervertebral disc herniation and other spinal problems. Although precautions are taken to avoid leaving such materials behind, mistakes do happen and the resultant foreign bodies can cause various clinical and radiological manifestations [1, 5, 8, 12]. In the early period after surgery, these forgotten materials can lead to infections and abscess formation. However, some remain clinically asymptomatic for many years, and then cause a foreign body reaction in the surrounding tissue, with new clinical signs indicating significant mass effect [3, 5, 6, 15, 22]. Cotton is not the only material that can lead to such problems. The literature contains reports of other hemostatic materials (such as gelfoam and Surgicel) causing foreign body reactions that could not be distinguished from recurrent tumors on MRI [9, 13].\nThe introduction of MRI has made it possible to diagnose most foreign bodies accurately. Findings differ according to the radiological modality that is used to investigate the patient. Cotton sponges and cotton fibers exhibit characteristic features on plain radiographs, whereas the findings on computerized tomography and ultrasonography are less diagnostic [3, 6, 11, 12, 14, 16, 20]. The MRI appearance of foreign materials that are left behind during surgery can differ greatly depending on the time since the operation and the type of foreign body reaction that occurs. There are two types of foreign body reactions: aseptic fibrous tissue reaction, which involves adhesion formation, encapsulation and granuloma formation, or the exudative-type tissue reaction, which leads to abscess formation [3, 6, 8, 14\u201316, 18, 22]. Karcnik et al. [7] reported a case of foreign body reaction that manifested in a later period after an anterior cervical fusion operation. MRI showed a granuloma that was hypointense on T1-weighted images and hyperintense on T2-weighted images, and that mimicked a solid tumor. In general, most lesions caused by foreign bodies are hypointense on T1-weighted images and hyperintense on T2-weighted images [3, 5, 8, 15, 16]. In our case, the MRI findings were consistent with an epidural abscess, but direct observations at surgery, microbiologic testing and pathologic examination revealed no infection.\nForeign bodies that are left behind during operations may organize and increase in size but such changes are not correlated with time. To date, the case reported by Taylor et al. [17] features the longest period from surgery to manifestation of symptoms. They detected an intrapulmonary foreign body 43\u00a0years after thoracotomy. The longest reported interval in the neurosurgery literature is 40\u00a0years. In that case, a cotton pad was left posterior to the lumbosacral vertebrae during a laminectomy operation, and the material eventually caused a cavitary lesion [16]. Our patient developed a textiloma 4\u00a0years after lumbar disc surgery.\nCivil lawsuits brought against surgeons for surgical complications are becoming more frequent, and this is prompting surgical teams to be even more careful. It is possible to to overlook cotton and gauze pads in the surgical field. Such materials should always have a tag that allows them to be easily located and removed, and all materials that are placed in the wound temporarily, must be counted many times with meticulous care. Cotton pads are not safe because they can break into fragments during manipulation; other materials are preferred for securing hemostasis. Once hemostasis is achieved, the operative site should be flushed with saline and carefully examined for foreign materials.","keyphrases":["foreign body","cotton","complication","lumbar disc surgery"],"prmu":["P","P","P","P"]}
{"id":"Osteoporos_Int-4-1-2267485","title":"FRAX\u2122 and the assessment of fracture probability in men and women from the UK\n","text":"Summary A fracture risk assessment tool (FRAX\u2122) is developed based on the use of clinical risk factors with or without bone mineral density tests applied to the UK.\nIntroduction\nSeveral multi-factorial diseases (e.g., diabetes, hypertension) are defined on the basis of important risk factors for the clinical outcome. Hypertension, for example, is defined from the measurement of blood pressure that provides information on the likelihood of stroke. Hypercholesterolaemia and osteoporosis are also examples. In the case of osteoporosis, bone mineral density (BMD) is measured both to provide a diagnosis, and to yield information on fracture risk [1].\nMany well-controlled prospective studies with dual energy X-ray absorptiometry (DXA), particularly in elderly women, indicate that the risk of fracture about doubles for each SD reduction in BMD [2, 3]. The measurement of a risk factor for diagnostic use, however, can only capture one aspect of the likelihood of the outcome when the disease is multifactorial, and in osteoporosis, assessment with BMD captures a minority of the fracture risk. For example, the annual incidence of hip fracture increases approximately 30-fold between the ages of 50 and 90\u00a0years; but, from the known relationship between BMD and fracture risk and the loss of bone with age, it is expected that hip fracture risk would rise only fourfold [4\u20136]. Thus, the increase in risk with age is approximately sevenfold greater than can be explained on the basis of BMD alone.\nThe imperfect capture of risk with BMD alone poses several problems for the clinical assessment of fracture risk. In the context of population screening with BMD alone, the performance characteristics of the test are less than optimal in terms of the trade-off between sensitivity and specificity [1, 7, 8]. Thus, osteoporotic fractures affect a substantial minority of the population, but intervention thresholds based on BMD alone lack sensitivity over most reasonable assumption, i.e., the detection rate is low. For example, at the age of 50\u00a0years the proportion of women with osteoporosis is approximately 5% [4]. The proportion of these who will fracture in the next 10\u00a0years (i.e., positive predictive value) is about 20%. The detection rate for these fractures (sensitivity) is, however, low, and 96% of fragility fractures would arise in women without osteoporosis given a test like BMD where the fracture risk doubled for each SD decrease [9]. Low sensitivity is one of the reasons why wide-spread population screening is not widely recommended in women at the menopause [1]. Moreover, a normal BMD measurement is no guarantee that a fracture will not occur.\nThe use of risk factors that add information on fracture risk independently of BMD improves the sensitivity of the assessment for any specificity [8, 9]. Over the past several years, we have undertaken a series of meta-analyses to identify clinical risk factors for fracture that provide independent information on fracture risk [3, 10\u201316]. The analyses were based on the primary data from prospective population based studies. This permits the inter-dependence of each of the candidate risk factors to be examined so that they can be accurately combined for clinical use. In the case of the clinical risk factors, BMI is used as a continuous variable, so that its distribution is preserved by the addition of dichotomous variables [8]. For hip fracture prediction, the gradient of risk (increase in fracture risk per standard deviation increase in risk score) for the CRFs is comparable to the use of DXA alone, and the gradient of risk is further enhanced by the addition of BMD to the CRFs [17]. These inter-relationships, assessed from multiple populations and validated in independent cohorts, permit the more accurate identification of individuals who will fracture, so that the average risk in any given proportion of the population identified for treatment will be higher [8].\nThese considerations indicate that assessment can be improved by the integration of clinical risk factors with or without BMD. In other words, treatment should be directed not only on the basis of T-score for BMD, but also on the independent contribution of other validated risk factors. However, the use of different metrics (the T-score, gradients of risk, risk ratios, etc) is confusing for clinicians and patients alike. For this reason, there has been interest in the development of algorithms that express absolute risk, or the probability of fracture within a given time period [18\u201320], as has been done for cardiovascular and other diseases [21\u201325]. The aim of the present study was to develop a model for the clinical assessment of fracture probability in men and women based on the epidemiology of the UK.\nMethods\nCohorts\nIn order to identify the relevant risk factors, we used baseline and follow-up data from nine prospective population-based cohorts comprising the Rotterdam Study, The European Vertebral Osteoporosis Study (later the European Prospective Osteoporosis Study (EVOS\/EPOS), The Canadian Multicentre Osteoporosis Study (CaMos), Rochester, Sheffield, Dubbo, a cohort from Hiroshima and two cohorts from Gothenburg. Details of each of the cohorts have been recently published elsewhere [10\u201316].\nBaseline and outcome variables\nHeight and weight were measured using standard techniques in all cohorts. Body mass index (BMI) was calculated as weight divided by height squared (kg\/m2) and used as a continuous variable. BMD was assessed at the femoral neck by DXA with the exception of the two Gothenburg cohorts in which BMD was measured elsewhere. Femoral neck BMD was used as a continuous variable (cohort-specific Z-scores excluding the two cohorts from Gothenburg). The clinical risk factors utilised were those identified from the previous meta-analyses [3, 10\u201316]. These comprised a parental history of hip fracture, exposure to systemic glucocorticoids, a prior history of fragility fracture, current smoking, high intake of alcohol (3 or more units daily on average) and the presence of rheumatoid arthritis as an indicator for secondary osteoporosis.\nFracture ascertainment in the primary cohorts was undertaken by self-report (Sheffield, EVOS\/EPOS, Hiroshima) and\/or verified from hospital or central data-bases (Gothenburg, CaMos, DOES, Sheffield, EVOS\/EPOS, Rochester, Rotterdam).\nModels used\nFour models were constructed from the risk factor analysis to compute fracture probabilities. These comprised the probability of hip fracture, with and without BMD, and the probability of other major osteoporotic fractures (clinical spine, forearm and proximal humerus), with and without BMD. For each model, fracture and death as continuous hazard functions were computed using a Poisson regression [26, 27] and detailed in the Appendix. In brief, for each risk factor, all significant interactions terms that were identified by meta-analysis were entered (with age, time, sex and the risk factor) with and without BMD [17]. Interactions that were significant for hip fracture risk were also entered into the model for other osteoporotic fractures, and also included in the model for death. Where interactions noted in the \u201cmega-analyses\u201d were no longer significant for both hip fracture and other osteoporotic fractures, these were omitted in a step-wise manner by dropping the interaction with the largest p value. For the death hazard, all significant interactions for fracture risk were included and thereafter omitted if appropriate in a step-wise manner, as described for the fracture hazard.\nEpidemiology\nThe incidence of hip, forearm and proximal humerus fractures in the UK was taken from Singer et al. [28]. Vertebral fractures can be classified as clinically overt fracture, that is, a symptomatic fracture that comes to clinical attention, or a morphometric fracture, which includes both symptomatic and asymptomatic fractures. For the purposes of this study the clinical definition of a vertebral fracture was used. Because data on vertebral fracture risk in the UK are scarce, with a great deal of variation in reported rates [29], the clinical vertebral fracture incidence was calculated by assuming that the ratio of clinical vertebral fracture to hip fracture would be similar in the UK compared to Sweden [30]. The validity of the assumption cannot be directly tested, but in the case of long bone fractures, there is a close concordance of relative incidence at different fracture sites despite marked differences in absolute risk [31]. Swedish fracture risk data were taken from Kanis et al. [32]. The models were then calibrated so that the mean hazard functions of fracture (and death) equalled that of the UK, as detailed in the Appendix.\nInput and output variables\nIndividual patient details comprise age (50 to 90\u00a0years), sex, weight (in kg) and height (in cm). BMI is automatically computed from height and weight. Dichotomised risk variables are then entered:\nA prior fragility fracture (yes\/no)Parental history of hip fracture (yes\/no)Current tobacco smoking (yes\/no)Ever long-term use of oral glucocorticoids (yes\/no)Rheumatoid arthritis (yes\/no)Other causes of secondary osteoporosis (yes\/no)Daily alcohol consumption of three or more units daily (yes\/no)\nA distinction is made between rheumatoid arthritis and other secondary causes of osteoporosis. Rheumatoid arthritis carries a fracture risk over and above that provided by BMD [11]. Whereas this may hold true for other secondary causes of osteoporosis, the evidence base is weak. Of the many secondary causes of osteoporosis, the following have been consistently documented to be associated with a significant increase in fracture risk:\nUntreated hypogonadism in men and women, e.g., bilateral oophorectomy or orchidectomy, anorexia nervosa, chemotherapy for breast cancer, hypopituitarism [33\u201340]Inflammatory bowel disease, e.g., Crohn\u2019s disease and ulcerative colitis [41\u201343]. It should be noted that the risk is in part dependent on the use of glucocorticoids, but an independent risk remains after adjustment for glucocorticoid exposure [44].Prolonged immobility, e.g., spinal cord injury, Parkinson\u2019s disease, stroke, muscular dystrophy, ankylosing spondylitis [45\u201350]Organ transplantation [51\u201354]Type I diabetes [55\u201358]Thyroid disorders, e.g., untreated hyperthyroidism, over-treated hypothyroidism [59\u201363]\nWhereas there is strong evidence for the association of these disorders and fracture risk, the independence of these risk factors from BMD is uncertain. It was conservatively assumed, therefore, that the fracture risk was mediated via low BMD, but with a risk ratio similar to that noted in rheumatoid arthritis. From an operational view, where the field for rheumatoid arthritis is entered as \u2018yes\u2019, a risk is computed with and without BMD. If the field for other secondary osteoporosis is also filled as \u2018yes\u2019 this does not contribute to the calculation of fracture probability. Conversely, where the field for rheumatoid arthritis entered as \u2018no\u2019, and the field for secondary osteoporosis is \u2018yes\u2019, the same \u03b2 coefficients as used for rheumatoid arthritis contribute to the computation of probability where BMD is not entered. In the presence of BMD, however, no additional risk is assumed in the presence of secondary osteoporosis, since its independence of BMD is uncertain.\nIf any of the fields for dichotomous variables is not completed, a negative response is assumed. Fractures probability can then be calculated. The output (without BMD) comprises the 10-year probability of hip, clinical spine, shoulder or wrist fracture and the 10-year probability of hip fracture (Fig.\u00a01).\nFig.\u00a01Input and output for the FRAX\u2122 model\nFemoral neck BMD can additionally be entered either as a Z-score or a T-score. The transformation of Z- to T-score and vice versa is derived for the NHANES III database for female Caucasians aged 20\u201329\u00a0years [64]. When entered, calculations give the 10-year probabilities as defined above with or without the inclusion of BMD.\nResults\nClinical risk factors\nThe contribution of single clinical risk factors is shown in Table\u00a01 for men and women aged 65\u00a0years. In the example, the BMI is set at 25\u00a0kg\/m2. In the absence of BMD, hip fracture probabilities were higher in women than in men. Each clinical risk factor had a different significance for hip fracture probability, with a family history having the least and a prior fracture the greatest weight in the absence of BMD. The rank order of weighting differed for the major osteoporotic fractures. For example, a parental history of hip fracture was a strong risk factor, close to that provided by a prior fragility fracture. The contribution of age to the assessment of probability is shown in Table\u00a02. In both men and women, there was greater than 100-fold difference in hip fracture probability between the age of 50\u00a0years (no risk factors) and 80\u00a0years (parental history of hip fracture).\nTable\u00a01Ten-year probability (%) of a major osteoporotic fracture or hip fracture in men and women aged 65\u00a0years according to the presence of a single clinical risk factor\u00a0Without BMDT-score \u22122.5 SDMenWomenMenWomenOsteoporoticaHipOsteoporoticaHipOsteoporoticaHipOsteoporoticaHipNo clinical risk factors4.90.88.61.39.83.612.43.0Parental history of hip fracture9.31.016.01.716.53.722.13.2Current cigarette smoking5.11.19.21.911.05.613.75.1Alcohol intake >2 units daily6.01.210.42.012.55.415.44.6Rheumatoid arthritis6.81.411.72.312.85.016.14.3Oral glucocorticoids7.51.513.72.715.06.119.75.5Previous fragility fracture9.61.916.43.216.05.920.25.0BMI is set at 25\u00a0kg\/m2. The right-hand panels show probabilities at a T-score of \u22122.5 SD at the femoral neckaHip, clinical spine, humeral or forearm fractureTable\u00a02Ten-year probability of fracture (%) at the sites shown for men and women with a BMI of 25\u00a0kg\/m2 according to age and the presence or absence of a single risk factor in the absence of BMD\u00a0Osteoporotic fractureaHip fracture5060708050607080(a) MenNo clinical risk factors2.83.95.77.20.10.41.33.4Parental history of hip fracture5.57.69.1150.20.62.811Current cigarette smoking2.84.15.97.50.20.71.84.2Alcohol intake >2 units daily3.34.77.19.50.20.72.05.1Rheumatoid arthritis3.75.38.0110.20.82.35.8Oral glucocorticoids4.46.18.5100.30.92.45.5Previous fragility fracture5.87.911120.51.32.75.2(b) WomenNo clinical risk factors3.56.011170.20.72.37.0Parental history of hip fracture6.91217310.30.95.022Current cigarette smoking3.66.512190.31.13.49.5Alcohol intake >2 units daily4.17.314220.31.13.610Rheumatoid arthritis4.78.215250.41.34.112Oral glucocorticoids5.69.818260.51.54.813Previous fragility fracture7.41220280.82.14.911aClinical spine, hip, humeral or forearm fracture\nThe presence of more than one risk factor increased fracture probability in an incremental manner. For example, in women aged 65\u00a0years with a BMI of 25\u00a0kg\/m2, the 10-year hip fracture probability was 1.3%. With one clinical risk factor, the probability ranged from 1.7% to 3.2%, depending on the risk factor. With two risk factors, the range was 2.5\u20136.6%, and with 3, 4 and 5 risk factors the ranges were 3.8\u201311%, 6.7\u201317% and 13\u201324%, respectively. In the presence of all six clinical risk factors, the 10-year probability of hip fracture was 30%.\nThe effect of several clinical risk factors on the 10-year probability of a major osteoporotic fracture is shown in Fig.\u00a02 for a woman aged 65\u00a0years and a BMI of 25\u00a0kg\/m2. In women with rheumatoid arthritis, there was a 33% increase in fracture probability compared with those without rheumatoid (from 9% to 12%). When women additionally took oral glucocorticoids and had a prior fracture there was a fourfold increase in fracture probability.\nFig.\u00a02Effect of combinations of clinical risk factors on the 10-year probability of a major osteoporotic fracture in women aged 65\u00a0years and a BMI of 25\u00a0kg\/m2. [05Ca201]\nA higher or lower BMI had a marked effect on fracture probability (Fig.\u00a03). For example, in women at the age of 65\u00a0years the 10-year hip fracture probability in the absence of risk factors was 2.3% with a BMI of 20\u00a0kg\/m2, but was fourfold lower (0.6%) at a BMI of 40\u00a0kg\/m2. At each level of BMI, fracture probability increased with an increasing number of clinical risk factors.\nFig.\u00a03Ten-year probability of hip fracture (%) in women aged 65\u00a0years according to the number of clinical risk factors with a BMI of 20\u00a0kg\/m2 (left-hand panels) and a BMI of 40\u00a0kg\/m2 (right-hand panels). The intervals reflect the different weights afforded by different risk factors and provide a range of probabilities. [05Ca070]\nBone mineral density\nThe relationship between fracture probability and BMD is shown in Table\u00a03. In general, fracture risk increased with increasing age and decreasing T-score. At low T-scores, however, fracture probabilities decreased with age, a phenomenon more marked in men than in women. For example, at the age of 50\u00a0years, the 10-year probability of hip fracture was 16.0% in men with a T-score of \u22124 SD and this fell progressively with age, so that at the age of 70\u00a0years, the probability was 12.9% and at the age of 90\u00a0years was 8.3%.\nTable\u00a03Ten-year fracture probability (%) in men and women from the UK with a BMI of 25\u00a0kg\/m2 and no clinical risk factors according to age and T-score at the femoral neckAgeT-score (SD)(years)+10\u22121\u22122\u22123\u22124(a) Hip fracture men50<0.10.10.41.34.716.0600.10.20.61.95.615.6700.20.51.12.65.912.9800.61.11.93.46.110.7901.21.82.53.85.68.3(b) Hip fracture women50<0.10.10.20.83.211.7600.10.20.41.44.413.5700.10.40.92.46.316.2800.51.02.14.49.519.7901.22.03.55.89.816.9(c) Osteoporotic fracturea - men502.52.73.55.19.220.9603.03.54.56.811.522.2703.64.35.58.212.820.9803.24.05.27.611.316.9903.13.95.16.99.713.5(d) Osteoporotic fracturea - women503.03.33.85.28.517.7604.24.85.67.912.623.0705.66.88.411.518.230.4805.87.510.113.821.734.3905.17.19.913.820.030.3aHip, clinical spine, humerus or forearm fracture\nThe differences in probabilities between men and women were much less marked for any given age and T-score than the differences seen with the use of clinical risk factors alone. At a fixed BMD (\u22122.5 SD in Table\u00a01), the difference in probability between men and women was less evident. For example, the hip fracture probability ratio (women\/men) with glucocorticoid use was 1.8 in the absence of BMD but 0.9 at a T-score of \u22122.5 SD. In the case of all major osteoporotic fractures, probabilities remained higher in women than in men at a T-score of \u22122.5 SD but the difference in probabilities between men and women was less marked.\nFigure\u00a04 compares the probability of hip fracture in men and women with a prior fracture according to age. In the absence of BMD there was approximately a twofold difference in probability between men and women. When the BMD was set at an average for women (Z-score\u2009=\u20090), there was little difference in probabilities up to the age of 75\u00a0years. Thereafter, men had a lower probability than women due to the higher death hazard.\nFig.\u00a04Ten-year hip fracture probability (%) in men and women with a prior fracture according to age. The left-hand panel gives probabilities in the absence of BMD. In the right-hand panel probabilities are shown at an average BMD for women at each specific age (i.e., a Z-score = 0). A BMI is set at 24\u00a0kg\/m2. [05Ca065]\nThe clinical risk factors added to the information provided by BMD. Isopleths for 10-year fracture probability are shown in Fig.\u00a05 for the combination of several of the clinical risk factors. The clinical risk factors were somewhat less predictive in the presence of BMD in the models. For example, in women aged 65\u00a0years and a BMI of 20\u00a0kg\/m2, the 10-year hip fracture probability in the absence of BMD ranged from 2.3% in the absence of clinical risk factors to 27.9% with four risk factors. When BMD was set constant at a T-score of \u22122.5 SD, the range was from 2.8% with no clinical risk factor to 19.7% with four risk factors (Fig.\u00a06). The effect of variations in BMI was even more markedly affected by BMD. For example, in women aged 65\u00a0years the 10-year hip fracture probability was 2.3% at a BMI of 20\u00a0kg\/m2 and decreased progressively at higher levels of BMI to 0.6% with a BMI of 40\u00a0kg\/m2. When BMD was fixed, hip fracture probability remained constant irrespective of BMI (Fig.\u00a07).\nFig.\u00a0510-year probability of a major osteoporotic fracture in men and women aged 65\u00a0years according to T-score and clinical risk factors. Body mass index is set at 25\u00a0kg\/m2. [05Ca136]Fig.\u00a06Ten-year probability of hip fracture (%) in women aged 65\u00a0years, according to the number of clinical risk factors. The left-hand panel shows the probabilities without BMD at a BMI fixed at 20\u00a0kg\/m2. The right-hand panel illustrates the effects with BMD fixed at the threshold for osteoporosis. [05Ca073]Fig.\u00a07Effect of variations in BMI on 10-year hip fracture probability (%) in women aged 65\u00a0years. Probabilities with BMD are computed at a T-score of \u22122.8 SD. [05Ca074]\nDiscussion\nThe present study provides a model for the assessment of fracture probability in men and women. The model (FRAX\u2122) uses data derived from nine cohorts from around the world, including centres from North America, Europe, Asia and Australia and has been validated in 11 independent cohorts with a similar geographic distribution [17]. The use of primary (but anonymized) data for the model construct permits the interaction of each of the risk factors to be determined to improve the accuracy whereby fracture probability can be computed. The large sample permitted the examination of the general relationship of each risk factor by age, sex, duration of follow up and, for continuous variables (BMD and BMI), the relationship of risk with the variable itself in a manner hitherto not possible. The use of primary data also eliminates the risk of publication bias. The validity of the clinical risk factors identified are supported by the expected relationships between BMD and fracture risk [3].\nIn the present study, the FRAX\u2122 model has been calibrated to the epidemiology of the UK, but could be calibrated to any country where the epidemiology of fracture and death is known (see Appendix). FRAX\u2122 models for the UK and some other countries are available through the web (http:\/\/www.shef.ac.uk\/FRAX\/index.htm). The approach uses easily obtained clinical risk factors to estimate risk. The estimate can be used alone or with BMD to enhance fracture risk prediction.\nSeveral previous studies have developed models to predict fracture risk from the combination of clinical risk factors and BMD [18, 65\u201378]. The risk factors used include activities of daily living, impaired cognition, liability to falls, poor overall health, history of stroke, seizure disorder and several different medications. A limitation of many of these studies is that, with the exception of the SOF study [18], and one of the GPRD studies [76], they have not been tested in other cohorts. The model described in this paper has been validated in 11 independent prospectively studied cohorts with in excess of one million patient years [17].\nThe use of risk factors for case finding presupposes that the risk so identified is responsive to a therapeutic intervention. To test this hypothesis, it would be necessary to recruit patients selected on the basis of the risk factor(s) to a randomised controlled trial (RCT). The risk factor that is best evaluated in this way is BMD, and indeed the vast majority of therapeutic studies have recruited patients on the basis of low BMD as recommended by regulatory agencies in the US and Europe [79, 80]. In recent years, other trials have recruited patients on the basis of age, gender, a prior vertebral fracture and current exposure to glucocorticoids irrespective of BMD, and have shown therapeutic effects similar to those noted in RCT\u2019s based on BMD selection [14, 81\u201383].\nFor other risk factors, comparable data are lacking. In the absence of empirical data, an alternative approach is to demonstrate that the presence (or absence) of a risk factor does not adversely influence therapeutic efficacy against fractures. Several studies have shown no significant interaction between response to treatment and the presence or absence of the risk factors used in the present study including age, height, family history of fracture, low body weight or BMI, smoking, alcohol intake or prior non-vertebral fracture [84\u201388]. In contrast, some risk factors may be associated with less therapeutic efficacy. For example, patients selected on the basis of risk factors for falling may respond less completely to agents that preserve bone mass than patients selected on the basis of low BMD [89]. This concern is greatest in models that omit BMD, because pharmacological agents may not be equally effective across the entire range of BMD [90].\nThe present model has several unique features. FRAX\u2122 uses Poisson regression to derive hazard functions of death and fracture. Such hazard functions are continuous as a function of time, unlike Cox\u2019s regression for which the corresponding hazard functions are zero except at the time points of a fracture or death. There are also several advantages of the Poisson model over logistic regression analysis. Logistic regression does not take account of when a fracture occurred, nor whether individuals without a fracture died or when death occurred. Secondly, for the assessment of 10-year probabilities by logistic regression, the observation period should be for 10\u00a0years. Moreover, information longer than the 10-year period cannot be used for analysis. The cost of ignoring information when fractures occur and whether and when deaths occur is on the precision of the estimate. In simulation experiments, the Poisson model gives the same precision as logistic regression with fewer numbers of individuals. In our own simulations in the present context (data on file), the Poisson model gave the same precision as logistic regression with half the number of individuals. Finally, the Poisson model allows adjustments to be made for time trends. The ability to use several Poisson models permits the use of data from different sources to integrate fracture and death hazards, and to calibrate to different countries.\nA further feature of the FRAX\u2122 model is that it takes account of deaths from all causes. In several recent models of disease probability, this has not been accounted for [23\u201325]. For example, the probability of stroke has been determined as a function of age, race, smoking, body mass index, atrial fibrillation, HbA1c, systolic blood pressure, ratio of total to HDL cholesterol and duration of diabetes [25], but the risk of dying from other reasons was not taken into account. In the context of osteoporosis, fracture probabilities are markedly underestimated when no account is taken of the competing death hazard [27, 35, 36]. For example, in a study of men followed after orchidectomy, the cumulative incidence of fractures was 19% after 15\u00a0years, but the figure was 40% when deaths were considered as a competing event [36].\nFRAX\u2122 also takes account of the impact of risk factors on the death hazard. For example, smoking and low BMD are risk factors for fracture but also significant risk factors for death. Thus, at very low T-scores for BMD, hip fracture probabilities decrease with age (see Table\u00a03), in part related to the higher mortality associated with the lower values for BMD.\nThere are several limitations that should be mentioned. As with nearly all randomly drawn populations, non-response bias may have occurred. The effect is likely to exclude sicker members of society, and may underestimate the absolute fracture risk for example by age. The analyses also have significant limitations that relate to the outcome variables and the characterisation of risk factors. The definition of what was considered to be an osteoporotic fracture was not the same in all cohorts, but the effect of this inconsistency is likely to weaken rather than strengthen the associations that were found. For the hip fracture outcome, the definition was similar in all cohorts, and may explain in part the higher risk ratios associated for this fracture rather than for osteoporotic fracture. Also, the analyses were confined to clinical fractures, and the results might differ from vertebral fractures diagnosed by morphometry or as an incidental radiographic finding.\nThere are also limitations with the risk factors themselves. In the case of BMI, this was chosen rather than weight as the measure for body composition. This has the advantage that there is less variability across countries and between sexes. A potential drawback is that BMI can be influenced by height loss associated with vertebral deformities. Therefore, in individuals with important loss of height, the risk conferred through BMI could be underestimated [91]. The use of maximal attained height, rather than current height, might be a solution in the future, if it were shown that fracture risk prediction could be improved.\nFurther problems relate to the construct of the questions to elicit the presence or absence of risk factors, which varied between cohorts. These included questions on family history, prior fracture, smoking and glucocorticoid use. The effect of this heterogeneity is likely to weaken rather than strengthen the associations found. Recall is also subject to errors and was not validated in any of these cohorts. This is particularly problematic in the elderly. In addition, the validity of self-reported alcohol intake is notoriously unreliable [92]. Indeed, alcohol consumption was significantly less in both men and women than that assessed in the UK [93]. Given that these studies were prospective, however, much of this error (with the exception of alcohol intake) should be random, giving rise to non-directional misclassification. Thus, the associations may actually be stronger than reported here. Any underestimate may have limited consequences for case-finding, since the populations to be tested are similar to the populations interrogated. Biases that arise have more significance where causality is inferred.\nA further limitation is that several of the clinical risk factors identified take no account of dose-response, but give risk ratios for an average dose or exposure. By contrast, there is good evidence that the risk associated with excess alcohol consumption and the use of glucocorticoids is dose-responsive [14, 94]. In addition, the risk of fracture increases progressively with the number of prior fractures [95]. These limitations are nearly all conservative.\nIt should be acknowledged that there are many other risk factors that might be considered for incorporation into assessment algorithms. These include BMD at other skeletal sites, ultrasonography, quantitative computed tomography and the biochemical indices of bone turnover. The available information was too sparse to provide a meta-analytic framework, but they should be incorporated into risk assessment algorithms when they are more adequately characterised. Notwithstanding, the present model provides a mechanism to enhance patient assessment by the integration of clinical risk factors alone and\/or in combination with BMD.\nThe application of this methodology to clinical practice will demand a consideration of the fracture probability at which to intervene, both for treatment (an intervention threshold) and for BMD testing (assessment thresholds). These are currently being developed for the UK, based on cost-effectiveness analyses [96]. Intervention thresholds developed for the United Kingdom may not be applicable to other countries. The 10-year probability of fracture varies markedly in different countries [97]. For countries with low hip fracture rates, as found in developing countries, the relative risk at which intervention is cost-effective will be higher, though the absolute risk at which intervention is cost-effective would not change assuming comparable costs. Intervention thresholds would, however, change with differences in costs, particularly fracture costs, which vary markedly world wide. There is also the issue of affordability or willingness to pay for a strategy. The gross domestic product (GDP) per capita provides an index of affordability. The GDP varies markedly in different regions of the world. In the UK, the GDP per capita is estimated at US$ 25,300 in 2002, as compared with US$ 7,000 in Turkey. Thus, for the same fracture risk and the same costs, treatment will be less affordable (at least to health services) in Turkey than in the UK. Nevertheless, individuals in Turkey, rather than society as a whole, may be willing to pay \u201cUnited Kingdom prices\u201d for health care. There is also a marked heterogeneity in the proportion of GDP devoted to health care, and in the proportion of the population at risk from osteoporotic fracture (i.e., elderly people) [98, 99]. For all these reasons, it is important to define intervention and assessment thresholds on a country by country basis that takes into account the setting for service provision and willingness to pay, as well as considerations of absolute costs.","keyphrases":["frax\u2122","fracture probability","clinical risk factors","osteoporotic fracture"],"prmu":["P","P","P","P"]}
{"id":"Clin_Auton_Res-3-1-1914255","title":"Leg blood flow measurements using venous occlusion plethysmography during head-up tilt\n","text":"We tested whether venous occlusion plethysmography (VOP) is an appropriate method to measure calf blood flow (CBF) during head-up tilt (HUT). CBF measured with VOP was compared with superficial femoral artery blood flow as measured by Doppler ultrasound during incremental tilt angles. Measurements of both methods correlated well (r = 0.86). Reproducibility of VOP was fair in supine position and 30\u00b0 HUT (CV: 11%\u201315%). This indicates that VOP is an applicable tool to measure leg blood flow during HUT, especially up to 30\u00b0 HUT.\nIntroduction\nThe main mechanism responsible for maintaining blood pressure during orthostatic stress is arteriolar vasoconstriction [6]. In order to quantify the response in vascular resistance to postural stress, in particular in the leg, it is necessary to measure leg blood flow accurately.\nVenous occlusion plethysmography (VOP) is a well-established method to measure calf blood flow, which has been used in a variety of conditions, i.e. exercise [13] and reactive hyperaemia [17]. However, its use for measuring leg blood flow in standing or head-up tilt (HUT) position remains controversial, since an empty venous system has been suggested to be requisite for this method [3, 4, 12]. Since in the upright posture veins are already distended, due to an increase in hydrostatic pressure, further collection of blood may be defined by venous compliance rather than arterial inflow. This may question the validity of measuring blood flow using VOP in dependent limbs or in the upright posture. Although VOP has been used during HUT, accuracy or reproducibility of this method has not been reported [22\u201324].\nTherefore, the purpose of this study was to assess the applicability and reproducibility of VOP for blood flow measurements in the calf (CBF) during HUT at different tilt angles (0\u00b0, 30\u00b0, 45\u00b0, and 70\u00b0). In a subgroup CBF measurements by VOP were compared with blood flow measurements using Doppler ultrasound. To assess reproducibility of VOP, measurements were performed twice.\nMaterials and methods\nSubjects\nIn total eighteen healthy, normotensive subjects aged 21\u201330\u00a0years volunteered to participate in this study. In eight subjects blood flow measurements using VOP were compared with blood flow measurements using Doppler ultrasound (DU). In nine other subjects the VOP measurements were repeated within two weeks to assess reproducibility.\nBaseline subject characteristics are illustrated in Table\u00a01. None of the subjects used cardiovascular medication or suffered from cardiovascular disease. All were non-smokers and had no history of syncope. All volunteers refrained from caffeine and alcohol for at least eighteen hours and from food intake for three hours prior to testing. The local medical ethical committee approved the study. All subjects gave their written informed consent.\nTable\u00a01Subject characteristicsMean\u00a0\u00b1\u00a0SDAge, years25\u00a0\u00b1\u00a04Length, cm187\u00a0\u00b1\u00a09Body mass, kg80\u00a0\u00b1\u00a011Calf circumference, cm38\u00a0\u00b1\u00a02Systolic blood pressure, mmHg125\u00a0\u00b1\u00a012Diastolic blood pressure, mmHg74\u00a0\u00b1\u00a05Heart rate, bpm64\u00a0\u00b1\u00a010\nMeasurements\nThe subjects lay in supine position on a manually driven tilt table and were supported by a saddle. The venous occlusion cuff was placed around the right thigh and connected to a rapid cuff inflator (Hokanson Stopler E-20, Bellevue, WA 98005, USA). The mercury-in-silastic strain gauge was placed around the thickest part of the calf and was connected to the plethysmograph. Red blood cell velocities and systolic and diastolic vessel diameter of the right superficial femoral artery were measured with a pulsed-colour Doppler device, which is described in detail elsewhere [8]. Reproducibility of DU in the superficial femoral artery was 1.5% for diameter, 14% for blood flow [9].\nProtocol\nSupine blood flow measurements using DU and VOP were performed after subjects were 30\u00a0minutes quietly in supine position. When the subject was 2\u00a0minutes into 30\u00b0 HUT, DU measurements started until 3.5\u00a0minutes where after CBF continued for another 3.5\u00a0minutes. The venous occlusion pressure was adjusted to the hydrostatic pressure column, which is derived from the vertical distance heart level\u2013thigh level and was calculated as the sinus of the tilt angle * actual distance heart\u2013thigh, and was 75\u00a0mmHg during 30\u00b0 HUT. The same procedure was repeated for 45\u00b0, and 70\u00b0 HUT using a venous occlusion pressure of 87, and 105\u00a0mmHg, respectively.\nData analysis\nCBF in ml\u00b7100\u00a0ml\u22121\u00a0\u00b7\u00a0minute\u22121 was calculated as described previously [25] and values from minute 3.5 until 7 were averaged to calculate CBF for each HUT position. Doppler ultrasound measurements were analyzed as described before [8] to obtain superficial femoral artery blood flow. To compare CBF measurements with superficial femoral artery blood flow, CBF measurements in ml\u00b7100\u00a0ml\u22121\u00a0minutes\u22121 were multiplied by lower leg volume (ml) as measured by water displacement.\nStatistics\nData are expressed as mean\u00a0\u00b1\u00a0standard deviation (SD).\nThe results of each method were correlated and agreement evaluated according to the method described by Bland and Altman [5]. The limits of agreement are defined as the mean of the relative differences between the two methods\u00a0\u00b1\u00a02 SD. Student\u2019s t-test was used to test for systemic differences between the two methods.\nReproducibility of the CBF was assessed by calculating the coefficient of variance (CV) from two measurements [25].\nTo determine whether hemodynamic responses were dependent on the angle of tilt one-way repeated measures ANOVA\u2019s were applied. If significant effects of tilt were observed post-hoc paired t-tests with Bonferroni correction for multiple testing were used. A P-value of <0.05 was considered to indicate significance.\nResults\nIn five volunteers, CBF could not be measured during 70\u00b0 head-up tilt (HUT), due to a poor plethysmography signal or near fainting of the subject.\nHemodynamic responses to HUT (Figure\u00a01)\nCBF and superficial femoral artery blood flow (BF SFA) decreased significantly from supine to 30\u00b0 with no further decrease with increasing tilt angle (Figure\u00a01). The relative decrease in CBF (46%\u00a0\u00b1\u00a011%) from supine to 30\u00b0 was significantly larger than the decrease in BF SFA (40%\u00a0\u00b1\u00a012%) (P\u00a0<\u00a00.001).\nFig.\u00a01Absolute values of calf blood flow (CBF) measured with venous occlusion plethysmography and superficial femoral artery blood flow (BF SFA), heart rate (HR) and mean arterial pressure (MAP) during supine position, 30\u00b0, 45\u00b0, and 70\u00b0 HUT. One-way repeated measures ANOVA showed a significant effect of tilt for all parameters. *P\u00a0<\u00a00.05 and indicates significantly different from the position before. \u25a0\u00a0=\u00a0supine; \u00a0=\u00a030\u00b0 HUT; \u00a0=\u00a045\u00b0 HUT; \u25a1\u00a0=\u00a070\u00b0 HUT\nAgreement VOP and DU \nThe Pearson correlation between the two methods was 0.86 (P\u00a0<\u00a00.001) for all data points (Figure\u00a02). The agreement between the two methods was evaluated by plotting the relative difference in each measurement against the mean for all data points and separated for the different tilt angles (Figure\u00a03). The relative mean difference ((VOP\u2013DU)\/DU) was \u221214%\u00a0\u00b1\u00a022% for all data points in supine position and HUT indicating that overall CBF (VOP) is lower than BF SFA (DU); for supine position the relative mean difference between VOP and DU was 1.5%\u00a0\u00b1\u00a024%; for 30\u00b0: \u221213%\u00a0\u00b1\u00a023%; for 45\u00b0: \u221223%\u00a0\u00b1\u00a019%; for 70\u00b0: \u221223%\u00a0\u00b1\u00a015%. Limits of agreement for all data points in supine position and during HUT were \u221258% to 31% and became smaller during HUT. The limits of agreement are reasonable, although CBF during HUT is lower than BF SFA.\nFig.\u00a02Blood flow in the superficial femoral artery (BF SFA) measured by Doppler ultraound during different angles of head-up tilt versus calf blood flow (CBF) measured by venous occlusion plethysmography corrected for lower leg volume. Pearson correlation coefficient is 0.86Fig.\u00a03Relative difference between the blood flow measured by venous occlusion plethysmography (VOP) and the superficial femoral artery blood flow measured by Doppler ultrasound (DU) versus the mean of both flow for each individual subject at different tilt angles\nReproducibility of VOP during head-up tilt\nThe coefficient of variation (CV) of CBF ranged between 8.7% and 15.0% (Table\u00a02). In 70\u00b0 HUT, the CV for both parameters was calculated over four subjects only.\nTable\u00a02Values of calf blood flow (n\u00a0=\u00a09)SubjectSupine30\u00b0 HUT45\u00b0 HUT70\u00b0 HUT (n\u00a0=\u00a04)test 1test 2test 1test 2test 1test 2test 1test 2Mean\u00a0\u00b1\u00a0SD2.6\u00a0\u00b1\u00a00.62.6\u00a0\u00b1\u00a00.81.3\u00a0\u00b1\u00a00.31.3\u00a0\u00b1\u00a00.3 1.1\u00a0\u00b1\u00a00.31.1\u00a0\u00b1\u00a00.21.1\u00a0\u00b1\u00a00.2 n\u00a0=\u00a041.2\u00a0\u00b1\u00a00.2 n\u00a0=\u00a04%change\u00a0\u00b1\u00a0SD\u221245\u00a0\u00b1\u00a011\u221249\u00a0\u00b1\u00a012\u221254\u00a0\u00b1\u00a011\u221257\u00a0\u00b1\u00a013\u221244\u00a0\u00b1\u00a017\u221244\u00a0\u00b1\u00a014CV15.0%(CI 10.1\u201329.1)11.0%(CI 7.4\u201321.3)14.9%(CI 10.0\u201328.9)8.7%(CI 4.9\u201333.2)Values of calf blood flow in ml\u00b7100\u00a0ml\u22121 \u00b7 minute\u22121 and mean absolute and relative data\u00a0\u00b1\u00a0SD in supine position, 30\u00b0, 45\u00b0, and 70\u00b0 head-up tilt (HUT) for the first and second test. Coefficients of Variation (CV). Missing data in 70\u00b0 HUT are due to near fainting or a poor plethysmography signal.\nDiscussion\nCalf blood flow (CBF) measured with VOP correlates well with superficial femoral artery blood flow (BF SFA) measured with DU, and can be measured reproducibly during HUT. Since the most profound changes in blood flow with both techniques were already measured in 30\u00b0 HUT, and the increase in hydrostatic and venous pressure, and concomitant technical difficulties are smallest from supine to 30\u00b0 HUT we recommend to use VOP for leg blood flow measurements during HUT up to 30\u00b0.\nThe decrease in leg blood flow assessed with VOP and DU, is comparable to tilt-induced blood flow changes in other studies using DU (33%\u201359%) [2, 7, 10, 11]. The strong relationship between VOP and DU blood flow measurements in the present study is in line with previous studies reporting correlation coefficients varying from 0.57 to 0.99 at rest and during exercise [14, 16, 27]. Head-up tilt affects muscle blood flow more than skin blood flow [21, 28]. Since skin blood flow contributes more to superficial femoral blood flow than to calf blood flow, this may explain the observed discrepancy between the decrease in CBF (\u223c48%) versus the decrease in superficial femoral artery blood flow (\u223c40%) in response to HUT.\nReproducibility of baseline CBF (15.0%) is in range with other studies using similar techniques to measure leg blood flow [1, 18, 25]. The coefficient of variation of CBF during HUT was even better (11.0%\u201317.9%), which indicates that VOP is a reproducible tool to measure tilt-induced vasoconstriction repetitively. The low coefficients of variation of CBF during 70\u00b0 (8.7%\u20138.9%) are not representative since these coefficients of variation were calculated over no more than 4 subjects. Not all subjects were able to abstain from moving their legs in 70\u00b0 HUT position, and some subjects fainted in this position. Moreover, the quality of the plethysmographic tracing became worse at 70\u00b0 HUT whereas at the lower tilt angles the plethysmography signal is of good quality indicated by the volume pulsations in the plethysmographic tracing for the period of venous occlusion.\nOur data and previous studies [15, 19, 20, 26] show that at 30\u00b0 HUT peripheral vascular responses are accomplished to a large extent. The increase in hydrostatic pressure and concomitant increase in venous pressure is low at 30\u00b0. From Figure\u00a04B it can be concluded that at 30\u00b0 HUT venous compliance is still at the steep portion of the venous compliance curve whereas during 45\u00b0, and 70\u00b0 HUT the venous compliance has shifted to the non-linear part. At 30\u00b0 HUT increase of the plethysmography signal during venous occlusion is linear while at 45\u00b0 and 70\u00b0 HUT, venous distensibility is reduced and consequently results in a non-linear increase in leg volume during inflation of the venous occlusion cuffs, which is illustrated in Figure\u00a05. We therefore recommend using VOP at 30\u00b0 HUT. For studies focussing on syncope at the endpoint of HUT, which requires larger tilt angles, other techniques to measure leg blood flow should be used.\nFig.\u00a04(A) Pressure-Volume curve and (B) Pressure Compliance curve based on data of a similar group of volunteers measured by venous occlusion plethysmography at different cuff occlusion pressures (for method and protocol see de Groot et al., Journal of applied Physiology, 2005). The increases in calf volume in response to 30\u00b0, 45\u00b0, and 70\u00b0 HUT are marked by the dotted lines in the pressure volume curve (A). The different tilting angles correspond with different venous pressures (x-axis). Transferring these venous pressures into the pressure-compliance curve (B) clearly demonstrate that during 30\u00b0 HUT venous compliance is still on the steep linear part of the curve, whereas during 45\u00b0, and 70\u00b0 HUT the venous compliance is compromisedFig.\u00a05Typical plethysmographic tracing of one individual subject during a complete experiment. Blood flow measurements at 30\u00b0 HUT start when the plethysmography signal does not change anymore, meaning that venous volume reached a steady state situation. Besides, looking at a typical VOP tracing at 30\u00b0 HUT, the increase in venous volume is linear during the first 5\u00a0seconds of cuff inflation, indicating that blood flow measurements using VOP during 30\u00b0 HUT are not compromised by a decrease in venous compliance\nLimitation\nUsing VOP, blood flow is defined as limb volume changes over time. During HUT, when the leg is below heart level, volume changes can still be measured using VOP, however, the physiological determinants of these volume changes are complex and it is no longer possible to say with reasonable certainty that a change in volume over time, which most likely reflects flow, is determined by resistance vessel tone. For example, limb blood flow measured using VOP in HUT position can decrease due to an increase in venous pressure, as a result of venous congestion and the associated fall in arterio-venous pressure gradient, without any increase in resistance at the arteriolar level.\nIn conclusion, this study demonstrates that CBF measured by VOP during HUT is suitable and reproducible. The method is easy applicable and recommended in tilt angles equal to 30\u00b0 to avoid high hydrostatic and leg venous pressures.","keyphrases":["calf blood flow","doppler ultrasound","strain gauge plethysmography","orthostasis","autonomic nervous system"],"prmu":["P","P","R","U","M"]}
{"id":"Aesthetic_Plast_Surg-4-1-2175019","title":"Cell-Assisted Lipotransfer for Cosmetic Breast Augmentation: Supportive Use of Adipose-Derived Stem\/Stromal Cells\n","text":"Background Lipoinjection is a promising treatment but has some problems, such as unpredictability and a low rate of graft survival due to partial necrosis.\nAutologous fat transplantation is one promising treatment for facial rejuvenation and soft tissue augmentation because it results in no incisional scar or complications associated with foreign materials. However, certain problems remain, such as unpredictability and a low rate of graft survival due to partial necrosis. Many innovations to overcome these problems have been reported [1, 2, 4\u20136, 18] and reviewed previously [4, 14]. On the basis of these reports, we tentatively concluded that we could harvest fat with a 2.5-mm cannula or 18-gauge needle at a vacuum lower than 700 mmHg and reinject it using an 18-gauge needle without significant adipocyte damage [14].\nLipoinjection can be used to treat facial changes associated with aging and to correct various types of depressed deformities such as hemifacial microsomia and pectus excavatum. It also has been used in breast augmentation by a limited number of plastic surgeons [3], although the use of autologous fat for breast augmentation has been controversial. Consensus is lacking on whether lipoinjection is safe and appropriate because of microcalcifications that may cause confusion in the evaluation of mammograms. Recently, autologous fat injection has been reevaluated as a potential alternative to artificial implants for breast augmentation [3, 15, 16, 19]. This reevaluation may reflect recent advances in autologous fat transfer and the radiologic detection of breast cancer.\nTo overcome the problems associated with autologous fat transfer, we use a novel strategy known as cell-assisted lipotransfer (CAL) (Fig.\u00a01). Findings have shown that tissue-specific progenitor cells in the adipose tissue have the capacity to differentiate into various cell lineages [21]. Thus, the progenitors, currently known as adipose-derived stem\/stromal cells (ASCs), are expected to become a valuable tool in a wide range of cell-based therapies.\nFig.\u00a01Scheme of cell-assisted lipotransfer. Relatively adipose-derived stem\/stromal cell (ASC)-poor aspirated fat is converted to ASC-rich fat by supplementing ASCs isolated from the other half of the aspirated fat. The ASCs are attached to the aspirated fat, which is used as a scaffold in this strategy\nThe therapeutic concept of CAL was described in our previous report on preclinical studies [9]. We found that aspirated fat has approximately half the number of ASCs found in excised whole fat. There are two main reasons for this relative deficiency. First, a major portion of the ASCs is located around large vessels and left in the donor site after liposuction [9]. Second, a part of the ASCs is released into the fluid portion of liposuction aspirates [20]. The relative deficiency of ASCs may induce postoperative long-term atrophy of injected fat, as partially confirmed in animal studies [8, 9, 11].\nWith the CAL strategy, autologous ASCs are used to enhance angiogenesis, to improve the survival rate of grafts, and to reduce postoperative atrophy. In CAL, half the volume of the aspirated fat is processed for isolation of the stromal vascular fraction (SVF) containing ASCs. During the isolation process, the other half of the aspirated fat is prepared for grafting. Freshly isolated SVF, which we characterized previously [20], is attached to the aspirated fat, with the fat acting as a living scaffold before transplantation. Finally, the SVF-supplemented fat is injected into the target sites. Thus, ASC-poor fat is converted to ASC-rich fat in the preparation process of the injectable material.\nIn this report, we describe the preliminary results experienced by patients who underwent CAL for cosmetic breast augmentation. This is the first report on the clinical use of ASCs for cosmetic purposes.\nMaterials and Methods\nPatients\nFrom 2003 to 2007, we performed CAL for 70 patients: in the breast for 60 patients (including 8 patients who had breast reconstruction after mastectomy), in the face for 12 patients, and in the hip for 1 patient. For three patients, CAL was performed at two sites. Informed consent was obtained from all the patients. The study protocol conformed to the guidelines of the 1975 Declaration of Helsinki and was approved by individual institutional review boards.\nIn this study, 40 patients with healthy thoraxes and breasts underwent CAL for purely cosmetic breast augmentation. Patients undergoing breast reconstruction for inborn anomaly or after mastectomy were not included. At this writing, 19 of these 40 patients have been followed for more than 6 months, and the maximum follow-up period has been 42 months. All the patients were Japanese women with a mean body mass index (BMI) of 19.1\u00a0\u00b1\u00a01.9. Their ages varied from 20 to 62 years (mean, 35.8\u00a0\u00b1\u00a09.1). The mean volume of injected fat was 268.1\u00a0\u00b1\u00a047.6 ml on the left side and 277.3\u00a0\u00b1\u00a039.1 ml on the right side. The demographic and surgical data for these patients are summarized in Table\u00a01.\nTable\u00a01Patient dataNo. of cases40Sex40 F, 0 MAge (years) 35.8\u00a0\u00b1\u00a09.1BMI19.7\u00a0\u00b1\u00a01.9 Surgical procedure\u00a0\u00a0Group A6\u00a0\u00a0Group B2\u00a0\u00a0Group C32Site of liposuction\u00a0\u00a0Thighs25\u00a0\u00a0Thighs and abdomen13\u00a0\u00a0Thighs and lower legs2Total volume of suctioned fat (ml) 1111.8\u00a0\u00b1\u00a0164.0Volume of injection (ml)\u00a0\u00a0Left268.1\u00a0\u00b1\u00a047.6\u00a0\u00a0Right277.3\u00a0\u00b1\u00a039.1Operation time (min)257.1\u00a0\u00b1\u00a039.1\nSurgical Techniques\nBefore the procedure began, the liposuction site was infiltrated with combined saline solution and diluted epinephrine (0.001%). With the patient under general anesthesia, adipose tissue was suctioned using a cannula with a 2.5-mm inner diameter and a conventional liposuction machine. Approximately half of the collected liposuction aspirate was used for isolation of the SVF from both the adipose and fluid portions of the liposuction aspirates, as described previously [20]. This cell processing procedure required about 90 min. During the processing period, the other half of the lipoaspirate was harvested as graft material.\nThe adipose portion of the liposuction aspirates was either washed several times and placed in an upright position to obtain clear separation of fluids and oil (groups A and B) or centrifuged at 700 g for 3 min without washing (group C), then put into a metal jar (500 ml), which was placed in water with crushed ice. In groups A and C, the fresh SVF isolated from both the adipose and fluid portions was added to the graft material. After gentle mixing and a wait of 10 to 15 min for cell adherence to the aspirated fat, the cell-supplemented fat was put into an injection syringe. In Group B, the freshly isolated SVF was resuspended in 60 ml of saline, then diffusely injected into the whole breast mounds separately (30 ml for each breast) immediately after conventional lipoinjection. There were 6 patients in group A, 2 patients in group B, and 32 patients in group C.\nFor the injection syringe, a 10-ml LeVeen inflator (Boston Scientific Corp., Boston, MA, USA) or our original 20-ml syringe was used because each is a screw-type syringe (with a threaded plunger), and the threaded connections fit both the connecting tube and the needle to allow precise control during injection. To reduce the time of the procedure, two syringes were used. While the one syringe was being used for an injection, the other was being filled with the graft material in preparation for the next injection. An 18-gauge needle (150 mm long) was used for lipoinjection and inserted subcutaneously at one of four points indicated in Figs.\u00a02A and B.\nFig.\u00a02Schematic instruction of the injection method. (A) A small amount of fat tissue is injected as small aliquots or as a thin string with a long needle, using a syringe with a threaded plunger, while the needle is continuously withdrawn. (B) The needle is inserted from either one of two points on the areola margin or one of two points at the inframammary fold in variable directions and planes to achieve a diffuse distribution\nThe operator took care to insert and place the needle horizontally (parallel to the body) to avoid damaging the pleura and causing a pneumothorax. The needle was inserted in several layers and directions. It was continuously and gradually retracted as the plunger was advanced. This technique was used to obtain a diffuse distribution of the graft material (Figs.\u00a02 and 3). The grafts were injected into the fatty layers on, around, and under the mammary glands, and also into the pectoralis muscles.\nFig.\u00a03A clinical view of injection. The injection needle is rigidly manipulated by an operator while an assistant rotates the plunger according to the operator\u2019s instruction. A high-pressure injection can be performed using a disposable syringe with a threaded plunger. A 150-mm-long, 18-gauge needle is connected to the syringe with a connecting tube threaded at both ends\nResults\nThe transplantation of adipose tissue was successfully performed in all cases, and the time of the injection process ranged from 35 to 60 min for both breasts. Subcutaneous bleeding, occasionally seen in some parts of the breasts, resolved in 1 to 2 weeks.\nTransplanted adipose tissue was gradually absorbed during the first 2 postoperative months (especially during the first month), and the breast volume showed a minimal change thereafter, although skin tension sometimes lessened after 2 months. Three representative surgical sites are shown in Figs.\u00a04 to 9. The difference in breast circumference (chest circumference at the nipple minus the chest circumference at the inframammary fold) had increased in all cases by 4 to 8 cm at 6 months, which corresponds to two to three brassiere cup sizes. This circumference increase appeared to correspond to the 100- to 200-ml increase in the volume of each breast mound, which was partially confirmed by our preliminary evaluation using a three-dimensional quantitative measurement system.\nFig.\u00a04Clinical views of a patient in group A (patient 1). Preoperative (left) and postoperative (right) views at 24 months. A 22-year-old woman underwent breast augmentation using cell-assisted lipotransfer (CAL) (290 ml in each breast), with satisfactory results at 24 months. Her breast circumference increased by 5 cm, and her augmented breast mounds remained soft and natural appearing without injection scars or subcutaneous indurationsFig.\u00a05Radiologic views showing the chest of patient 1. (A) A preoperative computed tomography (CT) image in the horizontal plane of the nipples. (B) A horizontal image 12 months after surgery. Note that the adipose tissue is augmented both subcutaneously and under the mammary glands. (C) Mammograms at 12 months show no calcification or other abnormal signsFig.\u00a06Clinical views of a patient in group C (patient 2). Preoperative (top) and postoperative (bottom) views at 12 months. A 32-year-old woman underwent breast augmentation with cell-assisted lipotransfer (CAL) (280 ml in each breast). Her breast circumference difference increased from 9 cm (baseline) to 14.5 cm (at 12 months). The breast mounds are soft and natural appearing with no visible injection scarsFig.\u00a07Radiologic views showing the chest of patient 2. (A) A preoperative computed tomography (CT) image in the horizontal plane at the level of the nipples. (B and C) Horizontal images by magnetic resonance imaging (MRI) 12 months after surgery: (B) T1-image; (C) T2-image. The adipose tissue is augmented around and under the mammary glands. A small cyst (<10 mm) appears in the fatty layer under the right mammary gland. (D) Mammograms at 12 months show no abnormal signs such as calcificationsFig.\u00a08Clinical views of a patient in group C (patient 3). Preoperative (top) and postoperative (bottom) views at 24 months. A 30-year-old woman underwent breast augmentation with cell-assisted lipotransfer (CAL) (310 ml in each breast). Her breasts were dramatically augmented with an increase in breast circumference difference by 8 cm at 24 months. The breast mounds were soft with no subcutaneous indurations. An original inframammary fold on the left breast is slightly visible, but injection scars are not visibleFig.\u00a09Radiologic views of patient 3. (A) A preoperative computed tomography (CT) image in the horizontal plane at the level of the nipples. Only a very thin fatty layer is observed around the mammary glands. (B) A horizontal magnetic resonance image (MRI) (T1 weighted) 24 months after surgery. Transplanted adipose tissues survived and formed thick layers around and under the mammary glands. (C) Mammograms 24 months after surgery show no abnormal signs\nCompared with breast augmentation using implants of the same size, augmentation with CAL showed a lower height but a more natural contour of the breasts. All cases but one (see later) showed natural softness of the breasts without any palpable nodules at 6 months, and all the patients were satisfied with the resulting texture, softness, contour, and absence of foreign materials despite the limited size increase possible with autologous tissue\u2014Cyst formation (<12 mm) was detected by magnetic resonance imaging (MRI) in two patients, and microcalcification was detected by mammogram in two patients at 24 months. In one of two patients in group B, fibrous breast tissue and fibrosis on the sternum were observed by computed tomography (CT) scan at 6 months, and the breasts were found to be harder than in other cases.\nDiscussion\nModifications of lipoinjection techniques to improve the survival rate for injected fat have been attempted. From these, it is well accepted that adipose tissue should be placed as small aliquots [3], preferably within an area 3 mm in diameter [1]. Because it takes a long time to perform ideally diffuse distribution of suctioned fat [3], we have used a disposable syringe with a threaded plunger and connections, a very long needle (150 mm), and an assistant to rotate the plunger. As a result, only 35 to 60 min are required for injection in both breasts. These devices are critical to performing large-volume lipoinjection safely and precisely in a short time.\nIn addition, the harvesting, preserving, and refining of graft materials also are important, as repeatedly indicated in the literature. We used a relatively large suction cannula, centrifuged the aspirated fat in some cases, and kept it cooled until transplantation. In this study, the clinical results (increase in breast circumference) appeared to be better in group C (using centrifuged fat) than in group A (using noncentrifuged fat), although quantitative measurement and statistical comparison were not done. In a previous study, we found that centrifugation of aspirated fat is substantially influential because centrifugation at 1,200 g decreases the fat volume by 30%, damaging 12% of the adipocytes and 0% of the ASCs. This leads to the concentration of cell numbers per volume of adipocytes and ASCs by 25% and 43%, respectively [7].\nIn addition, centrifugation may be especially beneficial in our treatment because water content in the graft material may disturb the adherence of ASCs to the adipose tissue and interfere with differentiation into expected lineages. Any ASCs floating in a solution, which is a nonphysiologic environment, may migrate over distances, penetrate into the lymphatic flow, and differentiate unexpectedly. We believe that such migration and altered cell differentiation caused the development of fibrotic tissue on the sternum of one patient in group B. Thus, we conclude that centrifuged fat combined with ASCs as cell pellets (group C procedure) was best among the three methods used in this study.\nAlthough small cystic formation and microcalcification were detected in some cases, the microcalcification was easily distinguished from that associated with breast cancer, and the overall cosmetic results were generally satisfactory and encouraging. Almost all the patients were satisfied with their enlarged and soft breasts with a natural contour. Both CT scans and MRI showed that transplanted fat tissue survived and formed a significant thickness of the fatty layer not only subcutaneously on and around the mammary glands, but also between the mammary glands and the pectoralis muscles. Breast volume stabilized 2 to 3 months after transplantation.\nMaximum breast augmentation using the described technique varied among the patients and appeared to be 100 to 200 ml. Although these volumes may be smaller than those achieved with large artificial implants, a definite advantage is that patients need not be concerned about postoperative complications induced by artificial implants such as rupture, infection, capsular contracture, unnatural contour, hardness, neurologic symptoms, and immune response. Compared with our dozens of patients who underwent conventional autologous lipoinjection to the breasts, augmentation effects were apparently higher with CAL. A 2- to 3-cm increase in breast circumference was common with the conventional procedure, compared with the 4- to 8-cm increase seen in this trial of CAL, although the augmentation effect varied among patients. The measurement system we recently devised may help to quantify the difference in augmented volume in the future.\nAdipose tissue contains not only adipogenic progenitor cells, but also multipotent stem cells, which can differentiate into fat, bone, cartilage, and types of tissue [21, 22]. Suctioned fat appears to lose a significant number of these precursors during liposuction and the preparation processes as compared with nonsuctioned adipose tissue [9]. This relative deficiency of precursors may contribute to the low survival rate and long-term atrophy of transplanted lipoaspirates. In CAL, the deficit of ASCs was compensated by supplementing ASCs. To maximize the biologic function and avoid unexpected behavior of ASCs, it seems important to ensure adherence of supplemented ASCs to adipocytes or connective tissue.\nWith this novel treatment, ASCs have four possible roles, which were partly confirmed in preclinical studies [8, 9, 11]. First, ASCs can differentiate into adipocytes and contribute to the regeneration of adipose tissue. Second, ASCs can differentiate into endothelial cells and also probably into vascular mural cells [8, 10, 12], resulting in the promotion of angiogenesis and graft survival. Third, ASCs are known to release angiogenic growth factors in response to hypoxia and other conditions [13], and these factors influence surrounding host tissue. In their final role, which may be the most influential, ASCs survive as original ASCs [9]. In the adipose tissue, ASCs reside between adipocytes or in the extracellular matrix, especially around vessels, and contribute to the turnover of adipose tissue, which is known to be very slow (2 years or more) [17]. However, adipose grafts probably turn over during the first 2 to 3 months after transplantation because they experience temporary ischemia followed by reperfusion injury. This turnover, the replacement process of the adipose tissue, is conducted by tissue-specific progenitor cells, which are ASCs. The relative deficiency of ASCs in aspirated fat may affect the replacement process and lead to postoperative atrophy of grafted fat, which is known to occur commonly during the first 6 months after lipoinjection.\nThe freshly isolated SVF used in CAL contains not only ASCs but also vascular endothelial cells, pericytes, blood cells (both white and red), and other cells, as previously described [20]. After transplantation, ASCs may interact with other cells such as vascular endothelial cells, and supplementation with the SVF may be superior to supplementation with ASCs alone in this treatment. However, further studies are needed to elucidate the synergistic effects of ASCs with other cells contained in the graft.\nIn this preliminary study, satisfactory clinical results were generally achieved without any major complications. Thus, we can conclude that CAL is sufficiently safe for continuation of the study, though controlled investigations and accumulated long-term results are needed to elucidate the overall safety and efficacy of the treatment. A variety of new innovations, including stem cell technology, may be developed and may contribute to the improvement of autologous tissue transplantation and regeneration. Further improvements of the technique may cause autologous tissue transfer to become the first choice for breast augmentation in the future.","keyphrases":["breast augmentation","adipose-derived stem\/stromal cells","lipoinjection","cell therapy","tissue engineering"],"prmu":["P","P","P","R","M"]}
{"id":"Behav_Genet-3-1-1824713","title":"The Association between Conduct Problems and the Initiation and Progression of Marijuana Use during Adolescence: A Genetic Analysis across Time\n","text":"The present study used a prospective, longitudinal design to investigate genetic and environmental influences on the association between earlier conduct problems and the initiation and progression of marijuana use during adolescence. Parent- and teacher-reported conduct problems assessed at Time 1 (1996) and self-reported marijuana use assessed at Time 2 (2004) were available for 1088 adolescent twin pairs participating in the Cardiff Study of All Wales and North West of England Twins (CaStANET). Using a novel approach to the modeling of initiation and progression dimensions in substance use, findings suggested that the initiation of marijuana use in adolescence was influenced by genetic, common and unique environmental factors. The progression (or frequency) of marijuana use was influenced by genetic and unique environmental factors. Findings for conduct problems indicated that while the presence or absence of conduct problems was largely heritable, the relative severity of conduct problems appeared to be more strongly environmentally influenced. Multivariate model fitting indicated that conduct problems in childhood and early adolescence made a small but significant contribution to the risk for marijuana use 8 years later.\nMarijuana use can pose a major risk to adolescent health and well-being, yet it remains the most commonly used illicit drug in both the United States and the United Kingdom. In the USA, both availability and rates of marijuana use among adolescents increased sharply during the 1990s, with only a slight decline in recent years (Johnston et\u00a0al. 2005). In the UK, there was a gradual increase in adolescents reporting marijuana use in the past 12\u00a0months, from 10% in 1998 to 13% in 2003, with a slight decline in 2004 to 11% (National Centre for Social Research 2005). In the United States, 50% of adolescents reported the use of marijuana by the age of 17 (Johnston et\u00a0al. 2005) while in the United Kingdom, 38% of 15\u201316\u00a0year olds had used marijuana (Hibell et\u00a0al. 2004). Marijuana use is related to a range of deleterious outcomes including lower academic achievement, criminality and mental health problems such as depression and suicidal behaviour (Fergusson et\u00a0al. 2002; Penning and Barnes 1982; van den Bree and Pickworth 2005). Recent research using a twin design has identified conduct problems in childhood and adolescence as a major risk factor for substance use, and marijuana use specifically (e.g. Miles et al. 2002; Silberg et al. 2003; Young et al. 2000).\nA series of studies have documented genetic and environmental influences on adolescent marijuana use (e.g. Maes et al. 1999; McGue et al. 2000; Miles et al. 2001). Given the complex processes thought to underlie the link between initiation and progression of drug use however, it is argued that the question should not be whether or not there is a genetic component to drug use per se, but rather what role genetic influences play in the aetiology of drug use over time (Rutter et al. 1999; e.g. Rhee et al. 2003). Recent research has highlighted that the initiation and progression of substances may not be characterised as a single liability of risk (e.g. Agrawal et al. 2005; Heath et al. 1991, 1997; Koopmans et al. 1999). Rather, the initiation and progression phases of substance use may be more appropriately characterised as partially overlapping (or even, independent; Heath et al. 1991). Indeed, while evidence from adult twin studies indicates that there are additive genetic, common environmental and unique environmental factors that are shared between the initiation and progression of marijuana use, there is also evidence of genetic and unique environmental influences that are specific to heavier use of marijuana (Agrawal et al. 2005). The increased influence of genetic factors for heavy substance use possibly reflects the increased involvement of biological processes in chronic use (Kender 2001; van den Bree 2005).\nFor many individuals, initiation and experimentation with substances occurs during adolescence (Fuller et al. 2005). Adolescents may however, have lower tolerance levels for substances, including marijuana, and become dependent at lower doses than adults (e.g. Chen et al. 1997). Furthermore, adolescent-onset substance misuse is characterized by more rapid development of multiple drug dependencies and more severe psychopathology (e.g. Clark et al. 1998). With this in mind, it is important to consider the relative role of genetic and environmental influences on initiation and progression of substance use in this age group and whether the relationship between these dimensions of use identified with adult samples is also observed in adolescents.\nAntisocial behaviour during childhood and adolescence has been linked to substance use in adolescence and early adulthood (e.g. Moffitt et al. 2002; Silberg et al. 2003). Moreover, in the context of other disruptive behaviour disorders, conduct disorder has been shown to be the most predictive of substance use and illicit drug use (e.g. Lynskey and Fergusson 1995; Moss and Lynch 2001). A recent follow-back study of a prospective, longitudinal cohort found that adults with a substance use disorder (alcoholism, marijuana and other drug dependence) were significantly more likely than those without such disorder to have had a conduct and\/or oppositional defiant disorder before 15-years old (Kim-Cohen et al. 2003). Strong associations have also been found between conduct disorder symptoms and marijuana use in adolescence for both males and females (Miles et al. 2002). This association was moderately influenced by genetic factors and to a lesser extent, by non-shared environmental factors (see also Young et al. 2000). The findings of a recent longitudinal twin study indicated that conduct problems in adolescence temporally preceded substance use and that the covariation between conduct problems and later substance use was influenced by genetic and common environmental factors (Silberg et al. 2003). These findings mark an important shift away from documenting cross-sectional associations between conduct problems and substance use, from which it is impossible to determine the direction of effects, toward the use of genetically sensitive research designs that assess relationships as they unfold over time. Indeed, in the absence of an experimental design, a primary way in which the nature of the relationship between conduct problems and the initiation and progression of marijuana use during adolescence can begin to be disentangled is to use a prospective, longitudinal twin design.\nThe present study is among the first to investigate whether the initiation and progression of marijuana use during adolescence reflects a common underlying liability. Extending previous research on the relationship between conduct problems and substance use, this study also examines whether genetic and environmental influences on conduct problems are related to the later initiation of marijuana use in adolescence and in addition, whether conduct problems are implicated in the continued use of marijuana once initiation has occurred. Bivariate genetic analyses were conducted to establish whether marijuana initiation and progression represented a single liability of risk or alternatively, whether these behaviours were more accurately represented as independent liabilities (see Fig.\u00a01). It was hypothesised that genetic, common and unique environmental influences would contribute to the initiation of marijuana use, consistent with the role of social factors in the initial experimentation phase of drug use (Rutter et al. 1999). In accordance with previous studies (Agrawal et al. 2005; Kendler et al. 1999; van den Bree et al. 1998) we hypothesized heavier use (i.e. the progression of marijuana use, rather than initiation) to be more strongly influenced by genetic factors with a reduced role for common environment. Multivariate analyses assessed the relationship between conduct problems and later marijuana use. It was hypothesised that earlier genetic and environmental influences on conduct problems would be related to the initiation of marijuana use 8\u00a0years later. Initiation of marijuana use was expected to remain the strongest predictor of progression of marijuana use after controlling for the influence of conduct problems.\nFig.\u00a01A conceptual model of the bivariate causal covariance contingency model. Note: Superscript i refers to factors for initiation. Superscript p relates to factors specific to progression\nMethod\nSample\nThe sample used in the present analyses was drawn from the second (1996) and fourth wave (2004) of data collection of the longitudinal Cardiff Study of All Wales and North West of England Twins (CaStANET; Rice et\u00a0al. 2002; van den Bree et\u00a0al. in press). The CaStANET register is a population-based twin register, including twins born between 1976 and 1991 in the Cardiff area of South Wales and between 1980 and 1991 for the rest of Wales and the North West of England. This twin register includes families from a systematically ascertained, population-based register of twin births between 1980 and 1991 in Wales and Greater Manchester, UK. Zygosity was assigned using a twin similarity questionnaire completed by parents that is more than 90% accurate in distinguishing monozygotic (MZ) from dizygotic (DZ) twins and, in a subsample, validated by genotyping DNA markers (Cohen et al. 1975; Nichols and Bilbro 1966; Payton et al. 2001). The CaStANET study received ethical approval from the Multi Centre Research Ethics Committee for Wales, UK.\nAt the second wave of data collection (1996), questionnaires were mailed to parents of twins aged 5\u201316\u00a0years old. Of 2846 families contacted, 2082 parents returned questionnaires, representing a response rate of 73% (see Thapar et al. 2000 for a more detailed description of the sample). Parental consent was also obtained to contact the twins\u2019 teacher. Of 2168 teachers contacted, 1913 returned questionnaires, representing a response rate of 88%. At the fourth wave of data collection in 2004, questionnaires assessing various aspects of family functioning, parent health and twin psychological adjustment were mailed to families (parents and twins) on the CaStANET twin register with twins aged 11\u201319\u00a0years. Families were sent a reminder postcard, reminder questionnaires and finally, a reminder letter. Non-responding families who might have moved address were traced through General Practitioners. Previous research has shown that adolescent smokers and marijuana users are less likely to respond at follow-up in longitudinal studies (Siddiqui et al. 1996). Extra efforts were therefore made to contact families via telephone where one twin had reported substance use. Of 1755 families with adolescent twins re-contacted in 2004, at least one family member from 1214 families returned questionnaires (1081 parents; 1125 twin pairs, where either or both twins replied) representing a response rate of 69%. Family members who returned questionnaires received a high street store voucher as a token of appreciation for their participation. Demographic statistics indicated that the sample was representative of British families living in the UK region of England and Wales with regard to family constitution, ethnicity, employment and economic factors (Social Trends 2004).\nThe present study is based on a subsample of 1088 adolescent twin pairs with information relating to conduct problems (parent and\/or teacher report in 1996) and marijuana use in 2004. For clarity, these assessments are referred to as Time 1 (1996) and Time 2 (2004), respectively from this point forward. Of this sample, there was complete information for 895 twin pairs. At Time 1 (1996), the sub-sample of twin pairs were aged 5\u201313\u00a0years (mean\u00a0=\u00a09.02\u00a0years old; SD\u00a0=\u00a01.98, range). At the Time 2 (2004), twin pairs were aged 11\u201320\u00a0years old (mean\u00a0=\u00a016.10\u00a0years old; SD\u00a0=\u00a01.94). There were approximately equal numbers of participating boys (n\u00a0=\u00a0960; 44.1%) and girls (n\u00a0=\u00a01216; 55.9%). The sample comprised 425 monozygotic twins (177 male, 248 female pairs) and 663 dizygotic twins (132 male, 189 female, 342 opposite sex pairs). The majority of twins lived with their biological mother and father (66.2%) with smaller proportions living with one biological parent and a stepparent (10.4%), a single parent (18.7%) and \u2018other\u2019 (4.6%). Twins classified as living with \u2018other\u2019 included those who were living with another relative (e.g. grandparents) and twins who lived apart or who were away at university for part of the year. Analyses conducted in which this last group were excluded resulted in a similar pattern of findings to those reported below.\nThe majority of parent questionnaires (Time 1) were completed by mothers (93.6%) with smaller numbers completed by fathers (5.3%) and others (1.1% e.g. stepparent; grandparent). There was no significant difference in mean levels of conduct problems as a function of the reporter of parent information. Tests indicated that twins who did not respond at the Time 2 follow-up had higher levels of Time 1 conduct problems than twins who did respond (t\u00a0=\u00a04.809, P\u00a0<\u00a00.001). This may indicate a possible bias, whereby adolescents with higher conduct problems who were less likely to respond at follow-up were also those adolescents more likely to use substances, including marijuana.\nMeasures\nConduct problems\nParent and teacher-reported conduct problems were assessed using five items from the Strengths and Difficulties Questionnaire (Goodman 1997; Goodman and Scott 1999). Items included, \u2018Often tells lies or cheats\u2019 \u2018Steals things\u2019, \u2018Fights or bullies other children\u2019, \u2018Often has temper tantrums or hot tempers\u2019 and \u2018Generally obedient, usually does what adults request\u2019 (recoded to reflect disobedient behaviour). Each item was rated, 0 \u2018Doesn\u2019t apply\u2019, 1 \u2018Applies somewhat\u2019 or 2 \u2018Certainly applies\u2019. These items are broadly equivalent to symptoms of conduct disorder as defined by the DSM-IV (American Psychological Association 1994). Parents and teachers reported on behaviour over the last six months. Recent evidence indicates that different informants provide related and unique information about children\u2019s antisocial behaviour (Arseneault et al. 2003). Moreover, because children\u2019s behaviour can vary between settings, the most valid measurement is that which includes data from more than one informant and from more than one setting or context (Scourfield et al. 2004). Consistent with this argument, conduct problem scores were combined for parent and teacher reports by counting symptoms reported by either parents or teachers as present, thereby capturing each of the five behaviours occurring in both the home and school context. The item-level correlations between parent and teacher reports of conduct problems ranged from r\u00a0=\u00a00.17 (stealing) to 0.56 (disobedience), P\u00a0<\u00a00.001. The highest rating given by either parent or teacher was taken as the score for that symptom, in other words the occurrence of the behaviour in one context (e.g. the home), was sufficient for it to be counted as present. The internal consistency estimate for the present sample was acceptable (\u03b1\u00a0=\u00a00.76) and the five items were added to give a total conduct problems score.\nUsing criteria outlined by Goodman (1997), conduct problems ranging from 0 to 2 were classified as \u2018normal\u2019, a score of 3\u20134 was classified as \u2018borderline\u2019 and scores ranging from 5 to 10 were classified as \u2018abnormal\u2019. Of respondents with conduct information at Time 1, 585 (73%) monozygotic twins were classified as normal, 154 (19%) as borderline and 67 (8%) as abnormal. Of the dizygotic twins, 877 (69%) were classified as normal, 227 (18%) as borderline and 166 (13%) as abnormal. Conduct problems were classified on two dimensions: (1) the presence of conduct problems was indexed as either \u20180\u2019 for \u2018normal range\u2019 or \u20181\u2019 for borderline or abnormal conduct problems (2) the second dimension labelled \u2018borderline\/abnormal\u2019, distinguished individuals in the borderline and abnormal range for conduct problems. Children in the normal range were coded as missing for borderline\/abnormal problems, borderline conduct problems was coded as \u20180\u2019 and abnormal conduct problems were coded \u20181\u2019.\nMarijuana use\nThe frequency of lifetime marijuana use was assessed using the following item from the Add Health questionnaire (Resnick et al. 1997), \u2018During your life, how many times have you used marijuana?\u2019 The six response options ranged from \u2018Never used marijuana in my life\u2019 to \u2018More than 30 times\u2019. Responses were collapsed to create two binary variables assessing initiation of use and frequency of use. Initiation was indexed as \u20180\u2019 for never having used marijuana and \u20181\u2019 for use. Frequency was indexed as light and heavy use with \u20180\u2019 representing use of marijuana 1\u20135 times and \u20181\u2019 representing use of marijuana six or more times. Individuals who had never used marijuana were coded as missing for frequency of use.\nStatistical analysis\nThe software package Mx (Neale 1997) was used for genetic model fitting. Analyses were conducted using a \u2018causal common contingent\u2019 model which facilitates the expression of marijuana use as a two-stage process incorporating an initiation stage (\u2018upstream\u2019, for example whether the individual has ever tried marijuana) that necessarily precedes a progression stage (\u2018downstream\u2019, for example, whether the individual uses the marijuana frequently; see Fig.\u00a01; Agrawal et al. 2005; Neale et al. 2006). The model estimates the magnitude of the relationship between initiation and progression by means of a beta pathway between these two stages (see Fig.\u00a01). If the beta coefficient is estimated to be zero, this suggests that the initiation and progression stages for a substance are entirely unrelated processes, i.e. genetic and environmental risk factors for initiation are completely independent from those for progression. Alternatively, if the beta coefficient is estimated to be 1, this indicates that initiation and progression are entirely overlapping dimensions with identical genetic and environmental risk factors. The 95% confidence intervals around the beta coefficient provide further information on the degree of overlap between the two stages. Lower limits closer to zero (or below) support independent liabilities and upper limits approaching 1 provide support for identical liabilities. The model also allows the estimation of: (1) additive genetic effects (a2), (2) common environmental effects (c2), and (3) unique environmental effects (e2), on both initiation and progression of substance use.\nAn important feature of this model is that it is uniquely suited to analysis of data from an adolescent age group, where individuals may have not yet engaged in marijuana use but will go on to become frequent users. The model takes into account the fact that some individuals may be above the liability threshold for progression of marijuana use (i.e. will become frequent users) but because they are not past the age of risk, have not yet initiated marijuana use. As such, their position on the liability distribution of progression is unknown. These individuals are treated as a special case of missing data for progression using the maximum likelihood approach for dealing with missing data in Mx (Neale 1997; Neale et al. 2006). Given the likelihood of an association between age and level of substance use, an age correction was also employed which adjusts the threshold for each twin according to his or her age at the time of questionnaire completion on the distribution of liability to conduct problems and marijuana use. Specifically, the threshold is modeled as a simple linear function:  where t is the population baseline threshold (for individuals of age zero), ta models the regression of the threshold on age, and ageiis the age in years of the individual i at assessment (Neale et al. 2006).\nModels were estimated using full information maximum likelihood (FIML) estimation with raw ordinal data, which included zygosity, twin age, and initiation and progression information relating to conduct problems and\/or marijuana use for each twin. The significance of parameters was evaluated using 95% confidence intervals (CIs), calculated using Mx (Neale and Miller 1997).\nResults\nThe rate of lifetime marijuana use for the total sample of adolescents was 21.6%. Examining the prevalence of marijuana use for younger and older adolescents highlighted an age-related difference in levels of use. Approximately 11% of 11\u201315\u00a0year olds had used marijuana compared with 32.5% of adolescents aged 16\u201320\u00a0years old. The average age of initiation of marijuana use was 14\u00a0years (mean\u00a0=\u00a014.73, SD\u00a0=\u00a01.67, range: 9\u201319\u00a0years). There was no difference in the prevalence of marijuana use between MZ (19.7%) and DZ twins (22.9%; \u03c72\u00a0=\u00a02.987, P\u00a0=\u00a00.084). Tests indicated greater variance in marijuana use for DZ twins compared to MZ twins (F\u00a0=\u00a04.220, P\u00a0=\u00a00.040). A mean difference for a combined estimate of parent and teacher-reported conduct problems between MZ and DZ twins was also found (MZ, mean\u00a0=\u00a01.68, SD\u00a0=\u00a01.92; DZ, mean\u00a0=\u00a01.89, SD\u00a0=\u00a02.13; t\u00a0=\u00a02.27, P\u00a0=\u00a00.024). Tests revealed greater variation around DZ conduct problems in comparison with MZ twins (F\u00a0=\u00a09.938, P\u00a0=\u00a00.002). The higher DZ variances for a parent and teacher rated measure of conduct problems may indicate contrast effects whereby one twin is rated as having more behaviour problems than the other twin. On the other hand, the greater variances for DZ twin marijuana use may indicate sibling interaction, for example where one twin is less likely to engage in substance use in response to the other twins\u2019 substance using behaviour. However, this is somewhat counterintuitive because research indicates that sibling deviance, and drug and alcohol use specifically, predicts increased substance use (e.g. Stormshak et al. 2004).\nTests of sex differences revealed no sex differences in levels of marijuana initiation or progression (initiation: males, mean\u00a0=\u00a00.22, SE\u00a0=\u00a00.01; females, mean\u00a0=\u00a00.21, SE\u00a0=\u00a00.02; t\u00a0=\u00a00.55, P\u00a0=\u00a00.583; progression: males, mean\u00a0=\u00a00.32, SE\u00a0=\u00a00.03; females, mean\u00a0=\u00a00.41, SE\u00a0=\u00a00.04; t\u00a0=\u00a01.75, P\u00a0=\u00a00.081) or for the presence and severity of conduct problems (presence: males, mean\u00a0=\u00a00.29, SE\u00a0=\u00a00.02; females, mean\u00a0=\u00a00.31, SE\u00a0=\u00a00.02; t\u00a0=\u00a00.78, P\u00a0=\u00a00.437; severity: males, mean\u00a0=\u00a00.38, SE\u00a0=\u00a00.03; females, mean\u00a0=\u00a00.37, SE\u00a0=\u00a00.03; t\u00a0=\u00a00.26, P\u00a0=\u00a00.792). All figures calculated for pooled twin data were conducted using the survey commands in STATA 9.0 (StataCorp 2005), appropriate for use with twin data when there is non-independence of observations. Preliminary analyses were also conducted to investigate the relationship between a continuously assessed index of conduct problems (1996) and marijuana use (2004). The phenotypic relationship between conduct problems and marijuana use 8\u00a0years later was significant (\u03b2\u00a0=\u00a00.19, P\u00a0<\u00a00.01, R2\u00a0=\u00a00.09). The magnitude of association between conduct problems and marijuana use for dizygotic twins and monozygotic twins was similar, suggesting common environmental influence (MZ twins, r\u00a0=\u00a00.18; DZ twins, r\u00a0=\u00a00.19). Examining the tetrachoric correlations using PRELIS 2.50 (Joreskog and Sorbom 1996) for each of the four constructs (initiation and progression of conduct problems and marijuana use, respectively) suggested genetic and shared environmental influence. The correlations for presence of conduct problems correlations were r\u00a0=\u00a00.77 for MZ twins and 0.46 for DZ twins indicating genetic and environmental influence, while the correlations for the borderline\/abnormal conduct problems construct were r\u00a0=\u00a00.21 for MZ twins and 0.30 for DZ twins, indicating stronger environmental influence. The results for initiation of marijuana use indicated genetic and shared environmental effects (MZ r\u00a0=\u00a00.80; DZ r\u00a0=\u00a00.70), while progression of marijuana use appeared to be more strongly genetically influenced (MZ r\u00a0=\u00a00.68; DZ r\u00a0=\u00a00.13).\nThe relationship between the liability to initiation and progression of conduct problems and marijuana use\nThe results of model tests for conduct problems and marijuana use, respectively, are presented in Table\u00a01.\nTable\u00a01CCC model tests for conduct problems and marijuana useInitiation Progression a2c2e2\u03b2a2c2e2Time 1Conduct problems0.69 (0.41, 0.86)0.08 (0.02, 0.32)0.22 (0.14, 0.32)0.74 (0.22, 0.95)0.00 (0.00, 0.00)0.27 (0.00, 0.37)0.73 (0.05, 0.76)(0.41, 0.86)(0.02, 0.32)(0.14, 0.32)(0.22, 0.95)(0.00, 0.00)(0.00, 0.37)(0.05, 0.76)Time 2Marijuana use0.35 (0.05, 0.63)0.47 (0.24, 0.71)0.18 (0.10, 0.36)0.88 (0.38, 0.99)0.64 (0.00, 0.65)0.00 (0.00, 0.00)0.36 (0.00, 0.48)(0.05, 0.63)(0.24, 0.71)(0.10, 0.36)(0.38, 0.99)(0.00, 0.65)(0.00, 0.00)(0.00, 0.48)\nConduct problems\nTo facilitate later multivariate tests of the relationship between earlier conduct problems (Time 1) and later marijuana use (Time 2), a CCC model was estimated for conduct problems. Given the differences found between MZ and DZ twins in levels of conduct problems, model tests were conducted in which thresholds for the different twin groups were allowed to vary. This provided a better fit to the data than a model in which thresholds were estimated to be the same between twin groups and these results are reported (\u03c72\u00a0=\u00a07.853, df\u00a0=\u00a02, P\u00a0<\u00a00.05). Substantively however, the pattern of results was the same. The liability to any conduct problems accounted for a large proportion, but not all, of the variance in the liability to be classified as borderline\/abnormal for conduct problems (\u03b2\u00a0=\u00a00.74; CI\u00a0=\u00a00.22, 0.95). The presence of conduct problems was mainly influenced by genetic (69%) and non-shared environmental factors (22%). Borderline\/abnormal conduct problems were influenced by common environmental (27%) and non-shared environmental (73%) factors. The threshold estimates were 0.47 for presence and 1.10 for severity. A comparison of goodness-of-fit indices indicated that modelling thresholds as a function of age did not significantly improve the fit of the model (\u03c72\u00a0=\u00a04.302, df\u00a0=\u00a02, P\u00a0>\u00a00.10).\nMarijuana use\nThe results for marijuana use indicated that the initiation of use was explained by a heritable component (35%), common environment (47%) and non-shared environment (18%). In contrast, the frequency of marijuana use was explained by a heritable component (64%) and a non-shared environment component (36%). The threshold estimates were 0.57 for initiation and 1.17 for frequency. The beta value (\u03b2\u00a0=\u00a00.88; CI\u00a0=\u00a00.38, 0.99) represents the genetic and environmental influences on initiation that are, in turn, transmitted to the frequency of marijuana use. This value, which is less than unity, indicated that while the liabilities for the initiation and frequency of marijuana use were not independent, neither could these dimensions be assumed to reflect a single liability of risk. In other words, the liability to initiate use of marijuana accounted for a substantial proportion (approximately 77%), but not all, of the variance in the liability to more frequent use of marijuana. The CI\u2019s around beta (0.38\u20130.99) further indicated that the two liabilities were moderately to strongly related but not identical. Goodness-of-fit statistics indicated that a model in which age-corrected thresholds were estimated provided a better fit to the data than a model that did not include age-corrected estimates (\u03c72\u00a0=\u00a056.155, df\u00a0=\u00a02, P\u00a0<\u00a00.001), corroborating our findings of a higher prevalence of increased marijuana use in older adolescents.\nMultivariate analyses\nAnalyses were conducted to examine whether the liability to conduct problems during childhood and early adolescence was a risk factor for the liability to initiate and use marijuana 8\u00a0years later. The model was again estimated using full information maximum likelihood (FIML) estimation with raw ordinal data, which included zygosity, twin age, and \u2018initiation\u2019 and \u2018progression\u2019 information relating to conduct problems and marijuana use for each twin. In addition to paths estimated from each initiation variable to the progression variable, paths were estimated from presence of and borderline\/abnormal conduct problems at Time 1 to marijuana initiation and progression at Time 2. Figure\u00a02 presents the results for the full model, in which all six pathways between conduct problems (Time 1) and marijuana use (Time 2) were estimated. This model provided similar estimates of the relationship between each classification of conduct problems and the initiation and frequency of marijuana use, respectively. Minor fluctuations were found between models (bivariate model to the full multivariate model) in the genetic and environmental estimates for conduct problems, which can be expected to be a result of the model estimation. Fixing estimates in the full model to their bivariate values would not change the substantive interpretation of the multivariate results, nor would it yield a significant difference in fit. However, the multivariate results should generally be regarded as superior, because they use more information.\nFig.\u00a02A causal covariance contingency model of the longitudinal relationship between conduct problems and marijuana use. Note: Superscript i refers to factors for initiation. Superscript p relates to factors specific to progression. 95% confidence intervals appear in brackets\nThe strongest beta-paths were observed within traits, between initiation and progression variables for both conduct problems and marijuana use. No significant effects were observed between conduct problems and later marijuana use (\u03b2 range\u00a0=\u00a00.04\u20130.17). In the context of strong effects from initiation to frequency of marijuana use (\u03b2\u00a0=\u00a00.75), associations between conduct problems and the frequency of marijuana use were relatively weak. As the bivariate analysis described above suggested however, there were also relatively high levels of covariation in the liabilities for presence and borderline\/abnormal conduct problems. This may have affected the power of either variable to predict initiation of marijuana use. To investigate this possibility, nested models were analysed to test the effect of dropping pathways between initiation and progression constructs and between conduct problems and marijuana use. Models were fitted whereby the paths between liability to conduct problems, severity of conduct problems, liability to marijuana initiation and frequency of use were dropped in turn and the deterioration in chi-square fit compared to a model in which all possible path coefficients from Time 1 conduct problems to Time 2 marijuana use were estimated (see Table\u00a02 for model fitting results). These results showed that when the path from liability to conduct problems and severity of conduct problems was dropped there was a significant reduction in model fit, compared to a full model. This was also the case when the path from liability to marijuana initiation and frequency of use was dropped. When the path from borderline\/abnormal conduct problems to initiation of marijuana use was dropped, the pathway from presence of conduct problems to the initiation of marijuana use became statistically significant (\u03b2\u00a0=\u00a00.23, CI\u00a0=\u00a00.13, 0.33). Likewise, when the path from presence of conduct problems to initiation of marijuana use was dropped, the pathway from borderline\/abnormal conduct problems to the initiation of marijuana use also became statistically significant (\u03b2\u00a0=\u00a00.24, CI\u00a0=\u00a00.22, 0.35). Inspecting the deterioration in model fit when each of these two paths was dropped indicated that when the pathway from presence of conduct problems to the initiation of marijuana use was dropped, there was a trend toward a significant reduction in model fit, (\u03c72\u00a0=\u00a03.02, df\u00a0=\u00a01, P\u00a0<\u00a00.10). In contrast, when the pathway from borderline\/abnormal conduct problems to the initiation of marijuana use was dropped, a significant drop in model fit was not observed (\u03c72\u00a0=\u00a00.17, df\u00a0=\u00a01, P\u00a0>\u00a00.10).\nTable\u00a02Multivariate model fitting for relations between conduct problems and marijuana useEstimated modelBeta coefficient set at zero-2LLdf\u03c72Model 1-Full theoretical model\u20135433.9455124\u2013Model 2\u03b2 2 15438.72051254.78*Model 3\u03b2 3 15436.96951253.02Model 4\u03b2 4 15433.84751250.10Model 5\u03b2 3 25433.77151250.17Model 6\u03b2 4 25433.82551250.12Model 7\u03b2 4 35442.25851258.31**Note: *\u00a0P\u00a0<\u00a00.05, **\u00a0P\u00a0<\u00a00.01\nDiscussion\nThis study is among the first to investigate the relationship between the initiation and progression dimensions of substance use with an adolescent twin sample and highlights that the initiation and progression of marijuana use, although strongly related, cannot be assumed to have a single liability of risk. The findings of this study also provide insight into the relationship between conduct problems and adolescent substance use. Specifically, the results indicate that in a UK sample, the longitudinal association between the liability to conduct problems during childhood and early adolescence and the liability to initiate and progress marijuana use in adolescence and early adulthood are not strongly related. Thus, other risk factors and the mechanisms through which they exert effects on the initiation and progression of marijuana use in adolescence should also be investigated.\nConsistent with previous research examining the relationship between initiation and progression (or use and abuse) of substances in adult populations, additive genetic, common and non-shared environmental influences were found for initiation of marijuana while factors specific to progression were influenced by genetic and non-shared environment with no role for common environment (e.g. Agrawal et al. 2005; Heath et al. 1997; Kendler and Prescott 1998; Koopmans et al. 1999). These findings concur with the view that genetically influenced biological mechanisms appear to play a more important role in the aetiology of problem use and substance dependence (Kendler et al. 1999; van den Bree 2005). Interestingly, when conduct problems were introduced as predictors of marijuana initiation and progression in the multivariate model, there was a slight reduction in the genetic estimate and an increase in the environmental influence. This was particularly the case for initiation of marijuana use. It is also interesting to note that while not independent, the initiation and frequency of marijuana use, to some degree, represent different liabilities of risk. Conceptually, this indicates that the use of marijuana by adolescents does not always and inevitably lead to more frequent use and that there may be different risk factors underlying initiation and progression to more frequent marijuana use. Studies into risk factors that predispose individuals to continued use of marijuana after first experimentation with the drug will be helpful in increasing insight into which adolescents are at greatest risk for problem use. For example, van den Bree and Pickworth (2005) found that some risk factors predict both initiation of experimental marijuana use and progression to regular use, while others are specific to each stage.\nThe results for conduct problems indicated that while the presence or absence of conduct problems were largely heritable, the relative severity of conduct problems, categorised as \u2018borderline\u2019 and \u2018abnormal\u2019 appeared to be more strongly environmentally influenced. However, we cannot rule out the possibility of greater measurement error at the upper end of the distribution contributing to a large non-shared environment estimate. The beta coefficient between the two dimensions suggested that they mostly, but not entirely overlap (\u03b2\u00a0=\u00a00.74; see Table\u00a01), with wide 95% CIs (0.22, 0.95). Conceptually and epidemiologically, recent evidence (e.g. Pickles et al. 2001; van den Oord et al. 2003) indicates that conduct problems may be best regarded as a single liability of risk where there is no demarcation between normality and psychopathology (Rutter 2003). The categorization of conduct problems into dichotomous variables may have affected the power to predict later marijuana use. Preliminary analyses indicated that conduct problems assessed as a continuous variable predicted later marijuana use, although the strength of association was not large (\u03b2\u00a0=\u00a00.19; P\u00a0<\u00a00.01). Likewise, model tests in which presence and severity of conduct problems were assessed separately indicated small but significant effects from conduct problems to the initiation of marijuana use. Although the trend toward a significant drop in model fit when the path from severity of conduct problems to initiation of marijuana use was removed suggests that it is the presence rather than severity of conduct problems that is important in the prediction of marijuana use initiation, this is speculative and should be replicated in independent samples of larger number. Moreover, it will be interesting in the future (as these models become available) to repeat these analyses including conduct problems as a dimensional measure with a single liability. The findings of the present study also suggest that this modelling technique could be applied to questions relating to the transition from symptoms to diagnosis, particularly when longitudinal data are available to examine this relationship over time (see Neale et al. 2006).\nA limitation of the present study was that the results of both the bivariate and multivariate model tests had wide confidence intervals around the progression variables (borderline\/abnormal conduct problems and frequency of marijuana use). These possibly reflect the low frequencies of adolescents with high levels of conduct problems and who had initiated marijuana use. For instance, preliminary analyses indicated that adolescents who did not respond at Time 2 (2004) had higher levels of conduct problems at Time 1 (1996). Thus, the present analyses may represent a conservative estimate of the influence of conduct problems on later initiation and progression of marijuana use. In addition, heterogeneity in the relationship between conduct problems and marijuana use as a function of the wide age range of the sample, together with a reliance on a single item to index marijuana use, may have affected the power to detect an association. In addition, in light of the strong relations between the initiation and progression variables, the cross-trait paths are relatively small and larger sample sizes than we currently have available are needed to obtain more conclusive nested model fitting results for the cross-trait paths. Finally, it should be noted that while the present study provides insights regarding the phenotypic relationship between conduct problems and the initiation and progression of marijuana use in adolescence together with sources of genetic and environmental influence on each index of conduct problems and marijuana use, it does not assess genetic and environmental sources of covariance between these behaviours (see Neale et al. 2006). In addition to addressing the caveats outlined above therefore, an important direction for further research is to assess these influences.\nPrevious research found sex differences in the prevalence of marijuana use (greater use among males; Johnston et al. 2005) and in the genetic and environmental influences on adolescent male and female marijuana initiation (e.g. Rhee et al. 2003). Recent research has also identified sex differences in the pattern of relations between the severity of conduct problems and the later initiation of marijuana use (Pedersen et al. 2001). The binary nature of the study variables together with the sample size did not permit model tests incorporating sex differences. Nevertheless, an important direction for future research is to test causal common contingent models for sex differences in the pattern of relations between conduct problems and marijuana use in an adolescent age group.\nNotwithstanding these limitations, the present study extends previous research by investigating longitudinal relationships between conduct problems and marijuana use during adolescence using a modelling approach that enabled age appropriate adjustments to estimates of the relationship between the initiation and progression dimensions of substance use. The findings are also consistent with previous research indicating stronger associations between conduct problems and substance use within than across-time (e.g. Miles et al. 2002; Silberg et al. 2003). As such, they suggest that interventions aimed at helping children with conduct problems in the years preceding the onset of substance use are unlikely to reduce the risk of marijuana use in adolescence and young adulthood. Other risk factors need to be investigated. An important direction for future research will be to develop models that can test these processes with greater precision. For example, models are required that permit the testing of risk factors assessed using continuous measures as predictors of initiation and progression of substance use and the investigation of moderators of the liability to initiate and progress in substance use when these reflect independent liabilities of risk. The findings of this prospective, longitudinal study represent a first step in examining the risk factors that influence the initiation of substance use and the progression to more frequent use during adolescence and young adulthood.","keyphrases":["conduct problems","initiation","marijuana use","adolescence","genetic","longitudinal","environmental"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Mar_Biotechnol_(NY)-3-1-2100433","title":"Understanding Marine Mussel Adhesion\n","text":"In addition to identifying the proteins that have a role in underwater adhesion by marine mussels, research efforts have focused on identifying the genes responsible for the adhesive proteins, environmental factors that may influence protein production, and strategies for producing natural adhesives similar to the native mussel adhesive proteins. The production-scale availability of recombinant mussel adhesive proteins will enable researchers to formulate adhesives that are water-impervious and ecologically safe and can bind materials ranging from glass, plastics, metals, and wood to materials, such as bone or teeth, biological organisms, and other chemicals or molecules. Unfortunately, as of yet scientists have been unable to duplicate the processes that marine mussels use to create adhesive structures. This study provides a background on adhesive proteins identified in the blue mussel, Mytilus edulis, and introduces our research interests and discusses the future for continued research related to mussel adhesion.\nIntroduction to Biological Adhesion\nBiomimetics\nBiomimetic materials are designed to mimic compounds with a biological origin. The biomaterial development field is actively pursuing the use of various synthetic and natural protein- and carbohydrate-containing compounds. The production of ecologically friendly materials also is of importance as a result of a rapid increase in the world\u2019s population and the subsequent environmental problems associated with product wastes (Arora and Arora 2004). The key interest in biomimetic adhesives is the interaction between the material and a surface, whether that surface is of biological or nonbiological origin. Scientific studies in the interdisciplinary field of biomaterial research include areas such as surface characterization, protein adsorption, and adhesion behavior (Kirkpatrick et al. 1997). Table\u00a01 briefly describes biomolecules with adhesive properties used for biomimetic research. Many of the proteins are scleroproteins, which contribute mechanical strength to supporting structures in animals. Waite (1983a, b) has commented on the durability of these proteins, in terms of relative insolubility, resistance to proteolytic hydrolysis, or other chemical dissolution. Repetitive amino acid motifs and intermolecular and intramolecular cross-linking are common; DOPA-containing scleroproteins produced through posttranslational modification of tyrosine by various organisms leads to further stabilization or curing of scleroproteins involved in adhesion and\/or protection of more flexible structural proteins.\nTable\u00a01.Research targets for biomaterial developmentBiological TargetDescriptionKeratinA hard, durable insoluble, structural protein that is the primary component of horns, hoofs, feathers, skin, hair, and nails; a scleroproteinElastinAn insoluble protein found in connective tissue and known for its elasticity and similarity to collagen; a scleroproteinCollagenA tough, insoluble, inelastic protein with high tensile strength that serves as the support structure in skin, tendons, and bone; a scleroproteinSilksHigh tensile strength protein fibers that contain various proteins (fibroin, spidroin); most commonly from spiders and silkwormsFibrin (and other coagulation system proteins)A sticky, insoluble, clot-forming protein formed by constituents in the blood; a scleroproteinChitinA specialized carbohydrate containing nitrogen (nitrogenous polysaccharide); found in the cell walls of certain fungi and in the exoskeletons of arthropodsCelluloseAn insoluble complex carbohydrate (polysaccharide composed of linked glucose units); main constituent of the cell walls of plantsMucinA nitrogenous, conjugated protein (protein linked to a sugar) found in mucous secretions; acts as a lubricant and protects body surfaces\nThe production of an underwater adhesive that mimics the properties of marine mussels is a challenge that has received considerable attention. Many of the mussel adhesive proteins identified to date are polyphenolic proteins. Polyphenolic proteins are nontoxic, biodegradable, and have low immunogenic qualities that make them highly attractive for environmental, medical, and industrial purposes.\nBiological Adhesion\nNaturally produced adhesives are common in many biological systems and are known for their superior strength and durability compared with man-made materials. Examples of specialized biological systems that generate a vast amount of adhesives research include bacteria, spiders, marine tubeworms, sea cucumbers, barnacles, and mussels. Many bacteria synthesize exopolysaccharides\u2014extracellular protective adhesive matrixes.\nBacteria can form layers, or biofilms, on a variety of surfaces by embedding cells in exopolysaccharides. The polysaccharide-based holdfast of the aquatic bacterium Caulobacter crescentus has been shown recently to demonstrate the strongest adhesive force measured in bacteria (Tsang et al. 2006). Spiders also express adhesive proteins by synthesizing different types of silk, all of which have high tensile strength, extensibility, and an energy-dissipative viscoelastic response that is not matched by synthetic polymers (Zhou et al. 2001; Vollrath 2000). Current research with spider silks involves the production of spider silk in other organisms, a technique known as recombinant protein expression (Piruzian et al. 2003).\nMany marine organisms have developed adhesive strategies to deal with the dynamic ocean environment, particularly at the tidal interface. Marine invertebrates attach permanently or temporarily to inanimate, and sometimes living, surfaces. Therefore, adhesive mechanisms have evolved to promote attachment. The marine polychaete Phragmatopoma californica is a tubeworm that builds protective \u201ctubes\u201d with secreted proteinaceous cement mixed with shells and sand particles from the sea floor. The cement adheres rapidly to a variety of materials in seawater (Stewart et al. 2004; Zhao et al. 2005). The sea cucumber (Holothuria forskali) reacts defensively through the discharge of Cuvierian tubules that ensnare their threat. Biochemical analysis of the tubules indicates a 3:2 protein to carbohydrate ratio, with a high proportion of highly insoluble protein. The soluble protein component appears to contain up to ten glycine and acidic amino acid-rich proteins ranging in size from 17 to 220 kilodaltons (DeMoor et al. 2003).\nBarnacles adhere directly and permanently to surfaces, such as underwater substrata (ship hulls, oil platforms, and pipelines). Initial attachment of barnacle cyprid larvae is via o-quinone cross-linking that resembles the dihydroxyphenylalanine (DOPA)-containing adhesive proteins of Mytilus spp. (Wiegemann 2005), but the adult barnacle cement is substantially different\u2014comprised of three groups of proteins that contain high levels of the amino acids serine, threonine, glycine, and alanine (Kamino et al. 2000; Wiegemann 2005). A pattern of short, alternating regions of hydrophobic and hydrophilic residues throughout the largest of the three groups of proteins has been noted and suggested that these alternating motifs may have a role in assembly in seawater (Kamino et al. 2000). Marine and freshwater mussel adhesive proteins differ from barnacle cement proteins because of the presence of repetitive amino acid motifs (characterized by a high polyphenolic content), high levels of the modified amino acid 3,4-DOPA, and chemical modifications (hydroxylations) to specific amino acids (Burzio et al. 1997). At least ten different adhesive-related proteins from the marine mussel M. edulis have been identified, reflecting the greater ease of working with the adhesive structures of this organism and also its popularity as a target for biomimetic research.\nMarine mussels, such as the blue mussel, M. edulis, attach to a variety of surfaces in an aqueous environment by using a natural adhesive that is incredibly strong and durable. There are no synthetic glues that can be similarly applied in an aqueous environment and are impervious to water and turbulent forces. Previous research has shown that one of the proteins in the adhesive, Mytilus edulis foot protein 1 (Mefp-1), bonds to glass, plastic, wood, concrete, and Teflon\u00ae. Nine other adhesive-related proteins from M. edulis have been identified to date. A tenth is implicated but has not been isolated. The precise mechanism for assembly of the ten proteins-Mefp-1, -2, -3, -4, -5; collagens such as precollagen-D, -P (variant P22 and P33), and -NG; proximal matrix thread protein (PMTP-1 and -1a); and a polyphenol oxidase\u2014is not understood. There also may be additional proteins involved in the formation of the adhesive. Figure\u00a01 illustrates adhesion of M. edulis to seaweed, other mussels, and a stainless steel surface.\nFigure\u00a01M. edulis attachment to (a) seaweed, (b) other mussels, and (c) a stainless steel surface.\nAdhesion Mechanisms in Mussels\nThe mollusk byssus evolved to anchor postlarval organisms during metamorphosis. Some species of mussels have retained the byssus in the adult animal, permitting a transition from a bottom-dwelling existence to an epibenthic lifestyle. Within the Mytilidae, examples of mussels across this range of marine lifestyles can be observed, from the burrowing Geukensia demissa, to Mytilus edulis and Bathymodiolus childressi, which live entirely exposed attached to hard surfaces, and intermediate species, such as Modiulus modiolus, which can survive under mixed conditions (Brazee and Carrington 2006). Common features of the byssus in the Mytilidae include a root attached to the byssal retractor muscle, a stem extending from the root, and individual byssal threads which are attached to overlapping cuffs of the root.\nThe strength of mussel adhesive scleroproteins is attributed to the introduction of cross-links between polymer chains of individual adhesive proteins (a process called \u201ccuring\u201d). Curing, or hardening, of the polyphenolic mussel proteins is believed to require a catecholic precursor (e.g., DOPA) and the presence of a catechol oxidase (Waite 1990).\nIndividual adhesive proteins from mussels are produced by the foot organ of the animal. The proteins are stockpiled in the foot then secreted or released into the byssal groove, which creates a template for thread and plaque formation to form strong attachments underwater. The reactive, oxidized form of DOPA, quinone, is thought to provide the moisture-resistance characteristic of mussel underwater adhesion (Yu and Deming 1998; Yu et al. 1999). DOPA is formed from the hydroxylation of tyrosine residues by a polyphenoloxidase (tyrosinase). DOPA can complex with metal ions and oxides (Fe3+, Mn3+) and semimetals, such as silicon, thus explaining its ability to adhere to rocks and glass (Sever et al. 2004). Sun and Waite (2005) have reported an incremental posttranslational modification of tyrosine to DOPA from the proximal end to distal tips of threads, coupled with a selective localization of metals (aluminum, calcium, iron, and silicon) and an associated iron gradient. The sequestering of iron occurs during feeding rather than after threads have been formed. Thus, iron is incorporated in a chemical gradient pattern similar to that of DOPA during secretion of byssal threads and plaques. The identification of 12 variants of the DOPA-rich adhesive foot protein 3 from Mytilus californianus (Mcfp-3) further supports the intricate role of DOPA in marine mussel adhesion (Zhao et al. 2006).\nThe catecholic content of mussel adhesive proteins has been linked to hydrogen-bonding and metal-liganding (strong chelating) capabilities (Monahan and Wilker 2003; Deming 1999). Examples of proteins undergoing the o-quinone intermediate process (quinone tanning) include collagen (mussel byssal threads), cellulose, chitin, and mineral deposits (mussel shells). Figure\u00a02 illustrates the chemical steps for hydroxylation of tyrosine and DOPA residues in M. edulis polyphenolic proteins.\nFigure\u00a02Hydroxylation of tyrosine residues in M. edulis polyphenolic proteins.\nOther constituents of mussel adhesive proteins include lysine and glycine. Lysine may contribute to adhesion via ionic bonding to negatively charged surfaces, such as collagen and acidic polysaccharides (Suci and Geesey 2000; Olivieri et al. 2002), and intermolecular cross-linking with o-quinones. Glycine may contribute to adhesion through the open, extended conformation it imparts on protein structures. Histidine, an amino acid exhibiting a gradient pattern and present in high concentrations in the distal region of byssal threads, has been correlated to transition metal content (zinc or copper) and adhesion in marine mussels (Waite et al. 2004). The exceptional strength of mussel adhesive proteins is undoubtedly the result of the repetitive nature of many of the individual proteins (decapeptide and hexapeptide repeats in Mefp-1 and hydroxyproline repeats in collagens), the modification of individual amino acids (e.g., hydroxylation of proline and tyrosine), and the gradient nature of byssal attachment devices.\nThis gradient nature was first noted by Brown in 1952 (referenced by Brazee and Carrington 2006), who observed corrugated proximal (to the organism) and smooth distal regions. The development of our understanding of the transition of mechanical properties along the thread length, from the more flexible proximal region close to the soft tissues of the mussel, through the collagenous main body of the thread, to the stiffer, less extensible distal region, and the exquisite fine tuning of protein components to impart these properties, has been concisely reviewed recently (Brazee and Carrington 2006). Total mussel adhesive strength is a function of the individual adhesive proteins secreted by the animal, their association with other proteins, their distribution along the thread, and their proximity to materials of differing moduli. Details of the individual adhesive protein components are described in the next section.\nComponents of Mussel Adhesion\nAttachment in Mussels: The Byssus\nThe byssus, an exogenous attachment structure, was first described in 1711 (Brown 1952). High concentrations of polyphenolic proteins (e.g., DOPA), the presence of collagen, and the presence of a catechol oxidase were among the first biochemical observations of byssal attachments. Environmental factors, such as salinity, temperature, pH, season, and substratum choice, as well as biological factors, such as age and metabolic state of the animal, affect the efficiency and strength of byssal attachment to surfaces (Van Winkle 1970; Crisp et al. 1985; Carrington 2002).\nThe stem is rooted in the byssal retractor muscles at the base of the foot organ. The byssal threads, flexible structures of variable dimensions (\u223c0.1\u00a0mm diameter, 2\u20134\u00a0cm length) and strength, originate from the stem. A byssal thread consists of a flexible, collagenous inner core surrounded by a hardened, cured polyphenolic protein. Numerous researchers photographed the collagen core in the 1930s (Brown 1952), well before three unique collagenous proteins were identified and characterized by Qin and Waite (1998, 1995). The outer polyphenolic protein, believed to undergo a quinone tanning-type reaction with a specialized catechol oxidase enzyme, is Mefp-1. Designation of this byssal thread polyphenolic adhesive protein, as well as subsequent adhesive proteins identified in Mytilus edulis, is preceded by the genus and species. The byssal structure culminates in a polyphasic plaque of varying size, dependent on both the size of the animal and the age of the byssus (Crisp et al. 1985). Plaques are commonly only \u223c0.15\u00a0mm in diameter where they meet the thread and \u223c2- to 3-mm diameter at the substrate interface. Plaque formation occurs from the deposition of proteins that originate in the foot organ. To date, five specialized adhesive proteins have been identified in byssal plaques from M. edulis: Mefp-1, -2, -3, -4, and -5. Figure\u00a03 illustrates the anatomy of M. edulis and the associated byssus structures. Figure\u00a04 illustrates the location of the adhesive-related proteins identified in the byssus of M. edulis.\nFigure\u00a03Anatomy of M. edulis mussel and byssus structures.Figure\u00a04Location of adhesive-related proteins identified in the byssus of M. edulis.\nMechanical Properties of Mussels\nThe most extensive research about the adhesive mechanical properties of mussels has been with M. edulis. The breaking energy of intact byssal threads is reported to be 12.5\u2009\u00d7\u2009106\u00a0Jm\u22123 versus tendon (2\u2009\u00d7\u2009106\u00a0Jm\u22123 to 5\u2009\u00d7\u2009106\u00a0Jm\u22123) and silk (50\u2009\u00d7\u2009106\u00a0Jm\u22123 to 180\u2009\u00d7\u2009106\u00a0Jm\u22123) (Denny 1988). Bond strengths are between 0.1 to 10\u2009\u00d7\u2009106\u00a0Nm\u22122, depending on the surface for adhesion (Waite 1999). Byssal thread strength at the distal portion of threads is as strong as a vertebrate tendon but three to five times more extensible (Qin and Waite 1998). Byssal thread strength at the proximal portion of threads is weaker but 15 to 20 times more extensible. Strain energy density of threads approaches that of silk, approximately six times tougher than a tendon (Smeathers and Vincent 1979; Coyne et al. 1997). Byssal threads can recover initial length and stiffness if given sufficient relaxation time (Bell and Gosline 1996).\nMefp-1, the most studied mussel adhesive protein, has adhesive properties comparable to synthetic cyanoacrylate and epoxy resins. These resin adhesives are popular because of their high bond strengths, quick polymerization, and ability to bond to a number of substrates (metals, glass, ceramic) (Savla 1977; Coover and McIntire 1977). Research related to the mechanical properties of individual M. edulis adhesive proteins is limited because of the difficulty in obtaining large quantities of isolated proteins for adhesive testing.\nByssal Thread Proteins\nByssal Thread Polyphenolic Protein: Mefp-1\nMefp-1 was the first polyphenolic protein to be identified in the mussel byssus (Waite and Tanzer 1981; Waite 1983b). The primary location of Mefp-1 is in the byssal threads, cross-linked via a polyphenol oxidase to form a hardened sheath around the flexible, collagenous inner core. Byssal plaques contain \u223c5% of Mefp-1 as well. Mefp-1 is a large (897 amino acids), basic hydrophilic protein with very little secondary structure and a molecular mass of 115\u00a0kDa, based on mass spectroscopy (Filpula et al. 1990). Decapeptide and hexapeptide repeats containing numerous posttranslational modifications (\u223c60\u201370% of the amino acid residues are hydroxylated) provided the first indication of an adhesive-related protein unlike any others identified in nature. The hexapeptide repeat is made up of the amino acids AKPTYK. The major decapeptide consensus repeat, consisting of the amino acids AKPSYP\u2019P\u2019\u2019TYK (in which P represents proline, Y represents DOPA, P\u2019 represents trans-2,3-cis-3,4-dihydroxyproline, and P\u2019\u2019 represents trans-4-hydroxy-L-proline) occurs \u223c80 times in Mefp-1. DOPA residues constitute 10\u201315% of the protein (Waite 1983b). The open conformation of the protein is believed to allow functional groups full accessibility for cross-linking interactions with other proteins and a variety of surfaces, including glass, Teflon\u00ae, and metals. Mepf-1 has been characterized as possessing random coil conformations with helix-like decapeptide segments under physiologic conditions (Haemers et al. 2005). Our laboratory recently has deduced complementary DNA (cDNA) sequences for Mefp-1 (GenBank Accession number: AY845258 and AY845259).\nOther Mytilus mussel species contain a protein analogous to Mefp-1, with differences in the decapeptide repeat frequency, residue composition, and nonrepetitive regions; these species include M. galloprovincialis (Mgfp-1) (Inoue and Odo 1994); M. coruscus (Mcfp-1) (Inoue et al. 1996b); M. trossulus (Inoue et al. 1995b); M. californianus Conrad, California mussel (Waite 1986); M. chilensis (Pardo et al. 1990); and M. sp. JHX-2002 (Wang et al. 2004).\nMefp-1\u2013like adhesive proteins from mussels outside of the Mytilus genus also have been identified: Dreissena polymorpha, zebra mussel (Dpfp-1) (Rzepecki and Waite 1993; Anderson and Waite 2000); Dreissena bugensis, quagga mussel (Dbfp-1) (Anderson and Waite 2002); Perna viridis, green mussel (Pvfp-1) (Ohkawa et al. 2004); Perna canaliculus, green shell mussel (Pcfp-1) (Zhao and Waite 2005); Guekensia demissa, ribbed mussel (Waite et al. 1989); Limnoperna fortunei, Asian freshwater mussel (Lffp-1) (Ohkawa et al. 1999); Aulacomya ater (Saez et al. 1991); and Choromytilus chorus (Pardo et al. 1990). Polyphenolic proteins from other marine mussels also have been compared with Mefp-1 (Rzepecki et al. 1991).\nMefp-1 requires oxidization of tyrosine residues by catechol oxidase, tyrosinase, or sodium periodate for conversion to the reactive DOPA residues required for strong adhesion. The enzyme catalyzing oxidation may serve as an oxidative agent and as a copolymer. Molecular oxygen can be used to further oxidize DOPA to a quinone. Possible cross-linking agents are oxygen, polyvalent metal ions (Fe3+ and Al3+), aldehydes, and many types of bi\/polyfunctional cross-linkers. The hydroxylation of DOPA from incubation with tyrosinase has been shown to form 3-, 4-, 5-trihydroxyphenylalanine (TOPA), in a decapeptide related to Mefp-1 (Burzio and Waite 2002). The prospective functions of TOPA isomers have included metal binding, wound healing, and adhesion.\nMefp-1 has been previously commercialized as a source for mussel adhesive protein. Companies supplying Mefp-1 have obtained adhesive protein from the byssal structures by using protein extraction techniques (Sigma-Aldrich: \u201cAdhesive Protein\u201d; Swedish BioScience Laboratory: \u201cMAP\u201d; BD Biosiences Clontech: Cell-Tak\u2122) and recombinant protein techniques using synthetic gene constructs (Genex Corp.: \u201cAdheraCell\u201d). However, currently there are no commercial sources for pure Mefp-1 because of the high cost of extraction methods and inconsistencies in quality of protein from recombinant protein techniques (Cell-Tak\u2122 Cell and Tissue Adhesive is a formulation of multiple polyphenolic proteins from Mytilus edulis). The laboratory-prepared products have not demonstrated comparable strength to the natural protein. Our laboratory is currently conducting research on the large-scale production of recombinant Mefp-1 protein (Silverman and Roberto 2006a).\nByssal Thread Polyphenol Oxidase\nThe Enzyme Commission of the International Union of Biochemistry classifies each enzyme into six major groups according to the reactions catalyzed. Enzymes are given specific nomenclature, with the letters \u201cEC\u201d prefacing the specific subclass with its major grouping (http:\/\/www.chem.qmul.ac.uk\/iubmb\/enzyme\/). Polyphenol oxidases fall under the broad enzyme grouping of \u201coxidoreductase.\u201d They are oxygen transferring, copper metalloproteins having both catecholase (act on diphenols) and cresolase (act on monophenols) activity. Polyphenol oxidases catalyze the orthohydroxylation of phenols to catechols and the dehydrogenation of catechols to orthoquinones. A monophenol monooxygenase (EC 1.14.18.1), more traditionally called tyrosinase, can oxidize tyrosine to L-DOPA and L-DOPA to o-quinone (Worthington 1993). A catechol oxidase (EC 1.10.3.1) can oxidize L-DOPA to o-quinone. The enzymatic formation of o-quinone is a browning\/tanning\/curing reaction that occurs in plants, bacteria, and animals (Waite 1990). The adhesive properties of marine mussels have been demonstrated by metal complex formation of DOPA and the intermolecular cross-linking of o-quinone with lysine residues.\nVery little definitive information exists about the polyphenol oxidase(s) present in M. edulis that is responsible for tyrosine and DOPA conversions in byssus formation. A cytochemical study of the enzyme gland in the foot organ of M. edulis demonstrated phenol oxidase activity in the Golgi complex but not the secretory granules (Zuccarello 1981). Waite (1985) measured catechol oxidase activity from enzyme gland extractions and whole byssus structures. The native enzyme was shown to prefer diphenols over monophenols as substrates. A single, purified protein was not obtained, and the enzyme was estimated to have subunits and a molecular weight of 120\u00a0kDa. Burzio (1996) extracted two catechol oxidases from M. edulis byssal threads, plaques, and feet. Both the byssal catechol oxidase-high molecular weight (BCO-H, 174\u00a0kDa) and byssal catechol oxidase-low molecular weight (BCO-L, 60\u00a0kDa) isozymes only oxidized catechols to o-quinones, and their amino acid composition was similar to other catechol oxidases found in nature. Again, homogenous purification was not possible. Recently, Hellio et al. (2000) purified a 34\u00a0kDa phenol oxidase monomer from mussel feet. The substrate specificity of the enzyme was greatest toward dihydroxyphenols (L-DOPA and catechol), followed by trihydroxyphenols, suggesting that there may be at least two forms of phenol oxidase involved in mussel adhesion. Extremely low relative activity was observed with monohydroxyphenols. Thus, the specific identification of the polyphenol oxidase enzyme(s) involved in byssus formation continues to elude researchers.\nVarious compounds have been found to inhibit the activity of the M. edulis phenol oxidases identified above: oxygen competitors (cyanide and nitrogen), metal chelators (diethyldithiocarbamate (DETC) and ethylenediaminetetraacetic acid (EDTA)), a reducing agent (L-ascorbic acid), and the competitive inhibitors benzoic acid and L-DOPA, with respect to 4-methylcatechol and L-DOPA (Waite 1985; Burzio 1996; Hellio et al. 2000). There is currently extensive research in the area of anti-fouling methods to prevent attachment of marine and\/or freshwater mussels to surfaces. These studies rely on understanding the biochemical properties of mussel adhesive proteins, including polyphenol oxidase, and the surface chemistry of paints, varnishes, and other materials.\nByssal Thread Collagens\nAll collagens in nature contain a triple-helical domain with the sequence repeat (Gly-X-Y)n, in which Gly is glycine, X often is proline, and Y is usually hydroxyproline. This triple-helical configuration provides mechanical strength but on its own offers little flexibility and elasticity. The collagens present in M. edulis byssal threads contain additional sequence domains that make the byssal thread approximately six times tougher than the human Achilles tendon (Smeathers and Vincent 1979; Coyne et al. 1997). Two M. edulis collagens appear in a gradient fashion in byssal threads, and a third is distributed evenly throughout the thread. Analogous byssal collagens have been identified in M. galloprovincialis (Lucas et al. 2002; Hassenkam et al. 2004).\nProximal Collagen (Col-P)\nThe proximal region of a byssal thread (the region closest to the animal) consists of a unique, natural co-polymer, termed proximal collagen (Col-P) (Coyne et al. 1997; Qin and Waite 1995). This protein is an incredible shock absorber with 160% extensibility. The precursor collagen (PreCol-P) has a molecular mass of 95\u00a0kDa and consists of seven domains: representative amino and carboxyl termini; a large, central collagenous domain (40\u00a0kDa) flanked by elastin-like domains (11\u00a0kDa each); and small, histidine-rich domains (5\u00a0kDa) that flank the elastin-like domains. A small acidic patch is present between the collagen and elastin-like domains at the carboxy end of the protein. The collagen domain, coupled with the presence of the two, elastin-like domains, provides for an incredibly tough and extensible proximal region in byssal threads. Histidine-rich domains are believed to function in metal binding. The transition metal zinc has been detected in M. edulis byssi, supporting theories of Zn2+-mediated cross-linking in the terminal domains of Col-P. Two variants of PreCol-P have been implicated in molecular dovetailing between the proximal byssal thread and the byssal stem (P22 and P33) (Coyne and Waite 2000). The presence of these variants suggests a mechanism in which mussels can mitigate stresses by overlapping different proteins, a phenomenon that occurs between tendon and bone (Fukuta et al. 1998) and in spiders from frame silk (web frame and radial supports) to viscid (glue-covered) silks (Guerette et al. 1996).\nDistal Collagen (Col-D)\nThe distal region of a byssal thread contains another natural co-polymer: distal collagen (Col-D). The Col-D is stiffer than the Col-P. The precursor collagen (PreCol-D) (molecular mass 97\u00a0kDa) also contains seven separate domains: representative amino and carboxyl termini; a central, collagenous domain larger than PreCol-P (45.5\u00a0kDa); silk fibroin-like domains flanking the collagen domain (5 and 15\u00a0kDa); and small, histidine-rich domains (7 and 5\u00a0kDa) that flank the silk-fibroin domains. A large collagen domain (175 repeats of Gly-X-Y), a small, acidic patch, and the histidine-rich domains are similar to the PreCol-P structure. The presence of silk-fibroin domains allow for extensibility in PreCol-D, as is the case for elastin in PreCol-P. However, the distal region of byssal threads is localized in straight bundles (strong and stiff), whereas the collagen fibers are coiled in the proximal region (pliable and elastic) (Waite et al. 1998). Before the characterization of PreCol-D, silk fibroin domains had been previously reported only in arthropods.\nPepsin-resistant Nongradient Collagen (Col-NG)\nThe entire length of a byssal thread contains a third block copolymer-like protein, pepsin-resistant nongradient collagen (Col-NG) (Qin and Waite 1998). The precursor pepsin-resistant nongradient collagen (PreCol-NG) (molecular mass 76\u00a0kDa) is believed to function as a mediator between PreCol-D and PreCol-P. It consists of a central collagenous domain (\u223c38\u00a0kDa), an acidic patch, and histidine-rich termini, similar to PreCol-D and PreCol-P. The regions flanking the collagen domain contain (X-Glyn)m repeats (which are similar motifs found in plant cell walls) in addition to the familiar silk fibroin-like domains that are present in PreCol-D. There is a larger distribution of tyrosine in the histidine domain of the N- and C- terminal sequences of PreCol-NG compared to the other two collagens.\nThe elastic domains of PreCol-P, the silk fibroin-like domains of PreCol-D, and the plant cell wall-like domains of PreCol-NG characterize the unique, collagenous block copolymers found in the byssal threads of M. edulis.\nProximal Thread Matrix Protein (PTMP)\nThe proximal portion of each byssal thread contains a water-soluble, noncollagenous protein designated proximal thread matrix protein (PTMP; Sun et al. 2002). This \u223c50\u00a0kDa matrix protein has a capacity for collagen binding and resembles the von Willebrand factor in amino acid sequence, antigenicity, and its stiffening effect. Two variants have been identified in M. edulis (PTMP1a and PTMP1b). A similar PTMP1-like cDNA has been found in M. galloprovincialis.\nByssal Plaque Polyphenolic Proteins\nMefp-2\nMefp-2 is found exclusively in byssal plaques, constituting from 25% to 40% of the total plaque proteins. Unlike Mefp-1, Mefp-2 is a smaller adhesive protein (molecular mass 42\u201347\u00a0kDa) with only 2 to 3\u00a0mol% DOPA and no hydroxylation of proline to trans-2,3-cis-3,4-dihydroxyproline or trans-4-hydroxy-L-proline. The DOPA residues occur primarily in the N- and C- terminal regions of the protein. Mefp-2 contains considerable secondary structure and is relatively resistant to a variety of proteases (compared with Mefp-1), an important characteristic for integrity of the byssal plaque. The high cysteine content (6\u20137\u00a0mol%), coupled with tandem, repetitive motifs similar to epidermal growth factor, suggests that Mefp-2 is an adhesive protein with a stabilization role in the byssus (Inoue et al. 1995a). A Mefp-2 multigene family may exist, based on evidence that at least three different repetitive motifs have been identified in the primary protein sequence (Rzepecki et al. 1992). A published full-length gene sequence for Mefp-2 is not available. However, an investigation of Mefp-2 cDNA sequences identified by our laboratory supports the multigene family premise (GenBank Submission numbers: AY845260, AY845261, and AY845262). Our laboratory is currently conducting research on the large-scale production of recombinant Mefp-2 protein (Silverman and Roberto 2006b).\nAs is the case with Mefp-1, other mussel species contain proteins analogous to Mefp-2: M. galloprovincialis (Mgfp-2) (Inoue et al. 1995b); M. coruscus (Mcfp-2) (Inoue et al. 2000); and D. polymorpha (Dpfp-2) (Rzepecki and Waite 1993). Stewart et al. (2004) reported that the tube cement of Phragmatopoma californica, a marine polychaete, forms solid foam (similar to Mefp-2) via cross-linking with DOPA.\nMefp-3\nMefp-3 is the smallest byssal adhesive protein identified to date, with a molecular mass of \u223c5 to 7\u00a0kDa (Papov et al. 1995; Warner and Waite 1999). Mefp-3 contains no repeats, 20 to 25\u00a0mol% DOPA, and a prevalence of 4-hydroxyarginine and tryptophan residues. Warner and Waite (1999) identified 20 gene variants (\u223c0.3\u00a0kB) of Mefp-3 in the foot organ; however, only four or five proteins have actually been detected in plaques deposited on glass or plastic. The presence of a gene family for Mefp-3 supports the primer-like function proposed for the protein in adhering to substrata. One hypothesis has been that deposition of a specific Mefp-3 variant is dependent on the surface used for attachment. However, Floriolli et al. (2000) reported no correlation between Mefp-3 expression and surface type (stainless steel, glass, polyethylene) in an individual mussel. Differences in cDNA transcripts were found between animals. Zhao et al. (2006) reported 12 different Mcfp-3 variants from a mussel population\u2014broken into \u201cfast\u201d or \u201cslow\u201d electrophoretic characteristics\u2014upon exposure to glass coverslips. The molecular mechanism(s) for the numerous Mefp-3 and Mcfp-3 variants is not known. Variables, such as exposure time to surfaces, water temperature, and the age of the animals, could influence expression levels.\nAs is the case with Mefp-1 and Mefp-2, other mussel species contain proteins analogous to Mefp-3 variants: M. galloprovincialis (Mgfp-3A and Mgfp-3B) (Inoue et al. 1996a) and M. californianus (Mcfp-3\u201312 variants, mentioned above) (Zhao et al. 2006).\nMefp-4\nMefp-4 is another protein identified in byssal plaques, with a molecular mass of 79\u00a0kDa (Weaver 1998; Vreeland et al. 1998; Warner and Waite 1999). Mefp-4 contains elevated levels of glycine, arginine, and histidine, as well as 4\u00a0mol% DOPA. A unique tyrosine-rich octapeptide is present, with variations in residue substitutions giving rise to a family of proteins. This very large protein most likely serves as a coupling agent in the thread-plaque junction designated by the precollagens and the byssal plaque protein Mefp-2. A gene sequence for Mefp-4 has not been identified; however, two-foot protein variants from M. californianus have recently been identified (Mcfp-4; Zhao and Waite 2006a).\nMefp-5\nMefp-5 is the most recent adhesive-related byssal plaque protein identified from Mytilus edulis (Waite and Qin 2001). Mefp-5 is a relatively small protein with a molecular mass of 9.5\u00a0kDa, a 27\u00a0mol% DOPA content, and the presence of phosphoserine. Phosphoserine is known to occur in acidic mineral-binding motifs of proteins that bind calcareous materials (e.g., osteopontin); therefore, its presence in byssal plaques may aid in adhesion of one animal to a neighboring mussel\u2019s shell. Mefp-5 was formerly associated with the Mefp-3 family of variants, and similarly, plays an interfacial role as a primer for substrate adhesion. An adhesive protein analogous to Mefp-5 has recently been reported to be present in M. galloprovincialis (Mgfp-5) (Hwang et al. 2004) and M. californianus (Mcfp-5-2 variants) (Zhao and Waite 2006b).\nDiscovery of Additional Foot Proteins: Mcfp-6\nMcfp-6 was identified along with Mcfp-5 by Zhao and Waite (2006b; 3 variants). This small protein (11.6\u00a0kDa) contains a relatively large amount of tyrosine and a small amount of DOPA. Its suggested role in adhesion may be to provide a link between the DOPA-rich proteins and the plaque proteins present in byssal attachment plaques.\nAdhesive Testing of Mussel Proteins\nA wide variety of adhesive tests have been applied to intact byssal threads, plaque, portions of threads, or materials bonded or coated with individual adhesive proteins. Gross comparative tests on byssal threads were described earlier in this review.\nMussels seem to prefer surfaces with higher critical surface energy, a phenomenon also observed in barnacles (Crisp et al. 1985; Waite 1987). Plaques were shown to attach more strongly to slate and glass than plastic acetal (acetate), paraffin wax, and polytetrafluoroethylene (PTFE). The high-energy, hydrophilic surfaces (glass, slate) had smaller plaque surfaces attached to substrates and smaller contact angles compared with the low-energy, hydrophobic surfaces (wax, PTFE).\nAttachment Failure\nByssal attachments can fail in a number of ways. A byssal plaque can cleanly peel away from a surface, the byssal thread itself can tear or fail, or the root can tear away from the animal. Failure of the thread is the most common cause of failure of byssal attachment. If byssal threads break or tear away from the animal, the true adhesive force is underestimated. Therefore, when analyzing adhesive forces, it is important to consider the type of failure. The surface for attachment, source of foot protein, age of animal, temperature, and season are important in evaluating the strength of byssal attachments (Crisp et al. 1985).\nAdhesive Techniques\nNumerous surfaces and techniques are used to evaluate the mechanical properties of mussel adhesive proteins. Atomic force microscopy (AFM; for surface topography), attenuated total reflection fourier transform infrared spectroscopy (ATR\/FT-IR; for information on molecular composition, bonding, conformation and orientation with respect to interfaces), surface plasmon resonance (SPR; to study interactions of samples and surfaces), and tensometer methods (for tensile strength and elastic properties) are common techniques used in adhesives research. Table\u00a02 provides examples of studies in which surfaces have been tested with mussel adhesive proteins or synthetic analogs containing repetitive motifs from mussel proteins\nTable\u00a02.Examples of materials testing with mussel adhesive proteins or synthetic analogs containing repetitive motifs from mussel proteinsSurfaces TestedMussel Protein or Synthetic AnalogReferencesSlateSynthetic recombinant Mefp-1Crisp et al. 1985Silica (glass)Plastic acetal (acetate)Paraffin waxPolytetrafluoroethylene (PTFE)PolystyreneRecombinant Mefp-1Filpula et al. 1990Limestone\/dolomite cobbleD. bugensis and D. polymorphaAckerman et al. 1992Mild steelStainless steelMarine concreteAckerman et al. 1995Marine plywoodPolyvinyl chloridePolymethylmethacrylate (Plexiglas\u00ae)Ackerman et al. 1996AluminumTeflon\u00aeD. bugensis and D. polymorphaDormon et al. 1997ConcreteMild steelPolyvinyl chlorideStainless steelSiliconeSynthetic recombinant Mefp-1Kitamura et al. 1999SilicaPolyethylene terephthalate (PET)Teflon\u00aeAluminumSynthetic polypeptide mimics of marine adhesivesYu and Deming 1998SteelSilicaPlasticsMicroporous apatite surfaceMefp-1Shirkhanzadeh 1998SilicaSynthetic homo- and copolypeptides of marine and related adhesive proteins (M. edulis, M. californianus, A. ater, G. demissa, liver fluke, pearl oyster)Yamamoto et al. 1999Polytetrafluoroethylene (PTFE)Teflon\u00aeNylonIronSoda glassMethyl- and oligo (ethylene oxide)-terminated, self-assembled monolayersMefp-1 (Cell-Tak\u2122) and fibrinogenHarder et al. 2000Germanium (oxide)Mefp-1, Mefp-2, and polylysineSuci and Geesey 2000PolystyrenePoly octadecyl methacrylateSuci and Geesey 2001SilicaMefp-1Frank and Belfort 2001Porcine skinM. edulis mussel feet extractNinan et al. 2003SilicaRecombinant Mgfp-5 and Mefp-1 (Cell-Tak\u2122)Hwang et al. 2004Polymethylmethacrylate (Plexiglas\u00ae)PolystyreneAluminum\nBrazee and Carrington (2006) recently provided an elegant and detailed study of whole byssal thread material properties comparing threads from a range of Mytilidae. This study also compared their results to those of previous studies, along with statistics to quantify the variation in the results. Quasi-static tensile strength tests and dynamic testing of elastic properties (extensibility, modulus, resilience, and recovery) were performed and correlated with morphometric measurements, including shell weight and dimensions, and thread morphometry. Several interesting findings were reported, including the elliptical cross-section (rather than a regular, round section) of the byssal thread, novel behavior of Modiolus modiolus threads (\u201cdouble yield\u201d under strain), the surprising strength of Dreissena polymorpha threads (although they lack a collagen core), and the overall greater strength of Mytilis californianus threads (suggested to be a factor in its success in living in the strong tidal environment of the Pacific coast).\nThe application of atomic force microscopy (AFM) is revolutionizing our understanding of the interactions of mussel adhesive proteins with surfaces and with other adhesive proteins that make up the byssal thread. It is now possible to measure adhesion energy and shear forces of individual adhesive proteins, such as has been done in comparing M. edulis foot proteins 1 and 3 (Lin et al. 2007). Even more remarkably, the cantilevers of an AFM can be coated with DOPA residues to perform single molecule measurements and quantify the differences in bond strength that might be expected between unmodified and DOPA-modified adhesives (Lee et al. 2006).\nAs our ability to perform sensitive measurements with small amounts of protein improves, along with the increased resolution of the techniques used, we can anticipate that our understanding of the interactions of adhesive proteins necessary to achieve robust adhesion will increase.\nFuture Supply of Adhesive Proteins by Recombinant Approaches\nBackground\nAn impediment to further understanding the unique adhesion system of M. edulis is the lack of availability of the individual protein components. Large quantities of M. edulis (or other mussel) adhesive proteins are needed to perform research and development for commercial adhesives. Current methods for obtaining the adhesive proteins of the mussel byssus rely largely on excision of the byssus followed by extraction of the proteins under acidic conditions. However, the chemical extraction process does not always yield pure or individual adhesive proteins. Approximately 10,000 M. edulis mussels are needed to produce 1\u00a0g of Mefp-1 adhesive from byssal structures (Strausberg and Link 1990; Hwang et al. 2007a). At least 100\u00a0mg of material is needed for performing small, conventional adhesive tests, such as tensile strength or wall-jet analyses. The sacrifice of such a large number of animals is neither environmentally friendly nor economically practical. Therefore, an alternate approach to obtain large amounts of adhesive protein components is necessary for further conventional adhesive testing formats and adhesive formulation development. Molecular and microbiology techniques can be used for the directed production of large quantities of many different proteins.\nProkaryotes and eukaryotes are used as hosts in recombinant protein expression systems. Common prokaryote hosts include bacteria (Escherichia coli), Saccharomyces cerevisiae (baker\u2019s yeast), Pichia pastoris (a methylotrophic yeast), or Kluyveromyces lactis (a lactic-acid\u2013producing yeast). Eukaryote hosts include plants (tobacco, potatoes), trees (loblolly-pine), and mammals (rabbit, mouse, goat). Cell culture techniques use insect, plant, and mammalian cells or tissue cultures rather than whole organisms. Variables important for successful recombinant protein expression systems include a codon-usage\u2013compatible host; promoter, transcriptional, and translational regulators; fine-tuned cultivation methods; targeted recombinant protein purification methodologies; posttranslational modification ability; and three-dimensional configuration of the recombinant protein.\nCodon usage refers to the predominant DNA bases that code for specific amino acids. Codon analysis can be performed for the source organism across all its known proteins or only for the specific protein or protein class of interest. Possible choices for mussel adhesive recombinant protein expression include a eukaryotic host, a special bacterial line (E. coli BL21 strains), or a plant source, such as tobacco (because plant cell wall proteins are repetitive proteins with hydroxylations).\nPromoter and transcriptional regulators are DNA sequences that direct gene expression. They can be native to the host, artificially added to the host DNA, or incorporated in vector systems. Translational stop signals can be incorporated into the DNA genes or are present on an expression vector. Prokaryotes and eukaryotes use different regulators for gene expression.\nCultivation factors, such as energy sources, aeration (oxygen), temperature, and induction protocols are specific to the host organism or cell type and are dependent on the quantity used for production. Small-scale cultivation generally involves flasks or petri dishes and volumes less than 1\u00a0L. Larger-scale cultivation in the research laboratory setting can use bioreactors with volumes as large as 100\u00a0L. Automated monitoring and control of cultivation variables is used in large-scale recombinant protein production.\nHeterologous recombinant protein production occurs simultaneously with the host\u2019s production of its own proteins. Thus, harvesting of a recombinant protein involves its identification and separation from the host\u2019s native proteins. Special tags that can be incorporated with a recombinant protein include specific amino acid repeats (histidine), fluorescent molecules (green fluorescent protein), biotinylation, or antibody recognition sequences (epitopes). The specific chemical properties of proteins also can be used for isolation and purification. For M. edulis adhesive proteins Mefp-1 and Mefp-2, acidic conditions in purification steps will exclude many of the host\u2019s native proteins while maintaining the integrity of the mussel proteins.\nOne caveat in heterologous protein production, however, is that correctly folded or modified proteins are not always produced. Prokaryotes and eukaryotes can modify their proteins following translation with specific disulfide bond formation for correct protein folding, cleavage of precursor protein forms to yield functional proteins, glycosylation of amino acid residues for protein stability, and other modifications to amino acids, such as phosphorylation, acetylation, or hydroxylation (as in the case with many M. edulis adhesive proteins). Posttranslational modifications do not always occur naturally in heterologous systems; therefore, in vitro methods often are required to obtain properly modified proteins. For example, heterologously produced, unhydroxylated Mefp-1 requires treatment with mushroom or bacterial tyrosinase to produce hydroxylated tyrosine (DOPA) at positions two and nine of the decapeptide repeat (Strausberg and Link 1990; Filpula et al. 1990; Kitamura et al. 1999). Other considerations for biologically functional, recombinant proteins include hyperglycosylation, inclusion bodies, and loss of expression plasmids with a scale-up in cultivation volumes.\nThe Recombinant Protein Approach\nThe recombinant expression of cDNAs or synthetic adhesive-related proteins modeled from organisms other than mussels has been reported: human blood coagulation protein Factor XIIIa in the yeast S. cerevisiae (Broker et al. 1991) and collagen and spider silk proteins in bacteria, yeast, insect, and mammalian cells and plants (Fahnestock et al. 2000; Kieliszewski and Lamport 1994; Scheller et al. 2001).\nResearchers have, to some extent, successfully produced Mytilus mussel adhesive proteins in bacteria, yeast, mammalian cells, and plants. Filpula et al. (1990) used a hybrid combination of promoters to direct the production of Mefp-1-like tandem repeats in S. cerevisiae. A single copy of the genomic cDNA clone for Mefp-1 encoded the carboxy terminus, including 19 decapeptide and one hexapeptide repeat sequences. Synthetic gene constructs carrying between one and four copies of a portion of the genomic sequence for Mefp-1 were shown to express proteins between 20 to 100\u00a0kDa. Amino acid analyses of the four different recombinant proteins expressed in the yeast system were similar to unhydroxylated natural Mefp-1. The recombinant protein constituted 2% to 5% of the total cell protein, and adhesive tests showed water-resistant bonding after in vitro modification of tyrosine residues to DOPA and then quinone. Salerno and Goldberg (1993) expressed a synthetic mussel adhesive protein analog in E. coli using a T7 promoter system and a repetitive gene cassette that encoded the consensus decapeptide repeat of Mefp-1. The synthetic polydecapeptide was produced from a 600 base pair gene (20 decapeptide repeats) and constituted up to 60% of total cell protein. Kitamura et al. (1999) also expressed a synthetic mussel adhesive protein analog in E. coli with a T7 promoter system and a repetitive gene cassette that encoded six repeats of the consensus decapeptide of Mefp-1. The researchers were able to obtain 10\u00a0mg of the model peptide per liter of growth medium. Surface strength and contact-angle were measured and used to calculate the value of work of adhesion (WA) for both the unmodified and mushroom-tyrosinase\u2013treated peptide. Adhesion to silicone, glass, polyethylene terephthalate, and Teflon\u00ae substrates was found to be dependent on sodium chloride concentration (e.g., sea water).\nOur laboratory has patented a technique for cloning and expressing recombinant Mefp-1 and Mefp-2 in S. cerevisiae from cDNA clones (Silverman and Roberto 2006a, b). We are currently producing rMefp-1, rMefp-2, and rMefp-3 in 20-L fermentation batches (unpublished data). Purification of each individual recombinant protein is in progress. The purified products will be analyzed by mass spectrometry (MS) for total intact mass followed by MS analysis (post-protease digestion). Adhesive studies will be performed in-house and\/or through interested parties after the confirmation of rMefp-1, rMefp-2, and rMefp-3.\nRecombinant mussel adhesive proteins from M. galloprovincialis also have been produced. Takeuchi et al. (1999) cultured mussel foot cells from M. galloprovincialis in Petri dishes. Gene-specific probes were used to identify the stage of development for different adhesive proteins. Probes for the genes encoding Mgfp-1, -2, and -3 were used to demonstrate that the expression of the byssal protein genes in M. galloprovincialis is morphologically programmed (Takeuchi et al. 1997). The findings suggested that a cell culture system for in vitro expression of byssal adhesive proteins might be a plausible alternative to yeast- or bacterial-based expression systems.\nHwang et al. (2004) produced a soluble, recombinant Mgfp-5 fused with a histidine tag in E. coli. A purification yield of \u223c7% was obtained from a 3\u00a0L working volume because of difficulties in removing the recombinant protein from the chromatographic resin used to bind histidine proteins. The adhesive properties of recombinant Mgfp-5 were compared to Cell-Tak\u2122 by using atomic force microscopy (AFM) , material surface coating (on glass, polymethylmethacrylate, polystyrene, a silicone-based antifouling agent-coated slide, and aluminum), and quartz crystal microbalance (QCM) techniques. Recombinant Mgfp-5 was shown to have adhesive abilities comparable to\u2014and sometimes exceeding\u2014those of Cell-Tak\u2122. The adhesion force of tyrosinase-treated Mgfp-5 (\u223c981\u00a0nN) was higher than both tyrosinase-treated Cell-Tak\u2122 (\u223c302\u00a0nN) and untreated Cell-Tak\u2122 (\u223c624\u00a0nN). The adhesion ability of recombinant Mgfp-5 to a glass slide and a poly-(methyl methacrylate) plate was comparable to Cell-Tak\u2122 but better than Cell-Tak\u2122 on a silicone-based antifouling agent coated slide. And, adsorption of Mgfp-5 to a gold surface was comparable to Cell-Tak\u2122.\nHwang et al. (2005) also produced recombinant Mgfp-3 (variant A) with a histidine tag in E. coli. The purification yield and solubility exceeded that of the above-mentioned recombinant Mgfp-5. The adsorption and adhesion force were comparable to Cell-Tak\u2122 but less than recombinant Mgfp-5 (e.g. \u223c230\u00a0nN for Mgfp-3, \u223c240\u00a0nN for Cell-Tak\u2122, and \u223c550\u00a0nN for Mgfp-5).\nHwang and colleagues noted the low production yields, low purification yields, and high levels of insolubility of rMgfp-3 and rMgfp-5 and sought to improve the technique for recombinant protein expression by designing novel fusion proteins (Hwang et al. 2007a, b). The first hybrid MAP mussel bioadhesive\u2014fp-151\u2014was produced from a fusion protein containing six Mgfp-1 decapeptide repeats at both the N- and C-termini of Mgfp-5 (Hwang et al. 2007a). The second hybrid MAP mussel bioadhesive\u2014fp-151-RGD\u2014was a fusion of the GRGDSP residues found in fibronectin (designated RGD) to the C-terminus of fp-151 (Hwang et al. 2007b). The authors reported greater production yields, easier purification, and improved solubility for both novel fusion proteins fp-151 and fp-151-RGD. In addition, the cell adhesion and spreading abilities of fp-151-RGD were superior to Cell-Tak\u2122, poly-L-lysine (PLL), a noncoated surface, and fp-151\u2014regardless of mammalian cell type tested (human or hamster).\nThe first non-Mytilus recombinant mussel adhesive protein, the Mefp-1\u2013like adhesive protein Dpfp-1 from the zebra mussel D. polymorpha, was expressed by Anderson and Waite (2000). Recombinant Dpfp-1 was produced as a maltose-fusion protein in E. coli. The recombinant protein was then used as an antigen for polyclonal antibody production. Immunologic studies definitively revealed the presence of Dpfp-1 in foot organs and byssal threads of D. polymorpha.\nWhole plants have been used to synthesize foreign\/recombinant proteins, such as antibodies, vaccines, and industrial enzymes (Doran 2000). An alternative to the use of whole plants is the use of plant tissue cultures. Tissue cultures provide a technology that may gain momentum because of the ease in manipulation of culture conditions for greater foreign protein levels. There is considerable expense in cell culture techniques, indicating that this scheme would not be optimal for industrial production. Tobacco and rice are the most commonly used plant cell cultures used to date. Correct posttranslational modification is an issue when using plants as hosts, similar to bacterial or yeast expression systems.\nHowever, plants may be very good hosts for production of the repetitive mussel adhesive proteins. Plant cell walls contain large, repetitive proteins similar to foot protein-1 analogs and the diverse collagens found in byssal threads. Dr. Simon McQueen-Mason (Department of Biology, University of York, United Kingdom) has investigated the ability of transgenic tobacco plants to produce recombinant spider silks and Mgfp-1 because of their similarity to the repetitive plant cell wall proteins expansin and extensin (http:\/\/www.cnap.org.uk\/). His group continues to research expansins and pectins from various plant species (Jones et al. 2005). Recently, Patel et al. (2007) reported the use of elastin-like polypeptide fusions (an ELP tag) to enhance the accumulation of a range of different recombinant proteins (human interleukin-10, murine interleukin-4, and the native major ampullate spidroin protein 2 gene from the spider Nephilla clavipes) in the leaves of a transgenic tobacco plant. The fusion protein concentrations in the plant leaves were significantly higher than the target recombinant proteins alone in all instances, suggesting that this type of fusion tag may be beneficial for producing large, repetitive recombinant proteins in plant hosts.\nCurrent and Future Research Areas Related to Mussel Adhesion and Adhesives\nCommercialization of Mussel Adhesive Proteins\nNumerous economic factors are important in the production and synthesis of foreign proteins, whether using microbial cell culture, animal cell culture, plant tissue culture, transgenic plants, or transgenic animals. Production costs (yield for cost comparisons), safety issues (for therapeutic use), and stability of the product (the potential for the protein to degrade or lose function during extraction\/purification procedures) are a few issues that require careful analysis before the method is chosen. Regulatory issues relevant to Good Manufacturing Practice (GMP) for production of therapeutic proteins must be followed. Any product containing recombinant mussel adhesive protein will require extensive testing and validation from health, environmental, and adhesives industries before commercialization.\nThe underwater adhesion of the marine mussel, M. edulis, has intrigued scientists for decades. Extensive progress has been made in elucidating the mechanisms responsible for adhesion, in understanding adhesion to a variety of substrates, and in producing recombinant proteins and materials that mimic the natural mussel adhesive. A selection of current and future research areas related to mussel adhesives is discussed below.\nBiofouling\nIn 1987, mussel biofouling cost the Navy more than $200M in the areas of hull scraping and excess fuel consumption from unnecessary drag on large vessels (Morgan 1990). Understandably, the Office of Naval Research and other organizations have invested considerably in the areas of antifouling and foul-release. Approaches to antifouling and bivalve control can generally be classified as chemical or physical (hull scraping) methodologies. Chemical methods have received the most attention. Until recently, bis(tributyltin)oxide was a popular antifouling component of boat paints. However, heavy metal-based and organotin coatings are now restricted because they are toxic to marine and freshwater organisms. Synthetic and natural biodegradable compounds are currently being investigated for their efficacy as antifouling agents. Imides (special types of amino acid-based compounds), terpenoids (multicyclic structures with basic carbon skeletons) from brown alga species and a marine sponge, proteases from a marine bacterium, phloroglucinol compounds (white, crystalline phenols) from Eucalyptus, and a variety of chemicals have all been shown to inhibit mussel attachment to surfaces (Zentz et al. 2001; Hellio et al. 2001; Sera et al. 1999; Venkateswaran and Dohmoto 2000; Cope et al. 1997). Experimentation with low-adhesion surfaces has included modified glass (Yamamoto et al. 1997), polyethylene glycol (PEG) (Dalsin et al. 2003), and various other polymeric surfaces (Frank and Belfort 2001). Antifouling and detachment research related to mussels and other sessile organisms is vitally important in addressing the economical problems associated with biofouling (Taylor 2006; Bellas 2006; Bellas et al. 2005).\nMussels from Extreme Environments\nHydrothermal vents and cold seeps from the Gulf of Mexico and the Japan Trench are home to unique combinations of sea life. New classes of animals, plants, and bacteria are rapidly being identified with the aid of special underwater submersibles (ALVIN, the Johnson Sea Link) and advances in molecular biology and microbiology. Bathymodiolinae is a subfamily of mussels that contains species harboring methanotrophic, chemoautotrophic, and\/or both types of bacterial endosymbionts within the mussel\u2019s gill tissue. Geological forces have caused cracks in the continental slope, allowing oil, natural gases, such as methane and hydrogen sulfide, and related substances to seep into the ocean environments. Bathymodiolinae mussels and other organisms from the Gulf of Mexico have been able to survive under anoxic, high-pressure, and high-saline (brine) environments for centuries. Figure\u00a05 illustrates the large shells and byssal plaques and threads from Bathymodiolus childressi mussels obtained by Dr. Charles Fisher from Brine Pool NR1 in the Gulf of Mexico (MacDonald and Fisher 1996) compared with M. edulis (Figure\u00a05a and c). The foot organs of M. edulis and B. childressi and M. edulis (Figure\u00a05b and d) also are shown for comparison. The morphologic differences in the foot organ, shell, and gill characteristics between the two marine mussel species, as well as the vast differences between the environmental conditions in which the two reside, may be important variables affecting byssus characteristics (material properties, adhesion mechanisms, individual proteins, etc.; Brazee and Carrington 2006). The identification of adhesive proteins from extreme mussels is an interesting task for prospective research.\nFigure\u00a05Gross appearance and internal organs of B. childressi and M. edulis mussels: (a) B. childressi shell, thread, and plaques, (b) B. childressi foot organ, (c) M. edulis shell, thread, and plaques, (d) M. edulis foot organ.\nNovel Applications for Mussel Adhesive Collagens and Polyphenol Oxidase\nImmobilization of mussel adhesive proteins (Mefp class and collagens) on solid supports may be exploited for uses in the design of biosensors, immunosensors, or artificial tissue scaffoldings and constructs: Mefp-1 with glucose (Saby and Luong 1998; Newman and Setford 2006), Mefp-1 with human chorionic gonadotrophin (Burzio et al. 1996), collagens as supports (BD\u2122 Three-Dimensional Collagen Composite Scaffold; BD Biosciences; San Jose, CA). The identification and subsequent use of the polyphenol oxidase derived from M. edulis byssal structures also could be exploited as a thickening agent for numerous industries\/applications (Yamada et al. 2000).\nMedical and Dental Adhesives\nCurrent adhesives approved for medical use in the United States include fibrin sealants, albumin-based compounds, glutaraldehyde glues, cyanoacrylates, hydrogels, and collagen-based compounds. Fibrin sealants are the most prevalent. They consist primarily of thrombin and fibrinogen, clotting factors in blood. Fibrin sealants have a wide range of uses and are bioabsorbed by the body. However, risks of products isolated from blood, albeit minimal, are a concern with this class of tissue adhesives. One albumin-based compound approved for use in the United States is BioGlue\u00ae Surgical Adhesive (CryoLife Inc.; Kennesaw, GA). It is glutaraldehyde-based, or gelatin-resorcinol-formaldehyde-glutaraldehyde-like, glue (without the formaldehyde) that has limited U.S. Food and Drug Administration approval because of known immunoreactivity problems. The cyanoacrylates approved for use in the United States are stronger than the fibrin sealants but not bioabsorbable. Their use is restricted to external or temporary applications because of associations with carcinogenicity, inflammation, and infection. Hydrogels are water-soluble polyethylene glycol polymers that are activated by light. Both protein-based and DOPA-based hydrogel formulations are being researched (Nowak et al. 2002; Mo et al. 2006; Liu and Li 2002). Currently, their use is limited because of the time required for application, setting, and bioabsorption.\nCollagen-based adhesives are the newest type of tissue adhesive available (Koob and Hernandez 2002). So far they work well and are inexpensive, thus adding an alternative to the use of fibrin sealants. Dr. Robin L. Garrell (Organic Chemistry Department, University of California, Los Angeles [UCLA]) has worked on elucidating the chemical mechanisms for adhesion and cohesion in Mefp-1 and other marine biopolymers to develop artificial tissues and bioreactors (Ooka and Garrell 2000; http:\/\/www.chem.ucla.edu\/dept\/Organic\/garrell.html). Future dental and medical adhesives may contain mussel adhesive proteins and\/or domains from mussel adhesives (Tay and Pashley 2002; Ninan et al. 2003; Fulkerson et al. 1990; Robin et al. 1988; Schmidt et al. 1994).\nConclusions\nScientists and laymen alike have been fascinated for years with the ability of mussels to cling to surfaces under water. The ability to adhere in an aqueous environment, withstand numerous environmental forces, and resist conventional approaches to detachment are factors that continue to intrigue researchers today. During the last two decades, considerable time and effort has been spent in identifying the proteins that contribute to underwater adhesion by marine mussels. The production-scale availability of recombinant mussel adhesive proteins will enable researchers to develop formulations for adhesives in which there exist endless applications for the commercialization of water-impervious, ecologically safe adhesives derived from mussels.","keyphrases":["adhesion","biomimetics","recombinant protein","marine mussel (mytilus edulis)"],"prmu":["P","P","P","R"]}
{"id":"Bioinformation-1-6-1891690","title":"DoD2006: molecular biology database update\n","text":"DoD2006, an updated version of Database of Databases is an online resource maintained collectively by ProGene Biosciences and Department of Inorganic and Analytical Chemistry, Andhra University. It links to all molecular biology databases that appeared in Nucleic Acids Research 2006 database issue. DoD2006 includes 873 databases, of which, 858 are derived from Nucleic Acids Research database issue and 15 are collected from In Silico Biology, Bioinformation journals and Google Scholar search. Each database has a search option, keyword help and a brief description with direct link to the database home page. The database is freely available online at http:\/\/www.progenebio.in\/DoD\/index.htm\nBackground\nOnline availability of databases is increasing due to their utility by structural and molecular biologists. These databases are generated using a variety of programming languages and they differ \nin content and access methods. [1] The information explosion in biology resulted in database proliferation and this necessitates the importance \nof data integration, storage and retrieval. Many new databases tend to appear every year, with ever increasing information or knowledge from biology, driven by genome sequencing projects and easy \ninternet access. [2]\nOn the contrary, fund crunching is a limitation for data collection, constant updates, infrastructure facilities and maintenance of databases. Biological databases with infrastructural funding are\nhosted by academic, governmental organizations and industry as well as non-profit organizations. [3] Collection and analysis of data from various databases is \nessential for computational biologists. But it is time consuming and proportional with increasing number of databases. In this context, there is a need to integrate all databases at a single interface, usually with \none search engine to query all databases. Therefore, we partially address the issue by maintaining the molecular biology databases at a single interface, with links to their respective pages. In practice, there are both \nlegal and technical impediments to cross-database communication which include digital locks, firewalls and the inability of search engines to recognize the data. [4]\nMethodology\nDoD2006 Content\nDoD2006 is an updated version of DoD. [5] It is a cross-database interface with 858 databases collected from Nucleic Acids Research \n[6], Bioinformatics [7], BMC Bioinformatics [8] and other \njournals [9] and 15 databases collected from In Silico Biology [10], Bioinformation [11] \njournals and Google Scholar [12] search. The current update with 873 databases against 719 reported in 2005 [5] represents additional \ndatabases that continue to grow despite the demise of a few. [13] A small number of inaccessible databases (MitoPD, ISSD [14]) reported in \n2005 are unchanged in DoD2006 so as to enable the users to know the type of database that once existed.\nThe complete list of databases is given in the website [15] and their categorization is given in Table 1. During the search, results are displayed in a separate window as this helps \nin further database scan without losing the home page. The screen-shot of nucleotide database is given in Figure 1. A hyperlink to \u2018Keyword help\u2019 lists keywords used to scan respective databases and the description of different links is given in \nour earlier publication. [5]\nFeatures of DoD2006\nNewly appended databases in DoD2006 are distinguished from the existing ones with a \u2018+\u2019 sign and inaccessible ones are recognized by a \u2018*\u2019 sign before the name. All databases in each category are given alphabetically. The appended databases \nare colored blue and the remaining in green color. Those who wish to have their database to be listed can contact the authors.\nDoD2006 provides an excellent and essential resource for molecular and computational biologists. Research through computational tools and software using databases has been recognized as a front-line research coupled with experimental design. Our \ndatabase suits to deliver the wide biological content through the cross-database interface. Updates to DoD shall be made on yearly basis.","keyphrases":["molecular biology","biological databases","database"],"prmu":["P","P","P"]}
{"id":"Photosynth_Res-3-1-2117338","title":"Chlorophylls, ligands and assembly of light-harvesting complexes in chloroplasts\n","text":"Chlorophyll (Chl) b serves an essential function in accumulation of light-harvesting complexes (LHCs) in plants. In this article, this role of Chl b is explored by considering the properties of Chls and the ligands with which they interact in the complexes. The overall properties of the Chls, not only their spectral features, are altered as consequences of chemical modifications on the periphery of the molecules. Important modifications are introduction of oxygen atoms at specific locations and reduction or desaturation of sidechains. These modifications influence formation of coordination bonds by which the central Mg atom, the Lewis acid, of Chl molecules interacts with amino acid sidechains, as the Lewis base, in proteins. Chl a is a versatile Lewis acid and interacts principally with imidazole groups but also with sidechain amides and water. The 7-formyl group on Chl b withdraws electron density toward the periphery of the molecule and consequently the positive Mg is less shielded by the molecular electron cloud than in Chl a. Chl b thus tends to form electrostatic bonds with Lewis bases with a fixed dipole, such as water and, in particular, peptide backbone carbonyl groups. The coordination bonds are enhanced by H-bonds between the protein and the 7-formyl group. These additional strong interactions with Chl b are necessary to achieve assembly of stable LHCs.\nIntroduction\nThe dramatic developmental transformation performed by the chloroplast has attracted broad interest over the past several decades (see Hoober and Argyroudi-Akoyunoglou 2004; Wise and Hoober 2006, for reviews). Although the organelle displays a variety of features among different organisms, as revealed by electron microscopy, its monophyletic origin by endosymbiosis of an ancient cyanobacterium has received increasingly strong support (Palmer 2003; Bhattacharya and Medlin 2004; Rodriguez-Ezpeleta et\u00a0al. 2005). Descendents of the primary endosymbiotic event branched into the glaucophytes, green algae, and plants, which contain chlorophyll (Chl) a and Chl b, and the red algae, which contain only Chl a. Tomitani et\u00a0al. (1999) provided evidence that the genes for chlorophyllide a oxygenase (CAO), the enzyme that catalyzes conversion of chlorophyllide (Chlide) a to Chlide b, also have a common origin. The evolutionary relationship of CAO in plants with the enzyme in the cyanobacterial prochlorophytes indicates that the original ancestor of plastids contained Chl b and that modern cyanobacteria, along with the red algae, lost this ability. Divergence from a secondary endosymbiotic event, also apparently singular, of a red alga gave rise to four major groups of chromophyte algae, the dinoflagellates, heterokonts, haptophytes, and cryptophytes, that contain Chl c as a major pigment in addition to Chl a (Bachvaroff et\u00a0al. 2005; Shalchian-Tabrizi et\u00a0al. 2006). This lineage suggests that the same fundamental mechanisms should underlie processes in chloroplast development in all plant species. Among these are expected to be the mechanisms that guide the interactions of Chls, proteins, and lipids during assembly of the thylakoid membrane. Even with the extensive studies already done on these processes, our understanding of many details of these mechanisms remains clouded.\nThis article will consider physicochemical factors that are likely fundamental in the assembly of light-harvesting complexes (LHCs) in the plastids of eukaryotic organisms. Particular emphasis is given to the properties of Chls a, b, and c and their interactions with ligands. Chls b and c occur essentially exclusively in LHCs. Whereas Chl a is ubiquitous, it alone is not sufficient for LHC assembly. Chl c seems to play the same role in LHC assembly in chromophyte algae as Chl b does in green algae and plants (Durnford et\u00a0al. 1999; De Martino et\u00a0al. 2000; Goss et\u00a0al. 2000). Thus a principal question is the step in LHC assembly for which synthesis of Chl b is required. It is likely that the plastids derived from the secondary endosymbiotic event solved this problem by finding a pathway to Chl c. The Chl-binding proteins in these organisms are evolutionarily related to those in green algae and plants (Schmitt et\u00a0al. 1994; Green and Durnford 1996). The relatively simple LHCs are well-defined structures and consequently are excellent systems to search for basic mechanisms. It is useful for illustration purposes to include Chl d, the most recently characterized member of the Chl family (Miyashita et\u00a0al. 1997; Akiyama et\u00a0al. 2002) found as the major Chl in the cyanobacterium Acaryochloris marina, which functions in core complexes in an analogous fashion to Chl a. Bacteriochlorophyll (BChl) a is also briefly mentioned for comparison.\nSummary of differences in chlorophylls\nThe spectral differences between the species of Chls in chloroplasts expand the ability of photosynthetic organisms to harvest light. However, the spectral range of Chl a alone is broadened by various micro-environments within complexes with proteins (Nishigaki et\u00a0al. 2001; Croce et\u00a0al. 2002; Linnanto et\u00a0al. 2006), which argues against spectral differences per se as the raison d\u2019\u00eatre for occurrence of the \u201csecondary\u201d Chls, in particular, Chls b and c. Overall properties, not only their spectral features, are altered as consequences of chemical modifications on the periphery of the molecules. Important modifications are desaturation of sidechains and introduction of oxygen atoms at specific locations. Oxygen is the most electronegative atom commonly found in biological systems and exerts significant effects on the electronic distribution in the Chl molecule.\nWe previously (Hoober and Eggink 1999; Eggink et\u00a0al. 2001, 2004; Chen et\u00a0al. 2005) proposed that modifications at the periphery of the Chl molecule influence the coordination chemistry of the central Mg atom and that this effect plays an important, if not major, role in the interaction of Chl b with LHC apoproteins (LHCPs) and thus in the assembly of LHCs. In particular, we proposed that, as a Lewis acid, the Mg atom in Chl b favors axial coordination bonds with harder Lewis bases than does Chl a. This proposal was supported experimentally by direct measurement of equilibrium constants of various tetrapyrrole derivatives with specific ligands (Tamiaki et\u00a0al. 1998). In an unbiased chemical context, Chls a and b should then prefer different ligands. Recent evidence demonstrated, however, that Chl a interacts with a broad range of ligands, from the imidazole group of histidine to water. Chl b, on the other hand, is found only with ligands containing an oxygen atom.\nChlorophylls a and d\nConversion of 3,8-divinyl-Chl a to 3-monovinyl-Chl a by reduction of the 8-vinyl group to an ethyl group is the final step in Chl a biosynthesis and yields the predominant form of Chl a (Nagata et\u00a0al. 2005). As a result, Chl a has electron-donating methyl and ethyl groups at positions 7 and 8, respectively (Fig.\u00a01). Along with reduction of the C17\u2013C18 double bond to a single bond, which converts the porphyrin precursor protochlorophyllide (Pchlide) to the chlorin ring system, these groups impose an electron density, from opposite sides of the molecule along the X axis, on the pyrrole nitrogens, which partially shields the positive charge of the central Mg atom. In addition, the 3-vinyl and 131-keto groups exert weak electron withdrawing effects on opposite ends of the Y axis.\nFig.\u00a01Structures of the major Chls. Except for the oxidation of the 7-methyl group in Chl a to the formyl group in Chl b, Chls a and b are identical. Chl d contains a formyl group at position 3. Chls a, b, and d include the 20-carbon isoprene alcohol, phytol (Ph), esterified to the carboxyl group at position 173. This carboxyl group remains unesterified in Chl c, which also contains double bonds in the sidechain between positions 171 and 172 and in the macrocycle between carbons 17 and 18. These additional double bonds extend conjugation of the macrocyclic \u03c0 system to the free carboxyl group. Chl c species differ at positions 7 and 8; c1: 7, \u2013CH3, 8, \u2013C2H5; c2: 7, \u2013CH3, 8, \u2013C2H3; c3: 7, \u2013COOCH3, 8, \u2013C2H3 (shown in figure)\nThe geometrical coordinates of the molecular framework for Chl a are shown in Fig.\u00a01. The primary X axis transects the molecule from the position of C17 to C7. The Y axis transects the molecule from C2 to C12. The experimentally determined, functional Qy transition-moment direction is a vector 70\u00b0 clockwise from the X axis (Fragata et\u00a0al. 1988; Simonetto et\u00a0al. 1999; Sundholm 2003; Cai et\u00a0al. 2006). This displacement from the geometrical Y axis places the functional Qy vector from near C1 to near C11. The functional Qx vector is within a few degrees of the geometrical X axis.\nChl d is synthesized by oxidation of the 3-vinyl group to a formyl group, whose electron-withdrawing character further extends the Qy vector but should also cause the transition direction to align more closely with the molecular Y axis. As a result, the lowest energy absorption band shifts from 665\u00a0nm (in methanol) for Chl a to a longer wavelength (lower energy) maximum of 697\u00a0nm for Chl d and increases the dipole strength, which is proportional to the molar absorption coefficient. The transition dipole is strengthened further in BChl a, which has an electron-withdrawing acetyl group on C3 and a single bond between C7 and C8. The Qy absorption maximum is shifted to 772\u00a0nm (in methanol), with a dipole of 7.2 Debye (D) (at a refractive index for the environment of 1.35) (Knox and Spring 2003). The electronic distribution in BChl a is more symmetrically aligned along the Y axis, with an elliptical electron density, and the Qy transition-moment direction is essentially perpendicular to the X axis, i.e., from C2 to C12 (Sundholm 2003).\nChlorophyll b\nSynthesis of Chl b involves incorporation of the electronegative oxygen atom to generate the 7-formyl group, which, as an aromatic aldehyde, is expected to have a dipole moment for the group of approximately 3.0\u00a0D (Desyatnyk et\u00a0al. 2005). The oxygen provides a significant pull on electrons away from the core of the molecule along the X axis, which weakens the Qy dipole strength of the molecule from 5.33\u00a0D for Chl a to 4.41\u00a0D in Chl b (at a refractive index for the environment of 1.35) (Knox and Spring 2003). Since the dipole strength determines the magnitude of the absorption coefficients, among other properties, the absorption coefficient of Chl b is only 50\u201362% (depending on solvent) of the Qy absorption peak of Chl a and is shifted to higher energy, with a maximum at 652\u00a0nm (in methanol). The Qy transition-moment direction is displaced further from the Y axis than in Chl a and is at an angle of only 61\u00b0 clockwise from the X axis (Simonetto et\u00a0al. 1999), described as a transect from near C20 to near C10.\nIntuitively, the Qx transition moment of Chl b should be stronger than that of Chl a. Computational analysis of molecular orbitals suggest that the Qx oscillator strength of Chl b is 2-fold greater than for Chl a, with a maximum at 538\u00a0nm (Linnanto and Korppi-Tommola 2004). The magnetic circular dichroism (MCD) spectrum of Chl b contains a weak negative transition at 540\u00a0nm and an intense negative transition at 625\u00a0nm (Frackowiak et\u00a0al. 1987). From the argument that a stronger molecular dipole is consistent with a longer wavelength absorption maximum, the longer-wavelength negative transition in the MCD spectrum of Chl b may correspond to the Qx transition.\nThe molecular orbital calculations suggest that, with weaker Qy and stronger Qx transitions, the electronic distribution in the Chl b molecule is essentially circular (Hoff and Amesz 1991). The lessened electron density around the pyrrole nitrogen atoms lowers the pK values for these atoms by about two pH units (Phillips 1963; Smith KM 1975). The withdrawal of electron density from the pyrrole nitrogens results in less shielding of the Mg atom of Chl b and allows it to more strongly express its positive point charge. Molecular orbital calculations give the Mg in Chl an atomic charge with a value of +0.7 to +1.3 (Linnanto and Korppi-Tommola 2004).\nChlorophyll c\nChl c is found with Chl a in chromophyte algae, where it functions as a light-harvesting pigment. Whereas most of these algae do not contain Chl b, some Prasinophycean algae, such as Montoniella squamata, accumulate a Chl c, 3,8-divinyl-Pchlide, along with Chls a and b (Schmitt et\u00a0al. 1994; Green and Durnford 1996). The red alga that contributed its plastid in the secondary endosymbiotic event apparently lacked Chl c. Ability to make Chl c was the solution these organisms found to solve the problem that was accomplished in chlorophytes with Chl b. As shown in Fig.\u00a01, the structure of Chl c3 has several unique features. The c-type Chls characteristically retain the C17\u2013C18 double bond that occurs in the porphyrin precursor, Pchlide a. In addition, a trans double bond is introduced between C171 and C172 in the sidechain, which extends conjugation of the ring \u03c0 system to the usually unesterified, electronegative C173 carboxyl group. Conversion of the propionate sidechain of Pchlide a to the acrylate sidechain of Chl c inhibits the ability of NADPH:Pchlide oxidoreductase to reduce the C17\u2013C18 double bond as occurs in Chl a synthesis (Helfrich et\u00a0al. 2003), which suggests that sidechain desaturation occurs prior to potential interaction of Pchlide a with the oxidoreductase. Thus Chl c retains the porphyrin ring system. The three major sub-types of Chl c (c1, c2 and c3) occur as the result of oxidation of the C7 and\/or C8 substituents. The C7 methyl group is modified to a methylcarboxylate (\u2013COOCH3) in Chl c3 and the C8 vinyl group remains unreduced (Porra 1997). These remarkable modifications all lie on the X axis of the molecule. Absorbance spectra of the series (c1, c2 and c3) suggest that the functional Qx transition moment increasingly dominates the long-wavelength absorption peaks (Jeffrey and Wright 1987; Helfrich et\u00a0al. 2003), further reduces the absorption coefficient and blue-shifts the Qy absorbance maximum to 630\u00a0nm for Chl c1 (in acetone). Additional modifications, such as esterification of the acrylate sidechain with galactosyl diacylglycerol, increase the number of minor forms of Chl c (Garrido et\u00a0al. 2000).\nA summary of ligands of chlorophylls in light-harvesting complexes\nCoordination bonds are formed between Lewis acids and bases. A Lewis acid has an unfilled orbital that can accept a pair of electrons. A Lewis base (ligand) has a pair of unshared electrons that are available for donation to the Lewis acid to form a donor-acceptor complex. Lewis acids and bases are characterized as \u201csoft\u201d or \u201chard\u201d according to their chemical properties (Jensen 1978). Soft species tend to bond by short-range orbital interactions, while hard species interact primarily by electrostatic forces.\nThe central Mg atom of Chl molecules, as the Lewis acid, interacts with proteins by formation of coordination bonds with an amino acid sidechain as the Lewis base. Compression of the electron cloud toward the Y axis of the Chl a molecule, as when the C17\u2013C18 double-bond and C8 vinyl group are reduced, tends to shield the Mg atom and effectively reduces the electronegativity of the metal. This results in weaker interaction with the negative end of a fixed dipole or even repulsion of negatively charged groups. In contrast, in Chl b the Mg atom is less shielded and more strongly expresses its positive charge. Electron density in Chl b is also pulled outward by H-bonds between the 7-formyl group and other structures, which further enhances the Qx transition moment. Substituting the central Mg in BChl a with other metals (e.g., Ni) of greater electronegativity strongly influences the Qx but not the Qy transition energies of the tetrapyrrole molecule (Hartwich et\u00a0al. 1998). In Chls a and b the metal is the same, but the argument can be applied in reverse, in which substitution of peripheral groups on the Qx axis alters the environment of the central Mg ion and thus its effective electronegativity.\nIf H+ is considered as a Lewis acid, the availability of electrons in a Lewis base should be reflected in its pK value (Jensen 1978) (pK values vary dramatically from those obtained in aqueous media when the ionizable group is located in a nonpolar micro-environment (Mehler er al. 2002)). The electron pair available on an amine nitrogen atom binds H+ strongly (pK \u223c9). Although the nonpolar micro-environment within a protein molecule may lower the pK of an amino group of lysine (Gunner et\u00a0al. 2000, 2006), the amine is usually protonated under physiological conditions, thus positively charged, and the electron pair is not available for coordination with Chl. Lysine amino groups are not ligands in membrane-spanning regions of Chl-binding proteins (Balaban et\u00a0al. 2002; Ferreira et\u00a0al. 2004; Liu et\u00a0al. 2004). The strength of other amino acid sidechains as Lewis bases should then decrease in the order of decreasing pK, e.g., imidazole (pK 7)\u00a0>\u00a0carboxylate (pK 4\u20135)\u00a0>\u00a0peptide backbone amide (pK \u22120.42)\u00a0>\u00a0sidechain amide (pK \u22120.62)\u00a0>\u00a0water (pK \u22121.74). However, formation of Chl\u2013ligand complexes does not follow this pattern. Chl a interacts as expected with imidazole, its predominant partner, but also with amide sidechains and water. Chl b does not interact significantly with imidazole, whereas the peptide backbone carbonyl group and water are favorable ligands. Orbital interactions may play a greater role in coordination bonds with ligands containing accessible electron pairs (i.e., high pK values) such as imidazole, whereas electrostatic interactions are more likely with ligands having a low pK value such as carbonyl groups. This comparison suggests that interactions of the Chls with ligands involve more than simply the availability of non-bonded electrons on the ligand. In addition to differences in the properties of the Lewis bases, the more exposed positively charged Mg ion in Chl b is more electronegative and acts as a harder Lewis acid than the metal in Chl a. Thus Chl b should favor electrostatic bonds with groups containing a strong, fixed dipole.\nWith the exception of the imidazole group, ligands to Chl contain oxygen. Lewis bases that contain oxygen atoms are polar, with a \u201cfixed\u201d dipole, and tend to be \u201chard\u201d Lewis bases. The structures listed in Table\u00a01, which are common or potential ligands of Chl, are ordered according to increasing dipole moment of the monomeric molecule, as calculated by ab initio methods, to emphasize the importance of this parameter. When these molecules interact by H-bonding with other molecules, as in a solution or a crystal structure, the dipole moments increase (Spackman 1992; Abramov et\u00a0al. 1999; Whitfield et\u00a0al. 2006). This effect is unlikely to be significant when ligands are isolated within the nonpolar environment in a membrane, although the dipole of a \u201cpolarizable\u201d ligand is affected by its interaction with the Mg of Chl. The dipole moment of an alcohol is too weak to effectively compete with water and thus hydroxyl groups are not common ligands. Each productive ligand is discussed in more detail in the following.\nTable\u00a01Values of dipole moments selected from the literature for potential ligands of ChlLigandpKaSolution Dipole (D)ab initio Dipole (D)Carboxyl group\u00a0\u00a0\u00a0\u00a0Formic acid3\u201351.41 (University of Southern Maine website 2006)1.52 (Dudev et\u00a0al. 1999)Alcohol\u223c \u22122 (Herschlag and Jencks 1989; Smith and March 2001)1.70 (University of Southern Maine website 2006)1.94 (Dudev et\u00a0al. 1999)Water\u22121.74 (Herschlag and Jencks 1989; Smith and March 2001)2.70 (Gregory et\u00a0al. 1997)1.868 (Gregory et\u00a0al. 1997)1.855 (Dyke and Muenter 1973)Imidazole6\u201373.96 (Spackman 1992)3.66 (Spackman 1992)4.80b (Spackman 1992)Amide\u00a0\u00a0\u00a0\u00a0Formamide3.84 (Spackman 1992)4.13 (Dudev et\u00a0al. 1999)4.83b (Spackman 1992)3.72 (Spackman 1992)\u00a0\u00a0\u00a0\u00a0Acetamide\u22120.62 (Grant et\u00a0al. 1983)3.87 (Spackman 1992)3.69 (Spackman 1992)4.95b (Spackman 1992)\u00a0\u00a0\u00a0\u00a0Sidechain3.46 (Antoine et\u00a0al. 2002)\u00a0\u00a0\u00a0\u00a0Peptide bond4.2 (Gunner et\u00a0al. 2000)\u00a0\u00a0\u00a0\u00a0N-Methylacetamide\u22120.42 (Grant et\u00a0al. 1983)4.2 (Whitfield et\u00a0al. 2006)3.73 (Whitfield et\u00a0al. 2006)\u223c6 (Whitfield et\u00a0al. 2006)Urea0.053 (Grant et\u00a0al. 1983)5.15 (Abramov et\u00a0al. 1999)4.56 (Spackman 1992)7.04b (Abramov et\u00a0al. 1999)a\u00a0pK for the conjugate acid reaction: AH(+) \u2194 A(\u2212)\u00a0+\u00a0H+b\u00a0Crystal formReferences are indicated in parenthesis\nWater\nWater is the Lewis base that seems to be a \u201cregulatory\u201d ligand because of its strong interaction with Chl b and its weaker interaction with Chl a (Ballschmitter et\u00a0al. 1969). In solution, where water is fully H-bonded (dielectric constant, 81), its dipole moment is 2.70 D (Table\u00a01); in ice, this value is 3.09\u00a0D (Batista et\u00a0al. 1998). In an environment in which the dielectric constant is 2\u20134, as occurs in a protein or membrane, the dipole of a water molecule is likely nearer to that in the gas phase, 1.85\u00a0D (Dyke and Muenter 1973). However, when associated with a positive charge such as the Mg in Chl, the dipole moment is probably near the H-bonded value. The charge at the negative end of the dipole of water provides an electrostatic contribution to the interaction.\nFor a functional group in a protein to form a coordination bond with the Mg atom in Chl, a water ligand, which is likely present throughout the latter steps in the biosynthetic pathway from Mg-protoporphyrin IX onward, must be displaced. It is interesting that three Chl b molecules retain water as a ligand and connect with the protein via a water bridge (see below).\nImidazole \nThe imidazole sidechain of histidine in the unprotonated form has an unshared pair of electrons on N(3) (designated as N\u03b52 by Standfuss et\u00a0al. 2005). H+ binds to the electron pair with a pK value that lies within the range of 5\u20138, depending upon the environment. Nonpolar environments stabilize the unprotonated form, and thus the electron-rich imidazole group is available for coordination with the Mg of Chl a within a membrane. The dipole moment for imidazole is between 3.66\u00a0D (gas phase) and 4.80\u00a0D (crystal structure), with the predominant contribution to the dipole provided by the N(1)-H bond (Spackman 1992). When the N(1) hydrogen is replaced with the electron-donating methyl group, the resulting coordination bond at N(3) is stronger (van Gammeren et\u00a0al. 2004). In aqueous solution, the dipole moment is enhanced to a value of 3.96\u00a0D by H-bonding (Table\u00a01). Both N atoms have a small negative charge, and the electron density is distributed nearly symmetrically (Fig.\u00a02). The aromatic character of imidazole allows the dipole to reorganize in response to interaction with another structure.\nFig.\u00a02(A) The structure of the imidazole group of histidine and (B) its electronic charge density, determined by X-ray diffraction at 103\u00a0K for the projection in (A) (adapted from Epstein et\u00a0al. 1982). In (A), R\u00a0=\u00a0remainder of the histidine molecule\nHis120, at the lumenal end of helix-2 in LHCII (see Fig.\u00a03), is not a ligand to Chl a, possibly because of its exposure to the thylakoid lumen where competition with water is greater than within the membrane. Also, the imidazole group may be protonated at the pH of the lumen during active photosynthesis, estimated to be near pH 5 (Kramer et\u00a0al. 1999; Sacksteder et\u00a0al. 2000). His212, a ligand to Chl a, is also near the lumenal surface of the membrane but is likely shielded from the aqueous lumen by helix-4 of the LHC protein.\nFig.\u00a03Model of the association of Chls with Lhcb1. The arrangement of the protein in thylakoid membranes is illustrated according to Green and Durnford (1996). The symbols designating the chlorin rings of the six Chl b molecules are filled (green). A water ligand for four Chl molecules is indicated by a central blue dot. The Chl a molecules are numbered 1\u20138 and the Chl b molecules 9\u201314, as designated by Standfuss et\u00a0al. (2005)\nThe imidazole group provides a good example of the attractive\/repulsive forces that limit the strength of the coordination bond. A repulsive force should exist between the \u03c0 clouds of Chl and the ligand. However, the pair of electrons on N3 is an attractive force that satisfies the needs of the Lewis acid, Mg. The concept of electronegativity equalization (Noy et\u00a0al. 2000) suggests that a partial charge (about 0.3e) is transferred from imidazole to the metal ion upon coordination (analyzed with Ni-BChl a). This shift in electron density should reduce the role of the dipole in the coordination bond and generate a partial positive charge on the ligand that is accommodated by the electron cloud of the conjugated \u03c0 system of Chl a. In contrast, the more exposed positive charge on Mg in Chl b likely repels the positive charge that develops on the ligand. Thus the attractive force is limited by the repulsive force created between Mg and the induced positive charge on the ligand.\nCarboxyl group\nTwo ligands for Chl a in LHCII are charge-compensated ion-pairs formed by electrostatic interaction between the sidechain carboxylate of glutamic acid and the guanidinium group of arginine. Although at pH 7 the carboxyl group has a negative charge and multiple pairs of unbonded electrons, these electrons are distributed between the two electronegative oxygen atoms in a resonance structure and are less available for protonation than in the imidazole group. This property is reflected in the higher-proton concentration (pH 3\u20135) required to protonate the carboxylate in solution. In proteins, its pK varies from a low of about 2, to a high of nearly 9 (Gunner et\u00a0al. 2000; Georgescu et\u00a0al. 2002; Laurents et\u00a0al. 2003; Li et\u00a0al. 2004). The higher pK values occur when the protonated carboxyl group is stabilized in a nonpolar environment (Mehler et\u00a0al. 2002). The protonated carboxyl group has a relatively low ab initio dipole moment of 1.52\u00a0D (Table\u00a01). When ionized, the negative charge is likely repelled by the \u03c0 electron cloud of Chl a. As the LHC apoprotein folds, the approach of a positively charged guanidinium group attracts electron density from the carboxylate group and the ion-pair is stabilized by the nonpolar environment. The resulting glutamate in this ion-pair is a soft Lewis base, with a dipole moment probably near that of a protonated carboxyl group.\nThe dipole moment of the ion-pair is possibly too low to displace a water molecule from Chl b. With its electron cloud pulled away from the central Mg (\u223c+1 charge), Chl b is expected to coordinate more readily with a carboxyl group (\u223c\u22121 charge) and thus form a bond with largely electrostatic character. In LHCII, Chl b is coordinated with the sidechain of Glu139 near the stromal end of helix-2, which nevertheless is sufficiently near Arg142 for at least partial charge-compensation (Standfuss et\u00a0al. 2005).\nThe ability of Chls to bind to imidazole and glutamate\/arginine ion-pair ligands was assayed experimentally by interaction with a synthetic peptide that mimicked helix-1 in LHCPs. Chls a and d bound with nearly equal affinity, assayed by F\u00f6rster resonance energy transfer from a tryptophan residue next to arginine (Chen et\u00a0al. 2005). In contrast, Chls b and c did not interact significantly with the peptide. A theoretical analysis of the interaction of the Chls with the peptide (Chen and Cai 2007) strongly supports the experimental data and indicates that bonding of Chls b and c with these ligands is thermodynamically unfavorable (Table\u00a02). In the presence of water, however, complex formation with Chl b is more favorable, which, as shown by molecular modeling, is the result of a water molecule bridging Chl b and the ligand (Chen and Cai 2007).\nTable\u00a02Heat of formation of Chl-peptide complexes calculated by molecular modelingSpeciesHeat of formation (kcal\/mol) In vacuumIn waterChl a\u2212706\u2212870Chl b770\u2212133Chl c11,001160Chl c21,115310Chl c387784Chl d\u2212806\u2212949BChl a\u2212892\u22121047The parametric method 5 was used to calculate the association of each of the species of Chl with a 16-mer maquette of helix-1 of Lhcb1 (Eggink and Hoober 2000) as described by Chen and Cai (2007). The more negative the value, the more thermodynamically stable the complex\nAmide group\nThe pK of an amide is \u22120.62 (Grant et\u00a0al. 1983), an indication that electrons on the oxygen or nitrogen are not readily available for bonding with H+. However, the group exhibits a relatively strong dipole, with the negative end on the oxygen atom. Sidechain amides in proteins have a dipole moment of 3.46 D (Table\u00a01). The dipole moments of the model compounds, formamide and acetamide, are about 3.8\u00a0D in solution. The dipole is sufficiently strong to displace a coordinated water molecule from Chl a and should also allow effective competition with water for Chl b within the environment of a membrane. In reconstituted complexes, several sites, including Gln197, have mixed occupancy (Bassi et\u00a0al. 1999; Remelli et\u00a0al. 1999), which suggests competition during folding. Yet the sidechain amide groups of Asn183 and Gln197 are ligands for only Chl a in LHCII in\u00a0vivo, which suggests that Chl a is more abundant during folding and competes effectively by mass action.\nPeptide bond\nBackbone amides are usually H-bonded within helical or \u03b2-sheet structures in proteins and therefore unavailable for interaction with Chls. However, proline residues occur at conserved regions within Chl-binding proteins, particularly in the N-terminal region of LHCPs (Jansson 1999), which preclude formation of H-bonds to nearby backbone carbonyl groups. The pK value for the model compound N-methylacetamide, an analog of the peptide bond, is \u22120.42 (Grant et\u00a0al. 1983), slightly more basic than a sidechain amide group. The electron-donating methyl group bonded to the nitrogen provides a larger \u201cpool\u201d of electrons for the carbonyl oxygen to draw from, which is reflected in the slightly higher pK value than of the sidechain amide group. This effect also increases the dipole moment of the carbonyl group. N-Methylacetamide has a calculated dipole of 3.73\u00a0D in the gas phase and a monomeric dipole moment of 4.2\u00a0D in liquid, which is the same as the calculated dipole moment of the peptide bond in proteins (Table\u00a01). The ab initio dipole moment of the fully H-bonded N-methylacetamide in liquid is calculated to have an average of 6\u00a0D, with a spread from 4 to 8\u00a0D (Whitfield et\u00a0al. 2006). Urea has an even stronger dipole moment than N-methylacetamide and also illustrates the effect of H-bonding. Its molecular dipole moment is 5.15\u00a0D and is increased to 7.04\u00a0D in the crystal form (Abramov et\u00a0al. 1999). An electrophilic center such as the Mg atom of Chl should have a similar effect to that of H-bonding. As expected from the strong dipole moment of urea, the negative point charge on its carbonyl oxygen may be repulsed by the electron density enclosing the Mg of Chl a, which prevents it from being an effective ligand. Urea does not seem to compete with imidazole or the glutamate\/arginine ion-pair, the most favorable ligands of Chl a (Eggink and Hoober 2000). However, it should effectively compete with other ligands for binding to Chl b.\nA peptide bond carbonyl in association with a polarizing, positively charged Mg should have a dipole moment at least as large as H-bonded N-methylacetamide and therefore should form a strong electrostatic bond with Chl b. A free backbone carbonyl group in helix-1 in Lhcb1 occurs at Gly78, because of nearby Pro82, within the interior of the membrane. This group should displace water from a Chl molecule but is probably sterically hindered from coordinating directly with the Mg atom by the adjacent, bulky amino acid sidechains of leucine and phenylalanine (see Fig.\u00a03). As a result, this ligand is bridged by a water molecule to Chl a6 in LHCII (Standfuss et\u00a0al. 2005). That this position is occupied by Chl a rather than Chl b may be determined by the availability and order of binding of the Chls during assembly of the complex. Repulsion of the strong dipole by the electron cloud of Chl a possibly limits its direct interaction with backbone carbonyl groups.\nOf particular interest is the finding that backbone carbonyls of proline residues provide ligands to Chl a in a water-soluble Chl-binding protein from Lepidium virginicum (Horigome et\u00a0al. 2007). In the complex, four Chl a molecules are bound in a solvent-excluded pocket at the interfaces of the tetrameric protein. This observation is evidence that Chl a can fulfill the full range of ligand coordination with sufficient support from the local environment. However, this arrangement is quite different from the interaction of monomeric Chls with LHCPs during LHC assembly.\nPhosphatidyl glycerol\nAn oxygen of the phosphodiester linkage in a phosphatidyl glycerol molecule serves as the ligand in LHCII to Chl a7, which resides near the stromal surface of the thylakoid membrane (Liu et\u00a0al. 2004). This rare ligand is also found in photosystem (PS) I (Jordan et\u00a0al. 2001). The side of the phosphodiester group opposite the Chl a molecule is H-bonded to sidechains of tyrosine and lysine residues (Liu et\u00a0al. 2004), which probably reduces the electron density on the ligand oxygen. Approach of the negative end of a strong dipole toward the Mg in Chl a should again be hindered by the electron density surrounding the metal.\nImplications for assembly of light-harvesting complexes\nThe concept of ligand preference was developed largely because Chl b is found only in LHCs, at specific sites, and with rare exceptions is not found in core complexes. X-ray diffraction studies of crystallized reaction centers (Fromme et\u00a0al. 2001; Jordan et\u00a0al. 2001; Loll et\u00a0al. 2005) and LHCII (Liu et\u00a0al. 2004; Standfuss et\u00a0al. 2005) revealed unambiguous Chl-ligand pairs, without mixed occupancy. Whereas a nitrogen atom of the imidazole group of histidine is the most common ligand, sidechain amide groups, water molecules and even a few carboxylate groups occur as ligands of Chl a in reaction centers of PS I and PS II (Jordan et\u00a0al. 2001; Balaban 2005; Balaban et\u00a0al. 2002; Oba and Tamiaki 2002, 2005; Ferreira et\u00a0al. 2004).\nAlthough Chl b expresses more strongly a positive charge on the central Mg than Chl a, and consequently interacts electrostatically more readily with hard Lewis bases, the above analysis indicates that Chl a can also bind to these ligands. However, a stable LHCII cannot be reconstituted in\u00a0vitro with only Chl a. Conversely, stable LHCII was reconstituted with only Chl b, and the number of Chl b molecules (13.5) was the same as when both Chls were present (13.7) (Kleima et\u00a0al. 1999; Reinsberg et\u00a0al. 2001; Schmid et\u00a0al. 2001). Thus sites normally occupied by Chl a can be occupied by Chl b. Whether Chl b molecules indeed interact directly with ligands of Chl a or are bridged by water molecules remains to be determined. As shown in Table\u00a02, calculated \u0394H of formation of complexes with a synthetic peptide containing the glutamate\/arginine ion-pair and a histidine residue indicated that complex formation with Chl b is thermodynamically unfavorable. However, insertion of a water molecule to bridge Chl b and the ligand dramatically increased thermodynamic stability (Chen and Cai 2007).\nThe overlap of ligand characteristics is substantial and too much may have been made about ligand selectivity with Chls. Therefore, unambiguous occupancy of specific binding sites as found in\u00a0vivo must involve more than these properties. Two recent publications shed light on how the LHCII complex is assembled in\u00a0vivo. Reinbothe et\u00a0al. (2006) showed that LHCPs are not imported at a detectable rate into plastids purified from a Chl b-less mutant of Arabidopsisthaliana. These authors also confirmed the localization of CAO on the inner membrane of the chloroplast envelope as reported by Eggink et\u00a0al. (2004). An alternate site for CAO activity was achieved by Hirashima et\u00a0al. (2006), who transformed the Chl b-less mutant of A. thaliana ch1-1 with the gene for CAO from the cyanobacterium Prochlorothrix hollandica to achieve active CAO on thylakoid membranes. In these plants, the higher Chl b content resulted in a Chl a\/b ratio that approached 1, much lower than the ratio of 3\u20134 in wild-type plants. In LHCII, the ratio was 0.8 in contrast to 1.3 in the complex from wild-type plants. Chl b was recovered in purified PSI and PSII core complexes, which normally lack Chl b, as well as in LHCs. Because of the widespread distribution of Chl b in the transformed plants, Hirashima et\u00a0al. (2006) concluded that the restrictive distribution of the Chls in wild-type plants is not the result of discriminatory binding affinities of Chl a and Chl b to ligands. Because the P. hollandica CAO on thylakoid membranes led to a widespread distribution of Chl b, the conclusion emerges that active CAO only on the envelope of chloroplasts leads to restriction of Chl b to LHCs. To achieve incorporation of Chl b selectively into LHCs, as found in wild-type plants, assembly of LHCs should therefore occur during import at the level of the envelope.\nIn Chl b-less mutants of higher plants, only a few of the apoproteins for LHCI and LHCII accumulate in the organelle in\u00a0vivo (Kr\u00f3l et\u00a0al. 1995; Bossmann et\u00a0al. 1997; Espineda et\u00a0al. 1999). This observation has traditionally been interpreted as an indication that the proteins are rapidly degraded upon entry into the chloroplast stroma unless Chl b is present to allow stable integration into the thylakoid membrane. However, as noted above, chloroplasts from a Chl b-less mutant of A. thaliana lacked the ability to import LHCPs. Accumulation of LHCPs in\u00a0vivo into the plastid of the alga Chlamydomonas reinhardtii was markedly reduced in the Chl b-less strain, cbn1-113 (Park and Hoober, 1997). Mature-sized proteins were detected in the cytosol, which indicated that import was aborted (White et\u00a0al. 1996; Park and Hoober 1997). In the absence of Chl, a condition achieved because the mutant strain was unable to synthesize Chl in the dark, accumulation of LHCPs in the plastid was not detected. However, the proteins were synthesized at the same rate as in cells greening in the light but accumulated in the cytosol and vacuoles. These results point to a requirement of Chl, and particularly of Chl b, for import and\/or retention of LHCPs in the organelle. Even in wild-type cells, excess LHCPs were shunted to vacuoles when the rate of Chl synthesis was insufficient to accommodate the rate of synthesis of the Chl-binding proteins (White et\u00a0al. 1996).\nModel of LHCII assembly\nFolding of a thylakoid membrane protein of cytosolic origin is a complex process, made more so by the environmental sensing of domains as the protein is threaded through the translocon in the chloroplast envelope. Popot and Engelman (2000) and Bowie (2005) described two steps of the process of folding of a membrane protein. The first involves achieving the correct location and topology by the initially inserted segments. Second is the folding and condensation of the protein from this starting point. The evidence indicates that LHCP precursors achieve location and topology as they are guided by the transit sequence through translocons on the outer and inner envelope membranes. The transit sequence is removed soon after the N-terminal domain gains access to the stroma (Soll and Schleiff 2004; Vothknecht and Soll 2006). Membrane-spanning, nonpolar sequences that serve as stop-transfer domains are minimally 14\u201316 amino acids in length (Davis and Model 1985; Adams and Rose 1985; Popot and Engelman 2000). In this respect, assembly of LHCs presents an interesting problem. Inspection of helix-1 of most LHCPs reveals that the length of the nonpolar sequence in the first membrane-spanning domain is only 10\u201312 amino acids long, which is on the short side of a significant stop-transfer signal. The nonpolar sequence is within the lagging half of the span, terminated by several charged amino acids (Green and Durnford 1996; Jansson 1999). Binding of Chl to amino acids in the leading half of the helix, which is untypically polar and charged for a membrane-spanning segment of a protein (see Fig.\u00a03), should increase the probability that this domain remains in the membrane.\nIt seems plausible to consider that as the N-terminal domain of LHCPs traverses the envelope inner membrane, sidechains of glutamate and arginine within the conserved sequence \u2013EVIHSR\u2013 in helix-1 form a looped ion-pair ligand for Chl a. The histidine residue provides a second ligand for Chl a, as described by Eggink and Hoober (2000). Kohorn (1990) showed that mutation of this sequence, to replace histidine with alanine, eliminated the ability of the chloroplast to import a LHCP precursor. Binding of Chl to these sidechains may allow this polar sequence to diffuse more readily into the nonpolar phase of the membrane. However, these interactions are insufficient to retain the protein in the membrane in the absence of Chl b. As shown by Chen et\u00a0al. (2005), Chls b and c bind poorly to imidazole or glutamate\/arginine ion pairs in\u00a0vitro.\nA possible ligand for the Chl b molecule that is necessary for retention of the protein in the plastid was suggested by the crystal structure of LHCII. Several backbone carbonyls near the N-terminus are precluded from H-bonding and formation of an \u03b1-helix because of the richness of proline residues in this region of the LHCP. The carbonyl of tyrosine (Tyr24 in spinach Lhcb1) resides three positions distant in the amino acid sequence from a proline residue and is thus free to form a coordination bond with Chl b (Liu et\u00a0al. 2004; Standfuss et\u00a0al. 2005). The unusual abundance of proline in the N-terminal domain also extends to an iron-deficiency-induced (Tidi) protein, a homolog of the light-harvesting Chl a\/b proteins, in Dunaliella, which increases the probability of interaction with Chl b during this stress condition that leads to chlorosis (Varsano et\u00a0al. 2006). The strong electrostatic bond formed by further polarization of the carbonyl dipole through interaction with Chl b may be essential to anchor a LHCP in the envelope membrane sufficiently long for the remainder of the protein to be transported from the cytosol to complete assembly (Fig.\u00a04).\nFig.\u00a04Model of LHCII assembly in the chloroplast envelope and the proposed role of Chl b. Several proposed intermediates are shown in the sequence, left to right. After synthesis in the cytosol, a LHCP precursor is imported sufficiently into the chloroplast stroma for removal of the transit sequence from the N-terminus and for the first membrane-spanning region to engage the inner membrane. Chl a (dark green rectangles) binds to ligands in the motif provided by the ion-pair of the sidechains of glutamate and arginine and the imidazole group of histidine (dotted line, a). However, binding to these sites is not sufficient to retain the protein in the envelope. Without Chl b the protein slips back into the cytosol for transfer to vacuoles and subsequent degradation. Chl b (light green rectangles) forms a strong coordination bond with the peptide bond carbonyl of Tyr24, near the N-terminus, and provides an additional hold on the protein (solid line, a\u00a0+\u00a0b). Along with the Chl a molecules that bind to the motifs in membrane-spanning helix 1, Chl b binds to Try24 and the peptide carbonyl of Val119 at the lumenal end of helix-2. These Chls retain the protein in the membrane sufficiently long for the remainder of the protein, including the conserved motif in membrane-spanning helix-3, to enter the membrane, bind additional Chl and xanthophylls molecules, and complete assembly (LHC). Other proteins in the membrane and stroma apparently assist assembly of the complete complex (see text)\nAs the remainder of LHCP is transferred through the translocon, a second Chl b possibly coordinates with the backbone carbonyl of Val119 (Liu et\u00a0al. 2004; Standfuss et\u00a0al. 2005). Helices 2 and 3 are then transferred through the membrane, which would complete the first step described above by Bowie (2005). The rather weak hydrophobic character of helix-2, and the short nonpolar sequence (again the lagging half) in helix-3, suggest that these domains may enter the membrane largely unassisted, as found for other membrane proteins containing transmembrane sequences that are only moderately hydrophobic (Brambillasca et\u00a0al. 2006). Other factors are also required at this step, probably to prevent helix-3 from escaping the membrane into the stroma (Fig.\u00a04). One of these factors is the chloroplast signal-recognition particle. The ability of this complex to bind with high affinity to the loop between helices 2 and 3 (Tu et\u00a0al. 2000) suggests that it plays an important role in this process (Sch\u00fcnemann 2003). Other proteins such as Albino3 have been identified as important in the integration of LHCPs into the membrane (Moore et\u00a0al. 2000; Bellafiore et\u00a0al. 2002), although their specific actions are not known. Gerdes et\u00a0al. (2006) found that Alb3 mutants were defective in chloroplast biogenesis but not in accumulation of LHCPs. With the overall disposition of the protein now achieved in the membrane, the glutamate\/arginine ion-pairs between helices 1 and 3 can now form, which stabilizes the protein.\nAlthough the pK values vary widely, the magnitude of the dipole moments of the ligands that selectively coordinate with Chl a or Chl b are not substantially different, except for the backbone carbonyl group. Therefore, as Hirashima et\u00a0al. (2006) conclude, highly specific ligand selectivity should not be expected. In\u00a0vitro reconstitution of LHCs has been remarkably successful in reflecting the innate stability of Chl-ligand pairs (Bassi et\u00a0al. 1999; Remelli et\u00a0al. 1999; Rogl and K\u00fchlbrandt 1999; Horn and Paulsen 2004) but these experiments did not fully achieve the selectivity of interaction that is found in complexes that are assembled in\u00a0vivo. Ligand selection can reasonably be considered by taking into account (i) the unique Lewis acid properties of Chl b, (ii) the preference of Chl b to form electrostatic bonds with hard ligands containing a fixed dipole (i.e., an oxygen atom), (iii) the micro-environments in which the interactions occur (i.e., the dielectric constant), and (iv) the order of addition of the pigment molecules, which is determined partly by the local concentrations of the two Chls. Interaction of Chl b with sidechain amide groups would be expected from the properties of the ligands, and both Chl a and b are found with these ligands after in\u00a0vitro reconstitution of LHCs (Remelli et\u00a0al. 1999). Yet these groups are not ligands of Chl b in the crystal structure of LHCII. Chl a is probably more abundant within the membrane and competes favorably for amide ligands by mass action during assembly. Thus, basing theoretical proposals for a specific Chl in each binding site on only the first three factors above is not sufficient. The lack of ambiguity, or mixed sites, found in complexes isolated from plants after in\u00a0vivo assembly is most likely achieved also by the order in which Chls bind, determined by the relative concentrations of each.\nAn important aspect in the interaction of Chls and ligands is the location of the ligand within the protein structure. Tyr24 is near the stromal surface of LHCII, in a region expected to have a membrane interface dielectric constant of 5\u201310 (Tanizaki and Feig 2005). As suggested below, association of Chl b9 with this residue may form prior to reaching this position in the membrane. Likewise, the carbonyl of Val119, non-H-bonded because of Pro116, is at the lumenal end of helix-2 in LHCII, also a region of relatively high dielectric constant. The backbone carbonyl group of Val119 coordinates with Chl b14. The Chl b molecules are possibly protected from the aqueous environment by the protein strand. H-Bonding of the 7-formyl group to Gln122 and Ser123 (Liu et\u00a0al., 2004) would increase the Lewis acid strength of Chl b14 and strengthen this bond. These two Chl b molecules possibly stabilize an intermediate in the assembly pathway of LHCII (Fig.\u00a04).\nFour Chl b molecules interact with helix-2 of Lhcb1. Chl b12 forms a coordination bond with Glu139, which as discussed above is likely charge-compensated by Arg142. The other three Chl b molecules (b10, b11 and b13) retain water ligands (Fig.\u00a03). H-Bonding of these Chl b molecules through the 7-formyl group would further enhance the electrostatic character of the Mg atom (Liu et\u00a0al. 2004). The 7-formyl group of Chl b11 is H-bonded to the peptide bond N of Leu148, and along with Chl b12 may help to retain the stromal end of helix-2 in the membrane. The 7-formyl group of Chl b13 is H-bonded to the water ligand of Chl b10, which is in turn H-bonded via its 7-formyl group to the amide N of Gln131. These Chl b molecules, as an aggregate, may fill a void in the protein between helices 2 and 3 as the protein folds. Such an aggregate of Chl b molecules would be considerably more stable than a similar complex composed of Chl a. These molecules would then enter the structure late in assembly, as Horn and Paulsen (2004) and Horn et\u00a0al. (2007) found during studies of the kinetics of reconstitution of the complex in\u00a0vitro.\nThe argument is then reduced to one or two critical Chl b molecules that are required for retention of LHCPs in the chloroplast envelop during assembly\u2013one that interacts with a backbone carbonyl near the N-terminus and possibly the second that binds to the lumenal end of helix-2. Since the catalytic center of CAO is on the envelope inner membrane facing the intermembrane space (Reinbothe et\u00a0al. 2006), the N-terminal domain of LHCP possibly binds to Chl b on that side of the membrane. As the protein is transported through the membrane, the bound Chl b would approach the stromal surface while the motif \u2013ExxHxR\u2013 in helix-1 enters the interior of the membrane and binds Chl a (Fig.\u00a04). These Chl molecules may then hold the N-terminal domain in the inner membrane sufficiently long for the remainder of the protein to be transported across the outer membrane of the envelope and become integrated into the inner membrane. This scenario ensures that Chl b enters the complex from the surface of the inner membrane that faces the outer membrane and thus occurs only in peripheral LHCs that are assembled with apoproteins synthesized in the cytosol.","keyphrases":["chlorophyll","ligands","light-harvesting complex","coordination bonds","lewis acid","lewis base","chlorophyllide a oxygenase","chloroplast development","chlorophyll b","dipole moments","chlorophyll c","chlorophyll molecular axes"],"prmu":["P","P","P","P","P","P","P","P","P","P","P","M"]}
{"id":"Qual_Life_Res-4-1-2358934","title":"Individual quality of life: adaptive conjoint analysis as an alternative for direct weighting?\n","text":"In the schedule for the evaluation of individual quality of life (SEIQoL) the weights for five individualized quality of life domains have been derived by judgment analysis and direct weighting (DW). We studied the feasibility and validity of adaptive conjoint analysis (ACA) as an alternative method to derive weights in 27 cancer patients and 20 patients with rheumatoid arthritis. Further, we assessed the convergence between direct weights and weights derived by ACA, and their correlation with global quality-of-life scores. All respondents finished the ACA task, but one in five respondents were upset about the ACA task. Further, the task was vulnerable to judgment \u2018errors\u2019, such as inconsistent answers. The agreement between the two weights was low. Both weighted index scores were strongly correlated to the unweighted index score. The relationships between the index score and scores on a visual analogue scale for global individual quality of life and global quality of life were similar whether or not the index score was calculated with DW weights, with ACA weights, or without using weights. We conclude that, because weights did not improve the correlation between the index score and global quality of life scores, it seems sufficient to use the unweighted index score as a measure for global individual quality of life.\nIntroduction\nQuality of life (QoL) instruments have traditionally been based on a needs model of QoL, with the same domains, assessment criteria, and weighting applied to all respondents [1]. Individual quality of life (iQoL) measures recognize that people define life domains in different ways, use different criteria to evaluate the domains, and place differing emphasis on their importance to overall quality of life [2, 3]. The schedule for the evaluation of individual quality of life (SEIQoL) is a method of assessing iQoL. It has been used in many contexts [3\u20137].\nThe SEIQoL consists of three stages of administration. The first is a semistructured interview, in which respondents are asked to nominate five areas of life (domains) that they consider most important to the overall quality of their lives [1]. In the second stage the respondents rate their functioning on each of these domains, and in the third the relative weights of the domains are quantified.\nIn the original version of the SEIQoL a method called judgment analysis (JA), based on social judgment theory, was used for stage 3 [2, 3]. In social judgment theory, linear models are used to explain the impact of important factors, or cues, and their weights, on judgments. For the SEIQoL, to quantify the relative weights, subjects were presented with 30 randomly generated profiles of hypothetical situations labeled with the five chosen domains [3]. Respondents were asked to rate the QoL they associated with each profile on a VAS. Ten of the 30 scenarios were replicates to allow for a measure of consistency of judgments. The judgments were modeled using multiple regression to produce five relative weights summing to 100 [2]. The authors stated that JA was a successful aid to the evaluation of QoL [2, 3, 8], but from a theoretical viewpoint the number of 20 scenarios was small. Thirty to 50 scenarios are recommended to model the five weights [9]. McGee reported the use of 40 scenarios in the first study on the SEIQoL [8], but in later studies only 30 scenarios were used, of which ten were replicates [1\u20133]. Further, the time required to administer the JA to respondents is long and the task cumbersome, especially for older and cognitively impaired people. It requires compensatory decision-making processes, i.e., the ability to make an overall judgment on the basis of weighted information. These drawbacks make JA unsuitable for application in most clinical practices as well as for frequent assessment over a short period of time [1]. Because of these problems with JA, Browne et\u00a0al. [1] developed a more easy to administer version of the SEIQoL, namely SEIQoL-direct weighting (SEIQoL-DW). Respondents are asked to fill in a pie chart in which the relative size of each sector of the pie represents the weight the respondent attaches to a QoL domain. The validity and reliability of the DW in comparison with the JA-based SEIQoL have only been investigated in two small studies. The first was done in a sample of 40 healthy volunteers [1]. The mean absolute difference between the 200 weights derived for each method, that is, 40\u00a0times five weights, was 7.8 at the first measurement, and 7.2 at the second measurement. When weights derived from the two methods were converted to ranks and compared for agreement, the \u03ba value was moderate at both measurements (0.40 and 0.44, respectively). The reliability of the weights was moderate for the DW (\u03ba\u00a0=\u00a00.51), and only fair for JA (\u03ba\u00a0=\u00a00.31) [1]. In a later study, Waldron et\u00a0al. compared the psychometric characteristics of the SEIQoL-DW with the SEIQoL-JA among 80 patients with advanced incurable cancer. The index scores generated by the two methods fell within a range of 14.9 [10]. These differences are large in clinical terms [3]. Therefore, the authors [10] concluded that the two methods are not interchangeable. Despite this lack of data on the DW, and its demonstrated lack of agreement with the JA, it has become the standard method for eliciting weights of the SEIQoL, due to it being simple to administer. However, a fundamental distinction in cognitive psychology is that between explicit and implicit thought [1], and previous evidence with weighting methods suggests that respondents may be unable to provide accurate implicit weights through a method such as the DW [1]. Indirect methods such as JA are based on more basic and simple judgmental tasks, such as paired comparisons, and thereby may reduce possible biases that may play a role in direct judgments. Further, indirect methods are more likely to avoid the social desirability effect according to which respondents bias their response toward the perceived values of the researcher\/clinician [11]. Another advantage is that indirect methods can provide measures of internal reliability and validity for individual interviews [1]. An alternative indirect weighting method might be conjoint analysis, which was developed in mathematical psychology and, like JA, has a strong theoretical basis [12\u201314]. As in JA, conjoint analysis is based on the premises that any treatment or health state can be described by its characteristics (or attributes) and that the extent to which an individual values a treatment or health state depends on the levels of these characteristics. The method can be used to estimate the relative importance of these attributes, and may therefore be suitable for eliciting the weights of domains of iQoL, since the domains can be seen as attributes of iQoL.\nIn conjoint analysis the number of paired comparisons needed to estimate the weights may be as high as the number of scenarios needed in JA, but nowadays software, adaptive conjoint analysis, is available that is adaptive to respondent\u2019s answers and can minimize the number of paired comparisons. As an indirect method, it may provide a feasible alternative to JA. ACA has been used to derive treatment preferences in patients with lupus nephritis, with HIV medication, and with cancer [15\u201317].\nThis study aims to assess the feasibility and the validity of the ACA to derive weights for iQoL domains. Furthermore, agreement of the weighting procedures performed by the ACA and the DW will be assessed. Because it would not be feasible to use the JA as well, the ACA was only compared with the DW. Since JA is rarely used and the scientific community has overwhelmingly embraced the DW, despite the lack of data on its validity, we wished to compare ACA and DW. Further, relationships of the resulting iQoL index scores with scores on a VAS for QoL and for iQoL may give more insight into the validity of both weighting methods.\nMethods\nPatients\nTo assess the feasibility and validity of the ACA to derive iQoL weights, a convenience sample of outpatients with rheumatoid arthritis or cancer who were treated at the Leiden University Medical Center were asked to participate in the study. We selected patients with rheumatoid arthritis (RA) who received multidisciplinary day treatment or had an appointment with the specialized nurse consultant about their treatment. Patients with cancer were selected if they received curative radiotherapy at the time of the study or had received curative radiotherapy in the 6 months before. The latter patients received a letter at home in which the head of the Department of Radiotherapy asked them to participate in the study. These groups were selected because they presented at the clinic with symptoms (RA) or were known to have side effects (cancer) impacting their quality of life. All patients were only included in the study after they had given their informed consent. The Medical Ethical Committee of the Leiden University Medical Center approved the research protocol.\nInterview and questionnaire\nPatients were interviewed either at the hospital or at home. First, patients had to rate their current global QoL on a horizontal VAS anchored at the two extremes by the terms \u2018best imaginable quality of life\u2019 and \u2018worst imaginable quality of life\u2019. Next, iQoL was assessed by the SEIQoL. If patients were unable to nominate five areas of life, they were presented with a list with predetermined domains, such as family, health, finances, living conditions, work, social, and leisure activities, to help them make a choice [3]. Next, patients rated their functioning on each of these domains on five adjacent 0\u2013100\u00a0mm vertical VAS scales anchored at the two extremes by the terms \u2018best possible\u2019 and \u2018worst possible\u2019. Further, patients had to rate their global iQoL, given their ratings on the five domains, on a VAS anchored at the two extremes by the terms \u2018the best life I can imagine\u2019 and \u2018the worst life I can imagine\u2019. In this article, the score for global iQoL on the VAS is named as SEIQoL-VAS. Finally, the weights of the IQoL domains were determined from the DW and the ACA.\nThe DW-pie consists of five stacked, centrally mounted, interlocking laminated discs. Each disc has a different color and is labeled with one of the five domains nominated by the individual. The discs can be rotated over each other to produce a dynamic pie chart where the relative size of each sector represents the weight the respondent attaches to a QoL domain [1].\nThe ACA is a computerized questionnaire [18]. At the start of the ACA survey, respondents were asked to indicate, using the mouse or key strokes, how important they considered the difference between the best and the worst level of functioning on each of the five SEIQoL domains on a seven-point Likert scale, with adjectives at the first (not important), third (somewhat important), fifth (quite important), and seventh (very important) radio button. Next they were presented with a series of paired comparisons. The pairs consisted of two scenarios, one on the left-hand side, and one on the right-hand side (see appendix). The scenarios differed with respect to the level of functioning on two or three domains of QoL. Although the use of scenarios with four or five domains would have provided for more precise estimates, we had to balance this against feasibility. We decided such scenarios were too difficult for this first experience using the ACA to assess SEIQoL weights. Each domain had four levels of functioning: very well, fairly reasonable, rather bad, and very bad. Respondents had to indicate their preference for the alternative on the left or right on a nine-point Likert scale. The number of pairs presented was based on the formula 3\u00d7(N\u00a0\u2212\u00a0n\u00a0\u2212\u00a01)\u00a0\u2212\u00a0N, where N is the number of levels across all domains and n is the number of domains, resulting in 3\u00d7(20\u00a0\u2212\u00a05\u00a0\u2212\u00a01)\u00a0\u2212\u00a020\u00a0=\u00a022 pairs [19]. This formula presents a rule of thumb leading to three times the number of observations as parameters available (for simulations on accuracy of prediction with various numbers of pairs, see [20]). We presented 25 paired comparisons, where scenarios were defined using two (pairs 1\u201315) or three (pairs 16\u201325) domains.\nFinally, patients filled out a questionnaire that addressed demographic factors such as age, sex, marital status, education, and religion.\nComputation of iQoL weights and index scores\nFor the DW, the relative weight of a domain is equal to the proportion of the pie chart that its sector represents, which can be read from a 100-point scale on the circumference. Relative weights for the ACA are calculated as follows. First, patient utilities for all levels of functioning on the domains are derived by ordinary least-squares regression analysis, from participants' answers to the pairwise comparisons, assuming a linear main effects additive model (for details see [18]). Next, the relative weights for the domains are calculated by dividing the range of each domain (utility of highest level \u2013 utility of lowest level) by the sum of ranges of all domains, and multiplying by 100 [16, 19]. The relative weights are expressed as percentages (the five weights add up to 100%) and reflect the extent to which the difference between the best and worst levels of each domain drives the decision to choose a specific scenario [16].\nThe index score for iQoL is a weighted score, calculated by multiplying the functioning scores for the domains with their corresponding weights as derived by the DW and the ACA method, and summing these. Further, an unweighted index score was calculated by simply summing up the functioning scores and dividing by 5.\nFeasibility and validity\nThe feasibility of the ACA was assessed by measuring the percentage of patients that were able to finish the task, by measuring the administration time, and by asking the patients how they evaluated the ACA with respect to difficulty and acceptability. We asked patients two quantitative items about the method being confronting (very, somewhat, not) or being unpleasant versus fun (1\u00a0=\u00a0very unpleasant, 5\u00a0=\u00a0much fun). Further, we also coded qualitative statements about the ACA being upsetting (comments such as \u2018nasty\u2019, \u2018mean\u2019, \u2018suicide questions\u2019, \u2018I felt like a prisoner\u2019). As a measure of difficulty we also assessed how often patients chose the worst option in a dominant pair, a pair in which one of the scenarios was on all domains better than the other.\nThe validity of the ACA was first studied by assessing the number of inconsistencies in the rank ordering of utilities, that is, the number of pairs in which the utilities for two levels of functioning were ranked opposite to the direction of the levels of functioning. We analyzed whether age, health status, and level of education were related to answers to dominant pairs and the number of inconsistencies by Pearson\u2019s correlation and analysis of variance. Next, we assessed whether patients were willing to trade off a decrease from the best to the second-best functioning level on their most important domain with the largest improvement on their second important domain. This was done by computing the ratio between the difference in utilities for the largest benefit in the second important domain and the difference in utilities for the two highest functioning levels of the most important domain. A value smaller than 1 was taken as indicating that the patient was not willing to trade off decline in the most important domain for any benefit in the second important domain. We similarly assessed whether patients were willing to trade off a decrease from the second to the third functioning level on their most important domain with the highest improvement on their second important domain.\nAgreement\nThe absolute differences between the weights assigned in the DW and in the ACA were described by means and standard deviations. Agreement between the weights was assessed by the intraclass correlation coefficient and Pearson\u2019s correlation coefficient. Pearson\u2019s correlation represents the linear association between two measures and is not strictly a measure of agreement. However, a Pearson\u2019s correlation which is much larger than the intraclass correlation would provide some indication of a systematic change between measures, as might occur if there were learning effects [21]. Correlations between the weighted scores and the unweighted score on the one hand, and the VAS scores for QoL and iQoL on the other hand, were assessed using Pearson\u2019s correlation coefficient. We used SPSS version 12.0 for Windows.\nResults\nPatients\nOf the 71 patients approached, 27 patients with cancer and 20 patients with rheumatoid arthritis were willing to participate (response rate 66%). Twenty cancer patients and four patients with rheumatoid arthritis declined to participate. Reasons not to participate were: too burdensome (n\u00a0=\u00a05), patients too busy with treatment or work (n\u00a0=\u00a03), and other (n\u00a0=\u00a03). In 13 cases the reason was unknown: two patients did not give a reason and 11 patients did not respond at all. Characteristics of the patients are described in Table\u00a01. Patients were, on average, 61\u00a0years old (SD 10\u00a0years). In 32 cases (68%), the interview was held at the hospital, the other interviews were held at home.\nTable\u00a01Characteristics of patients (N\u00a0=\u00a047)N (%)Sex\u00a0\u00a0\u00a0\u00a0Female24 (51) Living arrangement\u00a0\u00a0\u00a0\u00a0With partner41 (87) Education\u00a0\u00a0\u00a0\u00a0Lowa19 (40) Religion\u00a0\u00a0\u00a0\u00a0Religious25 (53) Diagnosis\u00a0\u00a0\u00a0\u00a0Rheumatoid arthritis20 (43) \u00a0\u00a0\u00a0\u00a0Breast cancer11 (23) \u00a0\u00a0\u00a0\u00a0Prostate cancer11 (23) \u00a0\u00a0\u00a0\u00a0Rectal cancer5 (11) Place of interview\u00a0\u00a0\u00a0\u00a0Hospital32 (68)\u00a0\u00a0\u00a0\u00a0At home15 (32)aLow education: lower vocational, lower secondary general education, or primary school; high education: intermediate vocational, higher secondary general education, higher vocational education, or university\nACA: feasibility and validity\nAll patients nominated five areas which they considered most important to their QoL, of whom two patients had to consult the list with predetermined domains. Domains of life mentioned most frequently were own health, relationships with partner and children, social contacts and friendships, hobbies and recreation, and work (Table\u00a02).\nTable\u00a02Nominated cues during the first stage of the SEIQoL (N\u00a0=\u00a047 patients)1N (%)Partner22 (47)Children12 (26)Partner and children13 (28)Family18 (38)Own health30 (64)Health of partner5 (11)Social contacts and friendship21 (45)Transportation10 (21)Independence5 (11)Hobbies and relaxation23 (49)Work21 (45)Feelings5 (11)Activities of daily life5 (11)Sports and holidays11 (23)Other34 (72)Total2351Only domains that were mentioned by five or more patients are reported, the others (such as sexuality and income) are grouped together in the final row\nThe ACA survey took on average 20\u00a0min (range 10\u201337\u00a0min). All patients were able to finish the ACA. Five patients (11%) were in some sense upset about the ACA survey, and a further three (6%) judged the questions as very confronting. An additional patient found it very unpleasant. For example, when a patient had nominated own health and relationship with the partner as domains, the ACA could offer one scenario in which the patient\u2019s health was very good whereas the relationship with the partner was poor, and another scenario in which the patient\u2019s health was very poor whereas the relationship with the partner was very good. Some patients became upset when they had to make a choice between such options.\nPatients were offered, on average, 2.9 dominant pairs (range 0\u20136). In such pairs, four times (3%) the worst option was chosen and once (1%) the patient had no preference. The four patients who chose the worst option had the same level of education and were of the same age as the other patients.\nWhen the utility assigned to a higher level of functioning on a particular domain is lower than that assigned to a lower level of functioning on that same domain, this is inconsistent. Because each domain had four functioning levels, six different pairs of levels [(4\u00d73)\/2] can be constructed for each domain, that is a total of 30 pairs of utilities per person. On average, patients\u2019 utilities were rank-ordered opposite to the level of functioning in 3.9 out of these 30 pairs (13%; Table\u00a03). For the most important domain, the mean number of inconsistencies was 0.2, whereas this was 1.7 for the least important domain, and the correlation between the inconsistencies and the domain weights was r\u00a0=\u00a0\u22120.50 (P\u00a0=\u00a00.000, n\u00a0=\u00a0235, 47 \u00d7 5 weights). The number of inconsistencies was lower in patients with a higher education level (Spearman\u2019s rho \u20130.30; P\u00a0=\u00a00.04), whereas it was not related to age or health status.\nTable\u00a03Consistency in utilities for levels of functioning of individual quality of life domainsaMost importantLeast importantTotal N\u00a0=\u00a047Domain 1Domain 2Domain 3Domain 4Domain 5Number of inconsistencies in rank order utilities out of six pairs per domainMean (SD)0.2 (0.6)0.2 (0.5)0.8 (1.0)1.0 (1.2)1.7 (1.1)3.9 (2.1)N(%)N(%)N(%)N(%)N(%)N(%)No inconsistencies41 (87)41 (87)25 (53)23 (49)8 (17)138 (59)One inconsistency2 (4)4 (9)7 (15)9 (19)11 (23)33 (14)Two inconsistencies4 (9)2 (4)13 (28)10 (21)17 (36)46 (20)Three inconsistencies2 (4)4 (9)9 (19)15 (6)Four inconsistencies1 (2)2 (4)3 (1)aThe content of the domains may vary between patients, according to what an individual patient evaluates as most important\nOne out of 43 patients was not willing to trade off a decline from the best to second-best functioning level on the most important domain for the largest benefit on the second important domain. Further, all patients were willing to trade off a decline from the second to the third level of the most important domain for the largest benefit on the second important domain.\nAgreement between DW weights and ACA weights\nThe mean absolute difference between the DW weights and the ACA weights varied from 4.4 to 7.5 for the five domains of iQoL, on a scale from 0\u2013100 (Table\u00a04). For all five domains together, 36 pairs (15%) differed by more than 10 points. For the most important domain according to the ACA weighting, 17 patients (36%) had a difference of more than 10 points between their DW weight and ACA weight.\nTable\u00a04Absolute differences between DW weights and ACA weightsMost important aLeast important aTotal (N\u00a0=\u00a0235)Domain 1Domain 2Domain 3Domain 4Domain 5Mean (SD)7.5 (5.3)4.7 (4.2)4.4 (3.5)4.6 (4.1)5.9 (4.9)P\u00a0=\u00a00.003Absolute difference between ACA and DW N (%)N (%)N (%)N (%)N (%)N (%)Less than 5 points17 (36)31 (66)28 (60)31 (66)26 (55)133 (57)5\u201310 points13 (28)12 (26)16 (34)13 (28)12 (26)66 (28)More than 10 points17 (36)4 (8)3 (6)3 (6)9 (19)36 (15)a Importance based on adaptive conjoint analysis procedure\nThe correlation between the DW weights and the ACA weights varied from 0.22 to 0.43, and the intraclass correlation coefficient varied from 0.18 to 0.33, both indicating a low agreement between the two weighting methods (Table\u00a05).\nTable\u00a05Agreement between DW weights and ACA weightsACA\u2013DW linear correlationACA\u2013DW intraclass agreementPearson rPICCbPDomain of individual quality of lifea\u00a0\u00a0\u00a0\u00a0Most important domain0.270.060.230.06\u00a0\u00a0\u00a0\u00a0Domain 20.300.040.280.03\u00a0\u00a0\u00a0\u00a0Domain 30.430.0030.330.01\u00a0\u00a0\u00a0\u00a0Domain 40.330.020.280.03\u00a0\u00a0\u00a0\u00a0Least important domain0.220.140.180.11ACA, adaptive conjoint analysis; DW, direct weightingaRank ordered according to weighting derived by the method of adaptive conjoint analysisbTwo-way mixed effects model where people effects are random and measure effects are fixed; f-test with true value 0\nConsequences of weighting method\nThe index score for iQoL calculated with the weights derived by the ACA (SEIQoL-ACA) was slightly higher than the index score of the SEIQoL-DW [mean score 71.6 (SD 11.5) versus 70.1 (SD 12.2); P\u00a0=\u00a00.02] and both were higher than the unweighted index (mean 67.2, SD 12.4). The SEIQoL-VAS score correlated strongly and almost equally with the SEIQoL-ACA, the SEIQoL-DW, and the unweighted score. The three index scores were also positively related to the global QoL VAS (Table\u00a06).\nTable\u00a06Impact of weighting procedure on index score for individual quality of life, and on correlations with global quality of life DW indexACA indexUnweighted indexrrrIndex score for individual quality of lifeDW-index score1.00.95**0.92**ACA-index score0.95**1.00.89**Unweighted index score0.92**0.89**1.0SEIQoL VAS0.62**0.54**0.63**QoL VAS0.40*0.36*0.33**P\u00a0<\u00a00.05; **P\u00a0<\u00a00.001DW, direct weighting; ACA, adaptive conjoint analysis; SEIQoL, schedule for evaluation of individual quality of life; VAS, visual analogue scale; QoL, quality of life\nDiscussion\nWe explored whether the ACA was a feasible and valid method to derive weights for iQoL domains. The iQoL community seems to have fully embraced the DW, despite its lack of agreement with the original JA method. Contrary to JA, the DW has no theoretical underpinning. Further, DW is a direct method, and as such may not capture implicit or unconscious thoughts or preferences. Since respondents will generally not be explicitly aware of the contributions of their life domains to their overall QoL, indirect methods may be of more value. JA is not feasible, however, in most situations, and for this reason we piloted another theory-based indirect method, the ACA. ACA and JA are based on the same premises. However, JA was initially developed for assessing expert opinion [22, 23], respondents have to rate many scenarios, and the task is cumbersome. Due to its adaptive nature, ACA is much less cumbersome than a full-scale conjoint analysis, which is a nonparametric form of JA. In addition to being indirect, ACA can provide measures of internal reliability and validity (inconsistencies, willingness to trade) for individual interviews. The agreement between DW weights and ACA weights may give insight into the validity of the two methods.\nACA was to some extent feasible, because the ACA took on average 20\u00a0min and all patients were able to finish it. However, one in five patients judged the ACA task as upsetting, very confronting, or very unpleasant. The paired comparison task, despite working well for some domains, turned out not to be appropriate for some others. Especially choosing between two domains that are dear to the patient turned out not to be feasible. Sometimes, the computer offered a dominant pair, mostly resulting from the fact that the utilities of the functioning levels were almost equal. Only seldom was the worst option chosen. This finding shows that almost all patients understood the task of paired comparisons and were able to make a valid choice.\nA limitation of our procedure was that patients did not rate scenarios with four or five domains. Although the use of such scenarios would have provided for more precise estimates, this had to be balanced against feasibility. Using all five attributes in the pairwise comparisons would also have allowed for the evaluation of full profiles (health states). The goal of this first study on the use of ACA for the SEIQoL was merely to assess its feasibility and to compare ACA weights with DW weights, not to assess full profiles. We therefore preferred to opt for the more feasible approach, which we deemed sufficiently difficult already.\nMany patients gave inconsistent answers, but these inconsistencies mostly occurred on domains 3\u00a0and lower, and especially on the least important domain. For the two most important domains, the large majority of patients had utilities ordered in the same direction as the corresponding levels of functioning. In the case of less important domains, the differences between utilities of successive functioning levels are probably small, which leads to inconsistent answers.\nA major premise of the ACA is that respondents are willing to use compensatory decision making. Our findings show that almost all people were willing to do so. The willingness to trade off the difference between a decline on the most important domain with the largest benefit on the second most important domain is consistent with the finding that, in patients with colorectal cancer, 95% were willing to trade off a 1% loss of survival [17].\nThe agreement between DW and ACA weights was low. Browne et\u00a0al. reported similar findings for the comparison between JA and DW [1]. Further, the weights derived from each method were only poorly or moderately correlated to each other. The differing weights from the DW and the ACA suggest that the two methods are not interchangeable.\nWeighting had almost no effect on relationships with other global measures of QoL, and the DW index score correlated 0.95 with the ACA index score, and both weighted index scores were also highly correlated to the unweighted index score. Weighting or not weighting domains of QoL has received much attention in the literature. Some studies have shown that there is no effect of weighting [24\u201328]. For iQoL, the findings of Wettergren et\u00a0al. suggest that the degree of importance is already built into the process of selection, when the participants themselves select the domains [28]. Further, it has been suggested that a satisfaction evaluation had incorporated the judgment of item importance [29]. Our findings indeed show that the weighted iQoL index scores were higher than the unweighted index score, which indicates that patients gave higher weights to domains with better functioning. Most likely, the patients already take into account the importance of a specific domain, first in the selection of the domain and next when they evaluate the functioning on that domain.\nOur study has some limitations. The number of patients was relatively small, due to the qualitative character and semistructured design of the SEIQoL interview. However, larger numbers would not have changed the conclusion of our paper. A problem in measuring the validity of the SEIQoL is that a gold standard for iQoL is lacking. JA has been considered the standard weighting method, but due to its complex nature has been replaced in the field by DW. Unfortunately, it was not possible to include both the JA and the ACA, because of the cognitive burden imposed on the patients.\nDespite the patients\u2019 reluctance to perform the ACA, our study gave clear insight into the problems of deriving weights for iQoL domains. Our findings show that weighting has almost no effect on the association between the SEIQoL and global iQoL, although incorporating weights for domain functioning led to slightly higher iQoL index scores than the unweighted index score. Selecting and weighting domains are clearly confounded. Because of the high correlations between the weighted and unweighted index scores, it seems sufficient to use the unweighted index score as a measure for global iQoL.","keyphrases":["individual quality of life","conjoint analysis","direct weighting","seiqol","patients"],"prmu":["P","P","P","P","P"]}
{"id":"J_Behav_Med-4-1-2346512","title":"Internet-administered cognitive behavior therapy for health problems: a systematic review\n","text":"Cognitive-behavioral interventions are the most extensively researched form of psychological treatment and are increasingly offered through the Internet. Internet-based interventions may save therapist time, reduce waiting-lists, cut traveling time, and reach populations with health problems who can not easily access other more traditional forms of treatments. We conducted a systematic review of twelve randomized controlled or comparative trials. Studies were identified through systematic searches in major bibliographical databases. Three studies focused on patients suffering from pain, three on headache, and six on other health problems. The effects found for Internet interventions targeting pain were comparable to the effects found for face-to-face treatments, and the same was true for interventions aimed at headache. The other interventions also showed some effects, although effects differed across target conditions. Internet-delivered cognitive-behavioral interventions are a promising addition and complement to existing treatments. The Internet will most likely assume a major role in the future delivery of cognitive-behavioral interventions to patients with health problems.\nIntroduction\nCognitive-behavioral interventions are probably the most extensively researched form of psychological treatment (Butler et\u00a0al. 2006). Cognitive-behavioral interventions are aimed at challenging negative automatic thoughts and dysfunctional underlying beliefs, and at changing behavioral patterns which are related to the problem being targeted in the therapy. More than 300 published controlled outcome studies, and probably many more, have examined the effects of cognitive-behavioral therapy (CBT) for a wide range of disorders and health problems, ranging from mental health disorders, such as depression (Hollon et\u00a0al. 2002), anxiety disorders (Barlow 2002), schizophrenia (Pilling et\u00a0al. 2002), to health conditions such as chronic pain (Morley et\u00a0al. 1999), sleep problems (Morin et\u00a0al. 1999), headache (Holroyd 2002), cancer (Moorey and Greer 2002) and many others. Most of these studies have shown that CBT has positive effects on these and several other health conditions. CBT is not only the most extensively researched form of psychotherapy, but also the most widely applied type of psychotherapy (Norcross et\u00a0al. 2005), and certainly the most widely applied \u2018evidence-based\u2019 type of psychological therapy.\nCBT is increasingly offered through the Internet. Internet-based interventions may have several advantages over other more traditional forms of delivery. They may save therapist time, reduce waiting-lists, allow patients to work at their own pace, abolish the need to schedule appointments with a therapist, save traveling time, reduce the stigma of going to a psychologist or therapist, and facilitate help for the hard-of-hearing as self-help treatments typically work with visual rather than auditory information (Marks et\u00a0al. 2007). Furthermore, Internet-delivered self-help may be programmed to enhance patients\u2019 motivation by presenting a wide range of attractive audiovisual information with voices giving instructions in whichever gender, age, accent, language and perhaps game format the client prefers. It can also quickly and automatically report patient progress and self-ratings.\nInternet-based interventions may reach populations with health problems, who can not be reached with other more traditional forms of treatments. For example, a considerable proportion of the patients with mental disorders are not reached with traditional forms of treatment (Bijl and Ravelli 2000) because of the stigma associated with mental disorder, prejudices about therapists, lack of willingness to talk to a stranger about personal problems, or because of physical obstacles like walking problems or long distances. For patients with somatic conditions there may be barriers to seek psychological treatment. Internet-guided interventions may reach a segment of this population who cannot be reached through traditional interventions.\nInternet-based psychological interventions for many health problems are commonly based on CBT techniques. One reason is that the effects of CBT have been shown in numerous trials. Another reason why CBT is often used in internet-interventions is that these techniques lend themselves to be operationalized in text. CBT interventions can very well be converted into a structured format, with psychoeducation, homework assignments and registration exercises presented via web pages (Ritterband et\u00a0al. 2006).\nDelivering CBT through the Internet does not, however, only have advantages. An online programme may not be suitable for technophobic patients and illiterates, nor can it answer all the possible questions users may ask; it can not detect subtle nonverbal and verbal clues to clients\u2019 misunderstandings; it may encourage clients to cherry-pick from a range of homework options presented; and not all clients find communicating via computers acceptable (Marks et\u00a0al. 2007). However, subtle text nuances may be detected and somewhat surprisingly, Internet interventions has been found to generate good working alliance between the patient and the therapist (Knaevelsrud and Maercker 2006).\nFurthermore, it is not yet very clear whether CBT interventions which have been proven to be effective when delivered in traditional format, are also effective when delivered through the internet. Recent studies, however, indicate that this might be the case for at least some patients and some conditions (Carlbring et\u00a0al. 2005).\nWhether or not an internet-based CBT is effective should be examined in randomized controlled trials, and can obviously not be based on the effects of traditionally delivered CBT (e.g., individual or group format). Since the Internet has become available to the broad public in many Western countries, several trials have examined the effects of cognitive behavioral interventions in randomized trials. In a recent meta-analysis, we examined the effects of internet-based treatments for depression and anxiety disorders, and found that these interventions had large effects (Cohen\u2019s d\u00a0=\u00a01.00) compared to control conditions, when some kind of guidance was given to the patients receiving the treatment (Spek et\u00a0al. 2007). This latter observation was also confirmed in a recent review in which a correlation of rho\u00a0=\u00a0.75, P\u00a0<\u00a0.005 was obtained between amount of contact spent with clients and the effect size (Palmqvist et\u00a0al. 2007). These large effect sizes suggest that Internet-administered CBT is as effective as face-to-face CBT, and that the format in which CBT is delivered may not be related to the effect sizes found. It is not known, however, whether Internet-administered CBT is also equally effective when other health problems are targeted.\nIn the current study, we will present the results of a systematic review of Internet-delivered CBT for health problems. In the review, we aim to establish for which health problems Internet-based CBT has been developed, and examined in randomized controlled or comparative trials, and whether these interventions were effective. We also examine the target groups and contents of these intervention, as well the quality of the studies.\nMethods\nSearch strategy and selection of studies\nStudies were traced through several methods. First, we conducted a comprehensive literature search in bibliographical databases (from 1966 to February 2007). We examined 1,608 abstracts in Pubmed (295 abstracts), Psycinfo (109), Embase (330) and the Cochrane Central Register of Controlled Trials (374). In order to find unpublished studies, we also searched Digital Dissertations (500 abstracts). We searched these databases by combining terms indicative of effect studies (randomized trials, controlled trials, clinical trials) and Internet (both keywords and text words). Second, we examined the references of earlier reviews of Internet-based interventions (Griffiths et\u00a0al. 2006; Wantland et\u00a0al. 2004), and we reviewed the reference lists of retrieved papers.\nStudies were included if they met the following criteria: (a) randomized controlled or comparative trials (b) examining interventions that were conducted through the Internet (at least 50% of the intervention), (c) based on CBT techniques, (d) aimed at behavior change (e) in patients with an existing disorder or health problem. We excluded studies aimed at mental disorders, because another recent systematic review was published about these studies (Spek et\u00a0al. 2007). We also excluded studies focusing on lifestyle (smoking, obesity, exercise, nutrition), because the character of these interventions differs strongly because the focus of these interventions is typically preventative.\nQuality assessment\nThere are at least 25 scales available to assess the validity and quality of randomized controlled trials (Higgins and Green 2005). There is no evidence, however, that these scales provide more reliable assessments of validity. We preferred therefore to use a simple approach for assessing the validity of the studies, as suggested in the Cochrane Handbook (Higgins and Green 2005).\nIn this context, the validity of a study can be defined as the extent to which its design and conduct are likely to prevent systematic errors (Moher 1995). Variation in validity can explain variation in the results of the studies included in a systematic review and may result in an erroneous conclusions that an intervention is effective if the less rigorous studies are biased toward overestimating an intervention\u2019s effectiveness (Higgins and Green 2005).\nWe assessed the validity of the studies using four basic criteria: allocation to conditions is conducted by an independent (third) party; adequacy of random allocation concealment to respondents; blinding of assessors of outcomes; and completeness of follow-up data.\nAnalyses\nWe examined the characteristics of the target populations, the interventions, and the design of the included studies.\nWe also examined which main outcome measures were used for each study, and we calculated standardized effect sizes for each of the main outcome measures. These effect sizes (d) were calculated by subtracting (at post-test) the average score of the control group (Mc) from the average score of the experimental group (Me) and dividing the result by the average of the standard deviations of the experimental and control group (SDec). An effect size of 0.5 thus indicates that the mean of the experimental group is half a standard deviation larger than the mean of the control group. Effect sizes of 0.8 can be assumed to be large, while effect sizes of 0.5 are moderate, and effect sizes of 0.2 are small (Cohen 1988).\nWhen sufficient effect sizes were available (at least three effect sizes examining the same outcome measure in the same health problem), we calculated pooled mean effect sizes. For these analyses, we used the computer program Comprehensive Meta-analysis (version 2.2.021), developed for support in meta-analysis. As we expected considerable heterogeneity, we decided to calculate mean effect sizes with the random effects model. In the random effects model, it is assumed that the included studies are drawn from \u2018populations\u2019 of studies that differ from each other systematically.\nIn these analyses, we tested whether there are genuine differences underlying the results of the studies (heterogeneity), or whether the variation in findings is compatible with chance alone (homogeneity; Higgins et\u00a0al. 2003). As an indicator of homogeneity, we calculated the Q-statistic. We also calculated the I2-statistic which is an indicator of heterogeneity in percentages as well. A value of 0% indicates no observed heterogeneity, and larger values show increasing heterogeneity, with 25% as low, 50% as moderate, and 75% as high heterogeneity (Higgins et\u00a0al. 2003).\nResults\nIncluded studies\nA total of 61 papers which possibly met our inclusion criteria were retrieved. Twelve studies (13 papers; two papers were published about the same study) met our inclusion criteria. The other studies were excluded because they did not examine a cognitive-behavioral intervention (30 papers), because they were not a randomized controlled or comparative trial (11 papers), or because they did not examine an Internet-based intervention (7 papers). Selected characteristics of the target groups, the intervention, and the general design of the twelve included studies are presented in Table\u00a01.\nTable\u00a01Selected characteristics of Internet-based cognitive behavioral interventions for health problemsParticipantsIntervention and conditionsStudyHealth conditionRecruitmentWomen (%)Age group (M)Conditions NInterventionContactPeriodMeasurementsDropout (%)CountryAndersson et\u00a0al. 2003HeadacheCommunity8218\u201359 (40)1. I-CBT\u00a0+\u00a0telephone24Psychoeducation, applied relaxation; problem solving; cognitive restructuringE-mail (at request)6\u00a0weeksPre, post32Sweden2. I-CBT20Weekly telephone callsDevineni and Blanchard 2005Tension-type, migraine-only or mixed headache Community83NR (42)1. I-CBT39Progressive muscle relaxation\u00a0+\u00a0cognitive stress coping therapy (tension-type), or autogenic training\u00a0+\u00a0PMR (migraine\/mixed)No therapist contact4\u00a0weeksPre, post38US2. Waiting-list47Str\u00f6m et\u00a0al. 2000 [33]Recurrent headacheCommunity68\u2265 18 (37)1. I-CBT51Psychoeducation, applied relaxation; problem solving; cognitive restructuringE-mail (at request)6\u00a0weeksPre, post61Sweden2. Waiting-list51Brattberg 2006Chronic pain\u00a0+\u00a0burn-out in patients on long-term sick leave Community9018\u201365 (47)1. I-CBT30Films\u00a0+\u00a0texts; psychoeducation\u00a0+\u00a0cognitive self-treatment (changing, coping with shame and guilt, depression, identity, etc.)Real live introduction meeting\u00a0+\u00a0weekly online sessions20\u00a0weeksPre, post, 1\u00a0year8Sweden2. Waiting-list30Buhrman et\u00a0al. 2004Chronic back pain Community6318\u201365 (45)1. I-CBT25Applied relaxation; physical exercise, coping strategiesE-mail (at request) weekly telephone calls8\u00a0weeksPre, post, 3\u00a0months8Sweden2. Waiting-list32Hicks et\u00a0al. 2006Pediatric recurrent pain\/headacheCommunity649\u201316 (12)1. I-CBT25Psychoeduation; relaxation; cognitive restructuringE-mail contact (5\u00a0times)\u00a0+\u00a0telephone contact (3\u00a0times)7\u00a0weeksPre, post, 3\u00a0months21Canada2. Waiting-list22Andersson et\u00a0al. 2002TinnitusCommunity4718\u201370 (48)1. I-CBT53Psychoeducation; applied relaxation; positive imagery; advice on noise sensitivity; cognitive restructuring; behavioral sleep management.Weekly report on progress, weekly encouraging e-mail 6\u00a0weeksPre, post, 1\u00a0year41Sweden2. Waiting-list64Hopps et\u00a0al. 2003People with physical disabilities who feel lonelyCommunity53\u2265 18 (34)1. I-CBT11Psychoeducation on communication; self-observation, role-playing, confrontation, positive verbalizations; social skills and assertiveness.Weekly sessions on the Internet12\u00a0weeksPre, post, 4\u00a0months14Canada2. Waiting-list11Lorig et\u00a0al. 2006Chronic diseases (heart, lung, or type 2 diabetes)Community71\u2265 18 (57)1. I-CBT457Exercise programs; relaxation; cognitive restructuring; psychoeducation; physician-patient communication; healthy eating; fatigue management; problem solving Web-based bulletin board discussion groups (trained peer moderators)6\u00a0weeksPre, post, 1\u00a0year18US2. Care-as-Usual501Owen et\u00a0al. 2005Early-stage breast cancerClinical100NR (52)1. I-CBT32Psychoeducation; coping advice for common physical symptoms such as pain and fatigue; structured coping-skills training exercisesBulletin board for asynchronous group discussion12\u00a0weeksPre, post15US2. Waiting-list30Str\u00f6m et\u00a0al. 2004InsomniaCommunity65\u2265 18 (44) 1. I-CBT54Psychoeducation; sleep restriction, stimulus control, cognitive restructuringE-mail (at request)5\u00a0weeksPre, post24Sweden2. Waiting-list55Wade et\u00a0al. 2006Pediatric brain injuryClinical385\u201316 (11)1. I-CBT20Problem solving; communication; behavior management skills; Videocontact with therapist14\u00a0weeksPre, post2US2. I-information20Abbreviations: I-CBT: Internet-CBT; NR: not reported\nIn the twelve included studies, a total of 1,704 patients participated, 841 in the Internet-CBT conditions, and 863 in the control conditions (mean number of respondents per study: 142; standard deviation: 258.7). More than half of these 1,704 patients (56%) participated in one study (Lorig et\u00a0al. 2006). In none of the other studies was the number of patients per condition larger than 64.\nEleven studies compared an Internet-based CBT intervention to a control condition, while one study compared two types of Internet-based CBT to each other (one with and one without weekly telephone calls). Nine of the eleven controlled studies used a waiting list control group, while one study used a care-as-usual control group, and the other one used an information control group. In all studies, participants were randomized to one of two conditions. In none of the studies was Internet-based CBT compared to a face-to-face intervention of another treatment. Six studies only presented pre-post data, while the other six also had a follow-up measurement (mean length of follow-up in these six studies was 7.67\u00a0months; standard deviation 4.76).\nSix studies were conducted in Sweden, four in the United States, and two in Canada. All included studies were conducted in the year 2000 or later (one in 2000 and another one in 2002, two in 2003, in 2004, and in 2005, and four in 2006).\nThe quality of studies varied. Three of the twelve studies reported that allocation to conditions was conducted by an independent party. Concealment of random allocation to respondents was not possible in any of the studies, while blinding of assessors was reported in none of the studies. Drop-out numbers ranged from 2% to 61%. In only one of the studies intention-to-treat analyses were conducted (Hicks et\u00a0al. 2006; the other studies were limited to completers-only analyses).\nThe target populations\nThree studies focused on patients suffering from pain, three on headache, and six on other health problems (tinnitus; physical disabilities; chronic diseases; breast cancer; insomnia; and pediatric brain injury). In ten of the twelve studies, patients were recruited through announcements on websites, referrals, and community recruitment. In the other two studies patients were recruited through screening of clinical samples. Ten studies were aimed at adults, two at children. None of the interventions were aimed at older adults, although three studies allowed older adults (\u226570\u00a0years) to participate. The other studies on adults only included younger adults for participation or did not report that they used an age limit.\nThe interventions\nThe character of the interventions differed from each other. One group of interventions consisted of self-help materials on the Internet, with supporting e-mails or telephone calls (5 studies). In two studies the intervention consisted of self-help materials on the Internet, but without the supporting e-mails or calls. In the other studies, the core of the intervention consists of online contact between a therapist or moderator and the patients (individual or in groups). Most interventions contained psychoeducation on the specific problem, and different CBT modules such as cognitive restructuring, relaxation techniques, and social skills training. The duration of the interventions ranged between 4 and 20\u00a0weeks.\nEffects of the interventions\nThe effects of the interventions on the main outcomes of each included study at post-test are presented in Table\u00a02.\nTable\u00a02Main outcomes of studies on Internet-based cognitive behavioral interventions for health problemsHealth conditionComparisonMain outcomeEffect size95% CIPainBrattberg 2006Chronic pain and burnoutRehabilitation course versus waiting list controlFunctional limitations0.48\u22120.03\u20130.99Buhrman et\u00a0al. 2004Chronic back painInternet-guided self-help versus waiting list controlCoping with pain0.790.22\u20131.36Hicks et\u00a0al. 2006 BPediatric recurrent pain\/headacheInternet-guided self-help versus waiting list controlPain0.47\u22120.24\u20131.18POOLED0.58a0.25\u20130.92HeadacheAndersson et\u00a0al. 2003HeadacheInternet self-help with support versus self-help onlyHeadache0.38\u22120.35\u20131.11Devineni and Blanchard 2005Chronic headacheInternet self-help versus waiting list controlHeadache0.560.13\u20130.99Str\u00f6m et\u00a0al. 2000Recurrent headacheInternet self-help versus waiting list controlHeadache0.19\u22120.40\u20130.78OtherAndersson et\u00a0al. 2002TinnitusInternet CBT versus waiting list controlDistress from tinnitus0.26\u22120.23\u20130.75Hopps et\u00a0al. 2003Physical disabilitiesGoal-oriented CBT chat-group teletherapy versus waiting list controlLoneliness0.46\u22120.45\u20131.37Lorig et\u00a0al. 2006Chronic diseases Online CBT workshops versus care-as-usualHealth indicators (only 1\u00a0year FU)0.10\u22120.04\u20130.24Owen et\u00a0al. 2005Early-stage breast cancerOnline CBT coping group versus waiting list controlHealth-related quality of life0.22\u22120.32\u20130.76Str\u00f6m et\u00a0al. 2004InsomniaInternet CBT versus waiting list controlWade et\u00a0al. 2006Pediatric brain injuryOnline family problem solving therapy versus Internet resources Parental mental health0.700.05\u20131.35aPooled with the random effects model; Z\u00a0=\u00a03.40, P\u00a0<\u00a00.001; Q\u00a0=\u00a00.75, n.s.; I2\u00a0=\u00a00\nWe pooled the three studies in which Internet-based CBT for pain was compared to control groups. The mean effect size on the main outcome measure was 0.58 (95% CI: 0.25\u20130.92; P\u00a0<\u00a00.001), indicating a moderate to large effect of the interventions compared to the control groups at post-test. Heterogeneity was very low (Q\u00a0=\u00a00.75, n.s.; I2\u00a0=\u00a00).\nThere were also three studies on headache, however one did not use a true control group (but compared two active interventions to each other). Therefore we did not pool the results of these studies. The effect sizes of these interventions were small (d\u00a0=\u00a00.19; Str\u00f6m et\u00a0al. 2000) to moderate (d\u00a0=\u00a056; Devineni et\u00a0al. 2005).\nThe effect sizes of the other studies were in the small to moderate range, varying from small (d\u00a0=\u00a00.10) for health indicators in chronic diseases at one-year follow-up, to somewhat larger for health-related quality of life in breast cancer patients (d\u00a0=\u00a00.22) and from tinnitus sufferers (d\u00a0=\u00a00.26), to moderate (loneliness in patients with physical disabilities; d\u00a0=\u00a00.46) and large (parental mental health in pediatric brain injury; d\u00a0=\u00a00.70). Most effect sizes did not significantly differ from zero because of the small sample sizes in the majority of the studies.\nDiscussion\nThis systematic review of controlled and comparative studies of Internet-based CBT for health problems showed that this field is developing fast. Since 2000, twelve randomized studies have examined interventions for pain, headache, and several other health problems. Half of these trials were published in 2005 and 2006, and it can be expected that the number of trials will rise sharply in the next few years. Overall, findings are promising but effects are slightly below the effect sizes found for Internet-delivered CBT for anxiety and depression (Spek et\u00a0al. 2007).\nAlthough several health problems were targeted in these Internet-based studies, there are gaps in the literature in terms of treatments for health problems which have been found to improve by means of CBT. For example, several studies have examined the effects of CBT for chronic fatigue syndrome (Knoop et\u00a0al. 2007), fibromyalgia (Garcia et\u00a0al. 2006), incontinence (Garley and Unwin 2006), or multiple sclerosis (Thomas et\u00a0al. 2006), but these have not yet been transformed into a web based intervention, although trials found positive effects of face-to-face CBT for these problems. Because the promising results of earlier studies, and because of the benefits of Internet-based interventions, we can expect development of new programs for these conditions in the future.\nOur review does not cover the whole field of internet interventions. While we focused on CBT for existing health problems, several other studies have examined CBT for mental health problems (Spek et\u00a0al. 2007), on internet-based preventive interventions aimed at a healthy lifestyle (weight loss, smoking, exercise; e.g., Swartz et\u00a0al. 2006; Mu\u00f1oz et\u00a0al. 2006; Oenema et\u00a0al. 2001; Tate et\u00a0al. 2006), and interventions using non-CBT methods (McMahon et\u00a0al. 2005; Edwards et\u00a0al. 2006; Gray et\u00a0al. 2000). However, as was shown in this review, research on CBT interventions has been growing fast in the past few years. Because CBT interventions are very well suited to be used through the internet, it can be expected that research in this area will continue to grow further in the next years.\nThe included studies do not yet\u00a0allow us to draw definite conclusions about whether CBT through the Internet are as effective as face-to-face interventions. For most health problems we found only one study examining the effects of an Internet-based CBT study. In fact, it was only for pain and headache did we find more than one studies. However, the effects found for Internet-based interventions aimed at pain are comparable to the effects found for face-to-face treatments for pain (Morley et\u00a0al. 1999), and the same is most likely true for the Internet-interventions aimed at headache (Bogaards and ter Kuile 1994). The other interventions also found some effects, although some effects were stronger than others. It does seem clear, however, that Internet-based CBT can have significant effects on some of the health problems described in this review. For at least one of the conditions\u2014tinnitus\u2014an effectiveness study has been published showing better results then the first controlled efficacy trial (Kaldo-Sandstr\u00f6m et\u00a0al. 2004).\nIt has been suggested self-help interventions be used as one of the first steps in stepped-care programs (Scogin et\u00a0al. 2003). Perhaps Internet-based interventions which are used in healthcare settings should also be placed within these stepped-care frameworks. In these cases, additional care is available if the Internet-based intervention does not reduce the problem of a patient sufficiently. On the other hand, Internet interventions can develop as well, and might at least for some patients be more suitable than face to face CBT. As many health conditions such as chronic pain and cancer require a multidisciplinary team approach for optimal treatment, we assume that future Internet interventions will take advantage of this possibility.\nThere is no consensus yet among researchers about the way CBT should be presented on the Internet, although standards are emerging. Most interventions used a guided self-help format in which the treatment protocol is presented on the Internet and the patient works it through more or less independently. The patients are supported by brief contacts with therapists through e-mail or telephone. However, other studies use a more traditional format in that the patients go online at the same time as the therapist and have a more or less regular treatment session. Group treatments can also be delivered in such a way.\nAnother difference between interventions concerns the additional elements on the Internet, apart from CBT. Some interventions have combined the cognitive behavioral interventions with other components, such as psychoeducation, films and texts to read, and a forum for users of the website. Other interventions do not provide such extras.\nOur review showed up several other important limitations of the current research in this area. First, most studies used waiting list control groups, and only very few used a care-as-usual or another control group. Subjects in waiting list control groups probably do not take constructive action to reduce their problems themselves during the waiting period, because they are expecting professional help in the future. This may result in an overestimation of the effects of an intervention, because there may be less spontaneous recovery.\nSecond, most studies recruited participants through the community and through other websites. This is not a problem for interventions that target the general population. But when such an intervention is effective this does not automatically mean that it is also effective in clinical settings. Subjects who are responding to community recruitment are probably very motivated which may improve their results compared to subjects who receive treatment.\nThird, none of the twelve identified trials compared Internet-based treatments to face-to-face or other treatments. This is, however, an important issue, because only direct comparisons can give evidence about the comparative effects of Internet-based treatments compared to more traditional treatments and the type of patients who can benefit from it.\nFourth, most studies were aimed at adults. Only two studies were aimed at children and adolescents, while these groups are probably the most familiar with the Internet. None of the studies were specifically aimed at older adults, while they suffer most from health conditions.\nFuture research should focus on these limitations of current research. More studies are needed with care-as-usual or other control groups, clinical recruitment strategies, comparisons with face-to-face treatments, and children or older adults as target populations. More research is also needed to examine how CBT should be presented on the Internet, and to examine reasons and solutions to the relatively high drop-out rates in several studies. Finally, it is also important to study how Internet-administered CBT can be integrated in stepped-care models of care.\nThis review has several limitations. First, the number of included studies is still very small. And the number of studies examining specific health problems is too small to integrate the results of these studies statistically into a meta-analysis. Second, the quality of the included studies is not optimal. Third, the drop-out rates reported are high in some studies. This is a concern for this type of intervention, as patients can very easily withdraw from the intervention. Remarkably, the studies in which more traditional therapies (live sessions with therapists) are delivered through the Internet have the lowest drop-out rates.\nDespite these limitations, however, there is no doubt that the number of studies in this area will increase considerably in the next few years, while the promising results of the studies in this review indicate that the Internet will assume a major role in the delivery of CBT to patients with health problems.","keyphrases":["internet","cognitive behavior therapy","systematic review","pain","headache","migraine"],"prmu":["P","P","P","P","P","P"]}
{"id":"Knee_Surg_Sports_Traumatol_Arthrosc-3-1-1915597","title":"The distal fascicle of the anterior inferior tibiofibular ligament as a cause of tibiotalar impingement syndrome: a current concepts review\n","text":"Impingement syndromes of the ankle involve either osseous or soft tissue impingement and can be anterior, anterolateral, or posterior. Ankle impingement syndromes are painful conditions caused by the friction of joint tissues, which are both the cause and the effect of altered joint biomechanics. The distal fascicle of the anterior inferior tibiofibular ligament (AITFL) is possible cause of anterior impingement. The objective of this article was to review the literature concerning the anatomy, pathogenesis, symptoms and treatment of the AITFL impingement and finally to formulate treatment recommendations. The AITFL starts from the distal tibia, 5 mm in average above the articular surface, and descends obliquely between the adjacent margins of the tibia and fibula, anterior to the syndesmosis to the anterior aspect of the lateral malleolus. The incidence of the accessory fascicle differs very widely in the several studies. The presence of the distal fascicle of the AITFL and also the contact with the anterolateral talus is probably a normal finding. It may become pathological, due to anatomical variations and\/or anterolateral instability of the ankle resulting from an anterior talofibular ligament injury. When observed during an ankle arthroscopy, the surgeon should look for the criteria described to decide whether it is pathological and considering resection of the distal fascicle. The presence of the AITFL and the contact with the talus is a normal finding. An impingement of the AITFL can result from an anatomical variant or anteroposterior instability of the ankle. The diagnosis of ligamentous impingement in the anterior aspect of the ankle should be considered in patients who have chronic ankle pain in the anterolateral aspect of the ankle after an inversion injury and have a stable ankle, normal plain radiographs, and isolated point tenderness on the anterolateral aspect of the talar dome and in the anteroinferior tibiofibular ligament. The impingement syndrome can be treated arthroscopically.\nIntroduction\nImpingement syndromes of the ankle involve either osseous or soft tissue impingement and can be anterior, anterolateral, or posterior [5, 25]. Ankle impingement syndromes are painful conditions caused by the friction of joint tissues, which are both the cause and the effect of altered joint biomechanics [14]. The leading causes of impingement lesions are post-traumatic ankle injuries, usually ankle sprains, resulting in chronic ankle pain [33]. According to the ISAKOS definition, anterior ankle impingement is a pain syndrome characterised by anterior ankle pain on activity. Recurrent dorsiflexion is often the cause. On investigation, there is pain on palpation at the antero\/medial and\/or anterolateral aspect of the ankle joint. Some swelling and\/or limitations of dorsiflexion are present [32]. The first reported impingement syndromes about the ankle have evolved osseous impingement and generally have been noted in athletes whose sports necessitated sudden acceleration, jumping, and extremes of dorsiflexion or plantar flexion. Impingement of the ankle was first described in the English language literature by Morris [20], in 1943, in five patients who had what he called the athlete\u2019s ankle. This was later called footballer\u2019s ankle in the report of McMurray [18].\nSoft tissue impingement of the ankle is a common cause of chronic ankle pain that usually arises at the lateral and\/or anterolateral compartment of the ankle joint following an inversion injury [8]. It is estimated that approximately 3% of ankle sprains may lead to anterolateral impingement [32]. Three types of intra-articular soft tissue lesions that lead to these complaints have been described: the meniscoid lesion, synovitis, and the distal fascicle of the anterior inferior tibiofibular ligament (AITFL) [4, 6, 8, 9, 12, 16, 19, 28]. The formation of tibiotalar spurs due to repetitive capsule traction therefore does not seem plausible reason for anterior ankle impingement [29].\nImpinging soft tissue pathology in the ankle was first described by Wolin et\u00a0al. as a \u201cmeniscoid mass or lesion\u201d [34]. They described the lesion as a membrane shaped hyalinised scar tissue obstructing the lateral talofibular articulation in nine patients with persistent symptoms over the anterolateral corner of the ankle after an inversion trauma [34]. Some authors thought this lesion was formed from the torn ends of the anterior tibiofibular ligament [3, 21]. However there was no ligamentous tissue found on histologic examination of this lesion [9]. Guhl thought the lesion was of synovial origin [11]. Irritation of the joint by bleeding and a torn capsule or a torn anterior tibiofibular ligament are thought to be the underlying factors for developing post-traumatic synovitis in the ankle [17] which in unstable ankles may lead to formation of fibrosis and granulation tissue. Although synovitis and the meniscoid lesion have been referred frequently, impingement by a separated fascicle of the AITFL is a relatively new and unknown pathology. Bassett et\u00a0al. [4] were the first who reported a ligamentous etiology of impingement of the anterior aspect of the ankle. The presence of the so-called accessory AITFL, as described by Nikolopoulos was confirmed by them [21]. However they called this accessory ligament, the distal fascicle of the normal AITFL [4]. The distal part of the fascicle rubs against the talus, and that this causes pain at the ankle [1, 2, 4, 8, 12, 22].\nThe objective of this article was to review the literature concerning the anatomy, pathogenesis, symptoms and treatment of the AITFL impingement and finally to formulate treatment recommendations.\nAnatomy of the anterior inferior tibiofibular ligament\nThe distal tibiofibular joint was classically described as a syndesmosis between the rough, triangular surface on the medial and distal aspect of the fibular shaft and the fibular notch of the distal end of the tibia. The tibiofibular syndesmosis is established by three ligaments: the interosseous ligament, the posterior inferior tibiofibular ligament, and the AITFL [7, 10, 15, 24, 26].\nThe AITFL is a flat band becoming thicker from superior to inferior [24]. The ligament starts from the distal tibia, 5\u00a0mm in average above the articular surface, and descends obliquely between the adjacent margins of the tibia and fibula, anterior to the syndesmosis to the anterior aspect of the lateral malleolus [7, 22]. Its length ranges from 12 to 20\u00a0mm [4, 22]; its thickness, from 1 to 3\u00a0mm [4, 22]; and its width ranges from 7 to 12\u00a0mm at the fibular insertion and 9 to 22\u00a0mm at the tibial insertion [22]. This ligament is the weakest of the syndesmotic ligaments and is the first to yield to forces that promote an external rotation of the fibula around its longitudinal axis [15]. Resection of the accessory ligament did not disturb the stability of the syndesmosis, a finding that was in accordance with the results reported by Rasmussen et\u00a0al. which suggests that mobility is minimally influenced by isolated cutting of the AITFL [23].\nNikolopoulos et\u00a0al. studied 24 cadaveric ankles to describe the anatomy of the AITFL [22]. In five ankles the AITFL appeared to consist of two layers, one superficial and one deep, which were separated by a thin fibrofatty septum [22, 24]. However, the total thickness of these two layers did not exceed the average thickness of the AITFL in the rest of the specimens [22]. In five ankles, the articulated surfaces of the distal tibia and fibula that formed the syndesmosis were covered by articular cartilage and were surrounded by synovial bursa [22]. A completely separate accessory anteroinferior tibiofibular ligament was present in 22 ankles (Fig.\u00a01). Nikolopoulos was the first to describe this accessory ligament [21]. However the results of his study were never published in English literature [26]. Nikolopoulos believed that this anatomical structure represents a separate ligament rather than a distal component of the AITFL [21]. The fact that there was clearly a fibrofatty septum that separates the AITFL from the accessory fascicle, similar to the one that covered the intermediate space between the interosseous and the anterior or the interosseous and the posterior tibiofibular ligaments, suggested that this fascicle could probably be considered an independent accessory ligament [22]. Some other authors described this accessory ligament as a complete separate distal fascicle of the AITFL [4, 15, 26].\nFig.\u00a01The distal fascicle in relation to the anterior inferior tibiofibular ligament\nRay and Kriz [24] founded in 10 of the 46 specimens an accessory ligament. This ligament was parallel and distal to the AITFL. The fibres of the accessory ligament descended obliquely from the lateral, anterior, and distal borders of the tibia to the anteromedial aspect of the lateral malleolus, approximating the fibular insertion of the anterior talofibular ligament (ATFL).\nIts length ranges from 17 to 22\u00a0mm, its thickness from 1 to 2\u00a0mm, and its width from 3 to 5\u00a0mm. The maximum, minimum, mean, and standard deviation for the length, width, gap, and angle of declination for the inferior fibre bundle are provided in Table\u00a01. The ligament was not covered by synovial tissue, ran deeper than the AITFL and was described as being intra-articular, although how this was determined was not stated by the authors. The accessory ligament crossed the superior lateral margin of the ankle joint. The accessory ligament was separated from the AITFL by a distinct fibrofatty septum. The ligament could even be divided in two, three, or more fascicles, increasing in length from superior to inferior [24].\nTable\u00a01Characteristics of the inferior fascicle of the AITFL [24]MaximumMinimumMeanLigament length (cm)3.42.12.6\u00a0\u00b1\u00a00.3Ligament width (cm)1.30.20.4\u00a0\u00b1\u00a00.2Gap (cm)0.50.10.2\u00a0\u00b1\u00a00.1Declination angle (deg)155.0121.5136.3\u00a0\u00b1\u00a07.4\nThe incidence of the accessory fascicle differs very widely in the several studies (21\u201392%) [2, 4, 22, 24]. The discrepancies in the incidence between the several studies were probably due to the different definitions of a separate fascicle. Ray and Kriz did not consider it a separate fascicle if any digitations were present between the fascicle and the main body of the AITFL [24]. The incidence was up to 92%, which implied that the ligament may be considered as a normal finding. Vessels from the anterior peroneal artery penetrate through the interlaminar spaces [26]. It is not uncommon to have branches from the anterior tibial artery or peroneal artery or a branch arising from the anastomosis between these two arteries pass between these fascicles [11]. The most inferior fibres of the ligament merged at their fibular attachment with the origin of the ATFL [24].\nRay and Kriz [24] studied 23 cadaveric ankles. A classification scheme was designed to categorise the anatomical variations observed in the AITFL (Table\u00a02).\nTable\u00a02AITFL classification system [24]Type 1Multiple fascicles (more than three) with or without small gaps between adjacent fascicles \u00a01AThe inferior fascicle is separated from the rest of the ligament by a gap and possesses its own distinct proximal and distal attachments \u00a01BThe inferior fascicle is not completely separated from the rest of the ligament by a gap. Either its proximal or its distal attachment is continuous with the rest of the ligament \u00a01CMultiple fascicles without gaps intervening between themType 2Three fascicles or less. A distinct inferior fascicle with both its proximal and distal attachments separate from the rest of the ligament. The inferior fascicle is separated completely from the main portion of the ligament by a gapType 3Three fascicles or less. A distinct inferior fascicle with either its proximal or its distal attachment continuous with the rest of the ligament. A gap does not completely separate the inferior fascicle from the rest of the ligamentType 4Three fascicles or less. The lower portion of the ligament possesses an inferior fascicle with both its proximal and distal attachments for the rest of the ligamentType 5Three fascicles or less. A ligament with no separations or gaps within its structure. It may or may not possess a fascicular arrangement\nPathomechanism of the impingement syndrome\nIt should be stressed that most studies were carried out in vitro and do not take muscular tonus and the effects of weightbearing into consideration, we performed a review of the pathomechanism of the impingement syndrome caused by the distal fascicle of the AITFL.\nBassett et\u00a0al. correlated the presence of this accessory ligament with pain from impingement in the anterolateral aspect of the ankle in patients with a history of inversion sprains [4]. They postulated that the post-traumatic anterolateral hyperlaxity, due to an injured ATFL, resulted in anterior extrusion of the talar dome with dorsiflexion, which now contacted the inferior fascicle of the AITFL with more pressure and friction [4]. This in turn might set up an environment favouring ligamentous impingement of the anterior aspect of the ankle [4].\nAkseki et\u00a0al. stated that the variations in width, length, and obliquity of the fascicle may be related to the pathological behaviour of the fascicle [2]. Mean width and length of the fascicle with bending during dorsiflexion and dorsiflexion-eversion were significantly higher than the fascicle without bending. Therefore this wider and longer fascicles had more potential to become pathological than thinner ones. They also conclude that if the fibular insertion point is far from the joint level, the fascicle has more potential to become pathological. These findings might be useful during arthroscopic procedures of the ankle.\nDuring dorsiflexion of the ankle the posterior shift of the anterior talus below the tibial plafond in ATFL intact cases was observed. Following incision of the ATFL this posterior shift of the talus was not observed; the talus remained in an anterior position and thus in contact with the fascicle [2]. Lesion of the ATFL led to bending of the fascicle with dorsiflexion and dorsiflexion-inversion in the specimens which the bending sign was negative. Lesion of the ATFL led also to contact between the talus and the fascicle in the ankles which showed no contact previously [2]. After sectioning the ATFL the anteroposterior laxity of the ankle increases especially in dorsiflexion [4, 13].\nIn 89.3% of the cases contact was observed between the ATFL and the anterolateral corner of the talus [2]. This contact may be normal in some of the cases. Contact was observed at an average of 12\u00b0 of dorsiflexion of the ankle [4]. Bending of the accessory ligament, indicating further tensioning of its ligamentous bands caused by impingement of the talus, this was noted in an extent of dorsiflexion that varied from 5\u00b0 to 10\u00b0 (average 7\u00b0) [22]. Maximum dorsiflexion of the foot relieved the contact between the ligament and the superolateral corner of the talus [22]. In other studies the contact was also observed in neutral position [2, 24].\nIn one study the contact between the fascicle and talar dome was observed throughout the whole range of motion of the ankle [1]. They called this the \u201carthroscopic impingement test,\u201d and it was positive in all of the cases with AITFL impingement they have studied [1]. Contact between the accessory fascicle and the talus was also observed during eversion of the foot, being more prominent when the foot was placed in a dorsiflexion-eversion position [22]. No interaction of the accessory ligament with the talus was noted during ankle plantar flexion [22].\nIn 82.9% of the cases with AITFL impingement Ray and Kriz [24] found an anterior abraded articular region of the talus. Akseki et\u00a0al. [2] found this in 17% of the cases. In the study of Basset et\u00a0al. [4] 71% of the patients, had an area of abraded articular cartilage on the anterolateral surface of the talar dome. There is no clarity if this abraded cartilage region is an anatomical variant of the talus or a pathological remodelling of the bone due to true ligamentous impingement [4].\nOn base of these studies we conclude that anterolateral hyperlaxity results in anterior extrusion of the talar dome with dorsiflexion, which contacts the inferior fascicle of the AITFL with more pressure and friction. This hyperlaxity was most likely due to an injured ATFL. Wider and longer fascicles and a fibular insertion point far from the joint level have more potential to become pathological. Often contact between the AITFL and the superolateral corner of the talus and an abraded region of the cartilage of the talus can be observed during arthroscopy.\nDiagnosis\nThe diagnosis of ligamentous impingement in the anterior aspect of the ankle should be considered in patients who have chronic ankle pain in the anterolateral aspect of the ankle after an inversion sprain and who have a stable ankle, normal plain radiographs, and isolated point tenderness on the anterolateral aspect of the talar dome and in the anteroinferior tibiofibular ligament [4]. A popping sensation and aggravation of pain with dorsiflexion and eversion were noted [2, 4]. This audible popping is usually not seen in other causes of impingement of the anterior aspect of the ankle [4]. Pain in the beginning of plantarflexion-inversion may alert the physician for an impinging AITFL [2].\nIt is usually impossible to distinguish the source of pain in patients with both instability and impingement. So if the patient complains of giving way and frequent sprains, the surgeon may choose a reconstructive procedure [1]. But it should be noted that a painful lesion in the joint (synovitis, meniscoid lesion, or an impinging fascicle of the AITFL) may cause reflex sprains [1]. This process may confuse decision making in this type of combined pathology. The differential diagnosis for pain in the lateral aspect of the ankle after an inversion sprain includes osteochondral fracture, instability of the lateral aspect of the ankle, synovitis or rupture of the peroneal tendon, and ligamentous impingement in the anterior aspect of the ankle, with the latter being the least common [4].\nTreatment\nPatients should be treated conservatively after an inversion sprain of the ankle for at least 6\u00a0months before operative intervention is considered [5]. This is with the exception of patients with an osteochondral fracture. Akseki et\u00a0al. observed that conservative management with physical therapy, NSAIDs and bracing in patients with instability symptoms, for a period of 3\u00a0months, failed in all 21 patients [1].\nNot every patient with chronic ankle instability needs a reconstructive procedure [1]. If the history, physical examination, and other diagnostic tests suggest impingement in addition to instability, arthroscopic debridement of the soft tissue lesion may be all that is needed [1. In cases with an area of abraded articular cartilage on the anterolateral surface of the talar dome, further debridement of this region was required [4]. At long-term follow-up arthroscopic excision of soft tissue overgrowth and osteophytes proved to be an effective way of treating anterior impingement of the ankle in patients who had no narrowing of the joint space [30]. Patients with less than 2\u00a0years of ankle pain before surgery for anterior ankle impingement showed significant better scores in pain, swelling, ability to work, and engagement in sports [31].\nThe decision to perform an arthroscopy of the ankle is typically based on the patient\u2019s history and physical examination [9, 19, 28]. Arthroscopically resection of the distal fascicle of the AITFL should be considered when there (Fig.\u00a02): (1) is contact between the AITFL and the talus was prominent also in the beginning of plantarflexion-inversion of the ankle, (2) is increased contact between the talus and the ligament and this continued until maximum dorsiflexion with abrasion of the articular cartilage (the surgeon should remember that this finding may be normal), (3) bending of the fascicle on the anterolateral edge of the talus with dorsiflexion and dorsiflexion-inversion, (4) is a distally inserting fascicle on the fibula, close to the origin of the ATFL on the fibula. This finding may be missed if the distraction is preserved throughout the procedure [2, 24]. For this reason, temporarily relieving distraction is advised, when a distal fascicle is seen during arthroscopy [2, 22].\nFig.\u00a02There is contact between the distal fascicle of AITFL and the talus in dorsiflexion with bending of this fascicle\nOnly three studies reported on the results of the treatment of AITFL impingement [1, 4, 12]. Akseki et\u00a0al. [1] arthroscopically resected the distal fascicle of the AITFL in 21 patients with chronic ankle pain after an ankle sprain. During the procedure an impinging distal fascicle of the AITFL was found in all cases. Following anterolateral synovectomie, the fascicle was excised. At an average follow-up of 3\u00a0years good to excellent results were obtained in 17 patients. Nineteen patients were satisfied with the procedure and 17 patients returned to previous level of activity. Two patients who had mild laxity were graded as poor because neuromas of the terminal branches of the superficial peroneal nerve. These patients became asymptomatic after an injection of steroids.\nBassett et\u00a0al. [4] treated seven patients who had talar impingement by an AITFL and they were followed for a mean of 39\u00a0months. Two patients had arthroscopy and five had an arthrotomy. In all patients, a thickened distal fascicle of the AITFL was resected. Five patients also needed debridement of an area of abraded hyaline cartilage on the anterolateral aspect of the talar dome at the point where the distal fascicle made contact. Post-operative management consisted of 2\u20134\u00a0days of immobilisation, followed by progressive weightbearing. Four patients reported no pain in their ankle or limitation in activity, and the results were considered to be excellent. Two patients reported improvement, with only occasional pain related to overuse, and the result was considered good. The last patient had been followed for only 6\u00a0months with excellent results.\nHorner and Liu [12] treated nine athletes with anterolateral ankle pain during and following athletic activities without history of ankle injury. After 3\u00a0months conservative management each patient was explored arthroscopically. Patients\u2019 outcomes were rated subjectively and objectively based on the UCLA ankle scoring system. Pathological characteristics were easily identifiable through arthroscopy. The structure may be resected during this procedure without sacrificing the stability of the ankle joint. They report that good to excellent results can be expected from this procedure including in the highest level of athleticism at an average follow-up of 18\u00a0months.\nThe decision to perform an arthroscopy of the ankle is typically based on the patient\u2019s history and physical examination. When an impinging distal fascicle of the AITFL and an abraded anterolateral region of the talus were observed during an ankle arthroscopy, the surgeon should look for the criteria described above to decide whether it is pathological and needs to be resected.\nConclusion\nBecause of the lack of evidence only preliminary conclusions can be drawn. The AITFL started from the distal tibia, 5\u00a0mm in average above the articular surface, and descended obliquely between the adjacent margins of the tibia and fibula, anterior to the syndesmosis to the anterior aspect of the lateral malleolus. The incidence of the accessory fascicle differs very widely in the several studies. The presence of the distal fascicle of the AITFL and also the contact with the anterolateral talus is probably a normal finding. It may become pathological, however, due to anatomical variations and\/or anterolateral instability of the ankle resulting from an ATFL injury. When observed during an ankle arthroscopy, the surgeon should look for the criteria described to decide whether it is pathological and considering resection of the distal fascicle.","keyphrases":["anterior inferior tibiofibular ligament","impingement syndrome","ankle","accessory fascicle"],"prmu":["P","P","P","P"]}
{"id":"Purinergic_Signal-3-4-2072926","title":"Adenosine A2A receptors modulate BDNF both in normal conditions and in experimental models of Huntington\u2019s disease\n","text":"Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, enhances synaptic transmission and regulates neuronal proliferation and survival. Functional interactions between adenosine A2A receptors (A2ARs) and BDNF have been recently reported. In this article, we report some recent findings from our group showing that A2ARs regulate both BDNF functions and levels in the brain. Whereas BDNF (10 ng\/ml) increased the slope of excitatory postsynaptic field potentials (fEPSPs) in hippocampal slices from wild-type (WT) mice, it was completely ineffective in slices taken from A2AR knock-out (KO) mice. Furthermore, enzyme immunoassay studies showed a significant reduction in hippocampal BDNF levels in A2AR KO vs. WT mice. Having found an even marked reduction in the striatum of A2AR KO mice, and as both BDNF and A2ARs have been implicated in the pathogenesis of Huntington\u2019s disease (HD), an inherited striatal neurodegenerative disease, we then evaluated whether the pharmacological blockade of A2ARs could influence striatal levels of BDNF in an experimental model of HD-like striatal degeneration (quinolinic acid-lesioned rats) and in a transgenic mice model of HD (R6\/2 mice). In both QA-lesioned rats and early symptomatic R6\/2 mice (8 weeks), the systemic administration of the A2AR antagonist SCH58261 significantly reduced striatal BDNF levels. These results indicate that the presence and the tonic activation of A2ARs are necessary to allow BDNF-induced potentiation of synaptic transmission and to sustain a normal BDNF tone. The possible functional consequences of reducing striatal BDNF levels in HD models need further investigation.\nIntroduction\nBrain-derived neurotrophic factor (BDNF) is an endogenous glycoprotein belonging to neurotrophins, a family of signalling molecules that play a key role in regulating neuronal proliferation, differentiation and survival [1\u20133]. Among the neurotrophins, BDNF has the widest distribution in the central nervous system [4, 5]. In the adult hippocampus, BDNF is critically involved in the regulation of synaptic plasticity [6] and facilitates long-term potentiation (LTP) [7\u201310; see 11 for review]. Most biological effects of BDNF are mediated by the tyrosine kinase receptor TrkB. Both BDNF and its receptors are highly expressed in the hippocampus, and the activation state of the complex BDNF\/TrkB appears critical for modulating synaptic efficacy [12] and the response to excitotoxic injury [13, 14].\nIn hippocampal neurones, adenosine has been reported to transactivate TrkB receptors, an effect involving the A2A receptor subtype (A2ARs) [15]. Furthermore, Diogenes et al. [16] reported that A2ARs facilitate the excitatory action of BDNF on hippocampal synaptic transmission.\nBesides its involvement in hippocampal functions, BDNF is also very important for the survival of striatal neurons and the activity of corticostriatal synapses [17]. Impairment in BDNF function is though to play a major role in Huntington\u2019s disease (HD), an inherited neurodegenerative disease caused by a mutation in the protein huntingtin and characterised by marked striatal degeneration (see [18] for review). Specifically, it has been shown that the activity of BDNF depends on the presence of normal huntingtin [19\u201321]. In vitro and in vivo data showed that wild-type huntingtin, but not the mutant protein, stimulates cortical BDNF production by acting at the level of Bdnf gene transcription [20, 21]. At the corticostriatal synapse, BDNF controls glutamate release and its exogenous administration allows striatal neurons to survive excitotoxin-induced neurodegeneration [22].\nMounting evidence also indicates an involvement of striatal A2A receptors in HD (see [23] and [24] for reviews). First, A2ARs are mainly localised on the neurons that degenerate early in HD [25]. Second, A2ARs and underlying signalling systems undergo profound changes in cellular and animal models of HD (see [24] for review). Thus, not only A2ARs seem to regulate BDNF functions in the brain, but both BDNF and A2ARs seem to be implicated in HD.\nIn this article we report and critically discuss some recent findings on the following issues obtained by our group: (1) the regulatory role exerted by hippocampal A2ARs on BDNF functions and levels as revealed by studies performed in A2AR KO mice, and (2) the effects of A2AR blockade on striatal BDNF levels in experimental models of HD or HD-like striatal degeneration.\nIn hippocampal slices from wild-type mice, the facilitatory effects exerted by BDNF on synaptic transmission require the endogenous activation of A2AR\nIn hippocampal slices from wild-type (WT) mice (400-\u03bcm thick, see [26] for a detailed methodological description), BDNF (10\u00a0ng\/ml over 30\u00a0min) induced a long-lasting increase in the slope of field excitatory postsynaptic potential (fEPSP) recorded in the CA1 area (Fig.\u00a01). This effect was abolished not only by the inhibitor of Trk phosphorylation K-252a, but also by the selective adenosine A2A antagonists ZM241385 and SCH58261 (Tebano et al., manuscript in preparation). The effects of A2ARs seem to involve the cyclic adenosine monophosphate\/protein kinase A (cAMP-PKA) pathway, the main transduction mechanism operated by A2ARs, as the selective PKA inhibitor KT 5720 abolished the excitatory action of BDNF (Tebano et al., manuscript in preparation).\nFig.\u00a01Brain-derived neurotrophic factor (BDNF) facilitates synaptic transmission in hippocampal slices from wild-type (WT) but not A2A receptor (R) knock-out (KO) mice. In hippocampal slices from WT mice, BDNF (10\u00a0ng\/ml) induces an increase of the excitatory postsynaptic field potentials (fEPSP) slope, whereas a lower concentration (5\u00a0ng\/ml) is ineffective. BDNF (5, 10 and 20\u00a0ng\/ml) was totally ineffective in hippocampal slices from A2AR KO mice. *P\u2009<\u20090.05 vs. baseline (paired t test)\nThe finding that BDNF enhances synaptic transmission in mice hippocampal slices confirms and strengthens previous data on the acute synaptic effects of BDNF at adult central synapses [17, 27]. Although the mechanisms responsible for such synaptic effects are not completely understood, they do not seem to be related to the neuroprotective ability of BDNF. That the above functions of BDNF recognise different molecular mechanisms is indicated by the fact that ligand-induced TrB translocation into lipid rafts is required for short-term synaptic modulation, but not neuronal survival, by BDNF [28]. Whatever the mechanisms, the blocking effect of ZM241385 and SCH58261 suggests that A2ARs have to be tonically activated by endogenous adenosine to manifest BDNF effects. To further explore the apparently \u201cpermissive\u201d role of adenosine A2ARs on the synaptic effects of BDNF, A2AR KO mice were used.\nBDNF is no longer able to facilitate synaptic transmission in hippocampal slices from A2A KO mice\nA2AR KO mice (A2AR-\/-) were generated, as previously described [29]. In hippocampal slices from A2AR KO mice, BDNF was no longer able to increase the fEPSP slope (Tebano et al., manuscript in preparation). As shown in Fig.\u00a01, none of the tested concentrations of BDNF (5, 10 and 20\u00a0ng\/ml) potentiated the synaptic response in slices from A2AR KO mice. Since the effectiveness of BDNF may depend on the proper expression of its receptors, we compared the levels of TrkB protein in WT and A2A KO mice by Western blotting. By using primary anti-TrkB antibody (Bioscience), we found that the levels of full-length TrkB isoforms were very similar in hippocampal extracts from WT and A2AR KO mice (Tebano et al., manuscript in preparation). Thus, the loss of BDNF activity observed in A2AR KO mice is not associated with a reduced density of TrkB receptors.\nBDNF protein levels are significantly reduced in the brain of A2AR KO vs. WT mice\nThe BDNF protein levels were then measured on hippocampal extracts from A2AR KO and WT mice using the BDNF immunoassay system [enzyme-linked immunosorbent assay (ELISA) kit; Promega]. Interestingly, hippocampal BDNF levels were significantly lower in the A2AR KO mice compared with the WT littermates. As shown in Fig.\u00a02, such an effect was not confined to the hippocampus, as a significant loss of BDNF levels was observed also in the striatum of A2A KO vs. WT mice.\nFig.\u00a02Brain-derived neurotrophic factor (BDNF) levels are significantly reduced in the hippocampus and striatum of A2A receptor (R) knock-out (KO) mice. BDNF concentration was significantly reduced in extracts of hippocampal and striatal tissues prepared from A2AR mice compared with wild-type WT mice. The amount of BDNF (total) is expressed as pg\/mg of wet tissue [means \u00b1 standard error of the mean (SEM) from five mice\/group]. *P\u2009<\u20090.05 vs. WT\nThis result indicates that the presence of A2ARs is required to maintain normal BDNF levels in the brain. Although the mechanism by which A2ARs regulate BDNF is yet undetermined, it is worth mentioning that the cAMP-PKA pathway (the main transduction system operated by A2ARs) has been implicated not only in \u201cgating\u201d BDNF functions [30], but also in modulating BDNF gene transcription [31] and release [32]. Furthermore, Kolarow et al. [33] showed that the PKA inhibitor Rp-cAMP significantly inhibited and delayed BDNF secretion in hippocampal neurons. Interestingly, the endogenous state of activation of A2ARs seems to be adequate to sustain a normal BDNF tone in WT mice, as no increase in the hippocampal levels of BDNF were observed in WT mice treated with a single (0.5\u00a0mg\/kg i.p sacrifice 24\u00a0h later) or with repeated (0.5\u00a0mg\/kg per day over 5\u00a0days) doses of the A2A agonist CGS 21680.\nOn their whole, the experiments performed in A2A KO mice indicate that A2ARs are essential for the tone and synaptic activity of BDNF. It should be noted, however, that the stimulation or the blockade of A2ARs can induce effects very different from those achieved after stimulation or blockade of the BDNF\/TRkB system. For instance, whereas BDNF is thought to play an important role in memory processes [34, 35], adenosine A2ARs seem to negatively influence them. Indeed, an improvement in spatial memory has been reported in A2AR KO mice [36], whereas working memory was impaired in rats overexpressing A2ARs [37]. Another consideration that apparently weakens the importance of the A2AR\/BDNF interaction is that A2ARs and TrkB Rs undergo opposite age-related changes in the hippocampus [38]. According to the authors\u2019 interpretation, however, the relationship between age-related changes in the density of TrkB and A2AR receptors does play a role in the modulation of BDNF effects, thus even reinforcing the hypothesis of a close functional interplay between A2ARs and BDNF.\nA2ARs regulate striatal BDNF levels in experimental models of HD or HD-like striatal degeneration\nTwo different models of HD were used: the quinolinic-acid (QA)-lesioned rats (pathogenetic model of HD-like striatal degeneration, [39]) and transgenic R6\/2 mice (genetic HD model, [40]). For the method of QA-induced striatal lesions, see [41]. Briefly, QA (180\u00a0nmol\/1\u00a0\u03bcl) was unilaterally injected in the striatum of anaesthetised rats, whereas vehicle [1\u00a0\u03bcl phosphate-buffered saline (PBS)] was injected in the other side. SCH58261 was administered at the doses of 0.01 and 1\u00a0mg\/kg i.p. 20\u00a0min before QA lesion. The levels of BDNF were measured 24\u00a0h after surgery. As shown in Fig.\u00a03, BDNF levels were significantly increased in the QA-lesioned vs. the control side. In animals pretreated with both doses of SCH58261 the rise in striatal BDNF was completely prevented. Worthy of note, SCH58261 markedly reduced BDNF levels in the control (vehicle-injected) side also (Fig.\u00a03). As the early rise in BDNF levels most probably reflects a compensatory mechanism following QA injection, one can consider that SCH58261 may worsen QA effects by preventing BDNF increase. Previous studies have, however, demonstrated that this is not the case. We found, indeed, that whereas SCH58261 exerted very clear neuroprotective effects towards QA at 0.01\u00a0mg\/kg i.p., it was no longer neuroprotective when administered at 1\u00a0mg\/kg [39]. The finding that SCH58261 reduces BDNF levels irrespective of the dose (neuroprotective or non-neuroprotective) indicates that early changes in the striatal BDNF levels do not seem to correlate with the clinical and neuropathological outcome in the QA model.\nFig.\u00a03The A2A receptor (R)-antagonist SCH58261 reduces striatal brain-derived neurotrophic factor (BDNF) levels in control and quinolinic-acid (QA)-lesioned rats. QA (180\u00a0nmol\/1\u00a0\u03bcl) was unilaterally injected in the striatum of anaesthetised rats, whereas vehicle [1\u00a0\u03bcl phosphate-buffered saline (PBS)] was injected in the other side. SCH58261 was administered at the doses of 0.01 and 1\u00a0mg\/kg i.p., 20\u00a0min before QA lesion. The levels of free mature BDNF were measured 24\u00a0h after surgery. BDNF levels were significantly increased in the QA-lesioned vs. the control side. In animals pretreated with both doses of SCH58261, the rise in striatal BDNF was completely prevented. N\u20095\/group. \u00b0P\u2009<\u20090.05 vs. PBS. *P\u2009<\u20090.05 vs. corresponding vehicle\nR6\/2 HD mice may represent a more suitable model to evaluate whether A2AR antagonists have a worsening influence through the reduction of striatal BDNF levels. Indeed, the administration of BDNF has been reported to have beneficial effects in transgenic models of HD [42, 43]. Considering that in the transgenic mice the neurodegenerative process progressively takes place over several weeks, to ascertain whether A2AR blockade could influence striatal BDNF levels, we decided to treat R6\/2 mice chronically with SCH58261. The drug was administered at the dose of 0.01\u00a0mg\/kg per day i.p. over 1 or 3\u00a0weeks starting from the fifth week of age. Another group of R6\/2 mice was treated with vehicle over 3\u00a0weeks. BDNF levels were then assayed at the beginning of the symptomatic phase (8\u00a0weeks of age). Three groups of age-matched WT littermates were used as controls and treated in the same way described as above. As shown in Fig.\u00a04, BDNF protein levels were not reduced, and showed instead a nonsignificant tendency to increase, in the striatum of early symptomatic R6\/2 vs. WT mice. This finding was unexpected, as a significant decrease in BDNF mRNA has been recently reported in the striatum of R6\/2 mice of the same age [43]. Although we have not explored the mechanisms responsible for such a discrepancy, a dissociation between the levels of BDNF protein and mRNA has already been observed in the brains of transgenic R6\/1 HD mice [44]. Another reason for which one would expect a reduction in BDNF levels in the striatum of R6\/2 mice is that a marked reduction in the expression of A2ARs (which, as mentioned above, are very important to maintain normal BDNF levels) was reported in these mice [45]. More recently, however, the A2AR-stimulated adenylyl cyclase, the A2AR density, and their affinity for the agonist CGS21680 were found unchanged in the striatum of clearly symptomatic R6\/2 mice [46].\nFig.\u00a04The A2A receptor (R)-antagonist SCH58261 reduces striatal brain-derived neurotrophic factor (BDNF) levels in R6\/2 HD mice SCH58261 was administered at the dose of 0.01\u00a0mg\/kg per day i.p. over 1 or 3\u00a0weeks starting from the fifth week of age. Free mature BDNF levels were then assayed at the beginning of the symptomatic phase (8\u00a0weeks of age). Three groups of age-matched wild-type (WT) littermates were used as controls and treated in the same way as described above. Both schedules (1 and 3\u00a0weeks) of SCH treatment significantly reduced BDNF levels in the striatum of either R6\/2 and WT mice. N\u2009\u20094\u20137\/group. *P\u2009<\u20090.05 vs. corresponding control group (two-tailed unpaired t test)\nAs for the influence of A2AR blockade, both schedules (1 and 3\u00a0weeks) of SCH treatment significantly reduced BDNF levels in the striatum of either R6\/2 and WT mice (Fig.\u00a04). Again, however, such an effect does not necessarily imply a negative influence of SCH58261 on HD mice. Indeed, in recent experiments, we found that SCH treatment (0.01\u00a0mg\/kg per day i.p. between the 5th and the 6th weeks of age) exerted some beneficial effects, namely, normalisation of emotional behaviour and restoration of a normal sensitivity to NMDA in corticostriatal slices (Domenici et al., manuscript in preparation). Thus, although the pharmacological blockade of A2ARs significantly reduces striatal BDNF levels in experimental models of HD or HD-like neurodegeneration, this does not seem to have a negative impact on the course of the disease. Of course, it is still possible that reducing BDNF levels at other time points of the pathological process has a much marked influence on the disease. This issue, as well as the evaluation of BDNF protein changes at different stages of the disease, will be the object of future studies.\nConclusions\nOver the last several years, BDNF has emerged not only as a potent neuromodulator but also as a substance exerting fast excitatory actions in neurons, controlling resting membrane potential, neuronal excitability and synaptic transmission and participating in the induction of long-term changes in synaptic transmission [47]. In agreement with the recent evidence that A2ARs play a major role in regulating BDNF functions [16, 48], data reported in this article indicate that the presence and tonic activation of A2ARs are necessary to allow BDNF-induced potentiation of synaptic transmission. As for the mechanisms responsible for the permissive effects of A2ARs, data indicate an involvement of the cAMP-PKA pathway, the main transduction mechanism operated by A2ARs. The finding of reduced BDNF levels in the brain of A2AR KO mice indicates that A2ARs are critical not only to allow the synaptic effects of BDNF but also to maintain normal BDNF levels. Findings also point out that A2ARs tonically regulate BDNF levels in different areas of the brain, irrespective of their levels of expression. The tonic effect exerted by A2ARs in the regulation of BDNF levels is also evident in experimental models of HD (R6\/2 mice) and of HD-like striatal degeneration (QA lesion in rats).\nGiven that BDNF delivery has beneficial effects in models of HD [42, 43], the possible functional consequences of reducing striatal BDNF levels in those models need to be clarified. In particular, further investigations on trophic factors and associated signal pathways are warranted before A2AR antagonists can be considered as a suitable therapeutic approach to HD [24].","keyphrases":["adenosine a2a receptors","huntington\u2019s disease","synaptic transmission","bdnf levels","striatum","hippocampus"],"prmu":["P","P","P","P","P","P"]}
{"id":"Histochem_Cell_Biol-4-1-2386529","title":"Intermediate filament cytoskeleton of the liver in health and disease\n","text":"Intermediate filaments (IFs) represent the largest cytoskeletal gene family comprising ~70 genes expressed in tissue specific manner. In addition to scaffolding function, they form complex signaling platforms and interact with various kinases, adaptor, and apoptotic proteins. IFs are established cytoprotectants and IF variants are associated with >30 human diseases. Furthermore, IF-containing inclusion bodies are characteristic features of several neurodegenerative, muscular, and other disorders. Acidic (type I) and basic keratins (type II) build obligatory type I and type II heteropolymers and are expressed in epithelial cells. Adult hepatocytes contain K8 and K18 as their only cytoplasmic IF pair, whereas cholangiocytes express K7 and K19 in addition. K8\/K18-deficient animals exhibit a marked susceptibility to various toxic agents and Fas-induced apoptosis. In humans, K8\/K18 variants predispose to development of end-stage liver disease and acute liver failure (ALF). K8\/K18 variants also associate with development of liver fibrosis in patients with chronic hepatitis C. Mallory-Denk bodies (MDBs) are protein aggregates consisting of ubiquitinated K8\/K18, chaperones and sequestosome1\/p62 (p62) as their major constituents. MDBs are found in various liver diseases including alcoholic and non-alcoholic steatohepatitis and can be formed in mice by feeding hepatotoxic substances griseofulvin and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). MDBs also arise in cell culture after transfection with K8\/K18, ubiquitin, and p62. Major factors that determine MDB formation in vivo are the type of stress (with oxidative stress as a major player), the extent of stress-induced protein misfolding and resulting chaperone, proteasome and autophagy overload, keratin 8 excess, transglutaminase activation with transamidation of keratin 8 and p62 upregulation.\nIntermediate filaments in health and disease\nTogether with the actin microfilaments and the microtubules, intermediate filaments (IFs) are the components of the cytoskeleton of eukaryotic cells, which is involved in the maintenance of cell shape, locomotion, intracellular organization, and transport (Bershadsky and Vasiliev 1988; Ku et al. 1999). IF proteins comprise a large family, which includes ~70 different genes (Omary et al. 2004; Kim and Coulombe 2007; Herrmann et al. 2007; Goldman et al. 2008). They are further divided into six subtypes (Table\u00a01), which are, at least in part, expressed in a cell type (and differentiation)-dependent manner (Omary et al. 2004; Kim and Coulombe 2007; Goldman et al. 2008). Accordingly, IFs serve as cell type markers and antibodies to IF proteins are widely used today in diagnostic pathology (Wick 2000; Barak et al. 2004). Individual IF proteins consist of a conserved central coiled-coil \u03b1-helical rod domain (interrupted by linkers) which is flanked by N-terminal (head) and C-terminal (tail) domains (Omary et al. 2006; Godsel et al. 2008; Kim and Coulombe 2007; Herrmann et al. 2007; Goldman et al. 2008). The N- and C-terminal domains contribute to the structural heterogeneity and are major sites of posttranslational modifications with phosphorylation being the best characterized one (Omary et al. 1998; 2006). This makes them important regulatory domains, since dynamic changes in phosphorylation status are responsible for alterations in IF dynamics, solubility, and organization (Omary et al. 2006).Table\u00a01Intermediate filament proteinsTypeName\/LocalizationDisease locationRemarksI (n\u00a0=\u00a028)K9-28 (epithelia)K10,14,16,17-skinAcidic keratinsK31-40 (hair\/nail)K12-cornea(pI\u00a0<\u00a05.7)K13-stratified mucosaType I\/II obligate 1:1 polymersK16,17-nailK18a-liverII (n\u00a0=\u00a026)K1-8, K71-80 (epithelia)K1,2e,5,9-skinBasic keratinsK81-86 (hair\/nail)K3-cornea(pI\u00a0\u2265\u00a06.0)K4-stratified mucosaType I\/II obligate 1:1K6a,6b-nailpolymersK8a-liverK75a,81,83,85,86-hairIIIDesmin (muscle)Muscle, heartDesmin, vimentin and GFAP are found in stellate cellsVimentin (mesenchymal)Peripherin (neurons)Braina, spinal cordaGFAP (astrocytes\/glia)BrainIVNF-L (neurons)Braina, spinal cord\u03b1-internexin forms polymers with NFsNF-M (neurons)Braina, spinal cordaNF-H (neurons)Brain, spinal cordaSynemin \u03b2 is also called desmuslin.\u03b1-internexin (CNS neurons)Nestin-stem cell markerSynemins (muscle)Syncoilin (muscle)NestinVLamin A\/C (ubiquitous)Heart, muscle, fat, premature aging, complex defectsThe only nuclear IFs, longer rod domainLamin B1\/2 (ubiquitous)OrphanPhakinin (lens)LensBeaded filaments in lens epitheliaFilensin (lens)Lensa Not a causative association, variants represent a risk factor. For an overview about the new keratin nomenclature, see Schweizer et al. (2006)\nIn addition to the posttranslational modification, IF function is modified and complemented through interaction with a variety of IF-associated proteins (IFAP; Table\u00a02; Green et al. 2005; Omary et al. 2006; Kim and Coulombe 2007). These proteins can be subdivided into several subgroups, which reflect multiple IF functions (Green et al. 2005). For example, IFs interact with a variety of anchoring proteins thereby forming transcellular networks which contribute to proper tissue architecture. IFAPs also include several cytolinker proteins, which provide the structural framework for coordinated cytoskeletal function (Table\u00a02). By doing that, they are important mechanical stabilizers and accordingly, IF disruption results in increased mechanical fragility (Omary et al. 2004; Ku et al. 2007; Herrmann et al. 2007). The scaffolding function of IFs is best seen in IF-deficient animals, who exhibit disrupted cellular architecture, protein mistargeting as well as alterations in organelle localization and function (Toivola et al. 2005). In the case of lamin deficiencies, the impairment of nuclear composition has profound impact on many aspects of normal nuclear functions such as epigenetic changes, chromatin organization or DNA transcription, and repair (Dechat et al. 2008).Table\u00a02Examples of IF-associated proteinsType of interactionExamplesFunctionAnchoringDesmoplakin, BPAG1, \u03b1-dystobrevinTissue architectureCytolinkerPlectin, Filaggrin,Cellular architectureChaperonesHsp27, \u03b1-B-crystallin, Hsp70Protein foldingKinasesPKC, Cdk5IF regulation, phosphate sink, cell cycleAdaptor proteins14-3-3 protein, AP-3Multiple effectsMembrane proteinsPolycystin-1UnknownApoptotic proteinsTRADD, TNFR2, C-Flip, Caspase3\/9Apoptosis regulationMotor proteinsDynein, KinesinMovement of IF components\nIFs are not just simple cellular scaffolds, they rather build complex signaling platforms (Pallari and Eriksson 2006; Kim and Coulombe 2007). IFs interact with a variety of enzymatic and adaptor proteins, thereby affecting a multitude of cellular functions. For example, keratins associate with 14-3-3 proteins in a phosphorylation-dependent manner and this interaction regulates cell growth and cell cycle progression (Ku et al. 2007; Kim and Coulombe 2007). IF phosphorylation through associated kinases does not only regulate IF properties, but IFs also serve as phosphate \u201csponge\u201d thereby preventing activation of other, potentially pro-apoptotic, substrates (Omary et al. 2006; Ku et al. 2007). In addition to that, IFs also directly participate in apoptosis regulation through binding of several apoptosis-related molecules (Marceau et al. 2007).\nIn contrast to the actin and tubulin system, IFs emerged later in the evolution and are important supportive elements of the cell rather than their essential components. Therefore, IF variants are observed in various human diseases, which reflect their tissue specific distribution, whereas only few actin and tubulin variants have been described so far, likely due to their embryolethality (Ku et al. 1999; Omary et al. 2004). Currently, more than 30 diseases are caused by\/associated with IF mutations (Omary et al. 2004). Among them, keratin-related- and lamin-related disorders are the best studied ones. For example, mutations in keratins 5\/14 cause a blistering skin disease termed Epidermolysis bullosa simplex, whereas mutations in lamins A and C result, among others, in different diseases including premature aging, cardiomyopathy, and lipodystrophy (Omary et al. 2004; Dechat et al. 2008).\nThe disease relevance of IFs is also highlighted by a variety of IF-containing inclusion bodies, which represent the pathological hallmarks of several neurodegenerative, muscular, and other disorders (Goebel 1998; Ross and Poirier 2004; Omary et al. 2004; Cairns et al. 2004). These aggregates share a variety of features such as presence of misfolded, ubiquitinated structural proteins together with variable amounts of chaperones and p62 (Kuusisto et al. 2001; Zatloukal et al. 2002; Ross and Poirier 2004). Among the IF-related inclusions, Mallory-Denk bodies (MDBs) are the most common and also the best studied ones due to the availability of animal MDB models (Denk et al. 1975; Yokoo et al. 1982; Jensen and Gluud 1994; Denk et al. 2000; Zatloukal et al. 2007). Therefore, one focus of our review will be to describe MDB as a prototype of IF-related inclusion body, which should offer useful insights into the formation of IF-related aggregates in multiple human diseases.\nKeratins as epithelially expressed IFs\nKeratins represent the largest subfamily of IFs consisting of >50 unique gene product members (Schweizer et al. 2006; Kim and Coulombe 2007; Godsel et al. 2008) which include 37 epithelial and 17 hair keratin members in humans (Table\u00a01; Schweizer et al. 2006). Based on their pI, epithelial keratins can be subdivided in types I (acidic) and II (basic) corresponding to keratins 9\u201320 (K9\u2212K28) and keratins 1\u20138 plus keratins 71\u201380 (K1\u2013K8; K71\u2013K80), respectively (Table\u00a01; Coulombe and Omary 2002; Schweizer et al. 2006). Keratins are found as obligatory type I and type II heteropolymers (i.e., consisting of at least one type I and one type II keratin) and a homozygous disruption of a keratin results in degradation of its keratin partner at the protein level (Ku and Omary 2000; Omary et al. 2004). Similarly to IFs, keratins are expressed in a tissue-specific manner, with different pairs being the major cellular IFs in different cell populations (Moll et al. 1982; Ku et al. 1999; Coulombe and Omary 2002). For example, \u201csimple\u201d (i.e., single layered) epithelia, as found in digestive organs, express K8 together with variable levels of K7, K18, K19, and K20 depending on the tissue (Moll et al. 1982; Ku et al. 1999; Coulombe and Omary 2002; Ku et al. 2007). In contrast, stratified epithelia, like epidermis, express K5\/K14 in the basal and K1\/K10 in the suprabasal keratinocytes, respectively (Moll et al. 1982; Lane and McLean 2004; Coulombe and Omary 2002). Despite their similar molecular composition, \u201csimple\u201d and \u201cstratified\u201d keratins are not interchangeable, as shown in K14-null mice, whose phenotype was only partially restored by addition of K18 (Hutton et al. 1998). Furthermore, keratins have their preferential binding partners in vivo and the lack of such partner leads to their rapid degradation (Magin 1998). This contrasts with the in vitro situation, where IF assembly is more promiscuous (Hatzfeld and Franke 1985).\nK8\/K18\/K19 are promising serological markers based on their high abundance (approximately 0.3% of total liver protein) and intracellular localization under basal conditions with release into blood upon liver injury (Omary et al. 2002; Ku et al. 2007). However, one important caveat is the fact, that K8\/K18\/K19 are expressed in most simple epithelial cells and are therefore not liver-specific (Moll et al. 1982; Ku et al. 1999).\nThe K8\/K18\/K19 epitopes used in serologic diagnosis can be divided into two classes, that is, non-specific and apoptosis-generated epitopes. The former class constitutes established tumor markers such as tissue polypeptide antigen (TPA, represents total K8\/K18\/K19), tissue polypeptide specific antigen (TPS, derived from K18), and CYtokeratin FRAgment 21-1 (CYFRA 21-1, derived from K19). Their original clinical use was to monitor treatment response and to detect recurring tumors (Barak et al. 2004). However, later studies showed that these epitope serum levels are also elevated in non-malignant diseases and might be a general marker of tissue injury (Gonzalez-Quintela et al. 2006a, b; Tarantino et al. 2007).\nThe apoptosis-specific keratin antibodies are based on the finding, that type I keratins are cleaved at a conserved VEMD\/VEVD residue during apoptosis. In addition to that, K18 posseses a second, K18 specific caspase-cleavage site at Asp396, which is an early event during apoptosis preceding the cleavage at the VEMD\/VEVD motif (Oshima 2002; Marceau et al. 2007). The cleavage of human K18 at Asp396 can be monitored using the M30 antibody (Leers et al. 1999) and M30-Ab ELISA has become a useful serologic test for determining liver disease severity. For example, elevated serum M30 titers can distinguish simple steatosis from non-alcoholic steatohepatitis (Wieckowska et al. 2006) and predict several important prognostic parameters in patients with chronic hepatitis C infection (Bantel et al. 2004; Volkmann et al. 2006).\nAs another tool for detecting apoptotic keratin fragments, an antibody specific to the conserved K18\/K19 cleavage site at Asp237 was recently generated (Tao et al. 2008). It detects both mouse and human K18\/K19 fragments and appears to be more sensitive than the established Asp396-Ab (Tao et al. 2008). Measuring the serum levels of apoptosis-specific keratin fragments should also improve our understanding of chronic liver disease, where apoptotic cell death is an important pathogenic feature (Malhi et al. 2006).\nIn addition to that, monitoring the phosphorylation status of the circulating keratin fragments might be useful. Keratins undergo dynamic phosphorylation during mitosis, apoptosis and a variety of stress situations (Omary et al. 1998; Omary et al. 2006) and their in situ phosphorylation status is a marker of human liver disease progression (Toivola et al. 2004). However, it is currently unknown whether the phosphorylation status of the circulating keratin fragments correlates with the situation in situ.\nHepatic phenotype in keratin-deficient transgenic animals\nThe liver consists of different cell types with characteristic IF composition (Table\u00a03; Omary et al. 2002). Adult hepatocytes are unique among simple epithelial cells in that they express exclusively K8 and K18, whereas other glandular epithelia exhibit a more complex keratin expression pattern (Omary et al. 2002; Ku et al. 2007). The hepatocytic keratin IF network is dense, particularly around bile canaliculi and at the cell periphery, and acts as cytoskeletal backbone to the functionally more dynamic and contractile actin microfilament system (Zatloukal et al. 2004). Biliary epithelial cells differ from hepatocytes by additional expression of keratin 7 and 19 (Omary et al. 2002; Zatloukal et al. 2004). Keratins in cholangiocytes, but not hepatocytes, exhibit polarized and compartment-specific expression pattern (Omary et al. 2002; Zatloukal et al. 2004). The biological significance of such an expression is enigmatic, but it may be related to polarity and secretory processes. Among non-epithelial cells, stellate cells express variable amounts of GFAP, desmin, vimentin, and nestin dependent on their activation status, localization, and other parameters (Table\u00a03; Geerts 2001).Table\u00a03IFs of liver cell populationsCell typeIF compositionHepatocyteK8\/K18aOval cellsK7\/K8\/K18\/K19CholangiocyteK7\/K8\/K18\/K19Kupffer cellVimentinStellate cellGFAP, Vimentin, Desmin, NestinbEndothelial cellVimentinModified from Omary et al. (2002)a During embryogenesis, hepatocytes also express variable levels of K19 (Vassy et al. 1997)b Stellate cells represent a highly heterogeneous population with variable IF expression dependent on species, activation status of the cell, location within the hepatic lobe and many other parameters (Geerts 2001)\nStudies in keratin knock-out mice revealed that the regular liver development does not require the presence of K8, K18, or K19 (Ku et al. 2007). However, K8\/K18 knockout mice exhibited mild chronic hepatitis, hepatocyte fragility and were markedly more sensitive to a variety of stress conditions (Omary et al. 2002; Zatloukal et al. 2004; Ku et al. 2007). Furthermore, K8\/K18 transgenic mice were developed, which over-express different single-amino-acid variants (Ku et al. 2007). Among them, the K18 R89C variant resulted in disruption of hepatocyte IF network and exhibited a phenotype reminiscent of the situation in K8\/K18-knockout mice (Ku et al. 1995, 1996). K18 R89C mice also predisposed to development of thioacetamide-induced liver fibrosis (Strnad et al. 2008). Ablations of different K8\/K18 phosphorylation sites usually led to a somewhat milder phenotype which became apparent in stress situations, but not under basal conditions (Ku et al. 2007). For example, K18 S52A variant resulted in increased sensitivity to microcystin-LR-induced liver injury, whereas K8 S73A mice were predisposed to Fas-induced liver apoptosis (Ku et al. 1998; Ku and Omary 2006). The ablation of K18 S33 phosphorylation site, which regulates the binding to 14-3-3 proteins, caused limited mitotic arrest and accumulation of abnormal mitotic figures after partial hepatectomy (Ku et al. 2002a).\nIn contrast, mice lacking K19 did not have an obvious liver phenotype (Tao et al. 2003), but surprisingly exhibited skeletal myopathy (Stone et al. 2007). GFAP\/vimentin-knockouts displayed compromised astrocytic function with attenuated reactive gliosis, but no obvious alteration in the in vitro activation of hepatic stellate cells despite the lack of IF network (Geerts et al. 2001; Pekny and Pekna 2004). However, in vivo studies are needed to conclusively address the fibrogenic potency of these transgenic mice.\nKeratin variants in liver disease\nThe large body of evidence from animal studies showing the importance of K8\/K18 for liver homeostasis led to a search for keratin mutations in patients with liver diseases. Several K8\/K18 variants were found to associate with the development of cryptogenic liver disease (Ku et al. 2001). In subsequent studies, K8\/K18 were shown to represent susceptibility genes for development of end-stage liver disease of multiple etiologies (Ku et al. 2003a, 2005). In particular, biologically significant K8\/K18 variants were found in 44 of 467 liver explants (12.4%), but only in 11 out of 349 analyzed blood bank donors, which were used as a control group (P\u00a0<\u00a00.0001, prevalence OR3.8; 95% CI\u00a0=\u00a02.1\u20137). Furthermore, K8\/K18 variants associate with liver fibrosis progression in patients with chronic hepatitis C (Strnad et al. 2006).\nK8 R340H represents the most common amino acid altering K8\/K18 variant and it is the only one, which was significantly associated with development of end-stage liver disease (Ku et al. 2005). Larger studies are needed to analyze the pathological significance of the other less common K8\/K18 variants (Ku et al. 2007).\nIn contrast to human association studies, experiments in K8\/K18-deficient mice were shown to predispose mainly to acute liver injury (Ku et al. 2007). To address this issue, we recently analyzed a large cohort of patients with acute liver failure (ALF). K8\/K18 variants were significantly more frequent in total ALF patient cohort (46\/345; 13.3%) and in patients with acetaminophen-induced ALF (21\/169; 12.4%) when compared to blood bank donors (11\/349; 3.7%; P\u00a0<\u00a00.002 for both comparisons). Among the single polymorphisms, the K8 R340H variant was found at significantly higher frequencies in the whole ALF cohort as well as in the acetaminophen-induced ALF subgroup (frequency 6.6 and 7.1%, respectively vs. 3.1% in the control group; P\u00a0<\u00a00.03 for both comparisons). In addition, transgenic mice over-expressing K8 R340 variants displayed augmented acetaminophen-induced liver toxicity. In conclusion, K8\/K18 are also susceptibility genes for development of ALF and K8\/K18 variants may predispose to drug-induced liver injury (Strnad et al., unpublished data).\nUp to date, only one published study analyzed the polymorphisms in K19 gene and found no association between K19 variants and inflammatory bowel disease (Tao et al. 2007). Interestingly, we recently observed K19 G17S variant in three out of 190 patients with primary biliary cirrhosis, but none was found in control blood bank donors (200 samples; Zhong et al., unpublished data). However, larger studies are needed to address the importance of K19 in biliary diseases.\nThe human K8\/K18 variants described above do not cause a particular liver disease per se, they just pose a risk factor for its development. This is different from the situation in stratified epithelia, where keratin mutations result in several monogenic keratin diseases (Lane and McLean 2004; Omary et al. 2004). This discrepancy may be caused either by the intrinsic difference between keratins of simple and stratified epithelia (Hutton et al. 1998) or by the different localization of the variants within the protein backbone. To that end, disease-causing keratin mutations in stratified epithelia are clustered in the highly conserved helix initiation and termination motif, whereas K8\/K18 disease-predisposing variants are observed in more variable domains (Owens and Lane 2004; Omary et al. 2004; Ku et al. 2007).\nThe disturbance in cytoprotective function of keratins is the likely mechanisms by which K8 and K18 variants predispose to liver disorders. For example, K8\/K18 are anti-apoptotic proteins and this ability is hampered in keratin-deficient animals (Oshima 2002; Ku and Omary 2006; Marceau et al. 2007; Ku et al. 2007). The ways of interaction between keratins and apoptosis are manifold. K8\/K18 bind to several apoptotic proteins and type I keratins are established caspase substrates (Oshima 2002; Green et al. 2005; Marceau et al. 2007). In addition, K8\/K18 serve as physiologic kinase substrates in vitro and in transgenic mice and an ablation of the K8 S73 phosphorylation site or introduction of the naturally occurring K8 G61C variant leads to increased apoptosis through increased phosphorylation of pro-apoptotic proteins (Ku et al. 2002b; Ku and Omary 2006). The keratin-mediated anti-apoptotic function may be highly relevant given the importance of apoptosis in liver disease and the pro-fibrogenic properties of elevated rate of apoptotic cell death (Friedman 2004; Malhi et al. 2006).\nKeratins exhibit anti-oxidative properties, as they sequester oxidatively damaged proteins, and similarly, the K18 R89C variant primes the liver towards oxidative injury (Tao et al. 2005; Zhou et al. 2005). This keratin property might be helpful in attenuating both liver fibrosis and acetaminophen-induced liver injury (Parola and Robino 2001; Jaeschke et al. 2003). Keratins are also established stress-inducible proteins, which are upregulated both in humans and mice under several, mainly cholestatic conditions (Fickert et al. 2002, 2003; Zatloukal et al. 2007; Ku et al. 2007; Strnad et al. 2008). Keratin variants may interfere with keratin upregulation or simply result in decreased keratin levels due to protein instability (as seen for K18 R89C variant in the liver; Ku et al. 1995).\nThe naturally occurring K8\/K18 variants interfere with basic IF properties such as K8\/K18 filament assembly and keratin solubility (Ku et al. 2001; Owens et al. 2004; Ku et al. 2007). Due to altered protein conformation, some of them impair potentially cytoprotective keratin phosphorylation at adjacent residues (as seen in K8 G61C and K8 G434S variant; Ku et al. 2005; Ku and Omary 2006).\nK8\/K18 variants may also affect organelle function, as seen in K8 knockout mice, which exhibit altered mitochondrial shape, localization, and alterations in several mitochondrial proteins (Toivola et al. 2005).\nDespite the various cytoprotective effects of keratins, their impact seems to be limited to certain conditions. For example, K18 R89C mice are predisposed to Fas but not TNF-induced apoptosis and the same mice develop more pronounced liver fibrosis after thioacetamide, but not after carbon tetrachloride injection (Ku et al. 2003b; Strnad et al. 2008). It is also unknown which one of the keratin-mediated effects is important in particular disease settings. The recently established transgenic mouse lines overexpressing the naturally occurring K8 G61C and R340H variants will likely offer valuable insights in this respect (Ku and Omary 2006; Zhou et al., unpublished data).\nKeratin network alteration in steatohepatitis of the alcoholic (ASH) and the non-alcoholic (NASH) type\nSteatohepatitis is characterized by hepatocyte \u201cballooning\u201d, that is, swelling and rounding with clearing of the cytoplasm, which prevails in the perivenular zone and is often associated with pericellular fibrosis, predominantly granulocytic inflammation (\u201csatellitosis\u201d), steatosis (usually of macrovesicular type), and cholestasis (Brunt 2004; Lefkowitch 2005). Ballooned cells are often associated with granular, rope- or clump-like cytoplasmic inclusions, called Mallory-Denk bodies (MDBs) (originally also designated alcoholic hyalin, which is, however, a misnomer since they are not specific for alcoholic etiology) together with derangement or even disappearance of the keratin IF cytoskeleton (Mallory 1911; Denk et al. 2000; Zatloukal et al. 2007; Fig.\u00a01a, b). The disappearance of the keratin immunostaining in ballooned hepatocytes is reasonably specific for ASH and NASH, since it is not seen in ballooned cells of viral hepatitis or toxic damage and can therefore be used as an objective morphologic parameter in grading of steatohepatitis (Lackner et al. 2008). However, ballooning (with concomitant cytoskeletal alterations) and MDB formation are not entirely interchangeable since not all ballooned hepatocytes contain MDBs.\nFig.\u00a01MDBs are readily detected by different methods. In clinical routine, MDBs are usually detected as eosinophilic aggregates in standard hematoxylin and eosin stained sections (a). After chromotrope aniline blue staining, MDBs appear as blue structures, often with red center (b). Immunofluorescence or immunohistochemical staining represents a more sensitive method for MDB detection than conventional histological stainings, but is strongly dependent on the antibody used as well as the staining protocol. MDBs can be reliably detected with antibodies against K8\/K18 [green and redchannel in (c) and (d), respectively] or p62 [red channel in (c)], whereas only some MDBs stain with antibodies to phosphorylated keratins such as K8 pS431 antibody [green channel in (d)]. In both immunofluorescence pictures, MDBs are seen as yellow structure due to co-localization of both visualized epitopes\nMDBs are typical morphological features of ASH and NASH, although NASH usually exhibits slightly less prominent MDBs than the ones seen in ASH (Brunt 2004; Zatloukal et al. 2007). MDBs can also be detected after intestinal bypass surgery for morbid obesity, in chronic cholestasis, particularly in late stages of primary biliary cirrhosis, Wilson disease and other types of copper toxicosis, various metabolic disturbances, and hepatocellular neoplasms (M\u00fcller et al. 2004; Zatloukal et al. 2007; Fig.\u00a02). In contrast, MDBs have not been observed in the context of acute cholestasis, acute viral hepatitis and a variety of acute toxic or drug-induced liver diseases (Jensen and Gluud 1994, Zatloukal et al. 2007; Ku et al. 2007). However, even in potentially MDB-forming liver diseases, MDBs are found only in a subset of patients, partially (but not completely) depending on the sensitivity of the detection method used. For example, when using immunohistochemistry for keratin or ubiquitin, MDBs were found in about 70% of ASH cases in contrast to 40% seen in hematoxilin-eosin-stained sections (Ray 1987).Fig.\u00a02MDBs are seen in various human liver diseases. Immunohistochemical staining with p62 antibody visualizes the presence of multiple irregularly shaped aggregates in patients with alcoholic steatohepatitis (a), non-alcoholic steatohepatitis (b), Indian childhood cirrhosis and (c), idiopathic copper toxicosis (d)\nThis suggests that MDBs require either a specific pathogenetic constellation or genetic predisposition for its formation, which is present only in a subset of patients.\nApart from MDBs, additional features may be observed in some chronic cholestatic conditions. For example, a low percentage of hepatocytes express keratin 7 and to a lesser extent keratin 19 which indicate that these cells acquire features of precursor cells which normally express keratin 8, 18, 7, and 19 during regeneration (Van Eyken et al. 1988; Zatloukal et al. 2004). In idiopathic copper toxicosis and hepatocellular carcinoma, MDBs may coincide with another type of cytoplasmic inclusions, termed intracellular hyaline bodies (IHBs), which share several components with MDBs, but do not contain keratins (Stumptner et al. 1999; Denk et al. 2006).\nThe ease of MDB detection makes them attractive morphologic markers. However, correlation between the clinical disease manifestation\/progression on one side and hepatocyte ballooning with MDB formation on the other is imperfect. For example, patients with severe clinical symptoms of ASH sometimes show only moderate histopathological alterations with few or no MDBs, whereas patients with pronounced histological alterations do not necessarily exhibit significant clinical and laboratory abnormalities (Zatloukal et al. 2007). Despite that, controlled clinical-pathologic studies comparing NASH patients with ambulatory and hospitalized alcoholics revealed that hepatocellular damage, presence of MDBs, inflammation, and fibrosis collectively correlated with disease severity (Cortez-Pinto et al. 2003). Also in other studies, hepatocellular ballooning and MDB formation was positively correlated with disease progression, development of fibrosis, and cirrhosis and liver-related mortality (Orrego et al. 1987; Matteoni et al. 1999; Gramlich et al. 2004; Mendler et al. 2005).\nMorphology and composition of MDBs\nMDBs are irregularly shaped, usually dense cytoplasmic inclusions of different sizes (Mallory 1911). Small MDBs arise in association with IF bundles throughout the cytoplasm, whereas larger MDBs are often seen in the perinuclear region (Denk et al. 2000; Zatloukal et al. 2007; Ku et al. 2007). Ultrastructurally, they usually consist of haphazardly arranged filamentous rods, approximately 10\u201315\u00a0nm in diameter, covered by a fuzzy coat (designated as type II by Yokoo et al. 1972). In addition, MDBs with an electron dense granular to amorphous center (designated type 3) are seen predominantly in older inclusions, and filaments in parallel arrangement were also described (designated as type I) but seem to be exceedingly rare (Yokoo et al. 1972). Keratins 8 and 18, sequestosome 1\/p62 (p62) and ubiquitin are major, and low and high molecular weight heat shock proteins (HSP 70, HSP 25), but also proteins of the protein degradation machinery, are minor constituents (Denk et al. 2000; Riley et al. 2002, 2003; Zatloukal et al. 2007; Ku et al. 2007; Figs.\u00a01c, d; Fig.\u00a02). Keratins 7, 19, and 20 have also occasionally been detected (Cadrin et al. 1990; Dinges et al. 1992). The K8\/K18 within MDBs exhibit increased \u03b2-sheet structure (Cadrin et al. 1991; Kachi et al. 1993), are hyperphosphorylated, partially degraded and cross-linked (Hazan et al. 1986; Zatloukal et al. 1992; Cadrin et al. 1995; Stumptner et al. 2000; Fig.\u00a01d). The list of MDB components is likely to grow since, in addition to intrinsic components, proteins non-specifically incorporated in the aggregate have to be expected.\nPathogenesis of MDBs\nStudies on MDB formation and composition are greatly enhanced by the availability of animal models. MDBs can be induced under standardized conditions and their fate followed in mouse liver by chronic griseofulvin or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) administration (Denk et al. 1975; Yokoo et al. 1982; Denk et al. 2000; Zatloukal et al. 2004, 2007; Fig.\u00a03c, d).Fig.\u00a03MDBs formed in DDC-fed animals resemble inclusion bodies observed in human diseases. Liver sections were double labeled with antibodies to K8\/K18 (green) and p62 (red). Samples from mouse fed DDC for 12\u00a0weeks (d) and from a patient with alcoholic steatohepatitis (b) exhibit multiple irregularly shaped inclusion bodies, which appear yellow due to presence of both epitopes. In contrast, control human (a) and mouse liver (c) display an unaffected keratin network with no apparent p62 staining. Note that DDC feeding leads to deposition of protoporphyrin, which can be seen as occasional blue pigment in (d)\nIn patients, MDB formation is usually a chronic process requiring several years of alcohol intoxication or metabolic imbalance, that is, in association with the metabolic syndrome, Wilson disease, other metabolic disorders, and chronic cholestasis. An exception is idiopathic copper toxicosis of children in which end stage liver disease associated with MDB formation occurs at very early age (M\u00fcller et al. 2004; Zatloukal et al. 2007). Surprisingly, in alcoholics recovered from ASH, an alcohol excess may almost instantaneously lead to MDB recurrence, a situation which has been compared to an immunologic response and termed \u201ctoxic memory\u201d (Jensen and Gluud 1994; Denk et al. 2000). The animal model of MDB formation does not only reproduce the structural and morphological features seen in humans, but also the phenomenon of \u201ctoxic memory\u201d (Fig.\u00a03; Denk et al. 2000; Zatloukal et al. 2004, 2007). To induce MDBs in mice, griseofulvin or DDC is usually fed for 2\u20133\u00a0months. MDB formation in mice is reversible, since MDBs disappear after recovery on standard diet for 1\u00a0month. However, after rechallenge of the recovered (\u201cprimed\u201d) animals, MDBs reappear within a few days (Denk et al. 2000; Zatloukal et al. 2004, 2007). Rapid MDBs formation in primed mice is rather non-specific, since it can be triggered by a variety of stress conditions including colchicine (but not by lumicolchicine), bile acids, bile duct ligation, several other toxins, and proteasome inhibitors which were unable to induce MDBs in the na\u00efve animal (Zatloukal et al. 2007, and references therein). The reasons for this phenomenon are as yet unclear but point to a final common pathway activated by the trigger.\nThe availability of cellular and animal models of MDB formation led to valuable insights into the mechanism of MDB formation. Several pathogenic mechanisms were implicated in this process (Fig.\u00a04; Dobson 2004):1.Enhanced oxidative stress2.Disproportional K8\/K18 expression together with keratin modifications3.Chaperone dysfunction4.Elevated p62 levels5.Insufficient protein degradation\nAd 1. The MDB-inducing conditions both in humans and mice cause elevated levels of oxidative stress (Tephly et al. 1981; Mehta et al. 2002; Dey and Cederbaum 2006; Farrell and Larter 2006) and MDBs themselves were shown to contain misfolded keratins with increased \u03b2-sheet formation (Cadrin et al. 1991; Kachi et al. 1993). Recent animal studies highlight the importance of altered methyl group metabolism and mitochondrial stress in this process (Li et al. 2008; Zatloukal et al., unpublished data). During DDC detoxification, N-methylprotoporphyrin is formed through transfer of a methyl group to heme moieties (Tephly et al. 1981). N-methylprotoporphyrin subsequently acts as a potent inhibitor of ferrochelatase, thereby causing porphyria (Tephly et al. 1980). Accordingly, MDB formation and DDC effects can be effectively attenuated by feeding S-adenosylmethionine, that is a compound involved in methyl group transfer (Li et al. 2008). This is reminiscent of the situation in ASH, which is also associated with disrupted methyl group metabolism (Schalinske and Nieman 2005).Fig.\u00a04MDBs formation results from a complex interplay of several contributing factors. Since the cytoplasm represents a hydrophilic milieu, all exposed hydrophobic molecules (depicted by red stretches within the protein) are predisposed to aggregation. Properly folded proteins usually hide their hydrophobic stretches inside, but these get exposed in nascent protein chains or after proteins become misfolded as a consequence of oxidative stress. Chaperones bind to these hydrophobic residues and facilitate protein refolding. Alternatively, damaged proteins become polyubiquitinated and degraded either by the proteasomal or autophagic system. MDB-causing agents typically generate extensive amount of oxidative stress with increased protein misfolding. In addition, chaperone levels are downregulated and\/or chaperone function is compromised. Dysbalanced K8\/K18 expression precedes MDB formation, likely increases keratin misfolding and predisposes to posttranslational modifications, which may interfere with keratin refolding and\/or repair. Accumulated misfolded proteins are sequestered as inclusions through the action of p62. p62 also shuttles polyubiquitinated proteins to degradative machineries. However, proteasomal degradation might be impaired by oxidative stress, may not be able to digest highly cross-linked protein species or might simply be overwhelmed by the excessive supply. On the other hand, autophagy is upregulated during MDB formation in mice and additional stimulation of autophagy attenuates MDB formation in certain conditions. Of note, supplementation of S-adenosylmethionine or mitochondrially targeted antioxidants effectively diminishes MDB formation, thereby pointing to a central role of oxidative stress in MDB generation\nDDC targets mitochondria, where it reacts with cytochrome P450 (Marks et al. 1985). An unbiased microarray analysis identified cytochrome P450 (Cyp) 2a5 as a major gene induced both after DDC exposure and particularly after DDC re-challenge. Moreover, Cyp2e5 overexpression spacially coincides with MDB formation (Zatloukal et al., unpublished data). Cyp2a5 is a \u201cleaky\u201d cytochrome which produces reactive oxygen species (Lewis et al. 1989). In that sense, it resembles human Cyp2E1, which was implicated as a source of oxidative stress in the pathogenesis of ASH and NASH (Villeneuve and Pichette 2004).\nTo address the role of mitochondrial stress in MDB formation, we re-fed DDC-primed mice with DDC alone or in combination with the mitochondria-targeted antioxidant mito Q (Smith et al. 2003). Mito Q co-administration attenuated both MDB formation and DDC-induced liver damage. Therefore, mitochondrial oxidative stress seems to be involved in MDB formation in mice which is in good concordance to the mitochondrial dysfunction seen both in ASH and NASH patients (Pessayre 2007; Mantena et al. 2008; Zatloukal et al., unpublished data).\nAd 2. Keratins are major constituents of MDBs and both altered K8\/K18 expression and keratin modification seems to affect MDB formation (Zatloukal et al. 2007; Ku et al. 2007). Griseofulvin\/DDC feeding leads to rapid induction of K8\/K18 expression with disproportional K8\u00a0>\u00a0K18 levels (Denk et al. 2000). The elevated K8\/K18 ratio is crucial for MDB formation as shown in K18-knockout and K8 overexpressing animals, who are predisposed to MDB formation already upon short exposure to DDC and even form MDBs spontaneously during aging (Magin et al. 1998; Nakamichi et al. 2005). Accordingly, K8-null or K18 overexpressing mice are resistant to MDB formation and the protective function of K18 is not affected by its phosphorylation status or mutation (Zatloukal et al. 2000; Harada et al. 2007). However, the exclusive MDB inducing property of K8 in vivo cannot be reproduced in vitro, where aggregates resembling MDBs can also be produced by transfection of K18 (Nakamichi et al. 2002; Stumptner et al. 2007).\nAmong posttranslational modifications, MDB formation is associated with K8 hyperphosphorylation and transamidation (Zatloukal et al. 1992; Stumptner et al. 2000). In a recent study, ablation of K8 S73 phosphorylation site in transgenic mice resulted in diminished MDB formation after DDC exposure (Harada et al., unpublished results). Several potential mechanisms might be responsible for this observation. K8 S73 is a well-known p38 kinase target site and p38 up-regulation induces keratin network reorganization with subsequent granule formation (Ku et al. 2002b; W\u00f6ll et al. 2007). P38 kinase-induced keratin network reorganization might be a necessary prerequisite for MDB formation, since p38 kinase inhibition prevented MDB formation in vitro (Nan et al. 2006). Alternatively, K8 hyperphosphorylation may induce MDB formation through inhibition of K8 degradation, which results in increased K8\/K18 ratio (Ku and Omary 2000).\nMDBs contain highly cross-linked keratins and the ablation of tissue transglutaminase effectively inhibits DDC-induced MDB formation (Zatloukal et al. 1992; Strnad et al. 2007). Since K8 is a much better in vitro transglutaminase substrate than K18 and highly cross-linked proteins found in MDB-forming mice contain K8, but not K18, it was suggested that excessive K8 gets preferentially transamidated and acts as a nucleus for MDB formation. This is supported by studies in transgenic mice, where K8 overexpression accelerates and K18 excess inhibits not only MDB, but also cross-link formation (Strnad et al. 2007).\nAd 3. MDBs were shown to contain misfolded keratins (Cadrin et al. 1991, Kachi et al. 1993). The protein misfolding is usually counteracted by the reparative function of chaperones (Ross and Poirier 2004; Macario and Conway de Macario 2005; Bukau et al. 2006), however, DDC feeding is associated with diminished chaperone expression, persistent chaperone modifications and impairment of chaperone function (Strnad et al. submitted). Similarly, chaperone function is impaired in a rat model of chronic alcoholic liver disease (Carbone et al. 2005).\nAd 4. P62 is a stress-inducible protein with multiple functions which is a constituent of multiple inclusion bodies termed sequestosomes (Shin 1998; Kuusisto et al. 2001; Zatloukal et al. 2002; Moscat et al. 2007). It binds proteins polyubiquitinated at lysine 63 and shuttles them for proteasomal or autophagic degradation (Vadlamudi et al. 1996; Bjorkoy et al. 2005; Seibenhener et al. 2004; Wooten et al. 2008). p62 has been shown to enhance aggregate formation (Donaldson et al. 2003; Wang et al. 2005; Komatsu et al. 2007; Gal et al. 2007; Nezis et al. 2008). p62 action is beneficial under normal conditions, since it prevents accumulation of abnormal proteins (Bjorkoy et al. 2005; Ramesh Babu et al. 2008), but may become harmful when protein degradation is inhibited (Komatsu et al. 2007).\nThere are several lines of evidence implicating p62 (containing the ubiquitin binding site) in MDB formation. In cell culture experiments, protein aggregates resembling MDBs formed only after p62 co-transfection, but not when K8, K18, and ubiquitin were transfected alone or in combination (Stumptner et al. 2007). Furthermore, p62 was rapidly induced in DDC-fed mice with p62-containing aggregates preceding the formation of MDBs (Stumptner et al. 2002). Furthermore, p62 inhibition attenuated, whereas p62 overexpression enhanced MDB formation in DDC-primed hepatocytes (Nan et al. 2006).\nAd 5. Accumulation of protein aggregates, as seen during MDB formation, is counteracted by proteasomal or autophagic degradation (Glickman and Ciechanover 2002; Williams et al. 2006) and both degradation machineries are active in the liver. For example, conditional knock-out of the autophagy-related gene 7 (Atg7) leads to development of ubiquitin-positive inclusions in hepatocytes (Komatsu et al. 2005). Similarly, proteasomes seem to interact with MDBs and inhibition of proteasomal degradation leads to formation of MDB-like structures both in vitro and in DDC-primed mice (Harada et al. 2003; Bardag-Gorce et al. 2002; Riley et al. 2002; French et al. 2001). Furthermore, autophagic degradation is upregulated in DDC-fed mice and its further stimulation with rapamycin attenuates spontaneous MDB formation in K8 overexpressing mice (Harada et al. 2008).\nIn contrast, MDB formation might be facilitated by proteasomal impairment, since MDBs are preferentially seen in proteasome-depleted hepatocytes (Bardag-Gorce et al. 2001). In addition, an aberrant form of ubiquitin, termed [UBB\u00a0+\u00a01] arising as a consequence of molecular misreading, is observed in MDBs and may inhibit proteasomal function (McPhaul et al. 2002; Lindsten et al. 2002). Similarly, alcohol and aging, both potential inducers of MDBs can be associated with decreased proteasome function (Hayashi and Goto 1998; Fataccioli et al. 1999).\nHowever, aggregate formation must not always be caused by a general inhibition in protein degradation. The degradative capacity may simply be overwhelmed by increased amounts of misfolded proteins. Alternatively, certain proteins may not be easily degradable, such as proteins with long polyglutamine stretches (Venkatraman et al. 2004). The latter scenario may apply to DDC-fed mice, which exhibit an accumulation of ubiquitinated protein only in the highly insoluble protein fraction (Strnad et al. 2007). This fraction is characterized by highly cross-linked protein species generated through extensive transamidation, which may make them resistant to proteolytic degradation (Strnad et al. 2007).\nPathologic significance of MDBs\nIn principle, cytoplasmic protein aggregates can be beneficial, detrimental or inert depending on the context. In many pathologic situations aggregation seems to be a protective response if the first lines of defense, that is, refolding and degradation, fail by sequestration of potentially harmful proteins (Arrasate et al. 2004; Bodner et al. 2006). On the other hand, large protein aggregates can be detrimental either by mechanical interference with cellular transport processes (e.g., shown with microtubule-dependent transport in neurons), deprivation of the cell of vital components by aggregation or coaggregation and by overwhelming\/inhibiting the capacity of the chaperone system and\/or the protein degradation machinery by indigestible material (Alonso et al. 1997; Stenoien et al. 1999; Suhr et al. 2001; Bence et al. 2001; Lee et al. 2004; Grune et al. 2004). It can be expected, therefore, that in different cell systems and pathologic conditions inclusion bodies differ regarding their cellular effects and consequences. MDBs per se do not seem to compromise the viability of transfected tissue culture cells (Stumptner et al. 2007) or hepatocytes in vivo, who even exhibit an activated phenotype (Denk et al. 2000). Further studies are needed to definitely characterize the molecular consequences of MDB formation.","keyphrases":["variant","inclusion","keratin","mallory-denk body","aggregate","ubiquitin","p62","steatohepatitis","oxidative stress"],"prmu":["P","P","P","P","P","P","P","P","P"]}
{"id":"Eur_J_Nucl_Med_Mol_Imaging-3-1-1914264","title":"Clinical radionuclide therapy dosimetry: the quest for the \u201cHoly Gray\u201d\n","text":"Introduction Radionuclide therapy has distinct similarities to, but also profound differences from external radiotherapy.\nIntroduction\nRadionuclide therapy (or \u201ctargeted\u201d, \u201cmetabolic-or \u201cmolecular-radiotherapy) may be defined as a radiation therapy that uses local, loco-regional or generally administered open (i.e. \u201cunsealed\u201d) radionuclides to achieve a transfer of radiation energy to a pathological target tissue and by this means to exert a destructive tissue effect. This \u201cinternal-radiation therapy has distinct similarities to, but also profound differences from the more commonly used external radiotherapy (EBRT).\nThe tissue effect is expressed as the absorbed radiation dose in grays (Gy), i.e. the amount of transferred energy in joules per unit mass of target tissue. The fundamental use of this unit is similar to that in EBRT, and there is a similar relationship between radiation dose and response in terms of cell killing\/survival. Therefore, calculation of the radiation absorbed dose to a targeted tissue makes sense at any point in treatment. In this respect, radionuclide therapy dosimetry may be considered an inherent part of radionuclide therapy in principle, as in EBRT.\nIn the literature, there is considerable confusion over the proper use of the term \u201cdose\u201d, which actually refers to the \u201cradiation dose-in the SI unit \u201cgrays\u201d. However, dose in the context of radionuclide therapy is frequently used when actually the \u201cadministered activity-in GBq or mCi is meant. To avoid confusion, the terms \u201cradiation dose-and \u201cabsorbed dose-are used when indicating Gy and the term \u201cactivity dose-when indicating GBq.\nIndividual patient dosimetry has the following goals [1]:\nTo establish individual minimum effective and maximum tolerated absorbed dosesTo establish a dose-response relation to predict tumour response and normal organ toxicity on the basis of pre-therapy dosimetryTo objectively compare the dose\u2013response results of different radionuclide therapies, either between different patients or between different radiopharmaceuticals, as well as to perform comparisons with the results routinely obtained with external radiotherapyTo increase the knowledge of clinical radionuclide radiobiology, in part with the aim of developing new approaches and regimens\nIn EBRT, the absorbed dose can be calculated relatively straightforwardly from the energy loss in the body from the point where the radiation enters the body to the target. Radiation in radionuclide therapy is directed to its target by a vehicle (hence \u201cradiopharmaceutical\u201d), which exhibits more or less specific binding to the target tissue. This is a highly dynamic metabolic process, both biochemically and physically, within the time interval of the decaying isotope, and entails a much more complex spatial and temporal radiation distribution than that in EBRT. Pharmacokinetics such as circulation, metabolisation, target expression heterogeneity and cellular uptake and release, as well as radiobiological phenomena such as biological or physical (\u201ccross-fire\u201d) bystander effects [2], play a determining role in the final radiation dose to the target. EBRT is typically a fractionated high dose rate radiotherapy in which episodes of radiation, aimed to cause as much lethal damage as possible, are interspersed with episodes of non-treatment, during which repair and repopulation occur. Radionuclide therapy is radiotherapy with a low and continuously decreasing dose rate, which requires a unique radiobiological approach [3, 4].\nIntegral activities can be determined numerically or by compartmental models [5] and presently rely heavily on the detection of the activity distribution by gamma cameras. Because the electron range is inferior to the spatial resolution of most molecular imaging devices, the \u201cperfect-dosimetric study cannot be achieved, and calculations are always an approximation. Using the temporal and spatial radionuclide distribution data, radiation doses to target organs have generally been calculated using the MIRD formalism, formerly used for calculation of the biodistribution of diagnostic radiopharmaceuticals. Commercially available software such as MIRDOSE3 or the newer OLINDA\/EXM [6, 7] is available for calculation of internal absorbed doses in organs and tumours. Although these models make important assumptions about anatomy (standard man and woman) and radiopharmaceutical distribution (uniformity of uptake in source and target) that are not necessarily valid in individual patients, they do provide a practical and standardised model for clinical end-users.\nNevertheless, to date the need for dosimetry to individually optimise the therapeutic activity to be administered has been far from self-evident. Radionuclide therapy dosimetry has not gained wide acceptance as a clinical tool among the (nuclear) medical community because of an imbalance between the accuracy and the complexity of the procedure. A number of studies have even completely discarded dosimetry, instead using fixed activities for all patients or activities based on kg or m2 body dimensions. [8-10]. To underline that this is unjustified, this review will describe the significant progress that has been achieved over recent years, especially in the fields of instrumentation (with provision of clinically useful instruments), physical modelling and radiobiology. Furthermore, the EURATOM Council Directive 97\/43 stipulated that in medical exposures for radiotherapeutic purposes, including nuclear medicine, \u201cexposures of target volumes shall be individually planned\u201d. In this context, the nuclear medicine physician is at present confronted with a \u201cdosimetric dilemma\u201d, because official guidelines and recommendations for most treatments do not include advanced dosimetric calculations. Therefore, this review primarily aims to provide the nuclear medicine practitioner with an up-to-date overview of clinically applied dosimetry techniques in radionuclide therapy. For those working at a more basic level of dosimetry, information is provided on the clinical application of methods and areas of further development. Given the large size of the field of radionuclide therapy, this review is limited to those oncology indications for which there is substantial literature concerning dose calculations, with in-depth discussion thereof. As it would be impossible to cover all areas of the complex subject of dosimetry, appropriate references will be provided where necessary, e.g. regarding basic physics, (radio)biology, radiopharmaceuticals and preclinical aspects.\nRadioiodine therapy in differentiated thyroid carcinoma\nRadioiodine therapy has proven to be a safe and effective method in the treatment of patients with differentiated thyroid carcinoma (DTC) after total or near-total thyroidectomy [11, 12]. It has been shown to be useful not only for ablation of benign remnant thyroid tissue, facilitating subsequent diagnostic testing, but also for treatment of any remaining cancerous cells either in the thyroid bed or at metastatic sites. Dosimetry plays an important role in answering questions concerning biodistribution since the biological half-time of radioactive iodine differs substantially between individual patients and even within distinct lesions of the same patient. It has proven exceptionally helpful in clinical trials of new drugs, for example retinoids, and in assessment of the use of recombinant TSH [13], as well as in the study of other questions such as the phenomenon of \u201cstunning\u201d. However, its possible role in extending therapy doses to the level of individual maximum effectiveness is currently less obvious.\nThere is at present no consensus on the activity dose of 131I to be administered in various settings according to recent European and American guidelines [14, 15], largely because of the lack of prospective, randomised data. In the majority of cases, 1.1-3.7 GBq (30-100 mCi) is prescribed empirically for the first radioiodine therapy after thyroidectomy in newly diagnosed DTC, to ablate the remaining glandular tissue. Activity doses as low as 1 GBq are used when the size of the thyroid remnant is small, as measured by the postoperative radioiodine uptake in the neck, and\/or to reduce local complications that could arise from radiation thyroiditis\/oedema, which has been reported in 10-20% of cases, although usually mild. The effectiveness of this ablative approach based on standard activities is reported to be about 80%. For the treatment of residual tumour, relapses or lymph node metastases, generally higher activities of 3.7-7.4 GBq (100-200 mCi) are used.\nApproaches in which quantitative dosimetry is performed to estimate the activity dose needed to deliver an effective radiation dose are scarce in the literature. Maxon and co-workers, using sequential planar scintigraphy, established in the early 1980s that an effective mean radiation dose of at least 300 Gy is required for successful remnant ablation [16]. The validity of this recommendation remains unclear as it conflicts somewhat with clinical experience. It also neglects microdosimetry of radioiodine and dose heterogeneity, which may well determine the overall response. As far as metastases are concerned, these authors concluded in the same study that a radiation dose of at least 80 Gy is associated with a significant increase in response, while a dose of less than 35 Gy offers little chance of success. In a later study [17], they found that when metastatic disease was present only in lymph nodes, a target radiation dose of at least 140 Gy was successful in 86% of patients and 90% of involved nodes. When nodal metastases were associated with either residual thyroid tissue or other metastatic foci, a single treatment calculated to deliver at least 85 Gy to the metastases proved successful in 74% of patients and 81% of nodes. The aforementioned doses were mean absorbed doses for the whole remnant or tumour; no data were provided on the dose distribution in smaller tumour parts. It is also unclear what causes the large difference between radiation dose values for remnants and metastases. This method requires sequential measurements with a scintillation camera 24, 48 and 72 h after administration of 74 MBq (2 mCi) of 131I. De Keizer et al. [18] recently calculated thyroid tumour doses with a similar methodology but with the use of recombinant TSH. A tumour dose of >80 Gy was found in only 20% of metastases visible on post-therapy 131I scanning. In 55% of patients, progressive disease was evident after 3 months and none of these patients had radiation doses to the tumour in excess of 30 Gy, confirming the existence of a clinical dose-response relation.\nIn cases of distant metastases, higher amounts of 131I are given in single doses and subsequent cumulative therapies. Most centres use a fixed dose of 7.4 GBq (200 mCi), but some use (much) higher activity doses. In order to avoid serious complications (bone marrow suppression, lung fibrosis), the commonly used \u201cmaximum safe-administered dose concept published by Benua et al. [19] restricts the blood absorbed dose to less than 2 Gy (200 rad) and the whole-body retention to 4.4 GBq (120 mCi) at 48 h in the absence of diffuse lung metastases. According to the protocol, activity concentration in the whole body as measured by 131I should not exceed 2,960 MBq (80 mCi) after 48 h when diffuse, functioning lung metastases are present. These measurements can be camera based without blood sampling. More recently, the blood dose formula derivation proposed by H\u00e4nscheid et al. [13] has provided a new tool for patient-specific blood dose assessment representing marrow dosimetry in DTC therapy. For more precise dosimetry, e.g. in dosimetric studies or for higher targeted blood absorbed doses, sequential blood sampling is recommended [20, 21]. The calculated radiation dose serves as a surrogate parameter for the organ at risk, the bone marrow, since to date direct determination of the bone marrow absorbed dose is not feasible. This method has been applied successfully in clinical practice. Dorn et al. [22] used a dosimetric study prior to therapy with 150-400 MBq 131I and obtained daily images up to 4-5 days. They maximised the administered activity to an absorbed dose of 3 Gy to the bone marrow or 30 Gy to the lungs, corresponding to the LD5\/5 in EBRT, while aiming at >100 Gy to all known metastases. This resulted in a mean of 22.1 GBq (597 mCi) and a range of 7.4-38.5 GBq (200-1,040 mCi) activity administered per treatment. Many institutions will find it difficult to deliver these amounts owing to radioprotection restrictions, limiting the applicability of this method. To date, bone marrow depression has rarely been reported, while the rate of dose-related leukaemia has been estimated to be approximately 1% after 5-10 years. Lung fibrosis as a consequence of micronodular iodine-avid metastases has been reported in 1% of patients suffering from pulmonary metastases. The downside of these high-dose strategies may be an increase in the occurrence of these side-effects. On the other hand, assuming this \u201cmaximum safe dose-to be an optimal dosing strategy, extrapolation to a \u201cfixed activity dose-strategy of 7.4 GBq (200 mCi), as is widely applied, would \u201cunder-treat-54% and \u201cover-treat-3% of patients [23]. To date, there are no randomised and prospective trials that directly compare a low- and high-activity dose strategy in patients with metastases.\nThe treatment of DTC in childhood varies substantially from the standard approach in adults mainly owing to the different tumour biology in this age group. Usually activities of 50-100 MBq\/kg are given, treatment data on children being scarce and non-systematic.\nThere are a number of potential drawbacks of dosimetry that may preclude its use in many centres. One problem is the uncertainty of volume determinations by neck ultrasound shortly after thyroid surgery since the differentiation between scar tissue, haematoma and thyroid remnant is often difficult. The same holds true for the use of computed tomography (CT), magnetic resonance imaging (MRI) and positron emission tomography (PET) in the evaluation of distant metastatic lesions, especially in the case of diffuse lung metastases. Also, it is difficult to predict radioiodine kinetics during therapy from prior diagnostic studies owing to the large difference in administered and measured activity and potential subsequent biological effects. PET studies using the cyclotron product 124I-NaI, if possible in combination with PET\/CT tomography, may prove to be particularly valuable in improving treatment planning and patient-specific dosimetry. Compared with conventional methods, including CT and 131I scanning, this technique was found to be superior in terms of lesion detection and functional assessment [24]. Using three-dimensional 124I-PET voxel-based dosimetry, Sgouros et al. [25] showed a wide range of mean absorbed dose values for individual tumours, from 1.2 to 540 Gy. Unfortunately, no correlation with response was reported in that study. However, this wide variation certainly implies that the assessment of meaningful dose-effect relationships and clinical dosimetry should include further development of a quantitative approach.\n131I-mIBG therapy in neuroblastoma and phaeochromocytoma\nFor more than two decades, mIBG labelled with the \u03b3\/\u03b2 emitter iodine-131 (131I-mIBG) has been used to treat neural crest-derived tumours, essentially neuroblastoma and phaeochromocytoma, both in relapsed and in newly diagnosed patients. In high-stage neuroblastoma, the treatment may represent (a) palliative therapy [26], (b) first-line therapy, as a single agent or combined with chemotherapy [27], (c) consolidation therapy after induction of a \u201cgood partial remission-[28] and, more recently, (d) second-line therapy after failed induction chemotherapy, combined with topotecan and stem cell rescue in children with metastatic neuroblastoma [29] or with myeloablative chemotherapy and autologous stem cell transplantation in refractory neuroblastoma [9]. In neuroblastoma, important efforts have been made to develop clinically useful dosimetry methods to predict the radiation dose to the red marrow as the critical organ for bone marrow toxicity, in order to apply optimal and individual dose maximisation in children with highly aggressive tumours. Three different methods of activity administration have evolved from the experience of treating such patients.\nThe first approach, developed in Amsterdam, the Netherlands, is to give fixed activity dose \u201cfractions-of 7.4-11.1 GBq (200-300 mCi), more recently combined with hyperbaric oxygen or topotecan. This approach has been proven to have a synergistic effect on cell killing in vitro when used in combination with 131I-mIBG [30]. Intervals between fractions are determined by blood count recovery and the treatment is continued to maximum response [31]. An advantage of this method is its simplicity, which may be viewed as important in very sick children and in those who require prompt treatment, especially when receiving first-line therapy. Another important advantage of lower activity doses is the shorter isolation and hospitalisation period required for reasons of radioprotection, which may be regarded as highly significant in these usually very sick children with a grave prognosis. As whole-body and tumour radiation doses are never calculated, any kind of dose-response assessment and optimisation on the basis of absorbed radiation dose is, however, impossible. Furthermore, the relatively low dose rate of radiation compared with (ultra-)high doses may be viewed as radiobiologically suboptimal. The clinical results of this approach need to be verified in a multicentre phase III trial that should include a dosimetric assessment of the whole-body dose.\nThe second approach originates from the United Kingdom, where there has been a long tradition of using pre-therapy dosimetry. In a phase I\/II study on advanced, chemo-refractory stage III\/IV patients, Lashford et al. observed that 80% of patients developed grade 3 or 4 haematotoxicity at a whole-body absorbed dose of 2.5 Gy established from a pre-therapy 131I-mIBG scan [32]. Monsieurs et al. used 123I-mIBG prior to therapy to the same effect [33]. Previously, Tristam et al. had shown a highly skewed distribution of tumour radiation doses after a fixed, diagnostic activity [34]. Matthay et al. found a good correlation between whole-body and tumour dose [35]. As radiation dose is principally related to response, this approach may lead to a more standardised whole-body and tumour absorbed dose [36] and may offer more possibilities of dose escalation, especially in the presence of an altered biodistribution, e.g. due to partial kidney failure or high tumour load. Recently, an ESIOP experimental mIBG protocol has been proposed in which the aim is to deliver a total whole-body absorbed dose of 4.0 Gy in two fractions, in combination with topotecan, after failure of induction chemotherapy. Post-therapy dosimetry is performed after a first fixed fraction of 440 MBq\/kg (12 mCi\/kg), with calculation of the activity dose to be administered with the subsequent second fraction in order to arrive at the desired total whole-body absorbed dose of 4 Gy. The importance of this protocol is that whole-body and tumour absorbed dose can be estimated and the relation to both toxicity and response can be established, unlike with the non-dosimetry protocols. The schedule of intensification of mIBG therapy by dose escalation and radiosensitisation with topotecan with a haematopoietic autograft has been shown to be safe and practicable [29]. The next step will be to prove that this approach will also lead to a better outcome in neuroblastoma patients.\nThe third approach can be derived from recent publications from the United States, in which patients received high activity doses of mIBG on the basis of an activity dose of 550-660 MBq per kilogram body weight (15-18 mCi\/kg), with stem cell support if necessary. In an early study, Sisson and co-workers examined predictors of toxicity and found the best correlation to be between whole-body absorbed dose and platelet ratio; however, comparable and statistically significant correlations were found with GBq\/kg body weight and GBq\/m2 body surface [37]. This observation was supported by Matthay and colleagues [35], who found the amount of 131I (GBq\/kg) and whole-body dose (Gy), but not administered activity dose (GBq), to be significantly correlated with platelet and neutrophil nadir. In a recent study by Dubois et al. [38], substantial haematotoxicity was seen at 660 MBq\/kg (18 mCi\/kg) in a group of patients with advanced, heavily pre-treated neuroblastoma, including myeloablative chemotherapy in 81%. Of these patients, 36% required autologous haematopoietic stem cell transplantation. The resulting median whole-body absorbed dose was 2.92 Gy with a median administered activity dose of 13.5 GBq (366 mCi, range 198-895 mCi). Interestingly, whole-body radiation dose was shown to correlate with failure to engraft platelets or red cells in those patients receiving autologous stem cell transplantation. An encouraging response rate of 27% was reported, however. The same authors recently established a maximum tolerated dose (MTD) of 440 MBq\/kg (12 mCi\/kg) with myeloablative chemotherapy and autologous stem cell transplantation [9]. The advantage is that there is no need for planning\/simulation in this high-dose protocol, which may increase in importance if multiple centres participate.\nMalignant, metastatic phaeochromocytoma is a very rare disease but can be excellently treated by 131I-mIBG in both a palliative and a curative setting. Literature data on dosimetry are scarce and casuistic. In 1997, Loh et al. reviewed the literature concerning 116 previously treated cases [39]. They found a cumulative activity dose of 3.6-85.9 GBq (96-2,322 mCi, mean 490 \u03b1 350 mCi) in 1-11 (mean 3.3 \u03b1 2.2) therapies, with a mean single activity dose of 5.8 GBq (158 mCi). Dose prescriptions were as a fixed activity dose of 3.7-11.1 GBq (100-300 mCi or 3-9 mCi\/kg). A recent study was performed in order to evaluate the performance of mIBG therapy with high activities after debulking surgery [40]. The median individual cumulative activity dose was 37.6 GBq (1,015 mCi), with a range of 14.3-62.5 GBq (386-1,690 mCi), in one to three consecutive therapies. Grade 3 platelet toxicity was observed after 79% of therapies, and grade 3 and 4 neutrophil toxicity after 53% and 19% of therapies, respectively. All patients had stem cells harvested before therapy, but 11 patients showed spontaneous reconstitution and only one required stem cell return. Response rate was 10\/12 patients (83%).\nRadiopeptide therapy for neuro-endocrine tumours\nSeveral clinical trials have investigated the use of peptide receptor radionuclide therapy (PRRT) with a radiolabelled somatostatin analogue as one of the newly developed targeted tools for neuro-endocrine tumours [41-44]. Individual pre-therapeutic dosimetry is necessary for patient selection and therapy planning because there are huge inter-patient differences in radiopeptide uptake in normal organs and tumour tissues. This may be related to varying somatostatin receptor densities on tumour cells, as well as to factors such as tumour volume, interstitial pressure and viability. The dosimetric studies that have been performed in this treatment have been the most sophisticated in the field and exemplory for other types of treatment. Initial studies were performed with the radiopeptide used in diagnostics, [111In-DTPA0]-octreotide, given in high activities. Results were encouraging, with symptomatic and biochemical responses in a large percentage of patients, although objective responses were rare [42]. The analogue [90Y-DOTA0,Tyr3]-octreotide has been investigated in various phase I-II trials [41, 43]. 90Y-labelled lanreotide has also been investigated (in the MAURITIUS trial) [45]. For dosimetry of 90Y from post-therapeutic bremsstrahlung, images that substantially lack quality must be quantified. Therefore, two alternative approaches have been developed as a surrogate for the original radiopeptide, namely therapy simulation with either the 111In-labelled peptide or the 86Y-labelled peptide [46, 47]. These methods are intended to provide pre-therapy assessment of the optimal activity administration [48-51] (Table 1). The variation that can be appreciated among the various combinations in the table may be primarily technical\/methodological in origin but also biological, based on differences in somatostatin receptors and isotope\/chelator affinity. The reader is referred to the respective papers for more detailed information.\nTable 1Absorbed doses to principal organs and to tumour (Gy\/GBq \u00b1SD), deriving from different radiopeptidesStabin 1997 [109], Kwekkeboom 2001 [50]Kwekkeboom 2001 [50]Cremonesi 1999 [47]Forster 2001 [110]Helisch 2004 [111]Forrer 2004 [112]Therapy[111In-DTPA0, Tyr3]-octreotide[177Lu-DOTA0, Tyr3]-octreotate[90Y-DOTA0, Tyr3]-octreotide[90Y-DOTA0, Tyr3]-octreotide[90Y-DOTA0, Tyr3]-octreotide[90Y-DOTA0, Tyr3]-octreotideDosimetry[111In-DTPA0, Tyr3]-octreotide[177Lu-DOTA0, Tyr3]-octreotate[111In-DOTA0, Tyr3]-octreotide[86Y-DOTA0, Tyr3]-octreotide[86Y-DOTA0, Tyr3]-octreotide[111In-DOTA0, Tyr3]-octreotidePatients16530385Kidneys0.52 \u03b1 0.241.65 \u03b1 0.473.9 \u03b1 1.9b\n2.73 \u03b1 1.412.84 \u03b1 0.64Kidneys + protection0.88 \u03b1 0.191.71 \u03b1 0.89Liver0.065 \u03b1 0.010.21 \u03b1 0.070.72 \u03b1 0.570.66 \u03b1 0.150.72 \u03b1 0.400.92 \u03b1 0.35Spleen0.34 \u03b1 0.162.15 \u03b1 0.397.62 \u03b1 6.302.32 \u03b1 1.972.19 \u03b1 1.116.57 \u03b1 5.25Red marrow0.03 \u03b1 0.010.07 \u03b1 0.0040.03 \u03b1 0.010.49 \u03b1 0.0020.06 \u03b1 0.020.17 \u03b1 0.02Tumour (range)0.72-6.8a\n3.9-37.91.4-313.21-19.582.1-29.52.4-41.7aFrom reference [50]bSeries enlarged from the original one, as in Bodei et al. [44]\nAlthough [90Y-DOTA0,Tyr3]-octreotide (or -lanreotide) and its imageable counterpart [111In-DOTA0,Tyr3]-octreotide (or -lanreotide) are not chemically identical, the latter has been used for dosimetric simulation, based on the hypothesis that the similar physical and biological half-lives yield a comparable in vivo pharmacokinetics and biodistribution, especially concerning the renal uptake, which depends on aspecific phenomena. A drawback of this method is that the small structural modification may affect the somatostatin receptor binding affinity [52]. Regarding biodistribution, the organs receiving the highest predicted absorbed doses, in a first series of 18 patients, included the spleen (7.6 \u03b1 6.3 mGy\/MBq), the kidneys (3.3 \u03b1 2.2 mGy\/MBq) and the tumour (1.4-31 mGy\/MBq, mean 10). When the series was enlarged to 30 patients, a slightly higher kidney absorbed dose (3.9 \u03b1 1.9 mGy\/MBq) was observed [43]. Shortcut approaches involving use of the commercially available molecule [111In-DTPA0]-octreotide or OctreoScan\u00ae have been proposed [53]. Nonetheless, probably as a result of the quite different biokinetics and receptor affinity of [90Y-DOTA0,Tyr3]-octreotide and [111In-DTPA0]-octreotide, which are indeed chemically different, comparative data obtained appear not to be sufficiently overlapping for this tracer.\nA thorough dosimetric study using PET with [86Y-DOTA0,Tyr3]-octreotide, biochemically identical to the therapeutic molecule, was carried out in 24 patients [54]. This offers substantial advantages in terms of spatial resolution and quantification, but the short half-life of the radionuclide leaves later phases of the biokinetics to estimates based on extrapolation.\nThe newest radiopeptide, [177Lu-DOTA0,Tyr3]-octreotate, offers further advantages. 177Lu has a lower energy (Emax 0.49 MeV) and penetration range (Rmax 2 mm) emission, but a longer half-life (6.7 days). The low abundance gamma emissions (113 and 208 keV) allow for dosimetry and imaging prior to as well as post therapy. Moreover, octreotate has a six- to ninefold higher affinity for somatostatin receptor 2, the somatostatin receptor most frequently expressed in neuro-endocrine tumours. Unfortunately, to date a thorough dosimetric analysis is lacking, but data deriving from a study comparing [177Lu-DOTA0,Tyr3]-octreotate with [111In-DTPA0]-octreotide indicate that, compared with [90Y-DOTA0,Tyr3]-octreotide, [177Lu-DOTA0,Tyr3]-octreotate delivers a lower burden to organs, with absorbed doses of 1.8-2.7 mGy\/MBq to the spleen, 1.0-2.2 mGy\/MBq to the kidneys (lowered to 0.7-1.1 mGy\/MBq with protection) and 0.1-0.3 mGy\/MBq to the liver. Red marrow absorbed dose, derived by the blood approach, ranged from 0.05 to 0.08 mGy\/MBq [44].\nDue to their marked radiosensitivity, the kidneys undoubtedly represent the critical organ, particularly after [90Y-DOTA0,Tyr3]-octreotide. Renal irradiation arises from the proximal tubular re-absorption of the radiopeptide and the resulting retention in the interstitium. According to EBRT studies, the renal maximum tolerated absorbed dose is conventionally considered to be in the range of 23-25 Gy. According to the National Council on Radiation Protection and Measurements (NCRPM) an absorbed dose of 23 Gy to the kidneys causes detrimental deterministic effects in 5% of patients within 5 years [55]. Sporadic cases of delayed renal failure, in some cases end-stage disease requiring dialysis, have been observed, especially in patients who have received an activity dose of more than 7.4 GBq\/m2 [56]. Nephrotoxicity is accelerated by other risk factors, such as pre-existing hypertension or diabetes. Given the high kidney retention of radiopeptides, positively charged molecules, such as L-lysine and L-arginine, are used to competitively inhibit the proximal tubular re-absorption of the radiopeptide. This results in a reduction in the renal absorbed dose of between 9% and 53% [57]. Doses are further reduced by up to 39% by prolonging infusion over 10 h and by up to 65% by prolonging it over 2 days after radiopeptide administration, thus covering more extensively the elimination phase through the kidneys [43]. Despite kidney protection, renal function loss may become clinically evident years after PRRT. A median decline in creatinine clearance of 7.3% per year was reported in patients treated with [90Y-DOTA0,Tyr3]-octreotide and of 3.8% per year in patients treated with [177Lu-DOTA0,Tyr3]-octreotate. Cumulative and per cycle renal absorbed dose, age, hypertension and diabetes are considered factors contributing to the decline of renal function after PRRT [58]. Clinical experience and dosimetric studies clearly indicate that the renal absorbed dose estimated by conventional dosimetry does not accurately correlate with the renal toxicity observed in patients treated with [90Y-DOTA0,Tyr3]-octreotide. Consideration of additional parameters, such as patient-specific kidney volume and distribution of the radionuclide, appears to give a better correlation with the clinical effects [59]. Assessment of individual kidney volume by CT scan yields a wide variability when compared with the standardised phantom. Moreover, autoradiographic studies, performed on human kidney after in vivo injection of 111In-peptides, have demonstrated that the majority of radioactivity is deposited within the renal cortex, mainly in the juxtamedullary region. This leads to a higher deposition of energy per unit mass, compared with conventional dosimetry. Hence, calculation of the kidney absorbed dose assuming a homogeneous renal distribution of radioactivity is inadequate. New techniques accounting for the difference in radioactivity placement in the kidneys, on the basis of a CT-based volumetric analysis, appear more realistic [60].\nEven if predicted absorbed doses are much lower than the threshold for toxicity, the other target organ that gives rise to concerns about acute and permanent toxicity after PRRT is the bone marrow, particularly in repeated administrations [41-43]. Acute haematological grade 3 or 4 toxicity is not uncommon, especially after [90Y-DOTA0,Tyr3]-octreotide, and sporadic cases of myelodysplastic syndromes or even overt acute myeloid leukaemia have been reported with all three therapeutic radio-compounds. Bone marrow dosimetry is usually modelled through a blood-based method, in which an equivalent distribution of the radioactivity from blood throughout the bone marrow is conservatively considered [61, 62]. Currently, the potential risk of kidney and red marrow limits the amount of radioactivity that may be administered. Indeed, when tumour masses are irradiated with suitable doses, volume reduction may be observed (Fig. 1) [63]. Tumour remission is positively correlated with high uptake during [111In-DTPA0] octreotide scintigraphy. Nevertheless, tumour radiation dose depends not only directly on the administered activity and the uptake versus time, but also on the tumour load. This is confirmed by clinical data regarding the response characteristics: patients with a limited number of liver metastases respond to PRRT, whilst patients with a high tumour load do not [45]. Mathematical models have shown that 177Lu is better in small tumours (optimal diameter 2 mm), whilst 90Y is better in larger ones (optimal diameter 34 mm): very small masses are likely not to absorb all the \u03b2-energy released in the tumour cells by 90Y, while larger tumours will suffer from lack of uniformity of activity distribution of 177Lu. Finally, differences in dose rate must be taken into account: the longer physical half-life of 177Lu means a longer period is needed to deliver the same radiation dose than when using 90Y. This may allow more time for tumour re-population [64]. Therefore, combination therapy with 90Y and 177Lu, either simultaneously or in distinct settings, has been suggested to overcome the difficulties of real clinical situations involving different sized lesions.\nFig. 1Tumour dose-response relationship in 13 patients treated with 90Y-DOTATOC. Tumour volumes were assessed by CT before and after treatment. Tumour dose estimates were derived from CT scan volume measurements and quantitative 86Y-DOTATOC imaging performed before treatment. Data were further computed using the MIRDOSE spherical model. Reprinted by permission of the Society of Nuclear Medicine from [63]\nTreatment of solid tumours by radiolabelled antibodies\nMost clinical radioimmunotherapy (RIT) studies have been performed in colorectal cancer, using antibodies against CEA, TAG-72, A33 and KSA. Other tumours studied have included ovarian cancer (anti-MX35, anti-folate receptor Mov18, anti-HMFG1), prostate cancer (anti-PMSA), breast cancer (anti-mucin BrE3, NR-LU-10), glioma (anti-tenascin) and renal cancer (anti-G250) [65].\nAlthough at later time points after injection of radiolabelled monoclonal antibodies, adequate to high uptake may serve to delineate deposits of solid tumours, the relatively unfavourable therapeutic window between the anti-tumour effect and toxicity hampers the introduction of these agents in the clinic. Solid tumours are generally more radioresistant than, for example, malignant lymphoma. The absorbed doses required to achieve a response of tumour deposits are higher than those needed to obtain a response in malignant lymphoma. Activity doses leading to adequate absorbed doses in tumour deposits therefore result in significant radiation-induced toxicity, primarily of the bone marrow, as the most radiation-sensitive organ; myeloablation may be the goal as well as the result of this.\nMost studies aim at administration of the MTD that results in acceptable toxicity to the bone marrow, as the organ responsible for dose-limiting toxicity. Several methods have been reported for calculation of the absorbed dose to the bone marrow, e.g. region of interest analysis of scintigraphic data or a model based on radioactivity in the blood. When radiolabelled antibodies bind to blood, bone and bone marrow components or when the radionuclide accumulates in bone or bone marrow upon metabolisation of the radionuclide-antibody complex, calculation of the red marrow dose is more complex than when using radiolabelled antibodies that lack these characteristics [66, 67]. The use of a model for bone marrow dosimetry using blood activity may result in better reproducibility of the bone marrow dosimetry. Wessels et al. [68] observed that historical variations as high as 200-700% between different institutions performing marrow absorbed dose calculations could be dramatically reduced to \u221229% to +20% by a central computing facility and the use of similar methodology, based on the standard American Association of Physicists in Medicine (AAPM)\/Sgouros blood model [66].\nIn a study using 131I-labelled murine monoclonal antibody G250 in patients with metastatic renal cell cancer, haematological toxicity correlated with whole-body absorbed radiation dose [69]. In a retrospective analysis of 114 patients who underwent 131I-labelled antibody therapy, absorbed dose-based definitions of MTD and escalation variables proved to be better than activity-based methods [70]. In a study in which therapeutic doses of 131I-cG250 (the chimeric variant of G250) were administered, no correlation was found between haematological toxicity and either the radiation absorbed dose to the whole body or bone marrow or the administered activity [71]. Juweid et al. observed that besides red marrow dose, baseline blood counts, multiple bone and\/or marrow metastases and recent chemotherapy are important factors related to haematological toxicity after radio-immunotherapy [72], making a dosimetric approach to identify the optimal radionuclide dose more complicated.\nOne way to overcome the unfavourable therapeutic index of tumour response and normal organ toxicity is to focus on treatment of patients with minimal residual disease. For example, patients with ovarian cancer in complete remission after debulking and chemotherapy have an approximately 50% chance of relapse. Compared with results in matched controls, Nicholson et al. observed an increase in 5-year survival from 55% to 80% following intraperitoneal administration of 90Y-labelled HMFG1 in patients with ovarian cancer in complete remission after chemotherapy [73]. However, a more recent randomised clinical trial in patients with ovarian cancer in complete remission after debulking and chemotherapy did not reveal a benefit in disease-free and overall survival after treatment with 90Y-labelled murine HMFG1 [74]. Introduction of 90Y-muHMFG1 did not delay the time to relapse and did not result in prolonged survival as compared with the control group. One can argue that 90Y is far from ideal for treatment of minimal residual disease owing to its high \u03b2-energy, and that radionuclides with lower range emissions, such as 177Lu, have more favourable characteristics for this purpose. Furthermore, relatively large antibody doses were used, which may have resulted in saturation of the epitopes on cancer cells and subsequently in low tumour-to-non-tumour ratios of radioactivity uptake. Obviously, clinical dosimetry could play an important role in explaining these differences in therapy results.\nA very significant advance that promises to increase the radiation dose to the tumour while reducing that to the bone marrow is the development of pre-targeting strategies. In pre-targeting, the tumour is first targeted with a specific non-radioactive monoclonal antibody construct. The antibody construct is allowed to accumulate in the tumour and to clear from the blood and non-target tissues. Subsequently, a small radioactive hapten is injected which has high affinity for the antibody construct. The radioactive hapten targets the antibody construct, while demonstrating rapid renal excretion. Examples of these approaches are the use of bispecific monoclonal antibodies and the biotin-avidin system [75, 76]. As pre-targeting increases the tumour\/normal organ uptake ratio [77], these approaches hold great promise for enhanced therapeutic efficacy. However, the use of multiple drugs poses the problem of fine-tuning dosing and timing of the interval between injections. Optimisation of the dosing and timing schedule is needed for optimal targeting of the tumour by the radioactive compound, while assuring low normal organ uptake. Robust dosimetric analysis of tumour and normal organ uptake of the radioactive small molecule is a requirement for the successful development and implementation of pre-targeting strategies [78, 79].\nRadioimmunotherapy of B-cell lymphoma\nResearch in RIT in lymphoma has resulted in two FDA-approved radiopharmaceuticals, 90Y-ibritumomab or Zevalin\u00ae (IDEC Pharmaceuticals and Schering AG) and 131I-tositumomab or Bexxar\u00ae (Glaxo Smith Kline), for the treatment of B-cell lymphoma [80, 81]. Both are directed against CD20, albeit not against the same epitope. They are both approved for the treatment of relapsed or refractory follicular\/low-grade or transformed B-cell lymphoma including rituximab-refractory follicular B-cell lymphoma in the US, but only Zevalin is approved in the EU and only for follicular lymphoma. Pre-treatment with unlabelled monoclonal antibody (=preload) as part of the treatment with Zevalin and Bexxar is current practice, as it leads to a more favourable biodistribution. This has been studied in animal models, as well as in the setting of myeloablative RIT [82-84]. The preload may clear peripheral B cells from the circulation, improving tumour targeting of subsequently administered radiolabelled monoclonal antibodies. This optimisation has been accomplished by diagnostic and dosimetric 111In or 131I tracer studies. On the other hand, the intrinsic therapeutic efficacy of the antibody is a confounding factor, making evaluation of the relation between absorbed dose and treatment response more difficult than for other radiopharmaceuticals [85, 86].\nBexxar has been developed using the whole-body absorbed dose as a substitute for bone marrow dosimetry by administering a trace amount of 131I-labelled CD20 antibody and determining the total body clearance prior to therapy in order to calculate the patient-specific injected activity dose that should deliver the specified absorbed dose to the whole body. The method has been further simplified to an estimation based on three points [87]. In a dose escalation study with absorbed doses to the whole body ranging in 0.1-Gy increments from 0.25 to 0.85 Gy, the MTD was found to be 0.75 Gy in patients who had not received prior high-dose chemotherapy with stem cell support and who had platelet counts of \u2265150 \u00d7 109\/litre. A comparison between the therapeutic activity calculated on the basis of the diagnostic pre-therapy tracer study and the amount calculated per kilogram body weight showed that, using the latter method, 50% of the patients would have been either over- or underdosed by 10% or more, and 16% of the patients by 25% or more. Interestingly, a correlation between the duration of complete remission (CR) and the absorbed dose to the whole body was found. Patients receiving a whole-body absorbed dose between 0.65 and 0.85 Gy showed longer CR than patients receiving between 0.25 and 0.55 Gy [88]. In contrast, Zevalin has been developed with the notion that dosimetry may be dispensable. In a randomised study comparing Zevalin with rituximab alone, a secondary objective was to determine whether dosimetry was required [10]. A tracer dose of 185 MBq (5 mCi) 111In was administered for dosimetric purposes with the first infusion, followed by a therapeutic activity dose of 0.4 mCi\/kg (15 MBq\/kg) Zevalin 7 days later. The absorbed doses to normal organs and the marrow were found to be within the specified limits of 20 Gy for solid organs and 3 Gy for red marrow in 72 patients. The median estimated absorbed dose to the tumour was 15 Gy (range 0.6-24 Gy). There was no significant correlation between dosimetric and pharmacokinetic parameters and haematological toxicity, although the correlation between nadir of the neutrophil count and the whole blood half-life of 90Y exhibited borderline statistical significance. It was concluded that dosimetry may be excluded for populations of Zevalin patients who meet certain criteria for pre-treatment platelet count and percentage of marrow involvement by tumour.\nA possible explanation for this discrepancy may be found in the photon energy emitted by 131I; this may account for a large part of the cross-absorbed dose to the bone marrow, which is almost independent of the amount of bone marrow involvement. It has been reported that the self-absorbed dose accounts for 64% of the whole absorbed dose to the red marrow [89], leaving 36% as the cross-absorbed dose. Scintigraphic assessment of absorbed dose to the bone marrow has its shortcomings owing to over- and underlying tissues as well as to the local variability in bone marrow involvement by the disease targeted. It is especially difficult to use pharmacokinetic data for calculation of the absorbed bone marrow dose, since bone marrow involvement is a strong confounder. Other important issues are the bone to bone marrow ratio and variable values for the activity concentration in the blood and red marrow [90, 91]. A fixed ratio between blood and bone marrow has been used in bone marrow dosimetry based on blood radioactivity levels [10, 64]. However, it has been suggested that the red marrow to blood ratio is not fixed, but increases continuously up to 72 h post injection in both patients and rats [92]. Furthermore, haematological toxicity, like tumour response, is a deterministic effect of radiation, characterised by a sigmoid rather than by a linear dose relationship [93]. A sigmoid relationship may be discernible in a reasonably homogeneous population, but is likely to be more difficult to identify if the population is heterogeneous. The bone marrow reserve may be more relevant to the magnitude of toxicity than is the absorbed dose. Another particular challenge to haematology RIT dosimetry may be tumour regression during energy deposition, as is frequently the case in rapidly responding lymphoma. This may lead to underestimation of absorbed dose, since dose is per definition energy per unit mass [94]. The opposite will be the case if the tumour grows during energy deposition. To avoid this pitfall, repeated volume assessments during therapy would be necessary, or alternatively one could use voxel-based dosimetry, which is likely to be less sensitive to changes in mass.\nMyeloablative RIT at the MTD for normal organs is by definition dependent on dosimetry. Dose-limiting organs may be the lungs, kidneys or liver. In high-dose RIT using the 131I-labelled mouse antibody tositumomab, the lungs were found to be the dose-limiting organ in 28 of 29 patients and the kidneys in the remaining patient [95]. The opposite was found when the pharmacokinetics of the chimeric 131I-labelled rituximab were studied, i.e. the kidneys were found to be the critical organ [96]. These differences may be due to the significant difference in half-life between the antibodies, the chimeric and murine antibodies having half-lives of 88 h and 56 h, respectively. The MTD for myeloablative 131I-tositumomab was established to be 25 Gy to the lungs. Studies of myeloablative RIT using 90Y are ongoing and encouraging results have been reported [97].\nIn several experimental studies, Auger emitters have been therapeutically superior to \u03b2 emitters when taken to the MTD [98, 99]. This is likely to be due primarily to lower bone marrow toxicity but is perhaps also attributable to the deposition of more energy in single cells or small tumour cell clusters. Auger emitters would thus be advantageous in an adjuvant setting, in leukaemia, but they may also be superior where there is tumour bulk. The challenge is how to estimate absorbed dose in single cells, but one may begin in patients with a significant amount of circulating tumour cells that can be studied ex vivo [100]. Interestingly, Kaminski reported that in a number of patients who relapsed following RIT the relapse occurred only at sites previously not known to be involved with tumour [101]; this indicates a possibility that small tumour manifestations receive lower absorbed doses than expected on the basis of antigen density and tumour diameter [102].\nDiscussion\nAs stated by DeNardo [103], \u201cclaims for specific dosimetry have to demonstrate that the frequency of excess toxicity and\/or tumour underdosing significantly decreases\u201d. Dosimetry should provide a quantification procedure that is primarily of additional benefit over empirical, fixed dosing with or without visual scintigraphic assessment. In standard oncology practice, a new therapeutic agent undergoes phase I, II and III testing before becoming a standard treatment. In a phase I study, the maximum tolerated dose is established and side-effects recorded. Dosimetry should play an essential role in this phase and establish a threshold dose above which clinically significant side-effects occur. In phase II, the new radionuclide therapy is evaluated in terms of effect on tumour response and survival. Here, dosimetry should enable the determination of a clinical dose-response relationship. In phase III, the new therapy is compared with the standard one. At this stage, dosimetry helps to elucidate the clinical effects in a larger patient group, e.g. it may be observed that some subgroups have a better or worse result. Moreover, multicentre trials offer the opportunity to compare results in different institutions and countries, as well as provide the opportunity to standardise the dosimetry procedure in a larger context. Evidence-based medicine entails randomised and prospective trials. However, after 60 years of treating thyroid cancer patients, international guidelines still cannot provide a consensus on the amount of radioiodine that should be given. Undoubtedly, the need for randomised trials will increase in the coming years, as in other areas. This underlines the importance of stepping up scientific efforts to include optimal dosimetry not only as an inherent part of radionuclide therapy, but also as an inherent part of these studies. In this context, there was a remarkable recent editorial in the Journal of Clinical Oncology [104], discussing the limitations of the body surface principle that forms the basis for chemotherapy dosing. Indeed, the bioavailability of chemotherapy, and hence the dose to the target, suffers from a similar metabolic variability as is observed for radionuclides, and this variability seems to be more important than can be accounted for by the body surface in square metres as the sole parameter. Interesting parallels may be drawn for translational research, for example in pharmacokinetic modelling and molecular imaging.\nMany modern gamma cameras are optimised for photons below 200 keV and are less suitable for radionuclide therapy dosimetry if higher energy photons are involved. SPECT overcomes the problem of superimposition of target and other activity, but spatial resolution at depth is always worse than in planar imaging, so quantification has to rely on more or less representative phantom models [105]. PET has a 30-40 times higher sensitivity for a given spatial resolution that is typically 5 \u00d7 5 mm. This is a major advantage over SPECT in pharmacokinetic dynamic modelling. Correction for attenuation has become relatively easy with the new PET\/CT and SPECT\/CT cameras, but other correction factors, e.g. for scatter, linearity and calibration, are also critical. Partial volume effects are a major issue in the quantification of small tumours and occur at below about twice the spatial resolution. This is thus more of a problem for SPECT (spatial resolution for 131I, 25-30 mm) than for PET (spatial resolution 6-8 mm). Finally, as discussed in the radiopeptide section, the radionuclides used for dosimetry must show similar biochemical and physical behaviour to those used for therapy. As previously highlighted, the use of 124I PET and 86Y-DOTATOC PET dosimetry [25, 59] has been a landmark development. PET\/CT and SPECT\/CT and \u201cmolecular-radiopharmaceutical developments offer major opportunities for radionuclide therapy dosimetry [1]. Further improvements in the performance of these cameras, and hence dosimetry, can be foreseen. Also, the proliferation of microsystems such as micro-PET\/SPECT\/CT\/MRI is allowing dynamic in vivo animal research, increasing our knowledge of radiopharmaceutical biodistribution, improving quantitation and permitting early selection of therapeutic radiopharmaceuticals. Table 2 lists some of the most important methodological issues involved in performing accurate dosimetry today.\nTable 2.Methodological issues in performing clinical dosimetry- Diagnostic tracer and\/or therapeutic activity study- Planar and\/or tomographic (SPECT and\/or PET) quantification- Dynamic and\/or multiple time point activity sampling- Linearity of detector response in low and\/or high activity- Correction factors for attenuation, scatter and\/or partial volume effects- Nuclear medicine and\/or radiological volume and response- Standard (MIRD,...) and\/or simulative (Monte Carlo,...) modelling- Tissue heterogeneity and\/or spatial resolution limits- Treatment of minimal residual disease and\/or partial volume effects- Disease-induced and\/or therapy-induced changes in parameters- Macro- and\/or microdosimetry techniques- Animal and\/or human dosimetry data\nRadiobiology is a science in itself. Nevertheless, up to now very little consideration has been given to the effects of radionuclide therapy at the cellular and molecular level [106]. Rather, extrapolations have been made from EBRT, despite the fundamental differences in radiation kinetics. The majority of observations in EBRT have been made under the condition of a high dose rate, while clinical radionuclide therapy entails a decreasing, low dose rate. The effect of fractionated EBRT is primarily influenced by the 4 R\u2019s of radiobiology: repair of DNA damage, repopulation of tissues, re-oxygenation of tumour and redistribution in the cell cycle. In radionuclide therapy, with its decreasing dose rate, tumour DNA repair takes place simultaneously with sublethal damage. Bystander effects, i.e. radiation-like effects in unhit cells, may be of significance in low-dose, low dose rate radiotherapy [2]. Furthermore, it is becoming increasingly apparent that the physical paradigm of direct cell killing by double-strand DNA breaks is insufficient. Non-DNA targets, such as cell membrane or RNA, may also be critical to target cell death or dysfunction. Moreover, new molecular-targeted oncology treatments may not produce direct cell death, but rather alter biochemical pathways or cellular homeostasis, that may interact with classical radiation targets or produce new radiation targets [107]. In this paradigm, the concept of radiation dose may need re-definition and the future radiation dose may be determined in terms of the biological and functional changes produced and observed, such as by blood markers and by PET, SPECT and\/or MRI. Biologically effective dose (BED) is a concept which has been successfully applied to radionuclide therapy through the landmarking studies of Barone et al. [59] using 86Y-labelled DOTATOC and kidney toxicity. BED is the product of the total physical dose multiplied by the \u201crelative effectiveness\u201d, which takes into account radiobiological parameters such as dose rate, radionuclide decay and tumour cell repair time, and allows direct quantitative comparison with EBRT. It should be noted that late-responding normal tissues and slow-growing tumours allow easier modelling than early-responding normal tissues and fast-growing tumours because of repopulation during treatment in the latter. Nevertheless, this may be regarded as a major development, stimulating further research [108] aimed towards the creation of a firm fundamental basis for radionuclide radiotoxicity and radiodosimetry. In future, it looks as if we shall no longer be looking at a \u201cHoly Gray\u201d, but rather at the worldly \u201cBEGray\u201d.\nIn conclusion, recent developments in molecular medicine, PET\/CT and SPECT\/CT cameras and radiobiology offer major scientific and clinical opportunities in radionuclide therapy dosimetry. However, only prospective, randomised trials with adequate methodology can provide the evidence that applied clinical dosimetry results in better patient outcome than is achieved with fixed activity dosing methods.","keyphrases":["radionuclide therapy","radiotherapy","thyroid carcinoma","neuro-endocrine tumours","solid tumours","lymphoma","radio-immunotherapy","radiation dosimetry"],"prmu":["P","P","P","P","P","P","P","R"]}
{"id":"Purinergic_Signal-3-1-2096766","title":"CD39 and control of cellular immune responses\n","text":"CD39 is the cell surface-located prototypic member of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) family. Biological actions of CD39 are a consequence (at least in part) of the regulated phosphohydrolytic activity on extracellular nucleotides. This ecto-enzymatic cascade in tandem with CD73 (ecto-5\u2013nucleotidase) also generates adenosine and has major effects on both P2 and adenosine receptor signalling. Despite the early recognition of CD39 as a B lymphocyte activation marker, little is known of the role of CD39 in humoral or cellular immune responses. There is preliminary evidence to suggest that CD39 may impact upon antibody affinity maturation. Pericellular nucleotide\/nucleoside fluxes caused by dendritic cell expressed CD39 are also involved in the recruitment, activation and polarization of na\u00efve T cells. We have recently explored the patterns of CD39 expression and the functional role of this ecto-nucleotidase within quiescent and activated T cell subsets. Our data indicate that CD39, together with CD73, efficiently distinguishes T regulatory cells (Treg) from other resting or activated T cells in mice (and humans). Furthermore, CD39 serves as an integral component of the suppressive machinery of Treg, acting, at least in part, through the modulation of pericellular levels of adenosine. We have also shown that the coordinated regulation of CD39\/CD73 expression and of the adenosine receptor A2A activates an immunoinhibitory loop that differentially regulates Th1 and Th2 responses. The in vivo relevance of this network is manifest in the phenotype of Cd39-null mice that spontaneously develop features of autoimmune diseases associated with Th1 immune deviation. These data indicate the potential of CD39 and modulated purinergic signalling in the co-ordination of immunoregulatory functions of dendritic and Treg cells. Our findings also suggest novel therapeutic strategies for immune-mediated diseases.\nIntroduction\nOur research interests have been in the purinergic modulation of vascular inflammatory and cellular immune responses in transplantation settings [1, 2]. It is generally accepted that extracellular nucleotides [e.g., ATP, uridine triphosphate (UTP), adenosine diphosphate (ADP)], and the derivative nucleosides (e.g., adenosine from ATP), are released in a regulated manner by most all cells to provide the primary components for purinergic responses [3]. High levels of ATP may be released by CD4+ and CD8+ T cells upon stimulation with Con A or anti-CD3 mAb and serve to activate cells [4]. Importantly, ATP [or uridine diphosphate (UDP)] stimulation of monocytes, lymphocytes and endothelium induces largely proinflammatory responses, such as the release of interleukin (IL)-1 (or IL-8) [4\u20136]. On dendritic cells (DC), exposure to extracellular ATP induces migration and differentiation to drive cellular immune responses [7]. Adenosine is also recognized as a bioactive agent in vascular inflammatory states, with effects mediated on both vascular cells and leukocytes [8]. In addition, adenosine has known anti-thrombotic effects, modulates the expression of anti-apoptotic genes and is immunosuppressive [9]. Adenosine is constitutively present in the extracellular space at low concentrations, but under metabolically stressful and hypoxic conditions, the levels rise dramatically [10]. Primary release of the mediator could occur ab initio, or this might follow conversion of released nucleotides to adenosine (see later).\nThe nucleotide\/nucleoside mediators alluded to above bind specific purinergic receptors that comprise the second requirement for this complicated signalling network. Almost all cells carry cell-surface type 2 purinergic (P2) receptors for nucleotides and adenosine or type 1 purinergic (P1) receptors [11]. There are seven ionotropic (P2X), at least eight metabotropic (P2Y) and four adenosine receptor subtypes that have been identified and characterized to date [12]. Multiple P2X and P2Y receptor subtypes are expressed by monocytes and dendritic cells, whereas lymphocytes express only P2Y receptors [11]. These various receptors operate in both auto- and paracrine loops and are considered to play a complex, important role in the regulation of vascular and immune cell-mediated responses. Depending on the P2 or adenosine receptor subtype, the cell types and signalling pathway involved, these receptors might preferentially trigger and mediate short-term (acute) processes that affect metabolism, adhesion, activation or migration. However, purinergic signalling also has profound impacts upon other more protracted reactions, including cell proliferation, differentiation and apoptosis, such as seen in several chronic inflammatory states [12]. These mechanisms could be also implicated in immune memory [9, 13].\nThe third, and final, component of purinergic signalling systems comprises ecto-nucleotidases [2, 14]. These ecto-enzymes hydrolyze extracellular nucleotides to generate nucleosides that in turn activate adenosine receptors, often with opposing effects to those seen with P2-mediated effects. Within the past decade, ecto-nucleotidases belonging to several enzyme families have been discovered, cloned and functionally characterized by pharmacological means.\nIn this review, we will focus on CD39, the prototype of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) family (EC 3.6.1.5) [15, 16]. These proteins comprise a group of ecto-enzymes that hydrolyze extracellular nucleoside tri- and diphosphates. The ecto-nucleotidase chain or cascade, as initiated by NTPDases, is terminated by ecto-5\u2013nucleotidase (CD73; EC 3.1.3.5) [2, 17]. Together, ecto-5\u2013nucleotidase and adenosine deaminase (ADA; EC 3.5.4.4), another ecto-enzyme that is involved in purine salvage pathways by converting adenosine to inosine, closely regulate local and pericellular extracellular concentrations of adenosine [2]. Most notably, however, in many tissues and cells, NTPDases also comprise dominant parts of a complex cell surface-located nucleotide hydrolyzing and interconverting machinery. Such ensembles also include the ecto-nucleotide pyrophosphatase phosphodiesterases (E-NPPs), NAD-glycohydrolases, CD38\/NADase activity, alkaline phosphatases, diadenosine polyphosphate hydrolases, adenylate kinase, nucleoside diphosphate kinase, and potentially ecto-F1-Fo ATP synthases [2].\nMany aspects of these ecto-nucleotidase families and detailed expositions of structure\/function relationships of the E-NTPDases and their role in the vasculature and nervous systems have been reviewed recently. The interested reader is referred to the recent special issue of Purinergic Signalling entitled \u2018Ecto-nucleotidases\u2013(PUSI 2, 2: 2006). This review will address only the immunobiology of CD39, focusing on T lymphocytes and alluding briefly to co-expression of CD73 by immunosuppressive T cell subsets.\nCD39 in the immune system\nRecently, almost all of the published investigative focus on CD39 has been on the thromboregulatory properties of CD39 with respect to platelet and endothelial activation [1]. However, CD39 was first described as a B lymphocyte activation marker [18]. This ecto-nucleotidase is also expressed on natural killer (NK) cells, monocytes, DC and subsets of activated T cells [19]. The relevance of the expression of CD39 by these cells is not yet clear.\nDevelopment of mutant mice either null for Cd39 or where human CD39 has been over-expressed have provided useful models to study the role of this ecto-nucleotidase in immune responses [20, 21]. In the Cd39-null mouse [20], B cell numbers [anti-CD45RA 14.8; anti-CD22.2 (Lyb-8.20; PharMingen, San Diego CA, USA)] in unstimulated blood and spleen have been noted to be normal (not shown). Somewhat surprisingly, elicited IgG xenoantibody responses to solubilized xenoantigens administered to Cd39-null mice were found to be markedly suppressed [14]. With Dr. M. Cascalho (Mayo Clinic, Rochester, MN, USA), we have investigated whether Cd39-null mice are capable of appropriate antibody responses. We have noted that Cd39-null mice exhibit impaired B cell memory responses to T-dependent antigens. Unexpectedly, however, these mutant mice show a significant increase in the frequency of somatic mutations post-immunization (not shown). These observations suggest that CD39 function may contribute to the affinity maturation of antibody responses and to facilitate post-germinal center terminal B cell differentiation (M. Cascalho et al., manuscript in preparation).\nAlthough CD39 has been shown to be the dominant ecto-nucleotidase expressed by NK and NK-T cells and to impact upon cytokine production ([19]; G. Beldi, unpublished), the relevance of this has not yet been fully elucidated.\nCD39 is also the major NTPDase expressed by monocyte-macrophages. Upregulation of tissue factor expression by these cells in vitro and alterations in splenic macrophage populations in vivo have been observed in Cd39-null mice. P2Y receptors that are impacted upon by CD39 are critically linked to monocyte and endothelial cell responsiveness [22, 23]. P2Y receptors on monocytes could be also subject to desensitization, comparable to what we have observed in platelet P2Y1 receptors in Cd39-null mice [20]. Therefore, we were interested to observe major defects in monocycle entry and migration into the substance of Matrigel plugs injected into the subcutaneous tissue of Cd39-null mice. In parallel, we also evaluated parameters of monocyte transendothelial migration influenced by ATP in vitro and noted failure of Cd39-null cells to migrate in response to exogenous nucleotides. This defect could be overcome by co-stimulation with serotonin, suggesting a degree of P2Y receptor desensitization in Cd39-null monocytes [14, 24].\nEcto-enzymes, specifically ecto-nucleotidases, are known to play an important role in leukocyte trafficking but this complex area will not be dealt with here (for an excellent review on this topic, see [25]). However, CD39 has been recognized as a critical control point in the regulation of leukocyte accumulation within hypoxic tissues and a sixfold increase in CD39\/CD73 tandem functioning in the setting of hypoxia has been demonstrated [26, 27].\nLangerhans cells (LC) are members of the DC family of antigen-presenting cells residing in the skin. NTPDase1 enzymatic function on antigen presenting DC is involved in the recruitment, activation and polarization of na\u00efve T cells. Mizumoto et al. established that LC from the Cd39-null mice do not hydrolyze ATP and ADP, unlike wild-type cells [28]. Cd39-null DC are also markedly unresponsive to ATP and are susceptible to cell death, but only after prolonged exposure to nucleotides [28].\nMutant mice null for Cd39 have amplified inflammatory responses to irritant chemicals. In these Cd39-null mice, there are major defects in dendritic cell formation, antigen presentation and T cell responses to haptens. These result in markedly attenuated responses to contact allergens in type IV hypersensitivity cutaneous responses that are also seen in inflammatory bowel disease models following haptenic stimulation (not shown).\nThese data suggest that Cd39 expression is required for optimal stimulation of hapten-reactive T cells in mice [28]. Somewhat paradoxically, CD39 also appears to function as an additional recognition structure on haptenated target immunocytes for HLA-A1-restricted, hapten-specific cytotoxic T cells [29]. Curiously, following deletion of Cd39, LC are fully functional with respect to homing and phenotypic maturation. However, the major defect is that these cells appear less able to stimulate T cells. These abnormalities are potentially also relevant to allograft rejection processes [14]. Sequelae of these putative immune abnormalities include the relative failure of Cd39-null mice to reject allografts under limited co-stimulation blockade [30]. These data indicate a previously unrecognized role of CD39 and the effects on nucleotide-mediated signalling in immunological responses [28].\nLocalization of CD39 within lipid rafts implies that this ecto-enzyme may be involved in cell-cell contacts and signalling [31]. Such membrane micro-domains serve as the \u2018message center\u2013in which numerous signalling molecules are concentrated and scaffolding developed [32]. The Cd39-null DC might exhibit defective functions because of putative defects in initiating and maintaining cell-cell contacts. This mechanism speculatively involves facilitating integrin associations by purinergic signals within the \u2018immunological synapse\u2013and would have parallels to the way chemokines have been implicated in this process [33\u201336]. CD39 on DC and\/or T cells may be likewise translocated to the immunological synapse during antigen presentation to facilitate intercellular signalling. More typically, CD39 is considered to play a cellular immunoregulatory role by hydrolyzing ATP (and perhaps ADP) released by T cells during antigen presentation and thereby generating adenosine, a known immunosuppressive molecule.\nRecent work has also indicated that regulatory CD4+ve CD25+ve T cells (Treg cells) play important roles in the suppression of immunological reactivity and maintenance of tolerance [37, 38]. Patterns of expression of CD39 by Treg and the possibility that the balance of extracellular nucleotides\/nucleosides influence(s) the function of these interesting cells have been a major focus of investigation in our laboratories of late.\nCD39 expression by immunosuppressive regulatory T cells\nTreg are central to the acquisition of immunological tolerance. Here the immune system does not mount cellular responses against specific antigens while reactivity towards other antigens is maintained. The precise mechanisms underlying the acquisition of tolerance are not fully understood. Transplanted graft outcomes in the absence of heavy immunosuppression depend on consistent balances between cytopathic effector cells and Treg [39]. Depletion of the cytopathic T cell clone has been proposed as one mechanism for the induction of tolerance. However, it is now clear that the long-term maintenance of tolerance is also dependent on self-perpetuating immunoregulatory mechanisms that limit or constrain alloresponses. The suppressive Treg populations are considered key to the development and maintenance of peripheral tolerance [33, 40].\nSignificant deficiencies are apparent in identifying Treg. Although traditionally defined by CD4+CD25+ expression, the latter marker along with other membrane proteins such as GITR and CTLA4 becomes non-specific and somewhat redundant following activation. Such markers are widely upregulated on other cells thereby losing specificity for the Treg population. The forkhead winged transcription factor FoxP3 is specific for Treg; however, its intracellular location limits its usefulness in the study of this population. Moreover, it is now apparent that CD25 does not encompass all Treg as defined by FoxP3+ expression [41].\nSimilarly, the mechanisms of Treg action are poorly defined. Putative mechanisms of suppression by Treg include cell-to-cell contact predominant in vitro and the release of soluble mediators that may predominate in vivo. As an example, IL-10 and transforming growth factor \u03b2 have been identified as soluble factors that may mediate Treg suppression. Our recent studies and data from other groups have indicated that adenosine is an important mediator generated by Treg cells, and appears responsible for, at least in part, their functions [42].\nPatterns of immune expression of CD39 by Treg were determined using standard techniques; using the Cd39-null mouse cells as negative controls. CD3+, CD8+, NK1.1+, B220+, CD11b+ and CD11c+ cells were positively selected from spleens and lymph nodes of 8- to 10-week-old C57BL6 mice through the use of MACS Sort magnetic beads in MACS LS Separation columns (Miltenyi Biotec, Bergisch Gladbach, Germany). T cells enriched for CD4 were obtained from lymph node and spleen preparations using CD4 T cell columns (R&D Systems, Minneapolis, MN, USA). CD4+\/CD25+ and CD4+\/CD39+ were positively selected using the relevant antibody and MACS Sort magnetic beads in MACS MS Separation columns (Miltenyi Biotech, Bergisch Gladbach, Germany). Where indicated, the same subsets were also sorted after staining the purified CD4+ cells with CD25 or CD39, using the MoFlo cell sorter (BD Biosciences, San Jose, CA, USA). In selected experiments, CD4+\/CD25+ cells were purified using the murine CD4+CD25+ T regulatory cell isolation kit from Miltenyi Biotech (Bergisch Gladbach, Germany). The purity of the different cell populations was verified by flow cytometry on FACSort (BD Biosciences, San Jose, CA, USA). Anti-mouse and anti-human CD4, CD8, CD19, B220, NK1.1 and CD25 were from eBiosciences (San Diego, CA, USA), as were anti-mouse CD5, CD62L, CD45RB, interferon (IFN)-\u03b3 and IL-4. Purified anti-mouse CD3 and CD28 were from PharMingen (BD Biosciences,San Diego CA, USA). Rabbit anti-mouse CD39 polyclonal antibody was used to stain cells purified from wild-type lymph nodes and from spleen, as described [43].\nUsing cells harvested from na\u00efve C57BL6 mice, CD39 was found to be expressed by the majority of monocytes and by subsets of lymphocytes, also inferred by gating on forward scatter (FSC) and side scatter (SSC; not shown). Among node-derived lymphocytes, the majority of CD39+ cells are found in CD19+ (or B220+) B cells. The remaining CD39+ cells were shown to reside almost totally within the CD4+ subset (Fig. 1a). There, they consistently range from 8 to 12% of all CD4+ cells in lymph node (and spleen). Further characterization of resting C57BL6 lymphocytes revealed that CD39 is selectively expressed on CD4+\/CD25+ T cells (Fig. 1a). CD39 is consistently and abundantly expressed in CD4+\/CD25high T cells, while the CD4+\/CD25dim populations show a dichotomic expression pattern of CD39, with ~50% of the cells positive. Less than 1% of CD39+ cells were found in the CD4+\/CD25\u2212 compartment. The majority of peripheral CD4+\/CD39+ cells are also CD45RBlow and mostly CD62Llow, with similar expression patterns observed in BALB\/c mice (not shown).\nFig. 1\na Expression of CD39 on mouse lymphoid cells. Lymph node cell suspensions were prepared from 8-week-old C57\/BL6 mice and were gated based on FSC and SSC parameters (not shown). CD4+ cells were gated based on CD25 expression. CD39 expression on the different subsets is shown in the histograms (open profiles) plotted against an irrelevant control (gray profiles). Differential expression of CD39 on CD4+ cells can be shown to be closely associated with CD25. b RT-PCR analysis of foxp3 mRNA expression. This was done in sorted cells with the following markers: Foxp3+\/CD39+, Foxp3+\/CD39-, Foxp3-\/CD39+ and Foxp3-\/CD39-. CD4+ cells obtained from the Foxp3-GFP \u2018knockin\u2013animals that had been generated by Mohamed Oukka and Vijay K. Kuchroo with colleagues (Ref). B lymphocytes were used as CD39+ control and included for comparison (striped bars). c RT-PCR analysis of CD73 mRNA expression. This was done in sorted cells with the following markers: Foxp3+\/CD39+, Foxp3+\/CD39-, Foxp3-\/CD39+ and Foxp3-\/CD39-. B lymphocytes were used as CD39+ control and included for comparison (striped bars). CD73 is also a useful immunophenotypic marker for Treg cells (not shown); and when combined with CD39 provides near-concordance with foxp3 expression. The Foxp3-\/CD39+ subset does not express CD73 and these cells resemble the memory phenotype (not shown)\nThese results were confirmed by reverse transcription polymerase chain reaction (RT-PCR) analysis of selected cell populations. Both CD11b+ and CD11c+ cells expressed high levels of CD39 mRNA, indicating that macrophages and dendritic cells constitutively express CD39. Within the T cell compartment, the highest expression levels of CD39 transcripts were found in CD4+ cells. Gene expression profiling of CD4+ T cells, sorted on the basis of CD39 cell surface expression, indicated that the CD4+\/CD39+, but not CD4+\/CD39\u2212, T cells robustly express markers of Treg cells [40], viz. Foxp3, CD25, GITR and CTLA-4. Further, CD4+\/CD39+ cells are anergic in the absence of IL-2 (not shown) and suppress T effector (CD4+\/CD25\u2212) proliferation with an efficacy similar to that observed with classic CD4+\/CD25+ Treg (S. Deaglio et al., submitted manuscript).\nWe have also confirmed the utility of CD39 in defining human Treg populations as comparable patterns of CD39 antigen expression in human cells are observed. The majority of human CD19+ B cells express CD39; however, within the T cell compartment CD39+ cells are present in the CD4+ subset (not shown). As in the mouse, CD39 is highly expressed in the CD4+\/CD25high T cells, with only ~50% of cells CD39+ within the CD4+\/CD25dim population. Negligible CD39 expression is found in the CD4+\/CD25\u2212 population (not shown). Gene expression profiling of human CD4+\/CD39+ T cells also confirms that these cells express Foxp3, GITR and CD25 in a pattern analogous to that of traditional CD4+\/CD25+ Treg (K. Dwyer et al., submitted). Our analysis demonstrates that CD39 expression in both murine and human T cells is restricted to a subpopulation of CD4+\/CD25+ cells that expresses markers associated with T regulatory function.\nWe have further examined the exact relationship between CD39 expression and the regulatory phenotype by using T cells from mutant mice with the green fluorescent protein (GFP) reporter gene introduced into the endogenous Foxp3 locus [designated as \u2018Foxp3+(GFP+) knockin\u2013cells; kindly provided for these experiments by M. Oukka and V. K. Kuchroo, Boston, MA, USA] [44]. Four populations could be defined by differential CD39 and Foxp3 expression: Foxp3+\/CD39+, Foxp3+\/CD39\u2212 (minor population grouping), Foxp3\u2212\/CD39+ and Foxp3\u2212\/CD39\u2212. These populations were sorted and gene expression profiles determined. The GFP+\/CD39+ fraction was shown to mirror the genetic profile of Treg, as defined by the presence of Foxp3 transcripts in this positive control (Fig. 1b). Interestingly, the next major subpopulation Foxp3\u2013\/CD39+ cells contain T lymphocytes that are not classic Treg and yet appear to be associated with the memory compartment (W. Gao, manuscript in preparation; Fig. 1b).\nFoxp3+(GFP+) Treg however were also found to co-express the ecto-nucleotidase CD73, a unique situation amongst T lymphocytes (Fig. 1c). Consistent with other phenotypic data (not shown), RT-PCR analysis confirms that Foxp3+\/CD39+ cells have high levels of gene expression of both CD39 and CD73 (Fig. 1b). CD73, which converts AMP to adenosine downstream of CD39, has been independently identified on CD25+ (FoxP3+) Treg and CD25\u2212 uncommitted primed precursor Th cells [42]. This recent work further supports our observations that the expression of ecto-nucleotidases and consequent adenosine generation play a role in the mediation of some of the suppressive capabilities of Treg cells. Hence, CD73 is co-expressed with CD39 as a cell surface marker of murine Tregs (Fig. 1c).\nAdenosine as a Treg effector molecule\nTregs from mutant mice deficient in CD39 have impaired regulatory function manifesting as a 50% decrease in the ability of Cd39-null Tregs to modulate effector T cell function in vitro and in vivo. These results indicate that CD39 expressed by Treg is the major and rate limiting ecto-nucleotidase responsible for the generation of adenosine and suggest that a putative CD39\/CD73-adenosinergic axis (i.e., generating adenosine) may contribute to the immunoregulatory function of Treg (S. Deaglio et al., manuscript submitted; also Fig. 2).\nFig. 2Schematic representation of Treg markers. The cellular phenotype of these suppressive T cells can be defined by FoxP3+\/CD39+\/CD73+ expression. Phosphohydrolysis of extracellular nucleotides by CD39 and CD73 generates adenosine, which exerts a component of the immunosuppressive effect\nAdenosine plays a central and direct role in the regulation of inflammatory responses and limiting inflammatory tissue destruction [9, 10, 45]. Potentially, close cell-cell contacts with pericellular generation of adenosine and regulated expression of adenosine receptors may be important modulatory factors directly suppressing T cell responses. Early in the immune response, adenosine favors recruitment of DC, which initiates specific immune responses [9, 10, 28]. The immunosuppressive effects of adenosine on T cells are thought to be mediated primarily through the A2A receptor. Adenosine inhibits the production of proinflammatory cytokines and superoxide anions. Adenosine activation of A2A receptor also induces heterologous desensitization of chemokine receptors, which are critical in leukocyte trafficking, through the activation of protein kinase A [46].\nIt is also feasible that the suppression mediated via Treg-generated adenosine could be exerted indirectly through downregulation of co-stimulatory molecules on DC or by competition for other non-characterized signal molecules [47]. Irrespective of the mechanism of action, we have shown that FoxP3+ Tregs are the only T cells that contain the full enzymatic machinery necessary to generate adenosine. This effect, in conjunction with the expression of the adenosine A2A receptor on effector (CD4+\/CD25\u2212) T cells, generates immunosuppressive loops limiting effector cell proliferation both in vitro and in vivo (S. Deaglio et al., submitted).\nImmune deviation and autoimmune diathesis in Cd39-null mice\nThe pathophysiological relevance of the CD39-adenosinergic loop was further tested by examining effects of exogenous, pharmacological adenosine A2A receptor agonists on T cells, committed to either Th1 or Th2 lineages [48]. Adenosine might contribute to the resolution of inflammation by facilitating Th2 pathways by the inhibition of Th1 cell functions [49]; this property has been addressed further in the Cd39-null mice. Subsets of na\u00efve CD4+ T cells were polarized to a Th1 or Th2 phenotype through the addition of IL-12 (10 ng\/ml) and anti-IL-4 (10 \u03bcg\/ml) or IL-4 (10 ng\/ml) and anti-IFN-\u03b3 (10 \u03bcg\/ml), respectively, following stimulation with plate-bound anti-CD3 (5 \u03bcg\/ml) and soluble anti-CD28 (2.5 \u03bcg\/ml). The polarization of cells was confirmed by intracellular cytokine staining and gene profiling via RT-PCR analysis.\nHeightened expression of the A2A receptor, as measured by RT-PCR, could be detected in CD4+\/CD25\u2212 cells polarized in vitro to Th1, but not Th2, phenotypes at day 3 (not shown). In keeping with this observation, the addition of the adenosine A2A receptor agonist, ATL146e (kind gift from J. Linden, Adenosine Therapeutics), at day 3 of culture, when all cells are already committed to either a Th1 or Th2 phenotype, selectively inhibits Th1 proliferation. This suggests once more that the A2A receptor is the critical immunomodulatory adenosine receptor [9]. No inhibition of the Th2 proliferation response by ATL146e [50] could be noted (Fig. 3a).\nFig. 3CD39 generation of adenosine preferentially regulates Th1 immune responses. a 3H-thymidine incorporation of CD4+ T cells purified from WT mice as polarized towards a Th1 or a Th2 phenotype for 5 days. The selective adenosine A2A agonist ATL146e was added after 3 days of culture. Data are expressed as % of inhibition and are the mean of duplicates; error bars represent the SEM of three independent experiments. b CD4+ T cells were purified from Cd39-null (filled histograms) or WT (open histograms) mice and polarized towards a Th1 phenotype. The mRNA was extracted at day 3 and assayed for IFN-g (left panel) by RT-PCR. Data are representative of more than four independent experiments. c CD4+ T cells were purified from Cd39-null (filled histograms) or WT (open histograms) mice and polarized towards a Th1 phenotype. The indicated adenosine receptor agonists and antagonists were added at the beginning of the experiment (left panel). d This panel shows the effects of apyrase on IFN-g production by Cd39-null CD4+ T cells polarized towards a Th1 phenotype. For these experiments, mRNA was extracted at day 3 and assayed for IFN-g (RT-PCR). Representative data are shown from three independent experiments for each. e Representative images of Cd39-null mouse on C57BL\/6\/129 SVJ (upper panel) or BALB\/c backgrounds (lower panel) manifesting alopecia. Histology of skin samples obtained from Cd39-null mice affected by alopecia. Uninvolved skin areas from the same animals as well as samples from WT mice were used for comparison. Biopsies were fixed and stained for CD4 cells. Original magnifications: \u00d710 for the upper middle panel, \u00d740 for the upper right and \u00d720 for the lower panels. f Skin samples were obtained from Cd39-null mice affected by alopecia, the tissue homogenized and mRNA extracted. Cytokine profiling was performed by RT-PCR, using a preamplification technique. Uninvolved skin from the same animal or a matched WT mouse used as controls. Representative data are from three animals\nConsistent with these results, in vitro polarization of CD4+ T cells towards a Th1 phenotype results in the increased production of IFN-\u03b3 by Cd39-null T cells, with a Th1-deviated phenotype (Fig. 3b). Addition of ATL146e [50], or soluble NTPDases (apyrase), strongly inhibits the Th1 response in both the wild-type and Cd39-null polarized cells confirming that A2A receptors are present and functional in both groups. These data suggest lack of substrate in the Cd39-null cells to be responsible for the observed Th1 bias (Fig. 3c). The A2A antagonist 8-(3-chloro-styryl) caffeine was shown to augment IFN-\u03b3 production in wild-type cells polarized to a Th1 phenotype (Fig. 3c), further supporting this hypothesis. In addition, reconstitution of Cd39-null cells with apyrase restores the catalytic potential of the cells, producing adenosine and inhibiting IFN-\u03b3 production in a dose-dependent manner, effects similar to that of ATL146e (Fig. 3d).\nAdditional evidence that the perturbation in adenosine generation causes Th1 deviation, is that Cd39-null mice spontaneously develop autoimmune alopecia. Fifteen per cent (9\/59) of designated Cd39-null animals develop skin lesions characterized by extensive and well-demarcated hair loss (Fig. 3e). No lesions are observed in age-matched WT controls (0\/28). Similar lesions were observed in Cd39-null mice on the BALB\/c background suggesting that the phenotype is strain independent (Fig. 3e). The alopecia typically appears at ~20\u20130 weeks of age in the facial region and thereafter extends out to include the trunk. The skin lesions are characterized by the presence of a population of CD4+ and CD8+ lymphocytes within the damaged hair follicles (Fig. 3e). Moreover, sixfold increases in IFN-\u03b3 transcript levels are noted in areas of skin with active disease, when compared to areas of uninvolved skin from the same animal or from wild type controls (Fig. 3f). In addition, there is increased expression of IL-2, Foxp3 and CTLA-4, consistent with the cellular infiltration. IL-4 and IL-10 were not detected (Fig. 3f). Autoimmune manifestations impacting other organ systems and on renal function are under evaluation (D. Friedman, not shown).\nThese data validate the importance of adenosine in directing T cell subset differentiation and support a role for CD39 in orchestrating Treg cell suppressive responses under both in vitro and in vivo conditions.\nSummation\nThis review summarizes components of extracellular nucleotide-mediated signalling pathway in T cells that are impacted upon largely by CD39, the prototypic member of the E-NTPDase family of ecto-nucleotidases. Modulated, distinct NTPDase expression appears to regulate nucleotide- and nucleoside-mediated signalling in the immune system. As the vasculature uses similar mediators to regulate blood fluidity and hemostasis, expression of CD39 on either endothelial or immune cells might allow for full integration of vascular inflammatory and immune cell reactions at sites of injury.\nThere is a wide field for future investigations of the role of nucleotides, nucleosides and ecto-nucleotidases in immune-mediated diseases. Increasing interest in this field may open up new avenues for investigation and the development of new treatment modalities for a large variety of illnesses, including atherosclerosis and the vascular or immune inflammation seen in transplant-related diseases.","keyphrases":["cd39","dendritic cells","t cells","ntpdase","vasculature","platelet","b cells","immunology","apyrase","ecto-atpase","e-ntpd","kidney","liver"],"prmu":["P","P","P","P","P","P","P","P","P","U","U","U","U"]}
{"id":"Purinergic_Signal-4-2-2377323","title":"Endogenous ATP release inhibits electrogenic Na+ absorption and stimulates Cl\u2212 secretion in MDCK cells\n","text":"Our previous studies with a line of Madin-Darby canine kidney (MDCK) cells (FL-MDCK) transfected with FLAG-labeled \u03b1, \u03b2, and \u03b3 subunits of epithelial Na+ channel (ENaC) showed that, although most of the short-circuit current (Isc) was amiloride sensitive (AS-Isc), there was also an amiloride-insensitive component (NS-Isc) due to Cl\u2212 secretion (Morris and Schafer, J Gen Physiol 120:71\u201385, 2002). In the present studies, we observed a progressive increase in NS-Isc and a corresponding decrease in AS-Isc during experiments. There was a significant negative correlation between AS-Isc and NS-Isc both in the presence and absence of treatment with cyclic adenosine monophosphate (cAMP). NS-Isc could be attributed to both cystic fibrosis transmembrane conductance regulator (CFTR) and a 4, 4'-diisothiocyano-2, 2'-disulfonic acid stilbene (DIDS)-sensitive Ca2+-activated Cl\u2212 channel (CaCC). Continuous perfusion of both sides of the Ussing chamber with fresh rather than recirculated bathing solutions, or addition of hexokinase (6 U\/ml), prevented the time-dependent changes and increased AS-Isc by 40\u201360%, with a proportional decrease in NS-Isc. Addition of 100 \u03bcM adenosine triphosphate (ATP) in the presence of luminal amiloride produced a transient four-fold increase in NS-Isc that was followed by a sustained increase of 50\u201360% above the basal level. ATP release from the monolayers, measured by bioluminescence, was found to occur across the apical but not the basolateral membrane, and the apical release was tripled by cAMP treatment. These data show that constitutive apical ATP release, which occurs under both basal and cAMP-stimulated conditions, underlies the time-dependent rise in Cl\u2212 secretion and the proportional fall in ENaC-mediated Na+ absorption in FL-MDCK cells. Thus, endogenous ATP release can introduce a significant confounding variable in experiments with this and similar epithelial cells, and it may underlie at least some of the observed interaction between Cl\u2212 secretion and Na+ absorption.\nIntroduction\nMadin-Darby canine kidney (MDCK, type I) cells grown as epithelial monolayers have been used extensively as a model of the mammalian collecting duct (CD). These monolayers exhibit many characteristics of the CD, including a high transepithelial resistance and amiloride-sensitive Na+ reabsorption that is mediated by the epithelial Na+ channel (ENaC) and stimulated by agonists that increase intracellular cyclic adenosine monophosphate (cAMP) [1, 2]. Similar cell lines that are derived from the CD, including mouse cortical CD (M-1) and inner medullary CD (mIMCD-K2) cells [3\u20135] and A6 cells from the Xenopus distal nephron [6, 7] share these characteristics. However, in contrast to the CD, all of these cell lines also have Cl\u2212 channels in their apical membranes, and, when studied under short-circuit conditions, exhibit Cl\u2212 secretion, which is observed as an amiloride-insensitive component of the short-circuit current (NS-Isc) that is also stimulated by cAMP [2, 3, 5\u20138].\nWe previously developed a line of MDCK cells (FL-MDCK) that had been retrovirally transfected with rat ENaC subunits containing the FLAG epitope in their extracellular loops [2]. Monolayers of these cells had higher rates of ENaC-mediated Na+ absorption, measured as the amiloride-sensitive short-circuit current (AS-Isc), than did the original MDCK cell line. Our previous studies with FL-MDCK monolayers in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) showed that cAMP treatment produced a rapid transient peak in the total short-circuit current (Isc) within 5\u00a0min, followed by a broad peak that decayed over 20\u00a0min [2]. This biphasic response to cAMP treatment has also been reported in A6 and M-1 cultures and has been attributed to rapid stimulation of Cl\u2212 secretion mediated by cystic fibrosis transmembrane conductance regulator (CFTR) followed by a slower activation of ENaC [6\u20139]. In FL-MDCK cells, the biphasic response to cAMP treatment was followed by a decay of Isc, which reflected a decrease in AS-Isc [2]. Morris and Schafer [2] found that this late fall in AS-Isc in FL-MDCK monolayer was prevented by the omission of Cl\u2212 from the bathing solution and attributed the effect to Cl\u2212 secretion via CFTR, which has subsequently been demonstrated to be present in this cell line [10].\nA large body of evidence indicates that CFTR is a cAMP-regulated Cl\u2212 channel as well as a conductance regulator, which is colocalized with ENaC in airway, colonic, and other epithelial tissues [11, 12]. It has been proposed that when cAMP activates CFTR, ENaC is inhibited, and this inhibitory effect of CFTR has been hypothesized to explain the pathophysiology of cystic fibrosis in airway epithelia [13, 14]. In Xenopus oocytes expressing ENaC, coexpression of CFTR reduces the ENaC-mediated Na+ current [15], an effect that might be attributed to a direct effect of CFTR on ENaC activity. However, Kunzelman and his collaborators [16, 17] have shown that the inhibitory effect of CFTR can be mimicked by coexpression of other anion channels or treatment with amphotericin B and is explained by an increase in intracellular Cl\u2212 ([Cl\u2212]i). We have shown that an increase in [Cl\u2212]i also inhibits ENaC in FL-MDCK cells; however, this effect cannot explain the effect of Cl\u2212 secretion on Na+ absorption with cAMP treatment, because stimulation of Cl\u2212 secretion by cAMP results in a fall rather than a rise in [Cl\u2212]i [10]. Moreover, in the Xenopus oocyte expression system, CFTR activation inhibits ENaC at a continuous holding potential at which CFTR mediates inward currents corresponding to Cl\u2212 efflux [18]. Under these conditions, activation of CFTR will not result in an increase in [Cl\u2212]i. Therefore, a change in [Cl\u2212]i is unlikely to fully explain the observed reciprocal regulation of ENaC and CFTR.\nIn the present studies, we show that adenosine triphosphate (ATP) accumulation in the apical solution produces a progressive stimulation of NS-Isc and a corresponding decrease in AS-Isc when FL-MDCK cells are studied in Ussing chambers. The possibility of constitutive ATP release raises an important issue when interpreting changes in ion transport in experiments such as these. More important in the present context, ATP release can explain at least part of the inverse relationship between Cl\u2212 secretion and Na+ reabsorption, and, as suggested by Wilson et al. [19], it may be involved in the conversion of CD cells from absorptive to secretory in polycystic kidney disease.\nMaterials and methods\nCell culture\nThe FL-MDCK cells used were a clone of the type-1 MDCK line that had been retrovirally transfected with flagged rat \u03b1-, \u03b2-, and \u03b3ENaC by Morris and Schafer [2]. The cells used were from passages 5\u201335 and were cultured in T-75 flasks at 37\u00b0C in DMEM (Life Technologies) supplemented with 10% fetal bovine serum (FBS), 50\u00a0mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (pH 7.4), 1% Pen\/strep-fungizone, and the selection antibiotics G418 (800\u00a0\u03bcg\/ml), hygromycin (300\u00a0\u03bcg\/ml), and puromycin (5\u00a0\u03bcg\/ml). For transepithelial transport studies, cells were seeded onto 24-mm Transwell inserts (Costar; Catalog No. 3412) at a density of \u223c105 cells\/cm2. For bioluminescence assays, cells were seeded on 12-mm collagen-coated Millicell inserts (Millipore; Catalog No. PHIP 012 50) at the same density. After seeding, cells were fed daily with DMEM containing FBS and HEPES but without selection antibiotics for 5\u20137\u00a0days. Before use in the experiments, the cell monolayers were induced with 1\u00a0\u03bcM dexamethasone plus 2\u00a0mM Na+ butyrate in the culture medium overnight.\nUssing chamber experiments\nFL-MDCK monolayers were carefully cut from the plastic insets and were mounted in Ussing-type chambers. In most experiments, both sides of the monolayers were bathed with Krebs-Ringer bicarbonate (KRB) solution, which contained (in mM): 113\u00a0NaCl, 1.2\u00a0Na2HPO4, 25\u00a0NaHCO3, 1.1\u00a0CaCl2, 1.2\u00a0MgCl2, 4.5\u00a0KCl, and 10 glucose. The KRB solutions were continuously gassed with a mixture of 95% O2-5% CO2 to give a pH of 7.40 at 37\u00b0C. As shown by the schematic diagram in Fig.\u00a01a, transepithelial short-circuit currents (Isc,\u00a0\u03bcA\/cm2) and voltages (Vte) were measured and conductances were evaluated every 30\u00a0s by measuring the current deflections induced by 4-s symmetrical voltage pulses of \u00b12\u00a0mV, as described previously [2, 10]. In most experiments, KRB was continuously recirculated on both sides of the epithelium from 10-ml reservoirs (Fig.\u00a01b); however, in the experiments involving \u201cfresh perfusion,\u201d as shown in Fig.\u00a01c, both sides of the monolayers were continuously perfused with fresh rather than recirculated KRB. In those experiments involving cAMP treatment, 100\u00a0\u03bcM 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) plus 100\u00a0\u03bcM isobutylmethylxanthine (IBMX) were added to both the apical and basolateral solutions. AS-Isc was defined as the change in Isc produced by the addition of 10\u00a0\u03bcM amiloride to the apical solution, and the remaining Isc was defined as NS-Isc. Because we observed time-dependent changes in AS-Isc and NS-Isc in the voltage clamp experiments, we conducted additional experiments in which the monolayers were left under open-circuit conditions during the course of the experiment, and the open-circuit transepithelial voltage (Vte, mV) was measured continuously except for intermittent voltage clamping to 0\u00a0mV and \u00b12\u00a0mV for a total of 4\u00a0s every 30\u00a0s to obtain Isc and conductance measurements.\nFig.\u00a01a Schematic diagram of the electrophysiological circuitry. The Ussing chamber is constructed of two lucite half-chambers, which are clamped together with the Madin-Darby canine kidney (MDCK) cell monolayer (on a Transwell membrane) between them. Voltage-sensing electrodes are positioned close to the center and on either side of the monolayer. Current passing electrodes, made from silver disks with a central hole to accommodate the voltage electrodes, are placed at the back of each half chamber and parallel to the monolayer. The transepithelial voltage (Vte) is measured by the voltage clamp\/analog to digital converter (called \u201cthe clamp\u201d). When no current is being passed, the voltage recorded is called the \u201copen circuit\u201d Vte. To short-circuit the epithelial monolayer, the clamp passes a current (I) that is sufficient to drive Vte to zero. The current measured in this situation is called the short-circuit current (Isc). b In experiments in which the Krebs-Ringer bicarbonate solutions (KRB) in the Ussing chambers were recirculated, 10-ml syringes were used as reservoirs, and a roller pump was used to continuously move the KRB from each half-chamber to its respective reservoir. c In experiments in which fresh KRB solutions were continuously perfused, large beakers served as the solution reservoirs from which KRB was constantly infused into each half-chamber by a roller pump and constantly removed by suction to a waste reservoir. In both the recirculation and fresh perfusion methods, the solutions in the reservoirs (10-ml syringes or beakers, respectively) were constantly bubbled with a 95% O2\u20135% CO2 gas mixture\nBioluminescence detection of ATP release\nATP released from the MDCK monolayers was measured as described by Taylor et al. [20]. Briefly, culture medium was removed from the inserts, and the cell monolayers were washed three times with phosphate-buffered saline (PBS) solution to remove any FBS present in the culture medium. Opti-minimal essential medium (MEM) with 2\u00a0mg\/ml luciferin-luciferase reagent (Sigma) was added to the apical or basolateral side of the monolayer. Each insert with cells contained 200\u00a0\u03bcl of solution, and the average light signal was measured for 10-s, nonintegrated photon collection periods with a TD-20\/20 luminometer (Turner Designs; Promega). Treatment with cAMP was applied by adding 1\u00a0\u03bcl of 20\u00a0mM CPT-cAMP and 20\u00a0mM IBMX stock solution to the 200\u00a0\u03bcl apical solution of the inserts (final concentration 100\u00a0\u03bcM for both), and control experiments were done with addition of the same volume of vehicle alone. The light output was measured for at least 2\u00a0min, and the average was taken for the calculation of ATP release. Individual batches of luciferin-luciferase reagent were tested for relative sensitivity by measuring the light output response to serial dilutions of an ATP stock solution with 2\u00a0mg\/ml luciferin-luciferase reagent in Opti-MEM.\nData analysis and statistics\nStatView for Macintosh (SAS Institute Inc.) was used for statistical analyses. Statistical significance (P\u2009<\u20090.05) was determined by analysis of variance (ANOVA) with Bonferroni\/Dunn post hoc testing for multiple comparisons and by paired or nonpaired t-tests, as appropriate, for single comparisons. Time dependence of transport was evaluated by linear regression analysis; the R value and significance of the slope are given. Correlation analysis was used to compare AS-Isc and NS-Isc and calculate a correlation coefficient, r.\nMaterials\nAll chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise noted.\nResults\nTime-dependent changes in Isc\nThe Isc in FL-MDCK cells was quite high and variable in the first 5\u00a0min after recording was begun using continuous short-circuiting. In six experiments, 10\u00a0\u03bcM amiloride added to the apical solution in the initial 5\u00a0min decreased Isc by 91\u2009\u00b1\u20092%, indicating that most of the current was due to ENaC-mediated Na+ absorption. The initial high Isc subsequently declined to a nadir at \u223c15\u00a0min (Fig.\u00a02a and b). Addition of amiloride at this nadir (Fig.\u00a02a) almost completely inhibited Isc; however, when the experiment was continued beyond the nadir, amiloride was less effective in reducing Isc (Fig.\u00a02b). In 35 experiments such as those in Fig.\u00a02a and b, the time interval between the nadir and amiloride addition to the apical solution was varied from 0 to 45\u00a0min. In these experiments, the average Isc at the nadir was 15.2\u2009\u00b1\u20090.5\u00a0\u03bcA\/cm2. Regression analysis showed that NS-Isc increased significantly (R\u2009=\u20090.76, P\u2009<\u2009.001) and AS-Isc decreased significantly (R\u2009=\u20090.53, P\u2009<\u2009.002) as a function of the time between the nadir and the addition of amiloride. The same high initial Isc and the time-dependent increase of NS-Isc were also observed in experiments using the intermittent short-circuit technique (see \u201cMaterials and methods\u201d), which indicated that neither effect is produced by prolonged short-circuiting of the epithelium (data not shown).\nFig.\u00a02Time course of the short-circuit current (Isc) and response to amiloride. Isc was measured across monolayers of FL-Madin-Darby canine kidney (FL-MDCK) cells induced overnight with 2\u00a0mM Na butyrate and 1\u00a0\u03bcM dexamethasone. Amiloride (10\u00a0\u03bcM) was added to the apical solution either early (A) or late (B) at the point indicated by the arrow. The transepithelial conductance (Gte) is proportional to the height of the deflections in Isc produced by transiently clamping the voltage to \u00b12\u00a0mV, as described in \u201cMaterials and methods\u201d\nAs shown in Fig.\u00a03, the effect of cAMP on Isc also depended on whether the treatment occurred early (at the nadir of Isc, Fig.\u00a03a) or late (\u223c40\u00a0min after the nadir, Fig.\u00a03b). With both early and late cAMP addition, Isc increased immediately, reaching a transient peak within 5\u00a0min; however, the magnitude of this initial peak was exaggerated with late cAMP treatment (Fig.\u00a03b). After the initial response, Isc showed a secondary broader increase that decayed more rapidly after late (Fig.\u00a03b) than after early (Fig.\u00a03a) cAMP treatment. In 29 experiments such as those in Fig.\u00a03, the time interval between the nadir in Isc and cAMP treatment was varied from 2 to 58\u00a0min, and 10\u00a0\u03bcM amiloride was added \u223c30\u00a0min after cAMP treatment. Regression analysis showed a significant increase in NS-Isc with the time between the nadir and cAMP treatment (R\u2009=\u20090.60, P\u2009<\u2009.002).\nFig.\u00a03Time course of Isc response to cyclic adenosine monophosphate (cAMP) treatment. A mixture of 100\u00a0\u03bcM 8-(4-chlorophenylthio) (CPT)-cAMP plus 100\u00a0\u03bcM isobutylmethylxanthine (IBMX) was added as indicated by the lower bar, either early (a) or late (b) to both sides of the monolayers, followed by 10\u00a0\u03bcM amiloride addition to the apical solution only (upper bar)\nFurther examination of the data from these experiments (see Fig.\u00a04) revealed a significant negative correlation between AS-Isc and NS-Isc, both with and without cAMP treatment. In other words, the time-dependent increase in NS-Isc observed in both sets of experiments was correlated with a corresponding decrease in AS-Isc. As expected, the average AS-Isc was significantly greater in those experiments with cAMP treatment (20.2\u2009\u00b1\u20091.1\u00a0\u03bcA\/cm2, n\u2009=\u200929) than in those without (12.8\u2009\u00b1\u20090.6, n\u2009=\u200935), but the correlation with NS-Isc was not significantly different between the two groups.\nFig.\u00a04Negative correlation between the amiloride-sensitive component of the short-circuit current ( Isc) (AS-Isc) and the nonamiloride-sensitive component (NS-Isc) in the absence and presence of cyclic adenosine monophosphate (cAMP). The 35 experiments in the first group (open circles, solid line) were conducted using the same protocol as the representative experiments in Fig.\u00a01 with no cAMP addition. The 29 experiments in the second group (solid dots, dashed line) followed the protocol of the representative experiments in Fig.\u00a02, with the addition of 100\u00a0\u03bcM 8-(4-chlorophenylthio) (CPT)-cAMP plus 100\u00a0\u03bcM isobutylmethylxanthine (IBMX) \u223c30\u00a0min before the addition of amiloride. In both groups, there was a significant correlation between AS-Isc and NS-Isc: \u2212cAMP group, r\u2009=\u20090.79, P\u2009<\u2009.001; +cAMP group, r\u2009=\u20090.65, P\u2009<\u2009.001\nEffect of cAMP and intracellular Ca2+ on NS-Isc\nIn the experiments shown in Fig.\u00a05, NS-Isc was measured throughout the experiments in the continuous presence of 10\u00a0\u03bcM amiloride in the apical solution. Treatment with cAMP produced an initial rapid peak in NS-Isc, followed by a sustained plateau. We then tested the effects of two inhibitors of Cl\u2212 transport pathways: glibenclamide, which is a well-established inhibitor of CFTR; and 4, 4'-diisothiocyano-2, 2'-disulfonic acid stilbene (DIDS), which is a less selective inhibitor of Cl\u2212 channels including the Ca2+-activated chloride channel (CaCC) and the chloride channel (CLC) family of Cl\u2212 channels but not CFTR. (See the Discussion for more detail about these inhibitors and references to their specificity.) Addition of 200\u00a0\u03bcM glibenclamide followed by 300\u00a0\u03bcM DIDS to the apical solution \u223c20\u00a0min after cAMP treatment significantly decreased NS-Isc to or below the magnitude prior to cAMP treatment (Fig.\u00a05a & b). In other experiments, addition of apical DIDS before cAMP treatment reduced the late plateau but not the initial peak of NS-Isc (Fig.\u00a05c and e), whereas apical glibenclamide significantly decreased the initial peak, with no effect on the late plateau (Fig.\u00a05d & e). These data suggest that at least two types of Cl\u2212 channels are stimulated by cAMP. One is sensitive to glibenclamide and responds to cAMP quickly while the other is sensitive to DIDS and responds to cAMP slowly.\nFig.\u00a05Effects of glibenclamide and 4, 4'-diisothiocyano-2, 2'-disulfonic acid stilbene (DIDS) on the nonamiloride-sensitive component (NS-Isc). In these experiments, 10\u00a0\u03bcM amiloride was continuously present in the apical solution. A Effect of cyclic adenosine monophosphate (cAMP) treatment [100\u00a0\u03bcM cAMP plus 100\u00a0\u03bcM isobutylmethylxanthine (IBMX), lowest bar] on NS-Isc. The middle and upper bars indicate the time of addition of 300\u00a0\u03bcM DIDS and 200\u00a0\u03bcM glibenclamide (Gliben.) to the apical solution. B Summary of 14 experiments such as that in A. The mean NS-Isc values immediately before cAMP treatment (point 1 in A), 3\u20135\u00a0min (\u201cinitial\u201d, point 2) and 15\u201320\u00a0min (\u201clate\u201d, point 3, just before DIDS or glibenclamide addition) after cAMP treatment, and after DIDS plus glibenclamide (point 4). * Significantly different from value before cAMP treatment, P\u2009<\u2009.001; \u2020 significantly different from the initial and late NS-Isc, P\u2009<\u2009.001. C Effect of 300\u00a0\u03bcM apical DIDS added before cAMP treatment. D Effect of 200\u00a0\u03bcM apical glibenclamide added before cAMP treatment. E Mean values of the change in NS-Isc (the cAMP-stimulated NS-Isc) produced by cAMP treatment in the initial (point 2 in A, C, and D) or late (point 3) response period in three sets of experiments: with no inhibitor (control, from summary of 15 experiments in B), DIDS before cAMP (as in C, n\u2009=\u20096) ,and glibenclamide before cAMP (as in D, n\u2009=\u20095). * Significant difference compared with the corresponding control value (calculated from the data in B), and between initial and late response to cAMP, P\u2009<\u2009.001\nThe Cl\u2212 channel that was quickly activated by cAMP and inhibited by glibenclamide has been attributed to CFTR, the expression and function of which has been identified by us in this same cell line [10]. Because cAMP increases intracellular Ca2+ ([Ca2+]i) in MDCK cells [21], we examined the hypothesis that CaCC contributes to the fraction of NS-Isc that is inhibited by DIDS by using thapsigargin to produce a modest but sustained elevation of [Ca2+]I [22, 23]. (Thapsigargin is known to elevate intracellular Ca2+ by releasing it from the endoplasmic reticulum [22].) In the experiments shown in Fig.\u00a06, NS-Isc was measured in the continuous presence of luminal amiloride. The addition of 1\u00a0\u03bcM thapsigargin to both sides of the monolayer produced a transient spike followed by a sustained increase in NS-Isc, most of which was sensitive to DIDS (Fig.\u00a06a). When 300\u00a0\u03bcM DIDS was added to apical solution before thapsigargin, it almost completely blocked the response of NS-Isc to thapsigargin (Fig.\u00a06b).\nFig.\u00a06Effect of thapsigargin on the nonamiloride-sensitive component (NS-Isc) and prevention of that effect by 4, 4'-diisothiocyano-2, 2'-disulfonic acid stilbene (DIDS). All experiments were performed in the presence of 10\u00a0\u03bcM luminal amiloride. A Mean NS-Isc values for seven experiments at four time points: just before adding thapsigargin to the apical and basolateral solutions, 3\u20135\u00a0min after addition of 1\u00a0\u03bcM thapsigargin to the apical and basolateral solutions (TG-initial), 15\u201320\u00a0min after thapsigargin addition (TG-late), and 3\u20135\u00a0min after the addition of 300\u00a0\u03bcM DIDS to the apical solution. * Significantly different compared with NS-Isc before thapsigargin treatment, P\u2009<\u2009.001; \u2020 significantly different compared with both the initial and late NS-Isc after thapsigargin, P\u2009<\u2009.001. B Mean values of the change in NS-Isc produced by thapsigargin: the initial peak (gray bars) and the late response (open bars) for control experiments (as in A) and in three experiments in which 300\u00a0\u03bcM DIDS was added before thapsigargin. * Significantly different compared with the corresponding control values, P\u2009<\u2009.001\nEffect of Ussing chamber perfusion on time-dependent changes in Isc\nIn all of the experiments presented up to this point, the KRB solutions that bathed the MDCK monolayers were recirculated as shown in Fig.\u00a01b. To examine whether the time-dependent changes in AS-Isc and NS-Isc might be caused by the accumulation of a secretagogue or metabolite in these recirculated solutions, we conducted paired experiments in which one Ussing chamber was perfused with recirculated KRB solutions and the other chamber was continuously perfused with fresh KRB solutions, as shown in Fig.\u00a01c. The representative control experiment shown in Fig.\u00a07a was conducted in the usual way: the apical and basolateral KRB solutions were recirculated through both sides of the Ussing chamber from 10-ml reservoirs. In the paired experiment in Fig.\u00a07b, we continuously perfused fresh KRB on both sides of the Ussing chamber. In both the recirculation and fresh perfusion experiments, cAMP treatment produced an initial transient spike of Isc lasting less than 6\u00a0min, followed by a broader peak; however, Isc decayed more rapidly in the recirculation experiment than it did in the fresh perfusion experiment. In four sets of paired experiments summarized in Fig.\u00a07c, AS-Isc was greater (39.8\u2009\u00b1\u20091.1\u00a0\u03bcA\/cm2 vs. 25.2\u2009\u00b1\u20093.3\u00a0\u03bcA\/cm2, P\u2009<\u20090.01) and NS-Isc was less (2.8\u2009\u00b1\u20090.2\u00a0\u03bcA\/cm2 vs. 7.7\u2009\u00b1\u20091.6\u00a0\u03bcA\/cm2, P\u2009<\u20090.01) with fresh perfusion than in the recirculation group, suggesting that some substance, which inhibited AS-Isc and stimulated NS-Isc, was accumulating in the recirculated bathing solution.\nFig.\u00a07Time course of the short-circuit current (Isc) in experiments with and without recirculation of the bathing solutions. The sequence of treatments was the same in these paired experiments: 100\u00a0\u03bcM cyclic adenosine monophosphate (cAMP) plus 100\u00a0\u03bcM isobutylmethylxanthine (IBMX) were added to both apical and basolateral solutions, followed by 10\u00a0\u03bcM amiloride to the apical solution. A Apical and basolateral Krebs-Ringer bicarbonate (KRB) solutions were recirculated through the two sides of the Ussing chamber from 10-ml reservoirs. 4, 4'-diisothiocyano-2, 2'-disulfonic acid stilbene (DIDS) (300\u00a0\u03bcM) was added to the apical solution near the end of the experiment. B In this paired experiment, fresh KRB solution was perfused through both sides of the Ussing chamber at the same rate (1\u00a0ml\/min) as for the recirculated solutions in A. C Summary results for four sets of paired experiments such as those in A and B. *Significantly different from control (with recirculation as in A), P\u2009<\u2009.001\nEffect of extracellular ATP on time-dependent changes in Isc\nWe hypothesized that ATP was released by the FL-MDCK cells and accumulated in the recirculated solutions. Extracellular nucleotides are known to act as autocrine and paracrine agents that affect Na+ absorption and Cl\u2212 secretion, and ATP has been shown to increase intracellular [Ca2+] [24, 25], which might augment the Ca2+-activated component of NS-Isc as well as inhibit ENaC activity. As shown in Fig.\u00a08a, in the presence of amiloride, the addition of 100\u00a0\u03bcM ATP to the apical solution increased NS-Isc, and, as shown in Fig.\u00a08b, this effect was almost completely abolished when the ATP was added together with hexokinase as an ATP scavenger [26]. (These experiments were conducted using the recirculated KRB method.) In five experiments such as that in Fig.\u00a08a, basal NS-Isc (just before ATP addition) averaged 4.1\u2009\u00b1\u20090.1\u00a0\u03bcA\/cm2. With the addition of ATP, NS-Isc reached a peak value of 15.4\u2009\u00b1\u20092.2\u00a0\u03bcA\/cm2 (P\u2009<\u20090.004) within 2\u20133\u00a0min, and then declined gradually to a plateau of 5.9\u2009\u00b1\u20090.3\u00a0\u03bcA\/cm2, which was still significantly larger than the basal level (P\u2009<\u20090.01). Addition of 300\u00a0\u03bcM DIDS to the apical solution before the addition of ATP had no effect on the peak amplitude (14.6\u2009\u00b1\u20090.7\u00a0\u03bcA\/cm2, n\u2009=\u20093), but decreased the plateau value to 4.5\u2009\u00b1\u20090.2\u00a0\u03bcA\/cm2 (n\u2009=\u20093), which was not significantly different from the basal value.\nFig.\u00a08Effect of adenosine triphosphate (ATP) on the nonamiloride-sensitive component (NS-Isc) and prevention of that effect by hexokinase. All experiments were performed in the presence of 10\u00a0\u03bcM apical amiloride. A Effect of 100\u00a0\u03bcM ATP added to the apical solution on NS-Isc. Mean values of five such experiments are given in the text. B Effect of 100\u00a0\u03bcM ATP on NS-Isc. In these experiments, 6\u00a0U\/ml hexokinase was continuously present in the bathing solution. The same results were obtained in three such experiments\nTo test whether endogenous ATP production by the epithelium might be involved in the time-dependent increase of NS-Isc and decrease of AS-Isc, we examined the effect of hydrolyzing extracellular ATP with hexokinase on the time course of NS-Isc and AS-Isc. Paired experiments without or with hexokinase were conducted as shown, respectively, in Fig.\u00a09a and b, both using the recirculated KRB method.. With hexokinase in the bathing solutions, the response of Isc to cAMP treatment was exaggerated, and the broader peak was more sustained (Fig.\u00a09b). As shown by the summary of four such sets of paired experiments (Fig.\u00a09c), hexokinase had no effect on the basal Isc measured just before cAMP addition; however, after cAMP, AS-Isc was significantly higher (40.4\u2009\u00b1\u20092.9\u00a0\u03bcA\/cm2 vs. 29.7\u2009\u00b1\u20092.8\u00a0\u03bcA\/cm2, P\u2009<\u20090.05) with hexokinase, whereas the DIDS-sensitive NS-Isc was not significantly different from zero and significantly less than in the control group (\u22120.3\u2009\u00b1\u20090.2\u00a0\u03bcA\/cm2 vs. 3.6\u2009\u00b1\u20090.7\u00a0\u03bcA\/cm2, P\u2009<\u20090.01). Thus, all of the above data were consistent with the hypothesis that the accumulation of extracellular ATP causes the time-dependent increase of NS-Isc and decrease of AS-Isc that were observed in those experiments in which the bathing solutions were recirculated.\nFig.\u00a09Time course of the short-circuit current (Isc) response to cyclic adenosine monophosphate (cAMP) treatment in the absence and presence of hexokinase. Both types of experiments followed the same general protocol: A mixture of 100\u00a0\u03bcM (4-chlorophenylthio) (CPT)-cAMP plus 100\u00a0\u03bcM isobutylmethylxanthine (IBMX) was added as indicated, followed by 10\u00a0\u03bcM apical amiloride and 300\u00a0\u03bcM apical 4, 4'-diisothiocyano-2, 2'-disulfonic acid stilbene (DIDS). A In this experiment, the Krebs-Ringer bicarbonate (KRB) solution had no hexokinase added. B A paired experiment in which 6\u00a0U\/ml hexokinase was continuously present in apical and basolateral bathing solution. C Average data for four paired experiments: the mean values of Isc immediately before cAMP was added (Basal-Isc), amiloride-sensitive (AS-Isc) at 30\u00a0min after cAMP addition (AS-Isc), and DIDS-sensitive Isc (DS-Isc). * Significantly different compared with the corresponding control values\nMeasurement of ATP released from MDCK cells\nATP released from polarized MDCK cells was determined by a bioluminescence assay, in which a luminometer was used to measure light production from ATP consumption in the luciferin-luciferase reaction [20]. MDCK cells were grown to confluence in 12-mm permeable supports (Millicell), and the ATP was measured in the apical or basolateral solution by adding the reagents only to that chamber. As shown in Fig.\u00a010, under basal conditions, apically directed ATP release produced a light emission of 16.4\u2009\u00b1\u20092.1 ALU (artificial light units), whereas basolaterally directed ATP release was nearly zero (0.7\u2009\u00b1\u20090.2 ALU). In ten paired experiments, addition of 100\u00a0\u03bcM cAMP and 100\u00a0\u03bcM IBMX to the apical solution significantly increased the apically directed ATP release from 16.0\u2009\u00b1\u20092.1 ALU (basal) to 51.1\u2009\u00b1\u20099.4 ALU (n\u2009=\u200910, P\u2009<\u20090.005), whereas addition of the same volume of solution without cAMP and IBMX (sham control for the possible effects of manipulating the monolayers) had no significant effect (17.4\u2009\u00b1\u20093.5 ALU basal vs. 15.1\u2009\u00b1\u20092.3 ALU sham; n\u2009=\u20094, P\u2009=\u20090.29). Treatment with cAMP had no significant effect on basolaterally directed ATP release (0.8\u2009\u00b1\u20090.3, n\u2009=\u20098, P\u2009>\u20090.5).\nFig.\u00a010Adenosine triphosphate (ATP) release across the apical and basolateral membranes of FL-Madin-Darby canine kidney (MDCK) monolayers. ATP release into the apical and basolateral solutions was measured for FL-MDCK monolayers in 12-mm culture inserts using the luciferin-luciferase assay. To measure apical ATP release, these reagents were present only in the apical solution (n\u2009=\u200914), and they were present only in the basolateral solution to measure basolateral release (n\u2009=\u200911). The monolayers were first incubated with 200\u00a0\u03bcl of standard Opti-minimal essential medium (MEM) solution on each side to measure basal ATP production. The monolayers were then removed from the instrument and either 1\u00a0\u03bcl of a stock solution of 8-(4-chlorophenylthio) (CPT)-cAMP + isobutylmethylxanthine (IBMX) stock solution (cAMP, n\u2009=\u200910; final concentrations: 100\u00a0\u03bcM cAMP and 100\u00a0\u03bcM IBMX) or 1\u00a0\u03bcl of plain Opti-MEM medium (Sham, n\u2009=\u20094) were added. Average values of the light emission in arbitrary light units (ALU)\u2009\u00b1\u2009standard error of the mean (SEM) are given. * Significantly different from basal and sham, P\u2009<\u20090.005\nDiscussion\nPrevious work [10] has shown that the triply transfected FL-MDCK cells used in these experiments (a subclone of \u03b1F\u03b2 F\u03b3F MDCK cells used in the experiments of Morris et al. [2]) expressed all three rat-ENaC subunits and CFTR, and that it was a very useful model epithelium for the study of ENaC regulation. In the current study, we examined in greater detail the relationship between Cl\u2212 secretion and Na+ absorption in this epithelium.\nTime-dependent changes in NS-Isc and AS-Isc\nFL-MDCK cells uniformly exhibited a high initial Isc, which was almost completely eliminated by 10\u00a0\u03bcM amiloride added to the apical solution. The initial Isc fell to a nadir within 15\u00a0min and then increased slowly (Figs.\u00a02b and 3b) due to an increase in NS-Isc. This progressive rise of NS-Isc occurred consistently regardless of whether amiloride was present or not and whether the epithelium was continuously short-circuited or was left in the open-circuit condition with only intermittent short-circuiting to measure Isc. Moreover, the time-dependent increase of NS-Isc could not be inhibited by the further addition of amiloride but was partially blocked by apical DIDS, which is consistent with anion secretion (see below).\nThe time course of the Isc response to cAMP using these FL-MDCK cells was like that observed in the previous studies of Morris and Schafer [2] and in other studies of similar epithelia [6, 9, 27]. There was an initial transient increase lasting less than 6\u00a0min, followed by a broader peak, which decayed over the next 30\u00a0min (Fig.\u00a03). Morris and Schafer [2] have shown that when these experiments are conducted in a Cl\u2212-free solution, the initial transient spike was absent and Isc was stable for at least 30\u00a0min. The initial transient and the broader peak of Isc were attributed to, respectively, stimulation of Cl\u2212 secretion and Na+ absorption by cAMP [2]. Interestingly, the effect of cAMP on Isc was also time dependent. With late-cAMP treatment (Fig.\u00a03b), the decay of Isc was faster and the magnitude of NS-Isc was larger. As shown in Fig.\u00a04, there was a significant inverse correlation between AS-Isc and NS-Isc both in the absence and in the presence of cAMP. This correlation does not indicate a causal relationship between the rise in NS-Isc and the fall in AS-Isc, but suggests that whatever process is responsible for the time dependence produces reciprocal changes in NS-Isc and AS-Isc.\nCl\u2212 channels associated with NS-Isc\nAs discussed previously [2], we attributed NS-Isc to Cl\u2212 secretion because in the presence of apical amiloride, Cl\u2212 was the only ion present in sufficient concentration to account for it. Furthermore, glibenclamide blocked the initial rise in NS-Isc in response to subsequent cAMP treatment, whereas DIDS blocked the broader late transport response but not the early peak (Fig.\u00a05). Glibenclamide is a well-established inhibitor of CFTR at the concentration (200\u00a0\u03bcM) used in these experiments. Although glibenclamide also inhibits ATP-activated K+ channels [28, 29], previous studies have shown that the apical membrane of MDCK cells has no measurable K+ conductance [10, 30]. Although DIDS effectively inhibits Cl\u2212 channels such as CaCC as well as the outwardly rectifying Cl\u2212 channel (ORCC), and the CLC family of Cl\u2212 channels, it does not inhibit CFTR [31, 32]. These results indicate that at least two types of Cl\u2212 channels contribute to NS-Isc and that they respond differently to cAMP.\nBased on these data, we have attributed the early, glibenclamide-sensitive, increase in NS-Isc after cAMP treatment to CFTR, which we have demonstrated is expressed in the apical membrane of these FL-MDCK cells [10]. CaCC appears to be responsible for the later increase in NS-Isc after cAMP treatment. As shown in Fig.\u00a06, we found that thapsigargin, which moderately increases [Ca2+]i [22], produced a transient spike and a sustained increase in NS-Isc that could be completely blocked by apical DIDS as expected for a CaCC [33]. Furthermore, cAMP increases [Ca2+]i in MDCK cells [21], and thus a CaCC may contribute to the DIDS-sensitive component of the NS-Isc response to cAMP. A CaCC has also been described in the M-1 and mIMCD-K2 cell lines [34, 35] and in primary cultures of rabbit proximal and distal tubule cells [36]. It should also be noted, however, that the molecular composition of CaCC is not as yet know. Thus, we were unable to provide unequivocal evidence for its existence in the FL-MDCK cell line.\nEndogenous ATP release inhibits amiloride-sensitive Na+ absorption\nWhen the MDCK monolayers were continuously perfused with fresh apical and basolateral KRB solutions rather than recirculated solutions, the time-dependent increase of NS-Isc and decrease of total Isc was prevented (Fig.\u00a07). AS-Isc was also significantly increased during the continuous perfusion, suggesting that inhibition of ENaC was prevented by washing out accumulated metabolites (Fig.\u00a07c). Furthermore, our experiments showed that hexokinase, a scavenger of extracellular ATP, produced the same effects on AS-Isc and NS-Isc as the perfusion of fresh KRB solutions (Fig.\u00a09). In the presence of hexokinase, AS-Isc was significantly higher but the DIDS-sensitive NS-Isc was significantly lower than in control experiments, indicating that extracellular ATP caused the inhibition of ENaC and activation of NS-Isc, the later possibly via a CaCC.\nExtracellular ATP is a well-established agonist for purinergic receptors, leading to an increase of intracellular [Ca2+] and activation Ca2+-activated Cl- channels [24, 37, 38]. In parallel, ATP attenuates amiloride-sensitive Na+ absorption in a variety of tissues including airway [39\u201341] and renal epithelia [42\u201344]. Similarly, activation of CFTR also increases Cl\u2212 secretion and inhibits ENaC in native epithelial tissues [14, 45]. Because CFTR has been suggested to potentiate ATP release [46, 47] and it has been shown to be expressed in this MDCK cell line [10], we examined a possible contribution of ATP release to the time-dependent transport in FL-MDCK cells. As shown in Fig.\u00a08a, addition of 100 \u03bcM ATP stimulated anion secretion, possibly via a Ca2+-activated Cl\u2212 channel, which was inhibited by DIDS, and this effect was prevented in the presence of hexokinase (Fig.\u00a08b).\nEndogenous release of ATP by the FL-MDCK monolayers was demonstrated by the bioluminescence assay experiments shown in Fig.\u00a010. Consistent with measurements in cortical CD (see [48]), ATP was shown to be released into the apical solution under basal conditions, and this release was augmented more than three-fold by cAMP treatment. In contrast, there was no significant release of ATP into the basolateral solution either without or with cAMP treatment.\nATP has been shown to inhibit AS-Isc and stimulate NS-Isc in other epithelia of distal nephron origin, including M-1 [43], mIMCK-K2 [42], and A6 cells [49]. Release of endogenous ATP into the extracellular medium has been proposed as a general response to hypotonicity and mechanical stimulation in many types of cells, including MDCK and A6 cells [49, 50]. Ostrom et al. [51] demonstrated that mechanical stimulation increases ATP release from MDCK cells as well as COS-7 and HEK-293 cells, and they proposed that ATP acts to alter the set point for a variety of signal transduction pathways. Praetorius et al. [50] also observed that mechanical stimulation caused ATP release in MDCK cells and that this release was associated with a transient increase in [Ca2+]i.\nBecause of the opposite actions of ATP in the extracellular apical membrane, it can serve to shift the balance between active NaCl absorption and active NaCl secretion in the MDCK cell, as shown by the hypothetical model in Fig.\u00a011. It is also quite important to note that the evidence we have presented for ATP accumulation in the extracellular fluid with longer incubation periods in Ussing chambers raises a caution for all such experiments, and it suggests that recirculation of the bathing solution, which has been in common use, should be avoided.\nFig.\u00a011Model for the role of adenosine triphosphate (ATP) in regulating Na+ absorption and Cl\u2212 secretion in Madin-Darby canine kidney (MDCK) epithelia. The epithelial Na+ channel (ENaC) mediates active Na+ absorption and produces the amiloride-sensitive component of the short-circuit current (AS-Isc). Because of the increased Na+ conductance produced by ENaC, it also depolarizes the apical membrane, producing a negative transepithelial voltage (Vte) of \u221230\u00a0mV. The basolateral membrane contains the ubiquitous Na, K-ATPase and, putatively, an Na-K-2Cl cotransporter, which actively accumulates Cl\u2212 in the cell. Cl\u2212 can then move across the apical membrane down its electrochemical potential gradient through Cl\u2212 channels, which include the glibenclamide-sensitive cystic fibrosis transmembrane conductance regulator (CFTR) channel, and a 4, 4'-diisothiocyano-2, 2'-disulfonic acid stilbene (DIDS)-sensitive, calcium-activated Cl- channel (CaCC). The net secretory movement of Cl\u2212 gives rise to the amiloride-insensitive component of the short-circuit current (NS-Isc). Our results show that ATP is secreted across the apical membrane. The pathway for ATP secretion is not known, but it has been proposed that it may also occur via CFTR [46]. When ATP accumulates in the apical extracellular space (e.g., when the solution bathing this space is restricted in volume or is recirculated), ATP inhibits ENaC and stimulates CaCC (and possibly CFTR or other Cl\u2212 channels), resulting in a fall in AS-Isc and an increase in NS-Isc\nImplications for renal pathophysiology\nIt must be recognized that the FL-MDCK cell line suffers from the limitations of any cell culture system when attempting to apply these results to an understanding of normal renal physiology or pathophysiology. Furthermore, even the type-1 MDCK cells used in these studies are heterologous, and their origin cannot be ascribed definitively to any nephron segment. Nevertheless, the ability of ATP to inhibit ENaC-mediated Na+ absorption and stimulate Cl\u2212 secretion, as shown in these experiments with FL-MDCK cells and in similar epithelia [42, 43, 49], suggests a role for such signaling in the nephron. Significant amounts of ATP are released from epithelial cells originating from all regions of the nephron, and multiple P2Y and P2X receptors have been identified in all segments of the nephron [48]. Thus, changes in the luminal ATP concentration in the cortical CD and other aldosterone-sensitive segments of the distal nephron, in which ENaC mediates Na+ transport, may be a physiologic signal for diminished Na+ reabsorption. Also, Wilson et al. [19] demonstrated significant accumulation of ATP in cyst fluid obtained from patients with autosomal dominant polycystic kidney disease. Thus, based on our observations in MDCK cells, it seems reasonable to speculate that ATP might favor cyst enlargement and consequent exacerbation of polycystic disease by inhibiting Na+ reabsorption and stimulating Cl\u2212 secretion [19, 52].","keyphrases":["atp","enac","cftr","clc","calcium-activated cl\u2212 channel","purinergic effects"],"prmu":["P","P","P","P","R","R"]}
{"id":"J_Mol_Biol-1-5-1950483","title":"Experiments Suggest that Simulations May Overestimate Electrostatic Contributions to the Mechanical Stability of a Fibronectin Type III Domain\n","text":"Steered molecular dynamics simulations have previously been used to investigate the mechanical properties of the extracellular matrix protein fibronectin. The simulations suggest that the mechanical stability of the tenth type III domain from fibronectin (FNfn10) is largely determined by a number of critical hydrogen bonds in the peripheral strands. Interestingly, the simulations predict that lowering the pH from 7 to \u223c4.7 will increase the mechanical stability of FNfn10 significantly (by \u223c33 %) due to the protonation of a few key acidic residues in the A and B strands. To test this simulation prediction, we used single-molecule atomic force microscopy (AFM) to investigate the mechanical stability of FNfn10 at neutral pH and at lower pH where these key residues have been shown to be protonated. Our AFM experimental results show no difference in the mechanical stability of FNfn10 at these different pH values. These results suggest that some simulations may overestimate the role played by electrostatic interactions in determining the mechanical stability of proteins.\nFibronectin is an extracellular matrix protein composed of three types of repeating domains (type I, type II and type III). The type III (fnIII) domains (which are ubiquitous in many multidomain mechanical proteins) in particular have been found to play a pivotal role in regulating and mediating physiological functions of cells. This is achieved through the interaction of various domains with the integrin family of cell surface receptors.1 One of the critical interactions in fibronectin is the binding of the RGD (Arg-Gly-Asp) motif in the tenth fnIII domain of fibronectin (FNfn10) to cell-surface integrins. A linear RGD-peptide has been shown to bind (with reduced binding strength and selectivity) to different members of the integrin family.2 It may be the dynamic structure of these domains that determines the functional states of the protein. Indeed, it has been suggested that force-induced conformational change of these fnIII domains may be crucial in transmitting cellular signals.3 Thus knowledge of how fnIII domains respond to mechanical forces and their mechanical resistance to conformational change is of importance to understand the function of fibronectin at the molecular level.\nA number of studies have investigated the mechanical properties of the fnIII domains of fibronectin by both experiment4,5 and simulation.6\u20138 Using steered molecular dynamics (SMD) simulations, Vogel and co-workers6 predicted a \u201cmechanical hierarchy\u201d of a number of fnIII domains that is in reasonable qualitative agreement with the hierarchy obtained from the atomic force microscopy (AFM) forced unfolding experiments.4 One of the most mechanically weak fnIII domains is the tenth fnIII domain of human fibronectin, FNfn10, although, interestingly, this is thermodynamically the most stable fnIII domain to have been studied to date.9,10 The simulations suggested that the mechanical stability of FNfn10 is largely determined by the hydrogen bonds between the A and B strands around Arg6 and Asp23 (Figure 1). Recent solution studies by Koide and co-workers10 showed that FNfn10 becomes more thermodynamically stable at lower pH (<5.0) as a consequence of the protonation of three negatively charged residues: Asp7, Asp23 and Glu9, which have raised pKa values of 5.54, 5.40 and 5.25, respectively; they are essentially fully protonated below pH 4.7. Using their simulations, Vogel and co-workers6 showed that protonation of these side-chains allows them to move closer together to form side-chain\u2013side-chain hydrogen bonds. They suggested that this stabilizes interactions between the A and B strands, resulting in a significant increase in the unfolding force in simulations at pH 4.7 even though it is well established that there is no correlation between mechanical stability and thermodynamic stability.11,12 This interesting result suggests that the mechanical stability of fibronectin might be modulated by a change in the pH of the tissues.\nWe tested this prediction by monitoring the unfolding force of FNfn10 at different pH values: pH 4.5 (50\u00a0mM acetate), pH 5.0 (50\u00a0mM acetate) and pH 7.0 (50\u00a0mM phosphate). It is important to note that Asp7, Asp23 and Glu9 are fully protonated at pH 4.5 as shown by NMR experiments carried out by Koide and co-workers.10 Any increase in mechanical stability due to the protonation of these three residues, as shown in the SMD simulation, should be observed in the AFM experiments. Forced unfolding experiments were performed for a polyprotein containing eight repeats of the FNfn10 domain.\nFigure 2 shows a \u201ctypical\u201d force-extension trace. As has previously been observed5 Fnfn10 unfolds either by a two-state unfolding mechanism (N\u00a0\u2192\u00a0U, where N is the native state and U the unfolded state) or a three-state unfolding mechanism via an intermediate (I) (N\u00a0\u2192\u00a0I\u00a0\u2192\u00a0U). The unfolding forces for three transitions could thus be determined: FN\u2192U, the force of unfolding of N directly to U; FN\u2192I, the force of unfolding of N to I; and FI\u2192U, the force of unfolding of I to U (Figure 2).\nThe intermediate, I, has been shown, using site-directed mutagenesis, to be a species with the A-strand detached.5 Thus one might expect, if the simulations of the unfolding of FNfn10 are correct, that both the N\u00a0\u2192\u00a0U and N\u00a0\u2192\u00a0I transitions will be at higher force at lower pH due to the new stabilising interactions between the A and B strands. However, since the A strand is detached in I, any stabilizing interactions between the A and B strands should not affect the mechanical stability of the intermediate, and FI\u2192U is expected to be unaffected by pH. Furthermore, the unfolding forces of N\u00a0\u2192\u00a0U are higher than those of N\u00a0\u2192\u00a0I. Li et al.5 suggested that this may be due to the \u201cstochastic nature\u201d of mechanical unfolding. They suggested that since N\u00a0\u2192\u00a0I unfolding is observed at higher unfolding forces than the subsequent I\u00a0\u2192\u00a0U unfolding, the I\u00a0\u2192\u00a0U unfolding may sometimes occur at a force that is too low to be seen in the AFM force-extension traces.5 That is, the unfolding is likely always to occur via this intermediate but it may not be observed, particularly when N unfolds at high forces. Thus if the simulations are correct we might expect to see fewer I\u00a0\u2192\u00a0U transitions at lower pH.\nTo our surprise, the unfolding forces at all pH values are the same within error (Figure 3). This is true at all pulling speeds. The dependence of the unfolding force on the pulling speed remains unchanged, suggesting that there has been no change in the unfolding pathway. Note also that the proportion of I\u00a0\u2192\u00a0U unfolding events remains about the same. Thus our results are in direct contradiction to the predictions from the steered molecular dynamics simulations.\nAFM combined with protein engineering \u0424-value analysis and molecular dynamics simulations has been previously used to solve the mechanical unfolding pathway of TNfn3, a homologous fnIII domain from the extracellular matrix protein tenascin.13 The results suggest that the unfolding of fnIII domains is a complicated multi-step process. The major barrier to a forced unfolding event in TNfn3 is the conformational transition from a twisted to an aligned state, which involves the breaking of several key hydrogen bonds along the peripheral strands (A-B strands and some between the G and F-strands). But there is also significant loss of hydrophobic side-chain packing interactions of residues in the A, B and G-strands, and importantly there is significant core re-packing. Thus, hydrophobic contacts of the buried residues and the hydrogen bonding interactions along peripheral strands are both apparently critical to the mechanical stability of TNfn3. TNfn3 and FNfn10 fold into essentially identical tertiary structures encompassing seven beta-strands running in two antiparallel beta sheets. A structural alignment of TNfn3 and FNfn10 reveals that these two fnIII domains have essentially the same hydrogen bonding patterns14 and molecular dynamics simulations have suggested that they have similar forced unfolding pathways. It is therefore reasonable to anticipate that these two fnIII domains will have the same molecular determinants for mechanical stability. We have made a \u201ccore-swap\u201d version of FNfn10, with the core of Tnfn3 which is significantly more stable than Fnfn10 itself.15 Considering these together, it seems that the mechanical stability of both FNfn10 and TNfn3 is likely to be associated with the complex interplay between key peripheral hydrogen bonds and hydrophobic effects from the packing of buried residues.\nMolecular dynamics simulations have previously proved to be of great value in predicting and understanding the behavior of proteins placed under mechanical stress.6,7,11,13,16,17 However, our results suggest that in the case of the simulations of forced unfolding of FNfn10 tested here,6 the relative strength and importance of electrostatic and hydrophobic components may not be adequately described. It is possible that this discrepancy lies in the different timescale of the simulations and the experiments. AFM experiments are typically performed at extension rates of \u223c1\u00a0\u03bcm s\u22121, whereas the timescale of molecular dymamics simulations allows for full extension of the protein in 1 or 2\u00a0ns, equivalent to a pulling speed of several m s\u22121, many orders of magnitude faster. Many slow conformational changes in proteins will not be observed in simulations on the nanoseconds timescale. Furthermore, the pathway of forced unfolding may vary as a function of the loading rate.18,19 Our study serves to emphasize the point that simulations need constantly to be benchmarked against experiment. However, the fact that simulators are prepared to make ab initio predictions that can be tested experimentally can only serve to improve our understanding of molecular mechanisms underlying mechanical strength in proteins.","keyphrases":["extracellular matrix","afm","forced unfolding","afm, atomic force microscopy","smd, steered molecular dynamics","fniii, fibronectin type iii","fnfn10, the tenth fniii domain of human fibronectin","tnfn3, the third fniii domain of human tenascin","md simulations","titin"],"prmu":["P","P","P","R","R","R","R","M","M","U"]}
{"id":"Eur_J_Health_Econ-3-1-1913175","title":"Modelling the 5-year cost effectiveness of tiotropium, salmeterol and ipratropium for the treatment of chronic obstructive pulmonary disease in Spain\n","text":"Our objective was to assess the 5-year cost effectiveness of bronchodilator therapy with tiotropium, salmeterol or ipratropium for chronic obstructive pulmonary disease (COPD) from the perspective of the Spanish National Health System (NHS). A probabilistic Markov model was designed wherein patients moved between moderate, severe or very severe COPD and had the risk of exacerbation and death. Probabilities were derived from clinical trials. Spanish healthcare utilisation, costs and utilities were estimated for each COPD and exacerbation state. Outcomes were exacerbations, exacerbation-free months, quality-adjusted life years (QALYs), and cost(-effectiveness). The mean (SE) 5-year number of exacerbations was 3.50 (0.14) for tiotropium, 4.16 (0.40) for salmeterol and 4.71 (0.54) for ipratropium. The mean (SE) number of QALYs was 3.15 (0.08), 3.02 (0.15) and 3.00 (0.20), respectively. Mean (SE) 5-year costs were \u20ac6,424 (\u20ac305) for tiotropium, \u20ac5,869 (\u20ac505) for salmeterol, and \u20ac5,181 (\u20ac682) for ipratropium (2005 values). Ipratropium and tiotropium formed the cost-effectiveness frontier, with tiotropium being preferred when willingness to pay (WTP) exceeded \u20ac639 per exacerbation-free month and \u20ac8,157 per QALY. In Spain, tiotropium demonstrated the highest expected net benefit for ratios of the willingness to pay per QALY, well within accepted limits.\nIntroduction\nOwing to the chronic progressing nature and increasing prevalence of chronic obstructive pulmonary disease (COPD), its treatment will undoubtedly increase pressure on the drug budgets of developed countries in the future. Several new and competing treatments for COPD have recently been introduced, or may soon become available [1]. Healthcare authorities will be forced to decide upon reimbursement of these medications and will require high-quality information about the costs and effectiveness of these new drugs. Pharmaco-economic evaluations provide this information and may guide the positioning of new drugs in the treatment spectrum of COPD.\nTo date, most economic evaluations of pharmacotherapy for COPD have been conducted alongside randomised controlled trials [2\u20137]. Consequently, their time horizon has been restricted to the duration of these trials, which is usually 6\u00a0months to 1\u00a0year, although economic evaluations of inhaled bronchodilators and corticosteroids piggybacked to clinical trials with durations of only 12\u201316\u00a0weeks have been reported [4, 5]. For reimbursement authorities and formulary decision makers, these short-term economic evaluations are of limited value, because COPD requires long-term maintenance treatment on a daily basis. Furthermore, these policy makers will need results that represent their national or regional setting. Because it is not feasible to conduct long-term empirical cost-effectiveness studies in all potential markets, some sort of modelling is required. This will allow expansion of the time horizon beyond that of a clinical trial, as well as the adapting and transferring of results from one setting to another. Decision analytical modelling has become an accepted approach to economic evaluations, and the use of modelling is supported by nearly all pharmaco-economic guidelines issued by health authorities involved in decision making about pricing and reimbursement of new pharmaceuticals, including Spain [8\u201311].\nThis study investigates the cost effectiveness of three bronchodilators for the maintenance treatment of COPD in Spain: tiotropium, salmeterol and ipratropium. Bronchodilators are central to the symptomatic management of COPD. Bronchodilators include three classes of medications: (1) the inhaled beta2-agonists, (2) the inhaled anticholinergics and (3) the oral methylxanthines. All three classes include drugs that are short acting or long acting. The most recently launched bronchodilator is tiotropium, an anticholinergic that provides 24\u00a0h bronchodilation with once-daily dosing. It was developed as a more effective and more convenient alternative to ipratropium, the most frequently used short-acting anticholinergic bronchodilator in COPD, which has to be used four times daily. At the time tiotropium became available, salmeterol, which is used twice daily, was the most effective and the most frequently used long-acting bronchodilator from the beta2-agonists class. Orally administered methylxanthines, such as theophylline, are not recommended for routine use due to their unfavourable risk\/benefit ratios. In a series of multi-national trials tiotropium was directly compared with either placebo [12], ipratropium [13] or salmeterol [14]. The above-mentioned clinical trials have shown that tiotropium provides sustained bronchodilation, reduces exacerbation rate, and improves dyspnoea and health-related quality of life when compared with placebo or ipratropium. Improvements in lung function have been shown to be significantly better with tiotropium than with salmeterol [14]. Because information on the long-term cost effectiveness of these bronchodilators is lacking, a decision analytical model was developed to determine the 5-year cost effectiveness of tiotropium, salmeterol and ipratropium for the treatment of COPD. Efficacy data were derived from the above-mentioned clinical trials, and the potential cost effectiveness of inhaled bronchodilator therapy was assessed in scenario analyses. The model was populated with Spanish epidemiological data on COPD, Spanish healthcare utilisation, and unit costs and utilities based on Spanish population values. Spain was chosen because cost-effectiveness data on bronchodilator therapy from a Southern European country were lacking.\nMethods\nThe model\nThis 5-year model can be characterised as a Markov model [15]. It builds on our earlier work in which we constructed a fully probabilistic Markov model to assess the 1-year cost effectiveness of tiotropium, salmeterol and ipratropium [16, 17]. All mathematical and technical details of this 1-year model have been published elsewhere [17]. In brief, all COPD patients were classified into three disease states of increasing severity, based on their pre-bronchodilator forced expiratory volume in 1\u00a0s (FEV1): moderate COPD (50% \u2264 the percentage of the predicted value (FEV1 % pred.) < 80%), severe COPD (30% \u2264\u00a0FEV1 % pred. < 50%) and very severe COPD (FEV1 % pred. < 30%). In pre-specified time intervals (Markov cycles) patients move between disease states and are at risk of experiencing an exacerbation, either severe or non-severe. All probabilities to move between disease states (transition probabilities) and to experience exacerbations that went into the 1-year model were obtained directly from six clinical trials comparing tiotropium with salmeterol, ipratropium or placebo (study codes: 205.114\/205.117, 205.115\/205.128, 205.122A\/205.126A, 205.122B\/205.126B, 205.130 and 205.137) [12\u201314]. Transition probabilities and exacerbation probabilities for tiotropium were based on the pooled patient-level data from all six clinical trials. To obtain the transition and exacerbation probabilities for the comparator arms, we applied the relative difference between tiotropium and the comparators in the individual trials to the probabilities of tiotropium that were derived from the pooled data [17].\nAll trials used similar inclusion and exclusion criteria. They enrolled patients with stable moderate-to-severe COPD who had an FEV1 <\u00a065% (salmeterol-controlled trials <\u00a060%) of predicted normal and FEV1\u00a0\u2264\u00a070% of forced vital capacity. The duration of these trials was 1\u00a0year for the those that compared tiotropium with placebo and ipratropium and 6\u00a0months for the trials that compared tiotropium with salmeterol. In total, 1,308 patients were randomly allocated to receive tiotropium, and 771 patients, 405 patients, and 179 patients were randomly selected to receive placebo, salmeterol and ipratropium. In all trials the same definitions of an exacerbation and its severity were used. An exacerbation was defined as a new onset or worsening of more than one symptom, such as cough, sputum, dyspnoea or wheeze, lasting for at least 3\u00a0days. Exacerbations were distinguished into severe and non-severe exacerbations. A severe exacerbation was defined as \u2018incapacitating or inability to do work or usual activity\u2019. All other exacerbations were non-severe. Severity was assessed by the clinical investigator. The risk of experiencing an exacerbation varied by disease state and treatment group. Given treatment group and disease state, exacerbation probabilities were assumed to be constant over time [17].\nThe current model expanded the time horizon from 1 year to 5\u00a0years and aimed to assess the cost effectiveness of tiotropium, salmeterol and ipratropium for treating COPD patients in Spain. To reflect the progressive nature of COPD in the long run and to incorporate mortality, an additional \u2018death state\u2019 was added to the model. The base-case analysis was performed from the perspective of the Spanish National Health Service (NHS) and included all costs covered by the NHS budget in euros at the 2005 value. A graphical presentation of the 5-year model is given in Fig.\u00a01. In addition, an analysis from the societal perspective was performed. The difference between the societal perspective and the NHS perspective was that the first additionally included patients\u2019 co-payments for medications and the costs of lost production from day\u00a01 of sick leave onwards; however, it excluded value added tax (VAT), as this does not represent a cost to society.Fig.\u00a01Graphical presentation of the Markov model. Backward transitions, i.e. from very severe to severe COPD and from severe to moderate COPD, were allowed during the first year, but not thereafter. The circles on the top left of each COPD severity state indicate that the patients can stay in the same state\nScenarios\nThe first year of the model incorporated the benefits of therapy as observed in the clinical studies for improvement in lung function, which delayed the progression to subsequent COPD severity states [17]. The first year of the model also incorporated the reduction in the number of exacerbations that were found in the clinical trials [17]. These first year probabilities are shown in the upper part of Table\u00a01 and in Table\u00a02. Because the clinical trials did not provide data on the probabilities to move between disease states and to experience exacerbations after the first year, the 5-year model was used to run three scenarios with different assumptions on transition and exacerbation probabilities for years 2 to 5: In the base-case scenario, the decline in FEV1 after the first year was assumed to be 52\u00a0ml\/year [18] in all treatment groups, whereas the exacerbation probabilities remained as they were for the first year. The decline of 52\u00a0ml was the mean annual change in FEV1 among smokers and ex-smokers in the Lung Health Study [18]. The lower part of Table\u00a01 shows the transition probabilities in the base-case scenario during years\u00a02 to 5, whereas Table\u00a02 shows the exacerbation probabilities.In the second scenario, both transition and exacerbation probabilities of the first year were assumed to remain constant during years\u00a02 to 5. In other words, the first year probabilities as shown in Table\u00a01 (under the heading \u201cSubsequent cycle probabilities, year 1\u201d) and 2 were applied to the later years.In the third scenario, it was assumed that disease progression and exacerbation risk no longer differed between treatment groups after the first year. Exacerbation probabilities for tiotropium, salmeterol and ipratropium were assumed to be equal to the first year probabilities of the ipratropium group (columns\u00a04 and 7 of Table\u00a02). The assumption on lung function decline was the same as in the base-case scenario. Note that the three scenarios do not differ with respect to the first year. During the first year, both forward transitions (transitions to worse disease states) and backward transitions (transitions to better disease states) are possible. In the base-case and third scenario, backward transitions during years\u00a02 to 5 were not allowed, reflecting the progressive nature of COPD in general. Further note that no differences between treatments in terms of mortality risk were assumed.Table\u00a01Mean (SE) probabilities to move between disease states in the base-case scenario. (These probabilities were also used in scenario\u00a03. In scenario\u00a02 the subsequent cycle probabilities for year 1 were applied to years\u00a02 to 5. The cycle length was 8\u00a0days for the very first cycle and 1\u00a0month for of all subsequent cycles)To \u21d2 From \u21d3\u00a0TiotropiumSalmeterolIpratropiumModerateSevereVery severeDeathModerateSevereVery severeDeathModerateSevereVery severeDeathFirst cycle probabilities\u00a0Moderate0.906 (0.018)0.092 (0.018)0.001 (0.002)0.001 (0.001)0.898 (0.033)0.100 (0.033)0.001 (0.003)0.001 (0.001)0.738 (0.092)0.257 (0.091)0.004 (0.013)0.001 (0.002)\u00a0Severe0.259 (0.017)0.715 (0.018)0.025 (0.006)0.001 (0.001)0.201 (0.028)0.765 (0.030)0.033 (0.013)0.001 (0.002)0.102 (0.028)0.841 (0.034)0.056 (0.022)0.001 (0.002)\u00a0Very severe0.010 (0.005)0.340 (0.024)0.648 (0.024)0.002 (0.002)0.001 (0.003)0.301 (0.042)0.696 (0.042)0.002 (0.004)0.005 (0.011)0.220 (0.066)0.773 (0.067)0.002 (0.007)Subsequent cycle probabilities, year 1\u00a0Moderate0.957 (0.010)0.040 (0.010)0.003 (0.003)0.001 (0.001)0.929 (0.025)0.066 (0.024)0.005 (0.007)0.001 (0.002)0.924 (0.050)0.073 (0.049)0.003 (0.010)0.001 (0.004)\u00a0Severe0.023 (0.006)0.952 (0.009)0.023 (0.006)0.002 (0.002)0.023 (0.011)0.916 (0.020)0.059 (0.017)0.002 (0.003)0.013 (0.011)0.948 (0.021)0.037 (0.018)0.002 (0.004)\u00a0Very severe0.001 (0.002)0.045 (0.012)0.947 (0.013)0.008 (0.005)0.006 (0.008)0.036 (0.019)0.951 (0.023)0.008 (0.009)0.003 (0.009)0.025 (0.026)0.964 (0.031)0.008 (0.015)Subsequent cycle probabilities, years\u00a02 to 5\u00a0Moderate0.984 (0.007)0.015 (0.007)0.000 (0.000)0.001 (0.001)0.981 (0.017)0.019 (0.017)0.000 (0.000)0.001 (0.003)0.978 (0.033)0.022 (0.033)0.000 (0.000)0.001 (0.005)\u00a0Severe0.000 (0.000)0.990 (0.004)0.008 (0.003)0.002 (0.002)0.000 (0.000)0.987 (0.008)0.011 (0.007)0.002 (0.003)0.000 (0.000)0.986 (0.012)0.011 (0.011)0.002 (0.005)\u00a0Very severe0.000 (0.000)0.000 (0.000)0.992 (0.005)0.008 (0.005)0.000 (0.000)0.000 (0.000)0.992 (0.007)0.008 (0.007)0.000 (0.000)0.000 (0.000)0.992 (0.011)0.008 (0.011)Table\u00a02Mean (SE) exacerbation probabilities in the base-case scenario. (These probabilities were also used in scenario\u00a02. In scenario\u00a03 the probabilities of ipratropium were also applied to tiotropium and salmeterol during years\u00a02 to 5)\u00a0Probability of experiencing an exacerbationProbability that the exacerbation will be severe, given that there is an exacerbationTiotropiumSalmeterolIpratropiumTiotropiumSalmeterolIpratropiumModerate0.051 (0.004)0.057 (0.013)0.080 (0.020)0.097 (0.024)0.030 (0.031)0.267 (0.114)Severe0.075 (0.003)0.089 (0.011)0.097 (0.013)0.136 (0.018)0.138 (0.033)0.188 (0.041)Very severe0.096 (0.005)0.104 (0.016)0.102 (0.022)0.192 (0.027)0.207 (0.048)0.186 (0.062)\nInput data: baseline distribution of patients among disease states\nThe baseline distribution of patients among the disease states was based on a Spanish study by Miravitlles et\u00a0al. [19] Re-analysis of the data according to the severity definition used in the model showed that, of 436 COPD patients, 55.2% had moderate disease, 34.9% had severe disease and 9.9% had very severe disease. A disease state for mild COPD was not included in the model, because mild COPD patients did not participate in the tiotropium trials.\nInput data: death state\nProbabilities of dying were based on all-cause mortality rates among Spanish patients with severe or very severe COPD as published by Miravitlles et\u00a0al. [20]. The data from this study were re-analysed using the same cut-off values for the FEV1% predicted of severe and very severe COPD that were used in the model. The 1-year all-cause mortality rate was found to be 25 per 1,000 among patients with severe COPD [20], and the relative mortality risk in patients with very severe COPD compared with severe COPD was found to be 4.96 for a mean decline in FEV1% predicted of 19.4%. Under the assumption of constant proportional hazards, the relative mortality risk of patients in the trials was estimated to be 3.754 for very severe COPD and 0.248 for moderate COPD compared with severe COPD. We applied these relative risks to the all-cause mortality rate of 25 per 1,000 among patients with severe COPD to derive the mortality rates and, subsequently, the probabilities of dying, among patients with moderate and very severe COPD. These probabilities were the same for all treatment groups.\nInput data: utilities\nTo reflect the impaired quality of life of patients with COPD, we attached utility weights to each disease severity state. These utilities were obtained from the EQ-5D scores at baseline in a subset of patients randomly accepted into the UPLIFT trial (n\u00a0=\u00a01,133) [21]. These scores were valued using the Spanish tariff [22]. A value of 1 represents perfect health, whereas 0 represents death. Mean (SE) utilities at baseline of the UPLIFT trial were calculated to be 0.809 (0.008) in patients with moderate disease, 0.762 (0.009) in patients with severe disease and 0.655 (0.024) in patients with very severe disease. During the months in which patients experienced an exacerbation, the utility value was reduced by 15% in the case of a non-severe exacerbation [23] and by 50% in the case of a severe exacerbation [24]. Each year, the number of quality-adjusted life-years (QALYs) was calculated as the sum of the multiplications of the number of months in a particular disease state and the utility of that disease state, divided by 12.\nInput data: costs\nThe details and results of the cost estimates are presented in Table\u00a03. Estimates of healthcare utilisation were primarily derived from two studies performed in Spain that had measured resource use and costs of the treatment of patients with COPD [25, 26]. Data from these studies were re-analysed to estimate resource use and costs by disease severity and exacerbation severity, using similar definitions as applied in the model. In the model, healthcare utilisation associated with COPD maintenance therapy varied by disease severity. Healthcare utilisation during exacerbations varied by the severity of the exacerbation (severe or non-severe). Calculations were conservative in assuming that costs per disease severity state and costs per severe or non-severe exacerbation were equal across treatment groups. Resources for maintenance therapy included visits to respiratory physicians inside and outside the hospital, visits to the general practitioner, pulmonary function tests, blood tests, imaging tests and respiratory medications. Resources associated with a non-severe exacerbation included general practitioner and respiratory physician visits and medications. Besides physician visits and outpatient medications, resources associated with severe exacerbations also included hospital admissions and visits to the emergency room (ER) department. Costs of pulmonary function and other tests, as well as costs of medications that were prescribed during inpatient stay or during ER visits, were not measured separately, because they were included in the overall costs per in-patient day and the costs per ER visit.Table\u00a03Mean healthcare utilisation (HU), unit costs, and mean (SE) total costs of maintenance therapy and exacerbations from the NHS perspective for Spain in euros at the 2005 value (HU healthcare utilisation, RP respiratory physician, GP general practitioner, ICU intensive care unit, exa exacerbation)Maintenance therapy\u00a0Unit costsModerate COPDSevere COPDVery severe COPDHU per patient per yearTotal costsHU per patient per yearTotal costsHU per patient per yearTotal costsVisit RP in hospital79.850.2722 (1)0.4435 (2)0.6350 (3)Visit RP outside hospital23.770.389 (1)0.5714 (1)0.6515 (1)Visit GP8.352.5922 (1)2.8824 (1)3.2727 (1)Thorax X-ray19.671.0320 (1)1.1823 (1)1.4929 (1)ECG22.530.8018 (1)0.8720 (1)1.1526 (1)Spirometry40.630.5522 (1)0.6627 (1)0.9237 (2)Blood analyses19.671.4128 (1)1.4328 (1)1.5831 (1)Blood gases27.250.339 (1)0.5615 (1)0.6718 (1)Influenza vaccination5.470.483 (1)0.644 (1)0.724 (1)Theophylline0.18122.0622 (8)161.7730 (4)159.0729 (5)Mucolytics0.3039.7411 (3)48.3114 (2)80.6024 (4)Oral corticosteroids0.2921.546 (3)23.737 (1)78.4823 (6)Inhaled corticosteroids0.61224.84138 (12)224.84138 (15)292.00179 (22)Oxygen3.7321.3279 (14)44.26165 (30)77.87290 (52)Other20 (3)44 (4)34 (5)Total costs per patient per year430 (24)587 (34)818 (58)ExacerbationsUnit costsNon-severe exacerbationSevere exacerbationStudy medication costs per dayHU per exaTotal costsHU per exaTotal costsICUa1,284\u2013\u20130.29374 (291)Tiotropium1.80Non-ICUa368\u2013\u20134.161,529 (307)Salmeterol1.20Emergency room115\u2013\u20130.94108 (11)Ipratropium0.19Visit GP8.351.6414 (1)1.008 (3)Visit RP in hospital79.85\u2013\u20130.5242 (10)Antibioticsb5.0011.0255 (7)7.5238 (7)Oral corticosteroidsb0.292.691 (1)4.981 (1)Inhaled corticosteroidsb0.617.014 (1)3.712 (1)Oxygenb3.732.058 (3)5.3220 (2)Other1 (1)1 (1)NHS sick leave benefitcs74.0913 (2)Total costs per exacerbation83 (7)2,136 (425)For medications and oxygen HU is expressed in number of days during which the medication or oxygen was usedaOf all severe exacerbations 52% required hospital admission. Of those, 14% were to the ICU. The length of stay on ICU is 4\u00a0days, and the length of stay on non-ICU is 8\u00a0days. Hence, 0.52\u00a0\u00d7 0.14\u00a0\u00d7 4\u00a0=\u00a00.29 and 0.52\u00a0\u00d7 8\u00a0=\u00a04.16bOnly medications prescribed in ambulatory settings. Costs of medications administered in hospital and at emergency rooms are included in costs of hospital stay\/emergency room visitcAverage labour costs per day in Spain are \u20ac74.09. Of Spanish COPD patients, 33.2% have paid employment; 0.332\u00a0\u00d7 \u20ac74.09\u00a0=\u00a0\u20ac24.60. The NHS covers 60% of these labour costs during days 16\u201320 of the sick leave episode and 75% from day 21 onwards. Of severe exacerbations, 7% are associated with sick leave longer than 15\u00a0days. The duration of these absence spells is 26\u00a0days. So, (5\u00a0\u00d7 0.6\u00a0\u00d7 \u20ac24.60\u00a0+\u00a06\u00a0\u00d7 0.75\u00a0\u00d7 \u20ac24.60)\u00a0\u00d7 0.07\u00a0=\u00a0\u20ac13.43\nUnit costs of healthcare resources were obtained from the SOIKOS health database and have been described in detail elsewhere [25]. They were updated to 2005 using the Spanish General Consumer Price Index. Acquisition costs of pulmonary drugs from the NHS perspective were based on public prices and calculated as the ex-factory prices multiplied by a mark-up of 1.596 to convert these prices to public prices [27]. The co-payment of 10% of the public price for the people in the work force was excluded. It was estimated that 33.2% of Spanish COPD patients had to pay these co-payments because they were still in the work force [27]. From a societal perspective, the drug costs included co-payment but excluded the 4% VAT that is included in the public prices. Costs of tiotropium, salmeterol and ipratropium from the NHS perspective were \u20ac1.80, \u20ac1.20 and \u20ac0.19 per day, respectively. From the societal perspective these costs were \u20ac1.76 for tiotropium, \u20ac1.18 for salmeterol and \u20ac0.18 for ipratropium.\nThe NHS covers the costs of absence from work due to illness from the 16th day of sick leave onwards. The NHS pays 60% of gross salary during days\u00a016 to 20 and 75% from day\u00a021 onwards. When we were calculating costs from the NHS perspective, these costs were added to the costs of all severe exacerbations with sick leave longer than 15\u00a0days (approximately 7% of all severe exacerbations). The costs from the societal perspective included the costs of lost production during all days of absence from paid work.\nThe resulting mean (SE) annual costs of maintenance therapy from an NHS perspective were \u20ac430 (\u20ac24) for a patient with moderate COPD, \u20ac587 (\u20ac34) for a patient with severe COPD and \u20ac818 (\u20ac58) for a patient with very severe COPD. The corresponding values from the societal perspective were \u20ac429 (\u20ac21), \u20ac586 (\u20ac34) and \u20ac816 (\u20ac58). The mean (SE) costs of exacerbations from the NHS perspective were \u20ac83 (\u20ac7) for a non-severe exacerbation and \u20ac2,176 (\u20ac425) for a severe exacerbation. From a societal perspective these costs were \u20ac121 (\u20ac7) and \u20ac3,912 (\u20ac543), respectively.\nCOPD severity\nTo investigate the impact of COPD severity on the cost effectiveness of the bronchodilators we ran sensitivity analyses with the base-case scenario, assuming that, at the start of the model, 100% of the patients had either moderate, severe or very severe COPD.\nDiscounting\nBecause of time preference, costs and effects that will arise in the future are usually valued lower than costs and effects in the present. Discounting is used to calculate the present value of future costs and effects. In accordance with proposed Spanish guidelines, an annual discount rate of 6% has been adopted [28]. In the base-case analysis, costs and health outcomes were discounted at the same rate, whereas, in sensitivity analyses, discount rates of 0% for both costs and effects, and 6% for costs combined with 3% for effects, were applied. In scenarios\u00a02 and 3 the same 6% discount rate was used as in the base-case scenario.\nProbabilistic sensitivity analysis\nTo facilitate the interpretation of results of economic analyses, the reporting of uncertainty associated with costs and outcomes is equally important as reporting the point estimates of these parameters. Hence, the model was designed fully probabilistic, and uncertainty around the probabilities to move between disease states, the probabilities to experience exacerbations, utilities and healthcare utilisation was addressed by defining a probability distribution for each input parameter [17]. The uncertainty around these input parameters was propagated through the model simultaneously by conducting second-order Monte Carlo simulations. This means making random draws of the uncertain parameters from their probability distribution, running the model for each set of parameters that is drawn, and collecting the outputs from each run [29]. The current results were based on 5,000 iterations.\nThe main outcome measures of the model were mean and standard error (SE, being the standard deviation of the 5,000 iterations) of the 5-year costs per patient, exacerbation-free months and quality-adjusted life years. The presentation of incremental cost effectiveness was based on the hierarchy of outcomes, i.e. each treatment option was compared with the next best treatment option in terms of effectiveness. The uncertainty around costs and effects was further explored by plotting of the 5,000 iterations on incremental cost-effectiveness planes (CE-planes) [30] and by presenting cost-effectiveness acceptability curves [30, 31] and frontiers [32]. The acceptability curve presents the probability that a treatment is the most cost effective of the three treatments at different threshold values for cost effectiveness (ceiling ratio), whereas the cost-effectiveness acceptability frontier demonstrates which of the three treatments should be adopted because it results in the highest expected net benefit, given the ceiling ratio. The net benefit is calculated as the total costs (C) minus the effects (E) multiplied by the ceiling ratio (C \u2212 (E \u00d7\u00a0ceiling ratio)) [33]. The model was programmed in Microsoft EXCEL.\nResults\nHealth outcomes\nThe results of the Markov simulation for the different scenarios are presented in Table\u00a04. In the base-case scenario, the mean (SE) number of exacerbations in 5\u00a0years was 3.50 (0.14) in the tiotropium group, 4.16 (0.40) in the salmeterol group and 4.71 (0.54) in the ipratropium group. The corresponding mean (SE) number of exacerbation-free months was 46.83 (1.11) in the tiotropium group, 45.29 (2.12) in the salmeterol group and 44.89 (2.86) in the ipratropium group. Estimates of the number (SE) of QALYs were 3.15 (0.08), 3.02 (0.15) and 3.00 (0.20), respectively. In all scenarios, differences in exacerbation-free months and QALYs between treatment groups were consistently in favour of the tiotropium group. Applying the first year probabilities to all subsequent years (scenario 2) increased the difference in exacerbation-free months between tiotropium and the other treatment groups to approximately 2.0 and the difference in QALYs to approximately 0.19. Assuming similar exacerbation probabilities across treatment groups in year 2 to 5 (scenario 3) reduced the differences between treatment groups considerably. In this analysis, the difference in exacerbation-free months between tiotropium and the other treatment groups was just above 1, and the difference in QALYs was about 0.13. There was almost no difference in health outcomes between ipratropium and salmeterol in this scenario.Table\u00a04Results of the Markov simulation for the base-case analysis from the NHS perspective covering a time period of 5\u00a0years. The table gives mean (SE) or mean (95% confidence interval). QALY quality-adjusted life year, Tio tiotropium, Salm salmeterol, Ipra Ipratropium, exa exacerbations, CE-ratio cost-effectiveness ratio, exa-free exacerbation-free\nCosts\nIn the base-case scenario from the NHS perspective, mean (SE) total costs over 5\u00a0years were lowest in the ipratropium group, with \u20ac5,181 (\u20ac682) per patient (Table\u00a04). Costs were \u20ac5,869 (\u20ac505) in the salmeterol group and \u20ac6,424 (\u20ac305) in the tiotropium group. The higher costs in the tiotropium and salmeterol group were largely due to the higher costs of study medication. Compared with those of ipratropium, the 5-year costs of study medication were \u20ac2,477 (\u20ac67) higher in the tiotropium group and \u20ac1,521 (\u20ac85) higher in the salmeterol group. Savings in other categories of costs, mainly exacerbation-related costs, offset approximately half of these additional study medication costs.\nBecause of a smaller difference in exacerbation rate between treatment groups in scenario\u00a03, the difference in total costs between ipratropium on the one hand and tiotropium and salmeterol on the other hand increased by 64% and 75%, respectively. The impact of applying the first-year transition probabilities to years\u00a02 to 5 (scenario\u00a02) on the 5-year differences in costs between treatment groups was much less (Table\u00a04).\nCost effectiveness\nIn the base-case scenario from the NHS perspective estimates of the incremental costs per exacerbation-free month were \u20ac360, when tiotropium was compared with salmeterol, and \u20ac1,711 when salmeterol was compared with ipratropium. The corresponding incremental costs per QALY were \u20ac4,118 and \u20ac38,931, respectively. The distribution of the results of the 5,000 model iterations on the CE-planes is also reported in Table\u00a04. The comparisons between tiotropium and salmeterol show that the majority of simulations (approximately 65%) lie in the upper-right quadrants, signifying better health outcomes and higher costs for tiotropium. The CE-planes comparing salmeterol with ipratropium show that about 35% of the dots are found in the upper-left and about 45% in the upper-right quadrant, signifying similar health outcomes and higher costs for salmeterol.\nFigure\u00a02 shows the cost-effectiveness acceptability curves in the base-case scenario from the NHS perspective. The curves show that, in terms of exacerbation-free months, ipratropium has the highest probability of being cost effective when the threshold value for cost per additional exacerbation-free month is below \u20ac1,050. In terms of QALYs, ipratropium has the highest probability of being the most cost effective for all threshold values for cost per QALY below \u20ac11,000. Above these values, tiotropium has the highest probability of being cost effective. However, when the distribution of the cost-effectiveness ratio is skewed, as is the case in the presented analyses, the treatment with the highest probability of being cost effective is not always the treatment with the highest expected net benefit [32]. In Fig.\u00a02, the cost-effectiveness acceptability frontier is drawn in bold. The frontier follows the curve of the treatment with the highest expected net benefit for a given value of the cost-effectiveness threshold. Hence, it indicates which treatment should be chosen because it is optimal. The bold curve demonstrates that both ipratropium and tiotropium are on the frontier. For any ceiling ratio above \u20ac639 per exacerbation-free month and above \u20ac8,157 per QALY, tiotropium is the preferred treatment option. In scenario\u00a02, these threshold values were lower: \u20ac551 and \u20ac6,226, respectively, whereas the corresponding values in scenario\u00a03 were \u20ac1,918 and \u20ac15,635. The point on the frontier where the most optimal treatment switches from ipratropium to tiotropium in Fig.\u00a02 corresponds to the base-case incremental cost-effectiveness ratio for the comparison between these two bronchodilators. Figure\u00a02 also shows that, although tiotropium has the highest probability of being the most optimal treatment, this probability is 58% at maximum. This 58% is the value at which the acceptability curve of tiotropium using QALYs becomes asymptotic.Fig.\u00a02Cost-effectiveness acceptability curves and frontier of the costs per exacerbation-free month and the cost per QALY. Ceiling ratio: threshold value for the cost-effectiveness ratio in euros. The curves in grey represent the probability that a treatment is cost effective for a given value of the ceiling ratio. In the case of skewed distributions of the ratio, the treatment with the highest probability of being cost effective is not always the treatment with the highest expected net benefit. The acceptability frontier (in black) shows which treatment is associated with the highest expected net benefit for each value of the ceiling ratio\nSocietal perspective\nFrom the societal perspective, overall mean (SE) costs increased in all treatment groups to \u20ac6,574 (\u20ac321) for tiotropium, \u20ac6,125 (\u20ac541) for salmeterol and \u20ac5,545 (\u20ac720) for ipratropium. However, the savings in exacerbation costs generated by tiotropium and salmeterol compared with ipratropium were higher from a societal perspective than from the NHS perspective. Consequently, from the societal perspective, the difference in overall costs between the treatment groups was reduced. Also, the incremental costs per exacerbation-free month were reduced to \u20ac308, for the comparison tiotropium versus salmeterol, and \u20ac1,375 for the comparison salmeterol versus ipratropium. The incremental cost per QALY were reduced to \u20ac3,483, for the comparison tiotropium versus salmeterol, and \u20ac35,158 for the comparison salmeterol versus ipratropium. Tiotropium had the highest expected net benefit for any value of the cost-effectiveness threshold above \u20ac547 per exacerbation-free month and above \u20ac7,076 per QALY. Below these values ipratropium is preferred.\nImpact of COPD severity\nWhen the model was run separately for patients with either moderate, severe or very severe COPD, it showed that the threshold value above which tiotropium had the highest expected net benefit increased with the severity of COPD. The threshold values for the costs per QALY above which tiotropium became the preferred option were \u20ac7,600 for moderate COPD, \u20ac8,800 for severe COPD and \u20ac12,500 for very severe COPD. Below these values ipratropium was preferred. Tiotropium had the highest expected net benefit when the ceiling ratios for cost per exacerbation free month were \u20ac560 for moderate COPD, \u20ac700 for severe COPD and \u20ac1,200 for very severe COPD. Below these values ipratropium had the highest expected net benefit.\nDiscount rates\nBecause discounting affects both health outcomes and costs, the effect of discounting in this study was small. Analyses based on discount rates of 3% and 0% showed almost similar cost-effectiveness ratios. Discounting health outcomes at a lower rate than costs led to cost-effectiveness ratios that were slightly more in favour of tiotropium.\nDiscussion\nBronchodilators form the main therapy for the symptomatic relief of respiratory symptoms in COPD patients. In this study we have constructed a decision analytical model to synthesise clinical trial data on the effectiveness of bronchodilator treatment. We have shown how scenario analyses can be used to extend the time horizon of the cost-effectiveness study beyond that of the clinical trials. In addition, we have shown how a model can be populated with country-specific data to obtain estimates of the cost-effectiveness of bronchodilators in the Spanish setting.\nThis comprehensive country adaptation and extension of the existing short-term Markov model [17] required much more than just the imputation of Spanish unit costs. We searched for Spanish sources for almost every type of input data, ranging from the distribution of COPD severity stages, mortality, the proportion of COPD patients in the work force, utilities, resource use and unit costs. We re-analysed patient-level data from previously published Spanish studies on prevalence, mortality, EQ-5D, and resource utilisation [19\u201321, 25, 26] to ensure that these Spanish data matched the model\u2019s definitions of COPD severity and exacerbation severity. This included obtaining point estimates as well as distributions of these input data. The strength of the Spanish data is also that mainly patients from general practices were included, thus reflecting the routine care setting closely. The majority of Spanish patients are treated by general practitioners, and the proportion of patients referred to specialist care by pulmonologists is smaller than in many other west European countries [34]. This may be related to the gate-keeping function of the general practitioner in Spain that is not enforced in some other European countries.\nThe base-case scenario showed that tiotropium was associated with an approximately 16% reduction in exacerbations when compared with salmeterol. Salmeterol was associated with 12% reduction in exacerbations, when compared with ipratropium. Differences between the three treatment groups in terms of QALYs were small. That was expected, given the 5-year time horizon and treatments that do not directly affect survival. The distribution of dots on the CE-planes showed that the higher effectiveness of salmeterol over ipratropium was associated with more uncertainty than the higher effectiveness of tiotropium over salmeterol, because, for the latter comparison, a larger proportion of the dots was found on the right side of the CE plane.\nBeside effectiveness, the choice between bronchodilators must involve many considerations, including economic considerations of costs and cost effectiveness. The final result of this cost-effectiveness analysis is an acceptability frontier that demonstrated that tiotropium has the highest expected net benefit for a threshold value of the costs per QALY that is well within the limits of other adopted therapies in Spain [35] and in other countries like the UK [36]. This threshold value increases with the severity of COPD, because the differences between treatments in the probabilities of developing a (severe) exacerbation decrease as COPD severity increases. Nevertheless, the cost-effectiveness threshold in the most severely affected patients is still relatively low.\nThe threshold value for costs per additional exacerbation-free month of \u20ac639 above which tiotropium becomes the optimal choice in Spain is higher than the ceiling ratios above which tiotropium became most cost effective in the Netherlands (\u20ac0) and Canada (\u20ac10) in the 1-year model [17]. Similarly, when QALYs were used as the outcome measure, the ceiling ratio of \u20ac8,157 per QALY above which tiotropium is the preferred option in Spain is also higher than in The Netherlands and Canada, where tiotropium became the preferred option when decision makers could afford to pay more than \u20ac0 and \u20ac120 for a QALY, respectively. This difference between these countries is largely driven by the relatively low acquisition costs of ipratropium in Spain, which is related to the fact that there is no widespread use of the more expensive dry powder formulation of ipratropium. In addition, the savings due to the reduction in exacerbations by tiotropium and salmeterol are less, because the costs of treating a severe exacerbation in Spain are lower than in The Netherlands and Canada. This is caused by a smaller proportion of patients hospitalised, as well as a shorter length of stay. As a result, tiotropium generated very small net savings in the Netherlands and similar costs to the other two bronchodilators in Canada, whereas it increased total costs in Spain. Nevertheless, in the three countries investigated, the economic evaluations indicated that the health benefits gained with tiotropium are either at almost no additional costs or at costs that appear reasonable and acceptable, given other adopted treatments [37, 38].\nOur base-case scenario was conservative with respect to lung function decline, as we assigned a similar decline of 52\u00a0ml\/year to the patients in all treatment groups after the first year. We chose this to be the best assumption for the base-case scenario, because there is little evidence in the literature that bronchodilators alter the rate of decline of lung function [39]. However, frequent exacerbations seem to accelerate the decline in lung function [40, 41]. Therefore, our base-case scenario might have underestimated the long-term effect of a bronchodilator that reduces the number and severity of exacerbations. For that reason, we developed the second scenario, which is the least conservative, because we assumed that the differences in lung function decline and exacerbation rates between the three treatments that were observed during the first year would remain during the 4\u00a0years thereafter. This scenario is most favourable for tiotropium, and we see the threshold value above which tiotropium becomes most cost effective drop to \u20ac551 per exacerbation-free month and \u20ac6,226 per QALY. In addition to this optimistic scenario, we developed a very conservative scenario by completely eliminating the difference in exacerbation rate between the treatment groups after the first year and assuming that exacerbation rates would resemble the rate observed in the ipratropium group. This third scenario also assumed a similar decline in lung function across treatment groups of 52\u00a0ml\/year. This third scenario is conservative because exacerbations were the main drivers of cost effectiveness. The threshold value above which tiotropium becomes most cost effective in this third scenario increases to \u20ac1,918 for an additional exacerbation-free month and \u20ac15,635 for a QALY, yet is still below acceptable limits reported for the Spanish setting. Below that, ipratropium was most cost effective. Altogether, these three scenarios give the range within which to expect the 5-year cost effectiveness of these bronchodilators. Note, that we had no scenarios assuming a differential effect of the treatments on, for example, utilities or costs of a single exacerbation, because the evidence of such effects is lacking. Furthermore, all scenarios had a time horizon of 5\u00a0years to meet the requirements of many European reimbursement authorities who prefer extensions to time horizons reflecting their budgeting process (typically for 3\u20135\u00a0years). Lifetime models that primarily aim to compare medications are soon outdated by the development of new therapeutic options.\nWhen the cost-effectiveness analysis was conducted from a societal perspective, the threshold value above which tiotropium is the preferred option is reduced considerably to \u20ac547 per exacerbation-free month and \u20ac7,076 per QALY. This improved cost effectiveness is primarily related to increasing exacerbation costs, because of the inclusion of the costs of production losses. Hence, a reduced exacerbation rate is associated with higher savings. The NHS perspective also included sick leave benefits paid for by the NHS from day\u00a016 onwards. However, this applies only to severe exacerbations where it was less than 1% of its costs. In contrast, from a societal perspective, costs of production losses are 10% of the costs of a severe exacerbation and 30% of the costs of a non-severe exacerbation.\nWe chose two outcome measures that are among the most relevant in the lives of patients with COPD: being free of exacerbations and having a good quality of life. The concept of an exacerbation-free month is comparable to the concept of a symptom-free day, which is a frequently used outcome measure in asthma [42]. As it is a positive outcome (i.e. more is better), its cost-effectiveness ratio is easier to interpret than the ratio \u2018cost per exacerbation avoided\u2019. In general, the number of exacerbations is not a good outcome measure to use in long-term models, especially when treatment improves survival. This outcome measure will bias the treatment which improves survival, because patients can experience exacerbations during the life years gained. In contrast, the number of exacerbation-free months takes account of the fact that the added years of life are partly lived free of exacerbations.\nThe 1-year model, as well as the currently presented long-term model, has been used in reimbursement negotiations in several countries. The major strength of this model is its transparency. We have fully disclosed the model structure as well as all the input. Costs per exacerbation and costs per disease state do not differ between the three treatment groups, and mortality rates by disease severity were also set equally for all treatment groups. Hence, the difference in cost effectiveness is driven by the difference in acquisition costs of the study drugs and the difference in their effectiveness, i.e. the difference in the probabilities to move between disease states and the differences in exacerbation risks. For the first year of the model, estimates of effectiveness were directly obtained from patient-level data from clinical trials. Hence, estimates of probabilities were not based on expert opinion or literature, as is often the case in modelling studies. We acknowledge that differences may exist between trial results from which we derived the model input and the effectiveness of therapy in routine daily practice. A model can be used to adapt the trial data to better represent daily practice. Particularly, compliance is known to be worse in daily practice. However, not accounting for compliance was conservative for tiotropium and salmeterol, since once-daily and twice-daily dosing is more likely to be accompanied by long-term compliance than is four-times daily dosing in chronic diseases [43]. Owing to the double-blind, double-dummy design of the clinical trials, the dosing frequency was the same in the treatment groups, and any impact of dosing frequency on outcomes was eliminated.\nIn conclusion, our model has demonstrated that tiotropium is the treatment with the highest expected net benefit, if decision makers can afford to spend additional budget to gain additional health benefits. The threshold value of the costs per QALY at which tiotropium becomes the preferred treatment is well within acceptable limits (i.e. \u20ac8,157 from the NHS perspective and \u20ac7,076 from the societal perspective).","keyphrases":["model","cost effectiveness","spain","bronchodilators","chronic obstructive pulmonary disease (copd)","quality-adjusted life year (qaly)"],"prmu":["P","P","P","P","P","P"]}
{"id":"Exp_Brain_Res-4-1-2373857","title":"The haptic perception of spatial orientations\n","text":"This review examines the isotropy of the perception of spatial orientations in the haptic system. It shows the existence of an oblique effect (i.e., a better perception of vertical and horizontal orientations than oblique orientations) in a spatial plane intrinsic to the haptic system, determined by the gravitational cues and the cognitive resources and defined in a subjective frame of reference. Similar results are observed from infancy to adulthood. In 3D space, the haptic processing of orientations is also anisotropic and seems to use both egocentric and allocentric cues. Taken together, these results revealed that the haptic oblique effect occurs when the sensory motor traces associated with exploratory movement are represented more abstractly at a cognitive level.\nIntroduction\nHaptic perception (or active touch) results from the stimulation of the mechanoreceptors in skin, muscles, tendons and joints generated by the manual exploration of an object in space (Gibson 1962; Revesz 1934, 1950). Haptic perception allows us, for example, to identify an object, or one of its features like its size, shape or weight, the position of its handle or the material of which it is made. A fundamental characteristic of the haptic system is that it depends on contact. The \u201ctactile perceptual field\u201d (i.e., the portion of the skin that is in contact with the external stimulus) has a limited size (the surface of both hands at maximum) and a limited reach (the length of the arm). It results from these characteristics of the tactile perceptual field that the perception of the spatial properties of the objects almost always involves some displacements of the arm and the hand to explore the stimulus. In fact, it is also known that the nature of these exploratory movements often depends on the specific property of the touched object (Lederman and Klatzky 1987, 1993). For example, one might follow the contour of an object with one finger to perceive its shape or squeeze it with the whole hand to perceive its compliance. Moreover, the haptic system must integrate information about the parts of the body touching the object with information about the position of the body parts in space. It results from these properties of the haptic system that the haptic perception of space is far removed from the proximal stimulation that occurs during the manual exploration, and depends on spatio-temporal integration of the kinesthetics and tactile inputs to build a representation of the stimulus.\nStudies on the haptic perception of space have generally focused on the perception of the elementary spatial properties of objects such as their length, curvature or orientation in space (cf. Hatwell et al. 2003; Henriques and Soechting 2005; Millar 2008). The hallmark of these studies is that the dimension of interest is systematically manipulated and that exploratory movements, if present, are very precisely monitored. In general, the aim of these studies is to measure the sensitivity of the corresponding perceptual channel. In addition, evidence such as a systematic bias or some variation in the sensitivity of parameters is used to understand the underlying processes. Altogether, these studies have shown that the haptic perception of simple spatial properties such as orientation and length are systematically distorted with respect to the physical reality. For example, it is well known that the haptic perception of the length between two points is influenced by numerous factors such as movement direction (radial versus tangential) or velocity (cf. Walsh et al. 1979; Imanaka and Abernethy 2002; Gentaz and Hatwell 2004). Others have found that straight lines are often perceived as curved and vice versa (Sanders and Kappers 2007; see also Henriques and Soechting 2003). In the same vein, Kappers (1999, 2002) and Kappers and Koenderink (1999) have observed that, two parallel (in physical space) bars in different spatial locations can be felt as being almost perpendicular. This remarkable effect remains even after various types of feedback. Finally, it is noteworthy that judgments of different spatial properties can be inconsistent among themselves. For example, Fasse et al. (2000) found that the biases in the perception of the acute angles of a right triangle were inconsistent with the observed biases in the judgments of the lengths of the adjacent and opposed edges. This study suggests that diverse spatial properties of an object might be processed independently from each other and do not necessarily refer to a unique underlying representation of space.\nThe present review is concerned by one very specific aspect of spatial perception: the haptic perception of spatial orientations. The perception of orientations is isotropic when the performances in orientation-adjustment tasks are similar regardless of the orientation values and anisotropic when performances differ according to these values. Historically, the orientation effect of a stimulus on the performance level was first studied in various detection, discrimination and reproduction tasks in the visual modality. A widespread observation, termed \u201coblique effect\u201d by Appelle (1972), is that the performance level is in general better when the stimulus is aligned with the vertical or horizontal orientation. The original motivation for studying the oblique effect in the haptic modality was to test possible explanations of the visual oblique effect. The existence of an oblique effect in the haptic modality would suggest that orientations are handled by the same processes in both modalities and, thus, would reinforce the view that high-level, possibly amodal (i.e., independent of sensorial modalities), processes could be responsible for the oblique effect. In contrast, its absence would indicate the existence of distinct orientation-processing streams in the haptic and visual modalities (cf. Gentaz 2000; Gentaz and Hatwell 2004; Heller 2000; Millar 1994). In this respect, we will show that orientation processing is generally anisotropic in the haptic modality for adults, children and even in infants. However, unlike the visual oblique effect where such anisotropy is almost always present, we will show that this effect depends on the gravitational cues and memory constraints that are very specific to haptics.\nThe second objective of this review is to identify the frame of reference associated with the haptic oblique effect. This question comes from the observation that the concept of orientation is by definition relative to a set of fixed axes, which define a coordinate system or frame of reference. For example, in the polar coordinate system, an orientation is defined by the angle between some features of the stimulus such as its main axis and an axis of the coordinate system. While the choice of this set of axes can be arbitrary, it is common to choose the vertical and its perpendicular (the horizontal) because of the permanent influence of the vertical gravity force on Earth. In fact, the concept of verticality is so dominant that even in a drawing put on a table that is on a horizontal plane, the verticality is projected as the line belonging to the sagittal plane and perpendicular to the axis of the body. However, the status of verticality must be questioned. In the visual modality, various studies have examined whether the oblique effect was tied to this geocentric reference frame or to a retinocentric frame of reference. In this respect, we will show that the haptic oblique effect observed in the frontal plane is defined in a subjective reference frame that combines ego and geocentric cues. We will also raise the question of which pair of angles is used to code the orientation of a stimulus in space, when the stimuli do not necessarily belong to a plane, where a single angle would suffice to code the orientation. As a matter of fact, it is important to note that different pairs of angles can be used to code an orientation in space in the same way as different coordinate systems can be used to code the position of a point in space.\nIn conclusion, we will argue that the haptic oblique effect occurs at a relatively late stage of orientation processing, when sensory motor traces associated with exploratory movements are transformed into a more abstract representation of the orientation, because the experimental factors that modify the haptic oblique effect's strength either contribute to establishing such a representation (e.g. gravitational cues) or favor its use (e.g., memory constraints). Finally, we will discuss various hypotheses about how the processes involved in the recoding of the sensory-motor traces might yield an anisotropy in the perception or recollection of orientations.\nHypotheses on the origins of the oblique effects\nNumerous studies have shown that vertically or horizontally oriented visual stimuli are generally perceived with greater precision than those oriented obliquely. While the existence of the visual oblique effect is well established, the explanations of this effect in vision are still debated today (e.g., Baowang et al. 2003; Essock 1980; Gentaz and Ballaz 2000; Gentaz and Tschopp 2002; Meng and Qian 2005; Westheimer 2003). Although orientation processing mainly involves the lower visual areas such as the Lateral Geniculate Nucleus (LGN) and the visual primary cortex (V1), it is completed at higher levels in the extra-striate and nonvisual areas (e.g., Goodale et al. 1991). The multitude of subcortical and cortical areas involved in orientation processing raises the question of where the processes responsible for the visual oblique effect specifically act in the anatomo-functional steps of visual processing. Two general hypotheses have been advanced to explain two types of oblique effect.\nThe first hypothesis has been in general advanced to explain the so-called \u201cClass 1 oblique effects,\u201d a term coined by Essock (1980), which are observed in tasks measuring the basic functional properties of the visual system (e.g., acuity and contrast). The hypothesis is that the visual oblique effect is generated at the lowest levels of the visual system, such as LGN and\/or V1 (for a discussion about the major role of V1, see Li et al. 2003). This hypothesis is supported by the very specific neurophysiological characteristics of orientation-selective neurons in these areas. In particular, it has been shown that neurons sensitive to the vertical and horizontal orientations are more numerous, sensitive and\/or narrowly tuned than neurons sensitive to the oblique orientations (e.g., Furmanski and Engel 2000; Li et al. 2003; Saarinen and Levi 1995). According to this hypothesis, the processes generating the visual oblique effect would therefore be specific to this modality.\nIn contrast, \u201cClass 2 oblique effects\u201d are observed in more cognitive tasks involving identifying, memorizing and categorizing orientations. Moreover, Class 2 oblique effects are influenced by factors, such as internal models (Morgan 1991), recognition (Heeley and Buchanan-Smith 1990) or attention (Shiu and Pashler 1992), that are not amenable to the characteristics of the early stages of visual processing. For example, the mere fact that an oblique effect is present in the reproduction of the orientation of two distant large dots is difficult to explain in terms of the properties of direction-sensitive neurons, since this stimulus in principle stimulates all orientations equally (Cecala and Garner 1986; Westheimer 2003). The second hypothesis is therefore that some oblique effects might occur not only at the low-level areas of the visual system such as LGN or V1 but also at higher level areas (from V2 to associative areas). This hypothesis suggests that the processes generating the visual oblique effect might not be specific to this sensory modality (Heeley et al. 1997). While some models of the Class 2 oblique effects make reference to neurophysiological mechanisms (e.g., McMahon and MacLeod 2003; Westheimer 2003), other explanations are formulated only in functional terms. For example, a widely shared idea is that orientations are represented in a reference frame having the vertical or horizontal orientations as principal axes at some later stage of visual processing. In this frame of reference, identification of these two orientations as principal axes can be carried out simply by identifying the stimulus with one of the two references axes, whereas the coding of oblique orientations would require the integration of information derived from both axes (Foster and Westland 1998; Gentaz and Hatwell 1996; Marendaz 1998; Regan and Price 1986). For example, the coding of oblique orientations might involve the computation of the ratio of the two values, which corresponds to the projections of the oblique on the references axes. According to this view, the oblique effect is caused by some additional processing needed to encode oblique orientations relatively to the reference axes. Alternatively, it has been suggested that all orientations are encoded with the same precision, but that participants have a remarkably accurate internal representation of the vertical and horizontal orientation, which can be used to produce the response when perceived orientation corresponds to these norms (Heeley et al. 1997). In other words, instead of assuming that some computation increases the error for oblique orientation, the higher level processes would bring additional information about the vertical and horizontal that decreases the variable error at the reference axes.\nAnother possibility, adopted by Huttenlocher et al., is that orientations are recorded in categorical terms (Huttenlocher et al. 1991, 1994; see also the work on Categorical Perception, Harnard 2003). According to the Category-Adjustment (CA) model, each orientation is categorized in one of the categories that partition the orientation space. This model was developed to explain the biases toward the diagonals observed in various position- and orientation-recall tasks (see also Spencer and Hund 2003; Haun et al. 2005). According to the CA model, the recall of an oblique orientation would be biased toward the closest diagonal because it would combine a low-level (presumably unbiased) representation of the orientation with the center of the category including the orientation, typically one of the four quadrants. In other words, the gist of the model is that the mental partitioning or labeling of the orientations into a limited number of categories is at the origin of systematic bias in the encoding and recall of an orientation. Finally, Spencer et al. have recently developed an alternative theoretical framework, the Dynamic Field Theory (DFT) of spatial cognition, to explain the presence of systematic biases in orientation-recall tasks (Spencer et al. 2006, 2007). The DFT is based on the dynamic properties of several topologically organized and interconnected layers of units and can provide insights about developmental processes such as the change in direction of the bias in orientation recall tasks between childhood and adulthood, and about real-time processes such as the increase of the recall bias with time. More specifically, the DFT includes a perceptual map that interacts with the environment, a working memory map and a long-term memory map. According to the DFT, repeated exposure to a reference axis results in the storage of this piece of information in the long-term memory, which can be at the origin of a shift of the stimulus position in the working memory even in absence of explicit contextual information in the environment. It is important to note that both the CA and DFT models were originally conceived to explain the results of experiments dealing with visual stimuli showing the presence of systematic biases toward the diagonals in various orientation or position recall tasks, a type of spatial anisotropy that, as we shall see, has not yet been studied in detail in the haptic modality. In the following section, we start to review experimental studies of the haptic oblique effect.\nIsotropic or anisotropic haptic perception of spatial orientations?\nExistence of an haptic oblique effect\nLechelt et al. (1976) were the first to show interest in the haptic oblique effect. The goal was to show the existence of an oblique effect in the haptic system analogous to the one observed in the visual system for the abovementioned reasons. To that end, they compared orientation perception in the visual and haptic systems by applying the same experimental paradigm to both systems. The visual and haptic stimuli were rods (30\u00a0cm\u00a0\u00d7\u00a00.6\u00a0cm), which could take one of six orientations: 0\u00b0, 45\u00b0, 90\u00b0, 135\u00b0, 225\u00b0, 315\u00b0. Adults were presented with two identical rods (a stimulus rod and a response rod) placed in the frontal plane, on either side and equidistant from their medial plane. In the visual condition, participants worked in a dark room with electroluminescent rods and gave verbal indications to adjust the response rod immediately after the stimulus rod had disappeared. In the haptic condition, participants were blindfolded and the response rod was adjusted with the contralateral hand immediately after the stimulus rod became unavailable.\nGlobally, the analysis of constant errors did not show any systematic deviation in either modality for all orientations. The analysis of absolute errors revealed a classical effect of modality: errors were greater in the haptic condition (from M\u00a0=\u00a04.8\u00b0 to M\u00a0=\u00a010.5\u00b0) than those in the visual condition (from M\u00a0=\u00a01.5\u00b0 to M\u00a0=\u00a04.2\u00b0). The effect of spatial orientations was significant and revealed an oblique effect in both modalities. In vision, the errors were lower for vertical and horizontal (M\u00a0=\u00a01.5\u00b0) than for the oblique orientations (M\u00a0=\u00a03.5\u00b0). In haptics, the errors were lower for vertical and horizontal (M\u00a0=\u00a05\u00b0) than for the oblique orientations (M\u00a0=\u00a09\u00b0). Lechelt and Verenka (1980) replicated these results with a slightly modified procedure, where the rod exploration time was fixed at 5\u00a0s for both perceptual conditions, where the delay between exploration and reproduction was set at 10\u00a0s, and where the orientations were no longer indicated by the experimenters before the test. The authors concluded that there was an oblique effect in the haptic system, analogous to that observed in the visual system.\nInitial hypotheses on the origin of the haptic oblique effect: the role of prior knowledge and the mode of reproduction of the orientation\nAppelle and Countryman (1986) questioned the abovementioned conclusions of Lechelt et al. (1976), and Lechelt and Verenka (1980). They argued that the haptic oblique effect might be induced by the prior visual perceptual experience and\/or the conditions of exploration\u2013reproduction movements. To show the possible effect of the first factor, the authors proposed two \u201cprior experience\u201d conditions: an \u201cinformed\u201d condition, in which standard orientations were verbally and visually presented to participants for as long as they wished; and an \u201cuninformed\u201d condition, in which participants did not know which standard orientations were being tested. On the basis of Appelle and Gravetter\u2019s (1985) results, they predicted that prior verbal or visual knowledge of standard orientations would lead participants to produce responses using internal orientation models as a reference (mainly the vertical and horizontal orientations), instead of the immediate haptic percept resulting from the exploration of the stimulus, and induce the haptic oblique effect. With respect to the second factor (conditions of exploration\u2013reproduction movements), these authors also argued that the haptic oblique effect observed in Lechelt et al.\u2019s experiments could be due to the use of the contralateral hand to reproduce the orientation. Indeed, when one hand explores the orientation of a standard rod and the other hand reproduces the same orientation on a test rod, the symmetrical disposition of the shoulder\u2013hand systems imposes nonhomologous movement patterns to explore and reproduce oblique orientations (agonist and antagonist muscles are mobilized differently). In contrast, exploration and reproduction movement patterns are homologous for vertical and horizontal orientations. Then, the authors proposed two exploration\u2013reproduction conditions: contralateral condition (one hand explores and the other reproduces) and an ipsilateral condition (the same hand explores and reproduces). In the latter case, the exploration\u2013reproduction movement patterns are homologous for all orientations and the oblique effect should not be present.\nHowever, Appelle and Countryman (1986) modified the experimental paradigm of Lechelt et al. (1976), and Lechelt and Verenka (1980). First, they used only four orientations (0\u00b0, 45\u00b0, 90\u00b0 and 135\u00b0). Earlier results justified this choice, as they showed that performances did not differ between 45\u00b0 and 225\u00b0 or between 135\u00b0 and 325\u00b0, for example. They introduced decoy orientations so that participants would not be aware that only four orientations were being tested. They presented the orientations in the horizontal plane (parallel to a table surface) instead of the frontal one. Finally, they fixed the delay between exploration and reproduction at 5\u00a0s. Absolute error analysis revealed that the performance on the oblique orientations, and therefore the amplitude of the haptic oblique effect, varied according to prior experience and exploration\u2013reproduction conditions. Vertical and horizontal orientations were stable. The extent of the haptic oblique effect was maximal in the informed contralateral condition (vertical\u2013horizontal: M\u00a0=\u00a05\u00b0 and obliques: M\u00a0=\u00a09\u00b0) and diminished in the uninformed contralateral condition (vertical\u2013horizontal: M\u00a0=\u00a05.5 and obliques: M\u00a0=\u00a07.5\u00b0). It was even smaller in the informed ipsilateral condition (vertical\u2013horizontal: M\u00a0=\u00a03.5\u00b0 and obliques: M\u00a0=\u00a05.5\u00b0). Finally, it was nil, and the oblique effect thus absent, in the uninformed ipsilateral condition (vertical\u2013horizontal: M\u00a0=\u00a04\u00b0 and obliques: M\u00a0=\u00a04.5\u00b0). In their study, Appelle and Countryman (1986) concluded that prior visual perceptual experience and exploration\u2013reproduction movements were responsible for the haptic oblique effect, contrary to the propositions of Lechelt et al. (1976), and Lechelt and Verenka (1980).\nRole of gravitational cues in the haptic oblique effect\nGentaz and Hatwell\u2019s (1995) study provides a test of Appelle and Countryman\u2019s (1986) hypothesis (second factor) regarding the existence of a difference between the one-handed exploratory movement patterns of an oblique orientation and the movement patterns needed to reproduce the oblique with the contralateral hand. As in Appelle and Countryman\u2019s (1986) study, the participants worked in a nonvisual and uninformed condition with a rod oriented at 0\u00b0, 45\u00b0, 90\u00b0 or 135\u00b0 and they reproduced the orientation of the standard rod after a 5-s delay. The standard rod was presented in the horizontal (as in Appelle et al.\u2019s experiment), frontal (parallel to the surface of a painting, as in Lechelt et al.\u2019s experiments) or sagittal (the median plane, perpendicular to the horizontal and frontal planes) planes. The task was performed ipsilaterally (with the same hand) or contralaterally (with the other hand). Given that, in the sagittal plane, the oblique orientation exploratory movements of one hand become homologous with those needed to reproduce the oblique orientations contralaterally; the oblique effect should also be absent in this plane if Appelle and Countryman\u2019s (1986) hypothesis (difference between exploration and reproduction movements) is determinant.\nAbsolute error analysis revealed that results did not concord with these predictions, because similar oblique effects were obtained in the frontal and sagittal planes under both ipsilateral and contralateral conditions (vertical\u2013horizontal: M\u00a0=\u00a05\u00b0 and 45\u2013135\u00b0 obliques: M\u00a0=\u00a08\u00b0). However, results in the horizontal plane replicated those of Appelle and Countryman (1986), i.e., an oblique effect was present in the contralateral condition (vertical\u2013horizontal: M\u00a0=\u00a08.9\u00b0 and 45\u2013135\u00b0 obliques: M\u00a0=\u00a012.9\u00b0) and absent in the ipsilateral one (vertical\u2013horizontal: M\u00a0=\u00a06.3\u00b0 and 45\u2013135\u00b0; obliques: M\u00a0=\u00a07.8\u00b0). Therefore, the presence or absence of an oblique effect does not appear to be due to the difference of movements between the two hands, but seems to be strongly linked to the type of spatial planes. To understand the results in the horizontal plane and, in particular, the lack of an oblique effect in the ipsilateral condition only, Gentaz and Hatwell (1995) asked blindfolded adults to symmetrically reproduce a previously presented orientation (a 45\u00b0 right standard oblique was to be reproduced at 45\u00b0 left and, similarly, a 45\u00b0 left standard oblique was to be reproduced at 45\u00b0 right) in the horizontal plane, either ipsilaterally or contralaterally. Given that the movements necessary for the exploration and the symmetrical reproduction of an oblique orientation become homologous in the contralateral condition and nonhomologous in the ipsilateral condition, the oblique effect should be present in the ipsilateral condition and absent in the contralateral condition if the Appelle and Contryman\u2019s hypothesis was valid. Results did not concord with these predictions, since an oblique effect was present in both reproduction conditions in the horizontal plane (vertical\u2013horizontal: M\u00a0=\u00a07\u00b0 and 45\u2013135\u00b0 obliques: M\u00a0=\u00a09.3\u00b0).\nTaken together, these results partially (see below) invalidate Appelle and Countryman\u2019s (1986) hypothesis about the role of exploration\u2013reproduction movements. Gentaz and Hatwell (1995) account for these results by suggesting that the haptic oblique effect was linked to the gravitational cues produced by the shoulder\u2013hand system. Indeed, the need to relatively move massive parts of the body such as the arm and forearm during the manual exploration of a stimulus brings antigravity forces into play. The role that gravity might play in the haptic system is clearly reduced in the visual system. The shoulder\u2013hand system is much more directly submitted to gravitational constraints than the ocular system. When it explores a stimulus, it has to produce antigravity forces and these, in return, provide what are known as \u201cgravitational cues.\u201d These gravitational cues provided by the proprioceptive system result from the deformation of cutaneous, muscular and articular tissues, and depend on the specific muscular forces needed to maintain or displace the shoulder\u2013hand system against gravity (Gentaz and Hatwell 1996). As such, these cues can provide a considerable amount of information on arm posture or motion, relative to some fixed \u201cgeocentric frame of reference\u201d (Paillard 1991) that is linked to gravity and exterior to the body.\nThis hypothesis was justified by an analysis of the task conditions proposed in the previous experiments. Indeed, the size of the stimulus rod (25\u00a0cm) and its position in relation to the participant (40\u00a0cm) required significant participation of the shoulder\u2013hand system during exploration. To explain the \u201cplane effect\u201d in the ipsilateral reproduction conditions observed above (i.e., the presence of an oblique effect in the frontal and sagittal planes and its absence in the horizontal plane), Gentaz and Hatwell (1996) hypothesized that participants favored gravitational cues in orientation processing, because these cues specify the gravitational vertical, which is used as a reference axis. The characteristics of these gravitational cues would differ according to exploratory conditions. In a normal situation, their average amplitude is practically the same in all three planes, since the arm is always influenced by the same gravitational forces. On the other hand, the variability of these cues is not the same depending on the plane the experiment is carried out in. There is a high variability in the frontal and sagittal planes, as the arm deploys considerable antigravitational forces when moving from bottom to top, but falls from top to bottom with little effort. On the contrary, in the horizontal plane, gravitational cues hardly vary during exploration as the arm constantly operates in a direction perpendicular to gravity.\nGentaz and Hatwell (1996) tested the hypothesis that the gravitational cues produced by the shoulder\u2013hand system during exploration were involved in the oblique effect and in the \u201cplane effect\u201d in the ipsilateral reproduction condition. The amplitude of these cues was modified by manipulating the level of gravitational constraint, and variability was modified by changing the plane the task was effectuated in. The oblique effect and the plane effect should diminish in conditions where gravitational cues are weak, and increase in those where these cues are greater. In one experiment, only the horizontal plane was tested. Participants explored the orientation of a rod with one hand and reproduced it with the same hand, either keeping their forearm\u2013wrist\u2013hand in the air (\u201cunsupported forearm\u201d condition), or resting their forearm\u2013wrist\u2013hand on the rod supporting surface (\u201csupported forearm\u201d condition). In the first condition, antigravitational forces were necessarily produced, whereas these forces were very much reduced in the second condition. Absolute error analysis revealed that the oblique effect was absent in the supported forearm condition (vertical\u2013horizontal: M\u00a0=\u00a04.7\u00b0 and 45\u2013135\u00b0 obliques: M\u00a0=\u00a04.2\u00b0) and was present in the unsupported forearm condition (vertical\u2013horizontal: M\u00a0=\u00a03\u00b0 and 45\u2013135\u00b0 obliques: M\u00a0=\u00a07\u00b0). In another experiment, blindfolded adults were tested in one of the three planes, either in a \u201cnatural\u201d condition, or in a \u201clightened\u201d condition in which gravitational cues had been reduced by a pulley system. In the latter case, the participant\u2019s forearm was attached to a display, which held it balanced in the air when the weight suspended on the pulley reached a certain value variable from one participant to another. The amplitude of the oblique effect was lower in the lightened condition (significant difference of 1.43\u00b0) than that in the natural condition (significant difference of 3.43\u00b0), and this is because that the accuracy of vertical and horizontal reproduction was deteriorated (thus increasing the difference with that of the oblique orientations, which remained stable). In addition, the exploratory plane had no effect. Taken together, these results revealed that gravitational cues are crucial when the haptic oblique effect is present in the three planes in blindfolded participants and in the ipsilateral reproduction condition.\nRole of previous visual experience and memory in the haptic oblique effect\nThe previous results do not, however, exclude an influence of high-level factors, such as visual experience or cognitive resources or both. As a matter of fact, it is well known that the haptic perception of the sighted working in a nonvisual condition can be fed by visual representations (Hatwell 1978; Thinus-Blanc and Gaunet 1997). Gentaz and Hatwell (1998) examined whether the participant\u2019s visual experience modified the action of the gravitational cues observed in sighted people. In this study, blindfolded sighted participants as well as early and late totally blind people were asked to explore a rod and to reproduce its orientation ipsilaterally. The magnitude of gravitational cues was modified by manipulating the level of gravitational constraints (natural and reduced) and their variability was modified by changing the task plane: horizontal (weak variability) and frontal (strong variability). By comparing the performances of the early and late totally blind, this study aimed at evaluating the role played by visual experience and visualization in the haptic oblique effect. If such is the case, the oblique effect should be present in the late blind and absent in the early blind. The comparison between blindfolded sighted and totally blind people, who are better-trained in the use of the haptic system, also allowed the authors to find out whether gravitational cues also played a role in the apparition of the haptic oblique effect in the blind as is the case with blindfolded persons. In this case, the oblique effect should be absent when cues are reduced and present when they are normal. Absolute error analysis showed that in the horizontal plane, the oblique effect was absent in both the early and late blind, regardless of gravitational constraints (vertical\u2013horizontal: M\u00a0=\u00a07.9\u00b0 and 45\u2013135\u00b0; obliques: M\u00a0=\u00a010\u00b0). In the frontal plane, the oblique effect was present in both groups of blind people, regardless of gravitational constraints (vertical\u2013horizontal: M\u00a0=\u00a05.8\u00b0 and 45\u2013135\u00b0; obliques: M\u00a0=\u00a09.9\u00b0). Furthermore, no difference was observed between the early and the late blind. In conclusion, these results confirmed that the variability of gravitational cues plays an important role in the presence or absence of the haptic oblique effect in the totally blind, although no visual experience effect was observed [first factor of Appelle and Countryman\u2019s (1986) hypothesis].\nGentaz and Hatwell (1999) examined further the role of attentional and cognitive resources on the haptic perception of orientations and the oblique effect by increasing memorization constraints. We know that the characteristics of the haptic system differ in fundamental ways from those of the visual system. The limited size of the tactile perceptual field requires that it be moved to gain knowledge about, for example, the shape of an object. In contrast, eye movements play only a secondary role in the visual perception of the position or shape of an object, since both pieces of information can be extracted from static images. This difference was recently re-emphasized by Henriques and Soechting (2005) who argued in a recent review that \u201cthe processing of haptic information differs fundamentally from visual processing in that the former requires the integration of information that evolves in time as well as space.\u201d It results therefore from this quality of proximal reception of the haptic system that haptic processing and haptic perception are consistently more sequential than visual ones. Moreover, haptic perception needs to rely more heavily on working memory to achieve the mental synthesis that is necessary to gain an unified representation of the object (Revesz 1950; cf. Hatwell et al. 2003).\nIn all previous research on the haptic perception of orientations, the delay between the stimulus exploration phase and the reproduction phase was 5-s long and was unoccupied. In Gentaz and Hatwell (1999), memorization conditions were thus altered\u2014the length and the nature of the gap was changed in the two exploratory conditions showing that the availability of gravitational cues affected orientation coding. Participants explored a rod in the horizontal plane with weak or natural gravitational cues and reproduced the rod\u2019s orientation ipsilaterally. The orientations were reproduced according to one of four memorization conditions: after unoccupied 5-s gaps or 30-s gaps and 30-s gaps occupied by interfering verbal or motor activities such as reciting the alphabet forwards or backwards (verbal) or haptically exploring a raised sinuous trajectory (motor). Absolute error analysis showed that when the gap was unoccupied (regardless of how long it was), haptic orientation treatment and the oblique effect depended on the exploratory conditions: the oblique effect was absent when gravitational cues were weak (vertical\u2013horizontal: M\u00a0=\u00a05.5\u00b0 and 45\u2013135\u00b0 obliques: M\u00a0=\u00a05.5\u00b0) and present when they were natural (vertical\u2013horizontal: M\u00a0=\u00a03.85\u00b0 and 45\u2013135\u00b0 obliques: M\u00a0=\u00a06.8\u00b0), like in Gentaz and Hatwell (1995, 1996). On the other hand, when the 30-s gap was occupied by interfering verbal or haptic activities, the haptic oblique effect was present in both experiments and regardless of available gravitational cues (vertical\u2013horizontal: M\u00a0=\u00a05.2\u00b0 and 45\u2013135\u00b0; obliques: M\u00a0=\u00a08.5\u00b0). Taken together, these results showed that high-level factors can indeed determine the presence of the haptic oblique effect, since the reduction of available attentional or cognitive resources by an interfering task caused an oblique effect in a condition in which it was not usually observed.\nIn conclusion, the presence of a haptic oblique effect in blindfolded sighted participants, as well as in the totally early blind, with natural exploration and ipsilateral reproduction in the frontal plane (regardless of gravitational constraints) invalidates Appelle and Countryman\u2019s (1986) hypothesis about the role of visual representations and shows the existence under very precise conditions of an oblique effect intrinsic to the haptic system (Table\u00a01).Table\u00a01Average absolute errors in the studies of the haptic oblique effect with blindfolded adultsExperimental conditionsOrientationsOblique effectReferencesTasksPlaneDelay (s)HandV\/HObliqueDifferenceReproduction task\u2002Free-time exploration and haptically informed about standard orientationsF0contra594*Lechelt et al. (1976)Reproduction task\u2002Limited-time exploration (5\u00a0s) and haptically informed about standard orientationsF0contra48.14.1*Lechelt and Verenka (1980)F10contra47.53.5*Production task\u2002Free-time production and verbally informed about orientationsF0one3.28.35.1*Appelle and Gravetter (1985)\u2002Free-time and haptically informed about orientationsF0one3.58.14.6*Reproduction task\u2002InformedH5Contra594*Appelle and Countryman (1986)\u2002UninformedH5Contra5.37.52.2*\u2002InformedH5ipsi3.55.52*\u2002UninformedH5ipsi44.50.5Reproduction task\u2002Free exploration and uninformed about orientationsF5contra583*Gentaz and Hatwell (1995)F5ipsi583*S5contra583*S5ipsi583*H5contra8.912.94*\u2002Supported explorationH5ipsi6.37.81.5\u2002Symmetric orientationH5contra79.32.3*\u2002Symmetric orientationH5ipsi79.32.3*Reproduction task\u2002Unsupported explorationH5ipsi374*Gentaz and Hatwell (1996)\u2002Supported explorationH5ipsi4.74.2\u22120.5\u2002Normal gravity cuesF5ipsi3.58.24.7*\u2002Reduced gravity cuesF5ipsi6.17.21.1*\u2002NormalS5ipsi3.67.14.5*\u2002ReducedS5ipsi5.77.31.6*\u2002NormalH5ipsi5.47.31.9*\u2002ReducedH5ipsi7.691.4*Reproduction task Early and late blind Pooled\u2002NormalF5ipsi5.89.94.1*Gentaz and Hatwell (1998)\u2002ReducedF5ipsi5.89.94.1*\u2002NormalH5ipsi7.9102.1\u2002ReducedH5ipsi7.9102.1Reproduction task\u2002Normal, unoccupied delayF5ipsi3.856.82.95*Gentaz and Hatwell (1999)\u2002Reduced, unoccupied delayF5ipsi5.55.50\u2002Normal, unoccupied delayF30ipsi3.856.82.95*\u2002Reduced, unoccupied delayF30ipsi5.55.50\u2002Normal, verbal taskF30ipsi5.28.53.3*\u2002Reduced, verbal taskF30ipsi5.28.53.3*\u2002Normal, motor taskF30ipsi5.28.53.3*\u2002Reduced, motor taskF30ipsi5.28.53.3*Reproduction task\u2002UprightF5ipsi2.55.53*Luyat et al. (2001)\u2002Body tiltedF5ipsi55.50.5Production task\u2002No context, smoothF5ipsi2.36.44.1*Luyat et al. (2005a, b)\u2002Congruent haptic cuesF5ipsi1.43.42*\u2002Incongruent haptic cuesF5ipsi1.84.83.05*The table reports the average absolute error for the vertical and horizontal orientations (V\/H column), for the 45\u00b0 and 135\u00b0 orientations (Oblique column), as well as the existence of an oblique effect, that is, of statistically significant difference between the two sets of orientation (marked by * in the Difference column) as function of experimental conditions. The reported studies mainly used a reproduction paradigm where the subject explored the orientation of a rod with one hand before to be reproduced with the same (ipsilateral condition) or other hand (contralateral condition, see Hand column). The Plane and Delay column specify the plane in which the orientations were presented and reproduced or produced as well as the delay between the presentation and reproduction. The first column specifies additional experimental factors that were manipulated in the corresponding studies (see text)\nHaptic orientations defined in a subjective reference frame\nAs previously indicated, the concept of orientation is by definition relative to one or more systems of coordinates with the cardinal vertical and horizontal orientations as norms of this system. However, on Earth, different verticals (gravitational, egocentric), which can constitute the norms for independent references, exist. Thus, the existence of an oblique effect raises the question of which kind of vertical is better perceived or in other words in which reference frame this effect is defined. A classical broad distinction is made between egocentric (referred to the participant\u2019s body) and allocentric (referred to environmental cues) spatial frames (Howard 1982; Rock 1990). The allocentric reference frame can be divided into a gravitational frame defined by the direction of the gravity pull (geocentric frame) and in pattern-centric reference frames defined by (visual or haptic or both) contextual cues. In natural conditions, the different reference frames are in general congruent. Consequently, mapping of orientations could result from either egocentric or allocentric reference frames or most likely from both. In darkness, tilting the body uncouples the gravitational and the egocentric reference frames and allows us to specify in which kind of reference frame orientations are defined.\nSubjective reference frame\nIn vision, certain studies have evidenced rather an egocentric (retinocentric) coding (Banks and Stolartz 1975; Chen and Levi 1996; Corwin et al. 1977), suggesting that the visual oblique effect could principally be accounted for by the properties of the orientation-selective neurons present in the primary visual cortex (see Introduction). Indeed, neurons tuned to vertical and horizontal retinal orientations are more numerous and feature particular response characteristics in relation to those tuned to oblique orientations. On the other hand, other studies have revealed the existence of gravitational orientation coding, implying that the origin of the oblique effect is probably more central, involving extraretinal information such as vestibular and somesthetic cues integrated at a higher level in the visual processing hierarchy (Attneave and Olson 1967; Buchanan-Smith and Heeley 1993; Ferrante et al. 1995; Lipshits and McIntyre 1999). However, very little research has been done concerning this topic in other perceptual modalities. In haptics, Luyat et al. (2001) tested the effect of tilting the body or the head on the haptic oblique effect. Blindfolded participants explored a standard rod in the frontal plane and reproduced its orientation with the same hand after a 5-s delay. Three conditions were examined: upright, inclined to the right (+45\u00b0) and to the left (+135\u00b0). Five orientations were tested: vertical, horizontal, 45\u00b0 oblique, 135\u00b0 oblique and the subjective vertical (SV). The SV is the individual\u2019s perception of the direction of gravitational force (cf. Howard 1982; Luyat 1997). The most commonly used paradigm consists of adjusting a stimulus (a rod) to the physical gravitational vertical. The deviation from the gravitational direction constituted a measure of the subjective vertical. In upright posture, the SV is very close to the physical vertical in participants with no vestibular disease or parietal lesion. However, in darkness, lateral head or body tilt provokes systematic deviations of the haptic SV in the opposite direction to that of the head: the M\u00fcller effect. A first experimental phase estimated each participant\u2019s SV in each postural condition and this SV was then tested in the exploration\u2013reproduction task. What is more, as an insurance against a possible fluctuation of the SV in the haptic perception of orientation, the SV was measured again after the exploration\u2013reproduction task.\nIf the haptic oblique effect is defined in a gravitational reference frame, then the reproduction of the vertical and the horizontal should be more accurate than the reproduction of oblique orientations, regardless of postural conditions. On the other hand, if the haptic oblique effect is defined in an egocentric reference frame, then the reproduction of the two diagonals (45\u00b0 and 135\u00b0), which are parallel and perpendicular to the body tilted to 45\u00b0 or 135\u00b0, should be more accurate than the reproduction of the gravitational vertical and the horizontal, which become oblique in relation to the body. Finally, knowing the effect of tilting the body on the vertical, the SV could constitute a reference axis when the body is tilted. In this case, the reproduction of the SV should be more accurate than all the other orientations in the tilted body conditions.\nThe results observed in the production task in which the participants had to manually orient the bar in the direction that they thought was vertical (phase 1, i.e., before the reproduction task and phase 2, i.e., after the reproduction task) showed systematic deviations of the haptic SV in the direction opposite to that of the tilted body (M\u00fcller effect). The comparison of phases 1 and 2 showed that SV perception was stable and faithful over time, thus justifying its study in the exploration\u2013reproduction task. Absolute error analysis in the exploration\u2013reproduction task showed that tilting the body affects the precision of reproduction, particularly of the gravitational vertical and the horizontal. Thus, an oblique effect was present in the upright condition (vertical\u2013horizontal: M\u00a0=\u00a02.5\u00b0 and 45\u2013135\u00b0; obliques: M\u00a0=\u00a05.5\u00b0), but absent in tilted conditions (vertical\u2013horizontal: M\u00a0=\u00a05\u00b0 and 45\u2013135\u00b0 obliques: M\u00a0=\u00a05.5\u00b0). It should be noted that the same results were observed in a complementary experiment in which only the head was tilted.\nIn conclusion, tilting affected vertical perception in the production task and also in orientation reproduction. This means that, in a tilted body condition, the gravitational vertical and horizontal orientations no longer appear to act as reference norms, as they were not better reproduced than the oblique orientations. Consequently, the hypothesis of a pure gravitational reference frame underlying the haptic oblique effect can be rejected. In the same way, the results did not confirm the hypothesis of a pure egocentric reference frame, as 45\u00b0 (or 135\u00b0) oblique orientations (which are vertical or horizontal in relation to the body) were not better reproduced than the gravitational vertical and the horizontal. On the other hand, results showed that the SV was better reproduced than the other four orientations, particularly in the tilted body conditions (M\u00a0=\u00a02.7\u00b0). In these conditions, the SV could constitute a norm for a subjective vertical reference frame.\nFinally, these results questioned the nature of the processes responsible for the oblique effect across sensory modalities (Hatwell 1994; Thinus-Blanc and Gaunet 1997). Several studies in the spatial domain show that visual representations largely feed tactile representations and vice versa. Thus, Luyat and Gentaz (2002) examined whether the visual oblique effect is also coded in a subjective gravitational reference frame, analogous to that used in haptics. The effect of inclining the whole body on the visual oblique effect was studied with the same paradigm as that proposed in haptics. The classic oblique effect observed in the upright body condition disappeared in the inclined body conditions because of a reduction in the accuracy of the reproduction of the gravitational vertical and the horizontal. In inclined conditions, the subjective vertical appears to be the best perceived orientation. Thus, the visual oblique effect seems also to be defined within a subjective vertical reference frame. However, results in the production task showed that inclining the body produces systematic deviations to the visual subjective vertical (VSV) in the direction of the body incline axis, whereas deviation is in the direction opposite to body inclined in the haptic modality. In another study, Gentaz et al. (2001) examined the oblique effects in the visual, haptic and somato-vestibular systems by asking adults to reproduce (after a 10-s delay) an orientation presented in the frontal plane. In the visual modality, participants reproduced the orientation of a luminous rod presented in a dark room. In the haptic modality, blindfolded participants explored a rod with one hand and reproduced its orientation ipsilaterally. In the somato-vestibular modality, blindfolded participants reproduced the incline of their body. Results revealed similar oblique effects in the three tasks. However, no significant correlation between the magnitude of the visual, haptic and somato-vestibular oblique effects was observed. Taken together, these results suggest the existence of both processes specific to the haptic system (different subjective reference frames in haptics and in vision) and processes similar to the corresponding visual processes (access to a subjective reference frame) but not common across sensory modalities (absence of correlations between modalities). Let us now look at the effect of contextual cues on the haptic perception of orientation in adults by comparing it to the corresponding visual one.\nHaptic contextual reference frame\nIn previous experiments, the stimulus consisted of a single rod without any contextual cues around it. In this case, it was not possible to determine whether the orientation perception in natural conditions could also be coded (at least partially) in a pattern-centric reference frame (defined by contextual cues). In vision, it is well known that spatial orientation is enhanced when vertical contextual cues are available, and as a consequence, it is strongly affected by roll or pitch of single tilted lines or by a more structured context such as a room (Groen et al. 2003; Luyat 1997). Recently, Luyat et al. (2005a) showed that the oblique effect decreased with visual contextual cues tilted 15\u00b0.\nAnother characteristic of the haptic system is that the shape and size of the tactile perceptual field can vary according to the mode of exploration adopted by the observer (e.g., with one or several fingers). This observation stands in sharp contrast with the visual system where the shape and size of visual field are essentially invariant. The ability of the haptic system to perceive contextual cues might vary drastically in accordance with the number of contacts that are established with the environment. For example, two-handed exploration might enlarge the haptic perceptual field sufficiently to facilitate the perception of contextual cues (on the role of reference cues, cf. Millar 1994; Millar and Al-Attar 2002).\nThe haptic perceptual field remains greatly reduced compared to the visual field even when the two hands are used with active exploratory movements. As a result, the perceptual cues, which are relevant in a task (or useful as a spatial exocentric reference frame), are less available in the haptic than in the visual modalities. Moreover, voluntary movements must be made to compensate for the smallness of the haptic perceptual field. As a result, haptic perception is highly sequential (cf. \u201cIntroduction\u201d). However, this latter property permits contextual effects to be reduced in haptic perception compared to visual perception. Indeed, it is not the case in vision because of the presence of simultaneous peripheral (contextual cues) and central (the target) stimulation.\nThe effect of contextual cues on the perception of orientations was investigated first by Walker (1972) in a short report in which the participant had to position a rod to the vertical while passing the right hand through a tilted grid. This author reported an effect, but unfortunately both precise data and important methodological aspects as the amount of tilt used were not provided. More recently, Luyat et al. (2005b) studied this question by asking blindfolded participants to position a rod with their right dominant hand to three different spatial orientations in their fronto-parallel plane: to the vertical and along two oblique orientations, respectively, at 45\u00b0 to the left and to the right of the vertical one. This production task was carried out in four different backgrounds: (1) in the absence of contextual cues (smooth), (2) in presence of congruent contextual cues (stripes parallel to the orientation to be produced), (3) in presence of tilted stripes context to the left and (4) in presence of tilted stripes context to the right of the orientation to be produced. Using at the same time, the right and left hands with one hand exploring the context and the other (dominant hand) adjusting the stimulus-rod to the expected orientation allows a simultaneous stimulation by both the context and the stimulus.\nThe analysis of the precision of adjustments (variable errors) revealed that the oblique effect obtained in the control condition (no context) (vertical: M\u00a0=\u00a02.3\u00b0 and 45\u2013135\u00b0 obliques: M\u00a0=\u00a06.4\u00b0) was similar to that obtained previously by Gentaz et al. (2002) with a similar method of direct estimation of orientations (production task). Indeed, most experiments on the haptic oblique effect have been carried out with a reproduction task, which requires memorizing an unknown but previously scanned orientation. This experiment confirmed that a haptic oblique effect in the fronto-parallel plane can be evidenced with a production task, which involves a cognitive representation of orientations probably based on an internal model of gravity. More interesting is the fact that tactile contextual cues had an influence on the production of oblique and vertical orientations, particularly on the oblique effect. When the context was congruent with the standard orientations, the precision of adjustments was significantly improved (vertical: M\u00a0=\u00a01.8\u00b0 and 45\u2013135\u00b0 obliques: M\u00a0=\u00a04.85\u00b0), an effect very similar to the effect exerted by visual vertical cues on spatial orientation. During noncongruent context scanning, the precision of orientations, particularly the obliques, also tended to be enhanced and, as a consequence, the oblique effect was significantly weakened. However, this improvement does not mean that the accuracy was enhanced, since analysis on constant error revealed that deviations erred in the direction of the stripes. Thus, the presence of a context, congruent or not, diminishes the variability of estimations and therefore enhances the precision of the production of orientations. Furthermore, the mismatch between the orientation to be produced and the context (noncongruent condition) revealed a tendency to adjust the rod in the direction of this context.\nIn conclusion, these results suggest that the haptic oblique effect is defined in a subjective reference frame, which may integrate not only vestibular and somesthetic inputs (head or whole body tilt) but also haptic contextual cues according to task conditions. Moreover, it should be emphasized that tasks such as pointing to remembered visual targets have also been found to involve processing information in multiple reference frames (see Carrozzo et al. 2002; McIntyre et al. 1997a, b). We propose that the subjective vertical and the subjective horizontal (probably) constitute the norms of this subjective vertical reference frame. At the neurophysiological level, the questions of how and where vestibular or contextual information dedicated to spatial cognition (specifically to the subjective perception of \u201cwhat is up\u201d) is processed in the cortex is far from perfectly known. Research in humans with functional magnetic resonance imaging (fMRI) (Fasold et al. 2002) or electrical stimulation in patients with epilepsy (Kahane et al. 2003) have shown several vestibular cortex areas, in particular insulo-parietal (analogous to the Parieto-Insular Vestibular Cortex in monkeys) and temporal areas with a right hemispheric dominance. The integration of gravitational and contextual cues in the oblique effect suggests that these associative areas with multimodal neurons could be implicated. A last question raised in current haptic studies is the presence of the haptic oblique effect in 3D space in adults.\nHaptic orientation in 3D space\nIn all previous experiments, we considered the haptic perception of orientations on a plane rather than in space. An orientation in space is defined by two parameters such as its azimuth and elevation. This observation raises the questions of whether the haptic oblique effect is still present in the absence of any spatial constraint when the participant needs to focus on at least two independent parameters to perform the task. More importantly, given the fact that there is a much larger set of alternative reference frames, which can be used to code an orientation in space than on a plane, the perception of orientations in 3D space raises the question of which reference frame is most adequate to describe the pattern of errors.\nTo examine this question, Baud-Bovy and Gentaz (2006) used a haptic display to present the stimuli and record the participant\u2019s hand movements to remove, if desired, the usual planar constraint imposed by the experimental apparatus used in most prior haptic studies. In a first experiment, the haptic reproduction of vertical, horizontal and diagonal orientations on a plane and in space was compared. Thus, participants were asked to explore the orientation of a \u201cvirtual rod\u201d with to-and-fro movements on the frontal plane and then they were asked to respond in two different conditions. In the 2D reproduction condition, the finger movements were constrained to the frontal plane like in the exploration phase. In the 3D reproduction condition, the movements of the finger were unconstrained and could move freely in space. The first finding was that the vertical and horizontal orientations were better reproduced than the diagonal orientations in the 3D condition. Thus, removing the constraint during the reproduction phase did not prevent the occurrence of the haptic oblique effect. Decomposing the angular error in the 3D condition in an in-plane component and in an out-of-plane component revealed that the anisotropy in this condition concerned mostly the in-plane component. As a matter of fact, the error pattern in the 2D condition was very similar to the in-plane component of the 3D condition, while the analysis of out-plane errors showed that their distribution was much more uniform across orientations. This finding suggests first that an orientation in space is coded by two parameters (i.e., the angle inside the frontal plane and the angle between target orientation and the frontal plane) and second, that these two parameters are processed independently.\nTo examine how orientations are coded in space in the absence of any sort of reference to a plane, Baud-Bovy and Gentaz (2006) tested thirteen target orientations, which belonged to different planes (three principal axes\u2014vertical, sagittal and lateral axes, the two 45\u00ba and 135\u00ba diagonals in the frontal, sagittal and horizontal plane, respectively, and four \u201c3D diagonals\u201d). As previously, blindfolded participants were asked to explore the orientation of a \u201cvirtual rod\u201d with to-and-fro movements and to respond in the 3D condition, i.e., without any constraint on the finger movements during the reproduction phase. The haptic processing of orientations was clearly anisotropic. The vertical orientation was reproduced most precisely. Once again the accuracy and consistency with which participants reproduced this orientation was striking. For the other orientations, the error pattern depended in a complex manner on participants, stimulus and error components, which makes its interpretation complex.\nOn the one hand, the three principal axes were most accurately reproduced when all error components were pooled together. This observation has a straightforward consequence in a \u201cplane-by-plane\u201d analysis, as it implies that the horizontal and vertical axes in the frontal and sagittal planes, as well as the lateral and sagittal axes in the horizontal plane, are more accurately reproduced than the diagonal orientations. In other words, this observation is akin to a demonstration of the classic oblique effect in each one of these planes. Regarding the frontal plane, the results of this experiment were similar to those of the previous experiment. The analysis of the different error components showed a clear oblique effect in both experiments, with the vertical and horizontal orientations being most accurately reproduced, even in the absence of any planar structure in the design of the experiment and of any constraint during the reproduction phase. On the other hand, the presence of the classical oblique effect was much less obvious when we considered the various error components separately. The average angular error inside the horizontal and sagittal planes did not exhibit the expected pattern. Decomposing the in-plane errors in systematic, intersubject and intrasubject error components confirmed this negative finding. Further studies are needed to understand these complex patterns of results observed in 3D space. Let us now look at the ontogenetic development of the haptic oblique effect by comparing it to the corresponding visual one.\nDevelopmental aspect of the oblique effects\nThe oblique effects in children and infancy\nIn vision, Leehey et al. (1975) tested the oblique effect in infants aged from 6 to 50\u00a0weeks using contrast sensitivity. The stimulus was a circular grating consisting of light and dark bars uniformly alternated. Bars were of five different widths. When spatial frequency was above threshold discrimination, the pattern appeared contrasted, whereas it looked like a homogeneous gray field when spatial frequency was below threshold. Two gratings of the same spatial frequency differing in orientations were simultaneously proposed. Each trial consisted of an oblique grating (45\u00b0 or 135\u00b0) and a vertical or a horizontal grating presented in a random fashion. As infants looked longer at the more contrasted of two patterns, a forced-choice judgment was used to determine which grating the young subject preferred. Given that the two stimuli have the same spatial frequency, an oblique effect would be present if, for a specific bar width, infants could discriminate the vertical or the horizontal lines but not the oblique lines. For this particular spatial frequency, infants should look preferentially at vertical or horizontal gratings rather than at oblique gratings. Results revealed an oblique effect. If the critical value of spatial frequency progressively decreased with age according to the development of visual acuity, an oblique effect was systematically observed at all ages. These findings were replicated by Gwiazda et al. (1978, 1984). According to Held and his colleagues, findings suggest that the visual oblique effect depends on endogenous maturation rather than exposure to a carpentered world, as assumed by other authors (Annis and Frost 1973; Ross 1992). Annis and Frost (1973) studied the oblique effect in Euro-Canadians raised in a carpentered environment characterized by the preponderance of cardinal contours, as well as in Cree Indians living in a traditional setting presenting more heterogeneous arrays of orientations. A classical matching task was used, subjects having to turn a test-rod until it matched the given standard-orientation. The four standard axes were tested: vertical, horizontal and the two diagonals. Prior knowledge of the tested orientations was given. The expected oblique effect was observed in Euro-Canadians, but it was absent in Cree Indians. In the same vein, Ross (1992) showed that its extent increases in an almost linear fashion between the ages of 7 and 12 years.\nIn haptics, the oblique effect has been studied in school-age children and in infants. In children (from 6\u00a0years old to 10\u00a0years old), results showed that the oblique effect may be present under certain conditions (like in adults), and that its extent did not change (Gentaz and Hatwell 1995). In infancy, Gentaz and Streri (2002) initially examined whether the 5-month-old babies are capable of haptically discriminating (without visual control) between a vertical rod and a 45\u00b0 oblique rod positioned in the fronto-parallel plane. The authors offered each baby a 90-s familiarization phase for one oriented rod. After this phase, a discrimination phase was proposed, in which the two orientations were alternately presented. By definition, haptic discrimination was said to occur when there was a significant difference between manual holding times in the discrimination phase for the familiar orientation (the one proposed in the familiarization phase) and for the novel orientation (for a discussion about the direction of preference, see Kerzerho et al. 2008). Results confirmed these predictions and thus showed that 5-month-old babies were capable of haptically discriminating between two orientations (vertical and 45\u00b0 oblique).\nThen, Gentaz and Streri (2004) studied whether a haptic oblique effect is present from the age of 5 months. To answer this question, the authors used a method based on a critical angular value, as did Leehey et al.\u2019s works in vision in babies. A haptic oblique effect is said to be present when the discrimination between two orientations does not only depend on their angular difference but also on the value of the tested orientation. In this case, the authors predicted that with the same angular difference (10\u00b0), discrimination should be observed between the vertical and a 10\u00b0 oblique orientation, whereas no discrimination should be observed between a 55\u00b0 oblique and a 45\u00b0 oblique. Results confirmed these predictions. These results showed that an oblique effect may be present when the shoulder\u2013arm\u2013hand system of the infants from the age of 5 months could actively move freely in the air to hold and to slightly explore the rod positioned in the frontal plane with the finger movements. This effect could be explained partially by the presence of the gravitational cues provided by the arm\u2013hand system. These cues could reinforce gravitational (vertical) direction as an important axis, which could be used by infants as a reference axis to define spatial orientations, as it is the case in children and in adults (Gentaz and Hatwell 1996).\nSubjective-reference frame in infancy\nAs in adults, the effect of body tilt on the oblique effect was investigated in the visual and haptic systems in infants. In vision, Jouen (1985) showed a classical oblique effect in the upright body position of 5-month-old infants, with longer looking at the vertical and horizontal gratings than at the 45\u00b0 and 135\u00b0 gratings. In contrast, in the tilted body position, infants looked longer at oblique stimuli aligned with their body orientation. These results showed that the visual oblique effect in infants is not defined in a gravitational reference frame but rather in a retinotopic reference frame. Consequently, this suggests that this effect would be directly related to the properties of orientation-selective neurons of the low-levels of the visual system where the retinotopical mapping of most orientation-selective neurons is preserved (cf. \u201cIntroduction\u201d).\nIn the haptic modality, Kerzerho et al. (2005) investigated the effect of whole body tilt on the haptic oblique effect by using the same method as Gentaz and Streri (2004). Two postural conditions were tested: upright body and body tilted 20\u00b0 to the left. Two orientations (vertical or +20\u00b0-left oblique), defined gravitationally, were proposed in the familiarization phase and four (vertical, 10\u00b0, 20\u00b0 or 30\u00b0-left oblique) in the test phase. The results showed that the body tilt had an effect on the infant\u2019s haptic discrimination of orientations. The understanding of this effect was complex, because it acted on both the spatial orientation discriminated and the direction preference of holding times. The pattern of results observed in the upright body position\u2014the presence of a discrimination between vertical and 10\u00b0-left-oblique rods and a failure between 20\u00b0-left oblique and 30\u00b0-left oblique rods\u2014confirmed Gentaz and Streri\u2019s (2004) previous observations and were in line with those found in vision by Held and his colleagues. However, the pattern of results observed in the tilted conditions showed the presence of discrimination between vertical and 10\u00b0-left-oblique rods and between 20\u00b0-left oblique and 30\u00b0-left oblique rods. In other words, the haptic oblique effect found in the upright posture disappeared when the body was tilted.\nThis effect of the body tilt on the patterns of results clearly showed that the hypothesis of a purely gravitational reference frame underlying the haptic oblique effect is not supported. Similarly, the results do not favor a purely egocentric reference frame, since a successful haptic discrimination was observed in the body tilted condition both (1) between the 20\u00b0 oblique rod that was vertical by reference to the body axis in the 20\u00b0 tilted position and the 30\u00b0 oblique rod and (2) between the gravitational vertical rod and 10\u00b0 oblique rod. In summary, the results suggest that spatial orientations are not defined only in a single reference frame. The results indicated that the haptic discrimination of the gravitational vertical (with the 10\u00b0 oblique) occurred in the two body positions. This result suggests that in tilted position, gravitational vertical seemed still to play the role of a reference axis at least partially. In contrast, the haptic discrimination of the 20\u00b0 oblique rod depended on the body position. It was absent in the upright position when the 20\u00b0 oblique rod was oblique by reference to the body axis, whereas it was present in the 20\u00b0 tilted body position when the 20\u00b0 oblique rod was vertical by reference to the body axis. This result suggests that, in the tilted position, the egocentric vertical also seemed to have the role of a reference axis, at least partially.\nIn conclusion, these results support the hypothesis of a reference frame, which integrates not only gravitational information but also egocentric information. As indicated before, this mixed-reference frame hypothesis underlying the orientation perception has already been proposed in the haptic (Kappers 2004) and visual (Lipshits et al. 2005; Lipshits and McIntyre 1999; Luyat et al. 2005a) systems in adults.\nGeneral discussion and perspectives\nThis review of experimental studies of the haptic oblique effect has showed that the perception of spatial orientations can be isotropic or anisotropic depending on various factors such as the presence of gravitational cues, the plane in which orientations are presented, the modality of response (ipsilateral or contralateral hand), etc. One of the most striking findings is the absence of an oblique effect in some conditions, which demonstrates that the haptic system can process all orientations isotropically. This observation stands in sharp contrast with vision where an oblique effect is observed much more systematically, probably because some form of directional anisotropy already emerges at the lowest levels of the visual system.\nOur general interpretation of these studies is that the haptic oblique effect is a classic Class 2 oblique effect, which occurs relatively late in the processing of sensory information. More precisely, we believe that these studies globally indicate that the oblique effect occurs when the sensory motor traces associated with the exploratory movements are transformed into a more abstract representation at the cognitive level, where presumably the perceived orientation is related to a frame in reference that favors the vertical and horizontal orientations. While the high-level representation would introduce some degrees of anisotropy, it would also be less taxing from a memory point of view and be more robust to motor interference tasks. In this framework, gravity would facilitate the transformation from the low-level to the high-level representation by adding information about the direction of the movement relative to the vertical in the sensory-motor traces. In absence of these cues, the oblique effect would be smaller or absent, because the axes of the reference frames would be known with less certainty. In addition, studies conducted in the haptic and visual modalities where the geo-centered and ego-centered reference frames was dissociated by tilting the body have shown that the axes of this reference frame were influenced by vestibular and somesthetic inputs. Thus, these findings suggest that orientations might be represented in a frame of reference based on the subjective vertical and horizontal, that mixes ego- and geo-centric cues. While it is tempting to postulate that this subjective reference frame is shared between the two sensory modalities, a detailed analysis of the results revealed a lack of correlation between the sizes of the oblique effect obtained in each modality (Luyat et al. 2001; see detailed review in \u201cHaptic orientations defined in a subjective reference frame\u2019\u2019). Additional studies are necessary to determine if this lack of correlation reflects only a difference of degree of anisotropy present in the visual and haptic processing of orientations or if it is due to a different positioning of the subjective axes in the modalities. In either case, the absence of a purely gravitational or egocentric frame of reference is a strong argument in favor of a high-level haptic oblique effect.\nThis review shows that gravity is a sufficient but not necessary condition for the occurrence of an oblique effect in the haptic modality. As a matter of fact, the haptic oblique effect can occur in absence of any gravitational cues. Moreover, the haptic oblique effect can also be strengthened by factors other than gravity. For example, some studies have shown the presence of an oblique effect with reduced gravitation cues when different hands were used in the exploration and reproduction phases (e.g., the contralateral condition in Appelle and Countryman 1986). To understand this result, it should be noted that the reproduction by the other hand of an oblique requires a complex transformation of the sensory-motor trace of the scanning movement given the symmetric arrangement of the two upper limbs. It is therefore plausible that the transformation from the low-level to the high-level representation is forced in this case, because the sensory-motor trace associated with the scanning arm is of little use for the reproduction phase and the haptic system must rely on the more abstract representation of the orientation (see Baud-Bovy and Viviani 1998 for a similar hypothesis in pointing context). Other studies have shown that an interference task during the delay between the presentation and the recall of the orientation (with the same hand) is responsible for an oblique effect in reduced gravity conditions or that it strengthens in normal conditions (Gentaz and Hatwell 1999). The idea is that higher attentional or cognitive demands decrease the amount of resources available to the haptic system. Such a situation would induce the transformation of sensory-motor trace or give more weight to a more abstract representation of the stimulus. Work in neighboring fields is consistent with this hypothesis. For example, Rossetti et al. (1996) showed that in proprioceptive pointing tasks, memorization leads participants to stop using sensory-motor representation to produce a movement and commits them to using a semantic spatial representation. In haptics, a similar shift in perception of parallelity was also observed by Zuidhoek et al. (2003). These authors tested the effect of a delay between the perception of a reference bar and the parallel setting of a test bar in the horizontal plane. They observed that a 10-s delay significantly improved performance. They suggest that a shift from the egocentric towards the allocentric reference frame during the delay period would lead to different spatial processing (for discussion, see also Faineteau et al. 2005; Gentaz and Gaunet 2006).\nThe nature of the more abstract representation and underlying processes is still an open question. One possibility is that the orientations would be mapped to one or more topologically organized layers of processing units, and that the perceived orientation would correspond to the most active units in one of these layers. The DFT of spatial cognition developed by Spencer and colleagues assumes such representational scheme (Spencer et al. 2006, 2007). According to this theoretical framework, the stimulus would propagate from perceptual maps to the working-memory map, where the oblique effect would progressively occur under the influence of neighboring units and of the long-term memory map. While it is out of scope to enter into the details of this model here, it is important to note that the dynamic properties of these maps could explain the presence of a systematic bias toward or away from the reference axes as well as the increase of precision observed along the reference axes. Work on this model has so far focused on the visual modality and has not yet addressed issues specific to the haptic modality, such as the role of gravity or the coding of an orientation in space. In particular, the problem of extracting an initial representation of the orientation from the complex afferent and efferent signals that correspond to the sensory-motor trace of the scanning movement seems daunting, but this observation also holds true for all other explanations of the haptic oblique effect.\nAnother possibility is that the orientation would be represented in categorical terms (Huttenlocher et al. 1991; see also the work on Categorical Perception; Harnard 2003). As described in \u201cHypotheses on the origins of the oblique effects\u201d, the Category-Adjustment model assumes the existence of a double representation of the stimulus. According to the CA model, the oblique effect occurs at a late stage of processing, when both representations are combined, with possible different weights, to produce the response. From a theoretical point of view, such a double representation can be advantageous when the reliability of the low-level representation decreases, though it might introduce biases. In this theoretical framework, variations of the strength of the oblique effect could be understood in terms of a relative reliability of abstract representation of the sensory motor traces. The idea of a double representation could also be useful to explain the complex error patterns observed in the reproduction of 3D orientations showing that a participant responded using a heterogeneous set of cues that included the low-level sensory-motor trace as well as some more abstract-coding scheme based on the two angles between the orientation and the sagittal or frontal plane (Baud-Bovy and Gentaz 2006). However, a problem with the CA model is that it predicts an increase of the variability of response at the category boundaries (G. Baud-Bovy, 2008, A critical commentary of the category adjustment model and the oblique effect. Psychol Rev, unpublished). This observation is problematic insofar that it is usually assumed that the category boundaries correspond to the horizontal or vertical orientations. A possible remedy would be to assume the existence of narrow categories around the vertical and horizontal orientations in addition to the usual oblique categories centered on the diagonals.\nTo conclude, it is important to note that both the DFT and the CA model were originally conceived to explain the presence of systematic biases toward the closest diagonal in various location-recall or position-recall tasks in the visual modality, which has not yet been observed in the haptic modality. As a matter of fact, the analysis of the constant errors in studies of the haptic oblique effect did not reveal systematic directional errors. This observation holds true whether the reproduction method (Appelle and Countryman 1986; Gentaz and Hatwell 1995, 1996, 1998, 1999; Lechelt et al. 1976; Lechelt and Verenka 1980) or the production method (Appelle and Gravetter 1985; Gentaz et al. 2002; Luyat et al. 2001) was used to assess the perceived position of the orientation in the haptic modality. This negative result does not, however, invalidate these models, since the aforementioned studies of the haptic oblique effect have used a limited set of stimuli (the vertical, horizontal and the two main diagonals) that precludes the observation of a bias toward the closest diagonal, since it can be observed only with oblique orientations that differ from the diagonals. The question of the accuracy of the perception, as opposed to its precision, has been so far neglected in studies of the haptic oblique effect and needs to be further investigated in future studies.","keyphrases":["haptic","orientations","oblique effect","space","hand","reference frame","categorization","development"],"prmu":["P","P","P","P","P","P","P","P"]}
{"id":"Virchows_Arch-4-1-2335297","title":"Epithelial atypia in biopsies performed for microcalcifications. Practical considerations about 2,833 serially sectioned surgical biopsies with a long follow-up\n","text":"This study analyzes the occurrence of epithelial atypia in 2,833 serially sectioned surgical breast biopsies (SB) performed for microcalcifications (median number of blocks per SB:26) and the occurrence of subsequent cancer after an initial diagnosis of epithelial atypia (median follow-up 160 months). Epithelial atypia (flat epithelial atypia, atypical ductal hyperplasia, and lobular neoplasia) were found in 971 SB, with and without a concomitant cancer in 301 (31%) and 670 (69%) SB, respectively. Thus, isolated epithelial atypia were found in 670 out of the 2,833 SB (23%). Concomitant cancers corresponded to ductal carcinomas in situ and micro-invasive (77%), invasive ductal carcinomas not otherwise specified (15%), invasive lobular carcinomas (4%), and tubular carcinomas (4%). Fifteen out of the 443 patients with isolated epithelial atypia developed a subsequent ipsilateral (n = 14) and contralateral (n = 1) invasive cancer. The high slide rating might explain the high percentages of epithelial atypia and concomitant cancers and the low percentage of subsequent cancer after a diagnosis of epithelial atypia as a single lesion. Epithelial atypia could be more a risk marker of concomitant than subsequent cancer.\nIntroduction\nBreast biopsies for infraclinical lesions are more frequent with mammographic screening programs, but the distribution of the corresponding histological lesions and their associations are still imprecise. Difficulties encountered in following up patients without cancer account for the fact that the clinical significance of certain non-malignant lesions and the management of patients are still debated. Moreover, the problem of surgical biopsy sampling has never been fully investigated and has added additional confusion in appreciating the distribution and clinical significance of such lesions. In 1981, breast epithelial atypia were hardly mentioned and not clearly defined in the World Health Organization\/International Union Against Cancer (WHO\/UICC) histologic classification of breast tumors [73]. The histologic classification of noncancerous lesions has been mainly based on studies analyzing for each lesion the associated risk of subsequent cancer. These studies were initiated by the survival studies of Dupont and Page [15, 46] based on lesions discovered by palpation before the era of mammography. Thereafter, further studies [6, 9, 17, 24, 31, 51, 62, 67] substantiated these results, which were ratified in 2003 by the new American Joint Committee on Cancer (AJCC)\/UICC classification of breast tumors [70]. Schematically, this classification differentiates benign epithelial lesions (usual ductal hyperplasia and other lesions) from atypical lesions of ductal or lobular type. Although this historical classification is challenged by a new classification [68], it remains the most widely used in practice. Interestingly, the occurrence of epithelial atypia was low in Page\u2019s study [46] and has increased with mammographic screening programs [60, 66] and with the development of percutaneous large core needle biopsy (CNB) methods using stereotactic mammography or ultrasound guidance. At present, CNB is frequently used for the initial evaluation of clinically occult breast lesions, thus generating dilemma for the subsequent management of certain noncancerous lesions. At our institution, surgical biopsies (SB) have always been managed in the same way, and most patients with atypical and malignant lesions have been followed. The objectives of our work were to analyze the occurrence of epithelial atypia and their association with a concomitant cancer in a large series of SB performed for microcalcifications without a palpable tumor and to assess the subsequent cancer probability in the group of patients with an initial diagnosis of epithelial atypia. Finally, we provide some practical considerations for the management of patients with epithelial atypia in this era of mammographic screening and CNB.\nMaterials and methods\nSelection of patients\nAt Institut Bergoni\u00e9, from January 1975 to December 2002, 3,166 breast biopsies for diagnostic purposes, 2,833 SB and 333 CNB, were performed for microcalcifications without any palpable mass in 2,708 patients (mean age 51.8\u00a0years, range 19.7\u201381\u00a0years). Among them, 248 (9%) had several biopsies in the same or contralateral breast. Since 1998, microcalcifications have been classified according to the classifications of the American College of Radiology [2]. SB for diagnostic purposes were defined before 1998 by the absence of a preoperative diagnosis based on the clinical\u2013mammographic\u2013cytologic triplet and by the absence of a positive frozen section and, since 1998, by the presence of epithelial atypia on CNB. Excluded from this study were 132 cancers and 139 non-atypical benign lesions diagnosed on CNB as well as 49 re-excisions performed elsewhere than in our center. Thus, 2,833 SB in 2,375 patients were available for analysis, among which 13 corresponded to re-excision after a CNB with epithelial atypia. Since 1989, needle localization, intraoperative specimen radiography, and post-excisional biopsy mammography have been performed in most cases.\nSurgical biopsies and tissue sampling: serial macroscopic sectioning\nSB was removed in one fragment and measured more than 3\u00a0cm in 94% of the cases (mean size 60\u00a0mm, 5\u2013250\u00a0mm). For SB margin assessment, either the surface of the specimen was inked or the surgeon during the same operation removed additional tissue in the remaining cavity after excision of the specimen (surgical margins). After fixation in Holland Bouin, SB and margin specimens were serially sectioned in their entirety into numbered slices every 2\u00a0mm [12]. In most cases (89%), careful macroscopic examination of the specimen failed to reveal any lesion. Each numbered slice was put in as many numbered separate cassettes as necessary and paraffin-embedded in sequence. The median number of blocks per SB was 26 (from 2 to 180) and 8 (from 1 to 44) for surgical margins. Each block was examined on one hematoxylin\u2013eosin\u2013safran stained slide.\nClassification of lesions and review of slides\nSince 1975, all patients have been prospectively included in our clinical, histologic, and biologic database by senior pathologists (IM, GMG, IS, JMC). For each SB and each lesion, we prospectively entered in our pathologic database morphological descriptive criteria by using 65 pathological items for noncancerous lesions and 181 for cancers. Definitions and terminologies given in the literature were used to report columnar cell lesions (CCL), non-atypical ductal hyperplasia, atypical ductal hyperplasia, ductal carcinoma in situ (DCIS), and lobular carcinoma in situ (LCIS) [1, 4, 7, 10, 17, 19, 21\u201328, 38, 43\u201345, 47, 49, 50, 56, 59, 61, 69, 72, 74, 75]. The interest of our database was to collect morphological descriptive criteria of nearly all the breast lesions without labeling them. In fact, labels and definitions of breast lesions have varied throughout the past 30\u00a0years, while neither lesions nor their corresponding descriptive criteria (i.e., size, type, architecture, cellular and nuclear features, etc...) have changed. The only changes during this period were the definitions and\/or the names given to these lesions. As we have listed for each lesion all the corresponding descriptive criteria among the 236 available items, we have been able to reclassify each lesion according to the \u201cnew\u201d criteria recommended by referent authorities for a new definition, by selecting in our database the \u201cnew\u201d correspondent descriptive criteria corresponding to this new definition. Consequently, this provided a homogeneous approach to the pathological lesions at the time of our study. For example, low-grade DCIS\u2009\u2264\u20092\u00a0mm have been reclassified as atypical ductal hyperplasia\/ductal intraepithelial neoplasia (ADH\/DIN) 1B (n\u2009=\u200930) according to the new AJCC\/UICC classification of breast tumors [70], and lesions that we used to term before 1997 [71] as clinging carcinoma of the monomorphic type [4] have been reclassified as flat epithelial atypia (FEA)\/DIN 1A or columnar cell change (CCC) with atypia (n\u2009=\u200984) [61]. About half of these 114 cases have been systematically reviewed by one (IM) or two senior pathologists (IM and GMG or IS), and there was a complete concordance between the second review and the initial descriptive criteria listed in the database. Similarly, lesions that we used to name LCIS before 1997 have been renamed lobular neoplasia (LN) since 1997, corresponding either to atypical lobular hyperplasia (ALH) or to LCIS. On the contrary, all the cases with micropapillary lesions were reviewed (n\u2009=\u2009155) because there was no item corresponding to precise descriptive criteria of micropapillations (number, topography around the duct, type).\nAtypical ductal hyperplasia: definition and sizing\nAmong the group of ADH\/DIN 1B, we individualized two morphologic types of ADH. Neither had any high-grade cytological atypia or necrosis.\nADH \u201cmimicking DCIS\u201d (Fig.\u00a01). In this type, architectural atypia were qualitatively insufficient to allow a diagnosis of DCIS, therefore this \u201cmimicking\u201d DCIS lesion was classified as ADH whatever its size. Tufts and short micropapillations formed by cells had a broad base and were cohesive. There was no polarization of cells, i.e., no true cribriform spaces. Pseudo-cribriform patterns comprised irregular or relatively round microlumina with incomplete polarization of surrounding epithelial cells. Cellular bridges were wavy without any cellular polarization. Cells corresponded either to columnar cells with uniform ovoid to elongated nuclei or to cells with a slight increase in the nuclear\/cytoplasmic ratio with more or less distinct cell borders and round or ovoid nuclei. These cells were sometimes admixed in the same lesion displaying a morphological gradient, but there was no regular arrangeent. Nuclear chromatin was evenly dispersed, homogeneous, or slightly marginated, and nucleoli were inconspicuous. Apical snouts, intraluminal secretions, and psammoma-type calcifications were frequently present.\nFig.\u00a01a\u2013d. ADH \u201cmimicking DCIS.\u201d a Tufts and short micropapillations with a broad base. b Pseudo-cribriform spaces. c Microlumen with incomplete polarization of surrounding epithelial cells. d Cellular bridges without cellular polarization. Cells are parallel to the axes (arrows)\nADH corresponding to \u201cmini DCIS\u201d (Fig.\u00a02). In this type, architectural and cytologic atypia corresponded to a low-grade DCIS but were quantitatively insufficient to allow a diagnosis of DCIS, therefore this \u201cmini\u201d DCIS lesion was classified as ADH when \u22642\u00a0mm. Tufts and short micropapillations had a tight base, were present on over all the periphery of the duct, and were non-cohesive with small free papillary tufts in the lumen. There were true cribriform patterns with a polar organization of cells around glandular spaces and\/or variants of cribriform patterns (i.e., trabecular bars, cartwheel formations, and Roman bridges, Fig.\u00a03) with polarized cells arranged perpendicular to the axes. Some solid areas with regular arrangement of cells were also present. Cells were often small, monomorphous, sometimes without a columnar change, with a distinct cytoplasmic membrane and a spaced regular round nucleus with uniformly dispersed chromatin without prominent nucleoli. Intraluminal secretions and calcifications (amorphous or psammoma-type) were also frequently present (Fig.\u00a04). When one mini DCIS focus was found in one partially or completely involved duct\/ductular cross-sections in one terminal ductal lobular unit (TDLU), it was classified as ADH when it measured \u22642\u00a0mm and as DCIS when it measured >2\u00a0mm. When there were several foci of \u201cmini\u201d DCIS in close duct\/ductular cross-sections in the same TDLU or in TDLUs located in the same field at low power magnification (2.5), the lesion was classified as ADH when its size, i.e., its largest diameter, was \u22642\u00a0mm and as DCIS when >2\u00a0mm. When there were several foci of \u201cmini\u201d DCIS in distant duct\/ductular cross-sections in the same TDLU or in close TDLUs, the size of each focus was assessed separately. FEA, rare and scattered single micropapillations, and cribriform variants were not taken into consideration for sizing, even if located in the same TDLU.\nFig.\u00a02a\u2013e. ADH corresponding to \u201cmini DCIS.\u201d a A solid mini DCIS focus measuring less than 2\u00a0mm in one TDLU. b Tufts and short micropapillations over the entire periphery of the duct with small free papillary tufts in the lumen. c Short micropapillations with a tight base. d True cribriform spaces. e Microlumen with complete polarization of surrounding epithelial cellsFig.\u00a03a\u2013c. Variants of cribriform patterns. Polarized cells arranged perpendicular to the axes. a Trabecular bars. b Cartwheel formations. c Roman bridgesFig.\u00a04a and b. a Mild cytologic atypia. b Columnar cells with uniform ovoid nuclei, intraluminal calcifications\nFEA were present either as a single lesion or in association with ADH in the same TDLU and since 1997 have been included in the ADH group. The distinction of FEA from columnar change without atypia was based on the criteria given by the WHO for the definition of FEA. Furthermore, columnar change without atypia was characterized by one or two layers of columnar cells without nuclear atypia, i.e., no increase in the nuclear\/cytoplasmic ratio, no prominent nucleoli. Nevertheless, some cases of columnar change with progesterone impregnation, especially in the second part of the cycle, might display a lobular distension with a secretory material and large nuclei with prominent nucleoli. In such cases, myoepithelial cells displayed the same alterations with clarified cytoplasms, thus facilitating the diagnosis. The distinction of ADH mimicking DCIS from usual hyperplasia (UDH) was based on morphological criteria. Architectural pattern and cytologic criteria of usual ductal hyperplasia were easy to identify in most cases. UDH corresponded to a proliferation of epithelial cells in solid or fenestrated areas without any polarization of surrounding cells. Cells were haphazardly arranged with overlapping nuclei or were parallel with characteristic streamings. They were elongated or pseudo epithelioid, but there was no columnar metaplasia. Cytoplasms were more or less abundant with indistinct borders. Nuclei had irregular size and shape and sometimes contained a prominent eosinophilic inclusion. In some rare cases, immunohistochemical staining with cytokeratin 5\/6 [41] was used and was negative in ADH mimicking DCIS and strongly positive in UDH. In some lesions, differential diagnosis between ADH and low-grade DCIS was all the more difficult because there were intermediate and intricated morphological aspects in the same TDLU. In practice, diagnosis of micropapillary lesions was often difficult. Extensive micropapillary lesions were classified as DCIS when quantitative and qualitative criteria were simultaneously present, i.e., lesion sizing more than 2\u00a0mm corresponding to micropapillations with a tight base over the entire periphery of the ducts. Additional sections could be useful for demonstrating more or less qualitative or quantitative diagnostic criteria. When malignancy remained equivocal, the case was classified as ADH. When a concomitant cancer was diagnosed, histologic size was assessed, and in DCIS, the percentage of blocks with cancer (\u201cpositive blocks\u201d) was specified [13]. Presence and topography of microcalcifications were also assessed. Lastly, when FEA and\/or \u201cmimicking\u201d DCIS foci were found on excision margins of a SB with DCIS, a further surgical resection was not performed.\nFollow-up of patients with epithelial atypia as a single lesion\nThere were 443 patients with epithelial atypia in one or several SB, without any previous or synchronous carcinoma in the same or contralateral breast and treated by biopsy alone (median follow-up 160\u00a0months, 7 to 315). Only 28\/443 (6%) were lost to follow-up. Among the 415 other patients, 180 were monitored at our institute and 235 outside by correspondent specialists working in close relationship with our institute. All patients received a clinical examination and mammography once a year. When a new biopsy was necessary, it was performed at our institute.\nStatistical analyses\nComparison of clinical and histologic characteristics was conducted by using the chi-square test. For women with epithelial atypia, the probability of developing in situ or invasive cancer was calculated from the date of the first biopsy to the earliest event: breast cancer (ipsi- or contralateral), death, or last contact (last consultation for the group monitored at our institute and checkpoint date, i.e., 1 March 2004, for the others). Probabilities were calculated according to the Kaplan\u2013Meier method (SPSSv11).\nResults\nOccurrence of epithelial atypia in the 2,833 surgical biopsies\nEpithelial atypia were recorded in 971\/2,833 SB (34%). They were found with and without a concomitant cancer in 301\/971 (31%) and 670\/971 (69%) of the cases, respectively. Thus, isolated epithelial atypia were found in 23% of the cases (670 out of the 2,833 SB). Calcifications were present at histologic examination in 98.6% of SB with cancer and were located in benign, cancerous, and both lesions in 10, 39, and 51% of the cases, respectively. In several cases, cancerous foci without any microcalcifications were located at points distant from those with calcifications detected by needle localization.\nTypes of epithelial atypia\nAmong the 971 SB with epithelial atypia, there were 101 SB with FEA as a single lesion (11%), 342 (35%) with ADH, 223 (23%) with LN, and 305 (31%) with ADH and LN. Thus, ADH was encountered in 647\/971 SB (66%).\nTypes of cancers associated with epithelial atypia\nCancers associated with epithelial atypia corresponded to DCIS and micro-invasive carcinoma (DCIS-MI) in 233 cases (77%). Among invasive carcinomas (n\u2009=\u200968), there were 13 (9%) lobular and 11 (6%) tubular carcinomas (Table\u00a01). Cancers were small (\u22645\u00a0mm in 46% of invasive carcinomas, fewer than half of the blocks positive in 76% of DCIS). They were non-high grade in 78 and 67% of DCIS and invasive carcinoma, respectively. In most cases, ADH and cancer were situated close to each other. FEA alone were less frequently associated with a concomitant cancer than ADH and\/or LN (p\u2009=\u20095\u2009\u00d7\u200910\u22124).\nTable\u00a01Types of concomitant cancers (n\u2009=\u2009301) in the 971 surgical biopsies with epithelial atypiaEpithelial atypiaFEA (n\u2009=\u2009101)ADH (n\u2009=\u2009342)LN (n\u2009=\u2009223)ADH\u2009+\u2009LN (n\u2009=\u2009305)No. of cases (%)No. of cases (%)No. of cases (%)No. of cases (%)Without cancer8483220641396222774With cancer17171223684387826DCIS\/DCIS-MI12121033058266020IDC\/NOS\u2013\u2013164.7178113ILC1110.36352TC44213121FEA Flat epithelial atypia; ADH atypical ductal hyperplasia; LN lobular neoplasia; DCIS ductal carcinoma in situ; DCIS-MI DCIS with micro-invasion; IDC infiltrating ductal carcinoma; ILC infiltrating lobular carcinoma; TC tubular carcinoma\nCancers without epithelial atypia (malignancy alone)\nThere were 821 malignant SB without epithelial atypia [590 micro-invasive carcinomas, 206 infiltrating ductal carcinomas (IDC), and 25 infiltrating lobular carcinomas (ILC)].\nSubsequent cancer in patients with an initial diagnosis of epithelial atypia as a single lesion\nAt 5 and 10\u00a0years, the probabilities of developing invasive breast cancer in the group of 443 patients with epithelial atypia were 2.8% [95%CI\u2009=\u20091.4 to 5.5] and 5.5% [95%CI\u2009=\u20093.3 to 9.9], respectively (Fig.\u00a05). Among the 18 subsequent carcinomas, 15 were invasive (11 IDC and 4 ILC), and 3 corresponded to DCIS. Most subsequent carcinomas were encountered in the homolateral breast (n\u2009=\u200914) and before 10\u00a0years (n\u2009=\u200916). Seven carcinomas occurred in the group of patients with an initial diagnosis of LN, in the same (n\u2009=\u20095) or contralateral (n\u2009=\u20092) breast. They corresponded to infiltrating ductal (n\u2009=\u20096) or lobular (n\u2009=\u20091) carcinomas. The interval of development was 4, 5, 6 (n\u2009=\u20092), 9, and 12 (n\u2009=\u20092) years. Seven carcinomas occurred in the group of patients with an initial diagnosis of ADH, in the same (n\u2009=\u20093) or contralateral (n\u2009=\u20094) breast. They corresponded to DCIS (n\u2009=\u20092) and to infiltrating ductal (n\u2009=\u20094) or lobular (n\u2009=\u20091) carcinomas. The interval of development was 1, 2, 3 (n\u2009=\u20093), 9, and 12\u00a0years. Four carcinomas occurred in the group of patients with an initial diagnosis of ADH associated with LN, in the same (n\u2009=\u20093) or contralateral (n\u2009=\u20091) breast. They corresponded to DCIS (n\u2009=\u20091) and to infiltrating ductal (n\u2009=\u20092) or lobular (n\u2009=\u20091) carcinomas. In the four cases, the interval of development was 4\u00a0years. There was no subsequent carcinoma in the group of patients with FEA.\nFig.\u00a05Probability of developing subsequent invasive breast cancer in the group of 415 patients with epithelial atypia (dotted line: confidence interval 95%)\nDiscussion\nApplication of the WHO classification: practical considerations\nFor a long time, DCIS was diagnosed even if the characteristic features were found in only one ductal space [6]. Thereafter, some authors introduced quantitative criteria for distinguishing between ADH and DCIS [46, 67], while others [23] rejected them. More recently, Rosen [57\u201359] and Schnitt and Vincent-Salomon [61] described CCL comprising CCC and columnar cell hyperplasia (CCH) with and without atypia. Nasser [40] challenged this classification based on columnar shape and limited the group of lesions to proliferations characterized by a low-grade atypicality, \u201catypical columnar cell lesions,\u201d (ACCL) rather by a columnar cell configuration. In the WHO classification, ADH includes various not clearly defined types of lesions (Table\u00a02). On one hand, there are lesions with arcades, moundings, and micropapillary formations, but without any true cribriform\/complex architectural patterns [34]. This type of ADH corresponds to the CCH with atypia of Schnitt and Vincent-Salomon [61] termed category 3 in Simpson\u2019s study [51], to the definition of ADH by Koerner [34] and to ADH \u201cmimicking\u201d DCIS in our study. On the other hand, there are lesions displaying architectural and cytologic atypia. This type of ADH corresponds to the complex architectural pattern with cytologic and architectural atypia of Schnitt and Vincent-Salomon [61] termed category 5 in Simpson\u2019s study [64], to the definition of \u201cmicrofocus of DCIS\u201d by Koerner [34] and to \u201cmini DCIS\u201d in our study. In Simpson\u2019s study, the ADH\/category 5 contained chromosomal changes and the same total mean number of changes to that observed in DCIS\/DIN IC, unlike the other CCL. Lastly, because there is still no consensus for measuring ADH, there is no clear-cut distinction between ADH and DCIS, and the cut-off at 2 or 3 [52] mm or at two completely involved spaces [70] seems arbitrary. While awaiting a definitive molecular classification, the simplest attitude could be recommended in routine practice. Only mini foci of ADH obviously similar to low-grade DCIS foci could be measured and classified as ADH when equal to or less than 2\u20133\u00a0mm [52] and as DCIS when more than 3\u00a0mm. Although the mode of measurement in our study is not under consensus, it is simple and can been routinely applied.\nTable\u00a02Terminologies used for intraductal proliferative lesions with low-grade cytologic atypia, so-called atypical columnar cell lesionsSpectrum of lesions1, 3\u20135 LayersNo polarization*With polarization\u00a0Occasional mounding\u00a0Mounding, arcades\u00a0Cribriform spaces and their variants\u00a0No or rare arcades and micropapillary formations\u00a0Cohesive micropapillary tufts with a broad base\u00a0Non-cohesive micropapillary tufts with a tight base[70]Flat epithelial atypia\/DIN 1AADH\/DIN 1B\u2009\u2264\u20092\u00a0mm; or in two spacesDCIS\/DIN 1C[59]Columnar cell hyperplasia with atypiaADH if not extensiveDCIS if extensive[61]Columnar cell change (CCC) with cytologic atypiaComplex structures with architectural and cytologic atypia[34]Columnar cell lesions + ADHMicroscopic focus of DCISDCISInstitut Bergoni\u00e9Ex-clinging carcinoma of monomorphic typeADH \u201cmimicking\u201d DCISADH corresponding to \u201cmini\u201d DCIS\u2009\u2264\u20092\u00a0mmDCIS[64]CCC with cytologic atypiaCCH with architectural atypiaCCH with architectural atypiaDCISOrAndCCH with cytologic atypiaCytologic atypiaRegrouping?DIN 1AADH\/DIN 1BADH\/DIN 1CDCIS\/DIN 1CNot measuredMeasured: \u22643\u00a0mm>3\u00a0mm*Or incomplete polarization\nOccurrence of epithelial atypia and their association with a concomitant cancer: practical considerations\nIn our study, the proportion of epithelial atypia is high (23%), a result difficult to compare to others in the literature, as the methodologies used by teams are different. In the Page and Dupont case-control studies [15, 46], ADH and ALH were found in 2.1 and 1.6% of the cases, respectively. In mammographic screening programs, epithelial atypia and cancers increase as the number of biopsies performed for microcalcifications increases, especially as ACR4\/ACR5 lesions are more often excised than ACR3. However, as underlined by Page [46], \u201cthe most direct relationship of epithelial atypia incidence is to slide rating.\u201d The number of slides per SB in our study (median 26) was higher than in the other studies on benign breast lesions: 1\u20135 in 93% of the cases in the study of Page et al. [46] (n\u2009=\u2009283), 3 (range 1\u201325, n\u2009=\u2009674) in the study of Shaaban et al. [62], and a mean of 1.6 slides per cm of tissue (n\u2009=\u2009199) in the study of Tavassoli and Norris [67]. In a recent study conducted in the south west of France in women aged between 50 and 75 with mammographically detected non-palpable breast lesions, a similar proportion of atypical lesions were found when biopsies were serially sectioned [39]. Furthermore, this high slide rating allowed the detection of small concomitant cancers in the vicinity of epithelial atypia in 31% of our cases, with a skew towards low-grade lesions (high proportions of DCIS and low-grade invasive carcinomas, especially tubular carcinomas). Our results strengthen the hypothesis that FEA and ADH are risk markers of low-grade cancers. This has been confirmed by the study of Simpson et al. [64] on molecular genetic profiles of CCL. In some of them, there are both a morphological and a molecular continuum in the degree of proliferation and atypia, supporting the hypothesis that \u201cCCL are a non-obligate, intermediary step in the development of some forms of low grade in situ and invasive carcinoma.\u201d The association of epithelial atypia with a concomitant cancer in nearly one third of the cases in our study parallels previous findings concerning the frequency of cancers found in SB performed for atypia in CNB. Thus, approximately 30% [20, 29] and 15 to 21% [5, 8, 14, 18, 35, 45, 53, 63, 76] of excisions after CNB with ADH and LN, respectively, were proven to have cancer. Consequently, excision is recommended [45] for all patients in whom ADH is identified on CNB and may be justified in patients with FEA, as they are included in the spectrum of ACCL. Excision remains a controversial issue in patients with LN. Some authors have advocated it [3, 18, 33, 63], while others have rebutted it [55], especially when LN is an incidental non-extensive finding [48] with no radiologic\u2013pathologic discordance [18] and without any synchronous mass lesion [37]. SB corresponding to re-excision should be processed in its entirety by serial macroscopic sectioning [32, 65]. When pathologic examination is exclusively focused on mammographic calcifications, the risk is to underestimate the DCIS size\/extension because cancerous foci without any calcification (10% in our study vs 6% in Owing\u2019study) [42] may be located at points distant from those with benign breast tissue containing calcifications.\nSubsequent cancer after an initial diagnostic of epithelial atypia as a single lesion: practical considerations\nIn the literature, 4 to 22% (average interval 8.3\u00a0years follow-up) [6, 42, 67] and 15 to 20% [16, 30, 56] of patients developed invasive carcinomas after a diagnosis of ADH and LN, respectively. The risk of developing cancer increases with extended follow-up, but many cancers after a diagnosis of LN have a good prognosis and a low mortality [36]. These results are difficult to compare to ours because the methodologies are different. The low probabilities of subsequent invasive cancer in our study could be due to the high slide rating, allowing the detection of small concomitant cancer that might have been missed with a low slide rating and inadequate patient management [11, 54].\nIn conclusion, when epithelial atypia are present, they are associated in nearly one third of the cases with a concomitant close cancer and are found as isolated lesions in nearly 23% of SB performed for microcalcifications. In practice, ADH should be more clearly defined with simple guidelines for measuring lesions. When malignancy remains equivocal and\/or when sizing is difficult, it is better to classify the lesion as ADH. Epithelial atypia could be more a \u201crisk factor\u201d of a concomitant geographically small close cancer than a risk marker for a subsequent cancer, as they form part of a spectrum of lesions [64].","keyphrases":["epithelial atypia","breast","cancer","atypical ductal hyperplasia","lobular neoplasia"],"prmu":["P","P","P","P","P"]}
{"id":"J_Antimicrob_Chemother-1-1-2386080","title":"HIV-1 drug resistance genotyping from dried blood spots stored for 1 year at 4\u00b0C\n","text":"Background Dried blood spots (DBSs) are an attractive alternative to plasma for HIV-1 drug resistance testing in resource-limited settings. We recently showed that HIV-1 can be efficiently genotyped from DBSs stored at \u221220\u00b0C for prolonged periods (0.5\u20134 years). Here, we evaluated the efficiency of genotyping from DBSs stored at 4\u00b0C for 1 year.\nIntroduction\nThe availability of clinical specimens that can be easily collected, stored and transported is advantageous in areas that lack appropriate infrastructure for blood processing. Whole blood samples collected by finger stick and dried onto a filter paper [dried blood spots (DBSs)] represent an attractive alternative to the conventional collection of blood in tubes. DBSs have been extensively used for HIV-1 antibody testing,1 molecular diagnostics2 and virus load (VL) quantification3\u20135 and are now considered a convenient alternative to plasma for HIV-1 drug resistance testing. We recently found that drug resistance genotypes generated from DBSs were similar to those derived from plasma in antiretroviral-naive and -experienced patients.6,7 We also found that resistance testing from DBSs can be as sensitive as with plasma when using a genotypic assay that amplifies a large (1.8 kb) pol fragment.6 The high success of amplification was noted using DBSs stored at \u221220\u00b0C in the presence of desiccant, suggesting that \u221220\u00b0C may represent a feasible storage temperature for DBSs. Although these findings were encouraging, storage at \u221220\u00b0C is not always possible in resource-limited settings where 4\u00b0C or room temperature may represent a more realistic alternative. Studies on HIV-1 VL determinations using DBSs have reported the efficient amplification of short HIV-1 RNA sequences from filter papers stored at 4\u00b0C or room temperature.3,8,9 However, genotypic assays usually rely on the amplification of large pol fragments and are thought to be particularly sensitive to HIV-1 RNA degradation that may occur under suboptimal storage conditions. Here, we report on the efficiency of amplification and drug resistance genotyping from DBSs stored at 4\u00b0C for 1 year.\nMethods\nPreparation and storage of DBSs\nWe prepared a total of 40 DBSs from residual diagnostic specimens collected from highly antiretroviral-experienced HIV-1 subtype B-infected persons. A more detailed description of the study population including antiretroviral drug treatment has been reported elsewhere.6 Drug resistance genotypes were available for the matched plasma specimens.6 The median plasma VL in these patients was 13 680 RNA copies\/mL (range 518\u2013676 694, Versant HIV-1 RNA 3.0 Assay, Bayer HealthCare Diagnostic Division, Tarrytown, NY, USA) (Table\u00a01). DBSs were prepared by pipetting 50 \u00b5L of whole blood onto pre-marked circles on 903 filter paper cards (Schleicher & Schuell, Keene, NH, USA). Cards were dried overnight at room temperature (25\u00b0C), placed in a gas-impermeable, sealable plastic bag (Fisher Scientific Company, Pittsburgh, PA, USA) containing a silica gel desiccant (Mini Pax Sorbent, Multisorb technologies, Buffalo, NY, USA) and stored at 4\u00b0C until they were shipped to CDC in dry ice. Upon arrival, individual plastic bags were put in a larger bag containing three extra desiccants and stored at 4\u00b0C. All desiccants were evaluated for humidity at 6 months; 21 of the 40 bags had evidence of humidity as indicated by the colour indicator in the desiccant. Therefore, a new desiccant was added to each individual bag after allowing the spots to equilibrate at room temperature for 30 min. None of the specimens had mould after 12 months of storage at 4\u00b0C.\nTable\u00a01\nFrequency of amplification of HIV-1 pol from plasma or DBSs stored at \u221220\u00b0C for 6 months or at 4\u00b0C for 12 months\nSpecimen ID\nPlasma VL (log10 copies\/mL)\nCD4 cells\/mm3\nPlasmaa\nDBS (\u221220\u00b0C, 6 months, ViroSeq assay)b\nDBS (4\u00b0C, 12 months, ViroSeq assay)\nDBS (4\u00b0C, 12 months, in-house assay)\n1\n676 694\n32\n+\n+\n+\n+\n2\n266 612\n195\n+\n+\n+\n+\n3\n214 330\n195\n+\n+\n+\n+\n4\n191 674\n48\n+\n+\n+\n+\n5\n170 289\n663\n+\n+\n+\n+\n6\n90 758\n324\n+\n+\n+\n+\n7\n63 261\n169\n+\n+\n+\n+\n8\n51 598\n324\n+\n+\n+\n+\n9\n52 342\n457\n+\n+\n+\n+\n10\n49 805\n450\n+\n+\n+\n+\n11\n37 010\n152\n+\n+\n+\n+\n13\n30 267\n525\n+\n+\n+\n+\n14\n28 742\n378\n+\n+\n+\n+\n16\n23 352\n221\n+\n+\n+\n+\n17\n23 321\n168\n+\n+\n+\n+\n18\n21 896\n391\n+\n+\n+\n+\n19\n17 563\n289\n+\n+\n+\n+\n20\n16 359\n609\n+\n+\nn.s\n+\n21\n14 622\n483\n+\n+\n+\n+\n22\n13 952\n396\n+\n+\n+\n+\n23\n13 407\n224\n+\n+\n+\n+\n24\n12 452\n374\n+\n+\n\u2212\n+\n25\n12 307\n465\n+\n+\n+\n+\n26\n12 264\n1320\n+\n+\n+\n+\n27\n11 999\n81\n+\n+\n\u2212\n+\n28\n11 663\n336\n+\n+\nn.s\n+\n29\n10 998\n442\n+\n+\nn.s.\n\u2212\n30\n9955\n342\n+\n+\n\u2212\n+\n31\n8316\n288\n+\n+\n\u2212\n+\n32\n4717\n+\n+\n\u2212\n+\n33\n4349\n990\n+\n+\n\u2212\n+\n34\n4213\n1080\n+\n+\n\u2212\n+\n36\n3880\n434\n+\n+\nn.s.\n+\n37\n3455\n468\n+\n+\n\u2212\n+\n38\n2240\n154\n+\n+\n+\n+\n39\n1929\n416\n+\n+\n\u2212\n+\n42\n1254\n858\n+\n+\n\u2212\nn.s.\n44\n1045\n360\n+\n+\n\u2212\n+\n45\n929\n63\n+\n+\nn.s.\n+\n46\n518\n240\n+\n+\n\u2212\n+\nn.s., HIV-1 RT and protease were amplified but failed to generate full-length sequences.\naPlasma genotypes were generated using the Viroseq assay except for 1, 3, 18, 32, 33, 39 and 42 (TrueGene assay).\nbAmplification from these DBSs stored at \u221220\u00b0C has been reported elsewhere.6\nNucleic acid extraction\nTotal nucleic acids were extracted from one spot after 1 year of storage at 4\u00b0C using a modification of the Nuclisens assay described previously.7 Briefly, a whole spot containing 50 \u00b5L of blood was cut with scissors and added into 9 mL of Nuclisens lysis buffer (bioM\u00e9rieux, Inc., Durham, NC, USA). Special care was taken to avoid contact of the scissors with the blood spots; scissors and forceps were sprayed with 70% ethanol after each use and wiped dry. After 2 h of incubation with lysis buffer at room temperature under gentle rotation, the supernatant was clarified by centrifugation at 250 g for 5 min and then transferred to a clean 15 mL conical tube. Nucleic acids were then extracted following the manufacturer\u2019s instructions, resuspended in 45 \u00b5L of elution buffer and stored at \u221280\u00b0C until use.\nDrug resistance testing\nResistance testing from DBSs was performed using the ViroSeq HIV-1 Genotyping System (Abbott Molecular, Des Plains, IL, USA) and an in-house reverse transcriptase (RT)-nested PCR method described previously.7 The ViroSeq assay amplifies a 1.8 kb pol fragment and has a sensitivity of detection of 2000 RNA copies\/mL plasma. The in-house assay amplifies a 1023 bp fragment of HIV-1 pol comprising amino acids 15\u201399 of the protease and 1\u2013256 of the RT. This assay has been validated only for HIV-1 subtype B viruses and has a sensitivity of detection of 1000 RNA copies\/mL of plasma.7 Genotypes were interpreted using the Stanford Genotypic Resistance Interpretation Algorithm (version 4.2.6) available at http:\/\/hivdb.stanford.edu\/pages\/algs\/HIVdb.html.\nResults\nWhen the ViroSeq assay was used, only 23 of the 40 (57.5%) DBS specimens stored at 4\u00b0C were successfully genotyped; 22 of the successful amplifications were from specimens that had plasma viraemia >10 000 RNA copies\/mL (Table 1). An additional five specimens produced amplicons that did not generate full-length RT and protease sequences. Visual inspection of the desiccant bags after 6 months of storage at 4\u00b0C showed evidence of humidity in 21 of the 40 specimens. Of these 21 specimens, only those with plasma VL higher than 14 000 RNA copies\/mL (12 specimens) were successfully genotyped by the ViroSeq assay at 12 months (data not shown).\nAs we previously noted using parallel DBSs stored at \u221220\u00b0C, resistance genotypes generated from DBSs and matched plasma specimens were highly concordant. Of the 163 drug resistance mutations identified in these 23 plasma sequences, 158 were also found in sequences generated from DBSs. Of the five mutations absent in DBSs, three were protease mutations, two were major (V82A and I54M) and one minor (L10IT), and two were polymorphisms at codon 333 of the RT (G333E and G333D) (data not shown). We also compared the efficiency of amplification seen in these DBSs with that previously seen in DBSs prepared in parallel from the same patients and stored at \u221220\u00b0C for 6 months.6 Under storage at \u221220\u00b0C, all of the 40 DBS specimens were successfully amplified and genotyped (Table 1).\nThe ViroSeq assay amplifies a large (1.8 kb) pol fragment in a single round of PCR and may be particularly sensitive to the degradation of HIV-1 nucleic acids that may occur during long-term storage. We, therefore, assessed if the rate of amplification from DBSs stored at 4\u00b0C could be improved by using an in-house assay that amplifies a smaller (1023 bp) fragment and uses a nested PCR step.7 Using this method, viruses from 38 of the 40 DBSs (95%) were successfully genotyped (Table 1). Despite the use of a different assay to genotype viruses from these 38 DBS specimens, resistance genotypes were highly concordant with those generated from plasma using the ViroSeq assay; 275 of the 291 mutations found in plasma viruses were also found in viruses from DBSs (data not shown).\nTo investigate if the decreased amplification success rate observed with the ViroSeq assay was due to insufficient or degraded HIV nucleic acids, we performed serial dilutions of RNA extracts from high VL specimens and tested them by both the in-house and the ViroSeq assays (Figure\u00a01). Although both assays have comparable sensitivity, amplification signals were only maintained when the shorter pol fragment was amplified by the in-house method. Figure\u00a01 shows that such signals were consistently lost when the larger pol fragment was amplified using the ViroSeq assay, suggesting the possibility of nucleic acid degradation during storage at 4\u00b0C. Such a possibility was further supported by parallel testing of serial dilutions of RNA extracts prepared from specimens stored at \u221220\u00b0C. Figure 1(c) shows that amplification signals generated from these specimens using the in-house assay were generally stronger than those obtained from the same DBS specimens stored at 4\u00b0C (Figure 1b).\nFigure 1\nAgarose gel showing the HIV-1 pol amplification signals obtained from DBS specimens stored at 4 or \u221220\u00b0C. Two serial 10-fold dilutions (1\/10 and 1\/100) of specimens 2 (266 612 RNA copies\/mL), 3 (214 330 RNA copies\/mL), 4 (191 674 RNA copies\/mL) and 10 (49 805 RNA copies\/mL) were prepared in Nuclisens elution buffer and were tested in parallel using the ViroSeq assay (1.8 kb) or the in-house RT-nested PCR method (1023 bp). (a) DBS specimens stored at 4\u00b0C and amplified using the ViroSeq assay. (b) DBS specimens stored at 4\u00b0C and amplified using the in-house method. (c) DBS specimens stored at \u221220\u00b0C and amplified using the in-house method.\nDiscussion\nOur study on a small number of specimens suggests that storage at 4\u00b0C may represent a feasible alternative to \u221220\u00b0C for long-term storage of DBSs. However, we noted that the efficient genotyping from these DBS specimens required the use of an in-house nested PCR assay and that the ViroSeq assay failed to genotype a substantial proportion of specimens. The results with the ViroSeq assay differed from our previous findings with this assay showing a high success of genotyping from DBSs stored at \u221220\u00b0C and suggest that some degradation of RNA may have occurred during long-term storage at 4\u00b0C possibly due to suboptimal storage temperature, humidity or both. Such a possibility was suggested by the weaker amplification signals observed in RNA extracts from specimens stored at 4\u00b0C compared with those stored at \u221220\u00b0C upon serial dilutions. Although different efficiencies of RNA extraction could partially explain these differences, we found that a high proportion of the specimens stored at 4\u00b0C had evidence of humidity at 6 months, whereas none of the specimens stored at \u221220\u00b0C did. High humidity conditions are thought to be detrimental to resistance testing from DBSs given the extreme sensitivity of HIV nucleic acids to degradation in the presence of humidity.10 Nonetheless, we showed that it is possible to overcome potential losses in HIV-1 RNA integrity and efficiently genotype from DBSs stored at 4\u00b0C by using an in-house nested PCR protocol that amplifies a smaller fragment using stringent, quality-controlled reagents. These results are encouraging and expand on our earlier findings showing the efficient genotyping of HIV-1 pol from DBSs stored at \u221220\u00b0C for up to 4 years.6,7 A high success of genotyping has also been noted from DBSs stored for 3 months at 37\u00b0C and 85% humidity in the presence of desiccant.10 Under these extreme conditions, viruses from \u223c85% of the DBS specimens could be genotyped by using in-house nested PCR assays that amplified small (700 bp), overlapping pol sequences.10 The use of nested PCR protocols and\/or the amplification of small fragments may likely result in higher amplification success rates from DBSs stored under less optimal conditions. Such a possibility was supported by our findings, showing a better maintenance of amplification efficiencies using the in-house method upon serial dilution of RNA extracts. Despite having comparable sensitivity, amplifications were rapidly lost when a larger fragment was amplified using the ViroSeq assay.\nThe main objective of our study was to evaluate the amplification success rate from DBSs stored at 4\u00b0C for a prolonged period of time. We used an in-house assay validated for HIV-1 subtype B since our study population included patients infected with subtype B viruses. It is important to note, however, that information on the performance of our assay with non-B subtypes is limited to a small number of specimens.7 Therefore, we caution that resistance testing from DBSs collected in areas with prevalent non-B subtypes will require in-house assays appropriately validated for other HIV subtypes.\nThe ability to collect blood samples on filter paper represents an advantage for HIV drug resistance surveillance and monitoring, particularly in areas that lack the appropriate infrastructure for plasma processing and transport. Much effort is currently underway to define the best conditions that will facilitate resistance testing from DBSs including the most appropriate storage temperature and time. The findings reported here suggest that 4\u00b0C may represent a feasible storage temperature for long-term storage of DBSs and add to the promise of DBSs as a convenient specimen type for HIV-1 drug resistance testing.\nFunding\nWork at the CDC was done with intramural funding. Work at Hospital Carlos III was supported by grants from the Fondo de Investigaci\u00f3n Sanitaria (FIS, CP06\/284 and PI06\/1826).\nTransparency declarations\nNone to declare.\nDisclaimers\nThe findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.\nUse of trade names is for identification only and does not constitute endorsement by the U.S. Department of Health and Human Services, the Public Health Service, or the Centers for Disease Control and Prevention.","keyphrases":["resistance testing","903 filter paper","viroseq assay"],"prmu":["P","P","P"]}
{"id":"Psychopharmacologia-3-1-1915624","title":"Effects of the noradrenergic agonist clonidine on temporal and spatial attention\n","text":"Rationale Recent theories posit an important role for the noradrenergic system in attentional selection in the temporal domain. In contrast, the spatially diffuse topographical projections of the noradrenergic system are inconsistent with a direct role in spatial selection.\nIntroduction\nThe locus coeruleus\u2013norepinephrine (LC\u2013NE) system is one of several brainstem neuromodulatory nuclei with widely distributed, ascending projections to the neocortex (Berridge and Waterhouse 2003). The conventional view of NE is that it has broad, nonspecific functions such as regulating arousal (Jouvet 1969). However, recent studies suggest that NE may have more specific cognitive functions, which are nevertheless closely related to the concept of arousal (Robbins 1997; Aston-Jones et al. 2000; Yu and Dayan 2005; Chamberlain et al. 2006). In particular, it has been proposed that NE has a key role in facilitating the responses to decision-making processes and in regulating the balance between exploitation and exploration (Aston-Jones and Cohen 2005).\nRecent views on the role of the LC\u2013NE system in decision making have been based on neuronal recordings of the primate LC during the performance of simple decision-making tasks. These recordings have indicated that under alert conditions (i.e., conditions typical of human subjects in our experiments), LC phasic responses are selectively observed after task-relevant and otherwise motivationally significant (e.g., highly salient or infrequent) stimuli (Aston-Jones et al. 2000). Furthermore, stimulus-elicited phasic LC bursts reliably precede (by \u223c200\u00a0ms) and are closely coupled in time with the behavioral response (Bouret and Sara 2004; Clayton et al. 2004). The noradrenergic innervation associated with such LC responses results in a system-wide, transient increase in the responsivity of efferent target neurons, which is thought to facilitate processing in response to the eliciting stimulus (Berridge and Waterhouse 2003). These observations have led to the suggestion that the LC phasic response can be thought of as a temporal attentional filter (1) that selects for the occurrence (i.e., timing) of task-relevant (Aston-Jones and Cohen 2005) or unexpected (Dayan and Yu 2006) events, much like cortical attentional systems filter the content (e.g., spatial characteristics or color) of stimuli and (2) that facilitates the processing of and responding to these stimuli to help optimize task performance (Aston-Jones and Cohen 2005; Nieuwenhuis et al. 2005a; see also Coull 1994).\nIn the current study, we evaluated the following two hypotheses: (1) According to the temporal filtering hypothesis outlined above, the LC\u2013NE system should be involved in temporal attention modulations operating at the phasic time scale (e.g., in the order of tens of seconds); (2) In contrast, because of the spatially nonspecific pattern of LC projections to the forebrain, the LC\u2013NE system cannot be directly involved in spatial attention shifts. That is, although the LC\u2013NE system projects to multiple brain areas, including many known to be involved in spatial attention, the diffuse character of these projections is inconsistent with the direct biasing of attention in any specific direction (i.e., the LC does not \u201cknow\u201d the relevant spatial location). To test these two hypotheses, we contrasted, in human subjects, the effects of a noradrenergic drug on measures of temporal and spatial attention. Most previous studies examining the functional significance of the LC\u2013NE system have employed drugs acting on \u03b12 receptors. In this study, we used the \u03b12 adrenoceptor agonist clonidine, which at low doses decreases LC firing and attenuates the release of NE from axon terminals (Svensson et al. 1975).\nPrevious studies have examined the effects of clonidine on temporal and spatial attention in a visuospatial cueing task. In this task, subjects are required to respond as quickly as possible to a series of stimuli presented to the left or right of fixation. Each stimulus is preceded by a cue indicating at which location the imperative stimulus is most likely to appear, and subjects are instructed to make use of this information by switching their attention to the location indicated by the cue. The comparison of reaction times on validly versus invalidly cued trials (the \u201cvalidity effect\u201d) provides an index of the efficiency of cue-induced spatial attention shifts. Fernandez-Duque and Posner (1997) extended this paradigm by including and contrasting reaction times on neutral-cue trials and no-cue trials (the \u201calerting effect\u201d), which provides an index of the transient increase in responsivity brought about by the mere presentation of the cue. The alerting effect thus serves as a measure of temporal (cue-evoked) changes in attention to the imperative stimulus.\nClonidine has been found to reduce the size of the alerting effect in humans (Coull et al. 2001) and monkeys (Witte and Marrocco 1997), suggesting that the temporal attentional effects of cues are mediated in part by the noradrenergic system. In contrast, studies have reported mixed results regarding the influence of clonidine on the validity effect: Two studies have reported a reduced validity effect (Clark et al. 1989; Coull et al. 2001), and one study has reported no effect of clonidine (Witte and Marrocco 1997). However, it is hard to interpret clonidine-related reductions in the validity effect, because they may reflect either a direct involvement of the noradrenergic system in spatial attention processes or an interaction between the alerting and attention-directing (i.e., spatial) effects of cues. Specifically, by increasing the responsivity of cortical representations after cue presentation, LC-induced phasic NE release can enhance the effects of target selection by location (e.g., the top-down modulation by prefrontal cortex), resulting in an increased validity effect. The effects of this amplification mechanism would be diminished by clonidine.\nIn contrast to previous studies, we used separate tasks for measuring the effects of clonidine on temporal and spatial attention: an attentional blink task, which requires the selection of targets in time and has no spatial component, and a visual search task, which requires the selection of targets in space but without the help of alerting cues.\nIn the attentional blink task, each trial starts with a rapid serial visual presentation (RSVP) stream containing two target stimuli (T1 and T2) and multiple distractors, each presented for about 100\u00a0ms. At the end of the RSVP stream, subjects are required to report the identity of T1 and T2.\nThe critical finding in this task is that subjects are heavily impaired at the identification of T2 for a few hundred milliseconds after correct identification of T1. This deficit, known as the \u201cattentional blink,\u201d is usually most severe around 200\u2013400\u00a0ms (or two to four items) after T1, after which, performance gradually recovers (Raymond et al. 1992; Chun and Potter 1995). In contrast, if T2 follows T1 without intervening distractors (at \u201clag 1\u201d), performance on T2 is often (partially) spared (Raymond et al. 1992; Hommel and Aky\u00fcrek 2005); this phenomenon has been labeled \u201clag-1 sparing.\u201d These and other findings suggest that the attentional blink task reveals important clues about the time course of fine-scale fluctuations in attention (Olivers 2007).\nRecently, a theory has been proposed that explains the attentional blink in terms of the temporal dynamics of the LC\u2013NE system (Nieuwenhuis et al. 2005b). The theory was inspired by the observation that the timing of the attentional blink coincides with the refractory period in LC activity that follows an LC phasic response to target stimuli. Specifically, although NE potentiates processing in cortical areas, local NE release within the LC is autoinhibitory because of the noradrenergic action at presynaptic and dendritic \u03b12 autoreceptors (Aghajanian et al. 1977). This autoinhibition results in a period after the LC phasic response (\u223c200\u2013450\u00a0ms after the eliciting stimulus), during which subsequent LC phasic discharge is rarely observed. According to the theory, the attentional blink may be mediated by this momentary unavailability of the LC phasic response (and attendant noradrenergic potentiation of information processing) after the LC response to T1. In other words, if T2 is presented during the LC refractory period associated with T1, it will not receive the benefit of NE-mediated facilitation and is therefore more likely to remain unidentified. A computational model implementing this theory was shown to accurately simulate the time course of the attentional blink, including lag-1 sparing (Nieuwenhuis et al. 2005b). In addition, the theory offers an account of the close relationship of the attentional blink to the P3 component of the event-related potential, an electrophysiological correlate of the phasic NE release (Nieuwenhuis et al. 2005a).\nTo assess the effect of clonidine on spatial attention, we used a visual search task. On each trial, subjects searched for a target among multiple distractors in a visual search array and indicated as quickly as possible whether the target was present or absent. The number of distractors (i.e., set size) was systematically varied across trials. In one condition (single-feature search), the target was defined by a salient basic feature, such that search was efficient and the number of distractors had little influence on reaction times. In another condition (conjunction search), the target was defined by a conjunction of two features, such that search was inefficient, and reaction times increased linearly with the number of distractors\u2014as if each item needed to be examined in turn. In this task condition, the efficiency of spatial attention processes is expressed in the function relating search reaction times to set size: The more visual attention is impaired, the steeper the slope of this function (as expressed in an increased effect of set size; Tales et al. 2002).\nOur predictions with regard to the effects of clonidine were the following: First, we expected that clonidine would impair performance on the attentional blink task, in particular through its stimulation of inhibitory \u03b12 autoreceptors in the LC. In accordance with the temporal filtering hypothesis, we expected that the clonidine-induced reduction in noradrenergic activity would lead to an impaired identification of T1 but especially of T2, because it should presumably exacerbate the lack of NE associated with T2 processing under normal conditions (Nieuwenhuis et al. 2005b). Second, aside from the well-documented main effect of clonidine on response speed (i.e., general slowing; e.g., Tiplady et al. 2005), we expected no drug effects on visual search efficiency, as indexed by the effects of distractor set size. This latter prediction was based on the notion that the effects of NE are topographically diffused and hence should not affect the efficiency of selection in the spatial domain.\nMaterials and methods\nSubjects\nThirty-two healthy young adults, aged 18\u201325\u00a0years, took part in a single experimental session in return for 60. Only subjects with a systolic blood pressure above 100\u00a0mmHg and a diastolic blood pressure above 70\u00a0mmHg were included in the study. All subjects underwent a medical screening and were considered to be in satisfactory health. The use of medication that could interfere with clonidine was stopped the day before. All subjects with color blindness were excluded from the study.\nSubjects received an oral dose of 150\u00a0\u03bcg of clonidine or placebo in a double-blind, between-subjects design. Clonidine has well-established antihypertensive properties; therefore, blood pressure and heart rate were monitored for subject safety. Measurements were taken every 15\u00a0min starting from t\u2009=\u2009\u221215 until t\u2009=\u2009120 and every 30\u00a0min from t\u2009=\u2009120 until t\u2009=\u2009240.\nThe clonidine group (N\u2009=\u200916, eight women, M age 21.3) and the control group (N\u2009=\u200916, eight women, M age 21.3) had similar mean ages. We also verified that the groups were similar in terms of intellectual functioning as assessed with the three-subtest version of the Groninger Intelligence Test (GIT), a reliable indicator of the full-scale IQ (Luteijn 1966). Estimated IQ scores were 117.1 for the clonidine group and 116.8 for the control group. The visual-search task data from one subject from the clonidine group were discarded because of an accidental failure to comply with the task instructions. The study was approved by the medical ethics committee of the VU University Medical Center and was conducted according to the Declaration of Helsinki. Informed consent was obtained from all subjects before their inclusion in the study.\nProcedure\nEach subject was tested at approximately the same time of the day (afternoon). Subjects were instructed to abstain from caffeine, nicotine, and other psycho-active substances from 15\u00a0h before the start of the session and to abstain from alcohol from 15\u00a0h before the start of the session until the day after the session. After the medical screening, subjects received a lactose-filled capsule with either clonidine or placebo. Immediately after that, the subtests of the GIT were administered. The two attentional tasks, described in detail below, were performed between t\u2009=\u200960 and t\u2009=\u2009120\u00a0min posttreatment. Half of the subjects in each group started with the attentional blink task. The other half started with the visual search task. After completion of the tasks, the subjects were debriefed and paid. At t\u2009=\u2009240, subjects were reevaluated and returned home by taxi if blood pressure was (near) normal.\nAttentional blink task\nStimulus generation and response recording in both tasks were controlled by E-Prime software (Psychology Software Tools, Pittsburgh, PA). Stimuli were presented in black against a light grey background. Each trial started with a fixation cross measuring 0.5\u2009\u00d7\u20090.5\u00b0, presented for 1,000\u00a0ms in the center of the display. Subsequently, the fixation cross was replaced by an RSVP stream of 15\u201322 uppercase letters, each measuring approximately 0.9\u2009\u00d7\u20090.9\u00b0. Each letter was randomly drawn (without replacement) from the alphabet and presented for 50\u00a0ms, followed by a 30-ms blank interval. This relatively rapid pace of stimulus presentation (i.e., 80\u00a0ms between two consecutive stimulus onsets) was chosen to avoid ceiling levels of performance and consequently to increase the sensitivity of the task to individual and group differences. \u201cI,\u201d \u201cO,\u201d \u201cQ,\u201d and \u201cS\u201d were left out as they resemble digits too much. On each trial, two of the letters were replaced with digits, randomly drawn without replacement from the set 2 to 9. T2 was presented three to six temporal positions from the end of the stream. The temporal distance between T1 and T2 was systematically varied from one to five items, corresponding to lags of 80, 160, 240, 320, and 400\u00a0ms. The subject\u2019s task was to identify both T1 and T2 by typing the digits in order on a standard keyboard after the end of the RSVP stream. Subjects were instructed to guess whenever they failed to identify a digit. The two keyboard entries were followed by the presentation of a feedback stimulus for 150\u00a0ms (e.g., \u2018+, \u2212\u2019 to indicate that T1 was correct and T2 was incorrect). After a 1,000-ms blank screen, the next trial started. Each subject started with 15 practice trials, three with each condition, randomly intermixed. This was followed by four blocks of 50 trials each with each block containing ten repetitions of each lag. All task instructions were automated and presented on screen.\nVisual search task\nOn each trial, subjects searched for a target among multiple distractors in a visual search array. On half of the trials, the target, a vertical red bar, was present in the array. On the other half of the trials, the target was absent. In one condition (single-feature search), the distractors in the search display were vertical green bars of equal size as the target. Because in this case the target is defined by a unique feature (color), it \u201cpops out\u201d from the display, resulting in a fast and efficient search process. In another condition (conjunction search), the distractors were vertical green bars and horizontal red bars. In this case, the target does not consist of a single identifying feature but is defined by a specific conjunction of features (color and orientation), resulting in a more time-consuming and error-prone search process.\nEach trial started with a white fixation cross measuring 0.9\u2009\u00d7\u20090.9\u00b0 against a dark background, presented for 500\u00a0ms in the center of the display. Subsequently, the fixation cross was replaced by the search display, which consisted of four, eight, or 16 items that were randomly plotted in the cells of an imaginary 6\u2009\u00d7\u20096 matrix (8.7\u00b0 horizontally\u2009\u00d7\u20099.6\u00b0 vertically) with some random jitter within the cells. The subject\u2019s task was to report whether or not the target (0.7\u2009\u00d7\u20091.3\u00b0) was present by giving a response with their left or right index finger using the \u2018z\u2019 and \u2018m\u2019 keys on the computer keyboard. The keyboard entry was immediately followed a 1,000-ms blank screen after which the next trial started.\nSubjects performed four blocks of 96 trials each, with each block containing 16 repetitions of the factorial combination of set size (4, 8, or 16) and trial type (target present or absent) presented in random order. Search condition (single-feature search or conjunction search) was varied across blocks in an ABBA-order. Half of the subjects started with single-feature search, and the other half started with conjunction search. Subjects received written instructions and 12 practice trials before entering the experimental phase. The task instructions encouraged subjects to respond as quickly as possible while minimizing the number of errors. Performance feedback was provided at the end of each block. All task instructions were automated and presented on screen.\nResults\nPhysiological measures\nFigure\u00a01 presents for both groups the absolute values of heart rate, systolic, and diastolic blood pressure as observed throughout the experimental session. All three measures showed a significant effect of time (all P\u2009<\u20090.001). Systolic [F(1, 30)\u2009=\u200911.8, P\u2009=\u20090.002] and diastolic blood pressure [F(1, 30)\u2009=\u20096.3, P\u2009=\u20090.018] both showed significant main effects of group. The main effects of time and group were qualified by significant time X group interactions for systolic [F(13, 390)\u2009=\u20099.8, P\u2009<\u20090.001] and diastolic blood pressure [F(13, 390)\u2009=\u20096.1, P\u2009<\u20090.001].\nFig.\u00a01Systolic and diasystolic blood pressure and heart rate following administration of placebo or 150\u00a0\u03bcg of clonidine (time\u2009=\u20090). The grey shaded area marks the time period during which the two cognitive tasks were administered\nAttentional blink task\nFigure\u00a02 shows the average T1 accuracy (left panel) and T2 accuracy (right panel; contingent on correct T1 identification) as a function of lag and group. A similar pattern of results was found if T2 accuracy was averaged across correct and incorrect T1 trials. Trials on which T1 and T2 were accurately identified but in the wrong order were treated as correct. As an analysis of variance (ANOVA) showed no significant effects of the order in which the two tasks were performed, this factor was omitted from the reported analyses.\nFig.\u00a02Average identification accuracy for the first (T1; left panel) and second (T2; right panel) of the two targets in the attentional blink task as a function of group (clonidine vs placebo) and the lag between T1 and T2. As is usual, T2 accuracy is reported contingent on accurate identification of T1\nT1 and T2 accuracy were entered in separate two-way mixed ANOVAs with lag and time-on-task (blocks 1,2 vs 3,4) as within-subject factors and group as a between-subject factor. The variable time-on-task was included to assess the possibility that a group effect emerged over time. T1 accuracy showed an increasing trend with lag [F(4, 120)\u2009=\u200912.3, P\u2009<\u20090.001]. The clonidine group (79.5% correct) performed slightly worse on T1 than the placebo group (84.6%), but the main effect of group did not reach significance [F(1, 30)\u2009=\u20092.0, P\u2009=\u20090.17]. The lag X group interaction was not significant [F(4, 120)\u2009=\u20091.0, P\u2009=\u20090.39].\nThe two T2 accuracy curves show a pattern that is characteristic of attentional blink research: lag-1 sparing, followed by a drop in performance for lags 2, 3, and 4 (i.e., the attentional blink), and followed by the initial recovery of performance at lag 5, at least in the clonidine group. This pattern was expressed in a significant effect of lag [F(4, 120)\u2009=\u200943.2, P\u2009<\u20090.001]. The most important for the present purposes is the finding that T2 accuracy did not reliably differ between the two groups [F(1, 30)\u2009<\u20091]. Furthermore, although at lag 4 there was a sizeable numerical effect of group in the expected direction, the lag X group interaction was not significant [F(4, 120)\u2009=\u20091.2, P\u2009=\u20090.30]. Time-on-task did not reliably affect T1 or T2 performance or interact with other variables.\nIn a separate analysis, we calculated the average T2 performance across lags 3\u20135 as a summary measure of attentional-blink magnitude and examined group differences in this measure. The two groups did not differ in attentional-blink magnitude, and performance at lags 3\u20135 did not improve with time-on-task [both F(1, 30)\u2009<\u20091]. Interestingly, there was a reliable interaction between group and time-on-task [F(1, 30)\u2009=\u20094.9, P\u2009=\u20090.034]: Whereas performance of the placebo group deteriorated over time (66.6 vs 62.2% correct; P\u2009=\u20090.65), performance of the clonidine group improved slightly (60.3 vs 62.6%; P\u2009=\u20090.28).\nIn a final analysis, we examined the percentage of order reversals at lag 1, the phenomenon that the two targets, when immediately succeeding each other, are often identified correctly but reported in the wrong order (Hommel and Aky\u00fcrek 2005). The percentage of such order reversals was roughly the same in the placebo group (34.4%) and in the clonidine group [35.4%; t(30)\u2009<\u20091].\nVisual search task: single-feature search\nThe total number of items in the search display (set size) was varied from trial to trial, allowing us to derive the function relating reaction time (RT) to set size. The slope of this function measures the cost for adding additional items to the display and is often interpreted as \u201csearch efficiency,\u201d with steeper slopes indicating slower, less efficient search. As expected and illustrated in Fig.\u00a03, slopes in the single-feature search condition were close to zero [average 1.6\u00a0ms\/item; t(30)\u2009=\u20092.6, P\u2009=\u20090.014].\nFig.\u00a03Average correct reaction times for the clonidine and placebo groups in the visual search task. Top panels show data from the single-feature search condition, and bottom panels show data from the conjunction search condition. Data for target-present trials are plotted in the left-hand panels, data for target-absent trials are plotted in the right-hand panels\nCorrect RTs were entered in a three-way mixed ANOVA with target presence (target present vs target absent) and set size (4, 8, or 16) as within-subject factors and group as between-subject factor. Despite the relatively shallow RT slopes, the main effect of set size was significant [F(2, 58)\u2009=\u20096.3, P\u2009=\u20090.006]. Furthermore, the clonidine group was overall slower than the placebo group [F(1, 29)\u2009=\u20095.6, P\u2009=\u20090.025]. Group did not interact with target presence [F(1, 29)\u2009=\u20091.2, P\u2009=\u20090.29] or set size [F(2, 58)\u2009=\u20092.5, P\u2009=\u20090.10]. The three-way interaction was also not significant [F(2, 58)\u2009<\u20091]. Error rates were generally low (average 2.3%). An ANOVA yielded only a significant main effect of target presence [F(1, 29)\u2009=\u20098.9, P\u2009=\u20090.006], with errors occurring more often on target-present trials (2.9%) than on target-absent trials (1.8%).\nVisual search task: conjunction search\nAs expected, slopes in the conjunction-search condition were sizeable [average 25.0\u00a0ms\/item; t(30)\u2009=\u200913.8, P\u2009<\u20090.001], indicating that visual search was time consuming and inefficient. A three-way mixed ANOVA yielded significant main effects of target presence [F(1, 29)\u2009=\u200931.5, P\u2009<\u20090.001] and set size [F(2, 58)\u2009=\u2009152.9, P\u2009<\u20090.001] and a significant interaction of these two variables [F(2, 58)\u2009=\u200926.7, P<\u20090.001], indicating that set-size effects were larger for target-absent trials. Importantly, the main effect of group was not significant [F(1, 29)\u2009=\u20092.1, P\u2009=\u20090.16], and group did not reliably interact with any of the task variables (all F\u2009<\u20091). Error rates were again low (average 3.3%). An ANOVA yielded the same pattern of effects as the RT analysis, indicating that there was no speed\u2013accuracy trade-off, and that there were no significant group differences.\nDiscussion\nThe main findings of the current study can be summarized as follows. First, in contrast to our predictions, clonidine did not have a deleterious effect on attentional blink performance. Although the numerical group differences in T1 and T2 accuracy were in the expected direction, these differences were small compared to the sizeable performance differences between the individuals within each group (for a discussion of these individual differences, see Martens et al. 2006). The results from the visual search task were as expected: Although clonidine slowed the overall response speed, it did not affect the efficiency of the visual search for a target in a two-dimensional array of stimuli. Below, we will discuss the implications of these principal findings.\nThe absence of a significant drug effect on attentional blink performance appears to be at odds with the temporal filtering hypothesis, which suggests that phasic activity of the LC\u2013NE system is important for the selection of important or infrequent stimuli in the temporal domain (Aston-Jones and Cohen 2005; Dayan and Yu 2006). The attentional blink results also seem inconsistent with the predictions of a recent theory that explains the attentional blink in terms of the temporal dynamics of the LC\u2013NE system (Nieuwenhuis et al. 2005b). One possible explanation for these discrepancies is that the predicted group differences do in fact exist, but that our task and experimental design were not sufficiently sensitive to reveal them. Some critical factors in this regard may be the choice of a between-subject design and the sample size of 16 subjects per group. However, previous studies using between-subject designs, similar group sizes, the same subject population, and the same task have had no difficulty detecting group-levels effect on attentional blink performance of various manipulations aimed at distracting subjects from the RSVP stream (Olivers and Nieuwenhuis 2005, 2006). Indeed, our choice of design was based on these previous studies and on the assumption that the effect of clonidine would be of at least the same size as the effect of these behavioral manipulations. In this context, it is worth noting that the interindividual variance in attentional blink performance was comparable to that in the Olivers and Nieuwenhuis studies, discounting increased variance as a reason for our failure to detect group differences.\nA more principled reason for the use of a between-subject design was that subjects can learn to adopt a \u2018mental state\u2019 that greatly benefits attentional blink performance (Olivers and Nieuwenhuis  2006), and we wanted to avoid the transfer of such learning between drug conditions to exclude a potential contaminating source of variance. In addition, Coull et al. (1995a, b) have found that in within-subject designs, the order of treatments (i.e., clonidine before or after placebo) can show complicated interactions with task performance, another source of variance that we aimed to exclude.\nAnother design feature that may have led to a failure to detect an existing effect is the timing of test administration relative to the treatment (t\u2009=\u200960\u2013120\u00a0min). In particular, the systolic and diastolic blood-pressure time series reveal a maximal drug effect after the test period, suggesting that cognitive effects may have peaked later too. However, there are at least three arguments against this possibility. First, Tiplady et al. (2005) have investigated the time course of effects of 150\u00a0\u03bcg of orally administered clonidine on cognitive test variables and found that such effects were already present at t\u2009=\u200945\u00a0min but were decreased at t\u2009=\u2009135\u00a0min. Second, our task-block comparisons show that, if anything, the group difference in attentional blink performance became smaller over time, a trend opposite from that expected if the effects of clonidine needed more time to develop. A third and related argument is that we found no significant interaction between group and task order, corroborating the notion that group effects did not become larger over time. Alternatively, we cannot rule out the possibility that group effects peaked earlier than our test period. However, our choice of test period seems justified by the finding of significant cognitive effects of clonidine in various previous studies using similar test periods (Halliday et al. 1989, 1994; Coull et al. 2001). Thus, our methods and results do not seem to contain any clear indication that the observed null effects reflect a lack of sensitivity. However, it is of course possible that a higher drug dose would yield more robust group differences. This is a possibility that can be addressed in future research (note that such research should take into account that the primary mode of action of clonidine changes with increasing dose; (Arnsten and Cai 1993). At low doses, presynaptic receptors and LC receptors are stimulated, leading to a reduced NE activity. In contrast, at higher doses, postsynaptic receptors are activated, resulting in a boost of NE activity. This has important implications for the expected effects of clonidine on cognitive function).\nAnother possible explanation for the absence of significant drug effects on attentional blink performance is that the attentional blink may not be mediated by the LC\u2013NE system. This possibility would be particularly bothersome for the theory of Nieuwenhuis et al. (2005b), which directly relates the attentional blink to a refractory period in activity of the LC\u2013NE system. Although there is substantial indirect evidence in support of this theory (e.g., the relationship between the attentional blink and the P3), the basic tenets of the theory are based on cell recordings in animals and are hard to validate directly in humans. Indeed, the current study is perhaps the most direct test to date of a relationship between the attentional blink and the LC\u2013NE system. In this context, it is important to note that another research group has reported preliminary evidence consistent with such a relationship (De Martino et al. 2005). This study, using a between-subject design, found that attentional blink performance was significantly impaired after intake of the \u00df-adrenergic receptor antagonist propranolol (40-mg oral dose compared to placebo). Although this finding is not predicted by the theory of Nieuwenhuis et al. (2005a, b), which relates the attentional blink to the dynamics of inhibitory \u03b12 autoreceptors in the LC, it will be informative to determine to what extent the current findings generalize to other task designs and other noradrenergic drugs. A further avenue for future research will be to test and evaluate the effects of noradrenergic drugs on temporal attention using alternative experimental paradigms, such as the temporal analogue of the spatial cueing paradigm (Posner et al. 1980; Coull and Nobre 1998; see Coull et al. 2001). Eventually, accumulating evidence obtained in such human psychopharmacological studies can be used to accommodate theories about the role of NE in temporal attention.\nThe results from the visual search task were consistent with our predictions: Although clonidine led to a general slowing of response speed, we found no evidence for drug effects on visual search efficiency as indexed by the effects of the distractor set size. This pattern of results was expected based on the notion that the effects of NE are topographically diffuse (i.e., not selective with regard to any specific spatial representations) and hence should not affect the efficiency of selection in the spatial domain. Previous studies using visuospatial cueing tasks have found that clonidine reduces the cue validity effect (Clark et al. 1989; Coull et al. 2001), which has led to the suggestion that the LC\u2013NE system is directly involved in visuospatial orienting. For example, it has been proposed that the LC\u2013NE system plays an important role in the disengagement of visuospatial attention from invalidly cued spaces (Clark et al. 1989; Posner and Petersen 1990). This proposal is consistent with the little available evidence, which suggests that the reduced validity effect after clonidine administration is mainly due to attenuated performance costs on invalidly cued trials. However, we propose an alternative interpretation for these results to reconcile them with our theoretical framework. According to our interpretation, the presentation of the cue causes a spatially nonspecific, transient LC\u2013NE response that boosts the processing of any stimuli presented for a brief period thereafter (i.e., the alerting effect). Although not supported by the current attentional blink findings, this assumption is based on various sources of evidence in the animal literature (Aston-Jones and Cohen 2005; Witte and Marrocco 1997). The cue-induced LC\u2013NE response speeds up cortical spatial selection processes involved in localizing the cue and\/or subsequent imperative stimulus. This improves performance on validly cued trials and impairs performance on invalidly cued trials thus increasing the validity effect. Administration of clonidine counteracts this amplifying effect of phasic LC\u2013NE activity thereby reducing the validity effect (note that this hypothesis predicts both improved performance on invalid trials and impaired performance on valid trials). This type of interaction between the alerting effect and spatial selection processes is absent in the visual search task, which has no clear alerting component. Therefore, the current results are consistent with the notion that the LC\u2013NE system is not directly involved in visuospatial orienting but can only indirectly modulate the effects of spatial attention mechanisms implemented elsewhere in the brain.\nOne drawback of the current study is that we did not include a task that has already been shown to be sensitive to clonidine in previous studies. Therefore, a possible interpretation of the reported null findings is that the administered dose of clonidine failed to affect general cognitive function in our subjects, and that the main effect of drug on visual search RT reflects drug-induced motor slowing rather than a central cognitive deficit. Although we deem this possibility unlikely, given that previous studies using similar doses of clonidine have found significant effects on cognitive function (Coull et al. 2001; Tiplady et al. 2005), a more extensive study with a larger task battery is needed to adequately address this issue.\nThere is rapidly growing interest in the specific role of the LC\u2013NE system in human cognition (Cohen and Aston-Jones 2005). Psychopharmacological data from humans will be of critical importance in testing and further developing the increasingly sophisticated hypotheses that are based on neurophysiological observations in animals (e.g., Yu and Dayan 2005). The current research provides a valuable contribution toward a better understanding of the role of the LC\u2013NE system in human attention, both in the spatial and the temporal domain.","keyphrases":["clonidine","attention","attentional blink","visual search","norepinephrine","locus coeruleus"],"prmu":["P","P","P","P","U","M"]}
{"id":"Eur_Child_Adolesc_Psychiatry-2-2-1705530","title":"The effect of birth-weight with genetic susceptibility on depressive symptoms in childhood and adolescence\n","text":"Low birth-weight has been associated with depression and related outcomes in adults, and with problem behaviours in children. This study aimed to examine the association between low birth-weight for gestation and depressive symptoms in children and adolescents and to examine whether the relationship is moderated by genetic risk for depression. An epidemiological, genetically sensitive design was used including 2,046 twins aged 8\u201317 years (1,023 families). Data were obtained by parental report and analysed using regression analysis. A small but significant association between birth-weight for gestation and early depressive symptoms was observed. The unit increase in depressive symptoms per unit decrease in birth-weight for gestation was greater for individuals at genetic or familial risk for depression. For low birth-weight children, genetic risk for depression moderated the influence of birth-weight for gestation in predicting early depressive symptoms. Birth-weight for gestation is moderated by genetic and familial risk for depression in influencing early depression symptoms. These observations have clinical implications in that the impact of being small for gestational age on depressive symptoms is greater in children at familial\/genetic risk although the association between birth weight and depression does not imply causality.\nIntroduction\nSeveral cohort studies have shown associations between indexes of foetal nutrition such as pre-natal famine [6] and low birth-weight (LBW) with depression and related phenomena in adults [3, 7, 49]. However, the association between birth-weight for gestation and depressive symptoms in childhood has not been well examined. Instead, studies of the association between birth-weight and difficulties in childhood have focused on total behavioural problems or externalising problems [20, 52, 56]. Follow-up studies of children who were LBW at birth have reported that these children show a number of difficulties, in particular disruptive behavioural problems and reduced IQ in comparison to children who were born with normal weight at term [4, 20]. Although there have been relatively few studies that have examined the prevalence of depression in LBW children, there are now a growing number of reports illustrating that LBW children also show elevated rates of depressive symptoms [5, 17]. In addition, one recent nested case\u2013control study of adolescent depression found increased rates of depressive disorder in adolescents who were either premature or LBW [32].\nPreliminary evidence suggests that early adversity may modify the link between birth weight and depression in adults, with one study finding a stronger association in those adults exposed to more adverse socio-economic conditions during childhood [15]. One other factor that may potentially modify the relationship between birth weight and depressive symptoms is familial or genetic risk for depression. Genetic factors are known to be important in the aetiology of depression [29, 37]. To our knowledge, no study has examined the impact of genetic risk for depression on the association between birth-weight and depression in early life. We used a population-based twin register to first examine the association between birth-weight and depressive symptoms and then to test whether this effect was moderated by familial and genetic risk for depression.\nMethods\nParticipants\nA total of 1,581 families, a sub-sample from a systematically ascertained, population-based register of all twin births between 1980 and 1991, the Cardiff Study of All-Wales and North-west England Twins (CASTANET) were invited to participate. Twins on this register were identified through child health databases and the characteristics of the sample have been described in detail elsewhere [38, 47] but are representative of the local population in terms of social class and ethnicity. The percentages of families from social class groups I, II, III, IV and V were 11.4%, 29.3%, 30.1%, 4.9% and 1.4%, respectively, with the remainder classified as at-home or unemployed. These figures are in keeping with those expected from a population from Greater Manchester [36]. The present wave of data collection took place in 2000 including twins from the register aged less than 17\u00a0years (range 8\u201317) and in full-time education born in Greater Manchester. The mean age of participating twins was 11.17 (standard error=0.068). Completed questionnaires were received from 1,023 families, giving an overall response rate of 65% (1,023\/1,581). Data on birth-weight, antenatal and perinatal factors and social class had been collected 3\u00a0years previously. There were 934\/1,545 twin pairs who took part at both assessment points (60%). (Note: There were 934 pairs who took part in both waves of the study but missing computed scores for some variables meant that they were not included in regression analyses). There were no significant differences between those individuals who had complete data available and those who did not take part at Time 2 in terms of birth weight (t=0.545, SE=0.15, P=0.586) or those who did not take part at time 1 in terms of depressive symptoms (t=0.730, SE=0.06, P=0.465). In addition, information on emotional symptoms as assessed by parental report on the Rutter questionnaire (40) at Time 1 did not reveal any significant differences between those with complete data and those who did not participate at Time 2 (t=1.114, SE=0.06, P=0.465). Multi-centre ethical approval was obtained. The study was described to participants and informed consent was obtained following return of the completed questionnaire package. Complete data on birth weight, gestation and depressive symptoms were available for 370 monozygotic (MZ) and 495 dizygotic (DZ) pairs. Zygosity was assigned according to parent responses to the twin similarity questionnaire, which has been found to be over 90% accurate in distinguishing MZ and DZ twins. An algorithm based on previous work was used [8, 47].\nMeasures\nMothers of the twins reported information on antenatal and obstetric factors using a questionnaire adapted from Lewis & Murray [27], which included the twins\u2019 birth-weights, number of weeks of gestation at birth, maternal age at birth and maternal smoking during pregnancy (yes\/no). Mother reports of birth weight and gestation have been found to be very accurate in comparison to antenatal records with correlations of over 0.90 on average [16, 51]. Social class information was also collected and classified according to Standard Occupational Classification [31]. Depression in the children and mothers was assessed 3\u00a0years later in the year 2000. Child depressive symptoms were assessed using the Mood and Feelings Questionnaire (MFQ) [11]. The MFQ is a 34-item scale based on DSM-III-R symptoms of depression that is a reliable quantitative measure of depression and a useful screening questionnaire for clinical depression in children in the community [11, 48] as well as in clinical populations [57]. Parent reports of children\u2019s depression symptoms were used as child reports are only reliably obtained for those over the age of 11\u00a0years. The mean depression score for the sample was 9.15 (range 0\u201360). Boys\u2019 scores (mean=8.63; range 0\u201343) were lower than those of girls (mean=9.59; range=0\u201360) although this difference was not significant (t=\u22121.45, SE=0.05, P=0.15). Given that there was no significant gender difference in mean symptom counts for the sample, and no evidence for gender differences in the magnitude of the genetic influence on parent-rated depressive symptoms in a previous analysis of this sample [38], the primary analyses were conducted using the combined sample rather than separately for each gender. Parents also completed the depression sub-scale of the Hospital Anxiety and Depression Scale (HADS) [59] about their own depression symptoms in the past week. The mean depression score for parents was 4.46 (range 0\u201321). The majority of analyses were based on total symptom scores given that there are strong arguments for regarding depression as a continuous dimension of risk rather than as a categorical disorder (affected\/unaffected) both in adults and in children. Firstly, studies of adults and children show that depressive symptoms behave as a continuum with no evidence of increased morbidity once clinical diagnostic criteria are met [23, 51]. For example, Pickles & colleagues [33] found that number of depressive symptoms but not functional impairment predicted future depressive episodes. Second, sub-clinical depressive symptoms are associated with impairment [2, 26]\u2014one of the main criteria for fulfilment of disorder. Third, sub-clinical symptoms are associated with service use and significantly increase the risk for depressive disorder in adulthood [21, 34]. Moreover, it appears that a continuous measure of depression is more indicative of genetic risk in children and adolescents. Several studies have found that the aetiology of very high levels of depressive symptoms is less influenced by genetic factors than depressive symptoms across the normal range [13, 14, 38]. Thus, at least in children, symptoms of depression may give a better indicator of genetic risk than categorical diagnoses. Nonetheless, because of the suggestion of aetiological heterogeneity, depressive symptoms were analysed both as a continuous and a categorical measure in this report. Birth weight was examined both as a dimensional measure and as a category since both birth weight across the continuum and LBW have been reported to be associated with depression in adults [7, 15, 32].\nStatistical analysis\nData were analysed using STATA [45] and SPSS [46]. We adopted two approaches for testing the effect of birth-weight for gestation and genetic\/familial risk. First, genetic regression analysis [22, 56] was used to test for genetic effects and for interaction of genetic risk and birth-weight. We made use of the twin design that relies on the observation that identical (monozygotic; MZ) twins share all of their genes in common (coefficient of relationship=1), while on average, non-identical (dizygotic; DZ twins) share only half of their genes in common (coefficient of relationship=0.5). Genetic regression analysis is undertaken using standard forced entry linear regression analysis with one twin\u2019s depression symptom scores being the dependent variable. The twin\u2019s depression score was predicted using the formula T=B0+B1 C+B2 R+B3 I+B4 M+B5 G where T=the twin depression score, B0=the regression constant, C=the co-twin depression score, R=a coefficient of genetic relatedness (0.5 for DZ twins; 1 for MZ twins), I is the interaction term (C*R*birth-weight corrected for gestation), M=the main effect of birth-weight (with the length of gestation regressed out) and G=twin gender. One twin\u2019s depression score was entered as the dependent variable, with co-twin depression, genetic relatedness and the interaction term as independent variables. Regression analyses were carried out using both continuous and categorical measures of birth-weight corrected for gestational age. Low birth-weight for gestation was classified as \u226410th centile since standard published cut points were not available for birth weights of twins.\nNext, linear regression analysis was used to test for a main effect of birth-weight on depressive symptoms. The survey commands in STATA were used to take account of clustering within twin pairs by likening the twin data to a two-stage cluster design with the twin pairs as the primary sampling unit. A robust variance matrix calculation is utilised that relaxes the assumption of independence within groups. Birth-weight was corrected for number of weeks gestation at birth by regressing out the effect of length of gestation and using the unstandardized residuals for further analysis. As boys tended to be heavier at birth than girls, and the association between birth-weight and depressive symptoms may differ by gender. The effect of sex was controlled for by including sex as a predictor variable in the regression model. A number of factors have been reported to be associated with LBW [12]. Information on three potential influences was available\u2014maternal smoking during pregnancy, social class and maternal age at birth. Social class was not associated with birth weight for gestation (b=\u22124.20, SE=3.32, P=0.206) or depressive symptoms (b=0.147, SE=0.107, P=0.169) in the present sample. Maternal pre-natal smoking (yes\/no) was inversely associated birth weight for gestation (b=\u2212141.62, SE=18.32, P=0.001) and positively associated with depressive symptoms in the offspring (b=2.16, SE=0.618, P=0.001). Maternal age at birth of child was negatively associated with child depressive symptoms (b=\u2212.143, SE=0.053, P=0.007) and positively associated with twin birth weight (b=6.68, SE=1.65, P=0.001). These three variables were entered as predictor variables in regression models in addition to child gender.\nThe second approach to test the effects of birth-weight and genetic\/familial risk was to use standard regression analysis but this time defining (1) genetic and (2) familial risk according to cut points on the depression questionnaires for (1) the co-twin and (2) the mother. Regression analysis was used to estimate the unit increase in depressive symptoms per unit decrease in birth weight for those individuals at genetic risk or family risk of depression and those not at risk. Both familial risk and genetic risk for depression was used for this analysis because to estimate the unit increase in symptoms by genetic risk meant restricting the analysis to MZ twins and thus a substantial reduction in sample size. Using familial risk allowed results to be replicated using the much larger full sample. Furthermore, using mother\u2019s depression to index familial risk rather than the DZ twins allowed the entire sample to be double entered i.e. each twin to be entered as an individual in the analysis. This is because maternal depressive symptoms is a variable that is obligatory shared for each member of a twin pair. Appropriate correction for the non-independence of twins was undertaken using the survey commands in STATA as described above. For genetic risk, birth-weight and gestation were entered as independent variables to predict MZ twin depressive symptoms for MZ twins individuals without an affected co-twin (MZ co-twin depressive symptoms <90th centile) and those with an affected co-twin (MZ co-twin depressive symptoms \u226590th centile). This analysis was then repeated for the whole sample using parent depression symptoms as the cut point (parental depression score \u226590th centile) as a measure of familial risk. Dummy coded groups [9] were then analysed to assess whether there was an interaction between genetic or familial risk for depression and birth weight in predicting early depressive symptoms. These dummy variables allow the difference between the beta coefficients for the low-risk and high-risk groups to be tested.\nResults\nDescriptives\nParent-rated MZ mean depression symptoms were lower than those of DZ twins but this was not significant (MZ mean=8.97, standard deviation=9.49; DZ mean=9.14, standard deviation=8.92; t=0.980, df=967, P=0.327). There were no significant differences on the ante-natal and labour complications assessed according to zygosity (admission to hospital for high blood pressure \u03c72=0.328, P=0.609, vaginal bleeding \u03c72=0.088, P=0.800, labour less than 3\u00a0h \u03c72=1.035, P=0.345, emergency Caesarean, \u03c72=2.270, P=0.151, maternal smoking during pregnancy, \u03c72=0.192, P=0.697, forceps or ventouse delivery? \u03c72=1.398, P=0.237).\nDescriptive statistics for the birth-weight and weeks gestation of twins are shown in Table\u00a01. It can be seen that second-born twins were lighter at birth than first-born twins. MZ twins were lighter than DZ twins although they also tended to be born earlier in pregnancy. The average birth-weight for the entire sample was 2469.81\u00a0g (standard deviation=553.38) and the average length of gestation was 36.36\u00a0weeks (standard deviation=2.87).\nTable\u00a01Birth-weight in grams and gestation in weeks by zygosity, gender and birth order for twins with complete data pointsNMeanStandard deviationFirst-born twin birth-weight8892484.55544.03Second-born twin birth-weight8892457.62562.17MZ first-born birth-weight3812404.32531.55MZ second-born birth-weight3812392.41545.44DZ first-born birth-weight5082544.65546.01DZ second-born birth-weight5082506.10570.11Girl first-born birth-weight4892445.43544.59Girl second-born birth-weight4892426.00560.75Boy first-born birth-weight4002532.18540.06Boy second-born birth-weight4002494.76562.45MZ duration of gestation (weeks)37136.443.00DZ duration of gestation (weeks)49735.992.75Girls weeks gestation48336.312.87Boys weeks gestation38536.182.87\nMain effect of birth-weight for gestation\u2014standard regression analysis\nLinear regression showed that birth-weight for gestation significantly predicted parent-rated child depression symptoms when controlling for child gender (b=\u22120.001, SE=0.001, P=0.003, 95% CI \u22120.003, \u22120.0006) (n=1,740). This remained significant when child gender, maternal age at birth, pre-natal smoking and social class were also included in the regression model (b=\u22120.001, SE=0.001, P=0.015, 95% CI=\u22120.003, \u22120.0003). This illustrates that children who were small for gestation had higher depressive symptoms in childhood and adolescence.\nGenetic regression analysis\nThe results of genetic regression analyses with depression as a continuous variable are shown in Table\u00a02. It can be seen from Table\u00a02 that birth-weight for gestation, genetic risk and maternal smoking during pregnancy showed main effects on depressive symptoms. When birth-weight for gestation was included as a continuous variable there was no birth-weight by genetic risk interaction effect although there was a slight trend (\u03b2=\u22120.048, P=0.096) (n=861). Data were also analysed using LBW as a categorical variable. Significant main effects of birth-weight for gestation, genetic risk, pre-natal smoking and a significant genetic risk by LBW interaction were found (\u03b2=0.394, P=0.001) (n=861). This relationship is illustrated graphically in Fig.\u00a01 and shows a stronger relationship between genetic risk (C*R) and depressive symtpoms for those individuals who were small for gestational age at birth (\u226410th centile). This suggests that it is LBW for gestational age that interacts with genetic risk for depression when depression is assessed as a continuous variable.\nTable\u00a02Genetic risk for depression and birth-weightBeta (birth-weight as a continuous variable)P valueBeta (low birth-weight for gestation as a categorical variable)P valueInteraction between birth-weight corrected for gestation & genetic risk\u22120.048a0.0960.394*0.001 Birth-weight for gestation\u22120.081*0.006\u22120.075*0.032Co-twin depressive symptoms0.541*0.001 0.212*0.002Twin gender0.044a0.1300.049a0.080Zygosity0.0270.349\u22120.096*0.010Maternal pre-natal smoking (yes\/no)0.063*0.0330.067*0.021Social class0.0110.7050.0110.700Maternal age at birth\u22120.0240.411\u22120.0180.529aP<0.1*P<0.05Fig.\u00a01Genetic risk and depressive symptoms by birth-weight for gestation\nAnalyses of depression as a categorical variable\u2014standard regression analysis\nWhen twins were classified at genetic risk of depression if they had an affected MZ co-twin, the coefficients derived showed that for those twins at genetic risk for depression (N=32), for each 1\u00a0kg decrease in birth weight corrected for gestation, gender, social class, maternal age at birth and pre-natal smoking there was an increase of 2 depressive symptoms. As birth weight was measured in grams, this was calculated by the regression coefficent multiplied by 1000 (\u22120.002\u00d71,000=\u22122). For those identical twins (N=338) not at genetic risk, the increase in depressive symptoms for each 1\u00a0kg decrease in birth weight was 1 symptom (\u22120.001\u00d71,000=\u22121). Thus, there was a two fold increase in the number of depressive symptoms for each 1\u00a0kg decrease in birth-weight for twins at genetic risk (2 vs. 1 symptom) when controlling for covariates. Similarly, for each decrease in birth-weight of 1\u00a0kg there was an increase of 7 depressive symptoms for those at familial risk of depression (N=196) (\u22120.007\u00d71,000=\u22127) and an increase of 1 depressive symptoms for those not at familial risk of depression (N=1478) (\u22120.001\u00d71,000=\u22121) representing a seven-fold increase in number of symptoms.\nRegression with dummy coded variables showed that the effect of birth-weight on depressive symptoms was not significantly different for those at high genetic risk of depression compared to those not at genetic risk of depression (for interaction term, \u03b2=0.009, P=0.853). It should be noted that regression with dummy coded variables for genetic risk of depression required restriction to MZ twins only and thus a substantial reduction in sample size. There was, however, a significant interaction according to familial risk (\u03b2=\u22120.086, t=\u22122.381, P=0.017).\nDiscussion\nDepressive symptoms in young people show strong similarities with depression in adulthood [18, 19] and are associated with a number of deleterious health outcomes including increased risk for depressive disorder in adulthood; impaired educational and social functioning; increased service use and deliberate self-harm [2, 18, 21, 26, 34]. We find that genetic risk interacts with LBW for gestation in influencing depression. Standard regression analysis treating birth-weight for gestation as a continuous variable also showed that familial risk but not genetic risk interacts with birth-weight. These results suggest that being at genetic or familial risk for depression increases the influence that being small for gestational age has on depression. This result suggests that there may be public health implications for the future mental health of infants who are small for gestational age if there is also a family history of depression.\nResults from genetic and standard regression analysis showed broadly similar results. However, results from genetic regression analysis suggested that LBW for gestation has the strongest effect on depressive symptoms in the context of genetic risk, rather than birth-weight for gestation across the continuum. It is possible that defining genetic risk according to cut points on the depression questionnaire may not be as robust an approach as examining depression as a continuum. Many studies of childhood depressive symptoms have reported estimates of genetic aetiology substantially lower for high depression scores than for scores within the normal range [13, 14, 38]. The results of studies reviewed earlier which suggest that depression may be viewed as a continuum [2, 21, 22, 26, 33, 34, 51] suggest that in the present study, results from analyses where depression is included as a continuous variable may therefore be more robust.\nIt is unclear whether birth-weight is a \u2018causal\u2019 risk factor for depression in children and adolescents, although some studies suggest that LBW is an important environmental risk factor for behavioural and cognitive problems [25, 52, 56]. However, the purpose of this paper was not to assess causality, but rather, to examine the association between birth-weight and depression and whether it was modified by genetic and familial risk for depression. Birth-weight has several properties that make it a useful risk factor for examining potential gene-environment interaction effects [25, 42]. First, birth-weight is primarily influenced by environmental rather than genetic factors. For example, in this sample, examination of the MZ and DZ correlations for birth-weight showed that birth-weight was influenced primarily by shared environmental rather than genetic factors as the MZ and DZ correlation coefficients are similar (rMZ=0.781; rDZ=0.697) (full model fitting results available from first author). Moreover, there was little evidence that there were common genes influencing both depressive symptoms and LBW as the cross-twin cross-trait correlations in this sample were near zero for both MZ and DZ twins. This is not true of other risk variables, indeed, there is consistent evidence that many important environmental risk factors (e.g. smoking in pregnancy) are not independent of genetic predisposition [42]. As many risk factors or indicators are influenced by genetic factors this can cause difficulty in detecting and interpreting gene by environment interactions [41, 44]. Second, LBW for gestation does not appear to be associated with maternal depression during pregnancy in developed countries [1, 35, 43, 58]. Indeed, there was no association between current maternal depressive symptoms and birth-weight corrected for gestation in this sample (rtwin 1 birth-weight=0.021, P=0.540; rtwin 2 birth-weight=0.001, P=0.975). This observation reduces the possibility that the influence of birth-weight on depressive symptoms observed in the present study is due to an association with maternal depression although it does not rule out the possibility. Birth weight is influenced by a number of psychosocial and maternal health characteristics including social class, pre-natal smoking, maternal height, weight, age and parity. There were three such factors that have previously been found to influence birth weight, for which data were available in this study (social class, pre-natal smoking, maternal age at birth of child). These were included as predictor variables in regression models in addition to child gender, thus effects for birth weight adjusted for gestation in the present study are independent of these variables although we were not able to control for all variables that have previously been associated with birth weight (e.g. maternal weight at birth).\nIn the present study, depression was assessed according to current symptoms. It is likely that this will underestimate the genetic risk for depression as life time ever rates of depression are significantly higher than point prevalence estimates [10, 24, 39]. We also relied on symptoms rather than diagnoses of depression. However, there is compelling evidence that depression appears as a continuum. Moreover, in particular for tests of interaction, statistical power is low [9, 42] and thus power is substantially increased with the inclusion of continuous rather than categorical measures. The reliance upon parental reports of birth-weight and antenatal risk factors in the present study is not ideal\u2014information from antenatal records would have been preferable. However, the retrospective recollection of birth-weight of children by parents has been found to be accurate in comparison to medical records with correlations of over 0.90 between the two data sources reported [16, 54]. In addition, retrospective recall of pregnancy related events has been shown to be reliable [50]. The reliance on parental reports of the twins\u2019 emotional state may have influenced results. Maternal reports of child psychopathology have been reported to be influenced by the mother\u2019s own mental state. For the most part, it has been found that this results in inflated levels of emotional problems in children being reported by mothers who have emotional difficulties themselves. This is consistent with evidence that depression runs in families, but also raises the possibility that familial risk for children may be partly attributable to rater effects. However, there is some evidence that depressed mothers can be more sensitive to depression in their children [55]. In addition, maternal reports of anxiety and depression are predictive of outcome in young adulthood, which lends some predictive validity to maternal reports of emotional problems in children [53]. Assortative mating has been reported for depression, though reported spousal correlations for depression have been relatively small [28]. Information on the father\u2019s mental state was not available in this study. Assortative mating for depression would result in an inflated DZ twin correlation and thus reduced evidence for genetic influences [30] and we have previously found a genetic influence for depressive symptoms in this sample [38]. One final potential criticism is that birth-weight in twins may not mean the same as in singletons. However, the twin design was necessary to allow for the effects of genetic influences and until susceptibility genes for depression are identified, molecular genetic indicators of risk for depression are not feasible.\nIn summary, the present results suggest that LBW for gestation and genetic\/familial factors co-act and interact in influencing early parent-rated depressive symptoms. These observations suggest that it may be useful to monitor children in cases where there is a family history of depression and the child was small for gestational age.","keyphrases":["gene","depression","child","twin","birth weight","interaction"],"prmu":["P","P","P","P","P","P"]}
{"id":"Crit_Care-8-6-1065076","title":"Tight blood glucose control: a recommendation applicable to any critically ill patient?\n","text":"The issue of tight glucose control with intensive insulin therapy in critically ill patients remains controversial. Although compelling evidence supports this strategy in postoperative patients who have undergone cardiac surgery, the use of tight glucose control has been challenged in other situations, including in medical critically ill patients and in those who have undergone non-cardiac surgery. Similarly, the mechanisms that underlie the effects of high-dose insulin are not fully elucidated. These arguments emphasize the need to study the effects of tight glucose control in a large heterogeneous cohort of intensive care unit patients.\nUntil the end of the past millenium, relatively little attention was given to control of blood sugar levels. In critically ill patients, hyperglycaemia was considered to be physiological because it results from the metabolic and hormonal changes that accompany the stress response to injury. In most intensive care units (ICUs), blood sugar was checked every 4\u20136 hours and hyperglycaemia (defined as blood sugar levels >10\u201312 mmol\/l [180\u2013216 mg\/dl]) was corrected by subcutaneous or intravenous insulin. The presence of pre-existing diabetes mellitus or post-neurosurgical status often prompted more intense control of hyperglycaemia. Furthermore, the issue of glucose control was discussed in few sessions or satellite symposia during intensive care meetings.\nThe deleterious effects of hyperglycaemia during critical illness have been characterized over the past few years, and include an increased susceptibility to infections and thromboses, macrovascular and microvascular changes, and delayed wound healing, among other effects (for review [1]). Renewed interest in control of hyperglycaemia in critically ill patients (Fig. 1) followed the publication of a study conducted by Van den Berghe and coworkers in 2001 [2]. Those investigators reported a 43% decrease in relative intensive care mortality as well as consistent decreases in several surrogate markers of disease severity in patients randomly assigned to tight glucose control by intensive intravenous insulin therapy. A post hoc multivariate logistic regression analysis of these data suggested that control of hyperglycaemia played a more important role than did the amount of insulin administered [3]. Interestingly enough, at least two recent retrospective, large-scale studies [4,5] confirmed that outcome was improved in patients whose average blood glucose was maintained below 8 mmol\/l (144 mg\/dl; Table 1).\nAlthough the findings reported by Van den Berghe and coworkers are impressive, some concern arose regarding the applicability of these results to other types of patients. Of the patients studied, 63% were admitted for follow up after cardiac surgery; this high proportion was felt to be consistent with a particular benefit from tight glucose control with intensive insulin in these patients, but there is uncertainty regarding whether tight glucose control is beneficial in patients who have not undergone cardiac surgery. Fear of life-threatening hypoglycaemia and increased workload and costs probably underlie the reluctance of many intensivists to launch systematic protocols of tight glucose control. Indeed, many intensivists still use a high glucose threshold (10 mmol\/l [180 mg\/dl]) [6]. In a European survey (unpublished data) we found considerable variation in the glycaemic thresholds employed in ICUs, which ranged from 6 to 11.1 mmol\/l (108\u2013200 mg\/dl).\nSome arguments against generalized use of tight glucose control are reported in the present issue of Critical Care by Vriesendorp and coworkers [7]. In a retrospective study performed at one centre in Amsterdam, those authors found that, after oesophageal surgery in patients without significant cardiovascular compromise (ASA class I\u2013II), postoperative hyperglycaemia was not a risk factor for infectious complications. Only by univariate analysis were they able to find an improvement in patients with blood glucose levels below 9.3 mmol\/l (167 mg\/dl) in terms of length of ICU stay. These findings differ strikingly from those of other studies [2,4,5]. Although the report by Vriesendorp and coworkers challenges the concept of tight glucose control, it can hardly be considered a major piece of evidence against it. Indeed, blood glucose concentrations were presented as means of values recorded only over 48 hours, whereas the ICU stay extended up to 71 days, with a median of 3 days. Insulin was administered to only 9% of the patients during the 48-hour period of observation. In addition, patients received a mean of only 22.5 g glucose\/day, and were fed early after surgery with an enteral solution of 'immunonutrients' \u2013 a potential confounding factor with respect to infectious morbidity. However, despite these limitations, as well as others that are acknowledged by the authors, the findings of the study support the hypothesis that tight glucose control could be of greater benefit to patients with cardiovascular disease than to those without.\nIn conclusion, as recently suggested by Van den Berghe [8], further studies are needed to confirm the benefits of tight blood glucose control with intensive insulin therapy in a heterogeneous population of ICU patients. Hence, a large randomized prospective multicentre trial is warranted. Such study will also help in determining the physiological importance of the effects of insulin and, more importantly, will provide intensive care workers with key information for guiding the management of blood glucose in critically ill patients.\nAbbreviation\nICU = intensive care unit.\nCompeting interests\nThe author(s) declare that they have no competing interests.","keyphrases":["critically ill","insulin","cardiac surgery","metabolism","hyperglycemia"],"prmu":["P","P","P","P","U"]}
{"id":"J_Mol_Biol-1-5-2291452","title":"Crosstalk between the Protein Surface and Hydrophobic Core in a Core-swapped Fibronectin Type III Domain\n","text":"Two homologous fibronectin type III (fnIII) domains, FNfn10 (the 10th fnIII domain of human fibronectin) and TNfn3 (the third fnIII domain of human tenascin), have essentially the same backbone structure, although they share only \u223c 24% sequence identity. While they share a similar folding mechanism with a common core of key residues in the folding transition state, they differ in many other physical properties. We use a chimeric protein, FNoTNc, to investigate the molecular basis for these differences. FNoTNc is a core-swapped protein, containing the \u201coutside\u201d (surface and loops) of FNfn10 and the hydrophobic core of TNfn3. Remarkably, FNoTNc retains the structure of the parent proteins despite the extent of redesign, allowing us to gain insight into which components of each parent protein are responsible for different aspects of its behaviour. Naively, one would expect properties that appear to depend principally on the core to be similar to TNfn3, for example, the response to mutations, folding kinetics and side-chain dynamics, while properties apparently determined by differences in the surface and loops, such as backbone dynamics, would be more like FNfn10. While this is broadly true, it is clear that there are also unexpected crosstalk effects between the core and the surface. For example, the anomalous response of FNfn10 to mutation is not solely a property of the core as we had previously suggested.\nIntroduction\nStudies of the folding of structurally related proteins have been a\npowerful tool for investigating conservation of folding pathways,1 identifying structurally important residues,2 and examining the role of highly conserved\nresidues.3 One of the most common folds in the SCOP database4 is the immunoglobulin (Ig)-like fold. Over 40,000 Ig-like\ndomains have been identified in the current PFam database5 and the fold is found in a number of different superfamilies,\nwhere there is no apparent sequence identity between superfamilies.4 In this study, rather than seeking to understand what related\nproteins have in common, we ask a different question\u2014can differences between\nclosely related proteins be explained?\nMany members of the Ig-like fold have been well characterised, leading to:\nidentification of the key residues essential for formation of the Greek key\nstructure,6,7 the observation of a correlation between stability and folding\nrate,8\u201311 the identification of a common folding pathway,12\u201316 and the identification of the role of conserved proline\nresidues3 and the conserved tyrosine corner motif.2 However, when two Ig-like domains from the fibronectin type III\n(fnIII) superfamily, the 10th fnIII domain of human fibronectin (FNfn10) and the\nthird fnIII domain of human tenascin (TNfn3), were studied in detail and\ncompared, they were found to differ markedly in several respects. FNfn10 and\nTNfn3 are essentially structurally identical (backbone RMSD is 1.2\u00a0\u00c5), but have\nlow sequence identity (24%). They differ in their stabilities17,18 and response to mutation19; they differ in their folding kinetics17,18,20 (although they share a common folding mechanism12,16); they display different backbone and side-chain\ndynamics21\u201323`; and, finally, they differ in their response to mechanical\nforce.24\u201326 Why do proteins that are structurally almost identical behave\nso differently? Which components of a protein are responsible for the various\naspects of its behaviour? How independent are the properties of the surface and\nhydrophobic core? We have addressed these questions by making a chimera of these\ntwo fnIII domains.\nThe chimera, FNoTNc, was created with the \u201coutside\u201d (surface and loops) of\nFNfn10 and the core of TNfn3.26 Fifteen mutations were made in the core of FNfn10 so that all\nburied residues (with <\u00a010% solvent-accessible surface\narea) are identical with those residues in the core of TNfn3 (total number of\ncore residues\u00a0=\u00a027) (Fig. 1). Thus, we\ncan test how transferable the properties of the core and surface of the\nrespective parents are when combined in this way. FNoTNc is a stable, folded\nprotein that is structurally almost identical with the parent proteins\n(Supplementary Fig. 1). FNoTNc has\nalso retained the cell-adhesion activity of the FNfn10 parent protein mediated\nby specific integrin binding.26 We have previously used this chimera to demonstrate that\nresistance to mechanical force is a core property: FNoTNc has mechanical\nunfolding properties indistinguishable from TNfn3 and very different from\nFNfn10.26 Here we investigate the stability, folding and dynamics of the\nnew, chimeric protein.\nMost protein engineering analyses concentrate on the hydrophobic core of\nproteins, since it was shown in 1959 that the hydrophobic interaction is the\nmajor factor involved in protein folding27 and surface mutations rarely affect protein stability by more\nthan \u223c\u00a01\u00a0kcal mol\u2212\u00a01. Our\nresults suggest, however, that the surface and loops can play a key (and\nsometimes unexpected) role in determining the biophysical properties of a\nprotein.\nResults\nThermodynamic stability\nWild-type FNoTNc\nFNoTNc has a free energy of unfolding\n(\u0394GD\u2013N) of 7.5\u00a0kcal\nmol\u2212\u00a01, intermediate between\nthe stabilities of FNfn10 and TNfn3 (9.4 and 6.7\u00a0kcal\nmol\u2212\u00a01, respectively) at pH\n5.0. We infer that the surface interactions and loop and turn regions of\nFNfn10 make a significant contribution to the overall stability of\nFNoTNc. As was shown for FNfn10, the\n\u0394GD\u2013N of FNoTNc is\nindependent of pH between pH 5.0 and pH 7.0, whereas TNfn3 is less\nstable at pH 7 (5.7\u00a0kcal mol\u2212\u00a01). The\ndependence of the stability of TNfn3 on pH has been shown to be the\nresult of the presence of patches of acidic residues on the surface of\nTNfn3, which are, of course, not present in FNoTNc.17\nAnomalous response of certain peripheral mutations\nA number of core residues were mutated in FNoTNc to investigate the\nresponse of the protein to mutation. These were positions that had\npreviously been investigated in the parent proteins FNfn10 and TNfn3.\nThe thermodynamic stability of the mutant proteins was determined by\nchemical denaturation in guanidinium chloride (GdmCl). The mutations can\nclearly be divided into two categories. A few peripheral mutations\n(mutations in the A and G strands and the B\u2013C loop) have little effect\non stability as was previously observed in FNfn10 (Fig. 2a),\nwhereas most other mutations were more typically destabilising. In the\nlatter case, the \u0394\u0394GD\u2013N was\nsimilar to (but in general slightly lower than) what had been observed\npreviously in TNfn3 (Fig. 2b).\nThe residues in each category are mapped onto a backbone ribbon\nrepresentation of FNoTNc in Fig.\n2c. \u0394\u0394GD\u2013N values\nfor all mutations are compared to those of FNfn10 and TNfn3 in\nSupplementary Table\n1.\nEquilibrium hydrogen exchange\nThe rates of hydrogen\u2013deuterium exchange were measured at pD 7.0.\nMeasured rate constants for exchange,\nkex, ranged between 9.3\u00a0\u00d7\u00a010\u2212\u00a02 and 1.6\u00a0\u00d7\u00a010\u2212\u00a04 min\u2212\u00a01. Values of\nkex and the apparent free\nenergies of exchange,\n\u0394Gexapp,\nare listed in Supplementary Table\n2. The\n\u0394Gexapp of\nFNoTNc, FNfn10 and TNfn3 are compared in Fig.\n3. The hydrogen\nexchange behaviour of FNoTNc clearly resembles that of FNfn10: both have\nmany residues that exchange in the experimental dead time, in particular\nin the edge strands (A, C\u2032, E and G strands) that are more protected in\nTNfn3. Note that this does not result from differences in the intrinsic\nstabilities of the domains\u2014TNfn3 is significantly less stable than\neither FNoTNc or FNfn10 under these experimental conditions.\nFolding kinetics\nWild-type FNoTNc\nThe rate constants of folding and unfolding were determined using\nstopped-flow measurements monitored by changes in fluorescence\n>\u00a0320\u00a0nm. The logarithm of the folding and\nunfolding rate constants was plotted against concentration of denaturant\n(Fig. 4). Both unfolding and refolding arms show a linear\ndependence on denaturant concentration. There is an additional refolding\nphase, which we attribute to proline isomerisation and do not consider\nfurther. (Both FNfn10 and TNfn3 have proline-isomerisation limited\nphases, and FNoTNc has eight Pro residues.) To compare the kinetics of\nFNoTNc with those of FNfn10 and TNfn3 (which have been studied using\ndifferent denaturants due to large differences in stability), the\nlogarithm of the observed rate constants is plotted against stability in\nFig. 4. The free energy of\nunfolding calculated from the ratio of the folding and unfolding rate\nconstants extrapolated to 0\u00a0M denaturant (7.7\u00a0\u00a0kcal mol\u2212\u00a01) and the kinetic m\nvalue (2.1\u00a0kcal mol\u2212\u00a01\nM\u2212\u00a01) are the same as the\nequilibrium \u0394GD\u2013N (7.5\u00a0kcal\nmol\u2212\u00a01) and\nm value (2.1\u00a0kcal mol\u2212\u00a01 M\u2212\u00a01) within\nerror\u2014consistent with the folding being a 2-state process, with no\nstable intermediates being populated. The relative compactness of the\ntransition state can be determined from the folding and unfolding\nm values. FNoTNc has a \u03b2T\nvalue of 0.6, similar to those of TNfn3 and FNfn10.\n\u03a6-value analysis\nThe folding kinetics of 18 variants of FNoTNc with nondisruptive\ndeletion mutations were measured as for the wild type. Few of these\nmutants were in the A, B and G strands due to very small changes in\nstability with mutation in general. In a few of the mutants, the\nunfolding arm of the chevron plots shows negative curvature at high\ndenaturant concentrations, which we attribute to the Hammond effect: the\nsimplest model that fits the data.28\u201330 All chevrons were fitted therefore to a two-state\nequation with a quadratic term in the unfolding arm. (All the chevron\nplots are shown in Supplementary Fig.\n2.) Note that the model chosen to fit the kinetic data\ndoes not affect our results because equilibrium values of\n\u0394\u0394GD\u2013N were used to\ndetermine \u03a6. In Table\n1, the \u03a6 values for\neach mutant are compared to those in TNfn3 and FNfn10. The \u03a6 values are\ngenerally low, closely resembling those of TNfn3.\nDynamics\nBackbone dynamics\n15N\nT1 and\nT2 relaxation time constants\nand 1H15N nuclear\nOverhauser enhancement parameters were measured using 1H\u201315N correlation spectroscopy\n(Supplementary Table 3).\nThe generalised order parameter,\nS2, and a conformational\nexchange broadening parameter,\nRex, were determined for each\nbackbone amide (Fig.\n5 and Supplementary Table 4). The\nS2 values are similar to\nthose of both the parents. However, the high values of\nRex that were seen in the\nA\/B region of FNfn10, have decreased in FNoTNc.\nSide-chain dynamics\nThe relaxation of the operator terms IzCzDz, IzCzDy and IzCz was measured to give deuterium relaxation time constants\nT1(D) and\nT2(D)31 (Supplementary Table\n5). Order parameters,\nS2, were determined for each\nmethyl group using the standard model-free formalism as previously\ndescribed22 (Supplementary Table\n6). The dynamics data for those methyl groups that had\noverlapping peaks were treated with caution, as the contribution from\nthe overlapped peaks cannot be separated. Nevertheless, we have some\nconfidence in these results due to the agreement of the order parameters\nfor residues that have both overlapped and well-resolved methyl groups.\nS2 ranges from zero to\nunity, with higher values of S2\nindicating greater conformational restriction. The methyl\nS2 values are shown\nprojected onto the protein structure and compared to the parent proteins\nin Fig. 6. Within the core of FNoTNc is a cluster of deeply\nburied residues, which have unusually low order parameters, as has\npreviously been observed in TNfn3.\nDiscussion\nThe core of FNoTNc is similar to that of TNfn3, but apparently less closely\npacked.\nEvidence from mutations\nFNoTNc is close in structure to both the parent domains (backbone RMSD,\nexcluding the mobile C\u2013C\u2032 and F\u2013G loops, is 0.95 and 0.89\u00a0\u00c5 compared to\nFNfn10 and TNfn3, respectively). From the crystal structure we would deduce\nthat the core of FNoTNc is essentially the same in terms of core packing and\nnumber of core contacts as TNfn3, the parent protein that donated the core\nresidues (Fig. 7a). However, the calculated free volume in the interior of\nFNoTNc is larger than for TNfn3 (128.4\u00a0\u00c53\nversus 118.1\u00a0\u00c53, respectively). When\nthe response of FNoTNc to mutation is compared to TNfn3, it is clear that\nthe \u0394\u0394GD\u2013N values of FNoTNc are\nslightly lower, on average \u223c\u00a080% of those in TNfn3\n(Fig. 7b). Since the loss of\nfree energy on mutation is strongly correlated with the number of side-chain\ncontacts deleted,32,33 we infer that the less tight packing of the core of FNoTNc\naccounts for the difference in\n\u0394\u0394GD\u2013N compared to TNfn3.\nEvidence from side-chain dynamics\nThe same conclusion can be drawn from the analysis of the side-chain\ndynamics. The core of TNfn3 has been shown to be exceptionally mobile, with\nseveral of the most buried residues falling several standard deviations\nbelow the expected order parameter for their residue type.22,23 FNfn10 has a core that is much more conformationally\nrestricted, with order parameters within the usual range for buried\nresidues. Our previous analysis of the side-chain dynamics of FNfn10 and\nTNfn3 led us to suggest that the differences in core dynamics between FNfn10\nand TNfn3 could, at least in part, be ascribed to the slightly lower core\npacking in TNfn3; the residues in TNfn3 with unusually low order parameters\nare also found to have packing volumes that are larger than expected. The\nburied side chains of FNoTNc were found to have, on average, even lower\norder parameters than the same side chains in TNfn3 (Fig. 6d).\nBehaviour of peripheral regions of the protein is modulated by\nthe surface and loops\nEvidence from mutation\nA number of sites in FNfn10 were identified where upon mutation the\n\u0394\u0394GD\u2013N is significantly\nlower than for the same (or an equivalent) mutation in\nTNfn3.19 These sites are at the periphery of the protein and\ninclude residues Pro5 and Leu8 (A strand), Pro25 (B\u2013C loop) and residues\nSer85 and Phe92 (G strand). Unexpectedly, in FNoTNc we see the same\nanomalous response to mutation for Pro5, Pro25 and Ser85, although Ile8A\nand Phe92A now have similar\n\u0394\u0394GD\u2013N values to TNfn3\n(Fig. 2a). Pro5, Pro25 and\nSer85 are all found in the same region of the molecule (Fig. 2c). The anomalous response to\nmutation in this peripheral region of the core of FNoTNc and FNfn10 is\nintriguing: apparently the \u201cplastic\u201d response of the protein to mutation\nof these three buried residues is modulated by the surface of the\nprotein and is not determined by the core alone. The additional\nplasticity at this end of the hydrophobic core may be due to its\nproximity to the longer FG loop in FNfn10 and FNoTNc, which restricts\nmotion less than the corresponding loop in TNfn3.\nEvidence from hydrogen exchange\nFNoTNc, FNfn10 and TNfn3 have very similar structures and hydrogen\nbonding patterns. Further evidence for \u201cplasticity\u201d of FNfn10 compared\nto TNfn3 came from the observation that the peripheral A, C\u2032, E and G\nstrands were significantly less well protected against amide exchange in\nFNfn10 than in TNfn3, despite the fact that FNfn10 is considerably more\nstable than TNfn3.19 Under the experimental conditions, hydrogen exchange is\nin the EX2 regime,34,35 meaning that exchange reflects local stability; that\nis, these peripheral regions of FNfn10 have lower local stabilities than\nTNfn3. Our experiments clearly show that the exchange behaviour of\nFNoTNc is similar to FNfn10\u2014with low protection of residues in the same\nperipheral strands (Fig. 3).\nAgain surprisingly, local instability appears to be a function of the\nsurface of the protein and not a property of the core; however, the\nplasticity inferred from response to mutation and local instability\nstill appear to be related, as was previously inferred. This is not,\nhowever, related to slow exchange motions of the backbone, as we had\npreviously suggested, since the millisecond time scale motions observed\nin the A\/B region of FNfn10 are not found in FNoTNc.\nThe stability of FNoTNc is modulated by both the core and the\nsurface\nIn summary, despite the evidence for FNoTNc having a less well packed\ncore than TNfn3, with the mutation of buried side chains having less effect\non the overall stability (Fig.\n7b), FNoTNc is significantly more stable than TNfn3,\nsuggesting again that the surface and loops of FNfn10 contribute\nsignificantly to the stability of FNoTNc. This is not a new observation; it\nhas been previously demonstrated that surface interactions can play a\nsignificant role in the stabilisation of proteins.36 Interestingly, however, the \u201clocal stability\u201d of the\nperipheral regions of FNoTNc is lower than the local stability of TNfn3, as\nmanifested in the hydrogen exchange experiments. The surface residues of\nFNfn10 are modulating the stability of these peripheral regions.\nA concurrent study by Siggers et\nal.37 investigated the stability and dynamics of a series of\nloop-swap mutants of FNfn10 and TNfn3: essentially these were mutants where\nthe C\u2013C\u2032 and F\u2013G loops were exchanged. Interestingly, both TNfn3 domains had\nlower thermal stabilities than the wild-type protein, whereas the thermal\nstability of FNfn10 was unaffected by the exchange. We infer that in our\nchimeric protein, interactions of the surface residues are most important in\nthe increase in stabilisation of FNoTNc beyond that of TNfn3, and not the\nnew C\u2013C\u2032 and F\u2013G loops.\nThe TNfn3 core governs the folding kinetics\nWild-type kinetics\nIn order to compare the kinetics of the three proteins directly,\nthe logarithm of the rate constant has been plotted against stability\n(Fig. 4). This unusual\nscale is used as each protein was characterised using a different\ndenaturant. FNoTNc folds at a rate intermediate between FNfn10 and\nTNfn3. In broad terms, the chevron of FNoTNc resembles that of TNfn3. It\nhas previously been pointed out that the folding rates of Ig-like\ndomains in water do not correlate with contact order (the average\nsequence separation of native contacts).8,38 For the present three proteins, which have almost\nidentical contact order (absolute contact order 15\u201316%, relative contact\norder 16\u201317%), the rate constants at the folding midpoint span 2.5\norders of magnitude. Thus, the variation in the folding rate cannot be\nexplained only by differences in contact order\u2014protein stability plays a\nkey role.8,39\nFNfn10 shows clear rollover in both the folding and unfolding arms.\nThe rollover in the folding arm has previously been ascribed to the\npresence of a populated folding intermediate.18 Neither FNoTNc nor TNfn3 show any evidence for\npopulation of a folding intermediate\u2014the presence of a stable folding\nintermediate in FNfn10 appears to be the result of core interactions.\nCurvature in the unfolding arm of a chevron plot has been ascribed\nto the presence of a high-energy intermediate, to Hammond effects, or to\npopulation of an unfolding intermediate.30,40,41 This has not been investigated for any of these fnIII\ndomains; indeed, it is often not possible to distinguish between the\nfirst two cases.42 Both FNfn10 and some mutants of FNoTNc display curvature\nin the unfolding arm that is not seen in wild-type TNfn3. It should be\nnoted, however, that unfolding curvature has been seen in a less stable\nform of TNfn3 (missing the final two C-terminal residues) and may simply\nnot be observed in this case because the unfolding arm in the stable\nform of TNfn3 used here is relatively short. In summary, FNoTNc has\nsimilar folding characteristics to TNfn3. This suggests that the core of\nthese proteins plays the major role in determining the folding\nbehaviour.\nHowever, FNoTNc is more stable than TNfn3. When we compare the\nfolding and unfolding rate constants in water we find that FNoTNc folds\nsome 10 times faster than TNfn3\n(kfH2O\u00a0=\u00a060 and 6\u00a0s\u2212\u00a01, respectively) but unfolds at approximately the same\nrate as TNfn3\n(kuH2O\u00a0=\u00a02\u00a0\u00d7\u00a010\u2212\u00a04 and 5\u00a0\u00d7\u00a010\u2212\u00a04 s\u2212\u00a01, respectively).\nThus, the stabilising surface interactions apparently stabilise the\ntransition state of FNoTNc as much as the native state, while still not\ncausing a folding intermediate to be populated. This, perhaps, allows us\nto pinpoint further which surface interactions are responsible for the\nadded stability of FNoTNc (over TNfn3). At the transition state, the\nloops and the peripheral A and G strands are largely unstructured, but\nthere is structure in the B, C, C\u2032, E and F strands, particularly\ntowards the centre of the core (see \u03a6 value section below). Interactions\nbetween surface residues in these strands are therefore the most likely\ncandidates for providing additional stability to the FNoTNc protein\n(both native and transition states), above those interactions between\nresidues that are packing in the core.\nIn contrast, FNoTNc folds some three times more slowly than FNfn10\nand the unfolding rate constant of FNfn10 in water is approximately an\norder of magnitude lower (\u223c\u00a02\u00a0\u00d7\u00a010\u2212\u00a05\ns\u2212\u00a01).\n\u03a6-value analysis\nThe \u03a6-value analysis reveals the extent of formation of structure\nin the transition state.43 The pattern of \u03a6 values in FNfn10 and TNfn3 are similar,\nsuggesting that they have a common folding mechanism. There are\ndifferences, however. The \u03a6 values of TNfn3 tend to be much lower than\nthose of FNfn10: TNfn3 has five \u03a6 values greater than 0.4,12 while FNfn10 has eight16; in FNfn10 the folding nucleus appears to be more\nextensive than in TNfn3, with more than one residue in the central C, E\nand F strands having high \u03a6 values. FNoTNc has even lower average \u03a6\nvalues than TNfn3 with only two \u03a6 values greater than 0.4 (Table 1) Both FNoTNc and TNfn3 have less\nextensive formation of structure in the transition state than FNfn10.\nHowever, \u03a6-value analysis is at its most powerful when patterns of\n\u03a6 values are compared in different proteins rather than by direct\ncomparison of \u03a6 values. Previous analysis of TNfn3 has identified a ring\nof interacting residues in the B, C, E and F strands as the folding\nnucleus, with residues in the C\u2032 strand packing onto these.12 The \u03a6 values of the peripheral A and G strands are all\nclose to 0, suggesting that they are unformed at the transition state. A\nsimilar pattern of \u03a6 values was seen in FNfn10, although this \u03a6-value\nanalysis was less complete.16 The \u03a6 values for FNoTNc were divided into three\ncategories of low (\u03a6\u00a0<\u00a00.25),\nmedium (0.25\u00a0<\u00a0\u03a6\u00a0<\u00a00.35) and high (\u03a6\u00a0>\u00a00.35) (Fig. 8)\nand compared to the pattern of \u03a6 values for the identical residues in\nTNfn3. The pattern of \u03a6 values is very similar to that of TNfn3,\nalthough there are slight qualitative differences. Again, the highest \u03a6\nvalues are found in the B, C, C\u2032, E and \u03a6 strands and the \u03a6 values in\nthe A and G strands are \u223c\u00a00. The folding mechanism of\nFNoTNc is unperturbed and the same as both the parents, although from\nthe magnitude and extent of the residues with higher \u03a6 values the\ntransition-state structure appears to be closer to that of TNfn3 than\nFNfn10.\nDynamics from NMR is determined by local interactions\nSide-chain dynamics\nThe side-chain dynamics appear to reflect core packing, as was\ndiscussed above, and so FNoTNc resembles TNfn3 more closely than\nFNfn10.\nBackbone dynamics\nThe S2 values of FNoTNc are\ngenerally very similar to those of both the parents (Fig. 5). It is interesting to compare\nour study with that of the loop swap mutants of FNfn10 and TNfn3 from\nPalmer and coworkers,37 where the F\u2013G loop of FNfn10 was grafted into TNfn3. In\nthat case, the S2 values in the\nloop region were more similar to those in FNfn10, than those in the\nequivalent positions in TNfn3. A similar result was seen in the protein\nwhere the C\u2013C\u2032 loop was grafted in. It is difficult to compare our\nresults directly to those of Siggers\net\u00a0al.37: their method of analysis leads to greater differences\nbetween the S2 values of FNfn10\nand TNfn3, resulting in more visible effects on the\nS2 values when the loops are\nswapped than we see in FNoTNc compared to the parent proteins. However,\nthere is a general agreement that the behaviour of the loops is a local\nproperty, rather than being a direct effect of either core or surface\ninteractions.\nConclusion\nWe have grafted the core of one fnIII domain (TNfn3) into the homologue\nFNfn10, creating a chimera, FNoTNc, which has retained the structure of the\nparent proteins. Using several different probes, we have shown that FNoTNc does\nnot behave like either one of the parent proteins alone. Instead, it has\nretained a number of properties of each. We find that each property investigated\nclearly resembles the behaviour of one of the parents, enabling us to separate\nthe contribution of the core and the surface of the protein in determining the\nbehaviour of the domain. Some of these are unsurprising, such as the pH\ndependence of stability, the core side-chain dynamics and the dependence of\nfolding on the composition of the core. However, the surface of the protein\nconfers significant stability not only on the native state, but also on the\ntransition state for folding. Others properties are less predictable. The\nsurface of the protein confers \u201cplasticity\u201d in peripheral regions of the\nproteins as detected by the anomalous response of some regions of the core to\nmutation and hydrogen exchange protection patterns.\nThis suggests that the surface of a domain may have a more significant\ncoupling with the core than we had previously considered. Since most biophysical\nstudies tend to focus on the core of a protein, this coupling is a relatively\nunexplored area of research.\nMaterials and Methods\nChemicals\nGdmCl was purchased from MP Biomedicals Inc., guanidine isothiocyanate\nfrom Gibco-BRL and urea from BDH Laboratory Supplies.\nProtein expression and purification\nSite-directed mutagenesis reactions were performed with the QuickChange\nkit from Stratagene using the FNoTNc plasmid. The identity of the mutants\nwas confirmed by DNA sequencing. The mutants studied in this work are listed\nin Table 1 and Supplementary Table 1. The nature of the\nmutation is indicated with the wild-type residue first (single-letter code),\nthe position of the mutation second and the mutant residue third. Expression\nand purification of FNoTNc and mutants was performed as described earlier\nfor TNfn3.20\nMeasurements of protein stability\nAll biophysical measurements were performed in 50\u00a0mM sodium acetate\nbuffer, pH 5.0 at 25\u00a0\u00b0C unless otherwise stated. The stability of FNoTNc and\nFNoTNc mutants were determined by equilibrium denaturation experiments using\nGdmCl and by standard methods using 1\u00a0\u03bcM protein.20\nKinetic measurements\nKinetics were measured using fluorescence stopped-flow measurements in\n50\u00a0mM sodium acetate buffer, pH 5.0, at 25\u00a0\u00b0C and were monitored by changes\nin fluorescence above 320\u00a0nm. Refolding measurements for FNoTNc were made in\n0\u00a0M denaturant by stopped-flow fluorescence using pH jumps from pH 12.4 to\npH 5.0 as previously described.12 (FNoTNc is acid stable.) NaOH (25\u00a0mM, pH 12.4) unfolds\nFNoTNc completely (data not shown).\n\u03a6-value analysis\n\u03a6 values were determined from refolding data at 1.0\u00a0M GdmCl, to avoid\nthe errors associated with long extrapolation, from Eq. (1):43whereand\nkfWT and\nkfmut are the\nrate constants for folding of wild-type and mutant proteins,\nrespectively.\nNMR sample preparation\nFNoTNc was expressed and purified by affinity chromatography as\npreviously described.17 Uniformly 15N labelled and\n13C and 15N\nlabelled samples were expressed in M9 minimal media containing 15NH4Cl and [U-13C]6-glucose as the sole nitrogen and carbon sources. Samples for\nside-chain dynamics were expressed as previously described.22\nChemical shift assignments\nBackbone assignment experiments were carried out on a double-labelled\n(13C, 15N) sample\nof FNoTNc, at an approximate concentration of 1\u20132\u00a0mM, in 50\u00a0mM imidazole\nbuffer at pH 7.0 in 10% D2O. Sodium azide (0.05%) was added\nto prevent microbial growth. The sample was centrifuged to remove insoluble\nprotein and degassed. Spectra were acquired at 298\u00a0K on a Bruker DRX500\nspectrometer with an inverse triple-resonance cryogenic probe. Backbone\nassignments were based on HNCACB, HNCO and CBCA(CO)NH experiments together\nwith the 1H\u201315N\nheteronuclear single quantum coherence (HSQC) spectrum. Side-chain 1H and 13C resonance\nassignments were obtained from 3-D HCCH\u2013total correlated spectroscopy\n(TOCSY) H(CCCO)NH and (H)CC(CO)NH preceding TOCSY spectra. The spectra were\nprocessed and analysed using NMRpipe and Sparky.44,45 The 1H\u201315N HSQC has excellent resolution, although resonances from 12\nresidues in loop regions cannot be detected at either pH 5 or pH 7\n(Supplementary Fig. 3 and\nSupplementary Table 7).\nAll samples for side-chain methyl assignment were prepared in 50\u00a0mM\nsodium acetate buffer at pH 5.0 in 10% D2O, at a\nconcentration of 1\u20132\u00a0mM. Sodium azide was added to prevent microbial growth.\nThe sample was centrifuged to remove insoluble protein and degassed. All\nexperiments were carried out as previously described.22,46,47\nChemical shift assignments of the side chains were made using standard\ntriple-resonance experiments. Many of the signals in the 1H\u201313C HSQC are overlapped, meaning\nthat 5 of the 64 methyl groups in the 1H\u201313C HSQC could not be assigned with confidence.\nAssignment of the leucine and valine methyl groups was made\nstereospecifically, based upon the phase of peaks in a 1H\u201313C HSQC acquired for a sample\nwith 10% 13C enrichment (Supplementary Fig. 4 and Supplementary Table 7).\nHydrogen exchange\nHydrogen exchange experiments were carried out under EX2 conditions on\na 15N-labelled sample of FNoTNc, at an\napproximate concentration of 1\u20132\u00a0mM, in 50\u00a0mM imidazole buffer at pD 7.0 in\n10% D2O. Sodium azide (0.05%) was added to prevent\nmicrobial growth. The exchange of amide protons was followed by the decay of\nintensity of peaks in HSQC spectra.48\nThe apparent free energy of exchange,\n\u0394Gexapp, was\ndetermined from the rate constant of exchange,\nkex, and the intrinsic rate\nconstant, kint, determined from\npeptide data to take account of the primary sequence of the protein and\nexchange conditions49,50 using Eq. (2) and\nintrinsic rate constants determined using the software Sphere\u2020.51\nBackbone 15N relaxation\nmeasurements\nBackbone dynamics were determined from 15N\nT1 and\nT2 relaxation times and the\nsteady-state heteronuclear 1H15N nuclear Overhauser enhancement at 500\u00a0MHz as previously\ndescribed.22 The data were analysed using standard protocols for backbone\ndynamics with the program TENSOR2.\nSide-chain methyl 2H relaxation\nmeasurements\nSide-chain deuterium relaxation times\nT1(D) and\nT1\u03c1(D) were determined by\nmeasuring the relaxation of the two- and three-spin operator terms,\nIzCz,\nIzCzDz\nand\nIzCzDy\nand analysed as previously described.22,31","keyphrases":["side-chain dynamics","immunoglobulin","fniii, fibronectin-type iii","fnfn10, 10th fniii domain of human fibronectin","tnfn3, third fniii domain of human tenascin","fnotnc, a core-swapped protein with the \u201coutside\u201d (surface and loops) of fnfn10 and the core of tnfn3","gdmcl, guanidinium chloride","hsqc, heteronuclear single quantum coherence","tocsy, total correlated spectroscopy","protein folding","extracellular matrix","protein design"],"prmu":["P","P","M","R","R","R","R","R","R","R","U","M"]}
{"id":"Int_J_Cardiovasc_Imaging-4-1-2233708","title":"3.0 T cardiovascular magnetic resonance in patients treated with coronary stenting for myocardial infarction: evaluation of short term safety and image quality\n","text":"Purpose To evaluate safety and image quality of cardiovascular magnetic resonance (CMR) at 3.0 T in patients with coronary stents after myocardial infarction (MI), in comparison to the clinical standard at 1.5 T. Methods Twenty-five patients (21 men; 55 \u00b1 9 years) with first MI treated with primary stenting, underwent 18 scans at 3.0 T and 18 scans at 1.5 T. Twenty-four scans were performed 4 \u00b1 2 days and 12 scans 125 \u00b1 23 days after MI. Cine (steady-state free precession) and late gadolinium-enhanced (LGE, segmented inversion-recovery gradient echo) images were acquired. Patient safety and image artifacts were evaluated, and in 16 patients stent position was assessed during repeat catheterization. Additionally, image quality was scored from 1 (poor quality) to 4 (excellent quality). Results There were no clinical events within 30 days of CMR at 3.0 T or 1.5 T, and no stent migration occurred. At 3.0 T, image quality of cine studies was clinically useful in all, but not sufficient for quantitative analysis in 44% of the scans, due to stent (6\/18 scans), flow (7\/18 scans) and\/or dark band artifacts (8\/18 scans). Image quality of LGE images at 3.0 T was not sufficient for quantitative analysis in 53%, and not clinically useful in 12%. At 1.5 T, all cine and LGE images were quantitatively analyzable. Conclusion 3.0 T is safe in the acute and chronic phase after MI treated with primary stenting. Although cine imaging at 3.0 T is suitable for clinical use, quantitative analysis and LGE imaging is less reliable than at 1.5 T. Further optimization of pulse sequences at 3.0 T is essential.\nIntroduction\nThe combination of functional cardiovascular magnetic resonance (CMR) and late gadolinium enhancement (LGE) is evolving as an important diagnostic [1, 2] and prognostic [3\u20135] modality in patients with ischemic heart disease. Because the availability of high field MR systems is increasing, the need arises to evaluate the performance and clinical value of these systems in cardiovascular disease. Earlier reports already suggested that 3.0\u00a0T MR systems offer higher temporal and spatial resolution, due to an increased signal-to-noise ratio, which would be especially advantageous in CMR scanning [6\u20138]. However, these studies were all assessed in healthy volunteers and in patients with non-ischemic heart disease or suspected coronary artery disease, and without the presence of coronary stents [6\u201310]. There is limited to no data about safety and image quality at 3.0\u00a0T CMR scanning in the acute or chronic phase after myocardial infarction (MI), in patients treated with percutaneous coronary intervention and primary stenting. In these patients B0 inhomogeneities (induced by the heart-lung interface or from coronary stents), flow artifacts, inhomogeneity of normal myocardial suppression in LGE (induced by B1 inhomogeneity) and poor cardiac triggering may interfere with the gain offered by the higher magnetic field.\nThe aim of this study was to test whether CMR scanning at 3.0\u00a0T is safe and feasible in patients with coronary stents in the acute and chronic phase after MI, and to prospectively compare image quality at 3.0\u00a0T with the current clinical standard at 1.5\u00a0T. Furthermore, the presence and significance of different image artifacts are considered.\nMethods\nPatient population\nPatients were eligible for the study if they had been admitted with a first ST-elevation acute MI, according to standard electrocardiographic and enzymatic criteria [11], and had undergone successful primary PCI with stent implantation. Exclusion criteria were electrocardiographic evidence of reinfarction, haemodynamic or other clinical instability or (relative) contraindications for CMR such as claustrophobia, pacemakers, intracerebral aneurysm clips or very irregular heart rhythm. Patients were treated with aspirin, heparin, abciximab, clopidogrel, statins, beta-blockade and ACE-inhibitors, according to ACC\/AHA practice guidelines [12]. The study was approved by the local ethics committee and all patients gave written informed consent.\nCMR parameters\nEighteen CMR scans were performed with a 3.0\u00a0T MR system (Intera, Philips, Best, The Netherlands), with a gradient performance of 30\u00a0mT\/m and slew rate of 150\u00a0T\/m\/s, using a six element cardiac phased array surface coil. Another 18 CMR scans were acquired with a 1.5\u00a0T MR system (Magnetom Sonata, Siemens, Erlangen, Germany), with a gradient performance of 40\u00a0mT\/m and slew rate of 200\u00a0T\/m\/s, using an eight element cardiac phased array surface coil.\nCine imaging was performed at both field strengths using a SSFP pulse sequence, without parallel imaging. Long axis views, as well as short axis views covering the entire left ventricle were acquired during repeated breath-holds in expiration. At 3.0\u00a0T, cine SSFP sequence parameters were a temporal resolution between 25\u00a0ms and 50\u00a0ms, excitation angle of 45\u00b0, receiver bandwidth 868\u00a0Hz\/pixel, TR\/TE of 3.8\/1.9\u00a0ms, matrix 192\u00a0\u00d7\u00a0155 and voxel size of 1.5\u00a0\u00d7\u00a01.8\u00a0\u00d7\u00a06.0\u00a0mm3. At 1.5\u00a0T, the temporal resolution was between 35\u00a0ms and 50\u00a0ms with an excitation angle of 60\u00b0, receiver bandwidth 930\u00a0Hz\/pixel, TR\/TE of 3.2\/1.6\u00a0ms, matrix 256\u00a0\u00d7\u00a0156 and voxel size of 1.4\u00a0\u00d7\u00a01.9\u00a0\u00d7\u00a06.0\u00a0mm3.\nLGE images were acquired in mid-diastole using a 2D segmented inversion-recovery gradient-echo pulse sequence, 10\u201315\u00a0min after intravenous injection of 0.2\u00a0mmol\/kg of a gadolinium chelate (Dotarem, Guerbet, Roissy, France). The LGE images were obtained in exactly the same orientation as the cine images. Sequence parameters at 3.0\u00a0T were an excitation angle of 25\u00b0, receiver bandwidth 434\u00a0Hz\/pixel, TR\/TE of 3.5\/1.3\u00a0ms, matrix 192\u00a0\u00d7\u00a0119, voxel size of 1.7\u00a0\u00d7\u00a02.1\u00a0\u00d7\u00a06.0\u00a0mm3, triggering every other heart beat, and an inversion time between 250\u00a0ms and 350\u00a0ms to null remote myocardium. At 1.5\u00a0T, sequence parameters were an excitation angle of 25\u00b0, receiver bandwidth 130\u00a0Hz\/pixel, TR\/TE of 9.6\/4.4\u00a0ms, matrix 256\u00a0\u00d7\u00a0166, voxel size of 1.4\u00a0\u00d7\u00a01.7\u00a0\u00d7\u00a06.0\u00a0mm3, triggering every other heart beat, and an inversion time between 220\u00a0ms and 300\u00a0ms to null remote myocardium.\nSafety and quality analysis\nTo evaluate patient safety, a physician was present at the CMR scanner throughout the scan. Heart rhythm was monitored continuously. The patient was asked to report any discomfort and symptoms during the scan procedure. Additionally, a repeat catheterization was performed in 16 patients who underwent CMR examination in the acute phase after MI (9 at 3.0\u00a0T, 7 at 1.5\u00a0T). Stent position and patency after CMR scanning were visually assessed on the repeat catheterization and compared to the primary PCI. Occurrence of repeat intervention or hospitalization within 30\u00a0days of the CMR examination was recorded.\nDifferent types of artifacts were reviewed and marked as being clinically relevant if the artifact interfered with visualisation of the myocardium. Flow related artifacts in SSFP imaging [13], and artifacts due to static field inhomogeneities such as the heart-lung interface were scored on cine images. Separately, we looked at artifacts caused by the coronary stent on the cine images, and measured the maximum artifact diameter, perpendicular to the length of the stent, using an appropriate cardiac view. In addition, the potential effect of B1 inhomogeneity on the homogeneous suppression of viable myocardium was visually assessed on LGE images.\nFurthermore, image quality of the cine and LGE images were scored on a separate workstation (Centricity Radiology v6.1, GE Medical Systems, Zeist, the Netherlands) by four independent observers, who were blinded for MR system and clinical history. To distinguish between image quality that is satisfactory for clinical use or high quality images for research purposes, images were scored on a scale from 1 to 4: 1 not clinically useful; 2 clinically useful, but of insufficient quality for quantitative analysis; 3 clinically useful and of sufficient quality for quantitative analysis; 4 excellent quality. The following definitions were used for evaluation of the cine images: 1 poor quality, extensive artifacts, completely obscuring endocardial borders; 2 moderate quality, assessment of global function is possible, partly using assumptions, but regional wall thickening is not possible in all segments, due to interfering artifacts; 3 good quality, assessment of global and regional function is possible, despite some small artifacts; 4 excellent quality, functional analysis is possible and there is no interference of artifacts. And for the evaluation of LGE images: 1 poor quality, extensive artifacts, infarcted myocardium is not visible; 2 moderate quality, infarcted myocardium is visible, but delineation is not possible in all segments; 3 good quality, infarcted myocardium is easy to distinguish from viable myocardium, despite some small artifacts; 4 excellent quality, no artifacts. Two scores were given for each scan: 1 for the set of cine images and 1 for the set of LGE images. After completing the independent image quality assessment, all 4 observers exchanged their scores for each case and agreed on a consensus score.\nStatistical analysis\nContinuous variables with normal distribution are expressed as mean\u00a0\u00b1\u00a0SD, or as median (25th\u201375th percentile). Comparison of the baseline characteristics and stent artifact diameter was done by using an unpaired Student\u2019s t-test. The consensus score of image quality of both field strengths was assessed for statistical difference by a Mann-Whitney U test. All statistical tests were two-sided with a significance level of P\u00a0<\u00a00.05. SPSS 12.0.1 for Windows (SPSS Inc., Chicago, USA) was used for analysis.\nResults\nTwenty-five consecutive patients with 36 CMR studies were included in the study. One patient was studied on both MR systems in the acute phase, 9 patients (5 on 3.0\u00a0T, 4 on 1.5\u00a0T) were studied both in the acute phase and in the chronic phase, and 1 patient (3.0\u00a0T) was studied twice in the chronic phase after MI. The baseline characteristics of all 25 patients are listed in Table\u00a01. On each system, 12 CMR scans were performed in the acute phase after MI, at 4\u00a0\u00b1\u00a02\u00a0days after primary PCI, and 6 CMR studies in the chronic phase, at 125\u00a0\u00b1\u00a023\u00a0days after primary PCI. One patient refused contrast injection during scanning in the chronic phase.\nTable\u00a01Patient characteristicsNumber of patients25Age (years)55\u00a0\u00b1\u00a09Men21 (84)BMI (kg\/m2)25.5\u00a0\u00b1\u00a01.9Risk factorsDiabetes mellitus0 (0)Hyperlipidaemia3 (12)Hypertension6 (24)Smoking16 (64)Family history of CAD8 (32)Maximum peak CK-MB (U\/L)308 (197\u2013464)Infarct-related arteryLAD18 (72)RCx3 (12)RCA4 (16)Values are presented as number (ratio in %), mean\u00a0\u00b1\u00a0standard deviation or median (25th\u201375th percentile)\nECG electrodes were positioned and displaced until an optimal ECG signal was acquired. Although it took more time to obtain a stable ECG waveform with a clearly delineated R wave at 3.0\u00a0T than at 1.5\u00a0T (10\u201315\u00a0min vs. <2\u00a0min respectively), it did not affect image quality, since scans were repeated when trigger problems occurred.\nStent safety and artifacts at 3.0\u00a0T\nNo patient reported any discomfort or symptoms during the CMR scan procedure. In addition, there were no clinical events during, or shortly after scanning at 3.0\u00a0T, and none of the patients underwent a repeat intervention or hospitalization for any reason within 30\u00a0days.\nThe mean number of stents implanted per patient per scan at 3.0\u00a0T was 1.3\u00a0\u00b1\u00a00.7, with a mean stent length of 19\u00a0\u00b1\u00a05\u00a0mm and diameter of 3.2\u00a0\u00b1\u00a00.4\u00a0mm. Further specifications of the implanted coronary stents are listed in Table\u00a02. Repeat catheterization was performed 2 (1\u20134) days after scanning in 9 of the 12 patients who underwent 3.0\u00a0T CMR scanning in the acute phase after MI. Angiographic evaluation revealed no differences in stent position and patency compared to the initial result after primary PCI.\nTable\u00a02Type and number of coronary stents used per CMR examinationField strength and timingNumber of patientsNumber and type of coronary stents present during CMRa3.0\u00a0T acute phase91\u00d7 Multi-Link Vision12\u00d7 Multi-Link Vision21\u00d7 Driver3.0\u00a0T chronic phase31\u00d7 Multi-Link Vision21\u00d7 Driver\/2x TAXUS Libert\u00e9b11\u00d7 Driver1.5\u00a0T acute phase41\u00d7 Prokinetic12\u00d7 Prokinetic21\u00d7 Lekton Motion11\u00d7 Lekton Motion\/2\u00d7 Multi-Link Vision11\u00d7 Multi-Link Vision11\u00d7 AVE11\u00d7 Multi-Link Zeta11\u00d7 CYPHERb1.5\u00a0T chronic phase21\u00d7 Lekton Motion12\u00d7 Lekton Motion11\u00d7 AVE11\u00d7 Multi-Link Zeta11\u00d7 CYPHERbaStent material and manufacturer\u2014Multi-Link Vision: cobalt chromium alloy, Abbott Vascular; Driver, cobalt chromium alloy, Medtronic; TAXUS Libert\u00e9, 316L stainless steel, Boston Scientific; Prokinetic, cobalt chromium alloy, Biotronik; Lekton Motion: 316L stainless steel, Biotronik; Multi-Link Zeta, 316L stainless steel, Abbott Vascular; AVE: 316L stainless steel, Medtronic; CYPHER: 316L stainless steel, CordisbDrug-eluting stent\nDuring cine imaging, the coronary stent was visible in 14 of the 18 scans (78%), with a susceptibility related signal loss of 12.8\u00a0\u00b1\u00a04.3\u00a0mm in diameter. Due to severe interference of the coronary stent in 6 scans (33%), analysis of surrounding myocardium was impossible (Fig.\u00a01A). Through-plane flow artifacts were present in 14 scans (from the aorta in 9 scans, from the pulmonary trunk in 7 scans, from the left ventricle in 3 scans), and hindered visualization of the myocardium in 7 scans (39%). In-plane flow artifacts were present in 4 scans, but were not clinically relevant. In the majority of the scans (94%) dark band artifacts appeared at the transition between myocardium and lung, with a predilection for the anterolateral wall, which resulted in severe image distortion in 8 scans (44%). Overall, artifacts were responsible for clinically relevant image deformation in 12 scans (67%), of which half was caused by the coronary stent (in 6 scans).\nFig.\u00a01Short axis cine SSFP images in different patients, demonstrating a signal void from a coronary stent with myocardial interference at 3.0\u00a0T (A, white arrow head), and a smaller signal void without interference at 1.5\u00a0T (B, white arrow head)\nNo effect could be observed of B1 inhomogeneity on the suppression of viable myocardium on LGE images at 3.0\u00a0T.\nStent safety and artifacts at 1.5\u00a0T\nAlso during scanning at 1.5\u00a0T, no patient reported any discomfort or symptoms, and there were no clinical events during, or shortly after scanning. None of the patients underwent a repeat intervention or hospitalization for any reason within 30\u00a0days after CMR scanning.\nThe mean number of implanted stents per patient per scan at 1.5\u00a0T was 1.2\u00a0\u00b1\u00a00.5 (see also Table\u00a02). The mean stent length was 19\u00a0\u00b1\u00a05\u00a0mm with a mean diameter of 3.2\u00a0\u00b1\u00a00.4\u00a0mm, which was comparable with the stent length and diameter scanned at 3.0\u00a0T (p\u00a0=\u00a0ns). In 7 of the 12 patients who underwent CMR scanning at 1.5\u00a0T in the acute phase, repeat catheterization at 2\u00a0(2\u20134) days after scanning revealed no differences in stent position and patency compared to post-PCI.\nDuring cine imaging, the coronary stent was visible in 6 of the 18 scans (33%), with a susceptibility related signal loss of 5.5\u00a0\u00b1\u00a00.3\u00a0mm in diameter, which was significantly smaller than at 3.0\u00a0T (P\u00a0<\u00a00.01). Furthermore, visible stents did not cause clinically important image deformation (Fig.\u00a01B). Besides the small signal void from coronary stents, no other artifacts were observed at 1.5\u00a0T.\nAlso at 1.5\u00a0T there was no effect of B1 inhomogeneity on the suppression of viable myocardium on LGE images.\nEvaluation of image quality\nWith scanning at 3.0\u00a0T, localized shimming was compulsory in every scan to obtain better image quality (Fig.\u00a02). At 1.5\u00a0T, there was no need for adjustments in sequence parameters or the use of local shimming to optimize image quality. At 3.0\u00a0T, in 44% of the cases, image quality was not sufficient for quantitative analysis and assessment of regional function of the left ventricle (Table\u00a03). For clinical purposes and global assessment of left ventricular function, image quality at 3.0\u00a0T and 1.5\u00a0T were comparable. Image quality of LGE images was not sufficient for quantitative analysis in 53% of the cases at 3.0\u00a0T, and 12% of the images were not useful for clinical purposes. Both image quality of cine and LGE imaging at 3.0\u00a0T were significantly lower compared to 1.5\u00a0T (P\u00a0<\u00a00.001).\nFig.\u00a02Four chamber cine SSFP images at 3.0\u00a0T in end-diastole (A & C) and end-systole (B & D), at the same slice position in one patient. Upper panels are without and lower panels with localized shimming. Dark band artifacts (apex) and flow artifacts (around the atrioventricular valves) are reduced with localized shimmingTable\u00a03Consensus score of image quality of cine and late gadolinium-enhanced images for 1.5\u00a0T and 3.0\u00a0TCine SSFP imagesLate gadolinium-enhanced images1.5\u00a0T3.0\u00a0T1.5\u00a0T3.0\u00a0T1. Not clinically useful0 (0%)0 (0%)0 (0%)2 (12%)2. Clinically useful, no quantitative analysis0 (0%)8 (44%)0 (0%)7 (41%)3. Clinically useful, quantitatively analyzable0 (0%)4 (22%)1 (6%)8 (47%)4. Excellent quality18 (100%)6 (33%)17 (94%)0 (0%)Values are presented as absolute numbers (percentage)\nDiscussion\nIn this paper we report on our initial experience with CMR scanning at 3.0\u00a0T in a clinical situation. We found that it is safe and feasible to perform CMR scanning at 3.0\u00a0T in the acute and chronic phase after MI in patients treated with primary stenting. Image quality of cine imaging at 3.0\u00a0T is of sufficient quality for global assessment of left ventricular function, however, quantitative analysis is not possible in almost half of the patients, due to dark band, flow and stent artifacts. Image quality of LGE studies were significantly better at 1.5\u00a0T. Therefore further optimization of pulse sequences at 3.0\u00a0T is essential.\nMR imaging is considered to be contraindicated in patients with ferromagnetic implants, primarily because of the potential risks associated with migration, the induction of an electrical current and heating of the implant [14, 15]. According to earlier reports and the American Society for Testing and Materials International, the overall magnetic field interaction and heating for coronary stents is limited or absent at 1.5\u00a0T and 3.0\u00a0T, as tested in vitro [16\u201318]. In vivo studies concerning stent safety at 1.5\u00a0T demonstrated that CMR scanning is safe in patients early after coronary artery stent placement [19\u201321]. In this in vivo study using both 1.5\u00a0T and 3.0\u00a0T, no clinical signs were observed by the attending physician, and no substantial side effects or clinical events occurred during or within 30\u00a0days of CMR scanning. In addition, in the patients who underwent a repeat catheterization there was no angiographic evidence of stent migration, confirming the in vitro data.\nAs discussed by Sch\u00e4r et al. [7], dark band and flow artifacts in SSFP cine imaging can be solved using optimized sequence parameters, localized shimming and correct water resonance frequency adjustment. In the clinical setting of the present study, it was not always possible to solve these artifact problems despite sequence optimization and localized shimming, especially in the presence of stent artifacts. The larger and diagnostically interfering artifacts of coronary stents at 3.0\u00a0T as opposed to the smaller stent artifacts at 1.5\u00a0T are an important issue in patients after MI, since primary PCI with stent implantation is the method of choice to re-establish coronary flow [22]. For clinical application, it may be more advantageous to return to spoiled gradient-echo cine imaging at 3.0\u00a0T, which includes longer acquisition times and lower contrast between blood and myocardium, but is less sensitive for off-resonance artifacts and equally accurate as SSFP cine imaging (Fig.\u00a03) [9, 23].\nFig.\u00a03Short axis cine SSFP image (A) and cine spoiled gradient-echo image (B), at the same slice position in one patient at 3.0\u00a0T. The coronary stent artifact on a SSFP image is larger than on a spoiled gradient-echo image (white arrows heads). Flow artifacts (A, asterisk) are less visible with spoiled gradient-echo imaging at 3.0\u00a0T\nWe initially intended to evaluate differences in signal- and contrast-to-noise ratios (SNR and CNR) of both pulse sequences as well. At 1.5\u00a0T good noise estimates could be made using the technique described by Constantinides et al. [24]. However, this method did not work at 3.0\u00a0T due to a different reconstruction algorithm, even without parallel imaging, and by switching off clear and image enhancement filters. Subtraction methods with two consecutive scans to estimate noise are inaccurate in a mobile tissue as the heart [25]. Other methods were beyond the scope of this initial study. Despite the fact that SNR\u2019s and CNR\u2019s were therefore omitted from this study, it is interesting to mention that an important reason for inferior quality of LGE images at 3.0\u00a0T was that infarcted myocardium was sometimes difficult to delineate from the left ventricular cavity (Fig.\u00a04).\nFig.\u00a04Short axis LGE images 15\u00a0min after injection of contrast at 3.0\u00a0T (A) and 1.5\u00a0T (B), in different patients. Visual assessment of infarct extent and location was more difficult at 3.0\u00a0T than at 1.5\u00a0T, since it was sometimes difficult to delineate infarcted myocardium from the left ventricular cavity at 3.0\u00a0T\nWe used the same contrast agent and dose at both field strengths. The contrast dose has been optimized for 1.5\u00a0T in the past [26, 27], but might be different at 3.0\u00a0T as T1 relaxation rates are in general field strength dependent. However, a recent study of Sharma and colleagues showed that there were only minor differences in post contrast myocardial T1 relaxation times between 1.5\u00a0T and 3.0\u00a0T, using a contrast dose of 0.2\u00a0mmol\/kg [28]. As long as inversion times are set appropriately at each field strength as done in this study, it is not to be expected that significant differences in LGE image quality are caused by contrast dose effects.\nIn conclusion, our study demonstrates that it is safe and feasible to perform CMR scanning at 3.0\u00a0T in the acute and chronic phase after MI in patients treated with primary stenting. Although cine imaging at 3.0\u00a0T is of sufficient quality for clinical use, quantitative assessment is less reliable compared to 1.5\u00a0T, mainly due to dark band, flow and stent artifacts. Further optimization of pulse sequences at 3.0\u00a0T is essential to make 3.0\u00a0T CMR scanning suitable for clinical cardiology.\nLimitations\nThe use of different MR systems, from different vendors, with different coils and sequence parameters, of course introduces confounding factors for a comparison. However, the sequences were optimized for their field strength to evaluate safety, feasibility and image quality rather than technical differences. A second limitation is that patients did not undergo a CMR examination at both 1.5\u00a0T and 3.0\u00a0T. Because the MR systems were on two different locations, it was not feasible to study the same patient twice in the acute phase after MI.","keyphrases":["3.0 t","cardiovascular magnetic resonance","safety","late gadolinium enhancement","acute myocardial infarction"],"prmu":["P","P","P","P","R"]}
{"id":"Diabetologia-4-1-2292420","title":"Brisk walking compared with an individualised medical fitness programme for patients with type 2 diabetes: a randomised controlled trial\n","text":"Aims\/hypothesis Structured exercise is considered a cornerstone in type 2 diabetes treatment. However, adherence to combined resistance and endurance type exercise or medical fitness intervention programmes is generally poor. Group-based brisk walking may represent an attractive alternative, but its long-term efficacy as compared with an individualised approach such as medical fitness intervention programmes is unknown. We compared the clinical benefits of a 12-month exercise intervention programme consisting of either brisk walking or a medical fitness programme in type 2 diabetes patients.\nIntroduction\nRegular exercise has been identified along with diet and medication as one of the three components of good diabetes therapy [1]. Structured exercise intervention programmes have been reported to be as effective as pharmaceutical strategies in improving glycaemic control [2\u20135] and\/or cardiovascular risk profile [6, 7] in type 2 diabetes patients. Despite the growing body of scientific evidence on the health benefits of exercise intervention, most meta-analyses report a lack of studies that have tried to assess the long-term efficacy of exercise intervention in type 2 diabetes patients [3\u20135, 7\u20139]. In general, exercise intervention studies implement endurance and resistance type exercise, supervised by a physical therapist [10\u201317]. The long-term adherence to these (so-called) medical fitness programmes has been shown to range between 10 and 80% [11, 18\u201320]. Financial costs per patient are considerable in such programmes. Brisk walking exercise has been proposed as a less expensive alternative, with a good clinical outcome when patients are frequently counselled by motivated, supportive physicians [6, 21, 22]. However, brisk walking programmes mainly consist of endurance type exercise activities. As combined endurance and resistance type exercise training has been reported to be of greater clinical benefit [3, 23], it is conceivable that the long-term efficacy of a medical fitness programme is greater than that of a brisk walking programme.\nIn the present study, we compared the changes in glycaemic control and cardiovascular risk profile following the prescription of 12\u00a0months of either supervised group-based brisk-walking or of a more individualised medical fitness programme in a large population of type 2 diabetes patients (n\u2009=\u200992) in a primary healthcare setting. We report our results for changes in HbA1c values as a primary outcome parameter, with blood pressure, plasma lipid levels, insulin sensitivity by homeostasis model assessment (HOMA), body composition, physical fitness, programme adherence and health-related quality of life as secondary outcome parameters. Given the greater improvements in glycaemic control following combined endurance and resistance type exercise training as opposed to each separately [3, 23], we hypothesised that a medical fitness programme would result in a significantly greater decline in HbA1c than brisk walking.\nMethods\nParticipants\nFrom June till December 2005, 493 patients with type 2 diabetes were preselected from a population of 12,197 patients attending a primary healthcare centre that offers structured and guideline-based [24] diabetes care programmes. After 5\u00a0months of active recruitment by two nurse practitioners and four general practitioners, a total of 76 (=15.4%) patients were willing to participate in supervised exercise sessions three times a week for a period of at least 1\u00a0year. The type 2 diabetes patients selected had been diagnosed more than 3\u00a0months prior to screening according to the WHO criteria [25]. Exclusion criteria were: presence of (silent) cardiac or peripheral vascular disease, orthopaedic limitations and\/or diabetic foot ulceration. To increase sample size, the exercise intervention programme was subsequently advertised in local newspapers, which yielded another 26 potential study candidates. In total, 99 type 2 diabetes patients agreed to participate in either brisk walking or medical fitness programmes. Potential study candidates performed a graded exercise test on a cycle ergometer (Medifit; Medifit Systems, Maarn, the Netherlands) using a ramp protocol [26]. Cardiac function was monitored using a 12-lead electrocardiogram and blood pressure was measured to detect malignant hypertension. Of the 99 selected patients, three did not show up and four were excluded because of an abnormal stress-ECG. The remaining 92 volunteers were randomly allocated to either brisk walking (n\u2009=\u200949) or medical fitness (n\u2009=\u200943) programmes, by subsequently drawing a sequentially numbered opaque, sealed envelope (Fig.\u00a01). Laboratory personnel and the physician in charge of the graded exercise tests and randomisation procedure were blinded to the selection outcome of each participant. Drug prescriptions of each patient were recorded through an electronic database system operated through a network of local pharmacies. As part of the diabetes protocol in the healthcare centre, all type 2 diabetes patients consulted a dietitian and\/or diabetes nurse educator annually for dietary guidance. Baseline participant characteristics are provided in Table\u00a01. The nature and the risks of the exercise intervention were explained to all participants before written informed consent was obtained. This study was approved by the local Medical Ethical Committee of the M\u00e1xima Medical Centre, Veldhoven, the Netherlands. Since the study protocol was planned before 2005, official pre-trial registration at international trial registers was not performed.\nFig.\u00a01Study flow diagramTable\u00a01Participants\u2019 baseline characteristicsCharacteristicTotal (n\u2009=\u200992)Brisk walking (n\u2009=\u200949)Medical fitness (n\u2009=\u200943)Age (years)60\u2009\u00b1\u2009961\u2009\u00b1\u2009959\u2009\u00b1\u20099Men\/women (n)47\/4527\/2220\/23Duration of diabetes (years)5\u2009\u00b1\u200955\u2009\u00b1\u200945\u2009\u00b1\u20095BW (kg)94.3\u2009\u00b1\u200918.494.1\u2009\u00b1\u200918.194.5\u2009\u00b1\u200919.0BMI (kg\/m2)32.3\u2009\u00b1\u20095.532.1\u2009\u00b1\u20095.232.5\u2009\u00b1\u20096.0Wmax (W)156\u2009\u00b1\u200947155\u2009\u00b1\u200949157\u2009\u00b1\u200945Estimated  (l\/min)2.1\u2009\u00b1\u20090.62.1\u2009\u00b1\u20090.62.1\u2009\u00b1\u20090.6%Pred. 82\u2009\u00b1\u20091182\u2009\u00b1\u20091083\u2009\u00b1\u200911Participants treated with:\u00a0Diet only (n)1156\u00a0Hypoglycaemic agents (n)\u00a0\u00a0Total814437\u00a0\u00a0Metformin432419\u00a0\u00a0Sulfonylurea251312\u00a0\u00a0Thiazolidinedione1046\u00a0\u00a0Insulin1367\u00a0Antihypertensive agents (n)\u00a0\u00a0Total593227\u00a0\u00a0ACEI\/ARB392019\u00a0\u00a0Beta-blocker17710\u00a0\u00a0Diuretic19109\u00a0\u00a0Calcium-channel blocker954\u00a0\u00a0Other312\u00a0Lipid-lowering agents (n)\u00a0\u00a0Total543024\u00a0\u00a0Statin532924\u00a0\u00a0Fibrate110\u00a0\u00a0Other000Data are n (number of participants) or means\u2009\u00b1\u2009SD.Estimated  was based on Wmax during cycling ergometry and the equation according to Storer et al. [30].Relative age-, height- and sex-adjusted cardio-respiratory fitness (%Pred. ) was based on the equation by Fairbarn et al. [31].ACEI, ACE inhibitor; ARB, angiotensin receptor blocker; BW, body weight\nExercise intervention\nOverview Two different exercise intervention programmes were implemented over a 12-month period, during which three brisk walking or medical fitness sessions were performed each week. The exercise load was progressive in nature and had components of resistance and endurance type exercise according to the American Diabetes Association\/American College of Sports Medicine guidelines [27].\nBrisk walking programme The weekly volume of brisk walking consisted of three 60\u00a0min exercise sessions. During the first 3\u00a0months participants were supervised by certified exercise trainers and a physical therapist. Group size varied between 15 and 25 patients. After 3\u00a0months, certified trainers guided and supervised the training sessions, while the physical therapist was visited on a consultation basis. The endurance type exercise consisted of brisk walking (5\u20136\u00a0km\/h), with a focus on interval type endurance exercise training. During the intervention period the intensity was gradually increased and averaged 75\u2009\u00b1\u20095% of maximum heart frequency, as determined during the maximal cycle ergometer test. The resistance type exercise training consisted of resistance and floor exercises using individual body weight and\/or elastic bands (The Hygenic Corporation, Akron, OH, USA). Further details on the brisk walking programme are provided in Electronic supplementary material (ESM) Table 1.\nMedical fitness programme The medical fitness programme consisted of three exercise sessions per week. Endurance type exercise consisted of interval type exercise on a home trainer, elliptical trainer or rowing ergometer with an average intensity of 73\u2009\u00b1\u20092% of maximum heart frequency. All training sessions were tailored to individual performance capacity. Resistance type exercise consisted of a selection of eight different exercises targeting upper and lower body muscle groups. Over a period of 6\u00a0months the training volume of the medical fitness programme was progressively increased from three times 30\u00a0min per week (90\u00a0min\/week) towards a total of 180 to 225\u00a0min per week. Further details on the medical fitness programme are provided in ESM Table 1.\nEnergy expenditure and monitoring blood glucose Based on average heart rate and indirect calorimetry measurements performed during either brisk walking [28] or circuit resistance training in elderly [29] participants, energy expenditure in both exercise interventions was estimated to range between 0.23 and 0.33\u00a0kJ kg\u22121 min\u22121. Given the participants\u2019 bodyweights, the exercise interventions should be regarded as moderate intensity. Participants were provided with blood glucose monitor systems and test strips (A. Menarini Diagnostics, Valkenswaard, the Netherlands) and during the first month of brisk walking or medical fitness were advised to assess capillary blood glucose levels \u223c30\u00a0min before and after exercise. Patients were instructed to report recurrent hypoglycaemic events to their diabetes nurse educator or general practitioner. For logistic reasons no systematic enquiries on mild hypoglycaemic events were performed.\nFinancial cost of the intervention programmes Direct yearly costs for the individualised medical fitness programme were Euro 853 per participant and consisted of an ECG-stress test, supervision and consultations by a physical therapist, and the use of fitness centre facilities. The direct costs of the group-based brisk walking programme averaged Euro 396 per participant. Costs consisted of an ECG-stress test, supervision and consultation by a physical therapist, and a brisk walking membership fee. Other healthcare-related costs were not assessed.\nOutcome variables\nResting heart rate and blood pressure Resting heart rate and blood pressure were determined in a supine position before and at 6 and 12\u00a0months after initiating the exercise programme. Heart rate was stored and averaged through a heart rate monitoring system (CO2ntrol; Tildesign, Zeewolde, the Netherlands). Average systolic and diastolic blood pressure were determined (HEM-907; Omron Healthcare, Hoofddorp, the Netherlands) from five successive measurements.\nPhysical fitness Before and at 12\u00a0months after the start of the exercise programme, peak oxygen uptake capacity () was estimated based on maximum workload capacity (Wmax) during cycling ergometry according to Storer et al. [30]. Relative age-, height- and sex-adjusted cardio-respiratory fitness (%Pred. ) was defined as the ratio between estimated  and predicted  as determined in a healthy non-diabetic population [31].\nBlood analyses Two weeks before and at 3, 6, 9 and 12\u00a0months after initiating the exercise programme, fasting blood samples were collected. Blood samples collected before and after 3 and 9\u00a0months of intervention were analysed for HbA1c and basal glucose concentration. On the evening prior to blood sampling, participants remained fasted from 00:00\u00a0hours onwards. They arrived at the laboratory at 08:00\u00a0hours, having travelled by car or public transport. After 5 to 10\u00a0min of rest, a venous blood sample was collected from an antecubital vein. Blood samples were collected in tubes containing a glycolytic inhibitor (sodium fluoride) and an anticoagulant (potassium oxalate), immediately centrifuged for 5\u00a0min at 1,000\u00d7g and 4\u00b0C, after which aliquots of plasma were immediately frozen in liquid nitrogen and stored at \u221280\u00b0C until analyses. Plasma glucose (dehydrogenase assay), triacylglycerol (lipase\/peroxidase assay) concentrations, and total serum cholesterol (peroxidase\/cholesterol-esterase assay), HDL-cholesterol (peroxidase esterase assay) and LDL-cholesterol (according to the Friedewald formula if triacylglycerol was <4.5\u00a0mmol\/l, otherwise through direct peroxidase esterase assay) were analysed (Synchron LX20; Beckman Coulter, Fullerton, CA, USA). Plasma insulin was determined in duplicate by electrochemiluminescence-immunoassay (Elecsys 2010; Roche, Mannheim, Germany). Because of cross-sensitivity in the latter assay, exogenous insulin users were excluded from this analysis. HOMA insulin resistance index [32] was assessed to monitor changes in insulin sensitivity [33]. To determine HbA1c, 3\u00a0ml blood samples were collected and analysed by high-performance liquid chromatography (HA8160 Menarini; A. Menarini Diagnostics, Florence, Italy).\nQuality-of-life assessment\nHealth-related quality-of-life was measured with the RAND 36-Item Health Survey 1.0 [34, 35] before and after 12\u00a0months of exercise intervention.\nStatistical analyses\nWe calculated that 74 persons (37 per group) were needed to have 80% power to detect a moderate 0.65\u00a0SD difference in HbA1c as primary endpoint parameter between brisk walking and medical fitness programme, with an \u03b1 of 0.05. Sample size was exceeded to allow for an estimated withdrawal of \u223c15%. Data were analysed according to the intention-to-treat (ITT) principle. To minimise type I errors and loss of power [36], endpoint analyses according to the last observation carried forward principle were performed for missing values at 12\u00a0months follow-up. To assess whether physiological differences existed between the long-term application of brisk walking and medical fitness programme, post hoc analyses were performed for individuals actively participating for 10\u00a0months or more. Data are expressed as means\u2009\u00b1\u2009SD. ANOVA repeated measures was used to determine differences between baseline and status after 12\u00a0months of exercise intervention. Unpaired Student\u2019s t, \u03c72 and Mann\u2013Whitney U tests were used to test whether long-term changes observed in brisk walking differed from those seen in the medical fitness programme. Significance was set at the 0.05 level of confidence. Pearson\u2019s correlation calculation was used to test for linear relationships between long-term changes in dependent variables. All statistical calculations were performed using SPSS 10.1 (SPSS, Chicago, IL, USA).\nResults\nCharacteristics of the study population\nFollowing randomisation there were no significant differences in baseline characteristics, sex and anthropometry between brisk walking and medical fitness programme groups (Table\u00a01).\nProgramme adherence, follow-up and adverse events\nAfter 6\u00a0months of intervention, 22 (45%) brisk walking participants and 13 (30%) medical fitness programme participants (p\u2009=\u20090.15) were no longer participating (ESM Table 2). After 12\u00a0months, 18 (37%) brisk walking and 19 (44%) medical fitness participants respectively were still actively participating with mean adherence levels of 75\u2009\u00b1\u200916% and 68\u2009\u00b1\u200913% (p\u2009>\u20090.05) for the 156 available exercise sessions (ESM Table 2). Besides motivational reasons (25%), orthopaedic-related co-morbidities, such as overuse injuries and\/or subclinical osteoarthritis of the lower extremities, formed the main reason for dropout in 48 and 50% of the brisk walking and medical fitness programme participants, respectively (Fig.\u00a01, Table\u00a02). The distribution of missing data points was similar and not statistically different between groups (p\u2009>\u20090.05).\nTable\u00a02Adverse medical eventsEventsBrisk walking (n\u2009=\u200949)Medical fitness (n\u2009=\u200943)Adverse events related to exercise intervention1512\u00a0Shoulder pain\/chronic tendinopathy of rotator cuff01\u00a0(Aggravation of) low back pain22\u00a0Aggravation of pre-existing osteoarthritis in hip or knee joint64\u00a0Shin splints\/lower leg pain10\u00a0Chronic tendinopathy of Achilles tendon\/plantar fascia31\u00a0Other\/generalised musculoskeletal discomfort33Adverse events not related to exercise intervention46\u00a0Medical event requiring hospitalisationa33\u00a0Other serious medical eventb13Participants with adverse event resulting in withdrawal1917Data are number of participating diabetes patients that experienced an adverse event throughout the 12\u00a0months follow-up periodaHospitalisations were related to: arthroscopic knee surgery (brisk walking, n\u2009=\u20091), elective surgery for chronic abdominal aortic aneurysm (brisk walking, n\u2009=\u20091), elective cataract surgery (brisk walking, n\u2009=\u20091), elective knee joint replacement (medical fitness, n\u2009=\u20091), bacterial pneumonia (medical fitness, n\u2009=\u20091), elective varicose veins surgery (medical fitness, n\u2009=\u20091, did not result in withdrawal from study).bOther serious medical events requiring medical attention were: otitis externa (brisk walking) n\u2009=\u20091, atrial fibrillation (medical fitness) n\u2009=\u20091, newly diagnosed myocardial ischaemia (medical fitness) n\u2009=\u20091, and newly diagnosed carcinoma of the mammary gland (medical fitness) n\u2009=\u20091\nGlycaemic control\nAccording to ITT analyses, changes in HbA1c values following the prescription of brisk walking or medical fitness intervention were identical (95% CI \u22120.42, 0.43; p\u2009=\u20090.99) (Table\u00a03). A total of 12 patients (brisk walking, n\u2009=\u20095; medical fitness, n\u2009=\u20097) had been prescribed higher doses of blood glucose-lowering medication throughout the follow-up period. When these participants were excluded from our ITT analysis, HbA1c increased by 0.05% (95% CI \u22120.41, 0.51; p\u2009=\u20090.82) in brisk walking as compared with medical fitness programme. Post hoc analyses of long-term active participants (\u226510\u00a0months with mean adherence level of 62\u2009\u00b1\u200917 and 70\u2009\u00b1\u200918% in brisk walking and medical fitness groups, respectively) showed that, independently of changes in prescribed blood glucose-lowering drugs, HbA1c in the brisk walking group increased by 0.53% (95% CI 0.07, 1.00; p\u2009=\u20090.025) as compared with the medical fitness group (ESM Table 3).\nTable\u00a03Changes in diabetes outcome (ITT analysis)VariableChange from baseline to 12\u00a0monthsMean\u2009\u00b1\u2009SDnMean\u2009\u00b1\u2009SDnDifference95% CIp ValueHbA1c (%)\u00a0Total7.13\u2009\u00b1\u20091.36926.99\u2009\u00b1\u20091.2671\u00a0\u00a0\u00a0Brisk walking group7.18\u2009\u00b1\u20091.42497.08\u2009\u00b1\u20091.3738\u00a0\u00a0\u00a0\u00a0Medical fitness group7.08\u2009\u00b1\u20091.29436.89\u2009\u00b1\u20091.1333\u00a0\u00a0\u00a0\u00a0Intragroup comparisons\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Intragroup comparison total\u00a0\u00a0\u00a0\u00a0\u22120.14\u22120.39, 0.030.153\u00a0\u00a0Intragroup comparison brisk walking\u00a0\u00a0\u00a0\u00a0\u22120.11\u22120.51, 0.150.464\u00a0\u00a0Intragroup comparison medical fitness\u00a0\u00a0\u00a0\u00a0\u22120.18\u22120.45, 0.090.176\u00a0Brisk walking vs medical fitness\u00a0\u00a0\u00a0\u00a00.00\u22120.42, 0.430.985BMI (kg\/m2)\u00a0Total32.3\u2009\u00b1\u20095.59231.8\u2009\u00b1\u20095.489\u00a0\u00a0\u00a0\u00a0Brisk walking group32.1\u2009\u00b1\u20095.24931.9\u2009\u00b1\u20095.048\u00a0\u00a0\u00a0\u00a0Medical fitness group32.5\u2009\u00b1\u20096.04331.7\u2009\u00b1\u20095.941\u00a0\u00a0\u00a0\u00a0Intragroup comparisons\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Intragroup comparison total\u00a0\u00a0\u00a0\u00a0\u22120.5\u22121.1, 0.10.108\u00a0\u00a0Intragroup comparison brisk walking\u00a0\u00a0\u00a0\u00a0\u22120.2\u22120.6, 0.30.471\u00a0\u00a0Intragroup comparison medical fitness\u00a0\u00a0\u00a0\u00a0\u22120.8\u22122.0, 0.30.152\u00a0Brisk walking vs medical fitness\u00a0\u00a0\u00a0\u00a00.7\u22120.5, 1.90.237Fasting plasma glucose (mmol\/l)\u00a0Total8.44\u2009\u00b1\u20092.81928.21\u2009\u00b1\u20092.3771\u00a0\u00a0\u00a0\u00a0Brisk walking group8.64\u2009\u00b1\u20092.83498.33\u2009\u00b1\u20092.6438\u00a0\u00a0\u00a0\u00a0Medical fitness group8.21\u2009\u00b1\u20092.80438.06\u2009\u00b1\u20092.0533\u00a0\u00a0\u00a0\u00a0Intragroup comparisons\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Intragroup comparison total\u00a0\u00a0\u00a0\u00a0\u22120.23\u22120.72, 0.250.345\u00a0\u00a0Intragroup comparison brisk walking\u00a0\u00a0\u00a0\u00a0\u22120.31\u22120.96, 0.340.344\u00a0\u00a0Intragroup comparison medical fitness\u00a0\u00a0\u00a0\u00a0\u22120.15\u22120.91, 0.610.699\u00a0Brisk walking vs. medical fitness\u00a0\u00a0\u00a0\u00a0\u22120.16\u22121.14, 0.820.744HOMAa\u00a0Total5.55\u2009\u00b1\u20093.68685.60\u2009\u00b1\u20093.3854\u00a0\u00a0\u00a0\u00a0Brisk walking group5.86\u2009\u00b1\u20093.56375.75\u2009\u00b1\u20093.2030\u00a0\u00a0\u00a0\u00a0Medical fitness group5.19\u2009\u00b1\u20093.58315.43\u2009\u00b1\u20093.6424\u00a0\u00a0\u00a0\u00a0Intragroup comparisons\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Intragroup comparison total\u00a0\u00a0\u00a0\u00a00.05\u22120.50, 0.620.833\u00a0\u00a0Intragroup comparison brisk walking\u00a0\u00a0\u00a0\u00a0\u22120.12\u22121.11, 0.850.794\u00a0\u00a0Intragroup comparison medical fitness\u00a0\u00a0\u00a0\u00a00.24\u22120.19, 0.740.241\u00a0Brisk walking vs medical fitness\u00a0\u00a0\u00a0\u00a0\u22120.40\u22121.52, 0.720.479Resting heart rate (bpm)\u00a0Total73.4\u2009\u00b1\u200911.89169.6\u2009\u00b1\u200913.977\u00a0\u00a0\u00a0\u00a0Brisk walking group72.6\u2009\u00b1\u200911.84868.3\u2009\u00b1\u200915.041\u00a0\u00a0\u00a0\u00a0Medical fitness group74.4\u2009\u00b1\u200911.84371.0\u2009\u00b1\u200912.436\u00a0\u00a0\u00a0\u00a0Intragroup comparisons\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Intragroup comparison total\u00a0\u00a0\u00a0\u00a0\u22123.9\u22125.9, \u22121.80.000\u00a0\u00a0Intragroup comparison brisk walking\u00a0\u00a0\u00a0\u00a0\u22124.3\u22127.4, \u22121.10.009\u00a0\u00a0Intragroup comparison medical fitness\u00a0\u00a0\u00a0\u00a0\u22123.4\u22125.9, \u22120.90.010\u00a0Brisk walking vs medical fitness\u00a0\u00a0\u00a0\u00a0\u22120.9\u22124.9, 3.20.678Systolic blood pressure (mmHg)\u00a0Total148.5\u2009\u00b1\u200918.191137.5\u2009\u00b1\u200915.577\u00a0\u00a0\u00a0\u00a0Brisk walking group150.3\u2009\u00b1\u200918.948138.9\u2009\u00b1\u200916.341\u00a0\u00a0\u00a0\u00a0Medical fitness group146.3\u2009\u00b1\u200917.143135.9\u2009\u00b1\u200914.636\u00a0\u00a0\u00a0\u00a0Intragroup comparisons\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Intragroup comparison total\u00a0\u00a0\u00a0\u00a0\u221210.9\u221214.0, \u22127.80.000\u00a0\u00a0Intragroup comparison brisk walking\u00a0\u00a0\u00a0\u00a0\u221211.4\u221215.9, \u22126.80.000\u00a0\u00a0Intragroup comparison medical fitness\u00a0\u00a0\u00a0\u00a0\u221210.4\u221214.7, 6.20.000\u00a0Brisk walking vs medical fitness\u00a0\u00a0\u00a0\u00a0\u22120.9\u22127.1, 5.3)0.768Diastolic blood pressure (mmHg)\u00a0Total81.9\u2009\u00b1\u200910.69176.6\u2009\u00b1\u20098.377\u00a0\u00a0\u00a0\u00a0Brisk walking group81.3\u2009\u00b1\u200910.84875.9\u2009\u00b1\u20098.841\u00a0\u00a0\u00a0\u00a0Medical fitness group82.6\u2009\u00b1\u200910.44377.4\u2009\u00b1\u20097.736\u00a0\u00a0\u00a0\u00a0Intragroup comparisons\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Intragroup comparison total\u00a0\u00a0\u00a0\u00a0\u22125.3\u22127.1, \u22123.60.000\u00a0\u00a0Intragroup comparison brisk walking\u00a0\u00a0\u00a0\u00a0\u22125.4\u22127.8, 2.90.000\u00a0\u00a0Intragroup comparison medical fitness\u00a0\u00a0\u00a0\u00a0\u22125.2\u22127.8, \u22122.70.000\u00a0Brisk walking vs medical fitness\u00a0\u00a0\u00a0\u00a0\u22120.1\u22123.6, 3.40.949Total cholesterol level (mmol\/l)\u00a0Total4.70\u2009\u00b1\u20090.86884.53\u2009\u00b1\u20090.9465\u00a0\u00a0\u00a0\u00a0Brisk walking group4.83\u2009\u00b1\u20090.89474.61\u2009\u00b1\u20090.9235\u00a0\u00a0\u00a0\u00a0Medical fitness group4.54\u2009\u00b1\u20090.12414.44\u2009\u00b1\u20090.9630\u00a0\u00a0\u00a0\u00a0Intragroup comparisons\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Intragroup comparison total\u00a0\u00a0\u00a0\u00a0\u22120.16\u22120.32, \u22120.010.035\u00a0\u00a0Intragroup comparison brisk walking\u00a0\u00a0\u00a0\u00a0\u22120.22\u22120.45, 0.010.064\u00a0\u00a0Intragroup comparison medical fitness\u00a0\u00a0\u00a0\u00a0\u22120.10\u22120.30, 0.100.307\u00a0Brisk walking vs medical fitness\u00a0\u00a0\u00a0\u00a0\u22120.12\u22120.42, 0.190.453LDL-cholesterol level (mmol\/l)\u00a0Total2.83\u2009\u00b1\u20090.76882.70\u2009\u00b1\u20090.7964\u00a0\u00a0\u00a0\u00a0Brisk walking group2.90\u2009\u00b1\u20090.82472.72\u2009\u00b1\u20090.8535\u00a0\u00a0\u00a0\u00a0Medical fitness group2.74\u2009\u00b1\u20090.69412.68\u2009\u00b1\u20090.7229\u00a0\u00a0\u00a0\u00a0Intragroup comparisons\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Intragroup comparison total\u00a0\u00a0\u00a0\u00a0\u22120.12\u22120.26, 0.010.070\u00a0\u00a0Intragroup comparison brisk walking\u00a0\u00a0\u00a0\u00a0\u22120.18\u22120.38, 0.020.080\u00a0\u00a0Intragroup comparison medical fitness\u00a0\u00a0\u00a0\u00a0\u22120.06\u20130.25, 0.120.493\u00a0Brisk walking vs medical fitness\u00a0\u00a0\u00a0\u00a0\u22120.11\u22120.38, 0.160.401HDL-cholesterol level (mmol\/l)\u00a0Total1.09\u2009\u00b1\u20090.28881.10\u2009\u00b1\u20090.2965\u00a0\u00a0\u00a0\u00a0Brisk walking group1.10\u2009\u00b1\u20090.23471.09\u2009\u00b1\u20090.2635\u00a0\u00a0\u00a0\u00a0Medical fitness group1.07\u2009\u00b1\u20090.33411.11\u2009\u00b1\u20090.3230\u00a0\u00a0\u00a0\u00a0Intragroup comparisons\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Intragroup comparison total\u00a0\u00a0\u00a0\u00a0\u22120.01\u20130.02, 0.040.570\u00a0\u00a0Intragroup comparison brisk walking\u00a0\u00a0\u00a0\u00a0\u22120.01\u22120.06, 0.040.629\u00a0\u00a0Intragroup comparison medical fitness\u00a0\u00a0\u00a0\u00a00.03\u22120.01, 0.080.129\u00a0Brisk walking vs medical fitness\u00a0\u00a0\u00a0\u00a0\u22120.05\u22120.12, 0.020.172Triacylglycerol level (mmol\/l)\u00a0Total1.88\u2009\u00b1\u20090.99881.79\u2009\u00b1\u20090.9164\u00a0\u00a0\u00a0\u00a0Brisk walking group2.00\u2009\u00b1\u20091.08471.94\u2009\u00b1\u20090.9835\u00a0\u00a0\u00a0\u00a0Medical fitness group1.74\u2009\u00b1\u20090.88411.61\u2009\u00b1\u20090.7929\u00a0\u00a0\u00a0\u00a0Intragroup comparisons\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Intragroup comparison total\u00a0\u00a0\u00a0\u00a0\u22120.09\u22120.25, 0.060.239\u00a0\u00a0Intragroup comparison brisk walking\u00a0\u00a0\u00a0\u00a0\u22120.06\u22120.33, 0.170.646\u00a0\u00a0Intragroup comparison medical fitness\u00a0\u00a0\u00a0\u00a0\u22120.13\u22120.27, 0.060.120\u00a0Brisk walking vs medical fitness\u00a0\u00a0\u00a0\u00a0\u22120.02\u22120.29, 0.330.892RAND-36 (score 0\u2013100 scale)\u00a0Total71\u2009\u00b1\u2009158970\u2009\u00b1\u20091558\u00a0\u00a0\u00a0\u00a0Brisk walking group69\u2009\u00b1\u2009154668\u2009\u00b1\u20091531\u00a0\u00a0\u00a0\u00a0Medical fitness group73\u2009\u00b1\u2009154371\u2009\u00b1\u20091427\u00a0\u00a0\u00a0\u00a0Intragroup comparisons\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Intragroup comparison total\u00a0\u00a0\u00a0\u00a0\u22120.6\u22122.4, 1.20.503\u00a0\u00a0Intragroup comparison brisk walking\u00a0\u00a0\u00a0\u00a00.1\u20132.7, 2.80.969\u00a0\u00a0Intragroup comparison medical fitness\u00a0\u00a0\u00a0\u00a0\u22121.4\u22123.8, 1.10.261\u00a0Brisk walking vs medical fitness\u00a0\u00a0\u00a0\u00a01.4\u22122.2, 5.00.438Data are means\u2009\u00b1\u2009SD. n=number of valid measurements. aFor HOMA calculations exogenous insulin users (n\u2009=\u200913) were excluded\nResting heart rate and blood pressure\nFollowing the 12\u00a0month exercise intervention, resting heart rate in the brisk walking group was reduced by 0.9 beats per min (bpm) (CI \u22127.1, 5.3; p\u2009=\u20090.68) as compared with medical fitness group. No significant differences in blood pressure response were observed between the two interventions (Table\u00a03). After excluding participants who were prescribed higher doses of antihypertensive drugs (brisk walking, n\u2009=\u20095; medical fitness, n\u2009=\u20097), mean arterial blood pressure decreased by 0.9\u00a0mmHg (95% CI \u22125.6, 3.9; p\u2009=\u20090.94) in the brisk walking as compared with medical fitness group. A post hoc analysis of 46 long-term active participants showed a non-significant difference in mean arterial blood pressure of \u22123.5\u00a0mmHg (95% CI \u22129.2, 2.2; p\u2009=\u20090.22) in the brisk walking compared with the medical fitness programme (ESM Table 3).\nBlood lipid profile\nAfter 12\u00a0months, the overall fasting lipid profile did not differ between the two groups (Table\u00a03). When participants (brisk walking, n\u2009=\u20099, medical fitness, n\u2009=\u20097) who were prescribed more blood lipid-lowering agents were excluded, HDL-cholesterol had changed by \u22120.05\u00a0mmol\/l (95% CI \u22120.12, 0.02; p\u2009=\u20090.17) in the medical fitness compared with the brisk walking group.\nBody composition and workload capacity\nBody composition (BMI) did not change (Table\u00a03). Workload capacity as measured during cycle ergometry averaged 155\u2009\u00b1\u200949 and 157\u2009\u00b1\u200945\u00a0W in the brisk walking and medical fitness programme groups, respectively, changing by \u22124.3\u00a0W (95% CI \u22129.1, 0.5; p\u2009=\u20090.078) in the former and by \u22122.5\u00a0W (95% CI \u221210, 5.6; p\u2009=\u20090.53) in the latter, with a non-significant difference of \u22121.8\u00a0W (95% CI \u221210.5, 6.9; p\u2009=\u20090.68) in brisk walking compared with medical fitness programme participants. Maximum heart rates recorded during ergometry averaged 146\u2009\u00b1\u200922 and 152\u2009\u00b1\u200919\u00a0bpm at baseline, and 152\u2009\u00b1\u200921 and 149\u2009\u00b1\u200922\u00a0bpm following 12\u00a0months of exercise intervention in the brisk walking and medical fitness groups, respectively (p\u2009>\u20090.05). In our long-term active brisk walking participants, Wmax changed by \u22126.3 W (95% CI \u221216.5, 3.9; p\u2009=\u20090.22) as compared with their medical fitness programme counterparts. No significant correlations were found between changes in HbA1c values and changes in workload capacity (\u0394Wmax, Pearson\u2019s R\u2009=\u20090.02, p\u2009=\u20090.93, n\u2009=\u200938) or body weight (Pearson\u2019s R\u2009=\u20090.18, p\u2009=\u20090.225, n\u2009=\u200945).\nQuality of life assessment\nA total of 89 and 58 participants completed the RAND-36 questionnaire at baseline and after 12\u00a0months of intervention, respectively. Reliability and internal consistency was excellent with Cronbach\u2019s alpha of 0.93 and 0.91 respectively. No significant difference in changes of total RAND-36 scores were observed between the two intervention groups (p\u2009>\u20090.05; Table\u00a03).\nDiscussion\nIt has been firmly established that physical activity counselling [37] and participation in structured exercise intervention programmes [15, 19] improves glycaemic control. However, long-term intervention studies on the clinical benefits of different types of exercise intervention programmes in type 2 diabetes patients are lacking. In the present study, we compared the clinical benefits of 12\u00a0months of a group-based brisk walking programme versus a more individualised medical fitness programme. ITT analysis showed that the prescription of a group-based, brisk walking programme is not necessarily inferior to a more individualised medical fitness programme with regard to glycaemic control and markers for cardiovascular risk profile (Table\u00a03). Our data therefore imply that most patients with type 2 diabetes will achieve more or less equal long-term therapeutic benefits from a low-impact brisk walking programme as opposed to a more individualised medical fitness programme. Nevertheless, long-term programme adherence and the possibility of dropout should be taken into consideration when prescribing either type of exercise intervention for a patient with type 2 diabetes. Independently of the provided level of guidance and infrastructure, 60% of the patients dropped out of the exercise programme during the 12\u00a0month intervention period, with no differences in adherence and dropout patterns between brisk walking and medical fitness programme. Although motivational factors explained 25% of the dropout in both exercise programmes, almost 50% of the dropout was attributed to orthopaedic-related co-morbidities and overuse injuries of the lower extremities. The latter was not anticipated, since clinically relevant orthopaedic limitations, such as a history of osteoarthritis of the hip or knee joint, were defined as exclusion criteria for participation in either exercise intervention programme. Apparently, subclinical joint disease and\/or degeneration of myotendineous structures become apparent when physical activity levels are increased. Stiffening of connective tissue due to non-enzymatic advanced glycation end-product formation [38], as well as decreased collagen turnover due to ageing and chronic disuse [39], could be responsible for the impaired response to increased musculoskeletal loading in these patients. Although more fundamental clinical research is warranted, the high incidence of overuse injuries in the present study adds to the results described in another study [23], suggesting that in unselected, non-study populations dropout will be even greater. Therefore, it might be advisable to implement a more gradual and less intense exercise regimen to allow myotendineous structures to adapt to increased loading. Moreover, before prescribing therapeutic exercise, diabetes healthcare workers should carefully consider obesity- and diabetes-related musculoskeletal deconditioning [40\u201342]. Such programmes should probably focus even more on resistance type exercise activities [43, 44] to bring patients to a level at which they are able to participate and adhere to more generic diabetes intervention programmes. It has also been suggested that psychological strategies such as motivational interviewing [45] or booster sessions [21, 46] might help to further improve programme adherence. Nevertheless, more long-term, tailored exercise intervention studies are warranted to further define the most effective and feasible exercise interventional strategy.\nThe absence of a significant decline in blood HbA1c levels in brisk walking participants does not imply that the prescription of low-impact, endurance type exercise has no therapeutic value [3, 7, 27, 47\u201349]. In fact, we observed significant improvements in blood pressure control following either type of exercise intervention (Table\u00a03). Although reliable assessment of actual drug use was not possible in the present study, the improvements in blood pressure occurred independently of changes in the prescription of antihypertensive medication. However, the lack of a non-exercise control group in the present study makes it impossible to speculate on cause and effect, since changes in blood pressure from baseline might also, or at least partly, be explained by the Hawthorne effect, for example, or by familiarisation with blood pressure measurement protocols. Nevertheless, our data suggest that the structured application of group-based brisk walking and supervised medical fitness programmes tends to have a similar impact on cardiovascular risk profile.\nIn conclusion, the prescription of group-based brisk walking represents an equally effective interventional strategy to modulate glycaemic control and cardiovascular risk profile in type 2 diabetes patients compared with a more individualised medical fitness programme. General deconditioning, musculoskeletal overuse injuries and lack of motivation limit the benefits of long-term exercise intervention as an add-on to a comprehensive diabetes care programme. Future diabetes exercise intervention programmes in primary healthcare settings should consider diabetes-related co-morbidities and patient motivation as important factors, which determine both long-term programme adherence and the associated clinical benefits.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nTable\u00a01\nExercise programme (PDF 60.7 KB)\nTable\u00a02\nIndividual follow-up data for primary outcome variable (HbA1c) and active programme participation (PDF 69.9 KB)\nTable\u00a03\nChanges in diabetes outcome in long-term active participants (PDF 160 KB)","keyphrases":["exercise","blood pressure","physical fitness","hyperglycaemia","lipid metabolism","randomised controlled clinical trial","type 2 diabetes mellitus"],"prmu":["P","P","P","U","M","R","M"]}
{"id":"J_Mol_Med-4-1-2262918","title":"Celastrol inhibits polyglutamine aggregation and toxicity though induction of the heat shock response\n","text":"Heat shock proteins (hsps) are protective against the harmful effects of mutant expanded polyglutamine repeat proteins that occur in diseases such as Huntington\u2019s, prompting the search for pharmacologic compounds that increase hsp expression in cells as potential treatments for this and related diseases. In this paper, we show that celastrol, a compound recently shown to up-regulate hsp gene expression, significantly decreases killing of cells expressing mutant polyglutamine protein. This effect requires the presence of the transcription factor responsible for mediating inducible hsp gene expression, HSF1, and is correlated with decreased amounts and increased sodium dodecyl sulfate (SDS) solubility of polyglutamine aggregates. These results suggest the potential of celastrol as a therapeutic agent in the treatment of human polyglutamine expansion diseases.\nIntroduction\nA large number of neurodegenerative diseases, including Huntington\u2019s and Kennedy\u2019s diseases, are characterized by expanded polyglutamine repeats in certain proteins leading to their aggregation and toxic effects on the cells expressing them [1, 2]. A number of results suggest that polyglutamine-containing aggregates are important both for the initiation and progression of these diseases [3, 4]. Polyglutamine protein aggregates in brains of patients and transgenic animals have been found to contain various molecular chaperones, ubiquitin, and components of the 20S proteasome [5, 6]. This suggests that neuronal cells recognize the protein aggregates as abnormally folded and try to disaggregate or degrade the mutant proteins by recruiting chaperones and proteasomal components [7]. Consistent with this view, increased expression of hsp70 and other heat shock proteins (hsps) has been found to be effective in reducing the toxicity of mutant polyglutamine proteins, suggesting the potential of pharmacological up-regulation of hsp gene regulation as a means for treating polyglutamine expansion diseases such as Huntington\u2019s [8\u201311].\nHeat Shock Factor 1, HSF1, is the transcription factor responsible for up-regulating the expression of hsp70 and other hsp genes in response to cellular stress [12, 13]. HSF activation involves stress-induced conversion of this factor to its trimeric DNA-binding form [14\u201317]. The activated trimeric HSF1 then binds to heat shock elements in the promoters of hsp genes to up-regulate their transcription, ultimately leading to elevated levels of cytoprotective hsps in these cells.\nThe function of HSF1 as a key positive regulator of hsp expression, coupled to the observed ability of hsp proteins to protect cells from polyglutamine toxicity prompted the hypothesis that interventions that lead to activation of HSF1 could provide protection for cells against this toxicity and possibly represent agents that could be useful in treating polyglutamine expansion diseases such as Huntington\u2019s [10, 18]. Consistent with this hypothesis, expression in cells of a constitutively active mutant HSF1 protein results in decreased polyglutamine toxicity and aggregate formation in both cell culture and animal models [19, 20]. These and other results have stimulated the search for pharmacological compounds capable of up-regulating hsp gene expression, as such compounds would theoretically have potential as treatments for these and other diseases of protein misfolding.\nRecently, it was demonstrated that a compound called celastrol, which is found in the Celastraceae family of plants and is already being used to treat diseases such as rheumatoid arthritis, bacterial infection, and fever [21, 22], is able to activate HSF1 and up-regulate hsp gene expression [18]. Celastrol has previously been found to inhibit the aggregation of purified Q58-huntingtin 1-171 amino terminal fragment in an in vitro assay, although this study did not examine effects of celastrol on polyglutamine aggregation in cells [23]. Celastrol has also been found to inhibit the cytotoxicity of expanded polyglutamine form of huntingtin exon 1 [24] and androgen receptor [25], but these studies did not examine the mechanism by which celastrol protects cells from expanded polyglutamine protein cytotoxicity.\nThe results of the experiments presented in this paper demonstrate that celastrol is effective in preventing both the aggregation and toxicity of polyglutamine expression in cells and that it mediates these effects via the HSF1-mediated gene expression pathway. These results support the potential of this drug as a possible therapeutic agent for treating polyglutamine expansion diseases. The results also suggest that other drugs that stimulate HSF1 activity leading to hsp gene expression may also have beneficial activity against these disease states as well as other human diseases that are caused by protein misfolding.\nMaterials and methods\nPlasmids, cell culture, and celastrol\nThe expression constructs encoding Q19-YFP, Q57-YFP, and Q81-YFP were kindly provided by Dr. James Burke (Duke University). The polyglutamine-YFP (Qn-YFP) vectors were constructed using CAG repeats that were synthesized by polymerase chain reaction (PCR) from human dentatorubral pallidoluysian atrophy (DRPLA) cDNAs containing different CAG repeats. HeLa cells were cultured in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM; Cellgro) supplemented with 10% fetal bovine serum (FBS) and 50\u00a0\u03bcg\/ml gentamicin. PC12 cells were cultured in DMEM medium (Cellgro) supplemented with 5% FBS and 10% heat-inactivated horse serum (Gibco), and 100 units\/ml Penicillin-Streptomycin (Gibco). HSF1+\/+ and HSF1\u2212\/\u2212 mouse embryo fibroblast (MEF) cells (kindly provided by Dr. Ivor Benjamin, University of Utah) were cultured in DMEM medium (Cellgro) supplemented with 10% FBS, 1\u00d7 Penicillin-streptomycin (Gibco), and 1\u00d7 Non-essential amino acids (Gibco). Transfection was performed using Effectene transfection reagent (Qiagen), following the manufacturer\u2019s protocol. Celastrol (Calbiochem) was dissolved in dimethyl sulphoxide at a stock concentration of 5.54\u00a0mM.\nTrypan blue cell viability assay\nCells were collected by centrifugation at 1,000\u00a0rpm for 10\u00a0min at 4\u00b0C, and the pellet was washed twice with 1\u00d7 phosphate-buffered saline (PBS). The cell pellet was then resuspended in 1\u00d7 PBS to a concentration of approximately 106 cells\/ml. A 1:1 dilution of the suspension was prepared using a solution containing 0.4% trypan blue stain (Gibco), and the suspension was then loaded into the counting chamber of a hemocytometer. The number of stained cells as well as the total number of cells was counted, and the percentage of stained cells was taken to represent the percentage of cell death. Experiments were repeated three times.\nFluorescence microscopy\nCells were seeded on coverslips, and 48\u00a0h after transfection, Hoechst 33342 and verapamil were added to the medium to final concentrations of 5 and 50\u00a0\u03bcg\/ml, respectively. After incubation at 37\u00b0C for 30\u00a0min, the medium was removed, and the coverslips washed with 1\u00d7 PBS for 5\u00a0min. A solution containing 3.7% paraformaldehyde in 1\u00d7 PBS was added, and after 20\u00a0min incubation at room temperature, coverslips were washed with 1\u00d7 PBS for 5\u00a0min. Coverslips were washed briefly three times in distilled water and mounted onto a slide spotted with 15\u00a0\u03bcl Vectashield (Vector Laboratories). Excess fluid was wicked from the coverslip and the edges of the coverslip sealed with fingernail polish. The fluorescence was then visualized using a Nikon fluorescent microscope and pictures taken with a Nikon Spotcam digital-imaging camera. To quantify the formation of polyglutamine aggregates, visual fields which contained similar numbers of cells (based on the density of nuclei stained by Hoechst) were chosen under 20\u00d7 objective, and then the number of cells that contained aggregates in each field of vision was counted. Three different visual fields were quantified in each case. There were approximately 300 cells in each visual field for the experiments involving HeLa cells and PC12 cells, and approximately 100 cells in the experiments involving MEF cells. We scored a cell as positive if it had any visible aggregates, and the aggregates in the majority of cells appeared to be intranuclear or perinuclear (example shown in Fig.\u00a0S1).\nExtract preparation and Western blot assay\nCell lysis was performed on ice for 30\u00a0min in 50\u00a0mM Tris\u2013HCl [pH\u00a08.0], 100\u00a0mM NaCl, 5\u00a0mM MgCl2, 0.5% NP40 lysis buffer [26] containing 1\u00d7 protease inhibitor cocktail (Roche), and 1\u00a0mM phenylmethanesulfonyl fluoride added. To examine hsp70 level, the cell lysate was cleared by centrifugation at 1,000\u00a0rpm at 4\u00b0C for 10\u00a0min, and the protein concentration of the supernatant was then determined by BioRad assay. Five micrograms of protein extract was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot following standard procedures. The antibodies and dilutions used to probe the Western blots were as follows. Anti-hsp70 (stress-induced form; Stressgen) was used at 1:10,000, anti-\u03b2-actin antibody (Sigma) was used at 1:10,000, and the anti-green fluorescent protein (GFP) polyclonal antibody (Bethyl labs) was used at a dilution of 1:4,000. Hsp70 Western blots were quantified using ImageQuant software.\nFiltration assay\nCell lysis was performed on ice for 30\u00a0min in the lysis buffer described above. The insoluble fraction was obtained by centrifugation of the cell lysate at 14,000\u00a0rpm at 4\u00b0C for 10\u00a0min. The pellet was resuspended in DNase buffer (20\u00a0mM Tris\u2013HCl [pH\u00a08.0], 15\u00a0mM MgCl2, and 0.5\u00a0mg\/ml DNase I), followed by incubation at 37\u00b0C for 1\u00a0h. After DNase treatment, the protein concentration was determined by analyzing an aliquot using the BioRad assay. The incubation was terminated by adjusting the mixtures to 20\u00a0mM ethylenediamine tetraacetic acid (EDTA), 2% sodium dodecyl sulfate (SDS), and 50\u00a0mM dithiothreitol and boiling for 5\u00a0min. Thirty micrograms of protein was diluted into 200\u00a0\u03bcl 2% SDS, and this was then filtered through a 0.2-\u03bcm pore size cellulose acetate membrane (Schleicher & Schuell) that was equilibrated with 2% SDS using a Dot-Blot system (Schleicher & Schuell). Filters were washed twice with 200\u00a0\u03bcl 0.1% SDS and then subjected to anti-GFP Western blot [27].\nSDS solubility assay\nTo perform the SDS solubility assay, cell lysates were prepared as described above for the filtration assay, including the 14,000\u00a0rpm centrifugation of the cell lysate and DNase treatment and protein concentration determination by BioRad assay. Forty micrograms of protein was then adjusted to 2\u00d7 SDS-PAGE sample buffer (62.5\u00a0mM Tris\u00b7HCl [pH\u00a06.8], 10% glycerol, 2% SDS, 5% \u03b2-mercaptoethanol) and boiled for 5\u00a0min after which the sample was analyzed by SDS-PAGE and Western blot with anti-GFP polyclonal antibody.\nStatistical analysis\nStatistical significance was determined using the Student\u2019s t test. A P value of <0.05 was considered to be statistically significant.\nResults\nCelastrol protects against polyglutamine toxicity\nThe experimental system we chose to test for effects of celastrol on polyglutamine aggregation and toxicity is transfection of a Q57-YFP fusion protein into cell lines (gift of Dr. James Burke), which is an established model for the aggregation of polyglutamine containing proteins in vivo [28]. Consistent with this, the Q57-YFP protein forms clearly visible aggregates upon expression in HeLa cells, while cells transfected with Q19-YFP exhibit a diffuse pattern (Fig.\u00a0S1). A previous study showed that 8\u00a0h of celastrol treatment leads to increased levels of the hsp70 protein [18]. As the studies described in this paper include cell death and aggregation assays performed at 48\u00a0h after transfection with the Q57-YFP construct, we wanted to ensure that cells treated with celastrol treatment for longer times also exhibit elevated hsp70 expression. As shown in Fig.\u00a01a (upper panel), celastrol treatment of HeLa cells for 24 or 48\u00a0h both result in a dose-dependent increase in expression of the hsp70 protein. Quantification of these hsp70 Western blot results indicates that the induction of hsp70 by each celastrol concentration is very similar at 24 and 48\u00a0h, showing that treatment with this drug results in a sustained increase in hsp70 levels (lower panel). Next, to test whether treatment with this drug confers protection from polyglutamine toxicity, HeLa cells were transfected with the Q57-YFP construct and then incubated in media containing different concentrations of celastrol. The results of this experiment, shown in Fig.\u00a01b, reveal that celastrol treatment is associated with a significant decrease in death of these cells expressing this mutant polyglutamine protein.\nFig.\u00a01Celastrol treatment reduces Q57-YFP cytotoxicity. a Hsp70 protein level is increased by celastrol treatment. HeLa cells were treated with the indicated concentrations of celastrol for 24 or 48\u00a0h, after which cell extracts were made and subjected to Western blot using antibodies against hsp70 or \u03b2-actin (upper panel). These results were quantified using the ImageQuant program, and the values for the two treatment times, grouped by celastrol concentration, were graphed (lower panel). b HeLa cells were transfected with Q57-YFP along with celastrol treatment at the concentrations indicated. After 48\u00a0h, the amount of cell death was determined by trypan blue assay. Data are shown as means \u00b1 SE (*P\u2009<\u20090.007, **P\u2009<\u20090.004, ***P\u2009<\u20090.0001, for each celastrol concentration treatment vs no celastrol)\nCelastrol protective effect are correlated with decreased number of cells containing polyglutamine aggregates\nTo determine whether the ability of celastrol to protect cells from polyQ toxicity could be mediated via effects on polyQ aggregates, we then examined whether celastrol treatment alters the number of polyQ aggregates in cells and\/or their solubility. The results of these experiments show that treatment with celastrol is associated both with a decrease in the number of cells containing Q57-YFP aggregates, as quantified by fluorescence microscopy (Fig.\u00a02a), and also with an increase in the amount of Q57-YFP that can be solubilized from aggregates by SDS treatment (Fig.\u00a02b). SDS solubility of aggregates is related with their toxicity, and data from previous studies suggests that molecular chaperones may ameliorate the neurodegenerative effect of mutant polyglutamine protein, at least in part, by increasing the solubility of these proteins [9, 29]. Together, the results shown in Figs.\u00a01 and 2 indicate that celastrol treatment decreases the death of cells expressing mutant polyglutamine protein and also decreases the number of cells containing Q57-YFP-aggregates and the insolubility of Q57-YFP-aggregates.\nFig.\u00a02Celastrol treatment reduces number of cells containing Q57-YFP aggregates and increases Q57-YFP solubility. HeLa cells were transfected with Q57-YFP along with celastrol treatment at the concentrations indicated. a After 48\u00a0h of transfection, the formation of Q57-YFP aggregates was quantified using fluorescence microscopy. Visual fields which contained similar numbers of cells (based on the density of nuclei stained by Hoechst) were chosen under 20\u00d7 objective, and then the number of aggregates in each field of vision was counted. Three different visual fields were quantified in each case, and data are shown as means \u00b1 SE (*P\u2009<\u20090.004, **P\u2009<\u20090.001, ***P\u2009<\u20090.0003, for each celastrol concentration treatment vs no celastrol). b To determine the amount of Q57-YFP monomer that could be solubilized from aggregates in lysates of the transfected cells by SDS treatment, the protein concentration of the insoluble fraction of the cell lysates was determined, and then 40\u00a0\u03bcg of protein was subjected to SDS solubilization treatment, followed by Western blot using anti-GFP antibody\nProtective effects of celastrol treatment in PC12 cells\nAs the deleterious effects of expression of mutant polyglutamine proteins in vivo are observed primarily in cells of neuronal origin, we next sought to test the effect of celastrol treatment on polyglutamine toxicity and aggregation in PC12 cells. As shown in Fig.\u00a03a, PC12 cells are very sensitive to killing by expression of the Q57-YFP protein, but as observed for HeLa cells in Fig.\u00a01b above, celastrol treatment results in a significant decrease in death of these cells. As was observed in the experiments using HeLa cells, treatment of PC12 cells with celastrol resulted in a significant decrease in the number of polyglutamine aggregates in the cells (Fig.\u00a03b) and an increase in the amount of Q57-YFP that can be solubilized from aggregates by SDS treatment (Fig.\u00a03c). Interestingly, the concentrations of celastrol required to achieve these effects on polyglutamine toxicity and number and solubility properties of aggregates in PC12 cells were significantly lower than that needed for HeLa cells.\nFig.\u00a03Celastrol effects on Q57-YFP toxicity and aggregates in PC12 cells. PC12 cells were transfected with Q57-YFP along with celastrol treatment at the concentrations indicated. After 48\u00a0h, the amount of cell death was determined by trypan blue assay (a), the number of cells containing Q57-YFP aggregates was quantified using fluorescence microscopy (b), and the amount of Q57-YFP monomer solubilized from aggregates by SDS treatment visualized by Western blot using anti-GFP antibody (c). In a and b, data are shown as means \u00b1 SE [*P\u2009<\u20090.0001 and **P\u2009<\u20090.0001 (a), *P\u2009<\u20090.001 and **P\u2009<\u20090.0001 (b), in each case for each celastrol concentration treatment vs no celastrol)\nHSF1\u2212\/\u2212 cells exhibit increased polyglutamine aggregation and toxicity\nInducible hsp expression is thought to be important for the ability of cells to decrease polyglutamine toxicity and aggregation [8\u201311]. Consistent with this hypothesis, HSF1\u2212\/\u2212 MEFs [30] that are transfected with Q57-YFP exhibit a higher incidence of cell death than HSF1+\/+ MEF cells (Fig.\u00a04a). The HSF1\u2212\/\u2212 MEFs also show increased Q57-YFP aggregation, both as measured by numbers of aggregates using fluorescence microscopy of cells (Fig.\u00a04b) and by amount of aggregated Q57-YFP protein present in cell extracts detected by filtration assay (Fig.\u00a04c).\nFig.\u00a04HSF1\u2212\/\u2212 cells exhibit higher Q57-YFP aggregation and cell death. HSF1\u2212\/\u2212 and wild-type MEF cells were transfected with Q57-YFP, and after 48\u00a0h, cell death was examined by trypan blue assay (a), and the number of cells containing Q57-YFP aggregates was quantified using fluorescence microscopy (b), or filtration assay, in which 30\u00a0\u03bcg of the insoluble fraction was filtered through 0.2\u00a0\u03bcm cellulose acetate membrane, and the aggregates retained on the membrane were immunoblotted using anti-GFP antibody (c). In a and b, data are shown as means \u00b1 SE. *P\u2009<\u20090.008 (a); *P\u2009<\u20090.003 (b)\nProtective effects of celastrol require HSF1\nBased on previous results showing the ability of celastrol to activate HSF1 leading to increased hsp gene expression [18], we hypothesized that this drug\u2019s ability to decrease polyglutamine toxicity and aggregation shown by the results of Figs.\u00a01, 2, and 3 above could be mediated via the HSF1-regulated gene expression pathway. To test this hypothesis, we compared the effect of celastrol treatment on polyglutamine toxicity and aggregation in the HSF1+\/+ vs HSF1\u2212\/\u2212 MEF cells. Celastrol does not exhibit any toxicity in HeLa cells at 1.6\u00a0\u03bcM, but we found its optimal concentration in MEF cells to be lower than that of HeLa cells. Thus, for these experiments, we used 0.4\u00a0\u03bcM celastrol treatment, which has no toxic effect in either HSF1\u2212\/\u2212 or wild-type MEFs (data not shown). First, Western blot analysis shows that the HSF1\u2212\/\u2212 cells are unable to up-regulate hsp70 protein expression in response to celastrol treatment, demonstrating that celastrol-induced up-regulation of hsp70 expression is mediated by the HSF1-regulated gene expression pathway (Fig.\u00a05a).\nFig.\u00a05Celastrol decreases Q57-YFP toxicity and aggregation in wild-type but not HSF1\u2212\/\u2212 MEF cells. a Celastrol treatment does not induce hsp70 expression in HSF1\u2212\/\u2212 cells. Wild-type and HSF1\u2212\/\u2212 MEF cells were treated with no celastrol or 0.4\u00a0\u03bcM celastrol, and after 12\u00a0h, cell extracts were made, and 5\u00a0\u03bcg protein was loaded into each lane for immunoblotting with anti-hsp70 antibodies. b and c Wild-type and HSF1\u2212\/\u2212 MEF cells were transfected with Q57-YFP, with celastrol at the indicated concentrations added at the same time. After 48\u00a0h, cell death was examined by trypan blue assay (b), with data shown as means \u00b1 SE (*P\u2009<\u20090.002 for HSF2+\/+ 0.4\u00a0\u03bcM celastrol vs no celastrol; *P\u2009<\u20090.002 for HSF2\u2212\/\u2212 0.4\u00a0\u03bcM celastrol vs no celastrol), and amount of aggregated Q57-YFP was determined by filtration assay followed by anti-GFP Western blot (c)\nThe results shown above (Figs.\u00a01, 2, 3, and 5a) indicate that celastrol treatment is effective in preventing polyglutamine aggregation and toxicity and that this drug up-regulates hsp70 expression by an HSF1-dependent mechanism. These results suggest that celastrol treatment protects cells by stimulating HSF1-dependent expression of hsps. However, celastrol could also modulate other cellular activities that could contribute to these protective effects. Thus, to test the contribution of HSF1-regulated pathways in these protective effects, we transfected HSF1+\/+ and HSF1\u2212\/\u2212 MEF cells with Q57-YFP in combination with celastrol treatment and then measured the amount of cell death and levels of aggregated Q57-YFP. The results of this experiment show that, as it did for the HeLa and PC12 cells in the experiments of Figs.\u00a01 and 3 above, celastrol treatment resulted in decreased death of HSF1+\/+ MEF cells transfected with Q57-YFP, but treatment with this drug did not protect the HSF1\u2212\/\u2212 cells (Fig.\u00a05b). Consistent with these results, the HSF1+\/+ cells, but not the HSF1\u2212\/\u2212 cells, showed decreased levels of aggregated Q57-YFP upon celastrol treatment, as measured by the filtration assay (Fig.\u00a05c). These results suggest that the HSF1 protein plays an important role in the protective effects of celastrol against polyglutamine toxicity and aggregation.\nDiscussion\nThe results described above indicate that the drug celastrol is able to decrease polyglutamine toxicity, supporting the proposal that this drug could potentially be useful in the treatment of Huntington\u2019s disease and possibly other human polyglutamine expansion disorders. In support of this possibility, it has been found that celastrol treatment of mice results in elevation of hsp70 levels in neurons in the brains of these mice [31]. The results also show that lower concentrations of celastrol are required for its protective effects against polyglutamine toxicity in PC12 cells, cells with neuronal characteristics, compared to other cell types. A particularly beneficial aspect of this drug is that it is already being used to treat people with other disorders [21, 22], and thus it could likely be adapted more quickly for use in treating polyglutamine expansion diseases than other drugs not currently being used in humans.\nResults presented in this paper indicate that the protective effect of celastrol against polyglutamine toxicity is associated with decreased numbers of cells containing aggregates as well as increased SDS-solubility of the mutant polyglutamine protein. This might seem to be at odds with findings that formation of polyglutamine aggregates can be protective for cells expressing these mutant proteins [32\u201334]. However, our results are consistent with the findings of a number of studies which indicate that molecular chaperones can reduce the formation of polyglutamine aggregates and increase the solubility of expanded polyglutamine proteins [8\u201311, 19, 20]. While one study found that hsp40 overexpression, but not hsp70 overexpression, was associated with reduced aggregation [35], the results of a number of studies do suggest that chaperones are able to reduce aggregation of mutant polyglutamine proteins. Molecular chaperones could be acting at multiple levels to reduce polyglutamine toxicity, but one possibility that has been proposed is that these chaperones, and by extension celastrol through its HSF1-mediated up-regulation of chaperones, may be acting at an early step to prevent formation of toxic intermediates before they can become part of larger aggregates [8\u201311, 19, 20]. This would explain both how celastrol treatment protects cells from polyglutamine toxicity and the effects we observe on polyglutamine aggregates in the celastrol-treated cells. An alternative explanation that must be considered, in keeping with previous studies indicating that aggregate formation can be protective for cells [32\u201334], is that the results we observe relating celastrol with the numbers of cells containing polyglutamine aggregates could be due, at least in part, to a potential ability of celastrol to enhance the survival of cells that do not contain aggregates, which would then lead to a perceived decrease in the numbers of cells containing aggregates in our experiments. Our data do indicate, however, the protective effects of celastrol with respect to polyglutamine toxicity.\nIn addition to its ability to its effects on HSF1 activity, celastrol has been found to affect other pathways in the cell, including the NF\u2013\u03baB pathway [18, 21, 22, 36]. However, our results suggest that the beneficial effects of celastrol treatment in decreasing polyglutamine toxicity are mediated through the action of the HSF1-regulated gene expression pathway, based on the finding that celastrol is not protective in Q57-YFP transfected HSF1\u2212\/\u2212 MEF cells (Fig.\u00a05b). This finding also suggests that other drugs that activate HSF1 could also have potential as candidate therapeutic agents for treating polyglutamine expansion diseases.\nElectronic supplementary material","keyphrases":["celastrol","polyglutamine","aggregates","heat shock proteins","hsf1","hsp70"],"prmu":["P","P","P","P","P","P"]}
{"id":"Naunyn_Schmiedebergs_Arch_Pharmacol-3-1-2020506","title":"Phospholipase D signaling: orchestration by PIP2 and small GTPases\n","text":"Hydrolysis of phosphatidylcholine by phospholipase D (PLD) leads to the generation of the versatile lipid second messenger, phosphatidic acid (PA), which is involved in fundamental cellular processes, including membrane trafficking, actin cytoskeleton remodeling, cell proliferation and cell survival. PLD activity can be dramatically stimulated by a large number of cell surface receptors and is elaborately regulated by intracellular factors, including protein kinase C isoforms, small GTPases of the ARF, Rho and Ras families and, particularly, by the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 is well known as substrate for the generation of second messengers by phospholipase C, but is now also understood to recruit and\/or activate a variety of actin regulatory proteins, ion channels and other signaling proteins, including PLD, by direct interaction. The synthesis of PIP2 by phosphoinositide 5-kinase (PIP5K) isoforms is tightly regulated by small GTPases and, interestingly, by PA as well, and the concerted formation of PIP2 and PA has been shown to mediate receptor-regulated cellular events. This review highlights the regulation of PLD by membrane receptors, and describes how the close encounter of PLD and PIP5K isoforms with small GTPases permits the execution of specific cellular functions.\nIntroduction\nThe activation of membrane receptors by hormones and growth factors results in the localized generation of intracellular second messengers. The hydrolysis of membrane phospholipids and the generation of biologically active products play important roles in the regulation of cell function and cell fate. Well known is the activation of phosphoinositide-specific phospholipase C (PLC) isoforms, which hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2), a membrane phospholipid found in all eukaryotic cells (Schmidt et al. 2004). Stimulation of PLC isoforms plays a major role in many early and late cellular responses to receptor activation, including smooth muscle contraction, secretion and neuronal signaling as well as fertilization, cell growth and differentiation (Berridge 2005; Nishizuka 2003). Phospholipase D (PLD) was first described 60\u00a0years ago as a distinct, phospholipid-specific phosphodiesterase activity in cabbage leaves (Hanahan and Chaikoff 1948). This pioneering research indicated that PLD hydrolyzes phosphatidylcholine to yield phosphatidic acid (PA) and choline. The recognition that PLD is rapidly and dramatically activated in response to extracellular stimuli in cultured animal cells, now 20\u00a0years ago (Bocckino et al. 1987; Cockcroft 1984), has brought PLD signaling to the very forefront of current biological and biomedical research. Meanwhile, phosphatidylcholine-hydrolyzing PLD has been identified in bacteria, protozoa, fungi, plants and animals, and, due to this widespread distribution, is assumed to be involved in the regulation of fundamental cellular functions. Indeed, it has now been established that activation of PLD and the generation of PA by a vast number of membrane receptors modulate such a wide array of cellular responses as calcium mobilization, secretion, superoxide production, endocytosis, exocytosis, vesicle trafficking, glucose transport, rearrangements of the actin cytoskeleton, mitogenesis and survival (Cockcroft 2001; Exton 2002b; Jenkins and Frohman 2005; Liscovitch et al. 2000).\nPIP2 is a critical cofactor for PLD, and profoundly affects the activity, membrane localization and receptor activation of both PLD isoforms, PLD1 and PLD2 (Brown et al. 1993; Hodgkin et al. 2000; Liscovitch et al. 1994; Pertile et al. 1995; Schmidt et al. 1996d). Thus, reduction of cellular PIP2 levels, for instance via scavenging of PIP2 by the actin-binding protein fodrin (Lukowski et al. 1998) or via forced PIP2 hydrolysis by the phosphatase synaptojanin (Chung et al. 1997), has been shown to inhibit PLD activity. Vice versa, the synthesis of PIP2 by phosphoinositide 5-kinase (PIP5K) isoforms can be directly stimulated by the PLD product PA (Jenkins et al. 1994; Moritz et al. 1992), and this regulation has also been confirmed to occur at the whole cell level (Divecha et al. 2000; Jones et al. 2000b; Skippen et al. 2002). It is now hypothesized that the reciprocal stimulation of PLD and PIP5K enzymes enables rapid feed-forward stimulation loops for a localized and explosive generation of PA and PIP2, which may then govern the recruitment and activation of proteins to execute specific cellular tasks, especially membrane trafficking, and changes in the organization of the actin cytoskeleton. The activity and localization of both PLD and PIP5K are under control of GTPases of the Arf and Rho families, which are well-defined regulators of membrane transport and actin-reorganization processes. The reciprocal stimulation of PIP5K and PLD, and the regulation of these enzymes by ARF and Rho GTPases, point to concerted mechanisms in cellular actions, involving acute, localized PIP2 and PA synthesis (Fig.\u00a01). This review will focus on the regulation of PLD enzymes by membrane receptors and monomeric GTPases, and on how PLD signaling is organized and connected by PIP2 metabolism.\nFig.\u00a01Regulation and cellular roles of PLD and PIP5K. Regulation of PLD and PIP5K by ARF and Rho family GTPases is essentially involved in the regulation of intracellular vesicle trafficking and actin cytoskeleton reorganization. Both PLD and PIP5K are stimulated by cell surface receptors and by conventional PKC isoforms, and the latter can become activated after receptor-induced hydrolysis of PIP2 by PLC. Positive feed-forward regulation is achieved by stimulation of PLD by PIP5K-derived PIP2, and of PIP5K by PLD-derived PA. Activation of ARF-GAPs by PIP2 accelerates the inactivation of ARF proteins, and may terminate a round of PA and PIP2 synthesis\nPhosphatidic acid and PLD isoforms\nMost cellular responses following PLD activation are probably mediated by the immediate reaction product PA. PA is a multifunctional lipid that can be further metabolized to the bioactive lipids, lysophosphatidic acid (LPA) and diacylglycerol (DAG), can by itself alter membrane curvature, and can serve as a protein attachment site and affect both cellular localization and activity of various proteins, including Raf-1 kinase, protein phosphatase 1, sphingosine kinase 1, and mTOR (mammalian target of rapamycin), a key regulator of cell growth and proliferation (Jenkins and Frohman 2005). PLD enzymes can catalyze a transphosphatidylation reaction in which the phosphatidyl moiety of phosphatidylcholine is accepted by primary alcohols, thereby producing stable phosphatidylalcohol instead of PA. This transphosphatidylation reaction is widely applied to measure PLD activity in biological samples, and quenching of PA synthesis by primary alcohols has proven extremely helpful to identify the involvement of PLD enzymes in cell physiology. In this way, a role for PLD has been demonstrated in a variety of signaling processes, such as activation of phosphoinositide (PI3K, PIP5K) and protein (Akt, ERK1\/2) kinases, calcium mobilization, cytoskeleton remodeling, endocytosis, exocytosis, membrane trafficking, superoxide production, glucose transport, cell migration, cell proliferation, and survival signaling (Exton 2002a; Foster and Xu 2003).\nThere are two mammalian PLD genes, PLD1 and PLD2. PLD1 has a low basal activity and is extensively regulated by conventional protein kinase C (PKC\u03b1, -\u03b2, -\u03b3) isozymes and small GTPases of the ARF (ARF1 - ARF6) and Rho (RhoA, Rac1, Cdc42) families (Henage et al. 2006). PLD2 has a higher basal activity than PLD1, but has been shown to respond to ARF and PKC as well (Chen and Exton 2004). PIP2 is recognized to be the most important cofactor for PLD, and both PLD isoforms are absolutely dependent on PIP2 for activity. Experiments utilizing inactive PLD mutants and RNA interference have discriminated isoform-specific PLD functions, and showed that PLD1 is involved in agonist-induced secretion, actin organization, and cell adhesion and migration (Exton 2002a; Iyer et al. 2006; Kim et al. 2006; Vitale et al. 2001), and PLD2 in endocytosis and recycling of membrane receptors (Du et al. 2004; Koch et al. 2006; Padr\u00f3n et al. 2006).\nThe PLD isoforms, both with two splice variants, share an ~50% amino-acid sequence identity (Colley et al. 1997; Hammond et al. 1995, 1997; Steed et al. 1998). The catalytic core of both PLD enzymes are composed of four conserved domains (domain I-IV), and the HKD motifs in the domains II and IV probably associate together to form a catalytic centre (Xie et al. 2000). PLD1 is characterized by a 116-amino acid loop region following domain II, which has been proposed to function as a negative regulatory element (Sung et al. 1999). PLD1 and PLD2 further possess N-terminal PH (pleckstrin homology) and PX (phox homology) domains. PIP2 binds to the PH domain (Hodgkin et al. 2000), but also to a polybasic PIP2 binding motif within the catalytic core (Sciorra et al. 1999), and interaction of PIP2 with both domains has been suggested to be involved in membrane targeting of PLD as well as stimulation of PLD catalytic activity (Du et al. 2003; Hodgkin et al. 2000; Sciorra et al. 2002). The PX domain of PLD1 has been reported to preferentially bind to phosphatidylinositol-3,4,5-trisphosphate (PIP3) (Lee et al. 2005; Stahelin et al. 2004), but interaction with PI5P has been observed as well (Du et al. 2003). Recently, it was shown that the PX domain of PLD has GTPase-activating protein (GAP) activity towards dynamin, and that PLD supports EGF receptor endocytosis (Lee et al. 2006). The PH and PX domains probably contribute to the proper localization of the PLD enzymes within cells. In line with a role for PLD enzymes in different cellular tasks, PLD1 and PLD2 show a diverse subcellular distribution. PLD1 is found throughout the cell, but primarily localizes to perinuclear endosomes and the Golgi apparatus (Brown et al. 1998; Freyberg et al. 2001; Hughes and Parker 2001). PLD2 is almost exclusively present at the plasma membrane in lipid raft fractions (Czarny et al. 1999). The localization of PLD1 does not seem to be static, and regulated translocation and recycling of the enzyme between cellular compartments may be crucial to its proper functioning. In an elegant study, coordinated subcellular targeting of the lipid binding motifs has been demonstrated to drive this subcellular cycling of PLD1 (Du et al. 2003). Upon stimulation, PLD1 was found to translocate from the intracellular compartments to the plasma membrane, and this process was probably dependent on the polybasic PIP2 binding site. The PH domain then facilitated entry of PLD1 into lipid rafts, a step critical for internalization of the enzyme, whereafter interaction of the PX domain with PI5P may control the efficient return of PLD1 to the endosomes.\nPIP2 and PIP5K isoforms\nPIP2 is an essential and versatile factor in cellular signaling. Hydrolysis of PIP2 by PLC into the second messengers, inositol-1,4,5-trisphosphate (IP3) and DAG, is a general and well-defined answer of cells in response to stimulation of many membrane receptors (Schmidt et al. 2004). Phosphorylation of PIP2 by PI3K results in the rapid accumulation of PIP3, which recruits and activates mediators involved in actin remodeling, mitogenesis and survival (Vanhaesebroeck et al. 2001). But it is now recognized that PIP2, as well as other phosphoinositides, are signaling molecules by themselves and can, by binding to unique phosphoinositide-binding sequences, such as the PH and PX domains, affect the activity and subcellular localization of many proteins, including many actin regulatory proteins, a wide range of ion channels, and PLD (Niggli 2005; Suh and Hille 2005; Yin and Janmey 2003). In this way, PIP2 can modulate a remarkable variety of cellular processes, including cortical actin organization, membrane ruffling, vesicle trafficking, gene expression, cell migration and cell survival (Ling et al. 2006; Oude Weernink et al. 2004b; Toker 2002). Subsequent dephosphorylation of PIP2 by inositol polyphosphate 5-phosphatases, such as synaptojanin, is believed to terminate local PIP2 signaling, for instance in the process of vesicle trafficking (Majerus et al. 1999).\nTo execute this variety of functions, PIP2 may be organized in discrete functional pools within cells, but the existence of PIP2 clusters in the plasma membrane is currently under debate. Using green fluorescent protein-tagged PH domains or antibodies to visualize PIP2, the lipid was found to concentrate in highly dynamic, actin-rich regions (Tall et al. 2000) and lipid rafts (Laux et al. 2000; Parmryd et al. 2003) in the plasma membrane, feeding the idea that spatially organized PIP2 synthesis regulates actin polymerization and other cellular processes. The localization of PIP2 in rafts is supported by biochemical data (Pike and Casey 1996); however, specific PIP2 clustering has been disputed (van Rheenen et al. 2005).\nPIP2 is generated after phosphorylation of phosphatidylinositol-4-phosphate by PIP5K. In mammals, cDNAs encoding three isoforms of PIP5K (designated I\u03b1, I\u03b2 and I\u03b3) with alternative splice variants have been cloned and characterized (Ishihara et al. 1996, 1998; Loijens and Anderson 1996). Sequence analysis has shown that PIP5K enzymes are related to PIP4K enzymes, but that they share no identity with most other lipid (PI3K and PI4K) or protein kinases. The sequence similarity between the PIP4Ks and PIP5Ks is clustered in the catalytic core of the kinases (Anderson et al. 1999; Hinchliffe et al. 1998). An activation loop spanning the catalytic domain has been shown to determine both substrate specificity and subcellular targeting of PIP5Ks, which can be swapped by substitution of a single amino acid within this loop (Kunz et al. 2002). In murine PIP5K-I\u03b2, two dimerization domains were identified, which may contribute to the proper subcellular localization and functioning of the enzyme (Galiano et al. 2002).\nThe identification of three PIP5K isoforms raised the expectation of a differential regulation of the enzymes by cellular signal transduction components, but up to now the regulatory properties of PIP5K-I\u03b1, I\u03b2 and I\u03b3 appear to be remarkably similar. All PIP5K isoforms are stimulated by PA, are extensively regulated by ARF and Rho GTPases, and inhibited by protein kinase A (PKA) and PI-stimulated autophosphorylation (Oude Weernink et al. 2004b). Nevertheless, evidence has been provided that PIP5K isoforms may selectively control functional PIP2 pools, which may support particular processes in different cell types. Thus, actin reorganization down-stream of Rac1 in platelets specifically involves murine PIP5K-I\u03b1 (Tolias et al. 2000). Human PIP5K-I\u03b1 was found to localize in Rac1-induced membrane ruffles, and the LIM protein Ajuba has been identified to interact with and stimulate PIP5K-I\u03b1 in leading-edge membrane ruffles in migrating cells (Kisseleva et al. 2005). Human PIPK-I\u03b2 was detected primarily in cytosolic vesicular structures (Doughman et al. 2003) and may synthesize the PIP2 pool involved in constitutive endocytosis (Padr\u00f3n et al. 2003). The long-splice variant of PIP5K-I\u03b3, PIP5K-I\u03b390, is enriched in neurons and is implicated in the regulation of clathrin coat recruitment, actin dynamics (Wenk et al. 2001) and focal adhesion formation (Di Paolo et al. 2002; Ling et al. 2002). In contrast, short PIP5K-I\u03b387 seems to be the major producer of the PIP2 pool that supports receptor-induced IP3 generation (Wang et al. 2004).\nThe execution of specific PIP2-modulated processes is very probably achieved by an orchestration of appropriate signaling partners within discrete subcellular microdomains, and PLD-derived PA as well as the PLD enzymes by themselves can contribute to this organization. Indeed, both PLD1 and PLD2 interact with PIP5K-I\u03b1, and PLD2 recruits PIP5K-I\u03b1 to a submembraneous vesicular compartment (Divecha et al. 2000). PLD2-derived PA was shown to stimulate PIP5K-I\u03b3 splice variants, and the subsequent formation of PIP2 to drive the initial stages of integrin-mediated cellular adhesion (Powner et al. 2005). In many processes, the temporal activation and correct localization of PLD and PIP5K isoforms by monomeric GTPases appears crucial to achieve the spatially organized production of PIP2 and PA (Santarius et al. 2006).\nARF GTPases and membrane traffic\nAlthough the direct interaction site on PLD for ARF has not yet been unequivocally defined, it is well established that ARF proteins, particularly ARF1 and ARF6, activate both PLD enzymes, but especially PLD1 (Hammond et al. 1995, 1997). ARF GTPases regulate intracellular vesicle trafficking and actin remodeling. ARF1 is localized to the Golgi complex, and is required for proper Golgi structure and function. The use of primary alcohols has also pointed to a role for PLD in vesicle transport to Golgi (Bi et al. 1997; Ktistakis et al. 1996). PLD activity has been shown to stimulate the release of nascent secretory vesicles from the trans-Golgi network (Chen et al. 1997), and to be required for maintaining the structural integrity and function of the Golgi apparatus, but the precise role for PLD in vesicle formation is still controversial. PIP5K is also a direct effector of ARF1, and an ARF1 mutant that selectively activates PIP5K, but not PLD activity, demonstrated that both PLD-derived PA and direct activation of PIP5K by ARF1 contribute to increased PIP2 synthesis (Skippen et al. 2002). In permeabilized cells, ARF1 has been shown to restore secretion by promoting PIP2 synthesis (Fensome et al. 1996), and ARF1-mediated PIP5K activation (Jones et al. 2000a) and recruitment to the Golgi complex (Godi et al. 1999) appears to be critical in Golgi functioning.\nARF6 regulates vesicular transport, secretion, and cortical actin reorganization. ARF6 activates PLD, and PA has been implicated in the mediation of the effects of ARF6 in vesicular trafficking events. A critical role for PLD1 in exocytosis has been established in different cell types, including neurons (Humeau et al. 2001), neuroendocrine cells (Vitale et al. 2001) and pancreatic \u03b2 cells (Hughes et al. 2004). PLD2 has recently emerged as a mediator of ARF-dependent internalization of the \u03bc-opioid receptor (Koch et al. 2003), and both PLD isoforms have been implicated in macrophage phagocytosis (Corrotte et al. 2006; Iyer et al. 2004). In addition, PIP5K colocalizes and interacts with, and is directly activated by ARF6 at the plasma membrane (Honda et al. 1999), and ARF6 and PIP2 colocalize on the plasma membrane and on endosomal structures (Brown et al. 2001). ARF6-organized PIP2 turnover at the plasma membrane is apparently involved in regulated secretion (Aikawa and Martin 2003; Brown et al. 2001; Lawrence and Birnbaum 2003). Focal and transient accumulation of PIP2 by PIP5K is required for phagocytosis as well (Botelho et al. 2000; Coppolino et al. 2002; Wong and Isberg 2003), and PIP2 hydrolysis probably dictates the remodeling of actin necessary for completion of phagocytosis (Scott et al. 2005). The synthesis of PIP2 is essential for priming the exocytotic apparatus, and the recruitment and activation of PLD1 by PIP2 seems the primary mechanism for the functional integration of PLD1 into the exocytotic pathway (Vitale et al. 2001; Waselle et al. 2005). Thus, CD16-induced cytolytic granule secretion mediated by ARF6 was shown to involve PIP5K-I\u03b1 membrane targeting and activation of both PIP5K and PLD (Galandrini et al. 2005). PIP2 also recruits additional proteins\u2014for instance the endocytic proteins AP-2, epsin and AP180\u2014to initiate clathrin-coat formation preceding endocytosis (Ford et al. 2001; Itoh et al. 2001; Padr\u00f3n et al. 2003), and CAPS (Grishanin et al. 2004) to initiate dense-core vesicle exocytosis. Direct activation of PIP5K-I\u03b3 by ARF6 has been shown to stimulate clathrin-coat recruitment to synaptic membranes to allow synaptic vesicle recycling (Krauss et al. 2003). PLD-derived PA may directly contribute to vesicle fusion in a biophysical manner, as PLD cleaves the non-fusogenic lipid, PC, to form the fusogenic lipid, PA. But PA also takes a function as an essential cofactor for PIP5K, and disruption of Golgi membranes (Sweeney et al. 2002), blockade of clathrin-coat assembly (Arneson et al. 1999) and inhibition of ARF1-reconstituted secretion (Way et al. 2000) after quenching of PA production could be attributed to inhibited PIP2 synthesis. Thus, both PLD and PIP2 synthesis seem necessary for membrane trafficking aspects in the endo- and exocytotic machinery. But PLD and PIP5K also mediate other processes down-stream of ARF6. Epidermal growth factor (EGF)-induced membrane ruffling requires ARF6-induced PIP5K-I\u03b1 translocation to the ruffles and local PIP2 production. This leads to the recruitment of PLD2, and PLD-derived PA and ARF6 may then synergistically activate PIP5K (Honda et al. 1999).\nThe relationship between ARF and PIP2 is also bidirectional, as phosphoinositides can regulate ARF activity by binding and activating both ARF-specific guanine nucleotide exchange factors (ARF-GEFs) (Klarlund et al. 1998; Paris et al. 1997) and ARF-GTPase-activating proteins (ARF-GAPs) (Kam et al. 2000; Nie et al. 2002) via their PH domains. The fact that ARF-GAPs bind PIP2 with high affinity and specificity offers an attractive feed-back mechanism for terminating ARF activation after a cycle of ARF-induced PIP2 synthesis.\nRho GTPases and actin dynamics\nPA formation, especially by PLD1, has been reported to induce stress fibre formation in specific cell types (Cross et al. 1996; Ha and Exton 1993; Kam and Exton 2001; Porcelli et al. 2002). Rho proteins, in particular RhoA, Rac1 and Cdc42, which control actin cytoskeleton reorganization, exclusively activate PLD1 by direct interaction with its C-terminus (Exton 2002b; Powner and Wakelam 2002). Thus, PLD stimulation by RhoA may happen by direct interaction, but may involve indirect, Rho-dependent mechanisms as well. Inactivation of Rho GTPases, with Clostridium difficile toxin B or Clostridium botulinum C3 exoenzyme, reduced cellular PIP2 levels, resulting in inhibiton of receptor-mediated PIP2 hydrolysis by PLC (Schmidt et al. 1996a) as well as diminished PLD stimulation (Schmidt et al. 1996d). As the inhibition of PLD signaling after Rho inactivation could be largely rescued by the addition of PIP2, Rho proteins do seem to affect PLD via PIP5K regulation (Schmidt et al. 1996c,d). PIP2 is well-known to associate with and regulate the activity of a plethora of actin-binding proteins that organize actin dynamics (Hilpela et al. 2004; Yin and Janmey 2003), and PA and PIP2 may act in concert to mediate Rho-dependent actin cytoskeleton remodeling. PIP5K isoforms are, like PLD, under direct control of Rho GTPases. PIP5K isoforms are markedly stimulated by RhoA, Rac1, and Cdc42 (Chong et al. 1994; Hartwig et al. 1995; Oude Weernink et al. 2004a), and physically associate with both RhoA (Ren et al. 1996) and Rac1 (Tolias et al. 2000), but not with Cdc42 (Oude Weernink et al. 2004a; van Hennik et al. 2003). PIP5K isoforms are now seen as critical mediators of RhoA- and Rac1-induced actin organization and remodeling (Doughman et al. 2003; Shibasaki et al. 1997; Tolias et al. 2000). The established Rho effector Rho-kinase, a serine\/threonine kinase, is apparently involved in Rho-dependent regulation of both PLD (Schmidt et al. 1999) and PIP5K activities (Oude Weernink et al. 2000), and PIP5K was found to play an essential role as down-stream effector of Rho and Rho-kinase in neurite remodeling (van Horck et al. 2002; Yamazaki et al. 2002) and platelet cytoskeleton assembly (Gratacap et al. 2001; Yang et al. 2004). But Rho may also directly signal to PIP5K independently of Rho-kinase, as RhoA-induced activation of ERM (ezrin, radixin, moesin) proteins, that cross-link actin filaments to plasma membranes, was found to be mediated by PIP5K, but not by Rho-kinase (Matsui et al. 1999). PLD and PIP5K were also demonstrated to collectively mediate Rho-induced changes in the actin cytoskeleton. Thus, myogenic differentiation induced by arginine-vasopressin, which involves actin fiber formation, is mediated by Rho proteins and PLD1, and involves PLD-induced PIP2 synthesis along the actin fibers (Komati et al. 2005). These findings suggest that PLD and PIP5K enzymes may co-operate down-stream of Rho in processes that depend on actin organization.\nAnother Rho effector, PKC-related protein kinase N (PKN), also directly interacts with PLD (Oishi et al. 2001) and mediates PLD activation by the \u03b11-adrenergic receptor (Parmentier et al. 2002). Interestingly, components of the actin regulatory machinery, \u03b2-actin and \u03b1-actinin, have been found to directly associate with and inhibit the activity of PLD isoforms (Lee et al. 2001; Park et al. 2000). PLD also binds to and is stimulated by filamentous F-actin, and PLD1 in particular may act as a signal transduction component responsive to dynamic changes of the actin cytoskeleton (Kusner et al. 2002). PKN interacts with \u03b1-actinin, and PKN may modulate PLD signaling by reversing the inhibitory effect of \u03b1-actinin on PLD1, and by direct interaction with PLD1.\nRegulation of PLD and PIP5K by membrane receptors\nIn line with the critical role of PA in cellular processes, the enzymatic activity of PLD is tightly regulated by a variety of hormones, neurotransmitters, and growth factors. Regulation of PLD enzymes by membrane receptors, including G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs), is complex and mediated by several cytosolic factors, including PKC as well as ARF, Rho and Ras GTPases (Exton 2002b; Liscovitch et al. 2000; L\u00f3pez De Jes\u00fas et al. 2006; Powner and Wakelam 2002). Most receptors that stimulate PLD also increase PLC activity, leading to activation of the PLD regulator PKC, and it was assumed that PLD activation might be secondary to PLC activation. A physical association between PLD with PKC isoforms has been reported, resulting in strong activation of in vitro PLD1 activity, and the major interaction site was identified within the N-terminus of PLD1 (Park et al. 1998). Indeed, inhibition of PKC was shown to reduce receptor-induced PLD responses, and PLD1 mutants unresponsive to PKC did respond poorly to activation of GPCRs (Zhang et al. 1999) or to active G\u03b1q proteins (Xie et al. 2002). However, stimulation of PLD in several receptor systems, including M3 muscarinic and \u03b11-adrenergic receptors, was actually PKC-independent (Balboa and Insel 1998; Muthalif et al. 2000; R\u00fcmenapp et al. 1997; Schmidt et al. 1994), suggesting that PLD stimulation must not necessarily be secondary to PLC stimulation.\nBrefeldin A, an inhibitor of certain ARF-GEFs, reduced receptor signaling to PLD in several cell types, indicating that ARF proteins participate in receptor-mediated PLD stimulation (Fensome et al. 1998; Mitchell et al. 1998; R\u00fcmenapp et al. 1995; Shome et al. 2000). Likewise, sequestration of ARF-GEFs by the ARF-related protein ARP inhibited M3 muscarinic receptor signaling to PLD (Sch\u00fcrmann et al. 1999). Clostridial toxins and enzymes that specifically inactivate Rho proteins and expression of inactive Rho mutants have been used to identify the role of Rho in signaling to PLD. Thus, Rho proteins were found to be involved in PLD stimulation by GPCRs (M3 muscarinic, bradykinin, sphingosine-1-phosphate and LPA), RTKs (PDGF, EGF), and immunoglobulin (Fc\u03b5RI) receptors (Hess et al. 1997; Ojio et al. 1996; Schmidt et al. 1996c).\nStimulation of PLD by GPCRs was shown to be mediated by both pertussis toxin (PTX)-insensitive (Gosau et al. 2002; Schmidt et al. 1994) and PTX-sensitive (Cummings et al. 2002; Fensome et al. 1998) heterotrimeric G proteins. G12 family proteins can stimulate PLD (Plonk et al. 1998), and RGS (regulators of G protein signaling) proteins, that act as \u03b1 subunit-specific GAPs, have been used to position G12 in PLD activation by the M3 muscarinic (R\u00fcmenapp et al. 2001), the PAR1 (Fahimi-Vahid et al. 2002), and the Ca2+-sensing receptor (Huang et al. 2004), as well as mechanical force (Ziembicki et al. 2005). As forskolin and cAMP were shown to cause activation of PLD via PKA and ERK1\/2 (Ginsberg et al. 1997; Yoon et al. 2005) or, alternatively, via the cAMP-activated GEF for Ras-like GTPases, Epac and R-Ras (L\u00f3pez De Jes\u00fas et al. 2006), Gs proteins also mediate stimulation of PLD. PLD activation is also controlled by \u03b2\u03b3-subunits, possibly via Src and\/or ARF6 (Le Stunff et al. 2000; Ushio-Fukai et al. 1999), but G\u03b2\u03b3 can also directly interact with and inhibit PLD (Preininger et al. 2006).\nAs the precise mechanism of PLD stimulation in intact cells was only poorly understood, during the last 10\u00a0years our laboratory in Essen has focused on the regulation of PLD activity by membrane receptors. In HEK-293 cells, signaling to PLD by a typical GPCR, the M3 muscarinic receptor, and an RTK, the EGF receptor, was studied and shown to be executed by several distinct pathways (Fig.\u00a02). In addition, by expressing inactive PLD mutants, the M3 muscarinic and the EGF receptors were found to signal to individual PLD isozymes and to selectively stimulate PLD1 and PLD2 respectively (Han et al. 2001). The M3 muscarinic receptor stimulates both PLC and PLD via PTX-insensitive mechanisms (Offermanns et al. 1994; Peralta et al. 1988; Schmidt et al. 1994). Interestingly, stimulation of PLD by the agonist carbachol was not affected by PKC inhibitors, suggesting that activation of PLD by the M3 muscarinic receptor was rather independent of PLC (R\u00fcmenapp et al. 1997; Schmidt et al. 1994). Expression of \u03b1-subunits of G proteins and of specific RGS proteins was used to identify the G proteins involved in these pathways, and demonstrated that whereas the M3 receptor signals to PLC via Gq proteins, activation of PLD is mediated by G12 family proteins (R\u00fcmenapp et al. 2001). PLD activation by the M3 receptor, but not by the EGF receptor, was further found to be under control of ARF (R\u00fcmenapp et al. 1995, 1997) as well as Rho proteins, particularly RhoA (Schmidt et al. 1996c,d). Likewise, regulation of mTOR by LPA, but not PDGF, involved PLD1 activation by Rho GTPases (Kam and Exton 2004). Both ARF1 and RhoA were found to become activated after M3 receptor activation (Keller et al. 1997; R\u00fcmenapp et al. 1995), and a role for Rho-kinase in RhoA-controlled PLD stimulation could be demonstrated (Schmidt et al. 1999). In further studies, it was shown that activation of PLD by RhoA and Rho-kinase is mediated by G12 and the tyrosine kinase Pyk2, whereas activation by ARF1 is mediated by G13, PI3K and the Arf-GEF ARNO (Han et al. 2003). In cardiomyocytes, Rho proteins were shown to affect signaling to PLD by both endothelin-1 and thrombin, apparently by controlling PIP2 synthesis, whereas ARF selectively affects signaling by the PAR1 receptor (Fahimi-Vahid et al. 2002).\nFig.\u00a02Regulation of PLD by the M3 muscarinic receptor and receptor tyrosine kinases in HEK-293 cells. In human embryonic kidney (HEK-293) cells, signaling to PLD by the M3 muscarinic receptor and by typical RTKs (EGF, PDGF, insulin) is organized into rather discrete pathways and channeled by particular heterotrimeric G proteins and small GTPases (orange), specific GEF proteins (pink) and further signaling components (green). AC, adenylyl cyclase; ROCK, Rho-kinase\nPLD can directly interact with RalA, and a Ras\/Ral signaling cascade was shown to regulate PLD responses. In HEK-293 cells, Ras and RalA\u2014but not Rho proteins\u2014were located in RTK signaling to PLD, and this Ras\/Ral-dependent signaling cascade was found to be dependent on PKC-\u03b1 and a Ral-specific GEF (Fig.\u00a02) (Schmidt et al. 1998; Voss et al. 1999). RalA apparently co-operates with ARF (Kim et al. 1998; Xu et al. 2003) and Rho proteins (Frankel et al. 1999; Wilde et al. 2002) to achieve full PLD activation. Likewise, Ras proteins were found to modulate PLD responses by PDGF (Lucas et al. 2000), and RalA to affect EGF receptor signaling to PLD (Lu et al. 2000). It was recently shown that direct activation of Ras-related R-Ras by Epac is involved in PLD stimulation by the M3 muscarinic receptor, apparently by coupling to Gs proteins (L\u00f3pez de Jes\u00fas et al. 2006), but a contribution of Ral proteins to GPCR-induced PLD activation has not been found (Meacci et al. 2002). Collectively, these data demonstrate that heterotrimeric G proteins as well as small GTPases co-ordinate PLD activation by specific membrane receptors in particular cell types, and these mechanisms probably contribute to the organization of agonist-induced PA production for the execution of diverse cellular signaling tasks.\nIn addition, the synthesis of PIP2 can be directly stimulated by GPCRs (thrombin, LPA, M3 muscarinic) as well as RTKs (Cochet et al. 1991; Nolan and Lapetina 1990; Pike and Eakes 1987). Receptor activation leads to increased association of PIP5K with the actin cytoskeleton (Grondin et al. 1991; Payrastre et al. 1991), and receptor-induced stimulation and cytoskeletal association of PIP5K may be directly involved in actin cytoskeletal regulation and initialize the assembly of enzymes into signaling complexes. GPCR-induced stimulation of PIP2 synthesis was found to be mediated by pertussis toxin-sensitive Gi proteins (Schmidt et al. 1996b; Stephens et al. 1993), but also by G12 and Gq proteins (Oude Weernink et al. 2003). Enhanced PIP2 synthesis is also caused by conventional PKC isoforms, which may increase PIP5K activity by stimulating PIP5K dephosphorylation by the okadaic acid-sensitive protein phosphatase 1 (Park et al. 2001).\nConcluding remarks\nIn the last decade, PLD has taken a firm position as all-round player in cellular signaling events. It is now appreciated that PLD and PIP5K act together to execute several important cellular functions, including vesicle transport, cytoskeleton dynamics and cell adhesion. Because of the reciprocal stimulation of their activities it seems inappropriate to generally assign a conventional \u201cupstairs-downstairs\u201d relationship to PLD and PIP5K isozymes. The localized generation of the lipid messengers by PLD and PIP5K, PA and PIP2, is clearly co-ordinated by small GTPases of the ARF, Rho and Ras families. The following picture emerges of how PLD and PIP5K may co-operate to execute their cellular tasks. Particular small GTPases, activated by membrane receptors or cellular factors, bind to PIP5K and recruit the enzyme to specific cellular compartments. Subsequent activation of PIP5K catalytic activity triggers the localized generation of PIP2, which now serves as an anchor for specific proteins, including PLD enzymes. The sequestered PLD is activated by PIP2 and the GTPases, and PLD-derived PA now, among other tasks, contributes to the activation of PIP5K. This feed-forward regulation loop depends on both PIP5K and PLD, and quenching of PA formation (by primary alcohols) or reduction of PIP2 levels (by PLC-mediated hydrolysis or dephosphorylation by phosphatases) can interrupt the snowball from rolling. PIP2 dephosphorylation may be important in the cell as a decisive mechanism to terminate the localized reactions before a cellular avalanche develops. Attractive candidates are further specific GEFs and GAPs for the GTPases, some of which have been shown to be directly regulated by PIP2. PIP2-dependent inactivation of the organizing GTPase may then provide the final turn-off signal.","keyphrases":["phospholipase d","pip2","phosphatidic acid","ras","arf","rho","phosphoinositide 5-kinase"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Purinergic_Signal-3-4-2072915","title":"New 2,6,9-trisubstituted adenines as adenosine receptor antagonists: a preliminary SAR profile\n","text":"A new series of 2,6,9-trisubstituted adenines (5\u201314) have been prepared and evaluated in radioligand binding studies for their affinity at the human A1, A2A and A3 adenosine receptors and in adenylyl cyclase experiments for their potency at the human A2B subtype. From this preliminary study the conclusion can be drawn that introduction of bulky chains at the N6 position of 9-propyladenine significantly increased binding affinity at the human A1 and A3 adenosine receptors, while the presence of a chlorine atom at the 2 position resulted in a not univocal effect, depending on the receptor subtype and\/or on the substituent present in the N6 position. However, in all cases, the presence in the 2 position of a chlorine atom favoured the interaction with the A2A subtype. These results demonstrated that, although the synthesized compounds were found to be quite inactive at the human A2B subtype, adenine is a useful template for further development of simplified adenosine receptor antagonists with distinct receptor selectivity profiles.\nIntroduction\nAdenosine, a naturally occurring nucleoside, is involved in a wide variety of physiological and pathophysiological processes [1]. Adenosine mediates these effects through the activation of at least four human receptor subtypes (P1), belonging to the superfamily of G protein-coupled receptors, which have been recently cloned [2] and classified as A1, A2A, A2B and A3 [3]. The subtypes are classified on the bases of coupling to second messengers and pharmacological profiles for agonists and antagonists. In fact, A1 and A3 adenosine receptor subtypes are linked to inhibition of adenylyl cyclase and A2A and A2B subtypes are linked to stimulation of the same enzyme [4].\nIn particular, A2B receptors have been implicated in several physiological functions such as the regulation of mast cell secretion [5, 6], gene expression [5, 7, 8], cell growth [9] and intestinal functions. A2B receptors may also play a role in asthma, since they mediate mast cell degranulation from human mast cells and are present in high density in human blood eosinophils [10, 11]. For this reason A2B antagonists could be considered potential antiasthmatic agents [10\u201312]. While the A1, A2A and A3 adenosine receptors have been pharmacologically characterized through the use of highly potent and selective agonists and\/or antagonists, an accurate investigation of the pathophysiological role of A2B receptors is precluded due to the lack of very selective ligands [13]. On the other hand, only recently radiolabelled adenosine antagonists have been used for binding assays at the A2B receptor subtype [1]. Recently, xanthine derivatives, such as compounds 1 and 2 in Fig.\u00a01, have been proposed as potent and selective adenosine receptor antagonists [14, 15]. On the other hand, in the non-xanthine family poor results have been obtained in recent years. However, mention should be made of the pyrazolo-triazolo-pyrimidine derivative 3, which showed promising binding affinity at the A2B adenosine receptor although the level of selectivity vs the human (h) A3 subtype was still poor [16]. Very recently, a bipyrimidyl derivative 4 has been proposed as an A2B adenosine receptor antagonist, with affinity in the same range of compound 3, while the selectivity vs the other receptor subtypes was found to be significantly better [17] (Fig.\u00a01).\nFig.\u00a01Structures and binding profile (Ki nM) of some A2B adenosine receptor antagonists\nA structural analysis of the derivatives 1 and 3 clearly shows the presence of bulky substituents such as aryloxyacetylamino-phenyl groups at the 8 position (compound 1) and arylacetyl moiety at the N5 position (compound 3).\nOn the other hand, in recent years a number of substituted adenines have been synthesized and tested at the four adenosine receptor subtypes, demonstrating that the introduction of different substituents at the 2, 8 and 9 positions of the adenine core resulted in high-affinity antagonists with distinct receptor selectivity profile [18\u201322]. At the A2B receptor the derivatives bearing an ethyl in the 9 position and linear chains in the 2 position showed potency in the \u03bcM range, while the presence of sterically hindered substituents in the same positions was detrimental for the potency. Furthermore, substitution of the 9-ethyl group with a propyl chain seems to favour the interaction with human A2B receptors [21].\nHence, on the basis of the results obtained with compound 3, introduction of a bulky substituent on the N6 amino group of the adenine and 2-chloroadenine moiety could increase potency and selectivity for the human A2B adenosine receptor subtypes.\nHence, bulky substituents such as arylacetyl or aryloxyphenylacetyl moieties were introduced on the N6 amino group of compounds 5 and 6 to obtain derivatives 7\u201314, with the aim of finding a new class of A2B antagonists (Fig.\u00a02).\nFig.\u00a02Structures of designed compounds\nChemistry\nThe designed compounds (5\u201314) have been synthesized as summarized in Schemes\u00a01 and 2. The starting 9-propyladenine (5) [23] was obtained by alkylation of commercially available adenine (15) with propyliodide in the presence of potassium carbonate. Flash chromatography led to the desired 9-substituted isomer 5 as the major product (yield 79%) together with the 7-isomer 5a (yield 9%). Isomeric structure of compounds 5 and 5a was assigned on the bases of 1D-1H-NOE difference spectra. In fact, irradiation of both CH2 groups of the propylic chain in compound 5a gave a NOE at both H-C(8) and 6-NH2 groups, demonstrating the 7 position as the alkylation site. On the contrary, a NOE at both H-C(8) and H-C(2), and not at the 6-NH2 group in compound 5, upon saturation of CH2 groups of the propylic chain, confirmed the 9 position as the alkylation site.\nScheme\u00a01a Synthesis of 9-propyladenine; b Synthesis of 2-chloro-9-propyladenine. Reagents: i: DMF, K2 CO3, propyliodide, RT; ii: liq. NH3, sealed tube, RTScheme\u00a02Reagents: i: CDI, dry THF, reflux\nThe 2-chloro derivative 6 was obtained by alkylation of commercially available 2,6-dichloropurine (16) with propyliodide, using the same procedure utilized for compound 5, to afford the 9-substituted isomer 17 as the major product (yield 75%) along with the 7-isomer 17a (yield 10%) [24]. [1H]-NMR spectra of 17 and 17a in CDCl3 are in agreement with those reported in the literature [24]; in the experimental part [1H]-NMR spectra of the same compounds are reported using dimethyl sulfoxide (DMSO) as the solvent. Compound 17 was reacted with liquid ammonia in a sealed tube at room temperature (RT) overnight to give the 2-chloro-9-propyladenine (6) [24] (Scheme\u00a01).\nFinal compounds 7\u201314 were obtained by condensation of amino compounds 5 or 6 with the appropriate acid 18\u201322 [25] in the presence of carbonyldiimidazole in tetrahydrofurane (THF) at reflux for 18\u00a0h (Scheme\u00a02).\nResults and discussion\nAll the compounds were evaluated at the human recombinant adenosine receptors, stably transfected into Chinese hamster ovary (CHO) cells, utilizing radioligand binding studies (A1, A2A, A3) or adenylyl cyclase activity assay (A2B). Receptor binding affinity was determined using [3H]CCPA (2-chloro-N6-cyclopentyladenosine) as the radioligand for A1 receptors, whereas [3H]NECA (5\u2019-N-ethylcarboxamidoadenosine) was used for the A2A and A3 subtypes. In the case of A2B receptors Ki values were calculated from IC50 values determined by inhibition of NECA-stimulated adenylyl cyclase activity. Ki values are in \u03bcM, with 95% confidence intervals in parentheses [26]. The results of binding and cyclase activity studies are reported in Table\u00a01.\nTable\u00a01Biological profile of synthesized compounds 5\u201314aDisplacement of specific [3H]-CCPA binding at human A1 receptors expressed in CHO cells. bDisplacement of specific [3H]-NECA binding at human A2A receptors expressed in CHO cells. cKi values of the inhibition of NECA-stimulated adenylyl cyclase activity in CHO cells expressing human A2B receptors. dDisplacement of specific [3H]-NECA binding at human A3 receptors expressed in CHO cells.\nAll the tested compounds 5\u201314 showed affinities at the human A1, A2A and A3 adenosine receptors in the \u03bcM range without significant levels of selectivity. At A2B receptors most compounds were found to be inactive when tested at a concentration up to 100\u00a0\u03bcM (Ki values > 30\u00a0\u03bcM). It is quite evident that the introduction of phenylacetic or aryloxyphenylacetic moieties at the N6 position of 9-propyladenine (5) or of 2-chloro-9-propyladenine (6) to give compounds 7\u201314 modifies the binding profile of the derivatives, although without significantly increasing binding affinity (Table\u00a01). On the other hand, the same substitutions were found to be detrimental for the activity at the A2B receptor subtype. In fact, the N6-unsubstituted derivative 6 proved to be the most potent of the series with Ki A2B\u2009=\u200911\u00a0\u03bcM.\nThe effect of the chlorine at the 2 position on binding affinity at the adenosine receptors it is not univocal, depending on the receptor subtype and\/or on the substituent in N6. Analysis of the binding profile of the N6-unsubstituted derivatives in more detail revealed that the presence of a chlorine atom at the 2 position (compound 6) increased the affinity (A1, A2A and A3) and potency (A2B) at adenosine receptors two- to threefold compared with the unsubstituted analogue 5.\nA quite similar profile could be observed when a 4-bromophenylacetic group was introduced at the N6 position (compare compound 8 with 7).\nAn opposite effect of the chlorine atom was detected at A1, A2B and A3 receptors when a bulkier substituent, such as the 4-aryloxyphenylacetic chain, was introduced at the N6 position. In fact, this kind of combination significantly reduced or did not modify the A1 and A3 affinity and the A2B potency (9: Ki A1\u2009=\u20091.4\u00a0\u03bcM, Ki A2B > 30\u00a0\u03bcM, Ki A3\u2009=\u20095.3\u00a0\u03bcM vs 10: Ki A1\u2009=\u200926\u00a0\u03bcM, Ki A2B\u2009>\u200930\u00a0\u03bcM, Ki A3\u2009=\u20094.9\u00a0\u03bcM and 12: Ki A1\u2009=\u200913\u00a0\u03bcM, Ki A2B\u2009=\u200922\u00a0\u03bcM, Ki A3\u2009=\u200910\u00a0\u03bcM vs 13: Ki A1\u2009=\u200922\u00a0\u03bcM, Ki A2B\u2009>\u200930\u00a0\u03bcM, Ki A3\u2009=\u200919\u00a0\u03bcM).\nHowever, in all cases, the presence of a chlorine atom in the 2 position favoured the interaction with the A2A subtype (compare A2A affinity of 5, 7, 9 and 12 with 6, 8, 10 and 13, respectively), while the presence of any substituent on the N6 position seems to somewhat reduce the affinity. In fact the compound endowed with the highest A2A affinity proved to be the 2-chloro-9-propyladenine (6: Ki A2A\u2009=\u20092.2\u00a0\u03bcM). These findings are in agreement with previous observations related to adenosine analogues strongly suggesting that the introduction of substituents in the N6 position dramatically reduces the A2A affinity [27\u201332].\nNevertheless, it should be underlined that the presence of a bulky chain at the N6 position significantly increased (20- to 70-fold) the affinity at the A1 and A3 subtypes in comparison with 9-propyladenine (9: Ki A1\u2009=\u20091.4\u00a0\u03bcM and 11: Ki A3\u2009=\u20091.4\u00a0\u03bcM vs 5: Ki A1\u2009=\u200924\u00a0\u03bcM and Ki A3\u2009>\u2009100\u00a0\u03bcM).\nThis increase of affinity seems also to be modulated by the substituent on the aryloxyphenylacetic group; in fact, substitution with a lipophilic bromine (9) or methyl group (11) at the para position is responsible for the increased A1 and A3 receptor affinity, respectively, while the presence of a hydrogen (12) or a methoxy group (14) did not positively influence the binding profile.\nConclusions\nIn conclusion the study herein presented, although it did not reach the proposed goal of obtaining A2B adenosine receptor antagonists, increased knowledge of the structure-activity relationships in adenine derivatives.\nMoreover, it was demonstrated that the introduction of bulky substituents at the N6 position of adenine derivatives significantly increased the affinity at the A1 and A3 adenosine receptors, while the presence of a chlorine atom in the 2 position favoured the interaction with the A2A subtype. These results demonstrated that, although the synthesized compounds were found to be quite inactive at the human A2B subtype, adenine is a useful template for further development of simplified adenosine receptor antagonists with distinct receptor selectivity profiles, opening up new chances to design structurally simplified A1 and A3 adenosine receptor antagonists.\nExperimental section\nChemistry\nGeneral: melting points were determined with a B\u00fcchi apparatus and are uncorrected. 1H NMR spectra were obtained with Varian VXR 300\u00a0MHz spectrometer; \u03b4 in ppm, J in Hz. All exchangeable protons were confirmed by addition of D2O. Thin layer chromatography (TLC) was carried out on precoated TLC plates with silica gel 60\u00a0F-254 (Merck). For column chromatography, silica gel 60 (Merck) was used. Elemental analyses were determined on Fisons Instruments Model EA 1108 CHNS-O model analyser and are within \u00b1 0.4% of theoretical values.\n9-Propyladenine (5) and 7-propyladenine (5a)\nTo a solution of adenine (15) (0.5\u00a0g, 3.7\u00a0mmol) in dry DMF (10\u00a0ml), under nitrogen, K2CO3 (0.83\u00a0g, 5.97\u00a0mmol) and propyliodide (0.433\u00a0ml, 4.44\u00a0mmol) were added. The mixture was stirred at RT for 16\u00a0h, then the solvent was removed under reduced pressure and the crude purified by flash chromatography (CHCl3-MeOH 98:2) to afford 5 [23] and 5a (yield 79 and 9%, respectively) as white solids, after crystallization from CH3OH.\n5: m.p. 173\u2013175\u00b0C. 1H-NMR (DMSO-d6) \u03b4 0.85 (t, 3H, J\u2009=\u20097.4, CH3); 1.82 (m, 2H, CH2-CH3); 4.11 (t, 2H, J\u2009=\u20097.0, CH2-N); 7.21 (bs, 2H, NH2); 8.15 (s, 2H, H-2 and H-8). Anal. Calcd. for C5H5N5 (177.2) C, 54.22; H, 6.26; N, 39.52; found: C, 54.54; H, 6.71; N, 39.33.\n5a: m.p. >250\u00b0C. 1H-NMR (DMSO-d6) \u03b4 0.86 (t, 3H, J\u2009=\u20097.3, CH3); 1.91 (m, 2H, CH2-CH3); 4.26 (t, 2H, J\u2009=\u20097.0, CH2-N); 7.75 (s, 1H, H-2); 7.85 (bs, 2H, NH2); 8.34 (s, 1H, H-8). Anal. Calcd. for C5H5N5 (177.2) C, 54.22; H, 6.26; N, 39.52; found: C, 54.45; H, 6.45; N, 39.45.\n2,6-Dichloro-9-propyl-9H-purine (17) and 2,6-dichloro-7-propyl-9H-purine (17a)\nTo a solution of 2,6-dichloropurine (16) (1\u00a0g, 5.29\u00a0mmol) in dry DMF (14\u00a0ml), under nitrogen, K2CO3 (1.18\u00a0g, 6.61\u00a0mmol) and propyliodide (0.59\u00a0ml, 6.08\u00a0mmol) were added. The mixture was stirred at RT overnight, then the solvent was removed under reduced pressure and the crude purified by flash chromatography (cC6H12-EtOAc 75:25) to afford 17 and 17a as white solids (yield 75 and 10%, respectively) [24].\n17: m.p. 58\u201359\u00b0C; 1H-NMR (DMSO- d6) \u03b4 0.86 (t, 3H, J\u2009=\u20097.5\u00a0Hz, CH2CH3), 1.85 (m, 2H, CH2CH3), 4.21 (t, 2H, J\u2009=\u20097.0\u00a0Hz, N-CH2), 8.76 (s, 1H, H-8). Anal. Calcd. for C8H8Cl2N4 (231.1) C, 41.58; H, 3.49; N, 24.25. Found: C, 41.85; H, 3.70; N, 24.10.\n17a: m.p. 103\u2013105\u00b0C; 1H-NMR (DMSO-d6) \u03b4 0.87 (t, 3H, J\u2009=\u20097.4\u00a0Hz, CH2CH3), 1.84 (m, 2H, CH2CH3), 4.40 (t, 2H, J\u2009=\u20097.2\u00a0Hz, N-CH2), 8.89 (s, 1H, H-8). Anal. Calcd. for C8H8Cl2N4 (231.1) C, 41.58; H, 3.49; N, 24.25. Found: C, 41.75; H, 3.55; N, 24.19.\n2-Chloro-9-propyladenine (6)\nLiquid ammonia (5\u00a0ml) and compound 17 (0.46\u00a0g, 1.97\u00a0mmol) were poured into a sealed tube and the resulting mixture was stirred at RT overnight. Ammonia was evaporated and the crude purified by flash chromatography (CHCl3-MeOH 99:1) to give 6 [24] as a white solid (yield 75%) m.p. 224\u2013226\u00b0C. 1H-NMR (DMSO-d6) \u03b4 0.84 (t, 3H, J\u2009=\u20097.3\u00a0Hz, CH2CH3), 1.79 (m, 2H, CH2CH3), 4.05 (t, 2H, J\u2009=\u20097.2\u00a0Hz, N-CH2), 7.72 (s, 2H, NH2), 8.15 (s, 1H, H-8). Anal. Calcd. for C8H10ClN5 (211.7) C, 45.40; H, 4.76; N, 33.09. Found: C, 45.75; H, 4.80; N, 32.87.\nGeneral procedure for the preparation of the N6-acylaminoadenine (7\u201314)\nA solution in dry THF (4\u00a0ml) of the appropriate acid (18\u201322) (0.46\u00a0mmol) and carbonyldiimidazole (83\u00a0mg, 0.51\u00a0mmol) was poured at reflux under nitrogen for 1\u00a0h. Then the amino compound 5 or 6 (0.46\u00a0mmol) was added and the resulting mixture was refluxed overnight. The solvent was removed under reduced pressure and the crude purified by flash chromatography to afford the desired final compounds 7\u201314.\n6-[(4-Bromophenyl)acetyl]amino-9-propyladenine (7)\nEluent for chromatography CHCl3-MeOH 95:5; yield 59%, white solid; m.p. 149\u2013151\u00b0C (dec.); 1H-NMR (DMSO-d6): \u03b4 0.83 (t, 3H, J\u2009=\u20097.2\u00a0Hz, CH2CH3), 1.84 (m, 2H, CH2CH3), 3.89 (s, 2H, CH2-CO), 4.19 (t, 2H, J\u2009=\u20097.1\u00a0Hz, N-CH2), 7.30 (d, 2H, J\u2009=\u20098.4\u00a0Hz, H-Ph), 7.51 (d, 2H, J\u2009=\u20098.4\u00a0Hz, H-Ph), 8.47 (s, 1H, H-8), 8.62 (s, 1H, H-2), 10.91 (s, 1H, NH). Anal. Calcd. for C16H16BrN5O (374.2) C, 51.35; H, 4.31; N, 18.71. Found: C, 51.65; H, 4.80; N, 18.50.\n6-[(4-Bromophenyl)acetyl]amino-2-chloro-9-propyladenine (8)\nEluent for chromatography CHCl3-cC6H12 80:20; yield 26%, white solid; m.p. 164\u2013166\u00b0C; 1H-NMR (DMSO-d6): \u03b4 0.84 (t, 3H, J\u2009=\u20097.5\u00a0Hz, CH2CH3), 1.83 (m, 2H, CH2CH3), 3.88 (s, 2H, CH2-CO), 4.15 (t, 2H, J\u2009=\u20096.9\u00a0Hz, N-CH2), 7.30 (d, 2H, J\u2009=\u20098.4\u00a0Hz, H-Ph), 7.53 (d, 2H, J\u2009=\u20098.4\u00a0Hz, H-Ph), 8.50 (s, 1H, H-8), 11.25 (s, 1H, NH). Anal. Calcd. for C16H15BrClN5O (408.7) C, 47.02; H, 3.70; N, 17.14. Found: C, 47.49; H, 3.83; N, 17.40.\n6-[(4-(4-Bromobenzyloxy)phenyl)acetyl]amino-9-propyladenine (9)\nEluent for chromatography CHCl3-MeOH 95:5; yield 58%, white solid; m.p. 154\u2013156\u00b0C; 1H-NMR (DMSO-d6): \u03b4 0.85 (t, 3H, J\u2009=\u20097.5\u00a0Hz, CH2CH3), 1.85 (m, 2H, CH2CH3), 3.82 (s, 2H, CH2-CO), 4.21 (t, 1H, J\u2009=\u20097.0\u00a0Hz, N-CH2), 5.07 (s, 2H, CH2-O), 6.95 (d, 2H, J\u2009=\u20098.8\u00a0Hz, H-Ph), 7.27 (d, 2H, J\u2009=\u20098.4\u00a0Hz, H-Ph), 7.40 (d, 2H, J\u2009=\u20098.4\u00a0Hz, H-Ph), 7.58 (d, 2H, J\u2009=\u20098.4\u00a0Hz, H-Ph), 8.48 (s, 1H, H-8), 8.62 (s, 1H, H-2), 10.81 (s, 1H, NH). Anal. Calcd. for C23H22BrN5O2 (480.4) C, 57.51; H, 4.62; N, 14.58. Found: C, 57.99; H, 4.66; N, 14.55.\n6-[(4-(4-Bromobenzyloxy)phenyl)acetyl]amino-2-chloro-9-propyladenine (10)\nEluent for chromatography CHCl3-cC6H12-MeOH 50:48:2; yield 14%, white solid; m.p. 176\u2013178\u00b0C; 1H-NMR (DMSO-d6): \u03b4 0.81 (t, 3H, J\u2009=\u20097.5\u00a0Hz, CH2CH3), 1.79 (m, 2H, CH2CH3), 3.76 (s, 2H, CH2-CO), 4.12 (t, 2H, J\u2009=\u20097.4\u00a0Hz, N-CH2), 5.03 (s, 2H, CH2-O), 6.92 (d, 2H, J\u2009=\u20098.6\u00a0Hz, H-Ph), 7.22 (d, 2H, J\u2009=\u20098.4\u00a0Hz, H-Ph), 7.36 (d, 2H, J\u2009=\u20098.4\u00a0Hz, H-Ph), 7.54 (d, 2H, J\u2009=\u20098.4\u00a0Hz, H-Ph), 8.46 (s, 1H, H-8), 11.11 (s, 1H, NH). Anal. Calcd. for C23H21BrClN5O2 (514.8) C, 53.66; H, 4.11; N, 13.60. Found: C, 53.75; H, 4.25; N, 13.29.\n6-[(4-(4-Methylbenzyloxy)phenyl)acetyl]amino-9-propyladenine (11)\nEluent for chromatography CHCl3-cC6H12-MeOH 70:28:2; yield 35%, white solid; m.p.131\u2013132\u00b0C; 1H-NMR (DMSO-d6): \u03b4 0.83 (t, 3H, J\u2009=\u20097.3\u00a0Hz, CH2CH3), 1.84 (m, 2H, CH2CH3), 2.28 (s, 3H, CH3-Ph), 3.80 (s, 2H, CH2-CO), 4.20 (t, 2H, J\u2009=\u20097.1\u00a0Hz, N-CH2), 5.02 (s, 2H, CH2-O), 6.93 (d, 2H, J\u2009=\u20098.4\u00a0Hz, H-Ph), 7.24 (m, 6H, H-Ph), 8.47 (s, 1H, H-8), 8.62 (s, 1H, H-2), 10.83 (s, 1H, NH). Anal. Calcd. for C24H25N5O2 (415.5) C, 69.38; H, 6.06; N, 16.86. Found: C, 69.74; H, 6.35; N, 16.54.\n6-[(4-Benzyloxyphenyl)acetyl]amino-9-propyladenine (12)\nEluent for chromatography CHCl3-MeOH 97:3; yield 52%, white solid; m.p. 121\u2013123\u00b0C; 1H-NMR (DMSO-d6): \u03b4 0.83(t, 3H, J\u2009=\u20097.5\u00a0Hz, CH2CH3), 1.84 (m, 2H, CH2CH3), 3.80 (s, 2H, CH2-CO), 4.19 (t, 2H, J\u2009=\u20097.1\u00a0Hz, N-CH2), 5.07 (s, 2H, CH2-O), 6.95 (d, 2H, J\u2009=\u20098.4\u00a0Hz, H-Ph), 7.25 (d, 2H, J\u2009=\u20098.8\u00a0Hz, H-Ph), 7.37 (m, 5H, H-Ph), 8.47 (s, 1H, H-8), 8.62 (s, 1H, H-2), 10.82 (s, 1H, NH). Anal. Calcd. for C23H23N5O2 (401.5) C, 68.81; H, 5.77; N, 17.44. Found: C, 68.97; H, 5.89; N, 17.35.\n6-[(4-Benzyloxyphenyl)acetyl]amino-2-chloro-9-propyladenine (13)\nEluent for chromatography CHCl3-MeOH 99:1; yield 17%, white solid; m.p. 150\u2013152\u00b0C; 1H-NMR (DMSO-d6): \u03b4 0.84 (t, 3H, J\u2009=\u20097.3\u00a0Hz, CH2CH3), 1.82 (m, 2H, CH2CH3), 3.79 (s, 2H, CH2-CO), 4.14 (t, 2H, J\u2009=\u20097.0\u00a0Hz, N-CH2), 5.07 (s, 2H, CH2-O), 6.95 (d, 2H, J\u2009=\u20098.6\u00a0Hz, H-Ph), 7.25 (d, 2H, J\u2009=\u20098.4\u00a0Hz, H-Ph), 7.37 (m, 5H, H-Ph), 8.49 (s, 1H, H-8), 11.16 (s, 1H, NH). Anal. Calcd. for C23H22ClN5O2 (435.9) C, 63.37; H, 5.09; N, 16.07. Found: C, 63.56; H, 5.14; N, 15.76.\n6-{[(4-Methoxybenzyloxy)phenyl]acetyl}amino-2-chloro-9-propyladenine (14)\nEluent for chromatography CHCl3-MeOH 99:1; yield 29%, white solid; m.p. 174\u2013176\u00b0C; 1H-NMR (DMSO-d6): \u03b4 0.84 (t, 3H, J\u2009=\u20097.4\u00a0Hz, CH2CH3), 1.82 (m, 2H, CH2CH3), 3.73 (s, 3H, CH3-O), 3.79 (s, 3H, CH2-CO), 4.14 (t, 2H, J\u2009=\u20097.0\u00a0Hz, N-CH2), 4.98 (s, 2H, CH2-O), 6.92 (d, 2H, J\u2009=\u20098.0\u00a0Hz, H-Ph), 6.93 (d, 2H, J\u2009=\u20098.4\u00a0Hz, H-Ph), 7.24 (d, 2H, J\u2009=\u20098.4\u00a0Hz, H-Ph), 7.35 (d, 2H, J\u2009=\u20098.4\u00a0Hz, H-Ph), 8.49 (s, 1H, H-8), 11.15 (s, 1H, NH). Anal. Calcd. for C24H24ClN5O3 (465.9) C, 61.87; H, 5.19; N, 15.03. Found: C, 61.99; H, 5.33; N, 14.91.\nBiology\nAll pharmacological methods followed the procedures as described earlier [26]. In brief, membranes for radioligand binding were prepared from CHO cells stably transfected with human adenosine receptor subtypes in a two-step procedure. In a first low-speed step (1,000\u00a0g) cell fragments and nuclei were removed. The crude membrane fraction was sedimented from the supernatant at 100,000\u00a0g. The membrane pellet was resuspended in the buffer used for the respective binding experiments, frozen in liquid nitrogen and stored at \u221280\u00b0C. For the measurement of adenylyl cyclase activity only one high speed centrifugation of the homogenate was used. The resulting crude membrane pellet was resuspended in 50\u00a0mM Tris\/HCl, pH 7.4 and immediately used for the cyclase assay.\nFor radioligand binding at A1 adenosine receptors 1\u00a0nM [3H]CCPA was used, whereas 30 and 10\u00a0nM [3H]NECA were used for A2A and A3 receptors, respectively. Non-specific binding of [3H]CCPA was determined in the presence of 1\u00a0mM theophylline; in the case of [3H]NECA 100\u00a0pM R-PIA was used. Ki values from competition experiments were calculated with the program SCTFIT [33]. At A2B adenosine receptors inhibition of NECA-stimulated adenylyl cyclase activity was used as a measurement of potency of the new compounds. IC50 values from these experiments were converted to Ki values with the Cheng and Prusoff equation [34].","keyphrases":["adenosine receptors","adenosine receptor antagonists","g protein-coupled receptors","adenine derivatives","adenosine receptor ligands"],"prmu":["P","P","P","P","R"]}
{"id":"J_Med_Internet_Res-7-5-1550689","title":"Architecture for Knowledge-Based and Federated Search of Online Clinical Evidence\n","text":"Background It is increasingly difficult for clinicians to keep up-to-date with the rapidly growing biomedical literature. Online evidence retrieval methods are now seen as a core tool to support evidence-based health practice. However, standard search engine technology is not designed to manage the many different types of evidence sources that are available or to handle the very different information needs of various clinical groups, who often work in widely different settings.\nIntroduction\nClinicians need to keep up-to-date with the biomedical literature in order to practice according to the best available evidence. However, this has become increasingly difficult as the amount of medical literature a clinician needs to consider grows exponentially [1,2]. As a result, the effort required to find a specific piece of evidence increases year after year [3]. Clinicians typically work under time pressure, which compounds the problem. The need to develop robust methods and tools to support evidence access is now widely recognized. Online evidence retrieval methods are increasingly seen as a core tool in support of evidence-based health care [4]. In the traditional model of online evidence services, clinicians have access to a number of online information sources, such as journals, databases, and Medline, each with its own idiosyncrasies and search interfaces. This means users need to know which resources are most suitable for their current question and how the search query must be formulated for a given resource. Interoperability standards for the efficient dissemination of content are being developed (eg, the Open Archive Initiative [5]), but until the majority of content adheres to such standards, there is still a need to search through heterogenous data sources.\nThe meta-search engine approach [6,7] addresses many of the limitations of these models by providing a mechanism to search all the available resources at one time and by translating user queries into the respective query languages of each resource. This typically uses a least-common-denominator approach, directly passing on user keywords to different information sources without regard for the specific capabilities or limitations of these resources. For example, a meta-search engine often disregards the rich query language available with some resources in order to simplify the overall meta-search process. Consequently, while the user expects the meta-search to return an integrated set of search results, the reality is that some resources would have been able to perform much better had they been queried individually; the user is unaware of the variations in search quality across the different resources that have been queried for them. Variants of the standard meta-search engine approach have been shown [8] to provide search capabilities beyond the least common denominator but still require users to select the resources they wish to search. One solution to this problem is to \u201cfederate\u201d the different resources so that they more genuinely behave as one uniform data source. A federated search system may perform a syntactic reformulation of a user query, translating it into queries that have been optimized for the native query language of individual evidence sources. Semantic reformulation is also possible [9]. For example, user keywords may be translated into equivalent keywords or phrases using a terminological system.\nHowever, a federated search can still produce an excessive number of candidate documents, or hits, many of them failing reasonable tests of relevance. One way to improve the chance of retrieving clinically relevant information is to pre-program a search system with specialist bibliographic knowledge using search filters. Search filters capture expert strategies for searching that are known to improve the precision of searches. For example, Medline offers a small set of \u201cclinical queries,\u201d which are pre-defined and validated search filters optimized to retrieve documents that are most likely to be clinically relevant, emphasizing disease etiology, diagnosis, therapy, or prognosis [10,11]. Such search filters are necessarily highly customized to the capabilities of individual information sources and their native search engines. For a federated search system to consistently use search filters, it would need to develop a generalized approach to search filters, or meta-search filters. Quick Clinical (QC) [4] is a federated evidence retrieval system designed to meet the specific needs of clinicians. Its design incorporates the novel use of meta-search filters to optimize search strategies, and it is based upon a wrapper-mediator architecture built around a universal query language. This paper describes the system architecture of QC and the technical challenges to the design of online evidence retrieval systems, and it reports on the technical performance of the system from a clinical trial with primary care physicians.\nMethods\nThe Quick Clinical System\nUser Interface\nIn the QC user model, a user is presented with a single query interface, which connects to an arbitrarily large number of federated knowledge sources and incorporates query specific meta-search filters called \u201cprofiles.\u201d QC guides users to first consider the purpose of their search through selection of a profile, and it then asks them to provide specific keywords related to that search task. As a consequence, users are guided through a process that structures their query for them and improves the chances that they will ask a well-formed query and receive an appropriate answer. Figure 1 depicts the QC search interface. On the left hand is a list of search filters that describe typical search tasks and that are customized to the specific information needs of different user groups. Figure 1 shows filters specifically designed for use by primary care physicians.\nFigure 1\nThe QC search query user interface\nIn QC, individual profiles are able to define different keyword types, such as \u201cdisease,\u201d which describe the keyword classes typically associated with that profile. Thus, on the right of the interface are four fields where users can provide keywords describing the specific attributes of their search. Selection of a different profile may thus alter the keyword types requested from the user for a given search. QC then translates and submits search queries to the sources specified in the chosen profile, collects and processes the results, and presents them to the user as a list of documents (Figure 2). The title of a document is followed by the link and a short abstract of the content. A user can drill-down into a specific group of results by source type (eg, journal articles or guidelines).\nFigure 2\nScreenshot of a QC results page\nQuick Clinical System Architecture\nOverview\nMost information sources such as websites, online texts, and databases have their own proprietary search interface, including query language and format for the display of results. Therefore, a federated meta-search engine that wishes to query a number of different information sources needs to first represent a user query using some internal query language [13] and then translate that internal query into the specific query languages of the relevant data sources. A well-documented [12] approach to this problem is to use a \u201cwrapper\u201d (Figure 3), which acts as an adapter between the proprietary language of individual information sources and the internal language used within a meta-search system. In QC, the internal query language is called the unified query language (UQL). Each information source known to QC has its own wrapper that translates queries from UQL into the native language of the source. As a result, internal components of QC only need to know UQL and not the individual query languages of the data sources. System maintenance is also simplified since the introduction of a new data source to the system only requires one new wrapper component to be generated. Once the results of a search are returned by an information source, the information must again be translated into a standard output format for presentation to the user, which, in QC, is called the unified response language (UReL). UReL also allows other components in the system to modify the presentation of search results without needing to understand the presentation format of individual sources (eg, to remove duplicate documents). In Figure 3, a search is initiated from the user interface, which forwards a query (in XML) to the mediator. The mediator splits the query into several subqueries and sends these to the appropriate wrapper (via a capability manager if required). Finally, the wrapper translates the query into the native query language of the data source (eg, in HTML for Web data sources). Similarly, the result from the data source gets translated back into the system\u2019s XML representation and sent back to the user interface.\nFigure 3\nArchitecture overview of Quick Clinical\nUnified Query Language\nUQL is used to represent queries obtained from users in a consistent internal way, and UQL statements identify query elements such as the external information sources to be searched and a set of search attributes used to delimit the search. For example, UQL expressions can store date range delimiters for a search. UQL also contains statements that indicate whether or not QC needs to process the query further. For example, we may wish to remove duplicate items obtained from different sources. In our current implementation, UQL is implemented using XML. To define the structure of the data within the XML document we use a data type definition (DTD), which allows various internal components of QC to validate the XML data received in the UQL query. The following example illustrates how a UQL query might look in XML.\n<QUERY keyword = \"iron AND deficiency\"\nprofile = \"treatment\"\nduplicateRemoval = \"yes\"\nsortBy = \"rank\"\nuseLexicalVariants = \"yes\"\ntimeout = \"20\"\ndateRangeBeginDay = \"1\"\ndateRangeBeginMonth = \"1\"\ndateRangeBeginYear = \"1999\" >\n<SOURCE name = \"PubMed\" \/>\n<SOURCE name = \"Harrison\u2019s online\" \/>\n<SOURCE name = \"Merck\" \/>\n<SOURCE name = \"MIMS\" \/>\n<\/QUERY>\nUnified Response Language\nSimilarly to the UQL, the unified response language (UReL) is used internally to guide display of information to users, also represented using XML. Each separate result, or \u201carticle,\u201d from a source can be broken up into smaller chunks and given meta-data labels to represent the different sections of the data (eg, abstracts from journal articles). Since the majority of sources accessed by QC are journals, the data that are retrieved typically contain document elements such as Title, Author(s), Journal Name, Date of Publication, and the URL where the electronic version of the paper is accessed. Other sources, such as drug descriptions from pharmaceutical compendia, have sections such as Drug Name and Manufacturer. These different document elements, based upon the typical sources QC expects to find, are defined as specific fields in the UReL definition. The following example illustrates how a set of documents retrieved by QC might be represented in UReL.\n<RESULT>\n<ARTICLE>\n<LINK>\n<HREF>\nhttp:\/\/www.ncbi.nlm.nih.gov:80\n\/entrez\/query.fcgi?cmd=Retrieve&db=PubMed\n&list_uids=12198020&dopt=Abstract\n<\/HREF>\n<LINKNAME>Abstract<\/LINKNAME>\n<\/LINK>\n<AUTHORLIST>Heath AL, \nSkeaff CM, \nGibson RS.\n<\/AUTHORLIST>\n<TITLE>\nDietary treatment of iron deficiency\n<\/TITLE>\n<DATE>\n<YEAR>2002<\/YEAR>\n<MONTH>9<\/MONTH>\n<\/DATE>\n<SOURCE>PubMed<\/SOURCE>\n<\/ARTICLE>\n<ARTICLE>\n<LINK>\n<HREF>\nhttp:\/\/mims.hcn.net.au\n\/ifmx-nsapi\/mims-data\/?MIval=2MIMS_abbr_pi\n&product_code=288\n&product_name=Ferrum+H+Injection\n<\/HREF>\n<LINKNAME>More Information<\/LINKNAME>\n<\/LINK>\n<AUTHORLIST>\nSigma Pharmaceuticals Pty Ltd.\n<\/AUTHORLIST>\n<TITLE>Ferrum H Injection<\/TITLE>\n<SOURCE>MIMS<\/SOURCE>\n<\/ARTICLE>\n<\/RESULT>\nWrappers\nFor every information source known to QC, there is a specific wrapper that translates a UQL query into the native query language and format of the source. The wrapper also extracts the relevant information from the HTML result pages returned by the search engine and re-expresses it in UReL. Figure 4 shows the basic architecture of wrappers in our current system. Each wrapper has three main components: a feeder, extraction rules, and a sieve. The feeder converts the user query into the native query language of the data source. The data source responds to the query and returns HTML raw data. The feeder passes the raw data to the sieve, which converts it to UReL in XML format by using the extraction rules for the data source. The UReL is then sent back via other components to the user interface, which can interpret the XML and display the results.\nFigure 4\nWrapper components\nMediator\nA key requirement of a multisource information retrieval system is the ability to perform concurrent searches on multiple sources with a single query [6,7]. The mediator addresses this requirement. The mediator first analyzes a query and determines how many sources are to be searched. It then creates a separate search job for each of these sources and forwards the search job to other system components. Additionally, the mediator collects individual results as they arrive and amalgamates them for the user into a single result. By introducing parallelism, the time to perform a search across a number of resources should be reduced to the duration of the slowest source. However, the potential drawback of parallel processing is the increased administration overhead of running multiple parallel processes within a system. As a rule of thumb, we would expect the benefits of parallel execution should increase with the number of sources queried, as response times for Web resources can be many seconds long, and computational execution of processes to manage parallel search are typically much less than one second.\nConnection speed and latency of response time from sources are, for practical purposes, nondeterministic in an Internet environment, and a meta-search engine can therefore experience large fluctuations in responses from the same source under different circumstances. Latency is subject to network traffic conditions, making it impossible to guarantee that all resources that are queried at a particular time will respond predictably and equally. To counter this, the mediator has a time-out feature. If a response is not received within the time-out specified by a profile, the mediator will cancel a subsearch and forward all the results currently available from other sources to the user interface. This effectively guarantees a defined response time irrespective of the state of the individual data sources and provides some control over the speed\/accuracy trade-off.\nCapability Manager\nSearch capabilities vary considerably between the search engines that QC might wish to interrogate, and some sources will have limitations in their ability to process search queries. One approach to this problem is to try to raise all sources to as high a level of common performance as possible by emulating missing capabilities locally, usually by modifying the query and\/or search result [13]. A trivial example is mimicking the ability to perform a Boolean search when a data source does not have this capability. To emulate a Boolean AND, a meta-search engine would perform two parallel individual searches on the source and then itself perform the Boolean operation on the two results.\nIn QC, a capability manager (CM) is responsible for mimicking a range of search capabilities and is located between the mediator and wrapper. The CM may modify a query and\/or the result depending on the capabilities of the sources about to be queried. Capabilities of the CM within the QC system included the following:\nDate-CM: search within a date range\nDuplicate-CM: remove document duplicates\nSort-CM: sort results by title, author, document rank, or date\nLexical-CM: expand a search term with lexical variants of the term. A lexical variant is a synonym, pluralization, hyphenation, or other modification that changes the text but not its meaning. Lexical variants are particularly important in the medical domain [14] because many concepts can be expressed in Latin or English (eg, cardio vs heart). Moreover, there is a common confusion between terms in American English versus British English (eg, hemoglobin vs haemoglobin, epinephrine vs adrenaline).\nQC uses a stacking mechanism to insert individual CMs into the processing of queries for wrappers and the processing of results from a source. A component called the search planner, containing simple rules, is responsible for stacking the CMs. This means that the sequence of CMs can be ordered to ensure the correct outcome of query or result translations. Theoretically, this corresponds to a composition of operations. A lexical variant CM, for example, has to replace the search terms in the query before the wrapper executes the search. The Date-CM, on the other hand, can only perform its job after the successful execution of the wrapper.\nSearch Filters\nExpert searchers typically will use search strategies that are more likely to accurately locate information, based upon an understanding of the specific capabilities of an evidence source. There is an increasing interest in the writing of search filters which capture such strategies, usually focusing on the major evidence repositories like Medline [10,11]. Search filters are designed for typical clinical queries such as \u201cdiagnosis\u201d or \u201cprescribing,\u201d and they are crafted to find evidence most likely to satisfy the query by first selectively searching resources identified to be of high quality and, second, by automatically adding specialist keywords to the general question posed by a user. Within QC, search filters are stored in the profiles function. For example, if a clinician selects the \u201cdiagnosis\u201d filter and enters the search term \u201casthma,\u201d QC can add in the additional terms when it queries Medline [10]:\nsensitivity and specificity [MESH] OR sensitivity [WORD] OR diagnosis [SH] OR diagnostic use [SH] OR specificity [WORD]\nThese terms have been shown to significantly enhance the quality of Medline results, but they are unlikely to be known to a typical clinical user.\nUnlike standard search filters, QC profiles are meta-search filters because they encode search filters for multiple different sources. Profiles thus encode expert search strategies that are most likely to answer a certain class of query, and they encode, among other things, the most appropriate content sources to search (Table 1). For a primary care physician, these search profiles might be for diagnosis, prescription, review, and treatment [4], but any set of profiles can be created within QC to meet the specific query types and search contexts of different users. In Table 1, the Treatment profile describes a set of nine separate source-specific search filters, which collectively describe the search strategy believed most likely to retrieve an accurate search result from each resource. The # symbol delimits keyword variables that are to be instantiated with user keywords. For example, #1# represents the keyword type \u201cdisease,\u201d and QC\u2019s mediator component will substitute the user-provided keywords for \u201cdisease\u201d throughout the profile, prior to sending the query to the individual wrappers for the different sources. More than one search string can be created for an individual source (eg, TGL 1 and TGL 2) as a single strategy may not always retrieve all the relevant documents.\nTable 1\nQuick Clinical meta-search filters\nSource\nSearch String\nTGL1\n(#1# AND #2# AND #3# AND #4#) AND+ (\"treatment\" OR \"therapy\" OR \"therapeutic use\")\nTGL2\n(#1# AND #3#) AND+ (\"treatment\" OR \"therapy\" OR \"therapeutic use\")\nHealthInsite3\n#1# AND #2# AND #3# AND #4#\nHealthInsite4\n#1# AND #3#\nPubMed5\n(#1# ATTR+ [Title] AND #3# ATTR+ [Title] AND #4# ATTR+ [Title] ATTR+ \/ther)English 10 years Human\nPubMed6\n#1# ATTR+ [Title] AND (#3# ATTR+ [Title] OR #4# ATTR+ [Title]) ATTR+ \/drug ATTR+ therEnglish 10 years Human\nMerck7\n((#1# AND #3# AND #4#) OR (#1# AND #3#)) AND+ (\"treatment\" OR \"therapy\")\nHarrison\u2019s8\nDisconnected\nHarrison\u2019s9\nDisconnected\nSystem Platform\nThe system was constructed using Java, the Struts Web application framework, and a MySQL database and is deployed on a RedHat Linux platform. The user interface (JSP, servlet, and HTML pages) is deployed through an Apache Web server connected to a Tomcat servlet engine. The Apache-Tomcat platform incorporates load balancing and fail-over and is suitable for scalability and large-scale deployment.\nTechnical Evaluation\nQC has undergone a series of clinical evaluations, which have been reported separately [4,15,16].\nIn total, 227 family physicians from across Australia participated in a trial of QC. Clinicians who had a computer with Internet access in their consulting rooms were recruited and asked to use QC for 4 weeks in routine care. Each participant was given a personal username and password to access the system. All clinicians completed an online pre-trial survey. QC was configured to search a set of eight sources, including remote sites such as PubMed, online journals such as BMJ and the Medical Journal of Australia (MJA), and locally cached sources such as The Merck Manual and Therapeutic Guidelines Australia.\nFor every search, the time from the request arriving at the system to the time when the results were sent back to the user was recorded (Figure 5; search time = system time + slowest source time). Note that there is a cap on search time when the time-out cuts in. Time-outs are search-profile dependent and were set at either 15 or 30 s. The time it took to conduct the search on the individual sources was also recorded. The time taken to send data between QC and the user\u2019s computer (user time) is not incorporated in these measurements.\nIn the following section we report on the technical performance of the architecture and then reflect on its suitability for supporting evidence retrieval in clinical practice.\nFigure 5\nSearch time metrics\nResults\nIn the pre-trial questionnaire, 40% of the clinicians reported having a broadband (ADSL, cable, satellite) connection, while 43% used a 56k or 64k modem connection. The remaining 17% either did not know the type of connection used or had a slower connection. A total of 1662 searches were performed over the trial.\nSearch Speed\nUnder local network conditions (LAN, 100MBit), the user time (from starting the search on a client computer to displaying the results) was approximately 1.5 s. However, since most users accessed the system through the Internet, latency was significantly longer and slowed down the overall search speed.\nThe average search time was 4.9 s, with a standard deviation of 3.2 s (N = 1662 searches). Figure 6 shows the distribution of all search times over the trial. There are four distinctive features in this chart. The first is a small peak at 1 s (ie, searches that took up to 1 s to complete). The second feature is a peak around the mean value. Third, there is a small peak at 15 s, and, fourth, there is a small peak at 30 s.\nFigure 6\nDistribution of search time for all 1662 searches\nSystem Time\nSystem time for a search was computed by subtracting the duration of the slowest source in every search from the search time (see Figure 5). From the system time histogram in Figure 7, it can be seen that for the majority of the searches the system takes between 100 ms and 130 ms (mean = 117.9 ms; SD = 68.4 ms; N = 1614 [48 searches had missing data, hence 1614 searches]).\nFigure 7\nDistribution of the system time for 1614 searches\nSystem Time Versus Number of Individual Sources Involved\nDepending on the search profile selected, the system will query a certain number of information sources and combine the results. To illustrate the dependency between system time and the number of sources queried, Table 2 shows average system time versus the number of sources queried in a search. The number of sources queried is predefined by the search profile, and none of the search profiles tested queried five, six, or eight sources.\nTable 2\nSystem time vs number of sources queried\nNumber of Sources Queried\nN\nAverage System Time (ms)\n1\n48\n18.1\n2\n9\n31.8\n3\n15\n73.3\n4\n7\n59.7\n5\n0\n-\n6\n0\n-\n7\n1373\n122.2\n8\n0\n-\n9\n162\n122.6\nSpeed and Reliability of Individual Data Sources\nIn addition to the performance measurements of the whole searches, the speed and reliability of the individual data sources was measured. Reliability was measured as the number of error cases (ie, queries that were not answered due to an error condition, such as a network error, an HTTP error, or queries that timed out). Reliability and speed figures are summarized in Table 3.\nTable 3\nReliability and speed of data sources\nSource\nType\nNumber of Searches\nNumber of Errors\nError (%)\nMean Speed (s)\nSD (s)\nMin (s)\nMax (s)\nMerck\nLocal\n2144\n0\n0.0\n0.06\n0.11\n0.01\n2.89\nTGL\nLocal\n2193\n0\n0.0\n0.05\n0.12\n0.01\n2.85\nBMJ\nRemote\n73\n1\n1.4\n4.55\n3.92\n0.99\n17.5\nHealthInsite\nRemote\n2993\n55\n1.8\n3.09\n1.08\n1.08\n22.3\nMedlinePlus\nRemote\n653\n0\n0.0\n1.87\n1.36\n1.09\n12.5\nMIMS\nRemote\n650\n3\n0.5\n0.98\n1.14\n0.28\n8.30\nMJA\nRemote\n58\n1\n1.7\n0.25\n0.31\n0.10\n1.73\nPubMed\nRemote\n3288\n39\n1.2\n3.76\n1.69\n1.87\n15.0\nTotal\n12052\n99\nMean\n0.8\n1.83\n0.63*\n0.68\n10.4\n* standard error of the mean\nThe most reliable sources were the locally indexed sources Merck (The Merck Manual) and TGL (Therapeutic Guidelines Australia), both which did not have any error cases. On the other end of the scale are HealthInsite (a national consumer site for health information) and MJA. The slowest source in the trial was BMJ, with an average of 4.55 s to process a query (SD = 3.92 s; N = 73). This was followed by PubMed, which returned results at an average of 3.76 s (SD = 1.69 s; N = 3288). The two locally indexed sources (Merck and TGL) returned search results within an average of 0.061 s and 0.047 s, respectively. However, the two local sources do have a relatively large standard deviation. Figure 8 shows the distribution of query times to the eight individual data sources.\nFigure 8\nHistogram of search times for each of the eight data sources (x-axis is time taken for a search, in ms; y-axis is number of searches)\nDiscussion\nSystem Time\nFrom the results of the system time versus source time, we can observe that system-processing time is only a fraction of the total search time. However, there are exceptions, namely when local data sources are used exclusively. From a user\u2019s perspective this still would not be an issue as the overall user time is greater by at least a factor of ten. It could, however, become a problem in a situation where many searches are dependant on the result of a previous search and have to be executed in series. System time has thus been kept relatively short, removing the initial reservation that too much parallelism could slow down the system excessively. From Table 3 it can be seen that the system time generally increases in line with the number of sources queried (with the exception of four sources queried). However, the order of this increase does not appear to be squared or even exponential, but rather linear.\nSearch Times\nThe four distinct features in the histogram of search times described in Figure 6 are due to the nature of the data sources and the value of the time-outs. The first small peak at 1 s is from search profiles that use exclusively local data sources. The second feature is a peak around the mean value and is caused by the six Internet resources. The small peak at 15 s is due to the large number of search profiles that have this value as a time-out. And finally, the tiny peak at 30 s is where the remaining searches time out.\nIt was to be expected that local sources would be more reliable and have a shorter latency in response time. This is due to the controlled environment, compared to the uncontrolled Internet environment of the external sources. It is interesting to note the difference between the six external data sources. While some sources are very popular (eg, PubMed) and therefore are expected to be busy, others might lack the resources to keep up with demand. The time-out value of individual data sources is a trade-off between speed and quality of results and is determined by the intended usage of the system. However, under certain circumstances there are optimizations that can be carried out without affecting quality of results. For example, the search duration histogram for HealthInsite (Figure 8; top right) reveals that if a search has not completed within 10\u00a0s it is highly unlikely it will complete within 15\u00a0s. Therefore, a time-out value of 15 s can safely be reduced to 10 s without significantly compromising search quality.\nFuture Work\nThe current QC architecture has demonstrated in trials that it meets the technical design goals set for it, and it provides good evidence that our general approach to federated searching is sustainable and maintainable. We intend to pursue research and development in areas of current interest to meta-search engines, information retrieval systems, and artificial intelligence. These include automatic wrapper generation [17,18] so that new data sources can be easily integrated into QC. Using this approach, a component could automatically generate a wrapper from knowledge of the data source query inputs and results. Another area of continued research will be automated data source consistency checking. Data sources often change in their formats, and this needs to be monitored with either automated or human intervention in order to modify wrappers accordingly. A third area will be intelligent search agents [19]. We envisage incorporating an intelligent agent that will guide users through the search process, using domain knowledge to help frame clinical questions and choose search parameters. This agent could learn to work with its user. An area of continued development will be semantic understanding of result sets. We would like QC to combine search results into a meaningful coherent story that presents a concise, relevant, and digestible response to the user [20]. These approaches, coupled with user support, will allow us to develop and improve the system with a view to it becoming an integral part of a clinician\u2019s daily practice. Even without these enhancements, we have demonstrated that the QC framework is a functional and useful approach for the delivery of online, just-in-time clinical evidence.","keyphrases":["meta-search filters","information retrieval","evidence-based medicine","clinical decision support systems"],"prmu":["P","P","M","M"]}
{"id":"J_Urban_Health-2-2-1705501","title":"Recruiting Injection Drug Users: A Three-Site Comparison of Results and Experiences with Respondent-Driven and Targeted Sampling Procedures\n","text":"Several recent studies have utilized respondent-driven sampling (RDS) methods to survey hidden populations such as commercial sex-workers, men who have sex with men (MSM) and injection drug users (IDU). Few studies, however, have provided a direct comparison between RDS and other more traditional sampling methods such as venue-based, targeted or time\/space sampling. The current study sampled injection drug users in three U.S. cities using RDS and targeted sampling (TS) methods and compared their effectiveness in terms of recruitment efficiency, logistics, and sample demographics. Both methods performed satisfactorily. The targeted method required more staff time per-recruited respondent and had a lower proportion of screened respondents who were eligible than RDS, while RDS respondents were offered higher incentives for participation.\nIntroduction\nInjection drug users (IDU) remain an important population in the study of HIV\/AIDS. The Centers for Disease Control and Prevention (CDC) estimates that 31.8% of cumulative male U.S. AIDS cases through 2004 and 40.7% of female cases were exposed through injection drug-use or male-to-male sexual contact and injection drug use.1 Unfortunately traditional sampling methods are inadequate when the population of interest consists of individuals who perform illegal or stigmatized behaviors. Injection drug users are one such population. Traditional probability-based sampling methods require the development of a sampling frame enumerating the entire population. This is prohibitive for these \u2018hidden\u2019 populations because their size is unknown, and their members are often reluctant to participate in studies due to legal issues or social stigmatization.2,3\nBecause of these difficulties, a number of alternative sampling methods have historically been employed to study hidden populations, including injection drug users.4 These methods have included convenience sampling, time\/space or venue-based sampling and chain-referral methods. Chain-referral or \u201csnow-ball\u201d sampling5,6 methods involve selection of an initial set of members of the target population, or \u2018seeds\u2019, who provide information on, or referrals for, subsequent waves of respondents. The number of waves and potential referrals, as well as the seed selection methods, may vary. Versions of these methods have proven successful at quickly and easily identifying members of hidden populations.7\u20139 However, results obtained are thought to be biased by the initial seed selection method as well as characteristics of the respondents' social network and have generally been classified as convenience samples.6,10,11\nAnother method proposed by Watters and Biernacki3 known as targeted sampling (TS) has had some success in recruiting injection drug users.12 Targeted sampling requires extensive ethnographic and formative research to describe the area and population of interest and to identify appropriate locations within that area for inclusion in a sampling plan.13 Existing secondary data sources are also reviewed to further describe the target population and geographic areas of interest. Qualitative data in the form of focus groups and in-depth interviews with key informants may be conducted to gain a more complete understanding of the population of interest as well as to identify possible specific locations for sampling. These locations are then systematically mapped and ethnographic observations are conducted. Based on all of this information a sampling frame of locations is developed and weighted by the density of observed indicators. Locations are randomly selected and recruitment is conducted within the selected locations. Targeted sampling, as well as other time\u2013space or venue-based sampling methods, may be biased to the extent that members of the population of interest do not attend the venues or areas that have been identified.\nHeckathorn10,14,15 has recently proposed a modified chain-referral method called respondent-driven sampling (RDS) as a promising strategy in surveying hidden populations. This approach retains the advantages of chain-referral sampling in terms of ease of respondent identification and recruitment while allowing population estimates and inferences to be made.10,15 Initial seeds are identified from known members of the population or through key informants. Information about the respondent's social network is collected, and seeds are provided with a set number of coupons for distribution to recruits within their own network. Eligible recruits are interviewed and provided coupons for distribution to the next \u2018wave\u2019. Recruitment continues in this way until the desired sample size is reached. Network information is then used to derive asymptotically unbiased population estimates for variables of survey interest.\nRDS has been shown to be a useful strategy for the recruitment of drug users.16\u201318 To date, however, no direct comparison of RDS with other sampling methods has been made in terms of the ability to recruit hidden populations such as IDUs, the \u201crepresentativeness\u201d of sample characteristics, and the practicality of administration.2,4 The objective of the current study is to compare and contrast RDS and TS methods on demographics, recruitment efficiency, and logistic concerns.\nMaterials and Methods\nThree sites in large Metropolitan Statistical Areas (MSA) (Detroit, Houston and New Orleans) that participated in the CDC-funded National HIV Behavioral Surveillance among injecting drug users (NHBS-IDU) conducted a pilot investigation comparing RDS and TS. Each site assessed the feasibility of recruiting at least 100 eligible IDUs utilizing RDS and TS methods over an 8-week period in late 2004. What follows is a brief presentation of the strategies used by each site to locate and recruit potential respondents using RDS and TS methods.\nTargeted Sampling\nFormative Research\nExtensive ethnographic and formative research was conducted at all sites prior to sampling. Each site completed a thorough review of secondary data sources dealing with the IDU population. Data were compiled and synthesized from several sources including number of persons living with HIV\/AIDS who have IDU as a reported mode of exposure, archival census data and findings from previous published and unpublished research.\nA total of 81 in-depth interviews were conducted with individuals who had knowledge of local injection drug-use communities. Those interviewed included local HIV prevention personnel and community planning group members as well as law enforcement representatives and current or former injectors. Ten focus groups with a total of 78 injection drug users were also conducted in order to provide specific knowledge about local injection habits and behaviors. These structured interviews were designed to provide a fuller understanding of the injection community at large as well as to solicit locations for possible sampling areas. Sites also utilized information on areas that had been previously identified by ethnographers with knowledge of the local IDU community or had been used in other local IDU studies.\nArea Identification and Recruitment\nField observations of the possible sampling areas were systematically conducted at varying times of day and week over the course of 5\u00a0months (March\u2013July 2004). Field staff conducted ethnographic observations within each area and recorded any IDU indicators that were present. IDU indicators included physical objects such as used syringes, baggies, balloons or injection works\/equipment as well as behavioral indicators such as copping activity, loitering or commercial sex work. Sites used a combination of ethnographic research, secondary data analysis, key informant interviews, and field observations to form expected yield ratings to identify areas where injection drug use was likely to occur and where potential survey respondents could be recruited.\nBased on highest expected yield rating, Detroit and Houston identified four and ten sampling areas, respectively, within seven ZIP codes. For these sites line based enumeration was then used to approach possible respondents. New Orleans identified 35 sampling areas within 16 ZIP codes of high, medium and low expected yield. These ZIP codes were stratified by the expected yield, and order of ZIP codes was then randomly selected without replacement at the beginning of each 2-week period. Interviewers approached all individuals within each of the prescribed three block sampling areas within each ZIP code.\nRespondent-Driven Sampling\nSeed Selection and Recruitment\nPossible seeds were identified through key informant referrals and street outreach. Seeds were purposefully selected to maximize diversity on race, sex and drug of choice. Each seed was screened, and eligible seeds were interviewed using the NHBS-IDU instrument and provided with a $20 cash value incentive. Each initial seed was provided with three recruitment coupons and instructed to give them to individuals they knew injected drugs. Seeds were also informed that they would be compensated with $10 cash value for each person that they recruited. Peer-recruited respondents returning these coupons were then screened for eligibility, interviewed and provided with three coupons they were instructed to give to someone who they knew injected drugs. They were told that they would be compensated $10 for each person they recruited. A specialized coupon tracking system database was used to track referrals and facilitate incentive provision.\nAnalysis\nAll RDS participants were asked to provide information about the size and characteristics of their social networks from which they would draw potential recruits. Specifically, each participant was asked to report the number, sex and race\/ethnicity of injectors they knew and had seen in the past 6\u00a0months. Relationship of the respondent to the recruiter (e.g., friend, injection partner, sex partner etc.) was also assessed. This information along with data from the coupon tracking system was analyzed using Respondent-Driven Sampling Analysis Tool (RDSAT) software (RDSAT (computer version). Version 5.4.0. Ithaca, New York: Volz E, Heckathorn DD; 2005) to provide estimated population proportions and confidence intervals for age, sex and race\/ethnicity variables.\nParticipants\nAll participants provided informed consent and were carefully screened for eligibility criteria. Eligibility criteria for study participation were that the respondent was at least 18\u00a0years of age, was resident of the study MSA and had injected illicit drugs within the past 12\u00a0months.\nTargeted Sampling\nIn Detroit 273 individuals were approached during TS. Two hundred twenty one individuals agreed to participate and were screened for eligibility. Of TS screened participants, 54% (n=120) were found to be eligible and completed the survey.\nIn Houston 174 individuals were approached during TS. One hundred forty eight respondents agreed to participate and were screened for eligibility. Of TS screened participants, 65% (n=97) were found to be eligible and completed the survey.\nIn New Orleans 560 individuals were approached during TS. Three hundred eighty-nine respondents agreed to participate and were screened for eligibility. Of TS screened participants, 35% (n=137) were found to be eligible and completed the survey.\nRespondent-Driven Sampling\nSix seeds were approached in Detroit for participation, and three of those individuals completed the survey and agreed to be seeds. One hundred sixty-nine recruitment coupons were distributed by Detroit during RDS over nine waves of recruitment; 106 of those individuals returned and were screened for eligibility. Of RDS screened participants, 96% (n=102) were found to be eligible and completed the survey.\nThirteen seeds were approached in Houston for participation, and one additional seed was recruited after being interviewed in the targeted sampling component. Of the 14 seeds recruited, 1 was found to be ineligible. One hundred sixty-eight recruitment coupons were distributed during RDS over seven waves of recruitment; 62 of those individuals returned and were screened for eligibility. Of RDS screened participants, 77% (n=48) were found to be eligible and completed the survey.\nTen seeds were approached in New Orleans for participation, and two of those individuals completed the survey and agreed to be seeds. Three hundred sixteen recruitment coupons were distributed during RDS over 19 waves of recruitment; 133 of those individuals returned and were screened for eligibility. Of RDS screened participants, 88% (n=118) were found to be eligible and completed the survey.\nMaterials\nThe standardized NHBS-IDU questionnaire was administered to all eligible participants. This 68-item questionnaire on HIV risk behaviors, requiring approximately 40\u00a0min, includes items on demographic characteristics, drug use, sexual risk behaviors, access to and use of health care and utilization of HIV prevention services. Variables of interest for this study included respondent demographic information, time required for survey completion, and cost of each survey.\nResults\nSample Characteristics\nDemographic characteristics of the samples obtained for each site by method are presented in Tables\u00a01, 2, and 3. Local HIV surveillance data on persons living with HIV\/AIDS who have injection drug use as a reported mode of exposure are presented for comparison purposes only. Chi-square tests indicated no significant association of sampling method and gender for any site (Detroit: , P=0.33; Houston: , P=0.38; New Orleans: , P=0.23). No significant association between age and sampling method was found (Detroit: , P=0.52; Houston: , P=0.14; New Orleans: , P=0.23). Two sites showed significant association between race\/ethnicity and sampling method (Detroit: , P=0.42; Houston: , P=0.01; New Orleans: , P<0.0001).\nTable\u00a01Demographic characteristics of Detroit IDUs obtained through RDS and TS, and persons living with HIV\/AIDS who are injection drug users (PLWH\/A-IDU)\u00a0TSRDSRDS pop. estimates, % (95% CI)PLWH\/A-IDU, (%)Race\/ethnicityN%N%African American101949696\u201391White 0011\u20135Hispanic1111\u20133Other6622\u20130Sex\/genderMale7469717163 (52\u201375)61Female3431242437 (25\u201348)39TransgenderAge18\u20133421220.1(0.2\u20130.7)435\u2013442423202017 (7\u201327)2245\u2013546560545451 (40\u201367)4755+1716242432 (16\u201343)27Table\u00a02Demographic characteristics of Houston IDUs obtained through RDS and TS, and persons living with HIV\/AIDS who are injection drug users (PLWH\/A-IDU)\u00a0TSRDSRDS pop. estimates, % (95% CI)PLWH\/A-IDU, (%)Race\/ethnicityN%N%African American42522745\u201369White22281423\u201320Hispanic10121932\u201310Other6700\u20131Sex\/genderMale5367467776 (45\u201396)56Female2025122024 (4\u201355)44Transgendera6823Age18\u2013342634122014 (0.1\u201331)1735\u2013442228254141 (33\u201386)4045+3039244045 (5\u201360)43aFemale and transgender categories were combined for purposes of Houston's RDS analyses.Table\u00a03Demographic characteristics of New Orleans IDUs obtained through RDS and TS, and persons living with HIV\/AIDS who are injection drug users (PLWH\/A-IDU)\u00a0TSRDSRDS pop. estimates, % (95% CI)PLWH\/A-IDU, (%)Race\/ethnicityN%N%African American12589675877 (64\u201390)65White54342920 (9\u201334)33Hispanic11223 (0.1\u20133)2Othera9613110Sex\/genderMale11985948174 (58\u201388)76Female1914221926 (12\u201342)24Transgender21Age18\u2013343726342927 (15\u201343)1635\u2013444431262227 (12\u201344)3545\u2013544029443839 (21\u201357)3755+191412107 (2\u201313)12aHispanic and other race\/ethnicities were combined for purposes of New Orleans' RDS analyses.\nAlso presented are the population estimates and confidence intervals obtained by RDSAT. No RDS population estimates were calculated for race\/ethnicity for Detroit or Houston due to lack of cross-group recruitment (e.g., only African Americans were recruiters of other race\/ethnicities in Detroit). For each RDS estimate presented, equilibrium was achieved.\nCost Effectiveness\nAcross all sites 47% of TS-screened respondents were found to be eligible IDUs, while 89% of screened respondents in RDS were eligible IDUs. TS required an average of 214\u00a0h performing ethnographic observation and mapping plus an average of approximately 2\u00a0h and 9\u00a0min of staff time locating, screening and interviewing each recruit (total = 4\u00a0h and\u00a06 min per recruit) while RDS required approximately 1\u00a0h and 13\u00a0min per recruit. TS methods provided each participant with an average of $20 in incentives, while RDS recruits received an average of $26.68. RDS incentives did not total $50 for every participant because some respondents did not produce the maximum of three recruits, and some recruiters did not return to receive the added incentives for recruitment efforts.\nLogistics\nStaffing requirements were similar for both methods, and sites were able to use the same staff to conduct RDS and TS concurrently. Some differences were evident as a result of method selection. Targeted sampling was conducted in the field while RDS was usually conducted from a single storefront. Interviewers reported that this led to a more controlled environment when using RDS. Increased privacy, comfort and perceptions of safety were reported, while distraction level decreased. Weather was also more of a concern with TS while interviewing outdoors.\nDiscussion\nThe use of multiple sources of ethnographic data was beneficial in identifying high-yield TS recruitment areas because areas with the highest number of eligible recruits were often not necessarily areas with obvious drug use indicators but areas identified through HIV surveillance or key-informant interviews. However, even with good ethnographic information, many areas that were expected to be high-yield were not, and less than half of all individuals approached were found to be eligible. This led to low interviewer morale in Detroit, where the interviewers were paid per interview completed. It also led to some bias in recruitment in Houston, where interviewers made the decision to recruit only individuals who looked like injection drug users on one particularly unsuccessful recruitment day.\nThe sample characteristics of those recruited through TS may not reflect the general IDU population but rather the population found during the recruitment times. New Orleans included low density areas and randomized area selection in the hopes of assembling a diverse sample, reflective of the general IDU population. However, this did not happen. The sample characteristics were reflective of the population found during recruitment times, and the sample may have missed less visible injectors, such as those with steady day time employment or child care responsibilities and those who remain indoors during the day or only come out at night. Houston and Detroit had similar problems. One solution might be to extend recruitment hours into the night or start very early in the morning, but this would further increase security concerns.\nTS offered less control in the working environment, compared to RDS. Safety was a concern: The interviewers tended to spread out in the targeted area and the interviewing process drew the attention of others. Groups often gathered to inquire about the interview and the incentive, heightening security concerns. Weather was also an important factor in recruitment success, as fewer individuals were on the streets during hot, cold, or rainy days, and the spring and summer of 2005 was particularly noteworthy, weather-wise. Overall the RDS storefront was preferable to conducting TS outdoors. Participants often scheduled interview appointments and came to a central location for their interviews. Adequate staffing and office space were essential. It was found to be best to have a general waiting area, private interview spaces for each interviewer, and an area for obtaining participants' coupon information and providing incentives. In addition, all sites concurred that a single staff person to monitor the flow of participants entering, waiting, and exiting the facility would have provided even more control of the environment.\nRDS success depends largely on the effective recruitment of seeds, an inter-connectedness of injection drug use networks, and the willingness of seeds and their recruits to travel to an interview location. Community partnerships were also important in the recruitment of successful seeds. In Detroit, seeds were recruited from two needle exchange locations in the city at the recommendation of staff familiar with the individuals' ability to recruit others. This worked well in Detroit, where three of six recruited seeds led to the recruitment of 106 eligible IDUs.\nIn Houston, the initial seeds were recruited from outreach workers who were conducting other research studies in drug-using populations. This was problematic, as some of these initial seeds were not actually linked to the injection drug using community but rather spent their days recruiting participants for these other research projects. These \u201cresearch hustler\u201d seeds were often very good at recruiting individuals (for money), just not at recruiting eligible individuals. This resulted in very short referral chains since few of their recruits were eligible.\nIn New Orleans, staff were only able to successfully recruit two seeds. Eight others identified through key informants agreed to participate but never came to the field location to be interviewed. The two seeds that were interviewed represented the two subgroups of the overall sample, as described in the formative research. One seed was an older African American male, the other a younger white female. The race\/ethnicity and age differences between the New Orleans RDS sample (29% white, 19% under 25) and the TS sample (4% white, 5% under 25) may have reflected the characteristics of the two seeds' networks rather than methodological differences.\nBased on lessons learned in this pilot, when NHBS-IDU shifted to full implementation, the sites made several adjustments to the RDS protocols. In New Orleans, prior to the discontinuation of the project due to Hurricane Katrina, seeds were recruited from among participants who were known to interviewers from the TS pilot and who encouraged others to be interviewed. Second, two field offices were established; one was close to downtown and easily accessible by public transportation and the other was near a large public housing project in an area that yielded high eligibility rates in TS. Finally, transportation tokens were purchased and made available to participants who came to the field office in an attempt to ease the burden of travel on respondents. In Houston, successful seeds were individuals the interviewers\/outreach workers found during the formative work for the targeted pilot. These were influential individuals or gatekeepers within small communities of injection drug users, often the individuals who were first to approach staff and inquire why interviewers were entering their geographic space.\nDiscontinuing RDS recruitment required some special considerations. While targeted sampling may easily stop on any given date or after a pre-determined number of participants have been recruited, the coupons distributed to potential RDS subjects represent a commitment on the part of the researcher. It was therefore important to carefully decrease the number of coupons distributed and shorten the coupon validity period so that recruitment would continue even as the end of the study date approached. Ending the distribution of coupons too early may result in not meeting the recruitment goal (in this case 100 participants), while ending it too late may lead to a disgruntled public.\nOverall, both RDS and TS proved effective in recruiting IDUs in the three pilot MSAs. Although no claim as to the actual representativeness of the obtained samples is made, both methods yielded samples that were similar in terms of many demographic characteristics (such as age, race and sex) as well as drug of choice (not presented here). Demographic results of the samples from both methods also compared favorably to each city's Census and HIV surveillance data from injection drug users. All three sites were able to reach the goal of 100 respondents in TS. Issues with initial seed selection and time constraints most likely resulted in the failure of one site to recruit 100 respondents with RDS. Total cost efficiency for the two methods was also comparable. Extensive formative research and low screened-to-eligible respondent ratios resulted in larger person-hour expenditures for TS, while RDS required higher incentive payouts to participants. Logistical concerns did differ considerably between the two methods. While RDS provided a more controlled interview environment, recruitment success seemed to be affected by the ability to identify quality seeds. Quality of ethnographic assessment and size and cultural qualities of the MSAs also affected targeted sampling success. While more research is needed in assessing the methodological impact of choice of sampling method in identifying hidden populations, these experiences should provide researchers some insight into the some of the practical strengths and weaknesses of these two methods.","keyphrases":["injection drug use","respondent-driven sampling","hidden populations","sampling methodology","targeted sampling."],"prmu":["P","P","P","R","R"]}
{"id":"Pediatr_Nephrol-4-1-2259258","title":"A genome search for primary vesicoureteral reflux shows further evidence for genetic heterogeneity\n","text":"Vesicoureteral reflux (VUR) is the most common disease of the urinary tract in children. In order to identify gene(s) involved in this complex disorder, we performed a genome-wide search in a selected sample of 31 patients with primary VUR from eight families originating from southern Italy. Sixteen additional families with 41 patients were included in a second stage. Nonparametric, affected-only linkage analysis identified four genomic areas on chromosomes 1, 3, and 4 (p < 0.05); the best result corresponded to the D3S3681-D3S1569 interval on chromosome 3 (nonparametric linkage score, NPL = 2.75, p = 0.008). This region was then saturated with 26 additional markers, tested in the complete group of 72 patients from 24 families (NPL = 2.01, p = 0.01). We identified a genomic area on 3q22.2\u201323, where 26 patients from six multiplex families shared overlapping haplotypes. However, we did not find evidence for a common ancestral haplotype. The region on chromosome 1 was delimited to 1p36.2\u201334.3 (D1S228-D1S255, max. NPL = 1.70, p = 0.03), after additional fine typing. Furthermore, on chromosome 22q11.22\u201312.3, patients from a single family showed excess allele sharing (NPL = 3.35, p = 0.015). Only the chromosome 3q region has been previously reported in the single genome-wide screening available for primary VUR. Our results suggest the presence of several novel loci for primary VUR, giving further evidence for the genetic heterogeneity of this disorder.\nIntroduction\nVesicoureteral reflux (VUR) (OMIM 193000) is the most common disease of the urinary tract in children and affects 1\u20132% of the Caucasian population [1]. VUR may be associated with both acquired postinfectious and congenital parenchymal damage, currently known as reflux nephropathy (RN) [2, 3]. The most serious consequence of RN is chronic renal insufficiency (CRI), leading to end-stage renal failure (ESRF), dialysis, and\/or renal transplantation: 25.4% of children affected with CRI have RN [4]. As a consequence, the impact of VUR on public health is considerable and, despite medical and surgical interventions for the past decades, the incidence of VUR-related renal failure has not decreased [5].\nThe reflux may occur isolated or in association with other congenital abnormalities of kidney\/urinary tract (CAKUT) or as part of syndromic entities, such as renal-coloboma or branchio-oto-renal syndromes [6\u20138]. The exact etiology of primary VUR is not known, but it is probably related to an abnormal morphogenesis of the ureteral bud, leading to a defect of the ureterovesical junction [9]. The initial evidence suggesting a genetic origin of primary VUR came from twin studies, showing an 80\u2013100% concordance for VUR in monozygotic twins vs. a 35\u201350% concordance in dizygotic twins [10, 11]. Subsequent evidence included familial clustering of VUR [12], ethnic differences between affected and nonaffected individuals [13], and an increased risk (30\u201350%) of developing VUR in first-degree relatives of an index case [14\u201316]. From family studies, a range of inheritance patterns was reported, including autosomal dominant with incomplete penetrance [17\u201320], autosomal recessive [21], polygenic [22], and even X-linked [23].\nPrevious studies suggest a urinary tract malformation locus on chromosome 6p [24, 25]. Studies of humans with chromosomal abnormalities also suggest candidate loci or genes on chromosomes 10q26 [26], 19q13 (USF2 gene) [27], and 13q33\u201334 [28]. Because mutations in PAX2 on 10q24 cause renal-coloboma syndrome, a rare autosomal dominant disease with kidney anomalies that include VUR, this gene was also proposed as a candidate [6, 7]. However, none of these loci or genes has been shown causally related to primary VUR [19, 20]. Recently, Lu et al. [29] showed that mutations in the ROBO2 gene contribute to the pathogenesis of VUR\/CAKUT in a small proportion of families. In the only genome-wide linkage study reported to date, Feather et al. [18] demonstrated linkage to chromosome 1p13 for primary VUR under a model of autosomal dominant inheritance with reduced penetrance.\nHere, we describe the results of the second genome-wide scan for primary VUR. Differently from previous studies and aiming to collect a homogeneous sample set, our patients were ascertained in a single geographic region. Our results suggest the presence of several novel loci for primary VUR, giving further evidence for the genetic heterogeneity of this disorder.\nMethods\nPatients and families\nFifty-one pedigrees with multiple patients with VUR coming from Campania (southern Italy) were enrolled in the study (Fig.\u00a01). All families were ascertained through an index case, with VUR documented by voiding cystourethrography (VCUG) in males and direct radionuclide cystography (RNC) in females and family members. Three pediatric nephrologists and one radiologist assessed the patients. RN was diagnosed by DMSA scintigraphy (dimercaptosuccinic acid labeled with Technetium-99\u00a0m) and defined as focal defects of radionuclide uptake and\/or by one-kidney differential uptake below 43% [30]. VUR grading was made according to the International Grading System of Vesicoureteral Reflux [31].\nFig.\u00a01Selection and distribution of families and patients included in the study. VUR vesicoureteral reflux\nAdditional family members were considered as \u201caffected\u201d based on the presence of reflux documented by VCUG\/RNC and\/or the diagnosis of RN, or the detection of ESRF\/renal replacement in absence of other known causes. As VUR may spontaneously disappear during childhood and adolescence [32], the finding of scintigraphic signs of RN in relatives of VUR patients strongly suggests the previous occurrence of reflux [33]. Individuals with renal symptoms indicative of VUR, such as previous urinary tract infections and\/or hypertension and\/or proteinuria, not supported by additional findings were classified as \u201cdiagnosis unknown\u201d. Patients with secondary VUR, i.e., neurogenic bladder and posterior urethral valves, or other urinary tract abnormalities, i.e., ureterocele and obstructive hydronephrosis, were excluded. The study focused on primary familial VUR. Eight families with 31 patients with VUR were selected for study phase 1 (genome scan) according to the following criteria: diagnosis of primary VUR in absence of any other malformation, two or more affected individuals per family, and a pattern of inheritance compatible with an autosomal dominant model. The second sample (follow-up) consisted of five affected relative pairs (parent\u2013child trios, ten patients) and 11 small families (31 patients) fulfilling the same criteria (Fig.\u00a01).\nInformed consent from patients and family members (parents for their children) and approval from the Ethic Committee at Second University of Naples were obtained previously.\nLaboratory analysis\nGenomic DNA was isolated from peripheral blood leukocytes by standard techniques and was sent from the Paediatrics Department of Second University of Naples to the Department of Clinical Genetics, Erasmus Medical Centre in Rotterdam. A systematic genome scan was performed using the ABI Prism MD-10 set (Applied Biosystems) consisting of 382 short-tandem-repeat polymorphisms markers (STRPs), average spaced 10\u00a0cM. Additional markers for further characterization of candidate regions were selected from the gender-average Marshfield genetic map. Information about marker order and distances were obtained from the National Center for Biotechnology Information (NCBI) physical map and Marshfield integrated genetic map. Polymerase chain reaction (PCR) products were resolved on an ABI3100 automated sequencer, and genotypes were analyzed using the GeneMapper software v.2.0 (Applied Biosystems).\nLinkage analysis\nOne thousand simulations were performed (SLINK, MSIM) [34] to investigate the statistical power of our sample set. An autosomal dominant mode of inheritance was assumed with a penetrance of 70%, a mutant allele frequency of 1%, and a phenocopy rate of 1%. The calculations were done under the assumption of genetic heterogeneity. Assuming 35% of unlinked families, a maximum total log of odds (LOD) score of 5.4 was obtained with a low average LOD of 1.58 [standard deviation (SD)\u2009=\u20091.26], after analyzing all eight pedigrees selected previously. Increasing the percentage of unlinked families to 50%, a maximum LOD score of 4.94 was observed (average 0.99, SD\u2009=\u20091.04). Families 1, 4, and 12 had the largest contribution to the total LOD score (LOD\u2009=\u20090.86, 1.04, and 1.62, respectively), followed by families 5, 7, and 13 (LOD\u2009=\u20090.57). Due to their small size, families 2 and 11 had less contribution to the final LOD (LOD\u2009=\u20090.29). Simulations were calculated in the replication group (11 families) after exclusion of five parent\u2013child trios, giving a maximum LOD of 4.57 (average 1.49, SD\u2009=\u20091.07) and a maximum LOD of 4.85 (average 1.14, SD\u2009=\u20091.08), under assumption of 35% and 50% unlinked families, respectively. Family 25 with seven patients was the main contributor to the total LOD score (LOD\u2009=\u20091.47). Finally, simulations were performed in all 19 families (genome scan group and replication group) reaching a maximum LOD of 9.19 (average of 2.76, SD\u2009=\u20091.78) and a maximum LOD of 7.36 (average of 1.71, SD\u2009=\u20091.34), under assumption of 35% and 50% unlinked families, respectively.\nMega2 [35] was used to process the genetic data, whereas the accuracy of allele segregation within the families was confirmed with Pedcheck. The program GENEHUNTER v.2.1 [36] was used to compute multipoint parametric and nonparametric (or model-free) linkage analysis. As VUR may often disappear with age, all individuals in whom VUR, RN, or both were not clinically proven were classified (and analyzed) as \u201cdiagnosis unknown\u201d. Thus, a conservative \u201caffected-only\u201d analysis was performed based on these criteria. Due to the uncertain pattern of inheritance, nonparametric analysis was computed. The rationale of a nonparametric analysis is that, among affected relatives, excess sharing of haplotypes identical by descent would be expected, irrespective of the mode of inheritance. The nonparametric linkage statistics examining all individuals simultaneously (NPL-all), is reported.\nA parametric analysis was performed as well. An autosomal dominant mode of inheritance with reduced penetrance, as described above (power calculations), was used. Marker allele frequencies were calculated using all spouses (unrelated individuals) coming from the same geographic area. Due to the genetic heterogeneity of the disease, the genome scan data was analyzed, maximizing the heterogeneity LOD score (HLOD) with respect to the proportion of linked families (\u03b1). Nonparametric (NPL) and parametric (LOD) scores were calculated for each of the families, and then total NPL and HLOD scores were obtained.\nResults\nPatients\nFifty-one pedigrees with multiple patients with VUR, originating from the same region of southern Italy (Campania), were available for the study. All persons in the study were ascertained through an index case documented by VCUG\/RNC. A total of 143 patients were detected. According to the phenotype, three groups of families were identified: primary VUR; VUR associated with additional abnormalities of kidney\/urinary tract such as duplicated collecting system, renal agenesis, or hypospadia; and VUR occurring in syndromes, such as renal-coloboma, branchio-oto-renal syndromes, and reflux associated with congenital ichthyosis (Fig.\u00a01).\nA total of 78 primary VUR patients belonging to 25 pedigrees were identified (33 based on a positive VCUG\/RNC, 18 based on detection of RN, 21 diagnosed as having both reflux and RN, and six with ESRF). During the course of the study, a new patient affected with RN was detected in family 1 and included in the second stage. Twenty-eight patients were males and 51 were females (ratio M\/F\u2009=\u20090.57). The median age at diagnosis was 3.5\u00a0years (range 1\u00a0month\u201368\u00a0years). Among the 79 patients, 19 were treated surgically, and four out of six who developed ESRF underwent renal transplantation.\nThe most informative pedigrees are shown in Fig.\u00a02a,b. The pedigrees showed patients in several generations, and male-to-male transmission was observed in some families, supporting an autosomal dominant mode of inheritance. As reported before for primary VUR, we observed several obligate carriers in our families, i.e., families 4, 6, 12, and 13. Family 6 is especially interesting: when assessing the parents (asymptomatic) of the four symptomatic children, both individuals were found to have RN. No consanguinity was reported, but history of ESRF in both branches was described. We are currently recruiting clinical information of those patients (family 6, second generation, Fig.\u00a02). Due to this bilinear transmission, this family was excluded from the linkage analysis.\nFig.\u00a02A selection of pedigrees enrolled in the study is shown. Squares indicate males and circles indicate females. Family members with unknown phenotype are in grey, whereas those unaffected (normal voiding cystourethrography or direct radionuclide cystography before age of 5\u00a0years and unrelated spouses) are in white. An asterisk highlights the individuals genotyped in the study. A number following the asterisk indicates the patients\/obligate carriers reported in Fig. 3. Individuals with urinary tract infections by history and \u201cobligate carriers\u201d are considered as unknown in all analyses. a Nine multiplex pedigrees included in the genome-wide scan are shown. An additional five parent\u2013child trios are not displayed. Family 6 was excluded from the linkage analysis due to the bilineal inheritance. b All 11 multiplex pedigrees included in the fine-typing stage are shown. VUR vesicoureteral reflux, RN reflux nephropathy, ESRF end-stage renal failure, UTIs urinary tract infections\nGenome search\nWe performed a systematic genome-wide scan using 382 microsatellite markers. Results from both parametric and nonparametric linkage analysis excluded most of the genome (data not shown). Furthermore, negative scores were found on both VUR loci, 1p13 [18] (NPL\u2009=\u2009\u22120.86, p\u2009=\u20090.86) and 13q33\u201334 [28] (NPL\u2009=\u2009\u22120.21, p\u2009=\u20090.58) previously reported.\nWe used a nominal p value of \u2009< 0.05 to decide whether a region was promising for further study. Table\u00a01 summarizes the genome-wide scan results, showing all regions that yielded a total NPL corresponding to p\u2009<\u20090.05. These regions were located on chromosome 1 (D1S468-D1S255, D1S213-D1S2785), chromosome 3 (D3S3681-D3S1569), and chromosome 4 (D4S402-D4S1597). Furthermore, on chromosome 22q11.22\u201312.3, patients from a single family showed excess allele sharing (D22S539-D22S280). The best evidence of linkage was observed on chromosome 3p12.3\u20133q24, which yielded the highest NPL score (2.75, p\u2009=\u20090.008, HLOD\u2009=\u20091.52, \u03b1\u2009=\u20090.76) and on chromosome 1p36.32\u20131p34.3 (NPL\u2009=\u20092.22, p\u2009=\u20090.02, HLOD\u2009=\u20091.13, \u03b1\u2009=\u20090.62). Recently, mutations in ROBO2 were described in two VUR\/CAKUT patients [29]. ROBO2, which is an ideal functional candidate gene for VUR\/CAKUT, maps on chromosome 3p12.3 at the border of our region. Thus, we retrospectively sequenced all gene exons in our index patients, and no mutations were found.\nTable\u00a01Summary of the genome-wide scan results. Total nonparametric linkage (NPL) scores with p values <0.05PedigreesD1S468-D1S255D1S213-D1S2785D3S3681-D3S1569D4S402-D4S15971p36.32\u20131p34.31q41\u20131q433p12.3\u20133q244q26\u20134q32.34\u201365\u00a0cM242\u2013266\u00a0cM109\u2013158\u00a0cM117\u2013169\u00a0cM1\u22120.80 (\u22121.09)0.59 (0.19)2.48 (0.87)\u22120.37 (0.07)21.34 (0.27)\u22120.42 (\u22120.76)1.34 (0.28)0.44 (0.27)41.14 (0.72)0.26 (\u22120.36)1.04 (\u22120.31)3.05 (1.3)51.9 (0.55)1.78 (0.55)1.89 (0.58)1.69 (0.57)71.59 (0.54)1.07 (0.42)1.75 (0.58)\u22120.33 (\u22121.37)111.34 (0.27)1.34 (0.27)1.34 (0.28)0.44 (0.28)121.47 (0.19)1.58 (0.35)0.65 (0.10)\u22120.04 (\u22121.66)131.25 (0.54)1.97 (0.54)1.25 (0.57)1.25 (0.57)Total NPL (p)2.22 (p\u2009=\u20090.02)2.35 (p\u2009=\u20090.02)2.75 (p\u2009=\u20090.008)2.15 (p\u2009=\u20090.02)Total HLOD (\u03b1)1.13 (0.62)0.61 (0.57)1.52 (0.76)1.31 (0.51)NPL and (LOD) scores per family are shown at each locus. Total NPL (p value) and HLOD (\u03b1) obtained from the analysis of all eight pedigrees are displayed. HLOD was obtained maximizing the \u201cheterogeneity\u201d LOD score with respect to the proportion of linked families (\u03b1). Negative NPL and LOD scores indicate no linkage or inconclusive resultNPL nonparametric LOD score, HLOD heterogeneity LOD score, \u03b1 proportion of \u201clinked\u201d families\nThe NPL score of 2.15 (p\u2009=\u20090.02) on chromosome 4q26\u201332.3 was mainly due to family 4 (NPL\u2009=\u20093.05, p\u2009=\u20090.062, LOD\u2009=\u20091.3). Yet, allele sharing among the patients of this family was observed also on chromosome 2p23.2\u20132p15 (D2S165-D2S337, NPL\u2009=\u20093.05, p\u2009=\u20090.062, LOD\u2009=\u20091.3). The only region in which patients from family 12 (Fig.\u00a02) showed excess allele sharing was on chromosome 22q11.22\u201312.3 (NPL\u2009=\u20093.65, p\u2009=\u20090.015, LOD\u2009=\u20091.49). We then closely inspected the haplotypes from the positive regions in all genotyped families. Families 1, 2, 5, 7, 11, and 13 displayed haplotype sharing among each family\u2019s patients on chromosome 3p12.3\u20133q24, supporting the linkage to this area. Thus, in six out of eight informative families, we observed allele\/haplotype sharing in an overlapping area on chromosome 3q. We noted also on chromosome 1p haplotype sharing in four out of eight multiplex families (families 2, 5, 7, and 11) extending from D1S2667 until D1S255. Therefore, we decided to explore these genomic areas.\nRefinement of the chromosome 3 locus\nThe chromosome 3q region between D3S3681 and D3S1569 was saturated with 26 additional microsatellites markers (average spaced approximately 2\u00a0cM). A maximum HLOD\u2009=\u20092.69 (\u03b1\u2009=\u20090.95) was reached with an NPL\u2009=\u20092.96, p\u2009=\u20090.001. In a second stage, the fine mapping was extended with 16 additional families with 41 patients. The expansion of pedigrees introduced additional unlinked families, which lowered the maximum HLOD to 1.24 (\u03b1\u2009=\u20090.36), giving a total NPL\u2009=\u20092.01 (p\u2009=\u20090.01) (Table\u00a02). Pedigree 25 was the main contributor to the total score, with an NPL\u2009=\u20094.63, p\u2009=\u20090.03, (LOD\u2009=\u20091.45). We then performed a detailed haplotype analysis in all 24 families. Patients from families 2, 5, 7, 13, 20, and 25 shared a common region delimited by markers D3S3641 and D3S1764, containing 36 genes according with the NCBI map, built 36.2. Instead, families 1 and 11 were sharing a more centromeric area not overlapping with the other families (Fig.\u00a03). We did not observe a common \u201cancestral\u201d haplotype shared across the families.\nFig.\u00a03Haplotype analysis in families supporting the chromosome 3 region after fine typing. A selection of microsatellite markers is shown. The shared genomic region is shown in grey; recombinants are shown in white blocksTable\u00a02Summary of fine-typing results in chromosome 1 and 3 (total NPL scores with p values <0.05)PedigreesD1S468-D1S255D3S3681-D3S15691p36.32\u20131p34.33p12.3\u20133q244\u201365\u00a0cM109\u2013158\u00a0cM1\u22120.79 (\u22122.19)1.43 (0.74)21.34 (0.28)1.34 (0.28)41.59 (1.17)0.46 (\u22120.43)51.89 (0.56)1.79 (0.58)71.6 (0.57)1.75 (0.57)111.34 (0.28)1.34 (0.28)121.51 (0.05)0.81(\u22120.73)131.26 (0.57)1.98 (0.57)141.41 (0.27)0.004 (0.001)15\u22120.24 (\u22120.07)1.41 (0.28)16\u22120.07 (\u22120.02)0.0004 (\u22120.008)171.41 (0.28)1.41 (0.28)18\u22120.37 (\u22120.32)\u22120.57 (\u22120.48)190.9 (0.28)\u22120.81 (\u22120.9)20\u22120.37 (\u22120.29)1.73 (0.48)21\u22120.48 (\u22120.45)\u22120.58(\u22120.48)221.37 (0.27)\u22120.57 (\u22120.48)232.43 (0.86)\u22121.41 (\u22120.96)250.22 (0.28)4.63 (1.45)Total NPL (p)1.70 (p\u2009=\u20090.03)2.01 (p\u2009=\u20090.01)Total HLOD (\u03b1)1.65 (0.55)1.24 (0.36)NPL and (LOD) scores per family are shown at each locus. Total NPL (p value) and HLOD (\u03b1) obtained from the analysis of all eight pedigrees are displayed. HLOD was obtained maximizing the \u201cheterogeneity\u201d LOD score with respect to the proportion of linked families (\u03b1). Negative NPL and LOD scores indicate no linkage or inconclusive resultNPL nonparametric LOD score, HLOD heterogeneity LOD score, \u03b1 proportion of \u201clinked\u201d families\nRefinement of chromosome 1 locus\nThe 1p36.22-p34.3 region between D1S468 (4.2\u00a0cM) and D1S255 (65.5\u00a0cM) was saturated with seven extra microsatellite markers (total 12, spaced 4\u20135\u00a0cM) in the complete group of 24 families (Table\u00a02). We observed a maximum HLOD of 1.65 (\u03b1\u2009=\u20090.55) and NPL of 1.7 (p\u2009=\u20090.03). The haplotype analysis showed allele sharing in families 2, 5, 7, 11, 19, and 23 between D1S228 (29.8\u00a0cM) and D1S255 (65.5\u00a0cM). Patients from families 13, 14, and 22 displayed haplotype sharing, but it was limited to the upper or lower part of the region, thus showing no overlapping with the rest of the families.\nCandidate genes\nThere were several interesting genes map on the chromosome 3q region. The PPP2R3A gene encodes the protein phosphatase 2A, one of four major protein phosphatases identified in eukaryotic cells, implicated in the regulation of most major metabolic pathways. It has been shown to be expressed ubiquitously in 15-day-old kidneys, regulating the mitogenic activity in the early embryonic kidney [37]. The FNDC6 gene encodes a type of fibronectin, expressed mainly in fetal kidney [38], implicated in the development of renal basement membrane [39]. RBP1 and RBP2 genes  (Retinol-binding-proteins type 1 and 2) are involved in the metabolism of vitamin A, which in its active form of retinoic acid plays a critical role during kidney development, even connecting ureters to bladder [40]. The AGTR1 gene encodes the angiotensin receptor II type 1 (AT1), shown to be related to gross abnormalities in renal morphogenesis in mutant mice (hydronephrosis) as well as a poor proliferation of ureteral smooth cells [41].\nDiscussion\nThis study reports the second genome-wide search for primary VUR. Our results suggest the existence of several loci mapping to chromosomes 1, 3, 4, and 22, further supporting the hypothesis that primary VUR is genetically heterogeneous.\nWe, as others, encountered several pitfalls when studying VUR. We observed clinical variability among and within families and the presence of obligate carriers (individuals who carry and transmit the disease allele but do not manifest any disease sign or symptom). Although the appropriate clinical investigations were performed in several apparently healthy individuals (carriers), no evidence of disease was found, indicating a reduced or age-dependent penetrance. As most of these individuals were recruited during their adulthood, we could not exclude an earlier disease condition that evolved to a spontaneous resolution. To overcome these problems, we first performed a careful clinical evaluation of patients and available relatives. All individuals older than 5 years of age with insufficient or no evidence of VUR were classified as diagnosis unknown, despite the consequent loss of power for the statistical analysis.\nWe performed the first genome search in VUR that includes families with the same ethnic origin. In order to strengthen the genetic homogeneity of the patients, all families included in this study originated from the same geographic area in the southern part of Italy. Yet, our results strongly support that primary VUR is a (highly) genetic heterogeneous condition. Whereas a large number of our patients and families supported the chromosome 3q locus, one relatively large pedigree (family 12) showed evidence of linkage on chromosome 22. Six of our families supported a locus on chromosome 3q22.2\u201323 (149.8\u2013153.2\u00a0cM). This region is fully overlapping with one of the loci reported by Feather et al. [18]. In their study, the interval on chromosome 3q (from GATA128C02 to D3S1763, 112\u2013176\u00a0cM) showed a high \u03b1 (0.98) with an NPL\u2009=\u20093, p\u2009=\u20090.008. This region was supported by one of their largest pedigrees (with seven patients) that was clearly not linked to the chromosome 1p13 locus. It is interesting that two independent studies have found the same genomic region in distinct groups of patients.\nBesides the number of patients included in our genome scan, the results of the statistical analysis are quite modest and do not reach the criteria suggested by Lander and Kruglyak [42] to declare significant linkage. Therefore, some of the identified loci may represent false positive hits and should be interpreted with caution.\nThe complex disease etiology of primary VUR has shown to be difficult to disentangle. Genetic heterogeneity and lack of knowledge of the true genetic model for VUR are probably the main difficulties in the identification of the genetic etiology of VUR. Although genetic studies in VUR are still in an early phase, we can presume that primary VUR is likely a complex disorder, with a number of not fully penetrant genes causing most of the familiar cases. Finally, primary VUR could be caused by simultaneous gene-environment interactions.\nIn conclusion, our results show further evidence for the genetic heterogeneity in primary VUR. We will next focus on the refinement of the identified genomic regions and the sequence analysis of the candidate genes according to their tissutal expression and biological function. Replication of the results in additional families will be essential, first to confirm and eventually to evaluate the contribution of these loci to the pathogenesis of primary, nonsyndromic VUR.","keyphrases":["vur","complex disorder","primary familial vesicoureteral reflux","genetic linkage"],"prmu":["P","P","R","R"]}
{"id":"Int_J_Cardiovasc_Imaging-4-1-2373860","title":"Calcium scoring using 64-slice MDCT, dual source CT and EBT: a comparative phantom study\n","text":"Purpose Assessment of calcium scoring (Ca-scoring) on a 64-slice multi-detector computed tomography (MDCT) scanner, a dual-source computed tomography (DSCT) scanner and an electron beam tomography (EBT) scanner with a moving cardiac phantom as a function of heart rate, slice thickness and calcium density. Methods and materials Three artificial arteries with inserted calcifications of different sizes and densities were scanned at rest (0 beats per minute) and at 50\u2013110 beats per minute (bpm) with an interval of 10 bpm using 64-slice MDCT, DSCT and EBT. Images were reconstructed with a slice thickness of 0.6 and 3.0 mm. Agatston score, volume score and equivalent mass score were determined for each artery. A cardiac motion susceptibility (CMS) index was introduced to assess the susceptibility of Ca-scoring to heart rate. In addition, a difference (\u0394) index was introduced to assess the difference of absolute Ca-scoring on MDCT and DSCT with EBT. Results Ca-score is relatively constant up to 60 bpm and starts to decrease or increase above 70 bpm, depending on scoring method, calcification density and slice thickness. EBT showed the least susceptibility to cardiac motion with the smallest average CMS-index (2.5). The average CMS-index of 64-slice MDCT (9.0) is approximately 2.5 times the average CMS-index of DSCT (3.6). The use of a smaller slice thickness decreases the CMS-index for both CT-modalities. The \u0394-index for DSCT at 0.6 mm (53.2) is approximately 30% lower than the \u0394-index for 64-slice MDCT at 0.6 mm (72.0). The \u0394-indexes at 3.0 mm are approximately equal for both modalities (96.9 and 102.0 for 64-slice MDCT and DSCT respectively). Conclusion Ca-scoring is influenced by heart rate, slice thickness and modality used. Ca-scoring on DSCT is approximately 50% less susceptible to cardiac motion as 64-slice MDCT. DSCT offers a better approximation of absolute calcium score on EBT than 64-slice MDCT when using a smaller slice thickness. A smaller slice thickness reduces the susceptibility to cardiac motion and reduces the difference between CT-data and EBT-data. The best approximation of EBT on CT is found for DSCT with a slice thickness of 0.6 mm.\nIntroduction\nThe presence of calcium in coronary arteries is known to be a strong indicator for coronary artery disease (CAD) [1]. It has been shown that quantification of coronary calcium enables the assessment of cardiac event risk stratification [1]. In 1990, Agatston et\u00a0al. described a method which determines the amount of coronary calcium from tomographic images [2]. This method, known as the Agatston score (AS), depends on the area and the maximum CT density of the calcification detected by electron beam tomography (EBT). Since then, EBT is generally accepted as the gold standard for determining the amount of coronary calcium. Alternative scoring methods have been proposed, such as volume scoring (VS), depending on the volume of the calcification, and equivalent mass (EM) scoring, which depends on the volume and the average density of the calcification [3\u20135].\nCalcium scoring (Ca-scoring) on EBT is known to be less susceptible to cardiac motion compared to other CT-modalities, because of its relatively high temporal resolution. However, since the appearance of multi-detector computed tomography (MDCT), scanners of this type are also widely used for Ca-scoring as an alternative to EBT. Although the temporal resolution of MDCT is lower than EBT, the spatial resolution is much higher (0.4 vs. 1.0\u00a0mm), enabling the detection of smaller lesions. Whereas Ca-scoring on EBT can only be used in sequential scanning mode, MDCT facilitates Ca-scoring in sequential and spiral mode. Spiral mode scanning has shown to decrease the variability of Ca-scoring when compared to sequential mode scanning [6]. With the development of dual source computed tomography (DSCT) in 2006, CT is finally approaching the temporal resolution of EBT combined with a high spatial resolution [7].\nIn order to use Ca-scoring as a useful diagnostic test, it must be demonstrated as accurate, clinically relevant and reproducible. Monitoring of coronary atherosclerosis by repeated scans is advocated by Callister et\u00a0al. to test the response to lipid-lowering pharmacologic therapy [8] and Budoff et\u00a0al. [9] showed that statin therapy induced a 61% reduction in coronary calcium progression rate. Therefore a highly reproducible scan-method independent of in-vivo conditions to test the accuracy of Ca-scoring is desirable. In this study a cardiac phantom was used to investigate the influence of cardiac motion on the absolute Ca-score for different kinds of scanners. To our knowledge no previous study has systematically investigated the influence of the heart rate on the absolute Ca-score using EBT, 64-slice MDCT and DSCT.\nThe purpose of this study was therefore to assess Ca-scoring on 64-slice MDCT and DSCT versus EBT on a moving cardiac phantom as a function of heart rate, slice thickness and calcification density using 3 different Ca-scoring methods.\nMethods and materials\nCardiac phantom\nA moving cardiac phantom (QRM, M\u00f6hrendorf, Germany) was used to simulate the movement of the coronary arteries (Fig.\u00a01, left) [7, 10]. The phantom consists of a robot arm which performs a pre-programmed motion (Fig.\u00a02). The robot arm moves in a water container inside a thorax phantom (QRM, M\u00f6hrendorf, Germany) [11]. Different inserts can be attached to the robot arm. The motion curves used in this study were based on velocity curves for the LAD given in literature in order to simulate the human coronary motion as realistically as possible [12]. Three different artificial arteries were investigated which were custom built by QRM. The artificial arteries were made of hydroxyapatite (HA) with a diameter of 4\u00a0mm and a length of 55\u00a0mm (Fig.\u00a01). Each artery contained three artificial calcifications with a length of 10\u00a0mm, a spacing of 5\u00a0mm and a thickness of 0.5, 1.0 and 2.0\u00a0mm, respectively. The density of the calcifications was different in each artery, one with high density calcifications (HDC), one with medium density calcifications (MDC) and one with low density calcifications (LDC). The concentration and density of the calcifications in the three artificial atereries is given in Table\u00a01. The artificial artery had a density of 50 Houndsfield Units (HU), simulating human blood.\nFig.\u00a01Left: the cardiac phantom. Right: schematic figure of the artificial artery, the dimensions are given in millimetersFig.\u00a02Motion curve of the phantom at 70\u00a0bpm. The curve is defined by the time-deflection points T1\u2013T8 and the reconstruction intervals of the DSCT and 64-slice MDCT are indicated by the grey areas. Other heart rates are obtained by a time scaling of the data points. For higher heart rates (>90\u00a0bpm) the data point T5 was omitted to reflect the relative larger diminishing of the diastolic phaseTable\u00a01The three artificial coronary arteries high, medium and low density calcification (HDC, MDC and LDC) with the properties of the inserted calcifications as specified by the manufacturerArtificial arteryConcentration (mgHA\/cm3)Density (g\/cm3)HDC7961.58MDC4011.30LDC1971.16\nData acquisition\nThe phantom was positioned at an angle of 45 degrees relative to the center axis of the scanner. Every scan was repeated five times with a small random translational (approximately 2\u00a0mm) and small random rotational repositioning (approximately 2\u00a0degrees) of the phantom after each scan. The ECG signal from the phantom was recorded during scanning to enable synchronization with the scanner. The scan parameters on the 64-slice MDCT (Somatom Sensation 64, Siemens, Forchheim, Germany) were: tube voltage 120\u00a0kV, tube current 250 mAs effective, collimation 64\u00a0\u00d7\u00a00.6\u00a0mm and rotation time 330 ms. A DSCT (Somatom Definition, Siemens, Forchheim, Germany) was used with similar scan parameters: tube voltage 120\u00a0kV, tube current 100\u00a0mAs\/rot (equivalent to the tube current of 64-slice MDCT), collimation of 64\u00a0\u00d7\u00a00.6\u00a0mm and rotation time 330\u00a0ms. A spiral scanning mode was used on both scanners for a better reproducibility. A standard hospital calcium scoring protocol was used on the EBT-scanner (e\u2212 Speed, GE Imatron, San Francisco, USA). This protocol uses a sequential mode with a tube voltage of 130\u00a0kV, a tube current of 44\u00a0mAs, a collimation of 3.0\u00a0mm and a scan speed of 50\u00a0ms.\nA standard calcium scoring kernel (B35f) was used for reconstruction of the CT-data. Images were retrospectively reconstructed with a slice thickness of 0.6\u00a0mm (increment 0.4\u00a0mm) and 3.0\u00a0mm (increment 3.0\u00a0mm) for both CT scanners. The phases with minimal motion were selected from the motion curves of the coronary arteries (Fig.\u00a02) and used for reconstruction of the raw data (Table\u00a02). The data from the EBT-scanner were reconstructed with a slice thickness of 3.0\u00a0mm (increment 3.0\u00a0mm) at 40% of the RR-interval with a standard calcium kernel according to the standard calcium scoring protocol used in our hospital.\nTable\u00a02Phases used for reconstruction of the images in percentage of the beat time at different heart rates used in beats per minute (bpm)Heart rates (bpm)506070809010011064-Slice MDCT-phase (%)76746058565351DSCT-phase (%)83827069696766\nCa-scoring was performed on the reconstructed image sets with commercially available software (Syngo CaScore, Siemens, Forchheim, Germany). Three different scoring methods were used: Agatston scoring, volume scoring and equivalent mass scoring. A standard scoring threshold of 130 HU was used during the procedure. Detailed descriptions of these scoring methods can be found extensively elsewhere [4, 11, 13\u201315]. The three calcifications of the arteries could not be detected individually at higher heart rates (at heart rates larger than 60\u00a0bpm for 64-slice MDCT and larger than 90\u00a0bpm for DSCT) combined with thin slices in some of the scans. Therefore the Ca-score of the total artery was used instead of the Ca-scores of the individual calcifications.\nData analysis\nTwo root mean square measures were used to analyze the scoring results. The first measure quantifies the susceptibility of the calcium score to cardiac motion. The second measure quantifies the deviation of the calcium score from the reference value.\nWe defined a cardiac motion susceptibility (CMS) index in order to assess the susceptibility to cardiac motion of the Ca-scoring methods: in which x0 is the Ca-scoring result at 0\u00a0bpm, xi is the scoring result at heart rate i and N is the total number of heart rates used. In the equation for the CMS-index a factor 1\/x0 is introduced to make the index independent of the absolute score which enables comparison of Ca-scores obtained at different slice thicknesses and with different scoring methods as a function of cardiac motion. A small CMS-index is equivalent to a low susceptibility of Ca-scores to cardiac motion.\nA second measure was introduced to compare the calcium score results of the two CT scanners to the results of the EBT scanner. The deviation of the calcium score on CT versus the reference value on EBT is defined using a \u0394-index: in which yi is the EBT-score at heart rate i, zi is the CT-score at heart rate i and yav is the average EBT-score over all heart rates. The normalization factor yav was inserted to make the \u0394-index independent of the absolute score and to enable comparison of \u0394-indexes obtained with different Ca-scoring methods and slice thicknesses. A low \u0394-index is equivalent to a small difference between Ca-scores on CT and EBT. The delta-index, as defined in Eq. 2, is used to quantify the difference in Agatston and volume scores on CT and EBT. For these scoring methods EBT provides the reference value. For the equivalent mass score, however, the reference value is given by the physical mass. The use of a phantom enables the possibility of calculating the true amount of calcium. Therefore the equivalent mass scoring results have been compared to the true values instead of the EBT-values, thus yi is the true value and zi is the CT\/EBT-score at heart rate i.\nNoise levels were measured using a standard Region of Interest (ROI) technique. The ROI was placed in a section of a slice containing only water. The standard deviation of the measured HU-values within the selected ROI was considered to be a measure for the noise level.\nAll measurements are considered to be normally distributed. Mean and standard deviation (sd) are given for each measurement.\nResults\nThe Ca-scoring results of the different arteries obtained with 64-slice MDCT, DSCT and EBT are shown in Fig.\u00a03 as a function of slice thickness and heart rate using the three different scoring methods. The scoring results are relatively constant at low heart rates (50\u201360\u00a0bpm). At heart rates higher than 60\u00a0bpm, however, the scores deviate from the values at lower heart rates and an increase or decrease of scoring results is observed depending on modality, slice thickness, calcification density and scoring method.\nFig.\u00a03Calcium scores as a function of heart rate in beats per minute using 64-slice MDCT at 0.6\u00a0mm (dotted line with circles), 64-slice MDCT at 3.0\u00a0mm (solid line with triangles), DSCT at 0.6\u00a0mm (dotted line with circles), DSCT at 3.0\u00a0mm (solid line with triangles), EBT (solid line with squares). Agatston score (AS), volume score (VS) and equivalent mass (EM) score from top to bottom; artificial arteries high density calcification (HDC), medium density calcification (MDC) and low density calcification (LDC) from left to right. The thick dotted black lines in the figures in the bottom row represent the physical amount of calcium\nThe results show a general underestimation of the Ca-score for Ca-scoring obtained at 3.0\u00a0mm slice thickness when comparing CT-data and EBT at all heart rates except for the Agatston and volume score of the high density calcifications at 70 and 80\u00a0bpm. In general, the Ca-scores obtained with 0.6\u00a0mm slice thickness on CT are overestimated compared to the EBT-data or are similar to the EBT-data at all heart rates.\nThe scores obtained with EBT (squares) increased at heart rates above 90\u00a0bpm for the artery containing the high density calcifications (Fig.\u00a03, left column), whereas the artery containing the medium density calcifications remained relatively constant throughout the whole range of heart rates (Fig.\u00a03, middle column). The artery containing the low density calcifications showed decreased scoring results at higher heart rates (Fig.\u00a03, right column).\nThe 64-slice MDCT with a slice thickness of 3.0\u00a0mm (solid lines with triangles) showed increased Ca-scores for the Agatston score at 70\u201390\u00a0bpm and for the volumes score at all heart rates for the high density calcification, whereas the equivalent mass score showed a slight decrease. The medium and low density calcification also showed a decrease in scoring results at higher heart rates.\nThe 64-slice MDCT with a slice thickness of 0.6\u00a0mm (dotted lines with circles) showed highly increased Ca-scores above 70\u00a0bpm for the high density calcification for all scoring methods. This is also seen for the medium density calcification for the volume score, whereas the equivelnt mass and Agatston score showed a peak in Ca-scores at 80\u00a0bpm. The low density calcification showed diminished results at higher heart rates for all scoring methods.\nThe Ca-scores of the medium and low density calcification obtained with DSCT with a slice thickness of 3.0\u00a0mm (solid lines with triangles) were decreased at elevated heart rates. The results of the high density calcification were relatively constant over the whole range of heart rates.\nFinally DSCT at 0.6\u00a0mm (dotted lines with circles) showed increased results for Agatston and volume score of the high density calcification. The Agatston score of the medium density calcification showed a small decrease and relatively constant results were observed for the equivalent mass score of the high density calcification and volume and equivalent mass score of the medium density calcification. Diminishing results with increasing heart rate were observed for all methods for the low density calcification.\nThe influence of cardiac motion on the Ca-score (CMS-index) using the different scoring methods is calculated using Eq. 1 and is summarized in Fig.\u00a04a\u2013c for all scanners and slice thicknesses. Looking at the results of the Agatston score, the average CMS-index for EBT was approximately similar to the CMS-index of DSCT at 0.6\u00a0mm, which was for its part approximately 60% smaller than the CMS-index of 64-slice MDCT at 0.6\u00a0mm. The CMS-index of DSCT at 3.0\u00a0mm was approximately 50% higher than the CMS-index at 0.6\u00a0mm. The CMS-index of 64-slice MDCT at 3.0\u00a0mm was approximately twice as large as the index of DSCT at 3.0\u00a0mm (Fig.\u00a04a). The results of the susceptibility to cardiac motion using volume and equivalent mass score were similar to the results obtained with the Agatston score, except for the relatively small CMS-index for 64-slice MDCT at 0.6\u00a0mm using for equivalent mass score. The absolute CMS-indexes using equivalent mass were approximately 10% lower compared to the other two methods. The CMS-indexes averaged over scoring method, slice thickness and calcification density were 2.5 for EBT, 3.6 for DSCT and 9.0 for 64-slice MDCT.\nFig.\u00a04Cardiac motion susceptibility-index (see text) determined with Agatston score (AS) (a), volume score (VS) (b) and equivalent mass (EM) (c) score for the high, medium, low density lesions and the average using EBT (with slice thickness of 3.0\u00a0mm), 64-slice MDCT (with slice thickness of 3.0 and 0.6\u00a0mm) and DSCT (with slice thickness of 3.0 and 0.6\u00a0mm). A small CMS-index represents a low susceptibility to cardiac motion. The standard deviations of the CSM-index are indicated by error bars. 64S\u00a0=\u00a064-slice MDCT; DS\u00a0=\u00a0Dual Source CT\nThe difference between the scoring results using Agatston and volume score of 64-slice MDCT and DSCT compared to EBT are calculated using Eq. 2 and are shown in Fig.\u00a05a\u2013b. For Agatston score (Fig.\u00a05a), the best \u0394-index was observed for DSCT with a slice thickness of 0.6\u00a0mm (35.9\u00a0\u00b1\u00a010.0 averaged over all densities). A \u0394-index approximately twice as large was observed for 64-slice MDCT at 0.6\u00a0mm (65.7\u00a0\u00b1\u00a09.0 averaged over all densities). Both CT-modalities at 3.0\u00a0mm had a \u0394-index approximately two times the \u0394-index of DSCT at 0.6\u00a0mm (91.0\u00a0\u00b1\u00a010.1 and 88.4\u00a0\u00b1\u00a09.1 for DSCT and 64-slice MDCT respectively averaged over all densities). Comparable results were observed for the volume score measurement (Fig.\u00a05b), although the \u0394-indexes for the measurements at 0.6\u00a0mm were higher with the highest \u0394-index for 64-slice MDCT at 0.6\u00a0mm.\nFig.\u00a05\u0394-index (see text) determined with Agatston score (AS) (a), volume score (VS) (b) and equivalent mass (EM) (c) score for the high, medium, low density lesions and the average using 64-slice MDCT and DSCT, both with slice thicknesses of 3.0 and 0.6\u00a0mm. For Agatston and volume score, EBT has been used as a reference value (a and b, respectively), whereas for equivalent mass score the physical mass has been used as a reference value (c). The equivalent mass measurement includes the EBT as well. A small \u0394-index represents a good correspondence with the EBT results. 64S\u00a0=\u00a064-slice MDCT; DS\u00a0=\u00a0Dual Source CT\nA \u0394-index was calculated for all scanners comparing the equivalent mass results to the theoretical true values. The results are shown in Fig.\u00a05c. The smallest \u0394-index was observed for 64-slice MDCT (55.9\u00a0\u00b1\u00a06.8) followed by higher indexes for DSCT (68.3\u00a0\u00b1\u00a08.3) and EBT (71.3\u00a0\u00b1\u00a07.9) both with a slice thickness of 0.6\u00a0mm, however the indexes of 64-slice MDCT and DSCT and the indexes of EBT and DSCT are within each margins of error shown by the error bars. Both CT-modalities at 3.0\u00a0mm showed \u0394-indexes approximately twice as large compared to the results at 0.6\u00a0mm (140.1\u00a0\u00b1\u00a07.8 and 131.1\u00a0\u00b1\u00a08.5 for DSCT and 64-slice MDCT respectively averaged over all densities).\nThe \u0394-indexes were 53.2 for DSCT and 72.0 for 64-slice MDCT both with a slice thickness of 0.6\u00a0mm averaged over the scoring methods and densities. The \u0394-indexes at 3.0\u00a0mm were 102.0 for DSCT and 96.9 for 64-slice MDCT averaged over the scoring methods and densities.\nNoise levels were as follows: 64-slice MDCT showed 36.1\u00a0\u00b1\u00a02.9 HU and 13.2\u00a0\u00b1\u00a01.2 HU for 0.6 and 3.0\u00a0mm slice thickness, respectively. DSCT showed 43.0\u00a0\u00b1\u00a01.6 HU and 16.1\u00a0\u00b1\u00a01.0 HU for 0.6 and 3.0\u00a0mm slice thickness, respectively. EBT with a slice thickness of 3.0\u00a0mm showed a noise level of 20.5\u00a0\u00b1\u00a00.8 HU. The noise did not vary at different heart rates.\nDiscussion\nAn assessment was made of Ca-scoring on 64-slice multi-detector computed tomography and dual-source computed tomography versus electron beam tomography on a moving cardiac phantom as a function of heart rate, slice thickness and calcification density using 3 different Ca-scoring methods. From the results it can be concluded that the Agatston, volume and equivalent mass scores depend on heart rate, slice thickness and the CT-system used. Furthermore DSCT is approximately 50% less susceptible to cardiac motion as 64-slice MDCT in Ca-scoring.\nIt has been shown in previous studies that the amount of calcium in coronary arteries is generally underestimated in MDCT with respect to the gold standard EBT. Stanford et\u00a0al. showed an underestimation of coronary calcium with 4-slice MDCT compared to EBT [16] and the same effect was reported by Horiguchi et\u00a0al. using 16-slice MDCT [14, 17]. Our results showed underestimation as well, but only for 3.0\u00a0mm slice thickness, whereas 0.6\u00a0mm showed an overestimation at all heart rates.\nSurprisingly the Agatston scores of the medium density calcification at rest using 3.0\u00a0mm slices are different for the 64-MDCT and DSCT, while similar scores are expected (approximately 165 for 64-slice MDCT and 135 for DSCT). The same effect is observed for heart rates of 50 and 60\u00a0bpm. A possible explanation for this phenomenon lies within the scoring algorithm of the Agatston score. For each calcification the maximum HU value within the calcification is obtained. Based on this maximum value the area of the calcification is multiplied by a weighting factor. For a maximum of more than 400 HU this factor is 4, for a maximum between 300 HU and 400 HU this factor is 3 [2]. The medium density calcification has a CT density of 400 HU. The difference in scoring results can be explained by a small difference in HU between the two scanners. Where the maximum CT density within the medium density calcification could be over 400 HU using the 64-slice MDCT, the maximum CT might have been below 400 using the DSCT. If this explanation is applied, the score obtained using 64-slice MDCT is more similar to the score obtained using DSCT (165*3\/4\u00a0=\u00a0124). Although the difference in HU is very small, the weighting factor of the Agatston algorithm can cause a large difference in scoring result.\nAt heart rates above 70\u00a0bpm Agatston, volume and equivalent mass score differ from the results at rest and at low heart rates. This difference depends on the density of the calcification as can be seen from Fig.\u00a03: calcifications with a high density show elevated scoring results, whereas low density calcifications are associated with diminished scoring results. A decrease of Agatston and equivalent mass score on increased heart rates using a calcification of 400 HU has also been reported by Ulzheimer et\u00a0al. using a 4-slice MDCT in accordance with our results [11]. We considered the influence of image blurring on the Ca-score as a function of the calcification density in Fig.\u00a06 for a possible explanation for this effect. Two calcifications of identical size are shown by black lines, one with a high density (X) and one with a low density (Y). The corresponding apparent images at a relatively low and high heart rate are given by the solid grey and dotted grey line, respectively. In addition, the default Ca-scoring threshold of 130 HU is shown by the dotted black line. At the level of the threshold the apparent width at high heart rates is larger than the apparent width at low heart rates for the high density object. The reverse effect is observed for the low density object; at high heart rates the apparent size is reduced compared to the apparent size at low heart rates. From this analysis it can be concluded that at high heart rates the apparent volume of high density objects is increased and the apparent volume of low density objects is decreased. With this model we can explain the increase of calcium score on increasing heart rate for high density calcifications, and a decrease of calcium score on increasing heart rate for low density calcifications, as observed in Fig.\u00a03. Decreasing scoring results with increasing movement have previously been reported on 4-slice CT [15].\nFig.\u00a06Theoretical estimated CT profiles for two objects (black) with high (X) and low density (Y) exhibiting a relatively low (solid grey) and high (dotted grey) movement. The dotted black line represents the standard Ca-scoring threshold of 130 HU\nThe susceptibility of calcium score on heart rate has been assessed by the CMS-index using the 3 different scoring methods available. The results show that the CMS-index of EBT is the lowest for all methods. Therefore it can be concluded that EBT is the least susceptible to cardiac motion. The CMS-index of DSCT is approximately half the CMS-index of 64-slice MDCT, showing a reduction of 50% of the influence of cardiac motion on Ca-scoring on DSCT with respect to 64-slice MDCT. These results can be explained with the improved temporal resolution of DSCT compared to 64-slice MDCT (83 vs. 165 ms). A reduction of the slice thickness also results in a lower CMS-index. Therefore we conclude that the use of a small slice thickness reduces the susceptibility to cardiac motion for both 64-slice MDCT and DSCT.\nThe difference between CT-data and EBT-data has been assessed by the \u0394-index using the Agatston and volume score, the equivalent mass results have been compared to the physical amount of calcium. The results show the lowest \u0394-index for DSCT with a slice thickness of 0.6\u00a0mm for Agatston and volume score. The CT modalities at 0.6\u00a0mm and EBT showed similar \u0394-indexes for the approximation to the physical mass. A reduction of the \u0394-index was observed comparing the two CT-modalities at 0.6\u00a0mm and 3.0\u00a0mm. The best resemblance between EBT and CT was observed for DSCT with a slice thickness of 0.6\u00a0mm.\nThe use of a smaller slice thickness has some disadvantages although it was beneficial to the scoring results in this phantom study. The noise measurements showed increased noise levels for the 0.6\u00a0mm slices compared to 3.0\u00a0mm slices. It is expected that for patient scanning the noise levels at 0.6\u00a0mm are too high to guarantee a reliable outcome of the Ca-scoring. To overcome these increased noise levels the tube current can be increased. However, this increases the patient dose as well. Although dose-reduction techniques have been investigated leading to dose-reductions up to 57% [18\u201321], a good balance between patient dose and accuracy of calcium scoring needs to be found.\nLimitations\nThe EBT-data acquisition of this study was performed with a standard hospital protocol using a tube voltage of 130\u00a0kV, whereas CT scanning was performed with a tube voltage of 120\u00a0kV. Although higher energies tend to show less density, Nelson et\u00a0al. reported very small differences between EBT at 130\u00a0kV and CT at 120\u00a0kV [22]. Therefore we expect that the influence of the difference in tube voltage can be neglected.\nThe pre-programmed movement of the calcified coronary arteries was 1-dimensional in contrast with the in\u00a0vivo situation where the motion of human coronary arteries is 3-dimensional and the direction and orientation of the human coronary arteries can vary. In our study the movement of the calcified coronary arteries was in the (x,z) plane with a 45o angle relative to the z-direction of the scanner. We expect that movement more perpendicular to the z-direction of the scanner will cause more blurring in the (x,y) plane and reduce blurring in the z-direction. In addition, we expect that movement more parallel to the z-direction of the scanner will be more subject to partial volume effects when using thick slices. Thin slices will be less subject to the PVE due to the isotropic resolution of 0.6\u00a0mm. The motion of the robot arm was programmed according to patient data [11] and therefore we expect that our analysis shows a good correspondence with a clinical situation, but a clinical validation is advocated.\nThe coronary artery we used for our simulation, the LAD, exhibits lesser motion than the LCX and especially the RCA, which exhibits very large motion swings especially in systole. In our study we, however, wanted to show the influence of motion on the coronary calcium score independent of a specific major coronary artery. We therefore have used motion curves with velocities similar to the LAD to simulate the motion, because if a dependency of calcium score on coronary motion could be proven for the lowest velocity of the LAD, we expect an even stronger dependence for the higher velocities of the LCX and RCA. In our study we have shown that for higher heart rates the under- or overestimation of the calcium score increases as a function of calcification density, independently of the absolute velocity of the artery, but depending on the relative heart rate difference\u00a0from 0\u00a0bpm. Because this motion dependent effect is pronounced visible for the relative low velocity of the LAD, we expect that the results can\u00a0also be applied to the vaster moving other major arteries.\nConclusion\nThe results of Ca-scoring are influenced by heart rate, slice thickness and modality used. DSCT is approximately 50% less susceptible to cardiac motion than 64-slice MDCT using a robot phantom. Susceptibility is further reduced with a smaller slice thickness. DSCT gives a better approximation of the absolute calcium score on EBT than results obtained with 64-slice MDCT when using a smaller slice thickness (0.6\u00a0mm). The two modalities show similar results when using larger slice thicknesses (3.0\u00a0mm). In general, the use of a smaller slice thickness further reduces the difference between CT-data and EBT-data. The best approximation to the physical amount of calcium was found using a small slice thickness, where 64-slice MDCT and DSCT show similar results. The best approximation of Ca-scoring on EBT is observed for DSCT with a slice thickness of 0.6\u00a0mm.","keyphrases":["calcium score","64-slice mdct","dual source ct","heart rate","electron beam ct"],"prmu":["P","P","P","P","R"]}
{"id":"Bioinformation-2-1-2139988","title":"A database for medicinal plants used in the treatment of diabetes and its secondary complications\n","text":"Effective treatment of diabetes is increasingly dependent on active constituents of medicinal plants capable of controlling hyperglycemia as well as its secondary complications. Sensing the importance of documenting such medicinal plants, here we describe a web database containing information (name, literature citation, active compounds and few related full text articles) of the diabetes medicinal plants exhibiting hypoglycemic, antioxidant and antimicrobial effects.\nBackground\nDiabetes mellitus is possibly the world's largest growing metabolic disorder and around 30 million people around the world suffer from this disorder.[1] Diabetes can be defined \nas a group of syndromes characterized by hyperglycemia, altered metabolism of lipids, carbohydrates and proteins.[2] The complications of diabetes include vascular diseases, eye \ndisorders, renal disorders and a host of secondary infections. [3] The treatment of diabetes with synthetic drugs is generally not preferred because of its high cost and the range \nof side effects caused. Hence development of traditional or alternative medicine is needed. Herbal drugs constitute an important part of traditional medicine and literature shows \nthat there are more than 400 plant species showing antidiabetic activity. [4,5] Our interest is to study these anti diabetic medicinal plants. Research findings suggest that many \nof these plants control diabetes by exhibiting hypoglycemic and antioxidant effects. The secondary infections associated with diabetes are also restricted to antimicrobial effect by \nplant products. Sensing the opportunities provided and in an effort to translate research into technology, this database \u2018DIAB\u2019 was created to document the research literature \navailable on these medicinal plants and their active compounds.\nMethodology\nStep 1: Data collection\nData of antidiabetic plants were collected from various literature sources such as PubMed [6], ScienceDirect [7], Biomed Central [8] \nSpringerlink [9], \nScirus [10], Wiley journals [11] and also from folklore medicinal usage. The current dataset contains information for about \n258 genus and 287 species of plants exhibiting antidiabetic effect. Of these 287 antidiabetic plants, 129 species show proven antioxidant effect and 53 species exhibit \nantimicrobial effect besides their hypoglycemic activity.\nStep 2: Database Design\nThe database is developed using MS Access as back-end and ASP.NET as front-end on a Windows platform and updated regularly.\nStep 3: Features of DIAB\nThree separate links (antidiabetic, antioxidant, and antimicrobial) are provided to access the literature citations of antidiabetic plants showing hypoglycemic, antioxidant and \nantimicrobial properties respectively. The record entry consists of the following literature: plant name, part investigated, authors and abstract. The \u2018Active principles\u2019 link provides \ninformation (plant name, part investigated, active compounds and properties) on the 46 compounds isolated from the antidiabetic medicinal plants. The \u2018Articles\u2019 link provides few free \ntext articles for further reading. A screen shot of the database is given in figure 1.\nUtility\nIn the current scenario there are several other databases which give information on medicinal plant name, distribution, drugs formulated and usage in treatment of diabetes. We believe \nthat the freely available database called DIAB will give supplementary information on the antidiabetic plants capable of controlling some secondary complications. This database finds \nutility to the scientific community for a quick review on plants for diabetes medicinal plant research and provides enormous scope for development of drugs.\nFuture development\nWe plan to refine and keep updating this database and hopefully a link solely for indigenous plants will be made available in the nearest future. The database will also be modified \nto develop provisions to search the database to identify plants of interest using keywords.","keyphrases":["database","medicinal plants","diabetes","literature","antioxidant","antimicrobial properties","active principles"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Liver_Int-1-_-2156150","title":"Interferon and lamivudine vs. interferon for hepatitis B e antigen-positive hepatitis B treatment: meta-analysis of randomized controlled trials\n","text":"Aims To compare interferon monotherapy with its combination with lamivudine for hepatitis B e antigen (HBeAg)-positive hepatitis B treatment.\nChronic hepatitis B is a common medical condition, affecting more than 400 million individuals worldwide, leading to hepatic inflammation and injury (1\u20134). This viral-triggered, immune-mediated condition predisposes those affected to cirrhosis and hepatocellular carcinoma, thus necessitating treatment (1\u20133). The treatment consists of individualized, single-agent therapy with interferon-\u03b1 or nucleoside analogues. Unfortunately, this treatment fails to yield long-lasting outcomes in majority of the treated population, prompting the notion of their use in combination to enhance the therapeutic efficacy (4\u20138).\nThe notion of combination therapy for chronic hepatitis B treatment has been previously examined, yielding inconclusive results (9\u201316). In our study, we aim to elucidate this topic comparing interferon monotherapy to its combination with the best-studied antiviral agent for that purpose, lamivudine. Furthermore, the focus of our analysis is hepatitis B e antigen (HBeAg)-positive patients, a subset of the patient population in which disease activity, risk of complications and the subsequent need of efficacious therapy are more pronounced.\nMethods\nLiterature search and study design\nTwo independent researchers conducted the literature search, study selection and data extraction, with any disagreements resolved by consensus among them.\nThe researchers conducted a systemic literature search using the electronic databases MEDLINE (1966 to January 2006), EMBASE (1980 to June 2006), OVID (1966 to January, week 3, 2006) and the Cochrane library clinical trials registry (issue 1, 2007). The following keywords were used: \u2018Hepatitis B\u2019, \u2018Interferon\u2019, \u2018Lamivudine\u2019 and \u2018combination therapy\u2019. In addition, a manual search using citations in previous publications was preformed. The following inclusion criteria were used: (i) study design: randomized controlled trials; (ii) study population: HBeAg-positive patients; (iii) intervention: interferon vs. interferon and lamivudine therapy. Our search was not restricted by language. The following exclusion criteria were used: (i) examining the nonadult population; (ii) not reporting any of the primary efficacy measures as defined by the authors. When several publications pertaining to a single study were identified, the most recent and complete publication was used.\nThe included studies were divided into two groups according to their use of conventional (CON) or pegylated (PEG) interferon-\u03b1, with patients within each group given interferon monotherapy, or interferon and lamivudine combination therapy. Data were extracted for study methodology and for the defined efficacy measures. Only data pertaining to the regimens in question were extracted, while data concerning other regimens were reviewed, and if found to be of significance to our study, were noted and discussed. Separate meta-analyses examining the defined efficacy measures were preformed. In addition, we compared the rates of sustained responses across groups aiming at the identification of a preferable regimen. Intention to treat analysis was used throughout this study, excluding histological response analysis, because of its low outcomes reporting rates.\nEfficacy measures and definitions\nEnd-of-follow-up (sustained) virological and biochemical response rates, and sustained HBeAg clearance and seroconversion rates were used as primary efficacy measures. Histological response, emergence of YMDD mutations, liver-related and all-cause mortality, and treatment safety were used as secondary efficacy measures. Virological response was defined as attainment of undetectable (or below 400 copies\/mL) levels of hepatitis B virus DNA, as determined by polymerase chain reaction, which was previously found to be the most accurate measure of virological response monitoring (2). Biochemical response was defined as normalization of alanine aminotransferase levels, HBeAg clearance as HBeAg disappearance and seroconversion as HBeAg antibodies appearance. Histological response was defined as a two-point reduction or increase in the histologic activity index score, signifying histological improvement and worsening respectively. Treatment safety was assessed using the occurrence rate of adverse effects necessitating treatment discontinuation.\nStudy quality and homogeneity\nThe included studies methodological quality was assessed using the Jadad quality scale (15), an established composite score evaluating randomization, concealment and reporting of patient withdrawal and dropout rates, with scores \u22653 signifying high-quality studies. Heterogeneity was assessed for each analysis.\nStatistical analysis\nQuantitative meta-analyses were performed to assess differences between monotherapy and combination groups. Statistical analysis was performed and the Forest plots were generated using the comprehensive meta analysis\u00ae software application, Version 2.0 (Biostat, Englewood, NJ, USA). The odds ratios (OR) were calculated along with their, respective, 95% confidence intervals (CI) and presented for each individual study as well as interferon type and across all studies. Subgroup analyses were presented using OR and their corresponding 95% CI. Heterogeneity was assessed for each of the meta-analyses by means of Q-statistics and their corresponding P-values.\nResults\nStudy selection and characteristics\nThe literature search yielded 13 studies. Five studies were excluded on account of the following: (i) examining the nonadult population (n=1); and (ii) not examining or reporting the sustained response rates (n=4). The eight remaining trials, involving 1321 patients, were included. Six trials used conventional interferon-\u03b1 (n=503) (16\u201321) and two trials used pegylated interferon-\u03b1 (n=808) (22, 23). Two of the CON group trials exclusively studied treatment-na\u00efve patients (16, 19), whereas the others studied a mixture of treatment-na\u00efve and previously treated patients (17, 18, 20\u201323). The majority of patients were treated for a period of 1 year (n=6) (16\u201323), with some treated for 6 months (n=2) (17, 18). Sustained response rates were obtained at 6 months following treatment completion in most studies (n=6) (17, 19\u201323), and at 40 or 54 weeks following treatment completion in some (n=2) (16, 18). The studies were of heterogeneous methodological quality (Jadad scores of 2\u20135). All studies were published as full publications, with six studies published in English (16\u201319, 22, 23) and two in Chinese (20, 21). One study used sequential therapy (18), six used concurrent therapy (16, 17, 19,21\u201323) and one used both (20) (see Tables 1 and 2).\nTable 2\nPatient selection criteria of studies included in the meta-analysis\nStudy\nInclusion criteria\nExclusion criteria\nAyaz (2006)\n1. HBsAg positive for >6 m and anti-HBeAg and HBsAg negative 2. Presence of HBV DNA 3. Evidence of inflammation on biopsy within 6 m of enrolment and ALT>1.5 NL\n1. Previous treatment with INF, antiviral or immunosuppressive agents 2. HIV, hepatitis C or D 3. Other aetiologies of liver disease, alcohol intake >40 g\/day, decompensated liver disease or cancer 4. No informed consent 5. Pregnancy 6. Any contraindications for INF use 7. Leucocytes, neutrophil or platelet count of <2500, <1000 and <100 000\/mL, respectively, or haemoglobin <10 g\/dL\nSong (2004)\n1. 19\u201365 years old 2. HBsAg positive for >6 m and HBeAg positive 3. HBV DNA>500 000 copies\/mL 4. Evidence of inflammation by 2 NL <ALT <500\nNot reported\nDeng (2003)\n1. 15\u201360 years old 2. HBeAg and HBV DNA positive for >6 m 3. HBV DNA>103 000 copies\/mL 4. Evidence of inflammation by ALT>2 NL\n1. Immunosuppressive or antiviral therapy within 6 m 2. Hepatitis of other aetiologies 3. Decompensated liver disease 4. Pregnancy or breast feeding\nYalcin (2002)\n1. 16\u201380 years old 2. HBeAg and HBsAg positive 3. HBV DNA positive 4. Evidence of inflammation by histology and 1.5<ALT<10 NL, on three occasions within 6 m\n1. Previous INF therapy, antiviral or immunosuppressive therapy, or contraindication for INF therapy 2. HIV, hepatitis C or D 3. Decompensated liver disease or carcinoma 4. Alcohol consumption >40 g\/day or other liver disease causes 5. Pregnancy 6. Leucocytes <2500\/mm3, neutrophils <1000\/mm3, platelets <100 000\/mm3, or haemoglobin <10g\/dL 7. Unable to obtain consent\nCindoruk (2002)\n1. Adults 2. HBeAg positive 3. HBV DNA positive 4. Evidence of inflammation by histology and by abnormal ALT levels for >6 m\n1. Previous INF therapy 2. HIV, hepatitis C or D 3. Decompensated liver disease 4. Diabetes, autoimmune, or other psychiatric or serious medical illness 5. High alcohol intake or current drug abuse 6. Pregnancy\nSchalm (2000)\n1. 16\u201370 years old 2. HBsAg and HBeAg positive at screening and at >6 and >3 m prior respectively 3. HBV DNA>500 000 copies\/mL 4. Evidence of inflammation by histology or persistently elevated ALT for >3 m\n1. Contraindication to or previous INF therapy, or antiviral therapy within 6 m 2. HIV, hepatitis C or D 3. Decompensated liver disease 4. Liver disease of other aetiology\nLau (2005)\n1. Adults 2. HBsAg positive for >6 m and HBeAg positive 3. HBV DNA>500 000 copies\/mL 4. Evidence of inflammation on biopsy and 1<ALT<10 NL\n1. Treatment within 6 m 2. HIV, hepatitis C or D 3. Decompensated liver disease 4. Serious medical or psychiatric illness 5. Alcohol or drug use within 1 y 4. Neutrophils <1500 g\/dL, platelets <90 000\/mm3, or creatinine >1.5 NL\nJanssen (2005)\n1. >16 years old 2. HBsAg positive for >6 m and HBeAg positive on two occasions within 8 w of randomization 3. Evidence of inflammation by two measurements of ALT>2 NL within 8 w of randomization\n1. Antiviral or immunosuppressive therapy within 6 m 2. HIV, hepatitis C or D 3. Advanced liver disease or carcinoma 4. Serious medical or psychiatric illness, or uncontrolled thyroid disease 5. Substance abuse within 2 y 6. Pregnancy or inadequate contraception 7. Leucocytes <3000\/mm3, neutrophils <1800\/mm3, or platelets <100 000\/mm3\nALT, alanine aminotransferase; HBV, hepatitis B virus; HBsAg, hepatitis B surface antigen; INF, interferon; m, months; NEG, HBeAg negative; NL, upper limit of normal; POS, HBeAg positive; w, weeks; y, years.\nTable 1\nCharacteristics of studies included in the meta-analysis\nStudy\nn\nStudy design\nJadad score\nTherapy period\nFollow-up period\nTherapy regimen\nConventional interferon-\u03b1\n\u2003Ayaz (2006)\n\u200268\nRCT\n2\n12 m\n\u20026 m\nINF-\u03b1-2a 9 MU \u00d7 3\/w with or without LMV 100 mg\/day\n\u2003Song (2004)\n\u200290\nRCT\n2\n12 m\n\u20026 m\nINF-\u03b1 3 MU \u00d7 3\/w with or without LMV 100 mg\/day\n\u2003Deng (2003)\n\u200262\nRCT\n2\n48 w\n24 w\nINF-\u03b1-1b 5 MU \u00d7 3\/w with or without LMV 100 mg\/day\n\u2003Yalcin (2002)\n\u200249\nRCT\n2\n52 w\n52 w\nINF-\u03b1-2b 10 MU \u00d7 3\/w with or without LMV 100 mg\/day\n\u2003Cindoruk (2002)\n100\nRCT\n2\n6 m\n\u20026 m\nINF-\u03b1 9 MU \u00d7 3\/w with or without LMV 100 mg\/day\n\u2003Schalm (2000)\n144\nRCT, DB\n4\n24 w\n40 w\nINF-\u03b1 10 MU \u00d7 3\/w with or without LMV 100 mg\/day\nPegylated interferon-\u03b1\n\u2003Lau (2005)\n542\nRCT, DB\n5\n48 w\n24 w\nPegINF-\u03b1-2a 180 \u03bcg \u00d7 1\/w with or without LMV 100 mg\/day\n\u2003Janssen (2005)\n266\nRCT, DB\n4\n52 w\n26 w\nPegINF-\u03b1-2b 100 \u03bcg \u00d7 1\/w with or without LMV 100 mg\/day\nDB, double blind; INF, conventional interferon; LMV, lamivudine; m, months; PegINF, pegylated interferon; RCT, randomized controlled; w, weeks.\nSustained virological response\nGreater sustained virological response rates were observed for patients given combination as compared with monotherapy in the CON group [61.1 vs. 35.4%, OR=11.7, 95% CI (7.8\u201317.6), P<0.0001], and overall [28.9 vs. 18.5%, OR=2.1, 95% CI (1.3\u20133.3), P=0.002], although not in the PEG group [12.2 vs. 11.8%, OR=1.1, 95% CI (0.5\u20132.3), P=0.8]. Heterogeneity was assessed and not found to be a concern (Q=3.5, P=0.06) (see Fig. 1).\nFig. 1\nSustained virological response. CON, conventional interferon monotherapy vs. its combination with lamivudine; PEG, pegylated interferon monotherapy vs. its combination with lamivudine. *Concurrent and sequential administration.\nSustained biochemical response\nGreater sustained biochemical response rates were observed for patients given combination as compared with monotherapy in the CON group [46.2 vs. 34.0%, OR=1.8, 95% CI (1.2\u20132.7), P=0.007], although not in the PEG group [37.9 vs. 38.1%, OR=1.0, 95% CI (0.7\u20131.3), P=0.94], or overall [36.1 vs. 36.7%, OR=1.2, 95% CI (0.9\u20131.5), P=0.15]. Heterogeneity was assessed and not found to be a concern (Q=3.3, P=0.07) (see Fig. 2).\nFig. 2\nSustained biochemical response. CON, conventional interferon monotherapy vs. its combination with lamivudine; PEG, pegylated interferon monotherapy vs. its combination with lamivudine.\nSustained hepatitis B e antigen clearance\nNo significant differences in sustained HBeAg clearance rates were observed between patients given combination and monotherapy in the CON group [33.5 vs. 24.0%, OR=1.6, 95% CI (0.9\u20132.7), P=0.09], PEG group [30.6 vs. 34.4%, OR=0.8, 95% CI (0.6\u20131.1), P=0.26] and overall [31.5 vs. 31.9%, OR=1.0, 95% CI (0.7\u20131.3), P=0.88]. Heterogeneity was assessed and not found to be a concern (Q=2.6, P=0.11) (see Fig. 3).\nFig. 3\nSustained hepatitis B e antigen (HBeAg) clearance. CON, conventional interferon monotherapy vs. its combination with lamivudine; PEG, pegylated interferon monotherapy vs. its combination with lamivudine. *Concurrent and sequential administration.\nSustained seroconversion\nGreater sustained seroconversion rates were observed for patients given combination as compared with monotherapy in the CON group [30.0 vs. 18.9%, OR=1.8, 95% CI (1.1\u20132.8), P=0.01], although not in the PEG group [27.9 vs. 31.0%, OR=0.9, 95% CI (0.6\u20131.2), P=0.34], or overall [28.7 vs. 27.1%, OR=1.1, 95% CI (0.8\u20131.4), P=0.59]. Heterogeneity was assessed and not found to be a concern (Q=3.5, P=0.06) (see Fig. 4).\nFig. 4\nSustained seroconversion. CON, conventional interferon monotherapy vs. its combination with lamivudine; PEG, pegylated interferon monotherapy vs. its combination with lamivudine. *Concurrent and sequential administration.\nHistological response\nGreater histological improvement rates were observed for patients given combination as compared with monotherapy in the CON group [83.8 vs. 26.6%, respectively, n=1, OR=14.3, 95% CI (3.3\u201361.3), P<0.001], although not in the PEG group [48.1 vs. 53.4%, respectively, n=1, OR=0.8, 95% CI (0.4\u20131.7), P=0.70], or overall [61.4 vs. 47.9%, respectively, n=2, OR=1.7, 95% CI (0.9\u20133.3), P=0.11]. No significant differences in histological worsening rates were observed between patients given combination and monotherapy in the CON group [0 vs. 6.7%, respectively, n=1, OR=0.23, 95% CI (0.02\u20133.3), P=0.16], PEG group [9.6 vs. 10.3%, respectively, n=1, OR=0.9, 95% CI (0.3\u20133.1), P=0.9] and overall [6.0 vs. 9.6%, respectively, n=2, OR=0.6, 95% CI (0.2\u20131.9), P=0.55].\nAll-cause and liver-related mortality\nThere were no reported deaths of any aetiology for either group (0% for both).\nSafety\nNo significant differences in safety rates were observed between patients given combination and monotherapy in the CON group [1.6 vs. 0.9%, OR=1.7, 95% CI (0.3\u20139.0), P=0.55, n=6], PEG group [6.0 vs. 4.2%, OR=1.7, 95% CI (0.9\u20133.2), P=0.12, n=2] and overall [4.1 vs. 2.7.%, OR=1.7, 95% CI (0.9\u20133.1), P=0.10]. Heterogeneity was assessed and not found to be a concern (Q=0.001, P=0.98).\nYMDD mutation emergence\nGreater YMDD mutation emergence rates were observed for patients given combination as compared with monotherapy in the PEG group [4.0 vs. 0%, OR=18.1, 95% CI (2.4\u2013136.6), P=0.005, n=2] and overall [4.5 vs. 0%, OR=14.8, 95% CI (2.8\u201377.6), P=0.001], although not in the CON group [5.9 vs. 0%, OR=9.9, 95% CI (0.5\u2013177.1), P=0.12, n=1]. Heterogeneity was assessed and not found to be a concern (Q=0.11, P=0.74).\nConventional interferon combination therapy vs. pegylated interferon monotherapy\nExcluding virological response [61.1 vs. 11.8%, OR=11.7, 95% CI (7.8\u201317.6), P<0.0001], no significant differences in rates of biochemical response [46.2 vs. 38.1%, OR=1.4, 95% CI (1.0\u20131.9), P=0.052], HBeAg clearance [33.5 vs. 34.9%, OR=0.9, 95% CI (0.6\u20131.4), P=0.77] or seroconversion [30.0 vs. 31.4%, OR=0.9, 95% CI (0.7\u20131.3), P=0.73] were observed between patients given combination therapy in the CON group and those given monotherapy in the PEG group. Significantly greater virological response rates were observed with monotherapy in the CON as compared with the PEG group [35.4 vs. 11.8%, OR=4.1, 95% CI (2.6\u20136.4), P<0.0001].\nDiscussion\nThe suboptimal outcomes of current hepatitis B therapies have prompted the notion of their use in combination to achieve a synergistic effect and decreased mutagenicity (2). Furthermore, it has been suggested that the enhanced efficacy of the combination will allow for the dose reduction of its components, thus decreasing the risk of potential adverse effects (2). In our study, we explored this notion in the subset of HBeAg-positive patients. Our study is the first to examine the combination of interferon and lamivudine for chronic hepatitis B treatment, pooling data from all pertinent randomized-controlled trials into meta-analysis. This analysis will aid in achieving evidence-based conclusions on the matter, resolving the controversy in its regard and directing future investigational efforts.\nIn our analysis, we found pegylated interferon monotherapy to be of comparable efficacy to its combination with lamivudine, providing similar rates of sustained virological and biochemical responses, and HBeAg clearance and seroconversion (P=0.66, 0.94, 0.26 and 0.34 respectively). Furthermore, while the pegylated interferon trials predominantly involved treatment-na\u00efve patients, analysis of previously treated patients within one of those studies yielded similar outcomes (24). In contrast, the addition of lamivudine to conventional interferon resulted in superior sustained virological, biochemical and seroconversion rates (P<0.001, P=0.007, 0.01 and 0.09 respectively), similarly observed with sequential and concurrent administration (20). A similar trend was observed with HBeAg clearance rates, although the sample size was insufficient to detect this effect (P=0.09). As with pegylated interferon, treatment-na\u00efve patients comprised the majority of the studied population and to a greater extent. Nonetheless, a controlled, nonrandomized trial of previously treated patients reported similar outcomes (25). These outcomes are corroborated by those of our histological analysis (P<0.001 and P=0.70 for histological improvement in the CON and PEG groups respectively) and by those of others (26). Importantly, our analysis provides an explanation to the discordance between the combinations' effectiveness with conventional and not with pegylated interferon, with lamivudine-induced mutagenicity suppressed with the former, while not with the latter (P=0.12, and 0.05 respectively).\nAccordingly, two possible regimens emerged from our analysis: pegylated interferon monotherapy, and conventional interferon and lamivudine combination therapy. A comparison between the two found them to be of comparable efficacy (P>0.05), with the exception of virological response (P<0.001). That said, it is the authors opinion that this combinations' favourable virological response should not prompt its use as the regimen of choice, as a greater portion of treatment-na\u00efve and thus easier to treat patients, comprised the CON as compared with the PEG group, with three CON group studies exclusively examining this patient population (16, 17, 19). Our hypothesis is further supported by the superior virological outcomes of conventional as compared with pegylated interferon monotherapy (P<0.0001), which is in conflict with current knowledge (27), and is easily explained by this hypothesis. Accordingly, we suggest that in comparable populations, pegylated interferon monotherapy is likely to be similarly or more efficacious than lamivudine and conventional interferon combination therapy. More so, the thrice-weekly injection therapy required with conventional interferon poses a risk of low-patient compliance rates (1\u20133, 28), with the risk further exacerbated by the addition of a second agent. The weekly administration of pegylated interferon monotherapy is likely to alleviate this concern, while carrying similar economic costs (29). Consequently, we conclude that pegylated interferon monotherapy is likely to be the treatment of choice for HBeAg-positive chronic hepatitis B, with this conclusion being supported by others (27). That said, when conventional interferon therapy is considered, particularly in highly compliant patients, its combination with lamivudine should be entertained.\nSimilarly, studies examining the HBeAg-negative hepatitis B population did not find the addition of lamivudine to pegylated (30), or conventional (31), interferon to be advantageous. In addition, while the superiority of the combination over lamivudine monotherapy was suggested in previous studies, this effect is likely to represent interferon's greater inherent efficacy as compared with lamivudine, rather than the enhanced properties of the combination, as demonstrated in those very studies (22, 30).\nOur study contains several limitations. Firstly, our use of intention to treat analysis, the methodological heterogeneity of the included studies, and the heterogeneity of their treatment and follow-up protocols, may have introduced some inaccuracies in our analysis. Notably, while the PEG group comprised large, carefully planned, well-executed studies, the CON group involved smaller, lower quality ones, thus weakening our conclusions in its regard. Secondly, the absence of adequate controls precluded the authors from studying the subsets of treatment-na\u00efve and previously treated populations. Those concerns, however, were alleviated by the low patient lost for follow-up rates, the lack of statistically significant heterogeneity across studies, the beneficial effects of the combination with conventional interferon across the measured indicators and the agreement between our conclusions and those of other studies.\nWhile the focus of our study was lamivudine and interferon combination therapy, a plethora of other combinations have been explored as well. Among those studied were combinations of interferon and various antiviral agents (32, 33), interleukin-12 (34) and prednisone (35). All yielded disappointing results. Additionally, studies investigating various antiviral combinations resulted in conflicting outcomes (36\u201340). Those results indicate the need for further study, as the goal of a safe and efficacious therapy is yet to be attained.\nConclusion\nPegylated interferon-\u03b1 monotherapy is the treatment of choice for HBeAg-positive chronic hepatitis B, with no added benefit with lamivudine addition. However, when conventional interferon therapy is considered, its combination with lamivudine should be entertained.","keyphrases":["interferon","lamivudine","hepatitis b","meta-analysis","therapy","combination"],"prmu":["P","P","P","P","P","P"]}
{"id":"J_Behav_Med-4-1-2413087","title":"Parental problem drinking, parenting, and adolescent alcohol use\n","text":"The present study examined whether parental problem drinking affected parenting (i.e., behavioral control, support, rule-setting, alcohol-specific behavioral control), and whether parental problem drinking and parenting affected subsequent adolescent alcohol use over time. A total of 428 families, consisting of both parents and two adolescents (mean age 13.4 and 15.2 years at Time 1) participated in a three-wave longitudinal study with annual waves. A series of path analyses were conducted using a structural equation modeling program (Mplus). Results demonstrated that, unexpectedly, parental problem drinking was in general not associated with parenting. For the younger adolescents, higher levels of both parenting and parental problem drinking were related to lower engagement in drinking over time. This implies that shared environment factors (parenting and modeling effects) influence the development of alcohol use in young adolescents. When adolescents grow older, and move out of the initiation phase, their drinking behavior may be more affected by other factors, such as genetic susceptibility, and peer drinking.\nIntroduction\nTime trends in Dutch epidemiological research show a significant increase in frequency and intensity of alcohol consumption among 12\u201315\u00a0year olds (Poelen et\u00a0al. 2005). Alarming high numbers of Dutch adolescents (75%) also report problem drinking behaviors such as binge drinking (consuming more than 5 amounts of alcohol on one occasion), when compared to their American counterparts (19%; Newes-Adeyi et\u00a0al. 2005; Van Dorsselaer et\u00a0al. 2007). In addition, previous studies show that high levels of alcohol-related problems such as social consequences of alcohol use (e.g., family problems) and dependence symptoms (e.g., loss of control) occur frequently in Western societies, with approximately 10% of both American and Dutch populations reporting 3 or more alcohol-related problems (Cornel et\u00a0al. 1994; NIAAA 1997; Van Dijck and Knibbe 2005; Wallitzer and Connors 1999).\nParental problem drinking increases risk for alcohol use in children (e.g., Chassin et\u00a0al. 1996; Hawkins et\u00a0al. 1992; Sher et\u00a0al. 1991). Children of alcoholics are not only at a higher risk for early alcohol initiation (Hill et\u00a0al. 2000), they also show a greater increase in alcohol consumption over time than adolescents without alcoholic parents (Chassin and Barrera 1993). In addition, children with a family history of alcoholism show more escalation of alcohol use (Lieb et\u00a0al. 2002), and more often develop alcohol disorders and dependence (Hill et\u00a0al. 2000) than children without a family history of alcoholic parents.\nIn an attempt to explain these associations, social theorists suggested a modeling effect (Bandura 1977) that causes youngsters to imitate their parents. Others have proposed that parental substance abuse may impair parenting (Sher 1991; Van der Vorst et\u00a0al. 2006; Van Zundert et\u00a0al. 2006), which subsequently may affect adolescent alcohol consumption. As Mayes and Truman (2002) pointed out, personality characteristics, disabilities, or impairments accompanying an addiction may affect the ability to raise a child. In addition, substance use alters the state of consciousness, memory, affect, and impulse control, each of which may impair the adult\u2019s parenting capacities. Indeed, empirical studies have shown that children of alcoholic parents receive less discipline (King and Chassin 2004) and less emotional support from their parents (Rutherford et\u00a0al. 1997). In addition, Chassin et\u00a0al. (1993) found that parental alcoholism decreased the amount of parental monitoring. This is all the more problematic, since discipline and rule setting, in turn, reduce the likelihood of youngsters\u2019 drunkenness (Engels and Van der Vorst 2003), and more parental monitoring is related to less heavy drinking in adolescents (Kerr and Stattin 2000; Van der Vorst et\u00a0al. 2006). In addition, parental support appears to prevent early onset of alcohol use, as well as frequent and heavy alcohol use among adolescents (Barnes et\u00a0al. 1994). Thus, numerous cross-sectional studies have demonstrated associations between parental alcohol use, parenting, and adolescent alcohol consumption (e.g., Chassin et\u00a0al. 1993; Kerr and Stattin 2000). However, with the exception of two prospective studies that showed that monitoring by fathers and parental discipline mediated between parental alcoholism and adolescents\u2019 alcohol use (Chassin et\u00a0al. 1996; King and Chassin 2004), longitudinal studies are lacking. In addition, to allow generalization of findings and to examine effects in potentially less severe cases, it is necessary to investigate community-based samples (Russell et\u00a0al. 1990). Accordingly, the central aim of the present study was to longitudinally examine the nature of the relations between parental problem drinking, parenting, and adolescent alcohol use in a three-wave community-based sample.\nAlcohol-specific parenting\nAlthough studies on parenting and adolescent alcohol use have been informative, two important issues have hardly been addressed. First, most studies on the link between parenting and adolescent alcohol use have focused on general parenting. However, alcohol-specific socialization, which refers to the actions parents undertake to discourage or prevent their offspring from drinking (Jackson et\u00a0al. 1999; Van der Vorst et\u00a0al. 2005), has received less attention in relation to parental drinking and adolescent alcohol use. Wood et al. (2004) found that late adolescents drank less alcohol when their parents disapproved of drinking. In addition, imposing strict rules prevented youngsters from heavy drinking (Jackson et\u00a0al. 1999; Van der Vorst et\u00a0al. 2005; Yu 2003). However, whether parental problem drinking affects alcohol-specific parenting has not yet been examined. From studies on smoking we know that parents who smoke are less frequently engaged in anti-smoking socialization practices than parents who do not smoke (Harakeh et\u00a0al. 2005). A similar process might be at work regarding alcohol-specific socialization, suggesting that parents with alcohol problems may engage less frequently in alcohol-specific socialization, and as such provide fewer alcohol-specific rules, are more permissive towards alcohol use and exert less alcohol-specific control.\nSecond, it is crucial to acknowledge that the association between parenting and adolescent problem behavior may be bidirectional: Parents do not only influence their children, but children\u2019s behavior also exerts an effect on parents. Indeed, recent longitudinal studies showed a bidirectional relation between parenting and adolescent substance use. Adolescent drinking, smoking or deviant behavior decreased the level of parental monitoring and rule setting (Huver et\u00a0al. 2006; Stice and Barrera 1995; Van der Vorst et\u00a0al. 2006). This implies that when these child effects are not taken into account, this may lead to an overestimation of parental influences (Kerr and Stattin 2003; Van der Vorst et\u00a0al. 2006).\nCurrent study and expectancies\nWe longitudinally investigated the direct effect of parental problem drinking on adolescent alcohol use, the role of alcohol-specific and general parenting practices in this relationship, and the reciprocal effects of adolescent alcohol use on parenting (see Fig.\u00a01). It was expected that parental problem drinking would have a direct positive effect on adolescent alcohol use, with more parental alcohol-related problems leading to more adolescent alcohol use. In addition, an indirect relationship was expected via parenting; more specifically, higher levels of parental problem drinking were thought to have a negative effect on both general and alcohol-specific parenting practices, which in turn would lead to more adolescent alcohol use. Moreover, the drinking behaviors of the adolescents were expected to influence parenting, with more adolescent drinking resulting in less parental discipline and monitoring.\nFig\u00a01Longitudinal Model of Parental Problem Drinking, Parenting, and Adolescent Alcohol Use\nMethods\nParticipants and recruitment\nThe data were derived from an ongoing Dutch longitudinal survey called \u2018Family and Health\u2019, which examines different socialization processes in relation to various health behaviors in adolescence (see Harakeh et\u00a0al. 2005; Van der Vorst et\u00a0al. 2005). A total of 428 Dutch families, consisting of mother, father, and two adolescent children, participated in our study in the first wave (2002\u20132003). Families were included when the parents were married or living together, and when all family members were biologically related. Families with twins, or with mentally or physically disabled offspring were excluded. Numbers of drop-outs were extremely low in the second (2003\u20132004) and third wave (2004\u20132005), with 416 (97%) and 404 (94%) participating families, respectively.\nThe majority of the families were of Dutch origin (>95%). The mean age of the participants at Time 1 was 15.2\u00a0years (SD\u00a0=\u00a0.60) for the older adolescents, 13.4\u00a0years (SD\u00a0=\u00a0.50) for the younger adolescents, 46.2\u00a0years (SD\u00a0=\u00a04.00) for the fathers, and 43.8\u00a0years (SD\u00a0=\u00a03.57) for the mothers. Of the older adolescents, 47% were girls, compared to 52% in the younger group. Concerning educational level, an equal distribution was realized, with about one third of the adolescents following low education, one third following intermediate general education, and one third following the highest level of secondary school. The different levels of the Dutch secondary school system are comparable with the different tracks within a middle class public high school in the USA, although they may not be completely interchangeable. In our sample, when compared to national Dutch figures, the intermediate general education is slightly underrepresented, while the low and high levels are slightly overrepresented (CBS 2007).\nProcedure\nThe families were visited at home by a trained interviewer. In his or her presence all four family members individually filled out an extensive questionnaire, which took about 2\u00a0h to complete. The participants were not allowed to consult each other or to discuss the answers. When all family members had completed the questionnaire, each family received 30\u00a0\u20ac (39\u00a0$). In addition, after completion of the first three waves of the project, 5 traveler cheques of 1,000\u00a0\u20ac (1,300\u00a0$) each were raffled among all participating families. Approval was obtained from the Central Committee on Research Involving Human Subjects on collecting the data.\nMeasure\nSelf-reports were used to measure parental problem drinking and adolescent alcohol use. The four parenting practices were based on adolescents\u2019 reports, reflecting how they perceived their parents\u2019 behaviors. The questions regarding the parenting variables were asked in such a way that the adolescents were able to discriminate between the parenting practices of their mothers and fathers.\nProblem drinking\nTo measure the severity of fathers\u2019 and mothers\u2019 alcohol-related problems, both parents completed the problem drinking list of Cornel et\u00a0al. (1994). The original scale was based on three commonly used instruments to measure problem drinking: CAGE (Cut down, Annoyed, Guilty, Eye-opener, CAGE is an acronym formed by taking the first letter of key words from each of the following questions; Mayfield et\u00a0al. 1974), Short Michigan Alcohol Screening Test (SMAST; Selzer et\u00a0al. 1975), and a shortened version of the Self-Administered Alcohol Screening Test (SAAST; Davis et\u00a0al. 1987). Seriousness of problem drinking was developed as a Rasch scale with items arranged in order of increasing severity. The more severe the items, the less frequently they are scored positively. Since all requirements of the Rasch model were met, the items form a reliable and unidimensional scale (Cornel et\u00a0al. 1994). Examples of items were \u2018Do you ever drink alcohol to forget your concerns?\u2019 (item 2) and \u2018Have you ever lost your job because of your drinking\u2019 (item 18). Respondents could respond 0 \u2018no\u2019, or 1, \u2018yes\u2019. Severity of problem drinking was reflected by the aggregated score with a maximum score of 18. Because of the skewness of the summed variable\u2019s distribution, scores were categorized into 3 meaningful groups: 1\u00a0=\u00a0never had problems due to alcohol; 2\u00a0=\u00a0has had problems due to alcohol a couple of times, 3\u00a0=\u00a0problem drinkers (see Cornel et\u00a0al. 1994).\nGeneral parenting\nTo measure parental behavioral control, we used a Dutch translation of the scale developed by Kerr and Stattin (2000). The scale consisted of 5 items with response categories ranging from 1 \u2018no, never\u2019 to 5 \u2018yes, always\u2019. Examples of items were: \u2018Do you need to have your mother\u2019s permission to stay out late on a weekday evening?\u2019 and \u2018Before you go out on a Saturday night, does your father require you to tell them where you are going and with whom?\u2019. Internal consistencies as measured with Cronbach\u2019s alphas ranged from .71 to .90 for the reports of both adolescents, about their mothers and fathers over the three waves.\nTo measure parental support, we used the Relationship Support Inventory (RSI; Scholte et\u00a0al. 2001) tapping several aspects of emotional and instrumental support. Examples of items were \u2018My mother shows me that she loves me\u2019 and \u2018My father supports me in what I do\u2019. The adolescents had to answer 12 items on a scale from 1 \u2018absolutely untrue\u2019 to 5 \u2018absolutely true\u2019. The amount of support was the mean score on 12 items. Cronbach\u2019s alpha coefficients were between .76 and .88 across the three waves.\nAlcohol-specific parenting\nVan der Vorst et\u00a0al. (2005) developed a 10-item scale to measure the degree to which parents permit their children to consume alcohol. Examples of items were: \u2018I am allowed to drink alcoholic consumptions when my mother\/father is at home\u2019 and \u2018I am allowed to drink alcohol on weekdays\u2019. Participants had to respond on a 5-point scale that ranged from 1 \u2018completely applicable\u2019 to 5 \u2018not applicable at all\u2019. The internal consistency was high, with Cronbach\u2019s alphas between .89 and .92 over the three waves.\nIn addition, the general behavioral control scale of Kerr and Stattin (2000) was adapted to measure behavioral control aimed at affecting adolescents\u2019 alcohol consumption. Examples of the 5 items were \u2018Do you need your mother\u2019s permission to drink alcohol on weekdays?\u2019 and \u2018Does your mother want to know whether your friends drink alcohol?\u2019. As in the original scale, the response categories ranged from 1 \u2018never\u2019 to 5 \u2018always\u2019. Cronbach\u2019s alpha coefficients were between .74 and .88 across the three waves.\nAdolescent alcohol use\nIntensity of drinking was assessed by questions that asked about the number of glasses consumed in the previous week, during weekdays and weekends, both outside and inside the house (Engels et\u00a0al. 1999). The aggregated score on these four questions was used as an indication of the adolescents\u2019 intensity of alcohol use (Van der Vorst et\u00a0al. 2005). Because of the skewness in the distribution of this variable, total scores were categorized into 7 groups (0\u00a0=\u00a00\u00a0glasses, 1\u00a0=\u00a01\u20132\u00a0glasses, 2\u00a0=\u00a03\u20135\u00a0glasses, 3\u00a0=\u00a06\u201310\u00a0glasses, 4\u00a0=\u00a011\u201320\u00a0glasses, 5\u00a0=\u00a021\u201330\u00a0glasses, 6\u00a0=\u00a031\u00a0glasses and above).\nStrategy of analyses\nFor the descriptive part of the analyses we applied t tests, Pearson correlations and general linear modeling with repeated measures (the latter to test changes over time in alcohol-related problems and alcohol use). We performed cross-lagged path analyses (see Fig.\u00a01), using version 4.1 of the Mplus statistical package (Muth\u00e9n and Muth\u00e9n 1998\u20132006), to test (a) to which degree parental alcohol-related problems, parental practices and alcohol use of adolescents were stable over time, (b) whether parental problem drinking was related to parental practices and alcohol use of the adolescent over time and (c) whether parental practices and alcohol use of the adolescent were cross-related over time (Finkel 1995).\nCross relations over time allow to test causal predominance: Are specific parenting practices the \u2018cause\u2019 of adolescent alcohol use, or does adolescent alcohol use provoke specific parenting practices (Byrne 1998)? Structural regression models are generally somewhat more sophisticated than the path models used in our study because they correct for measurement error (Kline 1998, p. 211). This controlling for error variance by means of latent variables that are measured by multiple manifest indicators plus their error variance, however, also increases the number of parameters to be estimated. In addition, more complex models, i.e., models with more parameters, require larger sample sizes than do more parsimonious models in order for the estimates to be comparably stable (Kline 1998, p. 111). Kline (1998) recommends a parameter\u2014subjects ratio of 1:10. As such, we used path models in which one manifest parameter represented all the individual items of one scale by means of the mean or sum score. The model depicted in Fig.\u00a01 was tested for each of the four parenting variables separately. A total of 4 (parenting variables)\u00a0\u00d7\u00a02 (fathers and mothers)\u00a0=\u00a08 models were tested. The variables at T1 and the disturbance terms of the variables at T2 and T3 were free to correlate. Because adolescent alcohol consumption and parental problem drinking were relatively skewed and the measurement level was ordered more categorical (ordinal) than interval, maximum likelihood estimation methods (demanding multivariate normal distributed variables) were less suited. We used the weighted least square method with adjusted mean- and variance chi-square (WLSMV) estimator, an estimation method specifically developed for ordered categorical dependent variables (Muth\u00e9n and Muth\u00e9n 1998\u20132006). To test model fit, standard chi-square tests as well as the number of degrees of freedom (df) were replaced by robust chi-square tests (mean- and variance-adjusted chi-squares) and estimates of df (Muth\u00e9n and Muth\u00e9n 1998\u20132004, pp. 19\u201320). The latter estimates are dependent on sample information and this explains why df with identical models can vary across different groups.\nTogether with the robust chi-square tests we used two fit measures: the Root Mean Square Error of Approximation (RMSEA; Byrne 1998; Steiger and Lind 1980), and the Comparative Fit Index (CFI) of Bentler (Bentler 1990). RMSEA is utilized to assess approximate fit preferably with values less than or equal to .05, but values between .05 and .08 are indicative of fair fit (Browne and Cudeck 1993). CFI is a comparative fit index, values above .95 are preferred (Kaplan 2000), but should not be lower than .90 (Kline 1998, see also Hu and Bentler 1999, and, for commentary on existing guidelines Marsh et al. 2004). Mplus has several possibilities to handle missing values depending on the estimation method used. In our case (using the WLSMV-estimator) all available information in the data was used by means of pair-wise information of each combination of two variables.\nResults\nDescriptives on alcohol consumption and problem drinking\nTable\u00a01 shows the means, standard deviations, and percentages of parental problem drinking. Fathers reported higher levels of problem drinking than mothers at all three waves, as was tested with separate t tests. (T1: t(424)\u00a0=\u00a08.11, p\u00a0<\u00a0.001; T2: t(426)\u00a0=\u00a08.13, p\u00a0<\u00a0.001; T3: t(424)\u00a0=\u00a07.93, p\u00a0<\u00a0.001). With general linear modeling repeated measures we tested whether maternal and paternal problem drinking differed over time (within factor). Both paternal and maternal problem drinking showed significant differences over time (for fathers: F(2, 421)\u00a0=\u00a023.18, p\u00a0<\u00a0.001, partial eta squared (PES)\u00a0=\u00a0.10, and for mothers: F(2, 424)\u00a0=\u00a059.10, p\u00a0<\u00a0.001, and PES\u00a0=\u00a0.22).1 Subsequently carried out repeated contrasts revealed significant differences over time only from T1 to T2, for both parents (p\u00a0<\u00a0.001), but not from T2 to T3. At T1, the older adolescents consumed on average 4.36 glasses in the past week (SD\u00a0=\u00a06.81; T2: M\u00a0=\u00a07.78, SD\u00a0=\u00a010.86; T3: M\u00a0=\u00a09.75, SD\u00a0=\u00a012.35), compared to 1.23 glasses (SD\u00a0=\u00a03.41) consumed in the past week by the younger siblings (T2: M\u00a0=\u00a03.70, SD\u00a0=\u00a08.99; T3: M\u00a0=\u00a06.22, SD\u00a0=\u00a010.32). Older adolescents reported significantly higher levels of alcohol consumption than younger adolescents at all three waves (T1: t(417)\u00a0=\u00a09.30, p\u00a0<\u00a0.001; T2: t(414)\u00a0=\u00a06.85, p\u00a0<\u00a0.001; T3: t(405)\u00a0=\u00a05.09, p\u00a0<\u00a0.001). Repeated measures showed a significant increase in alcohol consumption over time for both adolescents, for T1\u2013T2, and T2\u2013T3 (for older adolescents: F(2, 394)\u00a0=\u00a034.15, p\u00a0<\u00a0.001, PES\u00a0=\u00a0.15, and for younger adolescents: F(2, 414)\u00a0=\u00a042.64, p\u00a0<\u00a0.001, PES\u00a0=\u00a0.17).\nTable\u00a01Means, standard deviations and percentages of parental problem drinking (PD) and adolescent alcohol use (A) at Time 1 (T1), Time 2 (T2), and Time 3 (T3)T1T2T3M*SD%**M*SD%**M*SD%**PD father1.84a2.1819.42.33b1.9925.52.21b2.0022.7PD mother.87a1.575.61.44b1.518.41.42b1.49 9.1A younger adolescent1.22a3.41n.a.3.11b8.35n.a.5.27c9.76n.a.A older adolescent4.37a6.80n.a.7.15b10.62n.a.8.79c12.08n.a.Note: *M represents the mean score calculated from the aggregated scores of all 18 items (maximum score\u00a0=\u00a018) of which the problem drinking scale consists (Cornel and Knibbe 1994). **Percentages of problem drinkers are computed with a cut-off score\u00a0>\u00a03. n.a.\u00a0=\u00a0not applicable. Values for adolescent alcohol use (A) represent the intensity of alcohol use, i.e., the number of glasses of alcohol consumed in the past week. Fathers had significantly more alcohol-related problems than mothers at all three time points, with p\u00a0<\u00a0.01. Older adolescents reported significantly more alcohol than younger adolescents at all three time points. Means in the same row that do not share superscripts (a, b, c) are significantly different (p\u00a0<\u00a0.001)\nCorrelations between cross-sectional and longitudinal variables\nMaternal and paternal problem drinking correlated positively, but marginally with adolescents\u2019 alcohol consumption (.02\u00a0\u2264\u00a0r\u00a0\u2264\u00a0.19). Parental problem drinking correlated negatively with support (\u2212.18\u00a0\u2264\u00a0r\u00a0\u2264\u00a0\u2212.02), and alcohol-specific behavioral control (\u2212.22\u00a0\u2264\u00a0r\u00a0\u2264\u00a0\u2212.01), and positively with permissiveness (.03\u00a0\u2264\u00a0r\u00a0\u2264\u00a0.24), while both positive and negative correlations were found between parental problem drinking and general behavioral control (\u2212.13\u00a0\u2264\u00a0r\u00a0\u2264\u00a0.15). Adolescent alcohol use correlated low to moderately with general behavioral control (\u2212.24\u00a0\u2264\u00a0r\u00a0\u2264\u00a0\u2212.05), support (\u2212.15\u00a0\u2264\u00a0r\u00a0\u2264\u00a0\u2212.02) and alcohol-specific behavioral control (\u2212.29\u00a0\u2264\u00a0r\u00a0\u2264\u00a0\u2212.03), and positively with permissiveness (.18\u00a0\u2264\u00a0r\u00a0\u2264\u00a0.46). General behavioral control correlated positively with support (.11\u00a0\u2264\u00a0r\u00a0\u2264\u00a0.40) and negatively with permissiveness (\u2212.29\u00a0\u2264\u00a0r\u00a0\u2264\u00a0\u2212.02). Moderate correlations existed between the general behavioral control scale and the alcohol-specific behavioral control scale (.19\u00a0\u2264\u00a0r\u00a0\u2264\u00a0.55), indicating that they share the same basis, but can be seen as separate constructs. Correlation tables are available upon request.\nStructural equation models\nAll models showed an acceptable fit (Table\u00a02), with all Comparative Fit Indices (CFI) at least above .90 and all Root Mean Square Errors of Approximation (RMSEAs) below .08.\nTable\u00a02Fit indices for all modelsFatherMotherPMASBCSUPMASBCSUdf2630302627302928\u03c7\u00b236.1358.7376.0541.7847.9256.8078.4942.95p0.090.000.000.030.010.000.000.04CFI0.980.960.930.980.960.960.920.98RMSEA0.030.050.060.040.040.050.060.04Note: PM\u00a0=\u00a0Permissiveness, AS\u00a0=\u00a0Alcohol-specific behavioral control, BC\u00a0=\u00a0Behavioral control, SU\u00a0=\u00a0Support. Each column represents one model with a specific parenting variable, separately for mothers and fathers\nAlcohol-specific parenting: permissiveness and alcohol-specific behavioral control\nStandardized regression weights (\u03b2) of parental problem drinking, permissiveness, alcohol-specific behavioral control, and adolescent alcohol consumption showed a strong stability over time, with values between .36 and .88 (see Table\u00a03).\nTable\u00a03Structural parameters estimates of the alcohol-specific parenting practices, parental problem drinking (PD) and adolescent alcohol use (standardized beta weight)PermissivenessAlcohol-specific behavioral controlFathersMothersFathersMothersStability paths1. PD T1\u2013PD T2.79.76.80.762. PD T2\u2013PD T3.82.88.82.823. Parenting OA T1\u2013Parenting OA T2 .68.67.55.554. Parenting OA T2\u2013Parenting OA T3.73.73.64.645. Alcohol use OA T1\u2013Alcohol use OA T2.46.47.55.546. Alcohol use OA T2\u2013Alcohol OA T3.66.66.67.677. Parenting YA T1\u2013Parenting YA T2.75.76.50.498. Parenting YA T2\u2013Parenting YA T3 .76.76.49.489. Alcohol use YA T1\u2013Alcohol use YA T2.36.37.42.4210. Alcohol use YA T2\u2013Alcohol use YA T3.55.54.59.57Paths from PD11. PD T1\u2013Parenting OA T2.11**.12**.07\u2212.0812. PD T2\u2013Parenting OA T3.05.03\u2212.11**\u2212.0713. PD T1\u2013Alcohol use OA T2.08.16**.09.18***14. PD T2\u2013Alcohol use OA T3.02.02.03.0315. PD T1\u2013Parenting YA T2.07.03.05\u2212.0216. PD T2\u2013Parenting YA T3.05.04\u2212.14**\u2212.15**17. PD T1\u2013Alcohol use YA T2.00\u2212.02.00\u2212.0318. PD T2\u2013Alcohol use YA T3.19***.14**.20***.15**Cross-lagged paths19. Parenting OA T1\u2013Alcohol use OA T2.15**.12*.05.0420. Parenting OA T2\u2013Alcohol use OA T3.02.03\u2212.02\u2212.0721. Alcohol use OA T1\u2013Parenting OA T2\u2212.01.01\u2212.07\u2212.0522. Alcohol use OA T2\u2013Parenting OA T3\u2212.04\u2212.04\u2212.10*\u2212.0923. Parenting YA T1\u2013Alcohol use YA T2.17**.17**\u2212.23***\u2212.24***24. Parenting YA T2\u2013Alcohol use YA T3.04.05.05\u2212.0325. Alcohol use YA T1\u2013Parenting YA T2.01.02\u2212.02\u2212.10*26. Alcohol use YA T2\u2013Parenting YA T3.01.01\u2212.12*\u2212.10*27. Alcohol use OA T1\u2013Alcohol use YA T2.08.09.09.0928. Alcohol use OA T2\u2013Alcohol use YA T3.11*.12*.09.11*Note: PD\u00a0=\u00a0Parental Problem Drinking, OA\u00a0=\u00a0Older Adolescent, YA\u00a0=\u00a0Younger Adolescent. All stability paths are significant at p\u00a0<\u00a0.001. The numbered paths in the table correspond to the arrowed paths depicted in Fig.\u00a01*\u00a0p\u00a0<\u00a0.05, **\u00a0p\u00a0<\u00a0.01, ***\u00a0p\u00a0<\u00a0.001\nRegarding both the older and younger adolescents, no significant associations were found between parental problem drinking and parental permissiveness, with the exception of problem drinking of both fathers and mothers at T1 which was significantly and positively related to permissiveness towards the older adolescent at T2 (for fathers: \u03b2\u00a0=\u00a0.11, p\u00a0<\u00a0.01, for mothers: \u03b2\u00a0=\u00a0.12, p\u00a0<\u00a0.01). More problem drinking of the mother at T1 and T2 was significantly associated with more alcohol use of the older adolescent at T2 and drinking of the younger adolescent at T3, respectively (\u03b2\u00a0=\u00a0.16, p\u00a0<\u00a0.01; \u03b2\u00a0=\u00a0.14, p\u00a0<\u00a0.01). Problem drinking of the father at T2 was significantly and positively associated to alcohol use of the youngest adolescent at T3 (\u03b2\u00a0=\u00a0.19, p\u00a0<\u00a0.001). More parental permissiveness at T1 led to more alcohol use at T2 in both younger and older adolescents (.12\u00a0\u2264\u00a0\u03b2\u00a0\u2264\u00a0.17, p\u00a0<\u00a0.05). This significant relation was not found between T2 and T3. We did not find that parents adapt their levels of permissiveness in response to adolescent alcohol use.\nPaths from parental problem drinking at T2 on alcohol-specific behavioral control at T3 were significant for the father regarding both the older and younger adolescents (respectively \u03b2\u00a0=\u00a0\u2212.11 and \u03b2\u00a0=\u00a0\u2212.14, p\u00a0<\u00a0.01), and for the mother regarding the younger adolescent (\u03b2\u00a0=\u00a0\u2212.15, p\u00a0<\u00a0.01). Problem drinking of the father at T2 related substantially to alcohol use of the younger adolescent at T3 (\u03b2\u00a0=\u00a0.20, p\u00a0<\u00a0.001). Problem drinking of the mothers affected both the older (\u03b2\u00a0=\u00a0.18, p\u00a0<\u00a0.001) and the younger adolescents\u2019 alcohol consumption (\u03b2\u00a0=\u00a0.15, p\u00a0<\u00a0.01). For the younger, but not for the older adolescents, more alcohol-specific behavioral control at T1 was related to lower levels of adolescent alcohol use at T2 (\u03b2\u00a0=\u00a0\u2212.23, p\u00a0<\u00a0.001 for fathers, \u03b2\u00a0=\u00a0\u2212.24, p\u00a0<\u00a0.001 for mothers). Adolescent alcohol use negatively affected alcohol-specific behavioral control of both parents (.10\u00a0\u2264\u00a0\u03b2\u00a0\u2264\u00a0.12, p\u00a0<\u00a0.05)\nGeneral parenting: behavioral control and support\nThe standardized regression weights of parental problem drinking, support, behavioral control, and adolescent alcohol consumption showed a strong stability over time, with values between .42 and .85 (see Table\u00a04).\nTable\u00a04Structural parameters estimates of general parenting practices, parental problem drinking (PD) and adolescent alcohol use (standardized beta weight)Behavioral controlSupportFathersMothersFathersMothersStability paths1. PD T1\u2013PD T2.80.73.81.772. PD T2\u2013PD T3.83.82.85.803. Parenting OA T1\u2013Parenting OA T2 .61.64.76.734. Parenting OA T2\u2013Parenting OA T3.70.70.76.725. Alcohol use OA T1\u2013Alcohol use OA T2.54.53.53.536. Alcohol use OA T2\u2013Alcohol OA T3.66.66.67.677. Parenting YA T1\u2013Parenting YA T2.66.63.70.678. Parenting YA T2\u2013Parenting YA T3 .63.73.69.709. Alcohol use YA T1\u2013Alcohol use YA T2.43.42.43.4210. Alcohol use YA T2\u2013Alcohol use YA T3.58.57.59.57Paths from PD11. PD T1\u2013Parenting OA T2.03\u2212.06.00.0712. PD T2\u2013Parenting OA T3\u2212.05\u2212.04\u2212.06\u2212.0113. PD T1\u2013Alcohol use OA T2.09.18***.10.18***14. PD T2\u2013Alcohol use OA T3.03.00.03.0215. PD T1\u2013Parenting YA T2.06.00.08.0316. PD T2\u2013Parenting YA T3\u2212.04\u2212.12**\u2212.03\u2212.0617. PD T1\u2013Alcohol use YA T2.00.00.00\u2212.0118. PD T2\u2013Alcohol use YA T3.20***.15**.20***.14**Cross-lagged paths19. Parenting OA T1\u2013Alcohol use OA T2.04.03\u2212.04\u2212.0720. Parenting OA T2\u2013Alcohol use OA T3\u2212.11*\u2212.18***\u2212.02\u2212.0221. Alcohol use OA T1\u2013Parenting OA T2\u2212.07\u2212.05\u2212.01\u2212.0222. Alcohol use OA T2\u2013Parenting OA T3\u2212.12**\u2212.14**\u2212.06.0123. Parenting YA T1\u2013Alcohol use YA T2\u2212.15**\u2212.14**\u2212.10*\u2212.15**24. Parenting YA T2\u2013Alcohol use YA T3\u2212.05\u2212.08.07\u2212.0125. Alcohol use YA T1\u2013Parenting YA T2\u2212.04\u2212.09*\u2212.11**\u2212.08*26. Alcohol use YA T2\u2013Parenting YA T3\u2212.05\u2212.07.02\u2212.0227. Alcohol use OA T1\u2013Alcohol use YA T2.10*.09.09.1028. Alcohol use OA T2\u2013Alcohol use YA T3.10*.10*.11*.11*Note: PD\u00a0=\u00a0Parental Problem Drinking, OA\u00a0=\u00a0Older Adolescent, YA\u00a0=\u00a0Younger Adolescent. All stability paths are significant at p\u00a0<\u00a0.001. The numbered paths in the table correspond to the arrowed paths depicted in Fig.\u00a01*\u00a0p\u00a0<\u00a0.05, **\u00a0p\u00a0<\u00a0.01, ***\u00a0p\u00a0<\u00a0.001\nRegarding both the older and younger adolescents, paths of both maternal and paternal problem drinking with behavioral control were generally not significant. Only maternal problem drinking at T2 showed a significant association with behavioral control towards the youngest adolescent at T3 (\u03b2\u00a0=\u00a0\u2212.12, p\u00a0<\u00a0.01). Problem drinking of the mother at T1 and T2 was significantly associated with alcohol use of the older adolescent at T2 and drinking of the younger adolescent at T3, respectively (\u03b2\u00a0=\u00a0.18, p\u00a0<\u00a0.001, \u03b2\u00a0=\u00a0.15, p\u00a0<\u00a0.01). Problem drinking of the father at T2 related substantially to alcohol use of the younger adolescent at T3 (\u03b2\u00a0=\u00a0.20, p\u00a0<\u00a0.001). Considering the cross-lagged paths, more parental behavioral control at T2 was associated with less alcohol consumption of the older adolescents at T3 (\u03b2\u00a0=\u00a0\u2212.11, p\u00a0<\u00a0.05 for fathers, \u03b2\u00a0=\u00a0\u2212.18, p\u00a0<\u00a0.001 for mothers), but not from T1 to T2. Younger adolescents also consumed less alcohol at T2 when their parents exerted more behavioral control at T1 (\u03b2\u00a0=\u00a0\u2212.15, p\u00a0<\u00a0.01 for fathers, \u03b2\u00a0=\u00a0\u2212.14, p\u00a0<\u00a0.01 for mothers). In addition, older adolescents\u2019 drinking at T2 negatively affected parental behavioral control at T3 (\u03b2\u00a0=\u00a0\u2212.12, p\u00a0<\u00a0.01 for fathers, \u03b2\u00a0=\u00a0\u2212.14, p\u00a0<\u00a0.01 for mothers), indicating that when older adolescents drank more, parents exerted less general behavioral control. This result was not found for the younger adolescent.\nNo significant associations were found between parental problem drinking and the support parents provide to both the younger and older adolescent. Paternal problem drinking at T2 directly affected alcohol use of the younger adolescent at T3 (\u03b2\u00a0=\u00a0.20, p\u00a0<\u00a0.001). Mothers\u2019 problem drinking at T1 and T2 affected alcohol use of the older adolescent at T2, and drinking of the younger adolescent at T3, respectively (\u03b2\u00a0=\u00a0.18, p\u00a0<\u00a0.001; \u03b2\u00a0=\u00a0.14, p\u00a0<\u00a0.01). More parental support at T1 was related to less alcohol use of the younger adolescents at T2 (\u03b2\u00a0=\u00a0\u2212.10, p\u00a0<\u00a0.05 for fathers, \u03b2\u00a0=\u00a0\u2212.15, p\u00a0<\u00a0.01 for mothers). These associations were not found between T2 and T3, nor for the older adolescents. In addition, more alcohol use of the older adolescents at T1 was associated with less parental support at T2 (\u03b2\u00a0=\u00a0\u2212.11, p\u00a0<\u00a0.01 for father, \u03b2\u00a0=\u00a0\u2212.08, p\u00a0<\u00a0.05 for mothers).\nAdditional analyses\nWe also tested whether older adolescents influenced their younger siblings in drinking behavior. Results showed that alcohol consumption of the older adolescents tended to directly affect alcohol use of the younger adolescent (.10\u00a0\u2264\u00a0\u03b2\u00a0\u2264\u00a0.12, p\u00a0<\u00a0.05).\nDiscussion\nThe aim of the present study was to gain insight into the associations between parental problem drinking, parenting, and adolescent alcohol use in a sample of Dutch families. The first main finding shows that, except for alcohol-specific behavioral control, parental problem drinking does not structurally affect parenting over time. More alcohol-related problems did not result in less behavioral control, less general support, or higher permissiveness. Our results differ from those of other studies in which significant relations between parental problem drinking and parenting were found (Chassin et\u00a0al. 1993; King and Chassin 2004; Rutherford et\u00a0al. 1997). These differences could be due to methodological issues, as problem drinking or alcoholism in the latter studies were often diagnosed in conformity with the DSM-IV criteria, whereas we concentrated on a broader range of alcohol-related problems in a community sample. Thus, because of our assessment of problem drinking,2 instead of alcohol dependence or abuse, and because of our focus on a community sample instead of a clinical sample, the present study reflects the situation in the general population, and as such enhances the generalizability of the findings. Another explanation for the non-significant relation between parental problem drinking and general parenting comes from the buffering hypothesis, which states that \u201csupport protects persons from the potentially pathogenic influence of stressful events\u201d (Cohen and Wills 1985, p. 310). Accordingly, children of one problem-drinking parent may be protected from the parent\u2019s inadequate parenting by the support and adequate parenting of the other (non-problem drinking) parent. A buffering mechanism has been reported in the literature. For example, Van Aken and Asendorpf (1997) found that low support from one parent could be compensated by support from the other parent in affecting adolescent self-esteem. In addition, peer friendships, positive peer relations and family cohesion have each shown to be a protective factor against children\u2019s externalizing problems in family conflict situations (Criss et\u00a0al. 2002; Farrell et\u00a0al. 1995). With respect to problem drinking, the possibility of enhancing resiliency in children and adolescents, by protecting against possible harmful influences from one parent by a strong relationship with the other parent, siblings, or peers, should be a topic of examination in future research. In addition, with regard to the persons in our sample, being part of a stable, nuclear family that consists of two biological parents with two or more children living together may be protective in itself.\nOur findings do not imply that problem drinking has no effect on personal cognition or functioning, but suggest that parents are able to regulate their problem behavior with regard to their children and parenting practices. However, parental problem drinking may affect the way in which parents handle alcohol use within the family. Parents with more alcohol-related problems are not more permissive than parents who do not have these problems. Maintaining the set rules by means of alcohol-specific behavioral control, however, does appear to be a problem.\nOur second main finding is that higher levels of behavioral control, support, rules, and alcohol-specific behavioral control account for less alcohol consumption in mainly the younger adolescents, which corresponds with the literature on this topic (e.g., Kerr and Stattin 2000; Van der Vorst et\u00a0al. 2005, 2006). Alcohol-specific rule setting played an important role in drinking of both the younger and older adolescents. When parents were more permissive toward alcohol use, adolescents reported higher levels of drinking, which is in accordance with other studies on rule enforcement and adolescent alcohol use (Jackson et\u00a0al. 1999; Van der Vorst et\u00a0al. 2005; Van Zundert et\u00a0al. 2006; Yu 2003). However, support and both general and alcohol-specific behavioral control were only associated with less alcohol use of the younger adolescent, up to the age of about 14\u00a0years. For older adolescents the effect of parenting disappeared, and parental problem drinking was found to directly affect adolescent alcohol use. In explaining this pattern, it might be that parenting exerts influence before and during the initiation phase of alcohol use, which in Dutch adolescents takes place around the age of 14\u00a0years (Poelen et\u00a0al. 2005), but that parenting is no longer important once the habitual drinking pattern has been established (DeCourville 1995). Further, it has been suggested that genetic effects increase in importance over time during late adolescence, whereas environmental factors decrease in importance. Twin studies have indeed shown that shared environmental factors, such as parenting, play a profound role in the initiation of alcohol use, but that genetic factors are more important in frequency of alcohol use and problem drinking in young adulthood (see Hopfer et\u00a0al. 2003; Pagan et\u00a0al. 2006). Moreover, during adolescence, parental factors decrease in importance, whereas the influence of peers increases, making the latter a strong predictor for adolescent drinking (Fergusson et\u00a0al. 1995). As such, shared environmental factors, such as parenting and parental modeling, affect alcohol consumption in young adolescence. Drinking in later adolescence may be related to other, non-shared factors, such as genes and peer drinking. Accordingly, future research should apply a longitudinal, behavioral genetic design, preferably examining the different stages of adolescent drinking (initiation, frequency of consumption, drinking to intoxication) in order to partial out the different effects of genetic and environmental factors.\nOur third main finding considers the reciprocal associations between adolescent alcohol use and parental behaviors. More adolescent alcohol use made parents decrease their levels of general and alcohol-specific behavioral control over time. Levels of support were also negatively adjusted in response to adolescent alcohol use, but only in the younger adolescents. Our findings concur with recent studies that also reported bidirectional findings between parenting practices and adolescent substance use (Huver et\u00a0al. 2006; Stice and Barrera 1995; Van der Vorst et\u00a0al. 2006). However, parents did not adjust their levels of permissiveness in response to adolescent alcohol use. Perhaps rule-setting is more stable over time, and is not affected by fluctuations in adolescent alcohol use. The application of those rules, however, by means of exerting control, was influenced by the levels of alcohol that adolescents consumed. Since our study is one of the first to examine reciprocal effects between adolescent drinking and parental factors, more research on this topic is warranted.\nRegarding differences between fathers and mothers, we found that maternal, but not paternal problem drinking was directly associated with alcohol use of the oldest adolescent. This is a remarkable finding, considering the fact that in most parenting studies with a focus on alcoholism or problem drinking, women are underrepresented (e.g., Chassin et\u00a0al. 1993). Future research should specifically include mothers in studies regarding alcoholic or problem-drinking parents.\nLimitations\nDespite the advantages of our study, such as multi-informant data, longitudinal design and the testing of reciprocal associations in path analyses, some limitations should be addressed. First, we did not further examine relationships in subgroups (for example, sex differences) because of a lack of statistical power and, subsequently, the risk of making Type II errors. Nonetheless it should be stressed that in this type of longitudinal study with a full-family design, the sample size was substantial and the low attrition rates over the three waves were remarkable. Second, parental factors explained only a small part of the variance in adolescent drinking. However, finding small effects does not imply that parental factors are unimportant. The finding that parenting influences adolescent alcohol consumption can have large practical implications (see Abelson 1985). Third, parents may have under-reported their alcohol-related problems because of social desirability and adolescents may have under-reported their alcohol use because of the presence of their parents at home while filling in the questionnaires. In an attempt to anticipate these biases, and to ensure confidentiality, the questionnaires had to be completed individually and separately, without the possibility for family members to discuss the answers. In addition, studies have shown that self-reports concerning alcohol use are a reliable source of information (Engels et\u00a0al. 2007). Fourth, although the sample was carefully selected, the results cannot be generalized to the whole Dutch population, because of the lack of for example, single-parent families and step-families. Fifth, in the Netherlands the legal age to drink beer and wine is 16, and the legal age to drink liquor is 18. This may make it difficult to compare previous research from the USA, where the legal age to drink is 21, with our results. Sixth, it might be that the initial measurements triggered follow-up discussions about alcohol use within families, which might have acted as an intervention. However, since our study is longitudinal and ongoing, we did not want to encourage any speculations or thoughts on the content of the questionnaires which might affect the following measurements, and as such we did not consult the families on these matters. Seventh, in our questionnaires, the definition of \u201cglasses\u201d of alcohol was left up to the respondents to interpret. This may have lead to a reporter bias in the exact amount of alcohol consumed because of different (non-standard) glass sizes. However, measurement of the precise amount of alcohol consumed is rather difficult to realize in our current study design. Experimental designs or diary studies will be able to more accurately measure and control the precise quantity of consumed alcohol. See Kerr et\u00a0al. (2005) for an elaborated discussion on this topic.\nTaking these limitations into account, this study is the first to disentangle the prospective relations between parental alcohol-related problems, parenting, and adolescent alcohol use in a community sample using multi-informant data. The results show that parental problem drinking does not substantially and systematically affect parenting, and that parenting influences adolescent alcohol use, but only up to the age of about 14\u00a0years. This implies that shared environment factors (e.g., parenting and modeling effects) influence the development of alcohol use in young adolescents. When adolescents grow older, and move out of the initiation phase, their drinking behavior may be more affected by other factors, such as genetic susceptibility and peer drinking.","keyphrases":["parenting","problem drinking","adolescent","alcohol"],"prmu":["P","P","P","P"]}
{"id":"Bioinformation-2-4-2255066","title":"Database of neurodegenerative disorders\n","text":"A neurological disorder is a disorder caused by the deterioration of certain nerve cells called neurons. Changes in these cells cause them to function abnormally, eventually bringing about their death. In this paper we present a comprehensive database for neurodegenerative diseases, a first-of-its kind covering all known or suspected genes, proteins, pathways related to neurodegenerative diseases. This dynamically compiled database allows researchers to link neurological disorders to the candidate genes & proteins. It serves as a tool to navigate potential gene-protein-pathway relationships in the context of neurodegenerative diseases. The neurodegenerative disorder database covers more then 100 disease concepts including synonyms and research topics. The current version of the database provides links to 728 abstracts and over 203 unique genes\/proteins with 137 drugs. Also it is integrated well with other related databases. The aim of this database is to provide the researcher with a quick overview of potential links between genes and proteins with related neurodegenerative diseases. Thus DND providing a user-friendly interface is designed as a source to enhance research on neurodegenerative disorders.\nBackground\nNeurodegenerative disease leads to neurodegeneration and disabilities as a result of deterioration of neurons. [1] Progressive loss of motor, sensory neurons and the ability of the mind to refer \nsensory information to an external object is affected in different kinds of neurological disorders. [2] Mutations in the genes identified for these disorders leads to accumulation of misfolded protein \nresulting in protein aggregation and intracellular inclusions. [3] The knowledge gained from the genetic studies of many neurodegenerative disorders explains the disease mechanism that includes few pathways \nleading to neuronal death of cells. [4] Amyloid precursor proteins (APP), SNCA, Parkin, UCHL1, NR4A2, DJ1, PINK1 and LRRK2 are the genetic causes of some of the primary neurodegenerative disorders like \nAlzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and prion diseases. [5] The treatment strategies for these disorders include therapies and drugs. Because of the \ncomplexity involved in the neurodegenerative disease mechanism and treatment strategies, it is of interest to collate different Neurodegenerative Disorders in the form of a database. Hence we developed DND \n(Database of Neurodegenerative Disorders), an on-line web based database that contains more than 100 neuro related disease concepts and provides with a covering of all related genes, proteins, pathways and drug information.\nMethodology\nDatabase model and content\nDatabase of Neurodegenerative Disorders (DND) is developed as an open source software system using Mysql - 5.0.18 - Win32 [6] and PHP - 5.2.0. \n[7] DND uses the relational data model. A schema diagram describing \nthe DND is shown in the [figure 1]. MySQL tables are shown as rectangles. Mandatory attributes are in bold, optional are in italics. The relationships between tables in the database are shown as connecting \nlines. The public databases NCBI, SwissProt, Kegg and DrugBank are used for populating the database. The gene sequences related to various neurological disorders are obtained from NCBI. Both gene sequence and coding \nsequence can be obtained in Fasta format. Also HGNC links for each entry is provided. All protein sequences are extracted from UniProt, and fields that mapped to PDB, Pfam, Interpro, Prints and Smart are parsed and \nstored. Links from DND to these source databases are also provided for each entry. Fasta format of protein sequences too can be obtained. Pathway information for Neurodegenerative diseases as well as known neuro \nrelated drug compounds in KEGG is searched and the results obtained are incorporated in to the database. The articles are chosen to represent a diverse selection of reports on major Neurodegenerative diseases and \ncorresponding Pubmed ID is provided for all the entries. Neurological Associations and Organizations interested in neuroscience-related fields are tabulated with links to their corresponding web pages.\nSearching DND\nThere are three ways by which the user can query the database. The first one is the keyword search in home page that can be performed by giving keywords like Disease name, Gene name and Drug name to retrieve the \nrequired data. The second one is the advanced search option for specific requirements with two main entry points namely gene\/protein and drug\/drug properties. Gene\/protein include search options like Gene name, Gene id, \nOmim, Hgnc, Pubmed, Swissprot id, Pdb, Interpro, Pfam, Smart and Prints. The second entry point Drug\/drug properties include Drug name, Kegg id, DrugBank id, Pubchem, and IUPAC name and Molecular formula. Alternatively, \nthe users can also browse the database via the seven entry points namely browse by Diseases, Genes, Proteins, Drugs, Pathways, Related Articles and Organizations.\nUtility\nThe current version of the DND is comprehensive with enormous data related to every aspect of neurological disorders, providing public access to sequence, genetics, structural, and bibliographic information. Structures \nof drugs can be viewed using the browser plug in Chime [8] that allows chemical structures to rotate, reformat, and save in various file formats. A glossary describing the terms used in database is also provided to help \nthe end users. We believe that DND will assist the intended neurological researchers in understanding of fundamental molecular and genetic processes that control various neurodegenerative diseases.","keyphrases":["neurodegenerative disorder","neurological disorders","web database"],"prmu":["P","P","R"]}
{"id":"Pediatr_Nephrol-3-1-1805050","title":"Ambulatory blood pressure monitoring and renal functions in children with a solitary kidney\n","text":"The aim of this study is to investigate the blood pressure (BP) profile, microalbuminuria, renal functions, and relations with remaining normal kidney size in children with unilateral functioning solitary kidney (UFSK). Sixty-six children with UFSK were equally divided into three groups: unilateral renal agenesis (URA), unilateral atrophic kidney (UAK), and unilateral nephrectomy (UNP). Twenty-two age-, weight-, and height-matched healthy children were considered as a control group. The serum creatinine level and first-morning urine microalbumin and creatinine concentrations were determined by the standard methods. Also, the BP profile was determined by ambulatory blood pressure monitoring (ABPM). We found that the serum creatinine level was higher and creatinine clearance was lower in each patient groups compared to those of the control group (p < 0.05). Compared with the controls, each group of patients had mean office, 24-h, daytime, and night-time systolic and diastolic BP values similar to those of the controls (p > 0.05). An inverse correlation was found between the renal size standard deviation scores (SDS) of normal kidneys and 24-h systolic and diastolic BP load SDS in all of the patients (p < 0.05; r = \u22120.372, r = \u22120.295, respectively). The observed relationship between renal size SDS and 24-h mean arterial pressure (MAP), systolic and diastolic BP load SDS suggests that children with UFSK should be evaluated by using ABPM for the risk of hypertension.\nIntroduction\nArterial hypertension, proteinuria, and impaired renal functions are potential complications of unilateral functioning solitary kidney (UFSK), including unilateral renal agenesis (URA), unilateral atrophic kidney (UAK), and unilateral nephrectomy (UNP). The reduction of renal mass leads to compensatory hypertrophy of the remaining renal tissue in these patients [1\u20133]. Functionally, the remnant kidney will partly compensate for the lost function by increasing its workload, which is accompanied by increased glomerular blood flow and blood pressure (BP), so-called hyperfiltration [4]. It has been shown in animal models of unilateral renal ablation that the remaining kidney undergoes accelerated structural renal damage, generally in the form of glomerulosclerosis, with varying degrees of renal insufficiency, partly amenable to a variety of therapeutic interventions [5, 6]. A relatively small number of long-term follow-up studies in humans has documented that the surgical loss of renal functional mass in the presence of a normal remnant kidney rarely leads to renal insufficiency, although the incidence of mild to moderate proteinuria and hypertension has increased [7\u201310].\nKasiske et al. [11] reported that UNP does not cause progressive renal dysfunction, but it may be associated with a small increase in BP. On the other hand, Wikstad et al. [8] showed that adults born with URA or UNP in childhood did not have a marked increase in arterial BP or renal insufficiency. Some reports, however, indicate that patients with URA or with an UFSK left after nephrectomy may have proteinuria and focal glomerular sclerosis [5, 12]. Janda et al. [13] recorded marginal diastolic hypertension in one third of their subjects in a study of 40 children and\/or adolescents (23 URA, 17 UNP). Although some investigators have reported an increase in the prevalence of hypertension and\/or proteinuria, others have failed to document these abnormalities [11]. The discrepancy among the results of these studies may be caused due to a difference in the amount of renal mass removed, the age at the time of renal mass reduction, or unsuspected damage in the remaining kidney.\nIn the past few years, ABPM has become an accepted method for investigating the BP profile in children with renal disorders [14\u201316]. Over the years, ambulatory blood pressure monitoring (ABPM) has been increasingly used to investigate hypertension in different pediatric populations [17\u201320]. A careful review of the literature shows that there are few reports on ABPM, microalbuminuria, and renal functions in children with UFSK [21]. Furthermore, the utility of BP in children with UFSK is not well established. Therefore, the aim of this study was to investigate the BP profile by ABPM, microalbuminuria, renal functions, and relations with remaining normal kidney size in children with UFSK.\nMaterials and methods\nSixty-six children with a mean age of 8.32\u2009\u00b1\u20094.23 and range 0.5 and 18\u00a0years with UFSK were equally divided into three groups: URA group (n\u2009=\u200922), UAK group (n\u2009=\u200922), and UNP group (n\u2009=\u200922). Twenty-two Age-, weight-, and height-matched healthy children were considered as a control group (Table\u00a01). Renal ultrasound was normal in all controls. Informed consent was obtained from the children and their parents prior to the testing, and the study was approved by the local ethics committee of the School of Medicine, \u00c7ukurova University, Adana, Turkey. \nTable\u00a01Demographic data, anthropometric data, and renal functional parameters of the patients and control groupsParametersURA (n\u2009=\u200922)UAK (n\u2009=\u200922)UNP (n\u2009=\u200922)Controls (n\u2009=\u200922)Gender (M\/F)11\/1112\/1010\/1210\/12Age (years)8.63\u2009\u00b1\u20094.508.03\u2009\u00b1\u20094.308.31\u2009\u00b1\u20094.048.78\u2009\u00b1\u20093.49Weight (kg)28.44\u2009\u00b1\u200918.0727.01\u2009\u00b1\u200917.1426.57\u2009\u00b1\u200910.4829.19\u2009\u00b1\u200910.92Height (cm)124.55\u2009\u00b1\u200927.62126.05\u2009\u00b1\u200925.22123.96\u2009\u00b1\u200921.21128.36\u2009\u00b1\u200926.44BMI (kg\/m2)16.60\u2009\u00b1\u20092.9517.96\u2009\u00b1\u20093.1416.77\u2009\u00b1\u20092.0516.96\u2009\u00b1\u20091.91UMA (mg\/L)5.46\u2009\u00b1\u20093.914.80\u2009\u00b1\u20092.034.56\u2009\u00b1\u20098.115.18\u2009\u00b1\u20094.09UMA\/UCr (mg\/mg)0.23\u2009\u00b1\u20090.720.17\u2009\u00b1\u20090.300.07\u2009\u00b1\u20090.130.06\u2009\u00b1\u20090.06GFR (ml\/dk\/1.73\u00a0m2)1121.95\u2009\u00b1\u200934.19123.66\u2009\u00b1\u200943.67108.57\u2009\u00b1\u200926.71155.85\u2009\u00b1\u200941.27Serum Cr (mg\/dl)20.59\u2009\u00b1\u20090.130.60\u2009\u00b1\u20090.150.65\u2009\u00b1\u20090.100.47\u2009\u00b1\u20090.13Renal size (mm)100.32\u2009\u00b1\u200918.4291.32\u2009\u00b1\u200917.5697.23\u2009\u00b1\u200917.4992.36\u2009\u00b1\u200916.00Values are expressed as mean\u00b1SD, M: male, F: female, BMI: body mass index, UMA: urine microalbumin, UCr: urine creatinine, GFR: glomerular filtration rate1p\u2009<\u20090.05 URA vs. controls, UAK vs. controls, and UNP vs. controls2p\u2009<\u20090.05 URA vs. controls, UAK vs. controls, and UNP vs. controls\nBoth renal agenesis and atrophic kidney were established through renal ultrasound and scintigraphy. A non-functioning kidney is caused due to unilateral vesicoureteral reflux (in seven cases), ureteropelvic junction obstruction (in 10 cases), and multicystic dysplastic kidney (in five cases) in the UAK group. In contrast, indications for nephrectomy contained unilateral Wilms\u2019 tumor (in six cases), a non-functioning kidney is caused due to unilateral vesicoureteral reflux (in 10 cases) and ureteropelvic junction stenosis (in two cases), multicystic dysplastic kidney (in two cases), and nephrolithiasis (in two cases) in the UNP group. Patients with Wilms\u2019 tumor have favorable type and they did not receive chemotherapy. The mean time due to nephrectomy was 4.06\u2009\u00b1\u20093.87\u00a0years (range 0.5\u201317\u00a0years) in this group.\nAll patients had a normal renal function defined as a glomerular filtration rate (GFR) of over 90\u00a0ml\/min 1.73\u00a0m2 (as determined by Schwartz et al.\u2019s formula [22]) and normal urinary sediment. The remaining kidney has findings of normal ultrasound and normal scintigraphy in all patient groups. Only children without any anatomical abnormalities of the remaining kidney and with normal GFR were included in the study. No patient was treated with antihypertensive drugs or drugs interfering with BP. Neither the study patients nor children in the control groups had a positive (first-degree-relative) family history of hypertension. Patients with a renal scar in their solitary kidney or who have other urinary tract anomaly were withdrawn from the study. In all of the study group participants, urinalysis, serum uric acid, creatinine, cholesterol, protein and albumin levels, and first-morning urine microalbumin and creatinine concentrations were determined by the standard methods.\nIn all subjects, ABPM was performed over 24\u00a0h using the SpaceLabs 90217 oscillometric device (Redmond, Washington). The appropriately sized cuff, chosen based on arm width, was placed on the non-dominant arm. The BP was automatically recorded in every 15\u00a0min during the day and every 30\u00a0min at night. Only ABPM profiles with at least 30 recordings, including at least eight readings between midnight and 06.00\u00a0h, were accepted. Daytime was defined as 08.00\u201320.00\u00a0h and night-time as 00.00\u201306.00\u00a0h, according to the study of Soergel et al. [23]. For each subject, the BP values corresponding to the 95th percentile, according to sex and height, was determined [23, 24]. Hypertension was defined as a systolic BP and\/or diastolic BP>95th percentile. The percentage of systolic BP and diastolic BP readings above this value constituted a measurement of 24-h systolic and diastolic BP load, respectively. The dipping status was calculated by subtracting the mean night-time BP from the mean daytime BP, and then dividing this value by the mean daytime BP. Dipping was defined as a \u226510% drop in mean systolic BP or diastolic BP between daytime and night-time. Renal size and BP standard deviation scores (SDS) was calculated according the left main stem (LMS) method [24, 25].\nThe renal size of the normal kidney was evaluated by performing abdominal ultrasound and measuring the lower-to-upper-pole length in the supine longitudinal view by the same observer. Renal size SDS values were correlated to the BP SDS values for each patient. Renal size percentiles were determined and evaluated according to standard length-against-height-nomogram defined by Dinkel et al. [26]. Compensatory hypertrophy was accepted when the measured renal size was found to be greater than the 95th percentile, and its effect on the BP parameters was evaluated.\nAll data were stored and analyzed using the SPSS statistical package (SPSS Inc., Chicago, Illinois). The normality Kolmogorov-Smirnov test was performed to determine whether continuous variables were normally distributed or not. Differences between groups were analyzed using the non-parametric Kruskal-Wallis test and the Mann-Whitney U test. All values are expressed as mean\u00b1SD. The prevalence of hypertension was determined using Fisher\u2019s exact test. The relationships between variables were analyzed with the non-parametric Spearman\u2019s correlation test in each group. A value of p\u2009<\u20090.05 was considered to be statistically significant.\nResults\nThe demographic data and renal function parameters of all of the study groups and controls are shown in Table\u00a01. Age, height, weight, BMI, urine microalbumin, urine microalbumin to urine creatinine ratio, and renal size values are not different between each patient group and the controls (p\u2009>\u20090.05). The mean serum creatinine level was higher in all patient groups compared to that of the control group (p\u2009=\u20090.006 between URA and control, p\u2009=\u20090.004 between UAK and control, p\u2009<\u20090.001 between UNP and control) and the GFR was lower in all patient groups compared to that of the control group (p\u2009=\u20090.005 between URA and control, p\u2009=\u20090.016 between UAK and control, p\u2009<\u20090.001 between UNP and control).\nMean heart rates (24\u00a0h, daytime, night-time), mean systolic and diastolic BP parameters (office BP, 24\u00a0h, daytime, night-time), mean systolic and diastolic BP dipping, and 24-h systolic and diastolic BP loads are all given in Table\u00a02. When compared to the controls, each group with UFSK had mean office, 24-h, daytime, and night-time systolic and diastolic BP, and heart rate values similar to those of the controls (p\u2009>\u20090.05). Only the mean 24-h diastolic BP was higher in the URA group than that of the control group (p\u2009=\u20090.039). In addition, the diastolic load was higher in the UAK group than that of the control group (p\u2009=\u20090.012). There was no significant difference in the mean office, 24-h, daytime, and night-time systolic and diastolic BP, and heart rate between all of the patient groups (p\u2009>\u20090.05). The mean office systolic and diastolic BP, respectively, were higher than the mean daytime systolic and diastolic BP in all groups. The prevalence of hypertension in the URA group was 23% (5\/22), in the UAK group was 23% (5\/22), in the UNP group was 32% (7\/22) [in all patients, it was 26% (17\/66)], and in the controls it was 5% (1\/22). The prevalence of hypertension in patients was higher compared to that in the controls (p\u2009=\u20090.035). The prevalence of non-dipping phenomenon in the URA group was 23% (5\/22), in the UAK group was 36% (8\/22), in the UNP group was 32% (7\/22) [in all patients, it was 30% (20\/66)], in the controls it was 9% (2\/22). There was a statistically significant difference between patients and the controls (p\u2009=\u20090.049). \nTable\u00a02Average pulse rate and blood pressure (BP) of the patients and the control groupsParametersURA (n\u2009=\u200922)UAK (n\u2009=\u200922)UNP (n\u2009=\u200922)Controls (n\u2009=\u200922)Av. 24-h HR (beats\/min)89.36\u2009\u00b1\u200915.7994.64\u2009\u00b1\u200917.7488.18\u2009\u00b1\u200912.9590.82\u2009\u00b1\u200914.79Av. daytime HR (beats\/min)92.64\u2009\u00b1\u200915.5197.14\u2009\u00b1\u200917.2793.09\u2009\u00b1\u200912.9697.41\u2009\u00b1\u200915.55Av. night-time HR (beats\/min)80.23\u2009\u00b1\u200915.9986.46\u2009\u00b1\u200918.0177.86\u2009\u00b1\u200912.4382.50\u2009\u00b1\u200916.65Av. office SBP (mmHg)113.96\u2009\u00b1\u200916.00116.96\u2009\u00b1\u200911.24114.55\u2009\u00b1\u200914.83114.68\u2009\u00b1\u200912.66Av. office DBP (mmHg)74.73\u2009\u00b1\u200910.4871.50\u2009\u00b1\u200911.0671.05\u2009\u00b1\u200913.0072.00\u2009\u00b1\u20099.67Av. 24-h MAP (mmHg)80.00\u2009\u00b1\u20097.9278.82\u2009\u00b1\u20097.1678.41\u2009\u00b1\u20097.0678.27\u2009\u00b1\u20093.89Av. 24-h SBP (mmHg)106.64\u2009\u00b1\u200910.87105.59\u2009\u00b1\u20099.31104.22\u2009\u00b1\u20098.04105.05\u2009\u00b1\u20096.90Av. 24-h DBP (mmHg)165.91\u2009\u00b1\u20096.8264.55\u2009\u00b1\u20096.5964.09\u2009\u00b1\u20096.8763.41\u2009\u00b1\u20093.63Av. daytime MAP (mmHg)81.14\u2009\u00b1\u20099.1480.00\u2009\u00b1\u20098.0479.68\u2009\u00b1\u20097.3380.68\u2009\u00b1\u20094.86Av. daytime SBP (mmHg)109.59\u2009\u00b1\u200911.75107.18\u2009\u00b1\u200910.01106.55\u2009\u00b1\u20097.95108.14\u2009\u00b1\u20096.83Av. daytime DBP (mmHg)68.36\u2009\u00b1\u20098.3666.46\u2009\u00b1\u20097.1766.18\u2009\u00b1\u20097.2867.41\u2009\u00b1\u20094.36Av. night-time MAP (mmHg)72.86\u2009\u00b1\u20096.5571.86\u2009\u00b1\u20097.7072.09\u2009\u00b1\u20096.7673.64\u2009\u00b1\u20094.99Av. night-time SBP (mmHg)99.82\u2009\u00b1\u20099.3699.32\u2009\u00b1\u20099.9999.00\u2009\u00b1\u20097.7898.55\u2009\u00b1\u20096.60Av. night-time DBP (mmHg)59.00\u2009\u00b1\u20095.4658.23\u2009\u00b1\u20097.1858.09\u2009\u00b1\u20096.7357.59\u2009\u00b1\u20093.75Av. SBP dipping (%)8.26\u2009\u00b1\u20094.297.31\u2009\u00b1\u20094.686.58\u2009\u00b1\u20093.428.83\u2009\u00b1\u20093.74Av. DBP dipping (%)13.12\u2009\u00b1\u20096.5412.24\u2009\u00b1\u20097.6412.03\u2009\u00b1\u20097.1914.87\u2009\u00b1\u20096.32Av. 24-h SBP load (%)19.71\u2009\u00b1\u200925.7526.31\u2009\u00b1\u200925.1421.88\u2009\u00b1\u200920.0814.45\u2009\u00b1\u200913.81Av. 24-h DBP load (%)221.22\u2009\u00b1\u200917.1233.0.2\u2009\u00b1\u200928.9926.24\u2009\u00b1\u200924.5613.54\u2009\u00b1\u200913.75Values are expressed as mean\u00b1SD, Av.: average, HR: heart rate, MAP: mean arterial pressure, SBP: systolic blood pressure, DBP: diastolic blood pressure1p\u2009=\u20090.039 between URA and control groups2p\u2009=\u20090.012 between UAK and control groups\nCompensatory hypertrophy (renal size percentile >95%) was seen in 59% (39\/66) of patients. Age, height, weight, renal size, and GFR were statistically significantly higher in patients with compensatory hypertrophy than in patients without compensatory hypertrophy. In contrast, the mean heart rates were lower in patients with >95th percentile of renal size (Table\u00a03). \nTable\u00a03Some characteristics of the patients with unilateral functioning solitary kidney (USFK) according the percentile of renal sizeParametersRenal size <95th percentile (n\u2009=\u200927)Renal size >95th percentile (n\u2009=\u200939)pAge (year)5.91\u2009\u00b1\u20093.169.99\u2009\u00b1\u20094.09<0.001Height (cm)113.19\u2009\u00b1\u200921.24132.92\u2009\u00b1\u200923.47<0.001Weight (kg)19.80\u2009\u00b1\u20099.8932.56\u2009\u00b1\u200916.41<0.001Renal size (mm)80.37\u2009\u00b1\u200911.13107.31\u2009\u00b1\u200912.71<0.001GFR (ml\/dk\/1.73\u00a0m2)107.82\u2009\u00b1\u200937.31125.15\u2009\u00b1\u200933.09<0.05Av. 24-h HR (beat\/min)99.19\u2009\u00b1\u200915.4984.87\u2009\u00b1\u200912.96<0.001Av. daytime HR (beat\/min)102.00\u2009\u00b1\u200913.8188.95\u2009\u00b1\u200913.99<0.001Av. night-time HR (beat\/min)88.59\u2009\u00b1\u200917.0576.62\u2009\u00b1\u200913.02<0.05Values are expressed as mean\u00b1SD, Av.: average, GFR: glomerular filtration rate, HR: heart rate\nRenal size SDS was correlated with some BP SDS parameters in the patients and the controls. Statistically significant correlations are shown in Table\u00a04. There was no correlation between the renal size SDS and BP parameters, but an inverse correlation was found between the renal size SDS and 24-h MAP SDS, 24-h systolic and diastolic BP load SDS in all of the patients, but these correlations were not found in the controls. \nTable\u00a04Correlation between renal size standard deviation scores (SDS) and some parameters SDS in patients with UFSK and the controlsGroupsParametersrpPatients (n\u2009=\u200966)Av. 24-h MAP SDS\u22120.2840.021Av. 24-h SBP SDS\u22120.1750.159Av. 24-h DBP SDS\u22120.0340.788Av. 24-h SBP load SDS\u22120.3720.002Av. 24-h DBP load SDS\u22120.2950.016Controls (n\u2009=\u200922)Av. 24-h MAP SDS\u22120.0110.962Av. 24-h SBP SDS\u22120.0670.767Av. 24-h DBP SDS\u22120.3120.158Av. 24-h SBP load SDS\u22120.1480.510Av. 24-h DBP load SDS\u22120.0190.934SDS: standard deviation scores, Av.: average, MAP: mean arterial pressure, SBP: systolic blood pressure, DBP: diastolic blood pressure\nDiscussion\nThe UFSK is usually considerably enlarged, this constituting a compensatory diffuse but not partial, lobar enlargement, usually with a larger than normal pelvis [27]. The reduction of renal mass in the rat is associated with hyperfiltration and hypertrophy of the remaining nephrons, ultimately resulting in proteinuria, renal failure, and hypertension. Nagata et al. [28] reported that unilateral nephrectomy in young rats leads to a higher incidence of glomerular sclerosis than is observed in adult rats. A study conducted by Regazzoni et al. [29] in a follow-up of UNP in children has reported a decreased renal reserve capacity, but detected no evidence of hypertension or proteinuria.\nIn our study, subjects were screened with urine microalbumin, urine and serum creatinine, GFR, and urine microalbumin-to-creatinine ratios in first-morning urine for the detection of proteinuria and renal functions. No statistically significant difference was found between the urine microalbumin and microalbumin-to-creatinine ratios in the first-morning urine in all of the patient and control groups. However, the GFR was lower and the serum creatinine level was higher in the subgroups of patients than those of the control group. These results show that a mild renal dysfunction may be detected in patients with UFSK.\nAlthough some investigators have reported an increased prevalence of hypertension, others have failed to document these abnormalities in children with UFSK [11]. Janda et al. [13] recorded marginal diastolic hypertension in one third of their subjects in a study of 40 children and\/or adolescents (23 URA, 17 UNP). As we mentioned before, there are few reports which have utilized 24-h ABPM in UFSK children. Mei-Zahav et al. [21] showed that the presence of an UFSK, from whatever cause, leads to an increase in BP. This is particularly manifested in systolic BP and even more so in daytime systolic BP. Kasiske et al. [11] reported that UNP does not cause progressive renal dysfunction, but may be associated with a small increase in BP. On the other hand, Wikstad et al. [8] concluded that adults born with URA or UNP in childhood did not have a marked increase in arterial BP or renal insufficiency. We measured the ABPM in three groups of patient with UFSK and found that they have the same characteristics as in healthy controls. However, in our study, the mean office, 24-h systolic, daytime, and night-time systolic and diastolic BP, mean 24-h, daytime, and night-time heart rate, 24-h systolic BP load, systolic and diastolic BP dipping in all groups of patients were similar to those of the controls. In contrast, mean 24-h diastolic BP was higher in the URA group than those of the control group, but this finding showed no difference between the other patient groups and controls. The 24-h diastolic BP load was increased in the UAK group. In addition, an increased prevalence of hypertension was found in all of the patients groups. According to these results, we suggest that patients with a single kidney have potential risk factors for hypertension in the early childhood period.\nCompensatory hypertrophy of the remaining kidney may be seen in patients with UFSK. In adult animals, a compensatory increase in size following UNP is, for the most part, attributable to hypertrophy (an increase in cell size), which is predominantly of proximal tubular origin. In young animals, renal growth is achieved primarily by cell multiplication, that is, hyperplasia [3]. In the present study, an inverse correlation was found between renal size SDS and 24-h MAP SDS, 24-h systolic and diastolic BP load SDS in all of the patients, but was not seen in the controls. Therefore, we think that there is an increased risk of hypertension in children without compensatory hypertrophy.\nTo this end, we conclude that subtle alterations of renal function might be already present in children with UFSK, while no obvious pathological findings were made for BP or urinary protein excretion in the early period. However, an increased BP load was seen in these patients (particularly in patients without compensatory hypertrophy). Nevertheless, this might not exclude an increased long-term risk for the development of hypertension or impaired renal function in this patient group. Because patients in the present study have a small sample and a shorter follow-up period, further prospective studies with a larger sample size and of longer term are suggested to find the relationship between these parameters.","keyphrases":["blood pressure","renal function","solitary kidney"],"prmu":["P","P","P"]}
{"id":"Neurochem_Res-4-1-2413118","title":"Silencing \u03b1-Synuclein Gene Expression Enhances Tyrosine Hydroxylase Activity in MN9D Cells\n","text":"\u03b1-Synuclein has been implicated in the pathogenesis of Parkinson\u2019s disease (PD). Previous studies have shown that \u03b1-synuclein is involved in the regulation of dopamine (DA) metabolism, possibly by down-regulating the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in DA biosynthesis. In this study, we constructed \u03b1-synuclein stably silenced MN9D\/\u03b1-SYN\u2212 cells by vector mediated RNA interference and examined its effects on DA metabolism. We found that there were no significant differences in TH protein and mRNA levels between MN9D, MN9D\/\u03b1-SYN\u2212 and MN9D\/CON cells, suggesting that silencing \u03b1-synuclein expression does not affect TH gene expression. However, significant increases in phosphorylated TH, cytosolic 3, 4-dihydroxyphenylalanine (l-DOPA) and DA levels were observed in MN9D\/\u03b1-SYN\u2212 cells. Our data show that TH activity and DA biosynthesis were enhanced by down-regulation of \u03b1-synuclein, suggesting that \u03b1-synuclein may act as a negative regulator of cytosolic DA. With respect to PD pathology, a loss of functional \u03b1-synuclein may result in increased DA levels in neurons that may lead to cell injury or even death.\nIntroduction\n\u03b1-Synuclein is a 140-amino acid protein, which has been implicated in the pathogenesis of Parkinson\u2019s disease (PD) [20, 30]. Previous studies have shown that \u03b1-synuclein mutations (A53T, A30P, E46K) are associated with some autosomal-dominant PD [14, 15, 27] and that aggregated \u03b1-synuclein is the major component of Lewy bodies and Lewy neurites, pathological hallmarks of PD [2, 21, 34]. Although the normal function of \u03b1-synuclein and its role in the pathogenesis of PD remain unclear, several hypotheses have been proposed based on its physical properties or interacting partners [17\u201319, 25, 28, 31, 36]. One theory suggested that \u03b1-synuclein might be linked to PD via the regulation of dopamine (DA) homeostasis [1, 18, 25, 31, 41]. Several studies have shown that \u03b1-synuclein may be involved in regulating the biosynthesis, vesicular storage and release, as well as reuptake of DA [3, 9, 26, 32, 33, 38, 40, 42]. However, some of these findings remain controversial. It has also been reported that \u03b1-synuclein may down-regulate the gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme involved in DA biosynthesis [3, 42]. Others report that \u03b1-synuclein regulates DA biosynthesis by reducing TH activity [26]. Another study showed that the tyrosine-mediated enhancement of phosphorylated TH in nigrostriatal DA neurons was inhibited in \u03b1-synuclein knockout mice, suggesting that \u03b1-synuclein may either enhance TH phosphorylation or hinder TH inactivation during accelerated neuronal activity [9]. Although it has been established that \u03b1-synuclein plays an important role in enhancing TH activity, conflicting reports indicate that further studies are required to examine the role of \u03b1-synuclein in regulating DA metabolism.\nRNA interference (RNAi) is a new strategy for silencing gene expression that uses short double-stranded (ds) RNA to mediate the degradation of sequence specific mRNA [10\u201312]. It has been reported that introducing small interfering RNAs (siRNAs) or short hairpin RNA (shRNA) into mammalian cells could specifically silence the expression of target genes [7, 23]. The high specificity, efficiency and convenience of this technique means that it is a powerful tool for studying gene function.\nIn the present study, we silenced the expression of \u03b1-synuclein using vector mediated RNAi in dopaminergic MN9D cells and examined the effect of down-regulation of \u03b1-synuclein on TH and DA biosynthesis.\nMaterials and Methods\nConstruction of shRNA Expression Plasmids\nShort hairpin RNA were designed to target specific regions of mouse \u03b1-synuclein mRNA (GenBank accession no. AF179273). We identified two 19-nucleotide stretches within the coding region of the \u03b1-synuclein gene that were 40\u201350% GC rich, located within one exon and unique within the mouse genome. A nine-nucleotide loop structure was designed to form the hairpin. We designed three sets of oligonucleotides as instructed by the BLOCK-iT\u2122 Inducible H1 RNAi Entry Vector Kit (Invitrogen, Carlsbad, CA, USA): SH1\/\u03b1-SYN: 5\u2032-CACCGAGCAAGTGACAAATTTGTTCAAGAGACAACATTTGTCACTTGCTC-3\u2032 (forward), 5\u2032-AAAAGAGCAAGTGACAAATGTTGTCTCTTGAACAACATTTGTCACTTG CTC-3\u2032 (reverse); SH2\/\u03b1-SYN: 5\u2032-CACCAGGCTACCAAGACTATGAGTTCAAGAG ACTCATAGTCTTGGTAGCC-3\u2032 (forward), 5\u2032-AAAAGGCTACCAAGACTATGA GTCTCTTGAACTCATAGTCTTGGTAGCCT-3\u2032 (reverse); and SH\/CON: 5\u2032-CACCGGATCGCCAGAACAAGTATTTCAAGAGAATACTTGTTCTGGCGATCC-3\u2032 (forward), 5\u2032-AAAAGGATCGCCAGAACAAGTATTCTCTTGAAATACTTGTTCTGG CGATCC-3\u2032 (reverse). Single-stranded (ss) DNA oligos (BioSia, Co., Ltd, Shanghai, China) were synthesized. Equal amounts of each pair of ss oligos were annealed to generate ds oligos. The ds oligos were inserted into the pENTRTM\/H1\/TO vector (Invitrogen) using T4 DNA ligase and transformed into One Shot\u00ae TOP10 chemically competent Escherichiacoli (Invitrogen). The transformants were verified by PCR and sequencing. The positive plasmids were named pSH1\/\u03b1-SYN, pSH2\/\u03b1-SYN and pSH\/CON, respectively.\nCell Culture\nThe MN9D dopaminergic cell line was generated by fusion of rostral mesencephalic neurons from embryonic C57BL\/6J (embryonic day 14) mice with N18TG2 neuroblastoma cells [8] (gift from Dr Bastian Hengerer, Novartis AG). Cells were cultured in DMEM\/F12 media supplemented with 10% neonatal calf serum (GIBCO BRL, Grand Island, NY, USA; pH 7.2) and incubated at 37\u00b0C in an atmosphere of 5% CO2. MN9D cells endogenously express \u03b1-synuclein and TH, and produce measurable levels of DA.\nTransfection and Selection\nMN9D cells were transfected with pSH1\/\u03b1-SYN, pSH2\/\u03b1-SYN, pSH\/CON or pcDNA3.1 containing human wild-type (WT) \u03b1-synuclein cDNA (constructed in our laboratory). For stable transfection of pSH1\/\u03b1-SYN, pSH2\/\u03b1-SYN and pSH\/CON, cells were seeded in 6-well tissue culture plates pre-coated with poly-l-lysine (Sigma, St Louis, MO, USA) at 80\u201385% confluency. Transfections were performed using Lipofectamine\u2122 2000 (Invitrogen) according to the manufacturer\u2019s instructions. Four to six hours after transfection, the media was replaced with fresh growth media. The following day, cells were trypsinized and replated into a larger sized tissue culture format. Stably transfected MN9D cells were selected using 400\u00a0\u03bcg\/ml Zeocin\u2122 (Invitrogen) for 12\u00a0days. The Zeocin\u2122 resistant clones were then selected and expanded. Cells were maintained with media containing 50\u00a0\u03bcg\/ml Zeocin\u2122. Seventy-two hours post-transfection, cells transfected with pcDNA3.1 containing human WT \u03b1-synuclein cDNA were collected and named MN9D\/\u03b1-SYN+.\nRNA Isolation, RT-PCR and Real-time RT-PCR\nTotal RNA was extracted from pSH1\/\u03b1-SYN, pSH2\/\u03b1-SYN and pSH\/CON stably transfected MN9D cells and MN9D\/\u03b1-SYN+ cells using Trizol reagent. First-strand cDNA was synthesized from 5\u00a0\u03bcg of total RNA, using the SuperScript\u2122 First-strand synthesis System for RT-PCR (Invitrogen). The reaction mixture for PCR consisted of 1\u00a0\u03bcl of cDNA template, 17\u00a0\u03bcl of sterile ddH2O and 10\u00a0pmol (1\u00a0\u03bcl) of each specific primer. The following primers were used: \u03b1-synuclein: 5\u2032-ATAAGAATGCGGCCGCATGGATGTATTCATGAAAG-3\u2032 (forward) and 5\u2032-CCGCTCGAGGCTTCAGGTTCGTAGTCTTGA-3\u2032 (reverse). As an internal control, \u03b2-actin cDNA was co-amplified using the following primer sequences: 5\u2032-CCCATCTACGAGGGCTACGC-3\u2032 (forward) and 5\u2032-AGGAAGGAGGGCTGGAACA-3\u2032 (reverse). Each PCR was started by predenaturation at 96\u00b0C for 3\u00a0min. Each PCR cycle consisted of 94\u00b0C for 30\u00a0s, 50\u00b0C for 55\u00a0s and 72\u00b0C for 55\u00a0s, followed by a final 10\u00a0min extension at 72\u00b0C. PCR amplification was carried out for 35 cycles for \u03b1-synuclein and 22 cycles for \u03b2-actin using a Perkin\u2013Elmer DNA thermal cycler 480 (Applied Biosystems, Foster City, CA, USA).\nThe reaction mixture (20\u00a0\u03bcl) for real-time PCR consisted of 10\u00a0\u03bcl SYBR GREEN PCR Master Mix (Applied Biosystems), 0.5\u00a0\u03bcl of cDNA template, 0.4\u00a0\u03bcl of each specific primer (10\u00a0\u03bcM) and 9.7\u00a0\u03bcl of sterile ddH2O. The following primers were used: \u03b1-synuclein: 5\u2032-TGACGGGTGTGACAGCAGTAG-3\u2032 (forward) and 5\u2032-CAGTGGCTGCTGCAATGC-3\u2032 (reverse); TH: 5\u2032-CAGCCCTACCAAGATCAAAC-3\u2032 (forward) and 5\u2032-TACGGGTC AAACTTCACAGA-3\u2032 (reverse); and \u03b2-actin: 5\u2032-ACCACCATGTACCCAGGCATT-3\u2032 (forward) and 5\u2032-CCACACAGAGTACTTGCGCTCA-3\u2032 (reverse). Quantitative PCR was performed in duplicate or triplicate using a 7300 real-time PCR thermal cycler (Applied Biosystems). Each reaction was started by 50\u00b0C for 2\u00a0min, 95\u00b0C for 10\u00a0min and 96\u00b0C for 3\u00a0min, followed by 40 cycles of 94\u00b0C for 15\u00a0s, 59\u00b0C for 20\u00a0s and 72\u00b0C for 30\u00a0s.\nImmunofluorescence Staining\nCells were washed with 0.01\u00a0M PBS, fixed in 4% paraformaldehyde (30\u00a0min), permealized with 0.5% Triton-X (20\u00a0min) and blocked in 5% normal goat serum (30\u00a0min). Samples were incubated with a specific monoclonal antibody against \u03b1-synuclein (2E3; 1:1000) (gift from Dr Shun Yu, Xuanwu Hospital, Capital Medical University, Beijing, China) overnight at 4\u00b0C. Cells were washed with 0.01\u00a0M PBS and incubated with goat anti-mouse IgG conjugated to cyanine 3 (Cy3TM; 1:400, Sigma) at 37\u00b0C for 30\u00a0min. After a final wash, cells were examined by fluorescence microscopy.\nCell Viability Assay\nCell viability was assessed using a MTT Kit (Promega, WI, USA). Briefly, the transfected MN9D cells were plated into 96-well plates (1.0\u00a0\u00d7\u00a0104 cells per well) and cultured for 24\u00a0h prior to the assay. Sixteen wells were measured for each stably transfected MN9D cell line.\nWestern Blot Analysis\nLysates of MN9D cells were prepared as previously described [39]. Protein concentrations were determined using BCA relative to BSA protein standards according to the manufacturer (Pierce, Rockford, IL, USA). Whole cell protein extracts were resolved by SDS-PAGE and electrophoretically transferred to nitrocellulose membrane. Prestained protein standards were used to determine the relative molecular mass of proteins. The membranes were blocked in 5% nonfat dry milk in Tris-buffered saline, then incubated with the TH specific monoclonal antibody (1:5000, Sigma) or \u03b1-synuclein specific monoclonal antibody (2E3; 1:1000) at room temperature for 3\u00a0h, followed by a 1-h incubation with a peroxidase-conjugated secondary antibody (1:5000, ZhongShan, Beijing, China) at room temperature. The blots were washed and immunodetection was carried out by chemiluminescence using SuperSignal (Pierce). Blots were then stripped and re-probed with an antibody against \u03b2-actin (1:500, Zhongshan). Blots were scanned using Kodak Image Station (440 CF; Kodak, Rochester, MN, USA) and the optical densities of the bands relative to \u03b2-actin within each lane were obtained.\nHPLC for Assaying DA and 3, 4-Dihydroxyphenylalanine (l-DOPA) Levels\nMN9D cells were collected and re-suspended in HPLC buffer consisting of 0.1\u00a0N perchloric acid, 0.3\u00a0mM EDTA and 0.1% l-lysine. Cells were freeze-thawed three times, then centrifugated at 15,000\u00a0g for 15\u00a0min at 4\u00b0C to remove particulates. The supernatants were collected and stored at \u221280\u00b0C until analysis. Briefly, the supernatant samples (20\u201350\u00a0\u03bcl) were injected onto a Phase II Column (ODS, 3.2\u00a0\u00d7\u00a0100\u00a0mm cartridge, \u03a63.2\u00a0\u03bcm, Bioanalytical Systems, Inc, USA). The mobile phase consisted of 0.05\u00a0M CH3COONa, 0.05\u00a0M citric acid, 1\u00a0mM sodium octyl sulfate, 5\u00a0mM TEA, 0.075\u00a0mM Na2EDTA and 10% methanol (v\/v), pH 2.7. The mobile phase was pumped through the system at 0.4\u00a0ml\/min using a PM-80 pump (Bioanalytical Systems, Inc). Compounds were detected and quantified with an LC-4C detector (Bioanalytical Systems, Inc). The limit of detection for DA was 2\u00a0nmol\/20\u00a0\u03bcl. Peaks were identified by retention times set to known standards.\nTyrosine hydroxylase activity was assessed by measuring the accumulation of l-DOPA within cells treated with the aromatic l-amino acid decarboxylase (AADC) inhibitor n-hydroxybenzylhydrazine dihydrochloride (NSD-1015) and measured by HPLC as described above. Briefly, MN9D cells were cultured in 12-well tissue culture plates, washed twice in artificial CSF (ACSF containing 147\u00a0mM NaCl, 2.7\u00a0mM KCl, 1.2\u00a0mM CaCl2 and 1.0\u00a0mM MgCl2) and equilibrated for 20\u00a0min at 37\u00b0C before addition of 200\u00a0\u03bcM NSD-1015 in ACSF for 30\u00a0min. ACSF samples were collected and assayed by HPLC as described above.\nStatistical Analysis\nANOVA followed by Bonferroni post hoc multiple comparisons were used to examine the significance between differences among the experimental groups. A value of p\u00a0<\u00a00.05 was considered to be statistically significant. All data are expressed as mean\u00a0\u00b1\u00a0SE.\nResults\nConstruction of the shRNA Expression Plasmids\nAfter synthesis, each pair of the ss oligos was annealed to generate ds oligos. The annealing efficiency was analyzed by 4% agarose gel electrophoresis. Each ds oligo annealing reactant showed a clearly detectable molecular weight band around 50\u00a0bp, as expected for the length of the designed ds oligos. The 50\u00a0bp bands were brighter than the corresponding lower molecular weight band, which represented the unannealed ss oligos in the annealing reactant. These data indicate that each pair of ss oligos annealed effectively to form ds oligos. The ds oligos were ligated with linearized pENTR\u2122\/H1\/TO vectors and the positive clones were expanded and verified by PCR. Bands near 350\u00a0bp were found in all PCR reactants, indicating that the ds oligos were ligated into the pENTR\/H1\/TO vectors. Further analysis by sequencing confirmed that the orientation and sequence of the inserted ds oligos were correct.\n\u03b1-Synuclein Expression in MN9D Transfected Cells\nThe gene expression of \u03b1-synuclein in the transfected MN9D cells was assayed by RT-PCR, real-time RT-PCR, immunofluorescence and Western blot. RT-PCR analysis revealed that \u03b1-synuclein mRNA levels were decreased in pSH2\/\u03b1-SYN-transfected MN9D cells compared with MN9D and pSH\/CON-transfected MN9D cells (Fig.\u00a01a). However, \u03b1-synuclein mRNA levels in pSH1\/\u03b1-SYN-transfected cells were not significantly different to levels observed in control MN9D and pSH\/CON-transfected MN9D cells (Fig.\u00a01a). These data suggest that \u03b1-synuclein expression may be inhibited by transfection with pSH2\/\u03b1-SYN. In addition, real-time RT-PCR revealed that \u03b1-synuclein mRNA levels in pSH2\/\u03b1-SYN-transfected cells (9.8\u00a0\u00b1\u00a02.4%) were significantly lower than in MN9D and MN9D\/CON-transfected (81.6\u00a0\u00b1\u00a04.9%) cells (Fig.\u00a01b). The immunofluorescence data also indicated that \u03b1-synuclein gene expression was down-regulated in pSH2\/\u03b1-SYN-transfected cells (Fig.\u00a01c). Weak immunofluorescence staining was observed in pSH2\/\u03b1-SYN-transfected cells compared to strong \u03b1-synuclein staining in control MN9D, pSH\/CON-transfected and pSH1\/\u03b1-SYN-transfected MN9D cells. Furthermore, western blot analysis revealed that \u03b1-synuclein protein levels were significantly lower in pSH2\/\u03b1-SYN-transfected cells (0.150\u00a0\u00b1\u00a00.004) than in MN9D cells (0.401\u00a0\u00b1\u00a00.038) (Fig.\u00a01d,\u00a0e). Thus, \u03b1-synuclein expression was inhibited after transfection of MN9D cells with pSH2\/\u03b1-SYN. These data suggest that SH2\/\u03b1-SYN was an effective siRNA sequence and that \u03b1-synuclein expression was stably silenced in pSH2\/\u03b1-SYN-transfected MN9D cells. The pSH2\/\u03b1-SYN and pSH\/CON stably transfected cells were then named MN9D\/\u03b1-SYN- and MN9D\/CON, respectively.\nFig.\u00a01Inhibition of \u03b1-synuclein expression in pSH2\/\u03b1-SYN-transfected cells. (a) \u03b1-Synuclein mRNA levels were measured by RT-PCR. Lane 1 pSH1\/\u03b1-SYN-transfected MN9D cells; Lane 2 pSH2\/\u03b1-SYN-transfected MN9D cells; Lane 3 pSH\/CON-transfected MN9D cells; Lane 4 MN9D cells; Lane 5 100\u00a0bp DNA ladder. (b) Statistical analysis of \u03b1-synuclein gene expression by real-time RT-PCR. \u03b1-Synuclein mRNA levels were decreased by approximately 90% in pSH2\/\u03b1-SYN-transfected cells compared with normal MN9D cells [n\u00a0=\u00a05, F\u00a0=\u00a0216.167, df(total)\u00a0=\u00a014, **p\u00a0<\u00a00.001 compared with MN9D cells, **p\u00a0<\u00a00.001 compared with MN9D\/CON cells]. \u03b1-Synuclein mRNA levels were not significantly different in pSH1\/\u03b1-SYN-transfected cells compared with MN9D and MN9D\/CON cells. (c) Immunofluorescence staining of normal MN9D cells (a) and MN9D cells transfected with pSH1\/\u03b1-SYN (b), pSH\/CON (c) and pSH2\/\u03b1-SYN (d) using an antibody against \u03b1-synuclein. Strong \u03b1-synuclein staining was observed in MN9D, pSH1\/\u03b1-SYN-transfected and pSH\/CON-transfected MN9D cells, whereas weak staining was observed in pSH2\/\u03b1-SYN-transfected MN9D cells. Bar\u00a0=\u00a050\u00a0\u03bcm. (d) Western blot analysis of \u03b1-synuclein protein levels. Lane 1 MN9D cells; Lane 2 MN9D\/CON cells; Lane 3 MN9D\/\u03b1-SYN\u2212 cells. (e) Statistical analysis of \u03b1-synuclein protein levels in MN9D, MN9D\/CON and MN9D\/\u03b1-SYN\u2212 cells using the linear density ratio of \u03b1-synuclein\/\u03b2-actin. \u03b1-Synuclein protein levels were significantly decreased in MN9D\/\u03b1-SYN\u2212 cells (0.150\u00a0\u00b1\u00a00.004) compared with MN9D (0.401\u00a0\u00b1\u00a00.038) and MN9D\/CON (0.321\u00a0\u00b1\u00a00.011) cells [n\u00a0=\u00a05, F\u00a0=\u00a0162.034, df(total)\u00a0=\u00a014, **p\u00a0<\u00a00.001 compared with MN9D cells, **p\u00a0<\u00a00.001 compared with MN9D\/CON cells]\nEffects of Silencing \u03b1-Synuclein Expression on Cell Viability\nNo significant differences in cell morphology were observed between the three groups of MN9D cells (data not shown). As shown in Fig.\u00a02, the cell viability of MN9D\/\u03b1-SYN\u2212 cells was significantly decreased compared with control MN9D and MN9D\/CON cells [n\u00a0=\u00a05, F\u00a0=\u00a0421.457, df(total)\u00a0=\u00a014, **p\u00a0<\u00a00.001 compared with MN9D cells, **p\u00a0<\u00a00.001 compared with MN9D\/CON cells]. Cell viability was decreased by 58.3 and 50% in MN9D\/\u03b1-SYN\u2212 cells (0.091\u00a0\u00b1\u00a00.006) compared with MN9D (0.217\u00a0\u00b1\u00a00.012) and MN9D\/CON (0.189\u00a0\u00b1\u00a00.005) cells, respectively (Fig.\u00a02). These data suggest that \u03b1-synuclein may be important for the viability of MN9D cells.\nFig.\u00a02Effects of silencing \u03b1-synuclein expression on cell viability. Cell viability was measured using an MTT assay. Cell viability was decreased in MN9D\/\u03b1-SYN\u2212 cells compared with MN9D and MN9D\/CON cells [n\u00a0=\u00a05, F\u00a0=\u00a0421.457, df(total)\u00a0=\u00a014, **p\u00a0<\u00a00.001 compared with MN9D cells, **p\u00a0<\u00a00.001 compared with MN9D\/CON cells]\nTH Expression and Activity in MN9D Transfected Cells\nPrevious studies have reported that TH expression may be down-regulated by over-expression of \u03b1-synuclein in MES23.5 and M17 cells [3, 42]. Here, we examined whether a similar effect occurred after the silencing of \u03b1-synuclein expression in MN9D\/\u03b1-SYN\u2212 cells. TH mRNA, protein and phosphorylation levels were measured by real-time RT-PCR and Western blot analysis, respectively. Real-time RT-PCR showed that there were no significant differences in TH mRNA levels between MN9D, MN9D\/CON (116.00\u00a0\u00b1\u00a010.23%), MN9D\/\u03b1-SYN\u2212 (135.89\u00a0\u00b1\u00a014.58%) and MN9D\/\u03b1-SYN+ (85.67\u00a0\u00b1\u00a05.55%) cells (Fig.\u00a03a). These observations show that silencing \u03b1-synuclein expression did not alter TH gene expression, suggesting that \u03b1-synuclein does not regulate expression of TH. Similar TH protein levels were observed in MN9D, MN9D\/CON (103.55\u00a0\u00b1\u00a00.84%), MN9D\/\u03b1-SYN\u2212 (126.40\u00a0\u00b1\u00a010.81%) and MN9D\/\u03b1-SYN+ (83.89\u00a0\u00b1\u00a04.06%) cells by Western blot analysis (Fig.\u00a03b,\u00a0c). However, TH phosphorylation levels were significantly increased in MN9D\/\u03b1-SYN\u2212 (157.63\u00a0\u00b1\u00a08.36%) cells compared with MN9D and MN9D\/CON (97.45\u00a0\u00b1\u00a05.06%) cells, and significantly decreased in MN9D\/\u03b1-SYN+ cells (20.36\u00a0\u00b1\u00a02.86%) (Fig.\u00a03b,\u00a0d). To directly assess TH activity, we used NSD-1015 to inhibit AADC, thereby blocking the conversion of l-DOPA to DA. We found that l-DOPA levels were increased in MN9D\/\u03b1-SYN\u2212 cells (266.33\u00a0\u00b1\u00a011.05\u00a0nmol\/l) compared with MN9D (121.92\u00a0\u00b1\u00a05.61\u00a0nmol\/l) and MN9D\/CON (124.89\u00a0\u00b1\u00a08.94\u00a0nmol\/l) cells (Fig.\u00a04). In contrast, a significant decrease in l-DOPA levels was observed in MN9D\/\u03b1-SYN+ cells (41.14\u00a0\u00b1\u00a03.89\u00a0nmol\/l). Since accumulation of l-DOPA could only occur if TH actively converted tyrosine to l-DOPA, these data suggest that TH activity was increased in MN9D\/\u03b1-SYN\u2212 cells and diminished in cells over-expressing \u03b1-synuclein. These data support the hypothesis that silencing \u03b1-synuclein expression does not affect TH gene expression but can enhance TH activity by increasing TH Ser40 phosphorylation.\nFig.\u00a03Effects of silencing \u03b1-synuclein expression on TH and phospho-Ser40-TH levels. (a) Statistical analysis of TH gene expression by real-time RT-PCR. Differences in TH mRNA levels between the transfected MN9D cells were not statistically significant (n\u00a0=\u00a05). (b) Western blot analysis of TH, phospho-Ser40-TH and \u03b2-actin protein levels. Lane 1 normal MN9D; Lane 2 MN9D\/CON; Lane 3 MN9D\/\u03b1-SYN\u2212; Lane 4 MN9D\/\u03b1-SYN+ cells. (c) Statistical analysis of TH protein levels in MN9D, MN9D\/CON, MN9D\/\u03b1-SYN\u2212 and MN9D\/\u03b1-SYN+ cells using the linear density percent of TH\/\u03b2-actin. No significant differences in TH protein levels were observed between the transfected MN9D cells (n\u00a0=\u00a05). (d) Statistical analysis of phospho-Ser40-TH protein levels in MN9D, MN9D\/CON, MN9D\/\u03b1-SYN\u2212 and MN9D\/\u03b1-SYN+ cells using the linear density percent of phospho-Ser40-TH\/TH. Phospho-Ser40-TH protein levels were significantly increased in MN9D\/\u03b1-SYN\u2212 cells and significantly decreased in MN9D\/\u03b1-SYN+ cells compared with MN9D and MN9D\/CON cells [n\u00a0=\u00a05, F\u00a0=\u00a084.505, df(total)\u00a0=\u00a019, **p\u00a0<\u00a00.001 MN9D\/\u03b1-SYN\u2212 cells compared with MN9D cells, **p\u00a0<\u00a00.001 MN9D\/\u03b1-SYN\u2212 cells compared with MN9D\/CON cells; **p\u00a0<\u00a00.001 MN9D\/\u03b1-SYN+ cells compared with MN9D cells, **p\u00a0<\u00a00.001 MN9D\/\u03b1-SYN+ cells compared with MN9D\/CON cells]Fig.\u00a04Effects of silencing \u03b1-synuclein expression on l-DOPA levels. Cell lysates were prepared from transfected MN9D cells incubated in the presence of 200\u00a0\u03bcM NSD-1015 for 30\u00a0min. l-DOPA levels were measured by HPLC. l-DOPA levels were significantly increased in MN9D\/\u03b1-SYN\u2212 cells and significantly decreased in MN9D\/\u03b1-SYN+ cells compared with MN9D and MN9D\/CON cells [n\u00a0=\u00a06, F\u00a0=\u00a0140.944, df(total)\u00a0=\u00a023, **p\u00a0<\u00a00.001 MN9D\/\u03b1-SYN\u2212 cells compared with MN9D cells, **p\u00a0<\u00a00.001 MN9D\/\u03b1-SYN\u2212 cells compared with MN9D\/CON cells; **p\u00a0<\u00a00.001 MN9D\/\u03b1-SYN+ cells compared with MN9D cells, **p\u00a0<\u00a00.001 MN9D\/\u03b1-SYN+ cells compared with MN9D\/CON cells]\nEffects of Silencing \u03b1-Synuclein Expression on DA Levels\nTo assess differences in cytosolic DA levels between the transfected MN9D cells, we prepared cell extracts and measured DA content by HPLC with electrochemical detection. As shown in Fig.\u00a05, DA levels were significantly increased in MN9D\/\u03b1-SYN\u2212 cells compared with MN9D and MN9D\/CON cells. The DA content was approximately 4.8-fold higher in MN9D\/\u03b1-SYN\u2212 cells (834.72\u00a0\u00b1\u00a024.72\u00a0nmol\/l) than in normal MN9D cells (186.04\u00a0\u00b1\u00a031.38\u00a0nmol\/l) and 2.4-fold higher than in MN9D\/CON cells (341.87\u00a0\u00b1\u00a046.25\u00a0nmol\/l). No significant differences in DA content were observed between MN9D and MN9D\/CON cells (Fig.\u00a05). These results indicate that silencing \u03b1-synuclein expression leads to an increase in DA levels in MN9D cells.\nFig.\u00a05Effects of silencing \u03b1-synuclein expression on DA levels. Changes in DA levels were detected by HPLC. DA levels were significantly increased in MN9D\/\u03b1-SYN\u2212 cells compared with MN9D and MN9D\/CON cells [n\u00a0=\u00a06, F\u00a0=\u00a0161.774, df(total)\u00a0=\u00a017, **p\u00a0<\u00a00.001 compared with MN9D cells, **p\u00a0<\u00a00.001 compared with MN9D\/CON cells]. Differences in DA levels between the MN9D and MN9D\/CON cells were not statistically significant\nDiscussion\nIn the present study, we silenced the expression of \u03b1-synuclein in MN9D dopaminergic cells by vector mediated RNAi using the BLOCK-iT\u2122 inducible H1 RNAi entry vector. This vector was selected for several reasons. Firstly, the vector contains a kanamycin resistance gene for selection in E.coli and a ZeocinTM resistance marker to allow generation of stable cell lines that express the shRNA of interest. Secondly, the cloning site of the vector contains a 4-nucleotide overhang on the 5\u2032 end of each DNA strand, allowing directional cloning of the shRNA of interest. Thirdly, the H1 promoter of the vector is recognized by RNA polymerase III resulting in high-level, constitutive expression of shRNA in most mammalian cell types. We identified an effective targeting sequence for RNAi, which localized in the C-terminal coding sequence of the mouse \u03b1-synuclein gene, and used this to generate MN9D\/\u03b1-SYN\u2212 cells in which the expression of \u03b1-synuclein was stably silenced. Due to the endogenous expression of \u03b1-synuclein and the dopaminergic characteristics of MN9D cells, MN9D\/\u03b1-SYN\u2212 is a good cellular model for studying the normal function of \u03b1-synuclein and examining its role in PD pathogenesis.\nWe found that the cell viability of MN9D\/\u03b1-SYN\u2212 cells was significantly decreased compared with MN9D and MN9D\/CON cells, suggesting that loss of \u03b1-synuclein may induce cell injury directly or indirectly. Based on subsequent observations, we propose that a marked increase in cytosolic DA levels may account for decreased cell viability. DA can readily oxidize to generate hydrogen peroxide, superoxide and reactive DA quinones [16, 35], which are cytotoxic due to their inhibitory effects on the mitochondrial respiratory system [5, 13]. In addition, superfluous DA itself may be cytotoxic via inhibition of mitochondrial complex I activity [4]. Thus, it is also possible that increased cytosolic DA levels induced mitochondrial dysfunction resulting in decreased viability in MN9D\/\u03b1-SYN\u2212 cells.\n\u03b1-Synuclein may be involved in the regulation of TH, the rate-limiting enzyme of DA biosynthesis. Several studies have shown that \u03b1-synuclein regulates TH gene expression. In \u03b1-synuclein over-expressing MES23.5 dopaminergic cells, TH mRNA and protein levels were significantly reduced [42]. A similar effect was observed in M17 dopaminergic cells transfected with WT \u03b1-synuclein [3]. However, in the current study, no significant differences in TH mRNA and protein levels were observed between the four groups of MN9D cells. Interestingly, a previous study using MN9D cells reported that \u03b1-synuclein over-expression did not diminish endogenous TH levels [26], which is consistent with our results. We propose that the difference in TH levels between the present \u03b1-synuclein silenced system and the previously described \u03b1-synuclein over-expression system is mainly due to the different cell types and not due to the different ways in which the cells were manipulated.\nAlthough TH protein levels remained unchanged in MN9D\/\u03b1-SYN\u2212 cells, phosphorylation of TH at its Ser40 site was significantly enhanced. Furthermore, l-DOPA levels were significantly increased after NSD-1015 treatments. These results suggest that TH activity was enhanced in MN9D\/\u03b1-SYN\u2212 cells, which is consistent with previous reports [24, 26]. In addition, we also found that cytosolic DA levels were significantly increased in MN9D\/\u03b1-SYN\u2212 cells. Our data show that TH activity was enhanced in \u03b1-synuclein-silenced MN9D\/\u03b1-SYN\u2212 cells, and that this was associated with an increase in DA biosynthesis followed by increased cytosolic DA levels. The mechanisms by which \u03b1-synuclein may regulate TH activity have not been elucidated. It is known that only the phosphorylated form of TH is active, and that the phosphorylation and dephosphorylation of TH are important in regulating DA biosynthesis [29, 37]. Furthermore, phosphorylation of TH at Ser40 results in the most prominent activation of TH [37]. Thus, it has been suggested that \u03b1-synuclein regulates TH activity by regulating the phosphorylation of TH [24]. Protein phosphatase 2A (PP2A), an important enzyme required for dephosphorylation of TH, can be activated by \u03b1-synuclein, resulting in a significant increase in TH dephosphorylation and reduction in TH activity [6]. In addition, other factors may also be involved in the regulation of TH phosphorylation by \u03b1-synuclein. For example, 14-3-3 enhances TH activity by binding directly to phosphorylated TH. \u03b1-Synuclein may directly interact with 14-3-3 [22], thereby accelerating the dissociation of 14-3-3 from phosphorylated TH. \u03b1-Synuclein may also affect TH activity by directly interacting with TH [26].\nIn conclusion, our data demonstrate that in PD pathology, silencing of \u03b1-synuclein expression results in enhanced TH activity and increased DA levels in neurons. We propose that in cells that unable to remove excess DA from the cytosol, DA itself and its oxidized products including reactive oxygen species and reactive DA quinones may lead to cell injury or even death. Further understanding of the normal function of \u03b1-synuclein in maintaining DA homeostasis may help to identify novel preventative or therapeutic strategies for PD.","keyphrases":["\u03b1-synuclein","parkinson\u2019s disease","dopamine","rna interference"],"prmu":["P","P","P","P"]}
{"id":"Eur_Radiol-3-1-1914269","title":"A new approach to the assessment of lumen visibility of coronary artery stent at various heart rates using 64-slice MDCT\n","text":"Coronary artery stent lumen visibility was assessed as a function of cardiac movement and temporal resolution with an automated objective method using an anthropomorphic moving heart phantom. Nine different coronary stents filled with contrast fluid and surrounded by fat were scanned using 64-slice multi-detector computed tomography (MDCT) at 50\u2013100 beats\/min with the moving heart phantom. Image quality was assessed by measuring in-stent CT attenuation and by a dedicated tool in the longitudinal and axial plane. Images were scored by CT attenuation and lumen visibility and compared with theoretical scoring to analyse the effect of multi-segment reconstruction (MSR). An average increase in CT attenuation of 144 \u00b1 59 HU and average diminished lumen visibility of 29 \u00b1 12% was observed at higher heart rates in both planes. A negative correlation between image quality and heart rate was non-significant for the majority of measurements (P > 0.06). No improvement of image quality was observed in using MSR. In conclusion, in-stent CT attenuation increases and lumen visibility decreases at increasing heart rate. Results obtained with the automated tool show similar behaviour compared with attenuation measurements. Cardiac movement during data acquisition causes approximately twice as much blurring compared with the influence of temporal resolution on image quality.\nIntroduction\nThe introduction of multi-detector computed tomography (MDCT) has permitted the non-invasive visualisation of coronary arteries with sufficient temporal and spatial resolution. Moreover, MDCT has been used to research the assessment of coronary artery stent patency and discrimination between the presence of in-stent stenosis. Re-stenosis occurs in a substantial amount of patients which have been treated with stent implantation [1]. It has been shown that no direct visualisation of coronary in-stent re-stenosis is feasible using four-slice MDCT due to partial volume effects and beam hardening caused by metal artifacts of the stents [2\u20136]. Recently it has been shown that despite some limitations 16-slice MDCT is sufficiently useful for the assessment and detection of in-stent restenosis in patients with a high accuracy in comparison with conventional coronary angiography (CAG) [7]. With the emergence of 40 and 64-slice MDCT systems the assessment of lumen visibility and diagnostic accuracy of in-stent restenosis has been improved considerably with respect to the 16-slice MDCT systems [8, 9]. The artificial lumen narrowing shows a decrease of approximately 5% and the CT attenuation is reduced by approximately 35\u00a0HU [10].\nVisualisation of the in-stent lumen with 16-slice MDCT allows for the assessment of coronary artery stent patency based on the measured enhancement of contrast [11]. It has been shown that the best diagnostic quality images are obtained with a sharp edge-enhancing reconstruction kernel, although the quality of the obtained images is hampered by increased noise levels compared with standard kernels [11\u201313]. The results originate from the comparison of the lumen visibility images pairs, which are reconstructed with different reconstruction kernels. The apparent lumen width is measured using a digital measuring tool included in the visualisation software. This method, however, is subjective to the observer and it is difficult to determine the exact boundary of the stents in the gradient part of the image. In addition, the image quality of the stents is very sensitive to the movement of the heart and image quality diminishes at heart rates higher than 75 beats per min (bpm) [14, 15].\nIn previous studies, a patient population was used to investigate image quality of coronary artery stents at high and low heart rates on four- and 16-slice MDCT [14, 15]. To our knowledge no systematic study has been published about the image quality of coronary artery stents on 64-slice MDCT. In this study, we therefore aimed at describing the correlation of image quality of coronary artery stents and heart rate using 64-slice MDCT in an ex-vivo setting by using an anthropomorphic moving heart phantom.\nThe purpose of our study was to assess the lumen visibility of coronary artery stents at various heart rates with an automated objective method using a moving heart phantom on a 64-MDCT system.\nTheory\nCoronary imaging is hampered by motion artefacts originating from the relative large velocity scale of the coronary arteries within the cardiac cycle compared with the temporal resolution of the MDCT acquisition technique [16]. This movement results in motion artefacts in the reconstructed images, expressed as blurring. The amount of blurring depends on (1) the amount of movement of the imaging target MS and (2) the acquisition time (AT). The amount of movement of the coronary artery is a function of the heart rate (HR) of the patient. The temporal resolution of a MDCT system is a function of the reconstruction method used and HR [17, 18]. This means that the resulting image quality (IS) can be expressed as \nin which MS describes the amount of movement of the coronary artery as a function of heart rate, AT describes the acquisition time as a function of heart rate and g is a weight factor.\nThe heart rates can be ranked from low to high. With this ranking, a relative grading can be made for the function MS. In addition, the acquisition times can be ranked from high to low. With this ranking, a relative grading can be made for the function AT. The results of this grading are shown in Electronic supplementary material (ESM) Table\u00a01, where a high score implies a high image quality. Since there is no cardiac movement at 0\u00a0bpm, this heart rate was graded with the highest score for both factors.\nMaterials and methods\nNine commercially available stents were used and stent dimensions were measured using a digital calliper. The stent properties are summarised in ESM Table\u00a02.\nThe stents were inserted into plastic tubes and the tubes were filled with contrast fluid (Visipaque 320, Amersham Health, Little Chalfont, UK) diluted to an attenuation value of approximately 200\u00a0HU simulating contrast enhanced blood. The tubes were wrapped in horse fat with an attenuation value of \u2212100\u00a0HU to simulate the in vivo situation of epicardial fat (Fig.\u00a01left).\nFig.\u00a01Left: schematic figure showing the experimental setup of the contrast fluid filled tube surrounded by horse fat in the axial plane. The numbers depict the HU values of the different relevant structures. Right: moving anthropomorphic heart phantom with artificial coronary arteries\nNext, the stents were attached to a silicon, moving, anthropomorphic heart phantom (Limbs & Things, Bristol, UK) with artificial coronary arteries (Elastrat, Geneva, Switzerland) (Fig.\u00a01right). The phantom is connected to a respirator, which was used to control the heart rate. The heart rate was set at values of 0, 50, 60, 70, 80, 90 and 100\u00a0bpm.\nMeasurements have been performed on a 64-detector CT-scanner (Somatom Sensation 64, Siemens, Forchheim, Germany). The scan parameters were 120\u00a0kV, 120\u00a0mAs, 370\u00a0ms rotation time and 64\u2009\u00d7\u20090.6\u00a0mm collimation.\nImage reconstruction was performed using a sharp convolution kernel (Siemens B46f) with a reconstruction slice width of 0.75\u00a0mm and a 0.5-mm increment. ECG-gating was used during scanning and the images were retrospectively reconstructed at 25% of the RR-interval corresponding to maximum expansion of the heart phantom.\nAfterwards the stents were visualised on an Aquarius workstation version 3.3 (Terarecon, San Mateo, USA). We used two independent methods to asses the lumen visibility of the stents in the reconstructed images. In the first method, the average HU-value in the stent lumen was manually measured with a standard region of interest (ROI) technique. A window level\/width of 300\/800 was used for visualisation with an average ROI of 5.0\u00a0mm2 and 3.9\u00a0mm2 in the longitudinal and axial plane, respectively (Fig.\u00a02). The lumen measurements were used to calculate the mean CT attenuation in the stent lumen. In the second automated method, screenshots were taken from every stent in the longitudinal and the axial plane with a window level\/width of 512\/2,048. The screenshots were used to perform the automated lumen visibility measurements using a dedicated tool.\nFig.\u00a02Measuring the CT attenuation in the lumen. Left: measurements in the axial plane, Lekton Motion at 60\u00a0bpm. Right: measurements in the longitudinal plane, Lekton2 at 90\u00a0bpm. Calculated by the Aquarius workstation are mean HU-value, standard deviation and area of ROI\nThe automated lumen visibility measurements were performed with an Automatic Stent Visibility Calculation (ASVC), a custom-build Matlab tool (The Mathworks, Natick, USA). The tool constructs the average attenuation profile through the stent in the longitudinal and axial plane. The calculation of the average attenuation profile in the longitudinal plane is done as follows; attenuation profiles are calculated from position A to B for every image line perpendicular to the stent central axis, where the position A and B are determined by the user (Fig.\u00a03left). The average longitudinal profile is calculated from the individual longitudinal profiles. The calculation of the average attenuation profile in the axial plane is doen as follows: after determination of the centre of the stent by the user, 180 attenuation profiles are calculated evenly distributed around the centre (Fig.3right). The average axial attenuation profile is calculated from the individual axial profiles.\nFig.\u00a03Stent images (left: Jostent Stent Graft at 60\u00a0bpm; right: Taxus at 80\u00a0bpm) showing the positions of the individual profiles for calculation of the average stent profile in the longitudinal plane (left) and the axial plane (right)\nThe profile depth of the lumen (D), the full width at half maximum (FWHM) and the total width (TW) are calculated from both average profiles (ESM Fig.\u00a04).\nWe define the percentual width (Wp) as a percentage of the FWHM and TW \nThe percentual width is used instead of the absolute width to compensate for different magnifications of the stent images.\nFrom the profile depth (D) and Wp, the lumen visibility (LV) is defined by \nIf the left and right edge of the attenuation profile are at different heights, D is calculated from the average height.\nThe stent images at individual heart rates were ranked according to their CT attenuation (7=least attenuation, 1=most attenuation), FWHM (7=largest width, 1=smallest width) and D (7=largest depth, 1=smallest depth) in the axial and longitudinal plane.\nThe difference between the CT attenuation values in the axial and longitudinal plane were compared using the Wilcoxon test at a significance level of 5%. The correlation between heart rate and CT attenuation, and the correlation between heart rate and LV, were analysed by calculating the Spearman rank correlation coefficient at the significance level of 5%.\nResults\nCT attenuation in the stent lumen\nThe results for the HU-value measurements are summarised in ESM Tables\u00a03 and 4 for the longitudinal and axial plane, respectively. The CT attenuation in the stent lumen was calculated for each heart rate and the results are shown for the longitudinal and axial plane in ESM Figs.\u00a05 and 6, respectively. The error margins have been omitted to improve readability of both figures.\nAll stents showed increased attenuation with increasing heart rate in the longitudinal plane. The CT attenuation in the longitudinal plane showed an increase from 0 to 100\u00a0bpm between 75 and 221\u00a0HU for the Taxus and Bx Sonic stent, respectively. The average increase was 139\u2009\u00b1\u200949\u00a0HU. The average Spearman coefficient for all stents was 0.66.\nAll stents showed increased attenuation with increasing heart rates in the axial plane. The CT attenuation in the axial plane showed an increase from 0 to 100\u00a0bpm between 76 and 252\u00a0HU for the Bx Sonic, Multi-link Zeta and the Jostent, respectively. The average increase was 148\u2009\u00b1\u200969\u00a0HU from 0 to 100\u00a0bpm. The average Spearman coefficient in the axial plane was 0.58. The average increase of CT attenuation over both planes was 144\u2009\u00b1\u200959\u00a0HU.\nUsing the ASVC, the FWHM and depth of the stent profile were determined. The results for the FWHM are summarised in ESM Tables\u00a05 and 6; the results for the profile depth are summarised in ESM Tables\u00a07 and 8. The standard deviation for every measurement is given in parentheses.\nThe LV was calculated using equations\u00a02 and 3 at each HR using the measured width FWHM and depth D. The data are plotted in ESM Figs.\u00a07 and 8 for the longitudinal and axial plane, respectively, and have been fitted to a line. Error margins have been omitted to improve readability.\nAll stents showed decreasing LV with increasing heart rate in the longitudinal plane. The LV decreased from 0 to 100\u00a0bpm between 7.5% and 48.6% for the Taxus and Cypher stent respectively. The average decrease in LV was 26.8\u2009\u00b1\u200913.4%. The average Spearman coefficient was 0.47.\nAll stents showed decreasing LV with increasing heart rates in the axial plane. The LV decreased from 0 to 100\u00a0bpm between 19.8% and 48.8% for the Lekton and Multi-link Zeta stent respectively. The average decrease in LV was 30.9\u2009\u00b1\u200910.4%. The average Spearman coefficient was 0.58. The average decrease of LV over both planes was 29\u2009\u00b1\u200912%.\nImage scoring\nThe measurements are shown in ESM Fig.\u00a09 after grading and averaging as described in the methods section. From a least squares fit, we found an optimal value of g\u2009=\u20090.63\u2009\u00b1\u20090.06.\nDiscussion\nCoronary artery stenting is a successful method to treat stenosis. However, re-stenosis in the stent may happen after the procedure. This makes check-ups a necessary procedure to perform [19, 20]. MDCT is one the candidates for visualisation of the lumen of coronary artery stents. Although the results look promising, the image quality is hampered by cardiac movement and metal artefacts [21, 22]. This may result in blurred images and an exaggerated thickness of the stent struts compromising the visibility of the lumen. The 64-MDCT scanner has increased spatial and temporal resolution compared with previous CT scanner generations [23, 24] and increased LV of coronary stents is expected with these new scanners. Although 64-MDCT imaging of coronary artery stents show an improved image quality with respect to previous CT scanners, the images still show blooming artefacts and blurring [9].\nThe apparent width is measured in a generally accepted method to assess the stent lumen, but this method is very subjective to the user [25]. In contrast, our method, developed in this study to analyse the stent lumen, is an automated method. It enables the possibility of a systemic evaluation of the stent lumen at various heart rates. The in vivo conditions were approached as much as possible using contrast fluid and fat.\nInfluence of increasing heart rate\nTo our knowledge there have been no previous studies systematically investigating the relation between heart rate and image quality of coronary stents using 64-MDCT. There are, however, some studies with 16-slice MDCT assessing coronary arteries, which concluded that for successful cardiac imaging a heart rate below 75 or 70\u00a0bpm is necessary [14, 15, 26\u201329]. In a study with 40-MDCT by Gaspar et al. [8], patients were given an oral beta-blocker if their heart rate was higher than 65\u00a0bpm, and in a study with 64-MDCT, patients were given beta-blockers if their heart rate was higher then 70\u00a0bpm [30]. Ferencik et al. [23, 30] concluded that a low heart rate is an important prerequisite for excellent image quality.\nASVC\nTwo different methods were used to assess the relationship between heart rate and image quality of coronary stents. The first method, the CT attenuation measurement, is a commonly used method. The second method, the ASVC technique, is a new approach. Both methods show a negative correlation between image quality and heart rate as expressed by the linear fits. The fits, however, show small significance. This small significance can be explained by the fact that the image quality does not depend entirely on heart rate, which shows a linear behaviour, but also on temporal resolution, which shows a non-linear behaviour, as can be seen from ESM Fig.\u00a09. Despite the poor quality of the fit, the results obtained with this new ASVC method are in agreement with previous studies using generally accepted methods as stated before. Therefore, we conclude that our developed tool is a valid method for the analysis of coronary stent lumen.\nImage scoring\nThe image scoring results (Fig.\u00a09) show a varying resemblance between the theoretical scoring and the measured scoring. This resemblance depends on the proportion g and varies between 0 (IS completely depends on AT) and 1 (IS completely depends on MS). The best resemblance was found for g\u2009=\u20090.63. This implies that the cardiac movement causes almost twice as much blurring in the CT image compared with blurring caused by the limited temporal resolution of the acquisition method. Multi-segment reconstruction (MSR) has been developed to further increase the temporal resolution [6, 18, 32]. The data acquisition at a rotation time of 370\u00a0ms and a HR\u2009>\u200965\u00a0bpm has been performed during two consecutive heart cycles using a two-segment MSR. At heart rates of 70 and 90\u00a0bpm, the data acquisition is approximately evenly distributed over two consecutive heart cycles. At 80 and 100\u00a0bpm, however, the majority of data is acquired in the first heart cycle, and the remaining portion in the second heart cycle. From Fig.\u00a09, we can deduce that for HR of 70 and 90\u00a0bpm the measured IS is smaller than the theoretical IS. For HR of 80 and 100\u00a0bpm, the measured IS is larger than the measured IS. From this we can conclude that it is beneficial to the image quality to acquire during one cycle and that the MSR technique is not beneficial to image quality. In contrast to this, Halliburton et al. [31] showed that MSR is beneficial for 16-slice MDCT. Two other studies concluded that MSR was not beneficial to the image quality for four-slice MDCT, in accordance with our results [31, 32]. This can be explained by the fact that the coronary artery has to be in exactly the same position in the two heart cycles for the MSR algorithm to work properly. However, in general the starting position of the arteries at the beginning of the heart cycle will not exactly be the same as in the previous heart cycle. This phenomenon will introduce additional motion artefacts which result in extra blurring to the image, as has been shown before by Greuter et al. [33].\nReducing blurring\nCardiac movement and limited temporal resolution will introduce blurring in CT images of coronary arteries. The result for image scoring showed that the influence of cardiac movement is almost twice as large as the influence of the temporal resolution. To reduce blurring, it is therefore more efficient to reduce the heart rate than to increase the temporal resolution.\nLimitations\nThese results have been obtained using a moving heart phantom. It remains uncertain how well these results are applicable to patients. However, a previous study showed that the movement of the heart phantom is a good approximation to the in-vivo situation [18]. Furthermore, in order to approach the in-vivo situation as close as possible, contrast fluid and fat were used, with CT attenuation values equal to those in-vivo.\nScreenshots captured with the visualisation software were used to perform LV measurements. A loss of information is expected due to image compression used in the .jpg format of the screenshots. However, a comparison between the screenshot method and direct analysis using dicom files showed no observable difference in results.\nConclusion\nWe have shown that at increasing heart rates the CT attenuation in the stent increases and the lumen visibility decreases. A new approach to asses the stent lumen has been described which shows similar results to CT attenuation measurements and previous studies. The cardiac movement during data acquisition causes approximately twice as much blurring compared with the influence of temporal resolution. We conclude that a lowering of the heart rate is more beneficial to image quality than using a multi-sector reconstruction technique. In addition, we conclude that it is beneficial to image quality to acquire data in one cardiac cycle.\nElectronic Supplementary Material\nBelow is the link to the electronic supplentary material. \nFig.\u00a04\nFigure showinf the profile of the Bx Sonic at 80\u00a0bpm with the different variables which are determined by the ASVC; the dotted blue line depicts the error margins of the profile. TW total width; D depth; FWHM=full width half minimum (DOC 56 kb)\nFig.\u00a05\nCT attenuation measured in the stent lumen at various heart rates in the longitudinal plane (DOC 76 kb)\nFig.\u00a06\nCT attenuation measured in the stent lumen at various heart rates in the axial plane (DOC 77 kb)\nFig.\u00a07\nLumen visibility plotted versus heart rate in the longitudinal plane (DOC 75 kb)\nFig.\u00a08\nLumen visibility plotted versus heart rate in the axial plane (DOC 74 kb)\nFig.\u00a09\nThe measured IS (green), the theoretical IS for g\u2009=\u20090.63 (blue), the theoretical IS for g\u2009=\u20090 (dotted red), the theoretical IS for g\u2009=\u20091 (dotted orange) (DOC 53 kb)\nTable\u00a01\nAcquisition times for a 64-MDCT with 370\u00a0ms rotation time, number of cycles used and theoretical scores for every HR used in this study. AT acquisition time; MS the amount of movement of the imaging target (DOC 32 kb)\nTable\u00a02\nCommercial name, manufacturer, material, length and diameter of the stents used in the study (DOC 33 kb)\nTable\u00a03\nHU-value measurements in the longitudinal plane (DOC 34 kb)\nTable\u00a04\nHU-value measurements in the axial plane (DOC 34 kb)\nTable\u00a05\nResults of the measured FWHM in the longitudinal plane (DOC 34 kb)\nTable\u00a06\nResults of the measured FWHM in the axial plane (DOC 34 kb)\nTable\u00a07\nResults of the measured profile depth in the longitudinal plane (DOC 34 kb)\nTable\u00a08\nResults of the measured profile depth the axial plane (DOC 34 kb)","keyphrases":["64-slice mdct","heart phantom","coronary stent","image quality"],"prmu":["P","P","P","P"]}
{"id":"Clin_Oral_Investig-4-1-2238780","title":"Pathological or physiological erosion\u2014is there a relationship to age?\n","text":"This conventional literature review discusses whether pathological tooth wear is age dependant. It briefly reviews the components of tooth wear and the prevalence of tooth wear in children, adolescents and adults. The emphasis on terminology relating to tooth wear varies. In some countries, the role of erosion is considered the most important, whereas others consider the process to be a combination of erosion, attrition and abrasion often with one being more dominant. The importance of tooth wear or erosion indices in the assessment and the evidence for progression within subject and within lesions is described. The data from the few studies reporting pathological levels of wear reported in children and adults are discussed, in particular its relationship with age. There is little evidence to support the concept that pathological levels of erosion or wear are age dependant. There is, however, some evidence to suggest that normal levels of erosion or wear are age dependant.\nIntroduction\nThere has been considerable interest recently on the epidemiology and pathogenesis of dental erosion. Much of the epidemiology has been investigated in children and adolescents rather than adults and so the validity of any association between severity and age is based on clinical observations seen primarily in these young people [1, 3, 7, 17]. The aim of this paper is to consider and investigate the concept that pathological erosive tooth wear is age dependant.\nThere is some debate within the dental academic community on the appropriateness of terminology. Many academics recognise the term tooth wear as encompassing erosion, attrition and abrasion. However, other researchers focus upon acid erosion often using the term to describe what others would call tooth wear and use the term erosion in a context which others might not agree. Whilst the definitions of erosion, abrasion and attrition are accepted, the relative importance of these causes is not. Therefore, in this paper both terms are used where appropriate to convey meaning interpreted by many researchers working in this field.\nTooth wear and erosion\nThe causes of tooth wear are considered to be erosion, abrasion, attrition and possibly abfraction. Traditionally, erosion is associated with loss of enamel and dentine from acids either intrinsic [4] (gastric) or extrinsic (dietary) [29]. Abrasion is more commonly associated with wear from surfaces other than teeth, whereas attrition is wear from tooth to tooth contact [26]. Abfraction is a considered by some to be a component of tooth wear, but so far apart from some laboratory studies, there is little clinical evidence to provide support for its role [9].\nHowever, different countries interpret the aetiologies in different ways [8]. Many European countries focus attention upon erosive tooth wear, whereas in North American countries, abrasion or attrition is more commonly recognised whilst the role of erosion is less understood. In the United Kingdom, although the importance of erosion is acknowledged, the impact of abrasion and attrition is also recognised, and the term tooth wear is more commonly used. When interpreting studies from different parts of the world, some consideration of these subtle differences should be understood. The major difference seems to be the impact of attrition. For many clinical situations, the impact of wear on the teeth is a combination of attrition and erosion particularly on the incisal or occlusal surfaces of teeth (Figs.\u00a01 and 2) [10]. On the buccal or lingual surfaces, erosion is usually more dominant, but the impact of abrasion should not be forgotten. Since it is almost impossible to tell from the appearance of a lesion what is the underlying cause, it maybe more correct to use the term tooth wear. But there are circumstances in which one cause, more commonly erosion, is the dominant feature. Although the definitions of tooth wear and erosion are quite different, they can often be used to describe the same process.\nFig.\u00a01The wear on the occlusal\/incisal surfaces of the upper anterior teeth. The wear is caused by a combination of erosion and attrition. The \u201ccupped out or ditched\u201d areas result from the action of acids, whereas the flatter surfaces are caused by attritionFig.\u00a02The wear on the palatal\/lingual surfaces of the teeth has been caused by regurgitation erosion. It is unlikely that abrasion or attrition has contributed to the process\nPrevalence\nTooth wear indices grade the severity of wear by recording the surface characteristics of teeth with a numerical score. These data can be used to compare wear rates between individuals and between different populations. However, there is currently no agreed consensus on a universally accepted tooth wear index. Some indices [20, 24] appear to be more widely used than others, but even these have undergone many modifications since they were first published [22]. For the most part, indices record wear on all tooth surfaces: cervical, buccal, occlusal\/incisal and palatal\/lingual [24]. The Smith and Knight index [24] records wear on all surfaces, but no attempt is made to relate the aetiology to the outcome of the wear on the teeth. Smith and Knight and later Robb and Smith estimated acceptable levels of wear in each age cohort [23, 25].\nFor the majority of the population, any wear on teeth is often limited to enamel, and dentine involvement only occurs in a relatively small proportion of the population [25]. A study by Dugmore and Rock [17] reported that 59.7% of 1,753 12-year-old children had evidence of tooth wear of which 2.7% had exposed dentine and this rose to 8.9% by the age of 14\u00a0years. Another study by Bardsley et al. [3] reported that 53% of 2,385 14-year-old adolescents had exposed dentine, but this included assessment of incisal surfaces. This latter study reported higher values in contrast to most other studies [1, 7]. From most of these studies, it is clear that in children and adolescents wear of enamel is common, almost normal, but wear of dentine exposing more than 1\/3 of the tooth surface is less so.\nSmith and Robb observed that tooth wear in adults was an almost universal experience with up to 97% of all ages experiencing some wear on their teeth with the older aged cohorts, dentine exposure became more common [25]. In their study, Smith and Robb [25] identified that between 5\u20137% of 1,007 adults had wear that could justify treatment, in all age cohorts. This is the only study published to date, with a large sample, which provides data on tooth wear in all age groups in adults. Other studies have reported the levels in specific age cohorts [20]; Donachie and Walls [14] reported the degree of tooth wear in a sample of 586 adults aged 45 and over in Newcastle, UK and reported no correlation between severity of wear and age. The data available to guide our knowledge on the levels of tooth wear in adults are therefore limited.\nProgression of tooth wear\nThe term pathological tooth wear has been used to describe unacceptable levels of wear [18, 23, 25]. It is usually interpreted as meaning a level of wear that could justify operative treatment. However, finding a scientific way to interpret this concept is challenging. For pathological tooth wear to have meaning, the normal levels in different age groups are required to allow comparison, but the basic information on the pathogenesis of tooth wear is unknown. For instance, it is not known if tooth wear is episodic or continual throughout life. A few authors have reported data from longitudinal studies on adolescents and adults that indicate the development of new lesions [16, 19]. Lussi et al. [20] reported the results of a prevalence study on 26\u201330- and 46\u201350-year-old Swiss adults. In a later study, the same group of researchers reported the progression of wear in the same subjects over a 6-year period [19] and observed that wear progressed on the facial, occlusal and cervical surfaces and was more common in the older aged groups. However, the authors made no attempt to define what level of tooth wear was acceptable and what was not. Other studies have reported increase in the incidence of tooth wear in adolescents [16], but longitudinal data remain sparse.\nThere are no studies that report the progression of the same lesion within the same subject apart from one study which used a tooth wear index to record wear on study models taken from the subject's teeth [5]. They observed that only 7% of tooth surfaces showed any change over a median time of 26\u00a0months (IQR 14\u201350\u00a0months). The paucity of data on the severity of tooth wear in different age groups and its progression means it is difficult to be able to predict what is the pathophysiological behaviour of tooth wear.\nA number of authors have published methods to measure tooth wear and erosion using profilometric scanners to accurately map teeth [2, 13, 21, 28]. But these methods have reported their findings on relatively small numbers, and the methods are as yet unsuitable for larger studies. Often the time needed to scan a single tooth can take up to one hour and therefore they remain research tools for small studies. However, data from these studies suggest that wear on teeth may progress between 3.7\u00a0\u03bcm at 6\u00a0months [6] and 5.56\u00a0\u03bcm\/month [21] to 18.3\u00a0\u03bcm\/month [28]. There are no data to indicate if particular teeth or tooth surfaces have a greater potential to wear or whether abrasion, attrition or erosion is more important in the progression. Furthermore, correlating this rather limited data to the general population is not possible, and therefore, most researchers rely upon epidemiological studies to record the severity of wear.\nPathological tooth wear\nThe concept that an unacceptable level of wear for a particular age group was first proposed by Eccles, Smith and others [18, 23, 25]. These and other authors argued that tooth wear continued throughout life as it was part of a normal physiological process. Berry and Poole [11, 12] hypothesised that in common with other mammals, humans have compensatory mechanisms that adapt to wear of teeth. They based their argument on comparative anatomy of mammals, in particular large herbivores, and suggested that tooth wear was beneficial to the efficiency of mastication. A gradual reduction in height and shape of cusps on molar teeth, they argued, led to improved masticatory efficiency. Their hypothesis remains unique and unproven but raises a number of interesting concepts.\nSmith and coworkers [23, 27] introduced the term unacceptable levels of wear and later re-termed it as pathological tooth wear. The authors estimated unacceptable levels of wear based on their clinical judgement. Their so-called threshold levels were calculated for different age groups and used as an indicator for the necessity of operative intervention. However, these thresholds were judgements made by the authors, albeit based on clinical experience and previous research [25]. Smith and Robb [25] reported in their paper that the ultimate decision on the threshold values was the clinical experience of the first author. The data were analysed and re-analysed until the results matched the clinical judgement of the authors. Whilst this empirical assessment may have had some value at the time of publication, the ability to reproduce agreed thresholds in subsequent investigations would be challenging and have not been wholeheartedly supported by other researchers. Donachie and Walls [15] argued strongly that the threshold levels set for older age groups were inaccurate as they were based on a relatively small sample and so in their 1996 paper modified values were used [25].\nWe cannot rely upon the clinical judgement of one or two researchers to define what are pathological levels of tooth wear. Scientists need to have more defined and reproducible values. There are emerging data to suggest that tooth wear is common in all age groups [3, 17, 20, 25]. Smith and Robb [25] reported in their study of 1,007 subjects, aged 16 and over, that within each age cohort a proportion had higher grades of tooth wear [mean 5.1%] than the others. Virtually every other study on the prevalence of tooth wear also reports that a small proportion, generally between 2% and 10%, have higher levels of tooth wear than the majority. If these data are representative of the population at large, it is possible that a percentage of each age cohort has higher than normal levels of wear and this could be termed pathological.\nThe hypothesis that pathological tooth wear is age related depends on who is assessing the impact. State health care authorities, private insurance schemes, industry, dentists and patients all have different interpretations on what is pathological and what is not. For patients, loss of enamel could be considered pathological particularly if they are focussed upon the appearance of their teeth, whereas dentists may consider intervention is needed when dentine is involved, but their capacity to treat is affected by the limitations of restorative materials. On the other hand, state health care providers may take a much longer term view, with the assumption that a tooth remains functional, if not aesthetic, when operative care is not imperative.\nBased on the current data, it is too challenging to suggest that tooth wear is an age-related phenomenon. There is some justification to this hypothesis based on the current data from children, adolescents and adults. Clinical experience suggests that as adults age they tend to develop more wear on the occlusal and incisal surfaces of teeth. It is likely, therefore, that some progression of wear on teeth is age related. However, this assumption needs investigating. Evidence partly from prevalence studies and partly from accurate measurement of tooth wear by profilometry tends to give support to this hypothesis. It must be remembered, however, that the prevalence studies at best report on just over 1,000 subjects. This is extremely small considering the populations involved. Tooth wear indices remain the most convenient and reproducible method to grade severity but are limited by incompatible criteria. It is imperative that a consensus is developed to build a simple and reproducible index, used by researchers so that data on the prevalence of tooth wear, particularly in adults, can be investigated.\nConclusion\nThe physiological wear of teeth is probably an age-related phenomenon. As the teeth continue to function and be challenged by erosive, attritive and abrasive factors, there will be change to the surfaces of teeth. This is most commonly seen on the incisal edges of the upper and lower incisors. As the challenges continue throughout life, it is not surprising therefore to see, as reported in Smith and Robb's study, that most people have some evidence of wear [25]. Therefore, small changes or evidence of gradual wear throughout life is probably a feature of the ageing of the dentition.\nWhen pathological levels of wear are considered, the situation is less clear. There are insufficient data from epidemiological studies on adults to be definitive. But where data are present, it suggests similar proportions of severe levels of wear are observed in each age group, and it could be argued that this was independent of age [3, 17, 25]. It seems, therefore, that although tooth wear is an age-dependant phenomenon, severe tooth wear is not.","keyphrases":["erosion","tooth wear","attrition","abrasion","tooth wear index"],"prmu":["P","P","P","P","P"]}
{"id":"Anal_Bioanal_Chem-4-1-2359830","title":"Investigation into mercury bound to biothiols: structural identification using ESI\u2013ion-trap MS and introduction of a method for their HPLC separation with simultaneous detection by ICP-MS and ESI-MS\n","text":"Mercury in plants or animal tissue is supposed to occur in the form of complexes formed with biologically relevant thiols (biothiols), rather than as free cation. We describe a technique for the separation and molecular identification of mercury and methylmercury complexes derived from their reactions with cysteine (Cys) and glutathione (GS): Hg(Cys)2, Hg(GS)2, MeHgCys, MeHgGS. Complexes were characterised by electrospray mass spectrometry (MS) equipped with an ion trap and the fragmentation pattern of MeHgCys was explained by using MP2 and B3LYP calculations, showing the importance of mercury\u2013amine interactions in the gas phase. Chromatographic baseline separation was performed within 10 min with formic acid as the mobile phase on a reversed-phase column. Detection was done by online simultaneous coupling of ES-MS and inductively coupled plasma MS. When the mercury complexes were spiked in real samples (plant extracts), no perturbation of the separation and detection conditions was observed, suggesting that this method is capable of detecting mercury biothiol complexes in plants.\nIntroduction\nMercury is one of the most important elements to consider when environmental pollution is concerned, and ever since the Minamata disaster in 1959, the organic compound methylmercury has been in focus owing to its enhanced toxicity and its biomagnification properties in the food chain [1].\nFollowing the Minamata disaster, the differentiation of inorganic mercury in the form of Hg2+ from organic mercury, namely in the form of methylmercury, has become a major task. Mercury speciation in complex matrices is not easy to achieve [2], and more and more sophisticated analytical methods have been developed in the past few years in order to obtain reliable quantitative results. In particular, species-specific isotope dilution mass spectrometry (SSIDMS) employing stable mercury isotopes has been a major breakthrough [3, 4]. Otherwise, experiments with stable isotope tracers introduced into the environment have given invaluable information on the distribution and transformation behavior of mercury in the environment [5\u20137]. Mercury has also been identified as a global pollutant, and the importance of new analytical methods towards a more complete understanding of species distribution, pathways and global impact has been highlighted recently [8].\nAs of today, the most common application of mercury speciation in biota is focused on the determination and distinction between methylmercury and inorganic mercury in the forms of MeHg+ and Hg2+. To separate the mercury species from their matrix, usually extraction or soft digestion methods are applied, which conserve the C\u2013Hg bond, keeping the methylmercury moiety intact. However, it is more than unlikely that mercury and methylmercury occur as free cations, be it in biota or in the environment, and rather they are likely bound to sulfur-containing biomolecules, e.g. in fish [9], or present as chloride complexes, e.g. in seawater [10]. But, with the usual practice of mercury speciation, the counterion bound to the mercury species is lost and, even worse, completely disregarded. Thus, the information on the molecular entity of the mercury species is wasted\u2014strictly speaking, this is not really speciation, but rather a fractionation method.\nNow, in spite of the enormous progress made in mercury speciation, many questions relating to mercury and its behavior in biota remain unsolved mysteries: Why is methylmercury highly bioaccumulated, while Hg2+ is not? Why are different organs targeted by methylmercury as opposed to Hg2+, and how can the enormous latency period (up to several months!) for methylmercury intoxication be explained [1, 11]?\nLittle is known today about the mechanisms that enable mercury uptake into cells, and its subsequent distribution and bioaccumulation in biota. But, mercury and methylmercury are well known to bind to biothiols abundant in biota and form stable complexes via Hg\u2013S bonds. Especially, in a model system, mercury complexes with cysteine (Cys) as a ligand have been found to be able to cross cell membranes, and the Hg(Cys)2 complex has been defined as a likely form in which Hg2+ is present. Zalups [12] and Bridges and Zalups [13] proposed that Hg(Cys)2 mimics the cystine molecule, and thus may use the active cell transport systems usually used for cystine transport into the cell. Although the theory of molecular mimicry has recently been questioned using structural comparison obtained from X-ray absorption studies of the complexes concerned [14], it is still commonly agreed that mercury and methylmercury are most likely bound to and transported as complexes formed with biothiols abundant in biota. Other studies employing direct speciation methods in biota, like extended X-ray absorption fine structure and X-ray absorption near-edge structure, underpin the assumption that methylmercury and mercury are bound to sulfur in a complex matrix [9].\nAlthough mercury biothiol complexes have not been identified in vivo so far, mercury biothiols certainly play a key role in cell uptake, distribution and subsequently toxicity of mercury in biota [1, 11\u201313]. But, even state-of-the-art analytical methods for mercury speciation fail to provide us with all the necessary information, i.e. the complete molecular structure of the mercury compounds present in vivo.\nTherefore, our interest lies in the development of an analytical method that allows the direct detection of mercury and methylmercury biothiol complexes in biota.\nIn this paper, we describe an analytical method allowing the species identification of mercury and methylmercury biothiols as intact molecules. As example species, Cys and glutathione (GS) complexes with mercury and methylmercury were synthesised and analysed. Cys is a small sulfur-containing amino acid, while GS is a small peptide comprising only three amino acids: \u03b3-glutamyl (\u03b3-Glu), Cys and glycine (Gly). Like Cys, this compound is usually abundant in living cells in millimolar concentrations, and it is a key compound for the cell\u2019s redox state control: GS is the smallest building block for the synthesis of phytochelatins, which are produced in plants as a response to metal stress, and are believed to play a paramount role in metal detoxification in plants [15]. Plants are well known to accumulate mercury when grown on polluted soil [16, 17], and a future object of study for mercury exposure will be rice (Oryza sativa); therefore, we tested the method and the species stability in the matrix of a real plant extract derived from rice plants.\nExperimental\nInstrumentation\nThe instrumentation used for this work consisted of an Agilent Technologies (USA) suite comprising an 1100 series high-performance liquid chromatography (HPLC) system, an electrospray mass spectrometer (ES-MS) (XCT ion-trap mass spectrometer) and an inductively coupled plasma mass spectrometer (ICP-MS) (7500 c series). The HPLC system was equipped with an automatic degasser, a gradient pump, a thermostated autosampler tray and a thermostated column device.\nFor the chromatographic separation of the mercury biothiols, an HPLC method was adapted from that of Raab et al. [18]. Species separation was carried out using an Agilent Zorbax Eclipse XDB C-18 (4.6\u00a0mm\u2009\u00d7\u2009150\u00a0mm, 5\u2013\u03bcm) reversed-phase (RP) HPLC column using a gradient elution with eluent A being 0.1% formic acid in water and eluent B being 0.25% formic acid in methanol. The gradient programme went from 100% eluent A to 50% eluent B in 20\u00a0min with a flow rate of 1\u00a0mL\/min, and a volume of 50\u00a0\u03bcL was injected via the autosampler. The autosampler tray was cooled to 4\u00a0\u00b0C for all experiments.\nCoupling of the HPLC instrument to the ES-MS and ICP-MS system was performed either individually using PEEK capillary tubing (1.6-mm outer diameter, 0.3-mm inner diameter), or simultaneously via a micro flow splitter (Upchurch, UK). In split mode for simultaneous coupling, 80% of the HPLC eluent was directed into the ES-MS system, while 20% went into the ICP-MS system.\nHg(Cys)2, HgMeCys, Hg(GS)2 and HgMeGS solutions were prepared as described above in concentrations of 10\u00a0mg\/L (as Hg), each in 0.1% formic acid. From these solutions, a mixture containing all four compounds was prepared at equal concentrations, resulting in a solution with a concentration of 2.5\u00a0mg\/L (as Hg) of each compound.\nA continuous internal standard (Rh, 20\u00a0\u03bcg\/L) was mixed with the HPLC effluent prior to the ICP-MS nebuliser via a Teflon T-piece. This internal standard enables the monitoring of overall instrument sensitivity and plasma conditions, and may potentially be used to correct the mercury signal for changes in intensity due to matrix effects stemming from the introduction of methanol during gradient elution [19].\nThe ES-MS system was also used in direct injection mode for the species\u2019 fragmentation experiments. Here, the sample was introduced into the ES source via a syringe pump using a 1-mL glass syringe. The ES-MS and ICP-MS instrumentation parameters can be found in Table\u00a01.\nTable\u00a01ICP-MS and ES-MS parametersInstrumentSettingsICP-MSInstrumentAgilent Technologies 7500 cTorchStandard\u00a0Ar gas flows\u00a0\u00a0Cooling gas16\u00a0L\/min\u00a0Auxiliary gas1\u00a0L\/min\u00a0\u00a0Nebuliser gas0.95\u00a0L\/min\u00a0\u00a0Optional gas: (O2)5%\u00a0\u00a0Spray chamberScott, cooled (2\u00a0\u00b0C)\u00a0\u00a0NebuliserPFA, microconcentric \u00a0\u00a0Internal standardContinuous aspiration, 20\u00a0\u03bcg\/L Rh in 1% HNO3\u00a0\u00a0ConesPlatinum\u00a0\u00a0Isotopes monitored200Hg, 202Hg, 103Rh, 34S, 32S16OES-MSInstrumentAgilent Technologies XCT ion-trap mass spectrometerIon sourceElectrospray ionisationCapillary voltage4,500\u00a0VNebuliser pressureHPLC 50 psi (0.345\u00a0MPa); direct injection 20 psi (0.138\u00a0MPa)Drying gasHPLC 12\u00a0L\/min; direct injection 5\u00a0L\/min Gas temperature350\u00a0\u00b0CScan windowm\/z 100\u2013900MS2 fragmentation window m\/z 4PFA perfluoroalkoxy, HPLC high-performance liquid chromatography, MS2 Ion trap\nReagents\nAll reagents used were of analytical grade and were purchased from Sigma-Aldrich, UK, unless mentioned otherwise. The water used for all the preparations was of suprapure quality (18\u00a0M\u03a9, ELGA water system, UK). HPLC eluents were prepared from formic acid at 0.1% in H2O and 0.25% in methanol.\nAn inorganic mercury (Hg2+) standard stock solution at approximately 1,000\u00a0mg\/kg (as Hg) was prepared by dissolving solid HgCl2 in water. A methylmercury (MeHg+) standard stock solution at approximately 1,000\u00a0mg\/kg (as Hg) was prepared from solid MeHgCl in methanol. For the preparation of mercury and methylmercury biothiols, these stock solutions were further diluted in 0.1% formic acid. The water for the preparation of standards was degassed using ultrasonication (10\u00a0min) followed by purging with N2 (10\u00a0min at 200\u00a0mL\/min). Stock solutions were kept in a freezer at \u221220\u00a0\u00b0C; further dilutions were kept for a maximum of 1\u00a0week at 4\u00a0\u00b0C in the dark.\nReduced l-cysteine and reduced GS were dissolved in water at a concentration of 1\u00a0mg\/mL and were prepared fresh daily.\nSynthesis of HgMeCys, HgMeGS, Hg(Cys)2 and Hg(GS)2\nFrom solutions prepared as described above, mercury and methylmercury biothiols were synthesised by stoichiometrically adding the respective amounts of dissolved biothiol (GS or Cys) to a defined volume of mercury or methylmercury solution. All steps were carried out in a glove bag under a N2 atmosphere in order to avoid oxidation of the thiols. Usually, mercury\u2013thiol compounds were prepared fresh daily.\nModelling the MeHgCys conformation: ab initio and density functional theory calculations\nThe conformation of MeHgCys might involve bonding via either an oxygen atom in the carboxylic group or a nitrogen atom from the amino group, and either an uncharged or zwitterionic form could be possible. In an attempt to predict which, if any, of these conformations\/configurations might be favoured in the gas phase, a series of ab initio and density functional theory (DFT) calculations were performed on a variety of possible structures. Geometry optimisations were performed using the electronic structure software GAMESS-US [20] at both the DFT B3LYP and the ab initio MP2 levels of theory. The 7 September 2006 release of GAMESS-US was used for the DFT calculations and the 24 March 2007 release was used for the MP2 calculations. In both sets of calculations the 6-31G(d,p) basis set [21] was employed for all atoms with the exception of mercury, which was described by the SBKJC effective core potential basis [22]. A subsequent vibrational analysis at the optimised geometries showed them to be true minima and also provided zero-point energies (ZPEs) for use in correcting the energies obtained from the minimisations. The ZPEs were scaled by 0.961 (B3LYP) and 0.9646 (MP2) as suggested by the collected data available online from the CCCBDB database at http:\/\/srdata.nist.gov\/cccbdb\/. The relative populations of the conformers with the lowest ZPE-corrected energies were estimated by calculating the ratios of the Boltzmann factors [23] at 350\u00a0\u00b0C, which is the gas temperature inside the ES ion source.\nPlant preparation\nRice (Oryza sativa) was grown from seeds in vermiculite medium and fed with regular Hoagland solution [24] three times a week. After 2\u00a0months of growth, the rice plants were harvested. The vermiculite was washed off the roots, and the plants were sectioned into roots and shoots, cut into pieces of approximately 5 mm length. Extracts were prepared according to the method of Raab et al. [25]. Briefly, 1\u00a0g of plant material was ground in a mortar under addition of liquid N2, the sample was transferred into a 15-mL Greiner tube, 3\u00a0mL of 1% formic acid was added and the mixture was left standing in ice at 0\u00a0\u00b0C for 1\u00a0h.\nResults and discussion\nThe objective of this work was to develop an analytical method for the separation and identification of mercury and methylmercury bound to Cys and GS. Structural MS carried out here using an ES mass spectrometer equipped with an ion trap was used as a tool for the identification and structural characterisation on a molecular level. The information gained via the fragmentation pattern obtained from the ion-trap measurements proves invaluable for the identification of unknown molecules, along with some information on the molecules\u2019 conformation, while the simultaneous element-selective detection of mercury via ICP-MS is the key to identifying mercury-containing biomolecules in a complex matrix amidst a variety of other organic molecules.\nMore detailed information on the conformation of the HgMeCys molecule was obtained by a modelling approach, which underpins the findings of ion-trap ES-MS.\nIn this paper, we focus on the structural identification of the selected mercury and methylmercury biomolecules via ion-trap ES-MS after their separation using RP HPLC. Here, all mercury compounds were measured under the same detection conditions, and all ES-MS parameters were kept constant for all four species during direct injection as well as in HPLC injection mode. Simultaneous detection with ICP-MS was applied with HPLC separation for mercury-selective determination.\nIon-trap ES-MS measurements of HgMeCys, HgMeGS, Hg(Cys)2 and Hg(GS)2 using direct sample injection\nIn a first approach of identification, mercury and methylmercury complexes with Cys and GS, synthesised as described earlier in 0.1% formic acid at a concentration of 50\u00a0mg\/kg (as Hg) each, were injected as single standards into the ES source of the ion-trap MS instrument. The ES-MS parameters can be found in Table\u00a01.\nThe species identification was performed by using information obtained from the ES-MS and ion-trap MS data:\nThe MH+ molecule clusterThe characteristic isotope pattern dominated by the mercury isotope distributionIn-source fragmentation of the mercury biomolecule Cys and GS moietiesIon trap MS (MS2) of the MH+ molecular ion peak\nIn Fig.\u00a01, the ES-MS spectra measured for the molecular ions (MH+) of the four compounds are shown. The vertical black lines represent the theoretical isotope ratio pattern of the molecules according to their molecular structure.\nFig.\u00a01Electrospray (ES) mass spectrometry (MS) spectra of the MH+ peak cluster for four mercury biothiol compounds (direct injection of single standards). The theoretical isotope pattern is shown as vertical black lines. Molecular structures as ball-and-stick models are shown on the left (Hg violet, S yellow, O red, N green, C grey, H not shown). Theoretical isotope abundance is matched to the highest-abundance peak, set at 100%. The exact mass (theoretical) is given for the 100% peak. a MeHgCys. b MeHgGS. c Hg(Cys)2. d Hg(GS)2. Cys cysteine, GS glutathione\nThe mercury isotope pattern is very prominent, it dominates the overall isotope pattern of the molecules, and can therefore be used as a characteristic fingerprint: In small mercury biomolecules, the mercury isotope pattern is greatly preserved, even when the organic molecular structure, like in HgGS2, accounts for 75% of the molecular mass. The mass spectra show that under the ionisation conditions applied, the mercury compounds are protonated and detected by the ES-MS instrument as a molecule cluster. The experimentally obtained isotope cluster for MH+ match well with the theoretical isotopic molecule patterns calculated for each compound. The major isotope in the clusters is here referred to as the 100% abundance isotope, reflecting the usual practice in isotope abundance visualisation by defining the major mass as 100% and the less abundant isotopes as fractions of this.\nIn-source fragmentation of HgMeCys, HgMeGS, Hg(Cys)2 and Hg(GS)2\nMolecule fragmentation is usually observed during the ionisation step in the ES ion source. The fragmentation pattern obtained in this process gives information about the molecular structure of a compound, using the different fragments for reconstruction of the complete molecule. However, for our experiments, we are interested in keeping the in-source fragmentation as low as possible in order to get the main peak from the molecular ion MH+. One reason for this is that we try to obtain optimum detection limits for mercury biomolecules in real samples, and in-source fragmentation leads to a diminished MH+ intensity. Moreover, the MH+ peak gives information on the molecular mass and enables identification via the characteristic isotope pattern. Finally, where more information on the molecular structure is needed, e.g. to elucidate isomeric forms, further fragmentation is performed on selected masses within the ion-trap MS instrument, using collision-induced fragmentation.\nAll mercury compounds were synthesised in solution in stoichiometric amounts (i.e. two molecules of thiol for Hg2+, one molecule of thiol for MeHg+). In order to determine whether the mercury compounds had reacted quantitatively with the organic thiols, we monitored the protonated molecular ions (MH+) of Cys (m\/z 122) and GS (m\/z 308). As only the reduced thiols are able to bind to mercury, we also checked for the oxidised dimers of Cys and GS with m\/z 224 and m\/z 613. The ratio between the molecular ions of the mercury complexes and those of the free reduced or oxidised thiols gives an indication for the reaction yield in solution, as well as the stability of the complexes.\nIn Fig.\u00a02, the mass spectra (MS+) of the four mercury compounds investigated are shown, highlighting the in-source fragments observed. In-source fragmentation can be influenced by ionisation parameters, such as capillary voltage. Here, the parameters used were as detailed in Table\u00a01 for the direct-injection mode.\nFig.\u00a02ES-MS spectra (MS+) showing the in-source fragmentation for four mercury biothiol compounds (direct injection of single standards). a MeHgCys. b MeHgGS. c Hg(Cys)2. d Hg(GS)2\nFrom Fig.\u00a01 it is evident that from the four mercury compounds investigated only MeHgCys (Fig.\u00a01a) shows extensive in-source fragmentation, resulting in two clusters of approximately the same peak height: the MH+ cluster with m\/z 338 as the 100% abundance isotope and a second cluster with m\/z 321 as the 100% abundance isotope, showing a mass loss of 17 from the MH+ molecular ion cluster. This mass loss can be attributed to the loss of either OH or NH3. Apart from the two most prominent in-source fragments, only one fragment exhibiting a mercury-like isotope pattern can be found at m\/z 234. This may be attributed to a  or a MeHgOH moiety, and accounts for just 3% of the combined peak areas of the two main peaks. Similar fragmentation has been described by D\u2019Agostini et al. [26], who suggested the loss of NH3 from the MH+ ion, and hypothesised that two isomeric species of MeHgCys are present in solution, either exhibiting a Hg\u2013N or a Hg\u2013O interaction. Here, we show that the Hg\u2013N interaction is the most likely form present in the ion source, and that the in-source fragment at m\/z 321 is indeed most likely formed through loss of NH3. This was achieved through the ion-trap MS2 fragmentation pattern and a modelling approach, in which the most stable conformation for MeHgCys was calculated in the gas phase. These experiments are highlighted further below.\nA fragment with mass 119.5 may be attributed to free Cys or cystine (doubly charged), but occurs at less than 1% abundance compared with the two main clusters. The absence of free Cys also suggests that the complex is formed quantitatively in the solution.\nFigure\u00a02b shows the in-source fragmentation of MeHgGS during direct injection, clearly showing the MH+ molecular peak cluster as the dominant component. Two more fragment peaks can be identified. One fragment at m\/z 395, exhibiting a cluster with the typical mercury isotope pattern, can be attributed to a loss of the \u03b3-Glu moiety from GS. A second peak appears at m\/z 308, which can be clearly attributed to protonated GS. The spectrum shows the MeHgGS molecule peak prevailing with more than 95% abundance, while the two other fragments observed correspond to approximately 1% MH+ abundance only.\nFor Hg(Cys)2 (Fig.\u00a02c), MH+ is the most prominent peak in the spectrum. An in-source fragment obtained from a mass loss of 17 is also observed, but accounts for less than 6% of the MH+ peak only. Another fragment with the typical mercury isotope pattern and m\/z 339 the 100% abundance peak may be attributed to a  moiety, similar to the fragment observed for MeHgCys: a Cys group can obviously be cleaved from the molecule, leaving a Hg\u2013N bond behind. Two more peaks can be distinguished at m\/z 241 and m\/z 122, corresponding to protonated cystine (Cys\u2013Cys) and Cys. Cystine at m\/z 241 seems quite abundant, but accounts for only 10% of the MH+ cluster of Hg(Cys)2. Only traces of free Cys at m\/z 122 (less than 1% of the mercury molecular cluster) were found.\nThe spectrum of Hg(GS)2 (Fig.\u00a02d) shows two mercury-containing fragments at m\/z 544 and m\/z 508 besides the most prominent peak for MH+ at m\/z 815. While m\/z 508 can clearly be attributed to a protonated HgGS fragment after loss of one GS group, the cluster at m\/z 544 cannot be clearly identified, but might be a Hg\u2013GS cluster with two water molecules associated. A peak at m\/z 407 corresponds to the doubly charged molecule peak MH2+. Two more peaks can be distinguished: m\/z 613, corresponding to protonated oxidised GS, and m\/z 308, corresponding to free reduced GS.\nThe two mercury-containing fragments correspond to 10% (m\/z 544) and 15% (m\/z 508), respectively, of the overall abundance, while the protonated oxidised and reduced GS only account for approximately 1% each, and MH2+ accounts for less than 2%. MH+ is found to occur at more than 70%.\nThe ionisation efficiencies differ substantially between the four different compounds: While MeHgCys exhibits the lowest intensities, with 2\u2009\u00d7\u2009105 counts, the MeHgGS signal is tenfold higher. Hg(Cys)2 and Hg(GS)2, each with around 5\u2009\u00d7\u2009105 counts, show intensities between these two. Taking into account that MeHgCys forms an abundant in-source fragment with the same intensity as the MH+ ion, these three molecules appear to have similar ionisation behaviour, leading to comparable ratios of protonation in the ES source under the conditions applied.\nConformation of HgMeCys: ion-trap MS2 fragmentation and conformation modelling\nCollision-induced fragmentation was performed using the ion-trap device of the ES-MS instrument. The in-source fragmentation of MeHgCys showed an important fragment with a mass loss of 17 amu with abundance as high as the MH+ molecular ion. This mass loss can be explained by either loss of OH or NH3 from MH+, and both possibilities have been postulated before [26]. Here, we use the ion-trap MS2 information on the two prevailing peaks obtained combined with a modelling approach to find the most probable conformation of MeHgCys, which may explain the fragmentation behaviour observed.\nAs shown in Fig.\u00a03, the MS2 spectrum of the MeHgCys molecular ion (m\/z 336, 200Hg) shows a predominant fragment at m\/z 318 due to loss of H2O. A second fragment at m\/z 231 can be attributed to either  or MeHg\u2013O+, formed through rearrangement of the molecule under loss of the residual Cys moiety, and corresponds to the fragmentation pattern obtained in source. This finding is an indication that the Hg\u2013S bond is not the only interaction between Cys and HgMe, but that either Hg\u2013O or Hg\u2013N bonding is involved, i.e. either the carboxylic or the ammonia group forms a bond to the central metal.\nFig.\u00a03Ion trap MS (MS2) spectra as obtained from collision-induced fragmentation in the ion-trap device from the two main in-source fragments obtained from the direct injection of a single standard of MeHgCys. a MS2 of MH+ at m\/z 336 (Me200HgCys). b MS2 of MH+ at m\/z 319 (Me200HgCys\u2013OH or Me200HgCys\u2013NH3)\nA different picture is seen when MS2 is performed on the in-source fragment ion with m\/z 319 (formed through loss of OH or NH3 from 200HgMH+ at m\/z 336) . Here, we can distinguish several fragments: m\/z 301 (loss of H2O), m\/z 288 and m\/z 261, formed through the successive loss of parts of the Cys moiety, and m\/z 215, corresponding to MeHg+. In contrast to the MS2 of MH+ (m\/z 336), the fragment at m\/z 231 is not formed. This is an indication that the group forming this fragment has been lost during in-source fragmentation.\nModelling the MeHgCys conformation using ab initio and DFT calculations\nThe conformation of MeHgCys can involve either bonding via an oxygen atom in the carboxylic group or a nitrogen atom from the ammonia group, and can carry the proton at either group. A modelling approach was used to determine the total energies for the different possible conformations of HgMeCys, including correction for the ZPE.\nThe final conformations obtained from the geometry optimisations at the MP2 level are shown in Fig.\u00a04 (conformers 1\u20136). Those obtained from the B3LYP calculations differed only by small deviations in bond lengths and angles and are therefore not shown. Two of the geometry optimisations were started in zwitterionic configurations with either a carboxylic oxygen (conformer 1) or the amino nitrogen (conformer 2) positioned close to the mercury atom. The zwitterionic form of MeHgCys was found to be unstable and optimisations led to the neutral form regardless of the initial protonation state. Of the remaining calculations, conformer 3 and 4 were started with the amino nitrogen close to the mercury, whereas conformers 5 and 6 were started with a carboxylic oxygen in this position. In all of these optimisations the initial Hg\u2013X (X is O or N) interaction was retained throughout, suggesting that this is generally favoured in the conformational behaviour of MeHgCys.\nFig.\u00a04Modelling the MeHgCys conformation: final conformations of MeHgCys obtained from geometry optimisation at the MP2 level. Model numbers (1\u20136) as used in Table\u00a02\nThe pattern of relative conformational energies (Table\u00a02) is basically the same at both levels of theory, with the most energetically favoured conformations displaying the Hg\u2013N interaction. Those conformations displaying Hg\u2013O interaction make up the second-most thermodynamically stable grouping, whereas the extended conformer where the mercury atom is free from nonbonded interaction is considerably higher in energy than all the others. The energy differences are more pronounced at the MP2 level than at the B3LYP level and this is probably due to the fact that the perturbation theory treatment is capable of describing dispersion effects, whereas DFT is not. Dispersion effects are responsible for mediating nonbonded interactions such as hydrogen bonds and van der Waals \u201cbonds\u201d and therefore it would be expected that these would be important in this particular case. For this reason, the MP2 results are expected to be more reliable than those obtained with DFT since the former provide a more complete physical picture of the MeHgCys system.\nTable\u00a02Conformational energies obtained for the two different models usedConformerB3LYP\/6-31G(d,p) \u0394E (kJ\/mol)aMP2\/6-31G(d,p) \u0394E (kJ\/mol)b17.418.0212.730.330.00.047.66.9525.821.0613.721.4Scale factors obtained from the CCCBDB database at http:\/\/srdata.nist.gov\/cccbdb\/aZero-point energies scaled by 0.9610bZero-point energies scaled by 0.9646\nThe results of the Boltzmann factor calculations based on the corrected MP2 energies indicate that at the experimental temperature approximately 98% of the MeHgCys would be present as the form displaying the Hg\u2013N interaction, with the remaining 2% consisting almost exclusively of the Hg\u2013O forms. Using the B3LYP energies, these calculations suggest that the slightly lower figure of 87.5% for the Hg\u2013N forms, with one of the Hg\u2013O conformers making up most of the remaining population. As the MP2 energies are thought to be more accurate on the basis of physical considerations, it is reasonable to assume that the figure of 98% for the Hg\u2013N population is similarly more accurate; however, even at the B3LYP level the figures clearly point to this being the form of the molecule expected to be dominant in the gas phase at 350\u00a0\u00b0C. The use of larger and\/or more flexible basis sets might lead to alterations in these results owing to improved description of the Hg\u2013X interactions but it is felt that this would be unlikely to significantly alter the distribution of energies in this case.\nAssuming that the Hg\u2013N form is dominant in the ion source, it is reasonable to assume that the in-source fragment obtained at m\/z 234 (202Hg) corresponds to a (protonated)  ion. This is confirmed by the occurrence of the same fragment in MS2 at m\/z 231 (). The in-source fragment at m\/z 319 (200Hg) does not exhibit the m\/z 231 fragment during MS2, but loses the Cys completely, with MeHg+ remaining as the smallest mercury-incorporating fragment. This is suggestive of the possibility that the in-source fragment with m\/z 319 has lost NH3 rather than OH \u2013 a MeHg\u2013N fragment cannot be formed from this mother ion. However, it should be noted that besides the Hg\u2013N interaction, a small contribution of Hg\u2013O is possible.\nIon-trap MS2 of the MH+ cluster of Hg(Cys)2, HgMeGS and Hg(GS)2\nFigure\u00a05 depicts the ion-trap fragmentation patterns obtained for the three remaining mercury-containing biomolecules. The m\/z values of the MS2 spectra shown here in Fig.\u00a05 are each defined for the 200Hg isotope. However, the chosen trap window ion width of 4 amu ensures that a major part of the ion cluster is trapped and undergoes collision-induced fragmentation. Thus, easier identification of mercury-containing fragments is possible, as the mercury pattern is greatly conserved. However, the fragment incorporating 200Hg will be trapped with the highest abundance.\nFig.\u00a05MS2 spectra as obtained from collision-induced fragmentation of the MH+ molecular cluster in the ion-trap device from three mercury biothiols. a MeHgGS. b Hg(Cys)2. c Hg(GS)2\nHg(Cys)2\nThe ion-trap collision-induced fragmentation of Hg(Cys)2 exhibits a most abundant fragment at m\/z 434, corresponding to the loss of H2O, and a second important fragment at m\/z 337, which corresponds to the loss of one Cys molecule leaving a nitrogen atom bound to mercury (CysHg\u2013N+). This behaviour is similar to what was found for the fragmentation of MeHgCys, and seems to be typical for mercury bound to a Cys moiety. No other fragments containing mercury can be observed, and no unbound Cys molecules were detected.\nMeHgGS\nThe MS2 of MeHgGS shows one high-abundance fragment at m\/z 375, corresponding to the loss of a Glu moiety and OH, plus two low-abundance fragments at m\/z 509 (loss of H2O) and m\/z 393 (loss of Glu). GS is not lost during this fragmentation, but rather the Glu moiety is chopped off the GS. Interestingly, a MeHg+ ion fragment that it is formed during MS2 of HgMeCys cannot be detected either; hence, mercury is probably stabilised by the Gly\/Cys moiety.\nHg(GS)2\nThe MS2 of Hg(GS)2 shows two main fragments with m\/z 686 (loss of Glu) and m\/z 508 (loss of GS), plus four less abundant fragments: m\/z 795 (loss of H2O), and m\/z 666, m\/z 557 and m\/z 547 formed by successive loss of GS amino acids.\nHPLC separation of HgMeCys, HgMeGS, HgCys2 and HgGS2 with simultaneous ES-MS and ICP-MS detection\nFor the chromatographic separation of the mercury biomolecules, HPLC and the simultaneously coupled ES-MS and ICP-MS system was used as described in \u201cInstrumentation\u201d. Hg(Cys)2, HgMeCys, Hg(GS)2 and HgMeGS solutions were prepared accordingly, as well as a mixture of four compounds with a concentration of 2.5\u00a0mg\/L (as Hg) each.\nSingle injections of each standard revealed the retention times for each compound, and these injections were followed by injection of the mixture of the compounds. The elution order obtained was Hg(Cys)2, HgMeCys, Hg(GS)2 and HgMeGS, with HgMeGS being eluted at approximately 9.5\u00a0min. All compounds were eluted at a gradient composition of less than 15% eluent B, showing that relatively soft elution conditions can be applied. Figure\u00a06 shows the combined ICP-MS (202Hg, 103Rh) and ES-MS traces for the extracted ion chromatograms of m\/z for MH+ for the four mercury species.\nFig.\u00a06Simultaneous ICP-MS and ES-MS spectra from high-performance liquid chromatography injection of MeHgCys, MeHgGS, Hg(Cys)2 and Hg(GS)2\nThe ES-MS traces of the extracted ion chromatograms show distinct peaks of the protonated molecular ion masses. For each compound, the same isotope cluster was found as was obtained in the flow-injection experiments with the single species. Moreover, the MS+ spectra (in-source fragmentation) are identical to those obtained in the direct-injection experiment, as are the MS2 spectra of the MH+ molecular ions (data not shown).\nAll ion traces in ES-MS are on the same y-scale, and it is evident that the intensities measured in ES-MS are species-dependent, like in the direct-injection experiments. MeHgGS shows the highest intensity, approximately 10 times higher than MeHgCys. Although a methanol gradient was used in the HPLC experiment (12.5% methanol at 10\u00a0min retention time), the relative peak intensities are comparable with those obtained with the direct injections.\nA different picture is seen for the ICP-MS signal for mercury. In ICP-MS, all compounds should be completely destroyed by the hard ionisation conditions in the plasma. Therefore, the signal intensities for the different compounds should only be governed by the absolute mercury concentration. As all the compounds were made up in the same concentration (as Hg), the ICP-MS response should be very similar for all compounds. Figure\u00a06 shows that for the four compounds the intensities obtained are much more similar than those obtained by ES-MS, but are not ideally equal: the peaks corresponding to the first and the last species eluted (HgCys2 and MeHgGS) are approximately twice the peaks obtained for MeHgCys and HgGS2. Several reasons can be responsible for this finding:\nOverall intensity changes due to the input of methanol into the plasma. As shown in Fig.\u00a06, the signal for the internal standard is constant up to approximately 4\u00a0min of runtime; afterwards it decreases to about 50 % of the initial value at 10-min runtime, when the last mercury compound is eluted. If the mercury intensities are influenced by this process only, the signals for the first compounds eluted (both less than 4\u00a0min retention time) should be equal, whereas the signals for the GS complexes should be only about half. This is not the case; in fact the last compound eluted, MeHgGS, exhibits a signal equal to the first compound eluted. However, the behaviour of mercury with its high ionisation potential (10.4\u00a0eV) is greatly influenced by organic matter in the plasma, and the mercury signal intensity may change in a different way from the intensity of rhodium. The dependence of the mercury intensity on the methanol content of the eluent must be determined, but this was not performed during the experiments reported here.The volatilities of the compounds may be different, so different amounts of each compound may be introduced into the plasma during pneumatic nebulisation: this effect is well known for volatile species like HgMe2, and has to be investigated further for the compounds used in this experiment.MeHgCys and HgGS2 may partly decompose on the column, so not all of the compound injected may reach the plasma: in this case, we would expect broad, tailing peaks and substantial amounts of free Cys and reduced GS. However, these have not been observed.MeHgCys and HgGS2 are not present to 100% in the standard, and thus the concentrations of the mercury reaching the plasma are diminished: in this case, signals for unbound Cys and GS should show also significant intensity. In fact, some oxidised Cys and oxidised GS can be distinguished in the ES-MS, but is not clear whether the amounts may sum up to explain the intensity loss in MeHgCys and HgGS2. However, the corresponding mercury part of the molecules (Hg2+ and MeHg) should show up in the ICP-MS trace, and this is not the case. As shown further later for the spiking experiments (Fig.\u00a07), Hg2+ and HgMe+ are eluted from the column with distinct retention times differing from the retention times of the other complexes.Fig.\u00a07Overlaid chromatograms for injections of free and biothiol-complexed mercury species in a plant extract spiked with different Hg and HgMe biothiol complexes. Peak assignment: 1 Hg(Cys)2, 2 Hg2+, 3 MeHgCys, 4 MeHg+, 5 MeHgGS, 6 Hg(GS)2\nThus, a final explanation for the difference in intensity of the four compounds cannot be given at this point.\nCompound stability during spiking experiments of plant extracts\nThe mercury biomolecules synthesised and characterised here shall be detected and identified in real-world samples. One future application is the determination of such compounds in plants exposed to mercury contamination. Mercury can readily be taken up by plants, can accumulate in the roots and can be translocated into shoots and fruits [16, 17, 27, 28].\nFor the determination of such complexes in plant material, the latter has to be extracted using a minimally invasive method which does not destroy or transform the compounds we are looking for. Here, we tested the stability of the four example compounds in extracts from roots and shoots of rice plants.\nThe plant extracts, prepared as described earlier, were spiked with the four compounds and the signals obtained were compared with the corresponding signals when the compounds were prepared in 0.1% formic acid. Simultaneous ES-MS and ICP-MS detection was applied using the same setup as described earlier. Additionally, plant extracts were spiked with \u201cfree\u201d Hg2+ and HgMe+ in order to determine their retention behaviour on the column, and to see whether any complexes would form in situ with any biothiols possibly present in the plant extracts.\nIn Fig.\u00a07, the 202Hg traces monitored by ICP-MS are shown for MeHgCys, Hg2+, Hg(Cys)2, MeHg+, Hg(GS)2 and MeHgGS spiked into the shoot extract of a rice plant at a concentration of 10\u00a0mg\/kg (as Hg) each. It is evident that the elution order of the four mercury biothiol complexes matches very well the one obtained in a standard mixture (Fig.\u00a06). Surprisingly, Hg2+ is eluted later than HgCys2, whereas MeHg+ is eluted with a broad peak with a retention time of approximately 5\u00a0min between the Cys and GS complexes. The elution order and the retention times are practically the same for the standard, root and shoot extracts, and this also remains over time: a second injection of all samples, done 7\u00a0h after the first run, showed no significant change in retention times. A similar picture is seen for the peak areas. Except for Hg2+ and MeHgCys, the peak areas measured after 7\u00a0h are more than 80% of the initial value (peak areas corrected for instrumental drift using the internal standard signal). Surprisingly, MeHgCys turns out to be more stable in the extracts than in the standard solution. This might be due to the presence of some unidentified chelating agents. The peak area for Hg2+ varies considerably, showing a 50% rise for the standard in 0.1% formic acid, whereas a 30% decrease is observed in the plant extract. While no explanation can be given here for the intensity rise of the Hg2+ peak in the standard, in the shoot extract the formation of other, unidentified mercury complexes may be proposed.\nThe results for retention time and peak intensity reproducibility are summarised in Tables\u00a03 and 4.\nTable\u00a03Retention times for consecutive injection of six mercury compounds with 7\u00a0h differenceSampleHgCys2Hg2+MeHgCysMeHg+HgGS2MeHgGSStandard 1120.7152213.5302458.4529.1Standard 2120.7152218.5303454.1527.7Roots extract 1125.7NA213.5286457.8529.8Roots extract 2126NA212.8284.9452.7526.9Shoots extract 1125.7150.6213.5295.6457.7529.1Shoots extract 2125151.4214.2292.8455.5526.2Average123.9151.5214.3294.1456.0528.1\u03c32.50.662.17.72.31.4RSD (%)2.00.40.92.60.50.3NA not available, RSD relative standard deviationTable\u00a04Peak area (as percentage of first measurement) recoveries for consecutive injection of six mercury compounds with 7\u00a0h differenceSampleHgCys2Hg2+MeHgCysMeHg+HgGS2MeHgGSStandard 9115910989283Roots extract93NA67898786Shoots extract967749958686\nThe addition of MeHg+ to the extracts did not result in any additional peaks in the ICP-MS trace, while for the addition of Hg2+ a small, very broad peak can be distinguished around the retention time of HgMeGS. An elevated mercury background is seen after the introduction of Hg2+ to the standard and the extract, pointing to some kind of exchange on the HPLC column, possibly through formation of unidentified, labile complexes. However, the mercury counts are at the normal background level again prior to injection of the next sample. Regarding the ES-MS measurements (data not shown), the same MH+ ion cluster patterns are found for the four spiked mercury complexes as in the standards. As already seen from the ICP-MS traces, addition of Hg2+ or MeHg+ did not result in the formation of any other distinguishable mercury biothiol compounds. In the unspiked extracts, only trace amounts of free Cys or GS were found, but there was some oxidised and thus inactive GS.\nConclusion and outlook\nIn this work, we could successfully show the synthesis, structural identification and HPLC separation of mercury and methylmercury complexes with Cys and GS. An ES-MS system equipped with an ion trap for MS2 measurements revealed characteristic MH+ and MS2 fragmentation patterns for these compounds, enabling the identification of these or similar complexes in biota samples. The simultaneous coupling of HPLC with ES-MS and ICP-MS with its invaluable combined information on molecular structure and quantitative mercury detection proved to be suited for this kind of compounds. The method described seems to be acceptably robust and reproducible, and also suited for real samples. This was shown through spiking experiments of plant extracts.\nHowever, the quantification approach via the ICP-MS trace needs further investigation, especially in terms of mercury intensity changes due to the gradient introduction of methanol into the plasma, and also in terms of decomposition on column as has been described for As(GS)3 [18].\nThe work presented here is the first step in the determination of mercury species in biota on the molecular level.\nOngoing experiments are aimed at plant exposure to mercury and methylmercury. Here, we can also expect mercury complexes with larger biothiols, especially phytochelatins: phytochelatins are small peptides of the general structure (\u03b3-Glu\u2013Cys)n \u2013Gly (n\u2009=\u20092\u201311), which can bind to metal ions and play an important role in metal detoxification and translocation processes in plants. Other biota samples, e.g. fish, will be targeted as well.\nFinally, this study presents the first identification of mercury and methylmercury biothiols in spiked plant extracts; hence, this method is a novel tool to investigate whether mercury and methylmercury indeed form complexes with biothiols such as Cys and GS, or if they rather bind to larger entities such as phytochelatins in plants or proteins in fish and other biota.","keyphrases":["mercury","biothiols","methylmercury","electrospray mass spectrometry","high-performance liquid chromatography","conformation modelling","inductively coupled plasma mass spectrometry"],"prmu":["P","P","P","P","P","P","R"]}
{"id":"Anal_Bioanal_Chem-3-1-2117337","title":"A low perfusion rate microreactor for continuous monitoring of enzyme characteristics: application to glucose oxidase\n","text":"This report describes a versatile and robust microreactor for bioactive proteins physically immobilized on a polyether sulfone filter. The potential of the reactor is illustrated with glucose oxidase immobilized on a filter with a cut-off value of 30 kDa. A flow-injection system was used to deliver the reactants and the device was linked on-line to an electrochemical detector. The microreactor was used for on-line preparation of apoglucose oxidase in strong acid and its subsequent reactivation with flavin adenine dinucleotide. In addition we describe a miniaturized version of the microreactor used to assess several characteristics of femtomole to attomole amounts of glucose oxidase. A low negative potential over the electrodes was used when ferrocene was the mediator in combination with horseradish peroxidase, ensuring the absence of oxidation of electro-active compounds in biological fluids. A low backpressure at very low flow rates is an advantage, which increases the sensitivity. A variety of further applications of the microreactor are suggested.\nIntroduction\nConventionally, characterizing enzyme properties in terms of kinetics, conversion rate, and substrate specificity requires substantial amounts of pure enzyme. We aim to develop a versatile and robust microreactor to monitor on-line the activity of small amounts of enzymes, and that can be used under harsh conditions if required, for instance, to prepare modified forms of glucose oxidase (GOx) in which the flavin adenine dinucleotide (FAD) cofactor has been replaced by artificial flavins. GOx uses \u03b2-d-(+)-glucose with high specificity as substrate, converting it to gluconolactone and H2O2. Preparation of apoGOx with low residual activity requires partial unfolding of the protein under strongly acidic conditions followed by removal of the flavin by size-exclusion chromatography [1]. The activity of the enzyme can be restored by applying a solution of FAD or FAD analogues [1\u20134].\nEnzyme reactors can be created by covalent immobilization [5, 6]. Another option is to co-polymerise the enzyme in a flow system, trapping the enzyme in a polymer matrix [7, 8]. With these approaches assessment of enzyme properties, inhibitor screening [7], or conversion rates of stereoselective or enantiomeric substrates [9, 10] have been reported. Although convenient, covalent attachment and co-polymerisation require substantial amounts of protein to obtain a good response [11]. Upon immobilization, enzyme characteristics are often altered and enzyme activity may be lost. Especially when rare or expensive enzymes have to be used, it would be beneficial to have a system that uses far less enzyme, does not use chemical immobilization, and is easy to handle.\nWe propose a device where the enzyme is confined in a small space on top of one filter or between two filters and continuously perfused. The device, based on our previously described biosensor technique [12], is robust, easy to handle, and loss of activity and consumption of enzyme are kept to a minimum. With this device we were able to prepare apoGOx on-line by retaining GOx on a single polyether sulfone (PES) filter using acid treatment. Restoration of the enzyme activity was achieved in the same system using a solution of FAD. Furthermore, we were able to miniaturize the microreactor, using less enzyme, and increase the sensitivity of the system using a combination of ferrocene and horseradish peroxidase (HRP) as a mediator for electrochemical detection.\nExperimental\nMaterials\nGOx from Aspergillus niger (EC 1.1.3.4, grade 1) and horseradish peroxidase (HRP; EC 1.11.1.7, grade 1) were obtained from Roche (Almere, The Netherlands). Ferrocene carbolic acid was from Lancaster Chemicals (Lancaster Synthetics UK, Morecambe, Lancs, UK). Other chemicals were of pro analysis quality and purchased from Merck (Amsterdam, The Netherlands). Double quartz-distilled water was used for all aqueous solutions. Before and after acid treatment, the running buffer (a) of the microreactor was 0.15\u00a0mol L\u22121 sodium phosphate, pH 7.0, containing 1\u00a0mmol L\u22121 NaCl and 0.1% Kathon GC (Rohm and Haas, Croydon, Surrey, UK). Glucose solution (50\u00a0mmol L\u22121) prepared in running buffer was left to stand for several hours to reach mutarotational equilibrium. For electrochemical detection of GOx in combination with HRP, the running buffer (b) also contained 0.5\u00a0mmol L\u22121 ferrocene carbolic acid.\nDesign of the microreactors\nThe overall design of the microreactors is shown in Fig.\u00a01. They were micro machined from Delrin by the instrumental workshop of Groningen University. For preparation of apoGOx and reconstitution of GOx activity, a microreactor as described in Ref. [12] was used. In contrast to the original device, only one PES filter with a cut-off value of 30\u00a0kDa (Sartorius, G\u00f6ttingen, Germany) and punched to a diameter of 13\u00a0mm, was used. Performance studies were done with our miniaturized design. The dimensions of the miniaturized microreactor were: outer length 3\u00a0cm, outer diameter 1.5\u00a0cm. A PES membrane filter was punched to a diameter of 4\u00a0mm and placed in the microreactor (effective diameter 3\u00a0mm, effective volume 1\u00a0\u03bcL) and connected between the injection valve and the detector with fused-silica tubing (FST) (150\u00a0\u03bcm OD, 50\u00a0\u03bcm ID) (Polymicro Technologies, Phoenix, AZ, USA) pinched off in Teflon tubing 1\/16 inch OD and 170\u00a0\u03bcm ID (Aurora Borealis Control, Schoonebeek, The Netherlands) with use of finger-tight fittings, to avoid high dead volumes.\nFig.\u00a01Schematic design of the reaction compartment of the microreactor. (a) sketch; (b) schematic diagram. Shown here is the microreactor with one PES ultrafilter which is then connected to the flow system using FST and finger-tight fittings. The mesh screen is used to support the ultrafilter\nSet-up of the system\nThe perfusates of the microreactor were introduced into a flow-injection system and monitored electrochemically (VT-03 electrochemical flow cell and Decade Digital Electrochemical Amperometric Detector; Antec Leyden, Zoeterwoude, The Netherlands), essentially as previously described [13\u201315]. For the preparation of apoGOx and subsequent reconstitution of the holoenzyme a voltage of +0.5\u00a0V was applied between the working and reference electrodes.\nGOx\/HRP activity monitoring\nFor flows >1\u00a0\u03bcL min\u22121, a LC10ADvp solvent-delivery pump (Shimadzu Corporation, Kyoto, Japan) was used; for flows <1 \u03bcL min\u22121 a Harvard 22-syringe pump (Harvard Apparatus, Holliston, MA, USA) was used. A voltage of \u2212150\u00a0mV was applied between the working (glassy carbon) and reference (Ag\/AgCl) electrodes. The injection valve (Vici Cheminert C4, Valco Instruments, Houston, TX, USA) was equipped with a 20 nL internal loop; injection cycles were designed using software incorporated in the Decade system, which also thermostatically controlled the reaction temperature at 37\u00b0C. The current was registered using a flatbed recorder type BD41 (Kipp & Zonen, Delft, The Netherlands) and\/or the signal was integrated using Chromeleon Software (Dionex Corporation, Sunnyvale, CA, USA) on a PC using an RS232 interface. After the filter was installed in the system, it was left to equilibrate to a flow of 50\u00a0nL min\u22121. Glucose solution injection was sequenced to load\/inject 2\/3\u00a0min (cycle time 5\u00a0min) and repeated until a stable baseline was obtained.\nPreparation of apoGOx and restoration of the enzyme activity\nPreparation of apoGOx was essentially the same as described in Ref. [16], with a solution of 0.1\u00a0mol L\u22121 glycine, 0.1\u00a0mol L\u22121 NaCl, and 0.1\u00a0mol L\u22121 HCl, pH 1.5 mixed with glycerol to a final concentration of glycerol of 30% (v\/v), cooled in ice. This solution was applied to the GOx containing reactor. In detail, a 10 \u03bcL solution of 100\u00a0mg mL\u22121 GOx (625\u00a0\u03bcmol L\u22121) was pipetted on to the microreactor\u2019s PES filter, which was thus loaded with 6.25\u00a0nmol. After stabilisation of the signal, the initial enzyme activity was determined (=100%). The reactor was detached from the detector. The detached reactor was cooled in ice, and perfused with the cooled acidic glycerol solution at a rate of 25\u00a0\u03bcL min\u22121 for 15\u00a0min. After application of running buffer (a) until the effluent was pH 7, the reactor was taken out of the flow system, re-attached to the detector cell, and the residual activity of the apoGOx was measured in buffer system (a) with a flow of 25\u00a0\u03bcL min\u22121.\nAfter the reactor was released from the flow system, ca 150\u00a0\u03bcL of an FAD solution (3.4\u00a0mmol L\u22121) in 0.15\u00a0mol L\u22121 sodium phosphate, pH 7.0, at room temperature, was applied manually during ca 15\u00a0min, except for time-dependent tests. Afterwards, the reactor was re-attached to the detector cell and the restored activity of the holoenzyme was measured as described above.\nDetermination of several characteristics of the miniaturized enzyme reactor\nIncreasing amounts of GOx in running buffer (b) (range 625\u00a0amol to 625\u00a0pmol) were injected into the system, providing the amount of GOx retained on the membrane. After equilibration, a sequence of load\/inject of the glucose solution was applied. Subsequently, a solution of HRP in running buffer (b) was injected into the system providing 0.68\u00a0pmol (30\u00a0ng) of HRP. After equilibration the sequence of load\/inject of glucose was repeated. The efficiency of conversion at the electrode was calculated according to Eq. (1):\nwhere h = peak height (pA), \u03c3 = peak width at 60.7% (s), I = injection volume (\u03bcL), c = concentration (\u03bcmol L\u22121), n = number of electrons, F = 96485 (Faraday\u2019s constant, C mol\u22121) and \u221a(2\u03c0)h\u03c3 is peak area (C)\nIn Eq.\u00a0(1) the charge that is transferred to the electrode (area under the curve) is related to the real charge that is available in the reaction, e.g. the concentration of the substrate, the number of electrons involved, and Faraday\u2019s constant.\nResults\nPreparation of apoGOx and restoration of the enzyme activity\nWe followed the response to glucose, the residual enzyme activity of apoGOx after acid treatment, and restoration of the enzyme activity after application of a solution of FAD. After acid treatment, negligible activity of apoGOx was observed. Application of the FAD solution restored the enzyme activity. The time dependency of enzyme activity restoration is shown in Fig.\u00a02, composed of individual data from five apoGOx preparations. This time dependency of the reactivation is in accordance with Eq.\u00a0(2).\nwhere k = 0.035\u00a0min\u22121 and t is the reaction time (min).\nFig.\u00a02Restoration of apoGOx activity: circles, observed relative reactivation with FAD; solid line, fitting according to the equation f(t) = 1 \u2212 exp(\u22120.035t)\nApproximately 80% of the enzyme activity was restored within 60\u00a0min, which is above average compared to other procedures [1, 2].\nPerformance\nSeveral properties of the system were investigated. These included backpressure of the reactor at various protein loads, its stability and reproducibility, the efficiency at the electrode, system sensitivity, and ease of handling. The relationship of backpressure to the flow and the amount of protein retained on the filter are depicted in Figs.\u00a03a and 3b. The level of the backpressure is acceptable with these flows and these amounts of protein. The stability and reproducibility of the system, as tested with 625\u00a0fmol GOx on a series of glucose injections, showed an intra-assay standard deviation of less than 3%. The efficiency of the reaction is inversely proportional to the amount of protein on the filter. The sensitivity of the set-up was determined by retaining various amounts of GOx, ranging from 62.5\u00a0amol to 625\u00a0pmol on the ultrafilter, and testing the response. A measurable and reproducible response was obtained, even with the lowest amounts of enzyme tested (Table\u00a01). One load of enzyme in the microreactor can be used at least 10 times and the system can be regenerated simply by replacing the filter and injection of a fresh aliquot of protein solution.\nFig.\u00a03System backpressure. (a) Backpressure in relation to the flow at various protein loadings: diamonds, 0 \u03bcg; squares, 13 \u03bcg; triangles, 24 \u03bcg; circles, 39 \u03bcg; stars, 61 \u03bcg GOx. (b) Backpressure in relation to the amount of protein retained on the filter at various flow rates: circles 1 \u03bcL min\u22121; squares, 5 \u03bcL min\u22121; triangles, 10 \u03bcL min\u22121; diamonds, 25 \u03bcL min\u22121Table\u00a01Measured detector signal and relative conversion of glucose in relation to the amount of enzyme retained on the filter in the miniaturized microreactor. Indicated are the amounts of GOx (in fmol) on the PES filter and the signals of the detector (in nA) indicating the amount of converted glucose, calculated as described in the method sectionProtein (GOx) on the filter (fmol)Signal (nA)Conversion (%)0.06250.0160.040.6250.0280.0066.250.0510.0016250.530.00016250002.40.0000006\nConclusions\nThe low perfusion rate microreactor is suitable for the new application described here\u2014preparation of minute amounts of apoGOx and reconstitution of enzyme activity with a solution of FAD. In comparison to the conventional preparation of apoGOx using size-exclusion chromatography [1, 2, 16], this device requires neither complicated handling nor expensive materials and equipment. The enzyme is simply retained on the PES ultrafilter.\nA scale-up of the microreactor can easily be performed, allowing the preparation of larger amounts of apoGOx to be used, for example, in studies with modified FAD [2\u20134] or as the basis of a biosensor with which a wide variety of analytes can be detected [1]. The combination with immunological principles allows the analysis of small molecules when no specific enzyme is available, combining molecular recognition with enzyme enhancement of the signal. The sensitivity is improved even more by using electrochemical detection.\nLow perfusion rate microreactors may facilitate the study of enzymatic properties and modifications, including substrate inhibition, competitive and non-competitive inhibition, and allosteric activation. Because of the mild fixation conditions, they may enable investigation of receptor activity also. An advantage of the microreactor presented here is the small amount of protein required, thus allowing the use of expensive and rare enzymes. Applications using related devices, e.g. Refs. [7, 10, 17] usually require micrograms to milligrams of protein. Glucose sensors using glucose oxidase or glucose dehydrogenase have also been developed for clinical use. One example of such an application uses a volume of 300\u00a0nL blood for glucose measurements in diabetics [18]. Such reactors still use larger volumes as compared to the 20\u00a0nL we used here. Although we used electrochemical detection, other modes of detection including capillary electrophoresis, fluorescence, or chemiluminescence, are possible. In addition, with the current interest in proteomics, applications can be found where minute amounts of protein can be digested reproducibly and analysed directly by LC\u2013MS\u2013MS [19\u201321].","keyphrases":["glucose oxidase","apoglucose oxidase","flavin adenine dinucleotide","enzyme reactor","deflavination"],"prmu":["P","P","P","P","U"]}
{"id":"Emerg_Radiol-3-1-1914302","title":"Advanced Trauma Life Support\u00ae. ABCDE from a radiological point of view\n","text":"Accidents are the primary cause of death in patients aged 45 years or younger. In many countries, Advanced Trauma Life Support\u00ae (ATLS\u00ae) is the foundation on which trauma care is based. We will summarize the principles and the radiological aspects of the ATLS\u00ae, and we will discuss discrepancies with day to day practice and the radiological literature. Because the ATLS\u00ae is neither thorough nor up-to-date concerning several parts of radiology in trauma, it should not be adopted without serious attention to defining the indications and limitations pertaining to diagnostic imaging.\nIntroduction\nIn many countries, trauma care is based on Advanced Trauma Life Support\u00ae (ATLS\u00ae) [1]. Although the ATLS\u00ae manual and course are neither evidence based nor up-to-date concerning several parts of radiology in trauma, surgeons use the ATLS\u00ae recommendations, if present, routinely to support indications for diagnostic imaging. In addition, surgeons refer to the ATLS\u00ae unjustly with indications for imaging that are not supported by the ATLS\u00ae at all. Radiologists must be aware of this to intervene appropriately when sub-optimal imaging indications are presented. In this respect, knowing the content and the language of the ATLS can be helpful.\nThe objective of this review is to familiarize radiologist with the ATLS\u00ae. For this purpose, the rationale and indications of diagnostic imaging is assessed where it pertains to the ATLS\u00ae protocol. Instances of disagreement with the evidence in the literature and daily practice are highlighted [1].\nPurpose of ATLS\u00ae\nAccidents are the primary cause of death in patients aged 45\u00a0years or younger. In The Netherlands, 22 out of 100,000 people die each year because of accidental injury. For every one patient who dies, there are three survivors with serious disabilities [1, 2].\nThe purpose of adequate trauma care is to decrease this morbidity and mortality, which is expected to be achieved by fast, systematic, and effective assessment and treatment of the injured patient. Contrary to the ATLS guidelines, we think that imaging should play a prominent role in this process.\nHistory of ATLS\u00ae\nIn 1976, an airplane with an orthopedic surgeon, his wife and children crashed in a corn field in Nebraska. The wife died. The surgeon and three of his four children were seriously injured. Although they survived, he considered the standard of care in the local hospital insufficient and decided to develop a system to improve the care for trauma victims, and thus, ATLS\u00ae was born.\nSince the first ATLS\u00ae course in 1978, the concept has matured, has been disseminated around the world and has become the standard of emergency care in trauma patients in 46 countries [1].\nThe ATLS\u00ae concept is also used in the pre-hospital phase of trauma patient care and has been adopted for non-trauma medical emergencies and implemented in resuscitation protocols around the world.\nOriginally, ATLS\u00ae was designed for emergency situations where only one doctor and one nurse are present. Nowadays, ATLS\u00ae is also accepted as the standard of care for the first (golden) hour in level-1 trauma centers. The priorities of emergency trauma care according to the ATLS\u00ae principles are independent of the number of people caring for the patient.\nATLS\u00ae course\nThe ATLS\u00ae course is organized under license of the American College of Surgeons. Before the course, the students peruse the course manual. During a 2-day course, 16 students, mostly residents in surgery and anesthesiology, are trained by eight instructors. These instructors now number more than 100 in the Dutch ATLS\u00ae section, mostly surgeons and anesthesiologists but also two radiologists.\nDuring the course, all emergency measures are taught and reviewed. By means of observing, practicing, and repeating the ATLS\u00ae concepts, the object of the course is that the students are capable to perform the necessary measures independently with the correct priorities.\nThe course concludes with a written and practical examination, which has a pass rate of 80\u201390%.\nDuring the course, attention is also given to the multidisciplinary character of trauma care and the organization and logistics of trauma care in hospitals and surrounding area.\nRadiology has a minor part in the course There are only 50\u00a0min to teach the systematic evaluation of chest radiographs and another 50\u00a0min to teach in cervical spine radiographs, with the objective for the student to be able to identify life-threatening and potentially life-threatening injuries on chest radiographs and identify fractures on the radiographs of the spine. There is no lecture or skill set concerning computed tomography (CT).\nEssentials of ATLS\u00ae\nATLS\u00ae is a method to establish priorities in emergency trauma care. There are three underlying premises. (1) Treat the greatest threat to life first. (2) Indicated treatment must be applied even when a definitive diagnosis is not yet established. (3) A detailed history is not necessary to begin evaluation and treatment.\nTherefore, the assessment of a trauma patient is divided in a primary and a secondary survey. In the primary survey, life-threatening injuries are diagnosed and treated simultaneously. All other injuries are evaluated in the secondary survey.\nPrimary survey\nIn the primary survey, the mnemonic ABCDE is used to remember the order of assessment with the purpose to treat first that kills first (Table\u00a01). Airway obstruction kills quicker than difficulty of breathing caused by a pneumothorax, and a patient dies faster from bleeding from a splenic laceration then from a subdural hematoma.\nTable\u00a01In the primary survey, the mnemonic ABCDE is used to remember the order of assessment with the purpose to treat first that kills firstThe ABCDEAAirway and C-spine stabilizationBBreathingCCirculationDDisabilityEEnvironment and Exposure\nInjuries are diagnosed and treated according to the ABCDE sequence. Only when abnormalities belonging to a letter are evaluated and treated as efficacious as possible can one continue with the next letter. In case of deterioration of a patient\u2019s condition during assessment, one should return to \u2018A.\u2019 Imaging should not intervene with or postpone treatment.\nA: Airway\nThe airway is the first priority in trauma care. All patients get 100% oxygen through a non-rebreathing mask. The airway is not compromised when the patient talks normally. A hoarse voice or audible breathing is suspicious; facial fractures and soft tissue injury of the neck can compromise the airway, while patients in a coma are not capable of keeping their airway patent. Endotracheal intubation is the most definite way to secure the airway.\nIn \u2018A,\u2019 the cervical spine needs to be immobilized. As long as the cervical spine is not cleared by physical examination, with or without diagnostic imaging, the spine should remain stabilized.\nFor the evaluation of \u2018A,\u2019 no diagnostic imaging is necessary. Imaging of the cervical spine is just an adjunct to the primary survey and not part of the \u2018A,\u2019 specifically because, as long as the spine is immobilized, possible spinal injury is stabilized and diagnostic imaging can be postponed. When \u2018A\u2019 is secure, one can continue with \u2018B.\u2019\nB: Breathing\nBreathing is the second item to be evaluated in trauma care.\nTension pneumothorax, massive hemothorax, flail thorax accompanied by pulmonary contusion, and an open pneumothorax compromise breathing acutely and can be diagnosed with physical examination alone and should be treated immediately. Most clinical problems in \u2018B\u2019 can be treated with relatively simple measures as endotracheal intubation, mechanical ventilation, needle thoracocentesis, or tube thoracostomy. The lack of a definitive diagnosis should never delay an indicated treatment. To evaluate the efficiency of breathing, a pulse oximeter can be applied.\nInjuries, like a simple pneumothorax or hemothorax, rib fractures, and pulmonary contusion, are often more difficult to appreciate with physical examination. Because these conditions have less effect on the clinical condition of the patient, they can be identified in the secondary survey.\nA chest radiograph is an adjunct to the primary survey and can be helpful in evaluating breathing difficulties and is necessary to evaluate the position of tubes and lines. When \u2018B\u2019 is stabilized, one can continue with \u2018C.\u2019\nC: Circulation\nCirculation is the third priority in the primary survey. Circulatory problems in trauma patients are usually caused by hemorrhage. The first action should be to stop the bleeding. Hemorrhage can be external from extremity and facial injury or not visible from bleeding in chest, abdomen, and pelvis. Instable pelvic fractures can be temporarily stabilized with a pelvic band to decrease blood loss.\nBlood pressure and heart rate are measured; two intravenous lines are started, and blood is obtained for laboratory investigation.\nIn the search for internal blood loss, imaging can be very helpful. Radiological investigations such as a chest radiograph, when not already performed, ultrasound of the abdomen (focussed abdominal sonography in trauma, FAST) and a pelvic X-ray can suggest the localization of the bleeding.\nA tension pneumothorax can be the cause of circulatory distress but is usually diagnosed and treated in \u2018B.\u2019 When a patient\u2019s condition deteriorates, this diagnosis must be reconsidered. Hemodynamic instability can, infrequently, be caused by pericardial tamponade. Therefore, ultrasonography of the pericardial sac is part of a FAST examination. Other less frequently occurring causes of circulatory problems in trauma patients are myocardial contusion and loss of sympathetic tone caused by cervical and upper thoracic spinal cord injuries.\nWhen it is not possible to stabilize the patient in the trauma suite, other intervention like operation or embolization should be performed. The remainder of the primary survey will be finished thereafter. When \u2018C\u2019 is stabilized, one can continue with \u2018D.\u2019\nD: Disability\nDisability should be assessed as the fourth priority in the primary survey, and this includes assessment of the neurological status. The Glasgow coma score (GCS) is used to evaluate the severity of head injury. This score is arrived at by scoring eye opening, best motor response, and best verbal response. Patients who open their eyes spontaneously, obey commands, and are normally oriented score a total of 15 points. The worst score is 3 points. A decreased GCS can be caused by a focal brain injury, such as an epidural hematoma, a subdural hematoma, or a cerebral contusion, and by diffuse brain injuries ranging from a mild contusion to diffuse axonal injury. To prevent secondary injury to the brain, optimal oxygenation and circulation are important. Also, impaired consciousness can be caused or aggravated by hypoxia or hypotension for which ABC stabilization is essential.\nIf a cranial CT is indicated, it should be done in the secondary survey.\nE: Environment and exposure\nEnvironment and exposure represent hypothermia, burns, and possible exposure to chemical and radioactive substances and should be evaluated and treated as the fifth priority in the primary survey.\nAt the end of the primary survey, before continuing with the secondary survey, the ABCDEs should be re-evaluated and confirmed.\nSecondary survey\nDuring the secondary survey, the patient is examined from head to toe, and appropriate additional radiographs of the thoracic and lumbar spine and the extremities are performed when indicated. CT scans, when indicated, are also done in the secondary survey.\nIf, during the secondary survey, the patient\u2019s condition deteriorates, the primary survey should be repeated beginning with \u2018A.\u2019\nThe rigid spine board should be removed as early as possible because it is a serious risk for decubitus ulcer formation. Removing the hard backboard should not be delayed for the lone purpose of obtaining definitive spine radiographs.\nDiagnostic imaging\nRadiographs of the chest, pelvis, C-spine, and FAST are adjuncts to the primary survey.\nImaging is considered helpful but should be used judiciously and should not interrupt or delay the resuscitation process. When appropriate, radiography may be postponed until the secondary survey.\nCT, contrast studies, and radiographs of the thoracic spine, lumbar spine, and extremities are also adjuncts to the secondary survey.\nImaging is most useful and efficient if consulting with a radiologist becomes routine [3, 4]. We extrapolate this advice to the trauma setting and endorse consultation with clinicians strongly; however, consulting a radiologist is not mentioned once in the ATLS\u00ae manual!\nContrary to (ever-increasing) daily practice, CT plays a minor role in the ATLS\u00ae. With the increasing use of CT in the evaluation of trauma patients, radiation exposure should be a major issue in the field of emergency radiology. CT scanners using an automatic exposure control technique can help to reduce radiation dose [5, 6].\nBlunt trauma\nThorax\nA chest radiograph must be obtained to document the position of tubes and lines and to evaluate for pneumothorax or hemothorax and mediastinal abnormalities. When not obtained in the primary survey, it should be done in the secondary survey. From the ATLS\u00ae manual, it is not clear if a chest radiograph should be performed in every patient [1]. However, this is in accordance with the literature. At present, no clinical decision rule is available concerning the indication for chest radiography in trauma patients.\nA CT of the chest is considered an accurate screening method for traumatic aortic injury. If a contrast enhanced helical CT is negative for mediastinal hemorrhage and aortic injury, no additional diagnostic imaging is necessary [1].\nIf a CT is positive, the ATLS\u00ae manual states that the trauma surgeon is in the best position to determine which, if any other, diagnostic imaging is warranted. The possibility to construct multiplanar reconstructions (MPRs), maximum intensity projections (MIPs), volumetric, and virtual angioscopic three-dimensional views from MDCT data, making diagnostic angiography superfluous, is not stated [7]. The same post-processing tools can be used to differentiate between traumatic aortic injury and normal variants [7]. Neither consulting with a radiologist nor endovascular treatment of traumatic aortic injury are mentioned in the ATLS\u00ae manual.\nAlthough it is recognized that the severity of pulmonary contusions does not correlate very well with the chest radiograph, a CT for the evaluation of pulmonary contusion is not mentioned. The superiority of CT in the detection of pneumothoraces and evaluation of the position of chest tubes is not stated [8, 9].\nAbdomen\nFAST is used in hemodynamic abnormal patients as a rapid, non-invasive, bedside, repeatable method to document fluid in the pericardial sac, hepato-renal fossa, spleno-renal fossa, and pelvis or pouch of Douglas. When FAST is available, it replaces diagnostic peritoneal lavage (DPL) [10]. FAST is a good performing screening tool in evaluating hypotensive trauma patients to differentiate those patients who do need urgent laparotomy from those who do not [11].\nAn abdominal CT is the most sensitive and specific investigation for the diagnosis of visceral and vascular injury; however, according to the ATLS\u00ae, an abdominal CT can only be performed in hemodynamically normal patients because a CT is considered time consuming. This is no longer true with the helical CT available today. The rate-limiting step has become the movement of the patient to the CT suite and on and off the CT table [12].\nAccording to the ATLS\u00ae manual, an upper GI contrast study is the imaging method of choice in suspected diaphragm rupture. CT is not mentioned as an option. On the contrary, it is stated that CT misses diaphragmatic injuries. Although CT is not 100% sensitive, neither are GI contrast studies. A comparative study is not available. MDCT has the advantage that it is much easier and quicker to perform in trauma patients [1, 13]. Although no consensus of opinion exists, coronal, and sagittal multiplanar reconstructions (MPRs) might improve the accuracy of MDCT for the diagnosis of blunt traumatic diaphragm rupture [14, 15].\nA final omission, and contrary to daily practice in many hospitals, is that interventional radiology is not mentioned as an adjunct to non-operative management in patients with abdominal visceral injury [16, 17].\nPelvis\nIt is recommended that a pelvic radiograph should be performed when the mechanism of injury or the physical examination indicates the possibility of a pelvic fracture.\nEvaluation of the pelvis on an abdominal CT is not mentioned [18]. Compared to conventional radiography, CT has a higher sensitivity and specificity for the diagnosis of pelvic fractures, and MPRs can be used to delineate the full extend of the fracture [19, 20].\nIn a hemodynamically abnormal patient with a pelvic fracture and no indication for intra-abdominal hemorrhage on FAST or DPL, angiography with embolization is advised preceding surgical pelvic fixation.\nIn patients with an unstable pelvic fracture, inability to void, blood at the meatus, a scrotal hematoma, perineal ecchymoses, or a high-riding prostate, there is a suspicion of a urethral tear, and in these patients, a retrograde urethrogram should be performed before inserting a urinary catheter [1].\nTo exclude an intraperitoneal or extraperitoneal bladder rupture in patients with hematuria, a conventional or a CT cystogram can be performed [1, 21].\nCervical spine\nCervical spine radiographs are not indicated in patients who are awake, alert, sober, neurologically normal, have no neck pain or midline tenderness, can voluntary move their neck from side to side, and flex and extend without pain. In all other patients, a lateral, AP, and open-mouth odontoid view should be obtained. Although it is not mentioned in the ATLS\u00ae manual, this seems to be a combination of the Canadian C-spine rules and the Nexus criteria but the criterion \u2018painful distracted injury\u2019 has disappeared between the sixth and the seventh edition of the ATLS manual [1, 22\u201324]. Possibly, this was done because the definition of painful distracting injury is difficult, but if omitted, this reduces the sensitivity of the clinical decision rule [25].\nOn the lateral view of the cervical spine film, the base of the skull to the first thoracic vertebra must be assessed. If not all seven cervical vertebrae are visualized, a swimmers view must be obtained and is considered sufficient and safe [1]. Supine oblique views and, contrary to available literature, performing a CT scan of this area is not mentioned [26\u201328]. Further, of all suspicious areas and all not adequately visualized areas, an axial CT with 3-mm intervals should be obtained. In the cervical spine section, multidetector CT assessment with coronal and sagittal MPRs is not mentioned at all [1].\nPerforming a CT of the cervical spine without a preceding conventional radiograph as the screening method of choice is not mentioned. The recent ACR appropriateness criteria suggest otherwise [29].\nTo detect occult instability in patients without an altered level of consciousness, or those who complain of neck pain, flexion-extension radiographs of the C-spine may be obtained [1]. As flexion-extension radiographs are often non-diagnostic and necessitate movement of the spine that is potentially dangerous, at least, performing a CT first to exclude osseous injury or a magnetic resonance imaging (MRI) for the detection of ligamentous injury should be recommended today [27, 29, 30].\nMRI is recommended in patients with neurological deficits to detect an epidural hematoma or a traumatic herniated disc. Contrary to the ACR appropriateness criteria, the ATLS states that, when a MRI is not available, CT myelography may be used [1, 29].\nAngiography or CT angiography for the evaluation of injury to the carotid or vertebral artery is not mentioned [1].\nHead\nAgain, according to the protocol, a cranial CT should be considered in all head-injured patients with a focal neurologic deficit of which the cause can be localized in the brain, a Glasgow coma scale less than 15, amnesia, loss of consciousness of more than 5\u00a0min, or severe headaches. This is insufficient to detect all clinical relevant brain injury. There is no reference to evidence-based clinical decision rules such as the Canadian head CT rule, the New Orleans head CT rule or the CHIP prediction rule [1, 31\u201334].\nThoracic and lumbar spine\nThe indications for diagnostic imaging are the same as for the cervical spine. AP and lateral radiographs should be performed with additional CT of suspicious areas [1].\nIt is not mentioned that the thoracic and lumbar spine can be reliably evaluated on a CT of thorax and abdomen. When a CT of thorax and abdomen has already, or will be, performed, conventional radiography does not have any additional value especially when MPRs of the spine are obtained [35].\nPenetrating trauma\nChest\nPneumothorax and hemothorax can be diagnosed with a chest radiograph. Even in patients with a normal chest radiograph, a CT is advocated for the evaluation of heart, pericardium, and great vessels in patients with a suspicion of mediastinum transversing injury. For the heart and pericardial sac, a CT can be replaced by ultrasound, and for the major vessels, an angiography can be performed. For the evaluation of oesophageal injury, esophagography using a water-soluble contrast agent and complementary esophagoscopy should be performed. The trachea and bronchial tree can be evaluated by bronchoscopy.\nPatients with penetrating injury of the lower chest below the transnipple line anterior and the inferior tip of the scapula posterior are considered to have abdominal trauma as well until proven otherwise [1].\nAbdomen\nA hemodynamically abnormal patient with a penetrating abdominal wound does not need diagnostic imaging but should undergo laparotomy immediately.\nIn a hemodynamically normal patient, an upright chest radiograph can document intraperitoneal air and is useful to exclude hemothorax or pneumothorax. An abdominal radiograph (supine, upright, or lateral decubitus) may be useful in hemodynamically normal patients to detect extra-luminal air in the retroperitoneum or free air under the diaphragm.\nIn all patients with penetrating abdominal injury, an emergency laparotomy is a reasonable option, especially in patients with gunshot wounds. In initially asymptomatic patients with a lower chest wound or injuries to the back or flank, the ATLS\u00ae considers double or triple contrast CT, DPL, and serial physical examination less invasive diagnostic options, equivalent to each other [1].\nFor asymptomatic patients with anterior abdominal stab wounds, DPL, laparoscopy, and serial physical examination are mentioned as diagnostic options. However, although there is a significant body of evidence that this may not be optimal, CT is not mentioned as a diagnostic option in these patients [36].\nConclusion\nATLS\u00ae is a well-tried systematic approach for the assessment of trauma patients. In multidisciplinary trauma care, it is beneficial and, maybe, even mandatory for effective communication that all members of the trauma team, including the radiologist, speak the same ATLS\u00ae language.\nAlthough imaging should not intervene with or postpone treatment, a chest radiograph, pelvic radiograph, and FAST can direct treatment decisions and should be performed in the primary survey when indicated. Imaging of the cervical spine is also an adjunct to the primary survey but can be postponed as long as the spine is immobilized. All other imaging should be done in the secondary survey.\nUnfortunately, according to the ATLS\u00ae, CT plays a minor role in the evaluation of trauma victims. In the ATLS\u00ae, chest CT is only mentioned for the diagnosis of traumatic aortic injury but, in our experience, chest CT is valuable for the evaluation of pulmonary contusions and hemothorax and pneumothorax. Nowadays, abdominal CT is less time consuming than the ATLS\u00ae states and can be used to evaluate the extent of the abdominal injury in patients in whom no immediate laparotomy is indicated to evaluate the possibilities for non-operative management with or without endovascular embolization. The indications for head CT according to the ATLS\u00ae are insufficient to diagnose all patients with significant head injury. CT of the cervical spine can be used as a primary investigating tool and not only as an adjunct to conventional radiography. When a CT of the chest and abdomen is indicated, the thoracic and lumbar spine, as well as the pelvis, can be evaluated on the axial CT images combined with coronal and sagittal multiplanar reconstructions, and in these cases, conventional radiography of the spine and pelvis do not have any additional diagnostic value.\nBecause the ATLS\u00ae is neither thorough nor up-to-date concerning several parts of radiology in trauma, it should not be adopted without questions to define indications for diagnostic imaging. Consultation between clinicians and radiologists can improve the efficiency and quality of diagnostic imaging in trauma patients.","keyphrases":["trauma","radiology","diagnostic imaging","spine","thorax","abdomen"],"prmu":["P","P","P","P","P","P"]}
{"id":"Int_J_Hematol-4-1-2276242","title":"Nasal NK\/T-cell lymphoma: epidemiology and pathogenesis\n","text":"Nasal NK\/T-cell lymphoma (NKTCL) is an uncommon disease, but usually shows a highly aggressive clinical course. The disease is much more frequent in Asian and Latin American countries than in Western countries, and is universally associated with Epstein\u2013Barr virus (EBV) infection. Analyses of gene mutations, especially p53 and c-KIT, revealed the different frequencies by district. Epidemiological studies revealed the changes of the disease frequency in Korea during the period from 1977\u20131989 to 1990\u20131996. Case-control study showed that the exposure to pesticides and chemical solvents could be causative of NKTCL. Further studies including HLA antigen typing of patients is necessary to further clarify the disease mechanism.\nRapid destruction of the nose and face (midline) was firstly described by McBride in 1897 [1]. Macroscopically the lesions usually looked like necrotic granuloma and the patients showed aggressive and lethal course, therefore the term \u201clethal midline granuloma (LMG)\u201d or \u201cgranuloma gangrenescens\u201d were used for this condition. Later the term LMG is popularly used. It had became evident that the LMG is composed of three different types of histology, i.e. Wegener\u2019s granulomatosis (WG), polymorphic reticulosis (PR) or midline malignant reticulosis (MMR), and malignant lymphoma [2, 3]. These diseases could be histologically differentiated from each other by taking clinical findings into account. WG is usually characterized by generalized necrotizing vasculitis involving both arteries and veins and the presence of glomerulitis [4]. Cancer and specific inflammation such as tuberculosis also cause a condition indistinguishable from LMG.\nPR exhibits a polymorphous pattern of proliferation consisting of large atypical cells with mono- or multinucleus, small lymphocytes, plasma cells, benign-appearing macrophages, neutrophils and much less frequently eosinophils. PR had been considered as a variant of malignant lymphomas because the disease is frequently disseminated [2]. The nature of proliferating cells in PR had been controversial. Once the disease was termed as nasal T-cell lymphoma because the proliferating cells showed a positive immunoreactivity for polyclonal antibodies against T-cells [5, 6]. However, monoclonal rerrangement of the T-cell receptor genes was seldomly found in cases with \u201cnasal T-cell lymphoma\u201d [7]. Later Ng et al. [8] reported that the tumor cells showed a positive immunoreactivity for natural killer (NK) cell marker CD56. Subsequently it was shown that proliferating cells in PR had large granular lymphocyte morphology [9], which is a character of NK cells or cytotoxic T-lymphocytes. There have been accumulating evidences that PR is a neoplasm of activated NK cells [10\u201313]: the proliferating cells usually show CD2+ , CD56+ , CD3\u03b5+, CD7\u2212, CD16\u2212, cytotoxic granule-associated proteins+ and do not exhibit rearrangement of T-cell receptor or immunoglobulin genes (Fig. 1). Meanwhile rearrangement of T-cell receptor genes was recorded in the rare cases of PR [14, 15]. Although the upper respiratory tract, especially nasal region, is the common site of presentation, NK\/T-cell lymphoma of nasal type may present in diverse extranodal sites such as gastrointestinal tract, skin, testis, liver, and spleen [16]. In the World Health Organization classification, NK\/T-cell lymphoma encompasses the lymphomas involving nasal area and nonnasal area. Until present, there have been many review articles on the clinical, pathological, immunohistochemical, and immunogenetical aspects of NK\/T-cell lymphomas involving upper respiratory tract. This review focuses on the epidemiology and molecular pathogenesis of nasal NK\/T-cell lymphoma (NLTCL).\nFig. 1HE. Polymorphous pattern of proliferation in the nasal cavity. Large cells show positive immunoreactivity with CD3\u03b5, TIA-1, and CD56. ABC method, \u00d7400\nAssociation with Epstein\u2013Barr virus (EBV)\nThe association between EBV and human malignancies, including endemic Burkitt\u2019s lymphoma, Hodgkin lymphoma and non-Hodgkin\u2019s lymphoma (NHL) of either B- or T-immunophenotypes, has been reported. Etiological role of EBV for development of NKTCL was firstly reported in 1990 [17]. Subsequent study revealed the constant association of NKTCL with EBV in the world (Fig. 2) (Table 1) [18\u201323]. Sino-nasal lymphomas are immunophenotypically classified into NK\/T-(CD56+), T-, and B-cell type with distinctive clinical features [24]. CD56 positivity, therefore NKTCL, was closely associated with EBV positivity among sino-nasal lymphomas [22]. EBV could be subtyped based on the difference in sequence of EBNA2 region, i.e. type A and type B [25]. Almost all of the NKTCL in Korea and Japan had type A EBV, which is identical with the previous literature reporting that most cases of EBV-associated malignancies of immunocompetent patients in Asia had type A EBV [26]. Predominance of type A EBV was also found in NKTCL in Malaysia [27], indicating the predominance of type A EBV in NKTCL in Asia. Several studies indicated the occurrence of type B EBV in lymphoma of immunocompromised patients [28]. Borisch et al. [29] reported that three of six cases of NKTCL in Switzerland had type B EBV, although no findings suggestive of immunodeficiency were found in these patients. These findings suggest a geographic difference in the distribution of EBV subtype in the NKTCL.\nFig. 2In situ hybridization with EBER-1 probe reveals positive signals in the nucleus of proliferating cell\nTable 1EBV in nasal NK\/T-cell lymphomaSourceCountryEBV positive rate (%)EBV subtype Expression of latent[18]a\nFrance7\/7 (100)NA7\/7 LMP+[20]China21\/21 (100)NA5\/21 LMP-1+[21]Japan11\/12 (92)10\/11 typeA8\/12 LMP-1+[22]Korea15\/16 (94)15\/15 typeA7\/15 LMP-1+[24]Indonesia18\/20 (90)NANAEBV Epstein\u2013Barr virus, LMP latent membrane protein, NA not availableaFresh tissue samples were used in the analyses\nAs shown in Table 1, the proliferating cells in NKTCL frequently express latent membrane protein (LMP) as revealed by immunohistochemistry. Latent infection gene products of EBV, EBNA-2 and LMP-1, serve as target antigens for the elimination of EBV infected cells by host cytotoxic T-lymphocytes (CTL) [30]. Shen et al. [31] showed that NKTCL cells are able to provide target epitopes of EBV for CTL. In immunocompromised hosts, proliferating cells expressing EBNA-2 and LMP-1 can escape from immune surveillance by the host CTL, which might result in the development of malignant lymphomas [32]. However, systemic immunosuppression is not noted in patients with NKTCL, thus suggesting an unknown underlying mechanism for escape of LMP-1-expressing tumor cells from the CTL. Several mechanisms such as downregulation of the immunogenic EBV nuclear antigens and preferential selection of the deletion genotype of LMP-1 was reported by Chiang et al. [33, 34] and expression of IL-10, an immunosuppressive cytokine, was reported by Shen et al. [31]. The cells expressing viral antigens are eliminated primarily by CTL in a MHC-class-I-restricted manner [35]. Two CTL-epitopes were identified in LMP-1 that are possibly pan A*02-restricted [36]. It is possible that NKTCL patients show lower frequencies of A*02 allele compared with those in the normal population. Indeed high-resolution genetic typing revealed significantly lower frequency of HLA-A*0201 in NKTCL than in normal population [37]. These findings suggest that HLA-A*0201-restricted CTL responses may function in vivo to suppress the development of NKTCL, or in other words, role of EBV for NKTCL development.\nGenetical changes\nLymphoma arises from clonal expansion of lymphoid cells that are transformed by the accumulation of genetic lesions affecting oncogenes and tumor suppressor genes. In general, amount of samples from NKTCL lesions available for genetical analyses is small, and samples usually contain massive necrotic areas. Therefore information for genetical changes in NKTCL has been relatively limited until present.\nAlterations of tumor suppressor genes and oncogenes\nPolymerase chain reaction (PCR)\u2014single strand conformation polymorphism (SSCP) followed by direct sequence method was employed for analyses of gene alterations in NKTCL. The genes analyzed by this method were p53, k-ras, c-kit, and \u03b2-catenin on the NKTCL cases from Asian countries [38\u201342] and Mexico [43]. p53 is a well-known tumor suppressor gene that causes cells with damaged DNA to arrest at the G1 phase of cell cycle or stimulating expression of the BAX gene, the protein that promotes apoptosis [44]. In a wide variety of human cancers, p53 gene mutations have been detected mainly in exon 5 through 8 [45]. K-RAS, c-KIT, and \u03b2-catenin genes are oncogenes. Greenblatt et al. [46] identified 50 studies in which sequencing of the entire coding region of p53 had been reported. Of the 560 mutations reported in those papers, 87% were found in exons 5\u20138, and most of the others were in exon 4 (8%). In the studies for NKTCL in Asia, exons 4\u20138 or exons 5\u20138 were examined in one institute (Table 2). The frequency of p53 mutations was various by district: high in Japan and Indonesia and low in Mexico, Korea, and Shenyang. Shenyang situates at north China, which is adjacent to the Korean peninsula, suggesting that environmental and genetical factors might generate the differences in frequency. Transitions (G:C to A:T) were the predominant pattern of mutations except for Mexico, in which number of cases with mutations was only five [43], thus the data from Mexico seemed to be not conclusive. Predominance of transition mutations suggests that some \u201cendogenous\u201d mutagens act in lymphomagenesis. The transition pattern of p53 mutations is known to be more susceptible to spontaneous genetic instability than transversion. While genetic instability as revealed by widespread microsatellite instability was not found in the cases with NKTCL [47].\nTable 2p53 mutations in nasal NK\/T-cell lymphomaNumber of cases Exons examinedFrequency of mutations (%)Predominance of transition mutationAsia Japan [41]584\u2013862%yes Korea [41]424\u2013831%yes China Beijing, Chengdu [38]425\u2013848%yes Shenyang [40]204\u2013840%yes Indonesia [42]274\u2013863%yesMexico [43]215\u2013824%no\nQuintanilla-Martinez et al. reported the association of p53 overexpression with poor prognosis, and p53 mutations with large cell morphology and advanced stage [43, 48]. However these findings were not confirmed by other studies. [15, 38]\nThe c-KIT proto-oncogene encodes a receptor tyrosine kinase, which is involved in normal hematopoiesis, gametogenesis, and melanogenesis via the c-kit receptor-ligand system. [49]. Because the development of acute leukemia or malignant lymphoma was reported in transgenic mice expressing KIT [8, 14, 50], NKTCL in Asian countries was examined for the c-kit gene mutations [41]. Frequency of c-kit mutations was significantly higher in China (Beijing, Chengdu) (10 of 14 cases: 71.4%) [39] than in Japan (9 of 58 cases: 15.5%) [41], Korea (5 of 42 cases:11.9%) [41], northeast China (Shenyang) (2 of 20 cases:10%) [40], and Indonesia (3 of 27 cases :11.1%) [42]. These findings suggest that location-specific differences in etiological factors cause specific mutations in c-kit gene.\nFAS Gene Mutations\nFas (Apo-1\/CD95) is a 45 kDa membrane protein belonging to the tumor necrosis factor receptor family, and mediates programmed cell death (apoptosis) through binding of FAS ligand (Fas L) [51]. Fas consists of 325 amino acids with a single transmembrane domain, including signal peptide. The 80-amino acid portion in the cytoplasm, designated as a death signaling domain, is essential for the apoptotic signal transduction. FAS gene mutations were reported in about 10% cases with sporadic non-Hodgkin\u2019s lymphoma [52]. NKTCL frequently co-express FAS and FAS ligand (Fas L), but the tumor cells seldom undergo apoptosis. Some mechanisms for resistance to FAS\/FAS L - induced apoptosis might work in the development of NKTCL, thus FAS gene mutations could be one of the mechanisms. Two reports support this notion: NKTCL cells in 9 of 15 (60%) cases [53] and 7 of 14 (50%) cases [54] showed mutations of FAS gene. Mouse T-cell lymphoma cells transfected with mutated FAS genes were resistant to apoptosis (Fig. 3, Fig. 4) [54], indicating the mutations to be loss of function mutation. These findings suggest that accumulation of lymphoid cells with FAS mutations provides a basis for the development of NKTCL.\nFig. 3Summary of the FAS gene mutations found in patients with nasal NKTCL. The shaded rectangles at the COOH-terminus of the protein represents the small peptide added because of the frameshift in the gene encoding FAS.TM: transmembrane domain\nFig. 4The mouse WR19L cell line expressing recombinant human FAS protein with (T1102C, A978G, 1095 ins A) or without (wild type) mutations were incubated with various concentrations of anti-FAS antibody at 37.0\u00b0C for 16 h. Clones expressing FAS receptor with any mutations (A978G, 1095 ins A, T1102C) were resistant to apoptosis induced by the anti-FAS antibody\nOthers\nVarious cytogenetic alterations have been reported, of which deletions of 6q are the common [55].\nEpidemiological features\nIt had been reported that NKTCL seemed to be relatively common among non-Hodgkin\u2019s lymphoma in Hong Kong [56\u201358]: malignant lymphomas affecting nose and nasopharyngeal region constituted 7.2% of extranodal lymphomas [56] and 45 of 70 cases with malignant lymphomas in the nose, nasopharynx, and larynx had PR morphology [57]. This disease is occasionally encountered in the hospitals in Japan [5, 59, 60]. When one of the authors (KA) visited the USA in 1987, he noticed that the disease was quite rare in the USA, but they had consultation cases from Peru. Because Japanese and a part of Peruvian belong to Mongolian ethnic group, it was postulated that the Mongolian group might be much more frequently affected by this disease. Then they started to examine the frequency of NKTCL in Japan, Korea (Seoul), and China (Shanghai) during the period from 1987 to 1993. The results are summarized in Table 3 [61\u201363], in which the frequency of each disease constituting LMG is shown as the frequency per 100,000 patients who visited the Ear\u2013Nose\u2013Throat (ENT) clinic in 37 university hospitals in Japan, Ryukyu University Hospital in Okinawa, Japan, Yonsei University Hospital in Seoul, and Shanghai Medical University Hospital. All of the histological sections were reviewed by one of the authors (KA). Frequency of PR ranged from 8 to 40.8. That in the Institute of Laryngology and Otology, London (1966\u20131987) was four, showing two to ten times higher frequencies of the PR in the east Asian countries [61]. The disease is rare in the USA [64] and Europe [65]. The patients with NKTCL seem to be clustered also in Latin American countries and Indonesia [66, 67]. Information from other parts of the world such as Africa, the Middle and Near East, and Rusia is helpful to elucidate the etiology of this disease.\nTable 3Frequency of lethal midline granuloma in Japan, Korea (Seoul), and China (Shanghai)DiseaseNumber of patients (frequency per 100,000 ENT patients)Japan other than Okinawa (1965\u20131986)Okinawa (1973\u20131991)Seoul (1979\u20131989)Shanghai (1979\u20131990)Wegener\u2019s granulomatosis64(4)1(3)0(0)1(4)Polymorphic reticulosis114(8)9(27.4)56(40.8)73(9.8)Malignant lymphoma82(6)11(33.5)15(10.9)54(7.2)Others42(3)1(3)6(4.4)0(0)Total302(21)22(69.9)77(56)128(17)\nAt the late 1990s, otorhinolaryngologists in both Korea and Japan had the impression that the frequency of PR appeared to be decreasing. Then the changes in frequency of PR with time among cases from Seoul and 59 university hospitals in Japan were examined [68]: the frequency rate of PR per 100,000 outpatients of ENT clinics in Seoul decreased from 40 to 20 between the periods of 1977\u20131989 and 1990\u20131996. However, there were no significant changes in Japan during the period studies.\nLife-style and environmental factors\nEpidemiological studies have revealed that the NKTCL occurs much more frequently in Asian countries than in Western countries and it is closely associated with EBV infection. There are differences in frequencies of p53 and c-kit gene mutations among patients with NKTCL in Japan, China, and Korea. Recently the first case of familial NKTCL affecting a father and one of his six children was reported [69]. They used large amounts of pesticide in a green house. An increase in the risk of developing NHL among individuals exposed to pesticides was reported [70, 71]. In addition, a correlation of exposure to certain pesticides and organochlorines with increased titers of antibodies to EBV was reported [72]. All these findings might suggest a causative role for some genetical, environmental and life style factors in the development of NKTCL. Therefore, the epidemiological study to elucidate whether socioenvironmental ambient factors contribute to the development of NKTCL was conducted as a collaborative study of Japan, Korea and China (Table 4) [73]. The odd ratio (OR) of NKTCL was 4.15 (95% confidence interval (CI), 1.74\u20139.37) for farmers, 2.81 (CI 1.49\u20135.29) for producers of crops, and 4.01 (CI 1.99\u20138.09) for pesticide users. The ORs for crop producers, who minimized their exposure to pesticides by using gloves and glasses, and sprinkling downwind at the time of pesticide use, were 3.30 (CI 1.28\u20138.54), 1.18 (CI 0.11\u201312.13), and 2.20 (CI 0.88\u20135.53), respectively, which were lower than those for producers who did not take these precautions. Exposures to pesticides and chemical solvents could be causative factors for NKTCL. Previous studies showed the increased risk of NHL in individuals using pesticides, especially phenoxyacetic acid-type herbicides [74]. Association of pesticides with risk of developing t(14;18) positive-NHL, but not t(14;18) negative-NHL, was reported [75].\nTable 4Risk of nasal NK\/T-cell lymphoma in relation to cultivation of crops and pesticide useOdds ration (OR)a\nNumber of cases N = 88Number of controls N = 30595% CILowerUpperCultivation of crops At present2.8127361.495.29 More than 5 years5.0824192.4710.43Pesticides Users4.0123231.998.09Type of pesticide Herbicide3.1713161.367.38 Insecticide3.4520211.677.13 Fungicide6.051061.9818.46Precautions Gloves used3.3010111.288.54 Gloves not used 4.7613121.9311.72 Mask used5.4414102.2013.47 Mask not used 2.829131.087.37 Glasses used1.18130.1112.13 Glasses not used4.5222202.179.42 Sprinkling downward attended2.209160.885.53 Sprinkling downward not attended8.451473.0123.70CI confidence intervalaAdjusted for age (<40, 40\u201359, >60), sex (male\/female) and country (Japan, Korea and China)\nConclusions\nClinical course of patients with NKTCL is usually highly aggressive. Therefore clarification of risk factors for disease development is especially important to establish a strategy for disease prevention. Because EBV infection and pesticides could be risk factors for NKTCL, investigation on effects of pesticides for EBV activation is needed. Employment of the similar kind of the epidemiological study is desirable in other areas than East Asia. Patients with NKTCL cluster in Asia and Latin American countries, therefore some genetical factors might be involved in the disease development. Further studies including HLA antigen typing of patients is important to further clarify the mechanism for disease development.","keyphrases":["epidemiology","epstein\u2013barr virus","pesticide","p53 gene","c-kit gene","nasal lymphoma","nk\/t-cell type"],"prmu":["P","P","P","P","P","R","R"]}
{"id":"J_Hum_Genet-3-1-1915643","title":"The 894G>T variant in the endothelial nitric oxide synthase gene and spina bifida risk\n","text":"The 894G>T single nucleotide polymorphism (SNP) in the endothelial NOS (NOS3) gene, has recently been associated with embryonic spina bifida risk. In this study, a possible association between the NOS3 894G>T SNP and spina bifida risk in both mothers and children in a Dutch population was examined using both a case-control design and a transmission disequilibrium test (TDT). Possible interactions between the NOS3 894G>T SNP and the MTHFR 677C>T SNP, elevated plasma homocysteine, and decreased plasma folate concentrations were also studied. The NOS3 894TT genotype did not increase spina bifida risk in mothers or children (OR 1.50, 95%CI 0.71\u20133.19 and OR 1.78, 95%CI 0.75\u20134.25, respectively). The TDT demonstrated no preferential transmission of the NOS3 894T allele (\u03a72 = 0.06, P = 0.81). In combination with the MTHFR 677TT genotype or elevated plasma homocysteine concentrations, the NOS3 894GT\/TT genotype increased maternal spina bifida risk (OR 4.52, 95%CI 1.55\u201313.22 and OR 3.38, 95%CI 1.46\u20137.84, respectively). In our study population, the NOS3 894GT\/TT genotype might be a risk factor for having a spina bifida affected child in mothers who already have an impaired homocysteine metabolism.\nIntroduction\nNitric oxide synthase (NOS) catalyzes the generation of nitric oxide (NO), an important signaling molecule that mediates many of its biological effects by activating the enzyme guanylyl cyclase, thereby increasing cyclic GMP synthesis (Murad 2006). Besides its important role in cardiovascular control, NO has been suggested to play a role in development (Lee and Juchau 1994). NO has been demonstrated to be present in the neural tube of chick embryos at the time of neurulation (Traister et al. 2002) and regulates the balance between mitosis and programmed cell death (Plachta et al. 2003). Other studies show that NO can inhibit methionine synthase (MTR) enzyme activity, thereby interfering with homocysteine remethylation (Danishpajooh et al. 2001) and proper neurulation (Nachmany et al. 2006; Weil et al. 2004).\nThe 894G>T single nucleotide polymorphism (SNP) in the endothelial-derived nitric oxide synthase (NOS3) gene, identified by Hingorani et al. (1999), was shown to be associated with elevated plasma homocysteine levels in nonsmokers with low serum folate concentrations (Brown et al. 2003). More recently, it was demonstrated that the NOS3 894GT genotype is an embryonic risk factor for spina bifida (OR 1.63, 95%CI 1.09\u20132.42) (Brown et al. 2004).\nIn this study, we examined the relation between the NOS3 894G>T SNP and spina bifida risk in both mothers and children using a case-control design and a transmission disequilibrium test (TDT). An effect of the NOS3 894G>T SNP on plasma homocysteine or plasma folate concentration was examined as well as possible interactions between the NOS3 894G>T SNP and the MTHFR 677C>T SNP, elevated plasma homocysteine, and decreased plasma folate concentrations.\nMaterials and methods\nStudy population\nThe study population has been described in detail before (van der Linden et al. 2006). Briefly, the study population was recruited in collaboration with the BOSK and was extended by a group from the Pediatric Neurology Department of the Radboud University Nijmegen Medical Center. The study group included 109 spina bifida patients (61 girls and 48 boys, mean age 16.4\u00a0\u00b1\u00a011.3), their mothers (n\u00a0=\u00a0121, mean age 42.9\u00a0\u00b1\u00a010.9) and fathers (n\u00a0=\u00a0103, mean age 44.9\u00a0\u00b1\u00a010.2).\nThe control group consisted of 500 volunteers recruited from a general practice in The Hague (den Heijer et al. 1995). In the present study we only included the 292 women from this control group (mean age 50.6\u00a0\u00b1\u00a013.5). The pediatric control group included 234 children (119 girls and 115 boys, mean age 8.4\u00a0\u00b1\u00a06.4) (van Beynum et al. 1999). The children of secondary school age were healthy volunteers, and the younger children were, for ethical reasons, recruited in a hospital setting. The local medical ethics committee approved the study.\nFor the present study, DNA of 102 spina bifida patients, 116 mothers, 101 fathers, 265 control women and 211 pediatric controls was available for genotyping.\nBiochemical determinations\nPlasma total homocysteine concentration was measured in EDTA plasma by HPLC and fluorescence detection, as previously described by te Poele-Pothoff et al. (1995).\nPlasma folate concentration was determined using the Dualcount Solid Phase Boil Radio assay (Diagnostic Product Corporation, Los Angeles, CA).\nPCR amplification and genotype determination\nNOS3 894G>T genotyping was performed according to Hingorani et al. (1999). Primer annealing took place at a temperature of 58\u00b0C. The PCR product of 206\u00a0bp was cut with the restriction enzyme MboI, which cuts the T allele in fragments of 119 and 87\u00a0bp.\nStatistical analyses\nCase-control study\nOdds ratios (ORs) and their 95% confidence intervals (95%CI) were calculated by means of logistic regression analysis.\nThe effect of the NOS3 894G>T SNP on plasma homocysteine and plasma folate concentration was studied by means of linear regression analysis. Since the distributions of plasma homocysteine and plasma folate concentrations are positively skewed, these variables were logarithmically transformed prior to linear regression analysis.\nDeviation from Hardy\u2013Weinberg equilibrium was calculated to assess the accuracy of the genotyping assay.\nThe power of the case-control study was calculated using the Genetic Power Calculator (Purcell et al. 2003). All tests were performed using SPSS software package version 12.0.1. Statistical significance was accepted at a two-tailed P\u00a0<\u00a00.05 or 95%CI of an odds ratio that did not include 1.0.\nTransmission disequilibrium test\nThe relation between the NOS3 894G>T genotype of the spina bifida patients and spina bifida risk was assessed by evaluating the transmission of alleles from heterozygous parents to their affected offspring using the TDT, a family-based association test (Spielman et al. 1993). The power of the TDT was calculated using the Genetic Power Calculator (Purcell et al. 2003).\nResults\nCase-control study\nThe NOS3 894G>T genotype could be determined in 115 mothers of a spina bifida affected child and in 259 female controls. Genotype distribution did not differ from that expected under Hardy\u2013Weinberg equilibrium (P\u00a0=\u00a00.83), and the power to detect a twofold increase in maternal spina bifida risk was 59%. Table\u00a01 demonstrates that neither the NOS3 894GT genotype nor the NOS3 894TT genotype increased maternal spina bifida risk relative to the NOS3 894GG genotype. Since the effect of the NOS3 894GT genotype and the NOS3 894TT genotype on spina bifida risk was the same relative to the NOS3 894GG genotype, the NOS3 894GT and TT genotypes were combined to increase power, which resulted in a nonsignificant 36% increased risk of having a spina bifida affected child relative to the NOS3 894GG genotype (OR 1.36, 95%CI 0.87\u20132.11). The NOS3 894G>T SNP was not associated with plasma homocysteine or plasma folate concentrations in mothers and female controls (data not shown).\nTable\u00a01The NOS3 894G>T genotype distribution and unadjusted ORs in mothers of a spina bifida affected child and female controlsNOS3 894G>TMothers (%)Female controls (%)OR (95%CI)GG49 (42.6)130 (50.2)1aGT53 (46.1)106 (40.9)1.33 (0.83\u20132.11)TT13 (11.3)23 (8.9)1.50 (0.71\u20133.19)aReference group\nIn 95 spina bifida patients and 207 pediatric controls, NOS3 894G>T genotyping was performed successfully. The NOS3 894G>T genotype distribution did not differ from that expected under Hardy\u2013Weinberg equilibrium (P\u00a0=\u00a00.76), and the power of the study was 51%. The NOS3 894GT genotype increased spina bifida risk in children whereas the NOS3 894TT genotype did not (Table\u00a02). Combining the NOS3 894GT and 894TT genotypes resulted in a 66% increase in spina bifida risk relative to the NOS3 894GG genotype (OR 1.66, 95%CI 1.02\u20132.72). In the pediatric controls, the NOS3 894GT and 894TT genotypes were associated with increased plasma homocysteine concentrations relative to the NOS3 894GG genotype (14.7% increase, 95%CI 4.2\u201326.2% and 23.5% increase, 95%CI 3.9\u201346.8%, respectively). There was no association between the NOS3 894G>T SNP and plasma folate concentration in children (data not shown).\nTable\u00a02The NOS3 894G>T genotype distribution and unadjusted ORs in spina bifida patients and pediatric controlsNOS3 894G>TSB patients (%)Pediatric controls (%)OR (95%CI)GG39 (41.1)111 (53.6)1aGT46 (48.4)80 (38.6)1.64 (0.98\u20132.74)TT10 (10.5)16 (7.7)1.78 (0.75\u20134.25)aReference group\nTransmission disequilibrium test\nIn this study there were 75 complete mother\u2013father\u2013child triads, and the power to detect a twofold increase in spina bifida risk was 32%. In 36 out of 70 informative transmissions (51.4%), the NOS3 894T allele was transmitted to the child, which resulted in a \u03a72 of 0.06 (P\u00a0=\u00a00.81).\nGene-gene interaction\nData on both the NOS3 894G>T genotype and the MTHFR 677C>T genotype were present for 110 mothers of a spina bifida affected child and 259 control women. Since the MTHFR 677CT genotype did not increase spina bifida risk relative to the MTHFR 677CC genotype, we decided to combine the MTHFR 677CC and CT genotypes to increase power. In combination with the MTHFR 677TT genotype, the NOS3 894GT\/TT genotype increased the risk of having spina bifida affected offspring 4.5 times relative to the NOS3 894GG genotype combined with the MTHFR 677CC\/CT genotype (OR 4.52, 95%CI 1.55\u201313.22) (Table\u00a03).\nTable\u00a03Maternal spina bifida risk for the combination of the NOS3 894G>T SNP and the MTHFR 677C>T SNPNOS3 894G>TMTHFR 677C>TMothers (%)Female controls (%)OR (95%CI)GGCC\/CT42 (38.2)114 (44.0)1aGGTT6 (5.5)16 (6.2)1.02 (0.37\u20132.77)GT\/TTCC\/CT52 (47.3)123 (47.5)1.15 (0.71\u20131.85)GT\/TTTT10 (9.1)6 (2.3)4.52 (1.55\u201313.22)aReference group\nIn children, there was no interaction between the NOS3 894GT\/TT genotype and the MTHFR 677TT genotype (OR 1.23, 95%CI 0.31\u20134.91) (data not shown).\nGene-metabolite interactions\nSince the MTHFR 677C>T SNP is a genetic determinant of plasma folate and plasma homocysteine concentrations, possible interactions between the NOS3 894G>T SNP and these metabolites were examined in mothers. In combination with decreased plasma folate concentrations (<8.52\u00a0nmol\/L), the NOS3 894GT\/TT genotype did not increase maternal spina bifida risk relative to the NOS3 894GG genotype in combination with normal plasma folate concentrations (\u22658.52\u00a0nmol\/L) (OR 1.14, 95%CI 0.49\u20132.67) (data not shown). The risk of having spina bifida affected offspring increased to 3.4-fold when the NOS3 894GT\/TT genotype was present in combination with elevated plasma homocysteine concentrations (\u226512.56\u00a0\u03bcmol\/L) (OR 3.38, 95%CI 1.46\u20137.84) (Table\u00a04).\nTable\u00a04Maternal spina bifida risk for the combination of the NOS3 894G>T SNP and elevated plasma homocysteine concentration, adjusted for ageNOS3 894G>TPlasma Hcy conc (\u03bcmol\/L)aMothers (%)Female controls (%)OR (95%CI)GG<12.5642 (36.8)100 (38.6)1bGG\u226512.567 (6.1)30 (11.6)0.66 (0.26\u20131.65)GT\/TT<12.5647 (41.2)108 (41.7)1.17 (0.69\u20131.96)GT\/TT\u226512.5618 (15.8)21 (8.1)3.38 (1.46\u20137.84)aThe 80th percentile of the control plasma homocysteine distribution was used to define elevated plasma homocysteine concentrationsbReference group\nDiscussion\nIn this study, the NOS3 894TT genotype did not significantly increase spina bifida risk, and the TDT demonstrated no preferential transmission of the NOS3 894T allele. However, in combination with the MTHFR 677TT genotype or elevated plasma homocysteine concentrations, the NOS3 894GT\/TT genotype increased maternal spina bifida risk 4.5- and 3.4-fold, respectively.\nUsing a likelihood ratio test (LRT) for both proband and mother trios, Brown et al. (2004) demonstrated the NOS3 894GT genotype, but not the NOS3 894TT genotype, to be an embryonic risk factor for spina bifida (OR 1.63, 95%CI 1.09\u20132.42). Data from our case-control study might be in line with this observation of Brown et al. (2004); however, based on the effect of the heterozygous NOS3 894GT genotype on spina bifida risk, a larger effect of the NOS3 894TT genotype on spina bifida risk would be expected. The observation that the NOS3 894GT genotype increases spina bifida risk and the NOS3 894TT genotype does not could be explained as a chance finding, but could also be the result of lack of power, as also suggested by Brown et al. (2004).\nDespite the small numbers on which the analyses are based, the possible interactions between the NOS3 894GT\/TT genotype and the MTHFR 677TT genotype or elevated plasma homocysteine concentrations are interesting and may suggest a role for NOS3 uncoupling or S-nitrosation. The NOS3 894G>T SNP (Glu298Asp) has been suggested to impair NOS3 protein function and\/or activity resulting in decreased NO production (Sofowora et al. 2001; Tesauro et al. 2000), although results are contradictory (Fairchild et al. 2001). Elevated homocysteine concentrations have been demonstrated to switch the NOS3 enzyme to an uncoupled state directed to the synthesis of reactive oxygen species (ROS) rather than NO synthesis (Topal et al. 2004). The combination of the NOS3 894GT\/TT genotype and elevated plasma homocysteine concentrations may lead to such a decrease in NO levels that neurulation is hampered. NO can also react with homocysteine to form S-nitrosothiols, thereby modulating homocysteine availability (Stamler et al. 1993). As previously suggested by our group (Heil et al. 2004), decreased NO levels may result in decreased S-nitrosothiol formation, resulting in less capturing of homocysteine into S-nitrosoHcy. The subsequent increase in plasma homocysteine concentrations might influence proper neural tube closure, especially when plasma homocysteine concentrations are already high.\nThe results of our study suggest that the NOS3 894G>T SNP is a spina bifida risk factor in the mother and not in the child. Our results furthermore indicate a role for the homocysteine metabolism in the detrimental effects asserted by the NOS3 894G>T SNP. More studies on the association between the NOS3 894G>T SNP and spina bifida in larger populations are warranted.","keyphrases":["spina bifida","nos3 894g>t","transmission disequilibrium test"],"prmu":["P","P","P"]}
{"id":"Biochim_Biophys_Acta-2-1-2258316","title":"The microtubule-associated protein tau is phosphorylated by Syk\n","text":"Aberrant phosphorylation of tau protein on serine and threonine residues has been shown to be critical in neurodegenerative disorders called tauopathies. An increasing amount of data suggest that tyrosine phosphorylation of tau might play an equally important role in pathology, with at least three putative tyrosine kinases of tau identified to date. It was recently shown that the tyrosine kinase Syk could efficiently phosphorylate \u03b1-synuclein, the aggregated protein found in Parkinson's disease and other synucleinopathies. We report herein that Syk is also a tau kinase, phosphorylating tau in vitro and in CHO cells when both proteins are expressed exogenously. In CHO cells, we have also demonstrated by co-immunoprecipitation that Syk binds to tau. Finally, by site-directed mutagenesis substituting the tyrosine residues of tau with phenylalanine, we established that tyrosine 18 was the primary residue in tau phosphorylated by Syk. The identification of Syk as a common tyrosine kinase of both tau and \u03b1-synuclein may be of potential significance in neurodegenerative disorders and also in neuronal physiology. These results bring another clue to the intriguing overlaps between tauopathies and synucleinopathies and provide new insights into the role of Syk in neuronal physiology.\nMany neurodegenerative disorders are characterised by intracellular inclusions of highly insoluble proteins, and a classification based upon the main protein component of these aggregates is widely used. The term \u201csynucleinopathies\u201d, for instance, refers to a group of disorders including Parkinson's disease (PD) in which the synaptic protein \u03b1-synuclein (\u03b1-syn) forms neuronal or glial aggregates [1]. \u201cTauopathies\u201d are another pathological entity in which the microtubule-associated protein tau self-assembles into filamentous inclusions called neurofibrillary tangles (NFT) [2]. The tauopathies include Alzheimer's disease (AD), progressive supranuclear palsy and corticobasal degeneration.\nHuman brain tau consists of a family of six isoforms, generated by the alternative splicing of a single mRNA transcript. Tau proteins contain either three (tau 3R) or four (tau 4R) tubulin-binding domains depending on the splicing of exon 10, and either one (tau 1N) two (tau 2N) or no (tau 0N) N-terminal inserts depending on the inclusion of exon 2, exons 2 and 3, or exclusion of both, respectively. Their apparent molecular weights range from 45\u00a0kDa for tau 0N3R isoform to 65\u00a0kDa for tau 2N4R. When aggregated in NFT, tau is abnormally hyperphosphorylated by a series of serine\/threonine kinases, among which glycogen synthase kinase-3\u03b2, cyclin-dependent kinase 5 and casein kinase 1 appear to be the most relevant [3,4]. It has been hypothesized that this hyperphosphorylation contributes to neurodegeneration through the destabilisation of microtubules [5].\nRecent data suggest that phosphorylation of tau also occurs on tyrosine residues and this could be of potential significance in pathological conditions [6,7]. Human tau protein possesses five tyrosine residues on positions 18, 29, 197, 310 and 394 (numbered according to the longest 2N4R tau isoform). Tau extracted from NFT of AD patients was found to be phosphorylated on tyrosine 18 (Y18) by immunocytochemistry using a phosphospecific antibody [6], and on tyrosine 394 (Y394) by mass spectrometry [8]. Tyrosine 197 (Y197) has been shown to be phosphorylated in tau aggregates from transgenic mice overexpressing mutant tau [9]. Recently, the dual specificity kinase tau-tubulin kinase 1 has been shown to phosphorylate tau in vitro on Y197 [10]. While Abl was recently described as a candidate tyrosine kinase for Y394 [8], Fyn is considered to be the kinase for Y18 [6].\nThe tyrosine kinase Syk is known for its critical role in signalling through immune receptors in leucocytes [11]. Syk tyrosine kinase expression is however not confined to hematopoietic cells, and has been reported in a variety of tissues [12]. Syk expression has been confirmed in the central nervous system (CNS) by immunoblot analysis of mouse brain homogenates, and its localization in mouse neuronal cytoplasm affirmed by immunohistochemistry [12]. While its function in the CNS remains unclear, Syk was recently found to be a kinase for \u03b1-syn [13]. The direct interaction of Syk and \u03b1-syn was proven by confocal microscopy and a dual-hybrid system approach [13]. The intriguing overlap in the pathophysiology of synucleinopathies and tauopathies prompted us to assess whether tau protein was also a substrate for Syk tyrosine kinase [14]. We herein report that Syk can phosphorylate Tau on Y18, and that the kinase and its substrate directly interact.\nWe first investigated whether Syk can directly phosphorylate tau in vitro. Recombinant human tau 2N4R was incubated with or without recombinant Syk or Abl [8]. Tau was phosphorylated in vitro by Syk and by its previously recognized tyrosine kinase Abl as judged on Western blots probed with P-Tyr-100 anti-phosphotyrosine antibody (Cell Signaling), whereas no phosphorylation was observed in experiments where tau was incubated without Syk (Fig. 1). In the presence of Syk, an additional band of approximately 70\u00a0kDa, migrating just above tau, was observed on the phosphotyrosine immunoblot. This band is likely to contain Syk since the kinase is known to autophosphorylate. Taken together, these results demonstrate that Syk can directly catalyze tau phosphorylation.\nTo determine whether Syk can phosphorylate tau in cells, co-transfection experiments were performed in CHO cells using Syk expression vectors together with untagged or V5-tagged tau. Cells were lysed and tau was immunoprecipitated with either anti-V5 antibody (Invitrogen) or with anti-tau antibody (Tau-5, BD Biosciences). Western analysis was then performed on immunoprecipitated tau using the anti-phosphotyrosine (P-Tyr-100, Cell Signaling), anti-tau (Tau-5) or anti-Syk (N-19, Santa Cruz) antibodies as previously described [8]. In preliminary experiments, a V5-tagged tau 2N4R construct and wild-type Syk were transiently coexpressed in CHO cells. We have previously shown that V5-tagged tau 2N4R migrates at approximately 70\u00a0kDa on SDS-PAGE [8]. Because of the close molecular weights of Syk (72\u00a0kDa) and the V5-tau construct, the two proteins co-migrated using conventional denaturing gel electrophoresis (data not shown). To better resolve the two proteins, we used two complementary approaches. In a first set of experiments, we expressed V5-tagged tau 2N4R along with a Syk-GFP construct that migrates at 100\u00a0kDa. The Syk-GFP construct was proven to remain functionally active in previous studies [15]. In a second set of experiments, we coexpressed wild-type untagged Syk along with the shortest tau isoform (0N3R), which migrates at 45\u00a0kDa. Tau protein was phosphorylated on tyrosine when co-transfected with Syk-GFP construct or with its known tyrosine kinase Abl (Fig. 2A). Additional positive controls included tau-transfected cells treated with the tyrosine-phosphatase inhibitor pervanadate (100\u00a0\u03bcM for 30\u00a0min). When Syk-GFP was co-transfected with tau, tau immunoprecipitates contained a \u223c\u00a0100-kDa tyrosine-phosphorylated protein, likely to contain Syk-GFP since this enzyme is tyrosine phosphorylated (Fig. 2A). This was confirmed by blotting with Syk antibody after membrane stripping (Fig. 2A). This co-immunoprecipitation of Syk with tau is indicative of Syk and its substrate being in the same protein complex. In order to exclude any effects of the V5 and GFP tags fused with tau and Syk, we wished to repeat this experiment using vectors expressing native proteins. The untagged and shortest isoform of tau (0N3R) was phosphorylated on tyrosine when co-transfected with native 72\u00a0kDa Syk (Fig. 2B), as compared with the transfection of tau alone. As in Fig. 2A, an additional 72-kDa band corresponding to phospho-Syk was visible on the phosphotyrosine blot when Syk was co-transfected with tau. The co-immunoprecipitation is therefore confirmed with native proteins. Taken together, these results demonstrate that Syk tyrosine kinase can both phosphorylate and co-immunoprecipitate with tau in cells, consistent with the direct phosphorylation of the substrate demonstrated in vitro (Fig. 1). The binding of Syk to both the shortest and longest isoform of tau suggests that it is independent of tau splicing.\nTau has been shown to be tyrosine phosphorylated on residue 18, 197 and 394 [6,8\u201310]. To map the tyrosine residue(s) phosphorylated by Syk in tau, Syk was co-transfected into CHO cells along with wild-type or mutant tau constructs in which individual tyrosines were replaced by phenylalanine (Y18F, Y197F and Y394F). These cDNA constructs have been described in detail previously [8]. The only such tyrosine mutation that resulted in a strong decrease in tau tyrosine phosphorylation was Y18F (Fig. 3). Tyrosine phosphorylation of the Y197F and Y394F constructs were not significantly different from the wild-type control (Fig. 3). This clearly demonstrates that Y18 is the major tyrosine phosphorylation site for Syk. In addition, the binding of Syk was independent of the tyrosine phosphorylation state of tau (Fig. 3), suggesting that the SH2 domains of Syk are not involved in its binding to tau.\nAs Fyn and Syk phosphorylate the same tyrosine residue on tau, we decided to perform a time course of phosphorylation of tau by the two kinases under conditions designed to favour the measurement of stoichiometry. After 30\u00a0min, Syk had incorporated an average of 0.23\u00a0mol of phosphate\/mol of tau and Fyn had incorporated an average of 0.25\u00a0mol of phosphate\/mol of tau. These results show that Syk and Fyn phosphorylate tau with the same efficacy (Fig. 4).\nOur demonstration of binding of tau to Syk is consistent with tau being a specific substrate for Syk and for their involvement in a cell-signaling pathway in neurons. Interestingly, Syk plays an important role in signalling events of neurite induction and outgrowth in neuronal cell lines [16]. It is thus tempting to speculate that the phosphorylation of tau by Syk could be involved in neurite outgrowth. Our study also reinforces the role of Syk in neurodegenerative disorders. Remarkably, Y18 has been shown to be phosphorylated in NFT, suggesting that, like Fyn, Syk could be critical in the pathophysiology of AD [6,7,17]. Although early studies suggested a clear distinction between \u2018tauopathies\u2019 and \u2018synucleinopathies\u2019, more recent studies demonstrate that there is often an overlap in the pathological findings in these disorders [18\u201320]. Syk binds to and phosphorylates both tau and synuclein, suggesting that this kinase could be a link between synucleinopathies and tauopathies. In conclusion, our findings significantly contribute to the understanding of the signaling pathways involving tyrosine phosphorylation of tau in both physiological and pathological conditions. Further elucidation of the functional relationship between tau and the Syk in neurons could provide critical insights into the role of tau in cell signaling as well as the role of tau in neurodegenerative processes.","keyphrases":["tau","syk","tauopathy","tyrosine phosphorylation"],"prmu":["P","P","P","P"]}
{"id":"Eur_J_Health_Econ-_-_-1388087","title":"Spanish health benefits for services of curative care\n","text":"This contribution presents entitlements and benefits, decision criteria, and involved actors for services of curative care in Spain. It describes basic benefits included in the category of curative care defined by the central government and any additional benefits that some autonomous communities (ACs) have included to enlarge their own basket. It is concluded that there is no specific and explicit benefit catalogue. As no user charges exist for this category, waiting times serve as the main cost containment tool. There is a need for further legislation, as inequalities may increase across the territory as a matter of fact. Inequalities in access to health care resources between ACs are not due to differences in health baskets but mainly to the availability of technologies.\nThe principal actors responsible for defining of benefit catalogues from the services of curative care in Spain are the Central Government and the autonomous communities (ACs). Law 16\/2003 [1] states that the Spanish health benefit basket should serve as a minimum for all Spanish residents to ensure the equality of access and benefits among them irrespective of their location. Nevertheless, as ACs may vary the allocation of their funding, some may decide to offer additional or new benefits even including those that are explicitly excluded on national level by Royal Decree 63\/1995 [2] and Law 16\/2003.\nThe central government\u2019s main responsibilities are as follows: (a) general coordination and basic health legislation, (b) financing of the system and the regulation of the financial aspects of social security, (c) definition of a benefit basket guaranteed by the National Health System (NHS), (d) international health, (e) pharmaceutical policy, (f) health professionals undergraduate and postgraduate training, and (g) human resources policies regarding civil servants. A number of ministries (Economy and Finance, Public Administration, Labor and Social Affairs, Education and Culture, Environment, Justice, and Defense Ministries) share these responsibilities and therefore determine the health benefit basket, although the Ministry of Health and Consumer Affairs plays the main role in finally determining any health policies.\nRegional governments (ACs) have the responsibility for planning, financing, and providing health care services (including an enlargement of the basic health benefit basket), social and community care, and public health. Another important agent in coordinating and evaluating new benefits is the Institute of Health Carlos III. Local governments are responsible for home-based nursery and social services. Coordination takes place in the Inter-Territorial Council of the NHS, and advisory committee comprising representatives from the central and regional governments that is responsible for ensuring the equality of benefits among and access across regions.\nThe present contribution focuses on services of curative care, excluding services such as rehabilitative care and goods dispensed to outpatients. The other categories have been studied in a detailed report as part of the Health Basket Project and are presented in Tables\u00a01 and 2, including the benefit defining criteria. We first present an overview of the basic benefits that all ACs should provide and describe the additional benefits that some ACs include in their own basket. We then discuss the benefit catalogues and their actual implementation.Table\u00a01 Benefit-defining laws\/decrees and catalogues and the implicit regulation by sector. IGeneral Health ActRoyal Decree 63\/95Law 16\/03Ministerial Decree 30 April 1997Ministerial Decree 3 June 1998Ministerial Decree 3 March 1999Functional categories\u00a0\u00a0HC.1.1+++\u2013\u2013\u2013\u00a0\u00a0HC.1.2++\u2013\u2013\u2013\u00a0\u00a0HC.1.3+++\u2013\u2013\u2013\u00a0\u00a0HC.1.3.1+++\u2013\u2013\u2013\u00a0\u00a0HC.1.3.2++\u2013\u2013\u2013\u00a0\u00a0HC.1.3.3+++\u2013\u2013\u2013\u00a0\u00a0HC.1.3.4+++\u2013\u2013\u00a0\u00a0HC.1.4+++\u2013++\u00a0\u00a0HC.2.1+++\u2013\u2013\u2013\u00a0\u00a0HC.2.2+++\u2013\u2013\u2013\u00a0\u00a0HC.2.3+++\u2013\u2013\u2013\u00a0\u00a0HC.2.4\u2013\u2013\u2013\u2013\u2013\u2013\u00a0\u00a0HC.3\u2013++\u2013\u2013\u2013\u00a0\u00a0HC.4.1\u2013++\u2013\u2013\u2013\u00a0\u00a0HC.4.2\u2013++\u2013\u2013\u2013\u00a0\u00a0HC.4.3\u2013++\u2013\u2013\u2013Legal statusLawPresidential DecreeLawMinistry DecreeMinistry DecreeMinistry DecreeDecision makerParliamentGovernmentParliamentGovernmentGovernmentGovernmentDegree of explicitnessa132333Positive\/negative definition of benefitsPPPPPPOriginal purposeGeneral definition of entitlementsBasketEquality among citizens and ACsBasketBasketBasketCriteria used for defining benefits\u00a0\u00a0Need++++++\u00a0\u00a0Costs\u2013+++++\u00a0\u00a0Effectiveness\u2013+++++\u00a0\u00a0Cost-effectiveness\u2013\u2013\u2013\u2013\u2013\u2013\u00a0\u00a0Budget\u2013\u2013\u2013\u2013\u2013\u2013UpdatingNoNoNoRegularlyRegularlyRegularlya 1, all necessary; 2, areas of care; 3, itemsTable\u00a02 Benefit-defining laws\/decrees and catalogues and the implicit regulation by sector. IIGeneral Health ActRoyal D 1663\/1995Law 16\/2003Decree 3157\/1966Law 25\/1990Royal Decree 83\/1993Royal Decree 1663\/1998Royal Decree 1348\/2003Ministerial Decree 18 January 1996Functional categories\u00a0\u00a0HC.5.1+\u2013\u2013+++++\u2013\u00a0\u00a0HC.5.1.1\u2013\u2013\u2013\u2013+\u2013\u2013\u2013\u2013\u00a0\u00a0HC.5.1.2\u2013\u2013\u2013\u2013+\u2013\u2013\u2013\u2013\u00a0\u00a0HC.5.2\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u00a0\u00a0HC.5.2.1\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u00a0\u00a0HC.5.2.2\u2013+\u2013\u2013\u2013\u2013\u2013\u2013+\u00a0\u00a0HC.5.2.3\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013+\u00a0\u00a0HC.5.2.4\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013+\u00a0\u00a0HC.6.1\u2013++\u2013\u2013\u2013\u2013\u2013\u2013\u00a0\u00a0HC.6.2\u2013+\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u00a0\u00a0HC.6.3+++\u2013\u2013\u2013\u2013\u2013\u2013\u00a0\u00a0HC.6.4+++\u2013\u2013\u2013\u2013\u2013\u2013\u00a0\u00a0HC.6.5\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013Legal statusLawPresidential DecreeLawLawLawPresidential DecreePresidential DecreePresidential DecreeMinisterial DecreeDecision makerParliamentGovernmentParliamentParliamentParliamentGovernmentGovernmentGovernmentGovernmentDegree of explicitnessa132123333Positive\/negative definition of benefitsPPPPPNNNPOriginal purposeGeneral definition of entitlementBasketEquality among citizens and ACsGeneral definition of entitlementTarget ruleExclusionExclusionExclusionBasketCriteria used for defining benefits\u00a0\u00a0Need+++++\u2013\u2013\u2013+\u00a0\u00a0Costs\u2013++\u2013+++++\u00a0\u00a0Effectiveness\u2013++\u2013+++++\u00a0\u00a0Cost-effectiveness\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u00a0\u00a0Budget\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013UpdatingNoNoNoNoNoRegularyRegularyRegularyRegularya 1, all necessary; 2, areas of care; 3, items\nDefinition and structure of the main benefit basket for curative care\nWith regards to benefit coverage the Spanish Constitutional Act stipulates Spanish citizenship as the only precondition for access to the range of health care benefits. Parliamentary ratification on 18 December 1997 [3] extended coverage to the entire resident population. The major differences between ACs are related to foreigners.\nThe fundamental act on health benefit regulation is Royal Decree 63\/1995 (20 January) on the organization of health services provided by the NHS (Fig.\u00a01). It provides inclusion and exclusion criteria and defines the current level of provision as the guaranteed basic health care basket in a comprehensive form. It contains a checklist of services that constitute the entitlement, establishes certain exclusion criteria, and limits the provision of certain services due to the limited resources available to the NHS. It also stipulates conditions for the introduction of new technologies and procedures as well as new entitlements. Finally, it prescribes coordination between health and social care.Fig.\u00a01 Royal Decree 63\/1995\nServices, activities, and procedures are excluded under any of the following circumstances: (a) There is insufficient scientific evidence of their clinical safety and efficacy or if they are clearly outdated. (b) They have not sufficiently proven their effective contribution to prevention, treatment, or cure illnesses, preservation or improvement of life expectancy, self-help, or elimination or reduction of pain and suffering. (c) They are constitute merely activities such as those of leisure, rest, comfort, sport, cosmetic improvement, or spa.\nThe Spanish NHS has not yet in fact defined a basic basket of services, although Royal Decree 63\/1995 established a general framework of benefits, as did the Law 16\/2003 of 28 May (Fig.\u00a02). This law on \u201cCohesion and Quality of the NHS\u201d is a reform that built on the previous act with the purpose of coordinating a strongly decentralized activity. Its aim is to establish the legal framework for the coordination and cooperation of the Public Health Administrations guaranteeing equality, quality, and participation in the system. It also introduced the establishment of a general procedure for revisions on the content of the health benefit basket. Since 2002 all ACs have health service responsibilities. They manage their regional health care system and also have related legislative responsibilities. These regional health care baskets must include at least the benefits defined by the NHS. In this sense Law 16\/2003 is intended to sustain the coordination and the basic guarantees between all the territories.Fig.\u00a02 Law 16\/2003\nChapter I of Law 16\/2003 refers to health care benefits of the NHS and defines a catalogue that incorporates the benefits defined by Royal Decree 63\/1995, public health benefits. The catalogue of benefits is defined as a set of preventive, diagnostic, therapeutic, rehabilitative services, and direct health promotion to citizens, which includes public health, primary and specialized care, social and community care, dietetic urgencies, pharmacy, orthoprosthesis, products, and sanitary transport benefits. It guarantees the right of all citizens to obtain a second medical opinion, the right to receive medical assistance in one\u2019s own AC of residence within a maximum time along, and the right to receive the defined benefits of the NHS under the same conditions and guarantees as residents in other ACs.\nAs a further development of the 16\/2003 Law the Spanish Ministry of Health is currently drafting a Royal Decree to establish the NHS health benefit basket and the means for updating it. The purpose of the Royal Decree is to guarantee equality and appropriate care by the NHS. The ACs approve their own baskets by always including all the services of the NHS health benefit basket since these services are considered a basic need and common to all users of the NHS. They can enlarge their own baskets with additional technical services, technologies, and procedures. New benefits added to the basic basket are evaluated by the Ministry of Health through the Evaluation Agency of Health Technologies (\u201cAgencia de Evaluaci\u00f3n de Tecnolog\u00edas M\u00e9dicas\u201d) and the Carlos III Health Institute in collaboration with other evaluation agencies proposed by the ACs and the Inter-Territorial Council of the NHS.\nContents of the benefit baskets for curative care\nInpatient curative care\nThe above regulation affecting inpatient curative care defines the benefits covered by the NHS which ought to be provided by all ACs (free of any user payment) such as: specialized health care in hospitals, which includes medical attention, surgery, obstetric, and pediatric services for acute conditions, worsening of chronic conditions or delivery of recommended diagnostic treatments or procedures; organs, tissues and cells of human origin transplants according to the special legislation as long as there is the evidence for therapeutic effectiveness; palliative care to terminally ill patients; and mental health care and psychiatric assistance, which includes clinical diagnosis and follow-up and the prescribed group of family psychotherapy if it implies hospitalization when required. Hospitalizations of short or medium periods of time take place in the psychiatric departments of general hospitals. Inpatient mental long-term care services take place at psychiatric hospitals, which are part of the long-term care network (local authorities).\nThe main benefit exclusions are mental inpatient care (included only upon referral) and transplants of hair, nails, and placenta. Additional benefits on the part of ACs include sex\/gender change surgery intervention, such as that now offered in Andalusia. Andalusia also offers epidural anesthetics during birth. The other main benefit extension is related to euthanasia. Catalonia, Galicia, and Extremadura have regulated and included the vital testament as a benefit.\nDay cases of curative care\nThe entitlements concerning day cases of curative care define the service groups included in the Spanish benefits basket as: specialized ambulatory health care consultations that could include minor surgical procedures; specialized ambulatory health care delivered in \u201cday hospitals\u201d for patients in need of continuous specialized care, physician\u2019s or nursing services, including major surgery as long as no hospitalization is required; and hemotherapy. The following services are explicitly excluded: plastic surgery when it is not related to accident, disease, or congenital malformation; sex change surgery, except for those cases in which it is necessary to repair pathological intersexual conditions.\nServices of outpatient care\nOutpatient care is generally provided in primary care centers or specialist care centers (outpatient clinics). There is a wide disparity in the number and use of the latter among ACs. These categories of care are regulated both at national and AC levels. The outpatient benefit basket includes the following service groups (all free of user charges): health assistance for health consultation services and centers including basic medical and diagnostic services (curative) primary care; health services provided at home; indication or prescription and the compliance, in relevant cases, with examinations and basic diagnostic measures as indicated by the primary care physician; the administration of parenteral treatments, cures and minor surgery; initial mental assistance is provided in mental health centers, where patients are being evaluated, assisted, or referred to other assistance services. Benefits provided include: pharmacological and psychotherapy treatments, crisis interventions, etc. Mental emergencies can also be treated at the hospital emergency service; emergency primary health care which will be provided to persons of any age and delivered continuously 24\u00a0h a day, through medical and nursing services, on an outpatient basis or at the domicile of the patient if the situation requires to do so; primary dental health care, including dental health and hygiene information and education, preventive and assistance measures; application of topical fluoride, obstructions, sealing of fissures or other services for the infant population according to the annual financial budget and special programs for dental health, treatment of acute orthodontic problems including dental extractions, and preventive exploration of the oral cavity in pregnant women; renal lithotripsy, interventionist radiology, and radiotherapy. Certain services such as complex diet therapy are not free of charge but are also not explicitly excluded from the Spanish health care benefit basket. Other services such as spa treatments, rest cures, psychoanalysis, and hypnosis are explicitly excluded.\nIn this category of care many ACs offer additional services as part of their benefit catalogues. At primary care level the Basque Country, Extremadura, Castilla La Mancha, and Catalonia have passed several decrees; at specialized outpatient care level Navarre offers obstetrics, gynecological and family planning such as menopause care; Valencia provides reactive strips for determining glucose in blood and urine to diabetics; and at special treatment level the Balearic Islands offer neurological reflexology in cooperation with the Kovacs Foundation. It must also be noted that Catalonia has started a pilot experience in offering acupuncture for arthritis treatment.\nNine ACs also provide free dental care for some groups: Navarre, Basque Country, Andalusia, Extremadura, Castilla La Mancha, Cantabria, Castilla Le\u00f3n, Murcia, and Galicia. Catalonia offers periodic rinsing of a fluorine solutions program for primary school students and those taking the first two courses of secondary school. As all these catalogues are quite similar, we present below the dental care benefit catalogue of Castilla La Mancha (Fig.\u00a03). The Infant-Young Dental Care Plan defines the group of measures and activities, both preventive and welfare, as well as any procedures to provide dental care. Benefits are classified into: oral check-up, basic dental treatments, emergency dental treatments, special dental treatments, and orthodontics.Fig.\u00a03 Dental care catalogues: the example of Castilla La Mancha\nServices of curative home care\nAs part of the entitlements of services of curative home care, oxygen therapy service at home is provided at authorized and specialized centers capable of carrying out gasometry and spirometry. The Ministerial Decree of 3 March 1999 [4] elaborates these services, considering the following benefits: (a) oxygen therapy at home, (b) mechanical ventilation at home, (c) ventilation treatment of the sleep apnea symptoms, and (d) aerosol therapy. Enteral nutrition is also provided at home. This service is made available to patients who suffer from the following symptoms: swallowing, transit, digestion or absorption of foods in its natural form problems or when special requirements of energy and\/or nutrients exist that cannot be covered with foods of daily consumption.\nRoyal Decree 63\/1995 has been developed with regard to benefits with diet products in two ways: regulating complex diet therapy and regulating enteral nutrition at home. Complex diet therapy is dealt with in the Ministerial Decree of 30 April 1997 [5] which specifies the relationship of congenital metabolic upheavals of carbon hydrates and amino acids included in this benefit and the type of diet in each case. It also prescribes these treatments to be performed by a special physician in hospital units, specifically authorized by the appropriate Health Administration. Enteral nutrition at home is regulated by the Ministerial Decree of 2 June 1998 [6], which defines the requirements and the patient\u2019s clinical situation for this. There are also regulations at the national and interterritorial council levels as well as in six ACs that have developed their own dietetic regulations.\nThe Ministerial Decree of 3 March 1999 was passed upon the proposal of a panel of experts (Fig.\u00a04). This elaborates Royal Decree 63\/1995 regarding the techniques of oxygen therapy at home financed by the NHS and clarifies and standardizes the criteria, clinical situations, and other matters for the direction, prescription, and provision of these benefits. It defines the oxygen therapy techniques that can be carried out at home in terms of their financing by the NHS and establishes the basic requirements that justify their prescription. The Ministerial Decree of 3 March 1999 thus provides a common basis upon which the health service providers must develop their own procedure for providing and supervising these benefits. The Decree actually does not provide a benefit catalogue as such because the individual physician determines the specific treatment in each case. In addition there are also specific regional oxygen therapy regulations in four ACs (Andalusia, Canary Islands, Navarre and Valencia).Fig.\u00a04 Oxygen therapy at home\nServices of MUFACE catalogue\nAll civil servants and beneficiaries are entitled to receive benefits as determined by the MUFACE catalogue. This benefit basket is virtually equivalent to that of the NHS but include some additional benefits. The principal difference is related to copayments for pharmaceuticals. In addition to particular features for medical assistance abroad, orthoprosthetic, dental care, ocular, pharmaceutical, and sociosanitary benefits, services such as vaccinations are included free of charge. Specialized health care includes all the medical and surgical specialties, both in outpatient and inpatient sectors. The specialties are structured in four levels according to their complexity and geographic scope, sanitary transport, oxygen therapy, and aerosol therapy at home.\nDiscussion\nThe way in which the Spanish benefit catalogue is defined is characteristic of tax-financed NHS health care systems. A system such as that in Spain which guarantees universal coverage is not completely developed until it is specifies a minimum basket of health care services. However, as no specific and explicit benefit catalogue is being developed, there remains a need for further legislation. The Ministry of Health and Consumption is moving towards an explicit process of decision making in health benefit planning instead of defining a detailed benefit catalogue.\nAs is the case in many countries, the possibility of using regional plans as the basis for resource allocation and capacity planning has not yet been considered. An additional problem in Spain is the fact that the majority of health care plans offer unrealistically long lists of objectives which are often not sufficiently based on available epidemiological and cost-effectiveness evidence, and their efficacy is seldom evaluated. They cannot be considered as benefit catalogues since they are not sufficiently strong legal instruments, and therefore users are not able to claim the objectives contained in the health plans as benefit rights.\nThen purpose of the Spanish health benefits basket is twofold, as it was initially established to serve more as a budgetary than as a cost containment measure when created by the Social Security act [7]. However, on the other hand, as Royal Decree 63\/1995 and the 2003 Cohesion Law recognize, they are merely an ordering of benefits as they try only to describe what is being offered in practice.\nTwo major aspects of the Spanish health care system are: (a) A more explicit definition of the benefit basket is needed as the present vague definition leaves the final decisions to practitioners, making expenditure very volatile, and having as the sole restriction that of waiting times. (b) Greater transparency is needed in the process of approval\/rejection of new benefits, towards agents in the health sector, and towards citizenship along with the need to include economic considerations in the decision process. In Spain it still remains the case: \u201call for everyone and almost everything for free.\u201d This obviously leads to long waiting times and thus a dual system (public-private) for those able to skip the long queues and high public pressure over resources devoted to the NHS. Furthermore, since there are no user payments, waiting lists serve as the mechanisms restricting demand for health services. For the first time, the Catalan Health Service is now studying the possibility of introducing a copayment system in primary care (fixed payment per visit).\nThere are still some remaining problems in the Spanish benefit basket. In terms of equality, the fact that MUFACE-type schemes have not been fully integrated to the NHS provides additional benefits for these groups. There is a lack of real control over the managers of the health care services. Autonomy of decisions by practitioners may mean an obstacle to the early evaluation of new technologies since under uso tutelado (monitored use) it is physicians who start using them. In the present financing system there is no relationship between this and the benefits provided, nor a control mechanism over what is provided in reality.\nMoreover, the information systems are clearly the main problem in the decentralization process. To overcome this problem the Ministry of Health has recently introduced an institution for monitoring the health system. Even the appearance of new benefit differences across ACs will not affect equality such as it may affect clinical practices and access to equipment. Therefore the main purposes of the Spanish health policy are to promote common clinical guidelines and evaluation processes with a single registry on new infrastructure and the creation and regulation of a common procedure for new benefit approvals.\nIn the future the central government is not expected to increase benefits but rather the quality of the existing ones, through new technologies and clinical procedures. Improvements are expected regarding the provision of services (e.g., waiting times), better user information, and increased range of choices. Probably the most important task is the development of a long-term care or dependency system and a real network of mental health.\nFuture reforms taken by the different ACs will likely address the benefits basket. Dental care and natural and alternative medicines are probably the first services that will be addressed. The definition of a basic long-term social and community care benefit basket similar to that in health care will also be a priority for the central government. Nevertheless, as we can see in Table\u00a03, new benefits have very little impact on the health expenditure of ACs.Table\u00a03 Spending in the provision of health care services not included in the benefit catalogue of the National Health System (Royal Decree 63\/1995): proportion of total health care services spending19992000200120022003Total0.150.130.130.120.11Andalusia0.400.320.320.300.29Aragon\u2013\u2013\u2013\u2013\u2013Asturias\u2013\u2013\u20130.00030.0007Balearic Islands\u2013\u2013\u2013\u20130.02Canary Island\u2013\u2013\u2013\u2013\u2013Cantabria\u2013\u2013\u2013\u2013\u2013Castilla y Le\u00f3n\u2013\u2013\u2013\u2013\u2013Castilla\u2013La Mancha\u2013\u2013\u2013\u2013\u2013Catalonia0.400.380.360.320.27Valencia\u2013\u2013\u2013\u2013\u2013Extremadura\u2013\u2013\u2013\u20130.04Galicia\u2013\u2013\u2013\u2013\u2013Madrid\u2013\u2013\u2013\u2013\u2013Murcia\u2013\u2013\u2013\u20130.05Navarre0.470.440.470.460.49Basque Country0.200.180.190.180.19La Rioja\u2013\u2013\u2013\u2013\u2013Ceuta\u2013\u2013\u2013\u2013\u2013Melilla\u2013\u2013\u2013\u2013\u2013\nFinally, in Spain, the growth of public health expenditure is due to both the increase in input prices and the received average benefits per capita. The growth of public health care expenditure is largely attributable to the benefits provided. The yearly change in health care benefits was responsible for 1.86% of the 6.73% annual average increase in health expenditure between 1991 and 2003.","keyphrases":["spain","health services","health benefit plans","health priorities","national health programs"],"prmu":["P","P","P","R","R"]}
{"id":"Diabetologia-4-1-2270364","title":"Epigenetic regulation of PPARGC1A in human type 2 diabetic islets and effect on insulin secretion\n","text":"Aims\/hypothesis Insulin secretion in pancreatic islets is dependent upon mitochondrial function and production of ATP. The transcriptional coactivator peroxisome proliferator activated receptor gamma coactivator-1 alpha (protein PGC-1\u03b1; gene PPARGC1A) is a master regulator of mitochondrial genes and its expression is decreased and related to impaired oxidative phosphorylation in muscle from patients with type 2 diabetes. Whether it plays a similar role in human pancreatic islets is not known. We therefore investigated if PPARGC1A expression is altered in islets from patients with type 2 diabetes and whether this expression is influenced by genetic (PPARGC1A Gly482Ser polymorphism) and epigenetic (DNA methylation) factors. We also tested if experimental downregulation of PPARGC1A expression in human islets influenced insulin secretion.\nIntroduction\nType 2 diabetes is characterised by chronic hyperglycaemia as a result of impaired pancreatic beta cell function and insulin resistance in peripheral tissues, i.e. skeletal muscle, adipose tissue and liver. Although each of these pathogenic defects could be accounted for by specific mechanisms, impaired ATP production as a consequence of reduced oxidative phosphorylation might provide an intriguing common pathogenic pathway for all these defects. The transcriptional coactivator peroxisome proliferator activated receptor gamma coactivator-1 alpha (protein PGC-1\u03b1; gene PPARGC1A) is an important factor regulating the expression of genes for oxidative phosphorylation and ATP production in target tissues through coactivation of nuclear receptors [1]. We have previously shown that the expression of PPARGC1A and a set of genes involved in oxidative phosphorylation is reduced in skeletal muscle from patients with type 2 diabetes [2]. Furthermore, a common polymorphism, Gly482Ser, in the PPARGC1A gene has been associated with increased risk of type 2 diabetes and an age-related reduction in muscle PPARGC1A expression [3\u20135]. In addition, genetic variation in the PPARGC1A gene was associated with indices of beta cell function [6]. Despite the central role of ATP for insulin secretion, the function of PGC-1\u03b1 in human pancreatic islets and beta cells is less well established [7].\nObesity, reduced physical activity and ageing are well known risk factors for type 2 diabetes. However, all individuals exposed to an affluent environment do not develop the disease. One likely reason is that genetic variation modifies individual susceptibility to the environment. However, the environment could also modify genetic risk factors by influencing expression of a gene by DNA methylation or histone modifications. Cytosine residues occurring in CG dinucleotides are targets for DNA methylation and gene expression is usually reduced when DNA methylation takes place at a promoter. Whether DNA methylation influences gene expression in target tissues for type 2 diabetes and thereby the pathogenesis of the disease remains to be demonstrated.\nThe present study investigated: (1) whether expression of PPARGC1A is altered in human islets from patients with type 2 diabetes; (2) if this expression is influenced by genetic (the PPARGC1A Gly482Ser polymorphism) and epigenetic (DNA methylation) factors; and (3) if expression of PPARGC1A influences insulin secretion.\nMethods\nMulti-organ donors The characteristics of the 48 non-diabetic and 12 type 2 diabetic multi-organ donors, whose pancreases were processed for islet preparation, are presented in Table\u00a01. Pancreases were obtained and processed with the approval of the regional Ethics Committee.\nTable\u00a01Clinical characteristics of type 2 diabetic and non-diabetic donors\u00a0Non-diabetic donorsType 2 diabetic donorsp valuen (male\/female)48 (30\/18)12 (6\/6)\u00a09 (7\/2)a10 (5\/5)a\u00a0Age (years)53.2\u2009\u00b1\u20092.466.7\u2009\u00b1\u20092.4<0.0554.2\u2009\u00b1\u20093.5a65.1\u2009\u00b1\u20092.6aBMI (kg\/m2)24.8\u2009\u00b1\u20090.627.1\u2009\u00b1\u20091.0<0.0525.9\u2009\u00b1\u20091.3a26.9\u2009\u00b1\u20091.1aGly\/Gly (%)b53.327.3\u00a0Gly\/Ser+Ser\/Ser (%)b46.772.3\u00a0Data are expressed as mean\u2009\u00b1\u2009SEMaDonors used for DNA methylation analysisbPPARGC1A Gly482Ser polymorphism\nHuman pancreatic islets and experimental plan Isolated pancreatic islets were prepared by collagenase digestion and density gradient purification [8, 9]. After isolation, islets were cultured free floating in M199 culture medium (Sigma-Aldrich, St Louis, MO, USA) at 5.5\u00a0mmol\/l glucose concentration and studied within 3\u00a0days from isolation. Cell viability, measured by Trypan Blue exclusion, was higher than 90% in control and diabetic islets after 3\u00a0days in culture.\nInsulin secretion study Insulin secretion studies were performed as previously described [8, 9]. Following a 45\u00a0min pre-incubation period at 3.3\u00a0mmol\/l glucose, groups of 30 islets of comparable size were kept at 37\u00b0C for 45\u00a0min in KRB, 0.5% (wt\/wt) albumin, pH\u00a07.4, containing 3.3\u00a0mmol\/l glucose. At the end of this period, the medium was completely removed and replaced with KRB containing either 3.3, 16.7 or 3.3\u00a0mmol\/l glucose plus 20\u00a0mmol\/l arginine, or 3.3\u00a0mmol\/l glucose plus 100\u00a0\u03bcmol\/l glibenclamide. After an additional 45\u00a0min incubation period, the medium was removed. Media (500\u00a0\u03bcl aliquots from the 10\u00a0ml incubation volume) were stored at \u221220\u00b0C until insulin concentrations were measured by immunoradiometric assay (Pantec Forniture Biomediche, Turin, Italy).\nAnimals Streptozotocin (STZ)\u2013nicotinamide (NA)-treated male Wistar rats (2\u20133\u00a0months old) were administered 210\u00a0mg\/kg NA i.p. (Sigma, St Louis, MO, USA) dissolved in saline, 15\u00a0min before an i.v. injection of 60\u00a0mg\/kg STZ (Sigma) that had been dissolved in citrate buffer (pH\u00a04.5) immediately before use. Control rats were injected with vehicle alone. STZ\u2013NA-treated animals had stable hyperglycaemia (8.9\u201310.0\u00a0mmol\/l) and they were used for the experiments 5\u00a0weeks after diabetes was induced. Pancreatic islets were isolated by the collagenase method using the procedure of pancreatic duct cannulation and density gradient purification as described elsewhere [10, 11].\nGK rats were obtained from the Stockholm colony and bred as described [12]. Inbred, normoglycaemic F344 rats were purchased from Charles River Laboratories (Wilmington, MA, USA) and maintained by sister\u2013brother mating. Transfer of GK alleles onto the genome of F344 rats by repeated backcrossing (ten generations) established the homozygous congenic strains F344.GK-Niddm1f (NIDDM1F) and F344.GK-Niddm1i (NIDDM1I). NIDDM1F carries 0.5% of the GK genotype (8\u00a0cM), based on genetic distance, on a homozygous F344 genetic background. NIDDM1F rats display hyperglycaemia accompanied by fasting hyperinsulinaemia and increased epididymal fat, implicating insulin resistance. NIDDM1I carries 0.8% of the GK genotype (14\u00a0cM), and display hyperglycaemia and insulin secretion defects [13\u201315]. Backcrossing was designed to introduce mitochondrial DNA and chromosomes X plus Y from F344. The congenic strains were kept constant by sister\u2013brother mating for several generations. To avoid effects of the oestrous cycle and other sex-specific influences, only male rats were included in this study. Rats were maintained at constant temperature and humidity in a 12\u00a0h light\u2013dark cycle with free access to standard laboratory chow pellets and water. All experiments were approved by the local Ethics Committees. Isolated pancreatic islets were prepared from rats at 8\u00a0weeks of age after a 6\u00a0h fast (08:00\u201314:00 hours), by injection of a collagenase solution via the bile\u2013pancreatic duct [16].\nAnalysis of PPARGC1A mRNA expression in pancreatic islets Total RNA was extracted from human islets, after 3\u00a0days in culture, using the RNeasy Protect Mini Kit (Qiagen, Valencia, CA, USA). It was quantified by absorbance at A260\/A280 nm (ratio\u2009>\u20091.65) in a Perkin-Elmer spectrophotometer (Waltham, MA, USA) and its integrity was assessed after electrophoresis in 1.0% (wt\/wt) agarose gels by ethidium bromide staining. Human and rat PPARGC1A mRNA expression were quantified by RT-PCR [8]. Gene-specific probes and primer pairs for PPARGC1A (Assays-on-demands, human, Hs00173304_m1 and rat, Rn00580241_A1; Applied Biosystems, Foster City, CA, USA) were used. Each sample was run in duplicate and the transcript quantity was normalised to the mRNA level of cyclophilin A (human, 4326316E and rat Rn00574762_A1; Applied Biosystems). For each probe\/primer set, a standard curve was generated, which was confirmed to increase linearly with increasing amounts of cDNA.\nDownregulation of PGC-1\u03b1 in human islets using silencing RNA (siRNA) In order to test whether inhibition of PPARGC1A expression could directly modulate insulin release, isolated human islets were transfected with PPARGC1A siRNA by using Arrest-In Transfection (Open Biosystem; Celbio, Pero, Italy). This is a polymeric formulation developed and optimised for highly efficient delivery of siRNA into the nucleus of suspension cells in the presence of serum-containing medium. Pre-designed Silencer siRNAs and Silencer non-targeting siRNAs (negative control) for PPARGC1A (Ambion, Austin, TX, USA) were used. Transfection was performed according to the manufacturer\u2019s instructions. Briefly, islets obtained from five pancreases were washed 12 times in KRB and exposed for 10\u00a0min to free Ca2+- and Mg2+-KRB to allow cell disaggregation. At the end of the incubation period, 400 islets per study point were re-suspended in 800\u00a0\u03bcl M199 medium (Sigma-Aldrich) added with adult bovine serum. siRNA (80\u00a0nmol\/l) was diluted in 100\u00a0\u03bcl M199, while 20\u00a0\u03bcg Arrest-In solution (Celbio) was dissolved in 100\u00a0\u03bcl M199 and incubated for 10\u00a0min after rapid mixing to allow formation of transfection complexes. Finally, 200\u00a0\u03bcl of this solution were added into wells containing islets and incubation was allowed for 48\u00a0h in a CO2 incubator at 37\u00b0C. At the end of the incubation period well volume was doubled with M199 culture medium and samples were kept in the incubator for another 48\u00a0h, when islets function, viability, transfection efficiency (60% when measured by Polyfectamine tied to a fluorescent probe) and gene expression were evaluated.\nGenotyping Genomic DNA was extracted from pancreatic islets using a Wizard Genomic DNA Purification kit (Promega, Madison, WI, USA). The Gly482Ser (GGT\u2192AGT) polymorphism of PPARGC1A was genotyped using an allelic discrimination assay performed with an ABI 7900 system (Applied Biosystems), using PCR primers: 5\u2032-CACTTCGGTCATCCCAGTCAA-3\u2032 (forward) and 5\u2032-TTATCACTTTCATCTTCGCTGTCATC-3\u2032 (reverse), and TaqMan MGB probes: Fam-5\u2032-AGACAAGACCGGTGAA-3\u2032 and Vic-5\u2032-CAGACAAGACCAGTGAA-3\u2032 [5, 17].\nDNA methylation A sequence starting 5,000\u00a0bp upstream from the PPARGC1A translation start was used in MethPrimer (http:\/\/www.urogene.org\/methprimer\/index.html) to search for regions with CpG sites and PCR designs. A PPARGC1A sequence 986\u2013746 bases upstream from the translation start including four possible DNA-methylation sites and a putative hepatic nuclear factor 1 (HNF-1) binding site, was selected and analysed for DNA methylation (Fig.\u00a01c). Genomic DNA, isolated from pancreatic islets of nine non-diabetic and ten type 2 diabetic multi-organ donors, was treated with bisulphite using the EZ DNA Methylation Kit (Zymo Research, Orange, CA, USA). Bisulphite-modified DNA was amplified by nested PCR with primers designed using MethPrimer. Primer pair 1: 5\u2032-TAGGGTATTAGGGTTGGAATTTAATG-3\u2032 (forward) and 5\u2032-CCCATAACAATAAAAAATACCAACTC-3\u2032 (reverse), and primer pair 2 (used for nested PCR): 5\u2032-TATTTTAAGGTAGTTAGGGAGGAAA-3\u2032 (forward) and 5\u2032-ATAACAATAAAAAATACCAACTCCC-3\u2032 (reverse). The PCR products were then cloned into a vector (TOPO TA Cloning Kit for Sequencing, Invitrogen, Carlsbad, CA, USA) and ten colonies from each donor were purified with a Miniprep kit (Qiagen). These individual clones were sequenced using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The number of methylated sites was determined and divided by the total number of methylation sites and then multiplied by 100 to show the percentage of methylation for each donor.\nFig.\u00a01PPARGC1A mRNA expression in human pancreatic islets is influenced by type 2 diabetes, a PPARGC1A Gly482Ser polymorphism and DNA methylation. The influence of a type 2 diabetes and b the PPARGC1A Gly482Ser polymorphism on PPARGC1A mRNA expression in human pancreatic islets. c The PPARGC1A promoter sequence investigated, showing the four DNA methylation target sites; \u2212772, \u2212903, \u2212936 and \u2212961 and a putative binding-site for HNF-1. d The influence of type 2 diabetes on DNA methylation of the PPARGC1A promoter. The influence of e type 2 diabetes (T2D) and f the PPARGC1A Gly482Ser polymorphism on absolute insulin release (pmol islet\u22121 min\u22121) in response to 16.7\u00a0mmol\/l glucose. g Correlations between PPARGC1A mRNA expression and absolute insulin release (pmol islet\u22121 min\u22121) in response to 16.7\u00a0mmol\/l glucose in human pancreatic islets (r\u2009=\u20090.38, p\u2009<\u20090.05). Results are expressed as mean\u2009\u00b1\u2009SEM. *p\u2009<\u20090.05\nStatistical methods Differences in PPARGC1A mRNA expression, percentage of DNA methylation and insulin secretion between the different groups studied were analysed using Student\u2019s t test or the non-parametric Mann\u2013Whitney test, where appropriate. Correlations were calculated using Pearson correlation coefficients for normally distributed values and Spearman correlation coefficients when normality was rejected. Log values were used in the multivariate regression analysis. Differences in expression between GK, F344, NIDDM1F and NIDDM1I rats were analysed using one-way ANOVA, followed by a Kruskal\u2013Wallis Z test. All p values were two-tailed and p values less than 0.05 were considered significant. Statistical calculations were performed by NCSS software (NCSS Statistical Software, Kaysville, UT, USA).\nResults\nThe mRNA expression of PPARGC1A was markedly reduced in pancreatic islets from type 2 diabetic compared with non-diabetic donors (1.06\u2009\u00b1\u20090.63 vs 15.0\u2009\u00b1\u20093.0; p\u2009<\u20090.005; Fig.\u00a01a). Intriguingly, non-diabetic carriers of a PPARGC1A 482Ser allele, Gly\/Ser or Ser\/Ser genotype carriers had markedly lower PPARGC1A mRNA expression compared with carriers of the Gly\/Gly genotype [Gly\/Ser + Ser\/Ser 3.43\u2009\u00b1\u20090.94 (n\u2009=\u200917) vs Gly\/Gly 26.53\u2009\u00b1\u20094.42 (n\u2009=\u200917); p\u2009<\u20090.00005] (Fig.\u00a01b). However, there was no difference in PPARGC1A mRNA expression between non-diabetic carriers of the Gly\/Ser (3.9\u2009\u00b1\u20094.0; n\u2009=\u200911) or Ser\/Ser (2.7\u2009\u00b1\u20095.5; n\u2009=\u20096) genotypes. This demonstrates that the effect of the genotype on gene expression is seen already in the pre-diabetic state.\nThe diabetic donors were significantly older and had higher BMI than control donors (Table\u00a01). We therefore tested whether these factors might also relate to PPARGC1A expression in human islets using a multivariate regression analysis including age, BMI and disease status as covariates. Only disease status was significantly associated with PPARGC1A expression in this analysis (r\u2009=\u20091.04; p\u2009=\u20090.0065).\nWe next evaluated whether epigenetic phenomena such as DNA methylation would influence PPARGC1A expression in islets. Cytosine residues occurring in CG dinucleotides are targets for DNA methylation, and gene expression is usually reduced when DNA is methylated at the promoter. We used bisulphite sequencing to assess DNA methylation of four methylation target sites, \u2212772, \u2212903, \u2212936 and \u2212961 in the PPARGC1A promoter in pancreatic islets from the type 2 diabetic and non-diabetic human multi-organ donors (Fig.\u00a01c). Interestingly, there was an approximately twofold increase in DNA methylation of the PPARGC1A promoter of diabetic compared with non-diabetic human islets (10.5\u2009\u00b1\u20092.7 vs 4.7\u2009\u00b1\u20090.9%; p\u2009<\u20090.04; Fig.\u00a01d). Also in this subset of donors, the PPARGC1A mRNA expression was significantly reduced in diabetic compared with non-diabetic islets (p\u2009=\u20090.002). There was a trend towards an inverse correlation between the level of DNA methylation and PPARGC1A mRNA expression (r\u2009=\u2009\u22120.48; p\u2009=\u20090.08, after adjustment for disease status).\nInsulin release (pmol islet\u22121 min\u22121) in response to 16.7\u00a0mmol\/l glucose was reduced in type 2 diabetic compared with non-diabetic islets (0.32\u2009\u00b1\u20090.04 vs 0.55\u2009\u00b1\u20090.05; p\u2009<\u20090.01) as well as in non-diabetic islets carrying the PPARGC1A 482Ser allele (Gly\/Ser\u2009+\u2009Ser\/Ser) compared with carriers of the Gly\/Gly genotype (Gly\/Ser\u2009+\u2009Ser\/Ser 0.43\u2009\u00b1\u20090.06 vs Gly\/Gly 0.64\u2009\u00b1\u20090.07; p\u2009<\u20090.05; Fig.\u00a01e\u2013f). In addition, the mRNA expression of PPARGC1A correlated positively with glucose-mediated insulin release (r\u2009=\u20090.38; p\u2009<\u20090.05; Fig.\u00a01g). Moreover, the PPARGC1A Gly482Ser polymorphism did not significantly affect basal insulin secretion at 3.3\u00a0mmol\/l glucose (Gly\/Ser\u2009+\u2009Ser\/Ser, 0.23\u2009\u00b1\u20090.01 vs Gly\/Gly, 0.29\u2009\u00b1\u20090.02; p\u2009=\u20090.13), or insulin response to arginine (Gly\/Ser\u2009+\u2009Ser\/Ser 0.48\u2009\u00b1\u20090.05 vs Gly\/Gly 0.53\u2009\u00b1\u20090.08; p\u2009=\u20090.77) or glibenclamide (Gly\/Ser\u2009+\u2009Ser\/Ser 0.59\u2009\u00b1\u20090.1 vs Gly\/Gly 0.56\u2009\u00b1\u20090.09; p\u2009=\u20090.98).\nHaving seen the relationship between PPARGC1A expression and insulin secretion, we set out to determine whether modulation of PPARGC1A expression would influence insulin secretion. This was achieved by studying insulin secretion after experimental downregulation of PGC-1\u03b1 in human islets transfected with PPARGC1A siRNA (n\u2009=\u20093\u20135 experiments). As shown in Fig.\u00a02, transfection with siRNA was associated with a 45% reduction in the mRNA expression of PPARGC1A (p\u2009=\u20090.01), 32% reduction in insulin mRNA (p\u2009=\u20090.05) and 41% reduction of the insulin stimulation index (i.e. incremental folds above baseline insulin release; p\u2009=\u20090.01). Notably, no significant change was seen in glucagon mRNA expression, suggesting that the effect was primarily due to a downregulation of PGC-1\u03b1 in beta cells. As a negative control, transfection with scrambled siRNA was not associated with any changes in PPARGC1A (1.26\u2009\u00b1\u20090.25), insulin (1.29\u2009\u00b1\u20090.16) or glucagon (0.99\u2009\u00b1\u20090.15) mRNA expression (p\u2009>\u20090.05).\nFig.\u00a02Transfection of human pancreatic islets with PPARGC1A siRNA is associated with reduced mRNA levels of aPPARGC1A (n\u2009=\u20093) and b insulin (n\u2009=\u20095) and c concomitant reduction of the insulin stimulation index (ISI), i.e. incremental fold change above basal insulin release. d Conversely, inhibition of PPARGC1A expression in human pancreatic islets has no effect on glucagon mRNA expression (n\u2009=\u20095). Results are expressed as mean\u2009\u00b1\u2009SEM. *p\u2009<\u20090.05\nThe finding of reduced PPARGC1A expression in human diabetic islets was somewhat surprising given an earlier report of increased PGC-1\u03b1 levels in islets from diabetic mice (ob\/ob) and rat (ZDF and pancreatectomised rats) models [7]. To further explore species-specific differences we also measured PPARGC1A mRNA expression in pancreatic islets from two diabetic rat models. PPARGC1A expression was reduced in islets from STZ\u2013NA diabetic male Wistar rats compared with control animals (3.08\u2009\u00b1\u20091.07 vs 10.26\u2009\u00b1\u20091.12; p\u2009<\u20090.009; Fig.\u00a03a). Moreover, PPARGC1A expression was reduced in islets from the congenic NIDDM1I strain, which has insulin secretion defects, compared with the insulin-resistant NIDDM1F strain (7.18\u2009\u00b1\u20091.79 vs 11.30\u2009\u00b1\u20091.79; p\u2009<\u20090.02) of the GK rat, an animal model of polygenic type 2 diabetes (Fig.\u00a03b). However, no difference in PPARGC1A expression was found when comparing islets from F344 or GK rat strains (Fig.\u00a03b).\nFig.\u00a03PPARGC1A mRNA expression in rodent pancreatic islets. a Male Wistar rats (2\u20133\u00a0months old) were treated with STZ\u2013NA (n\u2009=\u20095) or vehicles (control; n\u2009=\u20095) [10, 11] and PPARGC1A mRNA expression together with the internal standard cyclophilin A was analysed in pancreatic islets isolated from STZ\u2013NA-treated animals showing a stable hyperglycaemia (8.9\u201310.0\u00a0mmol glucose\/l) and controls. bPPARGC1A mRNA expression together with the internal standard cyclophilin A was analysed in pancreatic islets prepared from rats at 8\u00a0weeks of age after a 6\u00a0h fast. GK rats (n\u2009=\u20096) were obtained from the Stockholm colony and bred as described [12]. Inbred, normoglycaemic F344 (n\u2009=\u20096) were purchased from Charles River Laboratories. Transfer of GK alleles onto the genome of F344 rats by repeated backcrossing (ten generations) established the homozygous congenic strains NIDDM1F and NIDDM1I. NIDDM1F rats (n\u2009=\u20096) carry 0.5% of the GK genotype, based on genetic distance, on a homozygous F344 genetic background (8\u00a0cM) and display hyperglycaemia accompanied by fasting hyperinsulinaemia, implicating insulin resistance. NIDDM1I (n\u2009=\u20096) carries 0.8% of the GK genotype (14\u00a0cM) and display insulin secretion defects [13\u201315]. Results are expressed as mean\u2009\u00b1\u2009SEM. *p\u2009<\u20090.05\nDiscussion\nThe key results from the present study were that PPARGC1A mRNA expression and insulin secretion were reduced in pancreatic islets of patients with type 2 diabetes and in non-diabetic carriers of a PPARGC1A 482Ser allele, a genotype previously associated with the disease. Moreover, we demonstrate that epigenetic mechanisms are likely to be operative in the pathogenesis of type 2 diabetes since DNA methylation of the PPARGC1A promoter was increased in human diabetic islets. Experimental downregulation of PPARGC1A expression in human islets by siRNA resulted in decreased insulin secretion thereby demonstrating a causal link between PGC-1\u03b1 levels and insulin secretion. Based on these observations we propose a model where combinations of genetic and epigenetic factors can influence the level of PGC-1\u03b1 in human islets and subsequently glucose-stimulated insulin secretion.\nThe transcriptional coactivator PGC-1\u03b1 is an important factor regulating the expression of genes for oxidative phosphorylation in a number of tissues including skeletal muscle, liver and adipose tissue. We and others have previously demonstrated reduced expression of PPARGC1A and a set of genes involved in oxidative phosphorylation in skeletal muscle from patients with type 2 diabetes [2, 18]. However, the level and role of PGC-1\u03b1 in pancreatic islets of patients with type 2 diabetes remained to be determined. The present results indicate that the situation in human islets mirrors the situation in skeletal muscle, since the expression level of PPARGC1A was reduced in human type 2 diabetic islets, thus providing evidence for a common defect that may simultaneously contribute to peripheral insulin resistance and impairment of beta cell function.\nIn contrast to the present results in humans, elevated PGC-1\u03b1 levels have been reported in islets from diabetic rodent models, ob\/ob mice, ZDF rats and pancreatectomised rats [7]. In these rodent models, the increase in PGC-1\u03b1 was associated with suppressed beta cell metabolism and insulin release. This could reflect true species differences, but also differences in experimental conditions. To address this issue, we determined: (1) the effect of inhibiting PGC-1\u03b1 production by transfecting human pancreatic islets with PPARGC1A siRNA; and (2) PPARGC1A expression in pancreatic islets of different rodent models with experimental diabetes, i.e. STZ-diabetic Wistar rats and congenic strains from the type 2 diabetes-like GK rat. Silencing PGC-1\u03b1 production was associated with concomitant reduction of insulin mRNA expression and insulin release in response to glucose. This effect appears to be highly specific for insulin regulation as it had no effect whatsoever on glucagon mRNA expression. The congenic strains have been bred from the GK rat based upon low insulin secretion or insulin resistance. In line with the results observed in human islets, PPARGC1A expression was reduced in islets from diabetic animals and in rat strains with suppressed insulin secretion. Collectively, these data support the notion that PPARGC1A expression is reduced in animal models of diabetes and human diabetes, and associated with impaired insulin secretion.\nThe risk of developing type 2 diabetes increases with high-fat\/high-energy diets, reduced physical activity and age. However, not all individuals respond to the environment in the same way. This variation in response to environmental factors has partially been ascribed to genetic factors. The common Gly482Ser variant in the PPARGC1A gene is a plausible candidate for such a genetic factor [3, 4]. We have previously shown that this polymorphism is associated with an age-related decline in PPARGC1A gene expression in human skeletal muscle [5]. The present study shows that the same PPARGC1A risk allele, 482Ser, is associated with reduced PPARGC1A expression and impaired insulin secretion in human islets. Thus, our results provide the ground for a common genetic predisposition towards type 2 diabetes contributing to both impaired insulin action and secretion.\nReduced PPARGC1A expression may not simply reflect an effect of the polymorphism per se but could also occur through epigenetic phenomena such as increased DNA methylation of the PPARGC1A promoter. Cytosine residues occurring in CG dinucleotides are targets for DNA methylation, and gene expression is usually reduced when DNA methylation takes place at a promoter. Epigenetic changes are well known to influence gene expression of suppressor genes and oncogenes in cancer cells and contribute to tumour growth [19]. Increased DNA methylation of the promoter of the PPARGC1A gene in type 2 diabetic human islets may contribute to beta cell dysfunction by similar mechanisms. To our knowledge, this is the first study to demonstrate that DNA methylation may play a role in regulating gene expression in human diabetic islets.\nOur data suggest that PGC-1\u03b1 can modulate glucose-mediated insulin secretion in human islets, most likely via an effect on ATP production as indicated by normal insulin release in response to arginine and glibenclamide, which stimulate insulin downstream of ATP. In support of such a role, PPARGC1A mRNA expression correlated with glucose-stimulated insulin release. Moreover, inhibition of PPARGC1A expression by siRNA transfection in human islets was associated with a decline in insulin mRNA and insulin release. Similarly, in human type 2 diabetic islets, reduced PPARGC1A mRNA levels were associated with impaired glucose-mediated insulin secretion. The PPARGC1A Gly482Ser polymorphism, which previously has been associated with type 2 diabetes, was also associated with reduced PPARGC1A expression and impaired insulin secretion in the human islets.\nOne caveat of this study could be that we did not measure the protein level of PGC-1\u03b1 in human islets. However, we have previously demonstrated a significant positive correlation between the PPARGC1A mRNA and PGC-1\u03b1 protein levels in human skeletal muscle [5] and we therefore assume that this might also be the case in human islets. The reduction in PPARGC1A expression in non-diabetic carriers of a 482Ser allele was strong and factors other than haploinsufficiency may therefore explain this reduction. Moreover, whether the genotype influences PPARGC1A expression in diabetic islets would be of interest; however, the present study lacks statistical power to perform this analysis. These relationships should be addressed in future ad hoc studies.\nIn conclusion, we have shown that PPARGC1A mRNA expression is reduced in islets from patients with type 2 diabetes and it is influenced by both genetic (Gly482Ser) and epigenetic (DNA methylation) factors. This reduction in PPARGC1A expression correlated with impaired glucose-stimulated insulin secretion, which is known to require ATP. Thereby, the present study provides two novel insights into the molecular mechanisms of islet dysfunction in type 2 diabetes; first that a master transcription regulator of oxidative phosphorylation, PGC-1\u03b1, may be involved and second, that epigenetic factors like DNA methylation should be considered when searching for the genetic causes of type 2 diabetes.","keyphrases":["epigenetic","genetic","ppargc1a","human","type 2 diabetes","pancreatic islets","pgc-1\u03b1","dna methylation","gene expression"],"prmu":["P","P","P","P","P","P","P","P","P"]}
{"id":"Soc_Psychiatry_Psychiatr_Epidemiol-2-2-1764204","title":"Regional differences in psychiatric disorders in Chile\n","text":"Background Psychiatric epidemiological surveys in developing countries are rare and are frequently conducted in regions that are not necessarily representative of the entire country. In addition, in large countries with dispersed populations national rates may have low value for estimating the need for mental health services and programs.\nCross-national psychiatric epidemiological prevalence studies using similar diagnostic instruments have resulted in disparate rates for specific disorders [1]. The reasons for these differences in rates have been attributed to methodological issues between studies; socio-demographic factors such as socio-economic status differences between countries; and cultural differences. Cross-national comparative studies [2, 3] attempt to correct for socio-demographic variability; yet, differences persist. In some regions of the world, such as prevalence rates in Chinese based studies [4\u20136], the rates are markedly different than the rest of the world. This either suggests true differences in the rates of pathology or lack of cultural appropriateness of the diagnostic tools and the Western diagnostic systems utilized [7]. Understanding these cross-national differences may provide clues into the etiology of psychopathology.\nRegional differences also exist within countries as evidenced by studies where the methodology is similar across geographic areas [8\u201311]. Regional differences in the presence of serious mental illness have been attributed to migration of the mentally ill [12, 13]; birth in urban areas [14]; and genetic pooling [15]. The most studied regional differences are those between urban and rural populations, where urban environmental adversity is argued to contribute to pathology [16, 17].\nAn understanding of regional differences in countries where the population is spread across large distances is relevant for health care planning. Potentially risk factors may differ across a country resulting in the need to address mental health needs on a regional basis rather than centrally. Furthermore, an understanding of geographic variability permits allocation of resources to be distributed in a proportional basis by need.\nThis issue is of particular importance to developing countries such as in Latin America, where frequently services are only provided centrally, or disproportionately to the wealthier regions of a country. In addition, epidemiological studies based on single regions of the country [18\u201320] are extrapolated to larger population bases for which they may or may not be representative.\nPopulation studies about psychiatric disorders in Latin America, as well as other developing regions of the world, are rare. They are important, however, for understanding variations in patterns of disorders, underlying determinants, and service needs. Chile, given its rather extraordinary geography provides an important test of variations in disorder rates across a spatially dispersed population, and offers perhaps the best case example of a country where national rates would seemingly have low value for estimating the need for mental health services and programs.\nThe Chile Psychiatric Prevalence Study (CPPS) was developed to address issues regarding the prevalence and risk factors for mental illness based on a nationally representative sample, and service utilization. Chile has a population of approximately 16\u00a0millions people. The country is composed of 51 provinces grouped in 13 regions covering an area spanning 2\u00a0million\u00a0km2 (including Antarctica and Insular Territories) over a length of 8,000\u00a0km. The large distances between major population centers resulted in the CPPS being conducted in four regions of the country, Bio Bio, the south-central region containing the second largest city, Metropolitana, the north-central region which includes the capital Santiago, Tarapaca, the north, and Araucania, the south of the country, in order to obtain a representative sampling of the population of the nation. This report focuses on whether regional differences in the prevalence of psychiatric disorders and service utilization, if present, are due to factors other than socio-demographic differences between population centers.\nMethods\nSample selection\nThe CPPS was based on a household stratified sample of people age 15 and older. A more detailed description of the methods used in the CPPS is available in earlier publications [21]. The sample frame was developed to be representative of the nation\u2019s population. Four regions and their most representative province and comunas were selected. These were subsequently subdivided into districts, and then randomly selected blocks. The number of households available on each block was enumerated. The 1992 census of each region was used to determine the number of households required on each block. A list of the inhabitants, age 15 and older, in each household was generated. Using pre-assigned Kish tables (Kish 1965) one person per household was selected from the list to be interviewed.\nThe survey was conducted by the University of Concepcion, Department of Psychiatry and Mental Health, between July 1992 and June 1999, with each site being completed in the following order based on funding: Bio Bio, Metropolitana, Tarapaca, and Araucania. A total of 2,987 individuals participated in the survey. Response rate did differ by site (\u03c72 = 11.08, df\u00a0=\u00a03, P\u00a0<\u00a00.02) with Metropolitana having the highest non-response rate 12.6% and Tarapaca the lowest 7.5%. A weight was used to account for the probability of the comuna, district, block, household, and respondent being selected. The data was adjusted to the 1992 census of each region based on age, gender, and marital status using a second weight.\nDiagnostic assessment\nComposite International Diagnostic Interview (CIDI) versions 1.0 and 1.1 [22] were used to generate the diagnoses using well-trained lay interviewers. DSM-III-R [23] diagnostic criteria were employed. A section on health service utilization in the 6-months prior to the interview was also included. The Spanish translation was conducted using the protocol outlined by the World Health Organization (WHO) [24]. A validation study of the Chilean CIDI was found to have kappas that ranged from 0.52 for somatiform disorders up to 0.94 for affective disorders [25] using a sample of patients and volunteers for each CIDI section. After double entry of data and verification for logical inconsistencies diagnoses were generated using the CIDI computer programs for 1.0 and 1.1 [26]. The DSM-III-R diagnoses included in this report are all affective disorders; all anxiety disorders defined as panic disorder, agoraphobia, and generalized anxiety disorder; substance use disorder which does not include nicotine dependence, and any diagnosis. Lifetime and 12-month prevalence rates were examined.\nInterviewers and training\nSocial science university students in their senior year underwent training following the WHO protocol at the University of Concepcion, a WHO CIDI training and reference center. The 64 interviewers received over 80\u00a0h of instruction and practice sessions. Each interviewer had to conduct practice interviews with volunteer adult subjects with and without psychiatric disorders selected from local clinics, as well as a pilot interview on an individual in a non-selected household in the community. Approximately 80% of the interviews were audiotaped following the subject\u2019s consent, and 20% randomly reviewed for quality control.\nAnalysis procedures\nThe SUDAAN statistical package [27], Taylor series linearization method, was used to estimate the standard errors due to the sample design and the need for weighting. The analysis was conducted using procedures without replacement for non-respondents. The comuna and district selected were used as the defined strata. Chi-square analyses were used to examine the association of disorders and service utilization between regions. Logistic regression was used to adjust for socio-demographic differences across regions accounting for differential rates or service utilization. Additional analyses were conducted to examine urban\u2013rural differences. The logistic regression analyses included gender, marital status, age group, education, and income as potential confounders. All results are presented as weighted data.\nResults\nThe distribution of income and marital status were found to differ across the four regions of the country (see Table\u00a01). The population of Bio Bio had significantly lower incomes than the other regions (\u03c72\u00a0=\u00a067.96, df\u00a0=\u00a09, P\u00a0<\u00a00.0001). In addition, Bio Bio had the lowest rate of individuals who were separated or had annulled marriages (\u03c72\u00a0=\u00a025.31, df\u00a0=\u00a012, P\u00a0<\u00a00.05). In two of the regions the rural population was under-represented relative to the census. In Bio Bio 22% of population was rural the sample only included 2.7%, and in Araucania 38% of the population was rural and the sample only included 7.7%.\nTable\u00a01Socio-demographic characteristics by regionDisordersBio Bio (N\u00a0=\u00a0800)Metropolitana (N\u00a0=\u00a01363)Tarapaca (N\u00a0=\u00a0306)Araucania (N\u00a0=\u00a0509)\u03c72dfP%SE%SE%SE%SEGender\u00a0\u00a0Male48.21.046.41.448.91.047.41.53.2330.38\u00a0\u00a0Female51.81.053.61.451.11.052.61.5Age\u00a0\u00a015\u20132426.71.624.92.127.32.327.07.47.3150.94\u00a0\u00a025\u20133425.41.626.11.427.10.623.24.6\u00a0\u00a035\u20134418.21.918.51.121.12.017.52.6\u00a0\u00a045\u20135412.11.412.81.211.80.412.31.2\u00a0\u00a055\u2013649.01.49.20.86.50.49.81.3\u00a0\u00a065+8.72.38.51.06.21.910.41.7Education\u00a0\u00a0No education2.71.01.10.20.30.21.31.011.1390.31\u00a0\u00a0Basic19.73.817.92.37.82.015.27.6\u00a0\u00a0Medium52.33.347.91.652.71.538.27.3\u00a0\u00a0High25.27.133.22.339.13.245.215.4Marital status\u00a0\u00a0Married54.72.852.82.353.73.252.35.825.31120.05\u00a0\u00a0Widowed5.21.14.70.73.31.35.80.2\u00a0\u00a0Separated\/anulled1.70.74.10.53.31.42.00.6\u00a0\u00a0Never Married34.41.932.62.433.91.136.18.0\u00a0\u00a0Common Law4.00.85.81.05.90.63.82.0Income\u00a0\u00a0U$100\u2013U$40070.57.154.53.912.24.847.715.467.9690.0001\u00a0\u00a0U$401\u2013U$80019.33.321.91.829.62.520.64.5\u00a0\u00a0U$801\u2013U$15006.91.911.91.335.63.312.54.0\u00a0\u00a0U$1501+3.32.511.72.822.73.619.27.2Urban\/rural\u00a0\u00a0Urban97.33.199.20.998.91.492.35.03.130.39\u00a0\u00a0Rural2.73.10.80.91.11.47.75.0\nPrior to adjusting for socio-demographic differences between the regions, a number of differences in prevalence rates were noted (see Tables\u00a02, 3). Lifetime rates for major depressive disorder were markedly elevated in Tarapaca, 17.2%, and lowest in Bio Bio, 11.6% (\u03c72\u00a0=\u00a09.76, df\u00a0=\u00a03, P\u00a0<\u00a00.04). Drug abuse, but not dependence, also had the highest prevalence rate in Tarapaca, 2.4% (\u03c72\u00a0=\u00a08.59, df\u00a0=\u00a03, P\u00a0<\u00a00.05). Interestingly, in Araucania the rate of non-affective psychosis was the lowest (\u03c72\u00a0=\u00a011.45, df\u00a0=\u00a03, P\u00a0<\u00a00.02). When females were examined the differential rates for major depressive disorder (\u03c72\u00a0=\u00a011.76, df\u00a0=\u00a03, P\u00a0<\u00a00.02) and substance use disorders were noted (\u03c72\u00a0=\u00a010.88, df\u00a0=\u00a03, P\u00a0<\u00a00.03). Among males the only lifetime difference in prevalence rates was for elevated alcohol abuse in Tarapaca (\u03c72\u00a0=\u00a09.17, df\u00a0=\u00a03, P\u00a0<\u00a00.04). For 12-month prevalence the increased risk for major depression in Tarapaca persisted (\u03c72\u00a0=\u00a08.78, df\u00a0=\u00a03, P\u00a0<\u00a00.05) for both genders combined and for females (\u03c72\u00a0=\u00a010.08, df\u00a0=\u00a03, P\u00a0<\u00a00.03). The differences noted in the prevalence of substance use disorders were no longer evident at 12-months.\nTable\u00a02Lifetime prevalence rates of DSM-III-R disorders by regionBio BioMetropolitanaTarapacaAraucaniaDisorders%SE%SE%SE%SE\u03c72PAffective disorders\u00a0\u00a0\u00a0\u00a0Major depressive episode7.11.311.60.817.22.49.80.89.760.04\u00a0\u00a0\u00a0\u00a0Manic episode2.20.71.40.41.80.31.51.31.110.78\u00a0\u00a0\u00a0\u00a0Dysthmia7.51.17.31.212.21.76.03.33.650.32\u00a0\u00a0\u00a0\u00a0Any affective disorder13.62.215.41.223.21.914.03.23.880.30Anxiety disorders\u00a0\u00a0\u00a0\u00a0Panic disorder1.20.61.30.34.30.81.10.51.160.77\u00a0\u00a0\u00a0\u00a0Agoraphobia without panic14.22.79.81.39.71.85.30.85.460.16\u00a0\u00a0\u00a0\u00a0Generalized anxiety disorder1.80.73.70.52.00.43.00.65.130.19\u00a0\u00a0\u00a0\u00a0Any anxiety disorder19.23.714.81.617.91.78.60.96.790.11Substance use disorders\u00a0\u00a0\u00a0\u00a0Alcohol abuse2.80.82.00.516.92.67.21.48.200.06\u00a0\u00a0\u00a0\u00a0Alcohol dependence7.01.76.40.86.30.55.00.83.630.32\u00a0\u00a0\u00a0\u00a0Drug abuse0.60.31.50.42.40.50.10.18.590.05\u00a0\u00a0\u00a0\u00a0Drug dependence2.20.73.31.02.20.31.20.17.720.07\u00a0\u00a0\u00a0\u00a0Nicotine dependence2.90.62.10.66.50.25.41.07.860.07\u00a0\u00a0\u00a0\u00a0Any alcohol or drug use disorder11.01.811.21.024.32.912.31.81.940.59\u00a0\u00a0\u00a0\u00a0Any substance use disorder13.02.012.91.129.62.914.61.72.430.50Other disorders\u00a0\u00a0\u00a0\u00a0Non-affective psychosis2.10.62.30.50.80.20.10.011.450.02\u00a0\u00a0\u00a0\u00a0Somatoform disorder2.71.14.40.83.10.63.50.71.750.63\u00a0\u00a0\u00a0\u00a0Cognitive disorder4.51.83.70.80.70.81.00.45.700.15Any CPPS disorder32.24.130.81.644.41.928.93.12.510.49FemaleAffective disorders\u00a0\u00a0\u00a0\u00a0Major depressive episode7.11.315.21.420.60.811.21.111.760.02\u00a0\u00a0\u00a0\u00a0Manic episode2.61.01.80.53.00.60.50.53.620.32\u00a0\u00a0\u00a0\u00a0Dysthmia10.72.011.61.917.21.38.75.12.430.50\u00a0\u00a0\u00a0\u00a0Any affective disorder16.42.621.01.728.30.916.04.93.600.32Anxiety disorders\u00a0\u00a0\u00a0\u00a0Panic disorder1.70.92.10.56.71.22.10.91.150.77\u00a0\u00a0\u00a0\u00a0Agoraphobia without panic20.33.213.62.012.20.27.02.44.880.20\u00a0\u00a0\u00a0\u00a0Generalized anxiety disorder2.51.06.41.03.80.84.40.45.980.13\u00a0\u00a0\u00a0\u00a0Any anxiety disorder26.43.920.92.524.82.112.32.05.190.18Substance use disorders\u00a0\u00a0\u00a0\u00a0Alcohol abuse0.50.41.10.62.60.41.00.42.060.57\u00a0\u00a0\u00a0\u00a0Alcohol dependence1.70.72.00.74.20.20.40.27.530.07\u00a0\u00a0\u00a0\u00a0Drug abuse0.00.00.80.32.30.50.00.07.380.08\u00a0\u00a0\u00a0\u00a0Drug dependence2.40.75.01.62.70.40.20.26.860.10\u00a0\u00a0\u00a0\u00a0Nicotine dependence2.50.72.30.810.00.62.60.42.450.49\u00a0\u00a0\u00a0\u00a0Any alcohol or drug use disorder3.60.97.11.56.90.61.60.611.800.02\u00a0\u00a0\u00a0\u00a0Any substance use disorder5.91.48.91.416.50.34.10.710.880.02Other disorders\u00a0\u00a0\u00a0\u00a0Non-affective psychosis2.40.71.90.61.60.30.10.17.810.07\u00a0\u00a0\u00a0\u00a0Somatoform disorder2.40.75.31.13.60.74.41.74.100.27Any CPPS disorder35.63.834.72.537.81.123.44.03.020.40MaleAffective disorders\u00a0\u00a0\u00a0\u00a0Major depressive episode7.11.57.41.313.64.68.31.56.840.10\u00a0\u00a0\u00a0\u00a0Manic episode1.81.01.00.40.50.62.62.21.180.76\u00a0\u00a0\u00a0\u00a0Dysthmia4.01.02.40.67.13.33.12.04.100.27\u00a0\u00a0\u00a0\u00a0Any affective disorder10.52.29.01.418.04.311.82.914.570.01Anxiety disorders\u00a0\u00a0\u00a0\u00a0Panic disorder0.70.50.40.21.80.30.00.04.390.24\u00a0\u00a0\u00a0\u00a0Agoraphobia without panic7.72.95.31.97.23.53.51.03.940.28\u00a0\u00a0\u00a0\u00a0Generalized anxiety disorder1.00.70.70.40.00.01.31.01.830.61\u00a0\u00a0\u00a0\u00a0Any anxiety disorder11.54.47.91.710.63.44.50.57.590.07Substance use disorders\u00a0\u00a0\u00a0\u00a0Alcohol abuse5.31.53.00.831.95.614.12.89.170.04\u00a0\u00a0\u00a0\u00a0Alcohol dependence12.53.711.61.98.41.210.21.81.110.77\u00a0\u00a0\u00a0\u00a0Drug abuse1.10.72.40.82.60.60.20.26.930.09\u00a0\u00a0\u00a0\u00a0Drug dependence1.91.11.50.61.60.42.30.51.400.71\u00a0\u00a0\u00a0\u00a0Nicotine dependence3.20.71.90.92.90.58.62.26.050.13\u00a0\u00a0\u00a0\u00a0Any alcohol or drug use disorder18.94.115.91.942.66.324.33.44.830.20\u00a0\u00a0\u00a0\u00a0Any substance use disorder20.54.317.62.143.35.826.22.95.550.16Other disorders\u00a0\u00a0\u00a0\u00a0Non-affective psychosis1.81.12.71.10.00.00.00.05.780.14\u00a0\u00a0\u00a0\u00a0Somatoform disorder3.01.73.21.32.60.52.60.90.230.97Any CPPS disorder28.55.326.42.351.24.035.05.04.460.23Non-affective psychosis includes schizophrenia, schizophreniform disorder, schizoaffective disorder, delusional disorder, and atypical psychosisAny CPSS disorder does not include nicotine dependence or cognitive disorder; \u03c72 df\u00a0=\u00a03Table\u00a0312-Month prevalence rates of DSM-III-R disorders by regionBio BioMetropolitanaTarapacaAraucaniaDisorders%SE%SE%SE%SE\u03c72PTotalAffective disorders\u00a0\u00a0\u00a0\u00a0Major depressive episode4.10.97.80.810.31.95.11.18.780.05\u00a0\u00a0\u00a0\u00a0Manic episode1.90.71.10.31.80.31.00.92.050.57\u00a0\u00a0\u00a0\u00a0Dysthmia3.00.74.11.27.92.23.12.26.100.13\u00a0\u00a0\u00a0\u00a0Any affective disorder7.81.610.81.315.01.67.52.95.190.18Anxiety disorders\u00a0\u00a0\u00a0\u00a0Panic disorder0.50.30.40.24.10.90.60.61.320.73\u00a0\u00a0\u00a0\u00a0Agoraphobia without panic7.01.86.11.57.52.02.20.94.440.24\u00a0\u00a0\u00a0\u00a0Generalized anxiety disorder1.20.52.30.61.10.31.70.52.410.50\u00a0\u00a0\u00a0\u00a0Any anxiety disorder11.11.79.21.611.91.63.81.35.690.15Substance use disorders\u00a0\u00a0\u00a0\u00a0Alcohol abuse2.10.61.80.55.81.83.30.84.050.28\u00a0\u00a0\u00a0\u00a0Alcohol dependence5.21.54.80.73.70.42.80.54.260.26\u00a0\u00a0\u00a0\u00a0Drug abuse0.20.20.40.21.50.40.00.03.990.28\u00a0\u00a0\u00a0\u00a0Drug dependence1.50.81.90.51.80.30.40.42.910.42\u00a0\u00a0\u00a0\u00a0Nicotine dependence2.10.51.90.65.60.34.41.04.180.26\u00a0\u00a0\u00a0\u00a0Any alcohol or drug use disorder8.41.67.90.910.72.06.21.22.840.43\u00a0\u00a0\u00a0\u00a0Any substance use disorder10.01.69.41.015.72.18.21.62.380.51Other disorders\u00a0\u00a0\u00a0\u00a0Non-affective psychosis1.10.41.40.30.80.20.00.08.450.06\u00a0\u00a0\u00a0\u00a0Somatoform disorder1.80.83.90.63.10.63.00.62.950.42Any CPPS disorder23.93.523.01.525.31.714.73.92.840.43FemaleAffective disorders\u00a0\u00a0\u00a0\u00a0Major depressive episode4.80.910.51.314.80.85.32.410.080.03\u00a0\u00a0\u00a0\u00a0Manic episode2.61.01.50.53.00.60.20.26.250.12\u00a0\u00a0\u00a0\u00a0Dysthmia4.31.46.92.38.81.73.93.26.920.09\u00a0\u00a0\u00a0\u00a0Any affective disorder9.61.815.22.220.00.98.25.35.720.15Anxiety disorders\u00a0\u00a0\u00a0\u00a0Panic disorder0.50.30.60.36.61.31.01.21.370.71\u00a0\u00a0\u00a0\u00a0Agoraphobia without panic11.93.08.42.410.40.33.41.05.780.14\u00a0\u00a0\u00a0\u00a0Generalized anxiety disorder1.40.83.91.02.20.62.70.73.240.37\u00a0\u00a0\u00a0\u00a0Any anxiety disorder17.32.313.02.517.51.26.31.75.900.14Substance use disorders\u00a0\u00a0\u00a0\u00a0Alcohol abuse0.50.41.00.50.90.20.60.50.930.82\u00a0\u00a0\u00a0\u00a0Alcohol dependence0.90.51.30.74.20.20.00.05.160.18\u00a0\u00a0\u00a0\u00a0Drug abuse0.00.00.20.20.50.10.00.02.480.49\u00a0\u00a0\u00a0\u00a0Drug dependence1.50.72.90.82.70.50.20.28.500.05\u00a0\u00a0\u00a0\u00a0Nicotine dependence2.00.72.10.88.50.42.00.81.710.64\u00a0\u00a0\u00a0\u00a0Any alcohol or drug use disorder2.91.04.50.85.10.40.80.77.980.06\u00a0\u00a0\u00a0\u00a0Any substance use disorder4.91.46.00.613.30.92.80.714.740.01Other disorders\u00a0\u00a0\u00a0\u00a0Non-affective psychosis1.50.71.20.51.60.30.00.07.050.09\u00a0\u00a0\u00a0\u00a0Somatoform disorder1.90.75.01.13.60.74.21.84.500.23Any CPPS disorder23.42.724.72.928.40.912.35.12.830.42MaleAffective disorders\u00a0\u00a0\u00a0\u00a0Major depressive episode3.41.14.71.15.53.65.00.41.300.73\u00a0\u00a0\u00a0\u00a0Manic episode1.10.90.70.30.50.61.81.60.680.88\u00a0\u00a0\u00a0\u00a0Dysthmia1.60.80.80.37.03.32.12.04.780.21\u00a0\u00a0\u00a0\u00a0Any affective disorder5.91.75.71.19.82.86.71.34.460.23Anxiety disorders\u00a0\u00a0\u00a0\u00a0Panic disorder0.60.40.20.21.60.40.00.03.070.39\u00a0\u00a0\u00a0\u00a0Agoraphobia without panic1.70.93.41.74.53.80.90.91.940.59\u00a0\u00a0\u00a0\u00a0Generalized anxiety disorder1.00.60.40.20.00.00.50.41.970.58\u00a0\u00a0\u00a0\u00a0Any anxiety disorder4.41.84.71.66.13.51.10.93.260.37Substance use disorders\u00a0\u00a0\u00a0\u00a0Alcohol abuse3.71.22.70.810.93.76.31.26.200.12\u00a0\u00a0\u00a0\u00a0Alcohol dependence9.72.88.81.83.11.05.90.93.880.29\u00a0\u00a0\u00a0\u00a0Drug abuse0.40.40.70.52.60.60.00.03.470.34\u00a0\u00a0\u00a0\u00a0Drug dependence1.51.20.80.50.90.20.60.80.330.95\u00a0\u00a0\u00a0\u00a0Nicotine dependence2.20.71.80.82.50.57.02.03.080.39\u00a0\u00a0\u00a0\u00a0Any alcohol or drug use disorder14.43.211.82.016.64.412.11.61.250.74\u00a0\u00a0\u00a0\u00a0Any substance use disorder15.43.213.32.118.24.014.12.81.070.78Other disorders\u00a0\u00a0\u00a0\u00a0Non-affective psychosis0.80.41.60.70.00.00.00.05.750.14\u00a0\u00a0\u00a0\u00a0Somatoform disorder1.61.22.61.02.60.51.80.82.050.57Any CPPS disorder21.85.320.52.522.03.017.33.41.120.77Non-affective psychosis includes schizophrenia, schizophreniform disorder, schizoaffective disorder, delusional disorder, and atypical psychosisAny CPSS disorder does not include nicotine dependence or cognitive disorder; \u03c72 df\u00a0=\u00a03Table\u00a04Mental health service utilization in the past 6-months by region among those with DSM-III-R 12-month prevalent disorderBio BioMetropolitanaTarapacaAraucaniaDisorders%SE%SE%SE%SE\u03c72PAny MH service17.61.121.12.525.90.312.01.910.090.03Non-specialized MH service15.21.017.11.824.30.410.71.612.630.02Specialized MH service5.20.96.71.35.61.13.41.14.260.26Substance service0.10.10.30.20.40.10.10.12.870.43\u03c72 df\u00a0=\u00a03, MH\u00a0=\u00a0Mental HealthNon-Specialized MH Services\u00a0=\u00a0primary care physicians; Specialized Mental Health Services\u00a0=\u00a0inpatient or outpatient services provided by a psychiatrist or a psychologist or formal substance abuse services; Substance abuse services included inpatient and outpatient detoxification or Alcoholics Anonymous\nUsing logistic regression controlling for socio-demographic variables the regional differences for major depression were maintained for both lifetime and 12-month prevalence, as well as among females in both prevalence periods. Males with lifetime prevalent affective disorders were also at increased risk in Tarapaca. In addition regional differences in lifetime prevalence for alcohol abuse, drug abuse, and nicotine dependence were found. Among females, regional differences in lifetime prevalence were noted for drug abuse, drug dependence, any alcohol or drug use disorder, and cognitive disorders, and among men alcohol abuse with increased risk among those residing in Tarapaca. The statistical differences in regional lifetime prevalence of non-affective psychosis for both genders combined, and females in both prevalence periods, persisted with the rates for Araucania remaining low. As the rates for not only major depression, but also alcohol and drug use disorders were elevated in Tarapaca, additional analyses were conducted controlling for comorbidity in the logistic regressions, the regional differences noted were not altered.\nDifferences in service utilization across the four regions were also found. Araucania had the lowest use of mental health services utilization (\u03c72\u00a0=\u00a00.03, df\u00a0=\u00a03, P\u00a0<\u00a00.03), in particular in the non-specialized health care sector (\u03c72\u00a0=\u00a012.63, df\u00a0=\u00a03, P\u00a0<\u00a00.02). The rates of service utilization by region are presented in Table\u00a04. The lower use of services persisted after controlling for socio-demographic variables in a logistic regression.\nWhen urban versus rural was examined across all sites no statistical differences in the rates of disorders were noted. In addition, there were no socio-demographic differences. The sample size of the rural population was small, 203.\nDiscussion\nRegional differences that persisted after adjusting for potential confounders persisted in the CPPS. Major depression and substance use disorders were highly prevalent in Tarapaca. The high rates of substance use disorders, especially drugs, and were not surprising as the region bordering Bolivia and Peru is heavily involved in the drug trade. The increased rates of major depression, however, could not be accounted for by substance use disorder comorbidity. The differences in rates for non-affective psychosis, although may simply be due to a type 1 error, are nonetheless surprising as the Araucania region\u2019s population and our sample has a sizable proportion of Mapuche indigenous people. The Mapuche in earlier psychiatric literature were thought to be at increased risk for psychosis [28]. The small sample size of the rural population precluded finding statistically significant differences.\nThe utilization of health service was lowest in Araucania and Bio Bio. This may be consistent with the inequities in availability and access that do exist in health and mental health resources across different regions of Chile. The southern half of the country is the poorest and has the least resources; therefore, the lower rates may be due to a lack of access rather than demand. For example across the regions the number of available mental health beds 2001 in the public health service per 100,000 were Araucania 2.2; Bio Bio 4.8; Metropolitana 34.2; and Tarapaca 47.2. The number of primary care physicians per 100,000 populations also was lowest in Araucania, 57.0, compared to Bio Bio with 169.7, Tarapaca with 61.6 and Metropolitana with 185.8.\nIt could be argued that these regional differences are simply artifact due to sampling differences. Clearly the population investigated in Tarapaca is small for a prevalence study and may have resulted in rates that may prove unstable. Another potential limitation is that the four regions were investigated sequentially, with the potential for socio-cultural influences to impact on the rates during the intervals between data collection. The high proportion of low-income individuals in the Bio Bio sample in comparison to the other sites and in particular Aracucania, the poorest region of the country, reflects the improved economic conditions in Chile during the course of data collection and supports a cohort effect. A cohort effect, however, is highly unlikely to explain the rates of psychopathology given that data collection was obtained from Araucania last, yet it has the highest rate of major depressive disorder.\nConclusion\nRegional differences across countries may exist that have both implications for prevalence rates and service utilization. Planning mental health services for population centers that span wide geographical areas based on studies conducted in a single region may be misleading, and may result in areas with high need being underserved. Psychiatric epidemiological studies that are nationally representative of developing nations are needed that have a sufficient sampling frame to examine populations believed to be at high risk and regions where increased inequities may exist. Even the most recent epidemiological studies representing Latin America [6, 29] have ignored large segments of the population, such as those countries and regions of countries with large indigenous populations or segments of the population that are very poor. Fewer studies in the region have examined service needs and none have addressed regional differences in services. Data that is more representative of the Latin American population is needed in order to improve mental health services planning and addresses the large under-estimated treatment gap.","keyphrases":["regional differences","psychiatric disorders","chile","community prevalences"],"prmu":["P","P","P","R"]}
{"id":"Support_Care_Cancer-3-1-2092409","title":"Temperament as a predictor of internalising and externalising problems in adolescent children of parents diagnosed with cancer\n","text":"Objective This study examined the relationship between temperament and internalising and externalising problems among children of parents diagnosed with cancer, beyond the effects of socio-demographics, illness-related variables and life events.\nIntroduction\nAll people are confronted with intense experiences during the course of their lifetime. Even children are not spared from them. Adolescent children, especially, are vulnerable to stressful events [1]. One stressful incident with which families with adolescent children are increasingly confronted is the diagnosis of cancer in one of the parents. Studies have shown that adolescent children in such situations, the daughters particularly, experience more emotional problems than their age peers do [2, 3]. Although the functioning of children of cancer patients has received increasing attention, research into risk and resilience factors is still in its infancy. Recent studies found evidence for the impact of parental characteristics [4], parent\u2013child communication [5] and family functioning [6] on child functioning. Increased attention is given to the different effect of parental cancer for boys and girls and children of different ages, but research into other child characteristics is lacking. Temperament is one of the factors that may have an important influence on how children cope with stressful events, and therefore why some children are more vulnerable to the development of problems than others [7]. The primary hypothesis of most models of temperament is that specific dimensions are related to prevalence of specific problems. Research into the role of temperament can provide insight into which children are at greater risk in such situations and which children are better equipped to handle the situation.\nThe developmental model of temperament as proposed by Rothbart and Derryberry is one of the few models that offers a measurement tool that is specifically designed for use with adolescents, and it was therefore used in the present study. Rothbart proposes that temperament is relatively stable, but that the expression of temperament can change as a result of maturation and (social) environmental influences, including life events [8, 9]. The model distinguishes between two concepts: reactivity and self-regulation. Reactivity is comprised of the physical and emotional differences that exist among individuals in reaction to stress. Children who have a high degree of reactivity are more easily upset and need more time to recover than other children do. The term \u201cself-regulating system\u201d refers to such processes as attention, activation and inhibition. These processes have an important influence on the regulation of emotions and behaviour. These children may be able to direct attention away from the stressor, such as parental cancer.\nTo our knowledge, no studies have reported on the effect of different temperament dimensions on the functioning of children of parents who have been diagnosed with cancer. The aim of the current study was to investigate the effects of temperament on prevalence of problems in these children, beyond the possible effects of socio-demographics (age, gender, educational level), illness-related variables (recurrent disease and time since diagnosis) and the number of negative life events that children experienced during the year before assessment.\nMaterials and methods\nProcedure\nBetween January 2001 and February 2003, written information on this study was offered to all cancer patients who were consecutively hospitalised or who visited the outpatient\u2019s clinic at the University Medical Centre Groningen by their physicians or oncology nurses. In addition, information was sent to patients and their family members who had contacted the researchers in response to media attention because they wished to participate in the study. Families were eligible if patients had been diagnosed between 1 to 5\u00a0years before study entry and if they had children between 4 to 18\u00a0years of age. Participants had to be fluent in Dutch. Patients discussed study participation with their partners (if present) and children. Informed consent was obtained according to the regulations of the Medical Ethical Committee of the University Medical Centre Groningen. After informed consent was received, questionnaires and prepaid return envelopes were provided.\nParticipants were guaranteed that answers were treated absolutely confident and will be described completely anonymous.\nParticipants\nThe current study is part of a larger study in which 476 families with children aged 4 to 18\u00a0years were approached, and information was mailed to 110 families who had contacted us for information about participation. Of these, 205 families from the first group and 89 families from the second group consented to participate (response: 43 and 81%, respectively). Ill parents who did not participate did not significantly differ from those who participated with respect to gender, tumour type and time since diagnosis.\nThe current study focused on the responses of children of 11\u00a0years of age and older, as they completed the questionnaires themselves. The sample for the present study consisted of 340 adolescent children (149 sons and 191 daughters), between the ages of 11 and 18\u00a0years (mean age=14.9\u00a0years, SD=2.3) and their 212 ill parents (80% mothers, mean age=45.4\u00a0years, SD=4.7). Twenty-one percent of the children were receiving education in primary school, 9% at the lower vocational level, 17% in lower general secondary education, 12% in intermediate vocational education, 38% in high school and 3% in higher vocational education or university. Nine percent of the children were from single-parent families. Parents (43 fathers; 169 mothers, mean age=45.4\u00a0years, SD=4.7) had been diagnosed with various types of cancer: breast (55%), haematological (9%), skin (9%), gynaecological (9%), urological (5%), bone tumours (4%), gastrointestinal (5%) or other cancers, such as cancer of the central nervous system or head and neck cancer (6%). The mean time since diagnosis was 2.6\u00a0years (SD=1.2). Twenty-nine percent of the parents had suffered relapses.\nChildren and parents approached in the hospital did not significantly differ in age or gender from children and parents who had volunteered for participation. Educational level of parents of the last-named group was significantly higher (t=5.8, p\u22640.001) than that of the first-named group, but this was not found for children\u2019s educational level. Furthermore, children and parents in both groups reported similar levels of internalising and externalising problems, and children did not differ significantly in temperament.\nMeasures\nTemperament Temperament was measured using the adolescent version of the Revised Early Adolescent Temperament Questionnaire (EATQ-R) of Rothbart and Derryberry [10\u201312]. The EATQ-R consists of 53 items and includes ten subscales that are designed to measure temperamental attention control, activation control, inhibitory control, high intensity pleasure, shyness, fear, frustration, affiliation, perceptual sensitivity and pleasure sensitivity. Answers were rated on a 5-point Likert-type scale (1 = \u201calmost always untrue\u201d to 5 = \u201calmost always true\u201d). Higher values represent a higher availability of the temperamental dimension concerned. The psychometric quality of the EATQ-R was reported to be sufficient among American adolescents with Cronbach\u2019s alpha\u2019s ranging from \u03b1=0.55 to \u03b1=0.78 [13]. In the present study, alpha scores ranged from \u03b1=0.36 to \u03b1=0.74, and mean inter-item correlations from r=0.11 to r=0.37. Five of the ten dimensions had mean inter-item correlations of <0.20. Therefore, factor analyses were carried out to study the extent to which the temperamental dimensions identified by Rothbart and colleagues emerged from the data of the present Dutch study (see preliminary analyses).\nInternalising and externalising problems The Youth Self-Report (YSR) and Child Behaviour Checklist (CBCL) were used to assess, respectively, adolescent children\u2019s self-reported and parent\u2019s reported behavioural and emotional problems in children [14\u201316]. The YSR\/CBCL consists of 102\/120 problem items with three response options (0 = not true, 1 = somewhat or sometimes true and 2 = very true or often true). Higher scores indicated more problems. The Internalising (TIS) and Externalising Behaviour Problem Scales (TES) were used in the present study. The internalising scale (32 items) consists of the syndrome subscales withdrawal, somatic complaints and anxiety\/depression. The externalising scale (30 items) consists of the syndrome subscales delinquent behaviour and aggressive behaviour. The YSR is one of the most commonly used questionnaires in adolescent research. Cronbach\u2019s alpha scores in the present study were high (\u03b1>0.80). The manual provides norm data based on a randomly selected Dutch sample of 560 adolescent boys and 564 adolescent girls.\nLife events The Questionnaire of Recently Experienced Events was used to ask children and parents about the number of life events experienced during the past year. This questionnaire is based on the Recent Life Change Questionnaire (RLCQ) developed by Rahe [17]. Questions measuring negative events (14 items; e.g., divorce, illness by other family members than the parent with cancer) were used.\nAnalyses\nFactor analyses of the EATQ-R were executed using Simultaneous Confirmatory Analysis (SCA) and Exploratory Principal Component Analysis (PCA) to investigate factor validity.\nChi-square and t tests were performed to compare children and parents who had been recruited in the hospital and those who had volunteered for participation on demographic characteristics and the problems reported.\nOne-sample t tests were performed to compare prevalence of internalising and externalising problems reported by children and parents diagnosed with cancer with those of the norm group.\nUnivariate statistics (t tests and pearson correlation analyses) were performed to investigate effects of study variables (age, gender, educational level, time since diagnosis, recurrence, number of negative life events, and temperament) and problems in children.\nHierarchical regression analyses were conducted to examine the contribution of temperament dimensions to prediction of internalising and externalising problems in children. Socio-demographics and illness-related variables (first step), and number of life events (second step) significantly related to temperament were entered into regression analyses to ensure that any effect found for temperament (third step) on children\u2019s problems would not be attributed to these variables. To examine whether multicollinearity exists between the independent variables, Pearson correlation analyses and Variance Inflation Factors (VIF) were performed. If the mean VIF is considerably larger than one and the largest VIF is greater than 10, multi-collinearity exists [18].\nThere is evidence that sons and daughters may respond differently to the cancer in the parent [19] and that they differ in temperament [13, 20]. Two-way-interaction terms (using standardised scores) were computed to examine whether the pattern of the relationship between temperament and prevalence of problems differ between sons and daughters. Only when the 2-way interaction accounted for a unique significant effect was it included in the model.\nOwing to the large number of comparisons in relation to the sample size significance was set at p\u22640.01.\nResults\nPreliminary analyses\nSimultaneous Confirmatory Analysis (SCA) was conducted to examine differences in the percentage of variance explained by the original structure and by the exploratory structure over the same number of factors. The difference in the variance explained by the original structure (41.0%) and by the exploratory structure (46.1%) was considered too large (>2% rule of thumb, [21]) to continue with the original structure. An exploratory Principal Component Analysis (PCA) was conducted using orthogonal rotation followed by varimax procedure. The number of constituting factors was determined based on the scree test. Items that loaded consistently low (<0.30) or on varying components were excluded. In addition, all items\u2014particularly those with loadings between 0.30 and 0.40\u2014were critically examined for the degree to which they formed a good reflection of the dimensions on which they loaded (face validity). Dimensions were assessed as consistent by an alpha above 0.60 and an inter-item correlation above 0.20. On the basis of these criteria, 17 items were excluded, including the entire inhibition-control (five items) and the affiliativeness scale (five items). Thirty-six items remained, distinguishing seven temperamental dimensions. The first dimension was called effortful control and consists of all five items from activation control and four of the six items from attention control. One of the attention control items was excluded and the other was added to the original four perceptual sensitivity items (\u201cI am good at keeping track of several things that are happening around me\u201d) that represent the second dimension. The third dimension is pleasure sensitivity; just one item did not meet the criteria and was excluded. The fear\/worry dimension forms the fourth dimensions. Three of the six items loaded on this scale. One of the other items was excluded, one was added to pleasure intensity (\u201cI get frightened when I ride with a person who likes to speed\u201d), and one was added to the original four items of the shyness scale (\u201cSome of the kids at school make me nervous...\u201d). Shyness was the fifth dimension. The sixth dimensions form the pleasure intensity. Three of the six items loaded low or were not consistent, and were therefore excluded. The seventh dimension forms frustration. One of the seven items did not meet the criteria and was excluded. The seven temperamental dimensions can be defined as follows. Effortful control measures the capacity to start and persist in an action and to focus attention. Pleasure sensitivity is the pleasure related to stimuli involving low intensity, complexity, novelty and incongruity. Perceptual sensitivity pointed to detection or perceptual awareness of slight, low-intensity stimulation in the environment. Pleasure intensity represents the pleasure derived from activities involving high intensity. Shyness is the behavioural inhibition to novelty and challenge, especially social. Frustration measures the negative effect related to interruption of ongoing tasks or goal blocking. Fear\/worry represents the worry for occurrence of unpleasant situations. The seven factors that were formed of remaining items explained 47.2% of the variation. Alpha coefficients from the other dimensions ranged from \u03b1=0.61 to \u03b1=0.79, and inter-item correlations ranged from r=0.21 to r=0.48 (Table\u00a01). \nTable\u00a01Cronbach\u2019s alpha EATQ-R dimensions of children in the current study and a control studyDimensionsCurrent studyControlCronbach\u2019s alphaMean inter-item correlationsCronbach\u2019s alphaMean inter-item correlationsNumber of itemsEffortful control0.750.260.740.259Pleasure sensitivity0.790.360.830.564Shyness0.690.300.630.255Frustration0.610.210.690.276Perceptual sensitivity0.620.250.700.315Pleasure intensity0.710.370.740.424Fear\/worry0.610.340.610.353\nCross validity The same research group conducted a prospective study among children of parents recently diagnosed (1\u201316\u00a0weeks ago) with cancer using a similar procedure in approaching families as was used in the current study. The adapted structure was tested among 144 adolescent children (54% daughters; mean age=14.2\u00a0years, SD=2.3\u00a0years) that participated in the prospective study (comparison group). Results of PCA among children in the prospective study were similar to those found in the current study. The internal consistency and mean inter-item correlations of the two groups were comparable (Table\u00a01). Results of the Principal Components Factor Analysis are available on request.\nInternalising and externalising problems in children\nT tests revealed no significant differences in prevalence of internalising and externalising problems between sons and norm group boys as reported by themselves and their ill parents. Daughters and their ill parents reported significantly more internalising problems than found in norm group girls (Table\u00a02). \nTable\u00a02Descriptive statistics of the YSR and one sample t tests for comparison of study and norm group\u00a0SonsNorm group boystDaughtersNorm group girlstMeanSDMeanSDMeanSDMeanSDInternalising problems YSR9.67.38.65.81.613.99.910.87.14.3*Externalising problems YSR11.46.211.56.7\u22120.210.86.210.06.11.7Internalising problems CBCL6.16.25.75.70.88.47.26.56.03.5*Externalising problems CBCL6.66.17.17.1\u22120.95.96.05.55.80.9*p<0.001\nChildren\u2019s problems and socio-demographics, illness-related variables and life events\nSocio-demographics Daughters experienced significantly more internalising problems than sons as was reported by ill parents and self-reports (t=2.9, p=.004; t=4.8, p\u22640.001). Neither children\u2019s age and educational level, nor parent\u2019s age, gender or educational level were significantly related to the prevalence of problems reported in children.\nIllness-related variables Parents and children reported more internalising problems in case of recurrent disease than in case of primary disease. Time since diagnosis was not significantly associated with reports of either internalising or externalising problems (even after controlling for recurrent disease).\nLife events The number of negative life events children or parents experienced during the preceding year was significantly positively correlated with internalising (r=0.36, p\u22640.001, r=0.25, p\u22640.001, respectively) and externalising problems (r=0.25, p\u22640.001) reported by or in children.\nChildren\u2019s temperament and socio-demographics, illness-related variables and life events\nSocio-demographics Sons had significantly higher mean scores on pleasure intensity (t=4.7, p\u22640.001) and significantly lower scores on shyness (t=\u22122.8, p=0.005) than daughters did. Age of children was significantly negatively related to effortful control (r=\u22120.18, p\u22640.001). No relationship was found between child\u2019s educational level and temperament.\nIllness-related variables No significant relationships were found between length of time since diagnosis and children\u2019s temperament. Children of parents who had recurrent disease differed significantly in fear\/worry from children of parents with primary disease (t=3.1, p=0.002).\nLife events Number of negative life events experienced was significantly positively correlated with pleasure sensitivity (r=0.15, p=0.007), perceptual sensitivity (r=0.15, p=0.005) and fear\/worry (r=0.30, p\u22640.001) and negatively with effortful control (r=\u22120.15, p=0.008).\nRelationships between temperament and problems in children\nAll temperament dimensions were significantly related to children\u2019s self-reported internalising problems. Shyness, pleasure intensity and fear\/worry were significantly related to internalising problems as reported by ill parents. Effortful control, frustration and fear\/worry were significantly associated with the prevalence of externalising problems in children as reported by children and parents (Table\u00a03). \nTable\u00a03Descriptive statistics of the temperament dimensions and correlations of these variables with internalising and externalising problemsTemperamentMeanSDChildrenIll parentsInternalisingExternalisingInternalisingExternalisingrrrrEffortful\u00a0control27.86.2\u22120.25**\u22120.44**\u22120.05\u22120.24**Pleasure sensitivity11.24.022**0.030.13\u22120.04Shyness12.03.80.30**0.000.19**\u22120.13Frustration18.43.80.29**0.36**0.100.14*Perceptual sensitivity16.43.50.15*0.07\u22120.01\u22120.09Pleasure intensity14.73.7\u22120.18**\u22120.02\u22120.19**0.10Fear\/worry7.42.70.46**0.29**0.23**0.16**p<0.01**p<0.001\nPredictors of internalising and externalising problems\nInternalising problems Child\u2019s gender and recurrent disease explained a significant percentage of the variance in the prevalence of internalising problems in child\u2019s (R2Ch=0.10) and parent\u2019s reports (R2Ch=0.08). Both variables appeared to have a significant negative independent effect. Life events accounted for a significant increment in explained variance in child\u2019s (R2Ch=0.10) and parent\u2019s reports (R2Ch=0.07). The temperament dimensions predicted a significant percentage of additional variance in children\u2019s (R2Ch=0.27) and parent\u2019s reports (R2Ch=0.04). Shyness, frustration, perceptual sensitivity and fear\/worry appeared to have a significant positive independent effect in children\u2019s reports. None of the variables appeared to have a significant independent effect in parent\u2019s reports. Interaction terms between temperament dimensions and child\u2019s gender failed to contribute significantly to the prediction of internalising problems in children and parent\u2019s reports. The final models accounted for 48 and 19% of the variance in child\u2019s and parents\u2019 models, respectively (Table\u00a04). VIFs ranged between 1.0\u20131.3, suggesting that there was no problem of collinearity. \nTable\u00a04Regression analyses examining temperament as a predictor of internalising problems reported by children an ill parents\u00a0ChildrenIll parentsInternalising problemsInternalising problemsBetaR2R2ChFChBetaR2R2ChFChStep 10.1017.1**0.0813.5**\u00a0Child\u2019s gender\u22120.25**\u22120.18**\u00a0Recurrent disease0.20**0.22**Step 20.200.1040.6**0.150.0724.4**\u00a0Negative life events0.33**0.26**Step 30.480.2722.8**0.190.045.3**\u00a0Effortful control\u22120.10\u2013\u00a0Pleasure sensitivity0.09\u2013\u00a0Shyness0.31**0.10\u00a0Frustration0.15**\u2013\u00a0Perceptual sensitivity0.12*\u2013\u00a0Pleasure intensity0.02\u22120.09\u00a0Fear\/worry0.25**0.13The dashes indicate that variables were not entered into the model because no significant univariate relationship was found.*p<0.01**p<0.001\nExternalising problems Life events accounted for a significant percentage of the explained variance in children\u2019s reports (R2=0.07). The temperament dimensions accounted for a significant increment in children\u2019s (R2Ch=0.22) and parent\u2019s reports (R2Ch=0.07). Effortful control had a significant unique negative effect for both children and parent, and frustration also for children\u2019s reported externalising problems. The interaction terms did not contribute significantly to the problems reported. The variables entered into children\u2019s and parents\u2019 models explained 29 and 7%, respectively, of the variance (Table\u00a05). VIFs in both models were acceptable, ranging between 1.1\u20131.2. \nTable\u00a05Regression analyses examining temperament as a predictor of externalising problems as reported by children and ill parents\u00a0ChildrenIll parentsExternalising problemsExternalising problemsBetaR2R2ChFChBetaR2R2ChFChStep 10722.6**\u00a0Negative life events0.26**\u2013Step 2290.2233.7**0.077.9**\u00a0Effortful control\u22120.33**\u22120.21**\u00a0Frustration0.23**0.06\u00a0Fear\/worry0.100.10The dash indicates that the variable was not entered into the model because no significant univariate relationship was found.*p<0.01**p<0.001\nDiscussion and conclusion\nThe present study is the first to examine the contribution of temperament to prevalence of problems among adolescent children of parents diagnosed with cancer. The findings suggest that temperament predicted internalising and externalising problems, beyond the effects of socio-demographics (child\u2019 gender), illness-related variables (recurrent disease) and number of negative life-events. The most powerful temperament dimensions for internalising problems were shyness and fear\/worry (reactive factor). To a lesser extent, frustration and perceptual sensitivity (children\u2019s reports only) heighten the risk for internalising problems. It was argued that shy and anxious children generally have a tendency to withdraw and are hesitant to seek support from their surroundings [22], whereas seeking support might be important, especially in situations in which a parent had cancer. High levels of frustration are related to reduced ability to regulate attention and emotions, whereby children are less able to relax and direct themselves toward matters other than the stressor [23]. Withdrawn behaviour, anxiety and depression might be consequences of these tendencies. Internalising problems reported by the child also increase when they were highly sensitive to things and people around them (perceptual sensitivity). Although, Rothbart and Bates (1998) hypothesised that children with this kind of sensitivity are more vulnerable for internalising and externalising problems, research on these dimensions is lacking [9]. The relationships between shyness, anxiety and frustration and internalising problems found in children reports have also been found in studies among the general population [9, 24\u201326].\nThose who have a temperament characterised by a low level of effortful control (regulative factor) and a high level of frustration are more at risk to develop externalising problems. The relationship between effortful control and externalising problems has been found in other studies as well [9, 11, 26\u201329]. Children who have more control show more initiative in undertaking activities, have more ability to shift their attention and to focus and are less easily distracted by circumstances [27]. The control that children have over their behaviour in this regard decreases the chance of externalising problems. As mentioned above, frustration decreases emotional regulation and can lead to externalising problems, in addition to manifesting itself in internalising problems [25]. No effect was found for pleasure intensity, which was in contrast to the results of a recent study among children in the general population. That study found that children who scored high on pleasure intensity experienced more externalising problems [26].\nWhether the relationship between temperament and problems is specific for situations in which children are exposed to a parent with cancer, is not clear. It is argued that reactive and regulative temperament factors really are important when the child experienced stressful events [30]. A stressful event, such as illness in the parent brings forth negative emotions, specifically in children high in emotionality. Children with low levels of effortful control may have difficulties to deal with these emotions, and may develop as a consequence emotional or behavioural problems [30].\nAlthough the sons and daughters differed in the problems experienced and in their temperament, the relationship between temperament and problems were similar for both genders, as was found in a previous study [26].\nSimilar patterns were found for parent\u2019s and self reported problems and temperament in the current study, according to a previous study [26]. Our results showed, however, a stronger relationship between temperament and the problems reported by children than parents\u2019 reports of problems, whilst Oldehinkel and colleagues found the opposite. This inconsistency may be attributed to the informant; Oldehinkel and colleagues used parents\u2019 reports to examine temperament, whereas the current study used self-reports from children. A number of studies found that parents and children perceived the level of problems of children under these circumstances different [19, 31, 48]. The differences in perception may have caused also the differences between parents and children found in the present study.\nAnother interesting result from this study is that, four of the seven temperament dimensions were related to the number of negative life events experienced. This is consistent with Rothbert\u2019s theory, which suggests that, despite its biological base, temperament is influenced by experiences. The influence of stressful environmental factors on the development of temperament is an interesting phenomenon. Because the current study uses a cross-sectional design, no causal statements can be derived from the results.\nThe current study is one of few to use self-reports of temperament from a large number of children to examine the relationship between temperament and internalising and externalising problems. It is generally assumed that self-description is an important source of information in the field of personality research. Nonetheless, it was faced with some difficulties. First, Rothbart and colleagues paid a lot of attention to the development of the theory around temperament. The empirical implementation of the EATQ-R, however, was limited. The original structure of the EATQ-R seemed not applicable for the Dutch children in the current study. A recent study among Dutch adolescents reported also some problems with the self-reported version of the EATQ-R [26] and used, therefore, the parent version. Furthermore, the comparison of results from the current study with those of studies that used other theories of temperament was difficult, due to differences in conceptualizing and in labelling with regard to corresponding dimensions. More research using reliable, validated instruments to investigate the temperament of children is necessary. Second, some of the items used to measure temperament resemble items on the problem scale and can cause item-overlap. Previous studies show that the association between constructs remains essentially the same when correcting for possible overlapping items [26, 32, 33] indicating that they are separate concepts. To examine whether patterns were similar among different informants, parent\u2019s reports were used also. Fourth, the current study is cross-sectional. Longitudinal designs may provide more insight into the causality of relationships. Fifth, most of the relations between temperament and problems found in the current study were similar to those found in other studies. The use of a control group may have provided more detailed information about whether the relationships between temperament and problems differed for children of parents with cancer and those of children in the general population. Finally, the current study did not pay attention to the interaction between children\u2019s temperament and other potential predictors, such as parenting, the parent\u2013child relationship and coping. Thomas and Chess introduced the \u201cgoodness-of-fit\u201d concept, which means that problems in children arise only when temperament and the expectations of the surroundings are not well adjusted to each other [34].\nThe fact that temperament of children can have an impact on the prevention of problems by children is important information. Health care providers can use this knowledge to assist parents to take the individual characteristics of the child into account and by means of this to understand their children\u2019s behaviour better. Additionally, parents might be supported to improve the \u2018fit\u2019 between the temperament of the child and the consequences of having a parent with cancer.","keyphrases":["temperament","internalising and externalising problems","adolescents","life events","parental cancer"],"prmu":["P","P","P","P","P"]}
{"id":"Skeletal_Radiol-3-1-2141652","title":"Ultrasound of the small joints of the hands and feet: current status\n","text":"The aim of this article was to review the current status of ultrasound imaging of patients with rheumatological disorders of the hands and feet. Ultrasound machines with high-resolution surface probes are readily available in most radiology departments and can be used to address important clinical questions posed by the rheumatologist and sports and rehabilitation physician. There is increasing evidence that ultrasound detects synovitis that is silent to clinical examination. Detection and classification of synovitis and the early detection of bone erosions are important in clinical decision making. Ultrasound has many advantages over other imaging techniques with which it is compared, particularly magnetic resonance. The ability to carry out a rapid assessment of many widely spaced joints, coupled with clinical correlation, the ability to move and stress musculoskeletal structures and the use of ultrasound to guide therapy accurately are principal amongst these. The use of colour flow Doppler studies provides a measure of neovascularisation within the synovial lining of joints and tendons, and within tendons themselves, that is not available with other imaging techniques. Disadvantages compared to MRI include small field of view, poor image presentation, and difficulty in demonstrating cartilage and deep joints in their entirety. Contrast-enhanced magnetic resonance provides a better measure of capillary permeability and extracellular fluid than does ultrasound. The ability to image simultaneously multiple small joints in the hands and feet and their enhancement characteristics cannot be matched with ultrasound, though future developments in 3-D ultrasound may narrow this gap. Magnetic resonance provides a more uniform and reproducible image for long-term follow-up studies.\nIntroduction\nThe small joints of the hands and feet play a central role in the diagnosis and classification of arthropathy. Ultrasound can be used to assess involvement in areas that are clinically occult as well as determine the precise structures involved. Whilst a systematic approach should include a full examination of extra-articular structures, including skin, subcutaneous tissue, the tendon and tendon sheath, the enthesis and associated bursae, this article focuses on intra-articular components of disease, specifically the early detection and classification of effusion, synovitis and erosions. In the patient with arthritis, serial examination can assess current activity and disease distribution, as well as monitoring progression or therapeutic response.\nTechnique\nThe hands are best examined with the patient and examiner seated. The examination table is adjusted to a comfortable height for both, taking account of coexistent shoulder and elbow disease. The examiner sits at 90\u00b0 to the patient, giving easy access to both the patient and the ultrasound controls. Good contact and near field resolution require liberal quantities of contact jelly, preferably warmed to body temperature (Fig.\u00a01). Many gel stand-off pads are rather awkward and can limit access to the lateral recesses of small joints; soft pads work best. Some operators like to immerse the patient\u2019s hand in warm water; if this method is used, it is preferable to leave the water to stand for some while so that all air bubbles are dissipated.\nFig.\u00a01Position for examination of the hands. The probe is in position for a sagittal examination of the metacarpo-phalangeal joint (MCPJ) on the extensor (a) and flexor (b) sides. The probe is held between thumb and forefinger, with the ulnar border of the hand used as support (arrow). Note the liberal quantity of gel used to provide some stand-off (arrowhead). c Position for examination of radial aspect of second MCPJ. d Probe position to examine the collateral ligaments and lateral compartments of the interphalangeal joints\nHigh-frequency linear-array probes are mandatory, probes with operating frequencies of 10\u00a0MHz or more providing the best images. The probe should be held lightly between thumb and forefinger (Fig.\u00a01), and the little finger allowed to rest on the table or patient to reduce contact pressure. Excessive probe pressure can obliterate small quantities of fluid, reduce the sensitivity for detection of blood flow and may obscure synovitis. Sagittal images are the mainstay for diagnosis, with axial [metacarpo-phalangeal joint (MCPJ)] and coronal [proximal interphalangeal joint (PIPJ)] images used in support. The superficial structures, including skin, subcutaneous tissue tendon and tendon sheath, should be assessed prior to the joints themselves. Within the joint, the capsule, extra-synovial connective tissue structures, synovium and visible cartilage are examined in turn. The joint should then be moved gently, as it is only during movement that some of the interfaces between the normal structures become sharply defined. Movement may also facilitate detection of low-volume synovial thickening, which bunches up in the proximal extensor recess on flexion.\nA comprehensive examination of the small joints of the hands and feet can be time-consuming; however, efficiency can be improved without compromising diagnostic impact, by omitting joints that are commonly negative or misleading. The first ray of both hands and feet often contain effusions, synovial thickening and osteophyte formation in the asymptomatic population. Clinically occult synovial thickening is found more commonly in the PIPJs than in the distal interphalangeal joints (DIPJs) of patients with rheumatoid arthritis (RA) [1]. Unless specifically symptomatic, the DIP joints are not routinely examined in patients with RA, but are included in patients with osteoarthritis or seronegative arthritis or where the diagnosis is unknown, in which cases the tendon insertions should also be included to look for enthesopathy.\nFor a routine rheumatological examination of the hand, the author examines the second to fifth rays. The first ray is included only in specific clinical situations (Fig.\u00a02). The extensor side of the metacarpo-phalangeal joints are examined first, followed by the extensor, ulnar and, in particular, the radial aspects of the IPJs, as indicated (Fig.\u00a01). The dorsal aspects of the wrist and extensor tendons are studied before turning to the palmar side. The flexor aspect of the metacarpophalangeal and proximal interphalangeal joints are examined, with particular attention paid to those that have been normal on the extensor side.\nFig.\u00a02Iatrogenic synovtis (arrowheads) of the carpo-metacarpal joint due to silastic implant (arrow)\nOther authors debate whether both the flexor and extensor sides need to be examined. Scheel and co-authors found that, in most cases, synovitis was detected in the palmar and proximal sites of the MCP and PIP joints, with only 14% of affected joints not showing synovitis in these locations [1] Because of this preponderance, they propose that the examination be streamlined to include only the palmar side. Not all authors agree, and other published work suggests that a significant proportion of synovitis would be overlooked if the examination were to be limited to either one or the other. Ostergaard and Szkudlarek found that only a third of patients had synovitis on both palmar and dorsal aspect of the PIPJ. In the majority, synovitis was limited to one or other compartment, with 43% limited to the palmar [2] and 27% to the extensor sides. The prevalence is reversed in the metacarpo-phalangeal joints; 80% of synovial thickening will be detected on the extensor aspect. The distribution of synovitis within MCPJs has also received attention [1\u20133]. Tan et al. used contrast-enhanced MRI and divided the volume of synovitis surrounding each MCP joint into eight sections. They detected a predilection for radial-sided synovitis in the second and third MCPJs, with equal distribution in both sides in the fourth and fifth MCPJs. The distribution on the dorsal versus palmar sides was not published. Hau and colleagues also found a predilection for radial-sided synovitis in the PIPJs [4]. Scheel et al. tested various combinations of joints to determine the most efficient method of providing an overall synovitis score. Synovitis and effusion were not differentiated. Of the various joint combinations tried, counting the medial four MCPJs only was least sensitive, counting the flexor side of the MCP and PIPJs of fingers 2-4 was most sensitive. Whilst this may work well for an overall synovitis score, omitting the fifth ray may reduce the sensitivity for detecting erosions, as the fifth MCPJ and fifth MTPJ are often involved early.\nNormal anatomy\nMetacarpo-phalangeal joints\nA sagittal image of the palmar aspect of the metacarpo-phalangeal joint is shown in Fig.\u00a03. Note the presence of a layer of contact gel between the probe and the underlying skin (Fig.\u00a03b). This provides good resolution of skin and subcutaneous tissues, as well as reducing the amount of probe pressure, which can interfere with the assessment of subtle joint effusion and blood flow. Abnormalities in the superficial layers include increased thickness in psoriatic arthritis and calcification.\nFig.\u00a03a Long axis (sagittal) view of the flexor aspect of the metacarpo-phalangeal joint. The flexor tendons (asterisks) are lying on the anterior aspect of the joint capsule (arrowheads) held in place by the cribriform (c) pulley (open arrowhead). The volar plate (arrow) and articular cartilage of the metacarpal head (open arrow) are visible. Extensor aspect in full extension (b) and flexion (c). Flexing the MCPJ compresses the connective tissue in the proximal recess (between arrowheads) and may make subtle synovial thickening more conspicuous. MC metacarpal, PP proximal phalanx\nDeep to this is the flexor tendon in its tendon sheath reinforced by the cruciform pulley. The superficial and deep flexor tendons should be independently identified as they pass over the metacarpo-phalangeal joints into the flexor tendon sheath of the fingers. Differences in tendon excursion on finger movement allows the two tendons to be separated easily. The tendon should pass in close proximity to the proximal phalanges where they are held in position by the annular (A2) pulley. The pulleys can be seen as thin poorly reflective linear structures (Fig.\u00a03). Small fluid collections are often seen in relation to them, which are not usually clinically significant.\nThe joint capsule on the flexor side is reinforced by several connective tissue structures, which can be identified on ultrasound. The collateral ligament runs obliquely from posterolateral to anterolateral and is best appreciated on coronal images (Fig.\u00a01). The accessory collateral ligament has its origin on the head of the proximal phalanx, between the collateral ligament and volar plate, with an insertion on the volar plate itself (Fig.\u00a04). The volar plate is a centrally positioned fibrocartilagenous structure that has a broad-based attachment to the base of the proximal phalanx (Fig.\u00a03). It inserts by two slips onto the neck of the adjacent metacarpal. These are called the check-rein ligaments. Within the joint, the proximal recess on the flexor aspect of the metacarpo-phalangeal joints is identified. Overlying this is the capsule of the MCP joint, which inserts on the adjacent metacarpal neck some distance from the joint surface. Hyporeflective articular cartilage is identified deep to the volar plate on the metacarpal head.\nFig.\u00a04Coronal image of the second metacarpo-phalangeal joint, showing the radial collateral ligament (arrow). MC metacarpal, PP proximal phalanx\nThe proximal recess is the area between the volar aspect of the metacarpal neck and the joint capsule, which abuts the deep surface of the overlying tendon. It is filled with intra-capsular but extra-synovial fat, which normally keeps the two layers of synovium closely approximated to one another. This layer of fat is very much more prominent in the proximal recess on the extensor aspect of the joint, which can extend up to 2\u00a0cm. from the joint level. This is to allow for finger flexion [5]. The distal recess is much smaller, as the extensor tendon conforms to the shape of the proximal phalanx.\nAbsolute measurements of normal joints have not been universally agreed, largely because different workers have used different joints, different parts of joints and different anatomical structures within the joint to define normal dimensions. The variation in the normal was demonstrated by Scheel and co-authors, who looked at the consensus between ultrasonologists involved in training programmes for the European League against Rheumatism (EULAR) [6]. Many of the differences in interpretation were the consequence of failure to appreciate normal findings. Schmidt et al. proposed a standard measurement, using the distance between the sub-chondral cortex on the head of the metacarpal and the volar plate in the second MCPJ. A mean of 1\u00a0mm was determined in a group of 102 asymptomatic volunteers [7], but variation was found to be quite wide. The extensor compartment was not examined, though this area is commonly screened for synovitis [8]. Furthermore, this measurement does not include the proximal recess, where early and prominent synovial thickening may occur. There are also differences between the proximal and distal recesses and some differences between radial and ulna sides, which cannot be encompassed by this single midline measurement.\nFor these reasons, others use the point of maximal joint distension and compare it with the normal measurement for that location. This, however, varies with the area of the joint being examined, but most authors agree that an increase in joint dimension of more than 1\u00a0mm above normal is sufficient to suggest abnormality. On the extensor aspect of the MCPJs, the proximal recess can be measured from bone to the deep surface of the extensor tendon, a distance of approximately 2.5\u00a0mm. The distal recess is much smaller, measuring less than 1.5\u00a0mm. In the transverse plane, the synovial space is limited on its lateral and medial sides, proximally by the extensor hood and distally by the extensor slips. There should be no posterior bulging of the hood or slips; this finding would suggest focal synovial disease. On the flexor side, the proximal recess is more distended and easier to visualise than the distal recess. The proximal recess measures approximately 3\u00a0mm from bone to the deep surface of the tendon. This space includes fat, capsule and two synovial layers, so the space may be increased by hypertrophy of any of these. On high-resolution equipment, the joint cavity itself can be identified between surrounding fat and connective tissue. Occasionally, a small quantity of fluid can be identified, separating the two synovial layers. With increasing practice, ultrasonologists quickly gain an appreciation of the range of normal for the small joints of the hands and feet.\nThe bony contours of the metacarpal head and proximal phalanx should be smooth, though it is not uncommon to identify a normal indentation on the dorsal aspect of the metacarpal head. This depression is smooth, well demarcated and has reflective bone at its base. It occurs at the site of the fused growth plate and is not associated with overlying synovial thickening. These features allow differentiation from a metacarpal head erosion.\nInterphalangeal joints\nThe flexor side anatomy of the proximal interphalangeal joints is similar to that of the metacarpophalangeal joints. The volar capsule is reinforced by similar connective tissue structures. A centrally positioned volar plate has a broad-based attachment to the base of the middle phalanx. It also inserts by two slips onto the neck of the adjacent proximal phalanx. On either side of this lie two collateral ligaments. The accessory collateral ligament has its origin on the head of the proximal phalanx between the collateral ligament and volar plate. The capsule and the flexor tendons lie superficial to these. The medial and lateral extensor tendon slips of the superficial flexor tendon can be followed as they insert just distal to the PIPJ (Fig.\u00a05). The deep flexor tendon can then be followed to its insertion on the base of the distal phalanx. In most cases, it is possible to identify a small quantity of fluid surrounding the tendon, seen as a poorly reflective halo. The thickness of this halo varies from person to person, and comparison with other tendons is helpful, assuming they are uninvolved. Another useful tip is that normal fluid surrounding the tendon is continuous on a longitudinal image, and there is no increase in vascularity, other than at the mesotenon.\nFig.\u00a05Long-axis parasagittal view of the proximal interphalangeal joint showing the insertion slips of the superficial flexor tendon (arrow), just distal to the proximal interphalangeal joint (asterisk).  PP proximal phalanx, MP middle phalanx\nLoose adipose tissue, which surrounds the synovial membrane, lies between flexor tendon and bone. Care must be taken not to misdiagnose this fat as synovial thickening, particularly when its reflectivity is artefactually reduced. On high-resolution equipment, the proximal recess on the flexor side can be differentiated from surrounding fat and connective tissue. The sagittal images are augmented by coronal views, which provide the best depiction of the medial and lateral joint recesses and collateral ligaments. The coronal images are obtained by asking the patient to hyperextend the MCP of the finger being examined (Fig.\u00a01d). Particular attention should be paid to the radial aspect of the joint, where synovial hypertrophy and erosions predominate.\nFor the PIPJ, measurements can be obtained from the flexor, extensor, radial and ulnar sides. On the extensor side, the distance between the proximal phalanx, at the junction with the head, and the deep surface of the extensor tendon is 1\u00a0mm in the central sagittal plane. On the flexor side the same measurement is 2\u00a0mm, with most of the space taken up by intra-capsular but extra-synovial fat. A small quantity of fluid is commonly encountered in this recess (Fig.\u00a06), and slight movement of the joint can make this more conspicuous. Under normal circumstances, the quantity of fluid should not be thicker than the joint capsule and should not extend out of the recess in which it is contained.\nFig.\u00a06Long-axis view of the second proximal interphalangeal joint flexor aspect. A small quantity of fluid can be present in the normal joint (arrow). The distal end of the A2 pulley is just visible (open arrow). The fluid is transonic, and the Doppler field shows no flow. PP proximal phalanx, MP middle phalanx, R2P annotation on image for right second PIPJ\nMeasuring the joint space on the radial and ulnar side is difficult. This is due to the variable appearances of the ulnar and radial collateral ligaments due to anisotropy. For this reason it is often best to measure from the bone surface to the outer aspect of the collateral ligament. Under normal circumstances this distance is less than 2.5\u00a0mm.\nThe wrist joint is often included in rheumatological examination of the hand, although a full description is outside the scope of this review. The joint is best appreciated from its dorsal aspect, where a small amount of fluid is frequently identified in the radiocarpal joint. Because of the undulating surface of the first carpal row there is normally a variation in the bone-to-capsule distance. To provide some standardisation, measurements can be obtained using the deep surface of the extensor tendons or bony structures as landmarks. The radius, lunate and capitate axis are particularly easy to identify (Fig.\u00a07). Normal ranges have not been established by large population studies; however, the author uses a guide measurement of 7\u00a0mm. It is emphasised that for larger joints, such as the wrist joint, measurements should only be used as a loose guideline, as normal variation is wide and synovial proliferation may be focal. Once again, comparison with the contralateral side may be of value in unilateral disease.\nFig.\u00a07Sagittal image of the normal wrist joint. Standard sections obtained along the third extensor tendon (asterisks) in the axis of the lunate (arrow) and capitate (arrowheads)\nThe small joints of the forefoot\nTechnique The metatarso-phalangeal joints and proximal interphalangeal joints of the toes are best examined from the extensor aspect. The big toe is usually not included in the general rheumatological screen, as effusion and bone irregularity are frequently encountered in the asymptomatic population.\nNormal anatomy On the dorsal aspect of the metatarso-phalangeal joint, extensor digitorum longus and brevis lie superficial to the joint. The extensor digitorum brevis tendon divides into four slips, one to each toe, which run alongside the extensor digitorum longus tendon before inserting into it. The longus tendon itself inserts into the dorsal aspects of the middle and distal phalanges, via the dorsal digital expansions. The dorsal digital expansions have three slips, one central and two collateral. The central slip inserts into the base of the middle phalanx. The collateral slips pass on either side of the inserting central slip and receive reinforcement from the interosseus muscles, the extensor digitorum brevis and the lumbricals. Deep to the extensor tendons, the metatarso-phalangeal joints are reinforced by collateral ligaments that run from dorsal to plantar, crossing the radial and ulnar aspects of the joint. These are difficult to identify individually.Flexor digitorum longus and brevis tendons lie on the plantar aspect of the forefoot. The flexor digitorum brevis divides into four tendons, one to each of the lateral four toes, and each of these in turn splits into two at the level of the base of the proximal phalanx. The two divisions reunite before dividing again to attach on either side of the middle phalanx. The first division of this tendon is to allow the deeper flexor digitorum longus to pass distally. The tendon sheaths are fibrous tunnels, reinforced by annular and cribriform condensations mimicking the pulleys of the fingers. Running between the metatarso-phalangeal joints are fibrous condensations termed transverse metatarsal bands. Taken as a unit, these are often referred to as the deep transverse metatarsal ligament. Lying directly on the plantar aspect of the joint capsule are the plantar plates (Fig.\u00a08), which are similar to the volar plates of the upper limb. On their deep surface, these blend with the joint capsule. The plantar plates are firmly attached distally to the base of the proximal phalanges. Proximally, there is a rather loose attachment just proximal to the articular cartilage of the head of the metatarsal.\nFig.\u00a08Long-axis (sagittal) view of the plantar aspect of the metatarso-phalangeal joint. The extensor tendon (asterisk) runs over the plantar plate (arrow). MT metatarsal head, PP proximal phalanx\nUltrasound pathology\nIntra-articular pathology\nEffusion and synovitis There is an increasing trend for early and more aggressive treatment of synovitis with disease-modifying anti-rheumatic drugs (DMARDs), which may demonstrate benefit when prescribed even within weeks to months of the onset of disease [9]. The emphasis in early disease detection has now moved from the detection of erosion to the earlier detection of synovitis, and erosions are now generally regarded as being a stage too late. The earliest detectable abnormality within the small joints of the hands and feet is effusion.\nDefinitions Simple effusion should be completely transonic, compressible, and with no increase in Doppler signal (Fig.\u00a09). Synovial hypertrophy is defined as non-displaceable, intra-articular, poorly compressible tissue, which may exhibit Doppler signal (Fig.\u00a010). Dynamic examination using the probe to compress the joint will cause fluid to be displaced away from the probe, whereas thickened synovium will be much less compressible. The most important pitfall is that normal anatomical structures may have low reflectivity and mimic synovitis if careful attention is not paid to technique, particularly with lower resolution equipment. In particularly, anisotropy of adjacent capsule ligaments and tendons should be avoided.\nFig.\u00a09Small effusion on the flexor aspect of the proximal interphalangeal joint. Fluid thickness is measured at 1.6\u00a0mm and extends out of the proximal recess (arrowheads). Compare with Fig.\u00a06. PP proximal phalanx, MP middle phalanxFig.\u00a010Moderate synovitis and bloodflow. Compare synovial thickening (arrowheads) with effusion (asterisk). Synovial thickening is hyper-reflective, non-compressible and demonstrates increased vascularity when compared with free fluid\nClassifying abnormal Synovial hypertrophy has been measured in a number of ways. Szuldarek categorised changes in synovial thickness by comparing it to bony structures. Grade\u00a01 is minimal synovial thickening (considered normal), grade\u00a02, synovial thickening bulging over the line linking the tops of the bones forming the joint without extension along the bone diaphyses, grade\u00a03, with extension to one of the metadiaphyses and grade\u00a04, extension to both metadiaphyses.The author classifies joint disease using semi-quantitative measurements of synovial thickness, vascularity and association with erosions. Synovial thickness is recorded on a 3-point scale (1\u20133\u2009=\u2009mild, moderate and severe), with moderate synovial thickening between 2\u00a0mm and 4\u00a0mm above normal. If desired, mild grades of synovitis can be further classified into focal and diffuse, with focal involvement limited to one recess. Moderate and severe synovial thickening is less often focal. This sub-classification may be of value in monitoring more subtle changes, compared with the more two-dimensional classification. A 3-point scale is also used to record blood flow: mild is defined as a few scattered vessels only (Fig.\u00a011), moderate as less than 50% vascularity in the synovium (Fig.\u00a012) and severe as more than 50% (Fig.\u00a013). Finally, 0 and 1 are used to denote the presence or absence of erosions, though where necessary for record or research purposes, a more detailed description of the proportion of bone involvement by erosions can be used. The score is recorded on the ultrasound image, together with annotation of the joint being measured. In summary, the second right MCPJ, with 3\u00a0mm of synovial thickening, diffusely involving the joint, with marked increased in blood flow and without erosions, would be classified as R3M 230. Images are printed as hard copy or stored on the department\u2019s picture-archiving communication system (PACS) for future comparison and to monitor the patient\u2019s progress.\nFig.\u00a011Sagittal image of the extensor aspect of an inflamed metacarpo-phalangeal joint. Mild hypervascularity with a few scattered vessels visible in the moderately thickened synovium. MC metacarpal head, PP proximal phalanxFig.\u00a012Sagittal image of the extensor aspect of an inflamed metacarpophalangeal joint (asterisk). Detectable blood flow on power Doppler is seen in less than 50% of the severely thickened synovium. MC metacarpal head, PP proximal phalanxFig.\u00a013Sagittal image of the extensor aspect of an inflamed metacarpo-phalangeal joint (asterisk). Detectable blood flow on power Doppler is seen in more than 50% of the moderately thickened synovium. Note the apparent defect in the metacarpal head (arrow). This is a normal finding and should not be missed or diagnosed as an erosion. Note the well-demarcated floor and lack of through sound transmissionSeveral studies have shown good inter- and intra-observer reliability of ultrasound classification. The classification by Szkudlarek et al. has been most tested and shown to have good inter-observer agreement. An inter-observer correlation coefficient ( ICC) of 0.61 was determined in a review of 150 small joints of the hand in 30 patients [10]. Naredo and co-workers coordinated a study of 22 ultrasonologists and 28 patients. For the detection of effusion and synovitis in the small joints of the hands and wrists, a mean kappa value of 0.61 was calculated [6]. In a study of 204 hands in normal subjects, Schmidt et al. found a reliability of 0.96 in repeat examinations of the same test subject [7]. Ultrasonography (US) classifications have also been shown to correlate reasonably well with findings on MRI in patients with synovitis, but there have been no studies of the variation that may be found in individuals with normal joints.\nAngiogenesis\nHigh-frequency US can differentiate solid from fluid elements within an enlarged joint space but is less effective at distinguishing inflamed synovium from inactive pannus, fibrous tissue and joint debris. One method of making this differentiation is to use Doppler colour flow, though there has been some variation in the reported findings in individuals with normal joints. Klauser et al. [11] found that healthy joints showed no intra-articular vascularisation, either before or after the administration of ultrasound contrast medium, while Terslev and colleagues [12] found vessels in the synovium of the MCPJs of 11 healthy subjects, in 18% before and 50% after administration of contrast agent. Different ultrasound equipment was used, with small differences in probe and Doppler frequencies. Though there is no method of determining for certain whether these differences account for the variation in findings, it is well recognised that sensitivities of ultrasound equipment from different manufacturers vary considerably, and the identification of the occasional blood vessel should probably not be regarded as abnormal when sensitive equipment is being used.\nResponse to inflammatory changes within the synovium results in the secretion of a large number of inflammatory mediators. Vascular endothelium growth factor (VEGF) is one of the pro-hormones secreted and results in proliferation of the vascular endothelium to form new vessels, a process termed angioneogenesis. Histological studies confirm that power Doppler changes correlate quite well with pathological changes in the synovium [13, 14], although great care should be taken to avoid interface artefact, which can be present at the bone synovium interface if gain settings are too high. Increased Doppler signal correlates especially with polymorphonuclear leucocyte infiltration and surface fibrin deposition [15], though, as yet, no direct correlation between serum VEGF measurement and neovascularisation has been identified.\nColour flow assessment should, therefore, accompany all ultrasound examinations of the swollen joint. The pulse repetition frequency should be kept low, and the region of interest should also be kept as small as possible, to maximise the detection of abnormal blood flow within the synovium. False positive readings can occur, due to patient or operator movement or at highly reflective interfaces, such as between bone and soft tissue (Fig.\u00a014).\nFig.\u00a014Static image from video-loop. Apparent Doppler signal from the bone surface of the proximal interphalangeal joint (arrow) in the patient with mild synovial thickening (asterisk). When colour flow settings are set to high sensitivity, flash artefacts can be identified where there are strong reflecting interfaces such as the bone surface. PP proximal phalanx, MP middle phalanx\nThere are two ways of measuring synovial blood flow, power Doppler ultrasonography (PDUS) and colour flow Doppler ultrasonography (CFDUS). Power Doppler is more sensitive but also more prone to artefact. It is sensitive to intravenously administered contrast agents which can be used to augment very slow rates of flow. The software that measures power Doppler is proprietary; therefore, results from one manufacturer may not be comparable with another. There is also a concern that serial studies on the same equipment may be incomparable following software or platform upgrades.\nUnlike CFDUS, PDUS does not carry specific directional information and will therefore register any flow within the region of interest. Although more recent advances in PDUS mean that some directional information can be imparted in the colour read out, quantification is still based on measuring the amount of colour pixels within the image, rather than an absolute measure of blood flow within the synovial tissue itself. These semi-quantitative measures can be scored either visually or by software analysis. Visual scales are most practicable in the clinical setting. As outlined above, a simple grading system is mild (few scattered vessels), moderate (<50%) or severe, >50% of the synovial exhibiting increased flow. Various other semi-quantitative scales have been proposed, including that of Klauser et al., who counted the number of visible vessels. Grade\u00a01 is 1\u20135 visible signals, grade\u00a02 6\u201310 and grade\u00a03 more than 11 vessels in the field of view [11]. Computerised methods involve capturing the ultrasound colour image, importing it into a proprietary photo-package and using the software to differentiate and count the number of colour pixels against the greyscale background [16]. The software method requires standardisation of magnification and field of view and is, therefore, troublesome to apply retrospectively.\nCFDUS differs from PDUS in that it is dependent on the direction of flow of blood (Fig.\u00a015). The signal acquired carries specific information about the vessel from which it has been obtained in the form of a pressure trace. The precise measurement that is most commonly calculated is the resistive index (RI) (Fig.\u00a016). RI is the difference between systolic and diastolic pressure divided by systolic pressure. Most manufacturers include software within the ultrasound system to calculate the resistive index. A good spectral trace from a vessel within the synovial hypertrophy is acquired. The operator selects one or two pulse cycles from within the trace for analysis. RI is a good marker of high flow resistance: under normal circumstances it is 1 and is reduced by angiogenesis induced by synovitis. In contrast to PDUS, resistive index is a physiological parameter and is, therefore, independent of the equipment used and manufacturer\u2019s software. Despite some of the limitations of power Doppler, particularly with regard to serial studies, it is a sensitive measure, which is easy to obtain and broadly reproducible between different operators on the same machine. It appears to correlate reasonably well with contrast-enhanced MRI and clinical findings and allows a rapid assessment, within days, of improvement following treatment.\nFig.\u00a015Colour flow Doppler image from a patient with synovitis of the third metatarso-phalangeal joint. Flow away from the ultrasound probe is depicted in blue and flow towards the probe is red. MT metatarsalFig.\u00a016Screen capture during colour Doppler examination of the finger , showing automated calculation of resistive index\nCFDUS is less sensitive to motion artefact and is a more objective measurement than is PDUS. Problems include difficulties in acquiring a good trace when angiogenesis is sparse and maintaining the fix as measurements are made, particularly if the equipment freezes the image during measurement. The use of beam steering can sometimes help with vessel fixation. CFDUS is insensitive to contrast agents, which diminishes its usefulness in patients with subtle or early synovitis. Changes in RI can also be induced by increasing probe pressure and by changes in temperature. Serial studies should be carried out at controlled temperature. In most cases this can be achieved simply by ensuring the patient has not just come from an abnormally hot or cold environment to room temperature, though some argue that the use of a water bath is the only means of ensuring reliable temperature control. Probe pressure can be controlled in the usual manner by ensuring that there is always a layer of contact gel between probe and skin.\nTerslev et al. demonstrated that Doppler ultrasound using changes in resistive index may be used as a quantitative measurement of blood flow and to detect changes following intra-articular steroid injection [17]. The same group also showed that estimates of synovial activity by Doppler ultrasound was comparable with post-contrast MRI, though there was no association between MRI, ultrasound and estimates of inflammation and pain visual analogue scale (VAS) scores. Changes in RI following treatment with anti-tumour necrosis factor (anti-TNF) have also been demonstrated [18]. Varsamidis and co-authors measured resistive index in the wrists of patients with rheumatoid arthritis. The RI improved as the patients went into clinical remission [19]. The authors also suggest that ultrasound may be able to predict relapse, as patients who had a flare up within 6\u00a0months had significantly lower RI following treatment than did patients who remained in stable remission.\nUltrasound contrast agents\nColour Doppler signal from small vessels within the synovium can be augmented by the intravenous administration of contrast agents. When mixed and injected, these agents release bubbles of gas that are sufficiently small to enter the microcirculation. The earliest compounds were mixtures of galactose and palmitic acid microparticles. When hydrated, galactose releases micro-bubbles of air, which adhere to the irregular surface of the palmitic acid microparticles. The air bubbles are approximately 8\u00a0\u03bcm in diameter and, consequently, are able to enter small capillaries. They remain as a stable level in the circulation for up to 5\u00a0min after injection and increase the Doppler signal by approximately 20\u00a0dB [20]. Second generation US contrast agents use gases other than air, for example sulphur hexafluoride, to provide ultrasonic contrast.\nBecause ultrasound involves assessment of a relatively small field of view, the short duration of optimal activity of micro-bubbles following a bolus injection means that there will be different concentrations in the synovium of a joint viewed at the beginning of an ultrasound examination compared with one examined at the end. This compares poorly with the single-time snapshot that can be achieved with intravenous administration of MRI contrast agent, where uptake in all of the joints and tendons within the larger field of view can be assessed simultaneously. There are other disadvantages of bolus administration of ultrasound contrast agents, which include a blooming artefact that can occur due to the sudden arrival of a large quantity of micro-bubbles into the ultrasound field and uneven destruction of micro-bubbles by the US wave acting on the large bolus itself. Administering the contrast agents by infusion rather than bolus injection can overcome some of these issues and yield more stable enhancement for up to 20\u00a0min. Three-dimensional ultrasound, with maximum intensity projection algorithms, may also help in overcoming some of these disadvantages and provide a better single-time snapshot image of synovial enhancement.\nContrast-enhanced power Doppler ultrasound (CEPDUS) has been used in a number of clinical studies to demonstrate improved detection,  over unenhanced ultrasound, of vascularised synovial tissue. Klauser et al. have done much of this work and have classified neovascularisation in patients with rheumatoid arthritis before and after the administration of contrast agent [11]. In the unenhanced study, 65% of subjects were classified as having normal synovial tissue, grade\u00a00, and 5% as severe, grade\u00a04. Following administration of contrast medium by the infusion technique, only 21% were deemed to have normal synovial tissue, whilst 22% increased to grade\u00a04, with increases in the intermediate groups. Other studies have also demonstrated synovial enhancement and increased detection of synovitis [21] [16]. CEPDUS shows improved correlation over unenhanced power Doppler compared with arthroscopy [22]. Despite this, the therapeutic impact and outcome value of contrast agents requires further study.\nBone erosions\nThe identification of bone erosion is a significant step in the natural history of erosive arthritis. The outcome measurement in rheumatoid arthritis and connective tissue (OMERACT) group has defined bone erosion as an intra-articular discontinuity of the bone surface that is visible in two perpendicular planes. Acute erosions generally have an irregular margin and a poorly defined base, which allows through transmission of sound (Figs.\u00a017 and 18) and are associated with active synovitis. Bone defects that do not have synovitis adjacent to them should be regarded with suspicion, though some may turn out to be true chronic erosions [23]. A common pitfall is the normal depression that is present on the dorsal aspect of the head of the metacarpal. This is smooth, with a clearly defined floor that does not allow through sound transmission and is unassociated with overlying synovitis (Fig.\u00a013).\nFig.\u00a017Sagittal image of a large erosion in the head of a metacarpal. Note the poorly defined floor of the erosion (open arrow) and through sound transmission (arrowhead). Compare with Fig.\u00a013. Note the adjacent synovial thickening (arrow) and the extensor tendon (asterisk). MT metatarsal head, PP proximal phalanxFig.\u00a018Moderate synovial thickening (arrowheads) with underlying metacarpal head (MC) erosion (arrow)\nErosions in RA are most commonly detected on the radial aspects of the heads of the metacarpals and on the bases of the phalanges. The DIPJs are least involved in RA [23], but more erosions are detected in sero-negative RA and osteoarthritis. In the hands, the second, third and fifth rays are most commonly affected, and, in the feet, the fifth, third and second show the greatest predilection. There are differences between individual joints in the ease by which ultrasound can detect erosions. In the hands, the second and fifth provide the greatest circumferential views at the level of the MCPJs (Fig.\u00a01c). The fourth is the most difficult to examine, especially on its radial and ulnar aspects. Scheel et al. found that ultrasound was superior to MRI in detecting erosions in the proximal interphalangeal joints but was less efficient at the metacarpo-phalangeal joints [24]. Conversely, in Backhaus and colleagues\u2019 study of patients with negative plain film findings, erosions dominated in the MCP joints.\nUltrasound appears to be particularly useful in detecting erosions in patients in the early stages of disease [25]. Erosions detected by US and MRI progress to radiographic detection in the majority of cases [24, 26] within 1 to 2\u00a0years. One of the earliest MRI signs of erosion is sub-chondral bone oedema, and there are, as yet, no studies to show that ultrasound is useful in detecting this. Although the contour of the cortex should be carefully examined for clarity and early defects, ultrasound will not appreciate intraosseous abnormalities that are not associated with an overlying surface defect.\nErosions in gout are larger, also irregular, and lie further away from the joint. Erosions must be distinguished from other causes of peri-articular bone irregularities. Entheseal new bone formation at the site of tendon and ligament insertions occur in seronegative arthritis and can give a similar appearance. They are particularly prevalent at the DIPJs, where erosive changes related to osteoarthritis are also found.\nSmall joint US following treatment\nChanges in synovial thickening and vascularisation following treatment can occur rapidly. A reduction in the number of vessels identified within the synovium has been shown by US within 3\u00a0days of intravenous corticosteroid administration [27]. Direct interarticular corticosteroid administration has also been shown to reduce the number of synovial vessels, which persist for several years, suggesting that they are a real response to therapy and not just a temporary alteration in flow characteristics induced by pressure changes within the joint [17, 28, 29]. Ultrasound has been used to track changes in total synovial volume by greyscale ultrasound imaging [30], power Doppler signal [31\u201333] and resistive index [31] in response to systemic anti-TNF therapy. Results show a consistent reduction in all these imaging parameters in the early stages of active treatment, though long-term results are less constant. Terslev and colleagues, in a study of wrist and small joint arthritis in patients with rheumatoid disease treated with Etanercept, noted that much of the initial reduction in synovial volume and vascularity and increasing resistive index was lost at 1\u00a0year. Conversely, Fiocco et al., in a study of 27 patients with knee involvement from either rheumatoid or psoriatic arthritis, noted that synovial thickening persisted at 3\u00a0months\u2019 follow-up but was reduced after 1\u00a0year. A reduction in vascularity of the synovium had a more rapid onset and also remained persistently low at 1\u00a0year [32].\nThe limited evidence would seem to suggest that changes in synovial vascularity can be seen very quickly, independent of the type of treatment used. Changes in synovial volume are slower to respond, and measurable differences may take some time to appear in larger joints. In most studies, these changes appear to persist, suggesting that ultrasound can be used as a marker of early improvement. Furthermore, ultrasound has detected improvements in joints felt to be clinical \u2018non-responders\u2019 [30], suggesting an important role for ultrasound in clinical decision making during the early stage of therapy. Most of these studies, however, are unblinded. Evidence that there is a link between early improvement in synovitis detected by US and a reduction in radiographic findings in later years is beginning to emerge [34].\nClinical application of ultrasound in small joint arthritis\nUltrasound versus clinical examination The role of ultrasound in the detection of sub-clinical synovitis has been examined in a number of studies, most of which demonstrate increased detection rates of intra-articular abnormalities. With MRI as the gold standard, the sensitivity, specificity, and accuracy of US for the detection of synovitis in the MTPJs were 0.87, 0.74, and 0.79, respectively, and for clinical examination, 0.43, 0.89, and 0.71, respectively [35]. Wakefield et al. examined the role of ultrasound in detecting sub-clinical synovitis in 1,470 joints in 80 patients with oligoarthritis. Of the clinically unaffected joints, 13% had synovitis detected by ultrasound; the majority, 79%, were metatarso-phalangeal joints, and 16% were metacarpo-phalangeal joints. This meant that one-third of the patients with clinical monoarthritis were upgraded to oligoarthritis (>1<6 joints) and a further quarter to polyarthritis (>6 joints) [36]. Szkudlarek and colleagues examined the metatarso-phalangeal joints in patients with rheumatoid arthritis and healthy controls. Joint effusion and synovitis were detected by ultrasound in 102 joints compared with positive findings in only eight joints on clinical examination [35]. In both studies non-specific findings, especially effusion, common in the first and second MTPJs, may have skewed those results. The finding of occult synovial disease using ultrasound has not been limited to rheumatoid arthritis but has also been extended to patients with systemic lupus erythematosis and other rheumatological conditions [37].\nUltrasound versus other imaging techniques Ultrasound has been compared with plain radiography and has shown significantly superior synovitis detection rates. This is unsurprising, as signs of synovitis on plain radiography, including peri-articular osteoporosis, joint space widening and soft tissue swelling, may be difficult to detect and are frequently overlooked. Backhaus et al. demonstrated a fourfold superiority with ultrasound, compared to the plain radiographic findings of peri-articular osteoporosis and soft tissue swelling [23]. In that study, patients with a variety of rheumatological disorders, who had negative findings on plain radiography, had synovitis demonstrated by ultrasound in all cases, whereas MRI was positive in 84%. In the same group, ultrasound detected erosions in 15 patients compared to 26 detected by MRI. More were found in the proximal interphalangeal joints than by MRI, whereas the reverse was true for metacarpo-phalangeal joints, though other authors have not confirmed this [38].The use of low-field MRI, sequence choice and the use of lower resolution dynamic rather than static post-contrast MR images may have contributed to the apparent superiority of ultrasound in detecting synovitis. Furthermore, both joint effusion and synovial thickening were interpreted as \u2018synovitis\u2019, which might have also increased detection rates for ultrasound over MRI [23]. Conversely, the analogue ultrasound equipment used would be regarded as relatively low specification by modern standards. Indeed, in a follow-up to that study, published 7\u00a0years after the original, ultrasound detected more erosions than MRI did, in 49% of patients (9% at baseline) compared to 32% detected by MRI (27% at baseline) [24]. In the follow-up study the original low-resolution analogue ultrasound equipment was replaced by a more advanced digital system. Although there was also an increase in the number of erosions detected by conventional radiography, from 5 to 33 joints, it is more likely that technological improvements in the ultrasound equipment accounted for the greater detection of erosions and that many had been missed at the baseline study. It is, therefore, imperative that superior resolution equipment is used for rheumatological examination. Other studies have confirmed the superiority of ultrasound over radiography in the detection of erosions [39], independent of the number of projections used [25].Contrast-enhanced ultrasound and MRI have been compared in a small number of studies. Terslev et al. compared Doppler ultrasound with quantification of both the colour fraction and RI with synovial thickness on post-contrast MRI [8]. Dynamic enhanced MRI detected more inflamed MCP joints than ultrasound did, but the reverse was true for the wrist. Szkudlarek and colleagues compared contrast-enhanced ultrasound using the bolus technique with post-contrast MRI in actively inflamed MCPJs [40]. The MRI enhancement rates at 55\u00a0s after injection were used, as the authors found this to correlate most closely with histological features of synovial inflammation [41\u201345]. They concluded that there was good correlation between PDUS and MRI, but noted that the diagnostic impact of contrast enhancement was limited to a relatively small number of patients.Although these studies have attempted to compare ultrasound and MRI contrast enhancement directly, it should be appreciated that there is an essential difference between enhancing synovium on MRI and measurable angioneogenesis on Doppler ultrasound. Enhanced MRI identifies contrast agent in the intravascular, extracellular and joint spaces, the latter depending on the time allowed for diffusion. CEPDUS recognises only new vessel formation within inflamed synovium. It is well recognized that synovial thickening (presumably detectable on enhanced MRI) can be present on ultrasound examination without associated increased vascularity. While it is, therefore, valid to draw some parallels between the two imaging techniques, direct comparison misses vital information on the physiology of the synovium, and differences between them should not be regarded as false negatives or false positives.Klarlund and co-workers compared MRI, plain radiographs and scintigraphy in 55 patients, followed-up for 1\u00a0year. MRI detected progression of erosions more often than radiography did, but ultrasound was not used in that study [46]. Low- and high-field MRI have also been directly compared with plain radiography [47]. The different field strengths had equivalent erosion detection rates, and both were superior to radiography.\nAdvantages and disadvantages of ultrasound\nThe current literature suggests that ultrasound is superior to MRI at detecting small quantities of fluid within the joint. There may be improved detection of erosions at the proximal interphalangeal joints and in the wrist and, although ultrasound struggles with some areas of MCP joints, particularly the radial and ulnar aspects of the fourth finger, overall erosion detection by US at the MCPJs is comparable to that by MRI [38]. US demonstrates angioneogenesis in active synovitis directly (Fig.\u00a019), whereas MRI requires injection of contrast medium. US is a dynamic technique; the examination can be tailored to include further involved joints following an up-to-date history with the patient present There are fewer problems with asymptomatic abnormalities, as these can be clinically correlated during the examination. Unlike MR, US is readily suited to guiding intervention, such as small joint injection or synovial biopsy (Figs.\u00a020, 21). There is also increasing evidence that patients prefer ultrasound to MRI [48]. This may be related to claustrophobia but equally to the human interaction that occurs during an ultrasound examination.\nFig.\u00a019Long axis-view on the dorsal aspect of the wrist deep to the extensor tendon (asterisks). Intense Doppler signal intensity is detected in the distal portion of the joint. DCR distal carpal row, PCR proximal carpal rowFig.\u00a020Needle introduced into the second metatarso-phalangeal joint (arrow) prior to aspiration and corticosteroid injection. MT metatarsal head, PP proximal phalanxFig.\u00a021A synovial mass (arrowheads) undergoing biopsy. Ultrasound allows accurate placement of a small biopsy needle within the synovial tissue\nConversely, ultrasound has difficulty with large deep joints, superficial joints when there are significant deformities, and in the assessment of articular cartilage. US is a relatively time-consuming procedure, especially if multiple joints are examined. The images acquired can be re-read and reclassified by other readers at a later date, but there is no ability to identify abnormalities overlooked and not imaged at the time of the original examination. MR images can be obtained remotely, reviewed, re-read and scored at a later date and preserved for serial comparison. Increasing use of ultrasound video-loops and 3-D ultrasound may assist in this regard in the future. MRI demonstrates single-time shot synovial enhancement in the entire field of view, which can include all the important small joints of the hands and wrists as well as adjacent tendons and ligaments. Although the time taken to review MRI images of the hand and wrist for diagnostic purposes is probably shorter than the time taken to complete an ultrasound examination, a full MRI score such as the rheumatoid arthritis MRI scoring system (RAMRIS), which is a more close approximation to ultrasound, takes considerably longer. Despite this, there is a strong argument that, for research studies of different therapeutic regimens in patients with rheumatoid arthritis, plain films and MRI provide more robust serial assessment and will remain the gold standard for some time.\nConclusions\nIn routine rheumatological practice, ultrasound of the small joints of the hands and feet accurately detects occult synovial disease and allows classification of the extent and activity of synovitis as well as superior detection of erosions in comparison with radiography. US can guide intra-articular therapy as well as assess its response and the response to systemic treatment. Future goals for ultrasound trials need to include further validation studies, studies of diagnostic and therapeutic impact and longer term outcomes from clinical and therapeutic decisions based on the ultrasound examinations.","keyphrases":["ultrasound","joint","synovitis","rheumatoid arthritis"],"prmu":["P","P","P","P"]}
{"id":"Diabetologia-4-1-2170457","title":"Effect of beta-adrenergic stimulation on whole-body and abdominal subcutaneous adipose tissue lipolysis in lean and obese men\n","text":"Aims\/hypothesis Obesity is characterised by increased triacylglycerol storage in adipose tissue. There is in vitro evidence for a blunted beta-adrenergically mediated lipolytic response in abdominal subcutaneous adipose tissue (SAT) of obese individuals and evidence for this at the whole-body level in vivo. We hypothesised that the beta-adrenergically mediated effect on lipolysis in abdominal SAT is also impaired in vivo in obese humans.\nIntroduction\nObesity is characterised by excess fat storage in adipose tissue, in the form of triacylglycerol (TAG). A blunted fat mobilisation, due to decreased adipose tissue lipolysis, might be an important factor contributing to the development or maintenance of the expanded adipose tissue mass in obesity. Fasting lipolysis per unit of lean body mass has been reported to be increased in obesity, whereas a decrease is reported when expressed per unit of fat mass (FM) [1]. The hormonal regulation of lipolysis is still under debate. There is strong evidence from in vitro and in vivo studies for the existence of lipolytic resistance to catecholamines in obese individuals. Blunted whole-body catecholamine-induced lipolysis has been shown in vivo in obese persons [2, 3]. This impaired lipolysis did not improve after weight reduction [4]. Furthermore, a decreased lipolytic response to catecholamines is a feature of childhood-onset obesity [5, 6] and is also present in adipocytes from first-degree relatives of obese persons [7]. These observations suggest that catecholamine resistance of lipolysis may be an important early, and perhaps primary factor, in the development of obesity. In vitro catecholamine resistance of lipolysis was reported in subcutaneous adipocytes of men and women with upper-body obesity, in relation with a decreased cell surface density of \u03b22-adrenoceptors [8]. We hypothesised that beta-adrenergically mediated lipolytic response at the whole-body and abdominal subcutaneous adipose tissue (SAT) level is impaired in vivo in obese persons. If so, this might contribute to the increased fat storage in this adipose tissue depot.\nTo obtain an accurate estimation of lipolysis, state-of-the-art [2H5]glycerol tracer methodology in combination with the measurement of arterio\u2013venous concentration differences across abdominal SAT was used. Obese and lean male control participants were investigated after an overnight fast and during catecholamine stimulation using the non-selective beta-adrenergic agonist isoprenaline. The primary outcome measures of the present study were glycerol rate of appearance (Ra) and glycerol exchange across abdominal SAT. A methodological issue that arises when determining local adipose tissue glycerol release is the possibility that adipose tissue may also take up small amounts of glycerol [9]. A pilot study was performed to determine the time period required to obtain a steady-state in glycerol enrichment in both arterialised and venous blood draining from adipose tissue, since a lack of isotopic equilibration may explain previous discrepant findings on glycerol uptake [10].\nMethods\nParticipants\nThree lean (two women and one man; BMI\u2009<\u200925\u00a0kg\/m2) participants took part in a pilot experiment, during which [2H5]glycerol enrichment was measured for 6\u00a0h to determine the time required for obtaining an isotopic steady-state. Thirteen lean (BMI\u2009<\u200925\u00a0kg\/m2) and ten obese (BMI\u2009\u2265\u200930\u00a0kg\/m2) non-smoking normotensive men participated in the actual SAT lipolysis study, during which [2H5]glycerol was infused for a 3\u00a0h period. Clinical characteristics of the participants are summarised in Table\u00a01. Body weight and body density were determined after an overnight fast, as previously described [11]. All participants were in good health as assessed by their medical history, free of any medication and spent no more than 3\u00a0h\/week in organised sports activities. The Medical Ethical Committee of Maastricht University approved the study protocol and all participants gave their written informed consent before participating in the study.\nTable\u00a01Clinical characteristics of the participants\u00a0Lean (n\u2009=\u200913)Obese (n\u2009=\u200910)Age (years)43\u2009\u00b1\u2009354\u2009\u00b1\u20093BMI (kg\/m2)23.0\u2009\u00b1\u20090.531.9\u2009\u00b1\u20090.6aFM (kg)15.2\u2009\u00b1\u20090.832.4\u2009\u00b1\u20091.2aWHR0.91\u2009\u00b1\u20090.011.01\u2009\u00b1\u20090.01aSystolic blood pressure (mmHg)126\u2009\u00b1\u20093137\u2009\u00b1\u20094Diastolic blood pressure (mmHg)77\u2009\u00b1\u2009285\u2009\u00b1\u20093HOMA-IR1.8\u2009\u00b1\u20090.23.4\u2009\u00b1\u20090.3aValues are mean\u2009\u00b1\u2009SEMap\u2009<\u20090.05 obese vs leanHOMA-IR, homeostatic model assessment of insulin resistance\nExperimental protocol\nPilot study The time course in [2H5]glycerol enrichment was determined in order to identify when steady-state levels were achieved (n\u2009=\u20093). Glycerol enrichment was measured in arterialised blood and blood draining the abdominal SAT (adipose vein) during primed (3\u00a0\u03bcmol\/kg) constant infusion for 6\u00a0h of [2H5]glycerol (0.20\u00a0\u03bcmol kg\u22121 min\u22121). Blood samples were taken simultaneously from the two sites, both at baseline before the start of the tracer infusion (t0\u00a0min), and at ten time points during glycerol infusion (t60, t90, t120, t150, t180, t210, t240, t330, t345, t360\u00a0min).\nWhole-body and SAT lipolysis study Glycerol enrichment and exchange across abdominal SAT were investigated during primed (3\u00a0\u03bcmol\/kg) constant infusion of [2H5]glycerol for 3\u00a0h (0.20\u00a0\u03bcmol kg\u22121 min\u22121). Following a 120\u00a0min baseline period, isoprenaline was infused at a rate of 20\u00a0ng [kg fat free mass (FFM)]\u22121 min\u22121 for 60\u00a0min. During the experiment, heart rate was recorded continuously by means of a three-lead ECG. When heart rate increased by more than 40 beats per min or in the event of ECG irregularities, isoprenaline infusion was stopped (n\u2009=\u20092, one lean and one obese participant). Before the start of the tracer infusion an arterialised blood sample was taken for background enrichment. Blood samples were also taken simultaneously from the arterialised and adipose vein at three baseline time points (t90, t105 and t120\u00a0min) and at three time points during the last 30\u00a0min of isoprenaline infusion (t150, t165 and t180\u00a0min). Adipose tissue blood flow (ATBF) was monitored continuously using the 133Xe washout technique [12]. ATBF results have been published previously in another context [13].\nClinical methods\nAll participants were asked to refrain from drinking alcohol and to perform no strenuous exercise for 24\u00a0h before the study. Participants came to the laboratory by car or bus at 08:00 hours after an overnight fast. Three cannulas were inserted before the start of the experiment. Arterialised venous blood was obtained through a 20-gauge cannula inserted retrogradely into a superficial dorsal hand vein. The hand was warmed in a hotbox, which was maintained at 60\u00b0C to achieve adequate arterialisation [14]. In the same arm, a second cannula was inserted in a forearm antecubital vein for the infusion of [2H5]glycerol tracer and the non-selective beta-adrenergic agonist isoprenaline at a rate of 20\u00a0ng (kg FFM)\u22121 min\u22121. At this infusion rate, plasma isoprenaline concentrations are comparable in lean and obese participants [3]. Finally, the veins on the anterior abdominal wall were identified by means of a fibre-optic light source. In order to obtain adipose tissue venous blood, a 10\u00a0cm 22-gauge catheter (Central venous catheter kit Seldinger technique; Becton Dickinson, Alphen aan den Rijn, the Netherlands) was introduced anterogradely over a guide wire into one of the superficial veins and threaded towards the groin, so that its tip lay just superior to the inguinal ligament [15]. This method provides the drainage from the adipose tissue of the abdomen, uncontaminated by muscle drainage and with only a minor contribution from skin [15]. The adipose vein was kept patent by continuous saline (9\u00a0g\/l NaCl) infusion at a rate of 80\u00a0ml\/h. The participants rested in a supine position for the duration of the study.\nAnalytical methods\nA small portion of blood was used to measure oxygen saturation (%HbO2) and ensure adequate arterialisation (ABL510; Radiometer, Copenhagen, Denmark). Blood was collected in tubes containing EDTA and immediately centrifuged for 10\u00a0min at 1,000g, 4\u00b0C. The plasma was removed for enzymatic colorimetric quantification of NEFA (NEFA C kit; Wako, Neuss, Germany), glycerol (Boehringer, Mannheim, Germany) and TAG (Sigma, St Louis, MO, USA) on a centrifugal spectrophotometer (Cobas Fara; Roche Diagnostica, Basel, Switzerland). Plasma glucose concentration (ABX Diagnostics, Montpellier, France) was measured on an automated spectrophotometer (Cobas Mira; Roche Diagnostica). Plasma insulin was measured with a double antibody radioimmunoassay (Linco Research, St Charles, MO, USA). Insulin sensitivity was assessed by the homeostasis model assessment index for insulin resistance, calculated from fasting glucose and insulin [16]. Packed cell volume was measured using a microcapillary system (Hirschmann Laborger\u00e4te, Eberstadt, Germany).\nIsotope enrichment\nTo determine isotopic enrichment of glycerol, samples first were derivatised. Acetone (1\u00a0ml) was added to 150\u00a0\u03bcl plasma and each tube was vortexed for 2\u00a0min and centrifuged for 20\u00a0min at 17,500g, 4\u00b0C. The supernatant fraction was transferred to a clean tube and dried under nitrogen at 37\u00b0C and derivatised by adding 80\u00a0\u03bcl ethyl acetate (cat. no. 45765; Sigma-Aldrich, Seelze, Germany) and 80\u00a0\u03bcl heptafluorobutyric acid anhydride (cat. no. 63164; Pierce Biotechnology, Rockford, IL, USA). The tubes were vortexed for 2\u00a0min and incubated for 1\u00a0h at 70\u00b0C. Samples were than rotated end over end for 5\u00a0min at 25\u00b0C and evaporated under nitrogen at room temperature. Ethyl acetate (70\u00a0\u03bcl) was added before injection into the GC-MS (Finnigan MAT 252, Bremen, Germany) for measurement of glycerol enrichment. Stable isotope enrichment was analysed by selectively monitoring the mass to charge ratio (m\/z) of molecular ions 253 and 257 for glycerol [17].\nCalculations\nThe net exchange (flux) of metabolites across abdominal SAT was calculated by multiplying the arterio\u2013venous concentration difference of metabolites by adipose tissue plasma flow. Plasma flow was calculated as ATBF\u00d7(1\u2013packed cell volume), with packed cell volume expressed as a fraction. A positive net flux indicates net uptake from plasma, whereas a negative net flux indicates net tissue release.\nThe expected adipose vein enrichment, in case of no glycerol uptake, was calculated as arterialised enrichment multiplied by arterialised glycerol concentration divided by the measured adipose vein enrichment.\nThe Ra of glycerol was calculated according to the following steady-state equation:\nwhere TTR is tracer\/tracee ratio and F is the isotope infusion rate (\u03bcmol kg\u22121 min\u22121).\nThe fractional extraction (fract) of glycerol across abdominal SAT was calculated by dividing the arterio\u2013venous concentration difference of [2H5]glycerol by the arterialised [2H5]glycerol concentration. Abdominal SAT total glycerol uptake was calculated as follows:\nwhere the units are nmol (100\u00a0g tissue)\u22121 min\u22121; (glycerolart) is arterialised glycerol concentration (\u03bcmol\/l); and ATBF is in ml (100\u00a0g tissue)\u22121 min\u22121. Abdominal SAT total glycerol release was calculated from the formula:\nStatistical analysis\nBaseline fasting values and changes (\u0394beta-adrenergic stimulation to baseline) were compared between groups (obese vs lean) using Student\u2019s unpaired t test. Statistical calculations were performed with SPSS for Macintosh (version 11.0; SPSS, Chicago, IL, USA). Data are presented as mean\u2009\u00b1\u2009SEM. A value of p\u2009<\u20090.05 was considered statistically significant.\nResults\nCharacteristics\nObese participants had significantly higher BMI, FM, waist to hip ratio, fasting circulating TAG and insulin concentrations than their lean counterparts (Tables\u00a01 and 2).\nTable\u00a02Circulating (arterialised) metabolite levels during baseline (fasting) and isoprenaline infusion in lean and obese participants\u00a0LeanObeseBaseline (n\u2009=\u200913)Isoprenaline (n\u2009=\u200910)Baseline (n\u2009=\u200910)Isoprenaline (n\u2009=\u20097)TAG (\u03bcmol\/l)701\u2009\u00b1\u200966648\u2009\u00b1\u2009641,464\u2009\u00b1\u2009190b1,667\u2009\u00b1\u2009217aNEFA (\u03bcmol\/l)661\u2009\u00b1\u200941942\u2009\u00b1\u200953638\u2009\u00b1\u2009421,124\u2009\u00b1\u200982aGlycerol (\u03bcmol\/l)102\u2009\u00b1\u20095118\u2009\u00b1\u20097106\u2009\u00b1\u20094147\u2009\u00b1\u200910aGlycerol Ra (\u03bcmol\/min)199\u2009\u00b1\u200912311\u2009\u00b1\u200928220\u2009\u00b1\u200915391\u2009\u00b1\u200930Glycerol Ra\/FM [\u03bcmol (kg FM)\u22121 min\u22121]13.1\u2009\u00b1\u20090.920.9\u2009\u00b1\u20091.67.3\u2009\u00b1\u20090.6b12.9\u2009\u00b1\u20091.1aGlucose (mmol\/l)5.3\u2009\u00b1\u20090.15.4\u2009\u00b1\u20090.15.5\u2009\u00b1\u20090.25.4\u2009\u00b1\u20090.1Insulin (pmol\/l)50\u2009\u00b1\u2009474\u2009\u00b1\u2009694\u2009\u00b1\u20097b167\u2009\u00b1\u200916aValues are mean\u2009\u00b1\u2009SEMbp\u2009<\u20090.05 baseline obese vs lean; ap\u2009<\u20090.05 change (\u0394) from baseline obese vs lean using unpaired Student\u2019s t test\nTracer\/tracee ratio\nIn the pilot experiment, the tracer\/tracee ratios (TTR) obtained during a 6\u00a0h [2H5]glycerol infusion after an overnight fast were examined (n\u2009=\u20093). Arterialised and adipose vein TTR reached a steady-state after 1\u00a0h of infusion (Fig.\u00a01). Mean values are presented, as all participants (n\u2009=\u20093) showed the same pattern. The measured adipose vein enrichment was consistently lower than the expected enrichment, implying uptake of glycerol by adipose tissue. In the actual SAT lipolysis experiment (3\u00a0h [2H5]glycerol infusion), TTR also reached a steady-state after 1\u00a0h and remained stable during isoprenaline infusion. Data in lean and obese men were comparable (data not shown).\nFig.\u00a01Plasma glycerol TTR during 6\u00a0h primed constant infusion of [2H5]glycerol (n\u2009=\u20093) in arterialised blood (squares) and blood draining from abdominal SAT (adipose vein; black circles). White circles, expected adipose vein enrichment. The measured adipose vein enrichment was consistently lower than the expected enrichment\nCirculating metabolites\nBeta-adrenergic stimulation with isoprenaline increased arterialised TAG concentrations in obese participants, while in lean participants TAG concentrations decreased during isoprenaline infusion (Table\u00a02). Thus, the change in arterialised TAG concentrations from baseline to isoprenaline was different between obese and lean participants (\u0394TAG obese vs lean, 100\u2009\u00b1\u200937 vs \u221245\u2009\u00b1\u200922\u00a0\u03bcmol\/l, p\u2009<\u20090.05; Table\u00a02).\nFurthermore, beta-adrenergic stimulation increased arterialised NEFA and glycerol concentrations in lean and obese participants. The beta-adrenergic mediated increase in arterialised NEFA (\u0394NEFA 454\u2009\u00b1\u200968 vs 271\u2009\u00b1\u200946\u00a0\u03bcmol\/l, p\u2009<\u20090.05) and glycerol concentration (\u0394glycerol 40\u2009\u00b1\u20098 vs 15\u2009\u00b1\u20095\u00a0\u03bcmol\/l, p\u2009<\u20090.05) was more pronounced in obese than in lean participants (Table\u00a02), suggesting a higher whole-body lipolytic response in the former. Indeed, beta-adrenergic stimulation increased whole-body glycerol Ra in lean and obese participants (Table\u00a02), this increase tending to be higher in the obese group (\u0394glycerol Ra obese vs lean: 172\u2009\u00b1\u200919 vs 109\u2009\u00b1\u200913\u00a0\u03bcmol\/min, p\u2009=\u20090.07; Table\u00a02). Expressed per unit of FM, fasting glycerol Ra was significantly reduced in obese compared with lean participants (p\u2009<\u20090.05; Table\u00a02). Interestingly, the beta-adrenergically mediated increase in glycerol Ra per unit of FM was significantly blunted in obese participants [\u0394glycerol Ra per unit FM: 5.4\u2009\u00b1\u20090.9 vs 7.7\u2009\u00b1\u20091.5\u00a0\u03bcmol (kg FM)\u22121 min\u22121, p\u2009<\u20090.05] (Table\u00a02), suggesting a blunted lipolytic response per unit of FM in obese participants.\nFinally, beta-adrenergic stimulation increased arterialised insulin concentrations in lean and obese participants (Table\u00a02). This increase in circulating insulin levels was significantly higher in obese than in lean participants (\u0394insulin 62\u2009\u00b1\u200913 vs 25\u2009\u00b1\u20094\u00a0pmol\/l, p\u2009<\u20090.05; Table\u00a02).\nAbdominal SAT lipolysis\nGlycerol uptake by abdominal SAT was observed in lean and obese participants after an overnight fast (Fig.\u00a02a). Fractional extraction of [2H5]glycerol from the circulation (lean vs obese, 16.6\u2009\u00b1\u20094.5 vs 13.9\u2009\u00b1\u20096.7%) and total glycerol uptake expressed relative to total glycerol release were very small (lean vs obese, 9.7\u2009\u00b1\u20093.4 vs 9.3\u2009\u00b1\u20092.5% of total release) with no significant difference between lean and obese participants (p\u2009=\u20090.74 and p\u2009=\u20090.92, respectively). Adipose tissue total glycerol uptake increased during beta-adrenergic stimulation in lean and obese participants, but this increase was not significantly different between groups [\u0394 total glycerol uptake obese vs lean, 4\u2009\u00b1\u20099 vs 21\u2009\u00b1\u20095\u00a0nmol (100\u00a0g tissue)\u22121 min\u22121, p\u2009=\u20090.15] (Fig.\u00a02a). The increased total glycerol uptake during beta-adrenergic stimulation appeared to be partly explained by the increase in ATBF (r\u2009=\u20090.633, p\u2009<\u20090.05).\nFig.\u00a02Total glycerol uptake (a) and release (b) across abdominal SAT after an overnight fast (black bars) and during beta-adrenergic stimulation (white bars) in obese vs lean participants during 3\u00a0h [2H5]glycerol infusion. Values are mean\u2009\u00b1\u2009SEM. *p\u2009<\u20090.05 for change (\u0394) from baseline obese vs lean\nFasting net glycerol and NEFA release across abdominal SAT were comparable between lean and obese participants (Table\u00a03). Beta-adrenergic stimulation increased net NEFA and glycerol release across abdominal SAT to a greater extent in lean than in obese participants, although changes were not significantly different between groups (Table\u00a03). In line with these findings, the beta-adrenergically mediated increase in total glycerol release across abdominal SAT was blunted in the obese group [\u0394 total glycerol release obese vs lean, 140\u2009\u00b1\u200971 vs 394\u2009\u00b1\u2009112\u00a0nmol (100\u00a0g tissue)\u22121 min\u22121, p\u2009<\u20090.05] (Fig.\u00a02b), suggesting a blunted lipolytic response per unit of abdominal SAT in obese participants. Finally, obese men tended to show an increased net TAG flux across abdominal SAT during beta-adrenergic stimulation [\u0394 net TAG flux obese vs lean, 75\u2009\u00b1\u200932 vs 16\u2009\u00b1\u200911\u00a0nmol (100\u00a0g tissue)\u22121 min\u22121, p\u2009=\u20090.06] (Table\u00a03).\nTable\u00a03Blood flow and net SAT fluxes during baseline (fasting) and isoprenaline infusion in lean and obese participants\u00a0LeanObeseBaseline (n\u2009=\u200913)Isoprenaline (n\u2009=\u200910)Baseline (n\u2009=\u200910)Isoprenaline (n\u2009=\u20097)ATBF [ml (100\u00a0g tissue)\u22121 min\u22121]2.2\u2009\u00b1\u20090.26.3\u2009\u00b1\u20091.21.4\u2009\u00b1\u20090.2a3.6\u2009\u00b1\u20090.6Net SAT fluxes [nmol (100\u00a0g tissue)\u22121 min\u22121]TAG25\u2009\u00b1\u2009843\u2009\u00b1\u20091734\u2009\u00b1\u200929113\u2009\u00b1\u200962NEFA\u2212780\u2009\u00b1\u2009160\u22122,101\u2009\u00b1\u2009371\u2212486\u2009\u00b1\u2009101\u22121,824\u2009\u00b1\u2009667Glycerol\u2212229\u2009\u00b1\u200949\u2212640\u2009\u00b1\u2009148\u2212211\u2009\u00b1\u200951\u2212486\u2009\u00b1\u2009128Glucose53\u2009\u00b1\u200949\u221269\u2009\u00b1\u2009151\u2212143\u2009\u00b1\u200996\u2212677\u2009\u00b1\u2009632Values are mean\u2009\u00b1\u2009SEMA positive net flux indicates net uptake from plasma, whereas a negative net flux indicates net tissue releaseap\u2009<\u20090.05 baseline obese vs lean using unpaired Student\u2019s t test\nDiscussion\nThe present study was designed to investigate in vivo whole-body and abdominal SAT lipolysis in obese and lean men. To our knowledge, this is the first study to show in vivo that obese participants have a blunted beta-adrenergically mediated lipolytic response per unit of adipose tissue.\nMethodological considerations\nA point of discussion with studies on glycerol uptake and release using tracer methodology is the infusion time of the labelled glycerol. In previous studies, the infusion time was relatively short (1\u20133\u00a0h), raising the question of whether equilibration between labelled glycerol and the adipose tissue glycerol pool is complete or not [9, 10]. We investigated glycerol enrichment during a 3\u00a0h and 6\u00a0h period of [2H5]glycerol infusion. Steady-state levels in labelled [2H5]glycerol were achieved in arterialised and adipose vein enrichment after 1\u00a0h and remained constant for the subsequent 5\u00a0h. Thus, our data support the use of a relatively short infusion time (1\u00a0h) for study of glycerol metabolism.\nGlycerol uptake\nThe present data show a slight glycerol uptake by abdominal SAT of lean and obese participants. Glycerol uptake was not significantly different between lean and obese participants. Uptake and dilution of [2H5]glycerol across abdominal SAT has been shown previously during 1\u00a0h [9] of tracer infusion. In this study, the dilution of the labelled glycerol was consistently greater than expected from the measured net release of glycerol, indicating significant uptake of glycerol by adipose tissue. In line with this study, we observed a two- to fivefold higher glycerol enrichment in arterialised than in venous blood draining adipose tissue. The observed enrichment in venous blood was universally lower than that predicted from the net addition of glycerol to venous blood. This indicates that an exchange must occur between enriched glycerol in the blood and the unenriched non-esterified glycerol pool in adipose tissue. In contrast, some studies were unable to detect significant uptake of glycerol by adipose tissue after 1\u00a0h of tracer infusion [10]. The reason for this apparent discrepancy remains to be elucidated. It should be mentioned that glycerol uptake is low in human adipose tissue as is the activity of the enzyme glycerol kinase [18]. This enzyme is responsible for the phosphorylation of glycerol into glycerol 3-phosphate, making it available for re-esterification.\nAbdominal SAT lipolytic response to beta-adrenergic stimulation\nA blunted isoprenaline-induced increase in total glycerol release per unit abdominal SAT was observed in obese men, indicating that in vivo beta-adrenergic mediated lipolytic response in abdominal SAT of obese participants is blunted. Our data are consistent with evidence of catecholamine resistance in vitro and in situ in obese individuals [2\u20134], in children with obesity [5, 6] and also in relatives of obese individuals [7]. Defects in catecholamine signal transduction have been observed at the \u03b22-adrenoceptor level and further downstream or directly involving hormone-sensitive lipase [8, 19\u201322]. However, from our experiments it is not possible to determine at which level the observed defect is located. Interestingly, catecholamine resistance has been observed in adipose tissue of first-degree relatives of obese participants [7] and persists after weight reduction [4], suggesting that catecholamine resistance may be a primary defect in obesity. Furthermore, plasma insulin concentrations may play an important role in regulating lipolysis [23]. Therefore, we cannot fully rule out the possibility that the blunted lipolytic response per unit adipose tissue mass that we observed is a secondary phenomenon, due to the higher degree of hyperinsulinaemia during beta-adrenergic stimulation in obese compared with lean participants. However, this explanation seems unlikely, since a blunted in situ lipolytic response in abdominal SAT of obese women was still observed when the confounding influence of hyperinsulinaemia had been excluded using a pancreatic hormonal clamp [2].\nIn contrast to the present study, two in situ microdialysis studies performed in men found that the increase in interstitial glycerol during isoprenaline administration did not differ between lean and obese individuals [24, 25]. A possible explanation for this is that in microdialysis studies interstitial glycerol is used as a measure of lipolysis. Since glycerol is taken up by adipose tissue, interstitial glycerol concentration may not reflect the overall rate of lipolysis. Rather, it may be the net result of TAG and glycerol metabolism, thus reflecting net glycerol turnover [26].\nWhole-body beta-adrenergically mediated lipolytic response\nWhole-body lipolytic response during isoprenaline infusion tended to be higher in obese participants. This was reflected by a higher increase in circulating NEFA and glycerol concentrations during beta-adrenergic stimulation in obese than in lean participants. Expressed per unit of FM, beta-adrenergically mediated lipolysis (glycerol Ra) was significantly lower in obese than in lean men. This suggests that the increased whole-body beta-adrenergically mediated lipolytic response in obese individuals is directly linked to the increased adipose tissue mass, as has been shown before in upper body obese women [2]. Increased release of NEFA into the circulation increases NEFA delivery to the liver, resulting in increased hepatic VLDL-TAG output and hence increased circulating TAG levels during beta-adrenergic stimulation, as was observed in our obese participants. The control of whole-body lipid metabolism is, to a large extent, dependent on the efficient regulation of lipid metabolism in adipose tissue and the liver. Moreover, hepatic VLDL-TAG is a precursor of TAG stored in adipose tissue [27, 28]. Consequently, a greater VLDL-TAG delivery to adipose tissue and greater lipoprotein lipase-mediated hydrolysis might explain the tendency towards increased positive TAG flux across abdominal SAT of obese participants during beta-adrenergic stimulation. Our observation is in agreement with a study by Samra et al. [29] showing an increased rate of action of lipoprotein lipase during epinephrine infusion. These in vivo findings are in contrast with in vitro studies showing that lipoprotein lipase expression and activity are suppressed by epinephrine [30, 31]. Future studies are needed to elucidate whether an increased TAG flux across SAT might contribute to the increased TAG storage in adipose tissue of obese participants.\nConclusion\nThe present study demonstrates in vivo that obese men have a blunted beta-adrenergically mediated lipolytic response in abdominal SAT. Therefore, a blunted lipolysis during beta-adrenergic stimulation may be an important factor in the development or maintenance of increased TAG stores and obesity.","keyphrases":["beta-adrenergic stimulation","adipose tissue","lipolysis","obesity","triacylglycerol","glycerol","in vivo","catecholamines","arterio\u2013venous differences"],"prmu":["P","P","P","P","P","P","P","P","R"]}
{"id":"Neurochem_Res-4-1-2270371","title":"Changes in Glial Cell Line-derived Neurotrophic Factor Expression in the Rostral and Caudal Stumps of the Transected Adult Rat Spinal Cord\n","text":"Limited information is available regarding the role of endogenous Glial cell line-derived neurotrophic factor (GDNF) in the spinal cord following transection injury. The present study investigated the possible role of GDNF in injured spinal cords following transection injury (T9\u2013T10) in adult rats. The locomotor function recovery of animals by the BBB (Basso, Beattie, Bresnahan) scale score showed that hindlimb support and stepping function increased gradually from 7 days post operation (dpo) to 21 dpo. However, the locomotion function in the hindlimbs decreased effectively in GDNF-antibody treated rats. GDNF immunoreactivty in neurons in the ventral horn of the rostral stump was stained strongly at 3 and 7 dpo, and in the caudal stump at 14 dpo, while immunostaining in astrocytes was also seen at all time-points after transection injury. Western blot showed that the level of GDNF protein underwent a rapid decrease at 7 dpo in both stumps, and was followed by a partial recovery at a later time-point, when compared with the sham-operated group. GDNF mRNA-positive signals were detected in neurons of the ventral horn, especially in lamina IX. No regenerative fibers from corticospinal tract can be seen in the caudal segment near the injury site using BDA tracing technique. No somatosensory evoked potentials (SEP) could be recorded throughout the experimental period as well. These findings suggested that intrinsic GDNF in the spinal cord could play an essential role in neuroplasticity. The mechanism may be that GDNF is involved in the regulation of local circuitry in transected spinal cords of adult rats.\nIntroduction\nSpinal cord injury (SCI), a common result of car accidents, high-altitude falls and crashes, and other violent injuries is increasing yearly. It has been reported that there are about 15\u201340 traumatic SCI cases per million persons annually population worldwide [1]. Once the spinal cord is damaged, it usually results in severe neurologic dysfunction and disability. Over the past several years, many extensive experiments have been done in order to find a way to promote the repair of the injured spinal cord. However, no good therapeutic approaches have yet been found. Recently, it has been shown that delivery of exogenous neurotrophic factors (NTFs) to the injured spinal cord has beneficial effects in improving functional recovery [2], indicating the potential value of NTFs in the treatment of SCI.\nGlial cell line-derived neurotrophic factor (GDNF), a distant member of the transforming growth factor-\u03b2 (TGF-\u03b2) family, was originally purified using an assay based on its ability to maintain the survival and function of embryonic ventral midbrain dopaminergic neurons in\u00a0vitro [3]. GDNF supports neural survival in several neuronal populations, including midbrain dopamine neurons and motoneurons in the central nervous system. It promotes the survival and the differentiation of many peripheral neurons, such as sympathetic, parasympathetic, and sensory neurons [3\u201310]. Following injury, GDNF showed a benefit effect to rescue motoneurons degeneration after spinal root avulsion and distal nerve axotomy [11], promotion of axonal regeneration [12] and enhancing regeneration of dorsal roots (DR) into the adult rat spinal cord [13]. In addition, the transplantation of GDNF-producing cells greatly enhances the survival of spinal cord motorneurons [11, 14, 15] and regeneration of several spinal systems, including dorsal column sensory, and regionally projecting propriospinal pathways [16, 17] after SCI. The biological action of GDNF is mediated by a two-component receptor GFR\u03b1-1 and protein tyrosine kinase Ret [18\u201321]. The GFR\u03b1-1 receptors expressed on a variety of neurons that project into the spinal cord, including supraspinal neurons, dorsal root ganglia, and local neurons. GDNF mRNAs are widely distributed in a variety of neuronal and non-neuronal tissues of embryos and adults [4, 22\u201325].\nAlthough exogenous GDNF is available partially to promote propriospinal axonal regeneration and locomotion functional recovery, the role of intrinsic GDNF in injured spinal cords is largely unknown. Therefore, the present study investigated the possible role of GDNF at various time intervals following spinal cord transection injury in adult rats, so as to gain insights into the contribution of endogenous GDNF in spinal neuroplasticity.\nMaterials and methods\nCharacterization of antibodies\nTo localize GDNF protein in the spinal cord, an affinity-purified rabbit polyclonal antibody (D-20, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used in this study. The specificity of antibody for GDNF was confirmed by Western blots using rat spinal cord homogenates.\nPreparation of probe for in\u00a0situ hybridization\nFor detection of GDNF cellular expression in\u00a0situ, we used Digoxigenin-labeled oligonucleotide probe designed by Primer premier 5.0 package which was complementary to the rats GDNF gene sequence (33 mer, 5\u2032-GCCCTACTTTGTCAC\u2013TCACCAGCCTTCTATTTC-3\u2032). This antisense DNA single-stranded oligonucleotide probe was synthesized by Takara Biotechnology Company.\nAnimal grouping and surgery\nSixty-five adult Sprague-Dawley rats of either sex (weighing 200\u2013220\u00a0g) were obtained from the Animal Experiment Center of Sichuan University of Medical Sciences. Fifty rats among all animals, designated as sham-operated group and spinal cord transection group that animals were allowed to survive 3, 7, 14 and 21\u00a0days post operation (dpo), were used for immunohistochemistry and in\u00a0situ hybridization (n\u00a0=\u00a05 in each group), and Western blot analysis (n\u00a0=\u00a05 in each group) respectively. Another five rats were intraperitoneally injected anti-GDNF solution (2.5\u00a0ml\/kg) once every 2\u00a0days until 21\u00a0dpo, designed as antibody neutralization group. Five animals, subjected to cord transaction, were injected with distilled water as the distilled water group. The last five operated rats were used to investigate corticospinal tract sprouting. Every effort was taken to reduce the number of animals and suffering during the experiments. All the experiments using animals were carried out according to the guidelines of the NIH Guide for Care and Use of Laboratory Animal. For the cord transection operation, the rats were anesthetized with 3.6% Chloral Hydrate (1\u00a0ml\/100\u00a0g). A midline incision was made in the back, then the T7\u2013T8 spinous processes and the vertebral laminae were removed to expose the spinal cord at T9\u2013T10 level. The cord was transected between T9 and T10 segments with a pair of microscissors. After surgery, the superficial back muscles and the skin were sutured along the midline.\nBehavior tests\nAll rats in each group were evaluated using the BBB (Basso, Beattie, Bresnahan) scale score [26]. The BBB score for evaluation of hindlimbs function after transected injury ranges from zero, which corresponds to flaccid paralysis, to 21, which is normal gait. Animals were allowed to walk around freely in a circular field (1.2\u00a0m in diameter) for 4\u00a0min while movements of the hindlimbs were closely observed. Ranking according to the scoring system described by Basso et al. [26] includes frequency and quality of hindlimb movement as well as forelimb\/hindlimb coordination.\nMeasurement of somatosensory evoked potentials (SEP)\nThe right peroneal nerve of the rats was dissected and a stimulation electrode was placed on it while a hole (3\u20134\u00a0mm in diameter) was made on the skull (2\u00a0mm to the left of the midline and 2\u00a0mm in front of the fonticulus posterior). An electrode was placed on the dura of brain cortex to record the SEP. The reference electrode was inserted at the nose epithelium. A ground electrode was inserted at the tail. The stimulus intensity was set high enough to produce a marked muscle twitch in the hind limb, about 1.1\u00a0mA, the amplitude was 0.2\u00a0ms and the frequency 3\u00a0Hz. The SEP Tracings represented the average of 200 responses.\nImmunohistochemistry\nAfter anesthesia with 3.6% Chloral Hydrate (1\u00a0ml\/100\u00a0g), rats were perfused with 500\u00a0ml of cold phosphate-buffered saline (PBS) for 5\u00a0min and 500\u00a0ml cold 4% paraformaldehyde solution for 30\u00a0min. The T9 and T10 spinal cord segments in the sham-operated group, and the cord stumps rostral and caudal to the injury site in the experimental group were harvested. This was followed by a postfix for 6\u201312\u00a0h, then immersed in 0.1\u00a0M PBS containing 20% sucrose overnight. Sections of 20\u00a0\u03bcm thickness were cut on a freezing microtome and used for freefloating GDNF immunostaining, then rinsed with 0.01\u00a0M PBS and soaked in PBS containing 3% H2O2 for 30\u00a0min at room temperature to quench the endogenous peroxidase activity. They were immersed in PBS containing 5% goat serum and 0.3% TritonX-100 solution at room temperature for 2\u00a0h, then incubated at 4\u00b0C for 24\u00a0h with anti-GDNF (1:1,000, Chemicon) rabbit polyclonal antibody. Sections were incubated with biotinylated goat anti-rabbit IgG antibody (1:100 dilution) for 1.5\u00a0h at room temperature, and by reaction with avidin-biotinylated peroxidase complexes (1:250, ABC Elite, Vector Labs). GDNF immunoreactivity was visualized with brown staining using diaminobenzidine (DAB) and H2O2 as substrate for 5\u00a0min, and observed with a light microscope. Negative control experiments in which PBS was substituted for the primary antibody were performed to ascertain the specificity of antibody staining.\nIn\u00a0situ hybridization\nSections from sham-operated rats of 25\u00a0\u03bcm thickness were also cut on a freezing microtome and used for in\u00a0situ hybridization. Sections were fixed in 4% paraformaldehyde in 0.1\u00a0M PBS, pH 7.2 (all treatments were performed at room temperature unless otherwise indicated), and further treated with 0.3% TritonX-100 solution for 10\u00a0min and proteinase K (5\u00a0\u03bcg\/ml) at 37\u00b0C for 25\u00a0min, refixed with 4% paraformaldehyde for 5\u00a0min, repeatedly immersed in 0.1\u00a0M PBS, then acetylated with 0.25% acetic anhydride in 0.1\u00a0M triethanolamine (pH 8.0) to prevent non-specific binding of the probes. The sections were washed with 2\u00d7 SSC (pH 7.0) and then prehybridized in a hybridization solution (50% formamide, 10% dextran sulfate, 1\u00d7 Denhardt\u2019s solution, 0.2\u00a0mg\/ml Herring sperm DNA, and 10\u00a0mM dithiothreitol) without probes at 37\u00b0C for 2\u00a0h before hybridization, then hybridized in 100\u00a0\u03bcl hybridization solution containing 1\u00a0\u03bcl probes at 37\u00b0C for 12\u201316\u00a0h in a moist chamber. This was followed by washing in decreasing concentrations of SSC, from 4\u00d7 SSC (pH 7.0) at 37\u00b0C for 20\u00a0min, 2\u00d7 SSC (pH 7.0) at 42\u00b0C for 20\u00a0min, 1\u00d7 SSC (pH 7.0) at 48\u00b0C for 20\u00a0min and ending with 0.5\u00d7 SSC (pH 7.0) at 50\u00b0C for 20\u00a0min. Then sections were incubated at 37\u00b0C in 1% blocking buffer (Roche) for 1\u00a0h, subsequently reacted in 1:1,000 sheep anti-digoxygenin-alkaline phosphatase (AP) antibody in 1% blocking buffer at 4\u00b0C overnight. Lastly, AP activity was detected using nitroblue tetrazolium (NBT)\/5-bromo-4-chloro-3-indolyl phosphate (BCIP) substrate (Roche). The sections were visualized with blue and purple sedimentation then observed with a light microscope.\nGFAP\/GDNF double-label immunohistochemistry\nTwo-color immunohistochemical staining for simultaneous detection of glial fibrillary acidic protein (GFAP)\/GDNF expression was performed as described above. Briefly, sections were stained with anti-GDNF rabbit polyclonal antibody (1:1,000), and using DAB solutions as a substrate. This was followed by second incubation with anti-GFAP rabbit polyclonal antibody (1:1,000, AB5804, Chemicon), and finally, reacted with TMB solutions. Double labeling showed a combination of brown and blue product.\nWestern blotting\nThe spinal cord from the rostral and caudal stumps was obtained. After carefully removing the spinal meninges, the cords were homogenized on ice in a Lysis Buffer containing 0.05\u00a0M Tris\u2013HCl (pH 7.4, Amresco), 0.5\u00a0M EDTA (Amresco), 30% TritonX-100 (Amresco), NaCl (Amresco), 10% SDS (Sigma) and 1\u00a0mM PMSF (Amresco), then centrifuged at 12,000g for 30\u00a0min. The supernatant was obtained and stored at \u221280\u00b0C for late use. Protein concentration was assayed with BCA reagent (Sigma, St. Louis, MO, USA). A 20\u00a0\u03bcl aliquot of the samples was loaded on to each lane and electrophoresed on 12% SDS-polyacrylamide gel (SDS-PAGE) for 2.5\u00a0h at a constant voltage of 120\u00a0V. Proteins were transferred from the gel to a nitrocellulose membrane for 435\u00a0min at 24\u00a0V. The membrane was blocked with phosphate-buffered saline containing 0.05% Tween-20 (PBST) with 10% non-fat dry milk overnight at 4\u00b0C. The membrane was rinsed with PBST and incubated with the primary antibody for GDNF (1:1,000) at 4\u00b0C. The membrane was incubated with a HRP-conjugated goat anti-rabbit IgG (1:5,000; Vector Laboratories, CA) for 2\u00a0h at room temperature. The membrane was developed in ECM kit and exposed against X-ray film in a darkroom. Densitometry analysis for the level of GDNF protein was performed by Bio-Gel Imagining system equipped with Genius synaptic gene tool software. \u03b2-actin (the primary antibody, 1:1,000, the secondary antibody, 1:2,000; Santa Cruz Biotechnology) was used as an internal control.\nBDA anterograde tracing\nAt 14\u00a0dpo, the animals for this part were anesthetized and fixed in a David Kopf Instruments (Tujunga, CA) stereotaxic head-holder device. Burr holes were made in the dorsal cranium, and biotinylated dextran amine (BDA) (10% BDA solution, Molecular Probes) was microinjected into eight sites at a depth of 0.7\u00a0mm from the cortical surface (0.5\u00a0\u03bcl\/site) to cover the hindlimb region. Animals were then sacrificed 2\u00a0weeks later to allow sufficient time for axonal transport of BDA in corticospinal tract. The spinal cords were removed and postfixed at 3\u00a0days in cold 4% paraformaldehyde in 0.1\u00a0M PBS (pH 7.2). Transverse sections (30\u00a0\u03bcm) of spinal cord at the injury site and neighboring rostral and caudal parts to the injury site were processed for the presence of BDA-labeled axons by incubation in avidin-HRP (Molecular Probes). Lastly, DAB stain was performed to visualize the positive fiber, as brown color staining.\nGDNF antibody neutralization\nAfter 14\u00a0dpo, each rat was intraperitoneally injected with 0.5\u00a0ml (30\u00a0mg\/ml, 30\u00a0mg of anti-GDNF diluted in 1\u00a0ml of distilled water) anti-GDNF solution once every 2\u00a0days until 21\u00a0dpo. GDNF antibody was the distilled water replace as control in another five rats. The locomotion in hindlimbs by BBB score was evaluated at 3, 7, 14, and 21\u00a0dpo.\nStatistical analysis\nAll data were expressed as the mean\u00a0\u00b1\u00a0S.E.M. They were analyzed using One-way ANOVA and LSD-q test by SPSS software package. The statistical significance was defined as P\u00a0<\u00a00.05.\nResults\nBehavior tests\nThe BBB score for locomotor function in sham-operated rats hindlimbs was 21. Compared with the sham-operation group, the BBB score of animals in the group with only the cord transection and distilled water group increased gradually from 7 to 21\u00a0dpo. A significant decrease in the BBB score for the GDNF-antibody treated group was observed (P\u00a0<\u00a00.05) (Table\u00a01).\nTable\u00a01Mean values of BBB scores in cord transected rats (mean\u00a0\u00b1\u00a0S.E.M)Group3\u00a0Days7\u00a0Days14\u00a0Days21\u00a0DaysSham-operation group5\u00a0\u00b1\u00a00.621\u00a0\u00b1\u00a0021\u00a0\u00b1\u00a0021\u00a0\u00b1\u00a00Transection group0\u00a0\u00b1\u00a000.8\u00a0\u00b1\u00a00.32.4\u00a0\u00b1\u00a00.73.6\u00a0\u00b1\u00a00.5Distilled water group0\u00a0\u00b1\u00a000.6\u00a0\u00b1\u00a00.72.0\u00a0\u00b1\u00a00.43.3\u00a0\u00b1\u00a00.3GDNF-antibody treated group0\u00a0\u00b1\u00a000.2\u00a0\u00b1\u00a00.40.6\u00a0\u00b1\u00a00.30.9\u00a0\u00b1\u00a00.5BBB score in rats subjected to the sham operation was constant. Following transection injury, the BBB score of animals increased significantly from 7 to 21\u00a0dpo (P\u00a0<\u00a00.05). There had no statistical significance in BBB score between cord transection group and distilled water group (P\u00a0>\u00a00.05), while a significant decrease of BBB score in GDNF-antibody treated group was observed (P\u00a0<\u00a00.05)\nSomatosensory evoked potentials (SEP)\nEvoked potentials are the electrical signals generated by the nervous system in response to sensory stimuli. Somatosensory evoked potentials consist of a series of waves that reflect sequential activation of neural structures along the somatosensory pathways following electrical stimulation of peripheral nerves. SEP (P1N1P2 type) was recorded in the control rats and shown in Fig.\u00a01. Throughout the experimental period, no SEP could be recorded in rats subjected to cord transection.\nFig.\u00a01Somatosensory evoked potential was recorded in normal adult rats (P1N1P2 type). The latency of SEP: P1 (10.3\u00a0\u00b1\u00a00.26)\u00a0ms, N1 (8.81\u00a0\u00b1\u00a00.34)\u00a0ms, P2 (15.5\u00a0\u00b1\u00a00.43)\u00a0ms, amplitude: P1 (2.34\u00a0\u00b1\u00a00.02), N1 (16.3\u00a0\u00b1\u00a00.14), P2 (\u22125.06\u00a0\u00b1\u00a00.05)\nThe GDNF immunostained intensity changes\nIn the control group, GDNF immunoreactivity was observed in the cytoplasm and nuclei of the neurons from the ventral horn in the gray matter (Fig.\u00a03a). Most of the small and medium-sized neurons in the intermediate zone and dorsal horn were stained faintly. GDNF positive profiles (Fig.\u00a03b) simultaneously labeled with GFAP, were present in the white matter (Fig.\u00a02b). In the negative control group, no specific immunopositive staining was detected (Fig.\u00a02a).\nFig.\u00a02In the negative control group, no specific immunopositive staining was detected (a). GDNF positive profiles, simultaneously label-GFAP, were present in the white matter (b)\nIn the rostral stump, strong immunostaining for GDNF was observed in neurons from the ventral horn at 3 and 7\u00a0dpo (Fig.\u00a03c, d; Table\u00a02). Moderate to intense immunostained neurons were seen at 14 and 21\u00a0dpo, as shown in Figs.\u00a03g, 4a and Table\u00a02. In the white matter, astrocytes showed strong immunoreactivity of GDNF from 3 to 21\u00a0dpo (Figs.\u00a03d\u2013f, 4b; Table\u00a02).\nFig.\u00a03Under higher magnification (400\u00d7), GDNF immunoreactivity (IR) was observed in neurons from the ventral horn of the gray matter in the normal spinal cord (a, arrow). GDNF (IR) in astrocytes was also seen in white matter (b, arrow). In the rostral stump, strong GDNF IR was detected in neurons from the ventral horn at 3 and 7\u00a0dpo (c, e, 400\u00d7, arrows). Moderate to intense immunostained neurons were labeled at 14 dpo (g, 400\u00d7, arrow). Intensely immunostained IR in astrocytes of the white matter were observed from 3 to 14\u00a0dpo (d, f, h, 400\u00d7, arrows)Table\u00a02GDNF immunostaining intensity in neurons and astrocytesNormal 3\u00a0Days7\u00a0Days14\u00a0Days21 \u00a0DaysNeurons+++++++++\u223c+++++\u223c+++Rostral part Astrocytes ++++++++++++++Caudal part Neurons++\u223c+++++++++Astrocytes++++++++++++Intensity: + weak, ++ moderate, +++ strongNeurons in the ventral horn of the rostral stump was strongly stained for GDNF at 3 and 7\u00a0dpo, and in the caudal stump at 14\u00a0dpo. The GDNF positive staining in astrocytes was also stronger at all time-points after transection injury than seen in control groupFig.\u00a04In the rostral stump, moderate to intense immunostained neurons were labeled at 21 dpo (a, 400\u00d7, arrow). Intensely immunostained IR in astrocytes of the white matter were also observed at 21 dpo (b, 400\u00d7, arrow). In the caudal stump, immunostained products were detected in neurons of the gray matter at 3 and 7\u00a0dpo (c, d, 400\u00d7, arrows). Strong immunostained IR for GDNF in neurons was observed at 14\u00a0dpo (e, 400\u00d7, arrow). Under higher magnification, immunoreactive structures of GDNF were localized in both cytoplasm and nuclei at 21\u00a0dpo (g, 400\u00d7, arrow), GDNF IR in astrocytes of the white matter was intensely immunostained at 14 and 21\u00a0dpo (f, h, 400\u00d7, arrows)\nIn the gray matter of the caudal stump, moderately immunostained neurons were detected at 3, 7 and 21\u00a0dpo, and more so at 14\u00a0dpo (Fig.\u00a04c\u2013e, g; Table\u00a02). The subcellular localization for GDNF was observed both in cytoplasm and nuclei at 21\u00a0dpo (Fig.\u00a04g). At both 14 and 21\u00a0dpo, astrocytes in the white matter strongly stain with GDNF immunoreactivity as shown in Fig.\u00a04f, h and Table\u00a02.\nLocalization of GDNF mRNA in spinal cord of rats\nSignals of hybridization for GDNF mRNA were markedly detected in the Central canal and lamina III, IV and V, in which small neurons were moderately NBT\/BCIP stained (Fig.\u00a05a). Medium-sized neurons were seen in the field of the intermediomedial nucleus and nucleus dorsalis (Fig.\u00a05a). In the ventral horn, GDNF mRNA-positive large neurons were also observed in lamina IX (Fig.\u00a05b). A few scattered glial cells were probe-labeled with light density in white matter (Fig.\u00a05c).\nFig.\u00a05In the spinal cord of adult rats, GDNF mRNA-positive product were markedly detected in neurons from the lamina III, IV and V and the central canal (a). Medium-sized neurons were seen in the field of intermediomedial nucleus and Nucleus dorsalis. In the ventral horn, GDNF mRNA-positive large neurons were also observed in lamina IX. Under higher magnification (200\u00d7), positive products with blue and purple staining were localized in the cytoplasm (b, arrows), also in white matter scattered glial cells were probe-labeled with light density (c, arrows)\nWestern blotting\nA single positive band was observed at a molecular weight of about 34,000, which corresponds to the molecular weight of GDNF.\nIn the rostral stump, the level of GDNF protein increased at 3\u00a0dpo, when compared with the control group (P\u00a0<\u00a00.05), then decreased quickly to its lowest level at 7\u00a0dpo (P\u00a0<\u00a00.05) which was less than that of the control level, and then began to increase again at 14\u00a0dpo. However, GDNF protein was higher than what was seen in the control group at 21\u00a0dpo (Figs.\u00a06a, 7). In the caudal stump, GDNF protein immediately decreased at 3\u00a0dpo, reached its lowest level at 7\u00a0dpo (P\u00a0<\u00a00.05), then increased from 14 to 21\u00a0dpo, but it did not go back to the control level (Figs.\u00a06b, 7).\nFig.\u00a06In the rostral stump, GDNF protein level increased at 3\u00a0dpo (P\u00a0<\u00a00.05), then reached its lowest point at 7\u00a0dpo which was below the control group (P\u00a0<\u00a00.05), then began to recover at 14\u00a0dpo, but it was less than that of normal level. This was followed by a continuous increase higher than the control group at 21\u00a0dpo (a). In the caudal stump, GDNF protein decreased at 3\u00a0dpo, reached its lowest point at 7\u00a0dpo (P\u00a0<\u00a00.05), then started to increase at 14\u00a0dpo, but it was lower than the normal level at 21\u00a0dpo (b)Fig.\u00a07GDNF levels were significantly different from each other (P\u00a0<\u00a00.05), except for the comparison between the 21\u00a0dpo and normal group, as well as the 21 and 3\u00a0dpo group in the rostral stump. The groups were significantly different from each other (P\u00a0<\u00a00.05), except for the comparison between the 3 and 14\u00a0dpo group in the caudal stump\nBDA tracing\nBDA-positive axons which stained as brown silkiness were detected in rostral cord sections of the injury site (Fig.\u00a08a), but not in the field of scar tissue (Fig.\u00a08b), and in the caudal segment near the injury site (Fig.\u00a08c).\nFig.\u00a08BDA-positive axons staining as brown silkiness were detected in rostral cord sections near the injury site (a), but not in the field of scar tissue (b), and caudal cord sections near the injury site (c)\nDiscussion\nIn the present study, GDNF immunoreactive products in neurons from the gray matter and in astrocytes from the white matter of the spinal cord were observed in the sham-operation group. This indicates that GDNF could be involved in either the physiological function or the maintenance of survival for all kinds of neurons and astrocytes. It has been shown that GDNF mRNAs widely distributed in a variety of neuronal and non-neuronal tissues of embryos and adults [4, 23\u201325]. In addition, GDNF mRNA positive signals were found in the ventral horns, the field of nucleus dorsalis, and the dorsal horns in the spinal cords of adult rats [2, 16, 27] and in the dorsal root ganglia in newborn rats [23]. Therefore, it raised the possibility that GDNF was synthesized by cells of the spinal cord and the GDNF expression may be changed after lesion, which implies that GDNF plays a potential role in both physiological and pathological conditions.\nFollowing cord transection, rats exhibited spinal cord dysfunction throughout the experimental period. There was an absence of SEP in surface recordings, indicating a loss of sensory function in the hindlimbs following transection injury. Comparatively, locomotion function recovery was observed from 7 to 21\u00a0dpo, which was parallel to the improvement in BBB score. It showed that locomotor functional plasticity in the hindlimbs of rats did occur following SCI. As corticospinal tract regeneration was not seen in the caudal part near the injury site and scar tissues, it appears impossible to reestablish connectivity between the areas rostral and caudal of the injury site. The possible reason for locomotor function improvement may be related to the modulation of spinal circuit activity from endogenous neurotrophins. In the present study, BBB scores in the GDNF-antibody treated rats was inferior to that of the group not receiving the antibody treatment, indicating intrinsic GDNF may be available for improving locomotor function recovery to some extent in the hindlimbs of rats [15, 28]. It has been reported that GDNF can promote the repair or survival of spinal motoneurons [4, 7, 29\u201332]. Several other studies have shown that the administration of GDNF could promote axonal regeneration, and enhance locomotion functional recovery [2, 15, 30, 33\u201336].\nGDNF immunoreactivity products in neurons from the ventral horn of the rostral stump were strongly stained at 3 and 7\u00a0dpo, and in the caudal stump at 14\u00a0dpo, suggesting that the GDNF expression in neurons was up-regulated following injury. This may be related to the locomotion functional recovery of hindlimbs. Glazner et\u00a0al. [37] showed that strong labeling for GDNFR-\u03b1 and c-ret mRNA was observed in large neurons from the ventral horn. Our results also supported the mRNA expression for GDNF in neurons of the ventral horn, as well as astrocytes in white matter using in\u00a0situ hybridization. RET expression was unregulated following peripheral axotomy in alpha-motoneurons [38], and topical application of GDNF 30\u00a0min after SCI significantly improved motor function and reduced blood\u2013spinal cord barrier (BSCB) breakdown, edema formation, and cell injury at 5\u00a0h has been reported [39]. Therefore, GDNF is available to assist in locomotion functional plasticity by exerting behavioral and anatomic neuroprotection following SCI.\nIn the present study, the expression patterns of endogenous GDNF indicated its possible role in maintaining motorneuron survival because GDNF mRNA-positive products were detected in neurons from the ventral horn by in\u00a0situ hybridization. Of course, GDNF retrograde axonal transport from a target tissue to neuronal cell bodies [40, 41] is not absolutely excluded. It has been reported that GDNF produced by skeletal muscle is taken up at the nerve terminals and retrogradely transported to motoneurons of the ventral horn by axons [30].\nComparing the results of immunostaining, the GDNF level varied throughout the spinal cord at 3\u00a0dpo. In the caudal stump, GDNF showed a significant decrease. This was probably due to the transection injury that triggers a process destructive to ascending and descending tracts conduction and that extends tissue loss. Differently, a rapid up-regulation of GDNF was observed in the rostral stump. This suggested that GDNF had accumulated around the transection site due to interruption of its transport within the axons. Satake et\u00a0al. [42] reported GDNF transcription began to increase within 30\u00a0min after injury and peaked within 3\u00a0h after spinal cord contusion injury. Interestingly, the level of GDNF protein reached its lowest point at 7\u00a0dpo in both stumps, probably due to the consequences of cell death or apoptosis and\/or interruption of the spinal cord pathways. Subsequently, GDNF protein levels gradually started to recover and surpassed the control level in the rostral stumps at 21\u00a0dpo, which may be explained by (1) GDNF transport within axons was strengthened, (2) inflammation leads to the increase of GDNF synthesis [36], and (3) induction of de novo synthesis of GDNF by neurons and\/or non-neuronal cells.\nNoticeably, the GDNF positive cells in the white matter were labeled by GFAP simultaneously, which identified the labeled cells as astrocytes. Indeed, an association between reactive astrocytes and regenerating nerve fibers in the white matter of the central nervous system in\u00a0vivo has been reported [43, 44]. Also, increased GFR-1 in astrocytes of degenerating white matter in adult rat spinal cords after mechanical injury has been shown [2]. These results indicated that GDNF expression in astrocytes may be involved in neuroplasticity following spinal cord transection injury.\nThe present study provides new evidence to understand the role of intrinsic GDNF in the rostral and caudal stumps of injured spinal cords. It suggests that GDNF plays an essential role in the neuroplasticity of the local circuitry, especially in the caudal stump of the transected spinal cords.","keyphrases":["rats","gdnf","antibody","western blot","spinal cord transection","immunohistochemistry","in situ hybridization"],"prmu":["P","P","P","P","P","P","R"]}
{"id":"Eur_Spine_J-2-2-1602200","title":"Bilateral pedicle stress fracture in the lumbar spine of a sedentary office worker\n","text":"A case of bilateral pedicle fracture in the lumbar spine of a sedentary office worker is being presented. No such case has been reported in the literature previously. Bilateral pedicle fracture is a rare entity. Few cases have been reported in literature. All the reported cases had some underlying causative factors like degenerative spine disease, previous spinal surgery or stress-related activities, e.g. athletes. Our case is a 36-year-old sedentary office worker with none of the factors mentioned. We present a case of a 36-year-old sedentary worker with long-standing low backache. There were no root tension signs. Plain radiographs were inconclusive. The patient had a CT scan. The CT scan revealed long-standing defects in the pedicles of L2 vertebra with pseudoarthrosis. Infiltration with anaesthetic relieved the symptoms. Our patient was managed conservatively with spine rehabilitation physiotherapy program. Pedicle fracture can develop due to abnormal stresses in the pedicle either because of previous spinal surgery or spondylitic changes in the spine. Bilateral pedicle fracture in the absence of these conditions is extremely rare.\nIntroduction\nIsolated pedicle fracture in the spine is uncommon. It has been reported in association with previous spine surgery, or in highly active athletic individuals. We report a case of bilateral pedicle fracture in a patient with chronic backache without the history of trauma or any of the above-mentioned conditions. The patient was a sedentary worker with minimal physical demands.\nCase report\nWe present a case of a 36-year-old female with a history of chronic backache. Her symptoms were of mild to moderate in intensity. Discomfort was mainly related to activity but she was performing her normal duties as an office manager without significant problems. On examination there were no neurological deficit or root tension signs. Plain radiographs were inconclusive. Clinical examination revealed localized deep tenderness at L2; therefore, a CT scan was performed. The CT scan showed bilateral long-standing defects through the pedicles of 2nd lumbar vertebra with pseudoarthrosis and sclerosis (Fig.\u00a01a, b). Infiltration with local anaesthetic, under the X-ray guidance, in the involved area relieved the symptoms. Surgical management was discussed with the patient, which was declined. The patient underwent spine rehabilitation physiotherapy program, which resulted in the improvement of the symptoms.\nFig.\u00a01a CT scan of the vertebra showing bilateral pedicle fracture. b CT scan showing bilateral pedicle fracture with pseudoarthrosis\nDiscussion\nBilateral pedicle stress fracture in the spine is a rare finding. Traughber and Havlina [10] reported the first case of a bilateral pedicle stress fracture. Cyron et al. [2] demonstrated that the parsintereticularis is thought to be the weakest site in the neural arch, followed by the pedicle. In cases with established unilateral stress fracture, there is a redistribution of forces in the neural arch, which leads to compensatory sclerosis of contralateral bony structures. A compensatory hypertrophy of the contralateral pedicle has been documented [1, 8]. This entity was first described by Wilkinson and Hall [11] in 1974. These authors reported on seven patients whose radiographs were suspicious for a neoplastic process occurring in a pedicle (e.g. osteoid osteoma or osteoblastoma). Further investigation revealed unilateral spondylolysis with sclerosis of the contralateral posterior elements. The incidence of bilateral pedicle stress fracture is unknown in an otherwise normal spine. Gunzburg and Fraser [3] introduced the term pediculolysis in 1991 in a patient with multilevel facet joint osteoarthritis and minimal spondylolisthesis at L4\u2013L5. Pedicle fracture has been reported in cases following posterolateral instrumented spinal fusion [4, 5, 7]. Bilateral pedicle stress fractures or pediculolysis have been documented in an athlete of high physical demand [6]. Stanley and Smith [9] reported a case of pedicle fracture following laminectomy. In our patient the fractures appear to be old with evidence of sclerosis at the fracture margins along with pseudoarthrosis. This type of bilateral pedicle stress fracture has not been documented in a normal spine of a sedentary office worker without a history of major trauma or surgery.\nConclusion\nPedicle fracture is uncommon but has been reported in association with degenerative spondylolisthesis, unilateral spondylolysis, following spinal surgery but not in an otherwise normal spine. Pedicle stress fracture may show either as a recent fracture or may have signs of established pseudoarthrosis. So far there is no case of bilateral pedicle stress fracture in an otherwise normal spine.","keyphrases":["bilateral","stress fracture","pedicle fracture","backache"],"prmu":["P","P","P","P"]}
{"id":"Pediatr_Radiol-3-1-1950217","title":"Pediatric DXA: clinical applications\n","text":"Normal bone mineral accrual requires adequate dietary intake of calcium, vitamin D and other nutrients; hepatic and renal activation of vitamin D; normal hormone levels (thyroid, parathyroid, reproductive and growth hormones); and neuromuscular functioning with sufficient stress upon the skeleton to induce bone deposition. The presence of genetic or acquired diseases and the therapies that are used to treat them can also impact bone health. Since the introduction of clinical DXA in pediatrics in the early 1990s, there has been considerable investigation into the causes of low bone mineral density (BMD) in children. Pediatricians have also become aware of the role adequate bone mass accrual in childhood has in preventing osteoporotic fractures in late adulthood. Additionally, the availability of medications to improve BMD has increased with the development of bisphosphonates. These factors have led to the increased utilization of DXA in pediatrics. This review summarizes much of the previous research regarding BMD in children and is meant to assist radiologists and clinicians with DXA utilization and interpretation.\nIntroduction\nPeak bone mineral accrual occurs during early puberty and peak bone mass is achieved in young adulthood. Low BMD can result from a wide variety of childhood diseases or be due to the effects of their treatment. Increasingly, pediatric specialists are requesting evaluation of BMD and radiologists are expected to be knowledgeable in the technique, interpretation and clinical applications of DXA. A review of the technical and interpretive aspects of DXA has been recently presented [1]. The clinical uses of DXA in pediatrics are exceedingly broad. This paper reviews the clinical data regarding pediatric DXA and can be used to assist radiologists and clinicians in DXA utilization and interpretation.\nGastrointestinal disorders\nGastrointestinal diseases may impact bone health in several ways. Poor calcium intake, as in patients with a milk allergy, and reduced calcium absorption, as in patients with untreated celiac disease, result in low BMD. Early correction of the underlying deficiency allows normal bone mineralization to occur. In addition to poor calcium absorption, inflammatory bowel disease likely impacts bone health through other factors including chronic diarrhea, decreased lean tissue mass, reduced physical activity, increased inflammatory cytokines, and CS therapy. As in all chronic conditions, correction of short stature and delayed maturation will allow better identification of those patients with abnormal DXA findings who will have significant bone mineral deficits.\nMilk allergy\nHenderson and Hayes [2] reported a positive correlation between LS and hip BMD and calcium intake in children with significant milk allergy. They found a 5\u20138% increase in LS Z-scores and a 9\u201313% increase in hip Z-scores in patients with daily calcium intake above the recommended dietary allowances. These findings were confirmed in children with reduced dairy intake due to a variety of other causes [3, 4]. There was a positive correlation between BMD and calcium intake with normal values in the subgroup with normal dietary calcium intake and decreasing BMD as the daily intake of calcium decreased. LS BMD was maintained, even with severe dairy restrictions when calcium intake was maintained through diet or supplementation.\nInflammatory bowel disease\nBoot et al. [5] found decreased LS and TBBMD in children with Crohn disease or ulcerative colitis. The decreases were statistically significant even when they accounted for the delayed bone age in their patients. Similarly, Ahmed et al. [6] found reduced LS and TBBMD Z-scores in nearly 70% of children with Crohn disease and, to a lesser extent, ulcerative colitis, when compared to healthy age-matched controls. However, when height was taken into account, only 22% of their patients had low BMD. Burnham et al. [7] found decreased height, lean tissue mass and TBBMC in children with Crohn disease when compared to normal controls. However, with correction for height, age, race, Tanner stage and lean tissue mass, no statistically significant differences were found between the study and control groups. The authors also found no correlation between CS treatment and height-corrected BMC. Similar findings were reported by Walther et al. [8]. Thus, in patients with inflammatory bowel disease, the apparent low LS BMD and TBBMC is largely a result of short stature, delayed maturation and decreased lean tissue mass.\nCeliac disease\nKalayci et al. [9] found lower LS BMD in untreated celiac disease patients compared to treated patients. They found that nearly all children with celiac disease whose treatment began before the age of 4\u00a0years reached normal BMD compared to only 50% of children whose treatment began after the age of 4\u00a0years. They also found that all patients who initially presented with gastrointestinal symptoms had normal BMD after 1\u00a0year of a gluten-free diet. In prepubertal children with celiac disease, Barera et al. [10], Tau et al. [11] and Szathmari et al. [12] found an increase in height, weight, BMC and BMD after 1.5\u00a0years of a gluten-free diet when compared to baseline measurements. These reports indicate that in patients with celiac disease, normalization of DXA findings can be expected with early diagnosis and treatment.\nLiver disease\nThe effects of liver dysfunction on bone health are complex and may involve vitamin malabsorption, failure of vitamin D activation, calcium malabsorption, bile salt deficiency and chronic malnutrition. Argao et al. [13] evaluated children with a variety of chronic cholestatic liver diseases and found distal radial BMC to fall quickly after birth and in infancy. The values remained low throughout childhood and reflected the severity of the underlying hepatic dysfunction. In a study of prepubertal children with Alagille syndrome, Olsen et al. [14] found reduced height, weight and TBBMC compared to healthy controls matched for age, gender, ethnicity and physical maturity. Malabsorption of fat-soluble vitamins, particularly vitamin D, significantly reduces LS and TBBMC. D\u2019Antiga et al. [15] found normal LS BMC and BMD in patients with childhood liver failure who were at least 1\u00a0year out from orthotopic liver transplantation. Return to normal values appeared to be unaffected by the severity of bone disease or cholestasis prior to transplantation. Thus, liver dysfunction with disordered vitamin D metabolism results in early decreased bone mineral accrual. With successful liver transplantation, normalization of DXA findings can be expected.\nRenal diseases\nChronic kidney disease results in abnormal bone metabolism via disturbances in calcium and phosphate handling, altered vitamin D and parathyroid hormone levels and function, and altered renal clearance of other metabolites. Additional factors that affect the BMD in these patients include malnutrition, metabolic acidosis, anemia and growth hormone abnormalities resulting in growth retardation. As with many chronic medical conditions, short stature will greatly affect BMD and needs to be accounted for in DXA interpretation.\nBakr [16] found reduced LS BMD in 60% of pre- and postdialysis patients. The degree of osteopenia correlated with biochemical markers of secondary hyperparathyroidism. However, the DXA BMD values were not corrected for height. Similar results were reported by Pluskiewicz et al. [17] following a 2-year longitudinal study of adolescents with chronic kidney disease. They found decreased or stable LS BMD (uncorrected for height) during a time when an increase would be expected. The most severe decreases in BMD were found in patients who were treated with CS. In contrast, Ahmed et al. [18] found normal TBBMD and TBBMC as well as normal LS BMD in children with chronic kidney disease when the DXA values were corrected for height. Similar findings were reported in children before transplantation [19]. Boot et al. [20] reported normal LS and TBBMD in patients with kidney disease of relatively short duration and in the setting of adequate vitamin D replacement. Two-year follow-up of these patients demonstrated continued maintenance of normal BMD [21]. Several authors found that the height-corrected LS BMD was reduced for up to 2\u00a0years after transplantation; this was felt to be due to high-dose CS treatment required to suppress transplant rejection. However, by 3\u00a0years after transplantation, LS BMD had recovered to normal values [18, 22\u201324].\nIdiopathic hypercalciuria results in excessive urinary excretion of calcium and has increasingly been identified as a cause of decreased BMD in children [25\u201328]. The degree of BMD reduction does not appear to correlate with the presence of urolithiasis or urinary uric acid concentration. Studies of children and their mothers with idiopathic hypercalciuria indicate that low LS and hip BMD persist into adulthood [25, 26].\nIn summary, the various metabolic disturbances resulting from altered renal physiology may compromise normal bone mineralization and remodeling, but children with chronic kidney disease often maintain normal BMD if calcium and vitamin D metabolism are normal. CS treatment, either before transplantation or when used to suppress transplant rejection, appears to contribute to low BMD but short-term follow-up indicates eventual normalization of LS BMD. Regardless of the nature of renal disease, correcting for height is essential for accurate interpretation of DXA results in these patients.\nEndocrinological diseases\nSkeletal development is significantly influenced by multiple hormone systems that are essential to normal bone mineral accrual. Both insufficient and excessive hormone levels can have deleterious effects, resulting in inadequate bone formation or disproportionate bone resorption. Pubertal hormones play a critical role in bone mass acquisition with major differences occurring based on sex and skeletal site [29]. Patients with early or delayed puberty will show early or delayed peak bone mass accrual, respectively [30]. Treatment of precocious puberty includes gonadotropin-releasing hormone analogs that could theoretically interfere with normal bone mineral acquisition. Short-term treatment does not interfere with normal BMD increases in these patients [31].\nDisorders of growth hormone, insulin and glucocorticoids\nAccurate DXA evaluation of children with growth hormone deficiency or idiopathic short stature must account for their smaller bones [1]. Children with growth hormone deficiency will present with low areal [32] and volumetric LS [33] BMD when first diagnosed. Children with idiopathic short stature have also been found to have low areal LS and hip BMD prior to therapy [34]. With sustained growth hormone replacement these values have been reported to remain stable [33] or normalize rapidly [34]. Children with growth hormone deficiency have a robust response to replacement therapy with increases in height, lean tissue mass and TBBMC [33]. Increases in BMD occur even after peak height is achieved, and this indicates the need to continue hormone therapy until peak BMD is achieved [35, 36].\nType 1 diabetes mellitus is a known risk factor for osteoporosis, but the underlying mechanisms are not fully determined. Adolescent patients have lower bone mass and bone size despite normal growth and maturation [37]. Studies suggest that children and adolescents with diabetes have reduced bone formation and increased bone resorption resulting in low bone mass and BMD [38, 39]. Hemoglobin A1C level, a measure of glycemic control, negatively correlates with BMD [40] and TBBMC [41].\nThe negative effects of chronic high-dose CS therapy on bone have been well documented. CS replacement therapy, as in congenital adrenal hyperplasia, has not been found to result in reduced BMD [42, 43]. However, young women with congenital adrenal hyperplasia are at risk of reduced BMD if there is biochemical evidence of androgen suppression due to CS therapy [44]. Patients with either childhood-onset or adult-onset Cushing disease often have severe reductions in BMD due to increased osteoclastic activity and bone resorption with normal or reduced bone deposition [45]. Additionally, concomitant reductions in sex and growth hormones contribute to their low BMD. Adolescents appear to be particularly vulnerable to reduced trabecular bone density as measured at the LS. BMD deficits and biochemical measures of bone turnover showed marked improvement after 2\u00a0years remission from hypercortisolism when compared to baseline values, but low LS BMD persisted in the majority of patients [46].\nDisorders of reproductive hormones\nReproductive hormones play an important role in the acquisition of bone mineral; estrogen is known to have a protective effect against the development of osteoporosis in postmenopausal women. The effects of hormonal birth control use on BMD in adolescent girls have been investigated by several researchers. The trend toward lower estrogen levels in oral contraceptives has been associated with decreased BMD in adolescent girls who use oral contraceptives [47, 48]. Progesterone-mediated contraceptives (Depo-Provera) have been shown to inhibit bone mineral acquisition in the LS and hip in adolescent girls [49\u201351]. The extent of this reduction appears to correlate with the duration of treatment and can be lessened with supplemental estrogen [51]. Partial or full BMD recovery can occur after cessation of treatment and can be facilitated with increased physical activity and adequate calcium and vitamin D intake.\nGirls with anorexia nervosa have been found to have decreased BMD at multiple skeletal sites [52\u201354]. These reductions are thought to be due to a combination of nutritional (decreased calcium and caloric intake), hormonal (decreased estrogen levels, delayed puberty), and mechanical (decreased lean tissue mass) factors. With resumption of normal caloric intake and return of menses, there is biochemical evidence of increased bone turnover; however, BMD levels remain low and Z-scores may continue to fall for at least 1\u00a0year [53]. It is not known if full BMD recovery will occur by early adulthood, but adult women who had adolescent-onset anorexia nervosa have more dramatic reductions in BMD than those with onset during adulthood [55, 56]. Estrogen replacement with oral contraceptives and vigorous exercise can protect against BMD loss in anorexia nervosa [57].\nUntreated congenital hypothyroidism can result in markedly reduced BMD. Children with adequate long-term thyroid replacement therapy show normal LS and hip BMD and BMC [58, 59] and normal findings continue into young adulthood [60]. Later in childhood, hypothyroidism is usually caused by Hashimoto thyroiditis. LS and distal radial BMD are expected to be normal at diagnosis and to be maintained with thyroid hormone therapy, and normal peak bone mass is to be expected in these patients [61]. Hyperthyroidism in Graves disease markedly decreases LS and hip BMD, but these normalize within 1\u00a0year following thyroid ablation and hormone therapy [62].\nIn summary, hormonal imbalances from any of a large number of endocrine disorders are associated with abnormal DXA findings and reduced BMD. With hormone replacement therapy, improved bone mineral accrual is to be expected in most of the endocrine deficiencies. However, with anorexia nervosa, especially with onset in adolescence, there appears to be a sustained reduction in BMD even after resumption of normal caloric intake and menses.\nRespiratory diseases\nCystic fibrosis\nIn cystic fibrosis (CF), low BMD may be caused by reduced gastrointestinal absorption of calcium and vitamin D, reduced testosterone levels, chronic hypoxia, chronic CS use, and reduced lean tissue mass. Because of the multifactorial nature of reduced BMD in CF, BMD Z-scores often reflect the severity of illness. Clinically stable children with mild CF have been shown to have normal BMD [63\u201365]. However, adolescents and adults with CF have accelerated bone loss over time reflecting disease progression [66, 67]. Henderson and Madsen [68] and Buntain et al. [66] found progressively decreasing BMD Z-scores with increasing age in CF patients. LS, hip and TBBMD in the prepubertal group were normal. TBBMD was reduced in adolescents; however, all other sites remained normal. In adult patients, BMD was reduced at all sites. In a follow-up study, Haworth et al. [69] reported that patients older than 25\u00a0years showed accelerated LS and hip BMD losses. Bhudhikanok et al. [70] found that LS, hip, and TBBMD were low at baseline and documented further bone loss at follow-up. CS use was identified as the most modifiable factor accounting for this bone loss and the authors suggested that bisphosphonates may be beneficial in these patients. The benefits of bisphosphonates in CF were confirmed by Aris et al. [71, 72] and Haworth et al. [73] who found increases in LS and hip BMD following treatment with intravenous or oral bisphosphonates, respectively.\nAsthma\nAsthma may inhibit normal bone metabolism due to chronic hypoxia and decreased physical activity. Additionally, asthmatic patients are treated with both oral and inhaled CS to reduce airway inflammation. Patients treated with low or moderate levels of inhaled CS have been shown to have normal BMD Z-scores when compared to CS-naive asthmatic controls but reduced BMD Z-scores when compared to normal controls when height is not taken into account [74\u201377]. Allen et al. [78] and Harris et al. [79] found significant reductions in BMD in asthmatic children receiving high doses of inhaled CS.\nHematological diseases\nAnemia and hemophilia\nChronic anemias can affect bone structure and density through bone marrow hyperplasia (resulting in expansion of the medullary space, trabecular coarsening and cortical thinning), and vasoocclusion (resulting in medullary and diaphyseal infarction). Additional factors for reduced BMD include reduced lean tissue mass, decreased physical activity and hypogonadism, and in the case of thalassemia, endocrine dysfunction. Brinker et al. [80] found low BMD in children with sickle cell anemia. These findings confirmed those of Soliman et al. [81] who reported low LS BMD in prepubertal children with sickle cell anemia. Lal et al. [82] and Buison et al. [83] noted markedly reduced BMD and BMC, which correlated with disease severity in these children. These changes were found to persist into adulthood [84]. Additionally, low calcium intake and low vitamin D levels may contribute to decreased BMD in these patients. Vogiatzi et al. [85] and Benigno et al. [86] evaluated children with thalassemia major who were fully treated with transfusion and chelation therapy and found reduced BMD at diagnosis and showed further reductions at follow-up.\nPatients with hemophilia are at risk for osteoporosis due to reduced levels of physical activity and sports, particularly those involving running, jumping and axial loading of the skeleton. The severity of hemophilic arthropathy can be quantitated using a clinically derived joint score, and this has been found to correlate with BMD [87]. Low BMD is independent of height and weight but correlates with disease severity and extent. These changes can be expected to persist into adulthood [88].\nOncological diseases\nAcute lymphocytic leukemia\nALL is the most common childhood malignancy, but because of its excellent prognosis, most children with ALL survive into adulthood. Arikoski et al. [89] found reduced BMD in ALL survivors up to 20\u00a0years after treatment. Of their 29 patients, 20 had received cranial irradiation and four males had received testicular irradiation. The use of high-dose methotrexate and cranial irradiation were found to be significantly correlated with low BMD. Differing results were presented by Brennan et al. [90] who also reported significantly reduced LS, hip and distal radial BMD in long-term survivors of ALL but found no statistically significant correlation of BMD with cranial irradiation, growth hormone status or height Z-scores. They suggested that the low BMD found in their patients was related to the effects of chemotherapy rather than cranial irradiation. Van der Sluis et al. [91] found normal LS and TBBMD a mean of 10\u00a0years after ALL treatment that included high-dose methotrexate and CS but not cranial irradiation. Their patients had all been prepubertal at the time of initial diagnosis and none had signs of significant gonadal or growth hormone dysfunction at the time of follow-up. The authors concluded that the deleterious effects of ALL and its treatment in childhood on BMD may be due to cranial irradiation but, in its absence, BMD will normalize by early adulthood. This conclusion is supported by other researchers [92, 93]. Jarfelt et al. [94] examined bone turnover and growth hormone status with respect to physical activity levels and BMD in adult survivors of childhood ALL. The patients were all prepubertal at the end of treatment and had been treated with high doses of CS and methotrexate. No patient had evidence for gonadal or endocrine dysfunction. The BMD values were normal for the group. Only the level of physical fitness at follow-up correlated positively with BMD. The authors stressed the importance of physical activity in restoring and maintaining normal BMD in survivors of childhood ALL.\nIn summary, children with ALL will have reduced BMD during treatment and shortly thereafter. The recuperative capacity in young children is high and normal BMD should be expected even after high-dose methotrexate and CS therapy. The role of adequate physical activity is being increasingly stressed as an important factor in normal BMD recovery. Children who have survived ALL may be at risk of persistently low BMD in adulthood if they have confounding factors such as gonadal dysfunction or if the course of their disease coincided with the period of expected rapid accrual of bone mineral that normally occurs during puberty.\nOther malignancies\nThere are only limited data regarding the effects of other childhood malignancies on BMD. Aisenberg et al. [95] and Vassilopoulou-Sellin et al. [96] found reduced femoral neck and TBBMD in young adult survivors of various childhood cancers. Gonadal dysfunction due to pelvic or cranial irradiation was the factor most strongly correlated with reduced BMD. CS treatment did not correlate with low BMD. Nysom et al. [97] reported normal size-adjusted TBBMC in adult survivors of childhood lymphoma and found no relationship between TBBMC and cumulative methotrexate or CS doses. Kelly et al. [98] evaluated adult survivors of various pediatric solid tumors and found reduced BMD in at least one site in half of their patients. Only the total number of chemotherapeutic agents correlated with reduced BMD. Five of six extremities involved with a bone sarcoma showed reduced BMD. Similar results were found in a group of sarcoma survivors reported by Ruza et al. [99]. Interestingly, they found that those diagnosed prior to puberty had more severe BMD reductions later in life. This may be due to the extensive physical disabilities associated with amputations and limb salvage procedures in these patients. Odame et al. [100] found that patients with childhood brain tumors treated with cranial irradiation had reduced LS and TBBMD and these were correlated with reduced physical activity and poorer quality of life. The authors postulated that the higher cranial radiation doses used for the treatment of brain tumors had a profound effect on long-term BMD compared to the relatively lower doses used in the treatment of ALL.\nIn summary, there are multiple factors that affect short- and long-term BMD in these patients\/survivors. These include pubertal status at diagnosis, type of malignancy, local (sarcomas, central nervous system tumors) versus systemic (leukemia) disease, initial (malnutrition, immobilization) versus prolonged (amputation) disease-related disability, types of chemotherapy, and radiation ports and dosages that may cause gonadal or growth hormonal dysfunction.\nNeurological diseases\nCerebral palsy\nBecause of decreased muscle development and ambulation, children with cerebral palsy are at increased risk of osteoporosis-associated fractures. Frequently, assessment of the LS and hips with DXA is impossible due to difficulties in positioning patients with contraction deformities, muscular spasm-induced motion artifacts or orthopedic hardware-related artifacts. Because of these limitations, Harcke et al. [101] suggested scanning the distal femur with these patients positioned on their side. Distal femoral BMD was found to be highly correlated with hip BMD and the technique yielded highly reproducible BMD data [102].\nHenderson et al. [103] demonstrated reduced LS and hip BMD in children with cerebral palsy. Measures of nutritional and ambulatory status were found to be the best predictors of BMD. In children with moderate to severe disease, Henderson et al. [104] found distal femur and LS BMD to be markedly reduced and both closely correlated with disease severity. Distal femoral BMD was negatively correlated with age, indicating progressive bone mineral deficits as these children grew older. The lack of a strong correlation between LS BMD and fracture risk in the lower extremities emphasizes the need for direct assessment of the distal femur with DXA in these patients. In a prospective longitudinal study of patients with cerebral palsy, Henderson et al. [105] found that low initial LS and distal femur BMD Z-scores were associated with poor growth, nutritional status and motor function, and these contributed to reduced bone mineralization at follow-up. Treatment with bisphosphonates for 1\u00a0year resulted in a substantial increase in distal femoral BMD in children with quadriplegic cerebral palsy [106]. The improvement was sustained for at least 6\u00a0months after the last treatment. However, Bachrach et al. [107] found that LS BMD returned to baseline values 2\u00a0years after bisphosphonate therapy was terminated. Importantly, despite the lack of sustained BMD improvement, no patient had a fracture during the treatment or follow-up periods.\nIn summary, children with CP show decreased bone mineral status that reflects the duration and severity of their disease. Distal femoral measurements better reflect the bone status at the sites more at risk of fracture. Bisphosphonate therapy increases BMD during treatment and it appears to have sustained benefits with reduced fracture rates even after treatment is terminated.\nMeningomyelocele\nQuan et al. [108] found reduced forearm BMD in children with meningomyelocele. Patients with a history of fracture had substantially lower forearm BMD than those without a fracture history. The reduced upper extremity BMD in these patients with preserved upper extremity function may indicate that both systemic and local factors affect bone mineralization. Hypercalciuria in nonambulatory patients with meningomyelocele may be such a factor. Quan et al. [109] later evaluated the effect of thiazide treatment for hypercalciuria in these patients and found no change after 1\u00a0year in forearm BMD when compared to placebo-treated controls. Valtonen et al. [110] found normal forearm and LS BMD in adult patients with meningomyelocele, but reduced hip BMD in one-third of the patients. There was a trend for low hip BMD in the nonambulatory compared to ambulatory patients but not for the LS. This dissociation of LS and hip BMD values was thought to be due to relatively preserved axial loading on the LS in upright patients with meningomyelocele. The normalization of forearm BMD in these adult patients was attributed to increased upper extremity muscular stress with the use of crutches and manual wheelchairs.\nConnective tissue diseases\nJuvenile rheumatoid arthritis and systemic lupus erythematosus\nRheumatoid diseases have long been known to affect bone health negatively. Disease extent, severity, subsequent disability and CS therapy negatively impact bone mineralization. Pepmueller et al. [111] and Pereira et al. [112] found decreased regional, LS, and TBBMD in patients with JRA. The decrease was more severe in children with longer disease duration and was similar for oligoarthritis and polyarthritis. Lien et al. [113] found decreased TBBMC and TBBMD in patients with juvenile idiopathic arthritis. Disease duration and severity correlated with TBBM but not CS therapy. Henderson et al. [114] found no statistically significant difference in TBBMD in prepubertal children with mild-to-moderate JRA and no history of CS treatment when compared to normal controls. When a similar study was performed on CS-naive older girls with mild-to-moderate JRA, Henderson et al. [115] found decreased TBBMC. As with other chronic diseases of childhood, it appears that persistent disease activity through puberty results in decreased BMC. Mul et al. [116] found marked reductions in LS BMD and BMC in children with rheumatic diseases treated for at least 1\u00a0year with high-dose CS. Bianchi et al. [117] found that long-term methotrexate for JRA did not result in reduced LS or TBBMD.\nIn summary, prepubertal children with JRA of mild or moderate severity without a history of CS treatment will have TBBMD similar to healthy children. With increasing disease severity and duration, especially through puberty, TBBMC will decrease when compared to normal children.\nThese changes are in contrast to the normal DXA findings reported in patients with juvenile systemic lupus erythematosus [118, 119]. Unlike patients with JRA, these patients typically do not have bone and joint involvement and thus are more likely to have preserved BMD. However, factors that may negatively impact bone health in these patients include immobility, limited exposure to sunlight, and CS therapy.\nMusculoskeletal diseases\nFor normal mineralization to occur, adequate muscle-induced mechanical stresses are required to induce bony remodeling. Insufficient muscle mass and activity result in poor bone accrual and low BMD. Additionally, therapy of primary muscle disorders with CS also inhibits normal bone mineralization.\nDuchenne muscular dystrophy and dermatomyositis\nIn a longitudinal study of boys with Duchenne muscular dystrophy, Larson and Henderson [120] found that LS and hip BMD correlated with functional mobility level with ambulatory boys having higher LS BMD than nonambulatory boys. Hip BMD was decreased before loss of ambulation and showed progressive reduction over time. Fractures occurred in nearly half of patients, most typically involving the lower extremity and frequently resulting in loss of ambulation. Bianchi et al. [121] found decreased LS and TBBMD in ambulatory boys with Duchenne muscular dystrophy when compared to healthy boys. LS BMD was more severely reduced in the subset of Duchenne muscular dystrophy patients treated with CS. The authors noted greater lower extremity than upper extremity bone mineral loss using regional DXA analysis, especially in the CS-treated patients, and they attributed this to reduced mechanical stress on the lower extremities. Hawker et al. [122] found an increase in LS and TBBMD after 2\u00a0years of bisphosphonate treatment, supplemental calcium, and vitamin D. Improvement in TBBMD was inversely related to age at baseline.\nIn summary, patients with Duchenne muscular dystrophy have reduced LS BMD, especially in the setting of CS treatment. The low LS and hip BMD worsen with loss of ambulatory status and are frequently associated with fractures of the lower extremities.\nStudies of a small number of children with dermatomyositis have found low LS BMD that worsens with ongoing CS therapy [123, 124]. Patients treated with bisphosphonates for osteoporosis-related compression fractures show increases in LS BMD [123].\nOsteogenesis imperfecta\nOsteogenesis imperfecta includes a spectrum of genetic disorders of collagen synthesis resulting in abnormal skeletal and connective tissues. Reduced BMD and other factors result in fragile bones and multiple fractures. Fracture occurrence and DXA findings in these children vary with subtype with few fractures and normal or near-normal DXA findings in type I osteogenesis imperfecta and more frequent fractures and marked reductions in BMD in types III and IV [125, 126]. (Type II typically results in perinatal demise.) Since as many as 40% of patients with osteogenesis imperfecta will have normal BMD and BMC, a normal DXA study does not preclude this diagnosis or distinguish these cases from non-accidental injuries [125]. In recent years, clinical trials using bisphosphonates in osteogenesis imperfecta have yielded impressive results with multiple studies demonstrating increases in LS, hip and TBBMD and improved quality of life [127\u2013130]. Patients with the lowest baseline bone mass experienced the most significant gains with treatment. Improved mobility, ambulation, muscle force and reduced chronic pain and fatigue lead to improved quality of life, and the stimulus of physical activity is known to be beneficial to bone. Increased bone mass in the skull following bisphosphonate therapy in these patients implies that the drug also has a direct effect on bone accrual irrespective of physical activity [129]. The theoretical concern that this therapy might negatively impact linear growth in these children has not been shown to be true [131].","keyphrases":["pediatrics","dxa","bone mineral density","osteoporosis"],"prmu":["P","P","P","P"]}
{"id":"Naturwissenschaften-3-1-2039849","title":"Why do house-hunting ants recruit in both directions?\n","text":"To perform tasks, organisms often use multiple procedures. Explaining the breadth of such behavioural repertoires is not always straightforward. During house hunting, colonies of Temnothorax albipennis ants use a range of behaviours to organise their emigrations. In particular, the ants use tandem running to recruit na\u00efve ants to potential nest sites. Initially, they use forward tandem runs (FTRs) in which one leader takes a single follower along the route from the old nest to the new one. Later, they use reverse tandem runs (RTRs) in the opposite direction. Tandem runs are used to teach active ants the route between the nests, so that they can be involved quickly in nest evaluation and subsequent recruitment. When a quorum of decision-makers at the new nest is reached, they switch to carrying nestmates. This is three times faster than tandem running. As a rule, having more FTRs early should thus mean faster emigrations, thereby reducing the colony\u2019s vulnerability. So why do ants use RTRs, which are both slow and late? It would seem quicker and simpler for the ants to use more FTRs (and higher quorums) to have enough knowledgeable ants to do all the carrying. In this study, we present the first testable theoretical explanation for the role of RTRs. We set out to find the theoretically fastest emigration strategy for a set of emigration conditions. We conclude that RTRs can have a positive effect on emigration speed if FTRs are limited. In these cases, low quorums together with lots of reverse tandem running give the fastest emigration.\nIntroduction\nOrganisms often employ more than one mechanism to accomplish a task. For instance, animals typically navigate with multiple \u2018input channels\u2019. The classic example is homing by the rock dove Columba livia, for which magnetic fields, the sun, landmarks and geophysical processes have all been shown to be used (Wiltschko and Wiltschko 2003).\nThe range of behaviours or mechanisms organisms employ may be puzzling. At times, an apparent simplicity is observed. Defence strategies against predators are a well-studied example here. Acacia trees (Acacia spp.) either have chemical defences to ward off herbivores or have symbiotic relationships with protective ants (Rehr et\u00a0al. 1973). Hosts parasitised by Eurasian cuckoos Cuculus canorus famously reject cuckoo eggs, but never reject cuckoo chicks (Davies 2000). This simplicity may be the result of evolutionary lag but, more interestingly, may also be caused by one strategy making another maladaptive by reducing predator abundance (Planqu\u00e9 et\u00a0al. 2002; Britton et\u00a0al. 2007).\nConversely, the portfolio of behaviours may be varied and complex. Different mechanisms may complement one another, and true redundancy is often hard to show (Able and Bingman 1987). Indeed, the existence of a suite of mechanisms against a broad ensemble of predators is readily understandable. One exemplar is the vertebrate immune system (Perelson 2002).\nAnother striking example of a system in which different mechanisms augment and complement one another, but now at a collective level, is house hunting in social insects. This has become one of the model systems to study distributed decision making in animals. When the nest is destroyed, the colony has to decide collectively where to settle next during a time of crisis (Franks et\u00a0al. 2003a). Individual ants or bees have been shown to combine sophisticated assessments of potential nest sites (Seeley 1977; Seeley and Morse 1978; Mallon and Franks 2000; Franks et\u00a0al. 2003b) with various recruitment mechanisms to collate information, and thus make collective decisions (Mallon et\u00a0al. 2001; Pratt et\u00a0al. 2002; Pratt 2005; Seeley and Visscher 2003, 2004; Visscher 2007).\nA typical emigration by a colony of Temnothorax albipennis may be described as follows. When the old nest is destroyed, a fraction of ants goes out scouting to find a new home. Upon finding a nest, the nest is assessed (Mallon and Franks 2000) and ants start recruiting other ants to it with a latency that is inversely proportional to the perceived nest quality (Mallon et\u00a0al. 2001), using a process called forward tandem running (M\u00f6glich et\u00a0al. 1974). During a forward tandem run, a knowledgeable ant teams up with a na\u00efve ant. The leader slowly progresses towards the new nest, each time waiting for the follower to catch up, thereby teaching her the way (Franks and Richardson 2006). Through this slow recruitment process, information on the location of the new nest spreads, and recruiter numbers increase. Once a nest population reaches a certain quorum threshold, the recruiters switch from slow tandem running to much faster social carrying, and transport the remaining passive ants and brood to the new nests (Pratt et\u00a0al. 2002; Pratt 2005).\nThis description has been the basis of several models (Pratt et\u00a0al. 2002; Pratt et\u00a0al. 2005; Marshall et\u00a0al. 2006; Planqu\u00e9 et\u00a0al. 2006). However, a behaviour commonly employed by these ants is usually not included (but see Pratt et\u00a0al. 2005) and has never been analysed. After the quorum, recruiter ants are not only engaged in social carrying, but also regularly perform tandem runs from the new back to the old nest. These so-called reverse tandem runs (M\u00f6glich 1978) are often more common than forward tandem runs (Mallon et\u00a0al. 2001; Pratt et\u00a0al. 2002), but their function is much less well understood.\nTo maximise fitness, the colony should emigrate as quickly as possible to avoid predation and other hazards. Therefore, during house hunting, a fast build up of recruiters is essential. Why then do ants mix fast carrying with slow reverse tandem running, when they already have forward tandem running at their disposal?\nIn this paper, the role of reverse tandem running is theoretically investigated. In particular, through the use of mathematical models, we explore under what circumstances reverse tandem running can have a positive influence on emigration speed.\nMaterials and methods\nWe present two mathematical models to investigate the possible role of reverse tandem runs in ant colony emigrations. Reverse tandem running does not contribute to the decision-making process of which new nest to choose (Pratt et\u00a0al. 2002; Franks et\u00a0al. 2003a). We, therefore, restrict ourselves to emigrations to one new nest only.\nWe model the emigration as follows (Fig.\u00a01). Only a small fraction of the ants in a colony are actively involved during emigrations (Pratt et\u00a0al. 2002; Langridge 2006). In this paper, we thus divide the colony\u2019s N ants into fractions FN of active ants A, and (1\u2009\u2212\u2009F)N passive ants P. This assumption to divide ants into active and passive is a crucial one, without which the models collapse. We will come back to this in \u201cDiscussion\u201d. Numbers of scouts and recruiters are denoted S and R, respectively. The rates at which active ants leave the nest and become scouts, and scouts recruiters, are given by \u03bc and k, respectively. Forward tandem running occurs at a rate \u03bb until the quorum Q is met, after which recruiters carry passive ants and brood at a rate \u03c6. To incorporate reverse tandem runs, we need to model which ants follow these tandem runs. Available data from nest-choice experiments (Mallon et\u00a0al. 2001) suggest two possibilities: Either the reverse tandem runs are followed by ants that have not found the new nest yet, or by ants that have been carried to the new nest. These two options are not necessarily mutually exclusive: the carried ants could have been scouts. We modelled both these two possible interpretations. Model 1 assumes that reverse tandem runs are followed by uncommitted scouts in the arena, and model 2 assumes they are followed by passive ants that were carried to the new nest.\nFig.\u00a01Diagrams of the two different models for which reverse tandem runs are hypothesised to increase speed. FTR Recruitment through forward tandem running; RTR recruitment through reverse tandem running. The parameters are explained in Table 1\nTo capture in detail the influence of reverse tandem runs on emigration dynamics, we need to consider the following points:\nBoth tandem running and social carrying involve a pair of ants from two different classes. Hence, recruitment can only occur if ants of both participating classes are available;Once the quorum has been met, recruiters cannot carry and perform reverse tandem runs simultaneously (we also assume recruiters are not involved in other activities than these two).\nThe interaction between different classes of ants has been modelled using simple interaction terms. We assume that ants of both classes are well mixed in the part of the arena (or nest) where they meet. With populations of ants of size X and Y meeting, the number of ants that on average meet is then proportional to XY\/(X\u2009+\u2009Y). Importantly, the smallest class limits the interaction rate, as is to be expected.\nWe thus also have to specify how much post-quorum time recruiters spend on carrying or reverse tandem running (they are assumed not to spend any time on other behaviours). Before the quorum is met, the rate at which active ants at the old nest, A, become recruiters, R, through tandem running is given by \u03bbRA\/(A\u2009+\u2009R). Now let f be the fraction of post-quorum time spent on reverse tandem runs, and the remainder 1\u2009\u2212\u2009f spent on social carrying. Then the mean number of scouts becoming recruiters through reverse tandem runs is\nFor carried ants becoming recruiters through reverse tandem running, we have by analogy\nSimilarly, the mean number of passive ants P that become carried ants is given by (1\u2009\u2212\u2009f) \u03c6RP\/(P\u2009+\u2009R). Recruiter ants should not perform reverse tandem runs when there are no scouts or carried ants left. Therefore, we replace f by  in Eqs.\u00a01 and 2, where  and min {Cf, f}, respectively. The min operation is for computational reasons only and ensures that  decreases continuously but rapidly to zero as S or C decreases, respectively. It has no influence on the models\u2019 predictions. We drop the bar on  in the rest of the paper.\nForward tandem running only occurs before the quorum is met and carrying and reverse tandem running only after. These are modelled with functions l, c and r, respectively, as follows.\nand, setting B to A for model 1, and to P for model 2,\nTo aid the reader, we state the full equations for both models. The equations for model 1 are given by\nwith initial conditions (A,S,R,P,C)(0)\u2009=\u2009(FN\u2009\u2212\u20092\u03b5, \u03b5,\u03b5,(1\u2009\u2212\u2009F)N,0). Model 2 is specified by\nwith the same initial conditions as model 1. Models 1 and 2 only differ in the placement of the term fr(\u03bb,R,Q,C). In both models, the \u03b5 in the initial conditions is necessary to avoid singularities in the denominators of the interaction functions l, r and c. We have used \u03b5\u2009=\u20090.01 throughout.\nWe also explored a number of other models in which some assumptions were relaxed. These are briefly discussed in the final section of this paper.\nThe main hypothesis we tested on both these models was: Reverse tandem runs speed up the emigration if recruiter numbers failed to increase early in the emigration; this occurs through a combination of the new nest being hard to find and forward tandem running being prohibited. We tested this hypothesis by finding the fastest emigration strategy for given parameter settings and determining whether reverse tandem runs formed part of this optimal strategy. We first fixed the scouting parameter, \u03bc, the fraction of active ants at the old nest, F, and k, the rate at which scouts become recruiters. Then quorum size Q and fraction of post-quorum reverse tandem running time f were varied to optimise emigration speed. Individual runs were performed in Matlab using a standard ode45 solver. Emigrations were termed completed when all passive ants and active ants at the old nest had disappeared. In particular, we set the threshold for emigration completeness at P\u2009+\u2009A\u2009=\u20090.01. Optimal strategies were found using the Nelder\u2013Mead simplex method (Nelder and Mead 1965). Parameters \u03bc and F were varied on an equidistant 20\u00d720 grid spanning [0.01,0.2] \u00d7[0.05,0.5]. Recruitment latency k was set at either 0.0001 or 0.001. The ranges of these parameters are inspired by experimental estimates in (Pratt et\u00a0al. 2002; Pratt 2005). Parameter values are summerised in Table\u00a01.\nTable\u00a01Values or ranges, where applicable, for the parameters used in models 1 and 2 depicted in Fig.\u00a01ParametersDescriptionValue\/rangeNColony size250FFraction of active ants[0.05,0.5]QQuorum thresholdn.a.fFraction of post-quorum reverse tandem running timen.a.\u03bcRate at which active ants at old nest become scouts (ant\u2009\u2212\u20091 min\u2009\u2212\u20091)[0.01,0.2]\u03bbRate at which ants following tandem runs become recruiters (ant\u2009\u2212\u20091 min\u2009\u2212\u20091)0.1\u03c6Rate at which passive ants are carried to new nest (ant\u2009\u2212\u20091 min\u2009\u2212\u20091)0.2kRate at which scouts independently become recruiters (ant\u2009\u2212\u20091 min\u2009\u2212\u20091){0.0001,0.001}Parameter choices for \u03bb, \u03c6 and N were taken from the ranges in Pratt et\u00a0al. (2005).\nResults\nFor both models, the optimal emigration strategy included reverse tandem runs for a wide range of parameters, together with low quorum thresholds (Fig.\u00a02). Fixing k whilst varying \u03bc and F, the optimal strategy often contained more reverse than forward tandem runs for a large part of the parameter range (Fig.\u00a02). The fraction of time spent on reverse tandem running f and the quorum threshold Q were negatively correlated. When either the fraction of active ants F decreased or the scouting parameter \u03bc increased, fraction f increased, and the quorum Q decreased. Choosing a higher recruitment latency by decreasing k gave more reverse tandem running and lower quorum thresholds (Fig.\u00a02).\nFig.\u00a02Optimal fractions of post-quorum time spent on reverse tandem runs (top figures), and optimal quorum thresholds (bottom figures) for models 1 (left two columns) and 2 (right two columns) for varying fractions of active ants F and scouting probabilities \u03bc and for two values of recruitment latencies k. Other parameter values used are given in Table\u00a01. See text for simulation details. For both values of k, reverse tandem runs are part of the optimal emigration strategy when scouting probability is high or when fraction of active ants is low. Lowering k enhances the use of reverse tandem running\nNote that, although models 1 and 2 broadly give similar predictions, they differ in the amount of post-quorum time spent on reverse tandem runs. In model 1, this reaches a full 100% in model 1, but never so in model 2.\nOverall, the models predict that reverse tandem running should be used more than forward tandem running, and the quorum threshold lowered, if the recruitment latency increases by decreasing k (scouting ants wait longer before starting their first recruitment act), in combination with either a decreasing fraction of active ants F, or an increasing scouting parameter \u03bc. For all but very large F, the optimal quorum threshold corresponded to the time when all active ants have left the old nest to go scouting. In the absence of multiple new nests, the decision when to switch from forward tandem running to social carrying is thus best made at the old nest. Recruiters should thus apply the following rule: Continue forward tandem running until there are no ants left to perform them with and then switch to social carrying; if few forward tandem runs have been performed (by the recruiters), combine carrying with reverse tandem runs; otherwise, do not.\nThe numbers of forward tandem runs, reverse tandem runs, the numbers of carried ants in the new nest and total emigration time were computed for each of the optimal strategies of models 1 and 2 (Fig.\u00a03). We note four points. First, for a large parameter range, there are more reverse tandem runs than forward tandem runs. This is broadly consistent with the experimental data from nest choice experiments in Mallon et\u00a0al. (2001). Numbers there range between 3 and 17 forward tandems, and between 9 and 25 reverse tandems, and reverse tandems were always performed more often than forward tandems. Second, as a validation of our optimisation method, note that the optimal emigration time varies smoothly under parameter changes, as is to be expected for this type of model. Third, for model 1, despite a clear drop in the post-quorum time spent on reverse tandem runs with increasing F (see Fig.\u00a02, top left), the number of reverse tandem runs in fact varies smoothly. Fourth, the number of carried ants that remained in the nest at the end of the emigration is clearly different between models 1 and 2. In model 1, this number is just (1\u2009\u2212\u2009F)N, the number of passive ants in the colony. In model 2, however, over half of the colony may end up being recruiters by drawing recruits from the carried class using reverse tandem runs.\nFig.\u00a03Numbers of forward and reverse tandem runs, number of carried ants and emigration time, computed for each of the optimal strategies for models 1 (top row) and 2 (bottom row), illustrated in Fig.\u00a02. See that figure for details and parameter choices. Here we have only illustrated k\u2009=\u20090.001\nDiscussion\nTo maximise their fitness, ants should try to achieve the fastest emigrations to minimise vulnerability (Franks et\u00a0al. 2003a; Franks et\u00a0al. 2003b). Therefore, the active ants either have to become scouts, discover a new site and then become recruiters or wait at the old nest until a recruiter leads them to the new nest. Both of these processes may be hampered: When all the active ants go out scouting, recruiter numbers slowly increase if the new nest is hard to find or if those few cannot find any active ant back at the old nest to tandem run with. In terms of the models, this could occur if scouts slowly become recruiters (low value for k), in combination with either a small class of active ants at the old nest (F is small) or all active ants having gone scouting (high value for scouting rate \u03bc). Under either or both of these circumstances, the model predicts that ants should not waste time trying to recruit by forward tandem runs but should do the next best thing and use a low quorum threshold to quickly switch to carrying. The recruiters should then invest a fraction of their time to recruit scouts or carried ants using reverse tandem runs, thus boosting the recruiter population and speeding up the emigration.\nConversely, the model also predicts that reverse tandem runs should not be used if either the new nest is easy to find (recruiter numbers then build quickly anyway), or when there are many ants to follow a forward tandem run.\nThese predictions fit quite well with previous experimental work. Ants have been shown to leave their intact old nest if the new nest is sufficiently better, but have lower standards when their nest is destroyed (Dornhaus et\u00a0al. 2004). In these experiments, reverse tandem runs were mainly observed when the old nest was destroyed, combined with few forward tandem runs. The model offers a simple explanation for this: The greater panic might have caused fewer scouts to remain at the old nest, thereby obstructing early recruitment.\nWhilst investigating speed\u2013accuracy trade-offs, Franks et\u00a0al. (2003a) found lower quorums under harsh than under mild conditions. This again fits with the models. On the other hand, the models also predict higher numbers of reverse tandem runs. In the experiments, this difference in reverse tandem running activity between mild and harsher conditions was found to be non-significant (Franks et\u00a0al. 2003a).\nCritique on model 2\nAlthough evidence for which ants follow reverse tandem runs is scarce, both models give the same qualitative predictions. Note, however, that the dynamics of the different ant classes during a simulated emigration in model 2 poorly match observed experimental dynamics (see, e.g., Planqu\u00e9 et\u00a0al. 2006). In many cases, the final number of ants active in an emigration is much greater than the original number of active ants. An example is given in Fig.\u00a04. These recruiter numbers far exceed observed numbers of active ants (available in Table 3 in Pratt et\u00a0al. 2005). Indeed, simulated emigrations often end with half the colony being recruiters and less than half carried into the new nest (Fig.\u00a03, bottom row, third from left).\nFig.\u00a04Examples of temporal dynamics for models 1 and 2. At \u03bc\u2009=\u20090.05, F\u2009=\u20090.1447, we have taken parameters optimal for models 1 and 2, respectively. Notice that, in model 1, the number of recruiters rises to about 35, but in model 2, there are no less than 100 recruiters at the end of the emigration, indicating that recruitment from the carried class (model 2) may give rise to very unrealistic emigration dynamics\nReverse tandem activity\nFigure\u00a02 (top left) shows that recruiters in model 1 should use a sequential strategy if F is small (and \u03bc is large): When the quorum is met, they first spend all of their time on reverse tandem runs until all scouts have become recruiters, and then switch to carrying. In contrast, when F is large, recruiters mix tandem running and carrying. This qualitative difference may be understood as follows.\nThe total number of recruiters is bounded by FN, the number of active ants in the colony. As F decreases, the remaining recruiters take longer to carry all the passive ants. Hence, the time costs for not having the recruiters increases and the time to recruit the remaining scouts decreases (as there are fewer scouts too). Hence, in this situation, recruiters should devote their post-quorum time, first, all on reverse tandem running before starting carrying.\nWhen F is large, the reverse argument applies. With less passive ants, there should be less emphasis on additional recruitment by reverse tandem running. One does not have to make many hands if the work was light to start with.\nNote that this behaviour for F \u21920 is different for model 2. In this paper, we never observe sequential strategies (Fig.\u00a02, top right), as there is no end to building recruiter numbers but by completing the entire emigration.\nThis argument also explains another difference between these models: The number of reverse tandem runs during an emigration. In model 1, there is a clear maximum for intermediate F, whereas in model 2 the number of reverse tandems strictly decreases with F (Fig.\u00a03, second from left, top and bottom).\nNonlinearities in the models and divisions between active and passive ants\nContrary to the models in this paper, two previous models of house hunting by T.\u00a0albipennis ants (Pratt et\u00a0al. 2002; Planqu\u00e9 et\u00a0al. 2006) assumed linear terms for tandem running and social carrying. There, these processes occurred at rates only proportional to the number of recruiters. The predictions of the current models proved to be strongly dependent on the assumption of non-linearity of these terms. The corresponding linear models predicted that reverse tandem running should not be used for practically any parameter choices in F and \u03bc or k.\nAnother ingredient in this model shared with the previous (Pratt et\u00a0al. 2002; Planqu\u00e9 et\u00a0al. 2006), models of this collective decision-making system is the division between active and passive ants. There is as yet little experimental evidence suggesting this division really exists. All we know at present is that a limited fraction of ants is actively engaged during an emigration. We have thus also explored models in which this division was absent, using both linear and non-linear interaction terms such as those in models 1 and 2 presented in this paper. In none of these models did reverse tandem running contribute to the optimal emigration speed. The division between active and passive ants is thus a crucial ingredient for reverse tandem running to have a positive impact on emigration speed, which should be experimentally validated.\nHypothesised explanations\nSeveral hypotheses on the potential role of reverse tandem running have been put forward (Pratt et\u00a0al. 2002). First, the ants might have a \u201chome nest\u201d, which changes during the emigration, thereby reversing the direction of any recruitment events from \u201chome\u201d to another nest (Pratt et\u00a0al. 2002). If true, this would predict a change of direction when about half of the colony had been displaced. This is not in agreement with the available data (Mallon et\u00a0al. 2001). Moreover, this hypothesis does not offer a suggestion why tandem running often occurs early in the emigration. In other words, it might explain the direction, but not the occurrence itself.\nSecond, it has been suggested that reverse tandem running may re-allocate recruitment (Pratt et\u00a0al. 2002). Again, this does not fit the available data from Mallon et\u00a0al. (2001). Reverse tandem runs were nearly always observed between the best nest and the old nest. The models in this paper do not incorporate choice between nest sites, but we conjecture that early flexible commitment (Planqu\u00e9 et\u00a0al. 2006) will be more efficient in redirecting ants to better nests than late recruitment. Other experimental results also corroborate that reverse tandem running does not influence the decision-making process (Franks et\u00a0al. 2003a).\nThe first models in which reverse tandem runs have been explicitly incorporated to analyse their role have yielded clear predictions: under a range of conditions, we expect a negative correlation between levels of early and late recruitment. This finding lends itself well to simple experiments, and we aim to present those in the near future.\nThe build up of recruiter numbers serves two purposes: to decide on a nest and to increase the number of ants actively involved in transport. The decision-making process and the implementation of this decision are thus conflated. This in itself is a side-effect of the distributed nature of this system. Reverse tandem running may thus be a logical extension to overcome this inherent problem. This suggests that such additional backup behaviours could be a common feature of decentralised collective decision-making systems.","keyphrases":["temnothorax albipennis","tandem running","social insects","recruitment methods","collective behaviour"],"prmu":["P","P","P","R","R"]}
{"id":"Matern_Child_Health_J-2-2-1592152","title":"The History of Preconception Care: Evolving Guidelines and Standards\n","text":"This article explores the history of the preconception movement in the United States and the current status of professional practice guidelines and standards. Professionals with varying backgrounds (nurses, nurse practitioners, family practice physicians, pediatricians, nurse midwives, obstetricians\/gynecologists) are in a position to provide preconception health services; standards and guidelines for numerous professional organizations, therefore, are explored. The professional nursing organization with the most highly developed preconception health standards is the American Academy of Nurse Midwives (ACNM); for physicians, it is the American College of Obstetricians and Gynecologists (ACOG). These guidelines and standards are discussed in detail.\nIntroduction\nFrom the earliest recorded time, women have been advised to increase their level of wellness and to avoid hazardous substances before becoming pregnant. Plutarch wrote that the ancient Spartans: \u201c...ordered the maidens to exercise themselves with wrestling, running, throwing the quoit and casting the dart, to the end that the fruit they conceived might, in strong and healthy bodies, take firmer root and find better growth\u201d [1]. In the Old Testament [2], the following passage appears: \u201cAnd the angel of the Lord appeared unto the woman, and said unto her, \u2018Behold now thou art barren, and bearest not: but thou shalt conceive and bear a son. Now, therefore beware, I pray thee, and drink not wine and strong drink, and eat not any unclean thing.\u2019\u2009\u201d\nAs the modern practice of obstetrics evolved, it became a specialty that separated women\u2019s prepregnancy wellness from prenatal care, but in the last quarter century this has begun to change. The dominant model of prenatal care as the main perinatal prevention strategy has been reexamined, and recognition of the importance of the prepregnancy period has emerged. Today the general public and health care professionals could easily be overwhelmed by the amount of information available which promotes prepregnancy or preconceptional health. A Google search (conducted April 25, 2005) of the term \u201cpreconception health\u201d identified 630,000 hits. On the same day a search of the National Library of Medicine\u2019s PubMed found 338 entries for \u201cpreconception health\u201d with the earliest published in 1978, and 103 articles for \u201cpreconceptional\u201d with the earliest published in 1982; in addition, most of the articles identified through the two search terms were unique. The nation\u2019s approach to women\u2019s health care may well be at the tipping point of redefining the perinatal period to include women\u2019s wellness across the reproductive life span as an appropriate and favored approach to impacting reducing poor pregnancy outcomes.\nStimulated by research findings which underscored the limitations of traditional prenatal care on decreasing the incidence of congenital anomalies, leaders in the health community, professional organizations, advocacy groups and federal government began actively encouraging redefinition of the United States\u2019 perinatal prevention paradigm in the early 1980s. Because the usual pathways to impact on perinatal outcomes (prenatal and neonatal care) often start too late to achieve primary prevention, the need to reach women with prevention opportunities before prenatal care was recognized and the concept of preconceptional health promotion emerged. This article explores the history of the preconception movement in the United States and the current status of professional practice guidelines and standards.\nThe decade of the eighties\nOne of the first federal position papers to acknowledge the need to change the nation\u2019s approach to prevention appeared in 1979; while focused on the need to develop a continuum of child health care, it included the concept of prepregnancy care [3]. The programmatic elements in the proposed comprehensive package of integrated services were identified as:Interconceptional carePrenatal carePerinatal careChild Health careServices for handicapped childrenAdolescent services\nInterest in moving away from categorical care to more integrated and comprehensive services has subsequently become a hallmark of current efforts to redefine the perinatal prevention paradigm. Why the list employed the word \u201cinterconceptional\u201d rather than preconceptional is not clear. Since 1979, \u201cinterconceptional\u201d care has come to represent efforts to address health status between pregnancies, birth spacing and intendedness of subsequent conceptions; \u201cpreconceptional\u201d generally refers to the woman\u2019s health status and risks before the first pregnancy and her health status shortly before any conception. \u201cPericonceptional\u201d usually refers to the time immediately before conception through the period of organogenesis. It is important to note that this nomenclature is inconsistently applied and not universally accepted. In England, for instance, \u201cpreconceptional\u201d is referred to as \u201cpreconceptual.\u201d\nBy 1985, the concept of preconceptional care and its potential advantages began to gain momentum. In that year, the Institute of Medicine published Preventing Low Birthweight [4] and noted that numerous opportunities exist before pregnancy to reduce the incidence of low birth weight, but that they are too often overlooked in favor of interventions during pregnancy. The Committee emphasized the importance of prepregnancy risk identification, counseling and risk reduction; health education related to pregnancy outcome in general, and to low birthweight, in particular. The Committee supported restructuring the perinatal prevention paradigm by noting:\u201cMuch of the literature about preventing low birthweight focuses on the period of pregnancy\u2014how to improve the content of prenatal care, how to motivate women to reduce risky habits while pregnant, how to encourage women to seek out and remain in prenatal care. By contrast, little attention is given to opportunities for prevention before pregnancy. Only casual attention has been given to the proposition that one of the best protections available against low birthweight and other poor pregnancy outcomes is to have a woman actively plan for pregnancy, enter pregnancy in good health with as few risk factors as possibly, and be fully informed about her reproductive and general health.\u201d (p. 119)\nThe IOM Committee advocated that family planning services be positioned as an essential component of effective preconceptional initiatives, thereby supporting the integration of services rather than reinforcing firm boundaries of categorical programs. It also recommended that the content of reproductive health education, particularly in schools and family planning settings, be expanded to introduce concepts of prepregnancy wellness. The third recommendation of the Committee was to develop the notion of preconceptional consultation to identify and reduce risks associated with poor pregnancy outcomes, particularly for women who had already experienced a poor outcome.\nIn 1989 another federally appointed committee, The Expert Panel on the Content of Prenatal Care, gave a strong endorsement to preconceptional health when it recommended that the preconception visit may be the single most important health care visit when viewed in the context of its effect on pregnancy [5]. The Panel went on to suggest that the last family planning visit should be the preconception visit and that the concept of preconception care should become a routine part of prenatal care with accompanying reimbursement and coverage included in all health insurance plans. However, the Panel indicated that preconception care is likely to be most effective when services are provided as part of general preventive care or during primary care visits for medical conditions. This approach has come to be referred to as \u201copportunistic care\u201d because it takes advantage of the opportunities afforded by visits for other reasons.\nSpecific components of preconception care were identified by the Panel as (1) risk assessment, (2) health promotion and (3) intervention and follow-up. The Panel advocated that the preconception assessment include a holistic range of investigations including: individual and social conditions, adverse health behaviors, medical, psychological and environmental conditions and barriers to family planning and early prenatal care enrollment.\nIn 1983, the American Academy of Pediatrics (AAP) and the American College of Obstetrics and Gynecologists (ACOG) in partnership with the March of Dimes Birth Defects Foundation published the first Guidelines for Perinatal Care [6]. Appendix C of those first guidelines notes that \u201cPreparation for parenthood should begin prior to conception. At the time of conception the couple should be in optimal physical health and emotionally prepared for parenthood\u201d (p. 257). The Guidelines indicated that when pregnancy is contemplated, the preconception visit should be part of a comprehensive gynecologic examination, and the history should include a detailed family history including ethnic background as well as investigation of lifestyle issues, religion, the home and work environments, hobbies, pets, immunizations, medications and dietary habits. The guidelines recommended use of an extensive checklist to systematically explore areas of potential significance. Based on the patient\u2019s history, the \u201ccounseling and instruction session\u201d could be focused in a variety of directions including testing for genetically transmitted diseases and the parent\u2019s carrier status, as well as counseling regarding potential teratogenic exposures, exploration and interventions to address medical problems prior to pregnancy, interconceptional care to address the likelihood of recurrence of any complications experienced in previous pregnancies, including congenital anomalies, and the importance of recording menstrual dates to allow the earliest possible initiation of prenatal care.\nIn 1985, ACOG provided a small grant to Moos and Cefalo to develop and test a checklist which came to be known as the preconceptional health appraisal [7, 8]. The appraisal was designed to aid providers in conducting comprehensive screening for preconceptional risks in a time-efficient manner and employed a self-assessment tool with built-in educational feedback. First introduced into local health department family planning clinics in North Carolina, it was adopted over the next several years by numerous state health departments across the country as well as private providers, large health maintenance organizations and several Canadian provinces. The commitment of public health organizations to introduce women to the concepts of preconceptional health during routine family planning visits placed the public sector in the position of being a leading innovator in the preconception movement. One state that demonstrated particular commitment to changing the perinatal prevention paradigm was Wisconsin. By the mid-1980\u2019s, the Wisconsin Association for Perinatal Care was calling for redefinition of perinatal care to include the preconceptional period [9], and it has been unwavering in its commitment to improve pregnancy outcomes through preconceptional health promotion strategies ever since.\nConcurrent with the above noted activities, additional credence to the benefits of preconceptional health promotion and primary prevention was provided when two nationally recognized obstetrician-gynecologists published books on the topic, one for the professional community and one for the consumer market [7, 10].\nThe decade of the nineties\nHealthy People 2000, the national health promotion and disease prevention objectives for the United States, published in 1990, moved preconceptional care into a standard expectation within the health care system when it defined it as a one of its service and protection objectives. The specific objective reads:Increase to at least 60% the proportion of primary care providers who provide age-appropriate preconception care and counseling (p. 199)\nSupporting rationale for the objective states that \u201cthe purpose of preconception care and counseling is to ensure that couples are healthy prior to pregnancy and prepared to assume the responsibilities of parenthood, thereby reducing the risk of poor pregnancy outcomes\u201d (p. 199). The remainder of the rationale not only echoes the position of the earlier 1989 Expert Panel on the Content of Prenatal Care regarding the opportunities of preconception care to address many medical conditions, personal behaviors and environmental conditions [5] but also stresses preconceptional health promotion as an opportunity to educate young people about the risks of sexual activity, including unintended pregnancies, disease-related infertility and cancers [11]. In 1993, the March of Dimes Birth Defects Foundation published Toward Improving the Outcome of Pregnancy: The 90s and Beyond which introduced the concept of \u201creproductive awareness\u201d as the basic health promotion strategy needed to improve pregnancy outcomes [12]. The document, often referred to as TIOP, reflects the position of the March of Dimes\u2019 Committee on Perinatal Health. Because awareness of reproductive risks, healthy behaviors and family planning options is essential to improving the outcome of pregnancy, the committee declared that \u201ca society-wide change in reproductive awareness is needed in the United States\u201d (p. 12) and called for \u201ca new strategy to reach each woman of childbearing age with reproductive awareness messages at every health encounter\u201d(p. 12). The committee called upon health care providers to move away from the expectation that the prevention of poor pregnancy outcomes is only the professional responsibility of obstetrical providers by noting:\u201cA woman\u2019s health status in relationship to pregnancy usually is not considered until the first prenatal visit, and prenatal care often is segregated from other health care by provider type and by payment mechanism. Yet women of childbearing age have many encounters with the health care system. These include visits to gynecologists, pediatricians, internists, family physicians, nurse midwives and nurse practitioners, in settings such as public health, school health, women\u2019s health, substance abuse treatment, family planning, and sexually transmitted disease clinics and private offices (p. 13).\u201d\nTIOP goes on to call for professional standards, structures and financing to be set in place to ensure an annual preconception or interconception risk reduction visit for every woman from menarche to menopause and that a prepregnancy planning visit become a standard component of maternity care, routinely available to all women and integrated into the perinatal care delivery system. TIOP also stressed that reproductive awareness is not sex or gender specific and that the need to reach out to males, especially during the preconception and interconception periods, requires thoughtful and innovative strategies.\nACOG published its first technical bulletin on preconception care in May, 1995 [13] The bulletin called for thorough and systematic identification of risks, the provision of education individualized to the patient\u2019s needs, and the initiation of any desired interventions. Risk identification was broadly defined to include the areas of medical, reproductive, and family histories, nutritional habits, drug and environmental exposures and social issues and these efforts were to be targeted at improving outcomes for both the mother and the fetus ACOG took the position that rather than ensuring healthy pregnancy outcomes as suggested by other position papers, preconception care allows the woman and her partner, if so desired, to make informed-decisions. It states:\u201cOnce information is available, the patient can be informed about the certainty or limitations of available information, what the risks of pregnancy may be, and precautions that may be beneficial. After women have been informed of the increased risks pregnancy may pose to their health or the health of a fetus or both, they can accept the increased risks, choose to modify their risks or opt to avoid childbearing (p. 2).\u201d\nACOG cautioned against over promising the benefits of preconceptional care to both providers and patients by specifically noting that preconceptional services do not guarantee good pregnancy outcomes. It noted that placing emphasis only on women who are planning their next conception or women who have been identified as high-risk will result in a significant number of missed opportunities for primary prevention. Women who experience an unintended pregnancy are at least as likely to have risk factors for poor pregnancy outcomes as women who consciously plan the timing of their pregnancy. Therefore, ACOG recommended that routine visits by women who may, at some time, become pregnant are important opportunities to emphasize the importance of preconceptional health and habits and the advantages of planned pregnancies. Finally, ACOG called for a coordinated multispecialty effort directed by the obstetrician-gynecologist as a means to provide a comprehensive framework for preconceptional health care for all women of childbearing potential.\nThe current decade\nIn 2002, the fifth edition of the AAP\/ACOG Guidelines for Perinatal Care was published [14]. In the preceding editions, preconception care had moved from an appendix item to the main text in preceding editions. This latest edition, however, reflects a shift away from framing preconceptional care as appropriately targeted toward prospective parents who are contemplating pregnancy to an emphasis on integration of preconceptional health promotion into all health encounters during a woman\u2019s reproductive years.\nWhile some efforts to operationalize a redefined perinatal prevention paradigm grew, others dropped back. For example, Healthy People 2010 eliminated preconceptional health as a specific objective [15]. Except for the objective, \u201cIncrease the proportion of pregnancies begun with an optimum folic acid level\u201d (target: 80%), the relevance of a woman\u2019s health status at the time of conception to pregnancy outcomes is not referenced in the document, and reliance on prenatal and neonatal care to impact on pregnancy outcomes is reinforced as the preferred strategy to meet the perinatal objectives.\nEvolving standards and guidelines from professional organizations\nAs the concept of preconception care has evolved in the health care community, several professional organizations have addressed its importance for their members, and some have developed specific guidelines or standards for preconception care.\nNursing organizations\nIn nursing, there are several professional organizations whose members care for reproductive age women. The AANP (American Academy of Nurse Practitioners) is the largest national professional organization for nurse practitioners of all specialties, with 15,000 members. While many nurse practitioners might be delivering preconception health services, AANP has not developed specific educational standards specific to this topical area, nor do their generic practice standards include preconception health. According to AANP Executive Director Dr. Judith Dempster (personal communication, April 11, 2005), members with questions about preconception health standards would be referred to specialty organizations such as AWHONN.\nAWHONN (Association of Women\u2019s Health, Obstetric and Neonatal Nurses) is the official professional organization for women\u2019s health and obstetric nurses, and is comprised of 22,000 nurses whose goals are to promote excellence in nursing practice. While AWHONN does not have specific standards of care for women during the preconception period, they do offer many products to their members which discuss preconception health, including a practice monograph which outlines the importance and components of preconception care [16]. They also publish a Clinical Position Statement which states that AWHONN supports legislation and policies that encourage women of childbearing age to consume 400 micrograms of synthetic folic acid every day [17]. In addition, AWHONN is also a member of the National Council on Folic Acid (NCFA), a partnership of over 80 national organizations and associations, state folic acid councils and government agencies. While not strictly a preconceptional project, AWHONN developed a specific Clinical Position Statement on Smoking and Childbearing, urging nurses to screen women for tobacco use and help women stop smoking [18]. Although nurses initiated this program during pregnancy, it had major interconceptional ramifications for the women involved, and has added to the literature on smoking cessation for women of reproductive age [19].\nACNM, the American College of Nurse Midwives is the professional organization for all nurse midwives in the United States, and represents over 7,000 nurse midwives who are providers of comprehensive women\u2019s health. In their Core Competencies for Basic Midwifery Practice (www.acnm.org\/prof\/display.cfm?id=137) they have specific educational and practice standards for preconception health for women. These standards are:\u201cV. Components of Midwifery Care: The Primary Health Care of WomenB. Applies knowledge of midwifery practice in the preconception period that includes, but is not limited to, the following:1. Assessment of individual and family readiness for pregnancy, including emotional, psychosocial and sexual factors2. Impact of health, family and genetic history on pregnancy outcomes3. Influence of environmental and occupational factors, health habits, and behavior on pregnancy planning4. Health and laboratory screening to evaluate the potential for a healthy pregnancy\u201d (p. 3)\nNAPNAP (the National Association of Pediatric Nurse Practitioners) does not publish any guidelines or standards for preconception care. Their members provide services for adolescents, but the organization has not yet addressed preconception care in a structured manner (personal communication Dr. Karen Kelly Thomas, Executive Director NAPNAP, April 13, 2005). They have, however, published at least one article in their official journal on the topic [20].\nIn terms of general nursing education, the AACN (The American Association of Colleges of Nursing) does not offer specific guidelines for what is to be taught to student nurses about preconception health, but rather sets standards for overall educational goals.\nOrganizations for all professionals\nThe March of Dimes, while not an organization which targets just one type of professional, has been producing numerous materials for health care professionals about preconception health for many years. Materials and products that the March of Dimes produces for providers of care include: their nursing module Preconception Health Promotion: A Focus for Women\u2019s Wellness [21], a Preconception Curriculum consisting of power point slides for Obstetricians, Family Medicine specialists, Pediatricians, and Internists which suggests that every visit to a health care provider should include elements of preconception care, a Preconception Screening and Counseling Tool, grants to the March of Dimes Chapters for preconception services, an educational program called Genetics and Your Practice (CD and online), and a Genetic screening pocket guide. Many of these can be found on their website www.marchofdimes.com, and their Spanish language website www.nacersano.org. They also produce an e-preconception newsletter (Spanish), an education program for Spanish women called Comenzando Bien, and pamphlets for patient education such as Are You Ready?, Think Ahead for a Healthy Baby, Folic Acid brochures, and Pre-Pregnancy Planning Fact Sheet.\nThe CDC, the March of Dimes, and the National Council on Folic Acid (NCFA) have organized a national folic acid promotion effort for the prevention of serious birth defects of the brain and spine (neural tube defects or NTDs). The goal of the effort is to teach all women about the importance of getting enough folic acid every day. The effort aims to reach every woman who could possibly become pregnant, as well as teach health care professionals and community advocacy groups about the importance of folic acid. The CDC, the March of Dimes, and NCFA have created messages, materials, websites, and other tools to reach providers and women with the folic acid message. In 2005, the CDC and March of Dimes collaborated to develop a national summit on preconception care in order to bring together all the stakeholders in the subject; this supplement issue of Maternal Child Health is one of the many consequences of these efforts by CDC\/MOD.\nPhysician organizations\nThe American Academy of Pediatrics represents 60,000 pediatricians, many of whom provide adolescent services to young women of reproductive age. They publish, along with the American College of Obstetricians and Gynecologists, Guidelines for Perinatal Care, the document which is generally accepted to be the outline for acceptable care during the perinatal period for women and infants. In the current edition, 5 pages are devoted to preconception care, which is described as \u201cthe identification of those conditions that could affect a future pregnancy or fetus, and that may be amenable to intervention (p. 73)\u201d [14]. The Guidelines note that all encounters with women of reproductive age should include counseling about health which could optimize pregnancy outcome. AAP is also a member of the National Organization on Fetal Alcohol Syndrome, which addresses medical school curriculum for fetal alcohol syndrome, and AAP supports numerous state-wide programs to enhance adolescent health before childbearing. In the American Board of Pediatrics study guide for the certifying examining for Adolescent Medicine certification, materials included are prevention of pregnancy, prevention of substance use, and general reproductive health care, all of which contribute to better preconception health (www.aap.org\/sections\/adol\/adol.pdf).\nThe American Academy of Family Physicians (AAFP) represents 94,000 family physicians, residents and medical students in the United States, and sets the professional standards for care delivered by family physicians. While no specific guidelines or standards for preconception health services developed by AAFP could be located, AAFP addresses the issue of preconception care in many articles in their official journal, and in other practice venues such as suggestions to increase use of folic acid [22, 23].\nCREOG is the Council on Resident Education in Obstetrics and Gynecology, and is charged with setting the educational standards for obstetricians and gynecologists in training. CREOG\u2019s Educational Objectives are contained in its core curriculum [24]. Regarding preconception care, CREOG\u2019s core curriculum states: \u201cIn evaluating patients for preconceptional care, obstetrician gynecologists must assess those factors of the history, physical examination, and diagnostic studies that pregnancy would alter; assess the patient\u2019s access to and compliance with a plan of prenatal care; and consult with or refer her to other experts on specific conditions that may arise during the pregnancy.\u201d(p. 39). Its standards are as follows:\u201c II. Antepartum Care\u2014Preconceptional Care1. Perform a thorough history, assessing historical and ongoing risks that my affect future pregnancy2. Counsel a patient regarding the impact of pregnancy on maternal medical conditions.3. Counsel a patient regarding the impact of maternal medical conditions on pregnancy4. Counsel a patient regarding appropriate lifestyle modification conducive to favorable pregnancy outcome.5. Counsel a patient regarding appropriate preconception testing.6. Counsel a patient regarding pregnancy associated risks and conditions, such as advanced age, hypertension, diabetes, genetic disorder, prior aneuploid or anomalous fetus\/newborn\u201d (p. 49)\nACOG, the American College of Obstetricians and Gynecologists represents 46,000 obstetricians and gynecologists in the United States, and in 2005 celebrated its 50th anniversary. ACOG has advocated for preconception care for several decades, and offers the most detailed guidelines for the provision of preconception services. ACOG\u2019s acknowledges that while ideal health prior to pregnancy does not guarantee a perfect or uncomplicated pregnancy, women who are considering pregnancy should undergo comprehensive preconception evaluation, examination, and counseling. However, preconception care is best when it is provided as part of primary health care, rather than as a discrete new category of health care, particularly since almost one-half of all pregnancies in the United States are unintended [25, 26].\nComponents of preconception care\u2014ACOG\nIn examining multiple documents produced by ACOG in recent years, certain components of preconception care can be delineated.\nPeriodic assessments\nPreconception counseling should occur at periodic assessments for all reproductively capable women. These assessments may be yearly or as appropriate [27]. Counseling should include discussions about the likelihood of pregnancy (planned or unplanned), counseling on appropriate medical care and behavior to optimize pregnancy outcomes, specific health risks the woman has that may predispose her to an adverse pregnancy outcome if not addressed, and how to either effectively prevent an unplanned pregnancy or to maximize her physical and emotional health prior to a planned or even unplanned event. Even clinicians that do not provide prenatal care should be able to provide preconception counseling and screening to their reproductively capable patients.\nThe patient\u2019s history should be reviewed and updated on an annual basis or more often if appropriate. The history should include a review of any medical conditions and medications the patient may be using. It is important to ask about prescription as well as over the counter drugs, herbs, and supplements. Inquiries about occupational and hobby exposures to chemicals, solvents, or heavy metals should be made. In this manner, potentially teratogenic agents may be identified.\nThe patient\u2019s reproductive history may provide important clues about future pregnancy risks. Recurrent miscarriages should raise suspicion of a possible genetic or chromosomal problem. The neonatal outcome of previous pregnancies should be noted but it is equally as important to ask women about the current health of any children they have as some congenital disorders do not become manifest until several months or even years after birth and may not be considered by the family to represent a congenital defect.\nGenetic disorders and genetic screening\nThe woman\u2019s family history and ethnicity for genetic disorders and malformations should be obtained. Carrier screening for Tay-Sachs disease is recommended for individuals of Eastern European (Ashkenazi) Jewish, French Canadian and Cajun descent [28]. ACOG recommends offering cystic fibrosis screening to individuals with a family history, reproductive partners of persons with cystic fibrosis, and to couples in whom one or both partners are Caucasian and are planning pregnancy [29].\nCarrier screening for Tay-Sachs, Canavan disease, cystic fibrosis, and familial dysautonomia should be offered to Ashkenazi Jewish individuals during preconception screening. These same individuals may be offered or may request screening for Gaucher disease, Niemann-Pick disease type A, Fanconi anemia group C, Bloom syndrome, or mucolipidosis IV as well. Carrier screening should be offered to any individual with a positive family history of one of these disorders; the screening should be for the specific disorder. When only one partner is of Ashkenazi Jewish descent, that individual should be screened first. Except for Tay-Sachs and cystic fibrosis, the carrier frequency and detection rate for the other disorders is unknown.\nIf an individual is found to be a carrier for Tay-Sachs or one of the other aforementioned diseases, their relatives are at risk for carrying the same mutation; carriers are encouraged to inform their relatives of the risk and availability of carrier screening [28].\nOther screening tests exist for genetic disorders with an increased incidence in specific ethnic groups, e.g., alpha or beta thalassemia screening in Orientals, beta thalassemia screening for those of Mediterranean descent, and sickle cell disease screening for African-Americans.\nWomen who have had a previous pregnancy complicated by autosomal trisomy, a sex chromosome aneuploidy, or if either partner has a chromosome translocation or inversion or aneuploidy should be given education about their increased risk of recurrence in future pregnancies and a referral for pre-pregnancy genetic counseling should be offered [30].\nImmunizations and infectious diseases\nThe woman\u2019s immunization status, history of childhood diseases, and risk for exposure to hepatitis, HIV, or other sexually transmitted infections needs to be updated at least annually. When practical, preconceptional immunization of women to prevent disease in the offspring is preferred to vaccination of pregnant women with certain vaccines [31].\nApproximately 30% of US adults have serologic evidence of prior exposure to toxoplasma gondii, a known teratogen. Screening for Toxoplasma gondii is controversial because evidence that treatment prevents congenital disease is lacking. All reproductively capable women should be advised to avoid eating undercooked or raw meat, wear gloves when working with soil, and practice safe-handling techniques when changing cat litter [32].\nCongenital cytomegalovirus (CMV) infection occurs in 1% of all live births in the United States and causes major neonatal illness in 5% to 10% of these cases. Women with young children or those who work with young children may be counseled about reducing the risk of CMV through universal precautions, e.g. the use of latex gloves and rigorous hand-washing after handling diapers or after exposure to respiratory secretions. There are currently no other specific recommendations as there is no evidence that screening and\/or treatment programs prevent infection [32, 33].\nAll women should be kept current with age-appropriate vaccines. Reproductive aged women should be asked about previous infection with varicella and offered vaccination if they report no known history of chickenpox. Conception should be delayed until 1 month after the second injection is given. Women who expect to be at least 3 months pregnant during the influenza season (November to April) should be vaccinated.\nBehavioral\/psychosocial factors\nA woman\u2019s lifestyle and social behaviors should be reviewed and updated frequently. The use of alcohol, smoking, or other substances may have significant effects on pregnancy. Smoking during pregnancy is associated with low birth weight, abruptio placenta, preeclampsia, and preterm labor [34]. A woman\u2019s personal situation has a significant effect on her health and often changes over time. Clinicians should ask all women about intimate partner violence and women should be aware that it may escalate during pregnancy. In addition, a woman\u2019s mental health history may provide clues about recurrence risks either during or after a pregnancy.\nA woman\u2019s dietary habits should be reviewed. Daily folic acid intake of 0.4\u00a0mg should begin at least 1 month before pregnancy and continue through the first trimester. For women who have had a child with a neural tube defect, a higher dose of folic acid (4.0\u00a0mg\/d) is recommended and has been shown to decrease the recurrence rate of neural tube defects [35]. Women who may become pregnant\u2014planned or unplanned\u2014should be aware of recent FDA warnings to avoid specific types of fish as they may be high in methyl mercury\u2014a known neurotoxin to a developing fetal nervous system. Women should also be questioned about the need for any dietary restrictions they may have required in childhood. Women with PKU in childhood may not currently be adhering to a phenylalanine free diet but hyperphenylananinemia is associated with neurological impairment in a developing fetus.\nMaternal prepregnancy weight is associated with several adverse outcomes. Women weighing more than >300\u00a0lbs (>136\u00a0kg) prior to pregnancy have a markedly increased risk for developing gestational diabetes, preeclampsia, and requiring a cesarean delivery compared with women weighing 100\u2013149\u00a0lbs (45\u201367\u00a0kg). In one study, even among a subsample of women who did not have any diabetic or hypertensive diseases, excess weight significantly increased the likelihood of macrosomia and NICU treatment [36]. The relationship between maternal obesity and perinatal outcomes is necessary when discussing routine care as well as specific preconception planning.\nMedical history and conditions\nWhile most reproductive aged women are healthy, there are specific medical conditions associated with adverse pregnancy outcomes if untreated or treated poorly. Some medical conditions are contraindications to pregnancy. Hypertension should be under control prior to initiation of a pregnancy and women using angiotensin-converting enzyme inhibitors and angiotensin II receptor antagonists who are considering pregnancy should be switched to other agents and adjusted on these medications prior to conception. Certain cardiac disorders, such as primary pulmonary hypertension, place women at a very high risk for morbidity or mortality during pregnancy [37]. There is an increased risk (3\u201310%, depending on the parent\u2019s condition) of congenital heart disease among children born to women who have a history of a congenital heard defect [38].\nWomen with a history of thromboembolism have an increased risk of recurrence during pregnancy. Women with a history of thrombosis should be offered testing for inherited or acquired thrombophilias, particularly if the results of the testing would affect the management of future pregnancies. Testing may be considered in women with a family history of thrombosis or a first degree relative with a specific mutation [39].\nIt is very important that women receive a thorough work-up and evaluation prior to being diagnosed with a seizure disorder and started on medication. Women diagnosed with seizures of unknown etiology should be told that even if they are on no medications at all, they have a two-fold higher risk for congenital malformations in their offspring than individuals without seizures. Anticonvulsant therapy use during pregnancy, particularly hydantoin or valproic acid, is associated with an increased risk for specific congenital defects. It is important to determine if anticonvulsant medication can be safely discontinued or to initiate therapeutic changes before pregnancy occurs. Women should be told that enzyme-inducing antiepileptic drugs may lower the efficacy of combination oral contraceptives, so an oral contraceptive containing a higher estrogenic concentration may be necessary. For women taking antiepileptic drugs who are considering a pregnancy, folic acid supplementation 5\u00a0mg\/day is recommended for one month prior to conception and until the end of the first trimester [40].\nWomen with poorly controlled asthma before pregnancy tend to have increased difficulty with asthma during pregnancy [41]. Severe, poorly controlled asthma may have an adverse effect on fetal outcome as a result of chronic or intermittent maternal hypoxaemia [42].\nWomen with quiescent autoimmune disease or a distant history of disease should be carefully evaluated and counseled about maternal and fetal risks. Patients should be counseled that the best time to attempt conception is during periods of inactive disease.\nWomen should be made aware of the deleterious effects of hypothyroidism in pregnancy, including an increased incidence of abortion, obstetric complications and fetal abnormalities in untreated women [43, 44]. Women being treated for hypothyroidism will require increased doses of thyroxine early and throughout pregnancy in order to maintain adequate levels; this is especially important during the first trimester [45]. Low maternal thyroxine concentrations may lead to impaired neurodevelopmental outcome in the neonate and child. Routine assessment for the presence of subclinical hypothyroidism is not recommended. ACOG recommends testing of thyroid function only in women with a personal history of thyroid disease or symptoms of thyroid disease [46].\nDiabetes mellitus represents a medical condition where preconception control has been proven to translate into improved maternal-fetal outcomes. Patients with poor glucose control during the first 8\u201310 weeks of pregnancy have a 2\u20133 fold higher risk of birth defects in their offspring than patients without diabetes. Good glycemic control before conception diminishes the risk of birth defects to a level comparable to that of the normal population [47].\nWomen should be asked about any ongoing dermatologic therapy she may be receiving. Isotretinoin (Accutane) is used for a variety of skin conditions and is a known teratogen. Some anti-psoriasis treatments (Acetretin X and methotrexate) are known teratogens, while some antiviral medications (podophyllum) are contraindicated in pregnancy.\nTable\u00a01 includes suggestions for the clinician during routine or periodic health assessments to optimize possible future pregnancy outcomes for all reproductively capable women.\nTable 1Suggestions for the clinician during routine or periodic health assessments to optimize possible future pregnancy outcomes for all reproductively capable women\u2022 Determine if the woman suffers from any undiagnosed or uncontrolled medical problems. If she does, provide recommendations for treatment of these conditions and when it would be best to attempt pregnancy.Make sure the patient is aware of any associations between the medical condition(s) and medications(s) she is taking and their impact on pregnancy outcomes.Ask the woman about her reproductive intentions at every visit, ascertain what her risk of an unplanned pregnancy may be; for women not actively seeking to become pregnant, discuss her current contraceptive method and any concerns or problems she may be having with it.Review the woman\u2019s family history\u2014including new births among family members\u2014annually as things change over time. Discuss any familial conditions that may herald an increased risk of adverse pregnancy outcome for the woman.Discuss the significance that nutrition can have on maternal fetal outcomes,e.g., the impact of 0.4\u00a0mg folic acid per day on neural tube defects in women with no family or previous history of a neural tube defect), the need to avoid excessive vitamin usage, especially vitamins A and D, and the additional measures women on restricted diets may need to take to optimize their health and the health of the developing fetus.Review the patient\u2019s social behaviors or lifestyle patterns\u2014such as smoking, alcohol, or other substance use or abuse\u2014that may affect pregnancy adversely and offer treatment options.Ascertain the immunity status of woman to rubella, hepatitis, and varicella. Ensure she is up to date on immunizations.\nConclusion\nThe concept of preconception care has gained momentum over the past two decades, and health care providers are increasingly being urged to provide such care by their professional organizations. It is hoped that, with its stated goal of improving birth outcomes, preconception care could help insure that the health status of all reproductively capable women would be optimized. In examining available standards and guidelines for preconception care, the professional nursing organization with the most highly developed preconception health standards is the American Academy of Nurse Midwives, and for physicians is the American College of Obstetricians and Gynecologists [48]. Other professional organizations examined did not have specific care standards for comprehensive preconception health, but have educated their members about one or more aspects of preconception health (smoking cessation, folic acid awareness).","keyphrases":["guidelines and standards","preconception health","women\u2019s health"],"prmu":["P","P","P"]}
{"id":"Biogerontology-3-1-2040177","title":"Heritability of a skeletal biomarker of biological aging\n","text":"Changes in the skeletal system, which include age-related bone and joint remodeling, can potentially be used as a biomarker of biological aging. The aim of the present study was to investigate the extent and mode of inheritance of skeletal biomarker of biological aging\u2014osseographic score (OSS), in a large sample of ethnically homogeneous pedigrees. The investigated cohort comprised 359 Chuvashian families and included 787 men aged 18\u201389 years (mean 46.9) and 723 women aged 18\u201390 years (mean 48.5). The TOSS - transformed OSS standardized in 5-year age groups for each sex, was analyzed as a BA index. We evaluated familial correlations and performed segregation analysis. Results of our study suggest the familial aggregations of TOSS variation in the Chuvashian pedigrees. In a segregation analysis we found a significant major gene (MG) effect in the individual\u2019s TOSS with a dominant most parsimonious model (H2 = 0.32). Genetic factors (MG genotypes) explained 47% of the residual OSS variance after age adjustment and after including sex-genotype interaction, they explained 52% of the residual variance. Results of our study also indicated that the inherited difference in the skeletal aging pattern in men lies mostly in the rate of aging, but in women in the age of the onset of the period of visible skeletal changes.\nIntroduction\nBiological age (BA) estimates the functional status of an individual in reference to his or her chronological peers on the basis of how well he or she functions in comparison with others of the same chronological age (Borkan and Norris 1986). Changes in the skeletal system, which include age-related bone and joint remodeling, can potentially be used as a biomarker of BA. Skeletal aging measures are strongly correlated with the status of the vital health systems (Gabriel et\u00a0al. 1999; Kiel et\u00a0al. 2001, Kadam et\u00a0al. 2004, Kalichman et\u00a0al. 2006b) and ultimately with survival rates (Johansson et\u00a0al. 1998; Haara et\u00a0al. 2003). The rate of degenerative changes in the skeleton may reflect an individual\u2019s biological resistance, immunity, functional or health status in reference to his or her chronological age. Therefore, age-related skeletal changes can possibly be used as an index of BA.\nRoentgenography is one of the common methods used to assess health status and age-related changes in bones and joints (Plato 1987; Plato et\u00a0al. 1994). Previously, Kobyliansky et\u00a0al. (1995) reported an osseographic scoring (OSS) system that uses hand radiographs to assess skeletal aging. OSS is a synthetic measure for assessing skeletal aging based on radiographic features of the hand, combining both osteoporotic and osteoarthritic changes of hand bones and joints. This system was originally suggested by Pavlovsky (1987) and has been used in BA evaluations in population studies (Kobyliansky et\u00a0al.1995; Livshits et\u00a0al. 1996; Pavlovsky and Kobyliansky 1997). Importantly, OSS has been shown to highly correlate with chronological age in adults of different ethnic groups (Karasik et\u00a0al. 1999, Kalichman et\u00a0al. 2002).\nAccumulating research shows that single gene changes in yeast, worms, and rodents can significantly alter life span (Kenyon et\u00a0al. 1993; Kaeberlein et\u00a0al. 1999; Cheng et\u00a0al. 2003; McBurney et\u00a0al. 2003). The observation that the first-degree relatives of human centenarians are both disease resistant and have longer than average life spans implies a genetic component to human longevity (Perls et\u00a0al. 2002; Atzmon et\u00a0al. 2004). Current estimates based on twin studies indicate that the human life span is 25% heritable (McGue et\u00a0al. 1993; Herskind et\u00a0al. 1996; Gudmundsson et\u00a0al. 2000; Perls et\u00a0al. 2002). Genetic factors may also significantly contribute to interindividual differences in BA (Karasik et\u00a0al. 2004, 2005). In variance component genetic analysis, performed in Karasik\u2019s study (2004), genetic factors explained 57% of the total variance of the BA index based on OSS measurements.\nThe aim of our study was to investigate the extent and mode of inheritance of a skeletal biomarker of biological aging (OSS) in a large sample of ethnically homogeneous pedigrees.\nMaterials and methods\nSample\nThe population sampled consisted of native Chuvashians residing in numerous small villages in the Chuvashia and Bashkartostan Autonomies of the Russian Federation. The data were gathered in three field studies during August\/September of 1994 (555 individuals), May\/June of 1999 (715 individuals), and September 2002 (240 individuals) by investigators from the Department of Anatomy and Anthropology, Sackler Faculty of Medicine, Tel Aviv University (Israel), and the Institute of Anthropology, Moscow University (Russia). The expeditions were part of a Chuvashia Skeletal Aging Study (ChuSAS) project whose main aim was to investigate the different aspects of skeletal aging within the Chuvashia population. All information collected and measurements taken during all expeditions were done by the same team of investigators. The investigated cohort comprised 359 families and included 787 men aged 18\u201389\u00a0years (mean 46.9) and 723 women aged 18\u201390\u00a0years (mean 48.5).\nThe Chuvashian population is characterized by a stable family structure with traditional relationships. For generations the Chuvashians have resided under the same environmental conditions and have not been exposed to an outside genetic flow (El\u2019chinova et\u00a0al. 2003). In the present study, we collected data from residents of a number of small villages (with a predominant Chuvashian population) located in the Volga region. A rural population is more homogeneous than urban in terms of ethnicity, occupation, and physical activity. The study participants shared similar living, economic, and professional conditions with most individuals employed in agriculture or in other occupations involving physical labor. Data from 80% to 90% of the families (including all family members who were living in the area at the time of the expedition) were obtained. Since almost every individual was related to one of the families, we were able to collect data on up to 90% of the population in each village. Data obtained from self-completed questionnaires and confirmed during the interview included sex, age, age at menarche, age at menopause and occupation. Menopause was defined as >12\u00a0months without menstruation. Age at last menstruation was considered as menopausal age. In order to minimize the possible recall bias, the investigators excluded from the analyses all women who had doubts about their age at menarche. The anthropometrical measurements (weight, stature, etc.) were also collected using standard methodology. In the studied sample, there were no individuals with known rheumatoid or psoriatic arthritis or users of steroid medication. Subjects with post-traumatic hand osteoarthritis were excluded from the study. All procedures involved were consensual. The subjects signed an informed consent form, and the entire project was approved by the Helsinki Ethics Committee of Tel-Aviv University.\nAdditional information about the studied population can be found in our previous publications: sex- and age-related variations of the somatotype (Kalichman and Kobyliansky 2006), reproductive indices, such as age at menarche and at menopause (Kalichman et\u00a0al. 2006 a, 2007a) and sex- and age-related variations of OSS (Kalichman et\u00a0al. 2007b).\nHand radiographs\nSingle plain radiograms of both hands were taken from each study participant in the postero-anterior position with the X-ray source located 60\u00a0cm above, using a standard radiographic technique, as described in detail by Livshits et\u00a0al. (1996). Hands were placed on the same film-containing plate to avoid any film or development variation. It was exposed for 5\u201310\u00a0s at 100\u2013150\u00a0mA without intensifying screens at 50\u00a0kV.\nOSS evaluation\nAn individual\u2019s OSS was determined by the occurrence of the following: (a) bone proliferations (spurs), including apiostoses (tufting of distal phalangeal tuberosity), osteophytes, enthesophytes of the juxta-articular area and at midshaft; (b) manifestations of bone porosity, such as resorption of trabeculae, development of lacuna (juxta-articular osteopenia) and erosion of cortex; (c) loci of osteosclerosis, defined as enostosis and sclerotic nuclei; and subchondral sclerosis; and (d) non-traumatic joint deformities, defined as narrowing of joint cavities and periarticular bone corrosion (Kobyliansky et\u00a0al. 1995; Pavlovsky and Kobyliansky 1997; Karasik et\u00a0al. 2005; Kalichman et\u00a0al. 2007b). Phalanges of the 2nd to 5th fingers of the left hand were examined. The first finger (thumb) does not directly project in a standard X-ray and was therefore not utilized. The presence of a given feature, but not its level of development, was documented for each OSS estimate. The overall number of those skeletal aging features was used as an OSS.\nEach radiograph was read by an experienced skeletal researcher according to the accepted protocol of OSS evaluation. Ten X-rays were read and then re-read by the investigator to estimate the intra-observer reliability of the readings.\u00a0All discrepancies were reviewed for systematic errors. This exercise continued until the reliability was high (k\u00a0>\u00a00.8). Afterwards, the investigator read the remaining X-rays, still blinded to patient identifiers. Before reading each batch of X-rays one re-read 5 X-rays, which had been previously read, to \u201ccalibrate\u201d one\u2019s readings to a standard. The intra-observer reliability (kappa statistics) was 0.82 (P\u00a0<\u00a00.01). The intraclass correlation for the OSS was 0.92 (P\u00a0<\u00a00.01).\nOSS reflects amount of skeletal changes occurring stochastically during the life-span, and therefore it is characterized by strong increase of trait variance with age (Kalichman et\u00a0al. 2007b and Fig.\u00a01). It is reasonable to assume that not the deviation of OSS from the age-group mean, but only relative rate of OSS accumulation with age can be inherited. Consequently we constructed a trait using the standardization in age-sex groups to equalize the means and variances in the whole age range for both sexes. The resulting trait, TOSS (transformed OSS) was further analyzed. The biological meaning of this trait is the relative position of individual (higher or lower than average values of OSS) in his\/her age-sex group expressed in the units of SD specific for each group. This accurately fit the definition of BA as the functional status of an individual in reference to his or her chronological peers (Borkan and Norris 1986).\nFig.\u00a01Mean OSS values and standard deviations (SD) in age groups of 5\u00a0years for men and women\nStatistical and genetic analysis\nPrior to any other evaluations, the descriptive statistics reflecting the basic characteristics of the studied sample were computed in the whole sample and within age groups of 5\u00a0years.\nAt the first stage we computed familial (spouses, parent-offspring and sib-sib) correlations between the corresponding TOSS levels to examine the potential genetic effect on the skeletal biomarker of BA.\nThe segregation analysis, as implemented in the statistical package MAN (Malkin and Ginsburg 2003), was utilized in this study to test a mode of inheritance of the skeletal biomarker of BA\u2014TOSS. Only individuals older than 29 were included in the analysis, since the high prevalence of zero OSS values in younger individuals make the standardization approach unacceptable. More precisely, in two 5-year age groups younger than 30\u00a0years the distribution of TOSS significantly deviated from normal (Chi-square\u03c72-test for frequencies gave P\u00a0<\u00a010\u20136), whereas in all older age groups did not (P\u00a0>\u00a00.01). Only pedigrees having at least one measured pair of relatives were included in the analysis. The mixed model of inheritance (Morton and MacLean 1974; Elston 1981; Lalouel 1983; Beaty 1997; Ginsburg and Livshits 1999) estimates the effects of a potential major gene and possible multifactorial effects, and is described in detail in numerous publications, including the aforementioned. The parameters that had been estimated in the general model are defined as follows: p\u2014the population frequency of allele A1 (assuming diallelic gene locus); \u03bcgs\u2014the average genotypic value of individuals having genotype g and sex s (g\u00a0=\u00a01, 2, and 3 corresponds to genotypes A1A1, A1A2, and A2A2, respectively); \u03c32g\u2014the trait variance in individuals having the same major gene genotype g, it estimates the trait variation resulting from the effect of all possible environmental factors and potential minor genes; \u03c4g\u2014the transmission probability parameter, which estimates the probability that a parent of genotype g transmits allele A1 to the next generation; \u03c1, \u03b2, and \u03b5 are partial correlations between the trait\u2019s residuals adjusted for a major gene effect, in spouses, parents\/offspring, and the siblings, respectively. A transmission probability test, described by Ginsburg and Livshits (1999), was used to estimate the significance of the major gene (MG) effect. A best-fitting and most parsimonious Mendelian model was established after dropping all non-significant parameters.\nUsing the procedure implemented in the package MAN (Malkin and Ginsburg 2003) and parameters estimated for the most parsimonious model, we determined the most likely MG genotype combination for each pedigree. We divided our sample into groups according to sex and the determined individual genotype of putative BA-related MG for genotypes having distinguishable genotypic values.\nTo analyze the age dependence of OSS, as a primary (directly measured) BA-related index, in groups of potentially different inherited BA, we used the stochastic model, described in detail by Kobyliansky et\u00a0al. (1995). We estimated for each group the following aging population parameters: t0\u2014the earliest age at which the first signs of OSS can be found, q\u2014the individual probability of revealing the first signs of OSS changes per chronological time unit, assuming an individual\u2019s age \u2265t0, and B\u2014the coefficient characterizing the rate of OSS changes per time unit (per year) after the process of aging has started. Next, we built the age-dependent expectation of the mean OSS level for each group according to sex and predicted MG genotype. Using the likelihood ratio test, we compared the extent of stochastic model approximation, both accounting for the MG genotype group and without it. Eventually we used ANOVA as implemented in STATISTICA 6.0 (StatSoft 2000) to test the differences between parameters of female organism development such as age at menarche and age at menopause in MG genotype groups.\nResults\nFigure\u00a01 shows the OSS mean values and SD in age groups of 5\u00a0years. Up to age 45, men have higher OSS scores than women. After age 45 OSS in women was higher, compared with men and the sex difference in the OSS scores increases up to the end of the age range. The OSS variance in age groups increases rapidly up to age 50.\nFamilial correlations of TOSS (Table\u00a01) showed no significant correlation for spouses, but parent-offspring and sibling correlations were significant. This indicates the existence of a clear familial aggregation of TOSS variation in the Chuvashian pedigrees, which cannot be explained by pure common environmental effects. We investigated the nature of TOSS inheritance later using segregation analysis. Because of the method by which TOSS was constructed (standardization within each sex-age group), in the segregation model the sex effects were not significant. The other results are presented in Table\u00a02 and support the MG hypothesis regarding trait inheritance. The most parsimonious Mendelian model was dominant. The recessive allele with a population frequency of 0.6 corresponded to relatively slow aging. For the most parsimonious Mendelian model, H2\u00a0=\u00a00.315; total proportion of TOSS variance explained by the model was 0.349. Using this model, we predicted the most likely MG genotype combination for each pedigree included in the analysis. Because of the dominant nature of the most parsimonious model, we divided all individuals into two groups with potentially different genotypic values: (1) homozygous on allele A1 and (2) all the rest. Using a stochastic model of aging (Kobyliansky et\u00a0al. 1995), we constructed the model of age dependence of directly measured OSS for a whole sample and for each predicted genotype group separately with and without considering the effects of sex. Table\u00a03 presents the parameters of four resulting models and a comparison between them. According to the likelihood ratio test (LRT), Model 1, which included only the effect of age, has a significantly worse extent of approximation than both Model 2, which included the effect of genotype-age, and Model 3, which included the effect of sex-age. The extent of approximation was significantly higher for the effect of genotype-age, which explained 47% of the residual OSS variance after only adjustment for age, and lower for the effect of sex-age (5%). Model 4, which includes all age-sex-genotype effects, showed a significantly higher extent of approximation in comparison with other models and explained 52% of the residual OSS variance after adjustment for age. After adjustment for age and sex, the residual parent-offspring correlation of OSS was significant, but became non-significant after adjustment for the sex-age-genotype.\nTable\u00a01Familial correlations of TOSSRelativesRN of pairsPSpouses0.083330.15Parent-offspring0.127550.002Sib pairs0.311890.0008Table\u00a02Segregation analysis for TOSS (OSS standardized in age groups of 5\u00a0years for each sex)ParameterMain modelsRestricted modelsGeneralMendelianEqual \u03c4-sArbitrary \u03c4-sMendelian most parsimoniousEqual \u03c4-s123456P0.6000.6280.2520.5580.598 \u00b1 0.0330.563\u03bc1\u22120.727\u22120.724\u22121.080\u22120.758\u22120.729 \u00b1 0.057\u22120.702\u03bc20.4810.525\u22120.3290.3800.429 \u00b1 0.0610.362\u03bc30.3620.3850.3830.380!0.429!0.362!\u03c3120.2990.3250.0740.2550.303 \u00b1 0.0450.289\u03c3221.0740.9880.5041.1651.067 \u00b1 0.103 1.181\u03c3320.2050.2321.0490.255!0.303!0.289!\u03c10.0850.1040.099[0.000][0.000][0.000]\u03b20.006\u22120.0290.033[0.000][0.000][0.000]\u03b50.3250.2280.3110.3270.249 \u00b1 0.0670.327\u03c410.910[1.000]0.2720.916[1.000]0.614\u03c420.632[0.500]0.272!0.618[0.500]0.614!\u03c430.001[0.000]0.272!0.020[0.000]0.614!Log LH\u22121425.66\u22121428.27\u22121435.91\u22121427.29\u22121429.61\u22121440.51\u03c72\u22125.22 NS (1)20.5** (1)1.63 NS (1)4.64 NS (4)26.44** (4)NS\u2014P\u00a0>\u00a00.05; ** P\u00a0<\u00a00.01Number in parentheses in the \u03c72\u2014row specifies a comparative columnConstraints denotation: [ ]\u2014 parameter is fixed to shown value; !\u2014 parameter is constrained to be equal to the parameter above in the tableTable\u00a03Parameters of stochastic aging models estimated for OSS data without grouping (1) and grouped according to predicted genotype (2), sex (3), or both sex and predicted genotype (4)ParameterAllA1A1A1A2 or A2A2Allt021.223\u00a0\u00b1\u00a00.93321.705\u00a0\u00b1\u00a00.88422.225\u00a0\u00b1\u00a01.179q0.118\u00a0\u00b1\u00a00.0150.077\u00a0\u00b1\u00a00.0100.217\u00a0\u00b1\u00a00.044B0.552\u00a0\u00b1\u00a00.0040.518\u00a0\u00b1\u00a00.0040.600\u00a0\u00b1\u00a00.004TM29.68934.61926.838Model 1 (3 d.f.)Model 2 (6 d.f.)R2=0.838R2=0.915Log LH=\u22123476.8Log LH=\u22123065.5\u2013\u03c72= 822.6 (1)Ment020.524\u00a0\u00b1\u00a01.23121.573\u00a0\u00b1\u00a01.14221.886\u00a0\u00b1\u00a01.683q0.127\u00a0\u00b1\u00a00.0240.098\u00a0\u00b1\u00a00.0140.233\u00a0\u00b1\u00a00.071B0.501\u00a0\u00b1\u00a00.0050.449\u00a0\u00b1\u00a00.0050.556\u00a0\u00b1\u00a00.005TM28.40031.74026.182Woment022.078\u00a0\u00b1\u00a01.34221.840\u00a0\u00b1\u00a01.16922.623\u00a0\u00b1\u00a01.601q0.107\u00a0\u00b1\u00a00.0190.057\u00a0\u00b1\u00a00.0000.187\u00a0\u00b1\u00a00.046B0.617\u00a0\u00b1\u00a00.0070.629\u00a0\u00b1\u00a00.0080.657\u00a0\u00b1\u00a00.007TM31.40039.37327.979Model 3 (6 d.f.)Model 4 (12 d.f.)R2=0.846R2=0.923Log LH=\u22123443.3Log LH=\u22123000.7\u03c72= 67.0 (1)\u03c72= 952.2 (1); 129.6 (2); 885.2(3);A1A1\u2014 genotype corresponding to lower values of TOSS in segregation analysis; t0\u2014 the minimal age at which initial bone changes occurred in a group; q\u2014the group probability that an individual will first develop involutive bone change after age t0; B\u2014the group rate of aging (bone change) per time unit; TM\u2014the expected mean age in a group in which the first bone change occurred; number in parentheses indicates the comparative model for LRT; all LRT values are significant with P\u00a0<\u00a010\u22126\nFigure\u00a02 shows a scatterplot of OSS-age for the predicted genotypes in both sexes and the corresponding curves of age-dependent OSS expectation given by the stochastic model. For women in the group of genotype A1A1 (with slow skeletal aging), the age at menarche was significantly higher (P\u00a0=\u00a00.012) than in the group of genotypes A1A2 or A2A2; the group mean difference was 0.45\u00a0year. The difference in the age at menopause was in the same direction, but not significant for the whole sample. Note that our sample includes women strongly influenced by difficult life conditions during World War II. In these women the variance in the age at menopause is increased in comparison with the normal population. For women whose year of birth \u22651937 (the normal part of the population), the age at menopause was higher in group A1A1 than in other group (P\u00a0=\u00a00.054); the group mean difference was 1.6\u00a0year.\nFig.\u00a02OSS values for predicted genotype groups: black\u2014A1A1, gray\u2014A1A2 or A2A2\nTo compare our findings with results of other authors, we quantitatively analyzed the similarity of differently constructed BA indices. The correlation between the BA index proposed by Karasik et\u00a0al. (2004) and TOSS was 0.82 in the Chuvashian population. The correlations between the BA index, used by Livshits et\u00a0al. (1996), versus the BA of Karasik et\u00a0al. (2004) and TOSS were estimated as 0.80 and 0.89, correspondingly. Though the resemblance of the indices may be influenced by population type, the aforementioned BA indices are sufficiently similar.\nDiscussion\nIn the present study, we evaluated the extent and mode of inheritance of a skeletal biomarker of BA. We constructed this biomarker (TOSS) to be maximally fitted according to the BA definition; it reflects the intensity of an individual\u2019s skeletal changes in relation to its coevals of the same sex, and the range of the index does not depend on the age cohort.\nWe previously evaluated the association between OSS, as an index of BA, with morbidity in a general Chuvasha population (Kalichman et\u00a0al. 2006b). Statistically significant association was found in the present study between age-adjusted OSS and rheumatic morbidity. Morbidities such as ischemic heart disease, pulmonary diseases, traumatic brain injuries and gynecological diseases also showed differences between mean values of OSS in affected vs. non-affected individuals. However, after correction for multiple testing, the statistical significance was insufficient. The association between OSS and diabetes mellitus was close to significant (P\u00a0=\u00a00.07) and we surmise that with a larger sample of diabetic individuals, a clear-cut association will be confirmed.\nIt is well-established that genetic factors play an important role in an individual\u2019s BA (Karasik et\u00a0al. 2005). Our finding suggests the familial aggregations of TOSS variation within the Chuvashian pedigrees. The evaluation of heritability of the skeletal biomarker of BA in segregation analysis suggested the existence of a MG effect with a most parsimonious dominant model. A number of BA indices, based on age-related skeletal changes, were previously proposed and analyzed in different populations. Livshits et\u00a0al. (1996) analyzed the OSS measurement in the Turkmenian population. They used the age-adjusted OSS values as a BA index. This index showed a significant familial parent-offspring correlation of 0.1\u00a0\u00b1\u00a00.09, which is approximately the same as for TOSS in our study (0.12). Nevertheless, in their segregation analysis of the age-adjusted OSS, the Mendelian model was rejected. A possible reason is that a simple age adjustment does not equalize the residual variance of OSS, which significantly increases with age. As a result, parents and offspring received a BA index with different amplitude and this difference probably prevented establishing MG inheritance in segregation analysis. Other phenotypes of BA, as well, are characterized by substantial heritability. Thus, functional age (other term for BA) has been assessed by a general linear regression model of many biomarkers in Minnesota twins (27\u201388\u00a0years old). A subset of these biomarkers (mostly physiological variables) identified by a factor analysis had heritability of \u223c59% (Finkel et\u00a0al. 1995). Duggirala et\u00a0al. (2002) have reported that BA, measured according to Uttley and Crawford (1994), had a substantial genetic component (heritability ranging from 0.27\u00a0\u00b1\u00a00.11 in one Mennonite geographical subsample to 0.47\u00a0\u00b1\u00a00.18 in the other). \nKarasik et\u00a0al. (2004) studied a sample that included 1,402 members of 288 pedigrees from the Framingham Heart Study; the log-transformation was used to reduce the growth of OSS variance with age. A special BA index was constructed by adjusting the individual\u2019s age for log-transformed OSS with cubic polynomial. The log-transformation automatically excludes from the analysis all individuals with zero OSS (note that for ages >30, the zero OSS value is meaningful and denotes a low BA). For the resulting trait, the genetic variance component analysis showed that sex, height, the body mass index, and, in women, menopausal status and estrogen use, jointly explained approximately 6% of the total variance of BA index. Genetic factors explained an additional 57%. The genome search for this BA index revealed a number of potential chromosomal regions in the two-point analysis, but for multipoint, only the region on chromosome 21 remained significant.\nThe variance component analysis, used by Karasik et\u00a0al. (2004), a priori assumes a polygene mode of inheritance. Our segregation analysis results confirm the findings about a significant genetic effect and furthermore suggest the existence of a MG effect in an individual\u2019s TOSS with a dominant most parsimonious model. The MG hypotheses may be, to some extent, supported by the fact that in the multipoint variance component linkage analysis by Karasik et\u00a0al. (2004) only one chromosomal region showed a significant LOD score >3 (likelihood ratio greater than 103). We found that genetic factors (MG genotypes) explained 47% of the residual OSS variance after adjustment for age, and after including the sex-genotype interaction, they explained 52% of the residual variance. For TOSS the MG effect was 32%.\nAdditionally, we compared the age dependence of OSS by directly measuring the individual\u2019s skeletal health index in predicted MG genotype groups. Kobyliansky et\u00a0al. (1995) proposed a two-stage stochastic statistical model of the population aging pattern for OSS. The implied stages are as follows: (1) the period of life between birth and the apparent onset of skeletal changes (t\u00a0<\u00a0ti), defined as the latent period, and (2) the period of visible changes beginning after ti (which is not the same in different individuals), when involutive changes occur continuously throughout the remaining life. Applying this model separately in predicted genotype groups, we compared their aging features by means of model parameters (t0, q, B). The earliest age of the first visible changes, t0, was almost the same in all sex-genotype groups, about 22\u00a0years. The probability per year of transition from the latent to the visible changes period, q, was for genotype A1A1 about 1\/3 of probability for other individuals. This was especially expressed for women and it results in greater values of the average transition age TM (39.3\u00a0years in A1A1-group and 31.7 for the others). The rate of aging, B, was also significantly different in the predicted genotype groups, but this difference was expressed more for men. Thus, we can conclude that the inherited difference in the aging pattern lies in men, mostly in the rate of aging, but in women, it is in the age of onset of the period of visible changes.\nAn additional finding was that in the group A1A1 (slow aging), for women, the age at menarche was significantly greater (P\u00a0=\u00a00.012) than in the group {A1A2 OR A2A2}. For women born after 1937 (those whose maturation period was not influenced by WW II or the subsequent rehabilitation period), the age at menopause was also greater in the group A1A1 (P\u00a0=\u00a00.054). Thus, there is a positive relation between the rate of sexual development and the rate of aging, characterized by degenerative skeletal changes. The rate of sexual development is positively associated with intensity of normal metabolism, which also may lead to accelerated aging. Early onset of menarche is also known to be linked to other aging conditions such as elevated blood pressure and glucose intolerance, compared with later maturing girls, independent of body composition (Remsberg et\u00a0al. 2005).\nThe main molecular characteristic of aging is the progressive accumulation of damages in macromolecules that result on macro level in heterogeneity of tissues and degenerative changes. Rattan (2006) pointed out three major sources of this damage: (1) reactive oxygen species (ROS) and free radicals formed due to external inducers of damage (for example ultra-violet rays), and as a consequence of cellular metabolism; (2) nutritional glucose and its metabolites, and their biochemical interactions with ROS; and (3) spontaneous errors in biochemical processes, such as DNA duplication, transcription, post-transcriptional processing, translation, and post-translational. Bessenyei et\u00a0al. (2004) reviewed data concerning SNPs associated with life span positioned in a variety of genes regulating metabolic processes, apoptosis, stress response (see also Singh et\u00a0al. 2006) and immune response. Several polymorphisms in ER-alpha (estrogen receptor that are known to play significant role in skeletal development) were reported to be associated with life span (Bessenyei et\u00a0al. 2004) and with both osteoarthritis (Jin et\u00a0al. 2004) and osteoporosis (Albagha et\u00a0al. 2005) related phenotypes. The VDR (vitamin D receptor) gene is also known to be associated with both types of degenerative changes in certain skeletal sites (Uitterlinden et\u00a0al. 1997; Yue et\u00a0al. 2005). A great number of genes with significant association signals were cited in recent reviews of Loughlin (2005) (osteoarthritis) and Liu et\u00a0al. (2006) (osteoporosis). de Boer et\u00a0al. (2002) described studies of mice with a mutation in XPD, a gene encoding a DNA helicase that functions in both repair and transcription. Mice with mutation were found to exhibit many symptoms of premature aging, including osteoporosis and kyphosis, osteosclerosis, early greying, cachexia, infertility, and reduced life-span. So OSS, the index comprising various accumulated degenerative skeletal changes, despite to different molecular mechanisms, which are involved in bone loss, cartilage loss and other OSS components, may be under genetic control of some polymorphic genes taking part in DNA repair pathways, transcription and translation control and\/or some polymorphic genes controlling intensity of normal metabolism, that influence the rate of molecular damage (Kirkwood and Austad 2000). This supports the assumption that OSS as a skeletal biomarker of BA can be used to evaluate the organism\u2019s general aging status.","keyphrases":["heritability","biological aging","osseographic score","chuvashia","skeletal aging"],"prmu":["P","P","P","P","P"]}
{"id":"Cancer_Causes_Control-2-2-1764866","title":"Transient caloric restriction and cancer risk (The Netherlands)\n","text":"Over the past century, many animal experiments have shown that caloric restriction can reduce the risk of cancer, a finding that proved to be highly reproducible. Many papers have been published on its potential for human health, but until know little evidence is available on its actual effects in humans. In Utrecht, The Netherlands, we have been investigating the effects of the 1944\u20131945 Dutch famine on breast cancer risk factors and breast cancer risk, and paradoxically the relatively short-term famine seemed to be related to increased breast cancer risk in later life. One of the differences between the famine situation and the large body of evidence from animal experiments is the duration of caloric restriction. Almost all animal experiments investigated sustained caloric restriction and information on the effects of short-term transient caloric restriction is very scarce. A search in the literature identified some animal experiments on short-term transient caloric restriction and these seemed to be at least supportive to the famine findings. Because caloric restriction in humans for preventive health measures would be mostly short-term, it is important to extend animal research on short-term caloric restriction.\nDuring the 20th century, a large body of experimental evidence has been accrued signifying that caloric restriction protects against cancer. Rodent data are abundant and consistently show (i) that caloric restriction lowers the incidence of a variety of spontaneous as well as induced or transplanted tumours; (ii) that this effect is directly proportional to the amount of caloric restriction; (iii) that the effects can largely be ascribed to caloric restriction per se, and not merely to the decrease in one or more dietary components; (iv) that caloric restriction initiated in early life as well as in later life is effective in cancer prevention; (v) that caloric restriction prolongs life [1\u20135]. A pooled quantification of the inhibitory effects of caloric restriction on spontaneous mammary tumour incidence in mice showed that caloric restriction lowered the incidence with 55% [6].\nPreliminary reports on energy restriction in non-human primates appear to be consistent with findings in rodents [7, 8]. What about humans? Obviously, this is difficult to investigate, but can lead to important insights in cancer aetiology and to opportunities for prevention. The 1944\u20131945 Dutch famine, a relatively short severe famine during World War II [9, 10], could shed some light on this issue, and has been used before as an \u201cexperiment of history\u201d [11\u201313].\nDuring the past years, we have conducted several studies on the effects of the 1944\u20131945 Dutch famine on breast cancer risk factors and breast cancer risk in Utrecht, The Netherlands. For these studies we used data on women who were between 2 and 33 years of age during the famine. Therefore we were not only able to investigate the long-term effects of caloric restriction, but could also relate those effects to specific time-windows in female development. Women were classified to their degree of famine experience on an individual basis, leading to a three-point famine score (\u2018absent\u2019, \u2018moderate\u2019, or \u2018severe\u2019 exposure) that enabled dose-response evaluation [14].\nIn brief, we found that the famine is associated with subsequent reduced reproductive ability in women who were exposed during childhood. This is manifested by decreased chance of childbirth and an earlier mean age at menopause [15, 16]. Strikingly, research on moderate early life diet restriction in Drosophila melanogaster by Tu and Tatar showed also a decrease in adult fecundity [17]. Furthermore, we found that hormone concentrations, measured postmenopausal, seem to be affected by the famine with increases in sex-steroid as well as insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-3 levels (resulting in unaffected IGF-I to IGFBP-3 ratios) [18, 19]. All our observations showed a dose-response to degree of famine exposure, with smaller but still observable associations in those moderately exposed.\nWith regard to breast cancer risk, the associations between the famine and the reproductive system would suggest a protective effect of the famine [20]. However, the associations with hormone concentrations would suggest the contrary [21, 22], with the exception of increased IGFBP-3 that may relate to decreased breast cancer risk [23].\nWhen we actually determined the effect of famine on breast cancer risk in this population, we found that the famine is related to increased risk, again in a dose response manner\u2014a finding that was stronger in women who were children during the 1944\u20131945 winter [24]. Body mass index at later ages did not confound this relation nor modified it (last data unpublished), suggesting that catch-up growth after the famine did not play an important role. Total cancer risk, exclusive of breast cancer, seemed not to be affected by the famine [25], which does not preclude that famine may have affected risk of specific types of cancer, but the numbers in our study are presently too small for analysis with more detail. Another famine study by Dirx et al. on breast cancer risk could not detect a protection against breast cancer from the famine either, and if anything, showed a moderate increase in risk in women that were exposed at young ages [11]. In addition, Dirx et al. showed that the famine was, non-significantly, associated with increased prostate cancer and decreased colon cancer risk [12, 13]. In these studies, Dirx et al. used place of residence as a measure of famine exposure whereas we used individual exposure data, based on recall.\nThese findings are not in line with the animal experiments consistently showing caloric restriction to prevent cancer. Several differences between the usually adopted caloric deprivation strategy in animal experiments and the 1944\u20131945 Dutch famine may explain these differences, such as the amount of restricted calories. The exact degree of caloric restriction during the 1944\u20131945 Dutch famine is difficult to ascertain, but rations dropped to about 30% of desired norms for adults and to about 50% for young children. The relative amounts of carbohydrates, fats and proteins remained more or less balanced, and supplementations to the rations were sometimes clandestinely available. Young children were relatively protected within families and by charity organisations [9, 10]. Although the amount of caloric restriction during this famine is larger than usually adopted in animal experiments, tumour incidence has been shown to decrease proportionally to degree of caloric restriction in animal experimental settings, amounting to an estimated 62% tumour reduction with 53% caloric restriction [2]. Therefore, it is unlikely that differences in the amount of caloric restriction explain the contradictory results of the famine studies and the animal experiments.\nMore notably, during the Dutch famine people were transiently exposed for a relatively short duration of time (6 months) [9, 10]. Therefore, this \u201cexperiment of history\u201d\u2014as most current famines due to crop disaster or war\u2014is only similar to experiments in rodents that studied short-term caloric restriction followed by ad libitum feeding, whereas the vast majority of these studies investigated dietary interventions that were sustained throughout the animals entire live.\nWe were able to identify eight animal experiments that investigated transient and mostly short-term caloric restriction, where after animals were allowed to eat at will [26\u201333]. Two of these experiments studied the effect of caloric restriction for different time periods during and after the chemically induction of breast cancer in female rats [28, 31]. Sustained caloric restriction dramatically decreased tumour incidence: from 50% in the ad libitum group to 20% in the continuously restricted group [31]. It also seemed that caloric restriction during tumour initiation\u2014from 1 week before until 1 week after 7,12-dimethylbenz-[\u03b1]anthracene administration\u2014potently reduced mammary tumour development [28]. However, restriction for any period after tumour initiation followed by ad libitum feeding, if anything, showed no substantial effect, and could even lead to increased breast cancer risk [28]. Furthermore, Kritchevsky noticed that when rats were returned from restricted to ad libitum feeding this resulted in hyperphagia, accelerated weight gain, transient mammary hypertrophy, and enhanced tumour growth [31].\nFour other experiments investigated the effect of caloric restriction for different periods on, amongst others, spontaneous overall tumour incidence in rats [26, 27, 30], and mice [29]. Again, these studies showed that sustained caloric restriction throughout life lowered cancer risk substantially. However, caloric restriction confined to early life did either not affect cancer risk [30], or actually seemed to increase it [26, 27, 29]. This feeding strategy did not materially increase lifespan, so this cannot explain the observed increase in cancer risk. The study of Cheney et al. in female mice provides some evidence that caloric restriction confined to mid-life may also eventually lead to increased cancer burden [29].\nRecently, interest was rekindled with two more studies on prolonged but transient caloric restriction and 1-methyl-1-nitrosaurea induced mammary carcinogenesis in rats [32, 33]. These studies both show that upon refeeding tumour incidence increased.\nThe disparity between our findings in humans after caloric restriction during a short and severe famine and the abundant literature on sustained caloric restriction and cancer risk in rodents may be ascribed to differences in exposure duration, as is corroborated by the abovementioned animal studies on transient caloric restriction that are in line with the famine observations.\nFrom a biological point of view, it may not be surprising that a short and transient period of caloric restriction in early life may increase human cancer risk. During caloric restriction, a range of responses can be seen, most of which could be directly beneficial to overall cancer risk but are unlikely to be of importance once the restriction is discontinued. Proliferation of cells is reduced with both increased rates of apoptosis together with decreased DNA synthesis and increased DNA repair, limiting the number of preneoplastic lesions. Oxidative stress is reduced, resulting in decreased reactive oxygen species that can damage DNA. Furthermore, of interest to hormone associated tumours, levels of a number of hormones and growth factors are altered during caloric restriction: glucocorticoids are increased whereas concentrations of IGF-I (and to a lesser extent IGFBP-3 resulting in decreased bioavailability of IGF-I), insulin, prolactin, estrogens and leptin are decreased [3\u20135, 34\u201336].\nChanges in leptin levels can interfere with sexual maturation [37, 38], a period in development during which the Dutch famine showed the largest impact on breast cancer risk in our study. It could be that the hypothalamo-pituitary axis, which is not matured in girls until a few years after menarche when regular menses are established [39], has erroneously adapted to the period of paucity, leading to inappropriate set-levels of hormones that could relate to hormone associated cancers [18, 19]. Such erroneous adaptations, also described as a \u201cthrifty phenotype\u201d [40], are the subject of the \u201cfoetal and infant origins of adult disease\u201d hypothesis by Barker and colleagues [41]. Evidence is mounting that such long-lasting effects indeed exist and may contribute considerably to later health [42]. The potential involvement of the hypothalamo-pituitary axis herein has been recognised [43].\nIn conclusion, the general notion that caloric restriction prevents cancer needs some amendment, even for rodents. Evidence is strong that during caloric restriction, cancer risk is decreased proportionally to the amount of restriction, and such interventions can be effective whether started in early life or later. However, a short and transient period of restriction followed by a \u201cnormal\u201d diet does not show such effects and could actually be detrimental.\nThompson et al. made a similar plea. If humans are advised to eat less as a means to prevent cancer, this would probably result in repetitive periods of weight loss followed by periods of weight gain. He warned that such weight cycling may be associated with a modest acceleration of the carcinogenic response [44].\nAnimal experiments on short-term interventions or weight-cycling diets are scarce and their results yet inconclusive. Such animal experiments are nevertheless highly relevant and need further investigation before any preventive strategy is researched in experimental settings, or actually adopted, in humans, as short-term interventions are the most feasible for the human situation but may bring along considerable hazards. It would be of importance to see if our observations can be replicated in such animal experiments and in human studies making use of other famine episodes, e.g. the 1959\u20131961 Chinese famine [45]. Other human studies may involve for instance children from countries with poor nutrition that are adopted by families in economically prosperous countries. These children have been exposed to adverse nutritional circumstances at young ages for a clearly demarcated period followed by nutritional abundance (in contrast to immigration studies of for example Japanese women to the United States who gradually adapt, often partly, to a Western lifestyle). Indian girls adopted in Sweden have for example been shown to reach menarche at younger ages compared to Indian standards [46]. It would be intriguing to see whether other physiologic changes occur in these children, e.g. with regard to hormonal levels.\nFurthermore, studies on cancer risk in patients with anorexia nervosa are also of interest. Two studies have reported on the topic and found a decreased risk of breast cancer [47, 48]. The generalisability of these observations is however troubled as factors underlying this disease may contribute to the decrease in breast cancer risk, so this may not merely be ascribed to a decreased caloric intake per se.\nCurrently, the relation between famine exposure in early life and risk of other types of cancer than that of the breast is largely unknown. It would be valuable to further investigate these relations to see whether associations are different between cancer types, e.g. comparing hormone to non-hormone associated cancer types. This would give further insight into whether general mechanisms in human cancer aetiology are involved or whether adaptation of hormonal axes leading to harmful hormone concentrations is the potential culprit.","keyphrases":["transient","caloric restriction","cancer","risk","human","famine"],"prmu":["P","P","P","P","P","P"]}
{"id":"Neurosci_Lett-2-1-2271123","title":"The effect of stress on the expression of the amyloid precursor protein in rat brain\n","text":"The abnormal processing of the amyloid precursor protein (APP) is a pivotal event in the development of the unique pathology that defines Alzheimer's disease (AD). Stress, and the associated increase in corticosteroids, appear to accelerate brain ageing and may increase vulnerability to Alzheimer's disease via altered APP processing. In this study, rats were repeatedly exposed to an unavoidable stressor, an open elevated platform. Previous studies in this laboratory have shown that a single exposure produces a marked increase in plasma corticosterone levels but animals develop tolerance to this effect between 10 and 20 daily sessions. Twenty-four hours after stress, there was an increase in the ratio of the deglycosylated form of APP in the particulate fraction of the brain, which subsequently habituated after 20 days. The levels of soluble APP (APPs) tended to be lower in the stress groups compared to controls except for a significant increase in the hippocampus after 20 days of platform exposure. Since APPs is reported to have neurotrophic properties, this increased release may represent a neuroprotective response to repeated stress. It is possible that the ability to mount this response decreases with age thus increasing the vulnerability to stress-induced AD-related pathology.\nThe role of amyloid precursor protein (APP), or its processed fragments, in normal brain function is not well understood. There is a significant body of evidence to suggest that the membrane-bound protein plays a role in cell\u2013cell interaction, neuronal plasticity and the formation and the consolidation of synaptic connections [9]. Soluble APP\u03b1 (APPs\u03b1) generated following cleavage by the \u03b1-secretase enzyme is thought to promote cell survival, neurite outgrowth and synaptogenesis [15]. The administration of APPs\u03b1 in vivo can reduce the level of neuronal damage following traumatic brain injury [23] and increase the number of EGF-responsive progenitor cells [6]. Lower levels of APPs in rats were associated with poor performance in the watermaze [1] whereas intracerebroventricular administration of sAPP enhanced the performance of mice in various learning tasks [17]. However if membrane-bound APP is cleaved by the \u03b2- and \u03b3-secretase protease enzymes it generates the A\u03b2 peptide that aggregates to form the neuritic plaques which define Alzheimer's disease pathology [9].\nFor a majority of AD cases it is not known what causes the shift to the amyloidogenic pathway although there is some preclinical and clinical evidence to suggest that stress may exacerbate the progression of AD and may be associated with the pathological hallmarks of the condition. In animals, exposure to isolation stress accelerated the age-dependent deposition of amyloid plaques in the Tg2576 mouse model of AD [11]. Chronic immobilisation stress had a similar effect on both memory impairment and amyloid deposition within the hippocampus and cortex of the APPV7171-CT100 mouse model [13]. These effects may be a direct consequence of the corticosteroids released during the response to stress as prolonged exposure to high levels of these hormones appears to accelerate age-related decrements in neuronal morphology and function [19]. Studies on patients with AD have shown that hypercortisolism is associated with greater cognitive deficits [25] and that increased plasma cortisol is associated with greater decline in both clinical and cognitive measures of dementia severity [10].\nThe aim of this study was to determine whether exposure to acute and repeated stress affected the levels of the two primary forms of APP in the normal rat brain and whether this was regionally selective.\nMale Sprague Dawley rats (Harlan, UK) weighing between 250 and 300\u00a0g were maintained on a 12:12\u00a0h light:dark cycle with lights on at 6.00 a.m. and access to food and water ad libitum. After 1 week they were divided into 4 treatment groups (n\u00a0=\u00a05 rats per group): a 20 days stress group, a 10 days stress group, an acute stress group (1 day) and a control group. Stress exposure involved placing the rats on an open elevated platform for 60 min. Acutely this procedure reliably increases the levels of corticosteroid hormones in the blood [2] but this response habituates after between 10 and 20 days exposure [18,22]. Control animals were not exposed to the stressor but were taken to the same laboratory and handled in an equivalent manner. All experimental procedures were subjected to local ethical review and covered by a U.K. Home Office project licence (PPL 60\/2845).\nRats were killed 24\u00a0h after the last stress exposure or handling session to measure the effect of stress-induced genomic changes on APP. Brains were removed and the frontal cortex, parietal cortex, striatum and hippocampus were dissected on ice and stored at \u221280\u00a0\u00b0C prior to analysis. The pathological changes that characterise Alzheimer's disease first appear in the cortical regions of the brain and the hippocampus. The striatum was included as an area also rich in cholinergic neurones that remains relatively free of pathology until advanced stages of the disease. The brain samples were then homogenised (10% w\/v) in homogenising buffer (1\u00a0mM EDTA, 1\u00a0mM EGTA, 0.32\u00a0M sucrose, 1\u00a0mM Tris, pH 8.0 containing protease inhibitor cocktail) at 4\u00a0\u00b0C. Homogenates were centrifuged at 100,000\u00a0\u00d7\u00a0g for 60\u00a0min at 4\u00a0\u00b0C and the resulting supernatant, containing the soluble brain fraction, was aliquoted into 1\u00a0ml fractions and stored at \u221220\u00a0\u00b0C. The pellet was resuspended in 1% (v\/v) Triton X-100 and re-centrifuged at 100,000\u00a0\u00d7\u00a0g for 60\u00a0min at 4\u00a0\u00b0C. The supernatant, containing the membrane, mitochondrial and nuclear fractions, was stored at \u221220\u00a0\u00b0C in 1.0\u00a0ml aliquots while the triton-insoluble pellet was discarded.\nThe total protein concentration of each sample was determined spectrophotometrically using the method of Bradford [4] and APP expression determined in 25\u00a0ug protein samples by Western blot analysis using a monoclonal primary antibody (MAB348 clone 22C11 Calibochem 1:1500) [16]. This recognises an N-terminal epitope (AA66-81) that is common to both APP and the amyloid precursor-like protein 2 (APLP2). The autoradiographs were scanned and band densities calculated using the SCION image (v3) software package. In order to determine whether there were any regional differences in APP expression between the groups the data were analysed by repeated measures analysis of variance using stress exposure as the between subject factor (GROUP) and brain region as the within subject factor (REGION). Post-hoc analysis of significant effects was carried out using one-way ANOVA or Dunnett t-tests where appropriate.\nIn the soluble brain fractions APPs was detected as a single band with a molecular weight of approximately 128\u00a0kDa (Fig. 1). The global analysis revealed that there was a significant effect of GROUP (F[3, 16]\u00a0=\u00a03.79, p\u00a0<\u00a00.05) and a significant interaction between GROUP and REGION (F[9, 48]\u00a0=\u00a03.58, p\u00a0<\u00a00.005) suggesting that stress had a regionally selective effect on the level of APPs. Subsequent analysis of this interaction revealed that this was due to the significant increase in APPs in the hippocampal region only following 20 days of stress (p\u00a0<\u00a00.05).\nIn the particulate brain fractions, APP was detected as a doublet with molecular weights of approximately 128 and 121\u00a0kDa (Fig. 2A). Global analysis of total APP revealed a significant effect of GROUP (F[3, 16]\u00a0=\u00a08.02, p\u00a0<\u00a00.005) but no interaction between GROUP and REGION suggesting that stress had influenced the level of APP but that this was not a regionally selective effect. Post-hoc testing confirmed that all stress groups expressed a significantly greater level of APP protein in the particulate brain fractions compared to controls (p\u00a0<\u00a00.05). Therefore, although the pattern appeared to be different in the parietal cortex compared to other regions, this effect was not large enough to influence the overall statistical analysis.\nThe 121\u00a0kDa molecular weight protein band has previously been demonstrated to represent the deglycosylated form of the protein [16]. When this band was expressed as a percentage of total APP (Fig. 2B) global analysis confirmed a significant effect of GROUP (F[3, 16]\u00a0=\u00a011.6, p\u00a0<\u00a00.001) but again no interaction between GROUP and REGION suggesting that stress had affected the APP band ratio but not in a regionally selective way. Post-hoc testing confirmed that, overall, only the acute and 10 days stress group had a significantly increased proportion of deglycosylated APP relative to control (p\u00a0<\u00a00.05). Close inspection of the data (Fig. 2B) suggests that the APP band ratio was still increased above control levels in the frontal cortex after 20 days stress but this effect did not significantly influence the global analysis.\nAlthough stress exposure appears to have altered the processing of APP with changes in the glycosylation patterns of the particulate form and the levels of APPs, it is not clear whether this is due to changes in the release of corticosterone over this time period [18,22] or if it is due to the action of other neurotransmitters, such as acetylcholine, that are altered following stress [14]. Other studies have shown that directly treating rats with the glucocorticoid agonist dexamethasone can also increase particulate APP levels in the rat cortex, cerebellum and brain stem without significant changes in APPs [5]. However, hippocampal APP expression was not specifically reported in that study. In this study, stress significantly elevated the proportion of deglycosylated APP in particulate brain samples for up to 10 days but after 20 days there was no significant difference from control. This follows the previously published pattern of habituation of the plasma corticosterone response in rats treated with exactly the same protocol [18,22]. Corticosteroids have also been shown to control the process of protein glycosylation and in the hippocampus sialyltranferase activity appears to be highly regulated by aldosterone [8]. The expression of the mineralocorticoid receptor, which has a relatively high affinity for aldosterone, is dynamically altered in the dorsal hippocampus during 20 days exposure to the elevated platform [18]. The stress-induced increase in the particulate deglycosylated form of the protein may therefore be a consequence of corticosteroid receptor changes. Although statistically, there was no significant difference between the ratio of deglycosylated to total APP in the hippocampus compared to other brain regions, this was the area that demonstrated the greatest change following acute exposure and was the only one where the ratio fell below control levels after 20 days of stress (Fig. 2B).\nThe hippocampus was also the only area where the amount of APPs increased following repeated stress. The hippocampus is involved in terminating the corticosteroid response to stress [20] and may also be important in habituating to repeated stress as previous studies have shown that 20 days exposure to the elevated platform produces a selective increase in GR immunoreactivity in the dorsal hippocampus [18]. The increase in APPs most likely represents an increase in soluble APPs\u03b1 that has been shown to exhibit neurotrophic properties. Since the hippocampus it also an area of the brain thought to be crucial for the formation of certain types of memory, the changes in APPs may have consequences for cognitive function. Previous studies have shown that stress can impair memory [21] or under certain circumstances can enhance it [3]. In particular, repeated exposure to the elevated platform has been shown to improve spatial memory using a watermaze task in young rats but only if they have been previously exposed to the apparatus [26].\nThe increase in deglycosylated APP seen following one or 10 days stress exposure may indicate an increased risk of A\u03b2 formation as the deglycosylated form of the protein is less likely to be trafficked to the cell membrane [16] for cleavage by \u03b1-secretase but rather may be retained in the endoplasmic reticulum\/golgi. Since \u03b2-secretase appears to be localised to the golgi and endosomal compartments of the cell [24] the deglycosylated APP is more likely to undergo amyloidogenic processing by \u03b2- and \u03b3-secretase pathway. Recent studies have shown that glucocorticoid treatment of either mouse neuronal cells or the 3XTg-AD mouse increases the formation of A\u03b2 [12]. This appears to be due to an increase in the expression levels of both APP and \u03b2-secretase. The formation of A\u03b2 peptide oligomers may also disrupt cognitive function, even prior to nerve cell death [7]. The increase in APPs within the hippocampus over the same period as habituation to a repeated stressor could therefore represent a compensatory response in relatively young adult rats. Further studies will be required to determine the exact mechanisms responsible for the changes in APP processing and whether an age-induced impairment in this neuroadaptation increases the risk of the stimulation of the AD-associated protein processing pathways.","keyphrases":["amyloid","alzheimer's disease","elevated platform","glycosylation","habituation","hippocampus"],"prmu":["P","P","P","P","P","P"]}
{"id":"Qual_Life_Res-3-1-2062490","title":"Response shift due to diagnosis and primary treatment of localized prostate cancer: a then-test and a vignette study\n","text":"Aim Whether a prostate cancer diagnosis induces response shift has not been established so far. Therefore, we assessed response shift in men who were diagnosed with localized prostate cancer.\nIntroduction\nResponse shift, defined as an adaptation to changing health [1], is a beneficial process for patients because it can help in adapting to a new situation. However, it may complicate the correct interpretation of change in health-related quality of life (QoL) scores over time in intervention studies, and therefore needs to be understood. Response shift refers to a change in the meaning of QoL over time [2] and can result from a change in one\u2019s internal standards of measurement (i.e. recalibration), a change in the importance attributed to the domains constituting QoL (i.e. change in values or reprioritization), or a change in the definition of the concept of QoL (i.e. reconceptualization) [3, 4]. These three forms of response shift are illustrated in the following example. Imagine a woman X. When asked to rate her QoL she thought of her (50-h a week) job, her partner and playing volleyball with friends, and rated her QoL as very good. Unfortunately she fell ill. For some months she was not able to work. Her partner and relatives supported her a great deal, which she appreciated enormously. Gradually she recovered and started working again for 20\u00a0h a week, but was no longer able to play volleyball. She rated her new QoL again as very good, but this time the main aspects of her QoL consisted of her partner, her family and work. Her ratings did not change, i.e. twice \u2018very good\u2019, but in fact three forms of response shift had occurred. \u2018Work\u2019 changed from a 50-h a week job to 20\u00a0h a week (recalibration), her partner became more important (reprioritization), and her concept of QoL has changed: no more sports, but relatives instead (reconceptualization).\nThis paper focuses on the assessment of response shift induced by a prostate cancer diagnosis. Schwartz and colleagues systematically addressed the state-of-the-art in the assessment and interpretation of response shift [5]. In a meta-analysis, following Cochrane guidelines, the magnitude and clinical relevance of response shifts across 19 longitudinal studies were evaluated. Most studies addressed global QoL and specific QoL domains such as fatigue, well-being, and pain, usually by conducting the then-test. Effect sizes, defined as the mean difference between tests divided by the standard deviation (SD) of the first assessment, were computed by the authors. These were generally small according to Cohen\u2019s criteria [6], with the largest effect sizes found for fatigue, followed by global QoL, physical role limitation, physical well-being, and pain. Effect sizes varied in direction, which complicated their interpretation. Schwartz et\u00a0al. concluded with recommendations for future response shift publications, such as explaining the meaning of the study results in terms of recalibration, reprioritization, and reconceptualization [5].\nResponse shift is more likely to occur when an intense and pervasive change in health is experienced [7]. A cancer diagnosis may have a large impact on a person\u2019s experienced health. Our group previously described the process of being diagnosed with prostate cancer through a screening process consisting of a Prostate Specific Antigen (PSA) test and, if indicated, a biopsy. Typically, localized prostate cancer diagnosed through screening is not associated with any physical symptoms. Men\u2019s mental health and the valuation of their own health decreased significantly after they received their diagnosis, and we concluded that being diagnosed with prostate cancer was a deeply felt change in health [8]. We started the present study because we expected that a prostate cancer diagnosis induces response shift. We hypothesized that the pre-diagnosis health state would be rated more positively in retrospect (i.e. if assessed after diagnosis) than at the reference point itself (i.e. pre-diagnosis).\nCollecting data on QoL before a cancer diagnosis is usually not feasible, since it is unknown who will develop cancer and when, so that the inclusion of a very large cohort would be required. However, the context of the European Randomized study for Screening on Prostate Cancer (ERSPC) [9] enabled us to include a cohort of men shortly before they were screened and subsequently diagnosed. We aimed at assessing the magnitude and direction of response shift effects after diagnosis and again after primary treatment. We employed two methods: the common then-test and a novel approach including rating of vignettes related to side effects of prostate cancer treatment (urinary, bowel, and erectile dysfunction).\nPatients and methods\nEthics approval and informed consent\nThe Ethics Committee of the Erasmus MC approved the research protocol. All participants gave additional written informed consent to be interviewed for the study.\nParent study\nInclusion of the ERSPC participants was initiated in 1994 among all male inhabitants of the Rotterdam region aged between 55 and 74\u00a0years. The only exclusion criterion was a previous prostate cancer diagnosis. Details on study recruitment for the ERSPC have been reported earlier [9].\nRespondents\nRandomly selected participants from the parent study were approached. All men who were due for the second (n\u00a0=\u00a02,798) or third screening round (n\u00a0=\u00a02,024) between January 2003 and May 2004 were sent a short questionnaire on health (see below) by mail. Men who were diagnosed through the screening process were interviewed twice by one of the authors (IK); one month post-diagnosis (but before treatment) and again 7\u00a0months post-diagnosis.\nAssessing response shift\nTo assess the magnitude and direction of response shift effects two methods were used: the then-test, and vignettes (a novel method in response shift research). For the resulting study scheme, see Fig.\u00a01.The then-test is a retrospective evaluation of an earlier assessment (retrospective pre-test-post-test design). At post-test respondents are asked to remember how they were doing at the reference point and to retrospectively rate their level of functioning or QoL at that time. The then-test was originally developed to measure recalibration. The method assumes that respondents will use their post-test internal standards when providing a re-evaluation or \u2018then-test\u2019 rating of their health at the reference point [5]. The comparison between the then-test and the post-test is thus assumed not to be confounded by recalibration and can be considered as an indication of true change [2]. The comparison of the mean pre-test, which is the assessment that was completed at the reference point, and then-test scores would reflect an estimate of the magnitude and direction of response shift [2]. Because respondents in our study were included before diagnosis, they could provide then-test scores relating to their health before diagnosis and to their health between diagnosis and treatment. Before completing the then-test, respondents were explicitly reminded about the period the then-test was referring to: e.g. the time when the respondents had not yet been diagnosed with prostate cancer and were unaware of having prostate cancer. Respondents were then asked to re-assess their health at that time. Three then-tests were conducted: two referencing pre-diagnosis health (measured at 1\u00a0month post-diagnosis and 7\u00a0months post-diagnosis) and one referencing 1-month post diagnosis health (measured at 7-months post-diagnosis), see Figs.\u00a02, 3, 4. The respondents completed generic QoL measures, i.e. the Short-Form 36 (SF-36) mental health and vitality, and the EQ-5D VAS for self-rated health, as a pre-test, post-test and then-test. The SF-36 consists of eight scales on physical and mental domains of health. We used the scales on mental health (five items on being nervous, down, peaceful, depressed and happy) and vitality (four items on being full of life, having a lot of energy, being worn out and tired). Higher scores (0\u2013100) indicate better mental health and vitality [10]. The EuroQol (EQ) 5D valuation of own health is a visual analog scale on current overall health, anchored at the lower end (0) by \u2018worst imaginable health state\u2019 and at the upper end (100) by \u2018best imaginable health state\u2019 [11].As a novel method to assess response shift we used vignettes that each described a health state relating to side effects of therapy for localized prostate cancer, i.e. urinary, bowel or erectile dysfunction. The vignettes contained items of the EQ-5D self-classifier complemented with items on dysfunction, for instance, \u2018Mr. A has no problems in walking about, has no problems washing or dressing himself, experiences urinary leakage daily, has no pain or discomfort, is not anxious or depressed\u2019. Respondents were asked to indicate how good or bad they evaluated these health states on visual analog scales anchored at the lower end (0) by \u2018very bad\u2019 and at the upper end (10) by \u2018very good\u2019. We used the vignettes to explore reprioritization. We hypothesized that men would value the health states as less detrimental after diagnosis than before. After diagnosis they knew they might experience these dysfunctions themselves in the context of prostate cancer treatment.Additionally, information on respondents\u2019 age and on the Gleason score (a clinical criterion for histological grading of the aggressiveness of the tumour) were obtained through the screening office.\nFig.\u00a01Study schemeFig.\u00a02Original and then-test scores of the EuroQol valuation of own health by prostate cancer patients (n\u00a0=\u00a052). If we measure only EQ-VAS preceding diagnosis and at 1-month post-diagnosis, the difference between these scores is regarded the \u2018observed change\u2019. However, if the retrospective pre-diagnosis assessment provides a more valid comparison with the post-diagnosis assessment, the \u2018true change\u2019 is reflected by the difference between the retrospective pre-diagnosis assessment and the post-diagnosis assessment. The difference between the pre-diagnosis assessment and the retrospective pre-diagnosis assessment provides an indication of the size and direction of the \u2018response shift\u2019 induced by the diagnosis. Similar explanations are valid for the other data points in the figureFig.\u00a03Original and then-test scores of the SF-36 mental health by prostate cancer patients (n\u00a0=\u00a052). \u2018Observed change\u2019, \u2018True change\u2019 and \u2018Response shift\u2019 refer to the differences in SF-36 mental health scores between the assessment at 2\u00a0months before diagnosis and post- and then-test at 1\u00a0month after diagnosis (for further explanation, see caption at Fig.\u00a02.)Fig.\u00a04Original and then-test scores of the SF-36 vitality by prostate cancer patients (n\u00a0=\u00a052). \u2018Observed change\u2019, \u2018True change\u2019 and \u2018Response shift\u2019 refer to the differences in SF-36 vitality scores between the assessment at 2\u00a0months before diagnosis and post- and thentestthen-test at 1\u00a0month after diagnosis (for further explanation, see caption at Fig.\u00a02.)\nStatistical analysis\nProcedures concerning imputation of missing responses in the SF-36 items were conducted according to the guidelines of the SF-36 Health Survey Manual [12]. Differences between assessments were tested with paired-samples t-tests. P-values\u00a0\u2264\u00a00.05 were considered statistically significant. The type I error rate, i.e. the ratio of significant findings to the number of comparisons, was calculated. To assess the magnitude of the differences between the assessments we used Cohen\u2019s effect sizes, defined as the mean difference between tests divided by the SD of the first assessment, and interpreted as follows: 0.2\u00a0<\u00a0d\u00a0<\u00a00.5 indicates a small, 0.5\u00a0\u2264\u00a0d\u00a0<\u00a00.8 a moderate, and d\u00a0\u2265\u00a00.8 a large effect size [6].\nThe minimal important difference (MID), which is the smallest change in a patient-reported outcome that is perceived by patients as beneficial or that would result in a change of treatment, was operationalized as a difference of at least half a SD [13].\nNon-response bias was analysed by testing differences between the respondents and the non-respondents with unpaired t-tests.\nResults\nOut of the 3,892 men who completed the initial questionnaire before screening on prostate cancer, 82 were subsequently diagnosed. Of these, 52 (response 63%) consented to participate in two additional telephone interviews at 1 and at 7\u00a0months post-diagnosis. All 52 respondents participated in the first interview, which took place before treatment had been initiated. Due to personal circumstances one respondent later refused the second telephone interview. Average age at screening was 67.3\u00a0years (SD 4.4), ranging from 60 to 74\u00a0years. The Gleason score was favourable in 42 of the 52 patients, i.e. below seven (Table\u00a01). In all respondents but one, treatment had been initiated at 7\u00a0months post-diagnosis, i.e. radical prostatectomy (n\u00a0=\u00a025), brachytherapy (n\u00a0=\u00a012), active surveillance (n\u00a0=\u00a010), external radiotherapy (n\u00a0=\u00a03), or hormonal treatment (n\u00a0=\u00a01), see Table\u00a01.\nTable\u00a01Gleason scores and treatment modality of the respondents (n\u00a0=\u00a052)Gleason score\u00a0<\u00a07 n\u00a0=\u00a042Gleason score\u00a0\u2265\u00a07 n\u00a0=\u00a010Total n\u00a0=\u00a052Radical prostatectomy18725External radiotherapy123Brachytherapy1313Active surveillance99Hormonal treatment 11No treatment choice yet11\nOriginal scores, i.e. scores relating to the respondents\u2019 health at the time of the assessment and interviews, and then-test scores relating to the two reference points are given in Table\u00a02. For example, \u201885.2\u2019 in the upper right corner of Table\u00a02 reflects the \u2018EQ valuation of own health\u2019 score of the then-test measured at 7\u00a0months post-diagnosis referencing 2\u00a0months pre-diagnosis. Mental and self-rated health scores worsened significantly from 2\u00a0months preceding diagnosis to 1\u00a0month post-diagnosis. The average mental health score, for instance, was 83.2 at 2\u00a0months pre-diagnosis, and 75.8 at 1-month post-diagnosis; a decrease of 7.4 that exceeds the MID. At 7\u00a0months post-diagnosis mental and own health scores had increased again, but not to their original level.\nTable\u00a02Mean health scores (standard deviation) of the respondents (n\u00a0=\u00a052) before and after diagnosis, original and thentests scoresReferring toOriginal scores (n\u00a0=\u00a052)P-value*Thentests1\u00a0month post-diagnosis (n\u00a0=\u00a052)P-value: thentest vs. originalEffect size: thentest vs original 7\u00a0months post-diagnosis (n\u00a0=\u00a051)P-value: thentest vs. originalEffect size: thentest vs. original2\u00a0months pre-diagnosisEuroQol own health80.2 (11.7)83.2 (9.0)0.058\u22120.2685.2 (7.6)0.002a\u22120.43SF-36 mental health83.2 (11.6)84.5 (11.0)0.304\u22120.1083.2 (10.9)1.000.01SF-36 vitality75.3 (15.6)79.6 (12.0)0.008\u22120.2879.4 (12.4)0.046\u22120.261\u00a0month post-diagnosis, before initiation of treatmentEuroQol own health74.5 (15.1)0.01074.1 (14.5)0.6180.03SF-36 mental health75.8 (16.8)0.001a72.8 (18.3)0.0420.17SF-36 vitality74.7 (14.2)0.77172.6 (13.7)0.1110.157\u00a0months post-diagnosis, after intiation of treatment in 80% of respondentsEuroQol own health77.6 (13.7)0.196SF-36 mental health80.4 (13.8)0.066SF-36 vitality73.1 (17.7)0.213*P-value of difference with previous original score (P\u00a0\u2264\u00a00.05 are considered significant)aChange exceeds the minimal important difference (operationalised as \u00bd standard deviation)\nOriginal scores of pre-diagnosis health were lower, indicating worse health than on the then-test scores. For example, the original pre-diagnosis mental health score was 83.2 on average, but the then-test score measured at 1\u00a0month post-diagnosis was 84.5, indicating a more positive judgement of pre-diagnosis mental health in retrospect. Original scores of health between diagnosis and treatment, on the other hand, were higher, indicating better health than on the then-test scores. The original vitality score, for instance, was 74.7 at 1\u00a0month post-diagnosis, but the then-test score measured at 7\u00a0months post-diagnosis was 72.6. This means that vitality between diagnosis and treatment was judged worse when measured in retrospect than when measured at the reference point itself. Original and then-test scores are presented in Figs.\u00a02\u20134, including estimates of the response shift effects, i.e. the difference between mean pre-test and then-test scores, and estimates of \u2018true\u2019 change, i.e. the difference between the mean post-test and then-test scores.\nEffect sizes of the differences between then-test and original scores were small (Table\u00a02).\nThe vignettes describing urinary, bowel and erectile dysfunction states were rated significantly higher (i.e. better) by respondents at 1\u00a0month post-diagnosis than at 2\u00a0months pre-diagnosis (P-values 0.038, 0.011, and <0.001, respectively). The valuation of erectile dysfunction showed the largest increase; i.e. from 5.3 to 6.7 on a 0\u201310 scale, with a moderate effect size of 0.57 (Table\u00a03). This implies that respondents considered especially erectile dysfunction less detrimental after diagnosis with prostate cancer than before diagnosis. The differences between pre- and post-diagnosis valuations of the vignettes exceeded the MID in 4 out of 6 cases (Table\u00a03).\nTable\u00a03Average valuation by VAS (SD) of prostate cancer specific vignettes, scale 0-10, P-values\u00a0\u2264\u00a00.05 were considered significantHealth state descriptionPre-diagnosis (n\u00a0=\u00a052)1\u00a0month post-diagnosis (n\u00a0=\u00a052)P-value pre-diagnosis vs. 1\u00a0month post-diagnosisEffect size pre-diagnosis vs. 1\u00a0month post-diagnosis7\u00a0months post diagnosis (n\u00a0=\u00a051)P-value pre-diagnosis vs. 7\u00a0months post-diagnosisEffect size pre-diagnosis vs. 7\u00a0months post-diagnosisDaily urinary leakage5.6 (2.1)6.3 (1.5)a0.038\u22120.325.9 (1.5)0.348\u22120.14Daily bowel cramps5.3 (2.0)6.0 (1.6) 0.011\u22120.416.2 (1.4)a0.012\u22120.39Serious erectile dysfunction5.3 (2.2)6.7 (1.8)a<0.001\u22120.576.5 (1.8)a0.005\u22120.47achange compared to previous score exceeds minimal important difference\nThe results of the then-test were significant in 4 out of 9 comparisons, the results of the vignettes in 5 out of 6. The overall type I error rate, which is the ratio of significant findings to the number of comparisons, was 0.6 (9 out of 15).\nNon-response analysis\nThe baseline average age in men who were diagnosed with prostate cancer but did not respond to the questionnaire (n\u00a0=\u00a030) was 66.7 (SD 4.3, range 59\u201373) years. Respondents and non-respondents did not differ significantly in age or other health measures (data not shown).\nDiscussion\nMen diagnosed with prostate cancer evaluated their pre-diagnosis health in retrospect as better than at the reference point itself. Post-diagnosis\u2013pre-treatment health was rated worse in retrospect than at the reference point. This suggests that \u2018true\u2019 changes in health between the first assessment before diagnosis and the second one at 1\u00a0month post-diagnosis were larger than the original scores disclosed, and that  response shifts were induced by first, the diagnosis, and second, subsequent treatment. The sizes of the response shifts induced by the diagnosis were larger than those induced by the treatment. The negligible to small effect sizes indicated that only some recalibration occurred. The directions of the effect sizes were interpretable and consistent with our hypotheses.\nAdditionally, men evaluated vignettes relating to side effects of prostate cancer treatment as less detrimental after they were diagnosed than before diagnosis. We interpreted this change as a reprioritization of respondents who became aware after being diagnosed with prostate cancer that they were at risk of experiencing these health states themselves as a consequence of being treated for prostate cancer. In this new context dysfunctional health states were evaluated as less bad than before. The effect sizes were moderate for erectile dysfunction and small in the two other ones, indicating that reprioritization also occurred. The directions of the effect sizes were interpretable and consistent with our hypotheses.\nThe overall type I error rate was 0.6, which indicated that the statistical significance is very unlikely to be caused by chance. This is an additional indication that our findings reflect real differences. We conclude that the results of the then-tests and the ratings of the vignettes both indicate the presence of a response shift and adaptation of the patients to their new situation.\nIn the meta-analysis of Schwartz et\u00a0al., the largest effect sizes on response shift (although still small) were found for the dimensions global QoL and fatigue [5]. These dimensions, represented in our study by EQ-5D on own health and the SF-36 vitality scale respectively, also resulted in small effect sizes.\nAn important criticism of the then-test approach is its susceptibility to recall bias. Respondents are supposed to be able to remember their previous health at the reference point, which is extremely difficult in case of a chronic disease with no obvious trend towards better or worse health [14]. However, in a study on response shift in cancer patients undergoing various forms of treatment, there was evidence that recall bias was absent [2]. We assume that in the case of a deeply felt change in health (such as being diagnosed with cancer or the initiation of cancer therapy) recall will not cause memory difficulties for most respondents. Therefore, in our study we expect that recall bias did not have a major influence on the results.\nThe then-test results in a retrospective judgement that subsequently is used to construct \u2018real change\u2019 since the reference point. This approach assumes that the information that was acquired after the original judgment was made leads to more accurate estimates of QoL than the original judgment itself. This assumption is, however, not always true; for example in the case that the newly acquired information is not correct [14].\nThe valuation of disease-specific vignettes (the second method used to assess response shift) has to our knowledge not been described before. It resulted in a moderate effect size considering the vignette on erectile dysfunction. The directions of the effect sizes were consistent with our hypotheses. Our results showed that response shift can be studied by using vignettes. We consider the valuation of vignettes as a useful addition to the already available collection of tools to assess response shift. Apart from this theoretical value, the results of the vignettes may also have implications for clinical practice. In case of a diagnosis of localized prostate cancer several treatment options are available. Since there is no consensus about which of these treatments has the best outcome in terms of survival and QoL, considerations of patient preferences regarding mode of treatment and side effects are an essential element in shared decision making on the choice of therapy. To elicit a patient\u2019s preferences and his individual trade-offs between benefits and side effects of various modes of treatment, vignettes can be useful [15].\nHowever, our study showed that patient preferences may change in the course of the diagnostic and treatment process, which illustrates how difficult it is for a patient to imagine the consequences of an intervention in advance. This finding confirms the point made by Cowen et\u00a0al. to recommend the use of individual utilities (\u201cactually prefer\u201d) instead of population\u2019s utilities (\u201cshould prefer\u201d) to optimise the choice of treatment for patients with prostate cancer [16].\nWe recommend further investigation of the vignettes method. The fact that being diagnosed with prostate cancer was found to induce response shift may be seen as an indication that men regard a prostate cancer diagnosis as a major life event, and is additional evidence for earlier findings [8].\nIn another study, men with metastic or locally advanced prostate cancer completed assessments on prostate symptoms shortly after diagnosis, and 3 and 6\u00a0months thereafter. The second and third assessments included then-tests. The presence of a response shift was suggested in patients and their spouses [17, 18]. The authors remarked that retrospective and prospective assessments cannot be used interchangeably.\nLepore and Eton tested two conceptual models of response shift among men newly diagnosed with prostate cancer to explain the frequently observed lack of association between health problems and QoL in cancer patients. No support was found for the suppressor model, according to which health change leads to response shift, which in turn leads to a change in QoL. Some evidence was found for the buffering model, according to which response shift effects moderate the negative association between health problems and QoL. Two aspects of response shift, recalibration and reprioritization, were assessed by then-tests and a measure of primary life goal changes, respectively. They were found to moderate the relation between negative changes in physical health and changes in QoL [19].\nIndications of response shift were also found in an earlier study on men treated for localized prostate cancer. Men stated, for instance, that they accepted the side effects of treatment because \u2018If they hadn\u2019t intervened, that operation, maybe I wouldn\u2019t be here anymore\u2019 [20].\nThe present study has several strengths and limitations. The study design is one of its strengths; the unique context of the ERSPC enabled the inclusion of respondents before they (or anyone else) were aware that they had prostate cancer, which is usually unfeasible. To our knowledge this is the first study to measure response shift in men who were diagnosed with cancer. An additional strength is the compliance of the respondents; 51 of the 52 respondents completed the 7-month assessment.\nFor the then-test we selected measures that are considered subjective (i.e. SF-36 mental health and vitality, and EQ-5D of own health) but no objective items, which can be considered a drawback of the study. Furthermore, we acknowledge that offering questionnaires in two different modes (self-administered questionnaires before diagnosis vs. telephone interviews afterwards) may have been less than optimal. This design was chosen based on practical considerations, because assessments by telephone in 3,892 screen participants was not feasible, and self-administered questionnaires at 1\u00a0month after diagnosis undesirable since we wanted these assessments to be completed before the initiation of treatment. The unavailability of information on marital status and education is also a drawback. Another potential limitation of our study is that the interval between the initiation of treatment and the assessment at 7-months post-diagnosis was not the same for all respondents; it is possible that response shift may vary according to the length of time that elapsed since treatment. However, information on this interval had been of limited use. The most common therapies for localized prostate cancer nowadays are surgery, radiotherapy, and active surveillance. These therapies differ greatly (by nature) in duration, the onset of side effects and their course over time.\nIt may be that particular groups may be more prone to response shift than others, e.g. depending on age or prognosis. We plan to address this issue further, preferably in a larger sample than used in the current study.\nConclusions\nUsing two complementary techniques we found that a diagnosis of prostate cancer induces response shift. From a methodology point of view, the vignette-method needs to be explored further.","keyphrases":["response shift","prostate cancer","quality of life","patient-reported outcome"],"prmu":["P","P","P","P"]}
{"id":"Mar_Drugs-5-4-2365694","title":"Evaluation of Harmful Algal Bloom Outreach Activities\n","text":"With an apparent increase of harmful algal blooms (HABs) worldwide, healthcare providers, public health personnel and coastal managers are struggling to provide scientifically-based appropriately-targeted HAB outreach and education. Since 1998, the Florida Poison Information Center-Miami, with its 24 hour\/365 day\/year free Aquatic Toxins Hotline (1\u2013888\u2013232\u20138635) available in several languages, has received over 25,000 HAB-related calls. As part of HAB surveillance, all possible cases of HAB-related illness among callers are reported to the Florida Health Department. This pilot study evaluated an automated call processing menu system that allows callers to access bilingual HAB information, and to speak directly with a trained Poison Information Specialist. The majority (68%) of callers reported satisfaction with the information, and many provided specific suggestions for improvement. This pilot study, the first known evaluation of use and satisfaction with HAB educational outreach materials, demonstrated that the automated system provided useful HAB-related information for the majority of callers, and decreased the routine informational call workload for the Poison Information Specialists, allowing them to focus on callers needing immediate assistance and their healthcare providers. These results will lead to improvement of this valuable HAB outreach, education and surveillance tool. Formal evaluation is recommended for future HAB outreach and educational materials.\n1. Introduction\nHarmful algal blooms (HABs) are caused by blooms of algae known as phytoplankton; these include organisms such as dinoflagellates, diatoms and cyanobacteria [1\u20136]. HABs can occur in all aquatic environments. In marine environments, they are also known as \u201cred tides\u201d because some of these organisms can change the color of the water to red or brown. HABs may cause harm to the environment and other organisms in two ways. First, through the severe overgrowth of the HAB organisms that depletes oxygen in the local environment, and second when the HAB organisms produce extremely potent natural toxins.\nPhytoplankton are the base of the marine food web; thus, the toxins they produce can bioconcentrate in organisms higher up in the food chain. Several human illnesses are caused by the ingestion of seafood contaminated with the natural toxins produced by the HAB organisms [1\u20136]. In addition to exposure through seafood ingestion, environmental exposures can occur through skin contact with contaminated water or by inhalation when the HAB organisms are broken up by waves and their toxins become aerosolized. For example, human exposure to aerosols containing brevetoxins from the Florida red tide dinoflagellate, Karenia brevis, has been associated with reports of respiratory distress, particularly but not exclusively in persons with asthma, as well as increased emergency room admissions for pneumonia, bronchitis and asthma among coastal residents during active Florida red tides [7\u201312].\nWith the increasing number of persons interacting with the coastal areas (both freshwater and marine) and with the apparent increase of HABs around the world, healthcare providers, public health personnel, and coastal managers are struggling to provide scientifically-based information [13\u201317]. There is a paucity of appropriately targeted outreach and educational materials for healthcare providers and persons with possible exposure to the marine and freshwater toxins, as well as coastal resource managers, the media, and the general public. A variety of educational outreach materials and services have been created, but there has been almost no formal evaluation to determine whether these materials are reaching their target audiences and meeting these audiences\u2019 expectations.\n1.1. Florida Poison Information Centers\nThe Florida Poison Information Center\/Miami (FPIC\/Miami) is one of three poison centers created in the State of Florida in 1989 by an act of the Florida Legislature (FS 395.038), and it has rapidly grown into a cost-effective model of a poison center system. The FPIC\/Miami is located at the University of Miami Miller School of Medicine and Jackson Health Systems, and directly serves the residents of Southeastern Florida (i.e. Miami-Dade, Broward, Monroe, Palm Beach, Lee and Collier counties) and beyond with toll-free 24 hour\/day services in several languages. For example, in 2006 the FPIC\/Miami responded to over 57,340 calls; of these calls, 35,741 (62%) were for known or suspected exposures to toxic substances. Approximately 80% of the patients calling from home were managed in the home, without the need for an emergency department visit. The FPIC\/Miami placed 18,833 (33%) follow-up calls to continually assess this home management. The FPIC\/Miami toxicologists provided over 1,705 consultations with Florida\u2019s physicians to assist in the care of hospitalized patients. In an attempt to prevent poisonings, the FPIC\/Miami educators coordinated 669 outreach and education programs for the lay public, health professional, and the media. They also distributed over 198,213 informational brochures, handouts and telephone stickers, reaching over 72,000 persons.\nSince 1998, with funding from the Florida Department of Health, the National Center for Environmental Health (NCEH) of the Centers for Disease Control and Prevention (CDC), and the University of Miami National Institute of Environmental Health Sciences (NIEHS) Marine and Freshwater Biomedical Sciences Center and the National Science Foundation (NSF)- NIEHS Oceans and Human Health Center, the FPIC-Miami established a toll-free hotline (1\u2013888\u2013232\u20138635) dedicated to providing information concerning aquatic health issues. All calls are answered by Poison Information Specialists, who are highly trained physicians and nurses. All calls are reported as a form of passive surveillance of HAB-related illness and information requests to the Aquatic Toxins Program (http:\/\/www.doh.state.fl.us\/ENVIRONMENT\/community\/aquatic\/) and to the Foodborne Illness Program of the Florida Department of Health.\nRecently, the Hotline was expanded to include additional options for callers. Using an automated call processing menu system, the \u201cAquatic Toxins Hotline,\u201d now allows callers to access information in English or Spanish about the possible health effects and locations of the Florida red tide (including the National Atmospheric and Oceanographic Administration (NOAA) Harmful Algae Bloom (HAB) Bulletin http:\/\/coastwatch.noaa.gov\/hab\/bulletins_ns.htm), information on ciguatera fish poisoning, blue green algae (cyanobacteria) and resources for learning about general marine toxin issues, as well as still giving the callers the opportunity to select to speak directly to a Poison Information Specialist if they wish. These materials were reviewed and developed with cooperation between the FPIC-Miami, the University of Miami, the Florida Department of Health, the CDC, Mote Marine Laboratory, and the Florida Fish and Wildlife Commission.\nConcurrent with this initiative, a number of academic, public health and community partners (including Solutions to Avoid Red Tide or START; http:\/\/www.start1.com\/) collaborated to create a series of informational signs advertising the Aquatic Toxins Hotline as a source of information for Florida red tide. After focus groups with public health personnel, beach managers, hotel and restaurant owners, and tourist boards, these signs were designed to be posted on and near beaches, and targeted at beach users including tourists (see Figure 1).\nThis Pilot Study evaluated whether the new automated system was used by callers and whether the callers considered the information to be useful to them, as well as to collect caller recommendations for the improvement of this service. The goal of this pilot study was to evaluate whether the Florida Aquatic Toxins Hotline was serving the self-identified needs of current callers.\n2. Methods\nThe FPIC-Miami receives the telephone numbers of all callers to the Aquatic Toxins Hotline, whether the caller speaks with a Poison Information Specialist or just the automated system. In this evaluation, telephone numbers from the calls made to the Aquatic Toxins Hotline from September 2006 through January 2007 were re-contacted. Of note, throughout this study time period, there was an active Florida red tide on the West Coast of Florida.\nThis evaluation aimed to determine:\nBasic Caller demographics\nWhat information\/services callers were seeking\nWhich menu selections were the most commonly chosen\nWhich menu options, in the caller\u2019s opinion, were the most helpful\nIf callers were satisfied with the service provided by the Aquatic Toxins Hotline\nIf callers would call again\nWhat would, in the callers\u2019 opinion, make the Aquatic Toxins Hotline service more useful.\nTrained interviewers contacted the Aquatic Toxins Hotline callers by phone in English and Spanish to explain the study, obtained verbal consent for participation and conducted a short scripted interview (available on request from the corresponding author). Specifically, the interviewers asked a brief series of questions about using the Aquatic Toxins Hotline and about the caller\u2019s perception of the system\u2019s usefulness. Study participants also had an opportunity to make suggestions to improve the system. No personal identifying information was collected. To maximize success in contacting callers, the interviewers called each telephone number three times (once during a week day, once during a week night, and once during the weekend) in an attempt to re-contact the specific person who had called the Aquatic Toxins Hotline.\nThis study was approved by both the University of Miami and Florida Department of Health Human Subject Committees. The data were collected into a Study Access Database. The data were analyzed using SAS version 9.1. The data were analyzed by frequencies as a descriptive process.\n3. Results\nThe Aquatic Toxins Hotline has experienced increasing use over the past few years, particularly during periods of active Florida red tides. Since its inception in 1998, there have been 25,000 calls to the Hotline, with 7,000 occurring between 2001 and 2006; during 2006 alone, 2,415 calls were made to the Aquatic Toxins Hotline. During the study period from Sept 2\/06 through Jan 31\/07, there were 1,163 calls made to the hotline, with 315 (27%) answered by a Poison Information Specialist; of these calls, 53% were for Florida red tide, 6% for ciguatera, 17% for jellyfish, 2% each for PSP\/pufferfish, scombroid, and stingrays, and 0.6% blue green algae. This means that only 27% of the callers selected to speak with a Poison Information Specialist from the automated HAB Menu in English or Spanish, while the vast majority (73%) only accessed the automated HAB menu.\nThe Interviewers attempted to contact each of the callers during the study period as described above. Even though the follow-up calls were made within two months of the original contact, only a small percentage (10%) of the callers was able to be re-contacted. Inability to re-contact these individuals was due to a variety of factors such as: no answer despite repeated calls, answering machines, and no real contact number (i.e. original call made from hotel, hospital, etc). Nevertheless, once contacted, all callers (100%) agreed to participate.\nA total of 118 callers were successfully re-contacted and agreed to participate. Of these, only 89 callers reported that they recalled making a call to the Florida Aquatic Toxins Hotline, and thus these 89 callers were considered the participants in this Pilot Study. Of note, both the actual number of respondents and the percentages are reported below because not all subjects answered all the questions.\nThe majority of the 89 participants were women (41 [61%]), self-described as white (82 [100%]) and non Hispanic (66 [94%]) whose primary language was English (80 [96%]) and with a mean age and standard deviation of 55.5 +\/\u2212 12.2 years (see Table 1). The participants were predominantly from Florida (50 [77%]), with the majority from the West coast of Florida (46 [60%]) particularly in Sarasota, the area of recurrent Florida red tide. There were 18 (23%) callers from other parts of the US, including 1 from the US Virgin Islands (data not shown).\nThe majority of the participants had heard about the Aquatic Toxins Hotline from a website (35 [39%]), newspaper or magazine article (16 [18%]), a friend or personal recommendation (13 [15%]), or a sign (7 [8%]). Many of the participants (38 [48%]) called with general information questions; of note, none of the participants reported calling because they or someone they cared for was sick or believed they had been exposed to a toxin.\nOverall, the majority of participants (59 [68%]) reported that they received an answer to the question that prompted their call to the Florida Aquatic Toxins Hotline (see Table 2). Furthermore, 59 (68%) reported that the Florida Aquatic Toxins Hotline was easy to use, with only 17 (20%) reporting otherwise. Finally, 69 (80%) of the participants reported that they would be likely to use the Florida Aquatic Toxins Hotline again, and 68 (79%) stated that they would recommend the Hotline to others.\nWith regards to the specific information supplied by the automated system, 34 (39%) of participants reported speaking with a Poison Information Specialist; among these participants, 23 (74%) were satisfied and only two (7%) were dissatisfied (see Table 3). Among those participants who used the automated HAB menu, the largest number (46 [54%]) reported listening to the NOAA HAB Bulletin which gives current and forecasted information on the location of the Florida red tides; among these participants, 30 (70%) were satisfied and seven (17%) were dissatisfied. The second largest group (37 [44%]) reported listening to general information about Florida red tides; among these participants, 26 (70%) expressed satisfaction, and 8 (22%) were dissatisfied. Participants also reported listening to information about other marine toxin issues (28 [33%]) with 20 (71%) expressing satisfaction; Ciguatera fish poisoning (11 [13%]) with 10 (90%) expressing satisfaction; and blue green algae (cyanobacteria) (10 [12%] with 9 (90%) expressing satisfaction.\nAt the end of the questionnaire, the participants were asked if there was anything in particular they thought should be done to improve the usefulness of the Florida Freshwater and Marine Health Hotline as an open ended question. The majority of the comments can be summarized as: a) wanting more up-to-date information (particularly geographic location of the Florida red tides) on even a daily basis; and b) wanting to speak with a person directly or listening to an automated system. Specifically, the participants recommended that the Hotline provide the geographic location of the Florida red tides on a map; provide more information to tourists in hotels; and make sure that the Poison Information Specialists were up-to-date with HAB and Florida red tide information.\n4. Discussion\nThis study was the first known systematic evaluation of the use of and satisfaction with educational outreach materials for HABs, specifically evaluating callers to an automated Florida Aquatic Toxins Hotline of the South Florida Poison Information Center. The majority (68%) of participants reported that they were satisfied with the information provided by the Aquatic Toxins Hotline; 80% that they would use the Hotline again; and 79% that they would recommend the Hotline to others. Furthermore, even when evaluating the specific services offered by the Aquatic Toxins Hotline automated system (including speaking directly with a Poison Information Specialist), the high level of satisfaction persisted. Of interest, only 8% of participants reporting hearing about the Aquatic Toxins Hotline from signs, with the rest learning about the hotline from a website (39%), newspaper or magazine article (18%), or friend (15%). Our evaluation demonstrated that the primary advantage of the new automated system is that it provided useful HAB information for the large majority of the callers, without requiring contact with a Poison Information Specialist. By decreasing the workload of purely informational calls for the Poison Information Specialists, the Hotline allows the Specialists to focus on those persons who need immediate assistance.\n4.1. Limitations\nAs seen in previous studies using the Poison Information data [18,19], only a small percentage (10%) of the callers were able to be re-contacted. This lack of participation in telephone surveys is part of an unfortunate and growing nationwide trend recognized by the evaluation research community [20,21]. Furthermore, the majority of the study participants (77%) gave zip codes of addresses within Florida; since the follow-up calls were made two months from the original call, it would appear that the majority of the participants were either part-time seasonal (i.e., \u201csnow-birds\u201d) or permanent residents. Based on the review of the zip codes and area codes of all Hotline callers, many were likely tourists, and thus were \u201clost to follow-up\u201d when they could not be reached two months after their original call to the Hotline [20,21].\nIn addition, these participants were not entirely representative of the overall Hotline caller population since a greater proportion (39%) reported speaking with a Poison Information Specialist than was reported (27%) for the entire population of callers to the Florida Aquatic Toxins Hotline during the study period. However, based on the limited demographic information, the participants interviewed were similar to those contacted in other studies which have used the overall South Florida Poison Information Center Caller Database, i.e., predominantly white non Hispanic older females [18,19]. We did not identify many Hispanic callers in this study. Of note, most of the relevant marine HAB activity is concentrated on the Gulf Coast, while the majority of Florida\u2019s Hispanic population is concentrated on the east coast of Florida. Finally, there were missing data (e.g. 22 out of 89 persons did not report their gender).\n5. Discussion and Recommendations\nThis Pilot Evaluation Study demonstrated that the additional outreach provided by the Hotline was successful in getting information to people who wanted it. It also suggested that there needs to be increased outreach and education efforts using a range of media to inform additional target populations about the Aquatic Toxins Hotline, such as residents and tourists, and their healthcare providers. For example, it has been shown in the case of ciguatera fish poisoning, that even in an endemic area such as Miami (Florida), the majority of healthcare providers do not recognize, nor (more important from the point of view of HAB surveillance) do they report cases of ciguatera to the Health Department even though it is a reportable illness throughout the US [22]. Therefore, additional outreach and education not only to the possible victims of HAB-related illness, but also for their healthcare providers is needed.\nRecommendations for improving the Aquatic Toxins Hotline automated HAB system include the following: 1) Existing automated materials need to be revised to incorporate an ongoing evaluation component, with a focus on tourist not just resident callers, and in coordination with ongoing efforts in providing geographic location [23]; 2) Existing materials will need constant updating; and 3) Ongoing training should be provided for the Poison Information Specialists (as well as other healthcare providers) concerning the possible exposures and health effects from the aquatic toxins as new HAB research data and resources are made available [13, 23\u201329]. In addition, as different HAB educational outreach materials and services are created, these materials and services need to be evaluated before and after implementation in the target populations to improve their quality and utility. Based on the participation rate in this study, rapid follow up is necessary to ensure an effective evaluation. This will require the incorporation of additional resources and collaboration with investigators with expertise in outreach, education and evaluation in future HAB activities.","keyphrases":["harmful algal bloom (hab)","outreach and education","poison information centers","cyanobacteria","brevetoxins","florida red tide","karenia brevis","ciguatera fish poisoning","blue green algae","ciguatoxins","human health effects","neurotoxic shellfish poisoning (nsp)","paralytic shellfish poisoning (psp)","solutions to avoid red tide (start)"],"prmu":["P","P","P","P","P","P","P","P","P","U","R","M","M","R"]}
{"id":"Eur_J_Clin_Pharmacol-3-1-2071959","title":"Adherence to HAART therapy measured by electronic monitoring in newly diagnosed HIV patients in Botswana\n","text":"Aims This pilot study was designed to evaluate the feasibility and benefits of electronic adherence monitoring of antiretroviral medications in HIV patients who recently started Highly Active Anti Retroviral Therapy (HAART) in Francistown, Botswana and to compare this with self-reporting.\nIntroduction\nBotswana is one of the countries worst affected by the HIV pandemic with a prevalence of approximately 17% of the entire population [1]. An estimated 38.5% of those aged 15\u201349\u00a0years are HIV-positive, and it is estimated that one in every eight children is born with HIV [2].\nSince 2002 the nation has embarked on the provision of antiretroviral drugs for all its eligible citizens by implementing the MASA program.(The national antiretroviral therapy program was given the name MASA, the Setswana world for \u201cdawn\u201d).\nThe use of potent antiretroviral combinations has provided unprecedented opportunities for effectively treating HIV disease by suppressing viral replication and has led to dramatic decline in HIV mortality [3, 4].\nAdherence to antiviral regimens in HIV infected patients is essential for adequate suppression of viral replication. When adherence falls below a certain level, intermittent viremia will occur, and this may increase the chance of the development of resistant strains possibly followed by therapy failure [5\u20137].\nIn contrast, nonadherence to the prescribed antiretroviral regimens is associated with a rapid selection of resistant HIV strains resulting in treatment failure [8, 9].\nThe required high level of antiretroviral drug adherence in a poor resource setting remains therefore a serious concern. Assessment of adherence in HIV patients such as in this pilot-study may also provide tools to allow feedback and education on an individual health care provider\u2013patient base.\nFor this reason patients in the Botswana Infectious Disease Care Centers (IDCC) treatment program are urged at each visit to the IDCC facility to comply with the prescribed ART regimen. This occurs in three stages: (1) in group instruction sessions together with other HIV patients and individual counselling by a trained nurse or pharmacist, (2) by their individual health care provider (physician), and (3) by the pharmacist. In addition, patients are usually accompanied by a close family member who is asked to assist or remind patients of the pill intake (adherence partner).\nReliable information about the actual tablet intake is a prerequisite for any form of management or modification of the adherence to therapy. It has been recognised that many of the traditional methods of assessing adherence such as pill counts, diaries, or self-reports are unreliable. Electronic monitoring enables the recording of the time points of pill bottle openings. This method also has drawbacks and may underestimate adherence [10] and, of course, it does not provide evidence of actual ingestion of the drug [11]. Despite these drawbacks it has been so far the closest to a gold standard for adherence measuring [12], although other methods remain of value.\nA new method to measure adherence to prescribed medication regimen is the use of electronic monitoring [10, 13, 14]. Such systems commonly rely upon a microprocessor located in the cap of the medication container, where time and date of each opening are recorded. Each cap opening and closing is assumed to reflect a single medication-taking event. The data stored in the microprocessor are transferred to a computer database and uploaded for analysis [11]. Other methods like self-reporting, pharmacy records, and pill counts tend to overestimate patient adherence by anywhere from 20\u201330% [15\u201321].\nThis study was designed as a pilot study to evaluate electronic adherence monitoring in an HIV infected patient group that was put on antiretroviral medication for the first time. A secondary objective was to compare the adherence measured by electronic monitoring with that of self-reporting by means of a medication diary.\nMethods\nPatients\nThirty consecutive patients were recruited into the study during the period 13\u201330 October 2005. All were patients with an AIDS-defining illnesses and\/or CD4 cell counts< 200 cells\/mm3 (uL) who were offered HAART according to the Botswana Guidelines on Antiretroviral Treatment [2]. All patients were ARV-na\u00efve.\nDesign\nThis was a trial in which treatment-na\u00efve patients were monitored with regard to adherence to prescribed anti-HIV medication. Patients were not informed about the use, blinded of the electronic monitoring system, and were only asked to return their pill-bottles at each visit to the pharmacy when they returned for a refill and a consultation. Patients were also supplied with a self-reporting form. The study did not involve study related interventions and the subjects were not required to change behaviour in any way. The subjects were informed that the medication was supplied in special containers that had to be returned to the clinic but not about the monitoring system to prevent bias. The study was approved by the hospital management and the chief physician of the department of internal medicine.\nTreatment regimens\nThe treatment regimen used consisted of 2-nucleoside reverse transcriptase inhibitors (NRTIs) plus 1 nonnucleoside reverse transcriptase inhibitor (NNRTI). Patients started the recommended first-line treatment with three different agents. These were zidovudine and lamivudine in a combination tablet Combivir (CBV) plus efavirenz (EFV) or nevirapine (NVP).\nMale and female patients who were not anaemic were prescribed Combivir medication whereas anaemic patients (Hb < 7.5\u00a0mmol) were given stavudine (d4T\/Zerit) and lamivudine medication. The stavudine dose was adjusted according to bodyweight (30 or 40\u00a0mg).\nAll patients except females in the reproductive age category were given once daily EFV. All females of the former category were prescribed NVP at a 200\u00a0mg once daily dose for 14\u00a0days which, after assessing the liver function parameters, was increased to 200\u00a0mg twice daily.\nThe continuation of NVP or EFV was dependent on the absence of significant rise in hepatic enzymes (AST and ALT). Approximately 90% of those who started the NVP or EFV treatment are able to continue this medication. Patients returned to the IDCC after a month for a medical check and refill of their prescription unless clinical events dictated earlier visits to the clinic.\nThe treatment starters were booked to see the doctor after the first 2\u00a0weeks of therapy. After seeing the doctor, the self recorded medication card and the electronic monitors were collected, and the data stored in the microprocessor were transferred to a database in the computer. Following this, each bottle was refilled and provided with a new label with medication instructions. Most patients received a refill for a period of 1 month, some however for a shorter period. The potential side effects were discussed with each patient. The results of the first analysis of the electronic monitors were not used in any counselling.\nThe electronic monitors were MEMS IV Track Cap devices (Aardex, Zug, Switzerland) with a MEMS IV Communicator for read-out of the results.\nStudy endpoints\nThe primary study endpoint was the adherence level measured (over a minimal period of 6\u00a0weeks) by the percentage of days on which the patients took a correct dosing over the monitored period. Adherence was also expressed as the number of pill-intakes recorded on a self-reporting forms designed to reflect the intended schedule and timing of treatment.\nIn the event a patient opened his\/her bottle more than was prescribed (surplus opening), it was assumed that the patient correctly took the prescribed pills. However, occasions on which a patient opened less than the prescribed dose frequency were considered as adherence failures.\nSequence and duration of trial period\nEach patient was immediately given counselling and made familiar with the ART treatment. During the counselling session emphasis was given with regard to the need of strict adherence of the prescribed medication and to methods to prevent disease transmission. They were also informed about the self-reporting form and given a pen to mark taking a treatment with a cross.\nART medication was started thereafter and the adherence to the pill intake schedule was monitored by means of using electronic monitors and self-recording for a period of 6\u00a0weeks. At the start of the treatment, electronic monitors containing medication for a period of 2\u00a0weeks were provided. After an evaluation by the doctor at day 14 of treatment the electronic monitors and self-reporting form were collected and the data in the microprocessor were entered in the database. The electronic monitors were subsequently refilled with medication for the next period of 1 month. A new self-reporting form was also given.\nPatients were given 2 (in case of Lamivudine\/Zidovudine + NVP or EVF) or 3 electronic monitors (in case D4T, 3TC, and NVP or EFV).\nPatients recruited in the study were asked to return the electronic caps and the self-reporting form on each occasion of a visit to the IDCC. A self-reporting form was issued at the start of the study. This form contains rows where the patient had to mark with x each time they took the pill at the correct time. As some people in Botswana could not read or write, this form was kept very basic. A pencil was given to every patient who participated in the study.\nResults\nA total of 30\u00a0HIV infected adults were enrolled in the study. In five patients full data could not be obtained because of various reasons. This leaves an evaluable group of 25 (9\u00a0male, 16\u00a0female; average age 35.6 years, range 22\u201355 years). Twenty patients completed the 6-week monitoring period and the mean follow-up period was 49\u00a0days (range 42\u201372 days).\nThe reasons for lack of follow-up in the five patients varied, but in three patients it was due to mortality. In these three cases, as the relatives or nursing staff did not know about the value of the medication bottle (due to the blinding of the patients), the bottles were not returned. One patient was admitted to hospital where the nursing staff discarded the pill bottles. One patient failed to return for follow-up. Full follow-up was not obtained in another five patients for various reasons. This included technical failure of the compliance monitor in two cases and a change in the return date of the subjects who then received a refill in a normal container. Adherence assessed from the dosing histories compiled by electronic monitoring are shown in Table\u00a01.\nTable\u00a01Adherence (%) of the patients (n\u2009=\u200925) by MEMS caps and self-reportingPatient numberTCFollow-up (days)MEMSSelf reportLamivudine \/zidovudine (bd)EFV (od)Lamivudine (bd)NVP (od\/bd)d4T (bd)Drop out\/non-retrievel11111001001001003227095100911003214100No data10010034151981009810054\u2212\u2212\u2212\u2212\u2212\u2212\u2212\u22121614471100719573457370928196814410010010010092449810010098102141001001001003111448810088981224393100939313144988998100141\u2212\u2212\u2212\u2212\u2212\u2212\u2212\u22122152449310098931621562100100623172\u2212\u2212\u2212\u2212\u2212\u2212\u2212\u22122\u20131182448410084861924598100981002019100No data1001003211\u2212\u2212\u2212\u2212\u2212\u2212\u2212\u2212122172751007594232442110021292424293100939525266971001009726143100100100100271\u2212\u2212\u2212\u2212\u2212\u2212\u2212\u221242814994No data969629156100100301472010020100avg4285988998928796Treatment codes (TC) are: 1 Lamivudine\/zidovudine, Efavirenz (EFV); 2 Lamivudine\/Zidovudine, Nevirapine (NVP); 3 Lamivudine, Nevirapine and stavudine (d4T); and 4 Lamivudine and Efavirenz. MEMS indicates the data combined for all different treatments. Patient numbers are not consecutive because patients who died have been omitted. Reasons for drop out and nonretrieval: 1 death; 2 MEMS thrown away; 3 missed by investigator (patients showed up on another date than the investigator expected); and 4 lost to follow-up\nAssuming that surplus opening of the bottles was associated with correct medication intake, the mean adherence level was 85% (SD\u2009=\u200923%, range 20\u2013100%). When surplus openings were calculated as incorrect, the adherence decreased to 70% (SD=23%, range 14\u2013100%) (data not shown). Seven patients (23%) had an adherence under 90%, a level at which virological failure increases substantially [10]. Examples of the medication records obtained from the electronic monitoring device are shown in Fig.\u00a01 for a patient with good adherence and a patient with low adherence to the regimen.\nFig.\u00a01Examples of data of from patients with good and respectively poor adherence\nAdherence assessed by means of self-reporting\nThe mean adherence assessed by means of self-reporting of medication intake was 98% with two (6%) patients recording adherence level lower than 90%. Adherence by self-reporting differed significantly from the adherence measured by the MEMS monitors method (p\u2009<\u20090.05, paired t-test). Three patients did not hand in their diary.\nDiscussion\nIn this pilot study we assessed the use of MEMS monitors to study the adherence to antiretroviral medication prescribed for HIV patients living in a low resource health care system. We demonstrated that assessment of adherence with this technique is feasible and may provide useful results. There was an approximately 30% drop-out rate of the recruited patients due to inability to recover data or early mortality. This may seem unacceptable in a well-resourced health care system. It is a reality in many countries where patients present with much more advanced disease, when there are sometimes great difficulties coming to the hospital, and patients can often not be reached by telephone or mail as they do not regularly have a postal address. It is likely that some association exists between failure to return to the hospital and adherence to the drug regimen and the current patient set therefore may reflect an overestimation of adherence.\nSelf-reporting of medication intake has been shown to be less reliable than the MEMS monitors [22, 23]. Therefore, it is not surprising that patients recorded adherence levels which were considerably higher compared with those assessed by the presumably more objective electronic monitors. Clearly, none of the methods used to measure adherence record actual intake of medication, and there are even indications that at least in some instances self-reporting is a more accurate record of adherence [24]. Despite these findings we consider the MEMS monitors more appropriate for a developing country with a larger potential for illiteracy. Patients knowledge about the monitoring of compliance will likely affect the absolute level of compliance [25], but there are no reasons why the relative ranking of compliance amongst patient groups is affected, and the device can still be used to improve adherence. In this study we chose not to inform the patients about the use of the monitoring device.\nOne of the critics often cited about the electronic monitor is that the opening of the bottle does not prove ingestion. However, it has been shown recently that projected plasma concentrations based on electronically compiled dosing histories correlates very well with directly measured concentrations [26]. It has also been shown that t in HIV patients electronically compiled dosing histories strongly correlates with viral suppression or the occurrence of virological failure [27]. It is therefore assumed that this method is a fair reflection of medication intake [28]. In this small study performed in HIV patients, some subjects opened their bottles more often than needed (surplus opening). Such surplus opening was not considered as nonadherence (in that case our reported adherence level would be lower). If surplus opening had led to extra intake of medication, it would not have contributed the endpoints of virological failure or the induction of resistance. We assume, therefore, that such surplus opening of the pill bottles had occurred for other reasons. Surplus openings occurred especially in the group of patients that had to take a once daily regimen together with twice daily regimens. These extra openings occurred often at the same time as when the prescribed twice-daily regimen was taken. This may have occurred because the patients were unsure about which bottle belonged to which medication. This may indicate that clearer labelling is essential, especially in an illiterate society. Under-opening most likely reflects nonadherence, although it cannot be excluded that in some cases patients removed several tablets at the same time. However, such behaviour is highly likely to increase the chance of erroneous medication intake and was for this reason registered as nonadherence.\nSeveral studies have indicated that medication adherence lower than approximately 90% increases the chance of virological failure and the development of resistant virus strains [12, 28, 29]. The result of this pilot study indicates that even after careful counseling and guidance, a significant number of patients did not manage to adhere sufficiently to the twice-daily pill intake regimen. This observation study was too short to allow an analysis of the clinical impact of the observed low adherence in such patients on the development of virological failure. Additionally, reliable measurement of viral load was impossible in the hospital. However, our data did help to identify some patients with low adherence at the start of treatment and allowed a diversion of scarce resources for extra counseling for such patients.\nThe outcome of monitoring pill intake by the electronic monitors may therefore assist in timely counseling of patients with regard to their medication intake and persistence with the prescribed regimen.\nAdherence measurements by means of using electronic monitors together with self and adherence partner recording of pill intake are likely to be useful in a much needed larger, long-term and more comprehensive adherence study in a low-resource health-care-system setting. Such a study can contribute to directed efforts to optimise the treatment of HIV-infected patients worldwide.","keyphrases":["adherence","haart","mems caps"],"prmu":["P","P","P"]}
{"id":"FEBS_Lett-1-5-1964784","title":"The RhoA transcriptional program in pre-T cells\n","text":"The GTPase RhoA is essential for the development of pre-T cells in the thymus. To investigate the mechanisms used by RhoA to control thymocyte development we have used Affymetrix gene profiling to identify RhoA regulated genes in T cell progenitors. The data show that RhoA plays a specific and essential role in pre-T cells because it is required for the expression of transcription factors of the Egr-1 and AP-1 families that have critical functions in thymocyte development. Loss of RhoA function in T cell progenitors causes a developmental block that pheno-copies the consequence of losing pre-TCR expression in Recombinase gene 2 (Rag2) null mice. Transcriptional profiling reveals both common and unique gene targets for RhoA and the pre-TCR indicating that RhoA participates in the pre-TCR induced transcriptional program but also mediates pre-TCR independent gene transcription.\n1\nIntroduction\nThe T cell antigen receptor (TCR) complex comprises variable \u03b1\/\u03b2 subunits that recognise peptide\/major histocompatibility (MHC) complexes and invariant signal transduction subunits of the CD3 antigen. A key stage in T cell development in the thymus is the selection of cells that have successfully rearranged their TCR-\u03b2 locus. This occurs in T cell precursors, which do not express of the MHC receptors, CD4 and CD8 (double negative (DN) thymocytes) [1]. The DN stages of intrathymic differentiation can be followed by the sequential pattern of expression of CD44 and CD25. T cell progenitors enter the thymus as CD44+CD25\u2212 cells (termed DN1); these then express CD25 (DN2) and begin to rearrange T-cell receptor \u03b2 loci. Cells then lose CD44 expression and continue \u03b2 chain rearrangements to completion (DN3). Cells that successfully rearrange their TCR-\u03b2 locus will express a functional receptor complex known as the pre-TCR comprising a TCR-\u03b2 chain, the p-T\u03b1 subunit and the signalling subunits of the CD3 antigen [2\u20134]. When the pre-TCR is expressed at the cell membrane it promotes cell survival and entry into the cell cycle [5]; cells downregulate CD25 and transit to the DN4 pre-T cell subset. DN4 T cells undergo proliferative expansion and differentiate to CD4+CD8+ double positive (DP) cells. These cells then undergo TCR \u03b1-chain gene rearrangements and upon expression of a functional \u03b1\/\u03b2 TCR complex are subjected to positive and negative selection to generate CD4+ or CD8+ single positive (SP) thymocytes [6\u20138].\nNormal pre-T cell development requires the coordination of a complex program of gene transcription by signal transduction networks mediated by the pre-TCR complex and cytokine and stromal signals [9,5]. Mice deficient for the recombinase activating (RAG) genes which are unable to rearrange TCR-\u03b2 subunits and express a pre-TCR are unable to progress beyond the DN3 stage of thymocyte development [10]. Similarly, the absence of pre-T\u03b1 or CD3 subunits blocks thymocyte development at the DN3 stage [11,3]. The transition of thymocytes beyond the pre-T cell stage of thymocyte differentiation is also dependent on signal transduction networks mediated by tyrosine and serine kinases [3,12,13]. In this context, crucial responses are mediated by Rho family guanine nucleotide binding proteins such as RhoA, Rac-1 and CDC42 [14\u201317].\nThe importance of RhoA for thymocyte development has been demonstrated by studies of transgenic mice that express Clostridium botulinum C3-transferase under the control of T cell specific promoters such as the p56lck and CD2 promoters [17,15,18]. This toxin selectively ADP-ribosylates RhoA within its effector-binding domain and abolishes its biological function. Transgenic mice that express C3-transferase under the control of the p56Lck promoter have a small thymus and severely reduced numbers of peripheral T cells [15,17]. This phenotype is caused by survival defects in DN2 and DN3 thymocytes that lack Rho function [15]. During embryogenesis no cells progress beyond the DN1 stage but in adult mice a few T cell progenitors survive and develop to DN4s and beyond [15]. This \u2018leakiness\u2019 either reflects selection of cells that compensate for loss of Rho function in DN2\/3 thymocytes or reflects heterogeneous and asynchronous expression of the lck promoter in adult DN thymocytes. The few DP thymocytes and mature T cells found in adult Lck-C3 transgenic mice have numerous defects including reduced survival, proliferation, integrin mediated cell adhesion and defective cell motility [15,19]. A complementary strategy to probe Rho function in the thymus used the CD2 locus control region (LCR) to target C3 transferase to T cell progenitors (CD2\u2013C3 mice) [18]. The inhibition of Rho function at the DN2\/3 stage in CD2\u2013C3 mice is not leaky and causes T cells to become blocked in differentiation at the DN3 stage of thymus development. CD2\u2013C3 mice thus have a thymic phenotype indistinguishable from the phenotype of Recombinase gene null mice or mice lacking key structural or signaling components of preTCR complex [10,18,20,21]: thymocyte development is blocked at the pre-T cell\/DN3 stage [18].\nThe basis for the failure of pre-T cell differentiation in CD2\u2013C3 transferase mice is not known. One way to address this issue is to use microarray gene expression profiling to determine the impact of losing Rho function on transcriptional responses in pre-T cells. Previous studies in transformed cell lines have identified a role for RhoA in regulating activating protein-1 (AP1) family of transcription factors [22\u201324] but transcriptional targets for RhoA signal transduction in primary non-transformed cells have not been explored. The present data show that RhoA regulates expression of genes encoding members of the Fos\/Jun and early growth response (Egr) family of transcription factors but also has an impact on expression of genes regulating diverse biological functions including serine\/threonine kinases, protein phosphatases, enzymes that regulate protein biosynthesis and proteins that regulate nuclear structure and function.\n2\nMaterials and methods\n2.1\nMice\nMice were bred and maintained under specific pathogen-free conditions in the transgenic animal unit. RAG2\u2212\/\u2212 and C3 transgenic mice, which selectively express the bacterial toxin C3-transferase under the control of CD2 promoter and locus control region in the thymus, have been described in detail elsewhere [10,18].\n2.2\nFlow cytometric analysis\nFluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), and biotin-conjugated antibodies were obtained from Pharmingen (San Diego, CA). Tricolour (PE\/Cy5 and APC\/Alexa750) conjugated antibodies and fluorophore-streptavidin conjugates were from Caltag (Burlingame, CA). Thymocytes were stained for cell surface markers and analysed on a FACS Calibur (Becton Dickinson Immunocytometry Systems Franklin Lakes CA). Data were analysed using CellQuest BDIS or FlowJo (Treestar Inc, Ashland OR) software. CD4\u2212CD8\u2212 double negative thymocyte subsets were analysed for CD44 and CD25 expression following lineage exclusion of mature DP and SP cells as well as non-T cell lineage cells using a cocktail of biotinylated antibodies (CD4, CD8, CD3 B220, Mac-1, NK, Gr-1, and \u03b3\u03b4) revealed with streptavidin-tricolour and costained with CD25-FITC, CD44-PE and Thy1.2-APC. Intracellular phospho-S6 and TCR-\u03b2 intracellular staining was carried out on thymocytes pre stained to identify DN3 and DN4 subpopulations. Cells were subsequently fixed in 1% paraformaldehyde for 10\u00a0min at room temperature, washed in PBS and permeabilized with saponin buffer (0.5% saponin, 5% FBS, 10\u00a0mM HEPES [pH 7.4] in PBS) for 10\u00a0min at room temperature. Permeabilized cells were incubated with PE-conjugated TCR\u03b2 antibody and phospho S6 antibody for 45\u00a0min at room temperature, washed in saponin buffer and subsequently stained with FITC conjugated donkey anti rabbit IgG (Jackson Immunoresearch, West Grove PA). Cells were analysed on an LSR Flow Cytometer (BDIS).\n2.3\nRNA extraction\nThymocytes were isolated from 4- to 6-week-old RAG2\u2212\/\u2212, CD2\u2013C3 and C57BL\/6 control mice. DN3 pre-T cells were purified firstly by removing CD4, CD8 DP cells using MACS CD4+ and -MACS CD8+ T cell isolation kits (Miltenyi Biotec, Ltd.) followed by magnetic autoMACS separation. CD4\/CD8 depleted thymocytes were then labelled with Thy1.2-APC, PE conjugated CD4, CD8, CD44 and TCR\u03b3\u03b4 antibodies, FITC conjugated CD25 antibodies and the DN3 subpopulation (Thy1+CD4\u2212\/CD8\u2212\/CD44\u2212\/CD25+) was purified by cell sorting on a FACS Vantage (BDIS) using Cell Quest software (BDIS). All sorted cells were analysed by flow cytometry and only used for experiments if the purity was 95\u201398%. Total RNA was extracted from sorted cells using the Qiagen RNeasy kit according to manufacturers instructions. Total RNA was quantified using the RNA 6000 Nano LabChip\u00ae kit and analyzed on an Agilent 2100 Bioanalyzer (Agilent Technologies). Total RNA from up to 10 thymi were pooled for microarray analysis and quantitative PCR.\n2.4\nMicroarray\nMicroarray analysis was carried out by the Finnish DNA Microarray Centre at the Centre for Biotechnology, Turku, Finland. For Affymetrix sample preparation, 100\u20131000\u00a0ng of total RNA was used as the starting material to synthesise target cRNA using the GeneChip\u00ae Eukaryotic Small Sample Target Labeling Assay Version II according to manufacturers\u2019 instructions (Affymetrix, Santa Clara, CA). The cRNA target sample was hybridised to the Affymetrix Mouse Genome 430 2.0 Array (45,101 probes sets and expressed sequence tag (EST) clusters). Data was analysed with Affymetrix Gene Chip Operating Software (GCOS) version 1.1 and filtered according to recommendations of the manufacturer. Comparison analysis was used to compare the expression profiles from two arrays \u2013 results were obtained by analyzing the experimental array in comparison to the baseline array. The global method of scaling was used for normalizing hybridization intensity between arrays. The statistical algorithm used by GCOS defined the expression status of each gene-specific probe set as not changed, increased, marginally increased, decreased or marginally decreased. Genes that were absent or unchanged between comparisons were excluded from the results. Comparison analysis defines a gene as up-regulated if the signal log ratio between the baseline sample and the experimental sample is larger than 1 (2-fold) and the experimental sample is present. Similarly changed genes were further analyzed\/classified using the gene ontology tool (www.Affymetrix.com) and represented by biological function. Gene lists were complied and sorted by genes that were either commonly or uniquely regulated in CD2-C3 and RAG2\u2212\/\u2212 thymocytes.\n2.5\nReal-time RT-PCR analysis\nPurified total RNA (200\u00a0ng) was reverse transcribed using the iScript\u2122 cDNA synthesis kit (BioRad). Real time RT-PCR was performed in a 96-well plate using iQ\u2122 SYBR Green based detection (BioRad) on a BioRad iCycler in 20\u00a0\u03bcl reaction volume containing 1\u00a0\u03bcl cDNA (20\u00a0ng), 0.8\u00a0\u03bcl 10\u03bcM sense and antisense primers, 10\u00a0\u03bcl iQ\u2122 SYBR Green supermix, and 4\u00a0\u03bcl nuclease free water. Each reaction was performed in duplicate and each experiment repeated in triplicate. 18S rRNA levels were used to normalise RNA concentrations between samples and the relative mRNA levels were calculated using the equation:where E is the efficiency of PCR, ct is the threshold cycle, u is the mRNA of interest, r is the reference gene (18S rRNA), s is the sample and c is the control sample. Primers used for RT-PCR were designed using Beacon Designer 2 software. Primer sequences are: Egr1 forward 5\u2032-ACAGAAGGACAAGAAAGCAGAC-3, reverse 5-CCAGGAGAGGAGTAGGAAGTG-3\u2032, Fos forward 5\u2032-CTACTGTGTTCCTGGCAATAGC-3\u2032, reverse 5\u2032-AACATTGACGCTGAAGGACTAC-3\u2032, Egr3 forward 5\u2032-TGACCAACGAGAAGCCCAATC-3\u2032, reverse 5\u2032-GCTAATGATGTTGTCCTGGCAC-3\u2032, Jun forward 5\u2032-CGCCTCGTTCCTCCAGTC-3\u2032 reverse 5\u2032-ACGTGAGAAGGTCCGAGTTC-3\u2032, JunB forward 5\u2032-CTTCTACGACGATGCCCTCAAC-3\u2032, reverse 5\u2032-GTTCAAGGTCATGCTCTGTTTTAGG-3\u2032, Nur77 forward 5\u2032-CCTGTTGCTAGAGTCTGCCTTC-3\u2032, reverse 5\u2032-CAATCCAACACCAAAGCCACG-3\u2032, 18S RNA forward 5\u2032-GTAACCCGTTGAACCCCATT-3\u2032, reverse 5\u2032-CCATCCAATCGTAGTA-3\u2032.\n3\nResults and discussion\nMice expressing the RhoA inhibitor Clostridium Botulinum C3 transferase under the control of the CD2 locus control region (LCR) have been described previously [18]. Loss of RhoA function in CD2-C3 mice results in loss of CD4\/CD8 double positive thymocytes (Fig. 1A) with a block in thymocyte development at the DN3 stage (Fig. 1B). Rag2\u2212\/\u2212 mice also block thymus development at the DN3 stage (Figs. 1A and B) due to failed expression of the pre-TCR complex. Despite this superficial similarity, loss of Rho function does not prevent TCR beta locus rearrangements although there is a reduced frequency of DN3 cells expressing intracellular TCR-\u03b2 subunits (Fig. 1C). Ectopic expression of transgenic TCR complexes cannot reverse the developmental block in CD2\u2013C3 thymocytes demonstrating that failed expression of TCR complexes does not explain the developmental defects caused by loss of Rho function [18]. Rho thus appears necessary for pre-TCR function although these results do not discriminate between a direct role for RhoA in pre-TCR signalling or a role for this GTPase in the cytokine\/stromal signalling pathways that synergise with pre-TCR signals to control pre-T cell differentiation [25\u201327].\nCD2\u2013C3 transferase mice provide a good model system to probe the immediate transcriptional consequences of losing RhoA function in pre-T cells since the CD2-LCR initiates expression of transgenes in T cell progenitors as they transit from the DN2 to DN3 stage of thymocyte development [18]. Accordingly, it is possible to isolate DN3 thymocytes ex vivo that have only just switched on expression of C3 transferase and prepare RNA for DNA microarray analysis. The Affymetrix Mouse Genome 430 2.0 array, representing 39\u00a0000 murine gene transcripts, was used to transcriptionally profile DN3 pre-T cells purified from the thymi of CD3\u2013C3 transgenic mice. In these experiments DN3 thymocytes from wild type control mice were transcriptionally profiled and used for comparisons.\nThe microarray data were analyzed with the Affymetrix Gene Chip Operating Software (GCOS, version 1.1) by comparing the gene expression profiles of CD2\u2013C3 pre-T cells to the profile of normal cells to reveal changes above or below wild type levels. DN3 pre-T cells expressed approximately 18\u00a0000 genes and there were only small changes in the transcriptional profile of CD2\u2013C3 DN3 thymocytes compared to control wild type DN3s. We focused our analysis on \u2a7e2-fold changes, which were further analyzed using the Affymetrix gene ontology tool. A full list of the genes regulated 2-fold or greater by loss of RhoA function in DN3 thymocytes is shown in Supplementary Fig. 1. There were approximately 18\u00a0000 genes expressed in wild type DN3 thymocytes: loss of RhoA function caused a \u2a7e2-fold decrease in expression of 383 genes and a \u2a7e2-fold increase in expression of 190 genes. RhoA regulated genes in DN3 thymocytes encode proteins with diverse biological functions including serine\/threonine kinases, protein phosphatases, enzymes that regulate protein biosynthesis.\nThis point is illustrated in Fig. 2A, which shows the genes whose expression is regulated more than 10-fold. The largest decrease was in expression of the gene encoding FosL2, a Fos family transcription factor (630-fold decrease) but other large changes included a subunit of eukaryotic translation initiation factor 2, Protein Kinase C beta and the membrane receptor CD69. There was also a loss of expression of genes encoding proteins that regulate nuclear structure and function such as genes encoding histones that are involved in nucleosome assembly. In a similar category there was reduced expression of mRNA encoding special AT-rich sequence binding protein 1 (SATB1) in CD2-C3 DN3s (Supplementary Fig. 1). SATB1 acts as a scaffold for chromatin modifiers and is known to regulate higher order chromatin structure and to regulate gene expression by acting as a \u201cdocking site\u201d for several chromatin remodeling enzymes [28\u201330].\nOf 55 genes whose expression was decreased \u2a7e5-fold, 23 encoded transcription factors; of 384 genes decreased \u2a7e2-fold, 63 were transcription factors (Fig. 2B). Transcription factors downregulated in CD2-C3 DN3 thymocytes included Kruppel family transcription factors KLF4, KLF6 and KLF9. There was also loss of expression of members of the immediate early gene family Egr1 and Egr3, AP-1 family members Fos, FosL2, FosB, Jun, JunB and downregulated expression of genes encoding Ets1 and Nurr77. To validate the microarray analysis, quantitative real-time PCR was used to compare expression of a selection of these different transcription factors in wild type versus CD2-C3 thymocytes (Fig. 2C). The experiments confirmed the reduction in Fos, Jun, JunB, Egr1 and Egr3 and NURR77 in CD2\u2013C3 DN3s compared to wild type controls.\nA number of gene targets for Egr transcription factors have been described [31] and in this context, RhoA inactivation caused loss of expression of multiple members of the MAP kinase phosphatase family DUSP 1, 2, 4, 6, 16. The latter are induced in negative feedback mechanisms that modulate MAP kinase activity and have been described as targets for Egr family transcription factors [31\u201333]. The loss of DUSP expression is thus almost certainly a secondary consequence of failed expression of Egr molecules. Egr1 and Egr3 are well characterised pre-TCR induced genes [34\u201338]. To verify this we compared the transcriptional profile of CD2-C3 DN3s with Recombinase gene 2\u2212\/\u2212 (RAG2\u2212\/\u2212) DN3s which lack expression of the pre-TCR complex because they fail to rearrange their TCR beta locus. Of the 23 transcription factors downregulated \u2a7e5-fold in CD2\u2013C3 thymocytes 18 were also downregulated in Rag2\u2212\/\u2212 DN3s (Fig. 2D) and these included Egr1, Egr3, Fos, Fosl2, FosB, Ets1, Nurr77. These microarray analyses were confirmed by quantitative real-time PCR analysis of wild type versus Rag2\u2212\/\u2212 thymocytes (data not shown). The common loss of expression of members of the Egr family in CD2\u2013C3 and Rag2\u2212\/\u2212 pre-T cells is thus consistent with a RhoA requirement for the pre-TCR induced gene transcription. However, comparisons of the total number of genes regulated uniquely in CD2\u2013C3 and Rag2\u2212\/\u2212 DN3s (Fig. 3) (VENN diagrams) indicate that there are a number of gene changes in CD2\u2013C3 cells that are not seen in Rag2\u2212\/\u2212 DN3s. The full list of genes that were commonly and uniquely regulated 2-fold and greater in CD2 C3 versus Rag2\u2212\/\u2212 pre-T cells is shown in Supplementary Fig. 2. In terms of \u2a7e 2-fold, gene repression, CD2-C3 and Rag2\u2212\/\u2212 DN3s had 180 gene targets in common with 202 genes being downregulated uniquely in CD2\u2013C3 pre-T cells and 120 genes downregulated uniquely in Rag2\u2212\/\u2212 cells. In terms of \u2a7e2-fold gene increases, CD2\u2013C3 and Rag2\u2212\/\u2212 DN3s had 65 gene targets in common, 129 genes that were increased uniquely in CD2-C3 DN3s and 182 increased uniquely in Rag2\u2212\/\u2212 cells (Fig. 3).\nThe common transcriptional changes caused by loss of the pre-TCR in Rag2\u2212\/\u2212 DN3s or loss of Rho function in CD2\u2013C3 mice indicates that Rho function is necessary for some pre-TCR induced transcriptional responses. However, there are genes downregulated in DN3s lacking RhoA function that are unchanged in Rag2\u2212\/\u2212 DN3s (Supplementary Fig. 3) indicating that RhoA is not just required to mediate the preTCR induced gene program but may also participate in cytokine\/stromal cell-initiated signalling pathways that control pre-T cell differentiation. The genes uniquely lost in CD3\u2013C3 DN3 thymocytes include serine kinases (Protein Kinase C beta and Map3k14); transcription factors (SpiB and Dlx1) and regulators of protein biosynthesis (Eif2s3y) (Supplementary Fig. 3). One gene of interest lost in CD2\u2013C3 DN3s but not Rag2 null DN3s was Hes-1 which is induced by Notch receptor-ligand interactions. Notch signalling is required throughout DN to DP stage of thymocyte development to support T cell metabolism and survival [39\u201342]. A RhoA requirement for Notch signalling has not been described but it is known that RhoA is necessary for integrin mediated cell adhesion and thymocyte cell migration [19]. Accordingly, decreased Hes-1 expression could reflect that pre-T cells lacking Rho function cannot make normal contacts with stromal cells that express the Notch ligands [43,42,44,45].\nThere were also unique transcriptional changes seen in Rag2\u2212\/\u2212 cells that were not found in the CD2\u2013C3 DN3s (Supplementary Fig. 4) which indicates that the pre-TCR can regulate a program of gene transcription via RhoA independent pathways The unique changes in Rag2\u2212\/\u2212 cells not surprisingly included loss of expression of rearranged TCR-\u03b2 subunits. One other gene downregulated in Rag2\u2212\/\u2212 DN3s but not CD2\u2013C3 DN3s was RPS6, which encodes the ribosomal S6 subunit. In this respect, we recently described high basal levels of S6 phosphorylation in ex vivo \u03b2 selected DN3s and no detectable S6 phosphorylation in ex vivo Rag2\u2212\/\u2212 DN3s [13]. The data in Fig. 4 quantify S6 phosphorylation in CD2\u2013C3 DN3s compared to wild type cells. Normal DN3s are heterogeneous for phosphoS6 with the majority of cells being phosphoS6low but a significant percentage of cells are phosphoS6high. The DN3 thymocyte subpopulation can be subdivided into cells that have not yet completed TCR-\u03b2 locus rearrangements and those that express a functional TCR-\u03b2 subunit that allows surface expression and signalling of the pre-TCR complex. Analysis of TCR\u03b2 expression by intracellular staining revealed that DN3s that express icTCR-\u03b2 chains are generally phosphoS6high whereas DN3s that are icTCR-\u03b2 null are uniformly phosphoS6low. In CD2\u2013C3 DN3s there were also high levels of phosphoS6 in TCR-\u03b2 high cells. The presence of normal S6 phosphorylation in \u03b2 selected DN3s lacking Rho function is a further indication that Rho is necessary for a subset of responses in DN3 thymocytes but does not globally block signalling.\nOne important question is whether the transcriptional changes in CD2\u2013C3 DN3s explain why loss of RhoA function results in failed thymocyte differentiation? In this respect, microarray analysis of Rag2\u2212\/\u2212 pre-T cells revealed decreased expression of TCR-\u03b2 subunits as a major defect (Supplementary Fig. 4) and failed expression of the TCR-\u03b2 subunit is indeed responsible for the DN3 developmental block in Rag2\u2212\/\u2212 mice. In CD2\u2013C3 DN3s there were several gene defects that would explain why RhoA function is essential for pre-T cell development. For example, RhoA is required for expression of Egr3 and Ets1 and loss of either of these transcription factors inhibits pre-T cell proliferation and is suboptimal for transition through early pre-TCR-dependent stages of thymocyte development [46,36]. Loss of individual Egr family or AP-1 transcription factors does not completely abrogate thymocyte differentiation but this reflects that there is considerable redundancy in AP-1 complexes due to the ability of different family members to pair as dimers [47\u201349]. The simultaneous elimination of Egr1, Egr3, Ets1 and multiple Fos\/Jun family members in CD2\u2013C3 DN3s would circumvent the possibility of redundancy [50,51]. This comprehensive loss of AP-1 activity in pre-T cell lacking RhoA function plus the decreased expression of other transcription factors would rapidly cause global changes in the transcriptional program of a cell. Moreover, the transcription factor defects would be exacerbated by the decreased expression of chromatin modifying enzymes such as SATB1.\nIn summary, loss of Rho function or failed expression of the pre-TCR complex in Rag2\u2212\/\u2212 null mice block T cell development at a common stage. The present data show that the genetic consequences of loss of Rho function versus loss of pre-TCR expression for T cell progenitors are not identical. The present results also show that loss of Rho function in pre-T cells results in downregulation of genes encoding members of the Fos\/Jun and Early growth response (Egr) family of transcription factors. The collective loss of these transcription factors and the resultant secondary genetic changes explains why loss of RhoA function prevents pre-T cell development.","keyphrases":["rhoa","thymus","egr","ap-1","pre-tcr"],"prmu":["P","P","P","P","P"]}
{"id":"Behav_Brain_Res-1-5-2148464","title":"Dorsal hippocampal N-methyl-d-aspartate receptors underlie spatial working memory performance during non-matching to place testing on the T-maze\n","text":"Previous lesion studies have suggested a functional dissociation along the septotemporal axis of the hippocampus. Whereas the dorsal hippocampus has been implicated in spatial memory processes, the ventral hippocampus may play a role in anxiety. However, these lesion studies are potentially confounded by demyelination of fibres passing through the lesion site, and the possibility of secondary, downstream changes in associated brain structures as a consequence of their chronic denervation following the lesion. In the present study, we have used the microinfusion of muscimol to temporarily inactivate either the dorsal or ventral hippocampus in order to re-examine the contribution of the hippocampal sub-regions to spatial memory. Microinfusion studies spare fibres of passage and offer fewer opportunities for compensatory changes because the effects are transient and short-lasting. Rats were infused prior to spatial working memory testing on a non-matching to place T-maze alternation task. Spatial working memory was impaired by dorsal but not ventral hippocampal inactivation. In a second experiment, infusion of the NMDAR antagonist, D-AP5, into dorsal hippocampus also impaired spatial working memory performance, suggesting that NMDAR function within the dorsal hippocampus makes an essential contribution to this aspect of hippocampal information processing.\n1\nIntroduction\nA role for the hippocampus in certain kinds of memory is well established. In rodents, the hippocampus has been particularly associated with spatial learning [1]. Complete hippocampal lesions have repeatedly been shown to produce robust and lasting impairments on spatial memory tasks [2,3]. More recently, however, these spatial memory functions have been ascribed specifically to the dorsal (or septal) portion of the rat hippocampus [4,5]. For example, selective cytotoxic lesions restricted to dorsal hippocampus produce spatial learning impairments on tasks like the Morris watermaze [6, 7, see also 8]. In contrast, lesions of the ventral hippocampus are without effect on spatial memory tasks, although conversely, they, but not dorsal hippocampal lesions, are associated with reduced anxiety [9\u201311]. This double dissociation suggests that different hippocampal sub-regions may mediate different aspects of hippocampal function [5].\nThus far, the conclusion that spatial learning is primarily a function of dorsal hippocampus has largely been based on the results of such cytotoxic lesion studies. However, this approach is potentially confounded in that any behavioural sequelae may be the result of indirect effects of the lesion procedure on other brain areas, rather than as a direct consequence of cell loss in the target region. For example, although the cytotoxic lesion approach is a clear improvement over more traditional techniques (e.g. aspiration, electrolytic or radiofrequency-generated lesions), in terms of sparing fibres of passage [12], there nevertheless remains some evidence of demyelination of nerve fibres passing through the lesion site even with this approach [13,14]. Furthermore, it is also possible that any behavioural effects of the lesion could reflect a secondary, downstream change in an associated brain structure as a consequence of the chronic denervation of that structure following the lesion [15]. It is therefore important to re-examine the dorsal\/ventral dissociation using alternative experimental approaches. For example, it is possible to produce a temporary, reversible inactivation of a specific brain region by microinfusing the GABAA agonist muscimol [16], which avoids many of the potential confounds associated with the lesion approach. Microinfusion studies offer fewer opportunities than lesion studies for compensatory changes because the effects of the manipulation are transient and short-lasting. They also permit within-subjects comparisons. Somewhat surprisingly, the effects of dorsal and ventral hippocampal inactivation on spatial working memory performance have never been fully assessed.\nIn the present study, we therefore compared the effects of dorsal and ventral hippocampal infusions of muscimol on spatial learning using a spatial working memory, non-matching to position (discrete trial, rewarded alternation) paradigm on the elevated T-maze [17]. This task is especially sensitive to hippocampal dysfunction (see effect sizes in [7], p. 1184), and is dramatically impaired by dorsal but not ventral hippocampal lesions [7,9,18]. We therefore compared dose-response functions for muscimol infusions into both hippocampal sub-regions (Experiment 1).\nIn a second experiment, the importance of N-methyl-d-aspartate receptor (NMDAR) function, specifically in the dorsal hippocampal subregion, for spatial working memory performance was assessed. The role of NMDARs in spatial working memory is well established [19,20]. Intracerebroventricular (i.c.v.) infusion of the NMDAR antagonist, d(\u2212)-2-amino-5-phosphonopentanoic acid (AP5) resulted in a delay-related impairment in choice accuracy on the same spatial, non-matching to place T-maze task [21]. However, the i.c.v. route of drug delivery results in NMDAR blockade in numerous brain regions, and not just the hippocampus [22]. In view of the proposal that it is the dorsal subregion of the hippocampus that is crucial for spatial working memory performance, we therefore also examined the effects on spatial non-matching to position of direct infusions of AP5 specifically into dorsal hippocampus (Experiment 2).\n2\nMethods\n2.1\nSubjects\nThirty male Lister-hooded rats (Harlan Olac, Bicester, UK; 250\u2013363\u00a0g at the time of surgery) took part in Experiment 1. Nine male Lister-hooded rats (270\u2013370\u00a0g at the time of surgery) took part in Experiment 2. All rats were experimentally na\u00efve and maintained on a 12\u00a0h light\/dark cycle (lights on at 7:00\u00a0a.m., with testing during the light phase). During the experiment, the animals were maintained at \u223c85% of their free-feeding weight but had access to water ad libitum. Rats were housed in groups of 2\u20133 prior to surgery but were singly housed after cannulae implantation. The experiments were conducted in accordance with the United Kingdom Animals Scientific Procedures Act (1986); under project license number PPL 30\/1505.\n2.2\nApparatus\nIn Experiment 1, rewarded alternation took place on an elevated (\u223c1\u00a0m above the floor), low-walled wooden T-maze consisting of a start arm (80\u00a0cm long; 10\u00a0cm wide) joined to two identical goal arms (60\u00a0cm\u00a0\u00d7\u00a010\u00a0cm), with each arm surrounded by a 1\u00a0cm ridge. In Experiment 2, rewarded alternation took place on an elevated Y-maze (\u223c80\u00a0cm above the floor). Each of the three arms (50\u00a0cm\u00a0\u00d7\u00a09\u00a0cm) was surrounded by a 0.5\u00a0cm ridge and extended from a hexagonal central platform (14\u00a0cm diameter). The T- and Y-mazes contained stainless-steel food wells at the far end of each goal arm and each maze was located in a well-lit testing room containing prominent extra-maze cues such as wall posters.\n2.3\nProcedure\nIn both experiments, rats were trained pre-operatively. Habituation to the maze took place over a five-day period. For the first three days rats were placed on the maze in pairs and left to explore and collect food rewards for 10\u00a0min. During days four and five, the rats were placed individually on the maze and allowed to explore freely for five minutes. By this stage all the rats ate from the food wells at the ends of the arms. Training on the rewarded alternation task then followed.\nEach trial in rewarded alternation had two runs: in the first, one of the goal arms was blocked, allowing the rat to enter only the other goal arm, whereupon it received 1 food pellet (45\u00a0mg Rodent Diet Formula A\/I, Noyes, Lancaster, NH). During the second run, the block was removed and the rat was placed on the maze and given a free choice of either arm. Rats received two pellets for choosing the previously unvisited arm (i.e. for alternating). Choosing the arm previously visited in the sample run yielded no reward. The time between the sample and the choice runs was approximately 10\u00a0s (Experiments 1 and 2a) or 30\u00a0s (Experiment 2b). Left\/right allocations for the sample and choice runs were pseudo-randomised over ten trials per day, with no more than three consecutive sample runs to the same side. The inter-trial interval was \u223c3\u20134\u00a0min. Prior to surgery, each rat was trained on the task until they achieved a criterion of at least 80% correct alternation over two consecutive days.\nIn Experiment 1, allocation to surgery groups (dorsal or ventral cannulation) was based on pre-operative performance. Rats were anaesthetised with Avertin (0.29\u00a0g\/kg; i.p.) and placed in a stereotaxic frame with the head level between bregma and lambda. An incision of the scalp was made along the midline, followed by a craniotomy. Stainless-steel guide cannulae (23-gauge, 12\u00a0mm) were implanted bilaterally into either the dorsal (n\u00a0=\u00a014; AP: \u22123.6 (from bregma); M\u2013L: \u00b12.8 (from bregma); D\u2013V: \u22120.8 (from brain surface)) or ventral HPC (n\u00a0=\u00a016; AP: \u22125.2 (from bregma); M\u2013L: \u00b14.9 (from bregma); D\u2013V: \u22123.6 (from brain surface)). In Experiment 2, cannulae were only implanted into the dorsal HPC (n\u00a0=\u00a09; coordinates as above). Cannulae were secured with three skull screws and acrylic dental cement. Microinjectors (31-gauge, 14\u00a0mm) were 2\u00a0mm longer than the guide cannulae giving a targeted D\u2013V coordinate (from brain surface) of either \u22122.8\u00a0mm (dorsal) or \u22125.6\u00a0mm (ventral). Stylets were inserted into the guide cannulae to prevent infection and blockages.\nRats were allowed a minimum of five days post-surgical recovery after which they received training in the absence of any infusions. Rats failing to score 80% correct alternation on these sessions were given additional training.\n2.4\nDrugs\nIn Experiment 1, muscimol (C4H6N2O2(1\/2 H2O); Tocris, Bristol, UK) was initially dissolved in normal saline (0.9% NaCl) at a concentration of 1\u00a0mg\/ml. This stock was then diluted to create individual aliquots at each of four concentrations (0.6\u00a0mg\/ml, 0.3\u00a0mg\/ml, 0.15\u00a0mg\/ml, and 0.075\u00a0mg\/ml), which were then frozen. Saline served as the vehicle. In Experiment 2, muscimol (0.3\u00a0mg\/ml) and AP5 (d(\u2212)-2-amino-5-phosphonopentanoic acid; 5.9\u00a0mg\/ml) were dissolved in PBS (pH 7.4), which served as vehicle.\nOn infusion days, the aliquots were defrosted and used to back-fill the microinjectors, which were connected via polyethylene tubing to a pair of Hamilton syringes (10\u00a0\u03bcl) driven by a microsyringe pump (SP250i, World Precision Instruments, England). Each rat was restrained firmly in a towel, the stylets were removed, and the microinjectors inserted through the guide cannulae into the hippocampus. Drug or vehicle (0.5\u00a0\u03bcl per side) was infused into the dorsal or ventral hippocampus at a rate of 1\u00a0\u03bcl\/min (i.e. for 30\u00a0s), and the microinjectors were kept in place for an additional 60\u00a0s to allow the drug to diffuse. Infusions into each hemisphere were made simultaneously and were given 15\u00a0min prior to testing. In Experiment 1, rats received five infusions in total (saline and four doses of muscimol: 0.3\u00a0\u03bcg\/side, 0.15\u00a0\u03bcg\/side, 0.075\u00a0\u03bcg\/side, 0.0375\u00a0\u03bcg\/side). In Experiment 2a, rats received three infusions (PBS, AP5: 2.95\u00a0\u03bcg\/side, muscimol: 0.15\u00a0\u03bcg\/side) and in Experiment 2b, the same rats received a further 4 infusions (PBS\u00a0\u00d7\u00a02, AP5: 2.95\u00a0\u03bcg\/side\u00a0\u00d7\u00a02), making seven infusions in total. Infusions were given in a counterbalanced, pseudo-random order with at least 48\u00a0h between each infusion. In addition, performance was assessed again 24\u00a0h after each infusion and rats failing to score 80% or more on these drug-free sessions received extra training before the next infusion was administered.\n2.5\nHistology\nOn completion of the experiment, rats were perfused transcardially and their brains were removed, sectioned, and stained with cresyl violet for verification of the injection sites.\n3\nResults\n3.1\nExperiment 1\nIn Experiment 1, of the 30 rats that began the experiment, 23 rats completed all testing and had accurate bilateral cannulae placements and were included in the final analyses (10 dorsal, 13 ventral; Fig. 1). The intra-hippocampal implantation of guide cannulae did not result in any post-surgical deficits in rewarded alternation in either dorsal (mean score\u00a0=\u00a094%) or ventral (mean score\u00a0=\u00a090%) cannulated rats. Indeed post-operative performance was slightly better than pre-operative performance in both groups. Student's t-tests carried out for each group confirmed that there were no differences between pre-operative and (uninfused) post-operative performance (dorsal: t(9)\u00a0=\u00a0\u22120.483; p\u00a0>\u00a00.2; ventral: t(12)\u00a0=\u00a0\u22121.15; p\u00a0>\u00a00.2).\nThe amount of alternation following saline infusion into either dorsal or ventral hippocampus was similar and comparable to uninfused performance (dorsal\u00a0=\u00a088%; ventral\u00a0=\u00a085%). In contrast, bilateral infusion of muscimol into the dorsal hippocampus produced a decrease in alternation levels (see Fig. 2). These data were analyzed using a repeated-measures ANOVA [model: cannula placement2\u00a0\u00d7\u00a0(drug treatment5\u00a0\u00d7\u00a0S23)]. The ANOVA revealed a significant effect of cannula placement (F1,21\u00a0=\u00a014.44; p\u00a0<\u00a00.001) and drug treatment (F4,84\u00a0=\u00a09.01; p\u00a0<\u00a00.001) but the interaction was not quite significant (F4,84\u00a0=\u00a02.05; p\u00a0=\u00a00.09).\nAnalysis of simple main effects and subsequent pairwise comparisons (using Sidak's method, see [23]) found an effect of cannula placement at three doses of muscimol: 0.0375\u00a0\u03bcg\/side (F1,21\u00a0=\u00a08.94; p\u00a0<\u00a00.01), 0.075\u00a0\u03bcg\/side (F1,21\u00a0=\u00a06.94; p\u00a0<\u00a00.05) and 0.3\u00a0\u03bcg\/side (F1,21\u00a0=\u00a04.45; p\u00a0<\u00a00.05) with the dorsal group exhibiting greater impairment than the ventral group. The groups did not differ following saline infusion (p\u00a0>\u00a00.2) or when the muscimol dose was 0.15\u00a0\u03bcg\/side (p\u00a0>\u00a00.1). Simple main effects analysis also revealed an effect of drug within the dorsal cannula placement group (F4,18\u00a0=\u00a06.86; p\u00a0<\u00a00.005), with all doses of muscimol leading to performance impairment compared to saline infusion. In contrast, there was no effect of drug within the ventral placement group (F4,18\u00a0=\u00a02.7; NS). In other words, although there was some suggestion that performance was decreasing as the dose of muscimol increased (see Fig. 2), no individual dose of muscimol into the ventral hippocampus significantly impaired performance compared to saline infusion.\nOn alternate days, the rats received 10 trials of rewarded alternation in the absence of any infusions to investigate the possibility of permanent or progressive damage to the targeted regions. Importantly, this data showed that rats from both groups alternated on more than 85% of trials 24\u00a0h after receiving infusions. Post-infusion performance of dorsal and ventral cannulated rats was investigated with a repeated-measures ANOVA [model: cannula placement2\u00a0\u00d7\u00a0(block5\u00a0\u00d7\u00a0S23)]. The ANOVA found no effect of cannula placement (F1,21\u00a0=\u00a02.37; NS), or block (F4,84\u00a0=\u00a00.49; NS), and there was no interaction (F4,84\u00a0=\u00a01.99; NS). This result demonstrates that the T-maze impairments identified in the previous analysis were reversible and only evident while the drug was actually present in the hippocampus.\n3.2\nExperiment 2\nIn Experiment 2, all nine rats had accurate bilateral dorsal hippocampal cannulae placements (Fig. 1). Experiment 2 was divided into two stages. Procedurally, Experiment 2a was identical to Experiment 1. Experiment 2b was identical to Experiment 2a except that a 30\u00a0s delay was interposed between the sample and choice runs of each trial of rewarded alternation (see [21]).\nIn Experiment 2a, the amount of alternation following PBS infusion into the dorsal hippocampus was similar and comparable to uninfused performance (uninfused\u00a0=\u00a090%, PBS\u00a0=\u00a083%). In contrast, there was a substantial drop in choice accuracy following the infusion of either muscimol (58%) or AP5 (67%). A one-way repeated-measures ANOVA [model: drug condition2\u00a0\u00d7\u00a0S9], found a main effect of drug condition (F2,16\u00a0=\u00a06.3; p\u00a0<\u00a00.01) and post hoc pairwise comparisons (Student's Newman\u2013Keuls) revealed significant differences between the vehicle and both drug conditions (p\u00a0<\u00a00.05) but not between the two drug conditions (p\u00a0>\u00a00.2).\nThe introduction of the 30\u00a0s delay in Experiment 2b did not lead to a further drop of performance in either infusion condition (PBS\u00a0=\u00a083%; AP5\u00a0=\u00a072%). Although not strictly appropriate (since the data were not derived in a counterbalanced manner), a repeated measures ANOVA was performed on the combined data of Experiments 2a and 2b (see Fig. 3). The ANOVA revealed a main effect of drug condition (F1,7\u00a0=\u00a011.75; p\u00a0<\u00a00.05) but no effect of delay or drug condition\u00a0\u00d7\u00a0delay interaction (both F\u00a0<\u00a01; NS).\nFinally, performance accuracy (90%) during drug-free testing carried out after the last infusion confirmed that there was no lasting damage due to cannulae implantation or the microinjection process.\n4\nDiscussion\nExperiment 1 showed that selective inactivation of the dorsal hippocampus results in significant impairment of spatial working memory performance whereas equivalent inactivation of ventral hippocampus does not. This result is consistent with previous lesions studies [7,18], but advances on these earlier findings by demonstrating that such dorsal\/ventral differences are not due to indirect effects such as demyelination or compensatory changes that result from permanent brain lesions. Also, the impairment resulting from infusion of AP5 directly into the dorsal subregion (Experiment 2) identifies the importance of N-methyl-d-aspartate receptor (NMDAR) function in the dorsal hippocampus for spatial working memory performance.\nThis data, and that obtained from previous cytotoxic lesions studies, are consistent with findings from a variety of different approaches which suggest a functional specialization along the septotemporal (dorsal\/ventral) axis of the hippocampus. Evidence from electrophysiological single unit recording studies in both rats [24,25] and primates [26], from c-fos activation studies [27], and from structural magnetic resonance imaging studies [28], are all consistent with a functional dissociation between dorsal (posterior in primates) and ventral (anterior in primates) hippocampus, and with a preferential role for dorsal hippocampus in spatial learning and memory.\nTo our knowledge, this experiment constitutes the first demonstration of differential effects of dorsal and ventral hippocampal inactivation on spatial working memory performance using T-maze rewarded alternation. It is worth noting, however, that the effects of selective inactivation of dorsal and ventral hippocampus on delayed alternation tasks have been examined previously using operant paradigms, but with mixed results. For example, Mao and Robinson [29] trained rats to press a lever on the left or right-hand side of the front wall of the operant chamber as indicated by the presence of a light stimulus (equivalent to the sample phase in the present study), then press a lever on the rear wall before being given a free choice of either front wall lever (equivalent to the choice phase in our study). The rats were required to alternate lever presses on the front wall in order to obtain food rewards. Although this task has some similarity to our T-maze task (both require alternation behaviour), bilateral infusions of muscimol into the dorsal hippocampus did not affect the proportion of correct responses. It is worth pointing out, however, that the doses used by Mao and Robinson were lower than the doses used in the present study (0.003\u20130.01\u00a0\u03bcg compared to 0.0375\u20130.3\u00a0\u03bcg in the present study). It is also worth noting that in their study dorsal infusions of higher doses of muscimol (0.03\u20130.2\u00a0\u03bcg) interfered with the rats\u2019 ability to perform various non-spatial aspects of task performance such as correct lever presses at the sample stage and the rear lever press between the choice and sample phases. In a separate study, Maruki et al. [30], trained rats to alternate between pressing the left and right lever on each trial. To prevent mediating strategies, the rat was required to make a touch response to the central food-well during the ITI. Using this continuous alternation paradigm, the authors found that infusions of muscimol (0.07\u00a0\u03bcg\/side) into the dorsal hippocampus reduced correct responding when the ITI was 20\u00a0s but not when it was 3\u00a0s. In a second experiment, they reported no effects of ventral infusions of muscimol relative to saline vehicle infusions on the same task. However, inspection of the data across the two experiments shows that the levels of performance in rats receiving muscimol infusions into dorsal hippocampus (70.9% correct) and in rats receiving muscimol into ventral hippocampus (72.3% correct) were virtually indistinguishable. The present study thus provides the first clear demonstration that spatial working memory performance is sensitive to dorsal but not ventral hippocampal inactivation.\nAs noted by Mao and Robinson [29] direct intra-hippocampal infusion carries the risk of non-specific effects on performance. For example, it is possible that unequal quantities of muscimol entering each hemisphere could lead to a turning bias. First, it is worth pointing out that in our study both hemispheres were infused simultaneously. Second, in Experiment 1, rats receiving dorsal HPC infusions exhibited side preferences (i.e. they exclusively entered the same goal arm on the choice phase of each trial of a session) in only 13 of the 40 sessions (10 rats\u00a0\u00d7\u00a04 muscimol doses). It is also worth pointing out that such side preferences are commonly observed in rats with permanent HPC lesions and in our experience these preferences are not dependent on the size of the lesion in one particular hemisphere. They are generally considered to be a consequence, rather than a cause, of the memory impairment. Furthermore, the fact that in Experiment 1, errors were made to both goal arms in almost 70% of sessions argues against a performance account based solely on side-preferences elicited by unequal quantities of muscimol entering each hemisphere.\nAn alternative account of the septotemporal differences found in Experiment 1 is that the spread of the drug within the hippocampus is simply greater when infused into dorsal than ventral HPC, effectively creating a larger dorsal than ventral HPC inactivation. Without autoradiography to determine the extent of muscimol diffusion we cannot rule out this possibility completely. However, when the data from the present study are considered along with the previous cytotoxic lesion studies, such an account seems unlikely. Evidence from permanent lesion studies suggests that lesion size itself is not the critical determinant of degree of dysfunction. For example, Moser and colleagues [6] found that rats with lesions encompassing 20\u201340% of HPC tissue starting at the ventral pole showed normal spatial learning in the watermaze, whereas rats with lesions of 20\u201340% of HPC tissue starting at the dorsal pole were profoundly impaired. Furthermore, we have previously tested rats with dorsal and ventral cytotoxic lesions of very similar sizes (50\u201355%) on the same T-maze rewarded alternation task as used in the present study, and have found that whereas dorsal lesioned animals resemble animals with complete hippocampal lesions, exhibiting chance levels of performance, ventral lesioned animals perform as well as sham operated controls [9]. These data strongly suggest that lesion location, and not lesion size, is the critical factor.\nIn the present study, although no dose of muscimol into the ventral HPC significantly impaired performance compared to vehicle, there was some suggestion of a dose related decline in performance (see Fig. 2). This could reflect a role, albeit more limited, for the ventral hippocampus in spatial information processing. However, it is also possible that this slight drop in performance may reflect the spread of muscimol into mid-hippocampal and\/or dorsal regions. In this regard, it is worth re-iterating that ventral hippocampal lesions, removing approximately 50% of the structure starting from the temporal pole, had absolutely no effect on performance on this very same spatial working memory, rewarded alternation task [7,9] Therefore, the evidence suggests that spatial working memory tasks such as rewarded alternation on the T-maze, like spatial reference memory tasks such as the standard fixed location version of the watermaze, are sensitive to dorsal but not equivalently sized ventral HPC lesions or inactivations.\nNevertheless, it may be unwise to completely exclude a role for the ventral HPC in spatial information processing. Single-unit studies have shown that cells selective for specific spatial locations (\u201cplace cells\u201d) do exist in ventral HPC, albeit they are fewer in number and their place fields are larger than those in dorsal HPC. Although the electrophysiological data are limited, McNaughton and colleagues have suggested a septotemporal gradient in spatial selectivity with higher resolution spatial discriminations processed in dorsal HPC [24,25]. In this way, cells in dorsal HPC might code for specific locations within a particular environment whereas ventral cells might code for different environments or contexts, although this may be just a subset of the contextual information that can be processed by the ventral subregion [5]. It remains to be seen if tasks requiring discrimination between different spatial environments or contexts are more sensitive to lesions of the ventral HPC.\nThe results of Experiment 2 show that NMDARs in dorsal hippocampus contribute to performance on spatial working memory tasks. Previous work in this laboratory showed that i.c.v. infusion of AP5 impaired performance on the same rewarded alternation task [21]. The present results now suggest that this effect is, at least in part, due to the blockade of NMDARs in dorsal hippocampus. Similar spatial working memory impairments have also been observed following AP5 infusions into dorsal hippocampus on a spatial, matching-to-position version of the watermaze task [19]. Both of these results are consistent with the hypothesis that NMDAR-mediated forms of synaptic plasticity, similar to experimentally induced long-term potentiation [LTP; 31, 32], may underlie a flexible, rapidly acquired form of spatial memory, as typified by these spatial working memory tasks [20,33].\nThe observation that introducing a further 20\u00a0s delay failed to exacerbate the spatial working memory deficit might be considered surprising. In contrast to the present results, Tonkiss and Rawlins [21] showed that increasing the delay by 20\u00a0s did in fact increase the impairment in rats infused with AP5 i.c.v., on the same T-maze spatial working memory task. However, it is worth pointing out that in both cases, the effect of increasing delay was not investigated as part of a fully counterbalanced design, and therefore some caution is required when interpreting these results. Steele and Morris [19] did report a delay-dependent impairment in their matching to position watermaze task with a fully counterbalanced design, although it is worth noting that performance at the shortest delay was not consistently spared in their study.\nWe have also recently tested genetically modified mice in which the NR1 subunit of the NMDAR has been selectively deleted, specifically from the dentate gyrus subfield of the hippocampus [34]. These mice exhibit deficits in LTP in both the medial and lateral aspects of the perforant path, whereas LTP in the CA1 subfield is normal. We assessed spatial memory on the radial maze in these mice, and found that they exhibited a very specific spatial working memory deficit. As with the present study, the animals displayed a spatial working memory deficit at the shortest-possible delays that we were able to test. It is of course possible that at even shorter delays performance may be spared in the AP5 rats and in the dentate gyrus NR1 knockout mice. Equally, it could be that by blocking NMDAR-dependent synaptic plasticity in the hippocampus, we have disrupted an existing spatial representation of the environment, or prevented the expression of memory in some way, for example, by impairing the ability to select the appropriate spatial response on the basis of information retrieved from memory (both of which might be expected to result in a delay-independent deficit). Against this, the dentate gyrus-specific NR1 knockout mice displayed perfectly normal acquisition and performance on the reference memory component of the radial maze task (i.e. learning which 3 arms were baited and which three arms were never baited). It may therefore be that disrupting hippocampal synaptic plasticity prevents the animals from rapidly forming a short-term spatial representation of the maze arm visited on the sample run. This ability to record or represent the recent experience of a particular spatial location or stimuli (i.e. the maze arm) is likely to be of crucial importance for efficient spatial working memory performance [35].\nIn conclusion, the present research has confirmed and extended previous findings from lesion studies, namely that the dorsal hippocampus has a greater involvement in spatial working memory than the ventral hippocampus. Furthermore, NMDAR activation within the dorsal hippocampus makes an essential contribution to this aspect of hippocampal information processing.","keyphrases":["dorsal","hippocampus","spatial memory","ventral","muscimol","ap5"],"prmu":["P","P","P","P","P","P"]}
{"id":"Osteoporos_Int-3-1-1820755","title":"Balance training program is highly effective in improving functional status and reducing the risk of falls in elderly women with osteoporosis: a randomized controlled trial\n","text":"Introduction The purpose of this study was to investigate the effect of a 12-month Balance Training Program on balance, mobility and falling frequency in women with osteoporosis.\nIntroduction\nOsteoporosis is a debilitating, widespread disease, which affects approximately 55% of the population above 50\u00a0years old in the USA [1]. Falls among the elderly, especially for those with osteoporosis, are associated with high morbidity and mortality and can involve high-cost medical intervention [2]. In fact, falls are responsible for 90% of the growing increase in hip fractures [3] and are the sixth cause of death among patients aged over 65 [4].\nFalls are multifactorial, and their causes are categorized as intrinsic (personal) and extrinsic (environmental) factors [5, 6]. Some examples of intrinsic factors include: altered balance, neurological diseases, sensory deterioration, musculoskeletal diseases, postural hypertension and the use of medication [7].\nResearch shows that altered balance is the greatest collaborator towards falls in the elderly [6, 8\u201312], with a high correlation between balance deficit and the incidence of falls [13, 14].\nFor this reason, studies regarding the risk of falling in osteoporosis are of high priority in clinical intervention. Diminishing the incidence of falls is a health priority, which reflects on both the quality of life and health costs [2].\nMoreover, evidence suggests that exercise reduces the risk of fractures, showing an effect on the maintenance of bone mass and, more importantly, improving postural stability, mobility and, consequently, diminishing the risk of falls [15]. Indeed, improving balance should be an objective in the prevention of falls [16].\nHowever, knowledge regarding balance deficit and the probability of falls is limited and controversial [17]. The literature shows that exercise may or may not be efficient in the control of falls [18], and the impact of prevention programs with balance training in diminishing falling frequency has yet to be established, principally in women with osteoporosis.\nBalance training has been investigated in healthy elderly individuals [3, 16, 19\u201321]; however, only one study regarding balance training in women with osteoporosis exists. Since patients with osteoporosis are at greater risk of fractures resulting from falls, further research in this group should be prioritized [22].\nThe purpose of the present study was to investigate the effect of a 12-month balance training program on functional and static balance, mobility and falling frequency in women with osteoporosis.\nPatients and methods\nPatient sample\nSixty-six elderly women aged over 65\u00a0years old were consecutively selected from patients of the Osteometabolic Disease Outpatient Clinic of the Rheumatology Division, University of S\u00e3o Paulo. Only patients with osteoporosis, classified according to the World Health Organization [23] were included; with a bone mineral density (BMD) T-score lower than \u22122.5 standard deviation (SD), in the lumbar spine, femoral neck or total femur region.\nThe following women were excluded: those with secondary osteoporosis, visual deficiency, severe auditive deficiency, or vestibular alteration of important clinical status, such as women who used assisted walking devices or who were unable to walk independently more than 10\u00a0meters [24]; those who planned to be out of town for more than 4\u00a0weeks during the 12-month study; and women who presented absolute or relative contraindications for exercise training according to the American College of Sports Medicine [25].\nThe patients were randomized consecutively into two groups: the group submitted for the Balance Training Program (Intervention Group), consisting of 34 patients; and the Control group, consisting of 32 patients without intervention. The Control group only received treatment for osteoporosis and orientation to prevent falls and return regularly (3-monthly follow-ups) to the Osteometabolic Disease Outpatient Clinic. All patients read and signed a term of free informed consent that described the procedures which would be realized during the research.\nMeasured variables: interview and medical chart records\nPersonal, family and clinical data were evaluated through an interview and medical chart records, with emphasis on the history of fractures, number of falls in the preceding year, use of medication for osteoporosis, and use of medication that favored the risk of falling, such as hypnotics, hypotensors and antidepressants.\nFunctional state evaluation\nStatic and dynamic balance and mobility were evaluated in all patients, before and at the end of the trial, by a physiotherapist who was blinded to the distinct group (Intervention, Control).\nFunctional balance\nThe Berg Balance Scale (BBS) is based on 14 items common to daily life activities used to evaluate functional balance [26]. The maximum score that can be achieved is 56, and each item possesses an ordinal scale of five alternatives which varies from 0 to 4 points.\nThe test is simple, easy to administer and accompanies the evolution of elderly patients. It only requires a ruler and a watch and takes approximately 15\u00a0minutes to execute [26]. A score lower than or equal to 45 is considered evidence of altered balance [27].\nStatic balance\nStatic balance was evaluated by the Clinical Test of Sensory Interaction for Balance (CTSIB), which consists of six sensory conditions (1: eyes open and firm surface, 2: eyes closed and firm surface, 3: eyes open, visual conflict and firm surface, 4: eyes open and unstable surface, 5: eyes closed and unstable surface, and 6: eyes open, visual conflict and unstable surface).\nStatic balance is considered to be altered when an individual cannot remain at least 30\u00a0seconds in each of the six conditions [28].\nImprovement in the test was defined as the capacity to complete the test during the final evaluation when unable to complete the same in the initial evaluation.\nFunctional mobility\nFunctional mobility was evaluated by the Timed \u201cUp & Go\u201d Test (TUGT) [29], which registers the time an individual takes to get up out of a chair, walk 3\u00a0meters, turn around, walk back and sit down again.\nElderly individuals without balance deficit are capable of completing the test in less than 10\u00a0seconds.\nFalls\nThe number of falls in the year prior to the study [30] was solicited and noted in the initial evaluation and at the end of the trial (final evaluation). During the year of the study, patients in both groups received a diary and were orientated to write down the days that they fell.\nAt the end of the study, the difference in the number of falls\/patient (final evaluation - initial evaluation) was compared between the Intervention Group and Control.\nIntervention\nThe Balance Training Program consisted of 1\u00a0hour of exercises realized once a week, with a total of 40 classes, supervised by an experienced physiotherapist. This program was realized in a club (Associa\u00e7\u00e3o Atl\u00e9tica Acad\u00eamica Oswaldo Cruz - AAAOC) belonging to the Clinics Hospital, School of Medicine, University of S\u00e3o Paulo, located near to the Hospital. The balance exercises described by Tinetti and Suzuki [3, 11] were used. The type and mild to moderate intensity of the exercises used in the present study were chosen so that they could also be performed by elderly patients at home [3]. A list of weekly attendance controlled the absences of each patient.\nBasic warm-up and stretching exercises\nPrior to training, the patients participated in 15\u00a0min of warm-up and stretching exercises, consisting of head rotation, shoulder rotation and stretching of the upper and lower limbs. Walking was performed for 15\u00a0min with the supervision of a physiotherapist, who associated exercises for the upper limbs throughout the walk.\nBalance training\nBalance was realized in dynamic and static positions for a period of 30\u00a0min. This consisted of walking in the tandem position (one foot in front of the other), walking on the tips of the toes and on the heel, walking sideways, walking while raising the leg and the contra-lateral arm, standing on one leg, and standing in the tandem position, while gradually increasing the period of permanence in these last two static positions [3, 31].\nHome-based exercises\nThe patients were instructed and encouraged to continue the same exercises at home at least three times a week for 30\u00a0min. A manual with instructions and illustrations for each exercise was distributed. The frequency of participation in the home-based exercises was noted each week by the physiotherapist.\nData analysis\nSixty-six consecutive patients were randomized in the present study. Data analysis was realized on 60 patients (30 Intervention Group and 30 Control), as six patients desisted (four Intervention Group and two Control). In the Intervention Group the reasons for desistance were: physical limitations as a result of falls (n\u2009=\u20091), foot pain (n\u2009=\u20091), personal reasons (n\u2009=\u20092); while in the Control group the reasons were: physical limitations as a result of falls (n\u2009=\u20091), personal reasons (n\u2009=\u20091).\u2009\u2009\nThe data were expressed as the mean and standard deviation (SD) for each variable and differences between the Intervention and Control groups were tested by the Student\u2019s t-test or Mann-Whitney test. The Chi-square test or Fisher\u2019s exact test was used to compare the number of patients in both groups (Intervention Group vs Control) for hypnotic and diuretic drug use, fracture history, CTSIB conditions (1\u20136) and improvement in CTSIB conditions. P values <0.05 were considered significant.\nResults\nThe basal characteristics of the patients of both groups were similar in relation to age, body mass index (BMI), fracture history, osteoporosis treatment, diuretics and hypnotics\/antidepressants use and bone mineral density (BMD), with no statistically significant differences between the two groups (Table\u00a01). \nTable\u00a01Data at the onset of the study in relation to anthropometric parameters, fracture history, medication use and bone mineral density values (T-score) in the Intervention and Control groupsVariableIntervention (n\u2009=\u200930)Control (n\u2009=\u200930)p-valueAge, years74.57\u2009\u00b1\u20094.8273.40\u2009\u00b1\u20094.610.342*BMI, kg\/m224.39\u2009\u00b1\u20094.4926.51\u2009\u00b1\u20095.320.100*Fracture history, n (%)13 (43.3)16 (53.3)0.438***Medication used for OP, n2.37\u2009\u00b1\u20091.502.30\u2009\u00b1\u20090.880.498**Hypnotics\/Antidepressants, n (%)6 (20.0)7 (23.3)0.754***Diuretics, n (%)14 (46.7)16 (53.3)0.606***Lumbar spine, T-score\u22122.83\u2009\u00b1\u20091.07\u22122.62\u2009\u00b1\u20091.120.470**Femur neck, T-score\u22122.70\u2009\u00b1\u20090.75\u22122.75\u2009\u00b1\u20090.900.821**Total femur, T-score\u22122.10\u2009\u00b1\u20091.26\u22122.10\u2009\u00b1\u20091.090.990**Data expressed in means \u00b1 SD or percentageBMI: body mass index, OP: Osteoporosis*Student\u2019s t-test**Mann-Whitney test***Chi-square test\nSimilarly, in the first evaluation, no differences occurred when comparing the Intervention Group and the Control Group in reference to: Berg Balance Scale (BBS) score, the number of patients that could not complete the Clinical Test of Sensory Interaction for Balance (CTSIB) in the six sensory conditions, the Timed \u201cUp & Go\u201d Test (TUGT), and the number of falls\/patients in the preceding year (p\u2009>\u20090.05) (Table\u00a02). \nTable\u00a02Data at the onset of the study for: Berg Balance Scale (BBS) score, number of patients that could not complete the Clinical Test of Sensory Interaction for Balance (CTSIB: condition 1: eyes open and firm surface; condition 2: eyes closed and firm surface; condition 3: eyes open, visual conflict and firm surface; condition 4: eyes open and unstable surface; condition 5: eyes closed and unstable surface; condition 6: eyes open, visual conflict and unstable surface), Timed \u201cUp & Go\u201d Test (TUGT), and number of falls\/patient in the preceding year in Intervention Group and Control\u00a0Intervention (n\u2009=\u200930)Control (n\u2009=\u200930)p valueBBS, score48.80\u2009\u00b1\u20094.1048.13\u2009\u00b1\u20095.360.900*CTSIB condition 1, n (%)0 (0.0)1 (3.3)1.000**CTSIB condition 2, n (%)2 (6.7)1 (3.3)1.000**CTSIB condition 3, n (%)2 (6.7)2 (6.7)1.000**CTSIB condition 4, n (%)4 (13.3)4 (13.3)1.000**CTSIB condition 5, n (%)15 (50.0)12 (40.0)0.604**CTSIB condition 6, n (%)12 (40.0)9 (30.0)0.589**TUGT, seconds14.31\u2009\u00b1\u20094.0313.86\u2009\u00b1\u20093.430.610*Falls\/patient preceding year, n1.20\u2009\u00b1\u20091.880.87\u2009\u00b1\u20090.860.745*Data expressed in means \u00b1 SD or percentage.*Mann-Whitney test**Fisher\u2019s exact test\nAdherence rate\nA high level of adherence was observed. Sixty percent of the patients participated in all of the exercise sessions at the club and absences occurred with the following justifications: doctor\u2019s appointment, the realization of laboratory exams or for personal reasons. In relation to home-based exercise, 76.67% of the patients realized exercises at least once a week, 40% of the patients exercised every day and 36.67% from one to four times a week.\nComparison between the Intervention Group and Control\nThe difference in BBS score (final\u2014initial evaluation) was greater in the group which suffered intervention (5.5\u2009\u00b1\u20095.67 vs \u2212 0.5\u2009\u00b1\u20094.88, p\u2009<\u20090.001) (Table\u00a03). \nTable\u00a03Differences (final evaluation\u2014initial evaluation) in: Balance Berg Scale (BBS) score, number of patients showing improvement in Clinical Test of Sensory Interaction for Balance (CTSIB condition 5: eyes closed and unstable surface; condition 6: eyes open, visual conflict and unstable surface), time of Timed \u201cUp & Go\u201d Test (TUGT), and number of falls\/patient in the Intervention Group and Control\u00a0Intervention (n\u2009=\u200930)Control (n\u2009=\u200930)p-valueDifference BBS, score 5.5\u2009\u00b1\u20095.67\u22120.5\u2009\u00b1\u20094.88<0.001*CTSIB condition 5, n (%)13 (43.3)1 (3.3)<0.001**CTSIB condition 6, n (%)12 (40.0)1 (3.3)0.001**Difference TUGT, seconds\u22123.65\u2009\u00b1\u20093.61+2.27\u2009\u00b1\u20097.18<0.001*Difference of falls\/patient, n\u22120.77\u2009\u00b1\u20091.76+0.03\u2009\u00b1\u20090.960.018*Data expressed in means \u00b1 SD or percentage.*Mann-Whitney test**Chi-square test\nSimilarly, the percentage of patients in the Intervention group whose static balance improved in two sensory conditions (CTSIB, condition 5: eyes closed, unstable surface; and condition 6: eyes open, visual conflict, unstable surface) was statistically significant when compared to Control (CTSIB condition 5: 13 patients vs 1, p\u2009\u2009<\u20090.001; CTSIB condition 6: 12 patients vs 1, p\u2009<\u20090.001) (Table\u00a03).\nEqually, a significant difference in the functional mobility, as measured by the TUGT (final\u2014initial evaluation) was observed in the Intervention Group compared to Control (\u22123.65\u2009\u00b1\u20093.61 vs +2.27\u2009\u00b1\u20097.18, p\u2009<\u20090.001) (Table\u00a03).\nParallel to these improvements in functional and static evaluation, a reduction in the number of falls\/patient (final-initial evaluation) was observed in the Intervention Group compared to Control (\u22120.77\u2009\u00b1\u20091.76 vs +0.03\u2009\u00b1\u20090.98, p\u2009=\u20090.018) (Table\u00a03).\nDiscussion\nFew studies have been developed regarding balance training in patients with osteoporosis. The present longitudinal prospective study demonstrated that a program of balance training realized over a period of 12\u00a0months was effective in improving the functional and static balance, mobility and diminishing the number of falls in elderly women with osteoporosis.\nIn the present work, improvement in relation to functional balance was demonstrated by an increase in the BBS score in the final evaluation in the group submitted to the balance training program (Intervention Group). Similar results were shown in a study by Melzer et al. [32], where the patients who participated in the balance training obtained 64% improvement in 3\u00a0months. The authors showed that the group who underwent the balance training demonstrated better performance than the group submitted to muscular strength training.\nPrograms that emphasize balance training are more effective at improving balance than those that consist primarily of aerobic, muscular strength or flexibility exercises [33].\nAlthough it is a complex issue to evaluate the effectiveness of different types of exercises [34], balance training has shown beneficial results, with diminished risks of falls [35].\nAnother positive result of the present study was the improvement in the two difficult conditions of the Clinical Test of Sensory Interaction for Balance (condition 5: eyes closed, unstable surface, and condition 6: eyes open, visual conflict, unstable surface) in almost half the patients. Carter et al. [24] obtained an improvement in static balance using muscular strength training rather than balance training, though only in 6.3% of the patients. Thus, the present findings suggest that balance training leads to more evident positive results in static balance than does muscular strength training.\nIn relation to functional mobility, improvement was demonstrated by a reduction in the TUGT time in the patients submitted to intervention. These results are relevant, since research shows that compromised mobility increases the risk of dependency three- to five-fold, in activities of daily life [36]. This is not surprising, considering that mobility is an important component of daily life activities, for example: going to the shopping mall, to the supermarket, to the doctor or the cinema. Increased dependency could lead to institutionalization and diminished quality of life. Good balance is considered fundamental for improving mobility and preventing falls [36].\nIn parallel to the improvement in functional evaluation, an important reduction in the frequency of falls was observed. Although the effect of exercise in the prevention of falls in the elderly is yet to be proved, some studies show that physical activity reduces the risk by 40% [37]. On the other hand, in a consensus on prevention of falls in the elderly, the only exercises recommended to prevent falls are those which specifically target balance training [38].\nMany studies that show improvement in balance and mobility present similar characteristics in their exercises. This suggests that the content and intensity of the exercise program could be more important than other intervention variables [39]. Moreover, differences in the administration of the exercise program, the professional experience of those who apply the exercises, the location in which the sessions are held and whether the exercise is conducted in group or realized individually, are all fundamental parameters which influence the success of the exercise program[39].\nAn important factor for the success of the exercise program is adherence. Contrary to the study by Forcan et al. [40], who stated that adherence to exercise in the elderly is weak, in the present study good adherence was observed, with more than half the patients present at all sessions. The current findings are similar to other studies which showed adherence up to 97.5% [17].\nThe success in adherence in the present study is probably due to the location where the exercises were performed (a pleasant, natural environment), to social interaction, and to the supervision of a physiotherapist. A secure environment, session supervision and the opportunity for social interaction reduce the feeling of isolation. A social support system is considered important in group activities, and helps sustain adherence and the effectiveness of the weekly exercise sessions and also the adherence to home-based exercises [35].\nSimilarly to our study, Robitaille et al. showed that an exercise program performed in groups improves the balance of the elderly in the community [41].\nAnother relevant factor was the use of a manual of instructions and illustrations for the realization of home-based exercises, which contributed to the continuity and adherence of the exercises performed. Each exercise was appropriately prescribed and illustrated by a physiotherapist, giving the patient adequate support. Descriptive and illustrated pamphlets have been used in some studies, complemented by a home-based exercise program, with positive results [3, 42].\nThe present positive results could also be related to the state of health of these patients, which was good in our patients. Buchner et al. [43] showed that exercise can have beneficial effects on health and on the risk of falls in certain subgroups of the elderly.\nThe physical and psychological benefits of the regularity of the sessions and the environment should never be ignored. Stimulating strategies and demonstrating ability in the transference from one exercise to another, consequently, maintaining enthusiasm during the exercises, can be practiced safely in groups [35].\nAlthough our study did not use laboratory equipment [44] to substantiate the results obtained, we showed by reliable and reproducible scales and tests [26, 28, 29] that balance training performed once a week, supervised by a physiotherapist, and complemented by home-based exercises, is very effective in the improvement of functional and static balance and mobility, and in the reduction of falls in elderly women with osteoporosis.","keyphrases":["balance","falls","osteoporosis","mobility","exercise program"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_Arch_Otorhinolaryngol-4-1-2279158","title":"Voice quality after endoscopic laser surgery and radiotherapy for early glottic cancer: objective measurements emphasizing the Voice Handicap Index\n","text":"We analyzed the functional outcome and self-evaluation of the voice of patients with T1 glottic carcinoma treated with endoscopic laser surgery and radiotherapy. We performed an objective voice evaluation, as well as a physical, emotional and functional well being assessment of 19 patients treated with laser surgery and 18 patients treated with radiotherapy. Voice quality is affected both by surgery and radiotherapy. Voice parameters only show differences in the maximum phonation time between both treatments. Results in the Voice Handicap Index show that radiotherapy has less effect on patient voice quality perception. There is a reduced impact on the patient\u2019s perception of voice quality after radiotherapy, despite there being no significant differences in vocal quality between radiotherapy and laser cordectomy.\nIntroduction\nSurgery and radiotherapy offer the same results in the treatment of early glottic carcinoma, with a cure rate of approximately 90% [1].\nThe introduction of endoscopic CO2 laser surgery in early glottic carcinoma (epidermoid carcinoma confined to the true vocal cords with normal mobility) has added controversy to the standard treatment choice. Laser surgery is quicker, which reduces cost considerably. However many institutions prefer to use radiotherapy in the belief that the voice will be better preserved after treatment [2]. Although the principal objective of oncology treatment is the complete eradication of the illness, normal voice preservation is another important consideration in the treatment choice of early glottic carcinoma. For this reason, post-treatment voice quality is a relevant factor to take into account when evaluating the results.\nQuantitative acoustic measurements are more regularly studied. These are obtained from tools that digitize and analyze the voice being investigated and quantify the characteristics that deviate from normality, understanding normality to mean when the voice is uniform in both amplitude and tone periodicity. The addition of noise to the voice signal is also a defect that affects voice quality, and this is the third acoustic characteristic that is studied using spectrography.\nAnother of the factors to take into account is the auditive perception that is generated in the listener and is evaluated by ways of perceived voice quality, making use of semi-objective scales such as the GRBAS scale, as described by Hirano [3]. It is recommended that these measurements are made by two experts, although the parameters have shown sufficient reliability (inter- and intra-observer reproducibility) when used in a clinical setting [4].\nAlthough all these measurements are important parameters in defining voice quality, they fail to provide information on the patient\u2019s perceived voice quality. For this there is a method available that permits the patient to describe the sensations their voice gives: the Voice Handicap Index [5]. It is a questionnaire that reviews situations grouped into three areas (functional, physiological and emotional) and gives an idea of the subjective impact that a vocal problem produces in a specific individual.\nIn this study, we present the objective and subjective analysis of voice quality following treatment of an early epidermoid glottic carcinoma. Results from the objective evaluation of the voice, along with the self-evaluation of voice quality quantified using the Voice Handicap Index of a group of patients treated with endoscopic laser surgery are compared with patients treated with radiotherapy.\nMaterials and methods\nVoice analysis and quality of life studies of 19 patients treated with laser surgery and 18 with radiotherapy, suffering from early glottic carcinoma, were evaluated. Lesions were classified according to the American Joint Committee on Cancer [6].\nIn patients who received radiotherapy as the primary treatment, this was performed using a 6\u00a0mv linear accelerator to bilateral ports and one in front, with field sizes ranging from 5\u00a0\u00d7\u00a05 to 6\u00a0\u00d7\u00a06\u00a0cm. Wedges were used to establish dose homogeneity.\nIn the case of patients treated with surgery, the treatment consisted of direct laryngoscopy for the complete resection (partial transmuscular cordectomy) of the lesion with CO2 laser. Objective voice analysis and voice quality studies, performed by both patients and examiners by means of an examination protocol including: perceptual analysis of dysphonia, acoustic analysis, aerodynamic efficiency and patient perception, were applied to both groups. In all patients, this protocol was undertaken at least 6\u00a0months after completing treatment.\nPerceptual analysis of dysphonia (GRBAS)\nPerceptual analysis of dysphonia was performed using the GRBAS scale [3]. Two experienced professionals evaluated the recorded voice samples simultaneously, classifying each sample from 0 to 3 (0\u00a0=\u00a0normal, 1\u00a0=\u00a0mild, 2\u00a0=\u00a0moderate, 3\u00a0=\u00a0severe). The severity of hoarseness is quantified under the parameter G (grade), which represents overall voice quality. B (breathiness): audible impression of turbulent air leakage through an insufficient glottic closure, which may include short aphonic moments (unvoiced segments). R (roughness or harshness): audible impression of irregular glottic pulses, abnormal fluctuations in F0, separately perceived acoustic impulses (as in vocal fry), includes diplophonia and register breaks. A (asthenicity): impression of weakness in the spontaneous phonation, hypokinetic or hypofunctional voice. S (strain, vocal tension): auditive impression of excessive force or tension associated with spontaneous phonation.\nAcoustic and spectrographic analysis\nThe acoustic analysis was performed using Doctor Speech V3 software for Windows 95. The acoustic signal was recorded using the Voice Assessment application. The computer used was a Pentium II at 100\u00a0MHz and with 16\u00a0Mb of RAM and a Sound Blaster 16 sound card. The sampling frequency was 44,100\u00a0Hz and a high frequency range microphone was used. The microphone was located 10\u00a0cm from the patient\u2019s mouth while they emitted the \u201ce\u201d sound at comfortable intensity and pitch levels in a soundproof chamber. The computer captured 3\u00a0s of sound. Once the signal was digitized, the computer calculated the following acoustic parameters: fundamental frequency (F0), jitter or frequency variation (%), shimmer or amplitude variation (%) and NNE or normalized noise energy, which measures the degree of noise produced by turbulent air escaping through the glottis during vocal emission.\nUsing the same digitized voice sample, a narrow band spectrogram was generated (45\u00a0Hz) using the Speech Analysis application. The spectrograms obtained were grouped into four types according to the Yanagihara criteria [7].\nAerodynamic efficiency analysis\nThis consisted in measuring the maximum phonation time (TMF) for the \u201ca\u201d vocal after instructing the patient to sustain this vocal for the longest time possible at a comfortable pitch and intensity. The patients were asked to repeat the test at least three times and the highest value was retained.\nPatient self-perception analysis (Voice Handicap Index)\nThe patients completed the \u201cVoice Handicap Index\u201d via a self-evaluation form comprising 30 questions covering three domains [5]: functional, physical and emotional, translated from the original work in English by one of the authors. Each question was assigned a score of 0\u20134 (from least disability to most). In each item, the maximum score was 40 points and we classified them as mild disability (less than 20), moderate (21\u201330) and severe (more that 30). Adding the three scores together, the maximum possible was 120, and we classified the vocal disability as mild (less than 30), moderate (31\u201360), severe (61\u201390) and very severe (91\u2013120).\nStatistical analysis\nThe data obtained was gathered in the statistical database SPSS 12.0. The statistical analysis used was the Student\u2019s t test in order to compare averages and the Chi-squared test for comparing proportions. The statistical differences were considered significant when P was lower than 0.05.\nResults\nIn the patient group treated with CO2 laser, the ages were in the range 44\u201386\u00a0years, with an average age of 64 and a mean follow-up of 30\u00a0months (12\u201348). In the patient group treated with radiotherapy, the ages were in the range 55\u201381\u00a0years, with an average age of 67 and a mean follow-up of 43\u00a0months (6\u201381). All patients in both groups were males.\nIn both groups, all lesions corresponded to stage I of the TNM classification. In the group of 19 patients treated with surgery, all the lesions were qualified as T1a and in the case of the 18 radiotherapy patients, 13 were classified as T1a and 5 as T1b. No metastases in the neck, or distant, were detected. At the time of the study, all patients were free from illness.\nIn the group of patients treated with radiotherapy, the nominal total dose was 6,525\u00a0cGy, with 225\u00a0cGy daily doses for a total of 29\u00a0days. Of the PTV volume, 100% received minimum doses of approximately 5,700\u00a0cGy, a maximum dose of approximately 6,720\u00a0cGy and an average dose of 6,520\u00a0cGy.\nAerodynamic efficiency and spectrographic analysis\nIn Table\u00a01, the maximum phonation times are presented for the \u201ca\u201d vocal along with the quantitative voice analysis of the vocal \u201ce\u201d obtained in the groups treated with laser and with radiotherapy. There was no significant difference between the parameters: fundamental frequency, (F0), jitter, shimmer and NNE on comparing both groups. However, there were significant differences in the maximum phonation time.\nTable\u00a01MPT and acoustic analysis of the vocal \/e\/ after treatment: laser (n\u00a0=\u00a019) or radiotherapy (n\u00a0=\u00a018)VariableLaserRadiotherapyMPT11.83\u00a0\u00b1\u00a05.288.63\u00a0\u00b1\u00a03.23P\u00a0<\u00a00.05F0 (Hz)173.39\u00a0\u00b1\u00a047.41199.04\u00a0\u00b1\u00a051.46NSJitter (%)0.44\u00a0\u00b1\u00a00.240.72\u00a0\u00b1\u00a00.91NSShimmer (%)5.08\u00a0\u00b1\u00a04.724.07\u00a0\u00b1\u00a04.04NSNNE (dB)\u22125.82\u00a0\u00b1\u00a02.98\u22125.02\u00a0\u00b1\u00a04.44NSMPT maximum phonation time, F0 (Hz) fundamental frequency, NNE normalized noise energy\nIn spectrographic terms, the 19 voice samples of patients treated with laser were classified as Grade I 4 (21%), Grade II 5 (25%), Grade III 7 (37%) and Grade IV 3 (15%). In the group of patients treated with radiotherapy, the 18 voice samples were classified as Grade I 1 (5%), Grade II 7 (39%), Grade III 5 (28%) and Grade IV 5 (28%). On comparing the spectrograms obtained in both groups, there were no statistically significant differences (P\u00a0=\u00a00.401).\nPerceptual dysphonia analysis (GRBAS)\nTable\u00a02 shows the results obtained using GRBAS scale on patients treated with CO2 laser surgery. Mild dysphonia was found in 31.5% of the cases, moderate dysphonia in 37% and severe dysphonias in the remaining 31.5%. Table\u00a03 shows the GRABS results of the patients treated with radiotherapy. Normal voices were observed in 11%, mild dysphonias in 44.4%, moderate dysphonia in 27.8% and severe dysphonia in 16.7% of the cases. On comparing the results obtained in the G domain of the GRABS scale, which corresponds with the degree of dysphonia, no significant difference was found between each group (P\u00a0=\u00a00.309).\nTable\u00a02Results obtained using GRBAS scale on the patients treated with CO2 laser surgery (n\u00a0=\u00a019)0123TotalG0%31.5% (6)37% (7)31.5 (6)100% (19)R10.5% (2)58% (11)26% (5)5.5% (1)100% (19)A21% (4)42% (8)21% (4)16% (3)100% (19)B84% (16)16% (3)0%0%100% (19)S31.5% (6)31.5% (6)21% (4)16% (3)100% (19)G grade, R roughness, A asthenicity, B breathiness, S strainTable\u00a03GRABS results of patients treated with radiotherapy (n\u00a0=\u00a018)0123TotalG11.1% (2)44.4% (8)27.8% (5)16.7%(3)100% (18)R55.6% (10)33.3% (6)5.6% (1)5.6% (1)100% (18)A55.6% (10)33.3% (6)11.1% (2)0%100% (18)B44.4% (8)27.8% (5)22.2% (4)5.6% (1)100% (18)S77.8% (14)16.7% (3)5.6% (1)0%100% (18)G grade, R roughness, A asthenicity, B breathiness, S strain\nPatients self-perception analysis (Voice Handicap Index)\nTable\u00a04 shows the averages obtained from the \u201cVoice Handicap Index\u201d questionnaire in the functional, physical and emotional scales, as well as the scores obtained in both groups. Upon completing the comparison between the two groups, the statistical difference is significant, in favor of the radiotherapy patients in the functional and emotional ratings as well as the global scores. No significant differences were found in the physical scales.\nTable\u00a04Averages obtained from the \u201cVoice Handicap Index\u201d questionnaire in the functional, physical and emotional scales, as well as the scores obtained in both groupsLaser RadiotherapyFunctional11.47 (0\u201332)2.83 (0\u201317)P\u00a0<\u00a00.05Physical12.68 (0\u201331)6.22 (0\u201318)NSEmotional4.63 (0\u201320)0.61 (0\u20135)P\u00a0<\u00a00.05Global28.79 (0\u201377)9.67 (0\u201329)P\u00a0<\u00a00.05\nDiscussion\nEarly glottic carcinoma can be treated using endoscopic surgery, radiotherapy or partial open surgery. Lesions limited to the vocal fold are normally treated with endoscopic surgery or partial open surgery, with local control results of between 80 and 90% [8]. In choosing one treatment or another, one should contemplate the cure rate, larynx preservation rate, post-treatment voice quality, morbidity and treatment cost [9].\nIn recent years, endoscopic CO2 laser surgery has made headway compared to radiotherapy, based on its good oncology results and reduced morbidity. CO2 laser treatment in early glottic carcinoma has greatly improved voice quality compared to that obtained following cordectomy via laryngofissure. as documented by some authors such as Keilmann et al. [10]. However in other published studies, such as Schindler [11], this improvement is not as evident. One of the advantages of laser surgery is its low cost when compared to radiotherapy, as well as the additional benefit of being able to opt for radiotherapy at a later stage to treat a relapse or second primary malignancy. In many institutions, these tumours are treated with external radiotherapy because of the supposedly better functional and quality of life results obtained compared to patients treated with surgery [12].\nVarious studies have been published comparing voice quality after both treatments [13\u201315]. In some studies, voice quality is similar, while other authors maintain that the voice is better after radiotherapy than after laser surgery [16\u201318]. However, there are fewer published works that include patient opinions with regard to the impact the illness and the treatment has had on their quality of life [19\u201321].\nIn our work, no significant differences in fundamental frequency (F0), jitter, shimmer and NNE were found between the two groups. There were, however, significant differences in maximum phonation time, favoring the patients treated with CO2 laser cordectomy. Other authors do not find significant differences in the maximum phonation time between the groups [22\u201324]. In the study by Tamura et al. [13] the fundamental frequency is higher for patients treated with CO2 laser surgery, which suggests that this surgery has a greater impact on the vocal fold function than radiotherapy. Krengli et al. [18] found statistically significant differences in the vocal acoustic parameters and fundamental frequency, favoring radiotherapy patients.\nIn terms of the spectrograms obtained in both groups, no statistically significant differences were found. According to our results, 48% of patients treated with laser, and 44% of radiotherapy patients have an aesthetically acceptable voice (type I and II dysphonia).\nThere were no significant differences found between the two groups on comparing the scores obtained using the G parameter of the GRABS scale, which corresponds to the degree of dysphonia. Other studies such as Loughran et al. [20] failed to find statistically significant differences on comparing GRABS between patients treated with each technique. In our work, there is an elevated percentage of patients treated with CO2 laser (84%) who obtained low scores in the breathy voice domain (B), which shows that apart from individual healing patterns, or treatment-induced secondary glottic defects, post-cordectomy phonetic compensating mechanisms are important in determining new voice quality. According to Sittel et al. [25], glottic phonation voice quality is better than that obtained by the non-glottic phonation. Although one could believe that these patients had benefited from voice therapy, there are contradictory opinions on this [26].\nWhile acoustic, physiological and perceptual measurements are important parameters in assessing vocal function, they do not provide information about the patient\u2019s perception of their own voice quality. Given that voice quality, because of its potential impact on quality of life, can be an important factor in the choice of treatment, it is important to include this information when evaluating results. Not only should dysphonia as a by-product of laryngeal physiology be considered, but also the effects on patient quality of life must be considered as well.\nIn our study, in order to quantify patient opinion as to the impact caused by treatment, the Voice Handicap Index has been employed. This is an instrument developed to help the professional in deciding the therapy, taking into account the patient\u2019s subjective sensations with respect to their problem. It is a post-treatment global result evaluation method, from the point of view of the patient\u2019s perceived global well being (physical, mental and social). Although it is a subjective evaluation based on the patient\u2019s own perception, it can provide valuable data as to the reasons why patients with similar dysphonia characteristics can have different handicap severity indices [27]. VHI allows investigators to obtain information regarding the patient\u2019s subjective perception as well as providing the physician with important information pre and post treatment. Given that the preservation of adequate phonation is an important consideration in early glottic carcinoma treatment selection and given equal oncologic results between the two treatments, the VHI proves to be a very important tool in the therapeutic decision-making process.\nIn our study, low scores were obtained for both groups in the three scales, functional, physical and emotional. This could imply reduced impact in the quality of life of patients treated either with laser surgery or radiotherapy. The statistically significant differences are in favor of radiotherapy patients in the functional and emotional scales, as well as the global scores. Although acoustic and perceptual voice analysis in both groups showed no significant differences, the radiotherapy group scored less in the VHI than the surgical group.\nLoughran et al. [20] performed a study where they compared the results between both therapies in terms of the Voice Handicap Index as well as two other questionnaires aimed at evaluating the patient\u2019s subjective voice quality (Vocal Performance Questionnaire, VPQ, and Voice Symptom Score, VoiSS). In this work, no significant differences were discovered between radiotherapy and CO2 laser surgery patients in any of the questionnaires, except for the emotional sub-scale inVoiSS.\nHowever, in other studies the average global score in the Voice Handicap Index questionnaire is lower for patients treated with endoscopic laser resection than in patients treated with radiotherapy. Peeters et al. [21] compared the results of both the Voice Handicap Index and a quality of life questionnaire (COOP\/WOONCA) following both treatments. In their results, they found statistically significant differences in favor of laser surgery for the VHI and none in the quality of life results. They found higher scores for global VHI results in 40% of patients treated using surgery, and 58% for those treated with radiotherapy. Their conclusions reveal that the treatment for T1 glottic carcinoma frequently generates vocal problems in day-to-day life, influencing negatively certain social activities. This data contradicts the results of our study where VHI scores of both groups were low and the differences found favored the radiotherapy patient group. Recently, Cohen et al. [28] published a meta-analysis in order to classify the quality of life related to voice in patients with TI glottic carcinomas treated with radiotherapy compared with CO2 laser resection. Six studies with a total of 208 patients treated with surgery and 91 with radiotherapy had similar scores for VHI, from which they concluded that both treatments result in comparable vocal handicap levels for patients with T1 glottic carcinomas [28].\nIn other works such as that by Stoeckli [19], which value long-term results with regard to quality of life (QOL) of early glottic carcinoma patients, no differences were found between the two treatments and they conclude that both therapies provide good results in terms of quality of life. They show in addition, differences in the relative scores for questions such as swallowing, xerostomy and dental problems, favoring patients treated surgically, and show no differences with regard to perceived voice quality. These results could be related to the fact that xerostomy, edema, mucositis and fibrosis increase the sensation of handicap to such an extent that these effects on patient quality of life could be greater than those coming from the dysphonia itself in laser surgery patients.\nFew are the patients that have a so-called \u201cnormal\u201d voice upon completing radiotherapy, above all taking into account that the larynx of these patients has undergone a surgical procedure in order to get a biopsy, has been intensely exposed to tobacco smoke and belongs generally to aged patients. All these factors negatively affect voice quality [29]. Despite this, the results of our work reveal that radiotherapy treatment for T1 glottic carcinoma generates lower repercussions in the subjective perception of residual dysphonia, as much in functional as in emotional aspects, when compared with CO2 laser surgery. The scores obtained in the VHI were low in the three domains for both groups, from which we can infer a scarce impact on the quality of life for laser surgery and radiotherapy for this type of patient.\nConclusions\nBoth CO2 laser resection and external radiotherapy as treatment for T1 vocal carcinoma offer similar objective measurement results (acoustic and spectrographic analysis) and subjective measurement results (GRABS scoring). However, in our series, the self-evaluation of the quality of voice, quantified by the VHI, shows a lower impact for radiotherapy patients.","keyphrases":["laser","radiotherapy","voice handicap index","glottic carcinoma","cordectomy"],"prmu":["P","P","P","P","P"]}
{"id":"Psychopharmacologia-3-1-1820758","title":"Dopamine efflux in the nucleus accumbens during within-session extinction, outcome-dependent, and habit-based instrumental responding for food reward\n","text":"Rationale Dopamine (DA) activity in the nucleus accumbens (NAc) is related to the general motivational effects of rewarding stimuli. Dickinson and colleagues have shown that initial acquisition of instrumental responding reflects action\u2013outcome relationships based on instrumental incentive learning, which establishes the value of an outcome. Given that the sensitivity of responding to outcome devaluation is not affected by NAc lesions, it is unlikely that incentive learning during the action\u2013outcome phase is mediated by DA activity in the NAc.\nIntroduction\nDespite more than 25\u00a0years of intensive research there is still a vigorous and ongoing debate concerning the specific aspects of complex and adaptive behaviors that are mediated by brain dopamine (DA) function (Ikemoto and Panksepp 1999; Redgrave et al. 1999; Salamone and Correa 2002; Schultz 2002; Berridge and Robinson 2003; Joseph et al. 2003; Wise 2004; Kelley et al. 2005; Salamone et al. 2005; Young et al. 2005). Much of the attention is focused on the nucleus accumbens (NAc) and a consensus has formed around the theory that DA innervation of this structure plays a key role in incentive motivation, a Pavlovian conditioned appetitive state that can influence the vigor of approach behavior (Fibiger and Phillips 1986; Robbins et al. 1989; Berridge and Robinson 1998; Cardinal et al. 2002; Parkinson et al. 2002). Consistent with the importance of conditional stimuli (CS+) in the initiation of approach behavior, visual and olfactory stimuli associated with natural rewards such as food or sexually receptive conspecifics can evoke significant increases in DA efflux in the NAc that precede similar changes observed during consummatory behavior (Fiorino et al. 1997; Ahn and Phillips 1999, 2002). Depletion or antagonism of DA function in the NAc eliminates or diminishes the influence of Pavlovian CSs on the level of instrumental responding, i.e., Pavlovian to instrumental transfer (Cardinal et al. 2002; Parkinson et al. 2002), and additionally, shifts response choice from that with higher work demand and a food reward of greater value to that with lower work cost and lower reward value (Salamone et al. 1994; Salamone and Correa 2002).\nContemporary views of instrumental conditioning propose that performance of an instrumental task is controlled by two distinct processes. Based on carefully designed experiments, Dickinson et al. (1995) have demonstrated that during initial learning trials (i.e., after exposure to 120 outcomes), an animal\u2019s performance is based on the knowledge (or expectation) that an instrumental action will lead to a specific biologically significant outcome. After many more trials (i.e., after exposure to 360 outcomes), responses gradually shift from being outcome-dependent to habit-based. It is only in the early action\u2013outcome controlled stage that instrumental performance is sensitive to manipulations that alter the incentive value of the outcome, underscoring the remarkable ability of rats to acquire and encode information relating current incentive value to an action\u2013outcome contingency in as little as 120 outcome trials. Using outcome devaluation and Pavlovian to instrumental transfer tests, lesions of the basolateral amygdala have been reported to impair the capacity of rats to encode the relation between a specific action and the value of an outcome (Corbit and Balleine 2005).\nGiven the generally accepted role of DA in the NAc in Pavlovian-based incentive motivation, the question arises as to whether DA activity in this region is involved in action\u2013outcome and\/or habit-based based stages of instrumental responding. A role for either the core or shell region of the NAc in the formation of action\u2013outcome associations has not been confirmed (Balleine and Killcross 1994; Corbit et al. 2001; de Borchgrave et al. 2002), but deficits in the acquisition of instrumental responding have been reported after blockade of the NMDA class of glutamate receptors in the NAc core (Kelley et al. 1997). In a recent study, Yin et al. (2006) examined DA function during early action\u2013outcome as distinct from later habit-based stages of instrumental responding. Mice, with a knockdown of the DA transporter and chronically elevated levels of DA, showed no deficits in acquisition of an instrumental task and responding early in training was still sensitive to tests of outcome devaluation (Yin et al. 2006). However, in rats with bilateral 6-OHDA lesions of the nigrostriatal DA system, responding on instrumental tasks remained sensitive to reward devaluation despite extensive training sessions (Faure et al. 2005). Thus, the nigrostriatal DA system, and more specifically, its innervation of the posterior lateral striatum, appears to be necessary for transition of instrumental conditioning from an action\u2013outcome stage to a habit-based stage.\nImportant insights into the role of DA transmission at different stages of instrumental behavior may be gained by examining in vivo changes in DA levels in major areas of DA innervation. Therefore, we conducted microdialysis experiments in the NAc and mediodorsal (MD) striatum at early (5th day) and later (16th day) stages of instrumental learning employing a protocol used successfully to demonstrate that responding after 120 but not 360 reinforced responses is sensitive to outcome devaluation and is therefore action\u2013outcome as distinct from habit-based (Dickinson et al. 1995). Given the finding that quinolinic acid or NMDA-induced cytotoxic lesions of the NAc failed to alter suppression of instrumental responding after outcome devaluation (de Borchgrave et al. 2002), it is unlikely that activity in the NAc is involved in instrumental incentive learning. Rather, the finding that lesions restricted only to the NAc completely abolished Pavlovian to instrumental transfer is consistent with the involvement of DA function in this nucleus in conditioned appetitive states. Therefore, we hypothesize that DA efflux in the NAc may be comparable during both early action\u2013outcome and later habit-based stages of instrumental responding. We also examined changes in DA levels in the MD striatum, as a control site for generalized activity; hence, we predicted no significant changes in DA efflux associated with instrumental responding. The design of this study also incorporated a \u201cwithin-trial\u201d extinction phase to test the hypothesis that DA efflux in the NAc but not MD striatum would be increased significantly by presentation of a Pavlovian CS+ paired previously with food pellets during instrumental responding in extinction. The relationship between response rate and magnitude of DA efflux in either NAc or MD striatum was also of interest.\nMaterials and methods\nSurgery\nLong\u2013Evans male rats (Charles River, Canada) weighing 280\u2013310\u00a0g were implanted bilaterally with stainless steel guide cannulae (19 gauge, 15\u00a0mm) under anesthesia induced by xylazine (7\u00a0mg\/kg) and ketamine hydrochloride (100\u00a0mg\/kg) delivered intraperitoneally. Cannulae were implanted 1\u00a0mm below dura immediately above the NAc [in millimeter: +1.7 anteroposteriorly (AP) and \u00b11.1 mediolaterally (ML) from bregma] or MD striatum (+1.2 AP, 2.5 ML) and secured with dental acrylic and four stainless steel screws. Stylets maintained patency of the cannulae until probe implantation. An additional sham-cannula was embedded within the back half of the acrylic head cap for purposes of habituation (see Behavioral apparatus and training).\nImmediately after surgery, rats were moved to a reverse light-cycle (lights on 7 a.m.to 7 p.m.) colony room maintained at 20\u00b0C and housed individually in plastic bins lined with corncob bedding. Novel objects (e.g., egg cartons and paper rolls) were placed weekly in the cages to promote exploratory and play behavior. Rats were handled and weighed by the experimenter on a daily basis. Four days after surgery, rats were placed on a food restriction schedule for the duration of the experiment, maintaining their body weight at \u223c85% of their free-feeding weight. The daily ration of food (20\u201325\u00a0g Purina Rat Chow) was given to the rats in their home cages following each day\u2019s operant responding session. Water was available at all times except during the operant training and testing sessions.\nBehavioral apparatus and training\nTraining and experiment sessions were conducted in a Plexiglas chamber (30\u00d723\u00d723\u00a0cm) with wire-mesh flooring. It was equipped with a retractable lever, a dispenser that delivered 45\u00a0mg Noyes pellets, and a light source (1\u00a0W, 12\u00a0V) located on the wall opposite the location of the lever. Pellets were dispensed into a photocell-equipped magazine located just left of the lever. The chamber was enclosed in a sound-attenuated ventilated box (Colbourn Instruments; Allentown, PA, USA) which had a small hole in the ceiling to allow passage of dialysis lines.\nTraining began 8\u00a0days into the food-deprivation schedule. Before a rat was placed in a testing chamber, a stainless steel coil was attached to the sham-cannula. This allowed the rat to habituate to the weight and feel of being tethered to the stainless steel coil which normally sheathes microdialysis lines during experiments. Each session began with rats being placed in operant chambers under baseline conditions (coil attached, lever retracted, and light off), the length of which was varied from day to day. Illumination of the light and insertion of the lever signaled the availability of pellets contingent on lever presses. A computer was used to control the equipment and record the number of lever presses and nose poke entries into the magazine.\nAt the start of the study, rats were trained to approach the magazine to retrieve 30 pellets delivered on a random-time 60-s schedule. Instrumental training sessions began the next day (day\u00a01) on a RI 2-s schedule of pellet delivery. The schedule was changed to RI 15-s on day\u00a02 and then to RI 30-s on day\u00a03, and remained so for the remainder of the training and microdialysis sessions. Each instrumental training session was terminated when rats had responded for 30 pellets, except on days\u00a04 and 15 when rats were allowed to press for 60 pellets (see Fig.\u00a02). To minimize the effect of stress before microdialysis tests, rats remained overnight in the chambers under baseline conditions with water, after the training session on day\u00a02.\nMicrodialysis probes and high pressure liquid chromatography system\nMicrodialysis probes were constructed as previously described (Ahn and Phillips 1999; Fiorino et al. 1997). They were concentric in design with a 2-mm semipermeable membrane (340\u00a0\u03bcm outer diameter, 65\u00a0kD MW cut-off, Filtral 12, Hospal; Neurnberg, Germany) with PE50 inlet and silica outlet tubing. A probe assembled in this manner typically had, at 21\u00b0C, in vitro recoveries of \u223c22% DA. Once a probe was implanted in the brain through the guide cannula (see \u201cMicrodialysis experiments\u201d section below for implant coordinates), a cylindrical brass collar secured the probe in place. The inlet tubing was connected to a liquid swivel (Instech 375s; Plymouth Meeting, PA, USA) which was mounted on top of the Colbourn box. Both inlet and outlet tubing were encased in a protective stainless steel coil which extended from the liquid swivel to the brass collar. A 2.5-ml gas-tight syringe (Unimetrics) and syringe pump (Model 22, Harvard Apparatus; South Natick, MA, USA) were used to perfuse a modified Ringer\u2019s solution (in millimolar: 10 sodium phosphate buffer, 1.3 CaCl2, 3.0 KCl, 1.0 MgCl2, 147 NaCl, pH\u00a07.4) through the probe at 1\u00a0\u03bcl\/min.\nDA content in microdialysis samples were separated by high pressure liquid chromatography (HPLC) and quantified by electrochemical detection. The HPLC system consisted of, in sequence of flow, a Bio-Rad pump (Hercules, CA, USA), an SSI pulse damper (State College, PA, USA) a Valco Instruments two-position auto-Injector (EC10W; Houston, TX, USA), a Beckman reverse-phase column (Ultrasphere, ODS 5\u00a0\u03bcm, 15\u00a0cm, 4.6\u00a0mm internal diameter; Fullerton, CA, USA), an ESA guard cell (Model 5020; Chemlsford, MA, USA), an ESA analytical cell (Model 5011), an ESA Coulochem II Electrochemical detector, and a dual channel chart recorder. The electrochemical detection parameters were: +450\u00a0mV for the oxidation channel, \u2212300\u00a0mV for the reduction channel, and \u2212450\u00a0mV for the guard cell output. The mobile phase (in millimolar: 83 sodium acetate buffer, 27 EDTA, and 1.30 sodium octyl sulfate at pH\u00a03.5, 10% methanol) flowed through the system at 1\u00a0ml\/min.\nMicrodialysis experiments\nIn all experiments, each rat was tested with microdialysis on days\u00a05 and 16. The side of probe implantation (i.e., left or right brain structure) was counterbalanced for each experiment day. The two microdialysis sessions were conducted using an identical protocol. The day before an experiment, rats were implanted unilaterally with probes in the NAc (exposed membrane spanned \u22126.0 to \u22128.0\u00a0mm DV from dura) or MD striatum (\u22124.0 to \u22126.0\u00a0mm DV from dura) after the day\u2019s training session and kept overnight (\u223c16\u201318\u00a0h) in the test chamber with water under baseline conditions. The next morning, microdialysis samples were collected at 10-min intervals (10\u00a0\u03bcl volume) and analyzed immediately with HPLC. Baseline conditions were continued until four consecutive samples showed stable DA levels (i.e., <10% fluctuation between samples; times 1\u20134).\nIn \u201cExperiment 1\u201d, the baseline period was followed by the concurrent illumination of the chamber and insertion of the lever. Rats were then allowed a 30-min period (times 5\u20137) during which they could lever press for food on a RI 30-s schedule. The session concluded with the retraction of the lever and a 40-min time-out period with the lights off (times 8\u201311). In \u201cExperiment 2\u201d, a 30-min extinction phase was incorporated (times 5\u20137) before a food reward phase. All aspects of the extinction phase were identical to previous rewarded training sessions except that lever presses did not lead to food delivery. A 20-min time-out period under baseline conditions (times 8\u20139) preceded a 10-min priming period when the light was on and five pellets were delivered randomly and noncontingently (time 10). The start of a food-rewarded phase (times 11\u201313) was marked by the insertion of the lever into the already illuminated chamber. During this period, rats were allowed to lever press for pellets on a RI 30-s schedule for 30\u00a0min. An experiment concluded with an additional 40-min postsession baseline period (times 14\u201317).\nHistology\nRats were deeply anesthetized with chloral hydrate and intracardially perfused first with 0.9% NaCl and then phosphate-buffered formalin (3.7% formaldehyde). The removed brains were stored in 15% (w\/v) sucrose in formalin for at least 24\u00a0h before being prepared as 50\u00a0\u03bcm coronal sections on 2% gel-coated slides. Cresyl violet staining was used to help verify placement of probe tracts. Only data from those rats with tracts in the shell\/core region of the NAc (16 of 18 rats) and MD striatum (six of seven rats) of both hemispheres were included in the statistical analyses.\nData analyses\nNeurochemical data were transformed into percentage of change from baseline (i.e., 0% representing the average concentration of the three samples preceding the final 4th baseline sample). Neurochemical data were analyzed using either a one-way (time) or two-way (day \u00d7 time) repeated measures ANOVA followed by the Dunnett method of multiple comparisons, using the final baseline sample (time 4) as the control sample. The Huynh\u2013Feldt correction for nonsphericity was applied to the degrees of freedom for all within-subject analyses. Comparisons between two means were assessed using paired t tests. A coefficient of determination (R2) was computed based on a linear regression of a scatter plot between lever presses\/10\u00a0min (y-axis) and corresponding percent changes in DA efflux (x-axis), for data obtained on test days\u00a05 and 16. Statistical analyses were performed using Systat or SPSS statistical packages.\nResults\nExperiment 1\nChanges in extracellular DA levels in the NAc were compared after limited and extended training sessions, while rats lever pressed for food reward on a RI 30-s schedule. As shown in Fig.\u00a01, the rates of responding approximately doubled from days\u00a05 to 16, even though rats obtained and consumed a similar number of rewards during the two sessions [43.3 pellets (\u223c2.0\u00a0g) on day\u00a05 and \u223c46.2 pellets (\u223c2.1\u00a0g) on day\u00a016]. The number of magazine entries also did not differ between test days. Separate one-way ANOVAs revealed a significant main effect of time on DA efflux on day\u00a05 (F7,49=12.038, p<0.001) and day\u00a016 (F7,49=5.325, p<0.044). Further analyses indicated that on both days, there was a significant increase in DA efflux above their respective baselines that remained elevated for the 30-min duration of instrumental responding for food pellets (Dunnett\u2019s, p<0.05). Despite the doubling of response rates from days\u00a05 to 16, the pattern and magnitudes of DA efflux on the two test days were not statistically different (maximal increase of 69\u00b116% on day\u00a05 and 71\u00b128% on day\u00a016), as a two-way repeated measures ANOVA failed to show a significant interaction of day \u00d7 time on DA efflux (F7,98=0.598, p=0.528). However, a paired t test showed that during the first 10\u00a0min (time 5), DA efflux was significantly higher on day\u00a016 than day\u00a05 (68\u00b124 vs 30\u00b112%, respectively; p<0.05). Basal values of DA in the NAc (uncorrected for recovery) were 2.72\u00b10.61 and 2.19\u00b10.29\u00a0nM for days\u00a05 and 16, respectively, and were not statistically different from each other.\nFig.\u00a01Change in DA efflux in the NAc (line graph) during instrumental conditioning on day\u00a05 (left panel) and day\u00a016 (right panel) in the same group of rats (n=8). Dotted lines highlight the Reward phase in which food pellets were delivered on a RI 30-s schedule (times 5\u20137). Lever presses (gray bars) and magazine entries (black bars) are shown per 10\u00a0min bin. Data are represented as mean\u00b1SEM. * indicates significant difference from the final baseline value (time 4) according to Dunnett\u2019s method of multiple comparisons, p<0.05. \u2020 indicates significant difference from the corresponding data point on day\u00a05 according to a paired t test, p<0.05\nExperiment 2\nInstrumental behavior As shown in Fig.\u00a02, all rats in the NAc and MD striatal microdialysis groups learned to lever press for food pellets on a RI 30-s schedule and to retrieve the pellets, as indicated by the number of magazine entries. Training data for one rat in the MD striatal group was lost and not included in the following analyses. Instrumental behavior became more efficient through the initial days of training, and during the third and fourth training sessions, rats made significantly more lever presses than magazine entries. This increase in ratio of lever presses to magazine entries, from limited to extended training experience, may be explained by proposing that rats learned to use the auditory click made by the dispenser with the delivery of a pellet. Thus, rats learned to enter the magazine only when they heard the click. On days (4 and 15) before microdialysis tests, rats were allowed to press for 60 pellets, rather than the normal 30 pellets available on other days, and accordingly increased the rate of lever presses on these days. The mean number of lever press responses per training session across the RI 30-s schedule tended to be higher in the NAc group (from 327 to 835) than in the MD striatal group (from 367 to 436). However, an ANOVA test revealed no main effect of group (F1,11=2.414, p=0.149). ANOVA of magazine entry data similarly indicated that counts were comparable between the NAc and MD striatal groups (F1,11=0.366, p=0.557).\nFig.\u00a02Instrumental performance of rats in the NAc or MD striatal group over 16 training sessions. Rats were trained to lever press for food pellets on a RI 2-s schedule the first day, RI 5-s the next day, and then RI 30-s for the remainder of the study. Each training session terminated upon delivery of 30 pellets (exception: *60 pellets delivered on days before test). Test days (day\u00a05 and 16) were composed of two phases, Extinction and Reward, which lasted 30\u00a0min each. Lever presses (gray and striped bars) and magazine entries (black bars) are shown per training session. Data are represented as mean+ SEM\nOn test days, rats were tested for instrumental responding in extinction and then with food reward. During the extinction phase, the pellet dispenser was disconnected from the magazine, but still produced an auditory click according to the RI 30-s schedule. During the rewarded phase, all rats consumed every pellet delivered during the lever press sessions. During the rewarded component of the experiment, rats consumed \u223c45.7 pellets (\u223c2.1\u00a0g) on day\u00a05 and \u223c46.2 pellets (\u223c2.1\u00a0g) on day\u00a016. The similarity in food consumption and difference in magnitude of DA efflux (see below), across days\u00a05 and 16, again supports the view that DA response is not a function of food reward. In both the NAc and MD striatal groups (Figs.\u00a03 and 4), rate of instrumental performance approximately doubled from day\u00a05 to day\u00a016, during both the extinction and rewarded phases of the sessions. Over the 30-min extinction phase, rats displayed a typical decline in rate of lever presses, whereas over the 30-min reward phase, there was a slight increase in rate of responding. In the NAc group, the number of lever presses was significantly higher on day\u00a016 than on day\u00a05 during the first 10\u00a0min of the extinction (t test, p=0.003) and rewarded (t test, p=0.002) phases. In the MD striatal group, the lever press rates during the first 10\u00a0min of the extinction phase were comparable on days\u00a05 and 16, but differed significantly between the 2\u00a0days during the first 10\u00a0min of the rewarded phase (p=0.038). In both the NAc and MD striatal groups, an ANOVA indicated that the number of magazine entries did not differ significantly between days\u00a05 and 16.\nFig.\u00a03Change in DA efflux in the NAc (line graph) during instrumental conditioning on day\u00a05 (left panel) and day\u00a016 (right panel) in the same group of rats (n=8). Dotted lines highlight the Extinction phase during which an auditory CS+ was activated on an RI 30-s schedule in the absence of food pellet rewards (times 5\u20137) and a Reward phase in which food pellets were delivered on a RI 30-s schedule (times 11\u201313). P represents the period during which five priming pellets were delivered noncontingently. Lever presses (gray bars) and magazine entries (black bars) are shown per 10\u00a0min bin. Data are represented as mean\u00b1SEM. * Indicates significant difference from the final baseline value (time 4) according to Dunnett\u2019s method of multiple comparisons, p<0.05. \u2020 Indicates significant difference from the corresponding data point on day\u00a05 according to a paired ttest, p<0.05. \u00a7 Indicates significant difference from the final baseline value (time 4; ttest, p<0.05)Fig.\u00a04Change in DA efflux in the MD striatum (line graph) during instrumental conditioning on day\u00a05 (left panel) and day\u00a016 (right panel) in the same group of rats (n=6). See Fig.\u00a03 for explanation of symbols. No statistically significant results were observed for neurochemical data set. \u2020 Indicates significant difference from the corresponding behavioral score on day\u00a05 according to a paired ttest, p<0.05\nDA efflux Basal values of DA in the NAc (uncorrected for recovery) were 2.58\u00b10.32 and 2.16\u00b10.16\u00a0nM for days\u00a05 and 16, respectively, and were not statistically different from each other. Basal values of DA in the MD striatum (uncorrected for recovery) were 2.71\u00b10.23 and 2.95\u00b10.34\u00a0nM for days\u00a05 and 16, respectively, and also did not differ statistically.\nIn the NAc, the overall pattern of DA efflux across the different phases of the microdialysis test on day\u00a05 appeared comparable to that observed on day\u00a016 (Fig.\u00a03), but statistical analyses revealed several key differences. Accordingly, an ANOVA identified a significant day \u00d7 time interaction on DA efflux (F13,182=2.760, p=0.022), with a significant simple main effect of Time on day\u00a05 (F13,91=8.690, p<0.001) and day\u00a016 (F13,91=14.649, p<0.001). On both days, there was an increase in NAc DA levels during the initial 10\u00a0min of responding in extinction (10\u00b17% above baseline on day\u00a05 and 19\u00b16% on day\u00a016), but this increase was only significant after extended training on day\u00a016 (paired samples t test, p=0.005). DA efflux then returned to baseline for the remainder of the extinction phase and time-out period. During the 10-min period preceding the rewarded responding phase, five pellets were noncontingently dispensed into the magazine; the purpose of this was to prime the rats to lever press again for food reward. During this period, DA levels did not differ from baseline values on day\u00a05 (4\u00b19%) but by day\u00a016, were increased significantly above baseline (27\u00b17%; Dunnett\u2019s test, p<0.05). The reward phase of the session was accompanied by significant elevation of DA efflux on both days (maximal increase of 37\u00b112% on day\u00a05 and +83\u00b115% on day\u00a016; Dunnett\u2019s test, p<0.05) that remained elevated after retraction of the lever and cessation of instrumental responding. DA levels then gradually declined towards baseline values over the remaining 60\u00a0min of the test session. During the initial 10\u00a0min of the reward phase (time 11), the magnitude of change on day\u00a016 (83\u00b115%) was significantly greater than the change in efflux observed on day\u00a05 (37\u00b112%; t test, p=0.013).\nIn the MD striatum, there were no statistically significant changes in DA levels during the entire instrumental responding session on both days\u00a05 and 16 (Fig.\u00a04). Despite performing lever presses and magazine entries at rates comparable to the NAc group, DA levels in this group showed only slight fluctuations around baseline.\nCorrelation between response rate and DA efflux\nBased on a linear regression of a scatter plot between lever presses\/10\u00a0min and percent change in DA efflux, R2 values of 0.0014 for data from day\u00a05 and 0.0065 on day\u00a016 indicated that <1% of the variation in lever presses could be explained by a linear correlation between lever presses and DA levels, on both test days (Fig.\u00a05).\nFig.\u00a05Correlation between instrumental response rates and NAc DA efflux. Scatter plot of lever presses\/10\u00a0min (y-axis) and corresponding percent change in DA efflux (x-axis) for data obtained on day\u00a05 (left panel) and day\u00a06 (right panel). Shown on each graph is the best fit linear regression line and R2 value\nHistology\nThe locations of all microdialysis probes are presented in Fig.\u00a06. The 2-mm lengths of the dialysis membrane were in the NAc (shell-core boundary) or medial aspect of the MD striatum (just dorsal and lateral of the anterior commissure).\nFig.\u00a06Location of microdialysis probes in the NAc and MD striatum. Black bars represent 2\u00a0mm length of dialysis membranes. Numbers beside each plate correspond to millimeter from bregma. Coronal drawings were modified from Paxinos and Watson (1997)\nDiscussion\nThe present study examined the role of DA activity in the NAc in behaviors maintained by instrumental and Pavlovian incentive learning. DA efflux in the NAc was increased significantly during both early and later training stages of an instrumental response for food on a RI-30-s schedule of reinforcement (Figs.\u00a01 and 3). It is important to note that this pattern of results was observed whether a period of extinction preceded a 30-min period of instrumental responding reinforced by food pellets. As such, the present findings confirmed previous reports of increased DA release in the NAc during lever pressing for food, employing fixed interval or ratio schedules of reinforcement (Salamone et al. 1994; Richardson and Gratton 1996; Cousins et al. 1999). Response actions during the early phase of training on interval schedules (i.e., in rats having received as few as 120 outcomes) have been characterized as goal- or outcome-directed (Adams and Dickinson 1981; Balleine and Dickinson 1992; Dickinson et al. 1995) and accordingly may represent instrumental incentives, as distinct from Pavlovian incentive processes related to incentive motivation (Parkinson et al. 2002). Continued performance of these instrumental actions leads to habitual responding which, unlike action\u2013outcome learning, is impervious to outcome devaluation or contingency degradation (Dickinson et al. 1995). We failed to observe a selective increase in DA efflux when rats had limited as compared to extended training experience, as might be expected if dopaminergic activity in the NAc is related to instrumental incentive learning. Indeed, the magnitude of DA efflux was significantly greater after extended training when behavior is said to be no longer controlled by incentive learning and is based instead on habit. These increases in DA efflux were site-specific, as no significant changes in medial MD striatal DA efflux were observed throughout the different phases of this experiment (Fig.\u00a04).\nPerformance on instrumental tasks is often conducted under nonrewarded or extinction conditions to evaluate the control of behavior by Pavlovian incentive stimuli, unconfounded by unconditioned reward stimuli. In the present study, a significant increase in DA efflux in the NAc was observed only during the initial 10-min sample of responding in extinction on training day\u00a016, but not on day\u00a05 (Fig.\u00a03). The specific CS+ present in this experiment was a distinct auditory \u201cclick\u201d of the pellet dispenser which occurred on a RI 30-s schedule, by itself during extinction or accompanied by delivery of food pellets into the magazine during rewarded responding. As such, these data are consistent with previous reports of increased DA efflux in the NAc elicited by a CS+ (Phillips et al. 1993; Datla et al. 2002). In a Pavlovian to instrumental transfer protocol, a CS+ previously paired noncontingently to food reward can facilitate the acquisition of an instrumental response (Parkinson et al. 2002). Systemic administration of DA receptor antagonists during Pavlovian pairings of a CS+ with food reward blocks Pavlovian to instrumental transfer (Beninger and Phillips 1981; Dickinson et al. 2000). Damage to the shell or core of the NAc spares the acquisition of Pavlovian to instrumental transfer, but disrupts the potentiation by intra-NAc amphetamine on responding for the CS+ (Parkinson et al. 2002). Together, these findings suggest that a phasic increase of DA in the NAc, shown to occur after treatment with amphetamine (Taepavarapruk and Phillips 2003; Brebner et al. 2005), may mediate the facilitatory effects of a Pavlovian CS+ on instrumental responding.\nIt is also of interest to note that the inclusion of an extinction session before a reinforced phase of instrumental responding attenuated the magnitude of DA efflux in the NAc observed when food reward was available on day\u00a05, but not on day\u00a016. This finding may be attributed to attenuation in the secondary reinforcement property of the CS+ associated with the delivery of food reward or possibly, an influence of frustrative nonreward engendered by extinction. In either case, it is apparent that these effects of extinction are restricted to the early phase of instrumental training.\nIn earlier studies, Salamone et al. (1994) proposed that an important aspect of dopaminergic activity in the NAc is related to behavioral activation, exertion of effort, and possibly cost benefit analyses relating effort to value of reward stimuli (Salamone et al. 2003, 2005). In support of this hypothesis, consumption of large quantities of freely available food pellets or lab chow was not accompanied by increased DA efflux. It must be noted in passing that consumption to satiety of a large meal of a palatable food such as fruit loops, onion rings (Ahn and Phillips 1999), or sucrose (Hajnal and Norgren 2002), is accompanied by a significant increase in DA efflux in both the NAc and medial prefrontal cortex. Salamone et al. (1994) also observed a significant relationship between response rates of individual rats and the magnitude of DA efflux in the NAc. Specifically, an increase in DA release was only observed in rats that responded at medium to high rates of responding, whereas in rats with low response output, this measure did not differ from controls.\nOur data are also relevant to the relationship between response output and DA activity. In both Experiments 1 and 2, there was no evidence of a simple relationship between response rates and magnitude of DA efflux. As shown in Fig.\u00a01, although the rate of lever presses were twice as high on day\u00a016 compared to day\u00a05, the magnitude of DA efflux did not different significantly between the 2\u00a0days. In Fig.\u00a03, on day\u00a05, initial response rates during the extinction and reward phases of the test were comparable during the first 10\u00a0min (times 5 and 11), yet the corresponding magnitude of DA efflux during the reward phase was three times greater than the extinction phase. A similar pattern was observed on day\u00a016. Thus, different rates of responding were associated with similar magnitudes of DA efflux, and similar rates of responding were associated with different magnitudes of DA activity. Accordingly, no positive correlations between rate of lever press responding and magnitude of DA efflux in the Nac were observed after limited and extended training (Fig.\u00a05). Finally, with respect to the appealing hypothesis that dopaminergic activity in the NAc is related to behavioral activation (Salamone et al. 2003, 2005), it must be emphasized that although the present data challenge this hypothesis, it cannot be refuted simply on the basis of the lack of a correlation between magnitude of DA efflux and intensity or degree of behavioral activation.\nThe failure to observe a significant increase in DA efflux in the MD striatum during either action\u2013outcome or habit-based instrumental responding provides a clear indication that DA transmission in this region of the striatum is not involved in instrumental conditioning or stimulus\u2013response habit formation. These data are consistent with the finding that neither excitotoxic lesions nor reversible inactivation of the anterior MD striatum had any effect on acquisition or expression of action\u2013outcome associations in instrumental conditioning (Yin et al. 2005). In contrast, lesions or inactivation of the MD striatum, posterior to the probe placements in the present study, impaired instrumental performance based on outcome\u2013expectancy (Yin et al. 2004). Blockade of NMDA receptors in the dorsomedial striatum also disrupted action\u2013outcome learning consistent with a role for glutamate-mediated synaptic plasticity in the encoding of action\u2013outcome associations (Yin et al. 2005). The MD (\u201cassociative\u201d) striatum receives inputs from association cortices (e.g., prelimbic region of the prefrontal cortex and premotor areas), as well as the basolateral amygdala, which appears to mediate the assignment of incentive value to the consequences of instrumental actions (Corbit and Balleine 2005).\nIntegrity of the dorsolateral striatum has been shown to be required for habit formation in instrumental learning, and furthermore, rats with damage to this region of the striatum reverted to a state in which instrumental actions were goal-directed (Yin et al. 2005). This finding implies that the system involving the dorsolateral striatum responsible for habit formation can inhibit the circuit that mediates action\u2013outcome or goal-directed instrumental actions. This in turn raises the possibility that the increase in NAc DA efflux observed in the present study after extended training provides a representation of instrumental incentive learning that is held in check by activity in the dorsolateral striatum.\nIn conclusion, the present findings provide neurochemical evidence in support of previous data questioning the role of DA in the NAc in coupling incentive value to representations of instrumental outcomes (de Borchgrave et al. 2002). The data showing elevated DA efflux in the NAc during extinction in the presence of a Pavlovian CS+, in turn are consistent with a role for the NAc in incentive motivation (Fibiger and Phillips 1986; Robbins et al. 1989; Phillips et al. 1993; Balleine and Killcross 1994; Berridge and Robinson 1998). Rats received an apportionment of \u223c60 food pellets during microdialysis test on days\u00a05 and 16, yet the magnitude of DA efflux was significantly greater after extended training sessions. These data refute the hypothesis that dopaminergic activity in the NAc is a reflection of either reward value or reinforcement of instrumental responses (Wise 2004). The present \u201cwithin subject\u201d design revealed a hitherto unappreciated effect of extended training of instrumental responding with an interval schedule on the magnitude of DA efflux in the NAc. For reasons discussed above, this does not appear to be related to motor responding per se. Rather, we speculate that this effect may reflect the specific condition of a random or variable interval schedule of outcome presentation, in which extended training is necessary to appreciate that the probability of receiving a beneficial outcome at any particular time in the 30-min test session is always unpredictable. This degree of uncertainty may be highly compatible with the optimal conditions for activating midbrain DA neurons (Fiorillo et al. 2003), which in turn would result in a sustained increase in DA release in the NAc throughout a period of random reinforcement. This pattern of DA release could play an important role in maintaining a high level of motivation at the service of a variety of response strategies available to ensure access to objects essential for survival.","keyphrases":["dopamine","nucleus accumbens","habit","food reward","action\u2013outcome","microdialysis","instrumental learning","uncertainty","mediodorsal striatum"],"prmu":["P","P","P","P","P","P","P","P","R"]}
{"id":"Mol_Immunol-1-5-1995235","title":"The mouse complement regulator CD59b is significantly expressed only in testis and plays roles in sperm acrosome activation and motility\n","text":"In mouse, genes encoding complement regulators CD55 and CD59 have been duplicated. The first described form of CD59, CD59a, is broadly distributed in mouse tissues, while the later identified CD59b was originally described as testis specific. Subsequent studies have been contradictory, some reporting widespread and abundant expression of CD59b. Resolution of the distribution patterns of the CD59 isoforms is important for interpretation of disease studies utilising CD59 knockout mice. Here we have performed a comprehensive distribution study of the CD59 isoforms at the mRNA and protein levels. These data confirm that expression of CD59b is essentially restricted to adult testis; trace expression in other tissues is a consequence of contamination with blood cells, shown previously to express CD59b at low level. In testis, onset of expression of CD59b coincided with puberty and was predominant on the spermatozoal acrosome. Ligation of CD59b, but not CD59a, markedly reduced spermatozoal motility, suggesting a specific role in reproductive function.\n1\nIntroduction\nCD59, a broadly distributed glycosyl phosphatidylinositol (GPI)-anchored protein, is the principal regulator of complement membrane attack complex (MAC) assembly on cell membranes (Meri et al., 1990). A decade ago, we identified the mouse analogue of CD59 and showed that it was broadly distributed on cells and tissues (Powell et al., 1997). Deletion of the gene encoding mouse CD59 caused minimal disturbance in unchallenged animals but markedly enhanced susceptibility to complement-driven pathologies (Holt et al., 2001; Turnberg et al., 2003, 2004; Lin et al., 2004; Mead et al., 2004; Williams et al., 2004). Others demonstrated that the gene encoding CD59 is duplicated in the mouse; the protein products of these two genes were then termed CD59a for the first described, and CD59b for the product of the new gene (Qian et al., 2000). In this first report, it was stated that CD59b message was significantly expressed only in testis. We developed monoclonal antibodies specific for CD59a and CD59b, enabling us to confirm the broad distribution of CD59a and testis restricted expression of CD59b at the protein level; we concluded that CD59a was the principle regulator of MAC assembly in mouse tissues (Harris et al., 2003). This conclusion was challenged in a re-analysis of the pattern of distribution of mRNA encoding the two forms of CD59; these studies suggested that both CD59a and CD59b were broadly expressed in the mouse and contended that the latter was the major regulator of MAC in the mouse (Qin et al., 2003). These contentions were bolstered by the demonstration that deleting the gene encoding CD59b in mice caused a severe, spontaneous phenotype with anaemia, platelet dysfunction and fertility problems. In light of these data we reassessed the distribution patterns at protein and mRNA levels using a combination of specific monoclonal antibodies and shared primers for PCR in high sensitivity assays (Baalasubramanian et al., 2004). These analyses confirmed our earlier work showing that CD59a was broadly expressed while significant expression of CD59b was restricted to testis. Trace amounts of CD59b message and protein were detected in erythrocytes but quantitative assays of protein expression showed that CD59b expression on erythrocytes was less that 5% that of CD59a. Further, we confirmed that CD59a was dominant in protection of erythrocytes from MAC lysis. The findings of these painstaking studies have recently been challenged yet again (Qin et al., 2006). The appropriateness of the techniques used and specificity of probes and primers have all been questioned. These authors concluded that CD59b was broadly expressed and an important regulator of MAC assembly on erythrocytes and in tissues.\nWe, and others, are using CD59a knockout mice in disease models to explore roles of MAC based on our evidence that CD59a is the principal regulator of the MAC in most tissues. If CD59b is indeed widely distributed then the value of studies in CD59a knockouts is in question. It is therefore essential that we test the evidence. Here using multiple methods we revisit these published studies and undertake new analyses to explore the expression patterns of CD59a and CD59b at the mRNA and protein level. We conclude that expression of CD59b at the mRNA and protein level is essentially absent in all tissues other than testis. Low level expression on blood cells was confirmed and trace detection of mRNA in tissues was shown to be likely due to blood contamination. We further analysed expression of CD59b in testis and showed that expression coincided with onset of puberty and was restricted to spermatozoa and their immediate precursors. Ligation of CD59b on spermatozoa with monoclonal antibody markedly inhibited sperm motility, suggesting a specific role in reproductive function.\n2\nMaterials and methods\n2.1\nMice\nAdult (8\u201316 weeks) and infant (1\u20135 weeks; pre-puberty) male mice, and embryos (day 5, 10 and 15) from C57BL\/6(H-2b) background mice were used in our investigation. All experimental procedures were performed in compliance with Home Office and local ethics committee regulations. cd59a\u2212\/\u2212 mice generated as described previously (Holt et al., 2001), and back-crossed 10 generation onto the same background were used as controls.\n2.2\nAntibodies and reagents\nRat anti-mouse CD59a (CD59a.1; IgG1) was generated in house (Harris et al., 2003). Mouse anti-mouse CD59b (CD59b.2; IgG1) was also made and characterized in house (Baalasubramanian et al., 2004). Separate aliquots of CD59a.1 and CD59b.2 were labelled with NHS-biotin (Sigma\u2013Aldrich, Gillingham, Dorset, UK) and FITC-NHS (Perbio Science UK Ltd., Cramlington, UK) according to manufacturer's protocols. As negative controls, rat IgG1 and mouse IgG1 (purified in-house) were labelled in the same manner. FITC-labelled streptavidin was purchased from DAKO (Ely, Cambridgeshire, UK). HRPO-labeled donkey anti-rat IgG and HRPO-labeled donkey anti-mouse IgG were purchased from Jackson ImmunoResearch Europe (Newmarket, Suffolk, UK).\n2.3\nSemi-quantitative RT-PCR\nTotal RNA was purified from all investigated mouse tissues using GenElute kit (Sigma\u2013Aldrich) and controlled for DNA contamination by RT-PCR without using reverse transcriptase and employing a \u03b2-actin specific primer pair (Table 1). Only samples that did not show amplification were used in all analyses. Aliquots of these RNAs (1\u00a0\u03bcg each) were reverse transcribed using random hexamers and multiscribe reverse transcriptase according to the manufacturer's instructions (Applied Biosystems, Warrington, UK). Primer pairs specific for either CD59a or CD59b were as described by Qin et al. (2006), and a new primer pair common for both CD59a and b and amplifying a sequence in exon 4 comprising 199\u00a0bp for CD59a and 162\u00a0bp for CD59b, were used in the subsequent amplifying reactions for detection of CD59a and CD59b mRNAs (Table 1). Either 25 or 35 amplification cycles were used, except where attempting simultaneous detection of both mRNAs when 40 cycles were performed. Amplified products were separated either in 1% agarose or in 5% poly-acrylamide gels (PAAG). To obtain semi-quantitative data for expression of the two genes, we used as templates either 2\u00a0\u03bcl of the original cDNA, or a series of dilutions as indicated in figures. Gels were scanned and band intensity was measured by densitometry using Quantity One 4.3.0 software (BioRad, Hemel Hempstead, Hertfordshire, UK). The quantifications were carried out in triplicate and means and errors calculated.\n2.4\nQuantitative real-time PCR analysis\nThe cDNAs from testis and liver prepared as described for the semi-quantitative RT-PCR, were subject to a Taqman assay as described previously (Baalasubramanian et al., 2004). One of the primer pairs was as described previously (Baalasubramanian et al., 2004), a perfect match for the sequence of CD59a mRNA and with a single internal nucleotide mismatch to CD59b mRNA; the second set was a perfect match for the same sequence in CD59b and with a single internal nucleotide mismatch to CD59a (Table 1).\nTo measure the relative number of mRNA copies for CD59b in liver, perfused liver and testis we used the primer pair reported by Qin et al. (2006) to specifically amplify CD59b in a quantitative PCR (QPCR). Because these primers were not designed specifically for this assay, they did not meet the optimal annealing temperature requirements. To correct for this anomaly, the standard amplification programme was modified to include a pre-annealing step at 56\u00a0\u00b0C for 20\u00a0s prior to the extension phase.\nPrimers were designed to \u03b2-actin (Table 1) as an internal control for normalization of starting cDNA levels. Quantitative PCR was performed on ABI PRISM 7000 using either TaqMan Universal PCR Master Mix or SYBR Green PCR Master Mix according to the manufacturer's instructions (Applied Biosystems) with 50 cycles of amplification.\n2.5\nPreparation of tissue lysates\nTo obtain tissue lysates, freshly harvested mouse organs were immediately chilled and homogenized with ice-cold lysis buffer (PBS containing 2% NP40, 1\u00a0mM phenylmethylsulfonyl fluoride, 10\u00a0mM EDTA, 1\u00a0\u03bcg\/ml leupeptin, and 1\u00a0\u03bcg\/ml pepstatin; 1\u00a0g of tissue\/1.5\u00a0ml of buffer) and incubated for 60\u00a0min on ice. Insoluble debris was removed by centrifugation (5000\u00a0\u00d7\u00a0g, 15\u00a0min at 4\u00a0\u00b0C) and the supernatants stored in aliquots at \u221280\u00a0\u00b0C until use. In some experiments the mouse was perfused with saline at the time of sacrifice to reduce blood contamination in harvested organs.\n2.6\nSDS-PAGE and Western blot analysis\nLysates were mixed 1:1 with sample buffer for SDS-PAGE and separated under non-reducing conditions in 15% gels. Separated proteins were transferred onto nitrocellulose membranes (Schleicher & Schuell, London, UK), and membranes blocked with 5% (w\/v) non-fat milk in PBS (PBS-M). Membranes were then probed with the primary mAb diluted in PBS-M, washed in PBS containing 0.1% Tween-20 (PBS-T), then probed with HRPO-conjugated donkey anti-rat Ig in PBS-M to detect the rat anti-CD59a or HRPO-conjugated donkey anti-mouse Ig (Jackson) to detect the mouse anti-CD59b. After further washing in PBS-T, bands were developed using ECL (Perbio Science UK Ltd.) and captured on autoradiographic film (Kodak Ltd., Hemel Hempstead, Hertfordshire, UK).\n2.7\nSpermatozoa preparation and analysis\nMotile cauda epididymal spermatozoa were obtained by a \u2018swim-up\u2019 technique as previously described (Mizuno et al., 2004, 2005). Briefly, two cauda epididymi from an adult C57BL\/6 mouse were roughly minced in 1\u00a0ml of DMEM (Invitrogen, Paisley, UK). This suspension was carefully overlayered with 1\u00a0ml DMEM and incubated at room temperature for 15\u00a0min. The upper layer was removed and spermatozoa pelleted by centrifugation (1000\u00a0\u00d7\u00a0g) for 5\u00a0min at room temperature. Cells were washed twice by gentle centrifugation as above. For immunofluorescence studies, the re-suspended cells were smeared onto glass slides, air-dried immediately, fixed in acetone at room temperature for 1\u00a0min and stored at \u221220\u00a0\u00b0C until use. To prepare lysate, the swim-up spermatozoa from 4 cauda epididymi were pelleted and incubated with mixing in 100\u00a0\u03bcl of lysis buffer for 30\u00a0min on ice. Insoluble debris was removed by centrifugation (15,000\u00a0\u00d7\u00a0g, 15\u00a0min at 4\u00a0\u00b0C) and the supernatant stored in aliquots at \u221280\u00a0\u00b0C.\nTo compare CD59b expression in unactivated and acrosome-reacted spermatozoa, the acrosome reaction was induced essentially as described (Mizuno et al., 2004). Briefly, swim-up spermatozoa (106\u00a0cells\/ml of DMEM) were incubated for 1\u00a0h at 37\u00a0\u00b0C with the calcium ionophore A23187 (Sigma\u2013Aldrich) at 1\u00a0\u03bcM to induce the acrosome reaction. Control cells were incubated without ionophore. Acrosome-reacted and control spermatozoa were smeared on glass slides and immediately air-dried. To observe CD59b distribution, the smears were incubated with FITC-labeled CD59b.2. Nuclei were counterstained with DAPI (4\u2032-6-diamino-2-phenylindole-2 HCl; 100\u00a0ng\/ml final concentration; Sigma\u2013Aldrich). On each slide, at least 100 cells were counted and the assay was carried out in triplicate.\n2.8\nFunctional inhibition assay of CD59a and CD59b in mouse sperm\nSpermatozoa harvested by swim-up from two epididymi, either from wild type or cd59a\u2212\/\u2212 mice, were washed and suspended in 2\u00a0ml DMEM. Paired aliquots (200\u00a0\u03bcl) were incubated at 37\u00a0\u00b0C with mAbs CD59a.1, CD59b.2, or isotype-matched control mAb, each at 10\u00a0\u03bcg\/ml. After 4\u00a0h of incubation, the sample was immediately placed on a glass haemocytometer slide and the total number of sperm and percentage remaining motile were counted under light microscopy. This experiment was performed in triplicate.\n2.9\nStatistical analysis\nAll values are expressed as mean\u00a0\u00b1\u00a0standard error (S.E.M.). The statistical analysis was performed by one-way ANOVA. When significant differences were observed, statistical analysis was further carried out using unpaired t-test between two groups. Significance between two groups was claimed when P\u00a0<\u00a00.05.\n3\nResults\n3.1\nCD59b mRNA is highly expressed only in testis\nWe have previously demonstrated that CD59a is the primary regulator of MAC assembly in mouse (Baalasubramanian et al., 2004). However, in a recent work (Qin et al., 2006), abundant expression of CD59b mRNA in a number of tissues was reported. In an attempt to clarify this issue, which is of key importance for complement studies in mice, we repeated some of the experiments carried out by Qin and co-authors using the primer pairs designed by them. Under routine conditions (25 cycles of amplification), CD59a mRNA was detected in all tissues tested while CD59b mRNA was found only in testis (Fig. 1A). Increasing the number of amplification cycles to 35 (Fig. 1B) revealed weak expression of mRNA for CD59b in liver, blood cells, heart and lung. Amplified sequence was confirmed to be CD59b by sequencing.\nWe next re-visited the primers used in our previous quantitative PCR analysis to assess expression of the two CD59 isoforms in different mouse tissues (Baalasubramanian et al., 2004). We had designed a primer pair that annealed to both isoforms and Taqman probes specific for either CD59a or CD59b to enable accurate quantitation. Qin et al. (2006) criticised these experiments on the basis that, while both primers used in our assay matched perfectly CD59a, each had a single internal nucleotide mismatch compared to the CD59b sequence (Table 1). They suggested, without evidence, that this mismatch favoured amplification of CD59a and caused our inability to detect CD59b in tissues other than testis and bone marrow. There is a large primer design literature that shows clearly that internal mismatches, unlike those at the 3\u2032 end, do not significantly influence amplification (Sommer and Tautz, 1989; Kwok et al., 1990; Christopherson et al., 1997; L\u00f6ffert et al., 1998). Nevertheless, we addressed further this issue by repeating our previous QPCR investigation but including a second primer pair from the same sequence matching perfectly CD59b mRNA but with a single mismatch to CD59a mRNA in the same position as in the original primer set (Table 1). Using the original primer pair that matched perfectly CD59a we obtained very similar results to those we previously published (Baalasubramanian et al., 2004). When we used the pair matching CD59b, there was a small increase in threshold cycle (Ct) for both CD59a and CD59b, likely a result of altered primer annealing temperature, but there was no difference in the calculated relative amounts for CD59a and CD59b mRNA in testis compared to the original primer set (Table 2). Of note, QPCR with either primer set did not detect any CD59b mRNA in liver, suggesting that the high cycle number PCR described above was detecting very small amounts of mRNA in these tissues. In summary, the data shows an 8-fold greater expression of CD59a mRNA in liver compared to testis, a 5-fold greater expression of CD59a mRNA compared to CD59b in testis and undetectable expression of CD59b in liver, regardless of primer set used.\nIn light of these results we developed a semi-quantitative PCR to quantify the low levels of CD59b mRNA detected in tissues. In order to be able directly to compare the expression of both isoforms, we designed a new primer pair within exon 4 (Table 1), which is 100% homologous to both mRNAs. Amplification of DNA-free mRNA with these primers will result in bands of 199\u00a0bp for CD59a and 162\u00a0bp for CD59b. In testis we detected both isoforms as expected (Fig. 2A). Densitometric comparison of band intensities at higher dilutions of template, when the amplification reaction is in the linear range, showed approximately 5-fold higher expression of CD59a mRNA as compared to CD59b mRNA in testis, a result compatible with the QPCR data (Table 2) and our published results (Baalasubramanian et al., 2004). However, in liver we failed to detect presence of CD59b even after 40 cycles of PCR (Fig. 2B). We reasoned that the large excess of CD59a mRNA in liver out-competes trace amounts of CD59b mRNA for limited reagents in the initial cycles of amplification, thereby reducing the chance for amplification of the low abundance CD59b mRNA. To test this reasoning we performed semi-quantitative PCR in separate tubes, using the primers specific for either CD59a or CD59b mRNA (Qin et al., 2006). Firstly, to optimise the annealing and amplification efficiency for both primer pairs, we performed this reaction for testis mRNA (Fig. 3A). Similar band intensities for CD59a and CD59b were obtained for 5\u00a0\u00d7\u00a0105-fold and 105-fold template dilution respectively (Fig. 3A and B). This indicated an approximate 5-fold higher expression of CD59a mRNA compared to CD59b mRNA, supporting our results described above (Table 2) and published (Baalasubramanian et al., 2004), and confirming similar amplification efficiency for the specific primer pairs. Amplification of liver mRNA using these primers, and comparison of band intensities in dilutions of templates showed that expression of CD59b mRNA in liver was approximately 105-fold less compared to that of CD59a mRNA (Fig. 3C and D similar band intensities for CD59a and CD59b at 5\u00a0\u00d7\u00a0105 and 5-fold dilution respectively). In these latter experiments we used liver perfused with saline at the time of sacrifice to remove the bulk of entrapped blood; nevertheless, the trace mRNA detected might be from residual blood cells, which clearly express CD59b protein at low level (Baalasubramanian et al., 2004; Qin et al., 2006). To clarify this issue, we compared mRNA from perfused and non-perfused liver by QPCR using CD59b-specific primers and found the amount of CD59b mRNA in liver was reduced 12-fold by perfusion (Fig. 3E). For comparison, expression of CD59b in testis was 2200-fold higher than in unperfused liver and approximately 25,000-fold higher than in perfused liver. Taken together these results strongly suggest that the detectable traces of CD59b mRNA in liver and likely other organs are a consequence of contamination with blood cells.\n3.2\nExpression of CD59b protein is testis restricted\nWe have previously reported, using immuofluorescence staining, that CD59b protein was highly expressed in testis but absent from all other organs tested (including liver, lungs, spleen, kidney and heart) (Baalasubramanian et al., 2004). We here extend these studies by Western blotting of testis, liver, lungs, plus aorta, chosen because of the suggested role of CD59 in vascular disease (Qin et al., 2004) (Fig. 4). Each organ was perfused with saline prior to preparation of protein lysates. CD59a was strongly detected in all the tissues; however, CD59b was detected only in testis lysates. These data confirm our published contention that organ expression of CD59b protein is restricted to testis.\n3.3\nExpression of CD59b mRNA in testis coincides with puberty and plays a role in spermatozoal motility\nWe previously reported that CD59b expression in testis was restricted to developing and mature spermatozoa (Baalasubramanian et al., 2004). To confirm this germ cell restricted pattern we examined expression of CD59b mRNA in testis from pre-pubertal mice. CD59b mRNA was absent in testes harvested from mice at days 10 and 20 post-partum and appeared only from day 30 on, coincident with puberty (Fig. 5A). In contrast, CD59a mRNA was present in testes at all timepoints. RNA extracted from embryos at days 5, 10 and 17 were all positive for CD59a but negative for CD59b mRNA (Fig. 5A). These observations were confirmed by Western blotting (Fig. 5B). Expression of CD59b protein was highest in spermatozoa, intermediate in adult testis and absent from infant testis. CD59a was present in all the lysates, albeit at low level in spermatozoa and with a reduced apparent molecular mass compared with the testis protein, suggesting modification during sperm maturation.\nWe previously reported that CD59b on spermatozoa was focussed on the head region in a highly granular pattern (Baalasubramanian et al., 2004). Here we have explored the expression pattern of CD59b in more detail. In freshly isolated spermatozoa, more than 80% showed this head\/granular staining pattern, a minority showing a more diffuse staining on the head region (Fig. 6). CD59b was weakly expressed on spermatozoal tails and, in about two thirds, strongly in the mid-piece. Initiation of the acrosome reaction (with A23187) caused a precipitous loss of CD59b expression on sperm heads, more than 60% of cells being negative by 30\u00a0min and 90% by 180\u00a0min post-initiation, indicating that CD59b was shed with the outer acrosomal membrane (Fig. 6). These data suggested that CD59b might play a role in acrosome function, an essential component of sperm capacitation for fertilisation. Therefore, we next investigated whether ligation of CD59b with antibody influenced spermatozoal mobility, a surrogate marker for fertilisation capacity (Fig. 7). Ligation of CD59b caused a significant suppression of motility of spermatozoa harvested from both wild type and cd59a\u2212\/\u2212 mice. As a control, CD59a was similarly ligated but did not alter spermatozoal motility compared with the effect of a control antibody. The data suggest that CD59b but not CD59a has a role in regulating spermatozoal motility. These findings are of particular relevance in that a major feature of the CD59b knockout mouse was spermatozoal dysfunction that included diminished motility and infertility (Qin et al., 2003).\n4\nDiscussion\nWe have recently examined the distribution patterns of the two isoforms of CD59 in the mouse and concluded, based upon its broad distribution, that CD59a is the primary regulator of MAC assembly in mouse (Baalasubramanian et al., 2004). We were unable to detect the expression of CD59b, either at mRNA or protein level, in brain, lungs, heart, liver, spleen and kidney. These data have recently been questioned by Qin et al. (2006) who first used a BLAST search of the mouse EST database and found ESTs matching the CD59b sequence from several tissues and organs. These in silico data were supported by RT-PCR analyses using primer sets specific for CD59a and CD59b, respectively that showed abundant expression of CD59b mRNA in all tissues tested, indeed, expression in testis was lower than in other organs. The apparent absence of CD59b mRNA in the CD59a knockout mouse in these published data was unexplained.\nHere we designed a number of quantitative and semi-quantitative assays to comprehensively address this controversial issue. Our data unambiguously demonstrated that expression of CD59b is essentially restricted to testis (Figs. 3 and 4). A comparative quantitative analysis of expression of CD59b mRNA in perfused and unperfused liver (Fig. 3) showed a 12-fold decrease in expression of CD59b following perfusion, strongly suggesting that the trace detection of this mRNA was a consequence of contamination with blood cells. This would explain why Qin et al. (2001) were able to detect CD59b in multiple mouse tissues using Northern analysis in which they loaded 10\u00a0\u03bcg of mRNA in each lane, a huge excess for this sensitive procedure. The contamination likely also explains the presence in multiple tissues of CD59b-specific EST sequences.\nWe further demonstrated that the testis expression of CD59b coincides with puberty, supporting our findings of expression only on spermatozoa and their immediate precursors (Fig. 5). We found that CD59b was released from spermatozoa heads upon acrosome activation (Fig. 6), an observation that strongly support a role for this protein in functioning of acrosome. Ligation experiments with antibodies showed that CD59b but not CD59a was involved in spermatozoa motility (Fig. 7), providing support for an earlier report describing decreased motility and viability of sperm in cd59b KO mice (Qin et al., 2005).\n5\nConcluding remarks\nThe data presented here, obtained using a broad panel of reagents and methods designed to give unbiased and unequivocal results, show that CD59b expression is limited. CD59b mRNA is abundantly expressed only on male germ cells and present in trace amounts in bone marrow and blood cells, but is absent from other organs where trace detection of mRNA is likely due to blood contamination. CD59b protein is abundant only on developing and mature spermatozoa. Erythrocytes express CD59b at low levels that we have previously quantified as less than 200 molecules per cell, irrelevant for protection from complement when CD59a is present at 2500 molecules per cell (Baalasubramanian et al., 2004). We show a unique distribution pattern of CD59b protein on spermatozoa, the precipitous loss of this protein with outer acrosomal membranes and effects of CD59b ligation on sperm motility. We conclude that CD59a is indeed the principal regulator of MAC expressed in the mouse and that the CD59a knockout is an appropriate model for studying the roles of MAC and its regulation in disease models. The CD59b knockout mouse might prove of value for studies of fertility; however, it should be noted that the mouse described by Halperin and co-workers is, for unexplained reasons, also markedly deficient in CD59a (Qin et al., 2006), limiting its utility for studying specific roles of CD59b in vivo.","keyphrases":["mouse","complement","cd59b","acrosome","cd59a","reproductive immunology"],"prmu":["P","P","P","P","P","M"]}
{"id":"Fam_Cancer-3-1-1914241","title":"Family history is neglected in the work-up of patients with colorectal cancer: a quality assessment using cancer registry data\n","text":"In the diagnostic work-up of hereditary non-polyposis colorectal cancer (HNPCC, Lynch syndrome), high-risk patients can be identified using information from the family history on cancer (\u2018Amsterdam criteria\u2019 and \u2018Bethesda guidelines\u2019). To investigate to what extent the medical specialists apply these criteria to patients with colorectal carcinoma and a suspicion of HNPCC, we collected information on diagnostic work-up of 224 patients of seven hospitals in the region of the Comprehensive Cancer Centre West in Leiden, The Netherlands. These patients were diagnosed with colorectal cancer between 1999 and 2001 and satisfied at least one of the Bethesda guidelines. A complete family history was recorded for 38 of the 244 patients (16%). Patients with a complete family history were more likely to be referred to the Clinical Genetic Centre than those with an incomplete or absent family history (53% vs. 13% and 4%, respectively; P < 0.0001), and more likely to be analyzed for microsatellite instability (MSI), which is a characteristic of HNPCC (34% vs. 6% and 1%, respectively; P < 0.0001). We conclude that the family history is neglected in the majority of patients with colorectal cancer and MSI-analysis is only performed in a small proportion of the patients that meet the guidelines for this analysis.\nIntroduction\nColorectal cancer (CRC) is one of the most common forms of cancer in Western society. Every year 9,500 patients in the Netherlands are diagnosed with the disease and almost half of them die from it (Dutch Cancer Registry, 2002). It is estimated that in around 20% of the patients with a colorectal tumour genetic factors play a role in the aetiology [1]. About 1\u20135% of the patients with CRC are thought to have hereditary non-polyposis colorectal cancer (HNPCC, Lynch Syndrome); a dominant hereditary disease, which is caused by a defect in one of the DNA-Mismatch Repair (MMR) genes [2]. The most important clinical characteristics of CRC associated with HNPCC, are the relatively young age at which patients are diagnosed with the disease (average <45\u00a0years old) and the proximal localisation of the tumour in the colon. Besides an increased risk of developing a tumour in the colon, there is an increased risk of developing a tumour elsewhere in the body, especially in the endometrium (lifetime risk: 50%), the small intestines, the ovaries, the brain, the urinary tract, the biliary tract and the development of a keratoacanthoma or a carcinoma of the sebaceous glands of the skin [3].\nThe identification of patients with hereditary colorectal carcinoma is of great importance for the patient, because the treatment and follow-up of the tumour differ from those with non-hereditary colorectal carcinoma [4]. Furthermore, the identification of these patients is important, because it offers efficient manners for the prevention of colorectal carcinoma and other forms of cancer for the patient himself as well as his family [5]. It has been shown that a colonoscopy every three years can lead to a decline in mortality of at least 65% [6].\nThe genetic defect in HNPCC can be detected by immunohistochemic staining of the MMR proteins in tumour cells [7]. Another method to select families for mutation analysis of the MMR genes is analysis of errors in repetitive DNA-sequences, i.e., Micro Satellite Instability (MSI) [8]. MSI is found in around 15% of the non-selected CRC and in more than 95% of the colorectal tumours associated with HNPCC [9]. In 1996, guidelines were formulated for patients whose tumours should be analyzed for MSI [10]. These so-called Bethesda-guidelines describe practically all situations, where there is a suspicion of HNPCC. Recently these guidelines have been revised (Table\u00a01) [11].\nTable\u00a01Guidelines for the performance of MSI-analysis of colorectal tumour [11]Revised Bethesda-guidelinesA person with colorectal carcinoma diagnosed at age \u226450A person with colorectal carcinoma and MSI-associated pathologya<60\u00a0yearsA person with colorectal carcinoma and a HNPCC associated tumourbA person with colorectal carcinoma and a first degree relative with a colorectal or HNPCC associated tumour; at least one of the tumours is diagnosed before the age of 50Three relatives diagnosed with colorectal carcinoma or a HNPCC associated tumour, diagnosed at any age; one patient needs to be a first degree relative of the other twoaThe presence of tumor-infiltrating lymphocytes, so called, \"Crohn's like lymfocyte reaction\", mucinous or signet ring cell carcinoma differentiation or medullary growth patternbCarcinoma of the endometrial tissue, stomach, small intestines, pancreatic gland, biliary tract, urinary tract, ovaries, brain, keratoacanthoma and carcinoma of the sebaceous glands\nBefore the discovery of the MMR-genes, the most common approach in the diagnostic work-up for HNPCC was to use the \u2018Amsterdam criteria\u2019. These criteria are met if there are, within one family, three individuals with a colorectal (or another HNPCC-associated kind of) tumour, of whom one person is a first degree family member of the other two and at least one carcinoma is diagnosed before the age of fifty [12]. To evaluate the Amsterdam criteria in patients with CRC, a complete history on cancer in the patient\u2019s family has to be obtained. Until now it is not known whether an adequate family history is taken of all patients with a colorectal carcinoma. We also do not know to what extent medical specialists use the above mentioned clinical Bethesda guidelines and if the tumours of all the patients who match the criteria are tested for MSI. The objective of the present study was to answer these questions using data of the Cancer Registry of the Comprehensive Cancer Centre West (CCCW) in Leiden, The Netherlands.\nPatients and methods\nWe selected patients who were diagnosed with a primary and invasive colorectal tumour in the period 1999\u20132001 from the Cancer Registry (CR) of the Comprehensive Cancer Centre West (CCCW) in The Netherlands. The patients had to satisfy one of the following two Bethesda guidelines: the patient had to have more than one tumour, i.e., one colorectal carcinoma and a second one (colorectal cancer or another HNPCC-associated kind of tumour), or the patient had to be fifty years or younger at diagnosis. The selected patients were considered to have an indication for the performance of MSI-analysis and\/or referral to the Clinical Genetic Centre (CGC). Patients with a carcinoma in situ or a carcino\u00efd of the appendix were not included in the analysis.\nBetween 1999 and 2001, 434 patients who complied with the above mentioned criteria were diagnosed with CRC in one of the twelve hospitals in the CCCW-region. Seven hospitals gave permission for the collection of information concerning family history, MSI-analysis and referral to the CGC. We extracted this information from the various (electronic) medical reports. The family history was considered complete if the medical records reported on cancer in the family, and if so, information about the age at the time of the diagnosis, the type of cancer and the occurrence of cancer within first-degree and second-degree family members.\nData were collected of 244 patients. Of these patients, 120 patients had multiple tumours, 109 patients were fifty years or younger at the time of diagnosis, and 15 patients had both characteristics. For comparisons between patients with multiple tumours and patients who were young at diagnosis, those with both characteristics were allocated to the \u201cmultiple tumours\u201d group. The data were analyzed using SPSS statistical software (version 12.0.1). Univariate comparisons of proportions between patient groups were performed by Chi-squared test. Multivariate logistic regression analysis was used to study whether the presence of a complete family history or referral to a CGC could be explained by age, sex, inclusion criterion (multiple tumours or young age at diagnosis), hospital or type of medical specialist.\nResults\nThe study group consisted of 244 persons, who complied with one of the Bethesda guidelines and therefore were considered to be referred for MSI-analysis and\/or genetic counselling. The male:female ratio was 49:51 and did not differ between the groups selected on the basis of multiple tumours or age \u226450\u00a0years at diagnosis.\nA complete family history was recorded in the medical records of 38 (16%) of the 244 patients. For 136 patients (55%) limited information on the family history was available, and for 70 (29%) patients no information on the family history was found in the medical records. In the seven participating hospitals, a family history was reported for 38\u201391% of the patients.\nOf the 38 patients with a complete family history, 20 (53%) were referred to the CGC. This percentage was higher than that of patients with an incomplete family history (13%) and that of patients without any information on family history (4%) (P\u00a0<\u00a00.0001, Table\u00a02). MSI-analysis was performed more often in the patients with a complete family history: 34% of patients with a complete family history compared to 6% of patients with an incomplete family history and 1% of patients without any family history (P\u00a0<\u00a00.0001) (Table\u00a02). Presence of a complete family history and the performance of MSI-analysis were not associated with age, sex, inclusion criterion (multiple tumours or young age at diagnosis), hospital or type of medical specialist (multivariate logistic regression analysis; data not shown).\nTable\u00a02Diagnostic work-up for HNPCC in 244 patients with colorectal cancer, by completeness of the family history as reported in the medical recordsDiagnostic workupFamily history complete (n\u00a0=\u00a038)Family history incomplete (n\u00a0=\u00a0136)Family history absent (n\u00a0=\u00a070)Referred to CGC20 (53%)17 (13%)3 (4%)MSI-analysis performed13 (34%)8 (6%)1 (1%)Results of MSI-analysis3\u00a0MSI, 10\u00a0stable7\u00a0stable, 1\u00a0unknown1\u00a0stableDiagnosis of HNPCC6 (16%)3 (2%)1 (1%)\nDiscussion\nWe used the Bethesda-guidelines to select a group of patients with a suspicion of HNPCC. These patients were diagnosed with colorectal cancer between 1999 and 2001, a period during which MSI-analysis and the Bethesda guidelines were already available. Therefore we expected that for these patients, physicians would have examined and reported their patients\u2019 family history and that MSI-analysis would have been performed. In our study group, however, the family history of the patients diagnosed with colorectal carcinoma was not sufficiently examined and reported in the medical records. For this reason, we believe the Bethesda-guidelines were not sufficiently applied by the physicians. As a consequence, MSI-analysis was performed on a small proportion of the tumours. More patients with a complete family history in their medical records were referred by their physicians to the CGC than patients without such a family history. MSI-analysis was also performed more often in this group. We expect that in a low-risk population, i.e., patients with colorectal cancer who do not meet the Bethesda guidelines, these results would be even more dramatic.\nOn the one hand, our results may appear to be better than they actually are. We collected our data using medical records from various medical specialties, while the treating physician will not have this overview in practice. On the other hand, it is possible that when a physician examined a family history and none of the family members was diagnosed with cancer, he did not report it in the medical records. In this case, the family history was considered as absent, although it in fact was examined. Nevertheless we expect that if MSI-analysis was performed or the patient was referred to the CGC, this would have certainly been reported.\nWe found that the attention for HNPCC in the diagnostic workup for CRC differed widely. For the seven participating hospitals, the proportion of patients with a reported family history on cancer ranged from 38% to 91%. Furthermore, only half of the approached hospitals were willing to cooperate. For these reasons, we cannot generalise our results for the whole CCCW-region. Nevertheless, we conclude that the family history appears to be neglected in the majority of patients with colorectal cancer in our study period, and that MSI-analysis was only performed in a small proportion of the patients that meet the guidelines for this analysis. Possibly, the attention for identification of patients with HNPCC has increased in more recent years. Our findings underscore the importance of implementation of family history and Bethesda guidelines in the physician education.","keyphrases":["family history","colorectal cancer","hereditary non-polyposis colorectal cancer","lynch syndrome","microsatellite instability","bethesda criteria"],"prmu":["P","P","P","P","P","R"]}
{"id":"Intensive_Care_Med-3-1-1915596","title":"Elimination of daily routine chest radiographs in a mixed medical\u2013surgical intensive care unit\n","text":"Objective To determine the impact of elimination of daily routine chest radiographs (CXRs) in a mixed medical\u2013surgical intensive care unit (ICU) on utility of on demand CXRs, length of stay (LOS) in ICU, readmission rate, and mortality rate.\nIntroduction\nChest radiographs (CXRs) are frequently obtained as a\u00a0complement to physical examination of critically ill patients\u00a0[1, 2]. There are two different schools of thought regarding the utility of CXRs in the intensive care unit (ICU): The CXRs should be made on indication only, specifically when there is a\u00a0sound reason to obtain a\u00a0film (so-called on demand CXRs); or CXRs should be obtained routinely every day, that is, without any specific reason (so-called daily routine CXRs). Argument for the latter strategy is the high prevalence of findings on CXRs of ICU patients\u00a0[3]; however, interpretation of studies on the usefulness of daily routine CXRs is hampered because of major differences in methodology\u00a0[4]. Importantly, most studies did not attempt to discriminate between clinically relevant and irrelevant findings. We recently demonstrated that daily routine CXRs hardly ever reveal potentially important abnormalities and seldom result in a\u00a0change in therapy\u00a0[5].\nWhile it can be recommended to discontinue a\u00a0daily routine CXR practice in ICU patients, elimination of these CXRs may have several disadvantages. Firstly, eliminating daily routine CXRs bears the risk that the number of on demand CXRs increases. In addition, elimination of daily routine CXRs might result in on demand CXRs being obtained more frequently during off-time hours, which may cause an inverse rise of costs. Secondly, length of stay (LOS) in ICU, readmission rate and mortality rate might be negatively influenced by this change in CXR practice.\nTo evaluate the impact of elimination of daily routine CXRs we determined the change in on demand CXR practice in our multidisciplinary ICU, where a\u00a0daily routine CXR strategy was applied until performance of this study. In addition, we evaluated the diagnostic and therapeutic value of on demand CXRs before and after this intervention. Finally, LOS in ICU, readmission rate, and mortality rate during a\u00a0daily routine CXR strategy were compared with those during an on demand CXR strategy.\nMaterials and Methods\nSubjects\nA\u00a0prospective, nonrandomized, controlled design with an intervention was used for this study. Of all patients, all CXRs taken in the adult ICU department of the Academic Medical Center in Amsterdam, Netherlands, from 1\u00a0March\u00a02004 to 31\u00a0July\u00a02004 and from 1\u00a0September\u00a02004 to 31\u00a0January\u00a02005 were studied. This department is a\u00a0closed-format tertiary care, referral, 28-bed multidisciplinary ICU. The patient population consists of cardiothoracic surgery patients, medical patients (including cardiology patients and pulmonary disease patients), and surgery patients (including trauma patients and neurosurgery patients). Patients who were admitted during the period in between phases\u00a01 and\u00a02, as well as patients that were readmitted, were not analyzed. The study protocol was approved by the local ethics committee.\nProtocol\nThe study period was divided into two parts: phase 1, a\u00a05-month phase before the intervention during which the daily routine CXR strategy was practiced; and phase\u00a02, a\u00a05-month phase which began 1\u00a0month after the intervention. The intervention consisted of a\u00a0change in the ordering practice of CXRs: no standing orders for daily routine CXRs; each (on demand) CXR required a\u00a0clinical indication, such as admittance to the ICU, insertion of central venous lines, intra-aortic balloon pump or tracheal and chest tubes, an increase in oxygen requirement, or a\u00a0change in pulmonary secretions with or without fever (see Table\u00a0E1).\nFor phases\u00a01 and 2, CXR volume data were collected prospectively. Type of, and reason for, admission was registered for all patients. Severity of illness was scored by means of acute physiology and chronic health evaluation (APACHE)\u00a0II for all patients. Data on LOS in ICU, readmission to ICU as well as ICU, and hospital mortality rate, were collected from the National Intensive Care Evaluation (NICE) database\u00a0[6]. The LOS was calculated from day and time of arrival at, and discharge from, ICU. The total number of hours in ICU were divided by 24\u00a0to determine the exact LOS in ICU in days.\nDiagnostic and therapeutic value of on demand CXR\nDiagnostic and therapeutic value of on demand CXRs was determined as described previously for daily routine CXRs\u00a0[5]. In short, the attending physician completed a\u00a0specially developed data sheet on radiological abnormalities which was printed on the back of the normal CXR request form. It was to be ticked whether a\u00a0certain finding was expected, and whether it was \u201cold\u201d (i.e., already present on preceding CXR) or \u201cnew\u201d (i.e., not present on preceding CXR). All CXRs were interpreted by an independent radiologist on the day the on demand CXR was performed. Similar to the ICU physicians, the radiologist structurally interpreted these on demand CXRs for each patient (i.e., the radiologist ticked whether radiological abnormalities were absent or present and, if an abnormality was present, whether it was judged to be an \u201cold\u201d or \u201cnew\u201d finding).\nIf a\u00a0predefined finding was unexpectedly found, then we determined whether any action was taken because of the new unexpected finding. To do this, two of us (M.G. and M.J.S.) and two independent observers carefully read the medical records, checked the patient data management system (Metavision, iMDsoft, Sassenheim, The Netherlands) and searched the hospital information system for the following: orders for sputum cultures or performance of a\u00a0bronchoalveolar lavage for culture, or start of, or a\u00a0change in, antimicrobial therapy in case of unexpected infiltrates on the CXR; repositioning of tubes in case of malposition of orotracheal tubes (ignoring planned extubations); ultrasound of the thorax in case of pleural effusion on the CXR, start or change in medication (diuretics); insertion of a\u00a0pleural drain; and repositioning of devices in the case of malposition of medical devices other than orotracheal tubes (ignoring planned changes such as removal of intravenous lines). The observers were not involved in the daily care of the patients, and ICU physicians were not aware of this part of the observation. As a\u00a0consequence, the clinical relevance of the predefined abnormalities could not be evaluated in some cases, specifically in cases of large atelectasis and severe pulmonary congestion, since start of physiotherapy, changes in levels of positive end-expiratory pressure, and the use of diuretics might have been triggered by other (clinical) findings.\nStatistical analysis\nAll data are expressed as means (\u00b1\u202fSD), or medians (interquartile ranges). A\u00a0Mann-Whitney U-test was used for analyzing continuous variables. A\u00a0chi-square test was used to compare the groups in phase\u00a01 and phase\u00a02. The incidences of expected and unexpected findings, and clinically important abnormalities, were compared by chi-square test. A\u00a0p-value <\u202f0.05 was considered to be statistically significant. All calculations were performed using SPSS version\u00a012.0.1 software (SPSS, Chicago, Ill.).\nResults\nStudy population\nWe evaluated 1376\u00a0patients over the two periods. Patient profiles on entering this study are summarized in Table\u202f1. A\u00a0total of 3894 CXRs were obtained from 754\u00a0patients in phase\u00a01; these included 2457 daily routine CXRs and 1437 on demand CXRs. A\u00a0total of 1267 CXRs were obtained from 622\u00a0patients in phase 2. These CXRs were, by definition, all on demand CXRs.\nTable\u00a01Demographic data. APACHE-II Acute Physiology and Chronic Health Evaluation\u00a0II, CXRs chest radiographs, CI confidence intervalPhase 1Phase 2Significance (p)No. of patients 754 622Age (years; mean, SD) 60 (16) 62 (16)0.02Gender (male; n) 475 (63%) 398 (64%)0.70CXRs while patients being mechanically ventilated (n)a3194 (82%)1115 (88%)<\u202f0.001APACHE-II score 16.4\u202f\u00b1\u202f6.9 16.4\u202f\u00b1\u202f7.21.00Patient subgroups Cardiac surgery (n) 317 (42%) 306 (49%)0.01 Medical (n) 197 (26%) 119 (19%) Surgical (n) 144 (19%) 131 (21%) Neurosurgical\/neurology (n) 69 (9%) 46 (7%) Other (n) 27 (4%) 20 (3%)Length of stay in ICU (days; median IQR)  1.9 (1.0\u20134.6)  1.9 (0.9\u20134.6)0.95Mortality ICU (n) 94 (12%) 62 (10%)0.49 Hospital (n) 132 (18%) 104 (17%)0.70 Predicted hospital mortality (%) 181 (24%) 155 (25%)0.69 Observed\/predicted ratio (95% CI)  0.73 (0.59\u20130.90)  0.67 (0.53\u20130.83)aAll patients were mechanically ventilated at any time during stay in ICU. Expressed is the percentage of CXRs during which patients were on the ventilator while the CXR was performed\nUtility of CXRs\nThe number of CXRs per day for the whole ICU declined from 22.6\u202f\u00b1\u202f4.9 to 8.2\u202f\u00b1\u202f3.2 (p\u202f<\u202f0.05; Fig.\u202f1). Adjusting for patient volume, the ratio of CXRs per patient day decreased from 1.1\u202f\u00b1\u202f0.3 to 0.6\u202f\u00b1\u202f0.4 after the intervention (p\u202f<\u202f0.05). The median number of CXRs per patient for the complete stay in ICU declined from 3 (range 2\u20135) during phase\u00a01, to 1 (range 1\u20132) after the intervention. The number of on demand CXRs increased minimally after the intervention, and the distribution over 24\u202fh, did not change (see ESM, Fig.\u00a0E1).\nFig.\u00a01Number of CXRs\/day during the study. Phase 1: daily routine CXR strategy, i.e., a\u00a0daily routine CXR was made every morning, from March to July; phase 2: on demand CXR strategy, i.e., each CXR required a\u00a0clinical indication, from September to January. Open bars: mean number (\u00b1\u202fSD) of on demand CXRs\/day; closed symbols: mean number (\u00b1\u202fSD) of all CXRs\/day\nDiagnostic and therapeutic value of on demand CXRs\nThe diagnostic and therapeutic value of on demand CXRs increased with elimination of daily routine CXRs (Tables\u202f2, 3). Before intervention, 38 expected predefined abnormalities were found (2.6% of all on demand CXRs in 4.9% of all patients), and after the intervention 64 expected predefined abnormalities were found (5.0%; p\u202f<\u202f0.05) in 9.5% of cases (p\u202f<\u202f0.05). All these findings led to a\u00a0change in therapy. Before intervention, 147 unexpected predefined abnormalities were found (10.2% of all on demand CXRs in 15.9% of all patients), of which 57 (4.0 in 6.4%) led to a\u00a0change in therapy. After intervention 156 unexpected predefined abnormalities were found (11.6% of all on demand CXRs in 19.1% of all patients), of which 64 (4.8 in 9.5%; p\u202f<\u202f0.05) led to a\u00a0change in therapy. Subgroup analysis revealed no differences between phases\u00a01 and\u00a02, except for medical patients, in which there was a\u00a0significant rise in the number of on demand CXRs that showed an unexpected predefined major abnormality (p\u202f<\u202f0.05 vs phase\u00a01; see ESM, Table\u00a0E2).\nTable\u00a02Expected and unexpected findings on on demand chest radiographsPhase 1 (n\u202f=\u202f1437)Phase 2 (n\u202f=\u202f1267)AbnormalitiesExpectedExpected+foundUnexpected+foundExpectedExpected+foundUnexpected+foundLarge atelectasis 37 (2.6) 2 (0.1) 13 (0.9) 49 (3.9) 3 (0.2) 15 (1.2)Large infiltrates 57 (4.0) 3 (0.2) 21 (1.5) 69 (5.4) 5 (0.4) 27 (2.1)Pulmonary congestion 98 (6.8) 8 (0.6) 25 (1.7)104 (8.2)14 (1.1) 22 (1.7)Pleural effusion 41 (2.9) 3 (0.2) 17 (1.2) 43 (3.4) 4 (0.3) 27 (2.1)Pneumothorax or pneumomediastinum 68 (4.7) 4 (0.3) 17 (1.2) 39 (3.1)c 3 (0.2) 12 (0.9)Malposition of invasive devices350 (24.4)18 (1.3) 54 (3.8)392 (30.9)c35 (2.7)c 52 (4.1)Total no. of abnormalities6513814769664c155Total no. of CXRs with abnormalitiesa641 (44.6)38 (2.6)133 (9.2)384 (30.3)c63 (5.0)c147 (11.6)cTotal no. of patients with CXRs with abnormalitiesb580 (76.9)37 (4.9)120 (15.9)223 (35.9)c58 (9.5)c119 (19.1)Numbers in parentheses are percentagesa\u202fAbsolute number of chest radiographs (CXRs; percentage of all daily routine CXRs)b\u202fAbsolute number of patients (percentage of all patients with on demand CXRs)c\u202fp\u202f<\u202f0.05 vs phase 1Table\u00a03Unexpected findings on on demand chest radiographs resulting in a\u00a0chance in therapy. ND not definedPhase 1 (n\u202f=\u202f1437)Phase 2 (n\u202f=\u202f1267)AbnormalitiesResulting in a change in therapyResulting in a change in therapyLarge atelectasisNDNDLarge infiltrates10 (0.7%)14 (1.1%)Pulmonary congestionNDNDPleural effusion11 (0.8%)12 (0.9%)Pneumothorax or pneumomediastinum11 (0.8%) 9 (0.7%)Malposition of invasive devices25 (1.7%)29 (2.3%)Total no. of abnormalities5764Total no. of CXRs with abnormalitiesa56 (3.9%)61 (4.8%)Total no. of patients with CXRs with abnormalitiesb48 (6.4%)59 (9.5%)ca\u202fAbsolute number of chest radiographs (CXRs; percentage of all daily routine CXRs)b\u202fAbsolute number of patients percentage of all patients with on demand CXRs)c\u202fp\u202f<\u202f0.05 vs phase 1\nLOS in ICU, readmission rate and mortality rate\nThe LOS in ICU was not different in phase\u00a01 as compared with phase\u00a02 (Table\u202f1). Total readmission rate was similar (8.4% in phase\u00a01 vs 7.6% and phase\u00a02, risk difference 0.8% (95% CI: 2.1\u20133.7%, P\u202f=\u202f0.6), and did not change with the intervention for the different subgroups. There were no statistically significant differences in ICU and hospital mortality rates before and after the intervention (Table\u202f1).\nDiscussion\nThe present study demonstrates the impact of elimination of daily routine CXRs in a\u00a0mixed medical\u2013surgical ICU. We found a\u00a0sharp decline in the total number of CXRs, while only a\u00a0minimal increase in the number of on demand CXRs was observed. In addition, the number of CXRs in off-hours was similar between the two periods. Elimination of daily routine CXRs did neither affect LOS in ICU and readmission rate nor ICU and hospital mortality rate.\nAlthough the diagnostic and therapeutic value of on demand CXRs was significantly higher after the intervention, we considered this difference clinically irrelevant. When one considers the increase in diagnostic and therapeutic value of on demand CXRs after elimination of daily routine CXRs indirect proof of the \u201cvalue\u201d of daily routine CXRs, one must also recognize its futility regarding the therapeutic value. Indeed, the percentage of CXRs with unexpected findings that truly led to a\u00a0change in therapy was similar in the two study phases. Since readmission rate and mortality rate remained unchanged after the intervention, we conclude that the true value of daily routine CXRs in our multidisciplinary ICU is very low. Interestingly, only in medical patients did the number of CXRs that showed an unexpected predefined major abnormality increase after elimination of daily routine CXRs. The reason for this finding remains unexplained. The distribution of abnormalities encountered on CXRs of these patients was similar in the two study phases; however, neither readmission rate nor differences in raw or risk-adjusted ICU and hospital mortality rates of medical patients was affected by the change in CXR practice.\nOne interesting finding was the decrease in abnormalities presumed to be present on CXRs. Indeed, a\u00a030% reduction in expected predefined findings was observed in phase\u00a02. This finding remains unexplained and we can only speculate on its cause. Firstly, it may be that physicians learned from experience that many of their expectations proved to be untrue during the actual carrying out of the study. This may have caused them to be more reluctant in scoring for expected findings. Alternatively, physicians may have become less enthusiastic about the study, which might have resulted in failure to comply with study rules at some moments (i.e., they did not fill in the back of the formal CXR request form); however, there was no change in expectations of physicians regarding abnormalities that truly led to a\u00a0change in therapy. More importantly, if the backside of the formal forms were not filled out, as a\u00a0rule the CXR was simply not obtained. Indeed, collection of data was complete regarding this issue, there were no on demand CXRs without a\u00a0completed form.\nOur study has, at least partially, overlap with two other studies\u00a0[7, 8]. Price et al. performed a\u00a0nonrandomized controlled study on the financial impact of elimination of daily routine CXRs\u00a0[7]. They showed that elimination of daily routine CXRs in a\u00a0pediatric ICU resulted in decreased variability in ordering practice, fewer CXRs per patient, and an accompanying cost savings, while not influencing LOS. In addition, cost reduction with the change in radiology policy was significant in their study. This is in line with our results, since we found a\u00a0substantial decline in radiology costs (see ESM). Besides the fact that this study was performed in a\u00a0pediatric ICU, making generalization of study results difficult, their study did not include all patient categories. Indeed, postoperative cardiovascular surgery patients continued to receive daily routine CXR. We specifically included this patient group in our study because cardiovascular patients form one of the largest categories in many adult ICU. Krivopal et al. performed a\u00a0randomized controlled trial to determine whether there is any difference in diagnostic, therapeutic, and outcome efficacy between protocols utilizing daily routine CXRs and those utilizing on demand CXRs in mechanically ventilated patients\u00a0[8]. In their study a\u00a0daily routine CXR strategy compared with an on demand CXR strategy was not associated with a\u00a0negative effect on LOS or mortality; however, this study was small, including not more than 94\u00a0patients.\nWe did not collect information on less evident findings on CXRs. Less evident findings (such as atelectasis less than two lobes, infiltrates less than one lobe, or small pleural effusions\u00a0[5]), however, might still influence daily management of ICU patients. Since LOS in ICU was not altered for the whole group, readmission rate and mortality rate remained unchanged after the intervention, we suggest that changes of less evident CXR findings are not at all important, at least in our ICU. In other ICUs, such as open-format ICUs, less evident findings might be of more clinical importance, however; therefore, our results must be interpreted with caution, it might be that our results are not easily translated to other types of ICU.\nSeveral important drawbacks of our study must be mentioned. Firstly, our study did not include a\u00a0strict method for tracking complications as a\u00a0result of elimination of daily routine CXRs. Indeed, several abnormalities might have been missed (or discovered too late) which might (or do) have impact on clinical outcome. Examples of these types of abnormalities include pneumothorax causing weaning problems, the malposition of devices such as central venous lines, causing extravasation of fluid, or orotracheal tubes, potentially causing injury to the vocal cords. Considering these examples, such a\u00a0strict method may mandate a\u00a0daily check of all invasive devices. Although possible complications of elimination of daily routine CXRs could be discussed in daily bedside rounds, daily radiology conferences and daily multidisciplinary meetings during the performance of our study, no clinically important complications were reported as the result of elimination of daily routine CXRs; thus, although we assume that the elimination of daily routine CXRs does not cause any complications, we cannot be certain that this was truly the case. Secondly, as mentioned previously, it is of importance to realize that results that come from one center may simply not be similar for other centers: differences in staffing; especially during off-hours, and differences in case\u00a0mix may be of great influence on outcome when abandoning daily routine CXRs. Thirdly, as mentioned previously, we found a\u00a0reduction in expected predefined finding in phase\u00a02. We assumed that the cause of this reduction might be that the physicians became less enthusiastic about the study, which might be seen as a\u00a0limitation of the study.\nConclusion\nIn conclusion, in our mixed medical\u2013surgical ICU elimination of daily routine CXRs leads to a\u00a0sharp decline in the total number of CXRs, while only minimally increasing the number of on demand CXRs. Although we cannot be certain whether we missed important findings by abandoning daily routine CXRs, its elimination did neither affect LOS in ICU, nor readmission rate and ICU and hospital mortality rates.\nElectronic supplementary material\nElectronic Supplementary Material (DOC 165K)","keyphrases":["daily routine","chest radiograph","icu","on demand","critical care"],"prmu":["P","P","P","P","R"]}
{"id":"Anal_Bioanal_Chem-4-1-2413088","title":"Disposable electrochemical flow cells for catalytic adsorptive stripping voltammetry (CAdSV) at a bismuth film electrode (BiFE)\n","text":"Catalytic adsorptive stripping voltammetry (CAdSV) has been demonstrated at a bismuth film electrode (BiFE) in an injection-moulded electrochemical micro-flow cell. The polystyrene three-electrode flow cell was fabricated with electrodes moulded from a conducting grade of polystyrene containing 40% carbon fibre, one of which was precoated with Ag to enable its use as an on-chip Ag\/AgCl reference electrode. CAdSV of Co(II) and Ni(II) in the presence of dimethylglyoxime (DMG) with nitrite employed as the catalyst was performed in order to assess the performance of the flow cell with an in-line plated BiFE. The injection-moulded electrodes were found to be suitable substrates for the formation of BiFEs. Key parameters such as the plating solution matrix, plating flow rate, analysis flow rate, solution composition and square-wave parameters have been characterised and optimal conditions selected for successful and rapid analysis of Co(II) and Ni(II) at the ppb level. The analytical response was linear over the range 1 to 20 ppb and deoxygenation of the sample solution was not required. The successful coupling of a microfluidic flow cell with a BiFE, thereby forming a \u201cmercury-free\u201d AdSV flow analysis sensor, shows promise for industrial and in-the-field applications where inexpensive, compact, and robust instrumentation capable of low-volume analysis is required.\nIntroduction\nMercury electrodes, normally in the form of mercury films for flow systems, have been widely used for performing stripping voltammetry as a result of their high sensitivity and reproducibility, but, due to increasing concern and legislation regarding the toxicity of mercury, alternative working electrode (WE) materials have been investigated. Metals such as Au [1, 2], Ag [2, 3], Ir [4] and even W [5] as well as bare and coated carbon electrodes [6, 7] have all been investigated for stripping analysis of metals.\nIn 2000, bismuth-film electrodes [8] were mooted as a replacement for mercury-film electrodes, as bismuth exhibits negligible toxicity and is more environmentally friendly in comparison. Mercury use is increasingly regulated, motivating the search for mercury-free methods. For example, Sweden banned the sale of switches, apparatuses and instruments containing mercury in 1993 (Ordinance SFS 1991:1290) and instructed the Swedish Chemicals Inspectorate (KemI) to look into a total ban on the handling of mercury in Sweden, including chemicals used for analysis. The Inspectorate proposed that mercury for chemical analysis should be exempted from the general ban until 31st December 2008 in order to enable the development and implementation of mercury-free analysis methods [9]. In 2005 the EU commission proposed a ban on mercury exports by 2011 as part of a strategy to reduce mercury emissions and protect against exposure [10], and recently MEPs voted to bring this date forward to 2010 [11].\nA variety of substrates have been used for bismuth films. While platinum [12\u201314] and gold [13] have both been investigated, the majority of films have been deposited on carbon substrates. Most studies have used glassy carbon [8, 13, 15\u201332], though wax-impregnated graphite [22, 24], pencil-lead graphite [33], carbon paste [13, 34\u201336], screen-printed carbon inks [21, 37, 38], carbon fibres [8, 13, 14] and boron-doped diamond [39] have all been employed as substrates. The use of Bi2O3 containing pastes [34, 40] and a bismuth bulk electrode [41] have also been reported.\nThe use of BiFE has recently been reviewed [42]. Bismuth-film electrodes have mainly been applied to anodic stripping voltammetry (ASV) and adsorptive stripping voltammetry (AdSV) techniques for metal ion analysis, although cathodic detection of nitrophenols [17, 27, 41] and amperometric detection of glucose [12] have also been reported. Cd [28], Pb [18, 37], Zn [31, 32] and mixtures of all three ions [8, 20, 22, 23, 33, 36, 41] have been the most extensively studied by ASV. Tl, Cu [16], In [16, 43], Mn [39] and Sn [44] have also been analysed. Fewer reports have been made employing AdSV, though Ni [15] and Co [28\u201330] and Ni with Co [24, 26] have been examined using dimethylglyoxime (DMG). Cr [21, 25, 45], U [46, 47], Al [48], V [49] and Mo [50] analysis by AdSV on bismuth-film electrodes has also been performed.\nStirred solutions have normally been used, though Economou et al. have reported the use of a rotating disc electrode [22\u201325] and a Nafion-coated BiFE in a thin layer flow cell (0.5\u00a0mm spacer) for sequential injection-ASV of metals [51, 52]. Hutton et al. generated an in-situ BiFE in a thin layer (0.3\u00a0mm spacer) flow cell (Bioanalytical Systems Inc., West Lafayette, IN, USA) for flow injection analysis of nitrophenols with cathodic amperometric detection [27].\nAdsorptive stripping voltammetry of DMG-complexed Co and Ni has found widespread use in trace metal analysis. The enhancement of the Co-DMG response using nitrite, i.e. catalytic adsorptive stripping voltammetry (CAdSV), has also been the subject of several publications, for example van den Berg et al. [53, 54] and the review of catalytic systems by Bobrowski and Zar\u0119bski [55]. CAdSV of Co on a BiFE was reported by Krolicka et al. [29, 30] using an ex situ plated film on glassy carbon. Most BiFEs have been plated from acetic buffers, although Krolicka et al. used a plating solution of Bi(NO3)3 (0.02\u00a0M), LiBr (0.5\u00a0M), and HCl (1\u00a0M) with a plating potential of \u22120.25\u00a0V for 45\u00a0s [addition of bromide and strong acid enabled a highly concentrated Bi(III) plating solution to be produced]. Stripping was typically carried out in 0.1\u00a0M ammonia buffer (pH 9.2) containing DMG (1\u2009\u00d7\u200910\u22124 M) and NaNO2 (0.5\u00a0M). Voltammograms were recorded in the differential pulse mode and a 15-fold enhancement of the Co-DMG stripping signal was reported.\nRecently the authors reported the use of 40% carbon fibre-filled nylon 6\/6 injection-moulded electrodes as working electrodes for the determination of Cu(II) by CV and ASV techniques [56].\nIn this work, the electrodes were moulded from a 40% carbon fibre-filled polystyrene, which was also found to be suitable for performing electrochemical detection. This paper presents the first report of the use of CAdSV at an in-line plated BiFE in an integrated three-electrode injection-moulded plastic micro flow cell.\nExperimental\nInstrumentation\nVoltammetric measurements were performed using an Autolab electrochemical system (PSTAT 10, Eco Chemie B.V., Utrecht, The Netherlands) controlled with GPES software (v4.6, Eco Chemie) running on a PC.\nRotating disc electrode experiments\nThe rotating disc electrode (RDE) was a glassy carbon electrode (6.1204.000, Metrohm, Buckingham, UK) 3\u00a0mm in diameter controlled with a variable speed (500\u20133000\u00a0rpm) electrode rotator (Part No. 628-10, Metrohm). The disc electrode was polished with an alumina slurry (0.3\u00a0\u03bcm Al2O3, Metrohm) before use. A standard (12\u00a0mm in diameter) glass double junction Ag\/AgCl\/KCl (3M) reference electrode was used (CR4\/DJ\/AG, Thermo Electron, Auchtermuchty, Fife, UK) with a Pt wire auxiliary 0.5\u00a0mm in diameter (99.9%, Aldrich, Gillingham, UK). The electrodes were mounted in a small-volume 50-ml beaker used as an electrochemical cell.\nFlow cell experiments\nA gravity-fed flow system was used comprising a reservoir formed from a disposable syringe body (20\u00a0ml), and narrow-bore PTFE tubing (0.032 in. i.d., Lee Products, Gerrards Cross, UK). The flow was controlled with a manual two-way valve (Part No. 2420, Omnifit, Cambridge, UK). Barbed (1\/16\u2033) polypropylene female Luer fittings (Cole-Parmer, Hanwell, London, UK) enabled connection of the flow system to the injection-moulded flow cell. Electrical connection to the Autolab electrochemical system was made using crocodile clips.\nChip fabrication\nThe injection-moulded flow cells were produced in-house. Figure 1 depicts the fabricated device showing the three integrated electrodes (WE, reference and auxiliary). Solid models created using AutoCAD (Mechanical Desktop 2004, Autodesk, San Jose, CA, USA) were converted into macro commands for a CNC milling machine (Datron CAT3D M6, Datron Technology, Milton Keynes, UK) to produce the milled aluminium moulds for the injection-moulding machines (Babyplast 6\/6 or 6\/10, Cronoplast SL, Barcelona, Spain). A four-stage process was required to manufacture a ready-to-use device. The polymer electrodes were injection moulded (moulding temperature of 220\u00a0\u00b0C) from a conducting polymer (40% carbon-fibre-filled high-impact polystyrene (HIPS), RTP 487, RTP Company (UK) Plastics Ltd., Bury, UK). The reference electrodes were coated with Ag (Ag evaporation slug, 99.99%, Aldrich) using an e-beam evaporator (Auto500 with EB3 e-beam unit, Edwards High Vacuum International, Crawley, West Sussex, UK) to a thickness of 125\u00a0nm. The WE, reference and auxiliary electrodes were then incorporated into the flow cell by an overmoulding procedure. The mould cavity for the flow cell design comprises a base plate into which the preformed electrodes are inserted in the appropriate recesses and a top plate with a relief pattern that forms the flow channel and integrated fluidic connectors. The flow cell was moulded (moulding temperature of 220\u00a0\u00b0C) from crystal polystyrene (Northern Industrial Plastics Ltd. Chadderton, UK). Using a 500-W ultrasonicwelder (Mini delta 9500, FFR Ultrasonics Ltd., Queniborough, Leicestershire, UK), with a weld time of 1\u00a0s and a hold time of 2\u00a0s, the top and base plates were sealed together to form a complete and ready-to-use device. The flow channel dimensions were 1\u00a0mm wide, 1\u00a0mm deep and 30\u00a0mm long. The working and auxiliary electrodes were 5\u00a0mm wide, the reference was 2\u00a0mm wide and the spacing between them was 2\u00a0mm.\nFig.\u00a01The injection-moulded electrochemical micro flow cell. Here (A) is the Ag-coated electrode, which forms the reference (Ag\/AgCl) element; (B) is the working electrode, on which the bismuth film electrode is formed; (C) is the auxiliary electrode; (D) is the device inlet and (E) is the device outlet. The flow channel is 1\u00a0mm\u2009\u00d7\u20091\u00a0mm and 30\u00a0mm long\nReagents and solutions\nAll aqueous solutions were prepared using >18M\u03a9 water (Elga Maxima Ultra Pure, Vivendi, High Wycombe, UK). The acetate buffer was prepared from acetic acid (glacial 99.8% AnalaR grade, BDH, Poole, UK) and sodium acetate (99+% ACS grade, Aldrich, Gillingham, UK). The ammonium buffer was prepared from ammonium chloride (99.8% AnalaR grade, BDH) and ammonium hydroxide (28\u201330% NH3 ACS grade, Aldrich). Bismuth plating solutions were prepared with bismuth(III) nitrate pentahydrate (99.999%, Aldrich,) either in acetate buffer or with lithium bromide (99+%, Aldrich) in hydrochloric acid (1\u00a0M volumetric standard, Riedel-de Ha\u00ebn, Gillingham, UK), as described in the \u201cProcedure\u201d section. The mercury plating solution was prepared in nitric acid from a 1000\u00a0ppm mercury(II) chloride (98+%, Aldrich) stock solution, as described in the \u201cProcedure\u201d section. Sample solutions were made from cobalt (995\u00a0\u03bcg\/l, Aldrich) and nickel (1\u00a0g\/l, Fluka, Gillingham, UK) atomic absorption standards with dimethylglyoxime (99+%, Acros, Loughborough, UK) and sodium nitrite (97+%, Aldrich). The saturated AgCl solution was prepared from silver nitrate (99.9% AnalaR grade, BDH) in potassium chloride (99.5% AnalaR grade, BDH). Nitric acid (2\u00a0M volumetric standard, Riedel-de Ha\u00ebn) was used for electrode cleaning, as described in the \u201cProcedure\u201d section.\nProcedure\nFormation of the Ag\/AgCl miniaturised reference electrode\nUsing an external auxiliary platinum wire located in the inlet of the microdevice, the Ag electrode was coated in AgCl (in saturated KCl solution that was also saturated with AgCl through the addition of a small quantity of AgNO3) at 2\u00a0V for 3\u00a0s.\nEx situ electroplating of the BiFE on the rotating disc electrode (RDE)\nTwo electroplating solutions were investigated, (A) and (B).\nAcetate buffer Bi(III) plating solution. 100\u00a0ppm Bi(III) in 1\u00a0M acetate buffer (pH 4.5). Plated for 5\u00a0mins at \u22121.0\u00a0V in a quiescent solution.LiBr\/ HCl Bi(III) plating solution. 0.02\u00a0M Bi(III) in 0.5\u00a0M LiBr and 1\u00a0M HCl. Plated for 60\u00a0s at \u22120.28\u00a0V in a quiescent solution.\nThe RDE was then carefully rinsed before transferring it to the analysis solution.\nIn-line electroplating of the BiFE on the flow cell working electrode\nTwo electroplating solutions were investigated, (A) and (B).\nAcetate buffer Bi(III) plating solution. 100\u00a0ppm Bi in 0.1\u00a0M acetate buffer (pH 4.5). Plated for 5\u00a0mins at \u22121.0\u00a0V at a flow rate of 37.1\u00a0\u03bcl\/s.LiBr\/ HCl Bi(III) plating solution. 0.02\u00a0M Bi(III) in 0.5\u00a0M LiBr and 1\u00a0M HCl. Plated for 120\u00a0s at \u22120.28\u00a0V at a flow rate of 37.1\u00a0\u03bcl\/s.\nAfter formation of the BiFE, the plating flow stream was flushed with water and switched to the analysis stream.\nBismuth film cleaning procedure\nThe Bi(III) film was cleaned of the remaining adsorbed complexes by holding the electrode at a potential of \u22121.3\u00a0V for 30\u00a0s in 0.1\u00a0M HCl.\nBismuth film removal\nBi(III) films were removed by applying a conditioning potential at +0.3\u00a0V, \u22120.5\u00a0V and 0\u00a0V for 2\u2009\u00d7\u200950\u00a0s in a flowing solution of 0.1\u00a0M HNO3.\nIn-line plating of MFE on the flow cell working electrode\nThe Hg plating solution was 200\u00a0ppm in 0.1\u00a0M HNO3. Plated for 120\u00a0s at \u22121\u00a0V at a flow rate of 9\u00a0\u03bcl\/s. After film formation the cell was flushed with water and switched to the analysis stream.\nMercury film cleaning procedure\nThe film was cleaned at a potential of \u22121.4\u00a0V for 10\u00a0s in 0.1\u00a0M HNO3.\nMercury film removal\nFilms were removed at +0.8\u00a0V in a flowing solution of 0.1\u00a0M HNO3 for 60\u00a0s.\nResults and discussion\nThe in-line prepared BiFE was assessed for its suitability in performing CAdSV of Co(II) and Ni(II). Various parameters, such as flow rates, solution composition, plating parameters and square-wave conditions, were studied to investigate the electrochemical behaviour of the Bi(III) films prepared on the injection-moulded carbon fibre electrodes.\nInvestigation of the effect of flow rate during the plating and accumulation stages\nUsing the acetate buffer-based plating solution, i.e. the 100\u00a0ppm Bi(III) in 0.1\u00a0M acetate buffer, the effect of varying the flow rate during BiFE plating procedure was examined at 20, 25 and 37\u00a0\u03bcl\/s by varying the height of the reservoir in the gravity-fed flow system (with sample conditions as given in Fig.\u00a02). The maximum response was obtained for the stripping peaks at a flow rate of 37\u00a0\u03bcl\/ s and so this rate was subsequently employed in this study.\nFig.\u00a02Influence of flow rate during the accumulation stage on the Ni(II) and Co(II) CAdSV peaks, where the flow rates are: (a) 5.8, (b) 15.7, (c) 25.6, (d) 37.1, (e) 42.9 and (f) 55.5\u00a0\u03bcl\/s respectively. Carried out with a 10\u00a0ppb sample of Co(II) and Ni(II) in 0.1\u00a0M ammonium buffer (pH 9.2) with 0.1\u00a0mM DMG and 0.5\u00a0M NaNO2. SW conditions: Conditioning potential \u22121.3\u00a0V (10\u00a0s), deposition potential \u22120.8\u00a0V (120\u00a0s), equilibration time 5\u00a0s, pulse frequency 15\u00a0Hz, pulse amplitude 40\u00a0mV and step potential 4\u00a0mV. Scan: initial \u22120.8\u00a0V to end potential \u22121.3\u00a0V. Data were smoothed with a level-2 Savitzky\u2013Golay filter\nThe effect of varying the flow rate during the accumulation stage on the peak heights of Co(II) and Ni(II) was studied over the range of 5.8\u201355.5\u00a0\u03bcl\/s; the results are depicted in Fig.\u00a02. The response for Co(II) increased linearly with increasing flow rate with a slope of \u22120.036\u00a0\u03bcAs\/ \u03bcl and an intercept of \u22121.26\u00a0\u03bcA with a correlation coefficient (R2\u2009=\u20090.96), whereas that for Ni(II) did not show significant variation. A flow rate of 37\u00a0\u03bcl\/s was selected, as this gave an acceptable response without adverse peak broadening and was compatible with the BiFE plating step flow rate, thereby simplifying the instrumentation.\nEffect of composition of the BiFE plating solution\nReported methods for AdSV of Co(II) and Ni(II) have generally employed acetate buffer (pH 4.5) containing 100\u00a0mg\/l Bi(III) [15, 24, 26, 28]. However, Kr\u00f3licka et al. [29, 30] suggested the use of a bromide-modified plating solution. Hence, both plating solutions were assessed using the RDE and flow cell setups. Figure 3 depicts the differing performances of the two plating solutions. The LiBr-containing plating solution gave the best stripping responses on both the RDE and the flow cell and was therefore used for subsequent studies.\nFig.\u00a03Comparison of different Bi(III) plating solutions. a Effect of (a) 100\u00a0ppm Bi(III) in acetate buffer (1\u00a0M, pH 4.5) and (b) 0.02\u00a0M Bi(III) in 0.5\u00a0M LiBr- and 1M HCl-based preplating solutions on RDE at 500\u00a0rpm. b Effect of (c) the acetate buffer (0.1\u00a0M, pH 4.5) and (d) LiBr\/HCl preplating solutions on the flow cell. c Comparision of acetate buffer pH\u00a04.5 Bi(III) preplating solution on (e) flow cell (0.1\u00a0M) and (f) RDE (1\u00a0M). d Comparison of LiBr preplating solution on (g) flow cell and (h) RDE. Carried out with a 10\u00a0ppb sample of Co(II) and Ni(II) in 0.1\u00a0M ammonium buffer (pH 9.2) with 0.1\u00a0mM DMG and 0.5\u00a0M NaNO2. SW conditions: Conditioning potential \u22121.3\u00a0V (10\u00a0s), deposition potential \u22120.8\u00a0V (120\u00a0s), equilibration time 10\u00a0s, pulse frequency 25\u00a0Hz, pulse amplitude 50\u00a0mV and step potential 5\u00a0mV (except trace (e) which was at 15\u00a0Hz, 40 and 4\u00a0mV respectively). Scan: initial \u22120.8\u00a0V to end potential \u22121.3\u00a0V\nEffect of solution parameters on the system behaviour\nBuffer concentration The effect of changing the ammonium buffer concentration over the range 0\u20130.5\u00a0M is shown in Fig.\u00a04a. Increasing the buffer concentration over the range 0\u20130.1\u00a0M had a marked effect on the height of the Co(II) peak, but increasing the concentration further did not improve the peak height due to the increased background signal. As Co(II) and Ni(II) were sufficiently resolved at 0.1\u00a0M, this concentration was selected for all further experiments.\nFig.\u00a04Effect of solution parameters on the CAdSV behaviour of a 10\u00a0ppb Co(II) and Ni(II) sample: (a) ammonium buffer (pH 9.2) concentration over the range 0\u20130.5\u00a0M, (b) DMG concentration over the range 0\u20130.35\u00a0M and (c) nitrite concentration over the range 0\u20130.8\u00a0M. Supporting electrolyte conditions were: (b and c) 0.1\u00a0M ammonium buffer (pH 9.2), (a and c) 0.1\u00a0mM DMG and (a and b) 0.5M NaNO2. SW conditions were: conditioning potential \u22121.3\u00a0V (10 s), deposition potential \u22120.8\u00a0V (120\u00a0s), equilibration time 10\u00a0s, pulse frequency 25\u00a0Hz, pulse amplitude 40\u00a0mV (except c: 50\u00a0mV) and step potential 4\u00a0mV (except c: 5\u00a0mV). Scan: initial \u22120.8\u00a0V to end potential \u22121.3\u00a0V\nEffect of DMG concentration Figure 4b shows the effect of increasing DMG concentration, over the range 0.01\u20130.35\u00a0mM, on the peak current. The Co(II) signal increases almost linearly with increasing DMG concentrations, whereas the signal for Ni(II) steadily decreased to 0.2\u00a0mM whereupon it levelled off. The signal for the Co(II) peak shifted to a slightly more negative potential (\u22124\u00a0mV) than that for Ni(II) with increasing concentration. A concentration of 0.3\u00a0mM was employed in the subsequent studies.\nEffect of nitrite concentration Figure 4c shows the influence of nitrite concentration on the sample signal. The Co(II) signal shows a rapid increase in signal to 0.3\u00a0M and a slower increase thereafter, whereas the Ni(II) peak height remained level to a concentration of 0.6\u00a0M. Concentrations of greater than 0.6\u00a0M lead to increased background current and so a nitrite concentration of 0.5\u00a0M was subsequently employed. Figure 5 shows the results obtained from the addition of nitrite to the sample system. Addition of nitrite to separate samples of Ni(II) and Co(II) results in increases in response for both metals, Fig.\u00a05a and b, whereas the addition of nitrite to a mixture of 10\u00a0ppb Co(II) and Ni(II) results in an almost unchanged Ni(II) peak height whilst that for Co(II) has increased elevenfold, Fig.\u00a05c. Figure 5d shows the reduction (16.6%) of the Co(II) peak height upon the addition of 10\u00a0ppb Ni(II), while Fig.\u00a05e displays the reduction (51.9%) of the Ni(II) peak height upon the addition of 10\u00a0ppb Co(II).\nFig.\u00a05a Effect of nitrite on stripping peak for 10\u00a0ppb Ni(II) when (a) no NaNO2 is present; (b) 0.5\u00a0M NaNO2 is present. b Effect of nitrite on stripping peak for 10\u00a0ppb Co(II) when (c) no NaNO2 is present; (d) 0.5\u00a0M NaNO2 is present. c Effect of nitrite on the stripping peaks for 10\u00a0ppb Co(II) and Ni(II) when (e) no NaNO2 is present; (f) 0.5\u00a0M NaNO2 is present. d Effect of addition of 10\u00a0ppb Ni(II) on the peak height for 10\u00a0ppb Co(II) in the presence of 0.5\u00a0M NaNO2 when (h) Co(II) only is present and (g) both Co(II) and Ni(II) are present. e Effect of addition of 10\u00a0ppb Co(II) on the peak height for 10 ppb Ni(II) in the presence of 0.5\u00a0M NaNO2 when (j) Ni(II) only is present and (k) Co(II) and Ni(II) are present. Carried out in 0.1\u00a0M ammonium buffer (pH 9.2) with 0.1\u00a0mM DMG. SW conditions: conditioning potential \u22121.3\u00a0V (10\u00a0s), deposition potential \u22120.8\u00a0V (120\u00a0s), equilibration time 5\u00a0s, pulse frequency 25\u00a0Hz, pulse amplitude 50\u00a0mV and step potential 5\u00a0mV. Scan: initial \u22120.8\u00a0V to end potential \u22121.3\u00a0V\nEffect of varying the square-wave (SW) stripping parameters\nFrequency The effect of frequency was studied in the range 12.5\u2013100\u00a0Hz, Fig.\u00a06a depicts the results obtained. The peak heights for both metals increased with increasing SW frequency (as expected from increasing the effective scan rate) and shifted towards more negative values. However, increasing the frequency leads to insufficient time for the background current to decay and leads to an increase in the contribution to the signal and hence a distortion in peak shape. A frequency of 25\u00a0Hz was generally the maximum employed.\nFig.\u00a06Effect of varying the SW stripping parameters on the CAdSV peaks of a 10\u00a0ppb Co(II) and Ni(II) sample. The variables are (a) frequency (12.5\u2013100\u00a0Hz), (b) step potential (0.15\u20137\u00a0mV), (c) amplitude (5\u201370\u00a0mV), (d) deposition potential (\u22120.3 to \u22120.9\u00a0V), (e) deposition time (30\u2013210\u00a0s). Supporting electrolyte: 0.1\u00a0M ammonium buffer (pH 9.2) with 0.1\u00a0mM DMG and 0.5\u00a0M NaNO2. SW conditions were (unless being investigated as above): conditioning potential \u22121.3\u00a0V (10\u00a0s), deposition potential \u22120.8\u00a0V (120\u00a0s), equilibration time 5\u00a0s, frequency 25\u00a0Hz, pulse amplitude 50\u00a0mV (except b and e: 40\u00a0mV) and step potential 5\u00a0mV (except e: 4\u00a0mV). Scan: initial \u22120.8\u00a0V to end potential \u22121.3\u00a0V\nStep potential Figure 6b shows the effect of the step potential on the peak heights. The response for Co(II) rose rapidly with rising step potentials (i.e. with an increase in effective scan rate) up to 3\u00a0mV and thereafter levelled off, whereas the response for Ni(II) increased slowly to 7\u00a0mV. Peak resolution remained the same across this step potential range, with both peaks shifting towards more negative potentials. Values of 4 or 5\u00a0mV were generally found to be ideal for adequate peak definition.\nAmplitude The effect of the SW pulse amplitude was studied over the range 5\u201380\u00a0mV. Both peaks shifted to more positive values, see Fig.\u00a06c and they both initially increased rapidly in height (5\u201320\u00a0mV) whereupon the responses for Co(II) and Ni(II) levelled off as a result of the increase in contribution from the background current. A SW pulse amplitude of either 40 or 50\u00a0mV was employed.\nDeposition potential Figure 6d shows the influence of the deposition potential on the peak currents. At potentials less than \u22120.4\u00a0V the Ni(II) signal is completely suppressed and the Co(II) signal was markedly reduced due to the oxidation of the bismuth film (at around \u22120.46\u00a0V in alkaline solutions). Both peaks gradually increase with potential in the range of \u22120.4\u00a0V to a maximum at \u22120.8\u00a0V and then exhibit a sharp reduction in adsorption at a more negative potential of \u22120.9\u00a0V resulting from further reduction of the analyte complex. A deposition potential of \u22120.8\u00a0V was selected, as this gave the maximum response for both metals.\nAccumulation time The influence of the accumulation time on the stripping signal was examined. Figure 6e depicts the response in peak currents for 10\u00a0ppb each of Ni(II) and Co(II). For both Co(II) and Ni(II) the response increases with accumulation time up to 150\u00a0s for Co(II) and 180\u00a0s for Ni (II), after which the response levels off, indicating saturation of the surface. An accumulation time of 120\u00a0s was used for subsequent measurements as this gave the best compromise between sensitivity and analysis time.\nAnalytical characterisation\nThe simultaneous determination of Co(II) and Ni(II) over the concentration range of 1\u201320\u00a0ppb was investigated. The determination was carried out in 0.1\u00a0M ammonium buffer (pH 9.2) with 0.1\u00a0mM DMG and 0.5\u00a0M NaNO2. SW conditions: conditioning potential \u22121.3\u00a0V (10\u00a0s), deposition potential \u22120.8\u00a0V (120\u00a0s), equilibration time 5\u00a0s, pulse frequency 25\u00a0Hz, pulse amplitude 50\u00a0mV and step potential 5\u00a0mV. Scan: initial \u22120.8\u00a0V to end potential \u22121.3\u00a0V. The BiFE exhibited a well-defined response to changing concentration and the peak current response (y, \u03bcA) was linear over the concentration range (x, ppb) of 1\u201320\u00a0ppb, as expressed in the calibration plots for Co(II),y\u2009=\u2009\u221211.4x \u2212 85.5 (R2\u2009=\u20090.96), and Ni(II),y\u2009=\u2009\u22121.64x \u2013 7.20 (R2\u2009=\u20090.92), respectively.\nThe stability of the BiFE formed on the injection-moulded substrates was investigated by performing replicate runs on the same film. Ten replicate runs gave an average peak height for Ni(II) of \u22121.05\u2009\u00b1\u20090.10\u2009\u00d7\u200910\u22125 A (rsd: 9.6%) and for Co(II) \u22128.19\u2009\u00b1\u20090.82\u2009\u00d7\u200910\u22125 A (rsd: 10.0%).\nTo compare the working electrode behaviour, AdSV and CAdSV of Co(II) and Ni(II) were also performed at a MFE in the flow cell. The solutions needed to be deaerated before analysis on the MFE. Neither AdSV or CAdSV of Co(II) on a MFE was possible in these particular flow cells due to a significant shift in the stripping peak to an extreme cathodic potential (<\u22121.4\u00a0V vs Ag\/AgCl), which led to analytical uncertainty at the edge of the supporting electrolyte potential window. However, AdSV of Ni(II) was possible, as illustrated in Fig.\u00a07. It has been noted previously by this group [57, 58] that the main drawback with MFEs is the difficulty involved in regenerating the active mercury surface after use, necessitating the inclusion of cleaning and mercury film renewal procedures. In contrast to the MFEs, the BiFEs formed in these flow cells enabled the generation of reproducible, quantitative analytical data, due to the enhanced stability of the bismuth films during the cleaning cycles, and the reduced cathodic potential for the cobalt stripping peak.\nFig.\u00a07AdSV of Ni(II) on a mercury film electrode. Here decimal numerals indicate the film number and roman numerals indicate replicate analyses carried out on the same film. Carried out with a 200\u00a0ppb sample in 0.1\u00a0M ammonium buffer (pH 9.3) with 0.1\u00a0mM DMG (deaerated) at a flow rate of 16\u00a0\u03bcl\/s. SW conditions: conditioning potential \u22121.4\u00a0V (10\u00a0s), deposition potential \u22120.5\u00a0V (200\u00a0s), equilibration time 5\u00a0s, pulse frequency 25\u00a0Hz, pulse amplitude 10\u00a0mV and step potential 4\u00a0mV. Scan: \u22120.5\u00a0V to end potential \u22121.4\u00a0V. Data were smoothed with a level-2 Savitzky\u2013Golay filter\nConclusions\nThe application of a BiFE for the determination of Co(II) and Ni(II) by square-wave CAdSV under flow conditions in a non-deaerated solution has been demonstrated. The injection-moulded carbon-fibre-filled polystyrene electrodes have proven to be suitable supports for the formation of bismuth films. The LiBr-containing plating solution was found to offer superior performance for both the RDE and the flow cell under the alkaline conditions required for CAdSV. The application of a flow stable BiFE in an inexpensive sensor system permits the possibility of autonomous on-site industrial and environmental monitoring. The inherent disposability of the devices coupled with their mercury-free status should advance the scope of the sensor to point-of-care clinical applications.","keyphrases":["flow cell","bismuth film electrode (bife)","dimethylglyoxime","sensor","electrochemical detection","injection moulding","channel","cobalt","nickel","square-wave adsorptive stripping voltammetry"],"prmu":["P","P","P","P","P","P","P","P","P","R"]}
{"id":"Glycoconj_J-4-1-2234451","title":"High-throughput screening of monoclonal antibodies against plant cell wall glycans by hierarchical clustering of their carbohydrate microarray binding profiles\n","text":"Antibody-producing hybridoma cell lines were created following immunisation with a crude extract of cell wall polymers from the plant Arabidopsis thaliana. In order to rapidly screen the specificities of individual monoclonal antibodies (mAbs), their binding to microarrays containing 50 cell wall glycans immobilized on nitrocellulose was assessed. Hierarchical clustering of microarray binding profiles from newly produced mAbs, together with the profiles for mAbs with previously defined specificities allowed the rapid assignments of mAb binding to antigen classes. mAb specificities were further investigated using subsequent immunochemical and biochemical analyses and two novel mAbs are described in detail. mAb LM13 binds to an arabinanase-sensitive pectic epitope and mAb LM14, binds to an epitope occurring on arabinogalactan-proteins. Both mAbs display novel patterns of recognition of cell walls in plant materials.\nIntroduction\nCarbohydrate microarrays provide a means of rapidly screening the interactions between glycans and other molecules [1\u20137]. Applications for this technology include the screening of protein\u2013glycan interactions, characterization of carbohydrate-active enzymes and the analysis of the specificities of monoclonal antibodies (mAbs) and carbohydrate-binding modules [8, 9]. mAbs are powerful tools for investigating the biological roles of glycans but producing them is time consuming, labour-intensive and costly. Hybridoma-based mAb production involves the fusion of spleen cells from an immunized animal with myeloma cells. The resulting hybridoma cell lines are both immortal and secrete antibody into the cell supernatant [10]. This procedure is conventionally performed in a one-by-one fashion, such that each animal is immunized with a single antigen and the resulting antibodies are screened for desired specificities using enzyme-linked immunosorbent assays (ELISAs). However ELISA-based screening is low throughput because only a limited amount (\u223c100\u00a0\u03bcl) of hybridoma supernatant is available for screening during the initial stages of mAb production and this is typically only sufficient to test each mAb against just one or two antigens. An alternative approach involves \u2018shotgun\u2019 immunisation with a mixture of antigens [11]. This potentially results in the simultaneous generation of antibodies with a range of specificities, but the time limiting step then becomes the detailed retrospective screening of their specificities. However, microarrays offer a means of rapidly screening limited amounts of hybridoma supernatant against multiple antigens and therefore greatly increasing throughput in the identification of valuable cell lines. Here we report the use of shotgun immunisation followed by microarray-based screening of hybridoma supernatants in order to produce mAbs against plant cell wall glycans.\nPlant cell walls are fibre composites that contain some of the most complex glycans known [12, 13]. In addition to their biological roles, many cell wall components have important industrial applications including as functional food ingredients, pharmaceuticals, nutriceuticals, fibres and increasingly, bio-fuels [14\u201316]. Cell wall glycans can be broadly grouped into cellulose, hemicelluloses, pectins and glycoproteins [17]. Cellulose microfibrils are cross-linked by hemicelluloses such as xyloglucans, xylans and mixed linkage glucans forming a tough load-bearing matrix which is embedded in pectic polysaccharides [18]. Pectins are the most complex and heterogeneous family of cell wall glycans and are comprised of a series of galacturonic acid-rich polymers including homogalacturonan (HG), rhamnogalacturonan I (RGI), rhamnogalacturonan-II (RG-II) and xylogalacturonans (XG). In addition, structurally complex arabinan, galactan and arabinogalactan polymers may be present as side chains to the galacturonan-rich backbone domains [18]. The protein moieties of cell wall glycoproteins are often rich in hydroxyproline (Hyp) and these polymers, which are collectively referred to as Hyp-rich glycoproteins (HRGPs), include the extensins and arabinogalactan-proteins (AGPs) [19\u201321].\nThe fine structures and relative amounts of cell wall components vary greatly not only among plants, but also between organs, tissues, cells, and even between different micro-domains within a single cell wall. This complexity and heterogeneity presents a major barrier to detailed analysis and our understanding of many aspects of plant cell wall structure and function is far from complete. Several mAbs with specificities for diverse plant cell wall components have been developed and these are powerful tools for the analysis of cell walls [22]. However, the repertoire of mAbs currently available covers only a small proportion of the glycan structures that have been identified and there is a pressing need for a wider range of mAbs to facilitate the further characterization of cell walls. We have developed a method of rapidly screening mAb specificities using microarrays of cell wall glycans including pectins with different degrees and patterns of methyl-esterification, pectic side chains (e.g. arabinan and galactan), hemicelluloses (e.g. xylans, mannans, and xyloglucans), HRGPs (e.g. AGP-rich gums), \u03b2-linked glucans (e.g. (1\u21923)(1\u21924)-\u03b2-glucan and (1\u21923)-\u03b2-glucan) and celluloses (e.g. hydroxylethyl cellulose and carboxylmethyl cellulose). A further 14 samples were sequentially extracted from A. thaliana using CDTA and NaOH which are known to predominately solublize pectic polymers and hemicelluloses respectively [23].\nExperimental procedures\nImmunisation and generation of monoclonal hybridomas\nCell wall polymers were isolated from 6\u00a0week old wild type A. thaliana plants, ecotype Col-0, grown in soil at 22\u00b0C with cycles of 10\u00a0h of light\/14\u00a0h of darkness. One g dry weight of a mixture of leaves, stems and roots was homogenized to a fine powder in liquid nitrogen. The homogenate was incubated with 20\u00a0ml 50\u00a0mM 1,2-Diaminocyclohexanetetraacetic acid (CDTA; pH\u00a07.5) for 3\u00a0h at 18\u00b0C and centrifuged for 20\u00a0min at 4,400\u00a0rpm. The supernatant was collected and dialyzed extensively against deionized water (dH2O) in dialysis tubing (6\u20138,000\u00a0kDa molecular weight cut off) to remove low molecular weight molecules and freeze dried. The material was dissolved in phosphate-buffered saline (PBS) to generate the immunogen. Rats were used for antibody production so as to make subsequent comparisons with existing mAbs, most of which were produced in rats, as valid as possible. The immunization of rats, hybridoma preparation and cloning procedures were as described previously [24]. Briefly, two male Wistar rats were each injected subcutaneously with 250\u00a0\u03bcl of an emulsion of the isolated cell wall material at 1\u00a0mg\/ml in PBS with an equal volume Freund\u2019s complete adjuvant on day\u00a00. On days\u00a040 and 79 the injections were repeated using incomplete adjuvant. Tail bleeds were taken 10\u00a0days after injections to assess the immune response. On day\u00a0198 a pre-fusion boost was given to the selected rat and 3\u00a0days later, the spleen was removed and lymphocytes were isolated and fused with rat myeloma cell line IR983F [25] using standard polyethylene glycol fusion of lymphocytes and myeloma cells. Hybridoma lines were initially screened by ELISA with the immunogen coated onto microtitre plates (MaxiSorp, Nunc, Roskilde, Denmark) at 50\u00a0\u03bcg\/ml.\nPreviously described monoclonal antibodies\nArrays were probed with 23 mAbs with previously described specificities and details of these are provided in Table\u00a01. All were rat antibodies except mAbs CCRC-M1, BS-400-02, BS-400-03, and BS-400-04 which were produced in mice and PAM1 which was produced by phage display.\nTable\u00a01Previously characterized monoclonal antibodies used to probe glycan arrays. HG, homogalacturonanSpecificityNameRef.Un-esterified HGPAM1[31]Un-esterified\/Calcium ion cross-linked HG2F4[34]Partially methyl-esterified HGJIM5[33]Partially methyl-esterified HGJIM7[33](1\u21924)-\u03b2-galactanLM5[37](1\u21925)-\u03b1-arabinanLM6[24]Fucosylated xyloglucanCCRC-M1[38]Non-fucosylated xyloglucanLM15(1\u21924)-\u03b2-mannan\/galacto-(1\u21924)-\u03b2-mannanBS-400-4[39](1\u21924)-\u03b2-xylanLM10[40](1\u21923)(1\u21924)-\u03b2-glucanBS 400-3[41](1\u21923)-\u03b2-glucanBS 400-2[32](1\u21924)-\u03b2-xylan\/arabinoxylanLM11[40]Arabinogalactan-proteinJIM4[43]Arabinogalactan-proteinLM2[42]Arabinogalactan-proteinMAC207[43]Arabinogalactan-proteinJIM8[44]Arabinogalactan-proteinJIM13[43]Arabinogalactan-proteinJIM16[43]Arabinogalactan-proteinJIM14[43]ExtensinLM1[45]ExtensinJIM19[46]ExtensinJIM20[46]\nGlycan samples used on the array\nDetails of the 50 defined glycans used are provided in Table\u00a02. Most glycans were dissolved in dH2O. Arabinoxylan and glucuronoxylan were prepared by boiling in dH2O for 10\u00a0min. and then standing for 3\u00a0h at 18\u00b0C before use. Glucomannan was prepared by wetting with 95% ethanol followed by addition of dH2O. The mixture was heated to boiling point and stirred for 20\u00a0min until dissolved. Pachyman was prepared by dissolution in a minimal volume of 10% (w\/v) sodium hydroxide followed by neutralization with acetic acid. 14 samples on the arrays were cell wall polymers extracted from A. thaliana organs listed in Table\u00a02 using CDTA and 4\u00a0M NaOH. Fifty milligrams (fresh weight) of each organ collected from at least four separate plants were homogenized to a fine powder prior to adding 300\u00a0\u03bcl of 50\u00a0mM CDTA (pH\u00a07.5). After incubating with rotation for 4\u00a0h at 20\u00b0C, the extracts were centrifuged at 4,400\u00a0rpm for 10\u00a0min and the supernatants (\u2018CDTA extracts\u2019) removed. Pellets were resuspended in 300\u00a0\u03bcl of 4\u00a0M NaOH and samples were incubated with rotation for 4\u00a0h at 20\u00b0C prior to centrifugation at 4,400\u00a0rpm for 10\u00a0min. Supernatants were \u2018NaOH extracts\u2019.\nTable\u00a02Samples included on the glycan arraysAlphanumerical codesSamplesA1Arabinan (sugar beet)B1Pectin (apple)C1Galactan (lupin)D1Homogalacturonan (sugar beet)E1Pectin (lime) B15F1Pectin (lime) B43G1Pectin (lime) B71H1Pectin (lime) 96A2Pectin (lime) F11B2Pectin (lime) F19C2Pectin (lime) F43D2Pectin (lime) F76E2Pectin (lime) P16F2Pectin (lime) P24G2Pectin (lime) P32H2Pectin (lime) P41A3Pectin (lime) P46B3Pectin (lime) P60C3Pectin (lime) P76D3RGI (soybean)E3RGII (A. thaliana)F3Xylogalacturonan (pea)G3MHR I (apple)H3MHR II (carrot)A4MHR III (potato)B4MHR HS1 (apple)C4MHR HS2 (apple)D4Xylogalacturonan (apple)E4AGP (P. patens)F4Seed mucilage (A. thaliana)G4Xyloglucan\/mannan (tomato)H4Glucomannan (konjac)A5Gum (guar)B5Gum (locust bean)C5Gum arabic (acacia)D5Gum (karaya)E5Gum (tragacanth)F5AGP (larch)G5Arabinoxylan (wheat)H5\u03b2(1-3),(1-4)-glucan (lichenan)A6Mannan (ivory nut)B6Xyloglucan (tamarind)C6Glucuronoarabinoxylan (maize)D6Hydroxyethyl celluloseE6\u03b2(1-4)-glucan (avicel)F6Carboxymethyl celluloseG6Alginic acidH6\u03b2(1-3),(1-6)-glucan (laminarin)A7\u03b2(1-3)-glucan (pachyman)B7\u03b2(1-4),(1-6)-glucan (pullulan)C7CDTA extract (A. thaliana flowers)D7CDTA extract (A. thaliana siliques)E7CDTA extract (A. thaliana stem top)F7CDTA extract (A. thaliana stem middle)G7CDTA extract (A. thaliana stem base)H7CDTA extract (A. thaliana leaves)A8CDTA extract (A. thaliana roots)B8NaOH extract (A. thaliana flowers)C8NaOH extract (A. thaliana siliques)D8NaOH extract (A. thaliana stem top)E8NaOH extract (A. thaliana stem middle)F8NaOH extract (A. thaliana stem base)G8NaOH extract (A. thaliana leaves)H8NaOH extract (A. thaliana roots)Alphanumerical codes refer to the position of samples on arrays. Source organisms are in parenthesesRGI Rhamnogalcturonan I; RGII rhamnogalacturonan II; MHR modified hairy region; AGP arabinogalactan-protein\nPost-printing modification of glycans\nGlycan samples on selected arrays were modified in situ after printing by enzymatic digestion. For the data in Fig.\u00a06 selected arrays were digested with endo-\u03b1(1-5)-L-arabinanase or endo-\u03b2(1-4-)-galactanase (both from Aspergillus niger, Megazyme (Bray, Ireland) used at 1\u00a0U\/ml in 200\u00a0mM sodium acetate pH\u00a04.0.\nPrinting of arrays\nGlycans were applied to nitrocellulose membrane (0.45\u00a0\u03bcm pore size, Schleicher and Schuell, Dassel, Germany) at two concentrations (0.2 and 0.04\u00a0mg\/ml) and in duplicate such that each sample was represented by four spots. CDTA and NaOH extracted A. thaliana material was printed as extracted and as a five fold dilution, also in duplicate. Printing was performed using a microarray robot (Microgrid II, Genomic Solutions, Ann Arbor, MI, USA) equipped with split pins (MicroSpot 2500, Genomic Solutions). Pins were washed twice in dH2O after deposition of each sample.\nProbing of arrays\nArrays were blocked by incubation for 1\u00a0h in PBS (140\u00a0mM NaCl, 2.7\u00a0mM KCl,10\u00a0mM Na2HPO4, 1.7\u00a0mM KH2PO4, pH\u00a07.5) containing 5% w\/v low fat milk powder (5%MPBS). Arrays were then probed for 2\u00a0h with antibodies diluted in 5%MPBS. All antibodies were used as 1\/10 dilutions except CCRC-M1 which was used at 1\/50, and BS-400-2, BS-400-3 and BS-400-4 which were used at 1\/200. After washing with PBS, arrays were incubated for 2\u00a0h in either anti-rat or anti-mouse secondary antibody conjugated to alkaline phosphatase (Sigma, Poole, UK) diluted 1\/5000 in 5%MPBS. After washing in PBS, arrays were developed using a substrate containing 5-bromo,4-chloro,3-indolylphosphate (BCIP) and nitroblue tetrazolium (NBT) in BCIP\/NBT buffer (100\u00a0mM NaCl, 5\u00a0mM MgCl2, 100\u00a0mM diethanolamine, pH\u00a09.5).\nScanning and analysis\nArrays were scanned, converted to 16 bit grey-scale TIFFs, transformed to negative images and uploaded into ImaGene 6.0 microarray analysis software (BioDiscovery, El Segundo, CA, USA). Semi-automatic gridding was used to create an analysis area for each spot, and a 5-pixel zone around each spot was used for calculation of local background signals. Individual spot signals were defined as the mean pixel value within each spot area (red zone in Fig.\u00a01d) minus the median pixel value in the surrounding local background area (green zone in Fig.\u00a01d). The mean of the four individual spot signals for each sample was defined as the \u2018mean sample value\u2019 (MSV) and \u2018total mean sample values\u2019 (TMSVs) were the means of MSVs from three separate experiments. Selected data sets (TMSVs) were used to generate heatmaps (Figs.\u00a02b and 5b) using online heatmapper software (http:\/\/bbc.botany.utoronto.ca\/ntools\/cgi-bin\/ntools_heatmapper.cgi). For each antibody in the heatmap, the maximal TMSV was set to 100% and all other values within that data set adjusted accordingly. A cut off of 5% of the maximal TMSV was imposed. Hierarchical cluster analysis was performed on TMSVs obtained for 23 previously characterized antibodies and 7 newly produced ones using Epclust software (http:\/\/ep.ebi.ac.uk\/EP\/EPCLUST\/). Clustering was based on correlation measure based distance and average linkage.\nFig.\u00a01Microarrays of plant cell wall glycans. a, b A library of 50 glycan polymers were spotted onto nitrocellulose membranes in an area 22\u00a0mm\u2009\u00d7\u200922\u00a0mm. Arrays included pectins, HRGPs, hemicelluloses and glucans (samples, A1\u2013B7) and the position of these classes of polymer on the arrays are indicated by colour coding in (b). Fourteen samples extracted from Arabidopsis thaliana using either CDTA (samples C7\u2013A8) or NaOH (samples B8\u2013H8), were also included. Details of the samples are provided in Table\u00a02. c Each sample was printed at the two concentrations indicated and in duplicate. d Microarray analysis software (ImaGene 6.0) was used to quantify spot signals and individual spot signals were defined as the mean pixel value within each spot area (red zone) minus the median pixel value in the surrounding local background area (green zone). e\u2013g Two pairs of three arrays were printed and probed with the anti-homogalacturonan mAb JIM5 on three separate occasions. The mean sample values (MSVs) for each pair were quantified and plotted against MSVs of an array produced on a different occasion. These are presented as scatter plots, with the R2-values for the plots indicatedFig.\u00a02Probing of cell wall glycan arrays with mAbs with known specificities. a Glycan arrays were probed with 23 mAbs with previously defined specificities and 5 representative examples are shown: PAM1 (anti-homogalacturonan); BS-400-2 (anti-(1\u21923)-\u03b2-glucan; LM5 (anti-(1\u21924)-\u03b2-galactan); JIM13 (anti-arabinogalactan-protein); JIM5 (anti-homogalacturonan). A control array was probed with secondary antibody, but no primary mAb. Arrays were scanned and converted into 16 bit greyscale TIFFs. b Heatmap showing the total mean sample values (TMSVs) for the binding of each mAb to each sample. The maximal TMSV for each mAb was set to 100 and all other values were adjusted accordingly. A cut off of 5% of the maximal TMSV was imposed and all values at, or below this are represented as black boxes\nIndirect immunofluorescence labeling of plant materials\nResin embedded and fresh plant material was sectioned and labelled with antibodies as described previously [26, 27]. LM14 and LM13 hybridoma supernatants were used as 1\/10 dilutions in 5%MPBS. Sections were counterstained for cellulose with Calcofluor white (fluorescent brightener 28, Sigma, Poole, UK) used as a 0.005% aqueous solution.\nCompetitive-inhibition ELISA assays\nThe ability of arabinose, galactose, rhamnose and oligoarabinosides to inhibit the binding of LM14 and LM13 to the immuogen in ELISAs was assessed as described previously [28].\nSugar composition analysis of modified pectic hairy regions (MHRs)\nSugar composition was determined using methanolysis as described previously [29]. MHRs were treated with 2\u00a0N HCl in dry methanol for 16\u00a0h at 80\u00b0C, followed by 1\u00a0h of 2\u00a0M CF3CO2H (TFA) at 121\u00b0C. The released sugars were analysed using high-performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) as described previously [30].\nResults\nProduction of microarrays of plant cell wall polymers\nThe layout of the arrays is shown in Fig.\u00a01 and colour coding indicates the distribution on the arrays of different classes of cell wall polymer (Fig.\u00a01a,b). A detailed list of the samples used is provided in Table\u00a02. Each sample was represented on the arrays by four spots (two concentrations, printed in duplicate) (Fig.\u00a01c) and microarray analysis software was used to quantify background-corrected spot signals (Fig.\u00a01d). As shown by the arrays in Figs.\u00a02a and 3, the consistency of the printing and probing replicates within arrays was high. However, reproducibility between arrays was also tested. A total of six arrays (3\u2009\u00d7\u20092 pairs) were printed and probed with the anti-HG mAb JIM5 on three separate occasions. The MSVs were quantified and the values for each array were plotted against those from an array produced on a different occasion. The R2-values obtained for these plots were all >0.98, indicating a high degree of reproducibility between arrays (Fig.\u00a01e\u2013g).\nFig.\u00a03Probing of cell wall glycan arrays with newly produced mAbs. Glycan arrays were probed with mono- or multiclonal antibodies generated following shotgun immunisation with plant cell wall polymers. Four representative examples are shown. Arrays were scanned and converted into 16 bit greyscale TIFFs\nProbing of plant cell wall glycan arrays\nArrays were probed with a range of mAbs with previously defined specificities for epitopes occurring on the major classes of cell wall polymers. Details of the mAbs used are provided in Table\u00a01. A total of 23 mAbs were tested and 5 representative examples are shown in Fig.\u00a02a. The TMSVs for the binding of these mAbs to each sample (derived from three independent experiments) are shown as a heatmap in Fig.\u00a02b, where spot signals are correlated to colour intensity. mAb binding profiles were in good agreement with the published results for the antibodies tested. For example, mAb PAM1 has specificity for HG with a low degree of methyl-esterification (DE) [31] and consistent with this, PAM1 bound to lime pectin samples with DEs of 11\u201319% (samples E1, A2 and B2). PAM1 did not bind above background to other pectin samples with higher DEs, or to any other cell wall glycans. mAb BS-400-2 is specific for (1\u21923)-\u03b2-glucan [32] and, of the defined samples, only bound to this polymer (sample A7) on the arrays. (1\u21923)-\u03b2-glucan is known to be extractable from plant cell walls using NaOH and consistent with this, BS-400-2 also bound to weakly to NaOH-solubilized extracts from A. thaliana (samples B8-H8). As expected, the epitopes recognized by some mAbs were detected on multiple samples and this was the case for mAbs LM5 (anti-(1\u21924)-\u03b2-galactan), JIM13 (anti-AGP) and JIM5. However, the binding profiles of these mAbs were also consistent with their known specificities. For example, JIM5 bound with greatest avidity to pectin samples that contained abundant HG domains (such as the lime pectins E1-C3), but not to pectic fragments lacking HG (such as the arabinan or galactan samples A1 and C1).\nTo generate a new series of cell wall-directed mAbs, rats were immunized with an immunogen consisting of a crude extract of cell wall material solubilized from A. thaliana using CDTA. 13 cell lines were selected by an initial ELISA analysis against the immunogen and these were subsequently probed against the cell wall polymer arrays and four representative examples are shown in Fig.\u00a03. Antibodies that bound with very low avidity, or appeared to have identical binding profiles to previously generated antibodies were not selected for further analysis whilst the data for the remaining seven were subjected to hierarchical cluster analysis.\nHierarchical clustering of antibody binding profiles\nCluster analysis was used to rapidly obtain information about the specificities of 7 new antibodies based on the similarly of their binding profiles on the arrays to 23 previously characterized mAbs (Fig.\u00a04). Clustering indicated that most of the antibodies tested separated into 4 broad clusters: HG-binders; pectic side chain-binders; HRGP-binders and hemicellulose-binders. Three antibodies with specificity for (1\u21923)(1\u21924)-\u03b2-glucan, (1\u21923)-\u03b2-glucan and (1\u21924)-\u03b2-mannan did not cluster with any other mAbs within the 0.55 (distance measurement units) cut off imposed. These broad clusters were in agreement with the previously defined specificities of the mAbs tested, and this was also the case within sub-clusters. For example, within the HG-binders cluster, mAbs JIM5 and JIM7, which both bind to partially methyl-esterified HG [33], clustered closely, whilst mAbs 2F4 and PAM1, which both bind to non-methyl-esterified HG also clustered closely [31, 34]. Similarly, within the hemicellulose-binders, the anti-xylan mAbs LM10 and LM11 formed a sub-cluster, as did the two anti-xyloglucan mAbs CCRC-M1 and LM15. One of the new mAbs (1H12, designated LM13 when cloned) was grouped with the pectic side chain-binders LM5 (anti-(1\u21924)-\u03b2-galactan) and LM6 (anti-(1\u21925)-\u03b1-arabinan). However, most of the new antibodies tested were grouped in the HRGP-binders cluster and within this, clustered most closely with the anti-AGP mAbs MAC207, JIM4 and JIM16. One of the new mAbs within this group (1B3, designated LM14 when cloned) was, together with LM13 subject to more detailed epitope characterization in order to further explore the evidence gained from the clustering analysis.\nFig.\u00a04Cluster analysis of antibody binding profiles to arrays. 23 mAbs with previously defined specificities, and 7 newly produced antibodies (red diamonds in black boxes) were analyzed using Epclust software. Hierarchical clustering was performed using clustered correlation measure based distance and average linkage. The binding profiles of all the mAbs analyzed clustered into four broad groups: 1 homogalacturonan-binders; 2 pectic side chain-binders; 3 HRGP-binders; and 4 hemicellulose-binders. Three mAbs were non-clustering outliers (NCO). The specificities of the 23 previously defined mAbs are listed in Table\u00a01\nDetailed characterization of the epitopes recognized by LM14 and LM13\nCluster analysis of LM13 and LM14 suggested that the epitopes recognized by these mAbs may contain neutral sugars such as arabinose and galactose, that are abundant in the side chains of both pectins and HRGPs. The glycan arrays contained five 5 samples of branched (or \u2018hairy\u2019) regions of pectic polymers modified by enzymatic digestion (\u2018modified hairy regions\u2019 or MHRs) that were rich in neutral sugars and for which monosaccharide composition was determined (Fig.\u00a05). The binding of LM14 and LM13 to these samples was therefore of particular interest. LM14 bound only to an MHR from carrot (sample H3), whilst LM13 bound to MHRs from potato and apple (samples G3, A4 and B4). It was noteworthy that the two MHR samples to which LM13 did not bind (samples H3 and C4) had the lowest arabinose content, suggesting that the epitope recognized by this mAb may contain arabinose. In order to explore this possibility further, the sensitivity of the LM13 epitope to arabinanase digestion was investigated. To do this, cell wall glycan microarrays were digested with arabinanase prior to probing with mAbs, and the TMSVs obtained with and without digestion were compared. These data are presented as scatter plots (Fig.\u00a06a\u2013d) in which signals from enzyme treated arrays were plotted against signals from untreated arrays. As a positive control, arabinanase digested and untreated arrays were probed with the anti-(1\u21925)-\u03b1-arabinan mAb LM6 [24]. As expected, the binding of LM6 to all samples was reduced to some extent by arabinanase digestion (Fig.\u00a06a). However, the effect of arabinanase digestion on LM13 binding was even more pronounced than for LM6 and LM13 binding to all samples on the arrays was essentially abolished (Fig.\u00a06b). This suggested that the epitope recognized by LM13 either contained arabinan and was degraded by arabinanase, or that the epitope was attached to arabinan and was released from arrays by arabinanase. In contrast, LM14 binding to all samples was essentially unaffected by arabinanase digestion (Fig.\u00a06c). The effect of galactanase digestion on mAb binding to arrayed samples was also assessed. LM13 binding was reduced to some extent by galactanase digestion, but the reduction was far less for than arabinanase digestion (Fig.\u00a06d). LM14 binding to all samples was unaffected by galactanase digestion, and the ability of this enzyme to degrade galactan was indicated by the fact that the binding of the anti-(1\u21924)-\u03b2-galactan mAb LM5 was essentially abolished by galactanase digestion (data not shown). The epitope recognized by LM13 was further characterized using competitive inhibition ELISA (ciELISA) assays (Fig.\u00a06e). The binding of LM13 to immobilized arabinan was inhibited to 50% of maximal binding by approximately 450\u00a0\u03bcg\/ml of both arabino-\u03b11,5-octaose and arabino-\u03b11,5-pentaose, but oligoarabinosides of lower degrees of polymerisation and rhamnose, galactose and glucuronic acid failed to inhibit binding at the highest concentration tested of 1\u00a0mg\/ml;. The binding of LM14 to immobilized antigen was not inhibited by any of the haptens tested at concentrations up to 1\u00a0mg\/ml (data not shown).\nFig.\u00a05Binding of LM13 and LM14 to samples to pectic modified hairy regions (MHRs). a Monosaccharide composition of 5 pectin modified hairy regions (MHRs) from apple (samples G3, B4 and C4), carrot (sample H3), potato (sample A4). b TMSVs for the binding of mAbs LM14 and LM13 to these samples. The maximal TMSV for LM13 and LM14 (to all samples on the array) was set to 100 and all other values adjusted accordingly. A cut off of 5% of the maximal TMSV was imposed and all values at, or below this are represented as black boxesFig.\u00a06The epitope recognized by LM13 is arabinanase sensitive. a\u2013d Scatter plots showing the effect of galactanse and or arabinanase on the binding of LM13, LM14 and LM6 (anti-(1\u21925)-\u03b1-arabinan) to glycan arrays. Arrays were incubated in enzymes after blocking but before probing. TMSVs were determined and plotted against each other. Spots that lie on the dotted line indicate that enzyme digestion had no effect on mAb binding, whereas spots that lie below the dotted line indicate where enzyme digestion reduced mAb binding. e The binding of LM13 to oligoarabinosides was tested by competitive inhibition ELISA, in which arabinan was the immobilized polymer. Oligoarabinosides with degrees of polymerization less than five failed to inhibit LM13 binding, even when used at 1\u00a0mg\/ml. Both arabino-\u03b11,5-octaose and arabino-\u03b11,5-pentaose inhibited the binding of LM13 by 50% of maximal binding when used at approximately 450\u00a0\u03bcg\/ml\nCluster analysis suggested that LM13 was most similar to that of mAbs with specificity for pectic side chains, whilst the binding of LM14 was most similar to that of anti-AGP mAbs. AGPs can be distinguished from pectic polymers using SDS-PAGE because whereas AGPs resolve as characteristic smears, pectic polymers typically do not enter gels [35]. Western blots were prepared using the same A. thaliana CDTA-extracted material that was used for immunisation and probed with LM13 or LM14 (Fig.\u00a07). LM13 did not bind to material on these blots whereas LM14 bound to a high molecular weight smear (\u223c70\u2013200\u00a0kDa) that is characteristic of AGPs.\nFig.\u00a07Immunoblot of monoclonal antibodies LM13 and LM14. The CDTA-soluble Arabidopsis thaliana fraction that was used as the immunogen was separated by SDS-PAGE with 30\u00a0\u03bcg protein loading per lane. A Western blot of this material was probed with LM13 and LM14. LM13 did not bind to the blot but LM14-probing produced a smear in the region of 70\u2013200\u00a0kDa, which is characteristic of AGPs\nImmunolocalization of the LM14 and LM13 epitopes\nIn order to further investigate the epitopes recognized by LM13 and LM14 in planta a range of plant materials were probed with these mAbs using indirect immunofluorescence labelling. In transverse resin-embedded sections of A. thaliana inflorescence stems, LM13 bound specifically to epidermal cells and most strongly to the tangential cell walls (Fig.\u00a08a). Plant cell wall glycans may be altered during extraction and\/or immobilization such that epitopes are changed from their native state in planta. In order to test if this was a factor in determining the arabinase sensitivity of the LM13 epitope, sections of A. thaliana stems were also digested with arabinanase prior to probing with LM13. As shown by comparison of Fig.\u00a08a,b, the binding of LM13 to A. thaliana stem epidermal cell walls was abolished following arabinanase digestion indicating that the epitope-structure immobilised on arrays recognized by LM13 reflected that observed in planta. In contrast to LM13, LM14 bound to some extent to all cell walls in an equivalent section through an A. thaliana stem (Fig.\u00a08d). Labelling of the surface of intact roots of A. thaliana seedlings indicated that LM13 bound very weakly whereas LM14 bound strongly to the surface of root hairs (Fig.\u00a08e and f). In transverse sections through tobacco stems, the LM13 epitope was associated with cell walls adjacent to metaxylem cells of the vascular tissue\u2014a distinctly different labelling pattern to that observed in A. thaliana stems (Fig.\u00a08g). In contrast, LM14 did not label any cell walls in tobacco stems (Fig.\u00a08h).\nFig.\u00a08Indirect immunofluorescence labelling of plant material with LM14 and LM13. LM13 bound strongly to inner and outer radial walls of epidermal cells in transverse sections of A. thaliana inflorescence stem sections. The section is counterstained with calcofluor (blue) that labels \u03b2-linked glucans. b LM13 binding to A. thaliana inflorescence stem sections when sections were treated with arabinanase. Arrowheads in a and b indicate the epidermal surface. c Control in which an equivalent stem to that in a was labelled with the absence of primary antibody. d, An equivalent section to that shown in a labelled with LM14 which bound to all cell walls. e LM13 bound very weakly to the intact surface of the roots of A. thaliana seedlings. f LM14 bound strongly to the surface of root hairs (arrowheads) of Arabidopsis seedling roots. g In transverse sections through tobacco stem LM13 bound strongly to cell walls adjacent to files of metaxylem cells (mx). h LM14 did not bind to equivalent sections through tobacco stems. Scale bars\u2009=\u200950\u00a0\u03bcm for a\u2013d, g and h; 20\u00a0\u03bcm for g inset, 50\u00a0\u03bcm for e and f\nDiscussion\nThe work presented here demonstrates the potential of glycan microarrays for overcoming a major bottleneck in anti-glycan mAb production. Specifically, the use of microarrays enabled 50\u00a0\u03bcl of hybridoma supernatant to be screened rapidly and simultaneously against >60 potential epitope-bearing target molecules. A novel aspect of this work was the use of cluster analysis of array data to rapidly predict antibody specificities by comparison with previously defined mAbs. Subsequent detailed analyses of the specificities of the new mAbs LM13 and LM14 indicated they bound to pectic and AGP class of polymers respectively, as was predicted by the cluster analysis. These results indicate that if a relatively large set of probes with defined specificities are available to serve as references, this is an effective method for high-throughput initial mAb screening. One potential limitation of shotgun immunisation could be immuno-dominance, such that an immune response is elicited against a limited subset of the injected antigens. In this study two mAbs, LM13 and LM14 were selected with specificity for two different classes of molecule. However, of the seven mAbs selected for cluster analysis of binding profiles, six clustered with mAbs with specificity for AGPs and one with mAbs to pectic side chains, suggesting that AGPs were the immuno-dominant antigens in this case. It is therefore likely that such a multi-antigen approach towards cell wall polymers may be most effective to obtain a panel of mAbs with a range of specificities within a single class of polymer.\nThe arrays we constructed were based on the non-covalent attachment of glycans to nitrocellulose. This approach has the advantage that molecules can be immobilized directly without the need to create functional groups, and nitrocellulose has been previously shown to be a effective substrate for the immobilization of diverse glyans [35, 36]. Non-covalent attachment has the potential drawback that variations in the effectiveness of immobilization may result from differences in the structural properties of the arrayed molecules [8]. All of the arrayed samples used in this work were recognized to some extent by at least one of the mAbs used (as shown in Fig.\u00a04), indicating that all were immobilized to some degree. Nevertheless, the spot signals obtained can only provide semi-quantitative information about mAb binding.\nTwo new probes, LM13 and LM14 were produced against cell wall polymers and both have distinctly different specificities to previously generated antibodies. Cluster analysis, ciELISA data immunoblotting and arabinanase sensitivity suggested that the epitope recognized by LM13 is an arabinan-containing structure that occurs as a side chain on pectic polymers. Pectic side chains typically consist of either galactan, arabinan or type I arabinogalactan chains in which arabinose usually occurs as a terminal sugar [18]. It was of note that LM13 binding to arrays was reduced to some extent by galactanase digestion. However, the galactanase used had very low arabinanase side activity (<0.03\u00a0U\/ml specific activity with an arabinan substrate compared to 780\u00a0U\/ml specific activity with a galactan substrate). It is possible therefore that galactanase digestion resulted in the indirect loss of the LM13 epitope by cleavage of galactan to which an arabinan-containing epitope is attached. The fact that LM14 bound to both pectin-derived MHR samples and to AGP-like material on a blot suggests that this mAb binds to an epitope of type II arabinogalactan that may occur on both pectins and AGPs. The novel patterns of recognition on A. thaliana and other plant materials indicates that these are useful new probes for the analysis of cell wall glycans polymers and complex cell wall architectures.","keyphrases":["monoclonal antibodies","plant cell walls","hierarchical clustering","carbohydrate microarrays"],"prmu":["P","P","P","P"]}
{"id":"Photosynth_Res-3-1-1769345","title":"The atypical iron-coordination geometry of cytochrome f remains unchanged upon binding to plastocyanin, as inferred by XAS\n","text":"The transient complex between cytochrome f and plastocyanin from the cyanobacterium Nostoc sp. PCC 7119 has been analysed by X-ray Absorption Spectroscopy in solution, using both proteins in their oxidized and reduced states. Fe K-edge data mainly shows that the atypical metal coordination geometry of cytochrome f, in which the N-terminal amino acid acts as an axial ligand of the heme group, remains unaltered upon binding to its redox partner, plastocyanin. This fact suggests that cytochrome f provides a stable binding site for plastocyanin and minimizes the reorganization energy required in the transient complex formation, which could facilitate the electron transfer between the two redox partners.\nIntroduction\nIn oxygen-evolving photosynthetic organisms, the cytochrome b6\u2013f complex couples proton translocation across the thylacoid membrane to the electron transport between photosystems I and II (Allen 2004). In this complex, cytochrome f (Cf) transfers electrons from the Rieske iron sulphur cluster to a soluble metalloprotein that acts as the immediate electron donor of P700 cofactor at Photosystem I.\nThe Cf subunit consists on a 28.2\u00a0kDa N-terminal soluble domain anchored to the membrane by C-terminal helix (Gray 1992). Its soluble domain is an atypical c-type cytochrome because both its \u03b2-sheet secondary structure and the unusual heme axial coordination (Martinez et al. 1994) with the N-terminal amino acid Tyr-1 acting as an axial ligand.\nThe most ubiquitous electron carrier between Cf and P700 is plastocyanin (Pc) (Sandman et al. 1983), which is a type I cupredoxin (Gough and Chotia 2004). Its structure consists on an anti-parallel \u03b2-barrel with a single copper atom (Coleman et al. 1978; Sykes 1985; Redinbo et al. 1994) coordinated by two nitrogen atoms and two sulphur ones from highly conserved residues (H39, C89, H92 and M97 in Nostoc cyanobacterium).\nThe mechanism of the electron transfer (ET) reaction between Pc and its physiological partners has been studied extensively (Hope 2000; Herv\u00e1s et al. 2003; D\u00edaz-Quintana et al. 2003) highlighting the role of electrostatic and hydrophobic interactions on binding. Also, the solution structures of several Pc\u2013Cf complexes have been reported (Ubbink 2004; D\u00edaz-Moreno et al. 2005a, Lange et al. 2005; Musiani et al. 2005). In all the cases, these structures are consistent with a single conformation ensemble in which the hydrophobic patch surrounding Tyr-1 in Cf docks the hydrophobic patch of Pc.\nDespite the large amount of data concerning the nature of the interactions influencing the binding between the redox partners, little is known about the effects of complex formation on the metal cofactors, and how they can modulate the ET process. An analysis about whether the Fe coordination in Cf is altered upon binding to Pc would be interesting to address the above-mentioned effects, especially due to the fact that the N-terminal amino acid Tyr-1, which is involved in the Pc\u2013Cf interface (Ubbink 2004; D\u00edaz-Moreno et al. 2005a, Lange et al. 2005; Musiani et al. 2005), acts as axial ligand. To get a deep insight on these subjects we have studied the Fe K-edge of free and Pc-bound Cf by X-ray absorption spectroscopy (XAS) in solution.\nMaterials and methods\nProtein samples\nNostoc sp. PCC 7119 Pc was purified from E. coli cells transformed with the pEAP-WT plasmid (Molina-Heredia et al. 1998). Production and purification of the soluble domain of Nostoc sp. PCC 7119 Cf were as previously described (Albarr\u00e1n et al. 2005).\nFor the XAS experiments, the Pc and Cf samples were concentrated to the required volume by ultrafiltration methods, and exchanged into 10\u00a0mM sodium phosphate pH 6.0. After concentration, stock solutions of 10\u00a0mM Pc and 4.5\u00a0mM Cf were obtained. Protein concentration was determined by optical spectroscopy using an absorption coefficient of 4.5 \u00a0mM\u22121\u00a0cm\u22121\u00a0at 598\u00a0nm for oxidized Pc (Molina-Heredia et al. 1998) and 31.5\u00a0mM\u22121\u00a0cm\u22121 at 556\u00a0nm for reduced Cf (Albarr\u00e1n et al. 2005). Oxidized Cf (Cf III) was prepared by adding an equimolecular amount of potassium ferricyanide, followed by gel filtration in a Pharmacia Superdex G75 column to remove ferrocyanide. Reduced Cf (Cf II) was obtained upon addition of a tenfold excess of sodium ascorbate to a resulting Cf III sample. Oxidized Pc (PcII) was obtained by addition of either ferricyanide, as described for Cf. The oxidized complex (PcII\u2013Cf III) was prepared by adding an aliquot of a 10\u00a0mM PcII onto a Cf III sample up to reach a final concentration of ca. 2\u00a0mM each. Under these conditions, the percentage of Pc bound to Cf was estimated to be ca. 87%, according to the binding constant calculated from NMR data (D\u00edaz-Moreno et al., 2005a). The reduced complex (PcI\u2013Cf II) was obtained upon addition of a tenfold excess of sodium ascorbate to a PcII\u2013Cf III sample prepared as above.\nXAS measurements\nThe X-ray absorption spectra were recorded at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France), which was operating with a ring current of 200\u00a0mA and energy of 6\u00a0GeV. The Fe K-edge (7112\u00a0eV) was measured at beam line BM29 using a double crystal monochromator fitted with a pair of flat Si (311) crystals detuned to 50% fwhm of the maximum transmission, for the suppression of high energy harmonics.\nAll measurements were made at room temperature in fluorescence mode, using a 13-element Canberra solid-state germanium detector. The samples were contained in a PTFE cell equipped with two 12\u00a0\u03bcm Kapton foil windows.\nEnergy calibration was achieved by measuring an iron foil for the Fe edge. The spectra of the foil were measured simultaneously, with the data in transmission mode, to be used as an internal reference. Ionization chambers filled with the appropriate mixture of gases were used as detectors.\nFor all measurements, each data point was collected for 4 s, and several scans were averaged to achieve a good signal-to-noise ratio. In no case protein photoreduction or damage was observed, even in those samples in which the measurements were made over several hours. For the oxidized forms free and Pc-bound Cf, the edge remained at fix energy position from the first to the last scan, thus confirming the absence of protein photoreduction. The lack of radiation-induced changes in the samples was also confirmed by UV-vis measurements before and after XAS measurements.\nXAS data analysis\nThe background subtraction required to obtain the extended X-ray absorption fine structure (EXAFS) functions \u03c7(k) from the measured X-ray absorption spectra were performed using the AUTOBK code from the University of Washington. E0 was defined as the maximum of the first derivative of the absorption edge.\nTo analyze the EXAFS spectra, the theoretical phases and amplitudes were calculated using the FEFF 6.0 code (Rehr et al. 1992; Newville et al. 1995). The fit to the experimental data was performed using the FEFFIT program (version 2.54) (Newville et al. 1995).\nResults and discussion\nXANES region\nFigure\u00a01 (upper panel) shows the X-ray absorption near edge structure (XANES) spectra corresponding to free Cf in its reduced and oxidized states. In both spectra there is a pre-edge feature corresponding to the forbidden transition 1s\u22123d, which is sensitive to the electronic and geometric structure of the atom (Fe) site (Westre et al. 1997). In fact, the intensity of this transition agrees with the octahedral environment around the iron atom in Cf (Martinez et al. 1994).\nFig.\u00a01XANES region of the Fe K-edge XAS spectra of free and Pc-bound Cf. Upper, experimental data for oxidized and reduced forms of free proteins, Cf III (continuous line) and Cf II (dashed line). Middle, data for the oxidized species, Cf III (continuous line) and PcII\u2013Cf III complex (dashed line). Lower, data for the reduced species, Cf II (continuous line) and PcI\u2013Cf II complex (dashed line). The presence of a pre-edge signal at 7113 eV is marked by arrows\nOn the other hand, the edge position, determined by the maximum in the first derivative of the absorption spectrum corresponding to free Cf II, appears ca. 1\u00a0eV below that of Cf III, as previously found for the reduced and oxidized states of two closely related proteins, as heart cytochrome c (Cheng et al. 1999) and Nostoc cytochrome c6 (D\u00edaz-Moreno et al. 2006). In case of cytochrome c (Cheng et al. 1999), photoreduction of the oxidized species was observed only after the 16th spectrum of the series. In our case, however, there is no photoreduction as inferred from the fact that the edge position remains fixed for all the spectra along each series i.e. reduced or oxidized, free or bound. The reason not to observe any sample photo-reduction is due to the moderate brightness of the EXAFS spectrometer deliberately chosen for this investigation. In addition, the same shift has also been reported upon oxidation from Fe2+ to Fe3+ at pH 7 in the heme protein cytochrome c (Shulman et al. 1976).\nMoreover, there are some differences in both XANES spectra, particularly in the first feature after the edge.\nFigure\u00a01 also shows the XANES regions of the absorption spectra of Cf bound to Pc in the oxidized and reduced state. The XANES region of the absorption spectra for PcII\u2013Cf III and Cf III are identical (Fig.\u00a01, middle panel), indicating that the electronic and geometrical configuration around the iron centre remains unchanged when bound to Pc. However, this is not the case for the reduced proteins (Fig.\u00a01, lower panel). In fact, the spectrum of the PcI\u2013Cf II complex exhibits some differences compared with that of free CfII. With regard to the pre-edge, a slight increase in the intensity of this feature is observed for CfII when bound to PcI, according to the decrease in electron density of the Fe atom. The shape of the first feature after the edge slightly changes too, thus suggesting certain modifications in the geometrical distribution of the ligands around the iron atom. It is worth noting that the spectrum of the reduced complex is very similar\u2013\u2013except for the pre-edge signal and edge position\u2013\u2013to that of the oxidized complex and free oxidized Cf, as shown in Fig.\u00a03. This suggests that the iron geometry in reduced Cf is distorted upon association to Pc, approaching to that of the iron centre in the oxidized form, either free or bound to Pc.\nEXAFS Region\nThe extracted EXAFS signals of Cf in the reduced and oxidized states are shown in Fig.\u00a02 (left panels) as k-weighted spectra. The choice of a k-weighting instead of a k3-weighting is based on the following facts. First, we were interested in determining the local environment around the absorbing atom and, in particular, at the first coordination shell. This shell is formed by iron and light elements, such as nitrogen, and their backscattering amplitude drops quickly at increasing energy (k value). Therefore, they have a larger contribution when the transforms are made in k rather than in k3. Second, if a k3-weighting is used, the highest energy part of the spectrum is emphasized and, in this case, the EXAFS signal would be dominated by the spectral region where the signal-to-noise ratio is poorer.\nFig.\u00a02EXAFS data (panels a and b) and their FT modules (panels c and d) at the Fe K-edge of Cf, either free or bound to Pc. The EXAFS spectra of free proteins (Cf II and CfIII) are shown in panela by continuous line for Cf II and dashed line for Cf III. In panel b, the EXAFS spectra of PcI\u2013Cf II and PcII\u2013Cf III complexes are represented by continuous and dashed lines, respectively. The corresponding FT modules of the EXAFS spectra for free Cf (Cf II and CfIII) are represented in panel c by closed and open circles, respectively. Panel d shows the FT modules for Pc-bound Cf forms: PcI\u2013Cf II by closed circles and PcII\u2013Cf III by open circles. The best fits of the FT data for both reduced (Cf II and PcI\u2013Cf II) and oxidized states (Cf III and PcII\u2013Cf III) are represented by continuous and dashed lines, respectively\nWe should note here that the XAS data were collected at room temperature in order to maintain consistency with previous room temperature NMR measurements made on the same systems (D\u00edaz-Moreno et al. 2005a, b). Although a better signal to noise should be obtained at cryogenic temperatures, the transient complexes we study are not stable upon freezing, unless the partners are chemically cross-linked. Similar experimental conditions are required by EPR. Although EPR measurements on free and Pc-bound Cf would help to study the iron geometry, any comparison between results inferred from NMR or XAS, on one hand, and EPR, on the other, would be unreliable.\nAs can be seen, the EXAFS function is not a simple wave but a combination of different waves corresponding to the contributions from the neighbouring atoms at different distances around the iron centre. Indeed, the EXAFS signals of Cf do not change upon binding to Pc, thereby indicating that the local coordination environment around the iron centre remains unchanged.\nThe Fourier Transform (FT) of the corresponding spectra and the best fits, obtained with the parameters included in Table\u00a01, are also shown in Fig.\u00a02 (right panels). As a starting model, the values obtained from the X-ray diffraction (XRD) structure of Phormidium Cf (Carrel et al. 1999) were used. In all cases there is a main signal around 1.5\u00a0\u00c5, which corresponds to the first coordination sphere formed by the four nitrogen atoms of the porphyrin ring (NA, NB, NC and ND), along with the nitrogen atoms of the two iron axial ligands Tyr-1 (N) and His-26 (N\u03b52). The intensities of the remaining signals are significantly lower than the first one because they correspond to contributions from lightweight elements lying at longer coordination distances. In order to fit the data, six simple scattering paths from the Fe atom to the six ligand N atoms were selected. \u0394r1 and \u0394r2 were used as two fitting parameters corresponding to changes in the distances of Fe to the two axial N atoms and to the four phorphyrin N atoms, respectively. The Debye\u2013Waller parameter, \u03c32, related to system dynamic disorder was also fitted, with \u03c312 accounting for the bonds between the Fe atom and the two axial N atoms and \u03c322 accounting for the bonds between the Fe atom and the four porphyrin N atoms. The fifth adjustable parameter was the internal potential correction (\u0394E0).\nTable\u00a01Best-fit structural parameters resulted from the EXAFS analysis for free and Pc-bound Cf, in both oxidized and reduced stateSystemLigand\u0394E0 (eV)r(M\u2013L) (\u00c5)\u03c32(M\u2013L) (\u00c52)CfIIIN (NC)\u22127\u00a0\u00b1\u00a041.96\u00a0\u00b1\u00a00.020.002\u00a0\u00b1\u00a00.001N (NA & ND)1.97\u00a0\u00b1\u00a00.02N (NB)1.98\u00a0\u00b1\u00a00.02N (N\u03b52)2.44\u00a0\u00b1\u00a00.060.010\u00a0\u00b1\u00a00.010N (N)2.49\u00a0\u00b1\u00a00.06CfIIN (NC)\u22126\u00a0\u00b1\u00a021.97\u00a0\u00b1\u00a00.010.002\u00a0\u00b1\u00a00.001N (NA & ND)1.99\u00a0\u00b1\u00a00.01N (NB)2.00\u00a0\u00b1\u00a00.01N (N\u03b52)2.43\u00a0\u00b1\u00a00.020.004\u00a0\u00b1\u00a00.003N (N)2.48\u00a0\u00b1\u00a00.02PcII\u2013CfIIIN (NC)\u22127\u00a0\u00b1\u00a031.97\u00a0\u00b1\u00a00.010.002\u00a0\u00b1\u00a00.001N (NA & ND)1.98\u00a0\u00b1\u00a00.01N (NB)1.99\u00a0\u00b1\u00a00.01N (N\u03b52)2.40\u00a0\u00b1\u00a00.030.011\u00a0\u00b1\u00a00.009N (N)2.45\u00a0\u00b1\u00a00.03PcI\u2013CfIIN (NC)\u22126\u00a0\u00b1\u00a031.97\u00a0\u00b1\u00a00.020.003\u00a0\u00b1\u00a00.001N (NA & ND)1.98\u00a0\u00b1\u00a00.02N (NB)1.99\u00a0\u00b1\u00a00.02N (N\u03b52)2.40\u00a0\u00b1\u00a00.030.010\u00a0\u00b1\u00a00.009N (N)2.45\u00a0\u00b1\u00a00.03S02, factor of amplitude of reduction; r(M\u2013L), metal\u2013ligand distanceS02\u00a0=\u00a01.0, \u0394k\u00a0=\u00a03.5\u201311 (1\/\u00c5), \u0394R\u00a0=\u00a01.1\u20132.1 (\u00c5), R factor (free Cf III)\u00a0=\u00a00.044, R factor (free Cf II)\u00a0=\u00a00.004, R factor (PcII-bound Cf III)\u00a0=\u00a00.004, R factor (PcI-bound Cf II)\u00a0=\u00a00.003\nAlthough there are no significant changes in the metal\u2013ligand distances, the Fe K-edge X-ray absorption data suggests a small distortion of the Fe2+ metal centre geometry when Cf II binds to PcI. The resulting Fe2+ geometry resembles that of Cf III, either free or bound to PcII (Fig.\u00a03). Although the Fe atom of Cf shows a well-defined position at the heme group, which provides a rigid frame, the small distortion on the Fe2+ metal centre when Cf II binds to PcI can be explained by the smaller size of Fe3+ compared to Fe2+: The Fe3+ atom fits well into the heme ring whereas the Fe2+ atom is somewhat shifted out of the plane (Schnackenberg et al. 1999); however, the Fe2+ atom could be driven into the ring upon complex formation, thus yielding a structure of PcI-bound Fe2+ similar to that of Fe3+ in free and PcII-bound Cf III.\nFig.\u00a03XANES region of the Fe K-edge XAS spectra of free CfIII and PcI\u2013bound CfII. Experimental data for Cf III and PcI\u2013Cf II are shown in continuous and dashed lines. The presence of a pre-edge signal at 7113\u00a0eV is marked by arrows\nThis tiny perturbation is consistent with the intensity changes at the pre-edge region of CfII in presence of PcI, because they correspond to transitions to the iron dz2 and dx2y2 orbitals, which are pointing towards the axial and equatorial ligands, respectively. As the reducing electron is not located in these orbitals, such subtle changes may have little influence on the redox potential. This situation is clearly different to that previously reported for cytochrome c6, which shows a perturbation on the pre-edge region corresponding to its oxidized species, instead of the reduced ones, upon binding its reaction partner, photosystem I (D\u00edaz-Moreno et al. 2006). In such case the pre-edge corresponds to transfer to a half-occupied non-bonding d orbital, which is that receiving the reducing electron, so the observed changes can be correlated with changes in the redox potential of the heme protein. Regarding to this point, it is important to note that the heme plane is normal to the interaction surface of cytochrome c6, but parallel in the case of Cf.\nThus, our results indicate that the bond between iron and the N-terminus nitrogen is strong enough to prevent a distortion of the iron-coordination geometry upon binding of Cf to Pc, as well as to provide a stable binding site to the copper protein. Such a stiffness of the coordination sphere minimises the reorganisation energy concomitant to charge transfer, thus optimising the ET process.","keyphrases":["cytochrome f","plastocyanin","transient complexes","x-ray absorption spectroscopy","electron transfer","metalloproteins"],"prmu":["P","P","P","P","P","P"]}
{"id":"Clin_Oral_Investig-3-1-2134843","title":"Nonsurgical and surgical periodontal therapy in single-rooted teeth\n","text":"The purpose of this study was to compare the effect of tooth related and patient related factors on the success of non-surgical and surgical periodontal therapy. In 41 patients (22 female) with untreated and\/or recurrent periodontitis, no therapy, scaling and root planing (SRP), or access flap (AF) were assigned according to probing pocket depth (PPD). PPD and vertical relative attachment level (RAL-V) were obtained initially, 3 and 6 months after therapy. Baseline data were compared according to therapy, jaw, tooth type, and site. Factors influencing clinical parameters were identified using multilevel analyses. Baseline PPDs were deeper interproximally, in the maxilla and at premolars compared to buccal\/oral sites, mandibular, and anterior teeth. At 6 months, PPD reduction and RAL-V gain were significantly greater at sites receiving SRP and AF as compared to untreated sites (p < 0.001). PPD reduction and RAL-V gain were significantly less (p < 0.005) in smokers as compared to nosmokers and at interproximal sites (p < 0.0001) as compared to buccal\/oral sites. RAL-V gain was less in aggressive periodontitis, and PPD reduction was less in the maxilla (p < 0.001). In sites with greater bone loss and infrabony defects, a poorer response was observed regarding RAL-V gain or PPD reduction, respectively. The conclusions of the study are the following: (1) Nonsurgical and surgical periodontal therapies are effective in single-rooted teeth; (2) severe interproximal bone loss and infrabony defects deteriorate clinical results; and (3) there seem to be more defect-associated (tooth, site) factors influencing treatment outcome than patient-associated factors.\nIntroduction\nThe major factor in the etiology of periodontitis is the dental biofilm, i.e., bacterial plaque. Thus, the aim of anti-infective periodontal therapy is the reduction in supra- and subgingival microbial plaque to resolve periodontal inflammation. Subgingival scaling and root planing (SRP) may result in resolution of inflammation, reduced probing pocket depths (PPD), and clinical attachment gain [1, 2, 16]. However, nonsurgical periodontal therapy, i.e., SRP, is not found to be successful at all treated sites. Although not all factors influencing failure of nonsurgical therapy are known, deep periodontal pockets [3, 21, 25, 28] and furcation involvement [6, 9, 23] are some of them. Recent meta-analyses have shown the efficacy of nonsurgical therapy in moderate and deep periodontal pockets. In deep pockets, access flap (AF) techniques result in better reduction in PPD and clinical attachment gain [14, 15]. However, these meta-analyses like many other studies on this issue before [3, 21, 25, 28] use mean values and are not able to reflect the benefit of a particular technique at teeth with certain defects, e.g., infrabony pockets. However, calculation of a mean value, e.g., for PPD reduction, across a patient in fact levels out tooth-related characteristics. Even if separate strata for different baseline PPD are considered, tooth-related factors other than baseline PPD are leveled out also. This may lead to quite similar mean values for nonsurgical and surgical therapy for a patient or across a whole sample because tooth-related differences between both approaches because of different amounts or types of bone loss, jaw, etc. are leveled out. More recent statistical methods such as the multilevel modeling are considering single-tooth or even site characteristics while still regarding the individual patient as a statistical unit. In a structured review of 2002, it was explicitly stated that the included studies showed similar results for nonsurgical and surgical therapy after 1\u00a0year. However, insufficient studies are available to evaluate the various treatment procedures, e.g., in angular defects. Further, it is stated that subject characteristics and their possible effect on treatment outcome was not addressed in the studies available at that time [14]. Thus, there is a need for more information on the effect of patient and tooth-related factors on nonsurgical and surgical periodontal therapy. Well known hypotheses and the use of these more recent statistical methods may lead to new information.\nClinical measurements in posterior are less reproducible than in anterior teeth [7]. Furcation involvement is a known factor to influence treatment outcome of nonsurgical and surgical therapy. To provide a high level of reproducibility of clinical measurements and to exclude a already known influencing factor, only single-rooted teeth were considered in this study.\nThe hypothesis behind this study is that different tooth-related factors (e.g., tooth type, site, plaque, residual inflammation, horizontal\/vertical bone loss) and patient-related factors (e.g., age, sex, diagnosis, smoking) determine the initial outcome of nonsurgical and surgical periodontal therapy in single-rooted teeth.\nThus, the aim of the present study was to identify patient and site factors that determine the initial outcome of nonsurgical and surgical periodontal therapy in single-rooted teeth using more recent statistical methods.\nMaterials and methods\nPatients\nThe study group consisted of 41 patients (22 female) suffering from moderate to severe periodontal disease. These patients had been scheduled for periodontal treatment within a randomized placebo-controlled clinical trial evaluating the effect of a subgingivally delivered 14% doxycycline gel at the Section of Periodontology, Department of Conservative Dentistry, Clinic for Oral, Dental and Maxillofacial Diseases, University Hospital Heidelberg. Each patient had to have three single-rooted teeth that qualified as test teeth for the doxycycline study; that is, only three teeth per patient were included into this study [8]. Inclusion criteria were at least 23\u00a0years of age, untreated moderate to severe periodontal disease or recurrent periodontitis without periodontal surgery at least for the last 24\u00a0months, effective individual oral hygiene (plaque control record\u2009<\u200935%) [24], and written informed consent. Exclusion criteria were local and\/or systemic antibiotic therapy within the last 6\u00a0months before the baseline examination of the study, known adverse reactions to any component of the test agent, anticoagulative therapy, liver, kidney, gastroenteral diseases, pregnancy, and women who were lactating. All patients were asked about current and past cigarette consumption. A patient was classified as a current smoker if he smoked one or more cigarettes per day and as a former smoker if he had quit smoking for at least 5\u00a0years. Patients who had quit smoking within the last 5\u00a0years were classified current smokers. No more than 50% of current smokers were allowed in the sample. All patients were classified as chronic or aggressive periodontitis according to the following criteria:\nAggressive periodontitis: Patient is clinically healthy; that is, systemic diseases predisposing for periodontitis are not known; radiographic bone loss greater than or equal to 50% in at least two different teeth and age less that or equal to 35\u00a0years at time of diagnosis.Moderate and severe chronic periodontitis: attachment loss greater than or equal to 3\u00a0mm and age greater than 35\u00a0years [19].\nThe study was approved by the Institutional Review Boards for Human Studies of the University of Heidelberg.\nClinical examination\nAfter completion of initial periodontal treatment including oral hygiene instruction and supragingival scaling, PPD were measured at four sites of every tooth. If a patient fulfilled the inclusion criteria, the study protocol, risks, benefits, and procedures were explained, and informed written consent was obtained. One week before test therapy, the Gingival Index (GI) and Plaque Index (PlI) [22] were assessed at six sites (mesiobuccal, midbuccal, distobuccal, distolingual, midlingual, mesiolingual) of every incisor, canine, and premolar. PPD and vertical relative attachment levels (RAL-V) were measured to the nearest 0.5\u00a0mm using a straight periodontal probe (PCPUNC 15, Hu Friedy, Chicago, IL) at six sites per tooth. A reference splint gave the location of probing by notches and served as reference for the RAL-V measurements. Bleeding on probing (BOP) was recorded approximately 30\u00a0s after probing. After the first course of measurements, all PPD and RAL-V measurements were repeated. The means of the pairs of double measurements were used for analysis. If a pair of PPD double measurements showed a difference greater than 2\u00a0mm, a third measurement was performed at the respective site (option 3). Then, the mean of the pair of the closer measurements was taken for analysis at the respective site. All clinical measurements before therapy were performed by one examiner (Eickholz) who also performed active periodontal therapy (subgingival debridement and surgery) in all patients. Within each patient, three test teeth with PPD\u2009=\u20095\u00a0mm and BOP or PPD\u2009\u2265\u20096\u00a0mm were selected for the doxycycline study. These teeth were excluded from this examination. Three and 6\u00a0months after therapy, the clinical examinations were repeated in the same manner by a second examiner (Kim) who did not have any information about baseline data and the therapy [8].\nRadiographic examination and evaluation\nBefore therapy, in 36 patients, full-mouth sets of periapical radiographs (Ektaspeed plus; Kodac, Rochester, NY) were obtained in XCP technique (XCP, Kentzler & Kaschner Dental, Ellwangen\/Jagst, Germany). Intraoral size 0 (maxillary second incisors and mandibular anteriors) and 2 (all other regions) dental films were exposed to an X-ray source (Heliodent DS\u00ae, 60\u00a0kV, 7\u00a0mA, Sirona, Bensheim, Germany) and developed under standardized conditions (XR 24 Nova\u00ae, D\u00fcrr Dental, Bietigheim\u2013Bissingen, Germany).\nAll radiographs were viewed in a darkened room using a radiograph screen (67-0420, Dentsply Rinn, Elgin, IL). Each interproximal surface of single-rooted teeth was classified for the type of bone loss (horizontal\/vertical). Further, the prevalence of a double contour of the root was noted that was taken as indicative for a mesial or distal groove. Relative bone loss in percent was assessed at two sites per tooth (mesial and distal) using a Schei ruler [27]. This ruler consisted of six straight lines that divert in the same angle. For each site, the basic line of the Schei ruler was placed to the cementoenamel junction (CEJ) of the respective tooth parallel to the occlusal plane. Then, the ruler was moved until the sixth line was placed tangentially to the apex. Finally, the alveolar crest was located. Location between the two coronal lines meant bone loss up to 20% of root length. Location between the second and third line meant between 20 and 40% bone loss and so forth. If the CEJ was destroyed or overlapped by interproximal restorations, the restoration margin was used as reference. If the alveolar crest could not be determined because of overlapping of adjacent teeth, the interproximal site was classified as \u201ccannot be assessed.\u201d If an interproximal surface showed a double contour, this was classified as an interproximal root groove. All radiographic assessments were performed by an examiner who was blinded to the clinical measurements and therapy assignments (Schenk).\nTherapy\nTeeth exhibiting PPD\u2009<\u20093\u00a0mm were treated neither by subgingival SRP nor by AF [26]. SRP was performed when PPD\u2009\u2265\u20093 and \u22646\u00a0mm were present. If periodontal surgery was necessary at teeth not adjacent to test teeth, this was performed 1\u00a0week after SRP: AF was chosen for teeth exhibiting PPD\u2009>\u20096\u00a0mm. However, SRP was performed at all test teeth (not included in this analysis) and the respective neighboring teeth under local anesthesia with hand instruments even for baseline PPD\u2009>\u20096\u00a0mm. Thus, also some sites with PPD\u2009>\u20096\u00a0mm were treated exclusively by SRP. Further, the decision to perform AF was taken per tooth and not per site. Thus, also sites with PPD\u2009\u2264\u20096\u00a0mm were treated by AF if the respective tooth exhibited PPD\u2009>\u20096\u00a0mm at one site. Thereby, effectiveness of SRP and AF maybe analyzed across the limits of indications as the ranges of PPD for the three treatment groups in Table\u00a04 demonstrate. Maintenance including oral hygiene instruction and professional tooth cleaning was done 3\u00a0months after test therapy.\nStatistical analysis\nData handling\nAll data were entered into a personal computer program (Systat\u2122 for Windows version 10, Systat, Evanston, IL; Schenk). Descriptive statistics were then calculated using another program (SPSS\u2122, version 10.0.1, SPSS, Chicago IL) by an independent statistician (Lungeanu). Multilevel analyses were modeled by another independent statistician (Reitmeir) using a third program (SAS\u00ae version 6.12, SAS Institute, Cary, NC).\nReproducibility\nThe replicate PPD and RAL assessments for all teeth were used to estimate the intraexaminer reproducibility. The intraexaminer reproducibility of PPD and RAL measurements was expressed as the standard deviation of single measurements [4].\nTreatment comparison\nThe individual patient was considered as a statistical unit in this analysis. The main outcome variable for the comparison of the therapeutic effects of no further therapy, SRP, or AF was chosen to be the change of PPD. RAL-V was considered as a secondary endpoint. All other clinical parameters (GI, PlI) were control variables.\nOnly sites were included that could be assessed at all three examinations. Additionally, baseline clinical parameters were calculated separately according to therapy (no therapy, SRP, AF), jaw, tooth type (anterior\/premolar), and site (interproximal\/buccal and oral) and compared using multilevel methodology [10, 11]. A probability p\u2009<\u20090.05 was accepted for a difference to be statistically significant. Because of multiple testing, a Bonferroni correction was performed: We tested 24 independent hypotheses on our set of data. Thus, the statistical significance level that should be used for each hypothesis separately is 1 of 24 times what it would be if only one hypothesis were tested, i.e., p\u2009=\u20090.002. For all analyses, the basic level \u201csite\u201d was nested in the upper level \u201ctooth,\u201d which itself was nested in the next upper level \u201cpatient,\u201d and patient effects on the outcome were assumed to be random. This technique allows the identification of single-tooth effects or parameters while still considering the individual patient as a statistical unit and the dependencies of site and tooth data within a patient [10, 11]. Multilevel regressions were calculated including only clinical parameters providing six measurements per tooth first (site level: n\u2009=\u20094,146; patient level: n\u2009=\u200940). Further analyses were performed that included also radiographic parameters. Radiographic parameters were only available for those patients contributing full-mouth radiographic examinations and could be related only to interproximal sites (mesiobuccal, mesiolingual, distolingual, mesiolingual), i.e., four sites per tooth (site level: n\u2009=\u20091,792; patient level: n\u2009=\u200936).\nTo identify factors influencing the treatment result, change of RAL-V and PPD were analyzed by application of a multilevel regression model using backward selection. Models were fitted for the dependent variables (1) RAL-V change after 6\u00a0months and (2) PPD change after 6\u00a0months including the following independent variables: (1) clinical: age, sex, actual smoking (yes\/no), pack years, diagnosis (aggressive\/chronic periodontitis), jaw (maxilla\/mandible), site (interproximal\/buccal or oral), tooth type (anterior\/premolar), therapy (none\/SRP\/AF), and PlI and GI at baseline, (2) radiographic: interproximal bone loss in percent, infrabony defect, and double contour of the root. For the analysis including radiographic parameters, the variable \u201csite\u201d (interproximal\/buccal and oral) dropped out because only interproximal sites remained in the analysis. A probability p\u2009<\u20090.15 was required for parameters to be kept within the models.\nResults\nAll 41 patients contributing 700 single-rooted teeth with 4,200 sites finished the 3-month re-examination. The patient characteristics (age, diagnosis, smoking status) are given in Table\u00a01. From baseline to the 3-month examination, two single-rooted teeth were lost: one to an acute periapical periodontitis and another because of prosthodontic planning. After the 3-month re-examination, one patient left the study. Only sites were included into the analysis, which could be assessed at all three examinations: n\u2009=\u20094,146 in 40 patients.\nTable\u00a01Study population as related to age, sex, smoking habit, and diagnosisVariableNumber (n\u2009=\u200941)PercentAge45.9\u2009\u00b1\u200910.9SexFemale2253.7SmokingActive1946.3Former1229.3Never1024.4DiagnosisAggressive819.5\nReproducibility\nThe results of the intraexaminer reproducibility of PPD and RAL measurements as standard deviation of single measurement for each examination time point are given in Table\u00a02.\nTable\u00a02Intraexaminer reproducibility at baseline, 3, and 6\u00a0months examinationExaminationMeasurements (n)s (RAL-V)s (PPD)Baseline8,4000.490.493\u00a0months8,3760.440.396\u00a0months8,2920.530.39s Standard deviations of a single measurement\nBaseline data\nThe mean clinical parameters (RAL-V, PPD, PlI, GI) at baseline over all sites are given in Table\u00a03. The respective data as related to therapy (no therapy, SRP, AF) are given in Table\u00a04. Whereas no statistically significant differences for PlI were observed between the groups, RAL-V, PPD, and GI of the SRP and AF sites revealed to be significantly (p\u2009<\u20090.001) greater as compared to untreated sites (Table\u00a04). Clinical parameters as related to jaw, tooth type, and site are given in Table\u00a05. Baseline RAL-V and PPD were statistically significantly (p\u2009<\u20090.001) greater in the maxilla than in the mandible, whereas for PlI, it was vice versa (Table\u00a05). For tooth type, just baseline PPD was statistically significantly (p\u2009<\u20090.001) different between premolars and anteriors with deeper pockets at the premolars (Table\u00a05). All baseline clinical parameters were observed to be statistically significantly (p\u2009<\u20090.001) greater at the interproximal than at the buccal and oral sites (Table\u00a05).\nTable\u00a03Clinical parameters at baseline, 3 and 6\u00a0months after therapy (mean\u2009\u00b1\u2009standard deviation)\u00a0Baseline3\u00a0months6\u00a0monthsSites4,1464,1464,146RAL-V (mm)5.12\u2009\u00b1\u20092.204.87\u2009\u00b1\u20091.874.92\u2009\u00b1\u20091.84PPD (mm)3.25\u2009\u00b1\u20091.832.67\u2009\u00b1\u20091.192.67\u2009\u00b1\u20091.20GI0.78\u2009\u00b1\u20090.970.51\u2009\u00b1\u20090.860.51\u2009\u00b1\u20090.86PlI0.16\u2009\u00b1\u20090.510.32\u2009\u00b1\u20090.700.37\u2009\u00b1\u20090.74Table\u00a04Clinical parameters at baseline as related to therapy (mean\u2009\u00b1\u2009standard deviation)SitesNo therapySRPAF1,440Range2,478Range198RangeRAL-V\/mm4.35\u2009\u00b1\u20091.82*1.25\u201319.005.55\u2009\u00b1\u20092.301.75\u201318.005.39\u2009\u00b1\u20092.022.25\u201312.00PPD\/mm2.45\u2009\u00b1\u20091.13*0.25\u20132.753.66\u2009\u00b1\u20091.980.50\u201312.253.98\u2009\u00b1\u20092.021.00\u201310.50GI0.48\u2009\u00b1\u20090.84*0.94\u2009\u00b1\u20091.001.02\u2009\u00b1\u20090.98PlI0.15\u2009\u00b1\u20090.490.16\u2009\u00b1\u20090.530.14\u2009\u00b1\u20090.49SRP Scaling and root planning, AF access flap*Statistically significantly different from SRP and AF with p\u2009<\u20090.001Table\u00a05Clinical parameters at baseline as related to jaw (maxilla\/mandible), tooth type (anterior\/premolar), and site (interproximal\/buccal and oral) (mean\u2009\u00b1\u2009standard deviation)\u00a0Maxillamandiblepremolaranteriorinterproximalbuccal\/oralSites1,9262,2201,5902,5662,7641,382RAL-V\/mm5.24\u2009\u00b1\u20092.14*5.00\u2009\u00b1\u20092.265.19\u2009\u00b1\u20092.20*5.07\u2009\u00b1\u20092.225.37\u2009\u00b1\u20092.26*4.60\u2009\u00b1\u20092.01PPD\/mm3.55\u2009\u00b1\u20091.88*3.00\u2009\u00b1\u20091.783.47\u2009\u00b1\u20091.88*3.12\u2009\u00b1\u20091.813.67\u2009\u00b1\u20091.82*2.43\u2009\u00b1\u20091.59GI0.80\u2009\u00b1\u20090.98*0.76\u2009\u00b1\u20090.960.83\u2009\u00b1\u20090.97*0.75\u2009\u00b1\u20090.960.89\u2009\u00b1\u20090.98*0.56\u2009\u00b1\u20090.89PlI0.11\u2009\u00b1\u20090.43*0.19\u2009\u00b1\u20090.570.14\u2009\u00b1\u20090.48*0.17\u2009\u00b1\u20090.530.18\u2009\u00b1\u20090.54*0.11\u2009\u00b1\u20090.44*Statistically significantly different with p\u2009<\u20090.001\nTreatment effect\nThe healing phase passed uneventfully for all patients. Clinical parameters as related to therapy after 3 and 6\u00a0months are given in Table\u00a06, and their change after 3 and 6\u00a0months is given in Table\u00a07. Whereas in untreated sites, the study failed to observe any significant change, SRP and AF resulted in PPD reduction and PAL-V gain after 3 and 6\u00a0months (p\u2009<\u20090.001; Table\u00a07).\nTable\u00a06Clinical parameters at 3 and 6\u00a0months after therapy as related to therapy (mean\u2009\u00b1\u2009standard deviation)\u00a03\u00a0months after therapy6\u00a0months after therapyNo therapySRPAFNo therapySRPAFSites1,4402,4781981,4402,478198RAL-V (mm)4.37\u2009\u00b1\u20091.765.13\u2009\u00b1\u20091.905.14\u2009\u00b1\u20091.644.47\u2009\u00b1\u20091.765.17\u2009\u00b1\u20091.855.11\u2009\u00b1\u20091.61PPD (mm)2.33\u2009\u00b1\u20090.962.84\u2009\u00b1\u20091.253.09\u2009\u00b1\u20091.342.35\u2009\u00b1\u20091.052.83\u2009\u00b1\u20091.242.99\u2009\u00b1\u20091.27GI0.36\u2009\u00b1\u20090.760.58\u2009\u00b1\u20090.900.69\u2009\u00b1\u20090.940.36\u2009\u00b1\u20090.760.58\u2009\u00b1\u20090.900.65\u2009\u00b1\u20090.93PlI0.22\u2009\u00b1\u20090.560.36\u2009\u00b1\u20090.740.52\u2009\u00b1\u20090.890.31\u2009\u00b1\u20090.680.41\u2009\u00b1\u20090.780.27\u2009\u00b1\u20090.65SRP Scaling and root planing, AF access flapTable\u00a07Change of clinical parameters at 3 and 6\u00a0months after therapy as related to therapy (mean\u2009\u00b1\u2009standard deviation)\u00a03\u00a0months after therapy6\u00a0months after therapyNo therapySRPAFNo therapySRPAFSites1,4402,4781981,4402,478198RAL-V (mm)0.03\u2009\u00b1\u20090.740.41\u2009\u00b1\u20091.11*0.26\u2009\u00b1\u20091.06*0.12\u2009\u00b1\u20090.940.38\u2009\u00b1\u20091.20*0.28\u2009\u00b1\u20091.18*PPD (mm)\u22120.11\u2009\u00b1\u20090.72\u22120.82\u2009\u00b1\u20091.33*\u22120.89\u2009\u00b1\u20091.25*0.09\u2009\u00b1\u20090.87\u22120.82\u2009\u00b1\u20091.40*\u22120.99\u2009\u00b1\u20091.41*GI\u22120.13\u2009\u00b1\u20090.97\u22120.36\u2009\u00b1\u20091.14*\u22120.33\u2009\u00b1\u20091.14*\u22120.13\u2009\u00b1\u20091.03\u22120.36\u2009\u00b1\u20091.18*\u22120.36\u2009\u00b1\u20091.44*PlI0.08\u2009\u00b1\u20090.690.20\u2009\u00b1\u20090.82*0.37\u2009\u00b1\u20090.97*0.16\u2009\u00b1\u20090.750.25\u2009\u00b1\u20090.87*0.13\u2009\u00b1\u20090.68*SRP Scaling and root planing, AF access flap*Statistically significantly different from no therapy with p\u2009<\u20090.001\nPrognostic factors\nRAL-V gain was less favorable in current smokers, in aggressive periodontitis, and at interproximal sites (Table\u00a08). PPD reduction was poorer in current smokers, at premolars, in the maxilla, and at interproximal sites (Table\u00a09). Whereas baseline PPD was related to better PPD reduction and RAL-V gain (p\u2009<\u20090.0001), baseline RAL-V was related to better RAL-V gain but to less favorable PPD reduction (Tables\u00a08 and 9). Baseline PlI was associated with poorer PPD reduction (p\u2009<\u20090.15; Table\u00a09). AF and SRP (p\u2009=\u20090.0114) were associated with better PPD reduction (Table\u00a09).\nTable\u00a08Backward multilevel linear regression analysis\u2014dependent variable: RAL-V reduction 6\u00a0months after therapy, n\u2009=\u200940 patients, 4,146 sites\u00a0EstimateSEdfF valuep valueIntercept\u22121.87760.106837Current smoking\u22120.28750.11063,4276.760.0094Site (interproximal)\u22120.23410.02773,42771.28<0.0001Aggressive periodontitis\u22120.29030.13083,4274.930.0265Baseline PPD0.27460.01583,427303.72<0.0001Baseline RAL-V0.18320.01413,427169.61<0.0001Table\u00a09Backward multilevel linear regression analysis\u2014dependent variable: PPD reduction 6\u00a0months after therapy, n\u2009=\u200940 patients, 4,146 sites\u00a0EstimateSEdfF valuep valueIntercept\u22121.64870.103738Current smoking\u22120.33120.10553,4519.860.0017Jaw (maxilla)\u22120.12990.03243,45116.05<0.0001Site (interproximal)\u22120.36640.02623,451195.10<0.0001Tooth type (premolar)\u22120.05010.03383,4512.200.1381Baseline PPD0.61620.01463,4511774.31<0.0001Baseline RAL-V\u22120.03540.01283,4517.630.0058Baseline Plaque Index\u22120.03680.02443,4512.270.1318Scaling and root planing0.11710.03923,4514.480.0114Access flap0.09970.08613,4514.480.0114\nTaking radiographic parameters into consideration, current smoking resulted in less favorable and baseline PPD in better RAL-V gain and PPD reduction, respectively (Tables\u00a010 and 11). Further, tooth type (premolar) and aggressive periodontitis were associated with poorer RAL-V gains (Table\u00a010). Location in the maxilla and baseline PlI resulted in less favorable PPD reduction (Table\u00a011). Generally, sites with greater interproximal bone loss demonstrated poorer RAL-V gain (p\u2009=\u20090.0001), and sites with infrabony defects were associated with inferior PPD reduction (p\u2009<\u20090.0001; Tables\u00a010 and 11).\nTable\u00a010Multilevel linear regression analysis\u2014dependent variable: RAL-V reduction 6\u00a0months after therapy, n\u2009=\u200936 patients, 1,796 sites\u00a0EstimateSEdfF valuep-valueIntercept\u22121.82900.138533Current smoking\u22120.37580.13341,3137.940.0049Tooth type (premolar)\u22120.07630.05021,3132.310.1292Aggressive periodontitis\u22120.33110.16351,3134.100.0431Baseline PPD0.23710.02561,31385.86<0.0001Baseline RAL-V0.26370.02421,313118.36<0.0001Bone loss in %\u22120.01150.00161,31350.58<0.0001Table\u00a011Multilevel linear regression analysis\u2014dependent variable: PPD reduction 6\u00a0months after therapy, n\u2009=\u200936 patients, 1,796 sites\u00a0EstimateSEdfF valuep valueIntercept\u22122.07710.136434Current smoking\u22120.46460.11971,31315.080.0001Jaw (maxilla)\u22120.14690.04331,31311.510.0007Baseline PPD0.62860.01191,3132791.95<0.0001Baseline Plaque Index\u22120.05500.03321,3132.750.0097Infrabony defect\u22120.29320.06711,31319.10<0.0001\nDiscussion\nStudy population\nPatients treated in this study ranged from 23 to 71\u00a0years of age with chronic and aggressive periodontitis. Original measurements were performed in the setting of a randomized placebo-controlled clinical trial evaluating the effect of a subgingivally delivered 14% doxycycline gel at the Section of Periodontology, Department of Conservative Dentistry, Clinic for Oral, Dental and Maxillofacial Diseases, University Hospital Heidelberg. Each patient had to provide three single-rooted teeth that qualified as test teeth for the doxycycline study. To blind the examiner for the test teeth, not only those but all single-rooted teeth were assessed in all patients [8]. Despite the test teeth and their direct neighbors, all single-rooted teeth were treated according to the following criteria: Teeth exhibiting PPD\u2009<\u20093\u00a0mm were treated neither by subgingival SRP nor by AF. SRP was performed when PPD\u2009\u2265\u20093\u00a0mm was present. AF was chosen for teeth exhibiting PPD\u2009>\u20096\u00a0mm [21]. However, teeth directly adjacent to test teeth were treated only by SRP irrespective of baseline PPD. Thus, also some sites with PPD\u2009>\u20096\u00a0mm were treated exclusively by SRP. Further, the decision to perform AF was taken per tooth and not per site. Thus, also sites with PPD\u2009\u2264\u20096\u00a0mm were treated by AF, if they were located at a tooth with one or more sites with PPD\u2009>\u20096\u00a0mm. Thereby, effectiveness of SRP and AF may be analyzed across the limits of indications as the ranges of PPD for the three treatment groups in Table\u00a04 demonstrate.\nHow could we exclude that the topically delivered doxycycline had any influence on the treatment results of the sites that were evaluated in this study? The subgingivally delivered agent or vehicle control may be partially absorbed and thereby have systemic effects at the other test teeth. A pharmacokinetic study had found doxycycline only in the blood of 1 of 16 patients [18]. Thus, a systemic additional effect on the other teeth is quite unlikely. It has to be kept in mind that in the sample investigated in this study, only one tooth of the whole dentition per patient received the active agent, i.e., doxycycline [8]. Further, amounts of the subgingivally delivered substance may leave the pocket via sulcus fluid and affect the other test teeth. Split-mouth studies designed to compare active agent and vehicle of topical antibiotics have made the provision that teeth selected as test sites should be separated by at least one tooth [8, 12], minimizing the interaction between therapy modes. In a pharmacokinetic study from control sites, much smaller amounts of doxycycline could be detected in the gingival crevicular fluid than from another site that had received the particular doxycycline gel subgingivally in the same dentition [20]. Thus, it is unlikely that the topical doxycycline had any additional effect on the treatment results of all teeth but the test teeth.\nTreatment effect\nThe results observed in this study after SRP are in agreement with observations reported by other authors 3 and 6\u00a0months after nonsurgical debridement of single-rooted teeth [2, 6]. Our PPD reductions and RAL-V gains achieved 6\u00a0months after therapy confirm data by a meta-analysis on nonsurgical vs surgical periodontal therapy that reports PPD reduction of 1.07\u00a0mm and vertical attachment (PAL-V) gains of 0.6\u00a0mm at sites with baseline PPD 4\u20136\u00a0mm 6\u00a0months after SRP. However, PPD reductions 6\u00a0months after AF for mean baseline PPD of 5.4\u00a0mm were 1.0\u00a0mm which is less than the meta analysis lists for baseline PPD 4\u20136\u00a0mm: PPD reduction 1.37\u00a0mm. PAL-V gain after AF was 0.3\u00a0mm which comes quite close to the 0.31\u00a0mm reported by the meta analysis [15]. Serino et al. report results 12\u00a0months after treatment [28]. For baseline PPD 4\u20135\u00a0mm they report PAL-V gains of 0.31\u00a0mm and PPD reduction of 1.1\u00a0mm after non-surgical as well as 0.21\u00a0mm and 1.6\u00a0mm after surgical therapy. This is quite similar to the results of this study regarding PAL-V gain and PPD reduction after SRP. However, the PPD reduction after surgical therapy is more favorable than ours [28].\nPrognostic factors\nCurrent smoking was generally associated with less favorable PPD reduction and RAL-V gain in all models. Smoking is the most significant external risk factor for periodontitis [5, 13, 29] and is known to deteriorate the results of periodontal therapy in general [7, 30]. However, smoking was the only patient-associated factor that was kept in all models. The diagnosis of aggressive periodontitis was associated with less favorable RAL-V gains, whereas it was not kept in the models to explain PPD reduction. It is interesting to note that the other patient-associated factors, i.e., age and sex, were kept in neither model. This confirms observations of D\u2019Aiuto et al. [5].\nIt is interesting to note that the diagnosis of aggressive periodontitis affected RAL-V gains but not PPD reduction. It seems that patients suffering from aggressive periodontitis have a higher risk for recessions than patients with chronic periodontitis.\nSeveral tooth- and site-associated factors were identified by multilevel analysis to influence clinical outcomes. Generally, baseline PPD was related to better PPD reduction and RAL-V gain (p\u2009<\u20090.0001). PPD reduction and RAL-V gain was less pronounced in the premolars and at interproximal sites (p\u2009<\u20090.15), and PPD reduction was less pronounced in the maxilla (p\u2009<\u20090.001). These results are plausible because compared to anterior teeth, premolars are less accessible and interproximal sites are more difficult to debride than oral and lingual sites. Less favorable PPD reduction in premolars than in incisors was also recently reported by other authors [5]. However, D\u2019Aiuto et al. observed more favorable PPD reduction at interproximal sites, whereas this study observed the contrary. The multilevel analysis performed in this study was adjusted for baseline PPD. Baseline pockets were deeper interproximally than at buccal or palatal\/lingual sites. This might explain more favorable PPD reduction at interproximal sites without adjusting for baseline PPD. Considering the different therapies, SRP and AF provided better PPD reduction than no therapy. However, the analysis failed to reveal statistically significant differences between both therapies in single-rooted teeth. It seems that AF provides no advantages over SRP in single-rooted teeth.\nTaking radiographic parameters into consideration, current smoking and baseline PPD were kept in the models. However, the greater the interproximal bone loss, the poorer the RAL-V gain (p\u2009<\u20090.0001), and infrabony defects were associated with inferior PPD reduction (p\u2009<\u20090.0001). Total amount of bone loss seems to be associated with poor prognosis of the respective tooth generally irrespective of the type of treatment. This has also been observed for long-term stability [17]. D\u2019Aiuto et al. [5] did not assess radiographic parameters. However, they considered tooth mobility and found mobility was correlated with less favorable PPD reduction. It is interesting to note that interproximal grooves that had been assessed as root double contours failed to influence the outcomes of therapy. Up to our best knowledge, this is the first analysis on the influence of baseline bone loss on the outcome of nonsurgical or surgical periodontal therapy that does not use patient means but individual tooth parameters.\nWithin the limitations of the present study the following conclusions may be drawn: (1) Nonsurgical and surgical periodontal therapies are effective in single-rooted teeth; (2) severe interproximal bone loss and infrabony defects deteriorate the clinical results, and (3) there seem to be more defect-associated (tooth, site) factors influencing treatment outcome than patient-associated factors.","keyphrases":["periodontal therapy","single-rooted teeth","smoking","clinical trial","surgical\/nonsurgical therapy"],"prmu":["P","P","P","P","R"]}
{"id":"J_Med_Internet_Res-6-3-1550604","title":"Internet Usage by Low-Literacy Adults Seeking Health Information: An Observational Analysis\n","text":"Background Adults with low literacy may encounter informational obstacles on the Internet when searching for health information, in part because most health Web sites require at least a high-school reading proficiency for optimal access.\nIntroduction\nAlthough a tremendous volume of educational health materials is disseminated in the United States, not all Americans find this information accessible or usable. In particular, adults with poor health and low functional literacy face great risks of poor health outcomes and preventable disease progression [1-4]. While many low-literacy adults could benefit from enhanced health knowledge, most current health education materials are written at a 10th grade or higher reading level [3]. Inability to access or understand health education materials inhibits important preventive or treatment measures, and may decrease the likelihood of identifying a symptom of disease. Low health literacy is also a barrier to enrollment in clinical trials [5,6] and minimizes adherence to instructions given by health professionals [7]. These obstacles are compounded by low income levels pervasive in the undereducated population [1], which can prevent individuals from pursuing regular primary care, paying health insurance premiums, or purchasing medications when prescribed. Collectively, these factors help to explain why low-literacy adults are twice as likely to be hospitalized as individuals with high functional literacy [8].\nThe expense of poor health and low functional literacy on the health system is estimated at $73 billion each year [9]. High cost estimates have encouraged many health-care providers to search for innovative ways to improve health literacy. The Internet has been embraced as an easy-to-use, convenient, and comprehensive clearinghouse for information on diseases, disorders, treatments, and preventions. Even when receiving physician care, between 40% and 54% of medical patients use the Internet to learn about treatment options and tobetter understand their medical conditions [10].\nHowever, the low-literacy population has largely been excluded from the veritable boom of Internet health resources. The expense of Internet services and personal computers may be too high for this population. In addition, most text-based health information on the Internet is too advanced to be optimally effective for low-literacy populations. On average, Internet health-education materials are written at a 10th grade or higher reading level, and 100% of English-language health Web sites examined in a 2001 study required at least high school-level reading proficiency [11,12]. Another study concluded that of 1000 Web sites reviewed, only 10 had a level of writing and content accessible to low-literacy adults [13]. Kalichman et al suggest that individuals who read English below a 6th grade level are not likely to make effective use of the Internet [14]. Further, Zarcadoolas et al report that complex Web features, such as animated links, may be challenging for low-literacy adults to identify and utilize [15]. The 1992 National Adult Literacy Survey (NALS) revealed that more than 90 million Americans either read at a low-literacy level or are functionally illiterate [1]; the paucity of Internet health resources appropriate for these individuals perpetuates discrepancies in health outcomes between the educated and undereducated.\nWhile no studies to date have determined how many low-literacy adults regularly use the Internet to find health information, the dearth of educational materials suitable for these individuals may impair optimal usage and navigation. One study has reported interventions that enabled low-income HIV-positive individuals to use the Internet and to critically evaluate information that they encountered [16]. Health-related Internet use has also been shown to enhance knowledge about HIV and to be correlated with active coping in a study of HIV-positive patients [17]. Although these studies focus on low-income status rather than low-literacy status, the established correlation between these two factors suggests that low-literacy adults may likewise benefit from augmented health education via the Internet.\nWe conducted an observational study of low-literacy adults to assess how they searched for Internet health information in as close to a natural setting as possible. Our investigative questions include the following: if low- to mid-level literacy adults are given access to the Internet, can they find basic health information that they can understand? Will their search strategies be effective in identifying information that they can use and comprehend? How do they rate current health Web sites in relation to their needs and interests? Will they be able to conduct successful self-directed searches? In our investigation, we also categorized navigational strategies used by low-literacy adults and the reading level of materials they accessed.\nMethods\nWe enrolled 13 adult literacy students (3rd to 8th grade reading levels) from Bidwell Training Center, a vocational school in Pittsburgh, Pennsylvania. The protocol used was approved by the University of Pittsburgh Institutional Review Board. Bidwell students are organized individually and\/or in small groups for reading instruction; they meet together once a week for program announcements. The literacy program coordinator introduced the study to 20 students in this large-group setting. Thirteen interested students then self-selected into the study. All subjects participated in a computer skills workshop in May 2003, where they were presented with basic search and navigation strategies and learned how to use the Google search engine. We selected Google because it is a widely used search engine and has a \"Did You Mean\u2026\" feature that corrects misspelled search terms. We anticipated that this might be a feature that low-literacy subjects would find particularly helpful. Among other topics, subjects were taught how to use the \"Back\" button and the \"Forward\" button, how to scroll down a page, how to identify links, and how to conduct basic searches. Each subject also filled out a brief questionnaire to give insight on their educational background, ethnicity, health insurance status, and previous experience with computers and the Internet. The questionnaire was written at a 3rd grade reading level (Flesch-Kincaid Reading Scale).\nAn investigator met individually with each of the participants within 3 weeks of the computer skills course for the observational portion of the study. Participants were 1) administered the REALM test (Rapid Estimate of Adult Literacy in Medicine) [18] to assess their health literacy level; 2) asked several questions to gauge their comfort level on the Internet and prior Internet experiences; and 3) taught how to \"think aloud,\" or continually express their thoughts while using the computer. Investigators engaged each participant in several think-aloud examples in order to actively illustrate this process.\nThe investigator then asked the participant to use the Internet and Google search engine and think aloud while finding information on a subject of his or her choice. This preliminary question allowed participants to practice and review their Internet searching techniques. Participants were permitted to ask the investigator technical and navigation-related questions during this part of the study. These questions included, but were not limited to, whether to put spaces between words in search terms and how to initiate a search once a search term had been specified.\nParticipants were then asked to find answers on the Internet to 3 health-related questions developed by members of the research team. Participants were instructed to use the Google search engine so that their answers could be standardized. A committee consisting of a physician, a faculty member specializing in human-computer interactions, a community health educator, and an information sciences specialist compiled various answers to these questions that would qualify as accurate and complete. Subjects who were able to generate any of these answers during their online searches were considered to have answered the questions correctly; subjects who were not able to generate these answers were determined to have answered the question either incorrectly or incompletely. Examples of responses for each question that would have been considered correct are included in the Results section\nThe investigator read the 3 questions aloud and also provided them to the participant in written form (Arial font, 20 pt):\n1. Think of a health question you are interested in for yourself or for someone you know. Find out information about this question on the Internet.\n2. Imagine that someone you care about has lung cancer. This person would like to know about treatments for lung cancer. Can you find out the three main types of treatments using the Internet?\n3. Imagine that you are at a doctor's office and you are told you have a disease called diabetes (sometimes called sugar). You are given a pill called Metformin to take for it. What does Metformin do?\nSubjects identified answers to the investigator, who then asked them to articulate the answers in their own words. Participants who seemed frustrated or unreceptive, or who asked to move to a new question were directed to the next task. Participants were allowed to use any Web sites they felt would help them answer the questions. Participants also were not provided with dictionaries-our objective was to examine how they navigated the Internet without assistance from external sources. Subjects were given up to 15 minutes to complete each task, as measured by the investigator. To minimize anxiety, they were not informed of the time limitation. After the 15-minute period, investigators used a series of prompts to gradually guide subjects, if necessary, to the next task.\nNext, investigators accessed the colon and rectum cancer Web page on the American Cancer Society (ACS) Web site [19]. Participants were asked to navigate through links on this page and find 2 ways to help prevent colon and rectum cancer. Investigators recorded the amount of time spent answering this question and the number of links participants clicked on to find the answers. After this task was completed, investigators asked the participants several subjective questions to qualify their experience on the Internet. Participants were then given $25 compensation, which ended their direct involvement in the study.\nInvestigators wrote notes on each participant's progress, and asked for participant feedback about the Internet both before and after searching the Internet. Investigators did not coach subjects on proper technical or navigational techniques after the initial practice question until subjects had completed their tasks. In 2 cases, investigators directed subjects to Google's \"Did You Mean\u2026\" search term correction option in order to adjust for spelling mistakes; these subjects had repeatedly demonstrated very poor spelling proficiency before this intervention.\nCamtasia Studio screen-capture software recorded individual keystrokes and think-aloud recordings. Questionnaires and think-aloud methods were used to ascertain the criteria used by participants in evaluating Internet health Web sites. Investigators also calculated the 1) literacy levels of Web sites accessed by the participants, 2) the amount of time spent on each Web site, 3) the number of questions answered thoroughly and correctly by each participant according to pre-determined standards, 4) the average number of sites used to answer each question, and 5) the number of participants who accessed sponsored sites, or paid advertisements appearing on the Google retrievals page, and how many used that information to answer questions.\nResults\nQualitative and quantitative results were analyzed in this study.\nParticipants\nIn this study, the subject population was reduced from 13 to 8. Two participants were excluded because they did not attend the one-on-one searching session with the investigator. Two other participants were excluded because they were non-native English speakers who did not understand the tasks presented to them. One participant was later excluded because technical problems prohibited the retrieval of her computer searches.\nThe average age of our 8 remaining participants was 41.5 years. Five subjects were male and 3 were female. Seven identified themselves as African Americans and 1 self-identified as of Asian descent. The Asian participant was an English-as-a-second language (ESL) speaker with a university education from his native country. Seven of the 8 participants reported having health insurance. Seven of the 8 also had at least some high school or trade school education; 1 participant did not report educational experience on the intake questionnaire.\nOf these subjects, 2 reported on the intake questionnaire that they had never previously used a computer or the Internet. Two reported that they had previously used a computer, but had not used the Internet. Subjects generally used computers with greater frequency than they used the Internet. Three participants reported on the questionnaire that they used the Internet 2 or more times a week; they later said verbalized that their main online interests were news, sports, cars, and\/or entertainment information. The other 5 participants reported on the questionnaire that they used the Internet either occasionally or not at all. Usage reports from the intake questionnaires are provided in Table 1.\nTable 1\nSelf-reported, written questionnaire responses about prior Internet and computer usage by subjects (n=8)\nSubject\nHave you ever used a computer? If so, how often?\nWhere do you use computers?\nDo you use the Internet? If so, how often?\nWhere do you use the Internet?\n1\nNo\n\"No where\" [sic]\nNo\n\"I've never used the Internet\"\n2\nLess than once a month\n\"When I was in jail\"\nNo\n(N\/A)\n3\n2 or more times a week\n\"At school, Bidwell Training Center in Ms. Cooper's class.\"\nNo\n\"At the Carnegie Library in Beechview where I live\"\n4\nNo\n\"No\"\nNo\n\"No\"\n5\n2 or more times a week\n\"To type\"\nYes; Less than once a month\n\"In school\"\n6\nNo; 2 or more times a week\n\"At home\"\nOnce a week\n\"At home\"\n7\n2 or more times a week\n\"Home\"\n2 or more times a week (at home)\n\"Home\"\n8\nOnce a week\n\"Different location\"\n2 or more times a week\n\"Different location\"\nAs seen in Table 1, the self reports of prior Web and computer experiences are unclear in several cases. Subject 3 reported no prior Internet usage in one part of the questionnaire, but reported in a subsequent answer Web usage at a local public library. In addition, as Table 1 indicates, subject 8 reported more frequent usage of the Internet than of computers; subject 6 (ESL student) first indicated no prior computer usage, then later reported on the questionnaire computer usage of twice a week. Because there were seemingly divergent perceptions of what constitutes a computer or Internet experience, perceived computer\/Web adeptness cannot be correlated with our participants' experience using this technology. Therefore, while this study will indicate differences in results between the 3 people with frequent Internet experiences (defined in this study as usage of at least once a week) and the 5 individuals without, the study will not attempt to conclude whether the skill level of subjects in the study correlated with the sustainability of their prior computer and Web experiences.\nSearch Engine Usage\nParticipants reviewed their navigational skills during their preliminary question, where they were encouraged to look for information on any subject that interested them. They used Google to search for a variety of topics, ranging from entertainment to health-related information. Participants occasionally searched for information on more than one topic.\nParticipants used the search items listed in Table 2 in order to answer the preliminary question and questions 1 to 3. Semicolons between words or phrases separate multiple search terms used by a subject to answer a question. The subjects are listed in Table 2 in the same order (ie, 1, 2, 3\u2026) as they appeared in Table 1.\nTable 2\nSearch terms used by subjects to answer preliminary questions and questions 1 to 3 (n=8)\nSubject\nPreliminary\nQuestion 1\nQuestion 2\nQuestion 3\n1\nlena horn\u2020\nLung cancer\nLung cancer\nMetformin\n2\nhealth care;health care mental\nSports and health\nhealth care about lung cancer\nA pill called metformin\n3\n(no clear search topic)\nHerpes\nCancer\nMetformin\n4\nWwwsoulfood; wwwsoulfoodcom; soulfood\nAIDS\nlung caner\u2020\nDiabetes\n5\nWill Smith; sipers\u2020; spiders\nHigh blood\nLung cancer\nMetformin\n6**\nBi;;;dwell training center\u2020\nHealth\nhealth lung caner\u2020\nHealth diabetes\n7\nsonny Rollins\nTuberculosis\nTreatments for lung cancer\nMetformin\n8\nBabyface recording artist\nPain\nCancer\nPdr*\n* Physicians' Desk Reference\n** English-as-a Second Language subject\n\u2020 misspellings for: \"lena horne,\" \"bidwell training center,\" \"lung cancer,\" and \"spiders\"; the Google correction option was used in two instances when the subject was prompted by investigators to amend search terms.\nQuestions 1 to 3 were given to our participants in writing, as well as orally; this may have affected their selection of search terms. For question 2, one participant wrote \"treatments for lung cancer\" in the search term box, a phrase that is written explicitly in that question. Another participant was similarly prompted by the wording of question 3 to write \"a pill called metformin\" as his search term.\nIndividuals who used the Internet at least once a week are labeled in Table 1 and subsequent tables as subjects 6 to 8. Search terms generated by these frequent Internet users did not differ greatly from search terms generated by individuals who had little Internet experience. The one exception was subject 8, who attempted to answer question 3 by using the online Physicians' Desk Reference, a site about which she had once heard good reviews.\nIn general, this group found generating original search terms to be somewhat challenging. Many did not initially remember whether to put spaces between the words in search terms. Even a subject who reported using the Internet once a week hesitated when writing the search term for question 1, finally stating, \"Yeah, you do have to space [between words]\u2026 I had to remember if you had to space.\" With one exception, participants were able to correct their terms by inserting the proper spaces.\nSpelling of search terms was generally a problem for only 2 participants, one of whom (subject 7) spoke English as a second language. Subjects tended to self-correct for spelling in the search term box before pressing the \"Google Search\" button or Enter key. Several participants also had difficulty understanding what type of terms to put in. When conducting a preliminary search for information on the television show, Soul Food, one participant typed into the search term box, \"wwwsoulfood.\" When this retrieved no results, the subject looked at the URL for guidance and then typed \"wwwsoulfoodcom\" into the Google search term box. This again did not yield any results. The participant next entered \"soulfood\" into the search term box. The investigator finally directed the subject to Google's \"Did You Mean\u2026\" option so that the subject could answer the question. However, this participant had continued difficulties generating correct search terms; later in the study, he used \"lungcaner\" as a search term to find information about lung cancer.\nNearly all participants retained skills such as scrolling and clicking on links from the computer workshop or previous Internet experiences. They also learned other navigational strategies through repetition and practice. For example, one participant who was conducting a preliminary search for information about Will Smith looked at the Google retrievals and stated, \"So it [search engine] must go to other Smiths ... I wonder if I was supposed to put in 'Will Smith the actor'?\" Quickly, the subject had learned that increasing the specificity of search terms generally improves the specificity of results.\nSix of the 8 participants did not venture past page 1 of the Google retrievals. One participant was surprised by the number of search results, saying, \"You find a lot of stuff on this thing [the Internet].\" Another participant explained why she stayed on page 1: \"Oh boy, I've got a lot to choose from. I don't want to go to the other ten [pages of retrievals] because it might give me other information I don't really need ... the first page gives me just enough of what I need to know.\" This participant had deduced that first-page retrievals typically have the most relevant sites to the particular search term used. Later, this subject stated, \"I didn't answer the questions, but I looked up the information, and it [Internet] gave me what it wanted me to have.\" This statement implies that the subject believed that the Internet was more in control of the searching than the subject, revealing a possible belief that the search engine and search terms selected are not the primary determinants of what type of information is retrieved.\nSites Accessed\nAbility to Answer Questions\nIn question 1, participants were asked to use information on the Internet to find the answer to a health-related query of their choice. Most participants identified only a subject area, and did not clearly articulate a specific question despite verbal prompting by the investigators. Several participants initially stated a topic, but changed it as they retrieved unrelated material that they found more interesting. While recordings from the think-alouds would have been helpful in designating the search topics, we found that despite investigators' prompts and encouragement, subjects were very reluctant to verbally report their real-time experiences navigating through the Web. As one subject stated, \"Shucks, I can't think aloud.\" It is therefore difficult to gauge whether participants were able to find adequate information for which they searched, especially during the unstructured searching period required to answer the first question.\nQuestion 2 required participants to locate the 3 main types of lung cancer treatments (acceptable answers: chemotherapy, surgery, radiation). This question models the navigation of a typical Internet health-information seeker who searches for disease-related information. Of all 8 participants, only subject 5 was able to answer this question accurately and completely. Subject 3 verbalized one viable option-chemotherapy-based on information accessed online. The remaining participants either did not answer the question or identified an alternative medicine as one of the principal types of lung cancer treatments available.\nQuestion 3 required participants to find out the role of metformin, or Glucophage, in diabetes treatment (one acceptable answer: metformin lowers sugar in the blood). This question models a doctor-patient interaction in which a patient who is prescribed an unfamiliar medication independently searches for information about its effects. Six of 8 participants were unable to find information on the Internet to answer the question. The 2 participants, subjects 3 and 7, who found the information, read directly from text on the site and did not articulate the information in their own words.\nSurprisingly, subjects who reported sustained prior Internet experience in the questionnaire were no more successful at answering questions than subjects with little Internet experience. This could have been a result of the generalized search terms that they used to look for answers. Prior Internet experience does not seem to lead to satisfactory search\/navigation skills for members of this group in searching for health information.\nInformation Accessed\nSites used by subjects 3, 5, and 7 to successfully answer questions 2 and 3 were written at a 12th grade reading level (Flesch-Kincaid). It is noteworthy that these subjects were able to identify the answer in the text and read it aloud. In 2 out of 3 cases, they were unable to express these answers in their own words, which suggests a minimal comprehension of the material accessed.\nSeven of the 8 participants accessed sponsored site information while attempting to answer questions. Businesses pay a service fee to Google to have their site names appear as sponsored sites when triggered by a particular search term or keyword. Sponsored sites are outlined in color and\/or appear in boxes on the right side and heading of the Google retrievals page. In general, alternative treatments and commercial therapies and medications appear under this listing; many of these sites may contain information that is uncorroborated by legitimate scientific sources.\nFive participants used information provided by the sponsored sites to answer questions. Two out of 3 of the subjects who used the Internet at least once a week also used this information to answer questions. Half of the participants searching for lung cancer cures arrived at the same site: an Asian dietary supplement site claiming to cure cancer by removing free radicals from the body [20]. Another popular sponsored site promoted a radio frequency technique to hinder cancer progression [21]. The titles of these sites as they appeared in the sponsored sites submenu were: \"New Cancer Treatment\" and \"Cancer Treatment.\" The Flesch-Kincaid formula indicated that the information on both sites was written at a 12th grade or higher reading level. Information on sponsored sites, therefore, was not necessarily any easier to read or interpret than information on non-sponsored sites accessed by subjects in this study.\nGeneral Site Profiles\nObservational logs and records on the Camtasia software show little correlation between our subjects' ability to identify answers and the amount of text on a page; analysis using the Camtasia software also showed little conclusive difference in the amount of time that the subjects spent on each site despite variances in the amount of text on the pages accessed. Therefore, subjects did not seem to prefer or navigate towards Web pages\/sites with less text.\nParticipants, on average, used between 1 and 2 Web sites to answer questions 1 to 3. Table 3 records the number of links from the Google retrievals page that were selected by subjects. The results for subjects 1 to 5-the participants with minimal prior Internet experience-are also presented separately from the results for participants with sustained prior Internet experience (subjects 6 to 8).\nThe Flesch-Kincaid reading scale used in this study scores text at a 1st to 12th grade reading level. Given this scale, sites ranked at the 12th grade level require at least that level of reading ability. That is, material scored at a 12th grade level may actually be written at a college level. In our study, the average site accessed required at least a 10th grade reading level.\nTable 3\nAverage number of links used to answer questions\nAvg. Number of Links Used (Average Total)\nAvg. Number of Links Used (Subjects 1-5)\nAvg. Number of Links Used (Subjects 6-8)\nPreliminary\n1.875\n2.4\n1.0\nQuestion 1\n1.14\n1.2\n1.67\nQuestion 2\n1.82\n1.8\n2\nQuestion 3\n1.5\n1.6\n1.33\nAVG.\n1.58\n1.75\n1.5\nTable 4\nAverage (rounded) reading level of sites accessed\nAvg. Reading Level of Sites Accessed\nAvg. Reading Level of Sites Accessed (Subjects 1-5)\nAvg. Reading Level of Sites Accessed (Subjects 6-8)\nPreliminary\n10.50\n10.7\n10.0\nQuestion 1\n10.50\n9.4\n11.2\nQuestion 2\n11.1\n11.3\n11.0\nQuestion 3\n11.8\n11.8\n11.9\nAVG.\n11.0\n10.8\n11.0\nTable 5\nAverage time spent on sites\nAvg. Total Time Spent Per Site (min)\nAvg. Total Time Spent Per Site (Subjects 1-5)\nAvg. Total Time Spent Per Site (Subjects 6-8)\nPreliminary\n7.2\n8.7\n4.7\nQuestion 1\n10.3\n10.6\n9.8\nQuestion 2\n8.7\n8.7\n8.7\nQuestion 3\n6.6\n8.3\n5.8\nAVG.\n8.2\n9.1\n7.25\nParticipants spent an overall average of 8.2 minutes on individual sites. All participants voluntarily finished answering questions 1 to 3 before the 15-minute time limit was reached.\nAfter completion of these first 3 questions, subjects were directed to a specific site; question 4 was posed about information directly linked to that site. We chose to use the ACS colon and rectum cancer Web page site, which contains links to a variety of prevention resources written at 6.3-12.0 grade levels (Flesch-Kincaid Reading Scale). The page to which we directed subjects consists of a listing of links to defined topic areas, one of which was closely related in wording to question 4. On the ACS site, 5 out of 8 people were able to answer question 4 correctly. Three of the 5 reported prior Internet experience; 2 reported none. These subjects used 3.8 links on average to answer the question. The 3 subjects who did not access the material used 6.5 sites on average before they were either stopped by the investigator or quit voluntarily. Two of these subjects had never used the Internet prior to enrollment in the study.\nAttitudes and Self-reporting\nWhile most participants were unable to answer all of the questions asked, 7 out of 8 reported feeling very comfortable or comfortable with their Internet searching experience. The eighth participant felt moderately comfortable. Also, 5 out of 8 found it at least moderately easy to find readable and understandable information on the Internet. Two of the remaining participants found it very difficult to find readable information, and one participant reported that finding understandable information is easy if the Web user has strong reading skills.\nDespite their dependence on sponsored sites and alternative Web sites to answer questions, 7 out of 8 subjects reported that they found it very easy to locate trustworthy information on the Internet. The eighth subject noted that it is moderately easy to find information that is trustworthy on the Internet. However, one subject said, \"I believe that on the Internet, you have your shysters ... just like anything.\" \nSubjects felt positive about continuing their online experiences, and all expressed some enthusiasm about improving their skills. One participant stated, \"I'm getting a computer ... it can help your typing skills.\" Another subject said, \"The computer is real interesting. I'm a see if I can get one so I can learn [how to use it].\" After the study was completed, many participants asked investigators to continue teaching them Internet skills or to continue helping them locate Internet resources on a variety of subjects.\nDiscussion\nThis observational study is the first to examine Internet use by low-literacy adults seeking health information [11]. Irrespective of prior experience using the Internet and\/or computers, low-literacy adults participating in our study did not use optimal search terms to answer questions, encountered difficulties finding health information at the appropriate reading level, and were unable to successfully interpret Internet health information as it was presented. While basic navigational skills (eg, using the \"Back\" button) were easily retained, areas that required reading and comprehension were problematic for most subjects-evidenced by their inability to answer questions and comments made during their think-alouds. Therefore, the literacy level needed to read health information on the Internet does appear to inhibit information-seeking efforts of low-literacy adults.\nSearching strategies were sub-optimal in several respects. First, the search terms used by subjects were predominately non-specific (Table 2). Although we anticipated that subjects who used the Internet more often would generate more specific search terms than did their peers, we did not observe this in the study.\nDifficulty Generating Search Terms\nWithout guidance, subjects had difficulty generating original search terms that would yield specific results. A recent study reveals that adolescents used similarly general search terms when searching the Internet for health information [22]; this corroborates results from another study, which found that among subjects with an average of 33 months of Internet experience, self-selected search terms to find health information were unexpectedly general [23]. These observations highlight search terms as a potential barrier to specific, targeted Internet health information for different types of Internet users with varying levels of Web expertise. A categorizing search engine might be particularly effective for use by these groups; it minimizes the need for individuals to both create a specific search term and independently read and assess all retrievals. A sample search to answer question 2 was conducted using the Vivisimo search engine [24]. The search term \"lung cancer\" yielded a series of folders about lung cancer separated by subject matter; one folder specifically focused on lung cancer treatments. Individuals clicking on that option could access all sites on lung cancer treatments retrieved by the engine, circumventing the need to sift through thousands of retrievals to locate treatment-focused sites. A future study could monitor the ease with which low-literacy individuals could conduct self-directed searches using an automatically sorting search engine.\nReluctance to Use Links\nSearch strategies observed in this study were also sub-optimal because most subjects exhibited some unwillingness to click on links to Web sites on the Google retrievals page. On average, subjects clicked on one to two links to answer questions. Even when the subjects did not appropriately answer questions or only partially answered questions, most seemed reluctant to click on additional links on the Google retrievals page, and 7 of 8 did not go to subsequent retrievals pages. These results did not seem to correlate with prior Internet experience. Subjects also rarely re-typed search terms in order to access more relevant retrievals. These results differ from those of a previous observational Internet study, whose participants preferred to choose links from page-one retrievals and then re-type original search terms if they were unable to find appropriate information [23]. As stated earlier, our subjects had such difficulty generating original search terms, figuring out appropriate spelling, and determining whether to place spaces between words in search terms, it is conceivable that this is why they avoided this strategy.\nAnother reason why subjects' generation of search terms and selection of links were so limited may have been because the subjects were not interested in the health materials or the questions. Subjects may have also found the Google retrievals page confusing and intimidating. While the think-alouds are inconclusive about which of these factors contributed most to the weak search strategies observed, the post-session questionnaire reveals that the majority of participants reported that it was easy to search the Internet. Future research may help to illuminate the factors that contribute to the inconsistencies between subjects' perceived unwillingness to explore the Internet's health resources and their positive feedback about navigating through these resources.\nHigh Literacy Levels of Health Web Sites\nThe health sites participants accessed to answer questions 1 to 3 had, on average, an 11th grade reading level (Flesch-Kincaid Reading Scale), which was consistent with the findings of previous studies [3,25]. Clearly, all of our subjects experienced difficulties using these sites to answer questions. The literacy level of the materials that the subjects did access may have limited their ability to read and understand materials as presented to them, and may have also impaired their ability to select the appropriate links for finding information. However, a majority of subjects were able to find specific information on the ACS Web site. As one subject reported about the site, \"This is a real good one 'cause it breaks it right down for you.\" This Web page consisted of a series of links: general links on the left and right sides of the page and links to colorectal cancer in the center. Subjects who were unable to answer the questions seemed to find the lists of links on the page confusing, and picked links that took them to unrelated pages on the ACS site rather than to specific pages containing colon and rectum cancer information. While the selection of only 1 link on the colon and rectum cancer Web page was necessary in order to answer the question, these subjects on average picked more than 6 separate links before quitting. Therefore, layout of health Web sites evidently affects the ability of low-literacy adults to find pertinent health information.\nDespite the navigational difficulties observed on the ACS Web page, the ability of 5 subjects to correctly answer question 4 probably resulted from the fact that the information needed to answer question 4 was written at an 8th grade reading level-significantly lower than the11th grade reading level required on average to read information retrieved in the first 3 searches. This suggests that low-literacy individuals can identify and utilize easier-to-read materials on Web sites. The Internet may indeed be a useful health resource to this population if materials are written at an appropriate reading level. Considering the navigational struggles of our subjects, the actual process of locating low-literacy sites on the Web may prove a more daunting challenge to this population.\nDifficulty Measuring Participants' Comprehension of Information\nWhile most were able to competently navigate through lower literacy materials, subjects' comprehension of Internet health information was difficult to measure in our study. Some participants found correct answers and read them to the investigators directly from the Web text, but none were able to articulate the answer in their own words when prompted. In their analysis of the1992 National Adult Literacy Survey (NALS) results, Kirsch et al reported that low-literacy adults may successfully perform simple comprehension exercises such as locating a single piece of information from text, but often find it more difficult to integrate and synthesize that information [1]. Furthermore, subjects in our study may have been able to use cues from sentence structure to locate an answer, and then relied on their pronunciation skills in order to read the answer as written. However, their ability to identify relevant health information within text is not necessarily a measure of their ability to comprehend that information.\nIn addition, several subjects seemed to compensate for their low literacy skills by using external information resources. One subject who examined a Web site on mental health law (12th grade level) expressed great enthusiasm about a particular topic that he said was presented on the site. A perusal of the site after the session showed that this topic was not addressed on any of the pages he had accessed. This participant may have compensated for his struggles in reading the site by citing facts with which he was personally familiar. Another subject used a similar approach when accessing a lung cancer site. When asked about the type of information he was reading, the subject responded that the page focused on smoking cessation. However, there were no smoking-related topics on the pages examined by the subject. The subject was able to correlate lung cancer with smoking, and may have relied on this information in order to answer the investigator's query. Overall, some subjects may have been able to rely less on actual comprehension skills and more on background knowledge in order to infer answers.\nPositive Web-site and performance feedback reported by most of the participants could have also been fueled by a desire to compensate for reading and comprehension difficulties. Participants were aware that the majority of the investigators were affiliated with a local hospital system; some may have felt compelled to answer positively about Internet health information because they were reporting to health-care professionals. Additionally, the participants may have been unwilling or ashamed to admit that they had difficulty understanding the information on the Internet. Individuals with low literacy tend to be embarrassed by their reading inadequacies [26]. Participants may have felt compelled to report more positively about their Internet experiences in order to de-emphasize their difficulties navigating the Web. These considerations might begin to explain that while most participants struggled when using the Internet, most 1) felt they did a good job searching for information, and 2) found information on the Internet readable and understandable. Collectively, then, poor comprehension of health information on the Internet coupled with a desire to compensate for self-perceived inadequacies in reading may have negatively affected the ability of our subjects to objectively evaluate Web sites. In this study, these factors may also have diminished the accuracy of their think-alouds and feedback in relation to their actual Internet experiences.\nInaccurate Self-assessment\nAn alternative reason why subjects reported positive experiences on the Internet could be that subjects were unaware of the magnitude of their Internet searching difficulties. A study by Moon et al indicates that 70% of subjects told investigators that they read \"really well,\" while in actuality, their mean REALM scores reflected a 7th to 8th grade reading level [27]. This suggests that individuals may actually overestimate their reading ability in relation to standard educational parameters; it may also relate to a similarly heightened perception of Internet competence. Furthermore, because the majority of our subjects had minimal Internet experience, they may not have been able to objectively gauge the limitations of their Internet skills in relation to the skills of more advanced users. While the investigators were able to categorize their searching as sub-optimal, our participants could have considered their searching strategies to be adequate, if not standard.\nPreference for Sponsored Sites\nSubjects' reliance on sponsored-site information to answer questions, regardless of the high literacy level required to read those sites, suggests that other factors promote the selective advantage of sponsored sites over non-sponsored sites. In fact, the design of sponsored sites on the Google retrieval page follows many of the guidelines for creating optimal layouts for health information targeted to low-literacy adults [28]. First, the sponsored sites are organized by topic, and are also segmented in colored boxes that stand out from the rest of the Google retrievals. They do not contain the \"teaser information\" and keywords associated with normal Google links, and minimize the amount of text used. Most are easier to read than the normal Google links, are automatically categorized by subject, and are visually stimulating. In addition, despite misspellings of search terms, sponsored sites are often applicable to the intended subject. For example, a search of \"lung caner\" instead of \"lung cancer\" yields sponsored sites on lung cancer, though most of the non-sponsored Google retrievals are irrelevant. When individuals misspell search terms, which the low-literacy subjects in our study did fairly commonly, they might easily gravitate to sponsored-site information to answer their health questions.\nOf concern is that subjects did not seem to differentiate between the information on the sponsored sites and information on non-sponsored sites. Subjects used these sites interchangeably to answer questions. One study suggests that critical interpretation of Web sites is based on the Internet acumen and interests of the information-seeker; if coupling the motivation to find a topic and the ability to do so successfully, the information-seeker will be well-equipped to evaluate Web sites objectively and perceptively [29]. This approach offers 3 possible explanations for our results. First, our questions may have been of little interest to our subjects; this may have diminished their motivation in answering questions and affected impacted their critical analysis of sites. Second, many of our subjects had little sustained exposure to various Web sites before the study. Those subjects in particular may not have been able to critically compare Web sites as readily as individuals who had previously seen both good and bad Web sites and developed their own rating system. In this context, most health information on the Internet may have seemed trustworthy and interchangeable to some of the subjects. Third, the searching problems observed even among those subjects with previous Internet experience underscore the fact that none of these subjects reported that their prior Web usage included searches for health information. While these subjects had successfully found items of personal interest in previous Web searches, they were unable to navigate to health materials that were any more accurate or easy-to-read than those found by the rest of the subjects. Therefore, health searches may present unique challenges to a low-literacy population that counter the ability to find accurate, trustworthy health information. This may result from the high literacy level required for reading health information and health Web sites in addition to the complexity of health terminology.\nLimitations of Methodology\nStandard methodologies used in this study to determine health literacy and to generate continual feedback were sub-optimal. First, REALM test results were inconclusive. Subjects were placed into the literacy program at Bidwell Training Center after taking the national Tests for Adult Basic Education (TABE). However, in our study, these subjects tested significantly higher on the REALM than expected for individuals with the reading levels indicated by their TABE scores as reported by Bidwell Training Center (3rd to 8th grade reading skills). Subjects may have strong phonetic skills that help compensate for poor word recognition and comprehension. This observation is supported in a study by Wilson et al [30], which similarly noted that lower literacy participants who used the REALM tested at several grade levels above their actual reading level. The REALM may not be an optimal tool for accurately determining the health literacy of low-literacy adults.\nWhereas complete think-alouds could have helped us better understand subjects' navigational priorities and comprehension levels, the protocols we used in this study were ineffective at prompting verbalization. None of the participants consistently articulated their step-by-step navigational process at all points during their searching session. Investigators continually prompted the subjects through the exercise, but were unable to stimulate free-thinking, consistent, and self-motivated think-alouds. One potential explanation originates from the observation that our study population was not uniformly familiar with the Internet. Therefore, some subjects may have felt overly challenged by simultaneously learning how to use the Internet and verbalizing their navigational strategies. According to previous studies [31], these subjects were probably in an \"acquisition role.\" Such studies disclosed that a learner who is new to a certain task focuses primarily on acclimatization, and finds it overwhelming to concurrently think aloud. Since traditional think-aloud protocols may be ineffective for this group, an interactive protocol may be of assistance for future studies. In such a protocol, subjects would directly be asked about specific site features, and asked to rate and make comparisons between health sites. This may highlight precise preferences the subjects might have for Web-site information, content, design, and presentation, and may result in a more cohesive rating system.\nOverall, however, our subjects were very enthusiastic about learning how to use the Internet, and all indicated an interest in improving their skills for future use. In this study and other studies [13,15], members of the low-literacy population have expressed excitement about using the Internet. In order for the Internet to further empower these individuals to make informed health decisions, the development of easy to read and easy to comprehend health materials is imperative. If Google's sponsored sites are usedas a guide, low-literacy adults prefer information that is aesthetically pleasing, has minimal text, and is organized by subject matter. Search engines that are able to consolidate these features for searches will probably be of greater use to this population. However, low-literacy adults must improve their navigation and searching skills to efficiently locate low-literacy materials on the Internet. With sufficient practice, they are likely to develop the skills to use the Internet to find specific health information, and learn to critically evaluate the information they access.\nIndications for Future Research\nOne caveat to the present study is that our sample size precluded the analysis of factors besides low literacy that could influence the results we observed. We believe, however, that our findings with this sample group in an observational study were representative of the way low-literacy adults interact with the Internet. It will be important to validate and analyze in a larger study the appeal of sponsored sites (as opposed to other retrieved links) to low-literacy adults. It will also be worthwhile to determine the relative importance of limited literacy in comparison to socioeconomic and cultural factors in effective use of the Internet by this population. Future work will identify the exact components of sites that engage and promote learning by low-literacy adults. Greater understanding of these factors will hasten the day when the Internet becomes an effective vehicle for optimizing the health knowledge and acumen for those at high risk of poor health outcomes.","keyphrases":["internet","literacy","health","reading","health education","health promotion","socioeconomic factors"],"prmu":["P","P","P","P","P","R","R"]}
{"id":"Arch_Orthop_Trauma_Surg-3-1-2092411","title":"Drilling k-wires, what about the osteocytes? An experimental study in rabbits\n","text":"Introduction The function of osteocytes regarding osteonecrosis has been underestimated for a long time. Recently it has been suggested that apoptosis of osteocytes results in strong osteoclastic bone resorption. Death of osteocytes due to drilling may therefore increase the risk of K-wire loosening. The purposes of our in vivo study were to assess the minimal drill time needed to notice disappearance of osteocytes and to measure the distance of the empty osteocyte lacunae surrounding the drill tract in relation with the insertion time, directly and 4 weeks after drilling Kirschner (K-) wires into the femur and tibia of rabbits.\nIntroduction\nExperiments concerning thermal damage to bone tissue due to drilling Kirschner wires (K-wires) show several variables interfering with heat generation [7, 9\u201314, 32]. These variables can be categorized into three groups: drilling technique, K-wire characteristics and external factors. The drilling technique is a subtle balance between drilling speed, insertion time and pressure [1, 2, 5, 30, 31, 34]. The characteristics of K-wires differ in diameter and K-wire tip [17, 23, 29]. The main external factor is irrigation with a water spray during drilling [3, 20, 24, 36].\nIt is believed that critical temperature for bone injury is around 56\u00b0C because alkaline phosphatase denaturates at this temperature. Osteocytes, however, may even be more sensitive to heat [23, 24]. Eriksson [9, 10, 12] observed that a temperature of 47\u00b0C for only 1\u00a0min results in bone resorption. Bone is capable of remodeling by bone resorbing osteoclasts, bone forming osteoblasts and osteocytes [15, 19, 22, 26]. The latter differentiate out of osteoblasts which have ceased bone production and have become embedded in the bone matrix, in vacuoles cushioned by fluid and large molecules, forming a network [8, 15, 19, 22, 25]. Despite the fact that the osteocytes are the major constituents of bone, their role in bone resorption regulation has remained controversial for a long time [4, 16, 21, 22, 27]. In the past decade, however, understanding of osteocyte physiology has increased dramatically and it has become clear that osteocytes play an important role in bone remodeling [6, 15, 18, 25, 26, 35]. Now we know that micro damage to bone, e.g. by drilling, is associated with an increase in osteocyte death by apoptosis [25]. This process triggers local bone resorption resulting in K-wire loosening, because death of osteocytes turns off the inhibition of osteoclasts [6, 15, 16, 22, 25, 35].\nThe status of osteocytes after drilling into bone has been investigated before. Thompson [33] describes the absence of osteocytes as an acute cellular reaction, which increases in severity with increase of drilling speed. Pallan et al. [28] continued Thompson\u2019s [33] work and describes the delayed cellular changes in bone after pin insertion which were left in situ for maximum 10\u00a0weeks. He also concluded that higher speeds produce relatively higher temperatures and increased tissue response after a long time period [28].\nIt is well known that in clinical daily practice K-wires are often drilled without proper cooling because most drilling devices do not have an incorporated cooling system and therefore cooling has to be done manually. In case percutaneous drilling is performed, the cooling effect on bone is minimal because of the surrounding soft tissues. An in vivo study was therefore designed which simulated daily practice concerning K-wire drilling to analyze the absence of osteocytes directly (t\u00a0=\u00a00) and 4\u00a0weeks (t\u00a0=\u00a04) after insertion. Our first aim was to measure the minimal drill time needed to notice disappearance of osteocytes. The second aim was to measure the distance of the disappeared osteocytes around the periphery of the drill holes in relation to drilling time.\nMaterials and methods\nAnimals and anesthesia\nA total of 14 healthy, New Zealand white rabbits of female sex weighing a mean of 2.81\u00a0kg (2.66\u20133.09\u00a0kg) were used in this investigation. The rabbits were solely housed on a 12\u00a0h\/12\u00a0h (light\/dark) cycle and provided with standard diet food and water ad libitum. All animals were housed in the Central Animal Laboratory, Utrecht University, Utrecht, The Netherlands and received care in compliance with the European Convention Guidelines.\nThe animals were pre-anesthetized with a combination of methadone (10\u00a0mg\/ml at a dose of 2.5\u20135.0\u00a0mg i.m.), ventraquil (10\u00a0mg\/ml at a dose of 2.5\u20135.0\u00a0mg i.m.) and etomidaat (2\u00a0mg\/ml at a dose of 2.0\u20138.0\u00a0mg i.v.).\nAfter introduction of anesthesia the rabbits were cuffed and mechanically ventilated with O2:N2O, proportion 1:1, and 2% isoflurane. During the surgical procedure methadone (2.0\u20135.0\u00a0mg i.v.) was given. At the end of the operation nalbuphine (10\u00a0mg\/ml at a dose of 1.0\u20132.0\u00a0mg\/kg i.v.) was administered. After surgery the rabbits were housed at the intensive care for the rest of the day and night. At the end of the study the rabbits were euthanized by an i.v. overdose of pentobarbital.\nSurgical technique\nAn operation device was created by the first author (Fig.\u00a01). The operation device consists of a base plate. On this base plate, a dynamic plate was fixed which could be moved up or down. In front of the entire length of this dynamic plate a sideways moving slide was fixed on the base plate. On this sideways moving slide another back-forward slide including the drill was fixed. Forward movement of the drill was initiated using a 1.5\u00a0kg weight.\nFig.\u00a01Operation device consisting of a base plate with a slide for sideways movement on top of this. On this slide another back-forward slide with a drill was fixed. The forward movement was initiated by a weight of 1.5\u00a0kg. Another dynamic plate was fixed on the base of the plate to move up and down. During surgery, the rabbit was fixed on this dynamic plate\nDuring surgery the rabbit was fixed on the dynamic plate. This plate made it possible that the femur or tibia were on the same height as the K-wire. The sideways moving slide was responsible for the exact position of the K-wire in front of the femur or tibia.\nAfter the rabbits were preanesthetized, X-rays were made to exclude deformities. Thereafter the animal\u2019s hind limb was carefully shaved and prepared with a povidone-iodine solution. After this procedure, the hind limb was fixed on the testing machine. With the animal surgically draped, a straight-line skin incision was made on the lateral aspect of the femur extending from just below the anterior\u2013inferior spine to the distal femur, followed by a straight-line skin incision on the lateral aspect of the tibia extending from just below the joint line proximally to about the joint line distally.\nDissection was carried out down to the periostium. Synthes Trocar tipped K-wires of 70\u00a0mm length and 0.6\u00a0mm thickness were drilled through the diaphysis. One K-wire was drilled into the femur and one into the tibia. Drilling was performed by a rotary engine fixed at 1,200 rpm. This is the maximum drilling speed used in our daily practice. Cooling was not performed. After insertion, the K-wires were cut short and the K-wire ends were bent to the cortex. After the wounds were closed in layers, X-rays were made to check the position of the K-wires and the condition of the bone. Insertion time could be measured very accurately by analyzing the operations recorded on video camera. All the experiments were performed by the same investigator.\nHistological technique\nAfter termination, the femur and tibia were removed from the hind limb and fixed in 4% formaldehyde solution. They were then decalcified, cut transversely next to the K-wire hole, after the K-wire was removed gently by a pair of tweezers, and embedded in paraffin according to standard procedures. Four micrometer thick serial sections were cut until the drill hole was visible, stained with hematoxylin and eosin and evaluated under a light microscope at 400\u00d7 magnification for the presence or absence of osteocytes in the osteocyte lacunae surrounding the drill holes by a single investigator. The best section was used for evaluation. The distance over which the osteocytes had disappeared perpendicular to the drill holes was measured with an interactive morphometry device (Q-PRODIT, Leica, Cambridge, UK). In each section, four distances from the drill hole to the first osteocyte bearing lacuna were measured and averaged.\nStatistics\nPearson\u2019s Chi-square test was used to determine the drilling time needed for osteocytes to disappear. Pearson correlation was used to highlight any significant correlation between the drilling time and the distance of the disappeared osteocytes surrounding the drill holes. A P-value <0.05 was considered statistically significant. The data were analyzed using SPSS 12.0.1 for windows.\nResults\nAfter surgery (t\u00a0=\u00a00) six rabbits were terminated. Both hind limbs were used resulting in 24 assessments. From the remaining eight rabbits only one hind limb was operated. Four weeks later at termination this resulted in 16 assessments (t\u00a0=\u00a04). Two t\u00a0=\u00a00 and one t\u00a0=\u00a04 assessments were lost because no sections could be produced showing the drill hole, leaving 22 t\u00a0=\u00a00 and 15 t\u00a0=\u00a04 assessments.\nHistological response to drilling was seen in most sections next to the drill hole (Fig.\u00a02). At t\u00a0=\u00a00 and t\u00a0=\u00a04, all osteocyte lacunae next to the drill holes were empty in 11\/22 (50.0%) and 13\/15 (86.7%) of bones, respectively. At t\u00a0=\u00a04, a drilling time longer than 27\u00a0s resulted in a significant loss of osteocytes surrounding the drill holes (P\u00a0=\u00a00.008). At t\u00a0=\u00a00, a drilling time longer than 37\u00a0s resulted in a significant loss of osteocytes surrounding the drill holes (P\u00a0=\u00a00.011).\nFig.\u00a02Status of osteocytes due to K-wire drilling at 1,200 rpm. Disappeared osteocytes due to drilling are marked by a dot. The osteocytes are encircled. The dotted line indicates the border between the present and disappeared osteocytes (hematoxylin and eosin)\nA statistically significant positive correlation was seen between the distance of the empty osteocyte lacunae surrounding the drill hole in relation with drilling time, directly (P\u00a0=\u00a00.008, Fig.\u00a03a) and 4\u00a0weeks after K-wire insertion (P\u00a0<\u00a00.000, Fig.\u00a03b).\nFig.\u00a03a Distance of empty osteocyte lacunae, in micrometers, surrounding the drill hole in correlation with drilling time, in seconds, at t\u00a0=\u00a00. b Distance of empty osteocyte lacunae, in micrometers, surrounding the drill hole in correlation with drilling time, in seconds, at t\u00a0=\u00a04\nDiscussion\nThe two most important findings of this experiment are that (1) osteocytes disappear especially beyond a drilling time of 27\u00a0s and (2) that there is a positive correlation between the distance of the empty osteocyte lacunae in relation with drilling time.\nAs far as we know there are no experiments studying the presence\/absence of osteocytes while using drilling time as a variable. Therefore it is interesting to notice that our results, representing daily practice by using a drilling speed of 1,200 rpm, clearly indicate that osteocytes disappear after a drilling time above 27\u00a0s in this animal model, when no cooling is used. Pallan [28] and Thompson[33] however, noticed absence of osteocytes already after a drilling time varying from 5 to 12\u00a0s. The limitation of their experiments is that the insertion force was not standardized. That could be a possible factor explaining the difference in insertion time with matching histological changes.\nThe effect on osteocytes in our study was more pronounced 4\u00a0weeks after insertion compared to the effect on osteocytes directly after surgery. This confirms the results from Thompson [33] where the effect on osteocytes was more pronounced 72\u00a0h after the operation than at either 24 or 48\u00a0h. Apparently, not all damage to osteocytes is acute and microscopically visible, directly after K-wire insertion. This can be explained by the study of Eriksson et al. [13]. They compared histology and histochemistry for detection of bone viability directly after surgery. The histological sections revealed a 200\u00a0\u03bcm wide zone of empty osteocyte lacunae around the periphery of the drill hole while histochemistry of the same bone specimens showed a zone of 500\u00a0\u03bcm lacking diaphorase enzyme activity. This indicates that the empty osteocyte lacunae directly after surgery underestimates the extent of the drilling trauma and becomes clear as time passes. Neovascularisation is probably a reason for the increase of osteocytes disappearance in time. During heating, due to drilling, blood flow will stop in minor vessels preventing the clearance of the osteocyte lacunae [10, 13]. After neovascularisation the osteocyte lacunae can be cleared finally.\nOur study also showed an interesting positive correlation between the distance of the empty osteocyte lacunae in relation with time. The zones of disappeared osteocytes varied from 106 to 1,285\u00a0\u03bcm and the drilling times from 41 to 262\u00a0s. A correlation between drill time and distance was not shown before. What we do know is that Eriksson et al. showed a 200\u00a0\u03bcm wide zone of empty osteocyte lacunae around the periphery of the hole after drilling into femurs of New Zealand white rabbits while conventional irrigation was administered[13]. These results are difficult to compare because Eriksson et al. did not measure time, irrigated with saline, did not standardize the insertion force and drilled with a running speed of 20,000\u00a0rpm. Furthermore, Pallan [28] showed that the histological changes, including absence of osteocytes, were never apparent more than 250 to 500\u00a0\u03bcm from the pins [28]. Like Eriksson they did not standardize insertion force and drilling time varied from 5 to 12\u00a0s.\nNo osteoclastic activity was noticed in the already existing cortex. This was expected as Pallan [28] hardly noticed osteoclastic activity after 6\u00a0weeks, and only slight osteoclastic activity after 8 and 10\u00a0weeks and our experiments covered a time span of only 4\u00a0weeks.\nWe are convinced that delayed tissue response corresponds with late K-wire loosening seen in daily practice. Although we did not compare drilling, with and without cooling, this study demonstrates the need for a short drill time, less than 27\u00a0s to prevent the disappearance of osteocytes and to limit the bone resorption cascade.","keyphrases":["drilling","insertion time","kirschner wire","animal","histology"],"prmu":["P","P","P","P","P"]}
{"id":"Purinergic_Signal-4-1-2246002","title":"The inflammatory effects of UDP-glucose in N9 microglia are not mediated by P2Y14 receptor activation\n","text":"In this study we evaluated the functionality and inflammatory effects of P2Y14 receptors in murine N9 microglia. The selective P2Y14 receptor agonist UDP-glucose (UDPG) derived from microbial sources dose dependently stimulated expression of cyclooxygenase-2 and inducible nitric oxide synthase, and potentiated the effects of bacterial lipopolysaccharide on nitric oxide production. However, another selective P2Y14 receptor agonist, UDP-galactose, did not affect these endpoints either alone or in combination with lipopolysaccharide. Interestingly, synthetic UDPG also had no detectable pro-inflammatory effects, although P2Y14 receptors are both expressed and functional in N9 microglia. While synthetic UDPG decreased levels of phosphorylated cyclic AMP response element binding protein, an effect that was blocked by pertussis toxin, the pro-inflammatory effects of microbial-derived UDPG were insensitive to pertussis toxin. These data suggest that the pro-inflammatory effects of microbial-derived UDPG are independent of P2Y14 receptors and imply that microbial-derived contaminants in the UDPG preparation may be involved in the observed inflammatory effects.","keyphrases":["microglia","p2y14","cyclic amp response element binding protein (creb)","cyclooxygenase-2 (cox-2)","inducible nitric oxide synthase (inos)","pertussis toxin (ptx)","udp galactose"],"prmu":["P","P","M","M","M","M","U"]}
{"id":"Mol_Syst_Biol-3-_-2174632","title":"A map of human cancer signaling\n","text":"We conducted a comprehensive analysis of a manually curated human signaling network containing 1634 nodes and 5089 signaling regulatory relations by integrating cancer-associated genetically and epigenetically altered genes. We find that cancer mutated genes are enriched in positive signaling regulatory loops, whereas the cancer-associated methylated genes are enriched in negative signaling regulatory loops. We further characterized an overall picture of the cancer-signaling architectural and functional organization. From the network, we extracted an oncogene-signaling map, which contains 326 nodes, 892 links and the interconnections of mutated and methylated genes. The map can be decomposed into 12 topological regions or oncogene-signaling blocks, including a few \u2018oncogene-signaling-dependent blocks' in which frequently used oncogene-signaling events are enriched. One such block, in which the genes are highly mutated and methylated, appears in most tumors and thus plays a central role in cancer signaling. Functional collaborations between two oncogene-signaling-dependent blocks occur in most tumors, although breast and lung tumors exhibit more complex collaborative patterns between multiple blocks than other cancer types. Benchmarking two data sets derived from systematic screening of mutations in tumors further reinforced our findings that, although the mutations are tremendously diverse and complex at the gene level, clear patterns of oncogene-signaling collaborations emerge recurrently at the network level. Finally, the mutated genes in the network could be used to discover novel cancer-associated genes and biomarkers.\nIntroduction\nCells use sophisticated communication between proteins in order to initiate and maintain basic cellular functions such as growth, survival, proliferation and development. Traditionally, cell signaling is described via linear diagrams and signaling pathways. As many more \u2018cross-talks' between signaling pathways have been identified (Natarajan et al, 2006), a network view of cell signaling emerged: the signaling proteins rarely operate in isolation through linear pathways, but rather through a large and complex network. As cell signaling is crucial to affect cell responses such as growth and survival, alterations of cellular signaling events, such as those that arise by mutations, can result in tumor development. Indeed, cancer is largely a genetic disease that is caused by acquiring genomic alterations in somatic cells. Alterations to the genes that encode key signaling proteins, such as RAS and PI3K, are commonly observed in many types of cancers. During tumor progression, it is proposed that a malignant tumor arises from a single cell, which undergoes a series of evolutionary processes of genetic or epigenetic changes and selections so that a cell within the population can acquire additional selective advantages for cellular growth or survival, resulting in progressive clonal expansion (Nowell, 1976).\nGenetic mutations of the signaling proteins might overactivate key cell-signaling properties such as cell proliferation or survival and then give rise to the cell with selective advantages for uncontrolled cellular growth and promoting tumor progression. In addition, mutations may also inhibit the function of tumor-suppressor proteins, resulting in a relief from normal constraints on growth. Furthermore, epigenetic alterations by promoter methylation, resulting in transcriptional repression of genes controlling tumor malignancy, is another important mechanism for the loss of gene function that can provide a selective advantage to tumor cells.\nEnormous efforts have been made over the past few decades to identify mutated genes that are causally implicated in human cancer. Furthermore, a genome-wide or large-scale sequencing of tumor samples across many kinds of cancers represents a largely unbiased overview of the spectrum of mutations in human cancers (Stephens et al, 2005; Sjoblom et al, 2006; Greenman et al, 2007; Thomas et al, 2007). Most of these efforts have been made by the Cancer Genome Project (CGP, http:\/\/www.sanger.ac.uk\/genetics\/CGP\/), which aims to identify cancer-mutated genes using genome-wide mutation-detection approaches. Similarly, genome-wide identification of epigenetic changes in cancer cells has been conducted recently (Ohm et al, 2007; Schlesinger et al, 2007; Widschwendter et al, 2007). These studies showed that a substantial fraction of the cancer-associated mutated and methylated genes is involved in cell signaling, which is in agreement with the previous finding that the most common domain encoded by cancer genes is the protein kinase domain (Futreal et al, 2004). Although there is a wealth of knowledge regarding molecular signaling in cancer, the complexity of human cancer prevents us from gaining an overall picture of the mechanisms by which these genetic and epigenetic events affect cancer cell signaling and tumor progression. Where are the oncogenic stimuli embedded in the network architecture? What are the principles by which genetic and epigenetic alterations trigger oncogene-signaling events? Given that so many genes have genetic and epigenetic aberrations in cancer signaling, what is the architecture of cancer signaling? Do any tumor-driven signaling events represent \u2018oncogenic dependence' (the phenomenon by which certain cancer cells become dependent on certain signaling cascades for growth or survival)? Who are the central players in oncogene signaling? Are there any signaling partnerships generally used to generate tumor phenotypes? To answer these questions, we conducted a comprehensive analysis of cancer mutated and methylated genes on a human signaling network, focusing on network structural aspects and quantitative analysis of gene mutations on the network.\nResults and discussion\nThe architecture and the relationships among the proteins of a signaling network are important for determining the sites at which oncogenic stimuli occur and through which oncogenic stimuli are transduced. Extensive signaling studies during the past decades have yielded an enormous amount of information regarding regulation of signaling proteins for more than 200 signaling pathways, most of which have been assembled and collected in public databases in diagrams. We manually curated the data on signaling proteins and their relations (activation and inhibitory and physical interactions) from the BioCarta database and the Cancer Cell Map database (see Materials and methods). We merged the curated data with another literature-mined signaling network that contains \u223c500 proteins (Ma'ayan et al, 2005). As a result, we have built a human signaling network containing 1634 nodes and 5089 links. Integrative network analyses have provided numerous biological insights (Wuchty et al, 2003; Han et al, 2004; Ihmels et al, 2004; Luscombe et al, 2004; Kharchenko et al, 2005; Wang and Purisima, 2005; Cui et al, 2006). Thus, the integration of the data on mutated and methylated cancer-associated genes onto the network will help us to identify critical sites involved in tumorigenesis and increase our understanding of the underlying mechanisms in cancer signaling.\nTo integrate mutated and methylated genes onto the network, we first collected the cancer mutated genes from the Catalogue Of Somatic Mutations In Cancer (COSMIC) database, which collects the cancer mutated genes through literature curation and large-scale sequencing of tumor samples in the CGP. We then combined these data with the cancer mutated genes derived from other genome-wide and high-throughput sequencing of tumor samples (Stephens et al, 2005; Sjoblom et al, 2006; Greenman et al, 2007; Thomas et al, 2007). The merged gene set represents a mixture of the past directed approach and current systematic screening of cancer mutations. The cancer-associated methylated genes were taken from the genome-wide identification of the DNA methylated genes in cancer stem cells (Ohm et al, 2007; Schlesinger et al, 2007; Widschwendter et al, 2007). Finally, 227 cancer mutated genes and 93 DNA methylated genes were mapped onto the network. Among the 227 cancer mutated genes, 218 (96%) and 55 (24%) genes were derived from large-scale gene sequencing of tumors and literature curation, respectively (see Materials and methods, Figure 1A). In general, cancer genes can be divided into two groups: positive regulators (oncogenes) that promote cancer cell proliferation and the negative regulators (tumor suppressors) that restrain it. By comparing the mutated genes with the known tumor suppressors, we found that only 6.6% (15 genes) of the mutated genes are known tumor suppressors and that the majority of the mutated genes are oncogenes (Supplementary Figure 1). On the other hand, methylated genes are mainly found to encode tumor suppressors in cancer cells (Supplementary Figure 1) (Ohm et al, 2007; Widschwendter et al, 2007).\nCancer mutated genes are enriched in signaling hubs but not in neutral hubs\nGenes that, when mutated or silenced, result in tumorigenesis often lead to the aberrant activation of certain downstream signaling nodes resulting in dysregulated growth, survival and\/or differentiation. The architecture of a signaling network is important for determining the site at which a genetic defect is involved in cancer. To discover where the critical tumor signaling stimuli occur on the network, we explored the network characteristics of the mutated and methylated genes. The signaling network is presented as a graph, in which nodes represent proteins. Directed links are operationally defined to represent effector actions such as activation or inhibition, whereas undirected links represent protein physical interactions that are not characterized as either activating or inhibitory. For example, scaffold proteins do not directly activate or inhibit other proteins but provide regional organization for activation or inhibition between other proteins through protein interactions. In this case, undirected links are used to represent the interactions between scaffold proteins and others. On the other hand, adaptor proteins are able to activate or inhibit other proteins through direct protein interactions. In this situation, directed links are used to represent these relations. There are two kinds of directed links. An incoming link represents a signal from another node. The sum of the number of incoming links of a node is called the indegree of that node. An outgoing link represents a signal to another node. The sum of the number of outgoing links of a node is called the outdegree of that node. We call incoming and outgoing links as signal links, whereas the physical links are neutral links. We first examined the characteristics of the nodes that represent mutated genes on the network. We compared the average indegree of the mutated genes with that of the nodes in the whole network. We found that the average indegree of the mutated nodes is significantly higher than that of the network nodes (P<1.1 \u00d7 10\u22126, Wilcoxon test, Supplementary Figure 2). A similar result was obtained for the average outdegree of the mutated nodes (P<6.0 \u00d7 10\u221214, Wilcoxon test, Supplementary Figure 2). In contrast, there is no difference of the average neutral degrees between the mutated nodes and other nodes in the network. To refine these results further, we calculated the correlations between the indegree, outdegree and neutral degree of the network nodes. We found a significant correlation between the indegree and the outdegree of the network nodes (R=0.41, P<2.2 \u00d7 10\u221216, Spearman's correlation), but no correlation between the indegree and neutral degree of the nodes (R=\u22120.02, P=0.54, Spearman's correlation). Taken together, these results suggest that cancer mutations most likely occur in signaling proteins that are acting as signaling hubs (i.e., RAS) actively sending or receiving signals rather than in nodes that are simply involved in passive physical interactions with other proteins. As these hubs are focal nodes that are shared by, and\/or are central in, many signaling pathways, alterations of these nodes, or signaling hubs, are predicted to affect more signaling events, resulting in cancer or other diseases. In previous studies, we found that cancer-associated genes are enriched in hubs (Awan et al, 2007). However, these results indicate that cancer-associated genes are enriched in signaling hubs but not neutral hubs.\nWe also investigated the relations between the node degree and the methylated genes in the network. Methylated gene nodes do not appear to differ significantly from the network nodes with regard to their indegree, outdegree and neutral degree, respectively (P=0.32, P=0.16, P=0.09, Supplementary Figure 2). These results suggest that cancer mutated genes and methylation-silenced genes have different regulatory mechanisms in oncogene signaling.\nActivating and inhibitory signals enhance and alleviate oncogene-signaling flows, respectively\nSignaling flow branching represents the splitting of one signal at a source node (Figure 1B), whereas signaling flow convergence represents the consolidation of the signals at a target node from two source nodes (Figure 1B). Both types of the signaling flows are the basic elements of the network architectural organization. In the network, when the upstream and downstream nodes of a particular signal transduction event get altered either genetically or epigenetically, we considered the transduction event (link) to be most likely selected and used in cancer signaling and defined it as an oncogenic signal transduction event (Figure 1B). If a particular oncogenic signal transduction event is frequently found in many tumor samples, we infer that the tumor cells are \u2018dependent' on this highly used signaling event and call it \u2018oncogene-signaling-dependent event' (Figure 1B). To investigate how cancer signaling is distributed on these signal transduction routes, we extracted all the branching and convergent signaling flow units that contain at least one oncogenic signal transduction event and conducted a quantitative analysis by overlaying the gene mutation frequency onto these units. The mutation frequency of a gene was defined as the number of tumor samples that contain that mutated gene divided by the total number of the tumor samples that are used to screen the mutations for that gene. The mutation frequency of each mutated gene was obtained by using the COSMIC database, which contains the data on more than 200 000 tumor samples screened for cancer gene mutations. For the signaling branching units, we divided the signaling flows into two groups: activating and inhibitory group (Figure 1B) and compared the gene mutation frequencies of the upstream nodes with those of the downstream nodes in each group. Interestingly, in the activating group, the upstream nodes often have lower mutation frequencies than those of the downstream nodes. In contrast, in the inhibitory group, the upstream nodes often have higher mutation frequencies than those of the downstream nodes (Table I). Statistical tests confirmed that these observations are statistically significant (Table I). Similar results were obtained for the signaling convergent units as well (Figure 1B, Table I). These results suggest that the oncogene-signaling event triggered by mutations is preferentially associated with activating downstream signaling paths or conduits. Conversely, oncogene-signaling event triggered by mutations are less likely to be associated with downstream inhibitory signaling paths.\nIn general, there are far more activating signaling flows than inhibitory ones in the network. Thus, we hypothesized that the downstream genes of the network, especially the genes of the output layer of the network, would have a higher mutation frequency. To test this possibility, we compared the average gene mutation frequency of the nuclear proteins, which represent the output layer members of the network, with that of the other network genes. Indeed, the nuclear genes have higher mutation frequency than others (P=0.01, Wilcoxon test), which complements with our previous finding that cancer-associated genes are enriched in the nuclear proteins (Awan et al, 2007). In contrast, the distributions of the methylated genes have no such preference, suggesting that DNA methylated genes do not tend to directly affect the output layer of the network. These results strongly suggest that the genes in the output layer of the network, which play direct and important roles in determining phenotypic outputs, are frequent targets for activating mutations. The importance of this output layer is reinforced by our previous observations that the expression of the output layer genes of the signaling network is heavily regulated by microRNAs (Cui et al, 2006).\nMutated and methylated genes are enriched in positive and negative regulatory loops, respectively\nThe complex architecture of signaling networks can be regarded as consisting of interacting network motifs, which are statistically overrepresented subgraphs that appear recurrently in networks. A signaling network motif, also known as regulatory loops in biology, is a group of interacting proteins capable of signal processing. They bear specific regulatory properties and mechanisms (Babu et al, 2004; Wang and Purisima, 2005). The structure and the intrinsic properties of the frequently occurring network regulatory motifs give us a functional view of the organization of signaling networks. Thus, the study of the distributions of the mutated and methylated genes in the network motifs will provide insights into cancer-signaling regulatory mechanisms. We first examined the mutated genes on the feed-forward loops, in which the first protein regulates the second protein, and both proteins regulate the third protein. We classified the feed-forward loops into four subgroups (labeled 0\u20133) based on the number of nodes that are mutated genes. We calculated the ratio (Ra) of positive (activating) links to the total directed (positive and negative) links in each subgroup and compared it with the average Ra in all the feed-forward loops, which is shown as a horizontal line in Supplementary Figure 3. The Ra (\u223c0.7) in subgroup 0 is less than the average Ra (\u223c0.74) of all the feed-forward loops (P<1.9 \u00d7 10\u22129, Fisher's test). However, as the number of mutated nodes rises, the Ra for the corresponding group increases to a maximum of \u223c0.93 (Supplementary Figure 3, Supplementary Table 1). We obtained similar results, when we extended the same analysis to all the 3-node- and 4-node-size network motifs (Figure 2, Supplementary Table 1). These motifs show a clear positive correlation between positive link ratio and the number of mutated genes in the motifs. These results suggest that cancer gene mutations occur preferentially in positive regulatory motifs. In contrast, all the 3-node and 4-node size motifs show an obviously negative correlation between positive link ratio and the number of methylated genes in the motifs (Figure 2, Supplementary Table 2). These results suggest that cancer gene methylation preferentially occurs in negative regulatory motifs. A similar trend was found for the 15 known tumor suppressors (Supplementary Figure 4a\u2013d), which is in agreement with the notion that cancer-associated methylated genes play roles as tumor suppressors. Collectively, these facts suggest that mutated and methylated genes have different regulatory mechanisms in cancer signaling and support the notion that gene mutations and methylations are strongly selected in tumor samples.\nSignaling information propagates through a series of built-in regulatory motifs to contribute to cellular phenotypic functions (Ma'ayan et al, 2005). The transition from a normal cellular state into a long-term deregulated state such as cancer is often driven by prolonged activation of downstream proteins, which are regulated by upstream proteins or regulatory motifs or circuits. Positive regulatory loops (Ferrell, 2002) could amplify signals, promote the persistence of signals, serve as information storage and evoke biological responses to generate phenotypes such as cancer. In cancer cells, constitutive activation of the oncogene signaling is necessary. Neutral mutations do not affect protein function, whereas missense mutations may have positive or negative effects on protein activity. The enrichment of gene mutations in positive regulatory loops suggests that the mutants in the motifs must have gain of function or increase their biochemical activities compared to the wild-type genes in order to constitutively activate downstream proteins. Indeed, a recent survey showed that 14 out of the 15 PI3K mutants in tumors have gain of function (Gymnopoulos et al, 2007). Gain-of-function mutants in a positive regulatory loop afford the amplification of weak input stimuli and serve as information storage to extend the duration of activation of the affected downstream proteins. This might allow the downstream signaling cascades to persistently hold and transfer information leading to tumor phenotypes.\nPromoter gene methylation is a known mechanism of inducing loss of function by inhibiting the expression of genes (Ohm et al, 2007; Widschwendter et al, 2007). Negative regulatory loops controlled by tumor-suppressor proteins repress positive signals and play an important role in maintaining cellular homeostasis and restraining the cellular state transitions (Ma'ayan et al, 2005). A loss of function of gene methylation in a negative regulatory loop could break the negative feedbacks, thereby releasing the restrained activation signals and promoting oncogenic state transitions. Homeostasis relies on the balance between positive and negative signals in crucial components of the network. Both the gain-of-function mutated genes in positive regulatory loops and the loss-of-function methylated genes in negative regulatory loops could break this delicate balance, thus promoting state transitions and generating tumor phenotypes. Therefore, both mutated and methylated genes and their regulatory loops (oncogenic regulatory loops) are critical components of the network where the oncogenic stimuli occur.\nAn oncogene-signaling map emerges from the network\nIn the language of networks, genes whose mutations or epigenetic silencing are crucial to trigger oncogene signaling might link together as components in the network. Identification of such components will help us to discover the relationships and structural organizations of the oncogenic proteins. To uncover the architecture of cancer signaling and to gain insights into the higher-order regulatory relationships among signaling proteins that govern oncogenic signal stimuli, we mapped all genetic mutations and epigenetically silenced genes onto the network. We found that most of these genes (67%) are connected together to form a giant, linked network component. Randomization tests confirmed that such a component is unlikely to be formed by chance (P<2 \u00d7 10\u22124). To build an oncogenic map, we included other mutated and methylated genes that are not present in the composition of the component into the giant network component based on node connectivity (see Materials and methods). The resulting oncogene-signaling map consists of approximately 20% of the signaling network nodes (326 nodes, 892 links) and includes almost 90% of the mutated and methylated genes (Figure 3). The map showed different network topological characteristics from the signaling network. For example, the average length of the map is less than that of the signaling network (5 versus 6, P<2 \u00d7 10\u221216, Wilcoxon test). On the other hand, the average clustering coefficient of the map is greater than that of the signaling network (0.08 versus 0.04, P=0.06, Wilcoxon test). These results suggest that oncogenic proteins tend to have more interactions and signaling regulatory relationships. The emerging oncogene-signaling map represents a \u2018hot area' where extensive oncogene-signaling events might occur. As a proof of concept, we found that the MAPK kinase and TGF\u03b2 pathways, which are well-known cancer-signaling pathways, are embedded in the map. For example, 50 out of 87 proteins in the MAPK kinase pathway (Supplementary Table 3) and 22 out of 52 proteins in the TGF\u03b2 pathway (Supplementary Table 4), respectively, are included in the map. More importantly, in addition to known oncogenic pathways, there are many other novel candidate cancer-signaling cascades present in the map. For a given gene mutation in a tumor, one could use this map to generate testable hypotheses to discover the underlying oncogene-signaling cascades in that tumor.\nAs mentioned above, oncogene-signaling-dependent events, which we define as the interactions between the cancer mutated or methylated genes, are frequently found in tumor samples and represent various oncogene-driving events that could play more critical roles in generating tumor phenotypes. To systematically identify such events and discover how they are organized in the map, we charted the gene mutation frequency onto the map and highlighted the signaling links between any two genes that have high mutation frequencies. Most genes have mutation frequencies lower than 2%, whereas a handful of genes have very high mutation frequencies, such as p53 (41%), PI3K (10%) and RAS (15%) (see Materials and methods). Therefore, a gene mutation frequency equal to or greater than 2% was considered as high. Interestingly, nearly 10% of the links in the map are oncogene-signaling-dependent events. Certain signaling events such as Pten-PI3K and RAS-PI3K in the map are well-known oncogene-signaling-dependent events\/cascades that are frequently used in various cancers.\nAs shown in Figure 3, most oncogene-signaling-dependent events are connected, and three major regions that contain densely connected oncogene-signaling-dependent events emerge in the map: the first region (p53 region) contains mainly tumor suppressors such as p53, Rb, BRCA1, BRCA2 and p14 (CDKN2A) etc.; the second region (RAS region) contains mainly well-known oncogenes such as RAS, EGFR and PI3K etc.; and the third region (TGF\u03b2 region) contains SMAD3, SMAD4 and a few other TGF\u03b2-signaling proteins. Interestingly, genes in the p53 and TGF\u03b2 regions are also heavily methylated in cancer stem cells, suggesting that these regions are involved in the early stage of oncogenesis. Other methylated genes are intertwined with the mutated genes in the map, suggesting that they share some oncogene-signaling cascades and might be regulated to cooperate in cancer signaling via gene mutation and\/or methylation. Notably, it seems that, in cancer stem cells, TGF\u03b2-signaling pathway is shut down, supporting its known role as a tumor suppressor in the early stages of tumorigenesis (Hanahan and Weinberg, 2000; Siegel and Massague, 2003). These results suggest that the crucial players of oncogene signaling tend to be closely clustered and regionalized. This map uncovers the architectural structure of the basic oncogene signaling and highlights the signaling events that are highly conserved in generating tumor phenotypes.\nFunctional collaboration of genes between oncogene-signaling blocks\nThe oncogene-signaling map can be decomposed into several network communities or network themes (Zhang et al, 2005), in which each network community contains a set of more closely linked nodes and ties to particular biological functions. To discover such network communities, we implemented and applied an algorithm that detects network communities to the map. As a result, 12 network communities, ranging in size from 11\u201365 nodes (Supplementary Table 5), called \u2018oncogene-signaling blocks', were found in the map. Structurally, the nodes within each block have more links and signaling regulatory relations among themselves than others. The genes in each block share similar biological functions such as cell proliferation, development and apoptosis (Supplementary Table 5). We further performed Gene Ontology (GO) enrichment analysis for each oncogene-signaling block using DAVID Tools (http:\/\/david.abcc.ncifcrf.gov\/home.jsp). Most of the oncogene-signaling blocks are enriched with protein serine\/threonine kinase activity (Supplementary Table 6), which is well known to take part in tumorigenesis. Notably, Block 1 is enriched with cell surface receptor-linked signaling, whereas Block 10 is enriched with intracellular signaling cascades. Block 11 is enriched with tumor suppressors and biological processes such as apoptosis and cell cycle. These results suggest that certain blocks are taking part in different parts\/kinds of signaling, that is, cell surface receptor-related signaling, intracellular signaling, cascade signaling and apoptotic signaling. However, three oncogene-signaling blocks have no GO enrichment detected. One of the reasons is that a fraction of the genes in these blocks is not well annotated yet. For example, about one-third of genes in Block 6 have no GO term associated.\nWe asked if the genes in each block could operate in a compensatory or concerted manner to govern a set of similar functions. Toward this end, we surveyed the gene mutations in tumor samples where at least two genes are screened for mutations. As a result, the co-occurrence in tumor samples of 25 mutated gene pairs is found to be statistically significant (Supplementary Table 7). Significantly, only three collaborative gene pairs came from the same block, whereas other collaborative gene pairs came from two different blocks, with predominantly one of them arising from Block 11 (defined as p53 block), which contains p53, Rb, p14, BRCA1, BRCA2 and several other genes involved in control of DNA damage repair and cell division. Collectively, these results suggest that the signaling genes from different blocks most likely work together in a complementary way to generate tumor phenotypes.\nWe further asked which oncogene-signaling blocks work together to produce a tumor phenotype. To address this question, we surveyed the gene mutations in the tumor samples where at least two gene mutations are found. In total, 592 tumor samples fit this criterion. We used the 592 samples to build a matrix (M) where samples are rows and the signaling blocks are columns. If a gene of a particular signaling block (b) gets mutated in a tumor sample(s), we set Ms,b to 1, otherwise we set Ms,b to 0. A heatmap was generated using the matrix (Figure 4A). If a sample contains statistically significant co-occurring mutated gene pairs (see Supplementary Table 7), these pairs were highlighted in the heatmap. Samples were organized based on the cancer types they belong to. Several cancer types such as breast, central nervous system, blood, lung, pancreas and skin tumors that have relatively more samples were also highlighted in the heatmap. As shown in Figure 4A, two signaling blocks have statistically significant enrichment of gene mutations (P<2 \u00d7 10\u22124, randomization tests), suggesting that genes in these two signaling blocks are predominantly used to generate tumor phenotypes. One oncogene-signaling block (Block 1, defined as RAS block) contains genes like RAS, EGFR and PI3K etc., which share similar biological functions such as cell proliferation, cell survival and cell growth, whereas the other is the p53 block, which share similar biological functions such as cell cycle checkpoint control, apoptosis and affecting genomic instability (Supplementary Table 5). These two blocks also represent the two oncogene-signaling-dependent regions (p53 and RAS regions) in Figure 3, respectively. When a tumor sample has a mutation in a gene from the RAS-signaling block, it is also most likely to contain a mutation in a gene from the p53 block (P<2 \u00d7 10\u22124). To check if this phenomenon is primarily due to a particular pair of genes, we calculated the likelihood of co-occurrence for each pair of the genes, of which one gene is mutated in one block and the other gene is mutated in the other block. We found that the P-values for gene pairs are always significantly greater than that for the pair of Blocks RAS and p53. For example, the P-value of co-occurrence of RAS (in Block RAS) and p53 (in Block p53) mutations is 0.01, which is greater than that of the two blocks (P<2 \u00d7 10\u22124). This indicates that these two oncogene-signaling blocks collaborate to generate tumor phenotypes for most tumors. Experimental examples have shown similar gene collaboration in tumorigenesis: activation of RAS (RAS block) and inactivation of p53 (p53 block) induce lung tumors (Meuwissen and Berns, 2005), whereas activation of RAS (RAS block) and inactivation of p16 (p53 block) induce pancreatic tumors (Obata et al, 1998). In general, tumor cells exhibit either elevated cell proliferation or reduced differentiation or apoptosis relative to normal cells. The oncogenic blocks we have identified, especially the RAS and p53 blocks, encode functions that are tumor-related, such as cell cycle control, cell proliferation and apoptosis (Supplementary Figure 5). Activation of genes in the RAS block promotes the cell proliferation, whereas inactivation of genes in the p53 block prevents apoptosis. Thus, a functional collaboration between the genes in these two blocks would promote synergistic cancer signaling and foster tumorigenesis.\nNotably, we found that at least one gene mutation in the p53 block had occurred in the tumor samples we examined. In other words, the p53 block is involved in generating tumors for most cancers. This result suggests that the p53 block is a central oncogene-signaling player and essential in tumorigenesis. This finding is further supported by the following observations. (a) To become oncogenic, tumor suppressors require loss-of-function mutations, which occurs more often than gain-of-function mutations (Gymnopoulos et al, 2007). Indeed, the average gene mutation frequency in the p53 block is higher than that of other signaling blocks including the RAS block. (b) The methylation of genes in the cancer stem cells resulting in long-term loss of expression represents the early stage of the tumorigenesis. In fact, most of the members of the p53 block are methylated in cancer stem cells. These facts further support that the p53 block might play an important role in the earlier stages of oncogenesis. (c) Gene methylation or inactivating mutations of DNA damage checkpoint genes such as p53 induce genome instability and thus increase the chance of other gene mutations, including the genes of other oncogene-signaling blocks that could functionally collaborate with the p53 block genes to generate tumor phenotypes.\nUsing the map as a framework, we benchmarked the mutated genes in the NCI-60 cell lines, which represent a panel of well characterized cancer cell lines and various cancer types. A systematic mutation analysis of 24 known cancer genes showed that most NCI-60 cell lines have at least two mutations among the cancer genes examined (Ikediobi et al, 2006). We built a matrix and constructed a heatmap using these cell lines and their mutated genes as described above (Figure 4B). Overall, the pattern obtained from the NCI-60 panel resembles that of the 592 tumor panel with both RAS and p53 blocks enriched with gene mutations and exhibiting statistically significant collaborations in these cell lines (Figure 4B, P<2 \u00d7 10\u22124), which is in agreement with the earlier observations. We also benchmarked the mutated genes derived from a genome-wide sequencing of 22 tumor samples (Sjoblom et al, 2006). Among these 22 samples, 10 breast and 10 colon tumor samples have at least two gene mutations in the map. As shown in Figure 4C and D, the p53 block is enriched with gene mutations. For the 10 colon tumor samples, collaboration between Block 6 and Block p53 is established, but for the 10 breast tumors, collaborative patterns between multiple blocks emerged.\nTo further examine the block collaborative patterns in individual tumor types in higher resolutions, from the heatmap (Figure 4A) we extracted the sub-heatmap for several tumor types that are better represented among the 592 tumor samples, that is, they have relatively more samples within the 592 samples (Figure 5). As shown in Figure 5, signaling block collaborative patterns are tissue dependent and are classified into two groups. One group contains pancreas, skin, central nervous system and blood tumors that have simple block collaborative patterns. In these tumors, signaling collaborations are mainly between Block p53, Block RAS with some minor contributions from Blocks 5, 6 or 7, suggesting that they predominantly use these oncogene-signaling routes to generate tumors resulting in relatively homogenous cancer cell types. The other group contains breast and lung tumors that also contain large proportions of mutations from the p53 block, but also have complex patterns of collaborations between assortments of multiple blocks, suggesting that these tumors may have a larger variety of oncogene-signaling routes, which may explain, in part, the heterogeneous nature of the tumor subtypes in this category. These results might also explain why both lung and breast cancers are the most common types of human tumors.\nIn this study, the cancer mutated genes were collected from a \u2018directed approach' (i.e., mutational analysis of specific genes, such as p53) and a \u2018large-scale approach' (i.e., large-scale sequencing of tumor samples). We tested whether the mutated genes from the directed approach introduce bias to our analysis. Literature-curated cancer mutated genes (directed approach) have been assembled in the Cancer Gene Census (Futreal et al, 2004), of which 115 genes were found in the human signaling network. As of November 30th, 2006, among the 115 Cancer Gene Census genes, mutations in 55 of them have been further validated by additional experimental evidence (i.e., other independent experiments confirming the mutation of these specific genes in cancer samples have been documented in the COSMIC database), whereas 60 of them have no such evidence in the COSMIC database (see Materials and methods). In fact, we included only these 55 literature-curated genes in the cancer mutated gene set (227 genes) used in all of our analyses above (see Materials and methods). Of the 55 literature-curated genes, only 9 were not already present in the output of large-scale sequencing of tumor samples (Figure 1A). We removed these 9 genes from the cancer mutated gene set (227 genes), mapped rest of the genes onto the human signaling network and rebuilt an oncogene-signaling map, oncogene-signaling blocks and a heatmap. On the other hand, we added the 60 literature-curated genes, which have no independent supporting evidence in the COSMIC database, to the 227-gene set and obtained 287 genes. Using these 287 genes, we reran the analyses mentioned above. In these two analyses, although the gene members of each oncogene-signaling block have some minor differences with those of the original blocks, the major collaboration patterns of oncogene-signaling blocks remain largely unchanged (Supplementary Figure 6a and b), suggesting that our findings are robust to addition or removal of the cancer mutated genes derived from the directed approach.\nThe mutated genes in the network provide a predictive power\nA substantial number (\u223c20%) of mutated genes were found in the network. We asked if a gene that has more links to mutated genes in the network is most likely to be cancer associated. To answer this question, we extracted the nonmutated genes that have more than one link to the mutated genes and then grouped them based on their link numbers to the mutated genes. We interrogated a cancer-associated gene set (Supplementary Table 8) compiled from literature mining (see Materials and methods) to find out how many genes in each group are cancer associated. As shown in Figure 6, the more mutated genes a gene links to, the more probably it is cancer associated. When the link number of the network genes is more than six, \u223c80% of them are cancer-associated genes. For example, SHC, a gene that has been implicated in cancer metastasis (Jackson et al, 2000), has numerous links to the mutated genes in the network. To further investigate the predictive power of the mutated genes in the network, we took the 14 network genes, which not only have at least four links to the mutated genes, but also are not implicated in cancer in the literature, to perform a survival analysis using a microarray data set that contains the gene expression profiles and survival information for 295 breast tumor samples. As a result, the expression profiles of 5 out of the 14 genes (36%) are able to discriminate \u2018good' and \u2018bad' tumors (i.e., patients having \u2018bad' tumors have higher chance of tumor recurrence and short survival time). Therefore, these genes are potentially novel biomarkers. In contrast, less than 10% of the nonmutated network genes have similar discriminatory power. These results suggest that the network genes, which have more links to the mutated genes, have more chance to be perturbed in tumorigenesis and be associated with cancer. Practically, the mutated genes in the network provide a predictive power that can be used to discover novel biomarkers of tumors.\nConcluding remarks\nAlthough a wide variety of genetic and epigenetic events contribute to the signaling of tumorigenesis, it has been challenging to gain a global view of where and how they affect the signaling alterations to generate tumors on the entire signaling network. By integrative analysis of the human signaling network with cancer-associated mutated and methylated genes, we uncovered an overall picture of the network architecture where the oncogenic stimuli occur and the regulatory mechanisms involving mutated and methylated genes. Mutations, the majority of which are activating, preferentially occur in the signaling hub genes (but not neutral hubs) and the genes of the positive regulatory loops, whereas methylated alterations tend to occur in the genes of the negative regulatory loops. Cancer and cell signaling have been well established, and extensive efforts have been made to illustrate cancer signaling during the past few decades. However, it has been a struggle to get clues of how the oncogene signaling is structurally and functionally organized. In this analysis, we extracted an oncogene-signaling map, which provides a blueprint of the oncogene signaling in cancer cells. From the map, we discerned that the oncogene-signaling-dependent events form three highly connected regions that resemble oncogene-signaling superhighways frequently used in tumorigenesis. Topologically, the map has been divided into 12 oncogene-signaling blocks. Functional collaborations between subsets of these blocks are underlying tumorigenesis. In most tumors, genes in both p53 and RAS blocks often get mutated, although the combinations of p53 with other signaling blocks are also found in a small fraction of tumors. Analysis of the NCI-60 cell line panel mutations showed the enrichment of gene mutations in p53 and RAS blocks, which is similar to the patterns found in the 592 samples. Furthermore, we can dissect some of this functional collaboration among different tumor types. These facts indicate that at least two signaling gene mutations, one from the p53 block and the other from another block, are necessary for tumorigenesis. This fact supports the notion that both the prevention of cell death (p53 block) and the promotion of cell proliferation (RAS or other blocks) are necessary to generate most tumors.\nAt present, a number of researchers doubt or even argue against the value of large-scale human cancer genome sequencing as a meaningful or efficient strategy in cancer research. Their arguments are based on the following observations (Chng, 2007): (a) previous large-scale human cancer genome sequencing revealed that each tumor has a different mutation pattern, and the prevalence and patterns of somatic mutations in human cancers are tremendously diverse and complex (Kaiser, 2006; Sjoblom et al, 2006; Greenman et al, 2007); (b) the interpretation of such complex somatic alterations is a formidable challenge (Chanock and Thomas, 2007; Thomas et al, 2007). We mapped the mutation data from the genome-wide sequencing tumor samples (Sjoblom et al, 2006) using the oncogene-signaling map as a framework. Although the number of mutated genes is impressive in toto, most signaling gene mutations are limited to 2\u20133 critical mutations, divided among several signaling blocks, per individual tumor. This result suggests that the mutations in the samples of the same tumor type might share a similar underlying signaling mechanism, because each oncogene-signaling block contains a set of genes linked together through shared regulatory relations and key input and\/or output signaling nodes that are involved in tumorigenesis. These findings imply that although the mutations seem tremendously diverse and complex at the gene level, clear patterns emerge recurrently at the network level in most tumors. Therefore, with proper bioinformatics analysis, large-scale cancer genome sequencing efforts would be fruitful in finding appropriate combinations of biological targets for cancer diagnostic and therapeutics.\nIn summary, this work revealed novel insights into the oncogenic regulatory mechanisms, oncogene-signaling network architecture and oncogene-signaling cooperative relationships that drive cancer development and progression. It also highlights the emergence of the central players in cancer signaling. Cancer studies have integrated microarray, knowledge, pathways and networks (Liu and Lemberger, 2007), but not genetic and epigenetic data yet. However, as the next generation of genome sequencing technology becomes more accessible and affordable, much more efforts involving genome-wide sequencing of large number of tumor genomes will be conducted. Our work provides a conceptual and technical framework for incorporating the genome sequencing outputs and other types of data such as microarray profiles to get more insights into the cancer-signaling mechanisms that will facilitate the identification of key genes for biomarkers and drug development.\nMaterials and methods\nData sets used in this study\nHuman signaling network\nTo build up the human signaling network, we manually curated the signaling molecules (most of them are proteins) and the interactions between these molecules from the most comprehensive signaling pathway database, BioCarta (http:\/\/www.biocarta.com\/). The pathways in the database are illustrated as diagrams. We manually recorded the names, functions, cellular locations, biochemical classifications and the regulatory (including activating and inhibitory) and interaction relations of the signaling molecules for each signaling pathway. To ensure the accuracy of the curation, all the data have been crosschecked four times by different researchers. After combining the curated information with another literature-mined signaling network that contains \u223c500 signaling molecules (Ma'ayan et al, 2005), we obtained a signaling network containing \u223c1100 proteins (Awan et al, 2007). We further extended this network by extracting and adding the signaling molecules and their relations from the Cancer Cell Map (http:\/\/cancer.cellmap.org\/cellmap\/), a database that contains 10 manually curated signaling pathways for cancer. As a result, the network contains 1634 nodes and 5089 links that include 2403 activation links (positive links), 741 inhibitory links (negative links), 1915 physical links (neutral links) and 30 links whose types are unknown (Supplementary Table 9). To our knowledge, this network is the biggest cellular signaling network at present.\nCancer mutated genes\nThe cancer mutated genes were taken from the COSMIC database (http:\/\/www.sanger.ac.uk\/genetics\/CGP\/cosmic\/) and other large-scale or genome-wide sequencing of tumor samples (Sjoblom et al, 2006; Greenman et al, 2007; Thomas et al, 2007). COSMIC database contains manually curated cancer mutated genes and the information of tumor samples, mutated sequences from literature and the output from the CGP's large-scale sequencing of tumor samples (Davies et al, 2005; Stephens et al, 2005; Greenman et al, 2007). The literature-curated genes were compiled as the Cancer Gene Census (Futreal et al, 2004), which is accessible in COSMIC database. The CGP is using human genome sequences and high-throughput mutation detection techniques to identify somatically acquired sequence mutations and hence to identify genes critical in the development of human cancers. A few recent publications (Davies et al, 2005; Stephens et al, 2005; Greenman et al, 2007) represent a small fraction of the CGP output. In addition, COSMIC database has provided mutation frequencies for most of the cancer mutated genes. The cancer gene mutation frequency of a gene is defined as the ratio of samples containing the mutated gene to the total samples screened for that gene. In the database, about one-third of the literature-curated mutated genes (Cancer Gene Census genes) have nonzero mutation frequencies, suggesting that the literature curation of these genes (i.e., included them into the Cancer Gene Census) has been supported by one or more other independent experiments.\nFor the network analysis in this study, we first intersected the network genes with the literature-curated mutated genes. As a result, we obtained 115 genes (Supplementary Table 10), of which 55 genes (Supplementary Table 10) have nonzero mutation frequencies. Meanwhile, we intersected the network genes with the mutated genes derived from the CGP large-scale sequencing output and several other genome-wide and high-throughput sequencing of tumor samples (Stephens et al, 2005; Sjoblom et al, 2006; Greenman et al, 2007; Thomas et al, 2007). As a result, we obtained another gene set containing 218 genes. Finally, we obtained 227 genes by merging the 55 genes and the 218 genes mentioned above. Among these 227 genes, 218 (96%) and 55 (24%) genes were collected from the large-scale sequencing of tumors and literature curation, respectively (Figure 1A). Notably, 46 genes (84%) of the literature-curated genes were overlapped with the mutated genes derived from the large-scale gene sequencing of tumors. The genes and their mutation frequencies from sequencing of tumors and literature were collected in Supplementary Table 10.\nMethylated genes in cancer stem cells\nWe obtained 287 DNA-methylated genes from the three recent genome-wide determinations of the methylated genes from cancer stem cells (Ohm et al, 2007; Schlesinger et al, 2007; Widschwendter et al, 2007). Out of the 287 genes, 93 were mapped onto the human signaling network (Supplementary Table 11).\nCancer-associated gene set\nThe cancer-associated gene set contains the following data sources: (a) the cancer mutated genes we mentioned above; (b) a literature-mined breast cancer gene set from plasmID database (http:\/\/plasmid.hms.harvard.edu\/GetCollectionList.do); (c) the genes extracted from the NCBI's Online Mendelian Inheritance in Man (OMIM) data set using the keywords such as \u2018cancer', \u2018tumor' and \u2018onco' etc. The cancer-associated gene list contains 2128 genes (Supplementary Table 8).\nMicroarray data\nGene expression profiles and the patients' survival data for the 295 breast tumor samples were obtained from Chang et al (2005).\nOncogenic map extraction\nTo extract an oncogenic map from the human signaling network, we mapped all the mutated and methylated genes onto the network. As a result, 67% of these genes are connected together to form a giant, linked network component. To include the mutated and methylated genes that are not present in this network component, we first found one shortest path between such a gene and a component node. If the length of the shortest path is 2 (i.e., the gene reaches one of the component nodes via a nonmutated network node), we linked that gene and the node on the shortest path into the component. A Java program had been written to implement this procedure (Supplementary File 1).\nNetwork analysis\nTo extract the members of the branching and convergent units and 3-node- and 4-node-size network motifs, mfinder program (Kashtan et al, 2004) was used. To detect the signaling network communities from the oncogene-signaling map, we applied a network community algorithm (Newman, 2006).\nAnalyzing the enrichment of the mutated and methylated genes in the network motifs\nWe mapped the mutated and methylated genes onto each type of the motifs. We then counted the number of mutated or methylated genes in each motif and classified each type of motif into several subgroups based on the number of nodes that are mutated or methylated genes. We then calculated the ratio (Ra) of the activation links to the total activation and inhibitory links in each subgroup.\nRandomization tests\nWe performed randomization tests to evaluate the statistical significance of the observations. A more detailed explanation of the randomization tests was described previously by Wang and Purisima (2005).\nSurvival analysis\nTo evaluate the prognostic value of a gene based on the gene expression profiles and the survival information of the tumor samples, we performed Kaplan\u2013Meier analysis by implementing the Cox\u2013Mantel log-rank test using R, a statistical computing language (http:\/\/www.r-project.org\/). If the P-value is less than 0.05, the gene was thought as statistically significant to classify the tumor samples into \u2018good' and \u2018bad' groups.\nSupplementary Material\nSupplementary Figures\nSupplementary Table 1\nSupplementary Table 2\nSupplementary Table 3\nSupplementary Table 4\nSupplementary Table 5\nSupplementary Table 6\nSupplementary Table 7\nSupplementary Table 8\nSupplementary Table 9\nSupplementary Table 10\nSupplementary Table 11\nSupplementary Java File\nSupplementary Tables and Java File Legends","keyphrases":["cancer signaling","signaling network","gene mutation","tumorigenesis","dna methylation","cancer-signaling map","oncogene-signaling dependence"],"prmu":["P","P","P","P","P","R","R"]}
{"id":"Mol_Neurobiol-3-1-2039784","title":"Neuronal Chemokines: Versatile Messengers In Central Nervous System Cell Interaction\n","text":"Whereas chemokines are well known for their ability to induce cell migration, only recently it became evident that chemokines also control a variety of other cell functions and are versatile messengers in the interaction between a diversity of cell types. In the central nervous system (CNS), chemokines are generally found under both physiological and pathological conditions. Whereas many reports describe chemokine expression in astrocytes and microglia and their role in the migration of leukocytes into the CNS, only few studies describe chemokine expression in neurons. Nevertheless, the expression of neuronal chemokines and the corresponding chemokine receptors in CNS cells under physiological and pathological conditions indicates that neuronal chemokines contribute to CNS cell interaction. In this study, we review recent studies describing neuronal chemokine expression and discuss potential roles of neuronal chemokines in neuron\u2013astrocyte, neuron\u2013microglia, and neuron\u2013neuron interaction.\nIntroduction\nChemokines are small proteins that are able to induce a chemotactic response in cells expressing the corresponding chemokine receptors. Since the discovery of the first protein with chemotactic activity [1], the chemokine family has expanded to approximately 50 chemokines [2] and 20 receptors [3]. Chemokines have been divided into four groups based on the position of four conserved cysteine residues in the N-terminal region of the protein. The two largest groups are CXC and CC. The first two cysteines in the CXC group are separated by one amino acid residue, whereas the first two cysteines in the CC group are adjacent to each other [4, 5]. The two small groups are the C chemokines, with only one cysteine in the N-terminal region, and the CX3C chemokine, where the first two cysteines are separated by three amino acid residues [5]. Chemokine receptors are designated according to the chemokine group they preferentially bind. For example, CC chemokines bind to CC receptors and so on. There has yet only been one exception reported, namely CCL21, that, in addition to CCR7, also binds to CXCR3 [6\u20138]. All chemokine receptors belong to the family of G-protein coupled receptors (GPCRs). In general, GPCRs can bind many different G-proteins, allowing for a great variety of intracellular signaling pathways (for excellent review, see [9]). The majority of chemokine-induced responses are inhibited by pertussis toxin (PTX), indicating that  mediate many effects [10]. Chemokine receptors can activate intracellular targets like adenylcyclase, phospholipases, GTPases like Rho, Rac, and Cdc42 and pathways of major kinases like mitogen-activated protein kinase (MAPK) and phosphatidyl inositol-3 kinase (PI3-K) [11, 12]. This diversity of intracellular signaling shows that chemokine receptors, in addition to pathways involved in cell migration, also activate other pathways and may, in that way, control a great spectrum of cellular functions [13, 14].\nChemokines are well-known regulators of peripheral immune cell trafficking under both physiological and pathological conditions (reviewed by [15\u201317]). In addition to chemo-attraction of immune cells, chemokines have been implicated in a variety of cell functions, such as early development, formation of secondary lymphoid organs, wound healing, angiogenesis and angiostasis, regulation of adhesion molecule expression, development of Th1\/Th2 profiles, tumor growth, and metastasis [5, 14, 18\u201324]. Thus, from being molecules thought to solely orchestrate immune cell migration, chemokines are now considered versatile messengers with the ability to control the interaction between a wide diversity of cell types.\nIn addition to their presence in the periphery, numerous studies have demonstrated that chemokines are also expressed in the central nervous system (CNS), where they play a crucial role in physiological and pathological conditions, such as development, synaptic transmission, homeostasis, injury, and disease-associated neuroinflammation [19, 25, 26]. Although astrocytes and microglia are the primary source of chemokines, there is evidence that neurons express and secrete chemokines as well, indicative of a neuronal contribution to chemokine signaling. In this paper, we review recent studies describing neuronal chemokine expression and discuss the potential roles of neuronal chemokines in neuron\u2013astrocyte, neuron\u2013microglia, and neuron\u2013neuron interaction.\nNeuronal Chemokine Expression\nApproximately 60 studies describe chemokine expression in neurons under physiological and pathological conditions (see Table\u00a01). These studies, of which the majority is published in the last 3\u00a0years, are reviewed in the following sections.\nTable\u00a01Neuronal chemokine expressionChemokineSpeciesConditionRNAProteinReferencesCCL2hBrain+[39]Spinal cord, ALS\u2191[35]Monoculture\u2191+[41]Cell line+[38]rBrain+[37]Brain, cranial nerve injury\u2191\u2191[30]Brain, ischemia\u2191\u2191[28]Retina, ischemia\u2191[168]Spinal cord, peripheral nerve injury\u2191\u2191[31\u201334]Monoculture+[164]mBrain, ischemia\u2191[27, 29]Spinal cord, ALS model\u2191[36]Monoculture, West Nile virus\u2191[43]CCL3hBrain, AD\u2248[40]Monoculture\u2191+[41]rRetina, ischemia\u2191[168]Monoculture+[51]mMonoculture\u2191[42]CCL4hMonoculture\u2191+[41]rRetina, ischemia\u2191[168]mMonoculture\u2191[42]CCL5hMonoculture\u2191+[41]mMonoculture\u2191\u2191[43, 44]CCL20rMonoculture+\/\u2191[51]Trigeminal neuron culture\u2193[169]CCL21mBrain, ischemia\u2191[45]Monoculture\u2191\u2191[45, 46]Neonatal hippocampal slice culture\u2191[46]CXCL1\/2\/3rMonoculture++[51]CXCL8hMonoculture+[41]CXCL9mMonoculture+[43]CXCL10hBrain, HIV\u2191[54]Mixed brain culture+[54]macBrain, HIV\u2191[54]rBrain, ischemia\u2191[52]mBrain, entorhinal cortex lesion\u2191[53]Brain, West Nile virus\u2191\u2191[43]Monoculture++[43]CXCL11mMonoculture+[43]CXCL12hBrain, HIV\u2191[59]Monoculture+[59]rBrain++[55, 60\u201362]Monoculture++[58, 60, 87]mBrain, ischemia\u2248\/\u2193[56]Brain, LPS injection\u2248[56]Mixed brain culture+[59]CX3CL1hBrain, MS\u2191[113]Brain, HIV\u2191[170]Spinal cord+[66]Monoculture\u2248\u2248\/\u2191\/\u2193[74, 75, 138]Cell line\u2248\u2191\/\u2193a[66, 75, 138]macBrain+[66]rBrain and spinal cord, EAE\u2248[63, 65, 66, 68]Spinal cord, peripheral nerve injury\u2248\u2248[171, 172]Brain, LPS injection\u2248[67]Brain, KA injection\u2248[67]Monoculture\u2248\u2191\/\u2193a[64, 65, 73, 76, 78, 118]mBrain++[69]Brain, prion disease\u2248[67]Brain, LPS injection\u2248[67]Brain, KA injection\u2248[67]Brain, EAE\u2248[66]Monoculture\u2248\u2248[77]Cell line\u2248\u2248[77]h Human, mac macaque, r rat, m mouse, ALS amyotrophic lateral sclerosis, AD Alzheimer\u2019s disease, HIV human immunodeficiency virus, MS multiple sclerosis, EAE experimental autoimmune encephalomyelitis, LPS lipopolysaccharide, KA kanaic acid; + present, \u2248 present without change in mentioned conditions, \u2191 present with increase in mentioned conditions, \u2193 present with decrease in mentioned conditions,aIncrease in soluble CX3CL1 and decrease in membrane-bound CX3CL1\nCC Chemokines\nCCL2\nCCL2 is currently the most extensively described neuronal chemokine. The majority of reports describing neuronal CCL2 expression are focused on pathological conditions. An induction of neuronal CCL2 expression was described upon ischemia [27\u201329], after axonal injury [30\u201334] and in motoneurons of patients with amyotrophic lateral sclerosis (ALS), and in mouse models for ALS [35, 36]. Interestingly, neuronal CCL2 expression in response to ischemia was detectable within 2\u00a0h, whereas CCL2 expression in astrocytes was detected only after 2\u00a0days [27]. Although most reports show induction of neuronal CCL2 expression under pathological conditions, a recent study has shown constitutive CCL2 expression in neurons throughout the rat brain [37]. This study demonstrated that, depending on the brain region, up to 100% of the neurons were positive for CCL2 [37]. CCL2 was mainly detected in neuronal cell bodies and costaining-depicted colocalization with various neurotransmitters and neuropeptides, corroborating a population-specific expression of CCL2 [37]. Constitutive neuronal CCL2 expression was also shown in a human neuronal cell line [38] and during human CNS development [39].\nCCL3, CCL4, and CCL5\nAt present, there is only one study describing neuronal CCL3 expression in situ, depicting protein expression in adult human brain [40]. Further, expression of CCL3, CCL4, and CCL5 was described in cultured forebrain neurons derived from human first trimester embryos. These chemokines showed increased expression after exposure to immunological stimuli [41]. CCL3 and CCL4 expression were induced in mouse cerebellar granule neurons after infection with Toxoplasma gondii [42], as was CCL5 expression after viral infections [43, 44].\nCCL21\nIn a middle cerebral artery occlusion (MCAO) mouse model of brain ischemia, cortical neurons rapidly expressed CCL21 in the penumbra of the ischemic core. Because control brain tissue did not express CCL21, CCL21 was assumed to be specifically expressed in endangered neurons [45]. In accordance with the in vivo findings, CCL21 expression was induced in cortical neurons in vitro within 2\u00a0h after excitotoxicity [45, 46]. The CCL21 expression in endangered neurons was rather surprising, as CCL21 is well known for its constitutive expression in secondary lymphoid organs, controlling the homing of mature dendritic cells and na\u00efve T cells [47] and is, therefore, generally considered a homeostatic chemokine linked to the development and maintenance of secondary lymphoid organs [48]. The rapid CCL21 expression in endangered neurons after injury indicates a brain specific role of CCL21. This assumption is corroborated by findings in transgenic mice in which CCL21 was expressed ectopically in various tissues. CCL21 expression in the brain induced a massive brain inflammation that killed the animals within 3\u00a0days after the expression onset [49], whereas CCL21 expression in the skin induced the formation of secondary lymphoid structures [50].\nOther CC Chemokines\nA single study has demonstrated a constitutive and inducible expression of CCL20 in rat cerebellar granule neurons in vitro, which was suggested to play a role in neuronal apoptosis [51]. Expression of other CC chemokines has not yet been observed in neurons.\nCXC Chemokines\nCXCL10\nCXCL10 expression was first described in cortical neurons in rat in response to MCAO-induced brain ischemia [52]. Remarkably, neuronal CXCL10 expression was transient and appeared rapidly after stroke (within 3\u201312\u00a0h), whereas CXCL10 expression in astrocytes was detectable later and persisted up to 15\u00a0days after MCAO [52]. Correspondingly, neurons also showed a rapid CXCL10 expression after entorhinal cortex lesion [53]. Further, neuronal CXCL10 expression and release was induced after viral infection in vitro and in vivo [43, 54].\nCXCL12\nThe CXCL12 gene contains three splice variants, termed stromal cell-derived factor-1 (SDF-1) \u03b1, \u03b2, and \u03b3. SDF-1\u03b3 was cloned from rat brain and showed constitutive neuronal mRNA expression with almost no change in level after peripheral nerve injury [55]. In addition, SDF-1\u03b1 showed neuronal mRNA expression with almost no change in level after brain ischemia or intracerebral LPS injection [56]. In contrast, SDF-1 \u03b2 mRNA expression was not detected in neurons [56]. As little is known about the role of SDF splice variants, and most studies did not specify the splice variants, CXCL12 is used for all SDF splice variants henceforth.\nLike CCL2, but in contrast to most of the other neuronal chemokines, CXCL12 is expressed constitutively in specific neuronal populations. Neuronal CXCL12 expression in vitro was observed in cultured cortical, hippocampal, and cerebellar neurons from human, rat, and mouse [57\u201360]. Neuronal CXCL12 expression in vivo was studied in detail in the adult rat brain, showing CXCL12 mRNA and protein expression in cholinergic, dopaminergic, and vasopressin containing neurons throughout the brain [61, 62].\nOther CXC Chemokines\nStudies describing the expression of other CXC chemokines in neurons are limited. Most notably, in vitro neuronal mRNA expression of CXCL1 [51], CXCL8 [41], CXCL9, and CXCL11 [43] has been illustrated. Expression of other CXC chemokines has not yet been described in neurons.\nCX3CL1\nCX3CL1 was the first chemokine shown to be expressed in neurons [63\u201366]. Because microglia were shown to express the corresponding receptor CX3CR1, a role of CX3CL1\u2013CX3CR1 signaling in neuron\u2013microglia interaction was suggested [63\u201365]. CX3CL1 is constitutively expressed in human, macaque, rat, and mouse neurons in vitro and in vivo, with high expression in cerebral cortex, hippocampus, caudate putamen, thalamus, and olfactory bulb [63, 65, 66, 68, 69]. CX3CL1 appears to be the only chemokine with a higher expression level in brain than in peripheral organs [70]. It is membrane bound and can be cleaved from the cell surface by proteases of the A Disintegrin and Metalloprotease (ADAM) family [71, 72]. The neuronal CX3CL1 mRNA expression remained relatively stable in response to both neuron-damaging stimuli in vitro [73\u201377] and during neuroinflammation in vivo [66], whereas in vitro neurons released CX3CL1 protein after glutamate-induced damage [73, 74, 78]. Furthermore, CX3CL1 concentrations higher than 300\u00a0pg\/mg were described in aqueous extracts of the brain [79], indicating that CX3CL1 can be cleaved from the neuronal membrane and released into the extracellular environment. It is yet unknown which ADAM protease cleaves CX3CL1 in neurons and whether CX3CL1 protein expression changes during in vivo neuroinflammation or degeneration.\nPotential Roles of Neuronal Chemokines in Neuron\u2013Astrocyte, Neuron\u2013Microglia, and Neuron\u2013Neuron Interaction\nAstrocytes, microglia, and neurons have been shown to express chemokine receptors in vitro under physiological and pathological conditions and in vivo. These would include CCR2 for CCL2, CXCR3 for CCL21 and CXCL10, CXCR4 for CXCL12, and CX3CR1 for CX3CL1. Studies describing the expression of these chemokine receptors on astrocytes, microglia, and neurons (see Table\u00a02) and studies indicating a role for these chemokine\u2013chemokine receptor pairs in CNS cell interaction are discussed in the following sections on neuron\u2013astrocyte, neuron\u2013microglia, and neuron\u2013neuron interaction.\nTable\u00a02Chemokine receptor expression in astrocytes, microglia, and neuronsChemokineReceptorCell typeSpeciesConditionRNAProteinReferencesCCL2CCR2astrocytehBrain, MS, HIV\u2191[96, 97, 173]Monoculture\u2191\u2191[83, 99, 174\u2013176]macMonoculture\u2191[175]rBrain, EAE, LPS injection\u2191[177, 178]microgliahBrain, MS, HIV\u2191[96, 173, 179]Monoculture\u2193+[173, 180]Glia culture+[99]rBrain, tumor, LPS injection, NMDA injection\u2191[177, 181, 182]Monoculture\u2191[88]mSpinal cord, peripheral nerve injury\u2191[183]neuronhBrain, HIV+[184]Monoculture++[38]Cell line++[38]rBrain and spinal cord++[161, 177, 185]Monoculture++[161, 164]CXCL10\/CCL21CXCR3astrocytehBrain, MS, HIV\u2191[95, 97, 179, 186]Astrocyte culture\u2191\u2191[84, 98, 175]Mixed glial culture+[95]macMonoculture+[175]mMonoculture+[84]microgliahMonoculture++[7, 84, 98, 114]Cell line\u2191\u2191[98]rCell line\u2191\/\u2193[187]mBrain, various infectious agents, axotomy\u2248\/\u2191\u2248\/\u2191[125]Monoculture+\u2193[45, 84]Cell line\u2191\/\u2193[188]neuronhBrain, AD+\u2248[95, 179, 189]Monoculture++[38]Cell line++[38]macBrain, HIV+[54]rMonoculture+[163]CXCL12CXCR4astrocytehBrain, HIV\u2191[173, 190, 191]Monoculture\u2191\u2191[85, 90, 98, 99, 175, 192\u2013196]macMonoculture\u2191\u2191[85, 175]rBrain+[197]Monoculture\u2191\/\u2193\u2191\/\u2193[57, 58, 102, 198]mMonoculture\u2191\/\u2193\u2191\/\u2193[86, 101, 104, 199, 200]microgliahBrain and spinal cord, HIV++[173, 179, 190, 191, 201, 202]Monoculture+\u2193[98, 99, 191, 202\u2013205]Cell line\u2248[98]babMonoculture\u2191[206]rBrain+[197]Monoculture\u2248\/\u2191+[58, 207, 198]mCell line+[86]neuronhBrain, HIV+\u2248\/\u2191[179, 184, 191, 197, 202, 204, 208]Monoculture++[38, 85]Mixed brain culture++[202]Cell line++[38, 191, 209]macBrain+[210, 211]Monoculture+[85]rBrain++[57]Monoculture++[64, 158]CX3CL1CX3CR1astrocytehBrain, MS\u2248[113]Monoculture\u2191+[113, 175]macMonoculture\u2191[175]rMonoculture\u2248\/\u2191\u2191[76, 198, 212]mMonoculture\u2193\u2193[77, 81, 200]microgliahBrain, MS\u2248[113]Brain, HIV\u2191[170]Monoculture++[75, 113]rBrain, ischemia, prion disease, cranial nerve injury, EAE\u2191\u2191[63, 67, 68, 213]Brain, LPS injection, KA injection\u2248[67]Spinal cord, peripheral nerve injury\u2191\u2191[171, 172]Monoculture\u2191\/\u2193\u2191[63, 65, 198, 212, 214]Cell line\u2193[187]Brain, LPS injection, KA injection\u2248[67]mMonoculture\u2248\u2248[77]neuronhMonoculture++[75]Cell line\u2248\u2191[75]rBrain, LPS injection, KA injection\u2248[67]Monoculture++[64, 139]mBrain, prion disease\u2193[67]Brain, LPS injection, KA injection\u2248[67]h human, mac macaque, bab baboon, r rat, m mouse, MS multiple sclerosis, HIV human immunodeficiency virus, EAE experimental autoimmune encephalomyelitis, LPS lipopolysaccharide, NMDAN-methyl-d-aspartatic acid, AD Alzheimer\u2019s disease, KA kainic acid; + present, \u2248 present without change in mentioned conditions, \u2191 present with increase in mentioned conditions, \u2193 present with decrease in mentioned conditions\nNeuron\u2013Astrocyte Interaction\nAstrocytes comprise the largest group of CNS-residing cells and are not only essential in development, homeostasis, maintenance of the blood\u2013brain barrier, and regulation of central blood flow but are also involved in the immune defense of the CNS. Furthermore, astrocytes are considered to be involved in neuronal information processing [80].\nIt is becoming clear that astrocytes play an active role in the intricate chemokine network of the CNS. Not only has it been shown that astrocytes express a wide variety of constitutive and inducible chemokines in vivo and in vitro, there is also extensive evidence that they express a repertoire of chemokine receptors under physiological and pathological conditions (see reviews [81, 82]).\nNeuronal Chemokines Induce Calcium Transients in Astrocytes\nThe activation of intracellular calcium transients is a hallmark in chemokine receptor signaling, a mechanism that also holds true for astrocytes [57, 76, 83\u201386]. Activation of GPCRs, including chemokine receptors, results in a rapid release of calcium from the endoplasmatic reticulum (ER) through the activation of inositol-1,4,5-triphosphate receptors on the ER membrane. One of the first chemokines described to induce calcium transients in astrocytes is CXCL12 [57, 85\u201387]. CXCL12 concentrations ranging from 0.1 to 100\u00a0ng\/ml [85, 86] or 10\u2013100\u00a0nM [57, 87] induced calcium fluxes in in vitro human, rat, and mouse astrocytes. In all cases, CXCL12-induced calcium mobilization was PTX-sensitive, indicating that this process is G\u03b1i-protein mediated. Similar results were found for CXCL10 [84], CCL2 [83, 88], and CX3CL1 [76].\nIn astrocytes, intracellular calcium transients not only function as a second messenger in multiple intracellular signaling pathways but are also implicated in astrocyte\u2013astrocyte signal propagation, astrocyte\u2013neuron synaptic transmission, and neurotransmitter release (see reviews [80, 89]). Recent findings corroborate that chemokines could also be involved in astrocyte-mediated neurotransmitter release. CXCL12 induced calcium-dependent release of glutamate from astrocytes in human and rat astrocyte cultures and rat hippocampal slice cultures [90]. Moreover, reports that investigated the effects of CXCL12 on the electrophysiological properties of neurons in brain slice cultures suggest that CXCL12-induced effects on neurons at least partly depend on astrocytic glutamate release [91\u201393]. Whether this astrocytic glutamate release was induced by CXCR4 activation or via other pathways was not investigated.\nNeuronal Chemokines Induce Astrocyte Proliferation and Migration in Vitro: Implications for Astrogliosis?\nAstrocytes respond to CNS injury or neuroinflammation by enhanced GFAP expression, proliferation, and possibly, migration, a process known as astrogliosis (see review [94]). In these reactive astrocytes, enhanced expression of chemokine receptors has been described under various pathological conditions, such as multiple sclerosis (MS), human immunodeficiency virus (HIV) infection, ischemia, and neoplasm [95\u201397]. Under these conditions, CXCR3 was mainly found in reactive astrocytes in the proximity of the lesion sites, suggesting that induction of CXCR3 expression in astrocytes is limited to damaged areas of the brain [95\u201397]. A comparable induction of CCR2 expression was found in reactive astrocytes in MS patients [97].\nInterestingly, both CCL2 and CXCL10 are implicated in astrocyte proliferation in vitro [98, 99]. In addition, CXCL12 has been shown to induce astrocyte proliferation in vitro, a process that is dependent on activation of extracellular signal-regulated kinases ERK1 and ERK2 [87, 100\u2013102]. Both CXCL12-induced astrocyte proliferation and ERK1\/2 activation was inhibited by PTX and wortmannin, suggesting that they are dependent on upstream activation of G\u03b1i proteins and PI3-K [87].\nAs chemokines are primarily known for their capacity to induce cell migration, migration assays have been used to determine chemokine receptor functionality in astrocytes [103]. Accordingly, astrocyte migration was demonstrated in vitro in response to CCL2, CXCL10, and CXCL12 [83, 84, 86, 103, 104]. Thus, reactive astrocytes express various chemokine receptors and activation of these receptors in vitro induces proliferation and migration, cellular reactions that are generally involved in astrogliosis. Therefore, it is tempting to speculate that chemokines are involved in the regulation of astrogliosis upon CNS injury or neuroinflammation. Whether neuronal chemokines are indeed responsible for either proliferation or migration of astrocytes in vivo is yet unknown.\nNeuron\u2013Microglia Interaction\nMicroglia in the healthy CNS are ramified cells that continually survey their environment by moving their processes. Upon injury, they quickly protrude their processes toward the damaged site and subsequently transform into amoeboid cells, reflecting a fast activation [105, 106]. Activated microglia form a first line of defense in CNS injury through their capacity to migrate, proliferate, secrete inflammatory and neurotrophic factors, phagocytose-damaged cells and debris, and present antigens [82, 107]. Although activated microglia were initially considered to be detrimental in CNS injury, recent findings indicate a prominent neuroprotective activity as well, suggesting a balance between neurotoxic and neuroprotective microglia activity (see for recent review, [108]). Therefore, it is of particular interest to gain insight into the process of microglia activation. Until now, it is largely unknown which environmental signals mediate microglia surveillance and activation. Almost 10\u00a0years ago, chemokines were indicated as promising candidates for neuron\u2013microglia signaling [63\u201365]. Because then, various studies have described constitutive chemokine expression in neurons and rapid changes in expression levels upon injury. Parallel to this, corresponding chemokine receptors were described in resting and\/or activated microglia. In addition, there is increasing evidence that neurons play an important role in microglia activity, which is at least partly mediated by chemokines.\nMicroglia Activity Upon Neuronal Damage\nUpon CNS injury, activated microglia retract their protrusions, transforming into amoeboid cells with migratory and\/or proliferative capacities [109\u2013111]. It is known that damaged neurons are accompanied by prominent activated microglia within hours after injury, suggesting that neurons emit signals that attract microglia [111]. Several findings support the notion that these signals are primarily chemokines. Microglia express various chemokine receptors, and cell migration is induced upon exposure to chemokines in vitro [7, 45, 84, 112\u2013115]. Moreover, damaged neurons in culture express and release chemokines like CX3CL1 [73, 74, 78], CCL21 [45, 46], and CXCL10 [43, 54], all of which are able to induce microglia migration [7, 45, 46, 73, 76, 84, 113, 115]. In accordance with this, inhibition of chemokine function diminished microglia migration in response to supernatants from damaged neurons [73]. Thus, in vitro results suggest a role of neuronal chemokines in neuron\u2013microglia activation.\nThe issue of chemokine-mediated neuron\u2013microglia activation has been further investigated using genetically modified mice. Mice deficient for either CX3CR1 [116] and CX3CL1 [117] have been studied in various CNS injury and neuroinflammation models. Although CX3CR1 deficiency did not influence microglia activity in response to facial nerve lesion [116], CX3CR1 deficiency was uniformly associated with higher levels of microglia activity in LPS-induced neuroinflammation, 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridin-induced neurotoxicity, and in the SOD1G93A-model of motoneuronal death in the spinal cord [79]. Interestingly, enhanced microglia activity in the last three models was accompanied by increased neuronal death, indicating that, in wild-type mice, neurotoxic microglia activity is inhibited by CX3CL1\u2013CX3CR1 signaling [79]. These findings are corroborated by several in vitro findings. Exposure of a neuron\u2013microglia coculture to CX3CL1 reduced inflammation-related neuronal death, accompanied by suppressed nitric oxide and proinflammatory cytokine production [118]. In conjunction with these findings, inhibition of endogenous CX3CL1 increased neuronal cell death in cocultures [77]. Moreover, in vitro exposure to CX3CL1 supported microglia survival under basal culture conditions and reduced Fas-ligand induced apoptosis considerably [119]. Thus, exposure of microglia to CX3CL1 reduced microglia toxicity and protected microglia from apoptosis under inflammatory conditions. In contrast to these results, CX3CL1 deficiency reduced the infarct volume and mortality after transient focal cerebral ischemia [117]. However, microglia activity in CX3CL1-deficient and wild-type mice was not compared in this study, making it difficult to determine whether disturbed neuron\u2013microglia signaling was responsible for the differences [117].\nIncreased microglia toxicity by CXCR3 and its ligands CXCL10 and\/or CCL21 is suggested by findings derived from the entorhinal cortex lesion (ECL) model, in which CXCR3 deficiency was associated with reduced microglia activity and reduced loss of secondary neurons in the hippocampal formation [53]. An interesting aspect of chemokines in neuron\u2013microglia signaling is acknowledged in the ECL model. In this paper, microglia activity is specifically found within the midmolecular layer of the dentate gyrus, which is the projection site of the transected neurons (see for review, [120]). The microglia activity at a site distant from the primary lesion indicates transport of the chemokine signal. Recent data reinforced this notion, showing that CCL2 that was induced in dorsal root ganglion (DRG) neurons after peripheral nerve injury was transported to afferent terminals in the spinal cord [34]. Moreover, in vitro neuronal CCL21 was sorted into vesicles, transported into neuronal processes, and even reached presynaptic terminals [46]. The finding of CCL21 protein in neuronal vesicles is a strong indication that neuronal chemokines may be the signals responsible for microglia activity at sites distant from the primary lesion, a phenomenon that has been observed also in humans [121, 122].\nA role of CCL2 in microglia activity after neuronal death is suggested by a delayed microglia activity in the thalamus of CCL2-deficient mice in response to cortical injury [123]. The delayed microglia activity was accompanied by a transient improvement of neuron survival in the thalamus, which may indicate that CCL2 is involved in neurotoxic microglia activity [123]. However, it is not yet clear if this effect is due to disturbed neuron\u2013microglia interaction. Damaged neurons are capable to express CCL2, as was found after axotomy in sympathetic ganglia [31] and facial nerve lesion [30], but cortical injury predominantly induced CCL2 mRNA expression in astrocytes [124]. Whether interference with CCL2 signaling would affect microglia activity in the first two models has not yet been investigated.\nIt is clear that the assumption that neuronal chemokines are involved in neuron\u2013microglia signaling is no longer based on the finding that damaged neurons rapidly alter chemokine expression patterns and that microglia express the corresponding receptors [63\u201365]. Studies now show that microglia activation is reduced in mice with genetically disturbed chemokine function, indicating an important role of chemokines in microglia activation. Recent data even suggest that neurosupportive and neurotoxic microglia activity are associated with chemokine receptor expression [125]. The ultimate effects of neuronal chemokines are likely dependent on injury type, brain region, and disruption of the blood\u2013brain barrier [53, 79, 116]. Whereas the exact role of neuronal chemokines in neuron\u2013microglia signaling remains obscure, their importance in regulating damage responses is becoming apparent.\nNeuron\u2013Neuron Interaction\nVarious reports indicate that chemokines influence neuronal development, differentiation [126, 127], survival [128\u2013130], electrophysiological properties [93, 131, 132], and synaptic transmission [26, 92, 133]. Because neurons can express numerous chemokines, autocrine and paracrine contributions of neuronal chemokines are likely.\nNeuroprotection\nNeuronal cell death, the ultimate consequence of all neuroinflammatory conditions, has been studied extensively in vitro. However, there are relatively few in vitro models that can be extrapolated to pathological conditions leading to neuronal death in vivo. One of the most prominent models is glutamate or NMDA-induced neurotoxicity, a model for excitotoxicity, which is most likely involved in various neurodegenerative diseases. \u03b2-amyloid-induced neuronal death serves as a model that may explain the loss of neurons in Alzheimer\u2019s disease, whereas exposure of neuronal cultures to HIV proteins gp120 or HIVtat are aimed to elucidate neuronal death in HIV-dependent neurodegeneration [134\u2013137]. Several reports indicate that neuronal chemokines may protect neurons from these toxic conditions. In vitro, CX3CL1 is known to protect neurons from glutamate-induced toxicity [78, 138], gp120-induced neuronal death [64, 139], and death induced by deprivation of trophic support [140].\nSimilar to CX3CL1, CCL2 exposure is shown to protect neurons from glutamate- and HIV-tat-induced neurotoxicity [141, 142]. However, CCL2 exposure was not protective in \u03b2-amyloid-dependent neuronal death [141]. As exposure of neuronal cells to chemokines is known to activate the putatively neuroprotective MEK\/ERK and PI3-K\/Akt signaling pathways [78, 138\u2013140], it is reasonable to argue that chemokine-dependent protection is mediated by these pathways. Indeed, inhibition of both pathways completely abolished the neuroprotective effects of CX3CL1 in gp120- and glutamate-induced neurotoxicity in hippocampal neurons [64, 78]. Interestingly, in case of glutamate-dependent neurotoxicity, the involvement of MEK\/ERK and PI3-K\/Akt signaling pathways was only evident when CX3CL1 was applied together with glutamate [78]. CX3CL1 exposure was shown to be protective even when the chemokine was applied up to 8\u00a0h after the glutamate stimulus. However, an inhibition of MEK\/ERK and PI3-K\/Akt pathways did not affect the protective activity of delayed CX3CL1 exposure, indicating that CX3CL1 may activate additional pathways in neurons that lead to neuroprotection [78]. The effect of CXCL12 on neuronal death is contradictory. Although several reports indicate that CXCL12 exposure may protect neurons from gp120-induced neuronal death, most papers describe a toxic effect of CXCL12 in neuronal cultures (see below) [64, 143].\nNeurotoxicity\nApproximately 10% of HIV-infected patients develop HIV-1 associated dementia (HAD). It has been shown that the viral protein gp120 itself is neurotoxic [144], indicating that the neuronal loss in HAD is not only due to inflammation occurring after the virus enters the brain but also because of direct toxic effects of viral proteins (see for recent review, [145]). It was shown in 1998 that the neurotoxic effect of gp120 is mediated via the chemokine receptor CXCR4 [146], findings that have been corroborated in subsequent years by various groups [64, 102, 147, 148]. The viral protein gp120 binds and activates CXCR4, the main coreceptor utilized by HIV-1 to infect T cells. CXCR4 has subsequently become the best investigated chemokine receptor with respect to neurotoxicity, and its involvement in neurotoxic signaling has been demonstrated by use of the specific CXCR4 antagonist AMD31000 [102, 147]. The HIV-derived protein gp120 shows agonist activity on CXCR4, and therefore, it is not surprising that its ligand CXCL12 has also been described to be neurotoxic [102, 143, 146, 148, 149]. Currently, there is little information on intracellular signaling pathways that are activated by CXCR4 and subsequently lead to neuronal death. One recent report indicates the involvement of Src activity in CXCL12-induced apoptosis in a neuronal cell line, whereas gp120-induced apoptosis in these cells was independent of Src activity [149]. Interestingly, CXCL12 and gp120 had different effects on ERK activation in neurons and astrocytes [102], indicating that CXCR4 signaling exerts both ligand and cell-type specific effects. The effect of CXCL12 is further complicated by matrix metalloproteinase-2, which was shown to remove the first four amino acids of CXCL12, resulting in a truncated form of CXCL12 [150]. This truncated form was found to be highly neurotoxic compared to the full-length CXCL12, which remarkably was not mediated by CXCR4 but by a yet unknown PTX-sensitive receptor [150]. Because MMP-2 has also been described in HIV-infected patients, it is reasonable to assume that truncated CXCL12 may be a neurotoxic player in HAD [151].\nCXCL12 is not the only neuronal chemokine that exerts neurotoxic effects. Neuronal cell lines and primary human neurons respond to high concentrations of CXCL10 with intracellular calcium transients, caspase activity, and apoptosis [54, 152, 153]. The direct involvement of CXCR3 was demonstrated by the use of an antibody that prevents the activation of CXCR3 and subsequently inhibited CXCL10-dependent neurotoxicity [153].\nChemokinergic Effects on Synaptic Transmission\nRecent data show that CXCR4 activation by either gp120 or CXCL12 significantly enhanced giant depolarizing potentials (GDP) in rat neonatal hippocampus [154]. These GDPs only occur in the developing hippocampus and are involved in growth and synapse formation. These data may explain why HIV infections have a greater impact in the developing brain than in adults [152] and show that neuronal chemokines may change the electrophysiological properties of neurons, thereby corroborating earlier findings [131, 132, 155, 156].\nThe electrophysiological properties of neuronal chemokine receptors have predominantly been studied in cultured primary neurons or neuronal cell lines and brain slice cultures [157]. Remarkably, in cultures of DRG, cerebellar granule or Purkinje neurons, and hippocampal pyramidal cells, chemokines induced changes in the electrophysiological properties of only 10\u201320% of the neurons [64, 140, 158, 159]. In addition, several effects of chemokines in neurons were not sensitive to PTX, in contrast to hematopoietic cells, suggesting that chemokine receptors in neurons, although generally accepted, are not solely coupled to G\u03b1i proteins [128, 140, 149]. Whether these chemokinergic PTX-insensitive effects are mediated by neuronal Gz-subunits is yet unclear [160]. Cultured cerebellar and DRG neurons respond to various chemokines with intracellular calcium transients [140]. In DRG neurons, exposure to CX3CL1 and CXCL12 also increased their excitability [158]. Although chemokinergic effects of CX3CL1, CXCL12, CCL2, and CXCL10 in neurons have been reported to modulate the frequency of both spontaneous and activity-dependent neuronal firing, a direct effect on the induction of action potentials has not yet been described [159, 161\u2013164]. Similar to the effects on isolated neurons, CX3CL1, CXCL12, and CXCL10 also affected neuronal signaling in brain slice cultures [26, 78, 91\u201393, 133, 134, 165, 166]. However, the presence of glia cells (astrocytes, microglia, and oligodendrocytes) in these slice cultures makes it difficult to determine whether the electrophysiological effects of chemokines are mediated by chemokine receptors on neurons and\/or on glia cells, as glia cells may also induce electrophysiological changes in neurons [26, 133, 156]. Whether the effects of CXCL12 in brain slice cultures are mediated via chemokine receptors on neurons and\/or on glia cells may depend on the concentration, as concentrations up to 1\u00a0nM caused a direct decrease in peak and discharge frequency of evoked action potentials in neurons and concentrations higher than 10\u00a0nM activated an indirect GABA-mediated hyperpolarization of neurons [92].\nFuture Directions\nAs discussed here, neuronal chemokines appear to be versatile messengers in CNS cell interaction. However, several important issues need to be addressed in future studies. To begin with, neuronal CX3CR1 expression in vivo remains controversial. Immunohistochemical analysis revealed CX3CR1 positive neurons in mouse brain sections with little changes under pathological conditions [67], whereas neuronal CX3CR1 expression was never described in studies using genetically modified mice in which CX3CR1-expressing cells are also positive for EGFP [116]. Different microscopic techniques and models of neurodegeneration have been explored in these mice, demonstrating only CX3CR1 expression in resting and activated microglia [79, 105, 106, 116]. An explanation may be that neuronal CX3CR1 expression is at such a low level that detection is difficult to achieve with microscopic techniques. This may also be the case for CXCR3 expression in microglia. Although CXCR3 expression has yet not been described in microglia in vivo, functional evidence derived from CXCR3-deficient mice strongly indicates that microglia do express CXCR3 in vivo [53, 115]. Therefore, it seems appropriate that future experiments concerning the expression of chemokine receptors in CNS cells in vivo also include functional analysis.\nAnother issue that needs to be addressed in more detail regards cellular localization. Neurons are highly polarized cells, as their function is largely dependent on their morphology and contacts with other cells (e.g. synapses with other neurons). Although neuronal signaling molecules, such as neurotransmitters, neuropeptides, and neurotrophins, are generally found at specific sites, most reports describing neuronal chemokine expression did not address this issue. Interestingly, a few recent publications do suggest a localized expression of chemokines comparable to other neuronal signaling molecules. Our group demonstrated that neuronal CCL21 is transported in vesicles, reaching presynaptic terminals in cortical neurons in vitro [46]. In subsequent studies, these vesicles appeared to be of the large-dense core type, in which other neuronal peptides are also found (e.g. neurotrophins; Stanulovic et al., manuscript in preparation). Moreover, it has been described for several neuronal populations in vivo that CCL2 and CXCL12 colocalize with other neurotransmitters and neuropeptides in synaptic regions [37, 62]. Like neuronal chemokine expression, a site-specific expression of chemokine receptors may exist, as is suggested by CXCR4 redistribution in the axonal and dendritic compartment of hippocampal neurons after prolonged CXCL12 exposure [167]. Because a localized expression of chemokines and their receptors may have a consequence for their role in cell interaction, future studies on neuronal chemokine expression may address this issue.\nAt last, as all reports indicating that chemokine exposure alters the excitability of neurons used exogenous chemokines, it is yet unknown whether chemokines released from neurons have similar effects.\nConclusion\nKnowledge on the spatial and temporal expression of neuronal chemokines and their regulation under physiological and pathological conditions is increasing rapidly. As CNS cells can express the corresponding chemokine receptors, contribution of these neuronal chemokines to CNS cell interaction is conceivable. This assumption is corroborated by various in vitro and in vivo studies. For example, the following effects of neuronal chemokines were observed in vitro: in astrocytes proliferation and migration, in microglia migration and neurotoxic and neuroprotective activity and in neurons electrophysiological changes, neurotoxicity, and neuroprotection. Further, the synaptic transmission between neurons seems to be influenced by the action of neuronal chemokines on neurons and\/or glia cells. In vivo studies support the important role of chemokines in migration and neurotoxic and neuroprotective activity of microglia upon CNS injury and neuroinflammation. Further exploration of the roles of neuronal chemokines in CNS cell interaction is needful, as insight into the role of neuronal chemokines in CNS injury and neuroinflammation may contribute to the development of therapeutic strategies.","keyphrases":["neurons","chemokines","central nervous system","cell interaction","cns","astrocytes","microglia"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Skeletal_Radiol-4-1-2424184","title":"Bone marrow edema in the knee in osteoarthrosis and association with total knee arthroplasty within a three-year follow-up\n","text":"Objective The purpose of this study was to determine if a correlation exists between magnetic resonance imaging (MRI) findings of bone marrow edema (BME) in osteoarthrosis (OA) of the knee joint and need for total knee arthroplasty (TKA) within a follow-up period of 3 years.\nIntroduction\nOsteoarthrosis (OA) is the most common cause of disability among the elderly population [1, 2]. OA is a condition where articular cartilage cannot maintain homeostasis in response to the forces acting on it. When homeostasis breaks down, there is a wide range of possible biologic responses based on the genetic background of the individual [3]. These responses may be anabolic or catabolic and involve cartilage, bone, and synovium. The exact pathogenesis of OA and the cause of the pain produced are unclear. OA is classically associated with degradation of the hyaline cartilage around the joint. However, cartilage contains no pain fibers. Other proposed sources of pain production in OA include periarticular structures and the underlying bone [3].\nSeveral recent studies have examined bone marrow edema (BME) lesions and their association with OA [1, 4\u201310]. BME has been defined as an area of low signal intensity on T1-weighted images, associated with intermediate or high signal intensity findings on T2-weighted images. The literature suggests that there are two distinct types of BME. The first type occurs traumatically with injury to a joint and tends to resolve over a period of weeks to months [11\u201314]. This type of BME does not appear to have any long-term implications [15, 16]. The second type of BME occurs without trauma and may be associated with rapidly progressing osteoarthrosis. Zanetti et al. [9] suggest a possible link between atraumatic BME and increased pain in OA. Felson et al. [7] have suggested a possible link between BME lesions on magnetic resonance imaging (MRI) and progression of OA findings on MRI over time. Hunter et al. found that bone marrow lesions that enlarge with time are associated with more cartilage loss, compared with bone marrow lesions that stay the same size over time [17].\nDespite the recent literature on BME, the implication of these findings on MRI and how they relate to clinical outcomes is unknown. Most studies have used the subjective complaint of pain as the only clinical correlate. There has been no measure of an objective clinical outcome related to the finding of BME. The purpose of this study was to determine if a correlation exists between MRI findings of BME of the knee joint and the incidence of total knee arthroplasty (TKA) within a follow-up period of 3\u00a0years.\nMaterials and methods\nThe entire database of knee MRI studies from 1995\u20131997 (over 4,000 studies) conducted at a large urban hospital system was used to select individuals with knee osteoarthrosis (OA). The study was reviewed and approved by the hospital\u2019s institutional review board. An initial random search was conducted within this database to identify two distinct groups of patients. The first group had MRI reports containing the phrase \u201cknee osteoarthrosis\/osteoarthritis,\u201d and the second group consisted of those containing the phrases \u201cknee osteoarthrosis\/osteoarthritis\u201d and \u201cbone edema.\u201d The search for \u201costeoarthrosis\/osteoarthritis\u201d yielded 235 cases for review. The search for \u201costeoarthrosis\/osteoarthritis\u201d and \u201cbone edema\u201d yielded 146 cases for review. After these two initial groups were identified, a chart review was conducted on all 381 patients to identify and include only those patients who had at least a 3-year clinical follow-up appointment from the time of the MRI study. Subjects were also excluded if the reason for referral to MRI was post-traumatic or post-surgical. An initial review of the images was completed by an experienced musculoskeletal radiologist to exclude any subjects with evidence of recent post-surgical or post-traumatic changes not mentioned in the report. After this chart review and initial radiology review process, there were 38 patients in the OA-only group and 35 in the OA with BME group.\nThe same musculoskeletal radiologist who provided the initial review was blinded to the original interpretation of the MRI studies and the patient outcome, and reviewed all 73 studies. The radiologist was aware of the study hypothesis at the time of interpretation. The radiologist assessed each knee for the presence or absence of BME and assigned each subject into either the BME group or the no-BME group for further evaluation. After this review of the images, there were a total of 25 patients with OA only and 48 with OA and BME.\nThe OA-only group consisted of four males and 21 females, with an age range of 28\u201375\u00a0years, and an average age of 49.3\u00a0years. Conventional radiographs consisting of anteroposterior, lateral and sunrise views were available for review with 13 of the 25 patients, 52% of the group.\nThe OA and BME group consisted of 23 men and 25 women, with an age range of 35\u201382\u00a0years and an average age of 53.5\u00a0years. Conventional radiographs, again consisting of anteroposterior, lateral, and sunrise views, were available for review with 33 of the 48 patients, 68.75% of the group.\nThe conventional radiographs, when available, were reviewed by the musculoskeletal radiologist. Radiographic evidence of OA on the radiographs was assessed using the Kellgren\u2013Lawrence scale [18] as follows:\nNoneDoubtfulMinimalModerateSevere\nAccording to this scale, evidence of OA includes (1) formation of osteophytes on joint margins or on the tibial spine, (2) periarticular ossicles, (3) narrowing of joint cartilage associated with sclerosis of the subchondral bone, (4) small pseudocystic areas with sclerotic walls situated usually in the subchondral bone, and (5) altered shape of the bone ends [18].\nAll patients had dedicated knee MR scans either on a GE 1.5-T magnet (23 in the OA-only group or 92% and 42 subjects in the OA with edema group or 87.5%) or a 1.0-T magnet (8% of the OA-only group, two subjects, and 12.5% of the OA with edema group, six patients).\nMRI films were reviewed and interpreted by the musculoskeletal radiologist for all patients in the study. For each patient, the sagittal proton density sequences (TR range, 2,666\u20134,100; TE range, 14\u201333; 44 conventional spin echo studies [ET\u2009=\u20090], 29 fast spin echo studies [ET\u2009=\u20094\u20138], 3- to 5-mm section thickness, 16\u2009\u00d7\u200916\u201318\u2009\u00d7\u200918 field of view [FOV], 256\u2009\u00d7\u2009128\u2013256 image matrix, bandwidth range 16\u201332), coronal proton density sequences (TR range, 2,316\u20133,950; TE range, 14\u201333; 44 conventional spin echo studies [ET\u2009=\u20090]; 29 fast spin echo studies[ET\u2009=\u20094\u20138]; 3-mm section thickness; 16\u2009\u00d7\u200916\u201318\u2009\u00d7\u200918 FOV; 256\u2009\u00d7\u2009128\u2013256 image matrix; bandwidth range, 16\u201332), and coronal fat suppressed T2-weighted images (TR range, 2,400\u20133,200; TE range, 56\u201380; 3- to 5-mm section thickness; 16\u2009\u00d7\u200916\u201318\u2009\u00d7\u200918 FOV; 256\u2009\u00d7\u2009128\u2013256 image matrix; bandwidth range, 15.6\u201316) were reviewed. Axial fat suppressed T2-weighted images (TR range, 3,000\u20133,600; TE range, 56\u201376; 1.5- to 10-mm section thickness; 16\u2009\u00d7\u200916\u201320\u2009\u00d7\u200920 FOV; 256\u2009\u00d7\u2009128\u2013256; bandwidth range, 15.6\u201316) were available and reviewed for 69 of the 73 knees. For the four subjects who had no axial T2 imaging available, BME in the patellofemoral joint was not assessed.\nThe medial, lateral, and patello-femoral compartments of the knee joint were assessed. Evidence of OA on MRI was assessed using a modified Noyes arthroscopic classification [19] as follows:\nNormalInternal changes only1\u201349% loss of articular cartilage50\u201399% loss of articular cartilage100% loss of cartilage with subchondral cortex intact100% loss of cartilage with ulcerated subchondral cortex\nThe most severe grade cartilage lesion within a compartment was utilized for the grading of that compartment. Also, the highest grade cartilage lesion on either side (femoral or tibial) of a compartment was used.\nThe radiologist then assessed each knee MRI for the presence or absence of BME. BME was defined as an area of intermediate or high-signal intensity findings on T2-weighted images. All patients in the BME group were then further classified into three separate, groups based on patterns of BME. Type I was termed \u201cfocal\u201d and contained a small area of edema adjacent to a cartilage defect (Figs.\u00a01 and 2). Type II was termed \u201cglobal\u201d pattern with marrow edema occupying most or all of a femoral or tibial condyle (Figs.\u00a03, 4, and 5), and type III was termed \u201ccystic\u201d and was associated with a subchondral geode with a small area of BME (Fig.\u00a06). The worst area of edema in a compartment was used in the classification. The presence or absence of meniscal tear was also noted.\nFig.\u00a01Focal edema with a total knee joint replacement in a 51-year-old female patient. The radiograph demonstrated moderate loss of cartilage joint space medially and spurring of the tibial spines. Coronal fast fat suppressed T2-weighted image (TR 3000\/TE 76\/256\u2009\u00d7\u2009192) shows the loss of hyaline cartilage and a small focal area of edema in the medial aspect of the medial femoral condyle (arrow)Fig.\u00a02Focal edema, no total knee joint replacement, in a 58-year-old male patient. Coronal fast fat suppressed T2-weighted image (TR 3000\/TE 56\/256\u2009\u00d7\u2009192) shows extrusion of the medial meniscus (curved arrow) and small adjacent areas of focal marrow edema (straight arrows). The medial meniscus has a degenerative horizontal cleavage tear approximating the tibial surface. Note also the moderate loss of hyaline cartilageFig.\u00a03Global edema with a total knee joint replacement in a 65-year-old male patient. The radiograph demonstrated absence of joint space in the medial and lateral compartments, and irregularity of the subchondral bone plate of the lateral femoral condyle. Coronal fast fat-suppressed T2-weighted image (TR 3000\/TE 72\/256\u2009\u00d7\u2009192) through the knee shows extensive edema in the lateral femoral and tibial condyles, extending into the metaphyses (straight arrows). Note also the small extruded body of the degenerative and torn lateral meniscus (curved arrow)Fig.\u00a04Global edema with a total knee joint replacement in a 72-year-old male patient. The radiograph demonstrated loss of cartilage joint space within the lateral compartment. Coronal fast fat-suppressed T2-weighted image (TR 3200\/TE 72\/256\u2009\u00d7\u2009192) shows extensive edema in the femoral and tibial condyles (arrows)Fig.\u00a05Global edema, no total knee joint replacement, in a 58-year-old male patient. Coronal fast fat suppressed T2-weighted image (TR 3000\/TE 76\/256\u2009\u00d7\u2009256) through the knee shows extensive edema in the lateral tibial condyle and edema in some of the lateral femoral condyle (arrows). Note also the almost complete absence of hyaline cartilageFig.\u00a06Cystic pattern with a total knee joint replacement in an 82-year-old female patient. The radiograph demonstrated subchondral lucency consistent with a geode and irregularity of the adjacent subchondral bone plate. Coronal fast fat-suppressed T2-weighted image (TR 3000\/TE 56\/256\u2009\u00d7\u2009192) shows complete loss of hyaline cartilage on the medial femoral condyle and adjacent moderate subchondral cystic areas consistent with geodes, and the adjacent bone marrow edema (arrows). Note also the extruded body of the medial meniscus containing a degenerative horizontal cleavage tear (curved arrow)\nGeneralized estimating equations (GEE) were used to determine which factors were related to receiving a TKA. There were eight subjects who had bilateral knee MRIs. GEE takes into account the correlation within an individual who had both knees involved, whereas a general linear model assumes that all observations are independent and does not allow for multiple observations from one individual. A multivariable logistic regression model using generalized estimation was also used to adjust for the age of the patients and to determine if the results were still significant after accounting for the age differences.\nResults\nThere were 65 subjects in this study, with a total of 73 knees. After the final classification by the radiologist, there were 48 knees in the group with BME. There were a total of 15 knees or 31.125% in this group that had a TKA. There were 34 knees with a focal pattern of BME, 12 with a global pattern, and two with a cystic pattern. Of those with a focal pattern, 20.5% [7] had a TKA (Fig.\u00a01). For those with a global pattern, 58.3% [7] had a TKA (Figs.\u00a03 and 4), and for those with a cystic pattern, 50% [1] had a TKA (Fig.\u00a06). These results are detailed in Table\u00a01.\nTable\u00a01Patterns of edema and percentage of those who subsequently received TKAPattern of edemaTotal numberNumber with TKAPercentage with TKA (%)Focal34720.5Global12758.3Cyst2150All patterns of edema481531.2None2528\nThere were 25 knees in the group with no BME. There were a total of two knees or 8% of those without BME who had a TKA (Fig.\u00a07; see Table\u00a01).\nFig.\u00a07No bone marrow edema with a total joint replacement in a 66-year-old female patient. Coronal fast fat-suppressed T2-weighted image (TR 2500\/TE 80\/256\u2009\u00d7\u2009256) shows almost complete loss of hyaline cartilage in the medial compartment and an extruded, degenerative torn medial meniscus. Note the absence of marrow edema\nFor statistical purposes, secondary to the small numbers of subjects in each subgroup, subjects were classified using the modified Noyes scale (0, 1, 2a, 2b, 3a, and 3b), and the data was grouped as cartilage damage less than 50% (0, 1, 2a) and cartilage damage greater than or equal to 50% (2b, 3a, 3b). For all three compartments of the knee, there were no statistical differences in rates of TKA between the groups that had less than 50% damage and the groups that had greater than 50% damage (see Table\u00a02).\nTable\u00a02Cartilage loss seen on MR, as graded by the modified Noyes scale, and its relationship to the occurrence of TKAVariableNo TKAYes TKAOR95% CIp ValueMedial cartilage loss <50%17 (30%)2 (12%)2.770.71\u201310.720.14Medial cartilage loss \u226550%39 (70%)15 (88%)Lateral cartilage loss <50%42 (75%)10 (59%)2.060.74\u20135.700.16Lateral cartilage loss \u226550%14 (25%)7 (41%)PF cartilage loss <50%15 (28%)3 (19%)1.680.45\u20136.290.44PF cartilage loss \u226550%38 (72%)13 (81%)\nFor statistical analysis of the radiographs using the Kellgren\u2013Lawrence scale, subjects who had scores of 0\u20132 (none, doubtful, or minimal) were compared with subjects who had a score of 3\u20134 (moderate, severe) to assess for statistical difference in the occurrence rate of TKA. Those subjects who had a Kellgren\u2013Lawrence score of 3\u20134 were no more likely to have a TKA performed than those who had a score of 0\u20132 (see Table\u00a03).\nTable\u00a03Radiographic findings, as graded by the Kellgren\u2013Lawrence scale, and their relationship to occurrence of TKAScoreNo knee replacement(N\u2009=\u200935)Knee replacement(N\u2009=\u200911)OR95% CIp Value0\u201328 (23%)1 (9%)1.00060.9996\u20131.00160.233\u2013427 (77%)10 (91%)\nTable\u00a04 shows the results of the subjects with BME, using the patterns of edema, compared to those without BME. Subjects who had BME of any pattern type were 5.48 times as likely to have a TKA when compared to subjects with no BME (p\u2009=\u20090.05). Upon further investigation, subjects with a global pattern of BME were 7.63 times as likely to have a TKA compared to subjects with focal, cyst, or no BME (p\u2009=\u20090.004). Subjects with global BME were 15.21 times as likely to get a TKA when compared to subjects without BME (p\u2009<\u20090.001).\nTable\u00a04Comparison of bone marrow edema patterns and their relationship to occurrence of TKAVariableNo TKAYes TKAOR95% CIp ValueNo BME23 (41%)2 (12%)5.480.98\u201330.680.05BME of any pattern33 (59%)15 (88%)Global BME5 (9%)7 (41%)7.631.89\u201328.340.004All other patterns (focal, cyst, none)51 (91%)10 (59%)Global BME5 (18%)7 (78%)15.212.38\u201397.10<0.001No BME23 (82%)2 (22%)First, no bone marrow edema is compared with BME of any pattern. Next global BME is compared to all other patterns, including no edema. Finally, global BME is compared with no BME.\nTable\u00a05 demonstrates the results of a univariate comparison between subjects with BME and without BME, and their scores on the modified Noyes and the Kellgren\u2013Lawrence classifications. For the medial compartment only, subjects who had high scores on the Noyes and Kellgren\u2013Lawrence scales tended to have BME, whereas those subjects with lower scores on the Noyes and Kellgren\u2013Lawrence scales tended to have no BME.\nTable\u00a05Univariate comparisons between those knees with BME and those without BME (t tests)VariableScoreNo BMEBMEp ValueModified Noyes score lateral compartment0\u20132a20 (80%)32 (67%)0.2322b\u20133b5 (20%)16 (33%)Modified Noyes score medial compartment0\u20132a11 (44%)8 (17%)0.0122b\u20133b14 (56%)40 (83%)Modified Noyes score patellofemoral compartment0\u20132a4 (17%)14 (31%)0.1932b\u20133b20 (83%)31 (69%)Kellgren\u2013Lawrence score lateral compartment0\u2013213 (100%)26 (79%)0.0713\u201340 (0%)7 (21%)Kellgren\u2013Lawrence score medial compartment0\u201329 (69%)11 (33%)0.0273\u201344 (31%)22 (67%)Kellgren\u2013Lawrence score patellofemoral compartment0\u201325 (42%)11 (33%)0.6063\u201347 (58%)22 (67%)\nThe presence of a meniscal tear did not correlate with subsequent TKA. There was no significant difference in the gender between the group that received a TKA and those that did not. The group of subjects who had a TKA were 12.6\u00a0years older than those who did not have a TKA (p\u2009=\u2009<0.001). In this study, the odds of having a TKA increase by about 1.11 times for each year increase in age.\nA multivariable logistic regression model using generalized estimation was performed to account for the age difference in the groups and assess whether or not the results were still significant despite the age difference. Table\u00a06 displays these results and demonstrates that, after accounting for the age difference, subjects who had BME of any pattern type were 8.95 times as likely to have a TKA when compared to subjects with no BME (p\u2009=\u20090.016). Subjects with global BME were 13.04 times as likely to get a TKA when compared to subjects without BME (p\u2009<\u20090.01). Subjects with a global pattern of BME were 5.45 times as likely to have a TKA compared to subjects with focal, cyst, or no BME (p\u2009<\u20090.05).\nTable\u00a06Results of the multivariable logistic regression model using generalized estimation to account for the age difference in the groups that received a TKA versus those that did not\u00a0Outcome: TKAVariableOR95% CIp-valueModel 1BME versus no BMEf8.951.49\u201353.680.0164Age1.131.07\u20131.20<0.0001Model 2Global BME versus No BME13.042.06\u201382.580.0064Age1.060.99\u20131.150.11Model 3Global BME versus all other patterns (focal, cyst, and no edema)5.451.02\u201328.960.0467Age1.1071.05\u20131.170.0002\nDiscussion\nBME has previously been noted in the musculoskeletal system in osteoarthrosis and other conditions [1, 4\u201311, 13, 14, 20]. Felson and colleagues have examined BME and its relation to progression of knee osteoarthrosis and noted that BME is a potent risk factor for structural deterioration in knee osteoarthrosis [1, 7, 17]. Its relation to progression is explained in part by its association with limb alignment. In two of his studies, medial bone marrow lesions were seen mostly in patients with varus limbs and lateral lesions were seen mostly in those with valgus limbs [7, 17].\nLink and colleagues [4] reviewed MR findings in osteoarthrosis and noted that cartilage lesions, BME patterns, and meniscal and ligamentous lesions were frequently demonstrated as MR changes in patients with advanced osteoarthrosis. However, in this study, there was no significant correlation between MR and clinical findings.\nA recent study conducted by Kornaat et al. [21] examined multiple imaging findings and their association with clinical symptoms. Their results suggest that only findings of a large-joint effusion or the presence of an osteophyte in the patellofemoral compartment were associated with pain and\/or stiffness. They found no association between BME and symptoms of pain or stiffness.\nThree studies have reviewed the MR appearances with histopathology findings [6, 9, 10]. Bergman and colleagues in their study found that subchondral bone marrow changes were present in seven of nine patients undergoing total knee replacement [10]. Histopathologically, those regions showed focal areas where fibrous tissue replaced fatty marrow in the subchondral trabecular space. Zanetti and colleagues reviewed the histopathological findings in 16 patients who had MR before total knee replacement. The BME pattern consisted of normal tissue and a smaller proportion of several abnormalities including bone marrow necrosis, bone marrow fibrosis, abnormal trabeculae, BME, and bone marrow bleeding. They concluded that a BME pattern in knees with osteoarthrosis represents a number of non-characteristic histological abnormalities [9].\nNolte\u2013Ernstein and colleagues examined the correlation between MR and histological findings of degenerative bone marrow lesions in experimental osteoartrhosis models in canine knee joints [6]. In these experimental lesions, the histopathology revealed 21 osteosclerotic lesions and 5 intraosseous cysts. Histopathological findings showed different degrees of osteosclerosis associated with bone marrow degeneration. Cystic lesions were of two types: subchondral epiphyseal cysts and synovial cysts within a large tibial osteophyte. High signal intensity on T2-weighted images and decreased signal intensity on T1-weighted images indicated high fluid content.\nNone of these prior studies specifically looked at the BME pattern on MR. Our classification of the patterns into global, focal, cystic, and absence of edema is an attempt to subdivide the presence or absence of edema in osteoarthrosis. However, this attempt is limited by the absence of histopathological findings.\nWe were surprised by the significantly increased risk of knee joint replacement with the global pattern of BME in relation to the other patterns. It appears that the more extensive and intense the BME, the more likely it is for the patient to have symptoms. The global pattern of BME was the best predictor of risk of TKA within 3\u00a0years, as those subjects with the global pattern were over five times as likely to receive a TKA when compared to those with any of the other patterns and over 13 times as likely to have a TKA when compared with subjects with no BME, after accounting for the age difference.\nWe were also surprised by the lack of association between cartilage loss and the likelihood of total knee replacement. Intuitively, one would think that the greater the cartilage loss, the more likely the possibility of total knee replacement. However, Link and colleagues [4] noted that clinical findings showed no significant correlation with the extent of cartilage loss on MR imaging. In this study, the lowest scores for pain and function loss were found in patients without cartilage lesions. However, the highest scores for pain, stiffness, and function loss were found in patients with less than 50% cartilage loss. The lowest scores for stiffness were found in patients with more than 50% cartilage loss and full-thickness lesions. These findings further support the theory that cartilage loss may not be the primary source of pain in patients with OA of the knee.\nWe did find, in the medial compartment only, that subjects with high grades of cartilage loss or advanced degenerative changes on radiographs tended to have some degree of BME. The lack of association between cartilage loss and the likelihood of TKA in our study could be a reflection of the limitations of the Noyes classification system. However, other investigators have also found that radiographic signs of cartilage loss may not relate to the degree of clinical symptoms. As mentioned above, Link and colleagues found no correlation with the extent of cartilage loss on MR and clinical findings. In their study, they also used a modified Noyes classification system [4]. Kornaat et al. also found no correlation between the extent of cartilage damage seen on MR imaging and clinical symptoms of pain and stiffness. They used a grading scale based on the maximum diameter of cartilage defects and the depth of the lesion based on the percentage of cartilage loss [21].\nIn clinical practice, OA is associated with the radiographic findings of joint space loss and subchondral sclerosis, presumably secondary to cartilage loss that results in pain. However, as stated earlier, there are no pain fibers in hyaline cartilage [3]. Furthermore, many patients have pain out of proportion to their radiographic findings. Our study may provide an insight into another possible mechanism for pain production in OA of the knee. While not every patient with OA warrants an MR scan, those patients without the classic presentation may benefit.\nWe did find that patients who had a TKA were 12.6\u00a0years older than those who did not have a TKA. Patients who are older are more likely to have OA and, in particular, more severe OA. Surgical replacement of the knee is more likely to occur in an older patient with OA than a younger patient with the same severity of OA. After further statistical analysis, we however found that our BME results were still significant despite the differences in age.\nThis paper has several limitations. Most patients who have a TKA do not get a pre-operative MR, and therefore, we may be dealing with a pre-selected population. What we have called \u201cBME\u201d on MR may not be true edema but may relate to other histologic findings as noted earlier. Our numbers are also small, and we had to group some of our subsets together for statistical analysis. Only one musculoskeletal radiologist was involved in the review of the radiology. There was also no arthroscopic correlation for cartilage defects or presence of meniscal tears. Our study is limited by the fact that it is retrospective. To generate a group of MR scans to review, we had to use a keyword search to identify potential subjects. We would have obviously missed all scans where the specific terminology \u201cbone edema\u201d or knee \u201costeoarthrosis\/osteoarthritis\u201d was not utilized. However, we feel that this is a relatively minor limitation, as we were able to generate 381 cases for review. Another potential limitation in the retrospective study design is the possibility of missing subjects who received follow-up at a different institution or those whose symptoms resolved without treatment.\nIn summary, we reviewed a series of knees in patients with osteoarthrosis and evaluated the pattern of BME, cartilage loss, radiographic findings when available, and the incidence of total knee joint replacement within a 3-year follow-up. Subjects with any bone marrow edema pattern were more likely to have a total knee joint replacement compared to subjects with no bone marrow edema. The worst prognostic pattern was the global pattern of bone marrow edema. Subjects who had a total knee replacement were also older than those who did not. However, even allowing for age, the global pattern of BME remained the variable with the highest statistically significant association with the incidence of total knee replacement.","keyphrases":["bone marrow edema","osteoarthrosis","total knee arthroplasty"],"prmu":["P","P","P"]}
{"id":"Crit_Care-5-4-37409","title":"Handheld computers in critical care\n","text":"Background Computing technology has the potential to improve health care management but is often underutilized. Handheld computers are versatile and relatively inexpensive, bringing the benefits of computers to the bedside. We evaluated the role of this technology for managing patient data and accessing medical reference information, in an academic intensive-care unit (ICU).\nIntroduction\nThe rapid development of computing technology has had a major impact on health care, particularly in technology-oriented areas such as critical care. Electronic patient records require a major commitment by the institution, in hardware, software, training, and support. In many places, bedside care of patients still relies on paper records or nonintegrated computer systems that do not take full advantage of their data-management capabilities [1]. Even where there are advanced computerized systems, the bedside clinician may still rely on written notes for patient management and billing, and refer to pocket textbooks or printed management algorithms.\nFor busy clinicians, the use of computers for hospital-based clinical care may be hampered by the computers' inaccessibility. Handheld computing technology is versatile and relatively inexpensive [2], combining many of the benefits of electronic patient records and paper charts. Handheld computers have been described in various medical situations; early reports describe programmable calculators used to make complex calculations in intensive-care units (ICUs) [3]. Handheld devices are increasingly being used by physicians for a variety of functions, such as scheduling, accessing drug reference information, patient data storage and billing. However, there are few published reports describing the benefits of this technology [4, 5, 6, 7].\nIn view of the potential advantages and increasing use of handheld computers in medicine, we evaluated the benefits and drawbacks associated with introducing this technology in an academic ICU.\nMaterials and methods\nHardware\nThe Palm III series handheld device (Palm device, Palm Canada Inc, Toronto, Ontario) was used, as some of our staff were familiar with this equipment. It is a pocket-sized (8 \u00d7 12 cm; 165 g) computer with a 4-Mb (PalmIIIx) or 8-Mb (PalmIIIxe) memory. It has an infrared data association (IrDA) port that allows transmission of data between Palm devices and other IrDA-compatible devices such as printers, laptop computers and cellphones. The device has a monochrome 160 \u00d7 160 pixel liquid-crystal display screen (Fig. 1) and allows the user to input data either by writing on the touch-sensitive screen with a stylus or by tapping on an on-screen keyboard. Handwriting is deciphered by Graffiti handwriting-recognition software (Palm Inc, Santa Clara, CA, USA), which requires the user to learn specific characters. For users who preferred to enter data using a keyboard, two GoType keyboards (LandWare Inc, Oradell, NJ, USA) were found in the ICU. When the Palm device was placed in this keyboard, the user could type in the standard way.\nSoftware\nEach personal digital assistant (PDA) was installed with medical reference information as well as hospital and ICU specific guidelines (Table 1). This occupied approximately 2 Mb of memory. The applications that come with the PDA (Addressbook, Datebook, Memopad, To Do list) were used for essential telephone numbers as well as call and teaching schedules, but additional software was required for medical databases. The spreadsheet database program JFile (Land-J Technologies, Orlando, FL, USA) was used for reference information, such as drug doses and laboratory reference ranges. The text readers AvantGo (AvantGo Inc, San Mateo, CA, USA) and iSilo (www.isilo.com), which convert word-processing and HTML documents, were used for textual medical reference information. CbasPad, a Tiny BASIC programming language interpreter and editor, was used to develop software to perform common critical-care calculations, such as calculated creatinine clearance and intravenous infusion rates. Additional software for medical reference data was introduced during the study period, including ePocrates qRx [8], a drug information database.\nPatient data were entered into the Memopad using a customized template generated with MemoPlus (Hands High Software Inc, Palo Alto, CA, USA). The information entered included demographic data, medical history, current diagnoses, therapy, procedures performed, and management plan. Data was transferred between medical personnel using the PDA's infrared beaming ability. As hospital policy requires a paper record, daily notes were generated by Palmprint software (Stevens Creek Software, Cupertino, CA, USA) using infrared transmission to an HP Laserjet 6P printer (Hewlett Packard, Palo Alto, CA, USA). Various software packages for patient data management (shareware or commercially available software) were evaluated during the study period.\nIn the light of focus-group feedback, a more comprehensive reference database was developed. The electronic files for the Critical Care Handbook of the Massachusetts General Hospital [9] were provided by the publishers, and converted to a PDA-readable (iSilo) format. This 1.4-Mb file contained the full text of the book, with multiple hyperlinks, and some of the images. Hard copies of the book were also obtained.\nStudy subjects\nPDAs were given to the ICU attending physicians, the rotating resident trainees, and other medical staff. Four to six residents (postgraduate years 2 to 4) worked in the ICU at any one time. On the first day of their ICU rotation, residents were taught how to use the PDA in a 1-hour seminar. The principle investigator and research team were available for further help and troubleshooting throughout the study The research team was responsible for installing and updating software and schedules. Patient data was entered by residents, either during morning rounds or when patients were admitted to the ICU. The updated database was beamed to the on-call resident in the evening and transmitted back to the team in the morning, with new admissions added.\nMethodology\nAn independent evaluation company (Smaller World Communications, Richmond Hill, Ontario) with experience in focus-group methodology was contracted to develop the qualitative methodology, collect data through focus-group meetings, and analyze the data [10, 11]. A preliminary moderator's guide was developed and tested on an expert panel, comprising two critical care physicians, an anaesthesiologist, three medical residents with experience in data management or PDAs, and a representative from Palm Canada Inc. The moderator's guide was designed to stimulate discussion about users' familiarity with the technology, the benefits to patient management, and the drawbacks encountered. Finally, ideas were generated for new applications for the technology and improvements to the hardware and software. Three focus groups were held with the residents and staff who used PDAs in the ICU. Tapes were transcribed verbatim and the notes were analyzed for themes by a research analyst [12]. Interim reports from the meetings were provided to the investigators. On the basis of this feedback, ongoing improvements were made to the medical databases and patient-management software.\nThe PDA reference database was evaluated objectively using a crossover study. The trainees' rotation was split into two 3-week periods. One of the periods was allocated as a control (PDA-free) block and in the other the PDA was available. Two groups of trainees were studied: in one the PDA period preceded the PDA-free period, and in the other, the order was reversed. During the PDA period, trainees had access to the full PDA database as well as the electronic version of the Critical Care Handbook of the Massachusetts General Hospital [9]. The printed copy of the handbook was given to trainees during the PDA-free period.\nObjective evaluation was accomplished using a pair of standardized clinical scenario tests made up of 20 questions answered over 30 minutes. The questions were about common critical-care problems, drawn randomly from a pool of questions written by physicians in our ICU and at other teaching hospitals in the Toronto area. Trainees made use of the textbook (control period) or PDA database (study period) during the examination. To standardize for the possible difference in difficulty between the two tests, 11 General Internal Medicine trainees, not involved in the PDA study, wrote both the tests. This generated a mean and standard deviation for each test. Study trainees' results were expressed as the standard deviation above or below this control mean, and compared using a permutation test, with P < 0.05 considered significant.\nResults\nDuring the 6-month study period, PDAs were used by 20 physicians (4 attending physicians, 1 research fellow, and 15 rotating medical residents) and 6 paramedical staff (3 respiratory therapists, 2 pharmacists, and 1 nurse educator). The three focus groups had a total of 19 participants. Two residents who were unable to attend participated in telephone interviews. Each focus group had six or seven participants, a number within the recommended range [11]. Only five of the users (19%) had previous experience with the PDA computing format.\nPhysical attributes\nUsers found the PDA to be a convenient pocket size, allowing it to be available at all times. The screen was clear and easy to read, although not ideal for long text documents or large tables. Many users became proficient in text entry using Graffiti, while others preferred to use the GoType keyboards. Of the 19 PDA units used during the 6-month study period, only one had a technical malfunction requiring replacement. Two were damaged after being dropped and needed to have their screens replaced. No other problems were encountered.\nMedical reference databases\nReference databases used regularly by medical residents included the critical-care drug dosing reference, ventilator weaning protocol, and electrolyte correction application. The calculation programs (creatinine clearance, ideal body weight) were found to be useful by the pharmacist and some residents. The ventilator weaning protocol was used by medical staff, as well as respiratory therapists, allowing regular assessment of whether patients met the criteria for extubation.\nMany databases were, however, not fully used. This appeared to relate more to inadequate training than to faults in the databases. In many cases, the PDA users were unaware that certain information was in their PDAs. This was because data were located on separate software programs (J-file, AvantGo, Cbas, Memopad) and may have been difficult to find. The PDA had a global 'Find' function to search for keywords, but this does not incorporate some of the added software programs, such as AvantGo. A unified database program with a search capability was suggested as a useful addition.\nPatient-management software\nPatient information was managed using the text-based MemoPlus software and a customized template. This required text entry on the PDA. Several modifications to the template were made during the study period. Residents responsible for patient data entry described difficulty entering data for new patients and keeping patient information updated during busy weekends. Attending staff found the patient data useful, particularly when they were taking over care of patients at the beginning of their on-call duties. Transferring the care of critically ill patients to a new physician is time-consuming and potentially stressful. The PDA patient database improved the staff's knowledge of patients, especially of previous medical problems in patients with complex conditions who had had a long stay in hospital. It also gave staff access to patient information when they were out of the ICU, aiding decision-making. During ICU rounds, the summarized chronological information was useful to find out how long intravenous lines had been in place and to review antibiotic therapy. Less benefit was noted in short-term patients. During night call, the patient summaries were of value when residents were called to see patients with whom they were not very familiar.\nIn our ICU, a daily physician note is written in the patient record. The print function to create a daily note reduced duplication of work, but the process for entering patient data was found to be time-consuming initially. While residents did not feel that the patient-management application (MemoPlus) improved efficiency, it did increase their knowledge of the patients.\nDuring the study period, other commercially available patient-management software systems were evaluated. These had the advantage of easy data input using single keystrokes for date entry and 'pop-up' lists of drugs and diagnoses. While this simplified data inputting, no system was found to be ideal for the ICU. Many of these systems did not support the infrared data transfer or printing functions.\nOther uses of the software\nStudy participants used a variety of other applications on a regular basis. Having the call and teaching schedules easily accessible was considered a benefit. The telephone list of hospital numbers was found to be valuable and the To Do list was used by most users to keep track of their work. Teaching rounds and morbidity and mortality rounds were facilitated by using archived patient data. Many participants used the Memopad to take notes in teaching seminars.\nSuggestions for change\nThe focus-group discussions generated a number of suggestions for improvement. The hardware unit was considered suitable, but a more robust one may be needed in view of the two damaged screens. Because most of the users had had no previous experience with the PDA, additional teaching sessions and follow-up training were suggested to make optimal use of the technology. This would have helped users to become more aware of the many databases available on their PDA. In this regard, the medical information on the PDA would clearly benefit from integration into a single, searchable program.\nThe patient-management software would be more user-friendly if the data could be entered with minimal effort, using customized pull-down lists of drugs, diagnoses, and procedures. The demographic data could be entered and updated daily by a ward clerk. Alarms were suggested - for example, to warn of prolonged intravenous line duration or the end of a course of antibiotic therapy. While transmission of data between staff by infrared was found to be useful, synchronization with the hospital electronic patient record was considered the optimal situation.\nObjective evaluation\nTwo groups of four trainees took part in each crossover study. Half of the residents had prior experience with PDAs. No difference was noted in their subjective preference for the PDA or printed copy of the handbook, and the individual's preference did not correlate with previous PDA experience. Comparison of the test scores revealed no difference between the scores in the PDA-assisted test and the paper-assisted test, analyzed after correction for difficulty using the control mean and standard deviation.\nDiscussion\nThis study prospectively evaluated the benefits and drawbacks associated with the introduction of handheld computers in an academic-critical care environment.\nWho benefitted most?\nThe introduction of handheld computers was well received by all users, despite differences in their familiarity with these devices. The most favourable response was from the more senior staff, namely, the attending physicians and fellows. This may be because of the longer time they were involved in the study, allowing more familiarity with the PDA platform. They were also more likely to benefit from having patient data available while on call outside the ICU. Furthermore, they were usually not responsible for entering patient data. Clearly, two conditions that might enhance the acceptance of these technological changes are adequate education and ease of data entry. Although an initial education session was held, it was when the junior medical staff in the study were beginning their rotation in an unfamiliar environment.\nMaking the devices more user-friendly\nThe patient data applications assessed were not ideal but did enable us to identify several criteria for a user-friendly system. These include ease of data entry using shortcuts and lists, limiting the range of data stored to that essential for patient management, and the ability to transmit data easily between staff. It is important that this computerized patient database should decrease workload and not cause duplication in work. In our study, enabling residents to print a daily note from their handheld computer offset the additional work of data entry. Ideally, the handheld system should be integrated with the hospital electronic patient record, allowing direct entry of demographic data as well as access to laboratory data.\nA wireless capability may also have significant benefits with respect to medical information databases. This would allow access to Medline searches and evidence-based guidelines. While internet access is available from desktop computers in the ICU, the ability to perform these searches on rounds or while consulting outside the ICU may be beneficial.\nDatabases on paper or on screen?\nThe comparison of paper and electronic databases did not reveal an advantage of one medium over the other. No significance difference was observed between the objective scenario test scores using the PDA or the paper database. The fact that equivalent results were obtained using this single database may suggest a potential benefit of using the PDA. The memory capability of the 8-Mb device would allow the trainees to carry five reference texts each of a size similar to that of the Critical Care Handbook of the Massachusetts General Hospital.\nWhat is needed\nCritical-care decision-making requires rapid access to strategic clinical data as well as to medical reference information. A patient in an ICU generates a large amount of data, and the number of information variables may exceed what clinicians can integrate and process [13]. Current information technology has the potential to realize the needs of the intensivist, but no customized product has been developed for this use. Handheld technology has a definite role to play, but systems need to be developed specifically for the critical-care environment to optimize real-time patient data management and communication between health care workers.\nAbbreviations\nHTML = hypertext markup language; ICU = intensive-care unit; IrDA = infrared data association; Mb = megabytes; PDA = personal digital assistant.","keyphrases":["computer communication networks","medical informatics","medical technology","microcomputers","point-of-care technology"],"prmu":["M","M","R","U","M"]}
{"id":"Brain_Struct_Funct-4-1-2248604","title":"Sensory and cognitive mechanisms of change detection in the context of speech\n","text":"The aim of this study was to dissociate the contributions of memory-based (cognitive) and adaptation-based (sensory) mechanisms underlying deviance detection in the context of natural speech. Twenty healthy right-handed native speakers of English participated in an event-related design scan in which natural speech stimuli, \/de:\/ (\u201cdeh\u201d) and \/deI\/ (\u201cday\u201d); (\/te:\/ (\u201cteh\u201d) and \/teI\/ (\u201ctay\u201d) served as standards and deviants within functional magnetic resonance imaging event-related \u201coddball\u201d paradigm designed to elicit the mismatch negativity component. Thus, \u201coddball\u201d blocks could involve either a word deviant (\u201cday\u201d) resulting in a \u201cword advantage\u201d effect, or a non-word deviant (\u201cdeh\u201d or \u201ctay\u201d). We utilized an experimental protocol controlling for refractoriness similar to that used previously when deviance detection was studied in the context of tones. Results showed that the cognitive and sensory mechanisms of deviance detection were located in the anterior and posterior auditory cortices, respectively, as was previously found in the context of tones. The cognitive effect, that was most robust for the word deviant, diminished in the \u201coddball\u201d condition. In addition, the results indicated that the lexical status of the speech stimulus interacts with acoustic factors exerting a top-down modulation of the extent to which novel sounds gain access to the subject\u2019s awareness through memory-based processes. Thus, the more salient the deviant stimulus is the more likely it is to be released from the effects of adaptation exerted by the posterior auditory cortex.\nIntroduction\nThe mismatch negativity (MMN) component of the event-related-potentials (ERPs) is assumed to reflect the operation of a pre-attentive memory-based comparison mechanism (e.g., N\u00e4\u00e4t\u00e4nen et al. 2005). For a wide range of stimuli, there is evidence that the MMN is elicited by a cognitive mechanism based on auditory sensory memory that compares between the incoming deviant stimulus and the standard template (N\u00e4\u00e4t\u00e4nen and Alho 1997; N\u00e4\u00e4t\u00e4nen et al. 2001).\nThe contrasting view is that attention switch to novel sounds is based on the transient frequency-specific adaptation of posterior auditory-cortex feature-detector neurons (Desimone 1992; N\u00e4\u00e4t\u00e4nen 1990, 1992; Ulanovsky et al. 2003; J\u00e4\u00e4skel\u00e4inen et al. 2004). Specifically, it has been suggested that MMN arises because of selective adaptation of the N1 response by preceding standard stimuli (sensory component) leading to its attenuation. This attenuation is interpreted in terms of neural refractoriness (N\u00e4\u00e4t\u00e4nen 1992). Thus, this account of MMN elicitation does not rely on memory representations and is sensorial in nature.\nPrevious research has shown that the MMN is sensitive to acoustic as well as to phonetic attributes of phonemes (e.g., Tampas et al. 2005). Furthermore, it was demonstrated that the MMN reflects the activation of memory networks for language sounds and spoken words (e.g., Pulverm\u00fcller et al. 2003; Shtyrov et al. 2005; Pulverm\u00fcller and Shtyrov 2006), the latter referred to as the \u201cword advantage\u201d effect (Pettigrew et al. 2004).\nThe design of the current functional magnetic resonance imaging (fMRI) study was a combination of modified protocols utilized in previous studies (i.e., Pettigrew et al. 2004; Opitz et al. 2005) that enabled to disentangle cognitive and sensory contributions to change detection (Opitz et al. 2005; Maess et al. 2007) when the deviant stimulus could be either a word (\u201cday\u201d) resulting in the \u201cword advantage\u201d effect, or a non-word deviant (\u201cdeh\u201d or \u201ctay\u201d).\nWe hypothesized that the sensory component will be located bilaterally in regions posterior to Heschl\u2019s gyrus (HG), including the posterior superior temporal gyrus (STG), regardless of the identity of the deviant stimulus (\u201cdeh\u201d, \u201cday\u201d or \u201ctay\u201d). This component relies on frequency-specific neurons in the auditory cortex responsible for the repetition-related decrement of N1 and its counterpart, the N1m (Romani et al. 1982; J\u00e4\u00e4skelainen et al. 2004; Opitz et al. 2005). Moreover, it was shown that the region posterior to HG is broadly tuned with respect to phonetic features (Ahveninen et al. 2006).\nIn contrast, it was shown that areas anterior to HG process sound-identity cues such as speech (Binder et al. 2000; Obleser et al. 2007; Ahveninen et al. 2006) and pitch (Warren and Griffiths 2003). Therefore, we assumed that the cognitive component which involves the representation of the current auditory event (Schr\u00f6ger 1997) will differentiate between the deviant word (\u201cday\u201d) and the non-words (\u201cteh\u201d and \u201ctay\u201d, i.e., the \u201cword advantage\u201d effect) and will be located anterior to HG.\nMethods\nSubjects\nTwenty right-handed adult healthy subjects, native speakers of English, 8 women and 12 men, participated in the study. Subjects ranged in age from 23 to 28. All subjects gave informed consent to participate in the study.\nExperimental protocol\nThe procedure is a modification of the one used by Opitz et al. (2005). Each subject participated in 12 functional imaging runs. The speech stimuli were grouped into two types of blocks \u201coddball\u201d and \u201ccontrol\u201d. In the first type of block, the \u201coddball\u201d block, deviant exemplars occurred quasi-randomly embedded within standard stimuli so that the frequency of occurrence throughout the block was balanced among exemplars. The interval between two successive deviants varied quasi-randomly with gaps of 4, 6, 8, 10 or 12 standards with the constraint that gap size was balanced throughout the block. In the \u201cOddball\u201d blocks deviants appeared 42\u00a0times out of a total of 354 stimuli (probability of occurrence\u00a0=\u00a012%).\nIn the \u201ccontrol\u201d blocks the same physical deviants and standards as in the \u201coddball\u201d blocks (deviant-counterparts and standard-counterparts, respectively), occurred quasi-randomly, while deviants were constrained by the same spacing rule mentioned above. However, each of the \u201ccontrol\u201d blocks contained eight different equiprobable stimuli, including the deviant and standard counterparts. Thus, in the control runs the seven stimuli beside the deviant-counterpart served as \u201cfiller\u201d or contextual stimuli which were added to the sequence so that the deviant-counterpart will appear at the same probability as any other stimulus in the sequence. Each of the stimuli in the \u201ccontrol\u201d block (a total of eight different stimuli) repeated 42 times and appeared with equal probability which was identical to that of the deviants in the \u201coddball\u201d block (12%). There were three blocks of each type (\u201coddball\u201d\/\u201ccontrol\u201d). Each block was repeated twice. In total, 12 blocks of approximately 6\u00a0min each were randomly presented for each subject within a session.\nThree non-words, \/de:\/ \u201cdeh\u201d; \/te:\/ \u201cteh\u201d; and \/teI\/ \u201ctay\u201d, and one word, \/deI\/ \u201cday\u201d, were recorded from a male native English speaker in a sound-proof chamber. These stimuli comprised the following standard-deviant pairs that resulted in the three \u201coddball\u201d runs: (1) deh (standard)\u2013day (deviant); (2) day\u2013deh and (3) teh\u2013tay. Thus, \u201cdeh\u201d and \u201cday\u201d swapped their roles as standard and deviant in the second \u201coddball\u201d block, whereas the third block controlled for the acoustic change associated with the transition from a monophthong (\/e:\/) to a diphthong (\/eI\/) occurring in the first standard-deviant pair. Additional four stimuli, \/pe:\/ \u201cpeh\u201d; \/peI\/ \u201cpay\u201d; \/be:\/ \u201cbeh\u201d; and \/beI\/ \u201cbay\u201d, together with the previous four mentioned above were embedded within three control runs, each containing a deviant-counterpart , either \u201cday\u201d, \u201cdeh\u201d or \u201ctay\u201d appearing with the same probability and obeying the same spacing rule as in the oddball blocks.\nThree exemplars for each stimulus (e.g., deh1, deh2, deh3) were selected (out of a pool of 24 recordings per stimulus) on the basis of acoustic similarity (see Table\u00a01 for the values of the lowest three formants). The parameters that were used to choose similar exemplars for each stimulus included the shape of the spectrogram at the voice onset, vowel durations, pitch and formant values (Hz) of the first three formants at the beginning (ca. 84\u00a0ms) and end (ca. 168\u00a0ms) of the \/e\/ segment (ca. 252\u00a0ms duration) of each stimulus. The stimuli were truncated to 280\u00a0ms and normalized to the same loudness level by using Adobe Audition 1.5 trial version software package. Spectral analysis of the stimuli was conducted by PRAAT software version 4.3.19 (http:\/\/www.praat.org).\nTable\u00a01Pitch and frequencies of the main speech stimuli (in Hz)StimulusF0F1 onset of \/e\/F1 end of \/e\/MeanF2 onset of \/e\/F2 end of \/e\/MeanF3 onset of \/e\/F3 end of \/e\/Meandeh11015546346031,7141,6671,7022,5482,6172,636deh2995396406241,7261,5931,6582,5762,5832,636deh31015396306141,7301,6001,6662,5852,5902,635day11044774064112,0762,2302,1422,6932,7062,703day21024924124191,9832,1372,0892,6452,6692,668day31034974624512,0182,0632,0512,6842,7032,685tay11015815275371,8581,9111,9422,5812,4952,641tay21026105666001,7871,8871,9062,6712,6352,670tay31036005896221,7901,8391,9152,6602,6222,681teh11036366237091,7091,6621,7472,5872,6252,695teh21017836267071,7501,6501,7352,5842,5252,639teh31018576157471,8051,6081,7482,5632,5312,655F0\u00a0=\u00a0pitch. F1, F2 and F3 indicate the frequencies (Hz) of the first, second and third formants, respectively. F1, F2 and F3 frequencies are given for the beginning and end of the \/e\/ segment common to the four main stimuli of the study. The mean frequency across the length of the common \/e\/ section is also indicated\nThe reason for using three tokens for each consonant-vowel stimulus was to control as much as possible for acoustic factors, other than those inherent in the structure of the stimulus, which could confound the elicitation of a deviant response. Using three different exemplars for each consonant-vowel (CV) stimulus diminished the likelihood of a contingency developing between a specific deviant-standard pair because of an uncontrolled acoustic facet associated with either the deviant or the standard.\nAs could be seen in Table\u00a01 the frequency parameters across homogeneous exemplars (i.e., the three representatives of the same CV) were very similar to each other. In addition, the F1\/F2 ratio in \u201cteh\u201d and \u201ctay\u201d is similar to the F1\/F2 ratio in \u201cdeh\u201d and \u201cday\u201d, respectively. Thus, the comparison between the responses to \u201cteh\u201d and \u201ctay\u201d could serve as a suitable control for the latter pair (Jacobsen et al. 2004).\nThe speech stimuli appeared randomly within each block and their occurrence was balanced throughout the block for standards as well as deviants. Stimuli were presented with an SOA (stimulus onset asynchrony) of 1\u00a0s at 95\u00a0dB SPL via headphones to subjects with ear-plugs (see Table\u00a02 for an example of a \u201ccontrol\u201d and \u201coddball\u201d blocks in case of the \u201cdeh\u201d\u2013\u201cday\u201d contrast). Stimulus presentation was carried out by E-Prime 1.1 (1.1.4.1) (Psychology Software Tools http:\/\/www.pstnet.com).\nTable\u00a02\u201cOddball\u201d and \u201ccontrol\u201d sequences for the \u201cdeh\u201d (standard)-\u201cday\u201d (deviant) pairOddballdeh1(A) deh1 deh3 deh2 day1(B) deh1 deh2 deh1 deh3 deh1 deh1 deh3 day3\u2026Controlbeh1 deh3(C) tay2 teh3 bay2 bay3 peh2 deh2 day2(D) peh3 bay1 teh3 pay1 day2\u2026.A\u2013D indicate the stimuli contrasted to evaluate: the deviance effect (B vs. A), the cognitive effect (B vs. D) and the sensory effect (A vs. C). The numbers attached to the stimuli indicate different exemplars of the same CV stimulus. Each contrast was computed across all exemplars of a specific CV. In the \u201coddball\u201d sequence \u201cday1\u201d serves as a deviant and \u201cdeh1\u201d functions as a standard. In the \u201ccontrol\u201d condition \u201cdeh3\u201d is a standard control counterpart and \u201cday2\u201d serves as a deviant control counterpart\nContrasting \u201coddball\u201d deviants and standards with the corresponding stimuli in \u201ccontrol\u201d runs (deviant and standard counterparts) allowed disentangling the two mechanisms of deviance detection, namely, the sensory one based on refractoriness (N\u00e4\u00e4t\u00e4nen and Picton 1987) from the cognitive one based on memory-based processes (N\u00e4\u00e4t\u00e4nen 1990; N\u00e4\u00e4t\u00e4nen and Winkler 1999).\nThe subjects were instructed to count every stimulus, press a button when they reached 100 and then reset their count and restart counting from zero again. This task was chosen to control for a possible contamination by N2b and P3b ERP-components which are elicited when deviants are being discriminated from standards (Donchin et al. 1997; Opitz et al. 2005).\nBehavioral assessment of discrimination between stimuli\nThirteen subjects (out of the 20 participating in the study) rated the stimuli in a behavioral session performed outside of the magnet. The behavioral assessment was carried out in a different session. Stimuli were presented simultaneously with recorded MR scanner noise. The stimuli and the scanner noise were both presented in 62\u00a0dB SPL which was a convenient hearing level for both the stimuli and the noise presented together.\nSubjects were presented with \u201ctriplets\u201d comprised of stimuli containing \/e:\/ and \/eI\/. In each block, 15 triplets were presented separated by a 2\u00a0s interval of silence allowing the subject to respond. The stimuli were the same as those presented in the fMRI experiment. There were several patterns of triplet presentation in each block, as follows. There were three triplets in which \u201cday\u201d was presented first followed by two \u201cdeh\u201d-s; three triplets in which \u201cday\u201d was presented last and preceded by two \u201cdeh\u201d-s; three triplets where \u201cdeh\u201d was presented first followed by two \u201cday\u201d-s and three triplets were \u201cdeh\u201d was last and preceded by two \u201cday\u201d-s. In addition, there were three triplets in which \u201cday\u201d, \u201cdeh\u201d and \u201ctay\u201d each appeared in the middle position, respectively, while the neighboring stimuli (in the first and third positions) where either the \/eI\/ or \/e:\/ counterparts (i.e., \u201cdeh\u201d \u201cday\u201d \u201cdeh\u201d; \u201cday\u201d \u201cdeh\u201d \u201cday\u201d and \u201cteh \u201ctay\u201d \u201cteh\u201d).\nOverall, four blocks of 15 triplets each were presented to the subjects. The order of triplets was randomized within each block. The three different exemplars representing each CV stimulus were balanced across the four blocks. The subjects were required to indicate the outlier in each triplet by pressing the key (either \u201c1\u201d,\u201d2\u201d or \u201c3\u201d on the keyboard) that corresponded to the position of the outlier in the triplet. The outliers were assigned mainly to the extreme positions (1 or 3) in the triplet to simulate more closely the \u201coddball\u201d design in which the deviant is surrounded by repetitive standards. The trials with the outlier appearing in the middle position were introduced to minimize the probability of guessing the identity of the third stimulus in the triplet after hearing the first two which were non-identical. Thus, subjects could guess the identity of the outlier with more confidence only after hearing two identical stimuli in a row.\nData acquisition parameters\nData was collected on a 3T Siemens Trio scanner. Each study began with two localizers: a three-plane localizer and a multiple-slice sagittal localizer. These were followed by the acquisition of twenty-five 6\u00a0mm T1-weighted axial slices (TR\u00a0=\u00a0300\u00a0ms, TE\u00a0=\u00a02.47\u00a0ms, flip angle\u00a0=\u00a060\u00b0, FOV\u00a0=\u00a0220\u00a0mm, 256 acquisition matrix). For each subject, 12 functional imaging scans were collected with slices in the same locations as the anatomical T1-weighted data. Functional images were recorded using a gradient-echo EPI sequence (TR\u00a0=\u00a01,550\u00a0ms, TE\u00a0=\u00a030\u00a0ms, flip angle\u00a0=\u00a080\u00b0, FOV\u00a0=\u00a0220\u00a0mm, 64 acquisition matrix). Each functional run involved the acquisition of 245 volumes with twenty-five 6-mm axial slices. Images were converted to analyze format and the first ten volumes of each functional series were removed to account for the approach to steady-state magnetization, leaving 235 volumes for analysis.\nImage analysis\nPreprocessing\nFirst, using sinc interpolation, the data from each slice were adjusted for slice acquisition time and then all data were motion corrected using SPM99 for six rigid body motions (displacement in the x, y, z direction and rotation: for pitch, roll, yaw). Flags were set for de-correlation and masking so a pixel was set to zero for every time point if it moved outside the volume. Functional image data were motion corrected by realigning the time sequence imaging to the first image in the middle run of the sequence using SPM99.\nIndividual subject data (responses to the deviants, standards and control stimuli) were analyzed using a General Linear Model on each voxel in the entire brain volume (Rajeevan et al. 2007). The data were normalized to a signal measure of 100 and were spatially smoothed with a 8.08\u00a0mm Gaussian kernel to account for variations in the location of activation across participants. The output maps are normalized beta-maps which are in the acquired space (3.44\u00a0mm\u00a0\u00d7\u00a03.44\u00a0mm\u00a0\u00d7\u00a06\u00a0mm).\nTo take these data into a common reference space, three registrations were calculated within the Yale BioImage Suite software package (http:\/\/www.bioimagesuite.org\/, Papademetris et al. 2006) using the intensity-only component of the method reported in Papademetris et al. (2004). The first registration performs a linear registration between the individual subject raw functional image and that subject\u2019s 2D anatomical image. The 2D anatomical image was then linearally registered to the individual\u2019s 3D anatomical image. The 3D differs from the 2D in that it has a 1\u00a0\u00d7\u00a01\u00a0\u00d7\u00a01\u00a0mm resolution whereas the 2D z-dimension is set by slice-thickness and its x\u2013y dimensions are set by voxel size. Finally, a non-linear registration is computed between the individual 3D anatomical image and a reference 3D image. The reference brain used was the Colin27 Brain (Holmes et al. 1998) commonly applied in SPM and other software packages. All three registrations were applied sequentially to the individual normalized beta-maps to bring all data into the common reference space.\nStatistical analyses\nWe used a two-stage random-effects model to analyze the data. In the first stage, statistical maps were calculated in the comparison of interest for each subject as described above. In the second stage, across subject analyses were conducted and the distribution of the individual subjects\u2019 statistics were tested for significance.\nWithin subject analyses\nFor each of the three deviants (\u201cdeh\u201d, \u201cday\u201d and \u201ctay\u201d) the following six types of maps were calculated: (1) Deviant maps computed from oddball runs extracting the response to low frequency stimuli (2) Standard maps computed from oddball runs extracting the response to high frequency stimuli (3) Deviant control maps computed from control runs extracting the response to the same physical stimuli that served as deviants in the oddball runs (deviant-counterparts) (4) Standard control maps computed from control runs extracting the response to the same physical stimuli that served as standards in the oddball runs (standard-counterparts). The contrasts extracting each deviant-counterpart and standard-counterpart stimuli were carried out against the same baseline that formed part of the \u201cfiller\u201d stimuli, i.e., \u201cbeh\u201d, \u201cpeh\u201d, \u201cbay\u201d and \u201cpay\u201d. The stimuli that served as deviants and standards (i.e., \u201cdeh\u201d, \u201cday\u201d, \u201cteh\u201d, \u201ctay\u201d) were taken out of the baseline so that their extraction could be conducted against a common baseline. (5) A Word map computed from control runs contrasting the response to the meaningful word (\u201cday\u201d) with the non-words that formed part of the \u201cfiller\u201d stimuli, i.e., \u201cdeh\u201d, \u201cbeh\u201d, \u201cpeh\u201d. (6) A Non-word map computed from control runs contrasting the response to the non-word (\u201ctay\u201d) with the non-words that formed part of the \u201cfiller\u201d stimuli (i.e., \u201cdeh\u201d, \u201cbeh\u201d, \u201cpeh\u201d.) The non-word \u201ctay\u201d was excluded from the baseline, since it was the only non-word with a diphthong (\/eI\/), and to avoid contrasting it with itself.\nAcross subject analyses\nComposite maps. Two types of composites were created: three composites of deviant maps, one for each deviant (\u201cdeh\u201d, \u201cday\u201d, \u201ctay\u201d) and three composites of the deviant-control maps, one composite for each control run that included either \u201cdeh\u201d, \u201cday\u201d or \u201ctay\u201d as a deviant-counterpart, respectively.\nPaired t tests. The contrast between deviants and their respective counterparts isolates the cognitive component of deviance detection. For this contrast, a paired t test was calculated between the Deviant map and the Deviant control map. The contrast between standards and their respective counterparts accounts for the sensory component based on refractoriness. For this contrast, a paired t test was calculated between the Standard map and the Standard control map.\nStatistical images were corrected for multiple comparisons over the whole brain using a magnitude threshold derived from Monte\u2013Carlo simulation that takes into account the number of contiguous activated voxels (Forman et al. 1995). Individual voxel thresholds were set at P\u00a0<\u00a00.001 for both the composites and paired t test maps. Data were corrected for multiple comparisons by spatial extent of contiguous supra-threshold individual voxels (experiment-wise P\u00a0<\u00a00.001 for a cluster). In a Monte\u2013Carlo simulation within the AFNI software package, using a smoothing kernel of 8.08\u00a0mm, a connection radius of 7.72\u00a0mm on 3.48\u00a0mm\u00a0\u00d7\u00a03.48\u00a0mm\u00a0\u00d7\u00a06\u00a0mm voxels, it was determined that an activation volume of 1,278\u00a0\u03bcl satisfied the P\u00a0<\u00a00.001 threshold.\nRegions of interest analyses. The different regions of interest (ROIs) were identified on the basis of the results obtained by the paired t test maps prior to cluster-size correction (P\u00a0<\u00a00.001) reflecting the cognitive and sensory mechanisms of change-detection. Then, each participant\u2019s model estimate of the percent signal change for each region of activation, averaged across voxels within the region was calculated for each of the six composite maps (three deviant maps and three deviant-control maps). The ROIs identified were analyzed in a 3 (ROI: anterior auditory cortex, posterior auditory cortex, superior\/middle frontal gyrus)\u00a0\u00d7\u00a02 (task-type: \u201coddball\u201d\/\u201ccontrol\u201d)\u00a0\u00d7\u00a03 (deviant: \u201cdeh\u201d, \u201cday\u201d, \u201ctay\u201d)\u00a0\u00d7\u00a02 (laterality: left\/right) repeated measures ANOVA with subjects (n\u00a0=\u00a020) as a random factor.\nROI analysis for the Word\/Non-word maps. To further investigate the \u201cword effect\u201d, each participant\u2019s model estimate of the percent signal change for each region of activation, averaged across voxels within the region was calculated for the Word map and Non-word map. Both types of maps were calculated from control blocks. Specifically, to create the Word map \u201cday\u201d was contrasted with a baseline comprised of \u201cfiller\u201d or contextual stimuli that were non-words (i.e., \u201cbeh\u201d, \u201cpeh\u201d, \u201cdeh\u201d, \u201cthe\u201d). To create the Non-Word map \u201ctay\u201d was contrasted with the same baseline. The delta blood-oxygen-level-dependent (BOLD) measures were subjected to a 2 (Laterality: left\/right)\u00a0\u00d7\u00a03 (ROI: anterior auditory cortex, posterior auditory cortex, superior\/middle frontal gyrus)\u00a0\u00d7\u00a02 [Lexical Status: word (\u201cday\u201d) vs. non-word (\u201ctay\u201d)] repeated measures ANOVA with subjects (n\u00a0=\u00a020) as a random factor.\nResults\nBehavioral results\nThere were no differences in response accuracy scores, computed across the three possible positions of the outlier, among the three CVs. The response accuracy for \u201cday\u201d was 95.88% (\u00b15.01), for \u201ctay\u201d 96.70 (\u00b15.15) and for \u201cdeh\u201d 95.33 (\u00b14.22) (the number in brackets denotes the standard deviation). A paired t test that was run on the individual response accuracy data for each CV confirmed that there was no difference in accuracy levels between CVs (in all three possible comparisons P\u00a0>\u00a00.1). We have also examined the response accuracy scores for the first and third positions in the triplet for each of the CVs. The response accuracy scores ranged between 98 and 99% among the three different CV stimuli as well as between the first and third positions (since there were only four triplets per block in which the outlier appeared in the middle, the middle position was excluded from the latter calculation).\nTable\u00a03 displays the reaction time data for each deviant and for each of the three possible positions of the outlier within a triplet. As expected, it could be seen that the reaction time for the third position in the triplet was the shortest. To evaluate this trend statistically a two-way ANOVA was run with Stimulus (\u201cday\u201d, \u201ctay\u201d, \u201cdeh\u201d) and Position (first, second, third) as factors and with Greenhouse\u2013Geisser adjustment to account for sphericity. Only the Position factor was significant [F(2,38)\u00a0=\u00a028.70, P\u00a0<\u00a00.001]. Paired comparisons (Bonferroni corrected, P\u00a0<\u00a00.05) confirmed that reaction time associated with stimuli in the first and second position in the triplet was prolonged relative to that associated with the third stimulus in the triplet. Taken together, there is dissociation between response accuracy and reaction time data. While no differences in response accuracy were found, reaction time was shorter when the outlier CV stimulus was in the third position in the triplet.\nTable\u00a03Reaction time (ms) in the behavioral triplet testStimulusPosition123day787.58\u00a0\u00b1\u00a0132.07907.01\u00a0\u00b1\u00a0240.71638.55\u00a0\u00b1\u00a0215.98tay858.59\u00a0\u00b1\u00a0180.23961.06\u00a0\u00b1\u00a0187.15650.98\u00a0\u00b1\u00a0212.58deh864.03\u00a0\u00b1\u00a0164.90847.17\u00a0\u00b1\u00a0158.27628.33\u00a0\u00b1\u00a0137.63Reaction time is given for each of the three positions in the triplet and for each stimulus that served as a deviant in the \u201coddball\u201d blocks. \u00b1Standard deviation\nThis confirms our prediction that reaction time will be shorter in case of an outlier presented in the third position following two identical stimuli. However, the fact that the main effect of Stimulus as well as the interaction between Stimulus and Position did not reach significance confirms that the stimuli were equally discriminable in the context of the \u201coddball\u201d paradigms used in this experiment.\nComposite maps\nDeviance effect\nThe spread of activation associated with \u201ctay\u201d deviant was very similar to that in response to \u201cday\u201d (Fig.\u00a01). To elucidate the positions and extents of the activated brain regions associated with the similar responses to the deviants \u201cday\u201d and \u201ctay\u201d, images in the coronal and sagittal planes were included (Fig.\u00a01, bottom panels).\nFig.\u00a01Deviant maps: brain regions in response to deviant stimuli embedded within the \u201coddball\u201d paradigms. BOLD contrasts were superimposed on a reference anatomical image (Holmes et al. 1998). Upper panels: axial sections displaying responses to the deviants \u201cdeh\u201d (left), \u201cday\u201d (middle) and \u201ctay\u201d (right). Bottom panels: coronal (two left panels) and sagittal (two right panels) sections of the responses to the deviants \u201cday\u201d and \u201ctay\u201d. Axial sections range from z\u00a0=\u00a0\u22126 to 46 (by increments of 4\u00a0mm). Coronal sections range from y\u00a0=\u00a0\u22121 to 50 (by increments of 5\u00a0mm). Sagittal sections range from x\u00a0=\u00a0\u221257 to 59 (by increments of 8\u00a0mm). Display follows radiological convention (left side of the brain is displayed on the right side of the screen)\nThe size of the right STG region activated in the word condition (i.e., \u201cday\u201d), was similar to that activated in the non-word condition (i.e., \u201ctay\u201d) (Table\u00a04). In contrast, the size of the left STG region activated in the word condition was almost twofold larger than that activated by the \u201ctay\u201d condition. In both hemispheres, the brain regions activated in the \u201cday\u201d and the \u201ctay\u201d conditions were larger than those observed for the \u201cdeh\u201d condition. Furthermore, the \u201cday\u201d, \u201ctay\u201d and \u201cdeh\u201d stimuli appear to activate different regions of the STG. The \u201cday\u201d stimulus activated parts of the posterior STG (i.e., BA 22\/42), whereas the \u201ctay\u201d and \u201cdeh\u201d stimuli activated large parts of the superior and middle temporal cortices (i.e., BA 22\/22) (Table\u00a04).\nTable\u00a04Brain regions activated in Deviant maps (Fig.\u00a01)StimulusVolume size (mm3)Mean t valueTalairach coordinates (mm)SideIdentified regionBAxyzday10,8034.7256\u2212224RTSTG22\/42day11,3494.73\u221259\u2212216LTSTG22\/42deh3,5624.9155\u2212224RTSTG21\/22deh1,5544.4\u221262\u2212234LTSTG22tay9,1074.6856\u2212182RTSTG21\/22tay6,0344.97\u221257\u2212183LTSTG21\/22Talairach coordinates (Talairach and Tournoux 1988) are given for the center of mass. The mean t value was computed across the voxels within an anatomical region. Maps were thresholded at P\u00a0<\u00a00.001, corrected for multiple comparisonsSTG superior temporal gyrus, RT right, LT left, BA Brodmann area(s)\nSimilar brain regions to those implicated in the deviance response were also found in the study of Opitz et al. (2005) in the context of tones. Specifically, in that study the deviance effect also implicated HG (primary auditory cortex) and the superior temporal plane (Talairach coordinates of peak location: \u221249, \u221214, 9; 53, \u221221, 10, respectively).\nDeviant control maps\nWhile activity extended from the STG across the lateral sulcus to the central sulcus in response to \u201cday\u201d, \u201cdeh\u201d had a similar but more focal response in the vicinity of the primary auditory cortex. To elucidate the positions and extents of the activated brain regions associated with the more similar responses to the deviants \u201cday\u201d and \u201cdeh\u201d, images in the coronal and sagittal planes were included (Fig.\u00a02, bottom panels). In response to \u201ctay\u201d activity in the STG did not reach significance and activation was only observed in the left precentral gyrus (Fig.\u00a02, Table\u00a05).\nFig.\u00a02Deviant control maps: brain regions in response to deviant-counterparts stimuli embedded within the \u201ccontrol\u201d paradigms. Upper panels: axial sections displaying responses to the deviant controls \u201cdeh\u201d (left), \u201cday\u201d (middle) and \u201ctay\u201d (right). Bottom panels: coronal (two left panels) and sagittal (two right panels) sections of the responses to the deviant controls \u201cday\u201d and \u201cdeh\u201d. Additional display details as in Fig.\u00a01Table\u00a05Brain regions activated in Deviant control maps (Fig.\u00a02)StimulusVolume size (mm3)Mean t valueTalairach coordinates (mm)SideIdentified regionBAxyzday21,5944.9353\u22121615RTPoCG\/PrCG STG40\/43\/6day27,9194.93\u221252\u22121816LTPoCG\/PrCG40\/43\/6day4,600\u22124.39\u2212244830LTSFrG9day2,860\u22124.2\u22129\u221280\u221214LTLT OCCP18\/19deh4,3904.6853\u2212219RTSTG42deh5,0214.4\u221251\u2212216LTSTG42tay3,4014.6\u221249\u2212838LTPrCG6PoCG postcentral gyrus, PrCG precentral gyrus, SFrG superior frontal gyrus, STG superior temporal gyrus, RT right, LT left, OCCP occipital pole, BA Brodmann area(s). Other details as in Table\u00a04\nThe size of STG activation was the most extensive over the left STG for deviant \u201cday\u201d in comparison to \u201ctay\u201d where it did not reach statistical significance, and \u201cdeh\u201d where it was more than five times smaller (Fig.\u00a02, Table\u00a05). In addition, in response to \u201cday\u201d a negative differential BOLD was evident in the left superior frontal gyrus and left occipital pole (Fig.\u00a02, Table\u00a05).\nPaired t test maps\nCognitive effect\nThe cognitive effect was derived by contrasting the Deviant maps with the corresponding Deviant control maps. Only the contrast map for \u201cday\u201d showed activations at the significance level used throughout this study (P\u00a0<\u00a00.001, corrected for multiple comparisons), reflecting the \u201cword-advantage\u201d effect. The negative differential BOLD associated with the cognitive effect implicated bilaterally the region extending from the postcentral gyrus to HG including parts of the Sylvian fissure, lateral sulcus and insula (Table\u00a06, Fig.\u00a03). The location of this region is proximal to the one obtained by Opitz et al. (2005) that was associated with the cognitive mechanism and located bilaterally in a non-primary auditory area within the lateral temporal lobe in the anterior rim of HG (Talairach coordinates of peak location: \u221242, \u221213, 6 and 49, \u221212, 7).\nTable\u00a06Brain regions implicated in the cognitive effect (Fig.\u00a03)StimulusVolume size (mm3)Mean t valueTalairach coordinates (mm)SideIdentified regionBAxyzday4,179\u22124.3741\u22121616LTLS\/INSa40\/43day3,802\u22124.5\u221250\u22122019RTLS\/INSa40\/43LS lateral sulcus, INS insulaaRegion chosen for ROI analysis. Other details as in Table\u00a04Fig.\u00a03Brain regions implicated in the cognitive effect, showing less activation in the Deviant map than in the Deviant control map for \u201cday\u201d. BOLD contrasts were superimposed on a reference anatomical image (Holmes et al. 1998). Axial sections range from z\u00a0=\u00a0\u22126 to 46 (by increments of 4\u00a0mm)\nRefractoriness effect\nBy looking at the t test images associated with refractoriness (Fig.\u00a04) a negative differential BOLD could be observed posterior to HG in response to each of the standard-deviant pairs (Table\u00a07). In case of the refractoriness effect when \u201cdeh\u201d served as the standard activity also extended to the superior temporal sulcus (Fig.\u00a04, left panel). Opitz et al. (2005) have also found activations in a proximal brain region in the lateral aspect of the posterior rim of HG bilaterally that was associated with the sensory mechanism of change detection (Talairach coordinates of peak location: \u221251, \u221218, 7 and 53, \u221219, 4).\nFig.\u00a04Brain regions implicated in the sensory (refractoriness) effect, showing less activation in Standard maps than in Standard control maps. Responses for the contrasts with \u201cdeh\u201d (left) \u201cday\u201d (middle) and \u201cteh\u201d are shown. BOLD contrasts were superimposed on a reference anatomical image (Holmes et al. 1998). Axial sections range from z\u00a0=\u00a0\u22126 to 46 (by increments of 4\u00a0mm)Table\u00a07Brain regions implicated in the sensory (refractoriness) effect (Fig.\u00a04)StimulusVolume size (mm3)Mean t valueTalairach coordinates (mm)SideIdentified regionBAxyzday2,777\u22124.2759\u2212187RTSTGa22\/42day4,473\u22124.39\u221259\u2212204LTSTGa22\/42deh7724.08274330RTS\/MFrGa8\/9deh2,5104.29\u2212334330LTS\/MFrGa8\/9deh3,1894.3131442MedialACC32deh2,726\u22124.4157\u2212263RTSTG22deh3,030\u22124.39\u221257\u2212183LTSTG22tay2,048\u22124.3559\u2212226RTSTG22\/42tay2,395\u22124.53\u221253\u2212174LTSTG22STG superior temporal gyrus, S\/MFrG superior\/middle frontal gyrus, ACC anterior cingulate cortexaRegion chosen for ROI analysis; other details as in Table\u00a04\nFrom here on, we will refer to the region posterior to HG (Fig.\u00a04, Table\u00a07) as posterior auditory cortex. This region was defined functionally as showing a refractoriness effect in the current study. The region anterior to HG extending from the postcentral gyrus to HG (Fig.\u00a03, Table\u00a06) will be referred to as anterior auditory cortex. This region was defined functionally as showing a cognitive effect in the current study. This terminology is based on the one used by Opitz et al. (2005) that described the regions implicated in the sensory and cognitive effects as residing in the vicinity of the posterior and anterior rim of HG, respectively.\nIn addition, when the sensory component was extracted for \u201cdeh\u201d standard (Fig.\u00a04, left panel and Table\u00a07) positive differential BOLD was observed in the superior\/middle frontal gyrus as well as in the anterior cingulate cortex (ACC). Thus, the positive differential BOLD observed in the ACC and in the frontal gyrus is a direct result of contrasting \u201cdeh\u201d with a baseline of \u201cday\u201d deviants to create the map reflecting the sensory component for \u201cdeh\u201d standard.\nROI analysis\nSix ROIs were chosen to be included in the ROI analysis. These ROIs were defined on the basis of previous findings and the data obtained in the current study. We have chosen the ROIs which were directly linked to the mechanisms associated with change detection, namely, the cognitive and sensory effects, or those that might be related to both change-detection and to the processing of linguistic stimuli. This is described in more detail in the section \u201cDiscussion\u201d.\nThe following ROIs were identified on the basis of the paired t test maps. From the paired t test map reflecting the cognitive effect (Fig.\u00a03) the anterior auditory cortex was chosen bilaterally. From the paired t test map reflecting the refractoriness effect when \u201cday\u201d served as a standard the posterior auditory cortex was chosen bilaterally (Fig.\u00a04, middle panel). From the paired t test map reflecting the sensory component when \u201cdeh\u201d served as the standard the superior\/middle frontal gyrus was chosen bilaterally (Fig.\u00a04, left panel, Table\u00a07). Note that all the ROIs were defined based on the t-maps prior to cluster-size correction (P\u00a0<\u00a00.001). All except the right superior\/middle frontal gyrus were apparent in the t test maps after cluster-size correction.\nFigure\u00a05 displays the average delta BOLD for each condition (\u201coddball\u201d\/\u201ccontrol\u201d) and hemisphere (left\/right) as a function of deviant stimulus and ROI. From Fig.\u00a05 it can be seen that the largest differences in delta BOLD between conditions (\u201coddball\u201d\/\u201dcontrol\u201d) are evident over the left hemisphere, mainly the anterior auditory cortex and superior frontal gyrus. These were larger for \u201cday\u201d than \u201cdeh\u201d and smallest for \u201ctay\u201d. The latter was associated with diminished levels of delta BOLD within the posterior auditory cortex in the control condition.\nFig.\u00a05Average delta BOLD for each condition (\u201coddball\u201d\/\u201ccontrol\u201d) and hemisphere (left\/right) as a function of ROI and deviant stimulus (\u201cdeh\u201d, \u201cday\u201d or \u201ctay\u201d). Anter_Aud anterior auditory cortex, Post_Aud posterior auditory cortex, Sup\/Mid_Frontal superior\/middle frontal gyrus\nThe ROI data was subjected to a four-way ANOVA with Laterality (Left, Right), ROI (anterior auditory cortex, posterior auditory cortex, superior\/middle frontal gyrus), Deviant (\u201cdeh\u201d, \u201cday\u201d, \u201ctay\u201d) and Task type (Control, Oddball) as independent factors, with an appropriate adjustment for sphericity (Greenhouse\u2013Geisser correction). A main effect of ROI was found [F(2,38)\u00a0=\u00a049.17, P\u00a0<\u00a00.001], as well as a Laterality\u00a0\u00d7\u00a0ROI [F(2,38)\u00a0=\u00a05.39, P\u00a0<\u00a00.05], Deviant\u00a0\u00d7\u00a0ROI [F(4,76)\u00a0=\u00a04.23, P\u00a0<\u00a00.05], Task Type\u00a0\u00d7\u00a0ROI [F(2,38)\u00a0=\u00a07.25, P\u00a0<\u00a00.01], and Deviant\u00a0\u00d7\u00a0Task Type\u00a0\u00d7\u00a0ROI [F(4,76)\u00a0=\u00a04.60, P\u00a0<\u00a00.01] interactions. The Laterality\u00a0\u00d7\u00a0ROI interaction was due to enhanced delta BOLD over the left hemisphere associated with the anterior auditory cortex (P\u00a0<\u00a00.05, Bonferroni corrected).\nThe Deviant\u00a0\u00d7\u00a0Task Type\u00a0\u00d7\u00a0ROI interaction is depicted in Fig.\u00a06. A set of paired comparisons (Bonferroni corrected, P\u00a0<\u00a00.05) were conducted separately within each task-type (\u201coddball\u201d\/\u201dcontrol\u201d). In the \u201coddball\u201d condition the comparisons were conducted among the three deviants for the posterior auditory cortex only, where a gradient of delta BOLD could be observed as a function of deviant type. In the \u201ccontrol\u201d condition comparisons were performed among the three deviants for each of the ROIs. In the \u201coddball\u201d condition a significantly larger delta BOLD was found in response to \u201ctay\u201d than to \u201cdeh\u201d in the posterior auditory cortex. In the \u201ccontrol\u201d condition delta BOLD to the word \u201cday\u201d in the anterior and posterior auditory cortices was significantly larger than that associated with the non-word \u201ctay\u201d.\nFig.\u00a06ROI \u00d7 stimulus (\"deh\", \"day\",\"tay\") \u00d7 condition (\u201coddball\u201d\/\u201ccontrol\u201d) interaction. The interaction was obtained by calculating the individual percent signal change in each ROI (across hemispheres) for each of the six composite maps (three Deviant maps and three Deviant-control maps). See text for the results of the a priori tests. Abbreviations as in Fig.\u00a05\nIn a separate set of paired comparisons (Bonferroni corrected, P\u00a0<\u00a00.05) the averages of delta BOLD between conditions (\u201coddball\u201d\/\u201dcontrol\u201d) were compared for each of the three deviant stimuli and each of the ROIs (across hemispheres). A larger delta BOLD was found for the response to \u201cday\u201d in the \u201ccontrol\u201d condition relative to the \u201coddball\u201d condition implicating the anterior auditory cortex.\nROI analysis for the Word and Non-word maps\nTo further investigate the \u201cword effect\u201d, each participant\u2019s model estimate of the percent signal change for each region of activation, was calculated separately for the Word map and Non-word map (Fig.\u00a07, Table\u00a08). The ROI data was subjected to a three-way ANOVA with Laterality (left, right), ROI (anterior auditory cortex, posterior auditory cortex, superior\/middle frontal gyrus) and Lexical Status [word (\u201cday\u201d), non-word (\u201ctay\u201d)] as independent factors, with an appropriate adjustment for sphericity (Greenhouse\u2013Geisser correction). A main effect of ROI was found [F(2,38)\u00a0=\u00a021.03, P\u00a0<\u00a00.001] as well as a Lexical Status\u00a0\u00d7\u00a0ROI interaction [F(2,38)\u00a0=\u00a06.05, P\u00a0<\u00a00.01] (Fig.\u00a08). Paired comparisons (Bonferroni corrected, P\u00a0<\u00a00.05) found a significant difference between \u201cday\u201d and \u201ctay\u201d only within the anterior auditory cortex.\nFig.\u00a07Word and Non-Word maps. Brain regions in response to \u201cday\u201d deviant-counterpart (Word map) and \u201ctay\u201d deviant-counterpart (Non-word map). Stimuli were contrasted against a baseline comprised of the non-word \u201cfillers\u201d (i.e., \u201cpeh\u201d, \u201cbeh\u201d, \u201cdeh\u201d, \u201cteh\u201d). Left panel: response to the Word-map. Right panel: response to the Non-word map. BOLD contrasts were superimposed on a reference anatomical image (Holmes et al. 1998). Axial sections range from z\u00a0=\u00a0\u22126 to 46 (by increments of 4\u00a0mm)Table\u00a08Brain regions activated in Word and Non-word maps in response to \u201cday\u201d and \u201ctay\u201d, respectively (Figs.\u00a07, 8)StimulusVolume size (mm3)Mean t valueTalairach coordinates (mm)SideIdentified regionBAxyzday (Word-map)12,5794.6355\u22121614RTPoCG\/PrCG STG40\/43\/6day (Word-map)14,2604.65\u221253\u22121813LTSTG42tay (Non-Word-map)4,8464.92\u221252\u2212834LTPrCG4PoCG postcentral gyrus, PrCG precentral gyrus, STG superior temporal gyrus, other details as in Table\u00a04Fig.\u00a08ROI\u00a0\u00d7\u00a0Lexical Status interaction. The interaction was obtained by calculating the individual percent signal change in each ROI for the Word and Non-word maps. Abbreviations as in Fig.\u00a05\nThe distribution of percent signal change across subjects\nTo learn more about the consistency and extent of activation of specific brain regions, data were analyzed for individual subjects. Specifically, the percent signal change of individual subjects was computed for each of the six ROIs chosen for the group level analysis as well as for additional two brain regions that might be implicated in speech perception, that is, the left occipital pole and the left precentral gyrus. The results showed that the majority of subjects within each statistical test showed the same pattern of activity (see Table\u00a09).\nTable\u00a09The distribution of percent signal change across subjectsBrain regionSideMap typeNo. of subj. displaying increases (+)\/decreases (\u2212) (n\u00a0=\u00a020)Mean\u00a0\u00b1\u00a0SD % signal changeLS\/INSRTCognitive (\u201cday\u201d), Fig.\u00a0317(\u2212)\u22120.09\u00a0\u00b1\u00a00.07LS\/INSLTCognitive (\u201cday\u201d), Fig.\u00a0318(\u2212)\u22120.09\u00a0\u00b1\u00a00.10STGRTRefractoriness (\u201cday\u201d), Fig.\u00a0418(\u2212)\u22120.15\u00a0\u00b1\u00a00.17STGLTRefractoriness (\u201cday\u201d), Fig.\u00a0417(\u2212)\u22120.16\u00a0\u00b1\u00a00.14S\/MFrGRTRefractoriness (\u201cdeh\u201d), Fig.\u00a0413(+)0.06\u00a0\u00b1\u00a00.12S\/MFrGLTRefractoriness (\u201cdeh\u201d), Fig.\u00a0413(+)0.07\u00a0\u00b1\u00a00.10PrCGLTDeviant control (\u201ctay\u201d), Fig.\u00a0219(+)0.13\u00a0\u00b1\u00a00.13OCCPLTDeviant control (\u201cday\u201d), Fig.\u00a0218(\u2212)\u22120.24\u00a0\u00b1\u00a00.20SD standard deviation. For each region and hemisphere the number of subjects presenting increases or decreases in percent signal change is indicated as well as the mean and SD across subjects. The stimulus in brackets denotes the specific map from which the brain region was selected. For the regions associated with the t test maps of the cognitive and refractoriness effects, data are reported for the difference percent signal change between the contrasted conditions in the t testLS lateral sulcus, INS insula, STG superior temporal gyrus, S\/MFrG superior\/middle frontal gyrus, PrCG precentral gyrus, OCCP occipital pole, RT right, LT left, No. of subj number of subjects, n total number of subjects that participated in the study\nDiscussion\nMain findings\nOverall, the results of the current study confirmed our hypotheses. Specifically, the sensory mechanism was located in the posterior auditory cortex (Fig.\u00a04, Table\u00a07) and the cognitive mechanism in the anterior auditory cortex (Fig.\u00a03, Table\u00a06). These locations match, respectively, those found in the context of tones (Opitz et al. 2005). Moreover, as expected, the cognitive effect was statistically significant only when the deviant was a word (i.e., \u201cday\u201d).\nThe similarity between the brain regions activated by speech in this study and the regions activated by tones (Opitz et al. 2005) could not be explained by the analysis of the physical sound features. First, the contribution of the acoustic component to the cognitive mechanism in this study was removed by contrasting deviants with their physical identical control counterparts. Second, our data suggest that the salience of the sounds, either tones or speech stimuli, accounts for the activation of the anterior auditory cortex (J\u00e4\u00e4skel\u00e4inen et al. 2004; Ahveninen et al. 2006) (see also \u2018Support for the existence of \u201cwhat\u201d and \u201cwhere\u201d auditory streams\u2019). Thus, the acoustic component could only account for the similar activation observed in the posterior HG. This is also in line with previous findings that link dorsal temporal brain areas including HG and planum temporale with the analysis of auditory features of complex sounds (Binder et al. 1996; Wessinger et al. 2001; Hall et al. 2002, 2003; Seifritz et al. 2002).\nActivation loci in superior and middle temporal areas that were found to be activated in the Deviant (Fig.\u00a01) and Deviant control maps (Fig.\u00a02) had also been earlier reported to contribute to lexical and semantic processing (Price 2000; Salmelin et al. 2000; Scott and Johnsrude 2003; Hickok and Poeppel 2004). Similarly, the left dominance for processing intelligible speech that was found in our study in the \u201coddball\u201d condition match previous results that link the left posterior STG to higher level linguistic processes (Narain et al. 2003).\nSupport for the existence of \u201cwhat\u201d and \u201cwhere\u201d auditory streams\nThe results of this study are in agreement with the segregation of the auditory system into \u201cwhat\u201d and \u201cwhere\u201d processing streams (J\u00e4\u00e4skel\u00e4inen et al. 2004; Ahveninen et al. 2006) associated with the analysis of auditory object content and location features that reside in areas anterior and posterior to primary auditory cortex, respectively (Rauschecker and Tian 2000). It was suggested (J\u00e4\u00e4skel\u00e4inen et al. 2004) that the \u201cwhere\u201d system is responsible both for fast analysis of sound location which is important for attentional orienting, and for detecting the degree of sound novelty, which affects its degree of distractibility. Specifically, the degree to which unattended novel sounds distracted visual forced-choice task performance coincided with the extent that the posterior N1 response was released from inhibition. On the other hand, the anterior N1 response was associated with the processing of fine object features (J\u00e4\u00e4skel\u00e4inen et al. 2004). Similarly, the results in our study indicate that the deviant word was released from inhibition in the \u201ccontrol\u201d condition because of its lexical status and that this \u201cword advantage effect\u201d was located anterior to HG. This is in agreement with the finding that neurons in the anterior auditory cortex are more sharply tuned to phonetic features of sounds (Ahveninen et al. 2006).\nThe effect of speech perception\nDespite the general similarity to the results obtained in the context of tones, some of the results are specific to speech perception, as follows. In the \u201coddball\u201d condition the brain regions activated in response to the deviants \u201cday\u201d and \u201ctay\u201d were larger than those observed for the \u201cdeh\u201d deviant (Deviant maps, Fig.\u00a01). In addition, in the control condition the precentral gyrus was activated in response to both \u201cday\u201d and \u201ctay\u201d (Fig.\u00a02). These similarities between \u201cday\u201d and \u201ctay\u201d may reflect an extra processing effort associated with the diphthong vowel \/eI\/ shared by \u201cday\u201d and \u201ctay\u201d (Sonty et al. 2003; Bohland and Guenther 2006). However, the fact that the similarity between the responses to \u201cday\u201d and \u201ctay\u201d diminished in the control condition (Fig.\u00a02) may indicate that the \/eI\/ diphthong associated with deviant \u201cday\u201d was more salient among repetitive \u201cdeh\u201d standards (\u201coddball\u201d condition) than among the variable filler stimuli (\u201ccontrol\u201d condition) (Nordby et al. 1994; Sabri and Campbell 2000).\nThe more similar responses between \u201cday\u201d and \u201cdeh\u201d in the control condition (Fig.\u00a02) may be explained by the shared \/e:\/ monophthong that might have elicited an expectation to hear a meaningful word (\u201cday\u201d) while presented with \u201cdeh\u201d. This expectation could account for the more extensive response in the STG relative to \u201ctay\u201d (Fig.\u00a02). The finding that activation in the left anterior auditory cortex dissociates the \u201coddball\u201d and \u201ccontrol\u201d conditions strengthens this interpretation (Scott et al. 2006) as well as the observed increase in the percent signal change in that region associated with both \u201cdeh\u201d and \u201cday\u201d (Fig.\u00a05, \u201cLeft\u201d panels). Thus, in the \u201ccontrol\u201d condition (Fig.\u00a02), an interaction between lexical and acoustic features affected the results.\nThere were additional findings indicating left hemisphere dominance, as follows. The size of STG activation was the most extensive over the left STG for \u201cday\u201d in comparison to \u201ctay\u201d where it did not reach statistical significance and \u201cdeh\u201d where it was more than five times smaller (Deviant control maps, Fig.\u00a02, Table\u00a05). Furthermore, the \u201cday\u201d control map revealed negative differential BOLD in the left superior frontal gyrus as well as in the left occipital pole (Fig.\u00a02, Table\u00a05). Left hemisphere dominance was also evident in the middle\/superior frontal gyrus in the sensory mechanism map for \u201cdeh\u201d (Fig.\u00a04, Table\u00a07). In addition, the four-way ANOVA (Fig.\u00a05) has shown.\nThis pattern of left hemisphere dominance is in agreement with a number of imaging and clinical studies (for a review see Tervaniemi and Hugdahl 2003). The left middle\/superior frontal gyrus activations are in line with previous findings implicating these brain regions with tasks engaging phonological working memory (Paulesu et al. 1993; Burton et al. 2000; Siok et al. 2003; LoCasto et al. 2004) and with the suggestion that a significant portion of active frontal areas is recruited for extracting acoustic information and maintaining it in memory (LoCasto et al. 2004). The negative differential activation evident in the left occipital pole associated with the \u201cday\u201d control map (Fig.\u00a02) may indicate that meaningful words are more likely to elicit activity in visual processing regions (Billingsley-Marshall et al. 2007).\nIt is noteworthy that the sensory mechanism map for \u201cdeh\u201d revealed a positive differential BOLD in the ACC (Fig.\u00a04, Table\u00a07). The ACC is implicated in initiating or inhibiting action and is considered to be part of a larger network that includes medial\/lateral frontal, prefrontal and temporal regions (Wang et al. 2005; Dias et al. 2006; Gold et al. 2006). Hence, it is possible that in our study inhibitory activations occurred in the ACC (as well as in the superior frontal gyrus) in response to \u201cday\u201d since it was a meaningful stimulus that interfered with the main counting task (see also Rinne et al. 2005). Since standard \u201cdeh\u201d was contrasted with deviant \u201cday\u201d to create the \u201cdeh\u201d standard map, the positive differential BOLD located at the ACC and middle\/superior frontal gyrus (Fig.\u00a04, \u201cdeh\u201d) might have been caused by a reduced activation associated with the response to deviant \u201cday\u201d.\nA possible effect of the lexical status in the \u201coddball condition\nIt appears that the lexical status of the deviant stimulus affected its processing not only in the \u201ccontrol\u201d condition but also in the \u201coddball\u201d condition in which the effects of adaptation caused by the repeating standards were more pronounced. Specifically, the \u201cday\u201d stimulus activated parts of the posterior STG (i.e., BA 22\/42), whereas the \u201ctay\u201d and \u201cdeh\u201d stimuli activated large parts of the superior and middle temporal cortices (i.e., BA 21\/22) (Table\u00a04). However, the size of the left STG region activated by \u201cday\u201d deviant was almost twofold larger than that activated by \u201ctay\u201d deviant (Deviant maps, Fig.\u00a02, Table\u00a04). This is in agreement with the assumption that the left posterior STG is the focus of a multi-modal network associated with language comprehension (Aboitiz and Garcia 1997; Narain et al. 2003). More strongly left-lateralized posterior superior-temporal activation is associated with analysis of speech sounds for mapping onto higher levels of language processing (e.g., syllable, word) (Price et al. 1992; Zatorre et al. 1996). Thus, although the word advantage effect was not salient in the \u201coddball\u201d condition, the different spread of activation between \u201cday\u201d and the other non-words may indicate differential processing based on the lexical status of the deviant stimulus.\nSummary\nTaken together, the results of the current study corroborate the existence of two independent mechanisms contributing to the change-detection response (Opitz et al. 2005; Hoshiyama et al. 2007; Maess et al. 2007): a sensory mechanism reflected by different refractory states of those subpopulations activated by the standard and the deviant and a cognitive mechanism which relies on auditory sensory memory representations which gives rise to the word advantage effect. Thus, on the one hand, our results support the view that the MMN represents a change-detection mechanism functionally and spatially distinct from an afferent input population (N1 generators) (N\u00e4\u00e4t\u00e4nen et al. 2005). On the other hand, our results indicate that a release from the inhibitory effects of adaptation is a prerequisite for the full realization of the significance of the deviant stimulus. This is in line with the adaptation hypothesis that assumes that the posterior auditory cortex gates novel sounds to awareness (J\u00e4\u00e4skel\u00e4inen et al. 2004).\nConclusion\nOur findings serve to unify the two opposing views suggested by J\u00e4\u00e4skel\u00e4inen et al. (2004) and N\u00e4\u00e4t\u00e4nen et al. (2005). Specifically, the gate to awareness for auditory deviation (J\u00e4\u00e4skel\u00e4inen et al. 2004; N\u00e4\u00e4t\u00e4nen et al. 2005) relies on adaptation that modulates the extent to which novel sounds will be accessible to memory-based processes. In case of the present study, the sensory component serves to modulate the salience of the speech stimulus by the degree to which it will be accessible to cortical memory traces for speech sounds (Pulverm\u00fcller and Shtyrov 2006). Furthermore, the lexical status of the speech stimulus interacts with acoustic factors exerting a top-down effect on the novelty value of the auditory object that affects, in turn, its degree of accessibility to the cognitive component.","keyphrases":["mismatch negativity (mmn)","functional magnetic resonance imaging (fmri)","speech perception","adaptation hypothesis"],"prmu":["P","P","P","P"]}
{"id":"J_Neurooncol-4-1-2174520","title":"Lovastatin sensitized human glioblastoma cells to TRAIL-induced apoptosis\n","text":"Synergy study with chemotherapeutic agents is a common in vitro strategy in the search for effective cancer therapy. For non-chemotherapeutic agents, efficacious synergistic effects are uncommon. Here, we have examined two non-chemotherapeutic agents for synergistic effects: lovastatin and Tumor Necrosis Factor (TNF)-related apoptosis-inducing ligand (TRAIL) for synergistic effects; on three human malignant glioblastoma cell lines, M059K, M59J, and A172. Cells treated with lovastatin plus TRAIL for 48 h showed 50% apoptotic cell death, whereas TRAIL alone (1,000 ng\/ml) did not, suggesting that lovastatin sensitized the glioblastoma cells to TRAIL attack. Cell cycle analysis indicated that lovastatin increased G0\u2013G1 arrest in these cells. Annexin V study demonstrated that apoptosis was the predominant mode of cell death. We conclude that the combination of lovastatin and TRAIL enhances apoptosis synergistically. Moreover, lovastatin sensitized glioblastoma cells to TRAIL, suggesting a new strategy to treat glioblastoma.\nIntroduction\nGlioblastomas are the most common intracranial brain tumors. Its prognosis is usually poor, with survival times of less than 15\u00a0months from first diagnosis [1]. Surgical resection and chemotherapy are common treatments [2]. Despite recent advances in the understanding of the molecular mechanism of tumourogenesis, the outcome of malignant glioma remains poor [3]. Thus, new effective forms of therapy are needed.\nThe Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) [4], a member of the TNF superfamily, can bind with death receptors, DR4 and DR5 [5, 6] and induces apoptosis in a wide range of cancer cells without harming normal cells. The specific property of TRAIL has attracted many researchers to look for new treatments by combining it with chemotherapeutic agents such as phenoxazine derivatives [7], doxorubicin and cisplatin [8]. Such combinations have shown synergistic effects on different types of cancer cells in\u00a0vitro.\nLovastatin, a 3-hydroxy-3-methlyglutaryl CoA (HMG CoA) reductase inhibitor is a commonly used cholesterol-lowering agent for prevention of atherosclerotic cardiovascular diseases [9, 10]. Lovastatin blocks the mevalonate pathway and reduces the formation of the downstream products, cholesterol, geranylgeranyl proteins, farnesylated [11]. Recently both in\u00a0vitro and in\u00a0vitro studies have found that lovastatin has antiproliferative, proapoptotic and anti-invasive properties in a wide range of cancer cell types [12]. Lovastatin is known to have an apoptotic effect on tumor cells and its combination with chemotherapeutics and cytokines often exert a synergistic effect against tumor growth [13\u201315]. The mechanism that leads to lovastatin-induced apoptosis is not yet clear but the main event is thought to be associated with the alteration of mitochondrial stress, which releases cytochrome C, activates pro-caspase cascade and finally leads to apoptotic cell death.\nEscape from apoptotic regulation is one of the major characteristics of cancer [16, 17], and many successful anti-cancer agents induce apoptosis by damaging DNA. Unfortunately such agents may also severely affect normal cells. Given the fact that both lovastatin and TRAIL are non-chemotherapeutic agents and capable of inducing apoptosis in different types of cancer cells, it is important to determine whether the combination of these two agents would produce synergistic effects that may be lighten for a novel therapeutic application in gliomas.\nWe therefore hypothesized that the combination of TRAIL and lovastatin, neither of which alone has noxious effects on healthy cells, could generate a regime that was effective in killing cancer cells but caused minimal insult to normal healthy cells. In this study we report the effects of TRAIL in combination with a non-chemotherapeutic drug, lovastatin, on glioblastoma cells.\nMaterials and methods\nReagents\n2-Methyl-1,2,3,7,8,8a-hexahdro-3,7-dimethyl-8-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-1-naphthalenyl ester butanoic acid (Lovastatin), DL-Mevalonic acid lactone, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (St Louis, MO). Lovastatin was dissolved in DMSO for stock and adjusted to final concentrations using complete medium or serum free medium. Soluble Human TRAIL (Apo2L) was affinity purified from lysates of bacteria transformed with pET plasmid containing TRAIL [18]. Cellular DNA fragmentation ELISA kit (Roche, Mannheim, Germany), RNeasy kit, DNA extraction kit (Qiagen, Germany) and RT-PCR kit (Promega, Madison, WI) were used. Three primary antibodies used were as follows: rabbit polyclonal antibody to DR4 (Chemicon International, 1:1,000 dilution), rabbit polyclonal antibody to DR5 (Cell Signaling Technology, 1:1,000 dilution), and rabbit polyclonal antibody to \u03b2-tubulin (Santa Cruz Biotechnology, 1:1,000 dilution). Goat anti-rabbit secondary antibody was obtained from Santa Cruz Biotechnology.\nCell culture\nThree human glioblastoma cell lines, A172, M059J, and M059K were purchased from American Type Culture Collection (Rockville, MD). The glioblastoma cells were kept in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM\/F12) (GIBCO BRL, Grand Island, NY) with or without 10% fetal bovine serum, 1% penicillin and streptomycin at 37\u00b0C under 5% CO2. Media were changed every 3\u00a0days.\nMeasurement of cell viability\nWe measured cell viability using the MTT assay. MTT is a water-soluble tetrazolium salt that is metabolized by viable cells to a colored, water-insoluble formazan salt. Thus the salt allows cell viability measurements. In short, 1\u00a0\u00d7\u00a0104 cells were cultured in the serum free DMEM\/F12 medium in the presence of lovastatin with or without TRAIL in a 96 well microtiter plate for designated time periods. The medium was aspirated and 100\u00a0\u03bcl MTT (0.5\u00a0mg\/ml in PBS) were added to each well and the cells incubated for 3\u00a0h. After MTT medium was aspirated, the cells were solubilized in 200\u00a0\u03bcl DMSO. The optical density of each sample at 570\u00a0nm (reference 630\u00a0nm) was measured using a microplate reader. The optical density of the media was proportional to the degree of viable cells.\nCell cycle analysis\nPropidium iodide (PI) staining and flow cytometry were used to determine the stage of the cell cycle. Cells were treated with 20\u00a0\u03bcM lovastatin for 48\u00a0h at 37\u00b0C and the control cells were treated with normal medium (DMSO) or serum free medium (DMSO). 2\u00a0\u00d7\u00a0106 treated cells were washed with 5\u00a0ml PBS and then were trypsinized at 37\u00b0C for 5\u201310\u00a0min. Cells were spun down and washed with 5\u00a0ml PBS. Finally cells were resuspended in 500\u00a0\u03bcl PBS and fixed with 4.5\u00a0ml 70% Ethanol with gentle vortexing. Cells were allowed to sit in \u221220\u00b0C for overnight. Fixed cells were spun down and washed with 5\u00a0ml PBS, and then cells were resuspended in 500\u00a0\u03bcl PI (2\u00a0\u03bcg\/ml)\/Triton X-100 (0.1% v\/v) staining solution with RNase A (200\u00a0\u03bcg\/ml) in dark and analyzed by a flow cytometer. The staining solution was purchased from Chemicon (Temecula, CA).\nApoptosis assay\nApoptotic cells were determined by two methods, Annexin-V and PI stained cells by flow cytometry and DNA fragmentation assay. During apoptosis, translocation of phosphatidylserine from inner membrane to outer membrane is a common phenomenon. Cells were stained with Annexin V for analysis of phosphoserine inversion, which was considered to be a sensitive marker of apoptosis. Using an Annexin V-FITC apoptosis detection kit (Molecular Probe Inc, Eugene, OR), the levels of binding of Annexin V and staining with PI were measured for the detection of early and late apoptosis respectively. All of the procedures were preformed under manufacturer\u2019s guidelines. Cells were treated with lovastatin or\/and TRAIL for 48\u00a0h and then stained with Annexin-V and PI. Viable cells were recognized as negative for both Annexin-V and PI; early apoptotic events were positive for Annexin-V but negative for PI staining. Late apoptotic events were positive to both Annexin V and PI. Necrotic cells were positive for PI staining only.\nDNA fragmentation determination, was carried out with the ELISA assay kit. After cells (1\u00a0\u00d7\u00a0104) were treated with lovastatin and\/or TRAIL for 48\u00a0h, DNA fragmentation was detected using 96 wells microplate reader.\nRT-PCR\nTotal RNA was isolated using Qiagen RNeasy extraction kit and performed according to the manufacturer\u2019s protocol. Total RNA (5\u00a0\u03bcg) was reversely transcribed using Promega RT-PCR kit and thermal program was set at 42\u00b0C for 15\u00a0min and 95\u00b0C for 5\u00a0min. PCR reaction was performed using the following primers, which have previously been tested successfully: TRAIL-R1, 5\u2032-CTG AGC AAC GCA GAC TCG CTG TCC AC-3\u2032 and 5\u2032-TCC AAG GAC ACG GCA GAG CCT GTG CCA T-3\u2032; TRAIL-R2, 5\u2032-GCC TCA TGG ACA ATG AGA TAA AGG TGG CT-3\u2032 and 5\u2032-CCA AAT CTC AAA GTA CGC ACA AAC GG-3\u2032; TRAIL-R3, 5\u2032-GAA GAA TTT GGT GCC AAT GCC ACT G-3\u2032 and 5\u2032-CTC TTG GAC TTG GCT GGG AGA TGT G-3\u2032; TRAIL-R4, 5\u2032-CTT TTC CGG CGG CGT TCA TGT CCT TC-3\u2032 and 5\u2032-GTT TCT TCC AGG CTG CTT CCC TTT GTA G-3\u2032. The thermal program was set up as one cycle at 94\u00b0C for 5\u00a0min, 30 cycles at 94\u00b0C for 1\u00a0min, 55\u00b0C for 1\u00a0min, 72\u00b0C for 2\u00a0min, and one cycle at 72\u00b0C for 5\u00a0min. PCR products were resolved and visualized on a 2% agarose gel stained with ethidium bromide.\nWestern blot analysis\nWestern blot was performed according to previous description [19, 20]. Briefly, after 2\u00a0\u00d7\u00a0106 cells were treated for 48\u00a0h, total protein was isolated and reacted with the relevant antibodies. The probed proteins were visualized using the enhanced chemiluminescence Western blotting detection system (ECL Western Blotting Detection, Amersham Biosciences).\nStatistics\nThe statistical significance was analyzed using one-way ANOVA analysis and Student\u2019s t-test. All statistical work was carried out using the SPSS software for Windows (Release 11.0.1, Chicago, IL). Differences were considered to be significant when P\u00a0<\u00a00.05.\nResults\nCell viability measured by MTT assay\nWe first examined the anti-proliferation effect of TRAIL on three glioblastoma cell lines. M059K and M059J glioblastoma cells were resistant to TRAIL-induced cell death and remained 100% cell viable following treatment in both normal and serum free conditions (Fig.\u00a01a, b). Only A172 showed minor cell death after TRAIL treatment (22%, Fig.\u00a01a).\nFig.\u00a01Anti-proliferation effects on glioblastoma cell lines A172, M059K, and M059J. MTT assays were preformed after 48\u00a0h incubation time treated with 0,\u00a0500, 1,000\u00a0ng\/ml TRAIL in normal medium (a) or serum free medium (b). A172 subjected to minor TRAIL-induced cell death while M059J and M059K cells were resistant to TRAIL. Same trend of data were obtained in both normal medium and serum free condition. Experiment set were repeated at least three times with triplicate wells for each condition (mean\u00a0\u00b1\u00a0SD)\nSynergistic effects by TRAIL and lovastatin were then examined. A172, M059J, and M059K glioblastoma cells were incubated in 1, 5, 20, and 40\u00a0\u03bcM lovastatin alone or together with 500\u00a0ng\/ml TRAIL for 48\u00a0h. In A172 glioblastoma cells, 5, 20, and 40\u00a0\u03bcM lovastatin with 500\u00a0ng\/ml TRAIL promoted significant cell death when compared with lovastatin control (Fig.\u00a02a). 5, 20 and 40\u00a0\u03bcM lovastatin alone induced 50%, 60% and 76% of cell death respectively, however in the presence of 500\u00a0ng\/ml TRAIL, lovastatin induced 78%, 94%, and 92% of cell death respectively. 1, 5, 20, and 40\u00a0\u03bcM lovastatin with 500\u00a0ng\/ml TRAIL also synergistically promoted cell death in M059J and M059K. In M059J cells, 1, 5, 20 and 40\u00a0\u03bcM lovastatin alone caused 40%, 55%, 40% and 60% cell death whereas in the presence of 500\u00a0ng\/ml TRAIL, lovastatin induced 83%, 94%, 95% and 95% cell death respectively (Fig.\u00a02b). There were 26%, 51%, 58%, and 71% cell death induced by 1, 5, 20, and 40\u00a0\u03bcM lovastatin alone in M059K, in the presence of 500\u00a0ng\/ml TRAIL, lovastatin at all concentration tested caused about 98% of cell death (Fig.\u00a02c). Collectively the combination of TRAIL and lovastatin was much more effective than lovastatin alone in the induction of cell death in all three glioblastoma cells tested and the result indicated that there was a synergistic effect when TRAIL and lovastatin were used together.\nFig.\u00a02Synergistic anti-proliferation effects on glioblastoma cell lines A172, M059J, and M059K. Glioblastoma cells were treated with 0, 5, 20 and 40\u00a0\u03bcM lovastatin alone or 0\u00a0\u03bcM lovastatin plus 500\u00a0ng\/ml TRAIL, 5\u00a0\u03bcM lovastatin plus 500\u00a0ng\/ml TRAIL, 20\u00a0\u03bcM lovastatin plus 500\u00a0ng\/ml TRAIL and 40\u00a0\u03bcM lovastatin plus 500\u00a0ng\/ml TRAIL for 48\u00a0h. Percentage of viable glioblastoma cells showed synergistic anti-proliferation effects by combined the two agents. In A172 glioblastoma cells (a), 500\u00a0ng\/ml TRAIL with 5, 20 and 40\u00a0\u03bcM lovastatin induced cell death significantly when compared with lovastatin only groups. In M059J (b) and M059K (c), 500\u00a0ng\/ml TRAIL with 1, 5, 20 and 40\u00a0\u03bcM lovastatin induced cell death significantly when compared with lovastatin only groups. ANOVA were used for statistics analysis and *P\u00a0<\u00a00.05, **P\u00a0<\u00a00.01. Experimental set were repeated for at least three times with triplicate wells for each condition (mean\u00a0\u00b1\u00a0SD)\nCell cycle determination by PI staining\nPropidium iodide staining and flow cytometry were used to determine the degree of cell synchronization by 20\u00a0\u03bcM lovastatin. Cells were incubated with normal serum medium, serum free medium and 20\u00a0\u03bcM lovastatin with normal serum medium for 48\u00a0h. PI-stained cells were analyzed using flow cytometry to quantify cells in certain cell cycle stages. A significant increase in cell population at G0\/G1 phase was observed when the cells were treated with 20\u00a0\u03bcM lovastatin indicating that lovastatin was able to arrest the cells at G0\/G1 stage. G0\/G1 cell population was also increased by serum free medium conditions (positive control) and the increase reached a significant level except in M059J cells (Fig.\u00a03).\nFig.\u00a03Lovastatin-induced glioblastoma cells arrested in G0\u2013G1 Phase. Propidium Iodide staining for cell cycle analysis were performed after glioblastoma cells were treated with normal medium without lovastatin, serum free medium without lovastatin and 20\u00a0\u03bcM lovastatin for 48\u00a0h. Serum free condition was used as a positive control which is commonly known to induce cell arrest in G0\u2013G1 phase. Serum free condition and lovastatin increased G0\u2013G1 cell arrest in all glioblastoma cells and reached significant level (except serum free condition in M059J). ANOVA were used for statistics analysis and *P\u00a0<\u00a00.05, **P\u00a0<\u00a00.01. Experimental set were repeated for at least three times with triplicate wells for each condition (mean\u00a0\u00b1\u00a0SD)\nApoptosis is the major mode of cell death\nIn order to determine the mode of cell death induced by lovastatin and TRAIL, Annexin V and PI staining was employed for this purpose. Cells were incubated with 5 or 20\u00a0\u03bcM of lovastatin in the presence or absence of 500\u00a0ng\/ml TRAIL for 48\u00a0h. The percentage of cell death of glioblastoma cells treated with both lovastatin and TRAIL was significantly higher than that with either agent alone (Fig.\u00a04a, b and c). A172 glioblastoma cells were vulnerable to TRAIL induced apoptosis (Fig.\u00a04a) but significantly more apoptotic cells were observed following treatment with both TRAIL and lovastatin (P\u00a0<\u00a00.005) (Fig.\u00a04a). TRAIL was able to induce apoptosis in 50% of A172 glioblastoma cells. However, TRAIL in combination with 5 and 20\u00a0\u03bcM could induce apoptosis in approximately 67% and 74% cells respectively, indicating a synergistic effect occurred. Similarly, a significant synergistic effect was also observed in M059K and M059J cells (P\u00a0<\u00a00.005) (Fig.\u00a04b, c), with up to nearly a 10-fold increase in apoptotic cells. 5\u00a0\u03bcM lovastatin only induced apoptosis in 5.3% and 2.3% M059J and M059K respectively, which were not different from the control (without any treatment). 5\u00a0\u03bcM lovastatin plus 500\u00a0ng\/ml TRAIL dramatically increased apoptotic cells to 47.8% and 61.4% in M059J and M059K respectively. M059J cells, which lack of DNA-dependent protein kinase expression [21], were less vulnerable to the two agents, implying that DNA-dependent protein kinase may play a role in apoptosis induced by TRAIL and lovastatin.\nFig.\u00a04The synergistic apoptotic effects were quantified by flow cytometry using Annexin V and PI staining. Glioblastoma cells were treated with DMSO (Control), 5\u00a0\u03bcM lovastatin, 20\u00a0\u03bcM lovastatin, 500\u00a0ng\/ml TRAIL, 5\u00a0\u03bcM lovastatin plus 500\u00a0ng\/ml TRAIL and 20\u00a0\u03bcM lovastatin plus 500\u00a0ng\/ml TRAIL for 48\u00a0h. Then cells were stained with Annexin V and PI to determine percentage of apoptotic cell death using flow cytometry. Synergistic apoptotic effects were observed in three glioblastoma cell lines A172 (a), M059J (b) and M059K (c), reached significant level. The portion of apoptotic cell death was indicated in low-right quarter of the flow-cytometry scatter plot and the trend of apoptotic cells between groups. ANOVA were used for statistics analysis and *P\u00a0<\u00a00.05, **P\u00a0<\u00a00.01 (compared to control group), +P\u00a0<\u00a00.05, ++P\u00a0<\u00a00.01 (compared to TRAIL only group). Experimental set were repeated for at least three times (mean\u00a0\u00b1\u00a0SD)\nDNA fragmentation in glioblastoma cells\nDNA fragmentation is one of the hallmarks when cells undergo apoptosis. We performed ELISA assay to confirm DNA fragmentation was induced in the cells treated with both lovastatin and TRAIL. A synergistic effect on DNA fragmentation by both agents was demonstrated in all three cell lines tested (Fig.\u00a05a, b and c). The level of DNA fragmentation was increased in treated A172 glioblastoma cells, but it was only statistically significant in the cells treated with TRAIL plus 20\u00a0\u03bcM lovastatin. In M059J and M059K cells, however, there was a dramatic elevation of DNA fragmentation in a dose-dependent manner when both of the agents were applied. This observation supports the argument that the cell death induced by the two agents was apoptotic and that both agents were able to function in a synergistic manner.\nFig.\u00a05DNA fragmentation was detected in combination of lovastatin and TRAIL. Same treatment in apoptotic cell staining was preformed for DNA fragmentation ELISA detection. Significant level of DNA fragmentation was detected in all glioblastoma cells when combined with lovastatin and TRAIL. ANOVA were used for statistics analysis and *P\u00a0<\u00a00.05, **P\u00a0<\u00a00.01 (compared to control group), +P\u00a0<\u00a00.05, ++P\u00a0<\u00a00.01 (compared to TRAIL only group). Experimental set were repeated for at least three times (mean\u00a0\u00b1\u00a0SD)\nThe expression of TRAIL receptors in glioblastoma cells\nResistance to cell death induced by TRAIL may be adapted by an altered level of TRAIL receptors. TRAIL-R3 (DcR1) and TRAIL-R4 (DcR2) are known to attenuate TRAIL-induced apoptosis whereas TRAIL-R1 (DR4) and TRAIL-R2 (DR5) promote TRAIL-induced apoptosis [5]. The effect of lovastatin treatment on the TRAIL receptors was not determined. RT-PCR was performed to investigate the expression profiles on three glioblastoma cell lines tested. Cells were treated with normal serum medium, serum free medium, serum medium with 5 or 20\u00a0\u03bcM lovastatin for 48\u00a0h. TRAIL-R3 and R4 were not detected in all conditions and cell types tested. TRAIL-R1 was only detected on M059J cells with serum free medium or lovastatin (Fig.\u00a06a). TRAIL-R2 was expressed on all cell types of cells with or without lovastatin and its level was not significantly different between lovastatin-treated cells and the controls. Therefore, the expression of TRAIL-R2 did not contribute to the lovastatin-induced cell death in glioblastoma cells tested.\nFig.\u00a06The expression of TRAIL receptor mRNA in glioblastoma cells. The cells were treated with normal serum medium, serum free medium, 5 and 20\u00a0\u03bcM lovastatin with normal serum medium. At the end of the treatment, RNA was isolated for the detection of TRAIL-R1 (DR4) (a) and TRAIL-R2 (DR5) (b) by RT-PCR\nAfter we tested the mRNA expression of TRAIL receptors on glioblastoma cells, the further investigation was performed to determine the active protein expression of TRAIL-R1 and matured TRAIL-R2 by Western blot. The result showed that the protein expression profiles of TRAIL-R1 and TRAIL-R2 were found in three glioblastoma cells with or without lovastatin, suggesting the protein of these two receptors was underwent post-translational modifications. The significantly differences were only found on the TRAIL-R1 expression of M059K cells with 20\u00a0\u03bcM lovastatin (P\u00a0=\u00a00.05) and the expression of TRAIL-R2 in A172 cells with lovastatin (P\u00a0<\u00a00.01) (Fig.\u00a07).\nFig.\u00a07The expression of TRAIL-R1 (DR4) and TRAIL-R2 (DR5) proteins in glioblastoma cells. The cells were treated with normal serum medium, serum free medium, 5 and 20\u00a0\u03bcM lovastatin with normal serum medium for 48\u00a0h. At the end of the treatment, proteins were isolated for the detection of TRAIL-R1 (a, b) and TRAIL-R2 (c, d) by Western blot. Representative Western blots were shown (a, c). The target bands were scanned and normalized to \u03b2-tubulin. The index of densities was calculated (b, d). *P\u00a0=\u00a00.05, **P\u00a0<\u00a00.01\nDiscussion\nMalignant glioblastoma is one of the major causes of brain tumors morbidity. Aggressive infiltration in the CNS ultimately leads to death in nearly all cases [2]. Malignant glioblastoma carries with aberrant biological and biochemical properties including several activating mutations that can lead to chemotherapeutics resistance [22]. Targeting apoptotic signaling machinery is thought to be a promising alternative for glioblastoma treatment [23]. The preliminary data presented in this report indicate that lovastatin, a blood cholesterol lowering medicine, sensitizes glioblastoma cells to TRAIL-mediated apoptosis. Earlier reports show statin-induced cell death through a mitochondrial-mediate pathway (intrinsic pathway) that is closely related to the Bcl-2 family protein Bid and activation of caspase 8, 9 and 3 [24\u201327]. However, the mechanism for statin activation of caspase 8 remains unknown because caspase 8 is normally activated by receptor-mediated signals, such as Fas ligand and TRAIL [5].\nLovastatin has been found to enhance TRAIL-induced cytotoxicity in a synergistic manner in glioblastoma cells. We demonstrated a synergistic effect produced by the combination of lovastatin and TRAIL on glioblastoma cells. Lovastatin was found to sensitize the cells to cell death induced by TRAIL. The mode of cell death induced by both agents in combination was apoptosis, as demonstrated by two different methods, Annexin V and PI staining and DNA fragmentation assay. We also demonstrated that two of the glioblastoma cell lines tested were resistant to TRAIL induced apoptosis. From this we inferred that lovastatin not only sensitized these glioblastoma cells through its effects on the TRAIL receptor pathway but also triggered an unknown mechanism: Lovastatin served as a cytostatic agent and turned on an unknown mechanism to support TRAIL-induced apoptosis in these glioblastoma cells. It has been shown that colon and lung tumor cells arrested in G0\u2013G1 stages are vulnerable to TRAIL-induced cell death [28]. However, our G0\u2013G1 arrested glioblastoma cell lines remained resistant to TRAIL-induced cell death in the serum free control. Our finding suggests that a combination of TRAIL and lovastatin together may form a new treatment for glioblastoma multiforme.\nThe mechanism by which lovastatin sensitizes glioblastoma cells to TRAIL induced-apoptosis remains unknown. In human glioblastoma, lovastatin has been shown to induce or to enhance apoptosis by altering a number of apoptotic molecules. For example, it can induce apoptosis or downregulate cell proliferation by targeting Ras in primary cultured human glioblastoma cells [29] and increase pro-apoptotic Bim in U87 and U251 glioblastoma cells [30]. Lovastatin has also been shown to downregulate RhoA and increase iNOS in T98G and A172 glioblastoma cells [31]. Additionally, Lovastatin may induce apoptosis by increasing p21 and the apoptosis induced can be prevented by the overexpression of Bcl-2 [32], suggesting a mitochondrial-related apoptosis. It is noted that this study by Schmidt et\u00a0al. fails to document that lovastatin can enhance death receptor (CD95)-mediated apoptosis in glioblastoma cells LN-18, LN-229, LN-308 and T98G [32]. The result from our study indicate that lovastatin in combination with TRAIL can synergistically induce apoptosis in A172, M059K and M059J glioblastoma cells. The apoptosis induced is associated with G0\u2013G1 arrest but not with pro-apoptotic Bid (data not shown). It is well known that TRAIL-induced apoptosis takes place via a death receptor-mediated pathway [5, 33].\nOur study showed that TRAIL-R1 mRNA expressed in M059J but was hardly detected in the other two cell lines. However, TRAIL-R1 protein was detectable in all three cell lines, suggesting that the TRAIL-R1 was modified by a post-translational mechanism in the cells tested. The level of TRAIL-R1 protein was higher in M059K cells treated with 20\u00a0\u03bcM of lovastatin. However, such an elevation of TRAIL-R1 is unlikely to be responsible for apoptosis induced by lovastatin because the apoptotic rate between M059K was not different from the other cell lines. TRAIL-R2 can be detected at both protein and RNA levels. The level of TRAIL-2 mRNA was not different in the cells treated with lovastatin but its protein level was much higher in A178 cells treated with either 5 or 10\u00a0mM lovastatin, the result of which indicated that TRAIL-R2 was modified by a post-translational mechanism in A172 cells. It is noted that the base level of TRAIL-2 in A172 cells does not differ from other two cell lines tested. Therefore, it is unlikely that TRAIL-2 could count for the relatively higher sensitivity of A172 cells to TRAIL stimulation than the other two cell lines. All three cell lines underwent a similar level of cell death when they were treated by lovastatin. Therefore, the contribution of lovastatin-induced TRAIL-2 protein to the cell death in A172 cells seems to be minimal. Such results appear to be in line with a study using mevastatin, a similar HMG-CoA reductase inhibitor. Mevastatin can significantly induce apoptosis of myeloma cells in a pathway independent of death receptors including TRAIL-R2 [34]. Nevertheless, further quantitative tests are needed in glioblastoma cells to verify the result obtained.\nApoptosis induced by lovastatin is generally considered to be via the mitochondrial-mediated pathway [30; 32]. It is possible for both pathways to talk to each other to amplify apoptotic signals and this is indeed a case for TRAIL, since TRAIL is capable of inducing either mitochondrial-independent or -dependent apoptotic pathways in some types of cells [17, 33]. The Bid is a molecule functions as a bridge that links death receptor- and mitochondrial-mediated pathways. Therefore, without involvement of Bid, apoptosis induced by TRAIL and lovastatin in combination in the present study seem to be separately mediated by these two pathways. However, considering the fact that two out of three glioblastoma cells tested are insensitive to TRAIL treatment but they become responsive in the presence of lovastatin, it can be hypothesized that lovastatin treatment may remove an unknown inhibitory factor(s) that overcomes the TRAIL-mediated pathway or that lovastatin may \u201cwake up\u201d an activator(s) that normally remains in a resting condition. One of inhibitory factors known to involve TRAIL-mediated pathways is its decoys, TRAIL-R3 and TRAIL-R4 [5, 33]. However, it can be seem from our study that this inhibitory factor(s), if any, should not be the decoys TRAIL-R3 and TRAIL-R4 since both are not detectable in the cells tested. One possible explanation for this synergistic effect is the activation of caspase 8, an initiator caspase in death receptor-mediated pathways. Lovastatin has been shown to enhance caspase 8 activity [35]. It is possible that caspase-8 is the activator factors waken up by lovastatin. Whatever it is, the mechanism accounting for the synergistic effect of TRAIL and lovastatin against glioblastoma cells in the present study is complicated and remains to be uncovered.\nMevalonate is a critical component of a complex biochemical pathway and its products are vital for a variety of important cellular functions including cell signaling, protein synthesis, and cell cycle regulation [11]. Little is known about the molecular events leading to apoptosis of cancer cells due to lovastatin exposure. It is likely that apoptosis is abrogated by mevalonate and GGPP and is partially reversed by FPP [12].\nOur results also reinforced the conclusion of our previous study, that DNA-dependent protein kinase (DNA-PK) plays an important role in cell apoptosis. M059J cells that lack of DNA-PK activity are resistant not only to total cell death but also apoptosis [19, 20]. Experts disagree over the functions of DNA-PK in cell regulation. DNA-PK has been reported to promote cell death by interacting with telomeres, whereas other reports suggest that DNA-PK protects cells from cell death via caspase-independent or p53 independent pathways. The reason for this paradoxical finding remains unknown.\nThis study demonstrated a synergistic interaction between lovastatin and TRAIL, but the mechanisms of action by which lovastatin sensitized glioblastoma cells remains unknown. Our results are in agreement with the concept of combined cancer therapeutic action via both intrinsic and extrinsic apoptotic cell death pathways. This combination of non-chemotherapeutic agents, TRAIL and lovastatin, may offer a potential regime for glioblastoma treatment.","keyphrases":["lovastatin","glioblastoma","apoptosis","tumor necrosis factor (tnf)-related apoptosis-inducing ligand (trail)"],"prmu":["P","P","P","P"]}
{"id":"Mol_Biochem_Parasitol-1-5-1964783","title":"TbARF1 influences lysosomal function but not endocytosis in procyclic stage Trypanosoma brucei\n","text":"The ADP ribosylation factors (Arfs) are a highly conserved subfamily of the Ras small GTPases with crucial roles in vesicle budding and membrane trafficking. Unlike in other eukaryotes, the orthologue of Arf1 in the host bloodstream form of Trypanosoma brucei is essential for the maintenance of endocytosis. In contrast, as shown in this study, knockdown of TbARF1 by RNA interference has no effect on fluid-phase endocytosis in the insect stage of the parasite. The protein remains essential for the viability of these procyclic cells but the major effect of TbARF1-depletion is enlargement of the lysosome. Our data indicate that protein trafficking and lysosomal function are differentially regulated by multiple factors, including TbARF1, during progression through the T. brucei lifecycle.\n1\nIntroduction\nThe secretory system plays a critical role in the efficient sorting and transport of a range of products to the surface of all eukaryotic cells. The small GTPase Arf1 is a vital component of this system, localizing to the Golgi apparatus in yeast and mammalian systems where it has highly conserved functions in vesicular assembly and the activation of phospholipid-modifying enzymes [1]. An exception is the orthologue of Arf1 (TbARF1) that has been recently characterized functionally in the bloodstream form (BSF) of the kinetoplastid parasite Trypanosoma brucei\n[2], a cell type which supports extremely rapid rates of internalisation and recycling of its major outer membrane protein, variant surface glycoprotein (VSG; [3]). Although TbARF1 localizes at or proximal to the Golgi apparatus in these parasites, the primary effect of TbARF1 knockdown is severe inhibition of the endocytic system, leading to cell death. In contrast, overexpression of a constitutively active GTP-bound form of TbARF1 inhibits protein trafficking from the Golgi apparatus to the lysosome [2].\nHere we show that the vital role of TbARF1 in endocytosis is not conserved throughout the T. brucei lifecycle. In the more sedentary insect procyclic form (PCF) of the parasite, ARF1 is required for cell viability but depletion of the protein by RNA interference (RNAi) has no effect on the uptake of dextran by fluid-phase endocytosis. Instead, the lysosome becomes enlarged, although degradation of the protein p67 within this organelle is not significantly impaired.\n2\nMaterials and methods\n2.1\nDisruption of TbARF1 expression by RNAi\nThe plasmid p2T7ARF1 [2] contains a non-conserved region of the T. brucei open reading frame (residues 101\u2013225) between two opposing T7 promoters under the control of tetracycline repressors. Mid-log phase parasites of T. brucei procyclic cell line 29\u201313 [4] were electroporated with 10\u00a0\u03bcg of NotI-digested p2T7ARF1 using methods described previously [5] to produce the stable cell line 29-13\/p2T7ARF1. Expression of ARF1-specific dsRNA was induced by incubating parasites in 100\u00a0ng\/ml tetracycline. Expression of TbARF1 was monitored by quantitative PCR, using SYBR Green Mastermix (Applied Biosystems). Total RNA was extracted from parasites using TRIzol Reagent (Invitrogen), treated with DNase I (Ambion) and reverse-transcribed using Omniscript RT (Qiagen). A 66\u00a0bp fragment of TbARF1 was amplified using SYBR Green Mastermix (Applied Biosystems) on a Prism7000 (Applied Biosystems) and compared to levels of a constitutively expressed control, \u03b1-tubulin. Oligonucleotides for amplification were: TbARF1 RTF1, 5\u2032-GGCTTCCGCTTTCAAATCC-3\u2032, TbARF1 RTR1, 5\u2032-CATCAAGGCCGACCATAAGAA-3\u2032, Tb\u03b1Tub RTF1, 5\u2032-CGTGAGGCTATCTGCATCCA-3\u2032 and Tb\u03b1Tub RTR1, 5\u2032-CCCAGCAGGCGTTACCAA-3\u2032.\n2.2\nMicroscopy\nIndirect immunofluorescence assays on parasites were performed as described [6]. Primary antibodies were gifts as follows: rabbit anti-TbGRASP from Graham Warren (Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA), mouse anti-p67 from Jay Bangs (Department of Medical Microbiology and Immunology, Madison, WI, USA). Primary antibodies were detected using Alexa-Fluor 488 or 633-conjugated secondary antibodies (Invitrogen). Samples were visualized by confocal microscopy using a Zeiss LSM 510 meta with a Plan-Apochromat 63x\/1.4 Oil DIC I objective lens and images acquired using LSM 510 version 3.2 software (Zeiss).\nFor transmission electron microscopy, cells were sequentially treated in 1% (w\/v) glutaraldehyde for 1\u00a0h, 1% (w\/v) tannic acid for 10\u00a0min, 0.5% (w\/v) osmium tetroxide for 45\u00a0min (all in 100\u00a0mM phosphate buffer), then in 1% (w\/v) aqueous uranyl acetate for 1\u00a0h. After dehydration in an acetone series, cells were embedded in Spurrs resin. Sections were cut on a Leica Ultracut, stained with saturated uranyl acetate in 50% ethanol and Reynolds lead citrate, and viewed with a Tecnai 12 BioTwin (FEI) at 120\u00a0kV. Images were acquired with a SIS MegaView III digital camera.\n2.3\nTrafficking assays\nTo monitor the uptake of dextran, cells were resuspended at 5\u00a0\u00d7\u00a0107\/ml in 1\u00a0mg\/ml Alexa-Fluor 488-conjugated dextran (Invitrogen) in SDM-79 medium and incubated for 20\u00a0min at 26\u00a0\u00b0C. For microscopy, parasites were then fixed in 4% paraformaldehyde and co-stained with DAPI, as described previously [7]. For FACS analysis, cells were fixed as above, then resuspended in PBS at a density of 5\u00a0\u00d7\u00a0107\/ml. Samples were then divided into two equal aliquots and anti-488 antibody (1:100, Invitrogen) added to one aliquot for 30\u00a0min at RT, before washing in PBS. Fluorescence was measured on a Dako Cytomation CyAn flow cytometer using the FL1 detector and results analyzed with Summit v4.1 software.\nER-to-lysosome trafficking and subsequent degradation of the protein p67 was analyzed by metabolic labelling of cells and immunoprecipitation as described previously [8].\n3\nResults\n3.1\nTbARF1 is essential for viability in T. brucei procyclic cells\nWe studied the functions of TbARF1 in PCF cells by knocking down expression using tetracycline-inducible RNAi. Detection of phenotypic effects arising from TbARF1 knockdown in these cells was considerably delayed in comparison to BSF parasites [2] in which cell death was evident by 24\u00a0h post-induction. This probably reflects a lower dependence on rapid protein trafficking in the PCF cell, as observed previously [9]. There was no significant decrease in PCF cell division until 90\u00a0h post-induction, when the cells started to become rounded and less motile, followed by cell death from 96\u00a0h (Fig. 1A, B). Quantitative PCR showed a significant loss of TbARF1-specific RNA by 16\u00a0h post-induction (Fig. 1C). Induced cells had no significant differences in nucleus and kinetoplast configurations between uninduced and induced cells over a 120\u00a0h time course (data not shown), indicating no major defects in the regulation of cell division.\n3.2\nLoss of ARF1 has no effect on fluid-phase endocytosis\nThe effects on fluid-phase endocytosis in these TbARF1-depleted procyclic cells were determined by analyzing the uptake of fluorescently labelled dextran. Analysis by microscopy showed internalization of dextran in both uninduced parasites and cells induced with tetracycline for 96\u00a0h (data not shown). We also used a novel FACS-based method to distinguish between external binding and internalization of dextran in these cells (Fig. 2A). Cells were incubated in the presence of Alexa Fluor 488-conjugated dextran, before fixing and incubation in anti-488 antibody which quenches the fluorescence associated with externally bound dextran. No significance differences were observed between uninduced and induced cells, either in total dextran fluorescence or internalized dextran alone (i.e. following antibody treatment) (Fig. 2A).\n3.3\nLoss of ARF1 causes an enlargement of the lysosome\nImmunofluorescence assays using the lysosomal marker, p67, showed that TbARF1-depleted procyclic parasites have an enlargement of the lysosome (Fig. 2B). Densitometric analysis of 20 cells per experimental group revealed that p67 staining in cells induced for 96\u00a0h covered an area approximately three times as large as in uninduced cells. No differences were seen in the size or position of the Golgi apparatus, as indicated by localization of the GRASP protein (Fig. 2B). The main defect visualized by transmission electron microscopy was the presence of a single electron-dense rosette-like structure near the flagellar pocket in a subset (30%) of cells by 72\u00a0h post-induction, before detection of a growth defect in these cells. This structure (Fig. 2C\u2013E) is composed of concentric layers of membrane and resembles that of membranous cytoplasmic bodies (MCBs) found in mammalian cells. These are lysosomal in origin and typically found in sphingolipidoses, a group of lysosomal storage diseases characterized by deficiencies in the lysosomal enzymes required for sphingolipid degradation [10]. Similar structures of mitochondrial origin have also been observed in T. brucei following depletion of the glycosomal PEX11-like protein GIM5B [11]. However, we found no obvious differences in mitochondrial structure in procyclic cells following the induction of TbARF1 RNAi, either by electron microscopy or fluorescent staining with Mitotracker (Invitrogen, data not shown). We therefore conclude that the structure detected in Fig. 2 is lysosomal in origin, although proof of this designation requires immuno-electron microscopy with a lysosomal probe.\n3.4\nEffects of ARF1 depletion on lysosomal trafficking and degradation of p67\nThe trafficking and degradation of p67 was analyzed by pulse-chase metabolic labelling of cells, followed by immunoprecipitation, as described previously [5,8]. In contrast to this process in BSF parasites, ER- synthesized p67 is not further modified in the Golgi in PCF cells, but proteolytic cleavage in the lysosome results in the generation of four glycosylated fragments: gp75, gp42, gp32 and gp28 [8]. In Fig. 2F, proteolytic fragments resulting from lysosomal degradation accumulated in uninduced and induced cells over the same time course, indicating that both trafficking of p67 and its subsequent degradation were unimpaired in cells depleted of TbARF1 protein (Fig. 2F).\n4\nDiscussion\nThe observations presented here and described previously [2] show that TbARF1 is essential for viability throughout the T. brucei lifecycle. However, the downstream effects of modulating expression of this protein differ between the two major life cycle stages. In the highly active bloodstream form, TbARF1 is required for both receptor-mediated and fluid-phase endocytosis, and depletion of the protein by RNAi causes a severe defect in these mechanisms, rapidly followed by cell death [2]. In contrast, procyclic cells significantly downregulate the expression of proteins associated with receptor-mediated endocytosis, including clathrin [12], and fluid-phase endocytosis is unaffected by the knockdown of TbARF1 expression. Rather than affecting the endocytic system, the absence of TbARF1 causes enlargement of the lysosome, indicating an accumulation of undegraded and\/or mistargeted material.\nAn interesting finding is that lysosomal targeting of p67 is not disrupted by knockdown of TbARF1 in either of the two life cycle stages but is inhibited by an increase in activated ARF1 protein in BSF parasites [2]. Lysosomal targeting in trypanosomes is not yet fully understood but must differ from higher eukaryotes in several respects. In mammals, most soluble lysosomal enzymes, including acid hydrolases, are sorted using mannose-6-phosphate signals [13] but there is no evidence to suggest that this mechanism is conserved in lower eukaryotes such as the kinetoplastids [8]. An alternative to the mannose-6 receptor, sortilin, has also been implicated in hydrolase sorting [13] but orthologues of this protein are not encoded by the T. brucei genome. The remaining class of identified signals, either tyrosine or dileucine-based motifs, are able to bind to GGA and AP adapter proteins, which then trigger the packaging of cargo for clathrin-mediated trafficking from the trans-Golgi network (TGN) to the lysosome. Both GGA and AP proteins are directly recruited to the Golgi apparatus by GTP-bound Arf1 [14]. However, genes encoding GGA proteins and AP-2 are absent from the T. brucei genome [2,15,16], implying either a greater dependence on the remaining AP-1 and AP-3 complexes or the operation of additional uncharacterized sorting mechanisms.\nMuch of our current knowledge of lysosomal targeting in T. brucei stems from studies on p67, a protein of unknown function that shares structural similarity but not sequence identity with the mammalian LAMP proteins [17]. This protein is trafficked from the ER via the Golgi to the lysosome, utilizing distinct targeting signals in different stages of the parasite life cycle. In PCF cells, the C-terminal cytoplasmic domain of p67 is necessary and sufficient for lysosomal targeting, whereas this region is not required in BSF cells [8]. While the cytoplasmic domain contains two putative dileucine motifs [8], a recent study has analyzed these regions functionally in PCF and demonstrated that both are required to support maximal targeting [18]. It is likely that p67 is sorted in PCF cells via binding to either AP-1 or AP-3 at the TGN, a process that could be inhibited by knockdown of ARF1. While the data presented here suggest that p67 continues to be targeted efficiently for degradation in the lysosome, this does not preclude a defect in the sorting or trafficking of other essential lysosomal factors.\nIn BSF parasites, the AP complexes may not be directly involved in p67 sorting, given the redundancy of the dileucine motifs in this life cycle stage [8]. As in PCF cells, knockdown of TbARF1 does not prevent p67 localization to the lysosome [2]. However, in contrast, the inducible expression of a constitutively active form of TbARF1 does not inhibit endocytosis but prevents p67 from reaching the lysosome, resulting in extremely rapid cell death [2]. In this situation, excess ARF1 may be sequestering effector proteins required for alternative pathways which would normally facilitate the correct localization of p67.\nGiven that forward biosynthetic trafficking to the lysosome is not quantitatively impaired in the absence of ARF1, this protein may alternatively influence the recycling of membranes from the lysosome, a defect that might generate multivesicular-type structures such as those observed at higher resolution in Fig. 2. Further studies are required to pinpoint the relationship between TbARF1 and lysosomal function, analysis which may reveal insights into the secretory pathways of lower eukaryotes.","keyphrases":["arf1","lysosome","endocytosis","trypanosoma brucei","arf, adp ribosylation factor","bsf, bloodstream form","pcf, procyclic form","rnai, rna interference","t. brucei, trypanosoma brucei"],"prmu":["P","P","P","P","R","R","R","R","R"]}
{"id":"Behav_Genet-4-1-2226021","title":"Evaluation of the Serotonergic Genes htr1A, htr1B, htr2A, and slc6A4 in Aggressive Behavior of Golden Retriever Dogs\n","text":"Aggressive behavior displays a high heritability in our study group of Golden Retriever dogs. Alterations in brain serotonin metabolism have been described in aggressive dogs before. Here, we evaluate whether four genes of the canine serotonergic system, coding for the serotonin receptors 1A, 1B, and 2A, and the serotonin transporter, could play a major role in aggression in Golden Retrievers. We performed mutation screens, linkage analysis, an association study, and a quantitative genetic analysis. There was no systematic difference between the coding DNA sequence of the candidate genes in aggressive and non-aggressive Golden Retrievers. An affecteds-only parametric linkage analysis revealed no strong major locus effect on human-directed aggression related to the candidate genes. An analysis of 41 single nucleotide polymorphisms (SNPs) in the 1 Mb regions flanking the genes in 49 unrelated human-directed aggressive and 49 unrelated non-aggressive dogs did not show association of SNP alleles, genotypes, or haplotypes with aggression at the candidate loci. We completed our analyses with a study of the effect of variation in the candidate genes on a collection of aggression-related phenotypic measures. The effects of the candidate gene haplotypes were estimated using the Restricted Maximum Likelihood method, with the haplotypes included as fixed effects in a linear animal model. We observed no effect of the candidate gene haplotypes on a range of aggression-related phenotypes, thus extending our conclusions to several types of aggressive behavior. We conclude that it is unlikely that these genes play a major role in the variation in aggression in the Golden Retrievers that we studied. Smaller phenotypic effects of these loci could not be ruled out with our sample size.\nIntroduction\nDogs have been living in close proximity to humans for at least 15,000\u00a0years (Clutton-Brock 1995). Behavior has been a strong selective factor in the domestication and breeding of dogs. According to the breed standard, Golden Retriever dogs should have a friendly character (http:\/\/www.goldenretrieverclub.nl; link accessed March 2007). However, there are reports of very aggressive Golden Retrievers (Galac and Knol 1997; Heath 1991). We recently described the behavioral phenotype of 110 Golden Retrievers referred to our clinic for aggression problems and 118 Golden Retrievers that were recruited because they were related to one or more of the aggressive dogs (van den Berg et\u00a0al. 2006). The phenotypes were based on mail questionnaires and on personal interviews with dog owners. In a quantitative genetic study including 325 Golden Retrievers, we found a heritability of 0.8 for the traits of human-directed aggression and dog-directed aggression (Liinamo et\u00a0al. 2007).\nThe influence of serotonin (5-hydroxytryptamine, 5-HT) on aggressive behavior has been studied extensively (reviewed by Berman and Coccaro 1998; Gingrich and Hen 2001; Lesch and Merschdorf 2000). There is evidence for a role of the 5-HT system in canine aggression as well. For instance, Reisner and colleagues (1996) reported decreased levels of 5-hydroxyindoleacetic acid (the major metabolite of 5-HT) in cerebrospinal fluid of dominant aggressive dogs. Badino et\u00a0al. (2004) found modifications of 5-HT receptor concentrations in brains of aggressive dogs. Domestication of silver foxes, which are taxonomically close relatives of dogs, seems to cause modifications in the 5-HT system (see Trut 2001 for a review). The role of 5-HT in canine aggression is further supported by two small clinical studies, where pharmacological or dietary intervention in the 5-HT system was shown to modulate aggressive behavior (DeNapoli et\u00a0al. 2000; Dodman et\u00a0al. 1996).\nFour genes that code for factors involved in serotonergic neurotransmission are particularly good candidates for the regulation of aggressive behavior: the serotonin receptor genes 1A (htr1A), 1B (htr1B), and 2A (htr2A), and the serotonin transporter gene (slc6A4). Serotonin receptor 1A plays a role in anxiety, stress response, and aggression (Olivier et\u00a0al. 1995). Htr1A knockout mice show increased anxiety and stress response and an antidepressant-like phenotype (Heisler et\u00a0al. 1998; Ramboz et\u00a0al. 1998). In the above-mentioned studies of silver foxes, the researchers observed a lower density of 5-HT1A receptors in the hypothalamus of tame foxes compared to their wild counterparts (Popova et\u00a0al. 1991). Many studies have suggested involvement of htr1B in the etiology of mental disorders. For instance, Huang et\u00a0al. (2003) and Sanders et\u00a0al. (2002) reported an association between one of the polymorphisms in the human HTR1B gene and alcoholism, suicidality, and obsessive-compulsive disorder. Knockout mice lacking htr1B display increased aggression (Saudou et\u00a0al. 1994). A mutation in the human HTR2A gene is associated with altered 5-HT binding, which has been implicated in schizophrenia, suicidal behavior, impaired impulse control, and aggression history (Abdolmaleky et\u00a0al. 2004; Bjork et\u00a0al. 2002; Khait et\u00a0al. 2005). Peremans and colleagues (2003) found an increased binding index of serotonin 2A receptors in cortical brain regions of impulsive aggressive dogs. A polymorphism in the promoter region of SLC6A4 influences serotonin transporter density in the brain and is associated with mental disorders in humans (Anguelova et\u00a0al. 2003; Hariri et\u00a0al. 2002; Lesch et\u00a0al. 1996). Slc6A4 knockout mice show reduced aggression (Holmes et\u00a0al. 2003).\nIn this paper, we test the hypothesis that there is a strong effect of variation in these genes on the variation in aggression in Golden Retrievers. We performed mutation screens of the coding DNA sequence in unrelated aggressive Golden Retrievers. In addition, we used linkage analysis to determine the likelihood of the presence of a strong aggression locus in or close to the genes in several dog families. Third, we used 50 unrelated aggressive Golden Retrievers and 50 unrelated non-aggressive Golden Retrievers to search for association of alleles of 41 SNPs flanking the candidate genes with the trait of human-directed aggression. To complete our analyses, we evaluated the effects of variation in the genes on a range of aggression-related phenotypes using the same models as in Liinamo et\u00a0al. (2007), extended to include the effects of the most common candidate gene haplotypes.\nMaterials and methods\nAnimals, DNA isolation, and phenotyping\nWe have collected behavioral information of 328 privately owned Golden Retrievers. This group includes 162 dogs that were referred to our clinic because of their aggressive behavior (\u201cprobands\u201d) and 166 relatives of 36 probands. DNA samples were available for 281 of these dogs. In addition, we collected DNA of a cohort of random privately owned Goldens that were born between July 2002 and February 2003. No phenotypes were available for these random dogs. Genomic DNA was isolated from whole blood leucocytes using a standard protocol (Miller et\u00a0al.1988). For each type of analysis (mutation analysis, linkage analysis, association analysis, and quantitative genetic analysis) we selected a study group that was suited for the study design. The study groups are described in the Supplementary Information I.\nWe have collected various quantitative measures of aggressiveness for the dogs (van den Berg et\u00a0al. 2003a, 2006). In the linkage and association analysis we focused on one of these measures: the dog owners impression on human-directed aggression. Owner impressions were collected in a personal interview. We asked the owners if their dog was aggressive towards humans and the status of the dog was coded in three classes: non-aggressive (score 1), threatens (score 2), or bites (score 3). We focused on human-directed aggression because the majority of the probands were referred to our clinic for human-directed aggressive behavior. Owner impressions were available for all dogs and the quantitative genetic analyses showed that the heritability of this trait was high in our population of dogs (Liinamo et\u00a0al. 2007).\nMutation screening\nWe analyzed the coding DNA sequence (CDS) of the four candidate genes in seven (htr1A and htr1B) or eight (htr2A and slc6A4) probands. The CDS were amplified and sequenced using overlapping primer pairs as described previously (van den Berg et\u00a0al. 2004, 2005). Possible functional effects of polymorphisms were predicted with POLYPHEN (http:\/\/www.genetics.bwh.harvard.edu\/cgi-bin\/pph\/polyphen.cgi). Effects of polymorphisms close to splice sites were predicted with three splice prediction programs: NetGene2 (Brunak et\u00a0al. 1991), Splice Prediction by Neural Network (Reese et\u00a0al. 1997), and SpliceSiteFinder (Shapiro and Senapathy 1987).\nLinkage analysis\nWe used nine families for linkage analysis (Figs. S1\u2013S9 of Supplementary Information I). DNA samples were available for 31 affected and 65 unaffected dogs from these families. We converted the owner impression about human-directed aggression into a dichotomous variable for the linkage analysis (see Supplementary Information I). We have described polymorphic markers for the candidate genes before (van den Berg et\u00a0al. 2003b, 2004, 2005). We selected three microsatellite markers and seven single nucleotide polymorphisms for linkage analysis (see Table\u00a01). Microsatellite markers were genotyped after PCR on an ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). PCR conditions were described by van den Berg et\u00a0al. (2004, 2005). GENESCAN 3.7 software was used for genotype assessment. Single nucleotide polymorphism genotyping was performed by DNA sequencing of PCR products on the ABI 3100 Genetic Analyzer (van den Berg et\u00a0al. 2004, 2005). The DNA sequence chromatograms were inspected using LASERGENE software (DNASTAR, Inc., Madison, WI USA). We combined several markers into haplotypes for the genes htr1B, htr2A, and slc6A4. For dogs that were heterozygous for multiple markers, we deduced the haplotypes from the data of relatives. If this was not possible, we assigned the most frequently observed possible haplotypes to these dogs.\nTable\u00a01Markers used for linkage analysisGeneaType of markerbPosition of markercAlleles observed (allele frequency)dHaplotypes observed (haplotype frequency)dhtr1A(CA)n(UU160O12)*7370297 (0.5)303 (0.5)\u2013htr1BA\/C SNPe157A (0.58)C (0.42)143-A-G-T-G (0.20)143-C-A-T-G (0.42)143-A-G-C-C (0.06)139-A-G-C-C (0.24)139-A-G-T-G (0.05)147-A-G-T-G (0.03)\nG\/A SNP246G (0.58)A (0.42)T\/C SNP955T (0.69)C (0.31)G\/C SNP1146G (0.69)C (0.31)(GA)n(UU18L8)\u221268395139 (0.29)143 (0.68)147 (0.03)htr2AC\/T SNPIVS 2-10C (0.85)T (0.15)128-C (0.07)130-C (0.41)132-C (0.37)132-T (0.15)(CA)n(UUHTR2AEX2)IVS2\u00a0+\u00a01439128 (0.07)130 (0.41)132 (0.52)slc6A4C\/T SNP411C (0.75)T (0.25)C-G (0.75)T-A (0.25)G\/A SNPIVS9-12G (0.75)A (0.25)ahtr1A, htr1B, htr2A\u00a0=\u00a0respectively serotonin receptor 1A, 1B, and 2A gene; slc6A4\u00a0=\u00a0serotonin transporter genebSNP\u00a0=\u00a0single nucleotide polymorphism. Names of microsatellite markers have been included in bracketscPosition refers to the coding sequence of the canine gene. We used the nomenclature recommended by den Dunnen and Antonarakis (2001): the A of the ATG start codon is designated number 1, the nucleotide 5\u2032 to this A is numbered \u22121, and the nucleotide 3\u2032 of the translation termination codon is *1. Positions in introns refer to the nearest exon. The nomenclature of the introns is based on the human gene structure. IVS\u00a0=\u00a0intervening sequencedAllele and haplotype frequencies were determined in a group of 27 (htr1A and htr2A), 31 (htr1B), or 26 (slc6A4) parentseThis polymorphism is nonsynonymous\nWe performed a parametric affecteds-only linkage analysis to determine whether the candidate gene haplotypes were linked to aggressive behavior in the Golden Retriever families. Marker haplotype frequencies were determined in a group of 27 (htr1A and htr2A), 31 (htr1B), or 26 (slc6A4) parent dogs (see Supplementary Information I). The mode of inheritance of the aggressive phenotype in our families is unclear. We therefore analyzed the data under both autosomal dominant and autosomal recessive models. The penetrance of the genotype at risk was set at 0.01. In this way, affected dogs are assumed to have the risk allele and the software calculated likelihood that aggressive dogs share alleles by descent from a common ancestor. Unaffected dogs with the genotype at risk have no effect on the outcome of the calculation. We assumed that there were no phenocopies in the families and we assumed genetic homogeneity because all probands were related to each other within a limited number of generations (not shown). The frequency of the aggression allele was set at 0.1 to allow for multiple transmitting ancestors in the pedigrees. SUPERLINK software was used to calculate two-point logarithm of the odds (LOD) scores (Fishelson and Geiger 2002, 2004).\nIn order to estimate the power of the pedigrees, we calculated the maximum obtainable LOD scores. Affected individuals were assigned haplotypes 2\/2 in these calculations; unaffected parents were assigned haplotypes 1\/2; and other unaffected individuals were assigned haplotypes 1\/1. We assigned haplotypes 0\/0 (unknown) to dogs from which we did not have a DNA sample. We assumed that there were four alleles of the hypothetical marker with equal allele frequencies.\nAssociation study\nTo test for a more complex genetic effect of variants of the candidate genes, we performed an association study. Fifty aggressive Golden Retrievers were selected from our database. The main selection criteria were high estimated breeding values for human-directed aggression and as little interrelationship among the cases as possible. Non-aggressive dogs were selected for low estimated breeding values for both human- and dog-directed aggression. We avoided an excess of relationship within either the case or the control group. The non-aggressive group was completed with 25 dogs from the random group. A more detailed description of cases and controls is provided in Supplementary Information I.\nWe genotyped a total number of 60,073 SNPs in these 100 Golden Retrievers using customized Affymetrix Genotyping Arrays. The SNPs were selected for the chip using a scoring system that optimized the SNPs accounting for low repeat content, low likelihood of SNPs in the assay probe sequences and their distribution over the genome as a whole (Lindblad-Toh 2007; personal communication). Twenty 32-mer probes interrogated each locus with genotyping calls made using the algorithm BRLMM (http:\/\/www.affymetrix.com) which analyses intensities for sets of probes that interrogate both forward and reverse sequences with perfect match and mismatched probes. Dogs with call rates of lower than 50% were discarded from the analysis. The total set of SNPs was filtered for genotype call probability, heterozygosity rate, and call rate across a large set of dogs, reducing the dataset to 26,625 SNPs. From this set, only SNPs within 1\u00a0Mb of the four candidate genes were used for the analysis described in this paper. There were 29 SNPs within 1\u00a0Mb of htr1A, 20 within 1\u00a0Mb of htr1B, 25 within 1\u00a0Mb of htr2A, and 20 within 1\u00a0Mb of slc6A4. From these SNPs, we selected 43 SNPs with a minor allele frequency >0.05 and call rates of >0.75.\nWe used Haploview software version 4.0 (Barrett et\u00a0al. 2005) for the analysis of the presence of Hardy-Weinberg equilibrium (HWE), the local association analysis, and the calculation of pairwise linkage disequilibrium (LD) between the SNPs. A HWE P-value cutoff of 0.001 was used. We used Bonferonni correction to account for multiple testing in the association analysis. Genotype frequencies in cases and controls were compared with Chi square tests using SPSS software. Two tailed Fisher\u2019s exact tests were used when the number of expected cases was less than 5 in more than 20% of the categories. Haplotype blocks were formed using three methods in Haploview (confidence intervals, four gamete rule, and solid spine of LD). For all possible combinations we performed 10,000 permutations to obtain empirical P values for haplotype association tests. We used the genetic power calculator prepared by Purcell to estimate the power of the association analysis (http:\/\/www.pngu.mgh.harvard.edu\/~purcell\/gpc\/). The following assumptions were made: high-risk allele frequency\u00a0=\u00a00.1; prevalence\u00a0=\u00a00.01. The mean pairwise D\u2032 between the SNPs flanking a candidate gene was used as an estimate of the local D\u2032 in these estimations. Calculations were performed for two different genotype relative risks: 2 (genotype relative risk Aa\u00a0=\u00a02; genotype relative risk AA\u00a0=\u00a04) and 5 (genotype relative risk Aa\u00a0=\u00a05; genotype relative risk AA\u00a0=\u00a010).\nQuantitative genetic analysis\nIn addition to the owner impression on human-directed aggression and dog-directed aggression, we collected a variety of other aggression-related behavioral measures using the canine behavioral assessment and research questionnaire (CBARQ; Hsu and Serpell 2003). As described in Liinamo et\u00a0al. (2007), these measures were of three types: original CBARQ items (27 items on the aggressiveness of the dog in various everyday situations), shortened CBARQ scores (scores based on questions that addressed stranger-directed, owner-directed, and strange dog-directed aggression), and CBARQ factors (scores based on questions about stranger-directed, owner-directed, and strange dog-directed, and familiar dog-directed aggression). For further explanation of the measures and the difference between shortened CBARQ scores and CBARQ factors, see Liinamo et\u00a0al. (2007).\nThe effects of the haplotypes of the serotonergic genes on the different aggression measures were estimated with Restricted Maximum Likelihood (REML) method (Patterson and Thompson 1971), using univariate analyses and an animal model with the VCE4.2.4 software (Groeneveld 1997). The analyses were an extension of the analyses outlined in Liinamo et\u00a0al. (2007), using similar linear animal model methodology, but this time also including the haplotype classes of the dogs for the four studied loci as additional fixed effects in the model. For instance, the linear animal model that was assumed in the analyses for owner impression traits was: where yijklmnois the observed value for the owner impression score for animal o;\u03bc the general mean in the population; sexithe fixed effect of the reproductive status (i\u00a0=\u00a01\u20134, with 1\u00a0=\u00a0intact male, 2\u00a0=\u00a0castrated male, 3\u00a0=\u00a0intact female, and 4\u00a0=\u00a0castrated female); agej the fixed effect of the age j (j\u00a0=\u00a01\u201311, with 1\u00a0=\u00a00.5\u20131\u00a0year old, 2\u00a0=\u00a01\u20132\u00a0years old,\u2026,10\u00a0=\u00a09\u201310\u00a0years old, and 11\u00a0=\u00a0over 10\u00a0years old); htr1ak , htr1bl, htr2am, and slc6a4n the fixed effects of the respective haplotype classes, aothe random additive genetic effect (i.e., polygenic breeding value) of the animal o, and eo the random residual effect related to the animal o. The age and reproductive status of the dogs had been recorded at the same time as the owner impressions. The haplotype classes were formed so that the most common haplotypes formed separate classes, the very rare haplotypes were all combined in one class, and the unknown haplotypes were classified as a separate class (see Table\u00a04).\nResults\nMutation screening of the coding DNA sequence\nThe coding DNA sequence of each candidate gene was scanned for mutations in seven (htr1A and htr1B) or eight (htr2A and slc6A4) aggressive Golden Retrievers. Analysis of the CDS in non-aggressive Golden Retrievers has been described by van den Berg et\u00a0al. (2004; 2005). There was no variation in the CDS of htr1A and htr2A in the Golden Retrievers. We observed five SNPs in the CDS of htr1B and one SNP in the CDS of slc6A4. The allele distribution of these SNPs in the two groups of Golden Retrievers did not indicate a role in aggressive behavior. In conclusion, there seems to be no systematic difference between the CDS of the candidate genes in aggressive and non-aggressive Golden Retrievers.\nLinkage analysis\nWe observed two alleles for htr1A marker UU160O12 (Table\u00a01). The four SNPs in htr1B were fully in LD (D\u2032\u00a0=\u00a01). The SNPs displayed six haplotypes in the Goldens, three of which were rare (frequency <0.1). We detected four haplotypes of htr2A in the Golden Retrievers. The two SNPs in slc6A4 were fully in LD and formed two haplotypes in the dogs. In the nine families that we used for linkage analysis, haplotypes were deduced with certainty in 86% of the dogs for htr1B, 100% for htr2A, and 87% for slc6A4. We calculated the maximum achievable LOD score using hypothetical genotypes. The maximum LOD score generated by our pedigrees was 2.8 at recombination fraction \u03b8\u00a0=\u00a00 assuming a dominant mode of inheritance (Table\u00a02). Under a recessive model, the maximum LOD score was 5.3 at \u03b8\u00a0=\u00a00. The families are therefore theoretically powerful enough to prove linkage under a recessive model and powerful enough to provide a good indication of the presence of linkage under a dominant model. There was no significant linkage of any of the candidate genes with the aggressive phenotype (Table\u00a02). LOD scores varied from \u22121.0 to +0.26 assuming dominant inheritance and from \u22122.3 to \u22120.30 assuming recessive inheritance. The highest LOD scores were obtained for htr1A (+0.26 under a dominant model and \u22120.30 under a recessive model).\nTable\u00a02Results from the ODDS (LOD) scoresaGeneAutosomal dominant Autosomal recessivehtr1A0.26\u22120.30htr1B\u22120.72 \u22122.3htr2A\u22121.0 \u22122.1slc6A40.030\u22121.2Maximum2.85.3aLOD scores were calculated with the following assumptions: frequency of the \u201caggression allele\u201d\u00a0=\u00a00.1; penetrance of the \u201caggression allele\u201d\u00a0=\u00a00.01; \u03b8\u00a0=\u00a00. Marker haplotype frequencies were deduced from a group of parents\nAssociation study\nTwo dogs (one case and one control) were discarded from the association analysis because they had call rates lower than 50%. Mean call rates in the other 98 dogs were 92% for SNPs flanking htr1A, 94% for htr1B, 93% for htr2A, and 93% for slc6A4. There were 43 SNPs with a minor allele frequency >0.05 and call rates of >0.75 that occurred within 1\u00a0Mb of the candidate genes. The genotype frequencies of BICF2P1093362 for htr1B and BICF2P969902 for slc6A4 deviated from HWE in control dogs (P\u00a0=\u00a04.42E-14 and P\u00a0=\u00a05.242E-12, respectively). All but one dog in both case and control group had heterozygous genotypes for these SNPs. We concluded that the data for these two SNPs was artefactual and they were excluded from further analyses. The observed genotype frequencies of the other 41 SNPs were in HWE in controls (P values are listed in Table S2 in Supplementary Information II). The final SNP set used for the association analysis consisted of 12\u00a0SNPs flanking htr1A, 11 flanking htr1B, 8 flanking htr2A, and 10 flanking slc6A4 (Table\u00a03).\nTable\u00a03Single nucleotide polymorphisms (SNPs) used for the association study, their allele frequencies in 49 aggressive cases and 49 control dogs, and results of chi-square tests for comparisons of case and control allele frequenciesSNP nameaChromosomal locationChromosomal position (Mb)bMinor allele frequency in controlsCorresponding allele frequency in cases\u03c72P-valueBICF2P546848251.800.290.300.0440.83BICF2P1051894251.960.0730.0281.60.21BICF2P1398268252.010.330.330.0110.92BICF2S23127755252.140.230.210.0780.78BICF2P1200391252.220.300.330.210.64BICF2P590055252.350.240.270.190.66BICF2S22939125252.440.480.530.540.46BICF2S23215863252.480.300.310.0250.87BICF2P25993252.730.490.450.190.66BICF2S23442706252.760.190.281.80.18Htr1A252.88\u201352.88\u2013\u2013\u2013\u2013BICF2P519607253.220.270.240.170.68BICF2P1341930253.620.490.540.520.47BICF2P11592411241.110.120.130.0120.91BICF2P5551301241.590.130.212.10.15BICF2S233262291241.630.240.321.30.25Htr1B1241.65\u201341.66\u2013\u2013\u2013BICF2P6703311241.800.150.232.20.14BICF2S231537601241.840.0390.123.20.073BICF2P14265221242.290.120.120.0130.91BICF2P275711242.340.120.100.210.65BICF2S234440661242.390.120.110.0320.86TIGRP2P164447_rs88059861242.510.0380.123.60.059BICF2P8121531242.570.0330.124.80.029BICF2P8554021242.620.0210.126.90.0086BICF2G630315581226.3830.100.140.830.36BICF2G630315746226.6110.100.140.450.50BICF2P1168502226.9730.100.110.00200.96Htr2A227.395\u20137.453\u2013\u2013\u2013\u2013BICF2P164280227.5090.430.542.00.16BICF2G630316047227.7090.440.531.70.19BICF2S23125159227.8660.190.210.0470.83BICF2S23661838228.2070.310.360.590.44BICF2S22954191228.4700.230.270.350.55BICF2P813837946.930.390.301.50.22BICF2S23018060947.060.330.370.220.64BICF2S23551918947.460.340.380.240.62Slc6A4947.55\u201347.57\u2013BICF2S23325050947.640.110.150.530.47BICF2S23124809947.790.180.220.380.54BICF2P950384947.880.110.120.0320.86BICF2S245135948.020.270.250.0740.79BICF2S2347312948.090.220.280.840.36BICF2S23154457948.130.310.241.20.28BICF2S23141984948.260.360.360.00700.93ahtr1A, htr1B, htr2A\u00a0=\u00a0respectively serotonin receptor 1A, 1B, and 2A gene; slc6A4\u00a0=\u00a0serotonin transporter gene. The genes are included in the table to show their position relative to the SNPsbSNP positions are based on the second version of the dog genome assembly, released in May 2005 (CanFam2.0) as can be viewed on \nhttp:\/\/www.broad.mit.edu\/ftp\/pub\/papers\/dog_genome\/snps_canfam2\/Positions of the genes are based on the second version of the dog genome assembly (CanFam2.0) as displayed in NCBI Map Viewer for Canis familiarishttp:\/\/www.ncbi.nlm.nih.gov\/mapview\/map_search.cgi?taxid=9615\nThe power of our association analysis depends on the local extent of LD. The mean r2 between the SNPs flanking htr1A was 0.36 and the mean D\u2032 between these SNPs was 0.89. Mean r2 values were 0.23, 0.25, and 0.21 for htr1B, htr2A, and slc6A4, respectively. Mean D\u2032 values were 0.80, 0.72, and 0.88 for htr1B, htr2A, and slc6A4, respectively. When the marker allele frequency is 0.1, the power to detect a variant with a relative risk of 5 with 49 cases and 49 controls would be 0.91 for htr1A, 0.85 for htr1B, 0.78 for htr2A, and 0.90 for slc6A4. Additional power estimations are provided in Fig.\u00a0S10 in Supplementary Information II.\nThe allele frequencies of the SNPs did not differ significantly between cases and controls after correction for multiple testing (Table\u00a03). Genotype frequencies also did not display significant differences between cases and controls (see Table\u00a0S2 in Supplementary Information II). We also analyzed the association of haplotypes with the phenotype. No significant associations were found (data not shown). In conclusion, there seemed to be no association between alleles, genotypes or haplotypes of SNPs that flank the candidate genes and human-directed aggression in the Golden Retrievers.\nQuantitative genetic analysis\nWe completed our analyses with a study of the effect of variation in the candidate genes on a collection of aggression-related phenotypic measures. The haplotype effects were studied on owner impressions on human- and dog-directed aggression, the original CBARQ items related to stranger- and owner-directed aggression, the shortened CBARQ scores and the CBARQ factors. The haplotypes did not have a significant effect on any of the studied measures, i.e. the heritability estimates of the measures remained similar to the results presented in Liinamo et\u00a0al. (2007) in spite of incorporation of the haplotypes in the mixed model. The results for owner impressions on human- and dog-directed aggression, which are the most reliable estimates due to the largest number of observations, are presented in Table\u00a04. In conclusion, the large genetic variability between the dogs could not be explained by the serotonergic genes studied in this paper.\nTable\u00a04The effects of the studied genotypes (htr1A) or haplotypes (htr1B, htr2A, and slc6A4) on owner impressions of human-directed aggression and dog-directed aggression in 320 dogs Genotype or haplotype classaNumber of animalsEffect relative to class \u2018unknown\u2019Human-directed aggressionDog-directed aggressionhtr1AUnknown440.000.00297\/297740.0280.11297\/3031110.240.054297\/305150.0120.089303\/303620.180.085303\/30510\u22120.11\u22120.067Other4\u22120.046\u22120.17htr1BUnknown1660.000.00143-A-G-T-G\/143-A-G-T-G20\u22120.730.28143-A-G-T-G \/143-C-A-T-G8\u22120.63\u22120.19143-A-G-T-G \/139-A-G-C-C190.110.19143-C-A-T-G\/143-C-A-T-G41\u22120.170.13143-C-A-T-G \/143-A-G-C-C10\u22120.0210.059143-C-A-T-G \/139-A-G-C-C300.00060.32139-A-G-C-C\/139-A-G-C-C90.240.11Other170.150.36htr2AUnknown1140.000.00132-C\/132-C40\u22120.15\u22120.19132-C\/130-C60\u22120.27\u22120.023132-C\/132-T28\u22120.220.073132-C\/128-C15\u22120.38\u22120.43130-C\/130-C21\u22120.33\u22120.072130-C\/132-T220.10\u22120.19130-C\/128-C9\u22120.52\u22120.29Other110.33\u22120.036slc6A4Unknown1030.000.00C-G\/C-G1160.025\u22120.23C-G\/T-A940.14\u22120.23Other70.079\u22120.10None of the effects was significantaNote that there are additional alleles compared to Table\u00a01 as a result of the larger study group. The htr1A class \u201cother\u201d contains genotypes 295\/297 (n\u00a0=\u00a02 dogs) and 305\/305 (n\u00a0=\u00a01 dog). For htr1B, the class \u201cother\u201d contains haplotypes 143-A-G-T-G\/143-A-G-C-C (n\u00a0=\u00a03 dogs), 143-A-G-T-G\/139-A-G-T-G (n\u00a0=\u00a01 dog), 143-A-G-T-G\/147-A-G-T-G (n\u00a0=\u00a02 dogs), 143-A-G-C-C \/139-A-G-C-C (n\u00a0=\u00a03 dogs), 143-A-G-C-C\/147-A-G-T-G (n\u00a0=\u00a01 dog), 139-A-G-C-C \/139-A-G-T-G (n\u00a0=\u00a03 dogs), and 143-C-A-T-G\/139-A-G-T-G (n\u00a0=\u00a04 dogs). For htr2A, \u201cother\u201d contains haplotypes 0\/130-C (n\u00a0=\u00a02 dogs), 0\/132-T (n\u00a0=\u00a01 dog), 132-T \/132-T (n\u00a0=\u00a06 dogs), and 132-T\/128-C (n\u00a0=\u00a02 dogs). For slc6A4, \u201cother\u201d contains C-G\/T-G (n\u00a0=\u00a01 dog) and T-A\/T-A (n\u00a0=\u00a06 dogs). Unknown genotypes and haplotypes are the result of failure of genotyping or of the absence of a DNA sample (n\u00a0=\u00a044 dogs)\nDiscussion\nWe collected behavioral information and DNA samples of 281 dogs over a period of 10\u00a0years. Dogs were selected from this collection to evaluate four genes involved in serotonin metabolism by four methods: DNA sequence analysis of the coding region of the genes, genetic linkage analysis, genetic association analysis, and quantitative genetic analysis. The results indicate that it is unlikely that there is a major locus effect of one of the genes on aggression in the Golden Retrievers that we studied.\nThe genetic study of the variation of aggression in Golden Retrievers is a promising tool to identify the molecular systems involved in aggression. The relative ease to find disease loci in the dog genome compared to the human genome is the result of the population structure of dog breeds. Within a breed, Lindblad-Toh and colleagues (2005) observed a limited number of common haplotypes per genomic region. In addition, LD in dog breeds extends over at least 50-fold greater distances than in human populations. These characteristics make the dog highly suited for molecular genetic studies of complex traits (Sutter and Ostrander 2004).\nWe did not detect mutations in the CDS of the genes specific for aggressive dogs. All SNPs except A157C in htr1B were synonymous. This A157C variation, resulting in an isoleucine\/leucine polymorphism of amino acid 53, was predicted to be functionally insignificant by POLYPHEN. The genes htr2A and slc6A4 contain SNPs close to splice sites (at position IVS2-10 and IVS9-12, respectively) that could theoretically affect splicing. However, the polymorphisms did not have a large effect on splice site prediction by three software programs. We performed mutation screening in a limited number of dogs and it is possible that we have missed rare alterations in the genes. Apart from this limitation, we conclude that there is no common variant acting on protein structure that contributes to the variation in aggression in our Golden Retriever sample.\nWe used linkage analysis to evaluate the likelihood that there is a major aggression-influencing variant in the chromosomal regions surrounding the coding exons. The affecteds only parameters that we used in the calculations are a simplification with the assumption that all affected dogs of a family have the genotype at risk but unaffected dogs can have any genotype at the aggression locus. A LOD score of 3 is usually considered as evidence for linkage, whereas LOD scores below \u22122 exclude the gene. A power calculation with simulated genotypes was not feasible in this study due to the complexity of some pedigrees with multiple loops. Instead, we calculated the maximum achievable LOD scores to get an impression of the power embodied in the pedigrees. We assumed full informativeness of the markers in these calculations, but in reality, we expect the markers to have limited informativeness. Realistic obtainable LOD scores would then be lower than the maximum values that we presented in Table\u00a02. These LOD scores are too low to obtain significant results, but they provide a means to set the obtained results into perspective. The LOD scores for htr1B and htr2A were low compared to the maximum obtainable scores. A major role of these genes is unlikely. The results for htr1A and slc6A4 are less conclusive. This is probably the result of the poor informativeness of the markers. For both genes, we observed only two alleles or haplotypes with high frequencies. Typing of additional markers might help to definitively exclude the genes. However, in the light of the observed low level of variation it is unlikely that htr1A and slc6A4 have a strong effect on aggression in the Golden Retriever families.\nOur linkage analysis does not account for genetic heterogeneity or phenocopies. We have thus only tested for a very strong major locus effect. In reality, the aggressiveness in the Golden Retrievers may be more complex. We therefore used a third study design to investigate the candidate genes: association analysis. For this analysis, we used data from a large-scale genotyping project in 100 Golden Retrievers. Our power calculations demonstrate that this sample size is expected to be sufficient to detect variants that confer a high relative risk for a range of marker allele frequencies. From the total set of 60,073 SNP genotypes, we selected 41 SNPs that flank the candidate genes. We found no association between alleles, genotypes or haplotypes of these SNPs flanking the candidate genes and human-directed aggression of the Golden Retrievers.\nWe focused on human-directed aggression in the affecteds-only linkage analysis and the association study. In our quantitative genetic analysis, we studied additional types of aggression. There is no consensus in the literature on how aggression should be subdivided (Houpt and Willis 2001; Jacobs et\u00a0al. 2003; Serpell and Jagoe 1995). There are indications that various types of aggression have a distinct genetic basis. For instance, selection of rats and silver foxes for reduced fear-induced aggression towards humans did not change predatory or inter-male aggression (Naumenko et\u00a0al. 1989; Popova et\u00a0al. 1993). This suggests that molecular genetic studies of aggressive behavior should focus on specific classes of aggression. However, reduced aggressiveness towards man in the rats and foxes was accompanied by reduced fear of novelties and irritable aggression, indicating that there is overlap between classes. As long as the genetic roots of aggressive behavior are poorly understood, it will remain impossible to design a classification that reflects the genetic basis.\nIn conclusion, none of the four methods of analysis provided evidence for a strong effect of variants of the candidate genes on aggression in the Golden Retrievers that we studied. These results seem to contradict reports of the involvement of the candidate genes in the regulation of aggressive behavior. However, the study designs that we used are not powerful enough to detect variants of small effect. We can therefore not rule out the possibility that variation in the candidate genes has a smaller genetic effect on aggression. In addition, our results cannot be construed as evidence against a major role for these genes in aggression in other dog breeds. Possibly, other genes in the serotonin pathway play a role. With the completion of the dog genome project, genome-wide association studies have become feasible in dogs (Lindblad-Toh et\u00a0al. 2005). This opens the opportunity for finding genes that have not been associated with aggression up to date. Such studies are in progress.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\n(DOC 174 kb)","keyphrases":["aggression","dog","serotonin","linkage","association","candidate gene"],"prmu":["P","P","P","P","P","P"]}
{"id":"J_Urban_Health-2-2-1705546","title":"Application of Respondent Driven Sampling to Collect Baseline Data on FSWs and MSM for HIV Risk Reduction Interventions in Two Urban Centres in Papua New Guinea\n","text":"The need to obtain unbiased information among hard\u2013to-reach and hidden populations for behavioural and biological surveillance, epidemiological studies, and intervention program evaluations has led researchers to search for a suitable sampling method. One method that has been tested among IDU and MSM recently is respondent-driven sampling (RDS). We used RDS to conduct a behavioural survey among FSWs and MSM in two urban centres in Papua New Guinea (PNG). In this paper we present the lessons learned implementing RDS in a developing country setting. We also present comparisons of RDSAT-adjusted versus unadjusted crude estimates of some key socio-demographic indicators as well as comparisons between the estimates from RDS and a hypothetical time\u2013location sample (TLS). Overall, the use of RDS among the MSM and FSWs in PNG had numerous advantages in terms of collecting a required sample size in a short time period, minimizing costs and maximising security for staff and respondents. Although there were a few problems these were easily remedied and we would recommend RDS for other similar studies in PNG and other developing countries.\nIntroduction\nPapua New Guinea is currently experiencing a major generalized HIV epidemic driven by high rates of unprotected sex, multiple sexual partners, early age of sexual debut, and high rates of STIs. Sexual violence and aggression is also widespread.1 PNG's HIV surveillance system has limited capacity, and valid scientific information is needed to guide community prevention efforts.2\nFemale sex workers (FSWs) and men who have sex with men (MSM) have been identified as groups at high\u2013risk for HIV throughout the world. There have been few published studies about FSWs in PNG.3,4 No previous quantitative research has been conducted with MSM in PNG, but qualitative research and unpublished reports indicate that MSM behaviour is prevalent in some areas of the country.5 Moreover, numerous anthropological studies indicate that many tribes in PNG have practised male\u2013male sexual behaviour in traditional rites and rituals.6\u20138\nThe major challenge in studying HIV\u2013risk behaviours and developing prevention efforts among high risk populations is gathering information from non\u2013biased samples.9 Members of population groups such as men who have sex with men (MSM), injecting drug users (IDU) and female sex workers (FSWs) are involved in stigmatized or illegal behaviour, leading individuals to hide their identity and to be reluctant to participate in research studies. No sampling frame exists for these groups, and the fact that creating one may be difficult and very costly makes them hard to sample with traditional probability\u2013based sampling methods.\nRespondent driven sampling (RDS) is a relatively new adaptation of chain\u2013referral sampling, where subsequent respondents are recruited by previous respondents through their network of acquaintances.9\u201311 It has several features that overcome the limitations of other sampling methods that allow it to provide unbiased and representative population\u2013based estimates. RDS has therefore been recommended as an alternative sampling approach for \u201chidden\u201d populations that do not congregate in identifiable or accessible locations.12\u201314 Because RDS relies on participants from the target population to recruit subsequent respondents, it has the advantages that it requires less detailed formative research, is less costly, can be completed in a shorter amount of time, and has greater external validity than other methods.\nThis paper presents lessons learned from applying RDS in a study to collect baseline behavioural data for HIV prevention programming among two high risk populations in two urban centres in Papua New Guinea. The research among FSWs in Goroka and Port Moresby and MSM in Port Moresby was linked to the Poro Sapot Project (PSP), which is an HIV prevention initiative of Save the Children in PNG (SCiPNG). The Papua New Guinea Institute of Medical Research (PNGIMR) partnered with SCiPNG and Family Health International (FHI) to conduct the research.\nReasons for Choosing RDS\nThe first phase of the research project was a qualitative evaluation of the two target populations. Qualitative research methods included field observations, focus group discussions, in\u2013depth interviews, and key informant interviews. In addition, a literature review of research studies on these populations in PNG was carried out.\nOur formative research showed that there was a relatively large and socially well connected population of MSM in Port Moresby, the capital and largest city of PNG. Although there were a number of commercial and public spaces where MSM met and socialised, none of them catered to a primarily MSM clientele. There were no formal community\u2013based or social organizations of gay or homosexual men. Due to social stigma and discrimination, the vast majority of MSM kept their sexual orientation hidden from their family, friends and co\u2013workers. The lack of MSM\u2013identified sites and the hidden nature of homosexuality in PNG meant that it would be difficult to use time\u2013location sampling (TLS) as the recruitment methodology.13,14\nThe decision to use RDS for the survey of FSWs came about following formative assessments in both locations. FSWs in PNG were found to be very mobile. Although a few illegal brothels did exist, only a small number of women worked in those locations. Most FSWs frequented formal establishments such as disco bars, hotels, motels, and guest houses in search of potential clients. In most locations FSWs mixed with the general population in a way that would have made it difficult to differentiate sex workers from other females.\nIn addition, security for the research staff and participants was a major concern. MSM informants had complained of physical and verbal abuse due to social stigmatization. The crime rate in Port Moresby was very high and had steadily deteriorated in the time period before the research was conducted. Since most MSM and FSWs went out at night to socialise or meet clients, it would have been difficult to ensure the security of research staff members if they had to contact potential respondents in public areas. RDS provided a safer alternative in this regard because respondents were not actively recruited by study interviewers but were instead referred by their peers to be interviewed at a safe study facility during a convenient time of the day.\nResearch Methods\nThe field work in Port Moresby began in February 2005 and was completed at the beginning of April 2005. Data collection in Goroka began in April 2005 and was completed in 2\u00a0weeks. The interviewers were all FSWs and MSM who received 1 and 1\/2\u00a0weeks of intensive training prior to the starting of field work.\nThe research teams at each site were comprised of a coupon manager, a research assistant and a group of peer interviewers. In Port Moresby we trained and used six MSM interviewers and four FSWs interviewers. In Goroka we trained and used five FSWs interviewers.\nThe Coupon Management System\nA coupon management system was developed in Microsoft FoxPro 2.6 and was used to track the relationships between the recruiters and their recruits. The same program was used to record biometric measurements (circumference of both wrists and length of both forearms), which were used to check for and prevent duplication of recruitment. The latter was done through the calculation of the index of differences between the candidate and all of the other biometric measures already entered.15\nRecruitment of Seeds and Study Participants\nThe FSWs seeds for the RDS sample were drawn from distinct geographic areas within Port Moresby and Goroka. The areas chosen in each location were the coverage areas that were mapped out and targeted for the intervention programs. Therefore eight seeds were chosen who lived and worked in five different areas of the city of Port Moresby, and six seeds were chosen from six different areas in Goroka. These areas and the number of seeds drawn for Port Moresby and Goroka are shown in Tables\u00a01 and 2, respectively.\nTable\u00a01Socio\u2013demographic characteristics of FSWs in Port Moresby\u00a0SeedsSample(n=8)Percent(n=245)Crude (%)RDSAT\u2013adjusted  (%)Absol. diff  (%)Age16\u201319112.54819.625.05.420\u201324112.53815.518.63.125\u201329112.55422.022.70.730+562.510542.933.59.4Marital statusEver married675.018575.566.98.8Currently married112.55823.717.26.5Currently married  cohabiting with  spouse00.072.93.91.0Education levelNo formal education00.05823.726.67.9Primary337.512149.447.51.9Secondary562.56124.922.22.7Other (eg. vocational)00.052.03.21.2Region of birthHighlands225.011346.142.73.4Southern562.511647.351.23.9Others112.5166.55.90.6Place of residenceTown\/Koki area225.06225.318.46.9Boroko area112.510040.823.617.27\u2013mile\/airport225.0156.14.61.5Gordons\/Hohola225.0197.88.81.0Waigani\/Gerehu112.54618.843.224.4Others00.031.21.11.2Table\u00a02Socio\u2013demographic characteristics of FSWs in Goroka\u00a0SeedsSample(n=6)Percent(n=249)Crude (%)RDSAT\u2013adjusted  (%)Absol. diff  (%)Age16\u201319116.74518.116.61.520\u201324116.78032.135.33.225\u201329116.76224.923.91.030+350.06224.924.00.9Marital statusEver married350.016465.965.80.1Currently married00.02710.812.41.6Currently married  cohabiting with  spouse00.000.00.00.0Education levelNo formal education233.38734.935.80.9Primary233.310843.443.20.2Secondary233.35321.322.00.7Other (eg. vocational)00.020.81.00.2Region of birthHighlands6100.023895.696.40.8Momase00.083.22.70.5Others00.031.20.80.4Place of residenceSeigu\/fish Wara116.77329.331.21.9Asariyufa116.76224.926.31.4North Goroka116.73514.111.13.0Kama116.7124.82.62.2Lofi\/Faniyufa116.7239.27.12.1Waterise116.73212.915.52.6Others00.0124.85.70.9\nIn some areas of Port Moresby two seeds were chosen: one seed was a younger FSWs while the other was an older one. Formative research had shown that the younger and older FSWs in some locations did not mix socially and therefore could be considered as separate populations. In other areas only one seed was used as in these areas the younger and older FSWs mixed together socially.\nThe eligibility criteria for the selection of FSWs seeds as well as their subsequent recruits were defined as \u201ca female over the age of 16 who has exchanged sex for money or other goods and services in the last 12\u00a0months and is not drunk at the time of the interview.\u201d\nFor the MSM study in Port Moresby we also attempted to recruit a diverse set of seeds based on self\u2013identified sexual orientation and region of origin.\nSeeds were recruited through SCiPNG, which had recently initiated a peer education program with MSM. The peer educators in the project also worked as interviewers for the research. These men tended to recruit MSM seeds from their own social group. As a result, eight of the ten seeds used were self\u2013identified as homosexual, and seven of the ten were from the southern region, which was the site of the research. As we will report later, the biases inherent within the initial group of MSM seeds were not present in the final sample population, in line with RDS theory.\nThe eligibility criteria for the selection of the MSM seeds as well as their subsequent recruits were defined as \u201ca man over the age of 16 who has had sex with another man in the last 12\u00a0months and is not drunk at the time of the interview.\u201d\nIn none of the three study groups was it found necessary to add additional seeds during the course of the study.\nIncentives\nSeeds and their recruits were offered monetary incentives as well as gift bags containing pamphlets, posters, free condoms and lubricant. Participants in Port Moresby were given 20 PNG Kina (K20, equal to U.S.$6.00) for successfully participating in the study and K10 (U.S.$3.00) each for every successfully recruited peer. The FSWs in Goroka were given K10 (U.S.$3.00) for participating and K5 (U.S.$1.50) for every successful recruitment made. The amount for incentive was less in Goroka because of the lower cost of living there compared to Port Moresby.\nInterviews and Setting\nAs part of the RDS field work in Port Moresby, the research team rented two private properties situated in secluded locations of the city. The FSWs study in Goroka was also carried out at a private property in a residential part of town. Data was collected by trained interviewers who were MSM and FSWs themselves and was directly supervised by the principal investigators. All interviews took place in private rooms with only the interviewer and subject present. The secure nature of the locations used also allowed the research staff to exclude non\u2013participants from the sites while recruitment and interviews were taking place.\nParticipants gave witnessed verbal consent to be part of the study. Interviews were conducted using a structured questionnaire. Information obtained included demographics, sexual behaviour, drug and alcohol use, knowledge and use of male and female condoms, knowledge and attitudes towards HIV\/AIDS and STI, history and treatment seeking behaviour of STI, experience with stigma and discrimination, and exposure to intervention programs. The MSM interviews included information about their sexual orientation. Additional information was collected that was specifically required for RDS methodology: personal network size, relationships to recruiters, and the number of recruitment refusals encountered.\nSample Sizes\nA total of 245 and 249 FSWs were interviewed in Port Moresby and Goroka, respectively. For the MSM study in Port Moresby, a total of 225 MSM participants were interviewed.\nA total of 471 and 621 coupons were given out to 157 and 207 FSWs in Port Moresby and Goroka, respectively, while 603 coupons were given out to 201 MSM. Participants in all sites were told in advance that recruitment would stop when the desired sample size was reached. Participants who had lost a coupon were not given any additional ones.\nFailure to meet the eligibility criteria resulted in 15 females (four from Goroka) and 35 males being refused participation in the study. Half of the women who were excluded in Port Moresby were under 16\u00a0years of age, while the other half were excluded because they reported that they had never sold sex. The four FSWs that were excluded in Goroka were all under 16\u00a0years of age. The bulk of the men that were excluded from the MSM study (33) did not meet the criteria of having had sex with another man in the previous 12\u00a0months, while two others were excluded for trying to repeat the interview.\nData Analysis Methods\nData was double entered using Microsoft FoxPro 2.6 and analysed using Stata 8.0 (Stata Corporation, Texas, USA), RDS Analysis Tool (RDSAT, Cornell University, NY, USA) and EpiInfo 6.04 (CDC, USA and WHO, Geneva). Adjusted analysis was carried out using RDSAT on single categorical variables whereas bivariate and multivariate analysis was done using EpiInfo and Stata. RDSAT adjusts the analysis of population estimates by taking into consideration the links between the recruiters and their recruits as well as their reported personal network sizes.11,12 A detailed description of the methods of calculation is beyond the scope of this paper.\nSome Key Results\nOf the 157 and 207 FSWs in Port Moresby and Goroka that were given coupons, 56.7 and 42.0%, respectively, recruited one or more of their peers into the study. For the MSM study, 57.2% of the 201 participants that were given coupons recruited one or more peers. Two questions were asked with regards to the number of coupons given out and the number of refusals encountered by each recruiter to determine refusal rates. However, the research assistants did not consistently record the answers to these questions. Nevertheless, the refusal rates appeared to be very low as indicated by the fact that 74.2 and 85.1% of the FSWs recruiters who successfully recruited other participants in Port Moresby and Goroka returned all three coupons. Of the successful MSM recruiters, 52.2% of them had all three coupons returned, 35.6% had two coupons returned and 12.2% had one coupon returned. Half of the MSM seeds (five) did not recruit any peers while three FSWs seeds (two from Port Moresby) did not recruit any peer.\nIt took a median of 4\u00a0days (Range=1\u201312) and 3.5\u00a0days (Range=1\u201310) for a coupon to be returned by FSWs in Port Moresby and Goroka, respectively. MSM returned the coupon at a median of 3\u00a0days (range=1\u201318). For both the FSWs and MSM, however, recruitment occurred at a much faster rate than had been anticipated and recruits presented to the study locations faster than they could be interviewed. It became necessary to slow down recruitment by scheduling interviews each day on a first\u2013come first\u2013served basis. Those that could not be interviewed the day they first presented were scheduled for the next day. Eventually the recruiters were told to bring their recruits in on specified dates in order to control the flow of new subjects to the study sites. The fast rate of recruitment for the three subpopulations enabled the sample sizes for FSWs in Port Moresby and Goroka to be reached in 17 and 20\u00a0days, respectively, while the sample size for MSM was reached in 22\u00a0days.\nThe main sociodemographic characteristics of the seeds and the overall samples for the three studies are presented in Tables\u00a01, 2 and 3. Comparisons between the RDSAT\u2013adjusted and crude analyses are given as well.\nTable\u00a03Socio\u2013demographic characteristics of MSM in Port Moresby\u00a0SeedsSample(n=10)Percent(n=225)Crude (%)RDSAT\u2013adjusted  (%)Absol. diff  (%)Age16\u201319220.04921.820.81.020\u201324110.08136.038.12.125\u201329660.06026.727.71.030+110.03515.613.22.4Education levelNo formal education00.0125.36.71.4Primary00.08738.749.510.8Secondary660.06930.740.49.7Secondary440.0156.73.13.6Employment statusUnemployed220.012054.052.02.0Employed330.02913.011.02.0Self\u2013employed550.06931.035.04.0Students00.042.02.00.0Region of birthHighlands220.03314.723.99.2Southern\u2014gulf only110.011450.754.13.4Southern\u2014others660.04821.310.710.6New Guinea islands00.0104.44.80.4Momase110.0208.96.32.6Sexual orientationHeterosexual110.02912.910.02.9Gay\/homosexual880.06528.923.05.9Bisexual110.013158.267.08.8Paid by man for sexYes990.016975.170.74.4No110.05624.929.24.3\nThe results for the FSWs study in Port Moresby show that, while the seeds where roughly comparable to the overall sample, there were still some major differences with regard to educational status and region of birth.\nFor the FSWs study in Goroka, there was less variation between the seeds and final sample (Table\u00a02), although the seeds tended to be older than overall sample.\nThe most striking differences between the initially selected seeds and the final sample were found for the MSM study in Port Moresby (Table\u00a03). The final sample was younger, less educated, and more often unemployed. In addition, while the majority of the seeds (80%) were gay identified, less than a third of the final sample self\u2013identified as homosexual and the majority of the final sample self\u2013identified as bisexual.\nFor all three studies (Tables\u00a01, 2 and 3), a common pattern in the findings that emerged with regard to comparisons of the characteristics of seeds and final samples, was that the overall sample was of lower socioeconomic status (especially for the MSM study). This suggests that RDS is able to penetrate \u201chidden\u201d populations that are often very different from the initially selected seeds.\nIn terms of the comparisons between the RDSAT\u2013adjusted and crude analysis in Tables\u00a01, 2 and 3, the greatest variations between the two analyses (as measured by the percent of absolute difference) were found for FSWs in Port Moresby. This was especially true with regard to place of residence, where up to 24.4% differences were found for the ones that reside in one part of the city (Table\u00a01). For FSWs in Goroka, considerably less variation was found with absolute differences ranging from 0 to 3.2% (Table\u00a02). For the MSM study, some considerable differences were also seen and absolute differences ranged from 0 to 10.8%.\nThe results presented in Tables\u00a04 and 5 were an attempt to understand the potential differences in respondent characteristics that would have existed between RDS and TLS, had this latter sampling method been used instead of RDS. The hypothetical TLS group for this analysis included FSWs and MSM respondents who reported that they \u2018always\u2019 looked for and met their sexual partners in visible locations that would have been likely included in a TLS sampling frame (i.e., nightclubs, bars, hotels, guesthouses and identifiable public venues). The results shown in Table\u00a04 indicate that the RDS samples for FSWs in both Port Moresby and Goroka were younger and less educated than the hypothetical TLS group. While reported condom use with clients was similar between the RDS and TLS groups, reported condom use with non\u2013paying partners was substantially lower among the RDS group. On the other hand, forced sex, knowledge, and reported STI symptoms were higher in the TLS group. Analysis of the key program exposure variable, contacted by peer educator, also revealed that program exposure was substantially lower among the RDS sample than hypothetical TLS sample. The analysis of the MSM study population in Port Moresby (Table\u00a05) showed striking differences in almost all variables examined. In comparison to the hypothetical TLS group, the RDS group was older, less educated, more likely to be unemployed, more likely to identify their sexual orientation as heterosexual or bisexual and less likely to use condoms for partnerships examined. This group was also less likely to use lubricant during anal sex and less likely to have been exposed to HIV prevention programming. On the other hand, the hypothetical TLS group was more likely to report forced sex, incidents of discrimination and symptoms of STIs.\nTable\u00a04RDS versus hypothesized TLS estimates of key indicators for FSWsKey indicatorsPort Moresby (%)Goroka (%)RDSa (n=235)TLS (n=103)RDSa (n=227)TLS (n=165)Age16\u20132443.636.951.949.725+56.263.147.950.3Education levelNo formal education26.621.435.830.1Primary47.553.343.244.6Secondary22.224.322.024.1Used condom with client at last sex86.088.279.077.7Used condom consistently with clientc62.661.231.331.3Used condom with non\u2013paying partner  at last sex48.375.743.062.0Used condom consistently with  non\u2013paying partnerc33.548.716.026.0Forced to have sexb58.466.766.570.5Thinks mosquitos can transmit HIV37.150.030.736.1Had vaginal dischargeb43.253.428.637.4Had sore in\/around vaginab20.524.312.119.4Never contacted by peerb educator24.418.540.939.6aRDSAT adjustedbIn the last 12\u00a0monthscIn the last 4\u00a0weeksTable\u00a05RDS versus hypothesized TLS estimates of key indicators for MSM\nIt is important to note that the observed differences between the two groups in Tables\u00a04 and 5 would have been even more pronounced if the comparisons had been made between the mutually exclusive categories\u2014the hypothetical TLS group and the RDS group restricted to respondents who would likely not have been sampled using TLS.\nDiscussion of the Lessons Learned\nSampling procedures should be capable of reaching all members of the population or subpopulation under surveillance in order to produce unbiased estimates of trends in HIV behavioural risks.12 In this respect, RDS was chosen to study the three subpopulations of interest in PNG after carefully considering its advantages and disadvantages for each target group. Many factors prompted us to opt for RDS instead of other sampling methods. For MSM, the main reasons why other sampling methods would not be adequate were that most of them were hidden due to social stigma and that no public venues existed to use as a sampling frame for TLS. For the FSWs, creation of a sampling frame for random sampling or TLS would be difficult and costly given their very high mobility and the illegal nature of sex work in PNG.\nFor all three target groups in both locations, security for both the subjects and the research staff was a major concern. This is an issue specific to PNG, but may also be an important issue in other developing countries with unstable economic and political situations. The ability to conduct interviews in a private and secure location during the daytime and have potential subjects come directly to the study location after being recruited by previous respondents was a major advantage of RDS in this study. In PNG, it would have been difficult to have ensured the safety of the research staff if they had to go out into the field at night time or may have required hiring security guards at considerable expense.\nRDS has been described by those studying more the difficult sub\u2013population of illicit drug users as a flexible and robust method that can produce a sample representative of the heterogeneity of the target population.16 We had a similar experience in implementing RDS among FSWs and MSM in a developing country. Although our original seeds were not as diverse as we intended them to be, a comparison of the seeds versus the final sample (Tables\u00a01, 2 and 3) shows that by using RDS, the characteristics of the final samples were often quite different from the initially selected seeds. Particularly striking was our finding that the overall samples in all three subpopulations were mainly lower socio\u2013economic status. A comparison of the RDSAT\u2013adjusted versus the crude estimates of the various socio\u2013demographic characteristics (Tables\u00a01, 2 and 3) showed mostly little variation.\nThe rapid rate of recruitment among all subpopulations suggests that the dual incentive system did work exceptionally well. We observed that the majority of the recruiters accompanied their recruits to the interview site. The reason for this was most likely that the recruiters wanted to collect their recruitment incentives immediately, but it also ensured that many of the recruited persons did in fact show up at the study sites in a relatively short amount of time.\nNot one FSWs or MSM subject used the phone number printed on the coupon to make an appointment. Telephone charges are relatively expensive, and mobile phones are not widespread among the general population in PNG. Therefore, it may have been easier and less expensive for recruits to simply show up at the research site than to have called ahead for an appointment. Thus the use of a telephone call to make appointments may not be necessary in this country in any future application of RDS for these or similar populations.\nThe results of the comparisons between the RDS and hypothetical TLS samples (Tables\u00a04 and 5) suggest that RDS allowed for greater penetration and representation of more vulnerable segments of the FSWs and MSM target populations. The use of dual financial incentives associated with RDS is also believed to have been important for reaching less educated and unemployed segments of the FSWs and MSM populations because they were more likely to find this a motivating factor for participation. During the RDS field work, it was observed anecdotally that the more economically desperate participants with ample disposable time were more likely to try to recruit individuals who did not meet the study eligibility criteria.\nCareful planning, staff training, and preparation for the field work enabled us to handle the onslaught of recruits and ensure the smooth implementation of RDS among FSWs and MSM in Papua New Guinea. The procedures checklist13 and practicing with the coupon management system were particularly important in preparing for the research. We agree with the view expressed by others that RDS is relatively easy to implement and less costly in that it did not require exhaustive mapping exercise to construct sampling frames.2,16 In our experience, the dual incentive system of RDS helped to reduce non\u2013response rates and fuelled recruitment.13\nFinally, in this study we relied on others to help select the original seeds. Some of the seeds failed to recruit any additional subjects. Half of the MSM seeds recommended to us simply did not recruit any other MSM, indicating that the recruitment of initial seeds in this study was not very effective. Problems were also encountered with potential recruits who misrepresented themselves in attempts to participate in the study in order to earn money. This was especially true in the MSM study, where many men who were not really MSM tried to participate. Similarly, a number of underage girls were refused entry into the study. This problem most likely resulted from the dual incentive system, in which a participant could potentially earn up to U.S. $15.00, a significant sum in a poor country like PNG. However, the screening and interview process in place was sufficient to track and preclude repeat participation or recruitment of individuals who did not meet the inclusion criteria.\nFor this study among FSWs and MSM in PNG, using RDS as the sampling methodology had numerous advantages in terms of collecting the required sample size in a short time period, minimizing study costs, and resulting in representative estimates of the target populations. Although there were a few problems in implementing a new research method in a resource\u2013poor setting, these were easily remedied, and we would recommend RDS for similar studies in PNG and other developing nations.","keyphrases":["sampling","papua new guinea","respondent-driven sampling","female sex workers","men who have sex with men"],"prmu":["P","P","P","P","P"]}
{"id":"Ann_Surg_Oncol-3-1-1914254","title":"Better Survival in Patients with Esophageal Cancer After Surgical Treatment in University Hospitals: A Plea for Performance by Surgical Oncologists\n","text":"Background In primary esophageal cancer, studies have frequently focused on surgical patients in an effort to link outcome to hospital- or surgeon-related experience, with operative mortality used as the main outcome measure. Many studies have found an inverse relationship between operative mortality and hospital volume and surgical expertise. This study aims to assess the influence of surgeon-related expertise and hospital volume on the relative survival of operated esophageal cancer patients.\nWith an incidence of 6.3 per 100,000 (European standardized rate) in the period 1994\u20131998, esophageal cancer ranked 13th among malignancies in men and 12th in women in the Netherlands. Incidence increased in both male and female patients, with an estimated annual percentage change of 3.1% and 2.0%, respectively. The mortality for men and women was, respectively, 9.4 per 100,000 and 3.1 per 100,000 in 2004, indicating that the prognosis of patients with esophageal cancer remained poor in this period (http:\/\/www.kankerregistratie.nl). The only curative option for esophageal cancer is surgery, which implies that improving the outcome of surgery is the best means of reducing mortality.1\nEsophageal cancer is one of the most challenging pathological conditions confronting the surgeon. It therefore seems reasonable to assume that concentration of esophageal surgery could improve outcome. Several studies have shown that various characteristics, including surgeon subspecialty certification, hospital setting, and the number of procedures performed, are associated with practice variation, complication rates, and even outcome.2\u20134 Evidence of improved outcome associated with specialist care exists for breast cancer,2,4 ovarian cancer,3,5 and malignant teratoma.6 However, there is no evidence of comparable quality for esophageal cancers. Several studies on esophageal cancer have focused on surgical patients in an effort to link outcome to experience, either on the part of the institution or the surgeon, by using postoperative mortality as the main outcome measure.7\u20139 A study on esophagectomies performed in England during the late 1980s found no independent association between operative mortality and hospital surgical volume,10 whereas two North American studies did demonstrate a lower mortality rate for esophagectomy when high-volume and low-volume hospitals were compared.11,12 Various smaller studies also found lower operative mortality rates among high-volume surgeons.13,14\nFew studies, if any, have attempted to relate esophageal cancer patient survival to surgeon expertise or hospital volume. To remedy this, in this study, we assessed the effect of surgeon-related expertise and hospital volume on the relative survival of operated esophageal cancer patients. We compared data from university, teaching nonuniversity, and nonteaching hospitals.\nPATIENTS AND METHODS\nPatients\nAll patients diagnosed with a primary invasive esophageal cancer in the region of the Comprehensive Cancer Centre North-Netherlands (CCCN) between January 1994 and January 2002 were eligible for entry onto the study. Patients with a history of cancer other than nonmelanoma skin cancer were excluded. The patients were selected through the population-based Regional Cancer Registry of the CCCN, which covers the northern part of the Netherlands, a mainly rural area with a population of approximately 2.1 million. The area is served by 17 community hospitals, 3 of which are teaching hospitals and 1 of which is a university hospital; the hospitals include four radiotherapy departments and seven pathology laboratories.\nData Collection by the Regional Cancer Registry\nPALGA, a Dutch nationwide network and registry of histopathology and cytopathology, regularly submits reports of all diagnosed malignancies to the cancer registry. The national hospital discharge data bank, which receives discharge diagnoses of admitted patients from all hospitals, completes case ascertainment. The cancer registry has no access to death certificates. After notification, trained registry personnel collect data on diagnosis and staging from the medical records, including pathology and radiology reports, in the hospitals. The cancer registry collected all data regarding the diagnosis and staging, but collected no data on specific surgical treatment in this patient population before 2002. The data collection occurs at least 4 months after diagnosis to comprehensively document all aspects of preoperative staging. All patients are staged according to the tumor, node, metastasis system (TNM) system for esophageal cancer in use during that period.15,16\nIn the Netherlands, the population registries of the municipality contain information on the vital status of their inhabitants. Vital status was established either through information derived from the patient\u2019s medical records or through linkage of cancer registry data with information from the population registries of the municipality within the registry areas or through linkage with the national death registry of the Central Bureau of Genealogy. The regional cancer registry of the CCCN checked vital status by active record linkage with municipal population registries in 2002\u20132003 and 2005 and with the national death registry of the Central Bureau of Genealogy in 2004.\nGuidelines for Staging and Treatment\nBy establishing multidisciplinary teams and cancer networks, the CCCN strives to improve the quality of cancer care. Within the CCCN area tumor working groups, comprising delegated specialists representing all regional hospitals, that have been developing and revising guidelines on diagnosis and treatment. The regional guidelines for esophageal cancer were based on the international TNM classification according to the International Union Against Cancer in use at that time.\nStatistics and Definitions of Variables\nThe \u03c72 test was used to compare the distribution over the patient population for categorical variables. For continuous variables, analysis of variance was used. Relative survival analysis was performed to estimate the effect of university, teaching nonuniversity, and nonteaching hospitals on the prognosis of operated patients with esophageal cancer. The a priori hypothesis was that patient volume and hospital expertise would increase from nonteaching to university teaching hospitals. Survival time was calculated from the date of diagnosis and ended at the date of death, including perioperative death, or the date of most recent linkage with the municipal population registries and\/or national death registry. The overall survival probability was estimated by the Kaplan-Meier method.\nThe expected survival probability was calculated by using age-, sex-, and period-matched mortality rates that were based on life expectancy tables in the Netherlands (http:\/\/www.statline.cbs.nl\/StatWeb\/) and was estimated by the Ederer 2 method.17 The cumulative relative survival (the ratio of the overall survival to the expected survival) was estimated by Stata (version 8.0) software and the strs function. The relative survival, which estimates the net esophageal cancer survival in the hypothetical situation that esophageal cancer is the only possible cause of death, has been shown to be a good estimator of disease-specific survival in the absence of information on the cause of death or in case information on the cause of death is inaccurate.\nThe excess mortality rate was calculated by subtracting the expected number of deaths, estimated from the expected survival probability, from the observed number of deaths in a subgroup or stratum and dividing the resulting excess number of deaths by the number of accumulated person-years, taking censoring into account. The relative excess risks (RER) of death were estimated as the ratio of excess mortality rates. RERs were estimated by a multivariate generalized linear model with a Poisson error structure, which was based on collapsed relative survival data, by using exact survival times.18 By use of this model, the effect of the type of hospital (university, teaching nonuniversity, and nonteaching) was studied, adjusting for the effect of various covariables on the excess mortality experienced by our cohort.\nVariables included in the final model were age (<50, 50\u201359, 60\u201369, >70), stage based on collapsed TNM data (stage 1,2a, 2b, 3\/4, unknown), hospital volume (<20 patients operated, \u226520 patients operated), frequency of referral (high referral [>33.3%], low referral [\u226433.3%]), and time since diagnosis (1-year intervals). The pathological stage was used whenever possible; in the absence of information about the pathological stage, the clinical stage was used.\nRESULTS\nTABLE\u00a01.\nCharacteristics of operated and nonoperated patients with esophageal cancer diagnosed 1994\u20132002\nCharacteristic\nTotal\nOperated\nNonoperated\nP value\nN\n%\nn\n%\nn\n%\nSex\n.465\nMale\n796\n69.1\n152\n71.4\n644\n68.6\nFemale\n353\n30.9\n61\n28.6\n292\n31.2\nHistology\n<.001\nSquamous cell carcinoma\n415\n36.1\n62\n29.1\n353\n37.7\nAdenocarcinoma\n592\n51.6\n140\n65.7\n453\n48.4\nOther\n141\n12.3\n11\n5.2\n130\n13.9\nTumor location\n<.001\nUpper thoracic\n82\n7.1\n3\n1.4\n79\n8.4\nMiddle thoracic\n210\n18.3\n36\n16.9\n174\n18.6\nLower thoracic\n770\n67.0\n169\n79.3\n601\n64.2\nOverlapping and unspecified\n87\n7.6\n5\n2.3\n82\n8.8\nAge at diagnosis (y)\n<.001\n<50\n86\n7.5\n24\n11.3\n62\n6.6\n50\u201359\n221\n19.2\n67\n31.5\n154\n16.5\n60\u201369\n319\n27.8\n80\n37.6\n239\n25.5\n70+\n523\n45.5\n42\n19.7\n481\n51.4\nStage\n<.001\n1\n52\n4.5\n32\n15.0\n20\n2.1\n2A\n174\n15.1\n65\n30.5\n109\n11.7\n2B\n69\n6.0\n26\n12.2\n43\n4.6\n3\n207\n18.0\n74\n34.7\n133\n14.2\n4\n316\n27.5\n8\n3.8\n308\n32.9\nUnknown\n331\n28.8\n8\n3.8\n323\n34.5\nTotal\n1149\n100.0\n213\n100.0\n935\n100.0\nIn the period 1994\u20132002, a total of 1149 patients were diagnosed with esophageal cancer, comprising 796 men and 353 (69.3%) women (30.7%). The median age was 68 years (range, 17\u2013103 years). Patient characteristics are described in Table\u00a01. A large proportion of the patients was diagnosed at an advanced stage of disease; 45.5% were stage III or higher. A further 28.8% were insufficiently staged. Of the 1149 patients, 85 patients (7.4%) were initially diagnosed in the university hospital, 428 patients (37.2%) in teaching nonuniversity hospitals. The remaining 636 patients (55.4%) were diagnosed in the nonteaching hospitals. Patients who were referred from the hospital of initial diagnosis for treatment may have subsequently undergone further diagnostic testing, and previously performed tests determined to be insufficient were repeated. The 5-year relative survival rate for men with esophageal cancer was 12.8% vs. 9.8% for women (P = .496). The 5-year relative survival markedly decreased as the stage advanced. The 5-year relative survival was 71.5% in stage I, 26.5% in stage IIA, 13.3% in stage IIB, 9.2% in stage III, 1.3% in stage IV, and 6.7% for patients with unknown stage (P < .0001).\nAs Table\u00a01 shows, only 213 patients (18.5%) underwent surgery. Older age (P < .001), advanced or unknown stage (P < .001), and proximal tumor location (P < .001) resulted in a lower probability of tumor resection. Squamous cell carcinoma was also associated with less surgery, but it was highly correlated with the tumor location. In all, 21.4% of the patients diagnosed in nonteaching hospitals underwent surgery, compared with 15.9% and 10.6% for teaching nonuniversity and university hospitals, respectively (P = .011). Adjusted for age, stage, and tumor location, the odds of operation was 1.89 (95% confidence interval [95% CI], 1.26\u20132.82) for patients diagnosed in a nonteaching hospital compared with patients diagnosed in a teaching nonuniversity hospital.\nOf all operated patients 45.1% were referred for surgery after diagnosis in the hospital of presentation (Table\u00a02). The nonteaching hospitals referred 57.4% of patients diagnosed in their hospitals for an operation elsewhere. Of the 14 nonteaching hospitals in which esophageal cancer patients were diagnosed, 12 referred nearly all (75%\u2013100%) patients, and two rarely (.0%\u20136.9%) referred patients. The teaching nonuniversity hospitals referred 26.5% of the patients diagnosed in their hospitals to a larger institution for therapy, with one hospital referring 63.2% of their patients.\nTABLE\u00a02.Referral pattern for esophageal cancer surgery per hospital in the North-Netherlands, 1994\u20132002HospitalOperated in hospital of diagnosis, n (%)Referred for surgery, n (%)Total (n)High-referral nonteaching hospitals 20 (20.8)76 (79.2)96Hospital A\u20132 (100.0)2Hospital B\u20133 (100.0)3Hospital C\u20133 (100.0)3Hospital D\u20139 (100.0)9Hospital E2 (11.8)15 (88.2)17Hospital F1 (14.3)6 (85.7)7Hospital G3 (18.8)13 (81.3)16Hospital H3(25.0)9 (75.0)12Hospital I3 (30.0)7 (70.0)10Hospital J1 (33.3)2 (66.7)3Hospital K1 (33.3)2 (66.7)3Hospital L6 (54.5)5 (45.5)11Low-referral nonteaching hospitals 38 (95.0)2 (5.0)40Hospital M27 (93.1)2 (6.9)29Hospital N11 (100.0)\u201311High-referral teaching, nonuniversity hospitals7 (36.8)12 (63.2)19Hospital O7 (36.8)12 (63.2)19Low-referral teaching, nonuniversity hospitals43 (87.8)6 (12.2)49Hospital P24 (82.8)5 (17.2)29Hospital Q19 (95.0)1 (5.0)20Low-referral university hospital9 (100.0)\u20139Hospital R9 (100.0)\u20139117 (54.9)96 (45.1)213\nTable\u00a03 compares characteristics of the operated patients in the three hospital types. Of the 213 operated patients, 95 underwent surgery in the university hospital; 86 of these patients were referrals. The three teaching nonuniversity hospitals provided surgery to 60 patients, including 10 referrals: one hospital performed surgery on 7 patients, with the two other hospitals operating on more than 20. The remaining 58 patients underwent surgery at one of the 14 nonteaching hospital after all were initially diagnosed in the same hospital; two low-referral hospitals operated on more than 10 patients, eight hospitals operated on 5 or fewer patients, and the remaining four high-referral hospitals did not perform any esophageal cancer surgery. There were no statistically significant differences in the distribution of age (P = .230), stage (P = .299), or tumor location (P = .130) between the hospital types, again showing little evidence of selective referral. However, of all operated patients per hospital, a slightly larger proportion of stage III\/IV tumors were operated on in the nonteaching (46.6%) and university hospitals (41.1%) compared with the teaching nonuniversity hospitals (26.7%), and the portion of stage IIA tumors was somewhat higher in nonteaching (31.0%) and teaching nonuniversity hospitals (38.3%) compared with the university hospital (25.3%).\nTABLE\u00a03.Characteristics for operated esophageal cancer patients diagnosed 1994\u20132002, according to hospital of surgeryCharacteristicTotalTeaching, nonuniversityUniversityNonteachingP valueN%n%n%n%StageStage 13215.51220.01414.7610.3.299aStage 2A6526.81838.32325.32431.0Stage 2B2612.2813.31212.6610.3Stage 3 + 48238.51626.73941.12746.6Unknown87.011.766.311.7Age at diagnosis (y)<502411.3813.31313.735.2.23050\u2013596731.51626.73637.91525.960\u2013698037.62541.73031.62543.170+4219.71118.31616.81525.9HistologySquamous cell carcinoma6229.12135.02728.41424.1.606Adenocarcinoma14065.73558.36366.34272.4Other115.246.755.323.4Tumor locationUpper and middle thoracic3918.31321.71717.9915.5.130Lower thoracic16979.34778.37376.84984.5Not stated52.3\u2013\u201355.3\u2013\u2013Total213100.060100.095100.058100.0a Excluding stage unknown.\nThe cumulative relative survival for university, teaching nonuniversity, and nonteaching hospitals is shown in Fig.\u00a01. Surprisingly, relative survival was markedly better in the university hospital compared with teaching nonuniversity and nonteaching hospitals. The 5-year relative survival was 49.2% for the university hospital versus 32.6% and 27.3% for teaching nonuniversity and nonteaching hospitals, respectively (P = .0039, Table\u00a04). In univariate analysis, the RER for university and teaching nonuniversity was, respectively, .48 (95% CI, .30\u2013.74) and .88 (95% CI, .55\u20131.42) compared with nonteaching hospitals. The proportion of operated patients who died within 3 months after diagnosis differed between university, teaching nonuniversity and nonteaching hospitals (4.2%, 13.3% and 19.0% respectively, P = .013). Excluding patients who died within the first 3 months, the RER for university and teaching nonuniversity was, respectively, .59 (95% CI, .35\u20131.00) and .97 (95% CI, .56\u20131.69) compared with nonteaching hospitals.\nFIG.\u00a01.Cumulative relative survival of patients operated for esophageal cancer diagnosed during 1994\u20132002 according to hospital type.TABLE\u00a04.Overall and relative 5-year survival and estimated excess risk (RER) of death with 95% confidence intervals (95% CI) for operated esophageal cancer patients diagnosed 1994\u20132002CharacteristicNOS (5 y)RS (5 y)OD (5 y)UnivariateMultivariate95% CIP valueED (5 y)RERa95% CIRERaStage<.0001Stage 1 3287.5%99.1%43.1.06.01\u2013.32.05.01\u2013.22Stage 2A6546.1%52.4%333.9.44.27\u2013.70.39.24\u2013.63Stage 2B2621.8%24.7%191.1.75.43\u20131.29.72.40\u20131.27Stage 3+4 (reference)8214.2%14.5%651.61.001.00Unknown816.7%18.0%6.2.99.41\u20132.351.62.65\u20134.01Hospital type.0126Nonteaching (reference)5824.9%27.3%402.31.001.00Teaching nonuniversity6029.7%32.6%391.9.89.55\u20131.421.32.79\u20132.22University9544.3%49.2%485.6.48.30\u2013.77.57.29\u20131.12Age (y).0467<50 (reference)2447.6%48.6%11.21.001.0050\u2013596732.1%33.6%421.21.73.87\u20133.441.51.74\u20133.0460\u2013698030.9%35.1%523.52.121.07\u20134.182.361.18\u20134.7070+4241.9%54.5%224.91.42.64\u20133.142.05.94\u20134.46Hospital volume.1125<20 patients operated3819.0%22.3%271.31.001.00\u226520 patients operated17537.8%41.7%1008.5.53.33\u2013.83.62.34\u20131.12Referral rate .8080High (>33.3%)11535.4%39.2%665.21.001.00Low (\u2264 33.3%) 9834.1%37.8%614.61.16.79\u20131.69.94.57\u20131.54OS, overall survival; RS, relative survival; OD, observed deaths; ED, expected deaths.a Adjusted for time since diagnosis.\nIn a multivariate analysis to adjust for the prognostic effect of patient age, tumor stage, tumor location, hospital volume, frequency of referral, and time since diagnosis, we found that stage, age, hospital type, and time since diagnosis were independently associated with the RER (Table\u00a04). The RER increased with more advanced stage. Patients aged <50 and patients aged \u226570 had a lower RER compared with patients aged 50\u201369 years. Adjusted for age, stage, and time since diagnosis, the RER for the university hospital was still considerably lower, at .57 (95% CI, .29\u20131.12), compared with nonteaching hospitals and .43 (95% CI, .24\u2013.76) compared with teaching nonuniversity hospitals (P = .0126). There was some evidence in the data for an independent effect of hospital volume, with a lower RER (.62; 95% CI, .34\u20131.12) if a hospital operated on \u226520 patients during the study period.\nIn our study 8.9% of the operated patients received some form of adjuvant therapy (Table\u00a05). Of these patients, 6.1% received preoperative chemotherapy and 2.8% received postoperative radiotherapy. Patients receiving chemotherapy were all treated in the university hospital. Comparing clinical and pathological stage, we found no evidence that chemotherapy led to a marked downstaging.\nTABLE\u00a05.Adjuvant therapies for patients operated for esophageal cancer diagnosed 1994\u20132002, according to hospital of treatmentTreatmentNonteachingTeaching, nonuniversityUniversityTotaln%n%N%N%Surgery5798.35896.77983.219491.1Surgery + radiotherapy11.7\u2013\u201355.362.8Surgery + chemotherapy\u2013\u201323.31111.6136.1Total58100.060100.095100.0213100.0\nDISCUSSION\nOur study shows that patients who underwent surgery for esophageal cancer in the university hospital had a markedly better relative survival, with a 50% lower risk of death compared with patients treated in nonuniversity hospitals. The risk of death did not differ for patients operated in teaching nonuniversity or nonteaching hospitals. In our study, we found that a higher hospital volume was weakly associated with better survival. Although hospital volume seems to influence better survival, it is unlikely that the difference between the three hospital types can be completely explained by hospital volume; other factors likely play a role. A recent British study found no effect of hospital volume on survival for the operated patients; further, this study did not find teaching hospital status independently associated with survival, but it is unclear whether this reflects the operated patients or the population as a whole.10\nOur study is one of the first studies to research the effect of a marker for hospital\/surgeon experience on patient survival. Most studies to date have evaluated 30-day postoperative or in-hospital mortality, and several demonstrated an inverse relationship between volume or surgical experience and operative mortality after esophageal resection.8,11,19 A Dutch study found that hospitals performing 1 to 10 operations per year for esophageal cancer and cancer of the gastroesophageal junction had an operative mortality of 12.2%, compared with 4.9% for hospitals performing >50 procedures per year.19 An American study had a similar result, with a mortality of 3.0% among high-volume hospitals and 12.2% among low-volume hospitals for both distal and proximal esophageal cancer,12 but this study used a cutoff point of five procedures per year to differentiate between high- and low-volume hospitals. What threshold distinguishes high-volume from low-volume hospitals remains matter of discussion. The results of these studies do suggest that centralization of esophageal surgery, so that only a few hospitals per region operate on esophageal cancer patients, may improve survival. The results of our study support the recommendation for referral of esophageal cancer patients to a center where there is a specific focus on esophageal cancer treatment. Combined with the fact that the surgical literature is increasingly advocating the need for centralization, we think that further research into the advantages of centralization of esophageal cancer treatment is warranted.\nOne of the possible pitfalls in our study remains selective referral. We showed that the likelihood of being operated on was 47% higher for patients diagnosed in a nonteaching hospital than for those diagnosed in teaching nonuniversity hospitals. Adjusted for age, stage, and tumor location, this was even higher, 73%, indicating that nonteaching hospitals considered patients with a worse prognosis possible candidates for surgery, or at least for referral to evaluate resectability. However, adding hospital referral frequency to our multivariate analyses showed no influence of referral on overall and relative survival or RER. The referral pattern showed little evidence for selective referral. Those nonteaching hospitals that referred patients referred almost all, and the teaching nonuniversity hospitals referred only few patients for surgery (Table\u00a02). The referral pattern of the nonteaching hospitals implies that the university hospital operated on a priori prognostically worse patients. This is a likely explanation for the higher number of stage III patients in the university hospital.\nTo minimize the eventual effect of any residual selection referral, the relative survival rate was adjusted for case mix, despite there being no statistically significant differences in the distribution of age, stage, and sex between the different types of hospital.\nOne may suggest that patients who underwent esophagectomy in the university hospital were mostly referred for treatment, thus adding a delay before surgery. A consequence could be that these patients had thus a slightly longer preoperative survival time, estimated to be between 2 and 4 weeks in our study. This short delay could mean that prognostically worse patients scheduled for surgery eventually fall out of the surgery category through disease progression during the delay period. However, little is known in the literature about the effect of longer preoperative delays on surgical outcome or eligibility in esophageal cancer. Although patients with advanced disease may miss surgery through stage progression, patients who do end up having surgery also progress, meaning the university hospital operates on patients with more advanced disease. This should negatively influence the survival outcome and would not explain the better performance by the university hospital. Furthermore, even if we were very conservative and excluded all patients who died in the first 3 months of our study, the university hospital still performs far better than teaching nonuniversity and nonteaching hospitals. So although we cannot discount early mortality as a factor in survival, we think that it is unlikely that the difference in performance can be fully explained by this.\nWe had no information about the operative procedure that had been performed. Treatment guidelines indicated a curative surgical approach for tumors encompassing \u22645 cm of the length of the esophagus, as based on ultrasonographic or radiological examination. Surgical resection could be attempted for tumors 5\u20138 cm in length. For this last group, neoadjuvant chemotherapy, which was provided in the university hospital after proof of locally advanced disease, could be attempted to improve resectability. For adenocarcinomas, a transthoracoabdominal approach with two-field lymphadenectomy was advised, combining a midline laparotomy and a right-sided thoracotomy. Alternatively a transhiatal blind esophagectomy could be performed with a cervical esophagogastrostomy. For distal adenocarcinomas without Barrett dysplasia, a left-sided thoracotomy with intrathoracic anastomosis was an alternative approach.\nIn general, patients in our population who were operated on in the university hospital underwent a transthoracic esophagectomy, with the exception of superficial T1 tumors, whereas patients treated in regional hospitals frequently underwent surgery with a transhiatal approach. Therefore, the two main operative strategies we encountered were transhiatal resection and transthoracoabdominal resection with a two-field lymph node dissection. There is no evidence in literature that the outcome differs for these two procedures,14,20 except for a tendency toward an improved long-term survival in the extended transthoracic group in the study of Hulscher et al.21 So the clinical outcome in our population is likely uninfluenced by differences in surgical procedure. However, there is possibly a stage migration effect between the more thorough pathological staging in operations with lymphadenectomy, as in the university hospital, and understaging in patients undergoing a transhiatal esophageal resection.\nA few of the operated patients received neoadjuvant therapy in our study, mostly in the university setting, which might account for a small part of the better survival in the university hospital. Separate analysis, however, showed that patients in our study, who received neoadjuvant therapy with surgical removal of the tumor, did not perform better than patients who underwent surgical removal alone in the university hospital. In other studies, the preoperative effect of cisplatin-based chemotherapy on both adenocarcinoma and squamous cell carcinoma showed no increase in overall survival.22\u201325 Preoperative chemotherapy or radiotherapy can result in downstaging and thus lead to a better resectability, but no clear downstaging was seen in our study when comparing pre- and postoperative clinical and pathological stage. In several randomized, controlled studies, postoperative radiotherapy demonstrated either no increase26,27 or a decrease28 in survival compared with resection alone. Postoperative chemotherapy has also been compared with surgical management alone in several randomized controlled trials, without demonstrating an improvement in survival.29 According to these results, the effect of perioperative treatment is not likely to influence our data.\nWe found a tentative relationship between higher volume and a better relative survival. However, this issue still is a debatable problem in determining treatment guidelines. Therefore, we suggest that guidelines concerning specific referral of esophageal cancer patients should be based on hospital outcomes, preferably in experienced centers, rather than on annual numbers of procedures as long as the factor that is determining patient survival is still unknown.\nThe individual surgeon could be an important parameter in determining the hospital outcome. Although the implications of the assertion that some surgeons have better outcomes than others make clinicians uncomfortable, there should be little doubt that it is true. Variation in performance has been shown to be related to surgeon characteristics, including surgical volume, subspecialization, and the hospital setting in which they operate.7,30 Individual surgical experience has been associated with the postoperative mortality of esophageal cancer. Sutton et al.9 showed a reduction in mortality from 6% to 3% after 150 procedures. Miller et al.14 published results in one center demonstrating an operative mortality of 22% among esophageal resections performed by a surgeon who performed fewer than six procedures a year. These results are widely quoted in the surgical literature as proving that surgeons without the necessary expertise should not perform esophageal resections.\nThere is some evidence that subspecialization improves outcomes. Herr et al.31 found that patients who underwent radical cystectomy by urology oncologists had substantially lower rates of local tumor recurrence than those who were operated on by general urologists (6% vs. 23% (P = .006). Dueck et al.32 reported that patients who underwent surgery for a ruptured abdominal aortic aneurysm had markedly better outcomes when the surgery was performed by a vascular surgeon rather than a general surgeon. The effect of subspecialization of the surgeon on the outcome of esophageal cancer has not yet been studied, but it may be a promising factor for decisions with regard to centralization.\nIt has been suggested in previous reports that the skill of the anesthesia and nursing staff affects morbidity and relative survival of esophagectomy patients and that it confounds the surgeon\u2019s personal outcome.7,9 Better critical-care experience of the support staff may explain a higher relative survival in university hospitals; staff may be more adept at caring for esophagectomy patients. Some authors have suggested that the expertise of the anesthesia and nurses in a hospital is directly correlated to the hospital and surgical load in that hospital.9 However, expertise can be acquired elsewhere, and expertise only develops through effective feedback, not only by number of patients.\nAlthough referral to dedicated centers possibly results in improved relative survival, the focus entails some disadvantages, which should be considered. Referral to centers means that many patients have to travel to distant sites, which can create hardship for the patient and his or her family. In-hospital family support and postoperative follow-up are more difficult when the hospital is farther away. Finlayson et al.33 demonstrated that 45% of the patients prefer to stay in their local area even if the projected operative mortality is doubled. However, that study represents the American situation, and it is questionable whether distance is perceived to be a problem in the Dutch situation. In our region, which has a relatively high density of hospitals, a recent patient survey showed that traveling distance was not considered a critical issue.\nIn conclusion, we demonstrated that in our region, the relative survival for patients operated on for esophageal cancer is better in the university hospital compared with teaching nonuniversity and nonteaching hospitals, emphasizing the need for referral to centers focused on the treatment of esophageal cancer. The underlying parameter for the observed difference remains unclear. We suggest that centers at least periodically review the morbidity and mortality rates of esophageal resections to assess their outcome and the possibility of referral. Eligibility for centers focused on esophageal cancer treatment should therefore be based on patient outcomes rather than on patient numbers.","keyphrases":["expertise","referral","survival benefit","esophageal carcinoma"],"prmu":["P","P","M","R"]}
{"id":"Purinergic_Signal-2-3-2096647","title":"Excitatory effect of ATP on rat area postrema neurons\n","text":"ATP-induced inward currents and increases in the cytosolic Ca2+ concentration ([Ca]in) were investigated in neurons acutely dissociated from rat area postrema using whole-cell patch-clamp recordings and fura-2 microfluorometry, respectively. The ATP-induced current (IATP) and [Ca]in increases were mimicked by 2-methylthio-ATP and ATP-\u03b3S, and were inhibited by P2X receptor (P2XR) antagonists. The current\u2013voltage relationship of the IATP exhibited a strong inward rectification, and the amplitude of the IATP was concentration-dependent. The IATP was markedly reduced in the absence of external Na+, and the addition of Ca2+ to Na+-free saline increased the IATP. ATP did not increase [Ca]in in the absence of external Ca2+, and Ca2+ channel antagonists partially inhibited the ATP-induced [Ca]in increase, indicating that ATP increases [Ca]in by Ca2+ influx through both P2XR channels and voltage-dependent Ca2+ channels. There was a negative interaction between P2XR- and nicotinic ACh receptor (nAChR)-channels, which depended on the amplitude and direction of current flow through either channel. Current occlusion was observed at Vhs between \u221270 and \u221210 mV when the IATP and ACh-induced current (IACh) were inward, but no occlusion was observed when these currents were outward at a Vh of +40 mV. The IATP was not inhibited by co-application of ACh when the IACh was markedly decreased either by removal of permeant cations, by setting Vh close to the equilibrium potential of IACh, or by the addition of d-tubocurarine or serotonin. These results suggest that the inhibitory interaction is attributable to inward current flow of cations through the activated P2XR- and nAChR-channels.\nIntroduction\nThe rat area postrema (AP) is a medullary circum-ventricular organ located in the hindbrain at the level of the obex, with a dense vascular supply devoid of a blood-brain barrier. The lack of a blood-brain barrier makes the AP ideally placed to act as a chemosensitive trigger zone involved in the control of vomiting in response to circulating emetic substances (Borison [1, 2]). Neurons in the AP are also responsive to changes in osmolarity or sodium concentration (Franchini et al. [3]), and can be activated by circulating vasoactive peptides such as angiotensin II (Fink et al. [4]), and arg-vasopressin (Undesser et al. [5]). Anatomical studies have revealed that the AP sends dense efferent projections to the nucleus tractus solitarius, parabrachial nucleus, nucleus ambiguus, and the dorsal motor nucleus of the vagus, and receives afferent inputs from the hypothalamic paraventricular and dorsomedial nucleii, and from the caudal nucleus tractus solitarius (Morest [6]; Kooy and Koda [7]; Shapiro and Miselis [8]). Thus, the AP is not only capable of responding to circulating hormones, but is also anatomically well situated to regulate a range of other central neurons, including those important in cardiovascular control (Sun and Spyer [9]). The low intrinsic firing rates of AP neurons in vivo (Papas et al. [10]) suggests that understanding and modulating excitatory inputs to AP could be particularly important in the functional output of AP neurons. A number of transmitters can evoke excitatory currents in the AP. Inward currents and increases in cytosolic Ca2+ ([Ca]in) via non-NMDA-glutamate receptors have been reported in rabbit and rat AP neurons, respectively (Jahn et al. [11]; Hay and Lindsley [12]). Our preliminary reports indicated that ATP also induces inward currents and [Ca]in increases via the activation of P2X receptor (P2XR) in isolated rat AP neurons (Sorimachi et al. [13, 14]). In addition, pre- and post-synaptic nicotinic ACh receptors (nAChR) have been demonstrated in the AP in rat brain slices (Funahashi et al. [15]), and we have recently reported the presence of nAChRs in dissociated rat AP neurons (Sorimachi and Wakamori [16]). During that study, we also noticed that many of these AP neurons also responded to ATP, which has prompted us to further investigate ATP responses in AP neurons, and potential interactions between nAChR and P2XR responses. In a variety of different peripheral neurons, including sympathetic neurons of bullfrog (Akasu and Koketsu [17]), rat (Nakazawa et al. [18]), and guinea-pig (Searl et al. [19]), cultured guinea-pig enteric and submucosal neurons (Zhou and Galligan [20] Barajas-Lopez et al. [21]), a negative interaction between P2XR- and nAChR responses has been reported. Such an interaction has also been observed for recombinant P2X2 and \u03b13\u03b24 nAChR channels in Xenopus oocytes and HEK cells (Khakh et al. [22, 23]; Boue-Grabot et al. [24]), where it has been recently suggested that this results from direct physical interactions between co-localized receptors (Khakh et al. [23]). In this study we more fully describe P2XR responses in AP neurons, demonstrate cross-inhibition between P2XRs and nAChRs and characterize some of the features of this cross-inhibition.\nExperimental procedures\nPreparation of AP neurons\nThe study was approved by the Committee on Animal Experimentation, Kagoshima University. Wistar rats (13\u201318 days-old) were anaesthetized with ether and decapitated. The brain was quickly removed from the skull and placed in ice-cold HEPES-buffered saline containing 150 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES and 5.5 mM glucose. The pH of the saline solution was adjusted to 7.4 by adding tris (hydroxymethyl) aminomethane. The brain was sliced at a thickness of 400 \u00b5m with a microslicer (DTK-1000, Dosaka, Kyoto, Japan), and the slices were kept in bicarbonate-buffered saline bubbled continuously with 95% O2\u20135% CO2 at room temperature (21\u201326 \u00b0C). The bicarbonate-buffered saline contained 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 20 mM NaHCO3, 2 mM KH2PO4 and 5.5 mM glucose. Following 30\u201360 min incubation, the slices were treated with pronase (0.2 mg\/ml) in bicarbonate-buffered saline for 20 min at 33 \u00b0C, followed by incubation for 30 min with bicarbonate-buffered saline containing thermolysin (0.12\u20130.15 mg\/ml). The bilateral AP regions were identified with a binocular microscope (Zeiss, Germany), and were cut out using the tip of an injection needle, and subsequently mechanically triturated with fire-polished glass pipettes of decreasing diameters. Dissociated neurons were placed on to the bottom of 35 mm culture dishes (Falcon, USA) for electrophysiological recordings, or on to glass coverslips (Matsunami, Japan) coated with poly-l-lysine for [Ca]in measurements.\nElectrophysiological recordings\nElectrical measurements were done using the whole-cell patch-clamp recording configuration under voltage-clamp conditions. Patch pipettes were fabricated from borosilicate glass tubes in five or six stages using a pipette puller (Model P-97, Sutter Instrument, San Rafael, CA, USA). The resistance between the recording electrode filled with the internal solution, and the reference electrode in the external solution, was 3\u20136 M\u03a9. Ionic currents were measured, and voltages controlled, using a patch-clamp amplifier (EPC-9, Heka, Darmstadt, Germany). All experiments were carried out at 24\u201326 \u00b0C. Culture dishes were placed on an inverted microscope (TE200, Nikon, Japan), and drugs were rapidly applied to single cells using a Y-tube perfusion device. The internal solution (patch pipette solution) contained 70 mM K-gluconate, 50 mM KCl, 10 mM NaCl, 0.5 mM CaCl2, 3 mM MgCl2, 10 mM HEPES, 10 mM EGTA, and 2 mM ATP. The pH was adjusted to 7.2 with KOH. For measuring current-voltage (I\u2013V) relationships, a Cs-based internal solution was employed, which contained 98 mM CsOH, 40 mM CsCl, 98 mM aspartate, 2 mM MgCl2, 5 mM HEPES, 5 mM EGTA, and 2 mM ATP. pH was adjusted to 7.2 with CsOH. The external solution was the HEPES-buffered saline, pH7.4, described above. When N-methyl-d-glucamine (NMDG+) or sucrose was substituted for external Na+ or NaCl, respectively, the osmolarity of the solution was kept constant as measured using an osmometer (Vogel OM801, Germany). To obtain the Ca2+\/Na+ permeability ratio (PCa\/PNa), we measured the reversal potential of the ATP-activated current (EATP) in the presence of 1 or 110 mM of external Ca2+ by stepping the holding potential (Vh) between 10 and 30 mV, using increments of 10 mV. The 1 and 110 mM Ca2+ solutions contained 155 or 0 mM NaCl, respectively, in addition to 10 mM HEPES with pH adjusted to 7.4 with NaOH or Ca(OH)2, respectively. The values of PCa\/PCs and PNa\/PCs were calculated using the constant field equation as described by Lewis ([25]), taking the activity coefficients of Na+, Cs+, and Ca2+ as 0.75, 0.75, and 0.3, respectively.\nMeasurement of [Ca]in\nDissociated AP neurons on a glass coverslip were incubated with 1\u20132 \u00b5M fura-2 acetoxymethyl ester (fura-2\/AM), 0.1% dimethyl sulfoxide, and 1% bovine serum albumin for 45 min at 37 \u00b0C. The coverslips were then mounted in a superfusion chamber and placed on the stage of an inverted microscope (Diaphot-TMD, Nikon, Japan). Cells were continuously superfused at a rate of 1 ml\/min with HEPES-buffered saline at 24\u201326 \u00b0C via a polyethylene tube placed 1\u20132 mm away from the cells.\nCells were viewed with a 40\u00d7 Fluor objective lens (Nikon), and a single cell (10\u201312 \u00b5m diameter) was fixed in a window positioned between the photomultiplier and the microscope. The changes in fluorescence ratios at 340 and 380 nm excitation wavelengths were measured using a CAM-200 spectrometer (Jasco, Japan). The absolute value of [Ca]in was calculated using the formulae as given by Grynkiewicz et al. [26]: . Calibration constants were determined in separate experiments with the same experimental set-up, as described previously (Sorimachi et al. [27]).\nStudent\u2019s paired t test was used to evaluate differences between mean values obtained from the same cells, and Student\u2019s unpaired t test was used for data obtained from different groups of cells.\nReagents\nThe following reagents were used: Fura-2\/AM [Dojindo, Kumamoto, Japan], ACh [Horai Chem. Co., Japan], PPADS (pyridoxal-phosphate-6-azophenyl-2\u2032, 4\u2032-disulphonic acid) [RBI, USA.], pronase [Calbiochem., USA], thermolysin, ATP, 2-methylthio-ATP, ATP\u03b3S, \u03b1,\u03b2-methylene-ATP, \u03b2,\u03b3-methylene-ATP, ADP, nitrendipine, nicardipine, suramin [all from Sigma, Aldrich, Tokyo, Japan], \u03c9-conotoxin-MVIIA, \u03c9-conotoxin-MVIIC and \u03c9-agatoxin-IVA [all from Peptide Institute, Osaka, Japan].\nResults\nATP-induced current\nRapid application of ATP (100 \u00b5M) to isolated AP neurons voltage clamped at a Vh of \u221270 mV induced an inward current, which desensitized slowly (Fig. 1A). The ATP-induced current (IATP) was recorded at various Vhs between \u221270 and +50 mV (Fig. 1: N = 6), and the relative current amplitude was plotted against Vh exhibiting a strong inward rectification. The EATP, estimated from the intersection of the current response and the zero voltage-axis (Fig. 1B), was 22.7 \u00b1 0.9 mV (N = 18).\nFig. 1Current-voltage relationship for ATP-induced currents. A) Representative currents in response to 200 \u00b5M ATP recorded at Vhs between \u221270 and +50 mV. B) Current-voltage relationship for the IATP. All responses are normalized to the peak current amplitude obtained at a Vh of \u221270 mV, and each point is the mean \u00b1 S.E.M. of five neurons. C) Averaged concentration-current relationships for ATP in the presence of 0.1, 2, and 10 mM external Ca2+. All responses were normalized to the mean of two control responses induced by 100 \u00b5M ATP in saline containing 2 mM external Ca2+ before and after the test response. Each point is the average \u00b1 S.E.M of responses from four to six neurons.\nTo investigate the ATP concentration-response relationship, care was taken to adjust the pH of the solutions containing higher concentrations of ATP to 7.3, since addition of ATP reduced pH and a small decrease in pH to 7.1 increased the IATP to 189 \u00b1 8% (N = 6) of control (pH7.3), as previously reported for recombinant P2X2 receptors (King et al. [28]). The responses to different ATP concentrations were flanked by responses to the control concentration of 100 \u00b5M, and normalized responses were expressed relative to the average of these control responses (Fig. 1C; Ca2mM). We also examined the effects of 0.1 and 10 mM Ca2+ on IATP. As summarized in Fig. 1C, an increase in the external concentration of Ca2+ shifted the concentration-response curve for ATP to the right, with the half-maximum effective concentration (EC50) values at 0.1, 2 and 10 mM Ca2+ being 30, 70, and 190 \u00b5M, respectively.\nThe purinergic agonists, 2-methylthio-ATP (50 \u00b5M) and ATP\u03b3S (50 \u00b5M) induced currents that were 41 \u00b1 3% (N = 4), and 41 \u00b1 4% (N = 5), respectively, of the IATP in response to 100 \u00b5M ATP. ADP (0.5 mM) did not produce any response. The IATP in response to 50 \u00b5M ATP was inhibited by the P2 antagonists; suramin (10 \u00b5M and 20 \u00b5M) and PPADS (50 \u00b5M), with the response being 35 \u00b1 2% (N = 5), 25 \u00b1 2% (N = 4), and 56 \u00b1 5% (N = 5), of the control IATP, respectively.\nATP-induced [Ca]in increase\nATP also increased [Ca]in in a dose-dependent manner (Fig. 2A). These responses were all recorded in the presence of 2 mM external Ca2+, and the [Ca]in increases by various concentrations of ATP were normalized to that induced by 100 \u00b5M ATP. The normalized and averaged responses to ATP, 2-methylthio-ATP and ATP\u03b3S are shown in Fig. 2B. There was no ATP-induced [Ca]in increase in the absence of external Ca2+ (N = 6; data not shown). Furthermore, neither ADP, \u03b1,\u03b2-methylene-ATP nor \u03b2,\u03b3-methylene-ATP increased [Ca]in when tested at concentrations of 200 \u00b5M(data not shown). The ATP-induced [Ca]in increases were inhibited by suramin and PPADS (Table 1).\nFig. 2Concentration-response relationships for the [Ca]in increases in response to ATP and ATP analogues. A) Representative [Ca]in increases induced by various concentrations of ATP in the presence of 2 mM external Ca2+. B) Averaged concentration-response relationships for ATP and ATP analogues. All responses were normalized to the mean of two control responses induced by 100 \u00b5M ATP before and after the test response. Each point is the mean \u00b1 S.E.M. of data from five to nine neurons. MeS-ATP: 2-methylthio-ATP.Table 1Effects of P2 receptor antagonists and Ca2+ channel blockers on the [Ca]in increases induced by ATP and high K+-salineStimulusBlockersPercent of controlATP100 MSuramin 10 M55 \u00b1 7 (N = 5)20 M30 \u00b1 5(N = 6)50 M13 \u00b1 3 (N = 5)PPADS 10 M96 \u00b1 6 (N = 3)20 M70 \u00b1 8 (N = 9)50 M25 \u00b1 5 (N = 6)100 M11 \u00b1 5 (N = 4)110 mMKClNitrendipine 2 M44 \u00b1 4 (N = 16)\u2212CT.M C 2 M61 \u00b1 4 (N = 16)\u2212CT.M A 2 M62 \u00b1 6 (N = 16)\u2212CT.M A 2 M + \u2212CT.M C 2 M58 \u00b1 5 (N = 10)ATP100 MNitrendipine 2 M56 \u00b1 4 (N = 21)Nicardipine 2 M47 \u00b1 9 (N = 10)Cd2+ 50 M27 \u00b1 3 (N = 14)\u2212CT.M A 2 M + \u2212CT.M C 2 M77 \u00b1 9 (N = 4)The mean [Ca]in increase induced by the first and third applications of ATP (100 \u03bcM) or 110 \u03bcM KCl were averaged and referred to as 100%, and the response in the presence of the P2 antagonist or Ca2+ channel blocker was expressed as a percentage of this control value. The cell was pre-treated for 30 s with the indicated agent before the second stimulation with 100 \u03bcM ATP or 110 mM KCl. Number of experiments is shown in parentheses. \u03c9-CT \u03c9-conotoxin.\nEffects of Ca2+ channel antagonists on ATP-induced [Ca]in increase\nPrevious results, using cultured rabbit AP neurons, demonstrated the presence of a \u03c9-conotoxin-M Csensitive Ca2+ response, but that did not involve L- or N-type Ca2+ channels (Hay et al. [29]). We also investigated the effects of various Ca2+ channel antagonists on [Ca]in increases induced by high KCl (110 mM) and ATP (100 \u00b5M). Antagonist for the L-type (nitrendipine, nicardipine), N-type (\u03c9-conotoxin-M A), or P\/Q-type (\u03c9-conotoxin-M C) Ca2+ channels each substantially inhibited these [Ca]in increases (Table 1). In contrast, the selective P-type Ca2+ channel antagonist, \u03c9-agatoxin IVA (1 \u00b5M), did not have any inhibitory effect on the [Ca]in increases (98 \u00b1 5% of control, N = 4).\nATP-induced current in the absence of external Na+\nWhen external Na+ was completely replaced by NMDG+, and in the absence of external Ca2+ (0 Ca2+ plus 0.5 mM EGTA), the IATP was markedly reduced to 8 \u00b1 1% of the control IATP recorded in the presence of external Ca2+ and 150 mM Na+ (N = 9, Fig. 3A). This current was further reduced to 4 \u00b1 1% (N = 3) in the presence of 20 \u00b5M suramin. The small suramin-sensitive, Na+-independent current supports the previous suggestion that the P2XR channel is permeable to glucosamine (Nakazawa [30]). To confirm this, ATP did not induce a current at all when sucrose (0.25 M) was substituted for NaCl (again in the absence of external Ca2+, N = 5). Addition of Ca2+ to the NMDG+-substituted saline further increased IATP. The currents induced by 500 \u00b5M ATP in the presence of 2 and 10 mM Ca2+, but in the absence of external Na+ (replaced by NMDG+) were 18 \u00b1 2% (N = 11) and 29 \u00b1 2% (N = 14, Fig. 3B) of the control IATP, respectively. Similarly, the currents induced by 500 \u00b5M ATP in the presence of 2 and 10 mM Ca2+ added to the sucrose-substituted saline were 6 \u00b1 1% (N = 6), and 13 \u00b1 2% (N = 16; Fig. 3C) of the control IATP, respectively.\nFig. 3ATP-induced current in Na+- and NaCl-free saline containing 0 and 10 mM Ca2+. A) A representative IATP recorded in Na+-free (Na+ replaced by NMDG+) and Ca2+-free saline (containing 0.5 mM EGTA) at a Vh of \u221270 mV. B) A representative IATP in Na+-free (NMDG+) saline containing 10 mM Ca2+. C) A representative IATP in NaCl-free (NaCl replaced by sucrose) saline containing 10 mM Ca2+.\nWe also measured the EATP of IATP in the presence of 1 mM external Ca2+ and 150 mM NaCl or 110 mM external Ca2+ and 0 mM NaCl. The EATP at 110 mM Ca2+ was 21.1 \u00b1 1.0 mV (N = 18), from which we calculated a PCa\/PCs of 6.3. The EATP at 1 mM Ca2+ was 20.2 \u00b1 1.0 mV (N = 13), from which we calculated a PNa\/PCs ratio of 2.1. From these, we obtained a PCa\/PNa ratio of 3.0, confirming the substantial Ca2+ permeability of P2XRs.\nNegative interaction between P2XR and nAChR channels\nRequirement of actual current flow through receptor channels for cross-inhibition\nIt has previously been reported that there is mutual occlusion between P2XR and nAChR in some neurons (Nakazawa et al. [18]; Nakazawa [30]; Searl et al. [19]; Zhou and Galligan [20]; Barajas-Lopez et al. [21]; Khakh et al. [22, 23]; Boue-Grabot et al. [24]). To examine whether there were negative interactions between the ACh-activated current (IACh) and IATP, one receptor agonist was added in the presence of the other. As shown in Fig. 4A, the IACh (100 \u00b5M) was markedly reduced when activated in the presence of ATP (50 \u00b5M). We next examined IACh (100 \u00b5M) in the presence of various concentrations of ATP. With 2, 10, 20, and 100 \u00b5M ATP, IACh was reduced to 95 \u00b1 1% (N = 20), 84 \u00b1 2% (N = 24), 60 \u00b1 4% (N = 15), and 46 \u00b1 6% (N = 7), respectively, of control (P < 0.01 except at 2 \u00b5M ATP). Thus, the IACh inhibition became stronger as the concentration of ATP was increased and with a higher agonist-receptor occupancy. In fact, when IATP at 100 \u00b5M ATP was markedly inhibited in the presence of 200 mM PPADS (8 \u00b1 2% of control, N = 6), IACh was not occluded (97 \u00b1 1% of control; N = 6). When the peak amplitude of IATP, evoked by various concentrations of ATP, was plotted against the ratio of IACh in the presence and absence of ATP, there was an inverse correlation between the amplitudes of these responses (Fig. 4B). Conversely, when ATP (50 \u00b5M) was applied in the presence of ACh (100 \u00b5M), IATP was also occluded (Fig. 4C). Again, when IACh at 200 mM ACh was nullified in the presence of 1 mM hexamethonium, a nAChR antagonist (0.2 \u00b1 0.2% of control, N = 6), IATP was 96 \u00b1 2% (N = 6) of control. IACh at 10, 20, 50, and 200 \u00b5M ACh were 6 \u00b1 2% (N = 5), 20 \u00b1 4% (N = 5), 61 \u00b1 8% (N = 6), and 139 \u00b1 8% (N = 6) of IACh at 100 \u00b5M. There was also an inverse correlation between the peak amplitude of IACh, evoked by various concentrations of ACh, and the ratio of IATP in the presence and absence of ACh (Fig. 4D). However, the inhibition of IATP by nAChR activation was weaker than that of IACh by P2XR activation. As the IACh desensitized faster than the IATP, the weaker inhibition of IATP by ACh could be due to the reduced amplitude of the IACh at the time of ATP application.\nFig. 4Inhibition of IACh and IATP, respectively, in the presence of ATP and ACh. A) Sample currents in response to ACh (100 \u00b5M) in the presence of ATP (50 \u00b5M). The responses to ACh applied in the presence of ATP were reduced. The extent of occlusion was normalized to the control ACh responses recorded before and after ATP application. This sequence was routinely repeated twice or three times, and the averaged amount of occlusion was plotted in B. B) Relative amplitude of the ACh current amplitude in the presence of ATP (ordinate) plotted against the peak amplitude of IATP obtained in the same neuron (abscissae). Note the negative correlation between these two variables. The line was drawn using KaleidaGraph (Synergy Software). Vh: \u221270 mV. C) The IATP was recorded in the presence of 100 \u00b5M ACh and this response was flanked by control ATP alone responses. This sequence was routinely obtained twice or three times in the same cell, and the averaged amount of occlusion was plotted in D. D) The ratio of the ATP current amplitude in the presence and absence of ACh (ordinate) is negatively correlated with the peak amplitude of IACh (abscissae). Vh : \u221270 mV.\nTo examine this possibility, IATP was measured in the presence of both ACh and d-tubocurarine (dTc, 10 \u00b5M) or serotonin (5HT, 50 \u00b5M), two experimental conditions which change the time course of the IACh response. The former agent, dTc, has been shown to be both an open channel blocker and a competitive antagonist of nAChR (Manalis [31]), and, as expected, a low concentration (10 \u00b5M) slightly reduced the peak amplitude of IACh (79 \u00b1 4% of control, N = 11) and caused the IACh to be terminated within 0.5 s (Fig. 5A, insert). Once the IACh response had returned to baseline (in the presence of ACh and 10 \u00b5M dTc) and ATP was applied, there was no inhibition of IATP (95 \u00b1 1% of control, N = 5; Fig. 5A). The IATP in the presence of ACh alone was 77 \u00b1 4% of control (N = 5, P < 0.01; Fig. 5A). When we measured the current (IACh+ATP+dTc) induced by a combination of ACh, ATP and dTc, the peak amplitude of IACh+ATP+dTc was occluded under these conditions, being 74 \u00b1 2% (N = 10) of the predicted sum of IATP and IACh+ dTc (Fig. 5B). The level of IACh+ATP+dTc at 0.5 s following ligand application, which corresponds to a time when the nAChR channels are completely blocked (Fig. 5A, insert), was 96 \u00b1 3% (N = 8) of the control IATP, measured immediately before and after (Fig. 5C). These results suggest that the inhibition of IATP by IACh disappears immediately after the IACh is abolished, although ACh is still present and bound to the nAChRs. 5HT has been shown to also accelerate the decay of IACh (Grassi et al. [32]; Garcia-Colunga and Miledi [33]; Sorimachi and Wakamori [34]), but not to the same extent as dTc. 5HT at 50 \u00b5M decreased the peak amplitude of IACh slightly, to 88 \u00b1 4% (N = 6) of control, and reduced the time constant (tau) of IACh decay from 2.02 \u00b1 0.18 to 1.06 \u00b1 0.19 s (N = 6). As shown in Fig. 5D, the extent of inhibition of IATP by ACh was smaller (to 92 \u00b1 2% of control, N = 6) in the presence of ACh and 5HT compared to that in the presence of ACh alone (to 73 \u00b1 3% of control, N = 6; P < 0.01).\nFig. 5Loss of the inhibition of IATP by ACh in the presence of dTc and 5HT. A) Representative IATP in control conditions (applied alone), in the presence of ACh and in the presence of ACh and dTc. This sequence was routinely repeated twice, and the averaged response was obtained. Note the very transient nature of IACh in the presence of dTc, with the current returning to baseline levels within 0.5 s (insert). Note also the lack of occlusion under these conditions. Vh: \u221270 mV. B) Effect of dTc on IACh+ATP. The sequence of applications was repeated multiple times in each neuron. The peak amplitude of IACh+ATP+dTc was smaller than the predicted sum of IATP and IACh+dTc. C) Rapid recovery of IATP from inhibition by ACh in the presence of dTc. The amplitude of IACh+ATP+dTc 0.5 s following application was equal to the mean of two control IATP at an equivalent time. Inset shows the rapid decline of IACh+ATP+dTc on an expanded time scale. D) Representative IATP in control conditions, in the presence of ACh, and in the presence of both ACh and 5HT. In this example, the tau of IACh in response to the first application was 2.7 s, while the tau of IACh+5HT was 0.9 s, showing the acceleration of the IACh decay by 5HT. Note the reduced occlusion under these conditions. Vh : \u221270 mV.\nNext, we measured the current induced by the concomitant applications of ACh and ATP (IACh+ATP) at a Vh of \u221270 mV. As shown in Fig. 6A, IACh+ATP was 75 \u00b1 1% (N = 44; P < 0.01) of the predicted sum of IACh and IATP. The decay phase of IACh, IATP and IACh+ATP were fit to a single exponential function, giving tau of 3.0 \u00b1 0.2, 7.5 \u00b1 0.6 and 3.7 \u00b1 0.2 s, respectively. When the decay of IACh was accelerated by the presence of 100 \u00b5M 5HT, the tau of IACh, IATP, and IACh+ATP were 0.8 \u00b1 0.3 s (N = 7), 7.4 \u00b1 1.4 s (N = 7) and 2.6 \u00b1 0.8 s (N = 7), respectively (the amplitude of IACh+ATP under these conditions was 76 \u00b1 2% of the predicted sum of IACh and IATP). Thus, IACh+ATP desensitization occurs with kinetics that cannot be explained by the desensitization kinetics of IACh or IATP alone, suggesting that IACh+ATP is mediated via both receptors.\nFig. 6Voltage dependence of IACh and of IATP occlusion. A) At a Vh of \u221270 mV, co-application of ACh (200 \u00b5M) and ATP (100 \u00b5M) induced an inward current that was smaller in amplitude than the predicted sum of the individual IACh and IATP. B) At a Vh of +40 mV, the amplitude of the outward current induced by the co-application of ACh (500 \u00b5M) and ATP (200 \u00b5M) was equal to the predicted sum of the individual IACh and IATP. C) Co-application of ATP and ACh does not markedly change EACh or EATP. Representative currents induced by ACh, ATP, and by co-application of ACh and ATP at a Vh of +10 mV. Note that IACh+ATP is composed of early outward and delayed inward current, corresponding to the ACh component and the ATP component, respectively.\nWhen Ca2+ was omitted from extracellular solution, the negative interaction between the two receptor channels was still obtained; with IACh+ATP being 77 \u00b1 1% (N = 5; P < 0.01) of the predicted sum of IACh and IATP. Hence negative interaction is not mediated by a Ca2+ influx-dependent mechanism, although both nAChR and P2XR are Ca2+-permeable cation channels (Fieber and Adams [35]; Rogers and Dani [36]). This result, however, does not necessarily rule out the possible involvement of [Ca]in in negative interaction.\nOcclusion was not only observed at a Vh of \u221270 mV; IACh+ATP at Vhs of \u221220, and \u221210 mV were 80 \u00b1 2% (N = 12; P < 0.01), and 76 \u00b1 2% (N = 8; P < 0.01), respectively, of the predicted sum of IACh and IATP. In sharp contrast, such occlusion was not observed at a positive potential; the outward current caused by coapplication of ACh and ATP at a Vh of +40 mV was 100 \u00b1 1% (N = 10) of the predicted sum of individual current (Fig. 6B).\nWe next investigated the possibility that the combined addition of ACh and ATP alters the driving force for Na+, by attempting to measure EACh+ATP. In these studies, currents in response to ACh, ATP and both ligands were measured at various fixed potentials close to the reversal potential for IACh and IATP. The EACh and EATP were 8.7 \u00b1 1.5 mV (N = 5) and 21.0 \u00b1 1.6 mV (N = 7), respectively, and IACh+ATP measured at a potential between these two reversal potentials (e.g.10 mV, Fig. 6C) showed a combination of both the ACh-induced outward current and the ATP-induced inward current. These results suggest that the driving force for permeant ions during combined ACh and ATP is similar to that during the application of each ligand separately.\nThe question arises as to whether occlusion is specific just for Na+ ions or whether inward currents carried by cations other than Na+ can also contribute to occlusion during co-activation of nAChRs and P2XRs. To address this question, we measured IACh, IATP, and IACh +ATP in the presence of Na+-free (NMDG+) saline containing 10 mM Ca2+ (Fig. 7). NMDG+ does not permeate through nAChR (Sorimachi and Wakamori [16]), and hence IACh would be mediated by only Ca2+ influx, whereas IATP would be mediated via both NMDG+ and Ca2+ influx (Fig. 3). Under these conditions, IACh+ATP was again occluded, being 70 \u00b1 3% of the predicted sum of IACh and IATP (N = 11; P < 0.01; Fig. 7).\nFig. 7Occlusion of IACh+ATP in the presence of Na+-free saline containing 10 mM Ca2+. IACh, IATP or IACh+ATP was recorded 20 s after switching to Na+-free (replaced by NMDG+) saline containing 10 mM Ca2+. The sequence of applications was repeated multiple times. Following each ligand application, the external solution was changed back to normal saline containing 150 mM Na+ before the next ligand application. Vh: \u221270 mV.\nIn a further attempt to distinguish whether channel activation or ion permeation was primarily responsible for occlusion of one channel by the other, we compared IATP with IACh+ATP under two experimental conditions, in which a higher concentration of ACh should activate nAChR but produce very little current. In the absence of Na+ and Ca2+ (replaced by NMDG+) application of ACh induces a negligible current (Sorimachi and Wakamori [16]), while ATP induces a substantial current under the same conditions (Fig. 3A). As shown in Fig. 8B, there was no occlusion in these conditions; the amplitude of IATP+ACh was 105 \u00b1 1% (N = 10) of the predicted sum of IATP and IACh. We also investigated currents in response to the ligands at a potential close to the EACh (7 \u00b1 2 mV in this experiment, N = 13), taking advantage of the more positive EATP (22.7 \u00b1 0.9 mV), so that at this potential only the IATP was observed. The amplitude of IATP+ACh was 105 \u00b1 3% (N = 13) of that of IATP, and IACh was close to zero (Fig. 8D). These results are, however, in contrast to that obtained in sympathetic neurons (Nakazawa[30]), in which IATP in the absence of both Na+ (replaced by glucosamine) and Ca2+ was inhibited in the presence of ACh, which caused no current (Nakazawa [30]). In these experiments, we only included data in which IATP was large enough to be clearly resolved, greater than 50 pA (corresponding to a control IATP at a Vh of \u221270 mV larger than 1.5 nA). These results suggest that actual current flow through both nAChR and P2XR is responsible for occlusion.\nFig. 8Lack of inhibition in response to co-application of ACh and ATP under two experimental conditions designed to virtually eliminate IACh. A) Control IACh recorded at a Vh of \u221270 mV in standard saline. B) Current responses recorded at \u221270 mV from 20 s after exchanging the standard saline for a Na+-free (replaced by NMDG+), Ca2+-free saline. This sequence was repeated twice or three times. Note the small amplitude of IACh, and the lack of occlusion of IACh+ATP. C) Control IACh recorded at a Vh of \u221270 mV in another neuron. D) Representative traces of IACh, IATP, and IACh+ATP recorded at a Vh of +7 mV, which is very close to EACh. Note the lack of occlusion at this potential, when current flow through the AChR is negligible.\nDiscussion\nIn this study, we found that ATP induced an inward current and increased [Ca]in in isolated rat AP neurons. The ATP-induced current and [Ca]in increase were mimicked by ATP\u03b3S and 2-methylthio-ATP, but not by \u03b1,\u03b2-methylene-ATP, \u03b2, \u03b3-methylene-ATP nor ADP, and was inhibited by suramin and PPADS, suggesting that it was mediated by P2XRs. These results are in good agreement with previous histochemical findings, which demonstrated the presence of P2X2, P2X4, and P2X6 receptor mRNAs (Collo et al. [37]), and of P2X2 receptor immunoreactivity in AP neurons (Kanjhan et al. [38]; Atkinson et al. [39]).\nThe IATP showed strong inward rectification and the EATP was 22.7 \u00b1 0.9 mV (Fig. 1B). The amplitude of IATP varied inversely with the extracellular Ca2+ concentrations (Fig. 1C). The inhibitory effect of increasing extracellular Ca2+ has also been reported in rat sensory neurons (Krishtal et al. [40]), PC-12 cells (Nakazawa et al. [18]), ventromedial hypothalamic neurons (Sorimachi et al. [41]), and in cells expressing recombinant P2X2 receptors (Evans et al. [42]; Virginio et al. [43]), where an allosteric alteration of the ATP binding sites has been suggested to be the underlying mechanism.\nThe IATP was markedly reduced, but still persisted when NMDG+ was substituted for external Na+ even in the absence of Ca2+ (Fig. 3A). Since ATP did not induce a current when sucrose was substituted for external NaCl, our result suggests that NMDG+ could permeate through P2XR. A substantial IATP has been similarly reported in PC-12 cells and sympathetic neurons when glucosamine was substituted for Na+ (Nakazawa et al. [18]; Nakazawa [30]). The addition of Ca2+ to NMDG-Cl- and sucrose-substituted saline increased the IATP (Fig. 3B and C, respectively), indicating that Ca2+ also permeates through P2XR channel. We quantified the relatively high Ca2+ permeability, obtaining a PCa\/PNa ratio of 3.0. A relatively high permeability of P2XR to Ca2+ has been reported in previous studies (Nakazawa et al. [18]; Sorimachi et al. [41]; Evans et al. [42]; Virginio et al. [43]). Although direct influx of extracellular Ca2+ through P2XR channel may thus contribute to the ATP-induced [Ca]in increase, membrane depolarization and the secondary activation of voltage-dependent Ca2+ channels could also make a significant contribution to the [Ca]in increase. In fact, the high K+- and ATP-induced [Ca]in increases were substantially inhibited by a range of Ca2+ channel antagonists, including those which block L- and N-type Ca2+ channels (Table 1). This is in contrast to a previous study, which reported an absence of the L- and N-type Ca2+ channels in rabbit AP neurons (Hay et al. [12]). The discrepancies between their results and ours could be, at least in part, accounted for by the use of different AP preparations (cultured rabbit neurons vs. acutely dissociated rat neurons).\nThe present results, in combination with our previous demonstration of nAChR on AP neurons (Sorimachi and Wakamori [16]), suggest that both ATP and ACh may act as excitatory neurotransmitters in AP neurons, although their release from presynaptic nerve terminals has not yet been reported. We also report a negative functional interaction between P2XRs and nAChRs in AP neurons, as has been observed in a variety of peripheral neurons and in recombinant expression systems (Nakazawa et al. [18]; Nakazawa [30]; Zhou and Galligan [20]; Barajas-Lopez et al. [21]; Searl et al. [19]; Khakh et al. [22, 23]; Boue-Grabot et al. [24]). This is the first report, that we are aware of, of such interactions occurring in central neurons. When ACh was applied in the presence of ATP, there was a positive correlation between the peak amplitude of inward IATP and the amount of occlusion of inward IACh (Fig. 4A and B). The converse was also true when ATP was applied in the presence of ACh (Fig. 4C and D). Non-additivity of the IACh+ATP was observed even when the inward current was carried by NMDG+ and\/or Ca2+ (Fig. 7). Co-application of two agonists did not seem to change the driving force for Na+, because at a Vh between EACh and EATP an outward current due to the activation of nAChR, followed by an inward current due to the activation of P2XR, was observed (Fig. 6C). Occlusion was also observed at a Vh of \u221210 mV, which is closer to EACh or EATP. Furthermore, the removal of external Ca2+ did not alter the occlusion, and thus a Ca2+-mediated mechanism does not contribute to the current occlusion.\nThe IACh+ATP occlusion was observed at all negative holding potentials when the current was inward but was not observed for outward currents at a Vh of +40 mV. Here the outward IACh+ATP was not different from the predicted sum of IACh and IATP (Fig. 6B). The same dependence on current direction has been reported in guinea-pig enteric and submucosal neurons (Zhou and Galligan [20]; Barajas-Lopez et al. [21]), suggesting that the current occlusion was triggered by the inward movement of cations through two channels.\nSome investigators have shown that the amplitude of IACh+ATP is equal to or even smaller than that of either IACh or IATP. For instance, the concentration of one agonist causing little or no inward current produced dramatic occlusion of the inward current generated by the other agonist (Searl et al. [19]). Khakh et al. [22] using Xenopus oocytes co-expressing P2X2 and \u03b13\u03b24 nAChR channels, provided several lines of evidence which indicated that occlusion was mostly mediated by the inhibition of the nAChR channel by activation of the P2XR. However, we found that concomitant application of two agonists always caused a larger current than either agonist (Fig. 6A), and that the amount of occlusion of one channel current in the presence of the other channel agonist was correlated with the amplitude of current through the other channel (Fig. 4). Furthermore, we found that IACh+ATP desensitizes faster than IATP, but more slowly than IACh, and IACh+ATP at a Vh between EACh and EATP showed a combination pattern of early outward IACh and delayed inward IATP (Fig. 6C). These results both strongly suggest that in AP neurons, IACh+ATP are carried through both nAChR and P2XR channels, and that inhibition between these channels is reciprocal.\nThe IATP was not inhibited by co-application of ACh when the inward IACh was markedly reduced in the absence of permeant cations (Fig. 8B) or at a Vh very close to EACh (Fig. 8D), and the inhibited IATP in the presence of ACh (Fig. 5B) recovered as soon as the nAChR channel closed in the presence of dTc (Fig. 5C). These results thus suggest that the inhibitory interaction not only requires the activation of both receptor channels, but also requires a substantial current to flow through these channels. These results are in contrast to that obtained in sympathetic neurons, in which the ATP-induced glucosamine influx was inhibited in the presence of ACh (Nakazawa [30]). The cause of this inconsistency remained unknown, but may reflect a real difference in the underlying mechanisms of occlusion in these two types of neurons.\nAltogether, our results have characterized the P2XR responses in AP neurons and the cross-inhibition between P2XRs and nAChRs in AP neurons. We show that the current flow through one receptor channel hinders the current flow through the other channel. These interactions support the notion that these channels are positioned very close to each other, as has been previously considered (Zhou and Galligan [20]; Barajas-Lopez et al. [21]; Khakh et al. [22]; Boue-Grabot et al. [24]) and more recently demonstrated for recombinant channels (Khakh et al. [23]). These results will be important to consider when designing ligands to modify excitability of ATP neurons and may have some physiological function during co-activation of P2XRs and nAChRs by synaptically released transmitters.","keyphrases":["atp","cytosolic ca2+ concentration","whole-cell patch-clamp recording","fura-2 microfluorometry","ach","negative interaction between nicotinic and p2x channels"],"prmu":["P","P","P","P","P","R"]}
{"id":"Breast_Cancer_Res_Treat-3-1-2096638","title":"Loss of expression of FANCD2 protein in sporadic and hereditary breast cancer\n","text":"Fanconi anemia (FA) is a recessive disorder associated with progressive pancytopenia, multiple developmental defects, and marked predisposition to malignancies. FA is genetically heterogeneous, comprising at least 12 complementation groups (A\u2013M). Activation of one of the FA proteins (FANCD2) by mono-ubiquitination is an essential step in DNA damage response. As FANCD2 interacts with BRCA1, is expressed in proliferating normal breast cells, and FANCD2 knockout mice develop breast tumors, we investigated the expression of FANCD2 in sporadic and hereditary invasive breast cancer patients to evaluate its possible role in breast carcinogenesis. Two tissue microarrays of 129 and 220 sporadic breast cancers and a tissue microarray containing 25 BRCA1 germline mutation-related invasive breast cancers were stained for FANCD2. Expression results were compared with several clinicopathological variables and tested for prognostic value. Eighteen of 96 (19%) sporadic breast cancers and two of 21 (10%) BRCA1-related breast cancers were completely FANCD2-negative, which, however, still showed proliferation. In the remaining cases, the percentage of FANCD2-expressing cells correlated strongly with mitotic index and percentage of cells positive for the proliferation markers Ki-67 and Cyclin A. In immunofluorescence double staining, coexpression of FANCD2 and Ki-67 was apparent. In survival analysis, high FANCD2 expression appeared to be prognostically unfavorable for overall survival (p = 0.03), independent from other major prognosticators (p = 0.026). In conclusion, FANCD2 expression is absent in 10\u201320% of sporadic and BRCA1-related breast cancers, indicating that somatic inactivating (epi)genetic events in FANCD2 may be important in both sporadic and hereditary breast carcinogenesis. FANCD2 is of independent prognostic value in sporadic breast cancer.\nIntroduction\nFanconi anemia (FA) is a recessive disease with both autosomal and X-linked inheritance. FA is associated with progressive pancytopenia, developmental defects, and marked predisposition to malignancies, especially acute myeloid leukemia and squamous cell carcinoma of the head and neck [1, 2]. FA cells are characterized by spontaneous chromosomal instability and hypersensitivity to DNA cross-linking agents such as mitomycin C (MMC). FA is genetically heterogeneous and comprises at least 12 complementation groups (A\u2013M). Eleven of the FA genes have been identified so far: FANCA, FANCB, FANCC, FANCD1\/BRCA2, FANCD2, FANCE, FANCF, FANCG, FANCJ, FANCL, and FANCM [3\u201318]. Eight FA proteins (A, B, C, E, F, G, L, and M) form a nuclear protein complex which is required for mono-ubiquitination of the downstream FA protein, FANCD2. Activation of FANCD2 by mono-ubiquitination is an essential step in the DNA damage response induced by MMC or ionizing irradiation [2, 10]. This DNA damage response pathway also includes the breast cancer susceptibility genes BRCA1 and BRCA2, also referred to as the FA-BRCA pathway. Following ionizing radiation, FANCD2 and BRCA1 accumulate and colocalize in nuclear foci, which reflect sites of DNA damage and repair [10, 19]. Like FA cells, cells lacking BRCA1\/2 proteins are hypersensitive to DNA cross-linking agents. D\u2019Andrea et al. showed that FANCD1 and BRCA2 are the same proteins. BRCA2 is a direct regulator of RAD51, a protein essential for homologous recombination repair [20]. Although BRCA1 is mainly involved in hereditary breast cancer [21], it has also been implicated in sporadic breast cancer [22]. In an immunohistochemical analysis, we have previously shown that FANCD2 is expressed in proliferating cells of different organs, including the premenopausal breast duct epithelium [23]. This is in line with the role of FANCD2 in DNA repair which is important to guarantee the integrity of the genome during cell replication [10]. As deregulation of proliferation is one of the crucial processes of carcinogenesis, these observations imply a potential role for FANCD2 in the pathogenesis of breast cancer. Indeed, FANCD2 knockout mice develop breast tumors [24]. These considerations prompted us to investigate the expression of FANCD2 in sporadic and hereditary invasive breast cancers by immunohistochemistry in relation to several other proliferation-related biomarkers and survival.\nMaterials and methods\nTissue microarray\nParaffin blocks containing formaldehyde-fixed breast cancer tissues of 129 cases of invasive breast cancer not selected for family history (further denoted \u201csporadic\u201d) were obtained from the archives of the Department of Pathology of the VU University Medical Center, Amsterdam. For all breast cancer cases, age, lymph node status, and tumor size were documented. A second array block was constructed containing 24 cases with a proven BRCA1 germline mutation identified through the Family Cancer Clinic of the VU University Medical Center as previously described [25]. Patient characteristics are shown in Table\u00a01. The hematoxylin\u2013eosin stainings were used to identify representative areas of tumor tissue in the blocks. A tissue microarray was then constructed by transferring tissue cylinders of 4\u20135\u00a0mm from the representative tumor area of each donor block to the recipient block using a tissue arrayer (Beecher Instruments, Sun Prairie, WI, USA) as described before [26]. A third tissue array block of 220 sporadic breast cancer patients with long-term follow-up was obtained from the archives of the Gerhard-Domagk Institute of Pathology, University of Muenster, as previously described [27].\nTable\u00a01Patient characteristics and histology of 120 sporadic and hereditary breast cancersSporadicBRCA1 mutationTotalNo. of patients9624120Age\u00a0\u00a0\u00a0Mean654262Lymph node status\u00a0\u00a0\u00a0Negative58967\u00a0\u00a0\u00a0Positive381149Tumor size\u00a0\u00a0\u00a0Mean2.342.782.40Histological type\u00a0\u00a0\u00a0Ductal8517102\u00a0\u00a0\u00a0Lobular718\u00a0\u00a0\u00a0Medullary123\u00a0\u00a0\u00a0Tubular1\u20131\u00a0\u00a0\u00a0Cribriform1\u20131\u00a0\u00a0\u00a0Apocrine1\u20131\u00a0\u00a0\u00a0Metaplastic\u201344\nSections of 4\u00a0\u03bcm were cut and transferred on SuperFrost+ (Menzel&Glaeser, Germany) slides for immunohistochemistry. Use of anonymous or coded left over material for scientific purposes is part of the standard treatment contract with patients in our hospital [28].\nImmunohistochemistry\nImmunohistochemical analysis had been previously performed on conventional sections for the following markers: Ki-67, Cyclin A, p21, p27, p53, estrogen receptor (ER), progesterone receptor (PR), HER-2\/neu, and EGF-receptor [29].\nRabbit polyclonal antiserum against FANCD2 was generated as previously described [23]. Tissue sections were deparaffinized and rehydrated. Endogenous peroxidase was blocked with methanol\/0.3% H2O2 (20\u00a0min). Sections were heated (30\u00a0min, 120\u00b0C) in 0.1\u00a0M citrate buffer pH 6. Unspecific binding was blocked with a 1:50 normal goat serum in PBS pH 7.4\/1% BSA. Polyclonal rabbit anti-FANCD2 (200\u00a0\u03bcg\/ml) was diluted 1:500 in PBS\/1% BSA, and sections were incubated overnight (4\u00b0C) in a humidified chamber. Subsequently, sections were incubated with HRP-conjugated secondary antibodies (EnVision, DAKO) and diaminobenzidin (10\u00a0min), counterstained with hematoxylin (20\u00a0s), dehydrated, and cover-slipped. Appropriate positive controls were used throughout, and negative controls were obtained by omission of the primary antibodies.\nPercentages of positively stained nuclei were estimated by an experienced observer (P.J.v.D.), except for HER-2\/neu and EGF-receptor where membrane staining was scored as positive. In addition, FANCD2 intensity was scored semiquantitatively as 0\u20133, and an FANCD2 score was calculated for each case by multiplying the % FANCD2-positive cells by the staining intensity.\nFor FANCD2\/Ki-67 double staining, anti-FANCD2 was diluted 1:150, incubated overnight, followed by incubation with swine anti-Rabbit HRP 1:200 (Dako, Glostrup, Denmark), and detected with the TSA\u2122 Tetramethylrhodamine system (PerkinElmer Life Sciences, Boston, USA). This was immediately followed by incubation with mouse anti-Ki-67 1:50 (MIB1, Immunotech, Marseille, France) followed by rabbit anti-mouse FITC 1:40 (Dako, Glostrup, Denmark). Nuclei were counterstained by incubation with TO-PRO-3 (Molecular Probes, Eugene, OR, USA) 1:5,000 as previously described [30].\nStatistics\nBivariate scatter plots were generated between the percentage of FANCD2-expressing cells and the other continuous features. For the proliferation-associated features Ki-67, Cyclin A, and MAI, the cases with no FANCD2 expression were excluded from the analysis, assuming that FANCD2 is, by some mechanisms, no longer expressed in these cases. By linear regression analysis, the correlation coefficient R and related p values were calculated. Student\u2019s t test was used to compare FANCD2 expression levels between the low-level vs. high-level groups for HER-2\/neu, the EGF-receptor, ER (cut off 10%) and PR (cut off 10%), p53 (cut off 10%), and Cyclin D1 (cut off 5%). Prognostic value of FANCD2 (Muenster cases) was assessed by computing Kaplan\u2013Meier curves, and differences between the curves were evaluated with the log-rank test. Multivariate survival analysis was performed by Cox regression.\nResults\nSuccessful FANCD2 staining was performed in 96 of the 129 cases of the VUmc sporadic array block. The drop outs were caused by damaged or detached cores during cutting, mounting, or staining, or did not contain tumor. Eighteen of these cases (19%) were completely FANCD2-negative. The other cases showed variable staining from 1 to 85% of the nuclei. In 21 of the 24 BRCA1 cases, FANCD2 staining was performed successfully. In two of these (9.5%), FANCD2 expression was completely negative. Figure\u00a01 shows some representative examples of FANCD2 staining.\nFig.\u00a01Examples of FANCD2 staining in sporadic (A\/B, A\u00a0=\u00a0negative control) and BRCA1-related breast cancers (C\/D, C\u00a0=\u00a0negative control)\nThe mean percentage of FANCD2-expressing cells was significantly higher in ER-negative patients (p\u00a0=\u00a00.019), PR-negative patients (p\u00a0=\u00a00.016), EGFR-positive patients (p\u00a0=\u00a00.002), Cyclin-D1-negative patients (p\u00a0=\u00a00.002), and p53-positive patients (p\u00a0<\u00a00.001) (Table\u00a02). No statistically significant difference was seen for HER-2\/neu. When analyzing the sporadic and hereditary subgroups, similar associations were seen. FANCD2 staining intensity yielded no useful correlations and no prognostic value, and the FANCD2 score yielded essentially the same correlations and prognostic value as the % FANCD2-positive cells (data not shown).\nTable\u00a02Mean percentage of FANCD2-positive cells in high- and low-level expression groups of hormone receptors, growth factor receptors, Cyclin D1, and p53 in sporadic and hereditary breast cancersNumber (%)Mean (SE) of FANCD2%p Value (t test)Total117 (100)ER\u00a0\u00a0\u00a0Low56 (48)14 (2.5)0.019\u00a0\u00a0\u00a0High61 (52)8 (1.6)PR\u00a0\u00a0\u00a0Low81 (69)17 (1.9)0.016\u00a0\u00a0\u00a0High36 (31)12 (1.9)HER-2\/neu\u00a0\u00a0\u00a0Negative93 (79)16 (1.6)0.797\u00a0\u00a0\u00a0Positive24 (21)17 (3.5)EGF receptor\u00a0\u00a0\u00a0Negative78 (66)13 (1.5)0.002\u00a0\u00a0\u00a0Positive39 (33)19 (3.1)Cyclin D1\u00a0\u00a0\u00a0Low85 (73)12 (1.9)0.002\u00a0\u00a0\u00a0High32 (27)7 (2.6)p53\u00a0\u00a0\u00a0Low88 (75)8 (1.3)<0.001\u00a0\u00a0\u00a0High29 (25)19 (4.3)ER estrogen receptor, PR progesterone receptor\nIn linear regression analysis (Table\u00a03), the percentage of FANCD2-expressing cells was significantly positively correlated to Ki-67 (R\u00a0=\u00a00.502, p\u00a0<\u00a00.0001), Cyclin A (R\u00a0=\u00a00.482, p\u00a0<\u00a00.0001), MAI (R\u00a0=\u00a00.506, p\u00a0<\u00a00.001), and p53 (R\u00a0=\u00a00.379, p\u00a0<\u00a00.0001), and significantly negatively correlated to age (R\u00a0=\u00a00.197, p\u00a0=\u00a00.033), ER (R\u00a0=\u00a00.221, p\u00a0=\u00a00.017), and PR (R\u00a0=\u00a00.204, p\u00a0=\u00a00.028). There was no correlation between FANCD2 and the other continuous features. Figure\u00a02 shows examples of FANCD2\/Ki-67 immunofluorescence double staining, underlining the coexpression of FANCD2 and Ki-67 in invasive breast cancers cells. In the Muenster cases, most of these correlations could be reproduced (age: p\u00a0=\u00a00.063, Ki-67: p\u00a0=\u00a00.001, p53: p\u00a0=\u00a00.003, ER: p\u00a0=\u00a00.034). Only PR was not significant here, and Cyclin A was not performed.\nTable\u00a03Correlation between mean percentage of FANCD2-expressing cells and other continuous clinicopathological variables in sporadic and hereditary breast cancersFeatureRp ValueMAIa0.506<0.001Ki-67a0.502<0.001Cyclin Aa0.482<0.001Age\u22120.1970.033ER\u22120.2210.017PR\u22120.2040.028Tumor size0.0180.852p27\u22120.0740.430p21\u22120.1710.065p530.379<0.001Cyclin D1\u22120.1260.176ER estrogen receptor, PR progesterone receptoraFANCD2-negative cases excludedFig.\u00a02FANCD2\/Ki-67 immunofluorescence double staining in a representative case of invasive breast cancer. Top left: Ki-67 staining. Top right: FANCD2 staining. Bottom left: TO-PRO staining. D: Triple exposure showing coexpression of FANCD2 and Ki-67\nIn the regression analysis between the percentages of FANCD2-expressing cells and Ki-67 and Cyclin A, the completely negative FANCD2 cases were excluded. These FANCD2-negative cases had Ki-67 values between 1 and 65 (mean 16%), Cyclin A values between 0 and 50 (mean 10%), and MAI values between 0 and 37 (mean 11), indicating that these cases had (sometimes even high) FANCD2-independent proliferation.\nIn survival analysis (sporadic Muenster cases), high FANCD2 expression appeared to be prognostically unfavorable (p\u00a0=\u00a00.03). Figure\u00a03 shows the survival curves. In Cox regression including tumor size, lymph node status, ER, and grade, FANCD2 staining appeared to have independent prognostic value for overall survival (p\u00a0=\u00a00.026).\nFig.\u00a03Prognostic value of FANCD2 expression in sporadic invasive breast cancer. Low expressors have a better survival than high expressors (p\u00a0=\u00a00.03, N\u00a0=\u00a0122)\nDiscussion\nThe aim of this study was to investigate the expression of FANCD2 in sporadic and hereditary breast cancers. This was inspired by several observations. First, FANCD2 and BRCA1\/2 are functionally closely linked in the DNA repair response, and BRCA1 and BRCA2 are implicated in hereditary and sporadic breast cancers [21, 22]. Second, targeted deletion of FANCD2 in mice resulted in an increased rate of breast tumors [24]. Third, we have shown that FANCD2 is expressed in proliferating cells in the duct epithelium of the normal breast [23].\nIt appeared that 19% of sporadic breast cancers completely lacked FANCD2 expression. Yet, these FANCD2-negative cases had high mean Ki-67, Cyclin A, and MAI values, indicating that the low FANCD2 levels in these cases cannot be explained by low proliferation. The fact that these FANCD2-negative cases stained for other proteins makes it quite unlikely that the FANCD2 negativity is due to fixation problems. In the FANCD2-negative cases, FANCD2 inactivation may, in view of its important function, have been a hit in carcinogenesis. BRCA1 germline mutation-related breast cancers showed lack of FANCD2 expression in only 9.5% of cases, which fits with the concept that a major hit in an important pathway (in these cases the BRCA1 germline mutation) is usually not associated with further hits in this pathway. It is yet unclear what the mechanism behind the lack of FANCD2 expression in these cases is. It needs to be further studied whether there are inactivating somatic mutations in these cases or whether promoter methylation plays a role.\nFANCD2 expression was strongly correlated with expression of the proliferation-associated features Ki-67, Cyclin A, and MAI, and FANCD2 and Ki-67 were coexpressed in invasive cancer cells. This is likely a reflection of the physiological function of FANCD2 in DNA repair of proliferating cells, rather than an independent overexpression of an altered gene, in line with our previous study where we found a coexpression of FANCD2 and Ki-67 in proliferating cells of various normal human tissues [23]. The observation that high FANCD2 expression indicated poor prognosis fits within the same concept, as rate of proliferation (and thereby FANCD2 expression) is a major cell biological phenomenon determining prognosis [31\u201333]. FANCD2 had prognostic value independent of stage and grade, which can be explained by the fact that proliferation and stage are not strongly correlated, and that grade includes nuclear atypia and tubule formation besides rate of proliferation as measured by mitotic index.\nAlthough heterozygosity for p53 was shown to accelerate epithelial tumor formation in Fancd2 knockout mice [34], a functional link between p53 and Fancd2 has not been described to explain the association found in the present study, which is likely caused by the fact that p53 mutated and thereby p53 protein accumulated tumors show, in general, higher proliferation and therefore more proliferating FANCD2-expressing cells. The same may also hold for the relation between FANCD2 and EGFR expressions, for which also no functional relationship has been described.\nThe negative relation between FANCD2 and age can likely be explained by the fact that BRCA1-related patients that have higher FANCD2 expression are younger.\nWithin the light of the above observations, the question remains why FANCD2 patients do not seem to be predisposed to breast cancer in clinical practice. FA itself is a rare genetic disease where the complementation group D2 constitutes only 1\u20132% of all FA cases and these patients generally have a more severe clinical course. They may therefore simply not live to get breast cancer in an apparent increased frequency. Our results do not indicate that somatic (epi)genetic changes in FANCD2 are a frequent secondary carcinogenetic event in BRCA1 germline-mutated patients, although this needs to be confirmed in a larger study group.\nIn conclusion, FANCD2 expression is absent in 10\u201320% of sporadic and BRCA1-related breast cancers, indicating that somatic inactivating (epi)genetic events in FANCD2 may be important in both sporadic and hereditary breast carcinogenesis. FANCD2 is of independent prognostic value in sporadic breast cancer.","keyphrases":["fancd2","breast cancer","brca1","proliferation","immunohistochemistry","prognosis"],"prmu":["P","P","P","P","P","P"]}
{"id":"Ann_Surg_Oncol-3-1-1914262","title":"The Impact on Post-surgical Treatment of Sentinel Lymph Node Biopsy of Internal Mammary Lymph Nodes in Patients with Breast Cancer\n","text":"Background Since the introduction of the sentinel lymph node (SLN) biopsy in breast cancer patients there is a renewed interest in lymphatic drainage to the internal mammary (IM) chain nodes. We evaluated the frequency of lymphatic drainage to the IM chain, the rate of SLNs that contain metastases and the clinical implications of IM LN metastases.\nThe internal mammary (IM) chain is well known as a site of metastases in patients with breast cancer. The presence of IM lymph node (LN) metastases is a poor prognostic sign as reflected by the UICC-TNM staging system that classifies IM LN metastases as stage N3.1 In earlier times dissection of the IM LNs, as part of an extended mastectomy with IM node dissection, was advocated by some authors but randomized trials failed to show any beneficial effect of the procedure that was accompanied by significant morbidity.2\u20134 Since then, interest in the IM LN chain has waned.\nSince the introduction of the sentinel lymph node (SLN) biopsy in breast cancer patients there has been a renewed interest in the IM LNs. Drainage to this basin is frequently seen on preoperative lymphoscintigraphy, while the localization of the primary tumor and the nanocolloid injection technique appear to influence the frequency of visualized IM nodes.5\u20137 Various authors advocate removal of these IM SLNs,8,9 but the clinical implications of IM LN biopsy are still unclear.\nIn this study we evaluated the frequency of lymphatic drainage to the IM chain, the rate of metastases in the IM SLN and the clinical implications of IM LN metastases.\nPATIENTS AND METHODS\nBetween June 1999 and April 2005, 523 consecutive patients underwent surgical treatment including SLN biopsy as a staging procedure for clinically stage T1-2N0 breast cancer. Data regarding all procedures were collected prospectively in a database of the nuclear medicine department.\nA diagnosis of invasive breast cancer was established preoperatively by fine-needle aspiration or image-guided large core needle biopsy. Sixteen patients who underwent SLN biopsy as a secondary operative procedure following previous excisional biopsy of the primary tumor and one patient who eventually turned out to have multicentric breast cancer were excluded from the study.\nThe study cohort consisted of 506 patients. At the time of the introduction of the sentinel node procedure the ethical committee of the hospital approved the routine use of the SLN biopsy as a staging procedure. All patients received written information regarding the SLN procedure and the possibility of SLNs in the IM chain. The unknown clinical implications of surgically removing IM LNs were discussed with the patients.\nOn the day of the operation, all patients received a combination of peritumoral intraparenchymal and subcutaneous injections of an average of 77.7\u00a0MBq (53\u2013150\u00a0MBq) of 99mTc nanocolloid in a total volume of 0.6\u00a0ml of physiologic saline, given in two to three equal doses. In case of a nonpalpable breast tumor the tracer was injected around the tumor using a 7.5-MHz ultrasound probe (Aloka). After injection the area was massaged until the appearance of the SLN. Continuous visualization was done and imaging started as soon as lymphatic drainage was visualized on the persistence scope and at 2\u20133\u00a0h after injection in both the anterior and lateral direction. Images were obtained during a 2-min imaging time on the Toshiba 901 HG single-head gamma camera, using low-energy high-resolution collimators. A skin marker was placed on the projection of the SN using a handheld \u03b3-ray detection probe (Europrobe, PI Medical diagnostic equipment BV).\nThe operative procedure was carried out in the afternoon of the same day of the 99mTc nanocolloid injection. A \u03b3-ray detection probe and a peritumoral injection of patent blue dye (Bleu patent\u00e9 V, Laboratoire Guerbet, Aulnay-sous-Bois, France) were intraoperatively used for SLN identification. During the operation axillary sentinel nodes were retrieved first and frozen section analysis was done to enable axillary dissection during the same operative procedure in case of LN metastases. When no axillary SLN was visualized on preoperative lymphoscintigraphy, the axilla was nonetheless explored in search for a blue dye containing SLN. When no axillary SLN was identified by either means, axillary LN dissection was performed. Following retrieval of axillary SLNs, surgical exploration for IM SLNs was done. When an IM SLN was visualized on preoperative lymphoscintigraphy, the \u03b3-ray detection probe was used to guide a parasternal intercostal incision through the best-suited intercostal space to harvest the visualized node. A partial rib resection was not routinely done for sentinel IM nodes that were localized behind the ribs.\nIn addition to frozen section analysis of the axillary SLNs, all collected SLNs were formalin-fixed, paraffin embedded and cut at five levels of 250\u00a0\u03bcm. Pathological evaluation followed hematoxylin-eosin and immunohistochemical cytokeratin-8 staining. The presence of axillary and IM LN metastases was classified according to the 2002 version of the UICC-TNM-classification.1\nTo assess the additional operative time of IM SLN biopsy we compared the time between incision and skin closure needed for lumpectomy and mastectomy with and without IM SLN biopsy. We selected four groups of ten patients: those who underwent lumpectomy with or without IM SLN biopsy and those who had had mastectomy with or without IM SLN biopsy. In all patients the operative procedure had started with axillary SLN biopsy. We only evaluated patients who did not have axillary dissection to avoid the potential bias of extra time awaiting the result of frozen section analysis. For each category we selected the last ten patients to avoid the learning curve effect.\nTo evaluate the impact of IM LN metastases on subsequent systemic treatment we assessed the proposed adjuvant treatment strategy in the absence and presence of metastases in these nodes, applying the Dutch national guidelines for treatment of breast cancer (version 2005, http\/\/www.oncoline.nl, summarized in Table\u00a01). Locoregional radiotherapy, comprising the affected breast and\/or thoracic wall, and the ipsilateral axillary, periclavicular and parasternal fields was indicated in patients with four or more metastatic axillary LNs. In patients with IM LN metastases with 0\u20133 tumor-positive axillary LNs a parasternal irradiation was given: in combination with irradiation of the breast in patients who had breast-conserving therapy or as parasternal radiotherapy following mastectomy.\nTABLE\u00a01.Indications for adjuvant chemo- and hormonal systemic therapy according to the Dutch national guidelines 2005Axillary lymph node metastasesPrimary tumor characteristicsTumor >3\u00a0cmTumor >2\u00a0cm and BR grade IITumor >1\u00a0cm and BR grade IIIOther conditionsAge <35\u00a0years: always systemic therapy 60-69 years: chemotherapy when ER- or \u22654 axillary lymph node metastases \u226570 years: no chemotherapyER statusHormonal therapy for the aforementioned indications at all ages if the tumor is ER receptor positive.BR, Bloom-Richardson; ER, estrogen receptor.\nChi-square analysis was performed to evaluate differences in IM SLN visualization rates between groups of patients with various clinicopathological variables and to explore the relation between IM and axillary LN metastases. The ANOVA test was used to explore the relation between age and the visualization of IM SLNs, as well as for the analysis of operative time differences.\nRESULTS\nThe median age of the 506 patients was 60\u00a0years (range 24\u201392\u00a0years) and there were three male patients (0.8%). The distribution of primary tumor characteristics is summarized in Table\u00a02.\nTABLE\u00a02.Comparison of characteristics of patients who had visualized internal mammary sentinel lymph nodes (IM SLNs) versus those who had not visualized IM SLNs on preoperative lymphoscintigraphyAll patientsIM SLN visualizedIM SLN not visualizedPn\u00a0=\u00a0506n\u00a0=\u00a0109n\u00a0=\u00a0397Median age (years) 60 (range 24\u201392) 57 (range 30\u201391)61 (range 24\u201392)0.016*Gender0.36\u00a0\u00a0Male 3(0.6)0(0)3(0.8)\u00a0\u00a0Female 503(99.4)109(100)394(99.2)T-stage0.47\u00a0\u00a0T1316(62.5)73(67.0)243(61.2)\u00a0\u00a0T2184(36.4)36(33.0) 148(37.3)\u00a0\u00a0T34(0.8)0(0)4(1.0)\u00a0\u00a0Tx2(0.4)0(0)2(0.5)Tumor localization <0.001\u00a0\u00a0Cranial47(9.3)11(10.1)36(9.1)\u00a0\u00a0Craniolateral230(45.5)30(27.5)200(50.4)\u00a0\u00a0Lateral25(4.9)2(1.8)23(5.8)\u00a0\u00a0Caudolateral40(7.9)10(9.2)30(7.6)\u00a0\u00a0Caudal18(3.6)1(3.6)17(4.3)\u00a0\u00a0Caudomedial30(5.9)13(11.9)17(4.3)\u00a0\u00a0Medial11(2.2)4(3.7)7(1.8)\u00a0\u00a0Craniomedial80(15.8)34(31.2)46(11.6)\u00a0\u00a0Central25(4.9)4(3.7)21(5.3)Malignancy grade0.63\u00a0\u00a0BRI206(40.7)48(44.0)158(39.8)\u00a0\u00a0BRII196(38.7)38(34.9)158(39.8)\u00a0\u00a0BRIII104(20.6)23(21.1)81(20.4)Estrogen receptor status 0.24\u00a0\u00a0Positive427(84.4)88(80.7)339(85.4)\u00a0\u00a0Negative79(15.6)21(19.3)58(14.6)Axillary lymph node involvement0.58\u00a0\u00a0No axillary metastases296(58.5)68(62.4)228(57.4)\u00a0\u00a01\u20133 lymph node metastases174(34.4)35(32.1)139(35.0)\u00a0\u00a0*4 lymph node metastases 36(7.1)6(5.5)30(7.6)Values in parentheses are percentages.BR, Bloom-Richardson grade.* Age difference between the groups was compared by ANOVA.\nVisualization and Retrieval of Sentinel Lymph Nodes in the IM Chain (Fig.\u00a01)\nSLNs were visualized on preoperative lymphoscintigraphy in 502 of the 506 patients (99%). Axillary SLNs were visualized in 499 of 502 patients (99%), while one or more IM SLNs were found in 109 patients (22%). Three of the 109 patients with visualized IM SLNs had IM sentinel nodes only (3%); in one of these latter patients, an axillary SN was removed following patent blue dye injection and axillary exploration. Location of the tumor in the craniomedial and caudomedial aspect of the breast was associated with drainage to the IM nodes (P\u00a0<\u00a00.001), and patients with visualized internal mammary nodes were younger (Table\u00a02).\nFIG.\u00a01.Summary of search for internal mammary sentinel lymph nodes (IM SLNs).\nIM SLNs could be retrieved through a parasternal intercostal incision in 85 of the 109 patients (78%). Parasternal exploration for IM SLNs added 16\u00a0min to the mean operative time in patients who underwent breast conservative therapy (P\u00a0=\u00a00.02). For patients who underwent mastectomy the operative time was not significantly prolonged (Table\u00a03). Complications (pleural breeching, internal mammary vessel damage) of the intercostal surgical exploration were rare; in particular there were no pneumothoraxes or bleeding complications necessitating drainage or reoperation (Table\u00a04).TABLE\u00a03.Operative time of surgical exploration for internal mammary sentinel lymph nodes (IM SLNs) (n\u00a0=\u00a040)Mean operative time in minutes (range)PAxillary SLN biopsy\/lumpectomy\/IM SLN biopsy60(27\u201376)0.02Axillary SLN biopsy\/lumpectomy\/no IM SLN biopsy44(32\u201383)Axillary SLN biopsy\/mastectomy\/IM SLN biopsy72(42\u2013104)0.8Axillary SLN biopsy\/mastecomy\/no IM SLN biopsy69(30\u2013100)TABLE\u00a04.Complications of surgical exploration for internal mammary sentinel lymph nodes (IM SLNs) (n\u00a0=\u00a0109)nIntraoperative complicationsPleural breeching4(4)Internal mammary vessel damage6(6)Postoperative complicationsPneumothorax\u2212Bleeding necessitating reoperation\u2212Values in parentheses are percentages.\nIM LN Metastases\nMetastases in the IM SLN were observed in 20 of the 85 patients who underwent successful IM SLN exploration (24%), and 42% of the patients with axillary SLNs (210\/499). There was a correlation between IM metastases and axillary metastases (P\u00a0=\u00a00.002). In 16 of the 20 patients with IM LN metastases, the axillary SLN contained metastases too (80%). Conversely, IM metastases were found in 1% of patients without axillary metastases (4\/297), in 7% of patients with 1\u20133 axillary metastases (13\/174) and in 8 % of patients with \u22654 axillary metastases (3\/36).\nClinical Implications of Metastases in the IM SLN (Table\u00a05)\nAdjuvant systemic therapy was already indicated in 14 of the 20 patients with IM metastases due to concomitant axillary LN metastases or unfavorable primary tumor characteristics: ten patients would be candidates for postoperative chemotherapy and 14 would receive hormonal treatment. Based on tumor-positive IM SLNs six additional patients would receive systemic treatment. This proportion reflects 7% of the patients in whom IM SLNs were removed. In four patients chemotherapy was indicated and in five patients hormonal therapy.\nTABLE\u00a05.Clinical postsurgical implications of internal mammary lymph nodes IM LN metastases (n\u00a0=\u00a020)No.Tumor characteristicsAxillary metastasesPostsurgical treatmentTreatment changed due to IM metastasesIM SLN not consideredIM SLN consideredSize (cm)Grade (BR)ER(n)CTHTRTCTHTRT Axilla N4+\/age<701) 63, BCT1.5I+7++LR++LRNo2) 45, BCT1.6III+4++LR++LRNo3) 47, mastectomy1.8I+4++LR++LRNoAxilla N1-3+\/age<704) 39, mastectomy2.5II+3++BCT++BCT+PSRT5) 46, BCT3.5III\u22123+\u2212BCT+\u2212BCT+PSRT6) 58, BCT1.8II+2++BCT++BCT+PSRT7) 45, BCT2.4I+2++BCT++BCT+PSRTAxilla N1-N1a or unfavorable primary tumor characteristics\/age<708) 43, BCT1.8I+1mi\u2212\u2212BCT++BCT+PSCT\/HT\/RT9) 54, BCT2.2II+1mi++BCT++BCT+PSRT10) 66, BCT2.5II+1mi\u2212+BCT\u2212+BCT+PSRT11) 50, BCT2.5III+1mi++BCT++BCT+PSRT12) 42, mastectomy3.0I+1++No++PSRTAxilla N0\/favorable tumor characteristics\/age<7012) 54, BCT2.1I+0\u2212\u2212BCT++BCT+PSCT\/HT\/RT14) 60, BCT2.5I+0\u2212\u2212BCT++BCT+PS CT\/HT\/RT15) 67, mastectomy0.8I+0\u2212\u2212No\u2212+PSHT\/RT16) 61, BCT1.1I\u22120\u2212\u2212BCT+\u2212BCT+PSCT\/RTAge>7017) 82, mastectomy2.8II+2\u2212+No\u2212+PS?RT?18) 71, BCT2.1II+1\u2212+BCT\u2212+BCT+PSRT19) 72, BCT2.1II+1mi\u2212+BCT\u2212+BCT+PSRT20) 85, mastectomy0.9I+1mi\u2212\u2212No\u2212+PS?HT\/RT?BCT, breast-conserving therapy; BR, Bloom-Richardson grade; ER, estrogen receptor status; Nax+, number of positive axillary lymph nodes; IM SLN, internal mammary sentinel lymph node; ST, systemic therapy; CT, chemotherapy; HT, hormonal therapy; RT, radiotherapy; PS, parasternal radiotherapy; 1mi, micrometastases.\nIn 3 of the 20 patients axillary tumor load (\u22654 tumor-positive lymph nodes) was a reason for locoregional radiotherapy including the IM lymphatic chain, leaving 17 patients in whom the radiotherapy field was adjusted because of metastases in the IM SLN. These 17 patients in whom the radiotherapeutic strategy was changed made up 20% of the patients in whom IM sentinel nodes were visualized.\nConversely, there were three patients with \u22654 axillary metastases and IM SLNs without metastases, and in these patients parasternal irradiation was omitted. In addition, parasternal irradiation could also be omitted in 30 patients with \u22654 axillary metastases who had no IM lymphatic drainage on preoperative lymphoscintigraphy.\nDISCUSSION\nIn the present study, SLNs in the IM chain were visualized in approximately one-fifth of the patients who underwent surgery for primary breast cancer. Retrieving these nodes by parasternal intercostal exploration was feasible in the majority of patients. One-fifth of the retrieved IM LNs contained metastases and radiation treatment was adjusted in most of these patients.\nIn this prospective cohort of patients who underwent SLN biopsy for clinically T1-2N0 breast cancer, IM SLNs were seen on the preoperative lymphoscintigraphy in 22% of the patients.5,6,8,10,11 The visualization rate in our study was rather high and the likely result of the tracer injection technique (Table\u00a06). We used a combination of a parenchymatous peritumoral and a subcutaneous injection technique of the 99mTc nanocolloid, and this technique is associated with a higher visualization rate of IM SLNs than the subcutaneous or periareolar injection of the radiotracer.5\u20137,12,13 Apart from the effect of the injection technique we observed a higher frequency of lymphatic drainage to the IM LNs in patients with cranio- and caudomedially located tumors, as reported by others,8,14,15 as well as an effect of age: IM SLNs were more common in young patients.\nTABLE\u00a06.Visualization and surgical extirpation rate of internal mammary sentinel lymph nodes (IM SLNs) in breast cancer patientsAuthorYear nMethod of tracer injectionVisualized IM SLNs (%)Surgically removed IM SLNs (%)*Madsen et al.2006506PT and SC2278Leidenius et al.102005984IT1488Paredes et al.62005383SC, later IT\/PT 0\u20131773Farrus et al.52004225SC, later IT\/PT 11\u20131769Estourgie et al.82003681IT2287van der Ent et al.112002256PT2563* Proportion of the visualized IM SLNs. SC, subcutaneous; PT, peritumoral; IT, intratumoral.\nRetrieving SLNs from the IM chain does not appear to be very troublesome. Approximately 15\u00a0min extraoperative time is needed during breast conservative surgery, while extra time is negligible in patients who undergo mastectomy. Although the success rate was lower than for SLNs in the axilla, 78% of the visualized IM nodes could be harvested by the described parasternal intercostal exploration. Others reported similar \u2018surgical identification\u2019 rates (69\u201388%, see Table\u00a06).5,6,8,10,11 It can be difficult to retrieve the usually very small IM SLNs when they are localized directly behind one of the ribs. \u2018Blind\u2019 retrocostal dissection is not without risks, and we consider a rib resection not justified as long as the place of parasternal lymph node exploration is not well defined. Although the pleural cavity was breeched occasionally during the procedure and a number of patients had postoperative hematomas, postoperative drainage of a pneumothorax or a reoperation for a bleeding complication of the parasternal wound was never necessary. Other studies also reported low morbidity rates.8,9,11,16,17\nIn the present study IM LN metastases were found in 24% of the patients who underwent surgical extirpation of these SLNs, while others usually reported lower rates of metastases containing IM SLNs (9\u201326%).8\u201311,18 In patients with IM metastases axillary metastases were common: 80% had concomitant axillary metastases.19 Conversely, axillary metastases were accompanied by IM metastases in 8% of the cases. Isolated IM metastases were rare affecting 5% of the patients with harvested IM SLNs and approximately 1% of patients without axillary LN metastases.8,10,19\nSuccessful exploration of SLNs from the IM chain had a substantial impact on subsequent radiotherapeutic treatment. In 20% of these patients adjustment of radiotherapy was considered necessary. Although randomized data about the use of radiotherapy in patients with IM metastases are lacking, adding a parasternal irradiation in these patients appears conceivable. After all radiotherapy does affect locoregional control in other high-risk patient groups such as patients with \u22654 axillary metastases and node-negative patients with young age, poor tumor differentation and large tumor size.20 We consider patients with IM metastases at risk for locoregional recurrence and therefore feel that parasternal irradiation is indicated in patients with IM metastases. Conversely, absence of IM lymphatic drainage or metastases in retrieved IM SLNs justified omission of parasternal radiotherapy in 7% of all the patients.\nSystemic treatment strategy was rarely influenced by IM metastases. Due to axillary metastases and unfavorable primary tumor characteristics, half of the patients already would have received chemotherapy and even more would have had hormonal therapy. In the remaining group of patients, old age and negative estrogen receptor status further limited the proportion of patients that would receive adjuvant systemic therapy based on IM LN metastases. However, since prognosis of patients with both axillary and IM metastases is poor when compared with axillary or IM metastases alone,3 patients with IM and axillary metastases might need different chemotherapy schedules. That would increase the proportion of patients in whom adjuvant systemic treatment would be adjusted.\nIs IM SLN biopsy worthwhile? One may argue that more extensive radiotherapy was indicated in less than 5% of all patients and additional systemic treatment in only 1%, and thus consider IM SLN biopsy hardly \u201cworth the effort.\u201d10 We feel that the group of patients with visualized SLNs should be taken as a reference, and that the proportion of patients in whom less radiotherapy was given should also be taken into account. As a consequence, we do consider the clinical implications of IM SLN biopsy substantial.\nIn conclusion, lymphatic drainage of breast cancer to IM LNs is a common feature and retrieving these nodes is relatively easy. The clinical impact of metastases in IM lymph nodes is substantial and justifies surgical exploration for these nodes. We advocate routine parasternal intercostal exploration for IM SLNs whenever preoperative lymphoscintigraphy visualizes IM SLNs. For that purpose we also advocate the (additional) intraparenchymatous tracer injection to optimize the visualization of IM SLNs.","keyphrases":["internal mammary lymph nodes","breast cancer","sentinel node","metastasis"],"prmu":["P","P","P","U"]}
{"id":"Bioprocess_Biosyst_Eng-2-2-1705518","title":"Development of a kinetic metabolic model: application to Catharanthus roseus hairy root\n","text":"A kinetic metabolic model describing Catharanthus roseus hairy root growth and nutrition was developed. The metabolic network includes glycolysis, pentose-phosphate pathway, TCA cycle and the catabolic reactions leading to cell building blocks such as amino acids, organic acids, organic phosphates, lipids and structural hexoses. The central primary metabolic network was taken at pseudo-steady state and metabolic flux analysis technique allowed reducing from 31 metabolic fluxes to 20 independent pathways. Hairy root specific growth rate was described as a function of intracellular concentration in cell building blocks. Intracellular transport and accumulation kinetics for major nutrients were included. The model uses intracellular nutrients as well as energy shuttles to describe metabolic regulation. Model calibration was performed using experimental data obtained from batch and medium exchange liquid cultures of C. roseus hairy root using a minimal medium in Petri dish. The model is efficient in estimating the growth rate.\nIntroduction\nIn vitro plant biotechnology offers a controlled environment and has been widely studied for phyto-pharmaceuticals and recombinant proteins production. However, the low productivity and the poor reproducibility of the cultures are still limiting the economical feasibility of such in vitro bioprocesses. Moreover, the lack of reproducibility of the cultures significantly complicates process validation and acceptance by the regulatory agencies, and thus the potential to rapidly put a product to market. Different approaches have succeeded in decreasing the technological risk associated with in vitro plant biotechnology. The introduction of elicitors such as chitin [1] and jasmonic acid [2] has shown to enhance significantly the production level for many plant species cultured as cell suspensions and hairy roots. The use of an extractive phase allowed the simplification of harvesting procedures [3, 4]. More recently, cell engineering studies have shown its potential in improving cell catalytic capacity towards the production of secondary metabolites [5\u20137] and in in vitro molecular farming for recombinant human proteins [8]. However, high variability levels in cell growth and production of biomolecules of interest are still observed. The genetic flexibility of plant cells may partly explain these phenomena [9] and inadequate culture management may also be involved.\nA plant cell has the ability to accumulate nutrients and metabolites which are involved in the regulation of its metabolic pathways. The key for improving plant cell culture reproducibility may thus rely on a better understanding of the links that are exerting between a plant cell physiological state and its potential towards growth and production of a biomolecule of interest. This understanding (following that of Bailey [10]) could take the form of a descriptive and predictive metabolic model. Such structured model may then be either useful to enhance our understanding of cell behaviour, in identifying possible regulatory roles [11], as well as being a tool for defining adequate controlled culture conditions. Metabolic modelling has been applied to plant cells for studying specific metabolic sub-networks such as photosynthesis [12, 13], respiration [14], cellulose biosynthesis [15] and lipid biosynthesis [16]. These studies have clearly showed the importance of the energy shuttles on the control of the metabolic pathways. In addition, some nutrients are known to be involved in the regulation of the cell metabolism. Intracellular inorganic phosphate (Pi) plays a central role in the regulation of enzymes activity through phosphorylation\/dephosphorylation processes, ATP\/ADP concentration ratio, starch synthesis and storage, and in the flux distribution between the glycolysis and the pentose-phosphate pathway (PPP). Intracellular nitrate and ammonium are known to affect amino acid (AA) production.\nThe large capacity of plant cells for nutrient and metabolite accumulation plays a crucial role in cell growth and biomolecule production, as observed for Daucus carota hairy root [17] and Eschscholtzia californica suspension cells [18]. Recently, we have developed a kinetic model based on intracellular nutrients such as inorganic phosphate, nitrate and sugars which showed to be efficient in simulating carrot hairy root growth for different culture media composition [17]. Therefore, the aim of this work was to include the description of metabolic pathways to the nutritional model in order to describe plant cells behaviour from the estimation of the cells physiological state, including nutritional and metabolic states. Catharanthus roseus was studied as a model biological system. Cell nutritional state in Pi, nitrogen (NO3\u2212 and NH4+) and carbohydrates (sucrose, fructose, glucose and starch) is described. The hypothesis of a central primary metabolism at steady state has been proposed based on literature [19, 20]. Using the metabolic flux analysis (MFA) approach, a model reduction [21] was applied on the central primary metabolism network and resulted in independent pathways. A second network includes transient fluxes such as for nutrient uptake and storage, energy shuttles management and root cells growth. Metabolic regulation of the fluxes from energy shuttles and nutrients is included. The hairy root specific growth rate is described as a function of the content in cell building blocks such as amino acids (including proteins), lipids (LIP), organic acids (ORA), organic phosphates (OP) (including nucleic acids) and structural hexoses (STH). Batch and medium exchange cultures of C. roseus hairy root were performed and the experimental data were used for model calibration.\nModel general structure\nThe model has been first developed by Tikhomiroff [22]. The cell metabolic network (Fig.\u00a01) is divided into two interlinked sub-networks as the stationary (SPMP) (Fig.\u00a02) and the transient (TPMP) primary metabolic pathways (Fig.\u00a01). The SPMP includes glycolysis, PPP, the TCA cycle and the catabolic reactions leading to the cell building blocks. The cell building blocks are amino acids and peptides which were taken as a unique pool of AA, ORA, OP, LIP and STH. The TPMP network is linked to the SPMP network and describes cells growth and nutrient transport between medium and intracellular volumes. Compartmentalization of nutrients and metabolites among the cytosol, the vacuole and other organelles is not included in the model and a single cell population was considered to describe the hairy root cells pools. This simplification already showed to be efficient to model hairy root growth and nutrition [17]. The secondary metabolism is simplified to fluxes leading to two pools, one accounting for the global pool in secondary metabolites derived from tryptamin (TRYSM), and one accounting for the global pool in secondary metabolites derived from secologanin (SECSM). The model is thus composed of interlinked metabolic networks that are at steady (SPMP) and transient (TPMP) states, and is described by a mass balance using the stoichiometric matrix and the hairy root specific growth rate:\nwhere S is the stoichiometric matrix, \u03a6 is a vector containing reaction fluxes,\u00a0\u03bc is the hairy root specific growth rate and M is a vector containing the concentration in cellular metabolites and nutrients. Root mass with time can then be estimated both kinetically and from a mass balance on all cell constituents.Fig.\u00a01The metabolic model global structure. Fluxes in the transient primary metabolic pathways (TPMP). Flux numbers refer to the stoichiometric biochemical reactions of Table\u00a03. Kinetic description of the resulting fluxes is presented in Table\u00a04Fig.\u00a02Fluxes in the stationary primary metabolic pathways (SPMP). Flux numbers refer to the stoichiometric biochemical reactions of Table\u00a01\nStationary primary metabolic pathways\nThe pseudo-steady-state assumption for the central metabolism was based on observations from Rontein et\u00a0al. [19] and Stitt and Fernie [20] and proposed to simplify the model development. The original SPMP metabolic network has 31 fluxes (Fig.\u00a02; Table\u00a01), which were reduced to 20 independent pathways (Fig.\u00a03; Table\u00a02) using the method proposed by Simpson et al. [21] and Stephanopoulos et\u00a0al. [23] and the following simplifications. Briefly, the minimal number of independent feasible metabolic pathways is determined with a group of metabolites assumed at steady state: G6P, F6P, R5P, G3P, E4P, CHO, PEP, PYR, A-CoA, OXO and OAA. The \u201cN\u201d matrix (11\u00d731) is first constructed from the 31 biochemical reactions and the 11 metabolites at steady state. The determination of the independent pathways then requires the knowledge of the kernel matrix \u201cK\u201d (31\u00d7number of independent pathways), which describe each pathway as a linear combination of the 31 biochemical reactions of the SPMP. This matrix is the non-trivial solution of the equation:\nSince this equation has an infinite number of solutions, both \u201cN\u201d and \u201cK\u201d matrices are decomposed each into two matrices:\nwith \u201c N1\u201d and \u201c K2\u201d selected as square invertible matrices. Starting using identity matrix for \u201cK2\u201d, \u201cK1\u201d can then be determined by modifying \u201cK2\u201d such that possible independent pathways can be identified:\nUsing the complete matrix K, we can build the independent pathways presented in Fig.\u00a03. In this work, the size of this matrix is 31\u00d720. The complete method to obtain the pathways can be found in Ref. [22].Table\u00a01Biochemical reactions of the stationary primary metabolic pathwaysFlux\u03bd(1)a\u2013c\u03bd(2)a\u2013c\u03bd(3)a\u03bd(4)a\u03bd(5)b\u03bd(6)b\u03bd(7)a,c\u03bd(8)a\u03bd(9)a\u03bd(10)b\u03bd(11)a\u03bd(12)b\u03bd(13)a\u03bd(14)a\u2013c\u03bd(15)a\u03bd(16)a\u2013c\u03bd(17)c,d\u03bd(18)a\u2013c\u03bd(19)c,d\u03bd(20)a\u2013c\u03bd(21)e\u03bd(22)b\u03bd(23)b\u03bd(24)a\u03bd(25)b\u03bd(26)e\u03bd(27)\u03bd(28)\u03bd(29)d\u03bd(30)a\u03bd(31)da[39]b[24]c[40]d[27]e[41]f[26]Fig.\u00a03Reduced independent biochemical pathways from the SPMP. Pathway numbers refer to the stoichiometric biochemical reactions of Table\u00a02. Kinetic description of the resulting fluxes is presented in Table\u00a04. The 20 independent biochemical pathways were obtained from the 31 biochemical reactions of the SPMP described in Fig.\u00a02 and Table\u00a01, using the reduction method of Simpson et al. [21] and Stephanopoulos et al. [23], as described in Sect. 3Table\u00a02Independent fluxes of the SPMPs after pathways reduction using MFA approachFluxa\u03bd(r1)\u03bd(r2)\u03bd(r3)\u03bd(r4)\u03bd(r5)\u03bd(r6)\u03bd(r7)\u03bd(r8)\u03bd(r9)\u03bd(r10)\u03bd(r11)\u03bd(r12)\u03bd(r13)\u03bd(r14)\u03bd(r15)\u03bd(r16)\u03bd(r17)\u03bd(r18)\u03bd(r19)\u03bd(r20)aThe biochemical reactions are denoted with an \"r\" to indicate that they are obtained from pathways reduction\nThe nutrient concentrations described are glucose (GLC), fructose (FRU), sucrose (SUC), ammonium (NH4) and inorganic phosphate (Pi). The anaplerotic pathways are simplified to the transformation of phosphoenolpyruvate (PEP) into oxaloacetate (OAA). The metabolite pools resulting from the SPMP are starch (STA), STH (cell wall and membrane constituents), OP (nucleotides, phospholipids, nucleic acids), total AA including that of peptides, ORA, pools from tryptamin (TRYSM) and from secologanin (SECSM). Starch biosynthesis (flux v(r14) in Table\u00a02) and catabolism (v(r13)) are included. AA (v(r5)) are assumed to be synthesized from oxoglutarate, since it is the site for ammonium fixation and thus the initiation step in the AA biosynthesis [24], even though there are AA synthesized from other pathways. Tryptamin (TRYSM) biosynthesis (v(r1)) has been explicitly described because this AA is a precursor, with secologanin (SECSM) (v(r2) and v(r10)), to the formation of the secondary metabolites. OP biosynthesis (v(r11)) has been simplified as illustrated in Fig.\u00a03 and is accounting for the four nucleotides. The biosynthesis of STH results from multiple biochemical reactions [25] but it is simplified to the condensation of G6P with R5P (v(r12)). The biosynthesis (v(r7)) and catabolism (v(r15)) of LIP is linked to acetyl-coenzyme A [26] as for ORA biosynthesis (v(r16)) and catabolism (v(r17)). The kinetic expressions of the fluxes are described in the next section.\nTransient primary metabolic pathways\nThe TPMP network includes the biochemical reactions that cannot satisfy the pseudo-steady-state hypothesis. These include nutrients transport and accumulation, root cells growth and energy shuttles dynamics. The general structure of the TPMP and its interactions with the SPMP are illustrated in Figs.\u00a01 and 2. The stoichiometric mass balances are presented in Table\u00a03 and the kinetic terms used in fluxes\u2019 regulation are described in Table\u00a04. Extracellular nutrients such as sucrose (ESUC), glucose (EGLC), fructose (EFRU), ammonium (ENH4), nitrate (ENO3) and Pi (EPi) are included. Sucrose is hydrolysed into fructose and glucose extracellularly by apoplastic invertases (Table\u00a03, v(28)), or intracellularly as described in the SPMP (Table\u00a02, v(r19)). It should be noted that sucrose biosynthesis (Table\u00a02, v(r20)) is not included into the model because there are no evidence that hairy roots have active chloroplasts. v(r19) then represents the net flux of sucrose hydrolysis. Alternative glycolytic pathways [27] are described and the carbohydrate sources include sucrose, glucose, fructose and starch. However, since all the biosynthesis pathways of the SPMP are defined with glucose as precursor, all available intracellular carbon sources have first to be converted into glucose. Fructose conversion (v(r3)), starch catabolism (v(r13)) and sucrose hydrolysis (v(r19)) processes are thus feeding the intracellular glucose pool. Plant cell adaptation mechanism to Pi deficiency is described with the fluxes for the degradation of pyrophosphate (v(26)) and OP (v(23)) into Pi. The plant cell storage capacity for sugars, NO3\u2212, NH4+, Pi and cell building blocks is described. Respiration is described (v(30)), however, oxygen is assumed to be non-limiting as discussed below. Energy loss associated with maintenance and other reactions that are not included into the SPMP is contained in flux v(29).Table\u00a03Biochemical reactions of the transient primary metabolic pathwaysFluxa\u03bd(21)\u03bd(22)\u03bd(23)\u03bd(24)\u03bd(25)\u03bd(26)\u03bd(27)\u03bd(28)\u03bd(29)\u03bd(30)\u03bd(31)aStoichiometric coefficients were taken from Ref. [24]Table\u00a04Kinetic expression of the fluxesFluxa\u2013c is fixed at 0 and no sucrose synthesis is consideredaReaction number refers to pathways of SPMP in Fig.\u00a03, and of TPMP in Fig.\u00a01bNot shown fluxes are related to exchange between the cytoplasm and the vacuole and were not included in this modelcUse of NTP, NDP, NADH, NAD, NADPH and NADP in flux kinetics was taken from Ref. [24]d[42]e[43]f[44]\nMetabolic regulation\nMetabolic regulation of two kinds is integrated into the model: that associated with the energetic status of the cells and that related to the nutritional state of the cells, variable denoted as \u201c\u03b8i\u201d below. Each flux (\u03bd) is regulated as follows, according to a multiplicative kinetic of each mechanism involved.\nThe order of the regulation kinetics can also be adjusted from the term \u03b1i to account for multi-steps mechanisms. Since we established the independent pathways for the SPMP with arbitrary conditions, it is not possible to compare the structure of the resulting kinetics with literature. However, the identified independent pathways have been selected for their feasibility, and their combination is mathematically equivalent to the whole metabolic system (31 fluxes), given that the pseudo-steady-state hypothesis is acceptable.\nFurthermore, a sigmoid function is used for nutrients acting like switches to avoid large discontinuities when solving the differential equations, which would have been the case using a simple on\/off switch type. Continuous functions are also more representative of biological behaviour [28]. The sigmoid function is also used for imposing maximum accumulation levels (see Table\u00a04):\nThe parameter \u201ca\u201d defines the steepness of the function, \u201cMi\u201d is the concentration of the nutrient involved in the regulation of a flux and \u201cMi,t\u201d is the nutrient concentration threshold.\nThe role of energy shuttles in the regulatory mechanisms is described in Table\u00a04. NADH, NADPH and NTP (the sum of ATP, GTP, CTP and UTP) as well as their reduced forms (NAD, NADP and NDP) are used. The sum in energy shuttles per g DW of roots in both oxidized and reduced forms (NADH + NAD; NDP + NTP; NADPH + NADP) are taken constant with time. However, the energetic status of the cells [NADH\/(NADH + NAD); NTP\/(NDP + NTP); NADPH\/(NADPH + NADP)] was not assumed constant. ATPase proton pumps that are linked to H+\/nutrient co-transport and involved in the control of intracellular pH are thus included. ATP consumption for transmembrane transport of glucose (v(21)), fructose (v(22)), sucrose (v(24)), nitrate (v(27)) and Pi (v(31)) is thus included. A unique value for the affinity constant for NTP (KmNTP) is used. The same strategy of using unique affinity constants for NAD, NADH, NADP and NADPH is also applied.\nRoot cells nutritional state in Pi, NH4 and in sugars are involved in metabolic flux regulation. Intracellular Pi is involved in enzyme activation\/deactivation processes through dephosphorylation\/phosphorylation, in the biosynthesis of NTP (v(30)) and of many other metabolites as described below. Intracellular NH4 is involved in AA biosynthesis (v(r5)) (Fig.\u00a03; Table\u00a04). Michaelis\u2013Menten kinetics with NH4 and Pi are at power two since a second-order mechanism is involved. NH4 has first to be transformed into NH2 radical, then into an AA which is finally integrated into a protein structure. Management of carbon source is crucial for plant cells since glucose, fructose, sucrose and starch can be used. However, since the model is based only on glucose, biochemical reactions converting other sugars into glucose are described (Fig.\u00a03; Table\u00a04). Starch biosynthesis (v(r14)) is controlled by the total intracellular available sugar concentration, and the sum in intracellular GLC, FRU and SUC has to be above 0.2\u00a0mmol\/g\u00a0DW for the STA biosynthesis to occur. A maximal STA storage capacity of 1.5\u00a0mmol\/g\u00a0DW was observed experimentally (see Sect. 7). Starch is degraded (v(r13)) when the GLC concentration is below 0.2\u00a0mmol\/g\u00a0DW. PPP (v(r4)) is regulated by NADP\/(NADP+NAPH) ratio, which has to be above 0.5 for the pathway to be active. Pyruvate kinase regulation (v(r6)) is under the control of intracellular Pi concentration with a flux increase at 1\u00a0mmol Pi\/g\u00a0DW and below. G3P conversion into PEP (v(r8)) is regulated by Pi concentration with a flux increase at 1\u00a0mmol Pi\/g\u00a0DW and below. TCA cycle (v(r9)) is regulated by the NAD\/(NAD+NADH) ratio, and a ratio that is above 0.5 will induce a flux increase. GLC (v(21)) and FRU (v(22)) uptake require a STA concentration that is below 0.07 and 0.02\u00a0mmol\/g\u00a0DW, respectively. OP synthesis (v(r11)) is regulated by intracellular Pi concentration with a flux increase at above 0.1\u00a0mmol\u00a0Pi\/g\u00a0DW. OP degradation (v(23)) is controlled by intracellular Pi concentration with a flux increase at below 0.045\u00a0mmol\u00a0Pi\/g\u00a0DW. LIP (v(r15)) and ORA (v(r17)) degradation into A-CoA is controlled by NADH concentration with fluxes increase at below 1\u00a0mmol NADH\/g\u00a0DW. Phosphofructokinase regulation (v(r18)) is controlled by intracellular Pi with a flux increase below 1\u00a0mmol\u00a0Pi\/g\u00a0DW. Respiration rate (v(30)) is controlled by NDP\/(NTP+NDP) ratio with a flux increase below a ratio of 0.5. Finally, regulation of root cells growth rate (v(32) is controlled by the intracellular concentration in the cell building blocks such as OP, LIP, amino acids and peptides (AA), ORA and STH. Monod model was used for LIP, AA and ORA. A hybrid Moser\u2013Monod model was used for OP and STH with each kinetic term at power 4 and 1.25, respectively (Table\u00a04). Since OP and STH are crucial to cell growth, the steepness of the affinity for both cell building blocks was increased.\nMaterials and methods\nCulture conditions\nLiquid cultures of hairy roots were performed in Petri dish as described in Jolicoeur et\u00a0al. [17]. The major problem in culturing hairy roots is the difficulty to obtain a representative sample of the root network since the roots develop a highly dense interlinked bed. Petri dish culture allowed distributing the roots inoculum in a series of dishes with each dish taken as a single sample. Root growth was also limited by the use of a low salt medium, thus preventing the occurrence of dense root network. Previous results in Petri dish [17] and in bioreactor [3] suggest that there was no oxygen limitation in the cultures of this work.\nHairy roots of C. roseus L. G. Don were established as described by Bhadra et\u00a0al. [29], with Agrobacterium rhizogenes strain A4. Hairy roots were transferred every month in Petri dishes in 25\u00a0mL of minimum medium [30] supplemented with 3% (w\/v) sucrose and with a threefold KH2PO4 (0.352\u00a0mM). In the batch culture experiments, approximately 0.125\u00a0g fresh weight (FW) of hairy roots was inoculated in each Petri dish containing 25\u00a0mL of minimum medium. In the medium exchange cultures, approximately 0.125\u00a0g FW of hairy roots was inoculated in Petri dish containing 25\u00a0mL of minimum medium. The medium was renewed at 3- or 2-day intervals to avoid depletion of Pi in the medium. Whole Petri dishes were harvested in triplicates (n=3) periodically and taken as distinct samples. Liquid from each dish was filtered at 0.45\u00a0\u03bcm (Millipore, Billerica, Massachusetts) and stored at \u221220\u00b0C for further analysis. Roots were filtered under vacuum on a glass fiber filter (47\u00a0mm diameter Glass Microfiber filters GF\/D, Whatman, #1823 047) and rinsed three times with 20\u00a0mL of de-ionized water. The filtered roots were removed from the filter and weighed for FW in a disposable aluminum dish (Fisher Scientific, # 08\u2013732) on a precision balance (Sartorius). Fresh roots were immediately frozen into liquid N2 and stored in liquid N2 for further analysis. Root samples were then freeze dried (Duratop and Duradry, FTS Systems Inc., Stone Ridge, NY, USA), weighted for DW measurement and grinded (mortar and pestle) for further analysis. All further analyses were performed using freeze-dried roots.\nAmino acids analysis\nApproximately 2\u00a0mg of freeze-dried roots were extracted in 1\u00a0mL of 2% w\/v 5-sulfosalicylic acid. The samples were sonicated for 15\u00a0min and then centrifuged at 12,000g for 5\u00a0min. The supernatant was analysed for AA by HPLC using a modified method from Gombert et\u00a0al. [31] as described in Benslimane et\u00a0al. [32]. The column is a high-efficiency Nova-Pak TM (C18, 4\u00a0\u03bcm). Precolumn derivatization of the AA was performed using AccQ.Fluor (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, or AQC), which is an N-hydroxysuccinimide-activated heterocyclic carbamate, and the derivatized AA were quantified via a fluorescence detector. \u03b1-Aminobutyric acid was used as an internal standard. The standard error of measurement using this method was routinely below 5%. AA concentration was then calculated since the peak for each individual AA accounted for the combination of free and that from peptides and proteins.\nExtraction of sugars\nApproximately 10\u00a0mg of freeze-dried roots was washed with 80% ethanol and then centrifuged at 16,000g for 5\u00a0min. This wash was done three times and each time the supernatant was kept for glucose, fructose and sucrose analyses. After the third wash the pellet was kept for starch analysis.\nAnalysis of sugars\nGlucose, fructose and sucrose analysis was performed with a Beckman Coulter HPLC (Beckman Coulter Canada Inc, Mississauga, Canada; Pump model 126, automatic injector model 508) equipped with a Gilson model 132 refractive index detector. A Prevail Carbohydrate ES column 4.6\u00a0mm\u00d7250\u00a0mm (Alltech Canada, Guelph, Ontario, Canada), coupled with a Prevail Carbohydrate ES All-Guard 4.6\u00a0mm\u00d77.5\u00a0mm guard column (Alltech Canada, Guelph, Ontario, Canada), was used at a column temperature of 35\u00b0C. The injection volume was 20\u00a0\u03bcL. The mobile phase consisted of acetonitrile and water 75:25 (v\/v) at 1.0\u00a0mL\/min. The cell pellet obtained from the soluble sugars extraction was re-suspended in 1\u00a0mL de-ionized water and sterilized at 121\u00b0C for 15\u00a0min together with a 1\u00a0mL 6\u00a0g\/L starch solution in de-ionized water. Samples were allowed to reach room temperature and their volume was readjusted to 1\u00a0mL with de-ionized water if necessary. Starch calibration standards from 0 to 6\u00a0g\/L were prepared by serial dilutions of the starch solution in de-ionized water. Calibration standards and samples were diluted 1:1 with an amyloglucosidase solution (Sigma, St. Louis, Missouri, USA, Cat. #S9144) and incubated for 15\u00a0min in an ultrasound bath at 60\u00b0C. Samples and standards were centrifuged for 10\u00a0min at 16,000g. A 10\u00a0\u03bcL aliquot of the supernatant was directly transferred into a spectrometric cuvette with 500\u00a0\u03bcL of \u201cGlucose Infinity\u201d reagent (Sigma, Cat. #17-25). After incubation (15\u00a0min) at room temperature, 500\u00a0\u03bcL of 100\u00a0mM KH2PO4 buffer pH\u00a07.5 was added followed by spectrophotometric reading at 340\u00a0nm.\nIons extraction\nIons were extracted from approximately 10\u00a0mg of freeze-dried roots in 1.5\u00a0mL of 5% (w\/v) trichloroacetic acid. This mixture was sonicated at 40\u00b0C for 30\u00a0min and then centrifuged at 16,000g for 10\u00a0min. The supernatant was filtered at 0.45\u00a0\u03bcm and analysed by HPLC.\nIons analysis\nCulture medium and intracellular contents in majors ions (Cl\u2212, NO3\u2212, H2PO4\u2212, SO42\u2212, NH4+, K+, Na+, Ca2+) were analysed using a Dionex HPLC system (Dionex Canada Ltd., Oakville, Canada) equipped with an isocratic pump, an automated sampler AS-3500 and a pulsed electrochemical detector in the conductivity mode, controlled by the Dionex A1-450 software for cations and the Dionex Peaknet software for anions. Anions were separated using a 4\u00d7250\u00a0mm IONPAC AS14A-SC analytical column, an IONPACAG14A-SC guard column and a ASRS-1 anion self-regeneration suppressor to improve the signal-to-noise ratio. The mobile phase consisted of an aqueous buffer of 2\u00a0mM Na2CO3\/1\u00a0mM NaHCO3 solution flowing at a rate of 1.0\u00a0mL\/min. Cations were separated using a 4\u00d7250 IONPAC CS-12 analytical column, a IONPAC CG-12 guard column and a CSRS-1 cation self-regenerating suppressor. The mobile phase was an aqueous 20\u00a0mM methanesulphonic acid solution flowing at a rate of 0.9\u00a0mL\/min.\nModel simulations\nThe model simulations were done using the Matlab software (The MathWorks Inc., Natick, MA, USA). The differential equations system was integrated through the Ordinary Differential Equation solver ode15s.m. The model error minimization was performed by means of manual and algorithm-based methods. The former was done to find good initial estimates based on literature (when available). The latter was then used to reduce the global error between the estimates and the experimental data. The algorithm used was the lsqcurvefit.m subroutine (Optimisation toolbox, Matlab) based on the Levenberg\u2013Marquardt algorithm [33].\nResults and discussion\nModel calibration and determination of the kinetic parameters\nModel calibration was performed using experimental data from C. roseus hairy root batch and medium exchange cultures. Each experiment (one batch and one medium exchange) was performed in triplicate. A total of 14 samples were taken during the batch experiment and 17 during the medium exchange. Parameter values were obtained from experimental data (maximum uptake rates, maximum growth rate, sucrose maximum hydrolysis rate and some of the maximal accumulation levels) and from literature, taking values from other plant species when unavailable for C. roseus (Tables\u00a05, 6, 7). Adjustment of the unknown parameters as well the values taken from literature was performed manually in parallel using a non-linear least-square algorithm (lsqcurvefit) from Matlab software. The least-squares criterion was applied on all the data points (triplicates taken as mean in (Mi)exp). The sum of squares were weighted as described below.\nwhereTable\u00a05Affinity constants (Km)ComponentValueLiteratureSpeciesUnitsReferencesAA0.0136Catharanthus roseusmmol\/g\u00a0DWModel calibrationFRU0.120C. roseusmmol\/g\u00a0DWModel calibrationGLC0.120C. roseusmmol\/g\u00a0DWModel calibrationLIP0.00254C. roseusmmol\/g\u00a0DWModel calibrationNAD0.00023C. roseusmmol\/g\u00a0DWModel calibrationNADH0.00030C. roseusmmol\/g\u00a0DWModel calibrationNADP0.000585C. roseusmmol\/g\u00a0DWModel calibrationNADPH0.00037C. roseusmmol\/g\u00a0DWModel calibrationNDP0.010C. roseusmmol\/g\u00a0DWModel calibrationNH40.2279C. roseusmmol\/g\u00a0DWModel calibrationNTP0.00625C. roseusmmol\/g\u00a0DWModel calibrationORA0.00807C. roseusmmol\/g\u00a0DWModel calibrationPi0.1997C. roseusmmol\/g\u00a0DWModel calibrationPPi0.0737C. roseusmmol\/g\u00a0DWModel calibrationSUC1.00C. roseusmmol\/g\u00a0DWModel calibrationSTA1.00C. roseusmmol\/g\u00a0DWModel calibrationSTH0.2C. roseusmmol\/g\u00a0DWModel calibrationOP0.0206C. roseusmmol\/g\u00a0DWModel calibrationEFRU0.04616Daucus carotamM[17]EGLC0.05356D. carotamM[17]ENO3_HA0.06770.281Citrus reticulatamM[45]0.005\u20130.2Arabidopsis thaliana[46]0.006\u20130.02Zea mays[43]ENO3_LA1.430.5A. thalianamM[46]0.02\u20130.1Z. mays[43]0.16D. carota[17]EPI_HA0.00260.007C. roseusmM[47]0.0056C. roseus[48]0.0035C. roseus[49]0.003C. roseus[49]0.0025Nicotinana tabacum[50]0.0079Lemma gibba[51]0.00049Z. mays[52]0.0018Z. mays[52]0.007Chara corallina[37]0.004C. corallina[37]0.025Nitella translucens[53]0.0024Neurospora crassa[54]0.0029N. crassa[54]0.0026D. carota[17]EPI_LA0.0800.900C. roseusmM[49]0.0463C. roseus[49]0.058Nicotinana glutinosa[55]0.076L. gibba[51]0.190C. corallina[37]0.220C. corallina[37]0.370N. crassa[54]1.029N. crassa[54]0.47D. carota[17]ESUC26.69C. roseusmMModel calibrationNO30.5181C. roseusmmol\/g\u00a0DWModel calibrationTable\u00a06Maximum reaction rates (Vmax)Reaction in TPMPCurrent value (mmol\/g\u00a0DW\/d)Literature (mmol\/g\u00a0DW\/d)SpeciesReferencer174.9934C. roseusModel calibrationr22C. roseusModel calibrationr30.764C. roseusModel calibrationr410,000C. roseusModel calibrationr5150C. roseusModel calibrationr61C. roseusModel calibrationr70.1C. roseusModel calibrationr83.0C. roseusModel calibrationr930.0C. roseusModel calibrationr102.00C. roseusModel calibrationr116.00C. roseusModel calibrationr120.05C. roseusModel calibrationr1315.0C. roseusModel calibrationr142.00C. roseusModel calibrationr165C. roseusModel calibrationr180.764C. roseusModel calibrationr190.1C. roseusModel calibrationr200.01C. roseusModel calibration2115.1C. roseusModel calibration224.6929C. roseusModel calibration230.4986C. roseusModel calibration240.100C. roseusModel calibration251.1377C. roseusModel calibration2633.5326C. roseusModel calibration27 HA0.00150.1555C. reticulata[45]0.072\u20130.1968Z. mays[43]27 LA0.1670.72\u20131.92Z. mays[43]281.001.69D. carota[17]290.382C. roseusModel calibration30900C. roseusModel calibration31 HA0.001753.80C. roseus[47]0.415C. roseus[48]0.127C. roseus[49]0.196C. roseus[49]1.96N. tabacum[50]0.0369N. tabacum[50]0.1728L. gibba[51]0.13824Z. mays[52]0.0092C. corallina[37]0.0576N. translucens[53]3.80N. crassa[54]0.0030D. carota[17]31 LA0.0401.268C. roseus[49]0.090C. roseus[49]0.046N. glutinosa[55]0.265L. gibba[51]0.023L. gibba[51]0.104C. corallina[37]0.0576C. corallina[37]10.4N. crassa[54]5.64N. crassa[54]0.045D. carota[17]GLC0.764C. roseusModel calibration320.1400.27D. carota[17]Table\u00a07State variables and initial values (t=0) used for model simulationsComponentValuesUnitsSpeciesReferencesAA0.818\/0.779mmol\/g\u00a0DWC. roseusBatch\/medium exchangeFRU0.0336\/0.0556mmol\/g\u00a0DWC. roseusBatch\/medium exchangeGLC0.1625\/0.1744mmol\/g\u00a0DWC. roseusBatch\/medium exchangeSEC0.00515mmol\/g\u00a0DWC. roseusUnpublished resultsLIP0.1mmol\/g\u00a0DWN. tabacum suspension cells[34]NAD7\u00d710\u22125mmol\/g\u00a0DWYoung photosynthetic tissue[56]NADH1.25\u00d710\u22125mmol\/g\u00a0DWYoung photosynthetic tissue[56]NADP8\u00d710\u22126mmol\/g\u00a0DWYoung photosynthetic tissue[56]NADPH1.12\u00d710\u22124mmol\/g\u00a0DWYoung photosynthetic tissue[56]NDP2\u00d710\u22123mmol\/g\u00a0DWYoung photosynthetic tissue[56]NH40.053\/0.041mmol\/g\u00a0DWC. roseusBatch\/medium exchangeNTP6.8\u00d710\u22123mmol\/g\u00a0DWYoung photosynthetic tissue[56]ORA0.16mmol\/g\u00a0DWN. tabacum suspension cells[34]Pi0.117\/0.100mmol\/g\u00a0DWC. roseusBatch\/medium exchangePPi0.0015mmol\/g\u00a0DWPotato tubers[34]SUC0.403\/0.330mmol\/g\u00a0DWC. roseusBatch\/medium exchangeSTA1.705\/1.137mmol\/g\u00a0DWC. roseusBatch\/medium exchangeSTH0.081mmol\/g\u00a0DWN. tabacum suspension cells[34]TRY0.001428mmol\/g\u00a0DWC. roseusUnpublished resultsOP0.01mmol\/g\u00a0DWC. roseusModel calibrationEFRU8.33\/29.6mMC. roseusBatch\/medium exchangeEGLC7.22\/23.0mMC. roseusBatch\/medium exchangeENH40mMC. roseusCulture mediumENO33.27\/3.27mMC. roseusBatch\/medium exchangeEPi0.100\/0.100mMC. roseusBatch\/medium exchangeESUC114.0\/149.0mMC. roseusBatch\/medium exchangeNO30.385\/0.345mmol\/g\u00a0DWC. roseusBatchX0.0197\/0.0180gDWC. roseusBatch\/medium exchange\nError terms were weighted using the maximum (max(Mi)exp) and the minimum (min(Mi)exp) experimental values (exp) from either batch or medium exchange cultures. This method was preferred to a method using experimental error because the experimental error was very high for some data points and varied with experimental data (Table\u00a08). This method has also showed previously to perform adequately to calibrate a nutritional model on hairy root data [17]. All the experimental data points from batch and medium exchange culture were used for the non-linear least-square regression. The total number of experimental data was then of 411 with 13 different measurements for each of the 31 samples plus eight measurements for intracellular AA. Many set of parameters were tested as initial guesses to improve the fitting. A combination of parameter values (Tables\u00a05, 6) showed to minimize the global error for combined data from batch and from medium exchange cultures, using a convergence criterion of 1\u00d710\u22126 on the variation of the global error. From the calculation of R2 values (Table\u00a08) for each of the 28 state variables that were fitted (14 state variables in two experiments), it seems that the best fit is obtained for cells growth and nutrient transport and storage (NO3, Pi, NH4). However, the sensitivity analysis also suggests that some parameters can still be optimized (Fig.\u00a04). A critical problem with such a descriptive model is a high number of parameters. The model has 35 maximum reaction rates and 26 affinity constants and the least-square regression was only performed on only 14 of the 31 state variables of the model. It is then possible that the identified solution corresponds to a local minimum. More measurements on metabolite concentration are thus necessary to improve the performance of the model and for being able to perform an adequate identifiability analysis on the parameters. In this context, the proposed model is probably over-parameterized. However, the goal of this work was not to establish a minimal model but a descriptive model that can be used as a tool useful at improving our understanding of plant cell culture.Table\u00a08Mean standard deviations on measured variables and correlation coefficients (R2)ComponentMean SD (%) (batch)Mean SD (%) (medium exchange)R2 (batch)R2 (medium exchange)AA11.8813.830.05210.0413FRU9.9234.960.00120.0415GLC10.8148.290.00580.1059NH49.8626.100.60500.8628Pi35.8512.010.75680.8303SUC25.7945.750.00320.0781STA9.0112.440.00100.0013EFRU15.0618.620.09130.0612EGLC13.2613.910.08380.0785ENO340.766.050.94560.9459EPi26.2743.200.98950.7737ESUC11.2610.830.91230.9461NO313.2624.960.65410.4103X14.7820.460.97500.9708Fig.\u00a04Sensitivity analysis on model parameters for batch (solid bar) and medium exchange (empty bar) cultures. Relative change in error is calculated as described in Sect. 7.1. Parameter adjustment of +50% (a). Parameter adjustment of \u221250% (b). Parameters not shown have absolute relative error changes that are below 0.05\nSimulation of the cells physiological state\nHairy root growth\nThe model allows estimating root growth from flux \u03bd(32) as well as from the total estimated mass of the root cells constituents per dish (\u03a3 Mi on a mass basis), taking average molecular weights for the cell building blocks (Fig.\u00a05). However, the total mass calculated at initial condition (t=0) was 25% lower than that of the inoculum with 0.0148\u00a0g\u00a0DW per dish as compared to 0.0197\u00a0g\u00a0DW per dish, respectively. The difference of \u22120.0049\u00a0g\u00a0DW per dish can be explained from the molecular species that are present in the cells but which are not included in the model. Therefore, the estimation of root mass with time obtained from \u03bd(32) was compared with that calculated from total cell mass, but adding a constant correction factor of +25%. Model simulations then describe hairy root growth for both batch and medium exchange cultures. Both root mass estimates from \u03bd(32) and \u03a3Mi are superimposed (Fig.\u00a05). However, the sum of the molecular species is overestimating measured biomass at the end of batch culture because of cell accumulation in diverse compounds while growth slowed down. Surprisingly, both batch and medium exchange cultures behaved similarly with a maximum specific growth rate of 0.035\u00a0d\u22121 and a growth cessation at around 40\u00a0d. Periodic medium renewal has avoided nutrient depletion in macronutrients and sugars (Fig.\u00a06). However, the plateau observed for the medium exchange culture may have resulted from a limitation in micronutrients, which were not measured. Therefore, only experimental data obtained before the occurrence of the plateau for the medium exchange culture were used for model calibration and other analyses. The strategy of describing cell growth as a function of intracellular content in building blocks thus showed to perform adequately. This result may support the global model structure as well as the hypothesis of pseudo-steady state for the SPMP. The central primary metabolic network, described here as SPMP, was observed at pseudo-steady state by Rontein et\u00a0al. [19] for most of in vitro culture duration for tomato suspension cells [20]. However, this hypothesis has to be further investigated.Fig.\u00a05Model simulation for Catharanthus roseus hairy root batch (filled square) and medium exchange (triangle) liquid cultures in Petri dish. a\u2013e. Intracellular concentration in cell building blocks with time. Batch (f) and medium exchange (g) cultures. The specific growth rate is described from \u03bd(32) as a function of intracellular concentration in cell building blocks. Model simulations for batch (solid lines) and medium exchange (dashed lines) cultures, and total mass for the molecular species accounted in the model increased of 0.0049\u00a0g\u00a0DW accounting for non-estimated molecular species (dashed line). Root mass was calculated using average MW for STA (180.15, based on glucose units); SEC (400); TRY (187); NAD\/NADH (712\/713); NADP\/NADPH (744.4\/745.4); NDP\/NTP (476\/507); ORA (809.75); STH (180.15, based on glucose units); OP (average of 305 from nucleotides); LIP (810, based on that of A-CoA); IPP (246) and AA (average of 136.75)Fig.\u00a06Extracellular concentration in nutrients with time. Experimental data for C. roseus hairy root batch (filled square) and medium exchange (triangle) liquid cultures. Concentration in sugars (a\u2013c), inorganic phosphate (d) and nitrate (e) in the culture media with time. Model simulations for batch (solid lines) and medium exchange (dashed lines) cultures\nThe cells building blocks\nThe measured total AA concentration decreased with time for the batch culture and reached a plateau for the medium exchange culture (Fig.\u00a05a). Simulated AA concentrations seem to follow the trend for experimental data but were overestimated for both batch and medium exchange cultures. The deviations cannot be explained, however, by exuded AA and proteins since no AA were detected in the used culture media. Values of 0.63\u00b10.10 to 0.82\u00b10.10\u00a0mmol\u00a0AA\/g\u00a0DW were measured for the batch culture which corresponds to 0.088\u00b10.014 and 0.12\u00b10.014\u00a0g\u00a0AA\/g\u00a0DW, respectively (taking an average amino acid MW of 140\u00a0g\/mol). Higher values were measured in the medium exchange culture with 0.63\u00b10.15 to 1.1\u00b10.14\u00a0mmol\u00a0AA\/g\u00a0DW, which represents 0.12\u00b10.021 and 0.16\u00b10.020\u00a0g\u00a0AA\/g\u00a0DW, respectively. Free AA content of 0.21\u00a0mmol\u00a0AA\/g\u00a0DW (estimated at 0.029\u00a0g\u00a0AA\/g\u00a0DW) is reported in literature for tobacco suspension cells using B5 medium [34] and 0.15\u00a0mmol\u00a0AA\/g\u00a0DW (estimated at 0.021\u00a0g\u00a0AA\/g\u00a0DW) in potato tuber [35, 36]. In tomato cell culture, the total protein content was of 0.1\u20130.4\u00a0g\u00a0proteins\/g\u00a0DW [19]. The total AA mass per root dry weight that have been measured, which represents the sum of free AA and that of peptides and proteins, is thus within the range reported in literature. The use of a minimal medium may explain a low value for total AA. The other cell building blocks were not measured but the model estimations were close to literature data. Total ORA concentration was estimated at 0.15\u00a0mmol\/g\u00a0DW as compared to 0.42\u00a0mmol\/g\u00a0DW reported by Farr\u00e9 et\u00a0al. [35]. Total STH were estimated at 0.14\u00a0mmol\/g\u00a0DW as compared to a range of 0.3\u20131.6\u00a0mmol\/g\u00a0DW (on the basis of glucose units). Maximal simulated rates of biosynthesis were compared with that from literature. The rate of AA biosynthesis (v(r5)) was estimated at 0.12\u00a0mmol\/g\u00a0DW\/d, which was within the range of 0.094\u20130.36\u00a0mmol\/g\u00a0DW\/d reported by Rontein et\u00a0al. [19] for tomato suspension cells. STH biosynthesis (v(r12)) was slow with 0.009\u00a0mmol\/g\u00a0DW\/d as compared to 0.36\u00a0mmol\/g\u00a0DW\/d [19]. Maximal net rate of starch (STA) biosynthesis (v(r14)) was 0.20\u00a0mmol\/g\u00a0DW\/d as compared to 0.15\u00a0mmol\/g\u00a0DW\/d [19]. Differences in metabolic fluxes may be explained from the differences in the specific growth rate. Rontein et\u00a0al. [19] have reported a specific growth rate of 0.4\u00a0d\u22121 for tomato suspension cells in B5 medium as compared to 0.035\u00a0d\u22121 in this study for C. roseus using a minimal medium. The same minimal medium has been reported previously to induce lower growth rate as compared to standard media for carrot hairy root [17].\nThe cells nutritional state\nExtracellular sucrose (ESUC) concentration profile was simulated by the model for both the batch and the medium exchange cultures (Fig.\u00a06), thus suggesting that the sucrose hydrolysis modelling strategy was adequate. In the case of glucose (EGLC) and fructose (EFRU), simulations for the batch culture followed the trend of experimental data but before growth cessation. Then, estimated glucose and fructose levels stayed high as compared to experimental data. This may suggest that the model is underestimating glucose and fructose uptake at reduced growth. However, simulated concentrations in intracellular sucrose (SUC) and glucose (GLC) were following similar trends than experimental data (Fig.\u00a07). However, model simulated constant starch (STA) concentration and overestimated fructose (FRU) concentration. In the case of the medium exchange culture the model simulated adequately experimental data on extracellular and intracellular sucrose and intracellular glucose. Extracellular glucose and fructose concentrations were underestimated and starch and fructose concentrations were overestimated. A maximal sucrose rate of hydrolysis (v(28)) of 0.72\u00a0mmol\/g\u00a0DW\/d was estimated as compared to 1.7\u00a0mmol\/g\u00a0DW\/d for D. carota hairy root using same minimal medium [17]. Contents of 0.25\u00a0mmol\u00a0SUC\/g\u00a0DW (batch) and 0.7\u00a0mmol\u00a0SUC\/g\u00a0DW (medium exchange) were obtained as compared to 0.05\u00a0mmol\u00a0SUC\/g\u00a0DW [19], 0.26\u00a0mmol\u00a0SUC\/g\u00a0DW [36] and 0.4\u00a0mmol\u00a0SUC\/g\u00a0DW [34]. For free glucose, cell contents of 0.3\u20130.05\u00a0mmol\u00a0GLC\/g\u00a0DW were obtained as compared to 0.6\u00a0mmol\u00a0GLC\/g\u00a0DW [19], 0.24\u00a0mmol\u00a0GLC\/g\u00a0DW [36] and 0.18\u00a0mmol\u00a0GLC\/g\u00a0DW [34]. For fructose, cell contents of 0.1\u20130.25\u00a0mmol\u00a0FRU\/g\u00a0DW were observed as compared to 0.009\u00a0mmol\u00a0FRU\/g\u00a0DW [35], 0.21\u00a0mmol\u00a0GLC\/g\u00a0DW [34] and 0.5\u00a0mmol\u00a0GLC\/g\u00a0DW [19]. Starch accumulation reached 1.25\u00a0mmol STA (based on glucose)\/g\u00a0DW as compared to 0.055 [34] and 0.1\u20130.6\u00a0mmol STA (based on glucose)\/g\u00a0DW [19].Fig.\u00a07Intracellular concentration in nutrients and metabolites with time. Experimental data for C. roseus hairy root batch (filled square) and medium exchange (triangle) liquid cultures. Intracellular concentration in sugars (a\u2013d), inorganic phosphate (e), nitrate (f), ammonium (g), and in secondary metabolites issued from tryptamin (h) and secologanin (i) with time. Model simulations for batch (solid lines) and medium exchange (dashed lines) cultures\nModel simulations of extracellular Pi and NO3\u2212 (ENO3) concentrations followed experimental data for the complete duration of the batch and the medium exchange cultures (Fig.\u00a06). Hairy root growth (simulated and experimental) has ceased concurrently to ENO3 depletion suggesting this ion to be limiting in the batch culture. Intracellular Pi and NH4 were simulated (Fig.\u00a07). It is interesting to note that the final intracellular Pi concentration in the batch culture seemed to reach a plateau at 0.05\u00a0mmol\/g\u00a0DW which was simulated. This plateau may be close to the Pi level that is essential to maintain the endogeneous metabolism [37]. A maximum Pi accumulation plateau was also observed for the medium exchange culture around 0.2\u00a0mmol\/g\u00a0DW. This value is close to the maximum value of 0.23\u00a0mmol\/g\u00a0DW measured for carrot hairy root [17, and references therein]. Maximum intracellular nitrate of 0.6\u00a0mmol\u00a0NO3\/g\u00a0DW was measured for both cultures. Intracellular nitrate was kept constant for the medium exchange and decreased to its initial value for the batch. Intracellular ammonium reached a value of 0.12\u00a0mmol\u00a0NH4\/g\u00a0DW for the medium exchange culture and decreased to 0.09\u00a0mmol\u00a0NH4\/g\u00a0DW after 30\u00a0d. For the batch culture, intracellular ammonium reached a maximum value (0.08\u00a0mmol\u00a0NH4\/g\u00a0DW) at day 5 and then decreased closely to its value at inoculation. The model overestimated intracellular NO3 concentration and simulated experimental intracellular concentrations in NH4 for both the batch and medium exchange cultures. A more precise description of the secondary metabolism and of the AA biosynthesis may be required to improve model simulation of nitrogenous compounds.\nThe cells energetic state\nModel has simulated energy rich cells with high levels in NTP and NADH at exponential growth (Fig.\u00a08). However, estimated cell content in NADPH showed an opposite trend with lower values at exponential growth. This result is interesting since NADPH is produced through the pentose-phosphate pathway, which is mostly active at secondary metabolism. The simulated cell energetic level decreased after 20\u00a0d at the initial value (inoculation) in batch culture and was constant for the medium exchange culture. Since the medium was renewed every 3\u00a0days for the medium exchange experiment, a pseudo-steady state was expected. NTP\/(NTP+NDP) and NADH\/(NAD+NADH) ratios of \u223c75% have been reported for tobacco suspension cells [34]. Farr\u00e9 et\u00a0al. [35] measured ratios of \u223c73% for NTP\/(NTP+NDP) in potato tubers. These authors have measured an NTP level of 0.00013\u00a0mmol\/g\u00a0DW as compared to 0.00143 and 0.00074\u00a0mmol\/g\u00a0DW that was obtained from simulations at exponential growth and stationary growth, respectively. We have recently measured by in vivo NMR ATP levels of 0.009\u00a0mmol\/g\u00a0DW at day 1 and 5, and of 0.0065\u00a0mmol\/g\u00a0DW at day 10 for suspension cells of E. californica cultured using B5 medium [38]. Energetic levels simulated by the model were then similar to that found in literature. However, variation of energy shuttles concentration has to be further investigated.Fig.\u00a08Energetic level of the cells with time. Intracellular NTP (a), NADH (b) and NADPH (c) relative levels with time. Model simulations for C. roseus hairy root batch (solid lines) and medium exchange (dashed lines) liquid cultures\nConclusion\nA metabolic model capable of describing hairy root growth from the estimation of the cells physiological state was developed. The model includes the central metabolism, the primary metabolic pathways (SPMP) assumed at steady state and a network for the TPMP. At this point, the model is simplified and intracellular compartmentalization processes into the different cell compartments and organelles are not included. Nevertheless, the metabolic model showed to perform efficiently in simulating hairy root growth and nutrition. The use of intracellular concentrations in nutrients and co-substrates as well as the cells energetic state seems an efficient strategy in describing regulation of the metabolic fluxes. However, more experimental is required for improving the model structure as well as parameter values which may be regulated with cell physiological state. Finally, the model will be applied to other plant species as well as cell suspensions, and it will be studied as a tool to describe transient processes such as metabolic regulation.","keyphrases":["metabolic model","metabolic regulation","plant cells","kinetic model","cell nutrition"],"prmu":["P","P","P","P","P"]}
{"id":"J_Biomol_NMR-3-1-2048825","title":"A global analysis of NMR distance constraints from the PDB\n","text":"Information obtained from Nuclear Magnetic Resonance (NMR) experiments is encoded as a set of constraint lists when calculating three-dimensional structures for a protein. With the amount of constraint data from the world wide Protein Data Bank (wwPDB) that is now available, it is possible to do a global, large-scale analysis using only information from the constraints, without taking the coordinate information into account. This article describes such an analysis of distance constraints from NOE data based on a set of 1834 NMR PDB entries containing 1909 protein chains. In order to best represent the quality and extent of the data that is currently deposited at the wwPDB, only the original data as deposited by the authors was used, and no attempt was made to \u2018clean up\u2019 and further interpret this information. Because the constraint lists provide a single set of data, and not an ensemble of structural solutions, they are easier to analyse and provide a reduced form of structural information that is relevant for NMR analysis only. The online resource resulting from this analysis (http:\/\/www.ebi.ac.uk\/msd\/srv\/docs\/NMR\/analysis\/results\/html\/comparison.html) makes it possible to check, for example, how often a particular contact occurs when assigning NOESY spectra, or to find out whether a particular sequence fragment is likely to be difficult to assign. In this respect it formalises information that scientists with experience in spectrum analysis are aware of but cannot necessarily quantify. The analysis described here illustrates the importance of depositing constraints (and all other possible NMR derived information) along with the structure coordinates, as this type of information can greatly assist the NMR community.\nIntroduction\nThe information contained in the world wide Protein Data Bank (wwPDB) (Berman et\u00a0al. 2007) is growing steadily, with increasing numbers of structures being deposited from both traditional single laboratory sources and recent structural genomics efforts. The two main methods to determine these structures are X-ray crystallography and Nuclear Magnetic Resonance (NMR). NMR structures account for around 15% of all entries in the wwPDB. While inherent size restrictions limit the method to molecules of lower molecular weight, NMR has still made a significant contribution to protein folds and molecular interaction data. However, NMR structures are less straightforward to use than structures determined by X-ray because they are often represented as \u2018ensembles\u2019 of structures, where the whole ensemble (and not individual structures by themselves) represents the solution of the structure determination problem based on the experimental data. The reason for this is that information derived from NMR is insufficient with respect to the structure calculation process and thus cannot lead to a single exact solution. For example, measured distance-related NOE data is ensemble and time averaged, so that the final observed NOE data for a set of multiple distinct conformations in fast exchange will be a degenerate mix of the distance information in each of those conformations. All calculated structures that conform to a set of criteria based on the fit to the experimental data (NMR derived constraints) and physical characteristics encoded in the calculation process (overall energy minimum, inter-atomic packing, \u2026) are therefore in principle equally valid (Spronk et\u00a0al. 2003). However, there is no \u2018standard\u2019 set of criteria, and different programs and researchers use different sets when selecting the \u2018best\u2019 structures out of the calculated ones. The quality of the NMR structures deposited at the wwPDB therefore varies widely (Nabuurs et\u00a0al. 2006). This problem is being addressed by novel structure determination methods like Inferential Structure Determination (ISD) (Rieping et\u00a0al. 2005) that perform more objective structure calculations and select statistically relevant ensembles as a representative solution to the experimental data. These methods are, however, computationally expensive.\nThe observed NMR data is used in structure calculations to constrain inter-atomic distances, relative bond orientations and\/or dihedral angles. Only just over half of the NMR entries in the wwPDB were deposited together with the constraint lists used in the structure calculation process. These constraint lists provide very valuable information, as, for example, they enable recalculation of the structures with better or different protocols, and a globally consistent comparison of the constraints to the original coordinates for validation purposes. However, a major problem in using this information is that constraint files come in many file formats, and that the atom naming and residue numbering in the coordinate and the constraint files often differs. To handle the problem of different file formats, constraint lists are now continuously converted into the NMR-STAR format at the BioMagResBank (BMRB) (Doreleijers et\u00a0al. 2003; Ulrich et\u00a0al. 1989). A further step, as part of the DOCR project (Doreleijers et\u00a0al. 2005), addresses the atom naming and residue numbering problem by directly relating the constraint atoms to the coordinate atoms and molecular system using the FormatConverter and CCPN data model (Fogh et\u00a0al. 2002; Vranken et\u00a0al. 2005). This data is then checked for consistency and redundant constraints are removed using the WATTOS software as part of the FRED project (Doreleijers et\u00a0al. 2005). In the original implementation the DOCR\/FRED project resulted in a set of more than 500 internally consistent constraint lists, molecular system information and structure coordinates. This data led to the construction of the RECOORD (Nederveen et\u00a0al. 2005) and DRESS (Nabuurs et\u00a0al. 2004) databanks, where structures from the PDB were respectively recalculated and re-refined based on the cleaned up constraint lists.\nThese efforts, as well as validation software like AQUA (Doreleijers et\u00a0al. 1998) or Procheck-NMR (Laskowski et\u00a0al. 1996), are applied on a per-entry basis, where the original or recalculated coordinates are related to the original or standardised constraint lists. With the amount of constraint data that is now available it has become possible to do a global, large-scale analysis using only the information from the constraints, without taking the coordinate information into account. The constraint lists are the \u2018final product\u2019 of the usually human analysis of NMR spectra, and as such they represent the experimental NMR-derived information that is relevant for the structure calculation. The constraint lists also provide a single set of data, whereas NMR structures are usually represented as ensembles that can be calculated in many different ways, which complicates their interpretation. Analysing the constraint data on a large scale can thus provide insights into the NMR data analysis process (e.g. which type of inter-atomic contact is often derived from the spectra), and the relation of the constraints to the coordinate data (e.g. does the structure calculation process add any distance information that is relevant for NMR).\nThe analysis described here relates only to distance constraints derived from NOE data, with a base set of 1834 NMR PDB entries containing 1909 protein chains. Only constraints between protons in protein chains were retained for analysis, and for validation purposes the base set was further divided into subsets for entries that contain intra-residue constraints and entries where all the original constraint and coordinate information was recognised and linked to each other. A coordinate data set based on the original coordinate files was also generated and used for comparison. This article explores some of the issues surrounding distance constraints and the NMR data they are derived from, and hopes to highlight the importance of depositing the constraint lists used for structure calculations along with the molecular coordinates.\nMaterials and methods\nThe data was obtained from two sources: the molecular system, coordinate, secondary structure and author information from the wwPDB, and the original constraint information as NMR-STAR files from the NMR Restraints Grid at the BioMagResBank. Each file was directly parsed and combined into the CCPN data model via the FormatConverter software (Vranken et\u00a0al. 2005), in a process that extends the procedure used in the original DOCR\/FRED project. To handle the larger number of entries an automatic mapping procedure was developed that maps the molecule sequence as derived from the PDB file to the atom information and sequence code numbering used within the constraint lists. For some entries manual mapping between the information from the PDB file and the constraint file was required. The original 409 mappings from the RECOORD project (by Aart Nederveen) and a further mappings 124 by the author and 17 by Jurgen Doreleijers were used to correctly set the PDB-constraint file mapping for the entries in the base set. Using the automatic or manual mapping the atom information from the constraints was then connected to that for the molecule. During this process the dependence on string-based atom names for the assignment (as used in all constraint files) was also removed, since the CCPN data model is object-based. The final CCPN project, in which all the information from the original files is now highly organised, was then written out. This process was completed for 2643 PDB entries.\nThree subsets of the base set were generated for comparison and validation purposes (Table\u00a01). Sets HIP and AHIP contain only entries with intra-residue constraints, sets AHP and AHIP only include entries where over 99% of relevant constraint information was assigned to atoms. In addition a set was generated based on the coordinates for the entries included in the HP set. Only the HP, HPC and AHIP sets are further referred to in this article, the HIP and AHP sets are available online for reference purposes.\nTable\u00a01Overview of the available data sets used in the analysisSet NameData typeDetails1HPConstraintsBase set2HIPConstraintsIntra-residue3AHPConstraintsHigh assignment4AHIPConstraintsIntra-residue, high assignment5HPCCoordinatesOriginal coordinate data\nA workflow based on Python (van Rossum 2003) scripts and dictionaries was then developed to handle the information from the CCPN project files for the 2643 entries (Fig.\u00a01). This workflow was run separately for the base set and each subset. In the first step, the original information was filtered so that only valid constraints between protons in protein chains were retained (Fig.\u00a01). Entries were removed for the following reasons (Table\u00a02): (1) No valid protein chains: the entry contained only chains that are sequence fragments or duplicates of other chains (only the chain with the highest number of valid constraints linked to it is retained in the data set), (2) No valid constraint lists: the originally deposited constraint lists could not be parsed or handled correctly, or (for sets HIP and AHIP), did not contain any intra-residue constraints, (3) Insufficient linking: less than 80% (sets HP, HIP) or 99.9% (sets AHP, AHIP) of the constraint information could be linked to the atom information (constraint information belonging to non-protein chains was ignored for this purpose), (4) Insufficient valid constraints: less then 20% of constraints remained for all lists after removing invalid constraints. Constraints were considered invalid if they did not have any valid items, no upper distance limit, an upper distance limit of larger than 10\u00a0\u00c5, or had only items between invalid atoms (non-proton, unlinked or non-protein). Note that some entries had specific distance constraint lists and\/or constraints removed for these reasons, but were still included in the analysed set.\nFig.\u00a01Overview of the workflow employed in the analysis. Grey boxes indicate files, white boxes Python scriptsTable\u00a02Overview, for each data set, of the number of removed and analysed entriesHPHIPAHPAHIPNo valid protein chains396396396396No valid constraint lists310409310409Insufficient linking4837677635Insufficient valid constraints55555555Total included entries1834174612031146Total included chains1909181712521192\nIn the second step, the data from all relevant entries was collated (Fig.\u00a01). After reading in the CCPN projects, non-relevant information was removed based on the filtering information, and the data reorganised into a set of Python dictionaries that contain the overall information on the entry, residue and constraint levels. In the final analysis step these dictionaries were read in, areas and data of interest were marked, and organised HTML output was produced for browsing. A Python dictionary with highly reduced information that can be further used for validation or constraint filtering purposes was also generated.\nThe same 1834 entries from the HP set were used to generate the HPC set. The original coordinates were analysed using r\u22126 distance averaging for equivalent and prochiral atom sets over all structures in the ensemble (Nilges 1995). Only individual distances of less then 7\u00a0\u00c5 were retained, and final averaged distances of less than 5\u00a0\u00c5 were considered to be equivalent to an observable constraint contact. Atoms without coordinates were ignored in the analysis.\nHighlights from the analysis are described in the results section, and complete details are available from a web site (http:\/\/www.ebi.ac.uk\/msd-srv\/docs\/NMR\/analysis\/results\/html\/comparison.html). For all statistical operations, the R package (Bates et\u00a0al. 2007) was accessed via the RPy Python module (Moreira and Warnes 2006). Since a contact is either observed or not observed, it was possible to use a binomial analysis to determine, for example, which secondary structure specific contacts were significantly less or more likely to be observed. Binomial analysis was used throughout with a confidence level of 0.99, meaning that only 1 out of every 100 determined outliers is a false positive. The correlations between the coordinate and the constraint data within a data set and correlations between data sets were plotted via RPy, with linear correlation coefficients determined by both the Spearman and Pearson methods. The Spearman method (Spearman 1904) is a non-parametric measure of correlation, which does not make assumptions about the frequency distribution of the variables, and does not require a linear relationship between the variables. The Pearson method (Pearson 1896) on the other hand assesses if the relationship between the variables is linear.\nIn the analysis, a residue is marked as \u2018assigned\u2019 when at least one proton belonging to it is linked to a constraint. The total number of times a particular inter-atomic contact is observed can be a fraction, as for ambiguous constraints each constraint item contributes a fraction of 1 to the total:\nThe total number of relevant distances for a particular inter-atomic contact (ndist) is always an integer. The occurrence for a particular inter-atomic contact is calculated as: where nactual is the number of times the contact occurs (with ambiguity taken into account), and npossible the total number of times this contact could occur between the relevant residues, either for all residues or only between \u2018assigned\u2019 residues. The occurrence is given in percent or as a fraction. The \u2018ambiguity\u2019 of an inter-atomic contact is defined as: \nIf the ambiguity is 0, this means that all contributing contacts are unambiguously assigned. The more highly ambiguous the contributing contacts are, the closer this number will be to 1, which also means it is less dependable. Within secondary structure combinations, the same definitions are used, except that npossible and nactual are now determined within that secondary structure combination as npossible,ss and nactual,ss. The uniqueness of a contact for a secondary structure combination, which indicates how unique the contact is within that combination, is defined as: \nFor prochiral and possible non-equivalent atom sets, the contacts are divided into the individual atom names (e.g. H\u03b22 and H\u03b23, or H\u03b41 and H\u03b42 for Phe) if they occur by themselves in a constraint. If both the atom names occur in different items within the same constraint to the same other atom, then they are grouped (e.g. H\u03b2*, or H\u03b4* for Phe). If they occur as a group in one constraint item they are used as is. In order to obtain total statistics for these contacts, they were also combined (e.g. (H\u03b22\u00a0+\u00a0H\u03b23), or (H\u03b41\u00a0+\u00a0H\u03b42) for Phe). To get the combined occurrence, the nactual for the group is added to the individual contribution with the highest nactual to obtain the nactual used to calculate the occurrence for the combined contact. The combined distance information is obtained from all contributions, and the ambiguity is then calculated from the total nactual of all contributions.\nResults\nInformation from analysis\nThe analysis of the data for each set is divided into separate categories (visible on the left-hand side menu in Fig.\u00a02): (1) Contact analysis: Arranges inter-atomic distances by residue\u2013residue combination, secondary structure of those residues, and contact type (intra-residue (i\u2013i), sequential (i\u2013i\u00a0+\u00a01), medium-range defined here as up to 6 residues separation (i\u2013i\u00a0+\u00a0n), and long range (i\u2013i\u00a0+\u00a06<)). (2) Protein secondary structure analysis: Groups inter-atomic distances by secondary structure combinations. (3) Residue atom analysis: Shows assignment percentages for the atoms in each amino acid. (4) Unassigned fragments breakdown: Lists unassigned sequence fragments. (5) Fragment analysis by residue: Analyses tripeptide fragments based on the assignment status of the central residue.\nFig.\u00a02Example web page for Ala\u00a0\u2192\u00a0Ala i\u00a0\u2192\u00a0i\u00a0+\u00a02 contact information from http:\/\/www.ebi.ac.uk\/msd-srv\/docs\/NMR\/analysis\/results\/html\/comparison.html. The different data sets, comparisons between them, and analysis categories can be accessed via the left hand side menu. In this contact information page, overall statistics for the individual Ala residues involved in this contact are shown in the \u2018Residue 1\u2019 and \u2018Residue 2\u2019 tables at the top, statistics for the combined Ala\u2013Ala residues in the \u2018Residue combination\u2019 table. The information on an atom level is listed in the \u2018Breakdown per contact\u2019 table. Outliers per secondary structure combination based on a binomial analysis are highlighted in red (higher than expected) and blue (lower than expected). This colour coding is used throughout the web pages for other types of analyses\nValidation of data sets\nThe relevance of the base set (HP) was validated by comparison with the most restricted AHIP set. This is necessary because the HP set contains entries without intra-residue constraints and entries where less constraint information is linked to atoms. The correlation between the occurrences of contacts between both sets is very high (0.97) (Fig.\u00a03), and in a breakdown per contact type no correlation is less than 0.91 (results on web pages). A further detailed analysis shows that out of 43,984 compared contacts only 5 differ significantly at a confidence level of 0.999. Of these, 3 are His\u00a0\u2192\u00a0His contacts where the differences are most likely introduced by changes in the number of His residues that are assigned in the data sets (from 71.66% in HP to 68.47% in AHIP). The other 2 are long-range contacts involving side chain protons between Leu\u00a0\u2192\u00a0Phe and Phe\u00a0\u2192\u00a0Val, which are uncommon, and these differences can be attributed to accidental variations in the number of entries that have these contacts and are included in the data set. Overall, this data shows that including the additional 688 entries does not introduce major changes in the occurrence levels. The HP set was therefore chosen as the reference set, as estimates of, for example, significant differences between contacts in secondary structure elements tend to become more reliable as more data is included. Information on correlations between all data sets is available from the web pages (Fig.\u00a02, the \u2018Comparison\u2019 link in the left-hand side menu).\nFig.\u00a03Correlation between the foccurrence for the HP and AHIP data sets (correlation Spearman 0.971, Pearson 0.996)\nA further way to validate the constraint information is that it should reproduce the typical contacts that are observed in secondary structure elements. The secondary structure definitions as determined by DSSP (Kabsch and Sander 1983) were taken from the PDB file header for each entry. Because of the size of the data that is generated by the analysis, only the Ala residue is used here as an example. The complete information can be accessed via the web pages (Fig.\u00a02). The numbers that are relevant in order to determine whether a contact is significant for a particular secondary structure element are the occurrence within that particular secondary structure element (which has to be significantly higher than expected from the overall occurrence), the ambiguity of the contact (if it is high it comes, by definition, from highly ambiguous constraints and is therefore unreliable), and the uniqueness of the contact (which indicates how often the contact is seen within that secondary structure element compared to the total number of times it is observed).\nFor intra-residue contacts there are not many significant differences in the observed occurrence within different secondary structure elements, although overall fewer contacts are observed when secondary structure is absent (Table\u00a03). This is likely due to higher signal overlap for atoms in \u2018random coil\u2019 fragments, which complicates assignment. The information from the coordinate-derived HPC set is provided throughout for comparison: there are, as expected when using a distance cutoff of 5\u00a0\u00c5, no differences for this HPC set between the occurrence for different secondary structure elements, and only minor differences in the average and mean distance are observed (data available on web pages).\nTable\u00a03foccurrence for selected backbone Ala\u2013Ala contactsTypeAtom 1Atom 2\u03b1-helix\u03b2-sheetNo secondary structurei\u00a0\u2192\u00a0iHH\u03b10.50\/1.000.51\/1.000.43\/1.00HH\u03b2*0.73\/1.000.68\/1.000.57\/1.00H\u03b1H\u03b2*0.32\/1.000.33\/1.000.29\/1.00i\u00a0\u2192\u00a0i\u00a0+\u00a01HH0.84\/1.000.50\/1.000.57\/1.00HH\u03b2*0.22\/0.990.07\/0.690.10\/0.83H\u03b1H0.52\/1.000.90\/1.000.67\/1.00H\u03b1H\u03b2*0.03\/0.420.30\/1.000.11\/0.93i\u00a0\u2192\u00a0i\u00a0+\u00a02HH0.47\/0.990.00\/0.000.16\/0.45HH\u03b2*0.09\/0.790.00\/0.000.03\/0.25H\u03b1H0.28\/0.970.04\/0.040.17\/0.60H\u03b1H\u03b2*0.02\/0.630.00\/0.000.02\/0.27H\u03b2*H0.22\/0.980.09\/0.450.17\/0.77H\u03b2*H\u03b2*0.02\/0.120.05\/0.700.01\/0.51i\u00a0\u2192\u00a0i\u00a0+\u00a03HH0.09\/0.860.04\/0.120.04\/0.16HH\u03b2*0.12\/0.840.04\/0.120.04\/0.22H\u03b1H0.66\/0.990.00\/0.040.14\/0.28H\u03b1H\u03b2*0.58\/0.990.00\/0.040.14\/0.33H\u03b2*H0.14\/0.910.04\/0.160.06\/0.34H\u03b2*H\u03b2*0.11\/0.970.04\/0.120.07\/0.46i\u00a0\u2192\u00a0i\u00a0+\u00a04 HH0.02\/0.010.00\/0.000.02\/0.03HH\u03b2*0.01\/0.010.05\/0.090.02\/0.07H\u03b1H0.32\/0.940.00\/0.000.07\/0.16H\u03b1H\u03b2*0.02\/0.790.00\/0.000.02\/0.23H\u03b2*H0.09\/0.850.00\/0.090.02\/0.22H\u03b2*H\u03b2*0.03\/0.930.00\/0.090.01\/0.37In the secondary structure columns, the first value is from the HP set, the second from the HPC set. A bold value indicates a contact that occurs significantly more than average, a italic value significantly less\nFor sequential Ala\u2013Ala contacts, the H\u2013H contact is observed significantly more in \u03b1-helices than in \u03b2-sheets (Table\u00a03), and is also highly unique (0.65). This situation is, as expected, reversed for the H\u03b1\u2013H contact, which is observed significantly less in \u03b1-helices and more in \u03b2-sheets. Overall, however, this contact is more uniquely observed in \u03b1-helices (0.49 compared to 0.08 for \u03b2-sheets), which is due to the prevalence of Ala\u2013Ala fragments in \u03b1-helices (56.2% compared to 5.35%). Interestingly, however, an H\u2013H\u03b2* contact is also observed significantly more in \u03b1-helices than in \u03b2-sheets or when no secondary structure is present, and has a uniqueness of 0.71, so within a sequential Ala\u2013Ala fragment this type of contact is highly predictive of \u03b1-helical structure. The sequential H\u03b1\u2013H\u03b2* contact is, on the other hand, highly predictive of \u03b2-sheet with a uniqueness of 0.20, although it is on average mostly observed when no secondary structure is present (0.38). This illustrates that the uniqueness of a contact is strongly related to the prevalence of a particular sequence combination in a particular secondary structure element, and is not necessarily indicative of the kind of secondary structure element a particular contact usually occurs in. The information from the constraints and the coordinates show clear differences, as illustrated by comparison with some of the contacts described earlier. The H\u2013H\u03b2* contact occurs quite often in \u03b2-sheets based on the coordinate data, with a median distance that is slightly higher than in \u03b1-helices (4.63\u00a0\u00c5 compared to 4.31\u00a0\u00c5). The H\u03b1\u2013H\u03b2* contact, which would generally be difficult to identify due to overlap in the aliphatic region of a NOESY spectrum, is always present based on the coordinate data, but is only seen in 0.30 cases based on the constraints. These differences illustrate that the NOE constraint data not only incorporates distance information, but also encodes NMR-specific information such as the difficulty with which a particular contact can be assigned.\nFor i\u2013i\u00a0+\u00a02 and i\u2013i\u00a0+\u00a03 contacts, as expected, the typical H\u2013H, H\u03b1\u2013H, H\u03b1\u2013H\u03b2* and H\u03b2*\u2013H contacts are highly prevalent for \u03b1-helices (Table\u00a03), with generally high uniqueness and low ambiguity. An H\u2013H\u03b2* contact is also more often observed than average (respectively in 0.09 and 0.12 cases), while H\u03b2*\u2013H\u03b2* contacts are very rarely seen for i\u2013i\u00a0+\u00a02 contacts in an \u03b1-helix. Note that based on the coordinate data the i\u2013i\u00a0+\u00a02 H\u03b2*\u2013H\u03b2* contact is highly relevant for \u03b2-sheets (0.70), but it is in practice rarely observed (0.05), probably because it falls in a densely populated region of a typical NOESY spectrum. For 310 helices the percentages are often similar to the \u03b1-helical ones, but there are often not enough data to determine whether a difference is significant (data on web pages). Also of interest is the i\u2013i\u00a0+\u00a03 contact between H\u03b2*\u2013H, which occurs in 0.04 cases for \u03b2-sheets and 0.06 cases where secondary structure is absent. The binomial analysis indicates that the 0.06 fraction is observed significantly less, while it does not mark the 0.04 fraction. This is because this type of analysis is dependent on sample size (only 1 sample for the 0.04 fraction, 12 for the 0.06 fraction).\nFor the i\u2013i\u00a0+\u00a04 contacts, H\u03b1\u2013H and H\u03b2*\u2013H connections are again highly prevalent for \u03b1-helices, while H\u03b1\u2013H\u03b2* and H\u03b2*\u2013H\u03b2* contacts are present in the coordinate set but are seldom observed in practice (Table\u00a03). All contacts with a separation of 5 residues or higher are very rarely observed in \u03b1-helices, but become highly prevalent for \u03b2-sheets (data not shown). The only exception to this are long range H\u03b1\u2013H\u03b2* and H\u03b2*\u2013H\u03b2* contacts, which are seen in significantly higher percentages between \u03b1-helices as compared to the average.\nContact data highlights\nTraditionally, and as described above, identifying secondary structure contacts is based on the commonly observed contacts between protons from the backbone and the \u03b2 position (W\u00fcthrich 1986). However, with experience in assigning NOE peaks comes the knowledge that other contacts are also often observed (e.g. i\u00a0\u2192\u00a0i\u00a0+\u00a02 contacts between side chain protons in a \u03b2-sheet). In this analysis, such contacts are readily observed (Table\u00a04). For example, the sequential Trp H\u03b53\u2013Gly H contact is quite common based on the coordinate data, but is in practice particularly observed in a \u03b2-sheet. The sequential Trp H\u03b53\u2013Phe H\u03b1 contact is seemingly more often observed in an \u03b1-helix, but the differences are not significant. This is a case that could be clarified if more relevant data were available. The Thr H\u03b32*\u2013Tyr H\u03b5* i\u2013i\u00a0+\u00a02 contact is clearly observed in mostly \u03b2-sheet. Interesting in this case is that according to the constraint data it can occur in an \u03b1-helix, while this is not the case based on the coordinate data, even though the ambiguity of the contact is 0.00. This is possible because this data point is based on one contact with an upper distance limit of 6\u00a0\u00c5, whereas the cutoff used for coordinates is 5\u00a0\u00c5. Generally speaking this type of situation can occur for highly ambiguous constraints, where other constraint items satisfy the upper distance limit. An H\u03b1\u2013H\u03b41* i\u2013i\u00a0+\u00a03 contact between Ala and Ile is observed almost exclusively in an \u03b1-helix (and never in a \u03b2-sheet), and is very unique (0.78). To be able to discern whether this contact is very common from the H\u03b1 of any amino acid to the H\u03b41* of an Ile, the data was joined as Xxx residues to produce generic information to and from each amino acid (data on web pages). The specific information for the i\u2013i\u00a0+\u00a03 H\u03b1\u2013H\u03b41* contact from all residues to Ile is shown in Table\u00a04 and shows that this contact is common in \u03b1-helices. Finally, some other interesting i\u2013i\u00a0+\u00a04 side chain contacts that often occur in helices are listed in Table\u00a04.\nTable\u00a04foccurrence for selected secondary structure related contactsTypeAtom 1Atom 2\u03b1-helix\u03b2-sheetNo secondary structurei\u00a0\u2192\u00a0i\u00a0+\u00a01Trp H\u03b53Gly H0.05\/0.450.56\/0.880.24\/0.63Trp H\u03b53Phe H\u03b10.55\/0.680.11\/0.160.13\/0.47i\u00a0\u2192\u00a0i\u00a0+\u00a02Thr H\u03b32*Tyr H\u03b5*0.02\/0.000.46\/0.800.12\/0.41i\u00a0\u2192\u00a0i\u00a0+\u00a03Ala H\u03b1Ile H\u03b410.66\/0.930.00\/0.000.11\/0.21Xxx H\u03b1Ile H\u03b410.57\/0.920.00\/0.020.09\/0.22i\u00a0\u2192\u00a0i\u00a0+\u00a04Trp H\u03b62Thr H\u03b320.52\/0.560.00\/0.000.00\/0.00Tyr H\u03b51\u00a0+\u00a0H\u03b52Val H\u03b320.47\/0.740.00\/0.050.06\/0.21In the secondary structure columns, the first value is from the HP set, the second from the HPC set. A bold value indicates a contact that occurs significantly more than average, an italic value significantly less\nIt is not possible to describe all information in detail in this article, and the web pages serve as the reference resource for any investigations. However, to provide a better overview of the overall trends in secondary structure elements, all backbone contacts were grouped by atom type (H, H\u03b1, H\u03b2) and secondary structure combinations (Table\u00a05, full data from web site). For intra-residue contacts, more contacts involving H\u03b2 protons are defined in \u03b2 sheets. Sequential contacts in \u03b1 helices originating from the H and H\u03b2 protons are observed significantly more often, whereas ones originating from the H\u03b1 proton are less common. This situation is reversed in \u03b2 sheets (except for H\u03b2\u2013H contacts). There are, only for sequential contacts, some discrepancies between the data from the HP and HPC sets, with, for example, the rate at which sequential H\u2013H\u03b1 contacts are observed being reversed in the HPC set as compared to the HP set. The reasons for this are not immediately clear, but are likely related to overlap. Most i\u2013i\u00a0+\u00a02 contacts are more common in \u03b1 helices, except for ones to the H\u03b1 proton and between H\u03b2 protons. The latter contact is more often observed in \u03b2 sheets. As expected most i\u2013i\u00a0+\u00a03 contacts are commonly observed in \u03b1 helices, except for H\u03b2\u2013H\u03b1 and H\u2013H\u03b1. The latter is again more frequently seen for \u03b2 sheets, but in this case this is expected to be between different strands in the hairpin area. The trends are not as clear for i\u2013i\u00a0+\u00a04 contacts, where H\u03b1\u2013H, H\u03b1\u2013H\u03b2 and H\u03b2\u2013H contacts are more frequent in \u03b1 helices, and all other ones more frequent in \u03b2 sheets. This is again likely related to hairpin contacts. All contacts from i\u00a0\u2192\u00a0i\u00a0+\u00a05 and more are very infrequent for \u03b1 helices but relatively very frequent for \u03b2 sheets.\nTable\u00a05Brief overview of general trends in joint secondary structure information for all contactsTypeSSH (i)H\u03b1 (i)H\u03b2 (i)HH\u03b1H\u03b2HH\u03b1H\u03b2HH\u03b1H\u03b2i\u2013iHelix. . + .. .\u2212 .\u2212 .+ .\u2212 .\u2212 .Sheet. .+ .. .. .+ .+ .+ .+ .i\u2013i\u00a0+\u00a01Helix+ .+ \u2212+ +\u2212 .\u2212 +\u2212\u2212+ .+ ++ +Sheet\u2212 .\u2212 +\u2212\u2212+ .+ ++ ++ .\u2212\u2212\u2212\u2212i\u2013i\u00a0+\u00a02Helix+ +\u2212\u2212+ ++ +- -. ++ +\u2212\u2212\u2212\u2212Sheet\u2212\u2212. \u2212\u2212\u2212\u2212\u2212. \u2212. \u2212\u2212\u2212+ .+ +i\u2013i\u00a0+\u00a03Helix+ +. \u2212+ ++ ++ ++ ++ +\u2212\u2212+ +Sheet\u2212\u2212+ +\u2212\u2212\u2212\u2212. \u2212\u2212\u2212\u2212\u2212. \u2212\u2212\u2212i\u2013i\u00a0+\u00a04Helix. .\u2212\u2212\u2212\u2212++. .+ ++ +\u2212\u2212. +Sheet+++ ++ .\u2212\u2212+ \u2212. \u2212. \u2212+ .+ \u2212i\u2013i\u00a0+\u00a04<Helix\u2212\u2212\u2212\u2212\u2212\u2212\u2212\u2212\u2212\u2212\u2212\u2212\u2212\u2212\u2212\u2212\u2212\u2212Sheet+ ++ ++ ++ ++ ++ ++ ++ ++ ++ Indicates that signals observed more than average, \u2212 less than average, . signifies that there is no trend. The first character in each cell contains the constraint HP set information, the second the coordinate HPC set\nAn analysis of the percentage of atoms that were assigned within each residue type (Residue atom analysis on the website) shows that generally atoms are significantly higher assigned in \u03b1-helices and \u03b2-sheets, and lower when no secondary structure is present. This finding is not surprising as the secondary structure elements are defined by constraints, and the atoms have to be assigned to obtain those. Another general trend is that prochiral methylenes are within \u03b1-helices significantly more degenerated, i.e. the HB2 and HB3 atoms exists as a QB pseudoatom or an HB* type atom set. This type of analysis can be significantly improved by cleaning up the stereospecific assignment status based on the coordinates.\nThe unique sequence fragments for which no assignments were found are also listed on the website (Unassigned fragments breakdown). An example from this data is that often no constraints are found for His tags. The LEHHHHH fragment, for example, was not assigned in 13 entries. A general overview of the percentage of residues that are assigned confirms that His residues, for example, are assigned in only 71.66% of cases, while Trp is assigned in 97.87% of cases (Fragment analysis by residue).\nTo examine this in more detail, all tripeptide fragments where a particular amino acid is the central residue are listed (for the N- and C-terminus these are dipeptide fragments). Listed for each fragment are (1) the total number of times the tripeptide fragment occurs in the data set, (2) the percentage of times it is unassigned compared to the total number of times the amino acid occurs, (3) the number of times the amino acid is not assigned, (4) the number of times the tripeptide fragment occurs overall, and (5) the assignment percentages per secondary structure element. The entries in which the fragments are unassigned are also listed. Continuing with His as an example, it is not assigned when part of the EHH fragment in 69% of cases, and when part of a C-terminal HH fragment it is not assigned in 96% of all times the fragment occurs. To get a better overall view of the sequence fragments that are difficult to assign, they were grouped by joining, respectively, the i\u00a0\u2212\u00a01 and i\u00a0+\u00a01 residues. The results for His and some selected other fragments are shown in Table\u00a06. A Ser residue preceded or followed by a Gly, for example, is often unassigned. This type of information could be useful for predicting which areas of a protein sequence are difficult to assign from an NMR perspective.\nTable\u00a06Selected sequence fragments where the central residue is often unassigned. The unassigned percentages are relative to the total number of times the fragment occursFragmentUnassigned (%)TotalXxx\u2013His\u2013His62756Xxx\u2013His\u2013Met583Gly\u2013His\u2013Xxx234His\u2013His\u2013Xxx63740Ser\u2013His\u2013Xxx8114Xxx\u2013Ser\u2013Gly18365Gly\u2013Ser\u2013Xxx19479Xxx\u2013Pro\u2013Ser8157\nDiscussion\nIn this analysis only the original data as deposited by the authors was used, and no attempt was made to \u2018clean up\u2019 and further interpret this information, except for linking the constraint with the coordinate data and removing identical sequences from the data set pool (where only the entry with the highest number of constraints linked to atoms was kept). This approach is intentional, as it best represents the quality and extent of the data that is currently deposited at the PDB. Only the distance constraint information was included in the analysis, and the information from dihedral, H-bond and RDC constraints was ignored. Even though these constraints contain important structural information, they were, as experimental data, recorded independently from the NOE data. They are used in the structure determination process, however, and it was not investigated whether their presence influences the quality of the final distance constraint lists. There are several other issues that can still be addressed, and although these are likely to improve some of the aspects of this type of study, it is also important to start with the original information so that a comparison point is available.\nThe first issue is that stereospecific assignments can be swapped or deassigned based on the original coordinates, similar to the approach in the RECOORD project. This could in principle reveal preferences related to stereospecifically assigned atoms in secondary structure elements. The second issue concerns the distances that were provided with the constraints. These are often \u2018binned\u2019 in weak\/medium\/strong classes with fixed distance cutoffs, so that the resulting distance distributions often show spikes at these distances. In Fig.\u00a04, for example, it is clear that spikes occur at 3.0, 3.5 and 5.0\u00a0\u00c5. Recalibrating the distances based on the deposited coordinates should improve the quality of the resulting information, and reveal relationships between distance and occurrence. The third issue concerns the sequences that are included: the current data sets include protein sequences with a high homology. This is clearly not an ideal situation, but there is currently not enough data available for a cleaner analysis. It is therefore important to check whether a particular contact appears in a large amount of entries, or the observed occurrence might be due to systematic error from homologous proteins produced by the same laboratory. The fourth problem is the identification of the secondary structure fragments. This is now based on the PDB DSSP analysis from the original coordinates for only the first or representative model so that secondary structure elements are not always identified properly. If chemical shifts were available an identification based on CSI (Wishart et\u00a0al. 1992) would become possible so secondary structure stretches can be included that are more flexible and less defined on the coordinate level. More refined secondary structure identifications from the coordinates could also reveal patterns related to, for example, turns. Overall, the best way to improve this analysis remains to increase the sample size by encouraging deposition of constraint lists and all related NMR information (peak lists, chemical shifts, spectra), and ensuring that the data is consistent when deposited by the authors. Efforts like the CCPN project (Fogh et\u00a0al. 2002, 2005), which allow data harvesting from NMR data collection to structure calculation, should provide this kind of data without requiring any additional effort by the scientists who produce the data.\nFig.\u00a04Distance distribution from the constraint information for sequential Ala\u2013Ala contacts between backbone H protons\nIn this analysis a particular inter-atomic contact between two residues from one PDB entry is either observed or not observed. The reason why a contact is observed (or not) implicitly includes distance information, peak overlap, water exchange line broadening, and all other factors that can lead to not observing or assigning a contact during analysis of a spectrum. This is different from the traditional meaning of an \u2018assigned atom\u2019 on the chemical shift level, where it means that the chemical shift value for the resonance that arises from the atom is known. However, this does not necessarily mean that these assigned atoms produce any valid inter-atomic distance information. Thus, an \u2018assigned atom\u2019 (or residue) on the constraint level means that a chemical shift assignment also produced useful and valid information related to the inter-atomic distances within the molecule.\nThe original study that defined the inter-atomic contacts relevant for assigning secondary structure elements with NMR used a set of 19 high-resolution protein crystal structures comprising about 3,200 residues (W\u00fcthrich et\u00a0al. 1984). In this study the extent of identification was defined as the percentage of times a distance was smaller than a particular cutoff value within a secondary structure element, while the uniqueness of identification is the percentage of times the distance is observed within a particular secondary structure element out of the total number of times it is observed. The extent is equivalent to the foccurence used in this study for the constraint sets, with the exception that no distance cutoff is used (although some distance information is, as mentioned, implicitly included because the reason a constraint is observed or not is very dependent on distance), and that values are labelled as significant based on a binomial analysis. The uniqueness has the same definition, except that again it is based on the amount of constraints that are observed.\nAlso of interest is the relationship between the information that comes directly from the deposited constraints and the information that comes from the deposited coordinates. Here, the constraint information is compared to the distances from the originally deposited coordinates. Although a set of recalculated coordinates (as in RECOORD) or X-ray structures could have equally well been used, the originally deposited coordinates were chosen because they should best match the content of the constraint lists. All comparisons between constraint and coordinate information are intended for informative purposes only: the constraints represent the experimental NMR side of the information contained in the coordinates, and are in effect only a subset of the information contained therein. However, a dependable determination of whether a particular NOE contact is observed or not is not possible based on an NMR structural ensemble, but is trivial based on the constraints because they inherently contain NMR-specific information like signal overlap, dynamics, etc.\nFrom Fig.\u00a05 it is clear that the contact occurrence is almost always higher for the HPC set compared to the HP set. This is related to the use of a direct distance cutoff of 5.0\u00a0\u00c5 in the HPC set: contacts with long distances could give rise to peaks that are too weak to be seen in a real spectrum but are still included. Also, many contacts have an foccurrence of 1.0 in the HPC set because of conformational constraints from covalent bonds. Not all of these contacts are seen in real spectra because of, for example, peak overlap or line broadening. The correlations between the occurrences overall are not very high (Spearman 0.770, Pearson 0.694), with especially the intra-residue contacts giving bad correlations (Spearman 0.451, Pearson 0.356, see web site), and i\u2013i\u00a0+\u00a02 (Spearman 0.720, Pearson 0.783) and i\u00a0+\u00a03 (Spearman 0.711, Pearson 0.659) contacts giving the best results. An indication that the main reason for the bad correlation between the occurrences is distance related comes from the large improvement that is observed in the overall correlations if only coordinate distances of less than 3.6\u00a0\u00c5 are considered (Spearman 0.906, Pearson 0.916). However, results from using both the HP and HPC information to filter ambiguous constraints lists show that both sets essentially give the same results (personal data), even though the constraints are available in a much more \u2018compressed\u2019 form than the coordinates, and no force field information was used.\nFig.\u00a05Correlation between the foccurrence from the HPC (Coordinates) and HP (Constraint) sets\nIn the KNOWNOE (Gronwald et\u00a0al. 2002) X-ray structure based approach to obtain probabilities for assignments, the distance distributions for inter-atomic contacts are used to generate volume-based probabilities in addition to the atom identity based probabilities. This approach improves the probabilities that are generated, but it does require that the original peak list with volumes is available. This is not possible within the current analysis, although this will be pursued if a meaningful way to recalibrate the distance constraint bounds is available. This would also allow a better comparison between the NMR constraint data and any coordinate data (from NMR or X-ray structures).\nConclusion\nA resource is now available where it is possible to check how likely a particular contact is when assigning NOESY spectra, or if a particular sequence fragment is likely to be difficult to assign. In this respect it formalises information that scientists with experience in spectrum analysis are aware of but cannot quantify. The amount of information provided here is extensive, however, and is even more useful when used as \u2018knowledge based\u2019 probabilities in automatic assignment strategies, to filter and\/or validate ambiguous constraint possibilities, and as a tool to rank assignment possibilities in spectrum analysis programs. These are being implemented as part of the CCPN framework.\nFinally, the NMR constraint lists encompass the experimental NMR data encoded in the NMR structural ensembles, and comprise a single set of data that is much easier to analyse than an ensemble of solutions. As such, they provide a reduced form of structural information that is relevant for NMR analysis only. For this reason, and to allow a basic level of scientific reproducibility and validation, it is important that constraints, and all other possible NMR derived information, are deposited along with the structure coordinates. It is very likely that a lot more information than described in this article can be gained from it, which in turn can assist the NMR community and can help to understand the relationships between NMR and structure.","keyphrases":["pdb","nuclear magnetic resonance (nmr)","constraint analysis","noe assignment","noe distances"],"prmu":["P","P","R","R","R"]}
{"id":"Anal_Bioanal_Chem-4-1-2259236","title":"High-precision frequency measurements: indispensable tools at the core of the molecular-level analysis of complex systems\n","text":"This perspective article provides an assessment of the state-of-the-art in the molecular-resolution analysis of complex organic materials. These materials can be divided into biomolecules in complex mixtures (which are amenable to successful separation into unambiguously defined molecular fractions) and complex nonrepetitive materials (which cannot be purified in the conventional sense because they are even more intricate). Molecular-level analyses of these complex systems critically depend on the integrated use of high-performance separation, high-resolution organic structural spectroscopy and mathematical data treatment. At present, only high-precision frequency-derived data exhibit sufficient resolution to overcome the otherwise common and detrimental effects of intrinsic averaging, which deteriorate spectral resolution to the degree of bulk-level rather than molecular-resolution analysis. High-precision frequency measurements are integral to the two most influential organic structural spectroscopic methods for the investigation of complex materials\u2014NMR spectroscopy (which provides unsurpassed detail on close-range molecular order) and FTICR mass spectrometry (which provides unrivalled resolution)\u2014and they can be translated into isotope-specific molecular-resolution data of unprecedented significance and richness. The quality of this standalone de novo molecular-level resolution data is of unparalleled mechanistic relevance and is sufficient to fundamentally advance our understanding of the structures and functions of complex biomolecular mixtures and nonrepetitive complex materials, such as natural organic matter (NOM), aerosols, and soil, plant and microbial extracts, all of which are currently poorly amenable to meaningful target analysis. The discrete analytical volumetric pixel space that is presently available to describe complex systems (defined by NMR, FT mass spectrometry and separation technologies) is in the range of 108\u201314 voxels, and is therefore capable of providing the necessary detail for a meaningful molecular-level analysis of very complex mixtures. Nonrepetitive complex materials exhibit mass spectral signatures in which the signal intensity often follows the number of chemically feasible isomers. This suggests that even the most strongly resolved FTICR mass spectra of complex materials represent simplified (e.g. isomer-filtered) projections of structural space.\nIntroduction\nStatus of the molecular-level analysis of complex materials\nNatural molecules can be classified into two main groups according to their functions and how they are synthesized (Fig.\u00a01). In living organisms, a genetic code initiates and controls the synthesis of functional, discrete molecules, which range in size from multienzyme complexes and molecular machines through mid-sized natural products to small molecules (e.g. NO). These molecules are often assigned to well-established distinct classes, such as proteins\/peptides, carbohydrates, lipids, and (less structurally related), natural products and metabolites. Groups of these molecules frequently act together in regulatory networks [1\u20134] so as to enable critical life functions. Typical biofluids and tissues are very complex mixtures which can be resolved into defined molecular fractions using current high-performance separation technologies.\nFig.\u00a01Natural complex organic materials divide into either functional biomolecules which eventually derive from a genetic code or complex biogeochemical nonrepetitive materials which are formed according to the general constraints of thermodynamics and kinetics from geochemical or ultimately biogenic molecules. While biomaterials are amenable to successful separation into unambiguously defined molecular fractions, complex nonrepetitive materials cannot be purified in the conventional meaning of purity due to their extreme intricacy; in fact, the molecular signatures of these supermixtures often approach the limitations imposed by the laws of chemical binding. Improvements in the resolution and sensitivity of analytical techniques combined with the use of minimal (non)invasive sampling techniques have enabled environmental and living systems to be observed to a degree of molecular resolution that was considered unthinkable only a few years ago. A molecular-level understanding of biogeochemical and life processes implies a key role for de novo structural analysis, which depends on the combined use of separation technology hyphenated to organic structural spectroscopy and integrated mathematical data analysis. Analyses of supermixtures depend even more on the mathematical analysis of correlated data obtained from complementary molecular-level precision analytical methods. The formation of NOM on the Earth preceded the evolution of life; the binding of NOM-derived prebiotic molecules to borate contributed to the synthesis of ribose, a crucial precursor of nucleotides, in good yield [5]. Later on in the Earth\u2019s history, coevolution occurred between prebiotic\/abiotic molecules, NOM and primitive and higher forms of life. The near-continuum of binding sites available to ions and organic molecules acts to buffer against environmental and chemical extremes in the geo- and biosphere, which could damage life because of their potent reactivity. This key supportive role of (for example) natural organic matter (NOM) in life processes is sustained by strong interactions between biological and geochemical cycles (Fig.\u00a02). Hence, plant and animal residues are key ingredients of NOM synthesis, while NOM itself, which defines the bioavailability of crucial organic and inorganic nutrients, is indispensable for the sustenance of the microbial life at the bottom of the food chainFig.\u00a02Natural organic matter (NOM) continuously interacts with a broad range of terrestrial, limnic and marine ecosystems. Common to all of these environments are the fundamental molecular aspects of life, and an availability of extended mineral surfaces for interactions with and binding of NOM. The dynamic equilibrium of NOM generation and decomposition spans timescales of many different orders of magnitude (from microseconds to hundreds of thousands of years), and it results from a combined action of biotic and abiotic reactions. NOM may be intrinsically recalcitrant because of the chemical structures of its organic molecules; alternatively, strong NOM\u2013mineral interactions could alter the reactivity of these organic molecules towards increased resistance to degradation. The physical protection of organic matter at interior mineral surfaces provides alternative pathways that enable the recalcitrance of NOM. Photochemical degradation, one of the most significant abiotic reactions of NOM, often results in small molecules like CO2, which are mobile and are easily distributed within various ecosystems. Biomolecules derived from photosynthesis or otherwise originating from a genetic code are eventually decomposed according to the general laws and constraints of thermodynamics and kinetics (Fig.\u00a01). Over very long timescales, the interactions of NOM with minerals at elevated temperatures result in the formation of geopolymers, like kerogen, coal, and oil shales. These ancient materials participate in bio- and geochemical cycling through natural and anthropogenic combustion and through weathering [6]Fig.\u00a03Hierarchical order of intricacy associated with the structural analysis of materials, in terms of polydispersity and molecular heterogeneity (see the main text). The structures (connectivities and stereochemistry) of monodisperse molecules are readily accessible (provided that sufficient amounts of the material are available) by organic structural spectroscopy [7\u20139]. Supramolecular structures [10\u201312] require an adequate definition of the covalently bonded molecules and of their noncovalent interactions [13\u201316]. The structural analysis of nonrepetitive complex unknowns, which feature substantial levels of both polydispersity and molecular heterogeneity [17, 18], is most demanding in terms of methodology and concepts [19\u201322]. Any highly resolved three-dimensional structure of a monodisperse biomolecule is based on a precise description of the unique chemical environment of any single atom [23, 24]. Currently, the molecular-level structural analysis of complex systems is primarily focused on covalent bond definition. Future high-quality structural analyses of these materials will have to assess both the (classes of) individual molecules and their interaction mechanisms [25]\nThe fate of the second vast group of molecules in the bio- and geosphere is governed by the rather fundamental restraints of thermodynamics and kinetics (Figs.\u00a01 and 2). In these intricate materials, the \u201cclassical\u201d signatures of the (geogenic or ultimately biogenic) precursor molecules, like lipids, glycans and proteins, have been attenuated [26, 27], often beyond recognition, during a succession of biotic and abiotic (e.g. photo- and redox chemistry) reactions. Because of this loss of a biochemical signature, these materials can be designated nonrepetitive complex systems. The quantity of molecules in the Earth\u2019s crust that can be attributed to these nonrepetitive complex materials, in the form of kerogens and natural organic matter (NOM) alone, exceeds the quantity of functional biomolecules by several orders of magnitude [28, 29]. Examples include freshwater, marine, and soil organic matter, kerogens and aerosols, among others. These materials typically exhibit an extremely complex array of chemical structures and interactions across a large range of size- and timescales, resulting in molecular signatures that reflect the fundamentals of chemical binding rather than those of their precursors. These novel signatures may in fact cover a sizable proportion of the theoretically feasible molecular composition space (Fig.\u00a09). This extraordinary heterogeneity of molecularly diverse species renders these materials refractory and also implies a limited probability of detecting identical molecules [18]. This contrasts sharply with even the most complex mixtures of biomolecules extracted from any living organism, from which molecularly pure fractions can be readily obtained.\nGiven these unique features, nonrepetitive complex systems epitomize supermixtures. The purification of a supermixture would, in the ultimate sense, approach a molecule-by-molecule separation\u2014a feat beyond our reach, both conceptually and practically. Therefore, these complex nonrepetitive systems are operationally defined according to their properties rather than according to their chemical structures, and their purification (in the conventional sense of the word) remains elusive [18].\nWhile the analysis of complex biomolecules has advanced to the degree that it is possible to obtain well-resolved three-dimensional molecular structures and even meaningful descriptions of dynamics and interactions [30\u201335], the molecular-level precision analysis of complex nonrepetitive materials remains rather rudimentary in comparison [20, 36\u201340]. First of all, theoretically well-founded approaches to numerically describe the complex, polydisperse and nonstoichiometric characteristics of nonrepetitive unknowns are missing at present, limiting our understanding of molecular structures and any application of quantitative structure\u2013activity relationships (QSAR) when modelling their properties. Novel approaches suitable for a quantitative description of various hierarchical levels of molecular organisation (e.g. elements, fragments, molecules) must be developed. Secondly, a meaningful molecular-level analysis of nonrepetitive systems\u2014such as aerosols, natural organic matter and native cell extracts\u2014obviously cannot rely on target analysis, as most of the chemical environments and linkages present are simply not known (Fig.\u00a03).\nConsequently, any comparative analysis of nonrepetitive unknowns with reference materials is very unlikely to provide satisfactory molecular resolution, because rather tiny variations in chemical binding may strongly and often unpredictably affect the properties commonly used for detection, such as retention times and spectral signatures. These fundamental restrictions that are intrinsic to comparative and target analysis are not easily circumvented and they necessitate an independent, spectroscopic \u201cbottom-up\u201d approach to the molecular-resolution characterisation of these complex unknowns.\nInformation transfer in organic structural spectroscopy and separation technologies\nInterestingly, this \u201cbottom-up\u201d approach to the molecular characterisation of complex systems and materials necessarily relies upon spectroscopic methods that translate high-precision frequency measurements into important molecular-level information. Frequencies can be measured to an accuracy of 15 digits, and recently (2005) a Nobel Prize in Physics was awarded for improvements of laser-based precision spectroscopy that enable even more exact frequency measurements to be obtained [41]. This high accuracy of frequency measurement translates directly into high resolution, itself a very useful and even indispensable feature when producing information-rich data. Well-resolved signatures are less susceptible to the detrimental consequences of intrinsic averaging, which is an often overlooked key feature of any low-resolution (spectroscopic and separation) method, when they are employed in the characterisation of complex systems (Fig.\u00a04).\nFig.\u00a04Information transfer in organic structural spectroscopy. These images define a three-dimensional space composed of the area of the image (pixel resolution along the x- and y-axes) and the depth of the color space. This three-dimensional space offers n options for depicting dissimilarity (n\u2009=\u2009x \u00d7\u00a0y \u00d7 color depth). Significant resolution is only attained if there is sufficient information to enable a meaningful assessment of data [here, the apparent differentiation of (a) a fish, (b) a mountain, (c) a human, and (d) a beetle is only possible at panels C and D]. In analogy with these considerations, a three-dimensional analytical volumetric pixel space comprising NMR spectroscopy, mass spectrometry and separation is developed in Fig.\u00a012. The current expansion of this analytical volumetric pixel space (log ) is sufficient to elaborate meaningful detail at molecular resolution from the most complex biological and biogeochemical mixtures\nBulk data of complex systems, like physical parameters, total acidity and elemental analyses, seem to be more precisely defined [42], but exhibit limited resolution. However, any sound structural model of these materials must conform to the constraints defined by these \u201chard\u201d bulk data. High-energy methods of organic structural organic spectroscopy, like XANES, UV\/VIS and infrared spectroscopy, exhibit intermediate structural resolution, which is sufficient, for example, for the characterisation of specific chemical environments [43]; for instance, functional group analysis (carbonyl derivatives, aromatics, heterocycles) in intricate materials.\nIn general, the degree of significant detail generated by a certain analytical technique will depend on both the intrinsic resolution of the respective method and the characteristics of the analysed material. Any inadequate relationship between the resolving power of the technique and intrinsic analyte properties will be wasteful. Investigations of near-featureless materials with methods of supreme resolution could result in unnecessary effort and expenditure. Insufficient resolution of any analytical method with respect to the properties of the analyte will inevitably result in intrinsic averaging, which typically results in poorly resolved properties (which affect the separation) and\/or poorly resolved chemical environments (which affect the spectra). Intrinsic averaging is visualized in Fig.\u00a04 in the form of images of ever-degrading resolution. Similarly, insufficient resolution deteriorates detail in spectra and chromatograms of complex nonrepetitive materials, producing low-resolution signatures and limited bandwidths of variance in bulk and spectral properties.\nHence, any organic structural spectroscopy with a limited peak capacity (Fig.\u00a05) will inevitably lead to a summary bulk-type description of complex materials and considerable averaging, rather than to a meaningful molecular-level resolution analysis. In the case of NOM, this inevitable relationship has been observed in many spectroscopic, separation and chemical experiments, resulting in data with a remarkably limited bandwidth of variance, even when advanced techniques (e.g. at the level of one-dimensional solid-state 13C NMR spectroscopy) are used [44, 45].\nAnalogously, the widespread use of the idiom HULIS (or humic-like substances) in the fields of, for instance, aerosol and remediation research [46\u201353] reflects the operational definition of humic materials as well as our current inability to perform a meaningful molecular-level analysis of complex unknowns, as materials currently denoted HULIS or humic-like substances undoubtedly encompass a wide range of very different species.\nDue to the huge peak capacity of FTICR mass spectrometry, FT mass spectra provide the most convincing direct experimental evidence for the extraordinary molecular diversity of complex materials at present. In these, the molecular-level intricacy of the complex unknowns is most adequately converted into very highly resolved and, consequently, extremely information-rich signatures.\nAnalogous considerations to those given here for spectroscopic characterisation also apply to the separation of complex materials [54, 55, 56].\nInstrumentation and methods\nThe 13C NMR spectrum of Suwannee river fulvic acid (SuwFA) shown in Fig.\u00a07 was acquired at the GSF with a Bruker (Bremen, Germany) AC 400\u00a0NMR spectrometer, operating at 100\u00a0MHz for 13C. FTICR mass spectra were acquired at Bruker\u2019s facilities with a 9.4-T APEXq FT mass spectrometer (data in Fig.\u00a09) and a 12-T APEXq FT mass spectrometer at the GSF (Fig.\u00a08). Here, FTMS spectra were acquired with a time domain size of 1\u00a0MWord (Fig.\u00a08a; Fig.\u00a09, typical resolution 3\u2009\u00d7\u2009105) or 4\u00a0MWord (Fig.\u00a08b, typical resolution 7\u2009\u00d7\u2009105). For Figs.\u00a08a and 8b, elemental compositions were computed with the DataAnalysis software, version 3.4 (Bruker), using the following restrictions: C, H, N, O, unlimited; S, P, 0\u20135; H\/C ratio < 3, mass error \u2264 0.5\u00a0ppm; observance of the nitrogen rule. Exactly one elemental formula was obtained for each peak. The elemental formulae of Fig.\u00a09 were batch-calculated using a software tool written in-house, as described elsewhere [36].\nFig.\u00a05\nCharacteristic resolutions (peak capacity: total range \/ half width [57]) of various separation technologies and organic structural spectroscopic methods (see the main text). This diagram represents a two-dimensional projection of the analytical volumetric pixel space\u2014comprising NMR spectroscopy, mass spectrometry and separation technologies\u2014that defines our current capacity to depict variance in complex systems with molecular resolution (see Fig.\u00a012)\nThe numbers of isomers, as displayed in Fig.\u00a011, were calculated by constructive enumeration using the software MOLGEN (Department of Mathematics, University of Bayreuth, Bayreuth, Germany). For this computation [58], certain restrictions were applied in order to exclude structures that are mathematically possible but are not likely to occur in materials of biogeochemical origin; this means that there are (1) no peroxides; i.e. no \u2013O\u2013O\u2013 connectivities; (2) no triple bonds (\u2013C\u2261C\u2013); (3) no three- or four-membered rings; and (4) no carbon with cumulative double bonds (=C=). The 1H and 13C NMR spectra of cholesterolacetate (section of 1H NMR spectrum in Fig.\u00a06, top panels) and 2-carboxypyrene (Fig.\u00a06, middle panels) were computed with ACD software (ACD\/HNMR and ACD\/CNMR Predictor, v. 5.0); the mass spectral isotope peaks of Ciguatoxin C60H86O19 (Fig.\u00a06, middle panels) were computed with Bruker Compass FTMS software.\nFig.\u00a06The two most significant methods of organic structural spectroscopy, nuclear magnetic resonance (NMR) and FTICR mass spectrometry, are based on high-precision frequency measurements. The top panels illustrate NMR and FTICR MS for atomic and molecular processes. The precessions of atomic magnetic moments in molecules are defined by the chemical environment, and this means that NMR yields unsurpassed resolution of short-range molecular order (in noncrystalline materials, for which X-ray crystallography is not available). In FT mass spectrometry, the orbital frequencies of ions depend on their molecular masses. Mid-size molecules (see middle panels) provide information-rich signatures in NMR and only single peaks in mass spectra (under conditions of non-fragmentation), while complex non-repetitive materials (see bottom panels) produce low-resolution signatures in NMR because of extensive peak overlap. High FT mass spectra resolution is retained for complex molecules, however, because of the extensive peak capacity of the technique (see Fig.\u00a05)\nHigh-precision frequency-derived organic structural spectroscopy\nThe two most influential organic structural spectroscopic methods for the investigation of complex materials, which depend upon high-precision frequency measurements, are NMR spectroscopy and FTICR mass spectrometry (Table\u00a01, Figs.\u00a06, 7). In NMR, the precession frequencies of individual atomic nuclei in an external magnetic field B0 are influenced by their respective chemical surroundings; in FTICR mass spectrometry, the orbital frequencies of ions in an ion trap cell depend on the mass and charge of the molecule of interest [59]. Both methods are isotope-specific, and the combination of NMR and FTICR mass spectral data provides more useful spectral information on complex unknowns at the molecular level than any other spectroscopic method at present.\nWhen studying typical organic molecules, NMR spectra provide more information-rich data than mass spectra, because any single atom within can produce an individual NMR signature (Figs.\u00a06 and 7) [7, 60\u201362]. In the absence of fragmentation, the mass spectrum of any particular molecule will contain only a single peak (in conjunction with its corresponding isotopic pattern [63, 64]). NMR spectra of ever more complex materials will eventually become near featureless because of the extensive overlap between individual NMR resonances [65]; however, the significance of the information is maintained because of the quantification reliability and insightful relationships between NMR chemical shift and extended substructures (at least for the \u201csmall\u201d NMR-active nuclei, e.g. 1H, 13C, 15N, 31P, which represent the key players in organic structural spectroscopy) [66]. Mass spectrometry retains its supreme resolution for extremely complex systems [63, 67\u201370], but will eventually become limited by the inability of mass spectrometry to (easily) discriminate between isomers [71]. Hence, mass spectra will at best represent isomer-filtered projections of the entire structural space of molecules (see Figs.\u00a07, 8, 9, 10, 11).\nFig.\u00a07Molecular-level resolution spectroscopic data represent projections of the vast total structural space of molecules, for which count estimates range from 1060 to 10200 [72]. The complementarity of NMR spectroscopy and mass spectrometry for the spectral characterisation of intricate materials is caused by the entirely different atomic and molecular processes that these methods rely upon (Fig.\u00a06). Mass spectra reflect the isomer-filtered complement of the entire space of molecular structures. The compositional space of molecules can be probed with ultrahigh-resolution FTICR mass spectroscopy, resulting in single peaks for molecules (in the absence of fragmentation). NMR spectra represent site- and isotope-specific projections of the molecular environments (the projected NMR spectrum given here shows a 13C NMR spectrum for an aquatic NOM). Accordingly, typical organic molecules exhibit single mass peaks (molecular ions given here: C9H9NO3) and more elaborate NMR signatures (13C NMR data are shown here for C9H9NO3; see also Fig.\u00a06). Because these atomic and molecular signatures are not entirely orthogonal, the data provided by NMR and MS show correlations that can be used to reconstruct chemical structures by empirical and mathematical back-projection. In mixture analysis, separation offers a (near-)orthogonal means of expanding the two-dimensional spectroscopic projection area into a three-dimensional analytical volumetric pixel space (see Fig.\u00a012). Following separation, molecules that often exhibit wide concentration variances in mixtures can be investigated by combined NMR\/MS, with acquisition parameters specifically adapted in order to maintain the optimum dynamic range, which is necessary for good-quality spectraFig.\u00a08FT mass spectral resolution and the C,H,O-compositional space (see the main text and Fig.\u00a09)Fig.\u00a09Van Krevelen diagram (which illustrates the C,H,O-compositional space) for consolidated ESI, APPI, APCI positive and negative ions of Suwannee river fulvic acid (SuwFA) in the mass range 200\u2013700\u00a0Da (see the main text and Fig.\u00a08)Fig.\u00a010Elucidating mass spectra of complex materials requires advanced means of data analysis, such as van Krevelen diagrams, which are based upon assigned molecular formulae, and fragment- or molecule-specific Kendrick mass defect analyses [73]. Any dot in the van Krevelen diagram of a complex material represents a projection of the elemental ratios derived from assigned molecular formulae, irrespective of molecular mass. Accordingly, any of these dots could represent an intrinsic superposition of all feasible isomers from possibly different molecular compositions, sharing only their respective elemental ratios. The numbers of chemically reasonable isomers easily account for many millions of isomers seen for moderately sized molecules (a few hundred Daltons), even when only a few double bond equivalents and heteroatoms (e.g. oxygen) are present (see Fig.\u00a011)Fig.\u00a011The relationship between the FTICR mass spectral intensity and the number of feasible isomers in complex materials. The distribution of (chemically relevant) C,H,O-isomer counts within the C,H,O-compositional space is visualized here in a van Krevelen diagram (left) for the eleven feasible molecular compositions CnHmOq that have an IUPAC nominal mass of 178 Da (see the main text). These molecules are arranged into three series (series 1a\u20131c with three members; series 2a\u20132f with six members; series 3a\u20133b with two members) of isobaric molecules, which are related by a formal exchange of CH4 against oxygen. For any molecular composition, the number of chemically relevant isomers is given in units of 104. Carboxyl-rich alicyclic molecules (CRAM), which represent a complex mixture of molecules with near-absent olefinic and aromatic unsaturation, have recently been identified in marine organic matter [18], and they occupy an area of C,H,O-compositional space for which the largest number of feasible C,H,O-isomers is expected (see the main text), suggesting that the actual number of different molecules in the mass spectra of CRAM for any given mass may correlate with mass spectral intensity patterns. The availability of aromatic moieties in Suwannee river fulvic acid (SuwFA) also allows chemically relevant structures at lower H\/C ratios (see the main text). The green circumfenced area denotes the van Krevelen compositional space of SuwFA, as provided in Fig. 9 (note that the SuwFA here represents consolidated positive and negative ion APCI+APPI+ESI FTICR mass spectra, as opposed to the negative ion ESI FTICR mass spectra given in the case of CRAM; see the main text). The panel on the right shows the CnHmOq isomers computed for an IUPAC nominal mass of 178 Da in an intensity versus mass display (analogous to a mass spectrum), denoted according to series 1\u20133 (see the main text). This pattern resembles the clusters of peaks observed in good-quality FT mass spectra of NOM (see Fig.\u00a06, bottom panels, and Fig.\u00a08), suggesting that the intensities of the C,H,O-derived mass spectral peaks of NOM follow the number of chemically relevant C,H,O-isomers computed\nTable\u00a01\nCharacteristics and significance of key molecular-level resolution techniques\nMolecular-level resolution technique\nAdvantages\nCurrent weaknesses and future developments\nNMR spectroscopy\nNMR spectroscopy provides isotope-specific information, in unsurpassed detail, on short-range molecular order (the arrangement of chemical bonds, including connectivities, stereochemistry and spatial proximity), dynamics [33, 74] and reactivity [35]\nRelative insensitivity compared with other analytical techniques\nNondestructive and isotope-specific [32] analysis across almost the entire periodic table combines with the most accurately defined near-quantitative relationship between the spin number and the area of the NMR signal. This key feature of NMR when applied to the analysis of complex systems implies the use of NMR spectrometry as a quantitative reference for other, complementary analytical methods [75\u201381]\nIntricate physics and chemistry of intra- and intermolecular interactions in complex mixtures may interfere with the direct relationship between chemical shift and molecular structure and, because of relaxation-induced variable line widths, quantification\nThe unique ability to generate and analyse data from multiple 1,2,3-D NMR experiments performed on a single sample enables the significance and authenticity of individual spectra to be assessed [82\u201384]\nNear-identical chemical shifts do not necessarily imply similar chemical structures [85]\nExtensive and far-reaching information can be obtained, even from ill-resolved NMR spectra, for \u201csmall\u201d nuclei (e.g. 1H, 13C, 15N, 31P) because of the plausible correspondence between chemical shift and extended substructures\nSensitivity and resolution increased by high-field magnets [86, 87], cryogenic [88] and micro- [89\u201392] probes, and by changing (to nanoliter) sample size [93, 94]\nThroughput increased by using fast higher dimensional spectroscopy with superior sensitivity and through the parallel acquisition of NMR spectra [95\u201399]\nFTICR mass spectrometry\nBest combination of spectral resolution and sensitivity, which allows miniaturisation [100] and hyphenation of mass spectrometry with high-performance separation techniques like capillary electrophoresis and UPLC (CE\/UPLC with mass-selective detection)\nMolecular-level structural information is mainly restricted to ionizable compounds\nDue to its supreme mass accuracy and resolution, molecular formulae from thousands of compounds can be obtained in a single experiment directly from mixtures [63, 69, 101\u2013104]\nIsomer differentiation is a nontrivial task [71]\nFragmentation provides further molecular-level structural information beyond molecular composition\nQuantification is difficult, even for identical molecules in mixtures, because of the variable ionization efficiencies of individual compounds, which strongly depend on the experimental conditions and mixture composition\nColumn adsorption and fractionation as well as electrochemical and redox reactions associated with the spray conditions may interfere with authentic sample representation\nA wide range of ionization techniques (electrospray, ESI; chemical ionization, CI; photoionization, PI; desorption ionization, DESI; field ionization, FI, among others, all performed in either positive or\/and negative modes) for mixtures is available under specifically adapted conditions [105\u2013110]\nMass-selective imaging is feasible with high spatial and mass resolution; qTOF mass spectrometry allows for very fast scan rates, and is perfectly suited for hyphenation with high-performance separation techniques (CE and UPLC) as well as mass-selective imaging\nFurther miniaturisation of separation and detection devices in conjunction with ultrahigh-resolution FTICR mass spectrometry will permit highly resolved and information-rich data to be obtained from tiny amounts of sample (chip-MS) [100]\nHigh-performance separation techniques (UPLC\/HPLC and capillary electrophoresis)\nLarge separation capacity and extensive miniaturisation; is cost-effective; can be highly automated\nGives only limited structure-specific information about the short-range molecular order [111, 112]\nElectrophoretic mobility and chromatographic retention time carry structure-specific information, which can be adapted to a wide range of experimental conditions in order to probe size, shape, charge characteristics and reactivity\nSensitive and versatile suite of separation methods and of structure-specific (and nondestructive) detection systems, such as (laser-induced) fluorescence, UV\/VIS, radioisotope or mass-selective detection\nCE complements NMR information about primary chemical structures (covalent bonds) by providing data on the corresponding secondary and tertiary structure\nFeasibility of up-scaling from capillary zone electrophoresis (CZE) to a preparative level by means of free flow electrophoresis (FFE) and from UPLC to any preparative LC method\nFurther miniaturisation offers hyphenation options down to single-cell analysis and compartments within\nMolecular-level resolution spectroscopic data represent projections of the vast total structural space of molecules, for which count estimates range from 1060 to 10200 [72]. The complementarity of NMR and mass spectrometry for the spectral characterisation of intricate materials is caused by the entirely different atomic and molecular processes these methods rely upon (Fig.\u00a06).\nMass spectra reflect the isomer-filtered complement of the entire space of molecular structures. The compositional space of molecules can be probed with ultrahigh-resolution FTICR mass spectroscopy, resulting in single peaks for molecules (in the absence of fragmentation). Two-dimensional projections of the structural space, like van Krevelen diagrams and Kendrick mass defect analyses, are indispensable tools for the evaluation of mass spectra of complex materials (Figs.\u00a08, 9, 10, 11) [73, 113, 114]. NMR spectra represent site- and isotope-specific projections of the molecular environments. Therefore, typical organic molecules exhibit single mass peaks (molecule ions) in mass spectra and more elaborate NMR signatures (Figs.\u00a06 and 7). Because these atomic and molecular signatures are not entirely orthogonal, the data provided by NMR and MS exhibit correlations that can be used to reconstruct chemical structures by empirical and mathematical back-projection.\nThe NMR and mass spectral data can be acquired via direct hyphenation or in separate experiments [115\u2013117]. In mixture analysis, separation offers a (near-)orthogonal means of expanding the two-dimensional spectroscopic projection area into a three-dimensional analytical volumetric pixel space (see Fig.\u00a012). Following separation, molecules that often exhibit wide concentration variances in mixtures can be investigated by combined NMR\/MS, with acquisition parameters specifically adapted to maintain the optimum dynamic range necessary for good quality spectra. Particularly in the case of mixture analysis, any joint mathematical evaluation of these correlated data will reveal hidden detail and will considerably enhance the resolution as well as the significance of molecular-level information (see also Figs.\u00a04 and 12).\nFig.\u00a012The currently accessible discrete volumetric pixel (voxel) space for the characterisation of complex materials is in the range of 108\u201314 voxels. Its expansion is defined by the significant resolution of the complementary techniques of nuclear magnetic resonance (102\u20135 buckets, depicting the short-range order of molecules), ultrahigh-resolution FTICR mass spectrometry (104\u20135 buckets, depicting molecular masses and formulae of gas-phase ions) and high-performance separation (102\u20134 buckets, depicting both ions and molecules; this provides a way to validate NMR against MS data); see also Fig.\u00a05 for even wider expansion. The various projections of this voxel space, like separation\/MS, separation\/NMR and NMR\/MS, can be realised in the form of direct hyphenation [104, 115, 117\u2013120] and via mathematical analysis [121, 122]\nFTICR mass spectra show supreme resolution, as indicated by the 12-T negative ionization ESI FT mass spectra of a barley extract (Fig.\u00a08a) and IHSS Suwannee River Natural Organic Matter (International Humic Substances Society NOM; Fig.\u00a08b). Here, CnHmOq molecules contribute most to the total ion count. These molecules can be arranged into series, which are related by the formal exchange of CH4 against oxygen. Figure\u00a08c denotes the mass peaks corresponding to the 37 theoretically possible and chemically reasonable\u00a0C,H,O-compositions depicted in Fig.\u00a08d that have a nominal mass of 301 Da. Note that negative M\u2013H+ ions (i.e. [M\u2212H+e]\u2212) are observed in the FTICR mass spectra (Fig.\u00a08b), and the C,H,O-compositions of molecules M are denoted in Fig.\u00a08c. M and M\u2013H+ differ in mass by one hydrogen (1.007825032 Da) minus an electron (0.000548625 Da); in Fig.\u00a08c this difference is decomposed into a mass shift of one (see the shift between the mass axes) and an additional small mass spacing \u0394m\u2009=\u20090.000233878 Da. The molecules in the barley extract exhibit mass peaks outside of the range accessible for any C,H,O-composition (dotted purple box in Fig.\u00a08a), indicating the presence of additional heteroatoms (e.g. N, P, S) in these ions.\nFigure\u00a08d denotes a van Krevelen diagram of the 37 chemically reasonable CnHmOq molecules, in which the 16\u00a0C,H,O-ions observed in Fig.\u00a08b are highlighted. The number of peaks identified corresponds to a coverage of 43% of the entire C,H,O-compositional space. These ions occupy an area for which the largest number of feasible C,H,O-isomers is expected (see Fig.\u00a011).\nMolecularly intricate materials, like natural organic matter (NOM), exhibit molecular signatures approaching the theoretical limits defined by the laws of chemical binding. In Fig.\u00a09, a van Krevelen diagram of Suwannee River fulvic acid (SuwFA) depicts the elemental ratios of CnHmOq ions (the ions shown represent a consolidation of the ions obtained by ESI, APCI and APPI positive and negative ionization from 9.4-T FTICR mass spectra; unpublished data). The peaks observed in the negative\/positive ionization mode only are coloured green\/orange; peaks observed in both positive and negative modes are depicted in black. The lack of signatures from biochemical precursor molecules [123] indicates the considerable level of processing typical of NOM. Within a mass range of 200\u2013700 Da and the given limits of the H\/C and O\/C ratios, the minimum consolidated number of individual C,H,O-molecular compositions (4270) represents a sizable fraction (23%) of the entire feasible compositional space of CnHmOq molecules (18414 in total; small grey dots). To further appreciate the remarkable intricacy of natural organic matter, it should be noted that any dot in the van Krevelen diagrams of these complex materials represents a projection of the elemental ratios derived from assigned molecular formulae, irrespective of molecular mass. Hence, the dots in the van Krevelen diagrams can represent multiple molecular formulae (Figs.\u00a010 and 11), while any identified molecular composition reflects an intrinsic superposition of all feasible isomers (Fig.\u00a011). Considering typical molecular weights of several hundreds of Daltons in the mass spectra of NOM (Fig.\u00a06, bottom panels), it is readily anticipated that the mass spectra of such systems represent simplified (e.g. isomer-filtered) projections of a still hugely more expansive structural space (Fig.\u00a07).\nKey trends relating molecular composition to the number of feasible isomers\nFor any exceedingly complex material, it is logical to postulate that many isomers will contribute to any given molecular formula. Analogously, the intensities of the mass spectral peaks, which superimpose all of the isomers present, will be a function of the abundances of these isomers in these materials and the ionization efficiency of each isomer under the given experimental conditions.\nFor molecules of a given mass composed of carbon, hydrogen, and oxygen, two major and independent trends are expected to define the number of feasible isomers. First, decreasing the H\/C ratio from fully saturated molecules (CnH2n+2) means removing hydrogen atoms, which is equivalent to introducing double bonds or (ali)cyclic structures (double bond equivalents, DBEs). Molecules with large H\/C ratios are structurally fairly uniform, consisting mainly of various branched chains of single bonds. Introducing large numbers of DBEs will lead to many new structures with double bonds and or (ali)cyclic structures in various positions. For an H\/C ratio of close to one, on average two carbons carry one DBE, and the introduction of further DBEs will lead to a lack of single bonds. Hence, the maximum number of feasible C,H,O-isomers is expected to occur for intermediate numbers of DBEs in a molecule, because the occurrence of a DBE (which solely depends on the H\/C ratio) enables double-bond displacement and the formation of (ali)cyclic structures, both of which greatly enlarge the number of feasible isomers. In contrast, only highly condensed structures can be assembled at very low H\/C ratios [37], and this constraint severely diminishes the number of feasible C,H,O-isomers (if mathematically possible but chemically unlikely isomers are excluded; see Fig.\u00a011).\nSecond, the insertion of oxygen into potentially any carbon\u2013carbon (creating C\u2013O\u2013C units) or carbon\u2013hydrogen bond (creating C\u2013OH functionalities) will result in many more feasible isomers at low O\/C ratios; in the presence of DBEs, \u201cterminal\u201d carbonyl derivatives (C=O) can also be constructed. At higher O\/C ratios, however, further insertion of oxygen decreases the number of feasible isomers for two reasons: oxygen provides fewer (two) options for forming (single) bonds with other partners than carbon (four); in addition, the higher mass of oxygen (16 Da) compared with that of carbon (12 Da) decreases the total number of \u201cheavy\u201d atoms available for the construction of CnHmOq molecules of a given mass.\nThese considerations imply that the number of feasible C,H,O-isomers for a given mass will reach maximum values at intermediate H\/C and O\/C ratios, and that these numbers will (sharply) decline at extreme (high and low) H\/C and O\/C ratios, respectively.\nThese dependencies are displayed in a van Krevelen diagram (Fig.\u00a011), in which the numbers of chemically relevant isomers for any given molecular composition CnHmOq of a single nominal IUPAC mass are provided. For any given nominal mass, the mathematically possible and chemically relevant structures composed solely of carbon, hydrogen and oxygen atoms can be constructively enumerated for each composition (molecular formula) [58]. By \u201cchemically relevant isomers\u201d, we mean all mathematically possible isomers (not counting stereoisomers) except for those containing O\u2013O bonds, C\u2261C bonds, three- or four-membered rings, or =C= fragments (cumulated double bonds), which are not assumed to occur in the materials of interest (natural organic matter here). These data are displayed in the right panel of Fig.\u00a011, where they are arranged according to actual mass.\nFor practical reasons, we have selected CnHmOq compositions with a nominal IUPAC mass of 178, for which the number of isomers can be computed within a reasonable time on a desktop computer; within the given limits of H\/C and O\/C elemental ratios, eleven feasible C,H,O-molecules are found, which are grouped into three series of isobaric molecules, related by a formal exchange of CH4 for oxygen (Fig.\u00a011).\nSeries 1 represents highly unsaturated molecules in which the number of isomers declines sharply with decreasing H\/C ratio. Series 2 presents the maximum number of isomers at intermediate H\/C (and O\/C) ratios and the decline in the number of isomers at both high and low H\/C (and O\/C) ratios, as anticipated (see above).\nThe maximum H\/C ratio found for a series 2 molecule amounts to almost 1.7, and the corresponding molecule C13H22 (2a) features three\u00a0DBEs, thereby allowing for a much larger array of unsaturation-related isomers than obtained for a fully saturated parent molecule. This is demonstrated by the variance in the isomer count when the fully saturated analogue C13H28 (184 Da, 802 isomers) is compared with the series 2 \u201cendmember\u201d C13H22 (2a; 178 Da, 1.7\u2009\u00d7\u2009105 isomers); analogous relationships are found for the series 1 \u201cendmember\u201d C14H10 (1a; 178 Da, 5.3\u2009\u00d7\u2009106 isomers) in comparison with its fully saturated parent molecule C14H30 (198 Da, 1858 isomers).\nA considerable fraction of the 16.6-fold increase in the isomer count observed when comparing C13H22 (2a; three\u00a0DBEs) and C12H18O (2b; four\u00a0DBEs) results from the ability to produce novel isomers with singly bonded oxygen and those with a C=O bond (carbonyl derivative). The maximum number of isomers (~1.1\u2009\u00d7\u2009107 each) is attained for the molecules C11H14O2 (2c; five\u00a0DBEs) and C10H10O3 (2d; six\u00a0DBEs), respectively. Further exchange of CH4 against oxygen again sharply decreases the number of feasible isomers [by a factor of 5.5 when proceeding from C10H10O3 (2d) to C9H6O4 (2e), and by a factor of 78 when changing from C9H6O4 (2e) to C8H2O5 (2f)].\nThe series 3 molecules C7H14O5 (3a) and C6H10O6 (3b) feature rather limited numbers of isomers because of their large O\/C ratios (see above). A comparison of C8H2O5 (2f) and C7H14O5 (3a) indicates that extreme hydrogen deficiency restricts the feasible number of isomers more severely than almost full saturation. Molecules C14H10 (1a; ten\u00a0DBEs, 5.3\u2009\u00d7\u2009106 isomers; series 1), C10H10O3 (3b; six\u00a0DBEs, 1.1\u2009\u00d7\u2009107 isomers; series 2) and C6H10O6 (3b; two DBEs, 6\u2009\u00d7\u2009104 isomers; series 3) are all related by a formal exchange of four carbons for three oxygen atoms. The introduction of oxygen initially outweighs the decrease in the number of carbon atoms and DBEs available because of (i) the reduced severity of unsaturation and (ii) the availability of oxygen to construct isomers (see above). Upon the transition from C10H10O3 (2d) to C6H10O6 (3b), however, both the lesser ability of oxygen to participate in chemical bonding (two bonds for any oxygen instead of four for any carbon) and the decline in available DBEs lead to a drastic decrease in the number of accessible isomers.\nMass spectral intensities and number of feasible isomers in marine and terrestrial NOM\nIn a highly processed and supposedly exceedingly complex material such as deep sea marine organic matter, most of the molecules of formula CnHmOq will contain an intermediate amount of unsaturation and numerous oxygen atoms [18, 20]. This flexibility to generate a potentially huge number of isomers implies that (in the absence of severe ion suppression) mass spectral intensities should correlate roughly with the number of feasible isomers for any given molecular composition. Recently, carboxyl-rich alicyclic molecules (CRAM) have been identified as prominent constituents of marine (and possibly freshwater and terrestrial) organic matter [18]. CRAM likely represent highly processed products of ultimately terpenoid origin and are expected to represent an extremely complex mixture of molecules. Based on the molecules of formulae CnHmOq and a recognition of FT mass spectral intensities, the CRAM that occur in deep ocean marine ultrafiltered organic matter comform mainly to the region inside the dotted ellipsoid in the van Krevelen diagram of Fig.\u00a011, which appears to coincide with the maximum number of feasible C,H,O-isomers.\nThe availability of aromatic structures in terrestrially and freshwater-derived NOM, such as that in Suwannee river fulvic acid (SuwFA; Fig. 9), opens up the compositional space of chemically relevant NOM molecules (see above) to significantly lower H\/C ratios than accessible solely on the basis of open-chain unsaturation (e.g. olefinic and carbonyl) and alicyclic double-bond equivalents (DBE). These dependencies are nicely illustrated by comparing the van Krevelen diagrams of marine ultrafiltered dissolved organic matter (UDOM) [18], a blackwater NOM [18, 124], and that of SuwFA (Figs. 9 and 11). While mass spectra of marine UDOM are dominated by carboxyl-rich alicyclic molecules (CRAM), composed mainly of carboxylic groups and alicyclic rings with only negligible aromatic and olefinic unsaturation [18], the significant terrestrial, aromatic-rich signature present in both blackwater NOM and SuwFA populates the compositional space with notably lower elemental H\/C ratios than feasible in marine UDOM [18].\nIt should be noted that oxygen-depleted molecules of formula CnHm are less likely to be ionized in standard ESI FTICR mass spectra in comparison with oxygenated molecules of formula CnHmOq. Carbohydrates, which are oxygen-rich, also are less efficiently ionized under standard ESI-FTICR mass spectral conditions than carboxyl-rich molecules like CRAM. CRAM therefore represent the most likely constituents of NOM to produce strong signals in ESI-FTICR mass spectra.\nThe compositional space of Suwannee river fulvic acid (SuwFA) given in Figs. 9 and 11 is derived from consolidated positive and negative ion FTICR mass spectra, obtained via APCI+APPI+ESI ionization modes, thereby facilitating the observation of oxygen-depleted molecules (Fig. 9).\nThe discrete analytical volumetric pixel space defines our current capacity to depict molecular-level variance in complex systems\nThe current capacity to describe complex materials at molecular resolution can be visualized in the form of an analytical space comprising individual volumetric pixels (voxels). The range of this discrete and quantized space is 108\u201314 voxels, as defined by the significant resolution of the complementary techniques of nuclear magnetic resonance (102\u20135 buckets, depicting the short-range order of molecules), ultrahigh-resolution FTICR mass spectrometry (104\u20135 buckets, depicting molecular masses and formulae of gas-phase ions) and high-performance separation (102\u20134 buckets, has the capacity to investigate both ions and molecules, and so provides a way to validate NMR against MS data).\nAn investigation of these correlated data is feasible at the level of the direct hyphenation of separation and spectroscopy [e.g. LC\/NMR and LC or CE\/MS; corresponding to the rear faces of the voxel space [119, 125\u2013128]) and by means of statistical heterospectroscopy (SHY) [129, 130]; corresponding to the top face (or any two faces) of the voxel space]. Any joint mathematical analysis of these correlated data will enhance the effective resolution of the data and the significance of the molecular-level analysis of complex unknowns [119, 121, 129].\nThis voxel space can be readily expanded to higher dimensions by including complementary data, like those derived from genomic and proteomic analyses [84, 131\u2013134] or by recognising selective chemical reaction products [135\u2013140]. Degradative approaches to the characterisation of complex systems produce limited amounts of unambiguously identifiable small molecules but lose crucial linkage information. Soft and selective biochemical and chemical reactions like mild hydrolysis, reduction, oxidation and derivatisation [141, 142] of complex systems will often result in larger fragments with valuable positional and stereochemical information for the assessment of synthesis and degradation pathways.\nThe chemical transformation of functional groups with NMR- and MS-recognisable labels enables isotope-specific functional group analysis based on structural rather than behavioural characteristics [143, 144]. Information concerning stereochemistry and stable isotope composition will become more important when assessing the origins and diagenesis of complex natural materials. Any progress in the determination of position-specific stable isotope composition (e.g. by NMR and MS methods) will be useful for advancing this field. Physical and chemical fractionation will greatly assist in these studies; further miniaturisation will enhance separation capacity and thereby improve the resolution of the analytical voxel space (Figs.\u00a05 and 12).\nIntegrated biomarker profiling approaches [145\u2013147] with higher resolutions, significances and accuracies will substantially improve the quality and relevance of current systems biology approaches in the health and environmental sciences. The great progress made in the molecular-level characterisation of complex systems over the last few years and foreseeable improvements in nascent technology and concepts will lead to strong synergetic effects that will further advance our understanding of any complex natural and living system whose properties and functioning depend on both strong (covalent) and weak (noncovalent) interactions.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nESM 1\n(PDF 12.9 MB)","keyphrases":["complexity","complex systems","resolution","separation","intrinsic averaging","nmr","ft mass spectrometry","isomers","compositional space"],"prmu":["P","P","P","P","P","P","P","P","P"]}
{"id":"Support_Care_Cancer-4-1-2206248","title":"Quality of life of parents with children living at home: when one parent has cancer\n","text":"Goals of work This study examined the quality of life (QoL) of cancer patients diagnosed 1\u20135 years previously and their spouses, with children 4\u201318 years living at home. Relationships between parents\u2019 QoL and the children\u2019s functioning were explored.\nIntroduction\nThe impact of cancer on a patient\u2019s psychosocial functioning has received much attention in the literature over the past decades. It is generally acknowledged that a patient\u2019s quality of life (QoL) decreases while they are battling their illness. Problems in QoL tend to diminish over time as a patient responds to treatment. However, a significant percentage of patients continues to experience clinically elevated levels of problems requiring professional treatment [40]. Furthermore, a growing number of studies provides evidence that cancer patients\u2019 spouses develop problems affecting their QoL [14, 17, 21, 23, 25, 27]. Spouses have reported psychological and physical distress, which have been found to correlate with the patient\u2019s dysfunction [17].\nHowever, the majority of studies examine patients and spouses in later adulthood [5, 6, 13, 25, 29]. Most incidences of cancer occur in older adults. According to the Dutch Comprehensive Cancer Center\u2019s database, 70% of all new incidences of cancer in The Netherlands in 2003 occurred in patients more than the age of 60 [11]. Parental cancer in younger families may be a more serious stressor than in later phases of life, as illness and death in later adulthood could be considered more natural or normative [37, 39]. It has been shown that older cancer patients report less anxiety and depression and better QoL than younger adult patients [26]. Couples in an earlier life phase with young children and adolescents at home lead busy lives. They juggle child-rearing responsibilities and the demands of sustaining a healthy marriage with trying to meet the individual (career) needs of each partner [28]. In the case of families with parental cancer, parents have the added strain of their loved one being ill. Additionally, their stress may be confounded when the patient may not be able to work or take care of the children as well as before the onset of the disease.\nOnly a few studies have examined the impact of cancer on the functioning of all members in families in this phase of life. In our previous investigations of families confronted with parental cancer, the main focus has been on the children\u2019s psychological well being [20, 36]. Results have shown clinically elevated levels of distress in 35% of adolescent daughters and 21% of adolescent sons [20] and higher levels of emotional problems in adolescent daughters and elementary school-aged sons of cancer patients [36]. It is possible that the children\u2019s emotional and behavioral functioning, which may be affected by their parent\u2019s cancer, affects how the parents function.\nThe current study had four main aims. The first aim was to examine health-related QoL of patients and spouses in families with young children 1\u20135\u00a0years after diagnosis. We hypothesized that cancer patients and their spouses\u2019 QoL would be lower than that of a norm group and that QoL would differ as a function of health status and gender (i.e., which parent was ill: the mother or father). Based on literature [30], we expected to find that female patients reported the lowest QoL, followed by female partners, male patients, and male partners. The second aim was to examine the effect of illness-related variables (time since diagnosis, recurrence, treatment intensity, and type of cancer) on parents\u2019 QoL. We expected that patients and spouses would report more problems more shortly after diagnosis, when confronted with recurrence of cancer and when treatment was intense and, also, that QoL would differ depending on type of cancer. Our third aim was to examine relationships between patient\u2019s and spouse\u2019s QoL; we hypothesized that they would be significantly related. Finally, the fourth aim was to explore possible relationships between the parents\u2019 QoL and the children\u2019s emotional and behavioral functioning.\nMaterials and methods\nProcedure\nBoth hospitalized cancer patients and those being treated or seen for follow-up visits at the outpatient clinic of the University Medical Center Groningen between January 2001 and February 2003 were approached by surgical, medical, and radiation oncologists and oncology nurses. Patients were eligible for the study if they were diagnosed with cancer 1\u20135\u00a0years before study entry and had children between the ages of 4 and 18\u00a0years at the time of parent\u2019s diagnosis who resided or had frequent contact with the diagnosed parent. Furthermore, both parents and children needed to be fluent in Dutch. A family could participate in the study if the patient and at least one child agreed to participate. Physicians and oncology nurses offered all eligible patients and spouses written information plus an information brochure adapted for the children. Informed consent was obtained from each family member separately, as regulated by the Medical Ethical Committee of the University Medical Center Groningen. After obtaining informed consent, researchers mailed packages with questionnaires and prepaid return envelopes to each family member. Cancer patients, spouses, and children were instructed to fill in questionnaires independently of each other and to not discuss their answers.\nInstruments\nDemographic data was gathered on: age, gender, level of education, length of relationship, number of children living at home, and who the primary caregiver in the family is. Patients provided information about when they were diagnosed, their type of cancer, treatment intensity, and recurrence. Education was measured on a seven-point scale from (1) elementary school only to (7) university degree.\nThe eight subscales and two component summary scores of the Dutch translation of the RAND-36 were used to measure QoL [8, 31, 38]. Scores on the subscales can range from 0 to 100, with higher scores indicating better functioning. When comparing patients and spouses to a reference group, norm scores were used from the Dutch manual for the RAND-36 [31]. The manual provides mean scores from a random sample of 1,063 people between the ages of 18 and 89 (65% women, age M\u2009=\u200944.1) from the population register of a municipality in The Netherlands. Norm scores are provided for each subscale for the entire sample and for men and women separately. Summary scores report physical functioning and psychosocial functioning and are standardized with a mean of 50 and a standard deviation of 10. The reliability and validity of the RAND-36 has been supported in a wide number of international and national studies [8, 32, 33]. Cronbach\u2019s alphas in this study on the eight subscales ranged between 0.72 and 0.90 for patients and 0.78 and 0.90 for partners.\nTo investigate how parents\u2019 QoL related to the children\u2019s functioning, parents were asked to complete the 120-item Child Behavior Checklist (CBCL) [1, 34]. Adolescents also completed the 102-item Youth Self-Report (YSR) [2, 35]. In this study, the internalizing (emotional functioning), externalizing (behavioral functioning), and total problem (total of internalizing, externalizing, and cognitive problems) scales were used to provide a picture of the problems occurring in the children of parents diagnosed with cancer. The CBCL\u2019s and YSR\u2019s reliability and validity have been supported in a great number of studies. In this study, Cronbach\u2019s alphas for the internalizing, externalizing, and total problem scales ranged from 0.84 to 0.94 for reports from patients, spouses, and adolescent children.\nAnalysis\nIn our analyses, a single variable was created to define treatment intensity. Patients were grouped into two categories based on the clinical expectation that surgical treatment alone (nonintense treatment) would be less distressing to the family because of less time away from home and fewer visible side effects. Other single-modal (chemo-, radiotherapy) and multimodal treatments (combination of surgery, chemotherapy, radiotherapy, hormone, or immunotherapy) were defined as intense. Time since diagnosis was calculated by subtracting the date on which the patient filled in the questionnaires from the date they were diagnosed. Descriptive analyses were performed on demographic information. Comparisons between patients, spouses, and the reference group were investigated using independent t tests. Results were corrected for the number of tests performed with a Bonferroni correction for multiple comparisons; p values less than or equal to 0.0015 were considered significant (0.05 of 32 tests). To assess clinical relevance, effect sizes (ESs) were calculated by dividing the difference between means by the square root of the average of the squared standard deviations [10]. ESs between 0.20 and 0.49 were considered small, 0.50 and 0.79 medium, and \u22650.80 large [10]. We considered that ESs greater than or equal to 0.50 indicated a clinically relevant difference [24]. A single variable was created to describe parents\u2019 gender and health status (ill mothers, ill fathers, healthy mothers, and healthy fathers). One-way analyses of variance (ANOVAs) with a Bonferroni post-hoc test were used to compare QoL of patient and spouse as a function of gender and health status. After the correction for multiple tests was applied, p values less than or equal to 0.005 were considered significant (p\u2009=\u20090.05 of ten tests). In analyses of QoL as a function of type of cancer, only groups of ten or more patients were included. An ANOVA and a Kruskal\u2013Wallis test (some patient groups were small) were performed to compare patients\u2019 and spouses\u2019 QoL as a function of type of cancer. After the correction for multiple tests was applied, p values less than or equal to 0.005 were considered significant (p\u2009=\u20090.05 of ten tests). The two QoL composite scores (physical summary score and psychosocial summary score) were used in correlational analyses to explore relationships between parents\u2019 QoL and between each parent\u2019s QoL and the children\u2019s problems, with separate analyses for elementary school-aged (4\u201310\u00a0years) and adolescent (11\u201318\u00a0years) children. Pearson\u2019s product-moment correlation coefficients were calculated; correlations with a coefficient less than 0.30 were considered weak, 0.30\u20130.50, moderately strong, and greater than 0.50, strong [10].\nResults\nParticipants\nA total of 476 families were approached for the study. Two hundred and nine agreed to participate (44%), including 336 children, with an average of 2.3 children per family. Because this study focused on couples, we selected data from families where both patient and spouse filled in questionnaires, resulting in a database of 166 couples and 304 children. Table\u00a01 summarizes the demographic information. The majority of cancer patients in this study were women (78%). Patients were diagnosed with various types of cancer (Table\u00a03), including breast (52%), gynecological (10%), hematological (9%), skin (9%), urological (6%), soft tissue and bone tumors (6%), head\/neck (4%), gastrointestinal (3%), and central nervous system (1%). Fourteen percent of patients had undergone nonintense treatment (N\u2009=\u200923), and 86% had undergone intense treatment (N\u2009=\u2009143).\nTable\u00a01Demographics\u00a0PatientSpouseChildrenMean age in years (SD)44.7 (4.9)45.8 (6.4)14.5 (4.2)Range32.8\u201357.831.2\u201365.74\u201323Gender, N (%)\u2014female129 (78)37 (22)167 (55)Gender, N (%)\u2014male37 (22)129 (78)137 (45)Mean length of relationship in years (SD), range 4\u20134121.3 (6.6)Mean education level (SD), range 1\u201373.8 (1.6)3.9 (1.8)Mean number of children per couple, range\u2009=\u20091\u201362.3Primary caregiver in family, N (%)\u00a0Mother105 (63)\u00a0Father5 (3)\u00a0Both parents54 (33)\u00a0Other2 (1)Mean time since diagnosis in years (SD)2.76 (1.2)Treatment intensity\u2014nonintense, N (%)23 (14)Treatment intensity\u2014intense, N (%)143 (86)Recurrence of cancer N (%)36 (22)\nThere were no significant differences between participating and nonparticipating parents regarding patient\u2019s gender, type of cancer, or time since diagnosis. Of the nonparticipants, 22% (N\u2009=\u200959) declined because of reasons directly related to the parents (e.g., parents had moved on with their lives or were too emotionally distressed). Reasons related to the children (e.g., children were not interested, children had not been informed of parent\u2019s illness) were given as the explanation for not wanting to be included in 20% (N\u2009=\u200953) of nonparticipating families. Twenty-five percent (N\u2009=\u200967) mentioned a variety of reasons, including another illness in the family or the parents\u2019 or children\u2019s busy-ness. The remaining 33% (N\u2009=\u200988) did not provide an explanation.\nPatients\u2019 and spouses\u2019 QoL in comparison with the norm\nResults are summarized in Table\u00a02. Clinically relevant ESs were found for male patients\u2019 social functioning, role limitations because of physical problems, and vitality. Both male and female patients scored statistically significantly lower QoL than the norm on one subscale: vitality. Female patients additionally scored lower on two subscales: social functioning and role limitations because of physical problems.\nTable\u00a02Descriptives of the RAND-36 scores by parents\u2019 gender and health status and comparison between groups and with the norm group\u00a0GenderPatientPartnerHealth status and genderNormMeanSDt (v. norm)Effect sizeMeanSDt (v. norm)Effect sizeANOVA FMeanSDPhysical functioningfemale76.122.2\u22122.120.2091.914.94.290.7512.9980.723.6male77.322.0\u22121.960.3389.116.92.440.2784.522.3Social functioningfemale78.524.4\u22123.160.3174.715.9\u22124.170.714.2886.120.9male72.628.4\u22123.310.5685.621.71.290.1388.419.6Role limitations\u2014physicalfemale64.941.2\u22123.440.3384.229.11.190.207.2078.336.5male57.445.6\u22123.130.5381.635.00.030.0081.533.6Role limitations\u2014emotionalfemale79.335.60.980.0981.933.9\u22120.090.021.4582.533.5male83.832.9\u22120.620.1187.828.90.160.0287.329.3Mental healthfemale72.516.8\u22121.820.1874.614.8\u22120.360.062.9675.518.9male78.514.5\u22120.370.0677.916.0\u22120.890.0979.417.3Vitalityfemale58.821.8\u22123.630.3463.817.6\u22120.840.146.5466.319.6male56.921.4\u22123.070.5969.118.7\u22120.230.0269.520.5Painfemale81.621.5\u22120.780.0785.318.3\u22121.690.292.3480.025.4male80.424.60.660.1187.719.22.140.2383.223.8General healthfemale66.321.9\u22122.250.2474.815.3\u22121.270.228.7171.521.8male58.525.9\u22122.920.4975.318.7\u22121.920.2171.423.3Physical summaryfemale47.29.8\u2013\u201349.710.2\u2013\u201311.38Not availablemale44.911.4\u2013\u201350.39.8\u2013Not availablePsychosocial summaryfemale49.710.2\u2013\u201353.68.6\u2013\u20132.31Not availablemale44.911.4\u2013\u201352.28.5\u2013\u2013Not availablep values were corrected for multiple comparisons.Significant values are shown in italics: p\u2009\u2264\u20090.0015 for comparisons with the norm; p\u2009\u2264\u20090.005 for comparisons of health status and gender.\nClinically relevant ESs were found for female spouses\u2019 physical functioning and social functioning. Female spouses reported statistically significantly higher scores than the norm on physical functioning. They also scored statistically significantly lower on social functioning than the norm.\nEffects of health status and gender, time since diagnosis, recurrence, treatment intensity, and type of cancer on QoL\nParents\u2019 health status and gender had a statistically significant effect on parents\u2019 physical summary score. A statistically significant effect was also found on parents\u2019 physical functioning, role limitations because of physical problems, vitality, and general health perception (Table\u00a02).\nA Bonferroni post-hoc test revealed the following. On the physical summary score, female patients scored lower than male spouses (p\u2009<\u20090.001), and male patients scored lower than female and male spouses (p\u2009<\u20090.001 for both). On physical functioning, female patients scored lower than female and male spouses (p\u2009<\u20090.001 for both). On role limitations because of physical problems, female and male patients scored significantly lower than male spouses (p\u2009=\u20090.003 and p\u2009=\u20090.005, respectively). On vitality, female patients scored significantly lower than male spouses (p\u2009<\u20090.001). Finally, on general health perception, female patients scored significantly lower than male spouses (p\u2009=\u20090.003), and male patients scored significantly lower than female and male spouses (p\u2009=\u20090.004, p\u2009<\u20090.001, respectively).\nTime since diagnosis did not correlate significantly with patients\u2019 QoL. Time since diagnosis correlated weakly with spouses\u2019 physical summary score (r\u2009=\u20090.19, p\u2009=\u20090.031). Patients with a recurrence scored clinically relevantly lower than patients without recurrence on the physical summary score (t\u2009=\u20093.44, ES\u2009=\u20090.82, p\u2009=\u20090.001), social functioning (t\u2009=\u20093.12, ES\u2009=\u20090.79, p\u2009=\u20090.003), role limitations because of physical problems (t\u2009=\u20093.72, ES\u2009=\u20090.72, p\u2009<\u20090.001), and general health perception (t\u2009=\u20093.69, ES\u2009=\u20090.82, p\u2009=\u20090.001). Spouses of patients with a recurrence scored clinically relevantly lower than spouses of patients without a recurrence on the psychosocial summary score (t\u2009=\u20092.23, ES\u2009=\u20090.94, p\u2009=\u20090.027) and the subscales: role limitations because of emotional problems (t\u2009=\u20092.72, ES\u2009=\u20090.78, p\u2009=\u20090.009), vitality (t\u2009=\u20093.56, ES\u2009=\u20090.69, p\u2009<\u20090.001), and general health perception (t\u2009=\u20092.19, ES\u2009=\u20090.52, p\u2009=\u20090.033).\nTreatment intensity was significantly related to patients\u2019 QoL. Patients who received intense treatment scored clinically relevantly lower than patients who received nonintense treatment on the physical summary score (t\u2009=\u20092.81, ES\u2009=\u20090.79, p\u2009=\u20090.004) and the following six subscales: physical functioning (t\u2009=\u20092.32, ES\u2009=\u20090.69, p\u2009=\u20090.02), social functioning (t\u2009=\u20092.33, ES\u2009=\u20090.76, p\u2009=\u20090.02), role limitations because of physical problems (t\u2009=\u20092.95, ES\u2009=\u20090.68, p\u2009=\u20090.006), vitality (t\u2009=\u20093.03, ES\u2009=\u20090.81, p\u2009=\u20090.003), pain (t\u2009=\u20093.19, ES\u2009=\u20090.78, p\u2009=\u20090.016), and general health perception (t\u2009=\u20092.60, ES\u2009=\u20090.69, p\u2009=\u20090.01). Treatment intensity was not significantly related to spouses\u2019 QoL.\nType of cancer had a statistically significant effect on patients\u2019 QoL as summarized in Table\u00a03. Statistically significant differences between patient groups were found on the physical summary scale and five subscales. Patients with skin cancer reported the highest QoL. One third of skin cancer patients received intense treatment. Patient with hematological cancer reported the lowest QoL levels. All 16 hematological cancer patients received intense treatment. A Kruskall\u2013Wallis test confirmed these findings. With regard to the spouses, QoL did not vary as a function of the patient\u2019s type of cancer.\nTable\u00a03Descriptives of the patients\u2019 RAND 36 scores by type of cancer and comparisons between groups\u00a0Gynecological tumorsBreast cancerUrological tumorSoft tissue\/boneHematologicalDermatologicalANOVAN\u2009=\u200916N\u2009=\u200986N\u2009=\u200910N\u2009=\u200910N\u2009=\u200916N\u2009=\u200915FMSDMSDMSDMSDMSDMSDPhysical functioning78.117.178.520.481.014.759.515.757.832.188.315.85.21Social functioning77.317.279.223.372.522.770.025.854.735.996.79.95.34Role limitations\u2014physical64.043.868.339.962.511.532.533.431.243.386.728.14.61Role limitations\u2014emotional91.725.879.534.796.710.576.741.760.442.5100.000.03.16Mental health73.015.073.616.577.616.469.525.974.517.776.013.80.29Vitality60.617.658.920.758.524.352.016.943.428.777.318.84.18Pain86.917.681.721.975.919.160.620.973.629.292.813.93.44Gen. health perception72.816.064.923.161.021.654.517.947.228.285.79.85.79Physical summary78.117.1178.520.481.014.759.515.757.832.188.315.86.48Psychosocial summary77.317.279.223.372.522.770.025.854.735.996.79.92.95p-values were corrected for multiple comparisons. Significant values are shown in italics: p\u2009\u2264\u20090.005\nFunctionality of patient in relation to functionality of spouse\nSpouses\u2019 psychosocial functioning was moderately strongly related to patients\u2019 psychosocial functioning (r\u2009=\u20090.44, p\u2009<\u20090.001) and weakly related to patients\u2019 physical functioning (r\u2009=\u20090.29, p\u2009<\u20090.001). No significant relationship was found between the spouse\u2019s physical functioning and the patient\u2019s psychosocial (r\u2009=\u20090.06) or physical functioning (r\u2009=\u20090.07).\nRelationship between patient and spouse\u2019s functioning and emotional and behavioral functioning of the children\nPatients\u2019 physical functioning was not significantly related to their elementary school-aged children\u2019s functioning but was weakly related to the adolescents\u2019 total problems and internalizing as reported by the patients (Table\u00a04). Patients\u2019 psychosocial functioning was moderately strongly to weakly related to their elementary school-aged children\u2019s internalizing, externalizing, and total problems and weakly related to the adolescents\u2019 internalizing, externalizing, and total problems as reported by the patient. The patients\u2019 physical and psychosocial functioning was moderately strongly to weakly related to the adolescents\u2019 internalizing and total problems according to adolescents\u2019 self-reports; their physical functioning was weakly related to adolescents\u2019 externalizing.\nTable\u00a04Correlations between parents QoL, as measured by RAND-36 composite scores, and children\u2019s functioning by age group as reported by parents (CBCL) and adolescents themselves (YSR)\u00a0Patient (N\u2009=\u2009162)Spouse (N\u2009=\u2009150)Physical functioningPsychosocial functioningPhysical functioningPsychosocial functioningElementary school-aged children (CBCL)Total\u22120.05\u22120.33**\u22120.19\u22120.11Internalization\u22120.03\u22120.36**\u22120.19\u22120.17Externalization\u22120.03\u22120.26**\u22120.16\u22120.05Adolescents (CBCL)Total\u22120.18**\u22120.24**\u22120.11\u22120.22**Internalization\u22120.19**\u22120.23**\u22120.15\u22120.27**Externalization\u22120.10\u22120.21**\u22120.09\u22120.09Adolescents (YSR)Total\u22120.32**\u22120.29**\u22120.11\u22120.15Internalization\u22120.31**\u22120.32**\u22120.11\u22120.19*Externalization\u22120.20*\u22120.13\u22120.09\u22120.02*p\u2009<\u20090.05**p\u2009<\u20090.01\nSpouses\u2019 psychosocial and physical functioning was not found to be significantly related to their elementary school-aged children\u2019s functioning as reported by the spouses. Additionally, their physical functioning was not significantly related to the adolescents\u2019 functioning. Spouses\u2019 psychosocial functioning was weakly related to the adolescents\u2019 internalization and total problems as reported by the spouses. Finally, the spouses\u2019 psychosocial functioning was weakly related to the adolescents\u2019 internalizing as reported by the adolescents (Table\u00a04).\nDiscussion\nThe aim of this study was to gain insight into the QoL of cancer patients in the child-rearing stage and their spouses. We found that cancer patients 1\u20135\u00a0years after diagnosis evaluate their QoL as clinically relevantly and\/or statistically significantly lower than the normal population on three of the eight domains, partially supporting our first hypothesis. This is in line with studies that have reported a decrease in patients\u2019 social and physical domains [7, 9]. However, our study did not find a decrease in patient\u2019s emotional functioning, in contrast to some reports [3, 4, 16]. Our study focused on a subgroup of cancer patients, namely those who are relatively young. The finding that our patient group scored similarly to the norm seems to indicate that these patients\u2019 lives have gotten fairly back to normal. These parents seem to be handling the unexpectedness of a cancer diagnosis during this life phase, the treatment, or the confrontation with a possible death at an early age, coupled with the responsibility of raising children fairly well. However, they still seem to experience problems in some areas, specifically social functioning, role limitations because of physical problems, and vitality.\nSpouses of cancer patients reported a QoL comparable to the norm group, and female spouses reported even better physical functioning than the norm. Only on one subscale (social functioning) did spouses (and then only women) report decreased functioning. Our findings largely negate our hypothesis and are in contrast with other studies where spouses have reported a decreased QoL [27]. An explanation for our findings may be that spouses in other studies, where the average age is higher, may have had a lower QoL simply because of their older age. As age increases, QoL scores decrease [33]. A second explanation could be that spouses viewed their QoL relative to their ill partner and therefore regarded their own health as good as or better than people generally may. It is also possible that we did not find a decreased QoL in the spouses because our patient group ranged from 1 to 5\u00a0years after diagnosis; time since diagnosis correlated significantly positively with spouses\u2019 physical functioning. The threat that the patient might not survive may be less prominent for spouses at this point in time.\nOur study found significant effects of gender and health status on the physical summary score and four of the eight subscales. To summarize our findings, patients scored lower than spouses, male spouses reported the highest QoL, and, on some scales, the female patient reported the lowest QoL and, on others, the male patient. In a study on gastrointestinal cancer, female patients and female spouses were both reported to suffer overall more distress than male patients [30], which our study did not find. They also reported that female spouses reported lower QoL than male patients and spouses. However, that study examined patients within 6\u00a0months after surgery. They also focused specifically on patients with gastrointestinal cancer, which is equally prevalent in men and women. That allowed them to more easily generalize that the differences they found were due to gender. As our study sample was diagnostically heterogeneous, it is difficult to differentiate whether the observed differences were due to gender or cancer site and consequent treatment received. Our study sample consisted of 78% women, which could seem skewed. However, according to the Dutch Comprehensive Cancer Center\u2019s database, cancer occurs more frequently in women during this age range; approximately 65% of cancer patients in this region of The Netherlands with invasive tumors are women [12].\nTo further explore our second aim, we analyzed QoL as a function of type of cancer and found differences depending on the type of tumor the patient had, supporting our hypothesis. Skin cancer patients in our study reported the highest QoL, while patients with hematological cancer reported the lowest QoL. These differences may be related to the kind of treatment the patient received. We found that patients who received nonintense treatment reported a better QoL than those who received intense treatment. The removal of a localized melanoma that only requires outpatient excision may affect QoL less than frequent and long hospital stays for courses of chemotherapy for hematological malignancies. Our findings are similar to another study reporting patient distress levels [40] that found that distress levels varied depending on cancer site. With regard to how spouses function, no significant QoL differences were found because of the type of cancer or treatment intensity. It would seem that tumor type or treatment intensity do not affect spouses\u2019 functioning. Whether the patient had a recurrence does seem to affect spouses\u2019 QoL; the ES of the psychosocial summary score was strikingly large. This may be due to, for those spouses, the still current threat that the patient may not survive. It is interesting to note that recurrence seems to have affected patients\u2019 physical and social functioning but not their mental functioning. Patients with recurrence do not report more mental distress than patients who have not had a recurrence.\nThirdly, we investigated relationships between patient and spouse QoL and found weak to moderate positive relationships between cancer patient\u2019s physical and psychosocial functioning and spouse\u2019s psychosocial functioning. These findings are in line with two studies reporting a moderate positive relationship between patients\u2019 and spouses\u2019 psychological distress [9, 18].\nFinally, the hypothesis that we would find a significant relationship between the parents\u2019 and the children\u2019s\u2019 functioning was supported, although the relationships were not strong. The patient\u2019s QoL related more often significantly with the children\u2019s functioning than the spouse\u2019s. Given that four of five of the patients were women, it could be that mothers were more often alert to possible problems in the children than fathers. This may be due to the large number of families in our sample where the mother, sick or healthy, is the primary caregiver. Mothers tend to orient themselves more toward others, whereas fathers tend be more self-oriented [18]; this could enable mothers to judge problems better. It is possible that illness plays a role in the patients\u2019 reports as distressed parents are likely to rate more behavioral and emotional problems in their children [22]. However, we found significant relationships between the adolescents\u2019 self-reports and parents\u2019 functioning. Unfortunately, the cross-sectional design of this study limits us in understanding the causal nature of this finding.\nWe found more significant, negative correlations with the children\u2019s internalizing and total problem scores than externalizing. It would seem that parents\u2019 QoL negatively relates to the children\u2019s emotional functioning, more than to their acting up. It is interesting to note that the parents\u2019 functioning related differently to adolescents and school-aged children. The patient\u2019s physical functioning correlated weakly with the adolescents\u2019 functioning but not significantly with the younger children\u2019s functioning. This was not found for spouses\u2014their physical functioning did not relate significantly to how children from either age group function. Patient\u2019s psychosocial functioning correlated weakly to moderately strongly with children in both age groups, while spouses\u2019 psychosocial functioning correlated weakly with the adolescents\u2019 functioning. Adolescent self-reports provided a similar picture; we found weak to moderately strong correlations between patients\u2019 physical and psychosocial functioning but only one weak correlation between spouses\u2019 psychosocial functioning and adolescents\u2019 internalization. Our finding that parents\u2019 functioning related more often significantly with the adolescents may be due to adolescents\u2019 stage of cognitive development. Adolescents are more able to understand the patient\u2019s illness and may pick up on physical problems more than elementary school-aged children [15].\nResearch considerations\nIn our investigation, being the first to evaluate family functioning in families in the child-rearing age, we purposely did not choose a homogenous cancer group (i.e., only breast cancer). Our results are statements for the general group of child-rearing families with cancer. Comparisons between patients in our research group with varying diagnoses showed differences between groups; however, the small subgroups make it difficult to generalize our findings. Future research will be required to investigate the effect of varying forms of cancer, the stage of cancer, its prognosis, and treatment on the functioning of individual family members.\nAdditionally, our response rate of 44% could mean that despite the fact that no differences were found between participants and nonparticipants in gender, type of cancer, or time since diagnosis, a sample bias may exist. We cannot be sure whether psychological problems were over- or under-reported; some nonrespondents stated still being overwhelmed by the illness as a reason for not participating, while others indicated they had moved on. Additionally, the cross-sectional nature of this study makes it impossible to accurately capture the dynamic processes present in family relationships or determine whether parents\u2019 QoL effects the children\u2019s functioning or vice versa. Furthermore, a longitudinal study could provide insight into the QoL of cancer patients and their family members over a period of time. This study found significant relationships between patient and spouse functioning and between parent\u2019s functioning and children\u2019s behavior. However, these relationships appeared to have modest predictive power. The patients\u2019 and spouses\u2019 functioning is likely more influenced by other factors not measured in this study, such as personality, social support, or family environment [19, 26].\nIn conclusion, cancer patients\u2019 QoL 1\u20135\u00a0years after diagnosis seems to be returning to normal, except in three domains. Their spouses seem to be doing well. The patients\u2019 QoL varied according to the type of cancer, how intense their treatment had been, and whether they had experienced a recurrence. Spouses\u2019 QoL seemed to be unaffected by the type of cancer and treatment intensity but did vary depending on whether the patient experienced a recurrence. A moderate positive relationship was found between the patient\u2019s functioning and his\/her spouse\u2019s. Parents\u2019 physical and psychosocial functioning was weakly to moderately strongly related to their children\u2019s functioning. The patients\u2019 functioning related more strongly to the children\u2019s functioning than the spouses\u2019 did. How cancer patients\u2019 families function may have an impact on the patient\u2019s functioning, up to 5\u00a0years after diagnosis. This is something that should be taken into account by clinicians.","keyphrases":["quality of life","children","patient","spouse","parental cancer"],"prmu":["P","P","P","P","P"]}
{"id":"Mol_Syst_Biol-4-_-2387231","title":"A map of human protein interactions derived from co-expression of human mRNAs and their orthologs\n","text":"The human protein interaction network will offer global insights into the molecular organization of cells and provide a framework for modeling human disease, but the network's large scale demands new approaches. We report a set of 7000 physical associations among human proteins inferred from indirect evidence: the comparison of human mRNA co-expression patterns with those of orthologous genes in five other eukaryotes, which we demonstrate identifies proteins in the same physical complexes. To evaluate the accuracy of the predicted physical associations, we apply quantitative mass spectrometry shotgun proteomics to measure elution profiles of 3013 human proteins during native biochemical fractionation, demonstrating systematically that putative interaction partners tend to co-sediment. We further validate uncharacterized proteins implicated by the associations in ribosome biogenesis, including WBSCR20C, associated with Williams\u2013Beuren syndrome. This meta-analysis therefore exploits non-protein-based data, but successfully predicts associations, including 5589 novel human physical protein associations, with measured accuracies of 54\u00b110%, comparable to direct large-scale interaction assays. The new associations' derivation from conserved in vivo phenomena argues strongly for their biological relevance.\nIntroduction\nAlthough considerable progress has been made in mapping the protein interaction network of yeast (Ito et al, 2000, 2001; Uetz et al, 2000; Ho et al, 2002; Gavin et al, 2006; Krogan et al, 2006), only minimal progress has been made on the interaction networks of higher eukaryotes, due primarily to their scale: for the \u223c20 000\u201325 000 human proteins, we expect a network of roughly 1\u2013400 000 interactions (Hart et al, 2006). Among the few methods scaleable to this size, the yeast two-hybrid assay has proven the most successful, with maps of \u223c20 000 interactions in fly (Giot et al, 2003), \u223c4000 in worm (Li et al, 2004), and more recently, assays of \u223c2800 and \u223c3200 human protein interactions (Rual et al, 2005; Stelzl et al, 2005). Direct mapping of protein complexes by mass spectrometry has also contributed another \u223c5000 interactions (Ewing et al, 2007). After including previously known human protein interactions (Bader et al, 2003; Lehner and Fraser, 2004; Peri et al, 2004; Joshi-Tope et al, 2005; Ramani et al, 2005), the human protein interaction map is currently perhaps 10\u201330% complete (Hart et al, 2006). It is therefore important to identify and employ methods for discovering interacting proteins without exhaustive experimental measurement of all pairs of proteins under each relevant condition or assay.\nProteins are evolved to interact under specific conditions in the cell, with the cell correspondingly optimized to facilitate these events, e.g. by expressing mRNAs before proteins are required, coordinating the expression of interacting partners, directing proteins to appropriate locations for their interactions, and so on. In this way, in vivo protein interactions are accompanied by corollary events that can be used to identify biologically relevant physical interaction partners.\nWe took advantage of two such corollary data types, the tendency for interacting proteins to have correlated mRNA expression patterns and the evolutionary conservation of such patterns, to identify new human protein interactions. It is well established that genes whose mRNA expression patterns are correlated across many diverse conditions can often be inferred to \u2018work together', i.e. to be functionally coupled (Eisen et al, 1998; Marcotte et al, 1999; Stuart et al, 2003; Lee et al, 2004a; Segal et al, 2004). Analyses of co-expression patterns of orthologous genes have shown that the conserved correlation in expression can also be used to transfer functional information across species (Teichmann and Babu, 2002; Stuart et al, 2003; van Noort et al, 2003; Bergmann et al, 2004; Snel et al, 2004). Transcriptional co-expression patterns have proved useful for inferring physical protein interactions (e.g. Deane et al, 2002; Jansen et al, 2003), with strongly co-expressed mRNAs more likely to indicate long-lived interactions (Ge et al, 2001; Jansen et al, 2002; Simonis et al, 2006). In general, we do not expect transcriptional data to distinguish between direct protein binding and membership in the same protein complex, and we term all such cases physically associated proteins.\nTo exploit these trends, we applied a supervised algorithm to discover physical associations among human proteins based upon the co-expression of their mRNAs and that of their orthologs in five organisms (the mustard plant Arabidopsis thaliana, the mouse Mus musculus, the fly Drosophila melanogaster, the nematode Caenorhabditis elegans, and the yeast Saccharomyces cerevisiae). By this approach, we mapped 7000 predicted human protein physical associations, of which 5589 are new to this analysis.\nResults\nPredicting physically associated proteins from patterns of conserved co-expression\nFigure 1 illustrates the overall method. We first identified orthologs for human genes in five other organisms using the InParanoid algorithm (Remm et al, 2001). We then compared the correlation in mRNA expression of each pair of human genes with the correlations in expression of each of their corresponding ortholog pairs from five organisms, in all calculating mRNA expression correlations for 5 708 925 human gene pairs on the basis of 3977 DNA microarrays. After removing 105 140 gene pairs likely to cross-hybridize on the microarrays (see Materials and methods) and filtering pairs with nonsignificant correlations, we employed a supervised algorithm on these data to identify those patterns of conserved co-expression (CCE) diagnostic of physical protein associations, based upon the correlations observed for known protein interactions versus random protein pairs. By searching for additional gene pairs exhibiting these patterns, we identified new associations.\nFigure 2 plots the derivation of the relationship between CCE and the tendency to be in the same physical complex, relying in this case on the comparison of human and C. elegans mRNA expression data. Briefly, the distribution of mRNA co-expression relationships was measured for 1769 gene pairs whose corresponding proteins are known to physically associate (Ramani et al, 2005), serving as positive training examples (Figure 2A); these 1769 pairs represent the subset of known human protein associations in the training set that also occur in the human\u2013worm co-expression data sets. Likewise, the distribution was measured for 642 295 gene pairs that are in the physical interaction training set but are not known to physically associate, serving as a negative training set (Figure 2B). Therefore, the log ratio of these two plots, corrected by prior expectation, represents the log likelihood for protein pairs to physically associate given any particular pattern of co-expression conservation (Figure 2C):\nwhere P(I\u2223D) and P(\u223cI\u2223D) are the frequencies of positive (I) and negative (\u223cI) training associations observed in the data set (D), respectively, while P(I) and P(\u223cI) represent the overall frequencies of positive and negative training associations, respectively. This score indicates how likely two proteins are to physically associate given their specific mRNA co-expression conservation in these data. The training set includes both direct interactions and protein pairs belonging to the same complex; we therefore consider this approach to support the more general case, i.e. proteins belonging to the same complex whether or not directly interacting. Note that the highest scores do not necessarily occur in the extreme top right corner of Figure 2C; lower counts of both positive and negative examples in the extreme corner, as well as filtration of highly correlated gene pairs where they may suffer from DNA microarray cross-hybridization (see Materials and methods), results in the highest scores occurring at correlation coefficients less than one.\nWe similarly analyzed co-expression patterns of human gene pairs with orthologs from four other organisms (A. thaliana, M. musculus, D. melanogaster, and S. cerevisiae), analyzing 3977 DNA microarray experiments in all. From each analysis (Figure 2C and Supplementary Figure 1) strongly co-expressing human genes with co-expressing orthologs are generally likely to encode physically associated proteins. The highest likelihoods of associating occur when the mRNA expression patterns of both human gene pairs and their orthologs are positively correlated, with odds of associating approaching 460:1 for C. elegans, 200:1 for A. thaliana, 100:1 for M. musculus, 25:1 for D. melanogaster, and 400:1 for S. cerevisiae. These learned relationships between mRNA expression profiles and physical associations were then applied to protein pairs not in the training set, thereby assigning a likelihood of physically associating to each untested protein pair. Each human gene pair discovered has at least one log likelihood score, to a maximum of five, from which the highest score was identified; pairs were ranked based on this score, then evaluated as a function of their rank.\nValidation of predicted physical protein associations using known interactions\nAs this assay relies upon indirect evidence, it is critical that putative physical associations discovered by this approach be carefully evaluated. We devised six tests for the enrichment of true physical associations, including direct experimental assay of physical association and of four proteins' functions suggested by the associations. First and most critically, to verify the accuracy of the co-expression-derived associations, we measured their likelihood to physically associate using an independent test set of 15 810 known physical associations, including both direct interactions and complexes (Ramani et al, 2005). Figure 3A shows that the CCE associations are highly enriched for true physical associations, varying from a likelihood ratio of \u223c60:1 to as high as \u223c400:1 of correctly capturing true physical associations. Importantly, the CCE pairs score \u223c25\u2013200 times higher than randomized pairs of the same proteins, as well as associations derived in the same manner but using only human (not ortholog) DNA microarray data (Figure 3A). Therefore, the data from orthologs enriched the signal for human physical protein associations considerably beyond the human data alone.\nSecond, we examined the functional relationships between the putative interaction partners. For this test, we compared the Gene Ontology (GO) and KEGG pathway database annotations of interacting partners, using a log likelihood framework (Lee et al, 2004b; Ramani et al, 2005) and testing the performance of the mapped associations with that of literature physical interactions (Figure 3B) (Bader et al, 2003; Peri et al, 2004; Joshi-Tope et al, 2005; Ramani et al, 2005). Literature associations score in the range of log likelihood ratio (LLR)=2.6\u20133.6, indicating high consistency with GO\/KEGG annotation. As expected, randomized interactions score near zero, and interactions derived from human-only co-expression data score lower (LLR=0.59\u20131.09). The CCE associations are comparable to the literature associations. Using interactions transferred from other organisms (orthology core set (Lehner and Fraser, 2004); LLR=2.2) to define a threshold of minimum acceptable quality and choosing associations (in bins of 1000) exceeding this threshold, we obtain 7000 associations from the present analysis, and all subsequent tests were performed on this set. These associations have a minimum likelihood of 9:1 (90%) of belonging to the same GO\/KEGG pathways. For consistency, subsequent tests include comparisons to the top-scoring 7000 associations derived from human-only mRNA co-expression, as well as to networks generated from the same proteins found in the CCE associations, but connected by 7000 random interactions (N=10 random networks).\nValidation of predicted physical protein associations by mass spectrometry\nWe next used quantitative mass spectrometry to test for physical associations between CCE partners (Figure 4A). Performing purifications under native conditions known to keep protein complexes intact (Dignam et al, 1983), HeLa cells were lysed, the cytoplasmic and nuclear\/mitochondrial fractions were separated, and their respective contents were fractionated biochemically on two sucrose density gradients. Proteins were quantified in each fraction by mass spectrometry. In all, 3013 proteins were quantified across 14 cytoplasmic and 14 nuclear\/mitochondrial sucrose density gradient fractions (Figure 4B). As proteins in the same complex should generally co-sediment, we expected physically associated proteins to often have correlated elution profiles.\nAnalysis of known protein complexes verified that components of a complex tended to co-elute (Figure 4C; additional controls in Supplementary Figures 3\u20135). For example, components of the TCP1 chaperone complex show strongly correlated elution profiles, as do core components of RNA polymerase II; the latter profiles are distinct from the former. Likewise, components of the NADH dehydrogenase 1b complex show strongly correlated elution profiles, eluting entirely in the nuclear\/mitochondrial fraction (Figure 4C). As an example of the utility of this approach, the protein GRIM-19, initially identified as a regulator of cell death induced by interferon-beta and retinoic acid, was later identified to be a subunit of the NADH dehydrogenase complex 1 (Fearnley et al, 2001); this association is clearly evident in the co-elution of GRIM-19 with other components of this complex.\nMore systematically, positive control human protein interaction partners known from literature (Joshi-Tope et al, 2005) show highly correlated elution profiles (Figure 5), unlike negative control random pairs (see histograms in Figure 6A). For cases in which both interaction partners were observed in the mass spectrometry experiment, 63% of the positive control pairs exhibited Pearson correlation coefficients >0.4, indicating a false-negative rate for identifying physical associations using the mass spectrometry-based elution profiles of 37% at this correlation threshold (28% if considering correlation coefficients >0.2). This agrees with the expectation that not all interacting proteins will co-sediment, with a probable bias toward stable complexes. Similarly, for proteins in the positive control set, protein pairs with the most correlated elution profiles showed \u223c40% probability of being in the same physical complex (Figure 5). Thus, correlated elution across these fractions is a strong indicator of direct physical association. As this assay is not used independently for discovery, but is confirmatory in nature, the false-negative and true-positive rates are sufficient for evaluating CCE associations.\nAlthough individual associations could be validated in this manner, by instead examining the aggregate distribution of elution profile correlation coefficients, we could directly estimate the error rate of the CCE associations. We calculated histograms of Pearson correlation coefficients from pairwise comparisons of elution profiles for CCE protein pairs, for protein pairs known from literature (Joshi-Tope et al, 2005) to be in the same complexes, and from random pairs of proteins (Figure 6A). We then fit the CCE histogram as a linear mixture of the positive and negative control histograms; the proportions that give the best fit thereby provided an estimate of the relative proportions of true and false associations in the CCE set. From this analysis, we estimate that 49\u201359% of the CCE associations correspond to true physical associations (Figure 6A, inset).\nComparing the CCE associations and the shotgun proteomics elution profiles reveals many interesting associations. For example, known complexes are correctly recovered, as for the DNA replication licensing factors MCM3, 5, 6, and 7, or for components of the proteasome. Figure 6B shows the example of the proteins prohibitin and prohibitin-2, known to form a large complex on the mitochondrial membrane that acts to suppress apoptosis, but which also shuttles to the nucleus in an estrogen-receptor-dependent manner and acts to repress transcription (Kasashima et al, 2006). New associations are also revealed: we observed a predicted physical association between MCM3 and MCM6 with the retinoblastoma-binding protein 4 (RBBP4). RBBP4 is known to participate in several chromosome replication and chromatin remodeling complexes, among them the chromatin assembly factor CAF-1 and a DNA replication-dependent chromatin assembly complex (Verreault et al, 1996). The CCE associations, supported by mass spectrometry, suggest direct physical association of RBBP4 with the replication initiation complex as well.\nFigure 6B illustrates two other such examples: first, we predict the ras oncogene-related small GTPase RAB5A, an essential component of receptor-mediated endocytosis (Bucci et al, 1992), to associate with the clathrin assembly lymphoid\u2013myeloid leukemia gene (CALM), a protein that helps recruit clathrin to endocytic vesicles. The CALM gene is a recurring site for chromosomal translocations in acute myeloid and mixed lineage leukemias (Wechsler et al, 2003). Physical association with RAB5A highlights a possible functional connection between these two endocytic components and is interesting in light of the leukemogenesis potential of chromosomal translocations involving CALM.\nLikewise, we predict the A-kinase anchor protein AKAP1 to be associated with the splicing factor SFRS9. AKAP1 is primarily involved in anchoring protein kinases, phosphatases, and a phosphodiesterase to specific cellular locations, but also contains KH and Tudor domains, motifs for single-strand RNA binding (Trendelenburg et al, 1996), which help target AKAP1 to well-defined nuclear foci in an RNA-dependent manner (Rogne et al, 2006). The association with SFRS9, which among other functions is involved in both constitutive and alternative splicing and can be specifically localized with other RNA processing proteins to nuclear stress bodies (Denegri et al, 2001), suggests that AKAP1 may also have a role in these processes or in mRNA localization, perhaps integrating RNA processing with signaling.\nQuantitative estimates of interaction accuracy\nWe further validated predicted physical associations by additional approaches. For each of these tests, we defined a standard curve relating a quantitative property of a protein pair with the tendency of the pair to physically associate, constructing the curve from control mixtures of literature (positive) and randomized (negative) physically associating proteins (Figure 7A). A set of all true-positive physical associations typical of those used to construct the curve scores high on these tests (\u223c100% for each standard curve); the addition of random interactions degrades the performance. The relationship between each test's performance and inclusion of false positives is unambiguous and well behaved, as judged by the quality of the standard curves, agreement between the tests, and performance curves from mixtures of known accuracy (Supplementary Figure 6). It is important to note that there are possible sources of bias for each test; however, taken as a whole, the tests are strong indicators for the enrichment of true physical associations.\nFirst, true human physical protein associations should be enriched for physical interactions among orthologous proteins in model organisms. We generated control association sets with known error rates by randomly selecting sets of 7000 interactions with varying proportions of true-positive and true-negative associations, ranging from 0% true positive (all 7000 interactions chosen from the true-negative set) to 100% true positive (all 7000 interactions chosen from the literature set), repeating the randomization 10 times. We measured the overlap of each control set with a benchmark set of 19 119 human protein pairs whose worm, yeast, or fly orthologs have been observed to interact by yeast two-hybrid (Ito et al, 2000, 2001; Uetz et al, 2000; Giot et al, 2003; Li et al, 2004) or affinity purification\/mass spectrometry assays (Ho et al, 2002; Gavin et al, 2006; Krogan et al, 2006). Figure 7B shows the resulting standard curve that relates enrichment for orthologous interactions to percentage of true physical associations. On the basis of standard curve, we estimated that \u223c37\u201341% of the CCE pairs correspond to true physical associations, somewhat lower than the value by mass spectrometry co-sedimentation but considerably higher than both randomized pairs and pairs derived from only human co-expression data.\nAs physically associated proteins often share similar functional annotation (von Mering et al, 2002), we also created a standard curve based upon the agreement of interaction partners' functional annotation, relating agreement of SwissProt keywords (Wu et al, 2006) to the percentage of true physical associations (Figure 7C). For each control set, we measured the average keyword overlap across 882 SwissProt keywords between the interaction partners. Keyword overlap varied from \u223c5% for the true-negative set to \u223c42% for the true-positive set. From this curve and measurements of the keyword overlap by the 7000 interactions, we estimate \u223c59\u201368% of the CCE set represent physical associations. Measurements of the percentage overlap of GO \u2018biological process' and KEGG pathway annotations result in comparable values (\u223c50\u201353%).\nFinally, legitimately associated proteins should be closer in a gene network (i.e. separated by fewer interactions) than random pairs. For each putative physical association, we calculated the distance between the genes' yeast orthologs in a functional gene network (Lee et al, 2007). We compared the distribution of path lengths to distributions from positive and negative control sets. Figure 7D shows that the interactions from CCE have a path length distribution more similar to the positive control set than the negative set, indicating strong enrichment for true-positive associations among the 7000 interactions. We fit the distribution of path lengths from the 7000 CCE associations as a linear combination of the positive and negative control distributions (as in Deane et al, 2002). The proportions from the best fit (Figure 7D) provide an estimate of the percentage of true physical associations. This approach estimates the CCE set at \u223c63\u00b13% true physical associations.\nTable I summarizes the measurements of physical association, along with comparisons to the randomized and human-only co-expression control sets. Estimates vary only minimally with changes in parameters (e.g. using percentage keyword overlap for Figure 7B versus Jaccard coefficient) or choice of control sets (e.g. employing alternative literature positive control sets for Figures 3 or 7; see Supplementary Table 2). Although individual tests may show some bias, we expect these biases to average out across the five tests; in fact, the estimates are similar across the five tests. These measures demonstrate that CCE associations are, on average, reasonably accurate (54\u00b110% true physical associations) and biologically relevant, are comparable in accuracy to direct large-scale experimental assays (Rual et al, 2005; Stelzl et al, 2005), and are significantly more enriched for physical associations than random controls.\nTo summarize benchmark support for individual CCE associations, we calculated a \u2018Binary Interaction Overlap Score (BIOS)' (Stelzl et al, 2005) for each association (Supplementary Figure 10). By this measure, 4354 (62%) of the 7000 associations have at least one line of additional evidence supporting them. Scores are reported in the supporting data file.\nDetailed evaluation of ribosome biogenesis proteins\nTo experimentally evaluate the quality of hypotheses arising from the CCE associations, and given a statistical enrichment for proteins of ribosome biogenesis (see below), we analyzed proteins predominantly linked to proteins of ribosome biogenesis, a pathway involving several hundred proteins yet still incomplete (Granneman and Baserga, 2004). We chose four proteins with yeast orthologs for direct validation: (i) WBSCR20C, named for Williams\u2013Beuren syndrome chromosome region 20C, shares high sequence similarity with duplicate genes WBSCR20A and WBSCR20B. This gene is deleted in Williams\u2013Beuren syndrome, a multi-system developmental disorder caused by deletion of genes at the 7q11.23 locus (Doll and Grzeschik, 2001). WBSCR20C encodes a conserved Nol1\/Nop2\/Sun family protein domain and is also a member of the COG0144 protein family, other members of which are tRNA and rRNA cytosine-C5-methylases involved in translation, ribosomal structure, and biogenesis. YNL022C, the yeast ortholog of WBSCR20C, is also uncharacterized. (ii) BCCIP, or \u2018BRCA2 and CDKN1A interacting protein', is an evolutionarily conserved nuclear protein with multiple protein interaction domains. This protein may be an important cofactor for BRCA2 in tumor suppression (Lu et al, 2005) and a modulator of CDK2 kinase activity via p21 (Meng et al, 2004). The yeast ortholog of this protein (Bcp1p) is an essential nuclear protein involved in nuclear export of lipid kinase Mss4p (Audhya and Emr, 2003). (iii) EPRS is predicted by sequence to be a multi-functional aminoacyl-tRNA synthetase. Its yeast ortholog YHR020W is essential, with sequence similarity to proline-tRNA ligase, but otherwise uncharacterized. (iv) LYAR is a nucleolar zinc-finger-containing protein (Su et al, 1993) whose yeast ortholog YCR087C-A is nucleolar (Huh et al, 2003), but uncharacterized.\nWe tested the ribosomal processing phenotypes of yeast strains with tetracycline-controlled downregulatable alleles of the genes (Mnaimneh et al, 2004). Two of the strains (TetO7-BCP1 and TetO7-YHR020W) show clear ribosomal processing defects upon downregulation of the genes (Figure 8B and C). From polysome profiles, Bcp1p (corresponding to human protein BCCIP) appears to participate in 60S ribosomal subunit biogenesis; loss of the protein results in the reduction in the 60S peak relative to the 40S peak. YHR020W (corresponding to human protein EPRS) appears to participate in 40S biogenesis, resulting in a decreased 40S\/60S ratio when depleted. We also tested each of the four proteins for co-sedimentation with the 40S, 60S, or 80S monoribosomes, which would provide additional support for the proteins' participation in ribosome processing. From crude cell lysates of yeast strains expressing TAP-tagged versions of each protein (Ghaemmaghami et al, 2003), we size-fractionated ribosomal subunits, free ribosomes, and polysomes using sucrose gradients. Three of the proteins (YCR087C-A, YNL022C, and Bcp1p) showed clear association with 40S and 60S subunits (Figure 8A), with Bcp1p and YCR087C-A associated with both 40S and 60S, and YNL022C showing preferential co-sedimentation with the 60S subunits. Mass spectrometry of untagged YHR020W, analyzing yeast lysate with the approach of Figure 4 (data not shown), indicates that YHR020W also co-sediments with 60S ribosomal subunits (Z Li and EM Marcotte, unpublished data). The Bcp1p-TAP co-sedimentation is less definitive than the controls; however, the polysome profile of the TAP-tagged Bcp1p strain indicates that the TAP tag itself disrupts Bcp1p activity (Figure 8B), causing a 60S ribosomal biogenesis defect and definitively implicating the protein in this process. The human BCCIP protein was also found by mass spectrometry to co-sediment with free cytosolic 40S and 60S ribosomal subunits (Supplementary Figure 4), raising the possibility of a role in ribosome recycling or nuclear export. All four genes assayed could therefore be implicated in ribosomal biogenesis.\nDiscussion\nCharacteristics of the newly mapped associations\nWe have described the prediction of 7000 human protein physical associations from indirect transcriptional evidence, as well as measurement of overall error rates and validation of specific associations. We further examined the associations for novelty, functional bias, and evidence for stable protein complexes. First, we compared the predicted interaction set directly to the existing human protein interaction data sets. Roughly 20% of the CCE associations can be directly verified from previously known interactions, while \u223c80% are new. Our analysis bears some relation to one reported by Stuart et al, which analyzed CCE, although not for the purpose of discovering physical interactions. However, we obtain a largely non-overlapping set of associations, sharing only 12% of associations (Supplementary Table 1). Differences arise primarily because we are explicitly learning physical associations using a supervised training framework; other differences include the choice of expression data, the methods for defining orthologs, the criterion used to define co-expression (we set a statistical significance threshold on the correlation coefficient; Stuart et al use correlation coefficients >0.2), and our removal of potential cross-hybridization artifacts, all of which contribute to producing largely distinct sets of associations. Only three CCE interactions are shared with large-scale yeast two-hybrid analyses of human proteins (Rual et al, 2005; Stelzl et al, 2005), 15 with affinity purification\/mass spectrometry analysis (Ewing et al, 2007), 195 with a previous computational analysis (Rhodes et al, 2005), and 211 with interactions inferred from other organisms in the OPHID database (Brown and Jurisica, 2005). These comparisons are summarized in Supplementary Table 1. In all, 5589 of the 7000 associations predicted in this analysis were not identified in previous high-throughput human protein interaction screens.\nBesides simply being novel associations, 80% of the interaction partners (66% of annotated interaction partners) share neither KEGG nor GO annotations. While this extent of annotation sharing across the set of partners is sufficient to imply high confidence associations (Table I), these results indicate that the inferred associations extend beyond trivial identification of new associations among proteins already known to be in the same pathways.\nWe examined the functions of the 2348 proteins in the set of 7000 associations (Supplementary Figure 7), using for this purpose the proteins' KOG annotations (Tatusov et al, 2003). We find the dominant class of proteins to be those for whom only general function is known (224 proteins); followed by 180 proteins participating in post-translational modifications, protein turnover, and chaperones; 163 in signal transduction; 141 in translation, ribosomal structure, and biogenesis; 141 in transcription; 117 of RNA processing and modification; and 87 of unknown function. Therefore, the proteins are not dominated by a single structure (e.g. the ribosome), but are generally informative for major cellular systems and uncharacterized proteins. Nonetheless, some specific functional biases occur among the CCE proteins (Supplementary Figures 8 and 9), mostly notable a statistical enrichment for proteins of membrane-bound organelles (e.g. nucleus, mitochondria, nucleolus, etc.), presumably reflecting evolutionary conservation of these proteins and their regulation, favoring detection by the CCE method. Likewise, proteins of RNA metabolism are enriched, especially ribosome biogenesis, with accompanying enrichment for nucleotide-binding protein domains. The overall trends among the proteins can be seen in a plot of the CCE associations, clustered by the spectrum of associations per protein (Figure 9), which shows that although several major clusters exist, many associations are binary protein pairs that are not otherwise seen to exist in larger assemblies, and thus lie far from the diagonal of the clustergram.\nThis clustering, along with the 1411 associations overlapping other data sets, provides some insight into the nature of the CCE associations. Both direct interactions, especially among members of larger complexes, as well as co-complex physical associations are observed. For example, interactions are observed between alpha- and beta-tubulin, which assemble into a heterodimer; SNRPE and SNRPF, known to bind directly in the core complex of spliceosomal U1, U2, U4, and U5 snRNPs (Camasses et al, 1998); and the E2 and E3 subunits of pyruvate dehydrogenase, which interact directly. A comparison of the 7000 CCE associations with experimentally determined protein interactions among components of the 20S proteasome, as determined from the X-ray crystal structure (Groll et al, 1997), reveals four interactions between proteins that directly contact each other in the proteasome (PSMA2\u2013PSMA6, PSMB3\u2013PSMB2, PSMA4\u2013PSMB2, and PSMA1\u2013PSMB1), and nine interactions between proteins that assemble into the same physical complex, but do not directly contact each other (PSMA3\u2013PSMA4, PSMA6\u2013PSMB2, PSMA4\u2013PSMA6, PSMA6\u2013PSMB3, PSMA2\u2013PSMB1, PSMA2\u2013PSMA1, PSMA2\u2013PSMB2, PSMA6\u2013PSMA5, and PSMA5\u2013PSMB2). Therefore, as expected, both direct binding and co-complex interactions can be found among the 7000 associations.\nIn fact, the CCE pairs are strongly statistically enriched for co-complex associations typical of affinity purification\/mass spectrometry interaction assays. If we consider only the subset of 2138 CCE pairs (out of the 7000) in which both proteins have yeast orthologs, 118 of these can be verified by the MIPS database as belonging to the same yeast complex (using the \u2018hand-curated' set of protein complexes; Guldener et al, 2005). This value is 21 standard deviations (P<10\u221298) above the mean of random trials: randomizing the 7000 interactions and repeating the comparison gives a mean of 23\u00b14.5 confirmed interactions for N=100 random trials. Similar enrichment can be seen by comparison with yeast protein complexes defined by affinity purification\/mass spectrometry (Gavin et al, 2006; Krogan et al, 2006): 74 of the 2138 CCE pairs can be confirmed, 8 standard deviations (P<10\u221215) above the mean of random trials (29\u00b15.5, N=100). Likewise, 392 of the 2138 CCE pairs can be confirmed by comparison to the full-matrix form of these data (i.e. considering both bait\u2013prey and prey\u2013prey interactions), 10 standard deviations (P<10\u221225) above the mean of random trials (214\u00b117, N=100).\nFinally, we looked for organismal bias among the CCE pairs, examining which model organism contributed the top LLR score for each interaction (Supplementary Table 3). The most associations were contributed from the comparison of human and C. elegans expression, accounting for 2949 of the 7000 associations, and the least (158) from mouse. The low number contributed by comparison to mouse may suggest the importance of employing more distant orthologs, especially to non-mammalian animals, in identifying interactions by this approach, but more probably stems from characteristics of the data employed, such as the smaller number of mouse microarray experiments analyzed (Supplementary Table 4).\nOne interesting aspect of the CCE assay is that it intrinsically samples all pairs of genes that are measured on the DNA microarrays. This has the effect of increasing the numbers of proteins for which interactions are observed, and thereby decreasing the number of interactions per protein (7000 interactions for 2348 proteins \u223c3 interactions per protein, somewhat lower than the 5\u201315 interactions per protein observed in other data sets (Ramani et al, 2005)).\nLimitations, false positives, and potential improvements\nGiven the derivation of CCE pairs from transcriptional evidence, there are important features and limitations to note. First, strong co-expression tends to coincide with stable, rather than transient, physical association (Jansen et al, 2002), and we expect CCE pairs to reflect this trend, with a correspondingly higher false-negative rate for transient interactions. Second, based on our measured error rates, there are still appreciable false-positive associations, although the false-positive and -negative error rates are comparable to the only direct experimental approaches\u2014yeast two-hybrid assays and mass spectrometry of cloned, epitope-tagged human proteins\u2014that have been applied to map physical associations on this scale. However, CCE false positives have unusual properties. As the CCE pairs were the highest scoring (top 0.1%) of >5 million tested gene pairs, the association partners are strongly co-regulated in an evolutionarily conserved manner, and thus are highly likely to function together, even if not physically associated. Finally, algorithmic improvements, such as better orthology assignment and alternative supervised learning frameworks, and application to additional DNA microarray data, e.g. tissue- and cell-type-specific data to learn tissue- and cell-type-specific associations, are certain to reveal new associations when applied in the general framework we have described. Thus, we expect new CCE associations can be identified by modifications to this method.\nSimilarly, the mass spectrometry data used to test the CCE associations have some important features and limitations. Primarily, co-sedimentation alone is not proof of physical association\u2014it is possible for unrelated complexes to co-sediment\u2014as reflected in the measured true-positive and false-negative rates for associations inferred solely from these data. These sedimentation-derived associations should thus not be viewed as standalone. However, as a benchmark applied in the manner we present (e.g. analyzed in aggregate form), or when considered in combination with other data, such as incorporated into the BIOS scores of the CCE associations, we find the mass spectrometry data to be extremely valuable. We suggest that benchmarks of this sort could be of great utility for evaluating physical complexes determined by other methods, and could be generally adopted for measuring assay accuracy.\nConclusions\nThe scale of the human interactome appears to be beyond any individual technique; a combination of complementary approaches will be needed to map the complete human protein\u2013protein interaction network. Although current methods for mapping interactions focus largely on direct experimental observations, sufficient functional genomics data exist that physical protein associations can also be indirectly identified from these data. We demonstrate that these approaches can be comparable in scale and quality, both in terms of false-positive and false-negative rates, to the current largest scale experimental screens. Finally, as CCE-based physical protein association mapping is based on conserved in vivo phenomena, this approach is likely to specifically discover associations relevant to in vivo biology.\nMaterials and methods\nMapping of orthologs\nOrthologs were obtained from the InParanoid database (Remm et al, 2001) as SwissProt identifiers for human proteins and their orthologs from five other organisms (A. thaliana, C. elegans, D. melanogaster, M. musculus and S. cerevisiae). Using ID-Serve (http:\/\/bioinformatics.icmb.utexas.edu\/idserve) and organism-specific databases, the SwissProt identifier for each gene was mapped to alternate identifiers: LocusLink identifiers (human), common names (M. musculus), WormBase identifiers (C. elegans), Locus codes (A. thaliana), Flybase gene identifiers (D. melanogaster), and standardized gene names (S. cerevisiae). Supplementary Table 5 lists the numbers of orthologous genes analyzed.\nmRNA expression data\nAll mRNA expression data (Supplementary Table 4) were obtained from the Stanford Microarray Database (Ball et al, 2005). It has previously been shown that extraction of co-expression relationships is improved by restricting comparisons to similar conditions and experiments (Lee et al, 2004a, 2004b; Segal et al, 2004). We therefore divided the available 1922 human DNA microarray experiments into 11 categories of experiments, as assigned by the Stanford Microarray Database, and restricted comparisons to experiments in the same category. Expression data for other organisms were treated as single categories. Each of the microarray expression vectors was mean centered (row and column) and normalized before carrying out correlation analysis.\nCalculation of co-expression\nFor each pair of human genes, as well as for their corresponding orthologs, the Pearson correlation coefficient was computed between the mRNA expression vectors. For each gene pair, this gives 11 measurements of correlation corresponding to the 11 categories of human expression data sets and up to 5 for the correlation between the orthologs in the other organisms. Paralogs (as defined by InParanoid) were excluded from being compared to each other, as they tend to have similar expression profiles and thus high correlation, which we empirically observe to substantially increase the false-positive rate. The significance of each correlation was computed based on t-test statistics as\nwhere r is the minimum significant correlation for n values in the two expression vectors being compared and t is the t-test value at a probability of P\u2a7d0.01 from a t-test table. Only statistically significant correlation coefficients were retained, thereby accounting for variability in the sparseness of expression vectors. For example, using expression vectors of 100 experiments with only 50 data points available for both genes being compared, the absolute value of correlation must be >0.36 for the comparison to be statistically significant at P\u2a7d0.01.\nRemoval of cross-hybridization artifacts\nCross-hybridization occurs when an mRNA probe binds to a non-cognate spot on the microarray instead of its perfect complement spot. This creates both false positives (due to additional signal at incorrect positions on the array) and false negatives (due to reduced signal in correct positions). Although cross-hybridization is well established in spotted cDNA-based DNA microarray experiments (Kane et al, 2000; Murray et al, 2001; Xu et al, 2001), there are no universal standards for filtering such effects. In this analysis, we expected that cross-hybridizing gene pairs would appear to have similar expression patterns and therefore contribute false positives to our analysis.\nTo filter out these potentially spurious interactions arising from cross-hybridization, we established a threshold for excluding cross-hybridization based upon analysis of the hybridization of four yeast genes (YPL274W, YLR467W, YIR039C, and YKL224) to their homologs on a yeast DNA microarray. The four genes were chosen such that BLAST-based comparisons of the genes' DNA sequences to other genes in the yeast genome yielded hits with percent identities to the query sequence in the range of 50\u2013100% and BLAST E-values \u2a7d10\u22124. The four query genes were amplified using standard PCR techniques and primers to flanking DNA, labeled with Cy5, mixed with Cy3-labeled reference DNA (Carlson, 2002), and hybridized to a yeast cDNA microarray containing \u223c12 000 spots comprising all the yeast genes and intergenic regions (Carlson, 2002; Hahn et al, 2004; Kim and Iyer, 2004). Standard microarray analysis was carried out to quantify hybridization strength as the mean of ratios of Cy5\/Cy3 fluorescence intensities across spots. By plotting hybridization strength against the DNA sequence identity of the genes (Supplementary Figure 2), we identified an operational threshold of BLAST E-value \u2a7d10\u22124 and DNA sequence identity \u2a7e70% within the aligned regions. Gene pairs that exceed this threshold (with either the human or model organism gene pair DNA sequences) were likely to cross-hybridize and were excluded from further analysis. This filter removes 47 145 protein pairs from the plant\u2013human analysis, 37 519 from the worm\u2013human, 26 724 from the fly\u2013human, 39 286 from the mouse\u2013human, and 2193 from the yeast\u2013human analysis. This filtration preferentially removes many false-positive interactions, as the average expression correlation of the filtered pairs was significantly higher than for the remaining pairs (e.g. the average expression correlation in the human\u2013plant analysis was 0.28, while the average for the filtered pairs was 0.56), with the maximum expression correlation among the removed pairs equal to 1.0 for all comparisons.\nTraining to extract physical protein associations\nWe used the 31 609 human protein interactions from Ramani et al (2005) as the physical association benchmark. The associations were randomly separated into testing and training data sets (15 810 and 15 799 associations, respectively). For each of the five human gene pair\/ortholog gene pair sets, the maximum expression correlation of the human genes from the 11 data sets was plotted along the x axis and the correlation of the orthologous genes plotted along the y axis (as in Figure 2). The fraction of gene pairs that showed a particular expression pattern was measured in bins of 0.1 \u00d7 0.1 units. Two-dimensional histograms were calculated for interacting proteins and for non-interacting proteins in the training set. The logarithm of the ratio of the histograms at a given position in the plot, corrected by the background likelihood of physical associations in the training set, gives the log likelihood estimate of physical association conditioned on the degree of co-expression of the human genes and their orthologs in that organism. To minimize possible errors due to orthology assignments, we further considered only counts in the upper right-hand quadrant of each analysis, corresponding to gene pairs for which the human and other organismal experiments describe similar co-expression trends. Protein pairs outside of the training set were then assigned log likelihood scores according to their expression patterns in these data sets. Similar analyses were performed for associations derived from comparison of human expression data with each of the four other organism-specific data sets, associating the maximum score from these five analyses as each protein pairs' estimated likelihood of associating physically. (The maximum score outperformed the na\u00efve Bayes sum of scores, suggesting that the five scores are not independent.) The 7000 top-scoring associations are listed in Supplementary information.\nThe human-only co-expression control set was generated by considering only the human DNA microarray data, ignoring contributions from other organisms and lifting the requirement for each member of a gene pair to have orthologs in the same second organism. Putative associations were identified as for the CCE case, but instead using the log likelihood framework to relate the correlation coefficients across only the human DNA microarray experiments to the likelihood of physically associating. All other calculations were performed identically to the CCE case, including calculation of correlation coefficients, significance testing of correlations, calculation of likelihood values, selection of priors, and filtration for cross-hybridization.\nTesting for enrichment of known physical associations\nWe measured enrichment for known physical associations using the independent test set of 15 810 physical associations and the same LLR framework used to initially derive the CCE associations. The 15 810 associations formed the positive test set; the negative test set was defined as all pairs of proteins chosen from the 15 810 associations set, omitting the 15 810 associations themselves. The prior odds ratio of interacting (P(I)\/P(\u223cI)) equaled the ratio of positive to negative test set examples (0.00085). For each query association network being tested (or for a given bin of 1000 associations selected from a rank-ordered list), we measured the fraction of query set associations shared with the positive test set (P(I\u2223D)), as well as the fraction shared with the negative test set (P(\u223cI\u2223D)). The posterior odds ratio was calculated as P(I\u2223D)\/P(\u223cI\u2223D), and the LLR calculated as indicated in the main text, equal to the posterior odds ratio divided by the prior odds ratio. For the purposes of Figure 3A, the log likelihood was calculated in a cumulative manner (i.e. aggregating successive bins of 1000 associations for analysis.).\nTesting for functional similarity\nWe measured functional similarity of interacting protein pairs by using the gene annotation information obtained from GO (Ashburner et al, 2000) process level 8 annotation and KEGG pathway annotation (Kanehisa et al, 2004). These databases provide specific pathway and biological process annotations for 7390 human genes, assigning them into 155 KEGG pathways (at the lowest level of KEGG) and 1356 GO pathways (at level 8 of the GO biological process annotation). Interactions were first rank-ordered by confidence scores. For each successive bin of 1000 interactions, the functional similarity was calculated in a cumulative manner by counting the number of pairs in that bin or previous bins that shared a functional annotation, dividing this by the number of pairs that did not share functional annotation, and correcting by the prior probability of annotated pairs sharing annotation (0.0589).\nConstruction of standard curves for estimating percentages of physical associations\nStandard curves were constructed as described in the main text. Positive control sets for Figure 7B and C were selected from the hand-curated protein complex assignments of Reactome (Joshi-Tope et al, 2005). For the analysis of Figure 7B, we restricted the analysis to the portion of each data set for which both interacting proteins have orthologs among the yeast, worm, or fly proteins sampled by the benchmark assays (i.e. considering only the subspace of interactions spanned by the assay bait\u2013prey pairs). For the standard curves of both Figure 7B and C, the derived percentages of physical associations do not strongly depend upon the sizes of the data sets or control sets, only upon their tendencies to share orthologous interactions or functional annotations (data not shown). Ranges of accuracies were derived directly from the standard curve (i.e. as empirically measured from replicate analysis of control mixtures of true- and false-positive interactions).\nFor the linear mixture model of Figure 7D, positive control associations were taken from Joshi-Tope et al (2005), only considering genes with yeast orthologs, and negative control associations taken as pairs of human genes from the positive control set that have yeast orthologs but do not have recorded interactions. To minimize possible circularity, we removed all functional linkages from the yeast network that were derived only from mRNA co-expression data. The variances associated with accuracy estimates in Figure 7D were derived from 10 replicate analyses of mixtures of known proportions of true- and false-positive interactions (Supplementary Figure 6).\nBinary interaction overlap score\nTo further assign confidence to each association, we have adopted the BIOS of Stelzl et al (2005): based upon the benchmark sets (Table I), we assign each association +1 if the protein pair is observed in the physical interaction benchmark, +1 for sharing GO\/KEGG keywords, +1 for sharing SwissProt keywords, +1 for sharing KOG annotation, +1 for being observed in the orthologous interaction benchmark, +1 for having a correlation coefficient >0.4 in the mass spectrometry elution profile experiments, and +1 for having yeast orthologs that are either directly connected or one link separated in the yeast functional network benchmark (with expression-only and orthology-derived links omitted). Each association is thus scored from 0 to 7 based on additional support for that association; the BIOS scores generally correlate with the LLR scores (Supplementary Figure 10) and are reported in the supporting data file.\nHuman cell culture and mass spectrometry\nHeLa S3 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37\u00b0C with 5% CO2. At about 80% confluency, cells were treated with 100 \u03bcg\/ml emetine for 10 min and harvested by scraping. Cells were centrifuged at 500 g for 10 min, washed three times with cold PBS buffer, and resuspended in five packed cell volumes of cold lysis buffer (10 mM Tris pH 7.4, 20 mM KCl, 5 mM MgCl2). After swelling on ice for 10 min, cells were centrifuged at 500 g for 10 min and resuspended in one packed cell volume cold lysis buffer supplemented with 1 \u00d7 protease inhibitor cocktail (Roche) and 100 \u03bcg\/ml emetine. After lysing the cells with a dounce homogenizer, nuclei were collected by centrifuging at 1000 g for 10 min. The supernatant was centrifuged at 15 000 g for 10 min to obtain the cytosolic fraction. Nuclei were suspended in lysis buffer and lysed by sonication, collecting the clarified supernatant after centrifugation at 15 000 g for 10 min.\nThe cytosolic and the nuclear fractions were each loaded onto continuous 7\u201347% sucrose gradients in lysis buffer. After a 2.5-h spin at 40 000 r.p.m. in a Beckman SW40 rotor, the sucrose gradient was fractionated using an ISCO gradient fractionation system. Proteins from each fraction were precipitated with 10% cold trichloroacetic acid (TCA) and washed with 100% cold acetone. The protein pellets were suspended in 100 mM pH 8.0 Tris buffer and digested with sequencing grade trypsin (Sigma). For each fraction, tryptic peptides were loaded onto a reverse-phase C18 column and washed with 95% water, 5% acetonitrile, and 0.1% formic acid. Peptides were eluted with a 240-min gradient from 5 to 40% acetonitrile and analyzed online with a nanoelectrospray ionization (300 nl\/min flow rate) LTQ-Orbitrap hybrid mass spectrometer (Thermo Electron) using data-dependent precursor ion selection. Each parent ion mass spectrum (MS) was analyzed at high resolution (100 000) with the Orbitrap; the top seven MS peaks were fragmented by helium collision-induced dissociation at 35 eV, analyzing the resulting MS\/MS spectra with the LTQ. Approximately 35 000 MS\/MS spectra were collected per fraction. Spectra were searched against the set of NCBI human protein sequences using TurboSequest (Bioworks v.3.2, Thermo Electron). Proteins from each fraction were identified at a 5% false detection rate using Peptide\/ProteinProphet (Keller et al, 2002; Nesvizhskii et al, 2003). The spectral count (number of total observations of MS\/MS spectra from a given protein in a given fraction) was used as an estimate of protein abundance (Liu et al, 2004), dividing the spectral count of a protein ( \u00d7 10 000) by the sum of spectral counts for all proteins identified in that fraction. Protein elution profiles are provided as Supplementary information.\nYeast media and strains\nAll yeast strains were cultured in YPD (1% yeast extract, 2% peptone, and 2% dextrose) at 30\u00b0C. Tetracycline promoter-controlled essential gene haploid MATa strains (Mnaimneh et al, 2004) and TAP-tagged haploid MATa strains (Ghaemmaghami et al, 2003) were obtained from Open Biosystems.\nPolysome profile analysis\nAll yeast strains were cultured to OD600 0.3\u20130.5. For tetracycline promoter-controlled alleles, overnight cultures were diluted to OD600 0.01, 10 \u03bcg\/ml doxycycline (Fisher Scientific) was added into the media, and cells were grown to OD600 0.3\u20130.5. Cycloheximide (100 \u03bcg\/ml) (Sigma) was added to each culture. Cultures were immediately cooled with ice, and all subsequent steps were performed on ice or at 4\u00b0C. Each cell pellet was washed once with lysis buffer (20 mM Tris pH 7.4, 20 mM KCl, 5 mM MgCl2, 100 \u03bcg\/ml cycloheximide, 12 mM \u03b2-mercaptoethanol, 2 \u03bcg\/ml leupeptin, 2 \u03bcg\/ml aprotinin, 1 \u03bcg\/ml bestatin, and 1 \u03bcg\/ml pepstatin A) without protease inhibitors (MP Biomedicals Inc.). The cells were pelleted, resuspended in one volume lysis buffer, and lysed with glass beads. Crude lysates were centrifuged at 15 000 g for 10 min. Fifteen OD260 units of each supernatant were loaded onto continuous 12 ml 7\u201347% sucrose gradients in polysome lysis buffer without protease inhibitors, as in Baim et al (1985). After a 2.5-h spin at 40 000 r.p.m. in a Beckman SW40 rotor, the sucrose gradient was fractionated and absorbance at 254 nm was measured. For TAP-tagged strains, fractions were collected, and proteins were precipitated with 10% cold TCA and washed with 100% cold acetone.\nImmunoblotting\nPrecipitated proteins were resuspended in 20 \u03bcl Laemmli buffer and 2 \u03bcl of each sample was deposited onto nitrocellulose membrane. TAP-tagged proteins were detected with PAP antibody (Rockland Immunochemicals Inc.) and chemiluminescence (ECL; Amersham Biosciences).\nSupplementary Material\nSupplementary Figures\nSupplementary Tables\nSupplementary Information 1\nSupplementary Information 2","keyphrases":["interactions","networks","mass spectrometry","proteomics","systems biology"],"prmu":["P","P","P","P","R"]}
{"id":"Environ_Health_Perspect-115-3-1849909","title":"Skin Exposure to Isocyanates: Reasons for Concern\n","text":"Objective Isocyanates (di- and poly-), important chemicals used worldwide to produce polyurethane products, are a leading cause of occupational asthma. Respiratory exposures have been reduced through improved hygiene controls and the use of less-volatile isocyanates. Yet isocyanate asthma continues to occur, not uncommonly in settings with minimal inhalation exposure but opportunity for skin exposure. In this review we evaluate the potential role of skin exposure in the development of isocyanate asthma.\nIsocyanates, a group of reactive chemicals [with the functional group N = C = O (NCO)] used extensively in the production of numerous polyurethane foams, coatings, and a wide array of consumer products, are a major cause of occupational asthma worldwide. The polyurethane industry has expanded dramatically, along with the number of workers and consumers at risk for exposure. Inhalation has long been considered the primary route of isocyanate exposure, induction of sensitization, and asthma; research, practice, and regulation have focused almost exclusively on understanding and preventing inhalation exposures. Airborne isocyanate exposures have been reduced through improved controls and use of less-volatile isocyanates. Yet isocyanate asthma continues to occur, not uncommonly in work settings where measured isocyanate respiratory exposures are very low or nondetectable, but where there is opportunity for skin exposure.\nIt has been > 25 years since Karol et al. (1981) demonstrated in guinea pigs that skin contact with isocyanates could lead to sensitization and subsequent asthmatic responses following inhalation exposure. However, knowledge and awareness remain limited regarding the potential for isocyanate skin exposure to contribute to the development of isocyanate asthma. For example, the literature on occupational asthma rarely mentions isocyanate skin exposure as a potential risk factor or target for prevention (Nicholson et al. 2005; Tarlo and Liss 2005). Over the past several years there has been a growing, but largely unrecognized, collection of animal, industrial hygiene, clinical, and epidemiologic data related to isocyanate skin exposure and its role in the development of isocyanate sensitization and asthma. Our primary purpose in this article is to review and synthesize this multi-disciplinary literature to address several key unresolved issues, including the extent of isocyanate skin exposures in the workplace, the effectiveness of personal protective equipment, and most importantly, whether human skin exposure contributes to the development of isocyanate asthma. The findings may be relevant to the larger issue of the role of skin as an important underrecognized site of exposure and sensitization for other environmental allergens.\nMethods\nDefinition of terms\nThe terms \u201cskin exposure\u201d and \u201cdermal exposure\u201d are used interchangeably to indicate exposure to the outermost layer of the epidermis or epicutaneous exposure, as is commonly done in the occupational and environmental literature. \u201cIsocyanates\u201d refers to diisocyanate monomers (two NCO groups) and their related polyisocyanates, which have similar health effects (Bello et al. 2004). The term \u201csensitization\u201d can generate misunderstanding. \u201cSensitization\u201d generally refers to priming of the immune system in response to a specific non-self antigen, a condition that involves immune memory, typically antigen-specific T cells and\/or antibodies. Subsequent reexposure to the antigen can result in an immunopathologic adverse reaction, such as a Th2 (T-helper 2)-type acute allergic reaction or asthmatic response, or contact hypersensitivity-type reaction, such as allergic contact dermatitis (Sheaarer and Fleisher 2003). Others consider sensitization any immunologic memory of exposure, regardless of its pathogenic potential (i.e., a specific IgG response) (Abbas et al. 2000). Skin or respiratory sensitization typically refers to the route of exposure that results in systemic sensitization, rather than localized immune responses at those sites.\nLiterature search\nWe have been active in the field of isocyanate research and have collected > 800 published and unpublished articles and documents from 1951 to the present; these articles and documents are related to isocyanates and span many disciplines. In addition, we performed computerized searches of the literature on Medline [1966 to the present (National Library of Medicine, Bethesda, MD)], National Institute for Occupational Safety and Health (NIOSH), Occupational Safety and Health Administration (OSHA) and U.S. Environmental Protection Agency (EPA) databases and Google (google.com) using the key words \u201cisocyanate,\u201d \u201cdiisocyanate,\u201d \u201casthma,\u201d \u201csensitization,\u201d \u201cexposure,\u201d \u201cdermal,\u201d \u201cskin,\u201d \u201coccupational,\u201d \u201cmethylene diphenyl diisocyanate,\u201d and other synonymous terms. Additional articles were identified from the reference lists of the selected relevant articles. We reviewed primarily English-language articles, as well as selected articles in German, Danish, and French. Human and animal articles that addressed isocyanate skin exposure, sensitization, and health effects were retained for further analysis. We also included clinical, epidemiologic, and biomarker studies and case reports that mentioned skin as a potential route of exposure or had low isocyanate air levels based on exposure data or work processes.\nResults and Discussion\nHealth effects of isocyanate exposure\nIsocyanates are considered potent respiratory allergens. Isocyanate asthma is the major health problem in isocyanate-exposed workers, affecting approximately 1\u201325% of the exposed population. The most important risk factor is isocyanate exposure, but the exposure characteristics and host factors involved remain unclear (Bernstein 1996; Wisnewski et al. 2006). Isocyanates can also cause hypersensitivity pneumonitis, contact dermatitis, and rhinitis, but these outcomes are less commonly reported (Baur et al. 1994; Musk et al. 1988).\nClinically isocyanate asthma presents similar to other types of allergic Th2-like asthma: Isocyanate asthma typically develops after repeated exposure for months to years, during which time sensitization to isocyanates is presumed to occur. Once sensitized, extremely low respiratory levels of isocyanate can elicit asthmatic responses. However, unlike for typical high molecular weight (MW) occupational allergens or environmental aeroallergens, research has failed to identify an isocyanate-specific immune response in most isocyanate asthmatics that indicates isocyanate Th2-like sensitization, such as a radioallergosorbent test (RAST) or skin prick test, thus hindering diagnosis. This difference may reflect involvement of non-IgE mechanisms in isocyanate asthma pathogenesis, and\/or may be related to the NCO functional group common to all isocyanates that renders them ideal cross-linking agents (Wisnewski et al. 2006). Unlike high-MW allergens, isocyanates can react with amino (NH2, NH), hydroxyl, and sulfhydryl groups of various proteins and peptides, including albumin and keratin, to form a number of different hapten\u2013protein complexes or antigens (Wisnewski et al. 1999, 2000). Human isocyanate skin-patch testing has been used to confirm sensitization in the uncommon person who develops contact hypersensitivity (allergic contact dermatitis) due to isocyanate skin exposure, but has not been helpful for detecting the Th2-like sensitization presumed to lead to isocyanate asthma (Kanerva et al. 2001). The mechanisms by which isocyanates cause asthma remain poorly defined, and lack of a good immunologic marker and unclear dose\u2013response relationships have hindered diagnosis and prevention (Wisnewski et al. 2006). The primary focus here is on the potential for skin exposure to contribute to the development of isocyanate asthma rather than pathogenic mechanisms. However, a better understanding of the role of skin exposure may help address these key problems.\nSeveral observations suggest that skin may also be an important site of exposure and sensitization. Isocyanate respiratory exposure alone, without any skin exposure, seems unlikely in most work settings. Isocyanate asthma occurs in settings with minimal documented respiratory exposures but clear potential for skin exposure, and splashes and spills have been reported by workers who subsequently develop isocyanate asthma (Bernstein et al. 1993; Lenaerts-Langanke 1992; Liss et al. 1988; Nemery and Lenaerts 1993; Zammit-Tabona et al. 1983).\nWorkplace and environmental isocyanate exposures\nThe chemical structures and important physicochemical properties of selected isocyanates are shown in Figure 1 and Table 1. The major commercial isocyanates are methylene diphenyl diisocyanate (MDI), toluene diisocyanate (TDI), and their nonvolatile polymeric forms pMDI and pTDI, followed by polymeric hexamethylene diisocyanate (pHDI) and isophorone diisocyanate (pIPDI). Isocyanates are reactive chemicals used extensively to make numerous polyurethane and other commercial products, such as poly-urethane foams, adhesives, and coatings. They are found in a wide range of industries, from construction to medical care. The increasing use of nonvolatile polyisocyanates has raised issues related to their measurement, exposure metrics, and regulation (Bello et al. 2004).\nOccupational exposures to isocyanates occur primarily in the many end-use settings, as well as in primary production facilities where exposures are generally better controlled. The total number of workers currently exposed to isocyanates is not known. NIOSH\u2019s estimate of 280,000 U.S. workers exposed or potentially exposed to isocyanates (NIOSH 1996) is undoubtedly higher today, given industry growth and new applications. Most commercial isocyanate products are complex isocyanate mixtures of variable molecular mass, volatility, and isocyanate content. Workplace exposures can occur in the form of isocyanate vapors, aerosols, or both, depending on the isocyanate type as well as the application method and other factors. Isocyanates are commonly mixed with various solvents, polyols, and other substances, such as catalysts and blowing agents, which may affect isocyanate reactivity, skin absorption, and health effects.\nAlthough data are limited, environmental exposures to unreacted isocyanates are also possible. A number of consumer products contain unreacted isocyanates, such as glues, polyurethane coatings, and foam insulation; domestic use of such products on rare occasion has been reported to trigger asthma symptoms in individuals previously sensitized (Carroll et al. 1976; Dietemann-Molard et al. 1991; Peters and Murphy 1971). Based on patch testing, a few cases of allergic contact dermatitis have been reported with the use of consumer products made from isocyanates and polyurethanes (Alomar 1986; Morgan and Haworth 2003; Vilaplana et al. 1987). Certain biomedical products, such as standard orthopedic casting material, may be a potential source of skin exposure to unreacted isocyanates for patients and cast technicians (Legris et al. 1995). Polyurethane foams and packaging have been reported to contain very small amounts of unreacted isocyanates (Gagne et al. 2003; Krone et al. 2003), but it is unclear whether these products have any potential to result in skin exposure. Environmental isocyanate exposures can also potentially occur from the release of isocyanates into the environment from primary or secondary production facilities (Darcey et al. 2002; De Zotti et al. 2000; Orloff et al. 1998), during the transport or storage of isocyanates or polyurethanes (Allport et al. 2003), or during the thermal degradation of overheated polyurethane products (Boutin et al. 2006), but these exposures are rarely reported. Isocyanate releases from end-users, such as auto body shops located in or near residential neighborhoods, are also possible but rarely reported. It has been hypothesized that environmental skin exposure to polyurethanes in childhood has contributed to the increased prevalence of childhood asthma (Krone and Klingner 2005), but human isocyanate exposure from such products has not been documented, and there are numerous other likely causative factors.\nThe primary isocyanate exposure routes are through the respiratory tract and the skin. Historically, the focus has been on inhalation exposures. Increased use of less-volatile MDI and polymeric isocyanates, as well as improved hygiene practices, have resulted in reductions in inhalation exposures to volatile monomer (Bello et al. 2004), thus potentially increasing the relative importance of skin exposure. Isocyanate skin exposure could contribute a significant part of the total body burden. For example, 1% skin absorption of a small MDI droplet (10 mg) would deliver a dose approximately 4.5-fold (450%) higher than the inhalation exposure at the current short-term UK occupational exposure limit (15-min, 70 \u03bcg NCO\/m3) or approximately 50% of the corresponding 8-hr (20 \u03bcg NCO\/m3) standard, assuming 100% lung retention and a ventilation rate of 7 L\/min (Bello et al. 2004).\nMeasuring isocyanate skin exposure\nQuantification of isocyanate skin exposure is important for research, prevention, and control. Assessment of skin exposure, in general, is much less developed than that of inhalation exposure. Skin exposure sampling methods typically are nonstandardized, have undergone limited validation, and can be technically challenging. Isocyanate skin sampling is further complicated by several factors, including the reactivity of NCO groups toward skin proteins, water, or other compounds, and the complexity of most isocyanate exposures. Biomarkers such as urinary metabolites, if available, could potentially be used to assess internal dose, but would not distinguish between skin and respiratory exposure.\nTechniques that have been used to detect isocyanate skin exposure include SWYPE pads (Colormetric Laboratories Inc., Des Plains, IL) (Liu et al. 2000), wipes (Bello et al., in press), and tape stripping (Fent et al. 2006). These methods rely on removal of isocyanates from the skin\u2014usually a period of time after initial exposure, and can underestimate exposure as a result of losses due to absorption, chemical reactions, and\/or poor removal efficiency (Bello et al. 2005; Wester et al. 1999). Techniques that quantitate isocyanate deposited on the skin, such as reagent-impregnated patches, may overcome some of these limitations. Recently, extraction of isocyanates from contaminated gloves was used to measure hand skin exposure to isocyanates (Pronk et al. 2006). Application of these skin exposure methods in the workplace has been limited, and findings have not been compared or validated. Workplace isocyanate skin exposure assessment is further complicated by the frequently sporadic nature of such exposures.\nWorkplace isocyanate skin exposure\nNumerous isocyanate end uses, such as spraying and application of foams and adhesives, provide opportunity for isocyanate skin exposure from deposition of aerosols and\/or absorption of vapors. Typical workplace isocyanate exposure levels are not irritating and give few warning signs, and skin protective equipment may not be worn, even when respiratory protection is used (NIOSH 1999). Skin exposure may result from direct contact of unprotected skin or the failure of personal protective equipment, such as gloves. Opportunities for isocyanate skin exposure, such as spills, cleanup, and contact with contaminated equipment, are well known to workers and field researchers. For example, NIOSH Health Hazard Evaluations of a variety of work settings have described workers with skin contact to isocyanates or uncured polyurethane products (NIOSH 1998, 1999).\nIsocyanate products that are not fully cured are another potential source of isocyanate skin exposure (Bello et al., in press). It is commonly believed that isocyanate-containing products polymerize rapidly, and once the product appears hardened, no unbound isocyanate species remain on the surface. However, there are few published data confirming this. A recent study demonstrated that curing proceeded more slowly than expected, with unbound isocyanate species detected on painted surfaces for prolonged periods of time (days to weeks) (Bello et al., in press). Thus, handling recently cured isocyanate products could be a source of isocyanate skin exposure. In addition, release of free isocyanates has been reported after heating of cured isocyanate products, as can occur when grinding, cutting, or sanding such products (Alliance for the Polyurethanes Industry 2005; Boutin et al. 2006; Littorin et al. 2002). Such tasks could represent another possible source of isocyanate skin exposure.\nIn spite of observational documentation of workplace isocyanate skin exposures, published data documenting such exposure are surprisingly limited. Isocyanate skin exposure has been documented qualitatively with colorimetric techniques in several work settings (Liu et al. 2000; NIOSH 1998). Recent studies have begun to quantitate such exposures. Fent et al. (2006) detected HDI monomer on tape strips from an auto-body painter\u2019s unprotected skin. Pronk et al. (2006) recently reported isocyanate exposure under gloves on both hands of auto body and industrial spray painters, with the highest median hand exposure detected during paint mixing (207 and 63 \u03bcg NCO, respectively).\nEffectiveness of personal protective equipment\nGloves and protective clothing remain a primary means of preventing skin exposure in the workplace, in addition to engineering and work practice controls. Gloves and protective clothing are presumed to protect against isocyanate skin exposure, with nitrile gloves considered preferable to latex. However, data on the workplace performance of protective gloves and clothing are limited, and there is evidence that isocyanates (Liu et al. 2000; Pronk et al. 2006) and solvents (Collin-Hansen et al. 2006) can be detected underneath gloves.\nSkin absorption of isocyanates\nAnimal studies employing radiolabeled 14C-MDI have demonstrated absorption of MDI after skin exposure (Leibold et al. 1999; Vock and Lutz 1997). However, quantitative data are limited and may have underestimated absorption due to technical issues, such as isocyanate binding to the dressing and skin. Studies documenting the disappearance of isocyanates from guinea pig skin with infrared spectroscopy also support isocyanate skin absorption (Bello et al. 2006).\nWe are not aware of published data directly confirming isocyanate skin absorption in humans; however, a number of studies indirectly demonstrate isocyanate skin absorption. For example, HDI-conjugated keratins have been identified from human skin biopsies obtained after epicutaneous application of HDI (Wisnewski et al. 2000). Patch testing with isocyanate (0.1\u20131%) can elicit an isocyanate-specific skin contact hypersensitivity reaction that implies isocyanate skin absorption.\nStudies investigating urinary biomarkers of isocyanate exposure (the corresponding diamines) have provided additional indirect evidence for isocyanate skin uptake (Creely et al. 2006; K\u00e4\u00e4ri\u00e4 et al. 2001; Maitre et al. 1996). Elevated levels of these urinary biomarkers have been detected in workers, in spite of very low or nondetectable documented inhalation exposures. Greater than 2-fold higher urinary metabolite concentrations have been reported for operators with likely skin contamination compared to those without (Creely et al. 2006). Maitre et al. (1996) measured urinary hexamethylene diamine (HDA) of two HDI-exposed coworkers. Both workers had similar inhalation exposure as confirmed by air measurements, but one had considerably greater HDI skin contact and a 10-fold increase in urinary levels of HDA. The authors concluded that HDI seemed to be readily absorbed through the skin.\nHuman isocyanate skin absorption likely depends on a number of factors, in addition to the extent of isocyanate skin exposure, and may vary between isocyanates because of differences in their physical and chemical properties, including molecular mass, fat solubility, and chemical reactivity. Skin absorption can be enhanced if the barrier properties of skin have been damaged, such as can occur with eczema, cuts, hand washing, cosmetics applications (shaving, waxing), and other conditions (Moody and Maibach 2006; Smith Pease et al. 2002). In addition, coexposures such as solvents used in the production of polyurethane foams, coatings, and spray applications can be absorbed through the skin (Boman and Maibach 2000). Such solvents may enhance isocyanate absorption and also break through the gloves.\nSkin sensitization\nNumerous substances\u2014primarily low-MW haptens, such as metals and chemicals, and less commonly proteins\u2014are known to initiate immune responses in the skin, most commonly hapten-induced contact hypersensitivity (Kimber and Dearman 2002). Contact hypersensitivity (allergic contact dermatitis) following skin exposure to isocyanates is well documented in animals and in the clinical dermatologic literature, with sensitization confirmed with patch testing (Goossens et al. 2002; Herrick 2002). Allergic contact dermatitis has been reported following skin exposure to isocyanates and polyurethane products in a number of different workplace and non-occupational settings, but has not been considered common, and is rarely reported in workers with isocyanate asthma (Alomar 1986; Frick et al. 2003; Goossens et al. 2002; Wilkinson et al. 1991). However, allergic contact dermatitis may be more common than suspected because symptoms can be mild, workers being evaluated for asthma are frequently not asked about skin problems, and patch testing can be falsely negative (Frick et al. 2004; Goossens et al. 2002).\nMuch less is known about the role of skin exposure in inducing Th2-type immune responses seen in asthma. Recent animal studies have documented that skin exposure to proteins such as ovalbumin or peanuts can induce systemic Th2-type sensitization and subsequent asthmatic responses (Herrick et al. 2003; Strid et al. 2005). Limited clinical and epidemiologic studies also support a role for skin exposure to allergens in the development of Th2-type sensitization and asthma, or of other immunologic lung diseases such as chronic beryllium disease (Cummings et al. 2006; Lack et al. 2003; Saloga and Knop 2000; Smith Pease et al. 2002; Tinkle et al. 2003).\nIsocyanate skin exposure, sensitization, and asthma\nAnimal models\nAnimal models using the three major isocyanates MDI, TDI, and HDI have all employed skin exposure to induce sensitization, with subsequent inhalation challenge, to create an asthmatic response in the lungs (Table 2). Skin sensitization with other chemicals, such as trimellitic anhydride, followed by inhalational challenge has also produced asthmatic responses (Vanoirbeek et al. 2006; Zhang et al. 2004).\nDifferent sensitization and challenge protocols (variable doses, frequency, timing, formulation, and route of exposure) using different isocyanates have resulted in variable pulmonary responses. In spite of these differences, several common themes emerge from these animal studies. First, skin has been a very effective route of inducing sensitization, sometimes more effective than inhalation (Ban et al. 2006; Rattray et al. 1994). For example, Ban et al. (2006) recently evaluated several different TDI sensitization scenarios (inhalation, subcutaneous, topical) and challenge protocols (vapor, tracheal instillation) in mice. Topical sensitization followed by tracheal instillation of TDI most closely reproduced the Th2-type lung inflammatory response seen in human asthma, whereas sensitization with vapor TDI was not effective.\nOf note, these isocyanate animal models also demonstrate that a single one-time or two-time skin exposure with relatively low concentrations of isocyanates can be sufficient to induce sensitization (Herrick et al. 2002; Karol et al. 1981; Rattray et al. 1994). Additionally, several models have shown paradoxical dose\u2013response relationships, such that a lower skin sensitization dose can result in greater lung inflammation upon inhalation challenge than a higher sensitization dose, and that isocyanate-specific antibody responses may not correlate with asthmatic responses (Herrick et al. 2002; Vanoirbeek et al. 2004). The effective skin sensitizing doses in these studies, typically in the order of 1\u2013100 \u03bcmol NCO (Table 2) delivered as diluted 1\u201310% isocyanate solution, represent a few droplets of a diluted isocyanate product. Comparable exposures likely occur in the workplace.\nThus, several different animal models in more than one species clearly demonstrate that isocyanate skin exposure can induce systemic sensitization, which with subsequent inhalation exposure can lead to asthma. Control experiments in which animals received only skin exposure demonstrate that isocyanate skin exposure alone does not cause asthmatic responses (Herrick et al. 2002; Karol et al. 1981; Vanoirbeek et al. 2004). Although issues of comparability with humans are inevitable, such as differences in skin permeability, much can be learned from animal models regarding exposure\u2013response relationships and the mechanisms that lead to skin sensitization and asthma.\nHuman studies\nAlthough patch testing has confirmed contact hypersensitivity following human isocyanate skin exposure, direct evidence that skin exposure leads to Th2-type sensitization and the subsequent development of asthma is limited. As noted above, there is no good test to identify isocyanate sensitization in humans. Isocyanate-specific IgE is present in less than half of isocyanate asthmatics (Mapp et al. 2005; Wisnewski et al. 2006), and isocyanate-specific IgG appears to be indicative primarily of exposure (Liss et al. 1988; Welinder et al. 1988; Wisnewski et al. 2006). Isocyanates are considered a potent respiratory sensitizer in humans, based largely on circumstantial evidence rather than on clear demonstration that respiratory exposure alone (without associated skin exposure) leads to isocyanate asthma.\nIndirect evidence from a growing number of case reports and clinical and epidemiologic studies suggests that isocyanate skin exposure occurs in the workplace and can increase the risk for sensitization and isocyanate asthma. Isocyanate asthma and\/or sensitization (MDI-IgE) have been reported in several case studies of workers who applied MDI-based orthopedic casts (Donnelly et al. 2004; Legris et al. 1995; Sommer et al. 2000). Allergic contact urticaria and asthma following direct hand contact with MDI glue has been documented based on a positive MDI-IgE, MDI patch test, and MDI inhalation challenge (Valks et al. 2003). Consistent across these case reports is the development of MDI asthma in settings where skin exposure to MDI occurred and where MDI air levels, if measured, were non-detectable or extremely low and opportunity for MDI respiratory exposure was very limited. These cases strongly suggest that skin exposure was the predominant exposure route and contributed to the development of isocyanate sensitization and asthma. They also demonstrate that isocyanate asthma can occur in settings where measured isocyanate respiratory exposures are below the level of detection, even when sensitive analytical methods are used.\nSkin exposure has also been implicated as a risk factor for isocyanate asthma in several epidemiologic studies of MDI-exposed workers, in which measured inhalation exposures were all very low or nondetectable. Petsonk et al. (2000) investigated a group of new workers exposed to MDI resins in a facility designed for minimal airborne exposures and noted new asthma-like respiratory symptoms in 27% of the workers with the highest potential for MDI exposure. These symptoms were associated with liquid MDI skin exposure, as evaluated by worker questionnaires and workplace observations.\nLeanerts-Langanke (1992) described an investigation of about 500 coal mine workers who used MDI for rock consolidation. Quantitative air sampling results were at or below the detection limit (1 ppb). MDI skin exposure was reported in about half the workers; MDI-IgE was detected in several workers; and 2 were diagnosed with isocyanate asthma based on a positive specific challenge. Elevated levels of MDI metabolites in urine were found in 6 of 8 workers seen after \u201cmassive skin contamination.\u201d The authors concluded that skin could be an important route of exposure, leading to sensitization and asthma.\nBernstein et al. (1993) studied a cohort of 243 workers in a urethane molding plant that consistently maintained low MDI airborne exposures (< 5 ppb). Isocyanate asthma and\/or sensitization (MDI-IgE) was diagnosed in several workers, most of whom were reported likely to have had MDI skin exposure.\nOur group has characterized workplace isocyanate exposures in a population of > 200 auto body shop workers with isocyanate-specific immune responses but without documented isocyanate asthma (Redlich et al. 2001; Woskie et al. 2004). Respiratory and skin exposures were estimated for each of the workers, based on exposure algorithms. HDI-specific IgG, present in 21% of the workers, was strongly associated with inhalation exposure, but skin exposure also contributed (Stowe et al. 2006). We have also detected MDI-specific IgG in > 30% of about 100 workers in a factory that uses MDI to produce polyurethane coated fabrics. MDI air monitoring data have consistently been very low, and MDI skin exposure has been documented by worker questionnaires and direct observation (Liss et al. 2006). In both work settings, isocyanate skin exposure appeared to contribute to the development of isocyanate-specific IgG, which has been associated with isocyanate exposure (Welinder et al. 1988; Wisnewski et al. 2004; Ye et al. 2006).\nStudies investigating the respiratory exposure conditions that lead to isocyanate asthma also suggest a potential role for skin exposure. Fewer cases of isocyanate asthma have generally been reported in settings with lower respiratory exposures, but cases continue to occur in settings with consistently low reported air levels (Baur 2003; Tarlo et al. 1997). Skin exposure (Bernstein et al. 1993; Leanerts-Langanke 1992) or intermittent peak exposures, which could also entail both respiratory and skin exposure (Tarlo et al. 1997), have been considered important contributing factors in such cases. Typically, such exposures are unpredictable and frequently accidental, making them difficult to investigate and quantify.\nFor cutaneous allergen exposure in general, the likelihood of sensitization in a susceptible person is thought to depend on several factors including the total dose and concentration of allergen, skin surface area, and the frequency of repeated contact with the skin, with determinants of individual susceptibility remaining poorly defined (Basketter et al. 2006; Boukhman and Maibach 2001). There are insufficient data to address isocyanate skin exposure\u2013response relationships in humans; however, as noted above, in animal models relatively small skin doses can induce sensitization, and dose\u2013response relationships may be variable.\nRegulatory standards for isocyanate skin exposure\nThe regulatory framework for skin exposure to chemicals is underdeveloped compared with inhalation exposures. Chemicals that pose a health hazard through skin exposure are commonly assigned two qualitative descriptors: a \u201cskin\u201d notation, referring to absorption of the chemical through the skin, and\/or a \u201csensitizer\u201d notation for an agent with the potential to produce sensitization regardless of the exposure route (respiratory, skin, or conjunctiva) [American Conference of Governmental Industrial Hygienists (ACGIH) 2006].\nIn the United States, although NIOSH recommends prevention of isocyanate skin exposure, skin exposure is not regulated. No \u201cskin\u201d notation exists for diisocyanates or polyisocyanates, except for the NIOSH (2005) recommendation for IPDI.\nAlthough data confirming the risks of human isocyanate skin exposure remain limited, there is sufficient information to recommend prevention of skin exposure. Such recommendations are now being made in material safety data sheets and are beginning to appear in the medical literature (Bakke et al. 2006; Baur 2003). Wider dissemination and improved hazard communication of this information by occupational and environmental health professionals, as well as better personal protection among workers, are needed. Inclusion of \u201cskin\u201d notation may encourage such protection for all isocyanates (diisocyanates and polyisocyanates).\nResearch needs\nIn this review we highlight several important areas for further research regarding isocyanate skin exposure, ideally using multidisciplinary approaches involving animal models and clinical and epidemiologic investigations. Such approaches should lead to a better understanding of the mechanistic pathways that result in isocyanate asthma and the role of skin exposure in this process. A key research need not unique to issues regarding skin exposure is the development of a good marker of isocyanate sensitization or \u201cpre-clinical\u201d asthma that correlates well with the subsequent development of asthma. Such a marker would greatly enhance isocyanate research, including elucidation of exposure\u2013response relationships, and facilitate diagnosis and prevention.\nThere is a need to better assess isocyanate skin exposures in the workplace and other environments and to incorporate these exposure data into epidemiologic and clinical studies. The typically more sporadic nature of such exposures further complicates real-world exposure assessment and requires algorithms that employ a combination of daily activities (diaries), questionnaires, and task-based exposure data. Development of route-specific bio-markers, such as those specific for skin or lung, would greatly facilitate isocyanate exposure assessment. Skin exposure methodologies and biomarkers of exposure can be further developed and validated using integrated animal models.\nNumerous host and environmental determinants of isocyanate skin exposure have barely been investigated. Isocyanate skin absorption likely depends on various factors including molecular size and coexposures (e.g., polyols, solvents, and other additives), which could enhance absorption. The role of host factors, such as history of eczema and hand washing, warrant further investigation, as does the effectiveness of gloves, protective clothing, and other preventive strategies.\nGiven the widespread and growing use of polyurethane consumer products, research is also needed to investigate potential environmental exposures from these consumer products, as well as exposures that may occur when isocyanate products are manipulated (ground, cut, drilled) or undergo thermal degradation as in fires. Further research investigating the curing process is also warranted because incompletely cured products could be a potential and unexpected source of skin exposure (Bello et al., in press).\nConclusion\nIsocyanates, primarily diisocyanate monomers and polyisocyanates, are a leading cause of occupational asthma. Human skin exposure to isocyanates has been underrecognized and can occur in various workplace and environmental settings. Multiple lines of evidence from animal studies and clinical, epidemiologic, and biomarker studies, as well as anecdotal evidence, indicate that in certain exposure settings human skin likely is an important route of isocyanate exposure and can contribute to the development of isocyanate asthma. This presumably occurs by isocyanate skin exposure inducing systemic sensitization, which then leads to isocyanate asthma after inhalation exposure; however, the mechanistic pathways involved remain poorly defined. Further research is needed to address issues regarding isocyanate skin exposure in the growing polyurethane industry. In spite of substantial research needs, sufficient evidence already exists to justify greater emphasis on the potential risks of isocyanate skin exposure and the need to prevent such exposures. We conclude with the visionary statement of Munn (1965):\nThe more one knows about these fascinating compounds [isocyanates] the more fascinated one becomes. So diverse are their uses, it is obvious that they are here to stay, and that their use will increase. So numerous have been the accounts of their effects, it is obvious not merely they are hazardous but that the nature and extent of their hazard has not always been fully appreciated.","keyphrases":["skin","isocyanates","asthma","sensitization","dermal exposure"],"prmu":["P","P","P","P","P"]}
{"id":"Int_Ophthalmol-4-1-2359829","title":"Eye bank issues: II. Preservation techniques: warm versus cold storage\n","text":"Most of the tissue used for penetrating keratoplasty is issued through eye banks that store the corneoscleral button either in hypothermic storage at 2\u20136\u00b0C or in organ culture at 31\u201337\u00b0C.\nIntroduction\nThroughout the world post-mortem eye tissue is used for keratoplasty. The generally accepted storage method for the whole globe is the \u201cmoist chamber\u201d; a moistened pot at 2\u20136\u00b0C introduced in 1935 [1]. The corneoscleral button is stored in tissue culture medium, either in the hypothermic storage method at 2\u20136\u00b0C introduced in 1974 [2] or in the organ culture method at 31\u201337\u00b0C introduced in 1976 [3]. Corneas cannot reliably be frozen.\nThe prevailing storing technique today is the storage of the corneoscleral button. The storage time can be extended by removing the corneoscleral button from the globe. A longer storage time permits greater flexibility in the use of the donor tissue and prevents wastage. In addition in many countries, for different reasons, legal or ethical, corneoscleral disc excision in situ is preferred to the removal of the whole globe. Moreover, the preparation of lamellae and mushroom-shaped grafts from a corneoscleral button is nowadays possible with help of an artificial anterior chamber. With the introduction of the preservation media, corneal surgeons have to rely on highly skilled technicians employed in eye banks selecting and storing the donor tissue.\nThe original hypothermic storage solution, the M\u2013K medium, has been succeeded by other solutions claiming better and longer maximum storage results. The hypothermic method is common all over the world.\nAlthough organ culture originates from the United States [3, 4] it has been strongly promoted by the Eye Bank of \u00c5rhus in Denmark [5, 6] and is now widely applied in Western Europe but not commonly used elsewhere. After some modifications shortly after its introduction the organ culture storage technique has stayed the same. The storage media became commercially available although some eye banks still prefer to prepare them themselves. They differ slightly in composition between banks and countries [7].\nBoth storage methods, hypothermic and organ culture will be compared considering technical aspects, tissue evaluation possibilities, storage time, microbiological safety, graft survival and future applications with regard to the increased interest in lamellar grafting.\nTechnical aspects\nGeneral\nProcurement and storage techniques have to be performed under aseptic conditions. Increasingly, eye banks have a formally established quality assurance program.\nThis and the increasing regulation may affect banks working on a smaller scale. For example in France the number of operating eye banks reduced form 226 in 1993 to 43 in 2004.\nHypothermic storage\nThe technique is simple: refrigerator storage with minimal handling. It requires no complex or expensive equipment. The storage solutions are commercially available and manufacturer recommendations should be followed for temperature, maximal storage time, expiry date and other factors. The vials may allow inspection of the endothelium by specular microscopy (Fig.\u00a01 left). During storage the cornea remains thin and, provided donor screening permits release of the tissue, it is directly available for surgery.Fig.\u00a01Corneoscleral buttons in different vials for hypothermic storage (left) and in the incubator during organ culture (right)\nThe medium consists of a tissue culture medium, supplemented with antibiotics, deturgescent agents like dextran and chondroitin sulphate to prevent corneal swelling in\u00a0vitro, and other additives such as energy sources, antioxidants, membrane stabilizing and growth factors to improve the storage capacity. Inspection of the tissue by slit-lamp and\/or specular microscope can be performed in a closed system.\nOrgan culture\nThe technique is relatively complicated, despite the fact that nowadays the storage solutions are commercially available.\nThe corneas are stored in an incubator at 30\u201337\u00b0 C in a tissue culture medium, supplemented with fetal or newborn calf serum, antibiotics and antimycotics (Fig.\u00a01 right). Dehydrating macromolecules, necessary to maintain normal hydration in\u00a0vitro, are ingested by the corneal cells at a physiological temperature and found accumulated in vacuoles in the cells and layers of the cornea [8, 9]. Therefore they are omitted from the storage solution. As a result the cornea swells to about twice its normal thickness during storage. The swelling should be reversed before transplantation. This is performed by placing the cornea in the storage medium supplemented with dextran. This so-called transport medium is also used for the transport of the cornea at room temperature. The extent of deswelling depends on the dextran concentration, varying from 4\u20138% in the different banks, resulting in a thickness of about 0.5 to 0.7\u00a0mm, depending on the preference of the surgeon(s) using the tissue from a certain bank. The maximum time the cornea spends in the transport medium also varies between banks; from less than one up to seven\u00a0days [7, 8]. The risk of the ingested dextran in relation to the export area and transport time is judged differently.\nDepending on the media used, renewal of the medium occurs after 10\u201314\u00a0days of storage [7]. For inspection of the endothelium, mandatory after storage, an invasive technique has to be used, which has to be performed under strict aseptic conditions.\nThe necessary transfer of the cornea from the storage to the transport solution averts a stored cornea from being directly available for use. Besides, a minimal storage period is required for microbiological testing. All this makes the technique more complicated than the hypothermic storage method.\nTissue evaluation\nGeneral\nIrrespective of the storage method(s) used the donor should be adequately screened. Tissue that is potentially hazardous to eye bank personnel and the recipient should be excluded in addition to tissue that poses a risk for the success of the surgery. Physical assessment, serologic testing and evaluation of medical and social history of the donor are mandatory.\nRoutine inspection of the endothelium is also part of the donor evaluation. Moreover, it can play an important role in setting higher and more-uniform quality standards for tissue acceptance. It may also help to increase the donor supply by assessing corneas that may otherwise be arbitrarily excluded for transplantation on the basis of age or time post mortem. The possibilities for evaluation of the endothelium are dependent of the storage method used.\nStudies linking graft outcome with morphometric parameters of solely the endothelium are still lacking. A model has been presented to calculate endothelial cell loss in the long run of 10\u201320\u00a0years after penetrating keratoplasty [10]. In this way it is possible to predict when cell density would reach levels that are incompatible with maintenance of transparency and graft function. The model provides a rationale for the setting of minimum donor cell densities. However, the definite cut-off points are still at the discretion of the bank and surgeon.\nHypothermic storage\nDetermination of endothelial cell density is a standard method of corneal tissue evaluation according to the Medical Standards of the Eye Bank Association of America, effective since December 2001. In general this will be a pre-storage evaluation of the endothelium by specular microscopy (Fig.\u00a02 left). Because the appearance of the endothelial cells varies with temperature, type and time of preservation and media, evaluation at room temperature is recommended. When it is impossible to obtain an endothelial cell count, this requirement may be waived on a case-by-case basis by the Medical Director.Fig.\u00a02Evaluation of the endothelium by specualr microscopy (left) and light microscopy after artificial swelling of the intercellular space (right)\nSelection criteria and cut-off points for the morphology of the corneal endothelium are not defined. In most cases only descriptions of the endothelial mosaic are used: swollen cells, dark spots, guttae, lysed cells, mild to severe polymegethism and pleomorphism [11].\nMost specular microscopes are equipped with software programs to determine parameters describing the endothelial mosaic in terms of variation in cell shape andthe percentage of hexagonal cells in addition to the assessment of the cell density. The obtained morphometric results may help to standardize descriptions, but only provided the microscopes are well calibrated and the necessary interactive manipulation is performed by an experienced observer.\nOrgan culture\nAfter organ culture specular microscopy is not suitable for visualization of the endothelium. Therefore light microscopy is applied, bright field or phase contrast. It is necessary to visualize the endothelial cells by swelling the intercellular space with a hypotonic solution. This allows inspection over the entire endothelial surface (Fig.\u00a02 right). The mechanism has been described by Kirk and Hassard [12] and was worked out for the corneal endothelium by Sperling [13]. Because it is an invasive technique, it must be performed under aseptic conditions. The swelling is transient, it disappears after a couple of minutes and is dependent on the integrity of the cellular membranes. In dead and necrotic cells and in their direct neighborhood swelling will not occur. Before and after storage balanced salt solution (BSS), phosphate buffered saline (PBS), 1.8% sucrose\u2014PBS mixture or hypo-osmotic BSS may induce swelling, while for tissue in solutions with dehydrating agents a stronger stimulus with 1.8% sucrose might be necessary [7]. Induction of swelling and the swelling pattern are dependent on storage time and medium [14]. Therefore the interpretation of images requires experience and constant working conditions.\nThe application of a vital stain such as trypan blue [15, 16] preceding the artificial swelling of the intercellular space may help to recognize dead or necrotic cells or denuded Descemet\u2019s membrane.\nMicroscopes should be well calibrated both for manual counting as for evaluation by image analysis programs [17]. These software programs are commercially available, either specifically designed for endothelial evaluation, or as general programs adapted to do this. All programs aim for automated cell analysis that is independent of the observer and experience. However, in general, reliable parameters for the endothelial mosaic are only obtained interactively. This still requires experienced observers. Manual counting by Gunderson\u2019s method [18] can provide reliable cell counts [19], but for parameters such as variation in cell size and the percentage of hexagonals image analysis is necessary.\nThe quality of the corneal stroma can be evaluated by light microscopy. The significance of the presence of lysed keratocytes for the survival of the graft has not yet been investigated [20].\nStorage time\nGeneral\nAs the endothelium is essential for graft clarity and survival, the maximum allowed storage time is predominantly determined by maintenance of the endothelial function and integrity [21]. Regression lines, coefficients and equations have been published for storage time and damage or loss of endothelial cells. In Fig.\u00a03 the regression lines are collected for different storage methods [22\u201324]. The referred studies have in common that the vital stain with trypan blue is used to assess endothelial damage. The figure clearly demonstrates the differences in endothelial viability and explains the differences in the allowed maximum storage period.Fig.\u00a03Regression lines showing endothelial cell damage and loss in different storage solutions assessed after staining with trypan. The regression formulas for the M\u2013K medium, the Optisol GS and the organ cultures are respectively: y\u00a0=\u00a011.8x\u00a0+\u00a011.6, y\u00a0\u00a0=\u00a00.19x\u00a0+\u00a04.2, and y\u00a0=\u00a00.11x\u00a0\u2212\u00a01.8\nHypothermic storage\nThe original M\u2013K medium claimed a storage period of up to 10\u00a0days. Solutions introduced later, such as the modified M\u2013K medium, K-sol, Dexsol, Likorol, Optisol (Plus, GS) claimed better storage capabilities and a maximum storage period of 14\u201316\u00a0days. Changes in the endothelium as a result of post-mortem time and other variables, such as cause of death, donor age, circumstances of death etc., might result in the transplantation of corneas of inferior quality after storage. Because degenerative changes will progress during hypothermic storage, endothelial cell loss has to be taken into account [25], and might even lead to complete cell death. The need for methods to detect corneas not tolerating the prolonged hypothermic storage has therefore been suggested [26]. To reduce the risk of primary graft failure, the recommended storage periods are kept far below the claimed maxima. The applied period increased from 2\u20133\u00a0days for the M\u2013K medium [27, 28] up to 7\u201310\u00a0days for Optisol [7, 25]. In addition the time interval from death of the donor to storage of the cornea is generally kept relatively short, within 12\u00a0h or shorter [7].\nOrgan culture\nWith organ culture the allowed storage period is longer. In addition the time interval between death and storage is generally extended to 24\u201348\u00a0h, because significant wound healing can still occur during storage [29].\nThe loss of endothelial cells during storage may differ between individual corneas (Fig.\u00a04) and is supposed to reflect differences in vitality as a result of post-mortem time and other variables such as cause of death, donor age, circumstances of death etc. In this respect, prolonged hypothermic storage might not be so different from organ culture. Severe endothelial cell loss during organ culture may also be caused by herpes simplex virus infection of the donor tissue [30, 31]. Organ culture is therefore considered a stress test [32, 33]. A storage period of up to 4\u20135\u00a0weeks is possible [32\u201335]. However, in order to detect tissue not tolerating the storage, according to European Eye Bank Rules, inspection of the endothelium after storage is mandatory.Fig.\u00a04Percentage endothelial cell loss (endothelial cell density before storage minus the cell density after divided by the cell density before multiplying by 100) during routine storage by organ culture is plotted for a given year\nMicrobiological safety\nGeneral\nDonor eye tissue is usually contaminated [36] and each eye bank has to cope with this. As a first step decontamination procedures are applied before enucleation or excision of the corneoscleral button. When properly performed these procedures are very effective at reducing the risk of contamination [37]. As a next step, antibiotics, and in the case of organ culture antimycotics, are present in the storage solutions. These are more effective if the contaminating microbes are metabolically active, which means that they are more effective in organ culture than during hypothermic storage. In addition the vulnerability of organ culture to microbial contamination can be exploited to detect microorganisms remaining from the donor and\/or introduced into the culture medium by the environment or personnel. Theoretically, in organ culture the risk of contamination is therefore lower than in hypothermic storage.\nHypothermic storage\nAntibiotics have little effect during hypothermic storage. Preoperative warming of the storage media to room temperature is important to enhance the decontamination effect. The optimal time period of room-temperature storage has yet to be established [38]. In addition, antibiotics accumulate in the tissue during storage [39] and become active in the eye after grafting as the temperature rises.\nDonor rims tested after grafting are positive for bacteria and fungi in 12\u201328% of the cases [40]. However, the value of routine donor rim cultures in clinical use is debated. On the one hand no relevance of infectious complications after keratoplasty has been reported [41, 42]. On the other hand a 22 times increased incidence of endophthalmitis in the case of a positive rim culture [43] to a fully associated fungal infection following penetrating keratoplasty has been described [44]. The overall incidence of postoperative keratitis and endophthalmitis caused by microbes transferred with the donor cornea is low and varies from 0.2% [43] to 0.41% [45] and to 1.3% [46].\nThe addition of alternative antibiotics to the current hypothermic storage solutions has been suggested, because donor corneas are often removed in hospitals and other settings where resistant bacteria may be thriving [38]. These may cause an increased incidence of endophthalmitis. From a microbiological point of view, however, it is disputable whether the addition of the latest antibiotics with the widest spectra is the best solution.\nOrgan culture\nContamination detected during organ culture varies between eye banks [7]. This may be dependent on the antibiotic cocktail present in the medium (narrow- to wide-spectrum antibiotics), collection procedures, in\u00a0situ excision or enucleation and the post-mortem time of collection and storage [47]. Microbiological testing of medium samples before surgery is mandatory as well as a quarantine period before issuing corneas. Microbiological safety of the tissue stored by organ culture is obtained by discarding contaminated tissue before grafting. The incidence of endophthalmitis reported after a properly performed organ culture procedure is 0\u20130.1% [7].\nSterility may be better with organ culture since microbial contamination will become more readily evident. Organ culture might be considered as the method of choice in circumstances where corneas are suspected of being at a higher risk of contamination.\nThe mandatory reporting of adverse reactions and the central registration of these within the EU area where both the hypothermic and the organ culture techniques are used will demonstrate in time whether the incidence of endophthalmitis really differs between organ culture and hypothermic storage.\nGraft survival\nOnly one prospective study is available comparing clinical results after grafting between the hypothermic storage and organ culture [48]. During the 1\u20132\u00a0year study period no statistically significant differences in visual acuity, corneal thickness or endothelial cell density were found in keratoconus patients. Bourne [49], however, found fewer endothelial cells after grafting on corneas stored by organ culture. At that time, M\u2013K medium was used to reverse the swelling, a method differing from the method used in Europe.\nRetrospective studies claim improved [33, 50] or comparable results with organ culture [34, 35, 51]. The improvement is not ascribed to the storage itself but to the extra selections routinely included in organ culture preservation. In addition most of the studies were performed at a time when old-fashioned hypothermic storage methods did not always include inspection of the endothelium.\nFuture aspects\nWith the advent of new surgical techniques such as lamellar grafting, the issues for eyebanks are also changing. The risk for immunological graft rejection is theoretically lower in lamellar grafting. This may reduce the interest in HLA matching of donor and recipient, an important reason to prefer organ culture as a storage method. Organ culture provides sufficient time for typing and matching and is also thought to immunologically modify the tissue through the loss of passenger leucocytes and epithelium [52].\nFor a long time stored corneosclereal buttons were used for penetrating keratoplasty whereas lamellar grafting was performed with tissue from whole globes stored in a moist chamber. With the introduction of the artificial anterior chamber, the microkeratome and the intralase or femtosecond laser, this is changing quickly. Corneoscleral buttons which have been precut in order to reduce the manipulation of the donor tissue at the time of surgery will be new products of eye banks. For a reliable production of these precut buttons the intralase or femtosecond laser technique seems very promising [53\u201355]. Whether the storage technique (hypothermic of organ culture) might affect the postoperative fate of the corneal lamella is still open for discussion.\nThe revival of the mushroom technique is another development, where the eye bank would be expected to prepare the corneal button [56, 57]. Hypothermic storage, where the thickness of the cornea is maintained and where the risk of epithelial ingrowth is low because there is no epithelial growth, seems to be more suitable for these indications.\nConclusions\nWith the more widespread use of the specular microcope and the introduction of hypothermic storage solutions claiming an intermediate storage period some of the advantages ascribed to organ culture [58], such as scheduling operations and minimizing wastage of donor tissue, have become less important. Others still remain: allowing time for tissue typing and matching and extensive testing, detection of residual micro-organisms before surgery, and selecting and dispatching corneas with a well-defined endothelial quality assessed after storage. In addition the pool of possible donors can be enlarged thanks to the possibility of wound healing during organ culture and the inspection of the tissue after storage. Because organ cullture involves extensive testing of the quality of the donor cornea, there are theoretically no preset limits on donor age and postmortem time.\nTo permit these possibilities the organ culture procedure is more complicated than the hypothermic storage. Well qualified personnel, advice of microbiological laboratory staff, and a well suited facility are essential for a proper organ culture procedure but may also be valuable for hypothermic storage. The higher costs of organ culture have to be balanced against the offered advantages and possibilities not permitted by the hypothermic storage.\nWhether organ culture will also be the proper storage technique for surgically manipulated corneoscleral buttons remains to be investigated.","keyphrases":["eye banking","hypothermic storage","organ culture"],"prmu":["P","P","P"]}
{"id":"Eur_Biophys_J-3-1-2082062","title":"Organisation of nucleosomal arrays reconstituted with repetitive African green monkey \u03b1-satellite DNA as analysed by atomic force microscopy\n","text":"Alpha-satellite DNA (AS) is part of centromeric DNA and could be relevant for centromeric chromatin structure: its repetitive character may generate a specifically ordered nucleosomal arrangement and thereby facilitate kinetochore protein binding and chromatin condensation. Although nucleosomal positioning on some satellite sequences had been shown, including AS from African green monkey (AGM), the sequence-dependent nucleosomal organisation of repetitive AS of this species has so far not been analysed. We therefore studied the positioning of reconstituted nucleosomes on AGM AS tandemly repeated DNA. Enzymatic analysis of nucleosome arrays formed on an AS heptamer as well as the localisation of mononucleosomes on an AS dimer by atomic force microscopy (AFM) showed one major positioning frame, in agreement with earlier results. The occupancy of this site was in the range of 45\u201350%, in quite good agreement with published in vivo observations. AFM measurements of internucleosomal distances formed on the heptamer indicated that the nucleosomal arrangement is governed by sequence-specific DNA-histone interactions yielding defined internucleosomal distances, which, nevertheless, are not compatible with a uniform phasing of the nucleosomes with the AGM AS repeats.\nIntroduction\nSatellite DNA consists of tandemly repeated sequences with monomer lengths of up to 500\u00a0bp and copy numbers of up to millions. It is found in all eukaryotic species where it is concentrated in the centromeric regions of the chromosomes, as shown for example in humans (Schueler et al. 2001). The association of satellite DNA with essential processes ensuring genome stability, such as the assembly of the kinetochore (Willard 1998), is still unclear since these processes are not strictly sequence-dependent (Amor and Choo 2002). \u03b1-satellite DNA (AS) might contribute to the formation of a centromere-specific chromatin structure. The presence of oligo-adenine tracts and intrinsic curvature are universal features of satellites, which therefore may have a role in positioning nucleosomes along DNA and in chromatin condensation (Fitzgerald et al. 1994). Indeed, sequence-dependent nucleosome positioning on satellite DNA fragments was observed in vitro (Linxweller and Horz 1985; Neubauer et al. 1986; Tanaka et al. 2005; Yoda et al. 1998). Also, satellite chromatin isolated from cell nuclei was found to differ from bulk chromatin by a higher compaction level which may be supported by the presence of a particularly ordered nucleosomal arrangement (Gilbert and Allan 2001).\nAlpha-satellite DNA is characterised by a monomer length of \u223c171\u00a0bp and is primate specific. This DNA allows for a simple phase relationship between nucleosome binding and the repeat length. In fact, AS chromatin of African green monkey (AGM) (Cercopithecus aethiops) was used earlier to verify this phase relationship in vivo. The initial finding of a simple phase, based on micrococcal nuclease (MNase) digestions of cell nuclei (Brown et al. 1979; Musich et al. 1982), was challenged by the finding that the MNase cleaves the AGM AS non-randomly (Fittler and Zachau 1979; Horz et al. 1983). More detailed studies showed the occupation of different nucleosomal positions relative to the AS sequence in AGM-cells where, however, one highly preferred site consistently emerged in different approaches (Smith and Lieberman 1984; Wu et al. 1983; Zhang et al. 1983). Neubauer et al. (1986) identified nucleosomal positioning sites similar to those occurring in vivo by in vitro reconstitution experiments using 250\u00a0bp AGM AS fragments. The major site was occupied by 80% of the nucleosomes on 250\u00a0bp DNA in vitro and by 35% in vivo (Neubauer et al. 1986; Zhang et al. 1983). These results argued for a strong contribution of sequence-dependent DNA\u2013histone interactions to the nucleosomal organisation in AS chromatin of this species, although nucleosomal positions and internucleosome spacing generally depend on many different factors in vivo. For instance, the sequence-dependent positioning signal contained in the 5S rDNA of Lytechinus variegatus can be overruled when nucleosomes are reconstituted in cell extracts (Blank and Becker 1996). Recently, Segal et al. (2006) found a high predicted occupancy of nucleosomes over centromeres indicating that centromere function requires enhanced stability of histone\u2013DNA interactions that are encoded in the genomic sequence.\nThe importance of a correct nucleosomal positioning at centromeric chromatin is also indicated by the observation that the foundation centromeric protein CENP-B influences the position of nucleosomes along AS in vitro (Tanaka et al. 2005). CENP-B is the only known inner kinetochore protein to bind to a specific centromere sequence, the 17\u00a0bp Cenp-B box, which occurs in a subset of \u03b1-satellite monomers in humans (Masumoto et al. 1989). AS arrays of humans are composed of a number of divergent AS monomers organised into higher order repeat units, whereas arrays of AGM exhibit a simple and homogeneous monomer organisation and, furthermore, lack Cenp-B-boxes (Alexandrov et al. 2001; Goldberg et al. 1996). Intriguingly, CENP-B was found to be absent from the centromeres of AGM although the protein is expressed (Goldberg et al. 1996). One interpretation for this result could be that CENP-B is dispensable in the centromeres of AGM, since the AS by itself efficiently defines nucleosomal positions (Goldberg et al. 1996), even though earlier the existence of a simple nucleosomal phase in vivo had been argued against, as mentioned above.\nThe motivation of the present work was to extend the earlier studies of nucleosome positioning on AS of this species. So far, the sequence-dependent assembly of nucleosome arrays has not been studied using repetitive AGM-AS, and nucleosomal positioning on DNA fragments containing the uninterrupted AGM-AS sequence has not been quantified. We therefore reconstituted oligonucleosome arrays with a heptamer and mononucleosomes with a dimer of the AGM AS and took advantage of atomic force microscopy (AFM) as single molecule approach to determine the nucleosomal organisation along the DNA. AFM can measure internucleosome distances with high precision (Allen et al. 1993; Mechelli et al. 2004; Yodh et al. 2002; Zlatanova et al. 1998) and also the quality of the reconstituted material can be characterised at the single molecule level. The height information, available through AFM imaging, gives molecular structural information (Nikova et al. 2004). Thus, the AFM approach circumvents drawbacks of enzymatic assays. Yet, we additionally used MNase and restriction enzyme digestion in order to find out whether chromatin reconstitution would give results similar to the earlier in vivo findings (Musich et al. 1982), taking into account that this approach may overestimate the extent of evenly spaced nucleosomes. Our AFM measurements of internucleosome distances show that the in vitro generated nucleosomal arrangement along the AGM AS heptamer is clearly defined by the sequence, yet deviates from a simple phasing. The preferred occupation of the known major positioning site was unambiguously detected by (1) MNase and EcoRI digestion and (2) by localising mononucleosomes reconstituted on the AGM AS dimer by AFM. The AS sequence repeat thus has a profound influence on the nucleosomal arrangement formed by reconstitution on this DNA and also the estimated occupancy of the major site was in a quite good agreement with the earlier in vivo findings.\nMaterial and methods\nPreparation of DNA\nAfrican green monkey \u03b1-satellite DNA was prepared from CV1-cells, a permanent line derived from C. aethiops kidney cells. Whole genomic DNA was isolated, deproteinised and treated with EcoRI restriction endonuclease essentially as described (Gruss and Sauer 1975). The DNA samples were run on 1.0% agarose gels and stained with ethidium. EcoRI produces one large smear in the upper region of the gel and a number of discrete bands below, corresponding to multiples of the AS-monomer (Gruss and Sauer 1975). Our attempts to clone repetitive AGM AS embarked on two strategies. One was to directly excise EcoRI-fragments containing different repeat numbers from low melting agarose gels (FMC) and ligate these with the cloning vector pBluescript-IIKS+. Escherichia Coli XL10 Gold cells (Stratagene) were transformed with the ligation products and cloned. By this approach, vectors containing AS-monomers and AS-dimers but no larger repeat numbers could be successfully cloned. It was verified by DNA-sequencing that the cloned DNA matches AGM AS: an isolated AS-dimer proved to be a tandem repeat of identical sequence. This yielded the plasmid pBluescript-IIKS-\u03b12. The second approach to obtain longer AS-arrays was a stepwise self-ligation of the already available \u03b12-insert. pBluescript-IIKS-\u03b12 was subjected to a partial EcoRI-digestion and the cleavage products were separated on a 1.4% low melting agarose gel to isolate the \u03b12-fragments. These were used for self-ligations at 4\u00b0C for 2\u20134 days using 10\u00a0U\/\u03bcl of T4 ligase. Afterwards, the DNA was ligated with pBluescript-KSII+ and we could obtain clones containing an AS-tetramer (pBluescript-KSII-\u03b14). The \u03b14-insert was used for the next self-ligation step carried out accordingly. This resulted in a clone containing an AS-heptamer (pBluescript-KSII-\u03b17). DNA-sequencing was used to verify the tandem orientation of the AS-repeats.\nA 208-6 construct was produced starting from the vector pPolI-5S-208-12 (a gift of Peter Becker) which contains a dodecamer of the 5S rDNA nucleosome positioning sequence (Blank and Becker 1996; Simpson et al. 1985). This DNA was subjected to a partial digestion with AvaI to produce linearised vector-DNA with different repeat numbers of the 208-sequence. The cleaved vector was re-ligated and cloned. A clone containing a hexamer of the 208-sequence was identified by 1.2% agarose gel electrophoresis after cleavage of the purified vector with PstI.\nTo prepare the target DNA-fragments for the chromatin reconstitution experiments, bacteria were grown in Luria Bertani medium at 37\u00b0C for 24\u00a0h and the cells were harvested by centrifugation at 6,000\u00a0rpm for 20\u00a0min at 4\u00b0C. The plasmids were isolated using the Nucleobond-kit (Machery-Nagel). AS-vectors were cleaved with NotI and XhoI to separate a fragment containing the AS-insert flanked on both sides by 33 and 37\u00a0bp, respectively (172-\u03b12, 415\u00a0bp and 172-\u03b17, 1,275\u00a0bp). The 208-6 DNA was separated from pPolI-5S-208-6 by cleavage with PstI and XbaI. The cleavage products were run on 1.4% low melting agarose gels, the target fragments were excised from the gels and purified using the gel purification-kit (Qiagen).\nChromatin reconstitution\nRecombinant histone proteins of Xenopus laevis were overexpressed and purified as described (Kepert et al. 2003; Luger et al. 1999). Histone octamers were formed by incubation of a stoichiometric mix of the histones for 30\u00a0min in unfolding buffer (7\u00a0M Urea, 20\u00a0mM Tris\u2013HCl, pH 7.4) and subsequent dialysis against refolding buffer (20\u00a0mM Tris\u2013HCl, pH 7.4; 0.1\u00a0mM EDTA; 0.5\u00a0mM \u03b2-mercaptoethanol; 2\u00a0M NaCl). An electrophoretic analysis of the used octamer preparation is shown elsewhere (Bussiek et al. 2005).\nNucleosome arrays with 172-\u03b17 and mononucleosomes with 172-\u03b12 were reconstituted by the salt dialysis method. Octamers were mixed with the DNA in 10\u00a0mM Tris\u2013HCl, pH 7.5; 2\u00a0M NaCl and kept at 30\u00b0C for 30\u00a0min. The mixture was transferred to mini dialysis tubes (Pierce, Rockford, USA) and dialysed against buffers with stepwise decreasing NaCl concentrations at 4\u00b0C (1.8, 1.4, 1.0, 0.8, 0.6, 0.4, 0.2 and 0.1\u00a0M for 1\u00a0h each and 0.005\u00a0M over night). The reconstitutions took place at different weight ratios of histone-octamer to DNA, ranging between values of 0.3\u20131.0. The DNA mass was 0.02\u00a0\u03bcg\/\u03bcl.\nAnalytical digestions with micrococcal nuclease and EcoRI restriction endonuclease\n172-\u03b17 DNA, either naked or reconstituted with histones, was digested with micrococcal nuclease (MNase). Reactions took place in 10\u00a0mM Tris\u2013HCl, pH 7.5; 5\u00a0mM NaCl; 0.5\u00a0mM CaCl2 using 0.3\u00a0mU\/\u03bcl MNase and 0.02\u00a0\u03bcg\/\u03bcl of the DNA. The reactions were stopped by mixing 50\u00a0\u03bcl aliquotes of the reaction with 5\u00a0mM EDTA after varying reaction times. Then, the DNA was isolated with phenol\/chloroform\/isoamylalcohol, precipitated with ethanol and resuspended in 15\u00a0\u03bcl Tris\u2013HCl, pH 7.5, 0.1\u00a0mM EDTA. The DNA was analysed on 1.2% agarose gels run in 1\u00a0\u00d7\u00a0TBE-buffer using ethidium staining.\nFor analytical digestion with EcoRI, another fraction of the reconstituted chromatin was first digested with MNase to produce mononucleosomal DNA. The reaction conditions and purification of the DNA was carried out as described above, where a total of 1.5\u00a0\u03bcg of DNA were digested. An aliquot corresponding to 1.0\u00a0\u03bcg of the purified DNA was subjected to EcoRI digestion for 2\u00a0h. The two DNA-samples (MNase and MNase\u00a0+\u00a0EcoRI) were run on a 1.7% agarose gel in 1\u00a0\u00d7\u00a0TBE, together with a 20\u00a0bp-ladder and stained with ethidium.\nAtomic force microscopy\nAtomic force microscopy was performed in air and in solution using a Multimode\u2122 (Digital Instruments) operated in tapping mode essentially as described (Bussiek et al. 2005). Mica modified with poly-L-lysine (PL) served as support for sample immobilisation. Freshly cleaved mica was pre-treated with 30\u00a0\u03bcl of an aqueous solution of PL (Sigma) at a concentration of 10\u00a0\u03bcg\/ml. After incubation for 30\u00a0s, unbound PL was removed by rinsing the mica disc with 4.0\u00a0ml of Millipore purified water followed by drying under a nitrogen stream. The chromatin was diluted to concentrations ranging between 0.5 and 2.0\u00a0nM (depending on the DNA size) in 10\u00a0mM Hepes-NaOH, pH 8.0, supplemented with salt as indicated in Sect.\u00a0\u201cResults\u201d. Thirty microlitres of the dissolved chromatin solution were placed on the PL-mica. For scanning directly in the adsorption buffer, silicon nitride probes (type NP-S20, Veeco Instruments) were used at drive frequencies of 8.0\u20139.5\u00a0kHz and a set point of 0.3\u20130.4\u00a0V. For scanning in air, the mica disc was washed carefully with 2.0\u00a0ml of Millipore water after incubating with the chromatin solution for 1\u00a0min. Then, the disc was dried with nitrogen and subsequently scanned using etched silicon probes (type NCH, Nanosensors) at drive frequencies of 280\u2013320\u00a0kHz and set point of 2.0\u20132.2\u00a0V. The images were recorded both in solution and in air at a scan diameter of 2\u00a0\u00d7\u00a02\u00a0\u03bcm, a scan rate of 1\u20132\u00a0Hz and a resolution of 512\u00a0\u00d7\u00a0512 pixels.\nImage analysis\nAtomic force microscopy images were flattened and zooms of individual complexes were produced using the Nanoscope IIIa software (version 5.12r3, Veeco Instruments).\nMeasurements of nucleosomal heights were done with the section analysis tool available in the same software package. This was also used to decide in some critical cases, whether a pair of two closely localised nucleosomes or a single one was present. When the cross-section showed two peaks or clearly a plateau next to a peak, two closely localised nucleosomes were assumed.\nLength measurements along DNA or chromatin contours were done using the freehand line tool in the program ImageJ (version 3.13, National Institute of Health). To measure the lengths of DNA fragments complexed into nucleosomes (Lcomplex), contours were traced along the entry and exit sites of the DNA strands into the nucleosomes. The nucleosome diameter measured by AFM is usually larger than the real diameter due to tip convolution, which means that the actual entry\/exit sites cannot be unambiguously localised. To account for this error, the apparent entry\/exit sites were always connected through the nucleosomal centres. In this way, the length of free DNA outside the nucleosomes (Lfree; free linker DNA and DNA termini) could be estimated by subtracting the known diameter of the nucleosome core particle (NCP) of 11\u00a0nm: where nncp is the number of NCPs per complex.\nInternucleosomal spacings between consecutive nucleosomes in the arrays were measured as centre-to-centre (cc) distances. The cc-distances were measured along DNA, when two nucleosomes were separated by a distance sufficiently long to make the intervening DNA visible. Otherwise, when two nucleosomes appeared close to one another, a straight line between the nucleosomal centres was assumed. The resolution limit that allows distinguishing nearby nucleosomes closely coincides with the NCP diameter. Since a correlation between the cc-distance and the internucleosomal separation along the DNA is not justified in this range of small distances, a minimum value of 11\u00a0nm was defined for the data analysis. The accuracy of the measurement as given by the pixel-size of the zooms used here is 0.5\u00a0nm. Pairs of nucleosomes had to fulfil some criteria in order to be included in the cc-distance measurements: it had to be unambiguous that the nucleosomes are ordered one after another along the DNA and the nucleosomes had to feature a certain minimal height (see below). Finally, nucleosomes localised directly at DNA termini were excluded from the cc-distance measurements.\nResults\nWe studied the positioning of reconstituted nucleosomes on AGM \u03b1 -satellite (AS) tandemly repeated DNA.\nThe cloned AGM AS\nWe constructed vectors containing AGM AS-DNA monomers as tandem repeats of up to seven. Figure\u00a01a shows the DNA-sequence of the isolated AS (Fig.\u00a01a, 1) aligned with the sequence (Figs.\u00a01a, 2) analysed earlier in nucleosome reconstitution experiments (Neubauer et al. 1986). This sequence did not contain the HindIII site that is present in the major fraction of AGM AS (Musich et al. 1982; Neubauer et al. 1986; Wu et al. 1983). The vectors containing the AS-fragments used for the reconstitution experiments (172-\u03b12 and 172-\u03b17) were NotI\/XhoI digested and analysed on the agarose gel in Fig.\u00a01b. For the reconstitution experiments, the target fragments were isolated from the agarose gels.\nFig.\u00a01Characterisation of the satellite DNA fragments used for chromatin reconstitutions. a The cloned AGM AS (1) is shown in upper case letters and aligned with the sequence (2) analysed earlier in reconstitutions experiments [adapted from Neubauer et al. 1986]. Lower case letters show flanking vector DNA. The EcoRI recognition sites are underlined. Numbering of sequence positions is according to Rosenberg et al. (1978). The sequence in brackets indicates the repetition of the 172\u00a0bp AS monomer starting and ending at the EcoRI site in our case (n\u00a0=\u00a02, 172-\u03b12 DNA; n\u00a0=\u00a07, 172-\u03b17 DNA). b Agarose-gel electrophoresis of the AS fragments after separation from the cloning vector with NotI and XhoI (lane1 172-\u03b12, 415\u00a0bp, lane 2 172-\u03b17, 1,275\u00a0bp). Note, that the target fragments were isolated from agarose gels for the reconstitution experiments. Here, the NotI\/XhoI digestion was directly loaded on the gel to also show the occurrence of a distribution of different AS repeat numbers around the major repeat length of n\u00a0=\u00a07 in the cell line containing pBluescript-KSII-\u03b17Fig.\u00a02Micrococcal nuclease (MNase) and EcoRI cleavage of reconstituted 172-\u03b17 nucleosome arrays. a Arrays (chrom) were treated with MNase for 1\u20135\u00a0min. Samples were loaded on the 1.2% agarose gel together with untreated arrays and naked 172-\u03b17 DNA, also either treated with MNase for 5\u00a0min or untreated. Untreated arrays and naked DNA migrate similarly in the agarose gel (DNA compaction and the increase in molecular weight by complexing with histones approximately compensate). First two lanes marker DNA. A photograph of the gel taken at higher light intensity is included underneath. b Magnified area of the same gel, marked by the frame in a.Arrows are shown to suggest a biphasic fragment pattern produced by the MNase. c An aliquot of the same arrays was digested with MNase for 7\u00a0min to obtain pure core particle length DNA. An aliquot of the cleavage product was digested with EcoRI and both samples were loaded on the 1.7% agarose gel (lane1 core particle DNA, lane2 EcoRI digestion) together with a 20\u00a0bp ladder. Both gels were stained with ethidium. d Densitometric trace of lane2 in c calculated with NIHimage software (version 1.63) after background subtraction\nDNA-sequencing verified the tandem orientation of the repetitive AS-monomers. Interestingly, all sequenced constructs so far obtained by self-ligation of AS-dimers or of AS-tetramers, contained tandemly oriented repeats (a total of 12 clones with various repeat numbers were sequenced), potentially due to an intolerance of the bacteria to inverted repeats. The formation of odd or even numbers of repeated monomers after self-ligation indicates that entire AS-monomer units can be excised during bacterial growth. The gel in Fig.\u00a01b also demonstrates that bacterial cell lines can contain a distribution of different AS repeat numbers centred around one prevailing length. Growing the bacteria for several days showed the stable maintenance of this distribution as well as the prevailing repeat number (data not shown). Preliminary results show that this distribution can be also used to generate successively longer repeats by isolating the largest fragments from gels (not shown).\nGel electrophoretic characterisation of chromatin reconstituted with 172-\u03b17\nA proper reconstitution of nucleosomes as well as a regular nucleosomal periodicity can be identified by a digestion with MNase. The cloned AS heptamer (172-\u03b17) was reconstituted with histones at an octamer to DNA weight ratio of 1.0. The chromatin was treated with MNase for three different periods of time, deproteinised and electrophoresed. Figure\u00a02a shows the expected protection of the nucleosomal core particle DNA, which is not observed when naked DNA was digested. Larger fragments were observed with lengths that are multiples of the AS-monomer length, suggesting the existence of regularly spaced nucleosomes. This ladder successively disappears with digestion time. On the other hand, a sequence-specific cleavage of the AGM AS may likewise have produced the ladder (Horz et al. 1983). The magnified area of the gel (Fig.\u00a02b) indeed suggested the presence of two phases and also a considerable smear superimposed the bands. In order to analyse nucleosomal binding further, pure core particle length DNA produced by MNase cleavage of the same reconstituted material was digested with EcoRI restriction endonuclease. This enzyme was chosen on the basis of earlier results that have localised the EcoRI cleavage site near the centre of a major nucleosomal positioning frame in vivo and in vitro (Musich et al. 1982; Neubauer et al. 1986; Zhang et al. 1983). The agarose gel in Fig.\u00a02c and the densitometric trace in Fig.\u00a02d demonstrate that EcoRI has produced discrete bands, indicating that the nucleosomes have occupied preferred sites along the 172-\u03b17 DNA. The largest fragment (Fig.\u00a02c, I) corresponds to core particle length (uncleaved) DNA, which must have originated from nucleosomes formed on one or different positions that do not incorporate the EcoRI site. Additionally, three smaller fragments were produced, one of which with size \u223c85\u00a0bp prevailed (Fig.\u00a02c, III). The size \u223c65\u00a0bp of another fragment (IV), when added to the 85\u00a0bp, approximately yields core particle length DNA. We therefore assume that these two fragments were produced by cleavage of the same nucleosomal core DNA. In support of this, the intensities of these two bands (Fig.\u00a02d) differ by a factor of 1.5, which roughly would be expected based on the molecular weight difference (factor of 1.3, assuming the 85 and 65\u00a0bp). Thus, the appearance of these fragments indicates a preferred nucleosomal binding frame, whose centre is located about 10\u00a0bp away from the EcoRI site, in good agreement with the earlier results (Musich et al. 1982; Neubauer et al. 1986; Zhang et al. 1983). Next, the gel indicates a second weaker positioning site by the presence of a \u223c120\u00a0bp fragment (Fig.\u00a02c, II). Here, however, the corresponding smaller fragment (expected size 27\u00a0bp to yield core particle length DNA) was faint. Based on the densitometric analysis, 50% of the nucleosomes were bound at the most preferred site. Note that the major site occurs six times in the 172-\u03b17 DNA and is interrupted at the fragment ends (since, in our case, monomers start and end at the EcoRI site).\nAFM imaging of 172-\u03b17 nucleosome arrays\nThe gel results support an at least partially regular nucleosomal organisation on the 172-\u03b17 template. It remains unclear, however, to what extent a fraction of the nucleosomes were positioned randomly. Therefore, AFM was used to determine distances between consecutive nucleosomes assembled along the 172-\u03b17 AS heptamer DNA. As a control, nucleosome arrays were reconstituted on a hexamer repeat of 208 5S rDNA to obtain a reference with well-documented properties. Tandem repeats of a 208\u00a0bp long DNA containing 5S rDNA of L. variegatus are often used as a model system for evenly spaced nucleosome arrays (Carruthers et al. 1998; Simpson et al. 1985) and their distribution of internucleosomal distances had been determined by AFM (Allen et al. 1993; Mechelli et al. 2004; Nikova et al. 2004; Yodh et al. 2002).\nScanning was done on air dried samples which ensured that all nucleosomes in the arrays were clearly distinguishable. The chromatin was immobilised at conditions that trap DNA on the surface irreversibly (Bussiek et al. 2003). We believe (and provide evidence) that this prevents the sample from extensive structural distortions once bound. Figure\u00a03a gives an example of the scans obtained and Fig.\u00a03b\u2013i shows zooms of individual nucleosome arrays reconstituted with 172-\u03b17 (B-G) and with 208-6 DNA (H, I). Adsorption of the arrays to the surface under low salt conditions yielded the expected extended conformations (Fig.\u00a03b\u2013e; H and I) and more compact conformations were observed in the presence of MgCl2 (Fig.\u00a03f,\u00a0g). Visual inspection suggested spaced nucleosomes on both sequences, although frequently nucleosomes appeared closely stacked on each other (arrows in Fig.\u00a03). Height measurements of those particles identified as nucleosomes yielded 2.6(\u00b10.7 SD)\u00a0nm, in agreement with previous measurements under the same scanning conditions in air (Bussiek et al. 2005). All particles with heights below 1.9\u00a0nm were rejected in order to ensure that only properly folded nucleosomes were considered in the data analysis. The DNA was not fully saturated with nucleosomes since we expected to identify \u2018gaps\u2019 corresponding to unoccupied repeats, if preferred nucleosomal binding sites existed. For comparison, arrays were also scanned in liquid, which yielded the same extended shapes in low salt buffer (not shown). A quantitative analysis after scanning in liquid was, however, not useful because nucleosomes and DNA were not imaged with the required sharpness. On contrary, scanning of mononucleosomes in liquid yielded high quality images, as shown below. We explain this difference by a more unstable scanning process of nucleosome arrays, because a local conglomeration of nucleosomes has the effect of a locally higher surface roughness.\nFig.\u00a03a\u2013i Atomic force microscopy imaging in air of nucleosome arrays. The chromatin was reconstituted at a nominal octamer to DNA weight ratio of 0.5 and adsorbed onto the surface in Hepes-NaOH buffer (10\u00a0mM, pH 8.0). a Overview scan of 172-\u03b17 arrays adsorbed in the buffer additionally containing 10\u00a0mM NaCl. b\u2013g Zooms of individual 172-\u03b17 arrays [adsorption buffer\u00a0+\u00a010\u00a0mM NaCl (b, c), no additional salt (d, e) and 10\u00a0mM NaCl, 2\u00a0mM MgCl2 (f, g)]. The arrows mark nucleosomes in a close contact. h, i Examples of 208-6 nucleosome arrays adsorbed in buffer\u00a0+\u00a010\u00a0mM NaCl. k Linear compaction of the DNA with number of NCPs per array (nncp). Blue, closed symbols, continuous line indicate 172-\u03b17 arrays in buffer\u00a0+\u00a010\u00a0mM NaCl, blue, open symbols, dashed line indicate 172-\u03b17 arrays in buffer without additional NaCl, green indicates 208-6 arrays in buffer\u00a0+\u00a010\u00a0mM NaCl, red indicates 172-\u03b17 arrays in buffer\u00a0+\u00a010\u00a0mM NaCl, 2\u00a0mM MgCl2. Error bars correspond to standard errors\nInternucleosomal distances were measured in a way that allowed a direct comparison with the expected nucleosomal repeat lengths of 172 and 208\u00a0bp. This was accomplished by determining the wrapped nucleosomal DNA length for the given experimental conditions. Low salt concentrations during the surface adsorption were selected so that nucleosomes formed extended conformations. As a consequence, linker DNAs could be well identified allowing for precise length measurements. However, it has to be taken into account that, due to repulsive electrostatic forces under low salt conditions, core particle DNA may partially unwrap from the histone surface to become linker DNA (Hamiche et al. 1996), a phenomenon that we also observed under the present experimental conditions (Bussiek et al. 2005). We therefore estimated the average length of wrapped DNA (Lncp) from the linear compaction of the DNA by complexing into nucleosomes. The 172-\u03b17 and the 208-6 nucleosome arrays were deposited onto the mica surface under the same low salt conditions (10\u00a0mM Hepes-NaOH, pH 8.0, 10\u00a0mM NaCl). All arrays with an unambiguous number and order of nucleosomes were included in these length measurements (a total of 106). Note that Fig.\u00a03f\u00a0and\u00a0g give examples of excluded arrays. These are shown to demonstrate the expected compaction in the presence of Mg2+. Figure\u00a03k shows that the nucleosome arrays formed on the DNA templates indeed shortened linearly with the number of nucleosomes nncp. Lfree measured as described in Sect.\u00a0\u201cMaterials and methods\u201d rather than the entire array length was plotted versus nncp to be able to include the length of the naked DNA fragments as data points for the calculation of linear regressions (433\u00a0nm for 172-\u03b17 DNA and 442\u00a0nm for 208-6 DNA). The slopes of the calculated regression lines, giving the wrapped DNA length, were about identical for the two nucleosomal arrays and removal of the NaCl from the buffer of one other sample had no effect, thus all three values obtained under these low salt conditions were averaged. This yielded a wrapped DNA length Lncp = 42\u00a0nm (or 123\u00a0bp for 0.34\u00a0nm\/bp), significantly less than the known core particle length of 147\u00a0bp. A wrapped DNA length of \u223c120\u00a0bp could be confirmed independently by measurements of mononucleosomes (see below). To further control the measurement accuracy, the wrapped DNA length was determined for a sample prepared in the presence of MgCl2, which yielded 48\u00a0nm (or 141\u00a0bp), very close to the core particle length.\nNext we determined the distribution of the centre-to-centre (cc) internucleosomal distances (Fig.\u00a04). The data reveal a non-random nucleosomal positioning along the 172-\u03b17 DNA. This is indicated by a clearly separated initial main peak followed by a recurring preference for a larger distance, consistent with the existence of preferred binding sites, a fraction of which was unoccupied (Yodh et al. 2002). The distribution includes three different samples with a combined average number of nucleosomes nncp-av = 4.2. Next, the cc-distances were plotted for only those complexes carrying less than five nucleosomes to obtain a subset sample with more unoccupied binding sites. Figure\u00a04b shows that the first recurring peak now is more accentuated and sharper compared to the distribution in Fig.\u00a04a. Even a second and a third recurring peak are suggested, but not statistically secure.\nFig.\u00a04Distributions of cc-distances in 172-\u03b17 and 208-6 nucleosome arrays, measured by AFM after adsorption under low salt conditions. acc-distances in 172-\u03b17 arrays measured in three samples with a combined average number of NCPs per array nncp-av = 4.2. bcc-distances for exclusively 172-\u03b17 nucleosome arrays with number of NCPs nncp <5, corresponding to nncp-av =3.2. ccc-distances measured in 208-6 nucleosome arrays (nncp-av =3.2). Numbers and arrow heads indicate cc-distances expected for a uniform nucleosomal phasing with the monomer lengths of 172\u00a0bp (a,\u00a0b) and 208\u00a0bp (c), as calculated assuming Eq.\u00a0(2). a is the number of unoccupied repeats between successive nucleosomes. The distance distribution begins at a value of 11\u00a0nm, the diameter of the NCP, meaning that a few measured distance values smaller than 11\u00a0nm were added to the first bin. The bin width of 12\u00a0nm corresponds to the length difference between monomer lengths of 172 and 208\u00a0bp (36\u00a0bp). Error bars indicate the square root of the number of observations in each bin\nFor comparison, nucleosome arrays were reconstituted with 208-6 DNA, expected to produce defined regular nucleosome arrays due to positioning in the 5S rDNA sequence (Carruthers et al. 1998; Meersseman et al. 1991; Yodh et al. 2002) and thus also serving as a control for appropriate experimental conditions. The reconstitution with the 208-6 DNA yielded an average number of nucleosomes that exactly matched the subset sample in Fig.\u00a04b (nncp-av = 3.2). Figure\u00a04c shows a sharply defined preference for cc-distances around the third bin (35\u201347\u00a0nm) for this sample. The peak, as also seen in Fig.\u00a04a, is differentiated from a recurring peak and additionally from a distinguishable preference for very small cc-distances (those found in the first bin). These correspond to the many contacting nucleosome pairs. Close contacts in the 172-\u03b17 arrays are as well reflected in a high number of counts in the first bin in Fig.\u00a04a although not as a separated peak. Examples can be seen in Fig.\u00a03b\u2013d. Importantly, positioning in the 5S rDNA led to a sharp initial peak, consistent with other reported AFM data (Mechelli et al. 2004; Nikova et al. 2004; Yodh et al. 2002).\nOn the basis of the average wrapped DNA length (Lncp \u223c120\u00a0bp), expected cc-distances (ccexp) for monomer lengths (Lmono) of 172 and 208\u00a0bp (58 and 71\u00a0nm, respectively) can be calculated assuming that where a is the number of unoccupied repeats between successive nucleosomes. These distances and their positions along the x-axes are indicated by numbers and arrowheads in Fig.\u00a04, indicating a good agreement with measured (and assignable) peak maxima. In the case of a uniform phasing along the AGM AS repeats, the initial peak for the 172-\u03b17 nucleosome arrays should be centred at about 30\u00a0nm (in the second bin) and not exceed values of \u223c40\u00a0nm, taking into account that variation in the wrapped DNA length likely broadens the cc-distance distribution. For an estimate of this variation we can assume a standard deviation of \u00b128\u00a0bp (corresponding to \u00b19\u00a0nm), as determined in 172-\u03b12 mononucleosomes (see below). The observed distances clearly exceed this boundary and actually show a high occurrence of internucleosomal separations of up to three times that expected for uniformly phased nucleosomes. This is different in the 208-6 nucleosome arrays: here, the width of the initial main peak suggests that most nucleosomes are bound at or near the same positioning site in the 5S rDNA repeats (neglecting close contacts). It is known for the the 5S rDNA that alternative positioning sites around the main site exist (e.g. Meersseman et al. 1991). The comparison of both sequences shows that multiple positioning in the 172-\u03b17 DNA leads to a higher variation between internucleosomal spacings, which can be due to a higher proportion of nucleosomes positioned out of the main phase and by larger distances between the alternative sites. Apart from this observation, the clear separation of the main and first recurring peaks observed for both sequences indicates clearly the strong sequence-dependence of the nucleosome array formation on repetitive AGM-AS.\nAFM imaging of 172-\u03b12 mononucleosomes\nTo further quantify the nucleosomal positioning along the AGM AS, mononucleosomes were reconstituted with the AS dimer containing 172-\u03b12 DNA (415\u00a0bp). Here, the complexes were imaged in liquid (10\u00a0mM Hepes-NaOH, pH 8.0, 10\u00a0mM NaCl) and the mica surface was modified with the same amount of polylysine that was used for immobilising the nucleosome arrays to ensure constant adsorption conditions. Figure\u00a05a shows a representative overview image displaying different kinds of complexes: mononucleosomes, a few dinucleosomes and uncomplexed DNA. Figure\u00a05b\u2013e shows zooms of individual complexes with different nucleosomal positions along the 172-\u03b12 fragment.\nFig.\u00a05Atomic force microscopy imaging in liquid of mononucleosomes reconstituted with 172-\u03b12 DNA. Scanning conditions were 10\u00a0mM Hepes-NaOH, pH 8.0, 10\u00a0mM NaCl. a Overview scan of a sample reconstituted at a nominal octamer to DNA weight ratio of 0.5. b\u2013e Zooms of individual mononucleosomes. f Height distribution of nucleosomal particles. Frequencies were fitted with the sum of two Gaussian functions to derive mean height values (see text). g Distribution of wrapped nucleosomal DNA lengths Lncp, analysed separately for particles <3.0\u00a0nm height (white bars) and \u22653.0\u00a0nm height (black bars). Mean values and standard deviation of Lncp were calculated by fitting Gaussian functions to both datasets (see text). For comparison with Fig.\u00a03, note that measured height values are larger in liquid than in air, presumably due to reduced sample\u2013probe interactions\nWe first characterised the heights of the mononucleosomes. The height values were distributed into two discrete peaks centred at 2.7 and 4.5\u00a0nm (Fig.\u00a05f). The larger peak value is only slightly smaller than the expected height of 5.0\u00a0nm for the NCP and also agrees with our previous height measurements under the same scanning conditions (Bussiek et al. 2005). Seventy-four percent of the small 2.7\u00a0nm particles were found at DNA termini (an example is given in Fig.\u00a05e). The sample was divided into two subsets, one containing the particles with less than 3.0\u00a0nm, the other those above 3.0\u00a0nm height. The wrapped DNA lengths (Lncp) of both subsets were determined after contour length measurements of the mononucleosomal fragments: the lengths of free DNA outside the nucleosomes were estimated according to Eq.\u00a0(1). These lengths were converted to base pairs, taking into account that measured DNA lengths on AFM images can deviate slightly from the expected length of 0.34\u00a0nm\/bp on PL-mica (Bussiek et al. 2003). A measurement of uncomplexed 172-\u03b12 DNAs, present in the same sample preparation, gave a mean contour length of 134.4\u00a0\u00b1\u00a04.3 (SD)\u00a0nm or 0.32\u00a0nm\/bp. For each individual complex, the free DNA length in base pairs was subtracted from the total fragment length of 415\u00a0bp to obtain Lncp. The small and the regular particles can be as well discriminated in terms of this length, since both samples yielded two distinct distributions of Lncp (Fig.\u00a05g). The peak centres of these distributions were at 119\u00a0\u00b1\u00a028 (SD)\u00a0bp for the larger particles (\u22653.0\u00a0nm height) and only 84\u00a0\u00b1\u00a037 (SD) bp for the smaller ones (<3.0\u00a0nm height) (P\u00a0< 0.0001). Importantly, the length of 119\u00a0bp is in close agreement with the 123\u00a0bp determined for the 172-\u03b17 and the 208-6 nucleosome arrays under the same salt conditions. This also provides some evidence that drying the arrays did not extensively distort the nucleosome conformation which we attribute to the irreversible trapping of the DNA to the PL-mica so that the nucleosomes become \u201cfixed\u201d after the binding to the surface.\nNucleosomal positions along the 172-\u03b12 AS dimer DNA were determined exclusively for mononucleosomes with particle heights larger than 3.0\u00a0nm. Positions were quantified as the ratio r, i.e. the distance from the nucleosomal centre to the nearest DNA terminus divided by the total mononucleosomal fragment length (Kepert et al. 2003). The distribution of r shown in Fig.\u00a06 clearly reveals that nucleosomes are preferably localised around the fragment centre, indicated by a defined peak at r\u00a0=\u00a00.4\u20130.5. An r-value of 0.5\u00a0\u00b1\u00a00.05 corresponds to a nucleosomal frame whose centre is located next to the EcoRI cleavage site at sequence position 37 (\u00b120)\u00a0bp (see Fig.\u00a01). Thus, taking account the error of r, the AFM measurement reveals the same prevailing binding site that was estimated by EcoRI cleavage (nucleosomal centre about 10\u00a0bp away from the EcoRI site, see above). The remaining nucleosomes (55%) were rather evenly distributed over the eight other bins (with some additional weak minor preferences) and there was no preference for regular size nucleosomes to be aligned with DNA termini.\nFig.\u00a06Positioning of nucleosomes along 172-\u03b12 DNA. Different nucleosomal positions near the termini that would correspond to r-values between 0 and 0.1 could not be distinguished due to the resolution limit. Therefore, the occurrences of r-values equal to 0 were evenly distributed over the first two bins (white bars). The error of r in the peak range approximately equals the bin size, since an error \u0394r\u00a0\u223c\u00a010% could be expected based on the measured standard deviation of the total mononucleosomal fragment length (\u00b1\u00a07.7%)\nDiscussion\nNucleosomal positioning along repetitive AGM AS\nWe reconstituted nucleosomes with dimers (172-\u03b12) and heptamers (172-\u03b17) of repetitive AGM AS by the salt dialysis method. Enzymatic digestions and atomic force microscopy were used to characterise the nucleosomal organisation along this DNA. The results demonstrate the formation of a well-defined (sequence-dependent) nucleosomal arrangement, essentially similar to the 5S rDNA model system. In the AGM AS arrays, however, the variation in the separation between neighbouring nucleosomes was rather large, and evenly spaced nucleosomes occurred only to a limited extent.\nA partial digestion of 172-\u03b17 nucleosome arrays with micrococcal nuclease (MNase) yielded a fragment ladder documenting the presence of phased rather than random nucleosome positions on 172-\u03b17 DNA. This result is very similar to MNase digestions of AGM AS chromatin in cell nuclei (Musich et al. 1982). Core particle length DNA derived from 172-\u03b17 nucleosome arrays was further digested with EcoRI. This produced discrete secondary fragments and thus verified the occupation of preferred nucleosomal binding sites. One of these sites was prevailing with a position consistent with published results (Musich et al. 1982; Neubauer et al. 1986; Zhang et al. 1983). One additional minor frame could be detected, which may explain a suggested biphasic cleavage pattern produced by the MNase. Musich et al. (1982) concluded that the MNase digestion of AGM AS chromatin depends on the nucleosomal organisation which includes the possibility that a sequence-specific cleavage coincidentally occurs in the internucleosomal linkers between nucleosomes bound at the mostly preferred site (Horz et al. 1983; Musich et al. 1982). This may lead to a selective representation of the fraction of phased nucleosomes and obscure deviations thereof. Although this source of error must be taken into account, the similar results for cellular AGM AS chromatin and reconstituted nucleosome arrays indicate similar properties, i.e. at least fractions of the nucleosomes are regularly arranged.\nAtomic force microscopy imaging was used to detect the spacing between all neighbouring nucleosome pairs in a sample. This circumvents possible artefacts caused by enzymatic analyses, although the precision of localising the nucleosomes is lower. Centre-to-centre (cc) internucleosomal distances measured by AFM imaging not only depend on nucleosomal positions relative to the DNA sequence but also on internucleosome interactions (which should be reduced under the low salt conditions in our measurements) and variations in the amount of wrapped nucleosomal DNA. An example is given by increasing cc-distances due to a release of nucleosomal DNA from the histone surface after removal of linker histones (Zlatanova et al. 1998). Especially the wrapped DNA length is susceptible to experimental conditions: a prevailing cc-distance of \u223c20\u00a0nm was previously measured in 5S positioning sequence repeat (208-12) nucleosome arrays that were adsorbed to naked mica in the presence of Mg2+ (Nikova et al. 2004). These measurements used arrays not fixed with glutaraldehyde. Fixed 208-12 nucleosome arrays adsorbed to either spermidine- or AP-mica under low salt conditions gave cc-distances of \u223c30\u00a0nm (Mechelli et al. 2004; Yodh et al. 2002). Our measurements in low salt conditions using PL-mica and unfixed material yielded a larger cc-distance for the 208-6 nucleosome arrays of 35\u201347\u00a0nm. An estimation of the wrapped DNA length at our conditions yielded \u223c120\u00a0bp, corresponding to \u223c1.4 superhelical turns around the octamer [full core particle length of 147\u00a0bp equals 1.7 turns (Davey et al. 2002; Furrer et al. 1995; Hamiche et al. 1996; Luger et al. 1997)]. The expected cc-distance for a nucleosomal repeat length of 208\u00a0bp on the basis of the 120\u00a0bp of wrapped DNA is \u223c40\u00a0nm, in agreement with the results obtained for the 208-6 arrays. The expected distances for additional unoccupied repeats matched the position of recurring peaks in the cc-distance distributions measured for the 208-6 and the 172-\u03b17 nucleosome arrays.\nEssentially, the shapes of the cc-distance distributions for the 208-6 and the 172-\u03b17 DNA shown in Fig.\u00a04b\u00a0and\u00a0c are very similar, which points to a sequence dependence of the nucleosomal organisation along repetitive AGM AS similarly strong as on the 5S sequence. However, in the case of the 172-\u03b17 DNA, the initial peak of the cc-distance distribution, corresponding to the distance between a nucleosome and its nearest neighbour located on an adjacent repeat, is significantly broader and shifted to larger distances than could be expected for evenly spaced nucleosomes. The distributions still show a high frequency of distances up to \u223c60\u00a0nm (corresponding to \u223c145\u00a0bp of linker DNA length), almost three times the linker length expected for a uniform phasing with the AGM AS repeats (18\u00a0nm or 52\u00a0bp, based on the 120\u00a0bp of wrapped nucleosomal DNA). Thus, many nucleosomal positions were separated by up to about 1.5 AS monomer lengths. The loading with nucleosomes did not significantly affect this result (compare Fig.\u00a04a,\u00a0b). 208-6 nucleosome arrays showed the expected distance distribution for nucleosomes predominantly bound at and near the main phase, showing that the broad initial peak seen in the 172-\u03b17 arrays was not simply due to a limited measurement accuracy or inappropriate reconstitution or sample preparation conditions. Thus, a strictly regular nucleosomal arrangement occurred in the AGM AS only to limited extent and variation in possible internucleosomal spacings is rather large. We conclude that the number of alternative sites is nevertheless small, because the gelelectrophoresis showed two identifiable sites and the initial peak in the cc-distance distribution is sharply separated from the first recurring peak.\nThe mapping of nucleosomal positions by AFM imaging of the 415\u00a0bp long 172-\u03b12 mononucleosomes was fully consistent with the occurrence of the known major positioning site, in addition to the identification of the same site in the nucleosome arrays by EcoRI digestion. The frequency of nucleosomes found at or near this major frame (45%) is lower than found by in vitro reconstitutions using a 250\u00a0bp AGM AS fragment (80%) (Neubauer et al. 1986), which is explained by the length difference of the DNA fragments used. The frequency is closer to the observed occupancy of the major site in vivo (35 %) (Zhang et al. 1983) and also closer to that estimated by gelelectrophoretic quantification of the EcoRI digested mononucleosomal DNA derived from the 172-\u03b17 arrays (upper limit of 50%). The DNA fragment used here for the reconstitution of the mononucleosomes is the minimal length that contains the AGM AS sequence uninterrupted, such that all possible binding sites are present. However, when comparing the positioning observed here and in vivo, it must be taken into account that a few possible binding sites occur twice in the 172-\u03b12 (i.e. those which do not incorporate the EcoRI site) and that some additional sites are present that include vector DNA at the ends (33 and 37\u00a0bp, respectively). In general, positioning on small DNA fragments can be influenced by a preference for DNA-ends (which was not observed here, as shown in Fig.\u00a06) and the length of the fragment used.\nA surprising feature was a bimodal height distribution of mononucleosomal particles reconstituted with the 172-\u03b12 DNA. The two height values were correlated with distinguishable wrapped DNA lengths. Nikova et al. (2004) obtained a very similar height distribution by AFM imaging of subsaturated 208-12 nucleosome arrays and presented evidence that particles with only \u223c75\u00a0bp of wrapped DNA and \u223c2.5\u00a0nm height correspond to sub-nucleosomal particles that do not contain the full histone octamer. In the present study, small particles of similar dimension (wrapped length of 85\u00a0bp and 2.7\u00a0nm height) were predominantly located at the termini of the 172-\u03b12 DNA (74%). In contrast, no preferential binding to termini was found for nucleosomes of regular size (4.5\u00a0nm height). We may speculate that this high frequency of the small particles at the fragment termini is as well associated with sequence properties of the AGM AS. In our case, the AS sequence starts and ends at the EcoRI site, which is located near the centre of the major binding site. This implies that the end regions of the fragment contain only parts of this binding site. Assuming that multiple DNA\u2013histone interaction along the entire core particle length are responsible for the binding specificity (Neubauer et al. 1986), it may be possible that histones interact preferentially even with parts of the major site. Then, a complete nucleosome assembly could be impeded at the fragment termini due to a lack of sufficient amounts of DNA in the required direction, leading to smaller particles.\nRelevance of AGM AS for centromeric chromatin structures\nThe kinetochore protein CENP-B is supposed to be involved in nucleosomal positioning in human AS (Tanaka et al. 2005). A structural motive consisting of the AS monomer, nucleosomal positioning and founding kinetochore protein binding may be involved in specifying the location of centromeric function. CENP-B is not present at the centromeres of AGM and therefore AS arrays of this species might compensate for the absence of CENP-B by influencing nucleosomal positioning by their sequence alone (Goldberg et al. 1996). We show here that the AGM AS indeed has a profound influence on the arrangement of oligonucleosomal arrays. This arrangement has similarities to the 5S rDNA model system, although multiple positioning leads to larger variation of internucleosome spacings in the AGM AS. The spacing of nucleosomes is expected to contribute to the higher order structuring of chromatin, therefore it is of interest, for example with regard to the design of particular synthetic chromatin structures, to what extent the in vitro produced nucleosomal arrangement along a DNA template resembles that occurring in vivo. Our MNase digestions suggested a high fraction of evenly spaced nucleosomes, similar to digestions of AGM AS in cell nuclei (Musich et al. 1982) whereas the AFM results are consistent with more detailed studies in vivo which demonstrated the occupation of different minor binding sites in addition to the major site (Zhang et al. 1983). The occupancy of the major binding site was in the range of 45\u201350% in our analysis which is also in a quite good agreement with the result of the cited in vivo work (35 %). This may further support that the AS sequence contributes significantly to the determination of AS chromatin and probably to centromeric chromatin structures. The DNA constructs built in this study will give the opportunity to investigate further the structure and biophysical properties of AS chromatin and centromeric chromatin. For example, it will be important to compare the nucleosomal arrangement on repetitive AGM and human AS (with and without CENP-B boxes). Nucleosomal phasing also occurs on human AS sequences (Tanaka et al. 2005; Yoda et al. 1998) and a preferred positioning site found in immunoprecipitated human centromeric AS indeed corresponded to that occurring in AGM AS (Vafa and Sullivan 1997).","keyphrases":["nucleosome arrays","green monkey","atomic force microscopy","alpha-satellite dna","centromere","chromatin","nucleosome positioning","5s rdna"],"prmu":["P","P","P","P","P","P","P","P"]}
{"id":"Oecologia-3-1-1915600","title":"Soil microorganisms control plant ectoparasitic nematodes in natural coastal foredunes\n","text":"Belowground herbivores can exert important controls on the composition of natural plant communities. Until now, relatively few studies have investigated which factors may control the abundance of belowground herbivores. In Dutch coastal foredunes, the root-feeding nematode Tylenchorhynchus ventralis is capable of reducing the performance of the dominant grass Ammophila arenaria (Marram grass). However, field surveys show that populations of this nematode usually are controlled to nondamaging densities, but the control mechanism is unknown. In the present study, we first established that T. ventralis populations are top-down controlled by soil biota. Then, selective removal of soil fauna suggested that soil microorganisms play an important role in controlling T. ventralis. This result was confirmed by an experiment where selective inoculation of microarthropods, nematodes and microbes together with T. ventralis into sterilized dune soil resulted in nematode control when microbes were present. Adding nematodes had some effect, whereas microarthropods did not have a significant effect on T. ventralis. Our results have important implications for the appreciation of herbivore controls in natural soils. Soil food web models assume that herbivorous nematodes are controlled by predaceous invertebrates, whereas many biological control studies focus on managing nematode abundance by soil microorganisms. We propose that soil microorganisms play a more important role than do carnivorous soil invertebrates in the top-down control of herbivorous ectoparasitic nematodes in natural ecosystems. This is opposite to many studies on factors controlling root-feeding insects, which are supposed to be controlled by carnivorous invertebrates, parasitoids, or entomopathogenic nematodes. Our conclusion is that the ectoparasitic nematode T. ventralis is potentially able to limit productivity of the dune grass A. arenaria but that soil organisms, mostly microorganisms, usually prevent the development of growth-reducing population densities.\nIntroduction\nRoot herbivores play an important role in shaping the composition of natural plant communities (Brown and Gange 1990). Nematodes and insects represent the vast majority of the belowground herbivores (Brown and Gange 1990; Stanton 1988). Nematodes are more abundant than soil insects, and in some grassland ecosystems, nematodes are the dominant herbivores (Ingham and Detling 1986). Root-feeding nematodes have been estimated to take up as much as one quarter of the net primary production of grassland vegetation (Stanton 1988), and they affect plant quality (Davis et\u00a0al. 1994; Troelstra et\u00a0al. 2001), plant diversity, and vegetation succession (de Deyn et\u00a0al. 2003). Root-feeding nematodes can also indirectly affect plant performance by their influence on bottom-up and top-down control of aboveground invertebrate herbivores (Bezemer et\u00a0al. 2005). However, in spite of the increasing knowledge on the significant role of belowground herbivores in the control of plant abundance and plant community composition, relatively few studies have investigated which factors control the abundance of the belowground herbivores in natural ecosystems (Strong et\u00a0al. 1996, 1999).\nHerbivore abundance can be influenced by natural enemies (top-down), by the host plant (bottom-up), and by competition with other herbivores (horizontal control). In (semi) natural ecosystems, most studies on the control of root-feeding nematodes have focused on plant quality (Yeates 1987), interspecific competition (Brinkman et\u00a0al. 2004, 2005), plant community composition (de Deyn et\u00a0al. 2004), plant succession and soil conditions (Verschoor et\u00a0al. 2002), and mycorrhizal fungi (de la Pe\u00f1a et\u00a0al. 2006). Soil food web models assume root-feeding nematodes to be controlled by carnivorous nematodes and microarthropods (Hunt et\u00a0al. 1987; Neutel et\u00a0al. 2002). However, most biological control studies in agricultural systems focus on managing nematode abundance by parasitic soil microorganisms (Kerry 2000; Sikora 1992) or mycorrhizal fungi (Hol and Cook 2005), suggesting that root-feeding nematodes are mainly controlled by microorganisms. Therefore, previous studies show little agreement and do not clearly predict how root-feeding nematodes will be controlled in natural ecosystems.\nEmpirical data for top-down mechanisms are rare for terrestrial ecosystems relative to the many studies in aquatic systems (Walker and Jones 2001). In general, trophic cascades have been argued to be less common on land than in water (Polis and Strong 1996). Nevertheless, there is empirical evidence supporting the existence of trophic cascades in terrestrial plant\u2013predator\u2013prey systems (Schmitz et\u00a0al. 2004). Tritrophic systems of plants, aboveground insect herbivores, and their natural aboveground enemies are the best-studied terrestrial examples of top-down and bottom-up herbivore controls (Carson and Root 1999; Rosenheim 1998). Below ground, tritrophic interactions may not essentially differ from what is known above ground (Bezemer and van Dam 2005), although rates of dispersal of organisms and chemical compounds will be lower than is mostly the case above ground (Rasmann et\u00a0al. 2005; van der Putten 2003). Therefore, the challenge is, similar to that above ground (Schmitz et\u00a0al. 2004), to assess what controls the abundance of root herbivores. This knowledge will enhance our understanding of belowground multitrophic interactions and their influences on plant performance and plant community composition.\nIn the present study, the role of microarthropods, nematodes, and microorganisms in controlling the abundance of the root-feeding nematode Tylenchorhynchus ventralis (Loof 1963) Fortuner and Luc (synonym Telotylenchus ventralis) was experimentally compared. This nematode is a polyphagous ectoparasite, which means that it is a quite generalistic root feeder that penetrates outer cortical cells with its stylet to collect and ingest cell contents (Yeates et\u00a0al. 1993). T. ventralis is a root parasite of the dominant coastal foredune grass Ammophila arenaria (Marram grass). In field soil, T. ventralis reaches densities that are 80 times lower than achieved when inoculated into sterilized dune soil (de Rooij van der Goes 1995). Whereas T. ventralis can strongly reduce growth of A. arenaria in sterilized soil, field densities in nonsterilized soil are too low to directly influence plant performance (de Rooij van der Goes 1995). The roots of A. arenaria are parasitized by an array of herbivorous nematodes ranging from ectoparasites to sedentary endoparasites (de Rooij van der Goes et\u00a0al. 1995). The control mechanisms of root herbivorous nematodes in dunes appear to highly depend on the feeding type of the nematode, and even on the species of nematode. Whereas the sedentary root knot nematode Meloidogyne maritima (Jepson 1987) Karssen, van Aelst and Cook is controlled by competition (Brinkman et\u00a0al. 2005), the sedentary cyst nematode Heterodera arenaria (Cooper 1955) Robinson, Stone, Hooper and Rowe appears to be controlled by bottom-up processes (van der Stoel et\u00a0al. 2006). The migratory endoparasitic root lesion nematode Pratylenchus penetrans (Cobb 1917) is controlled by arbuscular mycorrhizal fungi (de la Pe\u00f1a et\u00a0al. 2006). Thus far, the factors that control the ectoparasitic nematode T. ventralis associated with A. arenaria are unknown.\nPrevious studies showed bottom-up control of A. arenaria to occur only when the plants were severely growth reduced (de Rooij van der Goes et\u00a0al. 1995). Alternatively, competition with cyst and root lesion nematodes is a potential factor controlling ectoparasitic nematodes (Eisenback 1993). However, endoparasitic nematodes did not control abundance of T. ventralis (Brinkman et\u00a0al. 2004). In the present study, the top-down factors that may be involved in the control of T. ventralis populations were investigated in order to determine how belowground trophic interactions might influence plant performance and vegetation composition.\nTo assess the top-down control of T. ventralis, three experiments were performed. The aim of experiment 1 was to elucidate the potential top-down control of T. ventralis by the dune soil community. In experiment 2, the particular role of microorganisms was investigated by selective elimination of soil fauna (nematodes and microarthropods). In experiment 3, the hypothesis that emerged from experiment 2, that soil microorganisms are the main cause of top-down control of T. ventralis, was tested. Here, we applied Koch\u2019s postulates by collecting microorganisms, nematodes, and microarthropods from dune soil and adding them to sterilized soil inoculated with T. ventralis. New evidence that top-down control by soil microorganisms is the most important factor controlling the abundance of ectoparasitic nematodes in dune soil is presented and discussed.\nMaterials and methods\nSoil\nIn summer 2003, soil samples were collected from mobile and stable foredunes at Voorne, The Netherlands (Latitude 51\u00b055\u2032N to Longitude 04\u00b005\u2032E). The samples were collected along six transects parallel to the beach and 50\u00a0m apart. At each sampling station in the mobile and stable dune, 60\u00a0kg of soil was collected from the youngest root zone of A. arenaria. The soil was sieved (0.5-cm mesh size) to remove plant parts and debris and stored in plastic bags at 4\u00b0C until used (van der Stoel et\u00a0al. 2002).\nPlants\nSeeds of A. arenaria were collected from the same foredune area and stored dry until used. In order to obtain seedlings, the seeds were germinated for 2\u00a0weeks on moist glass beads in a climate room at a 16\/8\u00a0h light\/dark regime at a temperature of 25\/15\u00b0C, respectively. When the first leaf was 2\u20133\u00a0cm long, the seedlings were transplanted to 1.5-l plastic pots filled with 1,500\u00a0g of dune soil. In each pot, four seedlings of A. arenaria were planted, and the soil surface was covered with aluminum foil to protect the soil from desiccation. The soil moisture was adjusted to 10% w\/w and maintained at this level throughout the experiment by weighing the pots twice a week and resetting their initial weight using demineralized water. Once a week, full-strength Hoagland nutrient solution was added at a weekly rate of 12.5\u00a0ml\u00a0pot\u22121 for the first 3\u00a0weeks and then 25\u00a0ml\u00a0pot\u22121, subsequently (Brinkman et\u00a0al. 2004). This nutrient supply rate was effective to compensate for effects of nutrient release as a result of soil sterilization in dune soil (Troelstra et\u00a0al. 2001; van der Putten et\u00a0al. 1988). The experiments were carried out in a greenhouse at a day temperature of 21\u00b0C\u00a0\u00b1\u00a02\u00b0C (day length 16\u00a0h) with additional light (to maintain a minimum of 225\u00a0\u03bcmol\u00a0m\u22122\u00a0s\u22121 PAR with SON-T Agro lamps) and a night temperature of 16\u00b0C. These temperatures are comparable with summer conditions in the field and are optimal for both plant and nematode development (Troelstra and Wagenaar unpublished results).\nExperiments\nExperiment 1: multiplication of T. ventralis in sterilized and nonsterilized dune soil\nIn this experiment, the effect of soil origin (mobile and stable dunes) and soil organisms on multiplication of the ectoparasitic nematode T. ventralis was tested. Half of the soil was sterilized by gamma irradiation at an average dose of 25\u00a0kGray, which eliminates microorganisms and nematodes effectively from dune soil (de Rooij van der Goes et\u00a0al. 1998). One week after the seedlings of A. arenaria had been transplanted, half the pots were inoculated with 50 T. ventralis\u00a0pot\u22121. The noninoculated pots served as controls for effects of T. ventralis on plant biomass production. There were six replicates of each treatment.\nExperiment 2: reproduction of T. ventralis in partially sterilized soil\nMultiplication of T. ventralis was studied in soils from which microarthropods and nematodes had been selectively removed by stirring the soil for 15\u00a0min at 1,500\u00a0rpm. This method has proven to effectively kill the soil fauna (de Rooij van der Goes et\u00a0al. 1998). We confirmed this by inspecting the soil following stirring and found no live nematodes or microarthropods. The experiment was carried out as described above, but now the soil was completely sterilized by gamma irradiation (average 25\u00a0kGray), partially sterilized by stirring to remove the soil fauna, or nonstirred in order to have a nonsterilized control soil. Each soil was inoculated with 0, 25, or 250 T. ventralis pot\u22121 in order to examine any interaction between the effect of type of soil sterilization and nematode inoculation density. There were six replicates of each treatment.\nExperiment 3: reinoculation of microorganisms, nematodes, and microarthropods into sterilized soil with T. ventralis\nIn order to completely apply Koch\u2019s postulates, microarthropods, nematodes, and microorganisms were extracted from the soil of mobile and stable coastal foredunes and inoculated alone and in all factorial combinations into sterilized dune soil. Then, seedling plants of A. arenaria were grown as in the previous experiment, and every pot was inoculated with 50 T. ventralis. All treatments were carried out in six replicates.\nThe microorganisms were obtained by shaking soil samples of 100\u00a0g with demineralized water (1:1\u00a0w\/w) for 10\u00a0min and filtering the supernatant through a 20-\u03bcm mesh (Klironomos 2002). Prepared microbial filtrate contained no nematodes, but bacteria and fungi had readily passed through the filter. The pots with microorganisms were inoculated with 10\u00a0ml of the filtrate, which was 1\/15 of the original soil density. For each pot, nematodes had been extracted from 1,500\u00a0g of nonsterile soil by Cobb\u2019s method (Oostenbrink 1960) and added in a suspension of 10\u00a0ml\u00a0pot\u22121, so that nematode inoculation density corresponded with the density of nematodes in field soil. The nematode community added to the pots was analyzed microscopically (magnification 200\u00d7) and consisted of plant parasites (T. ventralis, T. microphasmis, Pratylenchus spp, Paratylenchus spp., Meloidogyne spp., Rotylenchus spp., Criconematidae), bacterivores (Acrobeles spp., Acrobeloides spp., Chiloplacus spp., Cephalobidae, Plectus spp.), omnivores (Aporcelaimellus spp., Microdorylaimus spp.), and carnivores (Choanolaimus spp.).\nMicroarthropods were collected from nonsterile dune soil by wet sieving through 180-\u03bcm mesh and added as 10\u00a0ml of suspension\u00a0pot\u22121, which corresponded with the field density of microarthropods. Demineralized water was added to all pots in equal amounts.\nAssessing the presence of microbial enemies on nematodes in field soil \nIn order to confirm whether microbial enemies may occur on T. ventralis in the field, we extracted mobile nematodes from 100\u00a0cm3 of the field soil from each of the sampling sites using an adaptation of the Tray method (Whitehead and Hemming 1965). Half of the resulting nematode suspension was inspected using an inverted microscope (magnification 200\u00d7), and the nematodes were checked for symptoms of infection by bacteria or fungi. Nematodes infected by fungi were picked from the suspension and transferred to a corn-meal agar plate with antibiotics to encourage sporulation (Smith and Onions 1994), making possible identification of fungi that were previously found in a vegetative state. Identification of fungal natural enemies was done by observing mycelia and spore structure morphology and comparing this with the descriptions of Barron (1977). Endospores of the parasitic bacterium Pasteuria spp. were recorded when observed attached to the nematode cuticle. Symptoms of infection by a nonlethal bacterial parasite Microbacterium nematophilum were assessed according to Sulston and Hodgkin (1988).\nTo detect whether nematode natural enemies may occur as dormant forms in the soil, nematode-baited sprinkle plates were used. Soil (1\u00a0g) from each of the samples was sprinkled on water agar (1%) in a 9-cm-diameter Petri dish. A concentrated suspension of an estimated 500 Caenorhabditis elegans synchronized in the young adult stage (Sulston and Hodgkin 1988) was added to the plates. A negative control containing nematodes only in water agar (1%) was used. The plates were sealed, kept at room temperature, and observed after 2\u00a0weeks and subsequently at weekly intervals up to 5\u00a0weeks (Barron 1977). Identification of fungal natural enemies was done as described above.\nHarvest\nAll three experiments were harvested 12\u00a0weeks after inoculation of T. ventralis, allowing this nematode to complete two reproductive cycles (de Rooij van der Goes 1995). The nematodes were extracted from soil by Cobb\u2019s decantation method and from the roots using a mistifier (Oostenbrink 1960). The numbers of T. ventralis were counted using a microscope (magnification 200\u00d7) and expressed as numbers 100\u00a0g\u22121 of dry soil. The roots and shoots of A. arenaria were dried for 48\u00a0h at 75\u00b0C and weighed.\nData analysis\nNormal distribution of data and homogeneity of variance were checked by inspection of the residuals after model fit (using the package Statistica 7). To obtain the normal distribution of data and homogeneity of variances, numbers of T. ventralis were log transformed in experiment 1 and square-root transformed in experiment 2. In all three experiments, the soil origin (stable or mobile dune) did not affect significantly (P\u00a0>\u00a00.05) the measured variables. Therefore, all data from treatments with those two soil origins was pooled, resulting in 12 replicates per treatment. Numbers of T. ventralis of experiment 1 were analyzed using one-way analysis of variance (ANOVA) with main the factor \u201csoil treatment.\u201d Two-way ANOVA with the main factors \u201csoil sterilization\u201d and \u201cnematode inoculation\u201d were performed for root and shoot biomass. Three-way ANOVA with the main factors \u201cstirring,\u201d \u201csterilization,\u201d and \u201cinoculation density\u201d were performed for analyzing the numbers of T. ventralis shoot and root biomass in experiment 2. Experiment 3 was analyzed by three-way ANOVA with the main factors \u201cinvertebrates,\u201d \u201cnematodes,\u201d and \u201cmicroorganisms.\u201d Treatments were compared by posthoc analysis using Tukey honestly significant difference (HSD) tests (P\u00a0<\u00a00.05).\nResults\nExperiment 1\nThe numbers of T. ventralis at harvest differed significantly between sterilized and nonsterilized soils (F2,33\u00a0=\u00a077.9 and P\u00a0<\u00a00.001). In the nonsterilized soil, addition of T. ventralis resulted in a significant increase of numbers at the end of the experiment compared with nonsterilized, noninoculated soil (Fig.\u00a01). However, there were five times more T. ventralis in the inoculated sterilized soil than in the inoculated nonsterilized soil (Fig.\u00a01; P\u00a0<\u00a00.05; sterilized soil without T. ventralis added was not included because the nematodes were absent). These results show that multiplication of T. ventralis in nonsterilized soil was significantly enhanced by inoculation but that T. ventralis multiplication was significantly reduced by some factor in the nonsterilized soil that could be excluded by soil sterilization.\nFig.\u00a01Numbers of Tylenchorhynchus ventralis in 100\u00a0g of nonsterilized and sterilized dune soil 12\u00a0weeks after inoculation with T. ventralis. Error bars indicate standard error, and different letters above the bars indicate significant difference at P\u00a0<\u00a00.05 (experiment 1)\nSoil sterilization influenced shoot biomass more than did T. ventralis inoculation (F1,44\u00a0=\u00a0117, P\u00a0<\u00a00.001 for soil sterilization and F1,44\u00a0=\u00a04.17, P\u00a0<\u00a00.05 for inoculation, Fig.\u00a02), and the effect of T. ventralis inoculation depended on soil sterilization (F1,44\u00a0=\u00a07.06, P\u00a0<\u00a00.05). Most shoot biomass was produced in sterilized soil, whereas T. ventralis inoculation significantly reduced shoot biomass (Fig.\u00a02). As expected, the least shoot biomass was produced in nonsterile soil; however, addition of T. ventralis caused no further reduction in growth (Fig.\u00a02). As expected, root biomass was also strongly influenced by soil sterilization (F1,44\u00a0=\u00a056.1 and P\u00a0<\u00a00.001), whereas the effect of T. ventralis addition was greater than for shoot biomass (F1,44\u00a0=\u00a016.8 and P\u00a0<\u00a00.001). As for shoot biomass, the effect of T. ventralis inoculation on root biomass depended on soil sterilization (F1,44\u00a0=\u00a09.87 and P\u00a0<\u00a00.005), which reflects that shoot biomass was significantly reduced by T. ventralis inoculation in the sterilized soil only (Fig.\u00a02).\nFig.\u00a02Shoot and root biomass of Ammophila arenaria in sterilized and nonsterilized soil after 12\u00a0weeks from inoculation with Tylenchorhynchus ventralis. Error bars and letters above indicate significant differences at P\u00a0<\u00a00.05 (experiment 1)\nExperiment 2\nSignificantly greater populations of T. ventralis developed in sterilized than in nonsterlilized soil at both inoculation densities (Tables\u00a01 and 2). At the low-inoculation density, the number of the nematodes in nonsterliized soil was 30 times less than in sterilized soil and 15 times less at the high-inoculation density. There was no significant effect of soil stirring on the numbers of T. ventralis, although there was a trend (P\u00a0=\u00a00.06) that stirring reduced T. ventralis multiplication.\nTable\u00a01Three-way analysis of variance (ANOVA) of the numbers of Tylenchorhynchus ventralis in nonsterilized and sterilized, and stirred and nonstirred dune soil at three inoculation rates (0, 25, 250\u00a0pot\u22121) after 12\u00a0weeks from inoculation to Ammophila arenaria. The data has been square-root transformed to achieve normal error distributiondfFPStirring (1)13.4810.06Sterilization (2)1137.41<0.001Inoculation density (3)295.55<0.0011\u00a0\u00d7\u00a0210.8580.361\u00a0\u00d7\u00a0321.2220.302\u00a0\u00d7\u00a03243.94<0.0011\u00a0\u00d7\u00a02\u00a0\u00d7\u00a0321.4990.23Error125Table\u00a02 Effects of soil sterilization on numbers of Tylenchorhynchus ventralis in 100\u00a0g of soil [\u00b11 standard error (SE)] 12\u00a0weeks from inoculation. Letters indicate significant differences at P\u00a0<\u00a00.05 (experiment 2)NonsterilizedSterilized0 T. ventralis added0.77\u00a0\u00b1\u00a00.22a0.08\u00a0\u00b1\u00a00.03a25 T. ventralis added12.3\u00a0\u00b1\u00a02.62b390\u00a0\u00b1\u00a073.03c250 T. ventralis added69.2\u00a0\u00b1\u00a016.76b,c1,162\u00a0\u00b1\u00a0203.7d\nAs expected, both soil stirring and sterilization influenced shoot biomass (Fig.\u00a03; Table\u00a03). Shoot biomass was greater in sterilized than in nonsterilized soil and in stirred than in nonstirred soil; however, inoculation density of T. ventralis did not influence the shoot biomass (Fig.\u00a03). Root biomass was affected by stirring, soil sterilization, and inoculation density of T. ventralis, whereas effects of inoculation density depended on soil stirring as well as on soil sterilization (Table\u00a03). If no nematodes were inoculated to the pots, soil sterilization almost doubled the root biomass. However, if inoculated with 25 or 250 T. ventralis per pot, the roots in sterilized soil with T. ventralis did not produce more biomass than those in nonsterilized soil (Fig.\u00a03). Root biomass was significantly increased by soil stirring when no or few (25\u00a0pot\u22121) T. ventralis were added to the pots, but there was no increase in root weight at the high inoculation rate.\nFig.\u00a03Effects of soil stirring, soil sterilization, and addition of Tylenchorhynchus ventralis on shoot and root biomass of Ammophila arenaria. Error bars and letters above indicate significant differences at P\u00a0<\u00a00.05 (experiment 2)Table\u00a03Shoot and root biomass of Ammophila arenaria 12\u00a0weeks after inoculation with nematodesdfShoot biomassRoot biomassFPFPStirring (1)114.13<0.0018.69<0.01Sterilization (2)130.49<0.000126.8<0.001Inoculation density (3)21.9980.13910.53<0.0011\u00a0\u00d7\u00a0213.2880.0721.910.1691\u00a0\u00d7\u00a0321.9160.1513.622<0.052\u00a0\u00d7\u00a0322.1450.1216.452<0.011\u00a0\u00d7\u00a02\u00a0\u00d7\u00a0321.9210.1511.9110.152Error132The results of a factorial analysis of variance (ANOVA) with factors \u201cstirring,\u201d \u201csterilization,\u201d and \u201cinoculation density\u201d (experiment 2)\nExperiment 3\nThe multiplication of T. ventralis numbers was significantly reduced by adding a mixture of soil nematodes; however, the effect of adding microorganisms was far greater (Fig.\u00a04; Table\u00a04). If microorganisms were present alone or in combination with other soil organisms, the number of T. ventralis was always less than when microorganisms were absent. On average, adding a suspension of nematodes reduced final numbers of T. ventralis by 15%, whereas adding microorganisms reduced numbers of T. ventralis by 55% (Fig.\u00a04). Therefore, the effect of adding nematodes on T. ventralis multiplication was substantially weaker than the effect of microorganisms. Microarthropods did not have a significant effect on the numbers of T. ventralis. Adding microarthropods, nematodes, and microorganisms did not influence shoot or root biomass (P\u00a0>\u00a00.05; data not shown).\nFig.\u00a04The effects of mixed nematode inoculum and microorganisms on Tylenchorhynchus ventralis multiplication. Error bars and letters above indicate significant differences at P\u00a0<\u00a00.05 (experiment 3)Table\u00a04The numbers of Tylenchorhynchus ventralis after 12\u00a0weeks from inoculationDegrees of freedom (df)F valueP valueMicroarthropods (1)10.00060.981Nematodes (2)15.167<0.05Microorganisms (3)187.84<0.00011\u00a0\u00d7\u00a0211.7360.1911\u00a0\u00d7\u00a0310.6180.4342\u00a0\u00d7\u00a0310.6720.4151\u00a0\u00d7\u00a02\u00a0\u00d7\u00a0310.0470.828Error88The results of a three-way analysis of variance (ANOVA) with factors \u201cmicroarthropods,\u201d \u201cnematodes,\u201d and \u201cmicroorganisms\u201d (experiment 3)\nIn the suspension of nematodes obtained from the pots to which microorganisms had been added, 47.3% showed signs of infection by culturable microbial enemies (Table\u00a05). The fungal parasite Catenaria spp. was found infecting 16 out of 110 T. ventralis inspected, and an unidentified fungus was detected inside 30 destroyed of 110 T. ventralis checked. Bacterial attachment to the cuticle was also observed for six nematodes, a Paenibacillus-like organism was found on four nematodes, and Pasteuria spp. on two out of 110 nematodes.\nTable\u00a05Microbial enemies in or attached to Tylenchorhynchus ventralis in a suspension obtained from microorganism treatment pots in experiment 3Microbial enemy detectedFraction of affected nematodes (%)Unidentified assimilative hyphae27.3Paenibacillus-like3.6Catenaria spp.14.5Pasteuria spp.1.8Total47.3(Healthy)52.7Total nematodes examined100 (n\u00a0=\u00a0110)\nAssessing the presence of microbial enemies on nematodes in field soil\nThe fungal natural enemies Catenaria spp., Harposporium spp., and Myzocytium spp. were found infecting nematodes extracted by the Tray method (Whitehead and Hemming 1965). The bacterium P.penetrans was attached to root knot nematodes (M. maritima). The fungal genera were also detected using nematode-baited sprinkle plates. Some of the nematodes on the plates had a swollen region behind the anus not observed in the original culture or in the negative control. This is a symptom of infection by a nonlethal bacterial parasite, M. nematophilum. An unidentified trapping fungus with nonconstricting rings was also detected in the sprinkle plates but could not be identified, as it did not sporulate. These identifications may not have been exhaustive, but they confirmed the presence of antagonistic microorganism species on T. ventralis as well as in the soil from the field.\nDiscussion\nIn coastal foredunes, the root-feeding ectoparasitic nematode T. ventralis would significantly influence the pioneer grass A. arenaria if the density of this nematode was not controlled naturally. Our study strongly suggests that the natural control of T. ventralis in coastal foredune soil is mostly due to soil microorganisms. When inoculated into sterilized soil, numbers of T. ventralis were more than five times greater than when inoculated into nonsterilized soil, whereas selective elimination of soil fauna by stirring did not affect nematode numbers. These results from selective elimination studies were confirmed by isolating microarthropods, nematodes, and microbes and adding these together with T. ventralis to sterilized soil. Inoculation with soil microorganisms reduced T. ventralis more strongly than did inoculation with a nematode community consisting of other plant parasites, bacterivores, omnivores, and carnivores. The negative effect of the nematode community on T. ventralis density might have been due to competition with other root feeders. However, competition between T. ventralis and endoparasitic nematodes (i.e. H. arenaria, M. maritima and P. penetrans) occurred only if numbers of the competitors strongly exceeded present field densities (Brinkman et\u00a0al. 2004). Therefore, the observed effect of adding nematodes on reducing T. ventralis was due to soil microorganisms cointroduced with the nematode suspension, carnivorous nematodes (Jairajpuri and Bilgrami 1990), or by effects of other non-plant-feeding nematodes. Our results highlight an important discrepancy in thinking about control mechanisms of plant-feeding (also called plant-parasitic) nematodes between biocontrol studies on the one hand and soil food web studies on the other. The majority of studies on the biocontrol of nematodes in agricultural ecosystems mostly focus on parasitic bacteria, such as P. penetrans and fungi (Kerry 2000), for example Arthrobotrys spp. and Pochonia spp. (Stirling and Smith 1998). Biological control practice usually does not consider microarthropods to be relevant for parasitic nematode control (Kerry and Gowen 1995), whereas the role of carnivorous nematodes has been considered (Mankau 1980; Yeates and Wardle 1996) but not successfully used. According to the food web model used by Neutel et\u00a0al. (2002), root-feeding nematodes in coastal ecosystems are affected by predaceous mites and carnivorous nematodes. The role of microorganisms in nematode control is generally ignored in prominent soil food-web models (de Ruiter et\u00a0al. 1993; Hunt et\u00a0al. 1987). In our study system, however, soil microorganisms appeared to play a more important role than soil fauna in the control of plant ectoparasitic nematodes. In dune grasslands, the densities of soil fauna are usually rather low, perhaps too low to control significantly the abundance of nematodes (Petersen and Luxton 1982). Our results, therefore, support the view of biological control studies more than it supports soil food-web models for the control of the ectoparasitic nematode T. ventralis.\nOur results suggest that top-down control by natural enemies is more important for ectoparasitic feeding generalists, such as T. ventralis, than are competition (Brinkman et\u00a0al. 2004) or mycorrhizal fungi (Pi\u015bkiewicz and Hol unpublished results), although these control mechanisms have been suggested for the other endoparasitic root-feeding nematode species in the same study system (Brinkman et\u00a0al. 2005; de la Pe\u00f1a et\u00a0al. 2006; van der Stoel et\u00a0al. 2006).\nThe control of T. ventralis in nonsterilized soil to which T. ventralis was added was not as good as in nonsterile soil, with only the background population of T. ventralis present. Earlier studies have shown that multiplication of T. ventralis is density and time dependent (de Rooij van der Goes 1995). Over time, a low inoculation density of nematodes may result in population increase, whereas a high inoculation density may result in population decline (de Rooij van der Goes 1995). However, we have no information as to how the microbial control shown in the present study may depend on nematode density, and subsequent long-term studies are required to further explore density dependence of nematode top-down control by microbes.\nThe two screening methods used to detect microbial enemies of nematodes in the soil yielded a diversity of fungal and bacterial antagonists. The Tray method (Whitehead and Hemming 1965) permitted the extraction of mobile stages of nematodes only, and therefore, dead or dying nematodes could not be screened. The nematode-baited sprinkle plate method, although useful for detecting microbial enemies in dormant forms in the soil, produced biased results that reflected the choice of nematode added, the bacterial-feeding nematode C. elegans. The fungal endoparasite Harposporium spp. infects nematodes that ingest its spores and therefore would not be able to infect plant parasites due to the narrow lumen of the stylet (Barron 1977). The bacterium M. nematophilum is thought to be a specialist parasite of C. elegans (Hodgkin et\u00a0al. 2000). T. ventralis was attacked by a subset of the microbial enemy genera found in the soil, which reflects some specificity in the action of these microbes. Some microorganisms, namely Pasteuria spp. and the Paenibacillus-like organism, have not been detected in the dune soil, although they are widespread in the dune sites. This may be due to their absence or to presence in small numbers that are below the level of detection. The unidentified assimilative hyphae could be the result of attack by generalist trapping fungi. This was a first assessment to confirm the presence of nematode antagonistic microorganisms on T. ventralis, and further studies are needed to isolate culture and inoculate those antagonists in order to evaluate their contribution in root-feeding nematode control.\nSoil sterilization always led to increased root and shoot biomass. Previous studies have already shown that soil biota may reduce performance of A. arenaria (van der Putten et\u00a0al. 1988; van der Stoel et\u00a0al. 2002). The effect of soil sterilization was greater for root biomass than for shoot biomass. In sterilized soil, enhanced plant growth can be caused by nutrient release as a result of the soil sterilization process (Troelstra et\u00a0al. 2001). We avoided different nutrient status of the sterilized and nonsterilized treatments by adding nutrient solution (van der Putten et\u00a0al. 1988). When added to sterilized soil, the root-feeding nematode T. ventralis reduced root and shoot biomass of A. arenaria, and the effect of nematodes on root biomass increased with increasing inoculation density. Addition of T. ventralis to the nonsterile soil did not change root biomass, showing that the contribution of this nematode species to growth reduction of A. arenaria is limited (de Rooij van der Goes 1995). Our study shows that the dune soil not only contains biotic factors that reduce growth of A. arenaria but that there are also (micro) organisms that control population abundance of T. ventralis. In prairie grassland ecosystems, root-feeding nematodes have been assessed to account for reducing 58% of aboveground biomass (Stanton 1988). These estimates are based on elimination trials using soil biocides. However, these studies did not verify the biocide effects by inoculation trials, and they also did not account for natural top-down control of the root-feeding nematodes. Our results suggest that when assessing the effects of root feeders on plant production, interactions of the root feeders with their natural predators needs to be taken into account as well.\nWe conclude that soil microorganisms contribute to controlling the plant ectoparasitic nematode T. ventralis at a low population density in natural coastal foredunes. When not controlled, T. ventralis would be a key control factor for A. arenaria performance. Addition of other soil fauna, i.e., nematodes and microarthropods, did not influence the abundance of T. ventralis as much as microorganism addition did, which confirms the marginal effects of soil fauna removal on reproduction of T. ventralis. Our results suggest that belowground multitrophic interactions can be crucial for plant performance. Revealing the precise identity of the microorganisms that have negative effects on the T. ventralis population, as well as mechanisms and involvement of the host plant, need further studies.","keyphrases":["tylenchorhynchus ventralis","ammophila arenaria","top-down control","multitrophic interactions","root herbivory"],"prmu":["P","P","P","P","M"]}
{"id":"Arch_Dermatol_Res-3-1-1910889","title":"Tolerance to coxibs in patients with intolerance to non-steroidal anti-inflammatory drugs (NSAIDs): a systematic structured review of the literature\n","text":"Adverse events triggered by non-steroidal anti-inflammatory drugs (NSAIDs) are among the most common drug-related intolerance reactions in medicine; they are possibly related to inhibition of cyclooxygenase-1. Coxibs, preferentially inhibiting cyclooxygenase-2, may therefore represent safe alternatives in patients with NSAID intolerance. We reviewed the literature in a systematic and structured manner to identify and evaluate studies on the tolerance of coxibs in patients with NSAID intolerance. We searched MEDLINE (1966\u20132006), the COCHRANE LIBRARY (4th Issue 2006) and EMBASE (1966\u20132006) up to December 9, 2006, and analysed all publications included using a predefined evaluation sheet. Symptoms and severity of adverse events to coxibs were analysed based on all articles comprising such information. Subsequently, the probability for adverse events triggered by coxibs was determined on analyses of double-blind prospective trials only. Among 3,304 patients with NSAID intolerance, 119 adverse events occurred under coxib medication. All adverse events, except two, have been allergic\/urticarial in nature; none was lethal, but two were graded as life-threatening (grade 4). The two non-allergic adverse events were described as a grade 1 upper respiratory tract haemorrhage, and a grade 1 gastrointestinal symptom, respectively. In 13 double-blind prospective studies comprising a total of 591 patients with NSAID intolerance, only 13 adverse reactions to coxib provocations were observed. The triggering coxibs were rofecoxib (2\/286), celecoxib (6\/208), etoricoxib (4\/56), and valdecoxib (1\/41). This review documents the good tolerability of coxibs in patients with NSAID intolerance, for whom access to this class of drugs for short-term treatment of pain and inflammation is advantageous.\nIntroduction\nNon-steroidal anti-inflammatory drugs (NSAIDs) are the most commonly used therapeutics in the outpatient management of pain and inflammation in a wide spectrum of diseases. Their primary mode of action is the blockade of prostaglandin synthesis by cyclooxygenases (COX): Constitutively expressed COX-1 is involved in fundamental mechanisms of homeostasis, whereas the inducible COX-2 mediates inflammation. Therapeutic effects of NSAIDs are primarily related to their ability to inhibit COX-2, whereas some of their most frequent adverse effects may be caused by COX-1 inhibition (Fig.\u00a01). In contrast to most \u201cclassic\u201d NSAIDs which block both isoforms, the so-called coxibs preferentially inhibit COX-2. This may result in better tolerability, namely reduction of gastrointestinal side effects [29, 85].\nFig.\u00a01Cyclooxygenase isoforms. Non-steroidal anti-inflammatory drugs (NSAIDs) block prostaglandin synthesis by cyclooxygenases (COX). Two isoforms exist of this enzyme: constitutively expressed COX-1 is involved in fundamental mechanisms of homeostasis, whereas the inducible isoform COX-2 mediates inflammation\nRespiratory and cutaneous adverse events triggered by NSAIDs are among the most common drug-related intolerance reactions in medicine. Typically, these manifest as asthma attacks or urticaria. Pathogenesis of these symptoms seems to be related to COX-1 inhibition [76]. Therefore, the hypothesis was put forward that coxibs may safely be used in patients with known NSAID intolerance. However, serious intolerance reactions to coxibs have also been observed, thus cautioning too euphoric expectations [71]. We therefore reviewed the relevant literature in a systematic and structured approach for evidence of coxib tolerance in patients with NSAID intolerance.\nMethods\nSearch strategy\nWe searched MEDLINE (1966\u20132006), the COCHRANE LIBRARY (4th Issue 2006) and EMBASE (1966\u20132006) up to December 9, 2006. The following search terms were used: rofecoxib OR celecoxib OR valdecoxib OR parecoxib OR etoricoxib, combined with hypersensitivity OR intolerance. Those coxibs used in veterinary medicine (deracoxib, tiracoxib and cimicoxib) were not included in the literature search. No language or publication restriction was predefined. All publications reporting individual patients with NSAID intolerance and subsequent exposure to a COX-2 Inhibitor were identified and the reference lists of these articles were hand-searched for further publications. If articles could not be retrieved in full text, a copy was requested from the corresponding author and\/or journal editor.\nInclusion criteria\nEach publication was appraised for inclusion in a stepwise approach (Fig.\u00a02). Only publications describing individual patients and providing a rational medication scheme as well as a sufficiently specific outcome report were included in this review. In a first step, evaluation focused on clinical symptoms and severity of adverse events in patients with NSAID intolerance. Therefore, all articles on this topic were included for this analysis. Subsequently, the probability for adverse events was analysed based exclusively on publications of double-blind prospective trials.\nFig.\u00a02Flow chart documenting the handling data (NSAID non-steroidal anti-inflammatory drugs)\nData extraction and synthesis\nAll articles were analysed using a predefined evaluation sheet. Uncertainties were resolved by consensus decisions among the investigators. Data synthesis was qualitative and descriptive. The Common Terminology Criteria for Adverse Events version 3.0 (CTCAE) was used to categorize adverse events.\nResults\nWe identified 230 publications on coxibs and\/or NSAID intolerance. Hundred and forty-six references not focusing on individual patients with NSAID intolerance were excluded. Unclear medication schemes or outcome reports led to exclusion of two articles. Eighty-four publications were evaluated for severity and type of adverse reactions to coxibs. Thirteen publications on double-blind studies were used to determine the probability of adverse reactions to coxibs.\nSeverity and type of adverse events to coxibs\nA total of 119 adverse events among 3,304 patients exposed to coxibs were identified [1\u201318, 20\u201324, 26\u201328, 30\u201335, 37\u201358, 60\u201375, 77\u201384, 86\u201389]. Adverse events were described for rofecoxib (48\/1,732), celecoxib (59\/1,148), etoricoxib (10\/328), and valdecoxib (2\/69), but not parecoxib (0\/27) (Table\u00a01). All adverse events, except two, have been allergic\/urticarial in nature, and none was lethal. One adverse event after administration of rofecoxib and one after celecoxib were reported as life-threatening (grade 4). The two non-allergic adverse events were described as a grade 1 upper respiratory tract haemorrhage, namely blood in nasal discharge after provocation with rofecoxib, and a grade 1 gastrointestinal symptom, namely nausea and abdominal pain.\nTable\u00a01Mode and severity of adverse event (CTCAE common terminology criteria for adverse events)PatientsEvents (total)Urticaria (grade)Allergic reactions (grade) Allergic rhinitis (grade)Airway obstruction (grade)Gastroin-testinal symtpms (grade)Pruritus (grade)Hemorrhage, upper respiratory (grade)Rofecoxib1,732489 (1)8 (2)1 (3)25 (3)1 (4)1 (2)2 (1)001 (1)Celecoxib1,1485919 (1)1 (2)1 (2)33 (3)1 (4)01 (1)03 (2)0Etoricoxib328103 (1)5 (2)1 (3)001 (1)00Valdecoxib6921 (1)1 (2)000000Parecoxib2700000000Total3,304119486213131Numbers and grade of adverse events (in parentheses) are indicated\nEvaluation of double-blind studies\nWe identified 13 double-blind studies evaluating tolerability of coxibs in a total of 591 NSAID intolerant patients (Table\u00a02). The coxibs tested in the context of these studies were rofecoxib (n\u00a0=\u00a0286), celecoxib (n\u00a0=\u00a0208), etoricoxib (n\u00a0=\u00a056) and valdecoxib (n\u00a0=\u00a041). Among the 591 patients described, 13 exhibited adverse reactions upon provocation with a coxib. The triggering coxibs were rofecoxib (2\/286), celecoxib (6\/208), etoricoxib (4\/56), and valdecoxib (1\/41) [13, 28, 48, 60, 69, 74, 75, 77, 78, 83, 84, 87, 88]. One of these reactions was clearly non-allergic, this one being the above-mentioned individual with grade 1 upper respiratory tract haemorrhage.\nTable\u00a02Snopsis of double blind studies (NSAID non-steroidal anti-inflammatory drugs)ReferencesSubstance (number of patients)DoseOral provocation with NSAIDFollow-upAdverse events (number of patients\/grade)[87]Celecoxib (17)Celecoxib 200\u00a0mgYesClinical examinations and forced expiratory volume hourly for 8\u00a0h.0[74]Celecoxib (12)\/rofecoxib (3)Celecoxib 200\u00a0mg\/rofecoxib 12.5\u00a0mgYesNo information0[75]Rofecoxib (60)Rofecoxib 37.5\u00a0mg total doseYesClinical examinations and flow\/volume recording hourly0[77]Rofecoxib (12)Rofecoxib 25\u00a0mgYesClinical observation every 30\u00a0min for 8\u00a0h and finally after 24\u00a0h.Traces of blood in nasal discharge on days 3\u20135 of rofecoxib administration (1\/1), transient urticaria for 30\u00a0min after administration of 5\u00a0mg rofecoxib, but not after further administration of higher doses of the drug (1\/1)[78]Celecoxib (14)Rofecoxib 10Celecoxib 200\u2013400\u00a0mg total dose Rofecoxib 37.5\u00a0mg total doseYesForced expiratory volume was recorded each hour for 3\u00a0h0[60]Rofecoxib (104)Rofecoxib 25\u00a0mg total doseYesClinical control for at least 6\u00a0h after each challenge and the next day.0[83]Celecoxib (60)Celecoxib 200\u00a0mgYesClinical examinations were conducted hourly0[28]Celecoxib (33)Celecoxib 130\u00a0mg total doseYesSpirometry and vital signs at 15- to 30-min intervals after each dose.0[88]Celecoxib (18)\/rofecoxib (18)Celecoxib 300\u00a0mg total dose\/rofecoxib 37.5\u00a0mg total dose1YesVital signs and forced expiratory volume every 15\u00a0min for 6\u00a0h.0[84]Rofecoxib (60)Rofecoxib 50\u00a0mgYesClinical examinations and forced expiratory volume hourly.0[13]Valdecoxib (41)Valedcoxib 30\u00a0mg total doseYesClinical examination and monitoring of pulse\/blood pressare until 16\u00a0h past last exposureGeneralised urticaria (1\/2)[69, 70]Celecoxib (54)Etoricoxib (56)Celecoxib 200\u00a0mgEtoricoxib 120\u00a0mgYesVital signs and pulmonary function was monitored at baseline and hourly for 3\u00a0h and the skin, nose, thorax were examined at the same intervalsCelecoxib: urticaria (3\/1), lip angioedema (1\/3), eyelid wheals and angioedema (1\/3), urticaria, rhinorrhea, and conjunctival injection (1\/1)etoricoxib: urticaria (3\/1), tongue, eyelid and lip angioedema (1\/3)[48]Rofecoxib (19)Rofecoxib 37.5\u00a0mg total doseYesSerial measurement of forced expiratory volume after 30, 60 and 120\u00a0min0\nDiscussion\nThis systematic review documents the low probability of allergic\/pseudo-allergic reactions induced by coxibs in patients with NSAID intolerance. To our knowledge, this is the first comprehensive analysis of data published on this topic. We have searched all three major medical databases available, namely MEDLINE, COCHRANE LIBRARY, and EMBASE using very broad and general search terms. Following identification of relevant publications, these were evaluated by means of a pre-defined evaluation form. The data available are described in the form of a structured review [19].\nNSAIDs are among the most commonly prescribed therapeutics in the world. Although generally considered safe, their wide and frequent use results in these drugs being among the most common causes of drug-related intolerance reactions. This may at least in part be due to their non-selective inhibition of both cyclooxygenase isoforms. In line with this hypothesis, NSAIDs characterized by pronounced COX-1 inhibition bear a high risk to trigger asthma attacks in patients with aspirin-sensitive asthma bronchiale, whereas preferentially COX-2 inhibiting NSAIDS are better tolerated by these patients [36]. Our analysis of published studies on this issue further supports this notion, as only 13 of 591 NSAID-intolerant patients showed adverse reactions upon provocation with coxibs in double-blind clinical studies; all of these were grade 3 or milder. Still, relatively selective COX-2 inhibitors have been identified as triggers of serious intolerance reactions [71]. This implies that our current understanding of NSAID-triggered intolerance is still imperfect, and its pathogenesis cannot be reduced to cyclooxygenase-mediated effects alone, but needs to take into account clinically relevant additional NSAID-mediated effects such as secretion of leukotrienes from mast cells and other leukocytes.\nIt has been suggested that coxibs may confer an elevated risk for acute myocardial infarction and sudden cardiac death, namely after long-term therapy [25]. As a reaction, several coxibs are no longer available despite a recommendation by the participants of an expert meeting organized by the Food and Drug Administration to grant further prescription of rofecoxib, celecoxib and valdecoxib in the US [59]. On the other hand, substantial evidence described here points towards a good tolerability of coxibs in patients with NSAID intolerance. Given the wide use of NSAIDs in the short-term treatment of trivial signs and symptoms, the availability of coxibs for these indications would be advantageous for this relevant subpopulation of patients, since this type of application is unlikely to increase cardiac risk.","keyphrases":["coxibs","intolerance","non-steroidal anti-inflammatory drugs","urticaria","adverse drug reaction"],"prmu":["P","P","P","P","R"]}
{"id":"Eur_Radiol-4-1-2220024","title":"CT colonography: optimisation, diagnostic performance and patient acceptability of reduced-laxative regimens using barium-based faecal tagging\n","text":"To establish the optimum barium-based reduced-laxative tagging regimen prior to CT colonography (CTC). Ninety-five subjects underwent reduced-laxative (13 g senna\/18 g magnesium citrate) CTC prior to same-day colonoscopy and were randomised to one of four tagging regimens using 20 ml 40%w\/v barium sulphate: regimen A: four doses, B: three doses, C: three doses plus 220 ml 2.1% barium sulphate, or D: three doses plus 15 ml diatriazoate megluamine. Patient experience was assessed immediately after CTC and 1 week later. Two radiologists graded residual stool (1: none\/scattered to 4: >50% circumference) and tagging efficacy for stool (1: untagged to 5: 100% tagged) and fluid (1: untagged, 2: layered, 3: tagged), noting the HU of tagged fluid. Preparation was good (76\u201394% segments graded 1), although best for regimen D (P = 0.02). Across all regimens, stool tagging quality was high (mean 3.7\u20134.5) and not significantly different among regimens. The HU of layered tagged fluid was higher for regimens C\/D than A\/B (P = 0.002). Detection of cancer (n = 2), polyps \u22656 mm (n = 21), and \u22645 mm (n = 72) was 100, 81 and 32% respectively, with only four false positives \u22656 mm. Reduced preparation was tolerated better than full endoscopic preparation by 61%. Reduced-laxative CTC with three doses of 20 ml 40% barium sulphate is as effective as more complex regimens, retaining adequate diagnostic accuracy.\nIntroduction\nFull bowel purgation remains a major cause of discomfort prior to any colonic investigation [1\u20133]. Furthermore, fluid and electrolyte imbalance may occur following aggressive cleansing [4]. A potential advantage of computed tomography colonography (CTC) over colonoscopy is the ability to reduce laxative requirements while maintaining diagnostic accuracy [5\u20137]. Reduced laxative regimens often incorporate orally ingested contrast agents to \u201ctag\u201d or \u201clabel\u201d residual fluid and faecal residue. The ideal tagging regimen remains controversial but must be safe, effective, simple and well tolerated.\nIn many respects, barium is an ideal tagging agent: it has an established safety profile, is relatively palatable, produces minimal side effects, and is effective for tagging solid residue [8]. Previous work has shown adequate tagging may be achieved using low volumes of 40% w\/v barium, although the ideal volume and dosing regimen has not been fully established [9]. Furthermore efficacy for fluid tagging has been questioned [9], with some investigators preferring iodine-based contrast either alone or in combination, claiming a more homogeneous fluid opacification better suited to digital subtraction [10]. We aimed to establish the optimum barium-based reduced-laxative faecal- and fluid-tagging regimen, to assess patient acceptability, and to document diagnostic accuracy compared to an enhanced colonoscopic reference standard.\nMaterials and methods\nFull ethical committee approval was obtained, and all subjects gave written informed consent.\nConsecutive patients were recruited from those scheduled to undergo afternoon diagnostic colonoscopy for symptoms suggestive of colorectal neoplasia (change in bowel habit, rectal bleeding, unexplained weight loss or palpable abdominal mass) from one of three institutions. Patients were excluded if below age 50 or if they had a known diagnosis of inflammatory bowel disease. Eligible patients were invited to undergo CTC at 8\u00a0am, prior to same day colonoscopy and were randomised via a computer random number generator to one of four reduced-laxative regimens using barium-based faecal tagging [20\u00a0ml 40% w\/v barium sulphate suspension (Tagitol V, EZEM, Lake Success, NY)] (Fig.\u00a01). For each of the regimens, patients followed a low-residue diet 2\u00a0days before the CTC (avoiding fatty food, milk and vegetables). The day prior to CTC, all patients were allowed a low-residue meal kit (Nutraprep, EZEM), and ingested the same reduced-laxative protocol [13\u00a0g sachet of senna granules (Reckitt Benckiser Healthcare, Hull, UK) at 7\u00a0am and 18\u00a0g magnesium citrate (Lo-So prep, EZEM) at 7\u00a0pm]. This laxative regimen was defined as \u201creduced\u201d given the normal laxative preparation prior to colonoscopy at the recruiting institutions includes an additional 18\u00a0g of magnesium citrate (see below). Overall fluid intake was restricted to 2.1\u00a0l the day prior to CTC for all four regimens.\nFig.\u00a01Details of reduced-laxative tagging regimens. Taken with meals from low residue meal kit (single star). Diluted in 250 mls of water (double stars)\nIn an attempt to improve fluid tagging, two regimens (C and D) included either an additional 250\u00a0ml 2.1% w\/v barium sulphate (Readi-Cat Banana Smoothie, EZEM) or 15\u00a0ml meglumine amidotrizoate (10\u00a0g sodium amidotrizoate\/66\u00a0g meglumine amidotrizoate per 100\u00a0ml; Gastrograffin, Schering) diluted in 250\u00a0ml of water, taken 2\u00a0h prior to CTC [11]. Patients otherwise took nothing by mouth the day of the CTC.\nStudy power\nThe study was powered to detect a 20% difference in tagging quality (see below) across the four regimens. Based on pilot data, the interclass correlation coefficient between colonic segments was calculated to be 0.30, and it was calculated that a sample size of 22 per group was required (alpha 0.05 at 80% power).\nCT colonography\nProne and supine CTC was performed with automated CO2 insufflation (Protocol pump, EZEM) [12], using either a 4-detector-row (GE Lightspeed Plus, GE, Milwaukee, WI, USA; 120\u00a0kV, 50\u00a0mA, 2.5\u00a0mm collimation, slice reconstruction 1.25\u00a0mm, pitch 1.5, n\u2009=\u200982 patients) or 64-detector-row scanner (Siemens Somatom Sensation 64, SEMS, Germany; 120\u00a0kV, 50\u00a0mA, 0.6\u00a0mm collimation, pitch 0.24, n\u2009=\u200913 patients).\nOne of three experienced radiologists (each with experience of at least 300 CTC cases with endoscopic validation) evaluated the datasets immediately after the examination using a dedicated workstation with proprietary software (Vitrea 3.8, Vital Images, Minnetonka, MN, USA), and noted the segmental location of any polyps or cancers on a study report sheet, together with lesion size measured using electronic callipers applied to the 2D MPR best showing the maximum diameter. The choice of reporting radiologist for each patient was dependent upon the particular recruiting institution and availability on the day of the scan. All three radiologists used a primary 2D approach with 3D reserved for problem solving.\nColonoscopy\nImmediately following CTC patients ingested a further 18\u00a0g of magnesium citrate in order to complete the normal endoscopic cleansing regimen prior to afternoon colonoscopy. Colonoscopy was performed as per usual practice by one of five experienced endoscopists, on average 6\u00a0h (range 4\u20138\u00a0h) after the CTC scan. Segmental unblinding was used as described previously [13, 14], using the sealed CTC report. In brief, the CTC report was handed to the endoscopy nurse accompanying the patient. CTC findings were revealed to the colonoscopist by the nurse on a segmental basis (caecum to rectum) once examination of each colonic segment was deemed complete during extubation of the colon. If CTC suggested a lesion had been missed, the segment was re-intubated and a second segmental examination performed. There was no time limit imposed on the colonoscopist for this second look. All polyps were photographed, their sizes estimated by direct comparison to adjacent open biopsy forceps, and then excised for histology where possible.\nPolyp correlation\nPolyps found at CTC were deemed true positive if a corresponding polyp was found in the same or adjacent segment at endoscopy and if the estimated size of the polyp was within 50% of the endoscopic measurement.\nPatient experience\nImmediately following CTC a questionnaire was administered [15] investigating patient experience of the reduced-laxative tagging regimen (Table\u00a01). Patient responses were compared to a historical cohort of 69 symptomatic patients recruited from the same endoscopy lists during a prior study comparing CTC to colonoscopy [16]. As part of this prior study, patients had undergone full bowel preparation [13\u00a0g senna granules and two doses of 18\u00a0g magnesium citrate (total 36\u00a0g) prior to CTC] and had completed the same questionnaire under similar circumstances. \nTable\u00a01Bowel-tolerance questionnaire questions and responses in comparison to historical controls undergoing full bowel preparationVariableResponseReduced preparationa, n (%)Historical controls [full preparation]b, n (%)P valueHow did you find understanding prep sheet?Easy56 (63)48 (70)0.39Fairly easy\/difficult33 (37)21 (30)How did you find swallowing medicine?Easy43 (48)44 (64)0.36Fairly easy36 (40)32 (32)Quite difficult\/difficult10 (11)3 (4)How did you find coping with special diet?No problem59 (66)42 (61)0.49Bit difficult24 (27)24 (35)Very difficult6 (7)3 (4)How did you feel after medicine?Fine65 (73)48 (70)0.96Unwell\/very unwell24 (27)21 (30)Did you have any abdominal pain?None31 (36)25 (36)0.37Mild39 (45)27 (39)Moderate\/severe16 (19)17 (25)Did you have any nausea\/vomiting?None58 (67)46 (67)0.92Mild22 (26)18 (26)Moderate\/severe6 (7)5 (7)Did you experience any faintness or dizziness?None70 (81)54 (78)0.63Mild\/moderate\/severe16 (29)15 (22)Did you experience any wind?None25 (29)26 (38)0.27Mild41 (48)32 (46)Moderate\/severe20 (23)11 (16)Did you experience any soreness?None37 (43)24 (35)0.15Mild37 (43)29 (42)Moderate\/severe12 (14)16 (23)Did you experience any incontinence?None68 (80)49 (71)0.2Mild8 (9)13 (19)Moderate\/severe9 (11)7 (10)Did you experience any sleep disturbance?None33 (39)41 (59)0.01Mild29 (34)20 (29)Moderate\/severe23 (27)8 (12)How many times did you open your bowel after starting the preparation?1\u201331 (1)N\/A3\u2013520 (22)>569 (77)N\/A Not applicable (not asked)a13\u00a0g senna plus 18\u00a0g magnesium citrateb13\u00a0g senna plus 36\u00a0g magnesium citrate\nOne week later a follow-up questionnaire (Table\u00a02) was mailed to the current study cohort investigating tolerance of the preparation prior to CT and preference, if any, over the full preparation required for subsequent colonoscopy. \nTable\u00a02Questions and responses to follow-up questionnaire pertaining to patient tolerance and preferencesVariableResponsePatient number (%)How did you find taking the low-residue diet?No problem46 (67%)Moderately inconvenient20 (29%)Very inconvenient3 (4%)How did you find drinking the tagging liquid?No problem57 (83%)Moderately inconvenient11 (16%)Very inconvenient1 (1%)How did you tolerate the preparation before CT?Well37 (53%)Fairly well29 (42%)Poorly3 (4%)How did you tolerate the additional preparation prior to colonoscopy, compared to that before the CT?No problem51 (74%)More uncomfortable12 (17%)Much worse6 (9%)How did you find the preparation before CTC compared to the full colonoscopy preparation?Much better18 (26%)Better24 (35%)No better26 (38%)\nGrading of bowel preparation\nTwo experienced radiologists (both with experience of over 700 validated CTC cases) in consensus retrospectively reviewed all CTC examinations, grading the quality of preparation and success of tagging. Observers were unaware of the tagging regimen used and divided the colon into six segments (rectum to caecum) for analysis [17].\nGrading of residual stool (irrespective of tagging status) was based on the percentage of total mucosal circumference coated on an axial image. For each colonic segment, the slice with the most residual stool was used to assign the score. Scores were as follows: 1: no residue or scattered residue, 2: coating of <25% or thin circumferential \u201cfilm\u201d less than 2\u00a0mm in depth, 3: coating of \u226525 to 50%, 4: >50% coating.\nGrading of residual fluid (irrespective of tagging status) was based on the maximum anteroposterior (AP) diameter of the colonic lumen submerged. For each colonic segment, the slice with the most residual fluid was used to assign the score. Scores were as follows: 1: no fluid, 2: <25% AP diameter, 3: \u226525 to 50% AP diameter, 4: >50% AP diameter.\nThe quality of tagging for solid residue and fluid was scored using a system adapted from Lefere at al. [9] and assigned for supine and prone positions combined for each colonic segment. Residual stool was divided into that measuring \u22645\u00a0mm and \u22656\u00a0mm (based on 2D measurement using electronic calipers), and the number of stool balls \u22656\u00a0mm was counted for each patient. Readers assessed the percentage of total residual stool volume (for each size category) that had been tagged for each colonic segment. Scores were assigned as follows: 1: all residual stool untagged, 2: 1 to <25% tagged, 3: 25 to <50% tagged, 4: 50 to <75% tagged, 5: 75 to 100% tagged.\nTagging of residual fluid was scored as follows: 1: fluid untagged, 2: layered tagging (a mix of tagging densities in one fluid level with a denser dependent layer and visibly less dense non-dependent layer (Fig.\u00a02), 3: fluid homogeneously tagged (single tagging density). The density of tagged fluid (in HU) was also recorded by taking the average of three regions of interest drawn in the deepest fluid pool for each colonic segment. If fluid was layered (Fig.\u00a02), the minimum and maximum HU was recorded. Finally observers recorded their confidence using a percentage score that, based on the bowel preparation, they would be able to exclude a polyp \u22656\u00a0mm in day-to-day clinical practice for each colonic segment.\nFig.\u00a02A 68-year-old female with change in bowel habit. Axial CT colonographic image demonstrating layering of contrast (arrow) within tagged fluid\nStatistical analysis\nPreliminary data analysis revealed skewed data for most bowel preparation and tagging variables, and thus scores were combined for subsequent analysis. For residue, scores were grouped into either a score of 1, or a score of 2 or more. For residual fluid, scores were grouped into scores of 1\/2, or scores of 3\/4. Scores for solid residue tagging were grouped into those of \u22644 or 5. Fluid tagging scores were grouped into scores of 1\/2, or score of 3. Logistic regression was then applied, adjusting for colonic segment, to compare the four regimens overall, and to compare the distal (rectum, sigmoid, descending) and proximal colon (transverse, ascending and caecum). In addition, the prevalence of layering within tagged fluid was compared on a per-patient basis using the chi-squared test. Tagged fluid attenuation was compared using linear regression following log transformation of the data. Confidence scores for excluding a polyp \u22656\u00a0mm were grouped as either <100% or 100%, and compared using logistic regression. For all regression analyses, robust standard errors were employed to account for interdependency between colonic segments and scan position (supine\/prone) in the same patient. Results were expressed as the odds of the outcome under consideration compared to regimen A.\nQuestionnaire responses were compared using Fischer\u2019s exact test.\nOverall categorical per-polyp and per-patient data are presented using descriptive statistics. Given the relatively low polyp incidence (and thus low statistical power), comparative statistics for polyp detection were not performed across regimens. False-positive numbers were compared using one-way ANOVA.\nResults\nA total of 95 patients were recruited (50 female, mean age 64\u00a0years, range 50\u201385\u00a0years), with 24, 25, 24 and 22 randomised to regimens A to D respectively. Seventy-seven, 13 and 5 patients were recruited from institutions 1, 2 and 3 respectively. Overall, 67 (71%) had a change in bowel habit, 18 (19%) had rectal bleeding, 7 (7%) had non-specific weight loss, and 3 (3%) had a clinically palpable abdominal mass.\nBowel preparation\nThe percentage of segments (supine and prone combined) assigned a score of 1 for residual solid residue (i.e. no residue or scattered) was 76% (218\/288), 82% (246\/300), 81% (232\/288) and 94% (247\/264), for regimens A to D respectively, and on average there were 2.5, 2.0, 2.8 and 1.1 stool balls \u22656\u00a0mm per patient. The improved quality of preparation in regimen D reached statistical significance [odds of score 2\u20134: 0.19 (95% CI: 0.07, 0.56), P\u2009=\u20090.02]. Across all regimens, the distal colon was significantly better prepared then the proximal colon (P\u2009=\u20090.006). For example the number of segments assigned at least a score of 2 for solid residue in the caecum was 42% (20\/48), 48% (24\/50), 35% (17\/48) and 23% (10\/44) for regimens A to D respectively, compared to 25% (12\/48), 16% (8\/50), 21% (10\/48) and 0% (0\/44) for the rectum.\nThe percentage of segments (supine and prone in total) assigned a score of 1 or 2 for residual fluid was 51% (148\/288), 59% (178\/300), 53% (153\/288) and 43% (114\/264) for regimens A to D respectively. There was no significance difference between groups either overall or between the proximal and distal colon (P\u2009=\u20090.22\u20130.37).\nTagging quality\nTagging quality was generally good (mean tagging score 3.7\u20134.5) (Fig.\u00a03). While there was weak evidence of improved proximal colonic tagging for residue \u22645\u00a0mm for regimens C and D (P\u2009=\u20090.08), overall there was no difference in the efficacy of solid residue tagging across regimens (Table\u00a03).\nFig.\u00a03A 54-year-old male with unexplained rectal bleeding. Axial CT colonographic image showing homogeneous tagging of stool \u22645\u00a0mm (arrow) and \u22656\u00a0mm (arrowhead) in sizeTable\u00a03Efficacy of tagging of solid residue according to size and regimenSolid residue sizeColon segmentsRegimenMean tagging score (SD)Odds ratio (95% CI)aP value\u22645\u00a0mmAll segmentsA4.3 (1.2)1B4.3 (1.2)0.94 (0.39, 2.32)C4.5 (1.1)1.47 (0.56, 3.87)D4.5 (1.2)1.67 (0.68, 4.14)0.56Distal colonbA1B0.97 (0.37, 2.52)C0.76 (0.26, 2.27)D1.00 (0.38, 2.61)0.97Proximal coloncA1B0.85 (0.26, 2.80)C4.06 (0.74, 22.1)D3.22 (0.95, 10.9)0.08\u22656\u00a0mmAll segmentsA4.1 (1.6)1B4.3 (1.5)1.82 (0.44, 7.62)C4.1 (1.7)1.56 (0.32, 7.76)D3.7 (1.8)0.43 (0.11, 1.65)0.24Distal colonbA1B1.30 (0.26, 2.52)C3.84 (0.37, 39.2)D0.17 (0.02, 1.27)0.12Proximal coloncA1B3.22 (0.26, 39.8)C0.94 (0.11, 7.87)D0.91 (0.11, 7.54)0.78SD Standard deviationaOdds of tagging score of 5 (best) compared to regimen AbRectum, sigmoid and descending colon combinedcTransverse, ascending colon and caecum combined\nThe average per-segment fluid tagging score was 2.5 (SD 0.8), 2.3 (SD 1.0), 2.5 (SD 0.9) and 2.4 (SD 1.2) for regimens A to D respectively. The odds of homogeneous tagging (i.e score 3) did not differ across the four regimens either overall or between the proximal and distal colon (P\u2009=\u20090.65\u20130.95). In total, 4% (6\/144), 1% (2\/150), 2% (3\/144) and 5% (6\/132) of segments respectively contained non-tagged fluid (score 1) (Fig.\u00a04).\nFig.\u00a04A 75-year-old female with change in bowel habit. Axial CT colonographic image showing failure of fluid tagging (arrow) (grade 1)\nOverall 33% (8\/24), 44% (11\/25), 38% (9\/24) and 36% (8\/22) of patients receiving regimens A to D respectively had at least one segment with layering of tagged fluid (P\u2009=\u20090.89) (Fig.\u00a02).\nFluid tagging density\nIn terms of mean HU of the tagged fluid (excluding the least tagged layer if tagging was layered), there was no significant difference among the four regimens, either overall or between the proximal\/distal colon (P\u2009=\u20090.14\u20130.86). The mean HU of tagged fluid was 522 (SD 283), 504 (SD 395), 430 (SD 268), and 491 (SD 269) for regimens A to D respectively.\nHowever in those segments with layering of contrast, the maximum attenuation (HU) of the least tagged layer was significantly higher for regimens C and D than for regimens A and B (P\u2009=\u20090.002) [77 (SD 105), 119 (SD 139), 155 (SD 118) and 174 (SD 122), for regimens A to D respectively].\nPatient experience\nA total of 89 of 95 (94%) patients completed the questionnaire (Table\u00a01), although responses were incomplete in 4. Demographic data for the present study cohort (50 female, mean age 64\u00a0years, range 50\u201385\u00a0years) were not significantly different from the 69 historical controls (36 female, mean 63\u00a0years, range 35\u201385\u00a0years) [16]. Other than sleep disturbance (worse after reduced preparation) (P\u2009=\u20090.01), there was no significant difference in reported tolerance of the reduced and full bowel preparation regimens for any of the factors tested.\nFollow-up questionnaire\nSixty-nine (73%) patients returned the follow-up questionnaire (Table\u00a02). Overall 95% tolerated the reduced preparation regimen well or fairly well, and most (83%) found drinking the tagging agents acceptable. Although most [51\/69 (74%)] found the additional preparation required for colonoscopy \u201cno problem\u201d, a majority (61%) found the reduced preparation regimen \u201cbetter\u201d or \u201cmuch better\u201d than the full preparation required for colonoscopy.\nDiagnostic performance\nFive patients (one each from regimens A, B, and C and two from regimen D) were excluded from the performance analysis due to a new diagnosis of inflammatory bowel disease (all presenting with rectal bleeding). A further patient (from regimen B) was excluded after refusing colonoscopy. Of the remaining 89 patients, 68 had either normal colonoscopy or diminutive polyps (\u22645\u00a0mm) only, and 21 had at least one polyp \u22656\u00a0mm or cancer. Colonoscopy was incomplete in 10\/89 (11%) reaching the transverse colon in 6, sigmoid in 3 and hepatic flexure in 1. Reasons for failure were obstructing stricture (1), severe diverticulosis (1), tortuous colon (5), and pain (3). Only those segments visualised at colonoscopy were included in the assessment of diagnostic performance.\nPer polyp analysis\nIn total there were 9 polyps \u226510\u00a0mm (all adenomatous), 12 polyps 6\u20139\u00a0mm (10 adenomatous, 2 hyperplastic) and 72 polyps \u22645\u00a0mm (46 were recovered for histology, of which 26 were adenomatous, 15 hyperplastic and 5 normal mucosa). One 6-mm polyp and two 5-mm polyps were found only on re-look endoscopy after segmental unblinding of the CTC report. No CTC-detected polyps were classified as false positives due to segmental or size mismatching with colonoscopy.\nSummed across regimens A\u2013D, detection of cancer, polyps \u226510\u00a0mm, 6\u20139\u00a0mm and \u22645\u00a0mm was 2\/2 (100%), 8\/9 (89%), 9\/12 (75%) and 23\/72 (32%) respectively (Fig.\u00a05) (Table\u00a04). In total there were only four false positives \u22656\u00a0mm (Figs.\u00a06 and 7), with no significant difference between regimens (P\u2009=\u20090.15).\nFig.\u00a05A 71-year-old male with rectal bleeding. Axial CT colonographic image demonstrating a 30-mm rectal cancer (arrow). Note the adjacent well-tagged residue (arrowheads)Table\u00a04Polyp detection overall and according to tagging regimenRegimenPatient numberDetection cancer, n (%)Detection 10\u00a0mm+, n (%)Detection 6\u20139\u00a0mm, n (%)Detection 1\u20135\u00a0mm, n (%)False positive 10\u00a0mm+, nFalse positive 6\u20139\u00a0mm, nFalse positive 1\u20135\u00a0mm, nP valueaA232\/2 (100%)1\/2 (50%)N\/A3\/10 (30%)0170.15B23N\/A3\/3 (100%)5\/6 (83%)10\/30 (33%)01b9C23N\/A1\/1 (100%)2\/2 (100%)6\/20 (30%)001D20N\/A3\/3 (100%)2\/4 (50%)4\/12 (33%)1c12Overall892\/2 (100%)8\/9 (89%)9\/12 (75%)23\/72 (32%)1319N\/A Not applicableaComparison of false positives across regimens using one-way ANOVAbIn patient with confirmed 8-mm polypcIn patient with confirmed 10-mm polypFig.\u00a06A 54-year-old male with change in bowel habit. Axial CT colonographic image of a 6-mm filling defect in the rectum reported as a polyp. No lesion was found at colonoscopy with segmental unblinding suggesting the lesion was untagged faecal residueFig.\u00a07Overall number of false positives according to size and regimen (n\u2009=\u200989)\nPer patient analysis\nAcross all regimens sensitivity, specificity, positive and negative predictive values for detection of patients with any lesion \u22656\u00a0mm were 96%, 97%, 0.9 and 0.96 respectively (Table\u00a05). \nTable\u00a05Per-patient performance overall and according to tagging regimenRegimenPatient numberPolyp \u22656\u00a0mm incl. cancer, n (%) [95% confidence limits]Polyp \u226510\u00a0mm incl. cancer, n (%) [95% confidence limits]SensitivitySpecificityPPVNPVSensitivitySpecificityPPVNPVA233\/4 (75%) [33\u2013100%]18\/19 (95%) [85\u2013100%]0.750.953\/4 (75%) [33\u2013100%]19\/19 (100%) [100\u2013100%]1.00.95B237\/7 (100%) [100\u2013100%]16\/16 (100%) [100\u2013100%]1.01.03\/3 (100%) [100\u2013100%]20\/20 (100%) [100\u2013100%]1.01.0C233\/3 (100%) [100\u2013100%]20\/20 (100%) [100\u2013100%]1.01.01\/1 (100%) [100\u2013100%]22\/22 (100%) [100\u2013100%]1.01.0D205\/7 (71%) [38\u2013100%]12\/13 (93%) [78\u2013100%]0.830.863\/3 (100%) [100\u2013100%]17\/17 (100%) [100\u2013100%]1.01.0Overall8918\/21 (96%) [93\u2013100%]66\/68 (97%) [93\u2013100%]0.9a0.9610\/11 (91%) [74\u2013100%]78\/78 (100%) [100\u2013100%]1.0b0.99NPV Negative predictive value, PPV positive predictive valueaPrevalence of abnormality\u2009=\u20090.24bPrevalence of abnormality\u2009=\u20090.12\nReader confidence\nThe mean segmental observer confidence for excluding a polyp \u22656\u00a0mm was 90, 89, 97 and 93% for regimens A to D respectively. There was no significant difference among the regimens, either overall or in the proximal\/distal colon (P\u2009=\u20090.15\u20130.57).\nDiscussion\nIn accordance with previous studies [6, 8, 10], we found patients in general preferred reduced preparation to the full purgation required for colonoscopy, although this preference was much less than expected (only 61% preferred the reduced preparation). We defined the laxation used as \u201creduced\u201d in comparison to the endoscopic preparation used at our institutions which includes an additional 18\u00a0g of magnesium citrate. We also used senna rather than bisacodyl as used by Lefere et al. [9]. Both are stimulant laxatives with a similar mode of action and effect at the doses administrated, although senna was preferred as it is widely used at our institutions. However a combination of 13\u00a0g of senna and 18\u00a0g magnesium citrate clearly produces relatively strong purgation, reflected in the induced symptoms (77% of patients opened their bowels over five times) and marginal patient preference. It would therefore seem reasonable to study reduced laxation further, perhaps omitting the senna and\/or reducing the dose of magnesium citrate. It is however important to realise that whereas normally patients took no solids by mouth for 24\u00a0h prior to colonoscopy, in the present study they were permitted to eat from a low-residue diet kit, an issue we did not specifically address with our questionnaires, thereby potentially underestimating this benefit.\nRecent data have questioned whether CTC is actually better tolerated than conventional colonoscopy [18], but because full bowel purgation is often cited by patients as the worst part of any colonic examination [1, 2], it is assumed that reduced laxative CTC will improve patient compliance (notably in a screening setting). However this assumption has not been proven in prospective trials, and we cannot extrapolate the preferences we found into increased compliance with CTC. Indeed it could be argued the laxative regimen we used would have relative little impact on compliance in a screening setting, given the side-effect profile.\nThe overall quality of bowel preparation was good, with all four regimens producing at least 76% of segments with either no or scattered residue only. This is similar to data reported by Lefere et al. [9], who combined 16.5\u00a0g of magnesium citrate with biscodyl tablets and suppository. Interestingly, regimen D, which included 15\u00a0ml meglumine amidotrizoate, resulted in significantly better preparation than the other regimens, possibility due to a \u201cwashing effect\u201d of the iodine-based contrast, incorporating solid residue into a more fluid solution [10]. Although we used a small dose, meglumine amidotrizoate is also known to have a laxative effect that may also have added to the superior cleansing.\nTagging of solid residue was in general good, with no significant difference between the regimens tested. This suggests barium-based tagging can be simplified to a 1-day regimen only, when combined with reduced cathartic preparation. Similarly all regimens produced an average tagged fluid density of around 500\u00a0HU. Recent phantom data suggest that although submerged polyp conspicuity is optimised at 700\u00a0HU, it remains good at 500\u00a0HU [19]. We did however demonstrate that fluid tagging could be manipulated by additional oral agents on the morning of CTC. As it is non-miscible with water, barium often produces a layering effect with lower attenuation fluid sitting above denser barium. Although there was no significant difference in layering among the regimens, the addition of morning 2.1% barium sulphate or meglumine amidotrizoate significantly increased the attenuation of the non-dependent fluid layer. It is arguable whether this will have significant impact clinically, not least because fluid moves between supine and prone positions, but it may have implications for subtraction software. Zalis et al. have recently demonstrated suboptimal subtraction when high-density barium is used as the sole tagging agent [10]. It is interesting to speculate whether manipulation of fluid tagging could optimise subtraction. It should also be noted our regimen mildly restricted fluid intake the day prior to CTC, which may have influenced our fluid tagging results.\nOne major advantage of barium as a tagging agent is its inert nature and safety. Although iodinated contrast medium produces more homogeneous fluid tagging, this is mainly of clinical importance if subtraction software is being used. While the risks of allergy to oral iodinated contrast are minimal, adverse events do occur [20, 21]. Our data suggest good results can be achieved using only barium.\nUltimately, diagnostic performance is the best measure of the success of any reduced-laxative protocol. Reassuringly overall, we prospectively found high diagnostic performance when compared to colonoscopy. Our data confirm again that full bowel preparation is not required to maintain diagnostic accuracy for CTC, assuming adequate reader training [22]. Importantly, there were only four false positive polyps over 6\u00a0mm, and across all regimens the positive predictive value for patients with any lesion \u22656\u00a0mm and \u226510\u00a0mm was 0.9 and 1.0 respectively (Table\u00a05).\nOur study does have weaknesses. It could be argued that we used a relatively harsh laxative regime given that good results have been reported without any laxative at all [5, 6]. However the vast majority of CTC worldwide is still performed using full bowel preparation, and in the authors\u2019 opinion, significant changes in practice are most likely to follow gradual reduction of purgation, which allows radiologists to become comfortable with this approach. However we acknowledge that if there was good evidence of acceptable performance data using CTC without prior laxation, this may well rapidly become widely implemented by the CTC community. We compared patient symptomatology with a historical cohort, and although well-matched in demographic terms, we cannot exclude bias completely. We did not instruct colonoscopists to grade the quality of bowel preparation and, in particular, the influence of retained barium on mucosal visibility, although no endoscopy failed due to incomplete bowel preparation. It could be argued that reduced preparation regimens are best suited to increase compliance in asymptomatic screening populations [23]. However for pragmatic reasons we used symptomatic patients, as we do not have access to a large screening population. Finally, although we were able to show high diagnostic performance for reduced-preparation CTC overall, given the relatively low number of polyps, we were unable to meaningfully compare across regimens.\nIn conclusion, a combination of reduced laxatives and a simple tagging regimen based on 40% barium sulphate the day prior to CTC maintains acceptable diagnostic accuracy. Three doses of 20\u00a0ml 40%w\/v barium sulphate are as effective as more complex regimens, but fluid tagging can be manipulated by addition of dilute barium or meglumine amidotrizoate on the morning of CTC, the latter also reducing the volume of residual stool.","keyphrases":["colonography","computed tomography","cathartics","barium sulfate"],"prmu":["P","P","P","M"]}
{"id":"Eur_J_Pediatr-4-1-2292480","title":"The efficacy of anakinra in an adolescent with colchicine-resistant familial Mediterranean fever\n","text":"Colchicine is the treatment of choice in familial Mediterranean fever (FMF) for the prevention of both attacks and secondary amyloidosis. The overall nonresponder rate is about 5\u201310%. Anakinra is known to have good effectiveness in a severe autoinflammatory syndrome [chronic infantile neurological cutaneous and articular (CINCA) syndrome] and other recurrent hereditary periodic fevers. Pyrin\u2014the protein involved in FMF\u2014has a role in activating the proinflammatory cytokine interleukin (IL)-1\u03b2. We report the effectiveness of the addition of an IL-1-receptor inhibitor (anakinra) to colchicine in controlling the febrile attacks and acute phase response in an adolescent with FMF resistant to colchicine.\nCase report\nA 15-year-old girl with familial Mediterranean fever (FMF) was admitted to our hospital because of the persistence of recurrent fever attacks (twice a month), accompanied by chest and abdominal pain with headache, despite taking colchicine at a high dose (2\u00a0mg\/kg per day). She suffered severe discomfort and complained of several school absences.\nAcute phase reactants [erythrocyte sedimentation rate (ESR) 110, C-reactive protein (CRP) 43\u00a0mg%] and serum interferon-\u03b3 (125.4\u00a0pg\/ml) were high during the crises. There was no proteinuria, but given the severity of the clinical picture, we performed a rectal biopsy to definitely exclude the presence of amyloidosis and it was negative.\nAnakinra was started at 50\u00a0mg\/day (1\u00a0mg\/kg per day) subcutaneously, without stopping colchicine. Approval was obtained from an Ethics Committee (Burlo Garofolo Health Authority).\nIn the following 3\u00a0months the patient developed only three mild episodes of abdominal pain without fever, which resolved in a few hours. During these episodes acute phase reactants were negative. Moreover, serum interferon-\u03b3 was low (23\u00a0pg\/ml).\nAfter 3\u00a0months, anakinra was discontinued to confirm its real efficacy, without stopping colchicine. Following this interruption, the girl complained of eight fever attacks in 3\u00a0months accompanied by abdominal pain, vomiting, chest pain, and headache. Anakinra was reintroduced. Since then (15\u00a0months of follow-up), the patient has been in good health, without any fever attacks or abdominal or chest pain.\nThere have been no adverse effects due to anakinra, apart from minor local stinging and erythema at the injection sites, which gradually diminished.\nDiscussion\nColchicine is the recommended treatment of FMF since it can prevent the febrile attacks and the development of amyloidosis, even though a few patients develop amyloidosis also when on colchicine treatment [3, 5]. However, approximately one third of the patients have a partial remission and about 5\u201310% are nonresponders [3]; another 2\u20135% do not tolerate the drug well [3]. In single case reports several drugs (interferon-\u03b1, infliximab, thalidomide, etanercept) have been suggested as a possible alternative treatment for these cases [3, 6].\nAnakinra is a recombinant form of the human interleukin (IL)-1-receptor antagonist that targets type I IL-1-receptor which is expressed in many tissues. There are some studies and case reports that show a good efficacy of anakinra in a severe autoinflammatory syndrome (CINCA, chronic infantile neurological cutaneous and articular syndrome) [2] and other recurrent hereditary periodic fevers [4]. Despite the elevated costs (approximately 50 \u20ac per 100\u00a0mg) its use appears justified in some inflammatory diseases that lack alternative treatments.\nSince pyrin\u2014the protein involved in FMF\u2014activates the precursor form of IL-1\u03b2 into its biologically active fragments there is a rationale for testing anakinra in FMF [1, 5]. Moreover, anakinra did appear to be effective in suppressing acute phase reactants in a patient with FMF (a nonresponder to the treatment with colchicine) and amyloidosis [1].\nIn our case, the addition of anakinra to colchicine had a dramatic therapeutic benefit without relevant side effects. As the febrile attacks recurred after discontinuing anakinra and disappeared again after reintroduction, we believe in the real therapeutic efficacy of this drug. In light of the present evidence [3, 5], colchicine should be continued in order to prevent further developments of amyloidosis.","keyphrases":["anakinra","familial mediterranean fever","colchicine resistance"],"prmu":["P","P","R"]}
{"id":"Mol_Imaging_Biol-3-1-1915656","title":"Attenuation-Corrected vs. Nonattenuation-Corrected 2-Deoxy-2-[F-18]fluoro-d-glucose-Positron Emission Tomography in Oncology, A Systematic Review\n","text":"Purpose To perform a systematic review and meta-analysis to determine the diagnostic accuracy of attenuation-corrected (AC) vs. nonattenuation-corrected (NAC) 2-deoxy-2-[F-18]fluoro-d-glucose-positron emission tomography (FDG-PET) in oncological patients.\nIntroduction\nThe attenuation of photons originating from the subject before they are detected by the camera is a generic limitation of nuclear medicine imaging. This attenuation can lead to image distortion and impairs adequate quantification. Attenuation correction has been commonly employed in 2-deoxy-2-[F-18]fluoro-d-glucose (FDG) imaging in an attempt to correct for these effects. With positron emission tomography (PET) scanners, this is accomplished by transmission scanning using a radionuclide source, such as germanium-68 or cesium-137, and with PET\/computed tomography (CT) using CT. With respect to visual interpretation of the images, the added value of attenuation correction has been controversial. Whereas attenuation correction provides a more realistic image of FDG distribution, its application significantly increases acquisition times on standard full-ring (FR) PET scanners. In addition, the performance of attenuation correction can introduce noise and even artifact. Paradoxically, even if the nuclear medicine community sees attenuation correction, or the lack of it, as a potential effect-modifier of test accuracy, its impact is rarely accounted for in systematic reviews on the diagnostic accuracy of PET. As a result, the impact of attenuation correction on lesion detectability and interpretation of PET for oncological purposes is not well established. With PET\/CT scanning, it is customary to evaluate either modality (primarily to account for artifacts), but one needs to know how to deal with discrepancies.\nThe objective of this systematic review and meta-analysis was to determine the diagnostic accuracy of nonattenuation-corrected (NAC) and attenuation-corrected (AC) FDG-PET in oncological patients. We studied the effects of attenuation correction for both FR-PET and dual-head coincidence PET (DH-PET), and as a function of different body locations (head\/neck, chest, abdomen\/pelvis).\nMaterials and Methods\nLiterature Search\nA computer-aided literature search was performed in both Medline and Embase databases without time range or language restrictions, applying controlled vocabulary (MeSH and EMTREE keywords, respectively) as well as free text words. The search date was February 10, 2006. The search strategy (Appendix) included terms for PET with FDG, modified from Mijnhout et al. [1] as well as search terms identifying both radionuclide and X-ray transmission, emission, attenuation correction, and oncological studies in humans. In addition, the reference lists of the eligible articles were reviewed to ensure that relevant articles had not been missed.\nStudy Selection\nFrom the list of retrieved articles, articles were initially evaluated for eligibility on the basis of title and abstract by two independent reviewers (UJ, PR). If there was uncertainty as to whether an article was eligible for inclusion, the entire article was reviewed. Inclusion criteria were (1) clinical studies evaluating FDG imaging with and without attenuation correction in oncology patients; (2) study population of at least ten patients; (3) sufficient detail to reconstruct a 2\u2009\u00d7\u20092 contingency table expressing FDG imaging results by disease status, or sufficient detail to reconstruct relative lesion detection measurement of AC vs. NAC imaging; and (4) studies utilizing FR-PET and\/or DH-PET. We excluded abstracts, editorials, and reviews, although the latter two categories were used for cross-referencing.\nMethodological Quality Assessment\nThe methodological quality of each article was independently assessed by each reviewer in terms of internal and external validity (Table\u00a01), based on the Cochrane Methods Group in Screening and Diagnostic Tests, modified for our area of interest [2]. Internal validity items focus on whether a valid reference test was used and whether this reference test was uniformly and independently applied and interpreted as well the type of study design. The external validity items evaluate the applicability of the results in terms of the type of patient population and spectrum, demographics, the inclusion\/exclusion criteria, the knowledge of previous test\/clinical information that could influence interpretation, and the index test characteristics. Items were scored as positive, negative, or unclear.\nTable\u00a01Methodological assessment of individual diagnostic studies: criteriaTestCriteriaA. Internal study validity\u00a0Al. Valid reference testHistology, AC FR or DH coincidence PET\u00a0A2. Blind measurement of reference test(s) without knowledge of index testAssessment of reference test independent of index test(s) results\u00a0A3. Avoidance of verification biasChoice of patients assessed by reference test independent of index test result\u00a0A4. Index test(s) interpreted independently of all clinical informationMentioned in publication\u00a0A5. Prospective studyMentioned in publicationB. External study validity\u00a0B1. Spectrum of diseasesLocalization of disease described (selected or general)\u00a0B2. Demographic informationAge and sex given\u00a0B3. Inclusion criteria describedMentioned in publication\u00a0B4. Exclusion criteria describedMentioned in publication\u00a0B5. Avoidance of selection biasConsecutive series of patients\u00a0B6. Standardized execution of index test(s)Described technical aspects of index test(s)C. Reproducibility describedMentioned in publication\nData Extraction and Quantitative Analysis\nIn addition to methodological quality assessment, data related to the type of camera, the FDG dose, the time interval between injection and imaging, the transmission and emission acquisition protocols, the reconstruction protocol, and the interpretation protocol were independently extracted from each study by each reviewer. For studies where it was possible, a contingency 2\u2009\u00d7\u20092 table was constructed. Disagreements were solved by consensus.\nFor studies using an independent gold standard (histopathology), we determined the sensitivity and specificity of the index tests using the number of true positive, false positive, true negative, and false negative results from the 2\u2009\u00d7\u20092 contingency table. Furthermore, we calculated the \u201crelative lesion detection,\u201d defined as the percentage of lesions scoring equally positive or negative with NAC vs. AC images. We performed a subgroup analysis for different locations of lesions and analyzed sensitivity, specificity, or relative lesion detection of NAC vs. AC for lesion location in the head and neck region, the chest, and the abdominopelvic region. In cases of discrepancy of relative lesion detection between NAC and AC, we extracted data to analyze whether this was related to lesion size and\/or intensity.\nThe statistical diagnostic heterogeneity of the sensitivity and specificity per index test across studies was tested by the chi-square test. In case of statistical heterogeneity of DH- or FR-FDG-PET imaging, a random effect model for pooling was used, whereas in case of statistical homogeneity, a fixed-effect model was used. Sensitivity, specificity, and relative lesion detection were pooled independently, all pooled estimates are presented with 95% confidence intervals (95% CI). The logit transformed sensitivity, specificity, relative lesion detection, and corresponding 95% CI of the index tests were compared using z-test statistics. A p value of less than 0.05 was considered significant.\nAll statistical analyses were performed with the SPSS 11.0.01 program for Windows (version 11.0.1., SPSS, Chicago, IL, USA).\nResults\nThe search strategy yielded 2,202 references, 1,477 in Medline and 725 in Embase on February 10, 2006. Of the Embase references, 370 were also included in Medline, leaving a total of 1,832 unduplicated references. On the basis of title and abstract alone, 1,806 references were excluded. After review of the full text of the remaining 26 articles, an additional 11 studies [3\u201312] proved to be ineligible because they did not perform a direct comparison of the yield of NAC vs. AC images in oncological patients. One study [13] was excluded because it was published in abstract form only. Another study [14] was excluded because it was published in Japanese and was not readily translatable. Finally, the study of Hustinx et al. [15], who evaluated the effect of attenuation correction in abdominal tumors for a sodium iodide crystal (NaI) PET scanner, was excluded because no 2\u2009\u00d7\u20092 contingency tables could be constructed. Eventually, we included 12 studies for review [16\u201327].\nA summary of the methodological quality assessment results can be found in Table\u00a02. Methodological quality was scored as negative when quality items were unclear or absent in the original article. In a minority (4\/12\u2009=\u200933%) of studies, histology served as the reference test. However, nine of the 12 studies provided a direct comparison of AC and NAC PET. In three studies, blind measurement of reference test was performed without knowledge of index test. All but one study avoided verification bias. In four studies, the index test(s) was evaluated independently of all clinical information. All studies provided information about the spectrum of diseases being evaluated and standardized the execution of the index test(s). Almost all studies (11\/12\u2009=\u200992%) described the demographics of the study population and inclusion criteria. However, only one study mentioned specific exclusion criteria. Six studies were prospective. Only two of the 12 studies specifically mentioned including consecutive patients, and only three studies specifically described the reproducibility of their results.\nTable\u00a02Quality assessment of included studiesStudyYearA1A2A3A4A5B1B2B3B4B5B6CBleckmann1999++++\u2212+\u2212+\u2212\u2212+\u2212Chan2001+\u2212+\u2212\u2212+++\u2212\u2212+\u2212Delbeke2001+\u2212+\u2212++++\u2212\u2212+\u2212Even-Sapir2004+\u2212+\u2212++++\u2212++\u2212Kotzerke1999+\u2212+\u2212\u2212+++\u2212+++Lonneux1999+\u2212+\u2212+++\u2212\u2212+++Nakamoto2002++++++++\u2212\u2212++Reinhardt2005+\u2212+\u2212\u2212++++\u2212+\u2212Schauwecker2003+\u2212\u2212\u2212\u2212+++\u2212\u2212++Weber1999+\u2212+\u2212++++\u2212\u2212+\u2212Zimny1999++++\u2212+++\u2212\u2212+\u2212Zimny2003+\u2212++++++\u2212\u2212+\u2212\nMeta-Analysis\nThree FR-PET studies were eligible for pooling of sensitivity on a patient basis [22, 24, 25]. The pooled sensitivities for AC and NAC FR-PET were 64% (95% CI 52\u201374%) and 62% (95% CI 51\u201373%), respectively (n\u2009=\u2009182 patients). Two FR-PET studies provided data that allowed pooled analysis of specificity [22, 24]. Weber et al. [25] could not be included as there were no patients without disease. The pooled specificities for AC and NAC FR-PET were 97% (95% CI 92\u201399%) and 99% (95% CI 95\u2013100%), respectively (n\u2009=\u2009155 patients). For DH-PET, only one study provided data on sensitivity and specificity [27].\nRelative lesion detection for NAC vs. AC PET was pooled for 11 studies, which are demonstrated in Fig.\u00a01. Lesion detection of NAC FR-PET vs. AC FR-PET was 98% (95% CI: 96\u201399%) for n\u2009=\u20091,012 lesions (pooling of n\u2009=\u20097 studies); 79% of which were classified FDG positive at AC FR-PET. Lesion detection of NAC DH-PET vs. AC DH-PET was 88% (95% CI: 81\u201394%) for n\u2009=\u2009288 lesions (pooling of n\u2009=\u20094 studies); 74% of which were classified as FDG-positive at AC DH-PET.\nFig.\u00a01Pooled lesion detection of NAC vs. AC images for FR-PET and DH-PET.\nIn addition, we evaluated the relative lesion detection depending on body location (head\/neck, chest, abdomen\/pelvis) in the four FR-PET [20, 21, 23, 26] and in the three DH-PET studies that provided sufficiently detailed information [17, 18, 26]. The relative sensitivity and specificity based on body location could not be calculated due to an insufficient number of studies. For FR-PET, we found similar relative lesion detection for the three body locations: 95% for head\/neck (95% CI 84\u201398%, n\u2009=\u200961 lesions), 97% for the chest (95% CI 94\u201399%, n\u2009=\u2009396 lesions), and 97% for the abdomen\/pelvis (95% CI 93\u20130.99%, n\u2009=\u2009205 lesions). For DH-PET, relative detection rates for NAC were not significantly different for the various body sites: 78% in the abdomen\/pelvis (95% CI 65\u201388%; 53 lesions), 84% in the chest (95% CI 74\u201391%; 136 lesions), and 90% in the head\/neck area (95% CI 73\u201397%; 38 lesions). However, in chest (p\u2009=\u20090.000089) and abdomen\/pelvis (p\u2009=\u20090.0037), the relative detection rates with NAC (vs. AC) for DH-PET were significantly lower than those obtained with FR-PET.\nA comprehensive analysis of the potential association of relative detection and lesion size and\/or intensity, for lesions with discrepant AC and NAC results, was not possible due to a lack of detailed information. We summarized the results in Table\u00a03: findings of Bleckmann et al. and Reinhardt et al. [16, 23] suggest that AC and NAC discrepancies may relate to (intrapulmonary) lesion size with more discrepancies occurring with smaller lesions at the subcentimeter level (an average of 3% of lesions were correctly detected with NAC and not with AC). The single discrepant lesion in the study of Weber et al. concerned a <1-cm lesion in the mediastinum [25]. However, the discrepant lesions in the studies of Nakamoto et al., Schauwecker et al., and Delbeke et al. included both small- and moderate-sized lesions (in relation to lesions included each study) [18, 22, 24]. In the study of Schauwecker et al., the discrepant lesions demonstrated SUVmax values ranging from 1.8 to 2.6, whereas, in the study of Delbeke et al., the two discrepant lesions demonstrated only mildly enhanced uptake on AC images and equivocal uptake on NAC images.\nTable\u00a03Evaluation of discrepant lesions between AC and NAC images with respect to lesion size and intensityStudyCamera typeNumber of discrepant lesionsSize rangeIntensity (semiquantitative or qualitative)Bleckmann et al.FR-PET5<1\u00a0cmNot givenNakamoto et al.aFR-PET11.8\u00a0cmNot givenReinhardt et al.FR-PET60.5\u20131.1\u00a0cm79\/174 lesions demonstrated discrepancy in qualitative lesion intensity: 72\/174 lesions demonstrating higher intensity (i.e., better visibility) on NAC imagesSchauwecker et al.aFR-PET40.8\u20133.9\u00a0cm31.8\u20132.6 (SUVmax)Weber et al.FR-PET10.8\u00a0cmNot givenDelbeke et al.DH-PET21.0\u20133.0\u00a0cm\u201cMild uptake\u201d at AC, \u201cequivocal uptake\u201d at NACaHistology used as gold standard and detailed information given only for true positive lesions\nDiscussion\nThe cumulated evidence summarized in this systematic review of oncological FDG imaging studies suggests that the accuracy of attenuation and nonattenuation corrected FR-PET are similar. However, with DH coincidence imaging NAC images detect 12% less lesions than AC images, without prominent differences between body areas.\nAlthough in the nuclear medicine field attenuation correction is generally seen as an important issue, it is surprising to find that several large systematic reviews did not thoroughly consider this as a potential effect-modifier. Gould et al. performed systematic reviews on FDG PET in pulmonary lesions [28] and mediastinal lymph node staging in non-small cell lung cancer [29]: in the former review, the item was not mentioned, and in the latter, attenuation correction was an item of study quality, but no analysis of potential impact was performed.\nThe choice of the reference test is obviously relevant in studies on test accuracy. In oncology, histopathology is the typical endpoint. Of the 12 eligible studies, four used histology as an independent gold standard. Meta-analysis of sensitivity and specificity was only possible for FR-PET, and we found no significant difference for either measure. We chose to use the AC detection rate as an alternative reference test, which defines the relative lesion detection of NAC vs. AC images. This choice theoretically biases in favor of AC: Bleckmann et al. and Reinhardt et al. [16, 23] reported an average of 3% more true positive lesions with NAC FR-PET. However, we expect that the resulting error is small because, in the comparison with histopathology, false positive rates were quite low for either modality. Despite this theoretical negative bias towards NAC, similar lesion detection rates were observed with both AC and NAC for FR-PET. Hence, attenuation correction may not contribute to the detection of malignancy using FR-PET. Conversely, with DH-PET, AC images demonstrated a significantly higher detection rate as opposed to NAC images, which is surprising given that AC images are usually significantly noisier than NAC images. We postulate that this may be secondary to differences in reconstruction\/filtering algorithms.\nIn addition, there are limitations associated with performing a meta-analysis and data pooling, such as the homogeneity of the data and the quality of the published studies. Homogeneous data have higher statistical strength than heterogeneous data. The data in our study were heterogeneous so that we used a random effect model for pooling. In addition, the statistical strength of the meta-analysis is limited by the quality of the published studies included in it. As mentioned earlier and summarized in Table\u00a02, the studies had several quality limitations. Finally, meta-analyses are limited by publication bias, which biases towards the publication of favorable results or popular subjects.\nWe were surprised by the limited number of good comparative studies evaluating the value of attenuation correction. It appears that attenuation correction has been accepted as the standard of practice without sound scientific evidence to support it.\nThe introduction of PET\/CT machines has made the time constraints associated with transmission scanning less of an issue. However, PET\/CT is not a panacea; X-ray transmission scanning has its own problems and numerous PET\/CT publications have demonstrated artifact that can be introduced with X-ray transmission scanning [5, 30\u201345]. Furthermore, in the study of Reinhardt et al. [23], a significantly improved visibility was demonstrated for 41% of lung metastases with NAC images as opposed to CT-AC images. This higher visibility for NAC images was even more pronounced for lesions smaller than 1\u00a0cm. These findings underline that even as PET\/CT use becomes more widespread, evaluation of both NAC and AC images should remain an integral part of image interpretation, and not just to recognize image artifacts. At the same time, NAC vs. AC discrepancies at PET\/CT offer an obvious opportunity for further investigation.\nConclusions\nIn this meta-analysis, we found no significant difference in sensitivity, specificity, or relative lesion detectability between AC and NAC FR FDG PET. However, attenuation correction improved lesion detection for DH coincidence imaging.","keyphrases":["positron emission tomography","attenuation correction","deoxyglucose (mesh)","tomography, x-ray computed (mesh)","tomography, emission-computed (mesh)","neoplasms (mesh)","human (mesh)","systematic review (mesh)"],"prmu":["P","P","M","R","M","M","R","R"]}
{"id":"Eur_J_Pediatr-3-1-2151774","title":"Pneumomediastinum: a rare complication of anorexia nervosa in children and adolescents. A case study and review of the literature\n","text":"Spontaneous pneumomediastinum is uncommon in paediatric practice. We describe two cases of spontaneous pneumomediastinum in a child and an adolescent with anorexia nervosa. Thorough investigation failed to reveal any underlying cause for secondary pneumomediastinum. Pneumomediastinum in anorexia nervosa can be caused by not only elevated intrathoracic pressures, but also by the poor quality of the alveolar walls due to malnutrition. The incidence of spontaneous pneumomediastinum in anorexia nervosa is probably higher than that recorded, since it resolves spontaneously and, therefore, it can remain undetected. We conclude that it is our considered opinion that malnutrition associated with anorexia nervosa predisposes for spontaneous pneumomediastinum due to weakness of the alveolar wall and the loss of connective tissue.\nIntroduction\nPneumomediastinum is rarely associated with anorexia nervosa (AN). Approximately 20 cases of pneumomediastinum in anorexia patients are described in the literature. Vomiting, a common symptom in AN, is a known cause of pneumomediastinum [13]. But of the cases reported in the literature, only a few (three) were preceded by vomiting [3, 4, 12]. So, there has to be another cause placing patients with AN at risk for spontaneous pneumomediastinum. We report one case with spontaneous pneumomediastinum as the presenting symptom of AN. In the other case, spontaneous pneumomediastinum is a complication of AN. A review of the literature is given and we discuss the possible pathophysiology of spontaneous pneumomediastinum in patients with AN.\nCase report\nCase 1\nA 13-year-old girl presented with increasing complaints of an unusual crackling sensation and sound in her neck during three days. At that time, she complained of neck pain and headache.\nWhen these complaints started, she had a sore throat with painful swallowing.\nFurther medical history mentioned a Cooper test, a test of physical fitness, at school two weeks earlier. There had been no trauma or injury and no coughing or vomiting.\nPhysical examination showed a respiratory rate of 16 per minute, cardiac rate of 51 per minute, blood pressure of 87\/55\u00a0mmHg and temperature of 36.5\u00b0C. Her height was 150\u00a0cm (SD \u22122), weight 34\u00a0kg (SD \u22121.5) and body mass index (BMI) 15\u00a0kg\/m2 (SD \u22122.5). There was a slight bilateral bulging of the skin of her neck from the mandible to both clavicles, with, on palpation, a crackling sensation as a diagnostic sign of subcutaneous emphysema. Apart from being extremely slim, the skin was normal, with no signs of injury. Cardiac evaluation showed normal heart sounds without cardiac murmur. There was no dyspnoea. On auscultation, there was normal bilateral vesicular breathing. Blood tests revealed no abnormalities. Chest X-ray and computed tomography (CT) scan revealed mediastinal emphysema with extension to the neck region (Fig.\u00a01). There was no pneumothorax and no extension to the retroperitoneum. An oesophageal gastric passage X-ray was normal, without signs of perforation. Laryngopharyngoscopy did not reveal mucosal lesions or signs of submucosal swelling. Electrocardiography was normal, except bradycardia. After admission to the hospital, she showed loss of appetite and further history revealed an anorectic episode, for which, she had been referred by her general practitioner to a psychologist. At the age of 13\u00a0years, she weighed 46\u00a0kg, diminishing to 37\u00a0kg 4\u00a0months later. At the time of admission to the hospital, her weight was 34\u00a0kg at the age of 13\u00a0years and 6\u00a0months. There was absolutely no history of vomiting.\nFig.\u00a01Coronal computed tomography (CT) reconstruction with mediastinal emphysema and subcutaneous emphysema in both axillas and lower neck regions in patient 1 with anorexia nervosa (AN)\nWithin a week, the subcutaneous emphysema disappeared. She was referred to an eating disorders clinic to manage her eating disorder. She never showed any signs of purging during treatment and recovered from her AN.\nCase 2\nA 17-year-old girl with known AN and vomiting presented with extreme malnutrition requiring refeeding. She complained of unusual crackling sensation in her neck during eight days, starting after a choking incident while drinking, followed by coughing. The same day, she felt pressure on her chest. During the following days, her neck became swollen and her voice became hoarse. The swelling had already subsided considerably at the time of presentation.\nPhysical examination showed an extreme cachectic girl of 17\u00a0years with a respiratory rate of 16 per minute, cardiac rate of 64 per minute, blood pressure of 85\/55\u00a0mmHg and temperature of 36.0\u00b0C. Her height was 170\u00a0cm (SD=0), weight 34.4\u00a0kg (SD<<\u22122) and BMI 12\u00a0kg\/m2 (SD<<\u22122.5).\nThere were signs of subcutaneous emphysema in the neck and in both arms. She had dry skin without signs of injury. Cardiac auscultation was normal without cardiac murmur. There was no dyspnoea. Pulmonary auscultation was also normal. Blood tests revealed no abnormalities. Chest X-ray and CT scan revealed subcutaneous emphysema in the neck and mediastinal emphysema with extension to the intra-abdominal, retroperitoneal and epidural regions (Fig.\u00a02). There was no pneumothorax.\nFig.\u00a02Chest CT showing epidural pneumatosis (arrow) in patient 2 with pneumomediastinum and AN\nLaryngopharyngoscopy did not reveal mucosal lesions. The laryngeal mucosa was slightly swollen.\nAfter refeeding, she was referred to an eating disorders clinic to manage her eating disorder.\nWithin two weeks, the subcutaneous emphysema further resolved spontaneously.\nDiscussion\nPneumomediastinum, the presence of free air contained within the mediastinum, usually results from spontaneous alveolar wall rupture and, far less commonly, from disruption of the upper airways or gastrointestinal tract [8].\nSpontaneous pneumomediastinum can be distinguished from pneumomediastinum secondary to traumatic events, such as chest trauma or thoracic surgery, endobronchial or oesophageal procedures, mechanical ventilation or other invasive procedures [13].\nA large and diverse group of factors has been implicated in the development of spontaneous pneumomediastinum. Various respiratory manoeuvres that have in common the development of high intrathoracic pressures may lead to pneumomediastinum. These include Valsalva manoeuvres, coughing, vigorous crying and vomiting. The mechanism of development of spontaneous pneumomediastinum was originally described by Macklin and Macklin as increased intra-alveolar pressure causing the rupture of alveoli with the escape of air into the mediastinum. The mediastinum is subsequently decompressed by further passage of the air into the retroperitoneal and subcutaneous spaces (the Macklin effect) [1, 9]. Whereas primary pneumomediastinum is a relatively benign and self-limited disorder, secondary pneumomediastinum due to oesophageal perforation or Boerhaave\u2019s syndrome is a potentially life-threatening disorder because of the inevitable development of mediastinitis. Primary pneumomediastinum rarely requires treatment as the alveolair rupture heals and the leaked air resolves spontaneously. By contrast, secondary pneumomediastinum due to oesophageal perforation often requires surgical intervention [8].\nPneumomediastinum typically manifests as sudden chest pain or dyspnoea and, less commonly, with dysphagia and hoarseness [8]. Our first patient, however, presented with a sore throat, painful swallowing, complaints of pain in the neck and headache. The second patient presented with initial chest pain and hoarseness. The detection of subcutaneous air in the neck and anterior chest wall and Hamman\u2019s sign, a crunching sound synchronous with the heartbeat, are important physical findings. Radiographically, pneumomediastinum manifests on the posteroanterior projection as a thin radiolucent line parallel to the outline of the heart, as the mediastinal pleura is displaced laterally, and on the lateral projection as the accumulation of air retrosternally [8].\nWhen chest pain and dyspnoea occur, panic attacks must be differentiated from pneumomediastinum. Panic disorders are often seen in patients with AN [6]. As a consequence, we believe that pneumomediastinum should be ruled out in anorexia patients presenting with signs of a first panic attack.\nSpontaneous pneumomediastinum is rarely associated with AN. Only 21 publications related to this association are reported by Pubmed.\nPurging behaviour, such as self-induced vomiting, is common among AN patients. Vomiting is a known cause of pneumomediastinum, placing anorexia patients, who frequently vomit, at risk for pneumomediastinum. However, in only a few of the cases described in the literature was vomiting was the preceding event of the pneumomediastinum. In most reports, like in our patients, pneumomediastinum in anorectic patients is not preceded by vomiting [1, 2, 4, 5, 7, 8].\nIn our patients, the diagnostic procedure did not reveal an oesophageal or upper airway perforation. The pathophysiologic mechanism of air entrance into the mediastinum can be explained by the rupture of an alveolar wall, which is mostly the cause of an air leak into the mediastinum [8]. Based on the literature and our observations, patients with AN are at risk for spontaneous pneumomediastinum, even if they are not vomiting.\nWe speculate that the state of malnutrition contributes to the risk of spontaneous pneumomediastinum.\nAnimal studies reveal that calorie restriction causes a loss of alveoli and a fall in gas-exchange tissue [10, 11] and thinner alveolar walls [10]. To survive periods of not eating, organisms sacrifice comparatively non-essential structures for gluconeogenesis to provide glucose for the brain and amino acids to maintain muscle. Because the total organismal and lung oxygen consumption fall during calorie restriction, some lung tissue is expendable [10].\nAlso in human studies, atrophic changes, such as large alveoli and thin alveolar walls, due to starvation were found in Jewish people in the Warsaw Ghetto [14].\nWith thinner alveolar walls and the loss of alveoli, malnourished individuals are at risk of alveolar wall rupture.\nSince known factors of increased intra-alveolar pressure were absent in our first case and since clinical examination and radiographic study did not reveal an esophageal or upper airway perforation, we must assume that subclinical alveolar leaks with subsequent air dissection, pneumomediastinum and diffuse soft tissue emphysema occurred because of weakness of the alveolar wall and thinning of the connective tissue caused by severe malnutrition. Therefore, even with a minimal increase of intra-alveolar pressure, such as that which may occur during usual daily activities, such as a choking incident, can become the cause of air leaks, as in our second case.","keyphrases":["pneumomediastinum","anorexia nervosa","adolescence","childhood"],"prmu":["P","P","P","U"]}
{"id":"Ann_Surg_Oncol-4-1-2234448","title":"Confirmation of Sentinel Lymph Node Identity by Analysis of Fine-Needle Biopsy Samples Using Inductively Coupled Plasma\u2013Mass Spectrometry\n","text":"Background The sentinel lymph node (SLN) biopsy technique is a reliable means of determining the tumor-harboring status of regional lymph nodes in melanoma patients. When technetium 99 m-labeled antimony trisulfide colloid (99 mTc-Sb2S3) particles are used to perform preoperative lymphoscintigraphy for SLN identification, they are retained in the SLN but are absent or present in only tiny amounts in non-SLNs. The present study investigated the potential for a novel means of assessing the accuracy of surgical identification of SLNs. This involved the use of inductively coupled plasma\u2013mass spectrometry (ICP-MS) to analyze antimony concentrations in fine-needle biopsy (FNB) samples from surgically procured lymph nodes.\nThe sentinel lymph node (SLN) biopsy technique, introduced in the early 1990s, has made it possible to establish the tumor-harboring status of the regional node field in melanoma patients with a minimally invasive procedure.1 Within 3\u00a0years of the publication describing the procedure, the accuracy of SLN biopsy was confirmed in two further studies in which the SLN status was found to accurately reflect the status of the entire lymph node field.2,3 The SLN biopsy technique has since become widely accepted as a method of staging the disease of patients with melanoma who have clinically negative lymph nodes. The status of the SLN is used to determine whether further surgical or adjuvant therapy is appropriate, and to assess patient prognosis. The tumor-harboring status of the SLN is the single most important prognostic factor for melanoma patients, surpassing Breslow thickness, ulceration, and mitotic rate.4\nTo provide accurate prognostic information and to guide appropriate management, it is imperative that all \u201ctrue\u201d SLNs are accurately identified, excised, and assessed for their tumor-harboring status. In a number of cases, a false-negative result may occur, whereby a patient with disease originally assessed as SLN-negative subsequently develops recurrence in a regional node field. It is disturbing to note that false-negative rates of up to 24.8% have been reported from some major melanoma treatment centers and cooperative groups.5\u201316 Available evidence suggests that these failures occur not because the SLN biopsy concept is flawed, but because of methodological and technical shortcomings in nuclear medicine, surgery, and histopathology.\nIn an attempt to identify how many of these false-negative cases might have been because of surgical failures\u2014that is, the removal of a lymph node that was not the SLN identified by the preoperative lymphoscintigraphy\u2014we have developed a technique for assaying antimony in tissue sections. This work is based on the concept that technetium 99\u00a0m\u2013labeled antimony trisulfide colloid (99\u00a0mTc-Sb2S3) particles used for preoperative lymphoscintigraphy in Australia are retained in the SLN but are absent or present in only tiny amounts in non-SLNs. It has been shown that antimony is preferentially retained by the SLN and can be measured in nodal tissue sections by inductively coupled plasma\u2013mass spectrometry (ICP-MS).17 It has also been demonstrated that human tissue contains only negligible amounts of antimony.17 ICP-MS enables validation of the SLN biopsy procedure26 and can identify false-negative results attributable to inaccurate SLN removal.18\nAlthough the morbidity associated with SLN biopsy is low,11 it is an invasive procedure, with a definite risk of complications and morbidity.19,20 In addition, the financial implications of SLN biopsy must be considered. One estimate of the cost of performing a SLN biopsy procedure on an outpatient basis was US$12,19321\u2014a high cost for providing melanoma treatment services to the community. The development of a minimally invasive technique for the identification of SLN metastases may minimize or eliminate the morbidity of the procedure and provide a potential cost benefit. It should be possible to identify SLNs by preoperative lymphoscintigraphy, localize the SLNs with ultrasound, and then perform a percutaneous fine-needle biopsy (FNB) under ultrasound control.22 Preliminary studies of proton magnetic resonance spectroscopy (MRS) analysis of FNB samples from lymph nodes indicate that this technique has the capacity to distinguish nodes containing metastatic melanoma from uninvolved nodes with high sensitivity, specificity, and accuracy.22,23 In addition to accurately determining the tumor-harboring status of the lymph node by this technique, it is also valuable to verify that the sampled lymph node is a \u201ctrue\u201d SLN, particularly if it has been assessed as being free of metastatic tumor.\nThe objective of this study was to develop a method for confirming that a FNB specimen was obtained from a true SLN by measuring the level of antimony (present in radiocolloid used during preoperative lymphoscintigraphy) in the FNB sample by ICP-MS.\nMaterials and Methods\nInstrumentation\nAll measurements were made with an Agilent 7500ce Inductively Coupled Plasma Mass Spectrometer equipped with a MicroMist glass concentric nebulizer, a Quartz-Scott spray chamber (Peltier cooled, 2\u00b0C), and an Agilent three-channel peristaltic pump. The operating conditions were optimized daily to ensure maximum sensitivity. Typical operating conditions are summarized in Table\u00a01. The mass spectrometer was operated in spectrum mode with an integration time of 300\u00a0ms on each of the following isotopes: 121Sb, 123Sb, 103Ru (internal standard\/control).\nTABLE\u00a01.Operating parameters for Agilent 7500ce Inductively Coupled Plasma\u2013Mass SpectrometryParameterConditionRadiofrequency power1500\u00a0WSample depth8\u00a0mmPlasma gas flow 15.0\u00a0L\u00a0min\u22121Carrier gas flow1.05\u00a0L\u00a0min\u22121Peristaltic pump.15 rpsSample read delay60\u00a0sRinsing time45\u00a0sDwell time100\u00a0msReplicates6rps, revolution per second.\nReagents and Chemicals\nAll reagents used were of the highest purity available. Seventy percent double-distilled nitric acid, 37% hydrochloric acid (Arastar), and 30% hydrogen peroxide were obtained from Sigma-Aldrich, Australia. Ultra-high-purity water was produced by passing distilled water through a Milli-Q deionizing system (Millipore, Australia).\nStandards and Certified Reference Materials\nCertified reference material GBW 07601 Human Hair Powder (Langfang, China) was selected because of its certified antimony levels. Multiple samples of the reference material were also analyzed with each batch of samples and during method development to ensure the accuracy and precision of the analytic technique.\nSLN Biopsy Procedure\nPreoperative lymphoscintigraphy was performed to identify the node fields receiving direct lymphatic drainage. This process involved intradermal injections of 99\u00a0mTc-Sb2S3 around the primary cutaneous melanoma site, followed by early and delayed imaging with a scintillation camera.24 The location of the SLNs was marked on the overlying skin by the nuclear medicine physician to assist the surgeon in locating SLNs during surgery.\nThe SLN biopsy procedure was performed within 24\u00a0hours of the radiocolloid injection, so that residual radioactivity in lymph nodes could be measured intraoperatively with a handheld gamma probe (Navigator GPS, RMD Instruments, Watertown, MA). Fifteen minutes before the operative procedure, multiple intradermal injections of Patent Blue V dye (Guerbet, Roissy, France) were made around the primary cutaneous melanoma site. SLN identification was based on visualization of the nodal blue dye staining and results from the preoperative lymphoscintigraphy, with gamma probe confirmation. The experimental protocols of this study were approved by the University of Sydney Ethics Review Board in accordance with the precepts established by the Declaration of Helsinki.\nFNB Samples\nThe FNB collection process involved puncturing the fresh SLN specimen within 30\u00a0minutes of its surgical removal with a 25-gauge needle attached to a 3-mL plastic syringe. Multiple passes were then made through each quarter of the specimen. A total of 47 FNB samples (from 32 presumptive SLNs and 15 nodes considered to be non-SLNs) were collected. All samples were placed in polypropylene vials containing 300\u00a0\u03bcL of phosphate-buffered saline (.27\u00a0mM of KCl, 13.69\u00a0mM of NaCl, 1.52\u00a0mM of KH2PO4; pH 7.2) made up in perdeuterated water (phosphate-buffered saline\u2013D2O) and immediately snap-frozen in liquid nitrogen and stored at \u221270\u00b0C.22\nMicrowave Digestion Procedure\nEach FNB sample was transferred to a polypropylene tube and prepared by the previously described microwave digestion procedure.17 Briefly, the sample was digested in a solution containing 300\u00a0\u03bcL of nitric acid, 300\u00a0\u03bcL of hydrochloric acid, and 500\u00a0\u03bcL of hydrogen peroxide. Each sample was digested five times in a 500\u00a0W microwave oven on the defrost setting for 30\u00a0seconds each time. The digest was then quantitatively transferred to a second polypropylene tube, made up to 10\u00a0g with a 1% nitric acid solution, and assayed by ICP-MS.\nStatistical Analysis\nThe unpaired t-test was used to analyze differences between the levels of antimony in the FNB digests from SLNs and non-SLNs. A P value of less than .05 was considered statistically significant.\nResults\nThe certified concentration of antimony in GBW 07601 is .095\u00a0\u00b1\u00a0.012\u00a0\u03bcg\/g (mean\u00a0\u00b1\u00a0SD). Replicate analyses of reference materials during method validation and sample analysis gave a mean value of .088\u00a0\u00b1\u00a0.007\u00a0\u03bcg\/g and a relative standard deviation of <9%, confirming the accuracy and precision of the method. The limit of detection (LOD) was evaluated by the 3\u03c3 criterion (the LOD is given by mb\u00a0+\u00a03\u03c3b, where mb is the blank measurement mean and \u03c3b is standard deviation of five blank measurements) and found to be .048 parts per billion (ppb).\nThe matrix-matched calibration standards were in the range of 0 to 20\u00a0ppb and gave an R2 value of >.9998 during method validation and sample analysis. The relative standard deviation of the slopes of the calibration curves was found to be <10%.\nThe concentration of antimony in the digested SLNs and non-SLNs was measured, and the results are presented in Tables\u00a02 and 3, respectively. The mean and median concentrations of antimony were .898 and .451\u00a0ppb, respectively, in the SLNs (range, <LOD\u22123.248) and .0145 and .0068\u00a0ppb in the non-SLNs (range, <LOD\u2212.1159). These results indicate that the levels of antimony in FNBs from SLNs were significantly greater than from non-SLNs (P\u00a0<\u00a0.00005) (Fig.\u00a01).\nTABLE\u00a02.Concentration of antimony (ppb) in fine-needle biopsy sample digests collected from sentinel lymph nodesSample No.Antimony concentration (ppb)1<LOD2<LOD3<LOD4<LOD5.06666.09797.17388.29859.302010.308511.325712.380713.381214.386415.390316.404517.497118.607919.640720.724321.8582221.091231.438241.444251.724261.760271.847282.013292.150302.298312.756323.248LOD, limit of detection (.048\u00a0ppb).TABLE\u00a03.Concentration of antimony (ppb) in fine-needle biopsy sample digests collected from nonsentinel lymph nodesSample No.Antimony concentration (ppb)1<LOD2<LOD3<LOD4<LOD5<LOD6<LOD7<LOD8<LOD9<LOD10<LOD11<LOD12<LOD13<LOD14<LOD15.1159LOD, limit of detection (.048\u00a0ppb).Fig.\u00a01.Antimony concentration in digested fine-needle biopsy samples from sentinel lymph nodes and nonsentinel lymph nodes.\nDiscussion\nCurrent standard clinical management of patients with primary cutaneous melanoma includes wide local excision of the primary tumor and SLN biopsy for patients considered to be at high risk of having regional node field metastases.25 Technical failures of the procedure could be attributable to errors in lymphoscintigraphy, sentinel lymphadenectomy, or histologic evaluation. In such cases, the potential for disease recurrence in a previously mapped lymph node basin exists.6,8\u201310,12,14\nA reliable technique to confirm the identity of a SLN has the potential to reduce false-negative results attributable to surgical errors. In a previous study, we demonstrated that the analysis of antimony in sectioned nodal tissue could be used to distinguish SLNs and non-SLNs removed from the same nodal basin.26 This technique can be used within the bounds of currently used histopathologic protocols and has the advantage of being applicable to archival paraffin-embedded tissue.\nIt has also been established that false-negative results may be caused by the removal of a lymph node incorrectly classified as a sentinel lymph node.18 Because neither blue dye nor radioisotope persist after procedures required for microscopic examination, they cannot be used retrospectively to confirm that the \u201ctrue\u201d SLN had been removed. The only other reported method to confirm SLN identity involves the injection, along with the blue dye, of carbon particles that are retained by the node.27,28 However, these large, dense carbon particles may hinder optimal histologic examination by obscuring metastatic melanoma cells. As far as we are aware, this method is not in routine clinical use.\nFNB digests from four nodes presumed to be SLNs (13%) were found to contain extremely low levels of antimony, below the LOD. In a previous study, we also identified SLNs that contained extremely low levels of antimony.26 The most likely reason for the low antimony levels would be inaccurate classification of the node that had been removed as a SLN, when it is in fact a non-SLNs. Other possible explanations to account for the low antimony levels include variable distribution of antimony throughout the node, limited migration of the colloid to the lymph nodes, or tumor deposits preventing colloid uptake. Haigh et al.27 investigated the distribution of carbon dye in SLNs and found a high concentration of carbon particles around the point of entry of afferent lymphatic channels. It is likely that antimony exhibits an analogous distribution pattern. If so, FNB samples that fail to include this region of the SLN may contain falsely low antimony levels. In view of these factors, it would be useful to set a criterion standard for classifying a given node as sentinel or not on the basis of antimony levels assessed by ICP-MS.\nHowever, defining such a criterion on the basis of our small sample set is difficult. In a previous study, we assessed antimony levels in archival tissue sections of paired tumor-positive SLNs and tumor-negative non-SLNs removed from the same regional node field during the same operative procedure from individual patients.26 The aims of this study were to determine whether antimony concentrations could be used to confirm whether removed SLNs were \u201ctrue\u201d SLNs and to differentiate SLNs from non-SLNs. The median concentration of antimony in the SLNs was .526\u00a0ppb and in non-SLNs was .043\u00a0ppb (P\u00a0=\u00a0.004). By using a cutoff point of .18\u00a0ppb (the median concentration of antimony in all SLNs and non-SLNs) to differentiate SLNs from non-SLNs, 20 of 24 SLNs and 20 of 24 non-SLNs were correctly identified by the SLN biopsy procedure.\nAlthough SLN biopsy is a highly accurate method for staging regional lymph nodes, it is an invasive surgical procedure that is costly and is associated with an inherent risk of complication and morbidity.19,20 Pathologic assessment of SLNs is laborious, time-consuming, and costly, and involves examination of multiple hematoxylin and eosin\u2013stained histopathologic sections as well as sections stained with immunohistochemical techniques. The development of a rapid nonsurgical technique that allows detection of metastatic tumor deposits would be of great benefit. It would eliminate the need for surgical excision of lymph nodes and reduce the complexity of pathologic assessment of SLNs. However, to ensure that potential false-negative results are minimized, it is imperative to ensure the accuracy of SLN identification.\nSeveral studies have identified proton MRS as a candidate for the nonsurgical assessment of lymph nodes.22,23 The technique monitors changes in the chemical composition of cells during tumor development and can identify differences that are not morphologically discernible. In these studies, the spectra collected from SLN FNB samples harboring tumor cells contain choline and taurine peaks, which are absent in disease-free samples.23 Another sensitive technique for the detection of melanoma metastases in SLNs is the assessment of tyrosinase mRNA by reverse transcriptase\u2013polymerase chain reaction (RT-PCR).29,30 However, as far as we are aware, RT-PCR assessment of tyrosinase mRNA has not been studied in FNB samples of SLNs. A major potential problem of RT-PCR analysis of FNB samples of SLNs is the issue of false-positive and false-negative results in such specimens and this would need to be assessed before the technique could be used in clinical practice.\nThe results of our preliminary work provide evidence that determining antimony levels in FNB specimens by ICP-MS can confirm SLN identity and can differentiate SLNs from non-SLNs. This technique may be a useful adjunct to other techniques assessing tumor-harboring status of SLNs on FNB specimens such as MRS. Potentially, proton MRS analysis of a FNB from a node confirmed to be a true SLN by ICP-MS could provide a reliable method for determining the tumor-harboring status of SLNs. Although the results are promising and raise the possibility that SLN assessment may be performed on FNB specimens (rather than histologic specimens of excised SLNs) in the future, further validation studies are necessary before such techniques are used in widespread clinical practice. Furthermore, the highly specialized and expensive nature of the equipment used for ICP-MS will likely limit the technique to specialist centers.","keyphrases":["sentinel lymph node","fine-needle biopsy","melanoma","clinical","pathology"],"prmu":["P","P","P","P","P"]}
{"id":"J_Gastrointest_Surg-3-1-1852387","title":"Platelet Function in Acute Experimental Pancreatitis\n","text":"Acute pancreatitis (AP) is characterized by disturbances of pancreatic microcirculation. It remains unclear whether platelets contribute to these perfusion disturbances. The aim of our study was to investigate platelet activation and function in experimental AP. Acute pancreatitis was induced in rats: (1) control (n = 18; Ringer\u2019s solution), (2) mild AP (n = 18; cerulein), and (3) severe AP (n = 18; glycodeoxycholic acid (GDOC) + cerulein). After 12 h, intravital microscopy was performed. Rhodamine-stained platelets were used to investigate velocity and endothelial adhesion in capillaries and venules. In addition, erythrocyte velocity and leukocyte adhesion were evaluated. Serum amylase, thromboxane A2, and histology were evaluated after 24 h in additional animals of each group. Results showed that 24 h after cerulein application, histology exhibited a mild AP, whereas GDOC induced severe necrotizing AP. Intravital microscopy showed significantly more platelet\u2013endothelium interaction, reduced erythrocyte velocity, and increased leukocyte adherence in animals with AP compared to control animals. Thromboxane levels were significantly elevated in all AP animals and correlated with the extent of platelet activation and severity of AP. In conclusion, platelet activation plays an important role in acute, especially necrotizing, pancreatitis. Mainly temporary platelet\u2013endothelium interaction is observed during mild AP, whereas severe AP is characterized by firm adhesion with consecutive coagulatory activation and perfusion failure.\nIntroduction\nAcute pancreatitis (AP) is characterized by an inflammatory affection of the exocrine pancreatic tissue and disturbances of pancreatic microcirculation.1 Depending on the severity of AP, irreversible perfusion failure with consecutive tissue hypoxia and necrosis can complicate the course of the disease and trigger systemic inflammatory and septic complications.2 The pathophysiology of AP has been investigated with regard to microcirculatory changes in several studies.1\u20135 Attention was paid especially to erythrocyte flow patterns, leukocyte\u2013endothelium interaction, and rheological approaches to improve perfusion and inhibit irreversible tissue damage.1,3\u20135 Leukocyte\u2013endothelium interaction as an early event of the inflammatory response has been characterized as a key step in the pathophysiology of AP.6 Besides, activation of the humoral coagulation cascade plays an important role in the development of microcirculatory disorders in AP.7,8 However, the role of platelets as the cellular elements of hemostasis that can functionally link inflammatory cells and humoral coagulation factors has not been investigated.\nThe aim of this study was to investigate platelet activation and function in experimental AP.\nMaterials and Methods\nAnimals The experiments were performed in 54 male Wistar rats weighing 270 to 335\u00a0g. Animals were fasted overnight with free access to water before the experiments. Care was provided in accordance with the guidelines published in the \u201cGuide for Care and Use of Laboratory Animals\u201d (National Institutes of Health, publication no. 85-23, 1985). Surgical anesthesia was induced with intraperitoneal injection of pentobarbital (25\u00a0mg\/kg) and intramuscular injection of ketamine (40\u00a0mg\/kg) for the procedures of catheter placement and induction of pancreatitis. Anesthesia during intravital microscopy was induced by intravenous injection of pentobarbital (10\u00a0mg\/kg). Polyethylene catheters (inner diameter 0.5\u00a0mm) were placed in the right jugular vein and left carotid artery, tunneled subcutaneously to the suprascapular area, and brought out through a steel tether that allowed the animals\u2019 free movement and access to water during the experiments.\nMonitoring blood samples Mean arterial pressure and heart rate were monitored during intravital microscopy by an electromechanical pressure transducer (Baxter Uniflow, Baxter Healthcare Cooperation, Deerfield, IL, USA). Arterial blood samples for determination of serum amylase were obtained before (baseline) and 24\u00a0h after (end point) pancreatitis was induced. Serum amylase was determined by standard laboratory methods (Hitachi automatic analyzer, Boehringer Mannheim, Germany).\nAnimal models Animals were divided into three groups. In each group, pancreatic microcirculation was evaluated in 12 animals by intravital microscopy, and morphological changes were assessed in six animals by histology. In the control group, animals underwent sham operation and received Ringer\u2019s solution only. Acute pancreatitis of graded severity was induced in the other groups either as mild or severe AP. Mild AP was induced by intraarterial infusion of cerulein (5\u00a0\u03bcg kg\u22121 h\u22121) for over 6\u00a0h. Cerulein was reconstituted in saline solution, and infusion volume was 4\u00a0ml\/kg\/h. Severe necrotizing pancreatitis was induced by infusion of bile salt (glycodeoxycholic acid [GDOC] 2.5\u00a0mM\/l) into the pancreatic duct in combination with intraarterial infusion of cerulein (5\u00a0\u03bcg kg\u22121 h\u22121) for over 6\u00a0h as described by Schmidt et al.9 in detail. Bile-salt infusion into the pancreatic duct was performed in a volume- (1.2\u00a0ml\/kg), time- (5\u00a0min), and pressure- (30\u00a0mmHg) controlled manner.In each of the models, animals received saline solution during the observation period (0.9%, 4\u00a0ml kg\u22121 h\u22121). Intravital microscopy was performed 12\u00a0h after the induction of pancreatic injury, and histological changes and blood samples were assessed 24\u00a0h after the infusions were started.\nPlatelet preparation One milliliter of whole blood was withdrawn before intravital microscopy. Platelets were separated and stained according to the method originally described by Massberg et al.10 Briefly, platelets were stained by rhodamine 6G and separated by 2\u00a0cycles of centrifugation under the addition of prostacyclin. After suspending and washing the separated platelets, blood cell count was performed to calculate the number of platelets per microliter and to rule out animal-specific differences in the number of platelets. Platelets were then reinjected, and intravital microscopy was performed.\nIntravital microscopy The abdomen was reopened, and the pancreas was carefully exteriorized in a horizontal position through the midline incision after the animal was placed on the right side. The duodenal loop with the head of the pancreas was carefully fixed on an anatomically designed stage in a temperature-controlled (37\u00b0C) Ringer\u2019s bath. Afterward, intravital microscopy was performed as described below. The animals were killed after the completion of intravital microscopy by a pentobarbital overdose.\nErythrocyte and leukocyte assessment A 0.5\u00a0ml\/kg of erythrocytes (hematocrit 50%) labeled with fluorescein isothiocyanate (FITC) as described before11 was applied intravenously. In addition, 1\u00a0ml\/kg of rhodamine-6G solution was applied intravenously to label leukocytes in vivo.12 Intravital microscopy was performed after an equilibration period of 15\u00a0min using a fluorescent microscope (Leitz, Wetzlar, Germany) with a 20-fold water immersion objective. An epi-illuminescent xenon lamp with an excitation filter of 450\u2013490\u00a0nm was used for visualization of FITC-labeled erythrocytes and an excitation filter of 540\u2013630\u00a0nm for rhodamine-labeled leukocytes.\nPlatelet assessment After platelet reinjection, intravital microscopy was performed by an epi-illuminescent xenon lamp with an excitation filter of 540\u2013630\u00a0nm.\nOff-line analysis Images were transferred to a monitor and simultaneously recorded on a videotape recorder. In each animal, five capillary fields of the exocrine pancreas and five postcapillary venules (20\u201340\u00a0\u03bcm) were recorded for 1\u00a0min. Off-line analysis was performed using a specially designed computer program (Capimage, Dr. Zeintl, Heidelberg, Germany). Erythrocyte velocity and platelet velocity were determined for 10 cells in each capillary field and venule. Additionally, temporarily (rolling) and permanently (sticking) adherent leukocytes and platelets were determined in pancreatic venules and capillary fields. Rolling cells were defined as cells with less than 66% of red blood cell velocity, whereas sticking cells are those that were adherent to the vessel wall for the whole observation period.13\nEdema A portion of pancreatic tissue was trimmed of fat and weighed. Pancreatic water content was determined by the ratio of the initial weight (wet weight) of the pancreas to its weight after incubation at 60\u00b0C for 72\u00a0h (dry weight).\nHistology The pancreas was immediately removed after killing and was fixed in 4% buffered formalin solution. It was then embedded in paraffin, cut, and stained with hematoxylin eosin. Histopathological evaluation was performed in a blinded fashion. For quantification of edema, inflammation, and necrosis, a modification of the scoring system originally described by Schmidt et al.9 was used, ranging from 0 to 3 (no pathological changes to severe injury).\nAssessment of thromboxane A2 Thromboxane A2 was measured in frozen serum by commercially available enzyme-linked immunosorbent assay (University of Freiburg, Germany).\nStatistical analysis Results are shown as mean\u2009\u00b1\u2009SEM. Student\u2019s t test was used when the data had a normal distribution, whereas Kruskal\u2013Wallis and Mann\u2013Whitney tests were utilized when the distribution was not normal. Statistical significance was accepted at the 5% level (p\u2009<\u20090.05).\nResults\nSerum amylase Twelve hours after the induction of AP, serum amylase increased significantly compared to control animals. Hyperamylasemia was comparable in both mild and severe AP indicating the presence of pancreatic cell damage. However, amylase was not a marker for the extent of tissue damage or disease severity (Table\u00a01). \nTable\u00a01Serum Parameters, Wet\u2013Dry Ratio, and Histopathology\u00a0ControlMild APSevere APSerum parametersAmylase (U\/l)586\u2009\u00b1\u200911627,200\u2009\u00b1\u20094,012*27,317\u2009\u00b1\u20093,220*Thromboxane A2 [pg\/50\u00a0\u03bcl]15.3\u2009\u00b1\u200910.347.8\u2009\u00b1\u200912.1*61.9\u2009\u00b1\u200915.8*Wet\u2013dry ratio2.87\u2009\u00b1\u20090.796.96\u2009\u00b1\u20090.95*4.77\u2009\u00b1\u20090.70HistopathologyInflammation0.25\u2009\u00b1\u20090.421.31\u2009\u00b1\u20090.08*1.95\u2009\u00b1\u20090.17*\u2020Necrosis0.08\u2009\u00b1\u20090.201.10\u2009\u00b1\u20090.11*1.70\u2009\u00b1\u20090.23*\u2020*p\u2009<\u20090.05 vs control group\u2020p\u2009<\u20090.05 vs mild acute pancreatitis\nSerum thromboxane A2 Thromboxane A2, as a marker of platelet activation, showed significantly higher levels in both AP groups compared to control animals after 24\u00a0h. Thromboxane liberation correlated with severity of AP, with the highest levels being present in animals with necrotizing AP (Table\u00a01).\nIntravital microscopy Erythrocyte velocity decreased significantly in mild as well as severe AP in both capillaries and venules compared to control animals. Platelets showed comparable flow features. Flow velocity decreased under both AP conditions, with a highly significant decrease in severe AP in venules and capillaries (Table\u00a02). These changes were paralleled by increased interaction between leukocytes and endothelium (Table\u00a02). Platelet adhesion in capillaries and venules increased significantly in both mild and severe AP (Figs.\u00a01 and 2). Reversible adhesion (rolling platelets) were comparable during both forms of AP, whereas the increase in irreversible adhesion (sticking platelets) depended on the severity of AP and showed peak platelet\u2013endothelium adherence in necrotizing AP (Figs.\u00a01 and 2). \nTable\u00a02Results of the Intravital MicroscopyIntravital microscopyControlMild APSevere APErythrocyte velocity (capillary) (mm\/s)0.65\/0.020.42\/0.01*0.36\/0.01*Erythrocyte velocity (venule) (mm\/s)0.93\/0.110.77\/0.170.58\/0.10*\u2020Platelet velocity (capillary) (mm\/s)0.54\u2009\u00b1\u20090.040.35\u2009\u00b1\u20090.03*0.29\u2009\u00b1\u20090.03*Platelet velocity (venule) (mm\/s)0.67\u2009\u00b1\u20090.050.63\u2009\u00b1\u20090.020.53\u2009\u00b1\u20090.05*Rolling leukocytes (capillary)1.3\u2009\u00b1\u20090.24.5\u2009\u00b1\u20091.4*9.0\u2009\u00b1\u20091.7*\u2020Rolling leukocytes (venule)1.3\u2009\u00b1\u20090.214.8\u2009\u00b1\u20091.2*18.9\u2009\u00b1\u20091.9*Sticking leukocytes (capillary)1.1\u2009\u00b1\u20090.310.2\u2009\u00b1\u20091.8*7.2\u2009\u00b1\u20090.7*Sticking leukocytes (venule)0.7\u2009\u00b1\u20090.15.6\u2009\u00b1\u20090.9*13.5\u2009\u00b1\u20092.0*\u2020*p\u2009<\u20090.05 vs control group\u2020p\u2009<\u20090.05 vs mild acute pancreatitisFigure\u00a01Intravital microscopy, capillary platelet adhesion (one per field). Control group (gray), mild acute pancreatitis (white), and severe acute pancreatitis (striped). Reversible platelet adhesion in mild and severe acute pancreatitis (left columns); irreversible platelet adhesion (right columns). *p\u2009<\u20090.05 vs control group, \u2020p\u2009<\u20090.05 vs mild acute pancreatitis.Figure\u00a02Intravital microscopy, venular platelet adhesion (one per 100\u00a0\u03bcm). Control group (gray), mild acute pancreatitis (white), and severe acute pancreatitis (striped). Reversible platelet adhesion in mild and severe acute pancreatitis (left columns); irreversible platelet adhesion (right columns). *p\u2009<\u20090.05 vs control group, \u2020p\u2009<\u20090.05 vs mild acute pancreatitis.\nTissue edema (wet\/dry ratio) Supramaximal cerulein stimulation induced a significant increase in pancreatic water content compared to control animals. In contrast, there was only a slight increase in tissue edema after GDOC treatment (Table\u00a01).\nHistopathology Control animals showed no histopathological changes after sham operation and 24\u00a0h infusion therapy. Histopathology of mild AP was characterized by significant edema formation, inflammatory tissue infiltration, and acinar cell necrosis. In severe AP, the changes regarding inflammation and necrosis were significantly more pronounced (Table\u00a01).\nDiscussion\nIn the present study, we have investigated platelet function in experimental models of AP. We chose two animal models to induce a mild edematous or a severe necrotizing course of AP. Both models are established, well characterized, and have been used in numerous studies.9,14,15 The induction of AP in these models results in a standardized grade of tissue damage, either mild or severe, with very little variance within each group. Therefore, the use of these models allows us to rule out the significant influence of preparatory or other methodological problems on the comparability of the results.\nAnalysis of platelet function by intravital microscopy has been established and standardized for examination of liver and small bowel perfusion by Massberg et al.10 We have modified this method to investigate the pancreas.15 In the present study, we could demonstrate that this method is not only suitable for the examination of healthy pancreas but also for the detailed analysis of pancreatic microcirculation in mild and severe AP.\nAcute pancreatitis is characterized by an impairment of microcirculation due to an activation of inflammatory cells with a consecutive increase of leukocyte\u2013endothelium interaction. These pathophysiological events mediate an inflammatory tissue infiltration, edema, and hemorrhagic lesions. While the inflammatory response is well investigated, the platelet function and the role of the coagulation cascade have not yet been investigated in detail.\nIt is well known that the inhibition of certain coagulatory steps, e.g., by applying hemodiluting or anticoagulatory substances, improves the outcome of AP.16,17 Coagulation and hemostasis comprise two interacting pathways: humoral coagulatory factors leading to the activation of fibrinogen as the final step of the coagulation cascade and cellular factors, which are represented by activated platelets. Different mechanisms of platelet interaction are responsible for their physiological function, namely, interactions with endothelium, leukocytes, and humoral coagulatory and inflammatory proteins.18,19\nIn the present study, we could demonstrate that the platelet\u2013endothelium interaction increases during AP and correlates with the degree of its severity. Comparable to leukocyte\u2013endothelium interaction, temporary and permanent adhesions of platelets to the vessel wall were evident in our experiments. This correlates with the activation patterns that have been observed in vivo in ischemia models of the liver and the pancreas,15,20 as well as in vitro.21 Therefore, it seems likely that these activation patterns reflect the severity of the pancreatic affection, leading to reversible adhesion in mild AP and irreversible adhesion in more severe organ affection. Especially, the firm adhesion of platelets contributes to microcirculatory disturbances and may induce perfusion failure and tissue necrosis in the progression to severe AP. The significantly elevated thromboxane levels correlate well with platelet activation and microcirculatory failure observed during intravital microscopy. The increase in serum thromboxane elucidates one mechanism of our results as it executes a direct platelet stimulation and leads to the conversion of \u201cresting\u201d to \u201cactivated\u201d platelets with the consecutive adhesive action. Furthermore, thromboxane does not only activate platelets but also acts as a complex pathophysiological mediator with multiple other targets. Its effects include leukocyte activation, upregulation of proinflammatory cytokines, and strong vasoconstricting effects. These are mediated via phosphatidylcholine and phosphatidylcholine-specific phospholipase-C pathway leading to a tonic contraction in smooth muscles and upregulating other vasoactive substances.22,23 Especially, this vasoconstrictor mechanism may additionally contribute to perfusion failure in the course of AP as observed in our study. How far platelet inhibition itself could be an approach to attenuate the course of AP experimentally or clinically is hypothetical but should certainly be addressed to in further studies. Possible aims could be adhesion molecules such as selectins or platelet receptors and also synthesis of thromboxane and prostaglandins.\nPlatelet activation was accompanied by leukocyte activation in the present study. An interaction between these two cell types has been demonstrated by the different authors in the past.24\u201326 Among others, P-selectin seems to be one of the most important adhesion molecules, which links the inflammatory and procoagulatory cascades and has the potency to activate leukocytes and platelets as the cellular elements of either pathway.18,19,25,26 Besides their adherence to endothelial cells, activated platelets form stable aggregates with leukocytes. This results in a combined inflammatory and coagulatory contribution to thrombus formation and is also mediated by P-selectin and beta-integrins.27\u201329 Especially, the formation of microthrombotic vessel occlusion with microcirculatory perfusion failure and consequent ischemia, hypoxia, and tissue necrosis was reflected by the intravital microscopic results in the present study.\nConclusion\nThe results of the present study show that activation and adhesion of platelets play an important role during AP. Platelet\u2013endothelium and platelet\u2013leukocyte interactions as well as thromboxane liberation show a correlation with the severity of experimental AP and seem to be of distinct importance in the progression from mild to severe necrotizing AP. A possible therapeutic use of these pathophysiological events should be evaluated in further studies.","keyphrases":["platelets","acute pancreatitis","microcirculation","leukocytes","coagulation","endothelium interaction"],"prmu":["P","P","P","P","P","R"]}
{"id":"Photosynth_Res-3-1-1769343","title":"Optical spectroscopic studies of light-harvesting by pigment-reconstituted peridinin-chlorophyll-proteins at cryogenic temperatures\n","text":"Low temperature, steady-state, optical spectroscopic methods were used to study the spectral features of peridinin-chlorophyll-protein (PCP) complexes in which recombinant apoprotein has been refolded in the presence of peridinin and either chlorophyll a (Chl a), chlorophyll b (Chl b), chlorophyll d (Chl d), 3-acetyl-chlorophyll a (3-acetyl-Chl a) or bacteriochlorophyll a (BChl a). Absorption spectra taken at 10 K provide better resolution of the spectroscopic bands than seen at room temperature and reveal specific pigment\u2013protein interactions responsible for the positions of the Qy bands of the chlorophylls. The study reveals that the functional groups attached to Ring I of the two protein-bound chlorophylls modulate the Qy and Soret transition energies. Fluorescence excitation spectra were used to compute energy transfer efficiencies of the various complexes at room temperature and these were correlated with previously reported ultrafast, time-resolved optical spectroscopic dynamics data. The results illustrate the robust nature and value of the PCP complex, which maintains a high efficiency of antenna function even in the presence of non-native chlorophyll species, as an effective tool for elucidating the molecular details of photosynthetic light-harvesting.\nIntroduction\nPhotosynthetic organisms have bioengineered a wide variety of protein-based structures to carry out photosynthetic light-harvesting (Frigaard et al. 2004). High-resolution molecular structures of many of these systems are providing important clues into the molecular features that control light-harvesting in photosynthesis (Germeroth et al. 1993; Hofmann et al. 1996; Hu et al. 1995; Koepke et al. 1996; K\u00fchlbrandt et al. 1994; Law et al. 2004; Liu et al. 2004; McDermott et al. 1995; Roszak et al. 2003). To understand fully how these naturally occurring pigment\u2013protein complexes harvest light so efficiently, it is necessary to employ spectroscopic tools that reveal directly the dynamics and efficiency of energy transfer and the nature of the excited energy states associated with the bound pigments. The spectroscopic studies are aided by the ability to make systematic alterations in the structures of the light-harvesting complexes, and then to examine how these changes manifest themselves in the spectroscopic observables, dynamics of energy transfer and efficiency of antenna function (Akahane et al. 2004; Cammarata et al. 1990; Chadwick et al. 1987; Crielaard et al. 1994; Croce et al. 1999; Davidson and Cogdell 1981; Frank 1999; Fraser et al. 1999; Morosinotto et al. 2002; Olivera et al. 1994; Olsen et al. 1997; Plumley and Schmidt 1987; Remelli et al. 1999; Struck and Scheer 1991; Struck et al. 1992).\nThe peridinin-chlorophyll-protein (PCP) complex from the dinoflagellae, Amphidinium carterae, is an example where many of these approaches have been applied. Atomic resolution structural analysis by X-ray diffraction to 2.0\u00a0\u00c5 resolution (Hofmann et al. 1996) has been augmented by pigment reconstitutions and protein refolding studies (Miller et al. 2005; Pol\u00edvka et al. 2005). Steady-state and time-resolved spectroscopy (Bautista et al. 1999a; Kleima et al. 2000a; Kleima et al. 2000b; Krueger et al. 2001; Linden et al. 2004; Shima et al. 2003; Zigmantas et al. 2002; Zimmermann et al. 2002) has been carried out on the excited singlet and triplet states associated with the bound pigments. The X-ray crystallographic analysis of PCP revealed a trimeric structure of subunits (Hofmann et al. 1996) where eight peridinins and two chlorophyll a (Chl a) molecules (Fig.\u00a01) bind non-covalently in a roughly C2 symmetrical arrangement in each of the three individual subunits. The peridinins are assembled in clusters of four within the subunits, surrounding, and in van der Waals contact with, one chlorophyll a (Chl a) (Fig.\u00a01). The two Chl molecules within a subunit have a center-to-center distance of 17.4\u00a0\u00c5. Steady-state and ultrafast optical spectroscopic investigations on peridinin in solution and on the main-form PCP (MFPCP) have explored its photoinduced excited-state processes and energy transfer pathways (Carbonera et al. 1999; Damjanovic et al. 2000; Kleima et al. 2000; Krueger et al. 2001; Ritz et al. 2000; Shima et al. 2003; Zimmermann et al. 2002). In addition, a variant form, the high-salt PCP (HSPCP), has been studied in the same manner (Ilagan et al. 2004; Sharples et al. 1996).\nFig.\u00a01Structure of the pigments in a monomeric subunit of the MFPCP complex. The coordinates were taken from Protein Data Bank with 1PPR code. Also shown are the structures of peridinin and the chlorophylls used in this study\nAdding to the arsenal of PCP complexes that can be used for structural and spectroscopic studies, Miller and coworkers (Miller et al. 2005) prepared and Pol\u00edvka and coworkers (Pol\u00edvka et al. 2005) studied the N-domain and full-length PCP apoproteins expressed in Escherichia coli and reconstituted with the total pigment extract from native PCP to produce a fully functional complex with a high peridinin-to-Chl a energy transfer efficiency. Also, they demonstrated that in the N-domain PCP apoprotein the bound Chl a molecules could be replaced by other Chls using the N-domain apoprotein incorporating the requisite stoichiometric amount of peridinin.\nIn this paper, we present a low temperature, steady-state, optical spectroscopic investigation of the spectral features of PCP complexes in which recombinant apoprotein has been refolded in the presence of peridinin and either chlorophyll a (Chl a), chlorophyll b (Chl b), chlorophyll d (Chl d), 3-acetyl-chlorophyll a (3-acetyl-Chl a) or bacteriochlorophyll a (BChl a). We use fluorescence excitation spectroscopy to examine in detail the peridinin-to-Chl energy transfer efficiencies of the various complexes at room temperature and correlate the results with previously reported ultrafast, time-resolved optical spectroscopic dynamics data (Pol\u00edvka et al. 2005). The low temperature data provide better resolution of the spectroscopic bands than seen at room temperature and reveal specific pigment\u2013protein interactions responsible for the observed Chl transition energies.\nMaterials and methods\nSample preparation\nThe PCP complexes were reconstituted from N-domain apoprotein with peridinin and either Chl a, Chl b, Chl d, 3-acetyl-Chl a or BChl a (Fig.\u00a01) as described (Miller et al. 2005). Size exclusion chromatography shows the reconstituted PCP to be approximately 40\u00a0kDa in apparent molecular mass; i.e., the same as that of the native PCP, suggesting that the samples used here consist of monomeric protein structures. The purified reconstituted PCP complexes were stored at 4\u00b0C until ready for use in the spectroscopic experiments. For the cryogenic experiments, the PCP complexes were dissolved in a buffer containing 50\u00a0mM Tricine, 20\u00a0mM KCl buffer and 60% (v\/v) glycerol at pH 7.6.\nChl a and Chl b were obtained from Sigma-Aldrich. Chl d and BChl a were extracted from the cyanobacterium, Acaryochloris marina, and the bacterium, Rhodospirillum rubrum, respectively. Total pigments were extracted from A. marina cells with 2-butanol and the extract dried in a gentle stream of nitrogen and Chl d was purified by HPLC as described below. BChl a was extracted from R. rubrum whole cells with 50:50 (v\/v) acetone:methanol. The extracted solution was filtered, dried and redissolved in 50:50 (v\/v) petroleum ether:methanol. The methanol phase, in which BChl a was dissolved, was collected and dried with nitrogen gas. The BChl a solution was subjected to an alumina column chromatography with increasing percent of acetone in hexane. 3-Acetyl-Chl a was obtained as an oxidation product of BChl a during its isolation from R. rubrum. All Chls were further purified using a Millipore Waters 600E HPLC system employing a Nova-Pak C18 column. For Chl a and Chl b, isocratic runs of acetone:methanol:water (75:20:5, v\/v\/v) were used as the mobile phase with a flow rate of 0.5\u00a0mL\u00a0min\u22121. For Chl d and BChl a, the mobile phase used a combination of solvent A (acetonitrile:methanol\u201450:50, v\/v) and solvent B (solvent A:water\u201450:50, v\/v). The run was programmed as follows: 0\u20135\u00a0min, linear gradient from 80% solvent A and 20% solvent B to 99% solvent A and 1% solvent B; and, 5\u201330\u00a0min, isocratic 99% solvent A and 1% solvent B with a flow rate of 1.0\u00a0mL\u00a0min\u22121. 3-acetyl-Chl a was separated from BChl a using solvent A (acetonitrile:methanol:water\u201487:10:3, v\/v\/v) and solvent B (hexane:propanol\u20144:1, v\/v) as the mobile phase. The mobile phase was programmed as follows: 0\u201320\u00a0min, linear gradient from 100% solvent A to 95% solvent A and 5% solvent B; and, 20\u201330\u00a0min, isocratic 95% solvent A and 5% solvent B with a flow rate of 1.0\u00a0mL\u00a0min\u22121. The collected samples were dried with a gentle stream of nitrogen gas and then redissolved in 2-methyl tetrahydrofuran (2-MTHF).\nSpectroscopy\nAbsorption spectra were taken at 10\u00a0K using a Cary 50 UV-visible spectrometer and a Janis model STVP100 helium vapor flow cryostat using 1\u00a0cm pathlength polymethacrylate cuvettes. The fluorescence and fluorescence excitation spectra of the PCP complexes were taken at 77\u00a0K using a Jobin Yvon Horiba Fluorolog-3 spectrofluorometer model FL3-22 on samples immersed in a custom built optical dewar (Kontes) as described (Ilagan et al. 2004).\nResults\nAbsorption spectroscopy\nThe 10\u00a0K absorption spectra of the PCP complexes reconstituted with peridinin and either Chl a, Chl b, Chl d, 3-acetyl-Chl a or BChl a are shown in Fig.\u00a02. All of the complexes exhibit strong absorption in the region 450\u2013575\u00a0nm associated with peridinin. For the complexes reconstituted with Chl a and Chl b, a double peak is evident in the Soret region (near 450\u00a0nm), but this is not seen in complexes containing Chl d, 3-acetyl-Chl a or BChl a. In the long-wavelength absorption region of the spectra, the Qy band of all the Chls is very sharp and appears at 646\u00a0nm for Chl b, 667\u00a0nm for Chl a, 682\u00a0nm for 3-acetyl-Chl a, 697\u00a0nm for Chl d and 782\u00a0nm for BChl a. All the Qy bands are blue-shifted compared to the spectra taken at room temperature. Also, except for Chl d that exhibits a weaker shift of 59\u00a0\u00b1\u00a06\u00a0cm\u22121, all are shifted by the same amount, 110\u00a0\u00b1\u00a015\u00a0cm\u22121. Also, in the PCP complex containing BChl a, a second peak on the long-wavelength side of the primary Qy feature is observed at 799\u00a0nm (Fig.\u00a02) and a trace of 3-acetyl-Chl a can be seen in the absorption spectrum of the complex with BChl a.\nFig.\u00a02Absorption spectra of the PCP complexes reconstituted with peridinin and either Chl a, Chl b, Chl d, aChl a (3-acetyl-Chl a) or BChl a. The spectra were taken at 10\u00a0K and were normalized at 500\u00a0nm\nFor comparison and analysis of the Chl spectra of the complexes, absorption spectra of the isolated and purified Chls in 2-MTHF were taken at 10\u00a0K. 2-MTHF forms a clear glass at that temperature and has been used previously in this type of analysis (Ilagan et al. 2004). Figure\u00a03 shows the 10\u00a0K absorption spectra of the Chl solutions in the Qy region plotted on a wavenumber scale. The spectra were analyzed by Gaussian deconvolution and were well fit using a sum of two components that were allowed to vary in amplitude, position and width built on a broad baseline. The Gaussian deconvolutions of the solution spectra were all very similar (Fig.\u00a03) and revealed a prominent spectral origin and a broad phonon wing at higher energy. The wavelengths of the prominent peaks computed from the wavenumber fits are 670\u00a0nm for Chl a, 654\u00a0nm for Chl b, 697\u00a0nm for Chl d, 689\u00a0nm for 3-acetyl-Chl a and 777\u00a0nm for BChl a. The results indicate the chlorophyll\u2013protein interaction induces a blue-shift in the case of Chl a, Chl b and 3-acetyl-Chl a, no shift for Chl d and a red-shift for BChl a.\nFig.\u00a03Gaussian deconvolutions of the 10K absorption spectra in the Qy region of purified Chls in 2-MTHF solvent. The fit (dashed line) is the sum of two Gaussian components built on a broad background. The data for the Gaussian deconvolution of the Chl a spectrum was taken from a previous study (Ilagan et al. 2004)\nFigure\u00a04 shows the 10\u00a0K absorption spectral profiles of the reconstituted PCP complexes in the Qy region on a wavenumber scale fitted using Gaussian functions that were also allowed to vary in amplitude, position and width. The spectra from the PCP complexes containing Chl a, Chl b and BChl a required four Gaussian components on a broad absorption background (Fig.\u00a04A,\u00a0B,\u00a0E) for a good fit. The spectra from the complexes having Chl d and 3-acetyl-Chl a required only two Gaussian functions on a broad absorption background (Fig.\u00a04C,\u00a0D) for a good fit.\nFig.\u00a04Gaussian deconvolutions of the 10K absorption spectra in the Qy region of PCP complexes reconstituted with peridinin and: (A) Chl a; (B) Chl b; (C) Chl d; (D) aChl a (3-acetyl-Chl a); and (E) BChl a\nSpectral reconstruction\nLinear combinations of the absorption spectra of peridinin and the various Chls taken in 2-MTHF at 10\u00a0K were used to reconstruct the spectral line shapes of the complexes in the region 375\u2013600\u00a0nm. According to previously published procedures (Ilagan et al. 2004), individual peridinin spectra were constrained to have identical intensities, but were allowed to vary with respect to wavelength. The spectra of the individual Chls were allowed to have any intensity and wavelength. For all of the complexes, two Chl and eight peridinin spectra were used to fit the lineshapes (Fig.\u00a05A\u2013E). The spectra of all the complexes were best fit using two blue-shifted peridinins and three separated pairs of peridinin spectra (Fig.\u00a05A\u2013E), one of which needed to be significantly red-shifted to accommodate the long-wavelength region of the spectra. In the complexes containing Chl a and Chl b, the Soret bands of the two Chls are split and have noticeably different intensities. This required two separate Chl spectra to reproduce the absorption lineshapes in this region (Fig.\u00a05A, B; Table\u00a01). No splitting of the Soret bands was evident for the complexes with Chl d, 3-acetyl-Chl a and BChl a, so the spectra were able to be fit in this region assuming that the two Chls were identical. A summary of the wavelengths of the spectral origins of the individual peridinins and the Chl bands assigned in this analysis is given in Table\u00a01.\nFig.\u00a05Reconstruction of the 10K absorption spectra of the reconstituted PCP complexes in the 375\u2013600nm region. The analysis was carried out by linearly combining individual 10\u00a0K absorption spectra of peridinin and Chl taken in 2-MTHF solvent as described in the textTable\u00a01Spectral origins of the eight peridinins and the positions of the Soret peaks and Qy bands of the two Chls derived from the spectral analysis of the 10\u00a0K absorption spectra of the reconstituted PCP complexesPCP complexPeridininChlReferenceSoretQy123456781212MFPCP470485522522526526544544434439665667(Ilagan et al. 2004)HSPCPna489533533533533533na436441667676(Ilagan et al. 2004)Chl a472\u00a0\u00b1\u00a02486\u00a0\u00b1\u00a02510\u00a0\u00b1\u00a01510\u00a0\u00b1\u00a01529\u00a0\u00b1\u00a01529\u00a0\u00b1\u00a01548\u00a0\u00b1\u00a01548\u00a0\u00b1\u00a01434\u00a0\u00b1\u00a01440\u00a0\u00b1\u00a01667668Chl b470\u00a0\u00b1\u00a03485\u00a0\u00b1\u00a02508\u00a0\u00b1\u00a01508\u00a0\u00b1\u00a01527\u00a0\u00b1\u00a02527\u00a0\u00b1\u00a02546\u00a0\u00b1\u00a02546\u00a0\u00b1\u00a02458\u00a0\u00b1\u00a01461\u00a0\u00b1\u00a01644647Chl d474\u00a0\u00b1\u00a02490\u00a0\u00b1\u00a02513\u00a0\u00b1\u00a01513\u00a0\u00b1\u00a01530\u00a0\u00b1\u00a01530\u00a0\u00b1\u00a01546\u00a0\u00b1\u00a01546\u00a0\u00b1\u00a01456\u00a0\u00b1\u00a02456\u00a0\u00b1\u00a02697697aChl a467\u00a0\u00b1\u00a02482\u00a0\u00b1\u00a02511\u00a0\u00b1\u00a02511\u00a0\u00b1\u00a02530\u00a0\u00b1\u00a02530\u00a0\u00b1\u00a02543\u00a0\u00b1\u00a01543\u00a0\u00b1\u00a01446\u00a0\u00b1\u00a01446\u00a0\u00b1\u00a01683683BChl a472\u00a0\u00b1\u00a02486\u00a0\u00b1\u00a02507\u00a0\u00b1\u00a02507\u00a0\u00b1\u00a02531\u00a0\u00b1\u00a01531\u00a0\u00b1\u00a01545\u00a0\u00b1\u00a01545\u00a0\u00b1\u00a01365\u00a0\u00b1\u00a01365\u00a0\u00b1\u00a01782782The numbers are in nm units and the uncertainties represent the range of values that give an acceptable fit to the dataMFPCP is main-form PCP, HSPCP is high-salt PCP, aChl a is 3-acetyl-Chl a, na is not applicable\nFluorescence spectroscopy\nFluorescence spectra of the reconstituted PCP complexes at 77\u00a0K are shown in Fig.\u00a06. The spectra display a prominent narrow band at 671\u00a0nm (full width at half maximum, FWHM\u00a0=\u00a08.5\u00a0nm) for Chl a, 647\u00a0nm (FWHM\u00a0=\u00a010\u00a0nm) for Chl b, 699\u00a0nm (FWHM\u00a0=\u00a08\u00a0nm) for Chl d, 686\u00a0nm (FWHM\u00a0=\u00a09.5\u00a0nm) for 3-acetyl-Chl a and 787\u00a0nm (FWHM\u00a0=\u00a014.5\u00a0nm) for BChl a. All of the spectra show structured vibronic features to the red of the main band. In the PCP complex with BChl a, a shoulder at 798\u00a0nm on the red side of the main peak is clearly evident.\nFig.\u00a06Fluorescence spectra of the PCP complexes reconstituted with peridinin and either Chl a, Chl b, Chl d, aChl a (3-acetyl-Chl a) or BChl a taken at 77\u00a0K. The spectra were normalized to their \u03bbmax values\nFluorescence excitation spectroscopy\nFigure\u00a07 shows the fluorescence excitation spectra of the complexes recorded at room temperature overlaid with their 1-T spectra, where T is transmittance. The Chl fluorescence was monitored at various wavelengths between 675 and 850\u00a0nm, and the fluorescence excitation spectra were found to be independent of the monitoring wavelength. The peridinin-to-Chl energy transfer efficiencies obtained from the spectra were averaged over the 450\u2013515\u00a0nm wavelength range and were: 94\u00a0\u00b1\u00a02% (Chl a), 92\u00a0\u00b1\u00a02% (Chl b), 96\u00a0\u00b1\u00a03% (Chl d), 99\u00a0\u00b1\u00a01% (3-acetyl-Chl a) and \u223c100% (BChl a).\nFig.\u00a07Overlay of the room temperature fluorescence excitation (ex) and 1-T (where T is transmittance) spectra of reconstituted PCP complexes containing: (A) Chl a; (B) Chl b; (C) Chl d; (D) aChl a (3-acetyl-Chl a); and (E) BChl a. The intensity of each fluorescence excitation spectrum was normalized to correspond to the intensity of its corresponding 1-T spectrum in the Qy region\nDiscussion\nLow temperature absorption spectra\nThe 10\u00a0K absorption spectra of the reconstituted PCP complexes (Fig.\u00a02) show significantly higher resolution than the spectra taken at room temperature (Pol\u00edvka et al. 2005). This allows a more detailed look at how changes in the structure of the incorporated Chl affect the spectral profiles. The 10\u00a0K lineshape of the PCP complex reconstituted with Chl a (Fig.\u00a02) is very similar to that from the native PCP complex (Ilagan et al. 2004), the major characteristics being the splitting of the Soret band near 435\u00a0nm and the shoulder on the red side of the peridinin maximum. These features are clearly evident (Figs.\u00a02,\u00a05A) in both reconstituted and native complexes (Ilagan et al. 2004). This supports the notion that the structures of these two complexes are very similar.\nThe 10\u00a0K absorption spectra of the reconstituted PCP complexes (Fig.\u00a02) also display blue-shifted Qy bands compared with thse spectra of the complexes taken at room temperature, and except for Chl d that exhibits a shift of 59\u00a0\u00b1\u00a06\u00a0cm\u22121, are all shifted by 110\u00a0\u00b1\u00a015\u00a0cm\u22121. This is opposite to the low temperature-induced red-shift observed for the 850\u00a0nm-absorbing BChl molecules in LH2 complexes from photosynthetic bacteria which was explained in terms of a structural change of the LH2 complex upon glass formation at low temperatures that increased excitonic coupling between the BChls (Wu et al. 1997). This cannot be the case for the PCP complexes here because the two Chl molecules are too far apart (17.4\u00a0\u00c5) to be excitonically coupled. However, the blue-shift may result from a slight change in the planarity of the Chl macrocycles. Assuming that glass formation induces a structural change (Wu et al. 1997) in the immediate environment of the Chls in the PCP complexes, one may expect a corresponding change in the conformation of the macrocycles. It has been shown that these conformational changes may induce either red- or blue-shifts of Chl Qy bands (Gudowska-Nowak et al. 1990). The fact that the shift is very similar (110\u00a0\u00b1\u00a015\u00a0cm\u22121) for all chlorophyll species except Chl d suggests that the structural change affects the macrocycle in the vicinity of Ring I because all the Chls except Chl d have a large substituent in the position R1 (Fig.\u00a01). Thus, if one assumes a low-temperature-induced protein structural change in the immediate environment of Ring I, this may lead to a distortion of the Chl macrocycle and consequently to the observed blue-shift. For Chl d, the smaller aldehyde substituent at position R1 may be less susceptible to protein structural changes and to inducing conformational distortion of the Chl macrocycle.\nThe Gaussian deconvolutions of the Chl solution spectra in the Qy region (Fig.\u00a03) serve as guides in the analysis of the absorption spectra of the reconstituted PCP complexes. Comparing the spectrum in the Qy region of the Chl a-reconstituted PCP (Fig.\u00a04A) with that of Chl a in solution (Fig.\u00a03A) shows that two distinct pairs of Chl a Gaussian functions built on a broad background are needed to reproduce the spectrum of that complex. Likewise, for the PCP complex containing Chl b, two pairs of Gaussian components having different prominent spectral peaks and zero-phonon lines built on a broad background were needed to reproduce the spectrum of the protein complex (Fig.\u00a04B). However, for the PCP complexes containing Chl d or 3-acetyl-Chl a, only two Gaussian components on a broad background were required to fit the spectra in the Qy band region indicating that Chl d and 3-acetyl-Chl a in these complexes are in highly symmetric environments. This conclusion is supported by the observation that the PCP complexes reconstituted with Chl a and Chl b show a splitting in the Soret band region (Fig.\u00a05A,\u00a0B), but all the other complexes do not. This indicates a less symmetric environment for the Chls bound in the complexes containing Chl a or b.\nThe observation that the spectra of the PCP complexes containing Chl a and Chl b show splittings in the Soret and Qy regions attributable to environmental asymmetry, but the spectra from the Chls in the other complexes do not, may also be traceable to the different functional groups on Ring I of the Chls. A view of the published structure of the MFPCP complex focusing on Chl 601 in the vicinity of Ring I shows that this part of the Chl macrocycle is in close proximity (within 4\u00a0\u00c5) to peridinins 612 and 614 (Fig.\u00a08). The distance between the vinyl group and the peridinins is closer than to any amino acid residue in the protein. Leucine-204 comes closest at \u223c5\u00a0\u00c5 (Fig.\u00a08). The splitting of the Soret and Qy spectral bands in the Chl a and Chl b PCP complexes implies that the vinyl group on Chl a and Chl b may adopt different configurations leading to distinct interactions between these Chls and the peridinins. In contrast, the carbonyl groups on Ring I of Chl d, 3-acetyl-Chl a and BChl a may have only a single configuration and may also sterically hinder the approach of peridinins 612 and 614 toward the Chl in those complexes, thereby minimizing the interaction between pigments, rendering the Chl spectra equivalent.\nFig.\u00a08A view of the structure of the MFPCP complex focusing on the Ring I of Chl 601. Per 612, Per 614 and Leu 204 are in close proximity to the Ring I of Chl 601. The coordinates of the structure were taken from Protein Data Bank with 1PPR code\nThe results from the deconvolution of the spectral features for the PCP complex containing BChl a are unusual. In addition to the main Qy absorption band at 782\u00a0nm (Figs.\u00a02,\u00a04E), a shoulder at 799\u00a0nm is observed which is highly reminiscent of the 4\u00a0K absorption spectrum of the antenna BChl a\u2013protein (Fenna\u2013Matthews\u2013Olson, FMO) complex from Prosthecochloris aestuarii (Johnson and Small 1991; Wendling et al. 2002; Whitten et al. 1978). The spectral features in the Qy region are attributed to a combination of site shifts and dipolar interactions among the seven BChl a molecules within one subunit of the FMO protein (Wendling et al. 2002). The crystal structure of this FMO complex has been determined to 2.8\u00a0\u00c5 resolution (Matthews et al. 1979) and later refined to 1.9\u00a0\u00c5 resolution (Tronrud et al. 1986). The FMO complex consists of three identical subunits and each subunit contains seven BChl a molecules where the center-to-center distances between BChls vary from 11 to 14\u00a0\u00c5 (Matthews et al. 1979). This is smaller than the center-to-center distance of 17.4\u00a0\u00c5 reported from the X-ray crystallographic analysis of the MFPCP complex containing Chl a (Hofmann et al. 1996). Why the BChl-containing complexes exhibit this long-wavelength feature in their spectra but the complexes containing the other Chl molecules do not, is unclear. Nevertheless, the Soret band and the Qy features of the BChl molecules in the PCP complex are reproduced (Figs.\u00a04E,\u00a05E) assuming only a single prominent feature and a phonon line. Thus, similar to the complexes containing Chl d or 3-acetyl-Chl a, this suggests a highly symmetric environment for the BChls bound in this reconstituted PCP complex.\nThe Chl\u2013protein interaction induces a blue-shift of the Qy band in the low temperature spectra of Chl a, Chl b and 3-acetyl-Chl a, no shift for Chl d, and a red-shift for BChl a compared to the spectra taken in 2-MTHF low temperature glasses (Figs.\u00a03,\u00a04). This may be explained in a manner similar to that used above to interpret the blue-shifts of the Qy bands induced upon lowering the temperature. Switching from a 2-MTHF glass to the protein may induce a conformational change of the macrocycle that leads to the observed shifts (Gudowska-Nowak et al. 1990). Structural differences between Chl species occur due to different functional groups attached at positions R1 and R2 (Fig.\u00a01), but again, it appears that the R1 site is key. The small aldehyde group of Chl d does not provide sufficient interaction leading to distortion of the macrocycle either in 2-MTHF glass or in the protein. Thus, essentially no protein-induced shift is observed. In contrast, the vinyl group on Ring I of Chl a and Chl b respond to the protein environment and induce a structural change that is manifested spectroscopically as a blue-shift of the Qy band. The red-shift of BChl a in the protein can be attributed to the acetyl group in the position R1. This functional group has been shown to cause a substantial red-shift of absorption spectra of BChl a in LH2 complexes from photosynthetic bacteria (Cogdell et al. 2002). The rotation of the carbonyl group with respect to the plane of Ring I causes changes in \u03c0-electron conjugation and this argument has been used to interpret the difference between the B820 and B850 absorbing BChls in different LH2 complexes (Cogdell et al. 2002). However, if this were the sole effect, a protein-induced red-shift would be expected of the 3-acetyl-Chl a that also has an acetyl group in the R1 site. This is not observed. Thus, we must assume that the saturation of the \u2013C=C\u2013 bond in Ring II for BChl also plays a role in determining the direction of the shift.\nEnergy transfer efficiencies\nLight absorption by peridinin promotes the molecule from its ground state, S0, to an excited state, denoted S2. The S2 state of peridinin then decays in \u223c100\u00a0fs to a lower-lying S1 state which cannot be directly accessed from S0 due to symmetry selection rules (Akimoto et al. 1996; Krueger et al. 2001; Zigmantas et al. 2003). Since the lifetime of the S1 state of peridinin in solution was found to be highly dependent on the polarity of the solvent (Bautista et al. 1999; Zigmantas et al. 2001; 2003), an intramolecular charge transfer (ICT) state was invoked to account for this behavior (Bautista et al. 1999), and quantum computations have supported this model (Vaswani et al. 2003). Moreover, it has been argued from pump-dump-probe experiments on peridinin in solution that S1 and ICT states are distinct but connected on a picosecond time-scale (Papagiannakis et al. 2004). However, other time-resolved and two-photon spectroscopic methods have suggested that it is the S1 itself that possesses the charge transfer character (Shima et al. 2003; Zigmantas et al. 2003). Since it is not completely resolved whether the S1 state in PCP is distinct from or the same as the ICT state, we shall use the notation S1\/ICT to indicate both possibilities.\nThe peridinin-to-Chl energy transfer efficiencies for the complexes can be computed assuming a two-state (S2 and S1\/ICT) donor model (Fig.\u00a09). The overall efficiency, \u03b5, is given by Desamero et al. 1998:  where kET2 and kET1 are the rate constants for energy transfer from the S2 and S1\/ICT states of peridinin, kIC2 is the rate constant for internal conversion from S2 to S1\/ICT state and kIC1 is the rate constant for S1\/ICT to S0 internal conversion. The rate constants obtained directly from the ultrafast, time-resolved optical spectroscopic experiments carried out (Pol\u00edvka et al. 2005) are shown in Table\u00a02. The value for the rate constant for S2 to S1\/ICT internal conversion, kIC2, is assumed to be 15\u00a0\u00b1\u00a01.5\u00a0ps\u22121 (\u03c4IC2\u00a0=\u00a066\u00a0\u00b1\u00a06\u00a0fs) from fluorescence up-conversion measurement on the native PCP complex (Linden et al. 2004). The value for kIC1 is assumed to be 0.063\u00a0ps\u22121 (\u03c4IC1\u00a0=\u00a016\u00a0ps) from a measurement of the efficiency and dynamics of energy transfer in the native PCP complex (Zigmantas et al. 2003). The overall energy transfer efficiencies calculated from Eq.\u00a01 are compared with the values obtained here from the steady-state measurements (Fig.\u00a07) and summarized in Table\u00a02. As one can see from the table, the efficiencies determined using both techniques are high in all cases and also in very reasonable agreement with each other.\nFig.\u00a09Schematic diagram of energy levels and energy transfer pathways between peridinin and Chl in the PCP complexes. kET1 and kET2 (solid lines) are rate constants for energy transfer from the S2 and S1\/ICT states of peridinin to Chl. kIC2 and kIC1 (dashed lines) are rate constants for internal conversion from S2 to S1\/ICT and S1\/ICT to S0, respectivelyTable\u00a02Rate constants and energy transfer efficiencies obtained from the ultrafast, time-resolved optical spectroscopic experiments on the PCP complexes reconstituted with various ChlsPCP complexkET2 (ps\u22121)kIC2 (ps\u22121)kET1 (ps\u22121)kIC1 (ps\u22121)\u03b5 (from fluorescence excitation), %\u03b5 (from time-resolved absorption), %Chl a10\u00a0\u00b1\u00a02.515\u00a0\u00b1\u00a01.50.34\u00a0\u00b1\u00a00.0340.06394\u00a0\u00b1\u00a0291\u00a0\u00b1\u00a07Chl b10\u00a0\u00b1\u00a02.515\u00a0\u00b1\u00a01.50.17\u00a0\u00b1\u00a00.0170.06392\u00a0\u00b1\u00a0284\u00a0\u00b1\u00a014Chl d13\u00a0\u00b1\u00a03.015\u00a0\u00b1\u00a01.50.59\u00a0\u00b1\u00a00.0590.06396\u00a0\u00b1\u00a0395\u00a0\u00b1\u00a02aChl a11\u00a0\u00b1\u00a02.815\u00a0\u00b1\u00a01.50.45\u00a0\u00b1\u00a00.0450.06399\u00a0\u00b1\u00a0193\u00a0\u00b1\u00a04BChl a10\u00a0\u00b1\u00a02.515\u00a0\u00b1\u00a01.52.22\u00a0\u00b1\u00a00.220.063\u223c10098\u00a0\u00b1\u00a02kET2 and kET1 are the rate constants for energy transfer from the S2 and S1\/ICT states of peridinin (Pol\u00edvka et al 2005). kIC2 and kIC1 are the rate constants for internal conversion from S2 to S1\/ICT and from S1\/ICT to S0, respectively. kIC2 was obtained from fluorescence up-conversion measurement on the native PCP complex (Linden et al 2004). kIC1 was deduced from the efficiency and dynamics of energy transfer in the native MFPCP given in Ref. (Zigmantas et al 2002). kIC2 and kIC1 were assumed to be unchanged in all the PCP complexes. The peridinin-to-Chl energy transfer efficiency, \u03b5, was obtained from steady-state fluorescence excitation experiments and from ultrafast, time-resolved absorption spectroscopic measurements at room temperature. The uncertainties in the values of \u03b5 from fluorescence excitation experiments were derived from the standard deviations obtained in the 450\u2013515\u00a0nm wavelength range. The uncertainties in the values of \u03b5 from time-resolved absorption measurements were obtained by propagating the published errors in the dynamicsaChl a 3-acetyl-Chl a\nIn this work, it was shown that optical spectroscopy carried out at low temperatures provides a more highly resolved view of the spectral features of light-harvesting complexes than seen at room temperature. The steady-state optical spectroscopic methods applied to PCP complexes in which recombinant apoprotein has been refolded in the presence of peridinin and either Chl a, Chl b, Chl d, 3-acetyl-Chl a or BChl a illustrate the robust character of the PCP protein which maintains high efficiency of peridinin-to-chlorophyll energy transfer function in the presence of non-native Chl species. The efficiencies of energy transfer deduced from the steady-state measurements are in good agreement with those from ultrafast, time-resolved experiments. The PCP complex continues to be an effective tool for elucidating the molecular details of photosynthetic light-harvesting via systematic modification of its structural components.","keyphrases":["reconstitution","chlorophyll","low temperature","absorption","fluorescence","fluorescence excitation","energy transfer efficiency","peridinin-chlorophyll a-protein"],"prmu":["P","P","P","P","P","P","P","U"]}
{"id":"Breast_Cancer_Res_Treat-3-1-2001218","title":"Letrozole as upfront endocrine therapy for postmenopausal women with hormone-sensitive breast cancer: BIG 1-98\n","text":"The BIG 1-98 trial is a large, randomized, independently conducted clinical trial designed to compare the efficacy of upfront letrozole versus tamoxifen monotherapy and to compare sequential or up-front use of letrozole and\/or tamoxifen as an early adjuvant therapy for patients with early breast cancer. We report on the results from the primary core analysis of the BIG 1-98 trial of 8,010 patients, which compares monotherapy with letrozole versus tamoxifen. This pre-planned core analysis allowed the use of patient data from the monotherapy arms of letrozole and tamoxifen and from the sequential arms prior to the drug switch point. Patients randomized to letrozole had a 19% improved disease-free survival (hazard ratio [HR] = 0.81; P = 0.003), due especially to reduced distant metastases (HR = 0.73; P = 0.001). A 14% risk reduction of fatal events in favor of letrozole was also observed (P = NS). The results from the monotherapy arms alone confirmed the findings from the primary core analysis. Based on the results from this trial, the aromatase inhibitor letrozole (Femara\u00ae) is currently recommended as a part of standard adjuvant therapy for postmenopausal women with endocrine-responsive breast cancer and has recently been approved in the early adjuvant setting in both Europe and the United States. A subsequent analysis after additional follow-up will address the question of monotherapy versus sequential therapy.\nIntroduction and rationale\nDuring the last century the management of primary breast cancer has evolved from gross surgical intervention to a sophisticated approach involving surgical, radiotherapeutic, hormonal, chemotherapeutic, and targeted biologic strategies. Currently, pharmacologic treatment is tailored according to disease and patient characteristics, including tumor size, histologic grade, lymph-node involvement, hormone receptor (HR) status, and human epidermal growth factor receptor-2 (HER2) overexpression. The benefits from adjuvant chemotherapy, adjuvant hormonal therapy, and adjuvant therapy with trastuzumab have been well-established [1, 2].\nPatients with early breast cancer presenting with estrogen receptor (ER)-positive and\/or progesterone receptor (PgR)-positive tumors typically receive adjuvant hormonal therapy. Adjuvant therapy with tamoxifen, a selective estrogen receptor modulator (SERM), significantly reduces the risk of breast cancer recurrence [1]; the Early Breast Cancer Trialists Collaborative Group reported that 5\u00a0years of tamoxifen reduces the annual breast cancer death rate by 31% and is significantly more effective than just 1\u20132\u00a0years of tamoxifen in ER-positive breast cancer [1]. However, intrinsic estrogenic activity [3\u20135] and increased risk of endometrial cancer [6] and thromboembolism [7, 8] have emerged as disadvantages when using tamoxifen. Based on current published data, extending the course of adjuvant tamoxifen beyond 5\u00a0years is not beneficial [9] despite the persistent risk of relapse in patients with HR+ breast cancer [10].\nIn the first-line treatment of advanced breast cancer, the third-generation aromatase inhibitors (AIs) have shown superior or equivalent efficacy compared with tamoxifen [11\u201315]. Letrozole demonstrated significant superiority in time to progression and overall response rate [12] and, in addition, an early survival benefit [11]. This indicates that a subset of the breast tumors is inherently less sensitive to tamoxifen [16] and that resistance to tamoxifen is acquired more quickly [17]. This superiority of letrozole over tamoxifen in the advanced setting led to the hypothesis that this AI may also be superior to tamoxifen when administered in the adjuvant setting.\nThe Breast International Group (BIG) 1-98 was designed, coordinated, analyzed, and reported by an independent academic group and currently is the largest ongoing adjuvant trial in breast cancer investigating the role of an AI. This review summarizes the design and results from the primary core analysis of the BIG 1-98 trial, which compared monotherapy with letrozole to monotherapy with tamoxifen and identified letrozole as a better alternative to tamoxifen in this setting. It also summarizes some of the additional published research using the BIG 1-98 database.\nTrial design and patients\nBIG 1-98 was a randomized, phase III, double-blind trial with two randomization options: two-arms (A or B) and four-arms (A, B, C or D) (see Fig.\u00a01) for women with operable invasive HR+ (ER+ and\/or PgR+) breast cancer. The treatment arms were: (A) initial therapy with tamoxifen for 5\u00a0years, (B) initial therapy with letrozole for 5\u00a0years, (C) initial therapy with tamoxifen for 2\u00a0years followed by letrozole for 3\u00a0years, or (D) initial therapy with letrozole for 2\u00a0years followed by tamoxifen for 3\u00a0years. The doses administered were 2.5\u00a0mg\/day of letrozole and 20\u00a0mg\/day of tamoxifen.\nFig.\u00a01BIG-98 trial design\nBetween March 1998 and March 2000, 1,835 women were randomly assigned to arms A or B, and between April 1999 and May 2003 a further 6,193 were assigned to arms A, B, C, or D. The primary core analysis compared treatment effects of patients randomized to receive letrozole initially (arms B and D) and those assigned to receive tamoxifen initially (arm A and C). In the sequential treatment groups (arms C and D), only events that occurred up to 30\u00a0days after switching treatments were included in the analysis. In total, 8,010 patients were included in the primary core analysis: 4,003 in the initial letrozole group (arms B and D) and 4,007 in the initial tamoxifen group (arms A and C) (see Fig.\u00a02). Patient characteristics were similar in the two treatment groups. Overall, the trial included 41.3% node-positive and 57.3% node-negative women, while 1.4% had unknown nodal status. Positive HR status was an eligibility criterion. The majority of patients (63.1%) had both ER+ and PgR+ tumors. A total of 25.3% of women received adjuvant or neoadjuvant chemotherapy.\nFig.\u00a02CONSORT (Consolidated Standards of Reporting Trials) flowchart of the BIG 1-98 trial. The primary core analysis includes all 8,010 assessable patients, but events and follow-up in the sequential treatment groups (L\u00a0\u2192\u00a0T and T\u00a0\u2192\u00a0L) are truncated at 30\u00a0days after switching to the other treatment. L denotes letrozole and T tamoxifen. Reprinted from [18, Supplementary Appendix]\nEnd points\nThe primary end point of the trial was disease-free survival (DFS), defined as the time from randomization to first occurrence of: invasive recurrence in ipsilateral breast, chest wall, regional site (internal mammary\/axilla), or distant site (including ipsilateral supraclavicular); contralateral breast cancer (invasive); second malignancy (non-breast); or death without prior cancer event. Protocol-specified secondary end points in the BIG 1-98 trial were: overall survival (OS), defined as time from randomization to death from any cause; systemic DFS, defined as time from randomization to distant recurrence; second non-breast malignancy or death from any cause (ignoring local and contralateral-breast events); and safety. Three additional end points were defined in the statistical analysis plan: (1) DFS excluding second, non-breast malignancies; (2) time to recurrence, defined as time from randomization to first breast cancer recurrence (excluding second, non-breast cancers and censoring data on patients who died without a prior cancer event); and (3) time to distant recurrence, defined as the time from randomization to the first breast cancer recurrence at a distant site.\nEfficacy analyses\nPrimary, secondary, and additional end points\nAt 25.8\u00a0months of follow-up, letrozole improved DFS by 19% (P\u00a0=\u00a00.003). The cumulative incidence of breast cancer relapse was significantly reduced with letrozole compared with tamoxifen (see Fig.\u00a03). The difference was evident from 1\u00a0year after randomization and at 5\u00a0years was 10.3% in the letrozole group compared with 13.6% in the tamoxifen group (P\u00a0<\u00a00.001). In addition, letrozole significantly improved systemic DFS compared with tamoxifen (hazard ratio 0.83; 95% CI 0.72\u20130.97). There was significant improvement with letrozole compared with tamoxifen for the additional end point of DFS excluding second non-breast cancers (hazard ratio 0.79; 95% CI 0.68\u20130.92). Letrozole was particularly effective in decreasing the risk of distant recurrence by 27% compared with tamoxifen (hazard ratio 0.73; 95% CI 0.60\u20130.88; P\u00a0=\u00a00.001) (see Fig.\u00a04). A non-significant 14% improvement in OS was observed in patients receiving letrozole. Thus, 166 deaths (4.1%) were observed in the letrozole group compared with 192 deaths (4.8%) in the tamoxifen group.\nFig.\u00a03Cumulative incidence of a breast cancer relapse in the BIG 1-98 trial. Reprinted from [18] with permission from the Massachusetts Medical SocietyFig.\u00a04Cox proportional-hazards model of data from the BIG 1-98 trial. The size of the boxes is inversely proportional to the standard error of the hazard ratio. The dashed vertical shows the hazard-ratio estimate for the overall analysis of the primary study end point (disease-free survival). Reprinted from [18] with permission from the Massachusetts Medical Society\nSubgroup analyses\nProspectively planned subgroup analyses, using the Cox proportional-hazards model, for primary, secondary, and additional end points are summarized in Fig.\u00a04. Subgroup analysis showed that the beneficial effect of letrozole on DFS was seen in ER+ tumors irrespective of PgR receptor status or patient age [18]. The role of ER and PgR in trials comparing AIs with tamoxifen remains an area of continued research [19, 20]. To explore this further, a central assessment of ER, PgR, and HER2 was recently completed for 6,500 patients in BIG 1-98. Results from the first 3,533 patients on the two monotherapy arms with ER+ tumors (by central assessment) and centrally assessed PgR and HER2 confirmed the results reported above from the local assessment of receptor status [18, 21], indicating that PgR status in ER+ tumors does not predict responsiveness to letrozole when compared with tamoxifen. The small group of patients with HER2 overexpression\/amplification in the tumor had a higher rate of recurrence with both treatments, but the superiority of letrozole over tamoxifen was similar irrespective of HER2 status [21].\nFor the primary end point of DFS, the relative risk reductions for letrozole compared with tamoxifen were 29% in patients with node-positive tumors (hazard ratio 0.71, 95% CI 0.59\u20130.85), 24% in patients with tumors >2\u00a0cm (hazard ratio 0.76, 95% CI 0.63\u20130.92), and 30% in patients with prior chemotherapy (hazard ratio 0.70, 95% CI 0.54\u20130.92). An additional logistic regression analysis of BIG 1-98 was performed to retrospectively identify clinical and pathological factors predictive of early breast cancer recurrence [22]. The final model, based on 5,980 patients from the four-arm option and 212 events, identified the following significant factors: tumor size (P\u00a0<\u00a00.001), ER\/PgR status (P\u00a0<\u00a00.001), node positivity (P\u00a0<\u00a00.001), and tumor grade (P\u00a0<\u00a00.001). There was a significant interaction between node positivity and treatment (P\u00a0=\u00a00.003). Patients with the greatest risk of recurrence had \u22654 positive nodes, tumors \u22655\u00a0cm, ER+\/PgR\u2212 tumors, and grade 3 tumors. The increase in risk associated with increased node positivity was greater for patients randomized to tamoxifen than to letrozole [22].\nLetrozole-only versus tamoxifen-only arms\nThe superiority of letrozole was confirmed in a protocol-defined supplementary analysis, which was restricted to the 4,922 patients randomized to the monotherapy tamoxifen or letrozole arms. At a median follow-up of 51\u00a0months, letrozole provided a significant benefit for the end points DFS (P\u00a0=\u00a00.007), DFS excluding secondary malignancy (P\u00a0=\u00a00.01), time to recurrence (P\u00a0=\u00a00.004), and time to distant recurrence (P\u00a0=\u00a00.03) (see Table\u00a01) [23].\nTable\u00a01Efficacy end points in patients randomized to treatment with letrozole (n\u00a0=\u00a02,463) or tamoxifen (n\u00a0=\u00a02,459) for 5\u00a0years in the BIG 1-98 trial [23]End pointEvents Hazard ratio95% CIP-valueLetTamDFS (primary protocol definition)3524180.820.71\u20130.950.007Overall survival1942110.910.75\u20131.110.35Systemic DFS3313740.870.75\u20131.010.07DFS (ignoring second non-breast cancer)3073640.830.71\u20130.960.01Time to recurrence2312910.780.65\u20130.920.004Time to distant recurrence1932340.810.67\u20130.980.03DFS disease-free survival, Let letrozole, Tam tamoxifen, CI confidence interval\nSafety\nAll adverse events were graded according to the Common Toxicity Criteria of the National Cancer Institute (version 2). Predefined adverse events were specifically asked and documented at each study visit. Furthermore, the IBCSG Coordinating Center conducted a medical review (reviewers were blinded to randomization) of all grade 3\u20135 cardiovascular events and other grade 3\u20135 adverse events that were considered clinically relevant but whose cause was unclear, and all deaths of women in whom there was no prior cancer-related event. The results of BIG 1-98 showed that letrozole was well-tolerated and had a safety profile different from tamoxifen (see Table\u00a02) [18]. A more detailed analysis of cardiovascular side effects, including baseline risk factors and cholesterol values over time, has recently been presented [24].\nTable\u00a02Cardiovascular adverse events and significant other adverse events among patients included in the BIG 1-98 safety analysis [18]Adverse event Incidence of any grade (%)P-valueLetrozole (n\u00a0=\u00a03,975)Tamoxifen (n\u00a0=\u00a03,988)Cerebrovascular accident or transient ischemic attack1.01.00.91Thromboembolic event1.53.5<0.001Cardiac event4.13.80.61Other cardiovascular event0.50.20.04Vaginal bleeding3.36.6<0.001Hot flashes33.538.0<0.001Night sweats13.916.20.004Fracture5.74.0<0.001Arthralgia20.312.3<0.001\nTamoxifen compared with letrozole was associated with an increased risk of thromboembolism, vaginal bleeding, and more endometrial biopsies (9.1% vs. 2.3%, respectively; P\u00a0<\u00a00.001), with a higher incidence of invasive endometrial cancers (0.3% vs. 0.1%, respectively; P\u00a0=\u00a00.18). In addition, tamoxifen was associated with a higher incidence of hot flushes (38.0% vs. 33.5%, respectively; P\u00a0<\u00a00.001) and night sweats (16.2% vs. 13.9%; respectively; P\u00a0=\u00a00.004).\nThere was a higher incidence of arthralgia and skeletal events with letrozole compared with tamoxifen, including a higher rate of fractures (5.7% vs. 4.0%, respectively; P\u00a0<\u00a00.001) and a shorter time to first fracture.\nHypercholesterolemia was among the adverse events listed on the case-report forms and was graded at each study visit during treatment [18]. A total of 43.6% of letrozole-treated and 19.2% of patients in the tamoxifen group had hypercholesterolemia, reported at least once during treatment [18]. Nevertheless, more than 80% of reported hypercholesterolemia was grade 1, and thus of uncertain clinical significance. In addition, serum total cholesterol values remained stable throughout the trial in the letrozole arm but decreased in the tamoxifen arm by approximately 13%, which is consistent with the known lipid-lowering effect of tamoxifen [25]. Thus, median changes in cholesterol values from baseline were 0, 0, and \u22121.8% at 6, 12, and 24\u00a0months in the letrozole group and \u221212.0, \u221213.5, and \u221214.1% in the tamoxifen group.\nThe overall incidence of cardiovascular events was similar for the letrozole and tamoxifen groups. Although there were higher incidences of grade 3\u20135 cardiac events and cardiac failure in the letrozole arm, the incidences were low in both groups (2.1% vs. 1.1%; P\u00a0<\u00a00.001 and 0.8% vs. 0.4%, P\u00a0=\u00a00.01, respectively) in this older patient population at competing risk for cardiovascular events [18].\nDiscussion \nClinical implications of BIG 1-98\nThe results of the primary core analysis [18], which included all available data from patients randomly assigned to the monotherapy arms and data from the sequential therapy arms censored at the time of the therapy switch, as well as the results from a recently published analysis limited to patients randomly assigned to the continuous therapy arms [23], demonstrate a significant benefit of letrozole over tamoxifen. Letrozole is at least as well-tolerated as tamoxifen, offering patients and physicians a true alternative. However, bone metabolism is differently affected by letrozole and tamoxifen. Patients receiving AIs are at increased risk for bone loss and osteoporosis and should therefore receive appropriate monitoring and medical intervention as part of daily practice.\nAs a result of the findings from BIG 1-98, letrozole was approved both in Europe and the United States as an early adjuvant treatment for postmenopausal women with HR+ breast cancer. The 2007 St. Gallen international consensus guidelines [26] and updated National Comprehensive Cancer Network (NCCN) guidelines [27] recommend letrozole as an option for adjuvant treatment of early breast cancer. In the 2007 St. Gallen Guidelines, use of an AI is considered as an alternative to tamoxifen for postmenopausal women with low-, intermediate-, or high-risk tumors that are classified as endocrine-responsive or endocrine response uncertain [26]. Similarly, in the NCCN guidelines, letrozole is a recommended adjuvant hormonal therapy for all postmenopausal women with hormone-responsive tumors, regardless of HER2 status [27]. The current guidelines do not recommend one AI over another but emphasize that treatment should be selected on the basis of clinical trial evidence in specific settings [27], nor do the guidelines provide recommendations as concerns the optimal use of the AIs as upfront monotherapy or sequenced with tamoxifen.\nResults from the Arimidex, Tamoxifen, Alone or in Combination (ATAC) trial provided evidence for superior DFS with anastrozole versus tamoxifen used as initial adjuvant hormonal therapy in postmenopausal women with HR+ breast cancer [28, 29]. At a median follow-up of 68\u00a0months, no survival advantage has been observed in the ATAC trial, and it remains an open question whether the DFS advantage observed in AI trials will translate into an OS advantage.\nBoth letrozole and anastrozole have demonstrated superiority over tamoxifen as initial adjuvant therapy [18, 28], but a direct comparison of letrozole with anastrozole awaits the results of a randomized head-to-head trial (Femara Anastrozole Clinical Evaluation [FACE]) [30, 31]. The adjuvant FACE trial compares upfront therapy with letrozole 2.5\u00a0mg with anastrozole 1\u00a0mg daily for up to 5\u00a0years in postmenopausal, HR+, node-positive breast cancer patients. In addition, recruitment of a direct comparison of anastrozole and exemestane (MA.27) has been completed recently.\nOther trials demonstrated better disease control when an AI was given after 2\u20133\u00a0years of adjuvant tamoxifen [32], but so far, no trial has reported on a regimen of an AI given 2\u20133\u00a0years before tamoxifen. Initial treatment with the \u201cgold standard\u201d tamoxifen followed by an AI may be a logical long-term strategy because of the lack of complete cross-resistance between these hormonal strategies. On the other hand, the greater anti-estrogenic potency and higher anti-tumor activity of AIs over tamoxifen, as demonstrated in preclinical models and randomized clinical trials [12, 17, 33, 34], may suggest that it is preferable to use an AI upfront to avoid early relapses that may occur while on tamoxifen therapy. Thus, the key question, \u201cshould AIs be given as initial therapy or used sequentially after tamoxifen?\u201d is as yet unanswered. Physicians often extrapolate data from switch trials, e.g., the Intergroup Exemestane Study [35, 36] or the MA.17 trial [33, 37] to sequential trials (e.g., BIG 1-98, Austrian Breast and Colorectal Cancer Study Group [ABCSG] 8 [38]). In these sequential trials, events are included in the analysis from treatment start and not from point of switch after 2\u20133\u00a0years of tamoxifen. Sequential and switch trials investigate obviously the same intervention but are conducted in different patient groups, thus, results are expected to be different and are different indeed.\nBest use of AIs remains an open question, at least until results of BIG 1-98 from the sequential use of letrozole and tamoxifen, in comparison with continuous monotherapy, as well as from the Tamoxifen and Exemestane Adjuvant Multicenter trial investigating exemestane monotherapy versus tamoxifen followed by exemestane [39] and from an updated analysis of ABCSG-8, become available.\nConclusions\nBIG 1-98 has shown that the AI letrozole results in better disease control than tamoxifen when given as initial endocrine therapy for postmenopausal women with hormone-responsive early breast cancer. Letrozole significantly reduced the risk of recurrence and of distant recurrence and has a reasonable safety profile [18]. The comparison between the monotherapy and the sequential treatment arms within the BIG 1-98 trial are eagerly awaited and are expected to have an important impact on the management of breast cancer.","keyphrases":["letrozole","breast cancer","big 1-98","tamoxifen","adjuvant therapy","aromatase inhibitor"],"prmu":["P","P","P","P","P","P"]}
{"id":"Biotechnol_Lett-4-1-2248218","title":"Purification and bioactivity of exendin-4, a peptide analogue of GLP-1, expressed in Pichia pastoris\n","text":"Exendin-4, a peptide analogue of glucagon-like peptide-1 (GLP-1), has been developed for treatment of type 2 diabetes. Herein, the secretive exendin-4 fusion protein, expressed by methanol induction in Pichia pastoris system, was purified to homogeneity by chromatography followed by enterokinase cleavage of the fusion protein and subsequent purification of the recombinant exendin-4. Purity of the recombinant exendin-4 was 95.6%. Bioactivity assay revealed that it had glucose-lowering and insulin-releasing action in vivo.\nIntroduction\nExendin-4 is a 39 amino acid peptide (53% structural homology to GLP-1) and was first isolated from the salivary secretions of Gila monster lizard (Heloderma suspectum) (Eng et\u00a0al. 1992). It shares many of the glucoregulatory actions with GLP-1 (Buse et\u00a0al. 2004; Kendall et\u00a0al. 2005) and has aroused great attention for its potential for the treatment of diabetes. Clinical and non-clinical studies have shown that exendin-4 has several beneficial anti-diabetic actions that include glucose-dependent enhancement of insulin secretion, glucose-dependent suppression of inappropriately high glucagon secretion, slowing of gastric emptying, reduction of food intake and body weight, and an increase in \u03b2-cell mass (Buse et\u00a0al. 2004; Edwards et\u00a0al. 2001; Kendall et\u00a0al. 2005; Kolterman et\u00a0al. 2003; Xu and Kaneto 2006).\nFor many years exendin-4 has been synthesized chemically at a high price and is thus unsuitable for mass production. It therefore needs to be produced in quantity by genetic recombinant technology. Exendin-4 has been successfully expressed in E. coli system and it proved having glucose-lowering action in vivo (Yi et\u00a0al. 2006; Yin et\u00a0al. 2005). However, no successful expression in other expression systems has been reported until now. For large-scale production, our laboratory recently constructed a recombinant Pichia pastoris that can produce exendin-4 constitutively and secrete it into the broth (Zhuang et\u00a0al. 2007). In this study, the purification and bioactivity of recombinant exendin-4 expressed in P. pastoris are reported.\nMaterials and methods\nMicroorganism and expression of exendin-4 fusion protein\nPichia pastoris GS115 was transformed with plasmid pPIC9 containing the gene encoding exendin-4, which was obtained from GenBank database (Accession No. AAB22006). The details of the vector construction and transformation were described elsewhere (Zhuang et\u00a0al. 2007). Recipes and fermentation procedures were all followed by the supplier\u2019s protocol and were described briefly as follows. Fermentation of the pPIC9\/exendin-4 transformant of was carried out using a 30\u00a0l fermentor. After 48\u00a0h of growth at 28\u00b0C, the transformant P. pastoris was induced by methanol for 72\u00a0h. The culture was centrifuged at 5,000g for 15\u00a0min to collect the supernatant which was used as the source of recombinant protein.\nPurification of fusion protein\nFor the purification of exendin-4, the total protein in the supernatant was first concentrated by ultrafiltration using a 10\u00a0kDa molecular mass cutoff membrane. The fusion protein was subsequently purified by ion-exchange chromatography, hydrophobic interaction chromatography (HIC) and size-exclusion chromatography. The void volume and eluted fractions from the three chromatographic columns were monitored at 280\u00a0nm. Fractions were collected and subjected to SDS-PAGE analysis. The purification steps were shown in Table\u00a01.\nTable\u00a01Summary of exendin-4 purificationPurification stepTotal protein (mg)Protein of interesta (mg)Recovery (%)Fusion exendin-4Broth supernatant1,640580100DEAE A-251,20039067Phenyl FF76028549G-7541021437Recombinant exendin-4G-25214264.5The concentrated broth supernatant was dialyzed with buffer A (20\u00a0mM Tris\/HCl, pH 6.0, 1\u00a0mM EDTA) and was applied to a DEAE Sephadex A-25 (DEAE A-25) (100\u00a0ml bed volume) equilibrated with buffer A beforehand, at 1\u00a0ml\/min. The column was washed with 300\u00a0ml buffer A and then eluted with a linear gradient of 0 to 1\u00a0M NaCl in buffer A. The eluted fractions containing exendin-4 were pooled and dialyzed. The desalted protein was mixed with ammonium sulfate at 1\u00a0M and applied onto a phenyl-Sepharose Fast Flow (Phenyl FF) column (50\u00a0ml bed volume) pre-equilibrated with buffer B [20\u00a0mM Tris\/HCl, pH 6.0, 1\u00a0M (NH4)2SO4, 1\u00a0mM EGTA] at 1\u00a0ml\/min, the column was washed with 150\u00a0ml buffer B and eluted with a linear (NH4)2SO4 gradient from 1 to 0\u00a0M in buffer B. The peak with exendin-4 was concentrated and dialyzed with buffer C (50\u00a0mM PBS, pH 6.0, 10% (v\/v) glycerol). The last step of purification was through a Sephadex G-75 column (1\u00a0\u00d7\u00a050\u00a0cm) pre-equilibrated with buffer C at 0.5\u00a0ml\/ min. For recombinant protein purification, the pool of fractions containing purified fusion protein from the three columns was dialyzed in 20\u00a0mM Tris\/HCl (pH 6.0)aThe amount of protein of interest was determined by quantifying the amount in each gel lane by densitometry (Totallab V1.11 software)\nCleavage of the fusion protein by enterokinase\nAfter desalting, the fusion protein was incubated with enterokinase (1\u00a0U\/1\u00a0mg fusion protein) at 37\u00b0C for 8\u00a0h to obtain the recombinant exendin-4. The reaction mixture was analyzed by Tricine SDS-PAGE. The recombinant exendin-4 was purified by Sepharose G-25 column and its purity was assayed by HPLC.\nSDS-PAGE, Tricine SDS-PAGE, immunoblotting and protein determination\nProteins were analyzed by SDS-PAGE under reducing conditions using 12% (v\/v) gels. Tricine SDS-PAGE was carried out with the supplier\u2019s protocol (Amersham Bioscience). The separated proteins were stained with Coomassie Brilliant Blue R-250 or electroblotted to nitrocellulose membrane for western blotting. After blocking with 3% (v\/v) non-fat milk in 0.05% Tween\/PBS (PBST), the membrane was washed 3 times with PBST for 10\u00a0min and incubated further with a 1:2,000 dilution of polyclonal rat anti exendin-4 antibody for 1\u00a0h, followed by washing as was described above. The membrane was incubated with a 1:1,000 dilution of HRP-conjugated goat anti-rat IgG as the second antibody, washed with the same procedure as above and revealed with DAB substrate (Sigma). Protein concentration was determined by the method of Bradford using bovine serum albumin as a standard.\nBiological activity assay\nThe effects of recombinant protein exendin-4 on plasma glucose concentration and insulin levels were examined using 8\u00a0week-old male Wistar rats. Animals were housed six per cage (three repetition) at 24\u00b0C environmental conditions with free access to food and water. They were allowed one week to adapt to their environment before the experiment. Food was withdrawn for 24\u00a0h before intraperitoneal injection of glucose (20\u00a0mM\/kg body weight) alone or in combination with exendin-4 (10\u00a0nmol\/kg body wt). Test solutions were administered in a final volume of 1\u00a0ml\/kg body weight (O\u2019Harte et\u00a0al. 2000). Blood samples were collected at 15, 30 and 60\u00a0min after the injection from the eye socket with capillary into chilled heparin Eppendorf tubes. Blood samples were centrifuged and plasma samples were stored at \u221220\u00b0C before determination.\nPlasma glucose and insulin level were assayed respectively by a glucose assay kit and insulin enzyme linked immunosorbent assay kit (Dingguo, Beijing). Results were expressed as mean\u00a0\u00b1\u00a0SE and were analyzed by one-way analysis of variance (ANOVA) by SPSS 11.0 software. Differences were considered significant at P\u00a0<\u00a00.05.\nResults\nExpression and detection\nThe results of expression and detection exendin-4 fusion protein are given in Fig.\u00a01.\nFig.\u00a01Expression and detection of exendin-4 fusion protein. (a) SDS-PAGE analysis of the expression level of fusion exendin-4 at different time in hours. After 56\u00a0h, the amount of the fusion protein expression reached the maximum (580\u00a0mg\/l). Fifteen micro liter of supernatant were applied to a 12% gel and then stained with R-250. Lane 1, control transformant GS155\/pPIC9 after 72\u00a0h culture; Lanes 2\u201310, recombinant GS115\/pPIC9\/exendin-4 after 8, 16, 24, 32, 40, 48, 56, 64 and 72\u00a0h of culture; The position of the target proteins induced by methanol is indicated by arrow. (b) Western blotting analysis of expressed protein in P. pastoris. Lanes 1\u20138 expressed protein of recombinant GS115\/pPIC9\/exendin-4 after 0, 8, 16, 24, 32, 40, 48 and 56\u00a0h, lane 9 expressed protein of control transformant GS115\/pPIC9\nPurifying the fusion protein and obtaining the recombinant exendin-4 by enterokinase cleave\nPurification of the fusion protein and production of the recombinant exendin-4 by enterokinase cleavage are given in Table\u00a01 and further details were shown in Fig.\u00a02.\nFig.\u00a02(a) Purification of the exendin-4 fusion protein by three chromatography steps. Lane 1, broth supernatant; lane 2, DEAE A-25; lane 3, Phenyl FF; lane 4, G-75; M is protein marker (KDa). (b) Purification of the recombinant protein exendin-4 by a G-25 column. Lane 1, reaction mixture of fusion protein cleaved by enterokinlase. Recombinant exendin-4 (4.3\u00a0kDa) is marked by arrow; lane 2, the non-target protein eluted from the G-25 column; lane 3, purified recombinant peptide exendin-4 from G-25 column. (c). The purity analyzed by HPLC was 95.6% which was performed on a 250\u00a0mm\u00a0\u00d7\u00a04.6\u00a0mm C5 column. The column was eluted with the linear gradient of acetonitrile (90\u20130%) in 0.1% trifluoroacetic acid for 35\u00a0min at 0.5\u00a0ml\/min. The target peak was at 18.3\u00a0min\nBioactivity of exendin-4 in vivo\nFigure\u00a03 showed the results of bioactivity of exendin-4 in vivo. Compared with the control group, plasma glucose concentration in GLP-1 group was significantly reduced (P\u00a0<\u00a00.05) at 30\u00a0min, and the same phenomenon was observed after 60\u00a0min in exendin-4 group (Fig.\u00a03a). There was no difference between the glucose-lowering capabilities of treatment group and control group before 15\u00a0min. The capabilities of stimulating insulin secretion showed significant response to GLP-1 and exendin-4 supplementation. The maximal increase in insulin in rat treated with GLP-1 was 2 times higher than the control group before 30\u00a0min. The plasma insulin concentration of exendin-4 treated group was also significantly raised (P\u00a0<\u00a00.05) at 30\u00a0min compared with the control group and the capabilities of stimulating insulin secretion by exendin-4 could keep to 60\u00a0min (Fig.\u00a03b).\nFig.\u00a03Biological activity assay of exendin-4 in rats. The capabilities of glucose-lowering (a) and insulin-releasing (b) after intraperitoneal glucose alone (20\u00a0mM\/kg) (control group), or glucose in combination with either exendin-4 (trial group) or GLP-1 (positive group) (10\u00a0nmol\/kg). The time of injection is indicated by the arrow (0\u00a0min). Values are mean\u00a0\u00b1\u00a0SE (n\u00a0=\u00a06). Asterisk denotes statistically significant differences (P\u00a0<\u00a00.05) between control and trial (or positive) groups by one-way ANOVA\nDiscussion\nFor many years, the production of exendin-4 was by chemical synthesis which necessitated a high cost. Recently, bioengineering methods of producing exendin-4 have appeared. Yi et\u00a0al (2006) and Yin et\u00a0al (2005) cloned the exendin-4 gene and obtained stable expression in E. coli. However, the E. coli-expressed exendin-4 existed initially in inclusion bodies and bioactivity was realized only after renaturation, which made the purification process complicated and led to a low yield. Furthermore, clinical application of bacterially produced products may be affected by the possible presence of endotoxins that sometimes contaminate protein preparations expressed by E. coli. We have several reasons to select P. pastoris as an alternative recombinant expression host for exendin-4 synthesis. P. pastoris has the advantages of large production, genetically stable expression strains, the potential to secrete recombinant proteins into culture medium, and simple inexpensive culture conditions (Patrick et\u00a0al. 2005). In this study, the highest expression level of fusion protein exendin-4 was estimated at about 580\u00a0mg\/l culture, which was higher than E. coli system. In addition, Pichia pastoris-synthesized recombinant exendin-4 was effectively purified from the culture medium through four chromatograph steps in this study. The whole purification process was simple and easy. These properties make P. pastoris a superior system to E. coli for preparation of exendin-4.\nTo the best of our knowledge, this is the first study to directly investigate the effects of recombinant exendin-4 expressed in P. pastoris on glucose-lowering and insulin-releasing in vivo. The results suggested that the ability of native peptide GLP-1 to reduce plasma glucose to a certain level was at 30\u00a0min, while the activity of exendin-4 appeared at 60\u00a0min (Fig.\u00a03a). It indicated that the exendin-4 has a longer duration of action and a greater potency than GLP-1 (Fig.\u00a03a). Insulin is one of the most important hormonal immune responses and is linked to glucose-lowering responses. Although reports on influence of administration of exenatides on insulin activity are not very consistent, this function can be enhanced by injection administration of exendin-4 or its analogue (Gedulin et\u00a0al. 2007; Kolterman et\u00a0al. 2003; Nielsen et\u00a0al. 2004; Xu and Kaneto 2006). The present study observed similar phenomenon. Furthermore, we observed that the capabilities of stimulating insulin secretion by GLP-1 was maintained up to 30\u00a0min, while exendin-4 was up to 60\u00a0min (Fig.\u00a03b). Thus the half-life of exendin-4 in plasma is longer than GLP-1. There are two main reasons for this. One reason is that the exendin-4 lacks many of the neutral endopeptidase substrate sites present in GLP-1 and has a nine-AA sequence at the C-terminus absent from GLP-1 (Hupe et\u00a0al. 1995). Another one is that the N-terminal sequence (His:Gly:Glu) of exendin-4 is not recognized by dipeptidylpeptidase (IV) (DPP (IV)), which rapidly cleaves the His:Ala:Glu sequence found on the N-terminus of GLP-1 (O\u2019Harte et\u00a0al. 2000). In our opinion, we should not rule out the presumption that the exendin-4 (4.3\u00a0kDa) has higher molecular weight than GLP-1 (3.1\u00a0KDa), which can reduce the glomerular filtration in vivo.\nIt is important to note that in the three independent experiments, the recombinant exendin-4 did not have obvious effects on one or two rats of the six. It was probably because of individual differences. The results of the three independent experiments were comparable. According to the research, the recombinant exendin-4 may also have effects at smaller doses but remains to be proved.\nIn summary, we have achieved high-level secretion of biologically active exendin-4 in P. pastoris. The result of the study indicated that although the heterogeneity-expressed exendin-4 in P. pastoris was slightly different from native GLP-1 in biological value, it still maintained good bioactivity. High purity and stability of the recombinant peptide of exendin-4 after purification makes it possible to be a therapeutical drug in the future. In the next step, we will focus on structure analysis and evaluation of the safety of exendin-4.","keyphrases":["exendin-4","pichia pastoris","diabetes","glucagon peptide analogue"],"prmu":["P","P","P","R"]}
{"id":"J_Abnorm_Child_Psychol-3-1-1915632","title":"Stability in Bullying and Victimization and its Association with Social Adjustment in Childhood and Adolescence\n","text":"This study examined the concurrent and longitudinal associations between stability in bullying and victimization, and social adjustment in childhood and adolescence. Participants were 189 girls and 328 boys who were studied in primary school and in secondary school. The mean age of the participants was 11.1 years in primary school and 14.1 years in secondary school. The measures consisted of peer reported social and personal characteristics. Children who bullied in childhood and adolescence were less liked and more disliked in childhood, and more aggressive and disruptive both in childhood and adolescence, than children who bullied only in childhood or adolescence. Children who bullied or who were victimized only in childhood did not differ largely in adolescence from the children that were never bullies or victims. Children who were victimized in adolescence closely resembled those who were victimized in childhood and adolescence in terms of being liked or disliked, being nominated as a friend, and shyness. The study stresses the need to distinguish between stable and transient bullies and victims.\n\u00a0\nBullying in school classes refers to negative physical or social actions that are repeated over time by one or more other persons towards a person that can not easily defend (Olweus, 1991). Bulling involvement seems relatively stable over time (Boulton & Smith, 1994; Kumpulainen, R\u00e4s\u00e4nen, & Henttonen, 1999) and has been related to various psychosocial adjustment problems in childhood and adolescence. Much research on bullying involvement, especially on victims, has focused on internalizing indicators of adjustment (see for a review Hawker & Boulton, 2000). Little is known about the overt, interpersonal behavioral characteristics of bullies and victims, and even less is known about the association between stability in bullying and victimization and these social characteristics. The aim of the present study was to examine the link between stability in bullying and victimization, and individual differences in social behaviors that are salient to the peer environment. More specifically, we were interested in differences in childhood and adolescent social adjustment of transient and stable bullies and victims.\nBullies, victims, and adjustment\nIn primary schools between 20% and 30% of the children are victims of bullying, while between 10% and 20% of the children are bullies (Smith et\u00a0al., 1999), both in Western and non-Western countries (Eslea et\u00a0al., 2003). Cross-sectional research on the adjustment of bullies revealed that bullies are more rejected and less popular (Boulton & Smith, 1994), and display more antisocial, aggressive and disruptive behavior than non-involved children (Pellegrini, Bartini, & Brooks, 1999; Rigby & Cox, 1996). Furthermore, longitudinal studies suggest that childhood bullying is associated with social maladjustment in adolescence (Kumpulainen & R\u00e4s\u00e4nen, 2000). Research on the adjustment of victims showed that these children are socially isolated and rejected, and have fewer friends (Hodges, Malone, & Perry, 1997; Hodges & Perry, 1997). In addition, victims tend to be more submissive in their interactions with peers (Schwartz, Dodge, & Coie, 1993) and show overt signs of helplessness and distress (Perry, Williard, & Perry, 1990).\nLongitudinal research on bullies, victims and adjustment usually linked being a bully or a victim in childhood to adjustment measures in adolescence, without accounting for the chronicity in bullying or victimization. For example, Kumpulainen and R\u00e4s\u00e4nen (2000), reported that children who bullied at age 8 and 12 displayed more externalizing behavior, hyperactivity, and relationship difficulties when they were 15 years old, while children who were victimized at age 12 were more likely to experience relationship problems such as being irritable, isolated, and rejected three years later. Camodeca, Goossens, Meerum Terwogt, and Schuengel (2002) found that stable victims were more reactively aggressive than transient victims. Neither of these studies, however, examined whether the participants were still bullies or victims at the follow-up assessment. Consequently, the longitudinal associations that have been reported may have been confounded by concurrent associations between adolescent bullying involvement and adolescent adjustment.\nAdequate descriptions of longitudinal associations between childhood bullying involvement and adolescent social adjustment may require distinguishing between children who remain involved in bullying from childhood to adolescence (i.e. stable bullies and victims) and those whose involvement is restricted to childhood (i.e., transient or unstable bullies and victims). Various studies showed that a substantial number of children display a stable pattern of bullying or victimization (e.g., Boulton & Smith, 1994; Camodeca et\u00a0al., 2002; Salmivalli, Lappalainen, & Lagerspetz, 1998). Boulton and Smith (1994) found correlations of around.60 between bullying at age 8 and one year later, while Kumpulainen, R\u00e4s\u00e4nen, and Henttonen (1999) reported that 25% of 8-year old bullies had turned into stable bullies by the time they were 12, and 15% of the victims had become stable victims. However, very few studies have examined the associations between stability in bullying involvement and interpersonal behavioral characteristics.\nStability in being a bully or a victim\nStability in being a bully or a victim in school can be caused by two mechanisms: continuity in social environment and continuity in children\u2019s interactional styles. According to Caspi, Elder, and Bem\u2019s cumulative continuity model (1987; Caspi, Bem, & Elder, 1989) and to Scarr\u2019s genotype environment correlations model (Scarr & McCartney, 1983; Scarr, 1985; Scarr, 1992), children\u2019s social maladaptive behaviors may direct them into social environments that perpetuate these behaviors. The idea is that children with specific behaviors select and create environments that promote and maintain their behavior. This may hold for bullies as well as victims. It is well known that antisocial boys affiliate with boys who are similarly aggressive and deviant (Cairns, Cairns, Neckerman, Gest, & Gariepy, 1988), and that victims are more likely to have friends who are less accepted by peers or who themselves are victimized (Hodges et\u00a0al., 1997; Salmivalli, Huttunen, & Lagerspetz, 1997), thus reinforcing their behavior. Additionally, peers may also reinforce bullying and victimization by conferring reputations that may lock bullies and victims into their specific roles (DeRosier, Cillessen, Coie, & Dodge, 1994). These reputations are difficult to change as long as the school class constellation remains unchanged (Hymel, Wagner, & Butler, 1990).\nThe social environment may reinforce bullies\u2019 behaviors yet in another way. Observational studies (Craig & Pepler, 1997; Pepler & Craig, 1995; Pepler, Craig, & Roberts, 1998) showed that peers often do not intervene in bullying. Bullies may perceive this lack of intervention as a signal that peers condone their bullying behavior. Furthermore, studies on participant roles reveal that some peers may even actively reinforce the bullies by encouraging gestures or by laughing during bullying episodes (Salmivalli, Lagerspetz, Bj\u00f6rkqvist, Osterman, & Kaukiainen, 1996).\nStability in being a bully or a victim may also be due to continuity in bullies\u2019 or victims\u2019 interactional styles (i.e., interactional continuity, Caspi et\u00a0al., 1987, 1989) that place them at risk for prolonged bullying or victimization, even after changes in the social environment. Being stably involved in bullying in the formative years of primary school may deprive children from positive social experiences, inhibiting the acquisition of prosocial skills and fostering social skills deficits. Because these children do not learn how to adequately react in social interactions, they may develop dysfunctional interactional styles which may make them prone to social adjustment problems later in life. In this way, a vicious cycle between bully\u2019s or victims\u2019 interactional styles and their bullying or victimization may be established (cf. Kochenderfer-Ladd & Wardrop, 2001). For example, Snyder et\u00a0al. (2003) showed that for some children victimization was situational, while for other children victimization became a trait like status, possibly because of this dysfunctional interactional style.\nStability in being a bully or a victim, and adjustment\nAlthough the processes contributing to stability in bullying or victimization are well described, very few studies have examined the behavioral profiles of different bully or victim trajectory types over time. Research has indicated that most bullies desist after some time, but a small group persists (Broidy et\u00a0al., 2003; Loeber & Hay, 1997; Le Blanc & Loeber, 1993). Because of their aggressive interactional styles and their lack of opportunities to develop social skills, these children are at risk for higher levels and more serious forms of aggression, poor peer relations and social maladjustment later in life (Pope & Bierman, 1999). This behavioral style reflects Olweus\u2019 (1991) notion that bullying is not an isolated phenomenon but rather a component of more general antisocial and rule-breaking behavioral patterns.\nRegarding victims, only two studies seem to have longitudinally examined the adjustment of stable and transient victim groups. Juvonen, Nishina, and Graham (2000) found that stable victims were lonelier and reported a lower self-worth than transient victims (ie. those who were victimized one year earlier but were no longer victims). Noteworthy, transient victims did not differ from stable non-victims on loneliness, self-worth or depressive symptoms. Covering a two-year period, Smith, Talamelli, Cowie, Naylor, and Chauhan (2004) found that stable victims had fewer friends at school and scored higher on self-reported and teacher-reported emotional problems and peer problems. As was found by Juvonen et\u00a0al. (2000), in general, the transient victims did not differ largely from the stable non-victims. The latter seems to indicate that psychosocial problems only occur at the time when the victimization takes place. It also supports the cessation hypothesis (Kochenderfer-Ladd & Wardrop, 2001) which states that the psychosocial problems are likely to disappear as soon as the victimization ends.\nThe present study\nThe present study investigated whether stable bullies or victims showed specific behavioral patterns that distinguished them from children who were involved in bullying only in childhood or adolescence. Whereas previous studies on bullying and victimization often focused on psychological adjustment in terms internal characteristics such as self-esteem, loneliness, or emotional stability, our aim was to examine the interpersonal, social adjustment as it was reported by the peers. The bully groups and victim groups were compared on social characteristics that have been identified in previous research as highly relevant correlates for describing bullies or victims in childhood or adolescence. These characteristics included being liked and being disliked (Hodges & Perry, 1999; Pellegrini et\u00a0al., 1999), friendships (Hodges et\u00a0al., 1997; Hodges & Perry, 1999), aggressive and disturbing behaviors (Boulton & Smith, 1994; Snyder et\u00a0al., 2003), help seeking, shyness (Boulton & Smith, 1994) and offering help and cooperation (Boulton & Smith, 1994). We used peer reports to assess these constructs for children may have distorted views of their own social competence (Lemerise & Arsenio, 2000) and self-reports may thus not always be the most reliable means of obtaining information about children\u2019s social functioning in peer groups.\nThe longitudinal group design allowed us to test a number of hypotheses. The hypotheses concerning bullies were in part guided by the work of Olweus (1991) and Loeber (Loeber & Hay, 1997; LeBlanc & Loeber, 1993) and the cumulative continuity hypothesis (Caspi et\u00a0al., 1987) and genotype-environment correlations model (Scarr, 1983, 1992; Scarr & McCartney, 1983). We hypothesized that due to the accumulative effects of their negative peer interactions stable bullies would exhibit a pattern of more severe, negative and hostile behaviors in childhood and adolescence, and would have poorer adjustment, compared to bullies who bullied for a restricted period of time (i.e., only in childhood or adolescence). Because we assumed that adolescents who first started to bully in adolescence may have done so in order to obtain dominance (Pellegrini et\u00a0al., 1999) and not because of a specific stable behavioral pattern, we expected that they would show specific maladaptive features in adolescence but not in childhood.\nConcerning the victims, we hypothesized that stable victims would show higher levels of peer perceived social problems in adolescence compared to childhood or adolescence-only victims. This was based on the idea that stable victims were exposed to victimization for a substantially longer period and thus have had fewer opportunities to acquire positive social skills and experiences. In line with the cessation hypothesis (Kochenderfer-Ladd & Wardrop, 2001) and the findings provided by Juvonen et\u00a0al. (2000) and Smith et\u00a0al. (2004) we expected that childhood-only victims would not differ from the stable non-involved children in adolescence. Although we did not have theoretically based hypotheses, we tested whether it would be possible to already predict in childhood which children would become new victims in adolescence (i.e., adolescence-only victims). Finally, we tested whether gender moderated the associations between stability and adjustment. Because we did not have theoretically derived hypotheses about the interaction the interaction effects of gender, testing these effects was entirely exploratory, and no research question specifically addressed this issue.\nMethod\nParticipants\nParticipants were 517 children (189 girls, 328 boys) who took part in the third and fourth wave of a longitudinal study that started in 1985. In the first two waves (1985 and 1986), 231 boys were examined (see for detailed information Cillessen, van Yzendoorn, van Lieshout, & Hartup, 1992). In waves 3 (1991) and 4 (1994), the initial boys were again assessed, but now the assessment also included the boys\u2019 classmates, resulting in total samples of 2521 and 3361 children, for Wave 3 and 4, respectively. The sample of the present study consisted of 517 children who were present both in Wave 3 (childhood data) as well as in Wave 4 (early adolescence data). Because the 231 initial longitudinal participants were boys, some of whom attended schools with a predominantly male population in secondary education (e.g., technical education), there were more boys than girls in the present study. Attrition bias checks using t-tests comparing the present sample with the larger childhood and adolescence samples that were dropped from this study (i.e., the Wave 3 and Wave 4 samples minus the participants who were in the longitudinal sample) revealed that the present study\u2019s participants did not differ on bullying or victimization, nor on any other study variable, from the larger cross-sectional childhood or adolescence sample. They did also not differ on ethnicity or education, nor on socioeconomic status of the parents. In 1991 (Wave 3), participants\u2019 age was 11 years, 1 month (SD\u00a0=\u00a011 months) and ranged from 10 to 13. The children came from 100 elementary school classes in the Nijmegen-Arnhem area in The Netherlands. In 1994 (Wave 4), all participants were enrolled in secondary education, and were distributed across 131 school classes. Their mean age was 14 years and one month (SD\u00a0=\u00a011 months), and ranged from 13 to 16. Ninety-five percent of the participants were of Dutch origin, while five percent of the adolescents were ethnic minorities: 1.5% came from Surinam, the Netherlands Antilles, and the Molucca Islands; 2% from Mediterranean countries; and 1.5% from elsewhere. Socio-economic status was based on the classification of the parents\u2019 occupations. The lower socio-economic status contained occupations such as factory workers, while middle socio-economic status consisted of occupations like for example teachers and nurses. The higher socio-economic status included occupations such as lawyers, and physicians. It showed that 22% of all children had a lower, 43% a middle, and 35% a higher socio-economic status. Overall, at the two waves the participants appeared to be representative of the Dutch school population in the geographic area in which the children lived and at the time the assessments were made.\nProcedure\nBoth the childhood data and adolescence data were obtained by classroom data collections, arranged separately for each school class (for detailed descriptions, see Haselager, Hartup, van Lieshout, & Riksen-Walraven, 1998, and Scholte, van Lieshout, & van Aken, 2001, for childhood and adolescent data assessment, respectively). In order to ensue that school staff and parents obtained as much information as they needed before the study was conducted, letters were sent to schools and children took home these letters for their parents to read. In these letters parents were informed about the study and were asked for passive consent. As in other large scale studies that we conducted in the Netherlands, there were no parents in the present study who did not allow their children to participate. Consent was also obtained from the children and adolescents themselves and from school authorities. Participants were guaranteed confidentiality in the collection and maintenance of the data. In both assessment waves the data collection session started with a brief introduction and class instruction, given by a trained examiner. During the one-hour assessment, the children were asked to fill out a questionnaire booklet. Before answering the bullying involvement questions, the children were provided a definition of \u201cbullying other children\u201d (Olweus, 1989) by the examiner.\nMeasures\nChildhood measures (peer nominations)\nWe developed a sociometric questionnaire containing 11 items on peer reported social behavior. This measure was similar to the widely used instrument developed by Coie, Dodge, and Coppotelli (1982). Children were asked to nominate a maximum of three classmates who best fitted the descriptions, in a rank ordered way such that the classmate who best fitted a description was nominated first, followed by the second and third best fitting classmate. Same- as well as cross-sex nominations were allowed on each description but self-nominations were not. The children were presented a roster of their classmates to use as a reference in making their nominations on the sociometric questions. Bullying other children was assessed by the question \u201cWhich children in class often bully other children, or pick on them.?\u201d Being bullied (victimization) was assessed by the question \u201cWhich classmates are often bullied and picked on by other children.?\u201d Being liked and being disliked were based on the questions \u201cWhich children in your class do you like most?\u201d and \u201cWhich children in your class do you like least,?\u201d respectively. Aggression referred to starting fights in class, while \u2018Disruption\u2019 assessed disturbing and disruptive behavior. Cooperation assessed being considerate and cooperative, while \u2018Offering help\u2019 indicated offering help to others. Seeking help assessed which children sought help often, while Shyness referred to being shy. Being nominated as a friend, finally, was based on the question \u201cWhich three children in you class are your friends.\u201d This variable was used as an indicator of a child\u2019s social integration in the class. Being nominated as a friend was not synonym with being liked because it was possible that children liked classmates without being friends with them.Table 1Intercorrelations among social adjustment measures in childhood123456781. Being liked2. Being disliked\u2212.38***3. Friend nominations.71***\u2212.35***4. Aggression\u2212.20***.65***\u2212.21***5. Disruption\u2212.14**.49***\u2212.15***.71***6. Cooperation.53***\u2212.36***.51***\u2212.30***\u2212.26***7. Shyness\u2212.16**\u2212.06\u2212.16***\u2212.13**\u2212.16***\u2212.048. Offering help.60***\u2212.21***.58***\u2212.06\u2212.04.61***\u2212.15**9. Help seeking\u2212.05.25\u2212.06.18***.18***\u2212.08.09.01**p\u00a0<\u00a0.01. ***p\u00a0<\u00a0.001.Table 2Correlations between social adjustment measures in childhood and adolescenceAdolescencechildhood1234567Insecurity1. Being liked.25***\u2212.16***.17***.01\u2212.06.17***\u2212.16***\u2212.14**2. Being disliked\u2212.17***.28***\u2212.13**.24***.19***.16***\u2212.05.073. Friend nominations.25***\u2212.14***.18***\u2212.04\u2212.08.21***\u2212.11*\u2212.11*4. Aggression.00.18***.02.38***.28***\u2212.14**\u2212.15***.005. Disruption.01.17***.05.40***.30***\u2212.14**\u2212.21***\u2212.016. Cooperation.18***\u2212.13***.11*\u2212.08\u2212.07.21***\u2212.02\u2212.087. Shyness\u2212.10*\u2212.00\u2212.09*\u2212.16***\u2212.12**\u2212.02.30***.15**8. Offering help.16***\u2212.09*.14**.06.01.15**\u2212.13**\u2212.13**9. Help seeking.10*.04.09*\u2212.03\u2212.01.03\u2212.05\u2212.09*p\u00a0<\u00a0.05.**p\u00a0<\u00a0.01.***p\u00a0<\u00a0.001.\nAdolescence measures (peer nominations)\nAdolescents were also presented a roster with the names of their classmates. In grades 1 through 3 of secondary education, adolescents were in the same group all year, and this group served as the nomination reference group. The reference group of the adolescents who were in grade 4 of secondary education consisted of their root class, which consists of those classmates with whom they spent most of the classes, and with whom they share the same mentor. The sociometric questionnaire administered in the adolescent sample contained 25 questions referring to liking and disliking, bullying and victimization, number of friends, and peer group behavior (Scholte et\u00a0al., 1997). Nine of the 25 items were similar to those asked in childhood (see below), while 16 new items assessed peer-perceived self-confidence, emotional stability, and school achievement. In the present study we only used items in adolescence that were similar to the items in childhood, added with the item on \u201cinsecurity\u2019 which in a way reflected the childhood question on seeking help.\nParticipants could nominate up to five classmates on each of the questions, and were asked to nominate first the classmate who fitted an item best, followed by the classmate who fitted an item second best et cetera. In the present study, only the first three nominations on each item were used in the analyses, which was similar to the number of nominations on each item in the childhood data. Same and cross-sex nominations were permitted, self-nominations were not allowed. Assistance was provided if needed. Bullying others and Being bullied were assessed by the questions \u201cWhich classmates bully others,\u201d and \u201cWhich classmates are being bullied,\u201d respectively. Being liked and being disliked, being nominated as a friend, aggression, disruption, cooperation, and shyness were assessed by similar questions as used in the childhood assessment. Insecurity was assessed by the item \u2018Which classmates are insecure and seem to lack self-confidence.\u2019 The intercorrelations among these indicators of social adjustment are given in Table 1.\nSociometric nominations were processed as follows. For each participant, scores on each item were determined by summing all received nominations from classmates on that item. These two raw scores were transformed into within-class probability scores (p-scores) assuming a generalized binomial distribution, thus correcting for unequal numbers of nominations made among children and differences in class size (Newcomb & Bukowski, 1983). The p-scores were then z-standardized across all participants.\nEach peer nomination scale in childhood and adolescence consisted of one item. Because peer nominations involve aggregating across multiple respondents, - in our study on average 23 per class- peer nominations based on one item tend to be reliable (Coie, Dodge, & Kupersmidt, 1990), because, in contrast to self-reports, they are based on the reports of many informants which may decrease the influence of individual bias (Boulton & Smith, 1994). In order to be able to describe the differences between the different groups of victims and bullies more clearly, the childhood and adolescence social adjustment variables were standardized within the total sample and these standardized scores were subsequently analyzed.\nResults\nThe correlations between the childhood measures and adolescence measures are presented in Table 2. As can be seen, correlations between the childhood and adolescence data were low to moderate, with the highest correlations between childhood aggression and disruption and adolescence aggression and disruption (r\u2019s between .28 and .40).\nClassification of bullies, victims and non-involved children\nTo determine which children and adolescents were bullies or victims, we used the peer nomination scores on Bullying others and Being bullied. Children and adolescents who scored 1 standard deviation above the mean on Bullying others were considered bullies, and children and adolescents who scored one standard deviation above the mean on Being bullied were regarded as victims. This procedure has been used in other studies (e.g., Pellegrini et\u00a0al., 1999; Solberg & Olweus, 2003).\nOf all children in childhood, 9 % were victims (n\u00a0=\u00a049; 17 girls of the total sample of girls, 32 boys of the total sample of boys), 19% were bullies (n\u00a0=\u00a0100; 6 girls), and 71% were not involved in bullying (n\u00a0=\u00a0368; 163 girls). In adolescence these percentages were 11% (n\u00a0=\u00a055; 14 girls) for victims, 20% (n\u00a0=\u00a0104; 12 girls) for bullies, and 69% (n\u00a0=\u00a0358; 160 girls) for non-involved children. Chi-square analyses indicated that while boys and girls were equally likely to be victims, boys were more likely to be bullies in childhood (\u03c72 (2) =\u00a044.57, p\u00a0<\u00a0.001) and in adolescence (\u03c72 (2) =\u00a037.34, p\u00a0<\u00a0.001). Because the focus of this study was on bullies or victims, children who scored one standard deviation above the mean on being bullied and bullying either in childhood or adolescence (i.e., bully-victims, n\u00a0=\u00a09) were not included in the analyses.\nTo examine the longitudinal involvement in bullying, a chi-square analysis was conducted with bully status in childhood and adolescence as factors. The chi-square analysis for the total sample (\u03c72(4, N\u00a0=\u00a0517) =\u00a0111.30, p\u00a0<\u00a0.001) and the subsequent test of standardized residuals (Haberman, 1973) showed that victimization and bullying were relatively stable across this three year period. Forty-six percent (n\u00a0=\u00a046) of the childhood bullies persisted into adolescence (i.e., Stable Bullies), whereas the others had either stopped being involved (i.e., Childhood Bullies, 45%, n\u00a0=\u00a045) or had turned into victims (9%, n\u00a0=\u00a09). Forty-three percent (n\u00a0=\u00a021) of the children who were victims in childhood were still victims in adolescence (i.e. Stable Victims), 51% of the childhood victims (n\u00a0=\u00a025) were not involved in bullying in adolescence (i.e. Childhood Only Victims), while 6% (n\u00a0=\u00a03) had turned into bullies. Of all the children not involved in bullying in childhood, 15% (n\u00a0=\u00a055) started bullying in adolescence (i.e., Adolescence Only Bully), and 7% (n\u00a0=\u00a025) became victims (i.e., Adolescence Only Victim). The children who were not involved in childhood nor adolescence were considered Non-involved, and served as a reference group. Gender differences in bullying and victimization continuity indicated that male and female childhood victims were equally likely to become a stable victim, but that the continuity of bullying other children was low in girls and high in boys (\u03c72 (6, N\u00a0=\u00a0517) =\u00a066,13, p\u00a0<\u00a0.001). Except for one girl, all stable bullies were boys.\nTo examine the association between the stability in bullying and victimization, and social adjustment, the three groups of bullies as well as the three groups of victims were compared. For reasons of conceptual clarity, children who bullied in childhood and had turned into victim in adolescence and those who were victims in childhood but became bullies in adolescence were dropped from further analyses. Bullying group by gender interactions were examined, as well as gender main effects. The latter were significant in childhood (Wilks \u039b\u00a0=\u00a0.93, F (9, 343) =\u00a02.94, p\u00a0<\u00a0.01) and adolescence (Wilks \u039b\u00a0=\u00a0.93), F (8, 342) =\u00a02.99, p\u00a0<\u00a0.01) and indicated that both in childhood and adolescence, boys were more disliked, more aggressive and disruptive, less cooperative, and less shy than girls. In addition, boys were less often nominated as a friend in childhood than girls did, and were less insecure in adolescence. Because the Stable Bullies included only one girl, gender by bully group interactions could not be examined in analyses that included the Stable Bullies group.Table 3Childhood and adolescent adjustment of childhood only, adolescence only, and stable bullies, and non-involved childrenBulliesChildhood only (n=45)Adolescence only (n=55)Stable (n=46)Non-involved (n=288)F-valuePartial eta squaredChildhoodBeing liked.08b.20b\u2212.37a.14b3.75*.03\u2003Being disliked.44c\u2212.28a1.39b\u2212.36a86.31***.38\u2003Friend nominations.06b.08b\u2212.38a.15b3.79*.03\u2003Aggression1.28b\u2212.28a1.78c\u2212.42a224.32***.61\u2003Disruption1.18c\u2212.12b1.69d\u2212.36a137.25***.49\u2003Offering help.03.11\u2212.10.08.49.00\u2003Cooperation\u2212.30a.09b\u2212.59a.20b10.80***.07\u2003Shyness\u2212.30ab\u2212.27ab\u2212.28a.06b4.40**.03\u2003Help seeking.35b\u2212.21a.02a\u2212.10a3.51*.02Adolescence\u2003Being liked.35.11\u2212.18.142.31.02\u2003Being disliked\u2212.37a.50b.87b\u2212.33a43.42***.23\u2003Friend nominations.28.11\u2212.05.12.86.01\u2003Aggression\u2212.26a.84b1.77c\u2212.34a118.42***.45\u2003Disruption\u2212.20a.54b1.26c\u2212.26a46.81***.26\u2003Cooperation.10b\u2212.28a\u2212.41a.19b7.27***.05\u2003Shyness\u2212.29a\u2212.28a\u2212.51a.02b6.85***.05\u2003Insecurity\u2212.16\u2212.14\u2212.21\u2212.011.01.01Note. All scores are z-scores. Means with different superscripts are significantly different from each other.*p\u00a0<\u00a0.05. **p\u00a0<\u00a0.01. ***p\u00a0<\u00a0.001.\nSocial adjustment of bullies in childhood\nTo examine whether the Stable Bullies experienced more social problems in childhood than the other bullies and non-involved children, and whether Adolescence Only Bullies showed signs of maladjustment already in childhood, a MANOVA was conducted with bullying groups (Childhood Only Bullies, Adolescence Only Bullies, Stable Bullies, and Non-involved) and gender as independent variables and the childhood adjustment scores as dependent variables.1 A significant multivariate relation was found, Wilk\u2019s \u039b\u00a0=\u00a0.32, approx F (27, 1230) =\u00a021.81, p\u00a0<\u00a0.001. Except for offering help, all univariate tests were significant (see Table 3). Childhood Only Bullies scored higher on help seeking behavior than the Stable Bullies. However, Stable Bullies were less liked and more disliked by their peers, were less often nominated as a friend, and displayed more aggression and disruption than all other children. Adolescence Only Bullies scored lower on being disliked, aggression, disruption, and help seeking, and higher on cooperation than Childhood Only Bullies.Table 4Childhood and adolescent adjustment of childhood only, adolescence only, and stable victims, and non-involved childrenVictimsChildhood only (n= 25)Adolescence only (n= 25)Stable (n=21)Non-involved (n=288)F-valuePartial eta squaredChildhood\u2003Being liked\u2212.52a\u2212.22ab\u2212.88a.14b9.28***.07\u2003Being disliked.63b\u2212.28a1.34c\u2212.36 a38.54***.25\u2003Friend nominations\u2212.60a\u2212.23ab\u2212.58a.15b7.44***.06\u2003Aggression.15b\u2212.32a.18b\u2212.42a14.82***.11\u2003Disruption\u2212.13\u2212.48\u2212.19\u2212.361.53.01\u2003Offering help\u2212.37a\u2212.33ab\u2212.44a.08b3.44*.03\u2003Cooperation\u2212.49a.02b\u2212.52a.20b5.87***.05\u2003Shyness.40.12.42.061.42.01\u2003Help seeking.30b\u2212.06a.87c\u2212.10a8.14***.07Adolescence\u2003Being liked\u2212.14b\u2212.92a\u2212.75a.14c10.61***.08\u2003Being disliked\u2212.21a.75b.81b\u2212.33a23.88***.17\u2003Friend nominations\u2212.07b\u2212.93a\u2212.80a.12b9.80***.08\u2003Aggression\u2212.28\u2212.25\u2212.29\u2212.34.09.00\u2003Disruption\u2212.26.05.04\u2212.262.12.02\u2003Cooperation\u2212.11ab\u2212.40a\u2212.58a.19b6.50***.05\u2003Shyness\u2212.19a1.03b1.27b.02a17.10***.13\u2003Insecurity\u2212.02a.37ab.88b\u2212.01a4.37**.04Note. All scores are z-scores. Means with different superscripts are significantly different from each other.*p\u00a0<\u00a0.01.**p\u00a0<\u00a0.01.***p\u00a0<\u00a0.001.\nSocial adjustment of bullies in adolescence\nNext, we wanted to answer the question whether the Stable Bullies differed from the other bullies and non-involved children in adolescence. To address this question, a MANOVA was conducted in which the three bullying groups and gender served as the independent variables, and the adolescent measures as dependent variables. A significant multivariate effect emerged for bully group (Wilk\u2019s \u039b\u00a0=\u00a0.49), approx. F (24, 1218) =\u00a014.14, p\u00a0<\u00a0.001. The results of the univariate and post hoc analyses (Student Newman-Keuls, p\u00a0<\u00a0.05) are presented in Table 3, and indicated no differences between the groups on being liked, number of friends, and insecurity. Childhood Only Bullies scored more positive on the other measures that did the other bullies, and in fact did not differ largely from the Non-involved children, except for their lower scores on shyness, indicating that they turned out to be normally functioning adolescents who did not display problematic social behavior. Stable Bullies turned out to be distinctive from the Adolescence Only Bullies in that they displayed higher levels of aggression and disruption. As the Stable Bullies consisted only of boys while the other groups also contained girls, the analyses were repeated comparing the Stable Bullies with only their male counterparts from the other groups. These analyses resulted in similar findings.\nSocial adjustment of victims in childhood\nThe next research goal was to examine whether the Stable Victims displayed lower social adjustment in childhood than the other victim groups and non-involved children and whether Adolescence Only Victims could already be identified in childhood. To address this goal, a MANOVA with the victim groups (Stable Victims, Childhood Only, and Adolescence Only Victims and non-involved children) and gender as independent, and childhood social adjustment as dependent variable were conducted. Results indicated a significant multivariate effect for group (Wilk\u2019s \u039b\u00a0=\u00a0.68), approx. F (27, 1002) =\u00a05.33, p\u00a0<\u00a0.001. Follow-up univariate analysis of variance were significant for all variables except for disturbance and shyness (Table 4). Compared to the Childhood Only Victims, Stable Victims scored significantly higher on being disliked and help seeking behaviors. In contrast to the other victims, Adolescent Only Victims did not differ from the Non-involved children on any of the behavioral characteristics. There was no significant multivariate gender by group interaction.\nSocial adjustment of victims in adolescence\nThe last question that we addressed was whether Stable Bullies showed more signs of maladjustment in adolescence than the other victim groups and whether those who were victimized only in childhood would show social maladjustment problems in adolescence. A MANOVA was run with the victim and noninvolved groups, and gender as independent variables, and the adolescent measures as dependent variables. Findings revealed a significant multivariate effect for victim group (Wilk\u2019s \u039b\u00a0=\u00a0.74) approx. F(24, 992) =\u00a04.58, p\u00a0<\u00a0.001. The results of the univariate tests and post hoc comparisons are given in Table 4. As can be seen, Childhood Only Victims scored more positively than the Stable and Adolescence Only Victims on being liked and being disliked, received friendship nominations, shyness, and insecurity. In fact, except on being liked, they did not differ from the Non-involved children, which indicated that being victimized only in childhood was not reflected in specific social behavior three years later. As Table 4 further shows, Stable Victims did not significantly differ from the Adolescence Only Victims on any of the peer nominated indicators of social adjustment. A multivariate victim group by gender interaction was found, (Wilk\u2019s \u039b\u00a0=\u00a0.88), approx. F (24, 992) =\u00a01.86, p\u00a0<\u00a0.01. Univariate analyses showed a significant group by gender interaction for shyness (F (3, 349) =\u00a05.07, p\u00a0<\u00a0.01 which revealed that in the Stable Victim group boys scored substantially higher on peer reported shyness than girls, whereas in the other three groups, boys scored lower.\nDiscussion\nThe findings of our study give insight into the peer perceived behavioral profiles of children who were bullies or victims for a restricted period of time and those who were involved over a period of three years. They show that stable bullies and victims displayed a behavioral pattern in childhood and adolescence that clearly distinguished them from the children whose bullying or victimization was restricted to childhood. The latter did not show social adjustment problems later in adolescence, while the former did. Children who were victimized only in adolescence showed similar signs of social maladjustment in adolescence as children who were consistently victimized.\nOur study shows that half of the childhood bullies turned into stable bullies, and half of the childhood victims into stable victims. In contrast to stability in victimization, continuity in bullying was very gender specific: Whereas only one out of 12 girls continued to bully in adolescence, this held true for almost half of the boys. An explanation for this gender specificity in stability may be that bullying behavior in our study referred more to overt aggression, which is more typical for boys, than to relational aggression (cf. Crick & Bigbee, 1998), which is more typical for girls. It is also possible that for girls, in contrast to boys, bullying may be more related to specific social situations rather than to an individual characteristic reflecting an underlying antisocial and aggressive personality pattern (Salmivally et\u00a0al., 1998).\nWith respect to the behavioral profile of bullies, this investigation revealed that, compared to Childhood Only Bullies, the Stable Bullies were less liked and more disliked, were less often nominated as a friend, were more aggressive and disruptive, and scored lower on help seeking in childhood. This finding supports our hypothesis which states that, because of their socially deviant behavioral profile, children who will continue to bully over time could already be distinguished in childhood from those who will desist after some time (Loeber & Hay, 1997). This negative behavioral pattern may result in accumulation of negative social consequences such as continued peer rejection and fewer opportunities to acquire adequate coping skills. Through this interactive continuity (Caspi et\u00a0al., 1987) the deviant behavioral pattern is likely to be maintained into adolescence. This was reflected in finding that the Stable Bullies showed signs of peer perceived social maladjustment that distinguished them from Adolescence Only Bullies, which was in line with our hypothesis. Olweus\u2019 (1991) notion that bullying reflects a stable aggressive and antisocial, rule-breaking personality pattern predisposing children to social maladjustment and delinquency in adolescence only matches the behavioral profile of the Stable Bullies. Given that Childhood Only Bullies did not differ much from the Non-involved children in adolescence in their peer perceived social adjustment, an important conclusion of our study is that half of the children who bully in childhood, may not constitute a group at risk for later social problems.\nThe possible role of friends in relation to bullying deserves closer attention. In childhood, Stable Bullies were less often nominated as a friend than the Childhood Only Bullies and Non-involved children. Although we do not know whether these nominations were reciprocated, they might suggest that Stable Bullies have fewer reciprocal friends or at least fewer peers who consider them to be friends. The explanation may be that because of their behavioral profile, these bullies are less attractive as a friend, which is in line with studies showing a negative association between socially deviant behavior and positive peer relations (Rose & Asher, 1999). However, caution must be taken in interpreting these findings, because the differences between the childhood only and stable bullies in the number of received friendship nominations were no longer present after the male only classes were removed. This finding might indicate that in male only classes stable bullies receive fewer friendship nominations than childhood only bullies, while this may not be true for classes where boys and girls are equally present. Because it is not yet clear why this is the case, further study is warranted.\nIn adolescence, although Stable Bullies (and Adolescence Only Bullies) were still more disliked than all other adolescents, they had similar number of peers who thought of them as friends, given that no differences existed between them and other adolescents in number of received friendship nominations. Recently, Cillessen and Mayeux (2004) showed that in adolescence antisocial behaviors including bullying are increasingly linked to social status, suggesting that bullying may become more accepted. Consequently, associating with and becoming friends with bullies may also become more accepted. Since friends may passively or actively encourage bullying (cf Salmivalli et\u00a0al., 1996) bullies may feel reinforced and continue with their behavior (cumulative continuity, Caspi et\u00a0al., 1987). The fact that Stable Bullies are as often nominated as a friend as other adolescents may reflect bistrategic orientations as described by Hawley (2003; Hawley, Little, & Pasupathi, 2002). That is, they display antisocial behavior but at the same time seem to be able to somehow convey to certain peers that they are their \u201cfriends\u201d even if the bully him or herself does not necessarily consider that person a friend. This suggests that these bullies are more likely to be skilled manipulators rather than to be socially inadequate (Sutton et\u00a0al., 1999). The environmental influences, combined with the Stable Bullies\u2019 behavioral pattern, may make them relatively resistant to behavior change.\nRegarding the victims, Stable Victims showed a pattern of peer perceived social maladjustment in childhood that clearly distinguished them from the Childhood Only Victims. They were more disliked by their peers and were more likely to be perceived as seeking help from others. As the study by Boulton and Smith (1994) suggests, a pattern of consistently seeking help may signal that these children lack social self confidence. This, rather than the lack of prosocial behaviors such as cooperation or offering help may predispose children to remain victimized from childhood to adolescence (Boulton & Smith, 1994; Egan & Perry, 1998). This feature may prevent them from successfully interacting with peers in childhood and adolescence (interactive continuity, Caspi et\u00a0al., 1987), depriving them of positive peer experiences. It may also affect the way they create their own environment (Caspi et\u00a0al., 1987; Scarr, 1985, 1992; Scarr & McCartney, 1983) in that they may be more inclined to withdraw from social interactions and make them prone to be targeted by bullies in the group throughout their school life.\nWe found that Stable Victims did not have a more problematic social behavioral profile in adolescence than Adolescence Only Victims did. This is in contrast to our expectation that the longer the victims experienced victimization, the more impaired their social behaviors would be (Kochenderfer-Ladd & Wardrop, 2001), and thus that Stable Victims would show the most problematic social adjustment. Because adolescents who are victimized only in adolescence show the same social adjustment problems as the adolescents who have been victimized for a long period of time, this finding might indicate that the duration of victimization is relatively unrelated to the severity or magnitude of peer reported social adjustment problems in adolescence. Our findings showed that in general, victimized or noninvolved boys were less shy than girls, however, those who were stably victimized scored higher on shyness in adolescence. This is consistent with other findings of negative social implications of shyness for boys (Kerr, 2000) and suggests that being shy in adolescence may place boys at particular risk for being victimized for a prolonged period.\nOne of the positive findings of the present study seems to be that Childhood Only Victims may become normally adjusted children in adolescence, at least in terms of their peer reported social adjustment. This suggests that victimization experiences that are restricted to childhood do not necessarily translate into impaired social functioning observed by the peer context. This finding does not support the widely held assumption that being a victim of bullying in childhood is related to social adjustment problems in adolescence (e.g., Kumpulainen & R\u00e4s\u00e4nen, 2000; see also Parker & Asher, 1987). It extends more recent studies because it shows that victimization is not only concurrently associated with psychological adjustment (Juvonen et\u00a0al., 2000; Smith et\u00a0al., 2004) but also with social adjustment. Like the findings in these other two studies, our findings may lend support for the cessation hypothesis (Kochenderfer-Ladd & Wardrop, 2001) and might indicate that social problems can disappear once the victimization is over. Nevertheless, it should be remembered that we assessed social adjustment as perceived by the peer group, and that findings might have been differed when more subjective experiences of feelings would have been used. Finally, the Adolescence Only Victims did not differ from the Non-involved children in their peer reported childhood social adjustment. This implies that children who will become victims later in their school career do not necessarily show specific social behavioral patterns that are salient to their peers and on the basis of which they could already be identified in childhood. An alternative explanation might be that becoming a victim in adolescence may actually have little to do with social behavior per se. Rather than because of their specific social behavior, adolescents may become victimized because of how they dress, how they look etc, which becomes more salient at this age. As the present study showed, these victims do have social adjustment problems in adolescence, but these problems may have resulted from being victimized rather than caused it.\nThe present study has a number of positive features. It is among the first to longitudinally examine stability in bullying and victimization in relation to peer perceived social adjustment, during the transition from primary to secondary education. Bullying and victimization have been studied in a cultural context (i.e., the Netherlands) that has not been reported on extensively. This issue may bear some importance given that dimensions of peer relations can have differing connotations by culture (e.g., Schneider, 2000). For example, a recent study revealed that large variations existed between western counties such as England, Spain, Italy, and Ireland not only in the prevalence of bullies and victims but also in how bullying and victimization were related to social relations (Eslea et\u00a0al., 2003). This indicates that findings from one western country may not be generalized to another. Our findings revealed that in general the behavioral profiles of bullies and victims in the Netherlands were similar to those reported in studies in other western cultures such as the USA (Hodges & Perry, 1999; Pellegrini & Long, 2002) and Great Britain (Boulton & Smith, 1994).\nNevertheless, several caveats should be kept in mind. First, we only examined bullies and victims, but not bully-victims. This latter group turned out to be very small in our sample (ie., 2%, n\u00a0=\u00a09), comparable to other studies (Boulton & Smith, 1994; Olafsen & Viemer\u00f6, 2000; Solberg & Olweus, 2003). Additional exploratory analyses that we conducted on the bully-victims showed that the only feature that distinguished them from the other victims was their aggressive and disturbing behavior in class, on which they scored higher than all other victims, both in childhood and adolescence. In fact they were as aggressive as the Stable Bullies in childhood and adolescence. Nevertheless, due to the small sample size these results are only exploratory, and more research seems warranted to further describe the social correlates of bully-victims. Second, because the Stable Bullies contained only one girl, gender interactions could not be examined in analyses comparing this group of bullies with all other groups of bullies and the findings regarding Stable Bullies may thus not generalize to female bullies. Third, although this study was longitudinal in nature, causality in terms of victims\u2019 social behavior triggering bullies bullying behavior or vice versa was not implied. Fourth, this study used peer reports to classify children and adolescents, and to assess the dependent variables. Using one source of information increases the risk of shared-method variance and inflated associations between independent and dependent variables. In addition, several behaviors assessed in our study were based on single items, which may raise some concerns about the validity of these behaviors. Finally, we have focused on the social adjustment of the bullies and victims as it is perceived and reported by their peer environment. Even though the use of peer reported social adjustment measures was informative, we could not identify which of the children who were not victimized in childhood became victims in adolescence. We did not examine children\u2019s subjective experiences such as self-esteem, social insecurity, and loneliness. Exploring these individual subjective experiences may prove more valuable in predicting who is at risk to become victimized in adolescence and may add to our understanding of the consequences of being a bully or victim.\nDespite these caveats the present investigation shows that it is highly relevant for future scientific research on bullying, victimization, and adjustment to distinguish between children who are only involved in childhood or adolescence, and those who are chronically involved from childhood into adolescence. The positive message of our study is that many of the childhood victims and bullies did not seem to show social adjustment problems in adolescence, as perceived by their peers. The more troublesome message is that between 40 and 50% of the childhood bullies and victims will continue to be involved in bullying in adolescence. These children are the ones who are likely to display peer perceived social behavioral problems in adolescence. However, while this study is among the first to examine the behavioral profiles of stable and transient bullies and victims, this suggestion may be premature, and more research on the stability of bullying and victimization is needed.","keyphrases":["stability","bullying","victimization","social adjustment"],"prmu":["P","P","P","P"]}
{"id":"Cell_Tissue_Res-4-1-2386754","title":"Effect of tissue-harvesting site on yield of stem cells derived from adipose tissue: implications for cell-based therapies\n","text":"The stromal vascular fraction (SVF) of adipose tissue contains an abundant population of multipotent adipose-tissue-derived stem cells (ASCs) that possess the capacity to differentiate into cells of the mesodermal lineage in vitro. For cell-based therapies, an advantageous approach would be to harvest these SVF cells and give them back to the patient within a single surgical procedure, thereby avoiding lengthy and costly in vitro culturing steps. However, this requires SVF-isolates to contain sufficient ASCs capable of differentiating into the desired cell lineage. We have investigated whether the yield and function of ASCs are affected by the anatomical sites most frequently used for harvesting adipose tissue: the abdomen and hip\/thigh region. The frequency of ASCs in the SVF of adipose tissue from the abdomen and hip\/thigh region was determined in limiting dilution and colony-forming unit (CFU) assays. The capacity of these ASCs to differentiate into the chondrogenic and osteogenic pathways was investigated by quantitative real-time polymerase chain reaction and (immuno)histochemistry. A significant difference (P = 0.0009) was seen in ASC frequency but not in the absolute number of nucleated cells between adipose tissue harvested from the abdomen (5.1 \u00b1 1.1%, mean \u00b1 SEM) and hip\/thigh region (1.2 \u00b1 0.7%). However, within the CFUs derived from both tissues, the frequency of CFUs having osteogenic differentiation potential was the same. When cultured, homogeneous cell populations were obtained with similar growth kinetics and phenotype. No differences were detected in differentiation capacity between ASCs from both tissue-harvesting sites. We conclude that the yield of ASCs, but not the total amount of nucleated cells per volume or the ASC proliferation and differentiation capacities, are dependent on the tissue-harvesting site. The abdomen seems to be preferable to the hip\/thigh region for harvesting adipose tissue, in particular when considering SVF cells for stem-cell-based therapies in one-step surgical procedures for skeletal tissue engineering.\nIntroduction\nTissue engineering is an emerging field in modern medicine. Therapies involve the combination of cells and scaffold materials that can be loaded with bioactive factors, ideally resulting in the regeneration or replacement of lost or damaged tissues and organs. Multiple cell sources have been investigated for their possible applicability in tissue engineering. Embryonal stem cells are the most potent stem cells; however, their use is controversial and has mayor ethical considerations (Dresser 2001). Mesenchymal stem cells (MSCs) can be obtained from the adult and are widely used because of their differentiation potential. In addition to bone marrow, periosteum (Nakahara et al. 1991), muscle (Asakura et al. 2001), and adipose tissue (Zuk et al. 2002) also appear to be sources of MSCs.\nSubcutaneous adipose tissue is a particularly attractive reservoir of progenitor cells, because it is easily accessible, abundant, and self-replenishing. It is derived from the mesodermal germ layer and contains a supportive stromal vascular fraction (SVF) that can be readily isolated (Gronthos et al. 2003; Zuk et al. 2001). This SVF from adipose tissue consists of a heterogeneous mixture of cells, including endothelial cells, smooth muscle cells, pericytes, leukocytes, mast cells, and pre-adipocytes (Oedayrajsingh-Varma et al. 2006; Peterson et al. 2005; Prunet-Marcassus et al. 2005). In addition to these cells, the SVF contains an abundant population of multipotent adipose-tissue-derived stem cells (ASCs) that possess the capacity to differentiate into cells of mesodermal origin in vitro, e.g., adipocytes, chondrocytes, osteoblasts, and (cardio)myocytes (Erickson et al. 2002; Guilak et al. 2004; Halvorsen et al. 2001; Hattori et al. 2004; Planat-Benard et al. 2004; Rangappa et al. 2003; Zuk et al. 2001). Because of these favorable characteristics, interest has been growing in the application of ASCs for cell-based therapies such as tissue engineering.\nFor clinical practice, an advantageous approach would be to harvest ASCs and immediately give them back to the patient within the same operation, the so called \u201cone-step surgical procedure\u201d (Helder et al. 2007). This overcomes long-lasting culture expansion of ASCs on the one hand, but necessitates the use of the SVF of adipose tissue on the other hand, since fast selection procedures for stem cells in the SVF are not yet available. Therefore, the success of this procedure requires SVF-isolates to contain sufficient ASCs capable of differentiating into the desired cell lineage. In view of this, we have previously investigated the effect of three different surgical procedures for the harvesting of adipose tissue, i.e., resection, tumescent or conventional liposuction, and ultrasound-assisted liposuction, on the yield and function of the stem cells. We have demonstrated that the SVF isolates from adipose tissue harvested by ultrasound-assisted liposuction contain fewer stem cells, and that the stem cells have a longer population doubling time, leading us to conclude that resection and tumescent liposuction are preferable to ultrasound-assisted liposuction for harvesting adipose tissue, if the cells are to be used for tissue-engineering purposes (Oedayrajsingh-Varma et al. 2006).\nIn the present study, we have investigated whether the yield and functional characteristics of ASCs in the SVF are affected by the most frequently used adipose-tissue-harvesting sites. We have previously demonstrated that the yield of nucleated cells in the SVF of the adipose tissue from these different tissue-harvesting sites is similar (Oedayrajsingh-Varma et al. 2006). In the current study, the frequency of ASCs in the SVF cell isolates has been determined by using limiting dilution and colony-forming unit (CFU)  assays. In addition, we have investigated the frequency of CFUs showing an osteogenic differentiation capacity. SVF cells have been subsequently cultured in order to obtain homogeneous cell populations and to acquire sufficient cells to determine their chondrogenic differentiation potential in micromass cultures. Homogeneity has been checked by determining the growth kinetics and phenotypic characteristics of the ASCs. To verify the maintenance of multidifferentiation potential, osteogenic and chondrogenic induction has been assessed in these homogeneous ASC cultures.\nMaterials and methods\nDonors\nSamples of human subcutaneous adipose tissue were obtained as waste material after elective tumescent liposuction or resection and donated after informed consent from healthy donors operated on at the Departments of Plastic Surgery of two clinics in Amsterdam, The Netherlands. Adipose tissue was taken from the abdomen (n\u2009=\u200912) and the hip\/thigh region (n\u2009=\u200910) during cosmetic surgery; 22 female donors were included in this study. The average age (mean age 40, range 24\u201362\u00a0years) and body mass index (BMI; mean BMI 25.5; range 22.2\u201329.6\u00a0kg\/m2) were similar for both groups (Table\u00a01).\nTable\u00a01Population characteristics (BMI body mass index, THS tissue-harvesting site, ASC adipose-derived stem cell, Res resection, T-LS tumescent liposuction, ND not determined)DonorAge (years)BMI (kg\/m2)THSProcedureASC yield (%)102-00016222.6AbdomenRes9.20203-00072624.4AbdomenT-LS8.50303-00184223.9AbdomenRes12.50403-00215729.6AbdomenRes4.40504-000337NDAbdomenT-LS2.20604-00133628.1AbdomenRes2.20704-00153726.9AbdomenRes0.93805-000439NDAbdomenT-LS1.50905-00074228.3AbdomenT-LS8.301006-00035026.5AbdomenT-LS3.61106-00064224.2AbdomenT-LS4.81206-00074630.5AbdomenT-LS9.71303-00102424.4Hip\/thighT-LS0.301404-000440NDHip\/thighT-LS0.821504-00082726.6Hip\/thighT-LS0.161604-000936NDHip\/thighT-LS0.161705-00053422.2Hip\/thighT-LS0.601805-00062823.2Hip\/thighT-LS0.211905-00084324.6Hip\/thighT-LS7.202006-00053323.2Hip\/thighT-LS0.102106-00105226.2Hip\/thighT-LS2.702206-00124223.7Hip\/thighT-LS0.16\nCell isolation and storage\nIsolation of the SVF from adipose tissue was performed as previously described (Oedayrajsingh-Varma et al. 2006). The isolation protocol included a Ficoll density centrifugation step to remove contaminating erythrocytes. After isolation, 4\u00d7106 SVF cells were resuspended in a mixture (1:1) of Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) and cryoprotective medium (Freezing Medium, BioWhittaker, Cambrex, Verviers, Belgium), frozen under \u201ccontrolled rate\u201d conditions in a Kryosave (HCI Cryogenics, Hedel, The Netherlands), and stored in the vapor phase of liquid nitrogen according to standard practice at the Department of Pathology of the VU University Medical Center and following the guidelines of current Good Manufacturing Practice.\nLimiting dilution assay\nTo assess the frequency of ASCs in the SVF of adipose tissue, SVF cells were seeded in normal culture medium, consisting of DMEM supplemented with 10% fetal bovine serum (FBS), 100\u00a0U\/ml penicillin, 100\u00a0\u03bcg\/ml streptomycin, and 2\u00a0mM L-glutamine (Invitrogen, Gibco, Calif., USA) in 96-well plates at 15\u00d7103 cells\/well in the upper row. Two-fold dilution steps of the cells were made in subsequent rows. All cultures were performed in duplicate. Medium was changed twice a week. After 3\u00a0weeks, each well was individually scored for the number of cells. A well containing a cluster of at least 10 adhered fibroblast-like cells was considered as being positive. The frequency of ASCs was calculated from the rows of cells for which 25%\u201375% of the wells were scored as positive.\nCFU assays\nCFU assays were performed to check the consistency of the limiting dilution assay method and to determine the frequency of CFU capable of differentiating into the osteogenic lineage from the abdomen (n\u2009=\u20097) and hip\/thigh region (n\u2009=\u20096). SVF cells were resuspended in normal culture medium, consisting of DMEM supplemented with 10% FBS, 100\u00a0U\/ml penicillin, 100\u00a0\u03bcg\/ml streptomycin, and 2\u00a0mM L-glutamine (Invitrogen, Gibco). Two 6-well plates were prepared in which the SVF was diluted ten-fold across both columns, resulting in a upper column containing 104 and a lower column containing 103 nucleated SVF cells.\nFor the CFU-fibroblast (CFU-F) assay, the fixation time was 11\u201314\u00a0days, depending on the amount and growth kinetics of the colonies (merging of colonies was avoided). At the appropriate time point, the medium was removed, and the cells were washed with phosphate-buffered saline (PBS), fixed with 4% formaldehyde for 10\u00a0min, and subsequently colored in a 0.2% toluidine blue solution in borax buffer for about 1\u00a0min. Excess stain was washed off with distilled water, and colonies were counted.\nCells of the duplicate 6-well plate were submitted to a CFU-alkaline phosphatase (CFU-ALP) assay. Cultures were performed in normal medium for 7\u00a0days in order to obtain colonies and to remove contaminating cells, after which osteogenic medium was added for 2\u00a0weeks. Following this period, cells in the CFU-ALP plate were rinsed with PBS, fixed in 4% formaldehyde, and incubated for 10\u00a0min in a 0.2\u00a0M TRIS-hydrochloride (pH 10), 0.2\u00a0M calcium chloride, 0.1\u00a0M magnesium chloride solution, whereafter a solution containing 0.2\u00a0M TRIS-hydrochloride (pH 10), 0.2\u00a0M calcium chloride, 0.1\u00a0M magnesium chloride, and 600\u00a0\u03bcl nitroblue tetrazolium\/5-bromo-4-chloro-3-indolyl-phosphate was added for 30\u00a0min. The percentage of the colonies staining positive for ALP was determined.\nCulturing of SVF cells\nASCs from the abdomen and hip\/thigh region were cultured up to passage 2 for an adequate and quantitative comparison of stem-cell proliferation and differentiation capacity. Single-cell suspensions of cryopreserved SVF cells were seeded at 5.0\u00d7106 nucleated cells\/cm2 in normal culture medium. The cultures were maintained in a 5% CO2 incubator at 37\u00b0C in a humidified atmosphere. The medium was changed twice a week. When reaching 80%\u201390% confluency, cells were detached with 0.5\u00a0mM EDTA\/0.05% trypsin (Invitrogen) for 5\u00a0min at 37\u00b0C and replated. Cell viability was assessed by using the trypan blue exclusion assay. A homogeneous population of ASCs was thus obtained from abdomen and hip\/thigh region and was subsequently checked by determining growth kinetics and by analyzing the surface-marker expression profile of the ASCs.\nGrowth kinetics of ASCs\nTo determine the growth kinetics of cultured ASCs, ten T25 flasks per donor were seeded with 1\u00d7105 cultured ASCs (passage 2 or 3). At several time points (between days 2 and 12) after seeding, cells from two duplicate flasks were harvested and counted. ASC numbers were plotted against the number of days cultured, and the exponential growing phase of the cells was determined. The population doubling time was calculated by using the formula:\nwhere N1 was the number of cells at the beginning of the exponential growing phase, and N2 was the number of cells at the end of the exponential growing phase.\nFlow cytometry\nSingle-cell suspensions of cultured ASCs from abdomen and hip\/thigh region were phenotypically characterized by using fluorescence-activated cell sorting (FACS; FACSCalibur, Becton Dickinson, USA) as previously described (Varma et al. 2007). All monoclonal antibodies (mAbs) were of the immunoglobulin G1 (IgG1) isotype. Cells were stained with fluorescently labeled antibodies (conjugated to fluorescein isothiocyanate, phycoerythrin, or allophycocyanin) against CD31, CD34, CD45, CD54, CD90, CD106, HLA-DR, and HLA-ABC (BD Biosciences, San Jos\u00e9, Calif.), CD166 (RDI Research Diagnostics, Flanders, N.J.), CD105 (Caltag Laboratories, Burlingame, Calif.), CD117 (PharMingen, San Diego, Calif.), and CD146 (Chemicon, Temecula, Calif.). Nonspecific fluorescence was determined by incubating the cells with conjugated mAb anti-human IgG1 (DakoCytomation, Glostrup, Denmark).\nChondrogenic and osteogenic differentiation\nThe chondrogenic and osteogenic differentiation capacities of the cultured ASCs from the abdomen (n\u2009=\u20094) and hip\/thigh region (n\u2009=\u20094) were studied. Chondrogenic differentiation was induced in cultured ASCs as previously described, with some modifications (Oedayrajsingh-Varma et al. 2006). In short, a 50-\u03bcl drop of a concentrated ASC cell suspension (8\u00d7106cells\/ml, passage 2) was applied to a glass slide and allowed to attach at 37\u00b0C for 1\u00a0h. Then, 750\u00a0\u03bcl chondrogenic medium, consisting of DMEM, plus ITS+ Premix (final concentration in medium when diluted 1:100 was 6.25\u00a0\u03bcg\/ml insulin, 6.25\u00a0\u03bcg\/ml transferrin, 6.25\u00a0ng\/ml selenous acid, 1.25\u00a0mg\/ml bovine serum albumin (BSA), 5.35\u00a0\u03bcg\/ml linoleic acid; BD, USA), 10\u00a0ng\/nl transforming growth factor- \u03b21 (TGF-\u03b21; Biovision, ITK-diagnostics), 1% FCS, 25\u00a0\u03bcM ascorbate-2-phosphate (Sigma, St. Louis, Mo.), 100\u00a0U\/ml penicillin, 100\u00a0\u03bcg\/ml streptomycin, and 2\u00a0mM L-glutamine, was overlaid gently. Cells were maintained in a 5% CO2\/1% oxygen custom-designed hypoxia workstation (T.C.P.S. Rotselaar, Belgium) at 37\u00b0C in a humidified atmosphere, as this was shown to enhance chondrogenic differentiation (data not shown). Chondrogenic media were changed every 2\u20133\u00a0days.\nFor osteogenic differentiation, ASCs (passage 2) were seeded at 5000 cells\/cm2 and cultured in monolayer in osteogenic medium, consisting of normal culture medium supplemented with 10\u00a0mM \u03b2-glycerol phosphate, 50\u00a0\u03bcg\/ml ascorbate-2-phosphate, and 100\u00a0ng\/ml bone morphogenetic protein 2 (BMP-2, Peprotech EC, London, UK). Osteogenic medium was changed twice a week.\n(Immuno)histochemistry\n(Immuno)histochemistry was performed as described previously (Oedayrajsingh-Varma et al. 2006). Cell nodules that formed under chondrogenic culture conditions were stained with Alcian blue (Sigma-Aldrich, Zwijndrecht, The Netherlands) at acidic pH for detection of proteoglycans. For the detection of collagen Type II, staining was performed with mouse monoclonal antibody II-II6B3 (1:50; Developmental Studies Hybridoma Bank, Iowa, USA) against human collagen Type II in PBS containing 1% BSA.\nOsteogenesis was visualized after 21\u00a0days of culture in osteogenic medium by Von Kossa staining to establish the formation of a calcified matrix, typical for mature osteoblasts. The protocol used was as described previously (Varma et al. 2007) with only one modification of counterstaining the cytoplasm of the cells using fast green. Calcified extracellular matrix was visualized as black spots.\nSpectrophotometric ALP activity\nEarly differentiation of MSCs into immature osteoblasts is characterized by ALP enzyme activity, with human MSCs expressing ALP as early as 4\u00a0days after induction, and maximum levels being observed at around 14\u00a0days after induction (Jaiswal et al. 1997). Therefore, cellular ALP activity was measured after culturing the ASCs in osteogenic medium for 14\u00a0days. Cells were lysed with distilled water, and the ALP activity and protein content were determined. To determine ALP activity, p-nitrophenyl phosphate (Merck, Darmstadt, Germany) at pH 10.3 was used as the substrate, as described by Lowry (1955). ALP activity was expressed as micromole per microgram of protein in the cell layer. The amount of protein was determined by using a BCA Protein Assay reagent Kit (Pierce, Rockford, Ill., USA), and the absorbance was read at 540\u00a0nm with a microplate reader (Biorad Laboratories, Hercules, Calif., USA).\nReal-time polymerase chain reaction\nRNA isolation and reverse transcription were performed as previously described (Oedayrajsingh-Varma et al. 2006). Real-time polymerase chain reactions (PCR) were performed by using the SYBRGreen reaction kit according to the manufacturer\u2019s instructions (Roche Diagnostics) in a LightCycler 480 (Roche Diagnostics). cDNA (approximately 5\u00a0ng) was used in a volume of 20\u00a0\u03bcl PCR mix (LightCycler DNA Master Fast Startplus Kit, Roche Diagnostics) containing a final concentration of 0.5\u00a0pmol primers. Relative housekeeping gene expression for 18\u00a0S-rRNA (18\u00a0S) and relative target gene expression for aggrecan (AGG), collagen Type II (COL2B), and collagen Type X (COL10\u03b11) regarding chondrogenic differentiation, and for collagen Type I (COL1\u03b1), osteopontin (OPN), and runt-related transcription factor 2 (RUNX-2) regarding osteogenic differentiation were determined.\nPrimers (Invitrogen) used for real-time PCR are listed in Table\u00a02. They were designed by using Clone Manager Suite software program version 6 (Scientific & Educational Software, Cary, N.C., USA). The amplified PCR fragment extended over at least one exon border, based on homology in conserved domains between human, mouse, rat, dog, and cow, except for the 18\u00a0S gene (encoded by one exon only). Amplified Col2B PCR products were electrophoresed on a 2% agarose gel and stained with ethidium bromide. For real-time PCR, the values of relative target gene expression were normalized to relative 18\u00a0S housekeeping gene expression.\nTable\u00a02\nPCR primer sets used for reverse transcription\/PCR (18\u00a0S 18\u00a0S subunit, AGG aggrecan, COL1\u03b1I collagen Type I, COL2B collagen Type II, COL10a1 collagen Type X, OPN osteopontin, RUNX-2 runt-related transcription factor 2)\nGene\nPrimer sets\nAccession number, product length (bp)\n18\u00a0S\nForward: 5\u2032 GTAACCCGTTGAACCCCATT\u2212 3\u2032\nHuman, NM_10098, 151\u00a0bp\nReverse: 5\u2032 CCATCCAATCGGTAGTAGCG 3\u2032\nAGG\nForward: 5\u2032CAACTACCCGGCCATCC 3\u2032\nHuman, NM_001135, 160\u00a0bp\nReverse: 5\u2032GATGGCTCTGTAATGGAACAC 3\u2032\nCOL1\u03b1I\nForward: 5\u2032 AAGCCGAATTCCTGGTCT 3\u2032\nHuman, NM_000088, 195\u00a0bp\nReverse: 5\u2032 TCCAACGAGATCGAGATCC 3\u2032\nCOL2B\nForward: 5\u2032 AGGGCCAGGATGTCCGGCA 3\u2032\nHuman, NM_033150, 195\u00a0bp\nReverse: 5\u2032 GGGTCCCAGGTTCTCCATCT 3\u2032\nCOL10a1\nForward: 5\u2032 CACTACCCAACACCAAGACA 3\u2032\nHuman, NM_000493, 225\u00a0bp\nReverse: 5\u2032 CTGGTTTCCCTACAGCTGAT\u2032\nOPN\nForward: 5\u2032 TTCCAAGTAAGTCCAACGAAAG 3\u2032\nHuman, AF_052124, 181\u00a0bp\nReverse: 5\u2032 GTGACCAGTTCATGAGATTCAT 3\u2032\nRUNX-2\nForward: 5\u2032 ATGCTTCATTCGCCTCAC 3\u2032\nHuman, NM_001024630, 156\u00a0bp\nReverse: 5\u2032 ACTGCTTGCAGCCTTAAAT 3\u2032\nReal-time PCR data analysis\nWith the Light Cycler software (version 4), the crossing points were assessed and plotted versus the serial dilution of known concentrations of the standards derived from each gene by the Fit Points method. PCR efficiency was calculated by Light Cycler software, and the data were used only if the calculated PCR efficiency was between 1.85\u20132.0.\nStatistics\nKolmogorov-Smirnov tests were used to determine the normalcy of measurements and, if appropriate, their logarithmics. For the evaluation of yield and growth kinetics, means between two groups in one variable were compared by using the independent sample two-tailed t-test. Partial correlation was expressed as the Pearson correlation coefficient, r. For evaluation of gene expression, a repeated measures analysis of variance was used to determine significant differences when increasing time-points in one donor within one variable were compared. If levels of gene expression were below the detection limit (0.05), values were set at 10\u22122 (or log level at \u22122). All statistical tests used a significance level of \u03b1\u2009=\u20090.05.\nResults\nEffects of tissue-harvesting site on frequency of ASCs\nIn a previous study, we demonstrated that the yield of nucleated cells in the SVF of adipose tissue from different tissue-harvesting sites was similar (Oedayrajsingh-Varma et al. 2006). To investigate whether the tissue-harvesting site affected the frequency of ASCs in the SVF in the present study, limiting dilution and CFU-F assays were performed. The outcomes of both types of assays were similar (Fig.\u00a01a, b). When combined, the SVF of adipose tissue harvested from the abdomen contained 5.1\u2009\u00b1\u20091.1% ASCs (mean\u2009\u00b1\u2009SEM), whereas the percentage of ASCs in the SVF of adipose tissue harvested from the hip\/thigh region was much lower (1.2\u2009\u00b1\u20090.7%; Fig.\u00a01c). This difference in ASC frequency between adipose tissue from the abdomen and hip\/thigh region was significant (P\u2009=\u20090.0009).\nFig.\u00a01Effect of adipose-tissue-harvesting site on the frequency of adipose-derived stem cells (ASCs) in the stromal vascular fraction (SVF). After isolation of the SVF from adipose tissue of both tissue-harvesting sites, the frequency of ASCs in the SVF isolates was determined by using: (a) a limiting dilution assay (LD)  and (b) a colony-forming unit (CFU) assay (CFU-F), with similar results. When combined (c), a significant difference was detected in ASC frequency between adipose tissue harvested from the abdomen and adipose tissue harvested from the hip\/thigh region (P-values: a limiting dilution: P\u2009=\u20090.003, b colony-forming unit: P\u2009=\u20090.05, c combined: P\u2009=\u20090.0009)\nEffect of tissue-harvesting site on frequency of CFUs having osteogenic differentiation potential\nParallel to the CFU-F assay (Fig.\u00a01b), CFU-ALP assays were also performed to determine the percentage of the CFUs capable of osteogenic differentiation. For adipose tissue from the abdomen, 54.9\u2009\u00b1\u200912.1% (mean\u2009\u00b1\u2009SEM) of the CFUs stained positive for ALP, whereas the CFUs from adipose tissue of the hip\/thigh region displayed an ALP positivity of 72.8\u2009\u00b1\u20098.1% (Fig.\u00a02). Apparently, no differences in osteogenic potential existed between ASCs from the two tissue-harvesting sites (P\u2009=\u20090.43).\nFig.\u00a02Effect of adipose-tissue-harvesting site on the osteogenic diffentiation capacity of CFU from the abdomen or hip\/thigh regions. No significant difference is apparent in the CFU-alkaline phosphatase (CFU-ALP) frequency from the abdomen and hip\/thigh region when corrected for CFU-fibroblast (CFU-F).\nPhenotypic characterization and growth kinetics of cultured ASCs\nCultured ASCs (passages 2 to 4) from abdomen and hip\/thigh regions were phenotypically characterized. ASCs from both tissue-harvesting sites were demonstrated to be homogeneous populations staining positive for stem-cell-associated markers CD34, CD54, CD90, CD105, CD166, and HLA-ABC, and negative for hematopoietic\/leukocytic\/endothelial markers such as CD31, CD45, CD106, CD146, and HLA-DR (Table\u00a03).\nTable\u00a03Surface-marker expression of human cultured ASC at passages 3\u20134. Results are expressed as mean fluorescence (MF), with isotype control <4.25 (\u2212  MF<8.5; +  8.5<MF<100; ++  100<MF<1000; +++ MF>1000).Cell-surface markerCultured ASC (n\u2009=\u20094)CD29++CD31\u2013CD34+CD45\u2013CD54++CD90+++CD105++CD106\u2013CD146\u2013CD166+HLA-ABC++HLA-DR\u2013\nTo determine growth kinetics, the population doubling time of ASCs from passages 2 to 3 was determined. When ASCs numbers were monitored over time, a cell growing curve was obtained showing an exponential growing phase, after which the cells reached confluency (Fig.\u00a03a,b). The mean population doubling time of the ASCs in the exponential growing phase was about 2\u00a0days when the adipose tissue was harvested from the abdomen and hip\/thigh regions (abdomen: 2.1\u2009\u00b1\u20090.8; hip\/thigh: 2.3\u2009\u00b1\u20090.3; mean\u2009\u00b1\u2009SEM; Fig.\u00a03c).\nFig.\u00a03Effect of the adipose-tissue-harvesting site on growth kinetics of ASCs in vitro. a Growth kinetics of ASCs of a representative donor when adipose tissue was harvested from the abdomen. b Growth kinetics of ASCs when adipose tissue was harvested from the hip\/thigh region. c Population doubling time was calculated from the exponential growing phase of the cells. There was no significant difference in population doubling time of ASCs from the abdomen and hip\/thigh region (P\u2009=\u20090.78, independent Student t-test).\nEffect of tissue-harvesting site on osteogenic differentiation potential of ASCs\nDifferentiation of cultured ASCs into the osteogenic lineage was induced by culturing the cells in monolayer in osteogenic medium containing BMP-2. Specific RNA expression of osteogenically induced ASCs from all donors tested increased over time, RUNX-2 being up-regulated 18-fold (P\u2009=\u20090.002) and COL1\u03b1 being up-regulated seven-fold (P\u2009=\u20090.024) after 7\u00a0days (Fig.\u00a04a,b). OPN gene expression was increased after 7\u00a0days; however, this increase was not significant (Fig.\u00a04c). No significant differences were detected in osteogenic gene expression between ASCs derived from the abdomen and ASCs derived from the hip\/thigh region, at all three time points tested.\nFig.\u00a04Effect of adipose-tissue-harvesting site on the osteogenic differentiation of cultured ASCs in vitro. a\u2013cRUNX-2 (runt-related transcription factor 2; P\u2009=\u20090.002), COL1\u03b11 (collagen type Ia; P\u2009=\u20090.024), and OPN (osteopontin; P\u2009=\u20090.38) gene expression was measured after 0, 4, and 7\u00a0days (d) of osteogenic induction, by using quantitative real-time polymerase chain reaction (qRT-PCR). No significant differences were detected in osteogenic gene expression between ASCs derived from the abdomen and ASCs derived from the hip\/thigh region, at all three time points tested. d ALP activity was significantly increased after 14\u00a0days in the osteogenically stimulated cells (stim) compared with that in unstimulated cells (con; P\u2009=\u20090.047). No statistically significant difference was apparent in ALP activity between ASCs derived from abdominal fat and ASCs from hip\/thigh fat. e\u2013g Von Kossa staining of ASCs from abdomen (e) and hip\/thigh region (f) after 21\u00a0days of culture in osteogenic medium and in control medium (g), showing mineralized matrix visible as black spots.\nALP activity in the ASCs was measured after 14\u00a0days of osteogenic stimulation. ASCs cultured in control medium served as negative controls. ALP activity in the osteogenically stimulated cells was significantly increased (P\u2009=\u20090.047) compared with that in control cells (Fig.\u00a04d). No statistically significant difference was apparent in ALP activity between ASCs derived from abdominal fat and ASCs from hip\/thigh fat. Calcification of the osteogenic matrix was confirmed by using Von Kossa staining; black spots could be observed after 3\u00a0weeks of osteogenic stimulation of ASCs from both origins (Fig.\u00a04e: abdomen, Fig.\u00a04f: hip\/thigh). No calcification was seen in ASC cultures expanded in control medium (Fig.\u00a04g).\nEffect of tissue-harvesting site on chondrogenic differentiation potential of ASCs\nThe chondrogenic differentiation potential of ASCs was analyzed after culturing the cells in a micromass in chondrogenic medium containing TGF-\u03b2. Within 24\u00a0h of culture, most of the cells formed nodules (n\u2009=\u20097).\nPCR-amplified COL2B mRNA expression was detectable but not quantifiable after 7\u00a0days in ASCs from both the abdomen and hip\/thigh region in most but not all donors (n\u2009=\u20095). As shown in Fig.\u00a05a, cells from both tissue-harvesting sites displayed COL2B mRNA. Under non-chondrogenic conditions, no COL2B could be detected. AGG and COL10\u03b11 mRNA expression in all donors tested increased over time, AGG being up-regulated 2.4-fold (P\u2009=\u20090.041) at day 7 when compared with day 4, and COL10\u03b11 being up-regulated four-fold (P\u2009=\u20090.024) after 7\u00a0days (Fig.\u00a05b,c). No significant differences were detected in chondrogenic gene expression between ASCs derived from the abdomen and ASCs derived from the hip\/thigh region, at all three time points tested.\nFig.\u00a05Effect of the tissue-harvesting site on the chondrogenic differentiation of cultured ASCs in vitro. a Both abdomen (lane\u00a01) and hip\/thigh region (lane\u00a02) display COL2B mRNA. Under nonchondrogenic conditions, no COL2B could be detected (lane\u00a03). b, c Aggrecan (AGG; P\u2009=\u20090.041) and collagen 10A (Col10a; P\u2009=\u20090.024) gene expression, respectively,  was up-regulated after 7\u00a0days (d), as measured by qRT-PCR. No significant differences were detected in chondrogenic gene expression between ASCs derived from the abdomen and ASCs derived from the hip\/thigh region, at all three time points tested. d, f Cartilaginous matrix expression was visualized in both tissue-harvesting sites by staining proteoglycans (Alcian blue) and COL2 (Col2-II6B3 antibody), respectively. e At higher magnification, the ASC nodules resembled cartilage-like tissue, composed of spherical cells surrounded by lacunae and lying in a proteoglycan-rich extracellular matrix.\nAlcian blue staining demonstrated proteoglycan deposition in the ASC nodules from both the hip\/thigh region and abdomen (Fig.\u00a05d). At higher magnification (Fig.\u00a05e), the ASC nodules resembled cartilage-like tissue, composed of round cells, surrounded by lacunae and lying in a proteoglycan-rich extracellular matrix that appeared positive for collagen Type II by immunostaining (Fig.\u00a05f).\nDiscussion\nIn this study, we have investigated whether the yield and functional characteristics of ASCs are affected by the adipose tissue-harvesting site, i.e., abdomen and hip\/thigh regions. We have found a difference in the frequency of ASCs between adipose tissue harvested from the abdomen and the hip\/thigh regions. SVF isolates derived from abdominal fat contain significantly higher frequencies of ASCs. When cultured, the growth kinetics and surface-marker expression of ASCs from both tissue-harvesting sites are similar. We have detected no differences in osteogenic or chondrogenic differentiation potential between these cultured ASCs from the two tissue-harvesting sites.\nAdipose tissue is a highly heterogeneous tissue, not only among individuals, but also when comparing different fat depots within one individual. Donor-dependent differences have been demonstrated to exist in (stem) cell yield, proliferation, and differentiation capacity, probably caused by differences in age (Hauner and Entenmann 1991; van Harmelen et al. 2003), BMI (Aust et al. 2004; Hauner et al. 1988; Jaiswal et al. 1997; van de Venter et al. 1994), and diseases such as osteoarthritis and diabetes (Barry 2003; Murphy et al. 2002; Ramsay et al. 1995). Donors used in this study for harvesting adipose tissue were healthy female donors up to 62\u00a0years old. Some patients who will benefit from tissue engineering of cartilage will be older or might suffer from disease, and males will also be affected. Therefore, future research should include these donor types to determine whether yields and functional characteristics are influenced by these variables.\nIn our donor population, no significant correlation has been detected between the frequency of ASCs and the age of the donor (P\u2009=\u20090.32, r\u2009=\u20090.27) or between the frequency of ASCs and BMI (P\u2009=\u20090.42, r\u2009=\u2009\u22120.22; data not shown). These data are in agreement with other studies that have demonstrated no correlation between BMI or age and numbers of ASCs per gram of adipose tissue (Hauner and Entenmann 1991; van Harmelen et al. 2003). Most importantly, this means that these variables cannot be responsible for the difference that we have found between the frequency of ASCs and the tissue-harvesting site.\nIn addition to donor-dependent heterogeneity, intra-individual differences between fat depots have been demonstrated, e.g., with regard to the metabolic response of adipose tissue to various hormonal and neurological stimuli (Guilak et al. 2004; Lacasa et al. 1997; Masuzaki et al. 1995; Monjo et al. 2003; Rodriguez-Cuenca et al. 2005) and the cellular composition of the adipose tissue (Peptan et al. 2006; Prunet-Marcassus et al. 2005). In this study, we have focused on the intra-individual differences in the yield and function of ASCs from two fat depots: the abdomen and the hip\/thigh region. We have demonstrated that adipose tissue derived from the abdomen contains significant higher frequencies of stem cells compared with adipose from the hip\/thigh region. Whereas SVF isolates from the abdomen in this study contain about 5.1% of ASCs, the frequency of ASCs in SVF from the hip\/thigh region is only 1.2%. Moreover, this 1.2% is the average of a population in which some donors hardly possess any adipose-derived stem cells (see spreading ASC frequency hip\/thigh in Fig.\u00a01). Despite this more than four-fold difference, the frequency of ASCs in adipose tissue from the hip\/thigh region is still much higher compared with the frequency of MSCs in the bone marrow compartment, which is as low as 0.001%\u20130.01% (Pittenger et al. 2000), thereby making the hip\/thigh region still a much more attractive stem-cell source for tissue engineering therapies.\nWhat are the implications of these stem-cell frequencies for clinical practice? We have shown that 0.5\u20132.0\u00d7108 SVF cells can be harvested from 100 g adipose tissue, an amount that can easily be obtained from a patient (Aust et al. 2004; De Ugarte et al. 2003; Oedayrajsingh-Varma et al. 2006; Zuk et al. 2001). With an ASC frequency of 5.1%, the SVF isolates contain between 2.6\u201310.2\u00d7106 stem cells, which is an amount that appears to be sufficient for cell-based therapies as compared with the amount of cells used by others (Erickson et al. 2002; Fan et al. 2006; Williams et al. 2003; Zheng et al. 2006). This implies that time-consuming culturing and expanding steps of the stem cells can be avoided. In comparison, a bone marrow transplant of 100\u00a0ml contains approximately 6.0\u00d7108 nucleated cells (Zuk et al. 2002), of which only 0.001%\u20130.01% (0.06\u20130.006\u00d7106 cells) are stem cells (Pittenger et al. 2000).\nTo determine the frequency of CFU capable of differentiating into the osteogenic lineage, a CFU-ALP assay has been performed on cells from the abdomen and hip\/thigh region. The frequencies of CFU-ALP (\u2009\u00b1\u20093%) are almost comparable with those of the CFU-F (Figs.\u00a01b, 2). This is higher than the value that Mitchell et al. (2006) have found in their clonogenic assay of freshly isolated stromal cells (\u00b10.5%). However, they have used a different assay method and shorter incubation period, and their frequency increases to the same level (5%) after progressive passaging (Mitchell et al. 2006).\nSVF cells have been cultured up to passage 3 to obtain a homogeneous population of ASCs as starting material for differentiation studies toward the chondrogenic lineage. When comparing the growth kinetics of these cultured ASCs from the abdomen and hip\/thigh region, the cell doubling time appears to be similar, being approximately 2\u00a0days. This is in accordance with the findings of others who have compared the replication rate of adipocyte precursor cells from various tissue-harvesting sites (Hauner et al. 1988; Pettersson et al. 1985; Roncari et al. 1981; Zuk et al. 2001). On the other hand, when comparing SVF cells from omental and subcutaneous fat, van Harmelen et al. (2004) have found a difference in cell proliferation rate; however, this may be explained by differences in methodological approach, since they use stromal cells instead of cultured ASCs. As we have shown that different fat depots contain different numbers of stem cells, these differences in proliferation rate may be caused by differences in initial stem-cell numbers when using SVF cells. This is reflected in the finding that, in our study, SVF cells derived from abdominal fat reach 80%\u201390% confluency within 5\u00a0days, whereas SVF cells derived from adipose tissue of the hip\/thigh region take more than 9\u00a0days to reach 80%\u201390% confluency when seeded in the same density (data not shown).\nIn addition to the determination of growth kinetics, we have phenotypically characterized the cultured ASCs. The surface-marker expression profile is in accordance with those found by others (Mitchell et al. 2006; Schaffler and Buchler 2007; Varma et al. 2007), making both tissue sites fully comparable with each other (Tables\u00a01, 3).\nThe homogeneous ASC population has been induced to the osteogenic and chondrogenic lineages. Having determined the osteogenic differentiation capacity of ASCs, we have shown significant up-regulation of osteogenic gene expression, ALP activity, and matrix mineralization. Interestingly, although no significant difference has been detected in ALP activity between ASCs from the abdomen and hip\/thigh regions, ASCs from the hip\/thigh region tend to show higher values of ALP activity after induction. This might be related to the underlying bone tissue, thereby implying that the ASCs of the hip\/thigh region are less multipotent and more committed to the osteogenic lineage.\nThe chondrogenic differentiation capacity of the ASCs has been demonstrated by the up-regulation of AGG and COL10\u03b11 gene expression and the production of matrix proteins. No difference has been detected in the chondrogenic differentiation potential between ASCs from the abdomen and hip\/thigh regions. However, in the PCR studies, we have not succeeded in quantifying COL2\u03b1 and Col2B mRNA expression for any of the donors tested. Others have obviously faced the same difficulty when trying to measure the up-regulation of COL2B genes in MSCs (Huang et al. 2004; Winter et al. 2003; Zuk et al. 2002). Although intending to measure collagen Type X mRNA expression as a hypertrophic and therefore late marker of chondrogenesis, we have noticed that this mRNA is expressed earlier than collagen Type II mRNA. This is surprising, as we would expect stem cells to have to differentiate into chondrocytes before they can become hypertrophic. However, this unexpected hierarchy of chondrogenic gene expression has also been found by Mwale et al. (2006). Moreover, both Mwale et al. (2006) and we have found AGG to be constitutively expressed in MSCs. Because of this constitutive expression of AGG and the early up-regulation of COL10\u03b11, Mwale et al. (2006) warn against using these molecules as markers for chondrogenesis and chondrocytic hypertrophy. We think that these genes can nevertheless be used as markers for differentiation into the chondrogenic lineage, albeit being exclusively shown as the quantitative up-regulation of gene expression, and always in combination with other chondrogenic markers (an awareness of the possible difference in the function of COL10\u03b11 in chondrogenesis in adult stem cells when compared with embryonic stem cells is also necessary).\nOur lack of detection of any significant differences in the osteogenic and chondrogenic differentiation potential when comparing ASCs from the two tissue-harvesting sites seems to be in contrast with studies of Hauner and Entenmann 1991) who have found differences in the adipogenic differentiation potential between SVF cells from abdominal and femoral adipose tissue. However, since Hauner and Entenmann 1991) have used fresh SVF cells instead of culture-passaged ASCs, the variation in differentiation potential might be attributable to differences in numbers of ASCs in the SVF isolates of the two regions, as we have shown in this study. Other factors responsible for the variation in differentiation potential can be ascribed to other specific histological characteristics of the adipose tissue at the anatomical site, such as vascularity and amount of fibrous tissue (Lennon et al. 2000; Peptan et al. 2006; Pittenger et al. 2000), and to differences in the regulation of gene expression (Djian et al. 1983; Peptan et al. 2006; Pittenger et al. 2000).\nWe therefore conclude that the yield of ASCs is dependent on the tissue-harvesting site. In planning the optimal one-stage procedure for the regeneration of cartilage tissue, factors that can positively influence the outcome of the operation must be taken into account. In view of this, the abdomen seems to be preferable to the hip\/thigh region for harvesting ASCs.","keyphrases":["tissue-harvesting site","adipose-tissue-derived stem cells","osteogenic differentiation","chondrogenic differentiation","human","colony-forming unit assay","stem-cell yield"],"prmu":["P","P","P","P","P","R","R"]}
{"id":"Int_Arch_Occup_Environ_Health-3-1-1915645","title":"Workdays, in-between workdays and the weekend: a diary study on effort and recovery\n","text":"Objectives Effort-recovery theory (Meijman and Mulder in Handbook of work and organizational psychology, Psychology Press\/Erlbaum, Hove, pp 5\u201333, 1998) proposes that effort expenditure may have adverse consequences for health in the absence of sufficient recovery opportunities. Thus, insight in the relationships between effort and recovery is imperative to understand work-related health. This study therefore focused on the relation between work-related effort and recovery (1) during workdays, (2) in-between workdays and (3) in the weekend. For these three time periods, we compared a group of employees reporting relatively low levels of work-related effort (\u201clow-effort group\u201d) and a group of employees reporting relatively high levels of work-related effort (\u201chigh-effort group\u201d) with respect to (1) activity patterns, (2) the experience of these activity patterns, and (3) health and well-being indicators.\nIntroduction\nMuch research has shown that high levels of job demands are related to increased levels of physical and psychological health problems across time (e.g., De Lange et\u00a0al. 2003). Despite this strong focus on the relations between job demands and health, relatively little attention has been paid to the psychological and physiological processes that may explain why health is adversely affected by high job demands. One notable exception is effort-recovery (ER) theory (Meijman and Mulder 1998; Geurts and Sonnentag 2006). ER theory argues that working inevitably requires effort as an appeal is made to workers\u2019 abilities and their willingness to dedicate these abilities to the work task. Expending effort at work (\u201cwork-related effort\u201d) produces two kinds of outcomes: the tangible result of work activities, i.e. a product or service, and the psychological and physiological \u201ccosts\u201d or load reactions (e.g., fatigue) associated with working. These load reactions are usually short-lived and reversible: they disappear after respite from work. However, under certain circumstances the recovery process may be insufficient or inadequate, and then short-term work-related load reactions may turn into adverse and more chronic health problems, such as prolonged fatigue, chronic tension, and sleep deprivation (\u00c5kerstedt 2006; H\u00e4rm\u00e4 2006; Sluiter et\u00a0al. 2001; Van Hooff et\u00a0al. 2005).\nRecovery opportunities after work may be inadequate in terms of quantity (time) and\/or quality. Recovery time may be insufficient in case of prolonged exposure to high demands, for instance, when workers continue to pursue job-related activities during non-work time (e.g., by working overtime) or engage in other demanding (e.g., domestic) activities. Recovery is particularly at stake when during private time an appeal is made upon the same psychophysiological systems that were activated on the job. The quality of recovery may be endangered when individuals\u2019 psychophysiological systems show prolonged activation even if not exposed to any special demands during the recovery period. This may happen when workers have difficulty to relax at home after a stressful working day. For example, Brosschot et al. (2005) showed that when workers worry in their private time about the past or upcoming working day, the psychophysiological systems that were activated on the job remain activated, thus impeding the recovery process (cf. Ursin and Erikson 2004). Due to repeated or prolonged activation of psychophysiological systems, these systems are in danger of chronic overactivity, producing lasting changes in homeostatic mechanisms (i.e., allostatic load, McEwen 1998). Consequently, these originally adaptive systems may start to malfunction by showing either hyperactivity (the systems fail to shut-off) or hypoactivity (the systems are not turned on when needed), constituting a serious health risk. For example, chronic stress may cause the immune system to be not sensitive enough (hypoactivity), allowing infectious agents (viruses and bacteria) to enter the body and cause infectious diseases. Alternatively, the system may become overreactive so that the immune system itself causes ill health (such as autoimmune diseases and allergic diseases, Clow 2001).\nThe present study\nEffort and recovery are nowadays salient research topics (Zijlstra and Sonnentag 2006). The present study builds on and extends this body of knowledge in at least four regards:\nFirstly, although the effort-recovery process is assumed to unfold on a daily basis, there is only a limited number of studies examining this process from such a day-to-day perspective (e.g., Cropley et al. 2006; Meijman et al. 1992; Rook and Zijlstra 2006; Sonnentag 2001; Sonnentag and Zijlstra 2006; Totterdell et al. 1995). The majority of research in this area still focuses on either cross-sectional or on global long-term relations between job demands, lack of recovery and health (e.g., Kompier 1988; Sluiter et\u00a0al. 2001). Thus, in order to obtain more insight in de day-to-day relations between effort and recovery, the present study examines the relation between work-related effort and recovery on a daily basis, both during and after working time.\nFurthermore, although weekends may offer important opportunities for recovery, they are hardly included in previous studies. Exceptions are Fritz and Sonnentag\u2019s (2005) diary study, which showed that well-being after the weekend was higher when individuals had engaged in social activities during the weekend. Also, Totterdell et\u00a0al. (1995) reported that sleep, mood and social satisfaction were worse on the first rest day following work shifts in comparison with subsequent rest days. In a study among shift-working nurses, Rook and Zijlstra (2006) found weekends to be important for recovery as well. To increase the understanding of the weekend as potential recovery period, the present study also included the weekend.\nThirdly, only limited attention has been given to actual activity patterns during work and non-work time in research on effort and recovery until now (see for exceptions: Fritz and Sonnentag 2005; Sonnentag 2001; Sonnentag and Bayer 2005). This is remarkable, as several work psychological theories (e.g., action theory, Frese and Zapf 1994; Taris and Kompier 2005) assume that job characteristics affect worker well-being through worker behavior: it is what people do that makes them feel tired or enthusiastic. Thus, in order to fully understand effort-recovery patterns during and in-between workdays, we must know how people spend their time on work as well as on home activities. Therefore, the present study provided a detailed assessment of employees\u2019 activity patterns during and in-between working time.\nFinally, what can be a burden for one individual may constitute a pleasure to the other. Consequently, insight in activity patterns in the work and private domain is insufficient to fully understand workers\u2019 effort-recovery patterns, and preferably workers\u2019 experience of the time spent on (non)work activities must be examined in this context (see also the recommendations by Sonnentag 2001). Until now, the extent to which workers experience their daily work and home activities as effortful and\/or pleasant has nonetheless remained largely ignored. Therefore, the present study provided a detailed assessment of how employees experience their activities during and in-between working time in terms of effort and pleasure.\nWe distinguished between workers who reported a relatively high level of work-related effort (i.e., who generally experienced their workdays as effortful) during a standard work week (further referred to as the \u201chigh-effort group\u201d) and workers who reported a relatively low level of work-related effort (\u201clow-effort group\u201d). This division of our sample was employed in order to maximize the contrast between the two subgroups in terms of reported effort. The two effort-groups were compared with respect to (1) activity patterns (i.e., the time spent on\/frequency of engaging in work activities, domestic activities, active leisure, and passive leisure), (2) experiences of activities (i.e., the specific effort and pleasure experienced while engaging in a specific work or home activity), and (3) health and well-being indicators (i.e., fatigue, sleep quality, sleep time, preoccupation with work, and work motivation). Fatigue is included an indicator of (lack of) recovery. As sleep provides the most \u201cnatural\u201d recovery opportunity for humans, sleep quality and sleep time are incorporated as well (\u00c5kerstedt 2006). Preoccupation with work is assessed, because it may prolong physiological activation and therefore interfere with the recovery process (Brosschot et al. 2005). Finally, to avoid focusing exclusively on the \u201cnegative\u201d consequences of working, work motivation is added in this study to acknowledge that work may be related to positive aspects of worker behavior as well. These constructs were measured in three time periods: (1) during work time, (2) in-between successive workdays, and (3) during the weekend. In order to minimize the amount of time elapsed between the occurrence and the reports of a certain activity or experience, we utilized a diary design covering five uninterrupted weekdays directly followed by two weekend days. In this vein, the risk of retrospection bias was reduced (Bolger et\u00a0al. 2003).\nThis study examines three interrelated research questions:How is work-related effort associated with (a) time spent on work activities, (b) experiences of work activities, and (c) health and well-being during the workday?As the distinction between the two groups is based on employees\u2019 reports of work-related effort, we expect that the high-effort group will also report to have expended higher effort on (at least some of) the specific work activities compared to the low-effort group (Hypothesis 1a). Support for this hypothesis is important from the perspective of validation of the effort-measure used to differentiate between the two effort-groups.\nAs the high-effort group should have invested higher levels of effort during the work day than the low-effort group, we expect to observe higher levels of fatigue at the end of the workday (Hypothesis 1b) as well as a (stronger) increase in fatigue during the workday (Hypothesis 1c) in the first group. We do not hold a priori expectations concerning the experiences of pleasure associated with work activities and with respect to the time spent on and the frequency of engaging in each work activity.How is work-related effort associated with (a) time spent on home activities, (b) experiences of home activities, and (c) health and well-being in-between successive workdays?We distinguish among four categories of home activities, i.e. (1) domestic activities (e.g., household chores), (2) overtime work, (3) active leisure activities (e.g., exercising), and (4) passive leisure activities (e.g., reading for pleasure, watching TV, listening to music; see also Sonnentag 2001). The latter category is considered as \u201cpassive\u201d, whereas the other three categories of activities demand effort to some extent, and are therefore labeled as \u201cactive\u201d. Based on ER theory, it can be argued that our capacity to expend effort is limited, and that the more effort is expended at work, the less remains for home activities. Accordingly, the high-effort group is expected to spend less time on and to engage less often in active home activities, and consequently, will spend more time on and engage more often in passive leisure activities (Hypothesis 2a). Because of the supposed limited amount of energy left in the high-effort group, we further expect that this group will experience engagement in active home activities as more effortful relative to the low-effort group (Hypothesis 2b). As we do not have a priori expectations regarding differences between the groups in the pleasure experienced in home activities, possible differences are examined in an exploratory fashion.\nFurther, we expect that those who have expended high effort on the job (high-effort group) will report higher levels of fatigue and more sleep complaints in-between workdays compared to the low-effort group (Hypothesis 2c). In order to obtain a full picture of the participants\u2019 recovery in-between workdays, sleep time is also examined. Finally, we assume that workers who have expended higher effort during working time, will also be more preoccupied with their job after work (Hypothesis 2d). This expectation is in line with Sonnentag and Bayer\u2019s (2005) finding that those who experienced high workload during the workday found it more difficult to detach from work during evenings than others. We do not formulate a priori expectations regarding possible differences in work motivation between the two effort-groups.How is work-related effort associated with (a) time spent on home activities, (b) experiences of home activities, and (c) health and well-being during the weekend days?The hypotheses formulated for the period in-between workdays (in \"Research question 2\") can be extended to the weekend. Hence, we expect that those who have spent high effort on the job during week days (the high-effort group), will\u2014during the weekend\u2014spend less time on and engage less often in active and will spend more time on and will engage more often in passive home activities (Hypothesis 3a), experience active home activities as more effortful (Hypothesis 3b), report more fatigue and more sleep complaints during the weekend (Hypothesis 3c), and will be more preoccupied with the upcoming workweek, than the low-effort group (Hypothesis 3d).\nMethod\nParticipants and procedure\nThis study was conducted in two stages among academic staff members of a medium-sized Dutch university. Of the 696 employees who were tenured and worked at least 3\u00a0days a week, only those could participate who (1) did not have a job outside this university (to keep the variation in work activities within acceptable limits), and (2) lived with a partner who worked at least 2.5\u00a0days a week (to increase the likelihood that the participants fulfilled at least some home obligations). Of the 146 employees who agreed to participate, 133 (19%) completed a general questionnaire (first stage of the study). Data from 13 of these 133 were removed, as they apparently did not meet one or both of the selection criteria. To already reduce the influence of one possible confounder (i.e., working hours) of the associations between work-related effort and the variables of interest, this study was restricted to employees who worked at least 32 contractual hours a week. As a result, our sample comprised 93 employees (69.6% male; 67.7% \u22651 child living in the household; Mage= 45.0\u00a0years, SD\u00a0= 7.6; 49.5% was assistant professor, 16.1% associate professor, 12.9% full professor, 21.5% other jobs, e.g. researcher or lecturer). Due to strict privacy regulations, we did not know how many of the employees that were approached for participation in the study actually met our inclusion criteria (i.e. had no job outside the university and lived together with a partner who worked at least 2.5\u00a0days a week). Therefore, we do not have insight in how many employees were in fact eligible for participation in the study, meaning that the overall response rate and the representativeness of our sample are unknown.\nIn the second stage of this study starting about ten days after the completion of the general questionnaire, the daily variables of interest were assessed by means of short questionnaires that were completed three times a day, from Monday to Sunday: (1) a morning questionnaire (to be completed after awaking in the morning, between 7:30 and 8:30 a.m.), (2) an afternoon questionnaire (to be completed around 6 p.m.), and (3) an evening questionnaire (to be completed before bedtime, between 10 and 11 p.m.). Only diaries that were completed within an acceptable time range around the requested time were included in the final database. We thus removed morning questionnaires that were completed more than 2\u00a0h after awakening; afternoon questionnaires that were completed before 4:30 p.m., after 8 p.m., or less than 3\u00a0h after the morning questionnaires; and evening questionnaires that were filled in less than 2\u00a0h after the afternoon questionnaire or after 3 a.m. This procedure resulted in 76.2% valid morning diaries, 73.4% valid afternoon diaries, and 72.5% valid evening diaries.\nVariables derived from the general questionnaire (general measures)\nJob types included \u201cassistant professor\u201d, \u201cassociate professor\u201d, \u201cfull professor\u201d, and \u201cother\u201d, such as researcher and teacher. Age was measured in years; Gender was coded as \u201c0\u201d for \u201cmale\u201d and \u201c1\u201d for \u201cfemale\u201d. Parental status was coded as \u201c0\u201d for having no children living in the household and \u201c1\u201d for having at least one child living in the household. The last three variables are potential confounders in the relationships of interest and are therefore included as covariates in further analyses.\nGeneral fatigue was assessed with the ten-item fatigue assessment scale (Michielsen et\u00a0al. 2003). An exemplary item is \u201cI am bothered by fatigue\u201d [1 \u201c(almost) never\u201d, 5 (almost) always\u201d], with higher scores reflecting higher levels of fatigue (\u03b1\u00a0=\u00a00.86).\nWork engagement was measured with five items adapted from Rothbard (2001). An example is \u201cWhen I am working, I often lose track of time\u201d (1 \u201cstrongly disagree\u201d, 5 \u201cstrongly agree\u201d; \u03b1\u00a0=\u00a00.78).\nJob pressure was measured with five items from the job content questionnaire (Karasek 1985), that were rephrased as questions [e.g., \u201cDo you have to work very fast?\u201d 1 \u201c(almost) never\u201d, 4 \u201c(almost) always\u201d; \u03b1\u00a0=\u00a00.74].\nJob control was measured with six items from Van Veldhoven et\u00a0al. (2002). An exemplary item is: \u201cCan you take a short break if you feel this is necessary?\u201d [1 \u201c(almost) never\u201d, 4 \u201c(almost) always\u201d; \u03b1\u00a0=\u00a00.67].\nSocial support from colleagues [e.g., \u201cMy colleagues show their appreciation for the way I do my job\u201d, 1 \u201c(almost) never\u201d, 4 \u201c(almost) always\u201d; \u03b1\u00a0=\u00a00.86], and Social support from supervisor [e.g., \u201cMy supervisor shows her\/his appreciation for the way I do my job\u201d, 1 \u201c(almost) never\u201d, 4 \u201c(almost) always\u201d; \u03b1\u00a0=\u00a00.90] were both measured with four items adapted from Geurts et\u00a0al. (1999).\nPositive affect and negative affect were measured by means of the positive and negative affect schedule (PANAS; Watson and Clark 1988). Following Rothbard (2001), we distinguished between positive and negative affect regarding work and positive and negative affect regarding family. Sample items for negative affect are \u201cupset\u201d and \u201cdistressed\u201d, and examples for positive affect are \u201centhusiastic\u201d and \u201cproud\u201d (1 very slightly or not at all, 5 extremely), with higher scores indicating higher negative or positive affect (negative affect: Cronbach\u2019s \u03b1\u00a0=\u00a00.83 for work and 0.84 for home; positive affect: Cronbach\u2019s \u03b1\u00a0=\u00a00.87 for work and 0.90 for home).\nLife events Participants could report for ten events (e.g., birth of a child, financial troubles, change of job) whether or not they had experienced this event during the past year. The number of events experienced was summed.\nMeasures derived from the daily questionnaires (daily measures)\nTo limit the participants\u2019 burden, the questionnaires contained a combination of validated scales as well as single-item report-marks.\nWork-related effort\nIn the afternoon questionnaire, participants were requested to indicate with a report mark the extent to which they considered the preceding workday as effortful (1 \u201cnot at all\u201d, 10 \u201cextremely\u201d).\nActivities\nTime spent daily on work activities\nParticipants received a list of 13 major work activities and indicated the time (0 \u201cnone\u201d, 1 \u201c<1\u00a0h\u201d, 2 \u201c1\u20132\u00a0h\u201d,..., and 7 \u201c>6\u00a0h\u201d) they had spent on each activity during regular work time, i.e., until 6 p.m. (afternoon questionnaire), and during nonwork time, i.e., after 6 p.m. (evening questionnaire). We recoded this time range to obtain an estimate of the actual time in hours by assuming that the actual time spent on an activity would lie halfway the two extremes (e.g., the category \u201c<1\u00a0h\u201d was recoded as \u201c0.5\u201d). Time spent on research activities by day comprised the time spent on \u201cconducting research\u201d, \u201cdata-analysis\u201d, \u201creading specialist literature\u201d, and \u201cwriting papers\u201d (until 6 p.m.). Time spent on teaching activities by day included the time spent on \u201cpreparing a lecture\u201d, \u201cgiving a lecture\u201d, \u201creading (Ph.D.) students\u2019 assignments\u201d, and \u201cappointments with (Ph.D) students\u201d (until 6 p.m.). Time spent on administrative activities by day consisted of time spent on \u201cpreparing a meeting\u201d, \u201cattending a meeting\u201d and \u201ce-mail\/phone\u201d. The category \u201cinformal contact with colleagues\u201d was entered in the analyses separately. A 13th activity, \u201cother\u201d, was not incorporated in further analyses, as on average only 0.42\u00a0h were devoted daily to these activities. All work activities are potentially relevant to all participants as in the Netherlands lecturers also have some research time, and researchers will usually also teach.\nOvertime work was computed by summing the time spent on all 13 work activities after 6 p.m. (this university did not offer evening classes) during weekdays, and by summing the total time spent on work activities before and after 6 p.m. on Saturday and on Sunday.\nTime spent daily on home activities\nParticipants indicated in both the afternoon (until 6 p.m.) and evening questionnaires (from 6 p.m.) the amount of time they spent that day on ten categories of home activities (largely based on those used in Sonnentag\u2019s (2001) diary study). Answer possibilities and recoding procedure were identical to those used for work activities. To ease interpretation of the categories, participants received examples of activities in each category. Time spent on domestic activities was calculated by summing the total time (i.e., before and after 6 p.m.) devoted each day to \u201chousehold activities\u201d, \u201cdoing odd jobs in or around the house\u201d, \u201cdoing the groceries\u201d, \u201ccare giving activities\u201d and \u201cbusinesslike activities\u201d. Time spent on active leisure activities comprised the total time spent daily on \u201cphysical activities\u201d, \u201ccreative activities\u201d and \u201csocial activities\u201d. The total time spent on passive leisure activities was computed by summing the time devoted to these activities (e.g., reading for pleasure, watching TV, listening to music) before and after 6 p.m. The tenth category, \u201cother\u201d, was omitted from further analysis as the mean time spent on these activities ranged from only 0.15\u00a0h on Saturday to 0.21\u00a0h during weekdays.\nExperiences\nParticipants indicated for each engaged work and home activity, the extent to which they considered it as effortful and as pleasant (1 \u201cnot at all\u201d, 10 \u201cextremely\u201d). An estimate of the average daily effort and pleasure for each category of activities was obtained by computing a weighted mean score. Thus, the summed product of hours spent on each activity within a category and the effort (pleasure) experienced while executing the activity was divided by the total hours spent on the activities in the respective category. By employing such a weighted score, the time spent on an activity is controlled for, assuring that the effort (pleasure) score really reflects effort (pleasure).\nHealth and well-being\nFatigue at work (weekdays\u2019 afternoon questionnaire) was measured with eight items adapted from Van Veldhoven et\u00a0al. (2002), for example \u201cI felt tired mentally\u201d (1 \u201cnot at all\u201d, 10 \u201cextremely\u201d). Participants rated each item twice: (1) with respect to the first hour of the workday (Cronbach\u2019s \u03b1\u00a0=\u00a00.87) and (2) with respect to the last hour of the workday (Cronbach\u2019s \u03b1\u00a0=\u00a00.86).\nFatigue was measured in the morning, afternoon and evening questionnaires. Participants rated their current state of fatigue (\u201cHow fatigued do you currently feel?\u201d) with a report mark varying from \u201c1\u201d (\u201cnot at all\u201d) to \u201c10\u201d (\u201cextremely\u201d).\nSleep complaints (each morning questionnaire) were assessed using a five-item sleep quality scale (Van Veldhoven et\u00a0al. 2002), slightly adapted to make it suitable for day-to-day measurement. An exemplary item is: \u201cLast night I woke up several times\u201d (1 \u201cyes\u201d, 0 \u201cno\u201d, \u03b1\u00a0=\u00a00.73 across all seven consecutive days). Note that each day\u2019s values for this scale refer to the previous night.\nSleep time (each morning questionnaire) was computed by calculating the self-reported number of hours in-between the time they went to sleep last night (\u201cwhat time did you go to sleep last night?\u201d) and the time they woke up this morning (\u201cwhat time did you wake up this morning?\u201d). Again, each day\u2019s values for this scale refer to the previous night.\nPreoccupation with work (each morning questionnaire) was assessed with one self-developed item: \u201cI am already mentally involved with the things I have to do at work today [next week]\u201d (1 \u201cnot at all\u201d, 5 \u201cextremely\u201d).\nWork motivation (each morning questionnaire) regarding the upcoming workday (during weekdays) or the next workweek (during weekend-days) was assessed with one self-developed item: \u201cI feel like starting the next workday [workweek]\u201d (1 \u201cnot at all\u201d, 5 \u201cextremely\u201d).\nA table with correlations between the study variables can be obtained from the first author on request.\nFigure\u00a01 gives an overview of the measurement structure.\nFig.\u00a01Overview of the measurement structure\nCreation of the two effort-groups\nThe global report mark for work-related effort as assessed in the afternoon questionnaire was used to create the two effort-groups. A workday was labeled as effortful if a report mark of six or higher was given. The number of effortful workdays was summed for each participant to obtain an estimate of how effortful he\/she considered the workweek. To increase reliability only participants who gave a report mark during at least three out of the five possible workdays were selected, resulting in a final sample of 72 of the 93 original participants. The low-effort group (n\u00a0=\u00a027; Meffort\u00a0=\u00a03.39) consisted of participants who considered none (out of three) or only one workday (out of four or five) as effortful (>6). The high-effort group (n\u00a0=\u00a024; Meffort\u00a0=\u00a06.77) included participants who labeled two or three (out of three), three or four (out of four) or four or five (out of five) workdays as effortful.\nStatistical analyses\nData were analyzed by means of (M)ANCOVA, which allows the examination of relationships between a categorical independent variable (the effort-subgroups) and continuous dependent variables (Maxwell and Delaney 2005). Gender, age, number of children in the household and number of contractual work hours (32 or more) were included as covariates in the analyses, because these may affect the relationships between work-related effort and the other variables of interest in this study. One key assumption of MANCOVA is that the criterion variables are multivariately normally distributed (Maxwell and Delaney 2005). To examine whether this assumption could be maintained, the distributions of the criterion variables were inspected for univariate normality, both for the total sample and for the low and high effort subgroups. The skewness of the criterion variables was for 61 out of 64 variables in the study lower than 1.00. As this number is already expected on basis of chance, this finding suggests that the assumption of a multivariate normal distribution of the criterion variables could be maintained for practical purposes.\nResults\nPreparatory analyses\nTable\u00a01 shows the descriptive statistics for the general measures for the total sample as well as for the two effort groups. The total sample can be characterized as middle-aged, rather engaged, and not very tired. The mean level of fatigue in the sample does not significantly differ from that in a heterogeneous sample of 1,123 employees (M\u00a0=\u00a01.97, SD\u00a0=\u00a00.57, T(1214)\u00a0=\u00a01.29, ns; Geurts et\u00a0al. 2005). Participants report relatively high levels of work pressure and job control. Levels of job control are higher than those in a heterogeneous sample of 1,740 employees (M\u00a0=\u00a02.54, SD\u00a0=\u00a00.63, T(1831)\u00a0=\u00a0\u221210.29, P\u00a0<\u00a00.001; Geurts et\u00a0al. 2005).\nTable\u00a01Means and standard deviations for the total sample and for the two effort-groups for the measures derived from the general questionnaireTotal sample (N\u00a0=\u00a093)Low-effort group (N\u00a0=\u00a027)High-effort group (N\u00a0=\u00a024)MSDMSDMSDAge44.957.6346.746.6944.177.80Work engagement3.940.693.920.634.050.67Fatigue1.890.611.870.572.040.60Work pressure2.470.542.230.532.490.60Job control3.220.423.250.463.300.39Social support colleagues2.590.652.620.532.600.66Social support supervisor2.350.872.490.792.190.90Positive affectivity work3.610.533.570.623.510.51Positive affectivity home3.590.633.740.713.470.52Negative affectivity work1.930.571.930.502.040.67Negative affectivity home1.830.561.830.591.950.47Life events1.981.532.041.342.171.81\nTo investigate possible differences in the composition of the two effort-groups, these groups were compared with respect to the general measures. No significant differences were observed regarding age (T\u00a0=\u00a01.27, df\u00a0=\u00a049, ns), gender (\u03c72\u00a0=\u00a00.07, df\u00a0=\u00a02, ns), parental status (\u03c72\u00a0=\u00a00.14, df\u00a0=\u00a01, ns), job type (\u03c72\u00a0=\u00a01.43, df\u00a0=\u00a03, ns), general fatigue (T\u00a0=\u00a0\u22121.00, df\u00a0=\u00a049, ns) and work engagement (T\u00a0=\u00a0\u22120.72, df\u00a0=\u00a049, ns). Also, the MANOVA executed with respect to job characteristics (job pressure, job control, social support from colleagues and supervisor) was not significant, F(4, 45)\u00a0=\u00a00.98, ns. Furthermore, the groups report comparable levels of positive and negative affect (work: T(49)\u00a0=\u00a0\u22120.71, ns; family: T(49)\u00a0=\u00a0\u22120.13, ns) and positive affect (work: T(49)\u00a0=\u00a0\u22120.39, ns; family: T(49)\u00a0=\u00a01.01, ns). Finally, the two effort-groups did not differ significantly regarding the number of life events experienced (T(49)\u00a0=\u00a0\u22120.29, ns). Thus, in sum, there were no significant differences between the two effort-groups with respect to the general measures.\nDifferences between Saturday and Sunday\nPreliminary analyses showed that the variables under study did not differ significantly between Saturday and Sunday (F-values ranged from F(1, 27)\u00a0=\u00a00.00, ns for effort reported for active leisure, to F(1, 8)\u00a0=\u00a01.95, ns for effort reported for overtime work). Therefore, further analyses are based on mean scores across the two weekend days.\nResearch question 1\nTable\u00a02 presents the means, standard deviations and F-statistics for the daily variables for the total sample and for each of the two effort-groups. As to work activities, two analyses were conducted. First, for each participant the percentage of days on which time was spent on each work activity was computed. MANCOVA revealed that these percentages did not differ significantly between the two effort-groups. Secondly, for each participant we computed the mean time they spent daily on each work activity across the five weekdays. Again, MANCOVA did not reveal any significant difference between the two effort-groups. Thus, the two effort-groups did not differ significantly in their work activity patterns during the workday.\nTable\u00a02Activity patterns, experiences and recovery indicators during the workdayHypothesisTotal sample (N\u00a0=\u00a093)Low-effort group (N\u00a0=\u00a027)High-effort group (N\u00a0=\u00a024)F (df)PMSDMSDMSD\u2013% Days\u00a0Multivariate 1.29 (4, 42)0.29\u00a0Research6133603762300.60 (1, 45)0.81\u00a0Teaching7530673779273.33 (1, 45)0.07\u00a0Administrative9016901686200.00 (1, 45) 0.99\u00a0Informal contacts5533553355300.32 (1, 45)0.57\u2013Time\u00a0Multivariate0.31 (4, 42)0.87\u00a0Research1.51.31.71.51.61.30.29 (1, 45)0.59\u00a0Teaching2.01.42.01.72.11.40.36 (1, 45)0.55\u00a0Administrative1.91.01.91.21.71.00.02 (1, 45)0.90\u00a0Informal contacts0.40.30.40.30.30.20.89 (1, 45)0.35Hypothesis 1aEffort (1\u201310) \u00a0Multivariate9.94 (4, 29)<0.001\u00a0Research4.872.203.241.666.441.7826.53 (1, 32)<0.001\u00a0Teaching4.471.763.241.495.871.2229.59 (1, 32)<0.001\u00a0Administrative4.051.892.741.345.441.8025.64 (1, 32)<0.001\u00a0Informal contacts2.601.481.851.183.301.627.76 (1, 32)<0.01\u2013Pleasure (1\u201310) \u00a0Multivariate 0.07 (4, 29)0.99\u00a0Research7.181.097.141.377.231.050.00 (1, 32)1.00\u00a0Teaching6.691.046.881.346.610.600.01 (1, 32)0.94\u00a0Administrative5.761.525.941.465.920.910.02 (1, 32)0.89\u00a0Informal contacts7.520.957.311.217.540.880.17 (1, 32)0.68Hypotheses 1b and Hypothesis 1cHealth and well-being\u00a0Work-related fatigue first hour1.891.031.690.651.960.91Time: 0.00 (1, 45)0.96Group: 4.63(1, 45)<0.05\u00a0Work-related fatigue last hour2.561.302.130.863.001.43Time\u00a0\u00d7\u00a0group: 6.09 (1, 45)<0.05F-statistics, P-values, and means and standard deviations for the total sample and for the two effort-groups\nTo study possible differences in experiences, two MANCOVA\u2019s were conducted, both based on mean scores across the five workdays. The first analysis revealed that the two effort-groups differed significantly in the average amount of effort reported with respect to the four work activities. Univariate tests showed that the high-effort group experienced each activity as more effortful (Hypothesis 1a supported). The second analysis revealed that the two effort groups did not differ significantly with respect to the pleasure they derived from their work activities.\nPossible differences between the two effort-groups in fatigue at work were examined in a 2 (Time: first hour vs. last hour)\u00a0\u00d7\u00a02 (Group: low vs. high effort) repeated-measures ANCOVA. The development of fatigue during the workday differed significantly between the two effort-groups (significant time\u00a0\u00d7\u00a0group interaction). Post-hoc analyses showed that there were no significant differences between the two groups in their level of fatigue during the first hour of the workday (T\u00a0=\u00a0\u22121.20, df\u00a0=\u00a049, ns). However, the high-effort group reported a significantly higher mean level of fatigue during the last hour of the workday (T\u00a0=\u00a0\u22122.66, df\u00a0=\u00a049, P\u00a0<\u00a00.05), indicating that the high-effort group reported more fatigue at the end of the workday (Hypothesis 1b supported), and showed a stronger increase in fatigue (Hypothesis 1c supported) during the workday.\nIn sum, the participants in the two effort-groups do not engage in different types of work activities, nor do they experience their work activities differently in terms of pleasantness. However, the high-effort group reports to spend significantly more effort on each of the work activities, experiences significantly higher work-related levels of fatigue at the end of the work day, as well as a stronger increase in fatigue during the workday.\nResearch question 2\nMeans, standard deviations and F-statistics are presented in Table\u00a03. As to home activity patterns, two analyses were performed. First, for every participant, we computed the percentage of workdays they spent time on each type of home activity (domestic, active leisure, overtime work, and passive leisure). For each of these activities, an ANCOVA was conducted. Results showed that the groups only differed significantly with respect to active leisure activities: whereas the high-effort group spent on less than half of the work days (43%) time on this type of activities, the low-effort group spent on more than half of the work days (62%) time on this type of activities. Secondly, we conducted four ANCOVA\u2019s based on each participant\u2019s mean time spent daily on each of the four activities during the five weekdays, but these revealed no differences between the two effort-groups. These results provide partial support for Hypothesis 2a by showing that participants in the high-effort group engage on average less often in active leisure activities.\nTable\u00a03Activity patterns, experiences, recovery indicators, work involvement and work motivation in-between workdaysHypothesisTotal sample (N\u00a0=\u00a093)Low-effort group (N\u00a0=\u00a027)High-effort group (N\u00a0=\u00a024)F (df)PMSDMSDMSDHypothesis 2a% Days\u00a0Domestic8620902283181.34 (1, 45)0.25\u00a0Active leisure 5633622643318.12 (1, 45)<0.01\u00a0Overtime4923432758344.92 (1, 45)<0.05\u00a0Passive leisure7327782870340.63 (1, 45)0.43Hypothesis 2aTime\u00a0Domestic 2.31.62.21.42.01.51.47 (1, 45)0.23\u00a0Active leisure0.90.70.90.50.70.71.90 (1, 45)0.18\u00a0Overtime0.90.80.80.71.11.02.42 (1, 45)0.13\u00a0Passive leisure1.31.11.30.81.20.80.36 (1, 45)0.55Hypothesis 2bEffort (1\u201310)\u00a0Multivariate 10.90 (4, 27)<0.001\u00a0Domestic 2.961.572.461.223.321.760.80 (1, 30)0.38\u00a0Active leisure3.581.762.911.364.561.997.24 (1, 30)<0.05\u00a0Overtime4.341.763.131.485.821.1526.11 (1, 30)<0.001\u00a0Passive leisure2.061.241.500.822.811.583.37 (1, 30)0.08\u2013Pleasure (1\u201310)\u00a0Multivariate 0.67 (4, 27)0.62\u00a0Domestic 5.791.406.081.345.601.210.38 (1, 30)0.54\u00a0Active leisure7.311.237.431.207.710.750.03 (1, 30)0.86\u00a0Overtime6.341.296.331.496.160.950.07 (1, 30)0.80\u00a0Passive leisure6.871.437.191.056.871.330.21 (1, 30)0.65Hypothesis 2c and 2dHealth and well-being\u00a0Fatigue t13.761.932.901.474.871.72Time: 2.16 (2, 44)0.13\u00a0Fatigue t24.921.743.901.565.881.40Group 22.46 (1, 45)<0.001\u00a0Fatigue t35.711.984.872.076.521.50Time\u00a0\u00d7\u00a0group 0.06 (2, 44)0.95\u00a0Sleep complaints1.541.121.251.001.950.954.12 (1, 45)<0.05\u00a0Sleep time7.090.886.851.057.130.701.85 (1, 45)0.18\u00a0Preoccupation work3.321.003.190.903.750.954.41 (1, 45)<0.05\u00a0Work motivation3.400.703.490.853.210.503.00 (1, 45)0.09F-statistics, P-values, and means and standard deviations for the total sample and for the two effort-groups\nIn order to investigate possible differences in their experiences of home activities, two MANCOVA\u2019s were computed, both based on mean scores across the five weekdays. The first analysis showed an overall significant difference between the two effort-groups in the extent to which they considered home activities as effortful. Univariate tests demonstrated that the high-effort group considered active leisure activities and overtime work as more effortful (Hypothesis 2b supported). The second analysis revealed that the two effort-groups did not differ significantly as to their pleasure regarding their home activities.\nRegarding health and well-being, three analyses were conducted, each based on mean scores across the five workdays. For fatigue, a 3 (time: morning vs. afternoon vs. evening)\u00a0\u00d7\u00a02 (group: low vs. high effort) repeated-measures ANCOVA indicated that fatigue did not vary significantly as a function of time. However, the two effort-groups did differ significantly in their average level of fatigue (main effect of Group). Post-hoc analyses demonstrated that the high-effort group reported higher levels of fatigue (M\u00a0=\u00a05.76) compared to the low-effort group (M\u00a0=\u00a03.89, T\u00a0=\u00a0\u22124.78, df\u00a0=\u00a049, P\u00a0<\u00a0.001; Hypothesis 2c supported for fatigue). The development of fatigue during the day did not vary significantly as a function of effort-group (time\u00a0\u00d7\u00a0group interaction, ns). Furthermore, ANCOVA revealed that the high-effort group reported significantly more sleep complaints (Hypothesis 2c supported for sleep complaints). The third analysis (ANCOVA) showed that the two effort-groups did not differ significantly with regard to sleep time. Concerning preoccupation with work, ANCOVA revealed that the high-effort group was significantly more preoccupied (Hypothesis 2d supported). A similar analysis conducted for work motivation did not reveal any significant differences between the two effort-groups.\nIn sum, the high-effort group engaged less often in active leisure activities in-between successive workdays, but did not differ significantly from the low effort-group regarding the experience of pleasure associated with these activities. Further, the high-effort group experienced the home activities as more effortful. In addition, we systematically observed higher levels of fatigue; more sleep complaints, and a higher preoccupation with work in the high-effort group in-between workdays.\nResearch question 3\nTable\u00a04 presents the relevant means, standard deviations and F-statistics for the total sample and for the two effort-groups. To map possible differences in activity patterns between the two effort-groups, two analyses were conducted. First, we computed for each participant the percentage of weekend days on which time was spent on each home activity: The four ANCOVA\u2019s (one for each percentage) conducted for these percentage revealed no differences between both effort-groups. Secondly, with respect to the time spent on the four types of home activities, also for each activity an ANCOVA was conducted. Results revealed one important difference in activity patterns: the high-effort group spent significantly more time on overtime work during the weekend compared to the low-effort group (Hypothesis 3a partially supported).\nTable\u00a04Activity patterns, experiences, recovery indicators, work involvement and work motivation during the weekendTotal sample (N\u00a0=\u00a093)Low-effort group (N\u00a0=\u00a027)High-effort group (N\u00a0=\u00a024)F (df)PMSDMSDMSDHypothesis 3a% Days\u00a0Domestic 9713981096140.13 (1, 45)0.74\u00a0Active leisure7032762963303.16 (1, 45)0.08\u00a0Passive leisure8726852779330.27 (1, 45)0.61\u00a0Overtime4339374150422.23 (1, 45)0.14Hypothesis 3aTime\u00a0Domestic 5.02.45.02.05.32.50.14 (1, 43)0.71\u00a0Active leisure2.52.02.41.62.42.60.11 (1, 43)0.74\u00a0Passive leisure2.61.62.71.52.71.90.38 (1, 43) 0.54\u00a0Overtime1.21.40.80.91.41.76.14 (1, 45)<0.05Hypothesis 3bEffort (1\u201310) \u00a0Domestic 3.331.802.651.443.911.975.87 (1, 45)<0.05\u00a0Active leisure3.241.922.411.643.872.136.50 (1, 42)<0.05\u00a0Overtime4.542.013.421.725.491.809.01 (1, 24)<0.01\u00a0Passive leisure2.091.421.731.242.651.595.49 (1, 41)<0.05\u2013Pleasure (1\u201310) \u00a0Domestic 6.241.106.201.316.071.020.15 (1, 45)0.70\u00a0Active leisure7.680.887.580.977.610.820.02 (1, 42)0.89\u00a0Overtime6.031.576.281.785.961.250.58 (1, 24)0.45\u00a0Passive leisure7.470.957.411.107.570.860.14 (1, 41)0.71Hypothesis 3c and 3dHealth and well-being\u00a0Fatigue (t1)3.432.102.691.694.002.24Time: 3.39 (2, 41)<0.05\u00a0Fatigue (t2)4.262.043.561.644.722.21Group: 7.80 (1, 42)<0.05\u00a0Fatigue (t3)5.692.094.562.196.381.57Time\u00a0\u00d7\u00a0group: 0.15 (2, 41)0.87\u00a0Sleep complaints1.041.110.941.071.401.311.44 (1, 44)0.24\u00a0Sleep time7.881.057.741.107.910.940.69 (1, 43)0.41\u00a0Preoccupation work2.411.072.090.942.691.152.28 (1, 44)0.14\u00a0Work motivation3.300.983.500.912.890.995.27 (1, 44)<0.05F-statistics, P-values, and means and standard deviations for the total sample and for the two effort-groups\nConducting multivariate analyses for \u201cpleasure\u201d and \u201ceffort\u201d would result in very restricted sample sizes (n\u00a0=\u00a013 in both groups). Therefore, only univariate tests were computed, revealing that the high-effort group considered all four activities significantly more effortful (Hypothesis 3b supported). Again, the two effort-groups did not differ significantly with respect to pleasure associated with their activities.\nThree analyses were conducted to examine possible differences between the two effort-groups regarding health and well-being indicators. A 3 (time: morning vs. afternoon vs. evening)\u00a0\u00d7\u00a02 (group: low vs. high-effort) repeated-measures ANCOVA revealed a main effect of Time. Post-hoc analyses indicated that fatigue increased significantly during the day, that is, was lowest in the morning (M\u00a0=\u00a03.32), somewhat higher in the afternoon (M\u00a0=\u00a04.08) and highest in the evening (M\u00a0=\u00a05.41). Furthermore, overall, the two effort-groups reported different levels of fatigue (significant main effect of group). Post-hoc analyses showed that the high-effort group reported significantly higher levels of fatigue (M\u00a0=\u00a05.03) than the low-effort group (M\u00a0=\u00a03.61; Hypothesis 3c supported for fatigue). Finally, fatigue did not vary significantly between the effort-groups as a function of time of the day (non-significant time\u00a0\u00d7\u00a0group interaction). Two ANCOVAs indicated that sleep complaints and sleep time did not vary significantly between the effort-groups (Hypothesis 3c rejected for sleep complaints).\nTwo additional ANCOVAs indicated that the two effort-groups did not significantly differ with respect to preoccupation with work during the weekend (Hypothesis 3d rejected), but that the high-effort group felt less like starting the next working week (work motivation).\nIn sum, the two effort-groups did not show significantly different activity patterns during the weekend regarding domestic work, active and passive leisure. However, the high-effort group spent significantly more hours on overtime work during the weekend than the low-effort group. Furthermore, the high-effort group experienced all home activities as significantly more effortful, although not as less pleasant, than the low effort-group. We also observed significantly higher levels of fatigue during the weekend and less motivation to start the upcoming workweek in the high-effort group.\nDiscussion\nThe present study was devised to enhance our insight in the associations between work-related effort and recovery from that effort. To this purpose, we compared two groups of employees reporting different levels of work-related effort (high vs. low) with respect to their activities, experiences, and health and well-being in three time-periods: (1) during work time, (2) in-between work days and (3) during the weekend.\nActivity patterns\nOur results revealed that the two effort-groups did not differ significantly in terms of their activity patterns at work. However, two significant differences were observed in the home domain. The first manifested itself in-between work days: the high-effort group performed active leisure activities on fewer days than the low-effort group, which is unfortunate, as active leisure activities seem to promote recovery (Sonnentag 2001).\nA second difference appeared during the weekend. Contrary to our expectations (Hypothesis 3c), employees in the high-effort group spent more time on working overtime in the weekend. This implies that these employees devote part of potential recovery time during the weekend to activities that may interfere with the recovery process (cf. Sonnentag 2001).\nThe amount of time devoted to domestic activities during the weekdays and weekend days did not vary significantly between the two effort-groups. This may be due to the fact that many domestic activities are obligatory in nature (e.g., it is difficult to circumvent doing the household chores). Finally, no significant differences between the groups emerged concerning low-effort activities, both during weekdays and weekend days.\nExperiences\nRegarding experiences, we distinguished between effort and pleasure. The high-effort group reported significantly more effort for all work activities. In the home domain, the high-effort group judged overtime and active leisure activities as more effortful during weekdays and judged all activities as more effortful during the weekend. No significant differences between the groups were observed with respect to pleasure, neither during work time nor in-between workdays, nor in the weekend. Hence, work-related effort is independent of the pleasure derived from work and home activities.\nHealth and well-being\nWe observed a stronger increase in work-related fatigue during the workday for the high-effort group than for the low-effort group. Thus, whereas the two groups did not differ significantly in work-related fatigue at the start of the working day, the high-effort group was more fatigued at the end of the working day. This difference persisted in-between workdays. This finding might explain why the high-effort group engaged less often in active leisure in-between workdays than the low-effort group. Also during the weekend, the high-effort group remained significantly more fatigued than the low-effort group. Possibly, this may be due to the fact that the former group spent more time on overtime.\nA somewhat different pattern of results was observed with respect to sleep complaints: The high-effort group reported more sleep complaints during the week, but not in the weekend. The additional finding that the two groups did not differ significantly with respect to sleep time suggests that work-related effort relates to sleep quality, but not sleep quantity.\nDuring the week, the high-effort group was apparently more preoccupied with work than the low-effort group. However, it cannot be excluded that this is partly due to our item wording. Although we asked participants to indicate the extent to which they were already preoccupied with the upcoming workday, it would seem possible that this measure (also) reflects the extent to which participants were still ruminating about their past working day. In the weekend, the two groups did not differ significantly in their preoccupation with the upcoming workweek. This is surprising, as the high-effort group spent more time on work-related activities during these days. The high-effort group nonetheless reported less work motivation than the low-effort group.\nLimitations and suggestions for future research\nSix issues with respect to the present study must be discussed. First, as we employed a single item report mark to create the two effort-subgroups, the reliability and validity of this measure can be questioned. However, we believe that there are good arguments in favor of employing this report mark: (1) we did not rely on a single observation of this measure, as each participant completed the item on at least three occasions; (2) employees in the high-effort group considered each of the four categories of work activities as more effortful than the low-effort group, thus suggesting that the report mark correctly reflects the effort experienced during the workday; and (3) there is a correlation of 0.85 (P\u00a0<\u00a00.001) between our single-item effort-measure and a weighted mean score of the effort experienced during the separate work activities. The latter was computed by first weighting the number of hours spent on each activity by the effort expended to this activity; the sum of these weighted scores was divided by the total number of work hours. Thus, our single-item report mark seems to measure a very similar quantity as a much more refined measure of effort.\nA second point of concern is the procedure used to create the two effort-subgroups. These groups were created based on the number of days participants considered their work as effortful. To probe the possibility that our findings are biased by this somewhat arbitrary procedure we repeated our analyses using a slightly different effort indicator. For each participant who completed the report mark of global work-related effort on at least three occasions, the mean score on this report mark across the week was computed. Based on these scores, two new subgroups were created: One including participants with scores in the highest tertile, and a second with participants having scores in the lowest tertile. Analyses were repeated for these two groups, yielding results that were highly similar to those found for the original subgroups (results can be obtained from the first author on request). Thus, our findings appear robust across different measures of effort expenditure.\nThirdly, our study relied exclusively on self-report measures, and this might have resulted in an overestimation of the associations among the variables due to common method variance. However, this should have inflated all relations studied and not just part of these: the fact that some relationships were found while others were not, argues against the influence of common method variance in our study. Besides, alternative measures such as observational or physiological measures are not free of error variance either, and should therefore not be considered superior to self-report measures (Semmer et\u00a0al. 2004; see also Kompier 2005). Furthermore, by demonstrating (1) that using self-reports does not guarantee finding significant results, (2) that potential biasing variables (social desirability, negative affectivity and acquiescence) do not generally inflate correlations among study variables and (3) that monomethod correlations are not by definition higher than multimethod correlations, Spector (2006) concludes that \u201cthe popular position suggesting common method variance automatically affects variables measured with the same method is a distortion and oversimplification of the true state of affairs\u201d (p 221). Thus, all in all we do not believe that common method bias severely biased our findings, although the use of physiological and performance measures in addition to self-reports could provide interesting insights in future research.\nA fourth issue is the impact of potential third variables. One might argue that differences between the two effort-groups regarding (experiences of) activities and health and well-being indicators might be due to personality characteristics or other person or work-related constructs, rather than to work-related effort. However, in our study we attempted to exclude the influence of these variables to our best ability: The two effort-groups turned out not to differ regarding the number of life events experienced, general work characteristics (work pressure, job control, social support), fatigue, work engagement, age and positive and negative affect. Of course this does not exclude the possibility that other third variables (e.g., other aspects of personality) may have acted as third variables in this study.\nFifth, this study did not offer insight in the intriguing question into the origin of the differences in work-related effort between the two subgroups studied. It may be that these differences are at least partly due to differences in participants\u2019 objective work performance (e.g., number of publications or student evaluations), but such measure was not incorporated in this study. Thus, it is unclear how the differences between the two effort-groups in their work-related effort are related to real output differences, and future studies on this topic should also include objective measures of task performance.\nFinally, the present research employed a very specific sample, consisting of academic staff members who worked at least 32\u00a0h a week and who lived together with a partner who worked at least 2.5\u00a0days a week, and who, as is common for tenured academics in the Netherlands, have relatively high job security and are not dependent on fund raising. Although we believe that our main findings on the relations among effort, recovery, health and well-being are not unique to this sample, it is desirable to replicate this study for employees in other professions, in other family situations and\/or with other working hours. Thus, future studies should employ samples from other contexts to broaden our understanding of effort and recovery patterns.\nAssets of this study\nIn spite of these limitations, we believe that the present study extends and enhances previous research on effort and recovery in at least four respects. First, this study is among the very few that examine effort and recovery from a day-to-day perspective, allowing us to demonstrate that work-related effort is related to various aspects of daily work and (potential) recovery time. In this vein, this study shows how effort expenditure at work is actually imbedded in everyday life, and how it relates to recovery during time-off-the job.\nSecondly, this study emphasized the importance of the weekend as a (potential) opportunity for recovery. Whereas some differences between the two effort-groups persisted throughout the weekend (e.g., higher levels of fatigue and effort-investment for the high-effort group), other differences manifested themselves only during the working week (i.e., less active leisure, more sleep complaints and more preoccupation with work for the high-effort group) or only during the weekend (i.e., more overtime work and less work motivation for the high-effort group). Thus, not all workers employ the recovery opportunities offered by the weekend in a similar fashion: some seem to employ the weekend as a means to catch up with their overdue tasks. These results suggest that it would be worthwhile to study the reasons why workers differ in the way they use their weekend.\nThirdly, by paying attention to employees\u2019 activity patterns at work and outside work, we were able to show that effort expenditure at work relates to activity patterns in the home domain. Namely, high levels of effort expenditure at work were associated with less engagement in active leisure and more overtime work. This finding thus suggests that for some workers, high effort expenditure at work is not compensated by a corresponding degree of participation in recovery activities. Given that an imbalance between effort and recovery is associated with adverse health outcomes, this particular group of workers may, in the long run, be a risk group for the development of ill health.\nFourthly, we demonstrated that experiences associated with engagement in work and home activities are important: higher effort investment at work is related to experiences of higher effort expenditure outside work, but not to experiences of less pleasure regarding work or home activities.\nPractical implications\nBased on our study\u2019s results, three practical suggestions can be formulated. Firstly, adequate control opportunities in the job setting will allow workers to adjust their work behavior to their current need for recovery and, thus, to prevent the development of negative load reactions during working. Secondly, employees should be encouraged to engage in leisure activities that potentially contribute to the recovery process, such as active leisure. Finally, the time spent on overtime work should be kept within acceptable limits, as overtime work impedes the recovery process. Employers should not to demand excessive overtime work from their employees, in order to guarantee sufficient (potential) recovery time (see also Beckers et\u00a0al. in press).\nTheoretical implications\nOur study revealed that workers who invest high effort at work differ in their off-the-job activity patterns from those who invest low effort at work: members of the first group are to a lesser degree engaged in active leisure during evenings in-between work days, and they spend more time on overtime work during the weekend. This different activity pattern may have consequences for the recovery process, as previous research suggests that active leisure promotes recovery, whereas overtime work impedes this process (Sonnentag 2001). That recovery is endangered in the high-effort group is also evidenced by the higher levels of fatigue during non-work time (evenings and weekends) and the lower sleep quality during the week. Apart from a different activity pattern, those expending high effort at work also expend high effort on home activities, which also may endanger the recovery process. Therefore, despite the fact that those investing high effort at work do not experience their activities as less pleasant than those expending low effort, they may be considered at risk for developing health problems in the long run.","keyphrases":["diary study","effort","recovery","university staff"],"prmu":["P","P","P","R"]}
{"id":"J_Med_Internet_Res-4-1-1761926","title":"A Framework for the Evaluation of Internet-based Diabetes Management\n","text":"Background While still in its infancy, Internet-based diabetes management shows great promise for growth. However, the following aspects must be considered: what are the key metrics for the evaluation of a diabetes management site? how should these sites grow in the future and what services should they offer?\nIntroduction\nManagement of patients with chronic conditions is a long-standing challenge for health care organizations. These conditions include diabetes, chronic heart failure (CHF), chronic obstructive pulmonary disease (COPD), Asthma, HIV\/AIDS, and cancer. Patients are required to adopt lifelong exercise, diet, and drug regimens to maintain optimal health and avoid the complications of the disease. These complications can arise suddenly and be life threatening; therefore, patients with chronic diseases must be monitored constantly [1].\nIn recent years, Internet-based home telemonitoring systems have become available [2]. These sites leverage the Internet to record, measure, monitor, manage, and deliver health care. These information-technology solutions are creating a link between patient and caregiver that enables patients to supply a steady stream of valuable health information to caregivers. For example, diabetics can report their blood glucose readings, thus creating a history of their glucose control, which caregivers can use to evaluate the impact of a therapy (eg, short acting insulin) or the need for a different one [1]. Conversely, caregivers have the ability to provide their patients with crucial information and feedback on the management of their disease. For example, patients can be notified about screening appointments for the complications of diabetes. Therefore, patients benefit from an improved control and understanding of the disease; the ability to self-monitor from home reduces the burden of the disease. These solutions have resulted in dramatic improvements in disease management as measured by hospitalizations [1] and in an overall reduction in costs [3]. Further, patients report higher levels of satisfaction and better control of their conditions [4].\nDiabetes is a chronic disease that affects 30 million people worldwide [5] and is the seventh leading cause of death in the United States [6]. The total annual economic cost of diabetes in 1997 was estimated to be US $98 billion. That includes US $44 billion in direct medical and treatment costs and US $54 billion for indirect costs attributed to disability and humanity [7] and a significant intrusion in the life of an individual. In managing diabetes, success is measured by positive change in prognostic indicators and outcomes. Below is a list of measurement criteria used in diabetes management [8,9,10].\nGreater patient self-efficacy\nGreater satisfaction with care, continuity, provider, quality of health outcome\nDecreased HbA 1cand blood glucose levels\nImproved diet and body weight control\nLowered cholesterol\nLowered perception of diabetes intrusiveness\nImproved quality of life\nLess depression\nDecreased incidence of diabetic complications.\nPrimarily, diabetes must be managed by the patient because it requires adherence to stringent dietary, physical, and medical regimes [8]. Internet-based diabetes management systems have the potential of reducing the burden of disease both to the patient and to the health care system. A recent study found that a high proportion of patients are willing to use Internet resources in the management of their disease [9]. The driving forces behind the proliferation of technology for disease management is the patients' demands to get real-time help, get real-time information, and keep in contact with their physician [1]. Not surprisingly, several diabetes-specific sites have recently appeared [10], including myDiabetes, Health Hero Network, LifeChart, LifeMasters, and Medifor.\nThe purpose of this paper is to review the patient's and the health care professional's needs in an Internet-based diabetes management solution and to examine how these needs are addressed in practice. An evaluation framework was constructed by grouping the requirements of an Internet-based diabetes management solution into 5 categories: Monitoring, Information, Personalization, Communication, and Technology. Two of the market leaders (myDiabetes and LifeMasters) were selected and evaluated to illustrate the use of the framework.\nMethods\nA literature search was conducted on medical databases (Medline, Pre-Medline, EMBASE, Cochrane, and PubMed) and a nonmedical database (Expanded Academic ASAP). The articles were identified by diabetes, chronic disease, internet, and technology. The searches were based on the following AND combinations of these keywords.\ndiabetes AND internet\ndiabetes AND technology\nchronic disease AND internet\nchronic disease AND technology\nThe exact search methodology differed among databases due to differences in their user interfaces. The methodology for each database is summarized in Table 1.\nThe abstracts of the articles retrieved by the searches were screened for relevance by the authors. The relevant articles were reviewed in order to compile a comprehensive list of requirements for an Internet-based diabetes management solution. These requirements were identified on the following basis:\nNo interdependence between requirements\nRequirements can be assessed as present or not present\nEqual implementation effort required to satisfy the requirements.\nThe implementation effort was quantified by the number of Use Cases as defined by the Universal Modeling Language (UML) [11,12]. The number of Use Cases ranged from 1 to 3 for each requirement. For example, the requirement defined as User defined parameter-Patient allows patients to define which health parameter they wish to monitor. This functionality requires 3 Use Cases: Identify User,\nRetrieve Parameters, and Save Parameters.\nThe requirements for Internet-based diabetes management were compiled into the criteria of an evaluation framework. The evaluation criteria were grouped into 5 categories: Monitoring, Information, Personalization, Communication, and Technology. The evaluation framework is presented in Table 2 and the evaluation criteria are discussed in detail in the \"Evaluation Criteria\" section of the \"Results\" section.\nTo illustrate the use of the evaluation framework, we have applied it to 2 existing Internet-based diabetes management systems: my Diabetes (www.myDiabetes.com) and LifeMasters (www.lifemasters.com). These 2 sites were selected because they were first movers in the arena of Internet-based diabetes management. MyDiabetes.com was one of the first sites going live in July 1999, shortly followed by LifeMasters.com in October 1999.\nThe sites were evaluated from November 1, 2001 through December 15, 2001. The evaluations were performed by 5 independent evaluators who were not aware of each other's ratings. All evaluators are computer literate and are familiar with the use of the Internet. The evaluators included a physician, 3 diabetic patients, and one author [CM]. All the evaluators registered separately with both sites (registration was free). Each evaluator was given a detailed description of the evaluation criteria, as described in the \"Results\" section, and Table 2, which describes the framework. The evaluators were also given an evaluation form to fill out (effectively Table 3 without results). For each criterion, the evaluators rated the sites as Yes if the criterion was satisfied or No if it was not satisfied. The evaluations were not supervised.\nFigure 1 and Figure 2 are screen shots of the entry forms for the daily glucose measurements forms at myDiabetes and LifeMasters respectively. This basic function of diabetes monitoring requires the user to input his or her blood glucose levels and the time of the readings. The data is stored, effectively creating a log of the glucose control of the patient. LifeMasters records glucose levels based on relative times such as Bedtime and asks for symptoms of high and low blood glucose as well as diabetic complications. Mydiabetes records the exact time of the blood glucose measurement but does not screen for any symptoms; this is done in another section of the site.\nTable 1\nSearch methodologies for databases\nDatabase\nSearch Methodology\nMedline (1966 to October week 5, 2001)\nAND\ndiabetes\nchronic disease\ninternet\ntechnology\n1 and 31 and 42 and 32 and 4The 4 terms were searched separately by entering the search string, exploding the subject, and selecting all subheadings. The search results were combined using the AND condition. The search history is described below: diabetes chronic disease internet technology 1 and 3 1 and 4 2 and 3 2 and 4\nCochrane and Pre-Medline\nAND\ndiabetes\nchronic disease\ninternet\ntechnology\n1 and 31 and 42 and 32 and 4The 4 terms were searched separately. The search results were combined using the AND condition. The search history is described below: diabetes chronic disease internet technology 1 and 3 1 and 4 2 and 3 2 and 4\nEMBASE (via ScienceDirect), Expanded Academic ASAP, PubMed\nAND All Fields All Years\ndiabetes AND internet\ndiabetes AND technology\nchronic disease AND internet\nchronic disease AND technology\nThe terms were searched in combination using the AND condition. The terms were searched in All Fields and for All Years indexed. diabetes AND internet diabetes AND technology chronic disease AND internet chronic disease AND technology\nFigure 1\nThe myDiabetes entry form for the daily glucose measurements\nFigure 2\nThe LifeMasters entry form for the daily glucose measurements\nTable 2\nEvaluation framework\nEvaluation Criteria\nDescription\nMonitoring\nUser defined parameters\nHealth care worker\nHealth care professionals can specify the parameters to monitor\nPatient\nPatients can specify the parameters to monitor\nUser defined parameter ranges\nHealth care worker\nHealth care professionals can specify the normal ranges for monitored parameters\nPatient\nnormal ranges for monitored parameters\nAutomated data collection\nVital data can be downloaded directly from the measurement device (eg, Glucometer)\nAlert algorithms to avoid false alarms\nEntry validation\nValidation that patient data is not the result of mistyping (eg, Realistic glucose levels)\nScreening of symptoms\nDetermine if changes in vital data is associated to symptoms indicative of an emergency\nPatient involvement in alert\nInvolving the patient in the decision to notify a health care professional\nMultidisciplinary approach\nMultiple aspects of disease management monitored\nThe monitoring is based on a multidisciplinary approach to diabetes\nPhysical\nMonitoring of physical parameters (blood glucose, weight, blood pressure, etc.)\nSocial\nMonitoring of the social aspects of diabetes (stigma, dieting, etc.)\nPsychological\nMonitoring of psychological aspects of diabetes (depression, loss of motivation, etc.)\nPatient access to multiple specialists\nAllowing for communication to multiple experts (dietitians, endocrinologists, etc.)\nProactive outreach\nNotification to patients\nmedications, health care appointments, etc\nNotifications to health care professionals\nare reminded of screening test and visits\nFeedback\nRetrieve and review medical information\nPatients can retrieve their medical data to monitor their progress (tabular or graphical format)\nRegular Feedback\ncontrol of diabetes is administered and stored\nInformation\nQuality of information\nsite should conform to an accepted level of standards\nPull\nNavigation\nNavigation should be based on a logical categorization of data\nSearch\nSearch Functionality availability\nPush\nNotifications\nThe system should notify its users of newly available information of interest based on their profile (eg. New research)\nNewsletter subscription\nUsers can subscribe to a specific newsletter that is delivered via e-mail of Web browser\nPersonalization\nAssessment and feedback\ndiabetes should be assessed using standard evaluation tools\nCollaborative goal setting\nmanagement should be clearly specified\nIdentification of barriers and supports\nUsing questionnaires to determine each patient's barrier and the appropriate support measures\nFollow-up support\nRe-iteration of support measures\nConstruction of personalized management plan\nTailored management plan as a central feature of the site (can be represented as schedules)\nModification of management plan\nThe ability for users to modify their plans\nLanguage and ethnicity\nMultilanguage delivery and culture conscious content\nCommunication\nHealth professional- patient\nSynchronous\nA channel for one-to-one synchronous communication (eg, videoconferencing)\nAsynchronous\nA channel for one-to-one asynchronous communication (eg, secure email)\nIndirect\nTechnical representation of the health care professional\nCommunity creation\nChat rooms\nSynchronous many-to-many communication channels\nNewsgroups \/ Forums\nAsynchronous many-to-many communication channels\nExpert moderation\nCommunication channels are based on the dialogue with an expert\nTechnology\nSecurity\nAuthentication\nIdentification of users (usually username\/password)\nEncryption\nData transmission security level (eg, 128-bit)\nUsability and user-acceptance\nEvaluation of usability and user-acceptance (achieved with questionnaires, usage monitoring etc.)\nReliability and availability\nService should be available at all times\nOpen architecture\non open standard technologies\nStatistical Analysis\nCohen's multi-rater kappa [13,14] was used to evaluate the agreement between raters for the evaluation framework as a whole. The multi-rater kappa was calculated with SPSS statistical software using the mkappasc procedure.\nResults\nEvaluation Criteria\nIn this section, we describe in detail the evaluation criteria presented in Table 2.\nMonitoring\nSuccessful patient monitoring is reliant on efficiently extracting the relevant information from a patient without excessive intrusiveness to both patient and health care professional. Several parameters can be monitored; some examples are blood glucose, weight, blood pressure, diet, foot care, smoking, and nutrition [4,15,16]. Health care professionals should be able to designate which parameters they want to monitor and specify the ranges for each patient. The health care professional should be able to indicate which course of action the system should take if the readings are outside the ranges (eg, notification, triage).\nPatients should also be able to designate parameters in an effort to improve self-management and goal setting (addressed in the \"Personalization\" section of \"Evaluation Criteria\") [17]; these, however, should be in addition to - and clearly differentiated from - the parameters specified by the health care professional. Patient-designated parameters should not be shared with the health care professional unless the patient desires that they be shared.\nThe degree of intrusiveness is a fundamental consideration when designing a diabetes management system. A major problem with many disease-management programs using information technology is that they try to collect too much data too often [1]. The desire to collect as much data as possible must be balanced with the disruption it may cause in a patient's life [4]. Successful strategies to reduce intrusiveness are based on automatic data gathering such as Glucometers that transmit glucose readings via the Internet and the use of simplified questionnaires for triage and screening. Intrusiveness to the health care provider is also an important consideration. If systems were designed to send alerts each time a patient's blood sugar readings are outside the normal parameters, the result would be many false alarms. Therefore, systems must have processes in place designed to not overwhelm health care professionals. These processes include entry validation, screening with the use of questionnaires, and patient involvement in the decision to launch an alert [1].\nEffective patient monitoring is not limited to the collection of health data, it also requires a multidisciplinary approach, proactive outreach, and feedback.\nMultidisciplinary Approach\nThe management of diabetes spans multiple medical specialties as evidenced by the use of multidisciplinary diabetes management teams. For example, an endocrinologist will manage medications and glucose levels, a dietitian will design an appropriate diet, and a psychologist will manage the mental aspect of dealing with diabetes. Internet-based diabetes management programs should be based on a multidisciplinary teamwork. This element consistently appears in successful chronic-disease management systems [18]. Patients should have the ability to interact with multiple specialists to manage each facet of their disease and the Internet can provide a communication channel to enhance this interaction. Successful evaluation tools have been created to effectively measure diabetes management outcomes along multiple dimensions (medical, social, psychological, etc.). Some examples of these tools are the Diabetes Quality of Life Measure (DQOL) developed for use in the Diabetes Control and Complications Trial (DCCT) [19] and the SF-36 [20].\nProactive Outreach\nProactive outreach and patient tracking are critical success factors for an Internet-based diabetes management system. Proactive outreach consists of notifications sent to patients to take their medication, visit the health care professional, or simply exercise once a day. The benefit of a proactive approach is well documented in the management of other chronic diseases such as chronic heart failure, where increased compliance and monitoring have resulted in a decrease in the number of hospitalizations for cardiovascular diagnoses and hospital days were reduced from 0.6 to 0.2 (P = .09) per patient per year [21]. Proactive outreach also applies to health care professionals. Reminders to physicians of routine testing for patients can be implemented in an Internet-based diabetes management system. A study determined that the use of a diabetes management system increases the likelihood of physicians ordering lipid-profile testing (19%) and retinal exams for their patients [22].\nFeedback\nThe role of the patient has become central in the management of chronic disease; therefore, monitoring must integrate the patient [22]. A crucial aspect of patient integration is feedback. Patients must have the ability to review their medical data at anytime. On-line graphical tools can allow patients to visualize their medical information in much the same way a physician would. Feedback also provides a valuable motivational tool that improves compliance [1] and system usage, both of which are linked to an improved outcome in diabetes management [23].\nInformation\nThe Internet has always served as a source of health information; 70 million of the 110 million American Internet users have searched the Web for health information in the past year. Currently they can choose from 20,000 health care sites with 1,500 more coming on-line each month [24]. A successful Internet-based diabetes management system should be a source of quality information for the patients who use it. The quality of information on the Internet is a source of great debate. The low barriers to publication on the Internet result in the presence of vast amounts of low-quality and inaccurate information. This misinformation or information that is out of date has the potential of misleading and even harming patients. Consequently, independent agencies such as the Health on the Net Foundation [25] were created to certify the content of medical information on the Internet. Information delivery is based on 2 models: pull and push.\nPull Model\nThe pull model relies on the patient retrieving the information he or she seeks. Two pathways are provided to this end. The patient can retrieve documents by navigating through the Web site or can retrieve information with a search engine.\nNavigation requires a clearly-defined information structure. This is effectively implemented with a hierarchical structure that users can follow to retrieve information of increasing level of detail. Navigation should be facilitated by a clear on-screen indication of the user's location in the information hierarchy.\nSearch engines allow users to search for documents based on keywords. Search engine technology is capable of cataloguing documents based on several criteria. In its simplest form, documents will be catalogued based on their text. Therefore, a search will yield all the documents containing the word that was searched for. However, a successful implementation of a search engine will categorize documents based on several criteria such as topic, author, date, and relevance. Users can then use these criteria to refine their searches.\nPush Model\nThe push model involves presenting the information to the patient who has opted to receive it. Relevant information could include new research or newly-released drugs for patients who have specified an interest. Interest can be formally expressed by the patient or can be inferred by the system in an effort to personalize the service (see the \"Personalization\" section of \"Evaluation Criteria\").\nInformation delivery in the push model can be implemented in several ways. Patients can be presented with the relevant information upon logging into the system. Alternatively, technologies such as mobile phones and pagers can be used for delivery. A successful Internet-based management system will implement both models of information delivery.\nPersonalization\nSelf-management Plan\nThe management of any chronic disease must be personalized to the individuals, as they are ultimately responsible for its success. Consequently, an Internet-based diabetes management system must allow patients to tailor the intervention to their specific needs. Patients benefit from a proactive approach to their management (in which they are not told what to do) and gain a valuable insight into the management options that may be available to them [17]. Patient involvement and contribution to disease management has demonstrated improved results and compliance [26].\nThe comprehensive management of diabetes can be based on several models. It is not the purpose of this paper to discuss these management models but rather their successful implementation as Internet-based diabetes management systems. One such model [17] (multilevel social-ecological model for self-management and support for behavior change) was implemented as a physical-activity intervention study [17]. This model is based on the creation of a personal action plan that is the result of both the patient's and health professional's requirements [27]. The creation of a personal action plan can be expressed as these self-management action steps: assessment and feedback, collaborative goal setting, identification of barriers and supports, individualized problem solving, follow-up support, and construction of a personal action plan. Glasgow and Bull have identified the strengths and limitations of interactive technologies such as the Internet for Self-Management Action Steps [17]. Nonetheless, a successful implementation of an Internet-based diabetes management system should provide the patient with the ability to navigate through each action step towards the creation of a personal action plan or the equivalent (depending on the disease-management model used).\nLanguage and Ethnicity\nPiette et al [28] demonstrated that an Automated Telephone Disease Management (ATDM) system produced positive results with an ethnically-diverse diabetic-patient population. Internet-based diabetes systems can reach different ethnicities by offering their services in multiple languages. In some groups where language may be a barrier to medical care, such systems may provide substantial benefits.\nInevitably, this opens the discussion of Internet demographics splitting patients between haves and have-nots. This is particularly relevant for type II Diabetes where some minority groups are disproportionately affected and have limited access to the Internet. However, the report from the National Telecommunications and Information Administration indicates a rapid change in Internet demographics that is reflective of the general population of the United States [29].\nCommunication\nCommunication Between Health Professional and Patient\nMost efforts in health care technology focus on assisting the doctor in diagnosing and treating a disease. This approach tends to omit a key component of the health care cycle: the patient. The new trend in medicine favors the inclusion of the patient as an integral part of the healing process. A review of 22 studies by Stewart et al [30] indicated a positive effect of communication on actual patient health outcome such as pain, recovery from symptom, anxiety, functional status, and physiologic measures of blood pressure and blood glucose.\nAn Internet-based diabetes management system must be a channel of communication between patients and their health care providers. The communication system can follow 3 models: synchronous, asynchronous, and indirect. Synchronous communication allows the patient and health care provider to communicate directly by using teleconferencing or videoconferencing. Traditionally, these services were in the realm of telemedicine [31] where specific technical equipment was installed to allow the communication to happen. However, the advent of multimedia on the Internet does allow for real-time voice-based and image-based communication. Although at its first steps, synchronous communication can be a valuable part of an Internet-based diabetes management system. Equally, the asynchronous communication model is a crucial part of a management system. Simple solutions such as secure text communication between patient and health care provider can be of great benefit in the management of diabetes. A study at the University of Pittsburgh describes a model of asynchronous communication between doctors and patients that reduced some of the differences in communication in terms of expectations, vocabulary used, and other factors [32]. This study was based on a communication system that allowed patients to familiarize themselves with the relevant domain terms at their own pace. The system also allowed physicians to request more information of patients while providing contextual information. This allowed patients to understand the underlying reasons for the questions.\nLastly, the indirect communication model is based on the concept of representation of the health care professional by technology. Such solutions have been implemented using software agents, a form of artificial intelligence that interacts with its environment and reacts to changes. In this case, the agent can interact with the patient and carry out a basic dialogue - and functions as information search and triage [33]. While still experimental, the use of indirect communication in Internet-based diabetes care shows great potential.\nCommunity Creation\nCommunity creation is based on a many-to-many communication channel compared with the one-to-one communication that occurs between health care professional and patient. Community support is a fundamental aspect of self-management of disease. Diabetes patients benefit from discussing topics that concern management of the disease, anxiety as to what the future holds, and interpersonal and social relationships.\nThe Internet can enable the creation of communities based on the same models of synchronous and asynchronous communication models. One study followed a diabetes chat room for 21 months and found that 79% of all respondents rated participation in the chat as having a positive effect on coping with diabetes [34]. Another study established a chat room for adolescents affected by diabetes and moderated by a diabetologist [35]. The results indicated a decrease in HbA 1cand an improved capacity for self-management. Anonymity undoubtedly favors a greater freedom of expression of individual problems. Community creation and maintenance should be an integral part of any Internet-based management systems. The implementation can be as synchronous chat rooms or as newsgroups where users communicate asynchronously by posting their comments. Further, experts can moderate chat rooms.\nTechnology\nThe complex network of human and machine relations involved in managing diabetes via an Internet-based system has strong implications for the design of such a service.\nSecurity\nOne of the main concerns with any medical informatics solution is security and privacy of the data. The success of any Internet-based diabetes management system is reliant on the user's trust that the user's data is secure, private, and confidential. This is possible with the recent availability of strong cryptographic tools used for 2 main purposes: authentication and encryption [23].\nAuthentication\nIdentification of users is a crucial step in gaining access to the system. Users are granted access to data based on their security profile. For example, only the treating physician can modify a specific patient's blood glucose ranges. Therefore, authentication is both the identification of a user (usually with a combination of username and password) and the enforcement of the security profile. Naturally, user identification is required for more-advanced functions like personalization as mentioned earlier.\nEncryption\nAll data transmitted between a patient and the system must be secure. Several encryption algorithms exist, with different strengths and speeds. Generally speaking, most Web servers can establish secure communication links using Netscape's Secure Socket Layer (SSL), which is de facto the Internet standard. Recently, 128-bit encryption has been made available worldwide. Any transmission of patient data should be encrypted at the highest level.\nUsability and User Acceptance\nTesting usability and user acceptance is a critical part of any computerized system and should be a continuous process during the life of the system. Typically, evaluation instruments have consisted of on-line questionnaires, on-line commenting (e-mail), telephone interviews, video-based testing, and tracking of system usage [36].\nMany physicians believe that the key success factor in managing diabetes is simplicity [1]. Consequently, the implementation of an Internet-based diabetes management system should strive towards simplicity for both patient and health care professional. Internet technologies can be a great supplement but if the implementation is not user-friendly, it can become a real barrier [1]. Although the technology has enormous potential, developers should not lose sight of the real purpose of these systems: to collect small amounts of data rapidly and efficiently. Therefore, an Internet-based diabetes management system will only be successful if implemented with a simple user interface used to collect the minimum amount of data from the patient (thus reducing its intrusiveness).\nReliability and Availability\nOne of the great advantages of the Internet is that it allows users to access systems anytime and from almost anywhere. This results in a need for systems to always be operational, that is, without downtime. Zero downtime (or close to it) requires fault-tolerant systems. Several technical solutions exist both at the software and hardware level. It is outside the scope of this paper to examine all the solutions; however, it is reasonable to expect an Internet-based diabetes management system to not require downtime for maintenance and to have a fault-tolerant hosting environment.\nOpen Platform\nOpen technologies are based on nonproprietary standards; therefore, a system can be built using technologies from multiple vendors. This is particularly useful for future expansions or medications to accommodate for increased scalability and functionality requirements. An Internet-based diabetes management system should be based on an open platform, particularly for data exchange. Open standards for data representation such as the eXtensible Markup Language (XML) are being adopted by multiple industries. Consequently, a system built using XML will be able to interface with multiple systems and devices. The same system could deliver its services via multiple devices (Internet, mobile phone, handheld computer, etc.) effectively making the Internet open platform the standard.\nEvaluation of 2 Existing Services\nTo illustrate the use of the evaluation framework, we have applied it to 2 existing Internet-based diabetes management systems: my Diabetes (www.myDiabetes.com) and LifeMasters (www.lifemasters.com).\nTo produce an overall evaluation, a criterion was considered satisfactory if the majority of the raters evaluated it positively (Yes rating). The results of the evaluations were numerically converted by assigning a value of 1 to all positive (Yes) ratings and a value of 0 to all negative (No) ratings. The results of all the evaluations are compiled in Table 3. The agreement level is reported for each individual criterion. This was calculated by dividing the number of ratings consistent with the overall rating (the majority) by the number of raters. For example, if a criterion was rated satisfactory or unsatisfactory by 4 out of the 5 raters, the criterion has an agreement level of 80% (4\/5).\nThe technology criteria registered the lowest agreement (60%-80%). The different levels of technical expertise of the evaluators may explain this difference. The Personalization criteria also showed lower levels of agreement between evaluators. This is due to the different interpretations of the criteria between evaluators. Personalization remains a difficult dimension to quantify and evaluate. The quality-of-information agreement levels were also low (60%-80%). Both sites displayed the HON code logo and stated that they subscribed to the HONCode principles. However, neither site was HON registered, although - as of December 14, 2001 - LifeMasters was under review process.\nThe multi-rater kappa for myDiabetes was 0.75 and for LifeMasters was 0.65, indicating a substantial level of agreement as defined by Landis and Koch [37]. There was an important difference between the kappa of MyDiabetes and the kappa of LifeMasters. Further testing is required to clarify the reasons for the difference.\nTable 3\nEvaluation Examples\nEvaluation Criteria\nmyDiabetes.com (Agreement Level)\nLifeMasters.com (Agreement Level)\nMonitoring\nUser defined parameters\nHealth care worker\nNo (100%)\nYes (100%)\nPatient\nYes (100%)\nYes (100%)\nUser defined parameter ranges\nHealth care worker\nNo (100%)\nYes (100%)\nPatient\nYes (100%)\nYes (100%)\nAutomated data collection\nNo (100%)\nNo (100%)\nAlert algorithms to avoid false alarms\nEntry validation\nYes (80%)\nYes (100%)\nScreening of symptoms\nYes (100%)\nYes (100%)\nPatient involvement in alert\nNo (100%)\nNo (100%)\nMultidisciplinary approach\nMultiple aspects of disease management monitored\nPhysical\nYes (100%)\nYes (100%)\nSocial\nYes (100%). Uses DQOL*\nYes (80%). Uses SF-36\nPsychological\nYes (100%). Uses DQOL\nYes (80%). Uses SF-36\nPatient access to multiple specialists\nNo (100%)\nYes (80%)\nProactive outreach\nNotification to patients\nYes (100%)\nYes (100%)\nNotifications to health care professionals\nNo (100%)\nYes (100%)\nFeedback\nRetrieve and review medical information\nYes (100%)\nYes (100%)\nRegular feedback\nYes (80%)\nYes (80%)\nInformation\nQuality of information\nYes (80%). Uses HON\nYes (60%). Uses HON\nPull\nNavigation\nYes (80%). Categorized\nYes (80%). Categorized\nSearch\nYes (100%)\nYes (100%)\nPush\nNotifications\nYes (100%)\nYes (100%)\nNewsletter subscription\nNo (100%)\nNo (100%)\nPersonalization\nAssessment and feedback\nYes (80%)\nYes (100%)\nCollaborative goal setting\nNo (100%)\nNo (80%)\nIdentification of barriers and supports\nNo (100%)\nYes (80%)\nFollow-up support\nNo (100%)\nYes (80%)\nConstruction of personalized management plan\nYes (80%)\nYes (80%)\nModification of management plan\nYes (100%)\nYes (100%)\nWeb site personalization\nYes (100%)\nYes (100%)\nLanguage and ethnicity\nNo (100%)\nNo (100%)\nCommunication\nHealth professional - patient\nSynchronous\nNo (100%)\nYes (80%)\nAsynchronous\nNo (100%)\nYes (80%)\nIndirect\nNo (100%)\nNo (100%)\nCommunity creation\nChat rooms\nYes (100%)\nNo (100%)\nNewsgroups \/ Forums\nYes (100%)\nYes (100%)\nExpert moderation\nYes (80%)\nYes (80%)\nTechnology\nSecurity\nAuthentication\nYes (100%). User and Password\nYes (100%). User and Password\nEncryption\nYes (100%). 128-bit\nYes (100%). 128-bit\nUsability and user acceptance\nYes (60%). Tested with forums\nNo (80%). Not actively tested\nReliability and availability\nNetscape compatible\nNetscape compatible\nOpen architecture\nNo (60%). IIS and ASP\nNo (60%). IIS and ASP\nTotal Positive Results\n25 out of 40\n32 out of 40\n* DQOL = Diabetes Quality of Life Measure\nFigure 3\nEvaluation of myDiabetes.com and LifeMasters.com. The value of each axis is normalized by conversion to a percentage of the maximum score\nGraphical Representation\nWe believe that a graphical representation of the evaluation results is particularly useful for comparing 2 systems and for determining in which direction the systems should expand their services. To this purpose, a radar graph with the 5 axes representing the 5 dimensions of Monitoring, Information, Personalization, Communication, and Technology is a useful representation. The value of each axis is normalized by conversion to a percentage of the maximum score. The evaluation of myDiabetes.com and LifeMasters.com is represented in Figure 3.\nThe results of the evaluation indicate that LifeMasters is a more-complete solution than myDiabetes in all dimensions - except Information, where both sites were equivalent. This is primarily due to LifeMaster's inclusion of the health care professional in the disease-management cycle. On the other hand, myDiabetes is uniquely interfaced with the patient and is quite good in providing a communication channel for community creation, however, communication with health care professional is lacking, hence the lower score than LifeMasters.\nDiscussion\nThe Internet will undoubtedly change the way we deliver health care services. Chronic disease management, which accounts for 60% of the U.S. medical care costs [38], is a desirable target for the efficiencies of the Internet. Chronic-disease management on the Internet is estimated to have a market potential of US $700 billion [24]. Already we are seeing several Internet-based chronic-disease-management sites arising; however, there is little evidence as to how these solutions are answering the needs of the consumer (the patient).\nConsumer health informatics research greatly contributes to the health care sector by attempting to systematize and codify consumer's needs, values, and preferences and by trying to build and evaluate information systems that interact directly with consumers and patients [39]. In this paper, we have attempted to catalogue the critical success factors for an Internet-based diabetes management system based on the available literature and the authors' experience. The result is a first step towards a comprehensive evaluation framework. The framework is based on the recognition that the management of diabetes via the Internet is based on several integrated dimensions, namely, Monitoring, Information, Personalization, Communication, and Technology. A successful diabetes management system should efficiently integrate all dimensions. Therefore, the framework provides a model for evaluation and, more importantly, for strategic growth planning for existing sites. For example, a site that is deficient in the communication dimension may enhance its offerings by adding a synchronous chat room.\nThis paper reports an initial evaluation of 2 sites. The results indicate a high-level inter-rater agreement as measured by Cohen's multi-rater kappa. However, this is based on a small sample of evaluations (5). Future research should focus on validation of the framework by consistency between larger samples of raters and on correlation with the success of the multiple sites available today. Key metrics for success include the number of enrolled patients; length of time managed; clinical, economic, and quality-of-life outcomes; and patient-satisfaction measures [24].","keyphrases":["evaluation","internet","diabetes","therapy","chronic disease management"],"prmu":["P","P","P","P","P"]}
{"id":"Neuroradiology-3-1-2082067","title":"Endovascular occlusion of high-flow intracranial arteriovenous shunts: technical note\n","text":"Endovascular closure of high-flow arteriovenous (AV) shunts in intracranial AV malformations or pial fistulas is technically challenging. In this paper, we illustrate two simple methods to occlude large high-flow AV shunts in a controlled manner.\nIntroduction\nEndovascular closure of high-flow arteriovenous (AV) shunts in intracranial AV malformations (AVM) or pial fistulas is technically challenging [1\u20133]. With the use of liquid embolic agents, the high flow through the shunt may cause migration of the embolic agent to the venous outflow tract with possible devastating consequences. In anatomical configurations with a very short arterial feeder, the shunt can only be occluded at the venous side. In this paper, we illustrate two simple methods to occlude large AV shunts in a controlled manner applied in three patients.\nCase 1\nA 26-year-old man was referred for treatment after haemorrhage of a large callosal AVM (Fig.\u00a01). Angiography demonstrated two high-flow AV shunts from the right anterior cerebral artery draining into enormously enlarged veins. Injection of Onyx 34 at the site of the largest shunt resulted in immediate migration to the venous side. Subsequently, a nondetachable microballoon (Magic B2, Balt, Montmorency, France) was flow-directed into the main feeder just proximal to the shunt and inflated to block the flow. At that time, Onyx injection was continued and resulted in gradual occlusion of the fistula in 7\u00a0min. Next, the second high-flow shunt was occluded in a similar way. Control angiography confirmed complete occlusion of both AV shunts. A second embolization procedure was scheduled to be performed 3\u00a0months after the first.\nFig.\u00a01A 26-year-old man with large callosal AVM. a Frontal and lateral right internal carotid angiogram shows an AVM with two high-flow AV shunts from the anterior cerebral artery (arrows). b Lateral subtracted fluoroscopy image obtained during injection of Onyx with a balloon catheter blocking the flow. The arrow points to the distal tip marker of the microcatheter delivering the Onyx. Onyx is deposited at the transition of artery to vein. c Lateral subtracted fluoroscopy image obtained during occlusion of the second shunt\nCase 2\nA 12-year-old boy was referred for treatment of a left frontal AVM discovered on MRI performed for seizures. Angiography under general anaesthesia demonstrated, besides a plexiform part of the nidus, two large AV shunts from the anterior cerebral artery (Fig.\u00a02). The largest shunt had a very short arterial feeder that precluded the use of Onyx, since reflux would have led to migration into the normal vasculature. A microcatheter was navigated through the shunt into the proximal venous part and detachable coils were inserted until the shunt was completely occluded. Next, the second AV shunt was occluded with simple injection of Onyx 34 and continued injection occluded the major part of the nidus. After embolization, still under general anaesthesia, the patient was transferred to the Gamma Knife unit and the small AVM remnant was irradiated.\nFig.\u00a02A 12-year-old boy with a left frontal AVM. a Lateral angiogram of the left internal carotid artery shows a large AV shunt with a very short arterial segment (arrow). b Lateral angiogram obtained after occlusion of the venous and arterial side of the shunt with detachable coils\nCase 3\nA 27-year-old woman was referred for treatment of an intracranial vascular disorder that had been discovered on CT scan performed for chronic headaches. Vertebral angiography revealed a high-flow single-hole side-wall pial fistula from the P3\u2013P4 segment of the right posterior cerebral artery draining into a dilated basal vein of Rosenthal (Fig.\u00a03). In addition, a flow aneurysm on the right superior cerebellar artery was present. A microcatheter was navigated through the shunt into the vein and the proximal part of the vein was occluded with detachable coils with preservation of continuity of the posterior cerebral artery. Subsequently, the flow aneurysm was occluded with coils. A follow-up angiogram obtained 6\u00a0months later showed continuing closure of the fistula and the aneurysm.\nFig.\u00a03A 27-year-old woman with pial fistula. a Frontal vertebral angiogram shows a high-flow single-hole side-wall pial fistula from the P3\u2013P4 segment of the right posterior cerebral artery draining into a dilated basal vein of Rosenthal. In addition, a flow aneurysm of the superior cerebellar artery is present (arrow). b Follow-up angiogram obtained 6\u00a0months after occlusion of the venous side of the fistula with detachable coils shows continuing occlusion of the fistula with preserved patency of the posterior cerebral artery\nDiscussion\nThe most commonly used embolic agent in brain AVMs is the fast polymerizing liquid adhesive n-butylcyanoacrylate (NBCA, Histoacryl; Braun, Melsungen, Germany). Pure NBCA polymerizes immediately after contact with blood. Slower polymerization is achieved when NBCA is mixed with iodinated oil (Lipiodol; Guerbet, Roissy, France). The use of a high concentration of NBCA or pure NBCA in high-flow shunts in brain AVMs may occlude the fistula within seconds, but this technique requires courage, experience and skill, since flow of NBCA is to some extent unpredictable [3]. Inadvertent migration of the glue into the draining veins may result in immediate haemorrhage by blocking venous outflow. Detachable coils may be inserted at the fistula site to decrease the flow and thereby facilitate the injection of NBCA [2]. Recently, the new liquid embolic agent Onyx has become available for embolization of brain AVMs (Onyx Liquid Embolic System; Microtherapeutics, Irvine, Calif.). Onyx is nonadhesive and polymerizes slowly. Onyx is available in several concentrations and the high-concentrated Onyx can be used to slowly occlude large AV shunts or pial AV fistulas in a more controlled way than that achieved with NBCA [4]. However, simple Onyx injection is not always feasible: in some very high-flow shunts Onyx may migrate through the fistula into the distal draining veins. We found the adjuvant use of a microballoon to block the flow to enable gradual occlusion of the large high-flow shunts with Onyx in the AVM of patient 1 a very useful approach.\nIn the other patients, one with a very short arterial feeder of an AV shunt in an AVM and one with a side-wall pial fistula, the use of liquid embolic agents was not possible and controlled delivery of detachable coils to the venous side of the shunt was technically easy with complete occlusion of the fistula. This technique is helpful in shunts with a moderately dilated venous outflow tract in which placement of coils is possible.\nIn conclusion, different types of high-flow AV shunts can safely be occluded with endovascular techniques tailored to the specific anatomical configuration of the shunt.","keyphrases":["pial fistula","av shunt","avm"],"prmu":["P","P","P"]}
{"id":"J_Biol_Inorg_Chem-3-1-2039866","title":"Zinc complexes of the biomimetic N,N,O ligand family of substituted 3,3-bis(1-alkylimidazol-2-yl)propionates: the formation of oxalate from pyruvate\n","text":"The coordination chemistry of the 2-His-1-carboxylate facial triad mimics 3,3-bis(1-methylimidazol-2-yl)propionate (MIm2Pr) and 3,3-bis(1-ethyl-4-isopropylimidazol-2-yl) propionate (iPrEtIm2Pr) towards ZnCl2 was studied both in solution and in the solid state. Different coordination modes were found depending both on the stoichiometry and on the ligand that was employed. In the 2:1 ligand-to-metal complex [Zn(MIm2Pr)2], the ligand coordinates in a tridentate, tripodal N,N,O fashion similar to the 2-His-1-carboxylate facial triad. However, the 1:1 ligand-to-metal complexes [Zn(MIm2Pr)Cl(H2O)] and [Zn(iPrEtIm2Pr)Cl] were crystallographically characterized and found to be polymeric in nature. A new, bridging coordination mode of the ligands was observed in both structures comprising N,N-bidentate coordination of the ligand to one zinc atom and O-monodentate coordination to a zinc second atom. A rather unique transformation of pyruvate into oxalate was found with [Zn(MIm2Pr)Cl], which resulted in the isolation of the new, oxalato bridged zinc coordination polymer [Zn2(MIm2Pr)2(ox)]\u00b76H2O, the structure of which was established by X-ray crystal structure determination.\nIntroduction\nThe 2-His-1-carboxylate facial triad is a structural motif of increasing prominence among structurally characterized metalloenzymes. Next to the rapidly growing subgroup of non-heme iron enzymes [1, 2], several mononuclear zinc enzymes also feature this triad at their active site [3]. The proteases thermolysin, carboxypeptidase, and neutral protease, for example, catalyze the hydrolysis of peptide bonds. The zinc ion in these enzymes is coordinated by three endogenous residues, i.e., one glutamate and two histidine residues (Fig.\u00a01). The fourth coordination site of the pseudo-tetrahedral zinc center is occupied by a catalytically important water or hydroxide ligand. This 2-His-1-carboxylate facial triad is a variation of the most commonly observed structural motif for the active sites of zinc-containing enzymes. Typically, the tetrahedral zinc centers in these enzymes are bound by a combination of histidine (N), glutamate or aspartate (O) and\/or cysteine (S) residues. The resulting NxOySz donor set determines its biological function [3]. Accordingly, it is important to understand how a particular combination of donor groups modulates the reactivity of the Zn(II) metal center. Fig.\u00a01Active site of a zinc-containing enzyme that features the 2-His-1-carboxylate facial triad: neutral protease from Bacillus cereus (Protein Data Bank accession code 1NPC.pdb) [4]\nThe study of small synthetic analogues has focused on establishing a basis for such a structure\u2013function relationship for these mononuclear zinc-containing enzymes [3, 5]. Initially, pioneering studies by the groups of Vahrenkamp, Parkin, Kitajima, and others employed all-N donor ligands such as the trispyrazolylborates [6\u20138]. In order to obtain close structural models for the 2-His-1-carboxylate facial triad containing zinc enzymes, one would preferentially like to construct small analogues based on a tripodal N,N,O ligand framework that incorporates the biologically relevant donor groups. Of the complexes with a mixed N\/O donor set reported to date [9\u201322], only a few make use of such tridentate, tripodal N,N,Ocarboxylato ligands. These tripodal ligands should be particularly well suited for the structural modeling of tetrahedral metal centers [23]. Dowling and Parkin [24] and Ghosh and Parkin [25] reported the first example of an N,N,O model complex with a carboxylato donor group, which resulted from the insertion of carbon dioxide into a borohydride bond. More recently, the groups of Burzlaff [9, 12] and Carrano [11, 18, 19] have reported studies on the zinc coordination chemistry of the bispyrazolylacetate ligand family. Beck et al. [9], for instance, reported that a 2:1 ligand\u2013zinc complex was obtained with bis(3,5-dimethylpyrazol-2-yl)acetate (bdmpza), a ligand closely related to 3,3-bis(1-methylimidazol-2-yl)propionate (MIm2Pr). These 2:1 complexes were obtained regardless of the ratio of reactants, i.e., even with stoichiometric amounts of ligand and zinc(II). Very recently, Friese et al. [22] constructed mononuclear zinc models via a new approach, i.e., by the use of sterically hindered carboxylato and N-donor ligand building blocks.\nWe have been studying the copper and iron coordination chemistry of the new ligand family of the substituted 3,3-bis(1-alkylimidazol-2-yl)propionates as accurate structural mimics of the 2-His-1-carboxylate facial triad [26\u201328]. These ligands incorporate the biologically relevant donor groups, i.e., two imidazoles and a carboxylate group, into a monoanionic, tripodal framework. Their general biomimetic potential has been illustrated by the structurally characterized copper [27, 28] and iron [26] complexes of these ligands. Here, our studies are expanded to the structural modeling of the zinc-containing enzymes and describe the zinc coordination chemistry of the ligands MIm2Pr and 3,3-bis(1-ethyl-4-isopropylimidazol-2-yl)propionate (iPrEtIm2Pr) (Fig.\u00a02). Furthermore, the attempted synthesis of zinc complexes with 3,3-bis(1-methylimidazol-2-yl)acetate (MIm2Ac), a direct analogue of the bispyrazolylacetates, is described. During the course of these studies, we found that zinc complexes of ligand MIm2Pr catalyze the unexpected and unprecedented conversion of pyruvate to oxalate in aqueous solution.Fig.\u00a02Ligands 3,3-bis(1-methylimidazol-2-yl)propionate (MIm2Pr), 3,3-bis(1-ethyl-4-isopropylimidazol-2-yl)propionate (iPrEtIm2Pr), and 3,3-bis(1-methylimidazol-2-yl)acetate (MIm2Ac) and the synthesis of the zinc complexes [Zn(MIm2Pr)2], [Zn(MIm2Pr)Cl], and [Zn(iPrEtIm2Pr)Cl]\nMaterials and methods\nAir-sensitive organic reactions were carried out under an atmosphere of dry, oxygen-free N2 using standard Schlenk techniques. Tetrahydrofuran (THF) and diethyl ether were dried over sodium benzophenone ketyl and distilled under N2 prior to use. Methanol was dried over magnesium methoxide and distilled under N2 prior to use. 1H and 13C NMR spectra were recorded using a Varian AS400, a Varian Inova 300, or a Varian Mercury 200 spectrometer, operating at 25\u00a0\u00b0C. IR spectra were recorded with a PerkinElmer Spectrum One Fourier transform IR instrument. Elemental microanalyses were carried out by the Microanalytisches Laboratorium Dornis & Kolbe, Mulheim a.d. Ruhr, Germany. ESI\u2013MS spectra were recorded using a Micromass liquid chromatography time-of-flight mass spectrometer at the Biomolecular Mass Spectrometry Group, Utrecht University. Bis(1-methylimidazol-2-yl)methane (MIm2CH2) [29,] 3,3-bis(1-methylimidazol-2-yl)propionic acid (HMIm2Pr), potassium and tetrabutylammonium 3,3-bis(1-methylimidazol-2-yl)propionate (K[MIm2Pr] and [Bu4N][MIm2Pr]) [27], potassium 3,3-bis(1-ethyl-4-isopropylimidazol-2-yl)propionate (K[iPrEtIm2Pr]) [26], and zinc bis(trimethylsilyl)amide [30] were prepared according to published procedures. All other chemicals were obtained commercially and used as received.\nBenzyl bis(1-methylimidazol-2-yl)acetate\nTo a cooled solution of MIm2CH2 (0.52\u00a0g, 3.0\u00a0mmol) in dry THF (50\u00a0mL) at \u221278\u00a0\u00b0C was added dropwise a solution of n-butyl lithium in hexane (2.0\u00a0mL, 3.2\u00a0mmol). The reaction mixture was stirred for 1\u00a0h at \u221278\u00a0\u00b0C, after which benzylchloroformate (1.19\u00a0mL, 3.5\u00a0mmol, 50\u00a0wt% solution in toluene) was added dropwise. During the addition, a white precipitate formed. The solution was allowed to warm to room temperature overnight. The reaction mixture was quenched with H2O (20\u00a0mL) and all volatiles were removed in vacuo. The aqueous layer was extracted with diethyl ether (4\u00a0\u00d7\u00a020\u00a0mL) and the combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The crude oil was purified by column chromatography (SiO2, ethyl acetate\u2013methanol 2:1). Benzyl bis(1-methylimidazol-2-yl)acetate (BnMIm2Ac) was isolated as a yellow oil in 62% yield (0.58\u00a0g, 1.9\u00a0mmol). 1H NMR (300\u00a0MHz, CD3CN, 25\u00a0\u00b0C): \u03b4\u00a0=\u00a03.44 (s, 6H, NCH3), 5.23 (s, 2H, CH2), 5.62 (s, 1H, CH), 6.85 (s, 2H, Him), 6.96 (s, 2H, Him), 7.35 (s, 5H, HPh) ppm. Anal. for C17H18N4O2 (310.35): calc. C 65.79, H 5.85, N 18.05; found C 65.88, H 5.74, N 17.92. IR (solid): \u03bd\u00a0=\u00a03,122.9, 2,911.5, 1,734.5, 1,547.4, 1,510.2, 1,454.4, 1,373.6, 1,263.4, 1,220.8, 1,150.4, 978.2, 958.3, 907.1, 847.8, 785.7, 776.5, 740.1, 696.7, 683.5, 665.3\u00a0cm\u22121. ESI\u2013MS: m\/z\u00a0=\u00a0409.0 ([M\u2013Cl]+, calc. 409.0).\n[Zn(MIm2Pr)2]\nTo a hot, colorless solution of HMIm2Pr (282\u00a0mg, 1.20\u00a0mmol) in dry acetonitrile (100\u00a0mL) was added a solution of Zn[N(SiMe3)2]2 (232\u00a0mg, 0.60\u00a0mmol) in dry diethyl ether (20\u00a0mL) via a cannula. The resulting colorless, clear solution was stirred for 60\u00a0h at elevated temperature, during which gradually a white precipitate formed. The reaction mixture was concentrated in vacuo to give the crude product as a white powder. Recrystallization from an acetone\u2013water mixture (4:1 v\/v) gave [Zn(MIm2Pr)2] as a colorless crystalline solid (230\u00a0mg, 72%). 1H NMR (300\u00a0MHz, D2O, 25\u00a0\u00b0C): \u03b4\u00a0=\u00a02.69 (d, 2H, J\u00a0=\u00a06.3\u00a0Hz, CHCH2), 3.74 (s, 6H, NCH3), 4.93 (t, 1H, J\u00a0=\u00a06.0\u00a0Hz, CHCH2), 6.69 (s, 2H, Him), 7.00 (s, 2H, Him) ppm. 13C{1H} NMR (50\u00a0MHz, D2O, 25\u00a0\u00b0C): \u03b4\u00a0=\u00a030.2, 33.4, 43.6, 122.7, 125.7, 146.8, 177.2\u00a0ppm. Anal. for C22H26ZnN8O4 (531.90): calc. C 49.68, H 4.93, N 21.07; found C 49.76, H 5.06, N 20.97. IR (solid): \u03bd\u00a0=\u00a03,120.2, 2,951.7, 2,907.1, 2,815.2, 1,580.5, 1,508.1, 1,427.1, 1,391.0, 1,306.4, 1,288.0, 1,229.3, 1,168.8, 1,143.3, 1,044.8, 956.9, 906.2, 769.3, 751.1\u00a0cm\u22121. ESI\u2013MS: m\/z\u00a0=\u00a0531.18 ([M\u00a0+\u00a0H]+, calc. 531.14), 553.18 ([M+Na]+, calc. 553.13), 569.16 ([M\u00a0+\u00a0K]+, calc. 569.10).\n[Zn(MIm2Pr)Cl]\nTo a hot, colorless solution of K[MIm2Pr] (255\u00a0mg, 0.93\u00a0mmol) in dry methanol (5\u00a0mL) was added a solution of ZnCl2 (127\u00a0mg, 0.93\u00a0mmol) in methanol (5\u00a0mL) via a cannula. Immediately upon addition a white precipitate formed and the suspension was stirred at elevated temperature for 1\u00a0h. The white precipitate was separated by centrifugation and washed with methanol (3\u00a0\u00d7\u00a020\u00a0mL). [Zn(MIm2Pr)Cl] was obtained as a white powder in almost quantitative yield (305\u00a0mg, 98%). 1H NMR (300\u00a0MHz, D2O, 25\u00a0\u00b0C): \u03b4\u00a0=\u00a02.80 (d, 3H, J\u00a0=\u00a03.3\u00a0Hz, CHCH2), 3.85 (s, 6H, NCH3), 4.99 (t, 1H, CH2CH), 6.99 (s, 2H, Him), 7.14 (s, 2H, Him) ppm. 13C{1H} NMR (50\u00a0MHz, D2O, 25\u00a0\u00b0C): \u03b4\u00a0=\u00a029.8, 33.4, 42.6, 122.5, 125.5, 146.1, 177.3\u00a0ppm. Anal. for C11H13ClN4O2Zn (334.11): calc. C 39.55, H 3.92, N 16.77; found: C 39.46, H 4.05, N 16.85. IR (solid): \u03bd\u00a0=\u00a03,138.0, 3,117.2, 1,614.8, 1,508.2, 1389.8, 1,299.8, 1,289.5, 1,151.7, 1,142.5, 1,089.7, 976.2, 949.7, 796.3, 765.3, 736.1\u00a0cm\u22121. ESI\u2013MS: m\/z\u00a0=\u00a0296.94 ([M\u2013Cl]+, calc. 297.03), 332.92 ([M\u00a0+\u00a0H]+, calc. 333.01), 413.97 ([3M\u2013Zn\u20133Cl\u00a0+\u00a0H]2+, calc. 414.09), 531.02 ([2M\u2013Zn\u20132Cl\u00a0+\u00a0H]+, calc. 531.15), 628.97 ([2M\u2013Cl]+, calc. 629.03), 827.10 ([3M\u2013Zn\u20133Cl]+, calc. 827.17).\n[Zn(MIm2Pr)Cl(H2O)]\nColorless crystals of [Zn(MIm2Pr)Cl(H2O)] suitable for X-ray diffraction were obtained upon standing of a concentrated solution of [Zn(MIm2Pr)Cl] in H2O for several weeks. The coordination polymer obtained is insoluble in all common organic solvents and water of neutral pH. Anal. for C11H15ClN4O3Zn (352.12): calc. C 37.52, H 4.29, 15.91; found C 37.44, H 4.27, N 15.97. IR (solid): \u03bd\u00a0=\u00a03,408.6, 3,113.5, 3,134.7, 1,594.5, 1,504.5, 1,403.1, 1,306.7, 1,255.5, 1,155.5, 1,136.3, 1,087.4, 983.7, 959.1, 934.9, 849.7, 764.6, 743.1, 717.6\u00a0cm\u22121.\n[Zn(iPrEtIm2Pr)Cl]\nTo a colorless solution of ZnCl2 (30\u00a0mg, 0.22\u00a0mmol) in dry methanol (10\u00a0mL) was added a solution of K[iPrEtIm2Pr] (85\u00a0mg, 0.22\u00a0mmol) in dry methanol (15\u00a0mL) via a cannula. The clear solution was stirred overnight at room temperature and evaporated in vacuo. The product was redissolved in dichloromethane and insoluble KCl was separated off by centrifugation. The solution was filtered over Celite and concentrated in vacuo to give [Zn(iPrEtIm2Pr)Cl] as a white powder (97\u00a0mg, 99%). Colorless crystals suitable for X-ray diffraction were obtained from a methanolic solution of [Zn(iPrEtIm2Pr)Cl] upon standing. 1H NMR (300\u00a0MHz, D2O, 25\u00a0\u00b0C): \u03b4\u00a0=\u00a01.22 (d, 6H, J\u00a0=\u00a07.2\u00a0Hz, CH3CHCH3), 1.25 (d, 6H, J\u00a0=\u00a06.8\u00a0Hz, CH3CHCH3), 1.44 (t, 6H, J\u00a0=\u00a07.2\u00a0Hz, CH3CH2), 2.88 (d, 2H, J\u00a0=\u00a06.0\u00a0Hz, CHCH2), 3.25 (m, 2H, CH3CHCH3), 4.21 (ABX3, 2H, J\u00a0=\u00a07.2, CHHCH3), 4.32 (ABX3, 2H, J\u00a0=\u00a07.2, CHHCH3), 5.02 (t, 1H, J\u00a0=\u00a05.7\u00a0Hz, CHCH2), 6.99 (s, 2H, Him) ppm. 13C{1H} NMR (100\u00a0MHz, CD3OD, 25\u00a0\u00b0C): \u03b4\u00a0=\u00a016.6, 22.1, 24.1, 28.0, 31.1, 42.9, 116.0, 146.2, 149.9, 175.7\u00a0ppm. Anal. for C19H29ClN4O2Zn (446.32): calc. C 51.13, H 6.55, N 12.55; found: C 51.05, H 6.43, N 12.37. IR (solid): \u03bd\u00a0=\u00a03,101.4, 2,967.9, 2,873.7, 1,626.6, 1,572.8, 1,494.8, 1,448.9, 1,464.1, 1,381.1, 1,324.8, 1,257.2, 1,175.8, 1,153.0, 1,027.6, 976.1, 801.2, 757.7, 660.5\u00a0cm\u22121. ESI\u2013MS: m\/z\u00a0=\u00a0409.13 ([2M\u20132Cl]2+, calc. 409.16), 445.11 ([M\u00a0+\u00a0H]+, calc. 445.13), 483.04 ([M\u00a0+\u00a0K]+, calc. 483.09), 631.21 ([3M\u20132Cl]2+, calc. 631.22), 853.29 ([2M\u2013Cl]+, calc. 853.29), 889.20 ([2M\u00a0+\u00a0H]+, calc. 889.26), 1297.02 ([3M\u00a0+\u00a0H]+, calc. 1297.42).\n[Zn(BnMIm2Ac)Cl2]\nTo a hot solution of BnMIm2Ac (259\u00a0mg, 0.84\u00a0mmol) in dry methanol (5\u00a0mL) was added a solution of anhydrous ZnCl2 (115\u00a0mg, 0.84\u00a0mmol) in dry MeOH (5\u00a0mL) via a cannula. The reaction mixture was stirred at elevated temperature for 1\u00a0h and gradually the product formed as a white precipitate. The product [Zn(BnMIm2Ac)Cl2] was separated by centrifugation and was obtained as a white powder in quantitative yield. Single crystals suitable for X-ray analysis were obtained by slow evaporation of a solution of [Zn(BnMIm2Ac)Cl2] in acetone. 1H NMR (300\u00a0MHz, CD3CN, 25\u00a0\u00b0C): \u03b4\u00a0=\u00a03.79 (2, 6H, CH3), 5.12 (s, 2H, CH2), 5.60 (s, 1H, CH), 7.13 (d, 2H, J\u00a0=\u00a00.75\u00a0Hz, Him), 7.22 (d, 2H, J\u00a0=\u00a00.75\u00a0Hz, Him), 7.25 (m, 2H, HPh), 7.33 (m, 3H, HPh) ppm. 13C{1H} NMR (100\u00a0MHz, CD3OD, 25\u00a0\u00b0C): \u03b4\u00a0=\u00a034.9, 40.6, 69.7, 124.5, 126.5, 129.1, 129.5, 129.7, 136.1, 142.3, 166.2\u00a0ppm. Anal. for C17H18Cl2N4O2Zn (446.67): calc. C 45.71, H 4.06, N 12.54; found C 45.64, H 3.95, N 12.61. IR (solid): \u03bd\u00a0=\u00a03,122.9, 2,911.5, 1,734.5, 1,547.4, 1,510.2, 1,454.4, 1,373.6, 1,263.4, 1,220.8, 1,150.4, 978.2, 958.3, 907.1, 847.8, 785.7, 776.5, 740.1, 696.7, 683.5, 665.3\u00a0cm\u22121. ESI\u2013MS: m\/z\u00a0=\u00a0409.0 ([M\u2013Cl]+, calc. 409.0).\n[Zn2(MIm2Pr)2(ox)] (direct synthesis)\nTo a solution of [Zn(MIm2Pr)Cl] (50\u00a0mg, 0.15\u00a0mmol) in water (5\u00a0mL) was added a solution of potassium oxalate hydrate (14\u00a0mg, 0.075\u00a0mmol) in water (3\u00a0mL). The solution was stirred overnight at room temperature, during which gradually a white precipitate formed. The precipitate was separated by centrifugation and washed three times with water (3\u00a0\u00d7\u00a020\u00a0mL). The product [Zn2(MIm2Pr)2(ox)] was dried in vacuo and obtained as a white powder in 82% yield (42\u00a0mg, 0.061\u00a0mmol). Anal. for C24H26N8O8Zn2\u00b76H2O (685.33): calc. C 36.33, H 4.83, N 14.12; found: C 36.45, H 4.50, N 14.25. IR (solid): \u03bd\u00a0=\u00a03,476.8, 3,135.4, 1,671.3, 1,651.6, 1,600.2, 1,544.9, 1,504.8, 1,423.6, 1,382.7, 1,320.1, 1,287.2, 1,158.1, 1,137.9, 985.1, 769.4, 745.1, 710.4\u00a0cm\u22121.\n[Zn2(MIm2Pr)2(ox)]\u00b76H2O, reaction of [Zn(MIm2Pr)Cl] with sodium pyruvate\nFor NMR investigation of reaction mixtures, stock solutions of [Zn(-MIm2Pr)Cl] (20\u00a0mg, 0.06\u00a0mmol) in H2O (1\u00a0mL) and sodium pyruvate (13.2\u00a0mg, 0.06\u00a0mmol) in H2O (2\u00a0mL) were prepared. NMR tubes were filled with 0.1\u00a0mL of each stock solution and kept either at room temperature or heated to 50\u00a0\u00b0C. The NMR samples were analyzed at different times after mixing the reagents. Before acquisition of the NMR spectrum, 0.4\u00a0mL D2O was added to the reaction mixture. Crystals usually deposited within 2 or 3\u00a0days in the NMR tube.\nIn the case of the catalytic runs, 0.1\u00a0mL of a solution containing 20 equiv of sodium pyruvate was added to 0.1\u00a0mL of the [Zn(MIm2Pr)Cl] stock solution and dimethyl sulfoxide (5\u00a0equiv, 11\u00a0\u03bcL) was added as an internal standard. The reaction mixture was kept at 50\u00a0\u00b0C and the NMR spectrum was recorded with addition of 0.4\u00a0mL D2O after 24\u00a0h.\nOn a preparative scale, to a solution of ZnCl2 (37\u00a0mg, 0.27\u00a0mmol) and K[MIm2Pr] (75\u00a0mg, 0.27\u00a0mmol) in H2O (10\u00a0mL) was added sodium pyruvate (30\u00a0mg, 0.27\u00a0mmol). The solution was heated to 50\u00a0\u00b0C and gradually an off-white precipitate was formed. After 1\u00a0week, the product was separated by centrifugation (29\u00a0mg, 31%). The IR spectrum of the product was identical to that of independently synthesized [Zn2(MIm2Pr)2(ox)].\nX-ray crystal structure determinations of [Zn(MIm2Pr)Cl(H2O)], [Zn(iPrEtIm2Pr)Cl], ZnCl2(BnMIm2Ac)], [ZnCl2(MIm2CH2)], and [Zn2(MIm2Pr)2(ox)]\u00b76H2O\nReflections were measured with a Nonius Kappa CCD diffractometer with a rotating anode (graphite monochromator, \u03bb\u00a0=\u00a00.71073\u00a0\u00c5) at \u2212123\u00a0\u00b0C. The structures were refined with SHELXL-97 [31] against F2 of all reflections. Non-hydrogen atoms were refined with anisotropic displacement parameters. Geometry calculations and checking for higher symmetry were performed with the PLATON program [32]. Further details are given in Table\u00a01. Table\u00a01Crystallographic data for compounds [Zn(MIm2Pr)Cl(H2O)], [Zn(iPrEtIm2Pr)Cl], ZnCl2(BnMIm2Ac)], [ZnCl2(MIm2CH2)], and [Zn2(MIm2Pr)2(ox)]\u00b76H2OCompound[Zn(MIm2Pr)Cl(H2O)][Zn(iPrEtIm2Pr)Cl] [ZnCl2(BnMIm2Ac)][ZnCl2(MIm2CH2)][Zn2(MIm2Pr)2(ox)]\u00b76H2O FormulaC11H15ClN4O3ZnC19H29ClN4O2ZnC17H18Cl2N4O2ZnC9H12Cl2N4ZnC24H26N8O8Zn2\u00b76H2OFW352.09446.28446.62312.50793.36Crystal size (mm3)0.30\u00a0\u00d7\u00a00.12\u00a0\u00d7\u00a00.090.18\u00a0\u00d7\u00a00.03\u00a0\u00d7\u00a00.030.60\u00a0\u00d7\u00a00.03\u00a0\u00d7\u00a00.030.30\u00a0\u00d7\u00a00.15\u00a0\u00d7\u00a00.060.24\u00a0\u00d7\u00a00.18\u00a0\u00d7\u00a00.09Crystal colorColorlessColorlessColorlessColorlessColorlessCrystal systemOrthorhombicMonoclinicMonoclinicMonoclinicTriclinicSpace groupP212121 (no. 19)Cc (no. 9)C2\/c (no. 15)P21\/c (no. 14)a (\u00c5)8.7159(1)10.1766(2)35.5761(4)7.3547(1)9.9634(7)b (\u00c5)10.1124(1)23.8561(6)7.4531(1)14.9538(2)12.0617(11)c (\u00c5)15.1177(2)9.1995(2)14.6106(2)12.3489(2)14.8889(9)\u03b1 (\u00b0)\u2013\u2013\u2013\u2013113.021(3)\u03b2 (\u00b0)\u2013107.0078(14)101.8858(5)117.5968(6)90.365(3)\u03b3 (\u00b0)\u2013\u2013\u2013\u2013102.513(3)V (\u00c5)1332.45(3)2135.72(8)3790.97(8)1203.62(3)1599.4(2)Z44842Dcalc. (g\u00a0cm\u22123)1.7551.3881.5651.7241.647\u03bc (mm\u22121)2.0561.2961.5972.4611.580Absorption correctionMultiscanMultiscanMultiscanMultiscanMultiscanAbsorption correction range0.71\u20130.840.86\u20130.960.85\u20130.960.74\u20130.860.64\u20130.87sin(\u03b8\/\u03bb)max (\u00c5\u22121)0.650.650.600.650.65Reflections (measured\/unique)21,618\/3,06311,758\/4,64428,566\/3,42427,341\/2,75935,709\/7,342Parameters\/restraints241\/0251\/2237\/0193\/0438\/0R1\/wR2 [I\u00a0>\u00a02\u03c3(I)]0.0182\/0.04470.0312\/0.06940.0309\/0.07040.0306\/0.07930.0389\/0.0867R1\/wR2 (all reflections)0.0190\/0.04520.0384\/0.07230.0512\/0.07860.0389\/0.08520.0540\/0.0947S1.0621.0411.0811.0891.083Flack parameter [37]\u22120.006(7)0.305(9)\u2013\u2013\u2013Residual density (e\u00a0\u00c5\u22123)\u22120.36\/0.19\u22120.27\/0.41\u22120.31\/0.43\u22120.54\/1.23\u22120.79\/0.51MIm2Pr 3,3-bis(1-methylimidazol-2-yl)propionate, iPrEtIm2Pr 3,3-bis(1-ethyl-4-isopropylimidazol-2-yl)propionate, and BnMIm2Ac benzyl bis(1-methylimidazol-2-yl)acetate, 22, bis(1-methylimidazol-2-yl)methane\nFor Zn(MIm2Pr)Cl(H2O)] the structure was solved with SHELXS-97 [33] using direct methods. All hydrogen atoms were located in the difference Fourier map and refined freely with isotropic displacement parameters.\nFor [Zn(iPrEtIm2Pr)Cl] the structure was solved with DIRDIF-99 [34] using automated Patterson methods. Refinement was performed as an inversion twin. All hydrogen atoms were located in the difference Fourier map and refined with a riding model.\nFor ZnCl2(BnMIm2Ac)] the structure was solved with DIRDIF-99 [34] using automated Patterson methods. All hydrogen atoms were located in the difference Fourier map and refined with a riding model.\nFor [ZnCl2(MIm2CH2)] the structure was solved with SIR-97 [35] using Direct methods. All hydrogen atoms were located in the difference Fourier map and refined freely with isotropic displacement parameters.\nFor [Zn2(MIm2Pr)2(ox)]\u00b76H2O the crystal was nonmerohedrally twinned with a twofold rotation about the crystallographic c-axis as a twin operation. This twin law was taken into account during the intensity integration using the program EvalCCD [36]. The structure was solved on the nonoverlapping reflections with DIRDIF-99 [34] using automated Patterson methods. All hydrogen atoms were located in the difference Fourier map and refined with a riding model. The twin fraction refined to 0.127(3).\nCrystallographic data (without structure factors) for the structures reported in this paper have been deposited with the Cambridge Crystallographic Data Centre (CCDC) as supplementary publication nos. CCDC-637452 for Zn(MIm2Pr)Cl(H2O)], CCDC-637453 for [Zn(iPrEtIm2Pr)Cl], CCDC-637454 for ZnCl2(BnMIm2Ac)], CCDC-637455 for [ZnCl2(MIm2CH2)], and CCDC-637456 for [Zn2(MIm2Pr)2(ox)]\u00b76H2O. Copies of the data can be obtained free of charge from the CCDC (12 Union Road, Cambridge CB2 1EZ, UK; Tel.: +44-1223-336408; Fax: +44-1223-336003; e-mail: deposit@ccdc.cam.ac.uk; Web site http:\/\/www.ccdc.cam.ac.uk).\nResults\nAs part of our efforts to study the coordination chemistry of the substituted 3,3-bis(1-alkylimidazol-2-yl)propionate ligand family, the complexes of MIm2Pr and iPrEtIm2Pr with ZnCl2 were synthesized and structurally characterized (Fig.\u00a02). MIm2Pr is the parent ligand of the family and iPrEtIm2Pr offers the most steric hindrance of the ligands studied so far. The coordination chemistry of MIm2Pr and iPrEtIm2Pr with zinc was found to be stoichiometry-dependent.\nZinc complexes of MIm2Pr\nThe formation of different species upon addition of either 0.5 or 1.0 equiv of ZnCl2 to a solution of the potassium salt of MIm2Pr in D2O is evidenced by the respective 1H NMR spectra in D2O (Fig.\u00a03). Fig.\u00a03400\u00a0MHz 1H NMR spectra of the titration of K[MIm2Pr] with ZnCl2 in D2O at room temperature: a K[MIm2Pr]; b K[MIm2Pr] and ZnCl2 2:1; c K[MIm2Pr] and ZnCl2 1:1. Asterisks denote the residual solvent peak\nAll four resonances associated with ligand MIm2Pr (spectrum a) shifted upon addition of 0.5\u00a0equiv of ZnCl2 (spectrum b). The observation of two sharp signals of equal intensity for the imidazole protons shows that all four imidazole groups are equivalent. This is indicative of symmetric binding of both ligands to the zinc ion. The resonances of the methyl groups and the methine proton at the bridging carbon shift downfield relative to the free ligand, whereas the propionate CH2 protons shift upfield. Note that the methine proton resonance of the free ligand is hidden under the residual solvent signal in spectrum a. The lack of any other resonances suggests the clean formation of a neutral, 2:1 [Zn(MIm2Pr)2] complex. Upon addition of another 0.5\u00a0equiv of ZnCl2 several resonances shift and signal broadening is observed (spectrum\u00a0c). Especially, one of the two imidazole signals is severely broadened. Again, no free ligand is observed in the spectrum. These observations indicate a dynamic system with an overall 1:1 ligand\u2013zinc stoichiometry.\nThe isolation and purification of the complex formed with 2\u00a0equiv of MIm2Pr with respect to ZnCl2 proved troublesome. The separation of the inorganic salts formed as a by-product in the synthesis by means of repetitive washing in all cases resulted in partial loss of coordinated ligand (as observed by NMR), regardless of the A[MIm2Pr] salt (A is K+, [Bu4N]+) originally employed. We therefore attempted the synthesis of this complex by the reaction of 2\u00a0equiv of the acid HMIm2Pr with Zn[N(SiMe3)2]2. Zinc bis(trimethylsilyl)amide can be synthesized in sizeable quantities [30] and was found to be a convenient reagent in this case. Indeed, the reaction of Zn[N(SiMe3)2]2 with 2\u00a0equiv of HMIm2Pr in acetonitrile resulted in the formation of a white precipitate, which was recrystallized from an acetonitrile\u2013water solution and was identified as the 2:1 complex [Zn(MIm2Pr)2].\nUnfortunately, all attempts at obtaining single crystals of [Zn(MIm2Pr)2] failed. On the basis of ESI\u2013MS, IR, and the previously described NMR analysis, we propose that [Zn(MIm2Pr)2] (Fig.\u00a04) is isostructural to the crystallographically characterized analogous complex [CuII(MIm2Pr)2] [27]. The position of the symmetric and asymmetric carbonyl stretching vibrations proved to be indicative of the binding mode of MIm2Pr (Bruijnincx et al., submitted) [27]. The \u03bdas(C=O) and \u03bds(C=O) are found at 1,581 and 1,391\u00a0cm\u22121, respectively, indicative of a monodentate binding mode of the carboxylate [\u0394(\u03bdas\u00a0\u2212\u00a0\u03bds)\u00a0=\u00a0190\u00a0cm\u22121, identical to \u0394ionic] [38]. Furthermore, the clean ESI\u2013MS spectrum of [Zn(MIm2Pr)2] confirmed the structure of a mononuclear, neutral 2:1 species with [M\u00a0+\u00a0X]+ (X is H, Na, K) as the prominent ions. Fig.\u00a04The 2:1 complex [Zn(MIm2Pr)2]\nThe 1:1 complex [Zn(MIm2Pr)Cl)] was synthesized by the reaction of equimolar amounts of ZnCl2 and K[MIm2Pr] in dry methanol. This led to the precipitation and isolation of [Zn(MIm2Pr)Cl] in almost quantitative yield. The broadened signals in the 1H NMR spectrum of [Zn(MIm2Pr)Cl)] in D2O prompted us to study this complex by variable-temperature NMR (Fig.\u00a05). As complex [Zn(MIm2Pr)Cl)] is insoluble in organic solvents, the variable-temperature NMR studies were limited to aqueous solutions, which restricted the temperature range that could be studied. Spectra were recorded in the range from 2 to 70\u00a0\u00b0C. Fig.\u00a051H NMR resonances of the imidazole rings of [Zn(MIm2Pr)Cl] at various temperatures in D2O\nFigure\u00a05 shows that at elevated temperatures two sharp imidazole resonances are observed. In the spectrum recorded at 70\u00a0\u00b0C, the (small) coupling between the two imidazole protons H1 and H2 can also be discerned. The imidazole signal at higher field broadens upon cooling and ultimately splits into two separate (broad) resonances. This implies that at higher temperature the imidazole H1 protons (Fig.\u00a03) become magnetically equivalent, whereas at lower temperature exchange slows down with the result that decoalesence into two broad resonances occurs. This behavior can be tentatively explained by the possibility of two different spatial orientations of the imidazole rings with respect to the carboxylate group of a second ligand upon coordination to zinc (vide infra), which would result in nonequivalent imidazole H1 protons at lower temperature. At higher temperatures these orientations average on the NMR timescale and only one signal is observed.\nThe asymmetric and symmetric carbonyl stretch vibrations of isolated [Zn(MIm2Pr)Cl] were observed at 1,615 and 1,390\u00a0cm\u22121 [\u0394(\u03bdas\u00a0\u2212\u00a0\u03bds)\u00a0=\u00a0225\u00a0cm\u22121], respectively. The asymmetric stretch thus shifted to higher wavenumbers compared with that observed for [Zn(MIm2Pr)Cl] and a different, but still monodentate coordination mode of the carboxylate must therefore be present in [Zn(MIm2Pr)Cl]. The ESI\u2013MS spectrum of [Zn(MIm2Pr)Cl] showed the presence of several different species, with ions corresponding to, e.g., [MIm2Pr\u00a0+\u00a0Zn]+, [2(MIm2Pr)\u00a0+\u00a0Zn\u00a0+\u00a0H]+, [2(MIm2Pr)\u00a0+\u00a02Zn\u00a0+\u00a0Cl]+, [3(MIm2Pr)\u00a0+2Zn\u00a0+\u00a0H]2+ and [3(MIm2Pr)\u00a0+\u00a02Zn]+. On the basis of the NMR, IR, and ESI\u2013MS data, the structure of [Zn(MIm2Pr)Cl] can be described as an oligomeric\/polymeric species both in the solid state and in solution. This was confirmed by the single-crystal structure determination of [Zn(MIm2Pr)Cl(H2O)].\nCrystal structure of [Zn(MIm2Pr)Cl(H2O)]\nColorless single crystals of complex [Zn(MIm2Pr)Cl(H2O)] suitable for X-ray diffraction were obtained from a solution of [Zn(MIm2Pr)Cl] in H2O upon standing. The molecular structure of [Zn(MIm2Pr)Cl(H2O)] is depicted in Fig.\u00a06, with selected bond lengths and angles presented in Table\u00a02. Fig.\u00a06Molecular structure of [Zn(MIm2Pr)Cl(H2O)] in the crystal, showing three molecules of the carboxylato-bridged coordination polymer, forming a one-dimensional chain in the crystallographic c-direction. C\u2013H hydrogen atoms have been omitted for clarity. Displacement ellipsoids are drawn at the 50% probability level. Symmetry operation a 3\/2\u00a0\u2212\u00a0x, 1\u00a0\u2212\u00a0y, z\u00a0\u2212\u00a01\/2Table\u00a02Selected bond lengths (angstroms) and bond angles (degrees) for [Zn(MIm2Pr)Cl(H2O)]BondBond lengthAngleBond angleAngleBond angleZn1\u2013N22.1203 (15)N4\u2013Zn1\u2013N288.05 (6)N2\u2013Zn1\u2013O1a92.41 (5)Zn1\u2013N42.0517 (14)N2\u2013Zn1\u2013O3173.58 (5)O1a\u2013Zn1\u2013O382.24 (5)Zn1\u2013O1a1.9975 (11)O3\u2013Zn1\u2013N490.38 (5)N4\u2013Zn1\u2013Cl1119.50 (4)Zn1\u2013Cl12.2844 (4)O3\u2013Zn1\u2013Cl190.23 (4)Cl1\u2013Zn1\u2013O1a130.40 (4)Zn1\u2013O32.1860 (13)Cl1\u2013Zn1\u2013N295.97 (4)O1a\u2013Zn1\u2013N4109.55 (5)Symmetry operation a 3\/2\u00a0\u2212\u00a0x, 1\u00a0\u2212\u00a0y, z\u00a0\u2212\u00a01\/2\nThe single-crystal structure determination reveals that the structure of the metal complex is that of a coordination polymer. The zinc atom is coordinated by two 1-methylimidazole groups of one MIm2Pr ligand, while the carboxylato group of this ligand coordinates to a neighboring zinc atom. In this way an infinite one-dimensional chain parallel to the crystallographic c-axis is formed. The chlorido anion and a coordinated water molecule complete the coordination sphere around each zinc center and render it five-coordinate. Thus, a new, bridging coordination mode of MIm2Pr is observed in the structure of [Zn(MIm2Pr)Cl(H2O)] comprising N,N-bidentate coordination to one metal center and O-monodentate coorrdination to a second one. This coordination mode complements the alternative N,N,O-tridentate facial capping mode of the ligand, which is commonly observed for divalent transition metals [26, 27, 39]. The coordination geometry around the five-coordinate Zn atom is distorted trigonal bipyrimidal (\u03c4\u00a0=\u00a00.72) [40]. The equatorial positions are occupied by N4 and the monoanionic carboxylato O1a and chlorido Cl1 ligands with O1a\u2013Zn1\u2013N4, Cl1\u2013Zn1\u2013O1a, and N4\u2013Zn1\u2013Cl1 angles of 109.55(5), 130.40(4), and 109.50(4)\u00b0, respectively (\u03a3angles\u00a0=\u00a0360\u00b0). The axial positions are occupied by O3 of the water molecule and imidazole N2 [O3\u2013Zn1\u2013N2 173.58(5)\u00b0]. The Zn1\u2013N2 and Zn1\u2013N4 bond lengths are different [2.1203(15) and 2.0517(14)\u00a0\u00c5], consistent with their respective axial and equatorial positions. The Zn1\u2013O1a distance of 1.9975(11)\u00a0\u00c5 is slightly longer than the Zn\u2013Ocarboxylato bond length observed in [Zn(bdatBu2,Me2)Cl]2 (1.942\u00a0\u00c5) [12] [bpatBu2,Me2 is (3,5-di-tert-butylpyrazol-1-yl)(3\u2032,5\u2032-dimethylpyrazol-1-yl)acetate], in which a carboxylato group is similarly coordinated to the metal center. The zinc-aquo distance of 2.1860(13)\u00a0\u00c5 in [Zn(MIm2Pr)Cl(H2O)] is rather long compared with most reported Zn\u2013OH2 distances (mean Zn\u2013OH2 distance for five-coordinate zinc complexes is 2.046\u00a0\u00c5; Cambridge Structural Database version 5.27) [41]. In the related coordination polymer [Zn(bpatBu,Me)(H2O)](ClO4) [11] [bpatBu,Me is bis(5-tert-butyl-3-methylpyrazol-2-yl)acetate], where the water molecule occupies a position in the basal plane of the trigonal bipyramid, the zinc\u2013water distance, for example, amounts to 1.961\u00a0\u00c5. [Zn(MIm2Pr)Cl(H2O)] crystallizes in the noncentrosymmetric space group P212121 and the determination of the Flack parameter indicates that the crystal is enantiomerically pure.\nThe one-dimensional linear chains in Zn(MIm2Pr)Cl(H2O)] are interconnected via hydrogen-bonding interactions into a two-dimensional network (Fig.\u00a07, Table\u00a03). The water molecule binds via its two hydrogen atoms by hydrogen bonding to a carbonyl O and a chlorido anion as hydrogen acceptors of a [Zn(MIm2Pr)Cl(H2O)] molecule in a neighboring strand. Fig.\u00a07Hydrogen-bonding network in [Zn(MIm2Pr)Cl(H2O)]. Hydrogen bonds connect the infinite one-dimensional chains into a two-dimensional network. C\u2013H hydrogen atoms have been omitted for clarity. Symmetry operations i 1\u00a0\u2212\u00a0x, 1\/2\u00a0+\u00a0y, 1\/2\u00a0\u2212\u00a0z; ii \u22121\/2\u00a0+\u00a0x, 3\/2\u00a0\u2212\u00a0y, \u2212zTable\u00a03Selected hydrogen bond lengths (angstroms) and angles (degrees) for [Zn(MIm2Pr)Cl(H2O)]Donor\u2013H\u00b7\u00b7\u00b7acceptorD\u2013HH\u00b7\u00b7\u00b7AD\u00b7\u00b7\u00b7AD\u2013H\u00b7\u00b7\u00b7AO3\u2013H1O\u00b7\u00b7\u00b7O2i0.78 (3)1.95 (3)2.7307 (19)176 (3)O3\u2013H2O\u00b7\u00b7\u00b7Cl1ii0.84 (3)2.31 (3)3.1472 (15)171 (3)Symmetry operations i 1\u00a0\u2212\u00a0x, 1\/2\u00a0+\u00a0y, 1\/2\u00a0\u2212\u00a0z; ii \u22121\/2\u00a0+\u00a0x, 3\/2\u00a0\u2212\u00a0y, \u2212z\nZinc complexes of iPrEtIm2Pr\nIn order to assess the influence of the presence of steric bulk in the ligand framework, we also studied the zinc coordination chemistry of ligand iPrEtIm2Pr. The ethyl and isopropyl substituents of this ligand provide significant steric hindrance and, moreover, increase the solubility of its complexes in organic solvents. The 1:1 complex [Zn(iPrEtIm2Pr)Cl] was synthesized by the reaction of equimolar amounts of ZnCl2 and K[iPrEtIm2Pr] in methanol. The complex was isolated in almost quantitative yield. The IR spectrum of [Zn(iPrEtIm2Pr)Cl] showed the asymmetric and symmetric carbonyl stretch vibrations at 1,627 and 1,381\u00a0cm\u22121 [\u0394(\u03bdas\u00a0\u2212\u00a0\u03bds)\u00a0=\u00a0246\u00a0cm\u22121], respectively. The structure of [Zn(iPrEtIm2Pr)Cl] was determined by X-ray crystal structure determination.\nCrystal structure of [Zn(iPrEtIm2Pr)Cl]\nColorless single crystals of [Zn(iPrEtIm2Pr)Cl] suitable for X-ray diffraction were obtained from a solution of [Zn(iPrEtIm2Pr)Cl] in methanol. The molecular structure of [Zn(iPrEtIm2Pr)Cl] is depicted in Fig.\u00a08, with selected bond lengths and angles presented in Table\u00a04. The crystal structure shows that whereas like [Zn(MIm2Pr)Cl(H2O)] [Zn(iPrEtIm2Pr)Cl] also crystallizes as a coordination polymer, the molecular structure of [Zn(iPrEtIm2Pr)Cl] is noticeably different. Unlike in the structure of [Zn(MIm2Pr)Cl(H2O)], no water molecule is found to coordinate to zinc in [Zn(iPrEtIm2Pr)Cl]. Instead of the five-coordinate structure in [Zn(MIm2Pr)Cl(H2O)], the zinc atom in [Zn(iPrEtIm2Pr)Cl] is four-coordinate. It has a pseudotetrahedral coordination geometry with two imidazole groups of one iPrEtIm2Pr ligand, a chlorido, and a carboxylato group of a neighboring ligand coordinated to it. The bridging mode of iPrEtIm2Pr observed in [Zn(iPrEtIm2Pr)Cl] is the same as found in [Zn(MIm2Pr)Cl(H2O)]. The deviation from ideal tetrahedral geometry is reflected in the observed bond angles [91.90(9)\u2013119.66(6)\u00b0]. The smallest angle (N2\u2013Zn1\u2013N4) is caused by the inherent geometrical restrictions imposed by the ligand and the largest angle (Cl1\u2013Zn1\u2013O1a) probably reflects the steric influence of the isopropyl groups. The Zn\u2013N, Zn\u2013O, and Zn\u2013Cl bond lengths in [Zn(iPrEtIm2Pr)Cl] are shorter than the corresponding distances in [Zn(MIm2Pr)Cl(H2O)], which reflects the stronger Lewis acidity of zinc(II) in a four-coordinate versus a five-coordinate geometry. The Zn1\u2013O1a bond length of 1.9628(19)\u00a0\u00c5 is comparable to values reported for other tetrahedral N,N,O zinc complexes with a monodentate carboxylate [9, 11, 12, 18]. The different coordination geometry at zinc results in a more folded zigzag polymer chain in [Zn(iPrEtIm2Pr)Cl], compared with the more stretched zigzag chain observed for [Zn(MIm2Pr)Cl(H2O)]. Fig.\u00a08Molecular structure of [Zn(iPrEtIm2Pr)Cl] in the crystal, showing three molecules of the carboxylato-bridged coordination polymer. C\u2013H hydrogen atoms have been omitted for clarity. Displacement ellipsoids are drawn at the 50% probability level. Symmetry operation a x, 1\u00a0\u2212\u00a0y, z\u00a0\u2212\u00a01\/2Table\u00a04Selected bond lengths (angstroms) and bond angles (degrees) for [Zn(iPrEtIm2Pr)Cl]BondBond lengthAngleBond angleAngleBond angleZn1\u2013N22.038 (2)Cl1\u2013Zn1\u2013N2116.85 (7)N2\u2013Zn1\u2013O1a108.85 (8)Zn1\u2013N42.045 (2)Cl1\u2013Zn1\u2013N4112.12 (7)N4\u2013Zn1\u2013O1a103.15 (9)Zn1\u2013O1a1.9628 (19)Cl1\u2013Zn1\u2013O1a119.66 (6)Zn1\u2013Cl12.2146 (7)N2\u2013Zn1\u2013N491.90 (9)Symmetry operation a x, 1\u00a0\u2212\u00a0y, z\u00a0\u2212\u00a01\/2\nThe structure of [Zn(iPrEtIm2Pr)Cl] and more generally the coordination chemistry of iPrEtIm2Pr with Zn in methanol solution were studied by 1H NMR spectroscopy and ESI\u2013MS. The 1H NMR spectra of a titration experiment comprising the addition of ZnCl2 to the potassium salt of iPrEtIm2Pr in methanol solution are shown in Fig.\u00a09. Fig.\u00a09400\u00a0MHz 1H NMR spectra of the titration of K[iPrEtIm2Pr] with ZnCl2 in CD3OD at room temperature: a K[iPrEtIm2Pr] and ZnCl2 1:0; b K[iPrEtIm2Pr] and ZnCl2 1:1; c K[iPrEtIm2Pr] and ZnCl2 2:1; d K[iPrEtIm2Pr] and ZnCl2 3:1. Asterisks denote the residual solvent peak and H2O\nComparison of the 1H NMR spectra of K[iPrEtIm2Pr] (Fig.\u00a09, spectrum a) with that of an equimolar K[iPrEtIm2Pr]\u2013ZnCl2 mixture shows significant shifts pointing to the formation of a [Zn(iPrEtIm2Pr)Cl] complex (spectrum b). In the 1H NMR spectrum of [Zn(iPrEtIm2Pr)Cl] one sharp resonance is observed for the imidazole protons at 6.99\u00a0ppm, which is shifted to lower field compared with that of the free ligand. The two imidazole rings are equivalent on the NMR timescale, suggesting that they are symmetrically bound to zinc. Importantly, the CH2 resonance of the ethyl groups is shifted and split into two signals of equal intensity (4.20 and 4.32\u00a0ppm). Similarly, the methyl groups of the isopropyl substituent now appear as two doublets of equal intensity. These splittings confirm that the CH2(Et) and CH3(iPr) groupings contain diastereotopic protons and methyl groups, respectively. No splitting or change in multiplicity of the resonances associated with the propionate backbone is observed, which is indicative of mirror symmetry of the complex in solution. Although at first sight the NMR data would allow an assignment of the solution structure for [Zn(iPrEtIm2Pr)Cl] as a mononuclear zinc complex with a facially capped ligand, a bridged oligomeric species (similar as the one discussed before for [Zn(MIm2Pr)Cl]) is preferred. ESI\u2013MS data support the latter description since trimeric, dimeric, and monomeric species are observed. Prominent ions corresponding to [Zn(iPrEtIm2Pr)Cl\u00a0+\u00a0H]+, [Zn2(iPrEtIm2Pr)2Cl2\u00a0+\u00a0H]+, and [Zn3(iPrEtIm2Pr)3Cl3\u00a0+\u00a0H]+ are, for instance, observed. The structure of [Zn(iPrEtIm2Pr)Cl] in solution can therefore best be described as a coordination polymer similar to [Zn(MIm2Pr)Cl].\nUpon addition of 2\u00a0equiv of K[iPrEtIm2Pr] to ZnCl2 in CD3OD (Fig.\u00a09, spectrum c), a second species is formed next to the 1:1 complex [Zn(iPrEtIm2Pr)Cl]. The fact that two species are observed, indicates that an equilibrium is obtained, i.e., the clean formation of a symmetrical 2:1 iPrEtIm2Pr to zinc complex, as previously observed for MIm2Pr, does not occur. Further inspection of the 1H NMR spectrum indicated that all resonances of the 1-ethyl-4-isopropylimidazole groups of the new species are split into two signals of equal intensity, e.g., the imidazole ring protons at 7.37 and 7.19\u00a0ppm. This suggests that the two imidazole groups are inequivalent and experience a drastically different magnetic environment. For instance, the two resonances of the CH protons of the two isopropyl groups are found at 2.96 and 1.03\u00a0ppm. Furthermore, the methine proton at the bridging carbon is still a single triplet, whereas the CH2 group of the propionate backbone has become diastereotopic. Variable-temperature NMR studies over a temperature range from \u221240 to 60\u00a0\u00b0C did not show significant spectral changes. The ESI\u2013MS spectrum of a 2:1 K[iPrEtIm2Pr]\u2013ZnCl2 solution showed prominent ions corresponding to the [Zn(iPrEtIm2Pr)2\u00a0+\u00a0H]+ and [Zn(iPrEtIm2Pr)2\u00a0+\u00a02H]2+ cations.\nOn the basis of these data, the structure of the new species is proposed to be a 2:1 ligand-to-zinc complex [Zn(iPrEtIm2Pr)2] in which each ligand is coordinated via its carboxylate group and one imidazole group, resulting in a four-coordinated zinc(II) ion (Fig.\u00a010). The coordination of two of the three donor atoms in a 2:1 ligand-to-metal complex has also been observed with bulky N,N,S(thiolate) ligands in the complexes [Zn(L2S)2] [L2SH is (3-tert-butyl-5-methyl-2-thiophenyl)bis(3,5-dimethyl pyrazolyl) methane] and [Zn(L2)2] [L2 is 1-methoxy-2-methyl-1,1-bis(1-methyl-4,5-diphenyl-1H-imidazol-2-yl)propane-2-thiol] [42, 43]. The addition of a third equivalent of K[iPrEtIm2Pr] (spectrum d) results in the disappearance of the signals associated with [Zn(iPrEtIm2Pr)Cl] and both free ligand K[iPrEtIm2Pr] and [Zn(iPrEtIm2Pr)2] are detected. No attempts to isolate [Zn(iPrEtIm2Pr)2] were undertaken. Fig.\u00a010Proposed structure for [Zn(iPrEtIm2Pr)2]. The structure was calculated by molecular mechanics geometry optimization (Spartan SGI [44] MMFF94)\nAttempted synthesis of a zinc bis(1-methylimidazol-2-yl)acetate complex\nRecently, the zinc coordination chemistry of bis(pyrazolyl)acetates (bpa) has been actively explored to mimic the 2-His-1-carboxylate facial triad found in several different zinc enzymes [9, 11, 12, 18, 19]. The backbone of bpa is more rigid than the backbone of the ligands MIm2Pr and iPrEtIm2Pr used in the present study. In particular, the bridging N,N-bidentate-O-monodentate coordination mode reported here is less favorable for bpa, for which a strong predisposition to an N,N,O facial capping mode is expected. The synthesis of zinc complexes of MIm2Ac would therefore be desirable, since it would allow a direct comparison of the respective pyrazole and imidazole donor groups (Fig.\u00a011).Fig.\u00a011Ligands benzyl bis(1-methylimidazol-2-yl)acetate (BnMIm2Ac), MIm2Ac, and bis(pyrazolyl)acetate (bpa) and the synthesis of complexes [ZnCl2(BnMIm2Ac)] and [ZnCl2(MIm2CH2)]\nWe attempted the synthesis of ligand MIm2Ac, but found that the common synthetic procedure, i.e., lithiation of MIm2CH2 followed by reaction with carbon dioxide [45], did not result in product formation in acceptable yields. Instead, it was found that the ligand or an ester precursor of the ligand decarboxylates rather easily, as was also observed by Peters et al. [39]. The benzyl ester of bis(1-methylimidazol-2-yl)acetic acid (BnMIm2Ac), however, was sufficiently stable and could be isolated and purified after reaction of lithio bis(1-methylimidazol-2-yl)methane with benzylchloroformate. Since hydrolysis and hydrogenolysis of the ester moiety resulted in decomposition of the ligand, the 1:1 zinc dichloride complex with BnMIm2Ac was synthesized in the hope that autocatalytic ester hydrolysis by the zinc complex would yield the desired zinc bis(1-methylimidazol-2-yl)acetate complex. A similar approach, involving the autocatalytic saponification of a zinc bis(picolyl)glycine ethyl ester complex, has been reported by Abufarag and Vahrenkamp [46].\nThe reaction of equimolar amounts of ZnCl2 and BnMIm2Ac gave the complex [ZnCl2(BnMIm2Ac)] as a white powder in quantitative yield. The structure of [ZnCl2(BnMIm2Ac)] was determined by X-ray diffraction studies and showed the BnMIm2Ac ligand bound to the zinc center in an N,N-bidentate way (Fig.\u00a012, top). However, stirring of [ZnCl2(BnMIm2Ac)] in H2O at room temperature in an attempt to obtain [Zn(MIm2Ac)Cl] resulted in decomposition of the ligand and formation of [ZnCl2(MIm2CH2)], which was isolated as one of the decomposition products. [ZnCl2(MIm2CH2)] was also characterized crystallographically (Fig.\u00a012, bottom). On the basis of these findings, it was concluded that the MIm2Ac monoanion is too unstable for practical studies and the pursuit of this ligand was abandoned. Fig.\u00a012Molecular structures of [ZnCl2(BnMIm2Ac)] (top) and [ZnCl2(MIm2CH2)] (bottom) in the crystal. Displacement ellipsoids are drawn at the 50% probability level. All hydrogen atoms have been omitted for clarity. Selected bond lengths (angstroms) as follows: [ZnCl2(BnMIm2Ac)], Zn1\u2013N2 2.011(2), Zn1\u2013N4 1.994(2), Zn1\u2013Cl1 2.2242(7), Zn1\u2013Cl2 2.2554(7); [ZnCl2(MIm2CH2)], Zn1\u2013N2 2.0247(17), Zn1\u2013N4 2.0222(17), Zn1\u2013Cl1 2.2110(6), Zn1\u2013Cl2 2.2504(6)\nReaction of [Zn(MIm2Pr)Cl] with sodium pyruvate: oxalate formation\nIn an attempt to prevent the formation of coordination polymers of MIm2Pr and to obtain mononuclear complexes with the desired N,N,O facial capping mode of the ligand, several different monoanionic, bidendate coligands were used. In the course of this study, we also investigated the reaction of [Zn(MIm2Pr)Cl] with sodium pyruvate. The addition of 1\u00a0equiv of sodium pyruvate to a solution of [Zn(MIm2Pr)Cl] in D2O resulted in the appearance of a new singlet at 2.38\u00a0ppm in the 1H NMR spectrum (Fig.\u00a013, top spectrum). The signal is not shifted with respect to the signal observed for free sodium pyruvate, which indicates that the interaction between [Zn(MIm2Pr)Cl] and the pyruvate anion is weak, at best. The ESI\u2013MS spectrum of an aqueous solution of equimolar amounts of [Zn(MIm2Pr)Cl] and sodium pyruvate, however, did show some major ions corresponding to a mononuclear {[Zn(MIm2Pr)(pyruvate)]\u00a0+\u00a0Na}+ and a dinuclear {[Zn2(MIm2Pr)2(pyruvate)]}+ cation, in addition to the ions observed in the ESI\u2013MS spectrum of a solution of [Zn(MIm2Pr)Cl] in water. Fig.\u00a0131H NMR spectra of a solution of [Zn(MIm2Pr)Cl] in D2O (asterisk) after addition of 1\u00a0equiv of sodium pyruvate and the same solution after 24\u00a0h\nHowever, when the mixture of [Zn(MIm2Pr)Cl] and pyruvate in D2O was followed in time by NMR spectroscopy, the gradual decrease in intensity of the signal of the pyruvate methyl group was noticed until it ultimately disappeared (Fig.\u00a013, bottom spectrum). After several days colorless crystals suitable for X-ray diffraction studies were formed in the NMR tube. To our surprise, the X-ray crystal structure showed the structure of the zinc complex [Zn2(MIm2Pr)2(ox)]\u00b76H2O. [Zn2(MIm2Pr)2(ox)]\u00b76H2O has the structure of a coordination polymer involving [Zn(MIm2Pr)]+ cations bridged by oxalato dianions.\nTo unequivocally prove the relevance of the structure obtained, zinc coordination polymer [Zn2(MIm2Pr)2(ox)] was also synthesized independently. Indeed, the addition of 0.5\u00a0equiv of K2C2O4\u00b7H2O to an aqueous solution of [Zn(MIm2Pr)Cl] yielded a white, insoluble powder. This powder was identified as [Zn2(MIm2Pr)2(ox)] by elemental analysis and IR spectroscopy. Typical features of the IR spectrum of [Zn2(MIm2Pr)2(ox)] include absorptions at 1,674, 1,638, 1,591, and 1,399\u00a0cm\u22121, which can be attributed to the oxalato and carboxylato groups in the polymer. Crystallization of [Zn2(MIm2Pr)2(ox)] proved difficult given its general insolubility. Single crystals could, however, be obtained via the gel crystallization method [47]. A gel formed from a solution of tetramethoxysilane and potassium oxalate hydrate in water was layered with an aqueous solution of K[MIm2Pr] and ZnCl2. Single crystals were obtained after several days and could be unequivocally identified as [Zn2(MIm2Pr)2(ox)] by X-ray crystal structure determination.\nCrystal structure of [Zn2(MIm2Pr)2(ox)]\u00b76H2O\nThe molecular structure of [Zn2(MIm2Pr)2(ox)]\u00b76H2O is shown in Fig.\u00a014, with selected bond lengths and angles presented in Table\u00a05.1 The polymeric structure of [Zn2(MIm2Pr)2(ox)]\u00b76H2O comprises two crystallographically inequivalent zinc atoms. Both zinc centers are five-coordinate and differ only in the slightly different bond lengths and angles. Each zinc atom is coordinated in an N,N-bidentate fashion by the 1-methylimidazole groups of one MIm2Pr ligand, two oxygen atoms of a bis O,O-bidentate bridging oxalato group, and a carboxylato O donor atom of a neighboring MIm2Pr ligand. The combination of the bridging oxalato and MIm2Pr ligands constructs a polymeric structure leading to the formation of two-dimensional sheets. Fig.\u00a014Molecular structure of [Zn2(MIm2Pr)2(ox)]\u00b76H2O in the crystal. C\u2013H hydrogen atoms and all solvent water molecules have been omitted for clarity. Displacement ellipsoids are drawn at the 50% probability levelTable\u00a05Selected bond lengths (angstroms) and angles (degrees) for [Zn2(MIm2Pr)2(ox)]\u00b76H2OBondBond lengthAngleBond angleAngleBond angleZn1\u2013O312.258 (2)O11\u2013Zn1\u2013O3189.48 (8)O21\u2013Zn2\u2013O4190.33 (8)Zn1\u2013O322.018 (2)O11\u2013Zn1\u2013O32124.09 (9)O21\u2013Zn2\u2013O42125.19 (8)Zn1\u2013O111.9694 (19)O11\u2013Zn1\u2013N22b108.91 (9)O21\u2013Zn2\u2013N12109.08 (9)Zn1\u2013N24b2.046 (2)O11\u2013Zn1\u2013N24b107.61 (9)O21\u2013Zn2\u2013N14107.85 (9)Zn1\u2013N22b2.088 (2)O31\u2013Zn1\u2013O3277.16 (9)O41\u2013Zn2\u2013O4277.84 (8)Zn2\u2013O412.233 (2)O31\u2013Zn1\u2013N22b161.43 (9)O41\u2013Zn2\u2013N12160.51 (8)Zn2\u2013O422.0179 (19)O31\u2013Zn1\u2013N24b87.69 (9)O41\u2013Zn2\u2013N1487.24 (9)Zn2\u2013O211.9630 (19)O32\u2013Zn1\u2013N22b89.94 (8)O42\u2013Zn2\u2013N1289.12 (8)Zn2\u2013N122.089 (2)O32\u2013Zn1\u2013N24b125.38 (9)O42\u2013Zn2\u2013N14124.42 (9)Zn2\u2013N142.052 (2)N22b\u2013Zn1\u2013N24b89.06 (9)N12\u2013Zn2\u2013N1488.34 (9)C31\u2013O311.248 (4)C41\u2013O411.250 (3)C31d\u2013O321.252 (4)C41c\u2013O421.254 (3)C31\u2013C31d1.544 (6)C41\u2013C41c1.537 (6)Symmetry operations b 1\u00a0+\u00a0x, y, z; c \u2212x, 1\u00a0\u2212\u00a0y, \u2212z; d 1\u00a0\u2212\u00a0x, 1\u00a0\u2212\u00a0y, 1\u00a0\u2212\u00a0z\nThe coordination geometry around each zinc center is best described as a severely distorted trigonal bipyramid, with \u03c4 values of 0.60 and 0.59 for Zn1 and Zn2, respectively [40]. The equatorial positions are occupied by N24b\/N14, and the anionic carboxylato O11\/O21 and oxalato O32\/O42 ligands for Zn1\/Zn2, respectively. The axial positions are occupied by O31\/O41 of the oxalato group and imidazole N22b\/N12 for Zn1\/Zn2 [O\u2013Zn\u2013N angles of 161.43(9)\u00b0\/160.51(8)\u00b0]. The planar oxalato moieties are rather asymmetrically bound to the metal centers, with the Zn1\u2013O31 and Zn1\u2013O32 distances amounting to 2.258(2) and 2.018(2) \u00c5, for instance. This asymmetry has also been observed in other bisbidentate bridged zinc\u2013oxalato complexes with five-coordinated metal centers and is consistent with the occupation of one equatorial and one axial site of the trigonal bipyramid [10, 48]. The shortest Zn\u00b7\u00b7\u00b7Zn distances via the bridging oxalato group are 5.5667(7)\u00a0\u00c5 for Zn1\u00b7\u00b7\u00b7Zn1d and 5.5332(7)\u00a0\u00c5 for Zn2\u00b7\u00b7\u00b7Zn2c. The distances of the metal centers to the donor atoms of MIm2Pr follow the same trend as observed in [Zn(MIm2Pr)Cl(H2O)]. The crystal structure of [Zn2(MIm2Pr)2(ox)]\u00b76H2O is further stabilized by 14 different hydrogen-bonding interactions, which involve the cocrystallized water molecules, the oxalato bridges, and the carboxylato groups (Fig.\u00a015). The oxalato groups are, for instance, involved in two chelated, three-centered hydrogen bonds with two water molecules via four O\u2013H\u00b7\u00b7\u00b7O hydrogen bonds. Fig.\u00a015Hydrogen bonding network and crystal packing of [Zn2(MIm2Pr)2(ox)]\u00b76H2O\nDiscussion\nThe aim of this investigation was to explore the biomimetic modeling potential of the MIm2Pr ligand family with respect to mononuclear zinc enzymes featuring the 2-His-1-carboxylate facial triad. Complex [Zn(MImPr)2] shows that the facial capping mode of MIm2Pr, as earlier observed with copper [27, 28] and iron (Bruijnincx et al., submitted) [26], is also accessible for zinc. The crystal structure of the 1:1 complex [Zn(MIm2Pr)Cl(H2O)], however, shows that MIm2Pr can also adopt other coordination modes. The formation of the oligomeric\/polymeric structure of [Zn(MIm2Pr)Cl], both in solution and in the solid state, can be attributed to the flexibility of the ligand due to the CH2 spacer of the propionate backbone. The formation of polymeric [Zn(MIm2Pr)Cl(H2O)] is further aided by the insolubility of the resulting coordination polymer. Two strategies were explored to circumvent polymer formation, i.e., increasing the steric bulk of the ligand and elimination of the CH2 spacer. The latter approach demanded the synthesis of the bis(1-methylimidazol-2-yl)acetate analogue of MIm2Pr, which was found to be rather unstable and its zinc complexes could not be obtained. The effect of more steric bulk was studied with iPrEtIm2Pr, which contains the sterically more demanding isopropyl and ethyl substituents. Zinc complexes of iPrEtIm2Pr are polymeric in nature as well, albeit with a different coordination geometry at the zinc atom as observed in [Zn(MIm2Pr)Cl(H2O)]. The first coordination spheres of [Zn(MIm2Pr)Cl(H2O)] and [Zn(iPrEtIm2Pr)Cl] are compared in Fig.\u00a016. The added steric bulk in iPrEtIm2Pr causes the zinc ion to adopt a pseudotetrahedral geometry in [Zn(iPrEtIm2Pr)Cl], instead of the distorted trigonal bipyramid observed for [Zn(MIm2Pr)Cl(H2O)]. The coordination around zinc in [Zn(iPrEtIm2Pr)Cl] resembles the first coordination sphere of the active sites of the zinc enzymes thermolysin, carboxypeptidase A, and neutral protease. The polymeric rather than monomeric nature of the complex, however, clearly limits its potential as a biological mimic. Fig.\u00a016Comparison of the first coordination spheres of the zinc atoms in the coordination polymers [Zn(MIm2Pr)Cl(H2O)] (top) and [Zn(iPrEtIm2Pr)Cl] (bottom)\nThe isolation of [Zn2(MIm2Pr)2(ox)] from the reaction of [Zn(MIm2Pr)Cl] with sodium pyruvate was rather unexpected and is, to the best of our knowledge, the first report of the nonoxidative formation of oxalate from pyruvate (Scheme\u00a01).2 Interestingly, Takeste and Vahrenkamp [51] very recently reported a trispyrazolylborato\u2013zinc complex with pyruvate, representing the first structurally characterized zinc complex of an \u03b1-keto acid. Although in their case the zinc\u2013pyruvate complex obtained was stable, unexpected cleavage reactions with other \u03b1-keto acids, such as benzoylformic acid, and \u03b1-diketones were observed.Scheme\u00a01The observed transformation of pyruvate to oxalate mediated by [Zn(MIm2Pr)Cl]\nA control experiment showed that an aqueous sodium pyruvate solution is stable under the reaction conditions. Apparently, [Zn(MIm2Pr)Cl] mediates the conversion of pyruvate to oxalate. The product [Zn2(MIm2Pr)2(ox)] invariably deposited as crystals in experiments done on the NMR scale. On a preparative scale (0.3\u00a0mmol), the reaction also takes place, yet the product was isolated as an insoluble white powder. The IR spectrum of this powder was identical to that obtained for independently synthesized [Zn2(MIm2Pr)2(ox)]. Most importantly, further experiments revealed that the reaction is catalytic in zinc. For example, addition of 20\u00a0equiv of sodium pyruvate to a solution of [Zn(MIm2Pr)Cl] in water under ambient conditions resulted in ten turnovers after 24\u00a0h. The disappearance of the pyruvate methyl signal was also observed in a solution containing ZnCl2 and sodium pyruvate without any further additives. Concomitantly, a precipitate was observed in the NMR tube. However, the composition of this precipitate, i.e., the formation of oxalate, could not be unambiguously established.\nSince it is known that pyruvate keto\u2013enol tautomerization is catalyzed by divalent metal ions such as Zn(II) [52, 53], the disappearance of the methyl signal could in principle also be attributed to consecutive H\/D-exchange steps. This would yield the deuterated pyruvate-d3 anion, which is also invisible in 1H NMR spectroscopy. To be able to exclude this possibility, experiments were performed in H2O and samples were diluted with 2\u00a0equiv of D2O just before the measurement. The same observations were made as previously, i.e., gradual disappearance of the methyl signal and slow crystallization of the product in the NMR tube. The presence of appreciable amounts of enolate should also lead to the detection of the dimerization product of pyruvate [52, 53]. This pyruvate dimer, which results from an aldol condensation reaction and has characteristic signals at 1.38 (singlet) and 3.35 (AB pattern)\u00a0ppm [52\u201354], was not observed under these conditions.\nPhenyl pyruvic acid (and other \u03b1-ketoacids) is oxidized by molecular oxygen to give benzaldehyde and oxalate or the carbon oxides (CO and\/or CO2) [55, 56]. This reaction is catalyzed by several metal ions, such as Mn(II), Fe(II), and Cu(II), but not by Zn(II) [57, 58], and has been attributed to the presence of the enol tautomer, which is capable of reacting with dioxygen [61]. In the absence of dioxygen, i.e., under an Ar atmosphere, pyruvate does react with [Zn(MIm2Pr)Cl] to form oxalate. The anaerobic reactivity cannot be attributed to any residual dioxygen traces, since phenyl pyruvate (a substrate more prone to the reaction with dioxygen) did not show any reactivity under these conditions.\nThe observed conversion of pyruvate to oxalate under anaerobic conditions clearly places it outside previously observed formation of oxalate from oxidative transformations of \u03b1-keto acids [55, 56, 59\u201363]. The observation is important, since pyruvate is a major metabolic junction and it is therefore of prime importance to understand its reactivity. At present no biological counterpart has been reported for the pyruvate\u2013oxalate conversion catalyzed by [Zn(MIm2Pr)Cl]. The chemical transformation as reported here, however, takes place at room temperature and physiological pH and should therefore be considered as a possible alternative pathway in the reactivity of pyruvate. The mechanism of this conversion is currently not clear. No other products could be detected by NMR during the reaction, which precludes the formulation of a mass balance for the reaction and clearly hampers a mechanistic interpretation. In any case, the pyruvate to oxalate conversion requires the scission of a C\u2013C bond. On the other hand, the reactivity of zinc enzymes and their model complexes is often of hydrolytic nature. This can be attributed to the generation of a nucleophilic hydroxide species and\/or the activation of a coordinated substrate towards attack of such a nucleophile [3]. The formation of oxalate from pyruvate via a hydrolytic mechanism is, however, difficult to envision. Further investigations have to be performed to address the mechanistic questions concerning this transformation.\nConclusions\nThe difference in applied steric bulk by the ligands MIm2Pr and iPrEtIm2Pr is reflected in the coordination chemistry of the two ligands towards ZnCl2. Octahedral (MIm2Pr) versus tetrahedral (iPrEtIm2Pr) and trigonal pyramidal (MIm2Pr) versus tetrahedral (iPrEtIm2Pr) coordination geometries were obtained in the 1:1 and 2:1 ligand-to-metal complexes, respectively. The bridging binding mode of the two crystallographically characterized coordination polymers illustrates the intrinsic flexibility of the 3,3-bis(imidazol-2-yl)propionate ligand framework. The polymeric nature of the complexes could limit their biological relevance with respect to the 2-His-1-carboxylate facial triad in the case of zinc. On the other hand, attempts aimed at obtaining structurally more faithful mononuclear complexes led to the observation of the unprecedented Zn-mediated pyruvate to oxalate conversion. This new, nonoxidative transformation of an important metabolic junction is intriguing and warrants further investigation.","keyphrases":["zinc","n,n,o ligands","pyruvic acid","oxalic acid"],"prmu":["P","P","P","R"]}
{"id":"Matern_Child_Health_J-2-2-1592139","title":"The Business Case for Preconception Care: Methods and Issues\n","text":"Only a limited number of economic evaluations have addressed the costs and benefits of preconception care. In order to persuade health care providers, payers, or purchasers to become actively involved in promoting preconception care, it is important to demonstrate the value of doing so through development of a \u201cbusiness case\u201d. Perceived benefits in terms of organizational reputation and market share can be influential in forming a business case. In addition, it is standard to include an economic analysis of financial costs and benefits from the perspective of the provider practice, payer, or purchaser in a business case. The methods, data needs, and other issues involved with preparing an economic analysis of the likely financial return on investment in preconception care are presented here. This is accompanied by a review or case study of economic evaluations of preconception care for women with recognized diabetes. Although the data are not sufficient to draw firm conclusions, there are indications that such care may yield positive financial benefits to health care organizations through reduction in maternal and infant hospitalizations. More work is needed to establish how costs and economic benefits are distributed among different types of organizations. Also, the optimum methods of delivering preconception care for women with diabetes need to be evaluated. Similar assessments should also be conducted for other forms of preconception care, including comprehensive care.\nIntroduction\nThe opportunity to assist a woman before she becomes pregnant to improve her chances of a healthy pregnancy is compelling. Adverse pregnancy outcomes, including birth defects, preterm birth, and perinatal complications, are the leading causes of infant mortality in the United States [1]. Further, developmental disabilities such as cerebral palsy result in direct and indirect economic costs that can exceed $1\u00a0million over a given child\u2019s lifetime [2], in addition to the social and emotional costs incurred by families. A number of additional conditions are associated with serious complications of pregnancy, such as uncontrolled diabetes, hypothyroidism, hypertension, use of alcohol or tobacco, and obesity. The economic burden of most of these conditions in conjunction with pregnancy has been poorly studied but is likely to be enormous.\nThis paper discusses the potential economic benefits of preconception care due to the prevention of adverse pregnancy outcomes with a focus on the data that would be needed to assess costs and benefits. Currently, the needed data and analyses are largely lacking. In the absence of information about the costs and benefits of comprehensive preconception care, which should ideally include nutritional counseling, evaluation of medications, and family history evaluation and genetic counseling, this paper focuses on principles and methods. Three published studies have analyzed the expected financial benefits from counseling women with diabetes prior to pregnancy. These studies are compared and lessons drawn, with discussion of implications of preconception care for women with other conditions.\nIn order to argue that preconception care is a \u201cgood buy,\u201d its costs and benefits must be assessed from the perspectives of a variety of stakeholders involved in health care services. This paper will first define a \u201cbusiness case\u201d analysis and outline the methods for determining the \u201creturn on investment\u201d from the perspective of various health care organizations. Similarities and differences between such an analysis and a standard cost-effectiveness analyses that calculate returns to the health care system as a whole or to society will be explained. Second, the paper reviews the literature on business case analyses from areas other than preconception care. Third, the paper discusses what data sources are available and needed to apply business case methods in preconception care with particular focus on women with diabetes. Finally, the paper draws conclusions about future steps needed to assess the business case for preconception care.\nWhat is a business case analysis?\nConcepts and definitions\nA business case is an argument for a financial investment couched in terms of potential economic or market advantages to an organization, most commonly a private corporation [3]. The core of a business case is usually a budget impact analysis, a calculation of the financial return on investment (ROI) made by the firm(s) or organization(s) [4]. In addition to financial returns, such as cost savings or cost avoidance, a business case can be based on perceived strategic advantages such as enhanced organizational reputation, market positioning, or increased employee and customer satisfaction. This paper focuses just on financial business case analyses.\nAccording to the Institute for Healthcare Improvement (IHI) and the Commonwealth Fund [3]:A business case for a health care improvement intervention exists if the entity that invests in the intervention realizes a financial return on its investment in a reasonable time frame, using a reasonable rate of discounting. This may be realized as \u201cbankable dollars\u201d (profit), a reduction in losses for a given program or population, or avoided costs. In addition, a business case may exist if the investing entity believes that a positive indirect effect on organizational function and sustainability will accrue within a reasonable time frame.\nA business case related to health care is based on one or several of the perspectives of specific payers, purchasers, or providers. Because health care providers, payers, and purchasers often have different, and sometimes conflicting, economic incentives, the business case for the same intervention will probably differ among these groups. For example, improved ambulatory care may lead to reduced inpatient admissions, which could result in financial savings for payers, lost revenue for hospitals, and higher costs for ambulatory care providers. The resulting misaligment of incentives in the health care system may, and often does, lead to inappropriate provision or use of certain services and underutilization of others [3].\nAlthough business case analyses by definition focus on businesses, they are relevant to other stakeholders as well. In particular, the consumer perspective is also important to keep in mind. If consumers do not consider a service such as preconception care to provide good value for their investment of time and money, low demand and utilization could render the business case unfavorable. Further, government agencies must meet budgets, and the short term financial impact of policy changes may influence funding decisions. In the long run, though, public health programs are expected to optimize population health subject to resource availability, which requires analysis of both short-term and long-term health outcomes and economic consequences.\nAnalytical techniques and outcome measures\nFinancial analyses that underlie a business case are variably referred to as budget impact and ROI analyses. It is important to situate such analyses in the context of two commonly used methods of economic evaluation, cost-effectiveness analysis (CEA) and cost-benefit analysis (CBA) [5]. A CEA calculates the net monetary cost per unit of health outcome achieved. Certain preventive services are both more effective than alternatives and less costly [6]. A CEA that yields evidence of net cost savings from the payer perspective could be used to frame a business case, but CEAs rarely report payer-specific costs. In a CBA, all outcomes are expressed in dollar values, and the summary measure is expressed as either a net economic benefit or benefit-cost ratio. For example, a CBA of prenatal care for undocumented immigrants in California estimated that public funding cost the state $58 million but saved $211 million in long-term costs of care, for a projected net benefit of $153 million and a benefit-cost ratio of 4.6:1 [7].\nA financial analysis of a business case can be viewed as a CBA conducted from the perspective of an individual firm or organization. One important distinction between financial analyses and standard economic evaluations is that the latter are supposed to reflect the opportunity cost or value of resources from a societal perspective whereas a financial analysis considers the direct monetary or budgetary impact to a specific organization.\nThe ROI metric is typically calculated as the ratio of dollars in increased revenue or decreased cost divided by investment cost, although this can also be expressed as net cost difference [8]. Various other metrics can be used in a financial analysis, such as payback period or internal rate of return. For example, a recent study estimated the number of years that could be required for employers to realize a positive net benefit from bariatric surgery for obese employees as a result of reduced medical costs (all of which were assumed to be paid by the employer) and loss of work time [9]. ROI analyses require payer-specific costs, which are rarely reported in standard CBA studies.\nTime horizon and discount rate\nBusiness case analyses generally employ a short time horizon; for example, only changes in use and costs over a period of 2 to 3 years following an intervention are typically considered. This reduces the problem of enrollee or employee turnover. When individuals switch plans or employers, the return on investments in the long-term health of that individual made by the former health plan or employer will be realized by someone else. This can make financial returns to employers or health plans substantially lower than the economic benefits to society [3, 10, 11]. A payback period ranging from 5 years to 10 years [9] is not likely to be attractive to employers.\nIn economic evaluations from the societal perspective, it is conventional to discount costs occurring in future years using a discount rate of 3%, which reflects the social return on capital [5]. For a business case analysis employing a short time horizon, long-term future costs or benefits may not need to be counted or discounted. In the short run, when ROI analysis does employ discounting, the discount rate should reflect the opportunity cost of capital in the private sector or individual firm.\nProspective and retrospective evaluations\nFinancial and health economic analyses of an intervention can be performed before or after its implementation. A prospective analysis predicts the financial return for a new initiative or program before it begins. This presumes that there is reliable information about the effectiveness of the intervention and accurate historical cost and revenue data. Analysts construct a simulation model that projects likely costs, health outcomes, and benefits based on a set of assumptions. The benefits include anticipated savings in health care costs and, for analyses from the employer perspective, worker productivity [12, 13].\nA limitation of prospective analyses is that key assumptions may turn out to be unrealistic. For example, analyses in the 1980s and early 1990s projected cost savings to Medicaid programs from reductions in the number of low birth weight (LBW) births if prenatal care coverage were expanded (e.g., [14\u201316]). In conjunction with effective political advocacy and policy changes, these arguments succeeded in more than doubling the number of pregnancies covered by Medicaid prenatal care between 1986 and 1991. Despite this expansion, little or no reduction in LBW rates could be confirmed to have occurred among eligible population groups [17]. The lack of an observed effect may reflect deficiencies in the effectiveness of the routine prenatal care that was offered, as well as barriers to access, and does not rule out a protective effect of an optimal prenatal care protocol. Although prenatal care expansion had other health benefits and can easily be defended, the cost-saving argument appears in retrospect to have been overstated [18].\nIn the absence of information about intervention effectiveness, a hypothetical calculation can be made regarding the potential magnitude of benefits if an effective intervention were to exist. One way to make such a calculation is to take cost-of-illness estimates of the economic burden of diseases and project the potential cost reduction that might occur as a result of prevention. Only short-term costs, which can be much smaller in magnitude than the long-term costs included in cost-of-illness studies, are likely to be relevant to employers or payers [19]. For example, one group of investigators calculated that poor birth outcomes such as preterm birth cost employer-sponsored health plans $5.6 billion in 1990 and projected that prevention of 10% of preterm births would lead to a cost savings of $560 million [20].\nA retrospective or ex post analysis is based on observed costs and benefits. For example, a recent ex post CBA of folic acid fortification calculated that the economic returns were substantially greater, and the costs somewhat lower, than had been projected in previously published analyses conducted prior to fortification [21]. Although actual data can yield more reliable results, it is challenging to collect data on multiple outcomes. Furthermore, interactions among the outcomes or confounding of results from other sources can make it difficult to precisely estimate the impact of the initiative or program alone.\nLessons from business case literature\nA number of business case studies of disease management and health promotion initiatives have been published [8, 22, 23]. However, there is a paucity of well-conducted analyses and studies that contain detailed data on intervention and outcome costs [24]. Readers of business case analyses should beware of publication bias [22] and \u201cwish bias\u201d leading to optimistic estimates of effectiveness and compliance with behavioral changes [10].\nOne group that developed seven business case studies for health care quality improvement initiatives, such as disease management, concluded that health care providers, payers, and purchasers cannot predictably expect to reap positive financial returns from initiatives to improve health care quality within short time horizons [3]. In the majority of case studies, short-term financial returns to health plan entities were negative. Furthermore, returns to health care providers were almost uniformly negative, as any cost savings were generally recouped by purchasers, and not by providers. In several cases, managers felt that there was a business case for investing in improved quality based on organizational benefits such as \u201cretained market share, increased staff loyalty, and reputation among employers\u201d rather than cost savings [3].\nSimilarly, the Child Health Business Case Working Group developed a set of business case studies [25]. For example, a case study of postpartum screening for risk factors in new mothers (e.g., depression, smoking, domestic violence) followed by referral to community resources found that such a program would lead to higher costs for the practice without additional reimbursements. A positive business case for providers may require a financial mechanism for tying reimbursements by payers to improved outcomes [3]. Integrated health service providers under capitation payments may have more opportunity to internalize the financial gains resulting from improved care [3, 25].\nSeveral studies reported positive financial returns to managed care organizations (MCOs) from prenatal or perinatal interventions targeted to high-risk pregnant women [26\u201329], although these reports require validation and replication in well-controlled studies. For example, a high-risk pregnancy management program in upstate New York implemented by a managed care program serving Medicaid and other lower-income beneficiaries reported that cost savings from reduced NICU (neonatal intensive care unit) admissions were more than twice as great as the costs of the intervention [28]. A similar program implemented in another upstate New York MCO during the same time period reported investment costs (including provider reimbursements) of $573,355, cost savings of $789,621 resulting from the estimated prevention of 48 LBW births, and a net benefit of $216,266 [27]. The authors reported an internal rate of return of 37% on the investment, which is equivalent to an ROI ratio of 1.37.\nEconomic analyses of preconception care\nFinally, we turn to the application of business case methods to preconception care. Preconception care refers to three types of services and target populations:Preventive services and screenings offered to women who expect to become pregnant in the near future;Interconception care for women who have given birth and intend to bear another child at some point in the future;Counseling about the impact of pre-existing health risks or conditions for affected women of childbearing age and their impact on pregnancy outcomes.\nFew economic evaluations of individual screenings or services in the pre-pregnancy period have been published. One CEA estimated that promoting folic acid supplementation to all women of reproductive age would be cost effective, although not cost saving [30]. If multivitamins containing folic acid were targeted instead to women intending pregnancy, supplementation might be cost saving. On the other hand, because most of the averted costs of caring for children with spina bifida occur years in the future and are shared by multiple payers, this type of analysis cannot be used to calculate the business case for folic acid supplementation strategies.\nOne preconception care service with published analyses of short-term financial costs and benefits is preconception counseling for women with diabetes. Women with poorly controlled diabetes before pregnancy have an elevated risk of having an infant with a birth defect [31], fetal death, or macrosomia. Women with poorly controlled diabetes prior to pregnancy can also experience medical risks during pregnancy, including diabetic retinopathy, nephropathy, and ketoacidosis, as well as pregnancy-induced hypertension and uteroplacental insufficiency [32].\nA substantial body of evidence on infant health outcomes associated with preconception counseling for women with diabetes yields consistent findings. According to a meta-analysis of 14 epidemiologic studies, pregnancies preceded by preconception care result in one third as many major congenital malformations as pregnancies not preceded by preconception care [31]. The Maine Diabetes in Pregnancy Program found 4-fold lower rates of both congenital anomalies and fetal and neonatal deaths among pregnancies with preconception care [33]. More recently, the Diabetes Complications Control Trial (DCCT) of 1441 adults with Type 1 diabetes randomly assigned to conventional or intensive therapy, including 180 women who became pregnant, reported 8 congenital malformations in the conventional therapy group and only 1 in the intensive therapy group [34]. Thus, in the case of maternal diabetes, randomized trial data confirm the findings of numerous observational studies that control of diabetes prior to pregnancy results in dramatically fewer birth defects.\nThree economic evaluations of preconception and pregnancy care programs for women with non-gestational diabetes were identified in the published literature [35\u201337]. All reported calculations of net short-term economic benefit based on reduced costs of maternal and neonatal hospitalizations and did not take into account long-term averted costs of care associated with the prevention of birth defects. Although the analyses were not developed as business case analyses, the focus on short-term costs and benefits is consistent with that approach. Regrettably, the lack of common metrics of cost and health outcomes makes it difficult to make direct comparisons of assumptions and findings of the three studies.\nThe first study is a prospective analysis of a hypothetical program of comprehensive preconception diabetes care consisting of 20 visits prior to prenatal care [35]. The preconception visits were projected to cost $2,638 per enrollee in 1989 dollars. The investigators calculated that reduced maternal and infant hospitalizations would result in net benefits of $1,720 per enrollee. The majority of the hospitalization cost savings were projected to result from an 8-fold reduction in major congenital malformations, from 3.2 to 0.4%. Using the cost of the intervention as the denominator, the benefit-cost ratio from the health care system perspective can be calculated to be 1.6. This means that, on average, for every $1 spent on the program, reduced hospitalization costs of $1.60 would be expected. Although the projected percentage reduction in major malformations appears high relative to the literature, the projected intervention cost was also rather high, since it is uncertain that 20 visits would be needed for preconception care for women with diabetes.\nThe second study, a retrospective analysis of data for the years 1986\u20131988 from the California Diabetes and Pregnancy Program (CDAPP), found that matched perinatal hospital charges for mother and child were 41% higher for controls than for program participants [36]. The benefit-cost ratio was 5.19, with reduced maternal and infant hospitalization costs of $5.19 observed for each $1 spent on the program. This finding likely contributed to the ongoing funding of the CDAPP. It is difficult to apply these findings to preconception care, though. Only 24 of 102 participants were enrolled in the program prior to 8 weeks of gestation, the time during which major congenital malformations are likely to occur. Although those participants had average charges that were 44% lower than those who enrolled after 8 weeks, women who enrolled in the program prior to conception incurred higher costs per delivery because of the longer time during which services were received and the fact that not all women who enrolled became pregnant. Therefore, positive net financial returns to the component of preconception care were not demonstrated in that study.\nThe third study is a retrospective evaluation of prospectively gathered data on pregnancy-related costs among women with type 1 diabetes who received both preconception care and prenatal care or prenatal care only at selected Michigan hospitals [37]. Women in the preconception care group received an additional two outpatient visits on average, at an added cost of $132. The rate of congenital malformation was 4.2% in the preconception care group compared with 13.5% in the prenatal care-only group, a reduction of 69%. The rate of maternal and neonatal intensive care unit (NICU) hospitalizations was 50% lower in the preconception care group. Combining maternal and child hospitalizations, cost savings averaged $34,000 per woman enrolled in both preconception and prenatal care compared to those who received standard prenatal care. Although no information was presented on the distribution of costs in this study, an implication of the findings is that an integrated health plan could likely achieve cost savings by paying for a modest number of preconception care visits for women with type 1 diabetes.\nDiscussion\nThe economic and business case literature on preconception care is quite limited. There is some evidence of positive financial returns for preconception counseling for women with diabetes, based on savings in hospitalization costs [35\u201337]. Although none of the studies involved randomized assignment, the relative differences in frequencies of birth defects are consistent with findings from other studies and increase the confidence that can be placed in the estimates of cost reductions. None of the studies indicate who was expected to bear the costs of counseling or the magnitude of cost savings to specific stakeholders. The studies are all based on relatively old data; newer studies with more complete accounting of costs would be more informative. Furthermore, determination of the optimal number and cost of preconception visits is essential.\nThe American Diabetes Association [38] and the American College of Obstetricians and Gynecologists [39] recommend that all women of childbearing age with diabetes receive counseling about the importance of glucose control before becoming pregnant. Despite these guidelines and positive economic analyses, many women with diabetes still do not receive such advice. A recent survey of women with diabetes ages 18 to 45 years enrolled in managed care plans found that only about half recalled receiving advice about the need for glucose control prior to pregnancy [40]. Research is needed to determine why some women with diabetes seek out preconception care and adopt intensive glucose control measures and others do not [41]. Potential barriers, such as opportunity cost of time, financial cost (e.g., co-pays), invasive testing, and medical risks [32] need to be analyzed from the consumer perspective. For example, it has been estimated that for patients with type 2 diabetes controlled by oral agents, following all self-care practices recommended by the American Diabetes Association could require more than 2 hours daily [42].\nBetter data are needed to establish the business case for preconception care. Although benefits of preconception care can be projected, data derived from the evaluation of actual preconception care initiatives are needed to make a compelling business case. Further, observational data comparing pregnancy outcomes among women who choose to use preconception care and those who do not are subject to biases due to non-randomized study design. A business case analysis of interventions with both demonstrated efficacy (works if followed) and effectiveness (behaviors are actually changed and lead to improved outcomes) is likely to be more persuasive. In order to develop a compelling business case for preconception care, studies of preconception care interventions accompanied by rigorous evaluations with randomized or matched groups of controls are required.\nPreparing a business case analysis based on intervention data poses challenges. It is logistically difficult but essential to prospectively collect data on costs and outcomes: \u201cTo understand which quality-enhancing interventions are likely to produce positive returns on investments, data collection and analysis must include tracking the investment and operating costs of implementing the intervention as well as the changes in revenues and costs that result from the intervention\u201d [24]. Easily accessible or extant data sets typically do not allow one to calculate costs from the perspective of an individual organization. This requires quantifying actual reimbursements, co-pays, deductibles, etc., rather than using standard charges for a clinic visit. It also requires tracking which patients are covered by different health plans and patterns of enrollment and disenrollment.\nConclusion and the way forward\nResearch demonstrating the effectiveness and financial returns to investments in preconception care is necessary to make a financial business case for preconception care. In particular, the only economic evaluations that have been published to date have been of specific components of preconception care, such as counseling women with diabetes or promoting preconception use of folic acid supplements. Evaluations of the costs and benefits of different \u201cbundles\u201d of preconception care services or comprehensive preconception care are needed. Further, such evaluations must include payer-specific cost data to be relevant to framing the business case for preconception care.\nAlthough more data are needed, such analyses may not be sufficient to ensure access to or provision of preconception care, as the diabetes preconception care experience suggests. Promotion of preconception care through carefully researched health marketing campaigns is also critical [41]. If, as a result of effective health marketing, consumers were to demand preconception care, providers and payers would be more likely to perceive a business case for providing it. Similarly, a business case might also be supported if health care professionals working with women of childbearing age were to advocate that their own organizations provide such services. Health care providers often lack the incentive to adopt a new service that improves outcomes; therefore, particular attention must be paid to the structure of incentives by both public and private payers and purchasers [3].\nPublic health programs should fund studies evaluating the effectiveness of preconception care and develop marketing strategies to promote awareness of strategies that are proven effective. Studies that test and evaluate preconception care strategies should collect the financial data needed to conduct business case analyses [24]. Controlled evaluations of preconception care need to be accompanied by financial analyses to help make the business case for providing and paying for such services. Better quality evidence of both the financial and health costs of poor pregnancy outcomes and the benefits from preconception and early prenatal care is needed to provide a compelling business case.","keyphrases":["economic evaluation","birth defects","cost-benefit analysis","prenatal care","diabetes in pregnancy"],"prmu":["P","P","P","P","P"]}
{"id":"Qual_Life_Res-3-1-2039824","title":"Health status, work limitations, and return-to-work trajectories in injured workers with musculoskeletal disorders\n","text":"Background The purpose of this study was to describe the health status and work limitations in injured workers with musculoskeletal disorders at 1 month post-injury, stratified by return-to-work status, and to document their return-to-work trajectories 6 months post-injury.\nIntroduction\nWork-related musculoskeletal (MSK) disorders are common health problems and a major contributor to disability and costs in working populations. In Canada, MSK disorders are responsible for 10% of the short-term disability costs and 39% of the estimated long-term disability costs [1]. The natural course of low back pain and other MSK disorders is characterized by recurrent disabling symptoms and can be described as chronic-episodic [2\u20136]. Similarly, the trajectory of return-to-work (RTW) following a period of work absence due to MSK disorders is a complex and dynamic process, frequently involving recurrences of work absence [7, 8]. Already a decade ago, it was shown that a first return to work after an injury is frequently followed by one or more recurrences of work absence, making a focus on first return to work a limited and potentially misguiding index of RTW outcomes, and one that does not address the important issue of sustainability of return to work [9, 10].\nRecently, Pransky et\u00a0al. [7] pointed out that \u201cdespite an abundance of RTW research, the concept of RTW is often poorly defined, and there is not substantial agreement about what constitutes a successful RTW outcome.\u201d Many studies have been focused on (first) return to work as the primary outcome measure, e.g., return to work is used as an indicator for a reduction in disability\u2014usually with the assumption that workers who return to work are completely recovered from the disabling effects of the injury [10]. However, several studies have demonstrated that workers who return to work are not fully recovered from their initial complaints or injury [10\u201315]. The traditional outcome measures of return to work and time lost from work do not capture important information about the burden of injury that can be shown by self-reported measures of disability and functional limitations. Hence, to obtain a complete picture of the complex RTW process, capturing the recurrences of work absence, the persistence of disability, and their consequences for work performance, it is important to use multiple outcome measures during follow-up. Although a few studies have addressed health outcomes, such as pain, functional status, and general health, in relation to RTW status [11, 12, 14], little is known about depressive symptoms, which have been suggested to increase the total numbers of days on benefit [16] and about limitations at work in injured workers. Furthermore, it is largely unclear how injured workers \u201ctransit\u201d in their RTW status over time. So far, we do know that a substantial proportion of workers with cumulative trauma disorders of the upper extremity [15] and compensated back pain (C\u00f4t\u00e9 et\u00a0al., submitted) experience multiple episodes of work absence.\nThe purpose of this study was to describe the health status, assessed by multiple outcome measures, and work limitations, in injured workers with MSK disorders 1\u00a0month post-injury stratified by RTW status, and to document their RTW trajectories over a period of 6\u00a0months post-injury.\nMaterials and methods\nStudy design\nThe present study was conducted within the sampling frame of a prospective study of Ontario workers with a back or upper extremity (UE) MSK disorder, who filed a Workplace Safety and Insurance Board (WSIB) lost-time injury claim. Data was collected from two sources: self-reports of participants and WSIB administrative data. The participants were interviewed by phone at baseline (1\u00a0month post-injury), and 6\u00a0months post-injury. Participants provided information on their RTW experience, workplace, healthcare provider, insurer, and physical and mental health. From the WSIB, administrative information on sociodemographics, workplaces, and claims (e.g., site of injury, claim status, time receiving wage replacement benefits) was obtained. This information was linked to the interview data, when the participants provided written consent for linkage. Ethical approval for the study was granted by the University of Toronto\u2019s Ethics Review Board. Participants were given the option to withdraw from the study at any point and to decline data linkage of their questionnaire responses with their WSIB data. It was made explicit to the respondents that study participation would in no way affect their claim with the insurer.\nParticipant recruitment and final study sample\nStudy eligibility required participants to have a new, accepted or pending, back or UE MSK lost-time claim, be absent from work for at least 5\u00a0days within the first 14 calendar days post-injury, and be 15\u00a0years of age or older. We excluded claimants with a fracture, amputation, burn, hernia, head injury, concussion, or electrocution, those who were not able to understand or speak English, and those with a security problem, who were incarcerated or received institutional care. From January to July 2005, a computer program run on WSIB weekly files resulted in the identification of 14,555 potential participants. Eligibility assessment and recruitment were conducted in three stages: at the WSIB tracking level, at the WSIB recruiting level, and the university-based research unit level (see Fig.\u00a01).\nFig.\u00a01Recruitment procedure and flow of participants\nA total of 632 participants completed the baseline interview 1\u00a0month post-injury. The overall response rate was 61% (632 out of 1,038 eligible and contacted potential participants). Verbal consent for the interview data was obtained from all participants. The mean time between the date of injury and the date of interview was 29.6\u00a0days (SD 6.2; median 29\u00a0days, range 15\u201346\u00a0days). Approximately 98% of the participants were interviewed within 6\u00a0weeks post-injury. For the linkage with WSIB data, written consent was obtained from 479 participants, for which WSIB wage replacement data was available for 431 participants. A consent-to-linkage analysis showed that consenters (n\u00a0=\u00a0479) and non-consenters were similar in terms of sociodemographic, workplace, health status, and work absence variables. However, consenters were more likely to have a higher level of education, and male consenters were more likely to be older than male non-consenters (Franche et\u00a0al., submitted).\nDefinition of the RTW status\nAt baseline, four mutually exclusive RTW status groups were constructed, based on the workers\u2019 responses to the following yes\/no questions: \u201cHave you gone back to work at any point since your injury (includes part-time or modified work)?\u201d and \u201cAre you currently working at any job right now?\u201d The four groups were: (1) sustained first return to work (RTW-S), (2) return to work with recurrence(s) of work absence and working at time of interview (RTW-R working), (3) return to work with recurrence(s) of work absence and not working at time of interview (RTW-R not working), and (4) no return to work (No RTW). In the analyses, we collapsed the two return to work with recurrence(s) groups into one group (RTW-R). RTW status was assessed at each follow-up.\nMeasurements: health outcomes and work limitations\nPain intensity\nWe used two items from the Von Korff Pain Scale [17, 18] to measure pain intensity. On a 10-point numerical rating scale (0\u00a0=\u00a0\u201cno pain\u201d to 10\u00a0=\u00a0\u201cpain as bad as could be\u201d), participants were asked to indicate their level of perceived pain from their workplace injury (1) at the present time and (2) on average in the past month.\nFunctional status\nFunctional disability associated with back pain was measured using the Roland\u2013Morris Disability Questionnaire [19], a 24-item questionnaire assessing the presence of activity limitations. Responses to individual items (yes\/no) are summed up and range from 0 (no disability) to 24 (severe disability). The score is averaged and\u2014for a better comparison with scores of other instruments\u2014transformed to a standardized score of 0\u2013100 (by multiplying each averaged score by 100), with a higher score indicating greater disability. The Roland\u2013Morris has been shown to have good psychometric properties [20\u201325]. In the baseline sample, the internal consistency (Cronbachs \u03b1) was 0.92.\nThe 11-item QuickDASH was used to assess physical function and symptoms in participants with MSK disorders of the upper limb [26]. The QuickDASH is a shortened version of the DASH Outcome Measure [27]. The items are scored from 1 to 5. Responses to the individual items are summed, averaged, and transformed to a standardized score of 0\u2013100, with a higher score indicating greater disability. Initial testing has shown that the QuickDASH has good psychometric properties [26]. The internal consistency in the present study was 0.90.\nWhen participants reported pain in both the back and UE, they completed both the Roland\u2013Morris and the QuickDASH. For these participants, scores from each instrument were converted into a z-score and the highest z-score was used as the index of functional status. For participants completing only one measure of functional status, the z-score of that measure was used as the index of functional status. In addition, for those completing both measures, determination of the main pain site, i.e., back or UE, was based on the highest z-score on the Roland\u2013Morris or the QuickDASH.\nGeneral health\nThe Short Form-12 (SF-12), a 12-item version of the SF-36, was used to measure physical (Physical Component Summary Scale Score; PCS12) and mental (Mental Component Summary Scale Score; MCS12) health-related quality of life [28, 29]. To calculate the PCS12 and MCS12, test items are scored and transformed in an algorithm to norm-based scores with a mean of 50 and a standard deviation of 10 [29]. PCS12 and MCS12 scores range from 0 to 100, a higher score indicating better health. The psychometric properties of the SF-12 are good: coefficients for test\u2013retest reliability, measured over 2\u00a0weeks, are 0.89 (PCS12) and 0.76 (MCS12) [28]. Moreover, Luo et\u00a0al. [30] reported good internal consistency, validity, and responsiveness in patients with low back pain. In the present study, the internal consistency was 0.89 (PCS12) and 0.86 (MCS12) at baseline.\nDepressive symptoms\nThe 20-item Center for Epidemiologic Studies Depression (CES-D) [31] scale was used to measure depressive symptoms. The items report the frequency of occurrence of symptoms in the past week on a 4-point rating scale ranging from \u201crarely or none of the time\u201d (<1\u00a0day) to \u201cmost of the time\u201d (5\u20137\u00a0days). The score ranges from 0 to 60 with a higher score denoting more depressive symptoms. CES-D scores \u226516 are indicative of individuals at risk for clinical depression [31]. The internal consistency was 0.92, measured in the baseline sample.\nWork limitations\nWe used the 16-item version of the Work Limitations Questionnaire (WLQ-16) to assess limitations at work due to injury or associated treatment [32\u201335]. The WLQ-16 covers four domains: output demands (4 items), mental demands (6 items), physical demands (4 items), and time management demands (2 items). Items are scored on a 5-point scale, ranging from \u201cnone of the time\u201d to \u201call of the time.\u201d The scores on the individual items are summed, averaged, and transformed to a standardized score of 0\u2013100, with a higher score indicative of more limitations. The internal consistency Cronbachs \u03b1\u2019s were 0.82 (output demands), 0.86 (mental demands), 0.78 (physical demands), and 0.76 (time management demands) at baseline.\nSociodemographics, days off work, and comorbidity\nParticipants provided information on age, gender, education, living status, number of children under the age of 18, and personal income. Information on occupational status was obtained from the WSIB database. One self-reported question assessed how many full days of work a participant had missed due to the injury. In addition, data on time receiving wage replacement benefits was obtained from the WSIB database. The Saskatchewan Comorbidity Scale was used to measure comorbidity (Jaroszynski et\u00a0al., unpublished work). The 16\/14-item (women\/men) self-report scale assesses the presence and severity of health problems. Participants are instructed to indicate whether they currently have a particular health problem\/disease and, if so how much it has affected their health in the last 6\u00a0months. The response options range from 1\u00a0=\u00a0\u201cnot at all\u201d to 4\u00a0=\u00a0\u201csevere.\u201d In the present study, two additional items pertaining to gynecological problems and pregnancy status were added for women. Responses were combined and categorized as: no comorbidity, comorbidity with no\/mild effect on health, and comorbidity with moderate\/severe effect on health.\nStatistical analyses\nUnivariate statistics (means, standard deviations, frequency counts) were used to describe participants, for the total cohort and by RTW status, in terms of their baseline sociodemographics, health outcomes, and work limitations. Differences in baseline characteristics between the three RTW status groups (RTW-S, RTW-R, and No RTW) were tested using a \u03c72 test or analysis of variance. Multiple comparisons, with RTW-S as reference group, were performed with a Tukey correction. Group differences in health outcomes and work limitations, adjusted for identified covariates were tested with GLM analyses, and multiple comparisons were performed with a Bonferroni correction. To describe the RTW trajectories, we used the self-reported RTW status at baseline and 6-month follow-up. All statistical analyses were performed with SPSS 13.0 [36].\nResults\nBaseline characteristics and selection bias analysis\nA total of 632 participants, 350 (55%) men and 282 (45%) women, completed the baseline interview 1\u00a0month post-injury. Table\u00a01 shows the sociodemographic factors for the total sample and by RTW status at baseline. The mean age of the total cohort was 42.2\u00a0years (SD 10.8) and approximately 69% lived with a partner. The mean duration of time receiving wage replacement benefits, based on WSIB data, was 19.1\u00a0days (SD 8.9; median 20\u00a0days). Sixty-six percent of the participants were primarily experiencing back pain and 34% UE pain.\nTable\u00a01Baseline (1\u00a0month post-injury) sociodemographic characteristics, days off work, pain site, and comorbidity for the total study sample (n\u00a0=\u00a0632) and by return-to-work status groupaTotalRTW-SRTW-RNo RTWN\u00a0=\u00a0632N\u00a0=\u00a0293 (46.9%)N\u00a0=\u00a088 (14.1%)N\u00a0=\u00a0244 (39.0%)GenderN(%)N(%)N(%)N(%)\u00a0\u00a0\u00a0\u00a0Female282(44.6)123(42.0)48(54.5)110 (45.1)\u00a0\u00a0\u00a0\u00a0Male350(55.4)170(58.0)40(45.5)134(54.9)Age categories\u00a0\u00a0\u00a0\u00a015\u201329\u00a0years93(14.7)45(15.4)9(10.2)35(14.4)\u00a0\u00a0\u00a0\u00a030\u201339\u00a0years137(21.7)58(19.8)29(33.0)48(19.8)\u00a0\u00a0\u00a0\u00a040\u201349\u00a0years228(36.1)109(37.2)31(35.2)87(35.8)\u00a0\u00a0\u00a0\u00a0\u226550\u00a0years173(27.4)81(27.6)19(21.6)73(30.0)Living with\/without partner\u00a0\u00a0\u00a0\u00a0Living with partner433(68.5)221(75.4)54(61.4)156(63.9)\u00a0\u00a0\u00a0\u00a0Not living with partner199(31.5)72(24.6)34(38.6)88(36.1)Children under age 18\u00a0\u00a0\u00a0\u00a0No children 341(54.0)158(53.9)48(54.5)131(53.7)\u00a0\u00a0\u00a0\u00a01 child118(18.7)53(18.1)17(19.3)47(19.3)\u00a0\u00a0\u00a0\u00a02 children118(18.7)57(19.5)15(17.0)44(18.0)\u00a0\u00a0\u00a0\u00a0\u22653 children 55(8.7)25(8.5)8(9.1)22(9.0)Education\u00a0\u00a0\u00a0\u00a0Some high school112(17.7)46(15.7)16(18.2)49(20.1)\u00a0\u00a0\u00a0\u00a0High school completed177(28.0)75(25.6)30(34.1)69(28.3)\u00a0\u00a0\u00a0\u00a0Some university or college130(20.6)70(23.9)14(15.9)45(18.4)\u00a0\u00a0\u00a0\u00a0University\/college completed213(33.7)102(34.8)28(31.8)81(33.2)Occupational status (N\u00a0=\u00a0479)b\u00a0\u00a0\u00a0\u00a0White collar89(18.6)47 (20.9)13(17.6)29(16.4)\u00a0\u00a0\u00a0\u00a0Pink collar156(32.6)71(31.6)26(35.1)58(32.8)\u00a0\u00a0\u00a0\u00a0Blue collar-indoor99(20.7)54(24.0)15(20.3)30(16.9)\u00a0\u00a0\u00a0\u00a0Blue collar-outdoor68(14.2)28(12.4)10(13.5)28(15.8)\u00a0\u00a0\u00a0\u00a0Missing67(14.0)25(11.1)10(13.5)32(18.1)Personal income\u00a0\u00a0\u00a0\u00a0<$20,00095(15.0)31(10.6)12(13.6)47(19.3)\u00a0\u00a0\u00a0\u00a0$20,000\u201339,999240(38.0)111(37.9)32(36.4)96(39.3)\u00a0\u00a0\u00a0\u00a0$40,000\u201359,999180(28.5)88(30.0)28(31.8)64(26.2)\u00a0\u00a0\u00a0\u00a0>$60,00081(12.8)46(15.7)9(10.2)25(10.2)\u00a0\u00a0\u00a0\u00a0Missing36(5.7)17(5.8)7(8.0)12(4.9)Number of working hours\/week at the time of the injury; n (%)\u00a0\u00a0\u00a0\u00a0\u226437.5179(28.3)84(28.7)24(27.3)69(28.3)\u00a0\u00a0\u00a0\u00a0>37.5\u201340.0281(44.5)128(43.7)40(45.5)110(45.1)\u00a0\u00a0\u00a0\u00a0>40.0172(27.2)81(27.6)24(27.3)65(26.6)Duration of receiving full wage replacement benefits within first 30\u00a0days post-injurycn\u00a0=\u00a0205n\u00a0=\u00a067n\u00a0=\u00a0156\u00a0\u00a0\u00a0\u00a0Days mean (SD)19.1(8.9)14.2(7.1)18.7(8.4)25.9(6.5)\u00a0\u00a0\u00a0\u00a0Days; median20.013.019.029.0Self-reported full days off work due to injury\u00a0\u00a0\u00a0\u00a0Days mean (SD)14.5(7.1)10.5(5.1)14.1(6.9)19.7(6.1)\u00a0\u00a0\u00a0\u00a0Days; median14.010.013.519Pain site; n (%)\u00a0\u00a0\u00a0\u00a0Back418(66.1)214(73.0)64(72.7)134(54.9)\u00a0\u00a0\u00a0\u00a0Upper extremities214(33.9)79(27.0)24(27.3)110(45.1)Comorbidity; n (%)\u00a0\u00a0\u00a0\u00a0No513(81.2)238(81.2)72(81.8)197(80.7)\u00a0\u00a0\u00a0\u00a0Yes, with no\/mild effect on health33(5.2)16(5.5)5(5.7)12(4.9)\u00a0\u00a0\u00a0\u00a0Yes, with moderate\/severe effect on health86(13.6)39(13.3)11(12.5)35(14.4)a\u00a0N\u00a0=\u00a0625 classified with regard to RTW status, N\u00a0=\u00a07 were working at time of the baseline interview, but were not asked item regarding recurrencesb\u00a0WSIB information available for participants who provided informed consent for linkage of WSIB data with questionnaire datac\u00a0N\u00a0=\u00a0431 for WSIB temporary total compensation within 30\u00a0days of accident; Missing data due to (1) no informed consent for linkage of WSIB data with questionnaire data (N\u00a0=\u00a0153) or (2) absence of data when claim was not a lost-time accepted claim (N\u00a0=\u00a048)\nTo examine a possible selection bias, we compared the cohort participants (n\u00a0=\u00a0632) to a group of algorithm-selected potential participants (n\u00a0=\u00a03,712) on characteristics extracted from the WSIB database, where the algorithm mimicked the inclusion criteria of our study.1 The time frame during which their injury occurred was the same as for our study sample. This analysis showed that participants were comparable to potential participants with regards to firm size, industrial sector, and income level (Table\u00a02). However, participants were more likely to be older and female. Women aged 40\u201349 were more likely to participate than women in the other age categories, and older men were more likely to participate than younger men. With respect to claim status, we compared only participants with accepted claims and available wage replacement data (n\u00a0=\u00a0559) with potential participants, since this data is not available for participants with pending, denied or abandoned claims. Participants were more likely to have a longer duration on wage replacement benefits at 1 and 6\u00a0months post-injury and a higher rate of wage replacement re-instatement at 6\u00a0months post-injury than potential participants, suggestive of more severe work disability in our cohort.\nTable\u00a02Comparison of baseline study participants with algorithm-selected potential study participantsVariableFull baseline cohort (n\u00a0=\u00a0632)Algorithm-selected potential participants (n\u00a0=\u00a03,712)Gender, n (%)\u00a0\u00a0\u00a0\u00a0Female282 (44.6)1,365 (36.8)\u00a0\u00a0\u00a0\u00a0Male350 (55.4)2,347 (63.2)Age at baseline interview, mean (SD) (median)42.2 (10.8) (43)40.3 (11.3) (40.7)Gender\u00a0\u00d7\u00a0age at baseline interview, n (%)\u00a0\u00a0\u00a0\u00a0Females: 15\u201329\u00a0years40 (6.3)262 (7.1)\u00a0\u00a0\u00a0\u00a0Females: 30\u201339\u00a0years59 (9.3)348 (9.4)\u00a0\u00a0\u00a0\u00a0Females: 40\u201349\u00a0years109 (17.2)428 (11.5)\u00a0\u00a0\u00a0\u00a0Females: \u226550\u00a0years74 (11.7)327 (8.8)\u00a0\u00a0\u00a0\u00a0Males: 15\u201329\u00a0years53 (8.4)524 (14.1)\u00a0\u00a0\u00a0\u00a0Males: 30\u201339\u00a0years78 (12.3)630 (17.0)\u00a0\u00a0\u00a0\u00a0Males: 40\u201349\u00a0years119 (18.8)714 (19.2)\u00a0\u00a0\u00a0\u00a0Males: \u226550\u00a0years99 (15.7)479 (12.9)Firm size, n (%)\u00a0\u00a0\u00a0\u00a0<20 employees58 (9.2)335 (9.0)\u00a0\u00a0\u00a0\u00a020\u201399 employees100 (15.8)680 (18.3)\u00a0\u00a0\u00a0\u00a0100\u2013999 employees190(30.1)1,166 (31.4)\u00a0\u00a0\u00a0\u00a0\u22651,000 employees150 (23.7)769 (20.7)\u00a0\u00a0\u00a0\u00a0Schedule 2134 (21.2)761 (20.5)Industrial sector, n (%)\u00a0\u00a0\u00a0\u00a0Automotive, manufacturing, steel109 (17.2)875 (23.6)\u00a0\u00a0\u00a0\u00a0Service127 (20.1)812 (21.9)\u00a0\u00a0\u00a0\u00a0Education, municipal, Schedule 2a146 (23.1)817 (22.0)\u00a0\u00a0\u00a0\u00a0Healthcare85 (13.4)425 (11.4)\u00a0\u00a0\u00a0\u00a0Transportation55 (8.7)333 (9.0)\u00a0\u00a0\u00a0\u00a0Chemical\/processing, electrical, food31 (4.9)212 (5.7)\u00a0\u00a0\u00a0\u00a0Construction14 (2.2)154 (4.1)\u00a0\u00a0\u00a0\u00a0Agriculture, forest, pulp and paper, mining15 (2.4)83 (2.2)\u00a0\u00a0\u00a0\u00a0Unknown50 (7.9)1 (0.0)Occupational status, n (%)b\u00a0\u00a0\u00a0\u00a0White collar109 (18.7)624 (16.8)\u00a0\u00a0\u00a0\u00a0Pink collar209 (35.9)1,112 (30.0)\u00a0\u00a0\u00a0\u00a0Blue collar-indoor132 (22.7)1,026 (27.6)\u00a0\u00a0\u00a0\u00a0Blue collar-outdoor86 (14.8)631 (17.0)\u00a0\u00a0\u00a0\u00a0Missing45 (7.7)319 (8.6)Weekly earnings in tertiles, n (%)\u00a0\u00a0\u00a0\u00a0\u2264$447.68116 (18.4)819 (22.1)\u00a0\u00a0\u00a0\u00a0$447.68\u2013\u2264$880.00281 (44.5)1,890 (50.9)\u00a0\u00a0\u00a0\u00a0\u2265$880.00157 (24.8)978 (26.3)\u00a0\u00a0\u00a0\u00a0Missing78 (12.3)25 (0.7)Duration of time receiving wage replacement benefits (30\u00a0days post-injury), mean (SD) (median)c20.6 (9.0) (25)15.9 (9.4) (13)Duration of time receiving wage replacement benefits (180\u00a0days post-injury), mean (SD) (median)d58.7 (57.2) (33)37.6 (50.2) (14)Occurrence of re-instatement of wage replacement benefits (30\u00a0days post-injury), n (%)c\u00a0\u00a0\u00a0\u00a0No507 (90.7)3,481 (93.8)\u00a0\u00a0\u00a0\u00a0Yes52 (9.3)231 (6.2)Occurrence of re-instatement of wage replacement benefits (180\u00a0days post-injury), n (%)d\u00a0\u00a0\u00a0\u00a0No463 (82.5)3,286 (88.5)\u00a0\u00a0\u00a0\u00a0Yes98 (17.5)426 (11.5)Claim status (30\u00a0days post-injury), n (%)\u00a0\u00a0\u00a0\u00a0Lost-time-accepted431 (68.2)2,893 (77.9)\u00a0\u00a0\u00a0\u00a0Lost-time-pending171 (27.1)765 (20.6)\u00a0\u00a0\u00a0\u00a0Lost-time-denied19 (3.0)43 (1.2)\u00a0\u00a0\u00a0\u00a0Lost-time-amalgamated5 (0.8)1 (0.0)\u00a0\u00a0\u00a0\u00a0No lost-time-accepted6 (0.9)6 (0.2)a\u00a0Schedule 2 firms do not operate under the collective liability insurance principle, and, as such, are individually responsible for the full cost of the injury\/illness claims filed by their workers. Schedule 2 employers include federal, provincial and municipal governments, railways, airlines, shipping, and telephone companiesb\u00a0Data on the full baseline cohort is restricted to participants with accepted claims (n\u00a0=\u00a0582)c\u00a0Data on the full baseline cohort is restricted to participants with accepted claims and available wage replacement data 30\u00a0days post-injury (n\u00a0=\u00a0559)d\u00a0Data on the full baseline cohort is restricted to participants with accepted claims and available wage replacement data 180\u00a0days post-injury (n\u00a0=\u00a0561)Note: Italics\u2013The main differences between the full baseline cohort and the algorithm-selected potential participants\nGroup differences in baseline characteristics\nA total of 625 participants were categorized into one of the four RTW status groups. The remaining seven participants were working when interviewed at baseline, but not asked about recurrence(s) due to an error in a skip pattern of the questionnaire, which was subsequently corrected. At baseline (approximately 1\u00a0month post-injury), 47% of the participants reported a sustained first return to work, 5% a return to work with recurrence(s) of work absence and working at time of interview, almost 9% a return to work with recurrence(s) of work absence and not working at time of interview, and 39% no return to work.\nWith regards to gender, age, children under age 18, education, occupational status, and working hours per week at the time of the injury no statistically significant differences were observed across the three RTW groups. However, participants who had a sustained first return to work reported more often that they lived with a partner (\u03c72\u00a0=\u00a011.0, p\u00a0=\u00a0.004) and reported a higher personal income (\u03c72\u00a0=\u00a011.7, p\u00a0=\u00a0.069) than those with a recurrence or no return to work. These variables were used as covariates in subsequent analyses.\nWith respect to the mean duration of time receiving wage replacement benefits, based on WSIB data, significant differences were observed across all three RTW groups (F\u00a0=\u00a0122.6, p\u00a0=\u00a0.000). Significant differences were also seen with self-reported full days off work due to the injury (F\u00a0=\u00a0169.7, p\u00a0=\u00a0.0000). With respect to pain site, a statistically significant difference was found across the RTW groups: participants with a sustained first return to work and those who experienced a recurrence reported low back pain more often, and participants who did not return to work reported pain in the UE more often (\u03c72\u00a0=\u00a021.6, p\u00a0=\u00a0<.0001). A total of 81% of the participants reported no comorbidity, whereas 5% reported no\/mild effects on health, and 14% reported moderate\/severe effects on health, with no statistically significant group differences.\nGroup differences in health outcomes and work limitations\nTable\u00a03 presents the adjusted (for age, gender, living status, and income level) estimated means for baseline health outcomes and work limitations by RTW status, with multiple comparison results. Participants with a sustained first return to work reported significantly less pain compared to those with a recurrence and no return to work. Moreover, they also reported significantly less pain in the past month compared to those with no return to work. In participants with back pain, those with a sustained first return to work reported significantly less functional disability compared to those with a recurrence and no return to work. In participants with UE pain, we observed that those with a sustained first return to work reported significantly less functional disability compared to those who did not return to work, but not compared to those who experienced a recurrence. With regards to physical and mental health as well as depressive symptoms, participants with a sustained first return to work reported significantly better health and fewer depressive symptoms than those with a recurrence and no return to work. It is interesting to note that high levels of depressive symptomatology, indicative of being at risk for clinical depression, were found in all participants, especially in those with a recurrence and those who did not return to work. For all outcomes, there were no significant differences between participants who experienced a recurrence and those who did not return to work. With regards to limitations at work, those with a sustained first return to work and those with a recurrence, reported limitations in all domains, but mainly for physical demands and time management demands. As expected, participants with a sustained first return to work reported significantly fewer limitations than those with a recurrence.\nTable\u00a03Estimated means (95% confidence intervals, CI) of baseline (1\u00a0month post-injury) health outcomes and limitations at work by return-to-work status group (N\u00a0=\u00a0625), adjusted for age, gender, living status, and personal incomeRTW-SaRTW-RNo RTWN\u00a0=\u00a0293 (46.9%)N\u00a0=\u00a088 (14.1%)N\u00a0=\u00a0244 (39.0%)Estimated mean (95% CI)Estimated mean (95% CI)pEstimated mean (95% CI)pPerceived painRange 0\u201310\u00a0\u00a0\u00a0\u00a0Pain at time of interview3.84 (3.54\u20134.13)5.19 (4.64\u20135.73).0005.94 (5.62\u20136.27).000\u00a0\u00a0\u00a0\u00a0Pain in the past month8.45 (8.25\u20139.13)8.78 (8.40\u20139.16).4218.90 (8.68\u20139.13).014Roland Morris\u00a0\u00a0\u00a0\u00a0Range 0\u2013100; N\u00a0=\u00a041852.73 (49.29\u201356.17)68.14 (61.75\u201374.54).00075.66 (71.30\u201380.03).000Quick DASH\u00a0\u00a0\u00a0\u00a0Range 0\u2013100; N\u00a0=\u00a021447.58 (42.61\u201352.55)54.94 (46.01\u201363.87).46963.15 (58.98\u201367.33).000Physical SF-12\u00a0\u00a0\u00a0\u00a0Range 0\u201310037.03 (35.98\u201338.09)32.75 (30.80\u201334.69) .00031.63 (30.47\u201332.78).000Mental SF-12\u00a0\u00a0\u00a0\u00a0Range 0\u201310048.55 (47.18\u201349.91)44.40 (41.88\u201346.92).01445.58 (44.08\u201347.07).013Depressive symptoms\u00a0\u00a0\u00a0\u00a0CES-D; Range 0\u20136013.16 (11.74\u201314.59)18.15 (15.53\u201320.78).00318.70 (17.14\u201320.26).000Work limitationsRange 0\u2013100\u00a0\u00a0\u00a0\u00a0Physical demands46.00 (42.79\u201349.22)62.81 (56.80\u201368.82) .000n.a.\u00a0\u00a0\u00a0\u00a0Mental demands17.12 (14.70\u201319.55)29.41 (24.88\u201333.95).000n.a.\u00a0\u00a0\u00a0\u00a0Output demands18.65 (15.95\u201321.35)35.59 (30.56\u201340.61).000n.a.\u00a0\u00a0\u00a0\u00a0Time management41.83 (38.43\u201345.23)61.90 (55.61\u201368.20).000n.a.a\u00a0The RTW-S group is the reference group for the multiple comparisons\nAttrition analysis\nSix months after injury, the 632 participants who had completed the baseline interview were approached again to complete the follow-up interview. Overall, 446 participants, 238 (53%) men and 208 (47%) women, completed the follow-up interview (retention rate of 70.6%). Reasons for non-response in the follow-up interview were \u201cunable to contact\u201d (n\u00a0=\u00a092), \u201cavoided contact\u201d (n\u00a0=\u00a049), and \u201crefused to participate\u201d (n\u00a0=\u00a045). An attrition analysis, comparing respondents (n\u00a0=\u00a0446) of the 6-month interview with non-respondents (n\u00a0=\u00a0186), revealed that non-respondents were more likely to be younger, to work longer hours at the time of injury, and to specify \u201cback\u201d as their primary pain site. Moreover, male non-respondents tended to be younger than male respondents, whereas in females differences in age were not statistically significant. Otherwise, non-respondents did not differ significantly with respect to other sociodemographic, workplace, health status, and work absence variables tested, including time receiving wage replacement benefits, re-instatement of wage replacement benefits 6\u00a0month post-injury, self-reported work absence duration 1\u00a0month post-injury, and claim status. Full details of the attrition bias analysis have been reported elsewhere (Franche et\u00a0al., submitted).\nRTW trajectories from baseline to 6\u00a0month follow-up\nFigure\u00a02 shows the RTW trajectories for 439 participants, based on self-reported RTW status at baseline and 6\u00a0month follow-up. The majority (73%) of workers with a sustained first return to work at baseline were still at work 6\u00a0months later. However, 27% had experienced at least one recurrence during that time period. All participants who experienced a recurrence remained, by definition, in this group. Of those participants who had not returned to work at baseline, 59% had a sustained first return to work 6\u00a0months later, 17% had made a RTW attempt with a recurrence, and 24% were still off work 6\u00a0months post-injury. Six months post-injury, the rate of recurrences of work absence in workers who had made at least one RTW attempt was 38% [n\u00a0=\u00a0153 recurrences\/(n\u00a0=\u00a0439 minus n\u00a0=\u00a040 with no RTW attempt)].\nFig.\u00a02Return-to-work trajectories based on self-reported return-to-work status at baseline (1\u00a0month post-injury) and 6-month follow-up (n\u00a0=\u00a0439). aRTW-S, sustained first return to work; bRTW-R working, return to work with recurrence(s) of work absence and working at time of interview; cRTW-R not working, return to work with recurrence(s) of work absence and not working at time of interview; dNo RTW, no return to work\nDiscussion\nThe findings of this cohort study suggest the presence of a pattern in baseline health states and work limitations specific to RTW status, 1\u00a0month post-injury. Workers with a sustained first return to work reported less pain, less functional disability, better physical and mental health, fewer depressive symptoms, and fewer work limitations compared to those who experienced a recurrence of work absence or who never returned to work. The study adds to the literature by demonstrating that depressive symptoms and limitations at work are prevalent in workers 1\u00a0month post-injury, including in those with a sustained first return to work. A substantial rate of recurrences of work absence over 6\u00a0months was found (38%), even in workers who had initially made a sustained first return to work at baseline (27%). Moreover, of those workers who did not return to work at baseline, 17% attempted to return and experienced a recurrence within 6\u00a0months of the injury, and 24% were still off work at 6\u00a0months post-injury.\nOur findings are consistent with previous research suggesting that a return to work does not translate into a complete recovery from a MSK disorder [11, 12, 14, 15]. For example, in a study among 205 workers with MSK injury, Evanoff et\u00a0al. [14] found that 88% of the workers had returned to work (with 83% working at full duty) at 6\u00a0month follow-up, and of these 24% reported continuing disability due to the injury (including 20% of those working at full duty). In our study, participants with a sustained first return to work at 1\u00a0month post-injury had SF-12 physical and mental health subscales scores below the healthy population average of 50 [29], and high levels of work limitations, specifically for physical demands and time management demands. Thus, the results suggest that many workers, while back at work, still have health problems and experience difficulties in meeting their work demands. Furthermore, the importance of measuring multiple health outcomes, in particular depressive symptoms, was shown by the high levels of depressive symptoms, indicative of being at risk for clinical depression, in injured workers suffering from MSK disorders, particularly in those who experienced a recurrence and those who did not return to work. Our finding of high levels of depressive symptoms in injured workers is in line with earlier studies showing that depressive symptoms are prevalent in MSK-injured workers [37, 38] and highlights the need to address and examine the mental health of workers suffering a workplace injury. Moreover, 6\u00a0months post-injury we found a high rate of recurrences of work absence (38%) in workers who had made at least one RTW attempt, which is consistent with studies suggesting that there is considerable movement in and out of work after the first return to work [15 C\u00f4t\u00e9 et\u00a0al., submitted]. In a study among 1,321 US workers who filed a workers compensation claim for back pain, C\u00f4t\u00e9 et\u00a0al. (submitted) observed that 23% and 30% of the workers, 6 and 12\u00a0months post-onset of back pain, respectively, experienced multiple work absences.\nWhen interpreting the results, the following methodological issues must be considered. Though reasonable for a study among claimants, the overall participation rate of 61% raises the question of selective participation, which may have biased the results. However, the cohort was shown to be representative of the most comparable claimant group with regards to basic demographic and workplace variables, but not with regards to duration of time receiving wage replacement benefits and rates of wage replacement re-instatement, suggesting the presence of more severe disability in the cohort. Hence, the generalizability of the results remains limited with respect to workers with less severe work disability. More importantly, the rates of self-reported recurrence of work absence may be inflated in our cohort.\nA related issue concerns the loss-to-follow-up of 29%. The attrition analysis demonstrated that non-respondents and respondents were similar with regards to time receiving wage replacement benefits, the occurrence of re-instatement of wage replacement benefits, and self-reported work absence duration. Non-respondents were younger males, worked longer hours, and were more likely to specify \u201cback\u201d as their primary pain site compared to respondents.\nFuture research should further explore the relationship between recurrence(s) of work absence, health outcomes, and work limitations over an extended period of time. We found that workers who experienced a recurrence after a first return to work clearly report more health problems and work limitations than those with a sustained first return to work, and their health status is often comparable to workers who do not return to work. Our trajectory analyses were based on a 6\u00a0months time window and it was not yet possible to examine multiple recurrences and their effects on health outcomes and work limitations over a longer period of time. However, 12\u00a0month follow-up data will make such analyses possible in the future. Moreover, we have to examine important and meaningful changes in health outcomes and work limitations between baseline and follow-up across the possible RTW trajectories [39], and also study them in relation to a broad range of factors (e.g., RTW interventions, disability management strategies) that might have influenced the RTW process and the outcomes considered. Finally, future research should identify early prognostic factors of the trajectories, particularly focusing on the \u201cproblematic\u201d trajectories (recurrences and persistent work absence), so that guidance for an optimal reintegration or for recurrence prevention can be provided.\nTo conclude, the results of this prospective study suggest that workers who had a sustained first return to work report a better health status and fewer work limitations than those who experienced a recurrence after a first return to work or who did not return to work. However, it is also demonstrated that a return to work is not equivalent to a complete recovery from MSK disorders. Workers who had a sustained first return to work still reported meaningful health limitations, as compared to the general population and significant work limitations. Given the considerable impact of disability on worker health and costs for the workers, employers, and society, the findings highlight the importance of considering multiple health outcomes, including depressive symptoms and work limitations, when studying the complex process of return to work and when developing RTW interventions and disability management strategies.","keyphrases":["work limitations","musculoskeletal disorders","return to work","health outcomes","workers\u2019 compensation"],"prmu":["P","P","P","P","R"]}
{"id":"Eur_Spine_J-2-2-1602204","title":"Dorsal dumb-bell melanotic schwannoma operated on by posterior and anterior approach: case report and a review of the literature\n","text":"The schwannomas are benign tumors originating from Schwann cells which constitute the nerve sheath. The dorsal pigmented type of schwannomas is relatively rare (Goldstein in Am J Med Genet 86:62\u201365, 2004; Kurtkaya-Yapicier in Histol Histopathol 18(3):925\u2013934, 2003). There are two distinct types of melanotic schwannoma: the sporadic melanotic schwannomas and the psammomatous melanotic schwannomas of Carney complex. We report a case of a patient harboring a right dorsal dumb-bell melanotic schwannoma and left adrenal mass. The patient underwent a surgical procedure for en bloc total removal of the mass by a posterior and anterior approach. Histopathological examination revealed the diagnosis of melanotic schwannoma. At present, we have no reliable marker of histopathological malignancy of melanotic schwannoma so the follow-up period ought to continue for a period of more than 5 years. As the total removal of the melanotic neurinomas is mandatory to prevent possible malignant transformation of the tumor recurrence or regrowth, we believe that the posterior and trans-thoracic approach are the most suitable one for the dorsal dumb-bell schwannoma.\nIntroduction\nDorsal dumb-bell melanotic schwannomas are very rare [13]. They are rather frequently associated to Carney\u2019s complex [8\u201310]. They present problems on different aspects, such as pre-operative diagnosis, surgical approach and post-operative course. We describe the case of a 53-year-old man who presented with left adrenal hyperplasia and right paravertebral mass suggesting a dumb-bell schwannoma.\nCase report\nPresentation and examination\nIn January 2003, a 53-year-old salesman, heavy smoker with an elevate level of arterial hypertension, began to complain about pain in his right chest and upper limb. Chest X-ray and thoracic CT scan in the following February revealed a D9\u2013D10 dumb-bell mass extending into the intrathoracic right space for about 2.5\u00a0cm. The patient was admitted to our clinic. A spinal dorsal MRI confirmed the presence of the voluminous D9\u2013D10 right paravertebral mass (Figs.\u00a01, 2). Abdomen MRI showed an almost 4\u00a0cm swelling of the left suprarenal gland, suggesting a pheochromocytoma (Fig.\u00a03). The neurologic examination and brain MRI were normal. The renal arterial echodoppler, the dosing of vanilmandelic acid, alpha-fetoprotein, carcinoembryonic antigen, urinary catecholamine and cortisol were normal. The renin activity and the plasmatic level of aldosteron resulted normal in orthostasis and increased in recumbency. The medullar adrenal gland scintigrams (123 IIs MIBG-148 MBqs e.v.) resulted negative for pheochromocytoma. Therefore, we excluded a pheochromocytoma and the patient underwent a surgical procedure for total removal of the dorsal mass by posterior and anterior approach in collaboration with the thoracic surgeons.\nFig.\u00a01MR images demonstrating a right dumb-bell intra-extradural mass at T9\u2013T10 level extending into the intrathoracic right space for about 2.5\u00a0cm. Coronal and axial T1-weighted images with Gd-DTPA enhancementFig.\u00a02Sagittal T2-weighted MRI showing T9\u2013T10 right intervertebral foramen occupied by the tumorFig.\u00a03Coronal abdominal spectral fat saturation inversion recovery (SPIR) MR images showing left suprarenal swelling\nOperation\nA D9\u2013D10 laminectomy after radioscopic control was performed. A right dumb-bell intra-extradural mass of blackish aspect was seen. En bloc total removal of a markedly melanotic intradural lesion was achieved using operating microscope. Then the patient was placed in the left lateral decubitus and by trans-thoracic approach the paravertebral mass was radically removed en bloc, also freeing the right D9\u2013D10 intervertebral foramen from the black and well-encapsulated neoformation. Accurate plastic of the paravertebral pleural wall was obtained. Histological examination revealed melanotic schwannoma with mitotic index not superior to one for ten fields on high magnification indicating low risk for malignancy.\nPostoperative course\nPost-operative course was uneventful, with marked improvement of the painful symptomatology and good healing of the surgical wounds. The patient was discharged from our ward after 10\u00a0days, with a recommendation for ichnographic controls of the left adrenal gland hyperplasia. During a 2-year follow-up period, no recurrence was evident on surveillance MRI.\nDiscussion\nThe schwannomas are benign tumors originating from Schwann cells which constitute the nerve sheath. The first case was described by Bjornboe [13, 15] in 1934 in a patient with neurofibromatosis type I. There are two distinct types of melanotic schwannoma: the sporadic and the psammomatous melanotic schwannomas of Carney complex [8, 14, 24, 27, 29, 36]. In 1985, Carney described a syndrome characterized by mixomas (heart, skin and breast), mucus-cutaneous spotty pigmentations and endocrine overactivity (due to thyroid dysfunction or hypophysial adenoma or Cushing syndrome secondary to adrenocortical pigmented primary nodular hyperplasia) [8, 9].\nA year later, a Mendelian dominant heredity was defined responsible for the symptomatologic complex [43]. About 50% of patients with melanotic psammomatous schwannomas are affected by the syndrome of Carney and one-fifth of them are carriers of multiple lesions [21, 42]. Generally, melanotic schwannoma affects the spinal nerves, the central nervous system and the autonomous nerve system, but extra-nervous locations can also exist. The dorsal location represents 30.5% of the spinal melanotic schwannoma [41]. Until today we were able to find 47 spinal cases of extramedullary melanotic neurinoma in literature (Table\u00a01) [1, 2, 3, 5, 6, 7, 11, 12, 13, 16, 18, 20, 21, 26, 28, 30, 31, 33, 34, 35, 38, 39, 44, 45]. The clinical presentation is not specific but similar to that of other extramedullary spinal tumors (radicular pain, dysesthesias, progressive sensorial-motor deficits). Preoperative diagnosis is based on magnetic resonance imaging (MRI): melanotic schwannomas appear hyperintense on T1-weighted sequences and hypointense on T2-weighted sequences compared to the nonmelanotic type of schwannomas that are hypo-isointense on T1 and hyperintense on T2 [1, 13, 17, 25, 37].\nTable\u00a01Review of 47 cases of spinal extramedullary melanotic schwannoma reported in literatureReferencesAgeGenderLevelAprile et al. [1]70FL3Bagchi et al. [2]40MT6\u2013T7Belak et al. [3]44\u2013DorsalBosman et al. [4]43ML4\u2013L5Bouziani et al. [5]46MLumbarBuhl et al. [6]28ML5\u2013S1Bunc et al. [7]\u2013\u2013T12\u2013L2Cornejo et al. [10]36ML3\u2013L4Di Gregorio et al. [11]\u2013\u2013SacralErlandson et al. [12]36ML5\u2013S1Goasguen et al. [13]66FC2\u2013C3Graziani et al. [15]40FT3Gregorios et al. [16]45FT2Iizuka et al. [18]58FT10Killeen et al. [19]26FS1Krichen et al. [20]27MC6\u2013C7Kuchelmeister et al. [21]53FC5\u2013C6Le Cam et al. [23]35FL4Leger et al. [24]36MC4Lowman et al. [26]17FT12\u2013L1Lowman et al. [26]26FC6Ludvikova et al. [27]56FSpineLudvikova et al. [27]58MSpineMandybur et al. [28]59MT7Martin-Reay et al. [29]32MSacrumMcGavran et al. [30]12FT2McGavran et al. [30]49FC8Mennemeyer et al. [31]23FL1Mennemeyer et al. [31]25MT7Mennemeyer et al. [31]36MS1Parent et al. [33]63FS1Paris et al. [34]49FC8Parker et al. [35]18FDorsalPrieto-Rodriguez et al. [36]38MT5Schmitz et al. [38]42FCervicalTawk et al. [39]61MT7Vallat-Decouvelaere et al. [41]5 casesF and MC\u2013T\u2013L\u2013SZhang et al. [44]5 casesF and MSpinal nerve rootZonenshayn et al. [45]27FL2 and L4\u2013L5\nThe histological differential diagnosis with the other spinal pigmented tumors is with the meningeal melanocitoma, the pigmented neurofibroma, the paraganglioma, the ganglioneuroma, the fibrous form of meningioma and above all with the primitive melanoma or relative metastasis [13, 15]. Even if the association of the left adrenal swelling and arterial hypertension with the dumb-bell thoracic melanotic schwannoma can be correlated to the Carney complex, in our opinion our patient did not harbor such a syndrome. In fact only the adrenocortical pigmented primary nodular hyperplasia is a characteristic stigmata of Carney complex. Moreover, our patient did not show cutaneous signs of neurofibromatosis and his family history was negative for Carney complex stigmata. The total body CT scan showed the left adrenal mass and excluded any other typical lesion of the syndrome. The increased plasmatic level of renin and aldosteron in recumbency in our patient, that was on heavy medical therapy for high level of arterial blood pressure, may be correlated to left adrenal hyperplasia or solitary adrenal adenoma [40] and not to adrenocortical pigmented nodular hyperplasia, which is the cause of ACTH-independent adrenal Cushing\u2019s syndrome [8] and normally do not trigger elevation of plasmatic aldosteron. Moreover, in our case the histological examination of the neurinoma did not show the most common, psammomatous aspect of Carney complex [8, 13]. In summary, we did not perform a left adrenal biopsy as we ruled out the Carney syndrome and malignancy of the adrenal mass by clinic and radiological investigations. Hypertension, adrenal mass and dumb-bell melanotic schwannoma do not imply Carney complex.\nIn our patient the hypertension was not correlated to the Carney complex, but to the hyperaldosteronism caused by left adrenal hyperplasia or solitary adrenal adenoma. Nevertheless, neurosurgeons ought to keep in mind the connection between the melanotic schwannoma and the Carney complex, since more than 50% of the cases described in literature have been related to such syndrome. A complete endocrinological evaluation and a total body CT should always be performed together with a genetic study in the suspicion of Carney complex. The risk of possible malignant transformation of the neurinoma should also be taken into consideration as cases of recurrences or metastases have been described even 13\u00a0years after surgery [4, 6, 19, 21, 23, 32, 41]. It is not possible to have any reliable histopathological indication of possible malignant transformation after the first surgical operation [42]. The follow-up ought to continue for a period of more than 5\u00a0years. We have both to check the primitive tumor regrowth or recurrence and the possible remote locations, particularly in the lungs.\nFrom the surgical point of view the total removal of the melanotic neurinomas is mandatory to prevent possible malignant transformation of the tumor remnants. The therapeutic potential of radiotherapy is not proven [42]. The prognosis depends, therefore, almost entirely on the extent of surgical removal of the tumor [3, 44]. So we believe that the posterior and anterior approach is the most suitable one to remove the dorsal dumb-bell schwannoma completely, both in the thoracic cavity and in the intervertebral foramen, and to avoid possible liquoral leakage.","keyphrases":["dorsal dumb-bell melanotic schwannoma","spinal tumor","carney syndrome","thoracic nerve sheath tumor"],"prmu":["P","P","P","R"]}
{"id":"Eur_J_Health_Econ-_-_-1388082","title":"Determining the \u201cHealth Benefit Basket\u201d of the Statutory Health Insurance scheme in Germany\n","text":"The issue of defining health benefit catalogues has recently gained new importance in Germany as a result of the creation of the new Institute for Quality and Efficiency. The Institute was designed to support the Federal Joint Committee conducting effectiveness studies for benefit coverage decisions. The Committee and the contractual partners (sickness funds and providers) define the benefit catalogues for the Statutory Health Insurance in the framework of Social Code Book V, Germany\u2019s most relevant health care scheme. Unlike other countries, the German federal government limits its regulatory role to defining procedures that determine the scope of Statutory Health Insurance services. The explicitness of the benefit catalogues varies greatly between different sectors. While benefits in outpatient care are rather explicitly defined, benefit definitions for inpatient care are vague. It is argued that the establishment of the new Institute and the development of the DRG system are initial steps towards a more effective and explicit benefit catalogue.\nOrganizational structure and actors involved in the definition of benefit catalogues\nA fundamental aspect of the German health care system is the sharing of decision-making powers between the federal government, the individual states, and designated self-governmental institutions. Responsibilities are traditionally delegated to membership-based, self-regulated institutions of payers and providers that are involved in financing and delivering health care. In the largest scheme (which covers 88% of the population), the Statutory Health Insurance (SHI), sickness funds, their associations and associations of SHI-affiliated physicians and dentists are recognized as quasipublic corporations. These corporatist bodies constitute the self-regulated structures that operate the financing and delivery of benefits covered by the SHI scheme within the legal framework of the Social Code Book (SGB) V [1].\nIn joint committees of payers (associations of sickness funds) and providers (associations of physicians and\/or dentists and\/or the Hospital Federation) legitimized actors define benefits, prices, and standards (federal level) and negotiate horizontal contracts to control and sanction their members (regional level). The vertical implementation of decisions taken at senior levels is combined with strong horizontal decision making and contracting among the legitimized actors involved in the various care sectors [2].\nPhysicians treating SHI-insured patients are organized into 17 regional physicians\u2019 associations. The Federal Association of SHI Physicians is responsible for cooperation on the federal level. SHI-accredited dentists are organized the same way as physicians through 17 dentists\u2019 associations and the Federal Association of SHI Dentists. The German Hospital Federation is also involved in the decision-making process.\nThe payers\u2019 side is made up of autonomous sickness funds organized on a regional and\/or federal basis. They are obliged to raise contributions from their members and to determine the contribution rate necessary to cover expenditures. Their responsibilities include contracting, negotiating prices, quantity and quality assurance measures. Services covered by such contracts are usually accessible to all fund members without any prior approval by the fund, except for preventive spa treatments, rehabilitative services and short-term home nursing care. If there is any doubt, the sickness funds must obtain an expert opinion on the medical necessity for treatment from the Medical Review Board, which serves as a joint institution of the sickness funds.\nThe most important body in the benefit negotiations between sickness funds and physicians concerning the scope of benefits is the Federal Joint Committee. Based on the legislative framework the Committee issues directives relating to all sectors of care. The main body of the Committee consists of nine representatives of the federal associations of sickness funds, nine representatives from provider groups, two neutral members with one proposed by each side, and a neutral chairperson-accepted by both sides. In addition, nine nonvoting representatives of formally accredited patient organizations have the right to participate in consultations, and to propose issues to be assessed and decided upon. The directives of the Committee are legally binding for all actors in the SHI scheme. These directives primarily concern the coverage of benefits and assure that SHI services are adequate, appropriate, and efficient.\nThe actual criteria defining benefits vary widely between sectors and types of catalogues. The most important benefit catalogues in the German SHI scheme and its underlying criteria are displayed in Tables\u00a01 and 2. This article concentrates on HC1 (services of curative care) of the International Classification for Health Accounts (ICHA) taxonomy [3].Table\u00a01 Catalogues and criteria used for defining benefitsLegal statusDecision maker(Original) purposePositive\/negative definition of benefitsDegree of explicitnessaIf itemized: goods, procedures only; linked to indicationsUpdatingCriteria used for defining benefitsNCECEBOtherSHI GBR LawParliamentEntitlementsP\/N1\/2\u2013When necessary+\u2013\u2013\u2013\u2013\u2013SHI FJC general directivesDirectiveFJCEntitlementsP2\u2013When necessary+++(+)b\u2013\u2013SHI FJC special directives: positiveDirectiveFJCEntitlementsP2\/3Goods, procedures, indicationWhen necessary+++(+)b\u2013\u2013SHI FJC appendices to directives: negativeDirectiveFJCEntitlementsN3Goods, procedures, indicationWhen necessary+++(+)b\u2013\u2013SHI DRGContractCommittee on Hospital PaymentReimbursementP3ProceduresEvery year+++\u2013\u2013\u2013SHI EBMContractValuation Committee (Physicians)ReimbursementP3ProceduresWhen necessary+++\u2013\u2013\u2013SHI BEMAContractValuation Committee (Dentists)ReimbursementP3ProceduresWhen necessary+++\u2013\u2013\u2013SHI BEL-IIContractValuation Committee (Dentists)ReimbursementP3GoodsWhen necessary+++\u2013\u2013\u2013Statutory long-term care insurance: GBRLawParliamentEntitlementsP\/N1\u2013\u2013\u2013+\u2013\u2013\u2013\u2013FJC Federal Joint Committee, SHI Statutory Health Insurance, GBR General Benefit Regulation, DRG diagnosis-related group,\nEBM Uniform Value Scale, BEMA Uniform Value Scale\u2013dentists, BEL-II Uniform Value Scale\u2013dental technicians, N need, C costs, E effectiveness,\nCE cost-effectiveness, B budgeta 1, \u201call necessary\u201d; 2, areas of care; 3, itemsb Although explicitly mentioned, it is provided principally for medical devicesTable\u00a02 Benefit-defining laws\/ decrees and cataloguesStatutory Health Insurance (SHI) - general benefit regulationSHI - general directives of the Federal Joint CommiteeSHI - special directives of the Federal Joint Commitee (positive)SHI - appendices to directives of the Federal Joint Commitee (negative)SHI - DRGSHI - EBMSHI - BEMASHI - BEL-IIStatutory long term care insurance - general benefit regulationCatalogue: type of document, actors and contentsHC.1.xxx (X)xHC.1.2xxx (X)xHC.1.3.1xxx (XI)xHC.1.3.2xxx (I)x (XII)xxHC.1.3.3xxx (II)x (XI)xHC.1.3.9xxx (III)x (XI)HC.2.1\/2.2xxHC.2.3xHC.3.1xxHC.3.2xxHC.3.3xx (IV)xHC.4.1xxxHC.4.2xxxxHC.4.3xx (V)HC.5.1.1xxHC.5.1.2xxx (VI)HC.5.2xxx (VII)HC.6.1xxx (VIII)HC.6.3xHC.6.4xxx (IX)xxHC.6.5xFJC Federal Joint Committee,\nSHI Statutory Health Insurance, GBR General Benefit Regulation, DRG diagnosis-related group, EBM Uniform Value Scale,\nBEMA Uniform Value Scale\u2013dentists, BEL-II Uniform Value Scale\u2013dental technicians,  I Directives on the Provision of Prosthetic Services II Directives on Psychotherapy\nIII Directives on Nonphysician Care\nIV Directives on Home Nursing Care V Directives on Patient Transport VI Directives on OTC\nVII Directives on Medical Aids VIII Directives on Maternity Care IX Directives on A. Early Detection of Cancer, B. Dental Prophylaxis & C. Medical Examinations for the Early Detection of Diseases X Appendix to Directive according to SGB\u00a0V, Sect. 137c (to evaluate hospital procedures)\nXI Appendix to Directive on Medical Procedures, XII Appendix to Directive on New Dental Procedures\nIn-patient curative care\nIf curative care (i.e., to detect, cure, prevent the worsening, or relieve the discomforts of accompanying diseases) cannot be achieved by ambulatory treatment (SGB\u00a0V, Sect. 39), the insured party is entitled to inpatient treatment in accordance with SGB\u00a0V, Sect. 27. This health care entitlement is linked to a copayment of \u20ac10 per calendar day to a maximum of 28 calendar days per year [SGB\u00a0V, Sect. 39(4)1].\nHospital services are granted in accordance with the care ability of each hospital and with the level of care assigned to each hospital. In each individual case the provision of services needs to be suitable and adequate for the insured. This includes medical treatment, nursing care, the provision of pharmaceuticals, cures and therapeutic appliances, as well as board and accommodation [4].\nHospital care may be only provided in hospitals included in the hospital plan of the respective federal state, in university hospitals, or in hospitals that have concluded a service provision contract with the sickness funds (SGB\u00a0V, Sect. 108). While the spectrum of services provided by the respective hospitals is indirectly determined by the hospital plan (which also determines governmental subsidies for investments), the reimbursement for the provided services is decided in negotiations between each hospital and the association of sickness funds.\nThe Federal Joint Committee presides over matters of exclusion of health care services, and\/or the evaluation and examination of treatment methods; the Committee handles these matters in response to requests from the federal associations of sickness funds and the German Hospital Federation. The method under examination will be scrutinized as to its suitability to provide adequate, expedient, and economical care for the insured persons, with general state-of-the-art medical knowledge taken into consideration. Should the examination reveal that the method does not meet the aforementioned, it may no longer be provided at the expense of the SHI system. In such instances, the Federal Joint Committee issues a corresponding directive according to SGB\u00a0V, Sect. 137c (1) (see Fig.\u00a01). Health care services in the framework of clinical studies are not subject to the directive. This means that all health care services that are not excluded by a directive of the Federal Joint Committee may be provided at SHI\u2019s expense.Fig.\u00a01 DRG Case Fees Catalogue for inpatient care\nThe SHI Reform Act 2000 required the selection and implementation of a case fee system for reimbursement effective as of 1 January 2003. On 27 June 2000 the federal associations of sickness funds, the Association of Private Health Insurance, and the German Hospital Federation adopted the Australian system of diagnosis-related groups (DRGs) as the basis for developing a German DRG system. On 10 May 2001 they founded the Institute for the Payment System in Hospitals (InEK) which is intended to support the introduction and the further development of the DRG system. The InEK is controlled and supervised by the Committee on Hospital Payment, an institution consisting of representatives from the contracting partners (see Fig.\u00a01). The matters addressed by the DRG Institute consist of defining the DRG case groups, the maintenance of the DRG system, and its severity classification system, the development of a coding directive and proposals for adapting German modifications of the International Classification of Diseases ICD-10 and the Operating Procedures System (OPS) into the DRG system. The Institute is also responsible for the calculation of DRG cost weights and individual adjustments within the DRG system.\nAs the basis for the new pricing system a uniform case fee catalogue with fixed payments for services and benefits, valid throughout Germany, was developed. The catalogue lists all procedures (services) performed in hospitals in accordance with respective clinical diagnoses. At the same time the DRG system constitutes the catalogue of services and benefits covered by the SHI scheme for inpatient care. The inclusion of new health care services in the DRG system is reflected at the beginning of each year when a new version of the OPS and the ICD-10 is made available and is linked to a DRG [5].\nThe Case Fees Catalogue of 2005 consists of 876 DRGs, of which 33 are not remunerable with a case fee, and an additional list of 71 negotiated extra remunerations. The German DRG system is subdivided into 23 major diagnosis categories (MDCs) which refer in principle to a body system or cause of a disease. The MDC category also defines the first of the four digits of a DRG. The second and third digits of a DRG indicate the respective partition. The partition differentiates between surgical procedures (01\u201339), other procedures (40\u201359), and medical (conservative) procedures (60\u201399) carried out during a hospital stay, thus linking a DRG to benefits provided in a hospital. The fourth digit further subdivides a DRG according to a patient\u2019s clinical complexity level, which is comprised of such factors as complexity of secondary diagnoses, cause of discharge and patient gender [6].\nFor inpatient services not covered by the DRG system (e.g., new methods of treatment), agreements are made with the hospitals concerned. The local contractual partners inform the contract partners at the federal level of such agreements, who may then decide to initiate an evaluation process in order to exclude these new services from the benefit package [SGB\u00a0V, Sect. 137c; V, Sect. 6 (2), Hospital Payment Act]. In principle however, as noted above, all health care services that are not explicitly excluded by a directive of the Federal Joint Committee can be provided at the expense of the SHI.\nOutpatient care\nThe provision of medical and dental care must be regulated and secured by agreements between the respective regional physicians\u2019 association\/regional dentists\u2019 association and the regional associations of the sickness funds (SGB\u00a0V, Sect. 72). Whereas, in accordance with SGB\u00a0V, Sect. 137c, medical care in hospitals shall be, \u201cadequate, expedient and cost-effective\u201d, for ambulatory care, in accordance with Sect. 135, the criteria to be applied are \u201cdiagnostic and therapeutic expedience, medical necessity and cost-effectiveness.\u201d Thus the inclusion and\/or exclusion of health care services from the benefit catalogues differ in the two sectors. In the outpatient sector a service provided must be confirmed to fulfill the criteria \u201cexpedience, necessity and cost-effectiveness\u201d in order to be included into the catalogue of services and benefits. In contrast to that, health care services in the inpatient sector is excluded from the benefit catalogue of the sickness funds only if the criteria are proven to be unfulfilled. For this reason it is possible that the health care services provided in the inpatient sector are not included in the benefit catalogue of the outpatient sector [7].\nBasic medical and diagnostic care\nInsured persons are entitled to preventive care, detection, and treatment of diseases [SGB\u00a0V, Sect. 28 (1)]. This entitlement also embraces complementary services by nonphysicians and practitioners, provided that they are prescribed by a physician. The legislative authority, however, does not define in detail the entitlements of the insured persons, but regulates the procedures with which the institutions of self-governance and the contractual partners determine the scope of SHI services [8].\nIn accordance with SGB\u00a0V, Sect. 92 (1), the Federal Joint Committee issues directives in respect of adequate, expedient and cost-effective medical care for the insured persons. The directives consist of a general part that explains their aim, their users and mentions the corresponding paragraph in the SGB V. After the initial section the directives become more detailed. For example, the Directive on Medical Procedures that regulates the in- and exclusion of benefits in the outpatient sector initially defines the term of a new service and the conditions an evaluation is depended upon. Thereafter it is stated that the regional physicians\u2019 associations, the Federal Association of SHI Physicians and the federal associations of sickness funds have the right to propose services for their inclusion. Then the criteria for the inclusion of services, the classification of evidence and the decision-making process are described in detail. The services included or excluded through the evaluation process are listed in the annexes [9].\nWhile the Federal Joint Committee decides on the in- and exclusion of services into the benefit package, the Valuation Committee, which consists of seven representatives of the Federal Association of SHI Physicians and representatives of the federal associations of sickness funds, defines the actual benefit catalogue for the insured, the Uniform Value Scale (EBM). The EBM defines, as an integral component of the Federal Framework Contract\u2013Physicians (BMV-\u00c4), the scope of medical care to be provided under the SHI throughout Germany. If the Valuation Committee fails to reach a consensus, at least two of its members or the Federal Ministry for Health and Social Security may demand that the extended Valuation Committee in accordance with SGB\u00a0V, Sect. 87 (4), be brought in to resolve a split decision. Resolutions are to be submitted to the Ministry of Health, which, in the event of unresolved objection, may define alternative executions.\nThe BMV-\u00c4 is concluded between the Federal Association of SHI Physicians and the federal associations of sickness funds (SGB\u00a0V, Sect. 82). In addition to the scope of health care provided under the SHI, the BMV-\u00c4 regulates participation in ambulatory care, the pertinent aspects of quality assurance, and entitlement to benefits. Thus the EBM and the directives of the Federal Joint Committee are both integral parts of this contract. In Sect. 2 of the BMV-\u00c4, the description of a service in the EBM is stipulated as a condition for the provision of the respective service. As a result, the EBM constitutes the catalogue of services and benefits covered by the SHI (see Fig.\u00a02).Fig.\u00a02 Uniform Value Scale for physicians for outpatient care\nThe broad structure and the contents of the EBM are stipulated in SGB\u00a0V, Sect. 87 [10]: (a) The EBM displays the health care services covered by the SHI scheme and their monetary value in relation to one another in the form of a points system. (b) A basic remuneration for general practitioners is defined. (c) Health care services are grouped into packages of similar services. (d) Differentiation is made between the health care services to be provided exclusively by general practitioners and those to be provided exclusively by specialists. (e) The respective health care services are assigned exclusively to the groups of specialists that are allowed to provide them.\nThe EBM catalogue is structured into six main chapters and various sections. Chapter I describes general regulations regarding the provision and reimbursement of health care services. Chapters II\u2013IV contain health care services related to different physician groups and\/or special criteria. Chapter V lists the general health care services provided by most physicians reimbursed with case fees. Chapter VI contains appendices (e.g., a list of services which are already contained in other services and are therefore not reimbursed additionally) [11].\nAs an appendix to the BMV-\u00c4 there is an agreement that applies to care provided by general practitioners under SGB\u00a0V, Sect. 73. It defines the provision of medical treatments and the early detection of diseases. The definition of individual services to be provided is included in the EBM. In addition to these central agreements, which are uniform for all sickness funds, there are numerous \u201csmall\u201d contracts determining the scope of the health care services covered by the German SHI scheme.\nOutpatient dental care\nWhile benefits for ambulatory physician services are legally defined in generic terms only, legislation regulating dental care is much more detailed in the SGB V. One reason for this is that the respective committee of the joint institutions until 2003 failed to provide more explicit definitions [12]. The basic entitlements of the insured to dental care are defined in SGB\u00a0V, Sect. 28 (2): The insured are entitled to prevention, early detection, and treatment of diseases of the teeth, the mouth, and the jaw. Consequently only prophylactic treatment, basic dental care, and dental prosthetic services are covered by the sickness funds [13].\nSimilar to the definition of benefits for basic medical care, the directives of the Federal Joint Committee broadly define when patients are entitled to a benefit. However, they do not define specific items that must be included. Therefore the Dental Valuation Committee, which consists of representatives of the federal associations of the sickness funds and the Federal Association of SHI Dentists defines the Uniform Value Scale for Dentists (BEMA; see Fig.\u00a03). The BEMA lists services that are reimbursed by the sickness funds, thereby explicitly defining the SHI benefit catalogue. The services of dental technicians producing the material needed for orthodontic or prosthetic services are listed in a similar framework, the Uniform Value Scale for Dental Technicians (BEL-II) which is negotiated by the same Committee.Fig.\u00a03 Uniform Value Scales for dentists and dental technicians\nOrthodontic treatments, except those for the treatment of abnormalities, are to begin during childhood and are excluded for insured parties over the age of 18\u00a0years (SGB\u00a0V, Sect. 29). To prevent overprovision of services dentists must prepare a cost schedule that is reviewed by the sickness funds. Prosthetic services are only partially covered by the sickness funds and are therefore defined more explicitly. The insured receive a so-called \u201csubsidy\u201d as a percentage of a \u201cstandard\u201d treatment, defined by the Federal Joint Committee in a directive according to SGB\u00a0V, Sect. 56. The directive currently in force defines a standard treatment for 52 findings. For each standard treatment all reimbursable services of the dentists and the dental technicians are listed separately according to the BEMA and the BEL-II [14].\nSickness funds usually cover 50% of the standard treatment costs. This proportion can increase to 70% or 80% if a patient can prove yearly preventive dental checkups over the past 5 or 10\u00a0years, and the patient\u2019s efforts for dental hygiene are observable. Higher payment levels, up to full coverage of the costs of the standard treatment, are provided only for persons of very low income. Patients are free to choose nonstandard treatments [SGB\u00a0V, Sect. 55 (5)] or include additional services [SGB\u00a0V, Sect. 55 (4)]; however, the amount of sickness funds\u2019 payments remains unchanged.\nOutpatient care performed by nonphysicians\nThe term \u201ccures\u201d subsumes health care services in Germany that are provided by nonmedical practitioners, which include professional, recognized therapists, such as physiotherapists and occupational therapists [15]. The entitlement of the insured to cures is found in SGB\u00a0V, Sect. 32. It is limited by copayments for insured parties over the age of 18\u00a0years under SGB\u00a0V, Sect. 61 (3).\nA further limitation on entitlements is imposed under SGB\u00a0V, Sect. 34 (4), \u201cExcluded Pharmaceuticals, Cures and Medical Aids.\u201d The Ministry of Health is entitled to exclude cures from the catalogue of services and benefits covered by the SHI through decrees, with the approval of the Federal Council (upper chamber of the federal Parliament). However, a corresponding legal decree does not exist at present.\nThe scope of services covered by the SHI scheme is explicitly described and regulated by the Directive on Non-physician Care issued by the Federal Joint Committee under SGB\u00a0V, Sect. 92 [16]. The prescription of more cost-effective measures with equal efficacy, for example, drugs and other therapeutic appliances that achieve the same therapeutic objective, is to be given precedence. The benefits are listed in the directive in connection with an indication. New benefits and\/or an extension of the indications for a given benefit may only be prescribed after the Federal Joint Committee has recognized their therapeutic value and included them into its directive (SGB\u00a0V, Sect. 138).\nThe federal associations of sickness funds and representatives of non-physicians compile a Catalogue of Non-physician Care. The catalogue facilitates the implementation of the directive on Non-physician Care issued by the Federal Joint Committee (see Fig.\u00a04; SGB\u00a0V, Sect. 125), which regulates: (a) the content, scope and frequency of cures, (b) further training measures and quality assurance (c) the content and scope of collaboration between non-physicians and the prescribing SHI physician, (d) measures to meet the aim of cost-effectiveness, and (e) specifications for remuneration structures.Fig.\u00a04 Catalogue of Non-physician Care\nConclusions\nDespite the existence of various catalogues and directives for the SHI scheme, the benefit package is not defined in detail because the obligation of the catalogues and their explicitness varies largely. Inpatient services not listed in the DRG catalogue can still be covered by the SHI scheme as long as they are not explicitly excluded by the Directive according to SGB\u00a0V, Sect. 237c. However in the ambulatory sector only those procedures listed in the SHI EBM or in the SHI BEMA are covered as benefits in the outpatient sector.\nWith the exception of the Catalogue of Non-physician Care the benefits described in the DRG, EBM, BEMA, and BEL-II are the aggregate results of decisions taken at various levels, and they are not linked to specific indications. The reason for this is that they were originally defined for reimbursement and were not meant to define the SHI benefit basket in full detail. For example, as DRGs aggregate multiple procedures and diagnoses, benefits (medical procedures) provided under one DRG will vary from case to case. Additionally, the patient clinical complexity level of a DRG is determined by diagnoses including comorbidities, gender and cause of discharge and not on the basis of the actual services provided. Therefore the scope of a DRG is very broad. Conversely, the development of a DRG catalogue can also be seen as a starting point towards a more explicitly defined benefit catalogue, and subsequently lead to benefit catalogues where all approved interventions are listed and grouped around the relevant diagnoses [2].\nIn recent years strong efforts have been made by the German government to move towards a more explicitly defined benefit basket. The creation of the Federal Joint Committee out of four smaller committees for the different sectors of care can be considered an improvement. The number of issued directives since the inception of the committee supports the assumption that it is more productive than its predecessors. This development suggests that the German health care system is moving towards a more explicitly defined benefit catalogue [17].\nUntil now the use of cost-effectiveness studies as part of the decision criteria for the inclusion of new benefits is widely lacking. The criteria of cost-effectiveness was only taken into consideration for benefit decisions on medical devices. However, it is likely that it will be considered for other benefits in the future as well. The creation of a supporting institute to the Federal Joint Committee, the Institute for Quality and Efficiency, in 2004, which increasingly commissions effectiveness studies, was one major step in that direction [2]. Although this will increase the information base for decisions [18, 19], the future impact of the cost-effectiveness criteria on the decision-making process and therefore on the structure of the health basket still remains unclear.","keyphrases":["germany","health benefit plans","health services","health priorities","national health programs"],"prmu":["P","R","R","M","M"]}
{"id":"Clin_Rev_Allergy_Immunol-3-1-2071970","title":"The Future of Biologic Agents in the Treatment of Sj\u00f6gren\u2019s Syndrome\n","text":"The gain in knowledge regarding the cellular mechanisms of T and B lymphocyte activity in the pathogenesis of Sj\u00f6gren\u2019s syndrome (SS) and the current availability of various biological agents (anti-TNF-\u03b1, IFN- \u03b1, anti-CD20, and anti-CD22) have resulted in new strategies for therapeutic intervention. In SS, various phase I and II studies have been performed to evaluate these new strategies. Currently, B cell-directed therapies seem to be more promising than T cell-related therapies. However, large, randomized, placebo-controlled clinical trials are needed to confirm the promising results of these early studies. When performing these trials, special attention has to be paid to prevent the occasional occurrence of the severe side effects.\nIntroduction\nSj\u00f6gren\u2019s syndrome (SS) is a chronic lymphoproliferative autoimmune disease with disturbances of T lymphocytes, B lymphocytes, and exocrine glandular cells [1]. SS can be primary (pSS) or secondary SS (sSS), the latter being associated with another autoimmune disease [e.g., rheumatoid arthritis, systemic lupus erythematosus (SLE)].\nLymphocytic infiltrates are a characteristic histopathological finding in SS. These infiltrates consist of T and B cells. The expression of different cytokines, such as tumor necrosis factor-\u03b1 (TNF-\u03b1) and interferon-\u03b1 (IFN-\u03b1), during the formation and proliferation of these infiltrates has been investigated. There is an overexpression of TNF-\u03b1, which is secreted by CD4+ T lymphocytes, mononuclear cells, and epithelial cells [2]. The intraglandular synthesis of TNF-\u03b1 causes destruction of acini by up-regulation of Fas at the surface of the glandular epithelial cells, stimulation of secretion of type 2 and 9 matrix metalloproteases by epithelial cells, and overexpression of different chemokines [3\u20135]. IFN-\u03b1 is produced by activated plasmacytoid dendritic cells in primary SS (pSS), and numerous IFN-\u03b1-producing cells have been detected in labial salivary glands [6]. IFN-\u03b1 promotes the autoimmune process by increasing autoantibody production and through the formation of endogenous IFN-\u00e1 inducers. IFNs have potent immunomodulating properties and are thought to trigger a systemic biological response [7].\nBesides the presence of proinflammatory cytokines, described in the previous paragraph, recent studies have shown an important role for B cells in the pathogenesis of SS. Presence of autoantibodies and hypergammaglobulinemia are both considered to reflect B cell hyperactivity. Systemic complications of SS are associated with this B cell hyperactivity [8]. Moreover, about 5% of SS patients develop malignant B cell lymphoma [9]. B cell activating factor (BAFF), also known as B lymphocyte stimulator (BLyS), is an important factor in local and systemic autoimmunity [1]. Dysregulated BAFF expression is implicated in disease progression and perpetuation of humoral autoimmunity. Overproduction of BAFF in transgenic mice has been shown to result in B cell proliferation and antibody production resulting in inflammation and destruction of the salivary glands, as well as kidney failure similar to observations seen in SLE [10]. In humans, circulating BAFF levels are increased in patients with pSS and correlate with disease activity [11].\nRecent insights in the cellular mechanisms of T and B lymphocyte activity in the pathogenesis of SS and the current availability of various biological agents have resulted in new strategies for therapeutic intervention. The use of these biological agents in the treatment of SS will be discussed in this review.\nBiological Agents\nCurrently, biological agents have been introduced in various systemic autoimmune diseases, as rheumatoid arthritis and SLE. Biological agents most frequently applied in autoimmune diseases are monoclonal antibodies, soluble receptors, and molecular imitators [12]. These biological agents enhance or replace conventional immunosuppressive therapy. In contrast to rheumatoid arthritis and SLE, no biological agent has been approved yet for the treatment of SS, but several phase II and III studies have been or are currently conducted. The biological agents used in SS trials are IFN-\u03b1 and agents targeting TNF-\u03b1 and B cells (anti-CD20, anti-CD22). Although no trials have been performed yet with BAFF antagonists, this might be a promising therapy [13] and will be discussed in this review, as well.\nAnti-TNF-\u03b1 Monoclonal Antibodies\nThere are three main biological agents targeting TNF-\u03b1: the chimeric monoclonal IgG1 antibody infliximab, the receptor fusion protein etanercept, and the fully humanized monoclonal antibody adalimumab.\nIn an open-label study, short-term treatment with infliximab was reported to be very effective in active pSS over a 3-month period [14]. Sixteen patients received three infusions (3\u00a0mg\/kg) at weeks 0, 2, and 6, which led to significant improvement in all clinical and functional parameters, including global assessments, erythrocyte sedimentation rate, whole salivary flow rate, tear secretion (Schirmer test), tender joint count, fatigue score, and sensation of dry eyes and dry mouth. Three patients, all with short disease duration (<3\u00a0years), were considered to be in complete remission up till 1\u00a0year. In 10 out of the 16 patients, SS symptoms, particularly mouth dryness, relapsed after a median of 9\u00a0weeks. In a follow-up study, a maintenance regimen of one infusion every 12\u00a0weeks was evaluated in these 10 patients. Retreatment induced an improvement of signs related to SS that was comparable with the effects from the three loading infusions [15]. To confirm these promising results from an uncontrolled study, the Trial of Remicade In Primary Sj\u00f6gren\u2019s Syndrome study was designed. In this multicenter, double-blinded, placebo-controlled, randomized clinical trial, 103 patients with active pSS were included and treated with infliximab infusions (5\u00a0mg\/kg) or placebo at weeks 0, 2, and 6. Follow-up was 22\u00a0weeks. Primary endpoint was an improvement of >30% of two of three VAS scores measuring joint pain, fatigue, and dry eyes. There were several secondary endpoints of which one was the basal salivary flow rate. In contrast to the previously mentioned uncontrolled studies, no evidence of efficacy of infliximab treatment on all clinical and functional parameters could be demonstrated in this randomized controlled clinical trial [2].\nA trial on 15 pSS patients (mean disease duration 3.6\u00a0years) with 25-mg etanercept, subcutaneously twice a week for 12\u00a0weeks, did not reveal a reduction in sicca symptoms and signs, neither did the repeated treatment for up to 26\u00a0weeks. Only in the subset of four patients with severe fatigue a decrease in fatigue was observed [16]. Another trial evaluating subcutaneous administration of etanercept vs placebo for 12\u00a0weeks (28 patients) also showed no clinical efficacy [17]. No trials of adalimumab treatment in pSS have been reported in the literature yet.\nIn conclusion, TNF-targeting treatment could not be proven to be of benefit in reducing the complaints of pSS patients.\nIFN-\u03b1\nIFNs are proteins with antiviral activity and potent immunomodulating properties. SS patients have an activated type I IFN system [6]. Such a role for IFN-\u03b1 appears to contradict the reports described below, that low doses of IFN-\u03b1 administered via the oromucosal route increase the unstimulated salivary output. However, it is hypothesized that oral IFN-\u03b1 treatment may act by increasing saliva secretion by up-regulation of aquaporin 5 transcription without significantly influencing the underlying autoimmune process [6, 7].\nIn a phase II study, treatment of pSS patients with IFN-\u03b1 administered via the oromucosal route (by dissolving lozenges) was demonstrated to be effective (improvement of salivary output, decreased complaints of xerostomia) and safe [18]. Based on these promising results, a randomized, parallel group, double-blinded, placebo-controlled clinical trial (497 pSS patients) was designed. Patients were randomized into two groups and received a 24-week daily treatment with either 450\u00a0IU IFN-\u03b1 (150\u00a0IU three times per day) or placebo in a ratio 3:2, administered by the oromucosal route. This randomized, controlled clinical trial failed to demonstrate a significant effect on the primary endpoints (VAS score for oral dryness and stimulated whole salivary flow) in the IFN-\u03b1 group relative to the placebo group. There was a significant increase in unstimulated whole saliva in the patients treated with IFN-\u03b1, which correlated positively and significantly with improvement in seven of eight symptoms associated with oral and ocular dryness. No adverse events were observed [7].\nIn conclusion, no clinical evidence for the efficacy of IFN-\u00e1 treatment in pSS patients has been shown yet; however, an improvement of unstimulated whole saliva was observed. Further research is needed to objectify the effect of IFN-\u00e1 on salivary gland tissue.\nAnti-CD20 Monoclonal Antibodies\nAnti-CD20 (rituximab) is a chimeric humanized monoclonal antibody specific for the B cell surface molecule CD20, which is expressed on the surface of normal and malignant pre-B and mature B lymphocytes. CD20 mediates B cell proliferation and differentiation. This antibody has been demonstrated to prevent B cells from proliferating and to induce lysis of B cells by complement-dependent and antibody-dependent cytotoxicity mechanisms as well as by direct induction of apoptosis [19].\nRituximab is currently used for the treatment of low-grade B cell lymphomas [20]. In controlled studies, it was shown to be safe and effective in the treatment of rheumatoid arthritis [21\u201323]. Moreover, open-label studies in SLE patients are promising [24].\nIn an open-label phase II study, 15 patients with pSS were treated with 4 infusions of rituximab (375\u00a0mg\/m2 once weekly) and followed up for a 3-month period. Eight of the 15 patients were early pSS patients (mean disease duration 28\u00a0months, all had residual salivary gland function at baseline), and 7 patients had a concomitant mucosa-associated lymphoid tissue (MALT) lymphoma (mean disease duration 79\u00a0months).\nIn the early pSS patients, rituximab treatment resulted in significant improvement of subjective symptoms and an increase in salivary gland function. All patients showed a rapid depletion of peripheral B cells within a few weeks, accompanied by a decrease in IgM-RF levels [8]. Repeated parotid gland biopsies in five of the early patients after treatment showed redifferentation of the lymphoepithelial duct lesions into normal striated ducts, possibly indicating regeneration of salivary gland tissue (unpublished data).\nFive of the eight pSS patients without a MALT lymphoma received a second course of rituximab (after 9\u201311\u00a0months) due to recurrence of symptoms. Retreatment resulted in the same significant improvement of the salivary flow rate and subjective symptoms compared to the results of the first treatment, together with a decrease in B cells and IgM-RF levels.\nSix of the seven MALT\/pSS patients were initially effectively treated with rituximab. The remaining MALT\/pSS patient had progressive MALT disease and severe extraglandular SS disease within 3\u00a0months after the start of rituximab treatment. Cyclophosphamide was added, which led to stable disease of both MALT and SS. One of the six patients initially responding had a recurrence of MALT lymphoma after 9\u00a0months and was successfully retreated with rituximab. The other patients are still in remission (unpublished data).\nIn another open-label study, 16 pSS patients received only two weekly rituximab infusions (375\u00a0mg\/m2), with a follow-up of 36\u00a0weeks. Again, treatment resulted in rapid complete depletion of peripheral B cells. At week 12, a significant improvement of VAS scores for fatigue and dryness was recorded, and at week 36, a significant improvement for VAS scores for global disease, fatigue, dry mouth, dry eyes, and dry vagina, but also in the number of tender joint and tender joint counts was seen [25]. Both in the study of Pijpe et al. [8] and the study of Devauchelle-Pensec et al. [25], patients with a short disease duration showed more improvements than patients with longer disease duration.\nTwo trials retrospectively evaluated the effect of rituximab (four infusions of 375\u00a0mg\/m2) in 18 pSS patients (mean disease duration 10\u00a0years) with systemic features. Self-reported dryness improved in six patients (VAS scores not known for three patients, no improvement in the other nine patients). Both studies reported good efficacy of the treatment on systemic features [26, 27].\nIn conclusion, in phase II trials, it has been shown that rituximab seems to be effective for at least 6\u20139\u00a0months in pSS patients with active disease, improving both subjective and objective complaints. Retreatment with rituximab resulted in a similar good clinical response. In pSS patients with longer disease duration, without residual salivary gland function, rituximab treatment seems to be effective for systemic features. To confirm these promising results, randomized placebo-controlled clinical trials are needed.\nAnti-CD22 Monoclonal Antibodies\nEpratuzumab is a fully humanized monoclonal antibody specific for the B cell surface molecule CD22. CD22 is expressed on the surface of normal mature and malignant B lymphocytes. CD22 appears to be involved in the regulation of B cell activation through B cell receptor signaling and cell adhesion [28]. In an open-label phase I\/II study, safety and efficacy of epratuzumab were investigated in 16 pSS patients. Follow-up was 6\u00a0months. These pSS patients received four doses of 360\u00a0mg\/m2 epratuzumab intravenously. Mean disease duration before therapy was 2.9\u00a0years, and none of the patients had received prior B cell-targeted therapy. Most improvements occurred in the Schirmer test, unstimulated whole salivary flow and the VAS score for fatigue. The new developed disease activity score consisted of the four domains: dryness of the eyes, dryness of the mouth, fatigue, and laboratory parameters. Based on this score, 53% achieved at least 20% improvement in at least two domains at 6\u00a0weeks. Corresponding rates for 10, 18, and 32\u00a0weeks are 53, 47, and 67%. Remarkably, the number of responders was higher 6\u00a0months after the treatment administration than earlier. Peripheral B cells decreased with a median decrease of 54 and 39% at 6 and 18\u00a0weeks, respectively.\nIn conclusion, epratuzumab seems to be an effective treatment. Randomized, placebo-controlled clinical trials are needed before epratuzumab can be advised for general treatment in pSS patients [29].\nAnti-BAFF\nBAFF is a B cell-activating factor that acts as a positive regulator of B cell function and expansion. BAFF levels were found elevated in serum and saliva in SS patients, but no correlation could be shown between serum and saliva levels [30]. However, circulating levels of BAFF in pSS patients were shown to be a marker for disease activity [11].\nTo the best of our knowledge, no trials have been performed with anti-BAFF treatment in SS yet, but such an approach might be considered for future trials. Currently, two human BAFF antagonists have been developed, a human antibody (anti-BLyS) that binds to soluble BAFF and a fusion protein of one of the BAFF receptors [31, 32]. Especially, SS patients with elevated BAFF levels, hypergammaglobulinemia, elevated levels of autoantibodies, and associated B cell lymphoma might be candidates for anti-BAFF treatment [33].\nSafety and Tolerability of Biological Agents\nThe most important side effects of treatment with biological agents are direct mild infusion reactions. Several patients developed a serum sickness-like disease a few days after the second infusion that might be related to the formation of antibodies against the biological agent [human anti-chimeric antibodies (HACAs) or human anti-human antibodies]. A few patients developed infections during treatment with a biological agent; however, some patients concomitantly used other immunosuppressive therapies. Therefore, the direct relation between the biological agent and the infection is unsure. All adverse events reported in the trials described in this review are reported in Table\u00a01. According to this table, the most frequent side effects of treatment with biological agents are mild infusion reactions. The most severe side effect of the various treatments used in SS patients was the development of a serum sickness-like disease. This adverse effect of treatment occurred in 16% (8 of 49) of the patients treated with rituximab. HACA formation was observed in patients developing a serum sickness-like disease and occurred only in patients receiving low-dose corticosteroids and no other immunosuppressive drugs. It is assumed that higher doses of corticosteroids during treatment might prevent the occurrence of serum sickness.\nTable\u00a01Adverse events after treatment with biological agents in SS\u00a0Agent\/doseNumber of patients in trial (number treated with the agent)Premedication\/concomitant immunosuppressive therapyInfusion reactionInfectionsSerum sicknessHACA\/HAHA formationOtherAnti-TNF-\u03b1 monoclonal antibodiesSteinfeld [14]Infliximab intravenous, 3\u00a0mg\/kg16 (16)n.r.\/no1 (6%)2 (13%) (respiratory tract)\u2013n.r.\u2013Steinfeld [15]Infliximab intravenous, 3\u00a0mg\/kg10 (10)n.r.\/no4 (40%)2 (20%) (enteritis, tonsillitis)\u2013n.r.\u2013Marriette [2]Infliximab intravenous, 5\u00a0mg\/kg103 (54)n.r.\/continuation of hydroxychloroquine and corticosteroids (\u226415\u00a0mg\/day)2 (4%)2 (4%) (1 cutaneous, 1 respiratory tract)\u2013n.r.2 (breast cancer, auto-immune hepatitis)aZandbelt [16]Etanercept subcutaneously, 25\u00a0mg15 (15)n.r.\/pilocarpine at a constant dose\u20131 (7%) (parotitis)\u2013n.r.\u2013Sankar [17]Etanercept subcutaneously, 25\u00a0mg28 (14)n.r.\/allowed to use long-term medication1 (7%)1 (7%) (skin lesion)b\u2013n.r.\u2013IFN-\u03b1Ship [18]IFN-\u03b1 oromucosal, 150\u00a0IU, 450\u00a0IU109 (87)n.r.\/non.a.\u2013\u2013n.r.\u2013cCummins [7]IFN-\u03b1 oromucosal, 450\u00a0IU497 (300)n.r.\/non.a.\u2013\u2013\u201323 (7.7%)d (34% gastrointestinal, 25% musculoskeletal)Anti-CD20Pijpe [8]Rituximab intravenous, 375\u00a0mg\/m215 (15)25\u00a0mg prednisolon intravenously\/patients with severe extraglandular manifestations (n\u2009=\u20093) received immunosuppressive therapy2 (13%)1 (7%) (zoster)4 (27%)e4 (27%)\u2013Devauchelle-Pensec [25]Rituximab intravenous, 375\u00a0mg\/m216 (16)n.r.\/no\u2013\u20131 (6%)n.r.\u2013Gottenberg [26]Rituximab intravenous, 375\u00a0mg\/m26 (6)n.r.\/hydroxychloroquine (n\u2009=\u20091), methylprednisolone (n\u2009=\u20093)1 (17%)\u20131 (17%)n.r.\u2013Seror [27]Rituximab intravenous, 375\u00a0mg\/m212 (12)n.r.\/cyclophosphamide (n\u2009=\u20091), hydroxychloroquine (n\u2009=\u20091), leflunomide (n\u2009=\u20091)1 (8%)\u20132 (17%)n.r.\u2013Anti-CD22Steinfeld [29]Epratuzumab intravenous, 360\u00a0mg\/m216 (16)0.5\u20131\u00a0g acetominophen, 25\u201350\u00a0mg antihistamine.\/no2 (13%)2 (13%) (sinusitis, dental abscess)\u20133 (19%)6 (38%) (TIA, osteoporotic fracture, diarrhea, dyspepsia, palpitations, paresthesia)n.a. Not applicable, n.r. not reported, HACA human anti-chimeric antibodies, HAHA human anti-human antibodiesaOne patient in the placebo group developed benign lymph node enlargementbOne patient in the placebo group developed a prolonged upper respiratory tract infectioncIn this study, there were mild adverse events; however, there were no significant differences between the groups. Adverse events were not specified.dEight patients (4.1%) in the placebo group developed adverse eventseOne of these 4 patients developed serum sickness after retreatment [8]\nFuture Perspectives\nBiological agents are promising therapies for SS. Randomized studies failed to show a clinical effect of anti-TNF-\u03b1 and IFN-\u03b1 in the treatment of SS. Notwithstanding the unfortunate results of anti-TNF-\u03b1 and IFN-\u03b1, B cell depletion (both anti-CD20 and anti-CD22) seems very promising. Again, this promising effect, as was previously also assumed for anti-TNF-\u03b1 and IFN-\u03b1, must be confirmed in larger randomized controlled clinical trials.\nHACAs have been reported to occur at a higher rate in patients with an autoimmune disease. It seems that monoclonal antibodies are more immunogenic in active autoimmune disease, independent of the type of disease. Additional use of immunosuppressive therapy in these patients might be mandatory to prevent serious side effects. These unwanted side effects might also be prevented by the use of fully humanized antibodies. The currently available humanized antibodies are promising, but need further study. Moreover, there is still a need for improved assessment parameters to monitor treatment effects, both subjectively and objectively. For studies on intervention of SS, evaluation of the parotid gland might be of use because function, composition of saliva, and histology can be evaluated on the same gland at different time points. Activity scores are currently under development by Bowman and Vitali [34, 35]. Finally, as soon as effective intervention treatments have been established, the cost-effectiveness of these currently very expensive antibodies needs to be analyzed to select those patients that might benefit the most from this kind of treatment.","keyphrases":["biological agent","treatment","sj\u00f6gren\u2019s syndrome","therapy","autoimmune disease","monoclonal antibody"],"prmu":["P","P","P","P","P","P"]}
{"id":"Oecologia-4-1-2311384","title":"The effect of temperature on growth and competition between Sphagnum species\n","text":"Peat bogs play a large role in the global sequestration of C, and are often dominated by different Sphagnum species. Therefore, it is crucial to understand how Sphagnum vegetation in peat bogs will respond to global warming. We performed a greenhouse experiment to study the effect of four temperature treatments (11.2, 14.7, 18.0 and 21.4\u00b0C) on the growth of four Sphagnum species: S. fuscum and S. balticum from a site in northern Sweden and S. magellanicum and S. cuspidatum from a site in southern Sweden. In addition, three combinations of these species were made to study the effect of temperature on competition. We found that all species increased their height increment and biomass production with an increase in temperature, while bulk densities were lower at higher temperatures. The hollow species S. cuspidatum was the least responsive species, whereas the hummock species S. fuscum increased biomass production 13-fold from the lowest to the highest temperature treatment in monocultures. Nutrient concentrations were higher at higher temperatures, especially N concentrations of S. fuscum and S. balticum increased compared to field values. Competition between S. cuspidatum and S. magellanicum was not influenced by temperature. The mixtures of S. balticum with S. fuscum and S. balticum with S. magellanicum showed that S. balticum was the stronger competitor, but it lost competitive advantage in the highest temperature treatment. These findings suggest that species abundances will shift in response to global warming, particularly at northern sites where hollow species will lose competitive strength relative to hummock species and southern species.\nIntroduction\nPeat bogs play a large role in the global sequestration of C. Although northern peatlands cover only 2% of the total land surface, they store about one-third of the world soil C in the form of peat (Gorham 1991). It is therefore important to know how ombrotrophic bog ecosystems, which form a large part of northern peatlands, will respond to predicted climate changes, especially since the rise in temperature is expected to be above the global average at high latitudes (Christensen et al. 2007) where the majority of peat bogs occur (Kivinen and Pakarinen 1980; Gunnarsson 2005).\nVegetation in ombrotrophic bogs is often dominated by different Sphagnum species. The Sphagnum species account for the bulk of the C sequestration in peat because of their recalcitrant litter (Coulson and Butterfield 1978; Clymo and Hayward 1982; Limpens et al. 2003). Therefore, it is crucial to understand how Sphagnum vegetation in bogs will respond to global warming in order to predict the role of bogs as C sinks in the future. Several studies have revealed differences in production (Lindholm and Vasander 1990; Gerdol 1995; Asada et al. 2003; Gunnarsson 2005) and in decomposition rate (Rochefort et al. 1990; Johnson and Damman 1993; Belyea 1996; Limpens and Berendse 2003) between different Sphagnum species. These differences between species are often related to differences in microhabitat preference (Gunnarsson 2005). Within a bog, different Sphagnum species occur at different heights above the water table and at different positions along pH and nutrient gradients (Andrus 1986; Sj\u00f6rs and Gunnarsson 2002; Limpens et al. 2003). The most obvious division in microhabitat preference is between hollow species, which grow in pools and at shallow water levels, and hummock species, which grow at deeper water levels. Hummock species can also grow at higher water levels, but they are then usually outgrown by hollow species (Rydin 1986, 1993, 1997). However, the competitive ability of species may differ between years, seasons and locations, and as such, competitive replacement occurs very slowly, if at all. This results in a relatively stable competitive balance between species (Rydin 1997).\nSeveral studies found a positive relation between Sphagnum productivity and temperature (Moore 1989; Sonesson et al. 2002; Gunnarsson 2005). The positive effect that increased temperature might have on C sequestration in bogs is, however, often diminished by the positive effect of temperature on decomposition rates (Hobbie 1996). Increased decomposition rates also lead to increased rates of nutrient release from the peat layer, enhancing production rates even further. A change in temperature influences not only the production and decomposition of individual Sphagnum species but also the competitive balance that exists between species. An important challenge facing ecologists is to predict how climate change will alter species distributions in ecosystems (Mooney 1991). Robroek et al. (2007b) already found different responses in biomass production among species when temperature was increased. It can be imagined that when a species with high production and\/or a low decomposition rate increases its relative abundance in a bog, this will increase the C storage capacity of the system.\nNot only are there different competing species within a bog, but there are also differences in dominant species between bogs when different climatic regions are compared. In this study, we used species from two different sites. At the site in northern Sweden, Sphagnum balticum and Sphagnum fuscum are the dominant species while at the site in southern Sweden these species also occur, but Sphagnum magellanicum and Sphagnum cuspidatum are the most abundant species. This corresponds with the general distribution of these species in Europe since both S. magellanicum and S. cuspidatum occur further south than S. fuscum and S. balticum (Daniels and Eddy 1985).\nTo examine the effect of temperature on the competition between species, we performed a greenhouse experiment in which we studied the effect of four temperature treatments on the growth of the four species: S. fuscum and S. balticum from a northern Swedish site and S. magellanicum and S. cuspidatum from a southern Swedish site. Three combinations of species were made to study the effects of temperature on interspecific competition. In our experiment we tried to answer the following questions:What is the effect of increased temperature on the growth of different Sphagnum species? We expect all species to show an increase in both height increment and biomass production as a direct result of increased temperature and indirectly through increased nutrient availability.What is the effect of temperature on competition between species? Since S. fuscum and S. magellanicum grow in drier and therefore also warmer microhabitats than S. balticum and S. cuspidatum respectively, we expect these species to be better adapted to higher temperatures. S. magellanicum grows at more southern sites than S. balticum, so we expect this species to be better adapted to higher temperatures. Consequently, we hypothesize that increased temperature will have a positive effect on competitive abilities of the hummock and southern species, leading to a relatively larger increase in height increment and biomass production with temperature than for hollow and northern species.\nMaterials and methods\nPlant material\nIn August 2004, Sphagnum cores (diameter 16\u00a0cm, height 18\u201322\u00a0cm) were collected at two different sites in Sweden. From the northern site Lappmyran (64\u00b009\u2032N, 19\u00b035\u2032E), 30 Sphagnum fuscum (Schimp) H. Klinggr cores and 40 Sphagnum balticum (Russ.) C. Jens. cores were collected. This site is a string flark mire or mixed mire with ridges of hummocks and hollows where S. fuscum is dominant on the hummocks and S. balticum in the dryer parts of the hollows. When identifying the species from this site in the lab, we found specimens of both S. balticum and Sphagnum angustifolium (Russ.) C. Jens. These species are difficult to distinguish, which both Russow (Smith 1978) and Klinggraff (Daniels and Eddy 1985) recognized when they identified both species as varietas of Sphagnum recurvum. In our samples, we were unable to quantify the exact percentages of S. balticum and S. angustifolium. As result, whenever we mention S. balticum, we are referring to a mixture of S balticum and S. angustifolium.\nFrom the southern site Saxn\u00e4s Mosse (56\u00b051\u2032N, 13\u00b027\u2032 E), 40 Sphagnum magellanicum (Brid.) cores and 30 Sphagnum cuspidatum (Hoffm.) cores were collected. At this site S. magellanicum occurs on the lawns and low hummocks and S. cuspidatum in the hollows and pools.\nCores were taken from monospecific stands of each Sphagnum species (>95%) with sparse vascular plant cover (<5%). The cores were placed in plastic containers (diameter 16\u00a0cm, height 22\u00a0cm). Vascular plants were clipped flush with the Sphagnum, and other Sphagnum species were removed with tweezers. The containers were brought to Wageningen and stored outside for 8 weeks before the greenhouse was available. As a result, containers from both sites could acclimate to the same climate to some extent.\nExperimental design\nAt the start of the experiment, the containers were brought into the greenhouse and randomly divided over the treatments and five replicate blocks, with seven species combinations (four monocultures and three mixtures) and four temperature treatments per block. All four species were kept in monoculture and the following three species combinations were made to study interspecific competition: two northern species S. fuscum with S. balticum, two southern species S. magellanicum with S. cuspidatum and a northern with a southern species S. balticum with S. magellanicum. To study competition between a northern and a southern species we chose the combination of S. balticum and S. magellanicum because they occur at similar water levels. To make the combinations, the cores were cut into four equal quarters and two quarters of both species were placed alternately in an empty container. In potting the species combinations, we made certain that the surface of the mixture was uniform. We did not cut monocultures in four quarters, but another experiment showed no difference in water content between cut and uncut monocultures (Robroek et al. 2007a).\nThe experiment was conducted in four adjacent climate controlled greenhouse compartments from November 2004 till April 2005 for a total of 154\u00a0days. Each compartment was assigned one of four temperature treatments. Treatments and containers were switched between compartments every 2\u00a0weeks to minimize any effect of the different compartments. The position of the blocks and the position of containers within the blocks were also switched every 2\u00a0weeks. The average day temperatures in the four temperature treatments were 11.2, 14.7, 18.0 and 21.4\u00b0C, respectively (Table\u00a01). During the dark period of 8\u00a0h, the day temperature was lowered by approximately 3\u20139.3, 11.6, 15.5 and 18.9\u00b0C, resulting in mean temperatures of 10.6, 13.7, 17.2 and 20.6\u00b0C in temperature treatments 1, 2, 3 and 4, respectively. In the region of the northern site, the mean temperature in July is 14.7\u00b0C (Alexandersson et al. 1991) and in the southern site the mean temperature in July is 17.0\u00b0C (Malmer et al. 2003). A light period of 16\u00a0h was applied. If light intensity was low during this period, SON-T AGRO 400 (Philips Powertone 400) lamps were used.\nTable\u00a01Day and night mean values of temperature (\u00b0C), relative humidity (%) and vapour pressure deficit (VPD) (kPa) of the four treatments, \u00b1SE, n\u00a0=\u00a0154TreatmentTemperatureRelative humidityVPDDayNightDayNightDayNightT111.2\u00a0\u00b1\u00a00.0493\u00a0\u00b1\u00a00.0581.9\u00a0\u00b1\u00a00.486.8\u00a0\u00b1\u00a00.40.24\u00a0\u00b1\u00a00.010.16\u00a0\u00b1\u00a00.01T214.7\u00a0\u00b1\u00a00.03116\u00a0\u00b1\u00a00.0576.9\u00a0\u00b1\u00a00.381.8\u00a0\u00b1\u00a00.50.39\u00a0\u00b1\u00a00.010.25\u00a0\u00b1\u00a00.01T318.0\u00a0\u00b1\u00a00.02155\u00a0\u00b1\u00a00.0375.2\u00a0\u00b1\u00a00.476.1\u00a0\u00b1\u00a00.40.51\u00a0\u00b1\u00a00.010.42\u00a0\u00b1\u00a00.01T421.4\u00a0\u00b1\u00a00.03189\u00a0\u00b1\u00a00.0472.7\u00a0\u00b1\u00a00.473.0\u00a0\u00b1\u00a00.50.70\u00a0\u00b1\u00a00.010.59\u00a0\u00b1\u00a00.01\nRelative humidity during the day was set at 75%. In the greenhouse it was not possible to keep the relative humidity exactly the same with all temperatures. The rise in temperature between treatments corresponded with a decrease in relative humidity, which caused an additional increase in vapour pressure deficit (VPD) with temperature (Table\u00a01). The difference in relative humidity only explained 33% of the increase in VPD with the highest temperature. If relative humidity would have been equal in all compartments, VPD would still have been twice as high at temperature 4 as at temperature 1. Our highest VPD of 0.7\u00a0kPa with a temperature of 21.4\u00b0C is actually still quite low compared to field conditions. Although not many data on VPD are published for similar ecosystems, Hobbie and Chapin (1998) did find the following values for VPD in Toolik Lake, Alaska, in June and July: in open field sites, 0.02\u20131.71\u00a0kPa with temperatures ranging from 5.9 to 22.5\u00b0C; and under small plastic greenhouses, 0.08\u20133.83\u00a0kPa with temperatures ranging from 6.4 to 31.1\u00b0C. Dorrepaal et al. (2003) measured a VPD of 1.54 under normal conditions and 1.41 in open-top chambers with an average temperature of 15\u00b0C in June and July in Abisko, Sweden.\nThe water level was set to 1\u00a0cm below capitula at the start of the experiment. All species were subjected to the same water level, so that temperature was the only changing environmental variable. This relatively high water level was used for all species because hollow species cannot grow at low water levels; while hummock species can survive the environmental conditions of hollows quite well; nonetheless, they are absent from these areas because of biotic factors (Rydin and McDonald 1985). Grosvernier et al. (1997) found that growth in height and dry weight is equal for S. fuscum grown at water levels of 1\u00a0cm and 40\u00a0cm below moss surface while, for S. magellanicum and especially for Sphagnum fallax, growth in height and dry weight is much greater with the high water level.\nDuring the experiment an artificial rainwater solution, an 8,000-fold dilution of a sea water solution (Garrels and Christ 1965), was added twice a week to bring the water level back to 1\u00a0cm below capitula. The amount of water added was used as a measure of evaporation. The drop in the water table was highest in the highest temperature treatment, but water level never dropped more than 6\u00a0cm below moss surface between two water additions. In a number of containers, Sphagnum grew higher than 1\u00a0cm above the container. To keep the water level at 1\u00a0cm below the moss surface, a plastic ridge was glued onto the containers and the crack was filled with silicone kit. Water content was measured using a theta probe (Delta-T Devices, Cambridge, UK) before the final harvest of the experiment. This was done 3\u20134\u00a0days after watering the containers for the last time, so the water content would reflect possible differences between treatments.\nMeasurements\nHeight increment of the Sphagnum carpet was measured non-destructively every month using a variation of the cranked wire method (Clymo 1970). We used plastic rods that were inserted to a depth of approximately 8\u00a0cm and anchored by plastic broom bristles, this method kept the cranked wires firm at the same place so they did not move with Sphagnum growth. Two plastic rods were inserted in the monocultures and one plastic rod was inserted in each quarter of the mixtures. The rods had a diameter of 1.5\u00a0mm and did not seem to interfere with the growth of the surrounding Sphagna.\nAt the end of the experiment, columns with a diameter of 5\u00a0cm were cut around each cranked wire and cut off at 5\u00a0cm length. Each column was put in a plastic Ziplock bag and fresh weight was determined. All bags were stored at 1\u00b0C till further measurements could be taken. Capitula were defined as the top 1\u00a0cm of each individual plant and stem as the 1\u20134\u00a0cm part. Capitula and stems were separated per column and oven dried at 70\u00b0C for at least 48\u00a0h and then weighed. The weight of the total sample was used to calculate bulk density and the biomass production per squared centimetre to account for changes in capitulum density. For nutrient analyses, samples of capitula were pooled per species for each container. Total N, P and K concentrations were determined by digesting 300\u00a0mg of homogeneous, milled material with H2SO4, salicylic acid, Se and H2O2. All samples were analysed for total N and P spectrophotometrically using an auto-analyzer (Skalar). K concentrations were measured with an atomic absorption spectrophotometer (Varian AAS). To compare capitulum bulk density and nutrient concentrations with field values, we collected five samples (d\u00a0=\u00a05\u00a0cm) from monocultures of the four Sphagnum species in the two Swedish sites in August 2006. Measurements on capitulum bulk density and nutrient concentrations were executed as described above.\nTo measure the change in cover of the species in the mixtures, digital pictures were made at the start and at the end of the experiment. In these pictures we measured the total surface cover per species in each pot with Image J (Abramoff et al. 2004). The biomass production per unit area (g\u00a0m\u22122) was calculated as follows: \nData analysis\nData were tested for normality and equality of variance. When necessary, data were square-root transformed to achieve homogeneous variances. Block effect was tested as random factor. When no block effect was detected, which was usually the case, block was omitted from the analysis to gain extra df. All analyses were conducted using the SPSS statistical package for Windows (12.0).\nOne container with a monoculture of S. balticum under temperature 4 was heavily affected by a fungal infection, probably Lyophyllum palustre. After 4\u00a0months, 90% of the plant material had died. This container was further omitted from the analyses.\nHeight increment in monocultures was tested per species with one-way ANOVA with temperature as independent factor. Height increment, biomass production, cover change, bulk density, water content and nutrient concentrations were tested per species. Two-way ANOVAs were performed with temperature and species combination as independent factors. The effect of temperature, species and competition on the concentrations of N, P and K was tested using a three-way ANOVA. Differences between the treatments were analysed using a Tukey post hoc test. To test how the environmental variables temperature, N concentration and water content influenced biomass production we performed a stepwise regression analysis per species.\nResults\nHeight increment\nIn the monocultures, the height increment increased with temperature in each species (Fig.\u00a01, Table\u00a02). For S. cuspidatum this effect was least distinct with the lowest height growth at temperature 2 and no difference in growth between temperatures 1, 3 and 4.\nFig.\u00a01Height increment of species in monocultures (cm). Data are mean values\u00a0\u00b1\u00a0SE, n\u00a0=\u00a05 except for Sphagnum balticum at T4, n\u00a0=\u00a04. Different letters indicate significant differences (P\u00a0<\u00a00.05) between temperature treatments within each species. For temperature treatments, see Table\u00a01Table\u00a02F-values and P-values and direction of main effects influencing height increment and biomass production per speciesa, corrected for cover, using a two-way ANOVATemperatureCombinationT\u00a0\u00d7\u00a0combinationFPEffectFPEffectFPHeight incrementSphagnum fuscum (+Sphagnum balticum)49.31<0.001+1.170.288+0.920.445S. balticum (+S. fuscum)51.19<0.001+9.130.005\u22120.610.616S. balticum (+Sphagnum magellanicum)19.58<0.001+10.320.003\u22123.240.035S. magellanicum (+S. balticum)28.71<0.001+8.870.006\u22121.880.154S. magellanicum (+Sphagnum cuspidatum)18.53<0.001+4.860.035\u22121.140.350S. cuspidatum (+ S. magellanicum)10.80<0.001+18.83<0.001\u22120.860.470Biomass productionS. fuscum (+S. balticum)20.99<0.001+18.57<0.001\u22122.310.095S. balticum (+S. fuscum)19.41<0.001+5.480.026\u22122.040.128S. balticum (+S. magellanicum)9.68<0.001+12.380.001\u22123.470.028S. magellanicum (+S. balticum)17.50<0.001+44.99<0.001\u22121.670.193S. magellanicum (+S. cuspidatum)11.75<0.001+23.36<0.001\u22122.000.133S. cuspidatum (+ S. magellanicum)5.150.005+2.200.148\u22120.180.913aNumber of observations n\u00a0=\u00a040, except for S. balticumn\u00a0=\u00a039\nOnly S. fuscum showed no difference in height increment between monoculture and mixture. All other species showed a growth reduction in mixtures (Table\u00a02). There were no significant interactions between the effect of temperature and competition on height increment, except for S. balticum in combination with S. magellanicum (Table\u00a02). Temperature increased height increment of S. balticum in monoculture, while there was no effect of temperature in mixture with S. magellanicum (Figs.\u00a01b,\u00a02b). When we compared height increment between the two species in each mixture, the \u201cwet\u201d species S. balticum and S. cuspidatum had higher values than the \u201cdry\u201d species S. fuscum and S. magellanicum at temperature treatments 1 and 2 (Fig.\u00a02). When temperature increased, the differences in height increment between the two species disappeared.\nFig.\u00a02Height increment of the individual species at each temperature treatment (see Table\u00a01) within the three mixtures of species: aSphagnum fuscum\u00a0+\u00a0S. balticum, bSphagnum magellanicum\u00a0+\u00a0S. balticum and cS. magellanicum\u00a0+\u00a0Sphagnum cuspidatum. Data are mean values\u00a0\u00b1\u00a0SE, n\u00a0=\u00a05. Different letters indicate significant differences between treatments (P\u00a0<\u00a00.05)\nCover\nThe hollow species S. balticum and S. cuspidatum increased in area in 53 out of 60 containers when growing in the mixtures. In the other seven containers (three with S. magellanicum\u00a0+\u00a0S. balticum, two with S. fuscum\u00a0+\u00a0S. balticum, two with S. magellanicum\u00a0+\u00a0S. cuspidatum), cover changed less than 5%. For S. balticum there were significant effects of temperature (F\u00a0=\u00a03.67, P\u00a0=\u00a00.023) and neighbouring species (F\u00a0=\u00a04.95, P\u00a0=\u00a00.033) on expansion (Fig.\u00a03a,\u00a0b). Maximum change in cover was for S. balticum in combination with S. fuscum with temperature 2 and 3. There was no effect of temperature on expansion of S. cuspidatum (Fig.\u00a03c, F\u00a0=\u00a00.88, P\u00a0=\u00a00.470).\nFig.\u00a03Cover change for hollow species: aS. balticum in combination with S. fuscum, bS. balticum in combination with S. magellanicum and cS. cuspidatum in combination with S. magellanicum. Data are mean values\u00a0\u00b1\u00a0SE, n\u00a0=\u00a05. There were no significant differences between treatments (P\u00a0<\u00a00.05). For temperature treatments, see Table\u00a01\nBiomass production\nThe response of biomass production to the temperature treatments was similar to the response of height increment (Table\u00a02). In all species biomass production increased with increased temperature. S. fuscum showed the strongest response; this species increased its biomass production 13-fold from the lowest to the highest temperature treatment in monocultures (Fig.\u00a04a). Only for S. balticum in combination with S. magellanicum was there a significant interaction between temperature and species combination in biomass production (Table\u00a02). Temperature increased biomass production of S. balticum in monoculture and in mixture with S. fuscum, while there was no effect of temperature in mixture with S. magellanicum.\nFig.\u00a04Biomass production corrected for change in cover for species in monocultures (mono) and in mixtures (mix) for each temperature treatment (see Table\u00a01) and species combination: aS. fuscum\u00a0(fus) +\u00a0S. balticum (bal), bS. magellanicum\u00a0(mag) +\u00a0S. balticum and cS. magellanicum\u00a0+\u00a0S. cuspidatum (cus). Data are mean values\u00a0\u00b1\u00a0SE, n\u00a0=\u00a04\u20135. Different letters indicate significant differences between treatments (P\u00a0<\u00a00.05)\nWhen differences in biomass production between monocultures and mixtures are compared per temperature treatment, it shows that S. fuscum suffered from competition with S. balticum at temperature 3 because production was lower in mixture than in monoculture, but it no longer suffered at temperature 4 (Fig.\u00a04a). At temperature 4 S. balticum does suffer from competition with S. fuscum, but not at lower temperatures. In the mixtures of S. magellanicum and S. balticum, S. magellanicum suffers from competition at temperature 1 and 2, whereas S. balticum suffers from competition at temperature 3 and 4 (Fig.\u00a04b). Biomass production of S. cuspidatum did not show any effect of competition with S. magellanicum, while biomass production of S. magellanicum did suffer from competition at temperatures 1, 2 and 3 (Fig.\u00a04c).\nIn the stepwise regression for biomass production of S. balticum, water content was selected as the most explanatory variable (R2\u00a0=\u00a00.36, P\u00a0<\u00a00.001). For biomass production of S. fuscum (R2\u00a0=\u00a00.52, P\u00a0<\u00a00.001), S. magellanicum (R2\u00a0=\u00a00.36, P\u00a0<\u00a00.001) and S. cuspidatum (R2\u00a0=\u00a00.12, P\u00a0=\u00a00.031), temperature was selected as the most explanatory variable.\nBulk density\nBulk densities were lower at higher temperatures for all species, except for bulk density of the capitula and stems of S. cuspidatum (Table\u00a03). Compared to field values, capitulum bulk density of S. fuscum and S. balticum decreased with temperature, while capitulum bulk density of S. magellanicum and S. cuspidatum seemed to increase at low temperatures.\nTable\u00a03Bulk density of capitula section, stem section and total sample. Data are mean values\u00a0\u00b1\u00a0SE, pooled within temperature treatment (see Table\u00a01) and within competition treatment. Different letters indicate significant differences between treatments within a species (P\u00a0<\u00a00.05). There were no significant interactions between temperature and competition treatments. fus S. fuscum, bal S. balticum, mag S. magellanicum, cus S. cuspidatumField valueTemperatureCombinationT1T2T3T4Monofus\u2013balmag\u2013balmag\u2013cusBulk density capitula (mg\u00a0cm\u22123)S. fuscum38\u00a0\u00b1\u00a0235\u00a0\u00b1\u00a02b37\u00a0\u00b1\u00a02b21\u00a0\u00b1\u00a01a17\u00a0\u00b1\u00a01a30\u00a0\u00b1\u00a02b25\u00a0\u00b1\u00a02a\u2013\u2013S. balticum32\u00a0\u00b1\u00a0126\u00a0\u00b1\u00a01b16\u00a0\u00b1\u00a01a14\u00a0\u00b1\u00a01a14\u00a0\u00b1\u00a01a20\u00a0\u00b1\u00a01b18\u00a0\u00b1\u00a02b15\u00a0\u00b1\u00a01a\u2013S. magellanicum19\u00a0\u00b1\u00a0121\u00a0\u00b1\u00a01b18\u00a0\u00b1\u00a01b13\u00a0\u00b1\u00a01a13\u00a0\u00b1\u00a01a17\u00a0\u00b1\u00a01\u201315\u00a0\u00b1\u00a0117\u00a0\u00b1\u00a01S. cuspidatum19\u00a0\u00b1\u00a0123\u00a0\u00b1\u00a0122\u00a0\u00b1\u00a0219\u00a0\u00b1\u00a0119\u00a0\u00b1\u00a0123\u00a0\u00b1\u00a01b\u2013\u201319\u00a0\u00b1\u00a01aBulk density stems (mg\u00a0cm\u22123) S. fuscum18\u00a0\u00b1\u00a01b16\u00a0\u00b1\u00a01b15\u00a0\u00b1\u00a02ab11\u00a0\u00b1\u00a02a17\u00a0\u00b1\u00a01b14\u00a0\u00b1\u00a01a\u2013\u2013S. balticum15\u00a0\u00b1\u00a01b11\u00a0\u00b1\u00a01a8\u00a0\u00b1\u00a01a9\u00a0\u00b1\u00a01a13\u00a0\u00b1\u00a01b9\u00a0\u00b1\u00a01a11\u00a0\u00b1\u00a01ab\u2013S. magellanicum11\u00a0\u00b1\u00a01c9\u00a0\u00b1\u00a00bc8\u00a0\u00b1\u00a01ab7\u00a0\u00b1\u00a00a9\u00a0\u00b1\u00a01\u20139\u00a0\u00b1\u00a018\u00a0\u00b1\u00a00S. cuspidatum10\u00a0\u00b1\u00a0110\u00a0\u00b1\u00a017\u00a0\u00b1\u00a017\u00a0\u00b1\u00a018\u00a0\u00b1\u00a01\u2013\u20139\u00a0\u00b1\u00a01Bulk density total sample (mg\u00a0cm\u22123) S. fuscum22\u00a0\u00b1\u00a01c21\u00a0\u00b1\u00a01bc17\u00a0\u00b1\u00a01ab13\u00a0\u00b1\u00a01a20\u00a0\u00b1\u00a01b16\u00a0\u00b1\u00a01a\u2013\u2013S. balticum17\u00a0\u00b1\u00a01b12\u00a0\u00b1\u00a01a10\u00a0\u00b1\u00a01a10\u00a0\u00b1\u00a01a14\u00a0\u00b1\u00a01b11\u00a0\u00b1\u00a01a12\u00a0\u00b1\u00a01ab\u2013S. magellanicum13\u00a0\u00b1\u00a01c11\u00a0\u00b1\u00a00b9\u00a0\u00b1\u00a01a8\u00a0\u00b1\u00a00a11\u00a0\u00b1\u00a01\u201310\u00a0\u00b1\u00a0110\u00a0\u00b1\u00a00S. cuspidatum12\u00a0\u00b1\u00a01c12\u00a0\u00b1\u00a00bc10\u00a0\u00b1\u00a01ab9\u00a0\u00b1\u00a00a11\u00a0\u00b1\u00a01\u2013\u201311\u00a0\u00b1\u00a00Values for material from the sites of origin (Field values, n\u00a0=\u00a05) are given for comparison. Number of observations for each species for each temperature treatment: S. fuscum and S. cuspidatum, n\u00a0=\u00a010; S. balticum, n\u00a0=\u00a014\u201315; S. magellanicum, n\u00a0=\u00a015. Number of observations for each species for each combination, n\u00a0=\u00a019\u201320\nBulk densities of both capitula and stems were lower in mixtures than in monocultures of S. fuscum and S. balticum. Similarly, in S. cuspidatum the bulk density of the capitula was lower in mixtures than in monocultures. There were no significant interactions between temperature and species combination.\nNutrient concentrations\nThe concentrations of all nutrients (N, P and K) differed between species and rose with increasing temperature. N concentration was also affected by the interactions of temperature by species (F\u00a0=\u00a02.66, P\u00a0=\u00a00.007) and species by combination (F\u00a0=\u00a03.73, P\u00a0=\u00a00.006). At temperatures 1 and 2, N concentrations were lower for S. fuscum and S. balticum than for S. magellanicum and S. cuspidatum, as was also the case in field values. At temperature 3, only the N concentration of S. balticum was lower than that of S. magellanicum. At temperature 4, N concentrations of S. balticum were lower than those of S. magellanicum and S. fuscum (Table\u00a04). Compared to field values, all species showed an increased N concentration with temperature.\nTable\u00a04N, P and K concentration (mg\u00a0g\u22121) of the capitula. Data are mean values\u00a0\u00b1\u00a0SE, pooled within temperature treatment (see Table\u00a01) and within competition treatment. Different letters indicate significant differences between treatments within a species (P\u00a0<\u00a00.05). There were no significant interactions between temperature and competition treatments. For abbreviations, see Table 3Field valueTemperatureCombinationT1T2T3T4Monofus\u2013balmag\u2013balmag\u2013cusN (mg\u00a0g\u22121)S. fuscum6.5\u00a0\u00b1\u00a00.28.4\u00a0\u00b1\u00a00.2a8.6\u00a0\u00b1\u00a00.4a11.3\u00a0\u00b1\u00a00.6b12.0\u00a0\u00b1\u00a00.7b9.8\u00a0\u00b1\u00a00.510.4\u00a0\u00b1\u00a00.5S. balticum5.3\u00a0\u00b1\u00a00.18.4\u00a0\u00b1\u00a00.38.7\u00a0\u00b1\u00a00.49.7\u00a0\u00b1\u00a00.59.6\u00a0\u00b1\u00a00.39.5\u00a0\u00b1\u00a00.48.7\u00a0\u00b1\u00a00.49.0\u00a0\u00b1\u00a00.3S. magellanicum11.1\u00a0\u00b1\u00a00.511.7\u00a0\u00b1\u00a00.512.6\u00a0\u00b1\u00a00.312.8\u00a0\u00b1\u00a00.312.9\u00a0\u00b1\u00a00.312.4\u00a0\u00b1\u00a00.312.3\u00a0\u00b1\u00a00.312.7\u00a0\u00b1\u00a00.3S. cuspidatum10.0\u00a0\u00b1\u00a00.39.8\u00a0\u00b1\u00a00.611.3\u00a0\u00b1\u00a00.610.5\u00a0\u00b1\u00a00.510.8\u00a0\u00b1\u00a00.811.5\u00a0\u00b1\u00a00.4b9.7\u00a0\u00b1\u00a00.4aP (mg\u00a0g\u22121) S. fuscum0.39\u00a0\u00b1\u00a00.030.19\u00a0\u00b1\u00a00.07a0.30\u00a0\u00b1\u00a00.02ab0.40\u00a0\u00b1\u00a00.03b0.41\u00a0\u00b1\u00a00.04b0.31\u00a0\u00b1\u00a00.040.34\u00a0\u00b1\u00a00.04S. balticum0.36\u00a0\u00b1\u00a00.030.13\u00a0\u00b1\u00a00.090.26\u00a0\u00b1\u00a00.040.29\u00a0\u00b1\u00a00.030.29\u00a0\u00b1\u00a00.020.32\u00a0\u00b1\u00a00.040.22\u00a0\u00b1\u00a00.050.19\u00a0\u00b1\u00a00.05S. magellanicum0.29\u00a0\u00b1\u00a00.010.10\u00a0\u00b1\u00a00.06a0.18\u00a0\u00b1\u00a00.04ab0.27\u00a0\u00b1\u00a00.04ab0.37\u00a0\u00b1\u00a00.08b0.26\u00a0\u00b1\u00a00.070.21\u00a0\u00b1\u00a00.050.21\u00a0\u00b1\u00a00.03S. cuspidatum0.31\u00a0\u00b1\u00a00.030.10\u00a0\u00b1\u00a00.080.18\u00a0\u00b1\u00a00.040.23\u00a0\u00b1\u00a00.020.26\u00a0\u00b1\u00a00.040.24\u00a0\u00b1\u00a00.040.15\u00a0\u00b1\u00a00.04K (mg\u00a0g\u22121)S. fuscum54\u00a0\u00b1\u00a00.24.9\u00a0\u00b1\u00a00.2a5.5\u00a0\u00b1\u00a00.3a7.1\u00a0\u00b1\u00a00.3b7.1\u00a0\u00b1\u00a00.2b6.4\u00a0\u00b1\u00a00.35.9\u00a0\u00b1\u00a00.3S. balticum4.7\u00a0\u00b1\u00a00.44.7\u00a0\u00b1\u00a00.3a5.2\u00a0\u00b1\u00a00.2a6.4\u00a0\u00b1\u00a00.3b6.5\u00a0\u00b1\u00a00.3b5.6\u00a0\u00b1\u00a00.35.9\u00a0\u00b1\u00a00.35.5\u00a0\u00b1\u00a00.3S. magellanicum2.2\u00a0\u00b1\u00a00.23.8\u00a0\u00b1\u00a00.1a4.7\u00a0\u00b1\u00a00.2b6.6\u00a0\u00b1\u00a00.2c6.9\u00a0\u00b1\u00a00.2c5.6\u00a0\u00b1\u00a00.35.3\u00a0\u00b1\u00a00.45.4\u00a0\u00b1\u00a00.3S. cuspidatum2.2\u00a0\u00b1\u00a00.34.5\u00a0\u00b1\u00a00.3a5.0\u00a0\u00b1\u00a00.2a6.6\u00a0\u00b1\u00a00.3b6.9\u00a0\u00b1\u00a00.4b6.1\u00a0\u00b1\u00a00.3b5.5\u00a0\u00b1\u00a00.3aField values (n\u00a0=\u00a05) are given for comparison. Number of observations for each species for each temperature treatment: S. fuscum and S. cuspidatum, n\u00a0=\u00a010; S. balticum: n\u00a0=\u00a014\u201315; S. magellanicum, n\u00a0=\u00a015. Number of observations for each species for each combination, n\u00a0=\u00a019\u201320\nP and K concentrations were affected by temperature (P concentration, F\u00a0=\u00a09.32, P\u00a0<\u00a00.001; K concentration, F\u00a0=\u00a082.77, P\u00a0<\u00a00.001) and species (P concentration, F\u00a0=\u00a03.05, P\u00a0=\u00a00.031; K concentration, F\u00a0=\u00a03.86, P\u00a0=\u00a00.011). P and K concentrations were higher at high temperature for all species (Table\u00a04). P-values decreased, compared to field values, while K concentration increased compared to field values.\nEvaporation and water content\nWater content decreased with temperature treatment (Table\u00a05). Water content was lowest in S. balticum in mixture with S. fuscum. Evaporation increased with temperature and therefore with VPD for all species (Table\u00a05). Evaporation in S. fuscum in both monoculture and mixture was lower than in other species.\nTable\u00a05Water content (% volume) at 5\u00a0cm depth and evaporation (mm\u00a0day\u22121) per temperature treatmenta (see Table\u00a01) and per species combinationb. Data are mean values\u00a0\u00b1\u00a0SE. Different letters indicate significant differences between treatments (P\u00a0<\u00a00.05). There were no significant interactions between temperature and competition treatmentsWater contentEvaporationTemperature 188\u00a0\u00b1\u00a02c11.2\u00a0\u00b1\u00a00.02a 279\u00a0\u00b1\u00a03b21.5\u00a0\u00b1\u00a00.03b 360\u00a0\u00b1\u00a03a31.7\u00a0\u00b1\u00a00.04c 460\u00a0\u00b1\u00a03a42.0\u00a0\u00b1\u00a00.05dMonoculturesS. fuscum83\u00a0\u00b1\u00a05cS. fuscum1.3\u00a0\u00b1\u00a00.05aS. balticum66\u00a0\u00b1\u00a06bS. balticum1.7\u00a0\u00b1\u00a00.08bcS. magellanicum74\u00a0\u00b1\u00a04bcS. magellanicum1.8\u00a0\u00b1\u00a00.08cS. cuspidatum78\u00a0\u00b1\u00a04bcS. cuspidatum1.8\u00a0\u00b1\u00a00.08cMixturesS. fuscum74\u00a0\u00b1\u00a05bcS. fuscum\u00a0+\u00a0S. balticum1.5\u00a0\u00b1\u00a00.07aS. balticum49\u00a0\u00b1\u00a05aS. magellanicum69\u00a0\u00b1\u00a05bS. magellanicum\u00a0+\u00a0S. balticum1.6\u00a0\u00b1\u00a00.07bS. balticum68\u00a0\u00b1\u00a05bS. magellanicum82\u00a0\u00b1\u00a05cS. magellanicum\u00a0+\u00a0S. cuspidatum1.7\u00a0\u00b1\u00a00.08bcS. cuspidatum73\u00a0\u00b1\u00a05bcNumber of observations for each temperature treatment: for water content, n\u00a0=\u00a050; for evaporation, n\u00a0=\u00a035. Number of observations for each competition treatment: for water content and for evaporation, n\u00a0=\u00a020\nDiscussion\nEffect of temperature on growth\nAs expected, all species in monoculture increased height increment with temperature (Fig.\u00a01). All species also showed a looser growth form with higher temperatures, which resulted in lower densities (Table\u00a03). Despite the lower bulk density at higher temperatures, biomass production still increased with temperature for all species (Table\u00a02, Fig.\u00a04). The response of height increment to temperature was about a factor 2 larger than the response of biomass production. Growth of Sphagnum was probably increased by a higher rate of photosynthesis and a higher N availability. Photosynthesis in Sphagnum has been shown to increase with temperature up to an optimum around 20\u201325\u00b0C (Skre and Oechel 1981; Harley et al. 1989). When temperature increased, the nutrient concentrations in Sphagnum were higher (Table\u00a04). Moreover, N and K concentration increased compared to field values (Table\u00a04). As biomass production also increased, N and K uptake must have increased considerably with increasing temperature. No nutrients were added with the rainwater solution, so the higher availability of nutrients must have come from Sphagnum itself and the peat below. Probably the lower Sphagnum parts in the containers decomposed faster when the temperature was higher, making more N and K available for growth. This temperature-induced stimulation of nutrient mineralization is probably larger than it would be in field conditions as the peat soil in the containers is surrounded by warm air on every side, resulting in relatively high soil temperatures in our experiment. However, it can be expected that increased temperature as a result of climate change will also enhance decomposition rates in field situations (Hobbie 1996), thereby increasing nutrient availability for both Sphagnum and vascular plants. The lower N concentrations of S. fuscum and S. balticum compared with S. magellanicum and S. cuspidatum at temperatures 1 and 2 are probably caused by the different sites of origin. In the northern site, the N deposition is much lower (approximately 0.2\u00a0g\u00a0N\u00a0m\u22122\u00a0year\u22121) than in the southern site (approximately 0.7\u00a0g\u00a0N\u00a0m\u22122\u00a0year\u22121) where S. magellanicum and S. cuspidatum were collected.\nIn this experiment we provided near optimal growing conditions for Sphagnum with high water level and high humidity, which explains why the Sphagnum plants could grow with a lower bulk density and still keep their capitula moist. However, Sphagnum water content still decreased with an increase in temperature. In field conditions it would probably not have been possible for the Sphagnum plants to change their density so much since it would have led to problems in maintaining the water content of the capitula. The effect of temperature on height increment might therefore also have been less. In a few field studies, no positive or even negative effects of temperature on production were found. However, in these experiments there were other factors besides temperature that could have had a negative effect on biomass production, like drought stress (Hobbie et al. 1999; Weltzin et al. 2001; Gunnarsson et al. 2004) and higher vascular plant cover, which could have resulted in etiolation of the Sphagnum plants and reduced biomass production because of increased shading (Gunnarsson et al. 2004).\nIn other studies also positive effects of temperature on height growth and production were found for different species, in both greenhouse (Robroek et al. 2007b) and field experiments (Moore 1989; Gerdol 1995; Sonesson et al. 2002; Dorrepaal et al. 2003). In accordance with our hypothesis, Asada et al. (2003) suggested that Sphagnum species respond positively to increased temperature and that hollow and lawn species are more sensitive to temperature than hummock species. This, however, does not correspond with the low response of S. cuspidatum to temperature in our experiment. Dorrepaal et al. (2003) also found a decrease in bulk density with increased temperature. Since the response of height increment and bulk density to temperature were in opposite directions, the dry matter production of S. fuscum did not show a significant increase with temperature. Our relatively high height increment and low density of S. fuscum compared to the values of Dorrepaal et al. (2003) can probably be explained by the much higher water levels in our experiment. Dorrepaal et al. (2003) used water levels of \u221220 to \u221240\u00a0cm below moss surface during their measurements. However, the Sphagnum densities we measured (Table\u00a03) agreed quite well with densities from other studies (Lindholm and Vasander 1990; Moore et al. 1998; Asada et al. 2003).\nFrom our results, we conclude that the height increment and biomass production of all Sphagnum species in bogs can increase with an increase in temperature. However, the literature shows us that, particularly under field conditions, this potential response may not be realized in instances of competition from vascular plants, drought stress or extreme temperature increases.\nEffect of temperature on competition\nWe hypothesized that increased temperature will have a positive effect on competitive abilities of S. fuscum and S. magellanicum. This hypothesis was confirmed in the competition with S. balticum, since the height increment and biomass production of S. balticum at the highest temperature treatment were no longer larger than those of S. fuscum and S. magellanicum (Figs.\u00a02,\u00a04). However, S. balticum still increased its cover at the expense of S. fuscum and S. magellanicum in all mixtures (Fig.\u00a03a,\u00a0b). This could be expected as the water level we used was relatively high and in the range of the natural habitat of these species. These results correspond to the findings from a field experiment of Rydin (1986), who found an expansion of S. balticum at the expense of S. fuscum at a water level of \u22121\u00a0cm. At water levels of \u22125 and \u221210\u00a0cm, he found a small expansion of S. balticum, but at a water level of \u221215\u00a0cm S. fuscum showed large expansion.\nIn agreement with the hypothesis, the difference in height increment and biomass production between S. fuscum and S. balticum disappeared as temperature increased (Figs.\u00a02a,\u00a04a). S. fuscum kept increasing height increment with temperature, but S. balticum already reached maximum height growth at temperature 2, which is comparable to the summer temperature in the site of origin of these species. Also, the expansion in cover became lower for S. balticum at temperature 4 (Fig.\u00a03a). As a result, the difference in biomass production between S. fuscum and S. balticum slowly decreased with an increase in temperature until there was no significant difference at the highest temperature. Apparently, the hummock species S. fuscum is better able to profit from a high increase in temperature than S. balticum, even at relatively high water levels. This is confirmed by the stepwise regression analysis which shows that biomass production of S. fuscum is correlated most to temperature, and biomass production of S. balticum to water content. As could be expected, S. fuscum as a hummock species has a higher water content than S. balticum, also at higher temperatures (Table\u00a05), and therefore can profit more from increased temperature, because water does not become limiting for growth. S. fuscum also seems to be the better competitor for nutrients. The N and P concentrations in S. fuscum tended to be higher in the mixtures than in the monocultures, whereas in S. balticum the opposite was the case (Table\u00a04). Indeed, S. fuscum is known for its high N uptake rate (Jauhiainen et al. 1998). Our results show that S. balticum loses competitive strength with increasing temperature. With a slight increase in temperature in northern bogs, S. balticum may remain the stronger competitor at high water levels, but at higher temperature increases, S. fuscum will gain a competitive advantage over S. balticum, also because increased temperature may lead to lower water tables in bogs. Recent climate change simulation models predict an increase of 2.5\u00b0C in summer temperature in northern Europe, with increasing precipitation in northern Scandinavia (Christensen et al. 2007). This limited rise in temperature, almost similar to the increase from temperature 2 to 3, would not affect competition between S. balticum and S. fuscum very strongly. However, the effect of a predicted increase in winter temperature of 5.0\u00b0C (Christensen et al. 2007) could shift the competitive balance between S. balticum and S. fuscum further, because increased snow cover and spring temperature have been shown to increase S. fuscum production (Dorrepaal et al. 2003).\nFor S. magellanicum and S. cuspidatum, temperature 3 is comparable to the summer temperature in their site of origin. There was no effect of a temperature increase from temperature 3 to 4 on biomass production and relative performance of the species. This implies that with the predicted increase in temperature of 2.5\u00b0C (Christensen et al. 2007), the competitive balance between S. magellanicum and S. cuspidatum will remain unchanged.\nThe mixtures of S. balticum and S. magellanicum show the largest difference in height increment and biomass production at temperature 2, which is comparable to the summer temperature in the site of origin of S. balticum. Also the expansion in cover of S. balticum was highest at this temperature. When temperature increases, the difference in height growth and biomass production became smaller and then disappeared in mixtures, whereas in monocultures the difference in biomass production between species remained. This implies that S. balticum as a northern species can enhance its production with an increase in temperature as long as it grows in monocultures. If S. magellanicum expanded its distribution further to the north as a result of global warming, it would be a strong competitor for S. balticum, thereby reducing S. balticum production. Gunnarsson et al. (2004) also showed that S. balticum performs less well under competition with increased temperatures. They performed a competition experiment in a poor fen, close to the site of origin of the S. balticum material. They found that when Sphagnum papillosum was transplanted to an area with S. balticum, S. papillosum decreased in area by 30%. However, when temperature was increased by 3.6\u00b0C, S. papillosum increased by 42%. This treatment is comparable to the increase from temperature 2 to temperature 3 in our experiment but with lower water tables, ranging from 5 to 17\u00a0cm under the moss surface.\nGenerally, we conclude from our experiment that an increase in temperature can favour hummock and lawn species compared to hollow species. This corresponds to the findings of Mauquoy et al. (2002), who showed from peat core analyses that in periods with lower temperature, due to decreased solar activity, there was a shift in representation from lawn and hummock species to hollow species in ombrotrophic mires in Denmark and the UK.\nIn conclusion, our findings suggest that production rates can increase and that Sphagnum species\u2019 abundances can shift in response to global warming, particularly at northern sites. There, hollow species such as S. balticum will lose competitive strength relative to hummock species such as S. fuscum and southern species such as S. magellanicum. What the consequences for the C balance of bog ecosystems will be depends not only on the production rates, but also on decomposition. In general, decomposition rates also increase with temperature and increased N availability (Hobbie 1996; Limpens and Berendse 2003) and hummock species decompose slower than hollow species (Rochefort et al. 1990; Johnson and Damman 1993; Limpens and Berendse 2003), but virtually nothing is known about the temperature sensitivity of decomposition of bog plant species.","keyphrases":["bog","global warming","greenhouse","cover change","species composition"],"prmu":["P","P","P","P","M"]}
{"id":"Pediatr_Radiol-3-1-2077923","title":"Fractional anisotropy in white matter tracts of very-low-birth-weight infants\n","text":"Background Advances in neonatal intensive care have not yet reduced the high incidence of neurodevelopmental disability among very-low-birth-weight (VLBW) infants. As neurological deficits are related to white-matter injury, early detection is important. Diffusion tensor imaging (DTI) could be an excellent tool for assessment of white-matter injury.\nIntroduction\nThe last decade has seen improved survival for very-low-birth-weight (VLBW) preterm infants [1, 2]. The survivors, however, still show a high incidence of neurodevelopmental disability, despite advances in fetal and neonatal intensive care [3\u20135]. These neurological deficits are usually related to white-matter injury [6, 7]. Thus early detection of abnormal white-matter maturation is important in the design of preventive, protective, and rehabilitative strategies for the management of the critically ill newborns [8, 9].\nThe clinical evaluation of these infants may not provide adequate diagnostic or prognostic information on white-matter injury. Various neuroimaging techniques have become available, however, that may be of help. Cranial US is such a technique used in premature infants, but it is not as sensitive as MR imaging, which shows better soft-tissue contrast [10]. Yet, conventional T1-weighted (T1-W) and T2-weighted (T2-W) MR imaging sequences also have limited value in the evaluation of the preterm brain, as they do not allow visualization of specific white-matter tracts before the onset of myelination. Most white-matter tracts of the premature cerebrum are unmyelinated except for a few pathways, e.g. the pyramidal tract that matures early as the infant approaches term-equivalent age [11, 12].\nDiffusion tensor imaging (DTI) is unique in its ability to visualize and quantify white-matter tracts in the human brain. It is superior to T1-W and T2-W imaging in detecting unmyelinated or premyelinated fibre tracts [13] and, therefore, likewise in assessing the microstructural organization of the developing white matter. DTI anisotropy measurements offer great potential in the study of white-matter damage in VLBW infants. So far, however, the clinical use of this technique is restricted by the lack of normal reference values for VLBW infants. Although there are reports on fractional anisotropy (FA) and apparent diffusion coefficient (ADC) values in preterm infants, only four groups have reported values for infants of gestational age less than 32\u00a0weeks without brain injury (Table\u00a01). Each of the populations studied included no more than four infants without white-matter damage on conventional imaging, scanned within days after birth and gestational age less than 32\u00a0weeks [14\u201317]. Because the sizes and shapes of the regions of interest (ROIs) in these studies differ, comparison between the studies is difficult. We therefore set up a retrospective study aimed at providing reference values for the clinical interpretation of DTI images in VLBW infants.\nTable\u00a01Overview of relevant literatureReferenceGestational age (weeks)Scan weekNo. of patientsNo. with gestational age <32\u00a0weeksMR field strength (T)ROI14<3728\u20134327<51.5FTa1524\u20133628\u20133917<51.5Manual1624\u20133628\u20133914<51.5Manual1726\u20133028\u2013406<51.5ManualaROI placement with fibre tracking.\nMaterials and methods\nSubjects\nThe Erasmus MC Ethical Review Board approved the study and written informed parental consent was obtained for each subject. The images used in this study had been obtained within the framework of a study in which premature infants of different gestational ages underwent serial conventional and DTI acquisitions to evaluate white-matter development. The inclusion criteria for our study were birth at gestational age 25\u201332\u00a0weeks, no evidence of white-matter injury on conventional MRI and scanned within 4\u00a0days of birth. Developmental outcome assessed at age 1\u20133\u00a0years needed to be normal. Gestational age was calculated from the mother\u2019s last menstrual period or estimated from early US (<18\u00a0weeks of pregnancy). Exclusion criteria were intraventricular haemorrhage, ventriculomegaly, congenital infection, brain malformation or a multiple congenital anomaly syndrome. Furthermore, preterm infants whose images showed severe motion degradation were excluded since evaluation of these scans was not possible. During the 25\u00a0months of the study period (March 2004 to April 2006), 41 infants were scanned and 32 met our study criteria. Four MR examinations were excluded due to severe motion artefacts, so 28 patients were included.\nNeurodevelopment monitoring\nThe infants\u2019 hospital charts were reviewed for neurodevelopmental outcome. They had all been neurologically assessed at 3, 6, 12 and 24\u00a0months, and Denver scores were used to monitor neurodevelopment [18]. At 24\u00a0months the Bayley scales of infant development, second edition (BSID-II) had also been applied [19]. All patients had shown normal neurodevelopment according to the most recent examination.\nConventional MR imaging\nImages were acquired using standard scanning protocols. All imaging was performed on a 1.5-T GE EchoSpeed scanner (GE Medical Systems, Milwaukee, Wis.). The imaging protocol included T1-W (spin echo, TR\/TE 500\/11\u00a0ms) and T2-W sequences (spin echo, TR\/TE 3,000\/120\u00a0ms) with a slice thickness of 4\u00a0mm and 0.4\u00a0mm gap. For DTI an echoplanar sequence with diffusion gradients (b\u2009=\u20091,000\u00a0s\/mm2) applied in 25 non-collinear directions was used with a slice thickness of 3\u00a0mm and no gap. An average of 20 slices was recorded within 4\u00a0min using TR\/TE 9,150\/98\u201391\u00a0ms. The FOV was 20\u00a0cm, the scan matrix 128\u00d7128\u00a0and the reconstruction matrix 256\u00d7256.\nAll patients were scanned using an MR-compatible incubator with a specialized high-sensitivity neonatal head coil (Lammers Medical Technology, Lubeck, Germany) that allowed DTI imaging at high spatial resolution and high signal-to-noise ratio (SNR). The incubator provided controlled temperature and humidity as well as MR-compatible pulse oximetry and ventilation. This set-up allowed imaging in this most vulnerable patient population in a stable and safe microenvironment [20\u201322]. We used mouldable earplugs and neonatal earmuffs to reduce the noise. No sedation was given. Pads around the infant\u2019s head kept movement to a minimum.\nDiffusion tensor postprocessing\nDiffusion tensor images were transferred to a GE Advantage Windows workstation (General Electric Medical Systems, Milwaukee, Wis.) for postprocessing using Functool 2000 software (General Electric). DTI measures the diffusion of water in each voxel and the extent to which water diffuses in particular directions as a result of the microstructural characteristics of the tissue imaged. High anisotropy indicates that the magnitude of diffusion is very unequal in different directions. The complex nature of anisotropic diffusion in the brain has been described by a diffusion tensor, which contains information about the magnitude of diffusion in different directions. Each tensor contains a set of three eigenvalues, which are related to the major, intermediate, and minor axes of a diffusion ellipsoid. The principal eigenvector (\u03bb1) specifies the direction in which water diffusion is greatest. FA measures the fraction of the magnitude of the diffusion tensor that can be ascribed to anisotropic diffusion [23\u201325]. For isotropic diffusion (\u03bb1\u2009=\u2009\u03bb2\u2009=\u2009\u03bb3), FA is zero, and in the case where there is a strongly preferred direction of diffusion (\u03bb1>>\u03bb2\u2009=\u2009\u03bb3), FA approaches a value of one. The colour maps were based on major eigenvector orientation with red representing right\u2013left, green representing anteroposterior, and blue representing superoinferior anatomical directions.\nThe tracts selected for quantization in the study included commissural tracts (corpus callosum: splenium and genu), projection tracts including those of the posterior limb of the internal capsule (PLIC), the anterior limb of the internal capsule (ALIC) and the optic radiation (OR), and association tracts (external capsule, EC).\nRegions of interest\nDTI measurements were taken from multiple ROIs positioned bilaterally within individual white-matter tracts. For the placement we used standard-size, round-shaped 16-pixel ROIs. To optimize our ROI placement we first detected the patient scan slice that represented slice \u201cO\u201d of the atlas of Wakana et al. [26]. We then placed the ROI, combining the FA, ADC and colour map to verify the right location. When the two researchers both agreed on the position, the ROI was used as a seed point to perform fibre tracking (minimum length 10\u00a0mm, minimum anisotropy 0.10). We compared the fibres that were visualized with the fibre bundles described in the atlas of Wakana et al. for verification (Figs.\u00a01, 2 and 3). We then looked for the maximum pixel value within the ROI (Figs.\u00a04 and 5). Two researchers experienced in neonatal DTI achieved consensus on ROI placement and measurements.\nFig.\u00a01Example of an FA map (gestational age 28\u00a0weeks) on which the ROIs are placedFig.\u00a02Confirmation of ROI placement on the colour map (gestational age 28\u00a0weeks)Fig.\u00a03Fibre tracking was performed in each of the ROIs to confirm the correct location. This is an example showing the corticospinal tracts (gestational age 28\u00a0weeks)Fig.\u00a04Delineation of the corpus callosum using free-hand ROI placement on an FA map (gestational age 28\u00a0weeks)Fig.\u00a05Automated calculation of the maximum FA pixel value within the ROI on an FA map (gestational age 28\u00a0weeks)\nStatistical analysis\nThe relationship between FA of the white matter tracts and gestational age was analysed by correlation analysis (Pearson product moment correlation, SPSS 13.0.1). Tract comparison was done using one-way analysis of variance (ANOVA).\nResults\nPatient characteristics\nGestational ages at birth ranged from 26 to 32\u00a0weeks (mean 28\u00a0weeks 5\u00a0days). Mean weight at birth was 1,148\u00a0g and mean head circumference was 26.4\u00a0cm. All patients had shown normal neurodevelopment as defined by the Denver or Bayley scoring system according to the most recent examination.\nFA and ADC of ROIs of standard pixel size\nWe plotted the average FA and ADC values of 16-pixel ROIs of the different white-matter tracts against gestational age. No statistically significant differences based on a two-tailed paired Student\u2019s t-test (at a significance level of P<0.05) were found comparing FA and ADC values between the left and right hemispheres in each subject. Therefore, the values obtained from the left and right were averaged to obtain the mean FA and ADC of the different ROIs. We found a significant correlation between gestational age and FA of the PLIC (r\u2009=\u20090.495, P<0.01; Fig.\u00a06). No significant correlations were found for the other tracts (Table\u00a02). We found no correlation between average ADC values and gestational age in the studied tracts (Table\u00a02).\nFig.\u00a06Average FA values of 16-pixel ROIs of the PLICTable\u00a02Tract statistics (FA and ADC mean values and standard deviations) and significant differences in FA and ADC between tractsStructureFAADCMean (SD)Tracts showing significant differences (P<0.05)Mean (SD)Tracts showing significant differences (P<0.05)Posterior limb of internal capsule0.349 (0.028)a, b, c1.09 (0.05)l, m, n, o, pAnterior limb of internal capsule0.242 (0.033)a, d, e, f1.27 (0.091)lExternal capsule0.175 (0.188)b, g, h, i1.29 (0.097)mOptic radiation0.270 (0.042)g, j, k1.30 (0.126)nCorpus callosum (genu)0.42 (0.048)d, e, h, j1.24 (0.111)oCorpus callosum (splenium)0.442 (0.056)c, f, i, k1.27 (0.131)p\nMaximum FA values of the PLIC\nThe maximum FA values of the ROI were plotted against gestational age and also showed a significant correlation between gestational age and FA of the PLIC (r\u2009=\u20090.569, P<0.01; Fig.\u00a07). We saw no other significant correlations between FA and gestational age. Compared with standard-size ROI mean FA values of the PLIC, the maximum values were a mean of 0.05 higher.\nFig.\u00a07Maximum FA ROI values of the PLIC\nComparison between tracts\nDTI values varied between white-matter structures (Table\u00a02). FA was highest in the commissural tracts of the corpus callosum (splenium>genu) and deep projection tracts (internal capsule). FA was lowest in association tracts (external capsule).\nDiscussion\nOur aim was to provide clinicians with reference values for the evaluation of diffusion tensor images of white-matter tracts in VLBW infants. A literature search identified four similar studies. Comparison with these studies seems irrelevant as each included no more than four infants evaluated below a gestational age of 32\u00a0weeks within the first days of life. We were able to study scans of 28 VLBW infants without white-matter damage within the first 4\u00a0days of life. We found a statistically significant correlation between gestational age and FA of the PLIC. Differences were observed between white-matter tracts: FA was highest in commissural, followed by deep projection and association tracts. Others have found the same hierarchy in the perinatal brain [16] and in fully myelinated adult tracts [27].\nDifferent anisotropy indexes can be used to study the anisotropy of white-matter tracts: FA, relative anisotropy (RA), axial anisotropy etc. We focused on FA, the most frequently used anisotropy index. FA has a high sensitivity for studying anisotropy in white-matter tracts [16]; it has a higher SNR than RA for anisotropic regions [28, 29]. Previous studies have shown that FA is the most sensitive and significant discriminating DTI parameter in pair-wise comparisons between different tracts in premature infants [16]. In most tracts FA increases significantly by the week, whereas weekly decreases in ADC values are smaller and less discriminative in most regions [16]. There are a growing number of reports of FA values of injured tracts [30\u201332]. Nevertheless, we should realize that FA is only one of the functions that describe the diffusion tensor. Although aberrant FA values seem to be a sensitive detector of abnormal tissue one could also use the relative magnitudes of the three principal eigenvectors to describe diffusion.\nWhat determines anisotropy in white-matter tracts? The value of the anisotropic index is determined by size, shape and composition of physical obstructions, as well as the space between them. Myelin, fast axonal transport, the axonal cytoskeleton (neurofilaments and microtubules), and local susceptibility gradients do not seem to have any major impact on anisotropic water diffusion [26, 33]. Indeed, intact membranes are thought to be the main contributing factor to anisotropy. The increasing anisotropy of white matter takes place before the histological appearance of myelin [15]. This increase has been attributed to the \u2018premyelinating state\u2019 [13, 34]. The corpus callosum, with a coherent parallel organization, has the highest FA values. Callosal fibre tracts, however, do not show a significant increase in FA with increasing gestational age, as the PLIC does. This might be due to the fact that myelination progresses more slowly in the callosal fibres than in the projection fibres at this age.\nDuring development a decrease in ADC values of the white-matter tracts is expected. We found no correlation between average ADC values and gestational age in the studied tracts. When looking at the available data reported previously we only saw the ADC values decrease significantly with gestational age when the ADC values of the infants older then 35\u00a0weeks were included. The fact that we could not find any significant correlation could be due to the limited number of infants scanned. The alternative is that at an earlier gestational age there is no significant decrease in ADC in the studied tracts.\nStudying DTI parameters of the white-matter tracts of VLBW infants is challenging for many reasons. One of these challenges is to determine a standard for the size and shape of the ROIs. A ROI is a controlled identification of a given area of an image for numerical analysis and the area of anatomy being scanned that is of particular importance in the image. Different authors have used different ways to set their ROIs. The reason we compared two techniques was to achieve better reproducibility. Maximum values (the maximum pixel value within a ROI) might be an alternative in which the value is given by the software. However, theoretically there is a bigger change of artefacts. Fibre tracking, colour maps and ADC maps are established but time-consuming techniques for the verification of tracts. We trust that automatic verification will become common practice in the future. A possible solution for ROI comparisons between researchers is the use of a neonatal brain atlas coordinate system. Individual brain images could then be transformed into a common coordinate space and the ROIs could be placed at specific topographic coordinates. Our research group is currently looking into this option.\nAnother serious challenge is the SNR and spatial resolution constraints due to the very low anisotropy of premyelinating white matter and the tiny size of white-matter tracts in premature newborns. Using a custom-made MR-compatible incubator with a high-sensitivity neonatal head coil that improved image quality, spatial resolution and patient comfort, we were able to overcome this challenge.\nIn this retrospective study a b value of 1,000\u00a0s\/mm2 was used to allow comparisons between early and later scans at different ages. We realize a lower b value would be optimal because the ADC value in neonatal white matter is higher than that of more mature brains. The optimal b value can be calculated as indicated by Jones et al. [35].\nLike all VLBW infants discharged from our NICU, our study patients were routinely seen by trained paediatricians and by paediatric physical therapists at the outpatient department for neurodevelopmental follow-up. Neurodevelopmental outcome was defined according to the most recent neurological examination and Denver and Bayley scores. The children\u2019s ages at the most recent assessments varied, making the results difficult to compare; also Denver and Bayley scores are limited in their prognostic value below the age of 2\u00a0years.\nBetter understanding of normal preterm white matter development is essential to encourage the use of DTI for evaluation and treatment of white-matter injury. Early diagnosis of white-matter abnormalities means that early intervention might be possible. We are exploring the feasibility of perinatal brain repair, and new MR imaging techniques such as DTI will enable us to improve our understanding of how the developing brain responds to our interventions.\nConclusion\nOur study gives anisotropy values for VLBW infants with normal outcome that can be used as reference values. This work adds to our understanding of normal preterm white-matter development.","keyphrases":["fractional anisotropy","diffusion tensor imaging","prematurity","mri"],"prmu":["P","P","P","P"]}
{"id":"Ann_Hematol-4-1-2226003","title":"Iron and thrombosis\n","text":"Although essential for cell physiology, an increase or depletion of body iron has harmful effects on health. Apart from iron deficiency anemia and iron overload-related organ tissue damage, there are increasing evidences that body iron status is implicated in atherosclerotic cardiovascular diseases. The hypothesis formulated in 1981 that iron depletion may protect against cardiovascular events is intriguing and has generated a significant debate in the last two decades. Indeed, to study this phenomenon, several investigators have tried to design appropriate experimental and clinical studies and to identify useful biochemical and genetic markers of iron status. The results of the literature on the effect of iron deficiency and overload on vascular health are critically reviewed in this study from a pathogenic and clinical point of view.\nIntroduction\nIron is an essential nutrient for living cells because of its role as a cofactor for enzymes in the mitochondrial respiration chain, in the DNA synthesis, and being the central molecule for binding and transport of oxygen by hemoglobin and myoglobin.\nWhile the lack of iron leads to growth arrest and anemia, an increased accumulation of this metal is associated with toxic radical formation and progressive tissue damage. It is interesting to note that both iron deficiency and excess have been associated with an increased risk of developing thromboembolic events [1\u20137].\nThis review will analyze, from a clinical and pathogenic point of view, the existing literature data on the relationship between iron and arterial and venous thrombosis.\nIron deficiency and thrombosis\nThere are several reports in the literature on thrombotic complications in iron-deficient children and adults [8\u201326]. Secondary thrombocytosis has been implicated in many cases. Indeed, iron deficiency is a cause of reactive thrombocytosis, usually mild [27]. For instance, within a study group of children with iron deficiency, reactive thrombocytosis was found in up to one-third of them [28].\nNagai et al. [29] reported a case of severe iron deficiency with marked thrombocytosis (1,020\u2009\u00d7\u2009109\/l) that was complicated by central retinal vein occlusion. By contrast, Kinoshita et al. [26] described two cases of cerebral venous sinus thrombosis associated with iron deficiency and normal platelet count. Hartfield et al. [13] reported six children with iron deficiency who developed an ischemic stroke or venous thrombosis. Four of them had a concomitant thrombocytosis. In a prospective case\u2013control study, Stolz et al. [30] found that severe anemia, along with thrombophilia and hypercholesterolemia, were independent risk factors for cerebral venous thrombosis.\nThe mechanisms causing reactive thrombocytosis in iron deficiency anemia are not completely understood. Iron is an important regulator of thrombopoiesis [31, 32]. Whereas normal iron levels are required to prevent thrombocytosis by inhibiting thrombopoiesis, a minimum amount of iron is required to maintain platelet production. Thus, while thrombocytosis is usually associated with a mild iron deficiency and is the result of a lack of inhibition of thrombopoiesis, a severe defect of this metal may be accompanied by thrombocytopenia. However, studies on thrombopoietic cytokines failed to show any effect on reactive thrombocytosis in iron deficiency [27]. For instance, Akan et al. [33] assayed the serum levels of thrombopoietin, erythropoietin, leukemia inhibitor factor, IL-6, and IL-11 in patients with iron-deficient anemia with or without elevated platelet count. Only erythropoietin level was elevated, correlated with thrombocytosis, and decreased with iron replacement. The other cytokines remained unchanged after therapy, suggesting that they probably do not play any significant role in iron deficiency-associated reactive thrombocytosis. Recently, Bilic and Bilic reported that the amino acid sequence homology of thrombopoietin and erythropoietin may explain the thrombocytosis in children with iron deficiency anemia [34]. By contrast, two other reports suggested that the relationship between iron deficiency and reactive thrombocytosis is more complex than a mere consequence of a crossreactivity between erythropoietin and thrombopoietin [35, 36]. In addition to the increased thrombotic risk associated with high platelet count, other authors have suggested that the decrease in antioxidant defense in iron deficiency anemia may cause increased oxidant stress, which in turn may result in a tendency toward platelet aggregation [37]. Thus, the abnormal platelet count and function observed in iron deficiency anemia could act synergistically to promote thrombus formation, especially in the setting of an underlying atherosclerotic disease [23].\nHowever, as not all cases of iron-related thrombotic events occur in patients with concomitant high platelet count [13, 26], other pathogenic mechanisms have been proposed in these last years. Thus, iron deficiency may contribute to a hypercoagulable state by affecting blood flow patterns within the vessels because of reduced deformability and increased viscosity of microcytic red blood cells [13]. Furthermore, anemic hypoxia secondary to iron deficiency could precipitate situations of increased metabolic stress (i.e., infections) in particularly vulnerable areas of the brain supplied by end arteries, such as the basal ganglia, thalamus, and hypothalamus [38]. This phenomenon could explain the association between iron-deficient anemia and reversible focal deficits and stroke found by some authors [1, 13, 39, 40].\nIron overload and thrombosis\nAccumulation of iron in excess of physiologic requirements has been implicated in the development of several chronic illnesses, including cardiovascular diseases [7]. As previously reported, iron is a prooxidant cofactor associated with an increased production of hydroxyl radical in cardiovascular tissues and increased progression of atherosclerosis in experimental models [41\u201344]. Indeed, in an experimental mouse thrombosis model, a moderate iron overload markedly accelerated thrombus formation, impaired vasoreactivity, and enhanced the production of reactive oxygen species and systemic markers of oxidative stress [41]. It is interesting to note that the administration of dl-cysteine, a reactive oxygen species scavenger, completely abrogated the iron load-induced thrombus formation thus corroborating the hypothesis that iron accelerates thrombosis through a prooxidant mechanism. Similarly, laboratory investigations have demonstrated iron-dependent generation of reactive oxygen species in endothelial cell cultures and increased aortic atherosclerosis in the apolipoprotein E-deficient mice and cholesterol-fed rabbits with increased iron intake [42\u201344].\nThe research in this field has focused on the evaluation of the impact of iron depletion and iron overload on cardiovascular outcomes. Sullivan first postulated in 1981 the \u201ciron hypothesis\u201d by which the chronic iron depletion has a protective effect against ischemic heart disease and may account for the reduced risk of cardiovascular events in menstruating women [45\u201347]. Basing on these observations, some investigators have studied the effect of serial blood donations on the coronary heart disease risk, but their results were inconsistent [48\u201353]. However, an important support to the hypothesis of a potential link between blood donation and reduced cardiovascular risk came from a recent study conducted by Zheng et al. [54] who found that high-frequency blood donors had decreased serum ferritin levels, a marker of body iron stores; decreased serum 3-nitrotyrosine levels, a marker of oxidative stress; and greater flow-mediated dilation in the brachial artery, a marker of vascular function. It is interesting to note that in a clinical study, the iron chelation with deferoxamine improved endothelial function in patients with coronary artery disease [55]. By contrast, in a multicenter, randomized controlled trial (the iron [Fe] and Atherosclerosis Study [FeAST]) on 1,277 patients with symptomatic peripheral arterial disease, the reduction of body iron stores by phlebotomy did not significantly decrease all-cause mortality or death plus nonfatal myocardial infarction and stroke [56]. Similarly, in a prospective analysis of the second National Health and Nutrition Examination Study (NHANES II), Sempos et al. [57] observed either no association (in Caucasian men) or a possible nonsignificant increased risk (in Caucasian women) of cardiovascular or coronary heart disease death among individuals with low ferritin concentrations.\nOn the other hand, the association between biochemical markers of body iron load and the risk of developing cardiovascular disease have been investigated by several studies [7, 58\u201372]. In the Kuopio Ischemic Heart Disease Risk Factor Study (KIHD), serum ferritin levels were found to be one of the strongest risk factors for acute myocardial infarction among Finnish men [61]. Similar findings were reported from a Canadian study [65] that observed an increased risk of myocardial infarction among subjects in the highest serum iron category and from the Bruneck study [64] in which a positive association between serum ferritin levels and ultrasound measures of progression of carotid atherosclerosis over a 5-year follow-up period was found. A relationship between serum ferritin levels and carotid atherosclerosis was also identified by a recent study conducted by Wolff et al. [71]. Haidari et al. [60] observed a significant correlation between serum ferritin levels and risk of coronary heart disease in male Iranian patients. However, a number of epidemiological studies did not find an association between iron status and coronary artery disease [61, 69, 70, 72]. For instance, Bozzini et al. [61] found that the mean serum ferritin concentrations were slightly higher in coronary heart disease patients than in controls, but this difference disappeared after adjusting for sex and C-reactive protein.\nThe 1996 discovery of HFE gene mutations responsible for most cases of hereditary hemochromatosis has led to the use of genetic markers of iron overload, which are not influenced by external factors such as inflammation, in epidemiologic studies. Thus, several authors have investigated in recent years the relationship between C282Y and H63D mutations in the HFE gene and the risk of cardiovascular diseases [73\u201388]. Three prospective population-based studies have reported an association between heterozygotes and vascular events [75\u201377].\nThe first study was from a subgroup of the original Finnish KIHD cohort [75]. Of 68 individuals, 8 (11.8%) were diagnosed with acute myocardial infarction, and of 1,150 noncoronary heart disease participants, 77 (6.7%) were carriers of C282Y. The crude relative risk of myocardial infarction was 2.0 (95%CI\u2009=\u20090.9\u20134.1) and the adjusted relative risk was 2.3 (95%CI\u2009=\u20091.1\u20134.8). In a cohort of 12,239 Dutch postmenopausal women, the C282Y carrier status was assessed among 531 women who died of cardiovascular disease and 555 randomly selected women who did not die of cardiovascular disease [76]. This study reported a relative risk of 1.6 (95%CI\u2009=\u20091.1\u20132.4) for total cardiovascular death. Finally, in the United States Atherosclerosis Risk in Communities (ARIC) study [77], a C282Y carrier frequency of 9.9% among 243 coronary heart disease cases and 6.1% among 535 controls was reported. The crude relative risk of coronary heart disease associated with C282Y carrier status was 1.6 (95%CI\u2009=\u20090.9\u20133.0) and was 2.7 (95%CI\u2009=\u20091.2\u20136.0) after being controlled for other risk factors. To support of these findings, Gaenzer et al. [78] found an association between increased iron stores and impaired endothelial function (measured as endothelium-dependent dilation and intima-media thickness) in patients homozygous for C282Y mutation. Iron-depletion therapy normalized the endothelial function in such patients thus reducing the increased risk of cardiovascular events.\nHowever, the majority of the studies disagreed with these results [61, 79\u201389]. Indeed, Bozzini et al. [61] found a similar rate of carriers of C282Y mutation among patients with coronary atherosclerotic disease and controls. In a case\u2013control study on 1,098 subjects, Rossi et al. [80] found that C282Y mutation was not a predictor of asymptomatic carotid atherosclerosis. Franco et al. [81] reported that the HFE genes were not associated with coronary or peripheral atherosclerosis in patients aged less than 50\u00a0years. Similarly, in the West of Scotland Coronary Prevention Study (WOSCOPS), Gunn et al. [86] found that the presence of a C282Y mutation in the HFE gene did not predict the occurrence of coronary events over a mean follow-up of 4.9\u00a0years. It is interesting to note that Yunker et al. [89] analyzed the relationship between biochemical and genetic markers of iron overload and carotid intima-media thickness and brachial flow-mediated vasodilation by high-resolution ultrasound in 907 males, but neither ferritin nor hemochromatosis genotype were related to brachial endothelial function and carotid atherosclerosis. In addition, a recent large study from Denmark found no increased risk of coronary heart disease among carriers of the C282Y mutation or individuals who had compound heterozygosity for the C282Y and H63D mutations [79].\nOther investigation have focused on the association between genetic markers of iron overload and idiopathic dilated cardiomyopathy and stroke, but their results were conflicting [90\u201394].\nFinally, some authors have suggested that HFE C282Y could interact with other predisposing factors for venous thromboembolism, such as factor V Leiden, thus exacerbating their prothrombotic effect [95].\nTable\u00a01 summarizes the most important studies on the association between genetic markers of iron overload and cardiovascular diseases.\nTable\u00a01Summary of the most important studies on the association between HFE gene mutations (C282Y and H63D) and the risk of cardiovascular diseasesAuthors [reference]Study designPopulationResultsTuomainen et al. [75]Prospective1,150 individualsC282Y heterozygosity is associated with a 2.3 RR for AMI compared with noncarriersRoest et al. [76]Prospective12,239 postmenopausal womenC282Y heterozygosity is associated with a 1.6 RR for TCD compared with noncarriersRasmussen et al. [77]Prospective243 CHD cases and 535 controlsC282Y heterozygosity is associated with a 2.7 RR for CHT compared with noncarriersGaenzer et al. [78]Case\u2013control41 C282Y\/C282Y cases and 51 controlsC282Y homozygosity is associated with impaired endothelial functionBozzini et al. [61]Case\u2013control546 CHD cases and 303 controlsC282Y mutation is not associated with CHDRossi et al. [80]Case\u2013control1,098 subjectsC282Y mutation is not a risk factor for asymptomatic carotid atherosclerosisFranco et al. [81]Case\u2013control256 CHD cases and 272 controlsC282Y and H63D mutations are not associated with CHDEllervik et al. [79]Prospective9,178 individualsC282Y and H63D mutations are not associated with CHDCase\u2013control2,441 CHD and 1,113 AMI cases vs 8,080 controlsC282Y and H63D mutations are not associated with CHDGunn et al. [86]Case\u2013control482 CHD cases and 1,104 controlsC282Y mutation is not associated with CHDCampbell et al. [88]Case\u2013control924 AMI cases and 1,029 controlsC282Y mutation is not associated with CHDYunker et al. [89]Case\u2013control907 individualsHFE genotype is not related to brachial endothelial function and carotid atherosclerosisAMI: acute myocardial infarction, CHD: coronary heart disease, RR: relative risk, TCD: total cardiovascular death\nConclusions\nIt is interesting to note that although with different pathogenic mechanisms, both iron deficiency and overload have been associated with an increased thrombotic risk in experimental and clinical studies.\nHowever, several aspects need to be still elucidated in this field. In particular, large prospective controlled trials are needed to elucidate the role of genetic markers of iron stores and the impact of long-term iron depletion on morbidity and mortality from cardiovascular events.","keyphrases":["iron","thrombosis","anemia"],"prmu":["P","P","P"]}
{"id":"Eur_Radiol-4-1-2292493","title":"Breast tumor characteristics of BRCA1 and BRCA2 gene mutation carriers on MRI\n","text":"The appearance of malignant lesions in BRCA1 and BRCA2 mutation carriers (BRCA-MCs) on mammography and magnetic resonance imaging (MRI) was evaluated. Thus, 29 BRCA-MCs with breast cancer were retrospectively evaluated and the results compared with an age, tumor size and tumor type matched control group of 29 sporadic breast cancer cases. Detection rates on both modalities were evaluated. Tumors were analyzed on morphology, density (mammography), enhancement pattern and kinetics (MRI). Overall detection was significantly better with MRI than with mammography (55\/58 vs 44\/57, P = 0.021). On mammography, lesions in the BRCA-MC group were significantly more described as rounded (12\/\/19 vs 3\/13, P = 0.036) and with sharp margins (9\/19 vs 1\/13, P = 0.024). On MRI lesions in the BRCA-MC group were significantly more described as rounded (16\/27 vs 7\/28, P = 0.010), with sharp margins (20\/27 vs 7\/28, P < 0.001) and with rim enhancement (7\/27 vs 1\/28, P = 0.025). No significant difference was found for enhancement kinetics (P = 0.667). Malignant lesions in BRCA-MC frequently have morphological characteristics commonly seen in benign lesions, like a rounded shape or sharp margins. This applies for both mammography and MRI. However the possibility of MRI to evaluate the enhancement pattern and kinetics enables the detection of characteristics suggestive for a malignancy.\nIntroduction\nBRCA1 and BRCA2 are the most well known gene mutations responsible for an increased risk for developing breast cancer. A BRCA1 or BRCA2 mutation carrier (BRCA-MC) has approximately a 3% risk of getting breast cancer before the age of 30. This risk increases to almost 50% when she reaches the age of 50 and becomes 50\u201380% at the age of 70 [1, 2]. To reduce this risk, these women can choose between bilateral prophylactic mastectomy [3], oophorectomy [4] or chemoprevention [5]. In breast cancer, close surveillance contributes to a more favorable stage of disease at detection and may reduce the rate of death from breast cancer [6, 7].\nIn the surveillance or general screening for breast cancer, mammography still plays a prominent role. However, due to the young age and thus in most cases dense breast tissue, the sensitivity for mammography is moderate. False-negative rates of up to 62% have been reported for mammography in screening gene mutation carriers [8, 9]. A malignant lesion in the breast is mammographically best detected if it presents itself as an ill-defined or spiculated mass, a group of microcalcifications or as an architectural distortion. A smoothly outlined well-defined mass detected on mammography has a chance of less than 1% of being malignant [10, 11]. Tilanus et al. [12] and Kaas et al. [13, 14] have evaluated the mammographic appearance of breast cancer in BRCA-MC. Tilanus and coworkers found the mammographic appearance suspicious for a malignancy in only 38% of the gene carriers in comparison with 71% in a control group. \u201cProminent pushing margins\u201d caused by a continuous front of tumor cells not separated by connective tissue were described in the BRCA-MC group as the main reason for a false-negative evaluation of mammograms [12]. Kaas and coworkers concluded in their study of 31 breast cancer cases in BRCA-MCs that all mammographically detected lesions should be further evaluated by ultrasound and biopsy regardless of their appearance [13]. Well-defined mammographic tumors correlated in 83% with histologic circumscribed tumor margins in BRCA1-MCs [14].\nFor women with an increased risk for developing breast cancer magnetic resonance imaging (MRI) should be included for close surveillance [15, 16]. The superior sensitivity of MRI (81%) for the detection of breast cancer in these women compared with mammography (40%) has been proven in literature [17]. However the classification of a lesion detected on MR as benign or malignant still remains a challenge. Morphological and dynamic features are important in breast MRI interpretation. Focal masses with smooth borders are associated with a high negative predictive value for malignancy [17]. An irregular lesion contour, inhomogeneous enhancement pattern and rim enhancement have been reported as features indicating malignancy [18]. The dynamic evaluation is often based on the enhancement characteristics 2\u20137\u00a0min after the injection of a paramagnetic contrast agent. In this approach, the decrease of signal intensity, often referred to as a type 3 curve or washout, is highly predictive for breast cancer, with a likelihood of malignancy of 87% [19]. Until now the appearance of breast malignancies in BRCA-MCs has only been investigated for mammography. In this study we analyzed the MRI characteristics of BRCA-MC-associated tumors compared with sporadic cases of breast cancer.\nMaterials and methods\nAll available (35) BRCA-MCs with a biopsy-proven malignancy, imaged with MRI for screening [9] or pre-operative evaluation in the period from July 2000 until November 2006, were included in the study: 23 BRCA1 carriers and 12 BRCA2 carriers. In order to compare tumor characteristics with sporadic cases of breast cancer an age, tumor type and tumor size matched control group was composed from 206 consecutive sporadic breast cancer cases imaged with MRI in the period from November 2001 until January 2007. All BRCA-MC cases were age matched within 5\u00a0years with sporadic breast cancer cases. Cases were also matched for tumor type (IDC, ILC or DCIS) and pathological tumor size. For size matching, the BRCA-MC cases were matched to the closest tumor size in the sporadic cases available, with a limit for the maximum size difference of 0.5\u00a0cm for tumors smaller than 1\u00a0cm, 1-cm difference for tumors up to 5\u00a0cm and 1.5-cm difference for tumors larger than 5\u00a0cm. BRCA-MC cases that could not be matched following these criteria were excluded.\nMammograms were obtained in the mediolateral oblique and craniocaudal direction on a digital mammographic unit (Senograph 2000 D or a Senograph DS, GE Healthcare, Wis., USA). Detection, density of the lesion compared with breast tissue, lesion morphology, and size were scored. In the morphologic assessment, lesion type was classified as either a mass, a calcifications or as an architectural distortion. Lesion shape was described as rounded, lobulated or irregular and lesion margins as sharp, vague or spiculated. The size of the tumor was measured by determining the longest axis through the displayed lesion. Spiculae surrounding a solid lesion were interpreted as desmoplastic reaction and not included in the measurement.\nMRI investigations were performed on a 1.5-Tesla system with a double breast coil (Magnetom Vision, Sonata or Symphony, Siemens, Erlangen, Germany). In the scanning, we used a coronally orientated three-dimensional fast low-angle shot (FLASH 3D) with the following parameters: TE 4\u00a0ms, TR 8.1\u00a0ms, FA 20\u00b0, FOV 360\u00a0mm, TA 96\u00a0s, image resolution 1.5\u00a0mm\u2009\u00d7\u20091.5\u00a0mm\u2009\u00d7\u20091.5\u00a0mm for all patients scanned prior to June 2004 and TE 4\u00a0ms, TR 7.5\u00a0ms, FA 8\u00b0, FOV 320\u00a0mm, TA 87\u00a0s, image resolution 1.3\u00a0mm\u2009\u00d7\u20091.3\u00a0mm\u2009\u00d7\u20091.3\u00a0mm for all patients scanned after June 2004.\nPrior to the MR examination, an intravenous catheter was inserted. All patients were placed in the prone position, with the breasts in the double breast coil and positioned at the isocenter of the magnet. After localizer images were obtained in three directions and a precontrast FLASH 3D series was recorded, 0.1\u00a0mmol\/kg bodyweight gadolinium chelate (Magnevist, Schering, Germany or Dotarem, Guerbet, The Netherlands) was administered using a power injector (Spectris, Medrad, USA) at 2.5\u00a0ml\/s followed by a 15-ml saline flush at the same injection rate. Thereafter, five post contrast FLASH 3D series were recorded.\nAll MRI examinations were retrospectively evaluated on a dedicated breast MRI workstation (Dynacad, Invivo, USA) scoring lesion detection, size, morphology and enhancement kinetics. Maximum intensity projections, coronal images and axial reconstructions of both the T1 weighted and subtracted images and time-intensity curves were displayed. The morphologic assessment included lesion shape, margin appearance and enhancement pattern. Lesion shape was classified as being rounded, lobulated or irregular. Margins were described as sharp, vague or spiculated. The enhancement pattern of a lesion was classified as homogeneous, heterogeneous or rim enhanced. Lesion enhancement kinetics were evaluated according to the criteria described by Kuhl et al. [19]. Type 1 shows persistent enhancement and is highly suggestive for a benign lesion. Type 2 shows a plateau after initial increased enhancement, where the maximum signal intensity is reached approximately 2\u20133\u00a0min after contrast injection and remains constant. This type of curve is seen in both benign and malignant lesions. In a type 3 curve, the peak enhancement is reached in the early postcontrast phase, and this is followed by a decrease of signal intensity (wash-out). The latter curve is strongly suggestive for a malignant lesion. The dynamic curves were evaluated based on a single voxel or by selecting a region of interest within the lesion, the workstation allowed the readers to use both methods. Because of the possible bias in this retrospective study, a BI-RADS classification [20] could not be scored objectively and was therefore not included in the evaluation.\nAll studies were evaluated retrospectively by two radiologists in conference and consensus. BRCA-MCs and controls were mixed during the evaluation. Except from the knowledge of a malignancy being present, the radiologists were blinded to any other clinical information. Mammography and MRI images were evaluated in separate sessions. From the histopathology reports, the tumor type, size and mitotic activity index (MAI) were recorded. The study was approved by the institutional review board; since the study was performed retrospectively, informed consent was not required according to the review board.\nIn the statistical evaluation, differences in patient and tumor characteristics between the BRCA-MC and control group were analyzed using an independent sample t-test if variables were continuous and normally distributed. For categorical variables, the Pearson chi-square test was used and we used Fisher\u2019s exact test when any of the expected values was less than five. Pearson\u2019s correlation coefficients were calculated for both mammographic size and MRI size versus pathologic size. All statistical analyses were performed using SPSS statistical software (version 12.0.1). P values\u2009<0.05 were considered to indicate statistical significance.\nResults\nSix BRCA-MC cases could not be matched according to the criteria defined; these cases were excluded from the study. Four BRCA-MCs were excluded because no match could be found based on patient\u2019s age; the other two were excluded because no match could be found based on tumor size.\nThe mean age and tumor size of the excluded cases were, respectively, 33\u00a0years (range 27\u201336, median 35, SD 3.4\u00a0years) and 1.4\u00a0cm (range 0.6\u20132.8\u00a0cm, median 1.1\u00a0cm, SD 0.8\u00a0cm). A total of 29 BRCA-MC cases were included for this study. In the BRCA-MC group, five women were symptomatic (17%); 21 women were symptomatic in the control group (83%).\nMean age in the BRCA-MC group was 42\u00a0years (range 32\u201368\u00a0years, median 40\u00a0years, SD 8.0\u00a0years); this was 44 (range 37\u201364\u00a0years, median 43\u00a0years, SD 5.6\u00a0years) for the control group. The mean pathological tumor size was 2.0\u00a0cm (range 0.4\u20137.0\u00a0cm, median 1.4\u00a0cm, SD 1.5\u00a0cm) in the BRCA-MC group and 2.3\u00a0cm (range 0.6\u20137.0\u00a0cm, median 1.9\u00a0cm, SD 1.7\u00a0cm) in the control group. No significant difference was found for patient age (P\u2009=\u20090.289) or maximal pathological tumor size (P\u2009=\u20090.371).\nThe mean tumor size on mammography was 2.1\u00a0cm (range 0.5\u20137.0\u00a0cm, median 1.5\u00a0cm, SD 1.49\u00a0cm). The mean tumor size on MRI was 2.4\u00a0cm (range 0.6\u20137.1\u00a0cm, median 1.8\u00a0cm, SD 1.75\u00a0cm).\nThere was a significant correlation between imaging measurements and pathological measurements; 0.664 (P\u2009<\u20090.001) for mammographic measurements and 0.808 (P\u2009<\u20090.001) for MRI measurements.\nIn both the BRCA-MC and control groups, 23 cases were based on invasive ductal carcinoma (IDC), two cases on invasive lobular carcinoma (ILC), one case on ductal carcinoma in situ grade 1 (DCIS1) and three cases on ductal carcinoma in situ grade 2 (DCIS2). In the BRCA-MC group, a mean MAI of 33.0 (range 6\u2013100, median 27, SD 27.1) was found, compared with 17.5 (range 1\u201360, median 14, SD 15.9) in the control group. The difference in MAI between the BRCA-MC and control groups was found to be significant (P\u2009=\u20090.044).\nIn the control group, one patient refused to undergo mammography because of implants. Overall mammography detected 44 of 57 lesions and MRI detected 55 of 58 lesions. Therefore, the overall detection is significantly better with MRI than with mammography (P\u2009=\u20090.021).\nMammography detected 22 (76%) lesions in the BRCA-MC group and 22 (79%) in the control group. No significant difference was found (P\u2009=\u20090.807). All lesions missed on mammography in the BRCA-MC were IDC. In the control group, five cases of IDC and one case of ILC were missed. Mammographic lesion characteristics are presented in Table\u00a01. Lesions in the BRCA-MC group were significantly more described as rounded (12\/\/19 vs 3\/13, P\u2009=\u20090.036) and were more often described to have sharp margins (9\/19 vs 1\/13, P\u2009=\u20090.024). Lesions in the control group were significantly more described as irregular (10\/13 vs 6\/19, P\u2009=\u20090.029). From the six BRCA-MCs that were excluded, only three lesions were detected on mammography. In two cases a mass was detected and in one case calcifications. Both these masses were described as rounded with sharp margins. \nTable\u00a01Mammographic lesion characteristics for both groups\u00a0BRCA-MC groupControl groupP valueLesions detectedn\u2009=\u200922n\u2009=\u200922Lesion typeMass19130.042Arch. distortion140.345Calcification250.216Lesion densityaHyperdense810.050Isodense11120.050Lesion morphologyaRounded1230.036Lobulated1\u20131.000Irregular6100.029Lesion marginsaSharp910.024Vague880.473Spiculated240.194aLesion density, morphology and margins of mass-like lesions only\nOn MRI, 27 lesions (93%) were detected in the BRCA-MC group, 28 (97%) in the control group. No significant difference was found (P\u2009=\u20090.553). The lesions missed in the BRCA-MC group were both cases of DCIS, one case of DCIS1, seen on mammography as an architectural distortion, and one case on DCIS2, seen on mammography as a mass. The lesion missed in the control group was based on DCIS2, seen on mammography as calcifications. Their was no significant difference found for the detection of breast cancer between mammography and MRI within the BRCA-MC (P\u2009=\u20090.18) or the control group (P\u2009=\u20090.13). Morphological and dynamic MR characteristics are presented in Table\u00a02. Lesions in the BRCA-MC group were significantly more often described as rounded (16\/27 vs 7\/28, P\u2009=\u20090.010), with sharp margins (20\/27 vs 7\/28, P\u2009<\u20090.001) and to show rim enhancement (7\/27 vs 1\/28, P\u2009=\u20090.025). Lesions in the control group were significantly more often described as irregular (18\/28 vs 8\/27, P\u2009=\u20090.010), with vague margins (15\/28 vs 6\/27, P\u2009=\u20090.017) and with a heterogeneous enhancement pattern (22\/28 vs 12\/27, P\u2009=\u20090.009). No significant difference between the two groups was found for enhancement kinetics (P\u2009=\u20090.667). From the six BRCA-MCs that were excluded, five were detected on MRI. Four of these lesions were described as rounded, one as irregular. The delineation was described as sharp in four and as vague in one of these cases. The enhancement pattern was described as homogeneous in two, heterogeneous in one and as rim-enhancement in two of these cases. All five cases showed a type 3 curve. \nTable\u00a02MRI lesion characteristics for both groups\u00a0BRCA-MC groupControl groupP valueLesions detectedn\u2009=\u200927n\u2009=\u200928Lesion morphologyRounded1670.010Lobulated331.000Irregular8180.010Lesion marginsSharp207<0.001Vague6150.017Spiculated160.049Enhancement patternHomogeneous850.304Heterogeneous12220.009Rim710.025Enhancement kineticsType 1121.000Type 2460.525Type 322200.380\nDiscussion\nIn this study, the overall false-negative rate for mammography was significantly higher compared with MRI. Although it is expected that the sensitivity for MRI in both the control group and the BRCA-MC is higher compared with mammography, no significant difference was found in this study within the two groups. This is probably due to the relatively small numbers of cases in this study. Kaas et al. [13] described in their mammographic study on BRCA1 and BRCA2 mutation carriers a sensitivity of 64% for the detection of a tumor in the original reports. In this study, 76% of the lesions were visible on mammography. Since this was a retrospective study and the radiologist was aware of the fact that a tumor was present at the time of the evaluation, no conclusion can be drawn from the difference in detection between both studies.\nOn mammography, the mass like lesions detected in the BRCA-MC group were significantly more often described as rounded. Also, lesions were found to differ in margin appearance; tumor margins in the BRCA-MC group were significantly more often described as sharp. A smooth, nonspiculated mass has previously been described by Tilanus et al. [12] as a reason for a false-negative mammographic evaluation in BRCA-MC. Thus, although Sickles et al. [21] described that nonpalpable, circumscribed, noncalcified breast masses (probably benign) should be managed with periodic mammographic surveillance regardless of lesion size and patient age, and Sardanelli et al. [22] published that both the well-defined margins and the rounded shape are more often associated with benign lesions, these findings are not applicable to the BRCA-MCs studied in this or other studies on this subject. Tilanus et al. [12] stated that any mammographic mass in BRCA-MCs must be regarded with suspicion. A similar conclusion was published by Kaas et al. [13]. An additional evaluation using ultrasound and biopsy of all lesions detected in BRCA-MC is mandatory, regardless of their morphological appearance. In their study of 28 BRCA-MCs, Hamilton et al. [23] also described the appearance of breast tumors on ultrasound. On ultrasound, 53% of the tumors were classified as either benign or indeterminate, making a biopsy of any detected mass inevitable. We are also of the opinion that any solid lesion detected in BRCA-MCs should be evaluated by a biopsy.\nIn the screening of women with an increased risk for developing breast cancer, more tumors are detected by MRI compared with mammography [8, 9]. In this study, all lesions missed on MRI were cases of DCIS. MRI is known to have a lower sensitivity in detecting DCIS compared with invasive carcinomas [24], especially in low-grade DCIS. The low or intermediate contrast uptake that is often observed in pure DCIS and the absence of a type 3 curve can result in a false-negative evaluation [24]. Kriege et al. [9] found in the screening of 1,909 women with an increased risk for developing breast cancer, including 358 carriers of germ-line mutations, that MRI missed five cases of DCIS that were detected on mammography, with six noninvasive tumors detected in total in the study. However, Kuhl et al. [25] reported MRI to be more sensitive for DCIS compared with mammography in a prospective study of women with an increased risk for developing breast cancer. Although MRI proved to be more sensitive for the detection of DCIS compared with mammography, not all cases of DCIS were detected by either modality. Therefore, at this point both modalities are still needed in the screening of women with an increased risk for developing breast cancer.\nSimilar to mammography, on MRI a significantly higher number of lesions were described as rounded and with sharp margins in the BRCA-MC group. Furthermore, the number of lesions with \u2018rim-enhancement\u2019 was found to be significantly higher in the BRCA-MC group. The presence of this enhancement pattern has been associated with malignant lesions [26, 27]. Because MRI enables the radiologist to evaluate the enhancement pattern of the lesion, where mammography does not, these lesions will become more suspectedly malignant, even though other morphologic features are more often seen in benign lesions. The association of rim-enhancement (Fig.\u00a01) with central necrosis or insufficient microvessel growth can be an indicator for the growth rate of tumors. Jimenez and coworkers have described centrally necrotizing carcinomas to have an accelerated clinical course and early systemic metastasis [28]. An accelerated growth rate can be associated with a high MAI. In this study, the MAI was found to be significantly different between both groups. This is in agreement with Tilanus et al. [12], who also found the mitotic count to be significantly higher in tumors found in gene mutation carriers. The higher rate of sharp tumor margins and rim-enhancement may thus be explained by the more aggressive nature of tumors in BRCA-MCs. Several authors have implied that, due to the rapid growth rate of tumors in gene mutation carriers, the screening frequency should be adjusted [13, 29]. Komenaka et al. [29] suggest a higher screening frequency in carriers because of the high number of interval cancers found in their group. Half of these interval malignancies were already positive for lymph-node involvement. The 13 carriers in their study were screened with mammography. Mammography in carriers is not sensitive, particularly because the women are young and thus more often have dense breasts [30, 31]. The use of MRI in screening has already been a step forward since MRI can detect smaller tumors, often occult for mammography, that are less likely to have progressed into lymph-node involvement [9].\nFig.\u00a01a An MLO mammogram from the right breast of a 42-year-old BRCA1-mutation carrier. b A coronal subtraction MR image of the same breast. An 11-mm sharply delineated rounded lesion is present on the mammogram projecting over the upper quadrants (arrow). On MRI, the same lesion was detected (arrow) with rim-enhancement. The rim-enhancement makes this lesion morphologically suspect malignant. Ultrasound guided core biopsy proved this lesion to be an invasive duct carcinoma\nRounded, homogeneous enhancing lesions found on MRI are in general not considered suspicious. A homogeneous enhancement pattern, found in nine BRCA-MCs, does not contribute to the malignant nature of these lesions. Therefore, enhancement kinetics are of value. In this study, the dynamic analysis showed in both the BRCA-MC and the control groups a type 3 curve in, respectively, 82% and 71% of the cases. As described by Kuhl et al. [19], a type 3 enhancement curve is highly indicative for malignancy. Using this characteristic, even rounded, sharply delineated, homogeneous enhancing lesions become suspect malignant (Fig.\u00a02).\nFig.\u00a02a A coronal subtraction MR image from the right breast of a 49-year-old BRCA1-mutation carrier. Lateral located in the right breast, a sharply delineated rounded, homogeneous enhancing lesion is visible with a longest diameter of 9\u00a0mm (arrow). b The relative enhancement versus time curve. The type 3 curve seen for this lesion was the only characteristic indicating a possible malignancy. Pathology proved this lesion to be an invasive duct carcinoma\nDespite all the findings discussed in this study, it remains questionable if the characterization of a lesion detected on either mammography or MRI in BRCA-MCs is even necessary. As the chance for these women to develop breast cancer is significantly increased, almost any detected lesion will in practice be classified as suspect malignant until proven otherwise. Sardanelli et al. [17] found a positive predictive value for MR in women with an increased risk of only 53% due to a high number of false positives. As stated previously for mammographically detected lesions, additional evaluation by core biopsy is the only definitive classification for lesions detected in this group of women. In the case of MRI screening in high risk women, short-term follow-up, target ultrasound or MRI-guided biopsies are therefore often indicated [32]. What the best strategy in this group of women will be, also in terms of cost effectiveness, needs to be further studied.\nWe conclude that in BRCA-MC malignant lesions frequently have morphological characteristics that are commonly seen in benign lesions, like a rounded morphology or a sharp delineation. This applies for both mammography and MRI. However, the possibility of MRI to evaluate the enhancement pattern and enhancement kinetics of lesions enables the radiologist to detect characteristics suggestive for a malignancy.","keyphrases":["breast","brca1","brca2","mri","mammography"],"prmu":["P","P","P","P","P"]}
{"id":"Mol_Cell_Biochem-4-1-2226060","title":"Modulation of calcification of vascular smooth muscle cells in culture by calcium antagonists, statins, and their combination\n","text":"Background Vascular calcification is an organized process in which vascular smooth muscle cells (VSMCs) are implicated primarily. The purpose of the present study was to assess the effects of calcium antagonists and statins on VSMC calcification in vitro. Methods VSMC calcification was stimulated by incubation in growth medium supplemented with 10 mmol\/l \u03b2-glycerophosphate, 8 mmol\/l CaCl2, 10 mmol\/l sodium pyruvate, 1 \u03bcmol\/l insulin, 50 \u03bcg\/ml ascorbic acid, and 100 nmol\/l dexamethasone (calcification medium). Calcification, proliferation, and apoptosis of VSMCs were quantified. Results Calcium deposition was stimulated dose-dependently by \u03b2-glycerophosphate, CaCl2, and ascorbic acid (all P < 0.01). Addition of amlodipine (0.01\u20131 \u03bcmol\/l) to the calcification medium did not affect VSMC calcification. However, atorvastatin (2\u201350 \u03bcmol\/l) stimulated calcium deposition dose-dependently. Combining treatments stimulated calcification to a degree similar to that observed with atorvastatin alone. Both atorvastatin and amlodipine inhibited VSMC proliferation at the highest concentration used. Only atorvastatin (50 \u03bcmol\/l) induced considerable apoptosis of VSMCs. Conclusion In vitro calcification of VSMCs is not affected by amlodipine, but is stimulated by atorvastatin at concentrations \u226510 \u03bcmol\/l, which could contribute to the plaque-stabilizing effect reported for statins.\nIntroduction\nAtherosclerosis is the principal cause of coronary artery disease, stroke, and peripheral artery disease, and is the major cause of mortality in the Western hemisphere. Vascular calcification is a prominent feature of atherosclerosis, and it is associated with an increased risk of myocardial infarction [1]. Vascular calcification refers to the deposition of calcium phosphate mineral, most often in the form of hydroxyapatite, in the vessel wall. Calcification of the vessel wall and heart valves is associated with ageing, diabetes, and uremia [2\u20134].\nVascular calcification is now considered to be an organized, regulated process comparable to bone mineralization. The presence of various components associated with bone mineralization, such as bone morphogenetic protein, osteocalcin, osteopontin, osteoblast-like cells, and matrix vesicles in atherosclerotic lesions supports this concept [5\u20137]. Vascular cells such as vascular smooth muscle cells (VSMCs) and pericyte-like cells play an important role in vascular calcification [8].\nThe lipophilic calcium antagonist (CA) amlodipine has been shown to restore cholesterol-induced membrane bilayer abnormalities in VSMCs derived from the atherosclerotic rabbit aorta [9, 10], thereby restoring normal calcium homeostasis. Other mechanisms, through which CAs may affect atherosclerosis development include inhibition of proliferation and migration of VSMCs [11, 12], and inhibition of lipoprotein oxidation [13]. In addition, CAs modify binding of monocytes to the endothelium, and activate synthesis of matrix components [14]. The effects of CAs on atherosclerotic calcification have not been widely studied, however.\nLipids may contribute to atherosclerotic calcification [15, 16]. Recently statins have been shown to decrease the progression of coronary artery calcification and aortic valve calcification [17, 18]. Therapy with a combination of a CA and a statin might be more atheroprotective than either treatment alone [19, 20]. Combination therapy with CA and statin was shown to improve endothelial function and arterial compliance, to diminish LDL atherogenicity, and to slow the progression of atherosclerosis. So far, the effects of the combination therapy with CA and statin on vascular calcification have not been studied extensively.\nWe used an in\u00a0vitro model of vascular calcification by neonatal rat VSMCs, isolated by outgrowth from aortic explants. The purpose of this investigation was to identify the factors, and their concentrations, that are mandatory to induce calcification of aortic VSMCs in\u00a0vitro. Since calcium is such a major component of advanced atherosclerotic lesions, we furthermore studied whether the CA amlodipine and the statin atorvastatin, alone and in combination, affected calcification of aortic VSMCs in\u00a0vitro.\nMaterials and methods\nCell culture\nVascular smooth muscle cells (VSMCs) were obtained from segments of aortas explanted from 2-day old Wistar rats. Aortic segments were obtained aseptically and cut open longitudinally. The endothelium was removed by gently rubbing the luminal side of the aortas over the surface of a tissue culture dish (Falcon). Subsequently, the aortas were placed, lumen side down, on the bottom of a tissue culture flask (Greiner), and allowed to adhere for approximately 3\u00a0h. Then tissues were immersed in growth medium consisting of Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Life Technologies), penicillin (100\u00a0U\/ml), and streptomycin (100\u00a0\u03bcg\/ml) (both supplied by BioWhittaker Europe). Seven days later the aortic tissues were removed, and the VSMCs that had grown out were detached by trypsinization. The detached cells were resuspended in growth medium and seeded in tissue culture flasks (Greiner), 6 or 12-well plates or on glass cover slips.\nImmunocytochemistry\nSubconfluent cultures at early passage were examined for the presence of \u03b1-smooth muscle actin, SM myosin heavy chain and calponin using specific antibodies (anti-SM actin clone 1A4 (1:400), Sigma; anti-SM myosin heavy chain (1:100), SanverTech; anti-calponin (1:10,000), Sigma). After fixation in 1% formalin in PBS on ice for 30\u00a0min, cells were permeabilized with 0.1% Triton X-100 for 30\u00a0min, and subsequently incubated with the primary antibody for several hours at 4\u00b0C. Secondary antibodies were conjugated with fluorescein isothiocyanate (FITC) or Cy3 (Sigma). To identify cell nuclei, the cells were also stained with Hoechst 33342 (10\u00a0\u03bcg\/ml; Molecular Probes). Immunofluorescent images were obtained using a fluorescence microscope (Nikon Eclipse) equipped with 20\u00d7, 40\u00d7, and 100\u00d7 objectives and a digital camera (Nikon DXM1200).\nIn\u00a0vitro calcification of VSMCs\nCalcification of VSMC cultures was induced by the method of Shioi et\u00a0al. [21] with minor modifications. When confluent, the incubation medium of VSMCs was switched from growth medium to calcification medium. Calcification medium consisted of DMEM (high glucose, 4.5\u00a0g\/l) supplemented with 15% FBS, penicillin (100\u00a0U\/ml), streptomycin (100\u00a0\u03bcg\/ml), 8\u00a0mmol\/l CaCl2, 10\u00a0mmol\/l sodium pyruvate, 1\u00a0\u03bcmol\/l insulin, 50\u00a0\u03bcg\/ml ascorbic acid, 10\u00a0mmol\/l \u03b2-glycerophosphate, and 100\u00a0nmol\/l dexamethasone. The medium was replaced with fresh medium every 2\u20133\u00a0days. In several experiments, the concentrations of CaCl2, ascorbic acid, \u03b2-glycerophosphate, and dexamethasone were varied.\nIn another series of experiments, calcification medium was supplemented with amlodipine (0.01\u20131\u00a0\u03bcmol\/l), atorvastatin (2\u201350\u00a0\u03bcmol\/l), or a combination of these drugs. The CA amlodipine was dissolved in absolute ethanol at a concentration of 1\u00a0mmol\/l. This stock solution was to be diluted at least 1:1,000 in culture medium. Atorvastatin, an inhibitor of HMG-CoA reductase, was dissolved in absolute ethanol at a concentration of 10\u00a0mmol\/l. This stock solution was to be diluted at least 1:200 in culture medium.\nCells treated with calcification medium without added drugs were used as controls. Appropriate amounts of solvent (ethanol) were added to these controls. Addition of ethanol at these levels did not affect cell growth. After 3\u00a0weeks of incubation, calcification was quantified.\nAssessment of calcium deposition\nVSMCs were decalcified with 0.6\u00a0N HCl for 24\u00a0h. The calcium contents of the supernatants were determined by spectrophotometer using the o-cresolphthalein method (Roche Diagnostics). After decalcification, the cells were washed with PBS and scraped from the culture plate. The protein content was measured using the BCA protein assay (Pierce). The calcium content of the cell layer was normalized to protein content.\nProliferation assay\nVSMCs were seeded at a density of 5\u00a0\u00d7\u00a0103\u20132\u00a0\u00d7\u00a0104\u00a0cells\/well in a 96-well plate and allowed to attach overnight. Subsequently, the standard culture medium (DMEM, 10% FBS, antibiotics) was replaced by calcification medium supplemented with amlodipine, atorvastatin, or a combination of both. Cell proliferation was assessed at day 4 and day 9 using the Cell Proliferation Kit II (XTT, Roche), which is a colorimetric assay for the non-radioactive quantification of cell proliferation and viability.\nDetection of apoptosis\nTo identify apoptotic cells by assessment of condensation of nuclear chromatin, VSMC cultures were stained with Hoechst 33342 (10\u00a0\u03bcg\/ml; Molecular Probes) for 10\u00a0min in the dark. Immunofluorescent images were obtained using a fluorescence microscope (Nikon Eclipse) equipped with 20x, 40x, and 100x objectives and a digital camera (Nikon DXM1200). In 6 frames per treatment, an average of 250 nuclei per frame were assessed for the presence of apoptosis.\nStatistical analysis\nResults are expressed as mean\u00a0\u00b1\u00a0SEM. For statistical analysis, SPSS 10.0 for Windows was used. Since the data were not normally distributed, non-parametric tests were used for comparisons between groups. Overall comparisons between groups were performed with the Kruskall\u2013Wallis test. If only two groups were compared, Mann\u2013Whitney rank sum tests were used. P values less than 0.05 were regarded as significant.\nResults\nVSMCs that have been cultured from rat aortic explants for approximately 7\u00a0days stained positive for \u03b1-smooth muscle-actin, smooth muscle myosin heavy chain, and calponin, confirming the smooth muscle cell nature of these cells (Fig.\u00a01, online supplement). VSMCs of passage 3\u20136 were subsequently used in calcification experiments.\nFig.\u00a01(A) Rat aortic VSMC isolated by outgrowth, phase contrast. Immunofluorescent staining of neonatal rat VSMC incubated with (B) (and insert) anti-smooth muscle actin Ab, (C) anti-smooth muscle myosin Ab, and (D) anti-calponin Ab. Original magnification B and C \u00d7100, D and insert \u00d7200. VSMC\u00a0=\u00a0vascular smooth muscle cell\nCalcium deposition and dependence of extracellular Ca2+ concentration\nCells were incubated with calcification medium supplemented with various Ca2+ concentrations for 21\u00a0days. At Ca2+ concentrations\u00a0\u22643\u00a0mmol\/l, hardly any calcium deposition was observed, but at Ca2+ concentrations >3\u00a0mmol\/l, a dose-dependent increase in calcium deposition was observed (Fig.\u00a02A). The amount of calcium deposition was positively correlated to calcium concentration in the culture medium (P\u00a0<\u00a00.01). We chose to continue with 8\u00a0mmol\/l of Ca2+-ions added to the calcification medium.\nFig.\u00a02(A) Dose-dependent effects of CaCl2 on calcification of neonatal rat VSMCs. (B) Dose-dependent effects of \u03b2-glycerophosphate on calcification of neonatal rat VSMCs. VSMCs were treated for 21\u00a0days with calcification medium containing varying concentrations of Ca2+ ions or \u03b2-glycerophosphate. Control cultures (=con) were incubated with DMEM, 10% FBS, and antibiotics. Calcium deposition was quantified by o-cresolphthalein method. The data are presented as mean\u00a0\u00b1\u00a0SEM (n\u00a0=\u00a03\u20139). *P\u00a0<\u00a00.05 vs. control cultures. \u25c6P\u00a0<\u00a00.05 vs. 2 and 3\u00a0mM calcium in culture medium. #P\u00a0<\u00a00.05 vs. all other treatments\nCalcium deposition and dependence of extracellular phosphate concentration\nShioi et\u00a0al. have demonstrated that \u03b2-glycerophosphate accelerates in\u00a0vitro calcification of VSMCs and induces extensive calcium deposition in a manner analogous to in\u00a0vitro mineralization by osteoblasts [21]. In our model of vascular calcification, the amount of calcium deposition was positively correlated to the \u03b2-glycerophosphate concentration in the calcification medium (P\u00a0<\u00a00.01; Fig.\u00a02B).\nCalcium deposition and dependence of extracellular dexamethasone concentration\nTo determine the contribution of added dexamethasone, we incubated the VSMCs with various concentrations of dexamethasone (10\u20131,000\u00a0nmol\/l). No significant differences in calcium deposition were observed between the cells incubated with 0, 10, 100, and 1,000\u00a0nmol\/l dexamethasone (Fig.\u00a03A).\nFig.\u00a03(A) Dose-dependent effects of dexamethasone on calcification of neonatal rat VSMCs. (B) Dose-dependent effects of ascorbic acid on calcification of neonatal rat VSMCs. VSMCs were treated for 21\u00a0days with calcification medium containing varying amounts of dexamethasone or ascorbic acid. Calcium deposition was quantified by o-cresolphthalein method. The data are presented as mean\u00a0\u00b1\u00a0SEM (n\u00a0=\u00a06\u201312). #P\u00a0<\u00a00.05 vs. all other treatments. \u25c6P\u00a0<\u00a00.05 vs. 0 and 5\u00a0\u03bcg\/ml ascorbic acid\nCalcium deposition and dependence of extracellular ascorbic acid concentration\nTo determine the contribution of ascorbic acid to calcium deposition, we incubated VSMCs with various concentrations of ascorbic acid (0, 5, 50, and 500\u00a0\u03bcg\/ml). At the highest concentration (500\u00a0\u03bcg\/ml) ascorbic acid was associated with significantly more calcium deposition than at all other concentrations (Fig.\u00a03B). However, 500\u00a0\u03bcg\/ml ascorbic acid caused a considerable decrease in pH of the culture medium. Since calcium deposition was positively correlated with ascorbic acid concentration (P\u00a0<\u00a00.01), we decided to use 50\u00a0\u03bcg\/ml ascorbic acid in future calcification studies, the highest concentration of ascorbic acid that did not cause acidification of the medium.\nEffects of amlodipine on in\u00a0vitro VSMC calcification\nTo study the effect of the CA amlodipine on VSMC calcification, VSMCs were incubated for 2\u20133\u00a0weeks with calcification medium supplemented with various concentrations of amlodipine (0.01\u20131\u00a0\u03bcmol\/l). Incubation of neonatal rat VSMCs with amlodipine had no effect on VSMC calcification, at none of the concentrations tested (Fig.\u00a04A).\nFig.\u00a04 (A) Dose-dependent effects of amlodipine on calcification of neonatal rat VSMCs. (B) Dose-dependent effects of atorvastatin on calcification of neonatal rat VSMCs. (C) Effects of amlodipine, atorvastatin and a combination of both treatments on neonatal rat VSMC calcification. VSMCs were treated for 21\u00a0days with calcification medium containing varying concentrations of atorvastatin or amlodipine, a combination of both, or none of them (control). Calcium deposition was quantified by o-cresolphthalein method. The data are presented as mean\u00a0\u00b1\u00a0SEM (n\u00a0=\u00a015). *P\u00a0<\u00a00.05 when compared to untreated control cultures. #P\u00a0<\u00a00.05 when compared to all other treatments\nEffects of atorvastatin on in\u00a0vitro VSMC calcification\nTo study the effect of the statin atorvastatin on VSMC calcification, VSMCs were incubated for 2\u20133\u00a0weeks with calcification medium supplemented with various concentrations of atorvastatin (2\u201350\u00a0\u03bcmol\/l). Atorvastatin increased VSMC calcification dose-dependently (Fig.\u00a04B). At a concentration of 2\u00a0\u03bcmol\/l atorvastatin, calcium deposition was increased by 30% (P\u00a0=\u00a00.04) when compared to VSMCs incubated with atorvastatin-free calcification medium. At concentrations of 10 and 50\u00a0\u03bcmol\/l atorvastatin, calcium deposition was increased 2.3-fold and 6.1-fold, respectively (P\u00a0<\u00a00.001) when compared to VSMCs incubated with atorvastatin-free calcification medium.\nEffect of a combination of CA and statin on in\u00a0vitro VSMC calcification\nTo study the effect of combining amlodipine and atorvastatin treatment on VSMC calcification, VSMCs were incubated for 2\u20133\u00a0weeks with calcification medium supplemented with 0.1\u00a0\u03bcmol\/l amlodipine, 10\u00a0\u03bcmol\/l atorvastatin, or a combination of these drugs in the same concentrations. Incubation of VSMCs with 0.1\u00a0\u03bcmol\/l amlodipine had no effect on VSMC calcification. Incubation of VSMCs with 10\u00a0\u03bcmol\/l atorvastatin resulted in a 2.2-fold increased calcium deposition when compared to control cultures treated with calcification medium only (Fig.\u00a04C) (P\u00a0<\u00a00.001). Treatment with a combination of amlodipine and atorvastatin also resulted in a 2.2-fold increased calcium deposition when compared to control cultures treated with calcification medium only (P\u00a0=\u00a00.026). The combination therapy resulted in significantly more calcium deposition than treatment with amlodipine alone (P\u00a0=\u00a00.003), and as much calcium deposition compared to treatment with atorvastatin alone (n.s.).\nEffects of amlodipine, atorvastatin, and their combination on VSMC proliferation\nVSMC proliferation was assessed at day 4 and day 9. After 4\u00a0days of incubation in calcification medium supplemented with amlodipine (1\u00a0\u03bcmol\/l), VSMC proliferation was decreased by 21% (P\u00a0<\u00a00.001) as compared to proliferation of VSMCs cultured in amlodipine-free calcification medium (Fig.\u00a06A, online supplement). After 9\u00a0days of incubation, VSMC proliferation had increased by 14% (P\u00a0<\u00a00.05) when treated with 0.01\u00a0\u03bcmol\/l amlodipine, but at an amlodipine concentration of 1\u00a0\u03bcmol\/l, VSMC proliferation had decreased by 26% (P\u00a0<\u00a00.01) as compared to proliferation of VSMCs cultured in amlodipine-free calcification medium (Fig.\u00a05A). Atorvastatin, at a concentration of 50\u00a0\u03bcmol\/l, decreased VSMC proliferation by 50% (P\u00a0<\u00a00.001; Fig.\u00a05B). Treatment of VSMCs with the combination of 0.1\u00a0\u03bcmol\/l amlodipine and 10\u00a0\u03bcmol\/l atorvastatin did not result in significant changes in cell proliferation compared to VSMCs incubated in the absence of these drugs.\nFig.\u00a05Effect of amlodipine and atorvastatin on VSMC proliferation. VSMC proliferation was assessed at day 4 and day 9 using the Cell Proliferation Kit II (XTT)(Roche). VSMC proliferation is depicted as percentage relative to untreated cells (=100%) (A) Effect of amlodipine. (B) Effect of atorvastatin. #P\u00a0<\u00a00.05 vs. control cultures. *P\u00a0<\u00a00.05 vs. control cultures\nEffects of amlodipine, atorvastatin, and their combination on VSMC apoptosis\nVSMCs incubated in calcification medium only and VSMCs incubated with amlodipine (0.01\u20131\u00a0\u03bcmol\/l) or atorvastatin (2 and 10\u00a0\u03bcmol\/l) in calcification medium for 72\u00a0h hardly contained any apoptotic nuclei. Amlodipine at the highest concentration tested (1\u00a0\u03bcmol\/l), caused apoptosis of on average 0.05% of the cells (n.s. compared to control). At the highest concentration of atorvastatin (50\u00a0\u03bcmol\/l), however, 5.6% of the nuclei were apoptotic (P\u00a0<\u00a00.05\u00a0vs. control, 2 and 10\u00a0\u03bcmol\/l atorvastatin; Fig.\u00a06).\nFig.\u00a06VSMCs were treated with 1\u00a0\u03bcmol\/l amlodipine (A, B) or 50\u00a0\u03bcmol\/l atorvastatin (C, D) for 72\u00a0h. Nuclei were visualized with Hoechst 33342. Apoptotic nuclei are circled in panel C and indicated with arrows in panel D. Microscopy: magnification A and C \u00d7100, B and D \u00d7400\nVSMCs treated with the combination of 0.1\u00a0\u03bcmol\/l amlodipine and 10\u00a0\u03bcmol\/l atorvastatin for 72\u00a0h had a number of apoptotic nuclei that did not significantly differ from those observed in 10\u00a0\u03bcmol\/l atorvastatin only, 0.1\u00a0\u03bcmol\/l amlodipine only, and VSMCs incubated in calcification medium in the absence of these drugs.\nDiscussion\nIn an in\u00a0vitro model of vascular calcification of rat aortic VSMCs, calcium deposition was dependent upon the extracellular concentration of organic phosphate (P\u00a0<\u00a00.01), calcium ions (P\u00a0<\u00a00.01), and ascorbic acid (P\u00a0<\u00a00.01). While studying the effect of the CA amlodipine and the statin atorvastatin, alone and in combination, on calcification, we found that, at none of the concentrations tested (0.01\u20131\u00a0\u03bcmol\/l) did amlodipine have any effect on VSMC calcification in this model, nor on development of apoptosis. In contrast, atorvastatin stimulated VSMC calcification at a concentration of \u226510\u00a0\u03bcmol\/l (P\u00a0<\u00a00.05), and 50\u00a0\u03bcmol\/l inhibited VSMC proliferation and induced apoptosis (P\u00a0<\u00a00.05).\nVSMCs retain remarkable plasticity, even in adult animals. VSMCs can undergo rapid and reversible changes in its phenotype in response to changes in local environmental conditions [22]. Due to production of a variety of cytokines by subendothelial macrophages, proliferation, and migration of VSMCs to the intimal layer can occur, thereby contributing to the process of intimal thickening and atherosclerosis.\nNeonatal VSMCs resemble VSMCs in atherosclerotic plaque, since they retain the proliferative phenotype. Neonatal aortic explants exhibit rapid outgrowth of VSMCs, and we chose to use these cells to develop a model of vascular calcification. VSMCs were shown to migrate from the aortic explant within several days, and were identified as VSMCs by immunohistochemistry [8].\nDialysis patients have accelerated atherosclerosis, with extensive calcification of both the intima and the media [23]. Hyperphosphatemia has been implicated in this process [24]. Inorganic phosphate (Pi) levels have previously been shown to regulate human aortic smooth muscle cell (HSMC) culture mineralization in\u00a0vitro [25, 26]. In the present study, the amount of calcium deposition in neonatal rat VSMCs correlated positively to the \u03b2-glycerophosphate concentration in the calcification medium.\nIn the present study, the amount of calcium deposition in rat aortic VSMCs correlated to the calcium concentration in the calcification medium. This observation was not a result of passive calcium deposition, since incubation of VSMCs with normal growth medium supplemented with 8\u00a0mmol\/l CaCl2 (final concentration) did not result in calcium deposition by these cells.\nShioi et\u00a0al. [21] have previously demonstrated that culturing bovine VSMCs in the presence of \u03b2-glycerophosphate, ascorbic acid, and insulin can induce diffuse calcification in a manner analogous to in\u00a0vitro mineralization of osteoblasts. The present study also showed that \u03b2-glycerophosphate is crucial for the induction of calcification in rat aortic VSMCs. Ascorbic acid augments this process. Dexamethasone was demonstrated to be less vital in this model of aortic VSMC calcification.\nStatins possess potent lipid-lowering effects. Besides, statins exert pleiotropic effects on vascular wall cells, which include improvement of endothelial function, stabilization of the atherosclerotic plaque, and suppression of inflammation [27, 28]. Although several studies have failed to demonstrate an effect of statins on the progression of calcific aortic stenosis [29, 30], statins have also been shown to decrease the progression of coronary artery calcification and aortic valve calcification [17, 18, 31, 32]. Accordingly, Kizu et\u00a0al. have demonstrated that statins inhibit calcification in an in\u00a0vitro model of inflammatory vascular calcification [33]. Using interferon-\u03b3, 1\u03b1, 25-dihydroxyvitamin D3, tumor necrosis factor-\u03b1, and oncostatin M to induce calcification in human VSMCs, it was demonstrated that cerivastatin and atorvastatin inhibited calcification dose-dependently. However, one should realize that this model differs from the model we have used in the present study. Whereas Kizu et\u00a0al. used inflammatory mediators to induce calcification of the VSMCs, we used increased levels of CaCl2 and organic phosphate in the culture medium to promote calcification. Statins are known to have anti-inflammatory effects, and therefore, their inhibitory effect on calcification induced by inflammatory mediators is to be expected. In addition, low doses of statins have also been shown to protect human aortic VSMCs from inorganic phosphate-induced calcification [34].\nIn contrast to the aforementioned studies, the present study showed a dose-dependent stimulatory effect of atorvastatin (10 and 50 \u03bcmol\/l) on calcification of VSMCs incubated in calcification medium. However, the dose of atorvastatin used in the present study was much higher than in the study described by Son et\u00a0al. (500x higher at the highest dose) [34], and most likely explains the discrepancy between the results obtained by Son et\u00a0al. and the results of the present study.\nReynolds et\u00a0al. [35] have demonstrated that human VSMCs undergo vesicle-mediated calcification in response to changes in calcium and phosphate concentrations in the culture medium. Elevated calcium and phosphate concentrations resulted in increased release of vesicles and stimulation of apoptosis. Calcification was initiated by release of membrane-bound matrix vesicles from living cells and also by apoptotic bodies from dying cells. Vesicles released by VSMCs after prolonged exposure to calcium and phosphate contained preformed basic calcium phosphate, and calcified extensively. The present study confirms that statins stimulate apoptosis of VSMCs. Apoptotic bodies can calcify extensively [36], so a likely mechanism of induction of calcification by atorvastatin is through induction of apoptosis. Indeed, atorvastatin stimulated both apoptosis and calcification in our model.\nMyocardial infarction and unstable angina usually arise from the disruption of mildly stenosed atherosclerotic lesions. Such vulnerable plaques typically contain a large amount of lipid and have a preponderance of inflammatory cells at the shoulders of the plaque and a thin fibrous cap. Calcium is found infrequently in the culprit lesion of ruptured plaque. Thus, plaque vulnerability is not associated with size, but with composition. Beckman et\u00a0al. [37] presented the concept \u201cthat calcium is not a critical substrate for plaque disruption and is, in fact, associated with more stable plaques.\u201d Therefore, statin-induced alterations in the plaque\u2019s composition from lipid to fibrous tissue without or with calcification would decrease the plaque\u2019s vulnerability to rupture, thereby decreasing the risk of acute coronary syndromes.\nProlonged intensive lipid-lowering has been demonstrated to increase calcium content of plaques as assessed by MRI in\u00a0vivo, and lowered plaque lipid content at about the same extent, without a pronounced effect on plaque size [38]. Since atherosclerotic plaque composition plays an important role in plaque stability, with lipids destabilizing the plaque and calcification stabilizing the plaque, the effect of statins on plaque composition may be favorable. In addition to lipid-lowering and calcification-inducing effects, we observed that statins have an anti-proliferative effect on VSMCs. Less proliferation of VSMCs may destabilize the plaque. However, this only occurred at high doses of atorvastatin, whereas calcification induction occurred at lower dose of atorvastatin.\nAlthough several in\u00a0vitro and in\u00a0vivo studies have demonstrated that combining CA and statin therapy might be more atheroprotective than either treatment alone [19, 20, 39], the present study found no difference between the combination of amlodipine and atorvastatin therapy, and atorvastatin therapy alone on VSMC calcification.\nIn conclusion, extracellular \u03b2-glycerophosphate and CaCl2 concentrations are important determinants of in\u00a0vitro calcification of rat aortic VSMCs. Ascorbic acid stimulates this process. Dexamethasone was demonstrated to be less vital in this model of VSMC calcification. In\u00a0vitro calcification of rat aortic VSMCs is not affected by amlodipine treatment, but is stimulated by atorvastatin treatment. As high concentrations of atorvastatin have a pro-apoptotic effect, the pro-calcification effect of atorvastatin may be explained by the production of apoptotic bodies that act as foci for calcium deposition. The latter finding may explain the plaque-stabilizing effect reported for statins.","keyphrases":["calcification","vascular smooth muscle cells","calcium antagonist","statin","atherosclerosis","in vitro model"],"prmu":["P","P","P","P","P","R"]}
{"id":"Pediatr_Nephrol-3-1-2100434","title":"Growth hormone axis in chronic kidney disease\n","text":"Chronic kidney disease (CKD) in children is associated with dramatic changes in the growth hormone (GH) and insulin-like growth factor (IGF-1) axis, resulting in growth retardation. Moderate-to-severe growth retardation in CKD is associated with increased morbidity and mortality. Renal failure is a state of GH resistance and not GH deficiency. Some mechanisms of GH resistance are: reduced density of GH receptors in target organs, impaired GH-activated post-receptor Janus kinase\/signal transducer and activator of transcription (JAK\/STAT) signaling, and reduced levels of free IGF-1 due to increased inhibitory IGF-binding proteins (IGFBPs). Treatment with recombinant human growth hormone (rhGH) has been proven to be safe and efficacious in children with CKD. Even though rhGH has been shown to improve catch-up growth and to allow the child to achieve normal adult height, the final adult height is still significantly below the genetic target. Growth retardation may persist after renal transplantation due to multiple factors, such as steroid use, decreased renal function and an abnormal GH\u2013IGF1 axis. Those below age 6 years are the ones to benefit most from transplantation in demonstrating acceleration in linear growth. Newer treatment modalities targeting the GH resistance with recombinant human IGF-1 (rhIGF-1), recombinant human IGFBP3 (rhIGFBP3) and IGFBP displacers are under investigation and may prove to be more effective in treating growth failure in CKD.\nLearning objectives:\nTo review the normal growth pattern in childhood and its alteration in chronic kidney disease.\nTo understand the mechanisms believed to be responsible for growth failure in chronic kidney disease.\nTo understand the new evidence supporting therapy for growth failure in chronic kidney disease.\nTo identify targets for future therapies for treatment of growth retardation in chronic kidney disease.\nIntroduction\nGrowth retardation is a common result of chronic kidney disease (CKD) in childhood. Children with CKD fail to achieve the final adult height consistent with their genetic potential [1]. Data from the North American Pediatric Renal Trials and Collaborative Studies (NAPRTCS) 2005 database revealed that 36.9% of children with CKD had statutory growth impairment. Even though the growth failure was correlated with the degree of renal impairment, those with mild reduction of glomerular filtration rate (GFR) also exhibited short stature. Children were short at initiation of dialysis, with younger age associated with more severe growth failure and little improvement in height standard deviation score (SDS). The mean height deficit for all renal failure patients was \u22121.85 (less than the third percentile of their healthy peers) at transplantation. This deficit was greater for male patients and for younger subjects. Those below age 6\u00a0years benefited most from transplantation in demonstrating acceleration in linear growth. For subjects aged 6\u201312\u00a0years, the linear growth remained stable, and those older than 12\u00a0years of age had no increase in their height deficit score [2\u20134].\nDespite advances in medical care, growth failure in CKD is associated with increased morbidity and mortality. Furthet al. demonstrated from the United States Renal Data System (USRDS) database that patients with severe-to-moderate growth failure had increased hospitalization rates and increased risk of death [5].\nGrowth retardation is assessed by the SDS or height deficit score, which measures the patient\u2019s height in comparison with that of unaffected children of similar age. Wong et al. demonstrated that, among pediatric patients on dialysis or after transplantation, each SDS decrease in height was associated with an increase in death by 14% [6].\nAlteration of normal growth patterns in CKD\nLinear growth is unique to childhood. Complex biological processes are responsible for maintaining normal growth.\nInfancy is the fastest growing period of childhood; one-third of the total growth occurs in the child\u2019s first 2\u00a0years of life, and this is mainly nutrient dependant. This growth is markedly decreased in congenital CKD, with the greatest height deficit occurring in the child\u2019s first year of life, especially during the first 4\u00a0months [7]. Thus, the earlier the onset of kidney disease, the more severe the growth disturbance.\nAdditional factors affecting growth are inadequate protein and calorie intake, water and electrolyte losses in polyuric and salt wasting conditions, anemia, metabolic acidosis, renal osteodystrophy, and resistance to hormones mediating growth. Early nutritional intervention and prevention of metabolic deficits of renal failure can preclude the development of growth failure in this period. Conservative treatment (no use of growth hormone) in pre-dialysis patients from birth to 3\u00a0years, revealed a favorable growth rate, with height velocity at 22.2\u00a0cm\/year, 10.9\u00a0cm\/year and 7.6\u00a0cm\/year for each year, respectively, all of which were higher than the lower two standard deviation scores [5]. Further discussion of these factors is beyond the scope of this article.\nDuring the mid-childhood period, a constant growth rate of 5\u20137\u00a0cm\/year is demonstrated, mainly under the control of growth hormone (GH) and thyroid hormone. Children with congenital CKD grow at the percentile achieved at the end of 2\u00a0years of life. Even though one of the most important predictors of growth impairment is the degree of renal insufficiency, significant short stature has been seen at all levels of renal function [3, 7].\nAt the onset of puberty, the growth hormone\/insulin-like growth factor (GH\/IGF-1) axis is activated by small increases in estrogen and testosterone in girls and boys, respectively. The onset of puberty is delayed in adolescents with CKD; peak height velocity is delayed by approximately 2.5\u00a0years. The pubertal growth spurt is delayed, shortened and associated with a reduced growth velocity. The pubertal height gain is about 65% of that seen in healthy children without CKD and is likely due to the shortened growth spurt [1]. These effects of delayed puberty in CKD are mediated by a loss of the normal pulsatile hypothalamic release of the gonadotropin-releasing hormone (GnRH).\nGH and IGF axis\nGrowth hormone is the key endocrine regulator of postnatal growth (Fig.\u00a01) [8]. The anterior pituitary secretes growth hormone in a pulsatile manner stimulated by the growth hormone releasing hormone (GHRH) and inhibited by somatostatin. GH mediates its somatotropic actions directly and mainly through IGF-1. Ghrelin, a growth hormone-releasing peptide expressed in the stomach and hypothalamus, is involved in hormonal and nutritional regulation of GH release [9].\nFig.\u00a01GH\/IGF-1 axis in CKD: deranged somatotropic axis in chronic renal failure. The GH\/IGF-I axis in CRF is changed markedly, compared with the normal axis shown here. In CRF the total concentrations of the hormones in the GH\/IGF-I axis are not reduced, but there is reduced effectiveness of endogenous GH and IGF-I, which probably plays a major role in reducing linear bone growth. The reduced effectiveness of endogenous IGF-I likely is due to decreased levels of free, bioactive IGF-I as levels of circulating inhibitory IGF-binding proteins (IGFBPs) are increased. In addition, less IGF-I is circulating in the complex with acid labile subunit (ALS) and IGFBP-3 as a result of increased proteolysis of IGFBP-3. Together, these lead to decreased IGF-I receptor activation and a decreased feedback to the hypothalamus and pituitary. Low free IGF-I and high IGFBP-1 and IGFBP-2 levels probably contribute to reduced renal function and lead to reduced stature. The direct effects of GH on bone, which are poorly understood, also are blunted. Reprinted from [8] with permission\nGrowth hormone and the IGF-1 axis play a major role in growth failure in CKD. Random fasting serum levels of GH are normal or increased in children and adults with CKD, depending on the extent of renal failure. The half-life of GH is prolonged, due to decreased metabolic clearance secondary to decreased functional renal mass in proportion to the degree of renal dysfunction. A high normal calculated GH secretion rate and amplified number of GH secretory bursts have been reported in pre-pubertal children with end-stage renal disease, likely due to attenuated IGF-1 feedback [8]. This has led to the concept of GH insensitivity or resistance in uremia.\nPubertal patients with advanced CKD have a reduced GH secretion rate, indicating altered sensitivity of somatotropic hormones to the stimulatory effects of sex steroids [10].\nGH resistance\nGH receptors One mechanism for GH resistance is a reduced density of GH receptors in target organs. Determination of the concentration of serum growth hormone binding protein (GHBP), which is a cleaved product of the GH receptor, may be used to assess GH receptor density in tissues, particularly liver, since GHBP is derived mainly, but not exclusively, from the liver. GHBP is low in children and adults with CKD and proportionate to the degree of renal dysfunction. Serum GHBP correlates with both spontaneous growth rate and response to GH therapy, and it is an indirect indicator of sensitivity to both exogenous and endogenous GH [11, 12]. However, there is controversy as to the reliability of serum GHBP level as a marker of GH receptor levels in specific tissues [13].\nJanus kinase\/signal transducer and activator of transcription signaling Another mechanism for GH resistance in uremia is a defect in post-receptor GH-activated Janus kinase\/signal transducer and activator of transcription (JAK\/STAT) signaling (Fig.\u00a02) [14]. GH action is mediated by the binding of GH to the growth hormone receptor (GHR), resulting in its dimerization and the auto-phosphorylation of the tyrosine kinases, Janus kinase\u00a02 (JAK2), which, in turn, stimulates phosphorylation of signaling proteins, STAT proteins STAT1, STAT3 and STAT5. Upon activation, these STAT proteins translocate to the nucleus and activate GH-regulated genes. An intact JAK2-STAT5b signaling pathway is essential for GH stimulation of IGF-1 gene expression. In uremia, a defect in the post-receptor GH-activated JAK2 signal transducer and STAT transduction is described as one of the mechanisms of GH resistance [14].\nFig.\u00a02Growth hormone-mediated JAK\/STAT signal transduction. GH activates several signaling pathways via JAK2, including the JAK\/STAT pathway [22, 23]. Binding of GH to its receptor (GHR) activates JAK2, which then self-phosphorylates. This is followed by phosphorylation of the GHR and, subsequently, STAT\u00a01a, STAT\u00a03, STAT\u00a05a, and STAT\u00a05b, members of a larger family of cytoplasmic transcription factors. These phosphorylated STATs form dimers that enter the nucleus, where they bind to specific DNA sequences and activate their target genes, IGF-1 and some suppressors of cytokine signaling (SOCS). Deletion of STAT5 expression leads to retarded body growth, and STAT5b is required for GH-mediated IGF-1 gene expression. In renal failure phosphorylation of JAK2 and the downstream signaling molecules STAT5, STAT3, and STAT1 is impaired, as are the nuclear levels of phosphorylated STAT proteins. This important cause of uremic GH resistance may result, in part, from up-regulation of SOCS2 and SOCS3 expression with suppressed GH signaling and also from increased protein tyrosine phosphatase activity, with enhanced dephosphorylation and deactivation of the signaling proteins. Reprinted from [14] with permissionThe JAK2\/STAT pathway is regulated, among other factors, by suppressor of cytokine signaling (SOCS) proteins, which is induced by GH. These proteins bind to JAK2 and inhibit STAT phosphorylation [15]. Up-regulation of SOCS has been described in inflammatory states and may play a similar role in CKD [16].\nIGF and IGFBP Daughaday et al. proposed the somatomedin hypothesis: GH leads to an increase in IGF-1 (formerly called somatomedin)\u00a0c) from the liver, which, in turn, reaches the growth plate to mediate its action [17]. However, it was later demonstrated that all the effects of GH are not mediated through IGF-1. Also, the liver is not the only source of IGF-1, and it is produced in other tissues. Circulating IGF-1 mainly derived from the liver acts as an endocrine hormone. IGF-1 produced locally acts as a paracrine\/autocrine hormone. The majority of circulating IGF in the serum exists as a 150\u00a0kDa complex, including the IGF molecule, IGF binding protein 3 (IGFBP3) and the acid labile subunit (ALS). IGF-1 gene knockout in mice results in miniature mice that die soon after birth. Knock-out of IGF-1 in the liver alone results in relatively normal sized mice with a 75% reduction in circulating IGF-1 levels, suggesting that local IGF-1 may be more important for body growth than liver IGF-1 [18]. ALS knock-out mice also exhibit relatively normal growth and development, despite a 65% reduction in circulating IGF-1 levels. Disruption of both liver IGF-1 and ALS genes results in 85\u201390% reduction in circulating IGF-1 levels and significant reduction in linear growth, suggesting that a threshold concentration of IGF-1 is necessary for normal growth [19].IGF-1 is the predominant IGF during the rapid growth period of puberty. The levels of IGFs (IGF-1 and IGF-2) are normal in pre-terminal renal failure. In end-stage renal disease IGF-1 level is normal or decreased and levels of IGF binding proteins (IGFBP) are increased, thus resulting in a net decrease in IGF bioactivity. Free IGF-1 is decreased in proportion to renal failure [20, 21].IGF-1 is an anabolic hormone; it binds to the IGF-1 receptor (IGF-1R) at the \u03b1 subunit, where it activates tyrosine kinase intrinsic to the IGF-1R\u03b2 subunit. In experiments with rats with CKD, skeletal muscles show decreased serum levels of IGF-1 and IGF-1 mRNA, while IGF-1R mRNA and IGF-1R numbers are increased, with normal binding affinity. However, there is resistance to post-receptor signal transduction; both auto-phosphorylation of the IGF-1R tyrosine kinase and activity of the IGF-1R tyrosine kinase to the exogenous insulin receptor substrate-1 (IRS-1), a natural substrate for IGF-1 receptor tyrosine kinase, are diminished in skeletal muscle of CKD rats. These abnormalities may account for the resistance of IGF-1 to protein turnover in skeletal muscles in CKD [22].IGFs are transported in plasma bound to IGF binding proteins (IGFBPs), which are responsible for extending their half-life and controlling their bioavailability. There are six different IGFBPs, based on their respective amino acid sequence. Of the IGFs in the serum, 75% circulate as a ternary complex bound to IGFBP3 and another subunit, acid labile subunit (ALS), to form major 150\u00a0kDa complexes. This acts as a reservoir and cannot cross the endothelial barrier. Some 20\u201325% of IGFs are present as smaller binary complexes with other IGFBPs forming the minor complex (35\u00a0kDa) [23]. Approximately 97% of IGF is bound; 1% of IGF-1 is in a free bioactive form. In healthy children there is a 25% excess of IGFBPs over IGF, while, in pre-terminal renal failure, IGFBPs are 150% in excess of IGFs, and in end-stage renal disease they are 200% in excess, thus reducing the bioavailability of IGF [21].IGFBP3 is the most abundant of all the binding proteins. IGFBP3 and IGFBP5 are similar in many aspects; structurally, they are both closely related, both potentiate IGF action and are up-regulated by GH. The serum level of intact IGFBP3 is normal in CKD, while the fragmented fraction (29\u00a0kDa) is increased but has a reduced affinity for peptides [24]. IGFBP5 level is not altered, but it exists mostly in the fragmented form.Intact IGFBP1, IGFBP2, IGFBP4 and IGFBP6 are elevated in CKD in relation to the degree of renal dysfunction [25]. IGFBP1, IGFBP2 and IGFBP6 are growth inhibitors of IGF-dependant proliferation of chondrocytes. Increased levels of IGFBP1 and IGFBP2 have been shown to correlate negatively with height [26]. Ulinski et al. analyzed serum levels of IGFBP4 and IGFBP5 in 89 CKD patients. There was a fourfold increase in IGFBP4, while IGFBP5 was not significantly increased. IGFBP4 level was inversely correlated with glomerular filtration rate and standard height. IGFBP5 level was positively correlated with standard height [25].\nGhrelin\nGhrelin is a newly discovered, 28-amino acid peptide, with growth hormone releasing properties [growth hormone secretagogue (GHS)], expressed most abundantly in the stomach and also in smaller quantities by pancreatic islet cells and hypothalamus. Ghrelin levels are increased by fasting, cholinergic stimulation, estrogen and recombinant hIGF-1, and are decreased by food intake [27].\nGhrelin has been shown to increase the release of GH which is amplified by co-administration of GHRH, and acts at the level of the hypothalamus and pituitary. The role of ghrelin in the growth abnormalities in CKD has yet to be defined.\nTreatment with recombinant human growth hormone\nStudies in the past have shown that therapy with recombinant human growth hormone (rhGH) in CKD is safe and efficacious, and that it increases growth rate and improves standardized height [28, 29]. Haffner et al. studied the effect of growth hormone treatment on final adult height of children with CKD (Fig.\u00a03) [1]. In contrast to the controls that had persistent growth failure, children treated with rhGH demonstrated sustained catch-up growth. In treated children the standardized height increased from baseline to a mean final adult height of 1.6\u2009\u00b1\u20091.2 SD below normal, while, in untreated control children, it decreased from baseline to a mean final adult height of 2.1\u2009\u00b1\u20091.2 SD below normal. Of the children treated with rhGH, 65% reached a final adult height within the normal range, although it was significantly below the genetic target [1].\nFig.\u00a03Growth hormone treatment in chronic kidney disease: change from initially predicted adult height at baseline in 38 children (32 boys and six girls) with chronic renal failure who received growth hormone treatment compared with 50 control children with chronic renal failure who did not receive growth hormone, according to gender. Values are means \u00b1 SD. Asterisks indicate significant differences from the previous period (P\u2009<\u20090.001) and daggers indicate significant differences from the children who were not treated with growth hormone (P\u2009<\u20090.001). Reprinted from [1] with permission\nPoor growth outcomes after renal transplantation are associated with corticosteroid use, persistent CKD and abnormalities in the GH-IGF-1 axis. The use of rhGH after transplantation leads to catch-up growth associated with increase in IGF-1 levels [26]. Using the NAPRTCS database of 513 renal transplant recipients receiving rhGH, Fine and Stablein demonstrated that final adult height was superior in rhGH treated patients compared to controls. Allograft function and graft failure rates were similar in both groups, with no increase in adverse events [30].\nRhGH treatment in renal failure is associated with an elevation in serum free IGF-1 [31]. RhGH increases IGFBP3, IGFBP4, and IGFBP5, decreases IGFBP1 and has no effect on IGFBP2 and IGFBP6. RhGH also increases IGFBP4, but does not correlate with change in height SDS, since most of the IGFBP4 in serum is fragmented [25, 31]. RhGH-mediated increase in IGFBP3, IGFBP5 and ALS results in increased ternary complexes and correlates positively with the increment in height SDS [31].\nAmong the growth-promoting effects of treatment with rhGH in CKD are anabolic effects, demonstrated by increase in body weight and mid-arm muscle circumference. The recommended dose approved for treatment of growth failure in CKD is 0.35\u00a0mg\/kg per week (28\u00a0IU\/m2 per week) [3].\nTargets for future therapy:\nTreatment with recombinant IGF-1\nThe GH resistance associated with CKD may be amenable to treatment with recombinant human IGF-1 (rhIGF-1). In children with GH-receptor deficiency or GH-inactivating antibodies, rhIGF-1 treatment resulted in a modest increase in growth velocity and height SDS, although less than that expected with rhGH [32].\nIn addition, short-term administration of rhIGF-1 has been shown to increase glomerular filtration rate and renal plasma flow in patients with end-stage renal disease and in healthy subjects. Vijayan et al. demonstrated sustained improvement in renal function in a 31-day randomized, double-blinded, placebo-controlled trial of 15 patients with advanced CKD, who had received intermittent doses of rhIGF-1 [33].\nOne reason for the use of IGF-1 treatment in CKD is that while patients are GH sufficient, they are GH resistant. Therefore, rhIGF-1 may be more beneficial than GH as therapy in CKD and merits further investigation as an agent for treatment of growth failure and improvement in renal function in CKD [34].\nCombined use of rhGH and rhIGF-1\nAnimal studies have shown that the combined use of rhGH and rhIGF-1 has an independent and additive effect. While whole-body growth in uremic rats was similar with treatment with either agent, an additive effect on longitudinal growth and anabolism was demonstrated when both agents were administered together [35].\nCombined use of rhIGF-1 and rhIGFBP3\nOne of the concerns with the use of rhIGF-1 in children with normal GH production is that rhIGF-1 may suppress endogenous GH, IGFBP3 and ALS production, which may have a detrimental effect on growth. The other adverse effect of the use of rhIGF-1 observed in GH receptor-deficient patients is hypoglycemia, due to low levels of IGFBP3 and increased level of free IGF-1 available for binding to insulin receptors [32]. Both these effects can be reduced by the combined use of rhIGF-1 and rhIGFBP3 [36].\nIGFBP displacers\nAt least two different IGF-1 analogs have been made, which have an affinity for IGFBPs but have no effect on IGF receptors, thus \u201cdisplacing\u201d IGF-1 from IGFBPs and elevating free IGF-1 levels. Animal studies in hypophysectomized rats and dwarf dw\/dw rats treated with IGFBP displacers demonstrated increased kidney size, improved renal function, and the stimulation of weight gain and bone growth. This effect was enhanced when treatment with IGFBP displacers was combined with rhIGF-1 [37].\nConclusion\nThis review of the literature lends support to the concept that CKD is associated with GH \u201cresistance\u201d. Despite adequate treatment with rhGH and improvement in catch-up growth, children with CKD display a final adult height that is often below the genetic target. The potential for newer therapies with rhIGF-1, combined use of rhGH and rhIGF-1, combined use of rhIGF-1 and rhIGFBP3 or IGFBP displacers to improve both the short and long term outcomes in the treatment of the disturbances in the GH\/IGF-1 axis in CKD awaits future investigations.","keyphrases":["growth hormone","insulin-like growth factor","treatment","growth failure","chronic kidney failure"],"prmu":["P","P","P","P","R"]}
{"id":"Eur_Spine_J-2-2-1602184","title":"Penetrating spinal injury with wooden fragments causing cauda equina syndrome: case report and literature review\n","text":"Study design: Case report Objective: To report an unusual case of cauda equina syndrome following penetrating injury to the lumbar spine by wooden fragments and to stress the importance of early magnetic resonance imaging (MRI) in similar cases. Summary of background data: A 22-year-old girl accidentally landed on wooden bannister and sustained a laceration to her back. She complained of back pain but had fully intact neurological function. The laceration in her back was explored and four large wooden pieces were removed. However 72 h later, she developed cauda equina syndrome. MRI demonstrated the presence of a foreign body between second and third lumbar spinal levels following which she underwent emergency decompressive laminectomy and the removal of the multiple wooden fragments that had penetrated the dura. Results: Post-operatively motor function in her lower limbs returned to normal but she continued to require a catheter for incontinence. At review 6 months later, she was mobilising independently but the incontinence remained unchanged. Conclusion: There are no reported cases in the literature of wooden fragments penetrating the dura from the back with or without the progression to cauda equina syndrome. The need for a high degree of suspicion and an early MRI scan to localise any embedded wooden fragments that may be separate from the site of laceration is emphasized even if initial neurology is intact.\nIntroduction\nPenetrating injuries to the spine, although less common than blunt trauma from motor vehicle accidents, are important causes of injury to the spinal cord [1, 2, 8, 12, 13, 18]. They are essentially of two varieties\u2014gunshot or stab wounds. Gunshot injuries to the spine are more commonly described and are associated with a higher incidence of neurological damage. On the contrary, the prognosis is better in stab wounds where surgery plays a greater role [9, 12, 14, 16, 18]. Very few case reports have been published on the onset of cauda equina syndrome (CES) following stab wounds. Injuries with sharp knifelike objects and rarely glass have been known to cause stab wounds to the spine [1, 10, 13, 14, 17]. However, there are no reports in the literature of penetrating injury by wood to the cord or cauda equina. We report a unique case of CES that developed in a patient almost 72\u00a0h following a penetrating injury to her back by a large wooden fragment.\nCase history\nA 22-year-old hairdresser accidentally slipped from the top of the stairs on to the wooden bannister which broke and as she landed at the bottom of the stairs, a piece of the wood caused a laceration to her back at the upper lumbar paraspinal region. She was taken to the nearby hospital where an open laceration to the area was found. She was complaining of severe back pain but had intact neurology in her legs. The wound was explored and debrided and four large pieces of wood were removed following which she was commenced on antibiotics. Her back pain persisted and 72\u00a0h later, she started complaining of incontinence with bilateral paraesthesia and pain down her legs. On examination, she had mildly reduced power (4\/5) distally in both legs with reduced sensation in her perineum.\nShe was transferred to the Neurosurgical unit and a MRI scan of the thoracolumbar region (Fig.\u00a01) was taken. This revealed an area of low signal intensity between the soft tissues extending towards the spinal canal into the conus at the level of the second and third lumbar spines (L 2 and L 3) with a high signal in the cord above. No obvious spinal haematoma was present. She underwent an emergency decompressive laminectomy of L 2 and L 3 with removal of several fragments of wood. At surgery, the spinous process and right hemilamina of L 2 were noted to be fractured and multiple wooden fragments were found to have entered the spinal canal (Fig.\u00a02) penetrating the dura in the midline causing contusion and division of some of the nerve roots of the conus. These wooden pieces were carefully removed using the microscope, the dura repaired and a closed drainage system left in. Swabs for microbiological examination were obtained from the surgical site and post-operatively prophylactic intravenous cefuroxime 750\u00a0mg, thrice a day for the next 7\u00a0days were commenced. She was advised strict bed rest in supine position for 5\u00a0days while the drain was left in situ. Over this period of time, the pain and weakness in her legs resolved completely but she continued to have numbness in the perineal region and continued to require the urinary catheter. A repeat post-operative MRI scan demonstrated the high signal in the cord as before with no residual foreign body in the canal. At discharge the wound appeared satisfactory and she was mobilising independently. When reviewed 6\u00a0months later, she was otherwise asymptomatic apart from persistent altered sensation in her perineal area.\nFig.\u00a01T 2 weighted Sagital MRI of the lumbar spine (left) demonstrates the wooden piece as an area of low signal intensity (arrow) within the soft tissues, extending into the spinal canal with an area of high signal in the cord above. T 2 weighted axial MRI (right) shows the wooden piece to have penetrated the theca on the left sideFig.\u00a02Per-operative view (left) demonstrates the wooden piece within the spinal canal (arrows) with size of the multiple wooden fragments demonstrated (right)\nDiscussion\nThe most widely reported cause of penetrating injury to spine is gunshot injury [1, 2, 8, 12, 13, 15, 18] with relatively few case reports describing injuries by glass or knifelike objects [1, 10, 13, 17]. Wooden foreign bodies have been reported to penetrate the cranium, orbit, face and limbs [3, 6]. However to our knowledge, there are no reports in the literature of PI to the spine by wooden fragments with or without development of CES. The only remote resemblance to our case is that by Lunawat et al. [9] who reported the presence of tiny pieces of wood in the spinal canal of an 18-year-old man who presented insidiously with weakness in his legs and who had suffered a PI to his abdomen 6\u00a0years ago.\nOur case is unique due to several reasons. It is the first report of a penetrating injury by wooden fragments into the lumar spinal canal producing CES. The mechanism of injury was the direct force of the sharp wooden fragment penetrating the upper lumbar paraspinal region. We believe that after piercing the skin and subcutaneous tissue, the piece of wood probably fragmented. While some of the fragments remained superficially in the deep subcutaneous tissue overlying the thoracolumbar fascia, others were pushed towards the spinous process and right hemilamina of L 2 which were fractured and subsequently the wooden fragments pierced the dura contusing the nerve roots of the conus. What is interesting is that she developed CES almost 72\u00a0h following the injury. Whether the fragments actually moved into the canal later as a result of her movement in bed following the initial wound debridement or whether she developed oedema at the site of nerve root injury is unknown.\nWe found MRI scan very useful to demonstrate and localise the foreign body and also to exclude any intra- or extradural haematoma or contusion in the cord or cauda equina. In our case some contusion was demonstrated in the cord (Fig.\u00a01). Previous reports of penetrated wooden pieces into the neck or extraspinal areas of the body have commented on the risk of misinterpretation of CT appearance of wood which appears as an area of high attenuation [3, 6]. Wood is highly echogenic and shows clear acoustic shadowing on sonography [10]. MRI is a useful adjunct to both CT and sonography for the detection of non-metallic foreign bodies such as wood [15].\nSurgery in PI is indicated for progressive neurological deficits, persistent cerebrospinal fluid (CSF) leak or for incomplete neurologic deficits with radiological evidence of compression [5]. However, surgery at the cauda equina, in such cases, may not be easy due to the dural tear associated with impingement of the foreign body with the contused nerve roots. In our patient urgent surgery was necessary as she developed progressive neurological deficit leading to CES with radiological evidence of a large foreign body causing thecal compression. Post-operatively, we took the extra precaution to prevent CSF leak from the wound by advising the patient a strict bed rest for 5\u00a0days. In our institute we advise 3\u20135\u00a0days of strict bed rest for patients with unintentional dural tears during Lumbar spinal surgery. This has been the practise by others too [19]. We do not routinely prescribe antibiotics for such cases. However, given the nature of this particular case, we decided to use prophylactic antibiotics in spite of negative bacteriological results. Incidentally it has been claimed that extraspinal sepsis is much more common than spinal (CSF) infection following PI to the spine [5]. We did not use methylprednisolone as there is no convincing evidence to suggest any advantage, and moreover there have been reports of myopathy following the administration of methylprednisolone in patients with acute spinal injury [7, 11].\nFinally, neurological recovery from injury to the cauda equina is unpredictable and may be influenced by several factors [4] such as patient age, energy transfer to the neurovascular structures and timing of neural decompression, although the latter is debated [12, 14, 15]. Late motor recovery has been reported to occur after PI to the cauda equina up to 16\u00a0years after injury in one study [8]. Our patient had excellent symptomatic relief of her back and bilateral leg pain following surgery with resolution of the motor weakness in her legs. However, the perineal numbness has persisted and we would have to wait and see if this improves in future.\nConclusion\nPenetrating wound to the upper lumbar spine is common with gunshot injuries but has never been described with wooden fragments. MRI scan provides excellent visualisation of the foreign body and also helps to exclude any underlying spinal haematoma or contusion. For all similar cases where a laceration containing wood is found, the authors recommend a high index of suspicion and a MRI scan must be performed early to look for separate fragments of wood deeper down. Neural decompression and removal of the foreign bodies should be performed to prevent neurological deterioration, infection and possible CSF leak.","keyphrases":["wood","cauda equina","penetrating injury"],"prmu":["P","P","P"]}
{"id":"Eur_J_Nucl_Med_Mol_Imaging-3-1-1914225","title":"Cardiovascular molecular imaging of apoptosis\n","text":"Introduction Molecular imaging strives to visualise processes at the molecular and cellular level in vivo. Understanding these processes supports diagnosis and evaluation of therapeutic efficacy on an individual basis and thereby makes personalised medicine possible.\nApoptosis\nThe term \u2018apoptosis\u2019 is derived from the Greek (apo = from and ptosis = falling, commonly pronounced ap-a-tow\u2019-sis) and denotes a regulated process of cell suicide resulting in a cell corpse that is distinct from the necrotic cell [1, 2]. Whereas necrosis is manifested by cellular swelling, plasma membrane rupture and rapid release of intracellular constituents into the environment, causing an inflammatory response [2], apoptosis is characterised by cell shrinkage, long-lasting maintenance of plasma membrane integrity and lack of inflammatory responses in the vicinity of the dying cell [2]. It has long been thought that apoptosis and necrosis are the only two modes of cell death counterbalancing mitosis [3]. However, recent insights have taught us that cells can follow more roads to death than only apoptosis and necrosis. In 2001, Leist and Ja\u00e4ttel\u00e4 suggested a model in which various forms of cell death could be positioned on a gliding scale between two extremes, apoptosis and necrosis [4]. The mode of execution depends on the cell type and the cell death-inducing trigger. Most knowledge about various modes of cell death has been generated in the field of oncology, where cell death plays an important role in the progression and treatment of cancer [5]. Several intermediate forms of cell death have been identified and characterised [6, 7], including mitotic catastrophe and autophagic cell death. Mitotic catastrophe can be the result of unsuccessful chromosome segregation, an event that requires malfunctioning of multiple cell cycle checkpoints. Catastrophic cells are large and non-viable cells with a compromised nuclear blueprint leading eventually to cell death [8]. Mitotic catastrophe appears to be a rare event in cardiovascular diseases. Autophagic cell death is characterised by the total destruction of the cell through autophagy. It is a process in which cytoplasmic constituents are degraded through the lysosomal machinery [9]. Autophagic cell death occurs both in oncology [10, 11] and in cardiovascular diseases [12]. The various described modes of cell death differ in morphology and biochemistry [13]. The caspase cascade is the most remarkable biochemical distinction [14]. It is activated during apoptosis but hardly, or not at all, during other modes of cell death.\nBiochemistry of apoptotic cell death\nTwo major biochemical routes dominate apoptotic cell death. They involve the activation of the caspase cascade through either death-receptor mediated signal transduction or stress-induced release of cytochrome C from mitochondria [15]. Caspases involved in apoptosis can be grouped into initiator (caspases 8 and 9) and effector caspases (caspases 3 and 7). The principal event of the caspase cascade is the activation of an inactive procaspase into a proteolytically active caspase. Activation of initiator caspases occurs through auto-activation, catalysed by the interaction with ligated death receptors (procaspase 8) or cytochrome C-containing apoptosome (procaspase 9). Effector caspases are activated through intra-chain cleavage by active initiator caspases [16]. In both cases, activation results in the exposure of active sites that selectively cleave peptide bonds after an aspartate residue in caspase substrates.\nSeveral checks and balances exist that control burst of the caspase cascade if triggered by minor undesired initiating events. Inhibitors of apoptotic proteins (IAPs) suppress proteolytic activity by binding to activated caspases. Inhibition of IAPs is in turn required to allow execution of apoptosis. Smac\/DIABLO residing in the mitochondria and co-released with cytochrome C inhibits IAPs and permits propagation and amplification of the proteolytic signal through the caspase cascade. Anti-apoptotic members of the Bcl-2 family such as Bcl-2 and Bcl-X prevent the release of cytochrome C and Smac\/DIABLO from mitochondria. Pro-apoptotic members such as Bax and tBid, which is the result of Bid cleavage by activated caspase 8, neutralise the protective effects of the anti-apoptotic members and provoke mitochondria to release their pro-apoptotic cargo.\nThe intricate scheme of the apoptotic machinery offers several potential targets for molecular imaging. For example, knowledge about the mechanism of caspase activation has triggered the design of several strategies to measure apoptosis [17\u201319]. The hurdle these methods have to take is entrance of the reporter of caspase activity into the cell. Consequently, in vivo applications of such strategies face unfavourable biodistribution profiles and high background levels. Targets embedded in the plasma membrane encompass a more promising set of molecules for molecular imaging.\nPhosphatidylserine\nAn essential part of the apoptotic program consists of the appearance of \u2018eat me\u2019 flags at the cell surface. Phagocytes recognise these flags and respond by engulfing the dying cell before it leaks pro-inflammatory components into the surrounding tissue. The \u2018eat me\u2019 flags are, alone or in combination, specific for the dying cell, allowing phagocytes to make the right choice in an environment filled with living cells.\nIn 1992, Valerie Fadok and co-workers reported that phosphatidylserine (PS) becomes exposed on the surface of apoptotic lymphocytes, where it functions as an \u2018eat me\u2019 signal for phagocytes [20]. PS, a negatively charged aminophospholipid, is predominantly found in the cytofacial membrane leaflets of living cells. The exofacial plasma membrane leaflet contains predominantly phosphatidylcholine and sphingomyelin while PS is almost completely lacking. The PS asymmetry results from the ATP-dependent action of the aminophospholipid translocase that transports PS from the outer to the inner leaflet [21, 22]. During apoptosis the aminophospholipid translocase is inhibited and concomitantly a scramblase is activated. The combined action results in the surface expression of PS whilst the plasma membrane integrity remains intact.\nSurface-expressed PS provides an attractive target for molecular imaging of apoptosis.\nAnnexin A5\nIn the past decade, a molecular imaging protocol was established to detect and measure apoptosis in vitro and in vivo in animal models and patients [23\u201326]. This molecular imaging protocol is based upon the protein annexin A5 (Anx A5), which binds with high selectivity and with great affinity (Ka\u2009=\u20097\u00a0nM) [27, 28] to the PS abnormally expressed on the cell membrane of apoptotic cells [21, 22, 29]. Anx A5 is a member of a multiprotein family of more than 160 proteins that share the property of Ca2+-dependent binding to negatively charged phospholipid surfaces [30]. The biophysical, biochemical and biological properties of Anx A5 and other members of the annexin family have been extensively described in a number of excellent reviews [30\u201334].\nDetecting apoptosis with Anx A5\nThe development of annexin-based detection of apoptosis started in 1992 when Fadok et al. revealed that PS is expressed on the cell surface of apoptotic cells [35]. This revelation led Koopman et al. to the design of an apoptosis detection assay on the basis of fluorescence (fluorescein isothiocyanate)-labelled Anx A5 [29]. This Anx A5 affinity assay was further developed by labelling Anx A5 with biotin or with several radionuclides to facilitate various protocols for measuring apoptosis both in vitro [36] and in vivo [23\u201325, 37\u201341] in animal models. The availability of 99mTc-labelled Anx A5 produced under GMP regulations led to the first studies of non-invasive detection of apoptosis in patients [26, 42\u201346].\nDetection of apoptosis using alternative methods\nIn addition to Anx A5, several other proteins may have a high specificity and tight binding to PS. If labelled with a fluorescent or nuclear probe, these proteins could possibly be used for in vivo detection of apoptotic cells in the field of cardiovascular disease and in other disciplines. In addition, other steps in the apoptotic signalling cascade, such as the activation of caspase-3, can be utilised to visualise activation of the apoptotic machinery (Fig.\u00a01).\nFig.\u00a01Targets for apoptosis detection. During apoptosis, initiator caspases are activated, via either cell death receptor-mediated or mitochondrial signalling. These initiator caspases, in turn, trigger the activation of effector caspases, such as caspase-3. The activation of caspase-3 results in the typical characteristics of apoptosis, such as DNA fragmentation, substrate cleavage of cytoplasmic proteins and cell membrane alterations. These apoptotic characteristics and the activation of caspase-3 offer targets for molecular imaging of apoptosis\nSynaptotagmin I\nOne such PS binding protein is the C2 domain of synaptotagmin I, which binds to anionic phospholipids in cell membranes [47]. By conjugation of the C2 domain, synaptotagmin can be used for in vivo molecular imaging of apoptosis. Zhao and co-workers conjugated the C2 domain with superparamagnetic iron oxide (SPIO) particles, a very effective T2 relaxing MRI contrast agent, and injected the product in tumour-bearing mice. They revealed that a murine lymphoma (EL4) tumour model treated with cyclophosphamide and etoposide showed an increase in apoptotic cells from a basal value of 4% to 32% during chemotherapy. This was readily detected with C2-labelled SPIO particles and T2W MRI. Tumour regions with the greatest MRI changes correlated with regions having the highest proportion of apoptotic and necrotic cells [47]. More recently, the same group labelled the C2A domain of synaptotagmin I radioactively (99mTc) or fluorescently (FITC) and used the labelled synaptotagmin in a reperfused acute myocardial infarction model (AMI) rat model. They found both ex vivo and in vivo accumulation of the radiotracer in the area at risk. Although some of the uptake was caused by passive leakage due to elevated vascular permeability in the area at risk, the majority was caused by specific binding to PS [48].\nBoth Anx A5 and synaptotagmin I detect apoptotic cells by selective binding to externalised PS. An alternative method to detect apoptosis may be to use a target more upstream in the apoptotic cascade, the effector caspases.\n5-Pyrrolidinylsulphonyl isatins\nIn one study the peptide-based irreversible pan-caspase inhibitor Z-VAD-fmk was radio-iodinated and suggested as a caspase imaging agent. However, due to poor cellular permeability, intracellular targeting of activated caspases was limited, preventing in vivo application [49]. In more recent work, non-peptidyl caspase inhibitors of the 5-pyrrolidinylsulphonyl isatin-type were proposed instead of a peptide-based caspase, such as Z-VAD-fmk. These caspase binding radioligands (CBRs) may be capable of directly targeting apoptosis in vivo. CBRs are expected to form intracellular enzyme inhibitor complexes by means of binding to the activated caspases. Kopka and co-workers succeeded in labelling several 5-pyrrolidinylsulphonyl isatins with iodine-125 [50]. However, none of these compounds have been used for non-invasive imaging in patients yet.\nApoSense\nThe ApoSense family is a group of low molecular weight amphipathic apoptosis markers targeting the cell membrane of apoptotic cells. ApoSense was developed by the Israel-based company NST. ApoSense has been used in vitro and in vivo in several disease models associated with cell death, such as radiation-induced lymphoma, renal ischaemia\/reperfusion and cerebral stroke [51, 52]. However, it is still not known to which cell membrane target ApoSense exactly binds. Therefore, it remains a challenge to link ApoSense binding to the signalling cascades in apoptosis signalling.\nRole and detection of apoptosis in CVD\nApoptosis of cardiomyocytes after myocardial infarction\nSeveral forms of cell death have been observed in the infarcted myocardium. The central ischaemic zone is dominated by necrotic cell death, whereas in the periphery of the area at risk mainly apoptotic cell death is observed. Apoptotic cell death in the infarct periphery is not the exclusive domain of the cardiomyocyte. Also non-myocytes, including endothelial cells, macrophages and blood cells, contribute to a comparable degree to apoptotic cell death [53]. Since the process of cell death can be pharmaceutically manipulated, thereby limiting the extent of myocardial cell loss, detection and quantification of apoptotic cells are of importance for determination of the full extent of reversible myocardial damage. Once the apoptotic program is activated, several targets are present both for imaging apoptosis and for therapeutic interventions.\nAs described earlier, the C2A domain of synaptotagmin I binds to the exposed PS on apoptotic cells. Zhao and co-workers developed a radiotracer for PS expression by labelling synaptotagmin via 2-iminothiolane thiolation with 99mTc, forming a technetium C2A glutathione S-transferase complex (99mTc-C2A-GST) [48]. In vivo planar imaging of AMI in rats was performed on a gamma camera using a parallel-hole collimator. The uptake of 99mTc-C2A-GST within the area at risk was quantified by direct gamma counting. The foremost finding was that in seven of seven rats the infarct was clearly identifiable as focal uptake in planar images. This and other findings led the authors to conclude that the C2A domain of synaptotagmin I labelled with a radioisotope binds to both apoptotic and necrotic cells. Ex vivo and in vivo data indicate that, because of elevated vascular permeability, both specific binding and passive leakage contribute to the accumulation of the radiotracer in the area at risk. However, the latter component alone is insufficient to achieve detectable target-to-background ratios using in vivo planar imaging.\nAnnexin A5 has been more widely used to target PS exposure. Hiller and co-workers [54] recently reported on a new Anx A5-based T1 contrast agent. They linked Anx A5 to gadolinium diethylenetriamine penta-acetate (Gd-DTPA)-coated liposomes. In their model of perfused isolated rat heart, ligation of the left coronary artery for 30\u00a0min was followed by reperfusion. T1 and T2* images were acquired using an 11.7-T magnet before and after intracoronary injection of the contrast agent. A significant increase in signal intensity, visible in those regions containing cardiomyocytes in the early stage of apoptosis, was found. The group of Weissleder also reported on Anx A5-based MRI contrast agents [55], demonstrating the construction of a magneto-optical nanoparticle AnxCLIO-Cy5.5, which was tested in a murine model of transient coronary artery (LAD) occlusion. The synthesis of the probe was achieved in three steps. First, the amino-CLIO nanoparticle was labelled with Cy5.5 and activated with SPDP [N-succinimidyl 3-(2-pyridyldithio)propionate], to yield a compound termed 2PySS-CLIO-Cy5.5. Second, Anx A5 was reacted with SATA (N-succinimidyl S-acetylthioacetate). In the final step of the process, SATAylated Anx A5 was linked with 2PySS-CLIO-Cy5.5 to yield the multimodal AnxCLIO-Cy5.5 nanoparticle. It was demonstrated that it is feasible to obtain high-resolution MR images of cardiomyocyte apoptosis in vivo with the use of the nanoparticle AnxCLIO-Cy5.5.\nIn early work preformed by our group a fluorescent invasive Anx A5-based imaging protocol for myocardial apoptosis was developed in mice [25]. The promising findings of this study encouraged use of modified human recombinant Anx A5 to construct (Anx A5)-n-1-imono-4-mercaptobutyl, labelled with pertechnetate for clinical studies. One milligram of 99mTc-Anx A5 (584 MBq) was injected intravenously 2\u00a0h after reperfusion in seven AMI patients. In six of the seven patients, increased uptake of 99mTc-Anx A5 was seen in the infarcted region of the heart on single-photon emission computed tomography (SPECT) images. No uptake was seen in the heart outside the infarcted area. In all individuals with increased uptake, a matching perfusion defect (on MIBI SPECT) was observed (Figs.\u00a02, 3). These results demonstrated the feasibility of non-invasive monitoring of apoptosis in patients. These techniques may allow measurement not only of the extent of reversible damage but also of the efficacy of novel therapies targeting apoptosis.\nFig.\u00a02Transverse tomographic images in a patient with acute anteroseptal infarction. a Arrow shows increased 99mTc-labelled Anx A5 uptake in the anteroseptal region 22\u00a0h after reperfusion. b Perfusion scintigraphy with sestamibi 6\u20138\u00a0weeks after discharge shows an irreversible perfusion defect which coincides with the area of increased 99mTc-labelled Anx A5 uptake (arrow). L liver. Reprinted with permission from Elsevier (The Lancet, 2000, 356, 211)Fig.\u00a03Transverse tomographic images in a patient with acute anterior wall myocardial infarction. a Arrow shows increased uptake of 99mTc-labelled Anx A5 in the infarct area 17.5\u00a0h after reperfusion. L liver. b Perfusion defect on sestamibi perfusion scintigraphy 6\u20138\u00a0weeks after discharge matches the uptake of Anx A5 (arrow). Reprinted with permission from Elsevier (The Lancet, 2000, 356, 211)\nAtherosclerotic lesions and plaque instability\nExtensive histopathological studies and investigations on the initiation of atherosclerosis in transgenic mice have generated a quite detailed picture of the development of atherosclerosis in the various stages of disease [56\u201363]. The onset of plaque formation is characterised by circulating levels of oxidised low-density lipoproteins, which are capable of penetrating the endothelial border while inflicting oxidative damage on the endothelial cells in the process. Subsequently, the reactionary expression of inflammatory markers on the endothelial cell surface attracts circulating monocytes to the site of injury, which, upon arrival at the scene, differentiate into macrophages and start ingesting the available lipid and other oxidised particles. Uninhibited intracellular lipid accumulation compromises the function of macrophages and converts them into foam cells [64]. The accompanying secretion of pro-inflammatory cytokines and matrix-metalloproteinases (MMP) likely contributes to the hostility of the plaque environment and weakening of collagen matrix structures, both indirectly through loss of smooth muscle cells and directly through MMP activity [65]. At a certain point, excess damage to macrophages and smooth muscle cells induces apoptosis, causing remnants of lipid-laden dead cells to form small extracellular lipid droplets.\nA luxating moment in atherogenesis is thought to occur when the remaining population of functional phagocytes becomes unable to engulf all apoptotic remnants of dying cells in the environment [65]. Once the lesion contains a large, necrotic, lipid pool that is covered by a fibrous cap, primarily consisting of SMCs and extracellular matrix, it is named an atheroma.\nA critical finding for vascular risk prediction is that although plaque rupture generally requires progression of lesion size, acute events resulting from rupture may just as well originate from only mildly stenotic lesions [66]. Coronary catheterisation studies in patients admitted with acute coronary syndromes have shown that most of the culprit lesions are below the 50% stenosis threshold [67\u201369]. In reality, the extent of the lesion may be underestimated, since conventional coronary catheterisation does not take the extent of outward remodelling of the coronary artery at the site of atherosclerotic lesions into account. The emerging cardiac multislice CT technology may be better equipped to take outward remodelling into account and to assess the total coronary plaque burden in patients. Nevertheless, these data imply that though plaque dimensions will in general be sufficient for imaging-target visualisation, focussing on mere plaque size entails unsatisfactory prognostic information for clinical use. From a biological perspective, several inflammatory parameters are associated with vulnerable lesions, such as elevated extracellular levels of MMP and pro-inflammatory cytokines, including a variety of interleukins and chemotactic proteins, as has been shown in both experimental and clinical studies [70\u201373]. Moreover, high-risk lesions are notorious for displaying high levels of inflammation and apoptotic cells; in particular, large amounts of apoptotic macrophages have been found to reside in fragile and ruptured fibrous caps [59, 65, 74]. In 2000, Kolodgie et al. [59] examined 40 culprit lesions from cases of sudden coronary death and reported apoptotic macrophages to constitute up to 50% of the total macrophage population, preferentially localising at the site of rupture. Also, once atheromas have formed, plaques have been found to be far more sensitive to SMC death than regular arterial walls, as recently demonstrated by the group of Bennett [61]. Since all these features share their relation to inflammation and more specifically apoptosis, detection of apoptosis in vivo seems a sensible approach to vulnerable plaque identification. This approach builds on the pivotal role that apoptosis plays in promoting plaque progression and disruption; however, the underlying mechanism is a complex interplay in which apoptosis affects endothelial cells, smooth muscle cells and macrophages in a different manner and with different consequences.\nGiven the firmly established direct causal relationship between apoptosis and lesion instability [59, 75], in vivo visualisation of the apoptotic plaque status could represent a significant leap forward in the field of therapeutic management for individual patients (Fig.\u00a04).\nFig.\u00a04Feasibility of non-invasive imaging of apoptosis by radiolabelled Anx A5. Left lateral oblique gamma images of experimental atherosclerotic (a\u2013c) and control (d\u2013f) rabbits injected with 99mTc-Anx A5; L and K indicate liver and kidney activities, respectively. Images at the time of injection (a, d) and at 2\u00a0h after injection (b, e) are shown. Although blood pool activity is seen at the time of injection (a) in the atherosclerotic animal, tracer uptake is clearly visible in the abdominal aorta (with lesions) at 2\u00a0h (b). c Ex vivo image of b shows intense 99mTc-Anx A5 uptake in the arch and abdominal region. Annexin-positive areas were confirmed to contain macrophage- and apoptosis-rich regions in the atherosclerotic plaque by histology. d\u2013f show the corresponding images in the control animal. Note that the aorta is indistinguishable from background at 2\u00a0h after injection (e). The blood pool at the time of injection in the control animal (d) is comparable to that in the atherosclerotic animal. f Ex vivo aortic image of the control animal demonstrates the absence of 99mTc-labelled Anx A5 uptake. From Kolodgie et al. [75]\nThe expanding knowledge about plaque biology has led to the identification of a variety of potentially suitable molecular markers for vulnerable plaque imaging [76]. Studies have been reported on the targeting of several inflammation-related molecular targets, including vascular adhesion molecules (ICAM1, VCAM1, E-selectin), neoangiogenesis molecules (\u03b1v\u03b23 integrin), scavenger receptors on macrophages, MMP proteolytic activity, macrophage metabolic activity and apoptosis (PS) [60, 62, 75\u201383]. Although some of these approaches have yielded promising experimental data, there is still a paucity of clinical studies confirming the feasibility of these targeted approaches in humans.\nOf the clinical results available, the most impressive have been achieved using 99mTc-Anx A5 SPECT imaging and [18F]fluorodeoxyglucose (18FDG) positron emission tomography (PET) imaging. The latter has been used for many years to detect metabolically hyperactive processes in nuclear oncology. Since the glucose analogue 18FDG is a marker for foci of increased metabolic activity [84] and it is known that macrophages involved in atherosclerotic plaques have a high metabolic rate, there is sound logic behind this strategy. In addition, 18FDG imaging capitalises on the merits of oncological medicine since 18FDG is a clinically approved tracer that can be detected with high sensitivity and accuracy by PET technology, whereas PET conjugates for most alternative plaque imaging targets are still in the process of development. 18FDG imaging of arterial inflammation has been performed with rather promising results in retrospective studies of oncological 18FDG scans, in experimental settings and in in vivo clinical studies by Rudd and colleagues [76, 82, 85, 86]. In an eight-patient pilot study, they were able to correlate 18FDG uptake in carotid lesions of patients scheduled for endarterectomy with histologically assessed macrophage density (p\u2009<\u20090.005). However, a practical difficulty of performing 18FDG imaging in the search for coronary lesion instability lies in the physical activity of the cardiac muscle, which results in high glucose requirements and thus severe background signal.\nIn addition to detection of the vulnerable plaque with 18FDG, imaging of the vulnerable plaque with 99mTc-Anx A5 has yielded some exciting results as well. The first demonstration of the feasibility of in vivo imaging of plaque instability with 99mTc-Anx A5, in animals, was given in 2003 by Kolodgie et al. It was demonstrated in a rabbit model of aortic de-endothelialisation followed by a high-fat diet that 99mTc-Anx A5 showed a ninefold higher binding to the atherosclerotic vessels than to the control vessels [75]. Further validation of the Anx A5 imaging concept in the vulnerable plaque was done by Johnson and colleagues in 2005 [87]. In a porcine model of atherosclerosis the feasibility of SPECT imaging with 99mTc-Anx A5 to assess atheroma progression in coronary arteries was demonstrated. This was ultimately challenging owing to the low mass of the imaging target and cardiopulmonary motion. As swine and human hearts have similar cardiac dimensions, these results indicate that non-invasive functional imaging of coronary plaque instability could be realistic in the near future. The first clinical demonstration of the feasibility of nuclear vulnerable plaque imaging with 99mTc-Anx A5 was given by Kietselaer et al. in 2004 [43]. In a pilot study, four patients were included who were scheduled for carotid endarterectomy and had a history of recent (n\u2009=\u20092) or remote (n\u2009=\u20092) transient ischaemic attack (TIA). Before surgery 99mTc-Anx A5 was administered and SPECT imaging was performed. In the two patients who had suffered a TIA more than 3 months before imaging and had since been treated with statins and antiplatelet agents, no tracer uptake of Anx A5 was observed (Fig.\u00a05). Histological analysis confirmed stable lesions with negligible macrophage infiltration and intra-plaque haemorrhage. In contrast, the two patients who had experienced transient ischaemia shortly before imaging (3 and 4\u00a0days, respectively) demonstrated marked uptake of Anx A5 in the culprit carotid vessel, while the contralateral arteries remained clear of tracer binding. Anx A5 binding was histologically localised to apoptotic macrophage membranes. An interesting finding during this study was that echo-Doppler examination in one of the patients with a recent TIA identified a severe stenotic lesion in the contralateral carotid artery prior to imaging. On the 99mTc-Anx A5 scan, however, the lesion was negative, though it might have been suspected as the culprit lesion by conventional techniques. This finding underlines the potential added value of functional versus anatomical imaging in predicting the likelihood of future thrombotic events.\nFig.\u00a05Images of unstable atherosclerotic carotid artery lesions obtained with radiolabelled Anx A5. a Transverse and coronal views obtained by SPECT in patient 1, who had a left-sided TIA 3 days before imaging. Although this patient had clinically significant stenosis of both carotid arteries, uptake of radiolabelled Anx A5 is evident only in the culprit lesion (arrows). b Histopathological analysis of an endarterectomy specimen from patient 1 (polyclonal rabbit anti-Anx A5 antibody, \u00d7400) shows substantial infiltration of macrophages into the neointima, with extensive binding of Anx A5 (brown). c In contrast, SPECT images of patient 3, who had had a right-sided TIA 3 months before imaging, do not show evidence of Anx A5 uptake in the carotid artery region on either side. Doppler ultrasonography revealed a clinically significant obstructive lesion on the affected side. d Histopathological analysis of an endarterectomy specimen from patient 3 (polyclonal rabbit anti-Anx A5 antibody, \u00d7400) shows a lesion rich in smooth muscle cells, with negligible binding of Anx A5. ANT anterior, L left. Copyright \u00a9 2004 Massachusetts Medical Society. All rights reserved. [43]\nAmong the discussed imaging modalities for vulnerable plaque detection, nuclear imaging (PET in particular) is far superior to MRI, being capable of detecting probes at the picomolar range [88]. PET further outperforms SPECT imaging regarding spatial resolution, with 4- to 6-mm versus 10- to 15-mm resolution in the centre of the field. The major difficulty in evaluating PET or SPECT usually originates from orientation and localisation of the measured signal in the body. To overcome this, nuclear imaging modalities can be co-registered and overlaid with conventional CT or MRI imaging, hence providing anatomical landmarks in relation to molecular information. Improvements in the availability and costs of PET\/CT or PET\/MRI imaging devices will therefore imply great advantages. Given the high costs of imaging, diagnostic algorithms in the future most likely will combine relatively cheap serum biomarkers that indicate plaque instability with the subsequent use of expensive imaging technology in selected cases. This indicates that the specific niche for both serum and imaging biomarkers needs to be defined in large prospective studies. In addition, much is to be gained from refinements of the radioligand with regard to blood clearance and biodistribution, since molecular imaging typically requires fast binding of the probe to the target, rapid subsequent clearance from the blood and not too short radioactive half-lives.\nHeart failure\nHeart failure (HF) is growing to epidemic proportions, partially due to the better treatment of AMI. The pathogenesis of heart failure is still poorly understood. There may be an important role for cell death in the progression of congestive HF. A number of papers have reported on the differences in the amount of cell death in healthy and failing hearts. The baseline rate of apoptosis in healthy human hearts is around one to ten cardiac myocytes per 105 nuclei. On the other hand, 80\u2013250 heart muscle cells per 105 cardiac nuclei undergo apoptosis at any given time in late-stage dilated cardiomyopathy. However, it remains unclear whether this cell death is a coincidental finding, a protective process or a causal component in the pathogenesis. In work done by Wencker and co-workers, the uncertainty of the role of cell death was addressed. In a transgenic mouse model with a conditionally active caspase expressed exclusively in the myocardium, they demonstrated that even very low levels of cardiomyocyte apoptosis (23 per 105 nuclei, compared with 1.5 per 105 nuclei in controls) caused a lethal dilated cardiomyopathy. Inhibition of this active caspase largely prevented the development of cardiac dilation and contractile dysfunction. These and other data suggest that cardiomyocyte apoptosis and necrosis may be a causal mechanism of heart failure, contributing to adverse left ventricle (LV) remodelling. Inhibition of this cell death process may constitute the basis for novel therapies. Consequently, anti-apoptotic agents have been developed and tested in animal models of HF with promising results [89]. \nAlthough, as discussed earlier, it is possible to detect cell death in MI leading to HF, the small amount of cell death present in heart failure (80\u2013250 cardiomyocytes per 105 cardiac nuclei) makes detection with the current techniques very challenging. However, the feasibility of detecting apoptosis in patients with progressive idiopathic dilated cardiomyopathy was demonstrated in a proof of principle study done by Kietselaer [90]. Nine patients with idiopathic dilated cardiomyopathy (IDCM) were evaluated, as well as two family members with hypertrophic cardiomyopathy in different stages of disease. Patients were imaged with 99mTc-labelled Anx A5. 201Tl images were used for orientation. Other causes of IDCM were excluded using laboratory testing, echocardiography and coronary angiography. It was found that five patients with IDCM showed focal uptake of radiolabelled Anx A5 and that one patient showed global uptake in the left ventricle (Fig.\u00a06, left panel). No enhanced uptake could be visualised in the other six patients (Fig.\u00a06) and controls. All cases that showed Anx A5 uptake in the heart displayed recent onset or recent worsening of left ventricular function and functional class. In cases that did not show Anx A5 uptake, LV function and clinical status remained stable. Kietselaer concluded that non-invasive imaging using 99mTc-Anx A5 detects apoptosis in patients suffering from IDCM, providing further evidence for the role of apoptosis in the pathogenesis of IDCM. In addition, Anx A5 imaging identifies high-risk patients who might benefit from cell death blocking strategies.\nFig.\u00a06Dual-isotope imaging using 201Tl for left ventricular contour detection and, simultaneously, radiolabeled annexin A5 in patients with dilated cardiomyopathy. a Dilated cardiomyopathy patient with rapid deterioration of left ventricular function. Note focal uptake in apex and lateral wall, and slight septal uptake. b Dilated cardiomyopathy patient in acute heart failure. Note global uptake of radiolabeled annexin A5. c Dilated cardiomyopathy patient in stable clinical condition. Uptake is absent even when image is enhanced to the extent that background radioactivity can be observed. d Family member of patient in panel b. No clinical evidence is seen of dilated cardiomyopathy. Note absence of uptake of radiolabeled annexin A5. ANT, anterior; INF, inferior; LAT, lateral; SEPT, septal. Reprinted by permission of the Society of Nuclear Medicine from [90]\nIn addition to imaging of apoptosis, the detection of the molecular substrates of early-stage alterations leading to HF, such as vascular remodelling in infarcted tissue, is an emerging technique. Vascular remodelling is associated with non-contractile scar tissue formation that may contribute to adverse LV remodelling. However, none of these molecular techniques has reached the clinical stage yet.\nCardiac allograft rejection\nUntil 1997, almost 46,300 heart transplantations had been carried out worldwide [48]. Despite this encouraging number, only 50% of cardiac transplant recipients will survive 10\u00a0years and almost all will die within 20\u00a0years unless re-transplantation is preformed [19]. One of the possible complications for patients who have received a donor heart is transplant rejection. Under the current medical guidelines, endomyocardial biopsies (EMBs) are recommended 15\u201320 times in the first year after transplantation to monitor potential allograft rejection. EMB is an invasive diagnostic procedure and not without risk of complications. Allograft rejection is pathologically characterised by perivascular and interstitial mononuclear inflammatory cell infiltration associated with myocyte necrosis and apoptosis [91, 92]. Because the process of apoptosis can be pharmaceutically manipulated, identifying apoptosis non-invasively is of critical importance [93].\nIn a study done by Narula and co-workers [42], 18 patients who had undergone heart transplantation within the past year were subjected to non-invasive imaging with Anx A5. Patients received an injected dose of 555\u20131,110\u00a0MBq (15\u201330 mCi) of 99mTc-Anx A5 and underwent SPECT imaging 1\u00a0h after injection (Fig.\u00a07). In 13 out of these 18 patients, no myocardial uptake of 99mTc-Anx A5 was observed. In the remaining five patients, uptake of 99mTc-Anx A5 in the myocardial region was observed, which was associated with at least moderate transplant rejection (Fig.\u00a08), of International Society of Heart and Lung Transplantation (ISHLT) grade 2\/4. Histological analysis revealed active caspase-3 in EMB specimens. This study demonstrated the clinical feasibility of detecting cardiac allograft rejection induced apoptosis, which, after confirming these findings in larger clinical studies, might lead to a reduction for the need of EMBs.\nFig.\u00a07SPECT images of 99mTc-labelled Anx A5 in three patients (10, 3 and 4) are demonstrated. a In patient 10, myocardial uptake (as outlined by solid circles) is clearly seen in all tomographic orientations and can be differentiated from the left ventricular cavity, especially in the transaxial slice. b In patient 3, by contrast, no Anx A5 uptake is observed, and only background activity is seen. c Patient 4 demonstrates Anx A5 activity in the blood pool originating from the great vessels (GV great cardiac vein, PA pulmonary artery,  Ao aorta) and ventricular contours, but no activity is observed in the myocardium. The scan of patient 10, with myocardial uptake, was further processed (Fig.\u00a08). RV right ventricle, LV left ventricle. Adapted by permission from Macmillan Publishers Ltd: Nature Medicine [42], copyright 2001Fig.\u00a08Diffuse myocardial uptake of 99mTc-labelled Anx A5 in cardiac allograft. Patient 10 underwent Anx A5 imaging 9\u00a0months after orthotopic heart transplantation. SPECT imaging 3\u00a0h after intravenous injection of radiolabelled Anx A5 demonstrated multifocal myocardial uptake of radiotracer. Smoothing of the images and cardiac SPECT processing in the short axis, vertical long axis and horizontal long axis revealed myocardial perfusion scan-like images with diffuse uptake in the whole myocardium, suggesting apoptosis in the myocardium. Adapted by permission from Macmillan Publishers Ltd: Nature Medicine [42], copyright 2001\nFuture perspectives\nOf all the apoptotic imaging agents, Anx A5 has made the most successful transition from the test tube to the clinical arena. Novel imaging modalities like PET\/CT and SPECT\/CT are making a significant contribution to the growing role of molecular imaging. The combination of anatomical imaging with CT and biological imaging using SPECT or PET yields a new synergistic imaging modality that provides detailed information on the molecular (patho)physiological processes in relation to exact anatomical orientation.\nThe next challenge is to assess the incremental clinical value of molecular imaging and its ability to change patient management decisions. For this purpose, prospective cohort studies need to be designed to evaluate the clinical meaning of molecular imaging scan results in relation to the progression of the underlying disease. For instance, data on the association between Anx A5 uptake in plaques in the carotid arteries and clinical event rate are still lacking. The availability of such large clinical datasets may allow for better stratification of patients and therefore more optimal treatment decisions.\nFinally, we and others have found that not only apoptotic cells but also viable cells are detectable with Anx A5. Our work on the vulnerable plaque showed that several processes leading to plaque instability are associated with PS expression, such as activated macrophages (inflammation) and aging red blood cells (intra-plaque haemorrhage). This ability of Anx A5 to visualise exposed PS in different biological conditions opens novel opportunities for imaging.","keyphrases":["molecular imaging","apoptosis","cardiovascular disease","annexin a5"],"prmu":["P","P","P","P"]}
{"id":"Cardiovasc_Eng-3-1-2137944","title":"Determination of myocardial energetic output for cardiac rhythm pacing\n","text":"This research is aimed to the determination of the changes in the cardiac energetic output for three different modes of cardiac rhythm pacing. The clinical investigation of thirteen patients with the permanent dual-chamber pacemaker implantation was carried out. The patients were taken to echocardiography examination conducted by way of three pacing modes (AAI, VVI and DDD). The myocardial energetic parameters\u2014the stroke work index (SWI) and the myocardial oxygen consumption (MVO2) are not directly measurable, however, their values can be determined using the numerical model of the human cardiovascular system. The 24-segment hemodynamical model (pulsating type) of the human cardiovascular system was used for the numerical simulation of the changes of myocardial workload for cardiac rhythm pacing. The model was fitted by well-measurable parameters for each patient. The calculated parameters were compared using the two-tailed Student\u2019s test. The differences of SWI and MVO2 between the modes AAI and VVI and the modes DDD and VVI are statistically significant (P < 0.05). On the other hand, the hemodynamic effects for the stimulation modes DDD and AAI are almost identical, i.e. the differences are statistically insignificant (P > 0.05).\nIntroduction\nThe medical management of cardiovascular diseases, especially of arrhythmia, has become increasingly reliant upon the use of implanted electronic devices for the treatment of bradycardia or tachycardia (Winters et\u00a0al. 2001; Gregoratos et\u00a0al. 2004). The simple pacemakers of the 1970 gave way to the more advanced devices capable of real-time diagnosis of arrhythmia-related disorders and their treatment. The pacemaker technology and clinical practice permits the use of either single-chamber ventricular pacemakers or dual-chamber pacemakers for patients who require cardiac pacing in some indications. The single-chamber ventricular pacemakers are less expensive and their implantation is simpler than the implantation of the dual-chamber pacemakers. Several randomized studies comparing the dual-chamber \u201cphysiological\u201d pacing to the ventricular pacing (Mattioli et\u00a0al. 1998; Connolly et\u00a0al. 2000; Lamas et\u00a0al. 2002) have shown that the physiological pacing was more beneficial in some subgroup of patients despite the fact that both variants caused mortality, stroke and heart failures were no significant differences.\nClinical studies focusing on the quality of life with respect to the type of the pacing system implantation were also conducted. The results of the studies confirmed that the implantation of a permanent pacemaker improves health and quality of life. The quality-of-life benefits associated with dual-chamber pacing as compared with the ventricular pacing were observed only in the subgroup of patients with the sinus-node dysfunction (Lamas et\u00a0al. 1998). The DAVID trial (Dual Chamber and VVI Implantable Defibrillator) demonstrated increase heart failure hospitalisation and mortality in patients programmed to the DDDR mode compared to ventricular backup pacing. The Multicenter Automatic Defibrillator Implantation Trial II (MADIT II) evoke the question of minimising the unnecessary right ventricular pacing (Wilkoff et\u00a0al. 2002; Moss et\u00a0al. 2002; Galley et\u00a0al. 2004).\nThe myocardial energy demands during the cardiac pacing were studied experimentally (Montgomery et\u00a0al. 1991; Portman and Ning 1995). The clinical data demonstrating the energetic changes during the pacing on the patients with dilated cardiomyopathy were shown by Baller et\u00a0al. (2004). Kerckhoffs et\u00a0al. (2005) carried out mathematical modelling of the right ventricular pacing and comparison with spontaneous depolarization.\nIn the present study we are using the numerical model of the human cardiovascular system developed in the Institute of Thermomechanics, Czech Academy of Sciences (P\u0159evorovsk\u00e1 and Mar\u0161\u00edk 2004) with the purpose to find the parameters of the myocardial energetics during the cardiac rhythm pacing.\nMethods and materials\nPatients\nFor our research purposes the patients were designated on the basis of selective criteria. The inclusion criteria were established sick sinus syndrome and pacemaker implantation lasting more than 6\u00a0months. The exclusion criteria were left ventricle dysfunction, clinical significant valvular disease and other pathological structural heart changes. The patients with other than sinus rhythm, AV block under 110\u00a0beat\/min during stimulation or bad image quality from echocardiography examination were also excluded. Our research finally involved thirteen patients with the sick sinus syndrome (eight males and five females with the average age 73\u00a0years, ranging 59\u201382\u00a0years). These patients with permanent dual-chamber pacemaker implantation were taken to echocardiography examination.\nStudy protocol\nThe numerical model of cardiovascular system was used to calculate the myocardial energetic parameters such as the stroke work index and the myocardial oxygen consumption for three types of pacing.\nThe stroke work index SWI (gm\/m2) is used as a measure of myocardial contractility or work performed by the heart at every contraction. The work performed by the heart can be simply by the fluid dynamic formula \nThe stroke work index of the left ventricle LVSWI is then determined by the stroke volume SV (ml), by the difference of the mean arterial pressure MAP (mmHg) and the pulmonary artery occlusion pressure PAOP (mmHg) (considered to be the indirect measure of the left atrial pressure) by the formula (http:\/\/www.lidco-ir.co.uk\/html\/clinical\/nhp.asp)1: the constant 0.0136 converts the units mmHg\u00a0l\/m2 to gm\/m2. BSA (m2) is the body surface area given by the DuBois formula (Wang et\u00a0al. 1992): where BW (kg) is the body weight and BH (cm) is the body height.\nThe myocardial oxygen consumption MVO2 (ml O2\/beat) is calculated by the relationship proposed by Suga 1990, which is based on the time-varying elastance model of the ventricle. This relationship represents linear correlation between the total pressure-volume area PVA (J\/beat) and MVO2 (1\u00a0ml O2\u00a0=\u00a020\u00a0J) where a is the slope coefficient and b is unloaded MVO2. PVA consists of the external mechanical work EW (J\/beat) within the contraction cycle and the potential energy PE (J\/beat) The values EW (representing the work done by the ventricle to eject blood) and PE (the work against the viscoelastic properties of the myocardium) can be estimated using the pressure-volume loops (see Fig.\u00a01) Fig.\u00a01Ventricular pressure\u2013volume loopsThe following symbols are used in Eq. 5:ppeakS (Pa) maximal value of the systolic ventricular pressureppED (Pa) end-diastolic ventricular pressureVED (m3) end-diastolic ventricular volumeVES (m3) end-systolic ventricular volumeVo (m3) ventricular volume at zero pressure (point of intersection on the volume axis, see Fig.\u00a01).\nThe input data for the numerical model, such as the anthropometrical data of the patients (weight, height) or the heart frequency, were obtained from their physical examination. The various model parameters, such as the calcium, sodium, potassium channel\u2019s conductance, were fitted to obtain the stroke volume, blood pressure and pulmonary artery pressure established by the echocardiography examination which was performed during the spontaneous heart rhythm and the AAI, VVI, DDD pacing modes. Each mode was examined for stimulation frequencies 70, 90 and 110\u00a0beat\/min. Each examination was performed 5\u00a0min after the mode change to ensure steady state. Standard views of the parasternal long-axis view and the Doppler outflow tract across the aortic valve were taken using the Agilent Sonos 5500 ultrasound device. The stroke volume was determined from the left ventricular outflow (aortic valve) diameter measured on the parasternal long-axis view and from the outflow velocity on the aortic valve measured from the apical five-chamber view (Huntsman et\u00a0al. 1983; Lewis et\u00a0al. 1984) by the Doppler probe. The blood pressure was measured by the sphyngomanometer for each stimulation mode. Then, the pulmonary pressure was measured by the Doppler echocardiography on tricuspid valve.\nMedical Ethical Committees of the University Hospital Kr\u00e1lovsk\u00e9 Vinohrady, Prague, approved the study protocol and the informed consent was obtained from all patients.\nMathematical model\nFor the modelling purposes, the real cardiovascular system was compartmentalised (see Fig.\u00a02). The 24-segment hemodynamical model (pulsating type) of the human cardiovascular system imitating the electrochemical and mechanical activity of the heart muscle was used for the numerical simulation of the myocardial workload changes during the cardiac rhythm pacing.\nFig.\u00a02Scheme of the human cardiovascular system applied to heart workload calculations\nThe system is approximated by twenty-four compartments, which are characterized by elasticity, resistivity and transfer of the relevant chemical components. The pulsating heart consists of the following compartments: RA\u2014Right Atrium, RV\u2014Right Ventricle, LA\u2014Left Atrium, LV\u2014Left Ventricle. The pulmonary circuit includes compartments representing the pulmonary artery (PA), arteries (Paa), capillaries (Pc), postcapillary venules (Pvv), and pulmonary veins (PV). Systemic circuit connects the aorta (Ao), systemic arteries (Saa), capillaries (Sc), head arteries (HA), capillaries (Hc), and head veins (HV). In addition, the systemic circuit includes the coronary circulation with coronary arteries (CA), capillaries (Cc), veins (CV), shunt (CS), and also the bronchial circulation with bronchial arteries (BA), left bronchial veins (BVL), capillaries (BRc), and right bronchial veins (BVR). The compartments of the pulmonary and systemic blood circulation are connected in series with the four pulsating heart compartments.\nThe suggested model describes the one-dimensional flow of incompressible blood through the network of elastic blood vessels. The heart compartments are considered to be made of anisotropic and viscoelastic incompressible material.\nThe mathematical formulation describing the blood flow in the cardiovascular system is based on the mass, energy and momentum balance. The behaviour of the cardiovascular system is described by the heart rate and by its hemodynamical variables, i.e., blood pressure, volume and flow, and by the cardiovascular parameters, such as the compliances and resistances in the corresponding compartments. The blood inertia and physicochemical variables, such as the cardiac action potential and the calcium, sodium and potassium ion concentrations, are included to support more accurate performance of the cardiovascular system. The Beeler-Reuter model (Beeler and Reuter 1977) describes the behaviour of excitable cardiac cells.\nDiscussion and conclusions\nAll experimental results obtained with the investigated patients and simulated values resulting from the applied numerical model were elaborated by the following statistical analysis. We use the null hypothesis to demonstrate the agreement or discrepancy between the values of the myocardial energetic parameters (stroke work index, myocardial oxygen consumption) obtained by the numerical simulation for the three pacing modes.\nAll comparisons of the data were made by the two-tailed Student\u2019s test. The statistical analysis was performed with 5% significance level and the degree of freedom df\u00a0=\u00a024. The data for the stroke work index are expressed as the mean value\u00a0\u00b1\u00a0standard deviation.\nThe calculated values of the stroke work index at the three pacing modes are shown in the Fig.\u00a03. It was shown that the differences in the compared pacing modes AAI and VVI and the modes DDD and VVI are statistically significant (P\u00a0<\u00a00.05). The comparison of the stimulation modes DDD and AAI shows that the differences are statistically insignificant (P\u00a0>\u00a00.05).\nFig.\u00a03Stroke work index calculated by the numerical model of cardiovascular and respiratory system\nThe calculated values of the myocardial oxygen consumption for three pacing modes are demonstrated in the Fig.\u00a04. The myocardial efficiency, which is an important factor for heart performance, changes according to the stimulation mode. The changes resulting from the hemodynamical model fitting by the well-measurable data, i.e., heart rate, stroke volume, systolic pulmonary artery pressure, systolic and diastolic aortic pressure. The conclusion is that the difference between the modes AAI and VVI and the modes DDD and VVI is statistically significant (P\u00a0<\u00a00.05). The statistical analysis of the AAI and DDD modes indicates that the compared data are not significantly different (P\u00a0>\u00a00.05).\nFig.\u00a04Myocardial oxygen consumption calculated by the numerical model of cardiovascular system in various pacemaker modes\nFrom the comparison of the obtained numerical values for the stroke work index between the AAI and VVI pacing modes and between DDD and VVI modes it follows that the differences are statistically significant (P\u00a0<\u00a00.05). The results are consistent with the published data (Leclercq et\u00a0al. 1995; Nielsen et\u00a0al. 1998). The results of the statistical analysis for the AAI and DDD stimulation modes show that the differences between the compared groups are statistically insignificant (P\u00a0>\u00a00.05). These results are consistent with the clinical observation where can achieve a similar clinical outcome in AAI and DDD modes in this group of patients (Masumoto et\u00a0al. 2004). The increase of the stroke work index in AAI or DDD modes was caused by saving the AV synchrony in the patient with the normal left ventricle systolic function. During the AAI pacing mode the implantation of the pacemaker is easier. This fact supports the clinical superiority of the AAI mode against the DDD mode in the sick-sinus group patients with preserved left ventricular function.\nThe results of the comparison of the obtained numerical values for the myocardial oxygen consumption between the AAI and VVI and between DDD and VVI modes show that the difference between the modes is statistically significant (P\u00a0<\u00a00.05). The increase of the myocardial oxygen consumption corresponds to the increase of the stroke work index. From the comparison of AAI and DDD modes we can see that the difference is statistically insignificant (P\u00a0>\u00a00.05).\nThe insufficient number of the designated subjects gives the limitation of our research study because very strict exclusion criteria exist for the concomitant disease. Many pacemaker patients were not eligible for echocardiography examination because they had hypertension, left ventricle dysfunction or valvular disease.\nWe conclude that mathematical modelling of the cardiovascular system is a recommended method for the estimation of the myocardial energy demand in patients with heart diseases.","keyphrases":["myocardial energetics","echocardiography examination","numerical simulation","heart arrhythmia","cardiac pacing modes"],"prmu":["P","P","P","R","R"]}
{"id":"Behav_Genet-3-1-1914301","title":"Variance Decomposition Using an IRT Measurement Model\n","text":"Large scale research projects in behaviour genetics and genetic epidemiology are often based on questionnaire or interview data. Typically, a number of items is presented to a number of subjects, the subjects\u2019 sum scores on the items are computed, and the variance of sum scores is decomposed into a number of variance components. This paper discusses several disadvantages of the approach of analysing sum scores, such as the attenuation of correlations amongst sum scores due to their unreliability. It is shown that the framework of Item Response Theory (IRT) offers a solution to most of these problems. We argue that an IRT approach in combination with Markov chain Monte Carlo (MCMC) estimation provides a flexible and efficient framework for modelling behavioural phenotypes. Next, we use data simulation to illustrate the potentially huge bias in estimating variance components on the basis of sum scores. We then apply the IRT approach with an analysis of attention problems in young adult twins where the variance decomposition model is extended with an IRT measurement model. We show that when estimating an IRT measurement model and a variance decomposition model simultaneously, the estimate for the heritability of attention problems increases from 40% (based on sum scores) to 73%.\nIntroduction\nIn quantitative genetics, one is interested in the extent to which variation in certain characteristics is heritable. Heritability is expressed in terms of the proportion of the variance of a trait in a population that can be attributed to genetic differences. This genetic variance component can be estimated in, for example, the classical twin design (Boomsma et\u00a0al. 2002a) in which the covariance structures of monozygotic and dizygotic twins are compared.\nHowever, it is not always straightforward to estimate variance components. A variance component is only meaningful when measures are expressed on a scale of at least interval level. Moreover, many statistical methods require the phenotype to be normally distributed. Many phenotypes are not expressed in clearly defined units and are at best ordinal in character (e.g., conservatism, extraversion). Some traits have even only a nominal character (e.g., psychiatric disorders). There are several ways of dealing with such nominal data. One possibility is to focus on concordance rates and compute recurrence risk ratios (Risch 1990, 2001). Alternatively, one might assume a latent continuous trait with a threshold above which individuals are affected and estimate the heritability on that latent trait (Lynch and Walsh 1998; Falconer 1965; Crittenden 1961). This method can also be used with ordinal data.\nFor some traits, it is convenient to have multiple indicators (items). For example one might have for a particular disease 10 symptoms that each can be scored as absent (0) or present (1). For each individual one can then compute a sum score that indicates to what extent the individual is affected by the disease. Such sum scores usually show a normal distribution or do so after an appropriate transformation. It is typically assumed that the normally distributed scores or transformations thereof reflect a continuous interval scale and the variance of the sum scores is subsequently decomposed. This approach follows classical test theory (CTT) where it is assumed that the observed score (the sum score) is the aggregate of a true score and a random component, usually referred to as measurement error. When decomposing the variance of sum scores, the measurement error variance (the unreliability) ends up as part of the non-shared environmental variance. As a result, when the reliability of a scale is low (i.e., the measurement error is large) and the analysis is based on sum scores, the heritability of the actual trait is significantly underestimated.\nModelling sum scores is appropriate if the sum scores are highly reliable (for instance because they are based on a large number of correlated items) and well validated. Furthermore, there should be enough variation and the distribution should be more or less normal. Finally, there should be no data missing. If these requirements do not hold, item response theory (IRT) provides a well-established alternative to classical test theory. This paper introduces the basics of the IRT framework, after which its advantages over a sum score approach are discussed. Next, it is argued that IRT models should be estimated simultaneously with the variance decomposition model, which can be done using a Bayesian approach with Markov-chain Monte Carlo estimation. Lastly, a simulation study shows the potential bias when estimating variance components on the basis of sum scores and the Bayesian method is illustrated with an empirical data set on attention problems.\nItem response theory models\nIn IRT models\u2014as opposed to CTT\u2014the influence of the items and the respondents are explicitly modelled by distinct sets of parameters. In these models, an assumed continuous latent variable \u03b8 reflects the trait and every item is identified by thresholds \u03b2 where a response in one category becomes more likely than a response in an adjacent category. It is usually assumed that the latent variables \u03b8j are drawn from a normal distribution, that is, \u03b8j are independently and identically distributed N(\u03bc, \u03c32), though this assumption is not always necessary to identify the model parameters. The probability of the presence of the symptom i in individual j, p(Yij\u00a0=\u00a01), is a function of the difference between the individual\u2019s trait score \u03b8j and the parameter \u03b2i, with \u03b2i indicating the location on the scale where the presence of a symptom becomes more probable than its absence. In the case of multiple symptoms, we have with \ndenoting the cumulative standard normal distribution function. That is, the probability of the presence of symptom i in person j is a function of both a person\u2019s liability score \u03b8j and a symptom (or item) parameter \u03b2i. In the IRT framework, this model is referred to as the one-parameter normal ogive model, or 1PNO (Lawley 1943; Lord 1952, 1953). This model is identified with a location restriction, for example, \u03bc\u00a0=\u00a00. The variance of the latent trait, \u03c32, can be estimated and can be interpreted as the covariance of the items: the larger the variance, the higher the reliability of the scale.\nAn alternative parameterisation replaces the normal ogive by a logistic curve, that is,\nwhere \nThis version of the model is known as the one-parameter logistic model (1PLM), or Rasch model (Rasch 1960). To illustrate the model, consider an individual with a score \u03b8j of 1 on the latent trait, and a particular item with parameter \u03b2\u00a0=\u00a01. Then the probability of a positive response from this individual on this item equals exp(1\u00a0\u2212\u00a01)\/(1\u00a0+\u00a0exp(1\u00a0\u2212\u00a01))\u00a0=\u00a0exp(0)\/(1\u00a0+\u00a0exp(0))\u00a0=\u00a01\/2\u00a0=\u00a050%. An individual with a score higher than 1 has a higher probability of showing a positive response, whereas an individual scoring lower than 1 has a lower probability. Individuals with a latent score of \u22121 have a probability of exp(\u22122)\/(1\u00a0+\u00a0exp(\u22122))\u00a0=\u00a012%. With a simple multiplicative transformation of the scale, the logistic and normal ogive curves are very similar and indistinguishable for all practical work (see, for instance, Lord 1980).\nIn the Rasch model, as well as in the 1PNO model, all items have the same correlation (\u2018\u2018factor loading\u2019\u2019) with the underlying latent trait. Analogous to factor models, it is possible to estimate factor loadings that differ across items. In the IRT framework these factor loadings are referred to as discrimination parameters \u03b1i. These parameters indicate the extent to which an item i loads onto the latent trait, and the model becomes\nAn alternative form in the literature replaces \u03b1\u03b8\u00a0\u2212\u00a0\u03b2 with \u03b1(\u03b8\u00a0\u2212\u00a0\u03b2). This leads to a somewhat different interpretation of the \u03b2-parameters (they are scaled differently) but it only involves a reparameterisation.\nEssentially, a one-parameter model can be described by a two-parameter model where all \u03b1 parameters are equal. In order to identify the model and estimate \u03b1, however, the variance of the latent trait should be fixed. Thus, a one-parameter model with a large variance of the latent trait is equivalent to a two-parameter model with large discrimination parameter \u03b1 that is equal for all items together with a fixed variance of the latent trait.\nThe two-parameter model must be identified by both a location and a scale restriction. The former can be the same restriction as above, that is, \u03bc\u00a0=\u00a00. The latter can be the additional restriction that the variance of the latent distribution is equal to one, that is, the model is identified by assuming a standard normal distribution, N(0,1), for the latent ability parameters \u03b8j. Alternatively one fixes one of the discrimination parameters to unity. Generally, however, this identification solution is not advisable, because the standard errors of the parameters blow up if the discrimination parameter chosen for the identification is poorly identified.\nIRT models for polytomous data\nOften, measurement is based on items or symptoms with more than two categories. For example, answers can be coded as 0 (not at all), 1 (somewhat, sometimes) and 2 (a lot, often). Typically in CTT approaches in behaviour genetics the sum of these item scores is regarded to represent a person\u2019s score on the trait of interest and is used for the statistical inference.\nThere are several IRT models for ordered categories (e.g., Samejima 1969; Masters 1982). These have different rationales and are not reparameterisations of each other, but the practical implications for preferring one over the other are often negligible. Here we describe a continuation-ratio model (Tutz 1990; Verhelst et\u00a0al. 1997). This model allows the transformation of a polytomous item into a set of dichotomous items, which facilitates model estimation. The response to a polytomous item is viewed as a set of responses to an ordered sequence of virtual dichotomous items: it is assumed that the respondent is administered virtual items until an incorrect or negative response is given. So, in this approach, an item with M categories labelled m\u00a0=\u00a00,..., M\u00a0\u2212\u00a01, the response is dummy-coded into M\u00a0\u2212\u00a01 dichotomous quasi-items. As an example, for an item with m\u00a0=\u00a03 categories we make two new virtual items. A score of 2 would be coded as correct responses to both virtual items. A score of 1 on the original item would be coded as a correct response to the first virtual item and an incorrect response to the second virtual item. A score of 0 would be coded as an incorrect response to the first virtual item and the second virtual item would be coded as not administered (missing). Now the responses to all virtual items can be modelled by an IRT model for dichotomous items, such as the models given by Eqs. 1, 2 or 3 and can be estimated by any IRT software package that can handle dichotomous items in combination with missing data. There are also IRT packages that estimate models for polytomous items directly (e.g., Multilog; Thissen et\u00a0al. 2003).\nAdvantages of using an IRT framework compared to analysing sum scores\nWe will discuss four advantages of using IRT: (1) it supports construct validity and the scoring rule (e.g., a scoring rule might consist of taking the unweighted sum of symptoms as an estimate of a person\u2019s liability), (2) it supports the use of incomplete item administration designs and handling of missing data, (3) it supports accounting for measurement error, and (4) it can handle floor and ceiling effects.\nAn IRT framework allows one to explicitly model the relationship between item scores and the phenotype of interest. Any combination of items can of course be summed (weighted or unweighted), but this does not guarantee that the sum score reflects a meaningful construct. The meaningfulness of the measurement can be directly assessed in an IRT framework. Fit to an IRT model is empirical evidence that the observed responses can be explained by an underlying structure. The latent variable of the IRT model should, of course, be an appropriate representation of the construct to be measured.\nThe IRT model that fits the data determines the score rule of the measurement instrument. If, for instance, a one-parameter model does not fit the data, but a two-parameter model does, the sum score where the items scores are weighted with their respective discrimination parameters is a sufficient statistic for \u03b8j (Lord and Novick 1968). So some items can be more important or sensitive indicators of a trait than others. Modelling the item data in a variance decomposition analysis allows the separate evaluation of model fit regarding the measurement model and the variance decomposition model.\nIn addition, group differences can be modelled, through differences in means, variances and variance components, and through differences in the way symptoms relate to the latent trait. For instance, one or more symptoms may show a higher incidence rate in one group (indicated by a difference in \u03b2-parameters across groups, e.g., females and males), or be a more sensitive indicator for the trait in a particular group (indicated by a difference in \u03b1-parameters across groups). Such violations of measurement invariance are usually referred to as differential item functioning (DIF).\nA practical advantage of the analysis of data using an IRT framework is the use of incomplete item administration designs and handling of missing data. In some situations, intentionally incomplete item administration designs can greatly improve the efficiency of data collection. With an IRT approach one can also effectively deal with problems specific to longitudinal research where items differ across waves. When using IRT models in a maximum likelihood or a Bayesian framework, it is easy to include individuals that have missing data on one or more items if the data are missing at random (Little and Rubin 1987). When data are not missing at random, the non-randomness can be modelled within an IRT framework by expanding the model with an IRT model that describes the pattern of the missing data (see, for instance, Moustaki and Knott 2000; Moustaki and O\u2019Muircheartaigh 2000; Holman and Glas 2005). The encompassing framework for handling missing data using IRT offers an important advantage over classical test theory. In classical test theory sum scores are only meaningful if the items are the same in all individuals and at all measurement waves.\nThe third advantage of the analysis of data using an IRT framework is that it accounts for measurement error. Unreliability suppresses the correlation between measurements (attenuation). Particularly when using a scale with only a few items, the correlations amongst sum score variables may be grossly attenuated. Clearly, this has important implications for the estimation of variance components in genetic research. In an IRT framework, the problem can be solved by, instead of focussing on sum scores, considering the correlations between latent variables (see, for instance, B\u00e9guin and Glas 2001; Fox and Glas 2003). These so-called latent correlations can be seen as estimates of correlations corrected for attenuation. A simulation study and an application of IRT to real data in a later section will show the possible extent of such attenuation effects on the estimation of heritability.\nThe fourth advantage of IRT has to do with floor and ceiling effects. A problem of analysing sum scores that represent indices of psychopathology is that these scores show a skewed distribution in the general population (Van den Oord et\u00a0al. 2003; Derks et\u00a0al. 2004). These skewed distributions result from the fact that many behavioural phenotypes are assessed using questions that relate to symptoms that are relatively rare in the population. These distributional violations may have important implications for the inference regarding relative variance components when analysing sum scores (Derks et\u00a0al. 2004). In an IRT framework one is essentially free to specify the distribution of the latent trait (in some cases, it can even be estimated). In most cases, with polygenic traits, a normal distribution seems the most reasonable alternative (a mixture approach may be more suitable for traits with only a few large QTL effects). When in turn the variance of the normally distributed latent trait is decomposed into genetic and non-genetic variance, the inference is unbiased if the assumptions of the model are correct.\nVariance decomposition: the one-step and the two-step approach\nIn IRT models, the latent scores \u03b8j are typically assumed to be random draws from a normal distribution. When we are interested in the extent to which individual differences on the latent trait are heritable, we only need to decompose the variance of the \u03b8js using, for example, the classical twin design. There are two approaches. The first approach is to first estimate the parameters of the IRT model using standard IRT software (such as, Bilog, Multilog, Parscale, Testfact, ConQuest, OPLM), and then to have the same software estimate each individual score on the latent trait. Next, one uses these estimates of the \u03b8js as observed values in a standard variance decomposition analysis. This we call the two-step approach.\nThere are several disadvantages to this two-step approach. First of all, in the IRT model fitting phase, the usual IRT estimation software cannot handle the dependency in the data inherent in twin and family designs. In some cases, with simple designs such as with sibling pairs only, weighting of the data would come a long way in solving this problem, but with more complex family designs, weighting is not a satisfactory solution.\nSecond, when estimating latent scores for each individual, the estimates of the \u03b8js, just like sum scores in the CTT tradition, are not simply observations but estimates with error variance. When computing the confidence intervals for the heritability estimates in the second phase, this uncertainty on the latent scores is not taken into account and the heritability confidence intervals are consequently too narrow and the estimates biased downwards. Moreover, in an IRT framework, the confidence intervals for estimates of individual latent scores are dependent on their location on the scale (actually, the number of items with \u03b2-parameters that are similar in magnitude to the person score \u03b8 and the items\u2019 discriminatory power, \u03b1), whereas in the variance decomposition, it is assumed that measurement error (as included in the non-shared environmental variance component) is independent of location (cf. CTT). For example, many psychopathology scales have only items that refer to relatively rare symptoms. As a consequence, many individuals in the general population score 0, which does not necessarily imply that all actually have the trait to the exact same degree. In other words, the scale provides very little information on the trait on the low end. In contrast, the upper end of the scale usually shows more variation, which may imply that the measures are more reliable (more items that discriminate between individuals). Thus, a priori it seems likely that psychopathological scales have more discriminatory power at the upper end of the scale than at the lower end. Of course, this is not a bad thing, since these scales were designed to discriminate between the healthy and the sick. Therefore it seems reasonable to forego the assumption of equal reliability across the scale and take differing reliabilities into account.\nActually, using the two-step approach the heritability coefficient estimate will be about the same as when the analysis is carried out on sum scores. This is because IRT estimates and sum scores correlate highly, well over 0.90 in the case of two-parameter models. When applying a one-parameter model, the correlation will be practically one, because a basic assumption of the Rasch model is that a sum score is a sufficient statistic for the score on the latent trait. Therefore, all persons with the same sum score will get the same estimate on the latent trait. Thus, a third problem of the two-step approach is that it neither solves the attenuation problem, nor the non-normality, nor the ceiling effects.\nIn order to take full advantage of the IRT approach, it is critical to estimate both the measurement model and the variance decomposition model simultaneously, using a one-step approach. However, computationally this is rather challenging. Below, it is shown how this can be done using software for Bayesian estimation procedures. In an application in a later section, we demonstrate the one-step approach for the estimation of heritability with both simulated and empirical data.\nBayesian estimation using a Markov chain Monte Carlo algorithm \nIn twin studies, a widespread method of estimating variance components is through structural equation modelling (SEM). For continuous traits with normal distributions, this is a flexible approach in that it is able to accommodate all linear models and allows for testing of equality of means, variances, covariances and variance components across subpopulations. However, with more elaborate models with discrete or categorical observed variables, SEM maximum likelihood (ML) estimation or ML procedures for estimating generalised linear mixed models such as GLAMM (Rabe-Hesketh and Skrondal 2005) soon reach computational boundaries. An alternative method is Bayesian statistical modelling with Markov chain Monte Carlo (MCMC) estimation algorithms (see also Eaves et\u00a0al. 2005).\nIn the Bayesian approach, inference is based on the posterior density of the model parameters, P(\u03b7|Y), where \u03b7 represents the vector of model parameters and Y the observed data. By Bayes\u2019 rule, the density P(\u03b7|Y) is proportional to the product of the likelihood of the data given the model parameters P(Y|\u03b7) and the marginal density for \u03b7, P(\u03b7), that is,\nThe marginal distribution of \u03b7 is termed the prior distribution (prior in the sense of before the data have been taken into account), and must be specified by the user. The model provides us with the likelihood function P(Y|\u03b7), and hence the posterior distribution of \u03b7 is determined (posterior in the sense of after the data have been taken into account). The posterior distribution is a description of the probabilities of possible values for \u03b7 given the observed data and forms the basis for statistical inference. We may, for example, take the mean or the median of this distribution as our point estimate for \u03b7. Further, the interval between the 2.5th and the 97.5th percentile of the posterior distribution provides the so-called central 95% credibility region, which is analogous to a 95% confidence interval in the ML framework. For more on Bayesian statistics, the reader is referred to the introductions by Box and Tiao (1973) and Gelman et\u00a0al. (2004).\nSometimes it is easy to compute the posterior distribution analytically, but very often this is not possible. One can then use computer simulation to draw a sample of \u03b7-values from the posterior distribution. The mean or median of the posterior distribution can then be approximated by the mean or median of the sampled \u03b7-values, and approximate credibility regions can be determined in a similar way. In practice, the joint posterior distribution of all model parameters is usually quite complicated. Therefore, the complete set of parameters is split up into a number of subsets in such a way that the conditional posterior distribution of each subset given all other parameters has a tractable form and can be easily sampled from. This approach is known as Gibbs sampling (Geman and Geman 1984; Gelfand et\u00a0al. 1990; Gelman et\u00a0al. 2004), which is a special case of an MCMC algorithm. When however the conditional posterior distribution of a subset of the parameters is not easy or even impossible to sample from directly, other MCMC algorithms can be used, where one samples from a similar proposal distribution and uses a decision rule to either accept or reject a sample so that the accepted values can be regarded drawings from the target distribution.\nIn each iteration of an MCMC algorithm, a sample is taken from each conditional posterior distribution for each subset of the parameter space, given the current values of the other parameters. After a number of so-called \u2018\u2018burn-in\u2019\u2019 iterations, necessary for a chain to achieve stationarity (i.e., approaching the target distribution: the joint posterior distribution) sufficiently closely, the subsequent draws can be regarded as sampled from the joint posterior distribution.\nThe application of the Bayesian approach with MCMC sampling to IRT models is mainly motivated by the fact that IRT models with complex dependency structures require the evaluation of multiple integrals to solve the estimation equations in a likelihood-based framework. This problem is avoided in an MCMC framework. In recent years, the fully Bayesian approach has been adopted to the estimation of IRT models with multiple raters, multiple item types, missing data (Patz and Junker 1999a, b), testlet structures (Bradlow et\u00a0al. 1999, Wainer et\u00a0al. 2000), latent classes (Hoijtink and Molenaar 1997), models with a multi-level structure on the ability parameters (Fox and Glas 2001, 2003) and the item parameters (Janssen et\u00a0al. 2000), and multidimensional IRT models (B\u00e9guin and Glas 2001). In behaviour genetics, the approach has been taken up by Eaves and his co-workers (Eaves et\u00a0al, 2005; Eaves et\u00a0al. 2004).\nIn IRT research, the Gibbs sampler is used in two versions: a version with a normal ogive representation such as in Eq. 1, introduced by Albert (1992), and a version with a logistic representation introduced by Patz and Junker (1999a). Below, a logistic version will be used for simulated and real data, implemented in the freely obtainable MCMC software package WinBUGS (http:\/\/www.mrc-bsu.cam.ac.uk\/bugs\/).\nGenetic models may be specified in WinBUGS as follows. Under the assumption that an ACE variance decomposition model (additive genetic, shared environmental and non-shared environmental effects) is appropriate for a latent trait \u03b8, the model can be parameterised as a linear random effects model (see also Van den Berg et\u00a0al. 2006a):\nwhere ck denotes the environmental effect for being a member of family k, and ejk denotes the environmental effect of being individual j in family k. The genetic component is split into a1 and a2 to model the different genetic correlations amongst monozygotic (MZ) and dizygotic (DZ) twins (cf. Jinks and Fulker 1970). The genetic correlation in MZ twins is usually assumed 1.0 and in DZ twins 0.5, in other words, the genetic covariance in MZ twins is twice as large as in DZ twins. Therefore, if we let the random effect a1 be constant within all families and we let a2 vary within families only for DZ twins (but be constant for MZ twins), and then fix the variances of a1 and a2 to be equal, the genetic covariance in MZ twins will be twice as large as in DZ twins. The variance of a1 and a2 together, VAR(a1)\u00a0+\u00a0VAR(a2)\u00a0=\u00a02 * VAR(a1) can then be interpreted as the variance due to additive genetic effects. We assume that a1\u00a0\u223c\u00a0N(0, \u00bd \u03c32a), a2\u00a0\u223c\u00a0N(0, \u00bd \u03c32a), c\u00a0\u223c\u00a0N(0, \u03c32c), and e\u00a0\u223c\u00a0N(0, \u03c32e).\nThe case for the ADE model can be derived similarly (Van den Berg et\u00a0al. 2006a). For some estimation problems, it might be computationally more convenient to model sum and differences scores, instead of the latent scores for the twins separately (Van den Berg et\u00a0al. 2006a; Robert and Casella 2004, p. 396; cf. Boomsma and Molenaar 1986).\nSimulation\nTo illustrate the effect of attenuation on heritability estimates, 101 datasets were generated consisting of 400 MZ twin pairs and 600 DZ twin pairs. A standard normally distributed latent trait was simulated with an additive genetic component of 72% and a non-shared environmental component of 28%. The 1PL IRT model was used to simulate responses to 14 dichotomous items, where the \u03b2 parameter values ranged from 0.5 to 3.5, with increments of 0.25. This corresponds to questionnaire items that are rarely endorsed by people. The simulated item data were fitted using a model with additive genetic and non-shared environmental effects (AE model) on a latent trait and a 1PL measurement model.\nNext, sum scores were computed and these were analysed with an AE model. Since the distribution of the sum scores is positively skewed, the AE analysis was also performed after a logarithmic transformation of the sum scores.\nThe simulations were carried out using the software package . For each replicated data set, we computed the twin correlations for the latent scores, the twin correlations of the sum scores and the twin correlations for the log-transformed sum scores. The three types of analyses were carried out in WinBUGS. After a burn-in phase of 1000 iterations, the characterisation of the posterior distribution for the model parameters was based on 1000 iterations from 2 independent Markov chains. From each of the 3 (analyses) * 101 (replicated data sets) marginal posterior distributions for the heritability we took the mean and the median as point estimates.\nFurther simulations were carried out to illustrate the attenuation effect and the bias in variance components. For simple genetic models, the twin correlations are sufficient statistics for the variance decomposition. Therefore it is enough to show how correlations based on sum scores behave as a function of number of items and beta parameters. Data were simulated using bivariate normally distributed latent values, with correlations 0.9, 0.7, 0.5, 0.3 and 0.1. These latent values were used to simulate corresponding sum scores using a one-parameter logistic IRT measurement model under a variety of conditions. First of all, we used different degrees of discrimination of the items (i.e., the variance of the latent trait: 0.676, 1 and 100). Second, we varied the way in which the items are distributed across the scale, either evenly scattered so that sum score distributions are symmetrical, or only scattered on the upper half part of the scale, that is, using only items that less than 50% of the population endorses, which results in positively skewed sum score distributions (cf. Derks et\u00a0al. 2004; van den Oord et\u00a0al. 2003). Third, we varied the number of items (5, 10, 20, 50, 100) to investigate attenuation.\nSimulation results\nTaking the median parameter values from the 101 data sets, the simulated latent data correlated 0.72 in MZ twins and 0.36 in DZ twins, just as would be expected. The sum scores correlated 0.45 in MZ twins and 0.21 in DZ twins (medians of the 101 data sets) and the log-transformed sum scores correlated 0.41 and 0.20, respectively. Thus, twin correlations are severely attenuated when analysing sum scores, even with 14 items.\nAnalysing the simulated item data with a 1PL IRT model, using the one-step approach, we recovered the true 72% value for the heritability coefficient closely (see Table\u00a01). When analysing the raw sum scores using a normal AE model, either with or without transformation, the heritability point estimate dropped considerably, to about 42%. Thus, when the true model is an IRT model and the number of items is limited, an analysis of raw or transformed sum scores can lead to extensive underestimation of heritability.\nTable\u00a01Simulation results. Reported heritability values are the medians of the 101 posterior means and medians, standard deviations between parenthesesMethod of analysisHeritability coefficient point estimatesPosterior meanPosterior median1PL IRT model0.7232 (0.0585)0.7245 (0.0589)Sum scores continuous model0.4364 (0.0393)0.4369 (0.0395)Log-transformed sum scores0.4046 (0.0403)0.4047 (0.0406)\nFor each condition of latent correlation, number of items, and variance of the latent variable, we simulated 100,000 twin pairs and correlated their sum scores. Figure\u00a01A shows the result for the condition where the variance was 1 and the items were nicely scattered across the distribution of the latent values, between \u22122\u00bd and 2\u00bd times the standard deviation (1). The attenuation effect is clearly dependent on the number of items: with 100 items, the correlation on the basis of the sum scores is very close to the true correlations. An analysis treating the sum scores as bivariately normal and applying a variance decomposition will approximate the true proportions. Moreover, the degree of the attenuation is proportional to the true correlation: with 5 items, a true correlation of 0.9 will be attenuated to a correlation of 0.55 (61%) and a true correlation of 0.1 will be attenuated to a correlation of 0.06 (60%). Therefore, when the analysis on 5 items is based on the sum score, and the true MZ correlation equals twice the DZ correlation, this ratio is maintained when analysing sum scores. Thus, when applying an AE model, heritability will be underestimated, but no artifactual shared environmental effects or dominance genetic effects will appear as a result of analysing sum scores.\nFig\u00a01Correlations of simulated sum scores as a function of true correlation at the latent level, variance of the latent trait (quality of the scale), and scatter of the item \u03b2 parameters (entire scale or only top half, i.e., all\u00a0>\u00a00)\nFigure\u00a01B shows the result for a scale with slightly worse discrimination: the variance of the trait is now only 0.767. The items are again nicely scattered, between \u22122\u00bd SD (\u22122.05) and 2\u00bd SD (2.05). Thus, we retain the spread of the \u03b2 values in terms of the SD, so that the expected proportion of individuals scoring a particular number of items remains equal across simulation situation; the resultant distribution of the sum scores will be equal. But now, due to the decreased sensitivity of the scale, the number of items has a more pronounced effect on the attenuation. The sum score correlations are now lower than under the model with variance\u00a0=\u00a01. However, the attenuation effect is still proportional to the true correlations.\nFigure\u00a01C shows an extreme situation where the items have high discriminatory power. The variance is now 100, and the items are evenly scattered between \u221225 and 25. Note that again, we retain the scatter of the beta values in terms of the SD, and again the sum score distribution will not be different from the earlier simulations. However, with such a sensitive scale, practically everybody that scores less than 1 SD below the mean will show a sum score of 16% of the total number of items. Everybody with a latent score higher than 1 SD below the mean will show a sum score of 84% of the number of items. Moreover, the data will show a scalogram pattern, for example with 3 items with increasing difficulty, the only observed patterns will be 111, 110, 100 and 000. Such a pattern will not be observed when the variance is 1, and even less so with a variance of 0.767: more individuals will then show patterns like 101 and 011, etc. Again, attenuation occurs when the number of items is limited, but the effect is much less pronounced, and again the attenuation is proportional across the different correlations. In this situation, an analysis of sum scores will yield reasonable estimates for the variance components given a sufficient number of items.\nActually, when the raw item data follow the scalogram pattern, the true correlations and the corresponding variance components will be recovered when applying a threshold model (Lynch and Walsh 1998). This is also true when the data follow a scalogram pattern but the items are not evenly scattered across the scale and the sum score distribution is skewed: applying a threshold model will recover the true correlations (cf. Derks et\u00a0al. 2004). However, when applying an ordinary variance component analysis, ignoring its non-normality will yield biased estimates, underestimating the effects of shared environment and overestimating the effects of dominance (cf. Derks et\u00a0al. 2004). This because when the items are not evenly scattered and the sum score distribution is skewed, the attenuation effect is no longer proportional to the true correlations (Fig.\u00a01D): small correlations are more severely attenuated than large correlations. In case the true DZ correlation equals half the true MZ correlation, DZ:MZ\u00a0=\u00a01:2, the correlations of the sum scores will show a smaller ratio, DZ:MZ\u00a0<\u00a01:2, usually an indication of dominance genetic effects or epistasis. This is hard to see from the Fig.\u00a01D, but with 5 items, the simulated sum score correlation is 0.83667 when the true correlation is 0.9 (92.96%), 0.42429 when the true correlation is 0.5 (84.86%), and 0.076575 when the true correlation is 0.1 (76.58%). Suppose we could analyse the true correlations, 0.9 and 0.5. One would then conclude that additive genetic variance accounts for 80% of the variance, non-shared environmental effects 10% and the shared environmental effects for the remaining 10%. Now if we would base our analysis on the observed sum score correlations 0.84 and 0.42, we would conclude that there are no shared environmental effects. One can imagine that when the true correlations are 0.90 and 0.45 one would conclude dominance effects to be absent, whereas if one would analyse observed sum scores correlations, one would find evidence for dominance genetic variance, the extent of which is dependent on the number of items.\nNow, scalogram pattern data that fit a Guttman scale model are extremely rare. More often, item data follow a pattern that can be explained by the more lenient IRT model. Figure\u00a01E shows the attenuation effect when the true model is a one-parameter IRT model with variance 1, where all items are endorsed by fewer than half the participants (i.e., all \u03b2 parameters larger than the average latent score). Again we see that under the usual IRT model, the attenuation effect depends on the number of items and again we see that due to the skewness of the sum score distribution, the attenuation is not proportional to the true correlation. For example, with five items the simulated sum score correlation equals 0.591256 for true correlation 0.9 (66%), 0.316222 for true correlation 0.5 (63%), and 0.056567 for true correlation 0.1 (57%). When true correlations are again 0.9 and 0.5, the most likely model would be, when based on an analysis of the sum scores with 5 items, 5% dominance genetic variance, 61% additive genetic variance and 34% non-shared environmental variance.\nThus, also under the IRT model, analysing sum scores leads to an underestimation of shared environmental effects and an overestimation of dominance genetic effects when the sum score distributions are skewed.\nAn application\nWe illustrate the decomposition of variance using an IRT measurement model with data from the Netherlands Twin Registry (NTR; Boomsma et\u00a0al. 2002b). Attention problems were measured with the Young Adult Self-Report (YASR; Achenbach 1997). We used data collected in the year 2000 from 460 males and 966 females from MZ twin pairs, 288 males from DZ same-sex twin pairs, 561 females from DZ same-sex twin pairs, and 305 males and 441 females from opposite sex twin pairs. All twins were between 18 and 30\u00a0years (inclusive). All available data were used, including data from incomplete pairs and individuals with several items missing. It was assumed that data were missing at random (cf. Van den Berg et\u00a0al. 2006c).\nThe attention problems (AP) subscale of the YASR consists of seven items (see Table\u00a02) with three ordered response categories (0\u00a0=\u00a0Not true, 1\u00a0=\u00a0Somewhat or Sometimes True, 2\u00a0=\u00a0Very True or Often True). In children, sum scores typically show a high heritability with a significant non-additive genetic component (Rietveld et\u00a0al. 2004). In young adults, AP sum scores also showed heritability (40%), but no non-additive genetic component (Van den Berg et\u00a0al. 2006c).\nTable\u00a02Items of the attention problems subscale of the young adult self-report (YASR; Achenbach 1997)ItemDescription1I act too young for my age2I have trouble concentrating or paying attention3I daydream a lot 4My school work or job performance is poor5I am too dependent on others 6I fail to finish things I should do7My behaviour is irresponsible\nHere, we estimate A and E variance components using a 1PL measurement model. A main effect of sex, \u03b4, was modelled on the latent trais. The seven original items with three response categories were transformed into 14 dichotomous dummy items for each individual as described above. A separate \u03b2-parameter was estimated for each dummy item, so that for each original item there are two \u03b2-parameters. For the variance components, locally non-informative (\u2018\u2018flat\u2019\u2019) inverse gamma priors were used, and for the \u03b2 and \u03b4 parameters we used locally non-informative normal priors. The parameterisation modelled the variances of sum and differences scores for the latent trait (Van den Berg et\u00a0al. 2006a). The appendix gives the WinBUGS script. Three independent MCMC chains were used with randomised starting values. The chains converged rapidly to the stationary distribution with relatively low autocorrelations. The first 1000 iterations were discarded as burn-in samples, and a further 1000 iterations were used for inference.\nResults \nTable\u00a03 gives the descriptives of the marginal posterior distribution of the parameter values. The estimate for heritability based on the mean of the posterior distribution is 73%. The main effect of sex on the latent trait, with females scoring higher than males, is just significant, as zero is not included in the central 95% credibility region. Values of the \u03b2-parameters are all around zero or positive, indicating that the AP scale is most sensitive for individuals with considerable attention problems but has a hard time discriminating individuals with relatively few problems with attention. This results in the severely skewed distributions of sum scores.\nTable\u00a03Descriptives of marginal posterior distributions for the AE variance decomposition model using the 1PL IRT model for polytomous items with a main effect for sexParameterMeanSD2\u00bdth percentileMedian97\u00bdth percentile\u03c32a0.840.070.710.840.99\u03c32e0.320.060.200.320.44\u03b4\u22120.130.05\u22120.24\u22120.13\u22120.02\u03b2110.250.050.150.250.34\u03b2122.760.102.562.762.96\u03b221\u22120.760.05\u22120.86\u22120.76\u22120.66\u03b2222.440.082.302.452.60\u03b231\u22120.430.05\u22120.53\u22120.43\u22120.33\u03b2321.840.081.711.841.98\u03b2411.900.061.781.902.02\u03b2423.960.203.583.964.36\u03b2510.220.050.130.220.32\u03b2523.030.102.833.023.23\u03b2610.620.050.530.620.73\u03b2623.900.153.633.904.19\u03b2712.570.072.442.572.71\u03b2724.610.314.054.605.27h20.730.050.630.720.82Note: First index of the betas refers to the item (see Table\u00a01) and the second to the threshold\nThe estimate for the heritability (73%) is much larger than the one reported earlier based on sum scores (40%, Van den Berg et\u00a0al. 2006c). In the current sample, twin correlations for sum scores are very much like those reported earlier (MZ:0.45, DZ:0.17). By applying an IRT measurement model the twin correlation estimates for the latent trait are much higher, 0.76 for MZ twins and 0.30 for DZ twins. For comparison, when using a two-step approach, first estimating IRT model parameters in Multilog and then estimating latent scores for each individual (correlation between sum score and IRT estimate: 0.98), the results showed twin correlations nearly identical to those based on sum scores.\nThe 1PL IRT measurement model could easily be extended to include discrimination parameters (\u2018\u2018factor loadings\u2019\u2019). It is most convenient to constrain these to be positive through the specification of lognormal priors where for instance \u03b1\u00a0=\u00a0exp(\u03b3) and \u03b3\u00a0\u223c\u00a0N(0, 100). In this case, the heritability estimate was not affected by this extension of the model (results not shown).\nDiscussion\nWe have compared an IRT model with a sum score approach with indirectly measured phenotypes. Under a range of conditions, the IRT framework is to be preferred over using sum scores. For example, in longitudinal studies with data missing by design or changing measurement instruments, when some items in a questionnaire change across birth cohorts or across different ages or when item data are missing, a sum score approach may no longer be appropriate, but in many cases the analysis can still be meaningfully carried out in an IRT framework using parameter expansion (see, for instance, Glas 1998).\nWhen a simple IRT model does not fit the data, one could consider deleting or changing bad fitting items, and\/or deleting bad fitting persons. Alternatively, one could consider using more general IRT models that offer many possibilities of obtaining model fit. General frameworks for multi-level and multi-dimensional IRT models are outlined in Skrondal and Rabe-Hesketh (2004) and De Boeck and Wilson (2004). In the specific context of genetic modelling, it might also occur that a particular subset of items show relatively high genetic correlations compared to the remaining items. In that case a more appropriate model would be an independent pathway model for categorical or ordinal traits (see for instance Van den Berg et\u00a0al. 2006b).\nGood fit to a one-dimensional IRT model is empirical evidence that the observed item responses can be explained by one continuous underlying trait. When it further can be concluded that the scale is meaningful (based on item analysis and association with external measures to assess its validity), and the assumption of measurement invariance across different subpopulations is tenable (Lubke et\u00a0al. 2004), the approach effectively deals with non-normal distributions of sum scores in for instance psychopathology (Van den Oord et\u00a0al. 2003).\nMoreover, when the measurement model and the variance decomposition model are estimated simultaneously, the variance decomposition deals appropriately with the dependency in the data when estimating IRT model parameters and testing the model\u2019s assumptions, and the IRT measurement model deals appropriately with the estimation of the heritability coefficient (correcting for attenuation to obtain an unbiased point estimate) and the reporting of the confidence intervals (correcting for location-dependent uncertainty of person scores on the latent trait).\nOur simulations showed the dramatic extent of the attenuation effect and the bias in estimating variance components due to imperfect measurement. Particularly when sum score distributions are skewed, underestimation of shared environmental effects and overestimation of dominance genetic effects may occur. The bias in variance components was also illustrated with an empirical data set: instead of finding a heritability estimate of 40% for attention problems with a sum score (Van den Berg et\u00a0al. 2006c), a heritability estimate of 73% was obtained when including a measurement model and estimating it simultaneously with the variance decomposition model. This example provides an additional illustration of the bias in variance components due to the analysis of sum scores. However, it should be noted that model fit was not assessed, nor was the assumption of measurement invariance tested. This requires further study.\nThe crucial element of the one-step approach that leads to unbiased point estimates is the inclusion of the appropriate probabilistic measurement model so that the estimation takes into account the unreliability of the measurement. The probabilistic modelling allows for the fact that twins with identical response patterns may have different scores on the latent trait, and also, that twins with non-identical response patterns may have exactly the same score on the latent trait. Discriminatory power of the items and the number of items are both crucial to the heritability estimated based on sum scores: the fewer the items and the worse the discrimination of the items (i.e., the smaller the variance of the latent trait in the one-parameter model; the smaller the factor loadings in the two-parameter model), the more biased the estimation will be when the analysis is performed on sum scores. High quality scales with a large number of items (say, more than 50) with high discriminatory power that are scattered across the entire scale can indeed be analysed with sum scores, but any other scale should be analysed using the IRT framework if one is interested in an unbiased heritability estimate with trustworthy confidence intervals.\nFuture work should focus on the assessment of model fit in the context of genetic models. It is only sensible to apply a one-step IRT approach when the data actually conform to an IRT measurement model. If data do not fit an IRT model, for instance when there is differential item functioning across subpopulations, the approach will still lead to biased estimates. A crucial first step therefore is assessing model fit and checking measurement invariance.","keyphrases":["measurement","sum scores","item response theory","mcmc","attention problems","bayesian statistics"],"prmu":["P","P","P","P","P","P"]}
{"id":"J_Comput_Aided_Mol_Des-4-1-2311385","title":"Recommendations for evaluation of computational methods\n","text":"The field of computational chemistry, particularly as applied to drug design, has become increasingly important in terms of the practical application of predictive modeling to pharmaceutical research and development. Tools for exploiting protein structures or sets of ligands known to bind particular targets can be used for binding-mode prediction, virtual screening, and prediction of activity. A serious weakness within the field is a lack of standards with respect to quantitative evaluation of methods, data set preparation, and data set sharing. Our goal should be to report new methods or comparative evaluations of methods in a manner that supports decision making for practical applications. Here we propose a modest beginning, with recommendations for requirements on statistical reporting, requirements for data sharing, and best practices for benchmark preparation and usage.\nIntroduction\nThe field of computational chemistry, particularly as applied to drug design, has become increasingly important in terms of the practical application of predictive modeling to pharmaceutical research and development. Tools for exploiting protein structures or sets of ligands known to bind particular targets can be used for binding-mode prediction, virtual screening, and quantitative prediction of activity. A serious weakness within the field is a lack of standards with respect to statistical evaluation of methods, data set preparation, and data set sharing. Our goal should be to report new methods or comparative evaluations of methods in a manner that supports decision making for practical applications. In this editorial, we propose a modest beginning, with recommendations for requirements on statistical reporting, requirements for data sharing, and best practices for benchmark preparation and usage.\nThere are two fundamental premises in making such a proposal. First, we must believe that the goal of reporting new methods or evaluations of existing methods is to communicate the likely real-world performance of the methods in practical application to the problems they are intended to solve. Ideally, the specific relationship between methodological advances and performance benefits will be clear in such reports. Second, we must understand that the utility of the methods of broad utility in pharmaceutical research application are predicting things that are not known at the time that the methods are applied. While this seems elementary, a substantial proportion of recent reports within the field run afoul of this observation in both subtle and unsubtle ways. Rejection of the first premise can reduce scientific reports to advertisements. Rejection (or just misunderstanding) the second premise can distort any conclusions as to practical utility.\nThis special issue of the Journal of Computer-Aided Molecular Design includes eleven papers, each of which makes a detailed study of at least one aspect of methodological evaluation [1\u201311]. The papers collected within this issue make the detailed case for the recommendations that follow; the recommendations are intended to provide guidance to editorial boards and reviewers of work submitted for publication in our field. In surveying the eleven papers, we feel there are three main areas of concern: data sharing, preparation of datasets, and reporting of results. Concerns within each area relate to three main subfields of molecule modeling, i.e. virtual screening, pose prediction, and affinity estimation, and to whether protein structural information is used or not. We describe the issues in each area and then present recommendations drawn from the papers herein.\nData sharing\nThe issues\nReports of new methods or evaluations of existing methods must include a commitment by the authors to make data publicly available except in cases where proprietary considerations prevent sharing. While the details are different across the spectrum of methods, the principle is the same: that sharing data promotes advancement of the field by ensuring study reproducibility and enhancing investigators\u2019 ability to directly compare methods. However, the details of this matter a great deal, both for docking methods and for ligand-based methods. Docking will be used to briefly illustrate the problem. Many reports make claims of sharing data by, for example, providing a list of PDB codes for a set of protein\u2013ligand complexes used in evaluating docking accuracy. In a very narrow sense, this might accommodate a notion of sharing. However, this is inadequate for four reasons:PDB structures do not contain all proton positions for proteins or ligands. Many docking approaches require all atoms, and nearly all require at least the positions of the polar protons. Without the precise protein structures used, in a widely used file format, it is not possible to reproduce the results of a report or make comparisons of other methods to those reported [7, 9, 11].Ligands within PDB structures do not contain bond order information and often do not even contain atom connectivity at all. Lacking this information, it is not possible to know what protonation state or tautomeric state was used to produce a particular result [4, 7\u20139].Docking methods have different sensitivities to input ligand geometries, both with respect to absolute pose and with respect to other aspects such as conformational strain and ring conformations. Since docking methods do not search ligand pose space exhaustively, absence of precise input ligand structures produces the same issue of reproduction and comparison as in (1) [4, 7\u20139].Different methods of protein structure preparation can yield subtle biases to different types of docking and scoring approaches. Very small changes in heavy atom or proton positions, as come with various relaxation strategies, can yield large changes in the positions of extrema for scoring functions. Provision of coordinates for all atoms allows other investigators to understand and differentiate the effects of methodology from the effects of protein structure preparation [4, 7\u20139].\nRecommendations on data sharing\nAuthors of reports on methodological advances or methods comparisons must provide usable primary data so that their results may be properly replicated and assessed by independent groups. By usable we mean in routinely parsable formats that include all atomic coordinates for proteins and ligands used as input to the methods subject to study. The commitment to share data should be made at the time of manuscript submission.\nExceptions to this should only be made in cases where proprietary data sets are involved for a valid scientific purpose. The defense of such an exception should take the form of a parallel analysis of publicly available data in the report in order to show that the proprietary data were required to make the salient points [8].\nPreparation of datasets\nThe issues\nAs stated earlier, the ultimate goal is predictions of things that we do not alreadyknow. For retrospective studies to be of value, the central issue is the relationship between the information available to a method (the input) to the information to be predicted (the output). If knowledge of the input creeps into the output either actively or passively, nominal test results may overestimate performance. Also, if the relationship between input and output in a test data set does not accurately reflect, in character or difficulty, the operational application of the method to be tested, the nominal reported performance might be unrelated to real world performance. Here, we will briefly frame the issue by discussing the differences between the operational use of methods and the construction of tests to measure and document their effectiveness for both protein structure, e.g. docking, and ligand-based methods in their areas of application.\nDocking\nPose prediction. Here the goal is to prove that a method can predict how a ligand may bind, but not whether it can bind. In the operational case, we typically have a protein structure in complex with a ligand (or several such examples). We desire accurate prediction of poses for novel ligands that are potentially quite different from those whose bound structures are known. For method evaluation, the construction of prediction tests varies, but there are two basic forms:\nCognate docking. The most common test of pose prediction involves a set of protein structures, each bound to a ligand, and with that ligand being the one to be tested. This represents the easiest form of the problem, since the conformation of the protein contains information pertinent to recovering the correct pose of the ligand. Most commonly, the protein coordinates are used as provided experimentally, with some variation in addition of protons, with the ligand in a randomized starting pose. Examples of information \u2018leak\u2019 include using of the cognate ligand pose as input [7], adding protons to the protein to favor the cognate pose [7, 9], choosing tautomer or charge states based on knowledge of the bound structure [8], and inappropriate use of bridging water molecules [9]. An extreme example would be optimizing the protein\u2013ligand complex under the same scoring function used for docking, and then using this new, non-crystallographic information as the \u201ctest\u201d data [7].Cross docking. The less common (but more relevant) formulation employs a protein structure with a bound ligand, but where the ligands to be predicted are different. The issue of similarity between the known ligand and the ligand being tested should be raised, but this is certainly more realistic, since the potential protein rearrangement from the apo form has been partially embedded in the structure but not optimized for each test ligand [7, 8].\nVirtual screening. Predicting whether a ligand will bind, but not its affinity or its pose. In an operational application, we typically have a protein structure (or several), and we may have a few ligands known to bind a site of interest. The goal is to find novel ligands from some library of compounds. Operationally, we do not have the bound structures of the ligands we are trying to find, nor do we generally have a specific protein structure in which we are guaranteed a hospitable geometry. Many of the same mistakes that can be made with pose prediction can also be made to prefer known ligands over decoys, but there are additional hazards:\nThe decoys do not form an adequate background [5\u20138, 10]. One of the frustrations in evaluating a study is how to judge the background against which a method is framed. It is very easy to generate a set of decoys that any method can tell apart from actives, and much more difficult to construct an informative collection.All the actives are chemically similar [2, 4, 5, 8, 10]. This is more relevant to ligand-based methods, but also applicable to docking because operationally finding chemically similar molecules as being potentially active is of little value in that these will likely be found by other methods.\nScoring. Prediction of affinity is the hardest problem for molecular modeling and is as yet unsolved. In the operational case, we typically have multiple protein structures with ligands and may also have a wealth of structure-activity data for multiple ligand series. Frequently the problem here is accurately predicting the activity of what may be considered an obvious analog in virtual screening. We do not know the precise bound geometry of the specific ligand whose activity we are predicting.\nAffinity prediction tests can be done absent any affinity data on related analogs. However, to date, successful predictions without prior affinity information have been so anecdotal and untransferable that the field seems willing to accept any input of prior structural information. Hence, inclusion of information as to the protein\u2019s disposition upon binding that is not available in an operational setting is considered acceptable.More typically, structural information and the activities of one or more closely related analogs are available. Here there are fairly regular reports of success, if given complete structural information. Chemical similarity is assumed, thus placing this technique in the domain of lead optimization, not lead discovery. As illustrated in at least one of the reports here [3], such methods are not currently successful when properly considered with control computations that include, for example, correlations of affinity with molecular weight.\nLigand-based modeling\nPose prediction. This is rarer than the use of ligand information in virtual screening but not operationally uncommon. The goal is to find the alignment of ligands to a protein using one or more known protein\u2013ligand complexes. If the known and predicted ligands are one and the same, then issues from cognate ligand apply, for instance using torsions from the crystal structure, rather than deriving such information. If the known and test ligands are different, then the caveats from cross-docking apply, for instance are the test ligands diverse enough to make this experiment meaningful.\nVirtual screening. We have some number of ligands known to bind a particular site competitively, or, minimally, a single compound that exhibits a desired activity. The goal is to find novel ligands from some library of compounds. The incremental value of obvious analogs of known ligands is small as such would typically be found from SAR expansion from the known active (and is relevant in the narrow case of expanding hits after, for example, an HTS screen).\nQuite frequently, test cases are constructed where both the input ligands and testing ligands are all trivial analogs of a common central structure [2, 8, 11]. This stems, in part, from the simple fact that the ligands available for constructing tests are most frequently synthesized as part of a design process in which creating analogs is a useful exercise. However, such test cases do not reflect a key feature of the practical application in lead discovery: ligands that are obvious analogs of existing lead compounds will not exist in libraries to be screened for new leads.The relevant test cases are those in which the ligands to be retrieved are not analogs of the input ligands. This is, to a degree, a subjective issue. However, construction of such cases can be done, for example, by choosing input ligands that were discovered long before the test ligands or by choosing input ligands that have substantially different overall biological properties (e.g. side effects) than the test ligands [2].\nScoring. Predicting affinities of ligands from the affinity of one or more ligands, whether relative or absolute. In practice, we generally have structure-activity data for multiple ligand series. Frequently the problem here is accurately predicting the activity of what would be considered an obvious analog in virtual screening. We do not generally know the bound geometry of the specific ligand whose activity is to be predicted. This methodological area of QSAR has its own set of relatively well-understood foibles and is not addressed in detail in this issue.\nThe descriptions of test case construction above involve different degrees of challenge in proportion to the amount of information provided to a method. The problems often encountered in reviewing or reading papers is that methods claim a lower level of information concerning the answers than is actually true. This is seldom intentional, no matter the provocation to believe otherwise, but a reflection of the difficulty in preparing a \u2018clean\u2019 test.\nRecommendations on dataset preparation\nProtein structure selection and preparation.\nProtein structure selection should take into account more than just the nominal resolution [4, 5, 9]. There are other measures such as coordinate precision that are more appropriate but require the use of structures where an R and Rfree are reported. In addition, checking to see if density actually exists for the poses being predicted is suggested, although this requires structure factors to have been deposited along with protein coordinates.Protein structure optimization must not be done by making use of the known geometry of the ligand that is the subject of a prediction [5, 7]. At most, selection of sensible protonation states, tautomers, and rotamers of ambiguous or underspecified groups should be done one time for each protein structure. Much fuller disclosure of preparation procedures is required than is typically seen.The most relevant tests of methods will employ proteins whose structure was determined with a ligand other than the one being predicted or a close analog thereof [8].The number and diversity of protein targets needs to be sufficient to enable to draw statistically robust conclusions [4, 6, 10, 11]. Some typical targets (e.g. HIV protease) are quite atypical [4] and in small datasets may dominate results [10, 11].\nDecoy set construction. There is clearly a consensus that decoy sets can have a significant impact on results [4\u20138, 10, 11]. The contributed papers here provide no clear consensus as to what constitutes an acceptable set of decoys, although there are lessons as to what not to do, for instance using molecules that might actually be actives, or have unusual properties compared to known actives. At present, the best suggestions seem to be to make decoys relatively \u2018drug-like\u2019, so as to mimic real, i.e. operational screens. We also recommend the practice of employing multiple decoy sets and including those developed by other investigators to facilitate study comparison and collation.\nActive ligand set construction. There is consensus that the degree of \u201cobvious similarity\u201d among actives has important effects, particularly in evaluating ligand-based methods [1, 2, 4, 7], but there is less agreement on how to either measure this or to control for it. Our recommendation is that such effects should be quantified in reports, where possible, by, for example, using 2D similarity methods to provide a baseline for the difficulty of a retrieval task or to provide a numerical characterization of the diversity of active ligand sets [2]. In addition, suggestions are made in this issue to either use only single representatives of a chemical class or to weight each according to its order of discovery [1, 4, 6]. Both ideas seem eminently worth further evaluation.\nLigand preparation. All ligands (whether active or decoys) must be prepared using automated procedures that are unbiased and which will not yield systematic differences between populations of molecules that will generate a systematic performance bias [7\u20139]. For instance, assign protonation states of ligands and decoys by the same protocol, and generate conformations from just connectivity records of both ligands and decoys.\nParameter tuning. Many papers in this issue show how the choice of parameters influences the apparent quality of results [3, 4, 9]. There is a dichotomy of opinion on whether \u201ctuned\u201d performance figures are relevant to future application of a method when the correct answer is unknown. Our recommendation is that if one chooses to report tuned performance, one must also report performance using standard parameters.\nEven within the constraints outlined above, data set preparation and parameter selection can yield a wide range of results. This is acceptable to illuminate which choices are of most benefit to users of the different methods. However, without strong requirements for data sharing (the subject of the previous section), this benefit will be diluted. Further, without baseline requirements for statistical reporting (the subject of the next section), this diversity will lead to an unacceptable degree of incomparability between different reports.\nReporting results\nThe issues\nThe issues surrounding what to report are substantially in dispute, and this has led to an alarming inability to compare multiple studies, except in the case where all primary data are available and where one is willing to make an independent analysis. Here there seem to be two schools of thought. The first is that molecular modeling is a special enterprise, distinct and different from other efforts at prediction. As such it is seen as a part of the process to select or invent measures that illustrate a particular point. The second school holds that molecular modeling is in fact similar to many other areas of science and commerce and that by ignoring standard practices in other, more established, fields, we do a disservice to modeling.Pose prediction. The almost universal measure for pose prediction is RMS, i.e. the root-mean-square difference between heavy atom positions seen in crystallographic refinement and predicted by a method, generally corrected to allow for internal symmetries within the ligand in question [8]. What is at issue is the manner in which RMS is reported. The desire, as with enrichment metrics, is for a single value to capture the performance of a method over a collection of test cases. The most commonly reported is the proportion of successes at some particular threshold of RMS (for instance, an arbitrary 2.0\u00a0\u00c5 RMS), but a number of investigators report average RMS instead. Neither is ideal, but mean RMS is less useful for two reasons. First, it can be skewed by small numbers of poor poses (each with very large RMS) [5]. Second, its magnitude can be directly manipulated by clever choice of poses against which to measure success [5, 7].Virtual screening. In many senses, this is the most disputatious area. The standard measure has been \u201cenrichment\u201d defined to be the ratio of the observed fraction of active compounds in the top few percent of a virtual screen to that expected by random selection. The reason enrichment is so prevalent is that it is synonymous with the purpose of virtual screening: to enable the selection of a subset of compounds with improved chances of drug discovery. However, by nearly all other considerations it is a poor measure. Most regrettable is its dependence on the ratio of actives to inactives, which makes enrichment a property of a method and an experimental set-up rather than an intrinsic property of the method [10]. A number of metrics have been proposed, many of which share this clearly undesirable quality [1, 7, 8, 10].Affinity estimation. Ideally the analysis of a prediction of affinity ought to be the simplest of tasks. Given a set of experimental values and a predicted set, merely calculate the average difference. If modeling could actually predict affinities, this might be a reasonable approach. As it is, the best generally hoped for is a rough correlation between activity and score and even in these cases there are obvious, and not so obvious, pitfalls. However, a rough correlation between activity and score is frequently obtained simply by equating activity with, for example, a monotonic function of molecular weight [3].General. There are other more subtle issues. One is the presentation of results where the answers have fed back to the input (training\/test set contamination). This is generally easy to spot and usually means a method is without merit. More subtle errors tend to be where forms for cross-validation are followed (proper separation into training and test systems), but where the true independence of these two sets is never called into question [10]. If the test set is not sufficiently different to the training set then there is no assurance against over-parameterized approaches. Finally, reports that profess to predict affinities seldom provide some reliable estimate of experimental affinity. The practice of combining results from multiple experiments is only acceptable if experimental conditions are similar. Anecdotal stories abound of different labs within the same company failing to be able to reproduce each other\u2019s binding affinities, often with difference of an order of magnitude or more. It seems sheer folly to think a test set from truly heterogonous sources can be called reliable.\nRecommendations for reporting results\nPose prediction. Success rates using multiple RMS thresholds should be reported. At a minimum we recommend 3.0, 2.0, 1.0, and 0.5\u00a0\u00c5. In fact, we encourage investigators to report full cumulative histograms of RMS performance for both top scoring and best-predicted poses. This will generally take very little additional space in a report, and it provides much more information to the reader. For example, if there are a large proportion of reported RMS values that appear to have greater precision than the experiment, this is detectable by inspection of the histogram [5, 7]. Statistically it is not impossible in a fair prediction for a measurement to be within, say, 0.1\u00a0\u00c5 of an experimental measurement that is only accurate to 0.5\u00a0\u00c5, but it is unlikely. We also suggest that if an estimate of the precision of the experimental coordinates is available that it must be reported. This, then, provides an excellent bulwark against over-fitting to the known results.\nVirtual screening. Based on multiple reports in this issue, we recommend reporting the area under the curve for ROC plots (AUC) [1, 3, 7\u201310]. These have for a long time been a standard metric for other fields and for good reasons. The argument against using AUC values to judge methods is that they are global measures, i.e. reflect the performance throughout a ranked list. Thus, the notion of \u201cearly enrichment\u201d may not be well characterized by just AUC, particularly when virtual screening methods yield AUC values short of the 0.8\u20131.0 range. Therefore we make two suggestions. First, enrichment percentages should be reported at the following four values: 0.5%, 1%, 2%, and 5%. Second, that a formulation of enrichment is used that reports the ratio of true positive rates (the Y axis in an ROC plot) to the false positive rates of 0.5%, 1%, 2%, and 5% (found on the X axis in an ROC plot). Thus \u201cenrichment at 1%\u201d becomes the fraction of actives seen along with the top 1% of known decoys (multiplied by 100). This removes the dependence on the ratio of actives and inactives and directly quantifies early enrichment. It also makes standard statistical analysis of error bars much simpler [10].\nAffinity estimation. First, standard correlation measures must be reported. We recommend Pearson\u2019s correlation (due to its intuitive appeal and ubiquity) as well as Kendall\u2019s Tau (due to its robustness in cases where Pearson\u2019s correlation can yield spurious values). Both are easy to calculate, and errors for both are simple to compute. Second, we recommend that papers claiming a correlation with affinity ought to also present the correlations achieved with simpler measures, to include molecular weight, cLogP, and hydrogen bond donor\/acceptor counts [3, 8]. Thirdly, authors must be held responsible for realistic estimates of the accuracy of experimental affinities, in particular when such results are from heterogeneous sources.\nGeneral. First, if data and dataset preparation are completely disclosed, then the issue of the precise manner of reporting in a paper becomes less vital. Authors may choose to emphasize whatever measures they wish but interested readers should be able to construct alternate measures. Secondly, the most lamentable aspect of reporting in our field is the lack of error bars on reported metrics and of the quantification of statistical significance more generally. This is the single simplest, most effective, and most needed reform that an editor can insist upon and that a reviewer should look for. Multiple papers here suggest approaches that should be applied [1, 5, 7, 10]. There can be no excuse for a paper on a modeling method to be published claiming one method is superior to another without proper statistical validation. Finally, we hold to the aforementioned second school of thought i.e. that molecular modeling should be held to the same standards as other fields. As such, our most general recommendation is to report standard metrics as a requirement and alternates as desired by authors.\nConclusions\nMolecular modeling is a relatively young field. As such, its growing pains include the slow development of standards. Our hope for this special issue of JCAMD is that with the help of the arguments made in the contributed papers, the modest recommendations made here will form the kernel of standards that will help us as a community to both improve the methods we develop and to reduce the disparity between reported performance and operational performance.","keyphrases":["benchmarking","statistical evaluation","docking","molecular similarity"],"prmu":["P","P","P","R"]}
{"id":"Dig_Dis_Sci-3-1-1914222","title":"Alterations in Epithelial and Mesenchymal Intestinal Gene Expression During Doxorubicin-Induced Mucositis in Mice\n","text":"In the current study we aimed to gain insight into epithelial-mesenchymal cross-talk and progenitor compartment modulation during doxorubicin (DOX)-induced mucositis in mice. Intestinal segments were collected on various days after DOX treatment. DOX-induced damage at day 1\u20132 was characterized by increased epithelial proliferation and apoptosis and a decrease in the expression of epithelial differentiation markers. Concurrently, T-cell factor-4 (TCF4) levels increased and the epithelial differentiation enhancing factor, bone morphogenic protein-4 (BMP4), decreased. During severe damage (day 3), BMP4 levels were significantly increased, which inversely correlated with epithelial proliferation. At the same time, the expression of the epithelial differentiation markers was increasing again. At day 7, BMP4 levels were down-regulated, while the levels of the epithelial differentiation markers and TCF4 were normalized again. These data suggest that in response to DOX-induced damage, BMP4 and TCF4 are modulated in such a way that homeostasis of the progenitor compartment is partly preserved.\nIntroduction\nThe highly proliferating small intestine is very susceptible to chemotherapy-induced damage [31, 47]. This side effect, often referred to as mucositis, is very painful and can be life threatening due to the enhanced risk of bacterial translocation caused by loss of epithelial barrier function. Each year, around 500,000 patients worldwide suffer from this side effect [7] for which there is still no definitive prophylaxis or treatment.\nDrug-induced damage to the highly proliferating stem cells and progenitors located in the crypts of Lieberk\u00fchn showed that different classes of cytostatic drugs affect epithelial crypt cells of different topographical and hierarchical status [12, 13, 24]. Methotrexate (MTX), 5-fluorouracil and vincristine for example, induce damage high in the proliferative compartment at crypt position 9\u201311. Damage induced by MTX is well characterized and shows severe morphological changes characterized by epithelial flattening, villus-atrophy, and specific de-differentiation of enterocytes. The last one is indicated by a decreased expression of the enterocyte-specific enzymes but a maintained expression of goblet and Paneth cell-specific genes [29, 38, 41, 45, 46]. Doxorubicin (DOX) is a cytostatic drug that also frequently causes severe mucositis. However, DOX-induced damage is not well characterized [28]. DOX preferentially attacks cells at crypt position 4\u20136, which is just above or at the same level as the intestinal stem cells [12, 32]. DOX is therefore thought to induce more severe damage, especially to the proliferating compartment at the bottom of the crypts, as for example MTX. In clinical practice, DOX is often used in treatment of solid tumors, leukemia, and lymphomas in both adult and childhood cancer patients [16, 18, 21, 28]. Recent studies revealed a dynamic cascade of events leading to chemotherapy-induced mucositis [35]. Not only are the specialized epithelial cells affected by cytostatic-drug treatment, but also the underlying submucosal connective tissue. Under normal physiological circumstances, the epithelium maintains a cross-talk with the mesenchyme. Epithelial-mesenchymal interactions are critical for the normal morphogenesis and maintenance of the crypt-villus axis [15, 23].\nBone morphogenic protein (BMP)- and Wnt-signaling pathways are two of the main pathways involved in embryonic and adult intestinal development [5, 33]. BMPs are morphogenes that constitute a large group of structurally and functionally related proteins of the TGF-beta superfamily. BMP signaling pathways have a key role in organogenesis [2, 37], gastrointestinal development and intestinal homeostasis in adults [8\u201311, 14, 27]. After intestinal development, BMP4 is exclusively expressed in the intravillus and intracrypt mesenchymal cells, including those adjacent to the intestinal stem cells [8, 10]. Paracrine BMP signaling occurs specifically in the villus from the mesenchyme to the adjacent epithelium [8], suggesting involvement in epithelial-mesenchymal interactions as a mesenchymal signaling molecule.\nThe Wnt-signaling pathway also has been implicated in the regulation of the intestinal epithelial proliferation\/differentiation balance in vitro [43]. In the intestine of mice deficient for transcription factor TCF4, the main Wnt pathway transcription factor in the intestinal epithelium, loss of proliferative compartments and epithelial cell differentiation are found [17]. Interestingly, the BMP- and Wnt pathways appear to be linked, as was shown by the fact that BMP signaling suppresses Wnt signaling to ensure a balanced control of stem cell proliferation and subsequent epithelial differentiation [8, 10].\nThe objective of this study was to develop an experimental mucositis mouse model to characterize DOX-induced intestinal damage and subsequent repair. In addition, we aimed to correlate the alterations in morphology, epithelial homeostasis, and gene expression with changes in BMP4 and TCF4 expression. This, in order to gain insight into possible modulation of the epithelial-mesenchymal cross-talk and progenitor compartment during chemotherapy-induced intestinal damage and regeneration.\nMaterials and methods\nAnimals\nAnimal experiments were performed with permission of the Animal Ethics Committee of the Erasmus MC-Sophia. Upon arrival at our institute, 10-week-old male BALB\/c mice (Harlan, Horst, The Netherlands) were housed individually during the whole experiment in micro-isolator cages under specific pathogen-free conditions with free access to a standard palletized diet (Hope farms, Woerden, The Netherlands) and water. After 1\u00a0week of adjustment to the new environment, the mice were divided into three groups and injected intravenously with doxorubicin (DOX) (Doxorubicin, Pharma Chemie, Haarlem, The Netherlands) on two subsequent days. At day \u22121 and 0, the first group of mice was injected with a low dose of DOX of 6 and 4\u00a0mg\/kg (low dose) respectively, a second group was injected with a medium dose of 8 and 5\u00a0mg\/kg (medium dose) and a third group was injected with a high dose of 10 and 6\u00a0mg\/kg (high dose). Controls were given equivalent volumes of 0.9% NaCl. Mice in the low- and high-dose group were sacrificed at day 1, 2, 3 and 7 after the final DOX injection; mice in the medium dose group were only sacrificed at day 3 and 7. One hour before sacrifice, the mice were injected with 120\u00a0\u03bcl 10\u00a0mg\/ml 5- Bromo-2\u2019deoxyUridine (BrdU) (Sigma-Aldrich, Zwijndrecht, The Netherlands), an uridine analog, to locate the proliferating cells. Per time point 4\u20136 DOX-treated animals and 2\u20134 control animals were sacrificed. Segments of mid-jejunum were collected and either processed immediately for histological analyses or snap-frozen in liquid nitrogen for storage at \u221280\u00b0C and subsequent protein isolation.\nHistochemistry\nFive-millimeter segments of mid-jejunum were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), dehydrated and embedded in Paraplast Plus (Sherwood Medical, Den Bosch, The Netherlands) as previously described [45]. Four \u03bcm sections were routinely stained with hematoxylin (Vector Laboratories, Burlingame, CA) and eosin (Sigma-Aldrich) to study morphological alterations of the crypts and villi. Immunohistochemistry was performed as described previously [45] with some minor modifications. The sections for BrdU staining required an extra adjustment to this protocol of HCL incubation, washing with borate buffer, and pepsin treatment as described before [34]. In short, the sections were blocked as described and incubated overnight with the following antibodies diluted in PBS: to visualize BrdU incorporation, mouse monoclonal anti-BrdU (1:250, Roche Applied Sciences, Indianapolis, IN) was used, as an enterocyte marker rabbit polyclonal anti-rat Sucrase-Isomaltase (SI) (1:9000 in PBS, kindly provided by Dr. K.Y. Yeh [49]) was used and as a goblet cell-specific marker rabbit polyclonal anti-rat trefoil factor family (TFF3: 1:3000, kindly provided by Prof. Dr. D.K. Podolsky) was used. Furthermore, BMP expression was visualized with anti-BMP4 (1:100, R&D Systems, Abingdon, UK). Immunoreactions were detected using Vectastain ABC Elite Kit (Vector Laboratories, Burlingame, CA) and 3,3\u2032-diaminobenzidine tetrahydrochloride (Sigma-Aldrich, Zwijndrecht, The Netherlands).\nCrypt and -villus length\nLongitudinal sections of crypts and their corresponding villi were selected so that the base (marked by Paneth cells), middle and top of the crypt were all in the plane of section and thus well orientated. The depth of ten crypts and the length of ten villi were measured on three slides per animal, four animals per time point, with the use of a Nikon Eclipse E800 microscope and IM 500 software.\nProtein dot blotting\nThe expression of enterocyte markers was detected and quantified as described previously [46]. Briefly, 5-mm segments of the mid-jejunum were homogenized and protein concentrations were measured using the BCA Protein Assay Reagent (Pierce, Rockford, IL) and 50\u00a0\u03bcg protein of each homogenate was dot-blotted on nitrocellulose (Protran BA83, 0.2\u00a0\u03bcm; Schleicher & Schuell, Dassel, Germany). Hereafter, blots were blocked for 1\u00a0h with blocking buffer containing 50\u00a0mM Tris, pH 7.8, 5% (wt\/vol) nonfat dry milk powder (Campina Melkunie, Eindhoven, The Netherlands), 2\u00a0mM CaCl2, 0.05% (vol\/vol) Nonidet P40 (BDH, Brunschwig Chemie, Amsterdam, The Netherlands) and 0.01% Antifoam B (Sigma-Aldrich). Blots were incubated overnight at 4\u00b0C with rabbit polyclonal anti-rat SI (1:1000 [49]), and rabbit polyclonal anti-rat trefoil factor family (TFF3: 1:1500) diluted in blocking buffer. After washing with blocking, the buffer blots were incubated with 125I-labeled protein A (specific activity 30\u00a0mCi\/mg, Amersham Biosciences, Roosendaal, The Netherlands) for 2\u00a0h at room temperature. Specific binding of 125I-labeled protein A to the enterocyte marker antibodies was measured using PhosphorImager detection. The elicited signal was quantified by ImageQuant software (Molecular Dynamics, B&L systems, Zoetermeer, The Netherlands) and the expression of TFF3 and SI was expressed per 50\u00a0\u03bcg protein of tissue. Average expression levels of TFF3 and SI in the mid-jejunum were calculated per mouse, followed by calculation of the mean expression of TFF3 and SI per time point studied. Subsequently, the average expression of TFF3 and SI of control mice was set at 100%. The specificity of the above-described antibodies was previously confirmed by Western-blot analysis [46].\nWestern-blot analysis\nThe same protein homogenate was used as described for protein dot blot analysis. Twenty\u00a0\u03bcg of protein was loaded per lane and run on a 12.5% SDS-PAGE. The separated proteins were transferred to nitrocellulose membranes (Protran BA83, 0.2\u00a0\u03bcm) and blocked for 1\u00a0h at room temperature in blocking buffer as described above. The blots were incubated overnight at 4\u00b0C with primary antibodies diluted in blocking buffer: mouse monoclonal anti-human PCNA, clone PC10 (1:250, Novo Castra Laboratories, Newcastle upon Tyne, UK) and mouse monoclonal anti-human BMP4, clone 3H2 (1:100), (Novocastra Laboratories, Newcastle upon Tyne, UK), rabbit polyclonal anti-human cleaved Caspase-3 antibody (1:1000, Cell Signaling, Beverly, MA), and mouse monoclonal anti-human TCF-4, clone 6H5-3 (1:250, Upstate, Waltham, MA). After washing with PBS, 0.2% Tween-20 blots bound antibodies were revealed using HRP conjugated goat anti-mouse or rabbit anti-goat secondary antibodies (1:1000) and SuperSignal West Femto Luminol Enhancer kit (Pierce, Rockford, IL). The signal was detected and quantified by the ChemiGenius gel documentation system (Syngene, Cambridge, UK) and the expression of the specific proteins analyzed was expressed per 20\u00a0\u03bcg protein of tissue. Average expression levels of PCNA, BMP4, TCF4, and caspase-3 in the mid-jejunum were calculated per mouse, followed by calculation of the mean expression of these specific proteins per time point studied. Subsequently, the average expression of PCNA, BMP4, TCF4, and caspase-3 in control mice was set at 100%.\nStatistical analysis\nChanges in protein expression levels during damage and regeneration were statistically analyzed using the Kruskal-Wallis H-test and the Mann-Whitney U-test. A p < 0.05 was considered statistically significant. Data are presented as the mean \u00b1 standard error of the mean (SEM).\nResults\nDose-response analysis of DOX-induced mucositis in an experimental mouse model\nTo optimize the dose of DOX necessary to induce severe intestinal damage (i.e., villus atrophy, crypt loss and flattening of the epithelial cells) a dose-response curve was performed. Thereto, mice were divided in three treatment groups: low dose, medium dose, and high dose (see Materials and methods, section Animals for details). In the high-dose treatment group, two of the six mice died at day 4. Necropsy showed an excess of fluid in the abdominal cavity of unknown source. Because of the elapsed time after death, morphological evaluation could not be performed.\nMorphological analysis of the low-dose treatment group at days 1 and 2 (data not shown), revealed only a slight increase in crypt length, which became more pronounced at day 3 (Fig.\u00a01B). An increase in crypt length was also seen in the medium dose group at day 3. (Note the medium-dose treatment group was not studied at days 1 and 2). No other morphological changes were seen in the low dose- (days 1\u20133) and medium-dose group (day 3). In contrast, in the high-dose treatment group we did see severe morphological damage (Fig.\u00a01D\u2013F). Specifically, severe villus atrophy, crypt lengthening, crypt loss, perturbation of crypt arrangement, flattening of crypt and villus epithelium and inflammation were observed at day 3 (Fig.\u00a01F). Moreover, the latter morphological damage was already seen, although in a milder degree, at days 1 and 2 after the high-dose DOX treatment (Fig.\u00a01D, day 1 and Fig.\u00a01E, day 2). At day 7, the intestinal morphology was completely restored in the low-dose and medium-dose group (data not shown), but was still affected in the high-dose group. As in the latter group, the crypt epithelium still showed signs of regeneration, like new crypt formation and less epithelial flattening (Fig.\u00a01G).\nQuantitative analysis of crypt and villus length\nTo quantify the amount of morphological damage induced by the different doses of DOX, the length of the crypts and villi were measured (Fig.\u00a01H\u2013I). In the high-dose group, but not in the low-dose or medium-dose group, a significant increase in crypt length was already observed at day 2 (data not shown). At day 3 the crypt length was significantly increased in each treatment group compared to the control group (Fig.\u00a01H). By day 7, the length of the crypts had returned to control levels in the low-dose and medium-dose group, but were still increased in the high-dose group. This increase in crypt length, however, showed a trend towards normal levels because the crypt length at day 3 was 41% above control level but at day 7 this was still only 13% above control level. The length of the villi did not change in the low-dose or the medium-dose treatment group on each day investigated. However, in the high-dose treatment group the length of the villi were significantly decreased on days 1\u20133 and 7 (Fig.\u00a01H, day 3 and 1I, day 7). Because severe intestinal mucositis (day 3) and morphological regeneration (day 7) were seen only in the high-dose treatment group, we continued our studies with the high-dose DOX treatment model.\nEffects of high-dose DOX treatment on enterocyte-specific gene expression\nTo gain insight into the functional capacity of the intestinal epithelium after DOX treatment, a sucrase-isomaltase (SI) immunohistochemical staining was performed. SI is an enterocyte-specific disaccharidase responsible for sucrose degradation. SI is expressed in the brush border of differentiated villus enterocytes directly after weaning [42] and is considered an intestinal epithelial differentiation marker. At day 1 and 2 after DOX treatment SI was expressed in the brush border of enterocytes along the entire villi, comparable to the control epithelium (Fig.\u00a02A, control; B, day 1; C, day 2). At day 3, SI staining showed a patchy pattern with weak staining on less affected parts of the epithelium and even absence of staining on severely damaged epithelial parts of the sections (Fig.\u00a02D). At day 7, during the regenerative phase, the SI staining pattern was comparable to control again (Fig.\u00a02E).\nTo quantify SI protein expression, protein dot-blot analyses were performed using SI-specific antibodies (Fig.\u00a02F). The SI protein expression level at day 1 was similar to the control situation. At day 2, SI expression levels were significantly decreased and remained significantly decreased at day 3. At day 7, the SI expression levels regained to control levels.\nEffects of DOX treatment on goblet cell-specific gene expression\nGoblet cell-specific gene expression after high-dose DOX treatment was analyzed by the expression of trefoil factor family 3 (TFF3). TFF3 is a bioactive peptide, produced and secreted by goblet cells, that is involved in epithelial protection and repair [6, 22]. Immunohistochemical staining of TFF3 showed TFF3 expression by goblet cells in crypts and villi of the jejunum (Fig.\u00a03). At day 1, the TFF3 staining was not visibly altered compared to the control situation (Fig.\u00a03B). At day 2, the immunohistochemical detection of TFF3 decreased visibly, especially in the goblet cells localized in the crypts and lower part of the villi where TFF3 protein could hardly be detected\/was absent (Fig.\u00a03C). At day 3, during most severe morphological damage, TFF3 staining by goblet cells in the crypt was reconstituted (Fig.\u00a03D). Day 7 showed complete regeneration of TFF3 (Fig.\u00a03E).\nSubsequently, a protein dot-blot analysis was performed (Fig.\u00a03F) using TFF3-specific antibodies to quantify TFF3 levels. At day 1, the TFF3 expression was already significantly decreased to 50% of control level and maintained at this low level at day 2. At day 3, concomitant with the most severe morphological damage, TFF3 expression returned to control level. TFF3 protein expression was again comparable to control situation at day 7.\nEffect of high-dose DOX treatment on epithelial proliferation\nLocalization of proliferation was studied by immunohistochemical detection of incorporated BrdU (Fig.\u00a04). In controls (Fig.\u00a04A) BrdU was mainly localized in cells from the bottom of the crypt up to three quarter of the crypt length. At day 1 and 2 the proliferative zone broadened and progressively moved upwards to the crypt-villus junction. At day 3, during severe morphological damage, BrdU-positive cells formed a scattered pattern throughout the remaining crypt structures of the small intestine. In the regenerative phase, at day 7 BrdU-positive cells were located in the lower part of the crypts again.Fig. 1Morphology of the murine small intestine after low, medium- or high-dose DOX treatment. Morphology of the jejunum of a control mouse (A), at day 3 after low-dose DOX (B) and at day 3 after medium-dose DOX (C), both were mildly affected by DOX treatment. Morphology of the jejunum at day 1 (D) and day 2 (E) after high-dose DOX treatment, which appeared progressively affected and severely deteriorated at day 3 (E). At day 7, the regenerative phase, the intestinal morphology of the mice in the low- and medium-dose group was completely restored to control situation (data not shown). The morphology at day 7 of the mice treated in the high-dose group was regenerating, showing new crypt formation but still villus atrophy. These photographs are representative examples of a group of four animals per time-point. To quantify the effect of these different doses of DOX treatment, crypt and villus lengths were measured. Crypt and villus lengths of control mice were set at 100%. At day 3 (A), the length of the crypts in all three DOX treatment groups was significantly increased in comparison to the control group. Villus length in the low- and medium-dose group remained at control level. The length of the villi of the high-dose group decreased significantly, indicating significant villus atrophy after high-dose DOX treatment. At day 7 (B) crypt and villus lengths of the low- and medium-dose DOX treatment groups showed complete restoration. The crypts of the high-dose treatment group were still significantly longer, but this increase in crypt length was declining compared to day 3 (day 3 41%, day 7 13%). The length of the villi was still significantly decreased at day 7, showing no signs of regeneration. *P < 0.05 versus control. The bars are expressed in mean + SEMFig. 2Effect of high-dose DOX treatment on the enterocyte-specific SI expression in the small intestinal epithelium. SI expression of the jejunum of a control mouse (A), at day 1 (B) and at day 2 (C). The brush border staining at day 1 and 2 is comparable to control situation. At day 3 (D), SI staining showed a patchy pattern; some parts of the brush border showed SI expression, on some other parts of the brush border SI expression was absent (\u2192). SI expression at day 7 (E) showed complete regeneration, and was equal to the control situation. SI protein expression levels were analyzed by dot-blot technique and the SI expression of control mice was set at 100% (F). The expression of SI remained stable at day 1, but decreased significantly at day 2 and 3. During the regenerative phase, at day 7, the SI expression reached control level again. *P < 0.05 day 2 and 3 versus control. The bars are expressed in mean + SEMFig. 3Effect of high-dose DOX treatment on the goblet-specific TFF3 expression in the small intestine. Immunohistochemical staining of the goblet cell-specific protein TFF3 (A\u2013E). TFF3 expression of the jejunum of a control mouse (A), at day 1 (B), at day 2 (C), at day 3 (D) and at day 7 (E). In the control situation, TFF3 was expressed in all goblet cells in the crypts and villi, at day 1 (B), the amount of goblet cells expressing TFF3 maintained at control level. At day 2 (C), TFF3 expression decreased visibly, the goblet cells localized in the crypts showed no staining. At day 3 (D), during severe morphological damage, TFF3 expression of goblet cells in the crypt was reconstituted. TFF3 expression at day 7 (E) was regenerated and comparable to control situation. TFF3 protein expression levels were analyzed by dot-blot technique, the TFF3 expression of control mice was set at 100% (F). TFF3 expression decreased significantly at day 1 and day 2, the expression was decreased to 50% of control level. During severe morphological damage at day 3, the TFF3 expression increased almost to control level. Day 7 showed TFF3 expression even above control level. *P < 0.05 day 1 and 2 versus control. The bars are expressed in mean + SEMFig. 4Effect of high-dose DOX treatment on epithelial proliferation. The localization of epithelial proliferation was visualized by detection of BrdU incorporation. BrdU incorporation of the jejunum of a control mouse (A) and mice at day 1 (B), at day 2 (C), at day 3 (D) and at day 7 (E). At day 1, the BrdU-positive cells were slightly migrated upwards in the crypt, at day 2 the BrdU-positive cells were seen along the entire length of the crypts. At day 3 the BrdU expression decreased and showed a scattered pattern on some crypts and villi. The location of BrdU-positive cells at day 7 was confined again to the lower part of the crypts. PCNA, Caspase-3, TCF4 and BMP4 protein expression levels were analyzed by Western-blot technique (F), the PCNA expression of control mice was set at 100% (G). The expression of PCNA protein showed a non-significant trend of increased expression at day 1, and a significant increase at day 2. PCNA expression at day 3 and 7 showed a decreasing trend to around 20% of control level. *P < 0.05 day 1 and 2 versus control. The bars are expressed in mean + SEM\nThe changes in proliferation induced by DOX were quantified by Western-blot analysis of PCNA protein expression [4, 40] using a specific monoclonal PCNA antibody. PCNA protein expression after MTX treatment (Fig.\u00a04F, G) showed a trend of increased expression at days 1. At day 2, this increased expression was significant compared to control levels, indicating that high DOX treatment did not induce an arrest in proliferation at day 1 and 2. At day 3, PCNA expression was decreased to around 20% of the normal level and was still 50% of control level at day 7. The latter data indicate that the inhibition of epithelial proliferation sustained during the regenerative phase. Overall, the PCNA levels correlated with the immunohistochemical data (Figs.\u00a04A\u2013E vs. 4G)\nEffect of high-dose DOX treatment on enterocyte apoptosis\nBesides proliferation, the influence of DOX treatment on apoptosis was quantified with a polyclonal antibody against cleaved Caspase-3 (Fig.\u00a04F, 5A). Caspase-3 is one of the key executioners of apoptosis [26]. The expression of cleaved Caspase-3 protein was significantly increased at day 1 and 2, at the beginning of DOX-induced morphological damage. At day 3, during severe morphological damage, and day 7, during regeneration, expression of cleaved caspase-3 was comparable to control level. Increased apoptosis at day 1 and 2 was primarily located along the crypt axis (Fig.\u00a05B showing day 1 after MTX treatment))Fig. 5Effect of high-dose DOX treatment on epithelial apoptosis. Caspase-3 protein expression of the control mice, analyzed by Western-blot technique (Fig\u00a0 4F), was set at 100% (A). At day 1 and 2, the Caspase-3 levels increased significantly in comparison to the control levels. At day 3, during severe morphological damage the caspase-3 expression maintained at control level, the same was seen at day 7, during the regenerative phase. The increase in apoptosis at day 1 is primarily located in the crypt region (B). *P < 0.05 day 1 and 2 versus control. The bars are expressed in mean + SEM. \u2192 indicates apoptotic cells\nEffect of high-dose DOX treatment on TCF-4 protein expression\nTo gain insight into the effects of DOX on the epithelial stem cell compartment, the expression of TCF-4 was quantified. TCF-4 is a transcription factor of the Wnt-signaling pathway and is expressed in the gut epithelium in a gradient fashion that is highest at the base of the crypts [1, 19]. TCF-4 \u2212\/\u2212 mice lose the intestinal epithelial progenitor and stem cell population before crypt formation can be established [17]. Therefore, TCF-4 has a role in intestinal epithelial stem cell maintenance. The TCF-4 expression (Figs.\u00a04F and 6) remained very stable despite DOX treatment. Only at day 1 the TCF-4 expression increased significantly, although little in absolute sense. But at day 2, day 3 and day 7, the TCF4 protein expression remained at control level.Fig. 6Effect of high-dose DOX treatment on TCF-4 protein expression. TCF-4 protein expression of control mice, analyzed by Western-blot technique (Fig.\u00a04F), was set at 100%. At day 1, TCF-4 protein expression increased significantly. At day 2, day 3, and day 7, the TCF-4 protein expression remained stable at control level. *P < 0.05 day 1 versus control. The bars are expressed in mean + SEM\nEffect of high-dose DOX treatment on expression of Bone morphogene 4 (BMP4)\nTo examine the role of BMP4 signaling in intestinal homeostasis the expression of BMP4 was quantified. BMP4 is crucial for epithelial-mesenchymal cross-talk and intestinal homeostasis. BMP4 is expressed in the intravillus and intracrypt mesenchymal cells [8, 10], inhibition of BMP signaling causes intestinal architectural abnormalities [8] and decreased differentiation as seen in tumorigenesis [27].\nBMP4 protein expression showed a mild decrease in expression level at day 1 after DOX treatment (Figs.\u00a04F, 7A). At day 2, this decrease in BMP4 expression level progressed and was almost significant (P=0.05) in comparison to the control level. Thereafter, BMP4 day 2 but not to control. Day 7 showed a non-significant decrease in expression compared to control mice. BMP4 was predominantly expressed in the intravillus mesenchym as shown by immunohistochemistry (Fig.\u00a07B, C), both in control as in MTX-treated mice.Fig. 7Effect of high-dose DOX treatment on expression of the morphogen BMP4. The BMP4 protein expression of the control mice, analyzed by Western-blot technique (Fig.\u00a04F), was set at 100% (A). Expression of the morphogen BMP4 protein showed a decreasing trend at day 1, which was at the brink of significance (P=0.05) at day 2 in comparison to control expression. At day 3, the BMP4 expression showed an increasing trend. This increase in expression was significant in comparison to day 2. Day 7 showed a decrease in expression again beneath the level of control expression. The localization of BMP4 expression did not change at day three (C) in comparison to control situation (B). *P < 0.05 day 2 versus control, **P < 0.05 day 2 versus day 3. The bars are expressed in mean + SEM\nDiscussion\nThis study revealed that DOX, in a dose of 10 and 6\u00a0mg\/kg induced severe morphological damage to the small intestine of mice within 3 days, which was almost completely regenerated by day 7. Moreover, it revealed that the intestine was virtually not or much less affected by lower doses of DOX. Mucositis induced by the chosen dose of DOX was characterized by an increasing degree of intestinal morphological damage at day 1 and 2, which correlated with a significant increase in both apoptosis and proliferation. During this phase of epithelial hyper-proliferation, the epithelial cells lost their highly differentiated status as measured by a significant down-regulation of epithelial-specific SI at days 2\u20133. The decreased expression of TFF3 at days 1\u20132 could be caused by a decrease in goblet cell differentiation, but on the other hand, could also be the result of increased TFF3 secretion. At day 3, the time-point when intestinal damage was most severe, the morphology was characterized by severe villus atrophy, a significant rise in crypt length, epithelial flattening, crypt loss, inhibition of proliferation and impaired epithelial differentiation. During morphological regeneration, at day 7, proliferation started to return to control level, and SI and TFF3 expression levels were normalized again.\nIn order to be able to prevent or treat chemotherapy-induced mucositis, it is essential to know if different cytostatic drugs induce the same or different kinds of intestinal damage. Potten et\u00a0al. demonstrated that there is a general tendency for antibiotics, like DOX, and radiation to damage the lower cell positions in the crypt near or at the position of the stem cells (position 4\u20136) [12]. Alkylating agents on the other hand mainly damage cells at position 6\u20138. Anti-metabolites like MTX and a microtubule dissociating agents act on higher cell positions [9\u201311]. It is, however, unclear if cytostatic drugs attacking at the lowest positions in the crypts cause a different kind of damage than drugs damaging cells at higher positions. If we compare the DOX-induced mucositis as studied presently with the well-characterized MTX-induced mucositis [34, 38, 44\u201346, 48], then it is clear that there are many similarities and few discrepancies between the two. Although the two drugs affected cells of different hierarchical height [12], they both caused apoptosis, villus atrophy, epithelial flattening, crypt loss and a temporary loss of SI expression and TFF3 expression [45, 46, 48]. Since SI is involved in carbohydrate metabolism and TFF3 is involved in mucosal repair [6, 22, 30, 42], these data suggest impaired absorption and mucosal repair after DOX as well as after MTX treatment. In contrast, both MTX [45] and DOX hardly affect the expression of lysozyme by Paneth cells in the crypts (data not shown).\nDecreased levels of TFF3 after both DOX and MTX was at the same time as epithelial hyper-proliferation [45, 48], but changes in proliferation, induced by MTX treatment followed a different time-line compared to DOX treatment. MTX causes proliferation inhibition within 1 day, followed by a period of hyper-proliferation during severe intestinal damage [34, 45]. DOX treatment leads to immediate hyper-proliferation (day 1 and 2) with subsequent inhibition of proliferation during severe morphological damage (day 3). Moreover, the cell-fate specific affect of MTX on goblet cells causing goblet cells to accumulate in the crypt and at the top of the villus [45, 46] was not seen after DOX treatment. The reason for these discrepancies remains to be further investigated, but might be directly related to the difference in topographical height (and thus status) of the cells vulnerable to the two different drugs. Overall, however, the similarities in intestinal responses after DOX or MTX treatment are striking. This suggests that there may be common pathways involved in intestinal damage and repair.\nHistorically, chemotherapy- or radiation-induced mucositis was believed to be solely due to damage to dividing epithelial cells at the bottom of the crypts [20]. However, recently it has become clear that other parts of the intestinal mucosa and submucosa might also be involved [35, 36]. Here we provide evidence for a mesenchymal contribution to the damage by showing that BMP4, a very important lamina propria derived-morphogen in the small intestine [8, 10], is affected by DOX treatment. BMP4 was modulated by DOX during the onset of damage at days 1 and 2. BMP4 expression decreased almost significant at day 2, which correlated well with an increasing degree of morphological damage, increased proliferation, and loss of epithelial differentiation as measured by the decreased SI and likely TFF3 expression. Very recently, a link between the BMP and the Wnt pathways has been demonstrated. It was shown that BMP signaling suppresses Wnt signaling to ensure a balanced control of stem cell proliferation and subsequent epithelial differentiation [8, 10]. Here we show that there indeed might be a close relationship between BMP and Wnt pathways, because at day 1 when BMP4 expression is decreased, expression of TCF4, a Wnt effector, increased significantly, which correlated well with increased proliferation and inhibited epithelial differentiation. At day 2, BMP4 remained low, whereas TCF4 returned to a normal level and remained at control level during the following days. At day 3, when damage was most severe, BMP4 was increased, which inversely correlated with proliferation, and correlated with epithelial differentiation, as suggested by the recovery of TFF3 expression level.\nBMP is involved in epithelial-mesenchymal signaling [8] and therefore we conclude that the data presented in this study indicate that epithelial-mesenchymal cross-talk is modulated during onset of DOX-induced damage in order to stimulate proliferation instead of differentiation and during severe intestinal damage to induce differentiation and inhibit proliferation. During the regenerative phase, at day 7, BMP4 expression level was down-regulated again, which could be a response to the shortage in number of crypts, since a blockage of BMP4 has been shown to cause stem cells to divide, leading to newly formed crypts [8, 10]. Therefore, our findings are in line with the roles of the BMP and Wnt\/TCF pathway in epithelial homeostasis\/morphogenesis. Furthermore, the decrease in BMP4 might also indirectly cause the observed decrease in SI expression, because inhibition of BMP4 stimulates Wnt signaling, and Wnt signaling itself induces SOX9, a negative regulator of Cdx2, which is a SI transcriptional activator [3, 10, 39]. In addition, BMP4 has been shown to be directly involved in HNF-1\u03b1 expression, also a well-known activator of SI transcription [25]. Thus, the decrease in BMP4 might result in a decrease in Cdx2 and HNF-1\u03b1 expression, two of the most important activators of SI transcription [39]. Currently, it is not known whether the Wnt or BMP4 signaling pathways regulate TFF3 gene expression.\nIn conclusion, high-dose DOX induces severe damage to the epithelium, which closely resembles damage induced by MTX, indicating that general mechanisms of damage and repair are involved. We show that signaling pathways involving BMP4 and TCF4 and thus epithelial-mesenchymal cross-talk are modulated by DOX-induced damage in such a way that homeostasis of the progenitor compartment is restored by initially inducing cell proliferation and inhibiting differentiation and subsequently inducing differentiation, inhibiting proliferation and promoting crypt fission. Understanding these mechanisms is essential to develop clinical strategies to prevent chemotherapy-induced mucositis.","keyphrases":["tcf4","morphogens","epithelial homeostasis","intestinal differentiation","bone morphogenetic proteins"],"prmu":["P","P","P","R","M"]}
{"id":"J_Biol_Inorg_Chem-4-1-2359827","title":"Spectroscopic evidence for an all-ferrous [4Fe\u20134S]0 cluster in the superreduced activator of 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans\n","text":"The key enzyme of the fermentation of glutamate by Acidaminococcus fermentans, 2-hydroxyglutarylcoenzyme A dehydratase, catalyzes the reversible syn-elimination of water from (R)-2-hydroxyglutaryl-coenzyme A, resulting in (E)-glutaconylcoenzyme A. The dehydratase system consists of two oxygen-sensitive protein components, the activator (HgdC) and the actual dehydratase (HgdAB). Previous biochemical and spectroscopic studies revealed that the reduced [4Fe\u20134S]+ cluster containing activator transfers one electron to the dehydratase driven by ATP hydrolysis, which activates the enzyme. With a tenfold excess of titanium(III) citrate at pH 8.0 the activator can be further reduced, yielding about 50% of a superreduced [4Fe\u20134S]0 cluster in the all-ferrous state. This is inferred from the appearance of a new M\u00f6ssbauer spectrum with parameters \u03b4 = 0.65 mm\/s and \u0394EQ = 1.51\u20132.19 mm\/s at 140 K, which are typical of Fe(II)S4 sites. Parallel-mode electron paramagnetic resonance (EPR) spectroscopy performed at temperatures between 3 and 20 K showed two sharp signals at g = 16 and 12, indicating an integer-spin system. The X-band EPR spectra and magnetic M\u00f6ssbauer spectra could be consistently simulated by adopting a total spin St = 4 for the all-ferrous cluster with weak zero-field splitting parameters D = \u22120.66 cm\u22121 and E\/D = 0.17. The superreduced cluster has apparent spectroscopic similarities with the corresponding [4Fe\u20134S]0 cluster described for the nitrogenase Fe-protein, but in detail their properties differ. While the all-ferrous Fe-protein is capable of transferring electrons to the MoFe-protein for dinitrogen reduction, a similar physiological role is elusive for the superreduced activator. This finding supports our model that only one-electron transfer steps are involved in dehydratase catalysis. Nevertheless we discuss a common basic mechanism of the two diverse systems, which are so far the only described examples of the all-ferrous [4Fe\u20134S]0 cluster found in biology.\nIntroduction\nThe anaerobic bacterium Acidaminococcus fermentans ferments glutamate via the 2-hydroxyglutarate pathway to ammonium, carbon dioxide, acetate, butyrate, and molecular hydrogen [1]. A key enzyme in this fermentation is 2-hydroxyglutaryl-CoA dehydratase (CoA is coenzyme\u00a0A), which catalyzes the reversible syn-elimination of water from (R)-2-hydroxyglutaryl-CoA, resulting in (E)-glutaconyl-CoA. This reaction is of considerable interest since a nonactivated \u03b2-C\u2013H bond has to be cleaved (pK\u00a0\u2248\u00a040); for reviews see [2\u20134]. The enzyme system from A. fermentans is composed of two protein components, the homodimeric activator or archerase (HgdC) and the heterodimeric dehydratase (HgdAB), whose genes have been cloned and sequenced. After overexpression of hgdC in Escherichia coli, the activator could be purified to homogeneity aided by a C-terminal Strep-tag. The protein [\u03b32, 2\u00a0\u00d7\u00a0(27\u00a0\u00b1\u00a01)\u00a0kDa] contains 4.0\u00a0\u00b1\u00a00.1 iron and 3.8\u00a0\u00b1\u00a00.1 acid-labile sulfur, which form one [4Fe\u20134S]+\/2+ cluster between the two subunits [5]. The activator can be readily reduced with dithionite or titanium(III) citrate. Most likely in vivo a reduced clostridial [4Fe\u20134S]-type ferredoxin [6] or the hydroquinone form of a flavodoxin [7], both isolated from A. fermentans, can act as reducing agents for the activator. The activator is extremely oxygen-sensitive; its half life under air is only about 10\u00a0s. The oxygen sensitivity results from destruction of the [4Fe\u20134S] cluster, which is exposed to the solvent [8]. Furthermore, the activator also precipitates in the presence of the dyes that are commonly used for the determination of redox potentials; herefore, the redox potential of the [4Fe\u20134S]+\/2+ cluster (E0\u2032 \u2248 \u2212320\u00a0mV) can only be roughly estimated from its almost complete reduction by ferredoxin or flavodoxin (E0\u2032\u00a0=\u00a0\u2212420\u00a0mV) [7]. The native 2-hydroxyglutaryl-CoA dehydratase HgdAB (\u03b1\u03b2, 54\u00a0+\u00a042\u00a0kDa) contains 4.5\u00a0\u00b1\u00a00.3 iron, 3.5\u00a0\u00b1\u00a00.4 acid-labile sulfur and 1.0 FMNH2 [5]. In order to obtain catalysis, incubation of the dehydratase with reduced activator in the [4Fe\u20134S]+ state, ATP, and MgCl2 is necessary. Spectroscopic studies revealed that thereby one electron is transferred from the activator to the [4Fe\u20134S] cluster of the dehydratase [5, 7, 9], which cannot be reduced by any other means, except by deazaflavin and light in the presence of EDTA as shown for the related 2-hydroxyisocarproyl-CoA dehydratase (J. Kim and W. Buckel, unpublished results).\nA mechanism has been proposed in which the dehydration is initiated by injection of this electron into the thiol ester carbonyl. The resulting substrate-derived ketyl radical eliminates the adjacent hydroxyl group. The enoxy radical formed can now be deprotonated to the product-related allylic ketyl radical, because radical formation has lowered the pK of the \u03b2-hydrogen by about 26\u00a0U [10]. In the last step the electron is returned from the allylic ketyl radical to the [4Fe\u20134S] cluster of the dehydratase and a new cycle can start. With the related 2-hydroxyisocaproyl-CoA dehydratase from Clostridium difficile this radical has been identified by electron paramagnetic resonance (EPR) spectroscopy [11] and it has been shown that such a cycle can endure for about 10,000 turnovers until a new \u2018shot\u2019 by the activator is required [12].\nHomologous activators with around 60% sequence identity have been characterized from 2-hydroxyglutaryl-CoA dehydratase in Fusobacterium nucleatum [13], lactyl-CoA dehydratase in C. propionicum (T. Selmer and W. Buckel, unpublished results), phenyllactyl-CoA dehydratase in C. sporogens [14, 15], and 2-hydroxyisocaproyl-CoA dehydratase in C. difficile [12]. Furthermore, each genome of almost all anaerobically thriving bacteria and archaea, even that of E. coli (yjiL gene), encodes up to four different homologues of the A. fermentans activator with 30\u201340% sequence identity. Only the function of the two homologues in Thauera aromatica and other anaerobic benzoate-degrading bacteria could be assessed as the \u03b1- and \u03b4-subunits of the four subunits comprising benzoyl-CoA reductase. The catalysis of this enzyme, which in addition requires ATP and reduced ferredoxin as substrates, has been proposed to proceed also via a ketyl radical [16].\nHence such activators, which probably all contain a single [4Fe\u20134S] cluster between two subunits with a helix\u2013cluster\u2013helix architecture [8], are widespread in the anaerobic world. Proteins with almost the same architecture but with unrelated sequences are the nitrogenase reductases, which appear to have the same function as the activators [17]. The proteins of both families increase the reductive power of an electron by ATP hydrolysis. Nitrogenase reductase from Azotobacter vinelandii, also called Fe-protein or Av2, has been shown to become superreduced to the all-ferrous state by titanium(III) citrate [18\u201326]. The energy efficiency of nitrogenase would be twofold enhanced if the catalysis proceeded via superreduced Av2, owing to the transfer of two electrons from the corresponding [4Fe\u20134S]0 cluster per hydrolysis of two ATP molecules [27, 28]. However, the low redox potential Em\u00a0=\u00a0\u2212790\u00a0mV reported for the (+1\/0) couple of the iron\u2013sulfur cluster virtually excludes any physiological relevance [measured with Cr(II)EDTA as a reductant] [29]. Interestingly, a much higher value, Em\u00a0=\u00a0\u2212460\u00a0mV, was initially obtained by using methyl viologen [26]. Moreover, a recent report indicates that nitrogenase catalysis actually can be supported by superreduced Av2 and may proceed via the (+2\/0) couple of the iron\u2013sulfur cluster, if the in vivo reductant flavodoxin hydroquinone (FldHQ) is used [30]. In this new state of the Fe protein, the all-ferrous [4Fe\u20134S]0 cluster is difficult to discriminate from the +1 and +2 redox states using the absorption spectra, and it appears to be diamagnetic. In contrast, the Av2 [4Fe\u20134S]0 cluster made with titanium(III) citrate has a distinct absorption spectrum [18], and an unusually high spin state S\u00a0=\u00a04, as was inferred from elaborate integer-spin EPR and magnetic M\u00f6ssbauer studies [18, 19, 31, 32] and theoretically explored by density functional theory studies [33, 34]. Here we report an all-ferrous state for the [4Fe\u20134S] cluster in the activator from A. fermentans after treatment with a tenfold excess of titanium(III) citrate. To our knowledge this is the second biological system which stabilizes a [4Fe\u20134S]0 cluster. Remarkable spectroscopic and biochemical similarities between activator and the A. vinelandii Fe-protein give reason to favor a common functional principle of these exciting proteins.\nMaterials and methods\nMetallic 57Fe was purchased from Chemotrade (D\u00fcsseldorf, Germany). The metal (20\u00a0mg) was dissolved in 0.5\u00a0mL 8\u00a0M HCl at 80\u00a0\u00b0C for 12\u00a0h. This stock solution was added directly to the medium. Standard I nutrient broth was from Merck (Darmstadt, Germany). E. coli strain XL1-blue was purchased from Stratagene (Heidelberg, Germany). Streptavidin\u2013Sepharose and the plasmid pASK-IBA3 were obtained from IBA (G\u00f6ttingen, Germany). All other chemicals and substances were from Sigma or Fluka (Deisenhofen, Germany).\nAnaerobic manipulations were performed in an anaerobic chamber (Coy Laboratories, Ann Arbor, MI, USA) with an atmosphere of 5% H2 in N2. X-band EPR measurements were performed using a Bruker E500 ELEXSYS continuous-wave spectrometer with the Bruker dual-mode cavity (ER4116DM) or a standard cavity (ER4102ST) and an Oxford Instruments helium-flow cryostat (ESR 910). Microwave frequencies were measured with a Hewlett\u2013Packard frequency counter (HP5352B), and the field control was calibrated with a Bruker NMR field probe (ER035M). The spectra were simulated on the basis of a spin-Hamiltonian description of the electronic ground state: where St is the spin of the coupled system and D and E\/D are the axial and rhombic zero-field parameters. The simulations were performed with a program which was developed from the S\u00a0=\u00a05\/2 routines of Gaffney and Silverstone [35] and which specifically makes use of the Newton\u2013Raphson iterative search method for transition fields as described there. Frequency-dependent Gaussian line shapes were used with full-width-at-half-maximum \u03c3\u03bd given in millitesla at g\u00a0=\u00a01. Alternatively also the Bruker XSOPHE suite [36] was used for the calculation of energy-level schemes and transition roadmaps. M\u00f6ssbauer spectra were recorded using two spectrometers with alternating constant acceleration. The minimum experimental line width was \u0393fwhm\u00a0=\u00a00.24\u00a0mm\/s (full width at half maximum). The sample temperature was maintained constant either in an Oxford Variox or an Oxford M\u00f6ssbauer-Spectromag cryostat. The latter is a split-pair superconducting magnet system for fields up to 8\u00a0T in which the sample temperature can be kept constant in the range 1.5\u2013250\u00a0K. The field is perpendicular to the \u03b3-beam. The 57Co\/Rh source was positioned at room temperature inside the gap of the magnet in a reentrant bore tube at a distance of about 85\u00a0mm from the sample. The field is zero at this position. All isomer shifts are quoted relative to iron metal at 300\u00a0K. Magnetic M\u00f6ssbauer spectra were simulated with a computer program based on the same spin-Hamiltonian for St\u00a0=\u00a04 as for the EPR simulations (Eq.\u00a01). The hyperfine interactions for 57Fe were calculated by using the usual nuclear Hamiltonian [37].\nPurification of recombinant activator from A. fermentans\nStandard I medium (8\u201310\u00a0L, Merck, Germany) was inoculated with 25\u00a0mL overnight culture of recombinant E. coli (XL1-blue\/hgdC in pASK-IBA3) and grown in tightly closed 2-L bottles at 30\u00a0\u00b0C. When an optical density at 600\u00a0nm of about 0.3 was reached, cells were induced with anhydrotetracycline (30\u00a0\u03bcg\/L). After anaerobic growth for a further 3\u00a0h, cells were harvested by centrifugation and suspended in 20\u00a0mM 3-(N-morpholino)propanesulfonic acid, pH 7.2, 100\u00a0mM NaCl, 10\u00a0mM MgCl2, 1\u00a0mM ATP, 2\u00a0mM dithiothreitol or 4\u00a0mM dithionite (buffer A). The bacteria were lysed anaerobically using a French press system. Cell debris and membranes were removed by ultracentrifugation for 60\u00a0min at 100,000g. The filtered supernatant was loaded with gravity flow onto an 8-mL streptavidin\u2013Sepharose column, which was equilibrated with buffer A. After complete binding, the column was washed with at least ten column volumes of buffer A. The activator was eluted with 2.5\u00a0mM d-desthiobiotin in buffer A, then concentrated with an Amicon stirred cell, and immediately used for further experiments.\nResults\nPreparation and characterization of superreduced activator\nThe activator as isolated was concentrated to 60\u201380\u00a0mg protein\/mL (1.2\u20131.6\u00a0mM) in 50\u00a0mM tris(hydroxymethyl)aminomethane hydrochloride pH 8.0. A tenfold excess of titanium(III) citrate was added, which yielded a color change from brown to red. Correspondingly, a new shoulder appeared in the UV\u2013vis spectrum at about 500\u00a0nm (Fig.\u00a01). The orange-red color apparently is a typical feature of superreduced [4Fe\u20134S]0 clusters, and does not originate from titanium(III) citrate directly, because this was removed by gel filtration on a Sephadex G-25 desalting column. It is noteworthy that the same color change was also observed upon full reduction of the nitrogenase Fe-protein from A. vinelandii [18].\nFig.\u00a01UV\u2013vis absorption spectrum of activator reduced with dithionite, and superreduced with titanium(III) citrate\nAs described previously, the activator had to be used right after purification for biochemical studies because of its tendency to precipitate within 4\u00a0days [5]. To our surprise, the superreduced activator was nearly as stable as the singly reduced or oxidized enzyme; the activity decayed only with a half life of 4\u20135\u00a0days or more. Remarkably, dehydratase activity could not be observed with 2-hydroxyglutaryl-CoA when titanium(III) citrate reduced activator was used in the activity assay [38].\nEPR spectroscopy\nIn a previous EPR and M\u00f6ssbauer study we demonstrated that dithionite-reduced activator1 possesses a [4Fe\u20134S]+ cluster with spin S\u00a0=\u00a03\/2 [5]. The unusual magnetic ground state was inferred from X-band EPR spectra as shown in the inset in Fig.\u00a02. The EPR resonances extend over a wide field range of at least 800\u00a0mT with a dominating strong absorption-like peak at g\u00a0\u2248\u00a04.5 and a broad trough at g\u00a0\u2248\u00a01.3\u20132. The large anisotropy and particularly the presence of high effective g values indicate the high spin multiplicity with large zero-field splitting (D\u00a0>\u00a0g\u03bcBB). Although the broad derivative spectrum does not have the classical appearance known for a quartet species from a transition metal complex [39], the pattern could be nicely simulated by using the usual spin-Hamiltonian for S\u00a0=\u00a03\/2 with reasonable zero-field splitting parameters D\u00a0=\u00a01.3\u00a0cm\u22121 and E\/D\u00a0=\u00a00.213. Just a simple Gaussian distribution of the rhombicity parameter with half width \u03c3(E\/D)\u00a0=\u00a00.11 was sufficient to account for the observed line broadening caused by g strain [5]. Moreover, a minor fraction of the [4Fe\u20134S]+ clusters (approximately 3%) was found to have the more common spin state S\u00a0=\u00a01\/2, which gives rise to the narrower resonances around g\u00a0=\u00a02. The corresponding simulations for both spin states of [4Fe\u20134S]+, S\u00a0=\u00a01\/2 and 3\/2, are shown as gray and blue lines in the inset in Fig.\u00a02.\nFig.\u00a02Normal-mode (B1\u22a5B0) X-band electron paramagnetic resonance (EPR) spectrum of titanium(III) citrate reduced activator (low-field part, microwave frequency 9.6337\u00a0GHz; power 1.6\u00a0mW; modulation 2\u00a0mT at 100\u00a0kHz). The simulation for St\u00a0=\u00a04 (green line) is obtained with D\u00a0=\u00a0\u22120.66\u00a0cm\u22121, E\/D\u00a0=\u00a00.165, g\u00a0=\u00a0(2.1, 1.99, 2.03) (Footnote\u00a02) and a frequency-dependent Gaussian line width of \u03c3\u03bd\u00a0=\u00a070\u00a0mT. The inset shows the full range X-band spectrum of dithionite-reduced activator at 10\u00a0K with simulations for the [4Fe\u20134S]+ clusters as published previously [5]. The arrows in the main panel and in the inset indicate the field position of the strongest integer-spin signal from [4Fe\u20134S]0 clusters with St\u00a0=\u00a04\nThe strong absorption-like band at g\u00a0\u2248\u00a04.5 from S\u00a0=\u00a03\/2 persists also in the EPR spectrum of titanium(III) citrate treated activator (Fig.\u00a02, bold trace). We assign it to [4Fe\u20134S]+ clusters remaining from incomplete conversion of the reduced activator. The relative abundance of this contribution is estimated by corresponding the M\u00f6ssbauer spectra to account for about 50% of the total iron (see below). Formation of superreduced clusters, however, is also discernible in the EPR spectrum from the appearance of two new lines in the low-field range at g\u00a0\u2248\u00a011 and g\u00a0\u2248\u00a016. Narrow absorption-like signals at such high g values without related strong derivative lines at higher field are completely unexpected for half-integer-spin systems, but rather suggest integer-spin transitions. The interpretation is corroborated by parallel-mode measurements (B1||B0) for which the new low-field resonances are the strongest signals, whereas the spectrum from the reduced activator, particularly the absorption-like band at g\u00a0\u2248\u00a04.5, is completely attenuated, as shown in Fig.\u00a03. The striking difference of the two detection modes results from the different selection rules \u0394m\u00a0=\u00a0\u00b11 for B1\u22a5B0 and \u0394m\u00a0=\u00a00 for B1||B0, which essentially discriminate half-integer-spin and integer-spin transitions [40, 41]. Therefore, the low-field transitions at g\u00a0\u2248\u00a011 and g\u00a0\u2248\u00a016 must be assigned to an all-ferrous [4Fe\u20134S]0, cluster for which coupling of the four Fe(II) sites with local spin SFe\u00a0=\u00a02 can yield only an integer total spin. This clearly indicates superreduction of the activator by titanium(III) citrate. The ground state for such a tetrameric all-ferrous cluster can have, in principle, any total spin ranging from St\u00a0=\u00a00 to St\u00a0=\u00a08, depending on the six spin coupling constants between the four ferrous ions. But sophisticated quantum chemical studies for the titanium(III) citrate reduced [4Fe\u20134S]0 cluster from the Fe protein suggest only St\u00a0=\u00a00, 4, or 8 as possible solutions for that particular system [33, 34]. Below we show that the all-ferrous [4Fe\u20134S]0 cluster in superreduced activator actually has total spin St\u00a0=\u00a04, with properties that closely resemble those of the superreduced state of the Fe protein from A. vinelandii [18, 31].\nFig.\u00a03Parallel-mode (B1||B0) X-band EPR spectra of titanium(III) citrate reduced activator recorded at 3, 10, and 20\u00a0K (microwave frequency 9.3404\u00a0GHz; power 1.6\u00a0mW; modulation 0.57\u00a0mT at 100\u00a0kHz). The green lines represent simulations for St\u00a0=\u00a04 with D\u00a0=\u00a0\u22120.66\u00a0cm\u22121, E\/D\u00a0=\u00a00.165, g\u00a0=\u00a0(2.1, 2.0, 2.1) (Footnote\u00a02), and a frequency-dependent Gaussian line width of \u03c3\u03bd\u00a0=\u00a030\u00a0mT\nThe integer-spin signals are remarkably sharp in the parallel-mode EPR spectra with full width at half maximum of only \u0393fwhm\u00a0\u2248\u00a04\u20135\u00a0mT for the main derivative peaks, but with very broad and asymmetric underlying shoulders. Such a superposition of broad and narrow powder signals may be caused by complex distributions of spin-Hamiltonian parameters, which owe their origin to microheterogeneity in the surrounding of the [4Fe\u20134S] clusters. In normal mode, however, the corresponding lines appear to be almost Gaussians of moderate 9\u201312\u00a0mT line width (Fig.\u00a02). The difference may be related to the different orientational selections that contribute to the respective powder spectra [41]. Other ms components of the wave functions determine the transition probabilities between the zero-field split sublevels of the integer-spin system for parallel-mode detection, and thus may respond differently on the structural distortions. In the simulations, however, we could not reproduce the complex experimental line shape of the parallel-mode spectra of the superreduced activator by a simple distribution model for any of the spin-Hamiltonian parameters. Since we wanted to refrain from further sophistications other than Gaussian distributions, we just focused on the main features of the EPR spectra, i.e., the well-resolved sharp lines in the parallel-mode spectra and the Gaussian lines of the normal-mode spectra recorded at base temperature.\nThe EPR spectra of superreduced activator recorded in normal and parallel mode can be consistently simulated with total spin St\u00a0=\u00a04, as shown in Figs.\u00a02 and 3. The zero-field parameters obtained from global optimization are D\u00a0=\u00a0\u22120.66 (\u00b10.1)\u00a0cm\u22121 and E\/D\u00a0=\u00a00.165 (\u00b10.01), with g\u00a0=\u00a0(2.2, 1.99, 2.03).2 Particularly the unusual temperature dependence of the two narrow parallel-mode signals in Fig.\u00a03 is basically well reproduced with those values, i.e., at 3\u00a0K the simulated line at g\u00a0\u2248\u00a011 is stronger than the line at g\u00a0\u2248\u00a016, whereas the intensity ratio is inverted at 20\u00a0K. According to the energy splitting scheme shown on the left in Fig.\u00a04, the derivative signals arise from transitions within the low-lying \u201cms\u00a0=\u00a0\u00b14\u201d and the excited \u201cms\u00a0=\u00a0\u00b13\u201d non-Kramers doublets, respectively. At the actual conditions of D and E\/D, both of these ms doublets are virtually unsplit at zero magnetic field. For fields applied in the molecular z-direction, their X-band resonances are expected at apparent g values of 16 and 12, respectively. However, the transition probability is zero for the \u201cms\u00a0=\u00a0\u00b14\u201d transition at exact B0||z and is very low for the \u201cms\u00a0=\u00a0\u00b13\u201d transition, but the Zeeman effect is very anisotropic and causes mixing of the ms wave functions for both doublets, so EPR transitions become allowed for B0 oriented between the principal axes. The major intensity in a parallel-mode powder spectrum actually arises from orientations close to the z-axis. The variation of the resonance field as a function of the field orientation is visualized by the transition roadmaps shown on the right in Fig.\u00a04. Powder derivative signals are expected from these diagrams only at the turning points of the respective traces. The corresponding g values for the \u201cms\u00a0=\u00a0\u00b13\u201d and \u201cms\u00a0=\u00a0\u00b14\u201d derivative spectra are indicated by the vertical dotted lines and g labels in diagram\u00a0B in Fig.\u00a04. Interestingly, there is a third resonance traced in the diagrams, coming from the highest pair of ms levels for the field close to the y-axis (marked with an asterisk). That also has a turning point at g\u00a0\u2248\u00a016, so the contribution in the EPR spectrum entirely overlaps the \u201cms\u00a0=\u00a0\u00b14\u201d transitions.\nFig.\u00a04A Energy level scheme for St\u00a0=\u00a04 with D\u00a0=\u00a0\u22120.66\u00a0cm\u22121, E\/D\u00a0=\u00a00.165, g\u00a0=\u00a02.03, and B0 applied in the z-direction. The ms values given in quotation marks are labels for the magnetic sublevels but do not reflect the true composition of the wave functions. The vertical bold bars indicate parallel-mode transitions for \u03bd\u00a0=\u00a09.6\u00a0GHz. B\u2013D Transition roadmap diagrams for the same system, which are plots of the resonance fields for hypothetical single-crystal rotation experiments. The resonances of the \u201cms\u00a0=\u00a0\u00b14\u201d and \u201cms\u00a0=\u00a0\u00b13\u201d non-Kramers doublets are shown in red and blue, whereas transitions within the highest two ms levels are marked with an asterisk: B rotation of B0 in the z\u00a0\u2192\u00a0x-plane; C rotation in the y\u00a0\u2192\u00a0x-plane; and D rotation in the y\u00a0\u2192\u00a0z-plane\nThe roadmap diagram also explains why the g\u00a0\u2248\u00a011 and the g\u00a0\u2248\u00a016 derivative signals have different intensities in the parallel-mode spectra at 20\u00a0K, although the Boltzmann law arrogates almost equal population of the corresponding non-Kramers doublets at this condition of temperature and field strength. Apparently, the ground-state \u201cms\u00a0=\u00a0\u00b14\u201d doublet has a higher magnetic anisotropy, so the resonances become spread over a wider field range than for the \u201cms\u00a0=\u00a0\u00b13\u201d transitions and the signal amplitudes are attenuated. Additionally, also lower intrinsic transition probabilities can be found at g\u00a0\u2248\u00a016, because higher ms functions become less mixed as a result of the competition between Zeeman- and zero-field interaction. At 3\u00a0K, in contrast, the g\u00a0\u2248\u00a011 signal fades out because the excited states become mostly depopulated. The effect yields substantial information about the magnitude of D and E\/D, although the constraint on the parameters obtained from the intensity variations in the parallel-mode spectra in Fig.\u00a03 may be somewhat weakened by the odd overall experimental line shapes of the two signals. However, the actual D and E\/D values are nicely supported by the successful simulation of the resonance lines in the normal-mode spectrum in Fig.\u00a02.\nThe parallel-mode spectra of the superreduced activator (Fig.\u00a03) resemble remarkably well the EPR features of the all-ferrous cluster in the Fe-protein [21, 24] with a ground-state spin St\u00a0=\u00a04 [18, 31]. This cluster also shows a very sharp line in the parallel-mode X-band spectra around g\u00a0\u2248\u00a016 (actually at g\u00a0=\u00a016.4), but at g\u00a0\u2248\u00a011 only a weak, broad feature is observed at elevated temperature, whereas for the activator the line at g\u00a0\u2248\u00a011 is sharp and appears already at 3\u00a0K as a strong sharp signal. The inherent similarity of the spectra illustrates the same spin state for the all-ferrous clusters of the activator and the Fe protein and reveals similar physical properties. The differences in the EPR spectra arise from only small differences in the zero-field interaction, which slightly modulate the properties of the resonating non-Kramers doublets as shown in Fig.\u00a04 (D\u00a0=\u00a0\u22120.75\u00a0cm\u22121, E\/D\u00a0=\u00a00.30 for the Fe protein [31] compared with \u22120.66\u00a0cm\u22121 and 0.165 for the activator). In view of the fact that the zero-field splitting of the cluster spin manifold is inherited from the single-ion properties of four ferrous sites [42], and that there are 15 different ways to obtain St\u00a0=\u00a04 from such a spin tetrade [31], the close spectral similarity suggests very similar electronic properties of the all-ferrous clusters from activator and the Fe protein.\nM\u00f6ssbauer spectroscopy\nWe used 57Fe M\u00f6ssbauer spectroscopy to quantify superreduction of the activator samples and to further substantiate the comparison with the Fe protein. Figure\u00a05 shows the zero-field M\u00f6ssbauer spectrum of titanium(III) citrate reduced activator recorded at 140\u00a0K. The asymmetric pattern could be fitted with three quadrupole doublets (I, IIa, and IIb), of which the prominent subspectrum I can be clearly assigned to [4Fe\u20134S]+ clusters from remaining starting material, because its isomer shift, \u03b4\u00a0=\u00a00.51\u00a0mm\/s, and quadrupole splitting, \u0394EQ\u00a0=\u00a00.94\u00a0mm\/s, are virtually identical to those observed for reduced activator at 80\u00a0K (\u03b4\u00a0=\u00a00.53\u00a0mm\/s, \u0394EQ\u00a0=\u00a00.95\u00a0mm\/s) [5]. The relative intensity of subspectrum I accounts for 54% of the total iron.\nFig.\u00a05Zero-field M\u00f6ssbauer spectrum of titanium(III) citrate reduced activator at 140\u00a0K. Subspectrum I and the superposition of subspectra IIa and IIb represent the contributions from reduced [4Fe\u20134S]+ and superreduced [4Fe\u20134S]0 clusters, respectively. \u03b4(I)\u00a0=\u00a00.51\u00a0mm\/s, \u0394EQ(I)\u00a0=\u00a00.94\u00a0mm\/s, 54% relative intensity; \u03b4(IIa,\u00a0IIb)\u00a0=\u00a00.65\u00a0mm\/s, \u0394EQ(IIa)\u00a0=\u00a01.51\u00a0mm\/s, \u0394EQ(IIb)\u00a0=\u00a02.59\u00a0mm\/s; intensity ratio of IIa and IIb fixed at 3:1\nSuperreduction of the activator and formation of all-ferrous [4Fe\u20134S]0 clusters is revealed by the presence of the wide split component with higher isomer shift shown as the shaded area in Fig.\u00a05. The corresponding all-ferrous [4Fe\u20134S]0 clusters from Av2 showed a similar pattern, but one that could be fitted with four subspectra according to four different iron sites. However, three subspectra were strongly overlapping and had similar quadrupole splittings in the range \u0394EQ\u00a0=\u00a01.2\u20131.7\u00a0mm\/s, but one deviated, showing \u0394EQ\u00a0=\u00a03.08\u00a0mm\/s [18, 31]. Such distinct differences cannot be resolved from the spectra of the all-ferrous cluster of the activator because of broad lines and limited experimental resolution, but the basic distribution pattern of three similar sites and one different site seems to persist also for the activator. We could fit our data with two Lorentzian doublets, IIa and IIb, having identical isomer shifts and a fixed intensity ratio of 3:1, so subspectrum IIa accounts for three similar iron sites of the all-ferrous cluster with unresolved signals, and subspectrum IIb represents a fourth, discernibly different site. Details on the significance of the fit model are given in the electronic supplementary material.3 The M\u00f6ssbauer parameters obtained are \u03b4\u00a0=\u00a00.65\u00a0mm\/s for IIa and IIb and \u0394EQ\u00a0=\u00a01.51\u00a0mm\/s for IIa and \u0394EQ\u00a0=\u00a02.59\u00a0mm\/s for IIb, respectively. The high isomer shift and large quadrupole splittings are typical for tetrahedral Fe(II)S4 sites with localized valences. Particularly the isomer shift, which is the key parameter for the assignment of valence states, compares very well with that of the monomeric Fe(II) centers in reduced rubredoxin (\u03b4\u00a0=\u00a00.70\u00a0mm\/s at 4.2\u00a0K) [32, 43] or with that of the diferrous [2Fe\u20132S] cluster in a plant-type ferredoxin (\u03b4\u00a0=\u00a00.71\u00a0mm\/s at 4.2\u00a0K) [44]. Finally it matches exactly the value found for the all-ferrous cubane clusters in the superreduced Fe-protein (\u03b4\u00a0=\u00a00.65\u00a0mm\/s at 130\u00a0K). We assume that the rather strong line broadening found for the activator, \u0393fwhm(IIa)\u00a0=\u00a00.7\u00a0mm\/s, \u0393fwhm(IIb)\u00a0=\u00a00.47\u00a0mm\/s, is caused by further heterogeneity of the cluster sites in the frozen protein sample. We can exclude broadening by paramagnetic relaxation at 140\u00a0K since basically the same pattern was observed at 80\u00a0K.\nIt was not possible to achieve higher yields of superreduced [4Fe\u20134S]0 clusters than the approximately 50% mixture with remaining [4Fe\u20134S]+ clusters observed in the M\u00f6ssbauer spectra shown here. Prolonged incubation with titanium(III) citrate or a higher excess of reducing agent did not improve the result. All attempts failed to fully convert a sample by the use of any chemical means, whereas photolytic reduction of the activator with deazaflavin led to cluster deterioration and precipitation of the protein. Because of this restriction, the midpoint of the [4Fe\u20134S]+\u00a0\u2192\u00a0[4Fe\u20134S]0 transition could not be exactly determined, but we estimate that the redox potential is lower than that of titanium(III) citrate, for which the value E0\u20321\/2\u00a0=\u00a0\u2212720\u00a0mV against the standard hydrogen electrode is reported [45] (E0\u2032\u00a0=\u00a0\u2212420\u00a0mV; or E0\u00a0=\u00a00\u00a0mV). This estimation is based on the fact that most likely two protons per electron are released during the oxidation of TiIII to TiIV, which forms a titanyl ion: Hence at pH 8.0, at which the all-ferrous cluster was prepared, the redox potential of titanium(III) citrate should be\u2014according to the Nernst equation\u20142\u00a0\u00d7\u00a060\u00a0mV more negative, i.e., E0\u2032\u00a0=\u00a0\u2212840\u00a0mV.\nMagnetic M\u00f6ssbauer measurements of titanium(III)-treated activator were performed at 4.2\u00a0K with applied fields of 0.2, 4, and 7\u00a0T. They yielded a complex magnetic hyperfine pattern with a large overall splitting of about 10\u00a0mm\/s as shown in Fig.\u00a06. On the basis of the model applied for the fit of the zero-field data, the spectra could be decomposed into three components. The inner part in the range from \u22122.5 to 3\u00a0mm\/s is mostly determined by the contribution from the reduced activator having [4Fe\u20134S]+ clusters with spin S\u00a0=\u00a03\/2 [5]. The component could be reasonably well simulated (subspectrum I, blue lines) by using isomer shift, quadrupole splitting, zero-field splitting, and magnetic hyperfine parameters as published previously for the S\u00a0=\u00a03\/2 species4 [5]. The relative intensity of subspectrum I was fixed to 54% as taken from the zero-field spectrum.\nFig.\u00a06Applied-field M\u00f6ssbauer spectra of titanium(III) citrate reduced activator recorded at 4.2\u00a0K with B\u00a0=\u00a00.2, 4 and 7\u00a0T. The blue lines are spin-Hamiltonian simulations for subspectrum I from reduced [4Fe\u20134S]+ clusters with spin S\u00a0=\u00a03\/2 [5] (Footnote\u00a03). The thick green lines and the shaded areas mark the superposition of the two subspectra IIa and IIb from superreduced [4Fe\u20134S]0 clusters with the parameters as given in Table\u00a01. The individual contribution of subspectrum IIb from the unique Fe(II) site with antiparallel aligned local spin is additionally shown as a thin green line above the other spectra\nThe outer hyperfine lines in the magnetic M\u00f6ssbauer spectra found at about \u22124 and +5.5\u00a0mm\/s are the best indicators for the paramagnetic contribution from the all-ferrous [4Fe\u20134S]0 clusters, which must have large internal fields at the iron nuclei because of the large spin. The magnetic splitting of the corresponding spectra is clearly outside the typical range of hyperfine splitting known for [4Fe\u20134S]+ clusters with spin S\u00a0=\u00a03\/2 or 1\/2. We could readily simulate that contribution from the superreduced activator (Fig.\u00a06, green lines with shaded areas) by using the spin-Hamiltonian for spin St\u00a0=\u00a04 (Eq.\u00a01), amended with the 57Fe hyperfine interaction with four sites grouped in the two subspectra IIa and IIb introduced above. Particularly the field dependence of the M\u00f6ssbauer spectra, which depends critically on the zero-field splitting, is well reproduced with the corresponding zero-field splitting parameters D\u00a0=\u00a0\u22120.66\u00a0cm\u22121 and E\/D\u00a0=\u00a00.165 obtained from the EPR analysis. The complete set of M\u00f6ssbauer parameters used in the simulation of the magnetic spectra are summarized in Table\u00a01. We have to mention, however, that particularly the results obtained for subspectrum IIb are not unique, because of the relatively poor signal-to-noise ratio of the broad spectra and the strong overlap with the remaining starting material, which perturb particularly the analysis of the otherwise informative weak-field spectra. Therefore, the solution given for the anisotropy of the A tensors and the components of the electric field gradient tensors (\u03b7 and the Euler angles \u03b1, \u03b2, and \u03b3) have to be regarded as generic. The main objective of presenting the analysis of the magnetic M\u00f6ssbauer spectra is to show that the data are nicely consistent with the EPR analysis.\nTable\u00a01M\u00f6ssbauer parameters for the all-ferrous [4Fe\u20134S]0 cluster of superreduced activator with spin St\u00a0=\u00a04 at 4.2\u00a0KSubspectrum IIa (three sites, 75% intensity)Subspectrum IIb (one site, 25% intensity)\u03b4 (mm\/s)0.71 (3)0.71 (6)\u0394EQ (mm\/s)+1.80 (5)+2.6 (1)\u03b70.8 (4)0.7 (4)\u03b1 (\u00b0)90a90a\u03b2 (\u00b0)54 (10)125 (40)Ax\/gN\u03b2N (T)\u221210.2 (1)+5.0 (5)Ay\/gN\u03b2N (T)\u22126.4 (1)+8.0 (5)Az\/gN\u03b2N (T)\u22127.23 (2)+7.8 (1)A0\/gN\u03b2N (T)b\u22127.95 (7)+6.6 (4)The zero-field parameters used for the simulation were D\u00a0=\u00a0\u22120.66\u00a0cm\u22121 and E\/D\u00a0=\u00a00.165. The Euler angles \u03b1, \u03b2, and \u03b3  rotate the electric field gradient tensor with respect to the principal axes system of the zero-field splitting. The numbers given in parentheses are rough estimates of the error margins obtained by single-parameter variation and visual inspectionaThe angles \u03b1 and \u03b3 were fixed at 90\u00b0 and 0\u00b0 to reduce the number of parametersbIsotropic value is 1\/3\u00a0tr(A)\nThe three iron sites of subspectrum IIa that are indistinguishable at zero field also could not be further discriminated with applied fields, neither by sign or size of the quadrupole splitting nor by the magnetic hyperfine interaction. The isomer shift and quadrupole splitting appear to be uniform, with values close to those at 140\u00a0K with appropriate corrections for the lower temperature, and the hyperfine splitting can be described by only a single A tensor. Any possible heterogeneity in subspectrum IIa is masked by the anisotropy of A and the rather poor experimental resolution. The isotropic part A0 of the hyperfine tensor is negative for subspectrum\u00a0IIa, which is best seen from the behavior of the resolved outer magnetic lines. They both have a strong component that is moving \u201cinward\u201d with increasing strength of the applied field from B\u00a0=\u00a00.2\u20137\u00a0T, owing to opposing internal and applied fields. Since the corresponding negative sign of A0 is like the sign intrinsically expected for iron(II) with respect to the local spin SFe\u00a0=\u00a02, the three iron sites forming subspectrum IIa must be basically coupled \u201cparallel\u201d to the total spin St\u00a0=\u00a04 of the [4Fe\u20134S]0 cluster.\nThe isotropic value A0 for subspectrum IIb, in contrast, is taken to be positive. This causes the increasing magnetic splitting of a part of the outer hyperfine lines moving outward with increasing applied field. For demonstration of this effect the corresponding subspectrum IIb is also shown separately as a thin green line above the superposition of subspectra\u00a0IIa and IIb and the experimental traces in Fig.\u00a06. Thus, the local spin of the corresponding unique Fe(II) site is predominantly opposite to the total spin of the [4Fe\u20134S]0 cluster. The specific scheme of negative and positive A0 values was adopted from the study of the [4Fe\u20134S]0 cluster in superreduced Fe-protein [31]. There it was also demonstrated that the occurrence of one positive and three negative A values is a necessary common property for any ground multiplet with total spin St\u00a0=\u00a04 that one can obtain from a tetrade of four spins SFe,i\u00a0=\u00a02 with isotropic exchange interaction [31].\nThe effective A-tensor components found for the [4Fe\u20134S]0 cluster of the superreduced activator are relatively weak compared with the intrinsic a0 value for high-spin Fe(II) sites like that of rubredoxins (isotropic part A0\/gN\u03b2N\u00a0=\u00a0\u22127.94 and 6.6\u00a0T for the activator cluster compared with a0\/gN\u03b2N\u00a0=\u00a0\u221216.2\u00a0T for rubredoxin) [32, 43], but they are in the range of those of the [4Fe\u20134S]0 centers from the Fe-protein [A0\u00a0=\u00a0\u221211.2 (\u00b10.2) and 5\u00a0MHz [31], which corresponds to A0\/gN\u03b2N\u00a0=\u00a0\u22128.1 (\u00b10.14) and +3.6\u00a0T, respectively]. The apparent reduction of the effective hyperfine coupling in the cubane clusters is caused by competing antiferromagnetic spin coupling. In the corresponding coupling scheme invoked for the all-ferrous cluster of the Fe-protein [31], the spin-projection factors Ki that describe the effective hyperfine interaction with respect to the total spin of the individual sites, A0(i)\u00a0=\u00a0Kiao(i), have been obtained as K1\u00a0=\u00a0K2\u00a0=\u00a0K3\u00a0=\u00a07\/15 (subsumed in subspectrum IIa here) and K4\u00a0=\u00a0\u22122\/5 (subspectrum IIb). With the a0 value for rubredoxin, a0\/gN\u03b2N\u00a0=\u00a0\u221216.2\u00a0T, one obtains A0(IIa)\/gN\u03b2N\u00a0=\u00a0\u22127.6\u00a0T and A0(IIb)\/gN\u03b2N\u00a0=\u00a06.5\u00a0T. Both values are in reasonable agreement with the experimental result found for the superreduced activator, similar to what was found for the Fe-protein. In summary the spectroscopic properties found for the all-ferrous [4Fe\u20134S]0 cluster from the superreduced activator resemble quite closely those of the superreduced Fe-protein from A. vinelandii nitrogenase.\nDiscussion\nIn the past, cubane [4Fe\u20134S]n+ clusters had only been known to undergo one-electron redox reactions using either the [4Fe\u20134S]+\/2+ couple or the [4Fe\u20134S]2+\/3+ couple, but not both [46]. According to which couple the clusters are supporting, the iron\u2013sulfur proteins were graded as ferredoxin-type proteins or high-potential iron proteins (HIPIPs) [47]. The latter are most abundant in phototrophic organisms. Except for the case of the [4Fe\u20134S]n+ cluster in the HIPIP from Chromatium vinosum, which can be artificially cycled from the +3 to the +1 state [48], the classification appeared to be unique and generally valid, until Watt and Reddy [26] reported that the [4Fe\u20134S]+ cluster of the nitrogenase Fe-protein from A. vinelandii could be reversibly reduced by methyl viologen to an all-ferrous [4Fe\u20134S]0 state where all irons have oxidation state +2. The midpoint potential for this reaction was initially given as \u22120.46\u00a0V versus the standard hydrogen electrode (SHE), but later studies with other reductants reported \u22120.79\u00a0V versus SHE [29], in contrast to \u22120.31\u00a0V for the first reduction (+2\u00a0\u2194\u00a0+1). Titanium(III) citrate reduced nitrogenase Fe-protein was the subject of elaborate EPR and M\u00f6ssbauer spectroscopic investigations, which yielded detailed insight in the electronic structure of the all-ferrous cluster and revealed spin S\u00a0=\u00a04 for the ground state [18, 31]. More recently, the formation of another superreduced state of Av2 with a diamagnetic [4Fe\u20134S]0 cluster and a different electronic absorption spectrum was observed, if the in vivo reductant FldHQ was used [30]. Notably, that system has a sufficiently high redox potential to support nitrogenase catalysis with two-electron transfer by using the (+2\/0) couple of the iron\u2013sulfur cluster.\nHitherto, nitrogenase Fe-protein was the only known enzyme which supported the all-ferrous [4Fe\u20134S]0 cluster. Also the first example of a synthetic all-ferrous [4Fe\u20134S]0 complex with ligands other than phosphane was reported only recently [49, 50]. It represents a very valuable analogue of the biological all-ferrous cluster, since it has been structurally and spectroscopically characterized. The M\u00f6ssbauer spectra of the compound closely match those of titanium(III) citrate reduced Av2. In this paper we have demonstrated that superreduction in biological systems is not a unique phenomenon of the Fe-protein, but also the activator from 2-hydroxyglutaryl-CoA-dehydratase is able to support the [4Fe\u20134S]2+\/+ as well as the [4Fe\u20134S]+\/0 redox cycles of a single cubane cluster.\nThe electronic structures of the superreduced [4Fe\u20134S]0 cluster from the Fe-protein and from the activator appear to have a series of remarkable spectroscopic similarities. First, the UV\u2013vis spectra of both systems exhibit a shoulder-type signal around 500\u2013530\u00a0nm which causes distinct color changes from brown to orange-red upon reduction. We note that reduced iron(II) rubredoxin does not exhibit a similar absorption in the visible region [51]. Moreover, both [4Fe\u20134S]0 clusters exhibit the same total spin St\u00a0=\u00a04 in their ground state, and even the zero-field-splitting parameters compare remarkably well (activator, D\u00a0=\u00a0\u22120.66\u00a0cm\u22121, E\/D\u00a0=\u00a00.165; Fe-protein, D\u00a0=\u00a0\u22120.75\u00a0cm\u22121, E\/D\u00a0=\u00a00.33 [31]). The moderate difference of the rhombicity parameters for the superreduced activator, deviating from the almost fully rhombic limit encountered with superreduced Fe-protein, gives rise to the two distinct lines in the parallel-mode EPR spectra of the activator instead of the strong single line found for the Fe protein.\nThe M\u00f6ssbauer isomer shifts for both centers are very similar and uniform for the respective cluster sites, owing to the same ferrous character of the metal ions. Our data are consistent with the specific distribution pattern of three iron(II) sites with similar quadrupole splitting and similar magnetic hyperfine coupling, and one different iron(II) site with larger quadrupole splitting and positive A tensor found for the Fe protein. In both enzymes the clusters are suspended at the interface of a protein dimer, but the spectra lack any indication of twofold symmetry, as one might expect from the respective protein structures [8, 52]. Since the electric field gradient, which gives rise to the quadrupole splitting, senses only local charge asymmetries, the more uniform appearance of the activator might reflect a smaller asymmetry of the actual cluster coordination in the protein and solvent moiety. Apart from that, the M\u00f6ssbauer lines of the superreduced activator are unusually broad. Therefore we suppose that the molecular structure is probably not very well defined in the frozen solutions and exhibits microheterogeneity at the cluster site which leads to a distribution of quadrupole splittings for each of the iron(II) sites.\nMicroheterogeneity seems also to be the reason for the unusual shape of the EPR signals measured from the superreduced activator. The resonances show a superposition of sharp lines and very broad shoulders centered at similar resonance fields. The corresponding distribution of spin-Hamiltonian parameters, like the distribution of electric field gradients, may be related to the location of the cluster at a sensitive position in the protein structure between two protein monomers [8], where variations in the conformational substates of the protein may have a particularly strong impact on the geometry and electronic properties of the cluster. Such a distribution of spin-Hamiltonian parameters has been observed previously also for the dithionite-reduced activator with [4Fe\u20134S]+ clusters having the unusual spin state S\u00a0=\u00a03\/2 [5] (the unusually broad EPR spectrum is redepicted in the inset in Fig.\u00a02). Here, it is interesting to mention that the [4Fe\u20134S]+ cluster of the Fe-protein has spin S\u00a0=\u00a01\/2 under physiologically reduction conditions, but it also changes to S\u00a0=\u00a03\/2 if it is reduced in the presence of urea [53].\nIn contrast to the situation for the Fe protein from nitrogenase, it was never possible to obtain a complete superreduction of the activator; at best a ratio close to 1:1 could be achieved for [4Fe\u20134S]+ and [4Fe\u20134S]0 clusters. One may speculate whether this is related to the conformational distributions invoked above for interpretation of the spectroscopic heterogeneities. Probably, some fraction within the ensemble of variations may impose such unfavorable conditions on the molecular structure of the cluster and the resulting electronic properties such that further reduction is practically impossible. This interpretation seems to be supported by the observation that hard photoreduction by using deazaflavin as a potent electron source leads to inactivation and precipitation of the protein. However, for the time being also the trivial explanation cannot be excluded, namely, the midpoint redox potential for the [4Fe\u20134S]+\/0 couple could be lower than that of titanium(III) citrate at pH 8.0 (see above), and thus even with a tenfold excess of this agent the cluster only becomes half reduced. We also cannot readily explain why titanium(III) citrate reduced activator is found to be enzymatically inactive, whereas the EPR and M\u00f6ssbauer spectra reveal the presence of about 50% [4Fe\u20134S]+ clusters remaining, which should be catalytically competent. We presume that in the enzymatic assay the activator is fully reduced to the [4Fe\u20134S]0 state because of the very low protein concentration in micromolar range, in contrast to the situation in the highly concentrated spectroscopic samples where the protein is in millimolar abundance.\nSince the ability of a [4Fe\u20134S] cluster to undergo two one-electron redox reactions using both the [4Fe\u20134S]+\/2+ and the [4Fe\u20134S]+\/0 couples should be basically related to its geometry, i.e., the metric details of molecular structure, as well as the (dielectric) properties of the protein site harboring the metal center. In this sense it may not be accidental that both proteins, the nitrogenase Fe-protein and the activator of 2-hydroxyglutaryl-CoA dehydratase, have very similar quaternary structures: homodimeric proteins in which a [4Fe\u20134S] cluster bridges the subunits. In both proteins an \u03b1-helix from each subunit points with its N-terminus towards the cluster and thus may stabilize with its dipole a negative charge, which should increase the redox potential. In the Fe-protein of nitrogenase this helix\u2013cluster\u2013helix architecture forms an angle of 150\u00b0, which upon binding of ATP and interaction with the iron\u2013molybdenum protein opens to 180\u00b0 [17]. Whether this conformational change further influences the redox potential of the cluster remains to be established. The helix\u2013cluster\u2013helix architecture of the activator of 2-hydroxyglutaryl-CoA dehydratase forms an angle of 105\u00b0, which is 45\u00b0 closer than that of the Fe-protein of nitrogenase [8]. Upon ATP binding, the activator angle is thought to open in a similar manner [54], but a crystal structure of the complex between the activator and dehydratase stabilized by AlF4\u2212-ADP (J. Kim and W. Buckel, unpublished results) is not available yet. Most likely the common electronic properties of the [4Fe\u20134S] clusters as well as the common structural features favor a related mechanism of both enzyme systems. Redox potentials are lowered by hydrolysis of ATP in order to achieve chemically difficult reductions of the substrates molecular nitrogen and 2-hydroxyglutaryl-CoA, respectively. However, owing to the observed inability of the superreduced activator to catalyze the enzyme reaction, the [4Fe\u20134S]2+\u00a0\u2192\u00a0[4Fe\u20134S]+ cluster transition and the subsequent electron transfer to the dehydratase component (HgdAB) appears to be the only biologically relevant charge transfer. The situation seems to be more complex for the nitrogenase mechanism. It was postulated that the all-ferrous nitrogenase Fe-protein indeed is an active redox state for nitrogen reduction. First, Watt and Reddy [26] reported that the ATP consumption for nitrogen fixation using all-ferrous Fe-protein is only half of that reported for the [4Fe\u20134S]+ cluster containing Fe-protein (one ATP per two transferred electrons compared with one ATP per one electron) [26]. In the meantime, the principal physiological relevance of the [4Fe\u20134S]0 cluster appears to have been corroborated for the nitrogenase system [30]. Maybe the enzyme can switch the redox pathway to choose between different electron donors for nitrogen reduction available in the cell. A similar role for the all-ferrous cluster of the 2-hydroxyglutaryl-CoA dehydratase system is still elusive.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nComparison of the fit model for the zero-field M\u00f6ssbauer spectra of Ti(III) citrate-treated activator\nwith one and two Lorentzian doublets for the all-ferrous cluster. (PDF 374 kb)","keyphrases":["all-ferrous","superreduced","activator","iron\u2013sulfur protein","2-hydroxyglutaryl-coenzyme a dehydratase"],"prmu":["P","P","P","P","M"]}
{"id":"Eur_J_Nucl_Med_Mol_Imaging-4-1-2275773","title":"99mTc Hynic-rh-Annexin V scintigraphy for in vivo imaging of apoptosis in patients with head and neck cancer treated with chemoradiotherapy\n","text":"Purpose The purpose of this study was to determine the value of 99mTc Hynic-rh-Annexin-V-Scintigraphy (TAVS), a non-invasive in vivo technique to demonstrate apoptosis in patients with head and neck squamous cell carcinoma.\nIntroduction\nApoptosis is an important mechanism of cell death in response to treatment with radiation and many chemotherapeutic agents [1]. To what extent apoptosis contributes to the overall cytotoxic effect of an anti-cancer treatment modality has been the topic of intense research during the last decade [2\u20134]. The relative contribution of apoptosis to the occurrence of cell death varies greatly both between different tumour types and normal tissues [5].\nRecently, in vivo imaging of apoptosis has proven to be feasible by using radiolabelled Annexin V [6\u201310]. This endogenous human protein has a high affinity for membrane-bound lipid phosphatidylserine (PS), which becomes exposed at the outer leaflet of the cell membrane bilayer at an early stage of the apoptotic process [11, 12]. PS then serves as a recognition site for macrophages that digest and remove apoptotic cells. The reproducibility of the 99mTc-Hynic-Annexin-V scintigraphy has been demonstrated in head and neck squamous cell carcinoma (HNSCC) patients by serial imaging in untreated patients [13] in which the mean difference in uptake was 6%. We have recently demonstrated that 99mTc-Hynic-rh-Annexin V scintigraphy (TAVS) correlates with radiation-induced cytologically confirmed apoptosis in non-Hodgkin lymphoma [14] and can be used to identify patients that have a favourable prognosis [15].\nThe current standard of practice for patients with advanced stage HNSCC is treatment with concurrent cisplatin-based chemoradiation [16, 17]. Although this is an effective treatment, it also is accompanied by more severe toxicity than radiation alone. We reasoned that TAVS early during treatment might be used to monitor apoptosis induction in tumour and normal tissue and to give an indication of the radiosensitivity of these structures. In future, this would also offer the possibility to adapt the treatment strategy at an early stage on a patient-by-patient basis.\nIn the present study, we therefore aimed at assessing the feasibility of TAVS as a non-invasive technique to demonstrate treatment-induced apoptosis in vivo, in patients with HNSCC, early during treatment with concurrent chemoradiation. The purpose was to determine the degree of uptake on TAVS in normal tissue, primary tumour and lymph node metastases and to evaluate the treatment-induced Annexin uptake in relation to radiation dose. Furthermore, we questioned whether the differences in uptake would correlate with treatment response.\nMaterials and methods\nPatients included in this study were recruited between January 2004 and March 2005 from a randomised phase III trial in advanced stage HNSCC, investigating the optimal route of cisplatin delivery during cisplatin-based chemoradiation (RADPLAT). Randomisation was between two treatment arms: Arm 1, standard intravenous (IV) administration of cisplatin 100\u00a0mg\/m2 (1\u00a0h before radiotherapy at days 1, 22 and 43) or arm 2, high-dose selective intra-arterial (IA) delivery of cisplatin at a dose of 150\u00a0mg\/m2 (on days 2, 9, 16 and 23, within 1\u00a0h after radiotherapy delivery). Main eligibility criteria included: inoperable squamous cell carcinoma of the oral cavity, oropharynx or hypopharynx, TNM stage T3-4 status of primary oral cavity or oropharyngeal tumours and T2-3-4 for hypopharyngeal tumours, with any N-status, (functionally) inoperable disease, no distant metastases, age at least 18\u00a0years, ability to give informed consent and no prior cerebro-vascular accident. Both the randomised trial and the TAVS study were approved by the medical ethics committee of the hospital. Patients were informed about the nature of the study protocol and signed informed consent before enrolment separately for both the randomised trial and the TAVS protocol.\nRadiotherapy was given with 4\u20136\u00a0MV photon linear accelerators. Target volume included the primary tumour and the bilateral neck for a dose of 46\u00a0Gy in 23 fractions. A boost was given to the known macroscopic tumour extensions at the primary tumour site and lymph node metastases to a dose of 24\u00a0Gy in 12 fractions. The total dose delivered was 70\u00a0Gy in 35 fractions, five fractions per week, with an overall treatment time of 7\u00a0weeks. The radiation technique was either a conventional three-field beam setup (using conventional simulation or virtual simulation with CT scan) or an intensity-modulated radiotherapy (IMRT) plan depending on resources and tumour extent. The IA cisplatin delivery was accomplished by a selective catheterisation procedure using the femoral artery according to the earlier described RADPLAT protocol [18, 19]. Concurrently, with IA cisplatin sodium-thiosulphate was administered to neutralise systemic cisplatin. In both arms, prehydration and posthydration were given.\n99mTc-Hynic-rh-Annexin V scintigraphy, diagnostic imaging, radiation treatment planning and image fusion\nBaseline TAVS was performed within 2\u00a0weeks before the start of concurrent chemoradiation (average interval 5\u00a0days, range 1\u20138). Post-treatment TAVS was done within 48\u00a0h after the start of cisplatin chemotherapy. At the time of post-treatment TAVS, patients had received 6\u00a0Gy of radiation in case of IV cisplatin, 8\u00a0Gy in case of IA cisplatin. Each patient received an average of 847\u00a0MBq (range 714\u20131032\u00a0MBq) of 99mTc-Hynic-rh-Annexin V (Theseus Imaging Corporation, Boston, USA) by slow intravenous injection 4\u00a0h before the planar imaging and single-photon emission tomography (SPECT) imaging. Planar images were used to assess the biodistribution. SPECT of the head and neck region was acquired by the step-and-shoot mode, one step per 3\u00b0, 30\u00a0s per frame, matrix size 128\u2009\u00d7\u2009128, using a dual-head gamma camera (Genesis, Philips, Best, The Netherlands) equipped with low-energy, high-resolution collimators. For SPECT reconstructions, an iterative algorithm was used and the images were postfiltered using a Butterworth filter (cutoff frequency 0.35, order 5). The use of absolute quantitative analysis of SPECT data was validated by comparison of iterative and FBP reconstruction methods. Transaxial, coronal and sagittal slices were visually examined to evaluate tracer uptake at the tumour sites and in normal tissues. The intensity of the obtained images was corrected by normalisation for the injected radioactive dose and body weight.\nBaseline diagnostic imaging with MRI (1.5-T system; Somatom; Siemens Medical Systems, Erlangen, Germany) or spiral CT (Tomoscan AVE1, Philips, Best, The Netherlands or HiSpeed CT, GE Medical Systems, USA) was performed within 3\u00a0weeks before the start of treatment and repeated 6\u20138\u00a0weeks after the end of the treatment for the evaluation of treatment response.\nRadiation treatment planning was done with our clinical treatment planning system (TPS; U-MPlan, University of Michigan, Ann Arbor, USA). Six patients were treated by IMRT, and the treatment plans were recalculated for the dose at the time of first post-treatment TAVS. The regions of interest (ROI) were delineated manually for each patient on the CT scan. These included the gross tumor volume (GTV) of the primary tumour and\/or lymph nodes, the parotid glands and the submandibular glands. In the other seven patients, a standard three-field technique was used by virtual simulation with a CT scan. In these latter patients, the CT scan was imported into the TPS in which the delineations were done. The treatment fields were reconstructed, and the clinically applied dose distribution at the time of TAVS was recalculated in the TPS. The primary tumour and lymph node volumes were calculated by 3-D reconstruction of the delineated GTV.\nSPECT, MRI and CT were performed separately. For the SPECT scan, we obtained reproducibility of the positioning of the head and neck as during RT by the use of immobilisation mask fixed to the table by adhesive tape. All images and the radiation dose data were transferred for image fusion to our in-house developed workstation for co-registration (Worldmatch Workstation) in DICOM format [20]. Keeping the limitations of the mask fixation in mind, it was decided that for more accuracy, matching was performed on different anatomical regions for different ROIs, e.g. for the analysis of parotid glands or oropharyngeal tumours, the co-registration was done in such a way that it ensured adequate matching for that area only. For co-registration of ROIs in the neck (e.g. lymph nodes or hypopharyngeal tumours), only the neck and vertebral column was used in the matching procedure. Accurate matching of body contours and bone structures was visually verified. SPECT, composite SPECT\/SPECT, CT and SPECT\/CT images, obtained using a colourwash technique, were simultaneously examined using linked cursor to evaluate Annexin V uptake in tumour and normal tissues.\nThe tumour and normal tissue uptake at baseline and post-treatment were calculated as follows. ROIs were delineated in the planning CT. Each ROI was projected using the registration transformation onto the baseline and post-treatment SPECT scans. The area was next sampled by 10.000 random points, and for each point, the Annexin uptake was determined in the SPECT scans by trilinear interpolation. Of these 10.000 samples, the maximum and mean values were computed. In this way, the uptake in the ROI was accurately sampled, although the pixel size in the SPECT scans is relatively large (about 5\u00a0mm). For the quantification of Annexin uptake, no attenuation correction was performed because the images were obtained on a conventional gamma camera without a hybrid system. Subsequently, the difference (\u0394U) between the post-treatment and baseline uptake was determined by subtraction of the baseline scan from the post-treatment scan. The subtraction was performed on a point by point basis for all 10.000 points. Then, the mean or maximum value of the difference of both scans (subtraction scan) was computed and expressed as \u0394U. No correction for background activity was made, as this was automatically eliminated by subtraction of the baseline activity from the post-treatment activity, assuming that the background activity is equal in the baseline and post-treatment scan. Radiation dose parameters (mean and maximum dose in cGy) within each ROI were calculated and correlated with corresponding \u0394U parameters. It was observed that the maximum \u0394U and the mean \u0394U were closely related: a linear relationship between both two parameters was found for each ROI (tumour: r\u2009=\u20090.93, p\u2009<\u20090.0001; parotid gland: r\u2009=\u20090.94, p\u2009<\u20090.0001; submandibular gland: r\u2009=\u20090.90, p\u2009<\u20090.0001; lymph node r\u2009=\u20090.94, p\u2009<\u20090.0001), indicating that both values would have been representative for the whole ROI.\nEvaluation of treatment\nSix to eight weeks after the end of treatment, the results of therapy were evaluated by means of radiological investigations (by MRI or CT scan and\/or ultrasound) and examination under general anaesthesia, with biopsies taken in case of suspicious findings. For residual disease in the neck at the time of evaluation, salvage neck dissection was performed if the patient was judged operable. Follow-up visits were planned every 3\u00a0months in the first year after therapy, every 4\u00a0months in the second year and less frequent thereafter. A follow-up chest X-ray was performed annually.\nStatistical analysis\nFor quantitative comparison of continuous data Student\u2019s t test was applied. Chi-squared and Fisher\u2019s exact test were used for analysis of categorical data. The Pearson and Spearman rank correlation coefficient r were used to calculate correlations between Annexin uptake and treatment parameters. Locoregional control and survival data were calculated from the start of treatment using the Kaplan\u2013Meier method and log rank testing. Two-sided P values of <0.05 were considered statistically significant.\nResults\nSixteen patients gave their consent for participating in the TAVS study. However, one patient refused post-treatment TAVS scans; in one patient, no CT scan in radiation treatment position was available, and in one patient, co-registration with SPECT was unsuccessful due to misalignment because no immobilisation mask and no tongue depressor could be used during SPECT due to pain (Fig.\u00a01). The number of patients included in this analysis on 99mTc-Hynic-rh-Annexin V scintigraphy in advanced stage HNSCC is therefore 13. The mean age of patients was 53\u00a0years (range 26\u201366\u00a0years). See Table\u00a01 for the patient, tumour and treatment characteristics.\nFig.\u00a01Sagittal projection of TAVS imaging co-registered with planning CT scan. The solid line represents the contour of the patient during RT-planning CT scan, including tongue depressor. The projected image is the TAVS. a Baseline TAVS; b post-treatment TAVS. Note the mismatch of contours of the head and neck mostly due to different position of the chin in b. The misalignment of the lower neck in a is due to the flexion of the neck. For the analysis, the matching procedure was focused on the mandibular region. This patient was excluded from analysis because of misalignment in bTable\u00a01Patient, tumour and treatment characteristicsPatient numberGenderAge (years)Tumor siteTNM stageTumor volume (cm3)Max \u0394U in primary tumourInterval cisplatin infusion and Annexin scan (h)RT techniqueMode of cisplatin administration1M64Oral cavityIII216252.2Conventional 3 fieldIntra-arterially2M58OropharynxIII4720350.3IMRTIntra-arterially3F65OropharynxIV2511253.1IMRTIntra-arterially4M55OropharynxIII263653.5IMRTIntra-arterially5M49HypopharynxII134353.3IMRTIntra-arterially6F66Oral cavityIV3213553.6Conventional 3 fieldIntra-arterially7M60OropharynxIV14719154.3Conventional 3 fieldIntra-arterially8M46OropharynxIV2817052.7Conventional 3 fieldIntra-arterially9M55OropharynxIV414249.9IMRTIntravenously10M57OropharynxIV255151.7IMRTIntravenously11M48OropharynxIV9111751.5Conventional 3 fieldIntravenously12M50HypopharynxIV10013250.3Conventional 3 fieldIntravenously13M26OropharynxIV1055250.1Conventional 3 fieldIntravenouslyM Male, F female, IMRT intensity modulated radiotherapy\nAnnexin uptake in normal tissue and tumour\nAt baseline scintigraphy, reproducible physiologic Annexin uptake was detected on the planar images in liver, kidneys, spleen, gall bladder, bone marrow, colon and urinary bladder, as described earlier [7, 14, 21].\nIn the parotid glands, weak baseline uptake was present in all cases. The post-treatment TAVS showed moderate to strong increase in uptake in 24 of 26 parotid glands (Fig.\u00a02a). The average of the mean number of counts increased from 55 to 88 (p\u2009<\u20090.001). Visual analysis of the increase in uptake showed that these changes were related to the radiation portals and the dose given to the parotid glands (Fig.\u00a03). The difference in Annexin uptake between the post-treatment and baseline TAVS (\u0394U) in each parotid gland was correlated with the radiation dose at the time of post-treatment scintigraphy (n\u2009=\u200913 patients, n\u2009=\u200926 parotids). The mean \u0394U showed a positive correlation with the mean radiation dose (Pearson coefficient r\u2009=\u20090.59, p\u2009=\u20090.002): parotid glands that received a higher dose of radiation showed a higher Annexin uptake (Fig.\u00a04a). The increase in Annexin uptake in parotid glands that were treated with parotid-sparing IMRT was less than in parotid glands that were treated with a conventional three-field technique (IMRT 29 counts, three-field technique 59 counts, p\u2009=\u20090.02). The given radiation dose at the time of post-treatment TAVS (6 or 8\u00a0Gy) and the mode of cisplatin administration (IA or IV) did not affect the uptake in parotid glands. As xerostomia scoring was not documented sufficiently detailed in the files, we interviewed patients alive at last follow-up for xerostomia grading for the purpose of this study. In nine patients alive at last follow-up, the treatment-induced Annexin uptake in parotid glands was related to the subjective xerostomia rating using the EORTC QLQ HN-35 questionnaire [22]. No relation could be established in this small set of patients (data not shown).\nFig.\u00a02Baseline (diamonds) and post-treatment (squares) Annexin activity in parotid glands (a), submandibular glands (b) and tumour (c) for all 13 patients. The data for 26 parotid and submandibular glands represent the right and left gland within each patientFig.\u00a03TAVS imaging co-registered with planning CT scan in frontal plane and axial plane, a and c at baseline, b and d after treatment, from patient number 2. The Annexin uptake is represented by colourwash. In b and d, the isodose lines show the dose distribution in relation to the parotid gland and primary tumour (isodose lines shown, 40, 60, 80 and 95% from outer to inner side of patient). Note the increased treatment-induced Annexin uptake in the right parotid gland, in correspondence to the higher radiation dose distribution, when compared to the left parotid gland. Also note the weak increase in primary tumour Annexin uptake in the right oropharynx after treatment and in the anterior floor of mouth (d)Fig.\u00a04Correlation plot of mean radiation dose (cGy) and the post-treatment increase in Annexin uptake, mean \u0394U, in parotid glands (a), normalised for tracer dose and body weight. Circles represent parotid glands treated with IMRT, triangles parotids treated with conventional technique. In b, the correlation between mean radiation dose (cGy) and the post-treatment increase in Annexin uptake, mean \u0394U, in submandibular glands is given\nFor the submandibular glands, a similar pattern as with the parotids was observed: absent\/weak baseline uptake and moderate to strong increase after the start of chemoradiation (Fig.\u00a02b). The average of the mean number of counts increased from 85 to 132 (p\u2009<\u20090.001). No correlation between the \u0394U and radiation dose was noted (Fig.\u00a04b), probably as all submandibular glands were located within the high-dose region.\nThe baseline TAVS showed moderate to strong Annexin uptake in the primary tumour in all patients, indicative of spontaneous apoptosis or necrosis. On the post-treatment TAVS, the uptake in tumour clearly increased in nine cases; in the other four, little or no changes occurred (Fig.\u00a02c). The average of the maximum number of counts increased from 127 to 163 (p\u2009=\u20090.007). The difference between post-treatment and baseline TAVS (\u0394U) in primary tumour was not correlated with primary tumour volume (Pearson coefficient r\u2009=\u20090.39, p\u2009=\u20090.21) nor with the mode of chemotherapy administration (p\u2009=\u20090.21). In nine patients, 17 lymph node metastases were present in the neck (mean lymph node volume 5\u00a0cm3, range 1\u201319). The treatment-induced Annexin uptake (maximum \u0394U) in primary tumour was positively correlated with the uptake in corresponding lymph node metastases (r\u2009=\u20090.73, p\u2009=\u20090.004) as shown in Fig.\u00a05. To verify that the observed effects of Annexin uptake in different structures could not be attributed solely to the administered radioactive dose per patient but was tissue-specific, we calculated the correlation between the \u0394U in tumour and parotid glands. No evidence of such an effect was found (r\u2009=\u20090.28, p\u2009=\u20090.18).\nFig.\u00a05Correlation plot of the treatment-induced increase in Annexin uptake, maximum \u0394U, in primary tumour and lymph node metastases. Each colour represents an individual patient. Some patients had multiple lymph node metastases originating from the same primary tumour. In these cases, multiple values of treatment-induced Annexin uptake in lymph nodes (on the y-axis) correspond to a single value of the primary tumor (on the x-axis)\nResponse to treatment, locoregional control and survival\nThe median follow-up of all patients alive at last follow-up was 30\u00a0months (range 24\u201338). The complete response (CR) rate at the primary tumour site was 85%. CR rate in the neck in the case of nodal metastases was 100%. The overall CR rate was 85%. The estimated locoregional (LR) tumour control rates were 68% at 2 and 3\u00a0years. The rates for disease-free survival (DFS) were 54% at 2 and 3\u00a0years. Overall survival (OS) rates at 2 and 3\u00a0years were 62 and 40%, respectively. In this small subset of 13 patients from a larger randomised phase III trial, there were no differences in response rates, LR control, DFS or OS between the two treatment arms (IV vs IA). No correlation could be established between baseline or treatment-induced Annexin uptake (\u0394U) and any outcome parameter (response rate, recurrences and\/or survival).\nDiscussion\nThis study represents our first clinical experience on in vivo imaging of apoptosis with TAVS in HNSCC patients in which we applied co-registration of multiple imaging modalities. By this, we were able to analyse treatment-induced changes in normal and tumour tissue, early during therapy, after one cycle of cisplatin chemotherapy and 6\u20138\u00a0Gy of radiotherapy. Treatment-induced Annexin uptake in the parotid glands could be visualised, indicative of early treatment related apoptosis at mean radiation doses as low as 3\u20138\u00a0Gy (see Fig.\u00a04a) and one course of cisplatin. The Annexin uptake in the parotids showed a radiation dose-response relationship: Glands that had received higher doses of radiation demonstrated increased Annexin uptake.\nLoss of parotid gland function, leading to xerostomia, is an important long-term side effect of radiotherapy, affecting quality of life of patients. It has well been shown by scintigraphy studies [23] and by salivary flow rate studies [24] that reduction of radiation dose to the parotid glands can maintain parotid function, decrease xerostomia and improve quality of life. Most studies on salivary gland function are performed after a full course of RT (typically 60\u201370\u00a0Gy in 6\u20137\u00a0weeks) [23\u201326]. Our study design with early in vivo imaging indicated that already after low dose of RT (6\u20138\u00a0Gy), parotid glands may be affected and that the physiologic process leading to loss of parotid gland function already starts early. This is in agreement with observations in experimental rodent studies in which apoptosis was induced early, after doses of up to 5\u00a0Gy [27]. Similar results were obtained in a monkey model, with early radiation induced apoptosis of serous acinar cells of salivary glands [28]. It was suggested that these early effects are mainly responsible for the acute sialoadenitis, which, at higher RT doses, might progress in chronic xerostomia by damage to and depletion of ductal stem cells within the gland. In the nine patients assessed for xerostomia, no relation could be established with subjective salivary gland function after radiotherapy and parotid gland Annexin uptake. However, the significance of this analysis is limited, as we assessed both parotid glands separately by TAVS, and xerostomia questionnaires give an overall impression of function. We did not include follow-up salivary flow studies. In short, our results indicate indirect evidence of early radiation-induced salivary gland damage and, in future studies, this needs to be correlated with functional outcome parameters.\nDue to the large inter-patient variability in the baseline TAVS (Fig.\u00a02c), the subtraction method for assessing treatment-induced changes was chosen over a relative method (e.g. percentage change). In this figure, it is important to notice that the maximum uptake value of the baseline and post-treatment scan might be located in different points within the tumour ROI. Therefore, these data cannot be simply subtracted, but for the calculation of the maximum \u0394U in primary tumour and lymph nodes, the baseline scan was first subtracted from the post-treatment scan, and, subsequently, these parameters were calculated from the subtraction scan. The treatment-induced Annexin uptake in primary tumour and pathological lymph nodes showed a positive correlation: Patients with primary tumours with a high Annexin uptake also appeared to have lymph node metastases with high Annexin uptakes. The \u0394U in primary tumour and lymph node metastases showed large inter-patient differences (Fig.\u00a05). This variation could not be attributed to differences in the route of cisplatin administration (intravenous or intra-arterial chemoradiation), nor to the radiation doses given (6 or 8\u00a0Gy), nor to the primary tumour volume. This positive correlation for \u0394U in primary tumour and lymph nodes as well as the large inter-patient differences might represent a tumour-specific apoptotic response.\nIn our series, repositioning of the patient during SPECT scan in RT position was standard procedure, but due to the limitations in fixation, not optimal. The repositioning facilitated the co-registration of SPECT and CT scan. In one patient, misalignment occurred because the positioning was not accurate due to the fact that the RT position could not be reproduced (Fig.\u00a01). So, for co-registration of (functional) imaging to RT-planning CT scan data in HNSCC, we recommend improvements from our protocol to obtain a reproducible positioning, like the use of an immobilisation mask fixed to the SPECT table, the use of the same mattress under the patient and a laser alignment system. In ongoing studies, we have now incorporated these. In the current series, no attenuation correction was applied because the images were obtained on a conventional gamma camera without a hybrid system. For the purpose of this study, the quantification of Annexin uptake remains valid, as we used the relative increase from baseline to post-treatment uptake, and the degree of attenuation will be equal on both scans.\nIn this small series of HNSCC, the treatment-induced Annexin uptake (\u0394U) in primary tumour did not predict outcome: No correlation between Annexin uptake and early response was observed probably partly due to the high initial response rates (85%). In addition, TAVS also did not predict the locoregional control rates within the first 2\u00a0years of follow-up. In our previous experience on TAVS in other tumour sites (mainly lymphoma), a strong correlation has been found between early response (within 3\u00a0months) and Annexin uptake [15]. One explanation for the lack of correlation might be that in the current series, these advanced stages HNSCC with large tumours harbour more necrosis, which is also detected by TAVS because of accessibility of PS at the necrotic cell membrane [29]. Van de Wiele et al. [8] showed that in HNSCC, Annexin scintigraphy correlated with histopathological apoptosis (using TUNEL assay) only in the absence of necrosis. Indeed, in this series, baseline Annexin uptake in the HNSCC patients was clearly demonstrated, whereas it was not present in our previous study in lymphoma patients [15]. This may suggest the presence of necrosis in large tumours from solid origin. Other factors that may affect Annexin uptake are the intra- and peri-tumoural lymphocyte infiltration [30]. For the future, we plan to increase the number of HNSCC patients to undergo the TAVS imaging to prove whether or not it can be used for outcome prediction as it was shown in patients with malignant lymphoma and non-small cell lung cancer [14, 15].\nIn conclusion, co-registration of Annexin V scintigraphy with radiotherapy-planning CT scan showed a radiation-dose-dependent uptake in parotid glands, indicative of early apoptosis during treatment. The inter-individual spread in Annexin uptake in primary tumours could not be related to differences in treatment schedule or tumour volume, but the Annexin uptake in tumour and lymph nodes were closely correlated. This effect might represent a tumour-specific apoptotic response.","keyphrases":["annexin","scintigraphy","apoptosis","head and neck cancer","parotid gland"],"prmu":["P","P","P","P","P"]}
{"id":"Med_Biol_Eng_Comput-3-1-2039845","title":"A newly developed tool for intra-tracheal temperature and humidity assessment in laryngectomized individuals: the Airway Climate Explorer (ACE)\n","text":"The aim of this study is to develop a postlaryngectomy airway climate explorer (ACE) for assessment of intratracheal temperature and humidity and of influence of heat and moisture exchangers (HMEs). Engineering goals were within-device condensation prevention and fast response time characteristics. The ACE consists of a small diameter, heated air-sampling catheter connected to a heated sensor house, containing a humidity sensor. Air is sucked through the catheter by a controlled-flow pump. Validation was performed in a climate chamber using a calibrated reference sensor and in a two-flow system. Additionally, the analyser was tested in vivo. Over the clinically relevant range of humidity values (5\u201342 mg H2O\/l air) the sensor output highly correlates with the reference sensor readings (R2 > 0.99). The 1\u20131\/e response times are all <0.5 s. A first in vivo pilot measurement was successful. The newly developed, verified, fast-responding ACE is suitable for postlaryngectomy airway climate assessment.\nIntroduction\nTotal laryngectomy results in a permanent disconnection of the upper and lower airways, and leads to an increase in chronic pulmonary complaints like frequent involuntary coughing, sputum production, and repeated daily forced expectoration in order to clear the airways [5]. Heat and moisture exchangers (HMEs) were developed in an attempt to compensate for the lost functions of the upper airway and have been found to reduce these symptoms and improve the quality of life [1, 4, 7].\nIn vitro, the moisture exchanging capacity of the HME can be specified with the ISO 9360 standard. However, this standard does not incorporate a standardized method for determining the heat exchange of the HME, and it is not possible to translate the results obtained with the lung model of the ISO 9360 standard to the true in vivo situation. Therefore several questions remain unanswered. Fundamentally: what is the in vivo heat and moisture exchanging capacity of the HME? And clinically, how does the in vivo heat and moisture exchanging capacity relate to that of the upper airway, i.e. where does the HME stand in the range from open stoma to nose breathing? Is a further improvement of the HMEs heat and moisture exchanging capacity possible and desirable, and does this result in a further decrease of the pulmonary complaints due to the use of this rehabilitation device?\nMeasuring intra airway temperature and humidity is a technically complex issue [15]. Assessing the temperature and humidity variations during normal breathing requires fast sensor response characteristics. No appropriate commercial systems for this purpose are available, and only a few self-constructed validated systems have been published [6, 8, 10, 11, 13, 14]. Two groups evaluated the influence of an HME on intra airway temperature and humidity in laryngectomees. In one study [10] the sensor response time was up to 9\u00a0s and in the other study [8, 9] a response time of 2\u00a0s is reported which has been shown to be sufficiently fast for normal breathing volunteers, but which might be insufficient for patients.\nOur goal was to develop a verified intra-tracheal airway climate analyser with response characteristics fast enough for assessment of end tidal intra-airway temperature and humidity variations during normal breathing in laryngectomized individuals, and for evaluation of the influence of HMEs on these parameters.\nDesign considerations\nPlacement of a humidity sensor in the airway itself was rejected. Humidity sensors must be kept clean, and a construction to protect a sensor would be so large that the aerodynamics within the airway would be disturbed. A high airflow around the sensor is required to minimize the response time of the sensor. A constant airflow is necessary for a reliable interpretation of the time dependent signal, and in addition facilitates the use of deconvolution to optimize system response characteristics. Therefore, a sensor environment had to be created in which airflow remains constant and high during the whole breathing cycle, i.e. in a \u201cchamber\u201d. In order to disturb airway aerodynamics as little as possible, a small diameter sample catheter for transportation of breathing air outside the airway to a humidity sensor is required. We decided that the lumen surface of the sample catheter should not exceed 10% of the lumen surface of the trachea. A decrease in lumen area of maximal 10% will result, by the law of mass conservation, in an increase in local flow velocity of 10%. This on its turn will lead to a transbronchial pressure drop of maximal 20% which is supposed to be small enough to avoid substantial deformation of the trachea wall. This means that based on a diameter of 2\u00a0cm, which is representative for the trachea of an adult person, the maximal diameter of the sample catheter should not be more than 6\u00a0mm.\nIn order to prevent condensation of water, the temperature of the measurement system must not be below the temperature of the breathing air. In addition, high humidity levels should be avoided to prevent hysteresis of the humidity sensor. Based on the maximal, i.e. end expiratory, temperature of about 36\u00b0C, with a relative humidity (RH) of about 97% [16], the breathing air must be heated to at least 38\u00b0C in order not to exceed 90% RH (http:\/\/www.thunderscientific.com\/web_humicalc\/index.php). On the other hand, a heated sample catheter should be thermally isolated in order to prevent patient discomfort and artificial heating of inspired air.\nWe decided to limit the sample rate to less than 10% of the respiratory minute volume. Based on a breathing frequency of at least 12\u00a0cycles\/minute and a tidal volume of 0.5\u00a0l for an adult human at rest, the sample rate should be 0.6\u00a0l\/min at most.\nFinally the catheter should be easy to manipulate and it should be possible to insert it into the trachea even if an HME filter is in situ. The primary measuring site for the sensor should be about 1\u00a0cm behind the HME, but preferentially, measurements deeper in the trachea should be possible as well.\nComponents\nAirway Climate Explorer\nBreathing air is sampled through a 0.8\u00a0mm diameter central canal of a multilumen tube (see Fig.\u00a01a, b.). Four of the six surrounding canals are threaded with two resistance wire loops in order to facilitate catheter heating. Resistance wire was chosen to minimize current load in case of unexpected patient contact. Two surrounding canals were threaded with a copper wire loop in order to measure temperature for the system heating control unit. A thermocouple (MLT1402 T-type Ultra Fast Thermocouple Probe (IT-23), ADInstruments Ltd, Oxfordshire, UK) was placed just inside the tip of the central canal, in order to establish a constant airflow around this sensor. The thermal response of the thermocouple is specified as 5\u00a0ms, accuracy \u00b10.1\u00b0C. The distal 2\u00a0cm of the sample catheter is not actively heated in order to prevent retrograde heating of the thermocouple. The multilumen tube is surrounded by a silicone isolation tube with an outer diameter of 5\u00a0mm. In between both tubes, a small layer of air increases thermal isolation. The length of the sample catheter is 30\u00a0cm.\nFig.\u00a01Drawings of the sample catheter; a 3D overview with enlargement of the tip. b cross section (total diameter 5\u00a0mm)\nThe proximal end of the catheter is connected to an aluminium sensor house, incorporating the humidity sensor (see Fig.\u00a02). The humidity sensor most suitable for our purposes appeared to be the capacitive humidity sensor of a radiosonde (RS92K, Vaisala Oyj, Helsinki, Finland). The technical specifications report a response time of <0.5\u00a0s at 20\u00b0C and <20\u00a0s at \u221240\u00b0C, both at an airflow of 6\u00a0m\/s. The humidity sensor is placed in an aluminium sensor house that is wrapped with copper wire for heating. The humidity sensor is mounted on a sensor beam and consists of a small chip with the sensor polymer and an aluminium wire that in the original radiosonde served as a heating wire, but which we use to measure the temperature of the humidity sensor in situ (see Fig.\u00a03). Apart from the humidity sensor chip, the sensor beam also contains a temperature sensor, but this was not used, as it is located relatively far from the humidity sensor chip.\nFig.\u00a02Cross section of the sensor house; I connection to catheter, II humidity sensor, III copper wire for heating of the sensor house, IV droplet interceptor with electrode for fluid detection, V valve for disconnection of pressure gradient in case of fluid detection in the droplet interceptor, VI thermocouple wireFig.\u00a03Details of the humidity sensor (dimensions 4\u00a0\u00d7\u00a01.5\u00a0mm2)\nThe sensor house is heated to avoid prolonged saturation of the sensor [12, 15]. The temperature is kept stable within 0.2\u00b0C. Therefore the output of the capacitive sensor which is sensitive for the relative humidity can be directly converted to absolute humidity. Because a radiosonde is not designed for long-term use, the sensor house was constructed such that it could be easily opened to replace the sensor beam. The diameter of the air canal that leads to the humidity sensor is 1.4\u00a0mm. The air inflow passes through a droplet interceptor in the sensor house in order to prevent sucked up sputum from being projected onto the sensor chip. In the bottom of the droplet interceptor an electrode is placed in order to detect fluid accumulation, in which case a mechanic valve instantly disconnects the pressure gradient through catheter and sensor house, which is established by a vacuum pump (DIVAC 0.6, Leybold Vacuum BV, Utrecht, the Netherlands). The flow rate is controlled and kept constant at 0.6\u00a0l\/min, by a mass flow meter\/controller (Smart Mass Flow Meter, type 5850S and Smart Control V1.4 Software, Brooks Instruments, Veenendaal, the Netherlands), which is placed between the sensor house and the pump.\nFigure\u00a04 summarizes the airway climate explorer in a block diagram. In this figure also the custom-made electronic module is shown. The main functions of the module are: processing of the humidity sensor signal and its associated temperature signal, i.e. heating of both the sample catheter via the resistance wire, and the sensor house via the copper wire, each to their own adjustable set points. The electronic principles of the design of the module are described in the appendix.\nFig.\u00a04Block diagram of the airway climate explorer\nFor patient safety, the total assembly is connected to a safety-isolating transformer.\nData acquisition and signal processing\nAll signals of the total assembly, i.e. voltage output of the sensor polymer and the aluminium wire on the humidity sensor of the ACE, temperature signal of the thermocouple, sample rate of the mass flow controller, and all ancillary equipment (see below), are read out simultaneously via a multichannel data acquisition system with additional software (Powerlab and Chart 5.4.1 software, ADInstruments Ltd, Oxfordshire, UK) on a PC. For deconvolution of the raw humidity output of the ACE, the software application LabVIEW (National Instruments Netherlands BV, Woerden, The Netherlands) is used.\nCleaning and disinfection protocol\nPrior to every in vivo measurement, the sample catheter is cleaned and disinfected with the following procedure. First the catheter is rinsed with tap water. Subsequently the catheter is cleaned with disinfection hand soap (Baktolin Basic, Bode Chemie, Hamburg, Germany) and rinsed again with tap water. Then the catheter is placed in a solution of Biotex (Sara Lee, Utrecht, The Netherlands) and tap water for 10\u00a0min, after which it is placed in a 70% alcohol\u2013water solution for 5\u00a0min. During the disinfection procedure, chemicals are prevented from entering the central canal of the sample catheter by establishing retrograde airflow, in order to protect the humidity sensor polymer. The central canal is not sterilized because during use there is continuous suction of air towards the vacuum pump. Finally the catheter is dried in room air.\nAncillary equipment for frequent in hospital calibration\nFor frequent, in-house, temperature calibrations a can of water is used in which the thermocouple is placed, simultaneously with a calibrated thermometer (Thermalite, Electronic Temperature Instruments Ltd, Worthing, UK), accuracy: \u00b10.4\u00b0C, acting as a secondary reference. By using water with melting ice or warm tap water, the full temperature range can be quickly calibrated.\nFor frequent humidity calibrations, a small climate room was constructed. This climate room consists of a plexiglas box of 26\u00a0\u00d7\u00a042\u00a0\u00d7\u00a016\u00a0cm3, in which moisturized air can be brought via the distal end of the tube of a neonatal respiratory humidifier (MR730 Respiratory humidifier, Fisher & Paykel Healthcare, Papendrecht, the Netherlands). Air is mixed via an electromotor-driven propeller (25\u00a0cm diameter). The box has entries for the tip of the sample catheter and for a calibrated heated humidity and temperature sensor accuracy \u00b10.6\u00b0C and \u00b12.5% relative humidity (RH), (Testo B.V., Almere, the Netherlands), which is used as a secondary humidity reference. The system can be operated in ambient air (30\u201360% RH), can be connected to the hospital oxygen supply, and can be set to deliver dry or 100% humidified gas at room temperature.\nAssessment of step-response characteristics was performed in a two-stream system, which consisted of two tubes that were mounted side by side, connected to\u2014in absence of compressed room air facilities\u2014the hospital\u2019s oxygen supply. Through one tube, dry oxygen flowed, and through the other the oxygen was heated and 100% humidified with the respiratory humidifier. Subsequently, the sample catheter tip was manually switched between both tube lumina.\nLeakage testing is performed by checking the time required to completely empty a flexible sack that is filled with a well-defined volume of air (1\u00a0l). This volume is pushed into the sack with a calibration syringe (Jaeger GmbH\/VIASYS Healthcare GmbH, Hoechberg, Germany).\nEnvironmental temperature and humidity are monitored with the heated humidity and the temperature sensor.\nCalibration and verification \nFull calibration of the system was performed with one single sensor beam. However, due to the disposable design of a radiosonde and the possibility of damaging the sensor during the measurements, most likely several sensor beams will be used over time. Therefore, response time behaviour was checked also for a second sensor beam, and the calibration procedure was designed such that these characteristics can be easily determined for a consecutive beam.\nOperating temperature\nRaising the temperatures of the system, and therefore of the humidity sensor, above room temperature, we observed an initial decrease in the response time, but above 40\u00b0C the response time increased. Therefore 40\u00b0C was chosen as the operating temperature. At this temperature, the sampled air in the catheter reaches 40\u00b0C well before arrival in the sensor house, and therefore the influence of breathing temperature variations on sensor temperature is negligible. For measurements at 1\u00a0cm deep in the trachea the heated part of the tube does not enter the trachea so that artificial heating of the inspired air will not occur.\nStatic calibration\nThe temperature of the sensor is kept stable within 0.2\u00b0C. Therefore the output of the capacitive sensor, which is sensitive for the relative humidity, can be directly converted to absolute humidity. In a climate cabinet (Heraeus Votsch HC7020), the humidity output of the ACE was plotted against a clinically relevant range of absolute humidity values, as measured with the secondary reference sensor, see Fig.\u00a05. Since this relation appeared to be linear, a 2-point calibration is considered appropriate for further frequent humidity calibrations in the hospital. Given the accuracy of the reference sensor and the somewhat limited range between the calibration points to avoid excessive condensation or temperature disequilibrium in the calibration device, the inaccuracy of the system for measuring absolute humidity values after the 2-point calibration is less than 5%.\nFig.\u00a05Output of the humidity sensor plotted against the secondary reference\nThe thermocouple measuring the temperature at the tip of the sample catheter was 2-point calibrated against the values of the reference thermometer in ice water and water at body temperature. This procedure can be performed in the hospital, prior to every in vivo measurement. Given the accuracy of the reference thermometer, the accuracy of the system for measuring temperature values after a 2-point calibration is 0.3\u00b0C.\nAssessing the role of condensation in the measuring system\nIn order to assess the necessity of heating the system in order to prevent condense formation within the device, step response measurements were performed with an unheated system and subsequently with the system heated to 40\u00b0C. With an unheated system, a delay of up to several seconds was observed in the humidity trace relative to the thermocouple trace after a downward step. The longer the sensor is kept in a high humidity environment, this delay increases; and it is indicative of the formation and subsequent evaporation of the condensate within the system. Even when simulating clinically relevant breathing frequencies, a noticeable delay of 0.15\u00a0s was observed.\nWhen measuring high humidity at operating temperature for a long time, after the downward step, a very fast response with a delay of less than 0.05\u00a0s was observed. This delay is caused by the transport of sampled air from the tip of the catheter towards the humidity sensor and is not relevant for the measurement of end tidal values. In the downward trace this fast response was followed by a shoulder at about 50% of the step, of which the length again increased with the duration at high humidity. This shoulder probably represents the evaporation of the condensate in the unheated tip of the sensor.\nDuring in vivo measurements, however, the system will never be exposed to high humidities for an extended period of time. Therefore we simulated a range of breathing frequencies with the two-stream system resulting in a series of block functions of approximately 9, 18, and 35\u00a0cpm (cycles per minute). At 9\u00a0cpm a short shoulder is still visible, but the condensate evaporates sufficiently fast that the \u201cend-inspiratory\u201d value is reached. At 18 and 35\u00a0cpm no shoulder was observed.\nRaw response time characteristics\nUsing the two-stream system the step response of the ACE was determined. Both for the temperature and the humidity signal we determined the 1\u20131\/e response time. For the downward humidity step we could not use a plain step function because condensation in the tip would occur. Therefore we used a block function with a frequency of 18\u00a0cpm (see Fig.\u00a06a). At this frequency the response time is still relatively short compared with the period of the block function and easy to determine from the humidity trace.\nFig.\u00a06Response to a block function; solid black line raw humidity trace, solid grey line deconvolved humidity trace, dashed line thermocouple trace. Vertical axis: \u00b0C\/mg\u00a0H2O\/l. Horizontal axis: seconds. a Response to a block function of approximately 18 cycles per minute. b Overview of the response to a block function with varying frequency (9 to 35\u00a0cpm)\nFor the thermocouple, the response to increasing and decreasing temperature steps was <0.2\u00a0s. For the first humidity sensor, the response to increasing humidity and decreasing humidity was <0.5 and <0.8\u00a0s, respectively, at a frequency of 18\u00a0cpm. We verified that the response characteristics at other frequencies were comparable. Both rise and fall times of the second humidity sensor were approximately 60% shorter.\nThe observed raw response times are slightly longer than we had expected based on the specifications of the Vaisala Humidity Sensor, probably because the airflow over the sensor is slower than specified. Although every attempt was made to minimize the volume of the sensor chamber we estimate that the airspeed over the sensor is between 2 and 3\u00a0m\/s instead of 6\u00a0m\/s.\nDeconvolution of the humidity signal\nSince the raw step response times of tested humidity sensors were considered too slow for patients with high breathing frequencies, it was decided to further process the raw humidity signal. Depending on the tested sensor, the raw step response can be closely approximated by a single or a double exponential function. Therefore it is possible to reconstruct the original signal by deconvolution. Although we observed a different response time for increasing and decreasing humidity steps, we nevertheless chose a single function because of its better numerical stability: \nFor the removal of excessive noise a low pass filter of <300\u00a0cpm was applied. Because the raw response characteristics varied in between the humidity sensors and over time, \u03c4 was adjusted for each individual measurement. The principle of adjustment of these parameters was to acquire a maximal value for \u03c4, such that no overshoot of the deconvolved humidity trace was visible. We used the thermocouple signal as reference for the true step function. For the first sensor, the deconvolved signal was stable and optimal for increasing humidity with \u03c4\u00a0=\u00a00.4\u00a0s. For the second humidity sensor a \u03c4 of 0.1\u00a0s appeared to be appropriate. We verified that the raw step response characteristics were reproducible, and that the deconvolution results were not influenced by the step size.\nAfter deconvolution of the humidity signal, the response characteristics improved, see Fig.\u00a06a. The 1\u20131\/e step response time to a block function for the first humidity sensor was <0.2\u00a0s for increasing humidity and <0.4\u00a0s for decreasing humidity at a frequency of 18\u00a0cpm. For the second sensor the deconvolved step response characteristics were similar after deconvolution.\nFigure\u00a06b shows the raw and deconvolved humidity response to a block function over a range of breathing frequencies. It is shown that the frequency dependence of the peak-to-peak values is reduced after deconvolution. At higher frequencies, we nevertheless see that the deconvolved signal increasingly deviates from the true peak-to-peak values. This deviation is more marked in the lower peak, due to the relatively long humidity fall time of the raw trace, compared to the rise time. The magnitude of the deviation in the lower peak ranges from about 1.5\u00a0mg\u00a0H2O\/l at a breathing frequency of 9\u00a0cpm to 5\u00a0mg\u00a0H2O\/l at 35\u00a0cpm. Since the step response characteristics of the deconvolved signal were comparable for both tested sensors, the deviations are of comparable magnitude too.\nAt 35\u00a0cpm even the amplitude of the fast thermocouple signal does not reach the true peak-to-peak values. The deviation is less than 0.5\u00b0C so that we do not consider a correction necessary for the thermocouple signal.\nThe in vivo measurement\nThe total assembly and the influence of a heat and moisture exchanger were tested in one laryngectomized volunteer. The tip of the sample catheter was pushed 1\u00a0cm through a small hole that was punched in an HME plaster (Fig.\u00a07). The diameter of the puncture was slightly smaller than the diameter of the sample catheter in order to avoid air leakage. Figure\u00a08 shows a segment of the raw and deconvolved humidity trace and the thermocouple signal. In this particular volunteer \u03c4 required adjustment to 0.35 in order to eliminate overshoot. During breathing in rest, intra airway temperature and humidity ranged from 28\/34\u00b0C and 15\/37\u00a0mg\u00a0H2O\/l (typical end inspiratory\/end expiratory values), respectively. During the total measurement session, breathing frequency ranged from 12 to 44\u00a0cpm (mean 17.8\u00a0cpm, SD 4.4\u00a0cpm). In addition, inspiration time may be relatively short compared to expiration time. See the beginning of the trace in Fig.\u00a08.\nFig.\u00a07Sample catheter in situ, pushed through a punched hole in the HME plaster, without HME filter in situFig.\u00a08In vivo humidity trace before and after deconvolution; black trace raw humidity signal, grey trace deconvolved humidity signal. Vertical axis: mg\u00a0H2O\/l. Horizontal axis: seconds\nDiscussion\nOur results show that we have succeeded in developing an intra-tracheal airway climate analyser with response characteristics fast enough for assessment of end tidal intra airway temperature and humidity variations during normal breathing in laryngectomized individuals. In the literature, only a few systems have been described that simultaneously measure temperature and humidity in the human airway [6, 8, 10, 11, 13, 14].\nIngelstedt [6] reported on intra airway climate in healthy individuals using a well- validated system based on psychrometry, with signal stability 0.3\u20130.4\u00a0s after sudden exposure to increased humidity. However, this method had a few disadvantages. The wet thermo element remained sufficiently moistened for only 5\u20138 respiratory cycles, after which it had to be retracted in order to be watered by a moistened wick again. Furthermore, even the slightest contact with tracheal secretion increased the thermal inertia of the psychrometer, after which it has to be cleaned in a 4-h lasting procedure.\nAlthough contact of the humidity sensor of the ACE by tracheal secretion is less likely, the central canal of the sample catheter is susceptible to obstruction by tracheal secretion. When this would occur, the sample catheter can be disconnected from the sensor house and subsequently the obstructive plug can be removed by establishing reversed airflow. The electrodes in the bottom of the droplet interceptor appeared to work effectively. If the humidity sensor would be damaged, it can easily be replaced. Due to the \u201cin hospital\u201d calibration procedures, the measurements can be continued within a relatively short time span.\nThe system of Primiano et al. [13] consisted of a sample catheter that was connected to a mass spectrometer. In order to prevent condensation, the tip of their sample catheter was constricted, establishing a decrease in air pressure. Extensive validation of the total assembly revealed impressive 10\u201390% response time characteristics of less that 0.25\u00a0s for humidity readings and less than 0.12\u00a0s for temperature readings, respectively. Since mass flow meters are expensive and require solid maintenance, the use of such a device was not considered feasible for our project. Although their measurement system has been used for assessment of anaesthesiology heat and moisture exchanger effectiveness, we are not aware that this system has been used for airway climate assessment in laryngectomees.\nMcRae et al. [10, 11] and Keck et al. [8, 9] have assessed tracheal climate and the influence of HMEs in laryngectomees. The set up of Keck et al. consisted of a sample catheter, connected to a sensor house that incorporated a capacitive humidity sensor.\nAs described in [9] the sensor house was heated with a heat mat to 34\u201335\u00b0C, which is slightly lower than the maximum end expiratory temperature observed (35.2\u00b0C) so that condensation still might be a possibility. However, the sample catheter was not heated and the verification of the total assembly has not been included in the report. In the present device, evidence of condensation\u2014probably precipitated in the unheated distal 2\u00a0cm of the catheter tip (which is in vivo somewhat heated by the body) is observed when offering block functions at a frequency of 9\u00a0cpm. Despite this, at 9\u00a0cpm the end values were not affected. It is therefore unlikely that also during in vivo measurements condensation will affect the amplitude of the peak-to-peak values. Moreover, the temperature and humidity steps will occur more gradually in the in vivo situation, theoretically decreasing the amount of condense formation in the sensor tip, compared to the offered block function.\nIn the studies by McRae et al. [10, 11], the response time of the humidity sensor used in the setup of McRae was up to 9\u00a0s. The frequency of human rest breathing is far too high compared to these response characteristics to obtain representative values for intra-tracheal humidity.\nHowever, as the two-stream block function experiment has shown, also in the present measuring device, the amplitude of the deconvolved humidity trace depends on the breathing frequency. The clinical implication of this finding is that, at higher breathing frequencies, the true peak-to-peak values are slightly, but progressively underestimated by the ACE, although probably less than in vitro due to more gradual humidity and temperature variation in the latter situation. The variation in end-inspiratory humidity values and temperature signals, as indicated by the deconvolved humidity signal and the thermocouple in the in-vivo experiment may therefore be partially due to a frequency dependency and not due to a real variation in humidity and temperature values.\nQuality control\nThe first sensor functioned correctly for 1\u00a0year and the second for half a year. Both sensors had to be replaced because of accidental damage during cleaning. The two humidity sensors appeared to have different response characteristics. Sensor age, variation in the production process of the humidity sensor etc. may result in variation between the characteristics of different humidity sensors, but also between the characteristics of one sensor measured over time. Linearity between reference humidity values and sensor output in all consecutive sensors is felt to be a prerequisite for serial production of radiosondes. The small climate room and the 2-point calibration is therefore considered appropriate for daily quality control. A potential source of error is leakage of air, causing dilution of sampled tracheal air with room air. Therefore the simple leakage test should be part of the regular quality control.\nAlthough both tested humidity sensors revealed the different step response times of the raw humidity signal, the peak-to-peak amplitude of deconvolved signal was comparable between both sensors. The two-stream step response set up facilitates regular control of the sensor response characteristics and, if necessary, adjustment of the deconvolution function can be easily performed.\nLimitations and further improvements\nDuring measurements deeper in the trachea (e.g. 4\u00a0cm), contact of the catheter tip with the airway wall may lead to irresistible cough. Preferably the distal end of the sample catheter should be better pliable in the shape of the trachea, in order to facilitate better control of its tip and reducing airway wall contact. Also visual control of the catheter tip may contribute to prevention of wall contact.\nThe response of the sensor when decreasing the humidity is slower than the response to increasing humidity. However, the same simple deconvolution function is used for increasing and decreasing humidity. A more advanced signal processing might further improve response characteristics and decrease the remaining frequency dependence of the peak tot peak amplitude. In particular for the end-inspiratory values this is important because of the relatively short time span of the inspiration time compared to the expiration time, combined with the relatively slow response characteristics to decreased humidity compared to increased humidity.\nIn conclusion, the Airway Climate Explorer is an easy to use, relatively inexpensive tool for intra airway temperature and humidity measurements. When looking at the end-inspiratory and end-expiratory humidity values as measured by our system, the accuracy is adequate for assessment of intratracheal climate.","keyphrases":["temperature","humidity","airway climate","heat and moisture exchanger","gas sampling probe"],"prmu":["P","P","P","P","R"]}
{"id":"Eur_J_Appl_Physiol-4-1-2374881","title":"Health-related physical fitness of adolescents and young adults with myelomeningocele\n","text":"To assess components of health-related physical fitness in adolescents and young adults with myelomeningocele (MMC), and to study relations between aerobic capacity and other health-related physical fitness components. This cross-sectional study included 50 adolescents and young adults with MMC, aged 16\u201330 years (25 males). Aerobic capacity was quantified by measuring peak oxygen uptake (peakVO2) during a maximal exercise test on a cycle or arm ergometer depending on the main mode of ambulation. Muscle strength of upper and lower extremity muscles was assessed using a hand-held dynamometer. Regarding flexibility, we assessed mobility of hip, knee and ankle joints. Body composition was assessed by measuring thickness of four skin-folds. Relations were studied using linear regression analyses. Average peakVO2 was 1.48 \u00b1 0.52 l\/min, 61% of the participants had subnormal muscle strength, 61% had mobility restrictions in at least one joint and average sum of four skin-folds was 74.8 \u00b1 38.8 mm. PeakVO2 was significantly related to gender, ambulatory status and muscle strength, explaining 55% of its variance. Adolescents and young adults with MMC have poor health-related physical fitness. Gender and ambulatory status are important determinants of peakVO2. In addition, we found a small, but significant relationship between peakVO2 and muscle strength.\nIntroduction\nDuring the last decades, life expectancy of persons with myelomeningocele (MMC) has increased and many will nowadays survive into adulthood (Bowman et al. 2001). As a consequence, lifestyle-related diseases, such as cardiovascular disease and diabetes mellitus, will be of increasing concern in this patient group. Therefore, more attention towards a healthy lifestyle is warranted. Similar to the general population, persons with MMC develop their own lifestyle during the transition from adolescence to adulthood. At this age, special attention should be paid to optimize the lifestyle in order to improve health throughout life.\nPhysical fitness is recognized as an important component of health (Lamb et al. 1988; Twisk et al. 2002) and it may be important for the performance of functional activities and quality of life (Noreau and Shephard 1995; Stewart et al. 1994). Low physical fitness may result in high physical strain during the performance of activities (Bruinings et al. 2007). As a consequence, activity levels may decrease due to fatigue and discomfort, exacerbating low physical fitness. Caspersen and co-workers defined several health-related components of physical fitness, i.e. aerobic capacity, muscle strength and endurance, flexibility and body composition (Caspersen et al. 1985).\nOnly a few studies are available on health-related physical fitness in persons with MMC. In a previous study in adolescents and young adults with MMC, we found that average peak oxygen uptake (peakVO2) was 42% lower than normative values of healthy peers, with lower values in non-ambulatory than in ambulatory persons (Buffart et al. in press). Several other studies have also reported low aerobic capacity (Agre et al. 1987; Sherman et al. 1997; van den Berg-Emons et al. 2003). Furthermore, children with MMC, and particularly non-ambulatory children, were found to have reduced strength of lower extremity muscles (Agre et al. 1987; McDonald et al. 1991; Schoenmakers et al. 2004). Hip and knee contractures have been reported in respectively 18% and 36% of adolescents and young adults with MMC and hydrocephalus (Verhoef et al. 2004). Finally, high levels of body fat have been found in persons with MMC (Bandini et al. 1991; Mita et al. 1993; Shepherd et al. 1991); previously we reported that 35% of adolescents and young adults with MMC were obese (Buffart et al. in press). Because these previous studies lack simultaneous assessment of several health-related physical fitness components, the relation between aerobic capacity and other components remains unclear. Insight in these inter-relations may show for example, whether focusing on peripheral factors such as muscle strength would have additional value for improving aerobic capacity.\nDue to the scarcity of studies in persons with MMC, the first aim of this study was to describe health-related physical fitness i.e. aerobic capacity, muscle strength, joint flexibility and body composition in a relatively large group of adolescents and young adults with MMC, allowing descriptions of subgroups regarding gender and ambulatory status. Secondly, we wanted to study the relation between aerobic capacity and other components of health-related physical fitness, controlled for relevant personal and disease-related characteristics. Studies in persons with other chronic conditions such as spinal cord injury have shown relations between aerobic capacity and muscle strength (Haisma et al. 2006; Janssen et al. 1993; Zoeller et al. 2005). Therefore, also in persons with MMC, we expected aerobic capacity to be related to muscle strength.\nMethod\nParticipants\nRecruitment\nAdolescents and young adults with MMC, aged between 16 and 30\u00a0years, were recruited from the university hospitals in Rotterdam, Leiden, Utrecht and Amsterdam and all rehabilitation centers in the Southwest of the Netherlands. Exclusion criteria were complete dependence on an electric wheelchair, presence of disorders other than MMC that affect daily physical activity (e.g. rheumatoid arthritis), and presence of disorders that contra-indicate a maximal exercise test (e.g. exercise-induced ischemia or arrhythmias, uncontrolled hypertension and exercise limitation due to chronic obstructive pulmonary disease). We invited 171 persons of whom 50 participated in the study (29%). Main reasons for non-participation were no interest, lack of time or duration of the measurements. No differences were found between participants and non-participants regarding age, gender, level of lesion and presence of hydrocephalus, as measured with an independent t-test or Chi square test (Buffart et al. in press). All participants and parents of adolescents aged less than 18\u00a0years gave written informed consent before participating in the study. The Medical Ethics Committee of the Erasmus MC Rotterdam and of all participating institutes approved the study.\nCharacteristics\nIn total, 25 males and 25 females (mean age 21.2\u00a0\u00b1\u00a04.5\u00a0years) participated in the study. Table\u00a01 presents personal and disease-related characteristics of participants. Neurological level of lesion and the presence of hydrocephalus were obtained from the medical records. Five categories of neurological level were distinguished: thoracic, thoracolumbar, lumbar, lumbosacral and sacral. We considered hydrocephalus to be present when a shunt was placed. Ambulatory status was determined according to the classification of Hoffer and co-workers (Hoffer et al. 1973): (1) community ambulator, walking indoors and outdoors, (2) household ambulator, walking only indoors, and (3) non(functional) ambulator. Non-functional ambulators walk only during therapy sessions and non-ambulators are completely wheelchair dependent. Since main mode of ambulation in daily life is similar between non-functional ambulators and non-ambulators, we combined these two groups. Educational level was categorized as low (pre-vocational practical education or lower level), medium (pre-vocational theoretical education and secondary education) or high (higher education and university) (Donkervoort et al. 2007).\nTable\u00a01Personal and disease-related characteristics of participants (n\u00a0=\u00a050) Gender (% male)50Age (mean\u00a0\u00b1\u00a0SD in years)21.2\u00a0\u00b1\u00a04.5Height (mean\u00a0\u00b1\u00a0SD in m)1.57\u00a0\u00b1\u00a00.12Body mass (mean\u00a0\u00b1\u00a0SD in kg)67.6\u00a0\u00b1\u00a015.6Level of lesion (%)\u00a0Thoracic2\u00a0Thoracolumbar14\u00a0Lumbar28\u00a0Lumbosacral42\u00a0Sacral14\u00a0Hydrocephalus (%)82Ambulatory status (%)a\u00a0Community ambulator30\u00a0Household ambulator14\u00a0Non (functional) ambulator56Educational level (%)\u00a0Low37\u00a0Medium39\u00a0High24aNo gender differences as tested with a Chi square test\nAerobic capacity\nAerobic capacity was measured in a progressive maximal exercise test, based on the McMaster All-Out Progressive Continuous Cycling and Arm test (Bar-Or 1983), on an electronically braked arm or cycle ergometer (Jaeger ER800SH and ER800 respectively; Jaeger Toennies, Breda, The Netherlands). Studying patients with cerebral palsy who were partly wheelchair-dependent, Bhambani and co-workers concluded that maximal exercise testing during the main mode of ambulation elicits the highest oxygen uptake (Bhambhani et al. 1992). Therefore, depending on their main mode of ambulation, participants performed an arm crank test (n\u00a0=\u00a033) while sitting in their own immobilized wheelchair with cranks at shoulder height, or a leg cycle exercise test (n\u00a0=\u00a017). The test was preceded by a 3-minute warm-up (5\u00a0W for arm ergometry and 20\u00a0W for cycle ergometry), followed by a resting period of 5\u00a0min. During the test, resistance was increased every 2\u00a0min with a variable load, ensuring that total individual exercise duration ranged from 8 to 12\u00a0min. The pedal\/crank rate was 60\u00a0rpm and strong verbal encouragement was given throughout the test. The test was terminated when the subject stopped due to exhaustion. Gas exchange was determined continuously using a breath-by-breath portable measurement system (K4b2, COSMED, Rome, Italy). Calibration was performed before each test with reference gases. Heart rate was measured continuously using a heart rate (HR) monitor which was attached to the system, and participants were fitted with a transmitter belt around the chest (Polar Electro, Finland). Aerobic capacity was defined as the mean oxygen uptake during the last 30\u00a0s of exercise (peakVO2, in l\/min). In addition, for those measured during cycle ergometry, values of aerobic capacity were expressed as percentage of reference values of Dutch able-bodied sedentary males and females of similar ages, as estimated from a submaximal exercise test on a cycle ergometer using the nomogram of \u00c5strand (Vos 2001). Peak work load (peakW) was defined as the highest work load maintained for at least 1\u00a0min.\nIn addition, the ventilatory anaerobic threshold (VAT) was estimated by the ventilatory equivalent method, when the ventilatory equivalent for O2 (Ve\/VO2) and the end-tidal O2 partial pressure (petO2) increased while ventilatory equivalent for CO2 (Ve\/VCO2) and end-tidal CO2 partial pressure (petCO2) remained stable (Reinhard et al. 1979; Wassermann et al. 1999). VAT was also expressed relative to the measured peakVO2 (VAT%). HR and respiratory exchange ratio (RER) were used as objective criteria for maximal exercise. Subjective strain was measured at the end of the final stage using the modified Borg scale of rating perceived exertion (RPE), which is a vertical scale labelled from 0 (no effort at all) to 10 (maximal effort) (Borg 1982; Mahler et al. 1987).\nMuscle strength\nWe measured strength of two large muscle groups of the lower and upper extremity with a hand-held dynamometer (MicroFet, Hoggan Health Industries) using the \u201cbreak\u201d testing method. We measured strength of hip flexors and knee extensors in persons whose main mode of ambulation was walking. In persons whose main mode of ambulation was wheelchair-driving, we measured strength of shoulder abductors and elbow extensors. The positions and the performance of the measurements were according to van der Ploeg and co-workers (van der Ploeg et al. 1991). The applicator of the dynamometer was held against the distal part of the limb segment, and participants were asked to build up their force to a maximum against it. The examiner applied sufficient resistance just to overcome the force exerted by the participant, and the applicator was then immediately moved away from the limb segment and the measured force was recorded. Each trial lasted approximately 4\u00a0s, and three repetitions were performed with 1\u00a0min rest in between. We used the average value of three repetitions of the dominant side for further analyses because we found no differences between the dominant and non-dominant side (tested with a paired samples t-test). To assess whether muscle strength was subnormal and to be able to compare strength of upper and lower extremity muscles, values were normalized to Z-scores using reference values of healthy males and females. For hip flexors, shoulder abductors and elbow extensors we used reference values of Phillips and co-workers for males and females aged 20\u201329\u00a0years (Phillips et al. 2000) and for knee extensors, we used reference values of Bohannon and co-workers (Bohannon 1997). In case the examiner could not resist the muscle strength, Z-score was set at 2. We used the lowest Z-score of the upper or lower extremity as indicator of muscle strength, and we considered muscle weakness to be present when Z-score \u2264\u22122.\nFlexibility\nAs indicator of flexibility of lower extremity we assessed passive mobility of hip and knee joint while participants were lying supine and of the ankle joint while they were sitting. We considered mobility to be restricted when extension of hip and knee joint and ankle dorsal flexion did not reach neutral position. Since no differences were found in mobility restrictions between dominant and non-dominant side (tested with the Wilcoxon signed rank test) we used the results of the dominant side to calculate a sum score of joint mobility ranging from 0 (no mobility restrictions in any joint) to 3 (all three joints have mobility restrictions).\nBody composition\nHeight was measured with a flexible tape while lying on a bed. In case of joint contractures, measurements were performed from joint to joint. Body mass of ambulatory persons was obtained while standing on a Seca scale and of non-ambulatory persons while sitting on an electronic scale (Cormier, France). Thickness of four skin-folds (biceps, triceps, subscapular, and suprailiac) was measured twice on the right side of the body with a Harpenden caliper (Burgess Hill, UK) and mean values were used for further analyses. In addition, sum of four skin-folds were expressed as percentage of reference values of Dutch sedentary males and females of similar ages (Vos 2001).\nStatistical analysis\nResults of health-related fitness components are presented as mean\u00a0\u00b1\u00a0standard deviation (SD) for the total group and for subgroups regarding gender and ambulatory status.\nSimple linear regression analyses were used to study relations between personal and disease-related characteristics and peakVO2 (in l\/min), in order to detect relevant determinants to include in the multiple regression models. Multiple linear regression analyses for peakVO2 were carried out in two steps. First, we built a model including relevant personal and disease-related characteristics. In the second step, we studied whether other health-related physical fitness components, i.e. muscle strength (Z-score), sum of four skin-folds (mm), and joint mobility (number of restricted joint), were significantly related to peakVO2, controlling for the personal and disease-related characteristics from step 1. We presented the standardized regression coefficients (\u03b2) and explained variance (R2) of the linear regression models. Statistical analyses were performed using SPSS 12.0 for Windows. P\u00a0\u2264\u00a00.05 were considered significant.\nIn the analyses, we pooled the data of peakVO2 measured during arm and cycle ergometry, and corrected for differences in exercise mode. Due to high collinearity between type of ergometer and ambulatory status (correlation coefficient (r)\u00a0=\u00a00.92, P\u00a0<\u00a00.001), we adjusted the analyses for ambulatory status as proxy for type of ergometer, because clinically, ambulatory status would be more meaningful.\nFurthermore, due to overlap between lesion level and ambulatory status (r\u00a0=\u00a00.58; P\u00a0<\u00a080.001), we chose to only correct for ambulatory status in the multiple regression analyses. Ambulatory status was determined during the study, whereas level of lesion was obtained from medical records. The national study on adolescents with spina bifida in the Netherlands (ASPINE) reported that the level of lesion as mentioned in medical records may be unreliable because lesions are determined at different ages, sometimes using the motor level and sometimes using the sensory level, and often lacking descriptions of methods (Verhoef et al. 2004).\nResults\nDescriptive results of aerobic capacity, muscle strength, joint mobility and body composition for the total group and for subgroups regarding gender and ambulatory status are presented in Table\u00a02. Average peakVO2 was 1.48\u00a0\u00b1\u00a00.52\u00a0l\/min. For persons measured during cycle ergometry (n\u00a0=\u00a017), peakVO2 corresponded to 67\u00a0\u00b1\u00a015% of reference values. Sixty-one percent of the participants had subnormal muscle strength as indicated by Z-scores and 61% had mobility restrictions in one or more joints. Average sum of four skin-folds was 74.8\u00a0\u00b1\u00a038.8\u00a0mm, corresponding to 159\u00a0\u00b1\u00a077% of normative values (Table\u00a02). According to objective and subjective criteria most participants reached their peak exercise performance. Average peakHR was 174\u00a0\u00b1\u00a019 beats per minute, which was 90.4\u00a0\u00b1\u00a09.6% of the age predicted maximum (220\u2014age for cycle ergometry; 210\u2014age for arm ergometry) and average peakRER was 1.17\u00a0\u00b1\u00a00.22. Average RPE was 6.2\u00a0\u00b1\u00a02.2, indicating that participants experienced the exercise as heavy to very heavy.\nTable\u00a02Descriptive results of health-related physical fitness components for the total group and for subgroups regarding gender and ambulatory statusAll (n\u00a0=\u00a050) GenderAmbulatory statusMale (n\u00a0=\u00a025) Female (n\u00a0=\u00a025) Community (n\u00a0=\u00a015) Household (n\u00a0=\u00a07) Non(functional) (n\u00a0=\u00a028) Aerobic capacity (mean\u00a0\u00b1\u00a0SD) PeakVO2 (l\/min) 1.48\u00a0\u00b1\u00a00.521.78\u00a0\u00b1\u00a00.511.18\u00a0\u00b1\u00a00.301.85\u00a0\u00b1\u00a00.571.44\u00a0\u00b1\u00a00.451.29\u00a0\u00b1\u00a00.40% of reference valuesa67\u00a0\u00b1\u00a01571\u00a0\u00b1\u00a01361\u00a0\u00b1\u00a01868\u00a0\u00b1\u00a01654\u00a0\u00b1\u00a02\u2013PeakVO2 (ml\/kg\u00a0min) 22.6\u00a0\u00b1\u00a08.228.1\u00a0\u00b1\u00a0 7.017.0\u00a0\u00b1\u00a04.729.0\u00a0\u00b1\u00a07.722.3\u00a0\u00b1\u00a06.619.2\u00a0\u00b1\u00a06.8Peak oxygen pulse (ml\/bpm)8.7\u00a0\u00b1\u00a03.010.1\u00a0\u00b1\u00a0 2.87.3\u00a0\u00b1\u00a02.410.7\u00a0\u00b1\u00a02.87.8\u00a0\u00b1\u00a02.47.8\u00a0\u00b1\u00a02.8PeakRER1.17\u00a0\u00b1\u00a00.221.17\u00a0\u00b1\u00a02.281.18\u00a0\u00b1\u00a00.201.15\u00a0\u00b1\u00a00.221.27\u00a0\u00b1\u00a00.161.16\u00a0\u00b1\u00a00.24PeakW (W)91\u00a0\u00b1\u00a042113\u00a0\u00b1\u00a04369\u00a0\u00b1\u00a028123\u00a0\u00b1\u00a04297\u00a0\u00b1\u00a03573\u00a0\u00b1\u00a034PeakHR (bpm)174\u00a0\u00b1\u00a019179\u00a0\u00b1\u00a016169\u00a0\u00b1\u00a020173\u00a0\u00b1\u00a021183\u00a0\u00b1\u00a014172\u00a0\u00b1\u00a018PeakHR % of predicted maximum90\u00a0\u00b1\u00a01092\u00a0\u00b1\u00a0889\u00a0\u00b1\u00a01087\u00a0\u00b1\u00a01095\u00a0\u00b1\u00a0891\u00a0\u00b1\u00a010VAT (l\/min)1.20\u00a0\u00b1\u00a00.431.39\u00a0\u00b1\u00a00.441.01\u00a0\u00b1\u00a00.321.55\u00a0\u00b1\u00a00.451.07\u00a0\u00b1\u00a00.291.05\u00a0\u00b1\u00a00.34VAT%82\u00a0\u00b1\u00a01580\u00a0\u00b1\u00a01486\u00a0\u00b1\u00a01684\u00a0\u00b1\u00a01077\u00a0\u00b1\u00a02283\u00a0\u00b1\u00a016Muscle strength (mean\u00a0\u00b1\u00a0SD) Z-score \u22122.1\u00a0\u00b1\u00a01.8\u22122.3\u00a0\u00b1\u00a02.1\u22121.9\u00a0\u00b1\u00a01.5\u22122.7\u00a0\u00b1\u00a02.2\u22122.0\u00a0\u00b1\u00a01.6\u22121.8\u00a0\u00b1\u00a01.7Weak strength: Z-score\u00a0\u2264\u00a0\u22122 (%)615864795754Joint mobility (median [range]) Number of restricted joints 1 [0\u20133]1 [0\u20133] 1 [0\u20133] 0 [0\u20132] 1 [0\u20132] 1.5 [0\u20133] Impaired mobility in any joint (%)615467295782Body composition (mean\u00a0\u00b1\u00a0SD)Sum of four skin-folds (mm) 74.8\u00a0\u00b1\u00a038.851.2\u00a0\u00b1\u00a024.6100.4\u00a0\u00b1\u00a035.159.1\u00a0\u00b1\u00a029.266.5\u00a0\u00b1\u00a034.786.0\u00a0\u00b1\u00a042.0% of reference values159\u00a0\u00b1\u00a077146\u00a0\u00b1\u00a079173\u00a0\u00b1\u00a073121\u00a0\u00b1\u00a052160\u00a0\u00b1\u00a0101181\u00a0\u00b1\u00a075aOnly for those measured on the cycle ergometer, n\u00a0=\u00a017 (ten males, seven females)\nSeveral personal and disease-related characteristics were related to peakVO2 (Table\u00a03). PeakVO2 was higher in males than in females (\u03b2\u00a0=\u00a0\u22120.61; P\u00a0<\u00a00.001), higher in community ambulatory persons than in household and non-ambulatory persons (\u03b2\u00a0=\u00a0\u22120.48; P\u00a0<\u00a00.001) and higher in persons with a lower level of lesion (\u03b2\u00a0=\u00a0\u22120.43; P\u00a0=\u00a00.002). Age, presence of hydrocephalus and educational level were not related to peakVO2.\nTable\u00a03Regression models for aerobic capacityIndependent variablesAerobic capacity (peakVO2, in\u00a0l\/min)\u03b2 P-valueR2Simple regression analysis\u00a0Personal characteristics\u00a0Gender\u22120.61<0.0010.36\u00a0Age\u22120.07 0.65\u2013\u00a0Lesion level\u22120.430.0020.17\u00a0Hydrocephalus\u22120.18 0.22\u2013\u00a0Ambulatory status\u22120.48<0.0010.22\u00a0Educational level0.130.39\u2013Multiple regression analysis\u00a0Step 1a\u00a0\u00a0Personal characteristics0.50\u00a0\u00a0Gender\u22120.55<0.001\u00a0\u00a0Ambulatory status\u22120.40<0.001\u00a0Step 2b\u00a0\u00a0Inter-relations\u00a0\u00a0Muscle strength (Z-score)0.220.040.55\u00a0\u00a0Body composition (sum of four skin-folds in mm)0.250.080.53\u00a0\u00a0Joint mobility (number of restricted joints)0.000.98\u2013Significant betas are presented in bold\u03b2 standardized regression coefficient, R2 explained variance, Gender male (0), female (1), Lesion level sacral (1), lumbosacral (2), lumbar (3), thoracolumbar (4), thoracic (5), Hydrocephalus no (0), yes (1), Ambulatory status community ambulator (1), household ambulator (2), non(functional) ambulator (3), Educational level: low (0), medium (1), high (2)aDue to large overlap between ambulatory status and lesion level, we only included ambulatory statusbRelations between aerobic capacity and other fitness components controlled for gender and ambulatory status\nFifty percent of the variance in peakVO2 was explained by gender and ambulatory status (Table\u00a03). In addition, when controlling for both variables, we found that participants with higher muscle strength had higher values of peakVO2 (\u03b2\u00a0=\u00a00.22; P\u00a0=\u00a00.04) explaining an additional 5% of the variance in peakVO2. Furthermore, we found that participants with higher sum of four skin-folds tended to have higher values of peakVO2 (\u03b2\u00a0=\u00a00.25; P\u00a0=\u00a00.08).\nDiscussion\nComponents of health-related physical fitness\nIn the present study, several health-related components of physical fitness were studied simultaneously in a relatively large group of adolescents and young adults with MMC. In general, most participants had poor health-related physical fitness.\nCompared to the general population and compared to other patient groups, persons with MMC had low aerobic capacity. Regarding community ambulatory persons with MMC, values of peakVO2 were 32% lower than the reference values for able-bodied people and 23% lower than the average peakVO2 of males with spastic diplegia measured during cycle ergometry (Lundberg 1978). However, peakVO2 is influenced by the amount of active muscle mass (Davies and Sargeant 1974,1975; Lewis et al. 1983), which may possibly be reduced due to paresis of lower extremity muscles. PeakVO2 in non-ambulatory persons with MMC was lower than in ambulatory persons which may be caused by the lower amount of active muscle mass during arm ergometry compared to cycling. In able-bodied people, arm exercise induces a peakVO2 ranging from 53 to 73% of that achieved with lower extremity exercise (Bar-Or and Zwiren 1975). If that ratio is also applicable to non-ambulatory persons who may be accustomed to using their arm and shoulder muscles, adapted values of average peakVO2 would range between 1.76 and 2.43\u00a0l\/min, which is of comparable range to the peakVO2 in ambulatory persons with MMC. Furthermore, compared to males with spinal cord injuries with lesions below T10 measured during arm ergometry (Janssen et al. 2002), the peakVO2 in non(functional)-ambulatory males with MMC was 22% lower. We therefore assumed that the poor aerobic capacity we found in persons with MMC may be influenced by reduced active muscle mass, but also deconditioning is likely to be present. This is supported by previous studies showing that adolescents and young adults with MMC were inactive, and that inactivity was associated with lower aerobic capacity (van den Berg-Emons et al. 2001), particularly in non-ambulatory persons with MMC (Buffart et al. in press).\nVAT is another indicator of aerobic capacity, which was strongly correlated to peakVO2 in the present study sample (r\u00a0=\u00a00.84; P\u00a0<\u00a00.001). In the general population, VAT roughly corresponds to 50\u201360% of VO2max during leg exercise (Davis et al. 1997), and values of 40\u201360% have been found in the able-bodied population during arm exercise (Flandrois et al. 1986; Lin et al. 1993; Schneider et al. 1999). Paradoxically, we found VAT to be at 82% of peakVO2 suggesting high aerobic capacity. Comparably, Coutts and McKenzie found that persons with tetraplegia had lower VAT than persons with paraplegia caused by smaller muscle mass, but they had higher VAT%, and it was suggested that values of peakVO2 were low in relation to VAT (Coutts and McKenzie 1995).\nFifty-four percent of the non-ambulators and 79% of the community ambulators had subnormal strength in at least one of the major muscle groups. The relatively better performance of non-ambulators compared to ambulators in this respect might be explained by their relatively higher muscle strength in the upper extremities due to the habituation of using their arms for wheelchair propulsion. Furthermore, in contrast to lower extremity muscles, the neurological level of lesion will not disturb innervations of upper extremity muscles, which suggests that the weak upper extremity strength we found in non-ambulators is due to atrophy of muscles as a consequence of disuse. Weak strength of lower extremity muscles in ambulatory persons with MMC corresponds to previous findings in children with MMC (Agre et al. 1987; McDonald et al. 1991; Schoenmakers et al. 2004) and might be related to disturbed innervations as well as to disuse of muscles.\nResults of the present study support the findings of Verhoef and co-workers that a large proportion of persons with MMC have reduced joint mobility (Verhoef et al. 2004). Similar to the results of Agre and co-workers restrictions in joint mobility in the lower extremities were mainly present in non-ambulatory persons with MMC (Agre et al. 1987).\nWe found high sum of four skin-folds, particularly in females and non-ambulatory, which is in agreement with high levels of body fat reported in previous studies (Bandini et al. 1991; Mita et al. 1993; Shepherd et al. 1991).\nRelations between aerobic capacity and other components of health-related physical fitness\nThe results of the regression analyses indicated muscle strength was associated to peakVO2 when controlling for gender and ambulatory status. This finding is in accordance with literature on persons with spinal cord injury (Haisma et al. 2006; Janssen et al. 1993; Zoeller et al. 2005). In contrast to gender and ambulatory status, muscle strength is modifiable. Strength training may increase muscle mass and thus metabolizing mass contributing to higher peakVO2 (Janssen et al. 1993). In persons with spinal cord injury, strength training resulted in increased peakVO2 (Cooney and Walker 1986). Because causality cannot be established with the cross-sectional design of the present study, future studies should confirm whether strength training results in increased aerobic capacity in persons with MMC. However, considering the small, but significant, contribution of muscle strength to the explained variance, we assume this specific effect to be small. In this respect, it seems that mainly aerobic training is needed in order to improve aerobic capacity; however, including strength training may have additional value.\nBody composition tended to be positively related to aerobic capacity, indicating that persons with more body fat had higher values of peakVO2. This relation may be caused by the greater fat-free mass of overweight persons concomitant with a greater body size (Unnithan et al. 2006).\nJoint mobility was not related to aerobic capacity. However, in non-ambulators we might have underestimated the relation because we did not measure mobility of upper extremity joints. Nevertheless, good flexibility of lower extremity is suggested to be important to prevent problems later in life, such as problems with personal hygiene and transfer capabilities (Agre et al. 1987).\nLimitations of the study\nThe methodology of the study had some limitations. First, because it is suggested that the primary mode of ambulation elicits the highest values of peakVO2 (Bhambhani et al. 1992), we used different exercise modes to assess aerobic capacity for ambulatory and non-ambulatory persons. However, due to large differences in active muscle mass, arm and leg exercise have different physiological responses. Therefore, we corrected for ambulatory status as proxy measure for type of ergometer when analysing relations between aerobic capacity and the other health-related fitness components. Furthermore, because the main mode of ambulation of ambulatory persons is walking rather than cycling, we may have underestimated peakVO2 in some ambulatory persons with MMC who rarely cycle. However, in clinical practice, the cycle ergometer seems to be more practical because physically disabled people may experience severe balance problems on the treadmill, and on the cycle it is possible to strap the feet to the pedals (Lundberg 1978). Peripheral local fatigue may have caused exercise cessation before reaching maximum oxygen uptake, however, based on the objective (peakHR and peakRER) and subjective criteria of maximal exercise (RPE), it may be concluded that values of peakVO2 are reasonable.\nFurthermore, muscle strength was measured using hand-held dynamometry. This method is known to be cheap, quickly applicable (van der Ploeg et al. 1984) and reliable (Bohannon 1997). However, for an average examiner, values above 250\u00a0N are considered too high to apply sufficient resistance (van der Ploeg et al. 1991), which may lead to less accurate results in strong muscle groups. Using absolute values of strength measured with an isokinetic device, instead of expressing muscle strength as Z-score, might provide more detailed insight into the relation between strength peakVO2. However, the current measurement method and use of Z-scores are considered adequate to determine weakness of major muscle groups.\nFinally, the response rate was low, which hampers generalization of results. However, personal and disease-related characteristics did not differ between participants and non-participants. Moreover, the prevalence of middle-level (lumbosacral) and high-level (lumbar or thoracolumbar) lesions of the present study sample was comparable to the persons who participated in the national ASPINE study (Verhoef et al. 2004). Despite, a selection bias may have occurred since it could be that the more active and more fit people had higher interest in participating than the less active and less fit ones, which may have led to an overestimation of health-related physical fitness components.\nIn conclusion, the results of the present study show that both ambulatory and non-ambulatory adolescents and young adults with MMC have poor health-related physical fitness. A large part of the variance in aerobic capacity is explained by gender and ambulatory status. Results showed a small but significant relationship between peakVO2 and muscle strength, suggesting that adding strength training to aerobic training may have additional value in increasing peakVO2.","keyphrases":["aerobic capacity","muscle strength","body fat","joint mobility","spina bifida"],"prmu":["P","P","P","P","P"]}
{"id":"Soc_Sci_Med-2-1-2430159","title":"Most of our social scientists are not institution based\u2026 they are there for hire\u2014Research consultancies and social science capacity for health research in East Africa\n","text":"There is a serious shortage of senior African social scientists to lead health-related research in Africa. This is despite the existence of many African social science graduates, and decades of Northern funded research programmes intended to develop local capacity. To investigate the barriers to developing health social science research capacity in East Africa, 29 in-depth interviews, informal conversations and a group discussion were conducted with professionals in this field.\nIntroduction\nThere is a serious shortage of senior African social scientists to lead or manage health-related research in Africa (World Bank, 2000). This is despite the graduation of many African social scientists, and decades of Northern funded research programmes intended to develop local capacity (Nchinda, 2002; Simon, 2000). Whilst weak research capacity probably affects all areas of health, the HIV epidemic \u2018offers a supreme test of how effectively African universities can respond to emerging challenges \u2026\u2019 (Zeleza, 2003, p. 84). Yet the shortage of senior social scientists is particularly apparent in sexual health. For instance, large-scale HIV\/AIDS research programmes in both Tanzania and Uganda have been unable to recruit local social scientists to senior posts on international salaries, despite having trained local junior social scientists for over 10 years. Furthermore, international debates on social dimensions of sexual health, such as the hypothesis of permissive African sexuality (Caldwell, J, Caldwell, P, & Quiggin, 1989), are dominated by Northern academics, despite their sensitivity.\nHealth-related social science research capacity is the ability to investigate and define the social dimensions of health problems, set objectives, identify solutions and build sustainable institutions (cf. Sitthi-amorn & Somrongthong, 2000). Limited capacity is problematic at several levels. Most obviously, it requires non-local researchers generally unfamiliar with local life, reliant on interpreters, and prone to cultural misunderstandings with local fieldworkers. Service providers and policy makers have to base decisions on more superficial analyses, but ex-patriate-initiated research is less likely to have such practical application anyway (Costello & Zumla, 2000). At the broadest level, limited social science capacity restricts intellectual sovereignty (Zeleza, 2003), undermining political autonomy (RAWOO, 2002; Sitthi-amorn & Somrongthong, 2000).\nThe main explanations for limited research capacity have been identified in the literature as: inadequate resources for education at every level (Nchinda, 2002; Sall, 2003; Sitthi-amorn & Somrongthong, 2000); the drain of expertise to the North (Pang, Lansang, & Haines, 2002; Ramsay, 2002; Sall, 2003; Zeleza, 2003); dependence on Northern research funding (Jentsch & Pilley, 2003; Lansang & Dennis, 2004); inequitable access to the literature (Lansang & Dennis, 2004); unbalanced North\u2013South research collaborations (Costello & Zumla, 2000; Jentsch & Pilley, 2003) and poor support from government (Nchinda, 2002; Sall, 2003; Sitthi-amorn & Somrongthong, 2000). Some see the perpetuation of inadequate research capacity as replicating the imbalance in global trade relationships (Zeleza, 2003) and essentially semi-colonial (e.g., Costello & Zumla, 2000). Others assume the good intentions of funders and research partners, but identify the perverse consequences of North\u2013South collaborations (Edejer, 1999), such as poaching senior researchers from local institutions. Either way, limited research capacity in Africa should be an ethical issue for Northern researchers working there.\nWhilst the main causes of weak research capacity are clearly macro-economic, the processes of conducting research might contribute to the problem and may be more readily modified (Green, 2003). Some reports have suggested that research consultancies, whilst augmenting meagre incomes, might marginalise teaching and research and undermine social science scholarship (Kwesiga, Mbago, & Chimanikire, 2000; Menken, Blanc, & Lloyd, 2002; Mkandawire, 1998; Sall, 2003). Being highly prescribed, consultancies have also been said to exacerbate the way African social science research is narrowly policy-bound (Allen, 1986; Sall, 2003), and increase Northern dominance of the research agenda (Mkandawire, 1998). \u2018\u2026consultants do not frequently choose to contradict the donor's agenda, \u2026 this would decrease their chances of \u2026 consultancies in the future.\u2019 (Rossi, 2004, p. 27). Consultancies have been described as particularly problematic in East Africa (Sall, 2003), the University of Nairobi being called a \u2018consultancy university\u2019 (Allen, 1986, p. 25).\nThere have been many attempts to strengthen research capacity in developing countries, leading sponsors being the WHO and other UN agencies (e.g., the Special Programme for Research and Training in Tropical Diseases (TDR); UNDP\/World Bank\/WHO, 2003), national development agencies (e.g., Swiss, Canadian and Japanese), foundations such as Rockefeller and NGOs such as the Population Council. However, there is little evidence about the most effective approach (Simon, 2000), and debates continue over, for instance, investing in individuals or institutions (Costello & Zumla, 2000; Nchinda, 2002), whether post-graduate training in the North exacerbates the brain drain (Nchinda, 2002), and Southern control of research budgets (Lansang & Dennis, 2004; Nchinda, 2002). The role of consultancies, however, has received scant attention and almost exclusively in the grey literature.\nIn order to investigate the poor capacity for health-related social science research in East Africa, the processes perpetuating it and possible ways to improve it, a small-scale exploratory study was conducted in Tanzania, Kenya and Uganda. The general findings have been reported elsewhere (Wight, 2005). This short report focuses on the individualised nature of research activity and the role of individual research consultancies in shaping research capacity.\nMethods\nIn 2003 and 2004, I conducted in-depth interviews with 29 leading professionals conducting, commissioning or supporting health-related social science research in East Africa (Table 1). The findings are biased towards Uganda, 18 interviewees being Ugandan, four Kenyan, three British, two North American, one Tanzanian and one Nigerian. This was primarily a snowball sample including seven senior social scientists from Makerere, the oldest university in East Africa with by far the largest research function in Uganda, and others from the London School of Hygiene and Tropical Medicine (three), the Universities of Nairobi (two) and Dar es Salaam (one) and from the leading independent research centres and research-supporting NGOs in Uganda and Kenya. None of the new universities were represented. To protect respondents\u2019 anonymity small institutions are not named.\nThe interview schedule covered leadership of local social science research, training and career paths, ways of strengthening research capacity and barriers to this. The full schedule is available (as Appendix A).\nInformal conversations were held with five senior and one junior researcher from the University of Dar es Salaam and National Institute for Medical Research, Tanzania, and nine junior researchers from Makerere and the MRC Programme on AIDS in Uganda. A group discussion was held with four of the Ugandans (three men, one woman), following the same schedule used for the in-depth interviews.\nThe interviews were summarised according to analytical themes. There was considerable concordance in accounts, which were not patterned by gender. Divergent opinions are considered in the Discussion. Interviewees were circulated the main report for comments and to confirm that their views were presented accurately. Four provided comments.\nFindings\nSeverity of the problem\nNearly all those interviewed thought that there is a serious shortage of social science research capacity in East Africa, the few really good social scientists being overworked and overwhelmed with requests for collaboration. Most academic health-related social science research in Uganda was said to be run by Northerners, yet Uganda was thought to have stronger capacity than Kenya, with Tanzania coming third. Particular limitations identified were in qualitative research, analysis and writing skills, and health-related specialisms.\nInterviewees stated that the vast bulk of social science research in East Africa is commissioned by NGOs or government departments, mostly funded from the North; consequently, it is highly applied and determined by external priorities. The few opportunities for academic research were said to come primarily from Northern researchers who win the funding, resulting in unbalanced collaborations.\nResearch processes: inter-collegiate support\nPoor social science capacity was primarily related to under-development and global economic inequalities: very poor schooling, talented students choosing high status vocational courses, poor university facilities and teaching, research funded through Northern institutions, and the drain of senior researchers abroad (Wight, 2005). The problem has been exacerbated by the death of many junior and mid-level Researchers from AIDS (Pfau & Barton, 2004, RAWOO, 2002, Zeleza, 2003). However, the individualised nature of departments and lack of collegiate support were also identified as unhelpful. This was primarily attributed to lack of resources and staff's reliance on individual research consultancies, resulting in no writing skills training and limited publishing experience to share. A head of department observed that senior staff rarely co-author papers with junior colleagues, due to \u2018the culture of individualism.\u2019 In her department there was no formal system to support junior researchers, though she was planning a mentoring system. Staff rarely seem to comment on their colleagues\u2019 draft papers; one senior respondent estimated that at Makerere only 1% of colleagues would have time.\nResearch processes: consultancies\nMost of the research work conducted by social scientists in East Africa is in the form of consultancies. The proportion of academics\u2019 time spent on them is unclear (Kwesiga et al., 2000), partly perhaps to disguise this from supervisors, but most estimates were around 50% of working time. Teaching takes up much of the rest, with very little for academic research.\u2026 in Makerere you can spend your entire time just working on very well paid, short-term consultancy studies for NGOs, \u2026 who want something done in three weeks, and will pay you very well \u2026 (Senior researcher, previously Uganda)\nExtremely low university salaries create a powerful incentive for consultancies. A research associate's salary might be $250\/month, while consultancies can pay $100\u2013$250\/day. In one research institute consultancies augment salaries from around $400\/month to about $5000. A head of department explained: \u2018\u2026 to rely on your salary would never make ends meet at all.\u2019 Furthermore, in contrast to regular salaries, most researchers can avoid declaring consultancy fees for tax (30%).\nResearch commissioners, predominantly government departments or NGOs, usually seek a contract with individuals, or sometimes consultancy firms, but rarely with university departments. Private consultancy firms, often constituted for a particular brief, usually employ university staff to help with the bid and subsequent research.\nCommissioning bodies are reportedly unwilling to pay overheads to institutions, and when they do, they are generally very low, e.g., 5\u201320% in Makerere departments, 20% at the University of Dar es Salaam, and a maximum of 15% at a Kampala independent research centre. The senior management at Makerere were said to encourage departments to become consulting firms and demand 30% overheads, but this leads university staff to work independently, undercutting university departments and earning more.\nThe predominance of research consultancies is critical for the development of research capacity. Financial insecurity leads researchers to take on any work available, and consequently:There are no research traditions being developed \u2026. We are social scientists but very few are specialists \u2026 (Faculty dean)\nConsultancy work also inevitably restricts academics\u2019 time for teaching and supervision. At Dar es Salaam and Makerere consultancies should not interfere with normal academic work, but this is difficult to enforce:He will leave you. And who loses? This is the person you have trained up to PhD level, and now he is leaving you, and you have no one to teach \u2026 (Head of department)\nWriting consultancy reports provides little incentive to develop analytical skills. Reports generally involve very tight timetables with little opportunity for peers\u2019 critical input, are descriptive and have limited dissemination (sometimes for internal use only). Several researchers said they do not publish from consultancies because they need the funder's permission, but none knew of it being refused. More plausibly, there is rarely time for such writing. Consequently, the CVs of highly experienced researchers often list numerous consultancy reports but very few journal publications, jeopardising their applications for senior jobs.\nThe conflict between consultancies and academic publications reportedly generates a professional culture in which: \u2018the point is to try and chase the quick money, and not take advantage of the chance of academic growth \u2026 people don\u2019t value it very much.\u2019 A faculty dean commented:Consultancies is not building the capacity of the person who is doing it. \u2026. [Some] have even refused scholarships to do PhDs because they were busy doing consultancies.\nOnly two interviewees questioned the inevitability that consultancies detract from publications or teaching: \u2018\u2026 consultancies can \u2026 be a source of writing \u2026 [and] training.\u2019 (Director large research programme).\nThe high remuneration from consultancies, and tensions with teaching responsibilities, might encourage researchers to become full-time consultants. However, few do this because commissioners seek the \u2018recognition and visibility\u2019 of \u2018high powered people\u2019 in established university posts. Furthermore, it would be too insecure: \u2018You would earn much more in the short-term, but then you would be unemployed in the long-term.\u2019\nStrengthening research capacity and likely barriers\nRespondents proposed many ideas to strengthen health-related social science research capacity (see Wight, 2005). Here I focus on those to modify the individualism of research practice and consultancies.\nFive senior interviewees identified the need to develop writing skills, for instance \u2018to guide you through \u2026 the very complicated processes \u2026 and requirements\u2019 to publish in international journals. Suggestions included experienced and in-experienced staff co-authoring, mentoring systems and support networks. The director of a research-facilitating NGO advocated posts dedicated to writing support, but with salaries adequate to prevent appointees taking on consultancies.\nThe potential advantages of institutional research consultancies were explored, and in particular establishing a norm of significant overheads, e.g., 30%. Everyone approved in principle. Overheads could be used for: libraries, computing and internet access; department-initiated research; disseminating reports; training staff and developing writing skills. Institutional consultancies might facilitate a more collective approach to research and assist management by departmental heads. Furthermore, paying overheads might benefit commissioners since they could require reports to be published, at least in an on-line journal.\nHowever, several objections to institutional consultancies were also raised.The culture of institutionalising things is not there. Many think the institution is a barrier to them. And \u2026 the bureaucracy, you know, many people would prefer to have the money in their own accounts \u2026. (Head of department)\nResearchers anticipated the frustrations of inefficient institutional administrations, with long delays in finalising contracts or being paid. It was feared that, since some universities do not allow departmental bank accounts, the central administration might appropriate funds raised through departmental consultancies. Furthermore, fees would not only have to be shared with the institution, but would have to be declared for tax. Consequently, the director of a large programme thought: \u2018\u2026 people will just try to get around it. They will get consultancies privately.\u2019\nSeveral interviewees said that commissioning agencies would not \u2018\u2026 want to pay the institutional fee.\u2019All the American universities [have] institutional overheads, but tell DfID that [they] have to be factored in [in Kampala] \u2026: \u201cOh, no!\u201d \u2026. How am I supposed to run the project without institutional overheads? \u2026 they have the mentality that they can do it on the cheap. Africa is poor, but it is not cheap! (Director of large programme)\nIt was also argued that some agencies want to commission specific individual researchers, and that individual consultancies incentivise good work produced on time.\nSome large donors practice their policies of strengthening institutional capacity by only contracting research through institutions, e.g., the Carnegie and Rockefeller Foundations, the World Bank, and the Swedish and Norwegian development agencies. However, some interviewees thought a concordat with all commissioning agencies to pay minimum overheads would be unrealistic, since East Africa is too dependent on donors.\nDiscussion\nAlthough most respondents were Ugandan, the data from Kenya and Tanzania suggest that these findings apply across East Africa, while the broader literature (e.g., Carlsson & Wohlgemuth, 1996; Sall, 2003; Zeleza, 2003) and contacts with researchers elsewhere suggest they are relevant to much of sub-Saharan Africa. There is a serious shortage of health-related social science research capacity in this region, as evidenced by the Northern intellectual leadership of most academic research. This perpetuates \u2018the international intellectual division of labour whereby African \u2026 social scientists \u2026 import appropriate \u2026 theory and, at best, export empirical data.\u2019 (Zeleza, 2003, p. 111) African countries\u2019 limited ability to define for themselves their problems and the solutions may have very practical consequences. For instance, Cleland and Watkins (2006, p. 2) argue that Africans\u2019 frustratingly slow response to the HIV epidemic is because \u2018the problem, and the remedies, were socially constructed in the West \u2026.\u2019\nLike most previous studies, this one points to global economic inequalities as the primary cause of limited research capacity. However, unremarked in nearly all the published literature, these findings also suggest that the problem is perpetuated by the highly individualised character of research in East Africa, fuelled by the dominance of individually-contracted consultancies. \u2018Most of our social scientists are not institution based, whether NGO or private. They are there for hire.\u2019 (Faculty dean) Such consultancies seem to stunt research capacity: reports are generally not disseminated, thus not contributing to collective understandings, university departments are denied overheads, and staff are diverted from teaching, supporting colleagues, or publishing. Furthermore, consultancies exacerbate the narrow policy orientation of African social science research (Allen, 1986; Rossi, 2004; Sall, 2003).\nGiven their prominence, this study explored the potential for research consultancies to be used to strengthen research capacity, which to date has been largely ignored in the wider literature. The principle that consultancies should be contracted with institutions, rather than individuals, with overheads of around 30%, was widely accepted. This could fund many initiatives to strengthen research capacity, facilitate a more collective approach to research, and in the longer term might mean commissioning agencies get better value. Some African universities and research centres already regulate the division of consultancy fees between researchers and their institution, e.g., the University of KwaZulu-Natal and the REACH Trust, Malawi (Theobald & Nhlema, in press). However, established individual consultants would probably oppose institutionalisation, given very low university salaries and lack of confidence in departmental administration, due to experience of patronage, mismanagement and corruption (Zeleza, 2003). Furthermore, commissioning agencies were said to prefer individual consultancies as cheaper and more straightforward.\nBy and large, initiatives to strengthen research capacity do not address the issue of research consultancies, although in practice they are in competition for researchers\u2019 commitment. This was clear in an academic research centre sponsored by an international NGO where researchers are prohibited from consultancy work. However, as noted above, some large donors further the development of institutional capacity by only contracting research through institutions, not individuals.\nUnsurprisingly, respondents\u2019 accounts were shaped by their professional and institutional positions, for instance leading them to defend their staff or externalise problems (see Wight, 2005). African interviewees gave more emphasis to economic factors, research commissioners\u2019 restrictions and exclusion from Northern-dominated academic networks, while Northern interviewees were more likely to contrast East African with Northern professional cultures. However, these cultural differences were usually attributed to underlying structural\/economic factors. Only one interviewee, an African, explicitly attributed inadequate research capacity to a global economy of academic research, in which Northern institutions actively maintain their dominance. Notably, the most critical reports of East African research came through informal conversations, rather than recorded interviews.\nThis has only been an exploratory study. Further research needs to clarify: the scale of individual consultancies across East Africa; whether revising commissioning practices would seriously contribute to research capacity; and, hardly represented here, the views of agencies commissioning consultancies.\nWhile the underlying causes of poor research capacity require global economic reform, this study also points to the importance of individually contracted research consultancies in perpetuating the problem. Although they greatly augment meagre university salaries, they also seem to divert university staff from academic research and training the next generation of researchers, stunt the institutional capacity of university departments, restrict the sharing of research findings and perpetuate donors\u2019 control of the research agenda. Commissioning bodies committed to strengthening research capacity should consider devising research contracts, and means to improve university administration, that ameliorate rather than exacerbate the problem.","keyphrases":["research consultancies","health social sciences","research capacity","sub-saharan africa","knowledge economy","east african universities"],"prmu":["P","P","P","P","M","R"]}
{"id":"Diabetologia-4-1-2292422","title":"A Kir6.2 mutation causing severe functional effects in vitro produces neonatal diabetes without the expected neurological complications\n","text":"Aims\/hypothesis Heterozygous activating mutations in the pancreatic ATP-sensitive K+ channel cause permanent neonatal diabetes mellitus (PNDM). This results from a decrease in the ability of ATP to close the channel, which thereby suppresses insulin secretion. PNDM mutations that cause a severe reduction in ATP inhibition may produce additional symptoms such as developmental delay and epilepsy. We identified a heterozygous mutation (L164P) in the pore-forming (Kir6.2) subunit of the channel in three unrelated patients and examined its functional effects.\nIntroduction\nATP-sensitive potassium (KATP) channels link cellular metabolism to membrane electrical activity by regulating K+ fluxes across the plasma membrane [1]. They are found in multiple tissues but are of particular importance in regulating insulin secretion from pancreatic beta cells [1, 2]. At substimulatory glucose concentrations, KATP channels are open so that the membrane potential is hyperpolarised and Ca2+ influx and insulin secretion are prevented [3]. Glucose metabolism enhances ATP production, resulting in closure of KATP channels, stimulation of electrical activity, opening of voltage-gated Ca2+ channels and exocytosis of insulin granules.\nKATP channels are hetero-octamers of Kir6.x and sulfonylurea receptor (SUR) subunits [4\u20136]. Four inwardly rectifying subunits (Kir6.2 in pancreatic beta cells) form the pore of the channel and four auxiliary SUR subunits (SUR1 in pancreatic beta cells) associate with the tetrameric pore and regulate its gating [7\u20139]. Binding and\/or hydrolysis of Mg-nucleotides by the intracellular nucleotide-binding domains (NBDs) of SUR produces channel opening [10\u201313]. It is believed that reciprocal changes in the intracellular concentrations of ATP and MgADP are involved in the metabolic regulation of KATP channels.\nOver the last 4\u00a0years, many different missense mutations in the genes encoding Kir6.2 (KCNJ11) and SUR1 (ABCC8) have been shown to cause permanent neonatal diabetes mellitus (PNDM). This is a rare disorder characterised by high blood glucose levels that manifests within the first 6\u00a0months of life. A subgroup of mutations were associated with a more severe clinical profile characterised by Delayed development of motor, intellectual and social skills, muscle weakness, Epilepsy, facial dysmorphism and Neonatal Diabetes (DEND syndrome) [14\u201316]. Mutations in Kir6.2 have also been found to cause a remitting relapsing form of neonatal diabetes that resembles transient neonatal diabetes mellitus [17, 18].\nAll Kir6.2 mutations analysed to date were heterozygous and most were de novo mutations [14, 19\u201321]. In most cases, the diabetes they caused could be successfully treated with sulfonylureas [14, 20, 22], which directly close KATP channels by binding to the SUR1 subunit of the channel [23]. All PNDM mutations result in a reduced KATP channel sensitivity to inhibition by MgATP in vitro [15, 24]. This is expected to cause an increased KATP current amplitude and reduced insulin secretion. Studies to date suggest that the severity of the clinical phenotype reflects the extent of the reduction of the channel ATP sensitivity. Thus, mutations that produce a small increase in KATP current in the presence of physiological concentrations of MgATP (1\u20135\u00a0mmol\/l) lead to PNDM, whereas mutations that cause a larger increase in KATP current give rise to DEND syndrome [15].\nIn this paper, we identify a KCNJ11 mutation (L164P) that causes neonatal diabetes without obvious neurological complications. In functional studies, we show that the L164P mutation produces a large increase in the resting whole-cell current and a marked reduction in KATP channel sensitivity to inhibition by ATP. These effects are a secondary consequence of an increase in the channel open probability (Po) produced by the mutation. Surprisingly, other mutations that produce a similar increase in Po cause DEND syndrome. The L164P mutant channel was also far less blocked by the sulfonylurea tolbutamide, which explains why the patients were unable to transfer to glibenclamide therapy.\nMethods\nParticipants Informed consent was obtained from all individuals investigated (or from their parents if they were children).\nMolecular genetic analysis Genomic DNA was extracted from peripheral lymphocytes using standard procedures. The KCNJ11 gene was amplified and sequenced as described [25]. Other family members were also tested for the novel mutation. Family relationships were confirmed using a combination of six microsatellites on chromosome 11: D11S902, D11S419, D11S1397, D11S1901, D11S921 and D11S1888.\nOocyte preparation Female Xenopus laevis were anaesthetised with ethyl 3-aminobenzoate methanesulfonate salt (MS222; 2\u00a0g\/l added to the water). One ovary was removed via a mini-laparotomy, the incision sutured and the animal allowed to recover. Subsequently, animals were operated on for a second time, but under terminal anaesthesia. Immature stage V\u2013VI oocytes were incubated for 60\u00a0min with 1\u00a0mg\/ml collagenase (Type V; Sigma, Poole, UK) and manually defolliculated. All procedures were carried out in accordance with UK Home Office Legislations and the University of Oxford ethical guidelines. Oocytes were coinjected with \u223c0.8\u00a0ng wild-type or mutant Kir6.2 mRNA and \u223c4\u00a0ng mRNA encoding SUR. The final injection volume was 50\u00a0nl per oocyte. Isolated oocytes were maintained in Barth\u2019s solution and studied 1\u20134\u00a0days after injection.\nElectrophysiology Wild-type or mutant Kir6.2 (GenBank D50581) were coexpressed with SUR1 in Xenopus oocytes (GenBank L40624) as described [26]. Because all the patients were heterozygous for the L164P mutation, their pancreatic beta cells will contain a mixture of wild-type and mutant Kir6.2 subunits. To simulate this heterozygosity, we coinjected Xenopus oocytes with a 1:1 mixture of mutant and wild-type Kir6.2 together with SUR1 mRNA. This is expected to give rise to a mixed population of channels composed of homomeric wild-type channels, homomeric mutant channels and heteromeric channels containing between one and three mutant subunits [21]. We refer to this global channel population as heterozygous channels.Whole-cell currents were recorded using a two-electrode voltage clamp in response to voltage steps of \u00b120\u00a0mV from a holding potential of \u221210\u00a0mV, filtered at 1\u00a0kHz and digitised at 4\u00a0kHz. Oocytes were perfused with a solution containing (in mmol\/l): 90\u00a0KCl, 1\u00a0MgCl2, 1.8\u00a0CaCl2 and 5\u00a0HEPES (pH\u00a07.4 with KOH). Metabolic inhibition was produced by 3\u00a0mmol\/l sodium azide.Macroscopic currents were recorded from giant inside-out patches using an EPC10 amplifier (List Medical Electronics, Darmstadt, Germany) controlled with Pulse v8.74 software (Heka Electronik, Lambrecht, Germany). Macroscopic currents were elicited by 3\u00a0s voltage ramps from \u2212110 to +100\u00a0mV (holding potential 0\u00a0mV), or recorded at a constant potential of \u221260\u00a0mV. They were filtered at 0.5\u00a0kHz and digitised at 1\u00a0kHz. The pipette solution contained (mmol\/l): 140\u00a0KCl, 1.2\u00a0MgCl2, 2.6\u00a0CaCl2, 10\u00a0HEPES (pH\u00a07.4 with KOH). The internal (bath) solution contained (mmol\/l): 107\u00a0KCl, 1\u00a0K2SO4, 2\u00a0MgCl2, 10\u00a0EGTA, 10\u00a0HEPES (pH\u00a07.2 with KOH) and Mg-nucleotides as indicated. Experiments were conducted at 20\u201322\u00b0C. Solutions were changed using a local perfusion system consisting of tubes of 200\u00a0\u03bcm diameter into which the tip of the patch pipette was inserted.Nucleotide concentration\u2013inhibition curves were fit with the Hill equation:\nwhere G is the KATP conductance in the presence of ATP, Gc is the KATP conductance in the absence of the nucleotide, [ATP] is the ATP concentration, IC50 is the nucleotide concentration at which inhibition is half maximal and h is the slope factor (Hill coefficient).Single-channel currents were measured at \u221260\u00a0mV, filtered at 5\u00a0kHz and digitised at 20\u00a0kHz. Unitary amplitude and Po were measured from the Gaussian fit to all-points amplitude histograms of tracts of current of 30\u201390\u00a0s duration.Data were analysed with in-house routines developed in the IgorPro platform (Wavematrics, Portland, OR, USA). Data are given as means\u2009\u00b1\u2009SEM in the text and in the figures. Statistical significance was evaluated using a two-tailed Student t test and p\u2009<\u20090.05 taken to indicate a significant difference.\nResults\nPatient characteristics and genetics Three unrelated probands with permanent neonatal diabetes were shown to be heterozygous for the KCNJ11 gene mutation L164P (c.491T>C, p.Leu164Pro). All were female and had unaffected parents of different ethnic origin. Two patients have been reported previously [27, 28]. Mutation testing and microsatellite analysis of DNA from both parents and the child for two of the cases confirmed that the mutation had arisen de novo (the parents of the third case were not available for testing). None of the patients had any neurological complications or obvious developmental delay.The first patient is currently 8\u00a0years old. She was born in Singapore from Sri Lankan parents, at 38\u00a0weeks of gestation with a birthweight of 2.6\u00a0kg. She developed diabetes at 30\u00a0weeks of age. When she came to Australia at the age of 6.5\u00a0years, her HbA1c level was elevated (7.4%) and her diabetes required insulin treatment (0.45\u00a0U kg\u22121 day\u22121). Glibenclamide treatment (1.0\u00a0mg kg\u22121 day\u22121) was trialled for 2\u00a0months (at 7\u00a0years of age) but did not produce a decrease in her insulin requirement. She continues to require insulin (up to 0.7\u00a0U kg\u22121 day\u22121) with an HbA1c ranging from 7.2 to 8.2%. She has normal developmental milestones, her neurological examination is normal and she has no evidence of diabetic complications.The second patient, born in Australia of Afghan origin, is currently 2.7\u00a0years old. She was born at term following an uneventful pregnancy, with a birthweight of 2.7\u00a0kg. She presented at 8\u00a0weeks in diabetic ketoacidosis and was subsequently treated (at 2.5\u00a0months for 4\u00a0weeks, then at 5\u00a0months for 3\u00a0months) with glibenclamide (up to 1.1\u00a0mg kg\u22121 day\u22121, regimens of two times per day or three times per day), but had no reduction in insulin requirement (0.4\u00a0U kg\u22121 day\u22121) [28]. Her mean HbA1c since diagnosis has been 7.6% (range 7.2\u20138.7%). Assessment at 5\u00a0months by a neurologist was entirely normal, with a normal EEG. Subsequently, she has had normal developmental milestones and her neurological examinations have been normal at all follow up visits every 3\u00a0months. Diabetic complications have not been assessed due to the young age of the patient.The third patient is from Slovakia [27]. She was born at term with a birthweight of 2.6\u00a0kg, and was diagnosed with diabetes at 5\u00a0weeks of age. She is currently 20\u00a0years old and is treated with insulin (1.24\u00a0U kg\u22121 day\u22121). Glibenclamide transfer was not attempted because the patient also suffers from hepatitis C. She has poor glycaemic control, with an HbA1c level of 15.2%, and various diabetic complications including retinopathy and nephropathy. Psychomotoric development in childhood was normal. A neurological examination suggested the presence of diabetic motoric polyneuropathy in the lower extremities but there were no further neurological findings. Although her IQ was not tested, she successfully completed a specialised business school training suggesting normal mental development.\nFunctional analysis: effects on whole-cell KATP currents We examined the functional effects of the L164P mutation by heterologous expression in Xenopus oocytes. When wild-type Kir6.2\/SUR1 channels are expressed in oocytes they are normally closed because they are inhibited by resting intracellular ATP concentrations ([ATP]i). They can be opened, however, by azide (3\u00a0mmol\/l; Fig.\u00a01), a metabolic inhibitor that lowers [ATP]i [29]. Heterozygous L164P (hetL164P) currents were about 18-fold larger at rest than wild-type currents, but increased twofold on metabolic inhibition indicating that the channel is only partially closed at resting ATP levels (Fig.\u00a01). In contrast, homomeric L164P (homL164P) channels displayed a much greater resting current and were little affected by metabolic inhibition (Fig.\u00a01).\nFig.\u00a01Whole-cell KATP current. Mean steady state whole-cell currents evoked by voltage steps from \u221210 to \u221230\u00a0mV before (control, white bars) and after application of 3\u00a0mmol\/l azide (grey bars) and in the presence of 3\u00a0mmol\/l azide plus 0.5\u00a0mmol\/l tolbutamide (black bars). The number of oocytes was five to seven in each case. G, KATP conductance; Gc, KATP conductance expressed relative to the conductance in the absence of the nucleotide. WT, wild-type; hetL164P and homL164P channels as indicated\nThe sulfonylurea tolbutamide (0.5\u00a0mmol\/l) blocked whole-cell KATP currents by 98% but had no effect on homL164P currents (4% block). HetL164P channels were blocked by only 54% (Fig.\u00a01).\nFunctional analysis: effects on KATP channel ATP sensitivity The increase in resting whole-cell KATP currents suggests that the L164P mutation may reduce the channel ATP sensitivity, as found for other PNDM mutations [21]. We first measured the ATP sensitivity of wild-type and mutant KATP currents in the absence of Mg2+, to isolate the effects of ATP on Kir6.2 (in the absence of Mg2+, ATP does not interact with SUR1 [11]). Both homL164P and hetL164P channels had severely impaired ATP sensitivity. HomL164P channels were not blocked at all by ATP, even at concentrations as high as 10\u00a0mmol\/l. The concentration\u2013inhibition curve for hetL164P showed a striking shift to higher ATP concentrations, with an IC50 of about 100\u00a0\u03bcmol\/l, and a marked pedestal of unblocked current at very high ATP concentrations. The data were best fitted by assuming that in the heterozygous state about 20% of channels are never closed by ATP (Fig.\u00a02a,b and Table\u00a01).\nFig.\u00a02ATP-inhibition of L164P channels is less that that of wild-type channels. a, c Currents recorded in inside-out patches excised from Xenopus oocytes expressing hetKir6.2-L164P\/SUR1 (hetL164P) or homKir6.2-L164P\/SUR1 (homL164P) channels, as indicated, in response to 3\u00a0s voltage ramps from \u2212110 to +100\u00a0mV. ATP (10\u00a0mmol\/l) was applied as indicated by the horizontal bars in the absence (a) or presence (c) of 2\u00a0mmol\/l Mg2+. b, d, mean relationship between [ATP] and KATP conductance (G), expressed relative to the conductance in the absence of the nucleotide (Gc), for wild-type (white circles, n\u2009=\u20099), hetL164P (white\/black circles, n\u2009=\u20096) or homL164P (black circles, n\u2009=\u20094) channels. Experiments were carried out in the absence (b) or presence (d) of 2\u00a0mmol\/l Mg2+. The continuous lines through the black circles were drawn by eye. The smooth curves are the best fit to the Hill equation with IC50 of 11\u00a0\u03bcmol\/l (wild-type) and 100\u00a0\u03bcmol\/l (hetL164P) (b, 0\u00a0mmol\/l Mg2+) or IC50 of 16\u00a0\u03bcmol\/l (wild-type) and 122\u00a0\u03bcmol\/l (hetL164P) (d, 2\u00a0mmol\/l Mg2+)Table\u00a01ATP sensitivity of wild-type and mutant channels\u00a0IC50 (Mg-free)h (Mg-free)IC50 (2\u00a0mmol\/l Mg2+)h (2\u00a0mmol\/l Mg2+)A (2\u00a0mmol\/l Mg2+)%Imax (3\u00a0mmol\/l MgATP)Wild-type9.6\u2009\u00b1\u20091.61.30\u2009\u00b1\u20090.0815.8\u2009\u00b1\u20093.00.99\u2009\u00b1\u20090.05n.a.0.01\u2009\u00b1\u20090.01hetL164P100\u2009\u00b1\u20098*1.10\u2009\u00b1\u20090.05118\u2009\u00b1\u200929*1.16\u2009\u00b1\u20090.340.29\u2009\u00b1\u20090.07*33.6\u2009\u00b1\u20093.4*homL164Pn.a.n.a.n.a.n.a.n.a.98\u2009\u00b1\u20091*Values are means\u2009\u00b1\u2009SEM. The number of patches was four to nine in each case.A The fraction of unblocked current used to fit the ATP concentration\u2013inhibition relationship; h Hill coefficient; IC50, ATP concentration (\u03bcmol\/l) producing half-maximal inhibition; %Imax the per cent unblocked current in the presence of 3\u00a0mmol\/l MgATP; n.a. not applicable (as no block).*p\u2009<\u20090.05 vs wild-type\nMolecular mechanism of the reduced ATP sensitivity Mutations that reduce the ATP sensitivity of the KATP channel can act in several ways. They may prevent ATP binding directly. They may impair the mechanism by which nucleotide binding is coupled to channel gating. They may also stabilise the intrinsic open state of the channel (i.e. that in the absence of ATP), which shifts the gating equilibrium in the presence of ATP towards channel opening and thus indirectly reduces the channel ATP sensitivity (e.g. [15, 30, 31]).In a structural model of Kir6.2 [32], L164P lies within the channel pore, at a considerable distance from the ATP-binding site (Fig.\u00a03). It is not predicted to interact directly with ATP. We therefore examined whether the L164P mutation alters intrinsic gating. Experiments were carried out in the absence of ATP, where intrinsic gating can be assessed. The L164P mutation had no effect on single-channel current amplitude (Table\u00a02). However, the intrinsic Po was markedly increased, being 0.86 (n\u2009=\u20096) for homL164P compared with 0.4 (n\u2009=\u20094) for wild-type channels (Fig.\u00a04 and Table\u00a02). This suggests that, at least in part, the L164P mutation alters channel ATP sensitivity indirectly, via an increase in Po.\nFig.\u00a03Homology model of Kir6.2 [32]. For clarity, only two subunits are shown. ATP (yellow) is shown in its binding site. Residue L164 is shown in redFig.\u00a04The L164P mutation enhances single-channel activity. Representative single KATP channel currents recorded at \u221260\u00a0mV in inside-out patches from oocytes expressing wild-type or homL164P channels, as indicated. Currents were recorded in the absence of Mg2+ and nucleotidesTable\u00a02Single-channel parameters for wild-type and homL164P channels\u00a0Poi (pA)Wild-type0.39\u2009\u00b1\u20090.054.0\u2009\u00b1\u20090.1homL164P0.86\u2009\u00b1\u20090.01*4.1\u2009\u00b1\u20090.1Mean\u2009\u00b1\u2009SEM values of intrinsic open probability (Po) and single-channel current (i) measured at \u221260\u00a0mVThe number of patches was five to six in each case* p\u2009<\u20090.05 vs wild-type\nEffects on KATP channel ATP sensitivity in the presence of Mg2+ Previous studies have shown that Kir6.2 mutations associated with neonatal diabetes may not only decrease the sensitivity of Kir6.2 to ATP, but can also enhance channel activation by Mg-nucleotides [24]. We therefore explored the effect of the L164P mutation on the ATP sensitivity in the presence of 2\u00a0mmol\/l Mg2+.Surprisingly, there was no difference in the effect of ATP in the presence and absence of Mg2+ on homL164P channels (Fig.\u00a02c,d). This contrasts with what has been observed for mutations in the ATP-binding site that completely abolished ATP inhibition in the absence of Mg2+, such as R50P and G334D: channels containing these mutations were activated by MgATP [33, 34]. The IC50 for ATP inhibition of hetL164P channels was also not significantly affected by Mg2+, although the pedestal of unblocked current at high ATP concentrations was increased by about 50% (Table\u00a01). At 3\u00a0mmol\/l MgATP, a concentration within the physiological range, the unblocked current was 34%, substantially greater than that found for wild-type channels (<1%; Table\u00a01).\nFunctional analysis: effects on MgADP sensitivity The lack of MgATP activation of hetL164P channels could be due to reduced functional coupling between SUR1 and Kir6.2-L164P, or to reduced MgATP binding\/hydrolysis at the NBDs of SUR1. To explore the former possibility, we measured the ability of MgADP to activate hetL164P channels preblocked by 100\u00a0\u03bcmol\/l MgATP. It was necessary to preblock the channels as the mutant channel has an open probability close to maximal and further activation is therefore not possible. Figure\u00a05 shows that 30\u00a0\u03bcmol\/l MgADP activates wild-type currents by 3.7\u2009\u00b1\u20091.8 fold (n\u2009=\u20093) and that 100\u00a0\u03bcmol\/l MgADP did not produce any further increase (3.7\u2009\u00b1\u20091.2-fold, n\u2009=\u20093). In contrast, hetL164P channels were activated less: 1.4\u2009\u00b1\u20090.1 fold (n\u2009=\u20093) by 30\u00a0\u03bcmol\/l MgADP and 1.5\u2009\u00b1\u20090.1 fold (n\u2009=\u20094) by 100\u00a0\u03bcmol\/l MgADP.\nFig.\u00a05Sensitivity to MgADP of wild-type and hetL164P channels. a Representative currents (I) recorded at \u221260\u00a0mV from inside-out excised membrane patches from Xenopus oocytes expressing wild-type or hetL164P channels, as indicated. Patches were exposed to 100\u00a0\u03bcmol\/l ADP in the continuous presence of 100\u00a0\u03bcmol\/l ATP: 2\u00a0mmol\/l Mg2+ was present throughout. b Mean current in the presence of 100\u00a0\u03bcmol\/l MgATP or 30 or 100\u00a0\u03bcmol\/l MgADP plus 100\u00a0\u03bcmol\/l MgATP, normalised to the current in the absence of nucleotides for wild-type (grey bars) and hetL164P (black bars) channels. Bars indicate means\u2009\u00b1\u2009SEM. The number of patches was three to four in each case\nDespite the fact that 100\u00a0\u03bcmol\/l MgATP blocked mutant channels less, the lower extent of activation of hetL164P channels by 30\u00a0\u03bcmol\/l MgADP is not due to the fact that channel activity is already maximal, as the amplitude is still significantly less than that in control solution (Fig.\u00a05).\nDiscussion\nWe describe the clinical and functional effects of a KCNJ11 mutation, L164P, associated with permanent neonatal diabetes. This mutation causes a marked reduction in KATP channel inhibition by ATP primarily by stabilising the open state of the channel. This leads to an increase in the whole-cell KATP current, and in beta cells is expected to result in a reduction in insulin secretion. Surprisingly, unlike other mutations that reduced the channel ATP sensitivity by a similar amount, no motor or mental developmental delay was associated with the L164P mutation.\nStructural considerations and molecular basis for reduced ATP sensitivity In a homology model of Kir6.2 [32], L164 lies partway along the permeation pathway, 35\u00a0\u00c5 away from the ATP-binding site (Fig.\u00a03). It is therefore unlikely that it acts by reducing ATP binding directly. The side-chains of L164 point into the pore, forming a hydrophobic girdle that is narrow enough to prevent the passage of water and hydrated K+ ions [35], which suggests that L164 may form a hydrophobic gate within the pore. Functional studies support this idea. Following mutation of L164 to cysteine, cadmium ions were able to block the KATP channel with high affinity [36, 37], suggesting that the four cysteines (one on each subunit) come together close enough to form a high-affinity binding site for Cd2+. These data indicate that the side-chain of L164 must face into the pore, and that the pore is very narrow at this position. Substitution of a proline for L164 is expected to produce a kink in the \u03b1-helix [38] and disrupt the hydrophobic gate. It is therefore not surprising that the L164P mutation caused a dramatic effect on the Po. Mutation of L164 to cysteine, alanine, valine, threonine or glycine also produces a very large increase in Po [30, 36, 37, 39], consistent with the importance of this residue in channel gating.\nThe fact that the L164P mutation enhanced Po can explain, in part, the reduced ATP sensitivity of the channel. It is also possible that the mutation may have effects additional to stabilisation of the channel open state.\nEffect of Mg2+ on Kir6.2-L164P\/SUR1 channel ATP sensitivity In contrast to all PNDM mutations studied to date [24], MgATP was unable to enhance the activity of either homL164P or hetL164P channels. The lack of MgATP activation of homL164P channels may be due to the fact that Po is already very high and thus there is no scope for further activation. However, little MgATP activation was also observed for hetL164P channels. This is in marked contrast to other mutations that caused enhanced Po (I296L, [16], V59G [15]) where MgATP activated both homomeric and heterozygous channels. It is therefore possible that the L164P mutation reduces the efficacy of coupling between SUR1 and Kir6.2, and thereby decreases the ability of MgATP to stimulate channel activity. In support of this idea, MgADP activation of hetL164P channels was also reduced. This may explain the inability of Mg2+ to reduce ATP inhibition of hetL164P channels, since it is well established that MgATP must be hydrolysed to MgADP to stimulate channel activity.The reason for the lack of Mg-nucleotide activation of hetL164P channels is unclear. Because L164 lies within the pore, far away from the NBDs of SUR1, the effect must be mediated allosterically. It is possible that this is mediated by an interaction between the backbone of L164 itself, or transmembrane domain 2 (within which L164 lies), and the transmembrane domains of SUR1. In the absence of an atomic resolution structure of the KATP channel complex, however, this cannot be definitely determined.HomL164P channels were not blocked by tolbutamide, as expected because of their high Po [40]. The lower efficacy of tolbutamide on hetL164P channels (about 50% block) may reflect the enhanced Po of channels within the heterozygous population containing mutant subunits. It could also reflect impaired coupling between SUR1 and Kir6.2, such as that found for MgADP.\nClinical implications Previous studies of KCNJ11 mutations have suggested that there is a good correlation between the percentage of current that remains unblocked in the presence of 3\u00a0mmol\/l MgATP and the clinical phenotype. Namely, currents that are >30% of maximal are associated with DEND syndrome, and those that lie between 5\u201310% of maximal with neonatal diabetes alone, compared with a current of <1% for wild-type channels (Fig.\u00a06). The L164P mutation does not conform to this simple relationship between the functional effects of the mutation and the clinical phenotype. The magnitude of the hetL164P current in 3\u00a0mmol\/l MgATP was 36%, yet none of the patients had extra-pancreatic symptoms.\nFig.\u00a06Mean KATP current (expressed as a % of maximum) measured in the presence of 3\u00a0mmol\/l MgATP from inside-out patches expressing heterozygous mutant channels as indicated. The dashed line indicates the maximal current amplitude normally associated with PNDM. iDEND, intermediate DEND syndrome (i.e. neonatal diabetes with developmental delay [21]). Data for wild-type (WT) and Kir6.2-R201H channels are from [14], for Y330C and F333I from [26], for Q52R, V59G and R201C from [15], for V59M from [24], for I296L from [16] and for G334D from [34]The reason for this anomaly is not clear, particularly as the resting whole-cell current was also very large. Previous studies have also indicated that such large whole-cell currents are invariably associated with more severe clinical symptoms. The lack of responsiveness of the patient to sulfonylureas is consistent with the very large increase in Po and the resting whole-cell current. It is therefore likely that hetL164P channels expressed in Xenopus oocytes are a reasonable model for the pancreatic beta cell KATP channels of the patients. We are therefore forced to conclude that compensatory mechanisms must ameliorate the extra-pancreatic effects of this mutation. The fact that the patients had very different ethnic origins suggests the compensatory mechanism is unlikely to reflect a shared genetic background: however, it does appear to be specific to the L164P mutation, which is unique (to date) in having a marked functional effect in vitro despite causing neonatal diabetes without neurological complications.Importantly, a maximally effective concentration of tolbutamide only blocked hetL164P channels by 50%. All patients to date whose channels are blocked by <65% have not been able to transfer from insulin treatment to sulfonylurea therapy. This suggested that our patients would be unable to transfer to sulfonylurea therapy, as indeed was found to be the case. This is likely to be due to the fact that L164P destabilises the long closed state of the channel, to which sulfonylureas preferentially bind, and which is rarely entered in channels with enhanced Po. Our results further suggest that not all patients with permanent neonatal diabetes will necessarily respond to sulfonylurea treatment, and indicates that knowledge of the functional effect of the mutation is helpful for predicting the drug response in patients.","keyphrases":["kir6.2","neonatal diabetes","katp channel","kcnj11","type 2 diabetes"],"prmu":["P","P","P","P","R"]}
{"id":"Behav_Brain_Res-2-1-2424131","title":"Effect of disconnecting the orbital prefrontal cortex from the nucleus accumbens core on inter-temporal choice behaviour: A quantitative analysis\n","text":"Previous experiments showed that destruction of the orbital prefrontal cortex (OPFC) or the nucleus accumbens core (AcbC) in rats altered choice between two delayed food reinforcers. Application of a quantitative model of inter-temporal choice suggested that lesions of either structure increased the delay-dependent degradation of reinforcer value (delay discounting); destruction of the OPFC (but not the AcbC) also increased the relative value of the larger reinforcer. This experiment examined the effect of disconnecting the OPFC from the AcbC on inter-temporal choice. Rats received excitotoxin-induced contralateral lesions of the OPFC and AcbC (disconnection), severing of the anterior corpus callosum (callosotomy), a combined lesion (disconnection + callosotomy) or sham lesions. They were trained in a discrete-trials progressive delay schedule to press levers A and B for a sucrose solution. Responses on A delivered 50 \u03bcl of the solution after a delay dA; responses on B delivered 100 \u03bcl after a delay dB. dB increased across blocks of trials; dA was manipulated across phases of the experiment. Indifference delay, dB(50) (value of dB corresponding to 50% choice of B), was estimated for each rat in each phase, and linear indifference functions (dB(50)vs. dA) were derived. The disconnection + callosotomy group showed a lower intercept of the indifference function (implying a higher rate of delay discounting) than the sham-lesioned group; the disconnection group showed a similar but less robust effect, whereas the callosotomy group did not differ significantly from the sham-lesioned group. The results suggest that OPFC\u2013AcbC connections are involved in delay discounting of food reinforcers, but provide no evidence for an involvement of OPFC\u2013AcbC connections in regulating sensitivity to reinforcer size.\n1\nIntroduction\nIt is widely accepted that the capacity of a reinforcer to exert control over operant behaviour is a direct function of its size and an inverse function of the delay that precedes its delivery. These two principles are placed in mutual opposition in inter-temporal choice schedules, in which the subject is required to choose between two reinforcers that differ along both dimensions. For example, the subject may be confronted with a choice between a smaller reinforcer, A, of size qA, delivered after a short delay, dA, and a larger reinforcer, B, of size qB, delivered after a longer delay, dB.\nInter-temporal choice schedules have provided valuable insights into the behavioural and neurobiological bases of \u2018delay discounting\u2019, the hypothetical process whereby the efficacy or \u2018value\u2019 of a reinforcer decays as a function of delay. Recent work has implicated the core of the nucleus accumbens (AcbC) [1,2,6,8] and the orbital region of the prefrontal cortex (OPFC) [23,26,42] in inter-temporal choice behaviour. However, the interpretation of results obtained using these schedules is complicated by the fact that magnitude and delay of reinforcement are generally manipulated simultaneously. Therefore it is often unclear whether the effect of an intervention on inter-temporal choice behaviour has been brought about by a change in the rate of delay discounting, by a change in the organism's sensitivity to relative reinforcer size, or both [8,19,31]. One approach to overcoming this problem is the application of null equations derived from quantitative models of inter-temporal choice [19,29\u201331]. For example, according to one such model [19], the overall value of a reinforcer, V, is determined by the multiplicative combination of two hyperbolic expressions that define the effects of delay and magnitude upon reinforcer value:where K is the delay-discounting parameter [29] and Q is a parameter expressing sensitivity to reinforcer size [19]. Faced with a choice between two reinforcers, A and B, the organism is presumed to select the reinforcer with the higher value. However, by experimentally manipulating the sizes and delays of the two reinforcers, it is possible to establish a state of \u2018indifference\u2019, in which A and B are selected with equal frequency. Indifference between A and B is taken to imply equality of value, i.e., VA\u00a0=\u00a0VB. Using Eq. (1) to define VA and VB and solving for dB(50) (the delay to reinforcer B at the point of indifference), yields the following null equation:in which dB(50) is linearly related to dA. By examining the effect of an intervention on this relation, it is possible to deduce whether the intervention has affected the organism's rate of delay discounting, its sensitivity to reinforcer size, or both. Since K makes no contribution to the slope of the function, a change in slope implies a change in sensitivity to reinforcer size, whereas a change in intercept in the absence of a concomitant change in slope implies a change in the rate of delay discounting [19].\nDestruction of the OPFC was found to increase the slope of the linear indifference function, indicating that the lesion affected sensitivity to reinforcer size; the lack of a concomitant increase in the intercept suggested that the rate of delay discounting had also been increased [26] (see Section 4 for further explanation). In contrast, lesions of AcbC reduced the intercept without significantly altering the slope of the function, implying a selective effect on delay discounting [2]. While these observations implicate both the OPFC and the AcbC in delay discounting, they leave unanswered the question of whether the two structures play independent roles in delay discounting, or whether they contribute to an integrated mechanism regulating inter-temporal choice behaviour. The present experiment was intended to address this question: the effect of functional disconnection of the OPFC and AcbC on inter-temporal choice behaviour was examined using the linear indifference relation epitomised by Eq. (2).\nThe principal anatomical link between the OPFC and the AcbC is an ipsilateral excitatory glutamatergic corticofugal pathway, which is believed to comprise one link in a cortico-striato-thalamo-cortical circuit. Inhibitory \u03b3-aminobutyric acid (GABA)ergic efferents from the AcbC project to the internal pallidum and substantia nigra pars reticulata. These structures in turn send inhibitory projections to diencephalic structures, whose excitatory corticopetal projections complete the circuit. In order to effect functional disconnection of the OPFC and AcbC without totally ablating either structure, a \u2018disconnection lesion\u2019 [12,14] was employed, which consisted of unilateral excitotoxic destruction of the OPFC in one hemisphere and of the AcbC in the other hemisphere. As recent evidence indicates that transcallosal fibres may make a significant functional contribution to cortico-striatal connections [9], the effects of callosotomy alone, and callosotomy combined with the disconnection lesion were also examined.\n2\nMethods\nThe experiment was carried out in accordance with UK Home Office regulations governing experiments on living animals.\n2.1\nSubjects\nSixty experimentally naive female Wistar rats (Charles River UK) approximately 4 months old and weighing 250\u2013300\u00a0g at the start of the experiment were used. They were housed individually under a constant cycle of 12\u00a0h light and 12\u00a0h darkness (light on 06:00\u201318:00\u00a0h), and were maintained at 80% of their initial free-feeding body weights throughout the experiment by providing a limited amount of standard rodent diet after each experimental session. Tap water was freely available in the home cages.\n2.2\nSurgery\nAnaesthesia was induced with halothane (4% in oxygen), and the rat positioned in a stereotaxic apparatus (David Kopf), with the upper incisor bar set 3.3\u00a0mm below the inter-aural line. Anaesthesia was maintained with 2% halothane in oxygen during surgery. Holes were drilled in the skull for introduction of a microinjection cannula or leucotome (see below). Disconnection: sixteen rats received unilateral lesions of the OPFC and the contralateral AcbC (the sides being counterbalanced across rats). The following coordinates (mm, measured from bregma) were used to locate the OPFC: site (i): AP +3.7, L \u00b11.2, DV \u22124.8; site (ii): AP +3.7, L \u00b12.8, DV \u22124.4. The coordinates for the AcbC were: AP +1.2, L \u00b11.8, V \u22127.1. Injections were given via a 0.3-mm diameter cannula connected by a polyethylene tube to a 10-\u03bcl Hamilton syringe. In each site, 0.5\u00a0\u03bcl of a 0.1-M solution of quinolinic acid (2,3-pyridinedicarboxylic acid) in phosphate-buffered 0.9% NaC1 (pH 7.0) was injected at a rate of 0.1\u00a0\u03bcl per 15\u00a0s. The cannula was left in position for 3\u00a0min after completion of the injection. Callosotomy: 15 rats underwent a midline leucotomy in order to sever the anterior corpus callosum. The leucotome, similar to that described by Gold et al. [17], was constructed from a 1-\u03bcl Hamilton microsyringe. A curved wire could be extruded from the tip of the syringe needle; when fully extended, the wire projected approximately 3\u00a0mm in the AP plane (i.e. at right angles to the needle). Two midline cuts were made: (i) the tip of the needle was positioned at AP 0.0, DV \u22125.0, the wire extruded in a rostral direction, and the tip slowly raised to DV \u22121.0; the wire was then retracted into the needle, and the needle was withdrawn from the brain; (ii) the tip of the needle was repositioned at AP +2.0, DV \u22124.0 and the procedure repeated. Disconnection\u00a0+\u00a0callosotomy: 15 rats underwent both the disconnection and callosotomy procedures described above. Sham lesion: fourteen rats underwent the same surgical procedures as the disconnection-lesioned group, except that the vehicle solution alone was injected into the target sites.\n2.3\nApparatus\nThe rats were trained in standard operant conditioning chambers (CeNeS Ltd., Cambridge, UK) of internal dimensions 25\u00a0cm\u00a0\u00d7\u00a025\u00a0cm\u00a0\u00d7\u00a022\u00a0cm. One wall of the chamber contained a recess into which a peristaltic pump could deliver a 0.6\u00a0M sucrose solution. Two apertures situated 5\u00a0cm above and 2.5\u00a0cm to either side of the recess, through which motor-operated retractable levers could be inserted into the chamber. The levers could be depressed by a force of approximately 0.2\u00a0N. A 2.8-W lamp was mounted 2.5\u00a0cm above each lever; a third lamp was mounted 10\u00a0cm above the central recess. Six red light-emitting diodes were mounted in a row, 4\u00a0cm apart, 5\u00a0cm above the levers. The operant chamber was enclosed in a sound-attenuating chest; masking noise was generated by a rotary fan. An Acorn microcomputer programmed in Arachnid BASIC (CeNeS Ltd., Cambridge, UK), located in an adjoining room, controlled the schedules and recorded the behavioural data.\n2.4\nBehavioural training\nTwo weeks after surgery, the food-deprivation regimen was introduced and the rats were gradually reduced to 80% of their free-feeding body weights. They were then trained to press two levers (A and B) for sucrose reinforcement, and were exposed to a discrete-trials continuous reinforcement schedule in which the two levers were presented in random sequence for three sessions. After this initial training, they underwent daily training sessions under the discrete-trials delayed reinforcement schedule for the remainder of the experiment. Each experimental session consisted of six blocks of six trials, except in phases 4 and 5 when sessions consisted of five blocks. The trials were 90\u00a0s in duration, with the exception of phase 5, in which the duration was increased to 120\u00a0s in order to accommodate the long delay to reinforcement (see below). The six blocks were signalled by illumination of the six light-emitting diodes: in block 1 the first (left-most) diode was illuminated, in block 2 the first and second diodes were illuminated, and so on. The first two trials of each block were forced-choice trials in which each lever was presented alone in random sequence. The other four trials were free-choice trials in which both levers were presented. The beginning of each trial was signalled by illumination of the central light above the reinforcer recess. After 2.5\u00a0s the lever or levers (depending on the type of trial) were inserted into the chamber. When a lever-press occurred, the lever(s) were withdrawn, the central light was extinguished, and the light located above the lever that had been depressed was illuminated. This light remained illuminated until the delivery of the reinforcer, and was then extinguished. The chamber remained in darkness until the start of the following trial. If no lever-press occurred within 5\u00a0s of the lever(s) being inserted, the lever(s) were retracted and the central light extinguished. (This seldom happened except during the first few training sessions.) A response on lever A initiated a fixed delay dA, following which 50\u00a0\u03bcl of the 0.6\u00a0M sucrose solution was delivered. A response on lever B initiated a variable delay dB, after which 100\u00a0\u03bcl of the same sucrose solution was delivered. The positions of levers A and B (left vs. right) were counterbalanced across subjects.\nThe experiment consisted of six phases, in which the value of dA was set at 1, 2, 4, 8, 12 and 0.5\u00a0s, respectively. In each phase, the value of dA was held constant. In each session the value of dB was set equal to dA in the first block of trials. In subsequent blocks dB was increased in increments of 75%. In phases 4 and 5, when dA was 8 and 12\u00a0s, respectively, computing five increments of 75% would have generated a value of dB that was longer than the duration of a trial in the sixth block of trials; therefore the number of blocks was limited to five in these phases. The first phase continued for 100 sessions, phase 2 for 50 sessions, and the remaining phases for 40 sessions.\nExperimental sessions were carried out 7 days a week, at the same time each day, during the light phase of the daily cycle (between 08:00 and 14:00\u00a0h).\n2.5\nHistology\nAt the end of the behavioural experiment, the rats were deeply anaesthetised with sodium pentobarbitone, and perfused transcardially with 0.9% sodium chloride, followed by 10% formol saline. The brains were removed from the skulls and fixed in formol saline for 1 week. Forty micrometer coronal sections were taken through the regions of the OPFC and AcbC (approximately from AP +5.0 to AP 0.0) using a freezing microtome.\n2.5.1\nCresyl violet staining\nThe procedure was similar to that described previously [26]. Alternate sections were mounted on chrome-gelatine-coated slides and air dried, hydrated by successive immersion in 95, 70 and 50% ethanol, stained in 0.25% cresyl violet for 2\u00a0min at room temperature, dehydrated by successive immersion in 50, 70, 95, and 100% ethanol and xylene, and mounted with DPX.\n2.5.2\nImmunocytochemistry\nIn the other sections neurone-specific nuclear protein (NeuN) was labelled as described by Jongen-Relo and Feldon [21]. Our protocol has been described elsewhere [2]. Briefly, freshly sliced sections were rinsed in 0.1\u00a0M phosphate buffered saline (PBS) and placed in 0.5% H2O2 in PBS for 30\u00a0min. After twice rinsing in PBS, they were placed for 1\u00a0h in a blocking solution (10% normal horse serum [Vector Laboratories, Peterborough, UK], 1% bovine serum albumin [BSA, Sigma\u2013Aldrich, Gillingham, UK] and 0.3% Triton X-100 [Sigma\u2013Aldrich] in PBS). They were incubated for 48\u00a0h at 4\u00a0\u00b0C with the primary antibody (monoclonal mouse anti-NeuN serum [1:5000, Chemicon, Chandlers Ford, UK] in 1% normal horse serum, 1% BSA and 0.3% Triton X-100 in PBS), washed twice in PBS, and incubated for 2\u00a0h at room temperature in biotinylated horse antimouse serum (Vector Laboratories) (1:1000 in 1% BSA and 0.3% Triton X-100 in PBS). After further rinsing in PBS, they were placed for 2\u00a0h in avidin\u2013biotin\u2013horseradish peroxidase complex (1:200, ABC-Elite, Vector Laboratories) in PBS. After two further rinses in PBS, they were placed in a chromagen solution (0.05% diaminobenzidine [Sigma\u2013Aldrich] and 0.01% H2O2 [Sigma\u2013Aldrich]) for 5\u00a0min. The reaction was observed visually and stopped by rinsing in PBS. The sections were floated on to chrome-gelatine-coated slides and mounted with DPX.\nAn investigator who was blind to the behavioural results performed the microscopic examination. Drawings of the area of the lesions were superimposed on the appropriate coronal sections in the stereotaxic atlas of Paxinos and Watson [34].\n2.6\nData analysis\nData from 5 of the 60 rats were discarded. Histological examination revealed two rats with misplaced excitotoxin-induced lesions; one rat in the disconnection\u00a0+\u00a0callosotomy group was discarded because of a failed callosotomy. Data from two further rats were discarded because they showed persistent exclusive responding on one lever. This left 13 rats in the sham-lesioned group, 14 in the disconnection group, 15 in the callosotomy group and 13 in the disconnection\u00a0+\u00a0callosotomy group.\n2.6.1\nPreference functions and linear indifference functions\nFor each rat, the percentage choice of lever B in the free-choice trials (%B) was computed for each block of trials from the pooled data from the last 10 sessions of each phase of the experiment. Plots of %B vs. dB were derived for each rat, and the indifference delay (dB(50): the value of dB corresponding to %B\u00a0=\u00a050%) was estimated by linear interpolation between the two delays which fell on either side of %B\u00a0=\u00a050% (i and j) using the formula: dB(50)\u00a0=\u00a0dB(i)\u00a0+\u00a0([dB(j)-dB(i)].[%Bi\u00a0\u2212\u00a050]\/[%Bi\u00a0\u2212\u00a0%Bj]) [39]. Plots of dB(50)\nvs. dA were obtained for each rat, and linear functions were fitted by the method of least squares; goodness of fit was expressed as r2, the proportion of the data variance accounted for by the fitted function. The slope and intercept of the linear indifference functions were analysed by two-factor ANOVA (presence\/absence of disconnection\u00a0\u00d7\u00a0presence\/absence of callosotomy) followed by multiple comparisons of the lesioned groups with the sham-lesioned group using Dunnett's test. Linear indifference functions (dB(50)\nvs. dA) were also derived for the group mean data. The slopes and elevations of these functions were analysed by one-factor ANOVA (group) followed by multiple comparisons of the lesioned groups with the sham-lesioned group using Dunnett's test, as described by Zar [43]: the slopes were first analysed, and in the absence of significant between-group variation in slope, a common weighted slope value was adopted in order to make comparisons among the elevations. (\u2018Elevation\u2019 refers to the y-axis location of the function taking the range of observed data into account, whereas \u2018intercept\u2019 refers to the intersection of the function with the y-axis location [43].)\n2.6.2\nPsychophysical analysis of preference functions\nLogistic functions were fitted to the group mean %B data and the %B data from each rat in each phase of the experiment: %B\u00a0=\u00a0100\/(1\u00a0+\u00a0[dB\/dB(50)]\u025b). This function defines a descending sigmoid curve which is symmetrical in semi-logarithmic co-ordinates; dB(50) and \u025b are parameters, dB(50) being the point of intersection of the logistic curve with the indifference line, and \u025b being the slope of the function. These parameters were used to derive the limen ([dB(25)\u00a0\u2212\u00a0dB(75)]\/2, where dB(25) and dB(75) are the estimated values of dB corresponding to %B\u00a0=\u00a025 and %B\u00a0=\u00a075, respectively), and the index of relative precision, the Weber fraction, was defined as limen\/dB(50). The Weber fraction was subjected to repeated-measures ANOVA (phase); as no significant effect of phase was revealed, the Weber fractions were averaged across phases and subjected to two-factor ANOVA (presence\/absence of disconnection\u00a0\u00d7\u00a0presence\/absence of callosotomy), as described above.\n3\nResults\n3.1\nBehavioural data\n3.1.1\nPreference functions and linear indifference functions\nPreference functions (%B vs. dB) derived for the four groups in all six phases of the experiment are shown in Fig. 1 (left-hand graphs). In all four groups, preference for lever B declined as a function of the delay to reinforcer B (dB). The horizontal lines in the graphs show the indifference level (i.e., %B\u00a0=\u00a050); the value of dB at which the preference function crossed this level (i.e. dB(50)) increased as a function of increasing values of dA, reflecting a progressive rightward displacement of the curve.\nFig. 2 shows indifference functions (dB(50)\nvs. dA) for the group mean data. In each group, the linear function accounted for more than 96% of the variance of the group mean data (r2\u00a0>\u00a00.96). Comparisons were made between the function derived for each of the lesioned groups and the function derived for the sham-lesioned group [43]. An initial test on the homogeneity of the slopes indicated that there was no significant effect of group upon the slope [F(3,20)\u00a0=\u00a01.8, P\u00a0>\u00a00.05], and the common (weighted) slope (3.16) was therefore adopted in statistical comparisons of the elevations of the functions. There was a significant effect of group on the elevation [F(3,23)\u00a0=\u00a024.4, P\u00a0<\u00a00.01]. Multiple comparisons (Dunnett's test) showed that the elevations of the functions derived for the disconnection and disconnection\u00a0+\u00a0callosotomy groups differed significantly from that of the sham-lesioned group [t(9)\u00a0=\u00a04.0, P\u00a0<\u00a00.05, and t(9)\u00a0=\u00a05.2, P\u00a0<\u00a00.01, respectively], whereas there was no significant difference between the elevations of the callosotomy and sham-lesioned groups [t(9)\u00a0=\u00a00.2, P\u00a0>\u00a00.3].\nLinear indifference functions were also fitted to the data from the individual rats. The group mean values (+S.E.M.) of the slope and intercept of the function are shown in Fig. 3. A two-factor analysis of variance (callosotomy\u00a0\u00d7\u00a0disconnection) showed no significant effect of either factor nor any significant interaction on the slope [all Fs\u00a0<\u00a01]. There was a significant effect of the disconnection lesion on the intercept [F(1,52)\u00a0=\u00a08.7, P\u00a0<\u00a00.005], but no significant effect of the callosotomy [F\u00a0<\u00a01] and no significant interaction [F\u00a0<\u00a01]. Multiple comparisons with the sham-lesioned group (Dunnett's test) showed that only the disconnection\u00a0+\u00a0callosotomy group differed significantly from the sham-lesioned group. The goodness of fit of the linear function did not vary significantly among the groups [F\u00a0<\u00a01]; the function accounted for >86% of the data variance for individual rats in all groups [r2\u00a0=\u00a00.865\u00a0\u00b1\u00a00.022].\n3.1.2\nPsychophysical analysis of preference functions\nThe logistic psychometric functions derived for the group mean data (Fig. 1, right-hand panels) accounted for 95% of the data variance (r2\u00a0>\u00a00.95) in all cases. The logistic function could be fitted to 311 of the 330 preference functions obtained for the individual rats in the six phases of the experiment (94.2%); functions could be fitted to the data from all six phases in 48 of the 55 rats. Analysis of variance (disconnection\u00a0\u00d7\u00a0callosotomy) indicated that the goodness of fit was not significantly affected by either the callosotomy [F\u00a0<\u00a01] or the disconnection lesion [F(1,51)\u00a0=\u00a01.2, P\u00a0>\u00a00.2], and there was no significant interaction [F\u00a0<\u00a01]. The overall mean (\u00b1S.E.M.) value of r2 derived from all rats in all phases of the experiment was 0.966\u00a0\u00b1\u00a00.005. There was good agreement between the values of dB(50) derived from the logistic functions (dB(50)logist.) and those derived by linear interpolation (dB(50)interp.). The slope of the regression of dB(50)logist.\nvs. dB(50)interp. (1.02\u00a0\u00b1\u00a00.01) did not deviate significantly from unity, and the intercept (\u22120.16\u00a0\u00b1\u00a00.31) did not deviate significantly from zero; the correlation (r) between the two estimates was 0.971.\nThe Weber fraction derived from the logistic function was not systematically related to the value of dA. A single-factor analysis of variance with repeated measures (incorporating data from the 48 rats that generated Weber fractions from all 6 phases) showed no significant effect of phase [F(5,235)\u00a0=\u00a01.9, P\u00a0>\u00a00.05]. The Weber fraction was therefore averaged across phases for each rat; the group mean values (+S.E.M.) are shown in Fig. 4 (left-hand histogram). Analysis of variance (callosotomy\u00a0\u00d7\u00a0disconnection) revealed a significant main effect of the disconnection lesion [F(1,51)\u00a0=\u00a06.6, P\u00a0<\u00a00.05], but no significant effect of the callosotomy [F(1,51)\u00a0=\u00a02.7, P\u00a0>\u00a00.1] and no significant interaction [F\u00a0<\u00a01]. Multiple comparisons (Dunnett's test) did not reveal any significant differences between the lesioned groups and the sham-lesioned group.\nThe group mean values of the slope of the logistic functions (\u025b) (+S.E.M.) are shown in Fig. 4 (right-hand histogram). Analysis of variance (callosotomy\u00a0\u00d7\u00a0disconnection) showed a significant main effect of the disconnection lesion [F(1,51)\u00a0=\u00a07.2, P\u00a0<\u00a00.05], but no significant main effect of the callosotomy [F\u00a0<\u00a01] and no significant interaction [F\u00a0<\u00a01]. None of the lesioned groups differed significantly from the sham-lesioned group (Dunnett's test).\n3.2\nHistology\nExamples of the lesions are shown in Fig. 5. OPFC: injection of quinolinic acid into the OPFC resulted in gliosis and atrophy of the ventral and lateral orbital regions. There was some damage to the medial prefrontal cortex (medial orbital, infralimbic and prelimbic cortices) in some rats. The AP extent of the lesion was from about +3.2 to +4.5; in no case did the lesion extend caudally as far as the anterior margin of the nucleus accumbens. AcbC: coronal sections showed ventricular dilatation and atrophy in the ventral striatal area. The NeuN labelled sections showed that there was extensive neuronal loss in the area of the AcbC of all lesioned animals, with some neuronal loss in the ventral and medial portions of the caudate-putamen in some animals; the shell region of the nucleus accumbens was essentially spared. Callosotomy: the corpus callosum was completely severed between AP +0.5 to +1.6 in most animals. There was some sparing of the corpus callosum posteriorly (caudal to AP +0.5), possibly reflecting the curvature of the leucotome. Destruction of the callosum was generally accompanied by some ventricular dilatation in the vicinity of the lesion, and the mesial surfaces of the cortex overlying the lesion showed some damage.\n4\nDiscussion\nInjections of quinolinic acid into the OPFC and AcbC produced lesions of similar extent to those seen in previous experiments in which excitotoxins have been used to lesion these structures [OPFC: 13, 23, 25, 26, and 42; AcbC: 1, 2, 3, 5, 6, and 35]. In the case of the OPFC lesion, the main area of damage included the ventral and lateral orbital (VO and LO) regions. There was also some intrusion into the medial orbital (MO), infralimbic (IL) and prelimbic (PrL) regions in some animals. According to the subregional classification recommended by Uylings and van Eden [40] and Kesner [22], the lesion embraced the ventral (VO) and lateral OPFC (LO), with some involvement of the medial PFC (MO, IL and PrL). In the case of the AcbC lesion, the area of destruction was mainly restricted to the target structure. Some additional damage was inflicted to the ventral portion of the caudate-putamen in some rats; and in some cases NeuN staining revealed a band of neuronal loss in the medial caudate-putamen adjacent to the lateral ventricle. The mesial shell region of the nucleus accumbens was spared. The callosotomy lesion was generally successful in severing the corpus callosum anterior to the caudal margin of the AcbC.\nThe discrete-trials schedule used in this study was an adaptation of the progressive delay schedule developed by Evenden and Ryan [10]. The rats in all four groups showed a progressive shift in preference from the larger to the smaller reinforcer as the delay to the larger of the two reinforcers (dB) was progressively increased across successive blocks of trials. This is consistent with previous studies that have used this schedule [2,6\u20138,10,11,23,24,26,33,42]. As in previous experiments [2,23,24,26], we used a geometric progression to determine the values of dB in successive blocks of trials, thereby allowing the range of values of dB to be adapted to the value of dA, which was systematically manipulated across the six phases of the experiment. The resulting preference functions (relation between %B and dB; see Fig. 1) were used to compute the indifference delays to the larger reinforcer (dB(50)) in each phase. This measure formed the basis of the linear indifference functions (see below).\nAs in previous studies employing Evenden and Ryan's [11] protocol [2,6\u20138,10,11,23,24,26,33,42], the preference functions seen in this experiment were characterized by a gradual reduction of %B as a function of dB. As noted by Bezzina et al. [2], this is apparently inconsistent with models of inter-temporal choice that are based on the computation of hypothetical \u2018values\u2019 of reinforcers. Such models generally assume that organisms should invariably select the more highly valued of two mutually exclusive reinforcers [18,19], leading to the prediction that the ideal preference function should be a step function, %B falling precipitously from near 100% to near 0% around the point at which VA\u00a0=\u00a0VB. Bezzina et al. [2] proposed that the gradual decline in preference generated by progressive delay schedules may represent a discrimination gradient for reinforcer value, and adopted a standard psychophysical approach to analyse the preference functions, an analytical approach that was also used in the present study. As in Bezzina et al.'s [2] experiment, a two-parameter logistic function adequately described the group mean preference functions (Fig. 1, right-hand panels) and more than 90% of the preference functions obtained from individual rats. The two parameters of this function define its slope (\u025b) and its locus on the abscissa (dB(50)). Combination of \u025b and dB(50) allows computation of the Weber fraction, the traditional measure of the relative precision of discrimination [15,16,20,28]. The data shown in Fig. 4 indicate that \u025b tended to be lower and the Weber fraction higher in the disconnection-lesioned and disconnection\u00a0+\u00a0callosotomy groups than in the sham-lesioned group. This resembles Bezzina et al.'s [2] finding with bilateral AcbC lesions, which induced a robust increase in the Weber fraction, consistent with an impairment of discriminative precision. It appears that functional disconnection of the OPFC from the AcbC had a similar effect on value discrimination as destruction of the AcbC.\nThe locus of the preference function is defined by dB(50), the delay to reinforcer B corresponding to indifference between the two reinforcers. According to hyperbolic models of inter-temporal choice [19,29], indifference implies equality of the values of the two reinforcers, which provides the basis for deriving the linear function expressed by Eq. (2). This equation offers a means of distinguishing between changes in inter-temporal choice behaviour brought about by effects on the hypothetical processes of delay discounting (K) and magnitude discounting (Q). The slope of the linear function reflects the physical magnitudes of the two reinforcers (qA and qB) and Q; a change in slope therefore implies a change in Q. The intercept of the function is influenced jointly by Q and K. Therefore, while an increase in Q causes an increase in both the slope and the intercept, an increase in K simply diminishes the intercept. If both parameters are increased, Q's effect on the intercept is countered by the change in K, whereas its impact on the slope remains unaltered [19].\nIn the present experiment, the indifference functions of the four groups did not differ significantly in terms of slope; however, there were significant between-group differences in the intercept. The disconnection-lesioned group showed a somewhat lower intercept than the sham-lesioned group. This effect was statistically significant when the functions fitted to the group mean data were compared; however it failed to reach significance when the functions fitted to the data from individual rats in the two groups were compared. A more robust effect on the intercept was seen in the case of the disconnection\u00a0+\u00a0callosotomy group, whose intercept was significantly lower than that of the sham-lesioned group, in the case of the functions fitted to the group mean data and also the functions fitted to the individual-subject data. The callosotomy had no significant effect on the intercept. These results indicate that functional disconnection of the AcbC from the OPFC selectively altered the delay discounting parameter, K. The lower intercept seen in the disconnection\u00a0+\u00a0callosotomy group implies a higher value of K, in other words, a higher rate of delay discounting, than that of the sham-lesioned group. The fact that callosotomy alone had no discernable effect on performance suggests that ipsilateral cortifugal fibres may be mainly responsible for the functional connection between the OPFC and AcbC; however, the fact that the combined disconnection\u00a0+\u00a0callosotomy lesion had a more robust effect on inter-temporal choice than the conventional lesion suggests that inter-hemispheric connections may also make a subsidiary contribution. The existence of inter-hemispheric cortico-striatal connections has been known for some time [32], although their functional importance has only recently been established. Dunnett and his colleagues [9,41] recently found that a combined disconnection\u00a0+\u00a0callosotomy lesion was needed to effect complete functional disconnection of the prefrontal cortex and dorsal striatum in the case of delayed alternation performance.\nPrevious experiments have examined the effects of selective lesions of the OPFC and AcbC on inter-temporal choice behaviour. Kheramin et al. [23,26] found that bilateral destruction of the OPFC increased the slope of the linear indifference function without altering the intercept, an effect that was attributed to an increase in the rate of delay discounting combined with increased sensitivity to relative reinforcer size (see also [25]). Cardinal et al. [6,8] first reported that bilateral lesions of the AcbC rendered rats more \u2018intolerant\u2019 to delay of reinforcement. This finding was recently confirmed and extended by Bezzina et al. [2], using the indifference-equation approach; these authors reported that destruction of the AcbC lowered the intercept of the linear indifference function without significantly altering the slope, implying an increase in the rate of delay discounting. These findings suggest that both the OPFC and AcbC may contribute to delay discounting. The present results further suggest that delay discounting may be regulated by an integrated mechanism that involves both these structures, and that the integrity of both structures and their connecting fibres may be important for the effective control of behaviour by delayed reinforcers. Interestingly, the present results provide no evidence for an involvement of OPFC\u2013AcbC connections in regulating sensitivity to reinforcer size. This suggests that while the OPFC may integrate information on multiple features of reinforcers, including both size and delay [37,38], its role in delay discounting may be more specifically related to its connections with the AcbC. Data consistent with this notion have recently been obtained using the progressive ratio schedule. Mathematical analysis of performance on this schedule [27] yields a quantitative index of instantaneous reinforcer value that is sensitive to variations of reinforcer size [4,36]. This index has been found to be reduced by lesions of the OPFC [25] but not by lesions of the AcbC [3], suggesting that the OPFC, but not the AcbC, may be involved in determining sensitivity to reinforcer size.","keyphrases":["orbital prefrontal cortex","nucleus accumbens core","inter-temporal choice","rat","delay discounting","disconnection lesion"],"prmu":["P","P","P","P","P","P"]}
{"id":"Pediatr_Nephrol-4-1-2413093","title":"How are podocytes affected in nail\u2013patella syndrome?\n","text":"Nail\u2013patella syndrome is an autosomal-dominant hereditary disease named for dysplastic fingernails and toenails and hypoplastic or absent kneecaps evident in patients with the syndrome. Prognosis is determined by the nephropathy that develops in many such patients. Besides podocyte foot-process effacement, pathognomonic changes in the kidney comprise electron-lucent areas and fibrillar inclusions in the glomerular basement membrane. These characteristic symptoms are caused by mutations in the gene encoding the transcription factor LMX1B, a member of the LIM-homeodomain gene family. Comparable with the human syndrome, homozygous Lmx1b knockout mice lack patellae and suffer from severe podocyte damage. In contrast, however, podocin and the \u03b13 and \u03b14 chains of collagen IV are absent in the glomeruli of Lmx1b knockout mice. Further studies with podocyte-specific Lmx1b knockout mice have confirmed the importance of LMX1B in podocytes, as these mice apparently develop foot processes initially but lose them later on. We therefore conclude that LMX1B is essential for the development of metanephric precursor cells into podocytes and possibly also for maintaining the differentiation status of podocytes. LMX1B can serve as a model system to elucidate a genetic program in podocytes.\nFor many years, the mesangial cell took the forefront of glomerular research (see, for example, [1, 2]), but its pedestal was first slowly shaken by the painstaking morphological investigations of Wilhelm Kriz (confer [3, 4] for an early and late review, respectively) and finally abruptly toppled by irrefutable genetic evidence pointing towards the podocyte as a crucial cell in the glomerulus [5]. Meanwhile, the podocyte is firmly rooted in pathogenetic models of glomerular diseases and it is hard to imagine that it will leave again. Although mutations in WT1, a gene encoding a transcription factor of the Cys2His2-zinc finger family, had been found responsible for the podocytopathies Denys-Drash syndrome [6], WAGR syndrome [7] and Frasier syndrome [8], the article by Karl Tryggvason\u2019s group on the identification of mutations in NPHS1 added another dimension [5]. NPHS1 is mutated in patients suffering from congenital nephrotic syndrome of the Finnish type. It codes for nephrin, a component of the slit diaphragm, and therefore is an essential part of the glomerular filtration barrier [9\u201311]. Since then several other genes have been cloned that when mutated lead to glomerular disease and which in the kidney are (almost) specifically expressed in podocytes. They are LMX1B [12\u201314], NPHS2, the gene encoding podocin [15]; ACTN4, the gene encoding \u03b1-actinin-4 [16]; CD2AP [17, 18] and TRPC6 [19, 20]. In the following years, an increasing amount of evidence has accumulated on the specific role of these proteins in the podocyte. Nephrin and podocin participate in the formation of the slit diaphragm complex, \u03b1-actinin-4 crosslinks actin filaments in podocytes, and TRPC6 belongs to a special class of cation channels. Very little, however, is known about how the podocyte-specific expression of these genes is achieved. The sparse evidence that has been published concerns WT1, which binds to sequences in the promoter regions of the Podxl gene (encoding podocalyxin) [21] and of the NPHS1\/Nphs1gene [22, 23]. Although WT1 activates the respective reporter constructs, the induction of the endogenous NPHS1\/Nphs1 gene by WT1 was described by one group [23] but not another [22].\nNail\u2013patella syndrome has been known for many decades as a hereditary disease and was one of the first genetic disorders for which linkage was established. In addition to the obvious limb abnormalities, nephrologic symptoms develop in \u223c40% of these patients over the course of several decades. On an ultrastructural level, the moth-eaten appearance of the glomerular basement membrane together with fibrillar deposits is considered typical of nail\u2013patella syndrome. In addition, podocytes lose their foot processes (for references, see [24]). In 1998, not only were the first mutations in the LMX1B gene published for patients suffering from nail\u2013patella syndrome [12\u201314], but a report also appeared on the first characterisation of the Lmx1b knock-out mouse [25]. A more careful analysis of the kidney phenotype in the Lmx1b knock-out mouse revealed pronounced retardation in the development of podocytes that did not elaborate foot processes and slit diaphragms. Consistent with this finding was the splitting of the glomerular basement membrane and the reduced number of endothelial fenestrations, because podocytes synthesise proteins of the glomerular basement membrane and control differentiation of glomerular endothelial cells [26, 27]. Attractive explanations for these morphological defects have come from the observations that the \u03b13 and \u03b14 chains of collagen IV are no longer detected in the glomerular basement membrane ([28] and personal observations), that the Nphs2 gene is no longer expressed in podocytes of homozygous Lmx1b knock-out mice, and that podocytes in homozygous Lmx1b knock-out mice produce less vascular endothelial growth factor (VEGF) [26, 27]. Further molecular analysis demonstrated that LMX1B bound to AT-rich sequences in the first intron of the COL4A4 gene [28] and in the promoter region of the NPHS2 gene [26, 27].\nAlthough the model that LMX1B activates the expression of COL4A4 and NPHS2, and that inactivating mutations in the LMX1B gene secondarily lead to the loss of collagen IV and podocin and therefore to the characteristic alterations in nail\u2013patella syndrome patients certainly is an attractive one, several caveats have to be mentioned as well. Firstly, we could not demonstrate activation of a reporter construct with 4.4\u00a0kbp of the NPHS2 promoter by LMX1B [27], although another group showed an approximate twofold activation of the reporter gene controlled by four concatemerised LMX1B binding sites from the NPHS2 promoter [26]. Secondly, when we stably transfected a human cervical carcinoma cell line HeLa cells (which admittedly bears only a very remote similarity to podocytes) with an LMX1B cDNA, we found no upregulation of the endogenous NPHS2 gene [27]. Thirdly, podocin and the \u03b13 and \u03b14 chains of collagen IV were still present in glomeruli from patients with nail\u2013patella syndrome [29]. And fourthly, the constitutive podocyte-specific inactivation of Lmx1b in the mouse does not lead to the loss of podocin or collagen IV [30]. What do these apparently discrepant results mean? If LMX1B already acts at a very early stage of podocyte development, specifically before the NPHS2, COL4A3 and COL4A4 genes are turned on (by other transcription factors?), the podocyte will just not have reached an advanced enough stage of development to produce podocin and collagen IV. In other words, LMX1B may rather exert a permissive influence and, for example, initiate the spreading of the foot processes upon which podocin would be produced and slit diaphragms be elaborated.\nThe constitutive podocyte-specific inactivation of Lmx1b represents a more comparable model for human nephropathy, but the mice only survive for \u223c2\u00a0weeks after birth [30], again limiting their usefulness. In those animals, podocin and collagen IV \u03b13\/\u03b14 are still present, and it appears as if foot processes and slit diaphragms are first elaborated and then lost secondarily (Fig.\u00a01). Does LMX1B therefore play a role not only for the initial development of podocytes but also in the maintenance of their differentiation status? Clearly, more elaborate mouse models with an inducible inactivation of Lmx1b in adult animals are needed to answer this question. Such mice will also have the additional advantage of permitting the isolation of sufficient amounts of glomeruli, which can be used for DNA microarrays and identification of LMX1B target genes. Mouse genetics has already provided us with the verification of LDB1 as an interaction partner of LMX1B [30], and it may in the end help us to identify a genetic hierarchy acting in podocytes by telling us what factors control the expression of LMX1B in podocytes, what other proteins LMX1B interacts with and what genes are regulated by LMX1B in this peculiar cell type.\nFig.\u00a01a\u2013f Ultrastructural and immunohistochemical characterisation of mice with podocyte-specific inactivation of Lmx1b. In 11-day-old mice, the podocyte-specific inactivation of Lmx1b leads to the loss of foot processes and to a thickened glomerular basement membrane (arrows in b). However, despite the inactivation of Lmx1b, podocin and the \u03b14 chain of collagen IV are still produced (d, f). +\/lox control mice with one wild-type and one floxed Lmx1b allele; lox\/lox mice with two floxed Lmx1b alleles; Cre presence of the Cre transgene under control of the human NPHS2 promoter. Bars: 5\u00a0\u03bcm (a, b), 20\u00a0\u03bcm (c\u2013f). With permission from [30]","keyphrases":["lmx1b","slit diaphragm","mouse model","target genes","podocyte foot process","promotor analysis"],"prmu":["P","P","P","P","R","M"]}
{"id":"Eur_Spine_J-2-2-1602201","title":"Intraoperative monitoring study of ipsilateral motor evoked potentials in scoliosis surgery\n","text":"Ipsilateral motor evoked potentials (MEPs) in spinal cord surgery intraoperative monitoring is not well studied. We show that ipsilateral MEPs have significantly larger amplitudes and were elicited with lower stimulation intensities than contralateral MEPs. The possible underlying mechanisms are discussed based on current knowledge of corticospinal pathways. Ipsilateral MEPs may provide additional information on the integrity of descending motor tracts during spinal surgery monitoring.\nIntroduction\nIntraoperative monitoring (IOM) of the motor pathways is a routine procedure for ensuring integrity of corticospinal tracts during scoliosis surgery. In combination with somatosensory evoked potentials, motor evoked potential (MEP) monitoring is widely utilized in operations with significant risks of spinal cord damage [2, 14].\nMEPs are most effectively obtained with multi-pulse cortical electrical stimulation during IOM [3]. However, anesthetic agents, which cause suppression of cortical and spinal motor neuron excitability, affect them [4, 5]. While most IOM protocols involving MEPs utilize total intravenous anesthesia (TIVA), we have previously reported success with desflurane as a halogenated inhalational anesthetic agent [15].\nIn IOM, MEPs are elicited mostly with contralateral cortical electrical stimulation. Ipsilateral MEP responses have not been adequately studied in this context. Our paper describes observations of ipsilateral and contralateral MEPs with bilateral recordings, in conjunction with TIVA or desflurane during IOM of scoliosis surgery.\nMethods\nWe studied nine patients (mean age 16.2\u00a0years; range 14\u201317\u00a0years; 1 male) over a 6-month period in a prospective manner. The local ethical committee has approved the study protocol. All patients did not have medical conditions contraindicating transcranial electrical stimulation. Apart from scoliosis, they were healthy and had normal neurological examinations.\nMulti-pulse transcranial electrical stimulation was performed using two constant-current stimulators connected in parallel configuration from a Dantec Keypoint EMG machine (Dantec, Skovlunde, Denmark). A train of five square wave stimuli 0.5\u00a0ms in duration was delivered at 4\u00a0ms (250\u00a0Hz) interstimulus intervals. Stimulating electrodes consisted of 9\u00a0mm gold-plated disc electrodes at C3C4 (International 10\u201320 system) affixed with collodion. Stimulation output was increased from 50\u00a0mA in steps of 5\u00a0mA until a reproducible MEP was elicited. The intensity was then increased and fixed at 10% above this threshold intensity to obtain a supramaximal MEP response. Each stimulator was capable of delivering a maximum output of 100\u00a0mA (200\u00a0mA in total). MEP recordings were obtained with 13\u00a0mm disposable subdermal needles (Technomed Europe, Beek, Netherlands) in the tibialis anterior (TA) bilaterally. Filter settings were set at 10\u00a0Hz and 2\u00a0kHz. Input impedance of stimulating and recording electrodes were maintained below 5\u00a0k\u03a9.\nFor induction of anesthesia, sodium thiopentone at 4\u00a0mg\/kg and fentanyl at 2\u00a0mcg\/kg was administered; 0.8\u00a0mg\/kg of intravenous atracurium was used to facilitate endotracheal intubation. No further doses of neuromuscular blocking agents were used subsequently. In the desflurane group, anesthesia was maintained using 60% nitrous oxide in oxygen. Desflurane was introduced through a calibrated vaporizer up to an end-tidal concentration of 2.1\u20134.3 %, with a mean concentration of 3.4% (approximately 0.5 maximum alveolar concentration). This was measured using an Ohmeda respiratory gas monitor 5250 (BOC Group, Louisville, USA). Closed circuit mechanical ventilation was adjusted to maintain end-tidal carbon dioxide levels between 32 and 35\u00a0mmHg.\nIn the TIVA group, anesthesia was maintained using the regime of 10\u00a0mg\/kg of propofol for the first 10\u00a0min, 8\u00a0mg\/kg for the next 10\u00a0min and 5\u00a0mg\/kg for the subsequent length of operation; 50% air in oxygen was administered. In both groups, morphine was titrated as required for pain relief.\nMonitoring included electrocardiography, pulse oximetry, capnography and direct radial artery pressures. All patients were kept nornothermic with a warming blanket. Normotensive anesthesia was maintained throughout the operation.\nAfter approximately 45\u00a0min post-induction, a train of four-twitch assessment was performed using a nerve stimulator (Fischer Paykel NS242, UK). Cortical stimulation was commenced only when the amplitude of the fourth was visibly similar to the first. An interval of 3\u20135\u00a0min was allowed between two trains of cortical stimulation. This alternated with monitoring of somatosensory evoked potentials from posterior tibial nerve stimulation.\nWe measured two parameters: MEP amplitude, onset latency and initial stimulation intensity. Peak to peak amplitudes (between two largest peaks opposite in polarity) and onset latency was utilized for all MEP responses recorded bilaterally. Hence, ipsilateral MEPs refer to MEPs recorded from the TA on the same side as cortical stimulation. Within each patient, ten consecutive supramaximal MEPs obtained before insertion of pedicle screws used as a baseline were averaged to obtain the first two parameters. The initial stimulation intensity was defined as the minimal intensity required to obtain five consistent visible MEP responses at a vertical gain of 20\u00a0\u03bcV per division.\nDuring insertion of pedicle screws and instrumentation, a 50% reduction of MEP amplitude or 10% prolongation of latency was brought to the surgeon\u2019s attention.\nStatistical analyses using Student\u2019s t-tests were obtained with Microsoft SPSS for Windows Version 10.1. Statistical significance was considered at P\u00a0<\u00a00.05.\nResults\nThere were no complaints of headache, seizures or skin burns postoperatively; all patients had normal neurological examination.\nMEPs were successfully obtained from all patients with TA recordings bilaterally. There were four patients in the TIVA group and five in the desflurane group. Mean ages for desflurane (16.2) and TIVA (15.7) groups were not significantly different (P\u00a0=\u00a00.6).\nNone of the patients had MEP amplitude or latency changes exceeding our set limits so as to require immediate surgical attention during and after pedicel screw insertion and spinal instrumentation.\nThe ipsilateral MEP amplitudes (standard deviation) were significantly larger than contralateral MEP amplitudes [68.9 (27.1) vs. 52.5 (15.7)\u00a0\u03bcV, P\u00a0<\u00a00.01, paired t-test]. The initial stimulation intensity to obtain ipsilateral MEPs was significantly lower than for contralateral MEPs [66.9 (12.3) vs. 74.4 (10.1)\u00a0mA, P\u00a0<\u00a00.05, paired t-test]. However, there were no significant latency differences for ipsilateral and contralateral MEPs [32.0 (2.1) vs. 31.5 (2.0)\u00a0ms, P\u00a0=\u00a00.3, paired t-test).\nThere was no significant difference between use of TIVA or desflurane anesthesia for MEP amplitudes obtained, with ipsilateral (P\u00a0=\u00a00.06, unpaired t-test) and contralateral (P\u00a0=\u00a00.09, unpaired t-test) stimulation. Additionally, there was also no significant difference between right and left sided MEP amplitudes, either with ipsilateral (P\u00a0=\u00a00.9, paired t-test) or contralateral (P\u00a0=\u00a00.7, paired t-test) stimulation.\nWe consecutively studied an additional 17 subjects monitored for scoliosis surgery using an identical protocol (1 men, mean age 16.1\u00a0years, range 14\u201322). All had the TIVA anesthetic regimen. With right cortex stimulation, mean initial stimulation intensity to obtain ipsilateral MEPs [39.7 (9.9)\u00a0mA] was significantly lower than to obtain contralateral MEPs [47.1 (11.3)\u00a0mA, paired t-test, P\u00a0<\u00a00.0005]. With left cortex stimulation, similar findings were obtained [40.6 (10.7) vs. 50.3 (11.8)\u00a0mA, paired t-test, P\u00a0<\u00a00.0005]. With right cortex stimulation, mean ipsilateral MEP amplitudes [107.1 (35.7)\u00a0\u03bcV] were significantly larger than mean contralateral MEP amplitudes [90 (37.1)\u00a0\u03bcV, paired t-test, P\u00a0=\u00a00.01]. With left cortex stimulation, similar findings were again observed [112.1 (37) vs. 82.3 (30.9)\u00a0\u03bcV, paired t-test, P\u00a0=\u00a00.0004]. With right cortex stimulation, mean ipsilateral MEP latencies [30.2 (2.4)\u00a0ms] were not significantly different from mean contralateral MEP latencies [30 (2.3)\u00a0ms, paired t-test, P\u00a0=\u00a00.3). With left cortex stimulation, similar findings were again observed [30 (2.1) vs. 30.4 (2.3)\u00a0ms, paired t-test, P\u00a0=\u00a00.2].\nExamples of MEPs obtained with both ipsilateral and contralateral stimulation are shown in schematically in Fig.\u00a01 and 2.\nFig.\u00a01Schematic diagram showing right cortical stimulation, resulting in ipsilaterally and transcallosally conducted corticospinal impulses activating the spinal cord anterior horn cell. The right ipsilateral MEP recording is from the TA. Summation of ipsilaterally conducted and transcallosally generated descending impulses may thus result in larger ipsilateral MEPs from right cortical stimulationFig.\u00a02Actual consecutive MEPs obtained from a patient, showing larger amplitude responses with ipsilateral stimulation. Both recordings were made from the TA at 70% stimulation intensity\nDiscussion\nThe present study showed that ipsilateral MEPs have significantly larger amplitudes and were elicited with significantly lower stimulation intensities than contralateral MEPs. However, onset MEP latencies were not significantly different.\nThe origin of ipsilateral MEPs in humans is not well understood. In animal studies, cat corticospinal neurons have been shown to evoke ipsilateral actions via ipsilaterally descending reticulospinal tracts, as well as via contralaterally descending reticulospinal neurons, both by synapsing spinal interneurons [9]. Tracer studies in rhesus monkeys have quantified ipsilateral corticospinal fibers as approximately 9\u201312% of the total descending corticospinal projections [14]. Thus, current evidence points to contralateral corticospinal fibers as the predominant pathway for spinal motorneuron activation.\nThe presence of ipsilateral MEPs has mostly been described in pathological conditions. Patients with congenital mirror movements [11], schizencephaly [12], and Kallmann\u2019s syndrome [13] show ipsilateral MEPs, which likely result from abnormal structure and function of ipsilateral corticospinal fibers. However, functional reorganization and unmasking of ipsilateral corticospinal pathways may contribute to the generation of ipsilateral MEPs after adult stroke [6] and congenital hemiparesis [18]. One study involving 50 normal children suggested that presence of ipsilateral MEPs might be a normal state of ontogeny. Their disappearance after 10 years old is likely due to increasing transcallosal inhibitory influences [16]. In our study, all patients were above 10\u00a0years of age, and did not have clinical features to suggest presence of underlying conditions mentioned above. Another study comparing healthy adults with stroke patients has suggested ipsilateral MEPs may be conducted via corticoreticulospinal or corticopropriospinal pathways in normal subjects [1].\nWhat are the possible underlying mechanisms, which explain our findings? Firstly, it is possible that ipsilateral MEPs may be solely due to transcallosal stimulation of the contralateral motor cortex. Additionally, the effects of anesthesia on corticospinal excitability may facilitate this, hence resulting in significantly lower initial stimulation intensity to obtain ipsilateral MEPs. While evidence to suggest this is scarce, rat brain studies have demonstrated widespread action of anesthesia at multiple binding sites [8]. Magnetic resonance brain imaging has also demonstrated increased callosal T2 changes with anesthesia, suggesting structural alterations at a molecular level [19]. It is also possible that longstanding scoliosis has led to spinal cord plasticity changes. Motor pathway reorganization and spinal cord plasticity have been well documented in response to cord injury [7] in an activity-dependent manner [10]. Thus, structural and postural changes of longstanding scoliosis may have resulted in reorganization of cortical or subcortical motor pathways, including ipsilateral corticoreticular fibres leading to our observations [17]. However, lack of lateralization of MEP amplitudes with ipsilateral or contralateral stimulation was not supportive of this hypothesis.\nAdditionally, lack of significant ipsilateral and contralateral latency differences suggest bilateral motor cortex stimulation has resulted in ipsilateral MEPs, which may have comprised early ipsilaterally conducted components and late transcallosally stimulated components (Fig.\u00a01). This might also explain the larger amplitudes of ipsilateral MEPs obtained than MEPs derived from contralateral motor cortex stimulation. Further studies clarifying the predominant mechanisms responsible would be interesting.\nAre ipsilateral MEP responses useful and relevant in clinical settings? Ipsilateral MEPs are readily elicited, as shown in this study. While the relative contributions of ipsilaterally and transcallosal conducted MEPs remain uncertain, bilateral MEP recordings during spinal surgery IOM may provide additional information regarding the integrity of descending motor tracts. Together with the electrophysiological findings presented here, future studies clarifying these aspects would be justified. It thus may be feasible to routinely monitor MEPs bilaterally in future IOM protocols for spinal surgery.","keyphrases":["intraoperative monitoring","ipsilateral","motor evoked potential","scoliosis","contralateral","corticospinal"],"prmu":["P","P","P","P","P","P"]}
{"id":"Clin_Rheumatol-4-1-2262146","title":"A 57-year-old man who developed arthritis during R-CHOP chemotherapy for non-Hodgkin lymphoma\n","text":"Rituximab is a chimeric human-mouse anti-CD20 monoclonal antibody, which is used in the treatment of both B-cell lymphomas and rheumatic diseases. We describe a case of a previously healthy 57-year-old man developing arthritis while being treated with rituximab-CHOP chemotherapy (R-CHOP) for a non-Hodgkin lymphoma. The remittant arthritis developed at successively shorter time-intervals after R-CHOP administration and only improved after rituximab was removed from the chemotherapy schedule, suggesting a rituximab-related phenomenon, as extensive diagnostic testing ruled out any other diagnosis.\nIntroduction\nRituximab is increasingly used in the treatment of both B-cell lymphomas as well as rheumatic diseases. Several side-effects of rituximab are known, but until now only two reports described the development of arthritis after rituximab therapy [1, 2]. Here we describe yet another case.\nCase\nA 57-year-old man was referred to the out-patient clinic of rheumatology because of arthritis of the right knee and left wrist.\nThe patient had been in perfect health until he noticed a swelling of 2.5\u00a0cm under his right jaw. He was referred to a specialized oncology center where he was diagnosed with an aggressive follicular B-cell non-Hodgkin lymphoma (NHL) stage IVA with involvement of submandibular and mesenteric lymph nodes and bone marrow. Treatment was instituted with R-CHOP chemotherapy, which consists of cyclophosphamide, doxorubicin, vincristine, prednisone, and rituximab, given at a 3-week interval. During this treatment, granisetron (Kytril\u00ae) was given and occasionally magnesiumoxide and paracetamol. Because this patient suffered from a low tumor burden, no tumor lysis profylaxis, i.e., allopurinol was prescribed.\nTwo and a half weeks after the first course of R-CHOP, the patient developed arthritis of his right knee and left wrist without morning stiffness or involvement of the small hand joints. He had no history of arthralgia, joint swelling, preceding trauma, fever, or infection nor did he have a history of inflammation of the eyes, Raynaud phenomenon, photosensitivity, sicca-syndrome, or inflammatory back pain. The further medical history disclosed several tick bites the year before, with a skin reaction. He rarely used alcohol (maximum of two glasses a day). Family history revealed no rheumatic diseases.\nPhysical examination showed a profusely swollen and painful right knee and left wrist. The other joints were not afflicted.\nLaboratory testing before the start of chemotherapy showed no abnormalities besides a erythrocyte sedimentation rate (ESR) of 21\u00a0mm\/h. At the time of presentation with arthritis, there was ESR of 41\u00a0mm\/h, C-reactive protein 256\u00a0mg\/l, leucocytes 11.1\u2009\u00d7\u2009109\/l, a normal liver and kidney function, and an IgM-rheumatoid factor (RF) 7\u00a0IE\/ml, anti-cyclic citrulline peptide (a-CCP) <1\u00a0AU\/ml, anti-nuclear factor (ANF) negative, and urate of 0.25\u00a0mmol\/l. A diagnostic puncture of synovial fluid from the right knee was performed, which revealed an inflammatory arthropathy (cell count 5,300\/mm3, 14% lymphocytes, 8% mononuclear cells, and 78% granulocytes) with no signs of malignant cells or crystals. Microbiological testing (Gram-stain, Ziehl\u2013Neelsen, and culture) of the synovial fluid showed no microorganisms. X-rays of both knee and wrist did not show bone involvement or chondrocalcinosis. The patient was treated with naproxen (500\u00a0mg, twice daily), with which both pain and swelling of the joints were reduced but not completely resolved.\nFour days after the second course of R-CHOP, the pain and swelling returned in all its severity and spontaneously regressed after 1\u00a0week.\nShortly after the third course of R-CHOP, the patient experienced the same painful joint swellings, now also involving the left knee. This was in spite of intra-articular kenacort injections and prednisone maintenance therapy (10\u00a0mg daily), which was started between the courses of chemotherapy. Repeated pathological and microbiological testing of synovial fluid showed no new results.\nAfter these three courses of R-CHOP, the effect on the NHL was evaluated and disclosed that the intra-abdominal NHL-mass was somewhat reduced, and the remnant of the submandibular lymph node had disappeared.\nSince the causal relationship between the (activity of) arthritis and the courses of R-CHOP and rituximab being the only component of R-CHOP of which the development of arthritis is described after the administration of this drug [1, 2], the decision was made to give the fourth and fifth course of chemotherapy without rituximab. This time there was no increase in pain or swelling of the joints.\nWell over a year after the first R-CHOP chemotherapy, the patient is fully recovered. Evaluation of his NHL shows a near complete remission, and no further treatment was indicated. The arthritis has fully resolved.\nDiscussion\nArthritis developing during the course of R-CHOP chemotherapy is very unusual. The arthritis developed after successively shorter time-intervals after the R-CHOP administration while no arthritis occurred when CHOP was given without rituximab. This suggests a probably rituximab related, immune-mediated phenomenon. We were not able to detect antibodies against rituximab or immune complexes that might strengthen this diagnosis. However, we did have strong arguments against the other differential diagnoses.\nThe pattern of arthritis in this patient is unusual for a rheumatoid arthritis; moreover, both IgM-RF and a-CCP were negative, while X-rays made of the afflicted joints disclosed no erosions.\nSpondylarthropathy very rarely develops after the age of 45\u00a0years. Moreover, inflammatory back pain was absent, and positive arguments for an infection causing reactive arthritis were lacking.\nOther autoimmune diseases like systemic lupus erythematosus (SLE) or Sj\u00f6gren\u2019s syndrome were also very unlikely looking at the clinical presentation, the absence of characteristic signs and symptoms, and the absence of the specific autoantibodies.\nA crystalarthropathy is a more likely candidate, as treatment of NHL can cause tumor lysis syndrome, which can give rise to gout. However, serum values of urine acid were repeatedly very low, and there where no other signs of tumor lysis syndrome. Moreover, repeated samples of synovial fluid failed to show any crystals, kidney function was normal; the patient barely used alcohol and did not use diuretics.\nThe X-rays of both the right knee and left wrist did not show signs of chondrocalcinosis.\nLatent infections provoked by the administration of R-CHOP were considered. However, repeated cultures, Gram-stain, and Ziehl\u2013Neelsen of synovial fluid did not disclose any bacteria or yeasts. Serologic testing for Borrelia Burgdorferi, Lues, Chlamydia trachomatis, Parvovirus B19 and PCR on Mycobacteria tuberculosis and Chlamydia trachomatis (both performed in the synovial fluid) proved to be negative.\nA wide spectrum of autoimmune manifestations, among others arthritis, is described in NHL [3]. However, arthritis is unlikely in this patient as autoimmune manifestation, as it developed during R-CHOP and the NHL responded well to this chemotherapy.\nFurthermore intra-articular localization of NHL was considered. However, both primary and secondary intra-articular localization of NHL is very rare [4], and the fact that more than one joint is afflicted in this patient makes it even more unlikely. Moreover, magnetic resonance imaging (MRI) of both knees did not show tumor mass, and repeated punctures of synovial fluid of both knees did not show any malignant cells. On top of this, a computed tomography (CT) scan of the abdomen revealed regression of the mesenteric NHL-tumor mass after chemotherapy, which means that in the case of any presumed intra-articular localization of NHL, one would expect relapse of joint involvement in the course of chemotherapy instead of progression.\nAn arthroscopy was considered but not performed because of the risks of infection in this immunocompromised patient, and the low expectancy of achieving the diagnosis by performing the arthroscopy as the presence of NHL intra-articular was considered to be very unlikely.\nBecause our patient received high dosages of prednisone, the diagnosis of avascular bone necrosis was considered but ruled out by a MRI of both knees. None of the other medications our patient used are known to give arthritis.\nRuling out the above-mentioned diagnoses and taking into account the relationship in time between the development of arthritis and the gifts of R-CHOP, one or more of the components of R-CHOP seem to be the most likely cause of the arthritis in our patient. Of all of the components of R-CHOP, only rituximab is known to be able to give arthritis [1, 2].\nPijpe et al. described the development of arthritis in three patients who received rituximab for primary Sj\u00f6gren\u2019s syndrome. All of these patients had developed human antichimeric antibodies (HACAs), which suggests an immune-mediated mechanism. Only one other patient tested positive for HACAs but was asymptomatic. The significance of HACAs being present is actually not quite sure as other studies show HACAs being present without specific clinical manifestations [5, 6] or show HACAs being present but later becoming undetectable [6].\nRepeated blood tests of our patient did not show any HACAs. The precise mechanism of action in the development of arthritis in our patient is still unknown.\nIn conclusion, we describe a patient developing severe oligo-arthritis at successively shorter time-intervals after receiving CHOP-rituximab for a NHL. The arthritis is probably caused by an immune-mediated reaction to rituximab. As it is to be expected that rituximab will be used more frequently in future to treat both NHL as rheumatological diseases, we think it is important for physicians to be aware that rituximab may cause severe (oligo-)arthritis as a side effect.","keyphrases":["arthritis","lymphoma","rituximab","adverse effects"],"prmu":["P","P","P","M"]}
{"id":"Eur_J_Epidemiol-4-1-2249618","title":"Use of recommended medications after myocardial infarction in Austria\n","text":"Guidelines recommend long-term use of beta-blockers (BB), statins, and angiotensin-converting-enzyme-inhibitors or angiotensin-receptor-blockers (ACEI\/ARB) after myocardial infarction (MI), but data on their use after discharge are scarce. From Austrian sickness funds claims, we identified all acute MI patients who were discharged within 30 days and who survived \u2265120 days after MI in 2004. We ascertained outpatient use of ACEI\/ARBs, BBs, statins, and aspirin from all filled prescriptions between discharge and 120 days post MI. Comorbidities were ascertained from use of indicator drugs during the preceding year. Multivariate logistic regression was used to evaluate the independent determinants of study drug use. We evaluated 4,105 MI patients, whose mean age was 68.8 (\u00b113.2) years; 59.5% were men. Within 120 days after MI, 67% filled prescriptions for ACE\/ARBs, 74% for BBs, and 67% for statin. While 41% received all these classes and 34% two, 25% of patients received only one or none of these drugs. Older age and presence of severe mental illness were associated with lower use of all drug classes. Diabetics had greater ACEI\/ARB use. Fewer BBs were used in patients with obstructive lung disease. Statin use was lower in patients using treatment for congestive heart failure (all P < 0.001). We conclude that recommended medications were underused in Austrian MI survivors. Quality indicators should be established and interventions be implemented to ensure maximum secondary prevention after MI.\nIntroduction\nCardiovascular disease is among the leading causes of premature death and morbidity in most Western societies. Fortunately, the prognosis of myocardial infarction (MI) has steadily improved over the past decades: case fatality after MI has decreased and long-term survival has increased [1\u20133], likely due to the introduction and increasing availability and use of acute invasive and non-invasive interventions as well as the introduction of powerful medications for secondary prevention [4].\nAmong the medications for long-term secondary intervention, aspirin, beta-receptor blockers (BB), 3-hydroxy-3-methyl-glutaryl-co-enzyme-A inhibitors (statins), and angiotensin converting enzyme (ACE) inhibitors have drawn the most attention. More recently, angiotensin receptor blockers (ARB) have become available for use in those patients who develop adverse events from ACE inhibitors; ACE inhibitors and ARBs have been shown to be equally efficacious and effective after MI [5\u20137].\nAll these interventions are highly efficacious in reducing the recurrence of MI, the subsequent use of coronary interventions, or the risk of death, and have been demonstrated to be highly attractive economic investments from a societal perspective [8\u201311]. Practice guidelines recommend the use of these medications for secondary prevention in patients with unstable angina or MI, regardless whether the MI is associated with ST-elevation [12\u201314]. Considerable variation has been observed, however, in the use of these beneficial interventions [3, 15\u201317], which has prompted the establishment of quality of care indicators in some countries with the goal to monitor appropriate use of these interventions during hospitalization, at discharge, and during outpatient follow-up after such an event [18\u201320]. Most relevant in the long run, however, is the outpatient use of these medications after discharge from MI. Several studies have indicated that these drugs are underused for secondary prevention. Most of these investigations were conducted in North America [21\u201323] and in Europe [24\u201329]. No such systematic assessment has been conducted in the Austrian health care system except for a small local study [30]. We sought to fill this void by studying proportions of BB, statin and ACE inhibitor or ARB use after MI in a large period cross-section of Austrian MI patients.\nMethods\nData sources\nAustria is a social welfare state that mandates universal health care to its residents [31]. Patients are assigned membership in one of several sickness funds dependent on their type and location of current or former employment. At the time covered by this study, there were 20 sickness funds in Austria; among those, the 9 provincial sickness funds covering more than three quarters of all residents (6.2 Mio. of 8.2 Mio. overall; data from 2004). Most inpatient and outpatient medical services are covered and each service encounter generates a claim to the health care system. Inpatient services are not paid directly by sickness funds, but by a separate entity, which, however, is partly funded by these. Thus, sickness funds are also informed about each encountered inpatient care. Similarly to most western health care systems, datasets provided by hospitals contain diagnosis codes, admission and discharge dates. Access to prescription drugs is equal across sickness funds and all medications that are deemed efficacious by a national panel are reimbursed. The copayment for medicines per package was \u20ac4.25 in 2003, \u20ac4.35 in 2004 and \u20ac4.45 in 2005. Packages of chronic medications usually contain 28, 30, or 50 pills. Sickness fund premiums, other coinsurance and copayments vary slightly, and indigent patients can apply for waiver of the usually modest copayment for prescription drugs. Few medications are subject to prior authorization by the sickness funds. Prescription claims contain a unique identifier for the specific drug, the dose, and the quantity dispensed.\nStudy population\nFor the purpose of this study, we used the complete claims data of individuals covered by several of these sickness funds: the provincial sickness funds (Gebietskrankenkasse) of Vienna, Lower Austria, Upper Austria, Styria, Burgenland, and Carinthia, as well as the funds covering all federal employees (Bundesversicherungsanstalt, BVA) and farmers (Sozialversicherung der Bauern, SVB), respectively. Cumulatively, these sickness funds cover approximately 6.1\u00a0million members of the total Austrian insured population of 8.2\u00a0million. Each sickness fund separately identified from their insurance claims all patients who were hospitalized and discharged with a primary diagnosis of acute MI in 2004 (International Classification of Diseases, ICD-9: 410.xx; ICD-10: I21) and provided us with their relevant anonymized health care claims data covering the period from January 1, 2003 to June 30, 2005. We only studied each patient\u2019s first hospitalization for MI (including direct transfers from one hospital to another) and required that the admission date be between January 1, and December 31, 2004. We retained only those patients whose hospitalization exceeded 3\u00a0days and who were admitted at an acute care hospital. Including patients who were hospitalized <4\u00a0days would open up the possibility of wrongly selecting patients into the study sample who had not experienced an MI (e.g., whose MI was ruled out or patients who were admitted for a diagnostic or therapeutic intervention and still coded for acute MI). Our approach of identifying patients with MI has been validated in similar claims data and found to be highly accurate (positive predictive value: 94%) [32]. Since we were interested in medication use after MI, we generated a uniform outcomes ascertainment window and required all patients to be discharged within 30\u00a0days and to have survived for \u2265120\u00a0days from their initial admission date (=index date). We also excluded those patients whose hospitalization occurred at a non-acute care hospital.\nOutcomes: use of recommended medications after myocardial infarction\nFrom all filled prescriptions, we recorded out-of-hospital use of several medications between date of discharge and 120\u00a0days after MI admission: aspirin, beta-blockers, statins, and ACE inhibitors or ARBs. We also assessed the total number of distinct medications that patients received among statin, BB, and ACE inhibitor\/ARB (minimum 0; maximum 3). Since aspirin was available for a price that was below the amount of the drug copayment (\u20ac4.35), it is possible that insurance claims data may lead to under-ascertainment of aspirin use. Thus, we decided to investigate aspirin separately, and only among patients who had their copayment waived based on income grounds. These indigent patients had a clear economic incentive to fill prescriptions for aspirin via the prescription route, thus generating a claim to the sickness fund.\nCovariates\nWe created variables indicating each patient\u2019s age in 2004, gender, the length of stay for their index admission, and whether a patient had their prescription copayments waived during the study period (NoCopay vs. Copay). Since claims from outpatient encounters did not contain any diagnosis codes, we ascertained medication use in the 365\u00a0days prior to the index date as proxies for several comorbidities using the anatomic therapeutic chemical (ATC) classification system: oral antidiabetic drugs, insulin, antigout medications, several cardiovascular drug classes (alpha blockers, BBs, calcium channel blockers, ACE inhibitors, ARBs, other antihypertensives, diuretics, nitrates, digitalis, vitamin K-antagonist, statins, fibrates), medications indicated for asthma or chronic obstructive pulmonary disease (COPD; inhaled corticosteroids, beta-receptor agonists), drugs reducing gastro-esophageal reflux or ulcer disease (histamine-H2-blockers, proton pump inhibitors, sucralfate, antacids), pain medications (including non-steroidal anti inflammatory drugs, selective COX-2 inhibitors, opioids, and others), oral corticosteroids, and several psychoactive drug classes (benzodiazepines or anxiolytics, antidepressants, antipsychotics). We also recorded the number of hospital days during the year prior to the index date (categorized into: no hospitalization, 1\u20137, 8\u201321, and >21\u00a0days).\nStatistical analysis\nWe plotted the unadjusted proportions of medication use for the overall population as well as by copayment status along with the corresponding 95% confidence intervals (CIs). We then used univariate and multivariate logistic regression to estimate the crude and multivariate adjusted odds of receiving a given study medication. Since none of our outcomes were rare, we were able to create full multivariate models that included all variables regardless of their statistical significance. In large datasets where outcomes are not rare, full multivariate models are superior to parsimonious models, because they provide better control for residual confounding compared to more restricted models. Odds ratios (OR) were presented with their 95% CIs. Additionally, we showed the population distribution of the number of different study drug classes received among statin, BB, and ACE inhibitor\/ARB (minimum 0; maximum 3). Multivariate ordinal logistic regression and linear regression were used to model the associations between covariates and the number of drugs received. All analyses were conducted in the full final study population as well as after restriction to new users of each study drug, i.e. patients who had not received the respective study drug in the year prior to admission for MI. We used the SAS for Windows (release 9.2) software for all statistical analyses (The SAS Institute, Cary, NC).\nResults\nStudy population\nWe first identified 8,416 hospitalizations with a discharge diagnosis indicating an MI. After excluding all repeat MI hospitalizations and those that occurred before or after 2004 (N\u00a0=\u00a01,977; 23.5%), we also excluded those patients who died within 120\u00a0days after admission for MI (N\u00a0=\u00a01,221; 14.5%). After further restricting the sample to those patients whose index hospitalization was \u22654, but \u226430\u00a0days and excluding admissions at non-acute care hospitals, we obtained a final study sample of 4,105 patients (48.8% of the original hospitalization sample drawn). The mean age was 68.8\u00a0years (standard deviation, SD: \u00b113.2), and 2,442 (59.5%) patients were men. Age differed considerably by gender: men (median: 66\u00a0years, interquartile range: 56\u201375) were substantially younger than women (median: 76\u00a0years, interquartile range: 67\u201382; P\u00a0<\u00a00.001). Overall, 654 (15.9%) had their prescription copayments waived on the grounds of low income (Table\u00a01). On average, they had been hospitalized for 6.7 (\u00b114.7) days in the year prior to their MI and the mean length of stay for their index admission was 10.9 (\u00b15.3) days. The proportions of selected prescription drugs used in the year prior to admission for MI are listed in detail in Table\u00a01.\nTable\u00a01Characteristics of study population (N\u00a0=\u00a04,105)VariableCount (%) or mean (\u00b1SD)Age68.8 (\u00b113.2)\u00a0\u00a0\u00a0\u00a0<50\u00a0years402 (9.8)\u00a0\u00a0\u00a0\u00a050\u201369\u00a0years1,515 (36.9)\u00a0\u00a0\u00a0\u00a070\u201389\u00a0years1,225 (29.8)\u00a0\u00a0\u00a0\u00a0\u226590\u00a0years963 (23.5)Male gender2,442 (59.5)Length of stay10.9 (\u00b15.3)Days of hospitalization in prior year6.7 (\u00b114.7)\u00a0\u00a0\u00a0\u00a0None2,500 (60.9)\u00a0\u00a0\u00a0\u00a01\u20137\u00a0days595 (14.5)\u00a0\u00a0\u00a0\u00a08\u201321\u00a0days612 (14.9)\u00a0\u00a0\u00a0\u00a0\u226521\u00a0days398 (9.7)Copayment waived654 (15.9%)Previous medication use\u00a0\u00a0\u00a0\u00a0Alpha blocker243 (5.9)\u00a0\u00a0\u00a0\u00a0ACE-inhibitor or ARB1,853 (45.1)\u00a0\u00a0\u00a0\u00a0Beta-blocker1,454 (35.4)\u00a0\u00a0\u00a0\u00a0Calcium channel blocker809 (19.7)\u00a0\u00a0\u00a0\u00a0Other antihypertensive734 (18.0)\u00a0\u00a0\u00a0\u00a0Diuretic927 (22.6)\u00a0\u00a0\u00a0\u00a0Nitrate1,032 (25.1)\u00a0\u00a0\u00a0\u00a0Digitalis347 (8.5)\u00a0\u00a0\u00a0\u00a0Acetylsalicylic acid1,169 (28.5)\u00a0\u00a0\u00a0\u00a0Clopidogrel or Ticlopidine385 (9.4)\u00a0\u00a0\u00a0\u00a0Vitamin K-antagonist249 (6.1)\u00a0\u00a0\u00a0\u00a0Statin1,043 (25.4)\u00a0\u00a0\u00a0\u00a0Fibrate116 (2.8)\u00a0\u00a0\u00a0\u00a0Oral hypoglycemic 620 (15.1)\u00a0\u00a0\u00a0\u00a0Insulin265 (6.5)\u00a0\u00a0\u00a0\u00a0Uric acid lowering drug559 (13.6)\u00a0\u00a0\u00a0\u00a0Pain medication1,934 (47.1)\u00a0\u00a0\u00a0\u00a0Gastroprotective drug1,584 (38.6)\u00a0\u00a0\u00a0\u00a0Asthma\/COPD610 (14.9)\u00a0\u00a0\u00a0\u00a0Corticosteroid379 (9.2) \u00a0\u00a0\u00a0\u00a0Benzodiazepine or anxiolytic491 (12.0)\u00a0\u00a0\u00a0\u00a0Antidepressant623 (15.2)\u00a0\u00a0\u00a0\u00a0Antipsychotics190 (4.6)\nSecondary prevention after myocardial infarction\nWithin 120\u00a0days of their admission for MI, 3,037 (74.0%; 95% CI: 72.6\u201375.3%) patients filled a prescription for a BB, 2,745 (66.9%; 95% CI: 65.5\u201368.3%) for an ACE inhibitor or an ARB, and 2,736 (66.7%; 95% CI: 65.3\u201368.1%) received a statin (Fig.\u00a01). Indigent patients who were not required to pay a copayment received more ACE inhibitors or ARBs (74 vs. 65.5%; P\u00a0<\u00a00.001), and slightly fewer statins (63.2 vs. 67.2%; P\u00a0=\u00a00.03), while the proportion of BB use was similar to patients without such a waiver (P\u00a0=\u00a00.86).\nFig.\u00a01Proportions of medication use after myocardial infarction\nThe distribution of number of distinct drug classes received in this population was as follows: 1,692 patients (41.2%) received a BB, statin, and an ACE inhibitor or ARB, while 1,391 (33.9%) received two of these drugs. Six hundred and sixty (16.1%) patients received only one of the study drugs whereas 362 (8.8%) patients received none of these medications for secondary prevention at all. This distribution did not differ between patients with vs. without copayment requirement (P\u00a0=\u00a00.33). The number of drugs received was slightly higher among men compared to women (2.1 vs. 2.0; P\u00a0=\u00a00.02) and decreased with higher age (P\u00a0<\u00a00.001). Among the 654 patients with waived copayment, 464 (71%) filled a prescription for aspirin within 120\u00a0days of admission for MI.\nIndependent predictors of study medication use\nAge was an independent predictor of recommended medication use after MI: compared to patients younger than 50\u00a0years, those between 70 and 89\u00a0years had 48% greater odds of receiving ACE inhibitors or ARBs (OR: 1.48), whereas the oldest patients (\u226590\u00a0years old) were least likely to receive these drugs (OR: 0.73; Table\u00a02). Similarly, these oldest patients were also markedly less likely to receive BBs and statins (Tables\u00a03 and 4). Number of days spent in the hospital in the year preceding the MI admission, a marker of preexisting comorbidity, was consistently and inversely associated with lower use of all these recommended medications (all P for trend <0.001). Patients who had their copayments waived were more likely to fill a prescription for an ACE inhibitor or an ARB after discharge from MI than patients with copayment required (OR: 1.35; 95% CI: 1.10\u20131.67), but BB or statin use did not differ between these groups. Angiotensin converting enzyme inhibitor or ARB use was further independently associated with previous calcium channel blocker use, use of an oral antidiabetic drug, and prior use of antipsychotic drugs (Table\u00a02). Beta-receptor blockers were more likely to be used in patients who had used other antihypertensive medications prior to MI (Table\u00a03). Lower use of BBs was observed in patients who had received diuretics, vitamin K-antagonist, insulin, antipsychotics, as well as in those patients who received inhaled medications for asthma or COPD (Table\u00a03). Medications that were inversely associated with statin use were diuretics, digitalis, clopidogrel, vitamin K-antagonist, and antipsychotics (Table\u00a04).\nTable\u00a02Independent determinants of ACE-inhibitor or ARB useVariableAll patients (N\u00a0=\u00a04,105)New users (N\u00a0=\u00a02,252)OR95% CIOR95% CIAge<50\u2013Referent\u2013Referent50\u2013691.190.96\u20131.491.230.96\u20131.5870\u2013891.481.19\u20131.851.541.19\u20132.00\u2265900.730.59\u20130.900.790.60\u20131.03Male gender1.070.91\u20131.251.190.98\u20131.44Length of stay1.021.00\u20131.031.031.01\u20131.05Hospital days*0\u2013Referent\u2013Referent1\u201370.680.56\u20130.840.620.49\u20130.808\u2013210.790.63\u20130.990.680.51\u20130.91>210.510.39\u20130.680.530.35\u20130.79Copayment waived1.351.10\u20131.671.341.03\u20131.74Alpha-blocker1.070.75\u20131.531.270.73\u20132.20ACE-inhibitor or ARB5.674.74\u20136.78\u2013\u2013Beta-blocker1.120.94\u20131.331.160.93\u20131.45Calcium channel-blocker1.351.10\u20131.661.341.01\u20131.79Other anti-hypertensive agents0.990.80\u20131.211.120.85\u20131.47Diuretic0.850.68\u20131.050.870.65\u20131.18Nitrate0.880.72\u20131.070.820.63\u20131.08Digitalis1.150.84\u20131.571.040.65\u20131.65Aspirin0.890.74\u20131.070.910.70\u20131.18Clopidogrel0.900.68\u20131.200.730.46\u20131.15Vitamin K-antagonist0.990.70\u20131.411.100.65\u20131.87Statin1.120.92\u20131.380.840.63\u20131.11Fibrate1.210.76\u20131.931.000.56\u20131.77Oral antidiabetic1.261.00\u20131.581.060.79\u20131.44Insulin1.020.73\u20131.441.200.68\u20132.10Asthma\/COPD1.070.86\u20131.341.100.83\u20131.47Benzodiazepines\/anxiolytics1.060.83\u20131.361.160.83\u20131.62Antidepressants0.870.70\u20131.090.850.64\u20131.13Antipsychotics0.640.45\u20130.910.650.41\u20131.04* P for trend <0.001Table\u00a03Independent determinants of beta-blocker useVariableAll patients (N\u00a0=\u00a04,105)New users (N\u00a0=\u00a02,651)OR95% CIOR95% CIAge <50\u2013Referent\u2013Referent50\u2013691.250.98\u20131.601.210.93\u20131.5970\u2013891.050.83\u20131.331.140.87\u20131.47\u2265900.620.51\u20130.760.540.43\u20130.69Male gender1.140.97\u20131.351.190.98\u20131.43Length of stay1.000.98\u20131.011.000.98\u20131.01Hospital days *0\u2013Referent\u2013Referent1\u201370.920.74\u20131.150.850.66\u20131.088\u2013210.770.61\u20130.960.780.60\u20131.02>210.570.44\u20130.750.610.43\u20130.84Copayment waived1.090.89\u20131.351.190.93\u20131.53Alpha-blocker1.541.07\u20132.201.731.12\u20132.67ACE-inhibitor or ARB1.221.02\u20131.451.160.94\u20131.41Beta-blocker4.363.55\u20135.35\u2013\u2013Calcium channel-blocker1.291.05\u20131.591.491.16\u20131.90Other anti-hypertensive agents0.830.68\u20131.010.870.69\u20131.10Diuretic0.800.65\u20130.980.770.60\u20130.99Nitrate1.030.84\u20131.261.040.81\u20131.33Digitalis0.790.60\u20131.040.680.48\u20130.96Aspirin0.960.79\u20131.160.880.70\u20131.12Clopidogrel0.790.59\u20131.070.650.43\u20130.97Vitamin K-antagonist0.670.49\u20130.930.440.29\u20130.66Statin0.940.76\u20131.160.850.67\u20131.10Fibrate1.140.70\u20131.861.210.65\u20132.25Oral antidiabetic1.060.85\u20131.321.070.82\u20131.39Insulin0.720.53\u20130.980.780.52\u20131.16Asthma\/COPD0.670.55\u20130.830.630.49\u20130.80Benzodiazepines\/anxiolytics1.331.04\u20131.711.270.94\u20131.72Antidepressants0.880.71\u20131.090.960.74\u20131.24Antipsychotics0.710.51\u20131.000.740.50\u20131.09* P for trend <0.001Table\u00a04Independent determinants of statin useVariableAll patients (N\u00a0=\u00a04,105)New users (N\u00a0=\u00a03,062)OR95% CIOR95% CIAge <50\u2013Referent\u2013Referent50\u2013691.381.09\u20131.761.401.09\u20131.8170\u2013891.080.86\u20131.361.090.85\u20131.39\u2265900.390.32\u20130.470.370.30\u20130.46Male gender1.100.94\u20131.291.130.95\u20131.35Length of stay0.980.97\u20130.990.990.97\u20131.00Hospital days*0\u2013Referent\u2013Referent1\u201370.880.71\u20131.090.820.66\u20131.048\u2013210.680.55\u20130.850.680.53\u20130.87>210.480.37\u20130.630.470.34\u20130.65Copayment waived1.090.89\u20131.341.070.85\u20131.34Alpha-blocker0.890.65\u20131.221.040.72\u20131.51ACE-inhibitor or ARB0.980.82\u20131.161.040.86\u20131.25Beta-blocker1.080.91\u20131.291.070.88\u20131.30Calcium channel-blocker1.010.83\u20131.230.980.79\u20131.23Other anti-hypertensive agents0.970.80\u20131.180.970.78\u20131.21Diuretic0.820.67\u20131.000.790.63\u20131.00Nitrate0.940.77\u20131.140.960.77\u20131.21Digitalis0.600.46\u20130.800.510.37\u20130.72Aspirin0.850.71\u20131.030.740.60\u20130.92Clopidogrel0.700.51\u20130.940.610.40\u20130.94Vitamin K-antagonist0.620.45\u20130.850.520.35\u20130.78Statin6.395.03\u20138.11\u2013\u2013Fibrate2.241.36\u20133.702.311.35\u20133.97Oral antidiabetic0.950.77\u20131.180.980.77\u20131.26Insulin0.760.56\u20131.040.710.48\u20131.05Asthma\/COPD0.870.71\u20131.070.840.66\u20131.06Benzodiazepines\/anxiolytics0.890.70\u20131.120.940.72\u20131.22Antidepressants1.130.91\u20131.401.080.85\u20131.39Antipsychotics0.480.34\u20130.680.530.36\u20130.79* P for trend <0.001\nNaturally, prior use of a medication class was highly predictive of use of that class after MI in the full study population. Results from the analyses restricted to new users of each study drug were very similar to the full population analyses, albeit with wider confidence intervals (Tables\u00a02\u20134). Analyses of independent predictors of number of recommended drugs received using linear or ordinal logistic regression confirmed what could be gleaned from the analyses of each drug class: older age, more hospital days in the previous year, indicator drugs for severe heart failure (digitalis, diuretics, vitamin K-antagonist), and antipsychotic use were all independently associated with fewer recommended medications received (results not shown). Gender, however, was not independently associated with number of medications received.\nDiscussion\nIn a large population-based study of patients who experienced an acute MI in Austria, we found that ambulatory use of several recommended medications after discharge was suboptimal. Within 120\u00a0days after their MI, only 74% patients received a BB, 67% a statin, and 67% filled a prescription for an ACE inhibitor or ARB. Only 41% of patients received all three interventions, while 25% of patients received only one of these beneficial drugs or even none at all. Underuse of these medications is unfortunate from both a patient\u2019s and a societal perspective: while optimal secondary prevention including these drugs prolongs the expected lifespan of a patient after MI, use of these interventions constitutes an attractive allocation of scarce economic resources. Thus, considerable room for improvement is present in the care of patients after MI in Austria, and interventions ought to be targeted towards increasing the prescribing and use of these medications.\nWhile this is the first large-scale study of the quality of post-MI care in Austria, similar investigations have been conducted in other countries. The most relevant comparison can be drawn with results from the EUROASPIRE studies [27\u201329]. These studies, three waves have been conducted to date, used surveys to evaluate coronary care in 9 (EUROASPIRE I), 15 (EUROASPIRE II), and 22 (EUROASPIRE III) countries, respectively; Austria did not participate in either of these. The most recent data available are from EUROASPIRE II, which covered the years 1999\/2000 [28]. Medication use was assessed at admission, discharge (abstracted from charts), and at least 6\u00a0months (from patient interview) after the acute admission or procedure. While some variation was observed across countries, data specifically for after MI drug use was only reported in aggregate across countries. Approximately 74% of MI patients received a BB at discharge and 68% >6\u00a0months after the event; these proportions are similar to the 74% observed in our study. For ACEI, the proportions at discharge and >6\u00a0months after the event were 49 and 45%, respectively, considerably lower than the 67% observed in Austria. It is unclear, however, whether ARB use was regarded equivalent to ACEIs and included in this quality measure in the EUROASPIRE study. Lipid-lowering drugs were prescribed to 42% of patients at discharge and used by 60% at >6\u00a0months after the event, compared to 67% in our study [28].\nA comparison of these reports, however, needs to be conducted with caution. Considerable differences exist between these and other studies, especially in their population selection criteria, ascertainment of medication use, and the year(s) studied. Several studies, specifically from the United States and Canada, investigated medication use only in older patients (>65\u00a0years) [33]. Results from elderly populations cannot be compared with findings from the general population, since age is a strong predictor of preventive medication use. Studies that measured medication use using discharge notes are likely to overestimate medication use, because patients may not have received a prescription or had chosen to not fill it. The increasing use of the study medications also needs to be taken into consideration and, in this respect, older reports considerably vary with recent ones. To this point, Gislason et al. [24] studied MI patients in Denmark and found that the proportion of patients who received an ACE inhibitor increased from 25% in 1995 to 36% in 2002. Similarly, use of BB increased from 38% to 68% during that time. This study differs from ours in that drug use was ascertained within 30\u00a0days following the discharge date from MI. While BB use is roughly similar to our findings, the use of ACE inhibitor was substantially lower in that Danish cohort. Statin or aspirin use was not part of that study, but a separate study using the same Danish database focused on this aspect of post MI care. In this report, statin use was ascertained within 6\u00a0months of discharge and increased from 13% in 1995 to 61% in 2002 [34]. This percentage is similar to the statin use of 67% in our Austrian sample, given that our data are slightly more recent; we did, however, use a shorter ascertainment window. Another report studied the use of preventive medications after MI in The Netherlands [26]. The authors confirmed a trend towards increased use of all drug classes from 1991 to 2000, with ACE inhibitor being dispensed to 44%, BB to 76%, and statins to 58% in the most recent study year (2000). Use of these medications was ascertained within 30\u00a0days after discharge, but patients were not required to survive for a minimum number of days after discharge to be eligible for study. Taking these study design differences into account, it appears that more patients received a BB after discharge in the Netherlands compared to Austria. Interestingly, neither the Dutch, nor the Danish study took ARB use into consideration as an established substitute for ACE inhibitors. Similarly, data from EUROASPIRE I and II confirms increasing use these preventive medications [29].\nIn the present study, we also identified several predictors of use for the study medications. In general, patients \u226590\u00a0years of age were substantially less likely to receive these recommended medications than younger patients. Similar evidence for under-treatment among the elderly was also found in other countries [24]. This behavior may constitute general treatment bias against very old patients, or reflect that these patients may not live long enough to reap the benefits of long-term preventive interventions. Indeed, specific evidence on the efficacy and cost-effectiveness of the study medications in very old patients is lacking. In general, old patients were excluded from participation in virtually all efficacy trials on which current clinical practice recommendations are based. Only the PROspective Study of Pravastatin in the Elderly at Risk (PROSPER) trial has addressed this issue and specifically studied older individuals. PROSPER demonstrated that statins are efficacious in prevention of coronary events in older patients, but the participants in PROSPER were still \u201conly\u201d 70\u201382\u00a0years at enrollment [35]. Further, statin therapy has been shown to be cost-effective in 75\u201384\u00a0years old patients [36]. We found that those indigent patients who had their copayment waived had at least the same level of medication use compared to relatively more affluent patients who were responsible for the copayment portion of their prescription (\u20ac4.35), thus indicating that such a waiver successfully removed the economic barriers to filling these prescriptions. Of note, patients whose copayment was waived were more likely to be women and previous recipients of antipsychotic drugs, but otherwise similar to those who were required to pay a copayment (detailed results not shown).\nThe number of hospital days in the year prior to MI was a strong negative predictor of medication use for all classes. This might reflect greater comorbidity or frailty in these patients, which both have been associated with lower use of and persistence with preventive medications. Similarly, patients receiving antipsychotic drugs had a lower likelihood to receive the study medications, likely indicating treatment bias regarding the mentally diseased. Beta-blocker use was significantly lower among patients who received any drugs for inhalation that are indicated in asthma or COPD, a plausible pattern, which may reflect presence of a relative contraindication or intolerance by the patient. Indeed, among patients without previous use of such asthma or COPD drugs (N\u00a0=\u00a03,495), BB use after MI was 75.4% (rather than 74.0% in the overall population), an only slightly higher proportion with respect to the goal of appropriately treating all patients free from contraindications. Statins were less likely to be used in patients who had previously used diuretics, digitalis, or coumadin, possibly indicating congestive heart failure or atrial fibrillation and thus, worse prognosis.\nIn theory, and barring presence of any absolute contraindications, 100% of patients could receive each of these medications. Clearly, the actual proportion that could be attained in practice is lower than that, since some patients will have an absolute contraindication for any given drug. For ACEI\/ARBs, this number would be very low, since the only absolute contraindication (other than pregnancy) is history of angioedema, whose incidence is below 1% among new ACEI users [37]. Beyond this, relative contraindications may exist such as hyperkalemia or advanced chronic kidney disease, but in most patients, at least a low-dose trial of an ACEI\/ARB should be attempted with appropriate clinical and laboratory monitoring in place, possibly in combination with a loop diuretic. Similarly, for statins, practically every one should receive this treatment and absolute contraindications are either very rare (active liver disease) or not to be expected in this MI population (pregnancy, lactation). Thus, at least an attempt at using statins and ACEI\/ARB in post-MI patients can be expected in probably >95% of patients. The situation is slightly different with BBs, since more contraindications exist whose classification as absolute vs. relative are uncertain [38]. Cardiogenic shock, hypotension, and certain bradycardic arrhythmias untreated with a pacemaker certainly constitute absolute contraindications, whereas a treatment attempt in patients with stable obstructive lung disease should be conducted. From our dataset, it is difficult to ascertain most of these conditions, but when eliminating all patients who had received inhalative corticosteroids or beta mimetics, the percentage of BB users increased slightly to 75.4% from 74.0% in the overall population.\nThis report needs to be read with several limitations in mind. Aspirin was available at a price below the copayment for prescription medications. Thus, aspirin use may be underascertained in non-indigent patients. We were, however, able to assess aspirin use in those patients who had their copayment waived and found that 71% of patients received this drug. While we cannot be certain that medications received in the pharmacy were actually taken by these patients, our way of ascertaining medication use is superior to other methods. Compared to notes in medical charts, patients may not fill the prescriptions they were given. In surveys, patients may report what they perceive as being desirable rather than their actual behavior. Both methods, may lead to inaccurately optimistic estimates of medication use. In our study, comorbidities were not ascertained from diagnosis codes, but rather from typical medications that are given for several comorbidities. It has been shown that the information from diagnosis codes and from medication claims provides only marginally inferior confounding control in administrative datasets [39]. On a similar note, we were unable to ascertain reliable information on contraindications for the study drugs. Since most of these contraindications are relative rather than absolute contraindications [37, 38] this aspect may be of minor importance. While not fully population based, our data differ from other studies in that a wide range of hospitals and typical care settings were studied rather than single departments in predominantly academic medical centers. We consider our data generalizable to the population level, since three quarters of the Austrian population were included in the study, and the preponderance of patients omitted (1.2 Mio.; data from 2004) was excluded because they lived in the three Western provinces (Salzburg, Tyrol, Vorarlberg), whose sickness funds did not participate in our data collection effort. The remaining patients that we were unable to capture were members of very small corporate sickness funds, railroad and mining workers, as well as self-employed individuals; all those, however, received care at the same hospitals and by the same physicians as the enrolled patients. Finally, the analytical dataset did not contain linkable pseudoanonymized hospital identifiers. Thus, we were unable to study variations in treatment behavior across the provider level. It has been shown that provider preference can be a stronger determinant of treatment received than actual patient level indications [40].\nIn summary, we provide evidence for underuse of several recommended medications after MI as recently as 2004 in the Austrian healthcare system. Our observations are in line with findings from other European and North American healthcare systems, despite the differences in data collection and time period studied. Educational efforts need to be directed at both physicians and patients, and the implementation of quality indicators should be considered. Maximizing secondary prevention after MI is highly desirable from an individual patient and the societal perspective.","keyphrases":["myocardial infarction","beta-blockers","statins","secondary prevention","angiotensin receptor blockers","angiotensin converting enzyme inhibitors"],"prmu":["P","P","P","P","P","P"]}
{"id":"Eur_J_Nucl_Med_Mol_Imaging-3-1-1998878","title":"Requirements regarding dose rate and exposure time for killing of tumour cells in beta particle radionuclide therapy\n","text":"Purpose The purpose of this study was to identify combinations of dose rate and exposure time that have the potential to provide curative treatment with targeted radionuclide therapy applying low dose rate beta irradiation.\nIntroduction\nMany types of tumour overexpress cell surface-associated antigens or receptors suitable as targets for radionuclide therapy, and many types of targeting agent have been suggested or are already being applied for such therapy. This therapy is currently employed for lymphomas [1, 2] using radiolabelled antibodies and also for neuroendocrine [3\u20135] and paediatric tumours [6, 7] using radiolabelled somatostatin analogues and meta-iodobenzylguanidine (mIBG), respectively. In the majority of these cases, beta emitters such as 90Y, 131I and 177Lu have been applied. The results have, so far, essentially shown palliative effects [1, 8\u201312], and there is hope that combinations of beta particle emitters, e.g. 90Y and 177Lu, will improve the therapy results [13].\nThe cell-killing capacity of low LET radiation, i.e. photons and electrons, is well known when applying high dose rates, typically 0.5\u20132.0\u00a0Gy\/min, as in external radiotherapy [14, 15]. However, the extensive experimental and clinical knowledge on the effects of external radiotherapy can be deployed to only a limited extent in understanding the effects of radionuclide therapy. A major difference is that the dose rate in radionuclide therapy is at least two orders of magnitude lower than in external radiotherapy [10, 16\u201319]. The lower dose rate allows for DNA repair and repopulation during the radiation exposure, which is not the case during high dose rate exposures. Basic radiobiological studies have shown that low dose rates, in the range of 0.1\u20131.0\u00a0Gy\/h, give a much lower biological effect (per dose unit) than high dose rates in the range 0.5\u20132.0\u00a0Gy\/min [15, 17, 20, 21]. It is also known that an inverse dose rate effect exists in that dose rates of 0.2\u20130.4\u00a0Gy\/h can give more cell kill than dose rates in the range 0.7\u20131.0\u00a0Gy\/h [15, 22].\nOnly crude estimates can be made from previous experiments to elucidate which combinations of low dose rate and exposure time can cure a metastasis containing, for example, 105 cells. Cell survival has most often been analysed after a cell cloning 1\u20132\u00a0weeks after the radiation exposure. For example, a total dose of about 30\u201350\u00a0Gy, given with 0.1\u20131.0\u00a0Gy\/h, seems necessary to decrease the single cell survival probability to 10\u22125 [23, 24], and thereby give a reasonable chance of killing 105 tumour cells. Furthermore, targeted radionuclide therapy is complicated, since it is not enough only to consider the macroscopic dose concept; different cellular and intracellular distributions of radionuclides can give different biological effects although the macroscopic dose is the same [25, 26].\nOne way to obtain solid information on which combinations of low dose rate and exposure time can give curative treatments with beta particles (which also is low-LET radiation) is, of course, through experiments and clinical trials. In this study we used an experimental model with the criterion that the low dose rate beta radiation must kill all 105 tumour cells in a culture dish in order to simulate a successful treatment. The follow-up period was 3\u00a0months.\nThe choice of 105 tumour cells is somewhat arbitrary and is based on two arguments. The first is that this number represents a small tumour cell cluster that normally cannot be identified by routine diagnostic procedures such as computed tomography or magnetic resonance imaging (unless the tumour cells cause macroscopic changes in the surrounding normal tissues). Furthermore, this number of tumour cells in most cases does not cause symptoms in the patient. Thus, a cluster of 105 tumour cells in a patient can be considered an \u201coccult\u201d or \u201csubclinical\u201d tumour or metastasis. The second argument is more practical, since the presence of 105 tumour cells in a normal cell culture dish or flask allows enough space for exponential growth and, at the same time, frequent cell\u2013cell contacts.\nOur ambition was not to simulate the dose rate variations in time and space that occur in radionuclide therapy. In the clinical setting, the dose rate varies with time, not only as a consequence of the physical half-life of the radionuclides, but also due to time-dependent changes in their spatial distribution [16, 17, 24, 26, 27]. Factors of importance are ongoing vascularisation processes, variations in vessel wall leakage and changes in blood flow. There are probably also various diffusion and convection conditions in different areas of tumours, resulting in variable penetration properties of the radiolabelled targeting agents. In addition, there might be variations in the expression of target structures on the tumour cells. All these time-dependent factors make it difficult to establish basic and reproducible dose rate\u2013response relations in vivo.\nOur experimental model was designed to give reproducible and controllable irradiation conditions, and we applied a model with a rather long physical half-life (32P sources with T1\/2=14.3\u00a0days), giving only a slow decrease in dose rate during the exposures. Relevant dose rates were selected through the amount of radionuclide placed in the irradiation chambers. The exposure times were selected to correspond to the effective half-lives of the radionuclides delivered by targeting agents of different types.\nIn targeted radionuclide therapy it is, of course, also necessary to consider unwanted side-effects on normal tissues. However, analyses of normal tissue effects were beyond the scope of this study. Hyperradiosensitivity [28, 29] at low doses, bystander effects [30\u201332] and low dose rate-induced apoptosis [33, 34] are all extensively studied processes. Our model allows these processes to work together, but we did not try to study them separately. The overall goal of the study was to find \u201cdose rate\u2013exposure time\u201d relations that could kill all of the exposed 105 tumour cells, so that no remaining cells would be observed after 3\u00a0months.\nMaterials and methods\nIrradiation chambers\nThe irradiation chambers have been described previously [35], so only a short description is given here. Three identical chambers were used and each was filled with 400\u00a0ml distilled water containing 0.74\u20132.22\u00a0GBq 32P. The water was boiled and degassed shortly before adding 32P and filling of the chambers, in order to avoid air bubbles. The beta emitter 32P (orthophosphate) (T1\/2=14.3\u00a0days) was obtained from Amersham Pharmacia Biotech (Amersham, UK).\nThe upper area of the chambers, where the cells were exposed to beta particles from 32P, was covered with a 0.5-mm thin transparent polycarbonate foil. The radiation protection walls surrounding the side and the bottom of the chambers, as well as the lid above the cell cultures, consisted of 15-mm transparent polycarbonate (Macrolon). 32P could not reach and be incorporated in the cells. The irradiated cells were grown in 3-cm-diameter culture dishes with a plastic bottom thickness of 1\u00a0mm. The culture dishes were placed directly on the thin foils above the 32P source. The chambers were kept in cell culture incubators at 37\u00b0C (Kebo Assab T304GF, Stockholm, Sweden) and supplied with 5% carbon dioxide. The lid above the cultures had side openings, allowing efficient passage of the incubator atmosphere.\nDosimetry\nThe dosimetry has also been described previously [35]. The dose rate for each chamber was controlled by measurements with a thin-walled parallel ion chamber [36]. The ion chamber was calibrated using an external 60Co source with the front wall of the ion chamber at the dose maximum depth, where the dose rate was 0.30\u00a0Gy\/min. The 60Co source was calibrated according to national standard procedures accepted for radiotherapy purposes. The thin-walled parallel ion chamber accurately measured the dose independent of dose rate down to at least 0.01\u00a0Gy\/h, if corrections were made for \u201cleakage current\u201d in the instrumentation.\nThe measurements of dose rates were performed with the thin-walled ion chamber placed in cell culture dishes standing on the 32P radiation chambers. This procedure allowed mimicking of the dose rate in the cell environment. All three chambers were measured repeatedly. Measurements were also made with a surface hand detector (RNI 10\/R Intensimeter, Nuklex, Uppsala, Sweden), and a calibration curve was constructed to facilitate repeated dose rate determinations during the cell culture periods.\nTumour cells\nThe cells used were HT-29 colorectal adenocarcinoma, A-431 cervical squamous carcinoma, SKBR-3 breast cancer, all from the American Type Culture Collection (ATCC), and the two gliomas U-118MG and U-373MG from the Department of Pathology, Uppsala University, Uppsala, Sweden. They were grown in Ham\u2019s F-10 medium supplemented with 10% fetal bovine serum, 2\u00a0mmol\/l L-glutamine, 100\u00a0\u03bcg\/ml streptomycin and 100\u00a0U\/ml penicillin, all components from Sigma AB (Stockholm, Sweden). The cells were normally grown in an incubator of type Galaxy S (LabRum Klimat AB, Stockholm, Sweden), and during the irradiations they were grown in a similar incubator (Kebo Assab T304GF, Stockholm, Sweden). Both incubators were run at 37\u00b0C and supplied with 5% carbon dioxide.\nThe cells in the study were selected for the following reasons. SKBR-3 cells are often studied since they express large amounts of HER2 receptors and are therefore applied in experiments on HER2-directed radionuclide targeting, using antibodies or affibody molecules [37, 38]. A-431 cells express both large amounts of EGFR and also rather large amounts of HER2 and are therefore often studied for radionuclide targeting with both EGF ligands [39] and anti-HER2 antibodies [40]. Both cell types are planned for experimental therapy using beta particle-mediated radionuclide therapy, especially 177Lu. The two glioma cell lines also express EGFR [41] to some degree but were, together with the HT-29 cells, selected because they have recently been studied at our laboratory with regard to short-term effects after low dose rate irradiations [35]. It was found that U-373MG cells showed radiation-induced apoptosis, while U-118MG cells did not. The HT-29 cells were intermediate in this respect [35]. Furthermore, U-373MG cells have previously been reported not to show hyperradiosensitivity at low doses, while both U-118MG and HT-29 cells do [28].\nChoice of dose rate and exposure time\nIn the previous study, with the same 32P chambers, we analysed effects on cells after exposure to initial dose rates of only 0.05\u20130.09\u00a0Gy\/h for 7\u00a0days [35]. The cells in that study were analysed for effects on cell number, apoptosis and cell cycle block at day 7 of continuous exposure. However, we also allowed cells to continue to grow for longer times in parallel dishes and found that all cultures recovered. Thus, we decided to apply initial dose rates from 0.1\u00a0Gy\/h up to 0.8\u00a0Gy\/h in the present study to have a reasonable chance of \u201csimulating\u201d curative treatment. We could not apply higher dose rates because of regulatory rules limiting the amount of 32P that could be handled in the cell culture laboratory. Furthermore, this dose rate range is the same as that applied in published studies on low dose rate effects [21\u201323], hyperradiosensitivity and low dose rate [29], and radionuclide treatment of gliomas in vitro [42] and in vivo [43], and actually covers the range up to the highest dose achievable in targeted radionuclide tumour therapy [10, 16, 18, 27, 44].\nFour cell dishes were placed in each irradiation chamber when the dose rate was as expected and the cells were then kept there for 24, 72 or 168\u00a0h (1, 3 or 7\u00a0days). The radiation exposure time was chosen not to be longer than a week, since it is known from numerous articles in the field of radionuclide therapy that the tumour cell retention of radioactivity is generally in the range of some days up to about 1 week (in some cases it is only a few hours). Furthermore, it is well known that the biological half-life of tumour targeting agents (ligands, antibodies and antibody fragments) in the systemic circulation is often shorter than a week (in the case of small ligands, it is only a few hours). Thus, the longest exposure time considered to be realistic was 1 week.\nCell counting\nThe medium was removed from the cell dishes and the cells were quickly washed with 0.5\u00a0ml trypsin-EDTA (0.25% trypsin\/0.02% EDTA solution in PBS, VWR, Stockholm, Sweden) and then incubated with 0.5\u00a0ml trypsin-EDTA (37\u00b0C, 5% CO2) until the cells detached. Next, 1.5\u00a0ml medium was added to each dish and the cells resuspended to a single cell solution. For cell counting, 19.5\u00a0ml PBS (pH 7.4) was added to 0.5\u00a0ml cell suspension and an electronic cell counter was used (Coulter Z2, 7\u201320\u00a0\u03bcm, Beckman Coulter, Stockholm, Sweden).\nCell culture conditions during and after irradiations\nCells were seeded sparsely in culture dishes (diameter 3.5\u00a0cm, surface 9.6\u00a0cm2, Nunc, Roskilde, Denmark) a few days before the start of the radiation exposure. They were seeded so that each culture dish contained about 105 cells at the start of the exposure. The first cell count was made at the start of each experiment, on representative culture dishes. If the cell number was about 105 per dish, four other, parallel culture dishes were placed in each of the three irradiation chambers. The irradiated cells were grown in the chambers with unirradiated control dishes placed on a near radiation-shielded shelf in the same incubator. The cells did not reach confluence during the irradiation period. After the radiation exposure, the cells were moved from the irradiation chamber incubator to another incubator and the cell growth was followed for several months. At the first subcultivation after the radiation exposure, the cells were transferred from culture dishes to culture flasks (25\u00a0cm2, Nunc, Roskilde, Denmark), and these flasks were then used throughout the growth period. The medium in all culture dishes and flasks was replaced three times a week. Cell counting was in most cases performed once a week, followed by reseeding of 105 cells in each new flask.\nGrowth curves\nThe growth curves were constructed as if all cells had been saved at each subcultivation. By calculating how many cells would have been obtained if all cells had been saved, such high cell numbers as 1015 (Fig.\u00a01a) and even up to 1025 (Fig.\u00a01b) were obtained. In reality, repeated dilutions were made to keep the number of cells in each culture flask in the range of 105\u2013106, thus allowing for exponential growth. Four parallel flasks were kept for each experiment. In order to analyse the significance of the differences between the groups, t tests were performed.\nCell kill versus regrowth\nThe criterion for killing of a cell culture with 105 cells was that no living cells could be seen in the phase contrast microscope and that no regrowth was observed after at least 3\u00a0months of follow-up, also applying electronic cell counting.\nGrowth delay\nGrowth delay was also analysed after 1, 3 or 7\u00a0days of continuous low dose rate exposure. The growth curves of the irradiated samples in some cases did not have the same slope as the control curves, so it was not possible to wait and measure growth delay when the recovered cells and the control cells had a similar growth rate. Instead, growth delay was defined as the time it took for the irradiated cells to reach the cell number 1010 in relation to the time it took for the control cultures to reach this number.\nLiterature survey\nThe Medline-based PubMed database was used to survey effects of targeted radionuclide therapy and of low dose rate therapy.\nResults\nGrowth curves\nFigure\u00a01 shows examples of growth curves for control cells and cells exposed to continuous low dose rate beta irradiation. In Fig.\u00a01a the initial dose rate was 0.415\u00a0Gy\/h and the exposure time 72\u00a0h. After 72\u00a0h the dose rate had decreased to 0.359\u00a0Gy\/h, and the exposure gave a total dose of about 27.8\u00a0Gy. The irradiated HT-29 cells recovered after irradiation and resumed, after about 50\u00a0days, a similar growth rate as the controls. In contrast, the U-373MG cells completely ceased to grow after the same radiation exposure. After more than 30\u00a0days, only a few giant cells could be identified in the microscope. They were too few to be counted with the electronic cell counter. However, there was a possibility that at least a few of these cells could grow later, so they were observed (with normal medium changes) for up to 3\u00a0months. Nevertheless, no recovery could be observed; instead, the cells disappeared. It was then concluded that the irradiation procedure had killed all U-373MG cells. Thus, the HT-29 cultures survived the treatment, while the U-373MG cells died.\nFig.\u00a01Examples of cell growth as a function of time after exposure to low dose rate irradiation. a HT-29 and U-373MG cells were exposed to 0.415\u00a0Gy\/h as the initial dose rate and then continuously exposed for 72\u00a0h. b A-431, U-118MG and SKBR-3 cells were exposed to 0.806\u00a0Gy\/h as the initial dose rate and then continuously exposed for 24\u00a0h. Mean values and maximal variations from four parallel samples are given\nFigure\u00a01b shows examples of growth curves for an initial dose rate of 0.806\u00a0Gy\/h and an exposure time of 24\u00a0h. After 24\u00a0h the dose rate had decreased to 0.768\u00a0Gy\/h and the exposure gave a total dose of about 18.9\u00a0Gy. The irradiated A431 cells continued to grow, but at a slower rate than the controls. The growth of irradiated U-118MG cells was arrested for up to about 40\u00a0days after the treatment. During those 40\u00a0days, resting cells were observed in the culture flasks, but there was no sign of growth. After 40\u00a0days a few mitotic cells were observed in the phase contrast microscope, and the number of cells was thereafter measured with the electronic cell counter. A clear regrowth was seen after 60\u00a0days. However, their growth rate was slower than that of the corresponding controls. The SKBR-3 cells looked severely damaged (fragmented, decreasing in number and forming giant cells) some days after the exposure and were too few to be counted with the electronic cell counter. Within 1 month, no surviving cells could be observed in the culture flasks. In this case, too, we continued to control the culture flasks for 3\u00a0months (with normal medium changes) and found that there were no remaining cells in these cultures. Thus, A-431 and U-118MG cells survived, while the SKBR-3 cells died.\nThe conclusions from the examples in Fig.\u00a01a and b are that four different responses were observed: \nThe HT-29 cells recovered to the control growth rate after a growth delay.The U-118MG cells recovered after a growth delay but continued to grow at a slower rate than the controls.The A-431 cells continued to grow without delay but at a slower rate than the controlThe U-373MG and SKBR-3 cells died.\nCell kill and regrowth versus dose rate and exposure time\nFigure\u00a02 summarises cell-killing and regrowth results from all performed low dose rate experiments. All five analysed cell types were irradiated with different initial low dose rates (0.1\u20130.8\u00a0Gy\/h) and were continuously exposed for 1, 3 or 7\u00a0days.\nFig.\u00a02Summary of all low dose rate experiments carried out for a U-118MG, b U-373MG, c HT-29, d A-431 cells and e SKBR-3 cells. The cells were irradiated with different initial dose rates and were then exposed to the radiation for 1, 3 or 7\u00a0days. The figures show at which combinations of dose rate and exposure time all cells were killed (area with no survivors), and at which at least some cells survived and displayed regrowth (the regrowth area). The separation between the two areas is indicated by bold solid lines. The total delivered radiation dose (Gy) is given in parentheses near each point. The 20-Gy isodose curve is indicated by a dashed line\nFigure\u00a02 parts a\u2013e show a similar and general pattern. When applying 7\u00a0days of continuous irradiation, low initial dose rates, about 0.2\u20130.3\u00a0Gy\/h, were enough to kill all cells. When cells were exposed for only 3 days, a dose rate in the order of 0.4\u20130.6\u00a0Gy\/h was necessary to kill all cells. Only the SKBR-3 cells were killed after 24-h exposure to about 0.8\u00a0Gy\/h. As mentioned in Materials and methods, higher dose rates could not be used in these experiments, so we do not know how high the dose rates would have to be during 24-h exposure in order to kill the other cell types. It was found that the regrowth pattern was the same in all four flasks.\nHowever, even if the results looked rather similar and independent of cell type, there were some cell type-dependent differences. The U-118MG cells (Fig.\u00a02a) required, during 7\u00a0days\u2019 exposure, at least about 0.3\u00a0Gy\/h as the initial dose rate to be completely growth inactivated. The U-373MG cells (Fig.\u00a02b) were completely growth inactivated after an initial dose rate of only about 0.15\u00a0Gy\/h and 7\u00a0days\u2019 exposure. Thus, the U-373MG cells were, in these cases, easier to kill than the U-118MG cells. Considering all cells, it seemed that the U-118MG cells were most resistant and U-373MG and SKBR-3 most sensitive, while A-431 and HT-29 were intermediate.\nCell kill and regrowth versus total dose\nThe 20-Gy isodose curve is drawn with a dashed line in Fig.\u00a02a\u2013e. For U-373MG and SKBR-3 cells, at least a total dose of 20\u00a0Gy was necessary to prevent regrowth. For HT-29 and A-431 cells an even higher total dose seemed necessary, and for the resistant U-118MG cells it seemed that total doses of at least 40\u00a0Gy were necessary to prevent regrowth.\nGrowth delay as a function of dose rate\nFigure\u00a03a\u2013e shows growth delays versus the initial dose rate after 1, 3 or 7\u00a0days of continuous low dose rate exposure. The growth delay curves in Fig.\u00a03a show that the radioresistant U-118MG cells had growth delays, after 24-h exposure, which increased slowly as a function of the initial dose rate. A growth delay of 2 months was obtained at about 0.7, 0.3 and 0.2\u00a0Gy\/h after 1, 3 and 7\u00a0days of continuous exposure, respectively. The growth delay curves for the more radiosensitive U-373MG cells increased more steeply (Fig.\u00a03b), and a growth delay of 2\u00a0months was obtained at about 0.5 and 0.2\u00a0Gy\/h after 1 and 3\u00a0days of continuous exposure, respectively. Thus, the U-373MG cells were more sensitive than the U-118MG cells also with regard to growth delay.\nFig.\u00a03Growth delay versus the initial dose rate after 1, 3 or 7\u00a0days of continuous low dose rate exposure for a U-118MG, b U-373MG, c HT-29, d A-431 and e SKBR-3 cells. The boxes at the top of each figure indicate at which dose rates and exposure times there was no regrowth (infinite growth delay or total kill of the cultures). Mean values and maximal variations from four samples are shown\nBoth the HT-29 cells (Fig.\u00a03c) and the SKBR-3 cells (Fig.\u00a03e) had a biphasic growth delay curve after 1 day\u2019s exposure. The A-431 cells (Fig.\u00a03d) had a slow increase in growth delay as a function of dose rate after 1 day\u2019s exposure.\nThe SKBR-3 cells had a growth delay that increased rather steeply as a function of the dose rate and reached a value of 2\u00a0months at about 0.50 and 0.15\u00a0Gy\/h after 1 and 7\u00a0days of continuous exposure, respectively (Fig.\u00a03e).\nGrowth delay as a function of total dose\nThe same growth delay data as in Fig.\u00a03a\u2013e are instead plotted as a function of the delivered total dose in Fig.\u00a04a\u2013c, but here, for clarity, without the maximal variations. In all cases, there was a general increasing growth delay as a function of dose. Growth delays in the order of 10\u00a0days were obtained for all cells after delivery of total doses in the range 5\u201315\u00a0Gy, independent of whether the dose was given during 1, 3 or 7\u00a0days. Delays around 100\u00a0days required total doses of about 15\u201320\u00a0Gy when given during 1 day and 25\u201340\u00a0Gy when given during 3 or 7\u00a0days, for all cells.\nFig.\u00a04Growth delay versus total dose after a 1, b 3 or c 7\u00a0days of continuous low dose rate exposure for U-118MG, U-373MG, HT-29, A-431 and SKBR-3 cells. The growth delay values are the same as in Fig.\u00a03 but, for clarity, without maximal variations\nDiscussion\nThe obtained results can hopefully serve as a guideline for the combinations of dose rate and exposure time necessary to kill tumour cells when applying low dose rate beta irradiation. The shift from regrowth to \u201ccure\u201d (no surviving cells) fell, for each cell type, within a narrow range of combinations.\nThe U-118MG cells were more resistant to the treatments than the U-373MG and SKBR-3 cells. The A-431 and HT-29 cells showed intermediate resistance. However, the differences between the cell lines were not dramatic, and it can be generally stated that when applying 7\u00a0days of continuous irradiation, initial dose rates of about 0.2\u20130.3\u00a0Gy\/h were enough to kill all cells in the cultures. When exposed for 3\u00a0days, an initial dose rate in the order of 0.4\u20130.6\u00a0Gy\/h was needed. When the cells were exposed for only 24\u00a0h it was not possible to kill all the cells (with the exception of SKBR-3 cells), even if the initial dose rate was as high as about 0.8\u00a0Gy\/h.\nThe studied dose rates are the highest that can be achieved in targeted radionuclide therapy [10, 16, 18]. The total doses achieved after 1, 3 or 7\u00a0days\u2019 exposure also correspond to the highest achievable doses in targeted radionuclide therapy [16], and most often total doses of no more than 10\u201320\u00a0Gy are obtained in targeting of B-cell lymphomas [19]. However, there are indications from preclinical studies that dramatic \u201cdose amplification\u201d per receptor interaction can be achieved by using effective residualising agents [45].\nThe obtained dose rates in beta particle-based radionuclide therapy are to a large extent a consequence not only of the amount of radionuclides bound to each tumour cell, but also of the cross-fire effect. This means that radionuclides bound to one cell also irradiate nearby cells owing to the long range of the radiation [44, 46]. This can increase the dose rate tenfold, and makes it reasonable to assume that dose rates in the range used in our model experiments can be achieved in tumours in patients. The dose rate will be lower for a single tumour cell considering only the radionuclides bound to that cell [25]. Beta particles with a long range will permit rather uniform dose distributions and hopefully deliver therapeutically relevant radiation doses also to non-targeted tumour cells.\nLower dose rates than we applied will probably not lead to curative treatments when beta particles are applied. From the obtained results it is obvious that, at least for the five types of tumour cell tested, we succeeded in finding \u201cdose rate\u2013exposure time\u201d combinations that could distinguish between \u201ccure\u201d (killing of 105 tumour cells) and \u201crelapse\u201d (recovery of tumour cells).\nThere may be cases in which only a fraction of the tumour cells have to be killed directly by radiation, since the remaining tumour cells may be killed through bystander effects [30\u201332] or other factors (e.g. limited nutrition supply, natural immune response, adjuvant chemo- or immunotherapy). However, our model can be applied under the assumption that 105 tumour cells have to be killed by radiation, even if other tumour cells are killed by other means.\nWhen considering radionuclide therapy it is, of course, also important to consider unwanted effects on normal tissues. The tolerance doses for most normal tissues are not known when exposure is to low dose rate irradiation. Targeted radionuclide therapy, using, for example, radiolabelled antibodies, fragments of antibodies or various receptor ligands, is expected to result in highly tumour-specific uptake of the therapeutic radionuclides. Thus, for a curative intent it is reasonable to establish the necessary tumour dose rates and exposure times. Another obvious question is which targeting agent should be tried for each type of tumour and, most importantly, whether the required conditions can be achieved without excessively severe side-effects on normal tissues. However, analyses of effects on normal tissues were beyond the scope of this study.\nHere we discuss the observed cell type-dependent differences. In a previous study [35] we published data on low dose rate acute effects, using three of the cells in the present study: U-118MG, U-373MG and HT-29. In that study the initial dose rate was only 0.05\u20130.09\u00a0Gy\/h and the exposure time, 7\u00a0days. As would be expected in view of our new data, all cultures did grow after such treatment. It was shown that the U-373MG cells had, at day 7, the most pronounced reduction in cell number owing to a combination of a G2 block and radiation-induced apoptosis. There were surprisingly low reductions in U-118MG and HT-29 cell numbers. U-118MG had a G2 block but no radiation-induced apoptosis. HT-29 had both a G2 block and some radiation-induced apoptosis, but the amount of apoptosis was smaller than for U-373MG cells. Thus, the results from that study indicated the U-373MG cells to be more sensitive than the other two cell lines owing to a higher degree of apoptosis. This is in agreement with the cell-killing results from the present study.\nThe intrinsic radiosensitivity measured as S2Gy, after exposure to a high dose rate (most often 0.5\u20132.0\u00a0Gy\/min) photons has previously been determined for four of our studied cell lines. No determination of S2Gy for SKBR-3 cells was found in the literature. The results are given in Table\u00a01 [28, 47\u201354]. \nTable\u00a01Survival at 2Gy after exposure to high dose ratesCell lineSurvival at 2GyReferencesU-118MG0.44\u20130.70[28], [47]U-373MG0.60\u20130.62[28], [48], [49]HT-290.55\u20130.78[28], [50], [51], [52], [53], [54]A-431\u22480.52[53]\nThere is also a review article on the intrinsic radiosensitivity, measured as cell survival at the dose 2Gy, S2Gy, for 694 human cell lines, of which 271 were from tumours [55]. The tumour cell lines were grouped according to tumour type, and the S2Gy values for the U-118MG and U-373MG gliomas and the A-431 cervical carcinoma used in this study fell within the expected range of values for the corresponding types of tumour. Thus, these cells can be considered typical for their tumour groups, at least regarding intrinsic radiosensitivity at high dose rate exposure. The HT-29 colorectal carcinoma is an exception, since it seems to be somewhat more radioresistant than most other colorectal carcinomas.\nConsidering the values in Table\u00a01, it is clear that there is no relation between S2Gy and the effects of low dose rate irradiations. For example, the U-118MG cells were found to be most radioresistant to a low dose rate, while their S2Gy values were in the same range as for the other cell lines. The U-373MG cells, which were considered most sensitive to a low dose rate, also had S2Gy values in the same range.\nOne possible explanation of the lack of agreement between intrinsic radiosensitivity measured as S2Gy and the effect of a low dose rate is that there are cell type-dependent differences in repopulation during low dose rate irradiations. Such differences can probably \u201covershadow\u201d the differences in intrinsic radiosensitivity. Another possible explanation might be cell type-dependent differences in the capacity for low dose rate-induced apoptosis. The latter is supported by our previous study [35], which showed that low dose rate-induced apoptosis was more frequent in U-373MG cells than in HT-29 cells and that no such apoptosis could be observed for U-118MG cells. Furthermore, it seems as if differences in hyperradiosensitivity (measured at low doses but at a high dose rate) are not of great importance, since the U-118MG and HT-29 cells have been reported to show hyperradiosensitivity, while U-373MG cells do not [28]. Had hyperradiosensitivity been of importance under the exposure conditions in this study, then U-373MG cells should have been more resistant than HT-29 and U-118MG cells.\nIt has recently been suggested that variations in radiosensitivity at low dose rates are related to the compactness of chromatin [56], but it is not known whether the cells in our study have any differences in this respect. In another recent experimental study, a good therapeutic effect at a low dose rate was reported; in fact, if the total delivered dose was in the range 1\u20132\u00a0Gy, the effect was as good as that achieved at a high dose rate, although the difference in dose rate was nearly three orders of magnitude [57]. This indicates that there are basic radiation biology aspects of low dose rate radiation that have to be analysed in much more detail. A clue to the molecular factors involved came from a recent report showing that activation or inhibition of the DNA damage sensor ATM is of importance [58]. It was found that DNA damage inflicted at a low rate failed to activate ATM; however, if ATM was activated by chloroquine, the cells survived the low dose rate much better.\nWe conclude that dose rates in the range 0.2\u20130.3\u00a0Gy\/h are necessary in order to kill 105 tumour cells during 1 week\u2019s exposure. Higher dose rates, such as 0.4\u20130.6\u00a0Gy\/h and >0.8\u00a0Gy\/h, are necessary if the exposure times are only 3\u00a0days and 1\u00a0day, respectively. If, in some cases, a good therapeutic effect cannot be obtained with beta particle radionuclide therapy because of too low dose rates and\/or too short exposure times, then repeated (fractionated) treatment is a possibility to improve the result. However, the cellular response to repeated low dose rate exposures has to be analysed in further studies, since there is a risk of radiation-induced changes in the growth pattern of the tumour cells surviving the first exposure, as shown in Fig.\u00a01b.","keyphrases":["radionuclide","low dose rate","tumour therapy","nuclear medicine","radiation effects"],"prmu":["P","P","P","U","R"]}
{"id":"Purinergic_Signal-3-4-2072914","title":"Autoregulation in PC12 cells via P2Y receptors: Evidence for non-exocytotic nucleotide release from neuroendocrine cells\n","text":"Nucleotides are released not only from neurons, but also from various other types of cells including fibroblasts, epithelial, endothelial and glial cells. While ATP release from non-neural cells is frequently Ca2+ independent and mostly non-vesicular, neuronal ATP release is generally believed to occur via exocytosis. To evaluate whether nucleotide release from neuroendocrine cells might involve a non-vesicular component, the autocrine\/paracrine activation of P2Y12 receptors was used as a biosensor for nucleotide release from PC12 cells. Expression of a plasmid coding for the botulinum toxin C1 light chain led to a decrease in syntaxin 1 detected in immunoblots of PC12 membranes. In parallel, spontaneous as well as depolarization-evoked release of previously incorporated [3H]noradrenaline from transfected cells was significantly reduced in comparison with the release from untransfected cells, thus indicating that exocytosis was impaired. In PC12 cells expressing the botulinum toxin C1 light chain, ADP reduced cyclic AMP synthesis to the same extent as in non-transfected cells. Likewise, the enhancement of cyclic AMP synthesis either due to the blockade of P2Y12 receptors or due to the degradation of extracellular neucleotides by apyrase was not different between non-transfected and botulinum toxin C1 light chain expressing cells. However, the inhibition of cyclic AMP synthesis caused by depolarization-evoked release of endogenous nucleotides was either abolished or greatly reduced in cells expressing the botulinum toxin C1 light chain. Together, these results show that spontaneous nucleotide release from neuroendocrine cells may occur independently of vesicle exocytosis, whereas depolarization-evoked nucleotide release relies predominantly on exocytotic mechanisms.\nIntroduction\nAdenine and uridine nucleotides are present in and released from all types of cells [2] and exert effects via P2X and\/or P2Y receptors: P2X receptors are ATP-gated cation channels [25]; P2Y receptors are G protein-coupled receptors, and eight different types have been identified (P2Y1, 2, 4, 6, 11, 12, 13, 14; [1]). P2Y1, 11, 12 and 13 are activated by adenine nucleotides, whereas P2Y6 is activated by uridine nucleotides. P2Y2 and 4 are sensitive to adenine and uridine nucleotides, and P2Y14 is activated by UDP-glucose. In a large number of non-neural tissues, e.g. liver, kidney, bones and blood vessels, released nucleotides subserve autocrine\/paracrine functions by activating certain P2Y receptors [32, 16, 38, 12, 30]. In a given cell, this autocrine\/paracrine regulation depends on the release of nucleotides, the presence of P2Y receptors and occasionally on the enzymatic activities of nucleotidases: in hepatoma cells [16], for instance, released ATP is degraded to ADP to stimulate P2Y1 receptors; in epithelial [32] and osteoblastic [38] cells, in contrast, ATP itself activates P2Y11 and P2Y2 receptors, respectively.\nAlthough ATP has first been described to be released from neurons [15] and to act as a neurotransmitter [6], available information about neuronal autoregulation via released nucleotides and P2Y receptors is limited. Early examples were the feedback inhibition of sympathetic transmitter release via presynaptic P2Y receptors (see [4]) and the ATP-mediated autocrine inhibition of voltage-activated Ca2+ channels in bovine chromaffin cells [9]. More recently, autoregulation via P2Y receptors has been described for the phaeochromocytoma cell line PC12 [24]. There, endogenous P2Y12 receptors were activated by spontaneous nucleotide release to achieve half-maximal inhibition of cyclic AMP (cAMP) synthesis. Depolarization of PC12 cells by 100\u00a0mM K+ further enhanced nucleotide release and thereby led to full inhibition of cAMP generation. This latter effect was prevented when voltage-activated Ca2+ channels were blocked by Cd2+ or when extracellular nucleotides were degraded by apyrase. Thus, the inhibition of cAMP synthesis in PC12 cells via P2Y receptors can be viewed as a biosensor for nucleotide release [24].\nNucleotides are released from various cells including fibroblast-like, epithelial, endothelial, glial and neuronal cells [36, 28, 21, 26, 35]. ATP release from non-neural cells can be elicited by different stimuli, such as hypotonic solutions [36], mechanical stimulation [28] or exchange of culture media [21, 26] and is frequently Ca2+ independent and therefore most probably non-vesicular. The mechanisms underlying this non-vesicular ATP release remained largely elusive, but ATP binding cassette transporters, stretch-activated cation channels as well as connexin hemichannels have been implicated [22]. In addition, evidence has been presented that indicates that nucleotide release from non-neural cells may to some extent be vesicular (e.g. [17, 8]).\nIn neurons and neuroendocrine cells, ATP is stored in vesicles either alone or together with classic neurotransmitters and is thus released by exocytosis [27]. Vesicle exocytosis occurs spontaneously at a slow rate and is largely accelerated when Ca2+ entry is triggered by depolarization-induced opening of voltage-activated Ca2+ channels [29]. Accordingly, depolarization-evoked ATP release from neurons and neuroendocrine cells is entirely Ca2+ dependent [35, 13], but it remained unknown whether all of the spontaneous nucleotide release from neuronal sources is also vesicular.\nVesicle exocytosis in neurons and neuroendocrine cells involves the SNARE proteins syntaxin, SNAP-25 and VAMP\/synaptobrevin. These proteins are cleaved by various serotypes of clostridial neurotoxins which thereby prevent exocytosis. In particular, botulinum neurotoxin C1 cleaves syntaxin, and in some cells also SNAP-25, and thereby impedes spontaneous as well as Ca2+-evoked vesicle exocytosis [5]. Transfection of PC12 cells with a plasmid coding for the botulinum toxin C1 light chain was reported to largely reduce spontaneous as well as depolarization-evoked release of co-transfected human growth hormone [10]. Here, we used an analogous method to investigate whether in PC12 cells the activation of P2Y12 receptors by endogenously released nucleotides relies on vesicle exocytosis.\nMaterials and methods\nMaterials\n[2,8\u22123H]adenine (specific activity 32\u00a0Ci mmol\u22121) and levo-[ring-2,5,6-3H]noradrenaline (specific activity 37\u00a0Ci mmol\u22121) were obtained from NEN (Vienna, Austria). Na-ADP, 4-(3-butoxy-4-methoxybenzyl)imidazoline-2-one (RO 20-1724), 3\u2032,5\u2032-cyclic AMP, apyrase (grade VII, with an approximately 1:1 ratio in ATPase and ADPase activity), 2-methylthio-AMP, and 2-p-(2-carboxyethyl)phenethylamino-5\u2032-N-ethylcarboxamido-adenosine (CGS 21680) were purchased from Sigma (Vienna, Austria).\nCell culture and transfection\nPC12 cells were maintained and subcultured as previously described in detail [24]. The cells were plated onto collagen-coated (Biomedical Technologies Inc., Stoughton, MA, USA) culture dishes (NUNC, Roskilde, Denmark) and were kept in OptiMEM (InVitrogen, Vienna, Austria) supplemented with 0.2\u00a0mM L-glutamine (HyClone, Aalst, Belgium), 25,000\u00a0IU l\u22121 penicillin and 25\u00a0mg l\u22121 streptomycin (Sigma, Vienna, Austria), 5% fetal calf serum and 10% horse serum (both InVitrogen, Vienna, Austria). Once per week, cell cultures were split; the medium was exchanged twice per week. To investigate the outflow of previously incorporated [3H]noradrenaline under continuous superfusion, PC12 cells were plated onto 5-mm discs, for assays of cAMP accumulation they were plated onto 6-well culture dishes and for membrane preparations for Western blots they were plated onto 100-mm culture dishes. All this tissue culture plastic was coated with collagen (as above).\nFor the generation of PC12 cell clones stably expressing the botulinum toxin C1 light chain (BoNT\/C1), 15\u00a0\u03bcg of a pcDNA3 vector harbouring the botulinum toxin C1 light chain (pBoNT\/C1; kindly provided by R.D. Burgoyne, Liverpool, UK) [10] were mixed with 50\u00a0\u03bcl of the TransFast transfection reagent (Promega, Mannheim, Germany) and added to semiconfluent PC12 cell cultures in serum-free medium. After a 1\u00a0h incubation at 37\u00b0C, two additional volumes of serum-free medium and the appropriate amount of serum (as above) were added. This medium was exchanged 48\u00a0h after transfection for a medium supplemented with 500\u00a0\u03bcg\/ml neomycin (G418) to allow for selection of drug resistance. This selection medium was replaced every 3\u20134\u00a0days until distinct islands of surviving cells were visible. Individual clones of antibiotic-resistant cells were transferred to 24-well plates and grown in medium containing 200\u00a0\u03bcg\/ml G418.\nImmunoblotting of SNARE proteins\nPreparation of protein extracts of PC12 membranes was performed as described previously [18]. Materials and methodology used for production and analysis of ECL-immunoblots were also essentially as described therein, with the following modifications: protein extracts in SDS sample buffer were incubated at 95\u00b0C for 5\u00a0min and subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) on 5% (stacking) and 10% (separating) gels. After electrophoretic transfer of proteins to nitrocellulose, membranes were cut horizontally along appropriate molecular weight markers in order to separate regions harbouring proteins with apparent molecular weights of 10\u201320\u00a0kD, 21\u201329\u00a0kD and 30\u201350\u00a0kD. These membrane strips were incubated in one of the following monoclonal SNARE antibodies: syntaxin 1 antibody clone 78.3, synaptobrevin 2 antibody clone 69.1 (both obtained from Synaptic Systems, G\u00f6ttingen, Germany) and SNAP-25 antibody MAB331 (purchased from Chemicon, Temecula, CA, USA).\nMeasurement of [3H]noradrenaline release\n[3H]noradrenaline uptake in and superfusion of PC12 cell cultures were performed as described previously [23]. Culture discs with PC12 cells were incubated in 0.1\u00a0\u03bcM [3H]noradrenaline in culture medium supplemented with 1\u00a0mM ascorbic acid at 36\u00b0C for 1\u00a0h. After labelling, culture discs were transferred to small chambers and superfused with a buffer containing (mM) NaCl (120), KCl (3.0), CaCl2 (2.0), MgCl2 (2.0), glucose (20), hydroxyethylpiperazine ethanesulphonic acid (HEPES) (10), fumaric acid (0.5), Na-pyruvate (5.0), ascorbic acid (0.57) and desipramine (0.001), adjusted to pH 7.4 with NaOH. Superfusion was performed at 25\u00b0C at a rate of about 1.0\u00a0ml\/min. Collection of 4-min superfusate fractions was started after a 60-min washout period to remove excess radioactivity.\nDepolarization-dependent overflow of previously incorporated [3H]noradrenaline was triggered by inclusion of 30 or 100\u00a0mM KCl in the superfusion buffer during two 30-s periods 20\u00a0min apart (Fig.\u00a02a). At the end of the experiments, the radioactivity remaining in the cells was extracted by immersion of the discs in 2% (v\/v) perchloric acid followed by sonication. Radioactivity in extracts and collected fractions was determined by liquid scintillation counting (Packard Tri-Carb 2100 TR).\nThe spontaneous (unstimulated) rate of [3H] outflow was obtained by expressing the radioactivity of a collected fraction as percentage of the total radioactivity in the cultures at the beginning of the corresponding collection period. Values obtained during the 4-min period directly preceding the application of 30\u00a0mM K+ were used for the comparison of spontaneous (basal) tritium outflow in wild-type and pBoNT\/C1 expressing PC12 cells. K+-evoked tritium overflow was calculated as the difference between the total [3H] outflow during and after stimulation and the estimated basal outflow which was assumed to decline linearly throughout the experiments. Therefore, basal outflow during periods of stimulation was assumed to equate the arithmetic mean of the samples preceding and those following stimulation, respectively. The difference between the total and the estimated basal outflow was expressed as a percentage of the total radioactivity in the cultures at the beginning of the respective stimulation (% of total activity).\nDetermination of cyclic AMP\nThe accumulation of cAMP in PC12 cell cultures was determined as described before [33]. After labelling of cellular purines with tritiated adenine (2.5\u00a0\u03bcCi\/ml for 12\u00a0h), the medium was replaced by a buffer (120\u00a0mM NaCl, 3\u00a0mM KCl, 2\u00a0mM MgCl2, 2\u00a0mM CaCl2, 20\u00a0mM glucose, 10\u00a0mM HEPES, 10\u00a0mM LiCl, adjusted to pH 7.4 with NaOH) supplemented with 100\u00a0\u03bcM of the phosphodiesterase inhibitor Ro-20-1724 [4-(3-butoxy-4-methoxybenzyl) imidazolidin-2-one] and 1\u00a0U\/ml adenosine deaminase. Dishes were then kept at room temperature for 105\u00a0min. During the last 15\u00a0min of this incubation period, the adenosine A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5\u2032-N-ethylcarboxamido-adenosine (CGS 21680), ADP (10\u00a0\u03bcM), apyrase (1\u00a0U\/ml) or 100\u00a0mM KCl (NaCl was reduced accordingly) were also included in the medium. When appropriate, the P2Y12 receptor antagonist 2MesAMP (100\u00a0\u03bcM; [14]) was present for the last 25\u00a0min. The incubation was terminated by exchanging the buffer for 1\u00a0ml of 2.5% perchloric acid containing 100\u00a0\u03bcM non-labelled cAMP followed by a 20-min incubation at 4\u00b0C. Subsequently, cAMP was separated from the other purines by a chromatographic procedure described before [33]. One tenth of each sample obtained as described above was used for the determination of the total radioactivity. The remaining 900\u00a0\u03bcl were neutralised by addition of 100\u00a0\u03bcl 4.2\u00a0M KOH and applied to Dowex 50 columns (AG 50W-X4; Bio-Rad, Vienna, Austria), which were then rinsed with 3\u00a0ml H2O. The eluate obtained by the subsequent application of 8\u00a0ml H2O was directly poured onto alumina columns (Bio-Rad, Vienna, Austria), which were then washed with 6\u00a0ml H2O. Finally, cAMP was eluted with 4\u00a0ml imidazole buffer (20\u00a0mM imidazole in 0.2\u00a0M NaCl; pH 7.45). Radioactivity within the samples obtained was determined by liquid scintillation counting. The radioactivity in the fraction of cAMP was expressed as percentage of the total radioactivity incorporated in the cells.\nStimulation of PC12 cells, whether they were clonal cell lines or wild-type cells, with the adenosine A2A receptor agonist CGS 21680 caused reproducible increases in the values of cAMP. However, the extent of basal and stimulated cAMP synthesis may vary between different preparations [33]. Hence, to be able to directly compare changes in CGS 21680-induced cAMP synthesis in different cell clones, values obtained in the presence of ADP, apyrase, 2MesAMP or KCl were expressed as percentage of the values obtained in the very same preparation, but in the absence of these agents (% of control).\nStatistics\nAll data represent arithmetic means\u00b1SEM; n represents numbers of culture dishes. Statistical significance of differences between single data points was evaluated by the non-parametric Mann-Whitney test, and p values\u2009<\u20090.05 were accepted as indicators of significance.\nResults\nTo assess the role of vesicle exocytosis in the release of endogenous nucleotides from neuroendocrine cells, this study employed the autocrine\/paracrine control of cAMP synthesis in PC12 cells via P2Y12 receptors. To prevent vesicle exocytosis, we generated cell clones stably expressing pBoNT\/C1. Depending on the cell types being investigated, this toxin cleaves one or more SNARE proteins and thereby prevents vesicular transmitter release or hormone secretion [5].\nCleavage of SNARE proteins by the expression of the BoNT\/C1 light chain\nThe major target of BoNT\/C1 is syntaxin 1, which is cleaved by this toxin in all types of preparations [5]. Therefore, several pBoNT\/C1 expressing clones were initially tested in immunoblots of harvested membranes for a lack of full-length syntaxin. Two of these pBoNT\/C1 expressing clones (BT2 and BT7) were selected for further analysis.\nIn bovine chromaffin cells, BoNT\/C1 was reported to cleave not only syntaxin 1, but also SNAP-25, even though the toxin failed to cleave purified and recombinant SNAP-25 [11]. To evaluate the efficiency of SNARE protein cleavage by the BoNT\/C1 light chain expressed in PC12 cells, the immunostaining of syntaxin and SNAP-25 in Western blots was compared with that of synaptobrevin, which is undoubtedly insensitive to BoNT\/C1 [11, 5]. As shown in Fig.\u00a01, the stable expression of pBoNT\/C1 markedly reduced or even abolished the staining of a protein with an apparent molecular weight of 37\u00a0kD by an anti-syntaxin antibody. In contrast, there were no clear-cut reductions in the staining of a 25-kD band by an anti-SNAP-25 antibody, and occasionally the immunostaining of this protein appeared to be even enhanced in pBoNT\/C1 expressing clones (Fig.\u00a01a). To quantify these changes in SNARE protein expression, the immunoblots were quantified by densitometry and the values obtained for the anti-syntaxin and anti-SNAP-25 immunoreactive bands were set in relation to those of the anti-synaptobrevin immunoreactive bands (Fig.\u00a01b). This procedure revealed a significant reduction of syntaxin in the pBoNT\/C1 expressing clones when compared with wild-type PC12 cells. SNAP-25 immunoreactivity, in contrast, was not significantly altered in these cell clones.\nFig.\u00a01Identification of SNARE proteins in membrane extracts from either wild-type (WT, untransfected) PC12 cells or from two PC12 cell clones stably expressing pBoNT\/C1 (BT2 and BT7). SDS-membrane extracts were separated on SDS-PAGE and transferred to nitrocellulose membranes. The membranes were cut horizontally to separate regions harbouring proteins with apparent molecular weights of 1\u201320\u00a0kD, 21\u201329\u00a0kD and 30\u201350\u00a0kD and immunostained with anti-syntaxin 1 antibody clone 78.3 (Stx), anti-SNAP-25 antibody MAB331 (S25) and anti-synaptobrevin 2 antibody clone 69.1 (Sb), respectively. a Representative blot obtained as described above. b The relation between the anti-syntaxin and the anti-synaptobrevin immunoreactivities (Stx\/Sb) as determined by densitometric analysis (n\u2009=\u20093). c The relation between the anti-SNAP-25 and the anti-synaptobrevin immunoreactivities (Stx\/Sb) as determined by densitometric analysis (n\u2009=\u20093). *Significant differences versus the results obtained with membranes from wild-type cells at p\u2009<\u20090.05\nInhibition of transmitter release by the expression of the BoNT\/C1 light chain\nPreviously, it has been shown that the transient expression of pBoNT\/C1 reduced stimulated vesicular catecholamine release from PC12 cells [10]. To evaluate whether catecholamine release was reduced in the pBoNT\/C1 expressing clones, clonal and wild-type PC12 cells were labelled with [3H]noradrenaline and the basal and depolarization-dependent outflow of radioactivity was determined. After washout of excess radioactivity during a 1-h washout period, all PC12 cell cultures steadily released small amounts of radioactivity in the superfusion buffer (Fig.\u00a02a). However, this spontaneous outflow of radioactivity was significantly less in the two pBoNT\/C1 expressing clones than in untransfected PC12 cells (Fig.\u00a02b).\nFig.\u00a02Basal and depolarization-evoked [3H]noradrenaline release from either wild-type (WT, untransfected) PC12 cells or from two PC12 cell clones stably expressing pBoNT\/C1 (BT2 and BT7). PC12 cell cultures were labelled with [3H]noradrenaline and superfused. Subsequent to a 60-min washout period, 4-min fractions of superfusate were collected. Tritium overflow was stimulated twice (S1 after 72\u00a0min and S2 after 92\u00a0min of superfusion) by the presence of KCl (30 or 100\u00a0mM, each for 30\u00a0s). a Comparison of the time courses of fractional [3H] outflow from the three different kinds of cell cultures (n\u2009=\u20095 to 6). b The amount of basal [3H] outflow from the three PC12 cell cultures (n\u2009=\u20098 to 9). c The amount of [3H] overflow from the three PC12 cell cultures triggered by 30\u00a0mM KCl (n\u2009=\u20098 to 9). d The amount of [3H] overflow from the three PC12 cell cultures triggered by 100\u00a0mM KCl (n\u2009=\u20098 to 9). *, **, ***Significant differences versus the results obtained with wild-type cells at p\u2009<\u20090.05, p\u2009<\u20090.01 and p\u2009<\u20090.001, respectively\nIn wild-type cells, the presence of 30\u00a0mM K+ (Na+ was reduced accordingly) for 30\u00a0s caused a reproducible increase in the outflow of radioactivity (Fig.\u00a02a), but such an effect was hardly observed in the pBoNT\/C1 expressing clones. Accordingly, the overflow of radioactivity triggered by 30\u00a0mM K+ was significantly reduced in the pBoNT\/C1 expressing clones in comparison with wild-type cells (Fig.\u00a02c). This confirms that the expression of the BoNT\/C1 light chain reduced spontaneous noradrenaline release and almost abolished release due to mild depolarizations.\nLarge intracellular Ca2+ increases have been reported to rescue transmitter release from cells poisoned with BoNT\/C1 [7]. Therefore, PC12 cultures were also exposed to 100\u00a0mM K+ (Na+ was again reduced) for 30\u00a0s. Although this stimulation paradigm caused unequivocal increases in the outflow of radioactivity even from pBoNT\/C1 expressing cells (Fig.\u00a02a), the amount of tritium overflow was still reduced when compared with the values obtained in untransfected PC12 cells (Fig.\u00a02d). Thus, the expression of the BoNT\/C1 light chain efficiently reduced spontaneous as well as depolarization-evoked vesicular transmitter release.\nChanges in the regulation of cAMP synthesis via P2Y receptors due to the expression of the BoNT\/C1 light chain\nIn PC12 cells, activation of endogenous P2Y12 receptors by the exposure to nucleotides causes an inhibition of adenylyl cyclase [33]. Furthermore, spontaneously released nucleotides are also sufficient to partially activate these receptors, and depolarization of the cells fully activates the receptors through enhanced nucleotide release to maximally suppress cAMP synthesis [24]. In accordance with these previous data, exposure of wild-type PC12 cells to 10\u00a0\u03bcM ADP significantly reduced the amount of cAMP generated due to the activation of A2A adenosine receptor by 1\u00a0\u03bcM CGS 21680 present for 15\u00a0min. In the two pBoNT\/C1 expressing clones, the inhibition of cAMP accumulation was as pronounced as in the wild-type cells (Fig.\u00a03a). Thus, the adenylyl cyclase inhibiting P2Y receptors were operating equally well in transfected as well as untransfected PC12 cells.\nFig.\u00a03Changes in cAMP synthesis caused by exogenous ADP or endogenously released nucleotides in either wild-type (WT, untransfected) PC12 cells or in two PC12 cell clones stably expressing pBoNT\/C1 (BT2 and BT7). After loading with [3H]adenine, PC12 cells were incubated in RO 20\u20131724 (100\u00a0\u03bcM) for 105\u00a0min. During the last 15\u00a0min of this incubation period, 1\u00a0\u03bcM CGS 21680 was present either alone or together with 10\u00a0\u03bcM ADP (a), 1\u00a0U\/ml apyrase (b), 100\u00a0\u03bcM MeSAMP (c) or 100\u00a0mM KCl (d). The amount of radioactivity retrieved within the fraction of cyclic AMP (cAMP) was calculated as percentage of the total radioactivity extracted from the cell cultures. In order to compare the results of different cell cultures, values obtained in the presence of ADP, apyrase, 2MesAMP or KCl were expressed as percentage of the values obtained in the very same preparation, but in the absence of these agents (% of control). a Results obtained in the presence of 10\u00a0\u03bcM ADP are expressed as percentage of the results obtained in its absence (% of control; n\u2009=\u20096 to 10). b Results obtained in the presence of 1\u00a0U\/ml apyrase are expressed as percentage of the results obtained in its absence (% of control; n\u2009=\u20099 to 10). c Results obtained in the presence of 100\u00a0\u03bcM MeSAMP are expressed as percentage of the results obtained in its absence (n\u2009=\u20096 to 9). d Results obtained in the presence of 100\u00a0mM KCl are expressed as percentage of the results obtained in its absence (n\u2009=\u20099; ***p\u2009<\u20090.001 versus the results obtained in wild-type cells; #, ###p\u2009<\u20090.05 and p\u2009<\u20090.001 versus the results obtained in the absence of KCl)\nIn the presence of apyrase (1\u00a0U\/ml) to degrade extracellular nucleotides, the cAMP accumulation triggered by CGS 21680 was almost doubled. Again, this effect was not different between wild-type and pBoNT\/C1 expressing cells (Fig.\u00a03b). In accordance with these latter results, the blockade of the receptors by the P2Y12 receptor antagonist 2-MeSAMP (100\u00a0\u03bcM; [14]) also enhanced the stimulation of cAMP about twofold and there were no significant differences between the three types of PC12 cultures (Fig.\u00a03b). Thus, the activation of the P2Y receptors negatively linked to adenylyl cyclase by spontaneously released nucleotides was the same whether pBoNT\/C1 was expressed or not.\nWhen wild-type PC12 cells were depolarized by 100\u00a0mM K+, the CGS 21680-induced cAMP accumulation was reduced to about the same extent as by 10\u00a0\u03bcM ADP. However, in one (BT2) of the two pBoNT\/C1 expressing clones, the depolarization of PC12 cells failed to cause any alteration in the CGS 21680-induced cAMP accumulation; and in the other clone (BT7), this inhibition was significantly weaker when compared with the values obtained in wild-type PC12 cells (Fig.\u00a03d). Thus, depolarization-dependent inhibition of the cAMP synthesis via P2Y12 receptors was greatly impaired in the pBoNT\/C1 expressing cells.\nDiscussion\nIn non-neural cells, release of nucleotides is mostly non-vesicular and involves, for instance, ATP binding cassette transporters, stretch-activated cation channels or connexin hemichannels [22]. In neurons, however, nucleotide release is generally believed to rely on vesicle exocytosis [27]. Here, we employed the autocrine\/paracrine activation of P2Y12 receptors in PC12 cells to investigate whether nucleotide release from neuroendocrine cells might involve a non-vesicular component.\nVesicle exocytosis involves the SNARE proteins syntaxin, SNAP-25 and synaptobrevin, and cleavage of one or more of these proteins by clostridial neurotoxins reduces or even abolishes exocytotic transmitter release [5]. The primary target of BoNT\/C1 is syntaxin, and its inhibitory effect on transmitter release has been correlated with the cleavage of this protein [3]. However, in cultures of bovine chromaffin cells [11], spinal cord [37] or hippocampal neurons [7], BoNT\/C1 was reported to cleave not only syntaxin, but also SNAP-25. In contrast, in sensory neurons the toxin was reported to act exclusively on syntaxin [31]. Furthermore, in cell-free assays BoNT\/C1 was found to leave either native SNAP-25 after subcellular fractionation or recombinant SNAP-25 unaltered [11, 37]. Thus, the effects of BoNT\/C1 on SNARE proteins appear to vary between different preparations. In this study, expression of the BoNT\/C1 light chain reduced syntaxin, but not SNAP-25 immunoreactivity detected in PC12 cells. Likewise, in the insulin secreting \u03b2-cell line HIT-T15 expression of the BoNT\/C1 light chain only affected syntaxin, even though application of recombinant toxin to permeabilized cells did lead to a reduction in SNAP-25 [20]. Hence, heterologous expression of the toxin\u2019s light chain in neuroendocrine cells does not entirely reproduce the effects of toxin application to these cells. Syntaxin was reported to be more sensitive to BoNT\/C1 light chain than SNAP-25 [20] and a 1000-fold higher light chain concentration of BoNT\/C was required for SNAP-25 cleavage when compared with the other SNAP-25-targeting serotypes BoNT\/A or BoNT\/E [34]. Thus, the resistance of SNAP-25 to heterologously expressed BoNT\/C1 light chain is likely due to the relatively low toxin affinity and insufficient expression levels of the peptide.\nNevertheless, in functional experiments, expression of the BoNT\/C1 light chain significantly reduced spontaneous as well as depolarization-evoked release of [3H]noradrenaline. On the one hand, this confirms that the cleavage of syntaxin alone is sufficient to reduce transmitter release [3]. On the other hand, this result corroborates that catecholamines are released from PC12 cells through vesicular mechanisms and these are largely impaired by the expression of the BoNT\/C1 light chain [10].\nConsidering that the heterologous expression of the BoNT\/C1 light chain did efficiently reduce vesicle exocytosis, the question arises as to how this manipulation altered the release of endogenous nucleotides. In PC12 cells, endogenously released nucleotides activate P2Y12 receptors, which mediate a decrease in the synthesis of cAMP triggered by the activation of A2A adenosine receptors [24]. In accordance with these previous results, exposure of non-transfected PC12 cells to the P2Y12 receptor antagonist 2-MesAMP enhanced the cAMP synthesis. An equivalent effect was achieved when the cells were incubated in the nucleotide degrading enzyme apyrase, thus indicating that spontaneously released nucleotides continuously activated the P2Y12 receptors.\nTo be able to evaluate whether the generation of PC12 clones stably expressing the BoNT\/C1 light chain might interfere with the above autocrine\/paracrine signalling loop only by affecting exocytosis, we had to establish that the function of the P2Y12 receptor was not compromised by the toxin\u2019s light chain or by the clonal selection procedure. Accordingly, 10\u00a0\u03bcM ADP, a saturating concentration of the nucleotide in the inhibition of cAMP synthesis in PC12 cells [33], were applied to non-transfected cells and to the two chosen cell clones (BT2 and BT7): the inhibition of cAMP accumulation was not different in the three cultures. Thus, the control of cAMP synthesis via the P2Y12 receptor was not altered by pBoNT\/C1 expression or by clonal selection. Previously, we had found that the control of voltage-gated Ca2+ channels via this receptor subtype was the same in a number of PC12 cell clones expressing various types of membrane receptors [19].\nKnowing that the P2Y12 receptors operated independently of the BoNT\/C1 light chain, the autocrine\/paracrine activation of the receptors was assessed. In the toxin expressing clones, the degradation of extracellular nucleotides by apyrase and the blockade of P2Y12 receptors by 2-MesAMP had identical effects on cAMP accumulation as in non-transfected cells. This demonstrates that the spontaneous release of nucleotides was not significantly affected by the BoNT\/C1 light chain.\nEnhancement of nucleotide release by depolarization of PC12 cells fully activates P2Y12 receptors and thereby reduces cAMP synthesis to about the same extent as 10\u00a0\u03bcM ADP. This phenomenon is based on Ca2+-dependent vesicle exocytosis as it is suppressed when Ca2+ influx is prevented by the blockade of voltage-gated Ca2+ channels or when extracellular nucleotides are degraded by apyrase [24]. In support of this conclusion, the inhibition of cAMP synthesis through a depolarization of PC12 cells by 100\u00a0mM K+ was either significantly attenuated or entirely abolished in the pBoNT\/C1 expressing clones. Hence, the K+-evoked and thus Ca2+-dependent release of nucleotides was largely reduced by the BoNT\/C1 light chain.\nTaken together, the present results show that the expression of the BoNT\/C1 light chain impairs vesicular transmitter release from PC12 cells, but does not alter the autocrine\/paracrine activation of P2Y receptors by spontaneously released nucleotides. Thus, the spontaneous nucleotide release from neuroendocrine cells may occur independently of vesicle exocytosis. The depolarization-evoked nucleotide release, in contrast, relies predominantly on exocytotic mechanisms.","keyphrases":["pc12 cells","p2y receptor","nucleotide release","cyclic amp","vesicle exocytosis"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_Arch_Otorhinolaryngol-4-1-2279159","title":"Dilatation tracheoscopy for laryngeal and tracheal stenosis in patients with Wegener\u2019s granulomatosis\n","text":"Wegener\u2019s granulomatosis (WG) frequently involves the subglottis and trachea and may compromise the upper airway. The objective of this study is to evaluate retrospectively the effect of treatment of subglottic stenosis (SGS) and tracheal stenosis (TS) by dilatation tracheoscopy (DT) in patients with WG. We performed a cohort study on all patients who underwent DT between February 2001 and September 2005 in our institution. From this cohort we identified a total of nine WG patients. In all patients, clinical, serological and histopathological data had been prospectively collected by a standardized protocol from the time point of diagnosis. In the nine patients that were identified with SGS or TS due to WG (eight women and one man), a total of 22 DT\u2019s were performed. Two patients needed a tracheostoma (one temporarily). The mean follow-up after the first DT was 25.4 \u00b1 14.1 months. Two patients did not experience a recurrence of SGS or TS. Six patients required a second DT without recurrence of local disease. The remaining patient underwent 8 DT's in a 4-year period. DT can offer a simple and repeatable solution to SGS and TS due to WG. Seven of the nine patients required more than one dilatation and some patients experience a functional restriction. One patient has a definitive tracheostoma.\nIntroduction\nWegener\u2019s granulomatosis (WG), first described by Friedrich Wegener in Stuttgart in 1936, is multi-system disease characterized by a necrotizing granulomatous arteritis of the upper and lower respiratory tract and a necrotizing crescentic glomerulonephritis [1]. Vasculitis may also affect other organs such as the eyes, skin, joints, heart and the nervous system [1]. Upper respiratory tract manifestations\u2014particularly sinusitis, ulcerations of the nasal mucosa and epistaxis\u2014are common and debilitating presentations. Subglottic stenosis (SGS) and tracheal stenosis (TS), however, are potentially life threatening presentations of WG. SGS has been found to occur in approximately 16\u201323% in patients carrying a diagnosis of WG [2, 3]. This narrowing of the upper airway at the level of cricoid cartilage and\/or upper tracheal rings presents a management dilemma. Dilatation tracheoscopy (DT) is one of the possible options for treatment of SGS and TS. The long-term effects of DT in patients with WG have never been reported.\nDT is a minimally invasive self-standing procedure. It can be used for elective and emergency intervention in patients with subglottic and tracheal stenosis. This has been described elsewhere (G.B. Halmos, F.G. Dikkers, Dilatation tracheoscopy in treatment of subglottic and tracheal stenosis, submitted for publication). Patients suffering from WG present differences (age, sex, response to treatment) compared to other aetiologies, which justifies separate publication. The objective of this study is therefore to evaluate the effect of the treatment of SGS and TS by DT in patients with WG.\nMethods\nWe retrospectively identified all patients from our institution that underwent a DT between February 2001 and September 2005. The total cohort of benign, grade II (Myer-Cotton) subglottic or tracheal stenosis consisted of patients with a history of posttraumatic tracheal stenosis, thermal tracheal stenosis, posttracheotomy cicatricial stenosis, tracheal rupture, postintubation stenosis, and WG, amongst others. As stated before, the cohort is described elsewhere.\nFrom the cohort we identified a total of nine WG patients. These nine patients had been diagnosed with WG between May 1990 and August 2003. During this period a total of 195 patients in our institution were diagnosed as having WG (including the nine patients who underwent DT). From this cohort, one additional patient underwent a DT for SGS in 1992. Three other patients were diagnosed with relatively mild SGS or TS that did not necessitate DT. The reference group for the comparison of demographic and serological data consisted of the total cohort, minus the patients who underwent DT and minus the three patients with mild tracheal stenosis who did not undergo DT. Differences in age between the DT group and the reference group were tested with the Mann Whitney U test; differences in the male\/female ratio and antineutrophil cytoplasmatic autoantibodies (ANCA) specificity were tested with chi-square test. Although this was a retrospective study, the clinical, serological and histopathological data of both the DT group and the control group had been prospectively collected by a standardised protocol starting at the time of the diagnosis of Wegener\u2019s granulomatosis. Extrarenal organ involvement was categorized as described previously [4]. ANCA was assayed with indirect immunofluorescence. Sera from all patients were assayed for the presence of antibodies against proteinase 3 (PR3\u2013ANCA), myeloperoxidase (MPO\u2013ANCA) and elastase as described previously [5].\nDT was performed when patients complained of progressive dyspnoea in combination with decrease of peak flows. DT is an endoscopically performed intervention. An intubation laryngoscope, a Groningen optical dilatation tracheoscope (Karl Storz 1033R) (Fig.\u00a01), telescope and suction tubes are required for this procedure. The tracheoscope has a length of 30\u00a0cm. The proximal end of the tracheoscope is designed in such a way that customary ventilation tubes and a 30\u00a0cm Hopkins\u00ae straight forward telescope (Karl Storz 27005AA) can be connected. The distal end of the instrument contains numerous lateral tiny openings, which enable air to come through in the centre of the stenosis. The dilatation tracheoscope is available with a diameter of 8 and 12\u00a0mm. The appropriate size of the instrument is determined by the size of the patient\u2019s larynx and the healthy portion of the trachea.\nFig.\u00a01Groningen dilatation tracheoscope\nThe intervention is carried out under general anaesthesia. Following the administration of the anaesthesia, with ventilation taking place via an anaesthesia mask, the dilatation tracheoscope is introduced under endoscopic control. The stenosis is then visible through the vocal cords (Fig.\u00a02). The bevelled design of the tip, which can be advanced forward through the stenosis, ensures that the ventilation is maintained during the process. The conical construction of the tip enables the instrument to be advanced up to the wider section of the tracheoscope (Fig.\u00a03), after which the tracheoscope remains in place for 5 to 10\u00a0min.\nFig.\u00a02Patient nr 9 pre dilatation. During laryngotracheoscopy a subglottic stenosis is clearly visible. The vocal cords can be seen bilaterallyFig.\u00a03Patient nr 9 during DT. The dilatation tracheoscope is introduced through the stenosis. Parts of the tissue protrude through the tiny distal openings of the tracheoscope\nThe constellation of the tracheoscope is suitable for most grade II (Myer-Cotton) subglottic or tracheal stenosis. The intervention can be repeated after any time interval.\nTypically, no antibiotics or corticosteroids are used. The use of mitomycin-C was considered in each case, but it was never used, because mitomycin-C should be used in fresh wounds. Patients are dismissed the day after DT. Peakflows were measured three times (Respironics Healthscan Inc., Cedar Groove, NJ, USA) in an upright position. The mean of the three measurements was taken.\nResults\nPatients\nThe total cohort of patients who underwent DT because of SGS and TS was 25. Nine of them were identified as patients with WG. Eight were female, one was male. The other 16 patients did not have vasculitis as cause of their stenosis. The causes in these 16 patients were posttraumatic [5], postintubation [4], idiopathic [4], post tracheotomy [2] and post thermal [1] injury. The mean age of the patient group at first presentation of WG was 41.6\u00a0\u00b1\u00a011.2\u00a0years (Table\u00a01). The interval between the presentation of WG and the first symptoms of laryngeal and tracheal stenosis varied between 0 and 122 months (mean 48.9\u00a0\u00b1\u00a044.1). The average age at of the time of presentation of the stenosis was 45.6\u00a0\u00b1\u00a011.9\u00a0years (Table\u00a01).\nTable\u00a01Patient characteristics of nine patients with Wegener\u2019s Granulomatosis (WG) and subglottic stenosis (SGS) or tracheal stenosis (TS). Clinical and laboratory findings. ANCA antineutrophil cytoplasmatic autoantibodies, PR3 proteinase 3, MPO myeloperoxidasePatient No.SexAge at presentation of WGOrgans involved at first presentation of WGANCA specificityNumber of relapses of WGOrgans involved at first relapse of WGFollow-up after diagnosis of WG (years)Age at first presentation of SGS or TSInterval between diagnosis of WG and first treatment of TS or SGS (months)1M44JointsPR312Joints, skin1751922F26Lungs, TracheaPR31Lungs, kidney142603F43TracheaMPO1Lungs124304F57JointsPR32Ear, eye, kidney, joints12671225F26TracheaPR31Kidney, eye1131606F51MastoidPR32Nose (concha inferior), mastoid1058817F42Ear drumAtypical0Lung, ear drum845358F52NosePR31Nose (septum and concha), joints, eye756489F33Nasal vesselsPR30Vessels4332\nAt diagnosis, all WG patients had additional organ involvement outside the otorhinolaryngologic area. The kidneys, lungs and eyes were affected three times. The joints were affected two times. The skin was affected once (Table\u00a01).\nAs shown in Table\u00a01, the number of relapses of WG varied between 0 and 12. There was no relation between the number of relapses and the occurrence of TS or SGS. In only one of the DT procedures there was endoscopic and\/or histopathological evidence of vasculitis activity. The TS or SGS occurred in 95% in periods where the disease appeared to be less active.\nIn Table\u00a02 the patient characteristics of the 9 patients who underwent DT for SGS or TS are compared with those patients who were diagnosed with WG in the same time frame but who did not have evidence of SGS or TS. In the DT group, 89% were female whereas 43% of patients in the reference group were female (P\u00a0<\u00a00.01). In the DT group the age at diagnosis of WG was significantly lower in comparison with the reference group (P\u00a0<\u00a00.05).\nTable\u00a02Patient characteristics of nine patients with Wegener\u2019s granulomatosis who underwent DT for SGS or TS compared with the reference group of 182 patients who were diagnosed with WG in the same period without evidence of SGS or TSWG patients who underwent DT (n\u00a0=\u00a09)WG patients without evidence of SGS or TS (n\u00a0=\u00a0182)P valueMale\/ female number (%)1\/8 (11%\/89%)104\/78 (57%\/43%) <0.01Age at diagnosis of WG (years, mean\u00a0\u00b1\u00a0SD)41.6\u00a0\u00b1\u00a011.253.3\u00a0\u00b1\u00a017.0<0.05ANCA specificityPR3-ANCA: n\u00a0=\u00a08 (89%)MPO-ANCA: n\u00a0=\u00a01 (11%)PR3-ANCA: n\u00a0=\u00a0160 (88%)MPO-ANCA: n\u00a0=\u00a013 (7.1%)HNE-ANCA: n\u00a0=\u00a01 (0.5%)ANCA-negative: n\u00a0=\u00a08 (4.4%)Not significantWG Wegener's granulomatosis, DT dilatation tracheoscopy, SGS subglottic stenosis, TS tracheal stenosis, ANCA antineutrophil cytoplasmatic autoantibodies, PR3 proteinase 3, MPO myeloperoxidase, HNE human neutrophil elastase\nFollow-up of the DT-group\nIn the nine patients identified with SGS or TS due to WG, a total of 22 DT\u2019s were performed (Table\u00a03). The mean follow-up after treatment was 25.4\u00a0\u00b1\u00a014.1\u00a0months (Table\u00a03). Two patients (numbers 7 and 8) did not experience a recurrence of significant stenosis. Six patients (numbers 1, 2, 3, 4, 6, 9) required a second DT without recurrence of local disease activity after the second DT. One of these patients (patient number 6) had acute WG in the trachea, proven by biopsy (Fig.\u00a04). She was treated with high dose prednisolone (intravenous methylprednisolone 1000 mg on three consecutive days) and intubation for a week. She could be detubated a week later (Fig.\u00a05). Two patients needed a tracheostoma (patient numbers 2 and 5). One patient (number 5) developed a cricoid stenosis 5\u00a0years after the diagnosis of WG. Initially the stenosis was treated with two DT\u2019s. During a pregnancy the patient required an emergency tracheotomy because of a threatened airway. After pregnancy she was treated with a CO2 laser and three DT\u2019s. Unfortunately, however, she required a definitive tracheostomy. Currently the process of decannulation is taking place. One patient (number 2) became respiratory insufficient at diagnosis of WG and had a tracheotomy for 3\u00a0months. She subsequently developed a cicatricial SGS, but she has enough lumen to live without a tracheostomy (Table\u00a03).\nTable\u00a03Follow-up after last DTPatient numberLocation of stenosisNumber of DT\u2019sFollow-up after last treatment (months)Tracheotomy required1Subglottic219No2Subglottic221Yes, temporary3Trachea243No4Trachea214No5Cricoid812Yes6Subglottic216No7Subglottic154No8Subglottic126No9Subglottic224NoFig.\u00a04Patient nr 6 with acute WG during biopsy of a subglottic stenosisFig.\u00a05Patient nr 6 after pulse therapy and intubation. The picture was taken one week after Fig.\u00a04\nThe effect of DT on peakflows was evaluated in five patients. In these patients the mean (\u00b1SD) peakflow increased from 164\u00a0\u00b1\u00a045.7\u00a0l\/min before DT to 226\u00a0\u00b1\u00a069.6\u00a0l\/min after dilatation. An example of the effect of DT on the peakflow is shown in Fig.\u00a06. Two months after this patient had been diagnosed with WG she developed complaints of dyspnoea. Tracheoscopy revealed a SGS with signs of active vasculitis. After treatment with high-dose corticosteroids she had a regression of the WG activity. Fourteen and 28 months after first diagnosis of SGS a dilatation tracheoscopy was performed successfully.\nFig.\u00a06Peak flow measurements after dilatation of patient number 9. The arrows indicate the DT procedures\nDiscussion\nThere are multiple causes of benign laryngeal or tracheal stenosis, the most common being traumatic. However, if there is no prior history of tracheal trauma, the aetiology of the stenosis may be obscure and difficult to determine, necessitating a systematic approach to make the diagnosis. Excluding trauma, the differential diagnosis of TS can be subdivided into four categories: congenital, neoplastic, infectious, and inflammatory.\nCongenital TS is really quite rare and is often the result of posterior fusion of the tracheal rings, thereby forming complete rings. Other causes of congenital stenosis include vascular rings and other congenital cardiovascular anomalies such as an anomalous subclavian artery. These patients typically present at young age.\nPrimary benign tumours of the trachea such as chondromas, fibromas, squamous papillomas, hemangiomas, and granular cell tumours are also unusual causes of stenosis. In addition, extrinsic compression of the trachea can occur by thyroid neoplasms and goiters.\nA number of infections of the bronchopulmonary tree can lead to TS. Fungal infections such as histoplasmosis and blastomycosis should always be considered when the aetiology of the stenosis is unclear. Serologic testing and histopathologic examination can be helpful in this regard. Other infectious causes of TS include rhinoscleroma, tuberculosis, syphilis, and diphtheria.\nNon-infectious, inflammatory causes of TS include sclerosing mediastinitis, primary amyloidosis, and sarcoidosis. WG and relapsing polychondritis can also cause TS, but they are almost always seen in combination with other, more classic hallmarks of these diseases.\nA laryngeal or tracheal stenosis is optimally diagnosed via tracheal visualization, which is generally performed by an otorhinolaryngologist. Indirect and fiberoptic laryngoscopy are non-invasive examination techniques that can be performed in the office, but usually do not show the entire trachea. A subglottic stenosis is not always visible, therefore direct rigid tracheoscopy in general anaesthesia is indicated in cases suspect of WG.\nRecurrent SGS is a well recognized but uncommon feature of WG. Patients with WG who develop laryngotracheal disease have usually already been diagnosed as such because of the presence of disease in other organs at the same time or at a previous occasion [6]. Isolated involvement of the subglottic larynx is conspicuously rare\u2014only a few cases have been reported in the literature [2, 7, 8]. In our series, however, three patients had SGS or TS as presenting symptom of their WG. We do not have an explanation for this rare presentation.\nAnother particular finding is that eight of our nine patients were female. This contrasts significantly with the male\/female ratio in the reference group that consisted of WG patients that were diagnosed in the same period as the DT group but who did not have evidence of SGS or TS. It is difficult to speculate on the reason for this female preponderance. Anatomically the female airway is narrower than the male, making it more prone for post-intubation stenosis. However, we even had females presenting with a SGS or TS. More research in larger series has to be performed to explain this phenomenon.\nIn cases where the diagnosis of WG is suspected, ANCA testing should be part of the routine laboratory evaluation [9]. C-ANCA has been shown to be a specific marker for WG with rare false-positive results; p-ANCA testing is much less specific. Only the particular laboratory findings of c-ANCA reacting with PR3 and p-ANCA reacting with myeloperoxidase (MPO) are specific for the autoimmune vasculitides [9, 10]. Notably, seven of our nine patients that underwent DT were PR3-ANCA positive and one patient was MPO-ANCA positive.\nAll patients had involvement of additional organs. In three patients the disease presented as a stenosis, and laryngotracheoscopy revealed the size and site of the lesion. Organs involved showed no pattern in which there should be additional suspicion for the development of SGS or TS. There was no relation between the number of relapses and the development of TS or SGS. The TS or SGS occurred more often in periods where the disease appeared to be less active.\nRemarkably, TS or SGS predominantly became manifest in periods in which WG appeared to be inactive. Interestingly, seven out of our nine patients who developed TS or SGS had not been diagnosed previously with tracheal involvement of WG, neither at first presentation nor at relapse(s). We speculate that during active disease a subclinical tracheal involvement occurs which may subsequently heal with scar formation. To examine this hypothesis in patients, elective tracheoscopy should be performed in all patients with WG. The time interval between presentation of clinical stenosis in these seven patients (mean 63\u00a0months, median 60, range 2\u2013122) favours watchful waiting.\nDT with the Groningen dilatation tracheoscope is a safe, minimally invasive procedure for the treatment of Cotton-Myer grade II subglottic or tracheal stenosis of various origin. It is an elegant, self-standing surgical intervention, where no additional interventions are needed. For an overview of the effect of DT the reader is referred elsewhere (G.B. Halmos, F.G. Dikkers, Dilatation tracheoscopy in treatment of subglottic and tracheal stenosis, submitted for publication).\nNowadays, mitomycin-C, an alkylating agent that inhibits cell division, protein synthesis, and fibroblast proliferation [11], is increasingly used as adjuvant treatment in the management of selected cases of laryngeal and tracheal stenosis, for example luminal obstruction in fresh circular sutured wounds. However, the laryngeal and tracheal stenoses of WG patients are almost always of older age and display advanced scarring at the time of diagnosis. Dilating such \u201cmature\u201d stenoses will inevitably lead to damage of tracheal epithelium. However, this damage is considerably different from granulomatous scarification in sutured lumina and, therefore, we do not expect an additional favourable response to mitomycin. Therefore, we have not applied mitomycin-C in our WG patient cohort.\nIn cases where TS or SGS develops, there is always the question whether or not to intervene, and the question of timing of the intervention. There are two main parameters in these patients: complaints, and physical signs. One might see this as a two-by-two table, with complaints on the x-axis, and peakflow values on the y-axis. Progressive complaints and progressive decline of peakflow values (+\/+) indicate intervention by DT. Absence of progressive complaints and unchanged peakflow values (\u2212\/\u2212) indicate watchful waiting. Absence of progressive complaints with decline of peakflow values (\u2212\/+) need to be addressed when the values reach a critical level, in which it can be expected that a common cold might lead to severe stenosis of the airways. Finally, progressive complaints with unchanged peakflow values (+\/\u2212) indicate that pulmonary function tests should be performed, and, if negative, indicate that the patient has an incorrect perception of his physical potentials. This can then be addressed. Unfortunately, in only five of the nine patients in this retrospective study, we have peak flow measurements. A reason is that two patients have or had a tracheostomy.\nWe have registered no complications or deaths during or because of DT. We have three reasons to regard dilatation tracheoscopy as a minimally invasive intervention. We have experienced no complications related to dilatation tracheoscopy. It requires short hospitalization (generally 3\u00a0days). The intervention is not straining for the patients.\nA variety of surgical techniques has been used to treat SGS or TS of other aetiologies [12, 13]. The success of these surgical techniques in upper airway stenosis related to WG has been variable [3, 14]. In our series two out of nine patients were treated once for upper airway stenosis with dilatation without any re-stenosis because of WG.\nUnfortunately, two patients required a tracheostomy. One was temporary, but one patient needed a permanent tracheotomy (11%): this was the pregnant woman needing an emergency procedure. This number equals that of Gluth et al. [9].\nConclusion\nWG as such is a rare disease, and SGS and TS are rare symptoms in patients with WG. DT can offer a simple and repeatable solution to this very serious symptom. Patient complaints and monitoring of peakflow values offer simple tools for the decision whether or not to intervene. However, a causative solution to WG should be the ultimate goal.","keyphrases":["dilatation tracheoscopy","tracheal stenosis","wegener\u2019s granulomatosis","subglottic stenosis","constriction"],"prmu":["P","P","P","P","U"]}
{"id":"Environ_Manage-4-1-2359833","title":"Public-Private Partnerships in China\u2019s Urban Water Sector\n","text":"During the past decades, the traditional state monopoly in urban water management has been debated heavily, resulting in different forms and degrees of private sector involvement across the globe. Since the 1990s, China has also started experiments with new modes of urban water service management and governance in which the private sector is involved. It is premature to conclude whether the various forms of private sector involvement will successfully overcome the major problems (capital shortage, inefficient operation, and service quality) in China\u2019s water sector. But at the same time, private sector involvement in water provisioning and waste water treatments seems to have become mainstream in transitional China.\nIntroduction\nIn the wake of the United Kingdom\u2019s water privatization in the 1980s, the 1990s witnessed the spreading of privatization and a variety of public-private partnership (PPP) constructions in developing countries, especially following the promotion and push by international development agencies such as the World Bank, the International Monetary Fund (IMF), the Asian Development Bank (ADB) and others (Nickson 1996, 1998; Kikeri and Kolo 2006). It was believed that private sector participation in the water sector would bring in much needed investment and improve service coverage, quality, and efficiency by replacing conventional public-sector systems suffering from under-investment and inefficiencies due to excessive political interference and rent-seeking behavior by vested state and bureaucratic interests (Hall and others 2005). During the past decades, a wide literature in economics, governance, and public management has provided theoretical and empirical arguments and evidence in favor of further private sector involvement in what used to be public utilities. At the same time, however, debate continues on the different partnership constructions, the division of tasks and responsibilities between public and private sectors, and the social effects coming along with these developments. Topics, such as the relationships between ownership (public or private) and efficiency (Vining and Boardman 1992; Spiller and Savedoff 1997; Birchall 2002; Afonso and others 2005; Anwandter and Jr.Ozuna 2002; Hart 2003), the classification of various public-private constructions and their characteristics (World Bank 2004; Sepp\u00e4l\u00e4 and others 2001; US National Research Council 2002), the consequences of privatization for governmental regulation (Nickson and Vargas 2002; Pongsiri 2002) and questions of equity and equality are still heavily debated, in particular with respect to the water sector and less so regarding other utilities.\nAlthough private sector participation in the water sector is one of the more controversial topics in public utility management today, this wave also spread to China at the turn of the millennium, where the government started to reform public sectors (water, electricity, roads, etc.) via introducing market functions. The so-called marketization reform expected to address the increase of several water problems (water shortage, insufficient infrastructure, water pollution, etc.) to meet the requirement posed by accelerated urbanization and high economic growth. As a late comer in this field of private sector involvement in the provision of water services, China is able to learn from numerous experiences of other countries, such as the United Kingdom, France, United States, Chile, Philippines, Mexico, Argentina, and Bolivia.\nSince the earlier attempts of applying the Build-Operate-Transfer (BOT) approach in the water sector in the 1990s and the full development of marketization reform in public sectors in 2002, China has applied different models of private sector involvement in over 300 water supply and wastewater projects. This marketization reform emphasizes the importance of the market, investment and financial liberalization, deregulation, decentralization, and a reduced role of the state in the water sector (also see Robison and Hewison 2005; Prasad 2006). Tariff reform with full-cost recovery, competitive bidding procedures, changing ownership structures (e.g., public and private, Sino and foreign), and restrictive fiscal policies are part of it.\nThis article reviews developments in private sector involvement in China\u2019s water management and assesses whether expected results of marketization in the Chinese water sector have been met: raising investment for infrastructure, increasing service coverage and improved efficiency in China\u2019s water supply and wastewater treatment. After interpreting further private sector involvement in China\u2019s urban water management in terms of modernizing water governance, this article provides a country-wide overview of current privatization developments in the Chinese water sector, and subsequently makes an in-depth investigation in three distinct cases with respect to the new roles and functions of the governments and private parties. The final section assesses the current status of privatization programs in China\u2019s water management and its implications of future research on water governance reform.\nPrivate Sector Participation as Part of Modernizing Urban Water Governance\nIn the debate on private sector participation in environmental governance in general, and urban water governance in particular, we can identify three \u2014 sometimes interrelated \u2014 discourses.\nFirst, private sector participation goes back to the literature on state failure in the early 1980s. State failure refers to the notion that the nation-state falls short in the provisioning of collective goods, in this case environmental services and quality. Some of the key publications in this regard come from Germany. Martin J\u00e4nicke\u2019s (1986) Staatsversagen analyzed the fundamental inability of the nation-state to protect the environment in the 1980s, and called for an innovation or modernization of environmental politics, later to be labeled political modernization (e.g., Tatenhove and others 2000; Mol 2002): a reorientation towards a more preventive, pro-active and flexible strategy using new instruments and closer cooperation with and participation of non-state actors. With a similar analysis of the environmental state\u2019s fundamental inabilities, Joseph Huber (1985) came to slightly different solutions with his strong plea for involving the private sector into environmental services and protection. Finally, around the same time Ulrich Beck (1986) formulated his Risk Society hypothesis and identified subpolitical arrangements (i.e., arrangements for environmental protection and service provision without and beyond the public state) as an alternative for the conventional environmental politics of the nation-state. Inspired by these and several other authors and ideas, from the mid 1980s onward environmental social science scholars started to develop ideas, investigate practices, and formulate theories on governing environmental problems, in which the environmental state was given a less dominant and monopolistic position.\nAround the same time (the second half of the 1980s) ideas of further private participation and involvement in the provisioning of environmental services (water, waste, energy, etc.) started to develop, especially in the United States and the United Kingdom. While also here the fundamental idea is involving the private sector in tasks traditionally fulfilled by the public sector, the orientation and literature is slightly different. The majority of the literature comes from the management and organization sciences and the orientation is less focused on state failures and governance, but rather on efficiency, the bringing in of new capital and the introduction of market logics. The dominant form of organizing urban infrastructure (water, energy, waste, transport) by state agencies has been replaced in many places by various PPP constructions, with different reasons put forward to legitimate such new constructions (cf. Linder 1999). At the same time, these partnerships led to considerable debate, most significantly on issues of equity and equality: who is involved in these partnerships, for who are these constructions bringing more effective and efficient services, are local governments able to balance the power of private capital coming in (especially in situations of Transnational Companies (TNCs) in developing countries) (e.g., Oppenheim and MacGregor 2004), and what does private sector involvement mean for affordability of environmental services for the poor?\nThirdly, in the 1990s, following the United Nations Conference on Environment and Development (1992, Rio de Janeiro), and even stronger after the Rio+10 conference (2002, Johannesburg), ideas and practices of public private partnerships started to emerge forcefully on the national and global agenda (cf. Mol 2007). In this literature, the emphasis is strongly on transnational partnering of public and private entities, with a strong focus on the role of civil society organizations. The main reason behind the recent attention to private sector participation in environmental protection and service delivery is related to tendencies of globalization and governance complexities. As Davies (2002) correctly summarizes, in this interpretation the notion of partnership has a positive rhetoric referring to inclusiveness, transparency, participation and dialogue, redistribution of power, and equity. And not so much to ideas of efficiencies, capital investment, market logics, and increased service coverage.\nIn reviewing the arguments and legitimacy of the push for private sector involvement in China\u2019s urban water governance, there is a strong relation to the second discourse on efficiency, capital investments and service coverage, while ideas of state-failure and political modernization incidentally emerge. By the same token, the Chinese discourse on private sector participation in urban water management hardly draws upon ideas of wide cross-sectoral partnerships and the positive logics of transparency, democracy, participation, and dialogue. Discussions on China\u2019s urban water governance reform argue for the advantages of effectiveness and efficiency, and debate the best organizational modes, division of responsibilities, and coordination structures. Potential negative outcomes of private sector participation \u2014 so strongly emerging in and dominating western debates \u2014 are much less emphasized: loss of decision-making autonomy of states and governments; unequal power relations and information asymmetry in public-private partnerships; problems around equity, access for the poor, participation and democracy in decision-making (e.g., Hancock 1998; Poncelet 2001; Miraftab 2004).\nAccording to the World Bank, China, Chile and Colombia are the only countries that remain active in water privatization after 2001 (Izaguirre and Hunt 2005). How to explain that, while the activities of water sector privatization intend to shrink in an increasing number of countries and international development agencies, such as the World Bank, start to slow down such privatization programs, China is actively promoting private sector involvement in urban water governance?Two interdependent arguments elucidate this. First, China\u2019s urban water management comes from a radically different starting position, where market principles and logics were almost absent. Water management was not just completely publicly organized but also highly inefficient, with large capital shortages, poor coverage, no economic incentives and demand side management, and highly centralized. This is a fundamental, rather than marginal, difference with most of the public utility systems in OECD countries before the privatization discourses and practices of the 1980s and 1990s. Under such Chinese conditions, private sector involvement in water management means more that just handing water business over to for-profit private companies. It most of all means building economic incentives and logics, safeguarding enough financial capital for infrastructure investments, and widening the service area. Second, private sector participation in China\u2019s urban water management is not just a matter of privatization. It is part of a much wider and complex modernization program in urban water governance, involving some of the critical issues that emerged in the privatization debates in OECD countries. The modernization of urban water governance also includes (see OECD 2003, 2004a, 2004b, 2004c, 2005a, 2005b):water tariff reforms, where costs of drinking water increasingly include full costs (also of wastewater treatment), but come along with safeguards for low income households to continue access to drinking water;transparency, accountability and control of the government;public participation in for instance water tariff setting, complaint systems on water pollution and corruption, public supervising committees on utility performance, public and media debates on water governance, disclosure of information to non-governmental actors (cf. Zhong and Mol 2007); anddecentralization of water tasks and responsibilities to the local level.In exploring the degree, nature, and forms of private sector participation in China\u2019s urban water governance in the following sections; we have to leave these wider \u2014 related \u2014 developments aside.\nPrivatization Policy in China\u2019s Water Sector\nIn China, the term \u201cprivate sector\u201d has been regarded as politically sensitive since 1949 when China started to establish a socialist regime characterized by the nationalization of ownership. The first breakthrough of the development of \u201cprivate sector,\u201d which was officially defined as \u201ceconomic organizations that aim at making profit, in which assets are privately owned and which have eight or more employees\u201d (Provisional Regulations of Private Enterprises in PRC, the State Council, June 25 of 1988), took place mainly in competitive sectors in accordance with the launch of China\u2019s economic reform in the late 1970s. The government remained in control of public sectors such as water services, energy provisioning, waste management, and public transport. In the mid 1990s, Chinese Government attempted to introduce the BOT approach into the field of urban infrastructures (thermal power, hydropower, highway, water supply, etc.) via promulgating the Circular on Attracting Foreign Investment through BOT Approach (No.89 Policy Paper of 1994, the former Ministry of Foreign Trade and Economic Cooperation, January 16 of 1995) and the Circular on Major Issues of Approval Administration of the Franchise Pilot Projects with Foreign Investment (No.208 Policy Paper of Foreign Investment, the former National Development and Planning Commission, the Ministry of Electric Power Industry, and the Ministry of Communications, 1995). These two policy papers formed the first legal ground for private sector involvement and foreign capital investment in Chinese urban infrastructure. Subsequently, the National Development and Reform Commission firstly approved three BOT infrastructure projects in 1996, including Chengdu No.6 Water Supply BOT Plant (B), Guangxi Laibin Power BOT Plant, and Changsha Wangcheng Power BOT Plant (failed).\nThe earlier experiences of BOT projects brought in needed capital and investment to develop China\u2019s urban water infrastructure. But it illustrated also many problems. The issue of the fixed investment return to investors was one of these problems. After intensifying control over foreign exchanges and loans in the late 1990s, the General Office of the State Council promulgated a specific circular in 2002 to correct foreign investment projects with fixed investment returns, by modifying the relevant contract terms, buying back all shares of foreign investors, transferring foreign investment into foreign loans, or dismantling contracts with often severe losses.\nThe full-fledged commitment of the Chinese government to private involvement in the water and other utility sectors dates from late 2002. In the December of 2002, the Opinions on Accelerating the Marketization of Public Utilities (No.272 Policy Paper of the MOC, 2002) started the marketization reform of water and other public sectors by opening public utilities to both foreign and domestic investors: multi-financing approaches, concession right and concession management, pricing mechanism, reduction of governmental monopolies and roles ended the traditional policies of public utilities. The subsequent Measures on Public Utilities Concession Management (No.126 Policy Paper of the MOC, 2004; in this policy, \u201cconcession management\u201d refers to all forms of private sector participation.) of 2004 specifies the procedure of how to involve the private sectors in public utilities through awarding concession right, but still relies heavily on BOT modes.\nThese steps proved more than just giving the private sector a permission to enter public utilities. It is a complex process involving among others ownership reforms, redefinition of the role of governments and operators, restructuring the tariff mechanism, reforming governmental regulation, and designing public participation. In the early years of marketization, the emphasis was especially on market opening and financing issues. With Opinions on Strengthening Regulation of Public Utilities (No. 154 Policy Paper, the MOC, 2005) the neglect of governmental regulation and the public good character of water in the previous policy papers was corrected. This policy paper emphasizes that the water sector provide basic public and social goods and that the governmental regulation remains essential (Fu and Zhong 2005). However, there is still a lack of a systematic and comprehensive regulatory framework for the Chinese urban water sectors in practice. The MOC is attempting to introduce and develop a competitive benchmarking system that might be helpful for further regulation, but this is not yet in place. During the authors\u2019 field surveys, the local officials of relevant water authorities are laboring under the lack of effective measurements for regulatory framework, have too much freedom of (non)regulation, and have sometimes an incorrect perception of the government role as a regulator. Fu and colleagues (2006) also refer to the fact that the government has paid some attention to assets regulation while restructuring ownership in the water sector, but neglected regulating water service quality.\nCompared to the exponential growth of water projects with significant private sector involvement, the legal basis under privatization developed quite slow and is still underdeveloped in China. Different from some water privatization forerunner countries (e.g., England and Wales, Philippines), which enacted specific laws before entering into privatization, the marketization reform of and private participation in the Chinese water sector is conducted under various governmental policy papers, but without specialized legislation. The current legal codification of public-private partnering in water services is largely a reactive process, where various policy papers address specific problems in the reform process due to the lack of a well-established legal framework. Thus, much room for improvement remains in the current legal basis, for instance on further economic regulation, stronger legislative sanctions, and public participation (cf. Tong 2005; Zhang 2006; Fu and Zhong 2005; Fu and others 2005).\nAs implementation problems were slowly or not adequately addressed or resolved at the national level, local governments started to issue local policy papers on specific water projects. For instance, the Interim Provision on Administrating Concession Right of Chengdu (No.131 Policy Paper of Chengdu Municipality, 2001) was issued for implementing the BOT project of Chengdu No.6 Water Supply Plant (B), which was the first water BOT pilot project approved by the NDRC. And the Measures on Public Utilities Concession Management of Shenzhen (No.124 Policy Paper of Shenzhen Municipality, 2003) guided the reform of Shenzhen Water Group, the largest water project with private sector involvement in China to date.\nThe Current Landscape of Private Sector Involvement\nIn China\u2019s water supply and wastewater services, four major types of private corporations are active (Fu and others 2006): (1) the water transnational corporations (e.g., VEOLIA and SUEZ); (2) Chinese investment developers (e.g., Beijing Capital Group and Tianjin Capital Environmental Protection Co. Ltd.); (3) liberalized water companies (e.g., Shenzhen Water Group and Beijing Sewerage Group); and (iv) environmental engineering corporations (e.g., Beijing Sound Group and Tsinghua Tongfang Water Engineering Corporation). In December of 2004, the Ministry of Construction called provincial-level authorities to summarize marketization of public sectors (e.g., water and wastewater, solid waste, gas, and public transportation). In July of 2005, a follow-up field survey was organized by the MOC, in which the authors have participated. All reported data of this section come from the reports of provincial-level authorities, supplemented by surveys of the Water Policy Research Center of Tsinghua University (in which authors participated).\nAccording to the MOC surveys, various forms of private sector participation can be identified in both water supply and waste water treatment: (1) commercialization of public utilities: it is the transformation of a public agency\/utility into an independent corporation; (2) management contract (or namely operations and maintenance contract): it refers to a contractual arrangement in which a private operator manages and maintains the service in a given period but does not have investment obligations; (3) lease contract: it is a short-term contract in which a private operator pays an agreed-upon fee to the government for the right to manage the facility; (4) Greenfield contract (such as BOT, TOT, BOOT, etc.): it means the government commits new investment projects to a private company, within the contract duration, the private operator manages the infrastructure and the government purchases the water by a contracted price (this price isn't necessarily determined by the actual water tariff); (5) concession contract: it is a long-term contract in which a private operator bears responsibilities for operations and maintenance and also assumes investment and service obligations; (6) Joint Venture: it is not a contract but, rather, an arrangement whereby a private company forms a legal entity with the public sector, in which both the private and the public parts share responsibilities and (investment) obligations; and (7) full sale (or full divesture): it is the sale of public assets to the private sector. Table\u00a01 summarizes the various forms of private sector participation and their characteristics. Until July 2005, a total of 152 water supply projects and 200 wastewater treatment projects involved private participation. The total water production capacity of the 152 water supply projects equaled about 17% of national water production capacity of 2004. The treatment capacity of the 200 wastewater projects was over 30 million m3 per day, equaling 67% of the national total wastewater treatment capacity of 2004.\nTable\u00a01Different forms of private sector participation in China\u2019s water sectorForm of private sector participationAsset ownershipCapital investmentOperations & maintenanceContract periodCommercialization of governmental enterprises\/utilitiesPublicPublicPublicIndefiniteManagement contractPublicPublicPrivate3\u20135yrLease contractPublicPublicPrivate8\u201315yrGreenfield (BOT-type)Private\/ publicPrivatePrivate20\u201330yrConcessionPublicPrivatePrivate25\u201330yrJoint ventureSharedSharedSharedIndefiniteSale or full divesturePrivatePrivatePrivateIndefinite\nFigure\u00a01 shows the prevalence of different forms of private sector participation in water supply and in wastewater projects. The joint venture approach (including the Sino-foreign joint ventures) has the largest share in the water supply sector with 51% of the 152 privatized projects. The Greenfield modes of private sector participation (including the BOT and TOT contracts) dominated in the wastewater sector, with 59% of the 200 projects. The commercialization of governmental utilities also plays an important role in both water supply (16% of 152 projects) and wastewater (13% of 200 projects). The differences in prevalence of private sector participation forms between water supply and wastewater have a close relation with the level of infrastructure development and with tariff levels. Compared to urban water supply (with service coverage of 88.8%), urban wastewater treatment lags behind, with a service coverage of 45.6% in 2004 (MOC 2005). Direct investment demand for urban wastewater infrastructure (including wastewater treatment, sewers, and sludge treatment) in China is expected to be over 30 billion US dollars between 2006 and 2010, to meet the objective of 60% municipal wastewater to be treated. Accordingly, local governments prefer direct private sector investment in and building of new wastewater infrastructure, resulting in high levels of the Greenfield modalities. In addition, the current low wastewater treatment charges result in a preference for Greenfield modes. In these modes, financing is based on negotiated prices between the government and the private sector and is less dependent to the user fee or charge; drinking water supply costs are much better represented in prices, making joint ventures more likely (Zhong and others 2006).\nFig.\u00a01Public sector participation in water: distribution over modalities (2005)\nFigure\u00a02 categorizes public sector participation into five groups, according to project capacity. The joint venture approach leads the reform of water supply sector in all size-categories, while the Greenfield approach dominates in wastewater sector, except for projects over 500,000 m3 per day. This might also be related to the different financial risks. Larger projects require much more direct capital investment from the private sector, increasing the financial risk for private investors and moving, then, rather toward joint venture approaches. Furthermore, the full sale\/divesture approach occurred more in the field of water sector and mainly in small projects in specific provinces (see Fig.\u00a03). And commercialization is more often found among larger projects. This might be related to not only the larger capital demands of bigger projects, but also huge labor redundancies within such large projects. Existing large water projects are traditionally run by state-owned enterprises with high levels of superfluous workers. For private investors it is often difficult to improve efficiency, because government contracts often do not allow firing existing workers following a commercialization process.\nFig.\u00a02Distribution of private sector participation in water projects by capacitiesFig.\u00a03Distribution of private sector participation in water projects by provinces\nFigure\u00a03 visualizes the provincial distribution of water projects with private sector participation. At least 25 provinces have private sector participation experience in water supply and 23 provinces in wastewater treatment. The form of private sector participation is determined by the level of development of water\/wastewater infrastructure, as well as the local economic, social and political conditions. With richer markets, more open economic policies and higher payment capacity of local residents, the southern coastal (e.g., Guangdong and Fujian) and the eastern coastal (e.g., Jiangsu) provinces witnessed high levels of reform in their water sector. Over 60% of foreign private sector investment in water supply projects and about 50% foreign private sector investment in wastewater projects were implemented in these coastal regions, according to the MOC survey. In the meanwhile, the first national BOT pilot project of Chengdu Water Supply (Sichuan Province) has triggered a wave of private sector participation in and around Sichuan Province (including Chongqing and Yunnan). Furthermore, the special environmental protection policies related to \u201cThe Three Gorges\u201d dam might have impelled private sector participation in wastewater sector of Sichuan Province and Chongqing.\nAs shown in Fig.\u00a03, in water supply the joint venture approach dominates in 19 provinces. In the wastewater sector, Greenfield projects (including BOT and TOT) dominate in 12 provinces. The commercialization of traditional state-owned water enterprises was adopted more widely in inland provinces (such as Gansu, Heilongjiang, Jilin, Sichuan, Xinjiang, Yunnan) than in coastal provinces. A joint venture approach for private sector involvement in the wastewater sector was only adopted in provinces with high wastewater treatment charges, such as Beijing, Fujian, Jiangsu, Zhejiang, and Shanghai.\nThree Case Studies of Public-Private Partnerships\nThe reported growing involvement of the private sector has led to radical changes in China\u2019s water management institutions. In this section, we report on fieldwork of three case studies with distinct modes of private sector involvement (a joint venture, a concession, and a Greenfield contract) to analyze in detail the new institutions and relationships between actors in these constructions. During fieldwork in Maanshan and Shanghai, we carried out face-to-face semi-structured interviews with relevant local officials (from the construction authority, price authority, planning and reform authority, state-owned assets administration authority, and environmental protection bureau) and managers of water service providers (water treatment plants\/companies, wastewater treatment plants\/companies). In the performance assessment project of Macau Water Company Ltd., the managers of relevant departments as well as the representative of Macau Government were interviewed. In total, around 30 interviews were held. While these three cases represent different forms of private sector involvement, they cannot be held representative. All three cases have been assessed positively by the Chinese government and independent researchers (see Fu and others 2006), making them rather best practices than representative cases. But together they illustrate the institutional transformations that come along private sector involvement.\nJoint Venture: Maanshan Water Supply\nMaanshan City is an industrial, prefecture-level city of 1686 square kilometers, and a population of 1.24 million (2004), of whom 46.8 per cent lives in urban areas. According to the 2004 MOC statistics, 88.7 per cent of the urban population has access to water supply. Water resources are abundant in Maanshan City due to its advantageous location on the south bank of the Yangtze River and abundant annual rainfall (1062\u20131092 mm). Maanshan Construction Commission (MASCC) is not only the competent authority for water supply and wastewater treatment and as such, plays a leading role in the water sector reform. It is also, as a so-called \u201cBig Construction Commission,\u201d the main governmental agency responsible for urban planning, construction, and management (cf Wu 2003).\nIn 2002, following the call of Central Government and Anhui Provincial Government, MASCC embarked upon marketization reform in water and other public utilities (e.g., gas and public transport), widely inviting business actors to become active and invest. The director of MASCC, Mr. Xu, argued that changing the current water institutions and increasing service quality were the most important reasons and objectives for embarking on marketization in the water sector in Maanshan, rather than bringing in nongovernmental capital (personal communication 2004). Marketization was expected to impel and accelerate the reform of converting the old Maanshan Water Supply Company (MASWSC, established in 1958 as state-owned and state-subsidized company with total assets of 4.37 million RMB in 2002, ca. 0.528 million US$ at the exchange rate of 1US$ = 8.277RMB) into a new institutional lay-out. After negotiating with several private companies, MASCC first started \u2014 as a kind of trial \u2014 a joint venture with Beijing Capital Group (BCG) for one water supply plant (WTP, BCG owning 60% of shares). This joint WTP sold purified water to MASWSC and performed significantly better than other WTPs managed by MASWSC alone. In 2004, MASCC expanded the joint venture cooperation with BCG to all WTPs of Maanshan City, in which BCG obtained a 60% share by bringing in 90 million RMB (ca. 10.875 million US$ at the exchange rate of 1US$ = 8.276RMB). The new joint venture company (MAS-BCWLC) was awarded a 30-year concession right. Both BCG (private sector) and MASWSC (public sector) bear responsibility of investment, operation, and maintenance of the WTPs (excluding the pipe networks) and service obligations (see Fig.\u00a04). With respect to the pipe networks, MAS-BCWLC manages and maintains the existing (pre-2004) network by signing a lease contract with MASWSC, which remained owner of the assets and bears the financial obligations (debts). In the meanwhile, MAS-BCWLC is requested to invest in new pipe infrastructure in new development areas and in nonpiped neighborhoods.\nFig.\u00a04Organizational structure of Maanshan water supply system\nWithin the new joint venture structure, the board of MAS-BCWLC (4 members from BCG and 3 from WASWSC) is the current decision-maker regarding planning (within the objectives set by the municipal master planning), investment and financing, partly replacing the tradition of government decision structures. According to the contract, the general manager of the joint venture company comes alternately from MASWSC and BCG. Taking into account the social dimensions of water provisioning, the government claimed three key conditions in the agreement with the concessionaire: first, the concessionaire (MAS-BCWLC) must ensure sufficient and safe water provision and the government can take over all facilities without any indemnity if the concessionaire fails; second, the concessionaire cannot change the public and social nature of water and should include relevant social responsibilities as governmental requirements (e.g., employing all personnel from the old water company, providing free water for firefighting, reducing\/subsidizing water bills of the poor); third, the government controls the water price.\nIn order to ensure high-quality water and service, MASCC regulates the performance of MAS-BCWLC via assessing annually the specified objectives approved by both the MAS-BCWLC board and MASCC. For instance, MAS-BCWLC was requested to achieve 12 key objectives in 2004: (1) investment of 18 million RMB (ca. 2.175 million US$ at the exchange rate of 1US$ = 8.276RMB); (2) selling 48 million cubic meter water or more and reclaiming >90% of water bills; (3) fulfilling indicators of water service quality (for instance, >99% of the control points should reach the required water quality standards; >98% control points should reach standards for water pressure; a maximum of 30% water loss; burst pipes repairs within maximum time limits); (4) fulfilling all MASCC indicators for safe work; (5) construction of the main body of the No.4 WTP and 25 kilometer new pipes; (6) fulfilling client service indicators (for instance, 100% good client service; >90% public satisfaction); (7) fulfilling the reconstruction of Xiangshan Town water supply system; (8) elaboration and submitting a water supply plan; (9) achieving the relevant objectives of National Civilized City Assessment System (which was proposed by Central Cultural and Ideological Building Commission in 2004; it includes 119 indicators); (10) submitting water supply plans to Municipal People\u2019s Congress and Municipal People\u2019s Political Consultative Conference; (11) responding adequately to complaints and reporting this information to the government; and (12) take anti-corruption measures.\nAfter establishing the joint venture in 2002, the total length of pipes and the volume of water provision have increased (see Fig.\u00a05) and MAS-BCWLC has been in compliance with all requirements of the government, according to interviews with local officials. From 2004 to 2005, MAS-BCWLC has invested about 90 million RMB (ca. 10.875 million US$ at the exchange rate of 1US$ = 8.276RMB) for building new infrastructure, updating old facilities and aged pipes, and establishing a customer service system. In the meanwhile, the government has stopped subsidizing WTPs after the involvement of BCG and the joint venture even turned over about 18.7 million RMB (ca. 2.260 million US$ at the exchange rate of 1US$ = 8.276RMB; including 2 million RMB of the rent fee for pipe networks, 4.7 million RMB of dividends, 7.7 million RMB of corporate income tax, 3 million RMB of value added tax, and 1.3 million RMB of the expense of other taxation; the total taxation of 12 million RMB is about 25% of the total turnover of MAS-BCWLC in 2004) to the local government in 2004. The improved service quality of water provision not only satisfied the consumers, but also resulted in government (and the price public hearing; cf. Zhong and Mol 2007) support for the first tariff reform after private sector involvement in 2004. Maanshan Government increased the water tariff from 0.83 to 1.08 RMB\/m3 (ca. 0.10 to 0.13 US$\/m3 at the exchange rate of 1US$ = 8.276RMB; rate for household consumers) and indirectly subsidized MAS-BCWLC by moving the additional tax of water provision (e.g., 0.05 RMB\/m3 for household consumers) to the income of the joint venture water company. In 2004, the per capita annual income of urban households of Maanshan was 10,189 RMB (ca. 1231.15 US$ at the exchange rate of 1US$ = 8.276RMB), of which around 1.16% was spent on water services (calculated based on daily household water use of 300 liters per capita).\nFig.\u00a05Total length of pipes and annual water provision in Maanshan (1995\u20132004)\nObviously, the involvement of BCG has brought in additional capital to develop Maanshan\u2019s water supply sector. But more importantly it has changed the institutional structure, improved the water service quality and quantity, as well as reduced the governmental input in this field (see Fig.\u00a06). In this structure, the government benefits both from the taxations and dividends of the joint venture company, while transferring part of the financial, building, and operational risks to the private sector. Following this model of Maanshan City, BCG has successfully expanded its activities to other cities, such as Huainan (Anhui Province), Baoji (Shanxi Province), and Yuyao (Zhejiang Province).\nFig.\u00a06Monetary flow within Maanshan water supply\nHowever, this private sector involvement practice of Maanshan is argued to have a (potential) political risk due to the lack of a sound legal basis. In transitional China, in particular, policies are perceived to be instable and insufficiently law-based. Until now, details on measures and rules to regulate private utility companies are still missing in current national and Anhui provincial policy papers (Maanshan has no legislation right). This is a common problem in Chinese marketization practices in the water sector, as argued by many lawyers and academics. For instance, Shenyang water supply has experienced several failed marketization practices due to the constantly changing policies and decisions of the local government during 1995\u20132000 (field survey 2004).\nConcession Contract: Macau Water Supply\nMacau is one of the two Special Administrative Regions of China, together with Hong Kong. Administrated by Portugal until 1999, it was the oldest European colony in China, dating back to the 16th century. As a small territory of 28 km2 on the southern coast of China, consisting of a peninsula and the islands of Taipa and Coloane, it has a population of 508,000 (2006).\nMacau has a long history in the private provision of drinking water, since the earliest Macau Water Company Ltd. (MWC) was founded in 1932 as a full private capital company invested by individuals. Three years later, MWC was taken over by a British Electricity Lighting Company for 10 years and since 1946 by the president of the Macau Economic Department and other individual shareholders. Due to lack of capital and advanced technologies, Macau had an inadequate water supply service with poor water quality and discontinuous water provision during the 1970s. In 1985, Macau Government, learning from the concession management practices in the French water sector, awarded a consortium of two private companies, NWS Holding Limits (Hong Kong) and SUEZ Environment (France), a 25-year concession contract. Macau Government remained owner of the existing, pre-1985, assets (plants and pipe networks), while the private Macau Water Supply Ltd. (MWSL, the former MWC) bears responsibilities for operations and maintenance of these assets, as well as for new investments and service obligations (see Fig.\u00a07). This concession contract is not only the first private sector participation construction in Chinese water sector, but also the first contract that seems to end with a positive result.\nFig.\u00a07Organizational structure in Macau water supply\nDistinct from the previous private owners, who had little experience in the field of water provision, SUEZ (France) brought in advanced water knowledge and technology. According to the concession contract, MWSL must provide high-quality water supply service, as well as bear several obligations, such as planning, investment, construction, operation, and maintenance of the infrastructure under the supervision of Macao Government. In practice, Macau Government has delegated tasks, responsibilities and obligations to a very large degree to MWSL.\nComing to the end of the 25-year concession contract, MWSL has fulfilled almost all terms of the initial contract. It has, among others, considerably improved water service quality by increasing service access and provision, decreased the loss of water leakage (see Fig.\u00a08), and kept water tariff (corrected for inflation) at a stable level (see Fig.\u00a09).\nFig.\u00a08Annual water demand-provision and water loss in Macau (1982\u20132005)Fig.\u00a09Water tariff rates of Macau (1982\u20132005)\nIn the concession contract, the government did not specify conditions and safeguards for the poor. But in practice, MWSL not only reduced the water bill for low-income, disabled and other vulnerable groups. For instance, MWSL has launched the \u201cElderly-In-Needs\u201d water subsidy program in 2001, which offers those aged over 55 free water consumption of 5 m3 per month. Since May 2005 the \u201cWater for All\u201d program offers free water consumption to other categories of people in needs, such as single-parent families and disabled. But also in addition, it built two potable \u201cWallace fountains\u201d (a special public fountain with potable water) in Macau, providing free potable water to tourists and citizens. MWSL has also been active in various social welfare and charity activities, providing total donations of 2.08 million MOP (1MOP = 0.965RMB, 2007; ca. 0.26 million US$ at the exchange rate of 1US$ = 8.276RMB) during 2002\u20132005. During 1985\u20132005, MSWL also charged discounted water tariffs for governmental agencies, and handed in over 260 million MOP (1MOP = 0.965RMB, 2007; ca. 32.56 million US$ at the exchange rate of 1US$ = 8.276RMB) of taxes and about 56 million MOP (1MOP = 0.965RMB, 2007; ca. 7.012 million US$ at the exchange rate of 1US$ = 8.276RMB) of concession fees to the government.\nIn both Maanshan and Macau, the water tariff is the main financial source for water companies, while governmental subsidies have been abandoned. Accordingly, whether the water tariff can cover the costs is significant. In the case of Macau, the Macau Government owns the pre-concession infrastructure assets, which demands a smaller first investment from the Consortium. The water tariff could easily cover the cost of operation and maintenance (and not the huge capital costs of existing assets). Unlike the joint venture construction in the Maanshan case, Macau Government leaves all financial responsibilities to the private sector after the concession, and benefits from taxes, concession fees and discounts on government water bills (see Fig.\u00a010). Due to the limited initial investments of the private consortium, sharp water tariff increases were avoided after privatization (often one of the major reasons for public resistance and failed private sector participation in other countries). The local government still owns part of the infrastructure assets, in particular the pipes system, with huge sunk-costs.\nFig.\u00a010Monetary flow within Macau water supply\nMacau is also an interesting case because of the unique regulatory system, which includes the water quality regulator (IACM), and a unique Government Delegate. IACM is in charge of the water quality regulation, and monitors and controls drinking water quality by random sampling and analysis of over 70 water samples around Macau everyday. The Government Delegate is not a government official, but an individual working in another public utility company and appointed by the government. Following Macau laws, Mr. Lin Runzhong, the Government Delegate for water supply, was appointed for a period of five years by the Macau Government, and is not only the regulator of MWSL, but also an important linkage between MWSL and the government. He participates at all MWSL board meetings and reports relevant information and documents to the government. The Government Delegate decides which information is considered relevant. He is also in charge of assessing the performance of MWSL, and comments on the five-year plans and tariff plans before MWSL sends these to the government for approval. The Macau government generally follows the comments and assessments of the Government Delegate. In this sense, the nongovernmental Government Delegate is defined a specified role and powerful position in governing the water sector. This institutional arrangement relates to the small size of Macau Government, where only a limited state capacity (in quantitative and qualitative terms) is available for numerous public tasks. In conclusion, it can be argued that after 1985 the Macau government has played a meager role in the drinking water management.\nGreenfield Contract: Shanghai Wastewater\nThe Greenfield contract (e.g., BOT, TOT) is the dominant form of private sector participation in wastewater sector reform throughout the country. Shanghai Zhuyuan No.1 WWTP project is one of the most famous Greenfield projects in China. It is presently one of the largest WWTP in China, with a treatment capacity of 1.7 million m3 per day and an advanced primary treatment, serving an area of 107 km2 and about 23.5 million inhabitants. But it also has become famous for the lowest service price: 0.22 RMB (ca. 0.0266US$ at the exchange rate of 1US$ = 8.276RMB) per cubic meter treated wastewater.\nIn 2002, the Youlian Consortium (consisting of Youlian Development Company with 45% shares, Huajin Information Investment Ltd. Company with 40% shares, and Shanghai Urban Construction Group with 15% shares) won the open tender for Zhuyuan No.1 WWTP project by bidding the lowest treatment costs. A Project Company (Shanghai Zhuyuan Youlian No.1 Wastewater Treatment Ltd. CO.) was established and awarded a 20-year concession agreement by Shanghai Water Authority. A service management contract was signed with Shanghai Sewerage Company (a fully state-owned company administrated by the government) including details of rights and obligations. Two years later, Youlian Development Company withdrew from this project by transferring the shares and obligations to InterChina Holdings Group (see Fig.\u00a011).\nFig.\u00a011Private sector involvement in Shanghai wastewater treatment\nAccording to the agreement between Shanghai Water Authority and the private company, Shanghai Water Authority should minimize its interventions in the construction, operation, and maintenance of WWTP and limit them to safeguarding public health and safety. All conditions and objectives with regard to water service quality are defined in the service contract between Shanghai Sewerage Company and the private company. Among others, the private company has to install an on-line monitoring system and is requested to invite an authorized third party for regular monitoring (on indicators such as BOD5, CODcr, SS, NH4-N, and phosphate). This should be paid by the private company, while reporting to the Shanghai Sewerage Company and should take place within five days. Shanghai Sewerage Company may conduct random water examination at any time. According to the local officials, Shanghai Zhuyuan WWTP has fulfilled all responsibilities and obligations required by the contract up till now, including meeting the water quality standards.\nIn the case of Shanghai Zhuyuan Greenfield project, the government has transferred its traditional responsibilities of investment, construction, operation, and maintenance (for the contract period) to the private Project Company, accompanied by paying a service fee (see Fig.\u00a012). Different from the joint venture construction in Maanshan and the concession construction in Macau, in which corporate profits directly depend on the water tariff, the private operator within a Greenfield contract is paid a service price negotiated between the government and the private sector. This service price depends on the investments and agreed performance levels, rather than on the user fee level, and which provides the private sector with the financial risks. Accordingly, the low service price of Zhuyuan No.1 WWTP (which was 42% less than the projected costs by government) presented in the public bidding, was argued to have a close relation to earlier governmental input in this project. Shanghai Water Assets Management Development CO. Ltd., a fully public-owned company, was in charge of the pre-phase design and invested about 30 million US dollars in the fixed infrastructure of this project, while the government provided the land free of charge to the operator. Strictly speaking, Shanghai Zhuyuan No.1 WWTP Greenfield project is a quasi-BOT project, due to the fact that part of the investment comes from the government.\nFig.\u00a012Monetary flow within Zhuyuan Greenfield project\nThe experience of Shanghai is an example of full governmental delegation of the daily management of WWTP to the private sector, while financial support via subsidies and preferential policies (e.g., land use) facilitate privatization with low service prices. It is, however, too early to fully assess the success of this project. Some BOT WWTP projects in other cities have met problems following gaps in the current national policy documents. For instance, projects in Foshan (Guangdong Province) could not run properly due to conflicts over current land use right. And projects in Beijing were delayed during the financing process because the domestic private actors met difficulties in obtaining loans from domestic banks due to the lack of a sound loan policy. The commercial banks couldn\u2019t provide long-term loans as required for BOT-types projects as their credit policies are restricted for the private sector (Zhong and Fu 2005), while the China Development Bank can provide long-term loans for BOT-types projects only for a limited number of clients (Chang and others 2006).\nConclusions\nWith the emergence and blossoming of various forms of private sector involvement in the Chinese water sector, the traditional structure of full governmental provision of water supply and wastewater treatment has changed dramatically. The analysis in this article has provided evidence of the contribution of these new modes to increased capital investment, and especially of more efficient operations and improved service provision. In that sense, the original goals of the Chinese government to embark upon private sector involvement in water provisioning and treatment have been met. However, the early stage that most contracts are in, and the not yet crystallized forms and modes of privatization, prevents us from drawing any final conclusions on the impact of private sector involvement in the Chinese water sector.\nFrom the three casestudy projects with private sector participation, we can draw some lessons for how to successfully involve the private sector into the provision of water services. Firstly, a balance between the water tariff level, profits of investor and governmental subsidies is required. As Hall and Lobina (2005) state, most practices of water privatization fail due to public resistance following sharp price increases and job losses. In China, this has not (yet) been the case, due to large increases in efficiencies and governmental support to fixed infrastructure assets, reducing financial risk of the private sector and limiting the need for large water tariff increases. At the same time, the significant economic growth levels enables local residents to cope with some tariff increases, the poor and disadvantaged have been subsidized by the government, job losses have been minimized following social policies, and public hearings have contributed to higher levels of legitimacy. This all contributed strongly to a relatively smooth transformation of China\u2019s water sector.\nSecondly, the selection of the PPP form has a close relation with the level of local water tariff. As illustrated by this article, Greenfield projects appear to be applied when tariffs are not sufficient, especially in the wastewater sector (see also Zhong and others 2006), while Joint Venture approaches are often used in cities with sufficiently high water tariff, in particularly in the water supply sector.\nThirdly, it is crucial to accelerate the establishment of systematic and comprehensive governmental regulatory framework, as the current ad hoc, fragmented and diverse regulatory system endangers efficiency in water service development and certainty and stability for foreign investors. Experiences in many countries have proven that regulation is a key aspect in successful privatization in the water sector and a competitive benchmarking system is regarded as useful in an effective regulatory approach. In late 2006, the MOC attempted to develop a Chinese water supply benchmarking system, which is still ongoing. However, the current private sector involvements in the Chinese water sector still face many legal and regulatory uncertainties. Too often local authorities experiment with systems of governmental regulation and control, or \u2014 as in Macau \u2014 seem to become marginalized. According to interviews with local officials during our fieldwork, the importance of establishing a workable regulatory and legal system is essential. Guaranteeing sufficient and safe water service to the public is jeopardized by the fact that governments can no longer fully control the planning, operation, and management of water services as before private sector participation. This might only be signs of uneasiness with the new water institutions and division of tasks and responsibilities, but can also be the heralds of an emerging debate on privatization in the Chinese water sector.\nFinally, but not least, it is important to identify the differences in risk allocations in the water (service) market between the public and private sectors within different modes of PPP. As Table\u00a01 and the three case-study projects illuminate, with the various forms of privatization, the government often transfers (smaller or larger parts of) financial risks, building risks, and operation and maintenance risks to the private sector. Meanwhile, in the end the government can always take over all facilities without paying an indemnity to the private sector if a concessionaire fails in obtaining the goals as formulated by governmental authorities, or some conflicts emerge in the further policies (e.g., the terminated contracts that are regarded as providing the private sector a fixed investment return). In that sense, the still unstable legal base in transitional China provides a major political and transfer risk for private investors.","keyphrases":["public-private partnership","china","water governance"],"prmu":["P","P","P"]}
{"id":"Pain-2-1-2258319","title":"How does the self-reported clinical management of patients with low back pain relate to the attitudes and beliefs of health care practitioners? A survey of UK general practitioners and physiotherapists\n","text":"Guidelines for the management of low back pain (LBP) have existed for many years, but adherence to these by health care practitioners (HCPs) remains suboptimal. The aim of this study was to measure the attitudes, beliefs and reported clinical behaviour of UK physiotherapists (PTs) and general practitioners (GPs) about LBP and to explore the associations between these. A cross-sectional postal survey of GPs (n = 2000) and PTs (n = 2000) was conducted that included the Pain Attitudes and Beliefs Scale (PABT.PT), and a vignette of a patient with non-specific LBP (NSLBP) with questions asking about recommendations for work, activity and bedrest. Data from 1022 respondents (442 GPs and 580 PTs) who had recently treated patients with LBP were analysed. Although the majority of HCPs reported providing advice for the vignette patient that was broadly in line with guideline recommendations, 28% reported they would advise this patient to remain off work. Work advice was significantly related to the PABS.PT scores with higher biomedical (F1,986 = 77.5, p < 0.0001) and lower behavioural (F1,981 = 31.9, p < 0.001) scores associated with advice to remain off work. We have demonstrated that the attitudes and reported practice behaviour of UK GPs and PTs for patients with NSLBP are diverse. Many HCPs held the belief that LBP necessitates some avoidance of activities and work. The attitudes and beliefs of these HCPs were associated with their self-reported clinical behaviour regarding advice about work. Future studies need to investigate whether approaches aimed at modifying these HCP factors can lead to improved patient outcomes.\n1\nIntroduction\nLow back pain (LBP) is common, affecting 38% of adults in any one year, of whom 1 in 4 experience significant disability [37]. Only 25% of patients consulting in primary care will be symptom free 12 months later [18]. The last two decades have also seen dramatic rises in work loss and sickness benefit payments, attributed to recurrent and persistent LBP [16,36].\nGuidelines for the clinical management of patients with LBP encourage health care practitioners (HCPs) to advise patients to stay active, avoid bed rest, stay at or return to work, and stress simple messages about self-management [3,31,45,47,49,50]. Previous studies have identified that HCPs do not always follow guideline recommendations for LBP [10,20,24,26] and so despite the abundance of guidelines for practice, the management of LBP poses considerable challenges and frustrations for both patients and practitioners [14] and it is increasingly clear that it is insufficient to study patient factors alone [25,48].\nA potentially important but relatively unexplored influence on patients\u2019 pain experiences is the attitudes and beliefs of the HCPs with whom they come into contact. HCPs are frequently asked to provide advice and recommendations about physical activities, work, and rest and HCPs\u2019 attitudes and beliefs may be an integral part of the health care process, influencing the success or failure of treatment. HCPs hold a range of attitudes and beliefs about back pain [17,19,28,32,39,42\u201344], and these attitudes appear to be associated with the work and activity recommendations that HCPs give to patients [17,28,42,44].\nIn the UK, approximately 98% of the population is registered with a National Health Service general practitioner (GP) [13]. GPs serve as gatekeepers to secondary care, selecting and referring patients for specialist investigations and treatment services. Physiotherapy is one of the most common services to which patients are referred, or which patients seek out privately [36], and LBP accounts for more than half of physiotherapists\u2019 workload in the UK [24].\nFew studies have explored HCP factors in the UK, but it has been shown that many physiotherapists (PTs) continue to advise limitations of work and activity levels, despite identifying when patients with LBP are at risk of chronicity [11] and an important proportion of therapists continue treating patients with LBP even when they fail to improve [41]. The aim of this study was to measure, in national random samples, the attitudes, beliefs and reported clinical behaviour of GPs and PTs about LBP, explore their associations and evaluate the implications for both clinical practice and future research.\n2\nMethods\n2.1\nDesign and setting\nWe conducted a cross-sectional, nationwide postal survey of UK GPs and PTs, involved in the management of patients with LBP, between April and November 2005. Ethical approval for the study was obtained from the West Midlands Multi-centre Research Ethics Committee (MREC). Written consent was not sought from each participant for use of survey data, but consent of respondents was assumed if they completed and returned the questionnaire.\n2.2\nQuestionnaire sample and mailing process\nWe used simple random sampling to obtain details of GPs (n\u00a0=\u00a02000) and PTs (n\u00a0=\u00a02000) from national databases (Binleys database for GPs, n\u00a0=\u00a046,000 GPs on the list; Chartered Society of Physiotherapy membership database, n\u00a0=\u00a032,000 PTs on the list). In the UK, all GPs working in the National Health Service are included on the Binleys database [7], which is produced in conjunction with the Royal College of General Practitioners. The Chartered Society of Physiotherapy (CSP) is the professional, educational and trade union body representing the UK\u2019s chartered physiotherapists and 98% of all PTs are members of the CSP.\nA sample size calculation indicated that a sample of 900 responders (450 GPs and 450 PTs) was required to allow us to find a minimum difference of 10% in the proportion of respondents with \u2018helpful\u2019 to \u2018unhelpful\u2019 beliefs by important practitioner characteristics at a significance level of 0.05 and a power of 90% [2]. A questionnaire package containing the questionnaire, a cover letter, an information sheet and a pre-paid envelope was mailed to each HCP. A single reminder was sent to all non-responders four weeks after the first mailing. In order to allow assessment of non-response bias within the survey estimates, a brief questionnaire was mailed to a random sample of non-responders. No incentives for completing the questionnaire were offered.\n2.3\nQuestionnaire\nA filter question was used to identify those HCPs who had treated at least one patient with non-specific LBP (NSLBP) in the previous six months, so that only respondents with recent experience of managing patients with LBP were included in the analysis.\n2.3.1\nDemographics and practice information\nA number of demographic and practice questions, relevant to each profession, were included. Some items were pertinent to both professions: gender; years since qualification; postgraduate training in LBP; clinical interests\/speciality and personal experience of back pain. Data gathered exclusively from GPs included whether they worked only in general practice and whether the practice was a single-handed or a group practice. Data gathered exclusively from PTs included how much of their clinical practice was based in the NHS, what proportion of their caseload was primary care patients, whether they worked alone or in a team, and grade of current job.\n2.3.2\nAttitudes and beliefs measure\nThe Pain Attitudes and Beliefs Scale (PABS.PT [28,39]) was included as a measure of HCPs\u2019 attitudes about LBP. This was selected following a systematic review of available tools for assessing the attitudes and beliefs of HCPs about LBP [12], in which the PABS.PT fared well on pre-defined quality criteria [34]. This tool was originally developed for use in physiotherapists, but more recently has been applied to a cohort of Dutch general practitioners [30]. In addition, the members of a multi-disciplinary clinical advisory group confirmed face and content validity of the PABS.PT for both GPs and PTs after recommending that the term \u2018therapy\u2019 was changed to \u2018treatment\u2019 in two of the items of the PABS.PT. The resulting minimally amended PABS.PT was used for both GPs and PTs.\nThe PABS.PT assesses the strength of treatment orientation on two subscales, \u2018biomedical\u2019 and \u2018behavioural\u2019. The biomedical orientation is described as one in which the HCP believes in a biomechanical model of disease, where disability and pain are a consequence of a specific pathology within the spinal tissues and treatment is aimed at treating the pathology and alleviating the pain. The behavioural orientation is where the HCP believes in a biopsychosocial model of disease in which pain does not have to be a consequence of tissue damage, and can be influenced by social and psychological factors. We used the amended PABS.PT [28], which consists of 19 items, each rated on a six point Likert scale (\u2018Totally disagree\u2019\u00a0=\u00a01 to \u2018Totally agree\u2019\u00a0=\u00a06), with ten items on the biomedical subscale (score range: 10\u201360) and nine on the behavioural subscale (score range: 9\u201354). Higher scores on each subscale indicate a stronger biomedical or behavioural treatment orientation, respectively.\n2.3.3\nClinical behaviour measures\nClinical behaviour was elicited by asking the HCPs about diagnostic investigations and for their recommendations about work, activity levels, and bedrest, for a patient with NSLBP described in a vignette. The vignette described a patient with uncomplicated NSLBP who was not at work as a result of their symptoms (Appendix A). Vignettes have been shown to be a useful measure of clinicians\u2019 practice behaviour and a more accurate assessment of clinical behaviour than data extracted from case notes when measured against the gold standard of standardised patients [40].\nThe clinical behaviour question regarding work was as follows:\n\u201cThe patient described in the vignette asks what your advice would be about her work. I would recommend this patient to: (Please tick the one response that best describes what you would recommend this patient to do)a.Be off work until pain has completely disappearedb.Return to part time or light dutiesc.Be off work for a further\u00a0\u2026\u00a0weeks (please state number of weeks)d.Return to normal worke.Be off work until pain has improved\u201d\nResponses for each of the work, activity and bedrest questions were subsequently classified by the authors as being \u2018strictly in line with guideline recommendations\u2019, \u2018broadly in line with guideline recommendations\u2019 and \u2018not in line with guidelines\u2019. For the work question given above, we considered option \u2018d\u2019 to be strictly in line with guideline recommendations, option \u2018b\u2019 to be broadly in line with guideline recommendations and options \u2018a\u2019, \u2018c\u2019 and \u2018e\u2019 to be not in line with guideline recommendations. This classification was based on a previously published expert consensus carried out on similar practice recommendations in a postal survey of physiotherapists, osteopaths and chiropractors in the UK [22].\n2.4\nBrief questionnaire\nThe brief questionnaire sent to a sample of non-responders contained the filter question to ensure that respondents recently involved in the management of patients with LBP could be identified. Alongside key demographic questions, we included four items from the PABS.PT (two from each subscale chosen on the basis of factor loadings described by the tool\u2019s developers and data from a pilot study), the vignette patient and the clinical behaviour questions related to work, activity and bedrest.\n2.5\nStatistical analysis\nScores for the PABS.PT were calculated according to methods specified by the questionnaire developers, i.e. a simple summation of the items in each subscale [39]. No method for dealing with missing data on this measure has been published so a pragmatic decision was made that if one value was missing from a subscale, a mean score based on the remaining values was substituted. If more than one value was missing the score for the whole subscale was classed as missing. A Pearson\u2019s correlation coefficient was calculated between the scores on the two subscales of the PABS.PT as previous work has shown that they are not totally independent [28,39]. We used descriptive statistics to summarise, by professional group, demographic, and practice data for both subscales of the PABS.PT. In addition, in response to the reviewer\u2019s suggestions, we conducted a subgroup analysis of work, activity and bedrest recommendations for those respondents who had high biomedical scores and low behavioural scores and vice versa. Unless differences occurred by profession, analyses were performed on the combined GP and PT dataset.\nThe relationship between attitudes and beliefs and clinical behaviour was examined using ANOVA to test for an overall relationship with clinical behaviour and, when appropriate, for a linear trend across clinical behaviour groups (strictly in line, broadly in line and not in line with guidelines). The effect of non-response was examined by comparing responses from all responders to the full questionnaire to those completing the brief questionnaire. All analyses were carried out using the Statistical Package for Social Scientists for Windows (SPSS Inc., Chicago, IL, version 13).\n3\nResults\nThe overall response rate was 38% (n\u00a0=\u00a01534), 22% (n\u00a0=\u00a0443) for GPs and 55% (n\u00a0=\u00a01091) for PTs. Of the respondents, 580 PTs and 442 GPs reported treating at least one patient with LBP in the previous six months and were included in the analysis.\n3.1\nCharacteristics of respondents\nThe demographic and professional characteristics of the respondents are summarised in Table 1. The majority of GPs worked exclusively in general practice, within group practices and had at least one specialist clinical interest. The majority of PTs worked within the NHS, with other HCPs, were of senior clinical grade or above, and had a patient caseload of more than 50% primary care patients. The PTs were qualified for a shorter length of time than GPs, were more likely to be female and to have postgraduate training in LBP.\n3.2\nAttitudes and beliefs\nScores for both of the PABS.PT subscales could be calculated for the majority of the 1022 responders (biomedical n\u00a0=\u00a01010, behavioural n\u00a0=\u00a01004). Mean (standard deviation, range) score for the biomedical subscale was 31.0 (6.4, 12\u201350) overall: GPs 30.9 (5.3); PTs 31.1 (7.2), and for the behavioural subscale was 33.0 (4.6, 15\u201348) overall: GPs 33.7 (4.2); PTs 32.5 (4.8). For both subscales and both professional groups, the mean observed scores were in the middle of the possible ranges. The Pearson\u2019s correlation coefficient (r\u00a0=\u00a0\u22120.38; p\u00a0<\u00a00.0001) showed a statistically significant level of dependence between the two subscales, suggesting that respondents who score higher on one subscale tend to score lower on the other subscale.\n3.3\nDiagnostic investigations\nIn response to the vignette patient, most HCPs reported that they would not want the patient described to have any diagnostic investigations. Of the GPs 33% (n\u00a0=\u00a0142) reported that they would request at least one investigation, compared with 24% (n\u00a0=\u00a0134) of PTs (Table 2). GPs were more likely to want laboratory tests and PTs were more likely to want an X-ray or special imaging procedure such as an MRI.\n3.4\nClinical behaviour\nThe responses to the clinical behaviour questions were classified according to whether these were \u2018strictly in line\u2019, \u2018broadly in line\u2019 or \u2018not in line\u2019 with guideline recommendations and the responses and the classifications are summarised in Table 3. The majority of respondents reported advice that was either \u2018strictly in line\u2019 or \u2018broadly in line\u2019 with guideline recommendations\u2019. Very small proportions of respondents reported they would provide advice that was \u2018not in line\u2019 with guideline recommendations for activity and bedrest, however, this figure was considerably higher for recommendations regarding work, with 28% of respondents reporting that they would recommend the patient in the vignette to remain off work.\nThe summary of responses of the two subgroups of high biomedical and low behavioural scores (n\u00a0=\u00a0187) and low biomedical and high behavioural scores (n\u00a0=\u00a0137), compared to the total sample, is also presented in Table 3. The proportion of practitioners recommending that the patient in the vignette remain off work, i.e. not in line with guideline recommendations, was substantially higher in those with high biomedical and low behavioural scores (44.9%) than those with high behavioural and low biomedical scores (11.9%). Similar differences were also seen for recommendations regarding activity and bedrest.\n3.5\nRelationship between attitudes and beliefs and clinical behaviour\nGiven the very small proportion of respondents whose advice was \u2018not in line with guidelines\u2019 for both activity and bedrest, associations with the PABS.PT scores were not examined. Fig. 1 shows the distributions of the PABS.PT biomedical and behavioural subscale scores for each of the reported work recommendation groups. With increasing disparity with guidelines, biomedical scores increased (mean scores: 28.3, 30.6, 33.5) and behavioural scores decreased (mean scores: 34.1, 33.3, 31.8). These associations were shown to have a significant linear trend for both the biomedical (F1,986\u00a0=\u00a077.5, p\u00a0<\u00a00.001) and behavioural (F1,981\u00a0=\u00a031.9, p\u00a0<\u00a00.001) subscale scores.\n3.6\nEffect of non-response\nIn order to assess the impact of non-response bias within the survey estimates, a brief questionnaire was mailed to a random sample of non-responders (GPs n\u00a0=\u00a0414, PTs n\u00a0=\u00a0243), and responses were received from 14% of GPs (n\u00a0=\u00a059) and 17% PTs (n\u00a0=\u00a040).\nFor the GPs, gender mix and years in practice were similar for those completing the full and brief questionnaire. For the PTs, those completing the brief questionnaire were slightly less experienced (mean of 12 years experience versus 15 years) and more likely to be male compared to the full questionnaire responders (25% vs. 19% male). Responses to both behavioural subscale PABS.PT items and one of the two items from the biomedical subscale were similar to those for the full questionnaire. Responders to the brief questionnaire, from both professions, were more likely to agree with the statement that \u2018patients with back pain should preferably practice only pain free movements\u2019, indicating a more biomedical orientation. The responses to the items regarding work and activity advice were similar for responders to the full and brief questionnaires. GPs responding to the brief questionnaire reported bedrest advice that was less in line with guideline recommendations than the responders to the full questionnaire (19.3% strictly in line with guidelines compared to 38.4%, respectively), whereas the PTs completing the brief questionnaire reported bedrest advice that was more in line with guideline recommendations than the initial responders (35.0% strictly in line with guidelines compared to 21.8%, respectively).\n4\nDiscussion\n4.1\nMain findings\nThis is the first national UK survey of LBP related attitudes, beliefs and reported clinical behaviour of GPs and PTs and results show that responses are diverse. The majority of respondents reported advice that was strictly or broadly in line with guideline recommendations about activity and bedrest, however, over a quarter of HCPs recommended that the vignette patient with NSLBP should remain off work. Reasons why adherence to guideline recommendations for work is lower than for activity and bedrest are unclear, but may be due to the complex nature of the clinical consultation, and previous studies have shown that GPs see sickness certification as a potential threat to the doctor\u2013patient relationship [15,29]. The attitudes and beliefs of HCPs were significantly associated with reported work advice for the patient described, i.e. HCPs with stronger biomedical and weaker behavioural treatment orientations were more likely to report advice, regarding work, which was \u2018not in line with clinical guidelines\u2019. The subgroup analysis supports this, although only a third of respondents could be categorized into these subgroups i.e. high biomedical and low behavioural scores on the PABS.PT or vice versa. The differences in the PABS.PT scores were small, and although statistically significant, no guidance is currently available to suggest whether these represent a clinically relevant difference.\nA considerable proportion of HCPs in the UK continue to provide advice to patients about work that is not in line with guideline recommendations. The associations between attitudes, beliefs and reported clinical behaviour suggest that some HCPs continue to practice predominantly within a biomedical model, placing most importance on the severity of tissue damage when determining a patient\u2019s level of pain and functional disability. Others have adopted a more behavioural approach to management, embracing the notion that the level of pain and functional loss may be influenced by psychological and social factors in addition to biomechanical factors.\n4.2\nComparison to other studies\nHCPs in this study had similar attitudes and beliefs to therapists in the Netherlands [28], with Dutch therapists having similar mean biomedical scores (29.5 vs. 31.0), but slightly higher behavioural scores (35.6 vs. 33.0) on the PABS.PT. Direct comparison of subscale scores with studies using the original PABS.PT is not possible due to a different number of items [30,39].\nThe attitudes and beliefs of HCPs were significantly associated with reported work advice for the vignette patient. Respondents reporting advice \u2018strictly in line with guidelines\u2019 demonstrated stronger behavioural and weaker biomedical orientations than those reporting advice \u2018not in line with guideline recommendations\u2019. Using a variety of measures, previous studies have demonstrated that advice to restrict work or activities is also associated with a biomedical treatment orientation [28], patho-anatomical focus of training courses [39], higher fear avoidance beliefs of HCPs [17,32,42] and a strong belief that pain and impairment are invariably linked [28,44]. Our study adds to this body of literature by showing a significant association between attitudes and beliefs and reported work advice in HCPs in the UK.\n4.3\nImplications for clinical practice and future research\nThe results suggest that the attitudes and beliefs of HCPs are linked to clinical practice and the recommendations provided to patients. These practitioner factors are thus part of the dynamic interaction within LBP care episodes, along with the LBP problem itself and the patient\u2019s own perceptions about their problem. This may help explain patient outcomes, although the mechanisms behind this are likely to be complex. It is probable that HCPs\u2019 attitudes and beliefs are expressed to patients in a variety of ways, with a range of possible consequences. By restricting activities and work, HCPs may reinforce patient\u2019s unhelpful illness perceptions and increase spinal vigilance. Alternatively, they may over-direct the patient by providing strict advice to perform only specific activities and exclude others, encouraging an over-reliance on the HCP [35], which may make it difficult to foster the patients\u2019 self-management skills, something recommended as part of best practice for patients with LBP.\nThe reported clinical behaviour of HCPs illustrates that the majority would provide advice that is strictly or broadly in line with guideline recommendations, however, nearly 30% reported they would advise the described patient to remain off work. Staying at work or an early return to work with NSLBP is recommended [50], as the longer someone is off work the likelihood of them returning steadily diminishes, with a 20% risk of long term disability for those off work for four to six weeks [51]. Although the management of LBP, in terms of advice about activity and bedrest, seems to be broadly in line with guideline recommendations, our results show that adherence about advising early return to work is suboptimal.\nAttitudes and beliefs held by HCPs may help explain why implementation of current LBP guidelines has been slow and difficult [6,8,20,23,33]. Changing clinical behaviour is recognised to be a challenge [27]. Evidence from recent clinical trials suggests that although modest intervention strategies can result in moderate changes in reported adherence to guideline recommendations [8], this does not lead to a corresponding improvement in patient outcomes [9,21,30]. A better understanding of the attitudes and beliefs of HCPs, what influences these and how these relate to outcomes of patients with LBP is needed to inform development of future implementation strategies.\nFuture work should further test the psychometric properties of the PABS.PT to assess responsiveness and determine appropriate cut offs for \u2018high\u2019 and \u2018low\u2019 scores on the subscales and what constitutes a clinically relevant change. Methods to assess HCP attitudes, beliefs and behaviours warrant further study. For example, the validity of using methods to measure implicit attitudes about LBP, such as those employing automatic responses, could be explored in an attempt to overcome potential social desirability bias in survey responses as HCPs become more aware of clinical guidelines.\n4.4\nStrengths and limitations\nThe strengths of this study include the large sample sizes, simple random sampling of UK GPs and PTs, use of a validated beliefs measure, and investigation of potential non-response bias. The response rate of GPs was low, but comparable to other postal surveys of GPs in the UK [4,5,38]. The sample size calculation took this into account and yielded the required sample size for the planned analyses. The response rate of PTs was in keeping with other studies [11,39,41]. Responses to the brief questionnaire were broadly similar to those completing the full questionnaire in terms of attitudes, and recommendations for work and activity. However, some differences in the advice for bedrest suggest that we cannot rule out non-response bias in our survey. Responses to one PABS.PT item showed a stronger biomedical treatment orientation for responders to the brief questionnaire. Also, GPs responding to the brief questionnaire reported advice for bedrest that was less in line with guideline recommendations than responders to the full questionnaire, so for GPs, where the potential for non-response bias is greatest, our survey may underestimate the strength of a biomedical treatment orientation and the numbers providing advice not in line with guideline recommendations.\nThis study captured self-reported behaviour rather than real clinical practice, which is very difficult to measure. To provide a context for the clinical behaviour questions we used a vignette of a patient with NSLBP, an approach shown previously to have acceptable validity [40,46]. Although we used established tools to assess attitudes, beliefs and clinical behaviours, there may be some overlap in the constructs they measure. We attempted to address this by the wording of instructions and the order of the tools within the questionnaire. The PABS.PT attitudes measure came first with instructions to respond to the general attitudinal type statements. The vignette and the behaviour questions came later with the instruction to consider the specific management of the patient described.\n5\nConclusion\nThis study shows the diversity of the attitudes and self-reported practice behaviour of UK GPs and PTs for patients with NSLBP. Many HCPs believed LBP necessitates some avoidance of activities and the need to be off work. For a patient with a history of being off work since onset of LBP four weeks previously, over a quarter of HCPs recommended further time off work. The attitudes and beliefs of HCPs were associated with their advice about return to work. Future studies need to investigate the associations between HCP factors and patient outcomes, and test if approaches aimed at modifying attitudes, beliefs and clinical behaviours of HCPs can be successful.","keyphrases":["low back pain","attitudes and beliefs","health care practitioners","survey","practice behaviour"],"prmu":["P","P","P","P","P"]}
{"id":"J_Immunol_Methods-2-1-2225449","title":"Quantitative imaging assay for NF-\u03baB nuclear translocation in primary human macrophages\n","text":"Quantitative measurement of NF-\u03baB nuclear translocation is an important research tool in cellular immunology. Established methodologies have a number of limitations, such as poor sensitivity, high cost or dependence on cell lines. Novel imaging methods to measure nuclear translocation of transcriptionally active components of NF-\u03baB are being used but are also partly limited by the need for specialist imaging equipment or image analysis software. Herein we present a method for quantitative detection of NF-\u03baB rel A nuclear translocation, using immunofluorescence microscopy and the public domain image analysis software ImageJ that can be easily adopted for cellular immunology research without the need for specialist image analysis expertise and at low cost. The method presented here is validated by demonstrating the time course and dose response of NF-\u03baB nuclear translocation in primary human macrophages stimulated with LPS, and by comparison with a commercial NF-\u03baB activation reporter cell line.\n1\nIntroduction\nNuclear factor kappa B (NF-\u03baB)\/rel represent a family of transcription factors, present in all eukaryotic cells, that regulate inducible expression of wide ranging genes involved in immune responses and cell-cycle regulation. Therefore activation of NF-\u03baB provides a key molecular switch that is relevant to many aspects of cellular immunology research. In immune cells NF-\u03baB is most abundant either as a heteromeric complex of two components, p65 (rel A) and p50, or as a p65\/p65 homodimer. The p65 component contains the main transactivating domain responsible for NF-\u03baB transcription factor function. Regulation of NF-\u03baB activity is dependent upon cytoplasmic sequestration in association with an inhibitory molecule, I\u03baB\u03b1. As a consequence of intracellular kinase signalling cascades I\u03baB\u03b1 is phosphorylated, and this leads to its degradation, allowing nuclear translocation of p65\/rel A and hence so-called \u201cactivation\u201d of NF-\u03baB (Ghosh et al., 1998). Conventional methods for testing NF-\u03baB nuclear translocation utilise a semi-quantitative electromobililty gel-shift assay. This involves incubation of nuclear extracts with 32P-labelled oligonucleotides of NF-\u03baB binding sites and separation from unbound probe by electrophoresis in a non-denaturing polyacrylamide gel. However, this assay is principally limited by sensitivity, and requires large scale cell culture (typically >\u00a010\u00a0\u00d7\u00a0106 cells), thus precluding its use with primary cells. Additional disadvantages are the labour-intensive protocol and the need for radioisotopes. Cell lines transfected with NF-\u03baB promoter\u2013reporter gene constructs are used widely to study cell signalling pathways, and are easy to quantitate with colorimetry or luminometry, but are not suitable for most primary cell culture studies. Commercial kits for enzyme linked immunosorbent assays of NF-\u03baB quantitation within nuclear extracts have also become available, but their expense may be prohibitive.\nAn attractive new strategy is to image NF-\u03baB translocation from the cytoplasm to the nucleus, using immunofluorescence staining. This can be performed on a small scale and at a single-cell level. Fluorescence microscopy, laser scanning cytometry and flow cytometry to quantify NF-\u03baB nuclear translocation have all been reported (Deptala et al., 1998; George et al., 2006; Rogers and Fuseler, 2007; Fuseler et al., 2006). Drawbacks of these previous reports are the need for expensive imaging equipment and\/or image analysis software. Here we present an alternative simple method for quantitative detection of NF-\u03baB rel A nuclear translocation which uses standard confocal immunofluorescence microscopy and the public domain Java image processing program, ImageJ. We suggest that the general availability of all three components\u2014fluorescence microscopy, immunostaining reagents, and the analytic protocol-provides a readily accessible method for the study of NF-\u03baB nuclear translocation in primary cell cultures.\nFor this study we have chosen to use a cell system that has been difficult to analyse previously, primary monocyte-derived macrophages that have been isolated from healthy human volunteers. In vivo macrophages are resident tissue mononuclear phagocytic cells derived from circulating monocytes. They function both as sensory cells of innate immunity, and as effectors, initiating early non-specific host defences, both by local recruitment of other immune cells and by induction of an acute phase response, which leads to systemic priming of the immune system. In vitro work on these cells often includes stimulation by model innate stimuli, now known to be Toll-like receptor (TLR) ligands, such as lipopolysaccharide (LPS) (TLR-4) and Pam3CSK4 (TLR-2). Activation of the NF-\u03baB pathway is a common downstream component of the cellular response to many different innate immune stimuli and is used frequently in these cells as a biochemical detection and quantification method to study innate immune cellular activation. Therefore analysis of the NF-\u03baB pathway in macrophages, and in related mononuclear phagocytic cells such as dendritic cells, is an important area of research interest. However, analysis in these cells has been hampered by the lack of suitable methods. Myeloid leukaemic cell lines (e.g. U937, THP-1) are the standard for these experiments, but unless they are very carefully differentiated (which itself may involve NF-\u03baB activation) they are not ideal models. This necessity for an accurate and reproducible primary macrophage system stimulated us to adapt the NF-\u03baB assay as described here.\n2\nMethods\n2.1\nMacrophage culture and innate immune stimulation\nHuman blood samples were obtained from healthy volunteers. The study was approved by the joint University College London\/University College London Hospitals NHS Trust Human Research Ethics Committee and written informed consent was obtained from all participants. Peripheral blood mononuclear cells (PBMC), consisting of monocytes and lymphocytes, were prepared by density-gradient centrifugation of heparinised blood with Lymphoprep\u2122 (Axis-Shield) according to the manufacturer's instructions. PBMC were resuspended in RPMI 1640 with l-glutamine (Gibco, Invitrogen) (NM) containing 5% heat inactivated (56\u00a0\u00b0C for 30\u00a0min) type AB normal human serum (NHS) (Sigma Aldrich) (107 cells \/ml) and seeded on to 13\u00a0mm (No 1.5) glass coverslips (VWR) using 2\u00a0\u00d7\u00a0105 cells\/cover slip. After 1\u00a0h at 37\u00a0\u00b0C non-adherent cells (lymphocytes) were removed by sequential washes with Hanks buffered saline solution (HBSS) (Gibco, Invitrogen). Adherent cells (monocytes) were incubated in NM containing 10% autologous heat-inactivated HS supplemented with 20\u00a0ng\/mL macrophage-colony stimulating factor (M-CSF) (R&D systems) for 3\u00a0days. Any remaining non-adherent cells were removed by further washes with HBSS, and NM\/HS was replaced, but without additional M-CSF. This protocol yields adherent macrophages by day six with less than 5% contamination by lymphocytes (data not shown). Ultra-pure LPS (Invivogen) and Pam3CSK4 (Invivogen) were used as prototypic innate immune stimuli at different concentrations as outlined below. Polymyxin B (Invivogen) was used as a specific inhibitor of LPS bioactivity.\n2.2\nImmunofluorescence staining\nFor immunostaining, rabbit polyclonal affinity purified antibody to rel A (C-20) (Santa Cruz Biotechnology) was used (2\u00a0\u03bcg\/ml) with a secondary antibody, Alexa-Fluor (AF)633\u2014conjugated F(ab')2 goat anti-rabbit IgG (Invitrogen) used at 4\u00a0\u03bcg\/ml. 10% normal goat serum (Sigma Aldrich) used as blocking buffer, eliminated all non-specific binding of the secondary antibody (data not shown). Cells cultured on glass coverslips were fixed with 3.7% paraformaldehyde (15\u00a0min, room temperature) and washed with Tris-buffered saline (TBS). Each coverslip was inverted on to 50\u00a0\u03bcl of solution placed on impermeable Nesco film (VWR). All reagents were diluted in TBS, and coverslips were washed by immersion into TBS between each staining step. Coverslips were incubated sequentially with 0.2% Triton-X100 (Sigma) (10\u00a0min, room temperature), blocking buffer (30\u00a0min, room temperature), primary antibody diluted in blocking buffer (overnight, 4\u00a0\u00b0C) and secondary antibody diluted in blocking buffer (1\u00a0h, room temperature). Nuclei were counterstained with 2\u00a0\u03bcg\/ml of the nuclear stain DAPI (Sigma Aldrich) for 5\u00a0min. Coverslips were mounted on to glass slides (VWR) using Vectashield hard-set mounting media (Vector).\n2.3\nImage acquisition and analysis\nFluorescence images were captured on a Leica SP2 confocal microscope. DAPI (excitation 405\u00a0nm, emission 400\u2013450\u00a0nm) and AF633 (excitation 633\u00a0nm, emission 650\u2013700\u00a0nm) fluorescence were captured using sequential acquisition to give separate image files for each (Fig. 1A). A pin hole of 1 Airy (114.5\u00a0\u03bcm), scan speed of 400\u00a0Hz and 4 frame averaging was used. Photomultiplier tube gain and offset were adjusted to give sub-saturating fluorescence intensity with optimal signal to noise ratio. Image analysis was performed using ImageJ v3.91 software (http:\/\/rsb.info.nih.gov\/ij). Five high power fields were selected for analysis of each stain. To avoid being biased by the NF-\u03baB staining, each field was selected by viewing nuclear (DAPI) staining only to identify near-confluent cells and thereby maximise the cell numbers included in the analysis. Preliminary experiments demonstrated that this approach provided data on at least 500 cells, and that the variance of the data was not changed by increasing the sample size further (data not shown). For each high power field, binary image masks were created of rel A and DAPI positive staining to define regions of interest (ROI) for analysis. This was done by applying a median filter (3\u00a0\u00d7\u00a03\u00a0pixel radius) to remove noise and to approximate the distribution of staining intensity to a median value (Fig. 1B). Automatic thresholding, using the Isodata algorithm (Ridler and Calvard, 1978) was then used to convert the image to a binary mask (Fig. 1C) that included all fluorescence data above background. The DAPI staining mask was used to define the nuclear ROI. Using the image calculator, the DAPI mask was subtracted from the rel A mask to create a staining mask defining the cytoplasmic ROI. Each of these ROI masks were then applied, by the image calculator, to the original rel A (AF633) staining images to separate nuclear and cytoplasmic staining within each high power field (Fig. 2A). Quantitative fluorescence data were exported from ImageJ generated histograms into Microsoft Excel software for further analysis and presentation (Fig. 2B). Measurement of nuclear fluorescence alone does not distinguish NF-\u03baB nuclear translocation from increased background levels of NF-\u03baB expression or artefactual differences in staining intensity. Therefore nuclear and cytoplasmic staining intensities were compared to give the nuclear:cytoplasmic ratio as a relative measure of rel A nuclear localisation. Nuclear and cytoplasmic histogram data were first normalised for the total number of data points included in the analysis and then comparison was made of the sum of staining intensities. In this way, NF-\u03baB nuclear translocation is represented by an increase in nuclear:cytoplasmic ratio of rel A staining. Multiple images can be batch processed simultaneously in this way by converting individual images into DAPI and NF-\u03baB rel A stacks.\n3\nData presentation and discussion\nTo validate this methodology and analysis, time course and dose response studies of LPS induced NF-\u03baB nuclear translocation were performed. Macrophages were stimulated with 0\u2013100\u00a0ng\/ml LPS in NM with 10% HS for up to 1\u00a0h. NF-\u03baB nuclear translocation was evident by 30\u00a0min and maximal at 60\u00a0min (Fig. 3). The expected increase in nuclear:cytoplasmic ratio was also evident across the LPS dose range (Fig. 4). In addition we tested the effect of Polymyxin B (PMB), a polypeptide that binds and neutralises the bioactive lipid A component of LPS. PMB completely abrogated NF-\u03baB nuclear translocation in response to LPS specifically, and had no effect on stimulation with the TLR2 ligand, Pam3CSK4 (Fig. 5).\nTo further evaluate the quantitation provided by this assay the results were compared with a commercial NF-\u03baB reporter gene assay using the HEK-293 cell line transfected with TLR2 and a secreted alkaline phosphatase NF-\u03baB reporter gene construct (Invivogen). Transfected cells were seeded on to glass coverslips as described above and allowed to adhere for 48\u00a0h. They were then stimulated with Pam3CSK4 (dose range 0\u20132\u00a0\u03bcg\/ml) diluted in the manufacturer's detection media. Cells were fixed and stained as above for immunofluorescence staining of NF-\u03baB after 1\u00a0h stimulation. Duplicate wells were allowed to incubate for 6\u00a0h to allow the reporter gene and substrate reaction to take place. Cell culture supernatants were then harvested to quantify the colorometric reaction spectrophotometrically at 630\u00a0nm. Quantitative comparison of NF-\u03baB activation using the reporter gene expression assay and NF-\u03baB nuclear translocation by confocal microscopy showed statistically significant correlation (Fig. 6). Importantly, reporter gene expression provides a measure of NF-\u03baB function that is dependent on its nuclear translocation, but not exclusively regulated by it. The difference between measurement of nuclear translocation and NF-\u03baB function is acknowledged and may in part explain the imperfect regression analysis (r2 0.83) between the two methods.\nThe method proposed here is most suitable for adherent cell cultures with relatively large cytoplasmic:nuclear area ratios that allow clear distinction between nuclear and cytoplasmic NF-\u03baB staining. It requires relatively few cells and can be used to study NF-\u03baB nuclear translocation at single-cell level or in mixed cultures. It can be readily applied to the study of NF-\u03baB activation in macrophages, dendritic cells, epithelial and endothelial cells, and fibroblastic cells. We have also been able to apply this method to monocytic cells in suspension (THP-1 and K562 cell lines) by air drying them onto coverslips for immunostaining (data not shown), albeit their typically small nuclear:cytoplasmic area ratio may limit the accuracy of quantitation. This generic image analysis methodology may be applied to quantitative analysis of other transcription factors and signalling events in which assessment of sub-cellular localisation is necessary. Where confocal microscopy facilities are available, this method overcomes the problems related to sensitivity, use of radioisotopes and cost. It can be easily adopted in current cellular immunology research, and given the ready accessibility of the public domain image analysis software, with further validation this methodology may serve as a universal standard that allows better comparison of data from separate experiments and different research groups.","keyphrases":["macrophages","image analysis","nuclear factor-kappa b","confocal immunofluoresence"],"prmu":["P","P","M","M"]}
{"id":"Environ_Manage-3-1-1866215","title":"Early Vegetation Development on an Exposed Reservoir: Implications for Dam Removal\n","text":"The 4-year drawdown of Horsetooth Reservoir, Colorado, for dam maintenance, provides a case study analog of vegetation response on sediment that might be exposed from removal of a tall dam. Early vegetation recovery on the exposed reservoir bottom was a combination of (1) vegetation colonization on bare, moist substrates typical of riparian zones and reservoir sediment of shallow dams and (2) a shift in moisture status from mesic to the xeric conditions associated with the pre-impoundment upland position of most of the drawdown zone. Plant communities changed rapidly during the first four years of exposure, but were still substantially different from the background upland plant community. Predictions from the recruitment box model about the locations of Populus deltoides subsp. monilifera (plains cottonwood) seedlings relative to the water surface were qualitatively confirmed with respect to optimum locations. However, the extreme vertical range of water surface elevations produced cottonwood seed regeneration well outside the predicted limits of drawdown rate and height above late summer stage. The establishment and survival of cottonwood at high elevations and the differences between the upland plant community and the community that had developed after four years of exposure suggest that vegetation recovery following tall dam removal will follow a trajectory very different from a simple reversal of the response to dam construction, involving not only long time scales of establishment and growth of upland vegetation, but also possibly decades of persistence of legacy vegetation established during the reservoir to upland transition.\nIntroduction\nAlthough more than 450 dams have been removed in the United States in the past century, dam removal has only recently received significant attention from the scientific community (Beyer 2002, Hart and Poff 2002, Graf 2003). Reasons for dam removal include unsafe conditions or loss of function associated with aging or sediment-filled structures and, more recently, environmental restoration (Hart et al. 2002, Pohl 2002). Dam removal decisions involve a tradeoff of multiple socioeconomic and ecological costs and benefits (Stanley and Doyle 2003). Potential environmental consequences of dam removal include (1) benefits from restoring more natural flow and sediment regimes (Poff et al. 1997, Kondolf 1997) and (2) removing barriers that block fish passage (Lenhart 2003) and fragment the river corridor (Nilsson et al. 2005). Potential negative effects include the impacts of releasing stored, and possibly contaminated, sediment and enhancing dispersal of undesirable species (Bednarek 2001). The aspect of dam removal examined here is the ecological fate of the land under the former reservoir pool. Trajectories of vegetation response on lands exposed by dam removal influence higher-order responses such as human and wildlife use and biogeochemical processes (Shafroth et al. 2002). In some cases, restoration of pre-dam vegetation may be a management goal. The extent to which this can be accomplished by natural colonization and subsequent vegetation change may significantly affect project costs. In some cases, natural colonization may be the default management action because of budgetary constraints, limited mandates, or lack of interest in restoration. Even in the absence of a specific restoration target for vegetation on the exposed surfaces, there are concerns about rapid dominance of these barren areas by undesirable, weedy, non-native species and the need to provide stabilizing vegetation to minimize erosion.\nSeveral sources of information support projections of likely future vegetation on land exposed by a dam removal. The first source includes studies of the plant communities in the surrounding upland landscapes, in the riparian zones of rivers, and in the margins of lakes and reservoirs. The description of these communities and the controls on their composition, especially vegetation dynamics on disturbed, bare ground sites, provide a coarse identification of possible states for the former reservoir pool. There is also a significant literature examining vegetation dynamics within periodically exposed lake or reservoir shorelines illustrating the importance of intra- and inter-annual water level fluctuation (Keddy and Rznicek 1982, 1986, Hill et al. 1998) and positive relations between species richness and both total cover and substrate fineness (Nilsson and Keddy 1988, Nilsson et al. 1997).\nStudies of floodplain vegetation colonization and dynamics provide information on likely pioneer species and subsequent changes associated with fluvial processes and geomorphic surfaces within river bottomlands in many regions. For example, Friedman et al. (1996) described patterns of vegetation change on the floodplain of Plum Creek in eastern Colorado, where species richness peaked at intermediate ages, older and higher surfaces were increasingly dominated by rhizomatous perennials, and the overall species list was 36% non-native. In the western United States, much of the focus on relations between streamflow and riparian vegetation has centered on cottonwood, which is the structurally dominant native tree. Populus deltoides subsp. monilifera (plains cottonwood) is a pioneer species with a relatively narrow regeneration niche. The requirements for a bare, moist surface with limited drawdown following germination have been represented in a formal recruitment box model describing the floodplain locations and patterns of water stage where establishment is likely for P. deltoides subsp. monilifera and other species of cottonwood and willow with similar establishment requirements (Mahoney and Rood 1998, Rood et al. 2005). This model is a clear example of how expectations derived from the study of riparian plant distributions and life history requirements can be used to inform an assessment of recolonization of the former reservoir pool following dam removal.\nThe second general source of information is observations from actual dam removals (Bednarek 2001, Stanley and Doyle 2002). In relatively few cases have environmental effects been evaluated following dam removal, and these studies were all of dams less than 17 m tall in relatively humid settings (Hart et al. 2002). Quantitative analysis of vegetation response to actual dam removal is rare, although two recent studies examine vegetation colonization and succession within the former reservoir pools of small dams removed in Wisconsin (Lenhart 2000, Orr and Stanley 2006).\nA final source of information is case studies from alterations at least partially analogous to dam removal, including breaching of beaver and debris dams, accidental human dam failures, and dam maintenance activities. For example, the episodic release of a large sediment pulse from dam maintenance has been analyzed as a surrogate for the downstream effects of the type of sediment pulse that might be produced by dam removal (Wohl and Cenderilli 2000, Zuellig et al. 2002). In this study, we examine vegetation colonization and early dynamics on areas exposed when a reservoir was drained for four years to facilitate dam repairs. We describe vegetation pattern in terms of time since exposure in this analog to dam removal, in order to supplement the sparse empirical database available to scientists, resource managers, and policy makers involved in dam removal evaluations.\nStudy Site\nHorsetooth Reservoir is located in the foothills of the Rocky Mountains, 7 km west of Fort Collins, Colorado, in the transition zone between two physiographic provinces: the Colorado Piedmont subdivision of the Great Plains to the east and the southern Rocky Mountains to the west (Fenneman 1931). At an elevation of 1,655 m asl, the study site is in the rain shadow of the Rocky Mountains, approximately 70 km east of the Continental Divide; mean annual precipitation ranges from 36\u201340 cm, more than 70% of which falls between April and September. Summer in the study area is typically hot, with a mean July maximum of 29\u00b0C. Fall is cool and typically dry, punctuated occasionally by wet and sometimes heavy upslope snowstorms. Winter is characteristically dry and cool to cold with a mean minimum January temperature of \u22123.2\u00b0C and extreme winter minimum temperatures as low as \u221240\u00b0C (Hansen et al. 1978).\nThe transition zone between these physiographic regions, described by Marr (1961) as the Grassland-Lower Montane ecotone, is characterized by a rapid change in elevation and a shift from grassland to forest. In this transition zone, localized differences in soil primarily determine the dominant vegetation type at a given location. Grasslands characteristically dominate deeper, finer textured soils, transitioning to shrublands and open stands of ponderosa pine (Pinus ponderosa) on shallow, rocky soils and fractured rock outcrops (Marr 1961). Existing vegetation on the steep slopes and ridges above the high water line of Horsetooth Reservoir, matches the transition from shrublands dominated by Cercocarpus montanus and Rhus trilobata (Rhus aromatica, Great Plains Flora Association 1986) to a Pinus ponderosa community type described by Peet (1981) for rocky slopes below 1,700 m. This open, xeric forest type is characterized by widely scattered Pinus ponderosa with a grass-dominated understory. Despite xeric site conditions, understory species diversity and cover are relatively high. Cercocarpus montanus, Rhus trilobata, and Yucca glauca dominate the shrub layer, while Stipa comata, Bromus tectorum, Helianthus pumilus, Sporobolus cryptandrus, Bouteloua hirsuta, and Verbascum thapsus are important in the herbaceous layer (Peet 1981). As seen in portions of the valley not inundated by Horsetooth Reservoir, grasslands dominate toeslopes above the valley margins as well as the valley floor, where finer-textured soils accumulate to greater depths. These grasslands are characteristically dominated by Agropyron smithii, Andropogon scoparius, Bouteloua curtipendula, B. gracilis, Bromus tectorum, and Stipa comata (Hansen and Dahl 1957). Narrow valley floors typically support scattered stands of riparian shrubs and trees, including Salix irrorata, Betula occidentalis, Populus deltoides subsp. monilifera, and Salix amygdaloides (Marr 1961).\nThe reservoir is situated between two sharp ridge crests or hogbacks, formed by steeply dipping layers of shales and sandstones. The reservoir is approximately 10 km long, and is formed by four large, earth-filled dams; Horsetooth Dam closes the northern end of the valley, and Soldier Canyon, Dixon Canyon, and Spring Canyon Dams close breaches in the eastern hogback ridge created by pre-existing cross-valley drainages. The structural heights of the dams are 47, 69, 73, and 67 m, respectively. Construction of the four dams creating Horsetooth Reservoir occurred between 1946 and 1949. With a total off-channel storage capacity of approximately 1.9 \u00d7 108 m3, Horsetooth is one of 12 storage reservoirs built as part of the Colorado-Big Thompson Project, which stores, regulates, and diverts approximately 3.2 \u00d7108 m3 of water annually from the Colorado River headwaters on the west slope of the Continental Divide to the more heavily populated east slope. The project provides water for irrigated agriculture, municipal and industrial use, hydroelectric power generation, and water-based recreation. Discovery of sinkholes and increased seepage from the reservoir prompted a dam modernization project and reservoir drawdown that began in the fall of 2000 (B. Boaz personal communication, U.S. Bureau of Reclamation 2005).\nMethods\nField Sampling\nWe sampled vegetation along 13 transects that began 5 m into upland vegetation (above the reservoir\u2019s high water mark of 1654.6 m above sea level) and extended down slope (perpendicular to the shoreline) to an elevation of 1621.5 m asl. Below 1621.5 m asl, the reservoir begins to separate into distinct pools with different water surface elevations. Transects were located randomly along the entire length of the shoreline, with the exception of the following excluded areas: small, shallow coves on the west side of the reservoir; the four dams; and 200 m on either side of each dam.\nAlong each transect, we estimated the percent cover of every species present in 1-m2 plots in mid-September of 2001 and 2002. Plants were identified to species when possible using local and regional floras (Great Plains Flora Association 1986, Weber 1990). Plots were spaced variably depending on the steepness of the slope so that they were evenly distributed along the elevational gradient. On steep slopes, a plot was sampled every meter; on progressively gentler slopes, plots were sampled at intervals of 2, 3, or 4 m in order to achieve as close as practicable to 3 plots per m of elevation change. Transect lengths ranged from 89 to 382 m. Following exclusion of plots without clear hydrologic history as described below, the numbers of plots analyzed per transect ranged from 80 to 142 with a total of 1,345 plots each year. The proportion of surface area at each plot that was occupied by cobble-sized particles or larger (>64 mm diameter) was estimated in the field. A single elevation of each plot was determined using a total station surveying instrument, registered to the water surface elevation and tied to the gage measuring long-term reservoir water levels. Substrate characteristics and topography were measured in 2001. General observations in 2002 suggested that there had been little change in substrate or topography between years.\nRecords of water level fluctuation during the study period were combined with plot elevations to estimate when plots were exposed. Using this information, we divided the transects into four basic zones (from highest elevation to lowest): UPL, an upland zone above the level of reservoir inundation; TOP, former reservoir bottom exposed during the 1999 growing season and not inundated again during the study; MID, former reservoir bottom exposed during the 2000 growing season and not inundated again during the study; and BTM, former reservoir bottom exposed temporarily during the 2000, 2001, and 2002 growing seasons (Figs.\u00a01 and 2). Areas excluded from analysis consisted of (1) portions of transects below 1621.5 m asl where separate pools began to form in various portions of the reservoir; (2) a wave action zone, above the high water mark of the reservoir, but still disturbed; and (3) a narrow band between the MID and BTM zones that was exposed in 2000 and 2001 but was not re-exposed in 2002. The wave action zone and the zone exposed only in 2000 and 2001 each spanned less than 0.5 m of the elevation gradient and had too few plots for meaningful analysis.\nFig.\u00a01Reservoir water surface elevation and drawdown zones. Vegetation analysis is limited to four distinct elevation zones: UPL was never inundated; TOP was first exposed in 1999 and not subsequently; MID was first exposed in 2000 and not subsequently; BTM was first exposed in 2000 and was subsequently re-flooded and re-exposed in both 2001 and 2002Fig.\u00a02Horsetooth Reservoir when sampled in September 2001. At this time, the TOP01 zone was in the third year of exposure, MID01 in the second year, and BTM01 in the first year. Second-year seedlings of Populus deltoides subsp. monilifera are evident in the MID01 zone and several mature individuals are present near the full pool elevation of the reservoir before drawdown. Melilotus spp. strongly dominates the UPL01 zone here\nData Analysis\nPlant characteristics and communities\nPlant characteristics are summarized by elevational zone and sampling year with each combination denoted by the zone (BTM, MID, TOP, and UPL) subscripted by the last two digits of the year of sampling (2001 and 2002). The resulting eight zone-year combinations are grouped into classes based on the number of years since the zone was last exposed or drawn down (1, 2, 3, 4 years and Upland). There was no preexisting, rooted vegetation in any of these zones prior to their first exposure and no apparent survival of vegetation in the BTM zone between the two successive years it was drawn down.\nFractional nativity, duration, and wetland index were based on the aggregate species list for each zone-year-transect combination. Nativity was calculated as the fraction of species classified as native and duration was calculated as the fraction of species classified as perennial based on McGregor et al. (1986) and USDA-NRCS (2004) For example, a value of 0.6 for nativity would mean 60% of the species were classified as native and a value of 0.4 for duration would mean 40% of the species were classified as purely perennial (as opposed to annual, biennial or with a mixed duration). Wetland indicators (Reed 1988, USDA-NRCS 2004) for individual plants of Obligate (OBL), Facultative Wetland (FACW), Facultative (FAC), Facultative Upland (FACU), and Upland (UPL) were assigned numeric values of 1 to 5, respectively (Tiner 1999). Thus, values of the wetland index range from 1.0 for a hydric community composed entirely of obligate wetland species to 5.0 for a xeric community composed entirely of upland species.\nTotal vegetative cover within each zone-year-transect combination was adjusted for differences in substrate by analysis of covariance using rockiness (proportion of surface occupied by cobble or larger particles) as a covariate. This analysis (Proc Mixed, SAS 2003) fit separate relationships between an arcsine square root transformation of cover and a square root transformation of rockiness for each zone-year combination. We report total vegetative cover means and 95% confidence intervals for each zone-year combination adjusted to the grand mean of the covariate.\nDirect comparisons of observed species richness across zones and transects were not possible because of the different areas and numbers of plots sampled. We pooled all plots sampled in each zone-year combination, without regard to transect, in order to develop comparable estimates of species richness. We used two procedures. A first-order jackknife estimate of total richness is based on the number of observed species, the number of sampled plots, and the number of species observed in only one plot (McCune and Grace 2002). We also used a bootstrap procedure to estimate the total number of species as the asymptote of a fitted species-area curve. For each zone-year combination, we drew 50 random samples of each number of plots (1 to n = total number of plots), averaged the total number of species for each number of plots, and then fit a curve to the data [Michaelis-Menten equation following Inouye (1998)].\nDifferences in dominant species were evaluated by calculating species cover relative to the total cover within each zone-year-transect combination. Differences in overall species composition between zone-year combinations were evaluated using relative Sorensen distance (McCune and Meford 1999, McCune and Grace 2002) expressing a percent dissimilarity of the distribution of cover across species, normalized to the total cover within a zone-year combination. This index was based on cover values for each zone-year obtained by first averaging all plots within a zone-year-transect and then averaging across transects within each zone-year combination.\nCottonwood seedling establishment\nWe examined a priori expectations derived from Mahoney and Rood\u2019s (1998) recruitment box model concerning (1) the position of new seedlings relative to the water\u2019s edge during the period of seed availability and (2) rates of water level decline that could be survived by new seedlings. For these analyses, we pooled plots across transects and years. We then calculated average cover of first-year Populus deltoides subsp. monilifera seedlings for each year in elevational zones relative to the elevations of the water surface each year during the seed release and germination window. We used the period of June 1 to July 7 for seed release and germination based on Segelquist et al. (1993) and our antecdotal observations in Fort Collins from 1990 to 2005. To examine the effects of drawdown rate, we focused on plots within the elevational zone corresponding to the location of the water\u2019s edge during the germination window. These plots were both predicted and observed to have the highest probabilities of establishment. For each of these plot-year combinations, we estimated the drawdown rate as the difference between the water surface elevation on the last day the plot was inundated and the water surface elevation 45 days later. In some cases, especially in 2002 when drawdown was very rapid, the water surface elevation 45 days later was below the limit of 1621.5 m asl at which pools formed in the reservoir bottom and reliable estimates of water surface at individual transects were not possible. In these cases, we used 1621.5 m asl as a lower bound of water surface elevation, thus producing a conservative (low) estimate of the rate of decline experienced by seedlings. Average cover of Populus deltoides subsp. monilifera seedlings was calculated for classes of drawdown rate by pooling observations of current year germinants in both 2001 and 2002.\nResults\nPlant Characteristics and Communities\nThe percentage of native species was relatively constant over time with median values ranging from 56% for BTM02 exposed for one year to 40% for TOP02 exposed for four years (Fig.\u00a03). There was no suggestion of a trend for the early colonizing communities to be shifting towards the higher proportions of native species associated with the upland that had median percentages of native species of 75% and 71% in the two years of sampling. In contrast, both duration and wetland index showed clear trends of increasing over the first four years of exposure toward values observed in the upland zone (Fig.\u00a03). Median percentages of perennial species were 20% and 21% in the first year of exposure (BTM01 and BTM02); 31% after two years (MID01); 32% and 38% after three years (MID02 and TOP01); and 46% after four years (TOP02). The median percentage of perennial species in the upland zone was 86% in both 2001 and 2002. Median values for wetland index in the first year of exposure were 2.4 and 2.6 (BTM01 and BTM02), which are in the range of wetland vegetation communities (Tiner 1999). Wetland index increased steadily over time to 4.2 in the fourth year (TOP02) approaching the xeric value of 4.7 for both 2001 and 2002 in the upland zone (Fig.\u00a03).\nFig.\u00a03Vegetation characteristics by drawdown zone and time exposed. The unit of replication is a transect within each zone-year combination. Wetland index, fraction native, and fraction perennial are calculated for each plot based on species presence and then averaged across plots for each transect within a zone-year combination. Tops and bottoms of boxes represent 75th and 25th percentiles, the horizontal line is the median, whiskers (vertical lines) include the minimum of 1.5 times the inter-quartile range and the range of the data, and values outside the whiskers are represented by asterisks\nPatterns in both species richness and cover were complicated by differences among sampling years in precipitation and among zones in substrate and sampled area. The second year of sampling, 2002, was drier than 2001, which contributed to lower total 2002 cover in the upland zone and lower numbers of identifiable species.\nWe recorded a total of 124 distinct taxa. In some cases these were combinations of species that could not be reliably separated in the field at the time of sampling. Examples among the more dominant species include conflation of Bromis japonicus and B. tectorum, and Melilotus spp. which included Melilotus alba and M. officinalis. Differences in estimation procedures for total species richness did not appreciably change the relative patterns (Fig.\u00a04). Based on the first-order jackknife estimate, species richness was 52 and 53 species in the first year following exposure (BTM01 and BTM02) compared to 78 and 53 species in the upland zone (UPL01 and UPL02). There was some suggestion of an intermediate peak in richness in the second (96 for MID01) and third (93 for MID02 and 59 for TOP01) years of exposure.\nFig.\u00a04Species richness and adjusted total cover by drawdown zone and time exposed. In the top graph, asterisks are the asymptotic richness from Michaelis-Menten species-area curves fit to bootstrapped sets of 1-m2 plots pooled across transects (Inouye 1998); open circles are first-order jack-knife estimates of richness (McCune and Grace 2002). In the bottom graph, solid circles are estimates of mean total cover adjusted to the grand mean substrate value from an analysis of covariance using transect within a zone-year combination as the unit of replication. The vertical lines from the solid circles are 95% confidence intervals for these estimated means\nThe substrate area composed of large particles (>64 mm diameter) was a significant predictor of total cover (P < 0.001), with more rocky substrate associated with lower total cover (Fig.\u00a04). Total plant cover, adjusted with analysis of covariance to the mean fraction of large particles, was 21\u201336% in the first year of exposure (BTM01 and BTM02). There was some suggestion of a decline in cover within the first four years of exposure, rather than a trend in the direction of the generally higher (37\u201358%) upland cover values.\nSpecies composition was most similar between zone-year combinations within a given time of exposure (Table\u00a01). Species composition of exposed areas became progressively more similar to the upland composition with increasing time of exposure. Composition at one year of exposure was 99% dissimilar to the uplands, and composition at four years of exposure was 83\u201384% dissimilar to the uplands. There was, however, substantial turnover of species within the first four years, with generally large dissimilarities between the different years (e.g., 94% dissimilarity between one and four years exposed).\nTable\u00a01Community dissimilarity matrix. Distance measure is a Sorensen percent dissimilarity using mean cover values for each zone-year combination and relativized to zone-year unit totals (McCune and Grace 2002). Values range from 0% for identical species cover composition to 100% for no similarity of species cover composition. Matrix is symmetrical around main diagonal. Main diagonal entries are indicated by dashes and are 0 by definintionYears exposedZone yearRelativized Sorenson distance (% dissimilarity)Years exposed1234UplandBTM01BTM02MID01MID02TOP01TOP02UPL01UPL021BTM01\u201415588087949999BTM0215\u20146579889499992MID015865\u201451587998983MID02807951\u201458799897TOP0187885858\u20145091924TOP029494795450\u20148483UplandUPL01999998979184\u201419UPL0299999897928319\u2014\nThere were also substantial shifts in the individual dominant species related to time exposed (Table\u00a02). The introduced annual Chenopodium glaucum, with a FACW wetland indicator value, strongly dominated the first-year communities, was the second most dominant species two years after exposure, but was strongly reduced in the third year and absent from fourth-year and upland zones. Panicum capillare, a native annual, was the second most dominant species in the first year of exposure, increased to the most dominant species in the second year, declined in the third and fourth years, and was absent from the upland zone. The native Rorippa curvipes is classified as an obligate (OBL) wetland plant and was important in the first year of exposure, declined to a very minor presence in the second year, and was absent in the third-year, fourth-year, and upland communities.\nTable\u00a02Relative cover of selected species by elevational zone and time exposed. Relative cover is calculated at the transect level for each zone-year combination and then averaged across transects. All species with cover ranks among the top four in any zone-year combination are included. Dashes indicate absenceSpeciesCharacteristicsRelative Cover (%) by Years Exposed and Sampled ZoneDurationNativityWetland indicator1234UplandBTM01BTM02MID01MID02TOP01TOP02UPL01UPL02Amaranthus albusANFACU2.27.13.72.6\u2014\u2014\u2014\u2014Ambrosia tomentosaPNUPL<0.1<0.14.69.3\u2014\u2014\u2014\u2014Bromus inermisPEUPL\u2014<0.1<0.1<0.1\u20140.313.011.0Bromus japonicus - B. tectorumAEUPL\u2014\u2014<0.10.21.74.324.718.7Cercocarpus montanusPNUPL\u2014\u2014\u2014\u20140.62.310.417.1Chenopodium glaucumAEFACW57.856.115.80.4\u2014\u2014\u2014\u2014Cirsium arvensePEFACU0.20.11.813.30.83.51.00.7Ericameria nauseosaPNUPL\u2014\u2014<0.10.210.39.21.71.8Lactuca serriolaAEFAC\u2014\u20142.02.912.011.7\u2014\u2014Melilotus spp.A\/BEFACU0.20.19.55.915.10.70.5\u2014Panicum capillareANFAC10.410.326.27.28.01.2\u2014\u2014Populus deltoides subsp. moniliferaPNFAC4.41.82.43.50.40.5\u2014\u2014Rhus aromaticaPNUPL\u2014\u2014\u2014\u2014\u2014\u201413.29.2Rorippa curvipesA\/B\/PEFACW5.30.9<0.1\u2014\u2014\u2014\u2014Salsola collinaAEUPL\u2014\u20140.915.12.913.3\u20140.3Suckleya suckleyanaANFACW3.57.20.10.2\u2014\u2014\u2014\u2014Verbascum thapsusBEUPL<0.1<0.11.32.511.111.7\u2014\u2014Verbena bracteataA\/PNUPL0.20.29.610.39.94.0<0.1\u2014A = annual, B = biennial, P = perennial, N = native, E = exotic, W = wetland, FACW = facultative wetland, FAC = facultative, FACU = facultative upland, UPL = upland\nSome of the dominant species in the second through fourth years of exposure were substantially less important or absent from both the first year of exposure zone and the upland zone. These included Cirsium arvense, Ericameria nauseosa, Lactuca serriola, Salsola collina, Verbascum thapsus, and Verbena bracteata. The conflated Bromus japonicus and B. tectorum, both introduced, dominated the upland zone. This group was absent in the first year of exposure and gradually increased in relative cover in the second through fourth years. The other most dominant species in the upland, Bromus inermis, Cercocarpus montanus, and Rhus aromatica, were unimportant or absent in essentially all of the first four years of exposure (Table\u00a02).\nCottonwood Seedling Establishment\nPlains cottonwood (Populus deltoides subsp. monilifera) was the dominant tree species colonizing the exposed surfaces (Table\u00a02). Mature individuals occurred in the upland near the margin of the full-pool reservoir, although none were sampled in the upland plots on randomly located transects. Seedlings of Salix amygdaloides, S. exigua, Populus angustifolia, P. tremuloides, and Tamarix ramossima were present, but rare in the drawdown zones along sampled transects. First-year cottonwood (Populus deltoides subsp. monilifera) seedlings were generally distinguishable from previous year germinants and root sprouts by their cotyledons and absence of bud scale scars when sampled in mid-September. New seedlings were strongly concentrated in, or slightly above, the elevational band occupied by the water\u2019s edge during the period of seed release of June1 to July 7 (Fig.\u00a05). However, smaller numbers of new seedlings were spread over a considerable range of elevations from 2 m above the zone exposed during the germination window to more than 5 m below the optimum zone. Drawdown rate had a strong influence on the cover of first-year seedlings as sampled near the end of the growing season (Fig.\u00a06). For seedlings established in the optimum band of the wetted edge during the germination window, cover decreased substantially at drawdown rates greater than 4\u20138 cm\/day. However, the range of drawdown rates survived by seedlings was wide, with new seedlings present with measurable cover at rates of greater than 24 cm\/day. The zone of maximum cover of current year germinants was 10.2 to 12.2 m above the water level on September 1 in 2001 and 7.2\u201311.7 m above in 2002, using the conservative 1621.5 m asl elevation at which separate pools formed in the reservoir. Actual observed water levels at the dam on September 1 were 1618.8 m asl in 2001 and 1618.1 m asl in 2002. Average elevations of all plots containing older than first-year Populus deltoides subsp. monilifera seedlings in 2001 were 9.6 m above the maximum water level in that year and 21.8 m above the conservative 1621.5 m asl value for September 1 water level; in 2002, older seedlings were 7.5 m above the maximum and 19.5 m above the conservative September 1 level.\nFig.\u00a05Elevational distribution of first-year Populus deltoides subsp. monilifera seedlings in relation to the elevations exposed during the germination window of June 1 to July 7. Means \u00b1 1 SE are based on plots in each elevational class pooled across transects and sampling yearsFig.\u00a06Cover of first-year Populus deltoides subsp. monilifera seedlings (established in the zone of the wetted edge during the germination window) in relation to rate of drawdown. Means \u00b1 1 SE are based on plots in each drawdown class pooled across transects and sampling years. Drawdown is calculated for the 45-day period following the date a plot was last exposed\nDiscussion\nNilsson et al. (1997) distinguished land as pre-upland or pre-riparian in an analysis of vegetation response to inundation from dam construction. This distinction is similarly useful in describing the response of vegetation to dam removal. Large fractions of the land under the reservoir pools of the shallow dam removal sites in Wisconsin examined by Orr and Stanley (2006) and Lenhart (2000) were pre-riparian. Contingent on changes in topography from sediment accumulated behind the dam and the geomorphic response to dam removal, pre-riparian areas might be expected to support marshland or riparian vegetation following dam removal (Shafroth et al. 2002).\nIn contrast, we sampled slightly more than 30 meters of drawdown at Horsetooth Reservoir. Essentially all of the area exposed was pre-upland. If this had been a permanent dam removal, the exposed area would not be expected to support a wetland or riparian plant community in the long term. The patterns of vegetation change we observed are a combination of the initial colonization of a riparian, bare ground disturbance patch and the vegetation response to a shift from mesic to xeric site conditions. The mesic to xeric transition is reflected in the wetland index value, which systematically increased from a hydric or wetland community in the first year of exposure to upland or xeric value in the fourth year of exposure, very similar to the upland values of the surrounding landscape vegetation. In contrast, Lenhart (2000) reported wetland index values at sites 3\u20135 years following removal of shallow dams in Wisconsin that were still indicative of hydric or wetland communities. The species composition at the Horsetooth site changed dramatically over the first four years of exposure (Tables\u00a01 and 2), but had not yet approached that of the surrounding upland plots.\nThe initial colonizing community of a bare surface might be expected to have higher fractions of short-lived and perhaps non-native species than later vegetation on the site. At the Horsetooth site, the fraction of perennials steadily increased through four years of exposure, whereas the fraction of native species was below that of the surrounding uplands and did not change appreciably or consistently. Substrate-adjusted mean values for total cover were lower than the upland and appeared to decline somewhat in the second through fourth years of exposure. A possible explanation for these patterns in cover and richness is that the mesic conditions in the first year of exposure produce a reasonably high plant cover composed heavily of annual species. This is followed by a period of reorganization, replacing annuals with deeper rooted perennials. Species richness peaks during this transition, whereas total cover tends to drop in the drier conditions of years two through four following exposure and only gradually increases to the ultimately higher cover of a slowly developing perennial community present on the dry upland sites.\nThe cottonwood recruitment box model of Mahoney and Rood (1998) expresses the regeneration niche of these disturbance-dependent, pioneer, riparian tree species and makes predictions about where recruitment will occur along rivers in central and western North America in relation to water surface elevations (Rood et al. 2005). Cottonwood seedling establishment requirements are well known from multiple laboratory (Fenner et al. 1984, Mahoney and Rood 1991, Segelquist et al. 1993, Amlin and Rood 2002) and field studies (Rood et al. 1998, Auble and Scott 1998, Shafroth et al. 1998, Rood and Mahoney 2000, Rood et al. 2005). Seedling recruitment tends to occur on bare ground sites that are moist during an early summer period of seed dispersal and germinability, and that remain moist enough for seedlings to survive drought stress. More specifically, for Populus deltoides subsp. monilifera, the model describes suitable establishment sites as (1) bare ground, largely created by fluvial disturbance; (2) wetted by water surface elevations during a 3- to 6-week window of seed release, dispersal, and germinability; (3) subject to water table declines of no more than 2.5 cm\/day; and (4) generally occurring at elevations from 60 to 200 cm above the water surface elevation of late summer base flow (Mahoney and Rood 1998).\nThe Horsetooth drawdown provided bare, wetted surfaces over a much greater elevational range than would be associated with riparian floodplain sites or removal of a shallow dam. Predictions of the recruitment box model were met in two respects. First, the highest cover of first-year P. deltoides subsp. monilifera occurred in the elevation zone corresponding to the water\u2019s edge during the estimated June 1 to July 7 period of estimated maximum seed release and germinability for the area (Fig.\u00a05). Second, cover of first-year seedlings dropped sharply at locations subject to drawdown rates >8 cm\/day (Fig.\u00a06). However, there was appreciable seedling establishment well outside the bounds predicted by the recruitment box model. Wave action, capillary rise, and plot heterogeneity in elevation are likely explanations for the substantial establishment in locations 0\u20131 m above the water\u2019s edge. The establishment at much lower elevations as far as 5\u20136 m below the water\u2019s edge on July 7 is more difficult to explain. It appears that the extreme drawdown rates created suitable bare moist sites far below the elevation of the optimum, but close enough to the period of maximum seed availability to allow some of the tail end of the distribution of germinable seed to establish.\nWe observed maximum cover at drawdown rates of 4\u20138 cm\/day compared to the upper limit of 2.5 cm\/day used in the recruitment box model, and we observed some seedlings surviving first-year drawdown rates of greater than 24 cm\/day. Some survival of seedlings at drawdown rates of 4 and 8 cm\/day has been reported in laboratory experiments (Mahoney and Rood 1991) and the value of 2.5 cm\/day is probably better viewed as the upper end of the most suitable range of rates rather than as an absolute limit. The rapid Horsetooth drawdown rates produced seedling establishment at high elevations relative to late summer water levels. The elevation zone with maximum cover of first-year seedlings was at least 10 m above the water level on September 1 in 2001 and at least 7 m above the September 1 level in 2002. This was not purely a first-year phenomenon as the average elevations of older than current-year seedlings were well above the range of current year water levels, more than 7 m above the maximum level of the current year and more than 19 m above the level on September 1 of the current year. Mahoney and Rood (1998) discuss several factors that may support establishment at high elevations relative to the water\u2019s edge including capillary rise, lateral subsurface water movement, and advantageous sequences of precipitation events. Given that some exposed surfaces at Horsetooth Reservoir were on the sides of sharp ridges with very limited contributing drainage area, we believe the most likely explanation for the high elevation establishment we observed is local variation in particle size and subsurface drainage (i.e., fine sediment in cracks between boulders) that create pockets of adequate moisture retained from inundation and channeled from rainwater-derived percolation.\nImplications for Dam Removal Decisions\nGiven the multiple dimensions in which dams differ (Poff and Hart 2002) and the relative paucity of long-term dam removal data sets, evaluations and decisions about dam removal in the near future will be based largely on case studies and site assessments rather than on statistical conclusions from a sampled population of previous removals. A first-order approximation of future vegetation within a former reservoir pool can be obtained by considering topography of the new surface in relation to the new water surfaces; substrate characteristics; the pre-dam vegetation and its character as pre-upland or pre-riparian; and typical wetland, riparian, and upland vegetation communities in the region (Shafroth et al. 2002). For shallow dams constituting the vast majority of dams removed to date (Hart and Poff 2002), the exposed surfaces will be at elevations close to the surface of the new river and a large fraction of the exposed area would have been riparian or wetland before inundation. For tall dams and the drawdown case we report, the vast majority of the exposed land is pre-upland, not significantly influenced by a river before dam construction, substantially above the water surface following removal or exposure, and likely to support upland vegetation typical of the region.\nThere are two primary ways such a first approximation could be misleading. The first is that the post-dam topography and substrate characteristics may have been altered substantially by the period of inundation. This is especially true if the dam has accumulated substantial quantities of trapped sediment that have altered topography, stored nutrients or contaminants, or altered organic content or particle size distributions. Our results showed a significant effect of substrate size proportions on total vegetation cover; however, Horsetooth Reservoir was not accumulating substantial sediment because of the diversion source of most of the impounded water.\nThe second way a first approximation might be misleading is that the character of the transition between extended inundation to post-dam site conditions will influence vegetation response over some time scale, at least on the order of the life spans of the initially colonizing plants. Erosion of accumulated sediment may produce substantial changes in post-dam topography, likely resulting in a topography intermediate between the pre-dam and immediately exposed conditions. This was not a major factor at Horsetooth Reservoir because of the relative absence of accumulated sediment. On the other hand, extended inundation is an abnormal source of disturbance to initiate a bare ground plant succession for pre-upland surfaces such as those exposed by the Horsetooth Reservoir. Thus, the initially colonizing plant community was considerably more mesic (lower wetland indicator score) than the community that might colonize a dry, bare-ground surface created by a disturbance such as fire. In semi-arid landscapes, establishment of pioneer riparian tree species such as P. deltoides subsp. monilifera is almost always associated with hydrologic disturbance and bare ground sites near the water\u2019s edge, although mature trees can often persist on much drier sites. Thus, successful establishment of riparian cottonwood during the drawdown may leave a transient legacy of vegetation on the exposed reservoir surfaces that would be atypical of the sites either before reservoir construction or over the long term following dam removal. These patterns are very much a function of the specifics of reservoir drawdown and are thus potentially amenable to management as part of a dam removal. For example, cottonwood would be unlikely to colonize the bulk of pre-upland first-year exposure sites if the drawdown occurred in winter and the area was dry during the early summer period of seed release. On the other hand, the summer drawdowns observed at Horsetooth probably increased total plant cover on the exposed surfaces, which might be desirable from the perspective of erosion mitigation.\nWeed control on the former reservoir pool is a consideration in many dam removal evaluations. Initial colonization of the reservoir pools at Horsetooth was by a ruderal community (Menges and Waller 1983, Grime 2001) with high fractions of short-lived species, including invasive, non-native weeds. This is a reasonable expectation of a riparian bare ground disturbance patch and generally for reservoir pool surfaces that might be exposed by dam removal where non-native species are common in the regional species pool (e.g., Orr and Stanley 2006, Lenhart 2000). Effective weed control is complicated in the case of a tall dam. We observed a dramatic turnover of dominant species during the first four years and substantial changes in wetland character of the vegetation in response to drier site conditions. This suggests that (1) species-focused weed control will have to be maintained over multiple years with shifting targets as some of the weeds are diminishing naturally and being replaced by other species; and (2) planting of a desired or transition crop to preempt space and resources from invading weeds will have to consider rapidly changing site conditions to be effective in both the initial drawdown and immediately subsequent years.\nStanley and Doyle (2003) suggested that dam removal is best viewed as a new disturbance with a consequent set of positive and negative effects, rather than a simple reversal of the original impacts of dam construction. Sediment transport is one clear example of hysteresis, where the pulse release of accumulated sediment can produce a post-removal response that is not a simple mirror image or reversal of the response to dam construction. Vegetation of the former reservoir pool is another such asymmetrical response. The existing plant community is eliminated quickly (weeks to months) following inundation of the reservoir pool from dam construction. Recovery follows a different path on a different time scale. Lenhart (2000) suggested that the initial establishment of stinging nettle (Utrica dioica) and rice-cut grass (Leersia oryzoides) on sediment of the former reservoir pool may be suppressing the development of woody vegetation on shallow dam removal sites in Wisconsin. We saw little indication of this type of effect, as the dominant species continued to shift rapidly in the first four years of the Horsetooth Reservoir drawdown. However, the changes we observed in the first four years suggest that recovery of the vegetation community will be on the time scale of years to decades. Vegetative cover, composition of dominant species, and proportions of native and perennial species were still changing after four years and were substantially different from the upland community representing the likely long-term vegetation. Furthermore, the establishment and survival of cottonwood in the drawdown zone suggests that the particular nature of the transition from decades of continuous inundation to a xeric condition may leave a legacy signature in the vegetation community at least on the decades to century time scale corresponding to the lifespan of cottonwood.","keyphrases":["dam removal","drawdown","horsetooth reservoir","colorado","riparian","recruitment box model","cottonwood","reservoir margin"],"prmu":["P","P","P","P","P","P","P","R"]}
{"id":"Histochem_Cell_Biol-4-1-2386534","title":"The human keratins: biology and pathology\n","text":"The keratins are the typical intermediate filament proteins of epithelia, showing an outstanding degree of molecular diversity. Heteropolymeric filaments are formed by pairing of type I and type II molecules. In humans 54 functional keratin genes exist. They are expressed in highly specific patterns related to the epithelial type and stage of cellular differentiation. About half of all keratins\u2014including numerous keratins characterized only recently\u2014are restricted to the various compartments of hair follicles. As part of the epithelial cytoskeleton, keratins are important for the mechanical stability and integrity of epithelial cells and tissues. Moreover, some keratins also have regulatory functions and are involved in intracellular signaling pathways, e.g. protection from stress, wound healing, and apoptosis. Applying the new consensus nomenclature, this article summarizes, for all human keratins, their cell type and tissue distribution and their functional significance in relation to transgenic mouse models and human hereditary keratin diseases. Furthermore, since keratins also exhibit characteristic expression patterns in human tumors, several of them (notably K5, K7, K8\/K18, K19, and K20) have great importance in immunohistochemical tumor diagnosis of carcinomas, in particular of unclear metastases and in precise classification and subtyping. Future research might open further fields of clinical application for this remarkable protein family.\nIntroduction\nMost eukaryotic cells contain in their cytoplasm a more or less elaborated cytoskeletal system consisting of intermediate filaments (IF), which are chemically very stable long and unbranched filaments of ~10\u00a0nm in diameter. Among the various families and subfamilies of IF proteins, that of the keratins is outstanding due to its high molecular diversity. The keratin gene family consists of the highest number of members in humans with 54 distinct functional genes. IF proteins are expressed in a highly cell type-specific manner, and herein keratins represent the typical IF category of epithelial cells. In some but not all epithelia, keratin filaments are conspicuously bundled as tonofilaments. Figure\u00a01 shows these keratin filament bundles at the light microscopical (Fig.\u00a01a, b) and the electron microscopical level (Fig.\u00a01c, d). Inside the cell they braid the nucleus (Fig.\u00a01a), span through the cytoplasm and are attached to the cytoplasmic plaques of the typical epithelial cell\u2013cell junctions, the desmosomes (Fig.\u00a01b, d; for a recent review, see Waschke 2008). This feature already suggests that keratins play a major functional role in the integrity and mechanical stability of both the single epithelial cells and, via cell\u2013cell contacts, of that of the epithelial tissues. Consequently, they are inherent part of the continuum of stability from the single cell to the tissue formation. Evidence for this main function of keratin filaments has been amply provided by the recognition of various hereditary keratin diseases and transgenic mouse models. In addition, however, various regulatory functions have been discovered more recently (for recent reviews, see Magin et al. 2007; Oshima 2007; Uitto et al. 2007; McLean and Irvine 2007).Fig.\u00a01Cytoskeleton of epithelial cells. a Immunofluorescence staining of keratin K18 (red, nuclei stained in blue by DAPI) in PLC (liver carcinoma) cells in vitro. b Keratin filaments (in red) and the desmosomal component desmoplakin (in green) are labeled in cultured keratinocytes of line HaCaT. c Electron microscopic image of tonofilament (keratin) bundles (arrowhead) of HaCaT keratinocytes. d Keratin intermediate filaments (black arrowhead) insert at desmosomes (white arrowhead) at cell\u2013cell contact sites of keratinocytes of the epidermal stratum spinosum (electron microscopy)\nHistorically, keratin research started with studies of sheep hair (wool) keratins (Crick 1952; Powell and Rogers 1986; Oshima 2007). Several important discoveries were made in the 1970s of the last century. One was the finding of the spontaneous self-assembly and polymerization of keratin filaments from denatured, soluble keratin proteins by dialysis in vitro (Steinert et al. 1976). Further milestones were the findings that antibodies against keratins from epidermis-type epithelia such as the bovine muzzle (\u201cprekeratin\u201d) react with tonofilaments in various epithelial cells including non-stratified \u201csimple\u201d epithelia of inner organs (Franke et al. 1978), and that keratins of various mammalian species exhibit a high degree of molecular diversity with differentiation-specific expression (Franke et al. 1981). Systematic protein biochemical analyses of human cells and tissues by one- and two-dimensional gel electrophoresis, Western blotting and peptide mapping disclosed the diversity of human (cyto)keratin polypeptides (Moll et al. 1982b; Tseng et al. 1982; Wu et al. 1982). From these data, in 1982, the catalog of human cytokeratins including 19 members was proposed (Moll et al. 1982b) which, although intended as provisional, has been widely accepted and used. Along with these studies and subsequently, the principle of separation of these proteins into type I (\u201cacidic\u201d) and type II (\u201cbasic to neutral\u201d) keratins (see below) also emerged. Another unique property of the keratins is that in contrast to the other IF proteins they only can constitute their filamentous stage by heteropolymeric pair formation of type I and type II (1:1) molecules. Later on, several new keratins were identified and added to the cytokeratin catalog, the most notable of these being the simple-epithelial keratin 20 (K20; Moll et al. 1990, 1992) and several keratins specific for distinct epithelia such as keratin K2e in the upper epidermis (appendix \u201ce\u201d; now K2), K2p in the upper hard palate epithelium (similar to K2e but with appendix \u201cp\u201d for palate; now K76) (Collin et al. 1992a, b), or several keratin K6 (K6a\u2013h) isoforms (Takahashi et al. 1995). Simultaneously, informations about the keratin gene sequences were revealed.\nMoreover, within the last 10\u00a0years a large number of hair follicle-specific epithelial keratins were discovered. This series started with K6hf (appendix \u201chf\u201d stands for \u201chair follicle expression\u201d, now K75), which was expressed in the hair follicle companion layer (Winter et al. 1998). K75 was the first epithelial keratin specifically expressed in the hair follicle. Surprisingly, there were much more epithelial keratins with hair follicle specificity, namely the type II keratins K6irs1, K6irs2, K6irs3 and K6irs4 (now K71\u2013K74) and type I keratins K25irs1, K25irs2, K25irs3 and K25irs4 (now K25\u2013K28), all of them specifically expressed in and closely restricted to the various compartments of the hair follicle inner root sheath (Langbein et al. 2002, 2003, 2006; for review see Langbein and Schweizer 2005). Besides the variety of epithelial (\u201csoft\u201d or \u201ccyto-\u201d) keratins, hairs and nails are built up from a somewhat separate subfamily of \u201chard\u201d or \u201ctrichocytic\u201d keratins, commonly designated as hair keratins (Heid et al. 1988a; Langbein et al. 1999, 2001, 2004; Langbein and Schweizer 2005; Schweizer et al. 2007). They differ from the epithelial keratins by their considerably higher sulfur content in their non-\u03b1-helical head and tail domains, which is mainly responsible for the extraordinary high degree of filamentous cross-linking by keratin-associated proteins (KAPs) (for review, see Rogers et al. 2006).\nVery recently, the \u201cKeratin Nomenclature Committee\u201d established the novel consensus nomenclature for mammalian keratin genes and proteins (Schweizer et al. 2006), relying upon and systematically extending the aforementioned 1982 catalog. This nomenclature is now in accordance with the nomenclature of the Human Genome Organization (HUGO) for both the gene and protein names. Following the unified new principles, several parts of the former nomenclature were implemented; the hair keratins (e.g. \u201cHa\u201d and \u201cHb\u201d) and the special epithelial keratin designations (e.g. K2p, K6hf, K6irs, K25irs) were equally integrated (see Table\u00a01), and the nomenclature system\u2014although now complete for humans\u2014is open to application in other mammalian species by following the same principles. Among human keratins, the new consensus nomenclature (Table\u00a01) comprises the type I keratins K9\u2013K10, K12\u2013K28, and K31\u2013K40 (including K33a and K33b) and the type II keratins K1\u2013K8 (including K6a, K6b and K6c) and K71\u2013K86. Thus, there are 28 type I keratin genes (17 epithelial keratins and 11 hair keratins) and 26 type II keratin genes (20 epithelial keratins and 6 hair keratins). All in all, out of the 54 human keratin genes, at least 26 (~50%) are specifically expressed in the hair follicle. In the human genome, the keratin genes are clustered at two different chromosomal sites: chromosome 17q21.2 (type I keratins, except K18) and chromosome 12q13.13 (type II keratins including K18). The keratin genes are designated as KRT1, KRT2, KRT3, etc. (Schweizer et al. 2006; Fig.\u00a02a).Fig.\u00a02Human keratins. a Organization of the human keratin genes in the genome. The type I and type II keratin gene subdomains are located on chromosomes 17 and 12, respectively. The type I keratin K18 is located in the type II cluster on chromosome 17 (arrow). b Two-dimensional catalog of the human keratin proteins according to molecular weights (MW) and isoelectric points (IEP) as calculated from amino acid sequences. The keratin genes are designated according to the new keratin nomenclature (Schweizer et al. 2006)Table\u00a01The new human keratin nomenclature (Schweizer et al. 2006)For K1\u2013K20 (in gray), the numbering of the original catalog (Moll et al. 1982b, 1990) has been maintained. The respective gene names (\u201cKRT\u201d) by the human genome consortium utilize the same numbers, e.g. \u201cKRT20\u201d\nAll these keratins belong to the family of IF proteins and therefore share common protein-structural characteristics. They contain a central rod domain of ~310 amino acids with \u03b1-helical conformation flanked by non-helical head and tail domains of variable length. The head domain consists of subdomains V1 and H1. The central \u03b1-helical rod domain is composed of subdomains 1A, 1B, 2A, and 2B connected by the linkers L1, L12, and L2. The tail domain then consists of subdomains H2 and V2 (Lane and McLean 2004; Parry et al. 2007; Geisler and Weber 1982). The molecular weight of human keratins ranges from ~44 to ~66\u00a0kDa (Fig.\u00a02b). A unique feature of keratins, including the hair keratins, is their pairing, i.e. the obligate formation of heterodimers between one type I keratin and one type II keratin. This occurs by association of the corresponding rod domains in \u03b1-helical coiled-coil conformation. The resulting heterodimers and -tetramers form the basic building units of the keratin filaments. Single keratin proteins deviating from equimolar type I\/type II amounts are rapidly degraded (Lu and Lane 1990).\nAs keratin filaments are important structural stabilizers of epithelial cells, there is unabatedly high interest in keratins in biology, embryology, pathology, and dermatology. Notably, this main cytoskeletal function transcends the single cell level. Typically, keratin filaments insert at desmosomes (Fig.\u00a01b, d) and hemidesmosomes. Thus, they contribute not only to the stability between epithelial cells itself but also to basement membrane attachment and insofar to the connective tissue compartment of a given epithelium. In the non-stratified (simple) epithelia of internal parenchymatous organs, which experience little mechanical stress, only very few keratin members form sparse and loosely distributed keratin filaments in the cytoplasm. Otherwise, considerably more members take part on the IF cytoskeletal composition of squamous epithelia which increases in the cornified stratified epithelia such as in the epidermis lining the outer body surface where they are abundant and densely bundled as tonofilaments. The loose filaments in the former case are composed of \u201csimple-epithelial keratins\u201d like K8\/K18 (and K19), while the bundled filaments (tonofilaments) in the latter case (Fig.\u00a01c, d) are built up from keratinocyte-type keratins such as K5\/K14 in the basal layer and\u2014with even more pronounced bundling\u2014K1\/K10 in the suprabasal layers and K2\/K10 in the uppermost ones. The \u201crule\u201d that the \u201cstronger\/harder\u201d the epithelial structure the more keratin members are involved culminates in the hair fiber where 17 keratins are sequentially expressed. This clearly underscores the importance of the keratins for the tissue integrity and the relevance of the molecular diversity of keratin proteins.\nThe important mechanical function of stratified epithelial- and epidermis-type keratins is evident and proven not only through knock-out mouse models but also through various human hereditary keratin diseases. Thus, point mutations of distinct keratin genes now widely explain the pathogenesis of several autosomal-dominant familial diseases, many of which are blistering skin diseases. The most well known of these inherited skin fragility disorders is epidermolysis bullosa simplex (EBS), the various variants of which are caused by a spectrum of point mutations of K5 or K14 (Lane and McLean 2004; McLean and Irvine 2007; Uitto et al. 2007). Nineteen different keratin genes including hair keratins and hair follicle-specific epithelial keratins have up to now been identified as being involved in pathogenic keratin mutations (Lane and McLean 2004; Schweizer et al. 2007); they will be discussed in the descriptions of the individual keratins below. Updated details may be retrieved from an Internet database (Human Intermediate Filament Database; http:\/\/www.interfil.org). Notably, the knock-out experiments and the genetic diseases demonstrated that mutations in keratin genes often (depending on the locus of mutation within the keratin molecule) cause more severe defects than the complete loss of a keratin gene whose failure might be compensated\u2014if available at this site\u2014by another\/other keratin\/s.\nMoreover, it has been recognized that keratins are not simply static intracellular skeletal structures but rather are highly dynamic. Along with this view, besides their mechanical function new functional roles of keratins have been defined and still emerge under special physiological conditions (Magin et al. 2007; Oshima 2007). These include the protection of the placental and trophoblast barrier function (K8\/K18\/K19: Jaquemar et al. 2003; Hesse et al. 2000), the protection from apoptosis (K8: Caulin et al. 2000; Ku et al. 2003b; K17: Tong and Coulombe 2006), the protection of the liver against stress and from injury (K8\/K18: Zatloukal et al. 2000; Ku et al. 2003a), and the regulation of protein synthesis and cell size during wound healing involving intracellular signaling pathways (K17: Kim et al. 2006). Keratins may also play a role in epithelial polarity and membrane traffic (Oriolo et al. 2007). Thus, keratins obviously exert widely varying signaling functions beyond their mechanical roles.\nBeyond their biological functions, keratin expression patterns not only characterize cells as \u201cepithelial\u201d, they are also characteristic for distinct\u2014including the terminal\u2014stages during cellular epithelial differentiation from embryonal to adult or of the internal maturation program during development. Epithelial tumors\u2014including metastases\u2014most widely retain the keratin patterns of their (normal) epithelial origin; thus, the determination of the keratin patterns of tumors are being widely exploited for cell and tumor typing. Therefore, keratins have evolved to be one of the most potent epithelial differentiation and tumor markers in cell biology, embryology, and surgical pathology. Specific antibodies against several keratins are routinely used world wide in pathology laboratories for immunohistochemical typing of carcinomas in tumor diagnostics. Numerous papers published since 1980 deal with the application of keratins as marker proteins in tumor pathology (Oshima 2007), and several previous review articles (e.g. Lane and Alexander 1990; Nagle 1994; Schaafsma and Ramaekers 1994; Moll 1998; Chu and Weiss 2002b) cover this field of application, which also will be especially considered in this review.\nAnother clinical application is the detection of soluble keratin protein fragments derived from K8, K18, and K19 in the circulation of cancer patients; such fragments\u2014released by carcinoma cells\u2014are increasingly used to monitor tumor load and disease progression in the case of certain carcinomas such as non-small cell lung cancer (Barak et al. 2004; Linder 2007). Through analysis of different K18 fragments in the serum it is also possible to assess the type of chemotherapy-induced tumor cell death and distinguish between apoptosis and necrosis, due to the fact that K18 is cleaved at specific sites during apoptosis and a monoclonal antibody (M30) specific for caspase-cleaved forms of K8 is available (Leers et al. 1999; Linder et al. 2004; Linder 2007).\nHuman keratins and their expression patterns\nIn the following, the different human keratins and keratin pairs will be discussed together with their cell type and tissue distribution (summarized in Table\u00a02) and their functional significance in relation to transgenic mouse models and human hereditary keratin diseases. Furthermore, characteristic expression patterns in human tumors (summarized in Table\u00a03) and their possible diagnostic relevance will be considered.Table\u00a02Characteristic expression patterns of typical keratins in selected human normal epithelial tissuesKeratins of simple epitheliaKeratins of stratified epitheliaType II keratins:K8K7K5K6K1K2K4Type I keratins:K18K19K10K14K15K16K17K10K9K13Parenchymatous epitheliaaAll cellsDuctal epithelia of parenchymatous organsbAll cellsK7 and K19 in all cellsSparse cellsSparse cellsK4 heterogeneously in pancreatic ductsGastrointestinal epitheliaAll cellsK19 in all cellsGastric foveolar epithelium, intestinal epitheliumcK4 heterogeneously in luminal cells; K13 in sparse luminal cellsRespiratory epitheliumPredominantly luminal cellsK7 in luminal cells; K19 in all cellsPredominantly basal cellsK6 in basal cellsPredominantly basal cellsUrotheliumAll cellsK7 and K19 in all cellsLuminal (umbrella) cellsBasal cellsFew basal cellsK13 in all basal and intermediate cellsNon-keratinizing stratified squamous epitheliaSome basal cellsK19 in many basal cellsBasal cell layer (predominantly)Basal cells layerSuprabasal compartmentAt some sites focal expression in suprabasal cellsSuprabasal compartmentEpidermisPredominantly basal cell layerBasal cell layerSuprabasal compartmentK2 in upper spinous and granular layer; K9 in palmoplantar epidermisdNot included are the corneal keratins K3\/K12, the gingival\/hard palate keratin K76, the eccrine sweat gland-specific keratin K77, and the hair follicle-specific epithelial and hair keratins (for keratin expression in eccrine sweat glands and in the hair follicle, see the schematic drawings in Figs.\u00a05 and 6)aIncluding hepatocytes, acinar cells of pancreas, proximal tubular cells of kidneybBile ducts, pancreatic ducts, renal collecting ductscMost villus- and surface-lining cells; scattered cells in cryptsdHeterogeneous expression in the suprabasal compartmentTable\u00a03Characteristic expression patterns of typical keratins in selected human carcinomasOnly diagnostically relevant data as detectable by monoclonal antibodies widely established in clinical pathology are presented (for references, see Moll 1998; Chu and Weiss 2002b; and text). Explanation of symbols: filled circle, extended staining of most tumor cases; open dotted circle, focal\/heterogeneous staining of some but not all cases; open circle, no stainingaIn rare cases focal staining may be observedbFocal or extended staining in a subpopulation of tumor cases, corresponding to the basal-like phenotypecNon-mucinous typesdPreferentially\/more extended in poorly differentiated caseseIn rare cases focal staining may be observed; however, squamous cell carcinomas of the cervix uteri may express K7 extendedly\nSimple (one-layered) epithelia\nK8\/K18: primary keratins of simple epithelial cells\nThe keratins K8 and K18 typically are co-expressed and constitute the primary keratin pair of simple epithelial cells, including various parenchymatous epithelia (Franke et al. 1981; Moll et al. 1982b; Owens and Lane 2003). They are the first keratins to appear in embryogenesis, as early as in pre-implantation embryos (Jackson et al. 1980), and also seem to be the oldest keratins during phylogenesis (Blumenberg 1988). In some epithelial cell types, K8 and K18 are the sole keratins present. The classical example is the liver, with K8\/K18 representing the characteristic and only keratin pair of normal hepatocytes. The same is true for other highly specialized parenchymatous epithelia such as acinar cells of the pancreas, proximal tubular epithelial cells of the kidney, and certain endocrine cells such as pancreatic islet cells. Ultrastructurally, keratin filaments of this simple composition are loosely distributed within the cytoplasm and show little bundling. In other simple, one-layered epithelia such as duct-lining cells, intestinal cells, and mesothelial cells, additional simple-epithelial keratins (K7, K19, and\/or K20; see below) are present in addition to the primary pair K8\/K18. Furthermore, K8\/K18 occur\u2014together with other keratins\u2014in various pseudostratified (e.g. respiratory) and complex (e.g. glandular) epithelia and in the urothelium; in these composite epithelial tissues, K8 and K18 are often most prominent in the lumen-lining cells. Even in non-keratinizing stratified squamous epithelia, K8 and K18 may be focally expressed in the basal cell layer, together with K19 and the constitutive stratified-epithelial keratins (Bosch et al. 1988; Moll 1993). Thus, K8 and K18 are widely distributed among normal epithelial tissues although they are absent in differentiating keratinocytes. It should be noted that K8 and K18 are not strictly epithelium-specific since expression of K8 and K18 may occur in rare mesenchymal cells (more frequently in fetal stages) such as certain smooth muscle cells and fibroblastic reticulum cells of lymph nodes as well as various mesenchymal tumors including rhabdo- and leiomyosarcomas (Huitfeldt and Brandtzaeg 1985; Franke and Moll 1987; van Muijen et al. 1987; Jahn and Franke 1989; Knapp and Franke 1989; Knapp et al. 1989; Jahn et al. 1993; Gould et al. 1995; Kuruc and Franke 1988; Langbein et al. 1989), where they are co-expressed with other intermediate filament types, notably vimentin and desmin.\nWhile highly specialized parenchymatous epithelial cells in their normal state, such as proximal tubular cells of the kidney, only express K8 and K18, this may change in reactive conditions. Upon various types of injury such as inflammation or atrophy these cells may additionally switch on K7 and K19, sometimes also K17 (as well as vimentin) and thus express four to five instead of two keratins (Moll et al. 1991). This increased keratin expression appears to parallel the reduction in the degree of differentiation. Thus, the keratin pattern of a given epithelial tissue may be modulated to some extent in the course of reactive changes, frequently resulting in higher complexity of keratin composition.\nAlready the tissue distribution of K8\/K18\u2014mainly in internal epithelia\u2014suggests that structural and mechanical functions are not their key roles, although their absence or dysfunction may be associated with hepatocyte and trophoblast fragility (for references, see Magin et al. 2007). Instead, genetic knock-out experiments have revealed distinct regulatory functions of these keratins (Magin et al. 2007; Oshima 2007). They play a role in protecting the placental barrier function (K8: Jaquemar et al. 2003) and protecting cells\u2014in particular liver cells\u2014from apoptosis (K8: Caulin et al. 2000; Ku et al. 2003b), against stress, and from injury (K8\/K18: Zatloukal et al. 2000; Ku et al. 2003a), possibly by functioning as a phosphate \u201csponge\u201d for stress-activated kinases (K8: Ku and Omary 2006). Interestingly, K8 and K18 may play a role in the regulation of the cell cycle, whereby phosphorylation of these keratins and binding of 14-3-3 adaptor proteins seem to be involved (Toivola et al. 2001; Ku et al. 2002; Margolis et al. 2006; Galarneau et al. 2007; Magin et al. 2007). In human pathology, defects in K8 and K18 may predispose to liver diseases, in particular cryptogenic liver cirrhosis (Ku et al. 2003a; Zatloukal et al. 2004), as well as to chronic pancreatitis and inflammatory bowel disease (for references, see Owens and Lane 2004). Altered K8 and K18 proteins, together with several stress proteins, in particular ubiquitin and p62, constitute the hyaline protein aggregates of hepatocytes of several (e.g. alcoholic) liver diseases now known as Mallory\u2013Denk bodies (Zatloukal et al. 2007).\nIn regard to malignant tumors, K8 and K18 are expressed in most carcinomas except for some differentiated squamous cell carcinomas. Therefore, K8 and K18 antibodies strongly stain most adenocarcinomas, hepatocellular carcinomas, renal cell carcinomas, and neuroendocrine carcinomas. Since highly sensitive monoclonal antibodies against these keratins are available, such as the classical mouse monoclonal CAM5.2 clone against K8 (Makin et al. 1984) and clone Ks18.04 against K18 (B\u00e1rtek et al. 1991), these keratins may be helpful in diagnostic immunohistochemistry in cases of carcinomas with low keratin content such as small-cell lung cancer, to prove their epithelial nature. Regarding carcinoma subtyping, negative or weak\/focal immunostaining for K8 and K18 may indicate squamous cell differentiation, although strong expression of these keratins can occur particularly in poorly differentiated squamous cell carcinomas (see below, chapter \u201cKeratins as diagnostic markers in tumor pathology\u201d). In the case of breast carcinomas, certain publications have reported a correlation between the level of K8 or K18 immunostaining and a favorable prognosis for the patients (see below, chapter \u201cKeratins as diagnostic markers in tumor pathology\u201d).\nAnother clinical application of K8\/K18 is the monitoring of fragments of these keratins in the serum as serological tumor markers to monitor cancer load, cancer progression, and response to therapy. Among the oldest of these markers are tissue polypeptide antigen (TPA) and tissue polypeptide-specific antigen (TPS) which have been recognized to correspond to a mixture of K8, K18, and K19 (Weber et al. 1984) and to K18 (Rydlander et al. 1996), respectively (for review, see Linder 2007). More recently, an apoptosis-specific fragment of K18 as detected by monoclonal antibody M30 (Leers et al. 1999) has become of increasing interest for distinguishing between necrosis and apoptosis and for the evaluation of the chemotherapy response of carcinomas by investigation of cancer patient serum, e.g. in prostate, breast, and lung cancer (Linder et al. 2004; Linder 2007).\nK7\/K19: secondary keratins of simple epithelial cells\nApart from K8\/K18, keratins K7 and K19 are \u201cadditional\u201d (secondary) and also widely distributed simple-epithelial keratins which are frequently but not always co-expressed. They typically occur as a keratin pair in simple ductal epithelia such as bile and pancreatic ducts (\u201cductal-type\u201d keratins). However, in several epithelia lacking K7 such as intestinal epithelium, the type I keratin K19 must form a pair with the sole type II keratin K8.\nThe type I keratin K19 is the smallest keratin and is exceptional since it widely lacks the non-\u03b1-helical tail domain typical for all other keratins (Bader et al. 1986). It may have evolved from keratinocyte keratins (Stasiak et al. 1989). As detectable by several specific and well-tested monoclonal antibodies (Karsten et al. 1985; B\u00e1rtek et al. 1986; Nagle et al. 1986), K19 exhibits a rather broad tissue distribution. It is expressed in most simple epithelia (excluding parenchymatous cells such as hepatocytes, pancreatic acinar cells, and renal proximal tubular cells), notably in various ductal epithelia, in small and large intestinal epithelium, in gastric foveolar epithelium, and in mesothelium. Furthermore, it is present in most cells of pseudostratified epithelia and urothelium as well as in basal cells of non-keratinizing stratified squamous epithelia.\nFunctionally, keratin K19 is dispensable since K19 knock-out mice were viable, fertile, and appeared normal (Harada et al. 1999). This is apparently due to functional compensation by K18, since only mice, double deficient for K18 and K19 exhibited a severe phenotype with trophoblast fragility and early embryonic lethality (Hesse et al. 2000). No mutation of the human K19 gene causing a disease has yet been found (Owens and Lane 2004).\nThe expression of K19 may be induced in certain epithelia that normally lack this keratin by pathological alterations. One example is damage to renal proximal tubular epithelia by various types of injury as discussed above (Moll et al. 1991). K19 induction is also observed in suprabasal stratified squamous epithelial cells of oral mucosa with epithelial dysplasia (Lindberg and Rheinwald 1989; for further references, see Moll 1998), but also with inflammation (Bosch et al. 1989; Moll 1993), so that K19 cannot be used as a specific marker for dysplasia in oral mucosa. In carcinomas, K19 is widely expressed in both adenocarcinomas and squamous cell carcinomas and therefore is not extensively used as an immunohistochemical marker for carcinoma subtyping. One example for such application may be, in liver tumors, the distinction of hepatocellular carcinomas, which show little expression of K19, from cholangiocarcinomas and adenocarcinoma metastases, which strongly stain for this keratin (Balaton et al. 1988; Goldstein and Bosler 2006; see Table\u00a03). The detection of soluble K19 fragments in the serum released by carcinoma cells by the CYFRA 21-1 assay has found broad clinical application as a marker to monitor treatment and evaluate response to therapy and has proven particularly useful in the case of squamous cell carcinomas of the lung (for review, see Barak et al. 2004, Gu and Coulombe 2007).\nThe type II keratin K7, another \u201cductal-type\u201d keratin, has a basically similar but somewhat more restricted tissue distribution as compared to K19 (Moll et al. 1982b; Ramaekers et al. 1990). Like K19, it is expressed in several simple ductal epithelia, in mesothelium, in pseudostratified epithelia (preferentially in luminal cells), and in urothelium but absent in parenchymatous cells such as hepatocytes. However, K7 is sparsely expressed or absent in gastric foveolar epithelium, intestinal epithelium, and stratified squamous epithelia. Human and mouse K7 genes have been characterized (Glass and Fuchs 1988; Smith et al. 2002), but mutations or disease associations have not yet been reported (Owens and Lane 2004).\nSeveral monoclonal antibodies against K7 have been described, some of which (e.g. Ks7.18, OV-TL12\/30) are well reactive with formalin-fixed, paraffin-embedded tissues. Of these, clone OV-TL12\/30 (van Niekerk et al. 1991) appears to elicit the broadest span of immunoreactivity and has found wide application in diagnostic tumor pathology, especially in cases where primary tumors or metastases are uncertain. Since the majority of carcinomas are K7 positive, negative reactions are of particular diagnostic significance. One main point of diagnostic utility of this keratin is the negative (or weak\/focal) K7 immunostaining in colorectal adenocarcinomas (see Fig.\u00a03c), in contrast to the strong staining in most other adenocarcinomas (see Fig.\u00a03e; Moll et al. 1992, 1993b; Chu et al. 2000; Chu and Weiss 2002b; Tot 2002). This is especially valuable for classifying adenocarcinoma metastases with regard to their possible primary tumor. Low K7 expression (negative or weak immunostaining, or staining of a minor proportion of tumor cells) is also a characteristic feature of conventional (clear-cell) renal cell carcinomas (as opposed to papillary and chromophobe carcinomas) (Moll 1998; Skinnider et al. 2005) and of (non-cervical) squamous cell carcinomas. The validity in predicting the primary tumor in cases of unclear metastases is significantly increased when K7 is used in combination with K20 (see below) since many carcinomas exhibit characteristic K7\/K20 phenotypes (Moll et al. 1992, Moll et al. 1993b; Chu et al. 2000; Chu and Weiss 2002b; Tot 2002; Dabbs 2006).Fig.\u00a03Keratins in simple epithelia and adenocarcinomas (paraffin sections of human tissues; avidin\u2013biotin complex peroxidase staining). Keratin K20 is a characteristic and prominent keratin of the foveolar epithelium of the gastric (a) and colorectal (b) mucosa. The K7\u2212\/K20+ phenotype of the normal mucosa is mostly maintained in primary and metastatic colorectal adenocarcinomas. This is shown here for a skin metastasis (on the head) of a poorly differentiated adenocarcinoma: the tumor cells are negative for K7 (c) but positive for K20 (d, left portion of the figure), even including a tumor cell cluster that has invaded a lymphatic capillary (d, right upper corner). This phenotype is strongly suggestive of colorectal origin. A primary tumor in the right colon was detected later. Liver metastasis of a ductal adenocarcinoma of the pancreas with typical keratin pattern, showing extended expression of K7 (e) and staining of scattered tumor cells for K20 (f). Magnifications: a, d \u00d780; b, c, e, f \u00d7140\nK20: keratin of gastrointestinal epithelium, urothelium, and Merkel cells\nK20 is the simple-epithelial keratin with the most restricted expression pattern. Although it appeared in our early cytoskeletal preparations of intestinal epithelial cells as a quite prominent protein spot of ~46\u00a0kDa, where we tentatively designated it as \u201cIT protein\u201d (from intestinal; Moll et al. 1982b), we succeeded rather late in identifying it as a type I keratin (Moll et al. 1990). For pair formation, its type II partner usually appears to be K8. The remarkable expression spectrum of K20 among normal tissues comprises gastric foveolar epithelium (Fig.\u00a03a) and small and large intestinal epithelium (Fig.\u00a03b; \u201cgastrointestinal-type\u201d keratin) and, in addition, the urothelium and certain neuroendocrine cells, in particular Merkel cells of the skin. In human embryogenesis, it appears in the small intestinal epithelium at embryonic week 8 (Moll et al. 1993b). Within the gastrointestinal and urothelial epithelial tissues, K20 is absent from the stem cell compartment and appears to be switched on during terminal differentiation and thus is most prominent in small intestinal villus-lining and large intestinal surface-lining epithelia (Fig.\u00a03b) and in urothelial umbrella cells. Although K20 is a keratin typically expressed in simple epithelia it is also found in the lone basally located Merkel cells of the epidermis and hair follicle outer root sheath (Moll et al. 1992, 1995).\nOur knowledge about the function of K20 still is limited. Mutations of human K20 or associated diseases have not yet been described (Owens and Lane 2004). Transgenic experiments suggest a role for K20 in maintaining keratin filaments in intestinal epithelia (Zhou et al. 2003). In mouse small intestinal epithelium, K20 phosphorylation on serine 13 is induced during apoptosis and tissue injury and thus may serve as stress marker (Zhou et al. 2006).\nAmong the K20-specific monoclonal antibodies available, clone Ks20.8 reacts well on routine paraffin sections (Moll et al. 1992) while clone Ks20.10 reacts with the K20 homologue of rodents (Moll 1993). K20 is a potent immunohistochemical marker in tumor pathology since its peculiar expression spectrum is essentially maintained in the corresponding primary and metastatic carcinomas (Moll et al. 1992), and the K20 clone Ks20.8 has become part of the routine antibody panel in most pathology laboratories. Most colorectal adenocarcinomas (Fig.\u00a03d), the majority of gastric adenocarcinomas, the majority of transitional cell carcinomas, as well as most Merkel cell carcinomas are K20-positive (Moll et al. 1992, 1993b; Miettinen 1995; Chu et al. 2000; Chu and Weiss 2002b; Goldstein and Bosler 2006). In a few other carcinoma types, variable and focal K20 expression is seen, notably in ductal adenocarcinomas of the pancreas (Fig.\u00a03f) and in adenocarcinomas of the biliary tract including cholangiocarcinomas of the liver (Moll et al. 1992; Miettinen 1995; Chu et al. 2000). Among ovarian carcinomas, K20 is mainly detected in the mucinous type. Most other carcinomas, including adenocarcinomas, irrespective of their morphology, are essentially negative for K20. Thus, significant K20 positivity of a metastatic adenocarcinoma is predictive of a primary tumor in the gastrointestinal or pancreaticobiliary tract. It should be noted that the diagnostic value is increased when the markers K20 and K7, are applied in combination. For example, a K7\u2212\/K20+ phenotype of an adenocarcinoma metastasis (Fig.\u00a03c, d) strongly favors a colorectal origin. Some colorectal adenocarcinomas co-express K7 in addition to K20, but as a general rule, the level of K20 immunostaining exceeds that of K7 (see below, chapter \u201cKeratins as diagnostic markers in tumor pathology\u201d).\nUsing RT-PCR analyses, K20 mRNA can be detected in cell preparations from bone marrow and peripheral blood from some colorectal cancer patients, indicating the presence of disseminated tumor cells that maintain K20 expression, and a number of clinical studies have shown that this is correlated with a worse prognosis (Soeth et al. 1996; Funaki et al. 1998; Wyld et al. 1998; Koch et al. 2005; Katsumata et al. 2006; Friederichs et al. 2007). In addition to patients with metastatic colorectal carcinoma, K20 expression in the peripheral blood was also detected in patients with metastatic gastric and pancreatic adenocarcinoma but hardly in patients with metastatic lung carcinoma (Chausovsky et al. 1999), underlining the tumor type-specific expression of this keratin.\nStratified epithelia\nK5\/K14: major keratins of basal keratinocytes\nThe type-II keratin K5 and the type-I keratin K14 form the primary keratin pair of the keratinocytes of stratified squamous epithelia, including the epidermis as well as mucosal non-keratinizing stratified squamous epithelia (Moll et al. 1982b). They are strongly expressed in the undifferentiated basal cell layer containing the stem cells and are down-regulated in the differentiating suprabasal cell layers (Fig.\u00a04a; Fuchs and Green 1980). Otherwise, in the widely well stratified follicular outer root sheath, K5 and K14 are uniformly expressed throughout all layers. Moreover, the follicular companion layer, which is directly adjacent to the outer root sheath (and formerly considered as \u201cinnermost layer of the outer root sheath\u201d), is completely negative for both of these keratins and expresses an own special keratin, K75 (see below). Ultrastructurally, K5\/K14 keratin filaments are bundled as tonofilaments and attached to desmosomes and hemidesmosomes. The mouse K5 promoter is frequently used in transgenic experiments to promote epidermis-specific expression of transgenes (e.g. Oki-Idouchi and Lorenzo 2007). In addition to their occurrence in keratinocytes, K5 and K14 are expressed in basal and myoepithelial cells of complex and glandular epithelial tissues (Purkis et al. 1990). On the other hand, these keratins are absent from most simple\/one-layered epithelia, with very few exceptions, notably the mesothelium lining serous cavities (Moll et al. 1989) and the amnion epithelium.Fig.\u00a04Keratins in stratified squamous epithelia and squamous cell carcinomas (paraffin sections of human tissues; avidin\u2013biotin complex peroxidase staining). In the epidermis as an example of a normal stratified squamous epithelium, the basal cell layer contains abundant keratin K5 (a) whereas the differentiating suprabasal compartment strongly stains for K10 (b; note the negative basal cell layer). Lymph node metastasis of a squamous cell carcinoma of the head and neck region, expressing K5 (c; more intensely in the peripheral tumor cell layers) as well as K6 (d; particularly strongly in central tumor cells) as signs of their keratinocyte origin. Keratin K5 is also maintained in a lymph node micrometastasis of a squamous cell carcinoma of the head and neck region (e) and in a lymph node metastasis of an undifferentiated nasopharyngeal carcinoma with dissociated growth pattern of the tumor cells (f), in these examples being a diagnostically helpful feature. Magnifications: a, b \u00d7160; c\u2013e \u00d780; f \u00d7140\nSeveral monoclonal antibodies (MAbs) specific for K5 and K14 have been described that helped to reveal their exact tissue distribution. These antibodies include MAb AE14 (Moll et al. 1989) against K5 and MAbs LL001 and LL002 against K14 (Purkis et al. 1990). The best performance on paraffin sections is displayed by MAb D5\/16B4 (Lobeck et al. 1989; Demirkesen et al. 1995) which\u2014although being often regarded as \u201cK5\/K6 antibody\u201d\u2014specifically recognizes K5 (B\u00f6cker et al. 2002).\nThe functional importance of K5 and K14 for the physical stability of the epidermis has become clearly evident by the recognition that dominant-negative mutations of the K5 or the K14 gene cause the hereditary blistering skin disease epidermolysis bullosa simplex (EBS) (for reviews, see Omary et al. 2004; Lane and McLean 2004). Most of these mutations are missense or small in-frame deletion mutations, primarily affecting the keratin rod domain. The presence of mutated K5 or K14 results in increased fragility of the basal keratinocytes so that even mild physical trauma leads to intraepidermal cytolysis of basal cells and the formation of fluid-filled blisters. These patient-related findings were preceded by the experimental demonstration that expression of mutant K14 in transgenic mice causes abnormalities similar to EBS (Vassar et al. 1991).\nThe expression spectrum of K5 and K14 in tumors corresponds well to the patterns in normal epithelia. Thus, most squamous cell carcinomas (Fig.\u00a04c, e) as well as malignant mesotheliomas strongly express these keratins whereas little, focal, or no expression is found in adenocarcinomas (Moll et al. 1982b, 1989; Moll 1998; Chu and Weiss 2002a, b). Hence, these keratins, in particular K5, have found several lines of diagnostic application in pathology, which has been aided by the availability of a highly sensitive and specific, robust, paraffin-suited MAb (D5\/16B4; see above). Pertinent examples are the recognition and diagnosis of poorly differentiated squamous cell carcinomas, including micrometastases in lymph nodes (Fig.\u00a04e), of undifferentiated nasopharyngeal carcinomas which may be diagnostically difficult due to their dissociated growth pattern (Fig.\u00a04f), and of malignant mesotheliomas. Thus, K5 immunostaining allows the distinction of the small cell type of squamous cell carcinoma of the lung, which is K5+, from a small cell carcinoma or a poorly differentiated adenocarcinoma, both of which are K5\u2212 (Moll 1998; Chu and Weiss 2002a), and the distinction of a malignant mesothelioma of the pleura (K5+) from a pulmonary adenocarcinoma with pleural involvement (K5\u2212) (Moll et al. 1989; Ordonez 1998; Yaziji et al. 2006). In well and moderately differentiated squamous cell carcinomas, K5 is preferentially localized in the peripheral layers of the tumor cell formations (Fig.\u00a04c), corresponding to the K5 expression in the basal cell layer of normal stratified squamous epithelia. Focal K5 expression may be observed in certain adenocarcinoma types, notably in adenocarcinomas of the endometrium, the ovary, and the pancreas, which seems to be related to their potency for focal squamous differentiation (Moll 1998; Chu and Weiss 2002a, 2002b). Much interest has evolved regarding the role of K5 in breast pathology in several aspects, including the identification of myoepithelial cells, the classification of proliferative lesions (Otterbach et al. 2000), and the recognition of a certain subtype of invasive ductal breast carcinoma (see below, chapter \u201cKeratins as diagnostic markers in tumor pathology\u201d). In prostate pathology, the diagnosis of prostatic adenocarcinoma is supported by the immunohistochemical demonstration of absence of K5-positive basal cells (Abrahams et al. 2002, 2003).\nK15: basal keratinocyte keratin and hair follicle stem cell \u201cmarker\u201d\nK15 was first identified as a minor keratin of human epidermis, by gel electrophoresis of cytoskeletal preparations (Moll et al. 1982b, c). Its sequence demonstrated its assignment to the type I keratins (Leube et al. 1988). Only later its cellular distribution was recognized, and it was uncovered that K15 is a specific basal cell component of the epidermis (Moll et al. 1993a; Lloyd et al. 1995) and other stratified squamous epithelia (Waseem et al. 1999) (see below for its significance in the hair follicle). Frequently, K5 and K14 can also be detected in the lower suprabasal cell layers (see above and Fig.\u00a04a). Whereas the mRNA synthesis of these keratins is restricted to the basal layer, the K5 and K14 proteins remain integrated in the complex keratin cytoskeleton for some time when cells leave the basal compartment. Thus, they may be stained by immunohistochemistry in more or less suprabasal layers depending on the epitope of the antibody used. In comparison, K15 seems completely restricted to the basal cell layer of stratified squamous epithelia (Lloyd et al. 1995; Waseem et al. 1999) where it can form heteropolymeric filaments with K5 (Lloyd et al. 1995).\nMAbs recognizing K15 include clone LHK15 (Waseem et al. 1999) and clone C8\/144B (Lyle et al. 1998, 1999). The latter in fact is a surprising and exciting MAb originally made against the lymphocyte antigen CD8 which cross-reacts with K15 especially in the basal keratinocytes of the hair follicle bulge region, where the respective stem cells are assumed. Also polyclonal K15 antibodies have furthermore been described (Moll et al. 1993a; Kurzen et al. 2001, Langbein et al. 2008). K15 expression in basal cells is downregulated in activated epidermal keratinocytes such as in organotypic cultures and in hyperproliferation (Waseem et al. 1999) or upon wounding (Porter et al. 2000). Human mutations or knock-out mice for K15 have not yet been described.\nAs to the occurrence of K15 in tumors, the literature is inconclusive as to whether there is differential expression of K15 among cutaneous tumors (benign follicular tumors versus basal cell carcinomas), possibly due to the use of different antibodies1 (see also above), and thus a putative diagnostic application of K15 immunohistochemistry in dermatopathology is still open (Kanitakis et al. 1999; Jih et al. 1999; Porter et al. 2000; Kurzen et al. 2001). Currently, the most interesting feature with K15 is the discovery that within the hair follicle at least one antibody against this keratin detects putative stem cells residing in the hair follicle bulge and thus might be used as a stem cell marker in hair follicle biology (Lyle et al. 1999).\nK6\/K16: keratins of hyperproliferative keratinocytes inducible in \u201cactivated\u201d epidermis\nBy gel electrophoresis, the type-II keratin K6 and the type-I keratin K16 have been identified in epidermis only in samples from plantar glabrous skin while in hairy skin these keratins appeared to be absent in interfollicular epidermis but were clearly present in hair follicle outer root sheath (Moll et al. 1982b, c; Langbein and Schweizer 2005) and companion layer (Winter et al. 1998; Langbein and Schweizer 2005; Gu and Coulombe 2007). In nail epithelia, K6 and K16 are constitutive components (Heid et al. 1988b; Lane and McLean 2004; Perrin et al. 2004). K6 and K16 are also consistently expressed in non-keratinizing stratified squamous epithelia (Moll et al. 1982b). They are often but not always co-expressed as a keratin pair (Moll et al. 1982b; Langbein et al. 2005). Molecular genetic studies have revealed that in humans three isoforms of K6 exist, K6a, K6b, and K6c, encoded by distinct genes (Rogers et al. 2005; Schweizer et al. 2006).\nMAb KA12 is an antibody which most probably stains at least keratin K6a isoform and reacts well with paraffin sections (Demirkesen et al. 1995; Langbein et al. 2003; Schmelz et al. 2005) although isoform-specific antibodies against K6a, K6b or K6c will hardly be made because of their extremely high peptide sequence homology. Using this MAb, plantar epidermis shows extended albeit heterogeneous expression of K6a, while interfollicular epidermis is negative or exhibits only some positive suprabasal cell groups (Demirkesen et al. 1995; Swensson et al. 1998). Non-keratinizing stratified squamous epithelia express K6 uniformly in all suprabasal cell layers. A MAb against K16 has also been described (Leigh et al. 1995).\nIn early immunohistochemical studies using keratin group-specific MAbs, Weiss et al. (1984) demonstrated the induction of K6 (56-kd keratin) and K16 (48-kd keratin) in various hyperproliferative epidermal disorders, suggesting that these keratins may be molecular markers for hyperproliferative keratinocytes. More recent experimental studies showed that after skin wounding, K6 and K16 are rapidly induced within 6\u00a0h in human keratinocytes at the wound edge, before migration and regeneration begins (Paladini et al. 1996). The particular cell biological properties of these keratins may confer to activated keratinocytes on the one hand a moderate level of mechanical scaffolding and on the other hand sufficient plasticity required during migration and re-epithelialization (Wawersik et al. 2001). K6a knockout mice showed delayed re-epithelialization after skin wounding (Wojcik et al. 2000). Upon K6a\/K6b double knock-out, mice exhibit epithelial disintegration and white plaques in the dorsal tongue epithelium (Wong et al. 2000; Wojcik et al. 2001). The absence of a hair and nail phenotype in these mice\u2014as might be expected from the occurrence of K6 in these appendages\u2014has been shown to be due to the presence and compensatory function of other K6-related keratins in hair follicles and nails, such as mK6hf (Wojcik et al. 2001; now mK75) or mK6irs1\u2014mK6irs4 (now mK71\u2013mK74; cf. Langbein et al. 2002, 2003).\nIn man, mutations in K6a or K16 have been proven to give rise to the hereditary disorder pachyonychia congenita type 1 (Jadassohn\u2013Lewandowsky form) that manifests with thickened nails, palmoplantar hyperkeratosis, and oral leukoplakias (McLean et al. 1995; for further references, see Lane and McLean 2004). Notably, the pathologic changes affect those tissues that constitutively express K6a and K16 (see above) but not the interfollicular epidermis.\nThus, K6\/K16 are constitutive keratins of stratified epithelia built up by keratinocytes of relatively high proliferative state such as mucosal tissues, palmoplantar epidermis, and certain skin appendages. On the other hand, they are \u201cstress-inducible\u201d keratins in interfollicular epidermis, being rapidly switched on e.g. after injury and UV-irradiation or being present also in inflammation and in hyperproliferative disorders.\nExpression of these keratins is not restricted to stratified squamous epithelia but may also be observed in certain glandular structures. Thus, K6 (most probably K6a) and K16 are expressed in ductal luminal cells as well as in some secretory cells of human eccrine sweat glands (Fig.\u00a05; Demirkesen et al. 1995; Langbein et al. 2005). K6 has been detected in subpopulations of luminal cells of the mouse mammary gland (Grimm et al. 2006) and the human mammary gland; in the latter, also in ductal myoepithelial cells (Hesse 2003; Langbein et al. 2005). Recently, a population of K6-positive cells in the prostate gland with high potential for proliferation and differentiation has been described (Schmelz et al. 2005).Fig.\u00a05Keratin K77 in eccrine sweat glands and adnexal tumors. K77 mRNA and protein by in situ hybridization (ISH, a, a\u2032) and indirect immunofluorescence (IIF, b-b\u2032\u2032) microscopy. This keratin is specifically expressed in the luminal cells (lc) of the intraglandular (igd), the intradermal (idd), the sweat duct ridge (sdr) and the intraepidermal\/acrosyringial (ied\/ac) duct of eccrine sweat glands of plantar skin. Peripheral duct cells are negative. The secretory portions (asterisks) are K77-negative. sb str. basale, ss str. spinosum, sg str. granulosum, sc str. corneum, gl glandular region with secretory and intraglandular duct portions. c Expression scheme of keratins of the eccrine sweat gland. d-d\u2032\u2032 Immunoperoxidase staining of K77 in the luminal cells of the eccrine sweat gland duct of the foot sole epidermis. K77 is also expressed in the tubular structures (arrows) of eccrine tumors such as syringoma (e) and cylindroma (f). Bars 100\u00a0\u03bcm\nIn the pseudostratified epithelia of respiratory mucosal tissues, K6 and K16 are highly upregulated in squamous metaplasia (Leube and Rustad 1991; Stosiek et al. 1992). Among tumors, K6 and K16 are typically and strongly expressed in squamous cell carcinomas of different sites (Moll et al. 1982b; Moll 1998), preferentially in inner, maturing layers of the tumor cell nests (Fig.\u00a04d). Although low expression of these keratins may be found in adenocarcinomas such as occasionally in adenocarcinomas of the uterine cervix (Smedts et al. 1993) and in less than 20% of invasive breast carcinomas (Wetzels et al. 1991), K6 as detected by MAb KA12 may be suitable\u2014in addition to K5\u2014as another immunohistochemical marker of squamous differentiation in poorly differentiated squamous cell carcinomas (Moll 1998).\nK17: keratin of basal\/myoepithelial cells and inducible in \u201cactivated\u201d keratinocytes\nThe type I keratin K17 was identified in our early gel electrophoretic studies as a major keratin of basal cell carcinomas of the skin which was also present in the normal pilosebaceous tract but not in normal epidermis (Moll et al. 1982c). Further protein analyses showed its presence in squamous cell carcinomas of various origins as well as in normal glandular tissues (such as sweat glands and breast) but its apparent absence also from non-keratinizing stratified squamous epithelia (Moll et al. 1982b, 1983). The unique cell type distribution of this keratin became apparent after establishment of a specific MAb, clone E3 (Troyanovsky et al. 1989). Broad tissue screening revealed its selective expression in basal and myoepithelial cells of complex tissues, including various glands, respiratory epithelium, and urothelium (Troyanovsky et al. 1989, 1992). Thus, K17 may be regarded as a \u201cbasal-\/myoepithelial cell keratin\u201d. In the hair follicle, confirming and extending previous biochemical findings (Moll et al. 1982c), K17 has been localized as a prominent component of the suprabasal cell layers of the outer follicular root sheath (Winter et al. 1998; Langbein and Schweizer 2005; Langbein et al. 2006; Tong and Coulombe 2006). It is also present in nail bed and nail matrix epithelia (McGowan and Coulombe 2000; Perrin et al. 2004). Immunohistochemistry also confirmed the essential absence of K17 from adult interfollicular epidermis, but, interestingly, revealed its specific expression in the specialized epidermal keratinocytes of the sensory Merkel cell-associated \u201chaarscheiben\u201d organs (Moll et al. 1993a); this keratin thus may be applied as a sensitive haarscheiben marker in studies of cutaneous neurobiology. In contrast to the adult, K17 is a prominent component of fetal epidermis (Moll et al. 1982a) as well as of cultured epidermal cells (Weiss et al. 1984).\nAnother interesting feature of K17 is its inducibility after skin injury: after K6\/K16 (see above), K17 is switched on in regenerating and migrating epidermal keratinocytes upon wound healing (Paladini et al. 1996). Its functional importance in wound healing is suggested by the observation that K17 knockout mouse embryos show a delay in the closure of surface ectoderm wounds (Mazzalupo et al. 2003). Recent transgenic experiments have shown that K17 can bind to the adaptor protein 14-3-3\u03c3 and influences cell growth and size of mouse keratinocytes by regulating protein synthesis (Kim et al. 2006).\nThe aforementioned expression of K17 in the pilosebaceous tract (in the follicular outer root sheath, companion layer, medulla and sebaceous gland; for review, see Langbein and Schweizer 2005) has also proven to be functionally relevant. K17 null mice develop transient severe alopecia in early postnatal life, correlating with hair fragility and apoptosis in hair matrix cells (McGowan et al. 2002). The same group later showed that K17 modulates hair follicle cycling by delaying apoptosis, whereby K17 is functionally linked with TNF\u03b1 signaling (Tong and Coulombe 2006).\nHereditary human diseases due to K17 mutations have been identified (for references, see Lane and McLean 2004), most notably pachyonychia congenita type 2 (Jackson\u2013Lawler form). The phenotype of this genodermatosis includes thickened nails and pilosebaceous cysts. Another condition related to K17 mutations is steatocystoma multiplex, in which patients present with multiple hair follicle-associated cysts. These genodermatoses obviously are related to the expression and functional importance of K17 in pilosebaceous and nail (Perrin et al. 2004; Langbein and Schweizer 2005) epithelia.\nSince in keratinocytes K17 is\u2014like K6 and K16 (see above)\u2014an inducible keratin upon stress, injury, or inflammation, it is not surprising that squamous cell carcinomas consistently express these three keratins (Moll et al. 1982b, 1983; Chu and Weiss 2002b). As most normal stratified squamous epithelia lack K17, its presence in the corresponding tumors may be regarded as neo-expression during tumorigenesis. In the uterine cervix, K17 is expressed in cervical intraepithelial neoplasia but since it is already present in endocervical reserve cells (Weikel et al. 1987) and immature squamous metaplasia, it has not yet become a routine diagnostic marker (Martens et al. 1999). Among adenocarcinomas, focal K17 expression seems to be particularly characteristic of pancreatic ductal adenocarcinomas (Real et al. 1993; Moll 1998), which may become useful for their distinction from other adenocarcinomas (Chu and Weiss 2002b). In ductal breast carcinomas, K17 is expressed in a minor subset of tumor cases (Malzahn et al. 1998), now recognized to correspond to the basal-like subtype as defined by global gene expression data (see below, chapter \u201cKeratins as diagnostic markers in tumor pathology\u201d).\nK1\/K10: major keratins of keratinocyte differentiation and keratinization\nIn the epidermis, the transition of keratinocytes from the proliferative basal cell layer to the postmitotic suprabasal spinous cell layers in the process of terminal differentiation and keratinization is characterized by a profound change in keratin expression. This involves a switch from expression of the basal cell keratins (K5, K14, K15) to the suprabasal epidermal keratins, the type II keratin K1 and subsequently the type I keratin K10 (Fig.\u00a04b; Fuchs and Green 1980; Moll et al. 1982b; Tseng et al. 1982, Weiss et al. 1984; Roop 1987, Stoler et al. 1988). This is one of the classical examples for the carefully regulated differentiation-specific expression of keratin proteins. Ultrastructurally, keratin filaments composed of the pair K1\/K10 form particularly dense bundles which are so characteristic of suprabasal epidermal keratinocytes (Fig.\u00a01d). Clearly, this imparts mechanical integrity to the cells and the whole epidermis. In addition, however, there seem to exist further functional roles, as experimental data have demonstrated that K10 specifically inhibits proliferation and cell cycle progression of keratinocytes (Paramio et al. 1999; Koch and Roop 2004) and loss of K10 leads to increased keratinocyte turnover (Reichelt et al. 2004; for review, see Magin et al. 2007).\nThe importance for epidermal integrity is underscored by the fact that point mutations in K1 and K10 are associated with the blistering disorder epidermolytic hyperkeratosis\/bullous congenital ichthyosiform erythroderma (BCIE), initially presenting with skin blisters but later with thickened ichthyotic skin (for reviews, see Lane and McLean 2004; Omary et al. 2004). As expected, the suprabasal cells become fragmented easily and, in addition, the epidermis becomes hyperproliferative and hyperkeratotic. K1 mutations are notably heterogeneous and may result in diverse overlapping, often relatively mild phenotypes (Lane and McLean 2004). In transgenic mouse experiments, mutation or knock-out of K10 results in a phenotype similar to human epidermolytic hyperkeratosis (Fuchs et al. 1992; Porter et al. 1996).\nDespite their association with terminal epidermal differentiation and keratinization, K1 and K10 may be focally expressed in suprabasal cells of internal noncornifying stratified squamous epithelia (for references, see Moll 1998). They are also a typical component of cells of eccrine sweat gland ducts (Fig.\u00a05; Langbein et al. 2005). Surprisingly, although the differentiated parts of the hair follicle, such as suprabasal outer root sheath, upper companion layer, upper inner root sheath or the hair fiber, are heavily keratinized structures, all of them are free of K1\/K10. Both typical epidermal keratins are completely lost in the infundibulum.\nAmong various antibodies against K1 and K10 described in the literature, MAb DE-K10 against K10 is particularly suitable for application with paraffin-embedded tissues (Ivanyi et al. 1989). In squamous cell carcinomas focal expression of K1 and K10, usually in relation to maturation and keratinization, can be observed regardless of whether they are derived from the skin or from internal organs (for references, see Moll 1998). They are more sparse in poorly differentiated tumors but may still be detected in about 50% of cases of oral and pharyngeal squamous cell carcinomas (Moll 1998). However, quantitatively, squamous cell carcinomas rather embark on an alternative maturation pathway characterized by abundant expression of K6 and K16 (see above; Fig.\u00a04d). Overall, K1 and K10 can be regarded as \u201ckeratinization markers\u201d of keratinocytes. These keratins have not yet been routinely applied to tumor diagnosis except for some special aspects of skin tumors (Yuspa et al. 1991).\nK9: palmoplantar epidermal differentiation keratin\nThe type I keratin K9 is a highly specific keratin of terminally differentiating keratinocytes of palmoplantar epidermis where it is abundantly, albeit heterogeneously expressed (Moll et al. 1987; Langbein et al. 1993). At other body sites, there may be extremely sparse and focal expression in upper epidermal layers (Moll et al. 1987). Thus K9, forming a pair with K1, appears to reflect a special program of keratinocyte differentiation associated with particular mechanical reinforcement (Swensson et al. 1998). Therefore it is not surprising that mutations in the K9 gene are associated with a disorder of the skin of the palms and soles, epidermolytic palmoplantar keratoderma, which manifests itself as cytolysis and epidermal thickening (Reis et al. 1994; Torchard et al. 1994; for review, see Lane and McLean 2004). In its sequence, K9 is most closely related to K10 (Langbein et al. 1993). MAbs against K9 (clones Ks9.70, Ks9.216; PROGEN, Heidelberg, Germany) are available (Langbein et al. 2005, 2006, 2008). Immunostaining for K9 has significance for characterization of palmoplantar keratinocyte direction of transplants (Compton et al. 1998; Stoner and Wood 1999) and of special genodermatoses (McLean and Irvine 2007) but there is no relevance of K9 in tumor diagnosis. It should be noted that the occurrence of keratin K9 (and K1 and K10) may give rise to problems in biochemical practice as biochemicals or buffers are sometimes contaminated with these keratins derived from abraded \u201chorny particles\u201d, i.e. terminally differentiated skin keratinocytes (\u201claboratory dust\u201d; Clark et al. 1971; Fox et al. 2008).\nK2: keratin of highly differentiated, advanced epidermal keratinocytes\nK2 (formerly K2e, see before and Table\u00a01) is another keratin specific for the advanced terminal differentiation process of epidermal keratinocytes. Being widely distributed over most body sites, this type II keratin is expressed late, at an advanced stage of differentiation, in the uppermost epidermal layers (upper stratum spinosum, stratum granulosum) to a variable extent (Collin et al. 1992a). K2 is not expressed in follicular skin adnexal structures like the late compartments of outer or inner root sheath. Correspondingly, mutations in K2 have been associated with ichthyosis bullosa of Siemens, a blistering disease showing cytolysis in superficial epidermal layers (for references, see Lane and McLean 2004). MAbs specific for K2 (clones Ks2.342.7.1, Ks2.398.3.1; PROGEN, Heidelberg, Germany) have been described (Langbein et al. 2005, 2006, 2008).\nK3\/K12: keratins of the corneal epithelium\nThe K3 (type II)\/K12 (type I) pair is the cell type-specific and differentiation-related keratin pair of the corneal epithelium. These keratins are expressed in all corneal epithelial cell layers, whereas in the limbus corneae only suprabasal cells are positive and the basally located corneal stem cells are K3\/K12 negative (Schermer et al. 1986; Pitz and Moll 2002). Mutations in these keratins give rise to Meesmann\u2019s corneal dystrophy characterized by intraepithelial microcysts in the corneal epithelium (Irvine et al. 1997; for further references, see Lane and McLean 2004). K12 knock-out mice have a mechanically fragile, easily detachable corneal epithelium (Kao et al. 1996). Antibodies against these corneal keratins described include MAb AE5 (PROGEN, Heidelberg, Germany) which reacts with K3 and additionally with the related K76 (formerly K2p; see below; Collin et al. 1992b) and MAb AK12 which recognizes K12 (Chaloin-Dufau et al. 1993).\nK4\/K13: keratins of mucosal stratified squamous epithelial cells\nIn internal stratified squamous epithelia which mostly are non-keratinizing, a highly characteristic keratin pair indicates the mucosal path of keratinocyte differentiation, i.e. the type II keratin K4 and the type I keratin K13 (Moll et al. 1982b; Cooper et al. 1985). Immunohistochemical studies using specific MAbs\u2014such as MAb 6B10 against K4 (van Muijen et al. 1986) and MAbs 1C7, 2D7 (van Muijen et al. 1986) and Ks13.1 (Moll et al. 1988b) against K13\u2014revealed the presence of K4 and K13 in the entire suprabasal compartment of mucosal stratified squamous epithelia, whereas the basal compartment is positive for K5\/K14. Interestingly, K4\/K13 is completely absent in the epidermis and adnexal structures. Keratin K13 is also expressed in the urothelium as a major component of the basal and intermediate cells whereas it is lost in the superficial umbrella cells (that switch on K20 expression; see above). Keratin K4 is also\u2014heterogeneously\u2014expressed in columnar luminal cells of the pseudostratified respiratory epithelium and in the non-stratified simple epithelial cells of e.g. pancreatic ducts (van Muijen et al. 1986; Moll 1993).\nFunctionally, K4 and K13 appear to be important particularly as components of mucosal stratified squamous epithelia. Mutations in these keratins, lying in the helix initiation or termination motifs (HIM or HTM, respectively), have been shown to cause the hereditary disorder white sponge nevus of Cannon (for references see Lane and McLean 2004). This mucosal disorder presents with white plaques mainly on the buccal mucosa, histologically showing thickened spongy epithelium with hydropic swelling of suprabasal epithelial cells. Here again, the clinical manifestation of pathological alterations of keratins well reflects their tissue distribution.\nIt is not surprising that squamous cell carcinomas derived from the epidermis essentially lack K4 and K13 (Kuruc et al. 1989; for further references see Moll 1998). However, in contrast to what might be expected, they are not major components of, but are only focally and variably expressed in squamous cell carcinomas of the head and neck, with more pronounced expression in poorly differentiated cases (Moll 1998). Instead, the predominant maturation-associated keratins expressed by these tumors are the hyperproliferative keratins K6 and 16 (see below, chapter \u201cKeratins as diagnostic markers in tumor pathology\u201d). Corresponding to its characteristic expression in normal urothelium, K13 is maintained\u2014at least focally\u2014in most transitional cell carcinomas of the urinary tract (Moll et al. 1988b; for further references, see Moll 1998). K13 may be part of a panel of markers (which also includes K20) useful in the histological diagnosis of metastatic transitional cell carcinomas (see below, chapter \u201cKeratins as diagnostic markers in tumor pathology\u201d). As to adenocarcinomas, it is noteworthy to mention the frequent expression of K4 in ductal adenocarcinomas of the pancreas (Sch\u00fcssler et al. 1992; Real et al. 1993; Moll 1998) and in a subpopulation of poorly differentiated invasive ductal breast carcinomas (Malzahn et al. 1998).\nK76, K77: keratins with very special expression sites\nK76 (previously designated K2p) is specifically expressed in suprabasal cell layers of oral masticatory epithelium, i.e. the slightly orthokeratinized stratified squamous epithelium lining the gingiva and the hard palate (Collin et al. 1992b). Because of the failure of specific antibodies for long time, which are now available (PROGEN, Heidelberg, made by L.L.), no tumor studies have been done yet.\nThe expression pattern of keratin K77 (previously designated K1b) was very surprising and extremely restricted. This keratin is exclusively expressed in and restricted to the luminal cells of eccrine sweat gland ducts (Fig.\u00a05a\u2013d). All other epithelia and glands tested so far, including apocrine sweat gland, were negative (Langbein et al. 2005). Based on the extensive investigation of the keratin pattern, new aspects of eccrine sweat gland differentiation could be achieved (Langbein et al. 2005 and supplemental data therein). The high specificity of expression makes this keratin recommendable for using as an \u201ceccrine duct marker\u201d in tumor diagnostics. In a first study, this assumption could be confirmed by the investigation of \u201ceccrine\u201d adnexal tumors such as syringoma (Fig.\u00a05e), poroma (Langbein et al. 2008) and cylindroma (Fig.\u00a05f).\nK25, K26, K27, K28, K71, K72, K73, K74, K75: hair follicle-specific epithelial keratins\nOnly recently it has become clear that some of the epithelial root sheaths of the hair follicle, the inner root sheath and the companion layer, are unique by their expression of a number of very special keratins (for review, see Langbein and Schweizer 2005; Langbein et al. 2006; Schweizer et al. 2007). Over the years, these keratins were not detected before by biochemical methods because of their quantitative \u201cunder-representation\u201d when compared to the masses of hair or epidermal keratins in the tissue. The first of these new special keratins described was K75. This keratin was originally called K6hf, with \u201chf\u201d indicating its expression site in the \u201chair follicle\u201d, not knowing that this aspect of designation would be unfeasible later. K75 (K6hf) is a type II keratin and was, although closely related in its peptide sequence to K5, following the principles of the former keratin designation (cf. Collin et al. 1992a, b) by using the electrophoretic properties, designated as a \u201cK6\u201d keratin (Winter et al. 1998). K75 is specifically expressed in the companion layer of the hair follicle (Fig.\u00a06a, h, i), a thin layer between the outer and the inner epithelial root sheath (Winter et al. 1998). As the expression of this keratin as monitored by its mRNA synthesis using in situ hybridization starts from the bulbar matricial compartment of the hair follicle and the protein is still existent in the upper differentiated part where the K75 mRNA is no longer synthesized, it was one first but doubtless indication that this structure is an own individual compartment of the hair follicle and not the \u201cinnermost layer of the outer root sheath\u201d (see Winter et al. 1998; Langbein and Schweizer 2005). The only further structures in which K75 has been detected are the hair medulla of, e.g. beard hairs, the nail bed, and fungiform papillae of the tongue (Wang et al. 2003; Perrin 2007; Langbein and Schweizer 2005). A mutation in K75 appears to predispose to the common hair disorder pseudofolliculitis barbae, which is characterized by ingrown beard hairs with inflammation, induced by shaving (Winter et al. 2004; Schweizer et al. 2007), and to the loose anagen hair syndrome (Chapalain et al. 2002). Immunostaining for K75 has been reported in trichoblastomas and basal cell carcinomas (Kurzen et al. 2001) and has recently been observed in some squamoid cells of pilomatricomas (M. Divo, L. Langbein and R. Moll, in preparation), indicating special focal differentiation in these diverse cutaneous tumors. We have recently found sparse and focal expression of K75 in certain squamous cell carcinomas of inner organs (M. Divo, L. Langbein and R. Moll, in preparation).Fig.\u00a06Immunofluorescence labeling of hair follicle-specific and hair keratins. The hair follicle-specific epithelial keratin K75 (a) is specifically found in the hair companion layer (cl) and in the medulla (med) of sexual (e.g. beard) hairs. K71 (b) is expressed in all compartments and K72 (c) in the cuticle (icu) of the hair inner root sheath (IRS). The hair keratin K85 (d) expression is found from the hair matrix to the upper cortex and the hair cuticle (cu), whereas K82 (e) is restricted to the hair cuticle. K86 (f) is an example for hair keratins expressed in the mid-to-upper hair cortex (co). g Hair keratin K81 is also expressed in the upper transitional cells of pilomatricoma. co cortex, dp dermal papilla, ORS outer root sheath. h, i Summary schemes of the expression of all hair and hair follicle-specific keratins in the human hair follicle. **K37 is found in the cortex of vellus hairs and medulla of sexual hairs. *K38 is heterogeneously expressed in the cortex. The keratin genes are designated according to the new keratin nomenclature (Schweizer et al. 2006)\nA set of four type I keratins (K25\u2013K28; previous designations K25irs1\u2013K25irs4, see Table \u00a01) and four type II keratins (K71\u2013K74; previous designations K6irs1\u2013K6irs4, see Table\u00a01) is highly specific for the inner root sheath (IRS) of the hair follicle (Fig.\u00a06h, i). These IRS keratins are differentially and partially sequentially expressed in the various IRS compartments, the Henle layer, the Huxley layer, and the IRS cuticle. The keratinocytes of all three compartments synthesize the IRS keratins K71 (Fig.\u00a06b, h, i), K25, K27 and K28. K74 is restricted to the Huxley layer, whereas K73, K72 (Fig.\u00a06c, h, i) and K28 are sequentially expressed in the IRS cuticle (Langbein et al. 2002, 2003, 2006; Langbein and Schweizer 2005; Fig.\u00a06h, i). Otherwise, no \u201cclassical\u201d epithelial keratins were evidenced without any doubts in the IRS and earlier reports most probably showed (at that time not expected) cross-reaction of antibodies with at least one of these IRS keratins (cf. Langbein et al. 2006). Some of the IRS keratins\u2014together with many others\u2014have also been found in the hair medulla (see Schweizer et al. 2007 and Langbein et al., in preparation). In mice, spontaneous hair disorders due to mutations in K71 have been identified (for references, see Schweizer et al. 2007). Human hair disorders related to the IRS keratins have not yet been discovered. Monospecific antisera against all of these keratins are available (PROGEN, Heidelberg, Germany; Langbein et al. 2004, 2006).\nK31, K32, K33a, K33b, K34, K35, K36, K37, K38, K39, K40, K81, K82, K83, K84, K85, K86: keratins of the hair fiber (hair keratins)\nIt is long known since the early period of keratin research that in the (hard) material of hairs, wool, nails, claws and feathers, the tremendous masses of keratin filaments (selection of the early literature: Odland 1953; Fraser et al. 1959; Rogers and Clarke 1965; Orfanos and Ruska 1968) are embedded in a matrix of cross-linking specialized keratin associated proteins (KAPs) with more than 85 genes in humans (for review, see Rogers et al. 2006). The special, more sulfur-rich keratin proteins constituting these filaments are the \u201chard\u201d or \u201ctrichocytic\u201d keratins. Originally, eight \u201cmajor\u201d (type I: Ha1-4, type II: Hb1-4) and two \u201cminor\u201d (Hax, Hbx) keratins were distinguished (Heid et al. 1986, 1988a, b). In the last 10\u00a0years many more, namely 17 members of this keratin subfamily have been identified first at their gene level (Rogers et al. 2004, 2005 and references therein) which are now generally referred to as the hair keratins (Langbein et al. 1999, 2001, 2007; Langbein and Schweizer 2005; Schweizer et al. 2006; see also Table\u00a01). The conspicuous abundance of these proteins comprises eleven type I hair keratins (K31\u2013K40; previous designations Ha1\u2013Ha8, Ka35, Ka36; Langbein et al. 1999, 2001, 2007, Langbein and Schweizer 2005; Schweizer et al. 2006; see also Table\u00a01) and six type II hair keratins (K81\u2013K86; previous designations Hb1\u2013Hb6; Langbein et al. 1999, 2001; Schweizer et al. 2006; see also Table\u00a01; Fig.\u00a06h, i). In the hair, they exhibit differential, complex and in many cases sequential expression patterns within the cuticle and the cortex (Langbein et al. 1999, 2001, 2007; Langbein and Schweizer 2005; Schweizer et al. 2007; for medulla: Langbein et al., in preparation). K35 and K85 (Fig.\u00a06d, h, i) are already expressed in the hair-forming matrix of the cortex and the hair cuticle. The other hair keratins [type I: K31, K33a, K33b, K34, K36, K38 (focally) and K39 (including hair cuticle); type II: K81 and K86] are sequentially switched on upon differentiation in the lower hair cortex and in particular the large \u201cbulk\u201d of hair keratins are expressed in the middle cortex (\u201ckeratinizing zone\u201d) of the ascending hair fiber (Fig.\u00a06f, h, i). Furthermore, K32, K83, K82 (Fig.\u00a06e, h, i) and K40 are sequentially expressed and restricted in the hair cuticle (Langbein and Schweizer 2005; Langbein et al. 2007). As exceptions, K37 was only found in the cortex of vellus hairs and K84, although a typical hair keratin was not detected in the hairs but specifically in the filiform papillae of the tongue (Langbein and Schweizer 2005).\nOne has to keep in mind that out of the 54 human keratins at least 26 (~50%) are expressed in the hair follicle. Therefore, it is most amazing that this extraordinary complex structure\u2014built up by a stratified outer root sheath, the companion layer, the inner root sheath comprising Henle, Huxley and IRS-cuticle layers, the hair cuticle, cortex, and sometimes a medulla\u2014differentiate from cells of an undifferentiated and pluripotent \u201cgerminative cell pool\u201d (Fig.\u00a06a, h, i). There must exist an incredible \u201cfine-tuning\u201d in the regulation of gene expression when e.g. one cell differentiates into the IRS-cuticle and the directly neighboring cell builds up the hair cuticle, both structures being only one cell wide. Unfortunately, up to now, our knowledge on this most complex regulation of hair keratin gene expression is rather fragmentary.\nHair keratins are also prominently expressed in the nail matrix and nail bed and contribute to the formation of the hard tissue of the nail plate (Perrin et al. 2004; Perrin 2007). Moreover, in the 1980s hair keratins have been additionally detected by immunohistochemistry in filiform papillae of the tongue and, intriguingly, in reticulum cells and Hassall\u2019s corpuscles of the thymus (Heid et al. 1988b).\nSome human mutations affecting hair keratin genes have been recognized. The most well-known of these is the congenital hair disease monilethrix, characterized by deformed hair shafts with a beaded appearance. Causative mutations for this disorder have been identified in the hair keratins K86, K81, and rarely in K83 (Winter et al. 1997; for further references, see Schweizer et al. 2007), all of which are expressed in the cortex of hair shafts and thus indicating that monilethrix is a disease of the hair cortex. A further, rare disease recently found to be related to a distinct mutation in the type II hair keratin K85 is ectodermal dysplasia of hair and nail type, characterized by total alopecia and severe nail dystrophy (Naeem et al. 2006). The expression of K85 already in the hair matrix from which hair fiber formation starts (Schweizer et al. 2007) may explain the severe hair phenotype of these patients.\nAs to tumors, hair keratins have\u2014up to now\u2014only been detected in pilomatricomas (Moll et al. 1988a; R\u00e9gnier et al. 1997) which are regarded as originating from hair matrix and undergoing true hair differentiation. The complex hair keratin expression in benign and malignant pilomatricoma (Cribier et al. 2001, 2004, 2006) confirms this notion at the molecular level. Monospecific antisera against all of these keratins are available (PROGEN, Heidelberg, Germany; Langbein et al. 2004, 2006).\nK23, K24, K78, K79, K80: keratins with still unknown expression pattern\nThese five, very different keratins complete the family of human keratin proteins. In principle, mainly the gene and cDNA sequences for the type I keratins K23 (Zhang et al. 2001) and K24 and the type II keratins K78 (formerly K5b), K79 (formerly K6l), and K80 (formerly Kb20) (Rogers et al. 2004, 2005) are known up to now. Unfortunately only very vague and preliminary (or no) expression data are available from Northern blot analyses, suggesting their expression in tongue (K78, K80) and skin (K79), respectively (Rogers et al. 2005). As they were obviously \u201coverlooked\u201d in former studies, their expression might be restricted to very special epithelia, distinct cellular differentiation stages or even transient expression phases. The investigation of the detailed expression pattern of these keratins will be one of the most urgent studies in this field.\nKeratins as diagnostic markers in tumor pathology\nOne of the important fields of \u201capplication\u201d of keratins (making use of the knowledge of their high number of gene family members combined with their special and characteristic expression patterns in distinct cell types, differentiation stages or functional states) is their use\u2014aided by specific antibodies\u2014as immunohistochemical markers in diagnostic tumor pathology. Epithelial tumors maintain\u2014at least widely\u2014specific features of the keratin expression patterns of the respective cell type of origin. Thus, in cases which on the basis of clinical data and conventional histopathology remain unclear, keratin typing may help to correctly identify and classify the tumor entity present. Keratin profiling is especially valuable for carcinomas of poorly differentiated histology, for carcinomas spreading over several organs, and in particular for metastases of an unknown primary tumor. Of the 54 human keratins, only a relatively small panel has attained diagnostic importance up to now, which might grow further with time and respective studies by including the keratins described within the last years. The carefully selected use of diagnostically relevant keratin antibodies\u2014if appropriate, as part of a panel together with other tumor type markers\u2014has become diagnostic standard in state-of-the-art clinical pathology (for recent overviews, see Chu and Weiss 2002b; Dabbs 2006). In the previous chapter on the individual keratins, some data on their expression patterns in tumors have already been presented. In the following, key diagnostic features of keratin typing concerning important tumor entities will be briefly summarized.\nAdenocarcinomas\nAdenocarcinomas are one of the largest groups of human malignant tumors and may arise in many organs and tissues. They comprise more than one-half of cases of cancer with unknown primary tumor. The identification of the specific origin\u2014e.g. colon, ovary, pancreas, or lung\u2014has become therapeutically important since effective chemotherapy schedules may be vastly different. As a group, adenocarcinomas are characterized by the predominance of simple-epithelial keratins, notably K8, K18 and K19, whereas K7 and K20 are variably expressed. It is this variability which may be diagnostically exploited, and thus staining for both K7 and K20 is the current practice resulting in different K7\/K20 phenotypes (Table\u00a03). The highest diagnostic significance of keratin typing is true for colorectal adenocarcinomas which\u2014like the normal mucosa\u2014almost always remain K20-positive (Fig.\u00a03d). Most cases, including metastases, exhibit a K7\u2212\/K20+ phenotype (Fig.\u00a03c, d) or express K7 at lower level as compared to K20 (Moll et al. 1992; Miettinen 1995; for further references, see Moll 1998, Chu and Weiss 2002b). K7 co-expression has been detected more frequently in advanced colorectal cancers (Hernandez et al. 2005). Although so characteristic of colorectal tumors, K20 may in certain situations be reduced. Thus, reduced expression of K20 has been found in a specific subset of colorectal carcinomas with high levels of microsatellite instability (McGregor et al. 2004); notably, K7 expression remained low in these tumors. Since K20 is a differentiation marker its reduction or even loss may also be due to dedifferentiation. In a large tissue microarray study, reduced expression of K8 and K20 was found to be associated with shorter patients\u2019 survival, possibly on the basis of epithelial-mesenchymal transition (Kn\u00f6sel et al. 2006). Adenocarcinomas of the stomach usually show heterogeneous expression of both K7 and K20, the latter being rather variable but yet expressed in the majority of cases (Moll 1998, Chu and Weiss 2002b). No systematic differences between intestinal and diffuse\/signet ring cell carcinomas have been described. In adenocarcinomas of the pancreas and the biliary tract, there is usually a clear predominance of K7 (Fig.\u00a03e) together with variable and focal (often minor) expression of K20 in up to 75% of cases (Fig.\u00a03f, Moll 1998; Chu and Weiss 2002b). Ductal adenocarcinomas of the pancreas in addition often express certain stratified-epithelial keratins, most notably K4 and K17 (Sch\u00fcssler et al. 1992; Real et al. 1993; Moll 1998). Finally, a K7+\/K20\u2212 phenotype is characteristic of adenocarcinomas of the ovary (except for the mucinous type), the endometrium and the lung. Endometrial adenocarcinomas often focally co-express certain stratified-epithelial keratins including K5, reflecting the potential of the tumor cells for stratification to develop squamous metaplasia.\nLike other adenocarcinomas, most breast carcinomas constitutively express K8, K18 and K19 (Altmannsberger et al. 1986; Malzahn et al. 1998). In some studies, however, a high level of K8 or K18 immunostaining, as detected by certain monoclonal antibodies, has been correlated with favorable prognosis and reduced or absent staining was associated with unfavorable outcome (Takei et al. 1995; Schaller et al. 1996; Woelfle et al. 2004). Microarray-based expression profiling of breast carcinomas has led to the definition of distinct subgroups. One of these has been designated as basal-like group (S\u00f8rlie et al. 2001) which is characterized by relatively poor prognosis. Interestingly, the typical expression profile of the basal-like cancers includes the basal cell-typical keratins K5, K14 and K17. Several older reports have already described the presence of such keratins in a subpopulation of breast carcinomas (Moll et al. 1983; Wetzels et al. 1991), and some did already point to a negative prognostic significance of basal cell keratin expression in invasive ductal breast cancers (Dairkee et al. 1987; Malzahn et al.1998). This has now been confirmed and extended by the growing body of recent microarray data, allowing to define a subgroup of sporadic breast cancers which exhibit bad prognosis and association with BRCA1 mutation or dysfunction (Gusterson et al. 2005; Diaz et al. 2007). For the recognition of this subgroup, immunostaining for keratins such as K5 may achieve importance (van de Rijn et al. 2002; for further references, see Gusterson et al. 2005).\nThe different subtypes of renal cell carcinomas (RCCs) exhibit some characteristic features in keratin expression which may support the precise classification of these tumors (Thoenes et al. 1988; Moll 1993; Chu and Weiss 2002b; Skinnider et al. 2005; Liu et al. 2007). Thus, conventional (clear cell) RCCs express a simple keratin pattern of mainly K8\/K18 together with variable, mostly minor expression of K19. In contrast, the papillary subtype of RCC is characterized by strong K19 expression as well as K7 expression in addition to the basic pair K8\/K18. Chromophobe RCCs, on the other hand, typically express K7 (but little K19) in addition to K8\/K18 (Thoenes et al. 1988). The benign oncocytomas, which histologically may resemble chromophobe RCC, are mostly K7-negative (Liu et al. 2007). The peculiar co-expression, together with keratins, of the mesenchymal intermediate filament protein vimentin in conventional (clear cell) and papillary RCCs but not in chromophobe RCCs and oncocytomas is another feature valuable in differential diagnosis. Another tumor category with characteristic co-expression of keratins and vimentin are malignant mesotheliomas. The epithelial type may be difficult to distinguish histologically from adenocarcinomas (e.g. pleural involvement by pulmonary adenocarcinomas or pleural\/peritoneal metastases of adenocarcinomas of various origins). In contrast to most adenocarcinomas, epithelial mesotheliomas consistently express keratinocyte-type keratins, notably K5, in addition to keratins K8, K18, K19, and K7 typical for simple epithelia (Moll et al. 1989; Ordonez 1998; Chu and Weiss 2002a; Yaziji et al. 2006). Thus, in recent years, K5 has been included in the battery of useful mesothelioma markers.\nNeuroendocrine tumors\nNeuroendocrine tumors, a large and heterogeneous tumor group, are generally characterized by the expression of keratins typical of simple epithelia (notably K8, K18, and\u2014more variably\u2014K19) and the complete lack of keratins K5\/K14 typically found in stratified epithelia. Particularly interesting in a diagnostic respect is the well-proven value of K20 as a consistent marker of Merkel cell carcinomas of the skin, allowing their delineation from metastatic small cell neuroendocrine carcinomas arising at other sites such as small cell lung carcinoma which\u2014although morphologically similar\u2014consistently lack K20 (Moll et al. 1992; Cheuk et al. 2001). Another interesting recent issue concerns endocrine tumors of the pancreas. Several studies suggest that the expression of K19 in these tumors may be correlated with a poor prognosis (Schmitt et al. 2007; La Rosa et al. 2007).\nTransitional cell carcinomas\nThe urothelium exhibits a unique, complex pattern of keratin expression. K8, K18, K19, and K7 are expressed in all cell layers. K5 and K17 are restricted to the basal cell layer. Particularly characteristic are K13 expressed in the basal and intermediate cell layers and K20 specific for the superficial (umbrella) cell layer. This urothelial keratin pattern is relatively well conserved in noninvasive and invasive transitional cell carcinomas (TCCs) (for detailed reviews, see Moll 1998; Southgate et al. 1999). K20 has been found to be retained in ~80% of TCCs (Moll et al. 1992). Indeed, the combined presence of keratins K8\/K18 (as well as K7 and K19) typical for simple epithelia together with K13 and K20 in a tumor is characteristic of urothelial origin, although K13 may be reduced or lost in poorly differentiated TCCs. Squamous metaplasia may modify the keratin expression pattern. Particularly interesting are the recent, prognostically relevant findings that noninvasive papillary TCCs may exhibit a normal K20 pattern (predominantly superficial) or an abnormal K20 pattern (all cell layers or negative) and that the normal pattern is predictive of tumor non-recurrence (Southgate et al. 1999).\nSquamous cell carcinomas\nSquamous cell carcinomas of different sites of origin are generally characterized by a predominance of stratified-epithelial\/keratinocyte-type keratins but may co-express certain simple-epithelial keratins (for details, see Moll 1998). Most of these tumors strongly express the keratins K5 (Fig.\u00a04c, e), K14 and K17 normally found in the basal layer as well as the keratins K6 (Fig.\u00a04d) and K16 characteristic for hyperproliferative keratinocytes. Focally, there may be expression of K1\/K10 (particularly in higher differentiated tumor cells which can end in the formation of horn pearls), and\u2014to a lesser extent\u2014K4 and K13. The co-expression of simple epithelia-typical keratins comprises K8, K18, and K19, and different studies have suggested that this co-expression seems to be more pronounced in poorly differentiated squamous cell carcinomas (for references, see Moll 1998). Recently it has been demonstrated that in squamous cell carcinomas of the oral cavity the expression of K8 and K18 is an independent prognostic marker and indicates a decreased overall and progression-free survival (Fillies et al. 2006).\nIn summary, stratified-epithelial keratins, in particular K5 and K6, are useful as general markers for squamous cell carcinomas in histologically uncertain, poorly differentiated, or metastatic tumor cases. Although certain differences between the keratin expression patterns of squamous cell carcinomas from different sites of origin have been noted, it is not yet possible to use keratins as specific site markers in cases of unclear metastases (Moll 1998).\nPerspectives\nIn the past 10\u00a0years, our knowledge on keratins has tremendously increased, regarding their molecular and cell biology as well as their application as markers in pathological diagnosis. The introduction of a new consensus nomenclature (Schweizer et al. 2006), which for the common (non-hair follicle specific) epithelial keratins widely conserves the older names established in the literature, makes this complex field much clearer than before and will facilitate future research. Today much is known about the structural functions of keratins, as proven by a wealth of human hereditary keratin diseases and transgenic mouse models. However, numerous questions still need to be answered, particularly concerning the regulatory functions of keratins. As reviewed in this article, recent experimental studies have pointed to newly recognized roles of certain keratins in apoptosis, cell growth, tissue polarity, wound response, and tissue remodeling. First, specific signaling pathways have already been described which seem to be associated with distinct keratins. The question of whether keratins might play a role in malignant transformation is particularly exciting, but at present a causal role in tumorigenesis has not been established for any keratin (for review, see Magin et al. 2007). However, keratins may in fact be linked to the cell cycle machinery (Margolis et al. 2006). Future research will hopefully answer the question why the human organism actually needs 54 different keratin proteins, all of which in the first instance form the seemingly \u201cprimitive\u201d, uniform structure of a 10\u00a0nm intermediate filament\u2014although with peculiarities in the head and tail domains of their single molecular components.\nOne may also expect for the future that the diagnostic application of keratins, although already established in tumor pathology, will be further refined and extended. Examples of very recent new developments in tumor classification are the recognition of the basal-like subtype of breast cancer on the basis of the expression of K5 and the prognostic relevance of K19 in endocrine pancreatic tumors. At the moment, the panel of keratins introduced into routine diagnosis as histopathological tumor markers is distinctly small, essentially consisting of K5, K7, K8\/K18, K19, and K20. Furthermore, the expression patterns of a battery of special keratins were detected only in the last few years and these keratins are just started to be involved into tumor diagnostic studies. Not to forget that at the time 5 out of the 54 human keratins are still uncharacterized in their expression sites and patterns and possibly, their expression in neoplasia might have a potential as diagnostic markers. Since all keratins have an exquisite cell type- and differentiation stage-specific regulation and expression pattern, it might well be that upon future research further keratins will be introduced as markers for certain diagnostic questions in pathology to widen and to refine the diagnostic potential of keratins.","keyphrases":["keratins","differentiation","cytoskeleton","tumor markers"],"prmu":["P","P","P","P"]}
{"id":"Bioinformation-1-7-1891696","title":"Database of cell signaling enzymes\n","text":"This paper describes a database for cell signaling enzymes. Our web database offers methods to study, interpret and compare cell-signaling enzymes. Searching and retrieving data from this database has been made easy and user friendly and it is well integrated with other related databases. We believe the end user will be benefited from this database.\nBackground\nA cell communicates with its neighbors and environment by sending and \nreceiving information in the form of chemical signals. These signals are \nconverted into intracellular second messenger signals that ultimately make the \ncells respond appropriately by dividing, moving or even dying. The external \nsignals may enter the cell through enzymes, G-protein coupled receptors, \nhydrophobic molecules and ion channels. When the receptor sensing the signal is \na catalyst such as enzyme, the response is amplified. Thus cell signaling \ngoverns the basic cellular activities and coordinates the cell action. Errors \nin cell signaling is the cause for many serious diseases\/disorders including \ncancer, autoimmune diseases, diabetes etc. By understanding cell signaling we \ncan treat these diseases effectively and potentially. [1] However, no attempts have been made \nso far to curate and catalog the enzymes involved in cell signaling.\nDCSE, the Database of Cell Signaling Enzymes covers a gamut of cell \nsignaling enzymes, which includes kinases, phosphatases, adenylyl cyclases, \ncaspases, phosphodiesterases, phospholipases, prenyltransferases etc. As \nmentioned above the defect in cell signaling mechanism is the major cause for \ndiseases and hence the cell signaling enzymes are considered to be potential \ntarget in rational drug design approach.\nMethodology\nDCSE was developed using MySQL [\n2], a relational database management system, at the back-end for \nstoring data. The database can be regularly updated. The data for the database \nwere collected from SwissProt [3\n], the repository of protein sequence information. PHP [4], a widely used general purpose \nscripting language that is especially suited for web development, was used to \ndesign the database interface.\nThe database can be accessed over the Internet. A screenshot of the home \npage is shown in Figure 1.The database can \nbe searched by specifying keywords such as name, Enzyme Commission Number and \nspecies. For each enzyme in the database a unique identifier called DCSE ID has \nbeen assigned. The ID consists of two parts. The first part tells about the \nenzyme class and the second part indicates the number of that enzyme in that \nclass. The two fields are separated by an underscore (_). Each enzyme has been \ndefined with its name, Enzyme commission number, and the species from which it \nhas been sequenced. Crossreference details to other databases are also \nprovided. The functional and other sequence related informations are provided \nby SwissProt, the domain classification and function is provided by InterPro \n[5], the protein family \nclassification is provided by Pfam [\n6] and the structural details are provided by PDB. [\n7] A hyperlink has been provided \nto corresponding entry page in the abovementioned databases.\nThe sequence is also displayed in raw-text format. One can retrieve the \nsequence in FASTA format using the \u2018Retrieve in Fasta format\u2019 \noption available with each entry. An advanced search can also be performed by \nfilling an advanced query form that takes input as DCSE id, name, EC number, \nSpecies, other database id such as SwissProt, InterPro, Pfam and PDB. The \nfields in the search form are joined together by AND operator. BLAST (Basic \nLocal Alignment Search Tool) [8\n] is an algorithm for comparing biological sequences. Given a library \nor database of sequences, a BLAST search enables a researcher to look for \nsequences that resemble a given sequence of interest. A standalone BLAST has \nbeen installed and it can be used for similarity search purpose. BLAST can be \ndone against SwissProt, DCSE or PD.\nUsability\/Accessibility\nThere are three ways in which a user can query the database. The first is \nthe \u2018keyword search\u2019 that can be done by specifying exact or \nlikely keywords such as name, Enzyme Commission Number and species. The second \nsearch option is the \u2018Advanced Search\u2019 wherein the user can \nfill a form by specifying the details for various fields such as DCSE id, \nEnzyme name, Enzyme commission number, SwissProt accession number, InterPro \ndomain ID, Pfam id or PDB id. The fields are joined together by AND operator. \nAlternatively the users can also browse the database by the 21 different \ncategories of cell signaling enzymes. Whenever the database is searched the \nsearch returns the result with number of hits found for that query along with a \nsummary of details for each entry with its id, name and species. The user can \nthen select the appropriate hit by following the link on it. This displays a \npage with all the available details of the enzyme in the database such as name, \nEC number, sequence information and cross-reference to already existing \ndatabases as shown in Figure 2. A \nhyperlink has been provided to corresponding entry page in the cross-referenced \ndatabases for easy access. The sequence is displayed in raw-text format. One \ncan retrieve the sequence in FASTA format using the \u2018Retrieve in Fasta \nformat\u2019 option available with each entry. A BLAST search can also be \ndone to search for similar sequences within the database or in general. A link \nhas been provided in the tools page. The BLAST input page and result page are \nshown in figure 3 and 4.\nUtility to the biological community\nSince DCSE gather together all the information on biological molecules, \nsequences, structures, functions, and biological reactions, which transfer the \ncellular signals, this database has the potential of becoming a major hub of \nresource for the biological community. This database provides a mechanism by \nwhich researchers and students can transform information about interactions \nbetween biomolecules into knowledge about a cellular process.\nCaveats\nThe database needs to be updated from time to time as the data has been \nobtained from other sources. Despite our efforts to collect information from \nvarious sources and check them for consistency, the quality of the information \ndepends heavily on the original source.\nFuture development\nThe coiled-coil structure is important for protein interaction. Since the \ncell signaling also involves protein interaction, a tool to predict coiled-coil \nstructure is of importance.\nThere are few tools for this purpose like MultiCoil. The Multicoil program \njust gives the probability for a residue to be in coiled-coil region. We intend \nto develop a coiled-coil prediction tool based on artificial neural networks \nthat could tell whether a residue is in coiled-coil region if it crosses a \nthreshold computed from a training set consisting of proteins with known \ncoiled-coil structure.","keyphrases":["cell signaling","enzymes","web database","pblast"],"prmu":["P","P","P","U"]}
{"id":"J_Occup_Rehabil-3-1-1915618","title":"Are Pain Intensity and Pain Related Fear Related to Functional Capacity Evaluation Performances of Patients with Chronic Low Back Pain?\n","text":"Introduction: Pain related fear and pain intensity have been identified as factors negatively influencing Functional Capacity Evaluation (FCE) performances in patients with CLBP. Conflicting results have been reported in the literature. The objective of this study was to analyze the relationships between pain intensity and pain-related fear on the one hand, and performances during an FCE on the other hand in two samples of patients with chronic low back pain (CLBP). Methods: Two cross sectional observation studies were performed with two samples of patients with CLBP (study 1: n = 79; study 2: n = 58). Pain related fears were operationally defined as the score on the Tampa Scale of Kinesiophobia in study 1, and the Fear Avoidance Beliefs Questionnaire (FABQ) in study 2. Pain intensity was measured with a Numeric Rating Scale in both studies. Avoidance behavior observed during FCE was in both studies operationally defined as the unwillingness to engage in high intensity performance levels of three different functional activities: high intensity lifting, prolonged standing in a forward bend position, and fast repetitive bending at the waist. Results: A total of 25 correlations between pain and pain related fear, and performance variables were calculated, out of which 7 were significant (p < 0.05). The strength of these significant correlations ranged from r = \u22120.23 to r = \u22120.50. Multivariate linear regression analyses revealed non-significant relationships in most instances. Pain and pain related fear contributed little if any to these models. Conclusions: The relation between pain and pain related fear and FCE performance is weak or non-existent in patients with CLBP.\nIntroduction\nFunctional Capacity Evaluations (FCEs) are batteries of tests designed to assess a person's functional capacity related to work [1\u20133]. To establish functional capacity, the patient is asked to perform a number of activities. Combined, these performances reflect a patient's ability to perform work-related activities. A patient's performance is determined by biological, psychological and social factors [4]. Pain related fear, also referred to as fear avoidance beliefs or fear of movement and reinjury [5], and pain [6] have been identified as examples of psychological factors influencing a patient's performance during an FCE.\nPain related fear refers to a condition in which the patient has an excessive, irrational, and debilitating fear of physical movement and activity, resulting in feelings of vulnerability to painful injury or reinjury [7, 8]. People who experience pain-related fear will avoid activities they associate with increased risk for pain or (re)injury. As such, pain-related fear should have a negative effect on the results of performance testing [5, 9]. It has been stated that \u2018a valid assessment of functional capacity cannot be carried out without controlling for fear avoidance beliefs\u2019 [8]. Several studies were performed to study the relationship between pain-related fear and different types of avoidance behavior in patients with chronic non-specific low back pain (CLBP). The strength of this relationship varied between the studies from r=0.06 to r=0.49 [8\u201310, 11]. The relationship between pain related fear and performance appeared stronger in studies where patients were observed under strictly controlled conditions [8, 10], and weaker in studies where patients were observed in a less controlled environment [11]. In a previous study performed in an admission phase for a pain rehabilitation program, no relationship was found in a sample of patients with CLBP, between lifting performance and pain-related fear [12]. Although the evidence from other studies did not overwhelmingly support the strength of the relationship, our results, presented in the Journal of Occupational Rehabilitation in 2003, were in contrast with other studies. It could not be determined whether the results found were weaker because of the operational definitions of pain-related fear (score on Tampa Scale for Kinesiophobia (TSK)) or avoidance behavior (maximum lifting performance). It was recommended that future studies should use additional operational definitions for pain-related fear and avoidance behaviors.\nIn a recent review pain intensity has been identified to consistently associate negatively with patient performances during FCEs [6]. The strength of the significant associations between pain intensity and FCE performances in patients with chronic pain vary from r\u00a0=\u00a0\u22120.25 to r\u00a0=\u00a0\u22120.56 [6]. In the study mentioned above, the relation between pain intensity and lifting performance was (somewhat) weaker and non-significant (r\u00a0=\u00a0\u22120.21) [12].\nAim of this study was to further analyze the relationships between pain intensity and pain-related fear on the one hand, and avoidance behaviors on the other hand. Two studies were performed with separate samples of patients with CLBP. Pain related fears were operationally defined as the score on the TSK in study 1, and the Fear Avoidance Beliefs Questionnaire (FABQ) in study 2. Avoidance behavior was in both studies operationally defined as the unwillingness to engage in high intensity performance levels of three different FCE tests: high intensity lifting, prolonged standing in a forward bend position, and fast repetitive bending at the waist. Non-significant or weak relationships were considered falsifications of the hypotheses that pain and pain-related fears would have clinically relevant impact on FCE performances in patients with CLBP.\nMaterials and methods\nPatients\nTwo separate cohorts of patients who were referred to an outpatient rehabilitation program of the Center for Rehabilitation of the Groningen University Medical Center, The Netherlands, were included. Both study samples consisted of patients diagnosed with CLBP, aged between 18 and 60 years, with symptoms lasting longer than 3 months. Excluded were patients with comorbidity with negative consequences for functioning (i.e. severe depression needing a psychiatric referral), and patients with specific pathology related to the lumbar spine (i.e. disc herniations, tumors, spondylolisthesis grade 3 or 4, etc.). Patients were not selected based on (high or low) levels of pain or pain related fears. Selection process of both study samples were equal, with the exception that patients in study sample 1 explicitly agreed to participate in a larger study in addition to regular clinical care [13]. The patients of sample 2 received regular clinical care only.\nProcedures\nPrior to the medical intake patients filled out a TSK [7, 8], a numeric rating scale to assess pain intensity (NRS), a Roland Morris Disability Questionnaire [14] (RMDQ) to assess disability, and a study consent form. The patients underwent the FCE approximately 2 weeks after the medical intake. Prior to the FCE, all patients were asked whether their pain and functional status was different compared to their status during medical intake. In study 2, patients filled out the FABQ [15] prior to the FCE. For study 1, approval was granted by the medical ethics committee of the University Medical Center Groningen, The Netherlands. The data of the patients of study sample 2 were collected as part of regular clinical procedures, for which institutional approval was received.\nMeasures\nPatients rated their current pain intensity on a NRS ranging from 0 to 10; 0 being no pain at all and 10 being the worst pain imaginable. Self reported disability was assessed by the RMDQ [14]. Scores on the RMDQ can range from 0 to 24; 0 indicating no disability and 24 indicating severe disability. The psychometric properties of the RMDQ are good [14, 16].\nPain related fear was assessed with a different questionnaire for each sample. In study 1, pain related fear, called fear of movement and reinjury, was assessed by the TSK [8]. The TSK consists of 17 items. Each item is provided with a 4-point Likert scale with scoring alternatives ranging from \u201cstrongly disagree\u201d to \u201cstrongly agree.\u201d Although the scale consists of 2 subscales, activity avoidance and somatic focus, a total score is usually presented. The total score ranges from 17 to 68. The reliability of the TSK is fair [8]. Criterion validity was established from correlations with other self-reported measures of fear (of bodily injury), anxiety, depression, and catastrophizing [8].\nIn study 2, pain related fear, called fear avoidance beliefs, was assessed by the FABQ [10, 15]. The FABQ consists of 16 items. Each item is provided with a 7-point Likert scale with scoring alternatives ranging from 0 to 6. The FABQ consists of 2 scales. The activity scale consists of 4 items (total score 0\u201324), the work scale consists of 6 items (total score 0\u201336). The remaining 4 items are not used. The reliability of the FABQ is good in patients with acute and subacute back pain [17], but has not been established in patients with CLBP.\nAvoidance behavior was assessed during an FCE. The patients were asked to perform 14 different activities to their maximum abilities (3 types of lifting, carrying, pushing, pulling, overhead work, stooping, crouching, kneeling, standing, walking, sitting, stair climbing), according to the protocols of Isernhagen Work Systems [18]. Selection of activities was based on the Dictionary of Occupational Titles (DOT), thus assuming construct validity with regard to work [19]. Three activities tested during the FCE were selected for this study, because they might be potentially harmful for patients with CLBP [20]. These activities were lifting, static bending and dynamic bending. Lifting was assessed by means of a standardized lifting task consisting of lifting a receptacle with incremental weights from a table (74 cm) to the floor and vice versa. \u2018Ergonomically correct\u2019 body mechanics were not emphasized [21]. The patient's maximum was reached in four to five increments. The maximal amount lifted five times within 90 seconds was recorded (kilograms). Test-retest reliability of the lifting task is good in patients with CLBP [22\u201324]. During lifting, heart rate was measured using a heart rate monitor. In study 2, the intensity level of lifting was observed by the evaluator, and recorded by means of a Borg CR-10 scale. The reliability and validity of this procedure is good [25]. These observations were not recorded in study 1. During the static bending capacity test the patient was asked to stand as long as possible with his \/ her trunk 30\u201360\u00b0 flexed forward, while performing a simple manipulation task. The time this position was held was recorded (seconds). Test-retest reliability of this test in patients with CLBP is good [22]. For the dynamic bending test, the patient is asked to bend at the hips and back as fast as possible, pick up a small object from the floor, and to remove this object to a shelf at crown level (top of head). This was repeated 20 times. The number of repetitions completed and the time needed to complete this activity was recorded (seconds). Test retest reliability of this test in patients with CLBP is moderate to good [22]. The construct validity of the tests has been established [26, 27]. Before testing, patients were instructed regarding termination of the test. Testing was terminated when one of the following occurred: the patient stated verbally that he\/she wished to terminate the activity, the heart rate reached 85% of the age-related maximum, indicated by a heart rate monitor, or the evaluator deemed further testing to be unsafe. No verbal reassurance was given during the testing procedures. The evaluator was blinded to the questionnaire scores.\nAnalysis\nDescriptive statistics were used to describe the study samples. All variables of interest were analyzed at interval level. A student t-test was used to analyze differences between males and females. When differences were significant, further analyses were performed separately for males and females. Pearson product moment correlations were calculated to express linear associations between pain intensity and measures of pain related fear on the one hand and performance variables on the other hand. Correlations were interpreted as follows: 0.25 or less little if any relationship, 0.26\u20130.49 poor relationship, 0.50\u20130.69 moderate relationship, 0.70\u20130.89 strong relationship, 0.90\u20131.00 very strong relationship [28]. Pain intensity and pain related fear, as well as gender, age and self-reported disability were entered as predictor variables in a multivariate linear regression model (method: enter) and performances as outcome variables. A p-value of <0.05 was considered statistically significant for all analyses.\nResults\nThe characteristics of the patients participating in study 1 and 2 are presented in Table 1. All patients declared that their pain and functional status had not changed between medical intake and the FCE. Scores on the TSK subscale in study 1 are: activity avoidance males mean 19.1 (SD 3.8), females mean 18.9 (3.3); somatic focus males mean 9.7 (2.5), females mean 9.2 (2.6). The 20 repetitions of the dynamic bending test were not completed by 10 (of 79, 13%) patients in study 1 and by 6 (of 58, 10%) patients in study 2. To correct for differences in number of repetitions, the time needed to complete one repetition was calculated and presented (time\/number of repetitions). The observed level of intensity of the maximal lifting performances in study 2 were for males mean 7.2 (SD 2.2) and for females 7.4 (SD 1.6) (Borg CR-10 scale). Observational data was unavailable in study 1. Within the study samples, differences between males and females were non-significant, with the exception of the maximum lifting performance. Between the study samples, none of the differences were significant.\nTable 1Age, pain intensity, self-reported disability, pain related fear (TSK or FABQ), and performance variables of 2 samples of patients with chronic low back painStudy 1 (n = 79)Study 2 (n = 58)Males (n = 49)Females (n = 30)Males (n = 39)Females (n = 19)Mean (SD)Mean (SD)Mean (SD)Mean (SD)Age (years)37.8 (9.0)37.8 (8.8)40.4(8.6)35.6 (8.3)Pain intensity (NRS; 0\u201310)4.7 (2.3)5.0 (1.6)4.5 (2.4)4.9 (2.2)Self-reported disability (RMDQ; 0\u201324)12.5 (4.4)12.4 (4.7)11.6 (4.4)11.2 (5.9)TSK (17\u201368)37.5 (5.3)36.0 (5.6)N\/AN\/AFABQ activity scale (0\u201324)N\/AN\/A13.4 (4.1)13.2 (5.6)FABQ work scale (0\u201336)N\/AN\/A19.3 (9.5)15.1 (11.6)Lifting performance (kg)*31.7 (14.7)18.9 (8.1)32.3 (3.8)20.5 (6.3)Lifting maximum heart rate (BPM)126.7 (15.8)129.7 (23.5)128.2 (14.5)128.8 (19.3)Static forward bend (sec)187.4 (148.9)230.6 (146.3)188.0 (115.8)237.3 (154.8)Dynamic forward bend (sec\/rep)2.8 (0.7)3.2 (1.3)3.2 (1.6)2.9 (0.8)SD: standard deviation; NRS: Numeric Rating Scale; RMDQ: Roland Morris Disability Questionnaire; TSK: Tampa Scale for Kinesiophobia; FABQ: Fear Avoidance Beliefs Questionnaire; BPM: beats per minute; N\/A: not assessed. *: Difference between males and females significant in both studies (p < 0.05).Note. Differences between the study samples were non-significant (p < 0.05).\nA total of 25 correlations between pain intensity, TSK (study 1) and FABQ (study 2) and performance variables are presented in Table 2. The strengths of the correlations were moderate in one occasion and otherwise weak\/poor or non-significant. Pain intensity explained 25% of the variance of the performances of male lifting performance in study 2 (r\u00a0=\u00a0\u22120.50). In all other analyses over both studies, the explained variance between pain intensity and performance variables was 8% (r\u00a0=\u00a0\u22120.29) or less. The FABQ work scale explained at best 13% of the variance of the performances (male lifting performance in study 2; r\u00a0=\u00a0\u22120.37). The correlation between either TSK subscales with any of the performance variables was non-significant. No differences were observed between patients who did or did not complete 20 repetitions of the dynamic bending test concerning their relationship with the TSK or the FABQ (all non-significant). The correlation between observed intensity of lifting and FABQ work scale was r\u00a0=\u00a0\u22120.27 (significant). The correlation between observed lifting intensity and FABQ activity scale was r\u00a0=\u00a0\u22120.07 (not significant).\nTable 2Relationships between performance variables and pain intensity, the Tampa Scale for Kinesiophobia (TSK, study 1; n=79) and the Fear Avoidance Beliefs Questionnaire (FABQ, study 2; n = 58) in patients with chronic low back pain. All relationships are expressed in Pearson's r (95% CI)Study 1Study 2PainTSKPainFABQ activity scaleFABQ work scaleLifting performance males (kg)\u22120.26 (\u22120.57 to 0.11)\u22120.12 (\u22120.46 to 0.25)\u22120.50* (\u22120.71 to \u22120.22)\u22120.13 (\u22120.43 to 0.19)\u22120.37* (0.06 to 0.61)Lifting performance females (kg)\u22120.06 (\u22120.34 to 0.23)\u22120.17 (\u22120.43 to 0.12)0.09 (\u22120.52 to 0.38)\u22120.07 (\u22120.51 to 0.40)\u22120.03 (\u22120.48 to 0.43)Lifting maximum heart rate (BPM)0.05 (\u22120.17 to 0.27)\u22120.10 (\u22120.31 to 0.12)\u22120.27* (\u22120.49 to \u22120.01)\u22120.24 (\u22120.47 to 0.02)\u22120.24 (\u22120.47 to 0.02)Static forward bend (sec)0.18 (\u22120.04 to 0.39)\u22120.23* (\u22120.43 to \u22120.01)\u22120.29* (\u22120.51 to \u22120.03)\u22120.33* (\u22120.54 to \u22120.08)\u22120.25 (\u22120.48 to 0.01)Dynamic forward bend (sec\/rep)\u22120.10 (\u22120.31 to 0.12)0.14 (\u22120.08 to 0.35)0.03 (\u22120.23 to 0.29)0.07 (\u22120.19 to 0.32)0.30* (0.05 to 0.52)Note. TSK: Tampa Scale for Kinesiophobia; FABQ: Fear Avoidance Beliefs Questionnaire; BPM: beats per minute; 95% CI: 95% confidence Interval.*p < 0.05.\nAnalyses were performed on the subgroups of patients scoring in the highest quartile of the pain intensity spectrum (study 1, NRS\u00a0\u2265\u00a06, n\u00a0=\u00a022; study 2, NRS\u00a0\u2265\u00a07, n\u00a0=\u00a017), the TSK (score\u00a0\u2265\u00a040, n\u00a0=\u00a022), and the FABQ (activity scale\u00a0\u2265\u00a017, work scale\u00a0\u2265\u00a025, n\u00a0=\u00a016). Analyses involving lifting performances were performed for males and females separately. Correlation coefficients between pain intensity or pain related fears and performance variables ranged between r\u00a0=\u00a00.00 and r\u00a0=\u00a00.23 in study 1, and between r\u00a0=\u00a00.00 and r\u00a0=\u00a00.26 in study 2. All correlations were non-significant. Scatter plots of the distribution of pain intensity or pain related fears and performance variables were created to visually analyze patterns in the datasets. As an example, the distribution of pain related fear (TSK) and lifting performances is presented in Fig. 1. Other distributions between pain intensity or pain related fears (TSK or FABQ) and performance variables were similar, but they are not presented.Fig. 1Plot of maximum lifting performance and scores on the Tampa Scale for Kinesiophobia (TSK) in 79 patients with chronic low back painTable 3Multivariate linear regression models predicting variance in performance in patients with chronic low back pain\u03b2 (95% CI)Betapr2 changeModel r2Study 1 (n = 79)Dependent variable: lifting\u2003\u2022Gender (female = 0, male = 1)14.45 (7.36 to 21.54)0.48<0.0010.230.28\u2003\u2022Constant17.01 (\u22129.60 to 43.62)\u20130.205\u2013Dependent variable: static forward bend\u2003\u2022Model r2 = 0.11; None of the independent variables contributed significantly to the regression equationDependent variable: dynamic forward bend\u2003\u2022Model r2 = 0.12; None of the independent variables contributed significantly to the regression equationStudy 2 (n = 58)Dependent variable: lifting\u2003\u2022Gender (female = 0, male = 1)14.94 (6.15 to 23.73)0.480.0010.230.37\u2003\u2022Pain intensity\u22121.83 (\u22123.65 to \u22120.01)\u22120.290.0490.08\u2003\u2022Constant (kg)45.70 (25.67 to 65.73)\u2013<0.001\u2013Dependent variable: static forward bend\u2003\u2022Constant (sec)549.15 (336.09 to 762.21)\u2013<0.001\u20130.27\u2003\u2022None of the other independent variables contributed significantly to the regression equationDependent variable: dynamic forward bend\u2003\u2022Model r2 = 0.18; None of the independent variables contributed significantly to the regression equation\nResults of the multivariate regression analyses are presented in Table 3. Study 1: Gender was the only variable that independently contributed significantly to the regression equation to predict lifting performance. The explained variance was 28%. Both models to predict the other two outcome variables, static forward bend and dynamic forward bend, were non-significant. Study 2: Gender and pain intensity (to a lesser extend) contributed significantly to the regression equation to predict lifting performances. The explained variance was 37%. In the regression analysis to predict static forward bend performances, none of the independent variables contributed significantly to the regression equation (explained variance 27%). In the regression analysis to predict dynamic forward bending performances none of the independent variables contributed significantly to the regression equation. Collinearity diagnostics were performed for the independent variables. In both studies the average variance inflating factors (VIF) were greater than 1, and none of the VIF-values were greater than 10. Specifically, the relationship between pain intensity and measures of pain related fear were analyzed. In study 1, the strength of the relation between pain and the TSK was r\u00a0=\u00a0\u22120.04 (ns). In study 2, the strength of the relation between pain intensity and FABQ activity scale was r\u00a0=\u00a00.07 (ns), and between pain intensity and FABQ work scale was r\u00a0=\u00a00.20 (ns).\nDiscussion\nThis study has demonstrated that the associations between pain intensity and pain related fears on the one hand and FCE performances on the other hand were generally weak or non-significant. Correlations between pain intensity and pain related fears and performances were significant in only 7 out of 25 analyses (Table 2). The strength of these significant relationships ranged from r\u00a0=\u00a0\u22120.23 to r\u00a0=\u00a0\u22120.50, explaining 5 to 25% of the variance (r2). Multivariate regression analyses were non-significant in 3 out of 6 instances. When significant, pain intensity or pain related fear contributed little if any to these models. Relationships are consistent throughout the intensity spectra (upper quarter analyses and Fig. 1). Thus, overseeing all the results of this study, it appears that the relationships between pain and pain related fear and performances in an FCE are generally weak or non-existent. Although we realize that interpretations of these results are open for discussion, we interpret the magnitude of the relationships between pain and pain related fears on the one hand and performances in an FCE on the other hand of limited clinical relevance. In comparison to our previous study [12], we have now used two new cohorts, two questionnaires instead of one to measure a wider range of pain related fears, and three performance tests instead of one to measure a wider range of avoidance behaviors. Additionally, we have added heart rate and observations as indices for effort level in lifting. Both pain intensity and pain related fear were poorly associated with indices for effort, explaining 7% or less of the variance. The results of the 2 new studies presented here are generally consistent with our previous study, which adds to the robustness of the results.\nThere may be several explanations for the weakness or non-existence of the associations between pain intensity and pain related fears on the one hand, and avoidance behaviors on the other hand. Pain intensity and pain related fears might have been inappropriately operationally defined by the NRS, the TSK and the FABQ, avoidance behavior might have been inappropriately operationally defined by the three performance measures, our study samples differ from samples reported elsewhere, or the variables were appropriately operationally defined, but the relationship was mediated by one or more currently unknown variables. Additionally, a combination of the above may explain our findings.\nThe NRS, TSK and the FABQ may be considered among the standards to measure pain intensity and pain related fears. The reliability and the validity of the measures for avoidance behavior and pain related fears have been established. Both the performance measure \u2018heavy lifting\u2019 and a postural tolerance test have been used previously in different studies as measures for avoidance behavior [8, 9, 12]. The activities selected are potentially harmful for patients with CLBP [20]. Thus, it seems unlikely that the operational definitions were inappropriate for the constructs measured. Both our study samples are similar to samples reported in other studies reporting on patients with CLBP concerning age, pain intensity, self-reported disability, and amount of fear avoidance beliefs [8, 10]. This similarity may rule out the plausibility the results of our study are caused by the selection of study samples. While the TSK and the FABQ may be considered among the consented standards to measure pain related fears, it may be possible that the domain measured might be too large and general to be of value in predicting specific behaviors such as the ones used in this study. This in itself could explain the findings of this study and the lack of consistent findings in previous research. However, if the measures are considered valid representations of the constructs pain intensity, pain related fear, and avoidance behavior, then a different explanation should be considered for our findings.\nOther variables may serve as a mediator between pain variables and avoidance behavior. As suggested in recent pain literature, two variables should be considered as mediators or confounders: motivation [29] and acceptance [30]. Within the motivational models [29, 31, 32], it is suggested that the value of the goal and the patient's belief in his ability (self-efficacy) to attain this goal is predictive for his behavior. When applied to the results of this study, patients were apparently willing to perform the tests regardless of their pain related fears. They were willing to perform, because they may have been convinced that this would have helped them to reach a valued goal (for example return to work). Contrarily, patients without pain related fears may perform poorly on the tests, because they do not see it to be of any value. Alternatively, a poor performance could be explained if the patient's goal would be to use the test results for financial gain (disability allowance) [33]. In that case, pain related fears would be of little importance to determine a patient's behavior. Within the acceptance model [30], it is suggested that patients may be able to accept their pain or fears as an inevitable part of their lives. Patients would report pain or have cognitions such as pain related fears, but are able to function normally with their pain or fears. Some empirical evidence is found to support the applicability of this model within chronic pain [34, 35]. If a mediating effect or confounding of one or more different variables is present, then poor or non-existent associations between pain intensity, pain related fears and test performance are very well imaginable. Future studies should be conducted to find empirical evidence for the proposition that constructs as motivation and acceptance are more important predictors for functional capacity than pain or pain related fears. Additionally, other currently unknown variables may have played a mediating role in the relationship between pain variables and avoidance behaviors.\nBoth self-report and performance measures assess effort-related performance [36]. As such, they cannot be defined independently of the person's behavior. Pain-related disability is a matter of human performance, whether it is observed\/measured or reported [36] and this may also apply to patient's behavior in an admission phase of a rehabilitation program. In this stage patients need not only reveal their cognitions and perception of disability, but at the same time present an image to the practitioner [37], to justify the need for treatment [38, 39] and perhaps to justify the fact that they are off work due to CLBP. The scores on the questionnaires may thus be an overrating of the \u2018real\u2019 pain or pain related fears. An FCE is a measure of demonstrated ability. Pain behaviors may prevent the patients to perform to their maximal physical capacity. The ratings of effort level indicate a sub maximal performance, suggesting that this behavior has occurred in study 2 (and probably also in study 1, but data were unavailable). Without these pain behaviors, the FCE results might have been higher. On average, the performance-based scores presented in this study may be considered an underrating of the physical abilities of the patients. Consequently, clinical interpretation of the outcomes of effort related assessments should be made with care. We propose that the scores on the questionnaires are interpreted as \u2018the patient reports that \u2026 (he is fearful), instead of \u2018the patient is\/feels\/perceives \u2026 (fearful\/fear). For the FCE, we propose that the scores are interpreted as the patient's performance, instead of the patient's capacity (FPE: Functional Performance Evaluation). The performance of a patient depends on his capacity and his willingness to produce [25]. The current study and our previous study show that report of pain and pain related fears explain little of the patient's performances during FCE. Statements that \u2018a valid assessment of functional capacity cannot be carried out without controlling for fear avoidance beliefs [8] and \u2018pain related fear is more disabling than pain itself\u201910 are not supported by the results of this study. The results of this study may not be generalizable to other FCEs or other groups of patients. From the results of the current study it should be concluded that future research aimed at unrevealing \u2018determinants of performance\u2019 in chronic pain should not be restricted to pain and pain related fears only, but include other avenues such as self efficacy [40, 41], motivation and acceptance as well.","keyphrases":["fear avoidance beliefs questionnaire","lifting","rehabilitation","tampa scale for kinesiophobia","pain cognitions","disability assessment"],"prmu":["P","P","P","P","R","R"]}
{"id":"Acta_Neuropathol-4-1-2386158","title":"Peroxiredoxin 6 in human brain: molecular forms, cellular distribution and association with Alzheimer\u2019s disease pathology\n","text":"Peroxiredoxin 6 is an antioxidant enzyme and is the 1-cys member of the peroxiredoxin family. Using two-dimensional electrophoresis and Western blotting, we have shown for the first time that, in human control and brain tissue of patient\u2019s with Alzheimer\u2019s disease (AD), this enzyme exists as three major and five minor forms with pIs from 5.3 to 6.1. Using specific cellular markers, we have shown that peroxiredoxin 6 is present in astrocytes with very low levels in neurons, but not detectable in microglia or oligodendrocytes. In control brains, there was a very low level of peroxiredoxin 6 staining in astrocytes that was confined to a \u201chalo\u201d around the nucleus. In AD, there were marked increases in the number and staining intensity of peroxiredoxin 6 positive astrocytes in both gray and white matter in the midfrontal cortex, cingulate, hippocampus and amygdala. Confocal microscopy using antibodies to A\u03b2 peptide, tau and peroxiredoxin 6 showed that peroxiredoxin 6 positive astrocytes are closely involved with diffuse plaques and to a lesser extent with neuritic plaques, suggesting that plaques are producing reactive oxygen species. There appeared to be little astrocytic response to tau containing neurons. Although peroxiredoxin 6 positive astrocytes were seen to make multiple contacts with tau positive neurons, there was no intraneuronal colocalization. In brain tissue of patients with AD, many blood vessels exhibited peroxiredoxin 6 staining that appeared to be due to the astrocytic foot processes. These results suggest that oxidative stress conditions exist in AD and that peroxiredoxin 6 is an important antioxidant enzyme in human brain defenses.\nIntroduction\nAlzheimer\u2019s disease (AD) is a neurodegenerative disorder characterized by the progressive decline in memory, language, cognition and motor functions. The neuropathological hallmarks of AD are the accumulation of extracellular amyloid plaques containing the amyloid beta peptide (A\u03b2) and intraneuronal neurofibrillary tangles containing hyperphosphorylated microtubule-associated protein tau. Although the initiating molecular event(s) in AD are not known, oxidative stress produced by the generation of reactive oxygen species (ROS) appears to be a fundamental process contributing to the neuropathophysiology [10]. Whether oxidative stress has a role in the cause or affect in AD has been debated for many years, but recent research has suggested that it occurs prior to the onset of symptoms [35]. The histopathological evidence for oxidative stress in AD includes lipid, protein and DNA oxidation. There is evidence to suggest that oxidative stress is generated peripherally [26] with the brain being particularly susceptible, but there is also a body of evidence to suggest that amyloid \u03b2 peptide (A\u03b242) may be linked to lipid peroxidation [5, 6], which may then be linked to the phosphorylation of tau [24]. Two reactive products of A\u03b242-induced lipid peroxidation are the production of four hydroxynonenal and acrolein [5], which have been shown to disrupt membrane asymmetry [7] and may be involved in the conformational changes to tau, promoting the formation of neurofibrillary tangles [24].\nOxidative damage occurs as part of the aging process, but it occurs at a much greater rate in most neurodegenerative diseases [3]. The hallmark of protein oxidation is the generation of protein carbonyls, which are markedly elevated in AD [3, 16]. There is overwhelming evidence to implicate oxidative stress in the pathogenesis of AD, and this is most likely an early event in the disease process. Less is known however of how the brain responds to oxidative insults. There are a number of cellular antioxidant defenses whose primary role is to convert the ROS into unreactive compounds. These defense mechanisms include superoxide dismutase, which works together with selenium-dependent glutathione peroxidase, catalase and more recently the peroxiredoxin family. These enzymes transform the superoxide radical to hydrogen peroxide, which in turn is converted to water. The distribution and changes in expression of the antioxidant enzymes have not been well studied as the other aspects of the disease process.\nCellular selenium-dependant glutathione peroxidase (GPx-1) has long been regarded as the major cellular antioxidant enzyme, but there are conflicting reports regarding the changes in activities of this enzyme in AD. Early reports suggest that there is no change in GPx-1 activities in AD brains [15, 20], while other researchers suggest that there is a decrease in GPx-1 activity [8, 27, 32]. There are no reports indicating compensatory increase in GPx-1 activity; however, cell models overexpressing GPx-1 are more resistant to A\u03b2-mediated toxicity, suggesting that increased levels of GPx-1 is protective [2].\nThere is considerable evidence to implicate A\u03b2 toxicity and the generation of ROS and in particular hydrogen peroxide as key steps in contributing to the neurotoxicity in AD. We have previously reported that peroxiredoxin 6, also termed 1 Cys-peroxiredoxin or nonselenium glutathione peroxidase, is upregulated in Parkinson\u2019s disease (PD) [30]. This enzyme has no amino acid homology to any known sequence of the glutathione peroxidase enzymes and has both antioxidant and phospholipase A2 activity and can reduce hydroxyperoxides [9]. Given that the brain is particularly vulnerable to oxidative damage due to its high energy and oxygen requirements, high levels of transition metals and peroxidizable fatty acids suggests that peroxiredoxin 6 could play a key antioxidant role in neuroprotection.\nThere are six members of the peroxiredoxin family at this stage with peroxiredoxins 1\u20135 being the 2-Cys members while peroxiredoxin 6 is the sole 1-Cys member [36]. This enzyme is bifunctional with 80% of its function as a peroxidase and the remainder as a phospholipase A2. Peroxiredoxin 6 has not specifically been examined in brain tissue of AD patients, and the aims of this study are to examine the molecular forms, cellular distribution and association with AD pathology. This study focused on the cingulate, amygdala, hippocampus and cortical regions in six AD and six control brains.\nMaterials and methods\nAntibodies\nPrimary antibodies\nPeroxiredoxin 6 (rabbit) was obtained from Antibody Technology Australia Pty, Ltd, Adelaide, Australia. Tau (mouse) was obtained from Novacastra Laboratories Ltd, Newcastle, UK. Tau (goat) was obtained from Santa Cruz Biotechnology Inc., California, USA. Amyloid beta (mouse) was obtained from Novacastra Laboratories Ltd, Newcastle, UK. Glial fibrillary acidic protein (GFAP) (mouse) was obtained from Novacastra Laboratories Ltd, Newcastle, UK. Human neuronal protein HuC\/HuD (mouse) was obtained from Molecular Probes Inc., Eugene, USA. Human HLA (MHC2) (mouse) was obtained from Dakocytomation, Denmark A\/S. Myelin basic protein (MBP) (mouse) was obtained from Novacastra Laboratories Ltd, Newcastle, UK.\nSecondary antibodies\nDonkey anti sheep Cy3 was obtained from Jackson Immunoresearch, West Grove, USA. Donkey antirabbit Cy3 and Cy5 were obtained from Jackson Immunoresearch, West Grove, USA. Goat antimouse Alexa 488 was obtained from Molecular Probes Inc., Eugene, USA. Goat antirabbit alkaline phosphatase was obtained from Sigma Chemical Company, St Louis, USA.\nBrain tissue\nThe brain tissue was obtained from the National Health and Medical Research Council South Australian Brain Bank. Brains were removed at autopsy from clinically diagnosed AD cases and confirmed pathologically postmortem. The control cases were obtained from people who died of unrelated causes without diagnosed neurological pathology that was confirmed postmortem. The brains were bisected and one half snap-frozen at \u221280\u00b0C and the other immersion fixed in buffered formaldehyde. Tissue blocks were processed and embedded in paraffin and 8\u00a0\u03bcm sections were cut and mounted onto gelatin-coated slides. Table\u00a01 provides a list of the brain regions examined and the case details.\nTable\u00a01List of cases used in cell countingCase numberSexAge (years)DiagnosisRegionPMI (h)Braak stageNIA ReaganAD1M65ADC, A, H, MC 35\/6HighAD2F84ADC, A, H, MC 165\/6HighAD3M63ADC, A, H, MC 335\/6HighAD4M59ADC, A, H, MC205\/6HighAD5F81ADC, A, H, MC195\/6HighAD6F69ADC, A, H, MC385\/6HighC1F79ADLC, A, H, MC4LowC2M69UnkC, A, H, MC48LowC3F61CFC, A, H, MC8LowC4F84SSC, A, H, MC15LowC5F86DMC, A, H, MC17LowC6F86HVDC, A, H, MC6LowPMI postmortem index, AD Alzheimer\u2019s disease, NIA Reagan high or low likely-hood of AD, M male, F female, C cingulate, A amygdala, H hippocampus, MC midfrontal cortex, C control, ADL adeno lung carcinoma, Unk unknown, CF cardiac failure, SS septic shock, DM disseminated malignancy, HVD hypertensive vascular disease\nBrain homogenate\nFrozen tissue from midfrontal cortex of three AD brains and three control cases were homogenized in 50\u00a0mM Tris, 5\u00a0mM EDTA, 0.1% sodium azide, 1\u00a0\u03bcl\/ml pepstatin and leupeptin and 0.3\u00a0mM of phenylmethylsulfonyl fluoride. Five milliliters of homogenization buffer was used per gram of brain tissue and homogenized using six passes of a motorized Wheaton Teflon pestle tissue grinder. Homogenates were centrifuged (500\u00d7g) for 10\u00a0min to remove particulate matter and snap-frozen at \u221280\u00b0C. Table\u00a02 provides the details of these cases.\nTable\u00a02List of cases used for 2D PAGE and Western blotsCase numberSexAge (years)DiagnosisRegionPMI (h)Braak stageNIA ReaganAD7F69ADMC225\/6HighAD8F79ADMC185\/6HighAD9F76ADMC235\/6HighC6F86HVDMC6LowC7F73ABDMC24LowC8M63SSMC38LowPMI postmortem index, AD Alzheimer\u2019s disease, F female, M male, MC midfrontal cortex, C control, HVD hypertensive vascular disease, ABD anoxic brain damage, SS septic shock\nSample preparation\nAliquots (100\u00a0\u03bcl) of Alzheimer\u2019s and control brain homogenates underwent a 2D clean up as per the Amersham Biosciences protocol. The samples were then resolubilized in 300\u00a0\u03bcl of TUC pH 4\u20137 buffer containing 7\u00a0M urea, 2\u00a0M thiourea, 4% CHAPS, 0.5% dithiothreitol, 0.5% IPG buffer pH 4\u20137. Samples were then assayed for total protein content. Prior to rehydration of the immobilized pH gradient strips, trace amounts of bromophenol blue was added to samples. Table\u00a02 provides a list of cases used in the 2D electrophoresis.\n2D polyacrylamide gel electrophoresis (PAGE)\nIsoelectric focusing\nIsoelectric focusing was performed on 13\u00a0cm immobilized pH gradient strips (pH 4\u20137NL, Amersham Pharmacia) using an IPGphor (Amersham Pharmacia) isoelectric focusing unit. Samples were applied in gel rehydration (50\u00a0V, overnight) using 7\u00a0M urea, 2\u00a0M thiourea, 4% CHAPS, 0.5% dithiothreitol, 0.5% pharmalyte pH 4\u20137 with a trace of bromophenol blue as the re-swell buffer with a total loading volume of 280\u00a0\u03bcl including sample. Stained gels received a 400\u00a0\u03bcg protein load and Western blotting gels received a 200\u00a0\u03bcg load. IEF was performed for \u223c60\u00a0kV\u00a0h (500\u00a0V 30\u00a0min, 1,000\u00a0V 30\u00a0min, 1,000\u20138,000\u00a0V 30\u00a0min, 8,000\u00a0V 5\u20137\u00a0h, 1,000\u00a0V hold to end).\nSDS PAGE\nSDS PAGE was performed using 12.5% acrylamide gels. Immobilized pH gradient strips were incubated in SDS equilibration buffer containing 1% w\/v dithiothreitol for 15\u00a0min followed by SDS equilibration buffer containing 4% w\/v iodoacetamide for 15\u00a0min. Strips were then placed above the second dimension gels and overlaid with 1% low melting point agarose in Tris glycine gel running buffer. Electrophoresis was performed at 350\u00a0V for 2.5\u20133\u00a0h at 10\u00b0C. Protein gels were visualized using SYPRO ruby fluorescent stain (incubated for 2\u00a0h) and scanned using a Typhoon 9400 Variable Mode Imager (Amersham Biosciences).\nWestern blotting\nSeparated proteins were transferred onto low fluorescence PVDF membrane by semi-dry electroblotting at 0.8\u00a0mA\/cm2 for 2\u00a0h. After blocking, the membranes were incubated in affinity-purified rabbit antiperoxiredoxin 6 antibodies (1\/2,000) overnight at 4\u00b0C followed by goat antirabbit alkaline phosphatase antibodies (1\/10,000) (Sigma) for 1\u00a0h and visualized using ECF substrate and a Typhoon 9400 Variable Mode Imager.\nImmunohistochemistry\nLocalization of peroxiredoxin 6\nImmunohistochemical staining of AD and control tissue was carried out as previously described [30]. The cellular distribution of peroxiredoxin 6 was compared with the staining obtained with antibodies to specific cellular markers. Antibodies to GFAP (astrocytes), HLA (MHC2) (microglia), MBP (oligodendrocytes) and HuC\/HuD (neurons) were used to map the distribution of peroxiredoxin 6.\nColocalization of peroxiredoxin 6, A\u03b2 and Tau\nBrain sections were incubated with rabbit antiperoxiredoxin 6, mouse anti A\u03b2 peptide and goat anti tau antibodies overnight and detected with secondary antibodies conjugated to fluorescent fluorophores Cy5, Alexa 488 and Cy3, respectively. Sections were examined using a Bio-Rad Confocal laser scanning microscope and Bio-Rad software package, as previously described [30].\nCell counting\nCell counting was carried out blind on masked sections, and peroxiredoxin 6 positive astrocytes were counted at 20\u00d7 magnification with a graticule eye piece (0.0625\u00a0mm2) using an Olympus HO-2 microscope on brain tissue from six AD and six control cases. In each case, five regions were randomly selected to give a uniform representation of the white and gray matter in the cingulate, hippocampus, amygdala and midfrontal cortex. The number of positive cells from each of the five areas was averaged and divided by the area of the graticule eyepiece and expressed as cells\/mm2. The data was then analyzed using a one-way ANOVA that examined the interactions listed in Table\u00a03. The significance was determined at 0.01 level of probability.\nTable\u00a03Interactions examined in cell counting dataInteractions examinedF valueCritical F (P\u00a0=\u00a00.01)Significance (>0.01)Are there differences in AD (C, A, H, MC) gray matter cell counts?1.004.94NSAre there differences in AD (C, A, H, MC) white matter cell counts?1.234.94NSAre there differences in control (C, A, H, MC) gray matter cell counts?0.624.94NSAre there differences in control (C, A, H, MC) white matter cell counts?0.254.94NSAre there differences between gray and white matter cell counts in AD tissue?89.37.21SAre there differences between gray and white matter cell counts in control tissue?14.57.21SAre there differences in gray matter cell counts between control and AD tissue?2997.21SAre there differences in white matter cell counts between control and AD tissue?28.07.21SAD Alzheimer\u2019s disease, NS not significant, S significant, C cingulate, A amygdala, H hippocampus, MC midfrontal cortex\nResults\nMolecular forms of peroxiredoxin 6 in human brain\nWe have previously shown that this antibody is specific for a 26\u00a0kDa monomer in rat lung and human brain, and the N-terminal sequence is identical to peroxiredoxin 6 [30, 31]. Two-dimensional PAGE and Western blotting of AD and control brain homogenates indicated that this protein exists as three major and five minor forms as shown in Fig.\u00a01a\u2013d. Although the loadings were consistent, the five minor forms could not be detected on all gels. For comparison, the spots were labeled 1\u20138 (Fig.\u00a02).\nFig.\u00a01a, b A two-dimensional PAGE gel and blot of AD brain tissue (Case AD7) and a corresponding control brain gel and blot panels (c, d) (Case C7). pI 4\u20136 is indicated between the gel and blots and the molecular weight of the standards (Std) in kilo Daltons is shown on the Y-axis. The gels were stained with SYPRO ruby fluorescent stain. The box indicates the location of the molecular forms of peroxiredoxin 6. Some spots are shown on the blots to indicate the range of peroxiredoxin 6 pIsFig.\u00a02A composite figure at higher magnification showing the range of molecular forms in three cases of AD tissue (a AD7, AD8 and AD9) and three cases of control tissue (b C7, C6 and C8). The range of molecular forms 1\u20138 is shown, but not all forms are present in all cases. Although there are slight variations between blots, the respective pIs are as follows: 1 (5.3), 2 (5.4), 3 (5.6), 4 (5.75), 5 (5.85), 6 (5.9), 7 (5.95), 8 (6.1)\nThe respective pIs of the eight forms are 5.3, 5.4, 5.6, 5.75, 5.85, 5.9, 5.95 and 6.1. The three major forms were 3, 6 and 7 and the average pIs of these were 5.59, 5.88 and 5.96 for AD tissue and 5.59, 5.80 and 5.86 for control tissue. These major forms were consistent in all homogenates. The intensity of the three major forms was analyzed using the Typhoon imager and the intensity of spots 6 and 7 was reversed in the AD and control homogenates (Fig.\u00a02). Minor forms 1 and 2 were present in AD homogenates but absent in the control tissue, while minor form 8 was present in control tissue but absent in AD tissue (Fig.\u00a02).\nCellular distribution of peroxiredoxin 6 in control and AD brain tissue\nUsing immunohistochemical markers for specific human brain cell types, we have shown that peroxiredoxin 6 is abundant in reactive astrocytes (Fig.\u00a03a\u2013c) in AD tissue and present in very low levels in neurons (Fig.\u00a03j\u2013l). We were not able to detect peroxiredoxin 6 in either microglia (Fig.\u00a03d\u2013f) or oligodendrocytes (Fig.\u00a03g\u2013i).\nFig.\u00a03Confocal localization of peroxiredoxin 6 (P6) with GFAP (astrocyte marker) (a P6, Cy2-green; b GFAP, Cy3-red; c merged image; bar\u00a025\u00a0\u03bcM), MHC2 (microglia marker) (d P6, Cy2-green; e MHC2, Cy3-red; f merged image; bar\u00a025\u00a0\u03bcM), MBP (oligodendrocyte marker) (g P6, Cy2-green; h MBP, Cy3-red; i merged image; bar\u00a010\u00a0\u03bcM), Hu (neuronal marker) (j P6, Cy2-green; k Hu, Cy3-red; l merged image; bar\u00a025\u00a0\u03bcM). Cell localization was performed using AD tissue\nThere was a marked contrast between the staining of control and reactive astrocytes in AD. Reactive astrocytes in AD were hypertrophied with abundant staining of peroxiredoxin 6, while in control astrocytes the staining was confined to a \u201chalo\u201d around the nucleus (Fig.\u00a04). The increased staining of peroxiredoxin 6 was seen in all regions examined and in both gray and white matter, but the staining was not evenly distributed and some regions on the same section were heavily labeled but adjacent regions showed little staining (Fig.\u00a05).\nFig.\u00a04Oil immersion image (\u00d71,000) of typical astrocyte seen in AD cortex (a) and in control cortex (b)Fig.\u00a05Light immunohistochemistry (DAB substrate) of peroxiredoxin 6 staining of astrocytes in AD gray matter (a), AD white matter (b), control tissue gray matter (c) and control tissue white matter (d). All images at \u00d7200 magnification\nCell counting\nIn AD and control tissue, peroxiredoxin 6 was colocalized with GFAP, indicating that the upregulation is occurring in astrocytes, which is similar to what was found in PD and dementia with Lewy bodies [30]. Cell counting was conducted on midfrontal cortex, cingulate, amygdala and hippocampus brain regions and in gray and white matter to determine if specific areas or matter type were specifically affected. These are summarized in Fig.\u00a06. A one-way ANOVA was performed comparing the cell counts between AD and control tissue in different regions by tissue and matter type. The results of the interactions examined are shown in Table\u00a03.\nFig.\u00a06A bar chart showing the range of cell counts in different brain regions in gray and white matter in control tissue and in AD tissue. WM white matter, GM gray matter, Cing cingulate, Amy amygdala, MFC midfrontal cortex, Hip hippocampus. Counts are shown as mean\u00a0\u00b1\u00a0SE, N\u00a0=\u00a06 in all cases\nColocalization of peroxiredoxin 6 and AD pathology\nThe major defining pathology in AD is the presence of extracellular plaques and intracellular hyperphosphorylated tau in the form of tangles. Plaques are present in brain tissue at different stages of development, ranging from diffuse plaques to fibrillar and neuritic plaques. It is generally considered that diffuse plaques do not contain tau, but neuritic plaques contain both A\u03b2 and tau. Colocalization of peroxiredoxin 6 and the A\u03b2 peptide, a major component of plaques, and tau was carried out to determine if peroxiredoxin 6 positive astrocytes were associated with AD pathology.\nTau pathology\nTau deposits in the form of neurofibrillary tangles were observed in many neurons and neurites, and were often surrounded by peroxiredoxin 6 astrocytes, which made multiple contacts (Fig.\u00a07a\u2013c). Although astrocytes made many contacts with neurons, most neurons had very low levels of peroxiredoxin 6 and it did not appear to be upregulated or to be colocalized with tau in AD.\nFig.\u00a07a\u2013c Confocal localization of tau within neurons (a Cy2-green) and peroxiredoxin 6 positive astrocytes (b Cy3-red) and (c) the merged image in AD brain tissue (bar\u00a0=\u00a010\u00a0\u03bcM). d\u2013f Confocal localization of A\u03b2 peptide (d Alexa 488, green) and peroxiredoxin 6 positive astrocytes (e Cy3-red) and (f) the merged image in a diffuse plaque in AD brain tissue (bar\u00a0=\u00a050\u00a0\u03bcM). g\u2013j Confocal localization of A\u03b2 peptide (Alexa 488, green) and peroxiredoxin 6 positive astrocytes (Cy5-light blue) in four different plaques in AD brain tissue (bar\u00a0=\u00a050\u00a0\u03bcM). k Immunohistochemical staining (DAB substrate) showing peroxiredoxin 6 staining of astrocytes and astrocyte foot processes in the walls of blood vessels in the midfrontal cortex in Alzheimer\u2019s disease (\u00d7400). Arrows indicate peroxiredoxin 6 positive astrocytic processes\nPlaques\nDiffuse plaques contained strongly stained aggregations of A\u03b2 peptide. Peroxiredoxin 6 staining was confined to astrocytes and their projections that were in close proximity to plaques (Fig.\u00a07d\u2013h). Some astrocytes were observed within plaques, but generally the astrocyte cell bodies were on the periphery with their processes projecting into the plaques. Some plaques contained the beginnings of a dense core of amyloid and took on a \u201cwagon wheel\u201d appearance (Fig.\u00a07i, j). Diffuse plaques and those with A\u03b2 beginning to aggregate appeared to elicit a stronger peroxiredoxin 6 astrocytic response than those with a dense core of A\u03b2.\nPeroxiredoxin 6 staining of blood vessels\nIn many regions of the AD brain, there appeared to be strong peroxiredoxin 6 staining in the walls of many blood vessels, which could be seen to have multiple contacts with strongly staining activated astrocytes (Fig.\u00a07k).\nDiscussion\nPeroxiredoxin 6, also known as 1-Cys peroxiredoxin or non-selenium glutathione peroxidase, is a human brain antioxidant enzyme that is abundant in activated astrocytes and present in low levels in neurons. On exposure to hydrogen peroxide, the N-terminal peroxiredoxin 6 corresponding to Cys 47 is readily oxidized, but as yet its redox partner has not been identified. Glutathione, lipoic acid and cyclophilin have all been implicated [13, 23, 29]. In addition to its peroxidase activity, peroxiredoxin 6 also has phospholipase A2 activity that might be significant in the brain, which has a high lipid content [9]. Peroxiredoxin 6 staining of astrocytes is markedly elevated in many brain regions in PD and dementia with Lewy bodies [30]. We now show that it is upregulated in the astrocytes in AD and describe for the first time the range of molecular forms of peroxiredoxin 6 in human brain in both control and AD tissue.\nThree major variants and five minor forms were identified with pIs ranging from 5.3 to 6.1. There were three major differences in the peroxiredoxin 6 variants between control and AD tissue. The AD tissue had the two more acidic forms 1 and 2 that were not present in control tissue; the intensity of the major forms 6 and 7 were reversed between AD and control tissue and minor form 8 was absent in AD tissue. We can only speculate on the significance of these findings at this stage, but different forms may have different affinities for different substrates that might be more abundant under various pathological conditions or the more acidic forms are the result of oxidation [11]. At this stage, we cannot determine whether these differences are true isoforms or post-translational modifications of the same protein, but would suspect the latter. These findings are in agreement with two previous proteomic studies in an AD mouse model and in human brain AD tissue, which reported elevated levels of peroxiredoxin 6 [33, 34]. A more recent study using PC12-resistant and PC12 cells treated with A\u03b2 also reported increased levels of peroxiredoxin 6 [11]. A\u03b2-treated cells contained an extra acidic form, which was suggested to be the result of oxidation of the catalytic cysteine to a sulfenic, sulfinic or sulfonic derivative. We found two minor acidic forms in AD tissue, which is also consistent with this report.\nTissue selection is also a variable that needs to be considered. While the AD tissue had all the hallmarks of AD pathology and was easy to define, age-matched control tissues are somewhat more variable. All the control tissue was from people without clinically defined AD, but most brains in this age group usually contain some neurodegenerative pathology. The age span of the two groups was similar, 59\u201384\u00a0years for the AD group and 61\u201386\u00a0years for the control group, but the mean age for the control group was 7\u00a0years older. We feel confident that, although some control tissue may have had some elements of subclinical AD pathology, the reduced level of astrocyte activation and the clear differences in the cell counting would indicate that all the control tissues were not experiencing high levels of oxidative stress as compared to the AD tissue.\nPreviously, we had shown that peroxiredoxin 6 was abundant in astrocytes in PD and dementia with Lewy body tissue [30]. We have now used cellular markers to other brain cell types to show that in addition to astrocytes there is a low level in the cytoplasm of most neurons. Unlike astrocytes, neurons do not appear to have increased peroxiredoxin 6 staining in AD tissue. We could not detect any peroxiredoxin 6 staining in microglia or oligodendrocytes.\nCell counting of peroxiredoxin 6 positive astrocytes indicated that there was a marked increase in both the gray and white matter in AD tissue compared with control tissue. Gray matter had more peroxiredoxin 6 positive astrocytes than white matter in both AD and control tissue, and this difference was more pronounced in AD tissue with F values of 89.0 and 14.5, respectively. Considering that the major pathology (plaques and tangles) is located in the gray matter, it would be expected that oxidative stress is more pronounced in this area. Cell counting indicated that the peroxiredoxin 6 positive astrocyte numbers were elevated in all regions examined, suggesting that the insult activating astrocytes is wide spread and not confined to any specific region in either the gray or white matter. Indeed, extracellular plaques were observed in all regions examined in AD tissue. Although all regions examined showed astrocyte activation, this was not uniformly distributed through all areas, with some areas showing extensive astrocyte activation while adjacent areas were similar to controls.\nThere are two defining pathologies in AD. These are the presence of extracellular plaques comprising abundant deposits of A\u03b2 peptides, which later aggregate to form amyloid [25] and intracellular hyperphosphorylated tau aggregations in the form of paired helical filaments, which are termed tangles [14, 22]. The two well-described forms of plaques have been termed diffuse plaques and neuritic or senile plaques [12, 18], although other intermediate forms such as fibrillar plaques have been described [12]. Diffuse plaques are spherical structures of less dense A\u03b2 deposits and are considered an early stage of plaque formation. In diffuse plaques, aggregations of A\u03b2 exists as nonamyloid deposits, but as the plaque progresses, the A\u03b2 and other protein aggregations change from the normal conformation into a \u03b2-sheet structure, termed amyloid, and take up specific dyes like Congo red and thioflavin [4]. These plaques do not generally contain tau; however, in many confocal images, diffuse plaques contain focal points of tau, which are probably enlarged neurites. Neuritic or senile plaques contain a dense core of amyloid enriched with A\u03b2 peptide with a corona of less dense amyloid. The diffuse plaques appeared to elicit a stronger astrocytic response based on the level of peroxiredoxin 6 staining than the neuritic plaques. Our images suggest that once the A\u03b2 forms amyloid and condenses, it is less stimulating toward astrocytes and perhaps the formation of amyloid is a protective mechanism to inactivate the toxicity of soluble A\u03b2. These observations are consistent with the findings of Kawaguchi-Niida et al., who reported that the highly reactive carbonyl crotonaldehyde generated during lipid peroxidation is localized to reactive astrocytes, microglia and diffuse plaques but undetectable in amyloid cores [19].\nAlthough the activation of astrocytes and upregulation of peroxiredoxin 6 would be considered a protective response, in cell culture, soluble A\u03b2 has been shown to stimulate nitric oxide synthetase and the production of nitric oxide, which is very damaging to neurons [17]. This response is further upregulated by other inflammatory cytokines such as interleukin 1\u03b2 and tumour necrosis factor-\u03b1, which are reported to be released from astrocytes [1]. At this stage, we are not sure of the net benefit to neural tissue of activated astrocytes with elevated levels of peroxiredoxin 6, concomitant with the increased production of nitric oxide.\nNeurofibrillary tangles comprising hyperphosphorylated tau were observed in many regions in AD tissue. Two antibodies were used to localize tau, a monoclonal antibody (Novacastra) and one specific for paired helical filaments (Santa Cruz) and both produced a similar staining pattern. Neurons containing tau were not surrounded by activated astrocytes as were the diffuse plaques and to a lesser extent the neuritic plaques, suggesting that these cells are not secreting toxic or activating products that are stimulating to astrocytes. In PD, Lewy bodies contained a dense core of peroxiredoxin 6 staining suggesting that it was trying to detoxify oxidative stress produced by Lewy bodies, but we did not observe any interaction between tau and peroxiredoxin 6 in AD tissue. Although there is considerable evidence to implicate oxidative stress in the hyperphosphorylation of tau, this does not appear to upregulate peroxiredoxin 6 in tau positive neurons.\nIn some regions, the walls of blood vessels exhibited strong peroxiredoxin 6 staining, which also elicited a strong astrocytic response in surrounding astrocytes. These activated astrocytes had many processes in contact with the endothelial cells. Although vascular amyloid deposits were seen in some sections and is a key feature of AD [28], the upregulation of peroxiredoxin 6 appeared to occur in regions without distinct A\u03b2 staining. A\u03b2 has been shown to induce vascular vasoconstriction in rat skin, which can be reversed by superoxide dismutase and catalase, indicating that A\u03b2 is capable of generating ROS within blood vessels [21]. The blood brain barrier consists of the capillary endothelial cells, and the foot processes of the astrocytes and the fused basal lamina of both cells. The peroxiredoxin 6 staining within the capillaries appears to be in the foot processes of the astrocytes. Whether this increased staining is due to ROS generated from within neural tissue or reflects a more systemic pathology derived from the circulation is unclear.\nIn conclusion, we have shown for the first time the range of peroxiredoxin 6 variants, which are either isoforms or post-translational modifications in human brain tissue. We have also shown that peroxiredoxin 6 is primarily an astrocytic enzyme with very low levels in neurons and is not detectable in microglia or oligodendrocytes. This enzyme is markedly elevated in astrocytes in both white and gray matter in AD. Strongly staining peroxiredoxin 6 astrocytes appear to be involved in the detoxification of diffuse plaques and to a lesser extent in neuritic plaques, but is not associated with tau aggregations within neurons. From this work and previous work on PD and dementia with Lewy bodies, we suggest that peroxiredoxin 6 is a major antioxidant enzyme in human neural tissue.","keyphrases":["peroxiredoxin 6","molecular forms","alzheimer\u2019s disease","astrocytes","oxidative stress"],"prmu":["P","P","P","P","P"]}
{"id":"Qual_Life_Res-3-1-1915604","title":"Quality of life in couples living with Huntington\u2019s disease: the role of patients\u2019 and partners\u2019 illness perceptions\n","text":"Research suggests that chronically ill patients and their partners perceive illness differently, and that these differences have a negative impact on patients\u2019 quality of life (QoL). This study assessed whether illness perceptions of patients with Huntington\u2019s disease (HD) differ from those of their partners, and examined whether spousal illness perceptions are important for the QoL of the couples (n = 51 couples). Partners reported that their HD-patient spouses suffered more symptoms and experienced less control than the patients themselves reported. Illness perceptions of patients and partners correlated significantly with patient QoL. Partners\u2019 beliefs in a long duration of the patients\u2019 illness and less belief in cure, were associated with patient vitality scores. Suggestions for future research emphasize the importance of qualitative research approaches in combination with cognitive-behavioural approaches.\nQuality of life research is increasingly being directed at both the impact of the social environment on the quality of life (QoL) of chronically ill individuals, and at the toll the illness exacts on the QoL of close family members [1] One of the most influential members of the social network of chronically ill individuals is the spouse. Research conducted in chronically ill individuals and their healthy spouses revealed that the spouses\u2019 role problems [2, 3], their unsupportive behaviour [4\u20136], and the marital relationship itself [7] are some of the factors influencing patients\u2019 QoL.\nStudies examining adaptation in caregivers have identified negative social support [8], patients\u2019 cognitive impairment [9, 10], and the caregivers\u2019 overestimation of the patient\u2019s functional disabilities [11] as some of the factors influencing the partners\u2019 QoL.\nRecent findings suggest that patients\u2019 QoL is also dependent on beliefs fostered by the social environment regarding the patient\u2019s illness. Illness beliefs or illness perceptions can be subdivided into five components: identity (the label the patient places on the illness and the symptoms he\/she experiences), cause (the personal ideas patients have about the cause of their illness), timeline (the duration of the illness according to the patient), consequences (the expected effects and outcome of the illness), and cure\/control (the curability or controllability of the illness according to the patient) [12]. In the area of physical health problems, illness perceptions have been found to be related to the ways patients react and cope with their illness. A strong illness identity combined with a perceived long duration and perceived more severe consequences of the disease for daily life have been associated with poor physical and psychological well-being. Stronger beliefs regarding the curability or controllability of a disease have been found to have positive effects on patients\u2019 QoL [13, 14].\nResearch shows that illness beliefs of spouses significantly affect patients\u2019 beliefs, the coping mechanisms they adopt to deal with their disease, and ultimately their functioning and well-being [15, 16]. Heijmans and colleagues found that dissimilarities between the illness perceptions of chronically ill patients (patients with Addison\u2019s disease and patients with Chronic Fatigue Syndrome) and those of their healthy spouses were associated with higher impairments in predicted patients\u2019 adaptive outcome [15]. In another study, Figueiras and Weinman explored whether the degree of similarity in patients\u2019 and spouses\u2019 illness perceptions was related to recovery following myocardial infarction [16].\nFew studies have focused on the role of illness perceptions for caregiver outcomes, and none have examined the role of patients\u2019 illness perceptions on the QoL of their healthy partners. Barrowclough et\u00a0al. [17] and Fortune et\u00a0al. [18] studied illness perceptions in carers of schizophrenia patients. Their findings suggest that carer perceptions about identity, consequences and control may have important implications for carer outcomes in schizophrenia. In this study we will focus on Huntington\u2019s disease (HD) which is an inherited neurodegenerative disorder, characterized by clearly defined clinical features such as involuntary movements and hypokinesia, dementia, and personality changes. The first symptoms of HD typically manifest between the ages of 35 and 45, and the disease has a mean duration of 16\u00a0years [19, 20]. At present, there is no cure for HD.\nLittle empirical data has been gathered on the psychosocial aspects of living with HD, but some research findings suggest that HD contributes to marital breakdown [21\u201323]. One of the most important reasons for this is believed to be the changes HD brings about in the relationship. Spouses of HD patients often consider their partners to be lost [23], particularly when dementia and changes in personality and behaviour develop [22, 24]. In addition, over time the spouse takes on an increasing nursing role, which creates a psychological distance between the patient and his\/her spouse [22]. HD greatly impacts the patients\u2019 physical and psychosocial well-being [25, 26], and places a heavy burden on families [27\u201329]. Interestingly, some research has indicated that there is a discrepancy between the aspects of HD the patient finds most disturbing and those the spouse or partner finds most disturbing. Partners are most disturbed by mental and personality changes in the patients [30, 31].\nIn all studies examining the role of spousal illness perceptions for patient outcomes, researchers have concentrated on the degree of similarity \/ dissimilarity in patients\u2019 and spouses\u2019 illness perceptions as a predictor of patient outcome, thereby ignoring alternative possible relationship patterns between spouses\u2019 illness beliefs and patient outcome. Results from Figueiras and Weinman [16] showed that not only similar positive perceptions in couples, but also conflicting representations were predictive of lower levels of disability (as compared to similar negative perceptions). This suggests that it might be more important that at least one member of a couple has positive perceptions. However, the results obtained in the Heijmans et\u00a0al. [15] study show that better patient adjustment in Addison\u2019s Disease is related to spouses\u2019 negative perceptions about timeline, while better patient adjustment in Chronic Fatigue Syndrome is related to spouses\u2019 positive perceptions of a short illness duration. From these studies it seems that both contrasting and concordant perceptions in couples can be related to better patient adjustment, and further study is needed to evaluate the extent to which spouses\u2019 illness perceptions can influence outcomes which have been found to be related to the patient\u2019s own perceptions.\nThus, the first aim of this study was to examine if patients and partners hold similar views about HD. Our second aim was to compare the relationship patterns between patients\u2019 and partners\u2019 own illness perceptions and QoL. The third aim was to contribute to the understanding of the cognitive factors related to QoL in couples dealing with HD, by examining which spousal illness beliefs are important in both patients\u2019 and partners\u2019 quality of life.\nMethod\nSample and procedure\nParticipants in this study were 51 couples (HD patients and their partners) who were recruited from the outpatient clinic of the Department of Neurology of the Leiden University Medical Centre (LUMC; n\u00a0=\u00a014 couples), and the Dutch Huntington Association (n\u00a0=\u00a037 couples). HD patients from the LUMC were selected if they had received a clinical diagnosis of HD at least 1\u00a0year prior to commencement of the study, and if they were capable (mentally, emotionally, and physically) of participating in an interview lasting approximately 2\u00a0h. The selected patients (n\u00a0=\u00a075) and their spouses were invited by post to participate. A total of 36 patients and 19 spouses agreed to participate. The main reason for not participating were the verbal communication difficulties that patients were experiencing, and having participated in other research projects before. Non-participants did not differ from participants with respect to age, sex, or duration of HD. HD patients and their spouses recruited from the Dutch Huntington Association were invited to participate by means of a letter sent to all the members of the association (n\u00a0=\u00a01,450). The selection criteria for participation were the same as those applied to the patients selected from the Department of Neurology of the LUMC. A total of 41 patients and 71 spouses agreed to participate. No information could be gathered on non-participants due to the fact that members of the Dutch Huntington Association are not registered on the basis of their patient status. Members include HD patients and their partners, as well as their family members, friends, and researchers in the field of HD. A total of 51 couples (married or living together) were identified. Patients and their partners were interviewed separately at their homes by a psychologist.\nMeasures\nDemographic variables\u2014HD patients and their partners were asked their age, sex, marital status, the duration of their relationship, number of children, their employment status, and the duration of HD.\nUnified Huntington Disease Rating Scale [32]\u2014We used the motor section of the UHDRS to assess HD patients\u2019 motor performance. The motor section is composed of 20 items rating ocular motor function, dysarthria, chorea, dystonia, gait, and postural stability. The Total Motor Score (TMS) is the sum of all the individual items, higher scores indicating worse motor performance (maximum score\u00a0=\u00a0124).\nMini-Mental State [33]\u2014We used this scale to assess HD patients\u2019 cognitive performance. It comprises 11 items covering a number of cognitive domains including: orientation, registration, attention, memory, language and visuo-constructional abilities. The maximum score is 30, lower scores indicating worse performance. Scores of 20 or less have been associated with dementia, delirium, schizophrenia or affective disorder [33].\nThe Illness Perception Questionnaire [34]\u2014This questionnaire was used to assess the illness perceptions of HD patients and those of their partners (not yet validated in Dutch). The latter were interviewed by means of a partner-version of the IPQ [35]. The IPQ consists of the following five subscales: \u201cIdentity\u201d, \u201cTimeline\u201d, \u201cCause\u201d, \u201cConsequences\u201d, and \u201cCure\/Control\u201d. In this study we divided the last subscale into \u201cCure\u201d and \u201cControl\u201d, thus creating a sixth scale. This is in line with current revisions of the IPQ in which these scales are generally separated [36]. The \u201cidentity\u201d scale was composed of 24 items, each corresponding to a symptom commonly reported in HD. Patients are asked to rate whether or not they have experienced each symptom since their illness began, and if they believe the symptom to be specifically related to their illness (yes or no). Partners were asked whether or not their partner (the patient) had experienced each symptom since onset of their illness, and to report if they believed the symptom to be specifically related to their partners\u2019 illness (yes or no). The summed yes-rated items on the second questions were divided by the number of items to form the illness identity scale, with higher scores indicating a stronger belief that the experienced symptoms are part of the patient\u2019s illness. Cronbach\u2019s alpha for patients and partners was 0.88 and 0.82, respectively.\nFor the remaining scales, patients and their partners were asked to indicate whether they agreed with statements on a five-point scale, ranging from \u201cstrongly agree\u201d to \u201cstrongly disagree\u201d. The \u201cTimeline\u201d scale contained two statements about the perceived duration of the disease, with high scores indicating strong beliefs in a chronic long-term disease. Cronbach\u2019s alpha for patients and partners was 0.45 and 0.72, respectively. \u201cConsequences\u201d consisted of five items assessing beliefs about the impact of HD on everyday life. High scores indicate stronger beliefs in serious consequences of the disease. Cronbach\u2019s alpha for patients and partners was 0.69 and 0.61, respectively. High scores on the \u201cCure\u201d scale (two items) indicate strong beliefs in the effectiveness of treatment. Cronbach\u2019s alpha for patients and partners was 0.77 and 0.66, respectively. \u201cControl\u201d contained two items pertaining to the degree to which patients and partners believe they have the ability to influence the course of illness. Cronbach\u2019s alpha for patients and partners was 0.85 and 0.83, respectively. The weight of all the items per scale were summed, and divided by the number of items, with the exception of those of the \u201cCause\u201d subscale. Each item in this subscale was considered individually because each causal item represents a specific causal belief.\nThe Medical Outcome Study 36-item Short Form Health Survey [37]\u2014We assessed the QoL of HD patients, and the QoL of partners by means of seven subscales of the MOS SF-36 (\u201cPhysical functioning\u201d, \u201cRole functioning-physical\u201d, \u201cGeneral health\u201d, \u201cVitality\u201d, \u201cSocial functioning\u201d, \u201cRole functioning-emotional\u201d, and \u201cMental health\u201d). The raw scores are transformed in order to obtain a 0\u2013100 scale, with higher scores indicating a better outcome.\nAnalysis plan\nFirst, the samples of HD patients and partners from the outpatient clinic and the Dutch Huntington Association involved in this study were compared for demographic and illness related variables (age, sex, marital status, duration of the relationship, number of children, employment status, duration of HD, patients\u2019 TMS, and patients\u2019 scores on the MMS) by means of t-tests. To examine the extent of (dis)agreement that HD patients and their partners held with regard to their beliefs about HD, Pearson correlations and paired sample t-tests were computed.\nTo assess whether spousal illness perceptions are related to patients\u2019 and partners\u2019 QoL, we conducted hierarchical regression analyses with scores on the MOS SF-36 as criteria. For patients, illness related variables (disease duration, TMS, and MMS scores) were entered as control variables, prior to the steps containing the patients\u2019 and partners\u2019 scores on the IPQ (step 2 and 3, respectively). For partners, parallel analyses were conducted, but no control variables were entered. The variables for the regression analyses were selected based on an examination of bivariate correlations between the illness perception dimensions and the QoL scales.\nResults\nSample characteristics\nCouples recruited from the Department of Neurology of the LUMC did not differ significantly in demographic or illness related variables from couples from the Dutch Huntington Association. Further analyses were thus conducted on the combined data.\nIn total, 51 HD patients (28 males, 23 females) and their partners (23 males, 28 females) were included in this study. The mean age of both patients and partners was 51\u00a0years (SD\u00a0=\u00a010). Forty-seven couples were married and four were living together. The mean duration of the relationship was 25\u00a0years (range: 2\u201348\u00a0years), and the mean number of children was 2 (range: 0\u20136). Eight couples did not have children. Thirty-nine patients (76.5%) were unemployed, of which 28 (54.9%; 19 males, 9 females) stopped working because of HD, whereas 10 (19.6%; 6 males, 4 females) were still working, and 2 (3.9%; males) were retired and receiving pension. Thirty-four partners (66.7%; 19 males, 15 females) were employed, 15 (29.4%) were unemployed (for different reasons including marriage and childbearing; 5 females had never had gainful employment), and 2 (3.9%; males) were retired and receiving a pension. The mean duration of HD was 7\u00a0years (SD\u00a0=\u00a05; range: 1\u201324). Patients\u2019 mean Total Motor Score (TMS) was 37.8 (SD\u00a0=\u00a029.5), and their mean score on the MMS was 23.5 (SD\u00a0=\u00a03.9).\nPatient-partner differences in illness perceptions\nHD patients and their partners did not differ significantly on the \u201cTimeline\u201d, \u201cConsequences\u201d, \u201cCure\u201d or \u201cCause\u201d subscales of the IPQ (see Table\u00a01). Both groups of respondents reported perceiving HD as having a long duration and having many consequences for their daily lives, without either of them believing in a cure for HD. Patients and partners (with the exception of four patients and one partner) attributed HD to genetic causes. Six HD patients (11.8%) reported believing that stress was an important contributing factor to the development of the symptoms of HD they were experiencing.\nTable\u00a01Means, standard deviations (SD), correlations and t-test statistics comparing Huntington\u2019s Disease (HD) patients and their partners on the Illness Perception Questionnaire (IPQ) IPQ subscalesHD patientsPartnersPatient-partner correlation coefficientaPatient-partner differencebMean (SD)Mean (SD)tIdentity.44 (.22).55 (.22).57***\u22123.59***Timeline4.70 (.63)4.50 (.81).101.44Consequences3.60 (1.19)3.82 (1.01).29*\u22121.16Cure1.72 (1.77)1.69 (1.69).90***.27Control3.11 (1.72)2.36 (1.58).30*2.73**aPearson correlation. b Paired t-test* P\u00a0<\u00a0.05; ** P\u00a0<\u00a0.01; *** P\u00a0<\u00a0.001\nPartners reported that the HD patients were suffering from significantly more symptoms (on the \u201cIdentity\u201d subscale) of HD than the patients did themselves, and reported experiencing significantly less control over HD than the patients did.\nCorrelations between illness perceptions and QoL\nTables\u00a02 and 3 display the bivariate correlations between the study variables. As expected, examination of the correlations between patients\u2019 and partners\u2019 illness perceptions and patients\u2019 QoL (presented in Table\u00a02) shows that patients\u2019 QoL was most strongly associated with their own illness perceptions. A higher QOL correlated with a less strong illness identity, a longer perceived illness (and thus life) duration, less perceived consequences, more control, and less belief in treatment. With regard to partners\u2019 perceptions, the same associations (albeit fewer) were found, with the exception of partners\u2019 cure perceptions. Partner, but not patient, belief in cure through treatment was significantly related to patients\u2019 vitality and social functioning ratings. Also, partners\u2019 identity and consequences perceptions were not significantly related to patients\u2019 vitality, social functioning, and mental health ratings. Except for the MOS subscales \u201cRole Functioning - emotional\u201d and \u201cMental Health\u201d, illness perceptions of both patients and partners correlated significantly with patient QoL. Thus, regression analyses were conducted on the remaining MOS subscales, including only the illness perceptions dimensions that correlated significantly with QoL.\nTable\u00a02Pearson correlations between Illness Perceptions (IPQ) and patients\u2019 QoL (MOS SF-36)IPQ patientsIPQ partnersIdentityTimelineConsequencesControlCureIdentityTimelineConsequencesControlCureMOS subscalesPhysical functioning\u2212.51***.48***\u2212.31*.37**\u2212.33*\u2212.43**.28*\u2212.31*.39**\u2212.39**Role functioning (physical)\u2212.31*\u2212.29*General health\u2212.39**\u2212.31*\u2212.31*Vitality\u2212.44**.34*\u2212.35*.33*\u2212.35*Social functioning\u2212.32*\u2212.38**\u2212.31*Role functioning (emotional)Mental health\u2212.40**\u2212.39**Note: Only significant correlations are depicted* P\u00a0<\u00a0.05; ** P\u00a0<\u00a0.01; *** P\u00a0<\u00a0.001\nBivariate correlations between illness perceptions and partners\u2019 QoL are presented in Table\u00a03. The quality of life of partners was associated with their own beliefs about the patients\u2019 illness, with a stronger belief in a long duration of the patients\u2019 illness being related to better physical functioning and more vitality, and less perceived consequences being related to better physical role functioning, more vitality, and to better mental health. Partners\u2019 vitality and mental health were also associated with illness perceptions of patients. Stronger patient beliefs in control over the illness, and less serious perceived consequences were related to better partner QoL. Interestingly, the only significant correlations between partners\u2019 ratings of general health and emotional role functioning, and illness perceptions were correlations with patient beliefs in control over the illness.\nTable\u00a03Pearson correlations between Partners\u2019 and Patients\u2019 Illness Perceptions (IPQ), and Partners\u2019 QoL (MOS SF-36)IPQ partnersIPQ patientsIdentityTimelineConsequencesControlCureIdentityTimelineConsequencesControlCureMOS subscalesPhysical functioning.29*Role functioning (physical)\u2212.38**General health.28*Vitality.33*\u2212.29*.30*Social functioningRole functioning (emotional).33*Mental health\u2212.40**\u2212.33*Note: Only significant correlations are depicted* P\u00a0<\u00a0.05; ** P\u00a0<\u00a0.01; *** P\u00a0<\u00a0.001\nImportance of spousal illness perceptions for patients\u2019 QoL\nTo determine the relative extent that partners\u2019 illness perceptions are predictive of patients\u2019 QoL, hierarchical stepwise multiple regression analyses were conducted for patients\u2019 physical functioning, role functioning (physical), general health, vitality and social functioning. The results of the regressions are summarized in Table\u00a04. After controlling for patients\u2019 illness related variables and their own scores on the IPQ, partners\u2019 IPQ scores added a significant amount of explained variance (13%) to patient scores on the \u201cVitality\u201d subscale only. A stronger partner belief in a long duration of the patients\u2019 illness (\u00df .30, P\u00a0<\u00a0.05), and less belief in cure through treatment (\u00df \u2212.33, P\u00a0<\u00a0.05) both added to the prediction of higher patient vitality.\nTable\u00a04Hierarchical regressions examining if spousal illness perceptions explain additional variance in patients\u2019 QoLStep and variablesAdj. R\u00b2R\u00b2 changeF for R\u00b2 changeMOS physical functioning1. Control variablesa.49.5217.05***2. Illness perceptions (patient): identity, timeline, consequences, control, cure.59.143.29*3. Illness perceptions (partner): identity, timeline, consequences, control, cure.64.082.06MOS role functioning (physical)1. Control variablesa.01.071.232. Illness perceptions (patient): cure.11.095.83*3. Illness perceptions (partner): cure .09.000.01MOS general health1. Control variablesa.10.152.84*2. Illness perceptions (patient): identity, consequences.18.113.24*3. Illness perceptions (partner): identity.16.000.08MOS vitality1. Control variablesa.12.183.35*2. Illness perceptions (patient): identity, timeline, consequences.29.204.65**3. Illness perceptions (partner): timeline, cure.41.13 5.62**MOS social functioning1. Control variablesa.01.050.832. Illness perceptions (patient): identity, consequences.11.154.18*3. Illness perceptions (partner): cure.16.063.51aDisease duration, TMS, and MMS scores * P\u00a0<\u00a0.05; ** P\u00a0<\u00a0.01; *** P\u00a0<\u00a0.001\nThe variance in patients\u2019 scores on the other subscales of the MOS SF-36 was explained mainly by patients\u2019 own IPQ scores, with the amount of added explained variance ranging from 9 to 20%. Less perceived consequences, a less strong illness identity, more control, and less belief in treatment contributed significantly to better QoL.\nImportance of patients\u2019 illness perceptions for partners\u2019 QoL\nTo determine the relative extent that patients\u2019 illness perceptions are predictive of partners\u2019 QoL, hierarchical stepwise multiple regression analyses were conducted for partners\u2019 vitality and mental health. The results of the regressions are summarized in Table\u00a05. Patients\u2019 IPQ scores added a significant amount of explained variance (8%) to partner scores on the \u201cVitality\u201d subscale only. Stronger patient beliefs in control over the illness (\u00df .29, P\u00a0<\u00a0.05) added to the prediction of higher partner vitality.\nTable\u00a05Hierarchical regressions examining if spousal illness perceptions explain additional variance in partners\u2019 QoLStep and variablesAdj. R\u00b2R\u00b2 changeF for R\u00b2 changeMOS vitality1. Illness perceptions (partner): timeline, consequences.17.216.26**2. Illness perceptions (patient): control.24.08 5.05*MOS mental health1. Illness perceptions (partner): consequences.14.169.04**2. Illness perceptions (patient): consequences.17.053.11* P\u00a0<\u00a0.05; ** P\u00a0<\u00a0.01; *** P\u00a0<\u00a0.001\nMost variance was explained by partners\u2019 own IPQ scores, with amount of explained variance ranging from 16 to 21%. A longer perceived illness (and thus life) duration, and less perceived consequences contributed significantly to better QoL.\nDiscussion\nOur results indicate that HD patients and their partners did not differ significantly in their beliefs regarding the duration, consequences, causality, and curability of the disease. HD was attributed, in all but four HD patients and one partner, to a genetic cause. There were however, significant differences between the illness identity of HD patients and that of their partners. Partners attributed significantly more symptoms to HD than patients. In addition, HD patients and their partners differed significantly in the degree of control they believed they had over the disease process. HD patients perceived their disease as being more controllable than their partners. In general, as was the case in couples dealing with Addison\u2019s disease [15], patients held more positive beliefs about HD than their partners did.\nExamining the relationships between spousal illness perceptions and patients\u2019 QoL, the results indicate that none of the partners\u2019 perceptions (whether consonant or dissimilar) are relevant in being associated with patients\u2019 quality of life in the areas of mental health and emotional role functioning. Partners\u2019 identity and consequences perceptions (whether consonant or dissimilar) are not associated with patients\u2019 quality of life in the areas of social functioning and vitality. Partners\u2019 consequences perceptions (whether consonant or dissimilar) are not relevant for patients\u2019 quality of life in the area of general health. Also, less partner (but not patient) belief in cure through treatment was related to higher patients\u2019 vitality and social functioning ratings. Thus, our results suggest that except for patients\u2019 physical functioning, it is of limited use to investigate (only) dissimilarities in illness perceptions of patients and partners as determinants of patient outcomes in HD.\nWith regard to the third aim of this study, although patients\u2019 and partners\u2019 own illness perceptions explained the largest amount of variance in QoL, spousal illness perceptions were related to patients\u2019 and partners\u2019 quality of life, but only to their vitality ratings. After controlling for patients\u2019 illness related variables and patients\u2019 own beliefs, a stronger partner belief in a long duration of the patients\u2019 illness and less belief in cure through treatment both added to the association with higher patient vitality. The results are in line with those of Heijmans et\u00a0al. [15], who found positive relationships between spousal maximization of illness duration and patients\u2019 vitality ratings in Addison\u2019s disease. Stronger patient beliefs in control over the illness added to higher vitality scores in partners. From both our results and those of Heijmans et\u00a0al., it appears that it is most beneficial to have a spouse who is realistic (albeit negative) about the possibilities for cure, and who expects the illness to be long-lasting. Research on how realistic optimism may impact on vitality in healthy persons and patient samples supports these findings [38, 39].\nThe results obtained in this study must be regarded in the light of some limitations. The lack of information on the non-response rate in the sample of members of the Dutch Huntington Association makes selection bias likely. Also, the patients involved in this study were in the early to middle stages of HD, as shown by their scores on the UHDRS and MMS. This prevents extrapolation of our results to other samples of patients with Huntington\u2019s disease. On the other hand, our results indicate that the patients in our study did not differ significantly from those patients with Huntington\u2019s Disease involved in other studies with regard to important disease related characteristics such as motor and cognitive functioning [40]. Our results could, therefore, be instrumental in future research on quality of life in patients (and their partners) who are in these stages of HD. The relatively small number of couples included and the relatively large number of variables entered into the regression analyses must be taken into account as well.\nMore research is justified on the unique contributions of the spouses\u2019 perceptions of the illness that may be associated with outcome in chronically ill patients and their partners. Examining these associations further and studying which psychological mechanisms may be involved, for instance by interviewing pairs in whom these associations are clearly discernable, are areas for future research. From a clinical perspective, an understanding of the cognitive factors that are related to quality of life in couples dealing with chronic illness will help to guide family interventions. Given the relative paucity of research on biopsychosocial aspects of Huntington\u2019s disease, qualititative research on quality of life may be helpful in future research, e.g., in the work by Brouwer-Dudokdewit et\u00a0al. [41], where qualitative research in a case-study format that was carefully embedded in a theoretical framework helped explore quality of life issues in pre-symptomatic testing for HD. These researchers emphasize the relevance of adding an existential and\/or spiritual approach in exploring quality of life issues in HD patients [42, 43]. Empirical studies in other neurological disorders support these suggestions: Hodgson et\u00a0al. [44], for example, describe the lives of 10 couples living with Parkinson\u2019s disease, and outline how these couples preferred a multidisciplinary approach to their treatment and believed in taking an active role in their health care. Qualitative approaches to assessing QoL in Huntington\u2019s disease spousal carers are described in a recent paper by Aubeeluck & Buchanan [29], where visual representations of QoL were gathered by using \u2018Photovoice\u2019: spousal carers photographed and described elements of their life. Finally, cognitive-behavioural approaches in patients with dementia and their caregivers were recently shown in a randomized controlled trial to result in improvements in patients\u2019 daily functioning and reduced burden in the caregiver. These studies and ours, illustrate possible directions for future research in patients (and their partners) who experience an extreme negative impact on quality of life [45].","keyphrases":["couples","huntington\u2019s disease","illness perceptions","patient qol","partner qol"],"prmu":["P","P","P","R","R"]}
{"id":"Sleep_Breath-4-1-2270921","title":"In-home evaluation of efficacy and titration of a mandibular advancement device for obstructive sleep apnea\n","text":"There is increasing evidence that mandibular advancement devices (MADs) can be an effective treatment for some patients with obstructive sleep apnea, a highly prevalent chronic disease. In this study, the objectives were to objectively assess the effectiveness of MAD therapy using a limited channel recorder, and to develop a model for identifying patients who may be appropriate for MAD therapy as the initial treatment option. Thirty patients were prospectively recruited and studied at two independent dentist offices and the participants\u2019 homes. Subjects wore the ARES Unicorder for two nights before insertion of the MAD, and again when the dentist determined that the patient had reached the titration endpoint. Self-reported measures of depression, sleepiness, and quality of life were obtained pre- and posttreatment. The reviewer was blinded to the study status while the physiological signals were being visually inspected. Significant reductions in the apnea\/hypopnea index (AHI), hypoxemia measures, and snoring level were observed posttreatment. Twenty-seven of the 30 (90%) patients had a posttreatment AHI (using a 4% desaturation for hypopneas) below a clinical cut-off of 10. All but one patient (97%) exhibited at least a 50% decrease in AHI or had a posttreatment AHI \u2264 10. Significant differences in body mass index, weight, and neck circumference in patients with posttreatment AHIs above and below a clinical cut-off of five were identified. The linear regression used to predict the posttreatment AHI using pretreatment data resulted in an R2 of 0.68. The model correctly predicted two patients who were unable to obtain a posttreatment AHI of 10 or less. This study was designed to demonstrate two models of collaboration between a dental sleep medicine specialist and a sleep medicine physician in the monitoring of a patient treated with a MAD. The outcome data suggest that the limited channel recording system can be used as an alternative to laboratory polysomnography to reduce the cost of MAD treatment, and to improve the quality and consistency of posttreatment patient care.\nIntroduction\nObstructive sleep apnea (OSA) is the most common disorder observed in the practice of sleep medicine and is responsible for more mortality and morbidity than any other sleep disorder [1]. Although characterized over 40\u00a0years ago [2, 3], OSA has only recently gained recognition as one of the world\u2019s most prevalent, underdiagnosed disorders [4, 5]. Due to associated morbidity, OSA has been identified as a major public health concern [6]. The disorder is characterized by frequent loud snoring and recurrent failures to breathe adequately during sleep (termed apneas or hypopneas), usually as a result of collapse of the upper airway.\nMandibular advancement devices (MADs) are being increasingly recognized as a treatment alternative to continuous positive airway pressure (CPAP), particularly for patients with mild to moderate OSA [7\u201312]. MADs are designed to protrude the mandible and thus the tongue and epiglottis during sleep, preventing airway occlusion [13]. Comparisons of MAD to CPAP have revealed that although MADs are less efficacious than CPAP in reducing the RDI they are, on average, used more frequently, preferred by more patients and more readily accepted than CPAP [14]. Recent studies demonstrated that both MADs and CPAP can reduce the 24-h diastolic blood pressure by an average of 3\u00a0mmHg after 4\u00a0weeks of treatment [15\u201317]. Similarly to CPAP, self-reported improvements in quality of life and decrease in sleepiness are reported in the majority of patients [14].\nThe primary goal of this study was to use a self-applied limited channel recorder to objectively assess the effectiveness of MAD therapy when the dentist determined the patient had reached the titration endpoint. A second goal was to determine factors which might predict successful treatment outcomes and provide a more refined method for identifying patients who may be appropriate for MAD therapy as the initial treatment option. Finally, we wanted to demonstrate two potential models of collaboration between the dentist and sleep medicine physician in monitoring MAD treatment outcomes.\nMaterials and methods\nEleven females and 19 males were recruited from two dental practices before treatment with an oral appliance and enrolled in the study. Twenty-five of the patients had failed CPAP therapy. After obtaining an informed consent (approved by the BioMed IRB, San Diego, CA, USA) patients completed a two-night pretreatment in-home study with the Apnea Risk Evaluation System (ARES TM) Unicorder  TM (Advanced Brain Monitoring, Carlsbad, CA, USA). This pretreatment recording was conducted between the time the dental impressions were taken and when the TAP II Mandibular Advancement Device (MAD) (Airway Management, Dallas, TX, USA) was fabricated and ready for insertion.\nThe TAP is a custom-made oral appliance with separate upper and lower appliances joined by a titration or advancement mechanism on the upper and a transverse bar or socket on the lower. The titration mechanism uses a hook to engage the bar or socket on the lower once each device is placed in the mouth. A jackscrew controls the position of the hook and thus the amount of protrusion. The patient can self-titrate the device using a removable hex key which engages the screw.\nOn the day of insertion of the MAD, patients completed the Beck depression index, Epworth sleepiness scale, and the Flemons\u2019 quality of life questionnaire. At the insertion appointment, both study sites attempted to achieve a starting MAD titration position whereby the patient could just hook the lower tray with the upper tray using active protrusion with both trays in place. In the rare occasion that this level of advancement was not tolerated by the patient, the starting protrusion was reduced. Patients were instructed to begin adjusting the MAD in one-half turn increments as soon as it was tolerable, until a cessation in snoring or the symptoms had resolved. As a result, the titration endpoint was determined by the dentist based on the patient\u2019s self-report. At the follow-up appointment, which was typically scheduled 3 to 4\u00a0weeks subsequent to the MAD insertion, the assessment questionnaires were completed again and the ARES Unicorder study was repeated. Twenty-seven of the 30 patients reached their endpoint within 34\u00a0days. The other three patients completed their endpoint in 40, 61, and 75\u00a0days; the delay in reaching the endpoint was due to patient illness unrelated to this study.\nIn between the time that treatment was initiated and the titration endpoint, each patient maintained a daily journal that recorded the time the appliance was inserted each night, and the time it was removed in the morning.\nThe pre- and posttreatment studies were conducted with the ARES Unicorder. From a single site on the forehead, the wireless recorder measures oxygen saturation, pulse rate, airflow, respiratory effort, snoring levels, head movement, and head position [18] (Fig.\u00a01). Reflectance oximetry is used to obtain the SpO2 and pulse rate signals. Respiratory effort is derived from the measurement of changes in forehead venous pressure acquired using a combination of photoplethysmography and changes in surface pressure of the forehead reflectance oximetry sensor. Airflow is obtained via a cannula and nasal pressure transducer. A calibrated acoustic microphone is used to acquire quantified snoring levels (dB). Actigraphy is used to measure head movement and derive head position. The recorder was designed to be affixed by the patient, and provide alerts during the study if poor quality airflow or SpO2 is detected so the device could be adjusted.\nFig.\u00a01Patient wearing a unicorder\nThe description and validation of this device in 284 valid comparisons of the in-laboratory simultaneous PSG and ARES and 187 valid comparisons of the in-laboratory PSG with a separate two nights unattended self-applied ARES Unicorder has been previously published [18]. Using a diagnostic AHI cutoff of >10, the concurrent in-lab comparison yielded a sensitivity of 97.4, a specificity of 85.6, a positive predictive value of 93.6, and a negative predictive value of 93.9. The in-home comparison sensitivity, specificity, positive predictive value, and negative predictive value were 91.5, 85.7, 91.5, and 85.7, respectively. The failure rate was 2%.\nIn an independent validation of 40 subjects (13 healthy controls and 27 patients with symptoms of EDS and\/or snoring) using a clinical cut-off of 15, the sensitivity of the ARES compared to concurrent PSG was 100% (25\/25 patients) and specificity 67% (10\/15 subjects). The sensitivity for two-nights of ARES in-home compared to the PSG was 96%; specificity was 80%. The failure rate was 5% [19].\nAutomated scoring algorithms were applied off-line to detect sleep disordered breathing. The AHI was computed using a time-in-bed measure based on recording time with acceptable signal quality minus periods when the patient was upright or presumed to be awake based on actigraphy. Apneas, based on a 10-s cessation of airflow detected by the automated algorithms, were included in all apnea\u2013hypopnea indexes (AHI). Hypopnea events required a 50% reduction and recovery in airflow and were further stratified based on the depth of desaturation. The AHI-4% criteria required a minimum 3.5% reduction in SpO2 and at least a 1.0% recovery. Hypopneas included in the AHI-3% and AHI-1% criteria required SpO2 desaturation and resaturation using stepped thresholds. For the AHI-3%, if the SpO2 at the point of maximum saturation before the event was greater than or equal to 95% then a 2.2% reduction and a 2.2% recovery in SpO2 was required. For maximum saturations of between 95\u201393% the required SpO2 change was a 2.5% reduction and 2.5% recovery; between 93\u201391% a 3.0% reduction and 2.7% recovery; between 91\u201388% a 3.5% reduction and 3.0% recovery; and below 88% a 4.0% reduction and 3.5% recovery. For the AHI-1%, if the point of maximum saturation before the event was greater than 93%, then a 1.0% reduction and 1.0% recovery was required; and for events with a starting SpO2 between 93\u201391%, a 1.2% reduction and 1.2% recovery was required. For an AHI-1% event to be called, the change in flow and desaturation needed to be associated with a behavioral arousal (i.e., an abrupt change in pulse rate, snoring sound or a head movement). After the automated scoring was applied, the full disclosure recordings were visually inspected by a sleep medicine physician to confirm the accuracy of the automated scoring, and to reclassify as central and\/or exclude autodetected events if necessary. At the time of the review, the clinician was blinded to the study status (i.e., pre- or posttreatment). The physiological data, including AHI values, percent time with SpO2 below 90, 85, and 80%, and percentage time snoring greater than 30, 40, 50, and 60\u00a0dB were then calculated.\nResponses to the Beck depression index and Flemons\u2019 quality of life questionnaire were tallied in accordance with published methods [20, 21].\nPaired t tests were used to identify significant changes in the pre- and posttreatment physiological data and questionnaire responses.\nTo identify anthropomorphic factors that may impact MAD treatment outcomes, patients were stratified into two groups. Group 1 included all patients with a posttreatment AHI-4%\u2009\u2264\u20095 (n\u2009=\u200918). The balance of patients with an AHI-4%\u2009>\u20095 were assigned to group 2 (n\u2009=\u200912). Paired t tests were used to identify significant group differences.\nTo develop and validate the prediction of the posttreatment AHI using pretreatment data, patients were paired and assigned into either the model development or cross validation group based on similarities in the pre- and post-4% AHI and 1% AHI. Correlation analysis was used to identify anthropomorphic variables and measures of obstructive breathing before treatment which might be useful in estimating the posttreatment 4% AHI (post-T 4%). Variables with significant correlations were then used in a linear regression to derive predicted posttreatment values (predict AHI).\nResults\nOverall effects of MAD treatment The mean \u00b1 SD and minimum pre- and posttreatment valid recording times were 9.6\u2009\u00b1\u20093.6 and 3.5\u00a0h; and 10.3\u2009\u00b1\u20092.4 and 4.6\u00a0h, respectively.Paired t tests revealed significant changes (p\u2009<\u20090.001) in all pre- vs posttreatment sleep disordered breathing measures, including: apnea\u2013hypopnea index with a 4% (AHI-4%) and 1% (AHI-1%) desaturation (Fig.\u00a02).\nFig.\u00a02Mean + SE changes in pre- and posttreatment AHI-4% and AHI-1%A decrease in hypoxemia measures pre- and posttreatment were also observed including the percentage of time below 90% SpO2 (T%\u2009<\u200990%) (p\u2009<\u20090.05) and the mean percentage change in SpO2 across all AHI events (% Dip)(p\u2009<\u20090.001) (see Fig.\u00a03).\nFig.\u00a03Mean + SE changes in pre- and posttreatment percentage of time the SpO2 was <90%, and the percentage change in SpO2 resulting from sleep disordered breathingSignificant reduction in the average snoring levels at 30 and 40\u00a0dB across the valid recording time were also observed (p\u2009<\u20090.001) (Fig.\u00a04).\nFig.\u00a04Mean + SE changes in pre- and posttreatment snoring loudness above 30 and 40\u00a0dBBased on conventional outcome measures, the MAD therapy was highly efficacious. Twenty-seven of the 30 (90%) patients had a posttreatment AHI-4%\u2009<\u200910 (Fig.\u00a05a). All but one patient (97%) exhibited at least a 50% decrease in AHI-4% or had a posttreatment AHI-4%\u2009\u2264\u200910.\nFig.\u00a05Individual changes in pre- and posttreatment sleep disordered breathing using: a AHI-4% desaturation and b AHI-1% desaturationUsing an alternative measure of AHI-1% (used to identify residual sleep disordered breathing\/upper airway resistance) 21 of the 30 patients (70%) had a posttreatment AHI-1%\u2009\u2264\u200915 (Fig.\u00a05b). Eighty-seven percent (26 of 30) of the patients exhibited at least a 50% decrease in AHI-1% or had a posttreatment AHI-1%\u2009\u2264\u200915.Paired t tests applied to the pre- and posttreatment scores revealed statistically significant differences for Beck depression index, Flemons\u2019 QOL, Epworth sleepiness score (all at the p\u2009\u2264\u20090.001 level) (Fig.\u00a06). Seventy-six percent (23\/30) of the patients showed a decrease in Epworth score; 60% had an Epworth score reduction of three or more. Seventy-three percent (22\/30) of the patients reported a posttreatment reduction in depression; 53% had a Beck depression index score reduction of three or more. Eighty-seven percent of the patients reported an increase in the Flemons\u2019 QOL index; 60% showed in improvement score of one or greater. All patients reported some level of subjective improvement on at least one of these subjective measures.\nFig.\u00a06Pre- and posttreatment changes in Beck depression index, Epworth sleepiness scale, and Flemons\u2019 quality of life indexThe correlation between the posttreatment percentage time snoring above 30\u00a0dB and the AHI-4% was computed because snoring is a principal measure used to assess the MAD titration endpoint. The results in Fig.\u00a07 suggest that snoring is a good predictor of outcome when objectively measured.\nFig.\u00a07Regression plot predicting posttreatment AHI-4% based on the posttreatment percentage of time the snoring loudness was above 30\u00a0dBDaily self-reported compliance for the MAD revealed that out of a total of 693 nights in bed recorded across all subjects, only 20 nights were reported as not wearing the MAD. The mean usage per night was 7.34\u2009\u00b1\u20091.3\u00a0h. Three of the 30 patients had incomplete paperwork due to either confusion on the part of the patient or problems in collecting the paperwork at the study sites. Although all three patients said that they had worn the MAD for the duration of the study, there was no written record available that allowed the investigators to assess compliance\/usage.\nFactors that affect treatment outcomes Analysis of variance was used to compare demographic and anthropomorphic measures of patients who had a posttreatment AHI-4%\u2009\u2264\u20095 and those who did not respond optimally to treatment (Table\u00a01). Significant differences in the two groups were observed in the body mass index (p\u2009<\u20090.01), weight (p\u2009<\u20090.01), and neck circumference (p\u2009<\u20090.02). \nTable\u00a01Characteristics of group that was treated optimally and the group that was not treated optionally with a MAD (mean + SE)\u00a0Treated optimally post-T AHI-4%\u2009<\u20095Did not treat optimally post-T AHI-4%\u2009>\u20095GenderFemales74Males118Age (years)48.3\u2009+\u20092.550.9\u2009+\u20093.4BMI (kg\/m)**27.6\u2009+\u20090.833.1\u2009+\u20092.1Weight (kg)**84.4\u2009+\u20093.0100.2\u2009+\u20095.0Neck circumference (cm)*40.0\u2009+\u20090.743.2\u2009+\u20091.0*p\u2009<\u20090.05**p\u2009<\u20090.01\nPrediction of successful treatment outcome Correlation analysis was applied to the entire data set (n\u2009=\u200930) identify pretreatment variables that had a significant relationship with the posttreatment AHI-4% (see Table\u00a02). \nTable\u00a02Correlations between posttreatment AHI-4% and pretreatment measuresPearson r (p\u2009<\u20090.01)Pearson r (p\u2009<\u20090.05)Pretreatment 4% AHIr\u2009=\u20090.53Snoring >30\u00a0dBr\u2009=\u20090.44Pretreatment 3% AHIr\u2009=\u20090.44Neck circumferencer\u2009=\u20090.45Pretreatment 1% AHIr\u2009=\u20090.54Body mass indexr\u2009=\u20090.45Snoring >40\u00a0dBr\u2009=\u20090.52Weightr\u2009=\u20090.45% Time <90% SpO2r\u2009=\u20090.56Various combinations of variables were submitted to linear regression using the model development data set in an effort to predict the posttreatment AHI-4%. Limiting the number of variables to four (due to the small sample size), the variables AHI-4%, AHI-3%, AHI-1%, and percent time snoring >30\u00a0dB resulted in an R2 of 0.68. The coefficients derived from the linear regression were applied to the model development and cross-validation data sets.Twenty-seven of the 30 patients had predict-AHI suggesting a successful outcome would be attained using a clinical cutoff post-T 4% AHI\u2009\u2264\u200910 (Fig.\u00a08). Two of the three patients were accurately predicted as unable to achieve a post-T AHI\u226410. The Bland\u2013Altman plots comparing the predicted-AHI and posttreatment AHI (Fig.\u00a09) suggest a reasonable fit of the data given the small sample size used for the model development and cross validation data sets.\nFig.\u00a08Comparison of pretreatment, posttreatment, and predicted posttreatment AHI-4% for a the model development, and b the cross validation groupsFig.\u00a09Bland\u2013Altman plot of the difference between the predicted posttreatment AHI-4% vs the actual posttreatment AHI for a the model development and b the cross validation groups\nDiscussion\nConsecutive patients who were referred to the dentist for a MAD were provided the opportunity to enroll in the study. The only exclusion criteria applied were ages less than 18 or older than 70\u00a0years. The inclusion of 25 subjects who had previously failed CPAP therapy contributed to the wide OSA severity range: nine of the subjects (30%) were considered to have severe OSA (i.e., AHI-4%\u2009\u2265\u200930) and an additional 30% were considered to have moderate OSA (AHI-4%>15 and <30). Seventy-seven percent of the patients (23\/30) had at AHI-1%>25, a clinical cut-off which was considered at least moderate.\nIn this study, a treatment efficacy rate for the MAD was 90% using an AHI-4% clinical cut-off of 10, and 97% when a 50% reduction in AHI was included in determining a successful outcome. Eighty-seven percent of the patients showed improvement using an AHI-1% with a clinical cut-off of 15 or a 50% reduction in AHI. Eighty percent of the patients (24\/30) had a posttreatment AHI-1%\u2009\u2264\u200916.\nA recent evidence-based review summarized results from 87 published studies (over 2,000 patients evaluated) including 15 Sackett Level I and Level II randomized controlled trials [14]. Using a definition of treatment success that required achieving a posttreatment respiratory disturbance index below 10, success was achieved on average across all studies for 52% of patients treated with oral appliances. When the more liberal criteria of a 50% reduction in RDI were applied to assess outcome, 65% of patients were shown to have a positive result with MAD.\nIn an attempt to compare these previous finding to this data set, a clinical cut-off of 10 was applied to the AHI-1%. The differences between the AHI-1% and RDI include a 50% reduction in flow vs 30%; a 1% desaturation vs no desaturation; arousal indicators based on changes in snoring, pulse rate, and head movement vs cortical arousals; and the use of time-in-bed vs total-sleep-time. Using this alternative clinical cut-off, 33% of the patients had a successful outcome. When a 50% reduction in AHI-1% was included, 77% had a successful outcome. Results are more consistent with the previous research.\nThe findings in this study were consistent with previous reports which found self-reported improvements in quality of life and decrease in sleepiness in the majority of patients [14]. No effort was made in this study to obtain objective measures of improvement. To date, only four studies have utilized objective measures of alertness or neurocognitive function to assess treatment outcomes with MAD therapy, with mixed results [22\u201325]. Further investigations need to be conducted to assess the effectiveness of MAD therapy in ameliorating cognitive impairments in OSA.\nThe strong correlation (r\u2009=\u20090.63) between posttreatment % time snoring >30\u00a0dB and the posttreatment AHI-4% suggests that the current clinical practice of relying on residual snoring to asses MAD titration is valid. It is interesting to note that there was no apparent correlation between snoring levels and pretreatment AHI values. Given the discrepancy between the self-reported and objectively measured snoring at the titration endpoint, it appears that the bed-partner or patient\u2019s observation may not provide the most accurate means for titrating a MAD.\nSeveral studies have reported increased efficacy for MADs in patients with supine positional OSA [7, 26]; however, the association has not been widely confirmed. This is due in part to the fact that the majority of studies on MAD therapy do not report the number of abnormal respiratory events by sleep position despite the recommendation of the ASDA Practice Parameters for separate calculations of supine and lateral RDIs [27, 28]. Supine positionality did not appear to influence the treatment outcome in our study, possibly, because there were so few MAD treatment failures. This may be a result of the appliance which was used or that the small sample size was not representative of the population treated with oral appliances.\nA recent evidence-based review reported that median MAD treatment compliance across patients was 77% after 1\u00a0year [14]. In this study, compliance was only monitored during the titration period which may have contributed to the favorable finding (i.e., 97%). Other factors that may have influenced this outcome were a relatively small sample size and\/or patient compensation being provided. Given that 80% of the study participants were CPAP failures, the influence of prior CPAP use on MAD compliance should be explored. While an objective measure of MAD compliance would eliminate potential bias contributed by self-reported use, practical methods are not currently available.\nThe suggested decrease in MAD efficacy with increasing body mass index (BMI) was confirmed [27, 28]. Weight and neck circumference also appeared to influence the posttreatment apnea\/hypopnea index. These variables make sense: the upper airway tends to be narrower in patients with more fatty tissue around the neck and the additional mass combines with gravity to contribute to greater collapsibility when sleeping supine.\nThe successful treatment outcome of patients with severe sleep apnea suggests that a more quantitative approach should be investigated to identify candidates appropriate for a MAD therapy. The results from the predictive model, once fully validated, could provide the guidance needed for sleep medicine physicians to recommend an oral appliance as an initial treatment option for more severe patients. Alternatively, substantial differences between the predicted and actual posttreatment AHI could help dentists determine when a patient has not been fully titrated.\nGiven the small sample size of the model development data set (n\u2009=\u200915), only four variables were included in the regression model even though the correlations presented in Table\u00a02 suggested additional variables would be predictive. It is expected that the error between the predicted and actual posttreatment AHI can be reduced with larger data sets. In addition to expanding the database used for the predictive model, future investigations should be conducted to determine if the accuracy of the predictive model is influenced by the type of MAD.\nDentists represent an important access point for identifying and treating patients with undiagnosed OSA. This study was designed to demonstrate two models of collaboration between a dental sleep medicine specialist and a sleep medicine physician. As recommended by the AASM, only patients with mild to moderate OSA were offered MAD therapy as the initial treatment choice in this study. Patients with severe sleep apnea were enrolled only after failure of CPAP therapy. In one model, the dentist referred the patient to the sleep medicine physician and his staff to obtain and review the pre- and posttreatment physiological data. In the second model, the dentist acquired the data and transmitted it to the sleep medicine physician for review. In both models, the physical and history was made available to the physician for interpretation of the data [29].\nA follow-up PSG is generally not affordable in cases where it is not covered by an insurance company or health ministry; sleep centers do not tend to offer less expensive level III studies as an alternative. This creates a situation whereby treatment outcomes are simply not assessed, or the dentist conducts the study independently, without the assistance or oversight of the sleep medicine professional. Left on their own, many dentists rely upon oximetry and other level IV devices to monitor treatment outcomes, in part because of their limited experience in interpreting the more sophisticated signals obtained by level III devices. Although several level III devices have automated scoring algorithms, the physiological data should still be reviewed by a trained professional. In two patients, the review of the full disclosure recording allowed recognition of complex sleep apnea (i.e., central sleep apnea was revealed posttreatment after the obstructive breathing was resolved) suggesting the importance of having an experienced professional review the data. This study suggests an alternative approach for assessing MAD treatment outcomes that is based on a collaborative relationship between dentists and sleep medicine physician using a limited channel recording system.","keyphrases":["mandibular advancement device","sleep apnea","sleep disordered breathing","home monitoring","outcome assessment"],"prmu":["P","P","P","R","R"]}
{"id":"Diabetologia-3-1-1820754","title":"Patients with type 2 diabetes have normal mitochondrial function in skeletal muscle\n","text":"Aims\/hypothesis Insulin resistance and type 2 diabetes are associated with mitochondrial dysfunction. The aim of the present study was to test the hypothesis that oxidative phosphorylation and electron transport capacity are diminished in the skeletal muscle of type 2 diabetic subjects, as a result of a reduction in the mitochondrial content.\nIntroduction\nIt has been reported that insulin resistance is associated with mitochondrial dysfunction in several tissues. While mitochondria are considered central to altered metabolic pathways, leading to pathogenic processes in type 2 diabetes, the mechanisms by which mitochondrial function contributes to the disease remain to be elucidated. Whether it is insulin resistance per se, chronic hyperglycaemia, or accumulation of intracellular lipid and associated alterations in metabolic pathways that affect mitochondrial function, or vice versa, is also unclear.\nIn skeletal muscle, evidence for reduced oxidative capacity in type 2 diabetes has been provided by findings of reduced oxidative enzyme levels [1\u20134] and mismatches between glycolytic and oxidative enzyme activities [3, 5]. The activities of rotenone-sensitive NADH:O2 oxidoreductase and citrate synthase have been shown to be reduced by 40% in the skeletal muscle of type 2 diabetic patients, and transmission electron microscopy has revealed that these mitochondria have a reduced size and an altered morphology [1, 6]. A decrease in the expression of genes involved in oxidative phosphorylation has also been reported in muscle of type 2 diabetic patients [7, 8] and men on a high-fat diet [9]. It has been stated that impaired muscle mitochondrial function is linked to excess intramuscular lipid accumulation and reduced fatty acid oxidation defined by rates of oxidative phosphorylation and ratios of inorganic phosphate:phosphocreatine determined by magnetic resonance studies [6]. These studies suggest mitochondrial dysfunction in type 2 diabetes; however, to date, no direct measurements of mitochondrial O2 flux capacity in intact cells in human type 2 diabetes have been reported. We hypothesised that oxidative phosphorylation and electron transport capacity are diminished in the skeletal muscle of type 2 diabetic subjects, and that these changes are attributable to a reduction in muscle mitochondrial content. In the present study we tested these hypotheses by using high-resolution respirometry to quantify oxidative phosphorylation and electron transport capacity in permeabilised muscle fibres from biopsy samples of the quadriceps in healthy subjects and patients with type 2 diabetes.\nSubjects and methods\nSubjects Informed consent was obtained from all subjects. The study was conducted in accordance with the principles of the Declaration of Helsinki, and was approved by the local ethics committee for Frederiksberg and Copenhagen County. Mitochondrial respiration was measured in permeabilised skeletal muscle fibres obtained from needle biopsies of the vastus lateralis in men with (n\u2009=\u200911) or without (control; n\u2009=\u20098) type 2 diabetes. The characteristics of the subjects are provided in Table\u00a01 and Fig.\u00a01. All subjects were in good health but classified as living a typical Westernised sedentary lifestyle, participating only in routine activities of daily living (walking, gardening, etc.) and not engaged in regular structured or individualised aerobic or strength training programmes or athletics. None of the control subjects had a family history of diabetes and none was receiving treatment for a disease. The diabetic patients were treated for their diabetes with diet or oral glucose-lowering medicine. All medications were withheld 24\u00a0h prior to the experiment. The patients with type 2 diabetes had no clinical signs of long-term diabetic complications and were representative of patients treated in the primary care sector. \nTable\u00a01Characteristics of the subjects\u00a0Type 2 diabetic subjects (n\u2009=\u200911)Control subjects (n\u2009=\u20098)Age (years)62\u2009\u00b1\u2009258\u2009\u00b1\u20091Height (cm)177\u2009\u00b1\u20093179\u2009\u00b1\u20091BMI (kg\/m2)32\u2009\u00b1\u20092*28\u2009\u00b1\u20091Time since diagnosis (years)5\u2009\u00b1\u20092\u2013Fasting insulin (pmol\/l)61\u2009\u00b1\u20099*34\u2009\u00b1\u20096Fasting glucose (mmol\/l)9.0\u2009\u00b1\u20090.5*5.4\u2009\u00b1\u20090.1Complex I activity (nmol min\u22121 mg protein\u22121)50.8\u2009\u00b1\u20096.058.3\u2009\u00b1\u20094.7 Citrate synthase activity (pmol mg\u22121 s\u22121)1.6\u2009\u00b1\u20090.12.0\u2009\u00b1\u20090.2mtDNA (copies\/\u03bcg tissue) \u00d7103119\u2009\u00b1\u20097*147\u2009\u00b1\u200912mtDNA\/genomic DNA2,773\u2009\u00b1\u20092523,030\u2009\u00b1\u2009185Data are means\u00b1SEM. *p\u2009<\u20090.05 vs control subjectsFig.\u00a01Glucose (a) and insulin (b) concentrations in venous plasma before (t\u2009=\u20090 min) and during an OGTT. The patients with type 2 diabetes had higher fasting glucose levels and were severely insulin resistant compared with healthy control subjects (*p\u2009<\u20090.05). Black and white symbols represent healthy control subjects and patients with type 2 diabetes, respectivelySubjects were fasted overnight prior to the experiment. A catheter was inserted into an antecubital vein for blood sampling. After local anaesthesia of the skin and the subcutis, a muscle biopsy was taken (Tru-Core; PBN-Medicals, Stenl\u00f8se, Denmark) and then a 120-min OGTT (75\u00a0g glucose dissolved in 300\u00a0ml of water) was performed. At t\u2009=\u200930\u00a0min, a second muscle biopsy was taken.A portion of the obtained muscle tissue was frozen immediately in liquid nitrogen and stored at \u221280\u00b0C for later analysis (see below), and a smaller piece (2\u20136\u00a0mg) was placed onto a Petri dish on ice with 1\u00a0ml of relaxing solution containing Ca2+\/EGTA buffer (10\u00a0mmol\/l), free calcium (0.1\u00a0\u03bcmol\/l), imidazole (20\u00a0mmol\/l), K+\/4-morpholinoethanesulfonic acid (MES) (50\u00a0mmol\/l), dithiothreitol (DTT; 0.5\u00a0mmol\/l), MgCl2 (6.56\u00a0mmol\/l), ATP (5.77\u00a0mmol\/l), phosphocreatine (15\u00a0mmol\/l), pH\u00a07.1, and individual fibre bundles were separated with two pairs of sharp forceps, achieving a high degree of fibre separation. The fibre bundles were permeabilised for 30\u00a0min in 3\u00a0ml of ice-cold relaxing solution containing saponin (50\u00a0\u03bcg\/ml) [10]. After rinsing in respiration medium (MiR05; Oroboros, Innsbruck, Austria) containing sucrose (110\u00a0mmol\/l), potassium lactobionate (60\u00a0mmol\/l), EGTA (0.5\u00a0mmol\/l), MgCl2.6H2O (3\u00a0mmol\/l), taurine (20\u00a0mmol\/l), KH2PO4 (10\u00a0mmol\/l), HEPES (20\u00a0mmol\/l), sucrose (110\u00a0mmol\/l), BSA (1\u00a0g\/l), pH\u00a07.1, the muscle bundles were blotted and measured for wet weight in a balance controlled for constant relative humidity, so that all biopsy samples were hydrated to the same degree. The muscle bundles were then immediately transferred into a respirometer (Oxygraph-2k; Oroboros) containing air-saturated respiration medium at 37\u00b0C.The Oxygraph-2k is a two-chamber titration-injection respirometer with a limit of oxygen flux detection of 1\u00a0pmol s\u22121 ml\u22121. The instrumentation allows for O2 flux measurements with only 0.04\u00a0mg of mitochondrial protein or 1.5\u00a0mg of muscle fibres (wet weight). Standardised instrumental and chemical calibrations were performed to correct for back-diffusion of O2 into the chamber from the various components, leak from the exterior, O2 consumption by the chemical medium, and sensor O2 consumption [11]. O2 flux was resolved by software capable of converting nonlinear changes in the negative time derivative of the oxygen concentration signal.\nAnalysis of muscle tissue Citrate synthase activity and complex I activity were measured spectrophotometrically at 37\u00b0C. Citrate synthase activity was determined as described previously [12], and complex I activity was assessed by measuring the oxidation of NADH (300\u00a0\u03bcmol\/l) using ubiquinone 1 (100\u00a0\u03bcmol\/l) as the acceptor. The complex I rotenone-sensitive activity was measured by the addition of rotenone (1\u00a0\u03bcmol\/l). The protein content, needed to calculate the specific activity, was measured using a commercially available assay (BCA, Sigma Chemicals, St Louis, MO, USA). For measurement of mitochondrial DNA (mtDNA) content, DNA was isolated from muscle biopsy samples (\u223c10\u00a0mg) by proteinase K digestion at 55\u00b0C for 3\u00a0days. The 100-\u03bcl digestion mix contained 50\u00a0mU proteinase K (PCR grade, Roche, Basel, Switzerland), 20\u00a0mmol\/l Tris-HCl (pH\u00a08.4) and 50\u00a0mmol\/l KCl. After incubation at 80\u00b0C for 45\u00a0min, the remains were spun down and the supernatant fraction diluted \u00d7200 in triethanolamine titanate (TE) plus 1\u00a0ng\/\u03bcl salmon sperm DNA (Sigma). 5\u00a0\u03bcl of this dilution was amplified in a 25\u00a0\u03bcl PCR reaction containing 1\u00d7Quantitect SYBR Green Master Mix (Qiagen, Hilden, Germany) and 100\u00a0nmol\/l of each primer. The amplification was monitored real-time using the MX3000P Real-time PCR machine (Stratagene, La Jolla, CA, USA). The primers were designed to target genomic DNA (Forward: AGG TGC TGT CAG GAA GCA AGG A, Reverse: TAG GGG GAG GAG GGA ACA AGG A) or mtDNA (Forward: CCC CTG CCA TAA CCC AAT ACC A, Reverse: CCA GCA GCT AGG ACT GGG AGA GA). The threshold cycle (Ct) values were related to a standard curve made with the cloned PCR products.\nRespirometry protocol All measurements of respiration were made in duplicate, simultaneously. Resting, routine respiration (state 2, absence of adenylates) was assessed by the addition of malate (1.5\u00a0mmol\/l) and glutamate (19\u00a0mmol\/l) as the complex I substrate supply, and then state 3 respiration was assessed by the addition of ADP (4.8\u00a0mmol\/l). The addition of succinate (9.5\u00a0mmol\/l) provided state 3 respiration with parallel electron input to complexes I and II. The integrity of the outer mitochondrial membrane was established by the addition of cytochrome c (19\u00a0\u03bcmol\/l); no stimulation of respiration was observed. We examined ADP control of coupled respiration and uncoupling control through addition of the protonophore carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP) (0.7\u00a0\u03bcmol\/l). The addition of rotenone (0.1\u00a0\u03bcmol\/l) resulted in inhibition of complex I for examination of O2 flux with complex II substrate alone, while antimycin A (12\u00a0\u03bcmol\/l) was added to inhibit complex III to observe non-mitochondrial respiration with small contributions from electron leak in the uncoupled state. The concentrations of substrates and inhibitors used were based on prior experiments conducted for optimisation of the titration protocols.\nData analysis All values are given as means\u00b1SEM for all experiments, run in duplicate or triplicate. For all statistical evaluations, a p value of less than 0.05 was considered significant. Statistical analysis of differences in oxygen flux between healthy control subjects and patients with type 2 diabetes was carried out with a two-way ANOVA for repeated measures. In the case of a significant main effect and interaction between the variables, the Holm-Sidak method was used for post hoc analysis. All other comparisons between the two groups were performed using the unpaired Student\u2019s t test. SigmaStat version 3.11 (Systat software, Richmond, CA, USA) was used in all analyses.\nResults\nThe sequential addition of substrates to the muscle tissue, obtained from both groups before an OGTT, always (p\u2009<\u20090.05) resulted in a stepwise increase in state 3 O2 flux (Fig.\u00a02). Notably, the addition of succinate (stimulating parallel electron input from complexes I+II) resulted in a marked increase in O2 flux in both groups. The O2 flux per muscle mass was significantly (p\u2009<\u20090.05) lower in the patients compared with the healthy subjects during complex I and complex I+II respiration (Fig.\u00a02). Further increases in flux capacity and preserved significant differences between the groups were observed with uncoupling by FCCP (109\u2009\u00b1\u20098 vs 86\u2009\u00b1\u20094\u00a0pmol mg\u22121 s\u22121 in control and diabetic subjects, respectively; p\u2009<\u20090.05). Subsequent inhibition of complex I and III with rotenone and antimycin A blunted the O2 flux (Fig.\u00a02). The addition of cytochrome c did not result in significant increases in O2 flux (data not shown).\nFig.\u00a02O2 flux in permeabilised skeletal muscle fibres from patients with type 2 diabetes and healthy control subjects. Data are shown as O2 flux per mg of tissue (a) and further normalised to the number of copies of mtDNA per \u03bcg of tissue \u00d710,000 (b). When data are expressed relative to mtDNA, any difference between the groups disappears. Data are means\u00b1SEM (*p\u2009<\u20090.05). Black and white bars represent healthy control subjects and patients with type 2 diabetes, respectively\nThe number of copies of mtDNA and citrate synthase activity (Table\u00a01) indicated a lower mitochondrial density in the patients with type 2 diabetes. The O2 flux data were therefore recalculated relative to mtDNA content (Fig.\u00a02b) and citrate synthase activity (data not shown). All differences in O2 flux between patients with type 2 diabetes and healthy control subjects disappeared following either normalisation procedure (Fig.\u00a02b).\nThe increase in O2 consumption when ADP was added (complex I respiratory control ratio [state 3:state 2 respiration]) was not different between healthy subjects and patients with type 2 diabetes (Fig.\u00a03).\nFig.\u00a03a Respiratory control ratio for complex I (NADH supply from substrates glutamate + malate) measured as the ratio of O2 flux with (state 3) and without (state 2) ADP. b Electron transport capacity measured as O2 flux after FCCP-induced uncoupling relative to coupled O2 flux at state 3 with malate + glutamate + ADP + succinate (parallel electron input into both complex I and II). No significant difference between the groups was noted. Data are means\u00b1SEM\nMitochondrial respiration may be influenced by the prevailing level of glucose and\/or insulin. However, none of the O2 flux rates measured with different substrates and inhibitors displayed significant correlations with these parameters, or the changes seen during the OGTT (data not shown).\nThe glucose and insulin concentrations were markedly increased in both groups at 30\u00a0min into the OGTT (Fig.\u00a01). However, the measured O2 flux in the muscle tissue obtained at this time point deviated to a lesser extent (mean +2.4\u2009\u00b1\u20093.5%) from the O2 flux measured in the biopsies obtained during fasting (data not shown).\nThe activity of complex I was affected by rotenone to a similar extent in the two groups (Table\u00a01). Furthermore, the biochemically measured activity of complex I was significantly (p\u2009<\u20090.02) correlated with O2 flux measured by respirometry during rotenone inhibition (r2\u2009=\u20090.37).\nDiscussion\nThe primary novel findings in this study are that: (1) ADP-stimulated state 3 mitochondrial O2 flux capacity with electron flux through either complex I or II, or with parallel electron input through both complexes I and II, is substantially reduced in type 2 diabetic patients when expressed per unit mass of skeletal muscle; and (2) when O2 flux is normalised for mitochondrial DNA content or citrate synthase activity, levels of both oxidative phosphorylation and electron transport capacity are similar to those observed in age-matched healthy control subjects. These results provide direct experimental evidence for normal function of muscle mitochondria in type 2 diabetes and do not support other investigations reporting mitochondrial dysfunction in diabetes. The reduced mitochondrial capacity per unit muscle mass observed in this study is consistent with the concept of reduced mitochondrial content and volume, oxidative enzyme levels, mtDNA and decreased levels of co-regulators of mitochondrial biogenesis-such as peroxisome proliferator-activated receptor-\u03b3 coactivator-1 (PGC-1), nuclear respiratory factor (NRF-1 and NRF-2) and mitochondrial transcription factor A (mtTFA)-in insulin-resistant states, as found in some [7, 8] but not all studies [13]. Therefore, it could be argued that specific cellular signals that alter levels of mitochondria, and thus reduce electron transport (and oxidative phosphorylation capacity) per unit muscle mass, contribute to a variety of aberrant metabolic pathways, including intracellular fat accumulation, insulin resistance and glucose intolerance.\nThe various substrate and inhibitor titrations employed in this study permitted an examination of various steps of oxidative phosphorylation and electron transport under resting respiration and maximally ADP-stimulated O2 flux by ADP (state 3). Both resting (state 2) and ADP-stimulated (state 3) coupled respiration were substantially reduced (18\u201328%) in diabetic subjects, as was uncoupled O2 flux with parallel electron supply from both NADH (complex I) and FADH2 (complex II). These responses indicate a blunting of oxidative phosphorylation linked to ATP synthase and a decreased maximal electron flux capacity in the uncoupled state induced by addition of FCCP, respectively. The key finding, however, is that these values are comparable with those in healthy control subjects when they are normalised for mtDNA content or citrate synthase activity, both of which are used as indices of mitochondrial density per unit muscle mass. The 30% reduction in state 3 O2 flux capacity per mg of muscle in diabetic subjects with parallel electron input (glutamate + malate + succinate) suggests an attenuation of cellular VO2max. While the prevailing view is that O2 delivery is a factor that influences whole-body maximal O2 consumption, it remains to be determined if, and to what extent, the observed decrement in muscle state 3 mitochondrial O2 flux capacity in diabetic patients contributes to the lower systemic VO2max and substrate utilisation.\nThe patients with type 2 diabetes were in a chronic hyperglycaemic state and were clearly insulin resistant, but even so, mitochondrial function\/mtDNA was not impaired. Furthermore, mitochondrial function was also measured in the biopsy samples obtained 30\u00a0min into the OGTT, at a time when both glucose and insulin were markedly increased (Fig.\u00a01). This acute metabolic perturbation did not have any significant effect on mitochondrial respiration in any of the subjects (data not shown).\nIt has been proposed that chronic hyperglycaemia associated with insulin resistance results in the alteration of several metabolic pathways [14]. A central hypothesis involving the mitochondria focuses on an effect of hyperglycaemia providing increased reducing equivalents to the electron transport chain, resulting in a higher membrane potential, with consequent flow of electrons between coenzyme Q and complex III, forming superoxides in the mitochondrial matrix. Excess superoxide production can induce damage to mitochondrial structures, including several electron transport complexes, the mitochondrial lipid bilayer and mtDNA. A role for excess superoxide production is supported by findings of reduced glutathione and metallothionein antioxidant defence systems in type 2 diabetic subjects [15]. The results of this study do not preclude these concepts, but rather suggest the possibility that many pathogenic pathways associated with mitochondrial function in cellular energetics may result from conditions leading to a reduced number of mitochondria, which in turn could place the existing mitochondria under stress with subsequent production of reactive O2 species, impaired metabolism of intracellular lipids, and glucose uptake.\nIt is well known that exercise training increases mitochondrial content in skeletal muscle [16\u201319], and recent work has provided evidence for increased levels of transcriptional regulators of mitochondrial biogenesis in response to exercise [20, 21]. In addition, physical training can play a significant role in the prevention of insulin resistance and type 2 diabetes [22\u201325]. Physical training also substantially improves skeletal muscle insulin sensitivity in patients with overt type 2 diabetes [26], and many cellular adaptations responsible for the effect of training on insulin sensitivity in skeletal muscle have been described [27\u201331]. The cellular and mitochondrial changes in response to exercise training occur in parallel. In obese insulin-resistant subjects, exercise training has been shown to increase the percentage of skeletal muscle fibre volume occupied by mitochondria [32]. Thus, the present data support the hypothesis that type 2 diabetes is, to a large extent, a lifestyle disease, with insufficient exercise-induced gene expression and a surplus of energy intake contributing to its pathogenesis. Accordingly, studies on the effects of \u2018de-training\u2019 have provided evidence of reduced mitochondrial content. After cessation of an endurance training program, citrate synthase and succinate dehydrogenase activities in human muscle have been shown to decline with a half-time of only 12\u201314\u00a0days [33]. Cytochrome c protein concentration in rat muscles declines with a half-life of only 7\u20138\u00a0days [34], and the activity in human muscles has a half-time of similar magnitude. These decreases in the activities of cytochrome c and succinate dehydrogenase do not exactly follow the detraining decline in VO2max [35]. Taken together, these studies support the notion that lack of physical activity lowers mitochondrial concentration. The independent influences of exercise training and detraining, hyperglycaemia, intramuscular lipid accumulation and obesity on mitochondrial function remain to be elucidated.\nIn this study, the ability of the mitochondria to respond with increased O2 consumption following the addition of ADP, represented by the respiratory control ratio, was preserved in the diabetic muscle (Fig.\u00a03). Thus, this index of mitochondrial phosphorylation capacity and coupling of electron transport to phosphorylation indicates that the respiratory chain of the mitochondria in type 2 diabetic subjects functions in a similar manner to that in the mitochondria in control subjects. This view is supported by similar data reported 40\u00a0years ago, albeit mostly from patients with \u2018juvenile diabetes\u2019 [36]. Similarly, an increase (1.3\u20131.4 fold) in O2 flux was seen in response to uncoupling by FCCP (Fig.\u00a03). The fact that the increase was similar in the two groups also testifies that electron transport capacity is not impaired in type 2 diabetes, and that the phosphorylation system (adenine nucleotide transporter, phosphate transporter and ATP synthase) exerts control over electron transport in patients and control subjects to the same degree.\nIn conclusion, the results of the present study provide the first direct evidence of normal mitochondrial function in the skeletal muscle of type 2 diabetic subjects. An apparent impairment of oxidative phosphorylation and electron transport capacity is fully accounted for by a diminished mitochondrial content in the diabetic muscle.","keyphrases":["diabetes","skeletal muscle","mitochondria"],"prmu":["P","P","P"]}
{"id":"FEMS_Microbiol_Ecol-1-_-2121141","title":"Multivariate analyses in microbial ecology\n","text":"Environmental microbiology is undergoing a dramatic revolution due to the increasing accumulation of biological information and contextual environmental parameters. This will not only enable a better identification of diversity patterns, but will also shed more light on the associated environmental conditions, spatial locations, and seasonal fluctuations, which could explain such patterns. Complex ecological questions may now be addressed using multivariate statistical analyses, which represent a vast potential of techniques that are still underexploited. Here, well-established exploratory and hypothesis-driven approaches are reviewed, so as to foster their addition to the microbial ecologist toolbox. Because such tools aim at reducing data set complexity, at identifying major patterns and putative causal factors, they will certainly find many applications in microbial ecology.\nIntroduction\nMicrobial ecology is undergoing a profound change because structure\u2013function relationships between communities and their environment are starting to be investigated at the field, regional, and even continental scales (e.g. Hughes Martiny et al., 2006; Ramette & Tiedje, 2007a, b). Because DNA sequences are being accumulated at an unprecedented rate due to high-throughput technologies such as pyrosequencing (Edwards et al., 2006a, b), single-cell genome sequencing (Zhang et al., 2006), or metagenomics (Venter et al., 2004; Field et al., 2006; Gill et al., 2006), future challenges will very likely consist of interpreting the observed diversity patterns as a function of contextual environmental parameters. This would help answer fundamental questions in microbial ecology such as whether microbial diversity responds qualitatively and quantitatively to the same factors as macroorganism diversity (Horner-Devine et al., 2004; van der Gast et al., 2005; Green & Bohannan, 2006; Hughes Martiny et al., 2006).\nMost obstacles encountered by microbial ecologists when they try to summarize and further explore large data sets concern the choice of the adequate numerical tools to further evaluate the data statistically and visually. Such tools, which have been developed by community ecologists to work on distribution and diversity patterns of plants and animals, could be readily applied in microbial ecology. Although multivariate analyses of community diversity patterns are well described in the literature, microbial ecologists have used multivariate analyses either rarely or mostly for exploratory purposes. A brief survey of the literature confirms this trend (Table 1; Fig. 1). Table 1 indicates that bacterial studies rank third after plant and fish studies for their use of multivariate analyses. Complex data sets are mostly explored via principal component analysis, or cluster analysis, and hypothesis-driven techniques such as redundancy analysis, canonical correspondence analysis (CCA), or Mantel tests are more rarely used (Fig. 1). Axis 1 (horizontal) clearly differentiates microscopic (bacteria, microorganisms, fungi) from macroscopic (fish, bird, plant, insect) life, and this may be related to the use of more exploratory methods (e.g. cluster analysis, PCA) in the first group. It is important to state that the figures presented in Table 1 and Fig. 1 have to be taken with caution because many articles do not include a description of statistical approaches in their titles or abstracts, and so the table is certainly biased and incomplete. However, the point of the table was both to identify some general trends in the literature and to give one example of the usefulness of multivariate analysis to analyze a data table.\nTable 1\nUsage (%) of multivariate methods in different fields\nExploratory analysis\nHypothesis-driven analysis\nKeywords\u2020\nCluster\nPCA\nMDS\nPCoA\nCCA\nRDA\nmanova\nMantel\nanosim\nCVA\nTotal number\u2021\nBacter*\n48.5\n38\n4.5\n0.4\n3.2\n1.8\n1.3\n0.4\n0.9\n1.1\n1141\nMicrob*\n45.8\n40.2\n3.9\n1.1\n2.2\n2.2\n1.1\n1.7\n0.6\n1.1\n\u2002179\nPlant*\n40.3\n28.5\n4.6\n1.7\n15.5\n3.7\n1.9\n2.3\n0.6\n0.9\n3335\nFung*\n54\n27.2\n2.8\n1.1\n8.5\n2.8\n0.9\n1.1\n0.2\n1.4\n\u2002563\nFish*\n30.1\n33.7\n9.8\n0.3\n13.5\n2.7\n3.6\n2.9\n2.3\n1.2\n1464\nBird*\n41\n20.5\n5.4\n0.7\n21.2\n3.5\n2.1\n4.2\n0.5\n0.9\n\u2002429\nInsect*\n54.3\n13.7\n6.1\n0.8\n11.5\n4.4\n3.5\n3\n1.1\n1.7\n\u2002637\nA literature search was performed with the Thomson ISI research tool with the following parameters (Doc type, all document types; language, all languages; databases, SCI-EXPANDED, SSCI, A&HCI; Timespan, 1900\u20132006) on December 13, 2006 in the titles and abstracts of the articles only.\n\u2020\nAsterisks were placed at the end of each keyword to accommodate for variations. Each keyword was additionally combined with the following technical designations: cluster, cluster analysis; PCA, principal component analysis; MDS, multidimensional scaling; PcoA, principal coordinate analysis; CCA, canonical correspondence analysis; RDA, redundancy analysis; Mantel, Mantel test, or CVA, canonical variate analysis.\n\u2021\nTotal number refers to the total number of publications identified by each keyword and all its combinations. The ordination based on correspondence analysis of the raw number is depicted in Fig. 1.\nFig. 1\nCorrespondence analysis of method usage in various scientific fields. In this symmetrical scaling of CA scores, the first two axes explained 47.3% and 35.8% of the total inertia of Table 1, respectively. The gray areas were drawn to facilitate the interpretation. Complete row names (scientific fields; full circles) and column names (methods; white triangles) are given in Table 1. Methods (triangles) located close to each other correspond to methods often occurring together in studies. The distance between a scientific field point and a method point approximates the probability of method usage in the field.\nThis review aims at presenting some common multivariate techniques in order to foster their integration into the microbial ecologist's toolbox. Indeed, \u2018it is no longer possible to gain a full understanding of Ecology and Systematics without some knowledge of multivariate analysis. Or, contrariwise, misunderstanding of the methods can inhibit advancement of the science\u2019 (James & McCulloch, 1990). Such a review is ambitious because it tries to provide a few guidelines for a very vast discipline that is still under development. For this reason, it cannot be exhaustive and does not pretend to offer in-depth coverage of all selected topics. The review is largely inspired by descriptions, comments, and suggestions originating from multiple, highly recommended sources (ter Braak & Prentice, 1988; James & McCulloch, 1990; Legendre & Legendre, 1998; Leps & Smilauer, 1999; ter Braak & Smilauer, 2002; Palmer, 2006), where detailed information about each technique can be obtained.\nIn the first part, data type and preparation are reviewed as a necessary basis for subsequent multivariate analyses. Second, common multivariate methods (i.e. cluster analysis, principal component analysis, correspondence analysis, multidimensional scaling) and a few statistical methods to test for significant differences between groups or clusters are described, focusing on the methods' main objectives, applications, and limitations. Beyond the mere identification of diversity patterns, microbial ecologists may wish to correlate or explain those patterns using measured environmental parameters, and this approach is addressed in the third part. Special emphasis is placed on a few methods that have proven useful in ecological studies, namely redundancy analysis, CCA, linear discriminant analysis, as well as variation partitioning. The final part provides practical considerations to help researchers avoid pitfalls and choose the most appropriate methods.\nData types and data preparation\nData sets\nThe initial multivariate data set may consist of a table of objects (e.g. samples, sites, time periods) in rows and measured variables for those objects in columns. This table structure is the standard used in the present review. When the latter variables are biological taxa, the columns will simply be designated as \u2018species\u2019 thereafter. It is critical to clearly identify what corresponds to objects and variables in the data set. Indeed, objects in one study may be species or operational taxonomic units (OTU) for which catabolic profiles, gene presence or polymorphism, etc. are measured. In another study where samples from different sites are compared based on, for instance, community fingerprinting techniques, objects can now be samples and species variables. This distinction is important because procedures that analyze relationships among objects or among variables are different. Objects are defined a priori by the sampling strategy before making observations and variable measurements. Besides, most multivariate analyses assume independence between objects (or samples), i.e. observations made on an object are not a priori dependent on those made on another object. Variables, however, can be found to be intercorrelated to various degrees, but this is not necessarily known in advance. Initial data sets can also consist of distance matrices where pairwise dissimilarities between objects are calculated. The original table of raw data is not always available, e.g. for DNA\u2013DNA hybridization values, phylogenetic distances, and thus specific multivariate techniques have to be considered to deal with data matrices.\nData transformations\nIn multivariate data tables, measured variables can be binary, quantitative, qualitative, rank-ordered, classes, frequencies, or even a mixture of those types. If variables do not have a uniform scale (e.g. environmental parameters measured in different units or scales) or an adequate format, variables have to be transformed before performing further analyses. Each qualitative variable has to be recoded as a set of numerical variables that replace it in the numerical calculations. One way to do so is to create a series of \u2018dummy\u2019 variables that correspond to all the states of the qualitative variable. For instance, if the variable \u2018season\u2019 has to be recoded, four associated variables will be constructed, and for each object the value 1 will be given to the corresponding season when it occurs, and 0 for the three other seasons when it is absent. Many statistical packages automatically perform this recoding.\nStandardization provides dimensionless variables and removes the undue influence of magnitude differences between scales or units. A common procedure is to apply the z-score transformation to the values of each variable. For each variable, it consists of (1) computing the difference between the original value and the mean of the variable (i.e. centering) and of (2) dividing this difference by the SD of the variable.\nNormalizing transformations aim at correcting the distribution shapes of certain variables, which depart from normality. One thus tries to obtain a homogeneous variances for variables, conditions under which multivariate procedures often perform better. Different mathematical transformations can be used to normalize the x values of a variable: for instance, the arcsin (\u221ax) transformation can be applied to percentages or proportions, log(x+c) to variables departing strongly from a normal distribution, and \u221a(x+c) to less problematic cases, with c being a constant that is added to avoid mathematically undefined computations. The c constant is generally chosen so that the smallest nonzero value is obtained by computing x+c in the former functions. The constant should also be of the same order of magnitude as the variable (Legendre & Legendre, 1998).\nTo make community composition (either presence\u2013absence or abundance) data containing many zeros suitable for analysis by linear methods such as principal component analysis (PCA) or canonical redundancy analysis (RDA), the Hellinger transformation [Eq. (1)] is one of five transformations that give good results (Legendre & Gallagher, 2001). The chord transformation is a useful transformation that also gives less weight to rare species in the species table [Eq. (2)]. The transformations are given by where yij is the original species value for site i and species j, yi+ represents the sum of all species values for site i (i.e. sum per row), p is the number of species in the table (number of columns), and  represents the resulting, transformed species value (Legendre & Gallagher, 2001). These transformations are particularly recommended when rare species are not truly rare, i.e. when they mostly occur because the sampling was performed blindly, as generally done in soil or marine microbial ecology. Further data transformations can be found in Sokal & Rohlf (1995) and Legendre & Legendre (1998).\nThe way to deal with missing data is a discipline on its own (Legendre & Legendre, 1998). Briefly, one can either delete rows or columns containing the missing value(s), or try to replace the missing values by mathematical estimates inferred from values obtained from other objects in the data set. In the latter case, it is still difficult to provide ecologically meaningful explanations for these estimates. In any case, the specific handling of missing data should be reported by the investigator.\nWhen dealing with matrices, it is possible to change a similarity matrix (S) into a dissimilarity matrix (D) by applying the following transformations: D=1\u2212S, D=\u221a(1\u2212S), or D=\u221a(1\u2212S2). To normalize any D matrix to the interval [0\u20131], one can compute D\/Dmax, or (D\u2212Dmin)\/(Dmax\u2212Dmin), where Dmax and Dmin represent the highest and lowest values of D, respectively (Legendre & Legendre, 1998).\nExploratory analyses\nVisualization and exploration of complex data sets\nThe basic aim of ordination and cluster analysis is to represent the (dis)similarity between objects (e.g. samples, sites) based on values of multiple variables (columns) associated with them, so that similar objects are depicted near from each other and dissimilar objects are found further apart from each other. Exploratory multivariate analyses are thus useful to reveal patterns in large data sets, but they do not directly explain why those patterns exist. This latter point is addressed in the third part of the review.\nCluster analysis and association coefficients\nCluster analysis encompasses several multivariate techniques that are used to group objects into categories based on their dissimilarities. The aim is both to minimize within-group variation and maximize between-group variation in order to reveal well-defined categories of objects, and therefore reduce the dimensionality of the data set to a few groups of rows (James & McCulloch, 1990; Legendre & Legendre, 1998). This approach is thus generally recommended when distinct discontinuities instead of continuous differences (i.e. gradients) are expected between samples (objects) because cluster analysis mostly aims at representing partitions in a data set (Legendre & Legendre, 1998).\nBecause distance matrices that are based on differences in DNA or amino acid sequences are commonly used to describe microbial diversity, cluster analysis has become very popular in microbial ecology (Table 1; Fig. 1). This is not surprising because the grouping of organisms based on their phenotypic or genotypic similarities in order to infer their taxonomic positioning is generally and historically based on cluster analysis (or at least based on a tree-like representation) and, as such, is central to biology and evolution (Avise, 2006). Typical microbial ecology questions that are addressed by cluster analysis are whether the clustering patterns of molecular sequences reflect sample origin or sampling time in order to reveal specific biogeographical or temporal patterns, respectively (Whitaker et al., 2003; Acinas et al., 2004). Those factors are generally hypothesized to be of a discontinuous nature, but the rationale of generally representing molecular differences as discontinuous clusters in microbial ecology and microbial genomic studies has only started to be questioned (Konstantinidis et al., 2006). Another common application consists of sorting out clones from environmental samples based on specific criteria (e.g. genetic or phenotypic markers) because clones or variants are expected to form tight clusters around their parental strains and to be more distinct from other lineages (Acinas et al., 2004). In microarray data analysis, cluster analysis has helped identify common expression patterns of groups of genes, which may shed light on functionally related genes or pathways (Eisen et al., 1998).\nCluster analysis of a data table proceeds in two steps. First, a relevant association coefficient has to be chosen to measure the association (similarity or dissimilarity) among objects or among variables. Second, the calculated association matrix is represented as a horizontal tree (hierarchical clustering) or as distinct groups of objects (k-means clustering), based on specific rules to aggregate objects. For ecologists, the power of cluster analysis derives from the existence of different types of (dis)similarity coefficients. The choice of appropriate and ecologically meaningful association coefficients is particularly important because it directly affects the values that are subsequently used for the categorization of objects.\nThe analysis of similarities among objects (rows) is designated as Q mode analysis, whereas when relationships among variables (columns) are the focus of the study, this is referred to as R mode analysis (Legendre & Legendre, 1998). Noticeably, the two modes of analysis do not generally use the same association coefficients. Although it is not possible to give a full review of all association coefficients here, it is useful to known that, for comparing objects (rows) based on their column attributes in Q mode analysis, coefficients may be chosen as a function of data type (quantitative, qualitative, ordinal, or mixed data, normalized data, presence-absence), importance given to rare species, weight given to each object, and calculation of associated probability levels. For comparing objects in a sample-by-environment table (e.g. water, soil chemistry), selection of appropriate coefficients generally depends on data type and unit homogeneity of the measured variables. In R mode analyses, in addition to the previously cited criteria, the choice of a coefficient may also depend on how the variables are related to each other (e.g. linearly, monotonically, qualitatively, ordered), and on how species absence is handled in the calculations. In most ecological studies, the absence of a species at two sites being compared is not considered as a measure of similarity between those sites. Indeed, a simultaneous species absence at two sites may be due to different reasons, e.g. the sites offer different physical\u2013chemical conditions and the species cannot exist under both conditions, and so there is no straightforward conclusion about site similarity that can be drawn in this case. Asymmetric coefficients are coefficients that do not take into account cases of double absences of species (\u2018double zeros\u2019) in the calculation of pairwise similarities among sites. Moreover, in microbial ecology where environmental communities are generally far from being exhaustively sampled, a double absence of an OTU has to be regarded more as a lack of information rather than a sign of common structure among samples, and asymmetric coefficients such as Jaccard (1901) or S\u00f8rensen (1948) should be preferred. More details about the calculation of association coefficients and their appropriateness can be found, for instance, in Chapter 7 of (Legendre & Legendre, 1998).\nWhen an association matrix is calculated, the relationships between objects or variables can be represented following specific aggregation rules. Three general approaches are commonly used: hierarchical clustering, k-means partitioning, and two-way joining. In hierarchical clustering, a linkage rule to form clusters and the numbers of clusters that best suit the data have to be determined a priori. Clusters, which are nested rather than mutually exclusive here, are either formed by progressively agglomerating objects from high to low similarity cutoff values (forward clustering), or using the converse strategy, i.e. grouping all cases together and progressing from low to high cutoff values in order to merge objects and clusters (backward clustering). These two strategies do not necessarily yield the same clusters. The merging of clusters is visualized using a tree format (generally horizontal) and is successful when well-defined clusters are identified in the data set (Sneath & Sokal, 1973).\nCommon linkage rules are, e.g. nearest neighbor (the distance between two clusters is the distance between their closest neighboring points), furthest neighbor (the distance between two clusters is the distance between their two furthest objects), and the widely used unweighted pair-group method using averages (UPGMA; Sneath & Sokal, 1973), where the distance between two clusters is the average distance between all intercluster pairs. When within-cluster homogeneity is desired, Ward's method, which merges clusters only if they increase the within-cluster variation the least, is recommended (Legendre & Legendre, 1998). Finally, equal weight can also be given to clusters that are expected to be of different sizes using the weighted arithmetic average clustering (WPGMA), which consists of giving less weight to the original similarities of the largest groups (Legendre & Legendre, 1998).\nIn k-means clustering, objects are assigned to k clusters (k being defined in advance), based on their nearest Euclidean distance to the mean of the clusters. The mean of the cluster is iteratively recalculated until no more assignments are made and cluster means fall below a predefined cut-off value or until the iteration limit is reached. Different means for each cluster are ideally obtained for each dimension used in the analysis, as indicated by high F-values from the respective analyses of variance. Unlike hierarchical clustering, k-means clustering does not require prior computation of dissimilarity matrix among objects and is therefore more adapted to large data sets (e.g. few thousand objects) where computing power is an issue. However, the method is quite sensitive to outliers, which are usually removed before performing the analyses (Legendre & Legendre, 1998).\nTwo-step cluster analysis may be useful to group objects into clusters when one or more of the variables are categorical (not interval or dichotomous). Objects are first grouped based on the categories, which are themselves hierarchically clustered as single cases. Because neither a proximity table nor iterative steps are required, the method is particularly suited for the analysis of very large data sets (Eisen et al., 1998).\nPrincipal component analysis (PCA)\nPCA has been applied to numerous phenotypic and genotypic (e.g. fingerprinting patterns) data sets, and it is one of the most popular exploratory analyses (Table 1), perhaps because the technique is generally the first multivariate approach to be explained in most data analysis manuals. However, this choice may not always be justified in ecology and recommendations for appropriate applications are provided at the end of this section and in the \u2018Practical considerations\u2019 part of the present review. Examples of use in microbial ecology concern the identification of patterns of microbial community change over seasons or geographic areas (e.g. Merrill & Halverson, 2002), or as those patterns relate to different plant compartments at different plant developmental stages (Mougel et al., 2006), or the reduction of the complexity of data sets involving hydrochemistry data, bacterial, and archeal community profiles in order to visualize and interpret complex multivariate data sets onto two-dimensional geographic maps of contaminated sites (Mouser et al., 2005).\nThe PCA procedure basically calculates new synthetic variables (principal components), which are linear combinations of the original variables (for instance, the species of a sample-by-species table), and that account for as much of the variance of the original data as possible (Hotelling, 1933). The aim is to represent the objects (rows) and variables (columns) of the data set in a new system of coordinates (generally on two or three axes or dimensions) where the maximum amount of variation from the original data set can be depicted. In practice, PCA is either performed on a variance\u2013covariance matrix or on a correlation matrix. The first approach is followed when the same units or data types are used (e.g. abundance of different species). The aim is then to preserve and to represent the relative positions of the objects and the magnitude of variation between variables in the reduced space. PCA on a correlation matrix is rather used when descriptor variables are measured in different units or on different scales (e.g. different environmental parameters) or when the aim is to display the correlations among (standardized) descriptor variables. The two approaches lead to different principal components and different distances between projected objects in the ordination; hence, the interpretation of the relationships must be made with care (Table 2). Indeed, for correlation matrices, variables are first standardized (i.e. they become independent of their original scales), and so distances between objects are also independent from the scales of the original variables. All variables thus contribute to the same extent to the ordination of objects, regardless of their original variance.\nTable 2\nInterpretation of ordination diagrams\nLinear methods (PCA, RDA)\nPCA, RDA\nRDA\nScaling 1\nScaling 2\nSamples\nSpecies\nENV\nNENV\nFocus on sample (rows) distance\nFocus on species (columns) correlation\n\u2713\nEuclidean distances among samples\n\u2013\n\u2713\n\u2013\nLinear correlations among species\n\u2713\nMarginal effects of ENV on ordination scores\nCorrelations among ENV\n\u2713\nEuclidean distance between sample classes\n\u2013\n\u2713\n\u2713\nAbundance values in species data\n\u2713\n\u2713\n\u2013\nValues of ENV in the samples\n\u2713\n\u2713\nMembership of samples in the classes\n\u2713\n\u2713\nLinear correlations between species and ENV\n\u2713\n\u2713\nMean species abundance within classes of nominal ENV\n\u2713\n\u2713\n\u2013\nAverage of ENV within classes\nUnimodal methods (CA, CCA)\nCA, CCA\nCCA\nFocus on sample (rows) distance and Hill's scaling\nFocus on species (columns) distances\n\u2713\nTurnover distances among samples\n\u03c72 distances between samples\n\u2713\n-\n\u03c72 distances among species distributions\n\u2713\nMarginal effects of ENV\nCorrelations among ENV\n\u2713\nTurnover distances between sample classes\n\u03c72 distances between sample classes\n\u2713\n\u2713\nRelative abundances of the species table\nRelative abundances of the species table\n\u2713\n\u2713\n\u2013\nValues of ENV in the samples\n\u2713\n\u2713\nMembership of samples in the classes\n\u2713\n\u2713\nWeighted averages \u2013 the species optima in respect to particular ENV\n\u2713\n\u2713\nRelative total abundances in the sample classes\n\u2713\n\u2713\n\u2013\nENV averages within sample classes\nThe interpretation of ordination diagrams depends on the focus of the study, because sample scores are rescaled as a function of the scaling choice. Approximate relationships between and among the different elements represented in biplots and triplots as species (represented as dots or arrows), samples (dots), environmental variables (ENV; arrows), and nominal (qualitative) environmental variables (NENV; dots). A meaningless interpretation (\u201c\u2013\u201d) happens when the suggested comparison is not optimal because of inappropriate scaling of the ordination scores. Adapted from ter Braak (1994); Leps & Smilauer (1999); ter Braak & Smilauer (2002).\nPCA results are generally displayed as a biplot (Jolicoeur & Mosimann, 1960), where the axes correspond to the new system of coordinates, and both samples (dots) and taxa (arrows) are represented (Fig. 1a). The direction of a species arrow indicates the greatest change in abundance, whereas its length may be related to a rate of change. Depending on whether a distance or a correlation biplot is chosen, different interpretations can be made from the ordination diagram (Table 2). The interpretation of the relationships between samples and species differs and is directly affected by the scaling chosen, i.e. whether the analysis mainly focuses on intersample relationships (scaling 1) or interspecies correlations (scaling 2). For instance, in scaling 1, the distances between objects are an approximation of their Euclidean distances in the multidimensional space, but this approximation is not valid if scaling 2 is chosen (Table 2). Projecting an object at a right angle on a species arrow in the ordination diagram approximates the position of the object along that species descriptor. The length of the species descriptor indicates its contribution to the formation of the ordination space. For correlation biplots, the length of the orthogonal projection of a species arrow on the axes approximates its SD on the respective axes. Angles between species arrows reflect their correlations, e.g. putative interactions between species (scaling 2). An erroneous interpretation of the biplot would be to use the proximity of an object point and the tip of a species arrow to deduce a relationship between them. Indeed, only right-angle projections of samples onto species arrows are correct to derive approximated species abundance in the samples.\nPCA should generally be used when the objects (sites or samples) cover very short gradients, i.e. when the same species are mostly identified everywhere in the study area (i.e., when samples mostly differ in species abundances), and when species linearly respond to environmental gradients. Because those conditions are often not met in ecological studies, other multivariate approaches have been progressively preferred over PCA (as also suggested by Table 1) such as correspondence analysis or multidimensional scaling.\nPCA is successful when most of the variance is accounted for by the largest (generally the first two or three) components. The amount of variance accounted for by each principal component is given by its \u2018eigenvalue.\u2019 The mathematical description of eigenvalue calculation steps goes beyond the aim of the present review but can be found in most linear algebra manuals. Eigenvalues derived from a PCA are generally considered to be significant when their values are larger than the average of all eigenvalues (Legendre & Legendre, 1998). The cumulative percentage of variance accounted for by the largest components indicates how much proportion of the total variance is depicted by the actual ordination. High absolute correlation values between the synthetic variables (principal components) and the original variables are useful to identify which variables mainly contribute to the variation in the data set, and this is referred to as the loading of the variables on a given axis. However, because the synthetic and original variables are linearly correlated (i.e. they are not independent), standard tests to determine the statistical significance of the correlations between them cannot be used.\nPrincipal coordinate analysis (PCoA)\nThe technique is more rarely used by microbial ecologists (Table 1), despite its usefulness at reducing and representing patterns present in distance matrices displaying dissimilarities among objects (Gower, 1966). Its objectives are very similar to those of PCA in that it uses a linear (Euclidean) mapping of the distance or dissimilarities between objects onto the ordination space (i.e. projection in a Cartesian space), and the algorithm attempts to explain most of the variance in the original data set. In microbial ecology, PCoA has been used, for instance, to test whether virulence profiles (i.e. presence or absence of specific genes) arising from pathogenic strains could differentiate either healthy or contaminated hosts (Chapman et al., 2006), or to determine whether PCoA axes could separate groups of Staphylococcus aureus isolates into bovine and human hosts when genetic relationships among them had been established by random amplified polymorphic DNA-PCR analysis (Reinoso et al., 2004).\nAs opposed to PCA, PCoA works with any dissimilarity measure and so specific association coefficients that better deal with the problem of the presence of many double zeros in data sets can be surmounted. Moreover, PCoA does not provide a direct link between the components and the original variables and so the interpretation of variable contribution may be more difficult. This is because PCoA components, instead of being linear combinations of the original variables as in PCA, are complex functions of the original variables depending on the selected dissimilarity measure. Besides, the non-Euclidean nature of some distance measures does not allow for a full representation of the extracted variation into a Euclidean ordination space. In that case, the non-Euclidean variation cannot be represented and the percent of total variance cannot be computed with exactness. The choice of the dissimilarity measure is thus of great importance, and subsequent transformation of the data to correct for negative eigenvalues is sometimes necessary (see Legendre & Legendre, 1998, section 9.2.4. for how to correct for such negative eigenvalues).\nObjects are represented as points in the ordination space. Eigenvalues are also used here to measure how much variance is accounted for by the largest synthetic variables on each PCoA synthetic axis. Although there is no direct, linear relationship between the components and the original variables, it is still possible to correlate object scores on the main axis (or axes) with the original variables to assess their contribution to the ordination.\nCorrespondence analysis (CA)\nA basic question that ecologists may want to address when facing a multidimensional table of sites (or samples) by species is whether certain species occur at specific sites, as a measure of their ecological preferences. CA has generally been used in microbial ecology to determine whether patterns in microbial OTU distribution could reflect differentiation in community composition as a function of seasons, geographic origin, or habitat structure (Olapade et al., 2005; Edwards et al., 2006a, b; Kent et al., 2007). The overall aim of the method is to compare the correspondence between samples and species from a table of counted data (or any dimensionally homogenous table) and to represent it in a reduced ordination space (Hill, 1974). Noticeably, instead of maximizing the amount of variance explained by the ordination, CA maximizes the correspondence between species scores and sample scores. Several algorithms exist and the most commonly described one is reciprocal averaging, which consists of (1) assigning arbitrary numbers to all species in the table (these are the initial species scores), (2) for each sample, a sample score is then determined as a weighted average of all species scores (this thus takes into account the abundance of each species at the site and the previously determined species scores), (3) for each species, a new species score is then calculated as the weighted average of all the sample scores, (4) both species scores and sample scores are standardized again to obtain a mean of zero and a SD of one, and (5) steps two to four are repeated until species and site scores converge towards stable solutions in successive iterations (Hill, 1974). The overall table variance (inertia) based on \u03c72 distances is decomposed into successive components that are uncorrelated to each other, as in the PCA or PCoA procedures. For each axis, the overall correspondence between species scores and sample scores is summarized by an eigenvalue, and the latter is thus equivalent to a correlation coefficient between species scores and sample scores (Gauch, 1982).\nThe technique is popular among ecologists because CA is particularly recommended when species display unimodal (bell shaped or Gaussian) relationships with environmental gradients (ter Braak, 1985), as it happens when a species favors specific values of a given environmental variable, which is revealed by a peak of abundance or presence when the optimal conditions are met (this can be visualized by plotting species abundance against the environmental parameter). The unimodal model that supports the concept of ecological niches has also been shown to be of the right order of complexity for the ordination of most ecological data (ter Braak & Prentice, 1988). Although examples of unimodal distributions along variables or environmental gradients exist with macroorganisms (ter Braak, 1985), the shape of the distribution of the abundance of microbial species along environmental parameters or gradients has not been extensively investigated (but see Ramette & Tiedje, 2007a, b). This may arise from the fact that, in microbial surveys, environmental sampling is mostly performed blindly in relation to environmental heterogeneity, and the abundance of target species is generally determined without systematically analyzing associated environmental parameters. Finally, another important feature of CA for microbial ecologists is that the reciprocal averaging algorithm disregards species double absences because the relationships between rows and columns of the table are quantified using the \u03c72 coefficient that excludes double absences (Legendre & Legendre, 1998).\nBoth samples and taxa are often jointly depicted in the ordination space (i.e. joint plot; Fig. 2b), where the center of inertia (centroid) of their scores corresponds to the zero for all axes. Depending on the choice of the scaling type, either the ordination of rows (samples) or the columns (species) is meaningful, and can be interpreted as an approximation of the \u03c72 distances between samples or species, respectively (see Table 2 for more details about interpretation). Sample points that are close to each other are similar with regard to the pattern of relative frequencies across species. It is important to remember that in such joint plots, either distances between sample points or distances between species points can be interpreted, but not the distances between sample and species points. Indeed, these distances are not simple Euclidean distances computed from the relative row or column frequencies, but rather they are weighted distances. The proximity between sample and species points in the plot can thus be understood as a probability of species occurrence or of a high abundance in the samples in the vicinity of a species point.\nFig. 2\nOrdination diagrams in two dimensions. (a) In a PCA biplot representation, samples are represented by dots and species by arrows. The arrows point in the direction of maximal variation in the species abundances, and their lengths are proportional to their maximal rate of change. Long arrows correspond to species contributing more to the data set variation. Right-angle projection of a sample dot on a species arrow gives approximate species abundance in the sample. (b) In a CA joint plot representation focusing on species distance, both samples and species are depicted as dots. Species dots correspond to the center of gravity (inertia) of the samples where they mostly occur. Distances between sample and species points give an indication of the probability of species composition in samples (see Table 2 for more details about diagram interpretation).\nIn scaling 2 (i.e. focus on species), species points found at the center of the ordination space should be carefully checked with the raw data to clarify whether the species ordination really corresponds to the optimal abundance or occurrence of the species, or whether the species is just badly represented by the main axes, as it is the case when other axes are more appropriate to represent the species. Rare species contribute little to the total table inertia (i.e. they only play a minor role in the overall table variance) and are hence positioned at the edges of the plot, next to the site(s) where they occur. In general, only the species points found away from the ordination center and not close to the edges of the ordination have more chances to be related to the ordination axes, i.e. to contribute to the overall variance (Legendre & Legendre, 1998).\nWhen the species composition of the sites progressively changes along the environmental gradient, sample positions may appear in the ordination plot as nonlinear configurations called \u2018arch\u2019 (Gauch, 1982) (or \u2018horseshoe\u2019 in the case of PCA), which may impair further ecological interpretation. In CA, the arch effect may be mathematically produced as a side-effect of the CA procedure that tries to obtain axes that both maximally separate species and that are uncorrelated to each other (ter Braak, 1987): when the first axis suffices to correctly order the sites and species, a second axis (uncorrelated with the former) can be obtained by folding the first axis in the middle and bringing its extremities together, thus resulting in an arch configuration. Further axes can be obtained by further dividing and folding the first axis into segments (Legendre & Legendre, 1998). To remove the arch effect in CA, a mathematical procedure, detrending, is used to flatten the distribution of the sites along the first CA axis without changing their ordination on that axis. The approach is then designated as detrended correspondence analysis (DCA). The review of different detrending algorithms such as using segments or polynomials goes beyond the scope of this review, but more information can be obtained in (ter Braak & Prentice, 1988; Legendre & Legendre, 1998). Some authors have also argued that the arch effect may not be an artifact but an expected feature of the analysis, especially when species turnover is high along environmental gradients (James & McCulloch, 1990). In that case, if the samples are meaningfully positioned along the arch, the ordination should be accepted as a valid result.\nNonmetric multidimensional scaling (NMDS)\nNMDS is generally efficient at identifying underlying gradients and at representing relationships based on various types of distance measures. Not surprisingly, NMDS has found an increasing number of applications in microbial ecology (Table 1). The technique has been generally applied to identify patterns among multiple samples that were subjected to molecular fingerprinting techniques. For instance, NMDS was used to analyze and to compare the reproducibility of various fingerprinting techniques such as ribosomal internal spacer analysis (RISA), terminal fragment length polymorphism (T-RFLP), and denaturing gradient gel electrophoresis (DGGE) between different laboratories when applied to samples chosen from a salinity gradient (Casamayor et al., 2002). NMDS was also used to compare diversity patterns of microbial communities (as determined by length heterogeneity-PCR) from samples undergoing different land management practices (Mills et al., 2006). Another example is the analysis of the bacterioplankton communities of four shallow eutrophic lakes that differed in nutrient load and food web structure using DGGE profiling, so as to determine the specificity of community signatures in each lake (Van der Gucht et al., 2005).\nThe NMDS algorithm ranks distances between objects, and uses these ranks to map the objects nonlinearly onto a simplified, two-dimensional ordination space so as to preserve their ranked differences, and not the original distances (Shepard, 1966). The procedure works as follows: the objects are first placed randomly in the ordination space (the desired number of dimensions has to be defined a priori), and their distances in this initial configuration are compared by monotonic regression with the distances in the original data matrix based on a stress function (values between 0 and 1). The latter indicates how different the ranks on the ordination configuration are from the ranks in the original distance matrix. Several iterations of the NMDS procedure are generally implemented so as to obtain the lowest stress value possible (i.e. the best goodness of fit) based on different random initial positions of the objects in the ordination space. For sample-by-species tables, simulations have shown that before applying NMDS, a standardization of each species by its maximum abundance, followed by the computation of distances between samples based on the Steinhaus or Kulczinski similarity coefficients yielded informative ordination results (Legendre & Legendre, 1998, p. 449).\nIn NMDS ordination, the proximity between objects corresponds to their similarity, but the ordination distances do not correspond to the original distances among objects. Because NMDS preserves the order of objects, NMDS ordination axes can be freely rescaled, rotated, or inverted, as needed for a better visualization or interpretation. Because of the iterative procedure, NMDS is more computer intensive than eigenanalyses such as PCoA, PCA, or CA. However, constant improvement in computing power makes this limitation less of a problem for small- to medium-sized matrices.\nTesting for significant differences between groups\nIn addition to representing objects in an ordination plot or as clusters of similar objects, another objective may be to test whether differences between groups of objects (rows) in a multivariate table are significantly different based on the set of their attributes (columns), i.e. to test whether similarities within groups are higher than those between groups. Here, nonparametric multivariate anova (npmanova) and analysis of similarities (anosim), which are commonly found in standard statistical packages, are briefly reviewed. It is also possible to use canonical analyses (\u2018Testing for significant differences between groups\u2019) to test for significant differences between groups of objects. These statistical tests, however, must not be used to assess the statistical difference among groups that were derived from a previous cluster analysis on the same variables because, under those conditions, the two approaches would not be independent from each other. Indeed, the groups derived from cluster analysis (which are themselves made to fit the data) would then be used for testing the null hypothesis that there is no difference among the groups. This hypothesis would then not be independent of the data used to test it, and would nearly always produce significant differences between the groups even if it is not the case (Legendre & Legendre, 1998).\nnpmanova\nThe method can be used to test for significant differences between the means of two or more groups of multivariate, quantitative data (Anderson, 2001). The null hypothesis of equality of means is tested based on Wilks' \u039b (lambda) statistic, which replaces the F-test normally used in univariate anova. When only two groups are compared, Hotelling's T2 test is more appropriate. The latter test can also be used, as a post hoc test, to assess the significance of pairwise comparisons statistically between groups, following an overall significant Wilks' test. Significance is generally computed by permutation of group membership, with several thousand replicates, alleviating concerns about multinormality of the data. Because multiple pairwise comparisons are made, the significance level of the pairwise Hotelling's tests needs, however, to be corrected. With the Bonferroni correction, for instance, the P-value usually chosen for significant differences between groups (i.e. 0.05) is replaced by a smaller P-value calculated by dividing the original P-value by the total number of pairwise comparisons that are performed. For instance, for 10 pairwise comparisons, the corrected P-value becomes 0.005. This correction is often judged to be rather conservative as it leads to significance for fewer pairwise comparisons (Legendre & Legendre, 1998).\nanosim\nThis nonparametric procedure tests for significant difference between two or more groups, based on any distance measure (Clarke, 1993). It compares the ranks of distances between groups with ranks of distances within groups. The means of those two types of ranks are compared, and the resulting R test statistic measures whether separation of community structure is found (R=1), or whether no separation occurs (R=0). R values >0.75 are commonly interpreted as well separated, R>0.5 as separated, but overlapping, and R<0.25 as barely separable (Clarke & Gorley, 2001). The test makes fewer assumptions than manova because it is based on the ranks of distances, and it is often used for sample-by-species tables, where groups of samples are compared. All groups should have comparable within-group dispersion to avoid finding falsely significant results (Legendre & Legendre, 1998).\nApplications in microbial ecology include testing for spatial differences, temporal changes, or environmental impacts on microbial assemblages. For instance, Kent et al. (2007) determined whether bacterial communities from the same lake were more similar in composition to each other than to communities in different lakes. The bacterial composition and diversity of samples from different geographic origins, habitats, and avian hosts were also compared using anosim based on a length heterogeneity (LH)-PCR (Bisson et al., 2007). Another example is the application of anosim to terminal restriction fragment length polymorphism (T-RFLP)-generated data to determine the impact of B and NaCl on soil microbial community structure in the wheat rhizosphere (Nelson & Mele, 2007).\nEnvironmental interpretation\nExploratory analyses may reveal the existence of clusters or groups of objects in a data set. When a supplementary table or matrix of environmental variables is available for those objects, it is then possible to examine whether the observed patterns are related to environmental gradients. Typical objectives may be, for instance, to reveal the existence of a relationship between community structure and habitat heterogeneity, between community structure and spatial distance, or to identify the main variables affecting bacterial communities when a large set of environmental variables has been conjointly collected.\nThe significance of the relationships between species patterns and environmental variables can generally be assessed by permutation techniques such as Monte Carlo permutation tests, which infer statistical properties from the data themselves. The order of data (generally the rows of one matrix) is permuted and the relationships between the observed patterns and environmental variables can be assessed for randomness. This approach is particularly suitable when variables do not follow a normal distribution (which is often the case with environmental or ecological data), as generally required by traditional statistical procedures (Legendre & Legendre, 1998).\nIndirect gradient analyses\nOrdination axes or clusters can be interpreted based on additional environmental variables (i.e. variables not used in the ordination or cluster analysis) that provide ecological knowledge about the studied sites or species ecological characteristics. When using exploratory ordination approaches on a sample-by-species table, samples are displayed along the axes of main variation in species composition. These axes are thus constructed without reference to environmental characteristics, but they can be hypothesized to represent underlying environmental gradients (e.g. environmental parameters, spatial or temporal variables, chemical gradients), which need to be subsequently identified. Such an approach is designated as \u2018indirect,\u2019 because synthetic variables (i.e. the axes) are first constructed and thereafter related to environmental variation. For instance, the scores of the objects on PCA or CA main components (axes) can be further related by standard statistical procedures (e.g. anova, regression analysis) to environmental variables. Likewise, in PCoA or NMDS, it is possible to statistically compare the ranks obtained by the objects on each axis and the ranks of those objects on additional environmental variables, using Spearman's rank correlation coefficients (Legendre & Legendre, 1998).\nA convenient method of interpretation is to represent the additional environmental variables as fitted arrows directly on the ordination diagram. These variables are added to the existing ordination by linear regression of their values onto the existing ordination axes. This procedure is implemented in various statistical packages (e.g. canoco, R). Hence, it is possible to assess the direction and magnitude of the most rapid change in the environmental variables and to determine whether they correspond to the observed patterns among objects (Oksanen, 2007). In cluster analysis, the magnitude of the absolute correlation value between an ordered clustering solution and environmental variables may also provide clues about putative environmental causes for the observed discontinuities in the data set.\nAnother convenient way of displaying additional information to help interpret the ordination is to use site symbols whose sizes are proportional to the values of the additional variable. Hence, one can visually assess whether the ordination of objects (samples, sites) matches specific trends in the additional variable. This strategy was, for instance, used on NMDS ordination plots inferred from DGGE profiles on which the values of five additional environmental variables were individually mapped as proportional circles in order to identify the main environmental factors related to the bacterial community structure in four freshwater lakes (Van der Gucht et al., 2005).\nDirect gradient analyses (constrained analyses)\nIn constrained (canonical) ordination analyses, only the variation in the species table that can be explained by the environmental variables is displayed and analyzed, and not all the variation in the species table. Gradients are supposed to be known and represented by the measured variables or their combinations, while species abundance or occurrence is considered to be a response to those gradients. Constrained ordinations are mostly based on multivariate linear models relating principal axes to the observed environmental variables, and the different techniques depend on data types (matrix or table), and on the hypothesis underlying species distribution in the gradients (i.e. linear or unimodal). Their aim is to find the best mathematical relationships between species composition and the measured environmental variables, and to assess whether, statistically, such a relationship could have been produced due to chance alone using permutation tests. The resulting ordination diagrams display samples, species, and environmental variables so that \u2018fitted species \u00d7 samples\u2019 and \u2018species \u00d7 environment\u2019 relationships can be derived as easily as possible from angles between arrows or distances between points and arrows (Table 2).\nRedundancy analysis (RDA)\nIn microbial ecology, RDA has been applied, for instance, to test whether the occurrence of biocontrol bacteria with specific carbon source utilization profiles was related to their origin from different root samples (Folman et al., 2003), to determine which environmental factors were the most significant to explain variation in microbial community composition in undisturbed native prairies and cropped agricultural field (McKinley et al., 2005), to examine the effects of sampling locations (longitude, latitude, altitude) on genetic diversity of plant pathogenic bacteria (Kolliker et al., 2006), or to assess the influence of season, farm management, and soil chemical, physical, and biological properties on nitrogen fluxes and bacterial community structure (Cookson et al., 2006).\nThis method can be considered as an extension of PCA in which the main axes (components) are constrained to be linear combinations of the environmental variables (Rao, 1964). Two tables are then necessary: one for the species data (\u2018dependent\u2019 variables) and one for the environmental variables (\u2018independent\u2019 variables). Multiple linear regressions are used to \u2018explain\u2019 variation between independent and dependent variables, and these calculations are performed within the iterative procedure to find the best ordination of the objects. The interest of such an approach is to represent not only the main patterns of species variation as much as they can be explained by the measured environmental variables but also to display correlation coefficients between each species and each environmental variable in the data set.\nWhen the data set consists of a matrix of distances between objects, distance-based RDA (db-RDA; Legendre & Anderson, 1999) can be applied to determine how well additional environmental parameters can explain the variation among objects in the matrix. The technique first applies a PCoA on the distance matrix to convert it back to a rectangular table containing rows of objects by columns of PCoA coordinates. Those new, uncorrelated coordinates thus correspond to synthetic \u2018species\u2019 variables that are then related to additional environmental parameters using a classical RDA. For instance, db-RDA was successfully used to determine how the variation in matrices of genomic distances among environmental strains could be explained by factors such as soil parameters, host plant species, and spatial scale, each factor being taken alone or in combination (Ramette & Tiedje, 2007b).\nMost software outputs provide the total variation in species composition as explained by the environmental axes, the cumulative percentage of variance of the species\u2013environment relationship, and the overall statistical significance of the relationships between the species and environmental tables. RDA can be represented by a triplot of samples (dots), species (arrows), and environmental variables (arrows for quantitative variables and dots for each level of qualitative or nominal variables), or by any combinations thereof (i.e. biplots) (ter Braak, 1994). Depending on the scaling chosen, i.e. whether the analysis mainly focuses on intersample relationships or interspecies correlations, the interpretation of the relationships between samples, species, and environmental variables differs (Table 2).\nCanonical correspondence analysis (CCA)\nThe approach is very similar to that of RDA, except that CCA is based on unimodal species\u2013environment relationships whereas RDA is based on linear models (ter Braak, 1986). CCA can be considered as the constrained form of CA in which the axes are linear combinations of the environmental variables. CCA uses the unimodal model to model species response to the environmental variation as a mathematical simplification to enable the estimation of a large number of parameters and the identification of a small number of ordination axes. This species model seems, however, to be robust even when some species display bimodal responses, unequal ranges, or unequal maxima along environmental gradients, and the technique is thus considered to be the method of choice by many ecologists (ter Braak & Smilauer, 2002). It is therefore particularly adapted for the environmental interpretation of tables of abundance and occurrence of species, and accommodates well the absence of species at certain sites in the data set. CCA is sensitive to rare species that occur in species-poor samples, and down-weighting of such species help reduce the problem (Legendre & Legendre, 1998). Software outputs are very similar to those of RDA and as for RDA, triplot and biplot representations and interpretation depend on the choice of the scaling type (Table 2). The same interpretation of the relationships between sample and species points is found in CA and CCA. Right-angle projection of these points on the environmental arrows leads to the correct approximation of the ranking of the points along environmental variables.\nCCA has been used in an increasing number of publications dealing with microbial assemblages in marine and soil ecosystems. Typical questions that are addressed concern the identification of environmental factors that influence the diversity of bacterial assemblages among large sets of candidate environmental parameters measured for the same samples, when the diversity is determined by culture-independent, genetic fingerprinting techniques such as automated ribosomal intergenic spacer analysis (ARISA) (Yannarell & Triplett, 2005), DGGE (Salles et al., 2004; Sapp et al., 2007), or T-RFLP (C\u00f3rdova-Kreylos et al., 2006; Klaus et al., 2007). Another interest in the technique comes from the possibility of determining the specific species or OTUs that respond to particular environmental variables, and as such that can be identified as candidate indicator species. Those species can then be subjected to further experiments so as to confirm their status of indicator species. For instance, the relationships, as determined by CCA, between bacterial community composition and 11 environmental variables for 30 lakes in Wisconsin, revealed that patterns in bacterial communities were best explained by regional- and landscape-level factors, as well as by specific seasons, pH, and water clarity (Yannarell & Triplett, 2005). CCA was also successfully used to demonstrate that former land use management affected the composition of the targeted soil microbial community (Burkholderia) to a larger extent than did plant species (Salles et al., 2004). Another interesting example in the marine ecosystem is the study of the interactions between various abiotic parameters and phytoplankton community data (biotic parameter) to explain bacterioplankton dynamics in the North Sea and the subsequent identification of the bacterial phylotypes responding more specifically to the factors (Sapp et al., 2007). Another example of using CCA to identify some microbial communities as pollution indicators can be found in (C\u00f3rdova-Kreylos et al., 2006).\nPartial ordination, variation partitioning\nWhen the effects of a particular environmental variable need to be tested after elimination of possible effects due to other (environmental) variables, partial ordination may be used (e.g. partial CCA, partial RDA). Such an approach is also referred to as \u2018partialling out\u2019 or \u2018controlling for\u2019 the effects of specific variables, which are specified as covariables in the constrained analysis. For instance, in a study dealing with the effects of environmental and pollutant variables on microbial communities, C\u00f3rdova-Kreylos et al. (2006) observed that variation in microbial communities was more due to spatial variation than to pollutants. The use of partial CCA to account for spatial variation in the biological data set revealed that metals had a greater effect on microbial community composition than organic pollutants.\nThis idea of controlling for the effects of specific variables can be extended to evaluate the effects of all the different sets (factors) of environmental variables present in a study so as to determine the relative contribution (amount of variation explained) and significance of each variable set on the total biological variance. The so-called variation partitioning procedure (Borcard et al., 1992) partitions the total variance of the species table into the respective contribution of each set of environmental variables and into their covariations using both standard and partial constrained ordinations (Fig. 3). Two methods have traditionally been used to partition the variation of community composition data, i.e. canonical partitioning and regression on distance matrices based on Mantel tests (Legendre & Legendre, 1998). The canonical approach has been shown to be more appropriate to partition the \u03b2 diversity correctly among sites and to test hypotheses about the origin and maintenance of its variation (Legendre et al., 2005).\nFig. 3\nPartitioning biological variation into the effects of two factors. The large rectangle represents the total variation in the biological data table, which is partitioned among two sets of explanatory variables (a, b). Fraction 4 shows the unexplained part of the biological variation. Fractions 1 and 3 are obtained by partial constrained ordination or partial regression, and can be tested for significance. For instance, fraction 1 corresponds to the amount of biological variation that can be exclusively explained by (a) effects when (b) effects are taken into consideration (i.e., when b is considered as a covariable). Fraction 2 [i.e., variation indifferently attributed to (a) and (b) or a covariation of (a) and (b)] is obtained by subtracting fractions 1 and 3 from the total explained variance, and cannot be tested for statistical significance.\nApplications of variation partitioning in microbial ecology include, for instance, the study by Ramette & Tiedje (2007b), which applied the technique in the context of RDA to disentangle the effects of space, environmental soil parameters, and plant species on Burkholderia community abundance and diversity. By quantifying the amount of biological variation that is left unexplained when all environmental variables had been considered, the study suggested that much less of the biological variation could be predicted at the intraspecific level compared with higher taxonomic levels. Another interesting example is the study of seasonal changes in bacterial community composition in shallow eutrophic lakes, in which top-down regulation (grazers) of bacterial community composition was examined after accounting for bottom-up regulation (resources) (Muylaert et al., 2002).\nLinear discriminant analysis (LDA)\nWhen groups or clusters of objects have been obtained by exploratory analyses for instance, LDA can be used to identify linear combinations of additional environmental variables that best discriminate those groups. In that respect, LDA can be seen as an extension of manova for two or more groups, in which environmental variables that specifically explain the grouping of objects are identified. Another application consists of assigning new objects to previously defined groups for prediction or classification purposes based on the calculated discriminant function. For instance, Fuhrman et al. (2006) used the technique to evidence the existence of repeatable temporal patterns in the community composition of marine bacterioplankton over 4.5 years.\nThe technique is mostly recommended for multinormal data for which attribute data are linearly related and for which variances and covariances of the variables are good summary statistics. A visual representation of LDA can be performed, and in the resulting ordination, the axes are then the discriminant functions. The distances between objects, which correspond to Mahalanobis distances that take into account the correlations among descriptors (Mahalanobis, 1936), are independent of the scale of measurement of the various descriptors and are mostly used to compare groups of sites or objects with each other (Legendre & Legendre, 1998).\nSelection of variables in regression models\nIn the previous constrained methods where linear combinations of environmental (explanatory) variables are used, the inclusion of too many explanatory variables to describe species distribution may lead to difficult ecological interpretations and to lower predictability of the models, due to intercorrelations among the explanatory variables (i.e. multicollinearity). Multicollinearity has the effects of inflating the variance of the regression coefficients in the models, leading to reduced precision in the prediction of the response variables (Legendre & Legendre, 1998). In order to only include in the model the environmental variables that mostly and significantly contribute to the variation of the species table, automatic selection procedures (forward selection, backward elimination, or stepwise selection) are often used. The selection depends on whether the partial correlation coefficients of the variables fall below a given significance level, the latter being generally assessed by Monte Carlo permutation tests.\nIn forward selection, the construction of the regression model starts with the variable that explains the most variation in the dependent variables (generally the species table). What remains of the biological variation to explain after fitting the first environmental variable (i.e. of the residual variation) is then used to choose the second environmental variable. The process of selection goes on until no more variables significantly explain the residual variation. In backward elimination, the construction of the regression model starts with all environmental variables and the least significant ones are excluded from the model, one at a time until a group of only \u2018significant\u2019 variables is obtained. To take advantage of the two approaches, stepwise regression mixes forward selection with backward elimination by performing a forward selection, but excluding the variables that no longer become significant after the introduction of new variables into the regression model.\nDespite the clear advantages of these variable selection strategies, most authors still caution that researchers should not blindly rely on automatic selection procedures to choose the relevant environmental variables in regression models because ecologically irrelevant models may also be obtained, or other variable combinations could also yield better models to explain species variation (Legendre & Legendre, 1998). Noticeably, the three selection strategies do not necessarily yield the same set of significant environmental variables, because they may be seen as heuristic methods to identify a significant model when all possible combinations of significant models are not possible to evaluate computationally. Another approach is thus to combine variables into biologically or environmentally meaningful sets, instead of relying on automatic selection procedures, and then to examine all possible regression models based on the reduced number of variable sets (James & McCulloch, 1990). For instance, before applying variation partitioning to different groups of variables representing spatial scales (15 variables), host species (four variables), and soil parameters (10 variables), Ramette & Tiedje (2007b) applied forward selection within each group to determine the variables significantly explaining the variation of microbial diversity and abundance at different taxonomic levels.\nMantel test\nThis test is appropriate to compare two matrices that were calculated for the same objects but that are based on two independent data sets (e.g. a species dissimilarity matrix and an environmental dissimilarity matrix for the same samples) (Mantel, 1967). It calculates the correlation coefficient between corresponding positions in the two matrices, and assesses its significance based on permutations of the objects in one of the matrices. In microbial ecology, the Mantel test has become popular especially for testing the relationships between molecular and geographic distance matrixes for a same set of organisms or to relate community diversity to environmental heterogeneity (e.g., Parker & Spoerke, 1998; Cho & Tiedje, 2000; Horner-Devine et al., 2004; Scortichini et al., 2006).\nAnother interesting application, called a goodness-of-fit Mantel test, corresponds to the case where one matrix is recoded to represent ecological hypotheses to be tested on the other matrix (Legendre & Legendre, 1998). For instance, if a matrix of molecular data is available for a set of strains and their habitat of origin is known, it is possible to determine whether the genetic distances are related to habitat type using the (goodness-of-fit) Mantel test. The matrix representing the ecological hypotheses should then consist of a series of 1 and 0 for isolates found in the same or different habitats, respectively. The Mantel test can thus determine whether the posited habitat distribution can significantly explain the structure of the molecular matrix. This test cannot be used, however, to test a hypothesis matrix that would be based on the results of a cluster analysis, for instance. Indeed, as indicated in \u2018Testing for significant differences between groups,\u2019 there would be a lack of independence between the hypothesis being tested and the data used to test the hypothesis.\nNote that the Mantel test is also used to compute Mantel correlograms, which are often found in biogeographical studies (e.g. Mantel correlograms are usually used to detect spatial structure in species assemblages based on grouping of the response data into specific spatial distance classes). Mantel tests are then applied to each group in order to detect significant correlations at a given scale, i.e. the scales at which the data are autocorrelated (Legendre & Legendre, 1998).\nPractical considerations\nChoice of an ordination method (Fig. 4)\nLinear methods such as multiple regression, LDA, PCA, or RDA are generally meant to be applied to continuous data. Their use is thus sometimes limited in Ecology where species generally display nonlinear, nonmonotone responses to environmental variables (ter Braak & Prentice, 1988; Legendre & Legendre, 1998). Different approaches can be undertaken to choose the most appropriate ecological model. Plot of species abundances along ordination axes or explanatory variables (also called coenocline) may help visualize whether species responses are linear or unimodal (ter Braak & Smilauer, 2002). Besides, the choice of linear (PCA, RDA) or unimodal (CA, CCA) species response models can be made on the basis of whether the underlying gradient length is short or long, respectively. Gradient length, as measured in SD units along the first ordination axis, can be estimated by DCA for unconstrained ordination and by detrended CCA (DCCA) for constrained ordination in, e.g. the software canoco (ter Braak & Smilauer, 2002). It is recommended to use linear methods when the gradient length is <3 SD, unimodal methods when it is >4 SD, and any method for intermediate gradient lengths (ter Braak & Smilauer, 2002).\nFig. 4\nRelationships between numerical methods. Exploratory tools such as PCA, CA, PCoA, NMDS, or cluster analysis can be applied to a sample-by-species table to extract the main patterns of variation, to identify groups or clusters of samples, or specific species interactions. Sample scores on the main axes of variation can be related to variation in environmental variables using indirect gradient analyses. When a constrained analysis is desired (i.e. direct gradient analysis), RDA, db-RDA, CCA, or linear discriminant analysis can be used as extensions of the unconstrained methods. Mantel tests are appropriate to test the significance of the correlation between two distance matrices (e.g. one based on species data and the other on environmental variables). Raw data may be transformed, normalised or standardised as appropriate before analysis.\nData type is also another important criterion. To represent absolute abundance values, linear-based methods (PCA, RDA), which produce weighted summations, are appropriate, whereas unimodal techniques (CCA, CA) are rather used to model relative abundances (because species scores are weighted averages of the samples scores, and vice versa), i.e. they model the dissimilarities between samples (\u03b2 diversity). They also accommodate well the presence of many zeros in the species table, in contrast to linear-based methods for which double zeros lead to inadequate estimates of sample distances.\nCluster analysis is the method of choice when relationships between objects are expected to be discontinuous and where defined categories or groups of objects are expected. On the contrary, ordination would be more useful when the variation between objects is posited to be continuous. Although NMDS is more computer intensive than PCoA, it is generally better at compressing the distance relationships among objects into a few dimensions. This is because NMDS can always lead to a Euclidean representation even for non-Euclidean embeddable distances (Legendre & Legendre, 1998). NMDS and PCoA can be compared using Shepard diagrams to decide which technique better represents the original distances.\nIf one assumes that species do not have a linear response to environmental gradients, NMDS is more appropriate than PCA. CA may also be an alternative to PCA when many zeros populate the data set and one strong gradient is present. With long ecological gradients, however, CA may produce the arch effect that can be corrected for using DCA. In terms of the underlying species model, the main difference between DCA and NMDS is that the former is based on a specific model of species distributions (unimodal model), while NMDS is not. Thus, DCA may be favored by ecologists who assume that the niche theory better fits their data set, while NMDS may be a method of choice if species composition is determined by factors other than position along a gradient (for instance if the habitat is known to be fragmented).\nIn constrained and unconstrained ordinations, all species are posited to react to different extents to the same composite gradients of environmental variables, whereas in a multiple regression approach, a different gradient could be modeled for each species separately. Because most species do not respond linearly to environmental gradients, fitting nonlinear models to individual species may be difficult, especially when dealing with a huge data set. Constrained ordinations thus provide a good summary of species\u2013environment relationships and can be very successful in ecological data analysis (ter Braak & Prentice, 1988). It is also useful to note that RDA is very similar to manova, but in contrast to the latter, RDA allows the consideration of any number of species (columns) (Legendre & Legendre, 1998).\nConstrained and unconstrained (exploratory) methods should be used in parallel (Fig. 4) because, with the former, only the biological variation that can be explained by the available environmental variables is represented on the main axes, whereas with unconstrained methods, the highest amount of variance is extracted from the biological data alone and represented on a few axes. If the constrained and unconstrained approaches yield the same ordination of the samples (objects), it thus means that the measured environmental variables explain most of the biological variation. In order to compare the results of different ordinations, a useful technique is Procrustes analysis (Gower, 1975), which estimates the concordance of scores in two ordinations after rotating, translating, and dilating them in order to obtain the best fit. A permutation procedure can also be used to test for the significance of the concordance between ordinations or matrices (Peres-Neto & Jackson, 2001).\nCluster analysis and ordination techniques can be combined to provide powerful visualization tools. For instance, hierarchical clustering can help obtain a better interpretation of ordination diagrams (Fig. 5). Because ordination diagrams represent most of the data set variation into a dimensionally reduced space, some relationships among objects can be distorted because only a few projection axes are considered. The addition of linkage results obtained from cluster analysis may help identify objects belonging to the same clusters even if their relative position in the ordination diagram is not ideal (Legendre & Legendre, 1998).\nFig. 5\nCombination of ordination and cluster analysis. On a same distance matrix, NMDS or PCOA can be applied to represent the major axes of variation among objects in a two-dimensional space. The superimposition of the results of cluster analysis (primary connections) onto the ordination diagram can help identify the structure in the data set as discontinuities (clusters) into a continuous space (ordination). Adapted from Legendre & Legendre (1998).\nOrdination and diversity indices\nThe measurement of diversity is generally performed using indices such as the Shannon or Simpson indices. The latter are often applied to measure different components of the diversity such as \u03b1, \u03b2, and \u03b3 diversity, corresponding to diversity within a particular site or ecosystem, to change in species composition from site to site (i.e. species turnover), and to the diversity at the landscape scale, respectively (Whittaker, 1972). The ordination approach sounds similar, in that variation among samples is compared based on their within-sample composition in species assemblages, and so some of the \u03b1 and \u03b2 diversity should be depicted on ordination diagrams. Because diversity indices pool the multispecies information into a single value for each observation, before comparing them, it is not surprising that complex diversity patterns may not be identified sometimes. For instance, Hartman & Widmer (2006) did not find significant changes in soil bacterial communities submitted to various soil managements when using diversity indices, while community structures were shown to have changed using community fingerprinting analysis.\nTo obtain a consistency between ordination techniques and diversity index measurements, two numerical strategies have been proposed: for species occurrence data, the CA-species richness strategy adapted for data set rich in rare species, and the Nonsymmetric CA \u2013 Simpson strategy, which is more appropriate for tables dominated by abundant species (Pelissier et al., 2003). These strategies attribute specific weights to the species data so that simple or constrained ordinations of the new species table represent the total inertia as \u03b1 and \u03b2 diversity, and would thus be consistent with the measures obtained by common diversity indices.\nMisconceptions about multivariate analyses\nIt is essential to reiterate that multivariate statistical procedures may suggest causes or factors, but investigators should bear in mind that the synthetic variables, axes, or clusters derived do not necessarily correspond to biological or ecological entities in nature (James & McCulloch, 1990). One should thus not overinterpret the data by relying on unjustified causality, especially in the absence of real experimentation. In theory, it would be necessary to validate the inferences and models made about pattern formation and putative causes by analyzing new data, but this is rarely performed in practice. Moreover, whether the originally collected data are typical of the situation to be described is most of the time not even questioned.\nAnother common misconception is that multivariate analyses alone can sort out all solutions of complex multivariate studies. Although exploratory analyses may help reveal interesting patterns in data sets, the interpretation and explanation of the observations ultimately rely on the researcher's hypotheses and previous knowledge of the ecological situation. Microbial ecologists themselves need to formulate ecologically sound hypotheses and test them.\nConclusions\nExciting questions in Ecology typically consist of determining whether community patterns are structured across space or time, of explaining how those patterns can be related to environmental heterogeneity, and of quantifying how much still remains unexplained when all significant, measured variables have been considered. Such questions can now start to be addressed in microbial ecology because numerical tools may help explore and test such ecological hypotheses. These are indeed exciting times because even larger and more complex databases are being created and in parallel, computing power gradually becomes less of an issue. If microbial ecologists want to test numerical methods, develop new ecological theories, or validate existing ones for the microbial case, access to diversity data and above all, to the relevant associated environmental parameters, becomes a central issue. It would thus be of great interest to make such complex data sets publicly available, such as microbial ecological databases, so that microbial diversity can be studied in its environmental context. This would indeed be a step toward making microbial ecology a central discipline in Ecology.","keyphrases":["multivariate","statistics","ordination","gradient","modeling"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_J_Appl_Physiol-4-1-2358938","title":"Characteristics of fast voluntary and electrically evoked isometric knee extensions during 56 days of bed rest with and without exercise countermeasure\n","text":"The contractile characteristics of fast voluntary and electrically evoked unilateral isometric knee extensions were followed in 16 healthy men during 56 days of horizontal bed rest and assessed at bed rest days 4, 7, 10, 17, 24, 38 and 56. Subjects were randomized to either an inactive control group (Ctrl, n = 8) or a resistive vibration exercise countermeasure group (RVE, n = 8). No changes were observed in neural activation, indicated by the amplitude of the surface electromyogram, or the initial rate of voluntary torque development in either group during bed rest. In contrast, for Ctrl, the force oscillation amplitude at 10 Hz stimulation increased by 48% (P < 0.01), the time to reach peak torque at 300 Hz stimulation decreased by 7% (P < 0.01), and the half relaxation time at 150 Hz stimulation tended to be slightly reduced by 3% (P = 0.056) after 56 days of bed rest. No changes were observed for RVE. Torque production at 10 Hz stimulation relative to maximal (150 Hz) stimulation was increased after bed rest for both Ctrl (15%; P < 0.05) and RVE (41%; P < 0.05). In conclusion, bed rest without exercise countermeasure resulted in intrinsic speed properties of a faster knee extensor group, which may have partly contributed to the preserved ability to perform fast voluntary contractions. The changes in intrinsic contractile properties were prevented by resistive vibration exercise, and voluntary motor performance remained unaltered for RVE subjects as well.\nIntroduction\nPrevious research has shown that exposure to actual or simulated spaceflight leads to pronounced muscle atrophy in humans. The associated muscle weakness significantly impairs the performance of various motor tasks (for reviews see Adams et al. 2003; Desplanches 1997; Edgerton and Roy 2000; Fitts et al. 2000). Muscle function (e.g., maximal isometric force) is often further impaired by adaptations in the central motor control system, as indicated by reductions in the amplitude of the surface electromyogram (EMG) during maximal voluntary contractions (Berg et al. 1997; Deschenes et al. 2002; di Prampero and Narici 2003; Gondin et al. 2004; Schulze et al. 2002).\nCompared to steady-state contractions, much higher levels of neural activation are needed for contractions where torque develops as rapidly as possible. This is seen for both voluntary (de Ruiter et al. 2004) as well as for electrically evoked contractions (de Haan 1998; de Ruiter et al. 1999). In daily life, elderly individuals who lack sufficient motor speed or possess poor lower extremity strength have an increased risk of fall-related bone fractures (Shigematsu et al. 2006). The same may hold for astronauts suffering from muscle atrophy, neural deconditioning and increased bone fragility following space missions (Smith and Heer 2002).\nThe adaptive physiological responses in the human body as a consequence of real or simulated space flight may be offset by means of efficient countermeasures. With respect to the neuromuscular system, it appears that resistance exercise (strength training) is effective to maintain, or at least to minimize changes in muscle mass and strength during bed rest (Akima et al. 2001; Ferrando et al. 1997; Kawakami et al. 2001). Previously, we have shown by means of the twitch interpolation technique, as well as by means of EMG recordings that neural deconditioning was absent during 56\u00a0days of bed rest for maximal steady-state contractions, regardless of whether subjects participated in an exercise countermeasure program (Mulder et al. 2006, 2007). Nonetheless, based on the above, it can be hypothesised that the neural activation of explosive isometric contractions is more deteriorated by bed rest than the activation of steady-state contractions (Mulder et al. 2007). The first aim of the study was to test the hypothesis that, in the absence of an exercise countermeasure, the maximal rate of voluntary isometric torque development would show a greater decrease during 56\u00a0days of bed rest than the maximal steady-state torque, due to reduced neural drive during the fast voluntary contractions. Such an effect was expected to be prevented by combining daily resistive exercise training with vibration training, which was hypothesized to provide a better protection against musculoskeletal deconditioning during bed rest than heavy-resistance training only (Rittweger et al. 2006). Although the individual merits of resistance training versus vibration training cannot be quantified with such a study design, the efficacy of this combined countermeasure to preserve neural activation and maximal steady-state torque is reported elsewhere (Mulder et al. 2007).\nApart from neural activation, the rate at which muscle force develops under voluntary command is also determined by peripheral factors, such as the intrinsic contractile muscle fibre speed (Andersen and Aagaard 2006) and the stiffness of the series elastic component (Bojsen-Moller et al. 2005). Both factors are known to be altered by exposure to actual or simulated space flight, but their effect is opposite. Whereas faster intrinsic contractile speed characteristics (Talmadge 2000) can partly or fully compensate for the effect of atrophy on power output of single muscle fibres (Widrick et al. 1998), the power output of whole muscles (with intact tendons) would be diminished by decrements in tendon stiffness (Kubo et al. 2000; Reeves et al. 2005). The second aim of the study was to assess whether changes in peripheral factors influenced the voluntary rate of torque development during bed rest. The functional change in peripheral factors, i.e., the combined effect of intrinsic muscle and tendon characteristics, were investigated by applying percutaneous muscle stimulation. This is a frequently used methodology to assess muscle properties irrespective of central neural influences (Binder-Macleod et al. 1995; de Haan et al. 2000; Gerrits et al. 2001; Harridge et al. 1996). We hypothesized that the muscle\u2013tendon complex of the knee extensors would acquire the intrinsic contractile properties of a faster muscle, which would be prevented by the current countermeasure design, conceivably due to the large number of contraction\u2013relaxation cycles during resistive vibration exercise (Blottner et al. 2006).\nMethods\nSubjects\nA total of 16 subjects participated in the present study. All subjects were in good health and were involved in normal physical activity before participation in the large-scale Berlin Bed Rest study (Rittweger et al. 2006). At the start of the study the subjects were randomly assigned to an experimental group or an inactive control group. The experimental group (RVE, n\u00a0=\u00a08; mean age, height and body mass\u00a0\u00b1\u00a0SD: 33.0\u00a0\u00b1\u00a01.9\u00a0years, 1.84\u00a0\u00b1\u00a00.03\u00a0cm and 79.5\u00a0\u00b1\u00a03.8\u00a0kg) participated in a progressive resistive vibration exercise (RVE) training program during the bed rest. The subjects of the inactive control group (Ctrl, n\u00a0=\u00a08; mean age, height and body mass\u00a0\u00b1\u00a0SD: 34.3\u00a0\u00b1\u00a02.5\u00a0years, 1.82\u00a0\u00b1\u00a00.02\u00a0cm and 76.8\u00a0\u00b1\u00a01.8\u00a0kg, respectively) were restricted to bed rest without exercise countermeasure. All subjects were familiarized with the concepts of the experiments, procedures, and the equipment during a familiarization session that was scheduled 3\u00a0days prior to the start of bed rest. The local Ethics committee of the Charit\u00e9, Campus Benjamin Franklin Berlin approved the study and all participants gave their written informed consent.\nGeneral design\nAll subjects underwent 56\u00a0days of strict horizontal bed rest at the Charit\u00e9 Benjamin Franklin Hospital, Berlin, Germany. During the bed rest, the subjects were not allowed to stand up, to lift their trunk in bed more than to 30\u00b0 of trunk flexion, to move their legs briskly, or to elicit large forces with their legs muscles other than during testing sessions or during training sessions. Adherence to this protocol was controlled for by continuous video surveillance and by force transducers in the frames of the bed. The diet was balanced using the Harris\u2013Benedict equation and ingestion of alcohol or nicotine, excessive doses of caffeine, as well as the regular intake of any drug or medication was prohibited (details in Rittweger et al. 2006).\nExercise countermeasure\nRVE subjects performed resistive exercises on a vibration system that was specifically developed for application under microgravity and bed rest conditions (Galileo Space; Novotec, Pforzheim, Germany). The applied equipment and protocol for countermeasure exercise are described in detail elsewhere (Rittweger et al. 2006). In short, the training device consists of a vibration platform, which is vertically suspended on a trolley. Elastic springs were attached to the trolley for the subjects to attach themselves through belts with their shoulders, hips, and hands. During bed rest, RVE subjects trained in the supine position, two times daily for 6\u00a0days\/week. In each training session, four resistive exercises were performed in the following order: squats, heel raises, toe raises and explosive squats. All exercises were performed while the platform was vibrated at a frequency of 19\u00a0Hz. Vibration frequency was progressively increased during the 56-day bed rest period to \u223c26\u00a0Hz at the end of bed rest (Rittweger et al. 2006).\nExperimental set-up\nIsometric force recordings were made from voluntary and electrically evoked contractions of the knee extensor group of the right leg. Subjects were tested in the supine position using the same equipment as previously described (Mulder et al. 2006). Force signals were digitized using a sampling rate of 1\u00a0kHz and stored on disc for immediate and off-line analysis. Torque (N\u00a0m) was off-line calculated as the product of force and external moment arm.\nExperimental procedures\nDuring bed rest, all subjects participated in seven experimental sessions, which were scheduled at days: 4, 7, 10, 17, 24, 38 and 56, the latter being the last day of the bed rest period. The baseline experiment was conducted on the fourth day of bed rest (BR4) for logistical reasons. Although this prevents us to address rapid initial changes in strength and contractile characteristics of the quadriceps (Berg and Tesch 1996), which might result in an underestimation of the effect of bed rest on the measured parameters, it does not compromise the comparison between groups during bed rest, because the RVE subjects started their exercise training program on the fourth day of bed rest, which was scheduled after the completion of the functional testing conducted on that day. In addition, in this way all experiments were conducted under methodologically similar conditions. That is, at the baseline experiment subjects were already minimally 72\u00a0h bedridden, each subject was tested at the same time of day, and subjects of the RVE group were always tested before their morning training session.\nTo minimize potential damage to muscle and tendon structures due to maximal and explosive contractions after a long period of strict muscle inactivity, subjects started each experimental session by performing a standardised warm-up set that consisted of eight to ten unloaded dynamic contractions (right leg not yet strapped to the force transducer), followed by eight sub-maximal isometric contractions at 70\u00b0 of knee flexion. Further force recordings were obtained at the individually determined pre-bed rest optimal knee flexion (either 60\u00b0 or 70\u00b0, whereby a knee flexion angle of 0\u00b0 corresponds to full knee extension; see Mulder et al. 2006 for details). Subjects were asked to perform two to three maximal voluntary contractions (MVC) of 2\u20134\u00a0s in duration. Attempts were interposed with 2\u00a0min of rest.\nAfter this procedure, the quadriceps muscle was stimulated through two self-adhesive surface electrodes (model 283100, Schwa-Medico, Nieuw Leusden, The Netherlands) of 80\u00a0mm\u00a0\u00d7\u00a0130\u00a0mm. The stimulation intensity was progressively increased until 40% of the MVC torque was obtained during a 700\u00a0ms tetanic contraction at 150\u00a0Hz (Mulder et al. 2006). The quadriceps muscle was then electrically stimulated with trains of the same single pulse intensity at low (7 pulses at 10\u00a0Hz) and high (24 pulses at 300\u00a0Hz) stimulation frequency. The trains were applied in this order and interposed with 1\u00a0min of rest.\nFollowing the electrically evoked contractions, the stimulation electrodes were removed and the skin over the lateral vastus muscle was re-prepared for the positioning of a high-density surface EMG system (HD-sEMG, Active One, BioSemi Inc., Amsterdam, The Netherlands). The system consisted of 130 densely spaced skin-surface electrodes, arranged in a rectangular 10\u00a0\u00d7\u00a013 matrix with 5\u00a0mm inter-electrode distance (Blok et al. 2002). Before mounting the grid to the skin, the skin was scrubbed with alcoholic pads and slightly rubbed with electrode paste. Prior to each test, the skin-electrode impedance was checked and, if necessary, the skin was re-prepared. The grid was positioned over the distal (third), anterio-lateral part of the right vastus lateralis muscle, such that the columns of 13 electrodes were aligned parallel to the muscle fibre orientation of the muscle and with the motor endplate zone around the centre of the columns of the grid (Mulder et al. 2007). The pre-amplified 130 monopolar signals (referenced to the patella) were bandpass filtered (16\u2013400\u00a0Hz) and simultaneously AD-converted (16 bits with a resolution of 1\u00a0\u03bcV\/bit at a rate of 2\u00a0kHz\/channel). Data were stored on hard disk for subsequent off-line processing.\nWith the sEMG system properly positioned, each subject performed another MVC of 2\u20134\u00a0s. If the torque deviated more than 5% from the highest torque attained during the previous MVC-task (i.e., the highest value of the attempts before the application of electrical stimulation), another attempt was made.\nLastly, each subject performed three fast (explosive) isometric contractions to assess the neural control of these contractions, as well as the initial rate of torque development during these attempts. Subjects were instructed to contract \u201cas fast and forcefully as possible\u201d on a given signal from the test leader (3-2-1 \u201cGo\u201d). Subjects were required to reach a minimum of 80% of the current MVT and to maintain torque at the highest attained level for approximately 1\u00a0s, i.e., \u2018kicks\u2019 were always disqualified. Attempts with an initial countermovement (identified by a drop in the torque signal exceeding 1\u00a0N just before the onset of torque development) were also disqualified (Aagaard et al. 2002; de Ruiter et al. 2004). Attempts were interposed with 2\u00a0min of rest.\nData analysis\nFast voluntary isometric knee extensions\nThe neural activation of muscle fibres at the start of a contraction greatly determines the performance of specific voluntary motor functions, such as the rate at which muscle force develops during fast and forceful voluntary isomeric contractions (de Ruiter et al. 2004). Even so, the maximal rate of isometric torque development did not differentiate subjects according to their ability to generate high neural activation levels at the very start of the contraction. Instead, the time torque integral, calculated as the area under the time torque curve over the first 40\u00a0ms after the onset of torque development, showed to be more sensitive to the initial level of neural activation (de Ruiter et al. 2004). Based on these findings, we calculated the voluntary time torque integral (vTTI40) as an estimate of maximal isometric tension development under voluntary command (Fig.\u00a01). The onset of torque development was thereby defined as the point at which the torque curve exceeded baseline torque by more than three standard deviations.\nFig.\u00a01Voluntary torque (thick line) and rectified surface EMG of the vastus lateralis muscle (thin line) time traces, obtained from a representative subject during an isometric voluntary knee extension performed as fast and forcefully as possible. The arrow and diamond at time 0\u00a0ms indicate the start of voluntary torque development. The shaded area under the voluntary torque trace reflects the vTTI40, calculated the first 40\u00a0ms after onset of torque development. The horizontal bar indicates the 40\u00a0ms immediately preceding the onset of torque development (i.e., from \u221240 to 0\u00a0ms) for which the root mean square (RMS) of the surface EMG was calculated. Subsequently, both vTTI40 and RMS\u221240\u20130 were normalized to the steady state maximal isometric knee extension condition at the day of testing\nVoluntary neural activation during the fast isometric knee extensions was assessed by averaging the amplitude (based on root mean square, RMS) of the monopolarly recorded sEMG signals (Fig.\u00a01) over 40\u00a0ms before the onset of torque development (RMS\u221240\u20130). Unlike a single bipolar recording, the HD-sEMG system allowed for the assessment of monopolar recordings, and allowed for the spatial selection of the grid column with the highest amplitude. The latter was based on the mean of all electrodes within one column.\nTo investigate how neural activation related to the relative maximal rate of torque development for each individual, vTTI40 values were corresponded to RMS\u221240\u20130 data, whereby each accepted contraction of each session was included. For the evaluation of both RMS\u221240\u20130 and vTTI40 as a function of bed rest duration, only the data of one single fast contraction per session were incorporated into the analyses. The contraction with highest level of EMG activity was selected for this purpose. Since we were interested to compare the neural activation during fast voluntary contractions with the neural activation during voluntary contractions where torque is developed more slowly, the contractile and electromyographic data were normalized to the steady-state maximal isometric knee extension condition at the day of testing. These data are also part of another study and are reported elsewhere (Mulder et al. 2007).\nElectrically evoked contractions\nFor each experimental session, the peak torque (T) attained during the 10\u00a0Hz (T10) train was expressed relative to the maximal tetanic torque reached during the 150\u00a0Hz tetanus (T150), i.e., expressed as a T10\/T150 ratio. The force profiles of the 10\u00a0Hz tetanus showed clear oscillations (Fig.\u00a02). The force oscillation amplitude (FOA) relative to the mean force was also calculated and used as a measure of the degree of force-fusion (Gerrits et al. 1999; V\u00f8llestad et al. 1997).\nFig.\u00a02Methods used for evaluation of contractile properties of the quadriceps femoris muscle evoked by electrically evoked muscle stimulation at 10\u00a0Hz. The torque elicited at 10\u00a0Hz stimulation was first expressed as a percentage of the maximal torque evoked at 150\u00a0Hz (T150). T10 was the peak value of the 10\u00a0Hz torque trace. The force oscillation amplitude (FOA) was determined by expressing the mean amplitude of the torque oscillation (Os) between the fourth and seventh stimulus as a fraction of the mean torque (Tm) during this time\nThe contractile characteristics at high pulse frequency stimulation (i.e., 300\u00a0Hz) were quantified by assessing the time to peak tension from the start of the evoked contraction (TPT300). The start of contraction was defined as the instant the first pulse of the 80\u00a0ms train was delivered. Half-relaxation time was determined for each session as the time needed for the elicited torque to decay to half the maximal value following the last pulse of the 150\u00a0Hz tetanus (HRT150). Force data were filtered using a fourth-order, 50\u00a0Hz low-pass filter. This filter was found not to affect the course of torque development; it only removed high-frequency noise from the signal.\nStatistical analysis\nValues are expressed as mean\u00a0\u00b1\u00a0SE (standard error of the mean). Independent-samples t tests were used to determine whether the absolute values of variables related to muscle strength, neural activation and contractile properties of the quadriceps femoris muscle differed at baseline (BR4). Changes in muscle strength and contractile properties after 56\u00a0days of bed rest were assessed by means of linear regression, and expressed as a percentage change with respect to the value at BR4. One-sample t tests were used to determine whether the normalized slope (slope\/intercept on y-axis) of the linear regression was significantly different from zero. Independent-sample t tests were used to determine whether the groups differed in their response to bed rest. Pearson\u2019s correlation coefficients were calculated to establish significance of correlation. The level of significance was set at P\u00a0<\u00a00.05.\nResults\nFast voluntary isometric knee extensions\nData of neural activation and steady state isometric strength are presented elsewhere (Mulder et al. 2006). Briefly, whereas neural activation remained unaltered for Ctrl, maximal voluntary isometric steady-state strength significantly declined after 56\u00a0days of bed rest by about 17%. For the RVE group neural activation increased by approximately 30%, whereas voluntary steady-state torque was maintained.\nThe ability to perform fast voluntary contractions showed a substantial variability during bed rest. There was considerable variation in vTTI40 as well as in RMS\u221240\u20130 during these contractions, both within one session as well as across sessions. Nonetheless, significant positive linear relationships (P\u00a0<\u00a00.01) between RMS\u221240\u20130 and the vTTI40 were obtained for 12 of the 16 subjects in the present study (4 RVE subjects and 8 Ctrl subjects). Significant Pearson\u2019s correlation coefficients (r) ranged from 0.52 to 0.90; with a median value of 0.74. When individual data were pooled per group, each group had a significant relationship between normalized EMG amplitude and normalized vTTI40 (RVE, r\u00a0=\u00a00.572; P\u00a0<\u00a00.01; Ctrl, r\u00a0=\u00a00.758; P\u00a0<\u00a00.01). However, as can be seen in Fig.\u00a03, for neither group did RMS\u221240\u20130, or vTTI40 change during the course of the bed rest.\nFig.\u00a03Mean values (\u00b1SE) of the voluntary time torque integral over the first 40\u00a0ms (TTI40) after torque development (vTTI40; a) and the sEMG amplitude 40\u00a0ms before the onset of torque development (RMS\u221240\u20130; b) obtained during 56\u00a0days of bed rest (BR). For each session, both vTTI40 and RMS\u221240\u20130 are expressed as a percentage of the corresponding values at the steady-state maximal voluntary torque (MVT) of that session\nIntrinsic contractile properties\nThe contractile properties obtained from electrically evoked contractions during bed rest are shown in Fig.\u00a04. The course of FOA during bed rest was significantly different for Ctrl and RVE subjects (P\u00a0<\u00a00.05). For Ctrl, the FOA increased substantially (by 47.5\u00a0\u00b1\u00a010.0%, P\u00a0<\u00a00.01) during the bed rest study (Fig.\u00a04a). In contrast, no change in the FOA was observed for RVE. The groups also differed in their response to bed rest with respect to contraction time as indicated by TPT300 (P\u00a0<\u00a00.01). TPT300 declined significantly by 6.8\u00a0\u00b1\u00a00.9% (P\u00a0<\u00a00.001) for Ctrl, whereas no changes were observed for RVE (Fig.\u00a04b). Furthermore, in the Ctrl group the HRT150 tended to be somewhat shortened by 2.5\u00a0\u00b1\u00a01.1% (P\u00a0=\u00a00.056) after 56\u00a0days of bed rest, whereas for RVE it again remained unaltered (Fig.\u00a04c). Group differences in HRT150 did, however, not reach significance. Interestingly, the T10\/T150 ratio increased for both Ctrl (by 15.0\u00a0\u00b1\u00a05.5%; P\u00a0<\u00a00.05) and RVE (by 40.6\u00a0\u00b1\u00a017.1%; P\u00a0<\u00a00.05) and these changes were not significantly different between groups (Fig.\u00a04d).\nFig.\u00a04Mean values (\u00b1SE) of the force oscillation amplitude (FOA; a) time to peak tension at 300\u00a0Hz stimulation (TPT300; b), half relaxation time at 150\u00a0Hz stimulation (HRT150), and peak torque at 10\u00a0Hz stimulation expressed as a fraction of the maximal torque obtained during tetanic stimulation at 150\u00a0Hz (T10\/T150) obtained during 56\u00a0days of bed rest (BR)\nDiscussion\nContractile characteristics of electrically evoked knee extensions\nThe rate at which muscle force develops during a fast voluntary contraction is influenced by both peripheral factors and neural activation properties. In order to separate these influences, the contractile response of the knee extensor group was also assessed by means of percutaneous sub-maximal muscle stimulation. This is a reliable method, which has been used to assess intrinsic muscle characteristics in various human populations (Binder-Macleod et al. 1995; de Haan et al. 2000; Gerrits et al. 2001; Harridge et al. 1996). Although it is not possible to assess the specific characteristics of the portion of the muscle fibre population that is recruited during the sub-maximal stimulation procedure of the present study, the activation of about 40% of the muscle fibres suffices to represent the global contractile characteristics of the quadriceps muscle. Previous studies have shown that the torque frequency relationship, as well as the contractile properties of the twitch are not intrinsically influenced by the absolute force level, provided that a force between 20 and 50% MVC is reached during maximal tetanic stimulation (Binder-Macleod et al. 1995).\nIn the absence of the exercise countermeasure, the knee extensors exhibited characteristics of a faster muscle following 56\u00a0days of bed rest. The degree of fusion at 10\u00a0Hz stimulation was decreased, the time to reach peak torque at 300\u00a0Hz stimulation was reduced and relaxation after tetanic stimulation at 150\u00a0Hz tended to be faster. The possibility of an error seems unlikely, since we found complementary changes towards enhanced contractile speed at all frequencies studied. In addition, these changes were observed only for the inactive control group.\nThe stiffness of the series elastic component is amongst the factors known to affect the rate of torque development (Bojsen-Moller et al. 2005). Although not measured in the present study, from previous other studies it appears that with muscle unloading tendon stiffness often decreases (Kubo et al. 2000; Reeves et al. 2005). This would tend to result in a reduced rate of torque development of the whole muscle\u2013tendon complex, opposite to what we observed. The increased rate of torque development, as well as the tendency towards a faster rate of relaxation in the present study could, however, be explained by an elevated rate of cross-bridge cycling (Unsworth et al. 1982). Although at odds with some previous findings (e.g., Davies et al. 1987; Gondin et al. 2004; Narici et al. 2003) such interpretation would be consistent with documented elevations in maximal unloaded shortening velocity (Caiozzo et al. 1994; Widrick et al. 2001; Yamashita-Goto et al. 2001) potentially linked to shifts in muscle fibre phenotype from slow to fast as a direct consequence of muscle unloading (Fitts et al. 2000; Gerrits et al. 2003; Ohira et al. 1999; Talmadge 2000; Trappe et al. 2004). Because the assessed muscle torque in the present study is the resultant of the entire muscle\u2013tendon complex, and given a likely increase in tendon compliance by bed rest, the observed enhanced contractile speed characteristics, might even underestimate the underlying intrinsic changes in the present study.\nDespite faster contractile properties, torque production at 10\u00a0Hz stimulation relative to maximal tetanic stimulation was increased after bed rest. Although consistent with previous research (Seki et al. 2001), the higher relative torque responses at low frequency stimulation did not result from a significant enhancement of twitch summation, as previously opted (Gondin et al. 2004). In contrast, the level of force fusion substantially decreased during bed rest period in the present study. At present, the exact processes responsible for these anomalous findings remain unclear, but a similar phenomenon was reported in the paralyzed muscles of individuals with spinal cord injury (Gerrits et al. 1999), which may be considered as an extreme model for muscle unloading.\nIn part, our data supports the hypothesis postulated by Rittweger et al. (2006) that the large number of contraction\u2013relaxation cycles during resistive vibration exercise (Blottner et al. 2006) may be effective in preserving muscle fibre contractile properties. Indeed, no changes in time to reach peak torque at 300\u00a0Hz stimulation, the rate of relaxation after tetanic stimulation at 150\u00a0Hz, or the level of force fusion at low stimulation frequency (i.e., the FOA) were observed in the exercise trained subjects. The significant difference between some baseline values, e.g., FOA and T10\/T150 ratio (Fig.\u00a04a, d), and the tendencies for TPT300 (P\u00a0=\u00a00.098) and HRT150 (P\u00a0=\u00a00.057) to be lower in RVE compared to Ctrl at baseline deserves attention, since it may point towards a difference between groups with respect to muscle fibre type at the start of the study, with RVE exhibiting a faster muscle. Nonetheless, at least for the FOA it has been demonstrated that it is still much higher (i.e., 0.65 in Gerrits et al. 1999) in paralyzed muscles of people with spinal cord injury. This makes it unlikely that the preservation of FOA in the present study resulted from a ceiling effect for RVE.\nDespite the observation that speed characteristics were unaltered for the exercise-trained group, the relative peak torque at low stimulation frequency also increased for this group. As the level of force fusion remained unaltered during bed rest for RVE, other factors are likely involved. Once possibility is that the peak torque during 10\u00a0Hz stimulation increased as a consequence of an enlarged twitch response. Although not directly measured in the present study, the torque developed during the first response of the 10\u00a0Hz tetanus increased during the course of the bed rest by about 26%, which was comparable to the elevation in 10\u00a0Hz peak torque (\u223c40%) for RVE. Interestingly, the relative torque production of the first response of the 10\u00a0Hz contraction also increased (by about 30%) for the inactive control group. However, the reduced fusion of successive individual twitches diminished the increase in peak torque at 10\u00a0Hz stimulation to about 15%. Despite the differences in contractile speed characteristics, the percent change in 10\u00a0Hz peak torque after 56\u00a0days of bed rest was not different between groups. At present, the selectivity of the exercise countermeasure paradigm to prevent changes in contractile speed properties, but not in the torque response at low frequencies of stimulation, remains difficult to explain. Indeed, more research is needed to determine the effectiveness of resistive vibration exercise as a countermeasure, since the individual merits of resistance training versus vibration training could not be quantified in the present study.\nFast voluntary isometric knee extensions at maximal effort\nAdequate preservation of the rate at which muscle torque develops during a forceful volitional contraction is imperative for astronauts, as neuromuscular deconditioning, coupled to a weakened load-bearing skeleton, increases the risk of fall-related bone fractures after prolonged space missions. To add to the concern, compared to steady-state contractions, much higher levels of neural activation are needed for contractions where torque develops as rapidly as possible (de Haan 1998; de Ruiter et al. 1999). As such, we hypothesised that the ability to perform fast and forceful voluntary contractions would be more deteriorated by bed rest confinement than the ability to perform maximal steady-state contractions. Surprisingly, and at odds with the finding of others (di Prampero and Narici 2003; Koryak 1998), we found no evidence for such bed rest induced functional impairment in either group (Fig.\u00a03).\nFor the same inactive control subjects as used in the present study, we previously reported an absence of neural deconditioning for maximal voluntary steady-state contractions, whether examined by the twitch interpolation technique, or by the assessment of electromyographic activity of the quadriceps femoris muscle (Mulder et al. 2006, 2007). We concluded that the preservation of neural activation of this specific motor task was likely associated with the repeated functional retesting sequence employed during bed rest. The observation that the percent reduction in maximal isometric knee extension strength of the left leg, which was not repeatedly retested during bed rest, exceeded the level of atrophy by a factor of two after 8\u00a0weeks of bed rest, strengthened this notion (Mulder et al. 2006). Based on the findings of the present study, we are inclined to suggest that the repeated retesting regime also served as a contributory factor in maintaining neural activation during fast isometric knee extensions. Although such an effect was not intended at the time, this supposition is important since it suggests that neural activation of different isometric motor tasks can be maintained during bed rest without rigorous exercise training regimes.\nIn part, the absence of loss of muscle functionality following bed rest might have resulted from changes in the contractile properties of unloaded muscles. Widrick et al. (1998) reported significant atrophy of single human soleus muscle fibres after 17\u00a0days of spaceflight. Absolute peak power of these fibres was, however, partly or fully preserved by an elevated contraction velocity. In the present study, the examined muscle group in the inactive control group also acquired mechanical characteristics of a faster muscle during the course of the bed rest. As selective neural deconditioning could not be demonstrated for the fast voluntary contractions, the expectation might arise of an increased rate of voluntary torque development for the inactive control group. Such a systematic change was not observed. Although it seems difficult to explain this absence based on the changes in intrinsic contractile characteristics, it is clear that neural activation and the subsequent mechanical response during voluntary contractions are much more variable than the activation and response when contractions are electrically evoked. To overcome this variability, larger alterations than those observed in intrinsic contractile characteristics would have been required to allow for detectable changes for the fast voluntary isometric actions.\nInterestingly, an increase in the rate of torque development could have also been expected for the exercise trained RVE group. For this group we previously found that the amplitude of the surface EMG during the maximal steady-state contractions was substantially increased at the end of bed rest (by \u223c30%; Mulder et al. 2007). The latter was suggested to result from an increase in the mean motor unit firing rate, due to a change in the excitability of the alpha motoneurons (Mulder et al. 2007). Such modulation has been associated with increased rate of force development after resistance training (Holtermann et al. 2007). However, in the present study the neural activation during the fast voluntary contractions and the subsequent initial rate of torque development remained unaltered for the exercise trained group. The current exercise-training regime consisted mainly of relatively slow dynamic loaded contractions while vibration was simultaneously applied to the feet. Such a motor task is quite different from the isometric contractions performed during the testing of the subjects. Based on the selectivity of training, and the fact that the number of exercise training sessions by far outweighed the number of testing sessions (89 vs. 7), it is likely that neural activation strategies employed by the subjects were more guided towards optimal performance during the training sessions, than optimal performance during the fast voluntary isometric actions during testing.\nIn conclusion, in the subjects who were confined to 8\u00a0weeks of bed rest without preventive measures the knee extensor muscle group acquired intrinsic contractile properties of a faster muscle. Resistive vibration exercise proved effective to counteract these changes at the muscle level. An unexpected finding of the present study was that neither group showed deterioration in the capacity to maximally activate the knee extensors at the very start of a voluntary contraction performed as fast and forcefully as possible. For the RVE group this might indicate an effective countermeasure design. However, considering that neural activation and voluntary muscle function were also maintained in the Ctrl group, it is also conceivable that the multiple retesting of the subjects resulted in or at least contributed to these preservations.","keyphrases":["force oscillation amplitude","emg","unloading","maximal rate of torque rise","time course"],"prmu":["P","P","P","M","R"]}
{"id":"Cancer_Immunol_Immunother-4-1-2335290","title":"Local therapy of cancer with free IL-2\n","text":"This is a position paper about the therapeutic effects of locally applied free IL-2 in the treatment of cancer. Local therapy: IL-2 therapy of cancer was originally introduced as a systemic therapy. This therapy led to about 20% objective responses. Systemic therapy however was very toxic due to the vascular leakage syndrome. Nevertheless, this treatment was a break-through in cancer immunotherapy and stimulated some interesting questions: Supposing that the mechanism of IL-2 treatment is both proliferation and tumoricidal activity of the tumor infiltrating cells, then locally applied IL-2 should result in a much higher local IL-2 concentration than systemic IL-2 application. Consequently a greater beneficial effect could be expected after local IL-2 application (peritumoral = juxtatumoral, intratumoral, intra-arterial, intracavitary, or intratracheal = inhalation). Free IL-2: Many groups have tried to prepare a more effective IL-2 formulation than free IL-2. Examples are slow release systems, insertion of the IL-2 gene into a tumor cell causing prolonged IL-2 release. However, logistically free IL-2 is much easier to apply; hence we concentrated in this review and in most of our experiments on the use of free IL-2. Local therapy with free IL-2 may be effective against transplanted tumors in experimental animals, and against various spontaneous carcinomas, sarcomas, and melanoma in veterinary and human cancer patients. It may induce rejection of very large, metastasized tumor loads, for instance advanced clinical tumors. The effects of even a single IL-2 application may be impressive. Not each tumor or tumor type is sensitive to local IL-2 application. For instance transplanted EL4 lymphoma or TLX9 lymphoma were not sensitive in our hands. Also the extent of sensitivity differs: In Bovine Ocular Squamous Cell Carcinoma (BOSCC) often a complete regression is obtained, whereas with the Bovine Vulval Papilloma and Carcinoma Complex (BVPCC) mainly stable disease is attained. Analysis of the results of local IL-2 therapy in 288 cases of cancer in human patients shows that there were 27% Complete Regressions (CR), 23% Partial Regressions (PR), 18% Stable Disease (SD), and 32% Progressive Disease (PD). In all tumors analyzed, local IL-2 therapy was more effective than systemic IL-2 treatment. Intratumoral IL-2 applications are more effective than peritumoral application or application at a distant site. Tumor regression induced by intratumoral IL-2 application may be a fast process (requiring about a week) in the case of a highly vascular tumor since IL-2 induces vascular leakage\/edema and consequently massive tumor necrosis. The latter then stimulates an immune response. In less vascular tumors or less vascular tumor sites, regression may require 9\u201320 months; this regression is mainly caused by a cytotoxic leukocyte reaction. Hence the disadvantageous vascular leakage syndrome complicating systemic treatment is however advantageous in local treatment, since local edema may initiate tumor necrosis. Thus the therapeutic effect of local IL-2 treatment is not primarily based on tumor immunity, but tumor immunity seems to be useful as a secondary component of the IL-2 induced local processes. If local IL-2 is combined with surgery, radiotherapy or local chemotherapy the therapeutic effect is usually greater than with either therapy alone. Hence local free IL-2 application can be recommended as an addition to standard treatment protocols. Local treatment with free IL-2 is straightforward and can readily be applied even during surgical interventions. Local IL-2 treatment is usually without serious side effects and besides minor complaints it is generally well supported. Only small quantities of IL-2 are required. Hence the therapy is relatively cheap. A single IL-2 application of 4.5 million U IL-2 costs about 70 Euros. Thus combined local treatment may offer an alternative in those circumstances when more expensive forms of treatment are not available, for instance in resource poor countries.\nSystemic IL-2 therapy of cancer\nRosenberg and co-workers were the first to treat cancer with IL-2. They showed that IL-2 renders white blood cells cytotoxic in vitro. These cells were coined Lymphokine Activated Killer cells (LAK cells). Rosenberg and co-workers treated transplanted lung metastases in mice. Injection of IL-2 plus LAK cells clearly reduced the number of metastases in the lung [82]. Next these authors treated 25 consecutive patients with different types of advanced cancer for whom no effective treatment was available. In 11 patients, objective responses were obtained after treatment with IL-2 and LAK cells [81]. Obviously this paper attracted worldwide attention, as it was a breakthrough in immunological treatment of cancer. In further studies, systemic application of IL-2 with or without LAK cells appeared useful in patients with metastasized renal cell carcinoma and metastasized melanoma. In both conditions about 20% objective responses were obtained; that is about 6% complete and about 14% partial tumor regressions [57]. Repeated cycles of systemic IL-2 administration were required to achieve systemic tumor-inhibitory effects. Grande et al. [46] recently reviewed the therapeutic effects of systemic IL-2 therapy.\nSystemic IL-2 application required bolus injections of IL-2 given every 8\u00a0h at a dose of 105\u00a0IU\/kg body weight for at least 5\u00a0days. These high doses were very toxic, as systemic IL-2 therapy induces a generalized vascular leakage syndrome [7, 83]. In addition the preparation and application of LAK cells was cumbersome indeed. Supposing that the tumor infiltrating leucocytes have to be stimulated by IL-2, we and other groups decided to focus on local IL-2 therapy, that is IL-2 application at the site of the tumor (peritumoral\u00a0=\u00a0juxtatumoral, intratumoral, intra-arterial, intracavitary infusion, or inhalation). Since local IL-2 application requires smaller doses of IL-2 than systemic treatment, less complications were expected. Locally applied relatively small doses of IL-2 leads to much higher IL-2 concentrations at the site of the tumor and to much lower concentrations elsewhere in the body.\nConclusion\nThe development of systemic IL-2 therapy was a break-through in cancer immunotherapy. We supposed that the drawbacks of systemic IL-2 application might be overcome by local IL-2 application.\nEffectiveness of local versus systemic IL-2 therapy\nSubject\nThe therapeutic effectiveness of local versus systemic IL-2 application.\nData\nSilagi et al. [86] studied mice with melanoma or sarcoma using combined cyclosphosphamide and IL-2. Cyclophosphamide was always applied systemically. Cyclophosphamide alone had no therapeutic effect but combined treatment of cyclophosphamide and IL-2 showed a synergistic effect. When either tumor was implanted s.c. at day 0, and IL-2 treatment was given at the site of the tumor beginning 1\u20133 days later, 87\u2013100% of the mice were cured. Only 35\u201350% were cured when IL-2 was administered i.p. Conversely, with i.p. treatment of i.p. tumors, 60\u201383% of the mice were tumor-free on day 50, as compared with only 17% with s.c.treatment.\nVaage [94] tested the therapeutic effects of 12 daily injections of 100\u2013300,000\u00a0U of human IL-2 against the syngeneic, immunogenic mammary carcinoma MC2 implanted s.c. into C3H\/He mice. A local therapeutic effect was observed after injecting tumors even with doses as low as 300\u00a0U of IL-2 per injection. Systemic IL-2 treatment however required at least 5,000\u00a0U per injection for obtaining discernable results.\nBelardelli et al. [10] treated mice with s.c. transplanted highly metastatic Friend leukemia, non-metastatic Friend leukemia, RBL-5 lymphoma, and HeJ16 fibrosarcoma. In all these tumor models, peritumoral injections of IL-2 were more effective in inhibiting tumor growth than systemic treatment.\nAnderson et al. [2] studied the therapeutic effects of IL-2 in C57BL\/6 mice with MCA-106 sarcoma pulmonary metastases. When mice were treated once daily with free IL-2 on days 5, 6, and 7 after tumor inoculation, the intrathoracic route was superior to the i.p. or s.c. routes.\nMaas et al. [68] treated DBA\/2 mice bearing i.p. and s.c. SL2 lymphoma. If IL-2 was injected i.p., then in 70% of the mice i.p. tumors regressed completely, and in 50% tumors regressed completely both i.p. and s.c. When mice had only a s.c. growing tumor and IL-2 was injected i.p., then in only 7% of the mice tumors regressed completely.\nDubinett et al. [33] transplanted s.c. Line 1 alveolar carcinoma cells in Balb\/c mice. On the third day following tumor implantation, mice received injections of IL-2 twice daily, either by i.p. or intratumoral injection, 5\u00a0days\/week for 3\u00a0weeks. Intratumoral injection of IL-2 significantly reduced the tumor volume, increased the median survival time, and resulted in a 23.5% cure rate. However no long-term survivors were among the i.p. treated mice.\nJacobs et al. [55] investigated human patients with nasopharyngeal carcinoma. Local IL-2 treatment was combined with standard irradiation therapy. Sixty three % of the patients showed a disease free survival during the next 5\u00a0years, whereas only 8% of the controls treated with irradiation alone were still disease free after 5\u00a0years. Interestingly, these results contrast with those of Chi et al. [23] who applied single modality systemic IL-2 therapy. They concluded that no response was observed. So, apparently, local IL-2 application is more effective than systemic application. It is important that these results of Jacobs et al. and Chi et al. were obtained in spontaneously occurring tumors, not with transplanted tumors.\nLocal IL-2 should even preferably be given intratumorally as IL-2 application adjacent to the tumor is far less effective. Jacobs et al. [56] compared the effect of peritumoral and intratumoral IL-2 therapy in mice with s.c. transplanted SL2 lymphoma. Intratumoral IL-2 was significantly more effective than peritumoral IL-2. Krastev et al. [61] treated 16 patients with various gastrointestinal tumors with intratumoral and\/or intraperitoneal IL-2 therapy. Six patients had a clinical response. All six belonged to the group of seven patients who received intratumoral therapy. No objective responses were obtained in patients treated only with intraperitoneal IL-2.\nIn an early review Bernsen et al. already concluded that locoregional IL-2 treatment was more effective than systemic treatment [13]. Since then an overwhelming amount of research has corroborated this conclusion.\nConclusion\nThe data show that local IL-2 therapy and in particular intratumoral IL-2 application is more effective than systemic IL-2 therapy.\nLocal IL-2 therapy leads to systemic therapeutic effects even curing metastatic disease\nSubject\nObviously a major problem of cancer therapy is inducing regression of metastases. Immunotherapy of cancer is an attractive concept, as systemic immunity may indeed cure metastases. Consequently, many groups have concentrated on the systemic therapeutic effects of local IL-2 therapy.\nData\nMaas et al. [67] have shown that i.p. injections of IL-2 can cure DBA\/2 mice with a large burden of i.p. transplanted and greatly disseminated SL2 lymphoma. This implied that tumor metastases can be successfully treated with local IL-2 therapy. In more detailed studies SL2 tumor cells were injected in mice both i.p. and s.c. on the flank resulting in i.p. and s.c. tumors [67]. About 50% of the mice treated i.p with IL-2 rejected both the i.p. tumor and the large distant s.c. tumor. In contrast, similar i.p. treatment cured only 7% of the mice bearing only a s.c. SL2 tumor. Thus, it was shown that IL-2 can induce systemic tumor rejection when injected at the site of tumor growth. This SL2 tumor rejection was specific, as mice that were rejecting i.p. and s.c. SL2 lymphoma did not reject P815 mastocytoma.\nVaage [95] tested the therapeutic effects of IL-2 against intramammary implants of an immunogenic, syngeneic C3H mammary carcinoma. Peritumoral injected IL-2 had almost equal local and systemic therapeutic effects, whereas systemically injected IL-2 was not therapeutically effective at all.\nImmunity also explains the long lasting absence (>54\u00a0months) of bladder carcinoma recurrences in two IL-2 treated patients who previously had a 7 and 11\u00a0years\u2019 history of recurrent bladder cancer [29].\nVan Es et al. [96] showed that peritumoral injections of IL-2 in a transplanted rabbit carcinoma model can induce complete regression of the treated tumors as well as untreated contralateral tumors in four out of 12 rabbits. Also metastases in the draining regional lymph nodes of both treated and untreated primary tumors regressed in three of these animals. So, local treatment of a tumor led to systemic effects curing untreated tumors at a distant site as well as metastases in the draining lymph nodes of both the treated and the untreated tumors. A second challenge with tumor cells of the cured animals was rejected.\nJacobs et al. [56] transplanted in mice tumors at two different sites. Rejection of the intratumoral IL-2 treated tumors was stronger than rejection of the untreated tumors.\nSystemic immunity is also likely in patients with nasopharyngeal carcinoma treated with radiotherapy and intratumoral IL-2 application [55]. Addition of IL-2 to the standard radiotherapy reduced the number of loco regional and distant recurrences.\nIn line with these observations is the finding that local or generalized effector dysfunction of the immune system can be reversed by IL-2 exposure in patients with advanced cancer [70].\nConclusion\nLocal IL-2 therapy can also cause systemic therapeutic effects, probably due to immune reactivity [68, 96].\nTherapeutic effects of local IL-2 therapy against transplanted tumors in laboratory animals (Tables\u00a01, 2)\nSubject\nFor studying the potential therapeutic effects of anticancer drugs one has to start in animal models using transplanted tumors, as well-performed and well-interpreted studies in animals have predictive value regarding the therapeutic effectiveness of a drug in human cancer patients [32].\nTable\u00a01Successful therapeutic effects of local IL-2 application on transplanted tumors: results published by other groupsTumorsSpeciesRoute of IL-2 applicationTherapeutic effectReferencesCarcinomasMammary carcinomasMousePeritumoral+[94, 95]Transitional cell carcinomaMouseIntratumoral+[87]Lung carcinomaMouseIntratumoral++[33]Prostate carcinomaRatIntratumoral+[50]SarcomasMC-induced sarcomasMousePeritumoral+[16\u201318]HeJ fibrosarcomaMousePeritumoral++[10]MCA106 sarcomaMousePeritumoral+[2]LymphomaRBL5 lymphomaMousePeritumoral+[10]MiscellaneousMyeloma X5563MouseLocal site++[69]MelanomaMouseTumor site++[86]Friend leukemiaMousePeritumoral+[10]HPV associated tumorMouseTumor site+[21]We have included data of all papers that we have found on therapeutic effects of local application of free IL-2 to transplanted cancer in animalsA well-recognized problem with a Table like this one is that usually positive results are published, in contrast to negative results. So, there is a positive publication bias.The message of Table\u00a01 is not that all transplanted tumors are sensitive, but that positive therapeutic effects have been obtained in many different models and many different tumor types+ Clear therapeutic effect++ Many cures were obtainedTable\u00a02Therapeutic effect of local IL-2 application on transplanted tumors: results from our groupTumorOrgan \/typeSpeciesPredictive modelRoute of applicationTherapeutic effectReferencesCarcinomasLine 10LiverGuinea pig+Intratumoral+[6, 72]M8013BreastMouse \u00a0Peritumoral+[58]MOTTeratomaMouse\u00a0Intratumoral-[14]MC-ASNBreastMouse+Peritumoral\u00b1[73]MC-BCBreastMouse+Peritumoral\u00b1[73]MC38ColonMouse+Peritumoral+[64]5D04StomachMouse\u00a0Intratumoral-\u00a0VX2 carcinomaHead and NeckRabbit+Peritumoral+[96]Recently arisen carcinomasX5BreastMouse\u00a0Intratumoral\u00b1[42]X6BreastMouse\u00a0Intratumoral\u00b1[42]X9BreastMouse\u00a0Intratumoral\u00b1[42]SarcomaMecho FibrosarcomaMouse\u00a0Intratumoral+\u00a0LymphomasSL2LymphomaMouse+Intratumoral++[12, 67, 68, 70]L5178YLymphomaMouse\u00a0Intratumoral++[67]L1210LymphomaMouse\u00a0Intratumoral+\u00a0RBL5LymphomaMouse\u00a0Intratumoral+\u00a0EL4LymphomaMouse\u00a0Intratumoral\u2212\u00a0MiscellaneousTLX9ThymomaMouse\u00a0Intratumoral\u2212\u00a0P815MastocytomaMouse+Intratumoral++[68]This Table presents all results obtained with models tested by our group; so there is no selection bias (in contrast to Table\u00a01)\u00b1 therapeutic effects just measurable, + therapeutic effects are clear; ++ animals can be cured from extensive tumor loadsThe four negative models were only tested in a few experiments, until we were convinced that therapeutic effects were not obtained in our hands. Three of the four negative models were not published for obvious reasons. On the other hand, SL2 lymphoma in syngeneic DBA\/2 mice is our standard model since the 1989 paper of Maas et al. [68]. Numerous studies were performed with this model. The therapeutic effect of local IL-2 application was always clear\nData\nAs early as in 1983, Bubenik and co-workers [18] established that peritumoral injections of rat lymphoid IL-2 suppressed or markedly inhibited the growth of methylcholanthrene-induced sarcomas in syngeneic mice. An equally effective inhibition of murine sarcoma transplants in syngeneic recipients could be obtained with crude lymphoid rat IL-2, with purified IL-2 of murine lymphoid origin, and with molecularly homogeneous human recombinant IL-2 [16\u201318].\nMaas et al. [67] injected DBA\/2 mice i.p. with SL2 cells and 10\u201314\u00a0days later these mice were treated with i.p. IL-2 injections. At the time of IL-2 injection the transplanted SL2 tumor had greatly expanded by growth, infiltration and metastasis. A mouse of 25 gram developed in 10\u00a0days a tumor load of at least 5\u00a0g, about 5\u00a0\u00d7\u00a0109\u00a0tumor cells. Nevertheless about 25% of these mice were cured by IL-2. This was a very important step forward in immunotherapy of cancer. The therapeutic effects were dose-dependent. These data were confirmed in more elaborated studies by Bernsen et al. [12] and by Everse et al. [37].\nTables\u00a01 and 2 summarize the therapeutic results of local IL-2 application to transplanted tumors. Table\u00a01 displays the results obtained by other groups and Table\u00a02 results by our group. Both Tables show that local IL-2 therapy may be effective in a broad range of tumor types such as carcinomas [6, 33, 42, 50, 58, 64, 72, 73, 87, 94, 96], sarcomas [10, 16\u201318, 86], a myeloma [69], lymphomas [10, 66, 67], leukemia [10], a mastocytoma [68], and HPV associated tumor [21].\nObviously not all cancers are sensitive to IL-2 therapy. Our group studied the effect of local IL-2 therapy in 19 models of transplanted tumors (Table\u00a02). In 15 models positive therapeutic results were obtained. IL-2 applied in breast cancers was only moderately effective. In four models no therapeutic effects were obtained, namely murine 5D04 stomach carcinoma [27], murine MOT teratoma [14], murine EL4 lymphoma [27] and murine TLX9 lymphoma [27]. Why some tumors are sensitive to local IL-2 therapy and other tumor models do not show any response, remains hitherto an enigma. The data summarized in Tables\u00a01 and 2 demonstrate that local IL-2 therapy has the capability to destroy tumor cells and to cure the hosts.\nAn animal model using transplanted tumors has predictive value for the therapeutic effect in human cancer if the model tumor comprises more than 1% of the body weight of the host and if this tumor is metastasized [32]. Eight models mentioned in Table\u00a02 fulfil these requirements, viz. the models marked in column 4 with +. So, the positive therapeutic results obtained with these models predict that local IL-2 therapy can be therapeutically effective in human patients with metastatic cancer.\nConclusion\nIn models with transplanted tumors in laboratory animals there is overwhelming evidence showing the therapeutic effect of local application of free IL-2. This therapy is effective against a broad range of tumors. In addition the magnitude of the therapeutic data (for instance [67]) suggests that this form of therapy can also induce objective therapeutic responses in human cancer patients.\nN.B. Not every transplanted tumor or tumor type is sensitive to local IL2 application. For instance transplanted EL4 lymphoma or TLX9 lymphoma are not sensitive in our hands (Table\u00a02).\nLocal IL-2 tumor treatment in veterinary patients (Table\u00a03)\nSubject\nPositive results in veterinary patients with spontaneous cancer are an important intermediate between experiments with transplanted cancer in laboratory animals and clinical application in human cancer cases. If therapeutic effects are positive in well-performed, well-interpreted experiments with transplanted cancer in laboratory animals as well as in spontaneous cancer in veterinary patients, then one can be almost certain that such a therapy will also be effective in human cancer patients.\nTable\u00a03Therapeutic effect of local IL-2 application veterinary cancer patientsType of cancerHostTherapeutic effectReferencesBovine ocular squamous cell carcinomaCattle++[30, 31, 84, 89, 90]Vulval papilloma and carcinoma complexCattle+[51]SarcoidsHorses+[88]FibrosarcomaDogs+[100]This Table summarizes to our knowledge all published studies on local application of free IL-2 in veterinary cancer patients+ Detectable effect++ Many CR cases\nData\nTable\u00a03 summarizes all published studies that we know on local IL-2 application in veterinary cancer patients.\nBovine Ocular Squamous Cell Carcinoma (BOSCC)\nBOSCC originates in the cornea, the third eyelid (membrana nictitans), the lower or the upper eyelid. Ultimately the tumor covers the whole eye. It also metastasizes to the draining lymph nodes. This tumor occurs frequently in tropical countries with intense solar radiation, particularly at high altitudes. BOSCC is for various reasons a very useful veterinary tumor model: The tumor is readily visible and can be directly treated with peritumoral or intratumoral IL-2 injection in field studies.\nIn The Netherlands BOSCC is a very rare disease. So our first tests of IL-2 sensitivity of BOSCC were performed in only five cows with BOSCC [84]. The results showed that BOSCC can be sensitive to local IL-2 therapy. In Zimbabwe about 10% of the cows are affected by BOSCC. This allows large-scale studies [31]. Added to this BOSCC causes an enormous economical burden. In our most extensive study in Zimbabwe [86] we treated 174 BOSCC cases with tumor areas ranging from 20 to 2,800\u00a0mm2. Peritumoral injections of various doses of IL-2 were applied during 2\u00a0\u00d7\u00a05\u00a0days. Nine months after treatment, the daily doses of 5\u00a0\u00d7\u00a0103, 2\u00a0\u00d7\u00a0104, 2\u00a0\u00d7\u00a0105, 5\u00a0\u00d7\u00a0105, 1\u00a0\u00d7\u00a0106, 2\u00a0\u00d7\u00a0106 U IL-2 had induced complete tumor regression in 82, 81, 56, 15, 44, and 35% of the animals, respectively. In the control animals the tumors had completely regressed in only 14% of the cases. After 20\u00a0months the comparable figures were 55, 52, 58, 50, 69 and 52%, respectively, and there had been no change in the control group. The tumors on the third eyelid and limbus were the most responsive [89]. Similar results were obtained in other studies [30, 90].\nEven large BOSCC tumors of up to 66\u00a0mm can regress completely by local IL-2 therapy [31].\nBovine Vulval Papilloma and Carcinoma Complex (BVPCC)\nBVPCC is a common disease in Bos taurus breeds of cattle kept at high altitude with high levels of solar radiation in Africa [19, 51]. It also occurs in other countries and continents with similar high levels of solar radiation. Burdin [19] originally described the pathogenesis of this neoplasm. Hill et al. [51] adapted this description to develop a useful system for clinical staging of the tumors. In Zimbabwe BVPCC occurs in about 10% of the cattle. It causes much animal suffering. In addition BVPCC usually proceeds to a more advanced stage. So BVPCC forms a real economical burden for the farmer and on a national scale.\nTwenty three papillomas and carcinomas of the bovine vulva were treated with local IL-2 therapy. Sixteen partial remissions and three complete remissions add to a tumor reduction in 83% of the treated cows [Stewart et al. to be published]. Remissions were striking in papillomas with a massive lymphocytic infiltrate in particular in those epithelial areas that showed marked dysplasia or (pre-)malignant changes.\nSarcoids\nSarcoids are fibro-epithelial skin tumors of horses, donkeys, and mules. Infiltrative growth is prominent but they seldom metastasize. After surgical removal they usually recur. Sarcoids were treated by intratumoral IL-2 injections for 5 or 10\u00a0days. There were 36 and 50% objective responses, respectively after 12\u00a0months [88].\nFibrosarcomas in dogs\nTumors often occur in cats and dogs as owners care for pets even into advanced age as members of the family. Like ageing humans they show a large variety of tumors in their later years. Preliminary data demonstrated that fibrosarcomas in dogs are sensitive to local IL-2 therapy [100].\nConclusion\nLocal IL-2 therapy can be effective against spontaneous veterinary tumors. Local IL-2 application has an enormous economical impact as BOSCC as well as BVPCC occur in about 10% of cattle in Zimbabwe and probably also in other tropical countries. Local IL-2 treatment of BOSCC leads to CR in the majority of the cases, and led to tumor reduction in the majority of BVPCC cases.\nFor ethical reasons specific immunity cannot be tested in veterinary patients. However specificity and systemic effects have been shown in mice [68] and rabbits [96] with transplanted tumors. Effective local IL-2 treatment greatly improves the quality of life by reducing suffering. These therapeutic effects in veterinary cancer patients make a strong case for the development and acceptance of local IL-2 therapy with free IL-2 in human cancer patients.\nResults of local IL-2 tumor treatment in human cancer patients (Table\u00a04)\nSubject\nThe final step in the experimental chain is the local application of free IL-2 to human cancer. Many research groups have pioneered in this field. We now summarize and discuss the results.\nTable\u00a04Therapeutic effect of local IL-2 application on human cancer patientsType of cancerNumber of patientsCRPRSDPDReferencesBasal cell carcinoma12831\u2013[59]Bladder carcinoma633\u2013\u2013[79]Bladder carcinoma51\u20134\u2013[53]Bladder carcinoma143\u201311\u2013[44, 45]Bladder carcinoma99\u2013\u2013\u2013[41]Bladder carcinoma108\u20132\u2013[29]Gastro-intestinal cancer16\u20136\u201310[61]Hepatocellular carcinoma5\u2013131[85]Lung cancer; pleural effusions2176\u20138[71]Melanoma265858[35]Melanoma23155\u20133[80]Mesothelioma21\u20134710[43]Mesothelioma2211137[5]Mesothelioma31161014[22]Mesothelioma11\u2013\u2013\u2013[62]Ovarian carcinoma3563719[34]Ovarian carcinoma1763\u20138[91]Neoplastic effusions1446\u20134[65]Total numbers28878655392\u00a0Percentage (%)10027231832\u00a0CR complete regression, PR partial regression, SD stable disease, PD progressive disease\nData\nBasal cell carcinoma\nKaplan et al. [59] treated basal carcinoma of the skin. A total of 12 tumors were treated in eight patients. Overall response rates were : complete response in 8 of 12 treated tumors, partial response in 3 out of 12 treated tumors, stable disease with no improvement in one tumor site.\nBladder carcinoma\nPizza et al. [79] obtained tumor regressions after intralesional injections of IL-2 in bladder cancer. Repeated injections of IL-2 under cystoscopic control resulted in complete regression of the tumor in three out of six patients and in partial regression of another three patients.\nHuland and Huland [53] obtained histologically confirmed complete remission lasting more than 6\u00a0months in one out of five patients with urinary bladder carcinoma after continuous IL-2 perfusion of the bladder for 5\u00a0days.\nGomella et al. [44, 45] treated 14 patients with superficial bladder carcinoma. Patients were treated first with transurethral resection leaving a marker lesion, followed by intravesical IL-2 instillation. There were three complete responses, one lasting more than 9\u00a0months.\nFerlazzo et al. [40, 41] treated superficial bladder cancer cases with intravesical infusions of IL-2. This gave similar clinical results as obtained by vesical instillation with BCG after transurethral resection.\nDen Otter et al. [26, 29] treated patients with recurrent bladder carcinoma stage T1, grades 1 to 2 with incomplete transurethral resection leaving a marker tumor of 0.5\u20131.0\u00a0cm. Two days after resection IL-2 was instilled for 2\u00a0h. Patients were asked to turn over regularly in order to ensure maximal exposure of IL-2 to the bladder wall. This procedure was repeated on five consecutive days. Two months later the effect was measured by cystoscopy examination. In eight out of ten patients the marker tumor had regressed completely. Four patients were still tumor free after 30 to 54\u00a0months. In one patient with a 7-years\u2019 history of bladder cancer requiring 23 cystoscopies, the marker had only partially regressed after 2\u00a0months. After removal of the remainder of the marker this patient was tumor free during the follow-up of 54\u00a0months. Also, a patient with an 11\u00a0years\u2019 history of recurrent bladder cancer remained tumor free during the whole follow-up period [29]. The finding that these patients remained tumor free for >54\u00a0months suggests that these patients were (locally) immune to the tumor after tumor regression.\nGrasso et al. [47] treated 27 patients with transitional bladder carcinoma Ta\/T1-G1\u20132 with intravesical instillations of IL-2 for 1\u00a0h during 5\u00a0days. After 2\u00a0months none of the lesions had disappeared or were clearly reduced. This result seems to contrast with the findings by Den Otter et al. [26, 29]. The different treatment protocols may be essential: Den Otter et al. started with an incomplete TUR followed by IL-2 instillations, whereas Grasso et al. did not perform a TUR before IL-2 instillation. TUR causes tumor cell damage, which may induce tumor immunity. Interestingly, Grasso et al. obtained 33.3% relapses after a median follow-up of 12\u00a0months; this contrasted to the restrospective analysis in which the historical recurrence rate per year was 95%.\nGastrointestinal tumors\nShirai et al. [85] treated five patients with hepatocellular carcinoma with intratumoral IL-2 injections. In two patients 32 and 57% tumor regression was observed.\nKrastev et al. [61] treated patients with different forms of stage III and IV gastrointestinal malignancies (primary or metastatic) for whom no further treatment options were available. With locoregionally applied IL-2 a modest but clinically worthwhile improvement was obtained in six out of 16 patients; remarkably these six all belonged to the group of seven patients that were treated with intratumoral IL-2.\nMelanoma\nRadny et al. [80] treated patients with skin and soft-tissue melanoma metastases with intralesional injection of IL-2. A total of 24 patients with AJCC stage III or IV melanoma and single or multiple skin and soft-tissue metastases were included. IL-2 was administered intralesionally into all cutaneous and soft-tissue metastases accessible from the skin, 2\u20133 times weekly, over 1\u201357\u00a0weeks. Response evaluation was confined to the intralesionally treated tumors. CR of the treated metastases was achieved in 15 patients, the longest remission lasting 38\u00a0months to date. In five patients a PR was achieved and in a further three PD (one patient was not assessable). A total of 245 metastases were treated. There was CR in 209 (85%), and PR in 21 (6%). The therapy was generally well tolerated; the observed adverse events were mainly of grade 1\u20132 severity.\nPfohler and coworkers [78] treated two patients with multiple cutaneous metastases of malignant melanoma with intra and perilesional application of interleukin-2 and achieved complete regression of these metastases.\nMesothelioma\nGoey et al. [43] treated patients with pleural mesothelioma stage I-IIA with continuous daily intrapleural infusion of IL-2. PR occurred in four out of 21 evaluable patients with a median time to progression of 12 months (range 5\u201337). SD occurred in seven patients with a median time to progression of 5\u00a0months (range 2\u20137). There were no CRs. The median overall survival time was 15.6\u00a0months (range 3.0\u201343).\nAstoul et al. [5] treated 22 patients with malignant pleural mesothelioma. The response rate was evaluated 36\u00a0days after treatment. There were one CR, 11 PR, three SD, and seven PD. The median survival time of responders differed significantly from that of the non-responders (28 versus 8\u00a0months).\nCastagneto et al. [22] treated 31 consecutive patients with unresectable malignant pleural mesothelioma with pleural effusion with intrapleural instillation of IL-2. In 90% of the patients there was no further or minimal asymptomatic pleural fluid collection. Median overall survival was 15\u00a0months whereas the expected survival range of patients with involvement of the visceral pleura is 9\u201312\u00a0months.\nKrastev et al. [62] treated a patient with a large abdominal mesothelioma with intratumoral IL-2 injections and IL-2 instillation in the peritoneal cavity. The tumor regressed completely; the patient was cured and is still healthy and working 6\u00a0years after publication.\nNeoplastic effusions\nMasotti et al. [71] treated neoplastic pleural effusions in 21 patients with non-small cell lung cancer with intrapleural administration of IL-2. CR was obtained in seven patients and PR in six patients.\nLissoni et al. [65] treated 14 patients with neoplastic effusions from a variety of solid tumors. There were four CR and six PR with a median duration of 4\u00a0months.\nCastagneto et al. [22] treated pleural effusions of mesothelioma as described in the previous section.\nOvarian carcinoma\nEdwards et al. [34] treated patients with ovarian carcinoma with infusions of IL-2. Eligibility criteria included six or more courses of prior platinum-based chemotherapy and laparotomy-confirmed persistent or recurrent ovarian cancer. Among 35 assessable patients, there were six laparotomy-confirmed CRs and three PRs. The median survival time of the cohort was 13.7\u00a0months and the overall 5-year survival probability was 13.9%. For the nine patients who demonstrated responses, the median survival time had not been reached at the time of publication (range 27 to 90+ months).\nTaylor et al. [91] treated patients with advanced ovarian cancer with intraperitoneal IL-2. Nine out of 17 patients showed an objective response.\nRenal cell carcinoma (lung metastases)\nHuland et al. [52] introduced IL-2 inhalation therapy for lung metastases of renal cell carcinoma. Progressive pulmonary metastases responded dramatically in 15% of the patients for a median of 15.5\u00a0months and were stabilized in 55% of patients for a median of 6.6\u00a0months. The overall median response duration was 9.6\u00a0months. Median survival was 11.8\u00a0months; expected survival according to risk analysis was 5.3\u00a0months [52].\nThe PortugeseSpanish Inhaled IL-2 Group [36] studied the effect of inhaled IL-2 on pulmonary metastases of renal cell carcinomas. They found 13.7% Objective Responses (OR), a median progression free survival of 8.6\u00a0months and an overall survival of 23\u00a0months.\nQuantification of therapeutic data\nTherapeutic data of Table\u00a04 were further analyzed regarding CR, PR, SD, and PD. This analysis was possible in 288 cancer patients treated with locally applied IL-2. There were 27% CR, 23% PR, 18% SD, and 32% PD. These data may be too optimistic as positive results are published more frequently than negative results.\nToxic effect of local IL-2 application\nTable\u00a05 shows the toxic effect of local IL-2 application to cancer patients. There were 24 papers with an abstract mentioning toxic side effects. In 20 papers there were no or minor side effects, one paper [41] reports some side effects, and three papers [22, 34, 43] mention more serious side effects. The latter papers have in common that very high IL-2 doses (see Table\u00a05) were used for intrapleural, intraperitoneal or subcutaneous administration. In essence these massive doses act in a pseudo-systemic manner. Much of these massive doses of IL-2 will be absorbed and as a consequence will give generalized effects similar to systemic treatment. Hence, toxicity mentioned in the latter three papers form the exception to the rule that local IL-2 application causes no or minimal side effects. In reference [43] side effects are due to the very high daily doses of 36\u00a0\u00d7\u00a0106\u00a0IU IL-2, as this was a Phase I-IIA study with escalating doses of IL-2. In this case IL-2 treatment may give systemic toxicity complications. In paper [22] patients were treated with repeated intrapleural instillations with 9\u00a0\u00d7\u00a0106 IU twice weekly for 4\u00a0weeks; in addition, in non-progressing patients 3\u00a0\u00d7\u00a0106 IU IL-2 were administered s.c. thrice weekly for up to 6\u00a0months. Obviously such high intrapleural doses are readily resorbed and reach the general circulation. Hence the toxicity (grade 3 fever and grade 3 cardiac toxicity) in 7\/31 patients is not surprising. In paper [34] dose-limiting toxicity was seen in patients treated with 7-days\u2019 infusions with the maximum tolerated dose; obviously there was toxicity per definition.\nTable\u00a05Toxic effects of local IL-2 application on human cancer patientsReference Toxic side effects[59]Local pain, swelling, erythema, in one patient fluelike symptoms[79]No early or late adverse clinical side effects[53]No evidence of side effects[44, 45]Toxicity from IL-2 given intravesically was minimal. One patient malaise for 24\u00a0h after each treatment. Two patients developed asymptomatic lower urinary tract infections[41]2\/9 patients had hematuria after the end of treatment; one patient had fever (grade I), and seven patients had hypotension (grade I-II)[29]No toxic effects[61]Negligible adverse effects[85]Abstract did not contain toxicity data[71]Treatment of pleural effusions of lung cancer was well tolerated[35]Side effects of treatment were minimal[80]Therapy was generally well tolerated; the observed adverse events were mainly of grade 1\u20132 severity[43]Intrapleural administration of IL-2 was associated with acceptable toxicity. Dose limiting toxicity was observed at 36\u00a0\u00d7\u00a0106 IU daily, and consisted of catheter infection, fever and flue-like symptoms.[5]Intrapleural administration of IL-2 was well tolerated[22]Patients with malignant pleural mesothelioma received intrapleurally 9\u00a0\u00d7\u00a0106 U IL-2 twice weekly for 4\u00a0weeks. In non progressing patients 3\u00a0\u00d7\u00a0106 U IL-2 were subcutaneously administered thrice weekly for up to 6\u00a0months. Toxicity (WHO criteria) with intrapleural IL-2 consisted of grade 3 fever in 6\/31 patients and cardiac toxicity (failure) grade 3 in one patient.[62]Abstract did not contain toxicity data[34]Significant locoregional dose-limiting toxicity was seen with 7\u00a0day infusionsb (including bowel perforation) with 6\u00a0\u00d7\u00a0105 IU\/m2\/day as the maximum tolerated dose, but catheter infection was the only significant complication seen with 24\u00a0hrs infusions[91]Abstract did not contain cytotoxicity data[65]No important toxicity in 14 patients with neoplastic effusions after intracavitary administration of IL-2[36]aInhaled IL-2: most common toxicities were cough (40% of cycles) and fatigue (7%). Grade 1 or 2 toxicities [40]aLocoregional perfusion of the bladder with IL-2 is safe[52]a116 patients. Toxicity associated with exclusive inhalation of IL-2 was local and consisted mainly of cough[93]aIntravesical IL-2 instillation. No evidence of toxicity[97]aRegionally administered IL-2 was well tolerated in patients with advanced squamous cell carcinoma of the head and neckToxicity as described in the abstracts of the papers mentioned in Table\u00a04aToxicity data from Abstracts that did not contain information for Table\u00a04bAlternating continuous 7\u00a0day infusions followed by 7-day intervals\nDose\u2013response\nFigure\u00a01 shows the total applied doses of IL-2 and the percentage of objective responses. Results suggest that (a) good therapeutic results can be obtained with low doses of IL-2 (103 to 105\u00a0IU); (b) that there is no dose-response effect in the range of total dose of 106 up to 109\u00a0IU IL-2. Of course, in this figure there are only a few data for different tumors treated slightly different by different groups. More detailed analysis with homogeneous groups of mice with transplanted SL2 lymphoma in DBA\/2 mice showed a dose response effect in the dose range of 5,000\u2013200,000\u00a0IU IL-2 given on five consecutive days [12]. It is not surprising that this was not reproduced with the very heterogeneous data in Fig.\u00a01. Nevertheless these data of Fig.\u00a01 are remarkable as total doses of 106\u2013109 IU of IL-2 can result in 20\u2013100% objective responses. We have analysed whether high and low percentages of objective responses are obtained with sensitive and non-sensitive tumor types, respectively. This seems not to be the case as bladder carcinoma, mesothelioma, and ovarian carcinoma were reported as sensitive (>60% OR) as well as non-sensitive (<60% OR) (Table\u00a04; Fig.\u00a01). Another hypothesis is offered by the work of Jacobs et al. [55]. He found that intratumoral IL-2 application is more effective than peritumoral IL-2 application. We therefore further analyzed whether the different therapeutic effects can be ascribed to differences in the localization of IL-2 application. Also this was not the case. Another possibility is that intratumoral IL-2 application leads to more intratumoral edema formation\/leakage causing hemodynamic stagnation and additional tumor necrosis. This may stimulate the antitumor response and consequently lead to superior therapeutic effects. Obviously this hypothesis is difficult to study in human cancer patients for logistical and ethical reasons. A more simple explanation of the absence of a dose-response effect is that different tumors have different sensitivity for local IL-2 therapy, e.g., due to different tumor associated antigenicity. Those that are well-responsive can be cured by lower doses, those that are less responsive are (automatically) treated with higher doses. Hence it is impossible to evaluate dose-response effects using different tumors.\nFig.\u00a01% Objective response versus total applied dose of IL-2. The data are derived from the abstracts of the papers mentioned in Table\u00a04\nConclusions\nLocal IL-2 therapy can be effective against a variety of human tumors.\nIn some studies results suggest that metastases are cured and that (systemic) immunity is obtained [29].\nThere is no obvious dose\u2013response with regard to therapeutic effects.\nGood therapeutic effects can be obtained with total doses of 103 to 105 IU IL-2; these doses cause no or negligible toxic effects. High total IL-2 doses of 108 IU and higher may exert toxic side effects as are described in systemic treatment.\nCombined therapy modalities (Table\u00a06)\nSubject\nCombination of various therapeutic modalities, like surgery, irradiation, chemotherapy, hormone therapy, etc., are standard in tumor treatment. For this reason several groups have also used local IL-2 therapy with free IL-2 combined with other treatment modalities.\nTable\u00a06Synergism of local IL-2 therapy with other therapeutic modalitiesTherapyTumorHostTherapeutic effectReferencesSurgery\u00a0FibrosarcomaDogCR for 12\u00a0months vs no CRa[100]Radiotherapy\u00a0LymphomaMouse93% vs. 17% disease free survival; P\u00a0<\u00a00.0001a[38, 58]\u00a0Nasopharyngeal carcinoma Man63% vs 8% disease free 5-years\u2019 survival a[55]Cytokine therapyInterleukin-1Friend leukemiaMouse Synergistic effect with IL-2, 60% survival[10]ChemotherapyCyclophosphamideMelanoma Mouse87\u201391 vs 67% curesa[86]Cis-platinumTerato carcinomaMouse20 vs 0% cures; 50% survival at ca 50 vs ca 25\u00a0daysa[14]DoxorubicinM109 adenocarcinomaMouseSynergistic effect with long term survivors[20]IfosfamideTC1MouseSubstantial inhibition of tumor residuab[54]Cis-platinumSarcoidsHorse53% CR, 27% PR vs 10\u201318% CR, 10\u201318% PRc[77]ImiquinodMelanoma metastasesMan 40.7% OR[48]aComparison of therapeutic effect of combined therapy and therapy without IL-2bTumor residua left after ifosfamide treatmentcCis-platinum\/IL-2 vs IL-2 only\nData\nSurgery\nZiekman [100] described cases of incomplete surgical removal of a tumor in veterinary patients, followed by intra-operative local IL-2 application. This led to complete tumor regression for instance in dogs with fibrosarcomas.\nIL-2 instillation in the bladder to treat bladder tumors seems only effective after a preceeding TUR (see above).\nRadiotherapy\nEverse et al. [38] treated mice with s.c. growing tumors with radiotherapy combined with IL-2 therapy. The combined therapy was more effective than radiotherapy or IL-2 therapy alone. Similar results were obtained by J\u0171rgenliemk-Schulz et al. [58].\nEarly nasopharyngeal carcinoma is not clinically apparent and hence these tumors are often diagnosed in an advanced stage. The standard treatment is irradiation, often complemented with chemotherapy. In The Netherlands usually WHO type 1 nasopharynx carcinoma occurs. Type 1 is very therapy resistant. That is, only about 10% of the patients treated with irradiation have a 5\u00a0years\u2019 tumor free survival [55]. In this study tumors of 10 patients were irradiated daily for 7\u00a0weeks. In weeks 2, 4, and 6 the tumors were (transnasally) injected with low doses of IL-2 from Monday through Friday. The 5\u00a0years\u2019 tumor free survival was 63% [55].\nCytokine therapy\nBelardelli et al. [10] described that combined treatment with IL-1 beta and IL-2 produced a synergistic antitumor effect: 60% of mice injected with highly metastasized Friend Leukemia Cells survived. T cells appeared to be essential for IL-1\/IL-2 therapy.\nChemotherapy\nEnk et al. [35] treated melanoma patients with pulmonary metastases with dacarbazine and concurrent inhalation of IL-2. The patients\u2019 condition previously had progressed on chemotherapy, predominantly on dacarbazine based regimens. Five of the 27 patients experienced a complete pulmonary remission. Eight patients achieved a partial pulmonary remission, and five patients experienced stabilization of the disease. Four of the five patients with a complete response and seven of the eight patients with a partial response were previously treated with dacarbazine and progressed. The complete responses all persisted during a follow-up of 12 months, whereas patients with partial responses or stable disease progressed when IL-2 was discontinued.\nSilagi et al. [86] found synergism between cyclophosphamide and IL-2 in the treatment of mice with sarcoma or melanoma.\nBernsen et al. [14] treated mice bearing Murine Ovarian Teratocarcinoma (MOT) with cis-platinum and IL-2. The effect of the combined treatment was greater than either therapies alone.\nSimilarly in horses with sarcoids (a characteristic equine fibro-epithelial tumor of the skin) the therapeutic effect of cis-platin and local IL-2 was greater than the therapeutic effect of either therapies alone [88].\nIndrova et al. [54] studied mice with HPV-16 associated tumors. Peritumoral IL-2 administration could substantially inhibit growth of tumor residua after chemotherapy with cyclophosphamide or ifosfamide derivatives.\nCabanes et al. [20] treated mice bearing M109 pulmonary adenocarcinoma with a combination of liposomal doxorubicin and free IL-2. Both the tumor and the IL-2 application were i.v. or both were i.p. There were synergistic effects with long-term survivors.\nGreen et al. [48] treated melanoma cases with multiple cutaneous and\/or subcutaneous metastases. Metastases were treated with imiquinod daily for 4\u00a0weeks before the introduction of intralesional IL-2. This was injected up to three times a week. 182 lesions in a total of 10 patients were treated. A clinical response was seen in 50.5% of the lesions with 40.7% being CR. Furthermore patients with clinically responding cutaneous lesions experienced a marked slowing of the appearance of new lesions. No cutaneous lesions that responded reappeared on cessation of the treatment. Imiquimod alone is often sufficient to elicit a response in purely cutaneous lesions. The addition of intralesional IL-2 however greatly increased the response rate in subcutaneous lesions, and in otherwise refractory cutaneous melanomas.\nConclusion\nCombined therapy of locally applied IL-2 and surgery, radiotherapy or chemotherapy may lead to a synergistic therapeutic effect. The durable complete responses of melanoma metastases in the lung after combined chemotherapy and IL-2 suggested that the metastases were eradicated [35].\nMechanism of tumor regression due to local IL-2 application \nOriginal hypothesis on the mechanism of IL-2 in tumor destruction\nThe original hypothesis about the function of systemically applied IL-2 in cancer therapy was that IL-2 induced Leucocyte Activated Killer cells (LAK cells; [15, 81, 82]. LAK cells were thought to invade the tumor and to kill tumor cells.The following data are incompatible with the cytotoxic LAK-cell hypothesis or any kind of cytotoxic cell hypothesis:Hardly any intravenously injected LAK-cell reaches the tumor [9].In the DBA\/2-SL2 lymphoma model it is impossible to induce cytotoxic LAK cells [67].Yet IL-2 therapy is very effective in this model.No general correlation was established between in vitro sensitivity to the cytolytic activity of LAK cells and the antitumor effects observed in vivo [10].Various types of leucocytes may dominate the tumor infiltrate after IL-2 application: macrophages, T cells, NK cells, NKT cells, plasma cells, neutrophilic, eosinophilic cells etc., [70]. The assumption that all these different types of cytotoxic cells are directly induced by IL-2 in different tumors is too complex to be true. In contrast IL-2 should induce a similar mechanism against a wide range of (sensitive) cancers.In addition, when SL2 lymphoma is growing both i.p. and s.c., and if IL-2 is injected i.p. at day 10 after tumor transplantation, then in about 50% of the cases the s.c. tumor regresses completely at about day 17, this in spite of the tumor containing only 0\u20132% leucocytes [70]. Furthermore, effector\/target cell ratios of cytotoxic T-cells are usually very low (in the order of 1:50). It is very unlikely that such a huge tumor is rejected by these few leukocytes. So, cytotoxic cells are not the major cause of tumor regression in this experiment [68].As this hypothesis was at least at some points wanting to explain the mechanism of the antitumor activity of IL-2, we started to study the histopathology of IL-2 induced tumor regression as study of the histopathology of the IL-2 induced reaction would be essential.\nIL-2 induced histopathology in normal tissue [60]\nThe study of the histopathological events during IL-2-induced tumor regression proved to be extremely difficult. The breakthrough came by serendipity. Professor Hennink and his group at the Faculty of Pharmacy, Utrecht University, are developing slow release systems (microspheres), among others for IL-2. To study the biocompatibility of these microspheres, we injected microspheres loaded with IL-2 subcutaneous in rats [60]. We discovered that the microspheres could be stained with PAS and thus these stained microspheres marked the IL-2 release site. This possibility of a precise localisation of IL-2 was a great tool to further analyse the function of the released IL-2. In a series of sections obtained during the IL-2 induced process we could trace a sequence of reactions. First a localized edema developed, related to swelling of the endothelial lining of the capillaries. After 3\u00a0days necrosis of the connective tissue was observed in the center of this edematous area. About a week later there were three zones around this necrotic area. Near the necrotic border was a zone of mixed inflammatory cells. The second zone was a large area of edematous connective tissue. The outer zone showed angiogenesis, a massive proliferation of macrophages around the newly formed blood vessels and also an influx of lymphocytes. About 3\u20135\u00a0week later these macrophages invaded the necrotic material showing features of phagocytosis. Finally also plasma cells and occasionally eosinophils appeared in the peri-necrotic inflammatory area. The plasma cells indicate the induction of an immune reaction.\nIn short two main phases could be ascertained in this process:an almost immediate marked edema due to leakage of the local blood vessels;a later and more complex reaction consisting of the induction of angiogenesis, a macrophage reaction, migration of the infiltrate into the seminecrotic tissue and finally clearance of tissue in combination with granulomatous processes.Next we again studied the histopathology of the mechanism of tumor regression induced by free IL-2. Of course the histopathological events after injection of IL-2 in or around a tumor are far more complex than the histopathological events after subcutaneous injection of IL-2 loaded microspheres in normal tissue in rats. But the events after injection of IL-2 at the site of tumors were similar to those after injection of IL-2 loaded microspheres in normal tissue. It is remarkable, however, that IL-2 induced tumor regression may be fast, requiring only some days, or slow, requiring several months, or intermediate. These differences can be related to histopathological differences of the tumor tissues. Before we describe these differences we have to pay attention to some hemodynamic features in tumors.\nSome hemodynamic features in tumors\nIn tumors the interstitial fluid pressure is higher in the center of the tumor mass than in surrounding tissue due to vessel leakage [98]. The increased vascular leakage in tumors is explained as follows:The tumor vessels usually lack a continuous basal membrane [26].Endothelial injury in ischemic tissue [49].Tumors have poor homeostatic control of the circulation due to lack of smooth muscle and lack of innervation [25].This vascular leakage causes increase of interstitial pressure. This pressure will cause vascular compression, particularly of the post-capillary venules. This leads to stagnation of the blood flow and vascular distension of the prestenotic vessels and sometimes thrombosis in smaller blood vessels. Stagnation of blood causes necrosis, in particular in the tumor center. A peripheral rim of vital tumor tissue often survives as it is just outside the area of deficient blood circulation.\nLocal IL-2 application into\/near the tumor appeared to induce additional edema in and around the tumor, just as in normal tissue. Increased edema exerts extra pressure that causes further stagnation of the blood flow and of the lymph drainage. Obviously, this leads to tumor necrosis and thrombosis within only a few days. This early edema is illustrated with photographs [8, 24, 60, 64].\nFast tumor regression of well-vascularized tumors (Fig.\u00a02) [8]\nIt is remarkable that IL-2 induced tumor regression is very quick in the case of (very) fast growing tumors like SL2 lymphoma and P815 mastocytoma [67, 68]. Similarly, relatively fast growing cases of mammary carcinoma [73] or BOSCC are more responsive to IL-2 than slower growing BOSCC [91]. In fast growing tumors the intratumoral vascular tree is relatively poorly developed and also the endothelial lining of the microvessels is abnormal. Local IL-2 application will further stimulate vascular leakage. So we assume that much fluid will leak from the tumor vessels, leading to edema, microthrombosis and extensive hemorrhages in the tumor. It is noteworthy that soon after IL-2 injection the tumor becomes firmer, indicating swelling of the tumor mass by edema. This leads to extensive necrosis.\nFig.\u00a02Mechanism of tumor regression caused by local IL-2 application of cancer\nOf course, this large necrotic tumor mass will induce an acute clearance reaction to remove this necrotic debris, which causes an early reduction in size of the tumor mass. This induces a marked immune reaction. It is also noteworthy that this tumor necrosis is accompanied by marked angiogenesis. Adjacent to many of the newly formed smaller blood vessels there is a cuff of proliferating macrophages, which ultimately move into the necrotic debris. As different tumors differ in tumor associated antigen make-up, they will induce also different types of immune reactions, dominated by macrophages, T cells, plasma cells, or eosinophilic cells. This phenomenon can be observed in biopsies taken late at the margin of the disappearing tumor. As a result of the increased tumor immunity isolated tumor strands may be enveloped by granulomatous inflammatory tissue destroying tumor cells. Specific antitumor immunity induced by IL-2 has been described by Maas et al. [67, 68]. After an animal is cured, a second implant of the same tumor is rejected [6, 68, 96].\nOur present hypothesis (Fig.\u00a02) seems now most straightforward to explain the fast tumor regression induced by IL-2 in fast growing tumors: The primary function of locally applied IL-2 is the induction of vascular leakage. This leads to an acute massive tumor necrosis and clearance of necrotic tumor material. As a consequence of massive liberation of antigenic tumor material an immune reaction develops. The different types of dominant cytotoxic cells (CTL, eosinophils, macrophages etc.,) depend on the character of the tumor-associated antigens released in the necrotic tumor debris.\nAccording to this view, maximal therapeutic results will be obtained with maximal edema formation within the tumor. In line with this view is the finding that IL-2 is more effective when injected directly into the tumor than when injected peritumorally [56].\nIt is interesting that it was originally thought that IL-2 killed tumor cells through induction of cytotoxic LAK cells and that the vascular leakage seemed a nasty side effect [7]. According to our present views, it is just the other way round: vascular leakage causing edema is the primary effect of IL-2; the development of systemic immunity [68, 96] seems the secondary effect.\nAn intriguing point was for many years that in the DBA\/2-SL2 model local IL-2 therapy is not effective 1\u201310 days after tumor cell transplantation, whereas it can be effective after 10\u00a0days until about 2\u00a0days before the expected death [37, 67]. Similarly, Maekawa et al. [69] found that local IL-2 therapy was only effective when IL-2 therapy was given seven or more days after transplantation of myeloma X5563. This can be explained by the observation that these days are required for developing a corona of angiogenesis around the tumor; this is essential for an effective local IL-2 therapy.\nSlow tumor regression of less vascularized tumors\nA number of tumors show a slow IL-2 induced regression. Examples are BOSCC [30, 84] and sarcoids in the skin of horses [88]. This regression pattern more closely follows the histological changes as observed in the model, in which the IL-2-releasing microspheres were deposited in normal subcutaneous tissue [60]. Also in these slowly growing tumors edema will be induced in the tumor, but this is more focal at the site of the injection and does not involve the entire tumor. Also the necrotic focus develops slower and the necrosis is less hemorrhagic. In these tumors the complete development of the IL-2 induced response takes at least some weeks. So, the tumor immunity will develop slower and consequently tumor regression will be slower. Moreover in BOSCC the tumor contains only about 50% of tumor cells; the other 50% is stroma. When a the tumor is reduced in size by 50%, then usually hardly any or no tumor cell is present anymore; the remaining 50% is stroma. After killing of the tumor cells, the final regression of the stroma requires several additional months.\nOf course both processes (edema and leukocyte infiltration after neoangiogenesis) are always present as a result of local IL-2 application. But in fast-regression models edema is more prevailing, in slow-regression models leukocyte and particularly macrophage infiltration dominate.\nSolid subcutaneous SL2 tumors expand by infiltrating the surrounding tissue. These tumor strands are the first targets of macrophages, as the tumor strands are in close proximity to surrounding tumor stroma and are progressively destroyed [8]. So, the body of the tumor and the infiltrating tumor strands are destroyed in two different ways, namely by edema due to vascular leakage and a granulomatous antitumor response originating in the tumor surrounding stroma, respectively.\nObviously IL-2 may stimulate the existing inflammation as often present in tumors and hence attracts more macrophages and inflammatory cells, and as a consequence intensifies the already existing cellular response to the tumor. Usually, there is in the perivascular zones a marked increase of varied types of inflammatory cells.\nThe reduction of the tumor can be accelerated by using IL-2 in combination with a tumor necrosis inducing agent, as is demonstrated by local cis-platin treatment of mice with MOT tumors [14] and sarcoids in horses [88]. Everse et al. [38] showed that irradiation stimulated the therapeutic effect of IL-2 therapy in mice. We assume that the necrotic material is phagocytosed, thus boosting an immune reaction.\nA remarkable finding is that IL-2 injected into the primary tumor can induce regression not only of the primary tumor but also the metastasis in the draining lymph node [6, 96]. However, after surgical removal of the primary tumor IL-2 cannot induce regression of the lymph node metastasis [6]. We assume that this depends on the differences between the histological structure of the primary tumors. Usually in primary tumors there is extensive vascularization and leucocyte infiltration and the tumor is in close contact with the surrounding stroma. In contrast, early tumors in lymphatic tissue often have a scanty vascularization and are situated within the lymph nodes without a direct stroma contact.\nTumor tolerance and local IL-2\nIn the previous sections histopathological techniques were used to study the mechanism of IL-2 induced tumor regression. In this section we will also pay some attention to immunological data.\nBesides stimulating immune responses, IL-2 may also inhibit immune responses. This is clear as deficiency of IL-2 leads to autoimmunity[1]. IL-2 inhibits the immune response by stimulation of CD4+CD25+ T regulatory cells (Treg). These cells suppress immune reactivity including antitumor immune responses [3, 75]. The presence of antigen [77] and IL-2 are important in the maintenance of CD4+CD25+ regulatory T lymphocytes. Depletion of CD4+CD25+ Treg is important for tumor rejection [99]. Depletion of Treg with anti-CD25 antibody stimulates tumor rejection [76].\nBecause of this tolerance inducing role of IL-2 through Treg, some authors have suggested to use IL-15 for cancer immunotherapy. IL-15 is a cytokine that is functionally closely related to IL-2, but it does not induce tolerance [74]. In 1995, the first animal studies for anti-cancer therapy were performed with IL-15. Although IL-15 therapy still lacks therapeutic successes in human clinic, some authors still suggest that IL-15 should replace IL-2 for immunotherapy of cancer [3].\nImportantly and in contrast to IL-15, IL-2 is capable of breaking tolerance [4, 11, 39, 70]. This may be crucial for effective anti-cancer immunotherapy. This reversal of tolerance is mediated through the activation of immature dendritic cells [63]. Activation of intratumoral dendritic cells and reversal of tolerance exerted through local regulatory CD4+CD25+ T lymphocytes, could be the link between local effects of intratumoral IL-2 therapy and systemic immunity. Stimulation of systemic immunity by local IL-2 therapy is also suggested by clinical data on cytokines in treated human patients. Pro-inflammatory cytokines (IFN-gamma, IL-5) are more increased after local IL-2 therapy than anti-inflammatory cytokines. In contrast, anti-inflammatory cytokines (IL-10) are increased after systemic IL-2 therapy [92]. These data highlight mechanistic differences between local and systemic IL-2 therapy.\nThe discussion above may lead to the question, whether the specific immune system is involved in IL-2 induced tumor rejection. Pleiotropic effects of high local concentrations of IL-2 could lead to aspecific activation of T cells, activation of NK cells and the generation of LAK cells. Nevertheless, the immune system must be involved as local IL-2 treatment causes systemic immunity, that resides in CD3+ lymphocytes or CD4+ and CD8+ lymphocytes [69, 70].\nConclusions\nLocal IL-2 application seems to induce severe vascular leakage in well-vascularized and fast growing tumors. This leakage in fast growing tumors induces a massive hemorrhagic necrosis which results in early clearance of the tumor tissue. In less-vascular tumor tissue the edema formation is more limited. Neoangiogenesis allows the arrival of especially macrophages that move to the tumor site. This leads to tumor cell killing and a specific immune reaction, resulting in tumor regression. The character of this immune reaction probably depends on the different types of tumor-associated antigens present in the seminecrotic tumor debris. According to our views the primary effects of local IL-2 application to a tumor are the vascular phenomena followed by a host of complex histological events clearing the tumor necrosis. The immune reactivity seems to be a secondary and presumably an indirect effect.\nOf course, this is an analysis at the histopathological level. The whole process is far more complicated as immune regulatory events\u2013\u2013including cells and cytokines are involved.\nCharacteristics of local IL-2 therapy\nLocal IL-2 treatment of cancer often cures also the metastases [67, 68, 96]\nThis systemic effect is probably the result of IL-2 induced systemic immunity [67, 68].\nLocal IL-2 therapy can cure large local tumors [31, 67, 68, 88] and even large metastasized tumor burdens [6, 67, 68, 96].\nLocal IL-2 therapy is effective against a large variety of spontaneous carcinomas, melanoma, fibrosarcoma, equine sarcoid, mesothelioma, lung metastases of renal cell carcinoma and melanoma (references see Tables\u00a03, 4). Of course, local IL-2 application is no panacea for all tumors and all types of cancer all the time.\nIntratumoral IL-2 application is more effective than peritumoral (juxtatumoral) application [55] or application at a more distant site [68].\nA single IL-2 application may be sufficient [28]. This can be advantageous during surgery when a tumor cannot be excised completely.\nLocal IL-2 therapy is particularly effective in fast growing tumors [67, 68, 73, 91].\nMost of the tumors that well respond to local IL-2 treatment are those with an already (pre-)existing peritumoral leukocytic infiltrate.\nLocal treatment of the primary tumor may result in regression of both the primary tumor and a metastasis in the draining regional lymph nodes [6, 96].\nIL-2 induced tumor regression can be fast [24, 67, 68], but it is often a slow process requiring months [30, 31, 88]. The fast tumor regression is often observed in fast growing highly vascularized tumors in which IL-2 causes vascular leakage, leading to edema and intravascular thrombosis followed by necrosis. This is also a consequence of the often abnormal endothelial lining of the intratumoral vascular tree. Slow tumor regression is often observed in poorly vascularized tumors with prominent macrophage infiltration after IL-2 induced neovascularization and an activation of the existing round cell infiltrate. Prior induction of tumor necrosis and local inflammation may accelerate the effect of IL-2.\nLocal IL-2 therapy can exert a synergistic therapeutic effect with surgery, radiotherapy and chemotherapy (references see Table\u00a06). Thus local IL-2 treatment can be a valuable addition to the standard oncotherapy.\nLocal IL-2 has minor side effects and is generally well tolerated (references see Table\u00a05). It can usually be applied without difficulty. It does not require complex technical procedures.\nLocal IL-2 does not hamper or interfere with the standard oncotherapy.\nThe local IL-2 therapy findings suggest to revisit our views on the relation between local and systemic oncotherapy at least as far as IL-2 therapy concerns\nSystemic chemotherapy\/hormone therapy, and later immunotherapy, were initially particularly reserved for patients in which adequate tumor treatment by surgery and radiotherapy was not possible. In most of these cases the tumor was inoperable or disseminated and often the patient was in the terminal phase of the disease. Now with modern therapeutic regimes the indications of systemic treatment have been extended, whereas local tumor treatment is becoming rare. Data described in this paper show that cancer treatment with local IL-2 application can lead to cure of metastases. We stress that there are good reasons to consider IL-2 tumor treatment also in early cancer.\nSurgical treatment remains the golden standard in malignant tumors. Local IL-2 therapy may be considered in local tumors if these tumors can not be resected completely. Occasionally a previously inoperable lesion might become fit for surgical interventions due to the reduction of the tumor size. In some cases local IL-2 application may be considered as an adjunct to the standard treatment particularly if recurrences are expected. An example of the latter is local IL-2 treatment in TUR-treated bladder tumors.\nLocal IL-2 therapy may also be used as an adjunct to treat a primary tumor showing metastases. In advanced tumor cases local IL-2 treatment of the primary tumor may induce a specific immune response and might also contribute to cure or decrease tumor size and sometimes also may have a positive effect on the metastases. IL-2 does not interfere with the standard oncotherapy and might be an useful adjunct to diminish suffering and expand lifespan.\nDose at the tumor site Only about 0.1% of the systemically applied dose reaches the tumor. In other words the same dose injected locally results at the site of the tumor in a 1,000 times higher concentration than a systemically applied dose.\nReduced side effects An advantage of local treatment is furthermore that side effects, both local and systemic, are modest if present at all, since the required local dose is only a fraction of the dose required in systemic treatment.\nNo immunologic overflow feed-back Systemic IL-2 therapy causes immuno suppression as indicated by systemic IL-10 production. This is probably the body\u2019s reaction to a generally activated immune system (cytokine storm). Local IL-2 avoids this down-regulating feedback, and leads to systemic increase of pro-inflammatory cytokines, like IFN-gamma and IL-5.\nReduced costs of locally applied low doses Systemic chemotherapy and immunotherapy in cows and horses is no option. Cattle and horses are so large that systemic oncotherapy is out of order; e.g., required doses of IL-2 would be very large and hence far too expensive in animal husbandry. This problem can be circumvented by local therapy. The results in cattle and horses show that local IL-2 therapy in these large animals is feasible, readily to apply, economic, and therapeutically effective.\nLocal IL-2 application: doses and duration of the treatment\nThe number of IL-2 injections required for optimal treatment is still a point of debate. A single injection containing 2 up to 16\u00a0million U IL-2 is sufficient in the DBA\/2\u2013SL2 model [28], whereas doses of only 5,000 U IL-2 were required in case daily injections on five consecutive days were given [67]. However, Kusnierczyk et al. [64] cured C57BL mice with s.c. solid colon carcinoma with multiple IL-2 treatments, whereas no cure was obtained by a single IL-2 injection. Treating spontaneous BOSCC with a single dose of 2\u00a0million U IL-2 [28] gave comparable results as daily IL-2 injection of the same dose on ten consecutive working days [30]. For logistic reasons most clinicians (both in human and in veterinary medicine) strongly prefer to apply one single dose instead of daily injections over five or ten consecutive working days. A single large dose can also be appropriate for tumor treatment during surgery when the tumor mass cannot completely be removed. But such a single dose should be much higher (about 4.5\u00a0\u00d7\u00a0106\u00a0IU IL-2) than doses on five or ten consecutive working days (ca 5,000\u00a0IU per day in mice).\nVery different IL-2 doses, time schedules and routes of application were used. In a human patient we might consider <106\u00a0U IL-2 as low dose, (1\u201318)\u00a0\u00d7\u00a0106 U as intermediate dose, and >18\u00a0\u00d7\u00a0106\u00a0U IL-2 high dose for local IL-2 application. But a systemically applied dose is far more toxic than the same dose after local application. In addition, the same dose given i.p. in a mouse can be regarded to be high, whereas this dose is low when it is given intravenously in a human patient. A systemically applied dose of 106\u00a0U IL-2 is intermediate. But if this dose is given daily for 100\u00a0days, then it is a large total dose.\nNowadays we usually treat cancer with a single local injection of 4.5\u00a0\u00d7\u00a0106\u00a0U IL-2. This dose is based on data obtained in mice, cattle and dogs [28]. It may be astounding that in local treatment the same dose is effective in a mouse as well as in cattle. In fact similar local processes have to be induced in these different species. In systemic application however the dose required by a cow has to be about 20,000 fold of that used in mice.\nWe preferably inject IL-2 inside the tumor. This leads to better therapeutic results than peritumoral IL-2 application [56].\nWebsite\nOur website on local IL-2 therapy is available at http:\/\/cancerimmunotherapy.net\/\nWe provide information targeted on medical professionals. Readers are invited to co-operation. Please reach us at w.denotter@uu.nl.","keyphrases":["immunotherapy","boscc","nasopharyngeal carcinoma","bladder carcinoma","sarcoids","interleukin-2"],"prmu":["P","P","P","P","P","P"]}
{"id":"Pflugers_Arch-3-1-2082653","title":"Restitution analysis of alternans and its relationship to arrhythmogenicity in hypokalaemic Langendorff-perfused murine hearts\n","text":"Alternans and arrhythmogenicity were studied in hypokalaemic (3.0 mM K+) Langendorff-perfused murine hearts paced at high rates. Epicardial and endocardial monophasic action potentials were recorded and durations quantified at 90% repolarization. Alternans and arrhythmia occurred in hypokalaemic, but not normokalaemic (5.2 mM K+) hearts (P < 0.01): this was prevented by treatment with lidocaine (10 \u03bcM, P < 0.01). Fourier analysis then confirmed transition from monomorphic to polymorphic waveforms for the first time in the murine heart. Alternans and arrhythmia were associated with increases in the slopes of restitution curves, obtained for the first time in the murine heart, while the anti-arrhythmic effect of lidocaine was associated with decreased slopes. Thus, hypokalaemia significantly increased (P < 0.05) maximal gradients (from 0.55 \u00b1 0.14 to 2.35 \u00b1 0.67 in the epicardium and from 0.67 \u00b1 0.13 to 1.87 \u00b1 0.28 in the endocardium) and critical diastolic intervals (DIs) at which gradients equalled unity (from \u22122.14 \u00b1 0.52 ms to 50.93 \u00b1 14.45 ms in the epicardium and from 8.14 \u00b1 1.49 ms to 44.64 \u00b1 5 ms in the endocardium). While treatment of normokalaemic hearts with lidocaine had no significant effect (P > 0.05) on either maximal gradients (0.78 \u00b1 0.27 in the epicardium and 0.83 \u00b1 0.45 in the endocardium) or critical DIs (6.06 \u00b1 2.10 ms and 7.04 \u00b1 3.82 ms in the endocardium), treatment of hypokalaemic hearts with lidocaine reduced (P < 0.05) both these parameters (1.05 \u00b1 0.30 in the epicardium and 0.89 \u00b1 0.36 in the endocardium and 30.38 \u00b1 8.88 ms in the epicardium and 31.65 \u00b1 4.78 ms in the endocardium, respectively). We thus demonstrate that alternans contributes a dynamic component to arrhythmic substrate during hypokalaemia, that restitution may furnish an underlying mechanism and that these phenomena are abolished by lidocaine, both recapitulating and clarifying clinical findings.\nIntroduction\nCorrelations between beat-to-beat alternations in electrocardiographic QT interval (QT or T-wave alternans) and cardiac arrhythmia have been described in classical work [13, 25] and subsequently confirmed in both clinical studies [2, 28, 38] and experimental models [7, 33, 52]. Indeed, the presence of such alternans provides a stronger predictor of arrhythmic risk than is offered by signal-averaged electrocardiography and a prediction of a similar value to that offered by invasive programmed stimulation procedures in current use [10] (but see [45]). The association between alternans and arrhythmogenicity is particularly marked at high heart rates (short baseline cycle lengths, BCLs) [15, 49] and in conditions of congenital and acquired electrocardiographic QT, and therefore action potential, prolongation [1, 34, 39, 46, 47, 64].\nThe relationship between heart rate and alternans has previously been analysed by constructing restitution curves which plot relationships between action potential duration (APD) and preceding diastolic interval (DI), as BCL is varied [12]. Clinical and experimental studies then associated alternans and arrhythmia observed at short BCLs with steeply sloping restitution curves with gradients greater than unity [14, 21, 24, 32]. The latter would reflect disproportionate decreases in mean APD with DI. Furthermore, manoeuvres which decrease the slopes of restitution curves, such as hyperkalaemia in a tachycardic canine endocardial preparation [22] and certain cardiotropic drugs in canine and porcine preparations [8, 31, 37], have been reported to both suppress alternans and exert anti-arrhythmic effects. In contrast, other studies have attributed such alternans to alteration in Ca2+ cycling involving the sarcoplasmic reticulum [35, 44, 54]. Whatever the underlying mechanism, alternans results in temporal variations in the wavelengths of propagating action potentials that could potentially lead to reentry and arrhythmogenesis [55].\nHypokalaemia is an important clinical cause of acquired QT prolongation and is associated with arrhythmia of various degrees of spatial organisation [12, 30, 57] suppressible by lidocaine [6, 36, 48]. The present study uses the monophasic action potential (MAP) technique [19, 23] and an established Langendorff-perfused murine model [18, 40\u201342] to explore the effects of hypokalaemia and of lidocaine on alternans and arrhythmogenicity for the first time in any species. These investigations of temporal heterogeneities in recovery after depolarization are performed on hearts paced at short BCLs, thereby complimenting recent work [40, 41] which contrastingly examined the contribution of spatial heterogeneities at long BCLs. This approach successfully demonstrates such phenomena in parallel with the clinical situation and proceeds for the first time to apply Fourier analysis to MAP waveforms and to recordings obtained during hypokalaemia. This analysis demonstrates evolution from a monomorphic to a polymorphic pattern as has previously been reported in other species [55, 58, 63], thereby further validating the murine model. The presence or absence of alternans and arrhythmogenicity is then related to alterations in the slopes of restitution curves. Thus, while the relationship between APD and BCL has previously been determined [20], the present study reports restitution curves relating APD to the preceding DI for the first time in a murine system. Use of this murine system further permits the comparison of findings with results obtained from genetically modified examples [26].\nTaken together, these novel findings implicate a dynamic component to arrhythmic substrate under conditions of hypokalaemia for which restitution may furnish a possible underlying mechanism.\nMaterials and methods\nExperimental animals\nMice were housed at 21\u2009\u00b1\u20091\u00b0C with 12-h light\/dark cycles, were fed sterile chow (RM3 Maintenance Diet, SDS, Witham, Essex, UK) and had free access to water. Wild-type 129\u00a0Sv mice aged 3\u20136\u00a0months were used in all experiments. All procedures complied with the UK Animals (Scientific Procedures) Act 1986.\nSolutions\nSolutions were based on bicarbonate-buffered Krebs\u2013Henseleit solution (mM: NaCl 119, NaHCO3 25, KCl 4, KH2PO4 1.2, MgCl2 1, CaCl2 1.8, glucose 10 and Na-pyruvate 2; pH adjusted to 7.4) and were bubbled with 95% O2\/5% CO2 (British Oxygen Company, Manchester, UK). Hypokalaemic (3.0\u00a0mM K+) solutions were prepared by reducing the amount of KCl added. Lidocaine-containing solutions were prepared by adding lidocaine (Sigma-Aldrich, Poole, UK) to a final concentration of 10\u00a0\u03bcM.\nPreparation\nA Langendorff perfusion protocol previously adapted for murine hearts [3] was used. In brief, mice were killed by cervical dislocation [Schedule 1: UK Animals (Scientific Procedures) Act 1986]. Hearts were then quickly excised and placed in ice-cold bicarbonate-buffered Krebs\u2013Henseleit solution. A short section of aorta was cannulated under the surface of the solution and attached to a custom-made 21-gauge cannula filled with the same solution using an aneurysm clip (Harvard Apparatus, Edenbridge, Kent, UK). Fresh Krebs\u2013Henseleit solution was then passed through 200- and 5-\u03bcm filters (Millipore, Watford, UK) and warmed (37\u00b0C) using a water jacket and circulator (Techne model C-85A, Cambridge, UK) before being used for constant-flow retrograde perfusion at 2\u20132.5\u00a0ml\/min using a peristaltic pump (Watson-Marlow Bredel model 505S, Falmouth, Cornwall, UK). Hearts were only regarded as suitable for experimentation if they regained a healthy pink colour and began to contract spontaneously on rewarming.\nElectrophysiological measurements\nAn epicardial MAP electrode (Hugo Sachs, Harvard Apparatus) was placed against the basal left ventricular epicardium. A small access window was created in the interventricular septum to allow access to the left ventricular endocardium [5]. A custom-made endocardial MAP electrode comprising two twisted strands of high-purity Teflon-coated silver wire of 0.25-mm diameter (Advent Research Materials, UK) was constructed. The Telflon coat was removed from the distal 1\u00a0mm of the electrode, which was then galvanically chlorided to eliminate DC offset, inserted and placed against the septal endocardial surface. MAPs were amplified, band-pass-filtered (0.5\u00a0Hz to 1\u00a0kHz: Gould 2400S, Gould-Nicolet Technologies, Ilford, Essex, UK) and digitised at a sampling frequency of 5\u00a0kHz (micro1401, Cambridge Electronic Design, Cambridge, UK). Analysis of MAPs in both the time and frequency domains was performed using Spike II (Cambridge Electronic Design).\nExperimental protocol\nA bipolar platinum stimulating electrode (1\u00a0mm inter-pole spacing) was placed on the basal surface of the right ventricular epicardium. Square-wave stimuli (Grass S48 stimulator, Grass-Telefactor, Slough, UK) of 2-ms duration and amplitudes of twice the excitation threshold were initially applied to hearts at a constant baseline cycle length of 125\u00a0ms until MAPs showed stable baselines, rapid upstroke phases that reached consistent amplitudes and smooth repolarisation phases [19] and for at least 10\u00a0min. Hearts were then exposed to test solutions for 20\u00a0min while stimulation was continued before subsequent recordings were made.\nIn initial experiments, MAPs were recorded during regular stimulation at baseline cycle lengths of 130, 100 and 70\u00a0ms. Hearts were then subjected to an adapted dynamic pacing protocol [9]. This comprised cycles each consisting of 100 stimuli delivered over a range of BCLs. Steady states were consistently reached during the first 50 responses in each cycle, and thus, mean epicardial and endocardial APD90 values and DIs were calculated from the final 50 action potentials of each cycle. With each successive cycle, BCL was decremented by 5-ms steps from an initial value of 175\u00a0ms. Cycles were continued until a reproducible sequence of consistently shaped waveforms was no longer obtained. These data were then used to construct restitution curves.\nAll data are presented as means\u2009\u00b1\u2009standard errors of the means and include the number of hearts studied. Comparisons between data sets used analysis of variance (significance threshold set at P\u2009\u2264\u20090.05). Curve fitting of particular function to data sets used a Levenberg\u2013Marquardt algorithm (OriginPro 7.5, OriginLab, MA, USA).\nResults\nThe experiments explored the consequences of increases in heart rate for arrhythmogenicity in hypokalaemic murine hearts, thereby extending a previous report that considered the effect of bradycardia [41]. They thus studied identical experimental groups, first exposing hearts to normokalaemic (5.2\u00a0mM K+) and hypokalaemic (3.0\u00a0mM K+) test solutions before and after addition of lidocaine (10\u00a0\u03bcM) for 20\u00a0min during regular stimulation at a BCL of 125\u00a0ms. Experiments then proceeded to study electrical activity through a range of steady state BCLs to assess propensity to spontaneous arrhythmia. Electrical traces from arrhythmic hearts were then quantitatively analysed in the frequency domain to empirically characterise their kinetics. Finally, hearts were subjected to a dynamic pacing protocol which explored the effect of varying BCLs [21] and permitted the analysis of alternans and its relationship to arrhythmogenicity.\nAlternans and arrhythmic activity occur in hypokalaemic hearts paced at a reduced baseline cycle length\nThe first series of experiments recorded MAPs [23] from the epicardia of hearts paced at BCLs of 130, 100 or 70\u00a0ms over 30-min recording periods (five hearts in each case; Fig.\u00a01). This demonstrated a tendency to both alternans, as reflected in alternating short-long-short sequences in action potential duration, and arrhythmogenesis at the shortest BCLs, corresponding to the highest heart rates, specifically in the hypokalaemic hearts and not in the other groups. Thus, MAPs obtained from normokalaemic hearts whether paced at BCLs of 130, 100 or 70\u00a0ms demonstrated consistent waveforms and stable rhythms during 30-min recording periods (five hearts in each case; Fig.\u00a01a). In contrast, while recordings from hypokalaemic hearts paced at BCLs of 130 and 100\u00a0ms demonstrated stable rhythms (five out of five hearts), alternans leading to periods of arrhythmic activity occurred during pacing at a BCL of 70\u00a0ms in all five cases (P\u2009<\u20090.01 as compared to normokalaemic hearts; Fig.\u00a01b). However, neither alternans nor arrhythmic activity occurred in either normokalaemic or hypokalaemic hearts treated with lidocaine during pacing at any BCL (five hearts in each case; P\u2009<\u20090.01 as compared to hypokalaemic hearts not treated with lidocaine; Fig.\u00a01c and d, respectively), confirming the anti-arrhythmic effect of lidocaine described in earlier studies [6, 41, 42, 48].\nFig.\u00a01Effect of baseline cycle length on presence of alternans and initiation of arrhythmic activity. Epicardial monophasic action potential recordings obtained from hearts exposed to normokalaemic (5.2\u00a0mM K+, a) and hypokalaemic (3.0\u00a0mM K+, b) test solutions, and normokalaemic (c) and hypokalaemic (d) test solutions containing lidocaine (10\u00a0\u03bcM) during regular stimulation at baseline cycle lengths of 130\u00a0ms (A), 100\u00a0ms (B) and 70\u00a0ms (C). Vertical lines indicate the timing of stimuli\nElectrophysiological waveforms after the initiation of arrhythmic activity\nWhen arrhythmic activity was observed in those hypokalaemic hearts paced at a BCL of 70\u00a0ms, this showed progressive degeneration from a regular monomorphic to a disorganised polymorphic pattern (four hearts), in common with earlier reports from the larger, porcine and human, hearts [16, 58, 63].\nThis trend was quantitatively apparent in a spectral analysis performed using a fast Fourier transform method [4] on \u223c10-s sequences of MAP waveforms recorded from the epicardia and endocardia of hypokalaemic hearts to which a Hanning window had first been applied. These waveforms had been sampled at a rate of 5\u00a0kHz with low- and high-frequency filter cutoffs of 0.5\u00a0Hz and 1\u00a0kHz, respectively. Each transform was performed on 16,384 (=216) points to give a spectral resolution of 0.30\u00a0Hz up to a maximum, Nyquist, frequency of 2.5\u00a0kHz. Relative root mean square powers were then extracted from the real and imaginary parts of the transforms.\nFigure\u00a02 thus compares typical waveforms in both the time (a) and frequency (b) domains. Waveforms recorded during the first \u223c5\u00a0min after the initiation of arrhythmic activity were monomorphic in appearance (A in Fig.\u00a02a). This was confirmed by spectral analysis which similarly demonstrated a single identical dominant frequency peak of 18.9\u2009\u00b1\u20091.8\u00a0Hz in both epicardial and endocardial recordings with harmonics whose frequencies were integral multiples of a dominant fundamental frequency and any given heart showed at least two such peaks (\u223c38.4, \u223c57.6, \u223c76.8\u00a0Hz, A in Fig.\u00a02b).\nFig.\u00a02Progression from monomorphic to polymorphic arrhythmic activity. Epicardial and endocardial monophasic action potential recordings obtained from hearts exposed to hypokalaemic (3.0\u00a0mM K+) test solution 5\u00a0min (A) and 10\u00a0min (B) after initiation of arrhythmic activity in the time (a) and frequency (b) domains\nIn contrast, waveforms recorded \u226510\u00a0min after the initiation of arrhythmic activity were consistently polymorphic in appearance (B in Fig.\u00a02a), with spectral analysis (B in Fig.\u00a02b) further demonstrating dissimilar epicardial and endocardial frequency spectra. The dominant frequency peak was then at 27.6\u2009\u00b1\u20095.6\u00a0Hz in the epicardium, while that in the endocardium was 26.5\u2009\u00b1\u20095.8\u00a0Hz. Furthermore, the higher order peaks, whether recorded from the epicardium or the endocardium, occurred at irregular frequency intervals that were not multiples of the dominant frequency.\nAlternans occurs in the epicardia and endocardia of hypokalaemic hearts paced at a baseline cycle length of 70 ms\nAs illustrated in C in Fig.\u00a01b, hypokalaemic hearts paced at a BCL of 70\u00a0ms demonstrated alternans. Figure\u00a03 goes on to show mean APD90 values of the alternating, odd-numbered (filled bars) and even-numbered (open bars) action potentials, and the difference between these values, giving the magnitude of any alternans (hashing), recorded from the epicardia (a) and endocardia (b) under normokalaemic (A) and hypokalaemic (B) conditions and under such conditions during treatment with lidocaine (C and D, respectively).\nFig.\u00a03Alternans during stimulation at a baseline cycle length of 70\u00a0ms. Durations of successive odd (filled bars) and even (open bars) numbered action potentials (at 90% repolarisation, APD90) and the difference between these values, giving the magnitude of alternans (hashing) in the epicardia (a) and endocardia (b) of hearts exposed to normokalaemic (5.2\u00a0mM K+, A) and hypokalaemic (3.0\u00a0mM K+, B) test solutions and normokalaemic (C) and hypokalaemic (D) test solutions containing lidocaine (10\u00a0\u03bcM) during regular stimulation at a baseline cycle length of 70\u00a0ms\nIn normokalaemic hearts (five hearts), alternans was not observed, the difference between the mean APD90 values of odd-number and even-numbered action potentials not reaching significance (P\u2009>\u20090.05) in either the epicardium (4.7\u2009\u00b1\u20092.7\u00a0ms) or the endocardium (5.6\u2009\u00b1\u20091.7\u00a0ms). In contrast, alternans did occur in hypokalaemic hearts (five hearts), the difference between the mean APD90 values of odd-number and even-numbered action potentials being statistically significant (P\u2009<\u20090.01) in both epicardium (14.9\u2009\u00b1\u20093.8\u00a0ms) and endocardium (15.6\u2009\u00b1\u20091.8\u00a0ms). Treatment with lidocaine had no significant (P\u2009>\u20090.05) effect on normokalaemic hearts (five hearts): The difference between the mean APD90 values of odd-number and even-numbered action potentials remained statistically insignificant (P\u2009>\u20090.05) in both epicardium (2.7\u2009\u00b1\u20093.4\u00a0ms) and endocardium (2.5\u2009\u00b1\u20094.6\u00a0ms). In contrast, treatment with lidocaine eliminated alternans in hypokalaemic hearts (five hearts). Hence, the difference between the mean APD90 values of odd-number and even-numbered action potentials did not reach significance (P\u2009>\u20090.05) in either epicardium (4.1\u2009\u00b1\u20094.6\u00a0ms) or endocardium (2.4\u2009\u00b1\u20093.2\u00a0ms).\nThe dynamic pacing protocol initiates alternans in hypokalaemic hearts at short baseline cycle lengths\nThe above findings concerning the presence or absence of alternans at the shortest BCL studied above prompted a detailed exploration of the effect of BCL upon the alternans phenomenon. To assess this relationship, hearts were subjected to a dynamic pacing protocol [21] consisting of cycles each lasting 100 stimuli at through a range of BCLs. In these procedures, steady states were consistently reached within the first 50 responses. Accordingly, mean values of epicardial and endocardial APD90 and DI were obtained from the final 50 action potentials. BCL was decremented in 5-ms steps with each cycle from an initial value of 175\u00a0ms until a reproducible sequence of consistently shaped action potential waveforms could no longer be obtained.\nFigure\u00a04a\u2013d shows typical recordings obtained from the epicardia of individual hearts under the four sets of experimental conditions during the dynamic pacing protocol at BCLs of 170, 130, 90, 85, 80, 75 and 70\u00a0ms when such steady states had been achieved. Figure\u00a05a\u2013d shows the corresponding relationships between the durations of successive odd- and even-numbered action potentials obtained from these same hearts during the dynamic pacing protocol over the full range of BCLs studied. Thus, alternans was not observed under normokalaemic conditions even at the shortest BCL studied (Fig.\u00a04a), resulting in points falling on the line y\u2009=\u2009x (Fig.\u00a05a). Conversely, alternans occurred under hypokalaemic conditions at short BCLs (Fig.\u00a04b), resulting in such points falling below the line of equality (Fig.\u00a05b). In contrast, alternans did not occur in either normokalaemic or hypokalaemic hearts during exposure to lidocaine (Fig.\u00a04c and d), again resulting in points falling on the line of equality (Fig.\u00a05c and d).\nFig.\u00a04Effect of baseline cycle length on action potential waveforms. Epicardial monophasic action potential recordings obtained from hearts exposed to normokalaemic (5.2\u00a0mM K+, a) and hypokalaemic (3.0\u00a0mM K+, b) test solutions and normokalaemic (c) and hypokalaemic (d) test solutions containing lidocaine (10\u00a0\u03bcM) during stimulation at baseline cycle lengths of 170, 130, 90, 85, 80, 75 and 70\u00a0ms. Vertical lines indicate the timing of stimuliFig.\u00a05 Relationships between durations of successive action potentials over a range of baseline cycle lengths. Relationships between the durations of successive odd- and even-numbered epicardial monophasic action potentials (at 90% repolarisation, APD90) in the same hearts exposed to normokalaemic (5.2\u00a0mM K+, a) and hypokalaemic (3.0\u00a0mM K+, b) test solutions and normokalaemic (c) and hypokalaemic (d) test solutions containing lidocaine (10\u00a0\u03bcM). These were obtained at baseline cycle lengths decremented in 5-ms steps from 175\u00a0ms until stimulation no longer resulted in a reproducible sequence of waveforms. Filled and open circles indicate data obtained at baseline cycle lengths at which arrhythmogenesis was and was not observed, respectively. Arrow indicates departure of points from the line y\u2009=\u2009x (broken line)\nAlterations in the slopes of restitution curves correlate with pro- and anti-arrhythmic effects\nThe findings described above prompted the construction and analysis of restitution curves relating APD90 and the preceding DI given by the difference between the BCL and the preceding APD90 [21, 29, 32, 55]. Alternans and arrhythmia have previously been associated with increases in the slopes of such restitution curves reflecting disproportionate decreases in mean APD90 with DI. Such curves (data points, left ordinate) were plotted using the APD90 (left ordinate) and DI data obtained from the epicardia (circles, Fig.\u00a06A) and endocardia (squares, Fig.\u00a06B) of hearts under these conditions (a\u2013d, respectively).\nFig.\u00a06Restitution curves obtained from epicardia and endocardia. Restitution curves plotting action potential duration (at 90% repolarization, APD90) against preceding diastolic interval (DI) obtained from the epicardia (circles, A) and endocardia (squares, B) of hearts exposed to normokalaemic (5.2\u00a0mM K+, a) and hypokalaemic (3.0\u00a0mM K+, b) test solutions and normokalaemic (c) and hypokalaemic (d) test solutions containing lidocaine (10\u00a0\u03bcM). Curves are fitted with mono-exponential growth functions obtained by least-squares fitting to the experimental values of APD90 and DI (solid lines, left ordinates). Gradients were obtained by differentiation of the fitted functions (broken lines, right axes). Shaded boxes indicate ranges of DI values at which such gradients exceed unity\nUnder all conditions, both epicardial and endocardial APD90 decreased as DI decreased. However, the slope of this relationship was greater under hypokalaemic (A and B in Figs.\u00a06b) than under normokalaemic conditions (A and B in Figs.\u00a06a). This paralleled both the alternans and arrhythmic activity that were observed under the hypokalaemic conditions. Such increases in the slopes of restitution curves were observed even when the DI was so short such that stimuli were delivered before 90% repolarisation was achieved. This would tend to result in the underestimation of APD90, and hence, the underestimation of the slopes of restitution curves.\nExposure to lidocaine had little effect on the slopes of the corresponding curves obtained under normokalaemic (A and B in Figs.\u00a06c), but markedly decreased the slopes of curves obtained under hypokalaemic conditions (A and B in Figs.\u00a06d), in precise agreement with its corresponding effects in suppressing alternans and arrhythmia.\nRestitution curves yield parameters predictive of arrhythmogenicity\nThe restitution curves obtained above proved amenable to quantitative analyses that has been described on previous occasions. For example, the simplest, mono-exponential growth function that has been used to describe data of this kind [13] takes the form:\nwhere y represents APD90, x represents DI and y0 and A and \u03c4 are constants obtained by least squares fitting to the experimental values of APD90 and DI in each case. Such curve-fits are used merely to empirically describe the data and should not be taken to imply any particular underlying mechanistic process. The corresponding gradient is then given by:\nand assumes its maximum value at the shortest BCL.\nFigure\u00a06 plots the original APD90 and DI values and superimposes optimisations of Eq. 1 to these data (solid lines, left-hand ordinates). Such optimisations gave reduced \u03c72 values indicative of better fits to data points obtained under normokalaemic than hypokalaemic conditions. It also shows the corresponding gradients derived from constants obtained from the optimisations (Eq. 2: broken lines, right-hand axes). Shaded regions where present indicate the range of DIs below a critical DI (DI < critical DI) at which these gradients exceed unity, obtained from Eq. 2:\nSuch a phenomenon is associated with an alternans initiated by incomplete recovery of one action potential and the consequent shortening of its subsequent DI. This would result in a disproportionate shortening of the subsequent action potential which would consequently permit its full recovery and a lengthening of the DI that follows, thereby reinitiating the cycle and resulting in a progressive decrease in mean APD90 relative to mean DI with decreasing BCL [29].\nThe resulting alternans would assume an amplitude determined by the gradient of the restitution curve at the given BCL, whether the latter is determined by altered external pacing rates or local ventricular arrhythmic activity. A BCL that gives a gradient of >1 will then initiate a shortening not only of mean APD90 but also of mean APD90 relative to mean DI. The resulting shortening of action potential wavelength would then predispose to reentry and arrhythmogenesis.\nThese parameters successfully predict the pro-arrhythmic effect of hypokalaemia\nFigure\u00a07 goes on to show these maximum gradients (a) and values of the critical DI (b) derived from the computed A and \u03c4 values in epicardia (filled bars) and endocardia (open bars). Under normokalaemic conditions (Fig.\u00a07A) A and \u03c4 took values of 56.3\u2009\u00b1\u20092.4\u00a0ms and 58.4\u2009\u00b1\u20099.8\u00a0ms in the epicardium and 73.8\u2009\u00b1\u20091.7\u00a0ms and 65.2\u2009\u00b1\u20098.4\u00a0ms in the endocardium, with reduced \u03c72 values from least-squares regression of \u2009=\u20091.4 and 1.2, respectively. This gave maximal gradients of 0.55\u2009\u00b1\u20090.14 in the epicardium and 0.67\u2009\u00b1\u20090.13 in the endocardium and critical DI values of \u22122.14\u2009\u00b1\u20090.52\u00a0ms and 8.14\u2009\u00b1\u20091.49\u00a0ms, respectively. None of the experimental DI values realised the critical DI in either the epicardium or the endocardium, paralleling the absence of arrhythmic activity (Fig.\u00a02a).\nFig.\u00a07Maximum gradients and critical diastolic intervals obtained from restitution curves. Maximum gradients (a) and critical diastolic intervals at which gradients equalled unity (b) obtained from epicardial (filled bars) and endocardial (open bars) restitution curves shown in Fig.\u00a06 under normokalaemic (5.2\u00a0mM K+, A) and hypokalaemic (3.0\u00a0mM K+, B) conditions and under normokalaemic (C) and hypokalaemic (D) conditions during exposure to lidocaine (10\u00a0\u03bcM). Asterisks indicate values that are significantly (P\u2009<\u20090.05) larger than those recorded in normokalaemic hearts\nHypokalaemia (Fig.\u00a07B) significantly (P\u2009<\u20090.05) increased the slopes of both epicardial and endocardial curves, concurring with the arrhythmogenic findings. Thus, A and \u03c4 took values of 166.7\u2009\u00b1\u20095.63\u00a0ms and 29.3\u2009\u00b1\u20098.29\u00a0ms in the epicardium and 133.3\u2009\u00b1\u20098.0\u00a0ms and 29.8\u2009\u00b1\u20092.1\u00a0ms in the endocardium, with  values of 8.2 and 2.5, respectively. This gave significantly (P\u2009<\u20090.05) increased maximum gradients of 2.35\u2009\u00b1\u20090.67 in the epicardium and 1.87\u2009\u00b1\u20090.28 in the endocardium and significantly (P\u2009<\u20090.05) increased the critical DI values of 50.93\u2009\u00b1\u200914.45\u00a0ms and 44.64\u2009\u00b1\u20095\u00a0ms, respectively. The critical DI was realised by the observed data points in both epicardium and endocardium. Furthermore, the DIs at which this occurred were in full agreement with the corresponding DIs at which alternans was observed. Thus, in the epicardium, alternans occurred during pacing at BCLs of \u226495\u00a0ms, corresponding to DIs of 46.7\u2009\u00b1\u20093.6\u00a0ms, while in the endocardium, alternans occurred during pacing at BCLs of \u226475\u00a0ms, corresponding to DIs of 44.0\u2009\u00b1\u20095.7\u00a0ms.\nThese parameters also predict the anti-arrhythmic effect of lidocaine\nLidocaine had no significant (P\u2009>\u20090.05) effect on these values when applied to normokalaemic hearts (Fig.\u00a07C). Thus, A and \u03c4 took values of 81.1\u2009\u00b1\u20093.05\u00a0ms and 56.92\u2009\u00b1\u200910.29\u00a0ms in the epicardium and 104.47\u2009\u00b1\u200913.69\u00a0ms and 97.17\u2009\u00b1\u200936.22\u00a0ms in the endocardium, with  values of 5.37 and 10.47, respectively. This gave maximum gradients of 0.78\u2009\u00b1\u20090.27 in the epicardium and 0.83\u2009\u00b1\u20090.45 in the endocardium and the critical DI values of 6.06\u2009\u00b1\u20092.10\u00a0ms and 7.04\u2009\u00b1\u20093.82\u00a0ms, respectively, which were not realised even at the shortest BCLs studied.\nIn contrast, treatment of hypokalaemic hearts with lidocaine (Fig.\u00a07D) significantly (P\u2009<\u20090.05) reduced the slopes of both epicardial and endocardial restitution curves, in parallel with its anti-arrhythmic effect. Thus A and \u03c4 took values of 109.90\u2009\u00b1\u200911.95\u00a0ms and 37.36\u2009\u00b1\u20095.97\u00a0ms in the epicardium and 231.72\u2009\u00b1\u200944.70\u00a0ms and 27.60\u2009\u00b1\u20094.44\u00a0ms in the endocardium, with  values of 6.83 and 15.68, respectively. This gave significantly decreased (P\u2009<\u20090.05) maximal gradients of 1.05\u2009\u00b1\u20090.30 in the epicardium and 0.89\u2009\u00b1\u20090.36 in the endocardium and significantly decreased (P\u2009<\u20090.05) critical DI values of 30.38\u2009\u00b1\u20098.88\u00a0ms and 31.65\u2009\u00b1\u20094.78\u00a0ms, respectively. The critical DI was realised only at the shortest BCLs studied and then only in the epicardium, in parallel with the substantial reduction in arrhythmogenicity produced by this drug.\nSlopes of restitution curves thus precisely correlate with the presence or absence of alternans and arrhythmogenesis under all the conditions explored in the hypokalaemic Langendorff-perfused murine heart.\nDiscussion\nElectrical alternans, beat-to-beat alternation in action potential duration in turn reflected in alternation in electrocardiographic QT interval (QT or T-wave alternans), is most commonly seen at high heart rates and has been associated with arrhythmogenesis in both clinical [2, 25, 28, 38] and experimental [7, 33, 52] studies. This association is especially marked in situations of preexisting action potential, and therefore electrocardiographic QT, prolongation [39, 46, 47, 49, 64] as might occur in the congenital long QT syndromes.\nAlternans phenomena have classically been analysed by the construction of restitution curves relating action potential duration to the preceding diastolic interval as heart rate is varied [29]. Restitution curves of slopes greater than unity have been associated with alternations in action potential durations of progressively increasing magnitude [14, 21, 24, 29, 32]. In contrast alternans has more recently been associated with disruption of normal cellular Ca2+ homeostasis leading to alternating short-long-short pulses of release from sarcoplasmic reticular stores [35, 44, 54]. Irrespective of the underlying mechanism, any spatial discordance in either phase or magnitude of alternans between myocardial regions would lead to spatial repolarisation gradients that might cause reentry and arrhythmogenesis [53, 56]. Furthermore, any resulting decrease in action potential wavelength, given by the product of action potential duration and conduction velocity, to less than a critical value, might break up the propagating wave of excitation and lead to reentry and arrhythmogenesis [55].\nRelationships between restitution curve slopes, alternans and arrhythmogenicity at high heart rates have been established in canine preparations made to model congenital long QT syndrome type 2 (LQT2) by application of the IKr blocking agent E-4031 [27, 61]. Correspondingly, there is a known association between sudden arousal, presumably resulting in increased heart rate, and arrhythmogenesis in human LQT2 [59]. This association has also been established in clinical hypokalaemia [11]. This common and pathophysiologically important condition similarly results in decreased repolarising K+ currents, together with action potential, and therefore electrocardiographic QT, prolongation [17, 62].\nThe experiments reported recorded MAP technique [19, 23] from an established intact hypokalaemic murine model [18, 40\u201342] and explored for alternans and arrhythmogenicity and the relationship between these before proceeding to study the effect of lidocaine upon these phenomena. Such studies of temporal heterogeneities in recovery were made at short BCLs in contrast to recent work [40, 41] which examined spatial heterogeneities at long BCLs. The murine system utilised facilitates the introduction of genetic modifications [26] and may therefore permit future comparisons with models replicating congenital arrhythmic syndromes. Indeed, alternans, in association with arrhythmogenicity, has previously been demonstrated in a murine model of LQT5 [50].\nWhile the murine heart is well established as a model for human disease, it must be noted that important interspecies differences exist. Thus, differences between humans and mice in the kinetics of the ion channels carrying the key repolarising currents (the delayed rectifier and slowly-inactivating delayed rectifier currents, IKr and IKs, in humans and the transient outward current, Ito in mice) result in shorter ventricular AP and the absence of a plateau phase in mice [2]. However, in both species, these key repolarising currents are K+-sensitive [6, 11, 16], making the murine heart well suited to modelling clinical hypokalaemia. Furthermore, in both species, depolarisation is rapid and attributable to the same fast Na+ current (INa,f) [4], making our model well suited to the study of Na+-channel-blocking agents such as lidocaine. While both species share similar differences in AP duration between epicardium and endocardium [7], M cells appear to be absent in the murine heart [1]. Nonetheless, relationships between APD and refractory period are similar between the two species [3, 8], and transmural conduction velocities are almost identical [5, 10]. With these caveats in mind, similarities between human and murine hearts make our murine model well suited for the study of the basic mechanisms of arrhythmogenicity.\nFirst, we confirm that alternans is absent in control normokalaemic hearts and that this is associated with the maintenance of stable rhythms even at short BCLs. Secondly, we report alternans in association with arrhythmogenesis at short BCLs for the first time in hypokalaemic murine hearts, in full agreement with previous clinical and experimental studies in other species [2, 7, 33, 38, 52].\nThirdly, we associate such alternans with increases in the slopes of restitution curves above unity [29, 55]. Fourthly, we demonstrate for the first time in any experimental model that exposure to the class I agent lidocaine reduces the slopes of restitution curves and suppresses alternans in association with its anti-arrhythmic effect. Taken together, these observations implicate a restitution mechanism in driving alternans in these hypokalaemic preparations. Furthermore, this extends previous reports that the anti-arrhythmic effects of both class III and IV agents are associated with decreases in the slopes of restitution curves [8, 31, 37]. This contrasts with a previous report that found lidocaine to have no effect on restitution curves obtained using an extrasystolic stimulation procedure or alternans phenomena in a normokalaemic excised canine papillary muscle preparation [43].\nFifthly, we show that such alternans and steeply sloping restitution curves are associated with the temporal evolution of the normal regular pattern of cardiac excitation into arrhythmic activity. The latter, in turn, was processed by a spectral Fourier transform analysis of MAP waveforms performed for the first time in any cardiac system. Arrhythmic MAP waveforms were initially monomorphic, and their corresponding spectra contained a single dominant frequency and integral multiple harmonics identical in epicardial and endocardial recordings. Such monomorphic arrhythmic waveforms subsequently degenerated into polymorphic waveforms whose spectra showed dissimilar epicardial and endocardial dominant frequencies and larger higher order peaks that were not integral multiples of the dominant frequency in full agreement with previous results from the porcine heart [63]. Spectra with similar features have previously been obtained from volume-conducted electrograms and pseudoelectrograms (as opposed to MAPs) from murine hearts during both monomorphic and polymorphic arrhythmic activity [51].\nTaken together, these results recapitulate in a murine system arrhythmogenic patterns classically described in clinical situations [16, 58] and implicate a dynamic component to arrhythmic substrate under conditions of hypokalaemia for which restitution may furnish a possible underlying mechanism. Furthermore, they disprove previous suggestions that small hearts are unable to sustain such arrhythmias [9, 60], and thus further validate the murine heart as a model for the study of clinical arrhythmia. The present findings might therefore form a basis for further explorations of the relationships between heart rate, restitution slopes, alternans and arrhythmogenicity in other states of QT prolongation, whether attributable to congenital or acquired factors.","keyphrases":["alternans","arrhythmia","monomorphic","polymorphic","restitution curve","hypokalaemia"],"prmu":["P","P","P","P","P","P"]}
{"id":"Breast_Cancer_Res_Treat-3-1-2092407","title":"Nuclear morphometric features in benign breast tissue and risk of subsequent breast cancer\n","text":"Certain nuclear morphometric features measured in breast tumor tissue have been shown to predict the prognosis of breast cancer patients. However, the application of these features to predicting risk of breast cancer development has received little attention. We conducted a case-control study to evaluate nuclear morphometric features in benign breast tissue in association with subsequent breast cancer risk. The study was nested within a cohort of 4,888 women with a histopathologic diagnosis of benign breast disease (BBD) and involved 61 cases and 71 controls, amongst whom there were 53 matched case-control sets. Conditional logistic regression models were fitted to assess various measurements of nuclear size and nuclear shape factors in relation to subsequent breast cancer risk. In multivariate analysis, subsequent breast cancer risk was positively associated with a nuclear shape factor that takes the shortest nuclear axis and the longest nuclear axis into consideration simultaneously (highest quartile versus lowest 3 quartiles: odds ratio = 3.07, 95% confidence limits = 1.61, 5.84). In contrast, there was no alteration in subsequent breast cancer risk in association with nuclear size features and other shape factors. In conclusion, our study results suggest that the shape factor that takes both the shortest nuclear axis and the longest nuclear axis into consideration might be of value to predict subsequent development of breast cancer among women with BBD.\nIntroduction\nBenign breast disease (BBD), in addition to certain hormonal, anthropometric, and lifestyle factors, is a well-established risk factor for breast cancer [1, 2]. However, BBD comprises a broad spectrum of histological entities [3]. Both epidemiologic and experimental studies suggest that non-atypical and atypical proliferative changes represent successive steps preceding the development of in situ cancer and then invasive carcinoma of the breast [4]. However, only a small fraction of women will eventually develop breast cancer after their diagnosis of BBD [5]. Therefore, it is important to differentiate BBD patients with a high risk of subsequent development of breast cancer from those with a low risk. Our understanding regarding this issue, however, is rather limited, although previous studies have suggested that factors such as type of histological subtype (e.g., atypical hyperplasia), menopausal status, and family history of breast cancer, might modify breast cancer risk among women with BBD [6].\nComputerized image analysis and morphometry can quantify a number of nuclear morphometric features such as nuclear size, nuclear shape, and chromatin texture [7]. The evaluation of these features may facilitate the diagnosis and management of breast cancer patients [8\u201310]. Indeed, certain nuclear morphometric features measured in breast tumor tissue have been shown to predict the prognosis of breast cancer patients [11\u201315]. Furthermore, a study by Mommers et\u00a0al. [16] observed that normal breast tissue or usual ductal hyperplasia harbored nuclear morphometric changes that might be used to predict subsequent development of breast cancer. In the study reported here, we conducted a nested case-control study to evaluate whether nuclear morphometric features as evaluated in tissue sections of BBD may be related to the risk of subsequent breast cancer among patients with BBD.\nMethods\nStudy population\nThe present investigation was undertaken using histological sections from a previous case-control study nested within the cohort of 4,888 women in the Canadian National Breast Screening Study (NBSS) who were diagnosed histopathologically with BBD during the active follow-up phase of the NBSS [17]. The NBSS is a multi-center randomized, controlled trial of screening for breast cancer among 89,835 women aged 40\u201359\u00a0years at recruitment. The design of the NBSS and population characteristics have been described in detail elsewhere [18, 19]. Recruitment took place between 1980 and 1985, and study subjects were followed actively until 1988. Eligibility for the study was restricted to women with no history of breast cancer (in situ or invasive). The NBSS was approved by the appropriate Institutional Review Boards, and the study described here involved the analysis of material and data from that study in accordance with the approved study design. Informed consent was obtained from all study participants.\nDiagnosis of BBD\nIn the NBSS, women who had clinical or radiologic evidence of breast lesion underwent either a needle aspiration or a biopsy. Diagnosis of BBD was performed by a reference pathologist. Our study was restricted to women who had no evidence of either in situ or invasive breast cancer on their initial surgical biopsy. Women with a history of BBD were not excluded from the analyses. During the follow-up period, we identified 4,888 women with a histopathologic diagnosis of BBD, who were followed up for the subsequent development of breast cancer.\nSelection of cases and controls\nIncident cases of breast cancer were ascertained by record linkage with the provincial cancer registries, and death clearance was performed by linkage to the Canadian National Mortality Database [18, 19]. The dates of the linkages varied by province, ranging from late 1988 to early 1991. A total of 16 subjects with ductal carcinoma in situ and 76 subjects with invasive carcinoma were ascertained among the cohort of women with BBD. Potential control subjects were women with BBD who had not developed breast cancer (but were alive at) by the date of diagnosis of the corresponding case subject. Five controls were selected randomly (with replacement) for each case from those non-cases available within strata defined by screening center, NBSS study arm, year of birth (if possible to the nearest year, and mostly within 2\u00a0years), and age at diagnosis of BBD. For the study reported here, 61 case subjects and 71 control subjects (including 53 matched case-control sets) were included.\nQuestionnaire\nUpon enrollment in the NBSS, all participants completed a questionnaire that sought information on demographic characteristics and risk factors for breast cancer, including menstrual and reproductive histories and family history of breast cancer.\nMorphometry\nMorphometric measurements were performed on H&E stained slides, using the QPRODIT interactive video-overlay system (Leica, Cambridge, UK). About 50 nuclei were selected in the most representative areas of the slide (selected by a breast pathologist), and their contours were traced manually using a 100\u00d7 objective (final magnification about 3,000\u00d7) [20]. Mean and standard deviation of nuclear area, perimeter, diameter, shortest axis, longest axis, and axis ratio were calculated, as well as different shape factors. The shape factors were calculated by the following formulas: Form_AR\u00a0=\u00a0(1\/4)\u00a0*\u00a0pi\u00a0*\u00a0longest axis * shortest axis; Form_PE\u00a0=\u00a04\u00a0*\u00a0pi\u00a0*\u00a0area\/(perimeter squared); Form_NCI\u00a0=\u00a0perimeter\/sqrt (area); Contour ratio\u00a0=\u00a0perimeter squared\/4\u00a0*\u00a0pi\u00a0*\u00a0area; and Roundness\u00a0=\u00a0perimeter\/(2*sqrt (pi\u00a0*\u00a0area)). All morphometric assessments were performed by one observer without knowledge of patient outcome.\nStatistical analysis\nMorphometric measurements were first compared between cases and controls using Student\u2019s t-test. Subsequently, the measurements were categorized by quartiles and then odds ratios (OR) and 95% confidence limits (CLs) were calculated for the risk of breast cancer for those in the highest quartile level compared to that for those in the lowest 3 quartile levels using conditional logistic regression. In multivariate analyses, we controlled for age at menarche (<13, 13, 14+), age at first live birth (nulliparous, <23, 23\u201326, 27+), menopausal status (pre-, peri-, post-), oral contraceptive use (ever versus never), postmenopausal estrogen use (ever vs. never), body mass index (<25, 25+), family history of breast cancer, and the presence of hyperplasia in the benign tissue. All statistical analyses were performed in SAS 9.1 (SAS Institute, Cary, NC). P-values were two-sided.\nResults\nTable\u00a01 summarizes the distribution of selected characteristics among the cases and controls. Overall, few differences between the cases and controls were observed for age at menarche, age at first live birth, menopausal status, oral contraceptive use, postmenopausal estrogen use, body mass index, family history of breast cancer, and the presence of hyperplasia in benign tissue.\nTable\u00a01.Distribution of selected characteristics among breast cancer cases and non-cases N (%)P-valueCasesControlsAge at menarche<1330 (49)26 (37)0.291313 (21)22 (31)14+18 (30)23 (32)Age at first live birthNulliparous11 (18)9 (13)0.84<2322 (36)29 (41)23\u20132619 (31)23 (32)27+9 (15)10 (14)Menopausal statusPre-30 (49)31 (44)0.71Peri-9 (15)14 (20)Post-22 (36)26 (36)Ever used oral contraceptivesYes35 (57)42 (60)0.76No26 (43)28 (40)Missing01Ever used postmenopausal estrogensYes15 (25)15 (22)0.70No46 (75)54 (78)Missing02Body mass index (kg\/m2)<2532 (53)41 (58)0.4225\u2013\u00a0<\u00a03027 (44)25 (35)30+2 (3)5 (7)Family history of breast cancerYes23 (38)28 (39)0.84No38 (62)43 (61)Hyperplasia in benign tissueAbsent34 (59)47 (68)0.27Present24 (41)22 (32)Missing32\nThere was little difference between the cases and controls with respect to nuclear morphometric features including mean area, standard deviation (SD) of area, perimeter, diameter, shortest axis, and longest axis, as well as such shape factors as Form_PE, Form_NCI, contour, and roundness (Table\u00a02). In contrast, the shape factor Form_AR was greater among cases than among controls. Furthermore, subjects with hyperplasia had greater measures of some nuclear size features including mean area, SD of area, perimeter, diameter, and longest axis, and the shape factor Form_AR than did subjects without hyperplasia (data not shown).\nTable\u00a02.Comparison of nuclear morphometric features in benign breast tissue between breast cancer cases and non-casesMorphometric measurementsMean (standard deviation)P-valueCases (n\u00a0=\u00a061)Controls (n\u00a0=\u00a071)Mean nuclear area (\u03bcm2)26.8 (7.5)25.3 (7.2)0.25SD of nuclear area (\u03bcm2)5.2 (1.8)5.0 (1.6)0.43Nuclear perimeter (\u03bcm)19.7 (2.7)19.3 (2.7)0.37Nuclear diameter (\u03bcm)5.8 (0.8)5.6 (0.8)0.23Shortest nuclear axis (\u03bcm)4.8 (0.7)4.6 (0.7)0.16Longest nuclear axis (\u03bcm)7.1 (1.0)7.0 (1.0)0.53Axis ratio1.5 (0.1)1.6 (0.2)0.15Form_AR0.984 (0.005)0.981 (0.007)0.0089Form_PE0.844 (0.037)0.831 (0.045)0.083Form_NCI3.874 (0.095)3.909 (0.122)0.071Contour1.198 (0.061)1.221 (0.080)0.068Roundness1.093 (0.027)1.103 (0.034)0.071\nQuartile analyses revealed that subsequent breast cancer risk was increased in association with the shape factor Form_AR, but not with the other nuclear morphometric measurements (Table\u00a03). Compared to BBD subjects with Form_AR equal to or less than 0.986, subjects with Form_AR greater than 0.986 had a more than three-fold increased risk of developing breast cancer subsequently (OR\u00a0=\u00a03.07, 95%CL\u00a0=\u00a01.61, 5.84). When the analyses were repeated using unconditional logistic regression, which enabled all the available cases and controls to be included, the results did not change substantially.\nTable\u00a03.Risk of Subsequent development of breast cancer in association with nuclear morphometric featuresaMorphometric measurementsCut-off valueOR (95% CL)Model 1bModel 2cMean nuclear area (\u03bcm2)31.21.28 (0.73, 2.25)0.94 (0.50, 1.78)SD of nuclear area (\u03bcm2)6.11.33 (0.76, 2.31)1.11 (0.59, 2.07)Nuclear perimeter (\u03bcm)21.41.14 (0.70, 1.93)0.85 (0.47, 1.55)Nuclear diameter (\u03bcm)6.31.29 (0.73, 2.27)0.95 (0.50, 1.79)Shortest nuclear axis (\u03bcm)5.21.62 (0.92, 2.86)1.18 (0.62, 2.26)Longest nuclear axis (\u03bcm)8.01.34 (0.75, 2.39)0.95 (0.50, 1.81)Axis ratio1.60.59 (0.30, 1.17)0.71 (0.33, 1.54)Form_AR0.9862.45 (1.42, 4.22)3.07 (1.61, 5.84)Form_PE0.8671.22 (0.71, 2.08)1.57 (0.83, 2.97)Form_NCI3.9351.07 (0.58, 1.98)1.18 (0.61, 2.27)Contour1.2361.13 (0.61, 2.10)1.22 (0.63, 2.35)Roundness1.1101.07 (0.58, 1.98)1.18 (0.61, 2.27)a\u00a0Analyses were conducted among 53 matched case-control sets by comparing the highest quartile versus the lowest 3 quartiles in conditional logistic regression modelsb\u00a0Adjusted for matching variablesc\u00a0Adjusted for matching variables, age at menarche (<13, 13, 14+), age at first live birth (nulliparous, <23, 23\u201326, 27+), menopausal status (pre-, peri-, post-), oral contraceptive use (ever vs. never), postmenopausal estrogen use (ever versus never), body mass index (<25, 25+), family history of breast cancer, and the presence of hyperplasia in the benign tissue\nDiscussion\nWe found that breast cancer risk in women with BBD was positively associated with the shape factor Form_AR, a measurement that takes the shortest nuclear axis and the longest nuclear axis into consideration simultaneously. In contrast, there was no alteration in risk in association with nuclear area, SD of nuclear area, nuclear perimeter, nuclear diameter, shortest nuclear axis, longest nuclear axis, and other shape factors. Although subjects with hyperplasia had greater measures of Form_AR than did subjects without hyperplasia, we adjusted for hyperplasia, suggesting that the association with Form_AR is independent of that due to the presence of hyperplasia.\nShape is one of the factors that pathologists use in assessing nuclear atypicality. Shape factors have been shown to have prognostic value in breast cancer [21\u201323], renal cell cancer [24], colorectal cancer [25], squamous cell carcinoma of the larynx [26], melanoma [27], and rhabdomyosarcoma [28]. Apparently, alterations in nuclear shape can already be present at the earliest stages of carcinogenesis. This has in the breast also been shown for nuclear chromatin patterns [29].\nTo date, only one study has been published that assessed morphometric features in association with subsequent development of breast cancer among women with BBD [16]. That study found positive associations for mean nuclear area, nuclear diameter, nuclear perimeter, and the longest nuclear axis, but no associations for SD of the nuclear area and the shortest nuclear axis; shape factors were not evaluated. However, potential confounding factors were not controlled for. In contrast to these findings, nuclear size features were not associated with risk in the present study, which may perhaps be explained by differences in tissue processing procedures.\nOur case-control study was nested in a cohort of patients with histopathologically confirmed BBD and our findings are likely to be internally valid. Biased measurement of the study exposures was not likely a source of error, given that the morphometric features were assessed without knowledge of the patient outcome status. Our study power, however, was limited by the relatively small sample size, due to which we were not able to evaluate modifying effects by well-documented risk factors of breast cancer. Moreover, residual confounding might still exist, although to minimize confounding we controlled for menstrual and reproductive history, exogenous estrogen use, body mass index, and family history of breast cancer in multivariate analyses.\nIn conclusion, our study results suggest that the shape factor that takes both shortest nuclear axis and longest nuclear axis into consideration might be of value to predict subsequent development of breast cancer among patients with BBD. Given the limitations of our study, larger studies are warranted to confirm our study results.","keyphrases":["breast cancer","benign breast disease","nuclear size","nuclear shape","morphometry"],"prmu":["P","P","P","P","P"]}
{"id":"Purinergic_Signal-1-3-2096543","title":"Tri-nucleotide receptors play a critical role in epithelial cell wound repair\n","text":"The cornea plays a major role in the refraction of light to the retina. Therefore, the integrity and transparency of the corneal epithelium are critical to vision. Following injury, a combination of rapid signal transduction events and long-term cell migration are essential for wound closure. We have demonstrated previously that injury resulted in the release of nucleotides that induce the propagation of a Ca2+ wave to neighboring cells. This suggests that nucleotides and their receptors are critical components of wound healing. Epidermal growth factor (EGF) and integrins also have been shown to play a role in injury. In this study, we demonstrate that pretreatment of cells with ATP and UTP inhibited the immediate wound response, while BzATP, ADP, and UDP did not affect this response. Tri-nucleotide pretreatment also reduced the EGF induced Ca2+ response. Additionally, lower EC50 concentrations of ATP and UTP triggered migration of cells that was enhanced further with EGF and was inhibited by the tripeptide, RGD. Results indicate that the desensitization induced by ATP and UTP was specific. While ADP and UDP cause a homologous desensitization of their own signal, they did not cause an inhibition of the wound response nor does BzATP. Neither Ca2+ wave propagation nor cell migration occurred in response to \u03b2,\u03b3-MeATP. Together these results lead us to hypothesize that corneal epithelial wound repair is mediated by both P2Y2 and P2Y4 receptors.\nIntroduction\nWound healing is a series of complex biological responses requiring cytoskeletal and extracellular matrix remodeling, signal cascades, and gene regulation. Initial and long-term signals after injury are communicated to the cells and promote wound healing. These signals and events must be understood to advance our knowledge of normal wound repair. Investigators have demonstrated that nucleotides are released from cells by mechanical stimulation, ligand binding, exocytotic release, or injury [1\u20135] Nucleotide stimulation can also lead to an increase in intracellular calcium. The increase in cytosolic calcium can be generated via activation of purinergic (P2Y or P2X) receptors through distinct mechanisms. In addition, purinergic receptors can promote cell migration, a critical component of wound repair. The exact role and identity of the various receptors and their importance in different systems has yet to be elucidated.\nNucleotides have been shown to accelerate wound closure [6]. Nucleotides can act as a chemotactic stimulus for cell migration in many cell types including corneal epithelial [7], arterial smooth muscle [8], microvascular endothelial [9], and immature dendritic cells [10]. P2 receptors also have been shown to play a role in repair after ischemia and renal tubular epithelial wound healing [11, 12]. Previously, we demonstrated that ATP plays a critical role in intercellular communication following mechanical injury. ATP induced cell migration, and Reactive Blue-2, a P2 antagonist, inhibited wound closure [7]. In addition, nucleotides induced calcium waves and phosphorylation of paxillin (pY118) and ERK1\/2 in a transient manner [7, 13]. Furthermore, neither occurred in the presence of \u03b2,\u03b3-MeATP [7, 13]. Our goal is to determine how epithelial cells respond to nucleotides in order to understand the events that occur after injury.\nP2Y and P2X receptors are expressed in a number of cell types. As G-protein coupled receptors (GPCR), P2Y receptors are seven pass transmembrane receptors coupled to heterotrimeric G-proteins (review in [14]). P2Y receptors couple to G\u03b1q and activate phospholipase C \u03b2 (PLC\u03b2), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to diacylglycerol (DAG) and phosphoinositol tri-phosphate (IP3). IP3 binds to Ca2+ channels on the endoplasmic reticulum and causes an increase in intracellular Ca2+. This family of receptors consists of eight members: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14. P2X receptors are ligand gated ion channels that open upon agonist binding allowing extracellular calcium to enter the cell. This family of receptors has seven members: P2X1\u20137. Each receptor has a specific agonist potency profile for different nucleotides with some showing preference for a specific nucleotide and others preferentially responding to tri-nucleotides or adenine based nucleotides [15]. We have shown that corneal epithelial cells express a number of P2YR subtypes [7], and an intracellular Ca2+ increase is seen in response to both nucleotides and injury [5].\nEpidermal growth factor receptor (EGFR) also plays an important role in injury response. The EGFR family has four receptor family members, ErbB1-4. These receptors can form both homo- and hetero-dimers when stimulated by the epidermal growth factor (EGF) or other ligands [16]. The EGFR becomes activated upon injury, and inhibition of ErbB phosphorylation limits proper wound closure in vitro [7]. EGF itself promotes cell migration and wound healing in vivo [17] and can up-regulate integrin expression [18]. Gross mechanical injury induces a Ca2+ wave that propagates out from the wound site. EGF has been shown to increase the intensity of the Ca2+ wave but is not required for propagation [5]. Recently, growth factor receptors and GPCRs have been demonstrated to participate in cross talk connecting signaling pathways through trans-activation, which can occur via enhancement or down-regulation of downstream signal cascades [19]. In addition, specific P2 receptors can associate with other signaling proteins. For example, P2Y2 has an arginine-glycine-aspartic acid (RGD) domain on its first extracellular loop that can associate with integrins [20]. Currently, no other P2Y receptor has an identified RGD domain that interacts with integrins. In addition, this RGD domain promotes signaling and interactions with epidermal growth factor receptors (EGFR) [21].\nOur goal is to determine if specific P2 receptors play a role in the injury response and wound healing in the cornea. Improper wound repair can alter refraction of light, one of the major roles of the cornea. Immediately after injury, corneal epithelial cells display an intracellular Ca2+ wave that propagates from the site of injury to neighboring cells [5]. This wave does not propagate via gap junctions but is mediated by the release of extracellular nucleotides activating P2Y receptors that lead to activation of intracellular signaling pathways such as ERK1\/2 [13]. We hypothesize that injury induces an immediate localized event that stimulates later events such as release of growth factors and long-term signals.\nIn this paper, we provide evidence that specific subtypes of purinergic receptors regulate the injury response. Pretreatment of cells with ATP and UTP inhibited the propagation of the injury induced Ca2+ wave while BzATP, ADP, and UDP did not participate in the desensitization. In addition, pre-stimulation with tri-nucleotides, but not BzATP or di-nucleotides, resulted in a decrease in the intracellular Ca2+ release induced by EGF. ATP and UTP preferentially enhanced later cellular events, including migration. Cellular migration was enhanced further when cells were co-stimulated with EGF. In addition, the ATP mediated migration was reduced by the tri-peptide, RGD. The results indicate that immediate and long-term components of the wound response are mediated by tri-nucleotide receptors.\nMaterials and methods\nReagents\nThe nucleotides [adenosine 5\u2032-triphosphate (ATP), uridine 5\u2032-triphosphate (UTP), adenosine 5\u2032-diphosphate (ADP), uridine 5\u2032-diphosphate (UDP), \u03b2,\u03b3-methyleneadenosine 5\u2032-triphosphate (\u03b2,\u03b3-MeATP), and 2\u2032-3\u2032-O-(4-Benzoylbenzoyl) adenosine 5\u2032-triphosphate (BzATP)], adenosine, apyrase, and Reactive Blue 2 were purchased from Sigma Chemical Company (St. Louis, MO). Human recombinant epidermal growth factor (EGF) and penicillin-streptomycin were purchased from Invitrogen Corporation (Carlsbad, CA). The fluorescent Ca2+ indicator dye Fluo-3AM and pluronic acid were from Molecular Probes, Inc. (Eugene, OR). 1,2-bis(o-aminophenoxy)ethane-N,N,N\u2032,N\u2032-tetraacetic (BAPTA) was purchased from Calbiochem (La Jolla, CA). RGD and RGE peptides were purchased from American Peptide (Sunnyvale, CA).\nCell culture\nPrimary rabbit corneal epithelial cells and an established human corneal cell line were used. Human epithelial cells transformed (HCE-Ts) were established by Araki-Sasaki et al. [22], using an SV-40 recombinant adenovirus vector. Primary corneal epithelial cells were isolated and cultured from freshly extracted whole rabbits eyes (Pel-Freeze Biologicals (Rogers, AR) [7].\nCa2+ imaging\nHCE-T cells and primary cells were grown to confluency on 22 mm2 coverslips and quiesced 18 to 24 h before experiments in unsupplemented serum-free keratinocyte medium (K-SFM). Cells were washed in a HEPES-buffered saline solution containing 137 mM NaCl, 5 mM KCl, 4 mM MgCl2, 3 mM CaCl2 \u00b7 2H2O, 25 mM glucose and 10 mM HEPES [23]. Cells were incubated 20\u201330 min in 5 \u00b5M Fluo-3AM resuspended in HEPES buffer supplemented with 0.02% pluronic acid in DMSO [5, 7, 13]. Coverslips were mounted in a flow through perfusion apparatus connected to inlet tubes with varying solutions and an outlet pump (Warner Instruments). The mounted coverslip was placed on the stage of a Zeiss Axiovert LSM 510 laser scanning confocal microscope, and cells were scanned with an Argon laser every 786 ms [7]. Cells were perfused with HEPES buffered saline at a rate that did not elicit a response, and baseline fluorescence was obtained. Cells were stimulated either by perfusing nucleotides or EGF prepared in HEPES buffered saline and\/or by manually perturbing cells [5].\nCa2+ data analysis\nCa2+ data was recorded by LSM software as average fluorescence intensity (F) for a region of interest over time. The region was either a 460 \\smm \\sx 460 \\smm field or single cells, over time. Raw data was entered into KaleidaGraph. Percent change in average fluorescence was calculated using the following formula  where F0 is initial average fluorescence for the region of interest [5, 7]. Data was either graphed as average maximal fluorescence of at least three different experiments or as percent change in average fluorescence for time course. For the injury experiments, the area cleared by the wound was excluded from calculations.\nMigration assay\nHCE-T cells were quiesced 18 to 24 h in unsupplemented K-SFM. Cells were washed in phosphate buffered saline (PBS), trypsinized, and the reaction was stopped with filtered soybean trypsin inhibitor as described previously [7]. Cells were resuspended at a concentration of 125,000 cells\/100 \u00b5l in binding buffer (unsupplemented K-SFM, 0.05% gelatin and 25 mM HEPES). Binding buffer, 600 \u00b5l, with or without added stimuli was placed in the wells of a 24 well plate. Costar Transwell inserts (6.5-mm diameter polycarbonate membranes, 8 mm pore size) were placed into the wells, and 100 \u00b5l of the cell suspension was added to the inserts. Experiments were conducted for 8 h at 37 \u00b0C.\nTo collect conditioned wound medium, cells were washed and incubated in binding buffer. Cells were scrape wounded as described previously [13]. The wound medium was collected immediately and used in migration assays. Unwounded medium was defined as binding buffer added to cells and collected without injury to cells. The conditioned wound medium was also treated with apyrase prior to the migration assays.\nTo determine migration, upper and lower chambers were aspirated and rinsed with PBS. Cells were fixed at room temperature for 10 min, rinsed with PBS, and non-migrated cells were removed. Migrated cells were stained with propidium iodide (5 \u00b5g\/ml) (Molecular Probes, Inc., Eugene, OR) for 5 min and rinsed with PBS. Membranes were mounted onto a slide with SlowFade Antifade (Molecular Probes, Inc., Eugene, OR). For each membrane (33.2 mm2), six random 10\u00d7 fields (one field = 1.37 mm \u00d7 1.08 mm, or 1.48 mm2) were photographed. Cells were counted and averaged for each membrane and experiments were performed in triplicate.\nStatistical analysis\nValues were given as the mean \\+- SD. Statistical comparisons were made using Student\u2019s t-test with a stringency of P > 0.01 for significance. Data were fit using KaleidaGraph to calculate concentration for half the maximal response (EC50) and maximal possible change in fluorescence for a given agonist (\\gD Fmax). A generalized single-site binding model was used as follows: , where \\gDF is the percent change in fluorescence and [L] is the concentration of the agonist.\nResults\nNucleotide dose response curves\nCa2+ dose response curves were determined for ATP, UTP, ADP, UDP, and BzATP over a concentration range of 10\u22127 to 10\u22123 M (Figure 1). The response to ATP and UTP was greater than that of ADP and UDP over the entire range. The BzATP response was similar to ADP and UDP at concentrations \u226410\u22124 but lower at higher concentrations. In addition, adenosine, an agonist for P1 receptors, and \u03b2,\u03b3-MeATP, an agonist for P2X receptors, were evaluated. Neither of these agonists induced a change in intracellular Ca2+ (data not shown). The EC50 for ATP and UTP were found to be (3.4 \u00b1 1.8) \u00d7 10\u22126 M and (3.4 \u00b1 2.0) \u00d7 10\u22126 M respectively. The EC50 for ADP and UDP were higher than the tri-nucleotides, (1.8 \u00b1 0.9) \u00d7 10\u22124 M and (1.83 \u00b1 0.12) \u00d7 10\u22124 M. respectively. The EC50 for BzATP was (8.6 \u00b1 5.9) sx 10\u22125. R values for line fit were 0.96 for ATP, UTP, and UDP, 0.93 for ADP, and 0.91 for BzATP.\nFigure 1Nucleotide induced Ca2+ dose response curves. HCE-T cells were incubated in 5 \u00b5M Fluo-3AM for 30 min and imaged every 786 ms using a flow through apparatus on an LSM 510 confocal. Cells were stimulated with the indicated concentration of nucleotide for 60 s. Maximal percent change in average fluorescence of a 460 \u00b5m \u00d7 460 \u00b5m field was determined. Data was fit to a generalized single-site binding model. Graph represents a minimum of three experiments at each concentration tested.\nTo verify that P2 receptors caused the intracellular Ca2+ increase, cells were pre-incubated for 30 min in 100 \u00b5M Reactive Blue 2 prior to experimentation. Cells were placed in a flow through system and washed for at least 50 s in HEPES buffered saline. Cells were stimulated with 100 \u00b5M of the indicated nucleotide for 100 s and washed again with HEPES buffer. Non-pretreated cells were used as control. Reactive Blue 2 lowered the Ca2+ response to nucleotides. The ATP and UTP responses were reduced by one-fifth, and the ADP and UDP responses were reduced by half.\nRole of nucleotides in injury response\nWe have demonstrated that epithelial cells respond to nucleotides in a saturable manner. However, these results did not identify which P2 receptors were involved in the injury response. To evaluate the wound response, cells were placed in a flow through apparatus. Background images were collected in HEPES buffered saline, and the cells perturbed manually (Figure 2a). Percent change in average fluorescence of individual cells adjacent to the wound (Figures 2b, 2c (#1\u20135)) are shown inside the large white circle and graphed (Figure 2c). These depict a rapid increase in fluorescence after injury that is followed by attenuation. The rate of attenuation of injured cells was variable. While these cells did respond they were not a part of the calcium wave that propagated from the site of injury [5]. We also examined cells not immediately adjacent to the wound, cells involved in the wave (Figures 2b, 2d). When individual cells that were not adjacent to the wound were evaluated [(Figures 2b, 2d (#6\u201310) (outside of white circle)], the percent response was lower on average and the response time was delayed (Figure 2d). The delay in response time could be accounted for by the time it took the Ca2+ wave to travel the distance to nonadjacent cells.\nFigure 2Pre-stimulation with ATP inhibits injury induced Ca2+ wave. Primary corneal epithelial cells were incubated in 5 \u00b5M Fluo-3AM for 30 min and imaged in a flow through apparatus on an LSM 510 confocal. Cells were washed in HEPES buffered saline with Ca2+ for at least 30 s, and stimulated by wounding or with ATP. Individual cells were analyzed for percent change in average fluorescence. Intensity scale is shown in (a) with red indicating highest Ca2+ levels and blue indicating lowest Ca2+ levels. The horizontal white bar in (a) represents 100 \u00b5m. a) Cells were washed in HEPES buffer containing Ca2+ and wounded. A series of images taken from a time course of a representative experiment of a wound (shown at asterisk) is presented. b) A single image taken after wounding is shown. Cells (#1\u20135) immediately adjacent to the wound (c) and cells away (#6\u201310) from the wound (d) were analyzed. e) Cells were washed in HEPES buffer containing Ca2+, stimulated with 100 \u00b5M ATP (80 s), and wounded (157 s). A series of images taken from a time course of a representative experiment is presented (wound shown at asterisk). f) A single image taken after wounding is shown. Cells (#1\u20135) immediately adjacent to the wound (g) and cells away (#6\u201310) from the wound (h) were analyzed. Images are representative of at least 10 independent experiments.\nTo evaluate what role ATP played in the initial injury response, cells were stimulated by ATP and wounded. To perform the experiment, cells were placed in the flow through apparatus, equilibrated with HEPES buffered saline, and stimulated with HEPES buffered saline containing 100 \u00b5M ATP. The response to ATP was allowed to attenuate (approx. 80 s), and then the cells were wounded (Figure 2e). The cells were analyzed for average change in percent fluorescence for individual cells adjacent to the wound (Figures 2f, 2g) and those involved in wave propagation (Figures 2f, 2h). As predicted from Figure 2a and d, cells adjacent to the wound responded (Figures 2f, 2g). The Ca2+ wave was absent when it followed pretreatment with ATP (Figures 2f, 2h). In its place was a minor inflection at the time of injury that resembled the wound response induced in the absence of intracellular Ca2+ [5] (see Figure 5). These responses indicate that the injury response was inhibited by the desensitization of specific P2Rs by ATP.\nFigure 5Ca2+ free media inhibits an injury response after ATP stimulation. Primary corneal epithelial cells were incubated in 5 \u00b5M Fluo-3AM for 30 min and imaged in a flow through apparatus on an LSM 510 confocal. Cells were washed in HEPES buffered saline for at least 30 s, and stimulated by wounding or ATP. Individual cells were analyzed for percent change in average fluorescence. Intensity scale is shown in (a) with red indicating highest Ca2+ levels and blue indicating lowest Ca2+ levels. The horizontal white bar in (a) represents 100 \u00b5m. a) Cells were pre incubated in BAPTA (100 \u00b5M) washed in HEPES buffer containing Ca2+ and wounded. A series of images taken from a time course of a wound (shown at asterisk) of a representative experiment is presented. b) A single image taken after wounding is shown. Cells (#1\u20135) immediately adjacent to the wound (c) and cells away (#6\u201310) from the wound (d) were analyzed. e) Cells were washed in Ca2+ free HEPES buffer containing EGTA, stimulated with 100 \u00b5M ATP, and wounded. A series of images taken from a representative time course is presented (wound shown at asterisk). f) A single image taken after wounding is shown. Cells (#1\u20135) immediately adjacent to the wound (g) and cells away (#6\u201310) from the wound (h) were analyzed. Images are representative of at least 10 independent experiments. The series of images are taken from Movie 2 (see online version of article at www.springeronline.com).\nTo determine which nucleotides could desensitize the Ca2+ release in response to wounding, cells were pretreated with nucleotides and injured (Figures 3 and 4). Both ATP and UTP pre-stimulation inhibited the wound-induced wave (Figure 3), while ADP, UDP, and BzATP did not inhibit the injury induced Ca2+ wave (Figure 4). Together, these results indicate that both ATP and UTP desensitize receptors and inhibit the injury response at an equivalent molar concentration.\nFigure 3Tri-nucleotides attenuate the injury induced Ca2+ wave. Primary corneal epithelial cells were incubated in 5 \u00b5M Fluo-3AM for 30 min and imaged in a flow through apparatus on an LSM 510 confocal. Cells were washed in HEPES buffered saline with Ca2+ for at least 30 s and stimulated with the indicated nucleotide and wounded. Intensity scale is shown in (a) with red indicating highest Ca2+ levels and blue indicating lowest Ca2+ levels. The horizontal white bar in (a) represents 100 \u00b5m. a, b) Cells were washed in HEPES buffer with Ca2+, stimulated with 100 \u00b5M of the indicated nucleotide, and wounded. A series of images taken from a time course of a representative experiment is presented (wound shown at asterisk). c, d) Percent change in average fluorescence for the whole field (460 \u00b5m \u00d7 460 \u00b5m) was calculated and graphed over the time course for each experiment. Images are representative of at least 10 independent experiments. Series of images are taken from Movie 1 (see online version of article at www.springeronline.com).Figure 4Di-nucleotides and BzATP do not attenuate the injury induced Ca2+ wave. Primary corneal epithelial cells were incubated in 5 \u00b5M Fluo-3AM for 30 min and imaged in a flow through apparatus on an LSM 510 confocal. Cells were washed in HEPES buffered saline with Ca2+ for at least 30 s and stimulated with the indicated nucleotide and wounded. Intensity scale is shown in (a) with red indicating highest Ca2+ levels and blue indicating lowest Ca2+ levels. The horizontal white bar in (a) represents 100 \u00b5m. (a\u2013c) Cells were washed in HEPES buffer with Ca2+, stimulated with 100 \u00b5M of the indicated nucleotide, and wounded. A series of images taken from a time course of a representative experiment is presented (wound shown at asterisk). (d\u2013f) Percent change in average fluorescence for the whole field (460 \u00b5m \u00d7 460 \u00b5m) was calculated and graphed over the time course for each experiment. Images are representative of at least 10 independent experiments. Series of images are taken from Movie 1 (see online version of article at www.springeronline.com).\nTo evaluate the inflection detected in Figures 2e\u2013h, cells were pretreated with BAPTA (100 \u00b5M) for 30 min and individual cells were analyzed (Figures 5a\u20135d). Cells responded to the injury in a manner similar to the cultures that had been pretreated with ATP (Figures 3 and 4). These results indicate that the cellular response adjacent to the wound did not occur via P2Y receptor signaling since the Ca2+ increase came from extracellular stores (Figures 5a\u20135d). This agrees with previous results using thapsigargin [5]. To evaluate the requirement for extracellular Ca2+, cultures were placed in the flow through apparatus in Ca2+ free HEPES buffer containing EGTA. These cells were stimulated with 100 \u00b5M ATP made in Ca2+ free HEPES buffered saline. Once the response had attenuated (approx. 100 s), the cells were wounded (Figure 5e), and individual cells were analyzed for average change in percent fluorescence in cells adjacent to the wound margin (Figures 5f, 5g) and those involved in wave propagation (Figures 5f, 5h; as in Figure 2). When the experiment was performed in Ca2+ free media, the inflection detected previously in the injury response was absent, completely eliminating any Ca2+ response to injury (Figures 5e\u20135h).\nNucleotides affect EGF induced Ca2+ response\nCross-talk between the EGF and ATP signaling pathways occurs in many cell systems. To determine if this was a property unique to ATP, the response to EGF was evaluated after pre-stimulation with other nucleotides. HCE-Ts were perfused with HEPES buffered saline to establish baseline fluorescence, and the cells were stimulated with 100 \u00b5M ATP, UTP, ADP, UDP or BzATP for 100 s. Cells were washed with HEPES for 100 s and stimulated with 8 nM EGF for 100 s. Maximal percent change in fluorescence of the entire 460 \u00b5m \u00d7 460 \u00b5m field was calculated (Figure 6a). The results were compared with cells washed with HEPES buffer and stimulated with 8 nm EGF alone for 100 s (\u2212 pretreatment). Cells exhibited an attenuated Ca2+ response upon EGF stimulation when they were pretreated with either ATP or UTP. In contrast, when the cells were pre-stimulated with BzATP, ADP, or UDP, there was no detectable desensitization (Figure 6a). The reverse experiment was performed where cells were pre-stimulated with EGF and then by specific nucleotides (data not shown). Pretreatment with EGF did not desensitize the cells indicating that EGFR stimulation with EGF did not inhibit the response to nucleotides.\nFigure 6Injury and tri-nucleotides decrease subsequent EGF induced Ca2+ response. HCE-T cells were incubated in 5 \u00b5M Fluo-3AM for 30 min and imaged every 786 ms in a flow through apparatus on an LSM 510 confocal. a) Cells were stimulated with 100 \u00b5M of the indicated nucleotide for 100 s, washed in HEPES buffered saline for 100 s, and then stimulated with 8 nM EGF or stimulated with EGF without pretreatment. Maximal percent change in average fluorescence of a 460 \u00b5m \u00d7 460 \u00b5m field was determined. b) Cells were either wounded and stimulated with 8 nM EGF or stimulated with EGF without injury. Individual cells were selected for analysis. In the wound model, cells that responded to the wound were selected. (t-test, * P < 0.0005) Data represent a minimum of three independent experiments.\nAs desensitization occurred after cells were pre-stimulated with ATP or UTP and wounded or stimulated with EGF, our goal was to determine if desensitization of the EGF receptor occurred in our wound model. Cells were placed in the flow through apparatus, perfused with HEPES buffered saline to establish baseline fluorescence, and injured. Once fluorescence returned to the base line, the cells were stimulated with EGF. The analysis was performed on individual cells that responded to both the injury and EGF (Figure 6b). Results were compared to individual cells stimulated with EGF alone. The Ca2+ response to EGF was significantly lowered after injury. The high standard deviation was due to the variable size of the wound.\nNucleotides and EGF induce cell migration\nThe long-term effects of P2 receptors on wound healing were examined using Transwell migration assays. Previously we showed that P2 receptors were necessary for wound repair in directed migration scratch assays [7]. Furthermore, we demonstrated that migration was optimal at 1 \u00b5M for ATP and 0.16 nM for EGF in Transwell assays [7]. In these experiments, migration to other nucleotides and adenosine were evaluated over a range of concentrations from 0.1 to 100 \u00b5M (Figure 7). Optimal migration for ADP and BzATP occurred at 10 \u00b5M (Figures 7a, b), while optimal migration for ATP and UTP occurred at 1 \u00b5M ([7] and data not shown). UDP and \u03b2,\u03b3-MeATP did not induce migration at any concentration (Figures 7c, d). To assess preferential migration to any one particular nucleotide or adenosine, the nucleotides and adenosine were evaluated and compared. The cellular response was tested at 1 \u00b5M, the optimal concentration for ATP and UTP. In addition, ADP was tested at 10 \u00b5M, its optimal concentration. ATP, UTP, and ADP induced comparable migration at their optimal concentrations (162 \u00b1 39, 170 \u00b1 70, and 188 \u00b1 47 cells\/1.48 mm2, respectively). However, ADP at 1 \u00b5M, UDP, adenosine, or binding buffer did not induce cell migration (23 \u00b1 7, 18 \u00b1 7, 31 \u00b1 6 and 29 T 11 cells\/1.48 mm2, respectively) (Figure 7e).\nFigure 7Cell migration is nucleotide specific. Transwell migrations were performed for 8 h at 37 \u00b0C with the indicated nucleotide. HCE-T cells were stained with propidium iodide, counted in six randomly chosen fields (1.46 mm2), and averaged. Dose response curves for a) ADP, b) BzATP, c) UDP, and d) \u03b2,\u03b3-MeATP are shown. Experiments were performed at least three times and representative curves were chosen. e) Cells were stimulated with binding buffer (neg), 1 \u00b5M ATP, UTP, ADP, UDP, or Adenosine (Ado), or 10 \u00b5M ADP. Experiments were performed in triplicate.\nWe evaluated the role of factors released into the wound media on cell motility and compared these effects to stimulation with nucleotides and EGF. Wound media was collected immediately after injury and added to migration assay chambers. Luciferase assays performed on wound media demonstrated presence of ATP [5]. Cell migration was 3.7-fold greater when cells were stimulated with wound media compared to wound media treated with apyrase or unwounded control media (Figure 8a). Previously we established that epithelial cells migrate to ATP or EGF in a dose dependent manner and optimal migration was determined to be 1 \u00b5M and 0.16 nM respectively [24]. To evaluate the combined effect of ATP and EGF, cells were co-stimulated with the optimal concentrations of each and compared. In a representative experiment an average of 369 (\u00b134) cells migrated\/1.48 mm2 in response to 0.16 nM EGF while an average of 314 (\u00b131) cells migrated\/1.48 mm2 in response to 1 \u00b5M ATP (Figure 8b). When cells were co-stimulated with 1 \u00b5M ATP and 0.16 nM EGF, migration increased more than threefold with an average of 1043 (\u00b166) cells\/1.48 mm2 (Figure 8b). Co-stimulation experiments were performed with UTP, ADP and UDP at their respective optimal concentrations (Figures 7 and 8c). Cells exposed to ADP or UDP (1 \u00b5M) and co-stimulated with EGF (527 \u00b1 61 and 637 \u00b1 51 cells\/1.48 mm2, respectively) showed migration similar to that of EGF alone (457 \u00b1 115 cells\/1.48 mm2). When cells were co-stimulated with UTP and EGF, migration was enhanced in an additive manner (801 \u00b1 133 cells\/1.48 mm2). In addition when cells were exposed to 10 \u00b5M ADP and EGF, migration was greater than either alone (753 \u00b1 173 cells\/1.48 mm2).\nFigure 8Wound media, nucleotides, and EGF mediate cell migration. Transwell migrations were performed for 8 h at 37 \u00b0C with the indicated nucleotides and\/or EGF. HCE-T cells were stained with propidium iodide, counted in six randomly chosen fields (1.46 mm2) and averaged. a) Migration of cells stimulated with wound media were compared to those stimulated with unwounded control media, wound media treated with apyrase and binding buffer (negative). b) A representative experiment is shown where cells were stimulated with optimal concentration of ATP (1 \u00b5M) and EGF (0.16 nM) and compared to co-stimulation with ATP and EGF and negative binding buffer. c) Cells were simulated with binding buffer (neg), 1 \u00b5M UTP, ADP, or UDP, or 10 \u00b5M ADP +\/\u2212 0.16 nm EGF.\nTo understand how nucleotides regulate cell migration, we asked whether integrins mediated the response. Integrins are known to play an important role in cell migration, and the integrin binding tri-peptide, RGD, that is found in the first extracellular loop of the P2Y2 receptor, has been shown to interact with integrins [20]. In addition, expression of \u03b16\u03b24 in corneal epithelial cells is up-regulated by EGFR activation [18]. To evaluate the role of integrin receptors in nucleotide mediated migration, RGD peptide was added to the cell suspension prior to its addition to the Transwell chamber. RGE peptide was used to control for non-specific binding. Cells were migrated to binding buffer or 1 \u00b5M ATP. When either the RGD or RGE peptide was added to the cells migrating to binding buffer, there was no detectable difference over control (Figure 9). Cells stimulated with ATP migrated 362 (\u00b192) per field. Interestingly, cell migration was reduced almost fourfold when RGD was present (96 \u00b1 43 cells\/1.48 mm2) (Figure 9). The control peptide did not decrease migration significantly from control (284 \u00b1 59). Since RGD peptide can inhibit migration, it is likely integrins play a role in ATP induced cell migration. The nucleotide receptors, EGFR, and integrins mediate calcium signaling and cell migration. Thus, interaction between these protein families may be critical in regulating the cellular signals after injury.\nFigure 9ATP and integrins mediate cell migration. Transwell migrations were performed for 8 h at 37 \u00b0C with the indicated nucleotides. HCE-T cells were stained with propidium iodide, counted in six randomly chosen fields (1.46 mm2) and averaged. RGD or RGE peptide, 1 \u00b5M, was added to the cell suspension. Cells were stimulated with binding buffer (control) or 1 \u00b5M ATP. (t-test, *P < 0.0005) Experiments were performed in triplicate.\nDiscussion\nThe results of these studies demonstrate that P2 receptors play a role in the initial cellular responses that occur after injury and the long-term signals that are necessary for wound closure in corneal epithelial cells. We have demonstrated that nucleotides are the active component released with injury and are responsible for the intercellular Ca2+ wave that is propagated in neighboring cells [5, 13]. We have shown that ATP, UTP, ADP, UDP and BzATP promote an intracellular Ca2+ increase, \u03b2,\u03b3Me-ATP does not. Our goal was to demonstrate that nucleotides play a prominent role in the cellular response to injury, in migration, and in the cross-talk with EGF.\nRecently involvement of P2 receptors in wound healing has begun to be demonstrated [2]. Desensitization experiments demonstrate the role of nucleotides in the injury response. We have shown pretreatment of cells with ATP and UTP inhibits the response to a subsequent wound (Figure 3). Several explanations are plausible: 1) Specific receptors desensitize via receptor internalization or signal transduction down regulation [25] or 2) When cells are exposed to a solution of a tri-nucleotide, ATP and\/or UTP receptors have bound agonist thus saturating the receptors. We predict that the amount of nucleotide used to desensitize the wound approximates the concentration needed for the maximal Ca2+ response (Figure 1). In either case, the result is that nucleotides that are released would not be able to induce any further effect upon the receptors. In addition, injury, ATP and UTP, induce a decrease in the EGF induced Ca2+ response (Figure 6). Since nucleotides are released with injury [13], we hypothesize that desensitization of the EGF receptor occurs via nucleotides. In contrast, while ADP and UDP cause homologous desensitization of their own Ca2+ signal [7], ADP, UDP, and BzATP do not affect either the EGF or wound response even when the receptors are saturated (Figures 4 and 6). Therefore, we hypothesize that P2Y1, P2Y6, and P2X7 receptors do not play a role in the Ca2+ wave that occurs after injury.\nWe have demonstrated that the source of the calcium for the injury induced wave is intracellular ([5] and Figure 5). Pretreatment with either thapsigargin or BAPTA inhibits the Ca2+ wave but not oscillations of cells at the wound margin. In addition, pretreatment with either ATP or UTP inhibits only the wave but not the Ca2+ increase in the cells adjacent to the wound. Cells adjacent to the wound edge utilize extracellular calcium (Figure 5), but the method of extracellular calcium entry is unknown. One possibility is that the plasma membranes of the injured cells are damaged, and Ca2+ freely enters. The response could also be mediated via stress receptors or P2X receptors, both of which are ion channels that permit entry of extracellular calcium [26]. While these cells are not involved in the propagation of the wave, we cannot rule out the possibility that they play a role in wound healing, and that is an area of current exploration.\nActivation of intracellular signaling pathways by P2 receptors is emerging as intricate patterns of cell specific processes. Involvement of \u03b1V\u03b23 integrins has been shown to be essential for both ATP induced intracellular Ca2+ increase and ERK1\/2 activation in 1321N1 P2Y2 transfected cells [20]. We have shown in our system that integrins play a role in the response to nucleotides (Figure 9). As integrins are known to play a role in cell migration and wound healing, their coordinated role in nucleotide signaling is of great interest. Purinergic signaling is also known to activate downstream signaling events using a number of receptors. We have shown that ATP, UTP, ADP, and UDP promote activation of ERK1\/2, but \u03b1,\u03b2-MeATP and \u03b2,\u03b3-MeATP do not [13]. P2X2 and P2Y1 receptors have been shown to play a role in activating ERK1\/2 in stretch induced injury of astrocytes in Ca2+ dependent manner [27]. In contrast in embryonic kidney cells, P2X7 activates ERK1\/2 in a Ca2+ independent manner [28]. Additionally PKC and PKD both have been shown to play role in P2X7 induced ERK1\/2 activation [29]. In pro-monocytic cells, activation of ERK1\/2 by P2Y2 has been shown to involve c-Src but not PKC [30].\nRecently, the P2YRs have been hypothesized to play a role in cross-talk with the EGFR [7, 13, 21, 31, 32]. There are a number of potential pathways, several of which may occur. One current hypothesis is cross-talk occurs via the triple membrane pass system where GPCR activated matrix metalloproteases (MMPs) cleave pro-heparin binding (HB)-EGF from the membrane and HB-EGF can then activate the EGFR [33, 34]. Another potential method for cross-talk is via Src. While Src is known to play a role in the phosphorylation of EGFR, its involvement in corneal epithelial injury has not been determined. GPCR stimulation has been found to lead to phosphorylation of the EGFR in a Src and\/or Pyk2 dependent manner in many cell types [21, 31, 35, 36]. Future experiments will be performed to test the hypothesis that Src and Pyk2 play an active role in nucleotide-mediated activation of the EGFR in corneal epithelial cells. Inhibition of the EGFR activation with tyrphostin has been shown to inhibit ATP induced cell migration in epithelial cells [7]. These indicate that cross talk between GPCR and the EGFR occur over the entire time course of wound response and repair.\nATP and UTP are the major players in Ca2+ waves after injury and the cross talk with the EGFR (Figures 3 and 6). However, this does not preclude that other nucleotides may mediate other facets of the wound response. While ADP and BzATP do not play a role in the injury induced Ca2+ wave, they can stimulate migration, albeit at a higher concentration than ATP or UTP (Figure 7). Though UDP does not stimulate migration, both ADP and UDP have the ability to activate downstream signals. We have shown that ERK1\/2 and paxillin become phosphorylated upon stimulation with both tri- and di-nucleotides [7, 13]. Additionally, BzATP has been shown to activate downstream signals such as ERK1\/2 in other cell systems [28, 29]. The signals propagated by di-nucleotides may be artifacts of saturating the system with larger than physiological concentrations [37]. Another possibility is that the signal is real and that after injury the receptors are available at a density that is unable to elicit a detectable downstream cascade. This may explain why ADP can induce migration at 10 \u00b5M. However, this does not take into account that high concentrations of ADP are still not able to inhibit the injury induced Ca2+ wave (data not shown). Proper signaling relies on optimal concentrations of nucleotides. At high concentrations where maximal Ca2+ release upon nucleotide exposure occurs (>100 \u00b5M), epithelial cells do not migrate toward the stimuli. The desensitization of the calcium response that occurs at 100 \u00b5M may explain why cell migration is minimal. In contrast, at lower concentrations where the maximal migration occurs, little desensitization occurs. This bell shape curve is not atypical and has been demonstrated to occur with nucleotide induced migration in epithelial and dendritic cells [7, 10].\nIt is likely that different signals are needed for the initial communication of an injury and the long-term need for wound healing, such as cell migration and changes in protein expression. The receptor specificity and the numerous signaling pathways provide an intricate network allowing for differential regulation of short versus long term signals. Our study provides evidence that P2Y2 and possibly P2Y4 play a role in the initial signals involved in the injury response and these receptors and other P2 receptors play a role in long term cell migration. Future studies will involve understanding the critical interactions between signaling pathways that play a role in wound healing.","keyphrases":["wound repair","epithelium","migration","epidermal growth factor","ca2+ imaging"],"prmu":["P","P","P","P","P"]}
{"id":"Anal_Bioanal_Chem-3-1-1914286","title":"Improvement of the liquid-chromatographic analysis of protein tryptic digests by the use of long-capillary monolithic columns with UV and MS detection\n","text":"Optimisation of peak capacity is an important strategy in gradient liquid chromatography (LC). This can be achieved by using either long columns or columns packed with small particles. Monolithic columns allow the use of long columns at relatively low back-pressure. The gain in peak capacity using long columns was evaluated by the separation of a tryptic bovine serum albumin digest with an LC\u2013UV\u2013mass spectrometry (MS) system and monolithic columns of different length (150 and 750 mm). Peak capacities were determined from UV chromatograms and MS\/MS data were used for Mascot database searching. Analyses with a similar gradient slope for the two columns produced ratios of the peak capacities that were close to the expected value of the square root of the column length ratio. Peak capacities of the short column were 12.6 and 25.0 with 3 and 15 min gradients, respectively, and 29.7 and 41.0 for the long column with 15 and 75 min gradients, respectively. Protein identification scores were also higher for the long column, 641 and 750 for the 3- and 15-min gradients with the short column and 1,376 and 993 for the 15- and 75-min gradients with the long column. Thus, the use of long monolithic columns provides improved peptide separation and increased reliability of protein identification.\nIntroduction\nIdentification of unknown proteins is a key step in proteome analysis. The standard method of protein identification consists of enzymatic digestion of the protein(s), usually by using trypsin, followed by mass spectrometry (MS) analysis of the resulting digest. When analysing a single protein, e.g. from an excised 2D gel spot, a peptide separation is usually unnecessary. When analysing a digest of a mixture of proteins, the resolution of only a mass spectrometer is usually not sufficient. In those cases, a peptide separation is applied before MS detection. In shotgun proteomics, where a whole proteome is digested without prior protein fractionation, 2D liquid chromatography (LC) is the method of choice owing to the high complexity of the peptide mixtures. A drawback of multidimensional methods is the time frame, as typical analysis times for 2D-LC analysis of peptide mixtures range from several hours to more than 1\u00a0day [1\u20134]. 1D-LC methods can be used for separation of less complex samples (digests of fewer than 100 proteins), but this requires very efficient separations in combination with the additional separation power of MS detection. Such high-efficiency separations have been reported for analyses with long, packed columns, using (ultra-) high pressure (about 70\u00a0MPa) systems [5, 6].\nPeak capacity is the primary parameter for evaluation of efficiency in gradient chromatography [7]. Peak capacity was first defined by Giddings [8] as the maximum number of peaks that can be separated by a phase system. This theory was later adapted for gradient chromatography by Horv\u00e1th and Lipsky [9]. There are generally two approaches for optimisation of the peak capacity. The first is increase of the gradient length; gradients of up to 10\u00a0h have been reported on single columns [10]. However, according to theory, peak capacity increases to a maximum and then decreases as the gradients become longer [11, 12]. The second approach is the use of longer columns, as the peak capacity increases linearly with the square root of the plate number and thus with the square root of the column length. Wang et al. [13] illustrated this by connecting several columns packed with a pellicular stationary phase. They showed that the ratio of the peak capacity and the square root of the column length was constant for columns ranging in length from 7.5 to 60\u00a0cm. The main limitation to simply increasing column length is the higher back-pressure. A possible solution for this problem is the use of monolithic columns. Monolithic columns have a higher permeability compared with packed columns, facilitating fast separations or the use of long columns [10, 14\u201316]. Wang et al. [13] reported a back-pressure of 28.5\u00a0MPa for a 60\u00a0cm\u2009\u00d7\u20092.1\u00a0mm packed column at a linear flow of 0.94\u00a0mm\/s. In contrast, other groups have obtained 3 times higher linear flow rates for monolithic columns of similar length [10, 17]. The higher permeability of monolithic columns makes it possible to operate long columns at higher flow rates while using conventional LC equipment. Luo et al. [10] separated a bacterial protein digest at a linear flow rate of 2.4\u00a0mm\/s with a 70-cm monolithic column at 34.5\u00a0MPa (5,000\u00a0psi). Tolstikov et al. [17] analysed plant metabolomic extracts with a flow rate of 2.6\u00a0mm\/s for a 60-cm column.\nThe quality of the analysis of a protein digest can be expressed in different ways. One way is to describe chromatographic efficiency in terms of parameters like peak width, peak capacity and resolution. Another way is to use the reliability of protein identification, like SEQUEST [18] or Mascot (http:\/\/www.matrixscience.com) [19] scores. Such an approach is also useful, as the mass spectrometer adds a second dimension to the separation, which is overlooked when only chromatographic efficiency is measured.\nIn this paper the evaluation of capillary monolithic silica columns of different lengths for the LC-UV-MS analysis of a bovine serum albumin (BSA) tryptic digest is described. Columns of 150- and 750-mm length were investigated using gradient times varying from 3 to 75\u00a0min. Chromatographic peak capacities, based on UV detection, and protein identification, based on Mascot scoring data of the MS detection, were determined as a measure for the efficiency of peptide separation.\nTheoretical aspects\nThe peak capacity is defined as the maximum number of bands that will fit within a chromatogram with a resolution of Rs\u2009=\u20091.0 [20]. In gradient LC, where peak width is about constant throughout the separation, the theoretical peak capacity is given by Eq.\u00a01: \nwhere tg is the gradient time and wav is the average baseline peak width. For large values of PC, this approaches tg\/wav. Because sample peaks often do not occupy the entire length of the gradient, the sample peak capacity can then be defined as \nwhere ta and tz are the retention times of the first and last eluting peaks, respectively.\nWang et al. [13] investigated the effect of column length on peak capacity in packed columns and found that the peak capacity is proportional to  if the gradient slope is proportional to L. The gradient slope can be defined as \nwhere \u0394\u03d5 is the change in organic modifier fraction during the gradient (0\u2264\u03d5\u22641) and t0 is the column dead time. If the linear flow rate is constant, t0 is only dependent on column length; therefore, tg should be varied proportionally to column length in order to keep the gradient slope constant.\nExperimental\nMaterials and reagents\nBSA, trypsin (porcine, type IX-S, EC 3.4.21.4) and 1,4-dithiothreitol (DTT) were purchased from Sigma (St. Louis, MO, USA), and iodoacetamide (IAA) and NH4HCO3 from Fluka (Buchs, Switzerland). Acetonitrile was high-performance LC gradient grade (Biosolve, Valkenswaard, the Netherlands), and spectroscopy-grade trifluoroacetic acid (TFA) was obtained from Merck (Darmstadt, Germany). All solutions were prepared using water from a Milli-Q water-purifying system (Millipore, Bedford, MA, USA).\nAll reagents for the digestion of BSA were prepared in 200\u00a0mM NH4HCO3 buffer, pH 8. The tryptic digest was prepared as follows. A 100-\u03bcl aliquot of BSA stock solution (3.5\u00a0\u03bcg\/\u03bcl in water) was set to pH 8 by adding 25\u00a0\u03bcl of 1\u00a0M NH4HCO3 buffer (pH 8). After addition of 25\u00a0\u03bcl of 10\u00a0mM DTT solution, the sample was incubated at 50\u00a0\u00b0C for 30\u00a0min to reduce disulfide bonds. After cooling to room temperature, 25\u00a0\u03bcl of 30\u00a0mM IAA solution was added and the sample was incubated in the dark for 60\u00a0min to alkylate the free thiols. Trypsin was dissolved in 10\u00a0\u03bcl of buffer to reach a trypsin-to-protein mass ratio of 1:50 in the final solution; the trypsin solution was added to the sample, which was incubated at 37\u00a0\u00b0C overnight for 15\u00a0h. The digestion was stopped by addition of 15\u00a0\u03bcl of 10% TFA. The sample was diluted to 200\u00a0ng\/\u03bcl (3\u00a0\u03bcM) with LC mobile phase A (water plus 0.05% TFA) and injected without further purification.\nApparatus and LC columns\nAll analyses were performed with an Agilent 1100 nanoLC system (Agilent Technologies, Waldbronn, Germany), consisting of a vacuum degasser, a binary Nano-Pump, a \u03bc-well plate sampler and a column switching module with a trapping column in the 1\u20134 position of the six-port column-switching valve. The trapping pump was a Gynkotek model 480 (Gynkotek, Germering, Germany). Detection was performed by UV and MS detection, with the detectors connected in series. The UV detector was an MU 701 UV\u2013VIS detector (ATAS GL International, Veldhoven, the Netherlands), equipped with an external optical-fibre flow cell (6\u00a0nl, 3-mm light path); peptides were detected at 215\u00a0nm. The mass spectrometer was an Agilent LC\/MSD Trap XCT (Agilent Technologies, Waldbronn, Germany) ion-trap mass spectrometer, equipped with an orthogonal electrospray ionisation (ESI) interface. The external flow cell of the UV detector allows minimal time delay and band-broadening between UV and MS detection.\nThe monolithic columns were provided by GL Sciences (Tokyo, Japan). The columns were a 150\u00a0mm\u2009\u00d7\u20090.1\u00a0mm MonoCap for nano-flow C18-silica monolithic column and a 750\u00a0mm\u2009\u00d7\u20090.2\u00a0mm MonoCap high resolution C18-silica monolithic column. For trapping of the digest a 5\u00a0mm\u2009\u00d7\u20090.3\u00a0mm column packed with 5\u00a0\u03bcm Zorbax 300 SB-C18 (Agilent Technologies, Waldbronn, Germany) was used.\nMethod and data analysis\nLC solvent A was water plus 0.05% TFA (v\/v); solvent B was acetonitrile plus 0.04% TFA (v\/v). The trapping solvent was a mixture of 5% (v\/v) solvent B in solvent A. After injection of the digest (0.25\u00a0\u03bcl for the 0.1-mm inner diameter column and 1.0\u00a0\u03bcl for the 0.2-mm inner diameter column), the sample was trapped on the trapping column at a flow rate of 5\u00a0\u03bcl\/min. After 5\u00a0min, the trapping column was switched on-line with the separation column and the gradient was started. All separations were performed at room temperature using a gradient of 5\u201350% solvent B with gradient times varying from 3 to 75\u00a0min. MS spectra were acquired in the positive ion mode over the 400\u20132,000\u00a0m\/z range, after which the two most intense ions (with a preference for doubly charged ions) were selected for fragmentation. MS\/MS fragmentation spectra were acquired over the 100\u20132200\u00a0m\/z range. An ESI spray voltage of -3\u00a0kV was used for all experiments.\nThe effect of separation efficiency on protein identification was evaluated using the Mascot search engine [19]. LC-MS\/MS data were converted to the Mascot generic format (.mgf file) using the data-analysis software, and the .mgf files were searched against the MSDB database using Mascot\u2019s MS\/MS ion search module. The database was searched for tryptic peptides from all entries in the database, allowing one missed cleavage per peptide and containing carbamidomethyl cysteine as a variable modification. Mass tolerances were set to default values: peptide mass tolerance \u00b12.0\u00a0Da, MS\/MS tolerance \u00b10.8\u00a0Da.\nResults and discussion\nLiquid chromatography\u2013UV analysis\nBecause of the difference in diameter, the 150\u00a0mm\u2009\u00d7\u20090.1\u00a0mm and the 750\u00a0mm\u2009\u00d7\u20090.2\u00a0mm columns were used with different flow rates. For the 150- and the 750-mm columns, the flow rates were 0.5 and 2.0\u00a0\u03bcl\/min, respectively, resulting in a linear flow rate of 1.06\u00a0mm\/s. Injection volumes were also proportional to the square of the column diameter, 0.25\u00a0\u03bcl of the digest for the 0.1-mm column and 1.0\u00a0\u03bcl onto the 0.2-mm column. During the gradient, the maximum back-pressure of the 750-mm column was below 20\u00a0Mpa, which is well below the manufacturers limit of 30\u00a0Mpa.\nFigure\u00a01 shows the LC-UV chromatograms of 3- and 15-min gradients run on the 150-mm column and 15- and 75-min gradients run on the 750-mm column. When the chromatograms of the analyses with similar gradient slopes are compared (Fig.\u00a01a,b, and Fig.\u00a01c,d), it is clear that an increase in column length improves the peptide separation. In order to quantify the efficiency of the separation, the sample peak capacity was calculated for all analyses. Because of the incomplete resolution of the digest, the peak capacity was estimated by using the average peak width of a selected number of peaks that appeared to contain only a single peptide. Using this method, we calculated peak capacities for all analyses and the results are summarised in Table\u00a01. The peak capacities found for the short column are comparable to those found in the literature for similar columns [21, 22]. As expected, the peak capacities of the long column are higher than those of the short column, but they are relatively low compared with the values reported in [10]. However, when gradient time is taken into consideration, the difference is significantly less: PC**\/tg is 0.55 peaks per minute with the present system (75-min gradient on the 750-mm column) and 1.62 peaks per minute for the 260-min gradient reported in [10]. A possible negative effect on the resolution of our system is the use of a trapping column, filled with a different stationary phase, which probably has a selectivity that differs from that of the analysis column.\nFig.\u00a01Liquid chromatography (LC)\u2013UV chromatograms of a tryptic bovine serum albumin (BSA) digest, separated by monolithic silica capillary columns of 150\u00a0mm\u2009\u00d7\u20090.1\u00a0mm (a, c) and 750\u00a0mm\u2009\u00d7\u20090.2\u00a0mm (b, d) using a gradient of 5\u201350% acetonitrile (0.04% v\/v trifluoroacetic acid, TFA) in water (0.05% v\/v TFA). a A 3-min gradient (15%\/min); b, c 15-min gradient (3%\/min); d 75-min gradient (0.6%\/min). The run times include 5 min of trapping and 2.5 and 12.5\u00a0min of gradient delay time for the 150-mm and 750-mm columns, respectivelyTable\u00a01Chromatographic parameters from liquid chromatography\u2013UV analysis\u00a0150\u00a0mm\u2009\u00d7\u20090.1\u00a0mm750\u00a0mm\u2009\u00d7\u20090.2\u00a0mmtg (min)wav\u0394tPC**wav\u0394tPC30.243.0512.60.283.1611.2150.4411.025.00.3911.629.7751.2737.629.60.7731.741.0tg gradient time, wav average width of seven to 12 peaks which were found to contain only a single peptide (based on mass spectrometry data), \u0394t elution window between the first and last eluting peptides, PC** sample peak capacity (\u0394t\/wav)\nIf chromatograms with the same gradient slope are compared (3- and 15-min gradients for the 150-mm column and 15- and 75-min gradients for the 750-mm column, respectively), the ratio of the peak capacities should be close to the square root of the column length ratio (2.24). For the 3-\/15-min gradient pair this ratio is 29.7\/12.6\u2009=\u20092.37; for the 15-\/75-min gradient pair the ratio is 41.0\/25.0\u2009=\u20091.64. For the short column, no increase in peak capacity is observed with a gradient of more than 15\u00a0min, but for the long column the peak capacity increases up to a 75-min gradient. A possible explanation can be found in the study of Stadalius et al. [11, 12], who demonstrated that peak capacity will increase with gradient time, until it reaches a maximum, after which it will even decrease. The gradient time at which this maximum is obtained is greater for longer columns.\nLiquid chromatography\u2013mass spectrometry analysis\nTo assess the influence of peptide separation on protein identification, MS\/MS data were investigated by database-searching using the MS\/MS ion search module from the Mascot search engine. The results are expressed as a Mascot score, the number of unique identified peptides and the percentage of the BSA amino acid sequence covered by these peptides (Table\u00a02). These results were compared with the scores obtained for direct infusion of the BSA digest at the same flow rates as for the LC separations. The protein identification parameters for the infusion experiments were about similar for both flow rates. All database searches gave bovine albumin as the top protein match and the only other significant matches were albumins from other species. \nTable\u00a02Mascot\u00ae database search results\u00a0150\u00a0mm\u2009\u00d7\u20090.1\u00a0mm750\u00a0mm\u2009\u00d7\u20090.2\u00a0mmtgMascot scorePeptidesaCoverageb (%)Mascot scorePeptidesaCoverageb (%)Infusionc31791737610183\u00a0min6411428787132515\u00a0min75016291,376243875\u00a0min69316299931929aNumber of unique peptides identified by database search.bPercentage of amino acid sequence covered by identified peptides.cDirect infusion of the digest into the mass spectrometer.\nFigure\u00a02 shows base peak chromatograms of separations with the same gradient slope, a 15-min gradient for the 150-mm column and a 75-min gradient for the 750-mm column. The Mascot scores for the 750-mm column are higher than those for the 150-mm column. The average score per identified peptide is between 52 and 60 for the long column and between 42 and 51 for the short column. Combined with the larger number of identified peptides on the long column, this adds up to a higher Mascot score (Table\u00a02).\nFig.\u00a02LC\u2013mass spectrometry (MS) base peak chromatograms and mass spectra of tryptic BSA digest, separated by monolithic silica columns of 150\u00a0mm\u2009\u00d7\u20090.1\u00a0mm (a) and 750\u00a0mm\u2009\u00d7\u20090.2\u00a0mm (b), using a gradient of 5\u201350% acetonitrile (0.04% v\/v TFA) in water (0.05% v\/v TFA). a A 15-min gradient (3%\/min); b 75-min gradient (0.6%\/min). The run times include 5 min of trapping and 2.5 and 12.5\u00a0min of gradient delay time for the 150-mm and 750-mm columns, respectively\nDespite providing only a low resolution, even the shortest gradients cause a considerable increase in the reliability of protein identification. Increase in gradient length leads to cleaner mass spectra (Fig.\u00a03) and a higher number of identified peptides and sequence coverage, compared with direct infusion. These numbers, however, decrease beyond a certain gradient time. This could possibly be attributed to the lower peak heights for longer gradient times. This result indicates that there is a gradient slope where an increase in chromatographic separation no longer improves protein identification.\nFig.\u00a03Averaged mass spectra of peptide YICDNQDTISSK (m\/z 722.32, M2H2+) as identified from extracted ion chromatograms in the LC-MS analysis of a tryptic BSA digest. a A 150-mm\u2009\u00d7\u20090.1-mm silica monolithic column, 15-min gradient of 5\u201350% acetonitrile (0.04% v\/v TFA) in water (0.05% v\/v TFA); b 750-mm\u2009\u00d7\u20090.2-mm column, 75-min gradient\nConclusions\nThe use of long silica-based capillary monolithic columns provides a clear advantage over use of shorter columns, i.e. an increase of chromatographic efficiency and reliability of protein identification. As expected from chromatography theory, a factor 5 longer column gives a 1.6\u20132.4 times increase in peak capacity for separations with similar gradient slope. The use of longer gradients also leads to an initial improvement of the protein identification score, but the score seems to have a maximum at longer gradient times.\nWhile the use of longer columns for the separation of peptides has a clear advantage because of the gain in chromatographic efficiency, this also gives a longer analysis time. As maximum protein identification scores for rather simple digests are reached at relatively short gradient times, it is important to find a compromise between chromatographic efficiency and analysis time. However, if the sample is more complex, the use of longer columns is more attractive as longer gradients are necessary to achieve sufficient separation.\nIn the near future, short and long columns of the same diameter (0.1-mm inner diameter) will be compared. Further improvement of the separation might be obtained by optimisation of the combination of the trap column and the analysis column. Moreover, the potential of longer monolithic capillary columns will be demonstrated by the analysis of more complex and real samples.","keyphrases":["monoliths","peak capacity","gradient liquid chromatography","long columns","protein identification"],"prmu":["P","P","P","P","P"]}
{"id":"J_Mol_Biol-2-1-2267215","title":"Structure of the Tandem Fibronectin Type 3 Domains of Neural Cell Adhesion Molecule\n","text":"Activation of the fibroblast growth factor receptor (FGFR) by neural cell adhesion molecule (NCAM) is essential for NCAM-mediated neurite outgrowth. Previous peptide studies have identified two regions in the fibronectin type 3 (FN3)-like domains of NCAM as being important for these activities. Here we report the crystal structure of the NCAM FN3 domain tandem, which reveals an acutely bent domain arrangement. Mutation of a non-conserved surface residue (M610R) led to a second crystal form showing a substantially different conformation. Thus, the FN3 domain linker is highly flexible, suggesting that it corresponds to the hinge seen in electron micrographs of NCAM. The two putative FGFR1-binding segments, one in each NCAM FN3 domain, are situated close to the domain interface. They form a contiguous patch in the more severely bent conformation but become separated upon straightening of the FN3 tandem, suggesting that conformational changes within NCAM may modulate FGFR1 activation. Surface plasmon resonance experiments demonstrated only a very weak interaction between the NCAM FN3 tandem and soluble FGFR1 proteins expressed in mammalian cells (dissociation constant > 100 \u03bcM). Thus, the NCAM\u2013FGFR1 interaction at the cell surface is likely to depend upon avidity effects due to receptor clustering.\nIntroduction\nThe neural cell adhesion molecule (NCAM) is the prototype and founding member of the immunoglobulin (Ig) superfamily cell adhesion molecules (CAMs).1\u20134 NCAM is present on the cell surface of neurons, astrocytes and oligodendrocytes, where it mediates homophilic and heterophilic cell adhesion. NCAM is involved in neuronal migration, axon growth and guidance, as well as in synaptic plasticity associated with learning and memory.5\u20137 Alternative splicing of the NCAM1 gene results in isoforms of three size classes that differ in their membrane attachment and cytosolic regions but have in common an extracellular domain consisting of five Igs and two fibronectin type 3 (FN3) domains.8 The two larger isoforms have a transmembrane helix and cytosolic domains of different sizes, while the smallest isoform has a glycophosphatidylinositol membrane anchor. Variable use of alternative exons in the extracellular domain results in small insertions into Ig4 or between the FN3 domains.9\u201312 NCAM function is further regulated by an unusual posttranslational modification, namely, the addition of polysialic acid to Ig5.13\nThe molecular basis of homophilic adhesion by NCAM has been a subject of intense study, and the results from biochemical and biophysical studies have not always been consistent.14,15 A crystal structure of NCAM Ig1\u2013Ig3 has led to a zipper model of adhesion, which postulates both cis and trans interactions by the Ig1\u2013Ig3 region of NCAM.16 While a conclusive picture has yet to emerge, it appears that there may be multiple modes of homophilic interaction.17\nNCAM is also engaged in heterophilic interactions. There is now convincing evidence that NCAM-mediated neurite outgrowth, as well as tumour development and progression, critically involves the activation of fibroblast growth factor receptor 1 (FGFR1), through a cis interaction of NCAM and FGFR1.18\u201322 The four FGFRs and their 23 growth factor ligands control a variety of cellular processes, including development, angiogenesis, hematopoiesis and tumourigenesis.23\u201326 Alternative splicing of the four FGFR genes results in at least 48 receptor isoforms that vary in their ligand binding profiles and kinase domains. The longest FGFR1 ectodomain variant consists of three Ig domains, D1\u2013D3, with a stretch of acidic amino acids (the \u201cacid box\u201d) inserted into the D1\u2013D2 linker; shorter forms lack D1 and the acid box. The NCAM\u2013FGFR1 interaction was originally proposed based on indirect biological evidence,27 but biochemical evidence has been obtained to suggest that the interaction is mediated by direct binding of the FN3 domains of NCAM to FGFRs (Fig. 1).20,28,29 We and others identified NCAM-derived peptides capable of stimulating FGFR1 signalling and inducing neurite outgrowth. One peptide (FRM peptide) is derived from the first FN3 domain of NCAM (1FN3),30 while another (FGL peptide) is derived from the second FN3 domain (2FN3).28 The structures of the 1FN3 and 2FN3 domains in isolation have been determined,28,31 but how the two domains cooperate in FGFR1 activation is unclear. In this study, we determined the crystal structure of the NCAM FN3 tandem (1FN3\u20132FN3) in two crystal forms. We report that 1FN3\u20132FN3 assumes a bent conformation in both forms, with evidence of substantial flexibility of the domain linker. In direct binding experiments with fully glycosylated proteins of mammalian origin, we observed only a very weak interaction (dissociation constant >\u00a0100\u00a0\u03bcM) of NCAM 1FN3\u20132FN3 with two FGFR1 ectodomain constructs. We conclude that the NCAM\u2013FGFR1 interaction at the cell surface may be transient or stabilised by avidity effects resulting from receptor clustering and that conformational changes within NCAM may have a profound role in FGFR1 activation.\nResults\nCrystal structure of the NCAM FN3 tandem\nTo obtain insight into the relative orientation of the two FN3 domains of NCAM, we determined the crystal structure of 1FN3\u20132FN3 at 2.3-\u00c5 resolution (Table 1). Both 1FN3 and 2FN3 adopt the typical \u03b2-sandwich fold of all FN3 domains consisting of seven strands arranged in two antiparallel sheets (ABE and GFCD) (Fig. 2a and b). Preceding strand A in both domains are short proline-rich segments that are integrated into the FN3 fold, with the proline tetrahydropyrrole rings pointing into the hydrophobic core (Pro500 and Pro503 in 1FN3; Pro601 and Pro604 in 2FN3). A similar feature has been observed in other FN3 domains (e.g., in gp13033 and titin34). 1FN3 contains an unusual \u03b1-helix situated between strands D and E, as reported previously.31\n1FN3 in our FN3 tandem structure matches the crystal structure of 1FN3 in isolation,31 with an r.m.s.d. of 0.50\u00a0\u00c5 for 100 C\u03b1 atoms. 2FN3 in our FN3 tandem matches the solution structure of 2FN3 in isolation,28 with an r.m.s.d. of 1.3\u00a0\u00c5 for 92 C\u03b1 atoms (the main differences are concentrated in the B\u2013C and C\u2013D loops).\nThe relative orientation of the two FN3 domains in the 1FN3\u20132FN3 tandem is characterised by an unusually bent conformation with an interdomain angle of \u223c\u00a080\u00b0 (calculated between the long axes of the two FN3 domains). The interface between 1FN3 and 2FN3 buries 630\u00a0\u00c52 of solvent-accessible surface (calculated with the CCP4 program AREAIMOL), which is in the typical range for rigid FN3 (and Ig) domain interfaces. The interface is dominated by polar interactions, between the A\u2013B loop of 1FN3 on the one hand and the domain linker and B\u2013C loop of 2FN3 on the other (Fig. 2c) The key interface residues (Tyr511, Ser513, Thr514, Pro601 and Asp625) are strictly conserved in all vertebrate NCAM sequences (Fig. 2d), suggesting that the bent conformation may be physiologically relevant. However, residues 511\u2013514 have also been implicated in FGFR1 binding,30 and the linker region could assume a very different structure when NCAM is bound to FGFR1 (see below).\nThe conformation of multidomain proteins often is influenced by the crystal lattice. In this regard, we noted that the asymmetric unit of our crystals contains two 1FN3\u20132FN3 molecules arranged in a tightly interlocked dimer (Fig. 2b). There are two main contacts responsible for dimer formation: the \u03b1-helix of 1FN3 packs against the GFCD sheet of 2FN3 of the other molecule, and the 2FN3 domains of the two molecules interact via their A and G strands. Altogether, these contacts bury as much as 3040\u00c52 of solvent-accessible surface. We do not think that the dimer observed in our crystals is physiologically relevant, as dimer interface residues are only poorly conserved (Fig. 2d) and NCAM dimers were never observed in electron microscopy studies.35,36 Because we were concerned that the tight association of 1FN3\u20132FN3 molecules in the dimer may have forced the unusually bent interdomain conformation, we sought to disrupt the dimer by mutagenesis and crystallise a mutant 1FN3\u20132FN3 protein in a different crystal form.\nStructure of the M610R mutant\nWe expressed three point mutants of NCAM 1FN3\u20132FN3 (M610R, Y672E and R690E), all of which should be incompatible with the dimer structure seen in crystals of the wild-type protein. Importantly, all three mutations target surface residues and are unlikely to have an effect on NCAM folding. Whether the mutations are functionally neutral could not be determined due to a lack of suitable assays. When examined by size-exclusion chromatography, all three mutants eluted as a mixture of monomers and dimers, similar to the wild-type protein (data not shown). We obtained crystals of the M610R mutant and determined its structure at 2.7-\u00c5 resolution by molecular replacement (Fig. 3a). The asymmetric unit of the crystals contains a hexamer of NCAM 1FN3\u20132FN3 M610R. The hexamer can be regarded as a trimer of dimers, with the dimers having a completely different mode of association compared with the wild-type structure (the individual domains are very similar, as expected, with r.m.s.d. values of 0.93 and 1.1\u00a0\u00c5 for 1FN3 and 2FN3, respectively). The relative orientation of the two FN3 domains in the M610R mutant is less severely bent than in the wild-type structure but still far from fully extended (interdomain angle \u223c\u00a0120\u00b0; Fig. 3b). The FN3 pair opens up in the M610R mutant, and 1FN3 is additionally twisted about its long axis. The combined hinge opening and 1FN3 twisting amounts to a pure rotation of 73\u00b0 for 1FN3 when the structures are superimposed on their 2FN3 domains. There is no domain interface to speak of in the mutant (240\u00a0\u00c52 buried), and the conformation appears to be stabilised entirely by the crystal lattice. Thus, crystal lattice forces can have a profound influence on the conformation of the NCAM FN3 tandem, suggesting that the 1FN3\u20132FN3 linker may act as a flexible hinge in native NCAM.\nWe compared the NCAM FN3 tandem with other FN3 tandems of known structure. Tandems from the extracellular matrix proteins fibronectin and tenascin generally assume an extended conformation.38\u201340 In contrast, many cytokine and hormone receptors (e.g., gp130)33 feature bent FN3 pairs that are superficially similar to the NCAM structures reported here (data not shown). Of particular interest is the structure of the FN3 pair of neuroglian, a Drosophila CAM.37 The neuroglian tandem, which has an extensive domain interface that incorporates a bound sodium ion, adopts a conformation that is intermediate between the two conformations we report for the NCAM tandem. Thus, a similar bend in the FN3 linker(s) may be a general feature of other animal CAMs containing FN3 domains in their membrane-proximal region.\nLocation of putative FGFR1 binding site\nPrevious studies have implicated two NCAM 1FN3\u20132FN3 regions in FGFR1 binding. Kiselyov et al. identified a bioactive peptide from the F\u2013G loop of 2FN3 (FGL peptide),28 and we identified a bioactive peptide from the A\u2013B loop of 1FN3 (FRM peptide).30 In agreement with our earlier prediction,30 the FRM and FGL loops are indeed located in close proximity on the same face of the wild-type NCAM molecule (Fig. 4a). However, in the M610R mutant, the opening up of the two domains combined with rotation of 1FN3 places the FRM and FGL loops much farther apart. Thus, conformational changes at the NCAM 1FN3\u20132FN3 hinge may modulate the interaction of NCAM with FGFR1.\nSurface plasmon resonance analysis of the NCAM\u2013FGFR1 interaction\nWe wanted to map the FGFR1 binding site on the NCAM FN3 tandem by structure-based mutagenesis and first sought to establish a suitable binding assay. A solid-phase assay with immobilised NCAM and Fc-tagged FGFR1 proteins did not show any appreciable interaction (data not shown). We therefore used surface plasmon resonance (SPR) to analyse the binding of NCAM 1FN3\u20132FN3 to two FGFR1 ectodomain constructs. The FGFR1 D1\u2013D3 construct used (residues 22\u2013364) spans essentially the full ectodomain and contains the acid box situated between domains D1 and D2. The FGFR1 D2\u2013D3 construct used (residues 151\u2013364) lacks D1 and the acid box but retains the binding site for FGFs; this construct is similar to the construct previously used by Kiselyov et al. in SPR studies.28 Both soluble FGFR1 proteins were produced by the 293-EBNA cells in good yields. Due to the presence of multiple N-linked glycosylation sites in FGFR1 (see below), the purified recombinant proteins migrate as diffuse bands of higher-than-calculated molecular mass on SDS-PAGE (Fig. 5). In a first set of experiments, the two FGFR1 constructs were immobilised on a CM5 sensor chip [8000\u00a0resonance units (RU) of D1\u2013D3 and 3850\u00a0RU of D2\u2013D3]. Recombinant FGF1 injected at a concentration of 100\u00a0nM produced sensorgrams characteristic of a high-affinity interaction, confirming that the immobilised proteins are functional (Fig. 6a and b). In contrast, wild-type NCAM 1FN3\u20132FN3 up to a concentration of 70\u00a0\u03bcM did not produce a signal on the FGFR1 D1\u2013D3 surface and showed only very weak binding to FGFR D2\u2013D3 (Fig. 6c and d). In a second set of experiments, the order of proteins was reversed. NCAM 1FN3\u20132FN3 proteins were immobilised on a CM4 sensor chip (1800\u00a0RU of wild-type protein and 1900\u00a0RU of M610R mutant), and the two soluble FGFR1 constructs were used as analytes up to a concentration of 100\u00a0\u03bcM. We again observed only weak interactions for all pairings (Fig. 6e\u2013h). Wild-type and M610R NCAM 1FN3\u20132FN3 behaved almost identically in these experiments, and, as before, it appeared that the affinity of NCAM for FGFR1 D2\u2013D3 was higher than that for FGFR1 D1\u2013D3. The fast association and dissociation steps in the sensorgrams prevented the fitting of kinetic constants. We used the plateau values at equilibrium to estimate a dissociation constant of >\u00a0100\u00a0\u03bcM for the interaction of NCAM 1FN3\u20132FN3 with FGFR1 D2\u2013D3 (not shown), but we emphasise that this value is very approximate given the weak resonance signals obtained. In view of the weakness of the NCAM\u2013FGFR1 interaction in our assay, we were unable to pursue our initial plans of mapping the binding site(s) by mutagenesis.\nDiscussion\nNCAM ectodomain structure\nThe current view of the NCAM ectodomain structure is based on early studies by rotary shadowing electron microscopy of tissue-derived NCAM.35 Electron microscopy visualised the NCAM ectodomain as \u223c\u00a028-nm rods bent at a flexible hinge located \u223c\u00a010\u00a0nm from the C-terminus; the angle between the two arms varied from 50\u00b0 to 140\u00b0 (average\u00a0=\u00a0100\u00b0). The hinge was attributed to the proline-rich linker between Ig5 and the first FN3 domain, and the long and short arms were described as rigid domain tandems (Ig1\u2013Ig5 and 1FN3\u20132FN3, respectively) in extended conformations.35 A crystal structure of NCAM Ig1\u2013Ig3 indeed showed a largely extended structure.16 In sharp contrast, the present crystal structure analysis of the NCAM 1FN3\u20132FN3 domain pair has revealed a prominent bend between the two domains in two independent crystal forms (Fig. 3b). This finding is difficult to reconcile with the uniformly straight appearance of the short arm in the electron micrographs and suggests that the hinge point may actually lie between 1FN3 and 2FN3. In this respect, we note that the Ig5\u20131FN3 linker is actually rather short and may well be rigid (in the Ig5\u20131FN3 linker sequence, ILVQADTPSSP, the isoleucine and valine residues are predicted to contribute to the hydrophobic core of Ig5 and the first proline is already part of the 1FN3 fold). If the hinge is instead situated in the 1FN3\u20132FN3 linker, the juxtamembrane domain of NCAM must have contributed to the short arm seen in electron micrographs, as a single FN3 domain would only account for half of the short arm length. The serine\/threonine-rich juxtamembrane domain of NCAM (sequence TSAQPTAIPANGSPTSGLSTGA) is predicted to be extensively modified by O-linked glycosylation (NetOGlyc 3.1 server\u2020) and could easily assume the extended and rigid conformation required to span the remaining \u223c\u00a05\u00a0nm.41 Further structural analysis, in particular, of the Ig5\u20131FN3 pair, is required to conclusively pinpoint the site of articulation within the NCAM ectodomain.\nNCAM splice variants\nInterestingly, the flexible hinge linking the two FN3 domains of NCAM 1FN3\u20132FN3 is known to be modified by alternative splicing. Our structure is of the shortest isoform (linker sequence TQPVREPSAP), whereas the underlined arginine residue is replaced by QG, HSPPPQG or even longer sequences in other variants.9,10,12 The HSPPPQG insertion has been suggested as a potential hinge region.1 The biological relevance of these splicing events is not well understood, but in vitro experiments have demonstrated that the NCAM isoforms differ in their capacity to support cell adhesion and spreading,42,43 as well as myoblast fusion.44 We prepared NCAM 1FN3\u20132FN3 proteins with QG or HSPPPQG inserts in the domain linker, but, unfortunately, these proteins were very prone to aggregation in physiological buffers and could not be used for SPR or structural analysis (data not shown).\nInteraction of NCAM with FGF\nHinge bending and alternative splicing at the 1FN3\u20132FN3 junction could affect NCAM function by modulating either its homophilic binding properties or its heterophilic interactions with other proteins. There is currently no evidence for the former scenario. In contrast, the functional interaction of NCAM with FGFR1 in cis (i.e., at the same cell membrane) is well established1,22,30 and would appear to be an attractive candidate for regulation by alternative splicing. Peptides from two regions of the NCAM 1FN3\u20132FN3 tandem, one in each domain, have been shown to modulate FGFR1-dependent neurite outgrowth.28,30 Intriguingly, the corresponding loop regions are in close proximity in the acutely bent conformation observed in crystals of the wild-type protein, suggesting that they are part of a larger FGFR1 binding site extending over the domain junction. In crystals of the M610R mutant, the two putative FGFR1-binding loops are farther apart and no longer on the same face of the 1FN3\u20132FN3 structure (Fig. 4). Thus, changes in the 1FN3\u20132FN3 conformation, either by biomechanical forces resulting from cell\u2013cell contact or by alternative splicing, could have a profound effect on the NCAM\u2013FGFR1 interaction. Other NCAM activities that might be affected by alternative splicing of the 1FN3\u20132FN3 linker include interactions with polysialyltransferases31 and prion protein,45 which both bind to the FN3 domains.\nPrevious biochemical studies reported a dissociation constant of \u223c\u00a010\u00a0\u03bcM for the interaction between NCAM 1FN3\u20132FN3 and FGFR1 D2\u2013D3.28,29 Another study, using cell-based assays, concluded that the acid box in the FGFR1 D1\u2013D2 linker was essential for the NCAM\u2013FGFR1 interaction.20 We wanted to use SPR binding experiments to identify NCAM residues involved in FGFR1 binding and test the effect of splice inserts in the 1FN3\u20132FN3 linker. Unfortunately, using our recombinant proteins expressed in mammalian cells (Fig. 5), we were unable to detect substantial binding between soluble FGFR1 and NCAM proteins, regardless of whether FGFR1 or NCAM was immobilised on the sensor chip. A very weak interaction (estimated dissociation constant >\u00a0100\u00a0\u03bcM) was evident between FGFR1 D2\u2013D3 and NCAM 1FN3\u20132FN3, but we observed no NCAM binding to the FGFR1 D1\u2013D3 construct containing the acid box (Fig. 6). The most likely explanation for the discrepancy between our findings and those of Christensen et al.29 is the difference in glycosylation of the FGFR1 proteins used. Christensen et al. expressed FGFR1 D2\u2013D3 in insect cells, which produce N-linked oligosaccharides of the high-mannose type, whereas our expression system (human embryonic kidney cells) produces complex-type oligosaccharides, which more closely resemble the glycan present on mammalian FGFRs. Human and rodent FGFR1 proteins are highly glycosylated, and the glycan is known to influence ligand binding.46 We think that there may be electrostatic repulsion between the acidic NCAM 1FN3\u20132FN3 protein (isoelectric point 5.0) and the terminal sialic acids present on FGFR1 expressed in human cells. The very weak interaction we observed between soluble NCAM and FGFR1 proteins does not preclude a critical role of this interaction at the cell surface, where avidity effects due to receptor clustering may be substantial. An attractive hypothesis is that the functional state of NCAM (i.e., whether it is engaged in a homophilic contact or not) is linked to FGFR1 binding and activation. It is tempting to speculate that the flexible 1FN3\u20132FN3 linkage revealed by our structural analysis could provide the molecular means for such a regulatory mechanism.\nExperimental Procedures\nExpression vectors\nNCAM constructs were made by PCR amplification from a bacterial expression vector coding for the FN3 pair of human NCAM. Our NCAM numbering scheme corresponds to SwissProt entry P13591 up to residue 598 but differs by \u2212\u00a01 from P13591 for all subsequent residues due to the replacement of Gln599\u2013Gly600 by Arg, a naturally occurring splice variant in the brain and muscle.12 The M610R mutation in NCAM was introduced by strand-overlap-extension PCR. FGFR1 constructs were made by PCR amplification from a complete cDNA clone of human FGFR1 (IIIc isoform; our numbering scheme corresponds to SwissProt entry P11362). The PCR products were cloned into a modified pCEP-Pu vector47 coding for proteins with a C-terminal His6 tag. The insert sequences of all expression vectors were verified by DNA sequencing. The domain boundaries of the constructs are as follows: NCAM 1FN3\u20132FN3, QADTP\u2026VFRTS (496\u2013692); FGFR1 D1\u2013D3, RPSPT\u2026EALEE (22\u2013364); and FGFR1 D2\u2013D3, VAPYW\u2026EALEE (151\u2013364). Vector-derived APLA and AAAHHHHHH sequences are additionally present at the N- and C-terminus, respectively.\nProtein expression and purification\nAll proteins were purified from the conditioned medium of episomally transfected 293-EBNA cells. Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Invitrogen), transfected using Fugene reagent (Roche Applied Science) and selected with 1\u00a0\u03bcg\/ml of puromycin (Sigma). Proteins were purified by a combination of affinity and size-exclusion chromatography performed on an \u00c4kta platform (GE Healthcare). Typically, 1.5\u00a0l of conditioned serum-free medium was loaded onto a 5-ml HisTrap column (GE Healthcare) equilibrated in phosphate-buffered saline (PBS) buffer, pH 7.45 (140\u00a0mM NaCl, 10\u00a0mM Na2PO4 and 3\u00a0mM KCl), and eluted with 500\u00a0mM imidazole in PBS. The eluate was concentrated using Vivaspin centrifugal devices (Sartorius AG) and further purified on a 24-ml Superdex 200 size-exclusion chromatography column (GE Healthcare) with Tris-buffered saline (TBS) buffer, pH 7.4, as the running buffer. Purified proteins were analysed by SDS-PAGE, quantified by measuring their absorption at 280\u00a0nm, concentrated to the final desired concentrations and flash-frozen in liquid nitrogen for storage at \u2212\u00a080\u00a0\u00b0C. Final yields were 10\u201320\u00a0mg of pure protein per litre of cell culture medium.\nCrystallisation and structure determination\nNCAM 1FN3\u20132FN3 was concentrated to 13\u00a0mg\/ml in TBS, and crystals were obtained by hanging drop vapour diffusion at room temperature using 2.2\u00a0M ammonium sulfate, 0.1\u00a0M sodium citrate, pH 5.2, 0.2\u00a0M potassium\/sodium tartrate and 3\u20135% ethanol as precipitant. Crystals grew within 2\u00a0days and belong to space group P212121 with unit cell dimensions a\u00a0=\u00a052.77\u00a0\u00c5, b\u00a0=\u00a071.35\u00a0\u00c5 and c\u00a0=\u00a098.22\u00a0\u00c5. There are two 1FN3\u20132FN3 molecules in the asymmetric unit, resulting in a solvent content of \u223c\u00a038%. Crystals were flash-frozen in liquid nitrogen after brief soaking in mother liquor supplemented with 20% glycerol. A crystal was soaked in mother liquor supplemented with 300\u00a0mM potassium iodide for 30\u00a0s before freezing to obtain a heavy atom derivative. Diffraction data from native and KI derivative crystals were collected at 100\u00a0K on station 14.1 at the Synchrotron Radiation Source (SRS) Daresbury and on station ID29 at the European Synchrotron Radiation Facility Grenoble, respectively. The NCAM 1FN3\u20132FN3 M610R mutant was concentrated to 14\u00a0mg\/ml in TBS, and crystals were obtained by sitting drop vapour diffusion at room temperature using 2\u00a0M ammonium sulfate and 0.1\u00a0M sodium acetate, pH 4.6, as precipitant. Crystals grew within 3\u20134\u00a0days and belong to space group P212121 with unit cell dimensions a\u00a0=\u00a092.74\u00a0\u00c5, b\u00a0=\u00a0107.49\u00a0\u00c5 and c\u00a0=\u00a0161.18\u00a0\u00c5. There are six copies of mutant 1FN3\u20132FN3 in the asymmetric unit, resulting in a solvent content of \u223c\u00a042%. Crystals were flash-frozen in liquid nitrogen after brief soaking in mother liquor supplemented with 20% glycerol, and diffraction data were collected at 100\u00a0K on station 10.1 at the SRS Daresbury. The diffraction data were processed with MOSFLM\u2021 and programs of the CCP4 suite.48 The structure of NCAM 1FN3\u20132FN3 was solved by single-wavelength anomalous dispersion phasing of a KI-soaked crystal using SHARP (Globalphasing Ltd., Cambridge) in full automatic mode. The structure was rebuilt with O49 and refined with Crystallography & NMR System.50 The structure of the NCAM M610R mutant was solved with some difficulty by molecular replacement with PHASER,51,52 using the isolated FN3 domains of the NCAM 1FN3\u20132FN3 structure as search models. Data collection, phasing and refinement statistics are summarised in Table 1. The figures were made with PyMOL\u00a7.\nSPR experiments\nBinding experiments were performed on a Biacore 3000 instrument (GE Healthcare) at 25\u00a0\u00b0C. Proteins were immobilised on activated CM4 or CM5 chips using standard amine coupling procedures following the manufacturer's instructions. Briefly, flow cells were activated with 20\u00a0\u03bcl of a mixture of 0.2\u00a0M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.05\u00a0M N-hydroxy-sulfosuccinimide at a flow rate of 5\u00a0\u03bcl\/min. The proteins to be immobilised (50\u2013100\u00a0\u03bcg\/ml in 10\u00a0mM sodium acetate, pH 4.5\u20135.5) were allowed to pass over activated flow cells to reach \u223c\u00a02000\u20138000\u00a0RU, after which unreacted groups were blocked with 20\u00a0\u03bcl of 1\u00a0M ethanolamine, pH 8.5. Reference flow cells without protein were treated identically. The chips were equilibrated in 10\u00a0mM Hepes, pH 7.4, 150\u00a0mM NaCl, 50\u00a0\u03bcM ethylenediaminetetraacetic acid and 0.005% surfactant P20 (HBS-EP buffer), and serial dilutions of analyte proteins in PBS (our recombinant proteins or FGF1 from PeproTech) were injected at 20\u00a0\u03bcl\/min for 300\u00a0s, followed by 200\u00a0s of pure buffer to monitor dissociation. Chips were regenerated using HBS-EP with 1\u00a0M glycine\u2013HCl, pH 3.5. The sensorgrams were analysed with the BiaEvaluation 4.1 software.\nProtein Data Bank accession codes\nCoordinates and structure factors for wild-type and M610R NCAM 1FN3\u20132FN3 have been deposited in the Protein Data Bank with codes 2vkw and 2vkx, respectively.","keyphrases":["cell adhesion","domain linker","cam, cell adhesion molecule","ncam, neural cell adhesion molecule","ig, immunoglobulin","fn3, fibronectin type 3","fgfr, fibroblast growth factor receptor","spr, surface plasmon resonance","ru, resonance units","pbs, phosphate-buffered saline","tbs, tris-buffered saline","crystal packing","protein interaction","x-ray crystallography"],"prmu":["P","P","R","R","R","R","R","R","R","R","R","R","R","M"]}
{"id":"Int_Arch_Occup_Environ_Health-4-1-2413125","title":"The influence of psychosocial factors at work and life style on health and work ability among professional workers\n","text":"Objectives The purpose of this article is to explore the associations of psychosocial factors at work, life style, and stressful life events on health and work ability among white-collar workers.\nIntroduction\nMany western countries face the challenge of an aging population, which also affects the workforce. From the biological perspective, aging means a progressive deterioration in various physiological systems, which is accompanied by changes in physical and mental capacities of workers (Ilmarinen, 1997). Aging of the workforce will result in an increased prevalence of work-relevant symptoms and diseases. Therefore, the role of (functional) health in working life is of interest, especially since modern welfare states are prolonging working life by increasing the statutory retirement age. A recent study on the relation between health and working life showed that a perceived poor health predicts staying or becoming unemployed (Schuring et al. 2007). This calls for better adjustments of the working life demands with the individual\u2019s health as a crucial element for a longer career at work. Within this framework, the concept of work ability has been developed as an important tool to identify workers at risk for imbalance between health, capabilities and demands at work.\nThe work ability concept is based on the assumption that work ability is determined by an individual\u2019s perception of the demands at work and the ability to cope with them. The work ability index (WAI) is a well-accepted instrument to conceptualize work ability. Several studies have shown that a low score on the index is highly predictive of work disability during follow-up (Liira et al. 2000; Tuomi et al. 1991). Previous research, predominantly in physically demanding jobs, showed that the WAI is negatively influenced by older age, high physical work demands, high psychosocial work demands (e.g. lack of possibilities to control one\u2019s own work), unhealthy lifestyle (lack of physical activity), and a poor physical fitness (Alavinia et al. 2007; Ilmarinen et al. 1997; Pohjonen, 2001a; Tuomi et al. 2001).\nFew studies have addressed determinants of work ability in occupational populations with predominantly mental demands at work. Among office workers Sj\u00f6gren-R\u00f6nk\u00e4 (2002) showed that low stress at work and a better self-confidence were directly related to higher work ability. Seniority in the job and job satisfaction were also associated with a better work ability among office workers (Martinez and Latorre 2006). However, the knowledge of determinants of work ability in mentally demanding occupations is scarce and hence, it remains unclear whether in these jobs the relative importance of personal and work-related factors is similar to their well-known contribution in physically demanding jobs.\nThe purpose of this study was to explore the associations of psychosocial factors at work, stressful life events, and life style on health and work ability among white-collar workers.\nMethods\nSubjects\nIn the period between 2003 and 2007 a total of 2,666 white-collar workers from six companies in commercial services were invited for a health examination. Twenty percent of the subjects were employed at three consultancy firms, 62% at two insurance companies and 18% at an information technology company. The health examination consisted of two parts, i.e. a questionnaire and a physical examination. Both parts were offered independently to workers and their participation was entirely voluntary. The response to the questionnaire was 69.4% (n\u00a0=\u00a01,850). The response to the physical examination was 67.8% (n\u00a0=\u00a01808). Selection of subjects with both a filled out questionnaire and a physical examination comprised the study population of 1141 (42.8%) subjects.\nWork ability\nWork ability was measured with the work ability index (WAI). The WAI consists of an assessment of the physical and mental demands on an individual in relation to his work, previously diagnosed diseases, limitations in work due to disease, sick leave, work ability prognosis, and psychological resources. The WAI consists of seven dimensions and the index is derived as the sum of the ratings on these dimensions. The range of the summative index is 7\u201349, which is classified into a poor (7\u201327), moderate (28\u201336), good (37\u201343), or excellent (44\u201349) work ability (Tuomi et al. 1998).\nFunctional health status\nFunctional health status was assessed using the Short-Form Health Survey (SF-12) version 2, the shortened alternative for the 36-item health survey. This measure provides two weighted summary scores assessing physical function (physical health component summary, PCS) and mental well-being (mental health component summary, MCS) (Ware et al., 2002). The mental health summary score ranges from 8 to 74, whereas the physical health summary score ranges from 4 to 73, with a higher score indicating a better health state.\nPsychosocial factors at work\nPsychosocial factors at work were measured by the stress monitor (Petri et al. 2001). The original monitor consists of four dimensions, whereas three dimensions (teamwork, stress handling, and self-development) were used in the current study. The three dimensions consist of 27 items on a five-point scale varying from \u201ctotally disagree\u201d to \u201ctotally agree\u201d. The dimension teamwork (Cronbach\u2019s alpha\u00a0=\u00a00.85) reflects social support and work spirit and consists of 12 items, e.g. \u201cI can rely on my colleagues and trust them\u201d and \u201cWe are not a team at work\u201d. The stress-handling dimension (Cronbach\u2019s alpha\u00a0=\u00a00.77) reflects active coping and self-efficacy and consists of seven items, such as \u201cIn difficult situations I do not wait and see, but take action\u201d and \u201cI can cope well with the demands of my job\u201d. The dimension self-development (Cronbach\u2019s alpha\u00a0=\u00a00.82) reflects possibilities for self-fulfilment and consists of eight items. Examples are: \u201cMy abilities are full employed\u201d and \u201cI need a new challenge\u201d. The scores on items within each dimension were transformed to a 0\u2013100 scale with a higher score indicating good teamwork, better stress handling, and more opportunities for self-development in work. The sum scores for the variables teamwork, stress handling, and self-development were not normally distributed. Tertiles were calculated to assign subjects into low, intermediate and high levels per dimension.\nStressful life events\nThe occurrence of stressful life events in the past 12\u00a0months was measured using a shortened social readjustment rating questionnaire (SRRQ) (Holmes and Rahe 1967). The original SRRQ consists of 43 life events (e.g., divorce, job change, death of family members and so forth), listed by rank order based on their mean life change values. Life change values classify the impact of the events and were obtained by scaling the life events based on the amount of coping required to deal with the event. The total score counts the life change values of all events in the past 12\u00a0months. In the current study the 25 events most appropriate for the population under study were selected. In theory, the total score can range from none of these events (0) up to all events (1077).\nLife style factors\nLife style factors were measured with the Dutch version of the Stanford wellness inventory (Sallis et al.  1985). Lifestyle factors of interest concerned moderate physical activity, vigorous activity, smoking, and alcohol use. The questionnaire has single questions on regular participation in moderate activities for 30\u00a0minutes or more and participation in vigorous activities for 20\u00a0min or more, both on a five-point scale ranging from \u201cnever\u201d to \u201c5\u00a0days or more per week\u201d. Those who reported moderate physical activity on at least 5\u00a0days per week were considered in agreement with the recommendation on moderate-intensity physical activity, and subjects with vigorous exercises at least 3\u00a0times per week were considered in agreement with the recommendation on vigorous-intensity physical activity (Pate et al. 1995). Current smoking was assessed with the question \u201cDo you smoke?\u201d. A five-point-response scale was used to assess alcohol drinking by average number of alcohol drinks per week (1\u20137, 8\u201314, 15\u201321, 22\u201328, more than 28). Problematic drinkers were defined as those who consumed more than 14 units of alcohol per week for women and more than 21 units for men (Health Council Netherlands    2006).\nPhysical examination\nPhysical examinations were performed using MicroFit equipment in accordance with the protocol of the American College of Sports Medicine (ACSM, 1975). During the physical examination biometry was recorded, including weight, height, biceps strength, and cardio respiratory fitness. The body mass index (BMI) was used to define subjects as normal (BMI\u00a0\u2264\u00a025), overweight (BMI 25\u201330), or obese (BMI\u00a0\u2265\u00a030). Maximal isometric muscular strength of the biceps was measured after one practice trial with a calibrated dynamometer with the subjects in standing position with 90\u00b0 flexion in the elbows for 3\u00a0s. The isometric biceps strength was calculated as the average of several hundred readings over the 3\u00a0s period. Cardio respiratory fitness was assessed by a 12\u00a0min sub-maximal bicycle ergometer test, supervised by instructors. Subjects pedaled at 60\u00a0rev.\u00a0min\u22121 for 12\u00a0min on the cycle ergometer, at an exercise intensity designed to produce a heart rate between 120 and 170\u00a0beats\u00a0min\u22121, in order to reach a level of 80% of the theoretical maximal heart rate of the participant for 3\u00a0min, after a warming up period of minimal 3\u00a0min. This level was sustained for 3\u00a0min and the heart rate was measured at the end of each minute. The VO2 max (mL\u00a0min\u22121\u00a0kg\u22121) was calculated by the work intensity (watts) and heart rates at the end of all the stages at exercise level.\nStatistics\nThe effects of individual characteristics (age and sex), life style, psychosocial factors at work, stressful life events, life style, and physical condition on the outcome variables work ability, and mental and physical health were investigated with linear regression analysis. Probability plots and Kolmogorov\u2013Smirnov tests showed that none of the determinants measured at continuous level were normally distributed. However, the evaluation of the distributions of residuals in the regression analyses showed that for those variables measured at ratio scale (i.e. age, VO2 max, and biceps strength), the assumption of linearity was not violated. These variables were included in the linear regression analyses as continuous variables. Due to considerable ceiling effects for the psychosocial variables and skewed distribution for life stress events, these variables were treated as categorical variables, defined by cut-off values based on tertiles.\nThe analysis started with univariate regression models to determine the single effects of all determinants of interest. A backward regression technique was used to determine the multivariate model with the best overall fit. In this analysis, independent variables with a P-value of 0.05 or less were retained in the final model.\nThe results of the regression analyses are presented by the regression coefficients and associated standard errors. A regression coefficient is an expression of the change in the work ability score due to a change in one unit of measurement of the independent variable of interest. For categorical variables, this reflects the effect on the work ability score of the presence of this determinant.\nThe regression analysis on determinants of work ability was stratified for three age groups. All significant determinants in the multivariate model for one age group were included in the models for other age groups as well in order to provide an appropriate comparison.\nAll analyses were carried out with the statistical package for social sciences version 11.0 for Windows (SPSS, 1999).\nResults\nThe study population included 769 men (67%) and 372 (33%) women in a variety of jobs (Table\u00a01). The median for age was 35.7\u00a0years (18\u201363). The distribution of excellent, good, moderate, and poor work ability was 42.8, 45.4, 9.7, and 2.1%, respectively. Subjects scored almost equal on mental health as on physical health, whereas the Pearson correlation coefficient between both measures of health was \u22120.20. The Pearson correlation coefficients between WAI and mental and physical health were 0.49 and 0.35, respectively. The three psychosocial factors at work were strongly interrelated with Pearson correlation coefficients varying from 0.45 to 0.57.\nTable\u00a01Characteristics of 1,141 commercial workers who participated in a voluntary medical examinationCharacteristicsCasesMedian (min\u2013max)Frequency (%)Individual characteristics\u00a0Age (year)1,14135.7 (18\u201363)67.4\u00a0Male769Work ability\u00a0Excellent (44\u201349)48842.1 (9\u201349)42.8\u00a0Good (37\u201343)51845.4\u00a0Moderate (28\u201336)1119.7\u00a0Poor (7\u201327)242.1Health\u00a0Mental health component summary (MCS) (8\u201374)1,14154.2 (10.9\u201367.9)\u00a0Physical health component summary (PCS) (4\u201373)1,14153.4 (18.2\u201370.6)Psychosocial factors at work\u00a0Teamwork (0\u2013100)1,13681.0 (27\u2013100)\u00a0Stress-handling (0\u2013100)1,13668.0 (11\u2013100)\u00a0Self-development (0\u2013100)1,13678.0 (9\u2013100)Stressful life events (0\u2013100)1,1365.5 (0\u221238.4)Life style\u00a0Lack of moderate physical activity (<5\u00a0days\u00a0week\u22121)79870.2\u00a0Lack of vigorous physical activity (<3times\u00a0week\u22121\u00a0)88678.0\u00a0Current smoker14512.8\u00a0Problematic alcohol use424.5Physical examination\u00a0Overweight (BMI 25\u201330\u00a0kg\u00a0m\u22122)37135.9 (11.4\u201361.7)34.6\u00a0Obesity (BMI\u00a0\u2265\u00a030\u00a0kg\u00a0m\u22122)5737.0 (8.0\u201394.0)5.3\u00a0VO2max (ml\u00a0kg\u00a0\u22121\u00a0min\u22121)1,117\u00a0Biceps strength (kg)1,134\nTable 2 shows mental health was statistically significant influenced by psychosocial factors at work, stressful life events, and, life style factors, whereas physical health was influenced by lifestyle factors and physical condition in the univariate model. The multivariate model explained 22% of the variance in mental health. An increase in age with one year increased the mental health score with 0.1 point, and decreased the physical health score with 0.1 point. In the multivariate analysis most determinants remained statistically significant, albeit with a lower regression coefficient, especially for teamwork and self-development. The multivariate model explained only 5% of the variance in physical health. It is of interest to note that neither problematic alcohol use nor overweight or obesity was associated with physical health.\nTable\u00a02Results of backward regression analysis: effects of psychosocial factors at work, stressful life events, lifestyle and physical condition on mental health and physical health among workers in commercial services (n\u00a0=\u00a01141)Mental Health (MCS)\nPhysical health (PCS)Univariate modelMultivariate modelUnivariate modelMultivariate model\u03b2SE\u03b2SE\u03b2SE\u03b2SEIndividual characteristics\u00a0Age (year)0.09*0.030.07*0.02\u20130.07*0.02\u20130.09*0.02\u00a0Male2.41*0.511.52*0.471.66*0.381.91*0.38Psychosocial factors at work\u00a0Low versus high teamwork \u20135.90*0.56\u20132.71*0.66\u20130.92*0.43n.s.\u00a0Intermediate high versus teamwork \u20132.39*0.57\u20130.700.58\u20130.680.45n.s.\u00a0Low versus high stress-handling \u20136.94*0.56\u20134.71*0.62\u20130.260.45n.s.\u00a0Intermediate versus  high stress-handling \u20132.39*0.58\u20131.42*0.590.010.46n.s.\u00a0Low versus  high self-development \u20135.44*0.60\u20132.12*0.65\u20130.730.46n.s.\u00a0Intermediate versus  high self-development \u20132.19*0.58\u20130.590.57\u20130.810.45n.s.Stressful life events\u00a0High versus  low stressful life events\u20133.13*0.59\u20133.13*0.54\u20130.620.44n.s.\u00a0Intermediate versus  low stressful life events\u20131.91*0.59\u20131.98*0.53\u20130.050.44n.s.Life style\u00a0Lack of moderate physical activity\u20130.140.53n.s.\u20130.010.39n.s.\u00a0Lack of vigorous physical activity\u20131.27*0.58\u20131.37*0.52\u20131.79*0.43\u20131.71*0.42\u00a0Current smoker\u20132.02*0.72\u20131.96*0.65\u20131.14*0.54n.s.\u00a0Problematic alcohol use\u20131.761.27n.s.\u20131.610.92n.s.Physical examination\u00a0Obesity (BMI\u00a0\u2265\u00a030) versus normal (BMI\u00a0<\u00a025)\u20130.841.14n.s\u20131.540.83n.s.\u00a0Overweight (BMI 25\u201330) versus normal 0.340.54n.s\u20130.300.39n.s.\u00a0VO2 max (ml\/kg\/min)\u20130.010.03n.s0.05*0.02n.s.\u00a0Biceps strength (kg)0.030.02n.s0.04*0.02n.s.n.s not significant, P\u00a0>\u00a00.05\nTable\u00a03 shows work ability was statistically significant influenced by psychosocial factor at work, stressful life events, lack of vigorous physical activity, and obesity in the univariate model. The multivariate model explained 29% of the variance in work ability. Again, in the multivariate model most determinants remained statistically significant, although with lower regression coefficients. The influence of stressful life events increased in the multivariate model.\nTable\u00a03Results of backward regression analysis: effects of psychosocial factors at work, stressful life events, lifestyle and physical condition on work ability among workers in commercial services (n\u00a0=\u00a01141)Work abilityWork abilityUnivariate modelMultivariate model\u03b2SE\u03b2SEIndividual characteristics\u00a0Age (year)\u20130.07*0.02\u20130.09*0.01\u00a0Male2.13*0.312.08*0.28Psychosocial factors at work\u00a0Low versus high teamwork\u20134.02*0.32\u20131.32*0.40\u00a0Intermediate versus high \u00a0teamwork\u20131.52*0.34\u20130.200.35\u00a0Lowversus versus high stress-handling\u20134.39*0.34\u20132.75*0.35\u00a0Intermediate versus high \u00a0stress-handling\u20131.41*0.35\u20130.79*0.35\u00a0Low versus high \u00a0self-development\u20134.11*0.35\u20132.20*0.39\u00a0Intermediate versus high \u00a0self-development \u20131.67*0.34\u20130.91*0.34Stressful life events\u00a0High versus low stressful life \u00a0events\u20131.36*0.36\u20132.01*0.32\u00a0Intermediate versus low stressful \u00a0life events\u20130.97*0.36\u20131.14*0.32Life style\u00a0Lack of moderate physical \u00a0activity0.490.32n.s0.31\u00a0Lack of vigorous physical \u00a0activity\u20130.71*0.35\u20130.71*\u00a0Current smoker\u20130.680.44n.s.\u00a0Problematic alcohol use\u20130.520.74n.s.Physical examination\u00a0Obesity (BMI\u00a0\u2265\u00a030) versus \u00a0normal (BMI\u00a0<\u00a025)\u20132.02*0.68\u20131.21*0.59\u00a0Overweight (BMI 25\u201330) versus \u00a0normal (BMI\u00a0<\u00a025)\u20130.490.32\u20130.320.28\u00a0VO2 max (ml\u00a0\u22121kg\u00a0\u22121min)0.030.02n.s.\u00a0Biceps strength (kg)0.030.01n.s.n.s\u00a0 not significant, P\u00a0>\u00a00.05\nNo significant interaction was observed for age, sex, and psychosocial factors at work.\nTable\u00a04 shows that in each age group sex, stress handling, and self-development were associated with the work ability index. Lifestyle factors were associated with work ability only in the oldest age group of workers, over 45\u00a0years. Obesity no longer was statistically significant.\nTable\u00a04Results on backward regression analysis per age-group: effects of psychosocial factors at work, stressful life events, lifestyle and physical condition on work ability among workers in commercial services (n\u00a0=\u00a01141)Work abilityMultivariate modelAge\u00a0\u2264\u00a032\u00a0year (n\u00a0=\u00a0335)Age 32\u201345\u00a0year (n\u00a0=\u00a0366)Age\u00a0>\u00a045\u00a0year (n\u00a0=\u00a0200)\u03b2SE\u03b2SE\u03b2SEIndividual characteristics\u00a0Male1.43*0.421.37*0.493.19*0.83Psychosocial factors at work\u00a0Low versus high teamwork\u20130.440.61\u20131.40*0.63\u20131.070.98\u00a0ntermediate versus high \u00a0teamwork\u20130.490.50\u20130.390.60\u20130.580.89\u00a0Low versus high \u00a0stress-handling\u20132.85*0.55\u20133.22*0.63\u20132.44*0.95\u00a0Intermediate versus high \u00a0stress-handling\u20130.960.52\u20131.080.61\u20130.830.86\u00a0Low versus high \u00a0self-development\u20132.59*0.64\u20131.64*0.59\u20133.57*1.01\u00a0Intermediate versus high \u00a0self-development \u20131.27*0.51\u20130.920.57\u20131.630.84Stressful life events\u00a0High versus low stressful life \u00a0events\u20131.25*0.49\u20131.67*0.51\u20132.60*0.84\u00a0Intermediate versus low \u00a0stressful life events\u20130.640.53\u20131.78*0.52\u20130.340.72Life style\u00a0Lack of moderate \u00a0physical activity\u20130.280.470.020.511.45*0.70\u00a0Lack of vigorous physical \u00a0activity\u20130.580.49\u20130.560.53\u20131.62*0.79\u00a0Problematic alcohol use1.700.93\u20130.851.19\u20132.62*1.33P\u00a0>\u00a00.05\nDiscussion\nThis study showed that work ability of white-collar workers in commercial services industry was strongly associated with psychosocial factors at work, such as teamwork, stress handling, self-development, and, to a lesser extent, with stressful life events, lack of physical activity, and obesity. Work ability was strongly associated with mental and physical health. Determinants of mental health were very similar to those of work ability, whereas physical health was influenced primarily by lack of life physical activity.\nSome limitations must be taken into account in this study. First, the cross-sectional design does not permit exploration of causal relationships between the determinants and work ability. Therefore, it remains unknown whether, for example, poor stress handling will decrease work ability or decreased work ability will cause a poorer stress handling. Nevertheless, the results are still of interest as they give a first insight into important factors for interventions among white-collar workers. Second, data were drawn from voluntary participants. Information on non-response for both measures showed that age and sex did not bias response. Non-response differences between questionnaire and physical examination did not show any bias; none of the questionnaire variables were associated with not participating in the physical examination; and also none of the physical examination variables were associated with not participating in the questionnaire. Third, the reliability of the physical examination highly depends on the professional skills of the instructor and the standardization of the examination. The maximum oxygen uptake was indirectly calculated using the heart rate, which can be easily increased by minor distractions, such as room temperature, and talking during the test.\nIn this study among white-collar workers in commercial services industry, the proportion of workers with poor work ability was 2.1% and the mean WAI was 41.1 (SD\u00a0=\u00a05.1). These results are slightly higher than the Finnish reference data in mentally demanding work (mean 39) (Tuomi et al. 1998).\nWork ability in this study population was influenced by sex, age, psychosocial factors at work, stressful life events, and life style factors. These factors together explained 29% of the total variance in work ability in this study population. Male sex increased work ability with two points, which means 4% of the maximum score. An increase in age of 40\u00a0years decreases the WAI score with four points, which is 7.3% of the maximum score, which indicates a rather modest influence of age on work ability. Psychosocial factors each had an effect on WAI comparable to sex, whereas the combined effect of the psychosocial factors is approximately 1.5-fold the effect of 40\u00a0years of aging. Lack of vigorous physical activity decreases the WAI score with only 0.7 points, which is no more than 1.5% of the maximum score. Obesity (5% of the population) compared to normal weight decreases the WAI score with 1.2 points, which is 2.4% of the maximum score.\nEach psychosocial factor at work was negatively associated with work ability. Univariate results showed comparable strength in associations, while the multivariate model showed lower regression coefficients, especially for teamwork. It seems that the association between teamwork and work ability was more influenced by other determinants included in the multivariate model, than the associations between work ability and stress handling and self-development.\nIn previous research, inconsistent results were found regarding the influence of psychosocial factors at work on work ability. For example, in the metal industry an increase in teamwork and increase in opportunities for development was not predictive of an increase in work ability during a 2-year follow-up (Tuomi et al. 2004). Negative associations between mental stress and work ability have been found among office workers (\u03b2\u00a0=\u00a0\u22120.17), but this association was minimized when including age in the regression model (Sj\u00f6gren-R\u00f6nk\u00e4, 2002). Among bus drivers, significant associations were observed for high control by superiors and lack of responsibility at work with lower WAI scores (Kloim\u00fcller et al. 2000).\nThe negative association of stressful life events with work ability in the current study is in agreement with earlier findings by Pohjonen (2001b), who found an increased risk for poor work ability [OR\u00a0=\u00a03.62 (2.2\u20135.9)] for a hard life situation outside of work.\nThe results showed that a lack of vigorous physical activity was associated with decreased work ability, whereas associations between work ability and biceps strength and maximum oxygen uptake were not found in the multivariate model. The lack of significant results for maximum oxygen uptake and biceps strength is in line with findings of Eskelinen et al. (1991), Nygard et al. (1991), and Pohjonen (2001a). It may be hypothesized that in mentally demanding jobs a good physical condition is not required to meet the work demands and, thus, will have no influence on work ability.\nStratification by age showed the importance of lifestyle in the oldest age group, but not among younger workers. This effect may be explained by the fact that health problems due to an unhealthy lifestyle, most notably diabetes mellitus and cardiovascular disease, occur primarily at older age. In the total study population, obesity was significantly associated with lower work ability, whereas no significant associations were found in the stratified analyses. This is partly due to lack of statistical power in these strata with smaller numbers of workers, since the magnitude of the regression coefficients were comparable but the standard errors increased substantially.\nThe Pearson correlation coefficient of mental and physical health was \u22120.20, which was in line with results of van Duijn et al. (2004). In a univariate analysis both mental health and physical health were associated with work ability. However, determinants of work ability were similar to determinants of mental health. This finding can be explained by the fact that the work setting of the white-collar workers in the current study is characterized by high mental demands. An exception to the similarity in factors influencing both mental health and work ability was, smoking. Smoking was related to mental health, but not to work ability.\nThe results of the current study outline the importance of work-related factors in white-collar workers, with regard to work ability. The combined impact of psychosocial factors is much stronger than is for individual factors, and is amenable to change, in contrast to individual factors as age, and sex.\nIn conclusion, among white-collar workers in commercial services industry psychosocial factors at work, stressful life events, lack of vigorous physical activity, and obesity were significant with regard to work ability. The strong associations between psychosocial factors at work and mental health and work ability suggest that in this study population health promotion should address working conditions rather than individual life style factors, although the importance of life style factors seems to increase with aging of the worker.","keyphrases":["psychosocial factors","work ability","physical activity","functional health"],"prmu":["P","P","P","P"]}
{"id":"Ann_Biomed_Eng-2-2-1705482","title":"Evaluation of Important Treatment Parameters in Supraphysiological Thermal Therapy of Human Liver Cancer HepG2 Cells\n","text":"This study was aimed at simulating the effect of various treatment parameters like heating rate (HR), peak temperature (PT) and hold\/total treatment time on the viability of human liver cancer HepG2 cells subjected to different thermal therapy conditions. The problem was approached by investigating the injury kinetics obtained using experimentally measured viability of the cells, heated to temperatures of 50\u201370\u00b0C for 0\u20139 min at HRs of 100, 200, 300 and 525\u00b0C min\u22121. An empirical expression obtained between the activation energy (E) and HR was extended to obtain the E values over a broad range of HRs from 5 to 600\u00b0C min\u22121 that mimic the actual conditions encountered in a typical thermal therapy protocol. Further, the effect of the HR (5\u2013600\u00b0C min\u22121) and PT (50\u201385\u00b0C) on the cell survival was studied over a range of hold times. A significant drop in survival from 90% to 0% with the simultaneous increase in HR and PT was observed as the hold time increased from 0 to 5 min. For complete cell death, the hold time increased with the increase in the HR for a given PT, while the total time showed presence of minima for 60, 65 and 70\u00b0C at HRs of 50, 100 and 200\u00b0C min\u22121, respectively.\nIntroduction\nPrimary liver cancer is the fifth most common malignancy in the world, with a global annual incidence of about one million new patients.6 In 2004, the American Cancer Society estimated 18,920 new cases of Hepatocellular Carcinoma (HCC) and the estimated deaths were 14,720.1 Surgical resection, the gold standard for the treatment of primary liver cancer, shows low success rate (20\u201337%) and a high recurrence (50\u201360%) rate due to many surgical complications.10,25 Liver transplantation is limited because of fewer donors.18 Combination treatment methods, using chemotherapy, embolization and chemoembolization have a limited effect even with the newer drugs available and their beneficial effects on the patient survival remain controversial in randomized studies.18,24 Use of radiation therapy is limited to alleviating the symptoms such as pain or just to shrink the tissue rather than destroying it. Ablation therapies that use heat to destroy the tumor are gaining increasing attention as an alternative because the treatment procedure is faster, simpler, less painful and cheaper.20\nThere are two known approaches for the application of these ablative procedures: hyperthermia, where the treatment modalities use temperatures ranging from 42 to 50\u00b0C for periods of 30\u00a0min to few hours. Another approach is thermal therapy, where the destruction of the tumor takes place by the application of heat at temperatures higher than 50\u00b0C within very short time periods of few minutes to seconds. The tumor is heated using any one of radiofrequency, microwave, laser or high intensity focused ultrasound energy sources. Several clinical trials have been performed to test the efficacy of these thermal ablative therapies in treatment of liver cancer using the above-mentioned energy sources. The results have shown survivals of 83\u201394% after 1\u00a0year, 50% after 2\u00a0years, 33% after 3\u00a0years and 33\u201340% after 5\u00a0years for hyperthermic radiofrequency ablation in studies conducted on 29\u2013123 patients.18 Survival of 86% after 1\u00a0year in 55 patients has been reported for hyperthermic interstitial laser therapy;12 survivals of 73\u201386% after 3\u00a0years, 18 50% after 4\u00a0years and 48.6% after 5\u00a0years17 have been reported for microwave coagulation therapy. In spite of all the above-mentioned clinical trials, no data exists showing the effect of thermal therapy on the survival of liver cancer cells. Effect of hyperthermic temperatures on human liver cancer cell lines has also been investigated in few in vitro studies. Callari et\u00a0al.7 used HTC hepatoma cells to study the action of retinol on viable cell recovery after in vitro hyperthermia at temperatures between 42\u00b0C and 44\u00b0C for 1\u00a0h. Hasumura et\u00a0al.13 studied the effect of TNF along with hyperthermia on JHH-4, JHH-5 and JHH-7 human hepatoma cells between temperatures of 41.4\u00b0C and 42.5\u00b0C. However, none of the studies demonstrated the effect of temperatures above 50\u00b0C any liver cancer cell lines.\nSince in thermal therapy, the injury accumulates at very high temperatures within a short period of time, knowledge of the injury kinetics is very useful for predicting the cell\/tumor damage15 for these thermal histories. Several thermal therapy studies have been performed to obtain the injury kinetics in different cell lines. Landry et\u00a0al.19 heated HeLa cells up to 55\u00b0C using water bath, with thermal equilibrium time of 1\u00a0min for 55\u00b0C. To reduce this equilibrium time Borrelli et\u00a0al.5 heated cells to 57\u00b0C on 0.025-mm thick mylar pieces. Cell injury kinetics was obtained by measuring the cell survival using clonogenic assay in both the studies. In addition, studies on T24 human bladder carcinoma,22 skeletal muscle,11 SN12 renal cell carcinoma15 and Dunning AT-1 prostate cancer cells4 in the temperature range of 40\u201370\u00b0C obtained cell injury kinetics using dye uptake assays and thus showed that dye uptake assay is a reasonable conservative estimate of the cell survival. Also, two recent in vitro studies in human benign prostatic hyperplasia tissue and rodent prostate cancer tissue showed that the cell injury kinetics measured by membrane integrity vital dye assay and histology assays are very similar.2,3 Therefore, a key observation from the above studies is that vital dye uptake assay can be used as an alternate and reliable conservative maker as it provides rapidity, automation and better control.15\nA key difference of high temperature\u2013short time thermal therapy protocols from the traditional hyperthermia protocols is the significant injury accumulation during the non-isothermal portion of the thermal history (heating up\/cooling down period). Therefore, injury accumulation tends to be a complex function of hold time as well ramp up and cooling time. Hence, accurate prediction of the cell injury kinetics requires the knowledge of heating\/cooling rates in addition to peak temperatures (PTs) and hold\/total time. This model also better represents the thermo-clinical applications because the rate at which the PTs are achieved at different locations inside the tumor vary with the applicator location and plays a paramount role in determining the amount of injury accumulated. The knowledge of the injury accumulated in reaching a PT or the PT required to obtain a desired injury under different heating rates (HRs) are some of the very important parameters in designing better and optimal clinical protocols.\nThis study was used to investigate experimentally and through simulations the parameters affecting thermal therapy of primary human liver cancer HepG2 cells between 50\u00b0C and 70\u00b0C using Ethd-1 uptake cell membrane integrity assay. The cells were thermally challenged on a programmable heating stage which can control and record the entire thermal history profile, allowing us to recreate typical thermal therapy protocols. Cell membrane integrity dye uptake (Ethd-1) was used as an irreversible injury marker. The first-order Arrhenius rate model was used to extract the kinetic parameters activation energy, E (kJ\u00a0mol\u22121) and frequency factor A (s\u22121).16 The survival data was fitted into the Arrhenius model for various HRs and the E and A values were predicted by minimizing the least square between the model predictions and the experimental data for suspended and attached cells. Based on the predicted E values, a relationship between the E and HR was obtained experimentally and was further used to simulate the survival map for different hold periods by varying the target PT and the HR. Finally, the hold time and the total injury time necessary for complete cell death (Survival\u00a0\u2264\u00a00.001%) was predicted for target PTs of 60\u201385\u00b0C and HRs of 5\u2013600\u00b0C\u00a0min\u22121 representing the typical transient thermo-clinical protocol.\nMaterials and methods\nHEPG2 Cell Culture and Sample Preparation\nHuman liver cancer Hepg2 cells (ATCC, VA) were propagated in MEM cell culture media with 10% Fetal Bovine Serum (Hyclone, UT) and 1% penicillin\/streptomycin (Invitrogen, CA). They were then incubated in 10% CO2 and 95% humidified air at 37\u00b0C in 75-cm2 T flasks. The cells were trypsinized with Trypsin-EDTA (ATCC, VA) for 5\u201310\u00a0min, centrifuged at 1200\u00a0rpm for 5\u00a0min and re-suspended in the media to an appropriate concentration. Suspended cell samples were prepared by placing 3\u00a0\u03bcl of cell suspension onto the center of the 12-mm diameter coverglass, covering it with another coverglass to prevent evaporation during heating. For attached cell studies, cell suspension was scattered over about 18\u201320, 12-mm cover glasses lying in a 110-mm diameter Petri dish. Overnight attachment of the cells on top of the coverglass was allowed in the incubator and then the cell sample was prepared in the same way as the suspended sample.\nHeating Stage\nAttached and suspended cell samples were heated on a programmable heating stage, at different HRs. Figure\u00a01 shows the setup diagram of the heating stage and the feedback control used. Cement T-type thermocouple (Omega Engg Inc., CT) was used for temperature detection, which is interfaced with a data acquisition board (DAB) (Keithly Inc., Cleveland, OH) connected to the computer port. The interface between the user and the DAB is through a Visual Basic code that calculates the output voltage based on the set point and instantaneous temperature and controls it based on proportional and derivative constants used in the feedback loop. Table\u00a01 shows the combinations of proportionality (kp) and derivative (kd) constants used in the feedback loop to achieve different HRs (100, 200, 300 and 525\u00b0C\u00a0min\u22121). An OPAMP (Analog Devices Inc, MA) circuit is used to amplify the feedback voltage signal from the DAB and heat the stage. The stage was calibrated using fixed temperature tempi labels and sticks (MSC Industrial Supply Co., NY) to an accuracy of \u00b10.3\u00b0C for all PTs at each HR used in the study.\nFigure\u00a01.Block diagram of the amplifying OPAMP circuit and feedback control of the heating stage. The OPAMP circuit amplifies the voltage signal from the data acquisition board to heat the stage, while the feedback control system, through the VB code, controls the amount of voltage based on the instantaneous temperature of the stage.Table\u00a01.Values of proportionality (kp) and derivative (kd) constants for different HRs to obtain desired PTs.HR (\u00b0C\u00a0min-1)PT (\u00b0C)kpkd100600.01550.0925650.01550.0925700.01550.0925200600.0350.108650.0350.108700.0350.121300600.0850.255650.0850.255700.09030.275\nHeating Studies and Ethd-1 Dye Uptake Assay \nSuspended and attached HepG2 cell samples were heated to different time\u2013temperature histories. Isothermal heating was carried out for suspended and attached cells at temperatures between 50\u00b0C and 70\u00b0C for 0.5\u20139\u00a0min. The cell sample was placed on the center of the heating stage after the PT was reached. The time for each cell sample to equilibrate to any given PT was experimentally calculated to be \u223c5\u00a0s and was added to the hold time during heating. HRs of 100, 200 and 300\u00b0C\u00a0min\u22121 were employed for heating attached cells non-isothermally between temperatures of 60\u00b0C and 70\u00b0C for a hold time of 0\u20133\u00a0min using the entire temperature\u2013time history, which includes the rise, hold and the cooling period. The HRs were assumed to be constant for the entire thermal history and were obtained by taking a tangent to the exponential curve of the temperature rise. Therefore, the cell sample was placed on the center of the heating stage before starting the heating cycle. The cooling time for each sample from a given PT was calculated based on the experimentally measured cooling rate of 100\u00b0C\u00a0min\u22121.\nCellular injury post heating for isothermal and non-isothermal studies was quantified using Ethd-1 (Sigma-Aldrich, MO) vital dye assay by counting the number of cells stained with Ethd-1 dye (dead only) and the total number of the cells stained with Hoechst (Sigma-Aldrich). Thermally treated cell samples were placed in a 50\u00a0\u03bcl drop of 2.5\u00a0\u03bcM Ethd-1 and 10\u00a0\u03bcM Hoechst dye solution in a 35-mm Petri dish and incubated for 3\u00a0h. The incubation time of 3\u00a0h was based on the experiments conducted after 1, 2 and 3\u00a0h of incubation (data not included), which confirmed that membrane damage equilibrates within this period and a significant amount of media is pulled between the cover glasses to stain the cells. Control samples underwent the same procedure without heating. After 3\u00a0h, the dead and the total number of cells were counted with a fluorescent microscope (Nikon Eclipse TS 100, Tokyo) using a 20\u00d7 objective. Multiple fields with at least 150\u2013300 cells were counted for each run.\nNormalized Cell Survival\nFor all the cell samples, the normalized cell survival (Se) was calculated as follows: \nNH and NE are the number of total and dead cells stained with Hoechst and Ethd-1 respectively; subscripts \u2018t\u2019 and \u2018c\u2019 represent thermally treated and control cells, respectively. The calculated cell survival was based on three separate experiments, with two runs for every data point in each experiment.\nThe Cell Injury Model\nThermally induced cellular injury was considered as a first-order irreversible process as shown below and as used in many previous heating studies; where V represents the viable state of a cell, I represents the injured state of a cell and k is the cell injury rate. The predicted cell viability (Sc) was calculated using the following equation: \u03b4 is the total treatment time (s).\nThe Arrhenius model16 was adopted in this study as it has been extensively used in previous studies to obtain the cell injury kinetics14,21 as shown below; A is frequency factor (s\u22121), E is activation energy (J\u00a0mol\u22121) and R is the universal gas constant (8.314\u00a0J\u00a0mol\u22121\u00a0K\u22121). A and E are related by an empirical formula, obtained by compiling the injury kinetics of various proteins, cells and tissues.15\nDetermination of Arrhenius Model Parameters\nThe kinetic parameters E and A were determined by fitting Eqs. (4) and (5) in Eq. (1) as obtained by He and Bischof15 They were further refined using a simple optimization subroutine to minimize the root mean square error between the experimental data and model prediction. \nN is the total number of data points, vector \u2018X\u2019 contains variables E and A. Se,i and Sc,i represent the ith measured data point and model fit, respectively. The goodness of the fit (R2) is calculated as follows: \nSav is the measured average survival from the Etdh-1 studies.\nResults\nThe results were generated by counting the number of total and dead cells from the micrographs of the cell samples as shown in Fig.\u00a02 and using Eq. (1) to calculate the normalized cell survival for each sample. Figure\u00a02a, b represent the control sample, where the number of dead cells, stained with Ethd-1 (red) dye, is very less compared to the total number of cells, stained with Hoechst (blue) dye and the cell survival is higher than 0.96. The cell survival decreases slightly as the number of dead cells compared to the total number of cells increases for the cell sample heated at 50\u00b0C for 9\u00a0min, as shown in Fig.\u00a02c, d. The survival drops dramatically with a sharp increase in the number of dead cells compared to total cells at 70\u00b0C as seen in Fig.\u00a02e, f.\nFigure\u00a02.Micrographs showing cell samples stained with Hoechst (Blue-all cells) and Ethd-1 (Red-dead cells only). (a and b) \u2013 control samples without heating; (c and d) \u2013 samples heated at 50\u00b0C for 9\u00a0min; (e and f) \u2013 samples heated at 70\u00b0C for 1\u00a0min.\nIsothermal Heating Studies\nThe isothermal heating studies were used to understand the effect of different temperature\u2013time histories on the viability of attached and suspended HepG2 cells and to quantify the cellular injury associated with these histories. All the cell samples reached the PTs in \u223c5\u00a0s at an approximate HR of 525\u00b0C\u00a0min\u22121 resulting in a negligible difference between the hold time and the total time of the treatment. Thus heating at the rate of 525\u00b0C\u00a0min\u22121 was referred as isothermal heating.\nThe data in Fig.\u00a03a, b represents the mean and the standard error for measured cell viability of attached and suspended HepG2 cells respectively using Ethd-1 dye uptake assay. Both the figures show that for a given PT, the survival drops with the increase in the hold time. At 50\u00b0C, the survival drops from 0.84 to 0.43 for suspended cells and from 0.89 to 0.62 for attached cells, as the hold time increases from 2 to 9\u00a0min. Similar trend was observed for heating at all the PTs. The figures also show that for a given hold time, the cell survival drops with the increase in the PT for suspended cells. For a hold time of 2\u00a0min, the cell survival drops from 0.84 to 0.52 and from 0.52 to 0.29 as the PT increases from 50\u00b0C to 55\u00b0C and from 55\u00b0C to 60\u00b0C, respectively. For a hold time of 1\u00a0min, the cell survival drops from 0.45 to 0.1 and from 0.1 to 0.003 as the PT increases for 50\u00b0C to 55\u00b0C and from 55\u00b0C to 60\u00b0C, respectively. The same trend is also seen for the attached cells but with slightly higher survival.\nFigure\u00a03.(a) Plot of experimentally measured and predicted survival of attached cells heated isothermally versus hold time in minutes for single cell heating at PTs of 50, 55, 60, 65 and 70\u00b0C. Cross (\u00d7), filled circles (\u2022), filled diamonds (\u2666), filled squares (\u25a0) and filled triangles (\u25b2) represents heating of samples at 50, 55, 60, 65 and 70\u00b0C, respectively. The lines represent the exponential fit through all the data points based on the predicted E and A values. (b) Plot of experimentally measured and predicted survival of suspended cells heated isothermally versus hold time in minutes for single cell heating at PTs of 50, 55, 60, 65 and 70\u00b0C. Cross (\u00d7), filled circles (\u2022), filled diamonds (\u2666), filled squares (\u25a0) and filled triangles (\u25b2) represents heating of samples at 50, 55, 60, 65 and 70\u00b0C respectively. The lines represent the exponential fit through all the data points based on the predicted E and A values. (c) Direct comparison between suspended and attached HePG2 cell viability, using student t-test assuming unequal variance. Each data point is an average of three separate runs using two different cell samples for each run. The error bar in the figure represents the standard error of the mean. Data points marked with asterisks indicate the data points where viability of suspended and attached cells is significantly different.\nThe figures also show the effect of PTs on the slope of the survival curve. The drop in the survival with the increase in the hold time is slow at low PTs. The survival curve gets steeper as the PT increases. When heated at 50\u00b0C for up to 9\u00a0min, the cell survival is still higher than 0.43 for both suspended and attached cells. This trend changes for 55\u00b0C, where the cell survival drops to 0.03 after only 6\u00a0min of heating. This trend magnifies sharply as the PTs increases to 60, 65 and 70\u00b0C. For suspended cells, the survival at 60\u00b0C varies from 0.58 to almost zero survival as the hold time increases from 0.5 to 3\u00a0min. The survival dropped from 0.2 to 0.003 for an increase in the hold time from 15\u00a0s to a min at 70\u00b0C. The attached cells also showed a similar trend with slightly higher survival than the suspended cells for all the data points.\nThe student t-test assuming unequal variance was used to observe the statistical significance between the experimentally measured cell viability of suspended and attached HepG2 cells as shown in Fig.\u00a03c. With p\u00a0=\u00a00.05 as the criterion, no significant difference in the cell viability was observed for most of the data points. The data points that were significantly different (p\u00a0<\u00a00.05) are 50\u00b0C \u2013 5\u00a0min, 50\u00b0C \u2013 9\u00a0min, 60\u00b0C \u2013 3\u00a0min and 70\u00b0C \u2013 1\u00a0min. This difference for data points of 60\u00b0C \u2013 3\u00a0min and 70\u00b0C \u2013 1\u00a0min can be neglected as the experimentally calculated cell viability in suspended and attached HepG2 for both these thermal insults is of the order 10\u22123. The p-value of 0.088 for 60\u00b0C \u2013 2\u00a0min data point suggests the difference in the cell viability is not significant, but is still different. As no significant difference between the survival of suspended and attached HepG2 cells was observed for most of the time\u2013temperature histories and since attached cells better describe the arrangement of the cells in a tissue, only attached cells were used for further non-isothermal studies.\nThe parameters of cell injury, E and A, were obtained by fitting the Arrhenius rate model into the survival data. This was done by minimizing the function in Eq. (6) using Eqs. (3) and (4). The predicted E and A values for suspended and attached cells for isothermal heating are shown in Table\u00a02. The E and A values obtained for suspended cells are 229.46 (kJ\u00a0mol\u22121) and 3.495\u00a0\u00d7\u00a01031 (s\u22121) and those for attached cells are 248.64 (kJ\u00a0mol\u22121) and 5.396\u00a0\u00d7\u00a01036 (s\u22121), respectively. The predicted survival based on the predicted E and A values for attached and suspended cells is shown by the solid lines in Fig.\u00a03a, b, respectively. The goodness of the fit between the predicted and experimentally measured values, obtained using Eq. (7) for suspended and attached cells, is 0.86 and 0.84, respectively.\nTable\u00a02.Activation energy E (kJ\u00a0mol\u22121) and frequency factor A (s\u22121) using all the data point for different HRs.HR (\u00b0C\u00a0min\u22121)Activation energy E (kJ\u00a0mol\u22121)Frequency factor A (s-1)100272.47.757\u00a0\u00d7\u00a01040200262.021.502\u00a0\u00d7\u00a01039300257.382.576\u00a0\u00d7\u00a01038525 (Isothermal \u2013 Attached)248.645.396\u00a0\u00d7\u00a01036525 (Isothermal \u2013 Suspended)229.463.495\u00a0\u00d7\u00a01031\nNon-Isothermal Heating Studies\nThe study focused next on the investigation of the effect of different HRs on the cell viability of attached HepG2 cells and extraction of kinetic parameters of the cellular injury associated with each HR. The HRs of 100, 200 and 300\u00b0C\u00a0min\u22121 were employed in this study, as they all showed a considerable difference in the rise-time at all the PTs. The total injury time (rise, hold and cooling time) was taken into account to quantify the thermal injury for all the HRs.\nThe data in Fig.\u00a04a\u2013c represents the mean and the standard error of the measured cell viability using Ethd-1 dye uptake assay for attached HepG2 cells at HRs of 100, 200 and 300\u00b0C\u00a0min\u22121, respectively. All the figures show that, for a given HR, the cell survival drops with the increase in the hold time and the PT, similar to that observed in isothermal heating. At the HR of 100\u00a0C\u00a0min\u22121, the survival drops from 0.82 for no hold time to 0.18 for a hold time of 2\u00a0min for a PT of 60\u00b0C. This trend remains the same for the PTs of 65\u00b0C and 70\u00b0C and for all the HRs. The survival drops from 0.82 at 60\u00b0C to 0.69 at 65\u00b0C and further to 0.11 at 70\u00b0C, for no hold time at HR of 100\u00b0C\u00a0min\u22121. This trend continues for all the HRs and at all the hold times. The figures also illustrates that for any given PT and for a given hold time, the cell survival increases with the increase in the HR. At 60\u00b0C for no hold time, the survival increases from 0.82 at 100\u00b0C\u00a0min\u22121 to 0.85 at 200\u00b0C\u00a0min\u22121 and further to 0.88 at 300\u00b0C\u00a0min\u22121. At 65\u00b0C for a hold time of 0.5\u00a0min, the survival increases from 0.15 at 100\u00b0C\u00a0min\u22121 to 0.19 at 200\u00b0C\u00a0min\u22121 and then to 0.22 at 300\u00b0C\u00a0min\u22121. This trend also continues for 70\u00b0C for any given hold time.\nFigure\u00a04.Plot of experimentally measured and predicted survival of attached cells heated at various heating rates versus time in minutes for single cell heating at temperature 60, 65 & 70\u00b0C. (a) 100\u00b0C min\u22121. (b) 200\u00b0C min\u22121. (c) 300\u00b0C min\u22121. Filled diamonds (\u2666), filled squares (\u25a0) and filled triangles (\u25b2) represents heating of samples at 60, 65 and 70\u00b0C respectively. The lines represent the exponential fit through all the data points based on the predicted E and A values.\nA trend was observed in the Arrhenius parameters obtained from the non-isothermal heating studies as the E, and the corresponding A value, decreased with the increase in the HR. Table\u00a02 shows the predicted E and A values for HRs of 100, 200 and 300\u00b0C\u00a0min\u22121. The activation energy decreases from 272.40\u00a0kJ\u00a0mol\u22121 at 100\u00b0C\u00a0min\u22121 to 262.02\u00a0kJ\u00a0mol\u22121 at 200\u00b0C\u00a0min\u22121 and then to 257.38\u00a0kJ\u00a0mol\u22121 at 300\u00b0C\u00a0min\u22121. The solid lines in the Fig.\u00a05a\u2013c represent the predicted cell survival based on above values respectively. The goodness of fit between the predicted and measured cell survival for all data points obtained using Eq. (7) is 0.97 for all the HRs used in this study.\nFigure\u00a05.Plot of the normalized survival of attached cells for various hold times at different peak temperatures and heating rates based on the predicted E and A values. (a) 0 min. (b) 0.25 min. (c) 1 min. (d) 5 min.\nRelation Between Activation Energy and HRs\nThe following relation was obtained by fitting a polynomial trend line through the data in Table\u00a02 that includes the isothermal heating at 525\u00b0C\u00a0min\u22121; \nE represents the activation energy and HR represents the HR. The above equation was then used to obtain the values of activation energy over a range of HRs from 5 to 600\u00b0C\u00a0min\u22121 in order to simulate the effects of various parameters on the cell survival. The values of the activation energy shows a small decrease from 282.09 to 248.29\u00a0kJ\u00a0mol\u22121 over a large increase in the HR increased from 5 to 600\u00b0C\u00a0min\u22121.\nEffects of PTs, HRs and Hold Time on the Cell Survival\nBased on the E and A values obtained at HRs of 5\u2013600\u00b0C\u00a0min\u22121 using the E\u2013HR relation, the cell survival was predicted at different hold times for PTs between 50\u00b0C and 85\u00b0C. The combined effect of the increase in the hold time and PT on the cell survival is shown in the Fig.\u00a05a\u2013d for all the HRs mentioned above.\nFor a given hold time and HR, the cell survival drops consistently with the increase in the PT. For a HR of 20\u00b0C\u00a0min\u22121, the cell survival drops from 0.98 at 50\u00b0C to 0.091 at 65\u00b0C and then to no cell survival at temperature above 75\u00b0C, when the hold time is 0\u00a0min. The trend remains the same for any HR and hold time combination. The drop in the cell survival for any hold time is lower when the PTs are 55\u00b0C and less. The survival drops slightly from 0.96 at 50\u00b0C to 0.84 at 55\u00b0C for heating at 200\u00b0C\u00a0min\u22121 for a hold time of 1\u00a0min. This drop is higher, from 0.84 to 0.48, when the temperature increases to 60\u00b0C. This trend is magnified with the increase in the PT to 65\u00b0C where the survival drops to 0.05 and to almost complete cell destruction for a further 5\u00b0C rise in the PT. At any given hold time and PT, the cell survival increases with the increase in the HR. The survival increased from 0.006 to 0.56 at 60\u00b0C for a hold time of 1\u00a0min and from 0 to 0.002 for a hold time of 3\u00a0min at 65\u00b0C as the HR increased from 5 to 600\u00b0C\u00a0min\u22121.\nFor a given PT and a HR, the cell survival drops with the increase in the hold time. At 60\u00b0C and 50\u00b0C\u00a0min\u22121 the cell survival drops from 0.76 to 0.008 when the hold time increases from 0 to 5\u00a0min. For the same increase in the hold time, the survival drops from 0.12 to zero at 70\u00b0C and 400\u00b0C\u00a0min\u22121. For any hold time, almost complete cell destruction takes place at all the HRs when the PTs are 75\u00b0C and above. For HRs of 50\u00b0C\u00a0min\u22121 and lower, the PT to acquire almost complete cell death decreases with the increase in hold time. For zero hold time, 70\u00b0C shows a survival of 0.004 and less for HRs of 50\u00b0C\u00a0min\u22121 and less. When the hold time is 1\u00a0min, complete cell death is observed at 65\u00b0C for all the HRs below 50\u00b0C\u00a0min\u22121 and at 60\u00b0C for HR of 5\u00b0C\u00a0min\u22121. The survival at 55\u00b0C decreases considerably with hold time at these low HRs but is still significantly high (>10%), while the survival at 50\u00b0C shows a very less drop. For a HR of 10\u00b0C\u00a0min\u22121, the survival at 55\u00b0C drops from 0.79 to 0.28 with the increase in the hold time from 0 to 5\u00a0min. At 50\u00b0C, this drop is from 0.96 to 0.79. This trend is depicted in Fig.\u00a05a\u2013d as the drop in the slope of the vertical portion with the increase in the hold time.\nHold and Total Injury Time for Complete Cell Destruction\nThe relationship between activation energies and HRs was further used to predict the hold time and total injury time required to attain complete cell destruction at different target PTs and HRs that represents different scenarios of a clinical thermal therapy protocol. Figure\u00a06a, b shows the calculated hold time and total time in minutes resulting in complete cell death at target PTs between 60\u00b0C and 85\u00b0C and HRs between 5 and 600\u00b0C\u00a0min\u22121, which represents the transient clinical problem strictly under thermal therapy conditions.\nFigure\u00a06.(a) Plot of hold time in minutes to attain complete destruction of attached HepG2 cells at various PTs between 50\u00b0C and 85\u00b0C and at HRs from 5 to 600\u00b0C\u00a0min\u22121. (b) Plot of total time (min) to attain complete destruction of attached HepG2 cells at various PTs between 50\u00b0C and 85\u00b0C and at HRs from 5 to 600\u00b0C\u00a0min\u22121 .\nFigure\u00a06a shows that, for a given target PT, the hold time increases with the increase in the HRs. It increases from 0.45 to 8.4\u00a0min, from 0 to 2.2\u00a0min, from 0 to 0.61\u00a0min, from 0 to 0.17\u00a0min and from 0 to 0.05\u00a0min at a target PT of 60, 65, 70, 75 and 80\u00b0C, respectively. The increase in the hold is negligible at 85\u00b0C. Further, the hold time decreases with the increase in target PT, for any given HR. At 300\u00b0C\u00a0min\u22121 the hold time for complete cell destruction decreases from 7.2\u00a0min at 60\u00b0C to 0.4\u00a0min at 80\u00b0C. For very low HRs of 5 and 10\u00b0C\u00a0min\u22121, complete cell destruction is achieved for target PTs of 65\u00b0C and above without any hold time. Similarly, no hold time is required when the target PTs are 70\u00b0C and above at 20\u00b0C\u00a0min\u22121 and 75\u00b0C and above at 50\u00b0C\u00a0min\u22121. Heating, targeted at PTs of 80\u00b0C and 85\u00b0C, kills all the cells without any hold period for any of the above used HR. These results demonstrate that significant cell death takes place during the ramp up and ramp down period.\nFigure\u00a06b shows the variation in the total injury time (rise, hold and cooling time) to achieve complete cell destruction with the change in the target PTs and HRs. For 60, 65 and 70\u00b0C, the total injury time showed a minima with the decrease in the HR. For example, the total time at the target PT of 60\u00b0C, decreases from 8.8 for 600\u00b0C\u00a0min\u22121 to 5.9 for 50\u00b0C\u00a0min\u22121 and then again increases to 7.8\u00a0min for 5\u00b0C\u00a0min\u22121. At 65\u00b0C, the total time decreases from 2.7\u00a0min to 2\u00a0min and then increases to 3.2\u00a0min for a decrease of HR from 600 to 100\u00b0C\u00a0min\u22121 and again to 20\u00b0C\u00a0min\u22121, respectively. The trend remains the same at 70\u00b0C, the transition of hold time being at a higher rate of 200\u00b0C\u00a0min\u22121. For target PTs of 75, 80 and 85\u00b0C, this trend reverses as the total injury time consistently increases with the decrease in the HR, unlike for all other target PTs. The total time increased almost twice from 0.76, 0.69 and 0.7 at 75, 80 and 85\u00b0C respectively, as the HR dropped from 600 to 50\u00b0C\u00a0min\u22121. For the target PTs (85\u00b0C to 65\u00b0C) where no hold time is required to attain complete cell death, total injury time was obtained as 1, 1.35, 2.4, 3.96 and 6.93\u00a0min at HRs of 100, 50, 20, 10 and 5\u00b0C\u00a0min\u22121, respectively. The trend in the change of the total injury time with change in target PT, for any given HR remains the same as that for the hold time.\nDiscussion\nInjury Kinetics of Attached and Suspended Cells\nThe comparison of the survival data for the suspended and the attached cells (Fig.\u00a03a, b) indicate that the suspended cells are a slightly more susceptible to heat than the attached ones. He and Bischof15 also obtained similar results for SN12 human renal cell carcinoma within the same temperature range, where the reason for this difference between suspended and attached cells was assumed to be the difference in their protein synthesis and gene expression. It may be assumed that some of the above differences also exist between attached and suspended HepG2 cells used in this study. The results shows a clear difference between the frequency factors for both the cell types, but the activation energies does not show much variation. But since the difference in the survival of suspended and attached cells is small either of the cell type can be used in studying the cell injury kinetics in HepG2 cells.\nComparison with Previous Hyperthermic Studies\nHyperthermic studies are mainly characterized by the presence of a shoulder region followed a slope in the survival curve. In these studies, the shoulder region is typically associated with considerably higher (\u223c30\u201340%) cell survival within a long heating period of few hours and has been observed in the temperature range of 42\u201345\u00b0C in many studies. Chinese hamster ovary (CHO) cells showed a small (15%) drop in the survival followed by reduction in the slope of the survival curve when heated at 42\u00b0C for 3\u00a0h.9 Human melanoma HTB-66 cells, when heated between 42\u00b0C and 45\u00b0C, also showed a considerably high survival (\u223c30%) followed by reduction in the slope at 42.5\u00b0C even after 5\u00a0h of heating.23 In this study, heating at 45\u00b0C for 15\u00a0min (data not included) and at 50\u00b0C for 9\u00a0min also shows a significantly higher (>42%) survival for attached and suspended HepG2 cells indicating the possibility of a shoulder region for these short time protocols. However, whether they biologically behave in a similar fashion to the hyperthermic shoulders by demonstrating thermotolerance has not been studied and further investigation is clearly warranted to verify these facts.\nAnother important aspect of the single cell heating studies is the presence of a break point, after which the slope of the Arrhenius plot drops significantly with the increasing temperature. The break point is believed to be the indication of an achieved thermotolerance and change in the mechanism\/target of the thermally induced cellular injury beyond it.9 Such break points have been observed at 43.5\u00b0C for human cell lines and 43\u00b0C for rodent cell lines. We have observed no significant break point for our studies between temperatures of 55\u201370\u00b0C. However, one interesting observation in our study was that heating at 60\u00b0C for 3\u00a0min for attached and suspended cells, shows a sudden change in the slope of the survival curve. The survival drops from \u223c0.25 at 2\u00a0min to 0.003 after 3\u00a0min of heating, suggesting the possibility of a distinct change in the response to heating at 60\u00b0C for HepG2 cells.\nComparison of Arrhenius Parameters\nThis study shows a linear relationship between activation energies and HRs for a temperature range of 50\u201370\u00b0C. The activation energies between 282.09\u00a0kJ\u00a0mol\u22121 and 248.29\u00a0kJ\u00a0mol\u22121 were obtained for HRs of 5\u2013600\u00b0C\u00a0min\u22121, which are within the range of the activation energies obtained in most of the dye uptake assays. Thermal injury in Dunning AT-1 attached prostate cancer cells from 40\u00b0C to 60\u00b0C gave activation energy of 245\u00a0kJ\u00a0mol\u22121.4 Activation energy values obtained in renal cell carcinoma studies are 290\u2013360\u00a0kJ\u00a0mol\u22121.15 and in skeletal muscle tissue is 230\u00a0kJ\u00a0mol\u22121.11 The activation energies for clonogenic assays for various studies like AT-1 prostate cancer cells (40\u201370\u00b0C),4 T24 human bladder carcinoma cells (48\u201365\u00b0C) and CHO (48\u201365\u00b0C)22 were found to be 500, 600 and 680\u00a0kJ\u00a0mol\u22121, respectively. These values are higher than the ones obtained in this study which is not surprising since clonogenic assay yields higher activation energies than dye uptake assays. Since the activation energies obtained in this study are in the same range of the values obtained for other dye uptake assays, we suggest that the mechanism of thermal injury and its measurement is consistent with other studies.\nClinical Relevance\nClinical thermal therapy is a transient conduction problem as the tumor gets heated by deposition of energy from the source and by diffusion of the heat from the applicator. Based on the size and heat absorption pattern of the tumor, different radial locations are heated to different PTs at different rates. The regions closer to the applicator gets heated to higher PTs at higher HRs than the ones that are further away resulting in different survival patterns at various locations inside the tumor. Thus, accurate prediction of the hold time of the applicator inside the tumor is very important to ensure desired cellular injury accumulation at every location. As in these high temperature\u2013short time thermal therapy protocols a considerable (>10%) injury accumulates within the non-isothermal portion of the thermal history, the determination of the total treatment time (rise, hold and cooling) is also necessary to obtain desired injury accumulation. Since the maximum size of a liver tumor that can be treated using various energy sources is limited to 4\u20136\u00a0cm diameter,8 the HRs between 600\u00b0C\u00a0min\u22121 and 5\u00b0C\u00a0min\u22121 are representative of all the locations within the tumor for any selected energy source, while the temperatures between 85\u00b0C and 50\u00b0C are descriptive of the actual temperature distribution inside the tumor during the thermal application. Temperatures higher than 85\u00b0C might result in carbonization near the applicator as well as overheating of the healthy tissue beyond the edge of the tumor, while temperatures lower than 50\u00b0C may result in insufficient injury accumulation within the short treatment time. Therefore, design of clinical thermal therapy protocols requires knowledge of the survival patterns for different hold times and precise prediction of hold and total treatment times to obtain desired cellular injury within the above mentioned range of HRs and PTs.\nSuch an attempt has been made in a previous study carried out by He and Bischof15 on SN12 renal cell carcinoma. The cell injury kinetics, based on the non-isothermal heating at an average rate of 100\u00b0C\u00a0min\u22121, were used to predict the PTs necessary to obtain a considerable (>10%) amount of cellular injury in cells during the rise period. This prediction was extended to various HR\u2013activation energy combinations just by selecting HRs between 2\u00b0C\u00a0min\u22121 and 200\u00b0C\u00a0min\u22121 and activation energies up to 1000\u00a0kJ\u00a0mol\u22121. However, experimental calculations to determine E as a function of HRs was not included in above study. The unique feature of this study is that for all PTs, prediction of survival and treatment time at any given HR is based on a specific experimentally calculated E\u2013A combination for that HR, which results in a close approximation of the actual cellular injury for these high temperatures-short time thermal treatments.\nAn important assumption made in this study is that the HR used to attain a target PT remains constant. The temperature rise on the heating stage, based on the constants kp and kd in the feedback loop, was found to be exponential. The HR was approximated by taking a tangent to the temperature\u2013time curve, resulting in a constant value for the entire temperature rise. However, inside a tumor, the bioheat equation is required to model the transient temperature rise which is rarely linear. But as an initial step in understanding the effect of important parameters governing thermal therapy, this assumption may be a good approximation to predict desired cellular injury and Figs.\u00a05 and 6 may provide important insights in this direction.\nThe predicted survival at different hold times in Fig.\u00a05a\u2013d shows that for any HR, the PT required to attain a considerable amount of injury accumulation (>90%) decreases as the hold time increases. A smaller flat portion representing complete cell death and the steeper vertical portion representing considerable (>10%) survival for zero hold time in Fig.\u00a05a intersects at 70\u00b0C and complete cell death is observed for HRs of 50\u00b0C\u00a0min\u22121 and lower. This suggests that for HRs of 50\u00b0C\u00a0min\u22121 and lower, typically at the boundary of the tumor, PTs of only 70\u00b0C and above are capable of accumulating significant amount of injury. This information is helpful in developing treatment protocols for small sized tumors, where the use of high target PTs will not significantly damage the healthy tissue beyond the range of the tumor, even though the temperatures at those locations are higher than 60\u00b0C. As the hold time increases the intersection shifts toward lower PTs increasing the flat portion of complete cell destruction. The decrease in steepness of the vertical portion also implies that sufficient cell damage can be obtained at tumor boundaries at these low temperatures. For example, when the hold time is 5\u00a0min, more than 80% survival is observed even at 55\u00b0C at the boundary of the tumor where the HR is as low as 5\u00b0C\u00a0min\u22121. This shows that thermal protocols with higher hold times are beneficial for cell injury in large size tumors. Since it is necessary that the intersection should coincide with the tumor boundary to maximize the damage within the tumor as well as minimize the damage of the surrounding healthy tissue, the contours obtained in Fig.\u00a05a\u2013d are very important in the selection of the hold times based on the size of the tumor.\nSince the goal of every clinical application is complete tumor destruction, it is very important to know the hold time required to destroy all the tumor cells for different PT\u2013HR combinations (that may represent different regions within a tumor depending upon its geometry and the energy source used). Figure\u00a06a shows the hold time necessary to acquire complete damage in attached HepG2 cells at PTs between 60\u00b0C and 85\u00b0C and HRs between 5\u00b0C\u00a0min\u22121 and 600\u00b0C\u00a0min\u22121 and suggests that the hold time increases with the simultaneous decrease in the PT and the HR. Therefore, as the distance from the applicator increases, decreasing the PT and the HR, the hold time for tumor destruction increases. For example, Fig.\u00a06a shows that the hold time increases from almost zero at 85\u00b0C \u2013 500\u00b0C\u00a0min\u22121 to 4.84\u00a0min at 60\u00b0C \u2013 50\u00b0C\u00a0min\u22121. Significant increase in the hold time for PTs of 60\u00b0C and 65\u00b0C suggests that in the regions away from the applicator (HRs of 100\u00b0C\u00a0min\u22121 and lower), the injury accumulation strongly depends on the hold time.\nHowever, the hold time is very less for PTs of 70\u00b0C and above even at high HRs and decreases to almost zero as the HR decreases, suggesting that the majority of the cell damage at these temperatures occurs just within the rise and cooling period. At 70\u00b0C and above for HRs of 20\u00b0C\u00a0min\u22121 and lower, the hold time completely vanishes and the thermally induced cellular injury is just a function of the non-isothermal portion of the thermal history. Therefore it is necessary to investigate the variation in the total treatment time (rise, hold and cooling) to attain complete cellular destruction at various PT\u2013HR combinations.\nFigure\u00a06b shows the variation in the total treatment time based on the temperature distribution within the tumor, depending upon its geometry and the energy source used. For PTs of 75\u00b0C and above, unlike hold time, the total treatment time increases with the decrease in the HR. This is due to the fact that for HRs above 50\u00b0C\u00a0min\u22121, the hold time is very small and negligible compared to the rise time to reach these high PTs and the time to cool down back to the room temperature. It was also observed that for a given target PT (>70\u00b0C)\u2013HR (<50\u00b0C\u00a0min\u22121) combination, where the hold time is zero, the total treatment time is lower than the calculated time. This suggests that if damage within a tumor is desired at very slower rate at PTs higher than 70\u00b0C, the tumor actually gets destroyed before even reaching the target PT at any location. But the interesting observation in Fig.\u00a06b is the presence of a minimal total treatment time at PTs of 70, 65 and 60\u00b0C for HRs of 200, 100 and 50\u00b0C\u00a0min\u22121, respectively. This implies that based on the size of the tumor and the energy source selected, if the temperature distribution within the tumor results in any of the above PT\u2013HR combinations at the boundary, complete cell death can be achieved in the entire tumor within a minimal total treatment time. For example, if the temperature distribution is such that the boundary of the tumor reaches 60\u00b0C at 50\u00b0C\u00a0min\u22121, the entire tumor can be destroyed in 5.9\u00a0min. Thus based on the size of the tumor, Fig.\u00a06a, b may help in selecting hold and total treatment time in order to design an optimal clinical protocol.\nSince this study is based on experimental single cell data to obtain the activation energies at different HR, it can serve as an accurate model for designing better thermo-surgical protocols using appropriate energy sources of heat delivery. Also, for a clinician practicing the technique, it can serve as a database, from where an optimal thermal therapy protocol can be selected for the treatment of liver cancer. By selecting appropriate blood flow and tissue properties and by using the bioheat equation, this in\u00a0vitro protocol can be extended to in\u00a0vivo clinical protocols.\nSummary\nThe investigation of injury kinetics in human liver cancer HepG2 cells between temperatures of 50\u00b0C and 70\u00b0C at HRs of 100, 200, 300 and 525\u00b0C\u00a0min\u22121 (isothermal) was used to extract Arrhenius parameters, E and A, that showed a minor decay with the increase in the HR. The extrapolation of the kinetic parameters over a range of heating conditions showed that the E value decreased from 282.09 to 248.29\u00a0kJ\u00a0mol\u22121 as the HR increases from 5 to 600\u00b0C\u00a0min\u22121. The measured and the predicted survival at all the HRs dropped as the PT and hold time increased. A sharp drop in the survival was observed after 2\u00a0min of heating at 60\u00b0C for all HRs. The measured and predicted cell survival increased with the increase in the HR at any PTs and hold time. This increase in the survival reduced significantly for target PTs of 65\u00b0C and above and for hold times of a minute and higher. The hold time for complete cell damage decreases with the increase in the target PT and increases with the increase in the HR. No hold time is required for target PTs of 75\u00b0C and above with the decrease in the HR below 100\u00b0C\u00a0min\u22121. The cellular damage is a dominant function of hold time at target PTs of 60\u00b0C and less and for HRs of 50\u00b0C\u00a0min\u22121 and higher. Total time (rise, hold and cooling time) plays a major role in accumulating the cellular injury when the temperatures are 70\u00b0C and above and when the HRs are between 0\u00b0C\u00a0min\u22121 and 50\u00b0C\u00a0min\u22121. Minimal total treatment time for complete cellular damage was observed at target PTs of 60, 65 and 70\u00b0C and HRs of 50, 50 and 20\u00b0C\u00a0min\u22121, respectively.","keyphrases":["heating rates","peak temperatures","total treatment time","injury kinetics","hold time","clinical protocols","arrhenius model","minimal treatment time"],"prmu":["P","P","P","P","P","P","P","R"]}
{"id":"Virchows_Arch-3-1-1888719","title":"TNM staging of foregut (neuro)endocrine tumors: a consensus proposal including a grading system\n","text":"The need for standards in the management of patients with endocrine tumors of the digestive system prompted the European Neuroendocrine Tumor Society (ENETS) to organize a first Consensus Conference, which was held in Frascati (Rome) and was based on the recently published ENETS guidelines on the diagnosis and treatment of digestive neuroendocrine tumors (NET). Here, we report the tumor\u2013node\u2013metastasis proposal for foregut NETs of the stomach, duodenum, and pancreas that was designed, discussed, and consensually approved at this conference. In addition, we report the proposal for a working formulation for the grading of digestive NETs based on mitotic count and Ki-67 index. This proposal, which needs to be validated, is meant to help clinicians in the stratification, treatment, and follow-up of patients.\nBackground\nIt has been known for a long time, and was finally defined within the World Health Organization (WHO) classification of endocrine and digestive tumors, that neuroendocrine tumors (NET) arising at different anatomical sites of the digestive system represent tumor entities that differ in their biology [5, 7, 9, 31]. Several recent publications focused on the application of the \u201cnew\u201d WHO classification and proved its effectiveness, supporting the concept that the different endocrine tumor types also differ in their clinical behavior [3, 4, 19, 20, 27, 32].\nMalignant gastroenteropancreatic NETs may be fatal, though at a significantly slower pace than their exocrine counterparts. A number of retrospective papers and epidemiological data solidly support such statements [8, 9, 12\u201314, 21, 24, 25, 32]. This peculiar clinical feature attracted the interest of pathologists very early and was the reason for the special designation of such tumors as \u201ccarcinoid\u201d by Oberndorfer [15].\nAs gastroenteropancreatic NETs are rare [9, 13, 14], it is tempting to lump them together and equate all digestive \u201ccarcinoids\u201d with the appendiceal \u201ccarcinoid,\u201d probably the best known NET with the most benign behavior [28]. However, in recent years it has become clear that gastroenteropancreatic NETs, especially foregut NETs, are heterogeneous in their morphological and biological features. In the last two decades efforts were therefore made by the WHO to define NET features that discriminate true benign behavior (low risk) from low-grade malignant well-differentiated NETs in the different parts of the digestive system. Although the new WHO classification is an important step toward defining the diverse tumor biology of NETs, further efforts are necessary to improve the prognostic assessment of the individual NET.\nThe demand for standards in the stratification and treatment of patients with gastroenteropancreatic NETs prompted the recently established European Neuroendocrine Tumor Society (ENETS) to define guidelines [22, 33]. Such guidelines underwent scrutiny for consensus in the first of two meetings entitled \u201cConsensus Conference on the ENETS Guidelines for the Diagnosis and Treatment of Neuroendocrine Gastrointestinal Tumors, Part 1: Foregut Tumors\u201d held in Frascati (Rome, Italy) from November 2\u20135, 2005. During this meeting the clinical need for a tumor\u2013node\u2013metastasis (TNM) classification of gastroenteropancreatic NETs was felt. Here we report the TNM staging classification proposal for foregut NETs that was approved at this consensus conference. In addition, we suggest a simple grading system with some pointers that may help to standardize the prognostic assessment of gastroenteropancreatic NETs.\nMaterials and methods\nSixty-two experts in the field of digestive endocrine tumors from 20 different countries attended the Consensus Conference. The attendees represented all medical branches involved in managing patients with gastroenteropancreatic NETs. They formed four working groups according to their specific clinical expertise: (1) pathology and genetics (11 participants, all listed as authors and G. Kl\u00f6ppel), (2) surgery (10 participants, including the coauthors H. Alhman and M. Falconi), (3) imaging and radiology (10 participants), (4) medicine and clinical pathology (31 participants, including the coauthors M. Caplin, W.W. de Herder, B. Erikssson, and B. Wiedenmann).\nThe Conference was divided sequentially into eight sessions devoted to specific topics on an anatomical basis (gastric NET sessions 1\u20132, duodenal NET, pancreatic NET sessions 1\u20134, and poorly differentiated endocrine carcinomas). A working booklet with the ENETS guidelines and specific queries had been prepared in advance by the Organizing Committee. The work was organized such that, after a short case presentation in a plenary session, each working group gathered separately to discuss group-specific questions. Once agreement was reached within each group, consensus statements were discussed and approved or rejected by all participants gathered in the plenary session. This procedure was followed for all eight sessions. The TNM staging proposal was made by the Pathology and Genetics working group and amended and approved by the plenary session of the consensus conference. The grading system was discussed and defined by the Pathology and Genetics working group only.\nResults and discussion\nThe consensus guidelines are reported elsewhere. Here, we report the TNM staging proposal for gastroenteropancreatic NETs of the foregut together with a grading system that may be relevant for the prognostic assessment by the pathologist. The foregut NETs were separated into gastric, duodenal (including ampulla and proximal jejunum), and pancreatic NETs, but were not distinguished according to specific functional activity, main tumor cell type, and specific genetic background.\nTNM staging proposal (see Tables\u00a01, 2 and 3)\nThe currently published TNM format was adopted as working template [29].\nTumor The proposed definition of tumor in situ applies to the stomach only and adheres to the literature [30]. No definition is given for the duodenum and pancreas because none has been agreed upon in spite of recent working proposals [1, 2]. The size limits indicated for T1 are those defined by the WHO for tumors with \u201cbenign behavior\u201d according to site-specific clinicopathological correlations [5, 7, 31]. Similarly, for T2 of the stomach and duodenum, the sizes are those indicated for tumors of \u201cuncertain behavior.\u201d In the pancreas the size limit given for T2 needs to be validated [5]. Deeply invasive tumors are included under the T3 and T4 definitions, taking into account site-specific features.\nNodes N1 indicates the presence of any single or multiple metastases in regional lymph nodes, according to TNM rules. Although the presence of regional lymph-node metastases is, per se, a negative prognostic factor in gastroenteropancreatic NETs [11], the prognostic significance of the number of metastatic nodes is not known. In light of this, stage 3B of Tables\u00a01, 2 and 3 is proposed to mark the N1 status for future validation. \nTable\u00a01Proposal for a TNM classification and disease staging for gastric endocrine tumorsTNMT\u2014primary tumor\u00a0TXPrimary tumor cannot be assessed\u00a0T0No evidence of primary tumor\u00a0TisIn situ tumor\/dysplasia (<0.5\u00a0mm)\u00a0T1Tumor invades lamina propria or submucosa and \u22641\u00a0cm\u00a0T2Tumor invades muscularis propria or subserosa or >1\u00a0cm\u00a0T3Tumor penetrates serosa\u00a0T4Tumor invades adjacent structuresFor any T, add (m) for multiple tumorsN\u2014regional lymph nodes\u00a0NXRegional lymph nodes cannot be assessed\u00a0N0No regional lymph node metastasis\u00a0N1Regional lymph node metastasisM\u2014distant metastasis\u00a0MXDistant metastasis cannot be assessed\u00a0M0No distant metastases\u00a0M1aDistant metastasisStage\u00a0Disease stages\u00a0\u00a0Stage 0TisN0M0\u00a0\u00a0Stage IT1N0M0\u00a0\u00a0Stage IIaT2N0M0\u00a0\u00a0\u00a0IIbT3N0M0\u00a0\u00a0Stage IIIaT4N0M0\u00a0\u00a0\u00a0IIIbAny TN1M0\u00a0\u00a0Stage IVAny TAny NM1aM1 specific sites defined according to Sobin and Wittekind [29]Table\u00a02Proposal for a TNM classification and disease staging for endocrine tumors of the duodenum\/ampulla\/proximal jejunumTNMT\u2014primary tumor\u00a0TXPrimary tumor cannot be assessed\u00a0T0No evidence of primary tumor\u00a0T1Tumor invades lamina propria or submucosa and size \u22641\u00a0cma\u00a0T2Tumor invades muscularis propria or size >1\u00a0cm\u00a0T3Tumor invades pancreas or retroperitoneum\u00a0T4Tumor invades peritoneum or other organsFor any T, add (m) for multiple tumorsN\u2014regional lymph nodes\u00a0NXRegional lymph nodes cannot be assessed\u00a0N0No regional lymph node metastasis\u00a0N1Regional lymph node metastasisM\u2014distant metastases\u00a0MXDistant metastasis cannot be assessed\u00a0M0No distant metastases\u00a0M1bDistant metastasisStage\u00a0Disease stages\u00a0\u00a0Stage IT1N0M0\u00a0\u00a0Stage IIaT2N0M0\u00a0\u00a0\u00a0\u00a0IIbT3N0M0\u00a0\u00a0Stage IIIaT4N0M0\u00a0\u00a0\u00a0\u00a0IIIbAny TN1M0\u00a0\u00a0Stage IVAny TAny NM1aTumor limited to ampulla of Vater for ampullary gangliocytic paragangliomabM1 specific sites defined according to Sobin and Wittekind [29]Table\u00a03Proposal for a TNM classification and disease staging for endocrine tumors of the pancreasTNMT\u2014primary tumor\u00a0TXPrimary tumor cannot be assessed\u00a0T0No evidence of primary tumor\u00a0T1Tumor limited to the pancreas and size <2\u00a0cm\u00a0T2Tumor limited to the pancreas and size 2\u20134\u00a0cm\u00a0T3Tumor limited to the pancreas and size >4\u00a0cm or invading duodenum or bile duct\u00a0T4Tumor invading adjacent organs (stomach, spleen, colon, adrenal gland) or the wall of large vessels (celiac axis or superior mesenteric artery)For any T, add (m) for multiple tumorsN\u2014regional lymph nodes\u00a0NXRegional lymph node cannot be assessed\u00a0N0No regional lymph node metastasis\u00a0N1Regional lymph node metastasisM\u2014distant metastases\u00a0MXDistant metastasis cannot be assessed\u00a0M0No distant metastases\u00a0M1aDistant metastasisStage\u00a0Disease stages\u00a0\u00a0Stage IT1N0M0\u00a0\u00a0Stage IIaT2N0M0\u00a0\u00a0\u00a0IIbT3N0M0\u00a0\u00a0Stage IIIaT4N0M0\u00a0\u00a0\u00a0IIIbAny TN1M0\u00a0\u00a0Stage IVAny TAny NM1aM1 specific sites defined according to Sobin and Wittekind [29]\nDistant metastasis M1 indicates the presence of any single or multiple metastases at any distant anatomical site (including nonregional nodes). Because there is evidence that extrahepatic bone metastases are a particularly ominous sign [6, 20], it is recommended to specify the anatomical site of the metastasis according to the TNM classification rules (PUL, pulmonary; HEP, hepatic; OSS, osseous; etc.) [29].\nStaging The proposed staging system lists stage 0 only for the stomach because this is the only anatomical site where Tis is defined. Stage I encompasses the T1 NETs with limited growth. Stage II identifies tumors that are larger in size or more invasive, either T2 or T3, though always in the absence of metastasis. At stage III the increased malignancy refers either to invasion into surrounding structures (stage IIIa) or to the presence of regional node metastasis (stage IIIb). Stage IV always implies the presence of distant metastasis.\nGrading proposal (see Table\u00a04)\nGrading It has been widely discussed and generally accepted that no histological grading system effectively predicts the behavior of well-differentiated endocrine tumors. The major obstacle to developing a practically effective grading system is the fact that severe cytological atypia, as, for instance, in pheochromocytomas, has no impact on the clinical behavior and malignancy of such tumors. However, recent studies in well differentiated NETs of the foregut, including the pancreas, and of the midgut have shown the usefulness of a grading system [10, 25, 32]. Thus, well-differentiated endocrine tumors with a more solid appearance and distinct proliferative activity, which also lead to difficulties in the differential diagnosis vs poorly differentiated endocrine carcinomas, seem to have a worse prognosis than NETs without these features [16\u201318, 22, 23]. It was therefore decided to introduce a grading system that could be of help in distinguishing the well-differentiated NETs into G1 and G2 categories.\nAs a working suggestion, we propose to apply to foregut NETs a grading system modified from that adopted by the WHO for endocrine tumors of the lung, though exclusively referring to the proliferation status. In brief (see Table\u00a04), three tumor categories are identified: G1, <2 mitosis per 2\u00a0mm2 [10 high power fields (HPF) 40\u00d7 magnification] and\/or Ki-67 index \u22642%; G2, 2\u201320 mitosis per 2\u00a0mm2 and\/or Ki-67 index between 3 and 20%; G3 with 21 or more mitosis per 2\u00a0mm2 and Ki-67 index >20%. \nTable\u00a04Grading proposal for foregut (neuro)endocrine tumorsGradeMitotic count (10 HPF)aKi-67 index (%)bG1<2\u22642G22\u2013203\u201320G3>20>20a10 HPF: high power field=2\u00a0mm2, at least 40 fields (at 40\u00d7 magnification) evaluated in areas of highest mitotic densitybMIB1 antibody; % of 2,000 tumor cells in areas of highest nuclear labeling\nIn general, G1 and G2 should refer to well-differentiated NETs displaying diffuse and intense expression of the two general immunohistochemical neuroendocrine markers, chromogranin A and synaptophysin [26]. Punctate necrosis is, per se, indicative of a more aggressive tumor, pointing to a G2 status, which, however, has to be confirmed by the mitotic count. G3 indicates a poorly differentiated neuroendocrine carcinoma. It has high mitotic counts\/Ki-67 index, is often associated with fields of necrosis, and shows significantly reduced chromogranin A expression, while maintaining intense staining for synaptophysin. It is relevant to remind here that the diagnosis of G3 carcinoma is based on a specific histologic pattern according to the current WHO criteria [5, 7, 31]. In addition, the clinical behavior of G3 poorly differentiated neuroendocrine carcinomas of the gastroenteropancreatic tract does not necessarily correspond to that of small cell cancers of the lung or of any other sites.\nMitotic count and Ki-67 index We propose that mitoses should be counted on hematoxylin and eosin-stained slides in at least 40 HPFs, where possible. The mitoses should be assessed in areas where they are most frequent after a general slide survey. For Ki-67 assessment, the MIB1 antibody is recommended at the conditions that have been established at the laboratory in question. The Ki-67 index should be assessed in 2,000 tumor cells in areas where the highest nuclear labeling is observed (often but not exclusively at the tumor periphery).\nConcluding remarks\nRequests for standardization in the management of patients with gastroenteropancreatic NETs recently resulted in the development of several guidelines, including those proposed by ENETS [16, 17, 22, 23]. However, it was never attempted to reach consensus on specific practical issues. The TNM staging system we propose here was developed to meet a clinical need, is based on the current WHO classifications of endocrine and digestive tumors, and is the result of a consensus conference held by specialists involved in the management of digestive endocrine tumor patients. Along the same line, the grading system described here attempts to close the gap between the advances of the most recent WHO classifications and the need for a better prognostic assessment of NETs. It is obvious, of course, that all our proposals have to be validated by future clinicopathological work.","keyphrases":["tnm","staging","grading","neuroendocrine tumors","pancreas","ki-67 index","gut","mitotic index"],"prmu":["P","P","P","P","P","P","U","R"]}
{"id":"Diabetologia-3-1-2039867","title":"Anthropometry, carbohydrate and lipid metabolism in the East Flanders Prospective Twin Survey: heritabilities\n","text":"Aims\/hypothesis We determined the genetic contribution of 18 anthropometric and metabolic risk factors of type 2 diabetes using a young healthy twin population.\nIntroduction\nType 2 diabetes is a heterogeneous disease that involves both genetic and environmental factors. Its incidence is rising rapidly worldwide and consequently much research is focused on the genetic components of the disorder, in order to get a better understanding of the pathogenesis and eventually to achieve better, more personalised diagnostics, treatment and prevention [1].\nThe starting point in the search for genes is to estimate the degree of heritability of intermediate traits leading to the disease in the studied population. Heritability is the proportion of phenotypic variation of a trait that can be attributed to genetic variation [2]. The degree of heritability is an important determinant of the power to detect and localise disease-related genes [3]. Although heritabilities can in principle be estimated from all kinds of related individuals, twin studies allow the variation to be split up into genetic, shared environmental and unique environmental components, thus offering one of the most valid estimations [2].\nA variety of studies estimating heritabilities of risk factors of type 2 diabetes in adult twins have been carried out. A summary of the larger studies (>200 twin pairs) on the risk factors obesity, glucose intolerance\/insulin resistance and dyslipidaemia is provided in Electronic Supplementary Material (ESM) Table\u00a01. The heritability of BMI, the most frequently used measure of obesity, has been estimated extensively and ranges between 40 and 90% [4\u201321]. For all the other traits, fewer studies have been performed; the total variation of fasting glucose and fasting insulin is explained by genetic factors for 12 to 50% [22, 23] and 14 to 54% [7, 18, 22\u201324], respectively; for total cholesterol, LDL-cholesterol, HDL-cholesterol and triacylglycerol heritability estimates range between 0 and 98% [7, 10, 13, 17, 21, 25\u201334]. In addition to sample numbers and statistical methodologies used, the large variation in heritability estimates may also be caused by the genetic background of the studied populations and the environmental exposures experienced (see ESM Table\u00a01). For the majority of intermediate traits related to type 2 diabetes, the number of larger studies performed is quite small and studies in which heritabilities of several risk factors of type 2 diabetes were estimated in the same population are even scarcer (ESM Table\u00a01).\nTo determine the genetic contribution to type 2 diabetes, we used variance components modelling to estimate the heritabilities of 18 anthropometric and metabolic risk factors of the disease, including parameters quantifying obesity, glucose intolerance\/insulin resistance and dyslipidaemia. In addition, we also determined the heritabilities of the hormones insulin-like growth factor binding protein-1 (IGFBP-1), which has been shown to be related to several cardiovascular risk factors, and leptin, which plays a role as satiety signal regulating body composition and energy expenditure [35]. The characteristics were measured in 756 healthy twins, divided into 240 monozygotic (MZ) and 138 dizygotic (DZ) twin pairs in the age range of 18 to 34\u00a0years, recruited from the East Flanders Prospective Twin Survey (EFPTS).\nMethods\nParticipants The EFPTS is a population-based twin register that started in 1964 and has recorded all multiple births in the Belgian Province of East Flanders until the present day. A detailed description of the EFPTS has been published [36]. Zygosity was determined using sequential analysis based on sex, fetal membranes, umbilical cord blood groups, placental alkaline phosphatase and DNA marker analysis. Between July 1964 and May 1982, the Twin Survey had registered 2,141 twin pairs who met the World Health Organization criteria for live born infants (birthweight \u2009\u2265\u2009500\u00a0g or gestational age \u2009\u2265\u200922\u00a0weeks if birthweight unknown). Pairs of whom one or both members were stillborn, died in neonatal or later life or suffered from major congenital malformations were excluded. We randomly contacted 803 pairs using an envelope system. To assure equally distributed groups, we stratified for birth year and zygosity. Since the twin survey was originally set up to determine chorionicity, the number of MZ twin pairs was over sampled. Eventually, 424 twin pairs (52.7%) volunteered to participate in the study. Sex, gestational age and birthweight did not differ between participants and non-participants (i.e. those who where eligible, but refused to participate or had not been contacted) (p\u2009>\u20090.05), although participants were slightly older than the non-participants and the proportion of monochorionic (MC) twins was higher in the group of participants (p\u2009<\u20090.05). A detailed description of the methods used to measure the anthropometric and metabolic characteristics of the twins is provided in the Electronic Supplementary Material. For the present analysis participants suffering from type 1 diabetes were excluded and only twin pairs of whom both members participated were incorporated. Consequently, phenotypic data were available for 378 complete pairs, consisting of 113 monozygotic men (MZM), 127 monozygotic women (MZF), 46\u00a0DZ men (DZM), 49\u00a0DZ women (DZF) and 43\u00a0DZ opposite sex (DZOS) pairs. Additionally, for the analysis of lipid and carbohydrate parameters, participants taking drugs with potential effects on lipid or carbohydrate metabolism were excluded (n\u2009=\u200911). Twins were randomly assigned to be the first or the second member of a pair. The Ethics Committee of the Faculty of Medicine of the Katholieke Universiteit Leuven approved the project and all participants gave informed consent.\nDescriptive statistical analysis Anthropometric and metabolic characteristics are expressed as mean\u2009\u00b1\u2009SD according to chorion type, for men and women separately. BMI, sum of four skinfold thicknesses (S4SF), fat mass, IGFBP-1, fasting insulin, insulin resistance, beta cell function, leptin, total cholesterol:HDL-cholesterol ratio and triacylglycerol values had a skewed distribution. After transforming these data into natural logarithms a normal distribution was obtained and the transformed data were used when performing statistical tests. Differences in means between MZ MC, MZ dichorionic (DC) and DZ twins were calculated using the PROC MIXED method implemented in SAS version 9.1 (SAS Institute, Cary, NC, USA). A random intercept model was used, where the intercept of each twin pair was modelled as a function of the population intercept plus a unique contribution of the twin pair. In addition, we allowed the variance\u2013covariance structure of the random intercept to differ between MZ MC, MZ DC and DZ pairs. Differences in means were considered significant if the 2 dfF test indicated p\u2009<\u20090.05.\nFor all traits, effects of potential covariates were also checked using the random intercept model of PROC MIXED, where the variance\u2013covariance structure of the random intercept was allowed to differ between MZ and DZ pairs. The variables BMI, WHR and S4SF were checked for the effect of potential confounding by sex and age; body mass, fat mass and lean body mass were checked for the effect of sex, age and height; the blood parameters were checked for the effect of sex, age and fat (BMI, WHR or S4SF). Covariates were considered significant if p\u2009<\u20090.05. Intra-pair correlation coefficients were calculated for MZ MC and MZ DC twin pairs and for each of the five sex by zygosity groups (MZM, MZF, DZM, DZF and DZOS) before and after adjustment for significant covariates using the Mx software package [37]. In addition, we used a linear regression analysis to test whether twin correlations differed between MZ MC and MZ DC twin pairs before and after adjusting for covariates.\nTwin model fitting Twin methodology makes use of the fact that MZ twins are genetically identical, whereas DZ twins share 50% of their segregating genes. Assuming that MZ and DZ twins share their common environment to the same extent, a higher concordance rate in MZ twins than in DZ twins reflects genetic influences. To estimate the genetic and environmental components of variance of the traits, twin model fitting of raw data was implemented using the statistical package Mx [37]. Scripts were downloaded from the GenomEUtwin Mx-script library (http:\/\/www.psy.vu.nl\/mxbib\/). Univariate twin analyses were performed, where the phenotypic variance can be decomposed into additive genetic (A, additive effects of genes on multiple loci), non-additive genetic (D, interactions between alleles at the same locus [dominance] or on different loci [epistasis]), common environmental (C, environmental effects shared by twins reared in the same family) and unique environmental effects (E, environmental effects unique to the individual). MZ twins are assumed to share the same A and D genetic variance; DZ pairs are assumed to share one-half of the A variance and one-quarter of the D variance. The C variance is assumed to be the same for both MZ and DZ twin pairs. The broad sense heritability (H2), which estimates the extent to which variation of a trait in a population can be explained by genetic variation, is defined as the proportion of genetic variance to total phenotypic variance. As non-additive genetic (D) and shared environmental effects (C) cannot be identified simultaneously in data from twins reared together, ACE and ADE models were fitted separately. The significance of variance components A, C or D in the model was tested by dropping these parameters and comparing the fit of the models [5].\nTo test whether genetic and environmental factors influence a trait to the same degree in men and women, we compared a quantitative heterogeneity model (variance components free to differ across sexes) with a homogeneity model (variance components equal for both sexes). In addition, we verified whether the distribution of a trait differed among men and women by testing a scalar model, which assumes that the female variance components are common multiples of the male variance components. In this model the variance components were constrained to be equal for both sexes, but total variances were allowed to differ between men and women (Fig.\u00a01) [5].\nFig.\u00a01Path diagram for a univariate quantitative heterogeneity (a), scalar (b) and homogeneity (c) model only presenting opposite sex pairs. Observed phenotypes (PM and PW) for the male and female twins are shown in rectangles, while latent factors (A, C and E) and latent phenotypes (LPM and LPW) are shown in circles. Path coefficients of observed variables on the different latent factors are shown in lower case: a, additive genetic effect; c, common environmental effect; e, unique environmental effect; k, scalar factor. The genetic correlation is represented by rg (1 for MZ and 0.5 for DZ twins) and the common environmental correlation is represented by rc (1 for MZ and DZ twins)\nIf for a certain trait the correlation of the opposite-sex pairs is smaller than the correlations for the like-sexed DZ pairs, it is suggested that the correlation between additive genetic factors in opposite-sex pairs is smaller than 0.5. This indicates that different genetic factors influence this trait in men and women. To test this, a full heterogeneity model (variance components free to differ across sexes, plus the correlation between the additive genetic factors in opposite-sex pairs free to be estimated between 0 and 0.5) was compared with a quantitative heterogeneity model.\nAll variance components were estimated both unadjusted and adjusted for the covariates. In the models, phenotypic means were adjusted for significant covariates by modelling them as definition variables in the means model. The difference in fit between the nested models was evaluated with the likelihood ratio \u03c72 test, which uses the difference between the \u22122log likelihood of the full and the restricted model. This difference is distributed as \u03c72. The df of the test were calculated as the difference in df between the models. When the \u03c72 was not statistically significant (p\u2009<\u20090.05), the most parsimonious model was selected, i.e. the model with the best fit given the number of df. When comparing the fit of non-nested models (e.g. ACE with ADE), the model with the lowest Akaike\u2019s information criterion was preferred.\nResults\nDescriptive statistical analysis The mean values of the anthropometric and metabolic characteristics of the twins are presented in Table\u00a01 according to chorion type for men and women separately. In men, fasting glucose levels were lower in DZ twins than in MZ MC and MZ DC twins. In women, MZ DC twins were younger and had lower total cholesterol and LDL-cholesterol levels than MZ MC and DZ twins (Table\u00a01).\nTable\u00a01Anthropometric and metabolic characteristics of the twin population according to chorion type for men and women separately in the EFPTSCharacteristicMenWomenMZ MCMZ DCDZp valueMZ MCMZ DCDZp valuen13492135142112141Age (years)a25.3\u2009\u00b1\u20094.525.0\u2009\u00b1\u20094.925.7\u2009\u00b1\u20094.70.3325.9\u2009\u00b1\u20094.424.0\u2009\u00b1\u20094.825.7\u2009\u00b1\u20094.60.02Body height (cm)178.0\u2009\u00b1\u20095.7 178.3\u2009\u00b1\u20097.2178.4\u2009\u00b1\u20096.50.92165.0\u2009\u00b1\u20096.3165.6\u2009\u00b1\u20096.1166.2\u2009\u00b1\u20096.40.59Body mass (kg)70.7\u2009\u00b1\u200910.569.6\u2009\u00b1\u20098.470.9\u2009\u00b1\u200910.20.6760.5\u2009\u00b1\u20099.760.5\u2009\u00b1\u200910.660.8\u2009\u00b1\u200910.20.98BMI (kg\/m2)22.1\u2009\u00b1\u20091.121.8\u2009\u00b1\u20091.122.1\u2009\u00b1\u20091.20.7322.0\u2009\u00b1\u20091.221.8\u2009\u00b1\u20091.221.8\u2009\u00b1\u20091.20.86WHR (%)83.4\u2009\u00b1\u20095.483.4\u2009\u00b1\u20095.882.8\u2009\u00b1\u20095.50.8473.4\u2009\u00b1\u20094.573.3\u2009\u00b1\u20094.672.5\u2009\u00b1\u20094.30.30S4SF (mm)b35.9\u2009\u00b1\u20091.534.6\u2009\u00b1\u20091.534.8\u2009\u00b1\u20091.50.8957.1\u2009\u00b1\u20091.455.6\u2009\u00b1\u20091.553.6\u2009\u00b1\u20091.40.50Fat mass (kg)b11.9\u2009\u00b1\u20091.611.2\u2009\u00b1\u20091.512.2\u2009\u00b1\u20091.50.3316.9\u2009\u00b1\u20091.316.7\u2009\u00b1\u20091.416.8\u2009\u00b1\u20091.30.95Lean body mass (kg)57.7\u2009\u00b1\u20096.957.6\u2009\u00b1\u20096.057.7\u2009\u00b1\u20096.60.9942.9\u2009\u00b1\u20095.143.1\u2009\u00b1\u20095.643.4\u2009\u00b1\u20096.00.89IGFBP-1 (ng\/ml)b11.0\u2009\u00b1\u20091.910.3\u2009\u00b1\u20091.811.5\u2009\u00b1\u20091.90.4616.8\u2009\u00b1\u20092.0 16.6\u2009\u00b1\u20092.218.4\u2009\u00b1\u20092.30.58Fasting insulin (pmol\/l)b33.5\u2009\u00b1\u20091.5 32.7\u2009\u00b1\u20091.632.7\u2009\u00b1\u20091.60.9438.6\u2009\u00b1\u20091.535.8\u2009\u00b1\u20091.640.1\u2009\u00b1\u20091.50.24Insulin resistance (HOMA)b1.2\u2009\u00b1\u20091.61.2\u2009\u00b1\u20091.61.2\u2009\u00b1\u20091.60.691.3\u2009\u00b1\u20091.51.2\u2009\u00b1\u20091.71.4\u2009\u00b1\u20091.50.26Fasting glucose (mmol\/l)5.0\u2009\u00b1\u20090.54.9\u2009\u00b1\u20090.44.8\u2009\u00b1\u20090.40.014.5\u2009\u00b1\u20090.34.6\u2009\u00b1\u20090.44.6\u2009\u00b1\u20090.40.23Beta cell function (HOMA)b77.2\u2009\u00b1\u20091.578.2\u2009\u00b1\u20091.686.5\u2009\u00b1\u20091.70.10135.2\u2009\u00b1\u20091.7115.7\u2009\u00b1\u20091.6126.7\u2009\u00b1\u20091.60.17Leptin (ng\/ml)b1.7\u2009\u00b1\u20093.01.4\u2009\u00b1\u20092.91.7\u2009\u00b1\u20093.00.4211.5\u2009\u00b1\u20092.111.2\u2009\u00b1\u20092.211.5\u2009\u00b1\u20091.90.92Total cholesterol (mmol\/l)4.8\u2009\u00b1\u20090.94.7\u2009\u00b1\u20091.04.9\u2009\u00b1\u20091.10.315.3\u2009\u00b1\u20090.94.9\u2009\u00b1\u20090.85.3\u2009\u00b1\u20091.00.006LDL-cholesterol (mmol\/l)3.0\u2009\u00b1\u20090.92.9\u2009\u00b1\u20090.93.1\u2009\u00b1\u20091.00.353.1\u2009\u00b1\u20090.82.7\u2009\u00b1\u20090.82.9\u2009\u00b1\u20090.80.012HDL-cholesterol (mmol\/l)1.3\u2009\u00b1\u20090.31.3\u2009\u00b1\u20090.31.4\u2009\u00b1\u20090.40.941.8\u2009\u00b1\u20090.41.8\u2009\u00b1\u20090.41.9\u2009\u00b1\u20090.40.39Total cholesterol:HDL-cholesterol ratiob3.6\u2009\u00b1\u20091.43.5\u2009\u00b1\u20091.33.7\u2009\u00b1\u20091.40.673.0\u2009\u00b1\u20091.32.8\u2009\u00b1\u20091.32.9\u2009\u00b1\u20091.30.34Triacylglycerol (mmol\/l)b0.9\u2009\u00b1\u20091.60.9\u2009\u00b1\u20091.50.9\u2009\u00b1\u20091.50.780.9\u2009\u00b1\u20091.50.9\u2009\u00b1\u20091.50.9\u2009\u00b1\u20091.50.83NEFA (mmol\/l)0.5\u2009\u00b1\u20090.20.5\u2009\u00b1\u20090.20.5\u2009\u00b1\u20090.20.050.7\u2009\u00b1\u20090.20.7\u2009\u00b1\u20090.30.7\u2009\u00b1\u20090.20.81Data are expressed as mean\u2009\u00b1\u2009SDHOMA, homeostasis model assessmentap value calculated using standard linear regression, because convergence criteria could not be met using a random intercept modelbGeometric mean\u2009\u00b1\u2009SDThe covariates adjusted for, the intra-pair correlations of MZ MC and MZ DC pairs, and the intra-pair correlations of each sex by zygosity group before and after adjustment are summarised in Table\u00a02. Intra-pair correlations of the anthropometric and metabolic characteristics did not differ between MZ MC and MZ DC pairs (p\u2009>\u20090.05). Adjusting for covariates strongly reduced the correlations of the leptin concentrations. The correlations of the other traits were only minimally affected by adjustment. Twin correlations for total cholesterol and LDL-cholesterol were high in both MZ and DZ twin pairs, indicating a common environmental effect. The DZ correlations of IGFBP-1, fasting insulin and insulin resistance were less then one-half of the MZ correlations, suggesting that non-additive genetic effects might be important. Correlations of the other traits were in agreement with a model containing additive genetic and unique environmental influences (Table\u00a02).\nTable\u00a02Intra-pair correlations of MZ MC and MZ DC pairs, and of each sex by zygosity group before and after adjusting for covariates in the EFPTSCharacteristicMonozygoticMonozygoticDizygoticCovariatesMCDCMenWomenMenWomenOSn (of pairs)138102113127464943Body mass 0.85\/0.790.84\/0.760.86\/0.820.76\/0.730.38\/0.280.58\/0.570.26\/0.35Sex, age, heightBMI0.80\/0.780.81\/0.770.86\/0.830.77\/0.740.46\/0.310.53\/0.560.47\/0.46AgeWHR0.87\/0.690.88\/0.710.79\/0.740.70\/0.660.39\/0.280.44\/0.480.31\/0.15Sex, ageS4SF0.82\/0.730.84\/0.750.81\/0.790.72\/0.680.46\/0.370.64\/0.630.36\/0.31Sex, ageFat mass0.85\/0.780.85\/0.750.85\/0.820.73\/0.690.46\/0.360.43\/0.460.42\/0.35Sex, ageLean body mass0.93\/0.810.93\/0.790.86\/0.820.79\/0.780.43\/0.390.65\/0.580.25\/0.39Sex, age, heightIGFBP-10.49\/0.390.60\/0.530.55\/0.510.45\/0.430.31\/0.210.08\/0.060.12\/\u22120.05Sex, age, BMIFasting insulin0.57\/0.480.52\/0.500.49\/0.450.58\/0.520.07\/0.130.18\/0.190.07\/\u22120.01Age, S4SFInsulin resistance0.54\/0.470.53\/0.500.49\/0.460.57\/0.510.03\/0.080.14\/0.170.04\/\u22120.05Sex, age, S4SFFasting glucose0.74\/0.660.73\/0.670.65\/0.650.70\/0.690.28\/0.240.57\/0.600.31\/0.32Sex, BMIBeta cell function0.71\/0.580.66\/0.600.52\/0.500.68\/0.660.32\/0.400.47\/0.460.37\/0.33Sex, age, S4SFLeptin0.85\/0.530.85\/0.570.70\/0.580.64\/0.520.35\/0.020.66\/0.310.38\/0.37Sex, age, S4SFTotal cholesterol0.76\/0.740.77\/0.720.78\/0.740.73\/0.730.52\/0.510.51\/0.440.63\/0.51Age, S4SFLDL-cholesterol0.78\/0.790.81\/0.740.81\/0.790.77\/0.750.52\/0.510.59\/0.520.68\/0.58Sex, age, S4SFHDL-cholesterol0.78\/0.710.84\/0.770.75\/0.760.74\/0.740.31\/0.300.44\/0.440.52\/0.52Sex, S4SFTotal cholesterol:HDL-cholesterol ratio0.81\/0.780.86\/0.810.84\/0.820.78\/0.760.50\/0.410.50\/0.490.65\/0.54Age, WHRTriacylglycerol0.58\/0.560.67\/0.600.59\/0.540.63\/0.600.29\/0.410.34\/0.340.16\/0.17Sex, S4SFNEFA0.49\/0.350.43\/0.390.39\/0.380.34\/0.370.10\/0.140.20\/0.180.25\/0.26Sex, S4SFValues are unadjusted intra-pair correlation\/adjusted intra-pair correlation.OS, opposite sex\nTwin model fitting The variance components and 95% CIs of the best fitting models before and after adjustment for covariates are presented in Table\u00a03. The best fitting model for lean body mass and the obesity parameters body mass, BMI, WHR, S4SF and fat mass was an AE model containing a major genetic component. For total cholesterol and LDL-cholesterol, the ACE model was the best fitting model. However, after adjusting for covariates, the AE model became the best fitting model. For IGFBP-1, fasting insulin and insulin resistance, a DE model containing a non-additive genetic and unique environmental component had the best fit. The variation of the remaining blood parameters, including fasting glucose, beta cell function, leptin, HDL-cholesterol, total cholesterol:HDL-cholesterol ratio, triacylglycerol and NEFA levels were best explained by an AE model (Table\u00a03).\nTable\u00a03Variance components estimates and 95% CIs of best-fitting models expressed in percentagesCharacteristicUnadjustedAdjusted for covariatesModelSexa2 (H2)c2d2 (H2)e2SModelSexa2 (H2)c2d2 (H2)e2SBody mass AEM90 (86\u201393)\u2013\u201310 (7\u201314)AEM84 (78\u201388)\u2013\u201316 (12\u201322)AEW80 (73\u201385)\u2013\u201320 (15\u201327)AEW74 (66\u201380)\u2013\u201326 (20\u201334)BMI AEM87 (82\u201390)\u2013\u201313 (10\u201318)AEM85 (79\u201389)\u2013\u201315 (11\u201321)AEW76 (68\u201382)\u2013\u201324 (18\u201332)AEW75 (67\u201381)\u2013\u201325 (19\u201333)WHRAEM94 (92\u201396)\u2013\u20136 (4\u20138)AE70 (63\u201375)\u2013\u201330 (25\u201337)M>WAEW70 (59\u201377)\u2013\u201330 (23\u201341)S4SFAEM88 (83\u201391)\u2013\u201312 (9\u201317)AE74 (68\u201379)\u2013\u201326 (21\u201332)M>WAEW75 (67\u201381)\u2013\u201325 (19\u201333)Fat mass AEM88 (84\u201392)\u2013\u201312 (8\u201316)AEM81 (74\u201386)\u2013\u201319 (14\u201326)AEW74 (65\u201380)\u2013\u201326 (20\u201335)AEW70 (60\u201377)\u2013\u201330 (23\u201340)Lean body mass AEM97 (96\u201398)\u2013\u20133 (2\u20134)AE81 (76\u201384)\u2013\u201319 (16\u201324)M>WAEW82 (76\u201387)\u2013\u201318 (13\u201324)IGFBP-1 DE\u2013\u201356 (47\u201364)44 (36\u201353)W>MDE\u2013\u201347 (36\u201356)53 (44\u201364)W>MFasting insulin DE\u2013\u201354 (45\u201362)46 (38\u201355)DE\u2013\u201349 (39\u201358)51 (42\u201361)Insulin resistanceDE\u2013\u201352 (42\u201360)48 (40\u201358)DE\u2013\u201348 (38\u201357)52 (43\u201362)Fasting glucose AE72 (66\u201377)\u2013\u201328 (23\u201334)M>WAE67 (60\u201373)\u2013\u201333 (27\u201340)M>WBeta cell function AE69 (63\u201375)\u2013\u201331 (25\u201337)AE62 (54\u201369)\u2013\u201338 (31\u201346)Leptin AEM92 (88\u201394)\u2013\u20138 (6\u201312)AE53 (44\u201361)\u2013\u201347 (39\u201356)M>WAE W63 (52\u201372)\u2013\u201337 (28\u201348)Total cholesterol ACE49 (28\u201374)29 (4\u201348)\u201322 (18\u201328)AE75 (69\u201379)\u2013\u201325 (21\u201331)LDL-cholesterol ACE43 (24\u201367)37 (14\u201354)\u201320 (16\u201325)M>WAE78 (73\u201382)\u2013\u201322 (18\u201327)HDL-cholesterol AE 82 (78\u201386)\u2013\u201318 (14\u201322)W>MAE76 (70\u201381)\u2013\u201324 (19\u201330)W>MTotal cholesterol:HDL-cholesterol ratioAEM88 (83\u201391)\u2013\u201312 (9\u201317)AE79 (74\u201383)\u2013\u201321 (17\u201326)M>WAEW79 (72\u201385)\u2013\u201321 (15\u201328)TriacylglycerolAE61 (52\u201368)\u2013\u201339 (32\u201348)AE58 (49\u201366)\u2013\u201342 (34\u201351)NEFAAE46 (36\u201354)\u2013\u201354 (46\u201364)W>MAE37 (25\u201347)\u2013\u201363 (53\u201375)W>MValues are variance components estimates (95% CI)M, men; W, women; a2, additive genetic variance; c2, common environmental variance; d2, non-additive genetic variance; e2, unique environmental variance; S, scalar effect; H2, broad heritabilityQuantitative sex differences were present in body mass, BMI, WHR, S4SF, fat mass, lean body mass, leptin and total cholesterol:HDL-cholesterol ratio, because variance components estimates were significantly different between men and women (Table\u00a03). The influences of additive genetic factors were larger in men than in women. For some traits, scalar sex differences were observed, implying that although variance components are equal across sexes, the total variances differ. As a result, total variance of IGFBP-1, HDL-cholesterol and NEFA levels in women was larger than in men, but smaller for fasting glucose and HDL-cholesterol levels (Table\u00a03).\nAfter adjusting for covariates, quantitative sex differences remained significant only for body mass, BMI and fat mass (Table\u00a03). In addition, scalar sex differences were significant for WHR, S4SF, lean body mass, IGFBP-1, fasting glucose, leptin, HDL-cholesterol, total cholesterol:HDL-cholesterol ratio and NEFA levels. Total variance of IGFBP-1, HDL-cholesterol and NEFA levels was larger in women than in men, but smaller for WHR, S4SF, lean body mass, fasting glucose, leptin and total cholesterol:HDL-cholesterol ratio (Table\u00a03).\nThe adjusted correlations of WHR, fasting insulin and triacylglycerol levels for opposite-sex pairs were smaller than the correlations for like-sexed DZ pairs, suggesting that different genetic factors influence this trait in men and women (Table\u00a02). However, the correlations between the additive genetic factors in opposite-sex pairs were not significantly smaller than 0.5 (p\u2009>\u20090.05).\nBroad-sense heritability estimates (encompassing both additive and non-additive genetic effects) were slightly lower after adjusting for covariates, with the exception of total cholesterol and LDL-cholesterol, which had a higher heritability after adjustment (Table\u00a03).\nIn summary, heritability estimates of body mass, BMI and fat mass were 84, 85 and 81% for men and 74, 75 and 70% for women, respectively. WHR, S4SF and lean body mass had heritability estimates of 70, 74 and 81%, correspondingly. For fasting glucose, fasting insulin, insulin resistance, beta cell function and IGFBP-1 levels, genetic factors explained 67, 49, 48, 62 and 47% of the total variation, respectively. Finally, heritability estimates of total cholesterol, triacylglycerol, HDL-cholesterol, LDL-cholesterol, total cholesterol:HDL-cholesterol ratio, NEFA and leptin levels were 75, 58, 76, 78, 79, 37 and 53%, respectively (Table\u00a03).\nDiscussion\nTo determine the genetic contribution to type 2 diabetes, we estimated the heritabilities of 18 anthropometric and metabolic characteristics related to this disease, including parameters quantifying obesity, glucose intolerance\/insulin resistance and dyslipidaemia. The traits were measured in 138\u00a0DZ and 240\u00a0MZ young healthy twin pairs recruited from the population-based EFPTS [36].\nObesity, defined as excess accumulation of adipose tissue, is a complicated trait to measure and numerous methods exist for its determination [38]. In the present study several parameters defining obesity, including body mass, BMI, S4SF, WHR and fat mass were used and heritability estimates ranged from 70 to 85%. According to ESM Table\u00a01, the heritabilities identified by us for BMI and body mass are in agreement with those reported in other twin studies, but the heritability identified by us for WHR is somewhat higher [7, 15, 18, 39]. Heritability estimates for S4SF and fat mass are not often reported and therefore we were not able to make a valid comparison. Despite the variety of measurements used to quantify obesity, the heritabilities of the different measurements were consistently in a high range, indicating that the contribution of genetic factors to total phenotypic variation in obesity is of great importance in the studied twin sample.\nQuantitative sex differences were significant for body mass, BMI and fat mass, with heritability estimates of 84, 85 and 81% in men and 74, 75 and 70% in women, respectively. Our results confirm those from other twin studies reporting quantitative sex differences in the heritability of body mass and BMI [4\u20136, 9, 20, 40]. However, in other twin studies the highest heritability estimates were not always observed in men (ESM Table\u00a01), indicating that the influence of environmental factors in the total variation of these traits according to sex may vary in different populations.\nIn the present study, 49% of the total variation of fasting insulin was explained by genetic factors, which is in agreement with other twin studies (ESM Table\u00a01) [41, 42]. The heritability of fasting glucose (H2\u2009=\u200967%) was higher than those observed in other twin studies, where it ranged between 12 and 58% (ESM Table\u00a01) [42, 43]. The heritabilities of beta cell function (H2\u2009=\u200962%) and insulin resistance (H2\u2009=\u200948%) was not higher than those of fasting glucose (H2\u2009=\u200967%) and fasting insulin levels (H2\u2009=\u200949%). This indicates that no additional power was obtained by calculating these alternative phenotypic markers with the homeostasis model assessment.\nThe variation of total cholesterol, triacylglycerol, HDL-cholesterol, LDL-cholesterol and total cholesterol:HDL-cholesterol ratio was primarily explained by additive genetic factors, accounting for 75, 58, 76, 78 and 79% of the total variance, respectively. No evidence was observed for quantitative sex differences in heritability estimates for these traits. The percentages are slightly higher than the heritability estimates reported in the literature, but are in the same high range (ESM Table\u00a01). To the best of our knowledge, ours is the first large twin study reporting the heritability of NEFA levels, which was 37%. This is somewhat surprising, since excessive circulating fatty acids represent a major contributor to the development of insulin resistance [44]. The heritability determined for NEFA levels in the current study was low compared with those reported for the other blood lipid parameters, indicating that environmental factors, e.g. nutrition, may be more important in determining the variation of NEFA levels than genetic factors.\nAdditive genetic variance explained 53% of the variation of total leptin concentration. In other western adult twin studies, heritabilities of total leptin concentrations ranged from 34 to 55% [45, 46], which is in agreement with our findings. Compared with other traits, the correlations and heritability estimates of leptin were strongly reduced after adjusting for covariates, which is probably caused by the adjustment for fat (using S4SF). This is not very surprising, since leptin is synthesised in and secreted from adipose tissue and plasma leptin levels are increased in obese humans in direct proportion to body fat mass [35]. The heritability of IGFBP-1 (H2\u2009=\u200947%) levels determined in our study was also relatively high, since the heritability in two other twin studies ranged from 0 to 36% [41, 47]. Interestingly, the heritabilities of IGFBP-1 levels in the latter studies were determined in older twins (mean age 63\u00a0years). This might suggest that the heritability of IGFBP-1 decreases with advancing age. Longitudinal twin studies need to be carried out to verify this observation.\nWe are the first to report that a model containing non-additive instead of additive genetic factors is the best-fitting model for insulin resistance, fasting insulin and IGFBP-1 levels [7, 18, 22, 24, 41\u201343]. In addition to the twin model fitting procedure, twin correlations can also give an impression of whether non-additive genetic effects might be involved, i.e. a DZ correlation of less than one-half of the MZ correlation suggests a contribution of non-additive genetic effects [18, 41\u201343]. The smaller sample size of some studies could be responsible for their not observing a DE model. Furthermore, the presence of non-additive genetic effects has not always been tested. However, if non-additive genetic effects are observed, this implies that a part of the variance is explained by interactions between alleles at the same locus or on different loci. Although this observation is likely to be the result of lack of power, we cannot exclude real non-additive genetic effects in the absence of additive genetic effects, as these three traits are closely related to or are a direct gene product.\nMZ MC twins have a more adverse intra-uterine environment than MZ DC and DZ twins, resulting in a significantly lower weight at birth and higher perinatal mortality and morbidity [36]. Hence, it has been hypothesised that for disorders like type 2 diabetes, in the development of which prenatal environment plays a role, the classical twin study might be an unreliable method of estimating heritabilities [48, 49]. In the current twin sample, intra-pair correlations did not differ between MZ MC and MZ DC twins, suggesting that the chorion type of MZ twins did not bias the heritability estimates of the studied traits.\nWith the exception of NEFA levels, the genetic contribution to the traits studied in our twin sample was high. When comparing the heritability values found by us with those reported in other twin studies (Electronic Supplementary Material Table\u00a01), our heritability estimates are in a slightly higher range. An important aspect affecting heritability estimates is the accuracy of the zygosity determination used in the present study via sequential analysis based on sex, fetal membranes, umbilical cord blood groups, placental alkaline phosphatase and DNA marker analysis. In other twin studies, zygosity was often determined using questionnaires [4], which sometimes leads to misclassification and consequently to underestimation of heritability [50]. Our high heritability estimates might also be a consequence of the homogeneous composition of the studied sample. All twins included were born in the Belgian province of East Flanders, which is a relatively small but rather densely populated area [36]. Another factor possibly contributing to the high heritability estimates is age, as shown by the fact that several large twin studies have reported a decrease in the heritability of obesity (BMI) in adults with increasing age [5, 12, 16]. Our twin sample is young (mean\u2009=\u200925\u00a0years) and the age range is small (18\u201334\u00a0years). As the power to detect quantitative trait loci is positively correlated with the size of the genetic effect [3], our study suggests that the search for candidate genes might be more efficient in a young homogeneous population.\nIn conclusion, this study is the first large study to give a comprehensive overview of the heritabilities of multiple risk factors related to type 2 diabetes within the same twin sample. The variation of traits related to obesity, glucose intolerance\/insulin resistance and dyslipidaemia in this Belgian twin sample is in general highly explained by genetic factors. These high heritabilities are very promising for follow-up to this research, in which, in order to explain some of the genetic variance observed, sib-pair linkage analyses will be performed using microsatellite markers located in the close vicinity of selected candidate genes.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nESM 1\n(PDF 64 kb)","keyphrases":["anthropometry","lipid metabolism","east flanders prospective twin survey","heritability","metabolic risk factors","type 2 diabetes","twin study","chorionicity","carbohydrate metabolism","structural equation modelling"],"prmu":["P","P","P","P","P","P","P","P","P","M"]}
{"id":"J_Med_Internet_Res-5-4-1550572","title":"Adolescents Searching for Health Information on the Internet: An Observational Study\n","text":"Background Adolescents' access to health information on the Internet is partly a function of their ability to search for and find answers to their health-related questions. Adolescents may have unique health and computer literacy needs. Although many surveys, interviews, and focus groups have been utilized to understand the information-seeking and information-retrieval behavior of adolescents looking for health information online, we were unable to locate observations of individual adolescents that have been conducted in this context.\nIntroduction\nThe Internet has become an important tool for many people with health concerns [1,2], especially for adolescents [3,4]. Concerns about confidentiality, coupled with the fact that many teenagers find accessing care through traditional providers difficult [5], make access to information via the Internet particularly important. Given rapidly-expanding Internet access among young people, it is not surprising, then, that more than 70% of 15 to 17 year-olds say they have used the Internet to look up health information (written communication, 2001 Dec; Generation RX.com Survey printouts; V. Rideout, Henry J. Kaiser Foundation, Menlo Park, CA). This percentage is likely to increase if Internet access from home continues to rise as it has in recent years [6].\nBecause of the enormous amount of unstructured online content, it is crucial to understand how youth navigate through the Web to find health information. Prior research, primarily from library and information science literature and education literature, has highlighted several search characteristics that are either unique or more pronounced in adolescents. For example, adolescents take more time to complete online tasks than college students [7], search less systematically [7- 10], have difficulty formulating search queries due to misspelling and problems with the level of specificity [8- 11], utilize less-advanced search syntax [7], and rarely consider the source of Web pages [8,9]. While informative, this literature is based primarily on adolescents searching for answers to homework questions rather than health information.\nSearching for online health information involves distinctive challenges including unfamiliar terminology [12]; encounters with pornography-blocking software (written communication, 2001 Dec; Generation RX.com Survey printouts; V. Rideout, Henry J. Kaiser Foundation, Menlo Park, CA), [13]; and the importance and difficulty of determining health information quality [14]. However, despite the need for research that details the online search behavior of health consumers, the authors were only able to locate a few articles in which health science researchers actually observed, recorded, and analyzed consumers of any age searching for health information [14- 16]. Instead, surveys (eg, written communication, 2001 Dec; Generation RX.com Survey printouts; V. Rideout, Henry J. Kaiser Foundation, Menlo Park, CA; and [1,2,4]) have been the predominant method used to understand health consumers' online searching behavior, despite problems with participant recall and the inability of surveys to capture specific search tactics. In addition, the authors found a handful of studies in the medical informatics literature that have also looked at log data from particular medical Web sites, but these studies are also limited in scope since they do not observe the actual searcher or see the broader context in which the searcher is acting [17] (see also [18] for similar studies performed on search engine data). The value of directly observing users was demonstrated in the Eysenbach study, which revealed that adults said they paid attention to the source of health sites during interviews, although this behavior was not found during the actual observations [15].\nObservational research specific to the adolescent age group and online search behavior for health information is also sparse. There have been some good surveys that answer many useful questions concerning why adolescents go to the Internet, what they search for, if they find it, and what they do with it (written communication, 2001 Dec; Generation RX.com Survey printouts; V. Rideout, Henry J. Kaiser Foundation, Menlo Park, CA;and [4,19]). The only observational study we were able to locate conducted 27 focus groups where groups of adolescents searched online for health information as they discussed their own experiences [14]. Many of the findings concerning adolescent search behavior found in the library and information science literature were confirmed and additional issues were raised, including concerns about low health literacy and trouble judging the quality of information, that may be more pronounced in adolescents than adults. However, that study only begins to paint a picture of adolescent search behavior for online health information, because the searches were performed in a group setting and the success, failure, and specific search tactics used were not coded or analyzed.\nThe study reported here provides a more in-depth understanding of how adolescents search for health information using the Internet and what implications this may have on access to health information. To capture enough detail, the study recorded specific actions taken by adolescents which were later coded and analyzed. Participants were encouraged to share their thought process out loud as they searched for answers to a list of predetermined health questions. The result was a rich set of both quantitative and qualitative data that was thoroughly analyzed for common themes and events. Specific questions of interest include, but are not limited to: What are the various search strategies used? What factors contribute to finding correct and useful answers? When using a search engine, how many results pages are viewed and utilized? What types of search strings are entered into search engines? Answers to these and related questions should be of interest to a number of parties including educators (eg, health educators, librarians, teachers), Web site and search engine designers, health care practitioners, and researchers (eg, to create a sample of URLs by simulating online searching behavior [20]).\nMethods\nSample\nTwelve students from 1 middle school (N= 4) and 2 high schools (N = 4 and N= 4) in southeast Michigan were recruited for this study. Staff at each school were asked to select 4 students who were (a) comfortable using computers, (b) comfortable searching for information on the Internet, and (c) strong students who could afford to miss one class period. Students received a University of Michigan T-shirt, valued at roughly $8, in return for their participation.\nThe parent or guardian of every student signed an informed consent document that described the purpose and procedure of the study. Students also signed separate assent forms with similar information. The University of Michigan Behavioral Science Institutional Review Board approved this study and the consent and assent documents.\nData Collection\nThree methods of data collection were used. First, one of the two members of the research team present during each of the observations coded searching behavior in real time while the second member of the research team interacted with the student. Second, TechSmith Camtasia 3.0.1 commercial tracking software [21] was installed on the computer. This software captured the students' voices and took pictures of the screen (screen captures) twice per second during the entire session. Finally, a video camera was positioned to capture the screen and the students' voices, but not the students' images. Observations coded in real time were used to develop a more detailed and systematic coding system for use when reviewing the tracking software records. It is data from the tracking software coding that is reported here.\nAll observations of adolescents were conducted during January 2002. Each school provided a room in which to conduct the observations. Students were brought to the observation room one at a time. Two researchers were present at every observation. For each student, one of the researchers first reviewed the assent form to introduce the project and obtain the student's permission to participate. The students were then asked 14 questions about demographics (age, race\/ethnicity, and gender) and their prior computer use (eg, how often they use computers or the Internet, what health topics they have searched, which search engines they used, and whether they have a computer and access to the Internet at home).\nOnce the brief interview had been completed, the observed searches began. To help the students understand the procedure and to reinforce the importance of thinking out loud while doing their searches, each student was first asked to do an easy non-health-related search looking for the next day's local weather forecast. As with the subsequent health-related searches, the local-weather question was first read to the student by a researcher and then a card with the question on it was set next to the computer in case the student needed to read it. As part of the think-aloud protocol, the experimenter asked the student to talk out loud about what they were doing, so that researchers could better understand the reasons behind the searching behavior. If a student stopped talking during the search, he or she was reminded by the observers to \"keep talking,\" but the experimenters did not ask students to elaborate on any specific thing they said. Concurrent verbal reports more accurately reflect a subject's mental state at the time of observed behaviors than do retrospective reflections, and this minimal think-aloud protocol has been shown to slow subjects down, but not to qualitatively change their problem solving behavior [22].\nAfter the students completed the practice local-weather search, they were given a sequence of up to 6 predetermined health information questions (see Table 1), 1 at a time. Questions were framed in a way that took into consideration the broader information concern that the question attempted to resolve. To eliminate confounding by learning effects between searches, we used a 6 x 6 Latin square to determine the order in which the questions were presented to the participants. The computer that students used was provided by the researchers, but connected to the school's network so that the students were protected from controversial or pornographic material by the same blocking or filtering software used by the school. The 3 different schools used 3 different filtering systems. Each observation session lasted one class period. No time limit was given for each question, but when the class period ended, any ongoing search was terminated and any remaining questions were skipped.\nTable 1\nHealth-related questions\nYour aunt was just told she has diabetes. She isn't sure what kinds of food she can or can't eat. Using the Internet, find some information for your aunt about what foods she should or should not eat.\nA friend recently started taking a drug called Paxil for depression. He seems to be tired all the time, and even falls asleep in class. Use the Internet to find out if the drug might be making him sleepy.\nYour older brother has a problem with drinking too much alcohol. He wants to go to a local Alcoholics Anonymous meeting. Use the Internet to help him find a local meeting.\nYou want to get an HIV test, but you don't want anyone to know. You also don't have any money to pay for it. Use the Internet to find a place to get a free and confidential HIV test.\nFor class, you need to learn about medicine that can help people stop smoking. Using the Internet, find the names of these medicines.\nYou are about to get a tattoo, but a friend warned you that some places spread infections like HIV and hepatitis. Use the Internet to find out if this is true.\nTopics for the health-related questions were chosen based upon responses to a survey of adolescents conducted by the Kaiser Family Foundation (written communication, 2001 Dec; Generation RX.com Survey printouts; V. Rideout, Henry J. Kaiser Foundation, Menlo Park, CA). Certain topics including homosexuality, teen pregnancy, and abortion were purposefully avoided so as not to expose participants to overly-controversial information.\nData Analysis\nAfter all the observations were completed, 3 researchers including a physician, health educator, and human-computer interface specialist met as a group to review the real-time coding results and to clarify or augment the coding scheme before the definitive final coding of the tracking-software records. The final coding scheme was designed to record data on the person searching, the question being asked, the time it took to find an answer, the search strategy utilized (eg, utilize search engine or directly type in URL); search strings used; number of search engine results pages reviewed; number of pages viewed within a particular site; and the use of menus, advertisements, and directories. One of the 3 coders was assigned as a primary reviewer for each of the observation sessions. The assigned primary reviewer was responsible for a detailed coding of the observation session and any coding problems were resolved in a second group discussion.\nThe reviewers classified each of the answers found by the students as correct or incorrect, complete or incomplete, and, for location questions only, useful or not\nuseful. To avoid being overly narrow in our classification of correct for the more open-ended questions such as the question on healthy foods for a person with diabetes, we used the following general rule for classification: to be considered correct, the content of the answer had to be the kind of information that might be discussed in a medical school or school of public health. This classification system was validated in previously-published work by the research team and resulted in a high inter-rater reliability (\u03ba = 0.84) [13]. The more-specific questions such as the question asking about a location for an Alcoholics Anonymous meeting were considered correct if the student found a Web page listing a meeting location and time or contact phone number. Answers were complete if the students were able to answer all parts of the question. For example, if the student found a discussion about HIV transmission by tattoo parlors, but did not find an answer about hepatitis it was classified as incomplete. Useful answers pertained to location questions. An Alcoholics Anonymous meeting in another state was not useful. A summary measure classifying each search as successful, partially successful, or unsuccessful was computed using the correct, complete, and useful ratings. To obtain a rating of successful, the answer had to be complete, correct, and useful. If the student gave up before finding an answer, the search was classified as unsuccessful.\nResults\nTwelve middle school students and high school students in southeast Michigan participated. Students ranged in age from 12 to 17 years old, with a mean of 14 years. Half of the students were female. Of the 12 students, 7 were white, 2 were African American, 1 was Indian American, 1 was Hispanic, and 1 was Asian American. Of the 12 students, only the 6 oldest students had searched for health information on the Internet before. The variation by age is consistent with other findings that youth age 15 to 17 years are significantly more likely to have looked up health information (32%) than youth age 12 to 14 years (18%) [23]. All of the students, however, had computers and access to the Internet at home. Students reported using a computer from 1 hour per week to 3 hours per day, with a mean of 12.3 hours per week.\nEleven students attempted all 6 searches, while the remaining student attempted 3, for a total of 69 searches. One search was not included since the Internet connection was not working properly, making a total of 68 searches that were analyzed. Searches took an average of 5 minutes and 41 seconds, ranging from just under a minute to nearly 24 minutes. This time frame is essentially the same as Eysenbach recorded for adults [15]. Although direct comparison is inappropriate since different questions were asked, the similar order of magnitude is suggestive.\nOverall Search Strategy\nAs students thought aloud, the researchers got a sense of what students were looking at on each page. Students seemed to skip around a lot, and didn't skim results pages or specific Web sites in any methodical or thorough ways, sometimes missing links or text that contained the answer to questions. This is also consistent with findings from non-health-related searching behavior as summarized in Hsieh-Yee [24].\nTable 2\nDistribution of pages viewed per site\nPages Viewed Per Site\nSites\nn\n%\nCumulative %\n1\n143\n70.4\n70.4\n2\n27\n13.3\n83.7\n3\n11\n5.4\n89.2\n4\n8\n3.9\n93.1\n5\n8\n3.9\n97.0\n6\n2\n1.0\n98.0\n8\n1\n0.5\n98.5\n9\n1\n0.5\n99.0\n15\n2\n1.0\n100.0\nTotal\n203\n100\nStudents used multiple methods to locate Web sites that they believed contained answers to the 68 questions. In 60 cases, the student started looking for an answer by visiting a search engine and entering in a search term or phrase. In 2 cases, the student started by selecting from directory menus (eg, choosing the topic health). In 6 cases, the student started by entering a URL (other than a search engine) directly into the browser address bar. In total, there were 215 attempts to access non-search-engine or directory Web sites. Nearly all of these attempts were made by following a link from a search engine either after a search or through the use of a directory. Of the 215 attempted site visits, 4 were broken links, 3 were blocked by the filters utilized at certain schools, and 5 were PDF files (read by Acrobat Reader) which students either could not download or chose not to download because downloading was too slow. This left 203 sites that were viewed with an average of 1.8 pages viewed per site. The distribution of pages visited per site is shown in Table 2. Note that the distribution is roughly consistent with a power law as observed in previous studies [25]. At a reviewer's request, this data was looked at on an individual student level. Students varied a great deal in the total number of visited sites. Eleven of the 12 students went only 1 page deep on the majority of visited sites. Although the individual-level data is not large enough to analyze more rigorously, the power law seems to operate on an individual level as well as the aggregate level.\nEven when students found a Web site that contained the answer to a question, they did not always find the answer. One example is the Alcoholics Anonymous site [26] where 8 of the 11 students ended up while searching for a local meeting. Although there was a link to a site that contained local information, only 3 of the 8 students were able to find the link, 1 of whom only found it on the second visit to the Alcoholics Anonymous site, after viewing a total of 16 pages within the site. Similarly, 6 of the 11 students who searched for whether or not Paxil causes drowsiness visited the official Paxil site [27]. Only 3 of the 6 students were able to successfully answer the question based upon the information they found at the site. Two of them failed to find the list of side effects and 1 of them found the list but did not understand it enough (or read it carefully enough) to answer the question correctly.\nSearch Engine Tactics\nSeven search engines were used, including 2 meta-search engines (Dogpile and Locate.com). The meta-search engine Locate.com offers the user a number of search engines to choose from. Searches performed from the Locate.com Web site that utilized another search engine (eg, Yahoo!) are reported as if the search occurred on the destination search engine (eg, Yahoo!). Table 3 summarizes the number of times that a particular search engine was used. If a search engine was used multiple times while searching for an answer to the same question, it is only counted once. Because students occasionally switched search engines while trying to answer the same question, there are more searches using a search engine (79) than there are attempts to answer questions (68). In total, 6 of the 12 students used only Google, 1 used only Yahoo!, and the remaining 5 changed search engines at some point.\nTable 3\nSearch engine usage\nSearch Engine\nTimes Used\nn\n%\nGoogle\n38\n48.1\nYahoo!\n13\n16.5\nAsk\n12\n15.2\nMSN\n7\n8.9\nHotbot\n6\n7.6\nDogpile\n2\n2.5\nAltaVista\n1\n1.3\nA total of 132 search phrases were entered into the various search engines. Only 104 of those search phrases were unique. The most-frequent 2 phrases used were \"diabetes\" and \"Paxil,\" each of which had 5 occurrences. There was an average of 3.6 words typed in per search phrase and 80% of the time there were 4 or fewer words per search phrase.\nTable 4\nDistribution of search-result links viewed\nBands of Search-Result Links Viewed\nChosen Links\nn\n%\nCumulative %\nResults 1-10\n137\n82.5\n82.5\nResults 11-20\n8\n4.8\n87.3\nResults 21-30\n11\n6.6\n94.0\nResults 31-40\n4\n2.4\n96.4\nResults 41-50\n4\n2.4\n98.8\nResults 51-60\n1\n0.6\n99.4\nResults 61 or more\n1\n0.6\n100.0\nOf the 132 search phrases, 30 contained at least 1 word that was misspelled (eg, \"tatoo,\" \"Alchoholics,\" or \"smokeing\"), despite the fact that students could read the correctly-spelled word on the index card containing the question. Some search engines (eg, Google) offer a feature that recommends an alternate search string with the correct spelling of a word. For example, if a student typed \"alchoholics anonymous,\" the first page of results began with, \"Do you mean 'alcoholics anonymous?'\" Students were offered a new search string with correct spelling on 15 separate occasions, but only noticed and used it 6 times. The remainder of the times they used the results that were offered for the incorrect spelling. Of the 7 students who were offered corrected spelling suggestions, only 2 ever used them.\nOnce a search string was entered into a search engine, students varied in the number of results pages that were viewed. Students viewed only the first results page 78% of the time and 4 pages or less of results 93% of the time. Because search engines report a different number of links per page of search results, Table 4 reports how often links were selected from the first 10 results, the second 10, and so on. Only 3 blocked links were encountered during all of the searches, suggesting that blocking software did not have a significant impact on these results.\nSuccessful Searching Characteristics\nOf the 68 questions that students attempted to answer, 7 searches were abandoned after the student gave up or, in 2 cases, when the class period ended. Of the remaining 61 searches, 47 were successful in finding a complete, correct, and useful answer to the health question and the remaining 14 were unsuccessful. Six of the unsuccessful answers were completely incorrect and not useful, 4 were useful but only partially correct, and 4 were fully correct but not useful.\nSeveral factors contributed to the success of finding a correct, complete, and useful answer. One important factor was the individual who was performing the search. Although every student answered at least 1 question correctly there was wide variation in the number of correct answers. Two students successfully answered 6 out of 6 questions, 3 students successfully answered 5 questions, 4 students successfully answered 4 questions, and the remaining 3 students only successfully answered 1 or 2 questions. While our sample of students was too small to draw conclusions from, no distinct patterns were observed that would indicate that race, gender, Internet experience, or health searching experience were significant determinants of success. However, the older adolescents (16-17 year olds) were successful 87% of the time (26 of 30) as compared to 68% (21 of 31) for the younger adolescents.\nAnother important factor was the difficulty level of the questions themselves. Table 5 shows the failure rate for each question. The 4 partially-correct answers were split evenly between the Alcoholics Anonymous and tattoo questions. All 4 of the correct but not useful answers resulted from the HIV test question.\nTable 5\nUnsuccessful searches by search topic\nSearch Topic\nUnsuccessful Searches\nn\n%\nHIV test\n8\n38.1\nPaxil\n4\n19.0\nAlcoholics Anonymous\n3\n14.3\ntattoo\n3\n14.3\nsmoking\n2\n9.5\ndiabetes\n1\n4.8\nTotal\n21\n100.0\nCertain search actions led to sites that contained the answer more often than others. Overall, students found answers on 22% of the sites they accessed (47 of 215). They accessed sites in 5 ways. Although not often taken, the action with the highest probability of success (47%; 7 of 15) was following a link from 1 non-search-engine site (eg, www.aa-intergroup.org) to another site (eg, www.alcoholics-anonymous.org). In most of these cases, the student accessed the first site directly from a search engine. Clicking on search engine results led to a site where students found an answer 21% of the time (35 of 166). Success rates were similar for following a recommended link from a list or menu provided by the search engine (18%; 4 of 22). Directly typing in a URL, bypassing search engines entirely, was successful only 9% of the time (1 of 11). A sponsored link from a search engine was followed only once, and the student found an incorrect answer on that site.\nAnother contributing factor related to success was misspelling of search terms. Of the 14 completed but unsuccessful searches, 29% (4 searches) had at least 1 misspelling compared to only 15% (7 searches) of the 47 successful searches. Perhaps even more telling, both successful and unsuccessful searches with misspellings took students 1.5 minutes longer on average than searches without misspellings. Observations confirmed that some students were unable to find an answer until they discovered and corrected their misspelling, resulting in higher quality and more-relevant results.\nOther search characteristics did not have statistically significant impacts on whether searches were successful, although this may have been due to small sample sizes. For example, the search engines were not significantly different in their percentages of successful searches. Similarly, the average number of words per search string was not significantly related to search success rate. (Data not shown.)\nQualitative Analysis\nCertain common behaviors of the adolescent searchers were observed which were not apparent from the quantitative analysis.\nFirst, the students were very comfortable and confident while searching online for health information. Most students knew where they wanted to start the search and navigated using quick mouse clicks and shortcut keys. However, this characteristic was likely over-represented in our population due to their strong academic performance and Internet proficiency.\nSecond, several searchers did not take much time in formulating a search strategy or (when applicable) choosing search terms. Instead, these searchers seemed to type in the first search string that came to mind. If the results were not what were anticipated, another search string was typed in, sometimes without even clicking on any results from the first search string. The overall approach was a trial-and-error method with frequent backtracking. The most-common problem with search strings was that they were not specific enough. For example, 2 different students typed in the search string \"hiv\" when looking for a place that administers free and confidential HIV tests.\nThird, most students quickly scanned pages, jumping from place to place within a page, rarely reading an entire paragraph. In some cases the answer to a question was contained on a page, but the student left before finding it. In other cases a link that would have led to the answer was missed. This finding supports prior research on adolescent search behavior related to nonhealth topics [7- 10].\nFourth, students mentioned that they purposefully avoided sponsored links and advertisements, despite the fact that many of the search engines present these results first. The qualitative data confirmed this practice, as only 1 sponsored link was ever selected.\nFinally, little to no attention was paid to the source of the answer. In the vast majority of cases, once an answer was located, it was simply assumed to be correct.\nDiscussion\nWhen compared with prior research, the findings of this study show many similarities and a few key differences between the behaviors of adolescents and adults while searching for health information. This study found that adolescents searching for health information utilized search engines nearly every time. This finding was similar to that for adults as described in the Eysenbach study [15]. These observational studies also suggest that after-the-fact survey questions concerning the use of search engines may underestimate this behavior. For example, 2 nationally-representative surveys reported that 58% of youth (written communication, 2001 Dec; Generation RX.com Survey printouts; V. Rideout, Henry J. Kaiser Foundation, Menlo Park, CA) and 81% of adults [1,2] started seeking health information at search engines. Our study found that adolescents relied upon links from only the first few results pages, and rarely explored far within any site. These results also were similar to adult searching behaviors [15], although youth seem to be more likely to search beyond the first 10 search results. Adolescents often chose search strings that were too general and\/or contained misspellings, so that they did not always find useful sites that were available. Eysenbach also reported search strings by adults that were too general [15], however, spelling seems to be more of a problem with youth. Adolescents were unsystematic in their reading of Web sites and some sites were poorly organized so that they did not always find the information they were looking for, even when it was present in a site they examined. Future research is needed to better understand if adolescents do not understand information provided on these sites, whether they simply have less patience, or some other explanation. In summary, many of the specific search tactics are similar for adults and adolescents, but a few issues related to spelling, browsing of Web sites, and understanding of content are notably different.\nSimulation of Searches\nThe results from this study have implications for anyone who simulates adolescent health searches, for providers of health information, and for educators. There are many reasons to simulate adolescent health searches. For example, an educator preparing a lesson plan may want to informally simulate searches in order to anticipate what students are likely to find if given certain particular search tasks. A researcher may want to simulate adolescent searches more systematically to evaluate the availability and accessibility of information on particular topics, to evaluate which search engines should be recommended to adolescents, or to evaluate whether the installation of filtering software will have a detrimental impact on accessibility of health information [13]. Because many of the search behaviors modeled by these simulations are similar for both adolescents and adults, results from studies that simulated one or the other group likely apply to both groups.\nThe results of this study suggest that such simulations can focus on the use of search engines, but that very-broad search terms and, especially for adolescents, common spelling errors should be considered. Ads and other nonresult links can be ignored. Since more than 80% of the links that were followed appeared in the top 10 results, and more than 95% were among the top 40, a search simulation need not consider result links beyond these.\nProviders of Internet Health Content\nGiven the patterns of adolescent searching behavior found in this study, providers of health content can do several things to increase the probability that adolescents will find their sites. Since adolescents rely primarily on the first few results from search engines and do not tend to look at ads, it is important to ensure that health sites appear near the top of the results for searches on health terms. Choices of keywords in the domain name, page title, meta tags, and the first few sentences, as well as links from other sites, can all affect placement in search results. It may also be useful to include some common misspellings in meta-tag keywords and in the body of the text in order to make a site appear in the results page of searches using those misspellings of related search terms. Because most major English-language search engines no longer use the keyword feature of meta tags, site designers are left with the difficult task of working misspelled words (eg, misspelt) into the text without coming across as poor spellers themselves. It is also important that the site descriptions displayed in search engines be attractive to adolescent searchers: while our study did not analyze the various reasons that adolescents chose to follow one link over another, we did observe that they made choices based upon the link descriptions and did not simply select the first link offered. Books and articles, software, and consulting services are all widely available to improve search engine placement and to influence the short summary text that search engines extract for display in search results [28,29]. Organizations that invest large amounts of money in developing sophisticated health-information sites would do well to spend a little bit more to ensure these sites are easily found.\nAnother area that Internet content providers should focus on is within-site navigation. Because students tend to skip around from place to place within a page and read little in sequence, it is important that sites with a significant adolescent audience are well organized, concise, and understandable. Long paragraphs, too many links, and difficult vocabulary all decrease the likelihood of adolescents finding health information they are seeking, even if it is contained within a site. Internet content producers should attempt to understand the needs of the site visitors and build hierarchal structures that reflect those needs. For example, if one of the primary needs of individuals visiting the Alcoholics Anonymous site is to find a local meeting, the first page of the site should include an obvious link (eg, \"Find an AA Meeting Near You\") that leads to another page that returns the nearest meetings after entering in a zip code or city name. While ease of within-site navigation is important for all visitors to health information sites, some information providers may want to develop sites targeted specifically to adolescents. While they might like the targeted information once they found it, we observed that adolescents tend to rely on general-purpose search engines. Thus, developing special youth-targeted versions of information sites may be of somewhat limited utility, unless also accompanied by advertising or education campaigns that make adolescents more likely to find such sites.\nRather than changing Web sites or their presentation in search engines, it may also be useful to undertake education campaigns to improve the search strategies and tactics that adolescents use when seeking health information. It may be helpful to guide them towards youth-oriented directories or search engines, rather than general-purpose search engines. For example, both Yahoo! and Google offer directories with subcategories of sites designed for teens that cover various health topics. This approach may be facilitated by including links to such resources on the Web browser's starting page in schools and libraries. Alternatively, adolescents might be taught techniques for formulating and refining search terms at general-purpose search engines, adding or dropping more-specific words based on the kinds of results returned. They might also be taught to notice potential search term misspellings based on surprising search results. Finally, adolescents might also be taught techniques for systematically exploring within a Web site to find the kind of information they are looking for.\nLimitations and Future Research\nThere are several important limitations to the interpretation of these results.\nFirst, this was not a representative or random sample of adolescents. It was a small convenience sample with a selection bias toward adolescents with strong Internet searching skills. While the results cannot be generalized to all adolescents and do not capture the full range of adolescent searching experience, we can assume that the average adolescent would have had even more trouble than our study participants in finding health information on the Internet.\nSecond, the health-related search questions were deliberately constructed to avoid controversial topics such as safe sex, abortion, and homosexuality. Given that adolescents are often faced with health problems related to sexuality, their actual search behavior and success at finding health information related to sexuality may not be reflected in our results. Another concern is that participants may have changed their search behavior because of the presence of observers and because they were aware that their search behaviors were being recorded. For example, students who had trouble finding an answer may have persisted in their search longer than they would have in a nonresearch setting. Alternatively, because students knew they had several search questions to answer during a single class period, they may not have been as persistent as they might have been with a more personally-relevant question and less-restricted search time. Thus, the data here reflect a rough estimate of persistence for an adolescent looking for health-related information. Also, searching was conducted individually, while in practice many searches both at home and at school are conducted with friends, teachers, or family close by. While it is difficult to know how this would affect searching behavior without future research, it is possible that students would act differently (eg, receive help with spelling).\nFinally, while components of our classification scheme for successful versus unsuccessful searching have been previously validated, the overall scheme was modified to more accurately code the search results as correct, complete, and useful. A more-systematic validation of coding schemes for health information search results is an important area for future research.\nMore research is needed to validate the results presented in this article, as well as determine if results vary for different populations (eg, age, race, and experience with health searching) and different health questions (eg, finding a practitioner versus finding the answer to a question). Additionally, instead of focusing on how adolescents currently search for health information, future studies may also want to explore interventions aimed at improving their searches. For example, should health portal sites designed for adolescents or online directories be used? Or would the current practice of using common search engines, but with adolescents learning improved search tactics be more effective? Also, which search strategies lead to sites that are the most likely to be accurate and influence adolescents to change their behavior?\nConclusions\nThis study provides a useful snapshot of current adolescent searching patterns. The results have implications for constructing realistic simulations of search behavior, and for both information providers and educators. Analyzing search behavior through actual observation should be a cornerstone in any effort to improve adolescents' access to health information.","keyphrases":["adolescent","internet","computer literacy","access to information","medical informatics","health education","computer simulation","search process","information seeking","information storage and retrieval"],"prmu":["P","P","P","P","P","P","R","R","R","M"]}
{"id":"Cardiovasc_Intervent_Radiol-3-1-1914265","title":"Subintimal Double-Barrel Restenting of an Occluded Primary Stented Superficial Femoral Artery\n","text":"In-stent re-stenosis is a frequent complication of endovascular stents, especially in the superficial femoral artery (SFA). Endovascular re-intervention of in- or peri-stent occlusive disease consists of recanilization through the occluded stent. In our case report, we describe the endovascular treatment of a previously placed stent in the SFA. We unintentionally passed the affected stent subintimally, in a double barrel fashion next to the first stent. The procedure was without any complications and with a successfull angiographic result. At one year follow-up the patient still has no complaints and the stent is still patent.\nIn-stent restenosis is a frequent complication, especially in the superficial femoral artery (SFA). If in-stent restenosis occurs there are several therapeutic options available such as conservative therapy and endovascular or surgical intervention. Endovascular reintervention of in- or peri-stent occlusive disease is one of the less invasive options. Endovascular therapy means passage through the occluded stent (recanalization) by additional means and redilatation or restenting of the lesion. In some cases passage of the occluded stent is not possible.\nIn this case report we describe the unsuccessful attempt to recanalize an occlusion in a stent in the SFA followed by the successful bypass of the stent using a subintimal technique. The concept of utilizing this secondary stent was to create enough radial force to keep open the neo-lumen next to the occluded stent. Although the per-interventional extraluminal recanalization (PIER) technique is usually performed for longer lesions of the SFA, in this case a shorter segment was successfully managed [1, 2].\nAn internet search for articles describing equivalent cases revealed no results. No publications on subintimal passage of a reoccluded stent and double-barrel stenting were found in the literature.\nCase Report\nA 75-year-old man underwent several percutaneous transluminal angioplasty (PTA) procedures in both upper leg vessels as well as stenting of a right SFA lesion in the adductor canal after an insufficient PTA result in an other facility (Figs.\u00a01 and 2).\nFig.\u00a01.\nSee text.\nFig.\u00a02.\nSee text.\nThe patient presented in 2004 with progressive complaints of the right leg. The patient\u2019s maximum walking distance was 800 m but he smoked 5\u201310 cigarettes a day. As consistent medication the patient was on antihypertensive drugs, 40 mg simvastatin (10 mg Zocor, MSD) as well as 100 mg carbasalaatcalcium (Ascal Cardio, Viatris). There was no cardiopulmonary comorbidity.\nRight pedal pulses were absent with a normal pulse at the right groin. Blood pressure was 160\/95 mmHg. The maximum walking distance was 160 m with a drop in the ankle\/brachial index (ABI) after excercise of 0.62 to 0.23 on the right side and 0.98 to 0.55 on the left side. In conclusion, this patient had peripheral arterial occlusive disease (PaOD) staged as Fontaine class 2a.\nA color Doppler ultrasound (CCDS) examination of the right femoropopliteal arteries revealed in- and peri-stent restenosis with a peak systolic volume (PSV) ratio of 5.7 and distal in-stent occlusion noted over 4 cm. At that time the patient could not be persuaded to undergo any intervention and was managed conservatively.\nIn March 2005 the patient presented again with unchanged symptoms. On physical examination the walking distance was now 240 m with an ABI drop after excercise of 0.44 to 0.22 on the right side and 0.91 to 0.52 on the left side. The patient and his attending physician discussed endovascular treatment such as recanalization of the affected vessel with PTA and\/or additional stenting as a proper next option for his PaOD Fontaine class 2a lifestyle-limiting symptoms.\nIn April 2005, an endovascular percutaneous intervention of the right SFA was performed at the Department of Radiology in our hospital. Because the patient was overweight an antegrade puncture was not a good option and the right SFA was approached by a crossover technique from the contralateral side. The right leg vessels were visualized angiographically demonstrating a stenosis of approximately 70% at the origin on the right SFA. More distally in the SFA an occlusion was noticed reaching from 4 cm proximal of the occluded stent to the upper border of the patella (The total length of this lesion was approximately 11 cm.) The 5 Fr Brite Tip sheath (Cordis, New York, USA) in the right femoral artery was changed for a long 6 Fr Balkin Up & Over flexorsheath (Cook, Bloomington, IN, USA). The sheath tip was placed in the left external iliac artery, just above the hip joint. First a PTA was performed at the origin of the SFA with a 6.0\/40.0 mm balloon (Pheron, Biotronik, Switzerland) after an intravenous (IV) injection of 2,500 IU heparin, with a satisfactory angiographic result. However, it proved impossible, after several catheter-guidewire manipulation efforts, to recanalize the occluded stent with a 0.035-inch J-shaped Terumo (Terumo, USA) guidewire. Next the occluded stent was bypassed subintimally PIER-wise and the native lumen successfully re-entered just distal to the occlusion. The neo-lumen thus created was predilated with an unsatisfactory result due to recoil and consequently stented with a self-expandable 6.0\/60.0 mm and a 6.0\/80.0 mm stent (Absolute, Guidant, Indianapolis, IN, USA). Post-dilatation was performed with a 6.0 mm Pheron balloon. Angiographic verification of the treated arterial segment revealed an acceptable angiographic result and good runoff to patent vessels below the knee. The 2,500 IU heparin was antagonized by an IV injection of 2,500 IU protamine. The angiographic sheath was withdrawn and the puncture site managed by manual compression for 10 min without any problems. The procedure was carried out without any minor or major events or complications.\nOne day after the procedure the patient underwent CCDS proving patency of the treated proximal SFA and newly inserted stents with no residual stenotic segments. The ABI on the right side was 1.0 and on the left side was 1.1. The patient reported a slightly painful sensation on the upper-medial side of his lower (treated) limb.\nOn his planned check-up at the department of surgery in May 2005 the patient reported himself very satisfied with the result. The pain sensations had disappeared and there were no more limitations to his walking distance.In August 2005 his ABI on the right side was 0.85 with a drop, after exercise, to 0.25. On the left side his ABI was 0.88 with a drop, after exercise, to 0.62. On color Doppler ultrasound investigation a patent secondary stent segment and no new stenotic lesions were observed and a normal flow pattern was described.\nDiscussion\nPeri- and in-stent restenosis is a common and well-known complication in the SFA region. There are several therapeutic options to solve this problem including endovascular treatment and bypass surgery. Because of our patient\u2019s physical condition and his lifestyle-hampering symptomatology we chose was endovascular treatment in an attempt to recanalize the affected stent.\nDuring the procedure it proved impossible after several attempts to recanalize the occluded stent in the SFA, probably because of already organized and fibrosed thrombus material in the stent. The crossover was not the cause of this failure, since the pushability and steerability of the guidewire-catheter assembly was sufficient. No angioplasty could be performed in the occluded stent. After several attempts we finally managed to pass the occluded stent subintimally, successfully re-entering the native lumen distally and creating a neo-lumen side-by-side with the primary occluded stent. The neo-lumen was stented to create enough radial force to prevent its elastic recoil. Two self-expandable stents were positioned in a double-barrel fashion alongside the previously occluded stent with proper clinical and angiographic result.\nA retrograde puncture was preferred because of the lower complication rate compared with an antegrade approach in this obese male. Moreover a crossover introducer sheath assembly creates more possibilities for easier catheter-guidewire manipulations.\nConclusion\nIf a patient presents with claudication symptoms caused by in-stent restenosis or occlusion, one can try to recanalize the occluded stent. This is often not possible, especially if the occlusion is of an older date and consists of fibrosed tissue, mainly because of reorganizing thrombus. Mechanical debridement is an option, but this is more successful in (sub)acute (re)stenosis [3]. Subintimal passage of a occluded stent using the PIER technique could be an option in such cases. The PIER technique is intended as a primary therapy, usually for longer lesions of the SFA and for patients with severe (critical) limb ischemia, and has a 1-year patency rate of 22% and a success rate of 62% [1, 2]. As shown in our case, PIER appears to be good not only for recanalizing occlusions but also for providing options for the treatment of nonpenetrable in-stent occlusions by trespassing PIER-wise and subintimal stenting of the neo-lumen.","keyphrases":["superficial femoral artery","endovascular stenting","pier","interventional radiology","reocclusion"],"prmu":["P","P","P","R","U"]}
{"id":"Cancer_Immunol_Immunother-4-1-2253649","title":"Clinical impact of HLA class I expression in rectal cancer\n","text":"Purpose To determine the clinical impact of human leukocyte antigen (HLA) class I expression in irradiated and non-irradiated rectal carcinomas.\nIntroduction\nThe immune system is thought to have an important function in controlling tumor growth and eliminating metastasizing tumor cells. The expression of human leukocyte antigen (HLA) class I, presenting tumor-associated antigens on the tumor cell surface, is considered as a prerequisite for an effective T cell immune response [34]. As a consequence, tumor cells with down-regulated HLA class I expression might escape this immune response, resulting in a selective outgrowth of these tumor cells.\nMany studies described HLA class I expression in cancer [10, 17, 22, 33]. Only limited studies have reported on the clinical impact of HLA class I expression in colorectal cancer with contrasting results. Some studies found no significant correlation between staining intensity and survival [1, 19, 20], while others found a prognostic correlation between HLA expression and survival [18, 35]. The latter two studies had in common that total absence of HLA class I resulted in a favorable prognosis as compared to patients with down-regulated expression of HLA class I of tumor cells. The discrepancy between these two studies is, whereas the one described high expression of HLA class I in tumor cells that resulted in a better prognosis as compared to the partial down-regulation of HLA class I [35], the other found the opposite [18]. These studies both analyzed a mixed population of colon and rectal cancer patients. For rectal cancer patients, the clinical impact of HLA class I expression is still unknown. Since HLA class I expression is often absent in microsatellite instable (MSI) tumors [9, 16] and MSI is more frequently observed in right-sided colon tumors than in rectal tumors [30], therefore the results obtained from a mixed population of colon and rectal cancer patients might not hold true for rectal cancer patients.\nThe purpose of this study was to analyze the clinical relevance of HLA class I expression for rectal cancer patients. In addition to determining the impact of MSI on HLA class I expression, the tumors most at risk for MSI i.e. HLA-negative tumors were examined for MSI by determining the expression of the mismatch repair proteins, mismatch mutL homolog 1 (MLH1) and postmeiotic segregation increased 2 (PMS2), that are most absent in sporadic MSI tumors [7, 36]. Radiotherapy has been described recently to increase cell surface expression of major histocompatibility complex (MHC) class I molecules in a murine colon adenocarcinoma cell line [26]. Therefore, our study also evaluated the effect of radiotherapy on HLA class I expression in rectal cancer patients. For these purposes, HLA class I expression was evaluated in a set of 1,135 formalin-fixed paraffin-embedded rectal cancer specimens. The tumors studied were obtained at the time of surgery from patients of a prospective multicenter trial, who were randomized between standardized preoperative radiotherapy treatment followed by surgery or surgery alone [15].\nMaterials and methods\nStudy population\nPatients were obtained from the Dutch TME trial, a multicenter trial that evaluated total mesorectal excision (TME) surgery with or without preoperative radiotherapy (5\u00a0\u00d7\u00a05 Gray) [15]. Radiotherapeutical, surgical and pathological procedures were standardized and quality-controlled [15, 21]. Tumor staging was determined using the tumor node metastasis (TNM) classification [29]. Patients with the hereditary Lynch syndrome also known as hereditary non-polyposis colorectal cancer (HNPCC) were excluded from the TME trial. Sufficient formalin-fixed paraffin-embedded tumor material was available for 1,135 Dutch patients. Three 2\u00a0mm cores of each tumor sample were arrayed into tissue microarrays (TMA) as previously described [5].\nImmunohistochemistry and microscopic analysis\nTMAs [5] were immunohistochemically stained for HLA class I using the mAb antibodies HCA2 and HC10 and the rabbit anti-\u03b22\u00a0m polyclonal Ab (A 072; DAKO Cytomation, Glostrup, Denmark). The HCA2 and HC10 antibodies were applied in immunohistochemistry as hybridoma culture supernatant, kindly provided by Prof. J.J. Neefjes from the Netherlands Cancer Institute (Amsterdam, The Netherlands). The reactivity spectrum of HCA2 includes HLA-A (except HLA-A24), HLA-B73 and HLA-C molecules as well as HLA-E, HLA-F and HLA-G antigens [27, 28, 31]. HC10 reacts with HLA-B and HLA-C molecules and HLA-A10, -A28, -A29, -A30, -A31, -A32 and -A33 heavy chains [13, 23, 31, 32]. The immunohistochemical procedures are described in detail elsewhere [18]. All tumor specimens were stained simultaneously to avoid intra-assay variation. Microscopic analysis was assessed by two independent observers (M.M. v. B. and M. v. V.) in a blinded manner. HCA2, HC10 and \u03b22\u00a0m stainings were scored in six categories. Essentially, the scoring was divided into quartiles but for tumors with less than 25% stained cells, there was a distinction made between those with 6\u201325% positively stained tumor cells, those with approximately 1\u20135% positively stained cells and those with absolute no positively stained tumor cells [3, 11]. Where discrepancies arose between the staining of cores from the same tumor, an average of the scores was taken, with confirmation by two observers using a double-headed microscope with a consensus decision taken in all cases. Tissue stromal cells, normal epithelium or lymph follicles served as positive internal controls to ascertain the quality of the staining. Patients were excluded if stromal cells of tumor were not stained for HCA2 or HC10. Twenty-five tumors with negative staining of the stromal cells for HCA2 were excluded. HC10 showed in all tumors staining of the stromal cells. Also TNM stage 0 patients, tumors lost due to technical failure and ineligible patients were excluded, leaving 1,092 tumors in which HC10, and 1,035 in which HCA2 could be evaluated. Combining the results for HCA2 and HC10 staining resulted in 1,008 eligible stage I\u2013IV rectal cancer patients for analyses of clinical impact of HLA class I expression.\nTumors negatively stained for HCA2 and\/or HC10 were stained for mismatch repair proteins MLH1 and PMS2. MLH1 and PMS2 are deficient in sporadic MSI tumors. Therefore, the expression of these proteins was used to differentiate MSI and MSS rectal cancers. Tissue stromal cells, normal epithelium or lymph follicles served as positive internal controls when analyzing MLH1, PMS2 expression. The expression of MLH1 and PMS2 was scored positive if tumor cells showed expression, and negative if tumor cells showed no expression of either MLH1 or PMS2, provided that and tissue stromal cells did show expression, indicating microsatellite stable (MSS) and microsatellite instable (MSI) tumors, respectively [7].\nStatistical analyses\nAll analyses were performed with SPSS statistical software (version 12.0 for Windows, SPSS Inc, Chicago, USA). Mann\u2013Whitney U, t test and \u03c72-tests were used to compare variables. Kaplan\u2013Meier analyses were performed to analyze patient survival. The entry date for the survival analyses was the time of surgery of the primary tumor. Events for time to local recurrence, distant recurrence, cancer-specific survival, disease-free and overall survival were defined as follows: from time of surgery to time of local disease relapse (for local recurrence), time of distant disease relapse (for distant recurrence), time of disease relapse or death by disease (for cancer specific survival), time of disease relapse or death (for disease free survival) and time of death, respectively (for overall survival). Non-irradiated and irradiated patients were first separately analyzed in univariate analysis and second, variables with a P value of <0.10 in the univariate analyses were subjected to a multivariate analysis. Multivariate analysis was performed on the whole group of irradiated and non-irradiated patients with the following variables: HLA class I, randomization for radiotherapy, TNM and circumferential margin. Cox\u2019 regression analyses were used to calculate hazard ratios (HR) with 95% confidence intervals (CI).\nResults\nScoring methods\nSeveral methods are described to analyze HLA class I expression in cancer. The standard is defined by the International HLA and Immunogenetics Workshop (IHIW) [3, 11]. A recent paper describing HLA class I expression in colorectal cancer used an adjusted form of this scoring method [35]. Our scoring was primarily adapted from IWIH, i.e. division into quartiles, but for tumors with less than 25% stained cells a distinction was made between those with 6\u201325% positive tumor cells, those with approximately 1\u20135% positive tumor cells and those with absolute no HLA class I positive-stained tumor cells. After scoring and analyzing this method we found that patients in the groups with absolute numbers, 1\u20135%, 6\u201325% and 26\u201350% HLA class I expression of tumor cells did not differ in prognosis but had a worse prognosis as compared to patients with HLA class I expression in groups with 50\u201375% and >75% of tumor cells expressing HLA class I. Therefore, we distinguished two categories. These two categories were (1) 0\u201350%; and (2) >50\u2013100% of tumor cells expressing HLA class I.\nHCA-2 and HC10 staining in rectal cancer\nImmunohistochemical staining with HCA2 and HC10 antibodies demonstrated strong positive membrane staining of stromal cells and tumor-infiltrating inflammatory cells, indicating the success of the staining. A total of 1,035 and 1,092 tumors were evaluated with HCA2 and HC10; 324 (65%) irradiated tumors and 312 (58%) non-irradiated tumors showed at least 50% of all tumor cells positive for HCA2. Staining with HC10 resulted in 403 (76%) irradiated tumors and 436 (77%) non-irradiated tumors that showed more than 50% positive tumor cells. The complete results are shown in Table\u00a01. Representative examples of the immunohistochemical stainings of tumors are displayed in Fig.\u00a01a\u2013f. These results show that about 35% of irradiated and 42% of non-irradiated patients showed less than 50% of the tumor cells expressing HCA2. HC10 is expressed in less than 50% of the tumor cells in about 25% of both irradiated and non-irradiated rectal cancer patients.\nTable\u00a01Most rectal tumors have high numbers of tumor cells positive for HCA2 or HC10Irradiated patients N (%)Non-irradiated patients N (%)HCA2\u00a0High324 (65%)312 (58%)\u00a0Low142 (28%)174 (32%)\u00a0Absence31 (6.2%)52 (9.7%)HC10\u00a0High403 (76%)436 (77%)\u00a0Low117 (22%)116 (21%)\u00a0Absence8 (1.5%)12 (2.1%)Numbers (N) of patients are indicated with percentages shown in parentheses, showing: expression of HCA2 and HC10 in more than 50% of the tumor cells (high), expression in less than 50% of the tumor cells (low) and total absence (absence)Fig.\u00a01Examples of HCA2 and HC10 immunohistochemical staining of rectal tumors; a\u2013c HCA2, d\u2013f HC10 expression. a, d Expression of HLA class I in >50% tumor cells; b, e expression of HLA class I in <50% tumor cells; c, f epithelial cells show total absence for HCA2 or HC10 and only stromal and infiltrative cells show positive staining for HCA2 or HC10; Original magnification \u00d720\nAnalysis of HLA class I expression in rectal tumors\nTogether, the results obtained with HCA2 and HC10 are expected to reflect HLA class I expression in rectal cancer. In a group of 64 tumors it was studied whether an additional staining for \u03b22\u00a0m would better define HLA class I expression. The results of the addition of \u03b22\u00a0m to HCA2 and HC10 were comparable to those obtained with HCA2 and HC10, i.e. only 1 of 64 tumors was differently classified. Therefore, \u03b22\u00a0m was not scored in the whole cohort and HLA class I expression was assessed by combining HCA2 and HC10.\nA total of 406 (85%) irradiated and 445 (84%) non-irradiated tumors exhibited expression of at least one of the two markers showing >50% positive staining of all tumor cells (further referred to as \u2018the HLA class I high expression group\u2019). A total of 70 (15%) irradiated and 87 (16%) non-irradiated tumors showed reduced numbers (\u226450%) of HLA class I positive tumor cells. Only 3 (0.6%) irradiated tumors and 8 (1.5%) non-irradiated tumors showed total loss of HLA class I (negative for both HCA2 and HC10). Survival results of patients with total absence of HLA class I on tumor cells did not show significant difference from patients with reduced numbers of HLA class I positive tumor cells. Therefore, these groups were combined and will be further referred to as \u2018the HLA class I low-expression group\u2019. The complete results are shown in Table\u00a02. The number of patients in the group of the HLA class I high-expression group and the HLA class I low-expression group was equally distributed between irradiated and non-irradiated tumors (\u03c72\u00a0=\u00a00.519, P\u00a0=\u00a00.471), indicating that irradiation had no effect on HLA class I expression in these patients.\nTable\u00a02Expression of HLA class I in rectal cancer using HCA2 and HC10 antibodiesHCA2HC10HLA class IHigh (N)Low\u00a0+\u00a0absence (N)N (%)IrradiatedHigh (N)27037406 (85%)Low\u00a0+\u00a0absence (N)997070 (15%)Non-irradiatedHigh (N)27732445 (84%)Low\u00a0+\u00a0absence (N)1368787 (16%)Expression of results of HCA2 and HC10 staining in a cross table for numbers (N) of irradiated and non-irradiated patients; expression of HCA2 and HC10 in more than 50% of the tumor cells (high) versus expression in less than 50% of the tumor cells (low) is shown. A significant correlation was noted between HCA2 and HC10 staining for both irradiated (\u03c72\u00a0=\u00a053.947, P\u00a0<\u00a00.001) and non-irradiated patients (\u03c72\u00a0=\u00a061.257, P\u00a0<\u00a00.001). The right side of the table displays HLA class I expression estimated on HCA2 and HC10 expression. A total of 406 (85%) irradiated and 445 (84%) non-irradiated tumors exhibited expression of at least one of the two markers showing >50% positive staining of all tumor cells. A total of 70 (15%) irradiated and 88 (16%) non-irradiated tumors showed reduced numbers (\u226450%) of HLA class I positive tumor cells. The number of patients in the group of the HLA class I high expression group and the HLA class I low expression group was equally distributed between irradiated and non-irradiated tumors (\u03c72\u00a0=\u00a00.519, P\u00a0=\u00a00.471)\nHLA class I negative cells and microsatellite instability\nIt has been described that a majority of MSI colorectal tumors do not express HLA class I, while only a minority of MSS tumors do not express HLA class I [8, 16]. Therefore, HLA class I negative rectal tumors are the most at risk to be MSI tumors. To evaluate the numbers of sporadic MSI tumors in our study, HCA2 or HC10 negative tumors were analyzed for the expression of PMS2 and MLH1. PMS2 and MLH1 are mismatch repairs proteins that are most frequently absent in MSI sporadic tumors [6]. Of the HLA class I negative tumors only 1 out of 11 tumors did not express PMS2 and MLH1. In the tumor group negative for only 1 of the 2 HLA class I markers, 2 of 81 tumors displayed no PMS2 and MLH1 staining. These results indicate that HLA class I down-regulation is not associated with MSI in rectal cancer and are in accordance with the previous findings that only a very small minority of rectal tumors are MSI [4, 25].\nHLA class I expression and clinicopathological parameters\nThe relationship between HLA class I expression and patient\/tumor characteristics was assessed (Table\u00a03). The HLA class I expression levels were distributed equally in non-irradiated and irradiated patients with regard to most clinical and pathological parameters. Three significant differences were observed. For the non-irradiated patients, significantly more men appeared in the HLA class I low-expression group (P\u00a0=\u00a00.03). The group of irradiated tumors with HLA class I low expression contained significantly more stage III and IV tumors (P\u00a0=\u00a00.01) and also more patients with a tumor-positive circumferential resection margin (P\u00a0=\u00a00.02) when compared with tumors with high HLA class I expression.\nTable\u00a03Clinicopathological characteristics of irradiated and non-irradiated patients with high or low numbers of HLA class I positive tumor cells Non-irradiated patientsIrradiated patientsHigh N\u00a0=\u00a0445Low N\u00a0=\u00a087P valueHigh N\u00a0=\u00a0406Low N\u00a0=\u00a070P valueGender\u00a0Male (%)63750.0365660.90Age\u00a0Median years65680.3265650.99TNM stage (%)\u00a0I31240.5233240.01\u00a0II27303021\u00a0III36383240\u00a0IV58514Circumferential margin\u00a0Negative (%)83770.2886740.02Distant from anal verge (%)\u00a0\u226510\u00a0cm28330.1727320.30\u00a05\u201310\u00a0cm41314636\u00a0< 5\u00a0cm31362732Operation type (%)\u00a0Low anterior resection66610.7765660.89\u00a0Abdomino-perineal resection29332930\u00a0Hartmann5664Number (N) of patients with expression of total HLA class I expression in more than 50% of the tumor cells (high) and expression in less than 50% of the tumor cells (low)*\u00a0Statistical significant P values are in bold\nExpression of HLA class I and clinical prognosis\nBecause radiotherapy might influence local tumor recurrences [15], irradiated and non-irradiated tumors were analyzed separately in order to evaluate the impact of HLA class I expression on tumor recurrence and patient survival. The HLA class I expression was not related with distant or local recurrence rates. The patients with low expression of HLA class I had a worse overall survival and disease-free survival when compared to patients with HLA class I high expression, irrespective of treatment (Fig.\u00a02; overall survival: P\u00a0=\u00a00.008 and P\u00a0=\u00a00.01; disease free survival: P\u00a0=\u00a00.01, P\u00a0=\u00a00.006 in irradiated and non-irradiated patients, respectively). Irradiated patients with low HLA class I expression also had a worse cancer-specific survival (P\u00a0=\u00a00.003). For non-irradiated patients, HLA class I expression had no significant effect on cancer-specific survival (Fig.\u00a02). All results of univariate analysis are shown in Table\u00a04. Univariate analysis showed a better outcome for overall survival and disease-free survival in patients with high HLA class I expression.\nFig.\u00a02Examples of Kaplan\u2013Meier curves showing overall survival and cancer specific survival for irradiated and non-irradiated patients (a\u2013d). Kaplan\u2013Meier curves for overall survival (a, b) and cancer specific survival (c, d); curves show prognosis for non-irradiated (a, c) and irradiated patients (b, d) for HLA class I expression in more than 50% of the tumor cells (high) versus expression in less than 50% of the tumor cells (low). P value is based on univariate log rank analysesTable\u00a04Both irradiated and non-irradiated patients with high expression of HLA class I have a better overall, and disease free survivalNon-irradiated patientsIrradiated patientsHigh (%)Low (%)P valueHigh (%)Low (%)P valueOverall survival65.558.50.01267.551.30.008Disease free survival62.253.50.01562.248.30.006Cancer specific survival74.371.40.4180.161.80.003Local recurrence8.913.70.224.73.20.72Distant recurrence26.728.70.8824.729.30.34Survival and recurrence rates indicated in percentages after 5-years of follow-up for non-irradiated and irradiated patients for HLA class I expression in more than 50% of the tumor cells (high) versus expression in less than 50% of the tumor cells (low). P value is based on univariate log rank analyses for overall survival, disease free survival, cancer specific survival, local recurrence and distant recurrence*\u00a0Statistical significant P values are in bold\nMultivariate analysis\nMultivariate analysis was performed to identify factors with independent prognostic significance and to calculate hazard ratios (HR). Analyses included TNM, circumferential margin, randomization for preoperative radiotherapy and HLA class I expression (low vs. high HLA class I positive tumor cells) (Table\u00a05). Advanced pathological (TNM) stage and tumor-positive circumferential resection margins retained their strength as independent prognostic factors in these survival analyses. HLA class I expression showed independent prognostic value for overall survival and disease-free survival (HR: 1.3, P\u00a0=\u00a00.042 and HR: 1.4, P\u00a0=\u00a00.006, respectively), but not for cancer-specific survival.\nTable\u00a05Multivariate analysis confirms independent better overall, and disease free survival for rectal cancer patients with high expression of HLA class IOverall survivalDisease free survivalCancer specific survivalHR (95% CI) P valueHR (95% CI) P valueHR (95% CI) P valueHLA\u00a0High10.04210.00610.653\u00a0Low1.3 (1.0\u20131.6)1.4 (1.1\u20131.8)1.1 (0.8\u20131.5)Randomization\u00a0TME10.63210.21410.282\u00a0TME\u00a0+\u00a0RT1 (0.8\u20131.2)0.9 (0.7\u20131.1)1.1 (0.9\u20131.5)TNM\u00a0I111\u00a0II2.2 (1.7\u20133.0)<0.0012.1 (1.6\u20132.8)<0.0013.5 (2.0\u20136.1)<0.001\u00a0III3.1 (2.4\u20134.1)<0.0013.1 (2.3\u20134.0)<0.0019.0 (5.4\u201314.9)<0.001\u00a0IV11.8 (8.1\u201317.1)<0.001\u2013\u201350.3 (28.5\u201389.1)<0.001CRM\u00a0Negative1<0.0011<0.0011<0.001\u00a0Positive1.3 (1.1\u20131.5)1.8 (1.4\u20132.2)1.3 (1.1\u20131.5)Multivariate analysis for cancer specific, overall and disease free survival was performed to identify factors with independent prognostic significance and to calculate hazard ratios (HR) with 95% confidence intervals (CI) shown in parentheses. HLA class I expression in more than 50% of the tumor cells (high) versus expression in less than 50% of the tumor cells (low), total mesorectal excision (TME), Radiotherapy (RT); circumferential margin (CRM); P value is based on Cox\u2019 regression analyses*\u00a0Statistical significant P values and HR are in bold\nDiscussion\nWe showed that rectal cancer patients from the HLA class I low expression group had an independent worse overall and disease-free survival when compared to patients from the HLA class I high-expression group. These data imply that the expression of HLA class I in tumor cells predicts survival for rectal cancer patients. Although significant better cancer-specific survival for irradiated patients with high HLA class I was found in univariate analysis, the predictive value was lost in multivariate analysis. This observation can be explained by the fact that the group with low HLA class I significantly included more stage III\/IV and more patients with a positive circumferential margin as compared to the group of patients with high expression of HLA class I. Also no predictive value of HLA class I expression was found with regard to recurrence-free survival of these patients. Therefore, we have no indications that support the notion that better survival of high HLA class I expression is due to the better antigen presenting function of these tumor cells, as has been suggested [18, 35].\nIn our study, no difference was found between irradiated and non-irradiated patients for HLA class I expression in tumor cells. It has been described that \u03b3-irradiation induces enhanced peptide production and surface expression of MHC class I in a colorectal mouse tumor cell line [26]. The fact that we could not find more HLA class I expression in irradiated tumors than in non-irradiated tumors indicates that radiotherapy does not induce HLA class I expression in vivo. Immunohistochemistry, however, is less suitable to measure subtle expression changes. Therefore, additional research is required to determine the impact of radiotherapy on expression levels of HLA class I in human tumors.\nIn our study, more tumors showed HLA class I down-regulation after immunohistochemical staining using HCA2 than using HC10. This difference might be due to differences in reactivity spectrum of both antibodies (see Materials and methods) or to the fact that HLA alleles are differently affected in colorectal cancer. If the latter is the case, our results suggest that HLA A alleles preferentially show down-regulation in rectal cancer.\nPrevious reports evaluated HLA class I expression in mixed patient populations of colon and rectal cancer patients [18, 35]. Watson et al. also found in a large group of colorectal cancer patients that patients with low expression of HLA class I had a poor prognosis [35]. However, in contrast to our results, both studies described a substantial population of patients with tumors showing absence of HLA class I. In addition, they described that absence of HLA class I was associated with better prognosis as compared to tumors expressing reduced numbers of HLA class I positive tumor cells. A relatively low number (1.1%) of HLA class I negative tumors was observed in our cohort of rectal cancer patients only. These patients showed no survival advantage when compared to patients with reduced numbers of HLA class I positive tumor cells. There are several explanations for the discrepancy in the number of HLA class I negative tumors between the study of Watson et al. and ours, like different definition of HLA class I expression, differences in staining techniques, different patient cohort and number of MSI tumors.\nWe showed that tumors that do not stain HC10 can stain positive for HCA2 and thus are still able to present antigens. Therefore, an explanation for the differences with the results of Watson et al. is that we used strict criteria to classify tumors as HLA class I absent (defined as both HCA2 and HC10 negative) as compared to Watson et al. (defined as negative for HC10 or negative for \u03b22\u00a0m instead of negative for both). Another important explanation is that we examined HLA class I expression in a relative more homogeneous population of patients with a rectal tumor, while the other cohorts are more heterogeneous, consisting of both colon and rectal cancer. Although combining results from colon and rectum is generally accepted when predicting prognosis, this might influence results [14].\nIn colon cancer patients, approximately 50% of all proximal colon tumors show MSI, whereas almost all distal colon and rectal cancers are MSS tumors [24, 30]. Loss of HLA class I has been described in at least 60% of all sporadic right-sided MSI colorectal tumors but in only 17% of MSS right-sided colon tumors loss of HLA class I is found [8, 16]. In our cohort, only 1 out of 11 HLA negative tumors and 2 out of 81 tumors negative for HCA2 or HC10 did not express MLH1 and PMS2 and were thus likely MSI tumors. This indicates that rectal cancers are mainly MSS tumors, as has previously been described [4, 14, 25]. Of the multiple mechanisms that have been shown to underlie defects in HLA class I expression in colorectal cancer (mutations in the individual HLA class I genes, mutations in \u03b22\u00a0m [16], and defects in components of the HLA class I-associated antigen processing machinery (APM) [2, 16]), only the first will result in allele-specific aberrancies while the other affect total HLA class I expression and may result in total absence in a tumor cell.\nThese observations imply that a population of colorectal tumors with total absence of HLA class I probably contains a disproportionate large number of MSI tumors when compared to colorectal tumors expressing HLA class I. In addition, MSI colorectal tumors have a better prognosis when compared to MSS colorectal tumors [12, 24]. Therefore, HLA class I negative tumors are more likely to be MSI tumors with a different clinical behavior as compared to MSS colorectal tumors. It is likely that MSI influences prognostic results when considering HLA class I expression in colorectal tumors.\nOur results show that HLA class I expression in rectal cancer affects the patient\u2019s prognosis. We hypothesize that both oncogenic pathway and HLA class I expression dictate clinical tumor progression. We suggest that in future prognostic studies, analyzing expression of HLA class I or other biomarkers in colorectal cancer, the impact of MSI should be considered.","keyphrases":["hla class i","rectal cancer","prognosis","immunohistochemistry","immunology"],"prmu":["P","P","P","P","U"]}
{"id":"Invest_New_Drugs-3-1-1915607","title":"Human mass balance study of the novel anticancer agent ixabepilone using accelerator mass spectrometry\n","text":"Summary\nIntroduction\nThe taxanes paclitaxel and docetaxel are important anti-cancer drugs [1]. However, their use is complicated by low oral bioavailability and the development of resistance due to MDR1 over-expression or \u03b2-tubulin mutation [2, 3]. A search for microtubule stabilizing agents with improved characteristics compared to taxanes led to the discovery of epothilone A and B in extracts of the myxobacterium Sorangium cellulosum [3], which were subsequently shown to bind to tubulin at the same binding site as paclitaxel [4]. In vitro, epothilone B was more active than in vivo (mouse models) [5], and in vivo hydrolysis of the lactone ring was suspected of producing inactive metabolites. To improve the metabolic stability of the naturally occurring epothilones, these were modified by replacing the ester bond with an amide bond [6,7]. In general, lactam analogues displayed tubulin-polymerizing and cytotoxic potencies inferior to their naturally occurring counterparts. The exception to this rule was the lactam analogue of epothilone B, ixabepilone (BMS-247550, NSC 71028, EpoB-lactam, aza-EpoB, 15-desoxy-15-aza-epothilone B), see Fig.\u00a01 for structure [6]. Ixabepilone has undergone a number of phase II clinical trials with varying schedules at doses from 6 to 40\u00a0mg\/m2 administered as an intravenous infusion over 1 to 3\u00a0h [8\u201311]. Response rates in metastatic breast (12\u201344%), non-small cell lung (14%), hormone-refractory metastatic prostate cancer (33%), and metastatic renal cell carcinoma (14%) were modest to promising and warrant further investigation [8]. The pharmacokinetics of ixabepilone have been described [9,12,13] and the contribution of two known chemical degradation products was shown to be negligible [14], however, other than that it is a CYP3A4\/5 substrate [15], little is known about the excretion and metabolic fate of ixabepilone.\nFig.\u00a01Chemical structures of epothilone B and its lactam analogue ixabepilone\nThe present investigation was aimed at determining the pharmacokinetics and excretory pathways of [14C]ixabepilone. To this end, we initiated a mass balance study of ixabepilone in humans. A mass balance is an elaborate pharmacokinetic investigation employing a radioactive tracer. It investigates the plasma pharmacokinetics and excretion of both the unchanged drug and the total radioactivity (drug and metabolites), and allows elucidation of the metabolic fate of a drug. The main objective is the maximum recovery of the radioactive dose in urine and faeces [16]. Due to autoradiolysis, [14C]ixabepilone at a typical specific activity of 1.5\u00a0\u03bcCi\/mg was unstable. However, [14C]ixabepilone at a specific activity of less than 10\u00a0nCi\/mg was found to be stable. Therefore a study was conducted with a low radioactive dose of 80\u00a0nCi of 14C-ixabepilone, as opposed to a more typical human radio-tracer dose of circa 100\u00a0\u03bcCi dosed in study. For detection of these very low levels of radioactivity we used an ultra-sensitive detection method, Accelerator Mass Spectrometry (AMS) [17\u201319], which measures the amount of 14C as if it were a stable isotope label instead of measuring its decay. AMS separates elemental isotopes through differences in mass, charge, and energy, resulting in a [14C]\/[12C] isotope ratio. Subtracting the natural [14C] background level and taking into account the total carbon content of the sample results in a total [14C] amount that is converted to a conventional unit for radioactivity (DPM, Bq, or Ci). Given the very low dose of radioactivity administered (80\u00a0nCi compared to 100\u00a0\u03bcCi in a typical radio-tracer study), administration would result in negligible radiation exposure.\nMaterials and methods\nStudy design\nThis was an open-label, single-dose study in which eight patients with histologically confirmed solid tumours received an intravenous fixed dose of 70\u00a0mg, 80\u00a0nCi [14C]ixabepilone over 3\u00a0h. The protocol and informed consent form were reviewed and approved by the Institutional Review Board of the Antoni van Leeuwenhoek Hospital\/The Netherlands Cancer Institute, and each patient gave written informed consent before receiving the study medication. Eligibility criteria most relevant to the mass balance study were: availability for in-house admittance during the first 8\u00a0days, adequate hepatic and renal function, a performance status (PS) of 0\u20132, and a life expectancy of at least 12\u00a0weeks.\nPatients\nEight patients (three male and five female) with a median age of 54.5\u00a0years and a WHO performance status of 0\u20132 were enrolled in the study. All patients had advanced cancer for which no standard anti-cancer therapy was available. The primary tumours were: squamous cell carcinoma, colon cancer (n\u2009=\u20092), gastric carcinoma, ovarian cancer, non-small cell lung cancer, sigmoid adenocarcinoma, and pancreas carcinoma.\nStudy medication\nIndividual vials of radiolabeled ixabepilone contained 20\u00a0mg, 22.8\u00a0nCi [14C]ixabepilone (specific activity 1.14\u00a0nCi\/mg). The [14C]ixabepilone was synthesized by Bristol-Myers Squibb by growing the myxobacterium Sorangium cellulosum in 14C containing medium. This resulted in labeling throughout the ixabepilone molecule. Radiochemical and chemical purity was confirmed at >99%. Vials were reconstituted using 10\u00a0ml Cremophor\u00aeEL:ethanol (1:1, v\/v) each. A 35\u00a0ml volume of the reconstituted [14C]ixabepilone was added to 140\u00a0ml Lactated Ringer\u2019s Infusion (LRI). After mixing, a 2\u00a0ml sample was taken for quantitation of radioactivity by liquid scintillation counting (LSC). LSC radioactivity values were recalculated to amounts of [14C]ixabepilone for use in calculating recovery from feces and urine. After the 3-h intravenous administration, infusion lines were flushed with 500\u00a0ml LRI. Gravimetric measurements of the infusion flask prior to, and after administration enabled calculation of the amount of radioactivity actually administered. To prevent Cremophor\u00aeEL-related hypersensitivity reactions, patients were pre-medicated with oral H1 and H2 blockers.\nSample collection\nBlood was collected at 0 (pre-dose), 1.5, 3 (prior to end of infusion), 3.25, 3.5, 3.75, 4, 5, 6, 8, 12, 24, 48, 72, 120, and 168\u00a0h after start of infusion. An indwelling catheter contra-lateral to the administration site was used for serial blood sampling. Separate, but simultaneous, blood samples were taken for the quantitation of total radioactivity and unchanged ixabepilone, respectively. Plasma was obtained by immediate centrifugation of the blood (10\u00a0min, 1000\u2009\u00d7\u2009g, 4\u00b0C). The plasma layer was aspirated and stored below \u221220\u00b0C until analysis. We collected complete urinary and faecal output up to 7\u00a0days after administration. Urine was collected over 24-h intervals in refrigerated collection jugs. At the end of each collection interval, the respective urine samples were mixed and total volume was recorded. Aliquots were stored below \u221220\u00b0C until analysis. Faecal samples were collected and stored at \u221220\u00b0C per portion and combined per 24-h interval. Faeces were homogenized after addition of water (2:1, w\/w). Aliquots were stored below \u221220\u00b0C until analysis.\nAMS analysis of total radioactivity (TRA) in plasma, urine and faeces\nTRA in plasma, urine and faeces was determined using AMS (Exceleron, York, UK) [20, 21]. Briefly, sample pre-treatment consisted of the conversion of the carbon within the samples to graphite via a two-step process of oxidation and reduction [22]. Oxidation (combustion) was performed by heating the dried sample with copper oxide under vacuum at 900\u00b0C for 2\u00a0h in a heat-sealed tube. Reduction (graphitisation) was performed by heating the carbon dioxide formed from the oxidised sample with zinc, titanium hydride and cobalt at 500\u00b0C for 4\u00a0h, followed by a further 6\u00a0h at 550\u00b0C. After the graphitisation process, the graphite was pressed into a cathode. In the AMS-procedure, this cathode was bombarded with high-energy caesium (Cs+) ions. The resulting negative carbon ion beam was introduced into the AMS instrument, accelerated by an electric field of 4.5 MV, and the carbon atoms were stripped of their valency electrons. This resulted in separation of the carbon isotopical elements as C4+ ions. The carbon isotopes 12C and 13C were quantitated using Faraday cups, while 14C was quantitated by a gas ionization detector. Because AMS provides an isotope ratio that is [14C]\/[12C] and not an absolute value, it is necessary to know the carbon content of the sample plus any added carrier. This was determined using a carbon, hydrogen, nitrogen analyser (Elemental Microanalysis Ltd., Okehampton, UK). The natural background [14C]\/[12C] level per matrix was subtracted to obtain the amount of ixabepilone-related 14C. The total carbon content and the specific activity of [14C]ixabepilone was used to convert the [14C]\/[12C] ratio to ixabepilone equivalents (ng\/ml sample or % of dose). Using this sophisticated technique, it was possible to quantitate <4 DPM\/ml plasma, 0.01 DPM\/ml urine and 0.1 DPM\/g faeces (1 DPM corresponds to 395\u00a0ng [14C]ixabepilone).\nAnalysis of ixabepilone in plasma and urine\nConcentrations of ixabepilone parent drug in plasma and urine were determined with a validated liquid chromatography assay equipped with tandem mass spectrometric detection (Bristol-Myers Squibb, Princeton, NJ). Briefly, after addition of internal standard (BMS-212188) to 0.2\u00a0ml of each sample, calibration standard and quality control sample, the samples were precipitated with acetone. The supernatant was further extracted with 1-chlorobutane. The organic layer was removed and evaporated to dryness. The residue was reconstituted and injected into the LC-MS\/MS system. Chromatographic separation was achieved, isocratically, on a YMC ODS-AQ column (4.6\u2009\u00d7\u200950\u00a0mm internal diameter) at a flow rate of 0.3\u00a0ml\/min with detection by electrospray tandem mass spectrometry. The mobile phase contained acetonitrile\u20140.01\u00a0M ammonium acetate (pH 5.0) (65:35, v\/v). The standard curve, which ranged from 2 to 500\u00a0ng\/ml, was constructed with a 1\/x weighted quadratic regression model. The within-run precision for ixabepilone in plasma and urine was within 15 and 9%, respectively. The between-run precision for ixabepilone in plasma and urine was within 13 and 9%, respectively. The accuracy was within 11 and 6% of the nominal values in plasma and urine, respectively.\nPharmacokinetic analyses\nThe pharmacokinetic analyses of the ixabepilone plasma concentration versus time data were performed by non-compartmental methods using Kinetica Version 4.2 (InnaPhase Corporation, Philadelphia, PA). The peak plasma concentration (Cmax) and the time to reach maximal concentration (Tmax) represent observed values. The area under the plasma concentration versus time curve (AUC) was calculated using a combination of linear and log trapezoidal summations. The AUC was extrapolated to infinity (AUCinf) by dividing the last measurable concentration by the terminal rate constant, k. The absolute value of k was used to estimate the apparent terminal half-life, t1\/2\u2009=\u2009ln 2\/ k. The total body clearance (Cl) was determined by dividing dose by AUCinf and the volume of distribution at steady-state (Vss) was calculated as dose \u00d7 area under the moment curve (AUMCinf)\/(AUCinf)2 with appropriate corrections for infusion duration. Renal clearance was calculated by dividing amount excreted by the AUC. Similarly, pseudopharmacokinetic parameters were calculated for TRA.\nOutliers were statistically assessed using Dixon\u2019s Q test.\nResults\nTo evaluate the pharmacokinetics and excretory pathways of [14C]ixabepilone, we performed a mass balance study in eight patients. Figure\u00a02 shows the mean plasma concentrations over time of both TRA and ixabepilone as determined by AMS and LC-MS\/MS, respectively.\nFig.\u00a02Mean (\u00b1SD) plasma concentration-time curves of [14C]ixabepilone derived radioactivity (filled square) and unchanged [14C]ixabepilone (open square) for eight patients\nBoth TRA and ixabepilone in plasma exhibited an initial distribution phase of approximately 10\u00a0h followed by a much slower terminal elimination phase with half-lives of 73.1 and 50.3\u00a0h, respectively. The time to maximum plasma concentration was typically around the end of infusion. Plasma ixabepilone concentrations were quantifiable through 168\u00a0h in all patients and plasma TRA concentrations were quantifiable through 168\u00a0h in all patients except in patient 1 in whom plasma TRA concentrations were only quantifiable for 48\u00a0h. Pharmacokinetic parameters are summarized in Table\u00a01. The ratio of the ixabepilone AUC to the total radioactivity AUC, or AUC-ratio, is a measure of exposure to [14C]ixabepilone relative to total [14C]ixabepilone related compounds. The AUC ratio of patient 1 was larger than unity. This was caused by the underestimated TRA AUC, which was based on measurements until only 48\u00a0h, after which plasma concentrations fell below the lower limit of quantitation. Besides the TRA AUC, the t1\/2 of patient 1 was also inaccurately estimated at 8.3\u00a0h. Therefore, patient 1 was omitted from calculation of mean plasma TRA parameters. Figure\u00a03 shows the plasma concentrations of ixabepilone relative to TRA for patients 1\u20137 and patient 8. Patient 8 experienced a blocked bile duct (reflected by a maximum plasma bilirubin concentration of 47\u00a0\u03bcmol\/l (3\u00a0\u00d7\u00a0upper limit of normal) and alkaline phosphatase of 500\u00a0U\/l (4\u00a0\u00d7\u00a0upper limit of normal) at day 7) resulting in a higher plasma TRA, Cmax and AUC. The higher TRA plasma concentrations in patient 8 resulted in an AUC ratio of plasma ixabepilone to TRA that was lower than that of the other patients. In contrast, unchanged ixabepilone plasma pharmacokinetics were not affected in this patient. \nTable\u00a01Plasma pharmacokinetic parameters of ixabepilone and total radioactivity in cancer patients after a 3\u00a0h intravenous infusion of 70\u00a0mg, 80\u00a0nCi [14C]ixabepiloneNrIxabepiloneRadioactivity (ixabepilone equiv.)AUClast ratioaCmax (ng\/ml)AUClast (\u03bcg*h\/ml)AUCinf (\u03bcg*h\/ml)t1\/2(h)Cl (l\/h)Vss (l)Cmax (ng\/ml)AUClast (\u03bcg*h\/ml)AUCinf (\u03bcg*h\/ml)t1\/2 (h)12773.063.3551.720.912753221.891.968.31.62b22872.752.9753.023.6117449711.011.848.70.2534332.832.9951.723.492968110.911.336.50.2644863.824.3267.416.2102794020.121.646.90.1952662.422.5223.527.875650320.044.3185.20.1262732.172.2936.730.591257311.916.399.80.1872012.252.4971.528.115204498.539.5961.20.2681871.932.2247.231.61635150232.534.434.40.06Mean3012.652.8950.325.3115468316.4b21.3b73.1b0.19bSD1050.600.6915.45.23083788.44b13.2b54.0b0.08baAUClast ratio is calculated by dividing AUClast ixabepilone by AUClast TRA and expresses the relative contribution of ixabepilone to TRA exposure in plasma.bPatient 1 was not included in the calculation because the radioactivity AUC and t1\/2 could not be determined accurately (see text). This also resulted in the deviating AUClast ratio (outlier).Fig.\u00a03Mean (+SD) [14C]ixabepilone to total radioactivity ratio in plasma over time for patients 1\u20137 (filled square) and patient 8 (open square)\nFigure\u00a04 displays the mean excretion of TRA in urine and faeces and the total, in time, and Fig.\u00a05 displays the mean cumulative urinary excretion of [14C]ixabepilone derived radioactivity and unchanged [14C]ixabepilone. Table\u00a02 shows individual 168\u00a0h excretions of [14C]ixabepilone and TRA in urine and faeces. The U\/F ratio is the ratio of urinary recovery relative to faecal recovery. Patient 3 displayed a rather low recovery of radioactivity from both urine (14.8%) and faeces (38.3%). Plasma pharmacokinetics were not different compared to the other patients. A low total recovery of 37.8% was also obtained for patient 4, attributable to a very low faecal recovery (12.7%), while urinary recovery was normal (25.1%). This patient only defecated at 24\u00a0h and at 168\u00a0h (a few grams), which almost certainly explains the observed low recovery. Although the plasma pharmacokinetics and urinary excretion did not deviate extremely from the other patients, patient 4 displayed a somewhat elevated plasma exposure to both radioactivity and ixabepilone, and the lowest clearance for unchanged ixabepilone. If we now look at the U\/F ratios (a measure of urinary versus faecal elimination), patient 4, who had the low fecal output, is an outlier (Q\u2009=\u20090.86, critical value p\u2009=\u20090.05: 0.57) compared to patients 1, 2, 3, 5, 6 and 7. Patient 8, who experienced the blocked bile duct stent, is also an outlier (Q\u2009=\u20090.91, critical value p\u2009=\u20090.05: 0.52) compared to the same patients. The urinary recovery of patient 8 was highest at 47.7% causing the large standard deviation of urinary recovery. However, the urinary recovery of unchanged ixabepilone of 4.0% was within the normal range. The faecal recovery of this patient was the lowest (excluding patient 4) at 15.6%. Apparently, there was no accumulation of the active drug despite a decreased biliary excretion.\nFig.\u00a04Mean urinary (open square, \u2212SD), faecal (open circle, +SD) and total (filled square, +SD) cumulative excretion (168\u00a0h) of [14C]ixabepilone derived radioactivity for eight patientsFig.\u00a05Mean (\u00b1SD) cumulative urinary excretion of [14C]ixabepilone derived radioactivity (filled square) and unchanged [14C]ixabepilone (open square) for eight patientsTable\u00a02Urinary and faecal recovery (0\u2013168\u00a0h) of ixabepilone and total radioactivity in cancer patients after a 3\u00a0h intravenous infusion of 70\u00a0mg, 80\u00a0nCi [14C]ixabepiloneNraRecovery (% of dose)Radioactivity (U\/F ratioc)Ixabepilone (Clrenal (l\/h))IxabepiloneTotal radioactivityUrineUrineFaecesTotal15.0714.5659.6974.250.241.1624.6623.2874.7898.060.311.1937.7614.7738.2953.060.391.9243.3225.0812.7137.791.970.6155.4619.6665.0284.680.301.5866.1230.7864.5995.370.481.9876.0325.0586.72111.770.291.8784.0247.6515.6263.273.051.46Mean5.3025.1052.1877.280.33b1.47SD1.3810.6327.1724.940.08b0.47aPatients 1, 2, and 8 were male.bPatient 4 was not included because of the extremely low production of faeces and patient 8 was not included in the calculation because his bile duct obstruction (relevance illustrated by increasing plasma bilirubin concentrations) may have reduced faecal excretion.cThe U\/F ratio is the ratio of urinary recovery relative to faecal recovery.\nOn average, more than 77% of the drug was excreted over the 7-day period with the majority of 52% being excreted in faeces and 25% in urine. Excretion in faeces occurred at a relatively constant rate over the entire collection period, with only 10 and 12% of dose recovered in the 0\u201324 and 24\u201348\u00a0h collection intervals, respectively. In contrast, urinary excretion displayed two distinct phases with 65% of total urinary radioactivity (corresponding to 17% of dose) and 76% of total urinary ixabepilone being excreted in the first 24\u00a0h. From 24 to 168\u00a0h, urinary excretion occurred at a relatively slow constant rate. This may reflect the biphasic plasma curve, with high concentrations in the distribution phase and lower concentrations in the terminal elimination phase. At first urinary excretion predominates and after 24\u00a0h, faecal excretion becomes the dominant route of excretion.\nDiscussion\nThe present investigation was aimed at determining the pharmacokinetics and excretory pathways of [14C]ixabepilone by performing a mass balance study.\nIn conventional human mass balance studies, approximately 100\u00a0\u03bcCi of [14C] is typically used to trace the fate of a drug [16]. Due to autoradiolysis of ixabepilone, this level of labeling was not possible. The level of radioactive labeling we used was more than 1,000-fold lower at 80 nCi. The most commonly used method for detection of radiotracers in mass balance studies is liquid scintillation counting (LSC). This method however, relies on the detection of \u03b2-emissions and therefore can only exploit a very small proportion of the isotope. 14C has a half-life of 5740\u00a0years and therefore only about 0.012% of the 14C atoms in a sample decay over the course of a year. AMS however, is a nuclear physics technique that detects the individual 14C atoms, as if they were stable isotopes, and is therefore much more sensitive than LSC [19]. Originally, AMS technology was developed in the 1970s for radiocarbon dating, but has recently found novel uses in biomedical research. It has been used to study DNA adducts of carcinogens, and it can replace decay counting as a method of analysis in biological samples [17, 23\u201325]. A first human mass balance using this ultra-sensitive technique has already been reported and is expected to be followed by many others [26].\nThe ixabepilone plasma pharmacokinetic parameters observed in the present investigation are similar to the values previously reported for a standard phase I dose escalation study in which 11 patients were administered 40\u00a0mg\/m2 of ixabepilone in an expansion of the recommended phase II dose [9]. However, in our study, the coefficient of variation for AUCinf and Cl of ixabepilone was 24 and 21% as compared to 46 and 38% in the phase I dose escalation study. The lower inter-patient variability may result from the greater rigor toward sample collection and processing that is possible in a mass balance study during which the patient is hospitalized. It should also be noted that the administration of ixabepilone as a flat 70\u00a0mg dose in the present study did not result in any increase in inter-patient variability compared with the dose, based on body surface area, which was administered during the phase I study.\nOur results show that ixabepilone is extensively metabolised. This is indicated by the plasma AUC ratio of 0.19 and confirmed by the recovery of ixabepilone relative to TRA in urine of approximately 0.21. Patient 8 experienced a blocked bile duct resulting in a higher plasma TRA, Cmax and AUC, but interestingly, unchanged ixabepilone plasma pharmacokinetics were not affected and excretion of ixabepilone metabolites was re-routed to urine.\nThe calculated half-life of TRA was longer than that of ixabepilone (t1\/2 of 73.1 and 50.3\u00a0h respectively), however, we do not conclude that TRA was eliminated more slowly than ixabepilone. Comparison of ixabepilone and TRA half-lives per patient revealed that in 5 cases (patient 2, 3, 4, 7, and 8) the TRA half-life was shorter than the ixabepilone half-life. The mean TRA half-life was disproportionally influenced by the long half-life observed in patient 5 (185\u00a0h). The very long half-life of TRA in this patient is not likely to be clinically relevant because this patient excreted 85% of the dose within 7\u00a0days. Based on the half-lives for plasma radioactivity observed in the other patients, in the once every 3\u00a0weeks administration schedule, accumulation of metabolites should not occur. The seemingly long ixabepilone half-life of over 50\u00a0h is not unusual when compared to other tubulin-interacting drugs. Half-lives of e.g. vinca-alkaloids range from 12\u201342\u00a0h for vindesine and up to a half-life of several days for vincristine.\nWith an average recovery of 77% of the drug excreted over the 7-day period, the recovery of radioactivity of this mass balance study is reasonably good [16]. The incomplete recovery is explained by the still ongoing excretion at the end of the 7\u00a0day collection period, and the long plasma half-life of TRA. Immediate assessment of the still appreciable amounts of radioactivity excreted on day 7 to possible extend the period of collecting excreta, as suggested in the literature [16], was not possible. The shipping and lag-time in quantitating radioactivity by AMS precluded this. Another contributing route of elimination not monitored in the current study is loss of radioactivity through expiration of 14C O2 [16]. At 52% of the dose versus 25%, faecal excretion is a quantitatively more important route of elimination than urinary excretion. Biliary excretion is governed by a molecular weight limit. In humans, polar compounds with a molecular weight above approximately 500\u00a0g\/mol are predominantly excreted via bile [27, 28]. Ixabepilone metabolites will probably be more polar (phase I metabolism, oxidation, hydrolysis etc.) and have higher masses (conjugates or phase II metabolites) than ixabepilone (Mwt 506.7\u00a0g\/mol, and rather hydrophobic). Whether this is the case for ixabepilone remains to be investigated by metabolic profiling of the excreta.\nGiven the normal ixabepilone plasma concentrations of patient 8 while experiencing a blocked bile duct, biliary excretion of unchanged ixabepilone into faeces may not be a very important elimination route, and the results suggest that ixabepilone was equally well metabolised but that the biliary excretion of these metabolites was hindered by the blocked bile duct stent, resulting in a shift to urinary excretion of total radioactivity.\nThe importance of metabolism in the elimination of ixabepilone has been confirmed by this study as apparent from the low contribution of unchanged ixabepilone to TRA in plasma and urine. Ixabepilone related radioactivity is predominantly excreted in the faeces. Future investigations must be aimed at elucidating the metabolic fate of ixabepilone, and determining the activity of the metabolites. Subsequent identification of drug metabolizing enzymes involved in ixabepilone metabolism may result in explaining pharmacokinetic variability of ixabepilone in individual patients.","keyphrases":["mass balance","ixabepilone","accelerator mass spectrometry","epothilone","anti-cancer agent"],"prmu":["P","P","P","P","R"]}
{"id":"Virchows_Arch-3-1-2039779","title":"Increased epithelial cell proliferation in the ileal pouch mucosa of patients with familial adenomatous polyposis\n","text":"To eliminate the risk of colorectal cancer in patients with familial adenomatous polyposis (FAP), reconstructive proctocolectomy is performed. Although most colonic mucosa is resected during the ileal pouch anal anastomosis, adenomas and carcinomas may develop in the pouch. This may be caused by altered cell kinetics due to intraluminal changes in the pouch. In 32 patients with FAP, biopsy specimens from the mucosa of the pouch and also of the afferent ileal loop were taken. Tissue sections were immunohistochemically processed with the monoclonal antibodies M30 and MIB-1 to assess apoptotic and proliferative indices, respectively. Cell proliferation was also assessed by a modified sign test. There were no significant differences in apoptotic rates between the mucosa of the pouch and the mucosa of the afferent ileal loop. However, cell proliferation was significantly higher in the mucosa of the pouch vs afferent ileal loop, both by using the quantitative (68.3% vs 61.6%, p = 0.001) and semiquantitative methods (p < 0.05). Our newly developed semiquantitative approach outperformed previously described methods. The higher cell proliferation in the pouch as compared to the afferent ileal loop may contribute to the increased risk for adenomas and carcinomas in the pouch of patients with FAP and emphasizes the need for regular endoscopic surveillance.\nIntroduction\nFamilial adenomatous polyposis (FAP) is an autosomal-dominant disease with an estimated prevalence of 1 in 5,000 to 1 in 7,500 [5]. It is characterized by the development of hundreds of adenomas in the large intestine. Without surgical intervention, virtually all patients will develop colorectal cancer at relatively young age.\nTo eliminate the risk of colorectal cancer, a restorative proctocolectomy with ileal pouch anal anastomosis (IPAA) is accepted as one of the surgical treatments of choice in these patients.\nIn the IPAA procedure, almost all colonic mucosa is resected, thus reducing cancer risk significantly. However in patients with FAP, adenomas are also present in the upper gastrointestinal tract; approximately 5% of the patients having gastric, 60 to 90% duodenal [4], 50% jejunal [14], and 9 to 25% ileal adenomas [14, 23, 34]. Occurrence of ileal adenomas is of special interest because the terminal ileum, which is used to construct the pouch, may already contain adenomas. There is accumulating evidence that adenomas develop in the pouch. The reported incidence after a follow-up of at least 5\u00a0years is 8 to 60%, increasing up to 75% in certain subgroups [14, 26, 33, 38], which is much higher as compared to the reported incidence of 9\u201325% in the preoperative ileum of patients with FAP [14, 23, 34]. Therefore, it seems that in the ileal pouch of patients with FAP, the development of adenomas is accelerated. At present, the malignant potential of the pouch adenomas is unclear. However, 13 patients with a carcinoma in the pouch have been recently described in the literature [2, 3, 7, 9, 13, 24, 25, 29, 35\u201337].\nStasis in the pouch causes a change in the luminal content, which is in close contact with the ileal mucosa. There is an increase in the concentration of short-chain fatty acids to colonic levels [10], an increase in anaerobic bacterial counts resulting in a more colonic type of flora, and an increased deconjugation and dehydroxylation of bile acids by anaerobic bacteria [20, 21]. The intestinal flora could play a role in initiation and promotion of colon cancer by activation of various classes of carcinogens, such as nitrosamines, polycyclic aromatic hydrocarbons, and glycosides [17]. A direct link between intestinal bacterial flora and tumor promotion in the APCMin\/+ mouse model for FAP has already been established [22].\nTherefore, we hypothesize that the intraluminal ecological changes in the pouch are responsible for enhanced tumor formation in the mucosa of the pouch. Alteration of cell kinetics in the mucosa, including a lower rate of apoptosis and a higher rate of cell proliferation, is associated with tumor initiation and promotion. In colorectal cancer, the balance between apoptosis, cell proliferation, and cell shedding is disturbed, and such changes may favor the development of adenomas [30]. Up to now, no data are available on changes in proliferation and apoptosis rates of the ileal mucosa of the pouch after IPAA in patients with FAP.\nThe aim of this study is to investigate changes in apoptosis and cell proliferation rates, occurring in the mucosa of the pouch of patients with FAP, in comparison with the ileum of the afferent loop. The results may contribute to a better understanding of the enhanced adenoma formation in the pouch compared to normal ileum.\nMaterials and methods\nPatients and tissues\nThe study was approved by the regional medical ethical commission, and informed consent was obtained from all patients.\nPatients with FAP and an IPAA, who were under surveillance in the Radboud University Nijmegen Medical Centre or regional affiliated hospitals, were invited to participate in this study. Thirty-two patients with FAP were included. The diagnosis FAP was based on either a clinical presentation of at least 100 colonic adenomas or a mutation in the APC gene. Data concerning the surgical procedures were obtained from medical records.\nFrom each patient, mucosal biopsy specimens of both the pouch and the afferent ileal loop were obtained during a regular surveillance endoscopy, in the period January 2002 until April 2004. Patients were fasted overnight. On the day of examination, patients were encouraged to drink liberally. No laxatives or cathartic enemas were given. To clear the pouch of fecal ruminants, two 250-ml water enemas were given before the endoscopy.\nThe endoscopy was performed with an Olympus GIF-1T140 video endoscope. From January 2002 until August 2003, a 2.8-mm diameter biopsy forceps (FB 13K-1 Olympus, Tokyo, Japan) was used, and from September 2003 until April 2004, a 3.0-mm diameter biopsy forceps (B102-C1-30.160 MedWork\/Treier Endoscopie GA, Berom\u00fcnster, Switzerland) was used. The afferent loop was introduced up to 20\u00a0cm proximal of the pouch. The mucosa was sprayed with 1% indigo carmine dye (Laboratoires SERB, Paris) at 1:1 dilution with water, where after photographs were taken to evaluate number and size of adenomas present.\nFor pathological examination, at least four biopsies were taken at random locations from the afferent ileal loop (10 to 20\u00a0cm proximal from the pouch), four biopsies from the pouch mucosa (at least 5\u00a0cm proximal from the anal verge), and four biopsies from adenomas if present. The biopsies from adenomas were used only for pathological evaluation, i.e., to exclude serious dysplasia in pouch adenomas, and were not used for research purposes. The biopsies were stretched on filter paper to maintain correct orientation of crypts, fixated in formalin, and embedded in paraffin.\nImmunohistochemistry\nCell proliferation and apoptosis were measured in the stretched formalin-fixed nonadenomatous tissue. The crypt cell proliferation activity was assessed after staining with the monoclonal antibody MIB-1 (Dako A\/S, Glostrup, Denmark), which recognizes the Ki-67 nuclear antigen of dividing cells in formalin-fixed paraffin-embedded tissue [8]. Apoptosis was assessed by staining with the monoclonal antibody M30 (Roche Diagnostics, Mannheim, Germany), which recognizes cleaved cytokeratin 18. Immunoreactivity of M30 is confined to the cytoplasm of apoptotic epithelial cells and is expressed during early apoptosis [18].\nTissue sections of 4-\u03bcm thickness were cut from paraffin blocks, mounted on electrostatic slides (Super Frost Plus, Menzel-Gl\u00e4ser, Germany), and dried overnight, followed by drying in a stove at 50\u00b0C for 15\u00a0min.\nSections were put in xylol for 10\u00a0min and taken from xylol through 100% alcohol to water. After deparaffinization, endogenous peroxidase was blocked by treatment with 3% hydrogen peroxide in phosphate-buffered saline (PBS) for 30\u00a0min. Pretreatment was performed by heating the tissue sections in citrate buffer (10\u00a0mmol\/l, pH\u00a06.0) at 180-W power in a microwave oven for 10\u00a0min. After cooling at room temperature for 1.5\u00a0h, sections were rinsed with PBS. Then, 20% normal horse serum (Vector Laboratories, Burlingame, CA, USA) was applied for 10\u00a0min. The sections were then incubated overnight at 4\u00b0C with either the mouse monoclonal antibodies MIB-1 at 1:1,000 dilution or M30 at 1:100 dilution.\nThereafter, the sections were rinsed in PBS for 10\u00a0min and incubated with biotinylated horse anti-mouse IgG (Vector) for 30\u00a0min. After rinsing for 10\u00a0min in PBS, incubation with \u2018avidin and biotinylated horseradish peroxidase macromolecular complex\u2019 (Vector) was performed for 45\u00a0min. Finally, 5\u00a0mg\/10\u00a0ml diaminobenzidine was used as chromogen, and the sections were put in 0.5% CuSO4\/0.9% NaCl for 5\u00a0min to intensify the staining. Mayer hematoxylin counterstaining was applied. Sections were dehydrated through 100% alcohol, cleared in xylol, and coverslipped with permount (Fisher Scientific, Fiar Lawn, NJ, USA).\nTissue sections of rectal carcinoma were used as positive controls.\nEvaluation of immunostaining results\nInvestigators were blinded for the origin of the tissue sections, regarding patient and biopsy location.\nFor evaluation of M30 staining, tissue sections were examined by light microscopy. M30 positivity was identified as brown cytoplasmic staining. M30-positive cells were marked by a first investigator (BvH) and reevaluated by an expert pathologist (IN). In all cases, the complete section was evaluated, and all M30-positive epithelial cells were counted. The apoptotic index was expressed as the number of M30-positive cells per tissue area in square millimeters. Tissue area was assessed by using a Zeiss KS400 computer-aided system.\nIn each MIB-1-immunostained tissue section, crypts whose entire length could be visualized were photographed under \u00d7400 magnification using a Zeiss KS400 computer-aided system. Crypts were excluded when they did not reach the muscularis mucosae or had multilayered bases.\nMIB-1 positivity was identified as brown nuclear staining. The number of MIB-1-positive epithelial cells and the total number of epithelial cells in up to five crypts per tissue section were counted from screen. The labeling index for each crypt was given by the ratio of MIB-1-positive cells and the total number of crypt epithelial cells and is expressed as percentage of total. For each patient, the labeling indices of pouch and afferent ileal loop were expressed as means of three to five counted crypts. If less than three crypts could be photographed for either pouch or ileal loop, the patient was excluded from analysis. The photographed crypts of five randomly selected patients were counted twice by one investigator (BvH) to determine intraobserver variability.\nAlthough great effort was made to obtain well-orientated mucosal crypts when using the quantitative method, however, not all biopsies reached the criteria mentioned above, and therefore, could not be examined. This problem mainly occurred in the biopsies taken from the pouch and might be due to friability of the pouch mucosa. We therefore developed a new semiquantitative scoring system. A representative part of the biopsies showing several complete crypt\/villous axes was photographed under \u00d7100 magnification. The photographs were judged pair-wise (pouch vs afferent loop) during which the investigators had to choose from four possible outcomes; one of the two locations showed most MIB-1 positivity. MIB-1 positivity did not differ or no judgement could be made. Judgement was based on relative length of the area of positive cells and the relative size of the stem cell compartment. Five investigators, two pathologists (IN, HvK), two gastroenterologists (PF, FN), and one junior investigator (BvH) independently compared the paired photographs of biopsies of pouch and afferent ileal loop of all patients. When three or more observers agreed in their judgement, this judgement was denoted as consensus judgement. If this criterion was not met, no consensus was reached. If the quality of the tissue sections was poor, no judgment was made. One investigator (BvH) judged the whole series twice for evaluation of intraobserver reliability.\nStatistical analyses\nValues for apoptosis and cell proliferation in the quantitative study were not expected to be normally distributed; therefore, they were presented as median and range. The Wilcoxon matched-pairs signed-ranks test was used to compare the paired observations in the apoptosis staining and the paired mean labeling indices in the cell proliferation study.\nConsensus judgements on the semiquantitative assessment of cell proliferation, favoring either pouch or ileal afferent loop, were compared with a Sign test. To evaluate the reliability of this semiquantitative method to assess cell proliferation, Cohen\u2019s kappa was calculated for the first and second series of judgements by the prime investigator to determine intraobserver reliability. Also, for each pair of investigators, a Cohen\u2019s kappa was calculated. The mean Cohen\u2019s kappa was taken as value for interobserver reliability.\nConsensus judgments were compared to the difference in mean labeling index between pouch and ileal afferent loop for each evaluable patient.\nA p value less than 0.05 was considered as significant (SPSS for Windows 11.0.1, 2001).\nResults\nPatient characteristics\nPatient characteristics are given in Table\u00a01. The median age of the 32 patients (19 men, 13 women) included in the study was 32 (range 16\u201372) years.\nTable\u00a01Patient characteristics\u00a0ApoptosisCell proliferationNumber of patients studied3220Male\/Female19\/1312\/8Median age in years (range)32 (16\u201372)29 (16\u201362)Median age at surgery in years (range)24 (10\u201355)20 (10\u201352)Median age pouch in months (range)96 (9\u2013216)105 (9\u2013216)IPAA: hand-sewn\/double-stapled\/ unknown9\/21\/25\/14\/1Carcinoma at surgery40Patients with adenomas at biopsy:yes\/no24\/815\/5For quantitative cell proliferation analysis, 12 patients had to be excluded from analysis (right column) due to absence of sufficient evaluable crypts (see under \u201cMaterials and methods\u201d).\nTwenty-three patients were operated in the Radboud University Nijmegen Medical Centre. The median age at the time of reconstructive colectomy was 24 (range 10\u201355) years. A mucosectomy with hand-sewn IPAA was performed in 9 patients, and a double-stapled IPAA was performed in 21 patients. For two patients, the information about the performed technical procedure could not be retrieved. At the moment of colectomy, four patients had a colorectal adenocarcinoma localized in the rectum, sigmoid, hepatic flexure, or appendix, respectively.\nAt the time of endoscopy, the median age of the pouch was 96 (range 9\u2013216) months. The medication used was loperamide by 18 patients, psyllium fibres by 3 patients, and iron, metoclopramide, colestyramine, omeprazole, sulindac, tramadol, tamoxifen, gosereline, nifedipine, metoprolol, furosemide, or losartan each by 1 patient. Thirteen patients were not on medication 3\u00a0months before endoscopy.\nOne patient used sulindac, a nonspecific cyclooxygenase inhibitor, which is thought to influence cell proliferation and especially apoptosis [15, 27]. Exclusion of this patient from analyses had no effect on the results.\nHistological examination revealed pouch adenomas in 24 patients (75%).\nApoptosis\nIn both pouch and afferent ileal loop, M30-positive cells were predominantly detected in the mucosal villi (Fig.\u00a01).\nFig.\u00a01Apoptotic epithelial cells (encircled) using M30 immunohistochemistry. Original magnification \u00d7200 (left) and \u00d7400 (right)\nIn the 32 pairs of biopsies from pouch and afferent ileal tissue investigated, the median absolute number of apoptotic cells in the pouch mucosa was two per tissue section (range 0\u20139), which was identical to the values in the afferent ileal loop (2, range 0\u201319). The median apoptotic index (expressed as number of M30-positive cells per mm2 tissue section area) did not differ between pouch (median 0.4\/mm2, range 0\u20132.9) and afferent ileal tissue (median 0.3, range 0\u20132.7; see Fig.\u00a02).\nFig.\u00a02Box-Whisker plots of apoptosis expressed as number of M30-positive epithelial cells per mm2 tissue section area of pouch and afferent ileum of 32 patients with FAP. Difference between groups was not significant. Values are given as median (fat line), lower to upper quartile (green box), and minimum and maximum values (error bars)\nCell proliferation\nQuantitative comparison\nThe intraobserver reliability for counting of the photographed crypts (Fig.\u00a03) was rs\u2009=\u20090.855, p\u2009=\u20090.002.\nFig.\u00a03Detection of proliferating crypt cells with MIB-1 immunohistochemistry. Brown stained nuclei are positive. Nonproliferative crypt stem cells are visualized at the base of the crypt. Original magnification \u00d7400\nIn 12 pouch tissue sections, less than three crypts were available for counting, and these samples were therefore excluded from analysis. For the same reason, two afferent ileal tissue sections were excluded. In the 20 pairs of pouch and afferent ileum tissue sections left for comparison, median labeling index (expressed as percentage of MIB-1-positive epithelial crypt cells) was significantly higher in the pouch as compared to the afferent ileum (median 68.3%, range 52.9\u201379.6% vs median 61.6%, range 38.0\u201373.9%; Wilcoxon signed-ranks test, p\u2009=\u20090.001; Fig.\u00a04).\nFig.\u00a04Box-Whisker plots of cell proliferation expressed as median labeling index (MIB-1-positive crypt cells per total number of crypt cells) of 3\u20135 crypts in tissue sections of pouch and afferent ileum of 20 patients with FAP. Difference between groups was significant (p\u2009=\u20090.001). Values are given as median (fat line), lower to upper quartile (red box), and minimum and maximum values (error bars)\nSemiquantitative comparison\nThe results of the semiquantitative comparison of the photographed tissue sections from the pouch and afferent ileum mucosae (Fig.\u00a05) are visualized in Fig.\u00a06. In 15 patients (47%), the proliferation was higher in the pouch; in 7 patients (22%), there was no difference; in 5 patients (16%), proliferation was higher in the afferent ileal loop; in 2 patients (6%), no consensus could be achieved; and in 3 patients (9%), no judgement was possible due to poor quality of the tissue sections. These differences were significant (p\u2009<\u20090.05).\nFig.\u00a05Photographs used for semiquantitative comparison of cell proliferation in MIB-1 immunohistochemically stained tissue sections of pouch (left) and afferent ileal mucosae (right; original magnification \u00d7100)Fig.\u00a06Histogram of semiquantitative comparison; 0 ileal mucosa shows more cell proliferation than pouch mucosa, 1 no difference in cell proliferation between pouch and ileal mucosae, 2 pouch mucosa shows more cell proliferation than ileal mucosa, 3 no judgement possible, 4 no consensus could be reached\nIntraobserver reliability was \u03ba\u2009=\u20090.87. Cohen\u2019s kappa for each pair of observers ranged from 0.26 to 0.59 with a mean Cohen\u2019s kappa of 0.38, which corresponds with \u201cfair agreement\u201d following Byrt\u2019s guidelines [6]. Thus, interobserver reliability was acceptable.\nIn cases in which a labeling index could be assigned to both pouch and afferent ileal loop, consensus judgment was available in 18 cases (Fig.\u00a07). In all cases in which the semiquantitative analyses showed more proliferation in the pouch (n\u2009=\u200911), this was confirmed by the quantitative method; the median value of the difference in proliferation between pouch and ileum was 0.107 (range 0.032\u20130.175). In the five cases where no difference was observed with the semiquantitative approach, the median value of the difference in proliferation between pouch and ileum was 0.023 (range \u22120.096 to 0.120). There were only three cases in which the semiquantitative method showed more proliferation in the afferent loop, and the median value of the difference in proliferation between pouch and ileum in these cases was 0.098 (range \u22120.021 to 0.262). The Spearman\u2019s correlation coefficient between the semiquantitative and the quantitative methods was 0.273 (p\u2009=\u20090.244).\nFig.\u00a07Semiquantitative comparison vs difference in mean labeling index of pouch minus afferent ileal mucosa; 0 ileal mucosa shows more cell proliferation compared to the pouch mucosa, 1 no difference in cell proliferation between pouch and ileal mucosae, 2 pouch mucosa shows more cell proliferation compared to the ileal mucosa\nDiscussion\nAlthough both the pouch and its afferent loop contain the same preexisting ileal mucosa, adenomas occur more frequently in the pouch of patients with FAP than in the afferent ileal loop, suggesting an accelerated adenoma formation in the pouch [14, 26, 33, 38]. To investigate the role of cell kinetics as a possible explanation for this observation, apoptosis and cell proliferation rates in the mucosa of the pouch were compared with those of the afferent ileal loop from the same patient to eliminate bias caused by interindividual differences. Cell proliferation was significantly higher in the pouch mucosa in comparison to mucosa of the afferent ileal loop. No significant difference in apoptosis was found in the mucosa of the pouch and afferent ileal loop.\nA low amount of apoptotic cells in the pouch as well as in the ileal mucosa was found. This might be caused by the APC mutation-induced apoptotic resistance [16]. Only a trend but no significant difference in apoptosis was found. Taking into account the broad range of apoptotic rates as found in our study samples, a significant difference might be found when a larger group of patients could be studied. There are no clear guidelines for estimation of ileal proliferation. For estimation of colonic proliferation, an accepted method is to count proliferating cells in five colonic crypts. Although most biopsy specimens were stretched and orientated directly after endoscopy, only a lower number of crypts could be counted completely along the longitudinal axis in most patients. For this reason, we accepted three full crypts as the minimal number to assess proliferation. Using this criterion, tissue sections from 12 patients still could not be used to determine the cell proliferation and had to be excluded from the study. Especially in the tissue sections of the pouch, this problem was evident and is possibly caused by a higher fragility of this tissue. In the remaining 20 pairs of tissue sections left for comparison, we found a significantly higher median labeling index in the pouch compared to the ileal afferent loop.\nTo overcome the problem of the relative low number of assessable crypts, another quicker but less quantitative method was applied in which five investigators compared photographs of tissue sections of pouch and ileal mucosae. In accordance with the results of the first method, we found significantly higher cell proliferation in the mucosa of the pouch compared to that of the ileal mucosa. Although no significant correlation could be found in a case-by-case comparison between both methods, in all cases in which the semiquantitative analyses showed more proliferation in the pouch, this was confirmed by the quantitative analyses of crypts. Furthermore, the inter- and intraobserver variability was good.\nIn addition, the semiquantitative method is far less time-consuming and can therefore give a relatively fast and easy impression of eventual differences in cell proliferation. Evaluation of this method in a larger study may further demonstrate its value.\nData on cell proliferation in ileal mucosa in patients with FAP are limited. Previously, de Silva et al. [11] reported a labeling index of 19.8% in afferent ileal loop mucosa of patients with FAP or ulcerative colitis (UC), far lower than the 61.6% that was found here. Their labeling index for cell proliferation in ileal pouch mucosa (33.6%) was also much lower than in the present study. Only the labeling index of 51.7% as found by them in pouches with pouchitis nears the values we obtained. However, the majority of patients included in the study of de Silva et al. were patients with UC (23 of the 26 patients), so direct comparison with our results therefore seems inappropriate. In addition, Goldberg et al. [12] reported a median labeling index of 34.9% in the ileal pouch of patients with FAP, which is much lower than the 68.3% we found. However, their labeling indices were based on a minimum of three counted crypts, and if this number could not be reached, halves of crypts were included in the analyses. Moreover, their study group consisted of only 5 patients with FAP, whereas in the present study, 3 to 5 whole crypts of 20 patients were counted.\nSeveral studies indicated that cell proliferation of normal-appearing colorectal mucosa of patients with adenomas or carcinomas was 19 to 86% higher compared to colorectal mucosa of healthy persons [1, 28, 30, 32]. These findings strongly suggest an association between the presence of adenomas or carcinomas and an increased mucosal cell proliferation. However, the difference in cell proliferation between pouch and afferent ileal mucosae of 6.7% as found in this study is less pronounced in comparison to the above referred findings. A possible explanation for this relatively small difference in cell proliferation between pouch and ileal mucosae in our study may be that intestinal epithelial cell proliferation is already very high in patients with FAP [19, 31], and these high cell proliferation rates make a further increase less pronounced.\nThe higher proliferation found in the pouch mucosa in comparison to mucosa of the afferent ileal loop can only be explained by intraluminal changes that occur after construction of the pouch. Whether changes in bacterial flora, bile acid composition, short-chain fatty acids, or other compounds are responsible for this finding remains unclear, but a better understanding of this process is necessary to find a possible treatment for this group of patients.\nIn conclusion, the increased cell proliferation in the ileal pouch mucosa compared to the mucosa of the afferent ileal loop may contribute to the enhanced risk for adenomas and carcinomas in the pouch of patients with FAP and emphasizes the need for regular endoscopical surveillance of the pouch in these patients. In addition, cell proliferation can be used as an early endpoint marker in chemopreventive studies in these patients.\nThe applied new method for semiquantitative evaluation of cell proliferation in immunohistochemically stained tissue sections seems promising, as it offers a relatively fast and easy means of assessment.","keyphrases":["cell proliferation","familial adenomatous polyposis","restorative proctocolectomy","apoptosis and carcinogenesis"],"prmu":["P","P","P","M"]}
{"id":"J_Med_Internet_Res-7-3-1550657","title":"Cancer Internet Search Activity on a Major Search Engine, United States 2001-2003\n","text":"Background To locate online health information, Internet users typically use a search engine, such as Yahoo! or Google. We studied Yahoo! search activity related to the 23 most common cancers in the United States.\nIntroduction\nHealth care providers [1-3] and their patients [4-7] regularly search for health information online. Internet users generally begin looking for health information using a search engine [8-12], an Internet tool that searches for Web pages containing terms specified by users [13]. In February 2004, Google and Yahoo! were the most popular search engines in the United States, with 38% and 32% of market share, respectively [14].\nTo date, few studies of Internet search activity related to health topics have been published. Most analyzed the proportion of health and non-health searches on various search engines and found that health searches constituted a small percentage of total searches [15-18]. This finding is not surprising given how infrequently Internet users search for health information compared with how often they look for news reports, product information, and other topics [19]. In any case, a small percentage of total Internet searches translates into thousands of health searches each day [16], and an estimated 95 million Americans have used the Internet at least once to search for health information [20].\nCancer appears to be a health topic of some interest to Internet users. Eysenbach and K\u00f6hler [16] found that searches for cancer information accounted for 5% of health-related searches on the search engine MetaCrawler. Phillipov and Phillips [18] found that \u201cskin cancer\u201d was one of only 17 health-related search terms among the most popular 300 Internet keywords identified by Wordtracker, a private research company. Bader and Theofanos [21] studied cancer searches conducted using the search engine AskJeeves during a 3-month period and found the most commonly searched cancers were digestive\/gastrointestinal\/bowel, breast, and skin. This study also compared the incidence of selected cancers with their associated search activity. While this relationship was not statistically tested, the authors observed that some rarer cancers constituted a higher proportion of total searches than their proportion of total cancer incidence. In addition, Bader and Theofanos, as well as Phillipov and Phillips, noted that media coverage appeared to prompt Internet searches for health topics, but they did not systematically investigate or test the relationship.\nThe study reported here builds on this prior work by analyzing cancer-related searches conducted in the United States from 2001 to 2003 using the search engine Yahoo! Specifically, we investigated three potential correlates of Yahoo! cancer search activity\u2014estimated cancer incidence, estimated cancer mortality, and the volume of cancer news coverage. Cancers that afflicted more individuals, claimed more lives, and generated more news coverage were expected to be associated with more Internet search activity than other cancers, given the interest generated by relevance and publicity. In addition, we assessed the periodicity of Yahoo! cancer search activity and examined sharp increases in Yahoo! search activity related to specific cancer types.\nMethods\nThis analysis included three types of 2001\u20132003 US data: Yahoo! cancer search activity, cancer burden (estimated incidence and mortality), and cancer news coverage. The study protocol was reviewed by the Institutional Review Board of the National Center for Chronic Disease Prevention and Health Promotion and was designated as \u201cresearch not involving human subjects.\u201d\nYahoo! Cancer Search Activity\nDuring 2001 (the beginning of the study period), Yahoo! was the most popular US search engine, with a 49% market share; however, in 2003 (the end of the study period), Google surpassed Yahoo! as the leading US search engine [22]. Yahoo! remains a widely used search engine; more than 45 million US Internet users conducted Yahoo! searches in February 2004 [14].\nYahoo! maintains a database of search activity called the Yahoo! Buzz Index [23]. This index provides a search activity score for individual search terms\u2014the words or characters that users type into the Yahoo! search box. Each point of a Yahoo! Buzz Index score equals 0.001% of users searching Yahoo! during the time period of interest (day, week, or month). For example, if 250 out of a total of 1 million users searched for \u201cbreast cancer\u201d on January 1, 2001, the Yahoo! \u201cbreast cancer\u201d search activity score on this day would be 25 (250\/1 million \u00d7 100000). For a search term to register a search activity score, it must generate at least 50 searches during the time period for which the score is calculated. Yahoo! search activity generated by search terms can be segmented by country, US state, or selected US cities. This study used daily US Yahoo! search activity data from January 1, 2001 (the earliest date for which Yahoo! search activity data are available) to December 31, 2003. We limited our analysis to Yahoo! searches because at the time of this study no other Internet search engine offered a dataset like the Yahoo! Buzz Index, which provides search activity scores adjusted for the size of the population searching for online information.\nYahoo! employs professional \u201csurfers\u201d or content indexers who manually classify Web pages into one of more than 2000 content categories, such as \u201cmovies,\u201d \u201cfootwear,\u201d \u201castrology,\u201d or \u201ccancer or neoplasms.\u201d The Yahoo! Buzz Index classifies search terms in the same content category as the first Web page link that a user \u201cclicks\u201d or activates after conducting a search. For instance, if a user entered the search term \u201ccolon\u201d and then clicked on a cancer website, \u201ccolon\u201d would be classified as a \u201ccancer or neoplasms\u201d search term. If the user clicked on a grammar website, however, \u201ccolon\u201d in that instance would be classified as an \u201ceducation\u201d search term. When a user does not click on a Web page link or when a user clicks on a Web page link that has not been classified, the Yahoo! Buzz Index categorizes the search term using a variety of algorithms that analyze recent content viewed by the user.\nTo identify commonly used Internet search terms related to specific cancers, we reviewed the search terms classified in the \u201ccancer or neoplasms\u201d category of the Yahoo! Buzz Index, which generated at least 50 searches in any month from January 2001 to December 2003. This search strategy identified 76 unique search terms, of which 23 were included in the analysis (Table 1). The remaining 53 terms were discarded because they did not relate to a specific cancer or included non-English words. Discarded terms included drug names or treatment modalities, such as \u201cchemotherapy\u201d (n = 19); the names of organizations or events, such as \u201cRace for the Cure\u201d (n = 16); general cancer or anatomy terms, such as \u201concology\u201d (n = 11); names of celebrities, such as \u201cGilda Radner\u201d (n = 5); and the carcinogen \u201cDES\u201d (n = 1). Also, \u201cleucemia\u201d (n = 1), the Spanish word for \u201cleukemia,\u201d was discarded because the Yahoo! Buzz Index does not consistently track foreign words, as it excludes search terms that contain non-English characters. For instance, the Yahoo! Buzz Index would not capture a search term with an accent mark, such as \u201cc\u00e1ncer colorectal\u201d (Spanish for \u201ccolorectal cancer\u201d).\nCancer Burden\nThe estimated incidence and mortality for 23 cancers during the study period were obtained from annual burden reports published by the American Cancer Society [24-26]. All cancers with at least 7500 new cases in 2001, 2002, or 2003 were included in the analysis (n = 23) whether or not they were associated with Yahoo! search activity.\nCancer News Coverage\nNews reports associated with specific cancer types were identified using the LexisNexis \u201cUS News\u201d database, which includes more than 400 national and regional newspapers, such as the Wall Street Journal and the Baltimore Sun, and a variety of newswire services, such as the Associated Press and UPI (United Press International). News reports published from January 1, 2001, to December 31, 2003, related to specific cancer types were found by locating reports with the identified Yahoo! search activity terms (eg, \u201cbreast cancer\u201d) in their headlines. In the case of cancers located in the esophagus and oral cavity, for which no Yahoo! search activity terms were associated, the terms \u201cesophageal cancer\u201d and \u201coral cancer\u201d were used as the headline search terms.\nAnalysis\nDescriptive statistics were calculated for the Yahoo! search activity score, estimated incidence, estimated morality, and news coverage volume associated with the cancers included in the study. Spearman rank correlations were used to establish the consistency of these variables across the study period, and the data were aggregated. Next, the relationships between Yahoo! search activity and the potential correlates of interest were tested using Spearman rank correlations.\nThe relationship between Yahoo! search activity and news coverage was further analyzed for the five cancers with the highest daily Yahoo! search activity. The number of news reports published each day was transformed into a categorical variable with four levels (0 news reports, 1\u20132 news reports, 3\u20134 news reports, and 5 or more news reports), and one-way analysis of variance (ANOVA) was used to compare mean daily Yahoo! search activity scores at increasing levels of news coverage. To detect possible periodicity effects, Yahoo! search activity data were visually inspected. Three possible periodicity effects were noted: a rise during weekdays (Monday\u2013Friday) compared with weekends; a rise during national cancer awareness months compared with other months; and a decline during summer months (June\u2013August) compared with other months. These possible effects were tested using t tests. Finally, the Yahoo! search activity associated with several cancers was marked by sharp increases of 100% or more from one day to the next. For these cancers, we identified the three highest peaks in 2003 Yahoo! search activity and investigated corresponding news events.\nResults\nWe found Internet search terms generating Yahoo! search activity associated with 21 of the 23 cancers included in the study (Table 1). Of these, 19 cancers were associated with only one Yahoo! search term each. The 2 remaining cancers were associated with two search terms each: cancers of the brain (\u201cbrain tumor\u201d and \u201cbrain cancer\u201d) and multiple myeloma (\u201cmultiple myeloma\u201d and \u201cmyeloma\u201d). In these cases, the daily Yahoo! search activity scores associated with each term were summed into a composite score for these cancers.\nThe highest mean daily Yahoo! search activity scores were generated by breast cancer (mean = 14.37), lung cancer (mean = 9.08), and leukemia (mean = 7.15). Cancers with the highest US 2001\u20132003 incidences were breast (n = 611300), prostate (n = 608000), and lung (n = 510800). For cancer mortality, lung (n = 469500), colorectal (n = 170400), and breast (n = 120800) cancer were the leading causes of death. Breast cancer (n = 5840), leukemia (n = 2143), and prostate cancer (n = 1822) were associated with the most US news reports from 2001 to 2003. Some cancers, such as leukemia, ovarian, and testicular, appeared to be associated with more Internet search activity than their burden would dictate.\nCancers were ranked by mean daily Yahoo! search activity score, estimated incidence, estimated mortality, and number of related news reports for each year in the study period (2001 to 2003). To explore the consistency of ranks over the study period within each variable, Spearman rank correlations were determined for each pair of years (2001 and 2002, 2002 and 2003, 2001 and 2003). We found statistically significant correlations (P < .001) between all year pairs tested (data not shown). Because the ranks associated with these variables were highly consistent from 2001 to 2003, we aggregated the data across the study period.\nTable 1\nMean daily Yahoo! search activity score (United States, 2001\u20132003), estimated incidence, estimated mortality, and number of news reports, by cancer\nCancer\nYahoo! Search Terms\nMean Daily Yahoo! Search Activity Score*\n(Rank)\nEstimated\nIncidence\n(Rank)\nEstimated\nMortality\n(Rank)\nNumber of News\nReports (Rank)\nBreast\n\u201cbreast cancer\u201d\n14.37 (1)\n611300 (1)\n120800 (3)\n5840 (1)\nLung\n\u201clung cancer\u201d\n9.08 (2)\n510800 (3)\n469500 (1)\n918 (5)\nLeukemia\n\u201cleukemia\u201d\n7.15 (3)\n92900 (10)\n65100 (7)\n2143 (2)\nColorectal\n\u201ccolon cancer\u201d\n7.08 (4)\n43120 (4)\n170400 (2)\n617 (6)\nProstate\n\u201cprostate cancer\u201d\n6.13 (5)\n608000 (2)\n90600 (4)\n1822 (3)\nOvary\n\u201covarian cancer\u201d\n3.71 (6)\n72100 (13)\n42100 (9)\n458 (8)\nLymphoma\n\u201clymphoma\u201d\n3.54 (7)\n185500 (5)\n78100 (6)\n480 (7)\nUterine, cervix\n\u201ccervical cancer\u201d\n2.53 (8)\n38100 (20)\n12600 (19)\n392 (9)\nMelanoma\n\u201cmelanoma\u201d\n2.25 (9)\n159200 (7)\n22800 (16)\n376 (10)\nBrain\n\u201cbrain tumor\u201d\u201cbrain cancer\u201d\n1.52 (10)\n52500 (16)\n39300 (10)\n925 (4)\nLiver\n\u201cliver cancer\u201d\n0.70 (11)\n50100 (17)\n42600 (8)\n110 (14)\nTestis\n\u201ctesticular cancer\u201d\n0.62 (12)\n22300 (23)\n1200 (23)\n50 (17)\nPancreas\n\u201cpancreatic cancer\u201d\n0.23 (13)\n90200 (11)\n88600 (5)\n185 (11)\nMultiple myeloma\n\u201cmultiple myeloma\u201d\u201cmyeloma\u201d\n0.11 (14)\n43600 (18)\n32900 (15)\n185 (11)\nStomach\n\u201cstomach cancer\u201d\n0.08 (15)\n65700 (14)\n37300 (13)\n50 (17)\nUterine, corpus\n\u201cuterine cancer\u201d\n0.012 (16)\n117700 (8)\n20000 (18)\n17 (22)\nLarynx\n\u201cthroat cancer\u201d\n0.012 (16)\n28400 (21)\n11500 (21)\n30 (20)\nBladder\n\u201cbladder cancer\u201d\n0.010 (18)\n168200 (6)\n37500 (12)\n118 (13)\nSoft tissue\n\u201csarcoma\u201d\n0.009 (19)\n25300 (22)\n12200 (20)\n25 (21)\nThyroid\n\u201cthyroid cancer\u201d\n0.002 (20)\n62200 (15)\n4000 (22)\n40 (19)\nKidney\n\u201ckidney cancer\u201d\n0.001 (21)\n94500 (9)\n35600 (14)\n77 (15)\nOral cavity\n-\n0.000 (22)\n86700 (12)\n22400 (17)\n69 (16)\nEsophagus\n-\n0.000 (22)\n40200 (19)\n38100 (11)\n13 (23)\n* Each point of a Yahoo! search activity score equals 0.001% of the population searching Yahoo! on any day.\nCorrelates of Yahoo! Cancer Search Activity\nWe tested the relationships between variables by determining Spearman rank correlations between each pair. Statistically significant correlations were found between all variable pairs (Table 2).\nTable 2\nSpearman rank correlations between mean daily Yahoo! search activity score (United States, 2001\u20132003), estimated incidence, estimated mortality, and number of news reports\nSpearman Rank Correlation*\nMean Daily Yahoo! Search Activity Score\nEstimated Incidence\nEstimated Mortality\nNumber of news reports\n.88\u2020\n.62\u2021\n.74\u2020\nEstimated mortality\n.66\u2020\n.71\u2020\n-\nEstimated incidence\n.50\u00a7\n-\n-\n* Spearman rank correlations were done on the rankings reported in Table 1.\n\u2020 \nP \u2264 .001\n\u2021 \nP = .002\n\u00a7 \nP = .015\nThe relationship between Yahoo! search activity and its most statistically significant correlate\u2014news coverage\u2014was further analyzed for the five cancers with the highest daily Yahoo! search activity (breast, lung, leukemia, colorectal, and prostate). For these cancers, the number of news reports published each day was transformed into a categorical variable with four levels. The mean daily Yahoo! search activity at each level was compared using ANOVA, and all tests were statistically significant (P \u2264 .001). For all five cancers, the mean daily search activity score was higher at each increasing level of news coverage (Table 3).\nTable 3\nMean daily Yahoo! search activity score (United States, 2001\u20132003), by number of news reports published daily and cancer\nCancer\nMean Daily Yahoo! Search Activity Score*\u2020\n(Number of News Reports )\nDays With\n0\nNews Reports\nDays With\n1\u20132\nNews Reports\nDays With\n3\u20134\nNews Reports\nDays With\n5+\nNews Reports\nBreast\n10.09 (81)\n11.49 (278)\n13.36 (252)\n17.27 (484)\nLung\n8.27 (633)\n10.00 (362)\n10.54 (71)\n11.71 (29)\nLeukemia\n6.89 (248)\n7.07 (523)\n7.18 (232)\n8.26 (92)\nColorectal\n6.72 (739)\n7.44 (297)\n8.25 (43)\n13.92 (16)\nProstate\n5.30 (390)\n6.40 (467)\n6.72 (150)\n7.43 (88)\n* Each point of a Yahoo! search activity score equals 0.001% of the population searching Yahoo! on any day.\n\u2020 ANOVA was used to compare the mean daily Yahoo! search activity at each level of news coverage. For all five cancer sites, a statistically significant difference (P \u2264 .001) was found.\nPeriodicity of Yahoo! Cancer Search Activity and News Coverage\nThree possible periodicity effects were tested: a rise during weekdays (Monday\u2013Friday) compared with weekends; a rise during national cancer awareness months compared with other months; and a decline during summer months (June\u2013August) compared with other months. To test for these potential effects, we used the five cancers with the highest daily mean Yahoo! search activity scores (breast, lung, leukemia, colorectal, and prostate) (Table 4). For all five cancers tested, both mean daily Yahoo! search activity scores and mean daily news reports were higher Monday\u2013Friday than they were Saturday\u2013Sunday (P < .001). Higher mean daily Yahoo! search activity scores were found for breast cancer (P < .001), lung cancer (P < .001), and colorectal cancer (P < .001) during their respective national awareness months. The number of mean daily news reports related to breast cancer (P < .001), colorectal cancer (P < .001), and prostate cancer (P = .007) rose during their respective national awareness months. Mean daily Yahoo! search activity scores for breast cancer (P < .001), lung cancer (P < .001), and leukemia (P < .001) were lower during the summer months than during the rest of the year. While mean daily news reports about breast cancer also decreased during the summer (P < .001), mean daily news reports about prostate cancer rose (P = .01).\nTable 4\nPeriodicity of mean daily Yahoo! search activity score (United States 2001\u20132003) and mean daily number of news reports, by cancer\nCancer\nWeekdays\nWeekends\nP value\nAwareness Month\nNon-Awareness Months\nP value\nSummer: June-August\nNon-Summer\nP value\nBreast\nMean Daily Yahoo! Search Activity Score*\n15.78\n10.84\n< .001\n26.33\n13.26\n< .001\n10.78\n15.58\n< .001\nMean Daily Number of News Reports\n6.26\n3.02\n< .001\n15.30\n4.41\n< .001\n4.19\n5.72\n< .001\nLung\nMean Daily Yahoo! Search Activity Score\n10.31\n6.00\n< .001\n11.69\n8.84\n< .001\n5.76\n10.20\n<.001\nMean Daily Number of News Reports\n1.03\n0.37\n< .001\n1.03\n0.82\n.226\n0.70\n0.89\n.086\nLeukemia\nMean Daily Yahoo! Search Activity Score\n8.13\n4.70\n< .001\n6.65\n7.20\n.093\n5.65\n7.66\n< .001\nMean Daily Number of News Reports\n2.20\n1.34\n< .001\n1.51\n2.00\n.036\n1.88\n1.98\n.506\nColorectal\nMean Daily Yahoo! Search Activity Score\n7.73\n5.44\n< .001\n10.46\n6.77\n< .001\n6.83\n7.17\n.081\nMean Daily Number of News Reports\n0.68\n0.27\n< .001\n1.55\n0.47\n< .001\n0.49\n0.59\n.214\nProstate\nMean Daily Yahoo! Search Activity Score\n6.82\n4.41\n< .001\n5.68\n6.18\n.044\n6.14\n6.13\n.997\nMean Daily Number of News Reports\n2.03\n0.74\n< .001\n2.39\n1.60\n.007\n2.14\n1.50\n.010\n* Each point of a Yahoo! search activity score equals 0.001% of the population searching Yahoo! on any day.\nPeaks in Yahoo! Cancer Search Activity and News Coverage\nOn several occasions, Yahoo! search activity scores associated with breast cancer, colon cancer, and prostate cancer were marked by sharp increases of 100% or more from one day to the next. We investigated news events that corresponded with the highest three spikes in 2003 Yahoo! search activity for these cancers. These peaks in \u201cbreast cancer\u201d and \u201ccolon cancer\u201d search activity all occurred during their respective national awareness months and appeared to be related to news coverage promoting the awareness months. The highest peak in \u201cprostate cancer\u201d search activity (22.34) occurred on July 17 after news reports of a study [27] exploring the association between sexual behavior and prostate cancer risk (Figure 1). These news reports generally focused on the possible protective benefit of masturbation. This study was not covered widely by the US news media, but it generated substantial news coverage in Australia and filtered onto the Internet via chat rooms, message boards, and medical news Web pages. While there was no corresponding spike in \u201cmasturbation\u201d search activity, there was a 117% increase in the search activity score (from 61.88 on July 16 to 133.08 on July 17) for \u201cmasterbation,\u201d a common misspelling. The second highest spike in \u201cprostate cancer\u201d search activity (14.59) occurred on October 21 after news broke that Academy-Award-winning actor Robert DeNiro had been diagnosed with prostate cancer. This story was widely covered by the US news media, and a 277% increase in \u201cRobert DeNiro\u201d search activity was observed on the same date (from 15.87 on October 20 to 59.90 on October 21). The third highest peak in \u201cprostate cancer\u201d search activity (12.41) occurred on December 29, when a study linking obesity with increased prostate cancer risk [28] was covered by several US news outlets. No corresponding rise in searches for the terms \u201cobesity,\u201d \u201coverweight,\u201d or \u201cweight loss\u201d was observed.\nFigure 12003 US prostate cancer Yahoo! search activity (each point of a Yahoo! search activity score equals 0.001% of the population searching Yahoo! on any day)\nDiscussion\nThis study suggests that media coverage plays a powerful role in prompting online cancer information seeking. News coverage correlated significantly with Yahoo! search activity (P < .001). Also, Yahoo! search activity was found to rise as news coverage increased, and sharp rises in search activity from one day to the next appeared to be associated with increases in relevant news coverage. This study also suggests that the Internet can rapidly disseminate health news: the highest spike in 2003 US \u201cprostate cancer\u201d Yahoo! search activity seemed to be generated largely by news coverage in Australia that rapidly filtered onto the Internet via chat rooms, message boards, and medical news Web pages. Thus, it possible that a news story does not necessarily have to be covered by the US news media in order to generate US Internet search activity.\nNews coverage volume also correlated with estimated cancer incidence (P = .015) and mortality (P < .001). This is interesting because past studies [29-33] on this topic have not generated consistent findings, with most [30-32] finding no relationship between disease burden and news coverage volume. However, none of the past studies focused on cancer, and none used our method for identifying news reports. While the news coverage of specific cancers generally matched their burden, we noted that some cancers, such as leukemia, ovarian cancer, and testicular cancer, were associated with more Internet search activity than their burden would suggest. A similar observation was reported by Bader and Theofanos [21], who suggested that this discrepancy may result from more searches being required to locate online information about less common cancers. The high correlation between cancer-specific news coverage and associated online search activity in the present study suggests another explanation: some cancers received a disproportionate share of news coverage relative to their incidence and mortality, and online search activity, often prompted by news coverage, reflects this imbalance.\nWe detected several periodicity effects in US Yahoo! cancer search activity, which tended to be higher on weekdays and during national cancer awareness months but lower during the summer months. It should be noted that these observations are not artifacts of the size of the online population during these periods because Yahoo! search activity scores are based on the percentage, not the number, of total users. One explanation for these results is that the volume of cancer news coverage tended to follow these trends. It is also possible that users tend to search for online cancer information from school or work settings. As a result, Yahoo! cancer search activity would be expected to drop during weekends when people are at home and over the summer months when many students are out of school and many workers go on vacation.\nAlthough Yahoo! is a leading US Internet search engine, the extent to which the findings of this study can be generalized to other search engines is not known. Also, we were unable to discern the motivations of Yahoo! users searching for cancer information. For instance, news coverage of a breast cancer drug might be associated with an increase in \u201cbreast cancer\u201d search activity. While the Yahoo! Buzz Index would detect this rise, it cannot tell how many searchers were breast cancer patients or family members and how many were investors interested in buying stock in the company developing the drug.\nInternet search activity offers an innovative tool for passive surveillance of health information\u2013seeking behavior. While our work focused on cancer, Internet search activity may be useful in gauging health information seeking related to other diseases. For example, the volume of Internet searches related to symptoms or conditions might be used to predict disease outbreaks (eg, influenza) or to assess mental health following a disaster. Researchers at the Centre for Global eHealth Innovation have begun to experiment with analyses of this type [34,35]. The Yahoo! Buzz Index is unique among Internet search datasets because it provides search activity scores adjusted for the size of the population searching for online information, which has steadily grown each year [19]. Perhaps in the future, other Internet search engines will offer databases similar to the Yahoo! Buzz Index, and research could be conducted using a combination of search engines.","keyphrases":["internet","neoplasms","health education"],"prmu":["P","P","R"]}
{"id":"Arch_Dermatol_Res-2-2-1705513","title":"The importance of disease associations and concomitant therapy for the long-term management of psoriasis patients\n","text":"It is well established that several inflammatory-type conditions, such as arthritis, diabetes, cardiovascular disease, and irritable bowel disease exist comorbidly and at an increased incidence in patients with psoriasis. Psoriasis and other associated diseases are thought to share common inflammatory pathways. Conditions such as these, with similar pathogenic mechanisms involving cytokine dysregulation, are referred to as immune-mediated inflammatory diseases (IMIDs). Considerable evidence for the genetic basis of cormobidities in psoriasis exists. The WHO has reported that the occurrence of chronic diseases, including IMIDs, are a rising global burden. In addition, conditions linked with psoriasis have been associated with increasing rates of considerable morbidity and mortality. The presence of comorbid conditions in psoriasis patients has important implications for clinical management. QoL, direct health care expenditures and pharmacokinetics of concomitant therapies are impacted by the presence of comorbid conditions. For example, methotrexate is contraindicated in hepatic impairment, while patients on ciclosporin should be monitored for kidney function. In addition, some agents, such as beta blockers, lithium, synthetic antimalarial drugs, NSAIDs and tetracycline antibiotics, have been implicated in the initiation or exacerbation of psoriasis. Consequently, collaboration between physicians in different specialties is essential to ensuring that psoriasis treatment benefits the patient without exacerbating associated conditions.\nPsoriasis: from gene to clinic\nA satellite symposium held at the Royal College of Physicians, London, United Kingdom, in December 2005 investigated the associations between psoriasis, comorbid diseases and concomitant therapy and their significance for long-term patient treatment. The programme generated substantial debate and raised some clinically significant issues, which are further outlined here.\nIntroduction\nPsoriasis is a common, chronic, immune-mediated inflammatory disease with a recognised genetic predisposition. It has been observed that, while the frequency of skin conditions such as acne, urticaria and atopic dermatitis are reduced in patients with psoriasis compared with expected frequencies in the general population, the frequency of some noncutaneous, although related, conditions is significantly increased [26, 53]. Perhaps the best known noncutaneous condition associated with psoriasis is joint disease, mostly expressed as psoriatic arthritis (PsA). In hospitalised psoriasis patients, a diagnosis of PsA [according to the European Spondyloarthropathy Study Group (ESSG) criteria] was present in 7.7% (71\/936) of patients; however, many more patients also reported paresthesias (12.3%), arthralgia (7%), stiffness (4.2%), swelling (3.7%) and ankylosis (1.2%) without meeting all of the ESSG criteria for PsA [19]. Further estimates place the occurrence of PsA in patients with psoriasis at upwards of 30% [31]. An additional study by Zachariae et al. [83] established even higher rates for psoriasis with joint involvement in 5,795 members of psoriasis associations from the Nordic countries and 702 patients seen by Nordic dermatologists. The result showed that upwards of 30% of all patients who presented with psoriasis were also diagnosed with arthritis by their dermatologist or a rheumatologist. In a recent study in a geographically defined population in Norway Madland et al. [41] found a prevalence of PsA in 2 per 1,000 inhabitants, which corresponds to about 10% in patients with psoriasis assuming a prevalence of psoriasis of 2%.\nThese results demonstrate that the prevalence of arthritis in patients with psoriasis may actually be higher than the previously accepted rate of 7% [83]. Inflammatory bowel disease is also more frequently observed in patients with psoriasis. An increased relative risk for Crohn\u2019s disease (CD) of 2.9 was found to be associated with a prior diagnosis of psoriasis in a Swedish case-control study [54]. Conversely, patients with CD are about seven times more likely to develop psoriasis than controls [5, 37, 82].\nAnalysis of data from more than 40,000 dermatological inpatients with psoriasis compared with control patients with nonpsoriatic skin diseases (adjusting for age and gender) revealed high ratios of observed versus expected frequencies for obesity (2.05, P\u00a0<\u00a00.05), hypertension (1.90, P\u00a0<\u00a00.01), heart failure (1.83, P\u00a0<\u00a00.001) and diabetes mellitus (1.47, P\u00a0<\u00a00.05) [26]. An additional analysis (retrospective chart review, 1997\u20132000) of 753 patients from an academic dermatology practice also confirms the presence of comorbidities in patients with psoriasis [53]. In fact, a comorbid diagnosis was confirmed in 551 patients (73%), with hypertension, dyslipidemia, diabetes and heart disease being the most common comorbidities identified [53]. Furthermore, patients with psoriasis appear to be at increased risk of atherosclerosis, which may at least in part be explained by an abnormal plasma lipid metabolism; serum lipoprotein (a) and triglycerides are significantly increased in patients with psoriasis compared with healthy control subjects [73], while high-density lipoprotein (HDL) cholesterol is significantly decreased [60]. Indeed, in patients with PsA, alterations of the lipid profile suggest similarities to the diabetic lipid profile, which is known to substantially increase cardiovascular risk [30].\nNearly 30\u00a0years ago it was reported that the rates of occlusive vascular disease were significantly higher in dermatological patients with psoriasis than those without and that the risk was greater in patients with predisposing factors [44]. Metabolic syndrome has since been identified as a clustering of metabolic abnormalities in individuals and is associated with a threefold increase in type 2 diabetes and a twofold increase in cardiovascular disease [86]. The most recent definition of metabolic syndrome requires central obesity (defined as body mass index >30\u00a0kg\/m2) plus any two of the following: elevated plasma triglycerides, reduced HDL cholesterol, elevated blood pressure and raised fasting plasma glucose (Table\u00a01). Such clustering of risk factors has been observed in patients with psoriasis [26].\nTable\u00a01Diseases associated with psoriasis [65]ConditionPrevalence in psoriasis n (%)Hospital controls OR (95% CI)RKI sample OR (95% CI)Diabetes mellitus type I11 (1.9)3.99 (1.30\u201312.2)*6.34 (2.80\u201314.3)***Diabetes mellitus type II68 (11.7)2.48 (1.70\u20133.61)**2.07 (1.50\u20132.85)***Arterial hypertension127 (21.9)3.27 (2.41\u20134.43)***1.39 (1.09\u20131.77)*Hyperlipoproteinemia30 (5.2)2.09 (1.23\u20133.54)**0.83 (0.54\u20131.28)fCoronary heart disease32 (5.5)1.77 (1.07\u20132.93)*0.93(0.60\u20131.43)fMetabolic syndrome25 (4.3)5.92 (2.78\u201312.8)***2.20 (1.41\u20133.43)**Alcohol consumption none vs. moderate246 (42.3)2.78 (2.14\u20133.62)***2.03 (1.62\u20132.55)***None vs. regularly75 (12.9)3.33 (2.20\u20135.05)***2.00 (1.45\u20132.77)***None vs. heavya24 (4.1)3.61 (1.85\u20137.07)***8.50 (5.28\u201316.8)***Cigarette smoking264 (45.4)2.96 (2.27\u20133.84)***2.49 (2.00\u20133.10)***The prevalence of diseases in plaque-type psoriasis patients (n\u00a0=\u00a0581) was compared with hospital-based controls (n\u00a0=\u00a01,044) and a population-based survey (RKI sample; n\u00a0=\u00a04,705). Common odds ratios adjusted for age and sex are presented with their 95% confidence intervals (95% CI), f\u00a0not significantaDue to low numbers, only adjusted for sex*P\u00a0<\u00a00.05; ** P\u00a0<\u00a00.01; ***P\u00a0<\u00a00.0001 by Mantel\u2013Haenszel test\nPsoriasis, especially in patients with more severe disease, has also been associated with other factors that can increase cardiovascular risk, thereby increasing morbidity and mortality. Alcoholism and liver cirrhosis are reportedly more common in psoriasis [39] with a reported prevalence of alcoholism of 18% among psoriatic patients compared with 2% in other dermatologic controls [22, 46]. Cigarette smoking has also been associated with psoriasis. A recent case-control study revealed that the risk of psoriasis was higher in ex-smokers and current smokers than in those who never smoked (odds ratios of 1.9 and 1.7, respectively), and smoking was strongly associated with the occurrence of pustular lesions (odds ratio\u00a0=\u00a05.3 for smokers) [48]. In a cross-sectional study, patients with psoriasis that enrolled in the prospective Utah Psoriasis Initiative had a significantly higher prevalence of obesity and smoking than the general population of Utah (P\u00a0<\u00a00.001 for both). The prevalence of obese smokers was significantly higher (P\u00a0<\u00a00.001) [27].\nA number of psychiatric\/psychological comorbidities have also been observed in patients with psoriasis [22]. An association between psoriasis and stressful life events in the year preceding diagnosis has been reported, suggesting that psychological stress may have a role in the pathogenesis of psoriasis [48]. Furthermore, psoriasis-related stress can play a role in the exacerbation of psoriasis, and greater stress reactivity has been associated with onset of psoriasis at an earlier age [22]. High depression\/anxiety scores, obsessionality and difficulties with verbal expression of emotions (especially anger) have been reported in various cross-sectional surveys [22]. Severely affected psoriatic inpatients (>30% body surface area affected) were likely to suffer clinical depression and suicidal ideation (7.2%); however, suicidal ideation was still relatively high (2.5%) in less severely affected outpatients (<30% total body surface area affected) and comparable to the reported prevalence among general medical patients (2.4\u20133.3%) [21].\nThese identified diseases associated with psoriasis have led clinicians to look at underlying mechanisms that might be involved and start considering how managing comorbid conditions may impact treatment selection and outcome for the psoriasis patient.\nCommon inflammatory pathways in psoriasis and associated diseases\nThe immune system has been strongly implicated in the pathogenesis of psoriasis that resembles a T cell-mediated, autoimmune, inflammatory disease [7]. T cells are found in the dermis and epidermis and are accompanied by increased numbers of dermal dendritic cells, macrophages and mast cells [51]. It is thought that a stimulus (such as trauma or infection) triggers a plexus of cellular events by inciting a cascade of cytokines, creating an inflammatory response. Dendritic cells and T cells become activated with the formation of an immunological synapse\u2014a multimolecular complex at the T cell-antigen-presenting cell interface that facilitates immune cell interactions. Once activated, dendritic antigen-presenting cells and T cells release cytokines, chemokines and growth factors, which trigger keratinocyte proliferation, altered differentiation and an angiogenic tissue response, giving rise to psoriatic lesions (Fig.\u00a01).\nFig.\u00a01Inflammatory pathway in psoriasis [50]. Working model for immunopathogenesis of psoriasis. Multiple stages are proposed for trafficking patterns of immunocytes, involving signals in which symptomless skin is converted into a psoriatic plaque. Key inflammatory events include intraepidermal trafficking by CD8+ T cells and neutrophils. Reproduced with permission from J Clin Invest (2004, 113:1664\u20131675). Copyright 2004, The American Society for Clinical Investigation\nOther inflammatory diseases, such as rheumatoid arthritis (RA) and CD, are thought to involve similar pathogenetic mechanisms. Recent research has shown that inflammation plays a key role in atherosclerosis [24]. A T-cell infiltrate is always present in atherosclerotic lesions, with activated T cells producing a type 1 helper (Th1) response. Psoriasis is up to seven times more common in patients with CD than in controls [37, 82] and, as discussed previously, there is often joint involvement in psoriasis. Such extracutaneous manifestations of the disease substantiate that psoriasis is a systemic disease and not just a skin disease [14].\nThe term immune-mediated inflammatory disease (I.M.I.D.) has been developed to describe conditions, such as psoriasis, CD and RA, with common inflammatory mechanisms described above involving cytokine dysregulation. This term also encompasses type 1 diabetes mellitus, ulcerative colitis, systemic lupus erythematosus, multiple sclerosis, ankylosing spondylitis and uveitis and may further be expanded to include asthma and atherosclerosis [79].\nAtherosclerosis and rheumatoid arthritis\nThe prevalence of coronary artery atherosclerosis has been found to be increased in patients with chronic inflammatory conditions, such as RA and systemic lupus erythematosus [3, 10]. Excess mortality due to coronary heart disease in RA patients was first observed in 1953 [11]. In a study of 236 RA patients, the age- and sex-adjusted incidence rate ratio of incident cardiovascular events was calculated to be 3.96 [16]. After adjusting for cardiovascular risk factors such as age, weight and smoking status, this figure decreased slightly to 3.17, suggesting that there are additional mechanisms responsible for cardiovascular disease in RA. The risk of cardiovascular disease increases with disease severity (eg, number of joints involved) and disease duration [64, 75]: severe coronary artery calcification is more likely in patients with established disease (>10\u00a0years) compared with controls after adjusting for cardiovascular risk factors (odds ratio\u00a0=\u00a03.42, P\u00a0=\u00a00.002) [10]. However, there is some emerging evidence for increased atherosclerotic disease in early RA [32].\nThe lipid profile in RA patients is directly affected by the systemic inflammatory response (active inflammation lowers HDL cholesterol, which leads to increased cardiovascular risk) [75]. Endothelial dysfunction, which underlies atherosclerotic plaque development, is associated with markers of systemic inflammation, such as C-reactive protein (CRP), soluble adhesion molecules (sICAM-1 and sE-selectin), IL-6 and TNF-alpha (TNF\u03b1) [24, 38, 78]. Indeed, treatment with the anti-TNF\u03b1 agent infliximab, which affects expression of all markers described above, has been shown to improve endothelial function in RA patients [28].\nAtherosclerosis and psoriasis\nAn increase in cardiovascular disease has been observed in psoriasis patients [39, 44] and has been shown to be related to disease severity and age of onset [42, 55, 62]. An increased incidence of traditional cardiovascular risk factors is frequently reported [26, 42, 56]. In psoriatic patients with a history of at least one hospital admission due to psoriasis, there is about a 50% increase in cardiovascular death as compared with the general population [standardised mortality ratio (SMR)\u00a0=\u00a01.52]. In those patients with 3 or more hospital admissions, the SMR increased to 1.82 and in those patients admitted at younger ages (20\u201339\u00a0years at first admission), the SMR increased to 2.62 [42]. However, the SMR was not increased in those treated as outpatients only, indicating a relationship to disease severity. In patients with PsA, an increase in mortality has also been observed (SMR\u00a0=\u00a01.65 in males and 1.59 in females) and the largest single cause of death is myocardial infarction (27.6%) [80]. \n\u00a0The increased cardiovascular risk in psoriasis may result from\u2022 The increased prevalence of associated risk factors, such as smoking, obesity, hypertension and alcohol misuse.\u2022 The use of dyslipidaemic therapies, such as corticosteroids, acitretin and ciclosporin.\u2022 An associated unfavourable lipid profile (high triglycerides, low HDL).\u2022 Endothelial dysfunction.\u2022 Uncontrolled inflammation. \u2022 Combination of some or all of the above.\nUnfortunately, there is a shortage of published studies corrected for traditional risk factors and no published study has formally assessed systemic vasculature inflammation in psoriasis.\nBoth psoriasis and atherosclerosis are T cell-mediated inflammatory conditions and a number of similarities exist between their pathogenetic mechanisms. Numerous factors have been shown to be increased in the serum of psoriatic patients including apolipoprotein E (ApoE), vascular endothelial growth factor (VEGF), sICAM-1, IL-8, soluble interleukin2 receptor (sIL-2R) and TNF\u03b1 [9, 12, 14, 15]. sICAM-1 and sIL-2R are soluble T-cell products [15]. ApoE plays a central role in lipoprotein metabolism and has been found to be significantly raised in patients with psoriasis, indicating that abnormal metabolism of lipoprotein exists in psoriasis patients [81]. Furthermore, the dyslipidaemic profile exhibited in psoriasis has been shown to modify dendritic cell functions in skin [1]. The impaired dendritic cell emigration from the skin parallels impeded migratory clearance of monocyte-derived dendritic cell-like cells from atherosclerotic plaques.\nVEGF mediates endothelial cell mitogenesis and enhances vascular permeability [4]. In psoriasis, VEGF is thought to promote localised angiogenesis and in severe psoriasis may act on renal vasculature to induce hyperpermeability with consequent proteinuria [12]. A role for VEGF has been postulated in the atherosclerotic process as changes in levels of VEGF and its receptors are present in atherosclerosis, and angiogenesis may play a role in plaque development and destabilisation [4].\nGenetic basis of comorbidities\nThere is considerable evidence for a genetic element in psoriasis. About 30% of individuals with psoriasis have an affected first-degree relative [69], and first-degree relatives are at a five to tenfold increased risk for developing psoriasis themselves [70]. Concordance rates for psoriasis are three to four times higher in monozygotic twins than dizygotic twins [6], and twin and family studies show that 60\u201390% of the variance in psoriasis liability is due to genetic factors. Finally, linkage and association to the major histocompatability complex (MHC) region has been repeatedly demonstrated [58]. However, the presence of many different phenotypes means that not all psoriasis is the same and polygenic inheritance is likely.\nInterestingly, by analysing gene polymorphisms of the receptor of advanced glycation end products (RAGE) in patients with plaque-psoriasis seems to be linked to a genetic risk for cardiovascular disorders in psoriasis patients [76].\nA combination of disease-specific and inflammatory modifying genes interacting with the environment likely determines the risk of comorbidity. A major susceptibility gene (Psoriasis Susceptibility 1 or PSORS1) is located in the MHC class I region and maps to an approximately 300-kb interval containing HLA-C and 10 other genes in strong linkage disequilibrium with it [25, 47, 77]. Very recently, through a combination of genomic sequencing and haplotype analysis, HLA-Cw6 itself has been identified as the major disease allele at PSORS1 [47].\nThe observation that bacterial infections are significantly less common in psoriasis patients with an age of onset \u226440\u00a0years while immunological skin disorders (allergic contact dermatitis, urticaria and atopic dermatitis) are reduced regardless of age of onset suggests a genetic link between resistance to infection and early-onset psoriasis [26].\nChronic tonsillitis has been observed over three times more frequently than would be expected in patients with psoriasis [26]. Interestingly, psoriasis is not associated with other forms of streptococcal infection, suggesting a specific role for the tonsils [20]. Recently, identical T-cell receptor rearrangements have been found in the tonsils and lesional skin of psoriatic patients [17]. The prevalence of streptococcal infection during sore throat was 10 times higher in people with psoriasis than in controls and can exacerbate chronic plaque psoriasis [20]. There was, however, no difference in asymptomatic carriage between psoriasis patients and controls [20]. These results suggest either that psoriatic patients are more susceptible to streptococcal infection or that they respond more vigorously to it and are more likely to develop symptoms.\nThis apparently defective response to streptococci combined with resistance to a variety of skin infections in psoriasis patients provides an interesting paradox. It is known that streptococcal (and other) infections may initiate psoriasis in genetically predisposed individuals, and psoriasis is characterised by strong Th1 immune deviation. Once the psoriatic cascade has been initiated, increased skin and\/or systemic inflammation directed by Th1 cells may be responsible for a lower risk of other infections. Interestingly, bacterial infection (principally with Chlamydia pneumoniae) has also been implicated in the pathogenesis of atherosclerosis [36, 63]. A phenomenon similar to that seen in psoriasis could contribute to increased risk of cardiovascular disease.\nClinical importance of comorbidities in psoriasis\nAccording to the World Health Organization (WHO), the occurrence of chronic disease is a rising global burden. In 2005, all chronic diseases accounted for 72% of the total global burden of disease in the population aged 30 years and older (Fig.\u00a02) [68]. More than 80% of the burden of chronic diseases occurs in people under age 70\u00a0years. While psoriasis causes significant morbidity, it is not generally associated with mortality; however, conditions linked with psoriasis are associated with excess mortality [56]. For example, the higher prevalence of smoking in the psoriatic population may explain why psoriasis has been associated with lung cancer in middle-aged women [39]. A registry of 5,687 patients admitted to the hospital for psoriasis between 1973 and 1984 who were followed for up to 22\u00a0years until 1995 found a significantly increased mortality for both alcohol- and smoking-related causes [56].\nFig.\u00a02WHO projected major causes of death 2005 [67]. The occurrence of chronic disease is a rising global burden and accounted for 72% of the total global burden of disease in the population aged 30 years and older in 2005. Reproduced with permission from Lancet (2005, 366:1578\u20131582). Copyright 2005, Elsevier Ltd\nAn increased rate of cardiovascular events has been observed in patients with psoriasis [39, 44], and it appears to be related to the severity of disease and age of onset [42, 55, 62]. Worldwide, cardiovascular disease is estimated to be the cause of 30% of all deaths in 2005 making it the leading single cause of death worldwide [68]. Identification and treatment of modifiable risk factors in patients with psoriasis could result in a lower associated cardiovascular morbidity and mortality [55]. Conceptually, the effective systemic control of inflammation in psoriasis could also lead to improvements in comorbid conditions driven by inflammation.\nImplications of comorbidities\nThe presence of comorbid conditions in psoriasis patients can have some important implications for clinical management. The general health status of the patient will be affected by all of these associated diseases, with substantial impact on quality of life (QoL) [33, 74]. The precise impact of comorbid conditions on QoL in psoriasis patients has not been quantified; however, it is known that the presence of joint involvement can significantly reduce health-related QoL [40] and that arthritis and arthralgia are important related factors affecting QoL in patients with psoriasis [83]. In fact, the effects of psoriasis on QoL are comparable to the effects observed from other major chronic diseases [59]. The authors performed an analysis of 317 patients with psoriasis and found that these patients reported a reduction in physical and mental functioning comparable to that observed in patients with cancer, arthritis, hypertension, heart disease and depression [59].\nA US study looking at the direct health care expenditures associated with psoriasis found that psoriasis patients treated with systemic therapy or phototherapy have significantly more comorbidities and higher mean total health care expenditures compared with non-psoriasis patients. Furthermore, selected comorbidities lead to significantly higher mean total health care expenditures in those patients with psoriasis than in those with the same condition without psoriasis [13].\nMany of the conditions associated with psoriasis could affect the way drugs are absorbed and eliminated from the body. For example, alcoholism impairs liver function, obesity affects drug distribution in the body, diabetes can impair kidney function, inflammatory bowel disease can reduce absorption from the gastrointestinal tract and smoking can impact the efficacy of some drugs [85]. Of course, the presence of these conditions will also lead to increased polypharmacy, increasing the likelihood of drug interactions and making it harder to implement an \u2018ideal\u2019 psoriasis treatment in individuals [34].\nThe implications of managing a patient with comorbid conditions areFocus on inflammation control.Consider impact on patient as a whole.Monitor all associated diseases.\nDrug interactions in psoriasis\nIt has been suggested that some drugs may trigger or exacerbate psoriasis. The most commonly proposed causative agents are beta blockers, lithium, synthetic antimalarial drugs, nonsteroidal anti-inflammatory drugs (NSAIDs) and tetracycline antibiotics [72]. Analysis of co-medication according to patients\u2019 files in 1203 consecutive inpatients hospitalised for treatment of severe psoriasis was conducted between 2003 and 2005 in 9 dermatology departments in Germany [84]. The mean age of patients was 50 years (55.9% female, 44.1% male). The mean Psoriasis Area and Severity Index was 26.0, average body mass index was 27.5 and mean age of first manifestation was 33.5\u00a0years. About one third of patients were found to be taking no systemic medication (34.4%), and 42.4% were taking three or fewer systemic agents. However, nearly a quarter (23.2%) were taking more than three systemic medications and of these, 11.1% were taking more than ten medications (Table\u00a02). Therefore, physicians caring for patients with psoriasis may consider withdrawal of agents that potentially exacerbate this condition. Further analysis suggests that comorbid cardiac and metabolic disorders are common in these patients: hypertension, 28.8%; type 2 diabetes, 10.5%; dyslipidaemia, 12.5%; hyperuricaemia, 5.3%; arrhythmias, 5.1%; coronary artery disease, 3.2%; and cardiac insufficiency, 2.1%. The presence of renal insufficiency (2.7%) and liver cirrhosis (2.4%) were also noteworthy.\nTable\u00a02Percentage of psoriasis patients receiving medications for comorbid disease [84]Drug classPatients (%) (n\u00a0=\u00a01,203)ACE inhibitors12.3Oral anticoagulants11.3Diuretics12.4Thyroid drugs9.9Beta blockers7.9Psycholeptics5.6NSAIDs7.0Lithium salts0.70Interferon-alpha0.25\nRisk of interactions with psoriasis therapies\nThere is a high risk of drug interactions with both ciclosporin and methotrexate, while retinoids and leflunomide exhibit a lower risk of drug\u2013drug interactions. Conversely, treatments with biologics, such as TNF\u03b1 inhibitors, and fumaric acid esters have no known risk of interactions. A number of medications can cause photosensitivity, which is an issue for any UV treatment of psoriasis (e.g., tetracyclines [doxycyclin] and thiazide diuretics) [67].\nEffect of psoriasis treatment on comorbidities\nThe importance of considering the impact of treatment of diseases associated with psoriasis cannot be stressed enough. Working with physicians in different specialties (e.g., rheumatology, gastroenterology and cardiology) would allow physicians an opportunity to ensure that the psoriatic treatments do not worsen the associated condition and where possible provide additional benefits.\nBenefits of psoriasis therapy on comorbid conditions\nSince many comorbid conditions have inflammatory mechanisms in common with psoriasis, drugs targeting inflammation and\/or suppressing the immune response are often effective in treating both psoriasis and related comorbidities. A number of treatments have shown some efficacy in treating both psoriasis and PsA including methotrexate, ciclosporin, leflunomide, etanercept and infliximab [2, 18, 31, 45]. The TNF\u03b1 inhibitors etanercept and infliximab have demonstrated halting of joint degradation [45].\nCrohn\u2019s disease\nThere are few treatments that are effective in both psoriasis and CD. Azathioprine and 6-mercaptopurine are often used in CD, and they have also shown some efficacy in PsA in small, uncontrolled trials; however, toxicity is likely to restrict their use [50]. Low-dose methotrexate has shown some efficacy in CD patients resistant to other therapies; however, its usefulness in the long term has yet to be established [66]. Infliximab has been shown to be effective in both attaining and maintaining remission in CD patients [23] as well as in the treatment of extra-intestinal cutaneous manifestations [61]. Conversely, etanercept has not been shown to be effective in the treatment of CD and may even worsen the condition [52]. The difference between the effects of TNF\u03b1 inhibitors on inflammation in CD are thought to be due in part to their different mechanisms of binding TNF\u03b1. Infliximab has higher affinity than etanercept, binding more forms of TNF\u03b1, and may induce cell apoptosis [35].\nCardiovascular disease\nEvidence for the improvement of psoriasis-associated cardiovascular risk following psoriasis treatment is limited to a trial of methotrexate in patients with psoriasis or RA [57]. Patients prescribed methotrexate had a significantly reduced risk of vascular disease compared with those who did not receive methotrexate (psoriasis, relative risk [RR]\u00a0=\u00a00.73; RA, RR\u00a0=\u00a00.83). Furthermore, risk reduction was greater in those who received a low cumulative dose of methotrexate (psoriasis, RR\u00a0=\u00a00.50; RA, RR\u00a0=\u00a00.65) and in those patients also receiving concomitant folic acid (psoriasis, RR\u00a0=\u00a00.56; RA, RR\u00a0=\u00a00.77). Further work in RA has suggested that anti-TNF\u03b1 therapy may provide some cardiovascular protection [29]. A comparison of two cohorts, one receiving TNF\u03b1 inhibitors and one receiving standard therapy (derived from patient registries), revealed a reduced age\u2013sex adjusted incidence rate of first cardiovascular event in those receiving TNF\u03b1 inhibitors compared with controls (14.0\/1,000 person\u2013years at risk vs. 35.4\/1,000 person\u2013years). After controlling for disability, the age\u2013sex adjusted rate ratio was 0.46 (P\u00a0=\u00a00.013) in the anti-TNF\u03b1-treated cohort versus the not-treated cohort.\nStatins are used to lower lipids in people at risk for cardiovascular events and have been shown to reduce cardiovascular morbidity and mortality; however, statins have also been shown to have anti-inflammatory properties [24, 43]. Statins down-regulate the expression of adhesion molecules and the expression of Th1 chemokine receptors on T cells, as well as block the expression of TNF\u03b1 and interferon-gamma in macrophages among other immunomodulatory actions [49]. A double-blind, placebo-controlled trial in 116 patients with RA demonstrated that 40 mg atorvastatin daily, in addition to existing RA therapy, produced a significant reduction in disease activity (P\u00a0=\u00a00.004), a significant reduction in inflammatory markers (CRP 50%, P\u00a0<\u00a00.0001; ESR 28%, P\u00a0=\u00a00.005) and a significant reduction in swollen joint count (\u22122.69 vs \u22120.53; P\u00a0=\u00a00.0058) compared with placebo [43]. In addition, simvastatin treatment has been shown to significantly reduce serum CRP and TNF\u03b1 in patients with RA and improve endothelial function [71]. Therefore, it has been suggested that statins may have beneficial effects in inflammatory dermatological conditions including psoriasis, and the results of controlled trials are awaited [49].\nPotential drawbacks of psoriasis therapy on comorbid conditions\nOther common comorbidities could influence the choice of psoriasis therapies available to the dermatologist. Ciclosporin can induce hyperglycaemia and acitretin can alter glucose tolerance; therefore, they are best avoided in diabetic patients. Methotrexate has been reported to precipitate diabetes and increase insulin requirements due to increased disease progression [8]. Ciclosporin is contraindicated in patients with uncontrolled hypertension and acitretin is contraindicated in hyperlipidaemia. Methotrexate and acitretin are contraindicated in the presence of hepatic impairment, which may result from psoriasis-associated alcohol misuse, while ciclosporin may need dosage adjustment and regular monitoring of kidney function since renal dysfunction is a known side effect [8].\nDrug interactions and precipitating disease in psoriasis\nA diagnosis of psoriasis also has implications regarding treatment for physicians from specialities other than dermatology. The potential of some drugs (eg, angiotensin-converting enzyme inhibitors, beta blockers, NSAIDs) to \u2018trigger\u2019 psoriasis means that these drugs should be avoided where possible and awareness of this potential should be raised. The potential for drug interactions with some psoriasis drugs is high and this should also be considered when designing therapeutic regimens. Furthermore, the increased risk of cardiovascular events associated with psoriasis should be considered when conducting global cardiovascular risk assessments. If possible, lower cut-off points for treatment should be used in psoriasis patients in an effort to take into consideration their overall cardiovascular risk.\nDiscussion and conclusions\nPsoriasis is a multifaceted systemic condition that is not restricted to cutaneous symptoms. There is a need for further research to fully understand the reasons for comorbidities and their implications on treatment and management of psoriasis patients. Data are required to fully characterise risk of cardiovascular disease in psoriasis patients and whether treatment can impact this level of risk. Could effective (systemic) treatment of psoriasis reduce the risk of comorbidities or, conversely, could effective treatment of comorbidities improve psoriasis? Investigation of the genetics of psoriasis and related inflammatory conditions may provide insight into their pathogenesis and points of commonality, which could reveal novel drug targets\nHowever, while research is conducted, there is a pressing need for education about comorbidities in psoriasis. Dermatologists need to be aware of the likelihood of comorbid conditions to ensure their detection and that psoriasis is managed with consideration of the possible impact on associated comorbid inflammatory conditions. Similarly, physicians from other specialities should be aware of the potential impact of their management strategies on psoriasis and take care not to exacerbate psoriasis when treating associated conditions. A focus on rapid and profound systemic control of inflammation in psoriasis may have added benefits by controlling inflammation associated with comorbid conditions. It is clear that TNF\u03b1 is a central mediator in psoriasis and many of the recognised associated diseases. Knowledge and awareness of the common inflammatory processes involved should provide the opportunity to more effectively treat the common causes of inflammatory conditions benefitting both physicians and patients. Managing the psoriasis patient as a whole with integrated therapy is likely to maximise outcomes.","keyphrases":["psoriasis","inflammation","comorbid","biologics","quality-of-life"],"prmu":["P","P","P","P","U"]}
{"id":"Eur_Spine_J-2-2-1602202","title":"Successful outcome of six-level cervicothoracic corpectomy and circumferential reconstruction: case report and review of literature on multilevel cervicothoracic corpectomy\n","text":"The authors report the successful outcome of a six-level corpectomy across the cervico-thoracic spine with circumferential reconstruction in a patient with extensive osteomyelitis of the cervical and upper thoracic spine. To the authors\u2019 knowledge, this is the first report of a corpectomy extending across six levels of the cervico-thoracic spine. Clinical relevance: the authors recommend anterior cage and plate-assisted reconstruction and additional posterior instrumentation using modern spinal surgical techniques and implants.\nIntroduction\nMulti (two or more)-level disease of the anterior cervical and upper thoracic spine can occur from degenerative, traumatic, infectious, and neoplastic causes. Successful treatment is dependent upon adequate decompression, immediate spinal reconstruction, and ultimately, the achievement of bony fusion. Decompression of the anterior cervical spinal canal can be achieved either by anterior cervical discectomy and fusion (ACDF) or anterior corpectomy and fusion (ACF). Across multiple levels, ACF offers several advantages over ACDF, including wider decompression and more complete removal of pathology causing spinal cord compression, and fewer graft-host interfaces that must consolidate for successful bony fusion. Corpectomies of the cervical and cervico-thoracic spine have been reported to extend across as many as four levels [5, 15, 16]. More extensive corpectomies have not been reported perhaps because of concerns for instrumentation failure and expected difficulty in obtaining adequate exposure of the anterior cervicothoracic spine.\nWe present a successful six-level cervicothoracic corpectomy and circumferential reconstruction in a patient with vertebral osteomyelitis and epidural abscess.\nCase report\nHistory and presentation\nA 50-year-old HIV-positive woman with a history of alcohol and drug abuse presented to our institution with a 7-month history of progressive neck pain, dysphagia, and radiculomyelopathy. Her past medical history was also significant for hepatitis B and multiple episodes of alcohol-induced acute pancreatitis. She denied a history of opportunistic infections. She had not been compliant with anti-retroviral therapy.\nPhysical examination\nOn physical examination, the patient was unable to extend her neck above the horizon and demonstrated 4+ out of\u00a05 motor strength throughout all muscle groups. Preoperative halo traction was not applied as the patient reported an intolerable amount of pain with any manipulation of her cervical spine.\nImaging studies\nPlain radiographs and computed tomography (CT) imaging demonstrated extensive bony destruction and a marked kyphotic angulation of the cervicothoracic spine (Fig.\u00a01). Magnetic resonance imaging revealed extensive prevertebral and epidural enhancement surrounding the cervical and upper thoracic spinal cord resulting in circumferential narrowing of the thecal sac (Fig.\u00a02). These findings were consistent with the diagnosis of vertebral osteomyelitis\/discitis and epidural abscess.\nFig.\u00a01(Left) Swimmer\u2019s view radiograph demonstrating kyphosis related to C4\u2013T2 osteomyelitis. (Right) Sagittal reformatted CT scan demonstrating extensive osseous erosion with kyphotic angulation from C6\u2013T2. Note that the apex of the kyphotic deformity is at the junction of the cervical and thoracic spineFig.\u00a02(Left) Sagittal T1 post-gadolinium MR sequence revealing extensive prevertebral and circumferential enhancing epidural abscess and enhancing vertebrae, compatible with osteomyelitis. (Right) Sagittal T2-weighted MR image demonstrating abnormal T2 signal within the spinal cord at the T1\u20132 level (arrow)\nOperation\nAn anterior approach to the cervical spine was performed first. The patient was positioned supine with traction on a halo ring in extension to partially reduce the kyphotic deformity. Next, a longitudinal incision along the medial border of the right sternocleidomastoid muscle was made and connected to a median sternotomy which was performed for exposure of the T1\u2013T3 vertebral bodies. Wide exposure of the C3\u2013T3 vertebral bodies was then carried out. Leksell rongeurs were used to remove the C4, C5, C6, C7, T1 and T2 vertebral bodies down to the posterior longitudinal ligament. Next, an appropriately-sized lordotic titanium cage (Pyramesh; Medtronic Sofamor Danek, Memphis, TN, USA) was packed with iliac crest autograft and recombinant human bone morphogenic protein (rhBMP) and impacted into position. A slotted dynamic Aesculap ABC plate (Aesculap AG, Tutlingen, Germany, and Aesculap Instrument Corp., South San Francisco, CA, USA) was then affixed to the spine with variable-angle screws into the C3 and T3 bodies.\nNext, the patient was turned prone and the halo head holder secured to a Mayfield frame. The posterior spine was dissected out from C1\u2013T5 and Stealth image guidance (Medtronic Sofamor Danek, Memphis, TN, USA) was then used to place pedicle screws bilaterally at C2 and from T1\u2013T4. Next, lateral mass screws were placed bilaterally at C3 and C4 and on the left side at C5. A laminectomy was performed from C4\u2013T2. Tapered rods were then attached to the lateral mass and pedicle screws and secured bilaterally. Thorough decortication of the facet joints from C2\u2013T4 was performed. Additional rhBMP and autograft was packed into the decorticated facets and lateral gutters. A lateral radiograph was obtained before closure to confirm satisfactory graft and hardware placement. After removal from the Mayfield frame, the halo brace and vest were then secured on the patient. Total blood loss was 2,000\u00a0cc and the total operating time was 8\u00a0h.\nPostoperative course\nPostoperative AP and lateral plain radiographs showed good hardware positioning with correction of the kyphotic deformity (Fig.\u00a03). The patient was kept in the halo brace postoperatively. She began ambulating with physical therapy on postoperative day (POD) #3. She experienced improvement in her neurological symptoms during the hospitalization. Due to the extensive dissection of her anterior cervical spine, a feeding tube was placed in the immediate postoperative period and was removed on POD #15. She was discharged to home on POD #30 in good condition to complete a 6-week course of intravenous antibiotics for Staphylococcus aureus osteomyelitis.\nFig.\u00a03Postoperative AP and lateral radiographs after six-level corpectomy from C4\u2013T2, anterior interbody contoured cage and anterior plating from C3\u2013T3. Posterior screw-rod fusion is evident from C2\u2013T4. Note that the T1 pedicle screws have been shortened to avoid breeching into the cage anteriorly\nFollow-up\nAt 4-month follow-up, the patient\u2019s neurological exam was improved from presentation and she demonstrated full strength throughout all muscle groups, normal sensation and resolution of neck pain. She was able to ambulate without assistance and denied any problems with breathing or swallowing. Plain flexion-extension radiographs of the cervico-thoracic spine were obtained after the patient was removed from the Halo vest (Fig.\u00a04). At last follow-up (2.5\u00a0years), the patient continued to remain neurologically intact without neck pain and no evidence of hardware failure.\nFig.\u00a04Flexion (left) and extension (right) plain radiographs obtained at 4-month follow-up demonstrate good hardware positioning without graft dislodgment. Note that the significant correction of sagittal plane alignment is maintained. There is no motion of the instrumented spinal levels\nDiscussion\nAnterior corpectomy and fusion is perhaps the most effective procedure for wide spinal cord decompression in patients with severe canal stenosis or pathology of the anterior spine [16]. Compared to more conventional surgical techniques for spinal cord decompression, such as ACDF, laminectomy, and laminoplasty, patients treated with ACF have better neurological recovery, less axial neck pain, and lower incidences of postoperative loss of sagittal plane alignment [21, 24, 25]. Extensive ACF involving three or more levels; however, involves more complicated surgical exposure and has been associated with increased rates of graft-related complications, including graft dislodgment, spinal cord compression, and pseudoarthrosis [12, 19, 20, 22]. That the anterior graft is subject to significant compressive forces after extensive ACF has been confirmed in biomechanical studies [8, 23]. Although each surgeon must take into account the clinical indications, risks and benefits of ACF in every patient, it seems logical to assert that the reported experience with ACF has been limited to four or fewer levels out of concern for these potential complications. Reconstruction after extensive ACF, therefore, is critical to prevent graft failure and allow for successful bony fusion.\nAnterior column reconstruction after extensive ACF may be performed using strut grafts or titanium mesh cages. Allo- or auto-graft struts may subside into the adjacent vertebral bodies or dislodge and cause cord compression [6, 12, 26]. Titanium mesh cages offer several advantages over strut grafts, including better correction of sagittal alignment (with pre-contoured cages), better endplate purchase, variable height and diameter options, and the ability to be packed with auto- or allograft bone [4, 7, 13]. Indeed, we were able to achieve significant correction of sagittal alignment in our patient with the use of a lordotic titanium cage. The addition of anterior plate fixation after interbody grafting enhances the rigidity and stability of the construct and therefore lowers the risk of graft-related complications [3, 5, 9, 16, 20]. Supplemental posterolateral fixation after extensive ACF has been found to further decrease the rates of graft-related complications, deformity correction loss, and pseudoarthrosis rates [8, 10, 16, 18, 20, 23]. Nevertheless, even with posterolateral fixation, in our experience there is inevitably a certain amount of subsidence of the anterior graft after extensive ACF. We used a slotted dynamic plate that allows for changes in the angle and alignment of the vertebral body screws to accommodate this expected subsidence (Fig.\u00a04). Other options to prevent subsidence-related failure of the anterior plate include using a cage only (no plate), buttress plates, or creating flanges in the cage that overlap the adjacent vertebral bodies and are secured with non-constraining screws [1].\nIt is important to note; however, that we did not perform postoperative CT to demonstrate bridging trabecular bone and therefore cannot definitively conclude that successfully bony fusion occurred in our patient. Nevertheless, we believe the absence of hardware motion on flexion-extension plain radiographs at 4-month follow-up does demonstrate the stability of this patient\u2019s extensive construct. Our patient has subsequently been lost to follow-up and it must be recognized that a pseudoarthrosis may manifest only after several years with a slowly progressive kyphosis.\nFinally, both in vitro and in vivo studies have shown that BMP and rhBMP can promote bony fusion and arthrodesis [2, 11, 14, 17, 27]. While fusion-promoting agents such as rhBMP are expensive and their use in the cervicothoracic spine is \u2018off-label, we believe it may improve the likelihood of fusion after the very extensive bone removal and instrumentation during this procedure and their use is therefore justified.\nConclusions\nA six-level corpectomy across the cervico-thoracic spine can be safely performed by combining a traditional longitudinal exposure of the cervical spine with a median sternotomy. Circumferential reconstruction with modern implants, such as an anterior contoured titanium cage-dynamic plate construct and posterior screw-rod fixation, is necessary to provide sufficient stability of the cervico-thoracic spine to allow for correction of sagittal alignment and to achieve bony fusion. While the lack of a CT scan demonstrating bridging trabecular bone precludes a conclusion that radiographic fusion occurred in our patient, we believe that the absence of new or worsening clinical symptoms at 2.5-year follow-up demonstrates the long-term stability and potential for successful fusion of this reconstruction. Fusion-promoting agents may be used to increase the fusion rate of these kinds of constructs with substantial bone loss and use of extensive instrumentation systems.","keyphrases":["corpectomy","reconstruction","fusion","cervical spine","internal fixation"],"prmu":["P","P","P","P","M"]}
{"id":"Arch_Orthop_Trauma_Surg-4-1-2225998","title":"An X-ray template assessment for distal radial fractures\n","text":"Background The current method for radiological measurements on plain X-rays of distal radius fractures is unreliable. We examined the reproducibility of a new X-ray assessment technique\u2014where the uninjured side is used as a template for the injured side\u2014compared to the conventional assessment technique.\nIntroduction\nThe important factors for decision-making in the treatment of distal radial fractures include the patient\u2019s vitality, fracture classification, fracture dislocation and soft tissue condition. Distal radial fracture deformities are quantified by X-ray measurements which are also used to classify the result after reduction (e.g., good or fair result) [1, 2]. However, the clinical usefulness of X-ray measurements is hampered by their limited reproducibility [3, 4]. It is also recognized that the modest reproducibility of X-ray deformations is one of the keystones for the lack of reproducibility in the treatment of distal radial fractures [3\u20135]. Clearly, when measurements cannot be made reliably, a straightforward clinical comparison of treatment techniques or reduction results is impossible, making it difficult to come to level 1 evidence-based treatment results and advices.\nOther studies that have evaluated sources of variance in the assessment of the quantitative X-ray measurements have reported difficulties with respect to the determination of the radial axis in the anterior\u2013posterior (AP) view, which is essential to assess the radial height and the radial inclination. This is obviously much less a problem in the lateral view, where the higher reproducibility value was found [6]. A second source of inaccuracy might be the use of the existing normal values for radial length (11\u00a0mm), radial angle (22\u00b0) and volar angulation (10\u00b0) [7\u201311], despite the normal variation that exists within the population [12].\nTo circumvent these difficulties, we designed a technique to assess fracture deformities of the distal radius that is based on the comparison of the injured side to the uninjured side. The approach is based on research findings that indicate that the patients\u2019 left and right side are comparable [13]. The aim of the study was to evaluate the reproducibility of the template assessment technique as compared with the direct quantitative measurement technique. We also examined whether the new technique resulted in a more reliable classification of reduction results for distal radius fractures.\nMaterials and methods\nInclusion criteria\nPatients aged 18\u00a0years or above with a unilateral dislocated distal radial fracture with the history of neither a distal radial fracture nor a distal radial operation were included after informed consent was taken. A stratification for the AO fracture types was performed to reflect the prevalence in clinical practice at a ratio of 5:2:3 for the AO type A, B and C distal radial fractures. The standard post-reduction (postero-anterior (PA) and lateral) X-rays and standard distal radius X-rays of the unaffected side of 30 consecutive patients visiting the emergency department were included after stratification for AO fracture type.\nEight experienced observers, 4 trauma surgeons, 3 orthopedic surgeons and 1 trauma radiologist, independently measured the X-rays and subsequently classified the reduction results using the Lidstr\u00f6m score (Fig.\u00a01).\nFig.\u00a01Anatomical radiological classification for distal radial fractures according to Lidstr\u00f6m\nIn the first direct measurement round, the x-rays were only measured quantitatively. The radial length and the radial inclination were measured on the posterior\u2013anterior plain X-ray film, while the dorsal\/volar angulation was measured on the lateral X-ray film. The measurements were performed with a goniometer and a ruler. The normal values for radial length (11\u00a0mm), radial inclination (22\u00b0) and volar angulation (10\u00b0) were used to assign a Lidstr\u00f6m score (Fig.\u00a02).\nFig.\u00a02Normal value measurement of the distal radius\nDuring the second round, scheduled 2\u00a0months later, the template measurement technique of comparing the fractured side with the unaffected side was performed by the same observers measuring the X-rays of the same patients. The observers were instructed to assign a Lidstr\u00f6m score using the X-rays of the unaffected distal radius as a template for the fractured side (Fig.\u00a03). The templates of the unaffected side were made by the investigators on transparent sheets, by tracing the distal radius, ulna and first carpal row from the unaffected side plain PA X-Ray film on a overlaid transparent sheet. For the lateral film, only the distal radius and the lunate bone from the unaffected side were drawn on to the overlaid transparent sheet. The reference points on the AP templates were the ulna and\/or the first carpal row (Fig.\u00a03). On the lateral template, the reference points were the radial shaft\/and or the lunatum. The measurements again were done with a goniometer and a ruler. And again the results were used to assign a Lidstr\u00f6m score.\nFig.\u00a03Measurements of distal radial fractures using the unaffected side as template\nStatistical analysis\nOverall inter-observer reproducibility of quantitative X-ray measurements for the difference in radial length, radial inclination and volar angulation for the eight observers was estimated using intraclass correlation coefficients (case 2, 1) [14]. The reproducibility of the ordinal Lidstr\u00f6m scoring was quantified using a weighted kappa coefficient (Kw). Since intraclass correlations (ICC) and Kw are computationally equivalent [15], both were interpreted using the arbitrary classification proposed by Landis and Koch [16]: <0.00 poor; 0.00\u20130.20 slight; 0.21\u20130.40 fair; 0.41\u20130.60 moderate; 0.61\u20130.80 substantial and 0.81\u20131.00 almost perfect agreement. Calculation of the reproducibility coefficients were carried out considering the pair-wise ratings of all possible combinations of observers. For example, observer 1 and 2, observer 1 and 3, observer 1 and 4, and so on to observer 1 and 8. Then observer 2 and 3, observer 2 and 4, observer 2 and 5 and so on to observer 2 and 8. This was done for each observer to the last observers 7 and 8. For 8 observers and 30 X-rays this resulted in 28 (8 \u00d7\u00a07\/2) possible pairs times 30 X-rays, totalling 840 pairs of observations. Sample size and number of observers were chosen to accommodate the precision of the reproducibility estimates we wanted to achieve. When the number of observations is 840, a 2-sided 95% confidence interval (CI) for a reproducibility coefficient arbitrarily set at 0.40 will extend 0.05 around the point estimate (from 0.35 to 0.45). Statistical evaluations were carried out using SPSS 11.5 and StatXact 3.02 for Windows.\nResults\nThirty patients with distal radial fractures were included. Of these patients, 15 fractures were of AO classification group A, 6 AO classification group B and 9 of AO classification group C.\nTable\u00a01, shows the reproducibility coefficients for the direct quantitative X-ray measures and the template assessment technique. Slight and insignificant increases in ICCs (95% CI) were observed for the radial length from 0.53 (0.48\/0.57) to 0.54 (0.49\/0.59) and volar angulation from 0.60 (0.56\/0.64) to 0.64 (0.60\/0.68). Marked improvements in reproducibility were observed for the radial inclination measurements. Intraclass correlations increased from 0.36 (0.30\/0.41) with the old technique to 0.49 (0.43\/0.55) with the template technique. The overall kappa for the Lidstr\u00f6m score in the old technique was 0.37 (0.31\/0.43) and improved in the template technique to 0.59 (0.52\/0.63).\nTable\u00a01Reproducibility coefficients for the X-ray measurements: old direct measurement technique versus template technique (N\u00a0=\u00a0840 observations)MeasureaOld technique (95% CI) Template technique (95% CI)Radial length 0.53 (0.48\/0.57)0.54 (0.49\/0.59)Radial inclination 0.36 (0.30\/0.41)0.49 (0.43\/0.55)Volar angulation 0.60 (0.56\/0.64)0.64 (0.60\/0.68)Lidstr\u00f6m0.37 (0.31\/0.43)0.59 (0.52\/0.63)aCategorical data, weighted kappa, numerical data, ICC\nDiscussion\nCompared to the traditional quantitative technique, the template assessment technique resulted only in an improved inter-observer reproducibility for the radial inclination. There were slight improvements in reproducibility of the radial length and volar angulation. However, there was a notable improvement in the reproducibility of reduction results as assessed using the Lidstr\u00f6m score.\nThe results of the quantative measurements of radial length and radial inclination are comparable to those reported by Kreder et al. They reported an inter-observer reliability coefficient of 0.49 for the radial length, and 0.32 for the AP radial angle [6]. As in our study, a better reproducibility was found for the volar angulation on the lateral view.\nIn this comparative study we had the same observers and the same patients with a 2-month interval and X-rays presented in another sequence order to overcome possible bias. The patients\u2019 sample was chosen to reflect the daily practice in which the AO type A fractures are more frequent than the Type B and C.\nOne could argue that the use of normal values for the traditional quantitative measurement technique would negatively affect its reproducibility since there is variation in anatomical proportions between individuals in the population. For example, the ulnar length varies within 30% of the population [12]. Therefore, the use of normal values may lead to inaccurate classification of the reduction result. However, in our study, the comparison with the normal values did not negatively influenc the reproducibility, since all observers used the same values. By doing so, the only source of error left was the direct measurement of the fractured side. When the normal side is used to compare with in the classical measurement way, there would be measurement errors of the fractured side and of the uninjured side.\nThe improved reproducibility of the fractures deformity classification as expressed by the Lidstr\u00f6m score (from 0.37 to 0.59) was primarily due to improvement in measurement of the radial inclination in the template technique. By using the template technique for the Lidstr\u00f6m score, one could expect the most improvements in both the radial length and radial inclinations. The volar angulation would not be expected to improve much since it is measured independently from the other side (Fig.\u00a01).\nSurprisingly, we could not demonstrate an influence of the template technique for the radial length in this study, although it is partly dependent on the position of the radial axis on the PA view and could therefore benefit from templating. An explanation for this phenomenon could be that for measuring the difference in radial height (as shown in Fig.\u00a03), there is still an axis needed. This becomes more eminent as the radial styloid is displaced more laterally (or rotated) and both tips of the radial styloid are not in line with the radial axis any more. Differences in placing the axis could be a source for variance, leading to a limited improvement in the Kappa value. In contrast, the radial inclination on the PA view in the template technique is basically independent of the radial axis and therefore more easy to measure with the template.\nA limitation of the template technique is that it may not be applicable in patients with a bilateral fracture or patients with a history of a distal radial fracture or deformity on the other side. In this study, only patients with a unilateral distal radial fracture were included, reflecting the most common occurrence of distal radial fractures.\nAnother limitation of this study was that, in order to avoid learning curves, only experienced observers were used, which may not always be the case in daily practice. Also, the templates of the unaffected side were made by the same investigator. In daily practice, making the template could also be a source of variation since the correct position of the templates may be more difficult in case of severe dislocation or with slight differences in projection of the bones due to the angle of the X-ray beam. Perhaps the use of digital subtraction may be helpful to overcome this.\nIn conclusion, by using the template technique for X-Ray measurement we improved the reproducibility of the classification of radiological dislocation of distal radius fractures which is a step to come to evidence based distal radial fracture treatment as outlined in two recent Cochrane reviews [4, 5]. This procedure can also be performed for other radiological parameters such as ulnar variance. Future research should focus on the improvement of the template technique and the use of digital measurement\u2014subtraction techniques\/software modules in this time of filmless X-Ray pictures.","keyphrases":["x-ray","distal radial fractures","radius","radius fracture","classification"],"prmu":["P","P","P","P","P"]}
{"id":"Acta_Neuropathol_(Berl)-3-1-2039791","title":"Pontocerebellar hypoplasia type 2: a neuropathological update\n","text":"Pontocerebellar hypoplasia type 2 (PCH-2; MIM 277470), an autosomal recessive neurodegeneration with fetal onset, was studied in six autopsies with ages at death ranging between 1 and 22 years. Three patients were distantly related. A case of olivopontocerebellar hypoplasia (OPCH; MIM 225753) was studied for comparison. Typical findings are: short cerebellar folia with poor branching (\u201chypoplasia\u201d), relative sparing of the vermis, sharply demarcated areas of full thickness loss of cerebellar cortex probably resulting from regression at an early stage of development, segmental loss of dentate nuclei with preserved islands and reactive changes, segmental loss in the inferior olivary nucleus with reactive changes, loss of ventral pontine nuclei with near absence of transverse pontine fibers and sparing of spinal anterior horn cells. Variable findings are: cystic cerebellar degeneration, found in two, with vascular changes limited to the cerebellum in one. Comparison to olivopontocerebellar hypoplasia (OPCH) strongly suggests a continuum of pathology between this disorder and PCH-2. Immunohistochemical evaluation of the endoplasmic reticulum stress response is negative. We conclude that the neuropathological findings in PCH-2 are sufficiently specific to enable an unequivocal diagnosis based on neuropathology.\nIntroduction\nThe term pontocerebellar hypoplasia (PCH) is applied to a group of autosomal recessively inherited neurodegenerations with prenatal onset. The distinguishing attribute of this group is the presence of combined pontocerebellar hypoplasia and atrophy present at the time of birth.\nSupratentorial structures are affected, though less prominently. The group has not been clarified genetically or biochemically. An initial proposal for classification [6] is based on two types in which type 1 (PCH-1) associates PCH with spinal anterior horn cell degeneration [14, 33], while type 2 (PCH-2) associates PCH with clinical extrapyramidal involvement and absence of anterior horn degeneration [5\u20137, 37, 44]. Early detailed neuropathological reports on PCH [9, 10, 26] in retrospect may have been cases of PCH-2, but cannot be matched with the customary classification because of lack of clinical details. Neuropathological finding in PCH-2 [5, 7, 37, 41] is microencephaly with severe cerebellar and ventral pontine hypoplasia. Typical pathological findings are: (1) subtotal loss of ventral pontine neurons and transverse pontine fibers with preservation of long fiber tracts; (2) atrophy and hypoplasia of the cerebellar hemispheres with relative sparing of the vermis and flocculi, variable loss of Purkinje cells and internal granule cells and reduction in folial length; (3) patchy loss of cerebellar dentate neurons, with the remaining dentate neurons grouped in \u201cislands\u201d; (4) loss of neurons in the inferior olivary nucleus with the winding pattern essentially spared; (5) absence of the medullary arcuate nuclei. Findings in PCH-1 are essentially similar, but preservation of spinal anterior horn cells distinguishes PCH-2 from PCH-1 [14]. Microscopic supratentorial findings in PCH-2 are non-specific with neuronal loss in various compartments, including the cerebral cortex, while myelin and myelination remain unaffected. Ultrastructural findings in a single reported neocortical biopsy are progressive neuronal loss in all layers and a peculiar degeneration in neurons in all layers that appear to start with patches of darkened endoplasmic reticulum [5]. Diagnosis during life is based on a combination of findings on MRI (Fig.\u00a01), a profile of clinical neurological deficits that includes severe cognitive delay, swallowing disturbance and chorea\/dystonia (less often, spasticity), exclusion of metabolic and chromosomal disorders with a similar MRI pattern and a family history compatible with autosomal recessive inheritance. In typical cases, behavioral and motor development is almost stagnant from the beginning, with onset of chorea\/dystonia during the first year and progressive microcephaly [7, 44]. Differential diagnosis requires exclusion of glycosylation disorders, especially congenital disorder of glycosylation type 1A (CDG1A) [2, 21, 23] and Muscle-Eye-Brain disease [16, 29], sequelae of extreme prematurity [28] and chromosomal disorders [4]. Overlapping neuropathological features exist between PCH-2 and olivopontocerebellar hypoplasia (OPCH), a more severe disorder with an essentially similar combination of hypoplasia and degeneration affecting the hindbrain [3, 11, 18, 35]. Autosomal recessive inheritance in OPCH is suggested by its recurrence pattern in families. Clinical findings are variable, but include such indicators of prenatal onset as polyhydramnios and contractures. Its main structural features are severe cerebellar hypoplasia with emphasis on the hemispheres, absence of neurons in the ventral pons, subtotal absence of cerebellar dentate nuclei, diminished or absent olivary winding and absence of spinal anterior horn involvement. In a recent PCH classification, the original types 1 and 2 have remained identical, while olivopontocerebellar hypoplasia has become type 4, PCH-4 [36]. Type 3, with optic atrophy as its main distinguishing clinical feature [38], has not been studied neuropathologically yet, while type 5 until now is represented by a single family. The number of papers addressing the neuropathology of PCH-2 with sufficient clinical detail to support a type 2 classification is limited [5, 37, 41] and results of immunohistochemistry have not been reported yet. The aim of the present report is to provide a range of pathological findings based on a series of six autopsies with ages at death varying between 15\u00a0months and 22\u00a0years and to provide an update on neurodegenerative features using more specific staining techniques. Special attention is given to variability in cerebellar degeneration, degenerative changes in pons and lower brainstem and the types of astroglial and microglial reaction. Three patients (no. 4, 5, 6) originate from an area with a high degree of intermarriage and are related by genealogy. A case of olivopontocerebellar hypoplasia (PCH-4) is included for comparison of its neuropathology to PCH-2.Fig.\u00a01MRI of patient 6 at 2\u00a0months. a Midsagittal inversion recovery sequence: flat aspect of ventral pons and moderate vermal hypoplasia. b Coronal T2-weighted image: severe hypoplasia of the cerebellar hemispheres (arrows) together filling only a fraction of the posterior fossa\nMaterials and methods\nStandard autopsies were performed on all patients mentioned except patient 1 who underwent a coroner\u2019s autopsy with only the cerebellum and brainstem made available for the present study (Table\u00a01).\nTable\u00a01Clinical profiles, macroscopic and principal microscopic findingsPatient no.1234a5a6a7SexFemaleMaleMaleMaleMaleFemaleMaleType of PCHPCH-2PCH-2PCH-2PCH-2PCH-2PCH-2OPCHParents, relatedNoNoYesYesYesUncertainYesNeonatal periodPoor swallowingPoor swallowing, seizureUnrestSeizurePoor swallowingPoor swallowing, unrestAbsent spontaneous respiration, tremor\/clonusHead circumference at birth\u22121 SD\u22122 SD\u22122 SDMicrocephaly progressive after birth++++++Motor development after birthNo motor control, some visual pursuit Minimal head control, some visual pursuitMinimal head control, some visual pursuitNo motor control, some visual pursuitNo motor control, no visual pursuitNo motor control, no visual pursuitChorea\/dystonia++++Spasticity+Forced flexion of extremitiesSeizures++++++\u2212Previous publicationPatient 9, [7]Patient 11, [7]Patient 7, [7]Patient 3, [7]\u2013\u2013\u2013Age at death1\u00a0year 2\u00a0months12\u00a0years22\u00a0years 10\u00a0months15\u00a0years 6\u00a0months2\u00a0years 4\u00a0months3\u00a0years 7\u00a0months2\u00a0daysCause of deathCot deathHyperthermiaCachexiaPneumoniaPneumoniaHyperthermia, rhabdomyolysisAbsent spontaneous respirationTotal brain weight (g)570720 1,110After fixation, 687660660After fixation, 172Control brain weight g\u00a0\u00b1\u00a0SD*940\u00a0\u00b1\u00a0120b1440\u00a0\u00b1\u00a010b1440\u00a0\u00b1\u00a020b1410\u00a0\u00b1\u00a010b1120\u00a0\u00b1\u00a0200b1090\u00a0\u00b1\u00a0230b433\u00a0\u00b1\u00a057cWeight of cerebellum\u00a0+\u00a0brainstem3216\u2013\u2013\u201312\u2013Cerebellar cortexSparse segmental lesions in hemispheres; diffuse loss of P- and granule cells Sparse segmental lesions in vermis and hemispheres; diffuse loss of all P- and granule cellsNear total destruction, only GFAP positive outlines remainingSegmental lesions in hemispheres; diffuse loss of all P and granule cellsSegmental lesions in vermis and hemispheres; diffuse loss of all P and granule cellsSegmental lesions in vermis and hemispheres; diffuse loss of all P and granule cellsMajor (dorsal) part of vermis and hemispheres \u201cdenuded\u201d, sparing of flocculus and nodulusDentate nucleusIslandsIislandsNo remnantsIslandsIslandsIslandsIslandsCerebellar cysts\u2212\u2212+\u2212+\u2212\u2212Inferior olivary nucleusReduced folding; segmental lesionsReduced folding; segmental lesionsTotal loss of structureReduced folding; segmental lesionsReduced folding; segmental lesionsReduced folding; segmental lesionsNo foldingVentral ponsLoss of nuclei, transverse fibersLoss of nuclei, transverse fibersLoss of nuclei, transverse fibersLoss of nuclei, transverse fibersLoss of nuclei, transverse fibersLoss of nuclei, transverse fibersLoss of nuclei, transverse fibersaRelated by common ancestry (Fig.\u00a02)bReference for fresh brain weights [12]cReference for fixed brain weights [15]\nTissue preparation and immunocytochemistry\nFormalin-fixed, paraffin-embedded tissue was sectioned at 6\u00a0\u03bcm and mounted on organosilane-coated slides (SIGMA, St Louis, MO, USA). Representative sections of all specimens were processed for immunocytochemical reactions to synaptophysin (polyclonal rabbit, DAKO, 1:200), microtubule-associated protein (MAP2; polyclonal rabbit, Sigma, 1:2,000), calbindin (mouse clone CL-300, Sigma, 1:400), SMI31 (Sternberger Monocl., IgG 1, 1:20,000), phosphorylated neurofilament (NF; mAb clone 2F11, DAKO, 1:500), myelin basic prote\u00efn (MBP; polyclonal, Neomarkers\/RB-1460-A, 1:400), vimentin (mouse clone V9, DAKO, Denmark, 1:1,000), glial fibrillary acidic protein (GFAP; polyclonal rabbit, DAKO, Denmark, 1:4,000), (HLA)-DP, DQ, DR (mouse clone CR3\/43, DAKO Glostrup), HLA-DR (mouse clone Tal1b5, Sigma, USA, 1:200), CD68 (KP1 clone, Novocastra), caspase-3 (polyclonal, 1:100 dilution, Cell Signalling Technology, Beverly, MA, USA), ubiquitine (polyclonal, DAKO\/Z458, 1:1,600), \u03b1-synuclein (polyclonal, Affiniti\/SA 3400, 1:2,000), BiP\/GrP\/78 [polyclonal rabbit, 1:100 dilution Santa Cruz Biotechnology (SCBT), Santa Cruz, CA, USA], phosphorylated (p)PERK (pThr981, polyclonal rabbit, 1:800 dilution, SCBT) and phosphorylated (p)eIF2\u03b1 (pSer51, polyclonal rabbit, 1:500 dilution, Sigma).\nParaffin sections were routinely stained with HE, LFB and Nissl. Cerebellar sections were silver stained according to Bielschowsky. Immunocytochemistry was carried out on paraffin-embedded tissue as previously described [2, 19]. The sections were incubated for 1\u00a0h at room temperature followed by incubation at 4\u00b0C overnight with primary antibodies. Single-label immunocytochemistry was performed using avidin-biotin peroxidase method. Chromogen 3,3-diaminobenzidine or 3-amino-9-ethyl-carbazole (AEC, Zymed, San Francisco, CA) was used and nuclei were stained with hematoxylin. Sections incubated without the primary Ab, with preimmune sera were essentially blank. For double labeling (with ubiquitin and HLA-DR), sections were incubated for 2\u00a0h at RT with Alexa Fluor\u00ae 568 and Alexa Fluor\u00ae 488 (anti-rabbit IgG, anti-mouse IgG; Molecular probes, Eugene, USA). The sections were then analyzed by means of a laser scanning confocal microscope (Bio-Rad, Hercules, CA, USA; MRC1024) equipped with an argon-ion laser.\nResults\nDetails of each patient are given in Table\u00a01. Figure\u00a02 shows the pedigree, which links patients 4, 5 and 6 to a common ancestry. Parents of patients 4 and 5 are consanguineous. The three parental couples originate from the Dutch community in Volendam, which has a high degree of consanguinity. They link genetically to one or two ancestral couples: I, 1\u00a0\u00d7\u00a0I, 2 and III, 4\u00a0\u00d7\u00a0III, 5 born in the eighteenth century. A genealogical link between these ancestral couples, who carry identical family names, is suggested but yet unproven.\nFig.\u00a02Pedigree linking patients 4, 5 and 6 in the present report; IX, 1 and IX, 3 were reported before [5]\nCerebellar cortex\nThe cerebellar hemispheres are severely affected in all. On macroscopic examination, all major lobes are reduced in width and dorsoventrally flattened. Size was best preserved in the transverse plane. This asymmetric diminution in size and the relative sparing of the vermis lend the cerebella a butterfly or batwing aspect (Fig.\u00a01b). Four of the six cerebella (cases 2, 4\u20136) are sectioned perpendicular to the folia with the line of the sectioning running from the culmen to the posterior pole. These sections include the flocculus. In two cases (1, 3), transverse sections of hemispheres and vermis are prepared. Low magnification (Fig.\u00a03) shows the diminished size of the cerebellar hemispheres, folial shortening and diminished number of folial branches compared to a control (Fig.\u00a03a, b). In the most affected case (Fig.\u00a03g, h), no branches are seen at all, while in a less affected case folial branches are reduced to two or less (Fig.\u00a03e, f). The other cerebella displayed regular folial branching (Fig.\u00a03c, d), though less in quantity than the control specimen (Fig.\u00a03a, b).\nFig.\u00a03Hypoplastic folia. Stains, synaptophysin. Sagittal sections of cerebellar hemispheres are seen on the left and detailed views on the right. Stunted folial growth with decreased number of folial branches. a, b Male, 8\u00a0years, with accidental death and normal findings on autopsy (control); c, d case 4; e, f case 6; g, h case 5. Marker bars on the left 1\u00a0cm, and on the right 1\u00a0mm\nThe vermis is fully developed in all cases with the individual lobules well developed in size and branching pattern. Sagittal sections of the vermis, available in four, show some inequality in thickness of the white matter cores of the lobules, with the declive\u2013folium\u2013tuber complex the best preserved part in three (cases 2, 4, 5) and the pyramis in one (case 5; Fig.\u00a04).\nFig.\u00a04Vermis cerebelli, case 5. Stains, a HE, b GFAP, c neurofilament protein, d vimentin. a\u2013c Adjacent sections with circumscript segments of loss of all layers and glial reaction. Arrows in (a) point to circumscript areas of lost cortex. d Sharply demarcated foci and replacing bands of astrocytes possibly modified Bergmann cells. Marker bar in (d)\u00a0=\u00a0500\u00a0\u03bcm\nOn microscopy, regressive changes are present in all, with the vermis relatively spared. In the vermis, diffuse but moderate loss of Purkinje cells, internal granule and folial white matter is seen in all lobules. Two cases present characteristic sharply delimited foci with full thickness loss of vermal cerebellar cortex with astroglial replacement (2, 5; Fig.\u00a04).\nThe hemispheric cerebellar cortex is affected in all cases by variable loss of Purkinje cells, thinning of the granule cell layer and inhomogeneous loss of myelinated fibers within folia and central white matter. The remaining axons are well myelinated and stain for SMI 131 and MBP. In addition to diffuse cortical changes, sharply delimited areas of up to 2\u00a0mm in width with loss of all cortical neurons are found in 5\/6 cases (1, 2, 4, 5, 6). These areas locate mostly, but not exclusively, to stretches of cortex connecting adjacent folia. The number of these areas varies between two per whole cerebellar hemispheric section (1, 2) to ten or more (5, 6; Fig.\u00a05). In one case (3), representing the oldest surviving patient, destruction of the whole cerebellum is almost complete with subtotal loss of all neuronal elements (Fig.\u00a010a). Even in this case, patches of whole thickness loss of cortex alternate with segments of cortex where gliosis still preserves the original frame of cortical structure (Fig.\u00a010a). In all cases, the flocculus belongs to the relatively spared areas of the hemispheric cortex. In one case, the sparing of the flocculus stands out in sharp contrast to the rest of the hemispheric cortex (Fig.\u00a06).\nFig.\u00a05Cerebellar hemispheric cortex with segmental lesions. Stains, a, h synaptophysin, b\u2013d vimentin, e, g, i GFAP, f MAP-2. Case 6: opposing arrows mark lesions with loss of all cortical layers (a) and glial replacement (b). c, d Case 5: segment of whole thickness loss of cortex is replaced by a single layer of evenly spaced astrocytes, their topography compatible with modified Bergmann cells. e, f, g Case 4: segmental loss of cortex with glial replacement marked by opposing arrows; in (f) higher magnification shows a group of remaining isolated granule cells. h, i Case 2: segmental loss of cortex on top of a folium in moderately hypoplastic cortex. Marker bars: a\u2013c, e, g\u2013i\u00a0=\u00a00.5\u00a0mm, d\u00a0=\u00a00.25\u00a0mm, f\u00a0=\u00a050\u00a0\u03bcmFig.\u00a06Flocculus. Cerebellar hemispheric cortex, case 2, synaptophysin. Floccular cortex shows broader folial cores and denser granule cell population than other parts of the cortex\nDentate nucleus\nThe dentate nucleus is affected in all cases by major loss of dentate neurons and by residual cells being grouped in \u201cislands\u201d (Figs.\u00a07, 8). \u201cIslands\u201d as well as individual neurons are surrounded by whorls of nerve fibers. Residual neurons and the proximal parts of their dendrites stain positive for MAP-2 and synaptophysin. Synaptophysin staining is abundant on neurons and proximal dendrites signifying retained network activity in surviving neurons. A peculiar vacuolization surrounds individual dentate neurons (Fig.\u00a08a, b). A proportion of these vacuoles stain positive for calbindin, suggestive of swollen axon terminals (Fig.\u00a08c, d). Cerebellar white matter surrounding the dentate nucleus on the inside and outside shows thickened axons with diameters up to 12\u00a0\u03bcm (Fig.\u00a08). The other internal cerebellar nuclei could not be identified with certainty.\nFig.\u00a07Dentate nucleus, case 6. MAP-2. Loss of normal outline with remaining dentate neurons clustered in \u201cislands\u201d. Marker bar\u00a0=\u00a01\u00a0mmFig.\u00a08Dentate nucleus, details. Stains: a MAP-2, b MBP, c, d calbindin, e synaptophysin, f SMI31. a Case 6: fine vacuolation surrounding neurons. b Case 1: myelinated fibers separate individual neurons and their surrounding vacuoles. c, d Cases 6, 1: some vacuoles are decorated by antibody; e Case 2: concentration of vesicles (dots) indicates high synaptic density on soma and dendrites of surviving neuron. f Case 1: thickened axons in hilus of dentate nucleus. Marker bars\u00a0=\u00a0100\u00a0\u03bcm\nCystic degeneration of the cerebellum\nIn two cases, destruction resulted in cyst formation (cases 3, 5). MR investigation shows cystic change in the cerebellar hemispheres of patient 5 at the age of 1\u00a0month (Fig.\u00a09b ). On autopsy the cysts are collapsed, and can only be retrieved by serial sectioning. The border of the cyst is lined by reactive astrocytes and macrophages (Fig.\u00a09a, c). In case 3, cyst formation compounds the subtotal loss of cortex, dentate nuclei and fiber tracts. A remarkable involvement of small sized vessels, not present in the other cases, consists of intimal proliferation and splitting of the elastica interna. This abnormality is only present in the cerebellum and its surrounding arachnoid vessels (Fig.\u00a010). Electron microscopy fails to show storage material or enlarged lysosomes (not shown). Double labeling for ubiquitin and HLA-DR shows that ubiquitin is mainly present in the vessel walls, and only in minor part associated with macrophages (HLA-DR; Fig.\u00a010c). Vascular abnormalities are absent in other parts of the brain and in internal organs, including the kidneys.\nFig.\u00a09Cerebellar cyst, case 5. a Vimentin, c HLA-DR. a Collapsed cyst seamed by astroglial reaction. b T1-weighted axial image of posterior fossa of patient at 1\u00a0month age shows the cysts in expanded state. c Macrophage reaction on the inside of the cystFig.\u00a010Regressive changes, case 3. Stains: a GFAP, b HLA-DR, c double labeling for HLA-DR (green) and ubiquitine (red), confocal laser microscopy, d ubiquitin. a Transverse cerebellar section with cortical and subcortical gliosis, loss of the subcortical and central cerebellar structure, in part cystic, broken cortical lining in places suggest segmental lesions similar to other cases before ultimate decay; b abundance of reactive macrophages, predominantly in and surrounding blood vessels with thickened vessel walls; c ubiquitin is mostly restricted to vessel wall and macrophages are partly positive for ubiquitin; d neuron of nucleus magnus raphes (pontine tegmentum) with cytoplasmic inclusion and positivity extending into the neurite. Marker bars: b\u00a0=\u00a0200\u00a0\u03bcm, c\u00a0=\u00a050\u00a0\u03bcm\nInferior olivary nuclei\nThe inferior olivary nucleus and the dorsal and medial accessory olivary nuclei are identified in each case. Mature folding of the inferior olivary nucleus is present in the lateral leaf and reduced in the medial leaf in one case (3; Fig.\u00a011). In all others, folding is reduced in both leaves, the medial leaf being more affected than the lateral. Discontinuous loss of parts of the nerve cell band is noted in each case, best shown by MAP-2 staining (Fig.\u00a011). In one case (3) nearly all olivary neurons, including the accessory olivary nuclei, are lost. Part of this loss is mirrored by vimentin positive astrocytes forming a ghost image of the lost neuronal framework (Fig.\u00a011c, d). Macrophages are present within and around the vanished structure, but not in a topographically distinct pattern. In all cases, internal and external olivary fibers are reduced or lost entirely.\nFig.\u00a011Inferior olivary nucleus. Stains: a\u2013c MAP-2, d vimentin. case 6 and 5: segmental loss of inferior olivary nucleus with intact dorsal (a) and medial (b) accessory nuclei. c Case 3. Complete loss of nucleus, including accessory nuclei, only sparse isolated neurons remaining, indicated by arrowheads; inset shows magnified solitary olivary neuron. d Case 3. Outline of lost lateral leaf reveals mature folding. Marker bars\u00a0=\u00a00.5\u00a0mm\nPons\nVentral pons\nSimilar findings are present in all cases. Diminution in size of the pons to about half or less of its size in transverse diameter is entirely due to loss of its ventral compartment. The majority of ventral pontine neurons and transverse pontine fibers are lost with sparing of the long motor and sensory tracts. Prominent gliosis (Vim and GFA) and microglial activation are found in the affected region. Staining for synaptophysin and calbindin in the remaining neurons does not reveal specific patterns. Caspase and ubiquitin staining is negative in the few remaining neurons.\nPontine tegmentum\nNo abnormalities are encountered in the cranial nerve nuclei, raphe or central tegmental tract. Ubiquitin staining in the reticular nuclei, especially the nucleus raphes magnus in case 3, reveals numerous neurons with ubiquitin-positive cytoplasmic inclusions. Caspase-3 staining is negative in all cases.\nOther findings in the brainstem\nTotal absence of the medullary arcuate nuclei is found in all cases. Widespread presence of lipopigment (lipofuscin) is observed in all cases, especially in larger neurons (dentate nuclei, olivary nuclei, pontine neurons, brainstem tegmentum and cerebral cortex), even in the youngest individuals. Staining for \u03b1-synuclein was negative in the brainstem as well as in the cerebellum in all cases.\nMidbrain, thalamus and basal ganglia\nNucleus ruber has diminished size and cell density in case 3. This case contrasts with other cases by its subtotal destruction of the cerebellum. In all available cases (2\u20136), the caudate nuclei are macroscopically atrophic. There is a general increase of astrocytes and microglia throughout the basal ganglia and thalami. Myelination is normal. No specific abnormalities are found. In one case severe necrotic changes are found in the globus pallidus, representing acute postischemic damage.\nCerebral cortex\nMacroscopic gyral atrophy is seen in three of five cortices available for study. The temporal lobes are affected in one (case 2), the frontal and temporal lobes in one (case 3) and all lobes in one (case 4). Myelination is normal in large sections (luxol fast blue), even in atrophic parts. Cortical development appears normal on routine staining. A diffuse increase of reactive astroglia and activated microglia is seen throughout the neocortex and subcortical and central white matter in all cases. Except for postischemic changes in one case (6), the hippocampal and dentate gyri are normal.\nOlivopontocerebellar hypoplasia\nCase 7: this male baby was born at 38\u00a0weeks ga. The amount of amniotic fluid was normal. Spontaneous respiration was absent. He had trismus, severe tremor\/clonus and varus deformity of the legs. On autopsy, the supratentorial parts of the brain are small size, but of normal appearance, while the cerebellum and brainstem are disproportionately small.\nThe cerebellum is remarkable for its complete sparing of the nodulus and flocculus, its severe cortical depletion on the dorsal side and hypoplastic cortical development on the ventral side of the hemispheres (Fig.\u00a012). Folial development of the neocerebellum is severely restricted with few folia on its dorsal parts. Microscopic examination of the flocculus and vermis is normal. Neocerebellar hemispheric cortex shows isomorphic gliosis through transformed Bergmann cells (Fig.\u00a012b). MAP-2 staining shows some retained neuronal elements (Fig.\u00a012c, d). The dentate nuclei are broken up into small islands that can only be retrieved by neuron-specific staining. This reveals a few small clusters with well-developed dentate neurons left in place (Fig.\u00a012e, f). The inferior olivary nuclei have the form of horseshoes without folding, and with the segments of the nucleus missing on both sides (Fig.\u00a013). The accessory olivary nuclei are spared. The ventral pons is severely underdeveloped with most of the pontine nuclei missing, dense gliosis and absence of CD68 staining. A diffuse astrocytic gliosis is found throughout the cerebral hemispheres, especially affecting the subcortical white matter and the fiber tracts of the striatum. The neocortex is unremarkable.\nFig.\u00a012OPCH, case 7. Stains: a, e synaptophysin, b vimentin, c, d, f MAP-2. a Whole mount transverse section of cerebellum and medulla oblongata: denuded hemispheric cerebellar cortex and striking preservation of nodulus and flocculi. b Hemispheric cortex lined by a single row of fibrous astrocytes. c \u201cEmpty\u201d cerebellar cortex, arrows indicate some remaining neurons. d Higher magnification of c, arrowhead indicates bipolar neuron, possibly basket cell; arrow indicates malpositioned Purkinje cell. e Overview of cerebellar hemisphere, arrows at residues of dentate nucleus. f Single residual island of dentate nucleus with well-developed neurons. Marker bars: b 200\u00a0m\u03bc, c 1\u00a0mm, d 100\u00a0m\u03bc, e 1\u00a0mm, f 100\u00a0m\u03bcFig.\u00a013OPCH, case 7. Stain, synaptophysin. Medulla oblongata with olivary nuclei. Inferior olivary nucleus with complete lack of folding. Lateral and medial leaves with segmental defects. Medial accessory olive is preserved in its dorsal part and shows discontinuities in its ventral part. Marker bar 0.5\u00a0mm\nUnfolded protein response \nWe previously observed morphological changes in the endoplasmic reticulum (ER) in a biopsy of a PCH-2 patient by electron microscopy. This led us to investigate whether the unfolded protein response (UPR)\u2014a stress response of the ER [40]\u2014is activated in neurons of PCH-2 patients. The UPR was studied in sections from the frontal cortex (5\/6 patients) and the cerebellar hemispheres, which included the dentate nucleus (6\/6 patients). As a marker of induction of the UPR, we used an antibody specific for the phosphorylated pancreatic ER kinase (pPERK), which is an ER stress transducer that is only phosphorylated during the UPR. Previously, we have used this method to show UPR activation in the brain of patients with Alzheimer\u2019s disease [19] or Parkinson\u2019s disease [20]. We find no immunopositive staining for pPERK in any of the patients, neither in the cortex, nor in the cerebellum. Eukaryotic initiation factor 2\u03b1 (eIF2\u03b1) is the substrate of pPERK and using a peIF2\u03b1 antibody, increased staining intensity is observed as compared to the control patient in the Purkinje cells of patients 2, 4 and 5. This is not found in patient 1 and severe degeneration in patients 3 and 6 precludes proper analysis. Apart from the fact that differences in intensities between pathological samples are not always readily interpretable, this does not provide evidence for UPR activation in the absence of pPERK reactivity, because other kinases can also phosphorylate eIF2\u03b1. The ER chaperone BiP is upregulated during the UPR, but in accordance with the pPERK data, no obvious differences in levels are observed. In summary, we find no evidence for activation of the UPR.\nDiscussion\nCerebellar cortex, dentate nucleus and olivary complex\nThe series of six PCH-2 cases presents developmental and regressive abnormalities of the cerebellar cortex, dentate nuclei and olivary nuclei. Cerebellar cortical findings range between short unbranched folia in the least-developed cerebellum (case 5) to shortened folia with a decreased number of branches. In the most affected cerebella, poorly developed folia alternate with flat stretches of surface that lack proper cortical lining. A similar observation is reported in PCH-1 cases [13, 14, 46], OPCH [35], a case classified as \u201cneocerebellar hypoplasia with combined olivo-ponto-dentatal degeneration\u201d [24], and PCH-2 with spasticity [42]. Reactive glial changes were reported in PCH-1 [46]. All \u201cempty\u201d segments of cortex in the present study have a similar appearance: a single evenly spaced row of transformed Bergmann cells, positively staining for vimentin. Synaptophysin and MAP-2 staining reveal the sparse presence of nerve fibers. This is the first time this patten is observed by immunohistochemistry. The earliest stages of cerebellar development are probably normal because they require the coordinate development and juxtaposition of two cell types: the Purkinje cells that arise by radial outward migration and the external granule cells that arise by tangential migration and enter the future cortex to become internal granule cells [39]. The microscopic structure of the \u201cempty\u201d segments betrays previous cortical development. The earliest estimate for the loss of cortex in these segments follows from the afolial stretches in patients 5 and 6 that represent a stage of cerebellar development prior to or coinciding with beginning folial outgrowth, around 25\u00a0weeks gestational age in the human fetus [39]. Cortical segmental loss, however, is not restricted to afolial segments, but is also seen in more developed hemispheric cortex (e.g., Fig.\u00a05h, i) and mature vermis. This suggests a time frame for segmental loss of cerebellar cortex from incipient foliation to encompass late cortical development. It cannot be established presently whether segmental loss is a completed process or still active at the time of demise. No microscopic vascular changes are seen in the vicinity of segmental lesions and their distribution is not restricted to arterial boundary zones. Also, cerebellar vascular changes observed in one case (3) are not found in any of the other cases and therefore offer no explanation for segmental cortical lesions. The spectrum of changes in the inferior olivary nucleus in this series similarly encompasses early and late developments. In five out of six cases, the degree of folding is immature, while in the sixth case (case 3), a mature folding pattern of the nucleus can be gauged by observing the outline of the astroglial scar. According to one study in human fetal material [30], the mature form of the nucleus is reached with a 300\u00a0mm crown\u2013heel length, i.e., 22\u201327\u00a0weeks. Segmental loss is observed in 5\/6 cases in the olivary nucleus, and the sequence of progressive loss may have been similar in the remaining oldest case (case 3) with loss of the entire nucleus. Segmental lesions in the inferior olivary nucleus in PCH were reported in 1926 [26]. The consistency of this finding is shown in the present series using immunohistochemistry. The third structure with a segmental pattern of loss is the dentate nucleus. We have no evidence in this series of a normal outline of the dentate nucleus during any phase of its development. One previous report [32] however relates a glial scar linking the \u201cislands\u201d of the dentate in PCH.\nWe examined a case classified as OPCH and on comparison to the PCH-2 series found important similarities: (1) flat stretches of cerebellar cortex and ghosts of primitive folia carrying microscopic remnants of a pre-existent developed cortex, (2) dentate islands with well-developed neurons, (3) segmental loss in the inferior olivary nucleus, (4) sparing of nodulus and flocculus. These findings reveal a remarkable analogy to the segmental cortical lesions in PCH-2. Differences between PCH-2 and OPCH are: (1) the horseshoe appearance of the inferior olivary nucleus compared to the diminished folding in PCH-2 indicates earlier onset of the pathological process in OPCH, (2) the extent of afolial cerebellar cortex.\nRegressive changes\nIn addition to developmental abnormalities, some findings indicate an ongoing process, such as cytoplasmic ubiquitin positivity in pontine tegmental neurons in case 3, the widespread presence of astrocytic and microglial reaction and the abundance of lipopigment, e.g., in cerebral cortical, cerebellar cortical, dentate and olivary neurons. Cyst formation in the cerebellar white matter (cases 3, 5) and vascular changes in the cerebellum (case 3) also signify a regressive element in PCH-2. A previously made observation on darkened stretches of endoplasmic reticulum in cortical neurons in a cortical biopsy [5] prompted us to investigate the ER stress response with negative results.\nComparison of PCH-2 to other neurodegenerative disorders\nThe nearest equivalent in neuropathological terms to PCH-2 is the group previously named olivo-ponto-cerebellar atrophy (OPCA), presently known as spinocerebellar ataxia (SCA). While olivary and\/or dentate nucleus may be affected, none of the typical features, especially dentate fragmentation, segmental lesions of the cerebellar cortex and olivary nucleus is part of the pathological spectrum of these disorders [8, 25]. Pathological studies on autosomal recessive or X-linked central nervous system disorders with onset in childhood and predominant impact on the cerebellum include the H\u00f8yeraal-Hreidarsson syndrome [1, 22], progressive encephalopathy, hypsarrhythmia, optic atrophy (PEHO) [16], primary granular cell degeneration of the cerebellum [31], and infantile onset spinocerebellar ataxia with neuropathy (IOSCA) [27]. These disorders display cerebellar atrophy rather than hypoplasia, lack the segmental cortical anomalies, the characteristic fragmentation of the dentate and the segmental loss of the olivary nucleus described above with the possible exception of IOSCA in which segmental loss of the olivary nucleus is documented [27]. Neurometabolic disorders of glycosylation, especially congenital disorder of glycosylation (CDG) type 1A and the alpha-dystroglycanopathies manifest cerebellar hypoplasia, often in combination with pontine hypoplasia. Neuropathological findings in CDG1A do not include circumscript dentate, olivary or cerebellar cortical defects [2, 21]. Alpha-dystroglycanopathies combine a severe type of congenital muscular dystrophy with cerebral, cerebellar and brainstem malformation and deficiency of alpha-dystroglycan (alpha DG) [29]. Alpha DG deficiency in this group probably causes a tangential neuronal migration defect affecting migration from the rhombic lip into the cerebellum and ventral pons. The resulting pathology [17] causes heterotopia and impaired foliation in the cerebellum. Summarizing previous and present data on PCH-2, this disorder bears a number of features not encountered in other disorders with the exception of PCH-1, which appears to be closely related but for the involvement of spinal anterior horn cells in the latter.\nNeural mechanisms\nA question may be raised whether segmental lesions in the three nuclear systems, as observed in PCH-2, could be effected through a single mechanism. Cerebellar cortex, olivary nuclei and dentate nuclei are functionally linked through the climbing fiber system, which is topographically organized in sagittal modules each formed by a segment of the olivary nuclear complex, its collaterals to the cerebellar nuclei, including the dentate nucleus, and a strip of cerebellar cortex [45]. A sagittal parcelleation of the cerebellum in biochemically distinct regions, however develops independent of the olivo-cerebellar projection [43]. Lesions of the olivary complex are not known to cause segmental lesions in other parts of the climbing fiber system. Experimental administration of the drugs harmaline or ibogaine to rats results in tremor and loss of sagittal strips of vermis. The role of the olivary nucleus in this type of segmental damage is highlighted by prior chemical ablation of the inferior olivary nucleus by 3-acetyl pyridine, which prevents the segmental damage from occurring. Overexcitation of the olivary nucleus rather than primary loss is apparently required for this type of lesion to occur [34]. The type of lesion reported in rodents is reminescent of the segmental lesions in PCH-2. One argument in support of a possible role of the olivo-cerebellar projection in PCH-2 is the selectivity of the cerebellar cortex involved, in which the vermis and flocculus are relatively spared. The climbing fiber output of the inferior olivary nucleus to the cerebellum is mainly directed to the cerebellar hemispheres. The flocculus and nodulus, both relatively spared in PCH-2 and in OPCH, are reciprocally connected to the vestibular nuclei [45]. We have not found abnormalities in the vestibular nuclei in our series.\nIn addition to cortical segmental loss, a more diffuse kind of degeneration is found, which includes loss of cortical elements such as Purkinje cells and granule cells. This lends a \u201cmoth-eaten\u201d aspect to the myelinated nerve bundles in the cores of folia and central cerebellar white matter, similar to the more common cerebellar degenerations such as in spino-cerebellar ataxias. Some findings such as gliosis in the central parts of the cerebellum and axonal swelling suggest an ongoing process. Study of the cell somata of the nucleus dentatus reveals some peculiarities in all patients where the nucleus could be identified, not seen in controls, such as perisomatic \u201cvacuolation\u201d that was even seen on routine HE staining. The presence of calbindin in some of these \u201cvacuoles\u201d suggests swollen nerve endings.\nGenetics and epigenetics\nVarying degrees of hypoplastic folial development are found in three patients (4, 5, 6) who share the same ancestors (Fig.\u00a03c\u2013h). This indicates that modifying factors bear on the expression of this genetic condition. Patient 3, the patient with the longest survival, who died at 22\u00a0years, differed from other patients in having vascular anomalies in the cerebellum. This phenomenon may be caused by an unknown age-bound complication of the disease. However, a genotype distinct from the others cannot be excluded pending the unraveling of the genetic basis of PCH-2.","keyphrases":["pontocerebellar hypoplasia","pons","olivary nucleus","cerebellum"],"prmu":["P","P","P","P"]}
{"id":"Diabetologia-3-1-2039861","title":"Cardiac contractile dysfunction in insulin-resistant rats fed a high-fat diet is associated with elevated CD36-mediated fatty acid uptake and esterification\n","text":"Aims\/hypothesis Changes in cardiac substrate utilisation leading to altered energy metabolism may underlie the development of diabetic cardiomyopathy. We studied cardiomyocyte substrate uptake and utilisation and the role of the fatty acid translocase CD36 in relation to in vivo cardiac function in rats fed a high-fat diet (HFD).\nIntroduction\nDiabetic cardiomyopathy (DCM) is a common complication in type 2 diabetes [1]. Changes in myocardial energy metabolism, due to altered substrate handling, characterise diabetes-related heart disease and DCM [1]. Studies in rodent models, including rats fed a high-fat diet (HFD) [2], leptin-deficient and -resistant animals [3\u20136] and transgenic mice with cardiac-restricted peroxisome proliferator activated receptor \u03b1 (PPAR\u03b1) overexpression [7], indicate that intramyocardial accumulation of triacylglycerol metabolites (lipotoxicity) and myocardial insulin resistance may underlie diabetes-related cardiac dysfunction [1, 8].\nAmong the putative causes of myocardial triacylglycerol accumulation are elevations in long-chain fatty acid (LCFA) uptake [6, 7, 9]. In the heart, approximately 50% of LCFA uptake is mediated by the fatty acid translocase CD36 [10, 11]. Under physiological conditions, both insulin and contraction stimulate LCFA uptake by inducing the translocation of CD36 from an intracellular pool to the sarcolemma, illustrating that CD36 is also directly involved in the dynamic utilisation of LCFA by cardiomyocytes. This acute translocation of CD36 involves activation of phosphatidylinositol 3\u2032-kinase (PI3K)\/protein kinase B (PKB\/Akt)- and AMP-kinase (AMPK)-dependent signalling cascades in response to insulin and contraction, respectively [11].\nInterestingly, CD36 deletion rescues the cardiac dysfunction, metabolic abnormalities and myocardial triacylglycerol accumulation observed in transgenic mice with cardiac-restricted PPAR\u03b1 overexpression [7], suggesting a critical role for CD36 in the development of diabetes-related heart disease [12]. We hypothesised that long-term alterations in the functional pool of CD36 lead to myocardial accumulation of (toxic) lipid metabolites and insulin resistance and contribute to the development of diabetes-related heart disease. To examine this, we first measured in vivo cardiac dimensions by echocardiography in rats exposed to a high-fat diet (HFD), which impairs myocardial insulin signalling [2, 13]. Subsequently, isolated cardiomyocytes were used to determine changes in substrate uptake and LCFA metabolism under basal conditions and after incubations with insulin and with oligomycin; the latter compound induces a contraction-like elevation of intracellular AMP levels and concomitant activation of AMPK [14]. Finally, we assessed whether the observed alterations could be ascribed to CD36 in hearts from rats on the HFD by analysing the effects of the CD36 inhibitor sulfo-N-succinimidyloleate (SSO) on myocardial LCFA uptake and utilisation in isolated cardiomyocytes, and by studying the expression and subcellular localisation of CD36 in cardiac ventricular tissue.\nMaterials and methods\nAnimals The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the NIH (NIH Publication No. 85-23, revised 1996) and was approved by the VU University Medical Centre Animal Care Committee. Adult male Wistar WU rats (n\u2009=\u200931; mean body weight 361\u2009\u00b1\u200924\u00a0g) were purchased from Harlan CBP (Horst, the Netherlands), and fed an HFD or an isocaloric low-fat diet (LFD) [2]. At week\u00a08, rats fasted overnight underwent an OGTT, and post-load blood glucose levels were determined 15, 30, 60, 90 and 120\u00a0min after ingestion of 2\u00a0g glucose\/kg body weight [2]. After 10\u00a0weeks on the diet, blood was collected for determination of glucose and insulin levels. Rats were then anaesthetised with an intraperitoneal injection of sodium pentobarbital, and hearts were removed for the isolation of cardiomyocytes [15].\nDiets Experimental diets were obtained from Hope Farms (Woerden, the Netherlands; HFD, catalogue no. 4148.02; LFD, catalogue no. 4148.01). The LFD consisted of 8% by weight of total fat, 22% by weight of protein and 60% by weight of carbohydrate; the HFD contained 25% by weight of fat, 32% by weight of protein and 25% by weight of carbohydrate, as well as more palmitate (91.12\u00a0g\/kg) and oleate (100.24\u00a0g\/kg) compared with the LFD (29.12 and 32.08\u00a0g\/kg, respectively). In the group fed the HFD, 50.4% of the ingested calories were derived from fat compared with 16.4% in the group fed the LFD.\nIn vivo cardiac function Non-invasive transthoracic echocardiograms were recorded at heart rates of 350\u2013400 beats per minute in rats anaesthetised with 1.5% isoflurane in a mixture of N2O\/O2 (1\/2, vol\/vol) [16] before and 8\u00a0weeks after initiation of the diet by an Aloka echo machine (ProSound SSD-4000) using a 13-MHz linear interfaced array transducer. M- and B-mode images were obtained in the parasternal long- and short-axis views of the left ventricle (LV) at mid-papillary level. At end systole (ES) and end diastole (ED), LV lumen diameter (D), LV ventricular diameter (VD) and posterior (PWT) and interventricular septum (IVSWT) wall thicknesses were determined for three cardiac cycles and averaged. The LV dimensions were used to calculate systolic parameters [17]: fractional shortening , where EDD is end diastolic diameter and ESD is end systolic diameter; end diastolic volume (EDV)\u2009=\u2009[EDD]3; end systolic volume (ESV)\u2009=\u2009[ESD]3; and ejection fraction . LV mass (LVM) was determined according to the uncorrected cube assumption: , where 1.05 is the specific gravity of the myocardium [18].\nPlasma and tissue determinations Blood glucose was measured from tail bleeds using a glucose analyser (HemoCue, Angelholm, Sweden). Plasma insulin was measured using ELISA (EZRMI-13K; Linco Research, St Charles, MO, USA). Myocardial triacylglycerol content was determined in ventricular lysates from a separate set of animals as described in [2].\nTreatment of isolated cardiomyocytes Cardiomyocytes were prepared using a Langendorff perfusion system as described in [15, 19]. Isolated cardiomyocytes were incubated for 30\u00a0min at room temperature with 0.5% DMSO or SSO [9]. The CD36-specific inhibitor SSO [20] was used to investigate the involvement of CD36 in basal LCFA uptake. SSO was applied at a concentration of 0.4\u00a0mmol\/l, which effectively inhibits CD36 [15, 21]. Then, cells were washed and incubation was continued for 15\u00a0min at 37\u00b0C with continuous shaking. Thereafter, the DMSO-treated cardiomyocytes were kept for another 15\u00a0min at 37\u00b0C and either kept untreated (basal) or incubated with insulin or oligomycin. To achieve optimal stimulation of glucose and LCFA uptake [22], insulin and oligomycin were used at a concentration of 10\u00a0nmol\/l and 30\u00a0\u03bcmol\/l, respectively. At this concentration, the ATP-synthase inhibitor oligomycin induces an increase in the intracellular AMP\/ATP ratio, thereby activating AMPK-mediated signalling pathways without inhibiting oxygen consumption [14].\nGlucose uptake, palmitate uptake and oxidation, and incorporation into intracellular lipid pools Following agonist treatment, [3H]-2-deoxyglucose and [1-14C] palmitate uptake were simultaneously determined during a 3-min incubation [15]. Rates of [1-14C] palmitate oxidation (measured as the production of 14CO2) and esterification (measured as incorporation of the 14C label into intracellular phospholipids [PL], and triacylglycerol) were determined after a 20-min incubation [9].\nWestern blotting Phosphorylation of proline-rich Akt-substrate 40 (PRAS40-Thr246) and acetyl-coenzyme A carboxylase-Ser79 (ACC-Ser79) was determined in homogenates of cardiomyocytes by western blotting using phospho-specific antibodies [9, 13]. PKB\/Akt-Ser473 phosphorylation was determined in ventricular homogenates as described in [2]. Expression levels of CD36 were determined by western blotting using the monoclonal CD36 MO25 antibody [23]. Immunoblots were quantified by densitometric analysis of the films [2].\nCardiac morphology and subcellular localisation of CD36 Cardiac ventricular tissue collected in a previous study [2] was routinely embedded in paraffin (Histowax; Leica Microsystems, Wetzlar, Germany). Sections (5\u00a0\u03bcm) were cut, and mounted on slides coated with 3-aminopropyltriethoxisilane (Menzal, Darmstadt, Germany). After deparaffinisation and rehydration, the slides were used for determination of cardiomyocyte cross-sectional area or immunohistochemical staining of CD36.Cross-sectional area was determined in randomly chosen fields in haematoxylin\/eosin-stained slides using NIH image analysis software (National Institutes of Health, Bethesda, MD, USA) for 20\u201330 cells per heart and normalised to sarcomere length [24].For CD36 localisation, sections were rinsed extensively with Tris-buffered saline (TBS; 50\u00a0mmol\/l Tris\u2013Cl, 500\u00a0mmol\/l NaCl, pH\u00a07.6), and incubated overnight with the monoclonal CD36 MO25 antibody [23] diluted 1:2,000 in TBS containing 0.5% Triton X-100, pH\u00a07.6 at 4\u00b0C. After several washes in TBS, sections were incubated for 1.5\u00a0h at room temperature with biotinylated anti-mouse IgG (Vector Laboratories, Burlingame, CA, USA), diluted 1:250 in TBS, followed by incubation for 1\u00a0h at room temperature with avidin\u2013biotin\u2013peroxidase (1:400; ABC Elite kit; Vector Laboratories). Tissue-bound peroxidase was visualised with the 3,3\u2032-diaminobenzidine tetrahydrochloride (DAB) chromogen reaction (7.5\u00a0mg DAB, 5\u00a0\u03bcl 30% H2O2 in 15\u00a0ml 50\u00a0mmol\/l Tris\u2013Cl, pH\u00a07.6) for 10\u00a0min at room temperature. Sections were then rinsed with distilled water, dehydrated in a graded series of ethanol, cleared in xylene, and coverslipped with Entellan (Merck, Darmstadt, Germany). Sections were analysed using a Leica DM-LB light microscope (Leica, Rijswijk, the Netherlands). Digital images were taken at \u00d7200 magnification using a Leica DC500 digital camera, and quantified using Quantity One software (Bio-Rad Laboratories, Veenendaal, the Netherlands).\nStatistical analysis Unless indicated otherwise, data are expressed as means\u00b1SE for the indicated number of animals (n). Statistical analysis was performed in SPSS for Mac OS X version 11.0.4 (SPSS Inc., Chicago, IL, USA). Differences between the LFD and HFD groups were determined using independent t tests. The paired sample t test was used to define the effect of agonist incubations on cardiomyocyte preparations. p\u2009<\u20090.05 was considered as statistically significant.\nResults\nHFD induces cardiac contractile dysfunction Feeding rats isocaloric LFD or HFD diets for 8\u00a0weeks induced similar weight gains in the two groups (Table\u00a01), but mild glucose intolerance, as determined by an oGTT, in HFD- vs LFD-rats (p\u2009<\u20090.05; Fig.\u00a01a). Figure\u00a01b shows the LV dimensions at the start and 8\u00a0weeks after initiation of the diet interventions. Baseline parameters were similar in the two groups (Table\u00a01). After 8\u00a0weeks on the diet, LVM, ED-VD, EDD and ED-IVSWT showed comparable increases in LFD and HFD hearts (all p\u2009<\u20090.05), whereas ED-PWT was unaffected by the diet (Table\u00a01). In the ES phase, both ES-PWT and ES-IVSWT were increased in LFD hearts only (both p\u2009<\u20090.005; Table\u00a01). ES-VD was similarly affected by LFD and HFD feeding. ESD was increased in LV from HFD-fed vs LFD-fed rats (p\u2009<\u20090.01; Table\u00a01). FS% was increased in LFD hearts (p\u2009<\u20090.05), and decreased in HFD hearts during the course of the diet intervention (p\u2009<\u20090.02; Table\u00a01). Accordingly, EF% was decreased in HFD compared with LFD hearts (p\u2009<\u20090.05; Table\u00a01). Collectively, these data show that HFD induced cardiac contractile dysfunction in rats.\nTable\u00a01In vivo cardiac characteristics of rats before and after 8\u00a0weeks on an HFD or a LFD\u00a0StartLFDHFD(n\u2009=\u200924)(n\u2009=\u20098)(n\u2009=\u200916)Physiological parameters\u00a0Body weight (g)295\u2009\u00b1\u20094476\u2009\u00b1\u20098*463\u2009\u00b1\u20098*\u00a0Left ventricular mass (mg)637\u2009\u00b1\u200915890\u2009\u00b1\u200918*882\u2009\u00b1\u200932*LV diastolic parameters\u00a0Posterior wall thickness (mm)1.63\u2009\u00b1\u20090.031.80\u2009\u00b1\u20090.071.71\u2009\u00b1\u20090.05\u00a0Lumen diameter (mm)6.92\u2009\u00b1\u20090.077.55\u2009\u00b1\u20090.20*7.68\u2009\u00b1\u20090.10*\u00a0Interventricular septum wall thickness (mm)1.37\u2009\u00b1\u20090.031.67\u2009\u00b1\u20090.08*1.54\u2009\u00b1\u20090.04*\u00a0Ventricular diameter (mm)9.80\u2009\u00b1\u20090.0610.86\u2009\u00b1\u20090.13*10.89\u2009\u00b1\u20090.12*LV systolic parameters\u00a0Posterior wall thickness (mm)2.93\u2009\u00b1\u20090.063.45\u2009\u00b1\u20090.12*3.00\u2009\u00b1\u20090.08**\u00a0Lumen diameter (mm)3.41\u2009\u00b1\u20090.103.32\u2009\u00b1\u20090.254.07\u2009\u00b1\u20090.12*,**\u00a0Interventricular septum wall thickness (mm)2.51\u2009\u00b1\u20090.052.85\u2009\u00b1\u20090.09*2.67\u2009\u00b1\u20090.06\u00a0Ventricular diameter (mm)8.81\u2009\u00b1\u20090.079.82\u2009\u00b1\u20090.12*9.81\u2009\u00b1\u20090.10*\u00a0Fractional shortening (%)50.8\u2009\u00b1\u20091.356.3\u2009\u00b1\u20092.4*47.1\u2009\u00b1\u20091.7*,**\u00a0Ejection fraction (%)87.5\u2009\u00b1\u20091.091.1\u2009\u00b1\u20091.484.9\u2009\u00b1\u20091.0*,**Data are means\u00b1SE*p\u2009<\u20090.05 vs start**p\u2009<\u20090.01 vs LFDFig.\u00a01a Blood glucose levels after an oral glucose load in rats fed the HFD (closed circles; n\u2009=\u200916) and the LFD (open circles; n\u2009=\u20098). Data are means\u00b1SE. b Representative echocardiographic M-mode images from at least three cardiac contractile cycles at the start of the diet intervention and 8\u00a0weeks after initiation of the LFD and HFD, respectively\nInsulin action, but not AMPK signalling, is impaired in HFD cardiomyocytes We used cardiomyocytes to unravel the diet-induced molecular alterations in energy substrate uptake and intracellular signalling pathways. Cardiomyocytes were isolated from a separate group of rats, fed either the LFD or the HFD under identical experimental conditions. At killing, body, heart and liver weights were similar between LFD- and HFD-fed rats, whereas perirenal fat pad weight was 50% higher in HFD- than in LFD-fed rats (p\u2009<\u20090.001; Table\u00a02). Furthermore, HFD feeding increased blood glucose levels (p\u2009<\u20090.02) and lowered plasma insulin levels (p\u2009<\u20090.05).\nTable\u00a02Characteristics of rats after 10\u00a0weeks on a high- or low-fat diet\u00a0LFDHFD(n\u2009=\u200914)(n\u2009=\u200917)Body composition\u00a0Body weight at killing (g)465\u2009\u00b1\u20095472\u2009\u00b1\u200910\u00a0Heart weight (% body weight)0.445\u2009\u00b1\u20090.0070.431\u2009\u00b1\u20090.012\u00a0Liver weight (% body weight)3.10\u2009\u00b1\u20090.073.02\u2009\u00b1\u20090.08\u00a0Perirenal fat pad weight (% body weight)1.63\u2009\u00b1\u20090.082.43\u2009\u00b1\u20090.14*Plasma characteristics\u00a0Blood glucose (mmol\/l)5.50\u2009\u00b1\u20090.085.78\u2009\u00b1\u20090.08**\u00a0Plasma insulin (pmol\/l)269\u2009\u00b1\u200918216\u2009\u00b1\u200916***Data are means\u00b1SE*p\u2009<\u20090.001 vs LFD**p\u2009<\u20090.02 vs LFD***p\u2009<\u20090.05 vs LFDBasal 2-deoxyglucose glucose uptake rates were 7.3\u2009\u00b1\u20090.8 and 11.0\u2009\u00b1\u20091.6\u00a0nmol min\u22121 [g wet cell mass]-1C in LFD and HFD cells, respectively (p\u2009=\u20090.063; Fig.\u00a02). Insulin stimulated 2-deoxyglucose uptake 3.7-fold in LFD cardiomyocytes and 2.2-fold in HFD cardiomyocytes (both p\u2009<\u20090.001 vs basal, p\u2009<\u20090.02 HFD vs LFD), indicating reduced myocardial insulin responsiveness. Oligomycin had similar stimulatory effects on 2-deoxyglucose uptake in HFD and LFD cardiomyocytes (HFD, 1.6-fold stimulation; LFD, 1.7-fold stimulation; both p\u2009<\u20090.05 vs basal).\nFig.\u00a02Rates of 2-deoxyglucose uptake in cardiomyocytes from rats fed the LFD (open bars) and the HFD (filled bars). Cardiomyocytes were incubated with DMSO (Basal), insulin (INS) or oligomycin (Oli) before measurement of 2-deoxyglucose uptake. Data are expressed as wet cell mass and are means\u00b1SE; n\u2009=\u20098. *p\u2009<\u20090.02, agonist effect; #p\u2009<\u20090.02, diet effectActivation of phosphatidylinositol 3\u2032-kinase (PI3K)\/protein kinase B (PKB\/Akt)- and AMP-kinase (AMPK)-dependent signalling cascades has been implicated in the stimulation of glucose uptake in response to insulin and oligomycin, respectively. We studied whether changes in the activation of these signalling pathways paralleled the observed alterations in glucose uptake rates in HFD cells. Therefore, phosphorylation of PRAS40-Thr246 and ACC-Ser79 were examined as respective distal determinants for PKB\/Akt and AMPK activity [13, 25], as the presence of bovine serum albumin (mol. mass 66 kDa) in the cardiomyocyte homogenates interfered with direct measurement of PKB\/Akt (mol. mass 64\u00a0kDa) and AMPK\u03b1 phosphorylation (mol. mass 62\u00a0kDa). Basal PRAS40-Thr246 phosphorylation was 1.7-fold higher in HFD than in LFD cells (p\u2009<\u20090.05; Fig.\u00a03a,b). Insulin stimulated PRAS40-Thr246 phosphorylation 12.0-fold in LFD cells (p\u2009<\u20090.001) and 4.0-fold in HFD cells (p\u2009<\u20090.001), thus confirming reduced insulin responsiveness. Oligomycin did not affect PRAS40-Thr246 phosphorylation.\nFig.\u00a03Immunoblots (a, c) and quantification of PRAS40-Thr246 (b) and ACC-Ser79 (d) phosphorylation after incubation of cardiomyocytes from LFD- and HFD-fed rats with DMSO (Basal), insulin (INS) or oligomycin (Oli). Data are means\u00b1SE; n\u2009=\u20095. *p\u2009<\u20090.05, agonist effect; #p\u2009<\u20090.05, diet effectACC-Ser79 phosphorylation was measured as a determinant of AMPK activity [25]. Basal levels of Ser79-phosphorylated ACC were similar in LFD and HFD cells (Fig.\u00a03c,d). Insulin had no effect on ACC-Ser79 phosphorylation, whereas oligomycin stimulated ACC-Ser79 phosphorylation in LFD and HFD cells to a similar extent (HFD, 3.0-fold stimulation; LFD, 2.9-fold stimulation; both p\u2009<\u20090.001 vs basal), indicating that AMPK signalling was not affected by high-fat feeding.\nHFD feeding results in increased CD36-mediated LCFA uptake Basal rates of LCFA uptake were increased 1.4-fold (p\u2009<\u20090.001) in HFD compared with LFD cells (Fig.\u00a04). The CD36 inhibitor SSO reduced basal LCFA uptake by 44% (p\u2009<\u20090.001) and 23% (p\u2009<\u20090.05) in HFD and LFD cardiomyocytes, respectively. Notably, the absolute rates of LCFA uptake were similar between SSO-treated LFD and HFD cells (Fig.\u00a04), suggesting that the increased basal LCFA uptake rates in HFD cells can be ascribed to changes in the sarcolemmal pool of CD36. Insulin stimulated LCFA uptake 1.3-fold in LFD cells (p\u2009<\u20090.02 vs basal) but had no effect in HFD cells (p\u2009<\u20090.02 vs LFD; Fig.\u00a04). Oligomycin increased LCFA uptake in LFD and HFD cardiomyocytes to the same extent (Fig.\u00a04; both p\u2009<\u20090.05 vs basal).\nFig.\u00a04Rates of palmitate uptake (a), oxidation (b) and esterification into intracellular triacylglycerols and phospholipids (c) in cardiomyocytes from LFD-fed rats (open bars) and HFD-fed rats (filled bars). Cardiomyocytes were incubated with DMSO (Basal), insulin (INS), oligomycin (Oli) or SSO before measurement of palmitate uptake. Data are wet cell mass and are means\u00b1SE; n\u2009=\u20098. *p\u2009<\u20090.05, agonist effect; #p\u2009<\u20090.05 diet effectWe next examined the metabolic fate of LCFA following their uptake by HFD and LFD cardiomyocytes. Basal rates of LCFA oxidation were reduced by 22% in HFD relative to LFD cells (Fig.\u00a04b; p\u2009<\u20090.01). SSO further lowered cardiomyocyte LCFA oxidation rates by 66% (p\u2009<\u20090.005) and 31% (p\u2009<\u20090.05) in LFD and HFD cells, respectively, but absolute rates of LCFA oxidation were comparable in SSO-treated LFD and HFD cells.The incorporation rate of LCFA into triacylglycerol was increased 1.4-fold (p\u2009<\u20090.05) in HFD compared with LFD cells under basal conditions (Fig.\u00a04c). SSO reduced triacylglycerol formation in both LFD and HFD cells (both p\u2009<\u20090.05 vs basal). Notably, the absolute triacylglycerol esterification rates in SSO-treated cells did not differ between HFD- and LFD-fed rats. Also, the incorporation of LCFA into phospholipids under basal conditions was increased 1.5-fold in HFD vs LFD cells (p\u2009<\u20090.05; Fig.\u00a04c), and was reduced by SSO in both LFD and HFD cells (both p\u2009<\u20090.05 vs basal). Myocardial triacylglycerol content was 181\u2009\u00b1\u200950\u00a0\u03bcg\/mg protein in LFD rats and 330\u2009\u00b1\u2009114\u00a0\u03bcg\/mg protein in HFD rats (p\u2009<\u20090.02) [2].\nHFD induces cardiomyocyte hypertrophy and alters the subcellular localisation of CD36 The inhibitory effect of SSO suggests that the increased basal LCFA uptake and esterification rates in HFD cells can be ascribed to changes in the sarcolemmal pool of CD36. Western blot analysis indicated that the levels of CD36 were similar between HFD and LFD hearts (Fig.\u00a05a). Immunohistochemical staining of cardiac LV tissue indicated cardiomyocyte hypertrophy, as reflected by an increased cross-sectional area (p\u2009<\u20090.05 vs LFD; Table\u00a03), and showed a more abundant presence of CD36 at the sarcolemmal membrane, particularly at the intercalated discs, in HFD hearts compared with LFD hearts (p\u2009<\u20090.002 vs LFD; Fig.\u00a05b,c and Table\u00a03).\nFig.\u00a05Expression and subcellular localisation of CD36. Expression of CD36 in ventricular lysates of HFD and LFD rats. Ponceau S staining confirmed that the filters contained equal amounts of ventricular extracts (a). Immunohistochemical staining for CD36 localisation of cardiac LV tissue sections from rats fed an LFD (b, d) or HFD (c, e) for 8\u00a0weeks. Rats received an i.p. injection of saline (b, c) or insulin (d, e) 30\u00a0min before killing. Photographs are representative of two independent experiments performed on three rats per experimental group. Arrows indicate intercalated discs. Scale bar indicates 25\u00a0\u03bcmTable\u00a03Cardiac morphology and subcellular localisation of CD36\u00a0LFDHFD(n\u2009=\u20096)(n\u2009=\u20098)Morphology\u00a0Cardiomyocyte cross-sectional area normalised to sarcomere length (\u03bcm2)214\u2009\u00b1\u20093.5276\u2009\u00b1\u200923*CD36 localisation\u00a0Sarcolemma\u00a0\u00a0Saline (%)61.7\u2009\u00b1\u2009374.8\u2009\u00b1\u20091**\u00a0\u00a0Insulin (%)75.9\u2009\u00b1\u20094***78.2\u2009\u00b1\u20093\u00a0Cytoplasm\u00a0\u00a0Saline (%)38.3\u2009\u00b1\u2009325.2\u2009\u00b1\u20091**\u00a0\u00a0Insulin (%)24.1\u2009\u00b1\u20094***21.8\u2009\u00b1\u20093Data are means\u00b1SE*p\u2009<\u20090.05 vs LFD**p\u2009<\u20090.002 vs LFD***p\u2009<\u20090.01 vs salineAs insulin stimulated LCFA uptake in isolated cardiomyocytes (Fig.\u00a04), we also examined the subcellular localisation of CD36 in hearts isolated 30\u00a0min following an intraperitoneal injection with insulin (10\u00a0U\/kg body weight) [2]. Figure\u00a05d shows that in vivo insulin treatment increased CD36 immunoreactivity at the intercalated discs in LFD hearts (p\u2009<\u20090.01 vs saline; Table\u00a03). In HFD hearts, insulin did not further recruit CD36 to the sarcolemma (Fig.\u00a05e).\nHFD-induced CD36 redistribution to the sarcolemma precedes the onset of cardiac contractile dysfunction and associates with elevated basal phosphorylation of PKB\/Akt We next analysed whether HFD-induced alterations in CD36 localisation precede the onset of cardiac contractile dysfunction, using rats fed an LFD or HFD for 4\u00a0weeks. This period was chosen based on serial echocardiographic measurements, which revealed a tendency towards decreased cardiac contractile function 4\u00a0weeks after initiation of the diet intervention. Specifically, FS% was 4.2% lower (p\u2009=\u20090.07) and EF% was 3.3% lower (p\u2009=\u20090.10) in HFD vs LFD hearts after 4\u00a0weeks on the diet (data not shown), compared with the 9.2 and 6.2% decreases observed after 8\u00a0weeks of the feeding regime (both p\u2009<\u20090.05; Table\u00a01). Despite the absence of significant effects on cardiac function after 4\u00a0weeks of HFD feeding, immunohistochemical staining of cardiac LV tissue showed that the abundance of CD36 at the sarcolemmal membrane was already markedly increased (Fig.\u00a06a,b) to levels almost similar to those observed after 8\u00a0weeks of HFD feeding (Fig.\u00a05b,c).\nFig.\u00a06Effect of the duration of HFD feeding on CD36 localisation and phosphorylation of PKB\/Akt. Immunohistochemical staining for CD36 localisation of cardiac LV tissue sections from saline-injected rats fed an LFD (a) or HFD (b) for 4\u00a0weeks. Photographs are representative of two or three independent experiments performed on six rats per experimental group. The arrows indicate intercalated discs. The scale bar indicates 25\u00a0\u03bcm. c Immunoblot and quantification d of PKB\/Akt-Ser473 phosphorylation levels in ventricular homogenates from saline-injected rats fed an LFD (open bars) or HFD (filled bars) for 4 and 8\u00a0weeks, respectively. Data are means\u00b1SE, n\u2009=\u20098. *p\u2009<\u20090.05, diet effectFinally, we analysed whether HFD feeding induced changes in the activity of signalling pathways implicated in CD36 trafficking. In the healthy myocardium, CD36 is stored in at least two endosomal storage pools that are regulated by AMPK and PKB\/Akt, respectively [26]. As insulin does not further recruit CD36 to the sarcolemma in HFD hearts (Fig.\u00a05e), we proposed that increases in basal activity of PKB\/Akt may underlie the redistribution of CD36 in HFD hearts. Phosphorylation of PKB\/Akt was determined in LV homogenates of the same group of saline-injected rats in which CD36 localisation was measured. As shown in Fig.\u00a06c,d, basal phosphorylation of PKB\/Akt was 1.7- and 2.6-fold higher after 4 and 8\u00a0weeks of HFD feeding, respectively (both p\u2009<\u20090.05). Collectively, these results indicate that increased activity of PKB\/Akt may underlie CD36 redistribution to the sarcolemma, and that these events precede the LV dysfunction induced by HFD feeding.\nDiscussion\nHere we report that HFD feeding induces cardiac contractile dysfunction in rats and that this is associated with a permanent relocation of CD36 to the sarcolemma. The continuous presence of CD36 at the sarcolemmal membrane results in enhanced rates of LCFA uptake and subsequent esterification. We propose that this contributes to a decrease in myocardial insulin action and the development of diabetes-related heart disease. In addition, we show that AMPK-mediated responses are not affected by the composition of the diet.\nA key observation in this study is that the alterations in cardiac contractile function in HFD hearts was associated with a continuous presence of CD36 at the sarcolemmal membrane. The present study provides the first morphological evidence for translocation of CD36 to the sarcolemmal membrane. Importantly, the amount of sarcolemmal CD36 closely correlated with enhanced LCFA uptake rates in isolated cardiomyocytes. The observation that SSO inhibited LCFA uptake in HFD cardiomyocytes to the same residual levels as measured in LFD cardiomyocytes provides further pharmacological evidence that the enhanced flux of LCFA in the heart of HFD-fed rats is a direct consequence of the relocalisation of CD36 to the sarcolemmal membrane.\nPreviously, a redistribution of CD36 to both subsarcolemmal and intramyofibrillar mitochondria has also been observed [27, 28]. Can the observed CD36 immunoreactivity be ascribed to subsarcolemmal mitochondria rather that the sarcolemma itself? Stimuli inducing translocation of CD36 to the mitochondria are expected not to discriminate between the subsarcolemmal and the intramyofibrillar mitochondria. Hence, if a stimulus or condition, in this case insulin or HFD feeding, were to increase the subsarcolemmal CD36 content, one would expect a similar increase in intramyofibrillar CD36 content. This, however, could not be confirmed in the immunohistochemical experiments. Furthermore, biochemical fractionations performed in previous studies substantiate the idea that a significant fraction of CD36 is present at the sarcolemma of hearts from insulin-resistant rats, and that only a minor portion of CD36 is found within the mitochondrial fractions [9, 22]. While insulin has been shown to translocate CD36 to the sarcolemma [9, 22], insulin-mediated CD36 translocation to the mitochondria has never been reported and even seems counterintuitive. Collectively, the available evidence strongly supports the idea that the observed CD36 immunoreactivity can be ascribed to CD36 located at the sarcolemma rather than in subsarcolemmal mitochondria.\nThe combined data of this and our earlier study on obese Zucker rats [9] suggest that relocalisation of CD36 is a general phenomenon in insulin-resistant hearts, and raises the question of what mechanism underlies the continuous presence of CD36 at the sarcolemmal membrane in HFD hearts. In the healthy myocardium, CD36 is stored in at least two endosomal storage pools that are regulated by AMPK and PI3K\/PKB\/Akt, respectively [26]. Activation of AMPK is critical for contraction- and oligomycin-mediated CD36 translocation, whereas PI3K\/PKB\/Akt is critical for insulin-mediated CD36 trafficking [10, 14, 26]. Basal phosphorylation of PKB\/Akt and its distal target, PRAS40, were elevated in HFD compared with LFD hearts, whereas AMPK activity was not affected by the diet. Furthermore, in cells from HFD-fed rats, the stimulatory effects of insulin on LCFA uptake, CD36 translocation and phosphorylation of PKB\/Akt and PRAS40 were abrogated, whereas oligomycin-induced AMPK activation and LCFA uptake was unimpaired between cardiomyocytes from LFD- and HFD-fed rats. Although we cannot exclude a contribution of other kinases or HFD-induced alterations in the as yet undisclosed trafficking machinery regulating the internalisation of CD36 [11, 26], our observations do not argue against the suggestion that increases in PKB\/Akt activity may contribute to the sustained sarcolemmal presence of CD36 in the heart of HFD-fed rats.\nPreviously, we suggested that increased plasma insulin might contribute to CD36 relocalisation in hearts from obese Zucker rats [9]. As systemic hyperinsulinaemia was not observed in HFD-fed rats [2, 13], it remains interesting to examine whether changes in the activity of other (insulin-independent) regulators of PKB\/Akt phosphorylation, such as PI3K\u03b3- and \u03b22-adrenergic receptor signalling pathways, Ca2+-calmodulin dependent kinase, protein phosphatase 2A and the sympathetic nervous system [29], can be linked to the observed increase in PKB\/Akt phosphorylation in HFD hearts.\nThe metabolic and biochemical data were paralleled by in vivo cardiac functional changes. Importantly, HFD-induced cardiac contractile dysfunction does not seem to be linked to diet-related elevations in blood pressure. Circadian haemodynamic parameters were monitored using implanted telemetry devices in an experiment performed in a separate group of animals. We found a slight increase in night-time (activity-related) heart rate in HFD rats, but no changes in blood pressure even after 10\u00a0weeks of exposure to the diet (Electronic supplementary material Table\u00a01). Others confirmed the absence of relevant hypertension after feeding rats a diet containing 74% fat for 17\u00a0weeks [30]. This underscores the possibility that alterations in metabolism, rather than in haemodynamics, underlie the impairment in cardiac contractile function in HFD rats.\nThe present study also suggests that HFD-induced alterations in the functional pool of CD36 may contribute to an imbalance in LCFA uptake and in oxidation and esterification rates in HFD vs LFD cells. Whereas LCFA uptake rates were increased 1.4-fold in HFD cells, the LCFA oxidation rates were not increased but rather modestly lower in HFD cells, while the rates of esterification into triacylglycerol and phospholipids were 1.4-fold higher in HFD vs LFD cardiomyocytes. Accordingly, triacylglycerol content was 1.9-fold increased in HFD compared with LFD hearts [2]. It seems unlikely that changes in malonylCoA levels underlie the reduced rates of LCFA oxidation in HFD hearts, as no diet-induced changes in ACC phosphorylation were found. Rather, degenerative changes in mitochondria, such as matrix dilution, cristolysis and mitochondria-associated lamellar bodies, have been observed in cardiomyocytes from HFD-fed rats [2] and have been linked to reduced oxidative capacity and lipid accumulation in skeletal muscle from patients with type 2 diabetes. However, further studies are required to examine whether these changes contribute to the reduced basal LCFA oxidation and increased LCFA esterification rates, or whether the extra LCFA taken up by HFD cells are stored as triacylglycerol and phospholipids as a consequence of HFD-induced changes in enzymes promoting LCFA esterification or inhibition of lipolysis.\nAn important finding of this study is that the HFD-induced CD36 redistribution preceded the onset of cardiac contractile dysfunction. Although we cannot unequivocally link CD36 redistribution to cardiac dysfunction, a recent report provided strong support for this notion as the absence of CD36 was found to prevent myocardial triacylglycerol accumulation in transgenic mice with cardiac PPAR\u03b1 overexpression, both under normal conditions and after HFD feeding [12]. Similarly, alterations in PKB\/Akt activity have been linked to cardiac dysfunction [31]. Myocardial biopsy samples obtained from patients with advanced heart failure or dilated cardiomyopathy show increased basal phosphorylation of PKB\/Akt [32, 33], and transgenic mouse models with constitutively activated PKB\/Akt in the heart develop hypertrophy, decreased cardiac function and impaired recovery from ischaemia\u2013reperfusion injury [31, 34]. Whereas chronic activation of PKB\/Akt increases basal glucose uptake rates in the heart [35], the effects on myocardial lipid metabolism have not been studied. Interestingly, PKB\/Akt signalling has been linked to palmitate-induced beta cell lipotoxicity [36]. Based on this report and the findings described in this study, it would be of interest to analyse animals expressing chronically active PKB\/Akt for translocation of CD36 to the sarcolemma and myocardial lipid accumulation.\nIt seems plausible that an enhanced CD36-mediated LCFA uptake rate may contribute to the development of cardiac contractile dysfunction. An increased supply of LCFA may influence excitation\u2013contraction coupling as well as other processes linked to cellular Ca2+ handling [37, 38]. Furthermore, triacylglycerol accumulation is strongly linked to cardiac insulin resistance and contractile dysfunction [1, 2, 8]. The dynamic equilibrium between triacylglycerol stores and triacylglycerol metabolites causes accumulation of ceramide and diacylglycerol during prolonged LCFA influx. Both ceramide and diacylglycerol have been implicated in the activation of serine\/threonine kinases, such as PKC, Jun N terminal kinase (JNK), and inhibitor of nuclear factor-\u03baB kinase (IKK), which counteract insulin signalling [39, 40]. Notably, chronic myocardial activation of Akt in transgenic mice has also been linked to feedback inhibition of PI3K activity [33] and blunted insulin stimulation of glucose uptake [35]. Importantly, reduced insulin responsiveness reduces the ability of insulin to regulate substrate handling, e.g. metabolic inflexibility [41]. In this study and previous reports [2, 13], we show that HFD feeding reduces insulin responsiveness and impairs metabolic flexibility, as illustrated by the blunted effects of insulin on the stimulation of glucose uptake, phosphorylation of PRAS40, LCFA uptake and translocation of CD36 to the sarcolemma.\nWe conclude that HFD feeding in rats induces cardiac contractile dysfunction, which is preceded by relocation of CD36 to the sarcolemma, and elevated basal levels of phosphorylated PKB\/Akt. The continuous presence of CD36 at the sarcolemma contributes to enhanced rates of fatty acid uptake, resulting in myocardial triacylglycerol accumulation and accompanying insulin resistance. Collectively, these data suggest that alterations in the subcellular localisation of CD36 may contribute to the development of diabetes-related heart disease and that CD36 may be a therapeutic target to prevent cardiac dysfunction and the development of heart failure in diabetes.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nESM Table 1\nHeart rate and blood pressure of rats after 10\u00a0weeks on an HFD or a LFD (PDF 22\u00a0kb)","keyphrases":["contractile dysfunction","cd36","diabetic cardiomyopathy","insulin resistance","glucose uptake","high-fat feeding","fatty acid utilisation"],"prmu":["P","P","P","P","P","P","R"]}
{"id":"Eur_Spine_J-4-1-2226059","title":"Complications and reoperations of the SB Charit\u00e9 lumbar disc prosthesis: experience in 75 patients\n","text":"Artificial disc prosthesis show fair to good short- and mid-term results. Long-term results are becoming apparent now, however, the incidence of late complications with this procedure remain poorly understood. In this report we will analyse late complications and discuss our experiences with salvage operations in patients with persistent pain after SB Charit\u00e9 disc prosthesis implantation. Seventy-five patients with persistent leg and back pain after insertion of an artificial disc prosthesis were enrolled in the study. In this negative selection frequently occurring late-complications were subsidence, wear, adjacent disc degeneration, facet joint degeneration and migration. In 15 patients we performed a posterior fusion without disc removal, and in 22 patients we removed 26 prostheses and performed a posterior and anterior fusion. The visual analogue scale (VAS) and Oswestry were examined before the salvage operation and after a follow-up period of at least 1 year, which is not yet available in all patients. The VAS and Oswestry decreased in the posterior group (n = 10) respectively from 8.0 (SD 0.9) to 6.3 (SD 2.1) and from 57.0 (SD 17.0) to 44.6 (SD 20.4); and in the disc removal group (n = 14) respectively from 8.0 (SD 0.9) to 5.6 (SD 2.7) and from 56.3 (SD 14.0) to 43.0 (SD 20.7). Serious late complications may occur following total disc replacement. Removal of the SB Charit\u00e9 artificial disc is feasible but with inherent risks. Removal of the disc prosthesis gives slightly better results than posterior fusion alone after a follow-up of at least 1 year.\nIntroduction\nDegenerative disc disease (DDD) is a major cause of pain and disability, with great social and financial impact, playing an increasing role in modern society [21]. Several surgical techniques have been developed to treat DDD. Spinal fusion is seen as the \u201cgold standard\u201d, but nowadays artificial disc prostheses are an alternative method [13]. The artificial disc should preserve motion, stability and normal function of a spinal segment. Also, less adjacent segment degeneration is expected [6, 7, 13, 15]. In theory, this procedure has many advantages over spinal fusion. A spinal fusion does eliminate motion and can cause overloading and early degeneration of the adjacent levels, although usually appearing only after 10\u201315\u00a0years [1, 11, 12, 17].\nResults after implantation of the SB Charit\u00e9 artificial disc prosthesis are diverse. short- and mid-term results are fair to good [3, 5, 13, 22]. The FDA-IDE study, in which the SB Charit\u00e9 artificial disc was tested for approval in the US, showed a FDA-defined, overall clinical success rate of 57.1% after 2-year follow-up [2, 15]. For the IDE study, the procedure was only judged to be successful if four criteria were satisfied: (1) greater than 25% improvement in Oswestry disability index; (2) no device failure; (3) no major complications; and (4) no neurological deterioration compared to preoperative status. Consequently, the composite definition of success employed in the IDE study makes it difficult to compare with other studies that employed different success criteria.\nIn a 10-year follow-up period, Lemaire et al. [13] found that 90% of patients had good to excellent clinical outcome. On the other hand, in the only available long-term study of Putzier et al. [16] with an average follow-up of 17\u00a0years, the investigators found no evidence that long-term results of the disc prosthesis were superior to spinal fusion [16]. It remains unclear whether the early fair to good results obtained with an artificial disc will be consistently maintained with a longer follow-up period [9, 16, 18, 20].\nIn the Netherlands, more than 1,000 patients have been implanted with a SB III Charit\u00e9 (Link, Germany) disc prosthesis starting in 1989. As these patients received the implant during routine clinical practice, they were not the subjects of a randomized trial. However, 50 patients (75 arthroplasties) were enrolled in a prospective observational trial, and at 2\u00a0years the clinical success rate was found to be 70% [22].\nSince 1989, there have been many changes in the available implant sizes, surgical instrumentation, and patient indications. Although the basic \u201cSB III\u201d design has remained the same throughout the past 18\u00a0years, there have also been evolutionary changes in the polyethylene (PE) resin, sterilization, and endplate fixation technology. Starting in 2004, the SB III design has been renamed the Charit\u00e9 artificial disc and is currently produced by a different manufacturer (DePuy Spine, Raynham, MA, USA). Despite these aforementioned changes, it remains crucially important to fully understand the long-term clinical failure modes of early total disc replacements, as they form the foundation for judging the performance of contemporary implant designs and treatment paradigms.\nAt present, we have treated 75 patients at our clinic with persistent leg and back pain after insertion of the SB Charit\u00e9 disc prosthesis. The incidence of complications following Charit\u00e9 artificial disc implantation at our institution has proven difficult to deduce because all operations were performed elsewhere. At 2\u00a0years of follow up, investigators from the hospital implanting the disc prosthesis reported that 17 out of 50 (34%) patients required secondary surgery, and there were three (6%) reported serious complications [22]. The purpose of this study is to analyse late-complications after insertion of a disc prosthesis, and to describe our experiences with salvage operations in this difficult patient group.\nMaterials and methods\nPatients\nApproximately more than 1,000 Dutch patients, suffering from serious and constant back and leg pain, have been implanted with a SB III Charit\u00e9 (Link, Germany) artificial disc prosthesis in the lower lumbar spine.\nOver the last 10\u00a0years, 75 patients of this cohort, with persisting back and leg pain and being unsatisfied with their clinical situation, were seen at the orthopaedic outpatient clinic of the University Hospital of Maastricht (UHM). They were seen by a different orthopaedic surgeon (AvO and LvR) than the surgeon who performed the prosthesis implantation. Twenty-seven of these cases have already been reported previously [20].\nForty-one patients were female with an average age at the time of disc implant of 42\u00a0years and 7\u00a0months (30\u201363\u00a0years) and 34 patients were male with an average age of 40\u00a0years and 9\u00a0months (30\u201351\u00a0years) at the time of disc implant (Table\u00a01). The operated levels and the year the patients received their artificial disc implantation is shown in Table\u00a01.\nTable\u00a01Patient characteristicsSexNAge at artificial disc insertionMales3440\u00a0years and 9\u00a0months (30\u201351)Females4142\u00a0years and 7\u00a0months (30\u201363)Operated level \u00a0First level\u00a0\u00a0L2\u2013L31\u00a0\u00a0L3\u2013L43\u00a0\u00a0L4\u2013L522\u00a0\u00a0L5\u2013S130\u00a0Second levels \u00a0\u00a0L3\u2013L4, L5\u2013S11\u00a0\u00a0L4\u2013L5, L5\u2013S116Third levels \u00a0\u00a0L3\u2013L4, L4\u2013L5, L5\u2013S11\u00a0\u00a0L2\u2013L3, L4\u2013L5, L5\u2013S11Time disc implantation\u00a01989\u2013199425\u00a01995\u2013199930\u00a02000\u2013200520\nForty-six out of these 75 patients needed one or more salvage operations after their artificial disc implant. Indicators for reoperations were primary absence of pain relief, new pathology according to radiography, CT-scan or MRI in adjacent segment(s), subsidence, facet joint arthrosis or migration of the prosthesis. In our hospital, we performed posterior fusion in 15 patients without disc removal, and in 22 patients we removed 26 prostheses and performed an anterior and posterior fusion. In addition, seven patients received posterior fusion elsewhere, and in two patients the disc prosthesis was removed elsewhere (Fig.\u00a01).\nFig.\u00a01Overview of reoperations after disc prosthesis implantation (UHM: University Hospital Maastricht)\nSurgical method of disc prosthesis removal\nThe disc prosthesis was removed by a team consisting of a vascular surgeon and an orthopaedic surgeon. The patient was placed under general anaesthesia and positioned in supine position (for level L5\u2013S1) or in a semilateral position (for level L4\u20135 or higher).\nA disc prosthesis at level L4\u20135 was removed via anterolateral lumbotomy, without mobilisation of the great vessels. A disc prosthesis at level L5\u2013S1 was removed through the original left retroperitoneal approach or alternatively via right retroperitoneal, between the great vessels. To remove the disc prosthesis one of the endplates was released by undercutting and removal of some bone after cleaning of fibrous tissue. The metal endplate was gripped and removed with twisting and pulling manoeuvres. Then the core and second endplate was easily released and removed.\nThe gap between the vertebral bodies after retrieval of the disc prosthesis was mostly 2.5\u20133\u00a0cm high. This was filled with an autologous strut graft in 2 patients, artificial bone in 3 patients and allograft strut grafts and allograft bone chips from femoral heads in 17 patients. We now favour the insertion of allograft strut grafts from the bone bank.\nClinical performance and complications\nBefore the orthopaedic surgeons in our outpatients clinic performed reoperation, they evaluated the images of X-ray, CT-scans, MRI and if necessary discography on the presence of late complications.\nClinical examination included the ten-point visual analogue scale (VAS), ranging from 0 points for \u201cno pain\u201d and 10 points for \u201csevere pain\u201d, and the Oswestry low back pain disability questionnaire. The VAS and Oswestry were examined before the salvage operation and after a follow-up period of at least 1\u00a0year after this operation. The 1\u00a0year follow-up VAS and Oswestry are not yet available in all patients.\nStatistical analysis\nAnalyses were performed using SPSS, release 12.0.1. Non-parametric tests, i.e. Mann\u2013Whitney and Wilcoxon, were used to test mean. Significance was indicated by P values less than 0.05.\nResults\nLate complications\nAn overview of late complications after disc implantation is shown in Table\u00a02. They included the following: 39 patients showed subsidence of the disc prosthesis, in 24 of these patients the disc prosthesis was considered too small. Thirty-six patients showed signs of adjacent disc degeneration, narrowing of the disc and osteophytes on conventional X-rays. In 17 patients this was not obvious before artificial disc insertion on plain X-rays and discography. Eleven patients with multi-level adjacent disc degeneration had developed degenerative lumbar scoliosis. In 25 patients facet joint degeneration was seen on CT-scans. In six patients the disc prosthesis showed anterior migration and in two patients posterior migration of the disc prosthesis occurred. In ten patients we discovered breakage of the metal wire around the core (Fig.\u00a02).\nTable\u00a02Overview of late complications after receiving a disc prosthesis (patients can have more than one complication)Late complicationsNumber of patientsSubsidenceDisc prosthesis too small3924Adjacent disc degeneration36Degenerative scoliosis11Facet joint degeneration on CT scan25Anterior migration6Posterior migration2Breakage metal wire10Wear5Severe osteolysis1Subluxation PE core1Fig.\u00a02Example of removed polyethylene core L4\u20135 (left) and L5\u2013S1 (right) (6.5\u00a0years after insertion) with a fracture of the metal wire and damage of disc prosthesis\nLess often the surgeons noticed the subsequent late complications. In one case severe osteolysis was seen in the sacrum in a ruptured and severely worn L5\u2013S1 case. In another case a subluxation of the PE core and an adjacent osteoporotic compression fracture was noticed. In five patients radiological wear of the disc prosthesis was obvious due to loss of height of the core, or sclerosis and cysts around the prosthesis on CT-scan.\nStudy population\nThe group of 15 patients receiving posterior fusions in our hospital without removing the prosthesis, consisted of 8 men and 7 women. Mean age at their revision surgery was 49\u00a0years and 9\u00a0months (34\u201376\u00a0years) and mean time-interval between their disc implant and revision surgery was 7\u00a0years and 11\u00a0months (2\u201315\u00a0years).\nFacet joint degeneration was noticed during all operations. Afterwards, we removed the disc prosthesis in four patients of this group because of persisting pain. Nowadays, we advise disc prosthesis removal in conjunction with fusion surgery, assuming that the disc prosthesis can remain a pain source even after solid posterior fusion.\nSo far, we retrieved 26 prostheses in 22 patients (17 females, 5 males). The additional posterior fusion took place in nine patients 2\u00a0weeks before or 2\u00a0weeks after the removal of the disc prosthesis. In the other 13 patients, posterior fusion was done as a second operation on the same day as the removal. Nowadays this is the standard procedure in our hospital. The mean interval between insertion and retrieval of the disc prosthesis was 8\u00a0years and 11\u00a0months (3\u201316\u00a0years). The mean age at retrieval of the disc prosthesis was 50\u00a0years (40\u201372\u00a0years).\nA pre- and post-operative radiograph of one case, in which we removed the disc prosthesis L4\u20135 and L5\u2013S1 and performed a posterior and anterior fusion, is shown in Figs.\u00a03 and 4.\nFig.\u00a03a Anteroposterior and b lateral radiograph of the lumbar spine 3\u00a0years and 9\u00a0months after disc replacement at L4\u20135 and L5\u2013S1Fig.\u00a04a Anteroposterior and b lateral radiograph of the lumbar spine 9\u00a0months after revison surgery\nIntraoperatively we twice encountered a lesion of the left common iliac vein (in L5\u2013S1 cases), once a lesion of the left common iliac artery (in a L4\u2013L5 case) and once a small incomplete colon lesion. These complications could all be controlled by the vascular surgeon with relatively little blood loss. Mean blood loss for the anterior procedure was 753\u00a0cc (60\u20135,100\u00a0cc). In one patient profound bleeding was encountered from the vertebral body bone and possibly the epidural plexus underneath the distal endplate of a L5\u2013S1 disc prosthesis. This was controlled by packing with bone bank chips and gel foam, however the total blood loss in this two-level case was 5,100\u00a0cc.\nIn another patient we planned to remove the disc prosthesis, however, due to a rupture of the small intestine during the access phase, we decided only to perform a posterior fusion.\nPreliminary clinical results\nGeneral clinical information for both revision strategies, with a follow-up period of at least 1\u00a0year, is shown in Table\u00a03.\nTable\u00a03Clinical information for both revision strategies (with a follow-up period of at least 1\u00a0year)NrSexYear index surgeryAge index surgeryLevelsYear re-operationRemoval\/fusionExtra procedures% improvement VAS% improvement OswestryClinically improved Oswestry (>25%)1F199344L2\u2013L32003FusionNone41.1827.50Yes2M199541L4\u2013L51997FusionNone\u221233.33\u221244.44No3F199263L5\u2013S12005FusionNone\u221214.29\u221224.34No4M199730L5\u2013S12001FusionNone27.7816.67No5F200042L4\u2013L52003FusionNone12.5014.71No6F199642L4\u2013L5, L5\u2013S12003FusionNone27.786.45No7F200238L4\u2013L5, L5\u2013S12003FusionNone29.4116.13No8M199748L5\u2013S12004FusionNone86.6782.76Yes9M199537L5\u2013S12005FusionNone18.750.00No10F199734L5\u2013S12002Fusion None0.0074.07Yes2005RemovalNone29.41MissingMissing11M199237L4\u2013L52005RemovalNone\u221212.5025.00Yes12F199533L4\u2013L52006Removal None\u221212.505.41No13M198944L4\u2013L52005RemovalNone12.5030.00Yes14F199139L4\u2013L52002RemovalNone87.5056.25Yes15F199539L5\u2013S12004RemovalNone76.4773.08Yes16F199946L4\u2013L5, L5\u2013S12005RemovalNone62.5062.50Yes17F199233L3\u2013L4, L5\u2013S12005RemovalNone11.1112.50No18M199846L4\u2013L52004RemovalNone9.0938.10Yes19F199232L2\u2013L3, L4\u2013L5, L5\u2013-S12004RemovalNone55.5616.80No20M200147L4\u2013L52005RemovalNone\u22126.670.00No21M199539L4\u2013L52005RemovalDynesys62.503.33No22F200237L4\u2013L5, L5\u2013S12005RemovalNone11.1110.00No23F199055L4\u2013L52006RemovalNone14.2913.97No\nTen patients receiving posterior fusion without removal of the prosthesis, have at this moment a follow-up period of more than 1\u00a0year since their posterior fusion. The mean VAS before posterior fusion was 8.0 (SD 0.9) and after posterior fusion 6.3 (SD 2.1) (Fig.\u00a05).\nFig.\u00a05VAS scores before and after salvage operation\nFurthermore, 14 patients had a follow-up period of more than 1\u00a0year since their disc prosthesis removal. Two disc removal patients had insertion of the Dynesys fixation system (one patient had a follow-up of at least 1\u00a0year), in addition to the fused disc prosthesis level, for multiple adjacent levels degeneration. This multilevel Dynesys instrumentation was recently removed in both patients due to screw loosening. The VAS score in this group decreased significantly from 8.0 (SD 0.9) before disc prosthesis removal to 5.6 (SD 2.7) after removal (P\u00a0<\u00a00.05) (Fig.\u00a05). The percentage of improvement after revision surgery in both groups is shown in Fig.\u00a06.\nFig.\u00a06Percentage change in VAS scores in both revision strategies\nThe mean Oswestry decreased in the posterior fusion group (n\u00a0=\u00a010) from 57.0 (SD 17.0) to 44.6 (SD 20.4), and in the disc removal group (n\u00a0=\u00a013) from 56.3 (SD 14.0) to 43.0 (SD 20.7) (Fig.\u00a07). This questionnaire is missing in one patient from the disc removal group. According to the abovementioned IDE-criteria, in which an improvement of \u226525% was considered to be clinically improved, 3 out of 10 patients in the fusion group and 6 out of 13 patients in the disc removal group were clinically improved (Table\u00a03; Fig.\u00a08).\nFig.\u00a07Oswestry scores before and after salvage operationFig.\u00a08Percentage change in Oswestry scores in both revision strategies\nPostoperative complications\nTwo patients from the posterior fusion group developed pseudo-arthrosis postoperatively.\nWe encountered five postoperative complications in the disc removal group. One patient developed deep venous thrombosis (DVT) of the left leg after suturing a left common iliac vein lesion. In two patients, decreased sensitivity in the left groin and upper leg was noticed, which was partially reversible. Two patients have severe pain and decreased diffuse strength in the left leg postoperatively. In one of these patients these complaints are diminishing at the moment. Presumably, excessive retraction of the lumbosacral nerves in the psoas muscle played a role in these left leg complications.\nDiscussion\nSeventy-five patients with persistent leg and back pain after insertion of SB Charit\u00e9 disc prosthesis were studied. The causes of persisting pain were thought to be related to the following late-complications: subsidence, migration, wear of the disc prosthesis, facet joint degeneration or adjacent degeneration in various combinations.\nGood placement and good sizing of the disc prosthesis appeared problematic for many of the patients in our series. In 39 patients we saw subsidence of the disc prosthesis, and in our judgement X-rays showed that in 24 of these patients the disc prosthesis was too small. Whether suboptimal sizing and placement resulted from initial surgical technique, or from historical limitations in instrumentation and\/or sizing availability remains unclear. Fixation of the disc prosthesis can be inadequate, giving subsidence and migration of the disc prosthesis. Migration can probably be prevented by coating of the metal plate, but subsidence probably not.\nPrevious studies suggested that adjacent degeneration is prevented after disc replacing surgery [4, 8, 14]. However, in our study 36 patients showed adjacent disc degeneration. This could be the result of the DDD itself, spreading to multiple levels of the spine, and\/or be the consequence of stresses on adjacent levels, generated from the unphysiological motion and functioning of the disc prosthesis.\nConcerning the 25 patients in which facet joint degeneration was visible on CT-scans, we hypothesized that an instability is created after removal of the anterior longitudinal ligament and the annulus fibrosis. Replacement with an unconstrained prosthesis will accentuate this instability, especially in extension and axial rotation. The facet joints will be the only restrictor of axial rotation and will degenerate with time.\nBecause of persisting back and leg pain we performed a posterior fusion without disc removal in 15 patients. Initially, the results were disappointing in most patients, and we therefore started to remove the disc prosthesis. In all patients with removal of the disc prosthesis, PE wear was observed in minor or major degree with surrounding inflammatory fibrous tissue containing PE wear debris. The association between periprosthetic tissue inflammatory reactions and clinical manifestations of pain is not clear at the present time and will be studied further. It is speculated that with removal of the disc prosthesis and the inflammatory periprosthetic tissue, the patient may obtain better pain relief than after only posterior fusion [19]. This hypothesis obviously needs further support with a larger number of more carefully selected patients.\nClinical results after disc prosthesis revision performed in our hospital were diverse. Patients with short segment fusions seemed to fare better than patients with long segment fusions or long flexible fixations with the Dynesys system. At present, the small number of patients and the large number of potential factors influencing the outcomes (Table\u00a03), precluded assessment of a significant association between revision strategy and outcome in the current study.\nThe mean VAS for pain after disc prosthesis removal was reduced significantly with 2.4 points, from 8.0 to 5.6, whereas the VAS in patients with posterior fusion showed a smaller decrease from 8.0 to 6.3. However, the analyses are based on a small number of cases and a greater number of patients with longer follow up are necessary for a more definitive conclusion.\nIn a collaborative study by Kurtz et al. [10] it was concluded that artificial discs exhibited wear mechanisms similar to artificial hips and knees. Since the operated patients are mostly between 30 and 50\u00a0years at the time of their disc implantation and these young patients will be likely very active, wear may become a clinically significant issue at long-term follow-up. The clinical relevance of wear in total disc replacements is the subject of intense, ongoing research at our institutions.\nA major point to consider is that, in contrast with a posterior fusion, retrieval of an artificial disc prosthesis can be dangerous because of nearby vascular structures and scar tissue from the original surgery. However, in our hands, disc removal surgery proved feasible in all but one case in this patient group (26\/27 disc retrievals, 96%). Due to the availability of a vascular surgeon during the retrieval surgery average blood loss could be restricted.\nIn conclusion, serious complications may arise following total disc replacement surgery, however, as yet relatively few data are available to provide the basis for treatment of patients with clinically failed artificial discs. Removal of the disc prosthesis with anterior and posterior fusion gives slightly better results than posterior fusion alone after a follow-up of at least 1\u00a0year. Removal of the SB Charit\u00e9 artificial disc was feasible but with inherent risks. As more data become available, perhaps revision artificial disc surgery may become a more common operation for spine surgeons in the near future.","keyphrases":["complications","artificial disc","retrieval surgery","risk of reoperation"],"prmu":["P","P","P","R"]}
{"id":"J_Gastrointest_Surg-3-1-1915599","title":"The Use of Animal Models to Study Bacterial Translocation During Acute Pancreatitis\n","text":"Infection of pancreatic necrosis with intestinal flora is accepted to be a main predictor of outcome during severe acute pancreatitis. Bacterial translocation is the process whereby luminal bacteria migrate to extraintestinal sites. Animal models were proven indispensable in detecting three major aspects of bacterial translocation: small bowel bacterial overgrowth, mucosal barrier failure, and disturbed immune responses. Despite the progress made in the knowledge of bacterial translocation, the exact mechanism, origin and route of bacteria, and the optimal prophylactic and treatment strategies remain unclear. Methodological restrictions of animal models are likely to be the cause of this uncertainty. A literature review of animal models used to study bacterial translocation during acute pancreatitis demonstrates that many experimental techniques per se interfere with intestinal flora, mucosal barrier function, or immune response. Interference with these major aspects of bacterial translocation complicates interpretation of study results. This paper addresses these and other issues of animal models most frequently used to study bacterial translocation during acute pancreatitis.\nIntroduction\nExperimental models of acute pancreatitis exist for almost 150\u00a0years, with Claude Bernard first describing experimental pancreatitis by injection of bile and olive oil into the pancreatic duct of a rabbit.1 Ever since, animal experiments were indispensable in providing insight in pathophysiology and treatment of acute pancreatitis. Experimental studies have major advantages over clinical studies, such as the availability of study subjects, standardization of disease severity, ability to perform invasive tests, extensive tissue sampling, and the possibility to test prophylactic treatment strategies.2 Despite these advantages, some major aspects of the pathophysiology of acute pancreatitis remain unclear, mortality in severe acute pancreatitis is still as high as 5\u201328%, and optimal treatment strategies remain a topic of debate.3,4\nIn 1986, Beger et al. demonstrated a link between the intestinal flora, infection of pancreatic necrosis, and clinical outcome in patients with severe acute pancreatitis.5 At the present time, infection of pancreatic necrosis is still regarded to be a main predictor of outcome during severe acute pancreatitis, and bacterial translocation of intestinal flora is considered to be the cause.4\nChanges in intestinal motility and the associated shift of intestinal flora, mucosal barrier function, and the immune system were identified as pivotal aspects of bacterial translocation during acute pancreatitis.6\u201311 This has greatly increased the understanding of bacterial translocation, but better insight into the exact mechanism of bacterial translocation and subsequent infection of pancreatic necrosis is needed to develop adequate prophylaxis and treatment strategies for patients with severe acute pancreatitis.\nA multitude of animal models were used to study the mechanism of bacterial translocation, including radiolabeling, plasmid-labeled bacteria, or fluorescent beads.12\u201315 Despite all these efforts, however, the exact origin, route, and mechanism of bacterial translocation causing infection of pancreatic necrosis are still unclear. The main reason for this uncertainty is the lack of an \u201cideal\u201d animal model of acute pancreatitis to study pathophysiology of bacterial translocation and its treatment. The ideal model should be minimally invasive, standardized, reproducible, and resemble etiology, pathophysiology, disease course, and outcome of clinical acute pancreatitis, including response to treatment.2 Experimental models used to study bacterial translocation in acute pancreatitis and its treatment all seem to have methodological restrictions that complicate the interpretation of study results. In 2000, Foitzik et al. reviewed the use of animal models of acute pancreatitis and their suitability for evaluating therapy and concluded that animal models should be designed to mimic etiology and clinical course of human pancreatitis to increase their value.2 In addition, we would like to discuss the value animals studies and experimental models of acute pancreatitis have in face of their interference with one or more of the known aspects of bacterial translocation: intestinal motility and flora, mucosal barrier function, or the immune system.\nThe aim of this paper is to provide useful insights into the use of animal models to study bacterial translocation during acute pancreatitis, in the light of current knowledge of pathophysiology.\nAnimal Species and Housing Conditions\nBefore the late 1970s, larger laboratory animals such as dogs were predominantly used to study acute pancreatitis. But since the introduction of models of acute pancreatitis in small laboratory animals, mice or rats are generally favored for financial and ethical or practical reasons. Because of physiological and anatomical differences between species, choice of laboratory animal has important implications on the study results and extrapolation to the human situation.\nIntestinal flora differs between animal species, largely depending on dietary demands and anatomical differences of the gastrointestinal tract and habits.16\u201318 The protein-rich diet of dogs or cats results in lower counts of endogenous lactobacilli and higher counts of potential pathogens (e.g., clostridia species), compared to rats or mice with fiber-rich diets. Coprophagy, demonstrated by most rodents, also influences intestinal flora, resulting in higher counts of gram-negative bacteria in the proximal gastrointestinal tract.19,20 Also, rats and mice are often bred and kept under specific pathogen-free conditions, introducing modifications of intestinal flora.\nIntestinal barrier function also differs between species. In an experiment comparing small intestinal permeability between humans and rats, significant interspecies variation in urinary recovery of orally delivered mannitol was observed.21\nAnatomical differences between species should also be considered. The relative size of the jejunum, ileum, cecum, and colon of different laboratory animals can influence origin and route of bacterial translocation during acute pancreatitis. In humans, retroperitoneal connections between the intestines and pancreas can greatly affect the clinical course of the disease.22 Similar to humans, the dog pancreas is situated retroperitoneally. Rat and mouse pancreata, however, are almost fully enveloped by peritoneum, resembling a more intraperitoneal localization. Variation in retroperitoneal connections between intestines and the pancreas offers different routes for bacteria to translocate without being exposed to intraperitoneal immune cells.23\nExperiments using small animals (e.g., mouse or rat) usually incorporate a larger number of animals compared to experiments with large laboratory animals (e.g., cat or dog). The use of a larger number of small laboratory animals improves statistical power of an experiment. On the other hand, the use of larger animals could resemble human pathophysiology better, but a smaller number of animals means lower statistical power and increased potential false negative or false positive results.\nModels of Acute Pancreatitis\nAn abundance of animal models of acute pancreatitis is used to investigate bacterial translocation. Only models most frequently used for this purpose will be discussed. Baseline characteristics of the discussed models and their potential effects on intestinal flora, mucosal barrier, and immune function are summarized in Tables\u00a01 and 2. \nTable\u00a01Characteristics of Several Animal Models of Acute PancreatitisModelAnimal SpeciesPancreatic NecrosisPancreatic InfectionMortalityInvasivenessDuodenal loop24,25RatNoConsiderableHighLaparotomyCholine-deficient diet30\u201332MouseYesLittleHighMinimalDuct ligation34\u201337Rat\/opossumNo\/YesLittleLowLaparotomyCerulein44Mouse\/ratYes\/NoLittleLowMinimalDuct perfusion48Rat\/dog\/pigYesConsiderableModerate to highLaparotomyDuct perfusion + cerulein52RatYesConsiderableModerateLaparotomyTable\u00a02Aspects of Bacterial Translocation and Potential Confounding Factors of Animal ModelsAspectConfounding FactorModelIntestinal motility and floraAnimal speciesPotentially all modelsHousing conditions (SPF)Potentially all modelsDietCDE dietAnalgesicsInvasive modelsLaparotomyInvasive modelsBile flowDuct ligationCeruleinCerulein modelsIntestinal manipulationInvasive modelsMucosal barrier functionStressPotentially all models DietCDE dietAnestheticsInvasive modelsPancreatic proteasesDuct ligationIntestinal manipulation\/punctureDuct perfusionImmune systemStressPotentially all modelsDietCDE dietDisease course\/severitySpecies-dependentObstructive jaundiceDuct ligation, duodenal loopIntestinal manipulationInvasive models\nDuodenal Loop\nClosing the duodenal lumen proximally and distally to the papilla of Vater results in reflux of the duodenal contents enclosed in the loop, including bile and pancreatic secretions, into the biliopancreatic duct.24 In rats, this leads to acute pancreatitis of varying severity.25 Discontinuation of the gastrointestinal tract leads to mucosal atrophy and functional changes to the mucosal barrier.26 Furthermore, obstruction of bile flow into the intestine was shown to reduce intestinal motility, causing small bowel bacterial overgrowth and increased bacterial translocation.27\u201329 Another major downside is the occurrence of reflux of duodenal contents, including bacteria, into the biliopancreatic duct. These obvious drawbacks of this model in experiments concerning bacterial translocation are the cause of its limited popularity.\nEthionine-supplemented Choline Deficiency\nLombardi et al.30 described severe acute pancreatitis in young female mice after feeding a choline-deficient, ethionine-supplemented (CDE) diet.31 Acute hemorrhagic pancreatitis ensues, as well as diffuse intraperitoneal fat necrosis and several systemic effects such as acidosis, hypoxia, and hypovolemia. In this model, mortality ranges from 0 to 100% after 4\u00a0days and can be controlled by varying the duration of the choline-deficient diet.32 To ensure homogeneity and reproducibility, sex, age, and weight of the mice have to be closely matched, as well as food intake of all animals.32\nApart from these practical downsides of the model, systemic complications unrelated to pancreatitis (e.g., parotitis and fatty liver disease) render the model less useful for investigating systemic events (e.g., immune response) of acute pancreatitis.31 Little is known of the effect of ethionine suppletion or choline deficiency on intestinal flora or mucosal barrier function. But the most important drawback of this model to study bacterial translocation is the low incidence of pancreatic infection (3\u20138%), even in severe necrotizing pancreatitis.33\nBiliopancreatic Duct Ligation\nIn the duct ligation model, the common biliopancreatic duct is surgically clipped or tied at the sphincter of Oddi complex. The resulting obstruction of pancreatic secretions and potential biliary reflux into the pancreatic duct produce moderate pancreatitis, characterized by edema, moderate inflammation and hemorrhage, fat necrosis, and minimal acinar cell necrosis. Only in the American opossum does biliopancreatic duct ligation leads to severe acute pancreatitis with considerable necrosis.34\u201337\nThis model of acute pancreatitis greatly interferes with the pathophysiology of bacterial translocation. Obstruction of bile flow into the intestine causes small bowel bacterial overgrowth and bacterial translocation.28 Also, exclusion of pancreatic proteases in the gut lumen alters intestinal permeability.38,39 Apart from effects on the intestinal flora and mucosal barrier function, obstruction-induced jaundice also causes impairment of the immunesystem.40\u201342 These effects complicate the interpretation of bacteriological results to study bacterial translocation.\nCerulein Infusion\nInfusion of low doses of cerulein, a cholecystokinin analog, enhances production of pancreatic exocrine cell secretions without cell necrosis. In most species, infusion of supramaximal doses results in a decrease of secretion and acute pancreatitis with interstitial edema and inflammatory cell infiltration.43 In mice, cerulein causes severe acute pancreatitis with necrosis of 40% of acinar cells.44 In rats and other animals, however, cerulein-induced pancreatitis is usually mild and generally self-limiting. Moreover, pigs are reported to be insensitive to cerulein hyperstimulation.45 It should be noted that cerulein is known to affect intestinal motility. Studies investigating the use of cerulein in man have shown absence of recognizable migrating motor complexes with decreased colonic transit time.46 In general, experimental acute pancreatitis is associated with reduced small bowel motility, resulting in small bowel bacterial overgrowth and increased bacterial translocation to extraintestinal sites.6,47 Thus, cerulein may interfere with intestinal flora by altering intestinal motility. Investigators should keep this in mind when designing a study and interpreting study results.\nBiliopancreatic Duct Perfusion\nDuct perfusion models are currently the most popular models of acute pancreatitis. Induction of acute pancreatitis involves infusion of bile, bile salts with or without bacteria, or activated pancreatic enzymes into the (bilio-)pancreatic duct. Early experiments mainly involved dogs, but currently, rats are used most frequently. Severity and reproducibility of acute pancreatitis and ensuing bacteriological results strongly depend on infusate, infusion pressure, volume, and time.48\nThe most commonly used infusates are solutions containing various concentrations of bile salts of varying hydrophobicity. Both chemical and pressure effects of infusion were suggested to play a major role in the pathogenesis of pancreatitis in perfusion models.48,49 In both chemical- and pressure-induced pancreatitis, destruction of the pancreatic duct mucosal barrier is the key event. This is followed by pancreatic edema, autolysis, reduction of pancreatic blood flow, and, in severe cases, destruction of pancreatic parenchyma and formation of pancreatic necrosis.50 Uncontrolled pressure-related damage causes variation in severity of the induced acute pancreatitis between study subjects, and thus should be avoided. Several experiments were performed to assess maximal pancreatic duct pressure before rupture of the duct epithelium causing increased and uncontrolled severity of acute pancreatitis. Data are conflicting, with rupture pressures varying from 15 to 82\u00a0mmHg.48,49,51,52 A maximum infusion pressure of 30 to 50\u00a0mmHg is currently accepted for rat models.\nPerfusion is usually performed by puncturing the duodenum and cannulating the papilla of Vater. The introduction of duodenal bacteria, through the papilla of Vater into the biliopancreatic duct could potentially be a confounding factor in transduodenal duct perfusion models. It was demonstrated, however, that significant bacterial infection of the pancreas (>1\u2009\u00d7\u2009102 colony forming units per gram) because of the surgical procedure does not occur.53\nAdvantages of this model are the quick procedure of acute pancreatitis induction and the reproducibility of results. Other than duodenal puncturing and intestinal handling during surgery, both potentially affecting mucosal barrier function, no direct effects on intestinal flora or immune function are expected in this model.\nBiliopancreatic Duct Injection and Cerulein Hyperstimulation\nThe combination of retrograde infusion of bile salts with superimposed cerulein hyperstimulation in rats was introduced by Schmidt et al. and was advocated as \u201ca better model for evaluating therapy.\u201d52 Although the disadvantages described for biliopancreatic duct injection and cerulein hyperstimulation all apply to this model, it was proven a very valuable model to examine bacterial translocation and treatment strategies. The major advantages are that histological and qualitative bacteriological results as well as reaction to treatment and disease course resemble human acute pancreatitis more closely than other models.2,52 Although proven a very valuable model, potential model-related confounding factors as described above should always be kept in mind when interpreting results.\nDisease Course\nEspecially in the severe form of acute pancreatitis, systemic events can be divided into two phases: early proinflammatory and late immunosuppressive.54 In severe acute pancreatitis, the early phase is associated with a systemic inflammatory response syndrome (SIRS), potentially leading to multiple organ failure and early mortality. The late phase is characterized by immunosuppression, providing opportunity for infectious complications (e.g., infection of pancreatic necrosis) associated with sepsis and late mortality.2,55 Laboratory animal species and experimental models, however, each show their own disease course of acute pancreatitis.\nAnimal models were mainly used to investigate the early phase of acute pancreatitis.56 However, the model described by Schmidt et al. seems the most appropriate to investigate early and late systemic complications, considering that both phases can be discerned.52,57 In this model, infection of pancreatic necrosis progresses at least until 96\u00a0h. When taking into account that disease course is more rapid in small rodents, timing could well correlate with data on the course of severe acute pancreatitis in humans, as described by Foitzik et al.2, Beger et al.,4 and Lankisch et al.58\nSeverity\nPancreatic necrosis is produced in several animal models of acute pancreatitis (Table\u00a01). On the other hand, only duct perfusion, with or without superimposed cerulein hyperstimulation, and murine CDE models demonstrate mortality comparable to human necrotizing acute pancreatitis.32,52,59 Models with high early mortality may be useful to investigate early phase systemic inflammatory response and organ failure, but are less adequate to investigate late infectious complications and associated (multiple) organ failure.\nIn most models, necrosis needs to be present for pancreatic infection to occur. It needs to be noted that this does not apply for the duodenal loop model in which reflux of duodenal contents into the biliopancreatic duct occurs.60 In contrast, the murine CDE model produces elaborate necrosis, but is associated with very low rates of pancreatic infection.33\nCulturing, Controls, and Route of Bacterial Translocation\nIn all animal models, factors such as analgesia, anesthesia, or surgical techniques can influence bacteriological results. Morphine-like analgesics have a significant effect on bowel motility and cause bacterial overgrowth and translocation to extraintestinal sites.61 The anesthetic pentobarbital was suspected to be a factor in promoting bacterial translocation in a model of hemorrhagic shock.62\nAlso, stress causes mucosal barrier failure and bacterial translocation.63 Surgical procedures are stressful events, but animal transport or handling alone could potentially cause stress-induced bacterial translocation. The influence of stress on adrenaline and corticosteroid levels could have its own effect on the function of the immune system, potentially influencing the systemic reaction to acute pancreatitis and bacterial translocation.\nProper sterile surgical techniques are very important when investigating bacterial translocation. If abdominal surgery is involved, control cultures of the peritoneal cavity to trace surgical contamination are of special importance. If peritoneal cultures are found to be positive, extra caution should be taken with interpretation of bacteriological analysis of abdominal organs. In case of surgical contamination or transperitoneal bacterial translocation, the peritoneal covering of the organ samples might be the cause of positive organ cultures, not the bacterial colonization in the organ itself (false positive culture).\nPuncturing the duodenum in duct infusion models hypothetically causes spillage of duodenal contents onto the peritoneum, covering all abdominal organs. In rats, however, duodenal contents usually have low bacterial counts, mainly consisting of nonpathogenic lactobacilli only. On the other hand, a duct infusion study by Cicalese et al. reported positive peritoneal cultures at time of induction of pancreatitis of 16.6 to 33.3% of the studied rats.15 Literature review of different animal models fairly frequently shows positive peritoneal cultures at the time of termination and organ sample collection of rats with acute pancreatitis. Positive peritoneal cultures are observed varying from 0\u201310% in minimally invasive models of acute pancreatitis (cerulein injection, CDE diet) to 8\u2013100% in more invasive models (duct perfusion with or without cerulein hyperstimulation).6,14,15,64\u201366\nDiscussion\nChanges in intestinal motility and flora, mucosal barrier function, and immune response were established as pivotal aspects in the process of bacterial translocation during acute pancreatitis. Early after the onset of acute pancreatitis, neurohormonal effects result in reduced small bowel motility.6 This causes stasis of luminal contents and small bowel bacterial overgrowth with potential pathogens, including Escherichia coli and Enterococcus species. The abundant presence of luminal pathogens forms a challenge for the mucosal barrier. Furthermore, pancreatitis-associated reduced intestinal blood flow results in mucosal ischemia and reperfusion damage.67\u201369 Luminal bacteria, normally held at bay by the mucosal barrier, now have opportunity to penetrate into the intestinal epithelium. Local intestinal inflammation follows, further compromising mucosal barrier function. Pancreatitis and ensuing intestinal inflammation both contribute to a systemic proinflammatory response (SIRS), with damaging effects on distant organs.70,71 If the systemic response is severe, multiple organ dysfunction syndrome (MODS) might follow.72,73 If the patient survives the early phase, counterregulatory immunological pathways releasing anti-inflammatory cytokines result in a refractory state characterized by immunosuppression.74,75 Persistent immunosuppression will render the patient liable for infection of pancreatic necrosis. Multiple organ dysfunction syndrome caused by infectious complications is considered accountable for so-called late mortality or \u201clate septic death.\u201d74,76\nAlthough animal models were proven indispensable in acute pancreatitis research, model-related problems are most likely the reason for important questions on pathophysiology and treatment strategies to remain unanswered. Current topics of debate include the route and origin of bacterial translocation and optimal prophylaxis and treatment strategies.\nSeveral different routes of bacterial translocation were described and have directed efforts for many prophylactic and therapeutic strategies. Webster et al. showed bacteremia to occur early after induction of acute pancreatitis in CDE-induced acute pancreatitis, suggesting a hematogenous route.77 Likewise, rapid passage of bacteria into the blood was found in other models of acute pancreatitis.78 On the other hand, Runkel et al. found bacteria migrating to lymph nodes before their translocation to distant sites in a duct ligation model, suggesting a lymphogenous route.79 Widdison et al. suggested transperitoneal translocation of bacteria originating from the colon in a feline model of severe necrotizing pancreatitis.80 Other study groups, including our own, have provided proof of the role of the small bowel in the pathophysiology of bacterial translocation in acute pancreatitis or after morphine administration.6,61,81\nThe model of duct perfusion and cerulein hyperstimulation described by Schmidt et al. was proven very useful because it resembles human disease quite well, considering its biphasic disease course, pancreatic histology, \u201cmoderate\u201d mortality, and the bacterial spectrum in pancreatic necrosis.52 However, whether a confounder is introduced by puncturing the duodenum and cannulating the biliopancreatic duct is unknown. Therefore, to ensure quality of the presented study results, control cultures of the peritoneal cavity should be done when organ samples are analyzed bacteriologically. Peritoneal bacteria can potentially affect bacteriological analysis of all abdominal tissues. Widdison et al. washed abdominal samples before analysis, but this is not commonly performed.80 A pilot study by Arendt et al. showed that washing removed 94\u201397% of intraperitoneally injected bacteria.23 Immunohistologically localizing bacteria can help clarify if positive cultures of abdominal tissues are because of peritoneally located bacteria or actual bacterial colonization in the underlying organ tissue.\nWhen experimentally evaluating therapy, treatment often starts before induction of acute pancreatitis. Obviously, this is an important reason why results cannot directly be translated to the clinical situation. On the other hand, these experimental studies provide proof of principle concerning the tested therapy. If prophylactically successful, the tested treatment strategy might be beneficial when started after the onset of acute pancreatitis and should therefore be further investigated. On the other hand, the faster course of acute pancreatitis in rodent models provides only a very short treatment window between the onset of the disease and early or late phase complications. This may lead to false negative effects of the therapy tested.\nIn conclusion, animal models of acute pancreatitis are indispensable tools, but model-related drawbacks often interfere with one or more pathophysiological aspects of bacterial translocation, complicating interpretation of results. When the ideal model of acute pancreatitis is not at hand, it is of major value that numerous alternatives are available. But with each experimental hypothesis, special care should be taken to select the most suitable model. Despite all the experimental work done, the route by which pancreatic infection occurs and gives rise to septic complications and mortality has not yet fully been elucidated. Optimal prophylactic and treatment strategies are also still widely debated. In the future, animal models will undoubtedly provide increasing understanding of these subjects, but model-related drawbacks should always be kept in mind when designing a study or when interpreting results.","keyphrases":["animal","model","bacterial translocation","pancreatitis"],"prmu":["P","P","P","P"]}
{"id":"J_Med_Internet_Res-8-3-2018828","title":"Characterizing Internet Searchers of Smoking Cessation Information\n","text":"Background The Internet is a viable channel to deliver evidence-based smoking cessation treatment that has the potential to make a large population impact on reducing smoking prevalence. There is high demand for smoking cessation information and support on the Internet. Approximately 7% (10.2 million) of adult American Internet users have searched for information on quitting smoking. Little is known about these individuals, their smoking status, what type of cessation services they are seeking on the Internet, or how frequently these searches for cessation information are conducted.\nIntroduction\nThe Internet has become the first source of health information for many people, primarily due to the ease of finding information [1]. In particular, there appears to be great demand for online information and services related to smoking cessation. In a random-digit dial survey conducted in 2004, 7% of Internet users in the United States reported using the Web to search for information on \u201chow to quit smoking\u201d [2]; more women reported to have looked then men (10% vs 7%), and unlike other health-related information seekers, they tended to be younger. At the time, this represented approximately 10.2 million people who had ever turned to the Internet for smoking cessation\u2013related information or services. Little is known about these individuals, including their basic demographic characteristics, smoking status (eg, current smokers seeking cessation treatment, recent quitters seeking support to maintain abstinence), readiness to quit, quitting history, and treatment preferences. With the proliferation of antismoking sentiments and restrictive smoking policies, a diverse group of individuals may be turning to the Internet for assistance. In order to provide individually tailored and effective cessation treatment services via the Internet, it is necessary to better understand the characteristics and needs of this population.\nThe Internet is a powerful delivery channel that has the potential to deliver behavior change interventions on a population-wide basis to help people modify risk factors such as smoking [3]. There are limited, but encouraging, data to indicate that Web-based cessation interventions are effective in controlled trials [4-6]. However, it is not known if these approaches are appealing to or appropriate for the broader population of Internet users seeking cessation assistance. For example, approximately 30% of visitors to a widely utilized smoking cessation website indicated that they had quit smoking within the past week [7]. These individuals would be excluded from most randomized clinical trials of smoking cessation treatment, but they may represent a sizable population in need of assistance to remain abstinent. Information and services may need to be specially tailored to address the unique needs of individuals searching for cessation information based on their smoking status, demographic characteristics, and quitting history.\nThe incidence of cessation-related Internet searches may provide an effective proxy for consumer demand for cessation services. To date, there is little information about the rate at which searches for smoking cessation information occur. Several different techniques have been used to estimate the frequency of general health-related Internet searches [8-10], with widely varying results. Analyzing the first 300 search terms of the Wordtracker Top 500 keyword list, Phillipov and Phillips found less than 1% to be health-related terms [10]. Eysenbach took repeated snapshots of current search terms used on a search engine over a 15-month period, analyzed a random subset of queries, and found that 3.6-5.3% could be classified as health related [8]. Fox found that 79% of surveyed individuals had ever searched for health or medical information, while 7% had searched for smoking cessation information [2].\nThe primary purpose of this study was to characterize individuals who search for smoking cessation information. Specifically, we sought to gather information about sociodemographic and smoking history variables, search patterns (eg, time of day, search terms used), and perceptions about specific types of cessation services. Additionally, we used publicly available data to estimate the incidence of these searches. This information will be critical to develop appropriate and effective online cessation treatment programs, to triage patients as part of a stepped-care treatment model, or to successfully recruit smokers into treatment via the Internet.\nMethods\nRecruitment and Eligibility\nOur recruitment strategy leveraged the prominent position of QuitNet (www.quitnet.com) on three of the largest Internet search engines. QuitNet is an established smoking cessation website [7] that is highly utilized, with over 600000 visitors and 97000 new registrants in 2004 from the United States alone. During the period of this study, it was listed in the top results for queries using \u201cquit smoking\u201d or \u201cstop smoking\u201d on three large search engines: Google, Yahoo!, and MSN (Appendix 1). In 2003, approximately 210000 (globally) and 110000 US individuals looking for information on quitting smoking arrived at QuitNet via these search engines. It has been estimated that 80% or more of Web users seeking health information start from search engines [11,12]. Research shows that Internet users read search engine results linearly, pay the most attention to the top three to five results, and click on the first promising link they find in the results [11,13]. Therefore, individuals who click on the link to QuitNet from a search engine results page are likely to be a representative sample of those individuals looking for cessation information on the Internet.\nWe recruited individuals based on four inclusion criteria: (1) use of the terms \u201cquit smoking,\u201d \u201cquitting smoking,\u201d \u201cstop smoking,\u201d or \u201cstopping smoking\u201d in a search engine query; (2) use of one of three major search engines (Google, Yahoo!, or MSN) to conduct these queries; (3) no prior visit to the QuitNet website (defined as www.quitnet.com or www.quitnet.org) as determined by the absence of a persistent (long-term) tracking cookie; and (4) location within the United States as determined by reverse lookup of IP (Internet protocol) addresses. When eligible Internet users clicked on the QuitNet link in the results of a search engine query, they were \u201cintercepted\u201d and recruited to participate in the study. The recruitment screen contained links to the survey and to the QuitNet website (Appendix 2). Those who accepted the invitation were directed to the QuitNet website following completion of the survey. Those who declined the survey invitation went directly to the QuitNet website. Recruitment for the survey was conducted for a total of 10 days: it began December 30, 2003, was suspended January 1 through January 3 due to technical concerns, and was completed January 12, 2004.\nGeneralizabilty was established from the complete panel of respondents, while we restricted further analysis to the respondents that reported any history of smoking and were seeking assistance for themselves.\nMeasures\nThe survey consisted of 10 questions that included basic demographic information (age, gender), reasons for searching for cessation information, current smoking status, readiness to quit, quitting history (number of past quit attempts, length of quit, quit methods used), information desired, and ratings of perceived helpfulness of various online cessation features (eg, bulletin board, assistance in setting a quit date). The survey questions were administered on three separate screens, with no more than three questions per screen. Date and time of survey completion were automatically logged to the database.\nData on utilization of QuitNet after survey administration were extracted, including registration and total time online. Time online was defined as the time between the first page view after completion of the survey through the time of the last page view.\nStatistical Analyses\nTo determine the generalizability of our final sample, we compared survey respondents to nonrespondents who went on to register and use the QuitNet website on the demographic, website utilization, and search pattern variables obtained from the QuitNet database. In addition, we sought to determine the generalizability of our sample to the broader population of individuals who search for online smoking cessation information throughout the year. To do this, we examined the percentage of participants referred from each search engine as well as the total volume of cessation search terms used in Internet search engine queries, using publicly available data from Nielsen\/NetRatings [14], Overture, and Wordtracker. Chi-square analyses were used to compare our sample to these national datasets.\nFor the 10-item survey, frequency tables were used to summarize the categorical data, and nonparametric tests were used to determine the statistical significance level. We used t tests for normally distributed continuous and ordinal variables.\nFinally, to estimate the incidence of cessation-related Internet searches each year, we replicated the technique used by Eysenbach and Kohler [8]. MetaSpy was queried several times per day over the course of 9 months and the active queries were logged. Duplicate results (defined as the same set of 10 search terms being returned in succession) were removed. Searches containing the key words \u201cquit[ing] smoking\u201d or \u201cstop[ing] smoking\u201d were classified as cessation related.\nResults\nRecruitment Outcomes\nDuring the 10-day study period, 2265 eligible US residents were intercepted. Of those, 35.8% (N = 811) clicked on the \u201csurvey\u201d link, 48% (N = 1088) clicked on the link to take them directly to the site (\u201cdeclined\u201d), and 16.2% (N = 366) did neither (\u201cabandoned\u201d). Of the 811 individuals who clicked through to the survey, 87.2% (N = 655) completed the full survey, yielding an overall response rate of 29% (Figure 1). Of the survey completers, 29 individuals reported having never smoked, leaving a final sample of 626 respondents.\nFigure 1\nEligibility and Recruitment Results\nGeneralizability\nTo assess generalizability, we compared all survey participants (N = 655) with nonrespondents who proceeded to register with QuitNet (N = 243). Overall, nonrespondents (N = 1454, abandoned and declined) were significantly less likely than survey respondents to register on QuitNet (16.7 vs 51.4%, \u03a72\n2 = 303.7, P < .001). Compared to survey respondents, nonrespondents spent less time on QuitNet (4.5 vs 12.0 minutes, t = 13.4, P < .001) and viewed fewer pages (5.9 vs 15.3 pages, t = 16.0, P < .001) on the website. Nonrespondents were more likely to be female (59.4 vs 51.9%, \u03a72\n2 = 4.2, P = .02) but did not differ by age, smoking status, time of survey invitation, or specific search engine used.\nAs shown in Table 1, the relative volume of participants referred from each search engine was consistent with national usage patterns (\u03a72\n2 = 1.06, P = .59). In this study, 57% of participants were referred from Google, 29% from Yahoo!, and 14% from MSN. At the time of this study, 60% of all Internet search queries were estimated to be conducted using Google, 23% with Yahoo!, and 17% with MSN [14].\nTable 1\nComparison of search engine usage to Nielsen\/NetRatings statistics\nRelative Reach of Search Engines\n\u00a0Search Engine\nSurvey Recruitment (%)\nNational Usage (%)\nGoogle\n57\n60\nYahoo!\n29\n23\nMSN\n14\n17\nTotal\n100\n100\nThe use of key search terms (\u201cquit smoking,\u201d \u201cquitting smoking,\u201d \u201cstop smoking,\u201d or \u201cstopping smoking\u201d) by survey respondents was also consistent with search patterns captured by Overture and Wordtracker. As shown in Table 2, the most commonly used search term was \u201cquit smoking,\u201d which constituted 52.9% of study queries, 59.1% of Overture queries, and 47.8% of Wordtracker queries. \u201cStop smoking\u201d was the second most frequently used search term, which constituted 24.9% of study queries, 31.1% of Overture queries, and 36.5% of Wordtracker queries.\nTable 2\nFrequency of smoking-related search terms in search engine queries\nSearch Term\nSearches (%)\nSurvey Participants (%)(\u03a724 = 3.35, P = .80)\nOverture (%)(\u03a724 = 152, P < .001)\nWordtracker (%)(\u03a724 = 138, P < .001)\nquit smoking\n52.9\n55.4\n59.1\n47.8\nstop smoking\n24.9\n23.9\n31.1\n36.5\nquitting smoking\n21.9\n20.4\n9.0\n13.4\nstopping smoking\n0.3\n0.4\n0.6\n1.8\ngiving up smoking\n0.00\n0.00\n0.2\n0.6\nParticipant Characteristics\nAs shown in Table 3, the majority of study participants were female (61.2%, n = 383) and between the ages of 26 and 44 years (62.7%, n = 393); 18.7% (n = 117) were aged 18-25 years, 17.1% (n = 107) were aged 45-64, and less than 1% were 65 or older (n = 4) or under age 18 (n = 5). Adjusted to local time of the participant, more than half (53.4%) of search engine queries for cessation information occurred during work hours (8 am-5 pm), 26.6% occurred between 5-9 pm, and 20% occurred at night (9 pm-6 am).\nParticipants were asked the reason they were searching for smoking cessation information. The majority of survey respondents (90.1%, n = 590) indicated that they were looking for help or support for themselves; 5.6% (n = 37) were looking for general information; 3.4% (n = 22) were looking for help for someone else; and 1% (n = 6) were health professionals or researchers looking for information. Further analyses were limited to individuals looking for cessation help or support for themselves or for general cessation information (N = 626). Among these individuals, 75.4% (n = 472) were current smokers, 17.4% (n = 109) had quit within 7 days (\u201crecent quitters\u201d), and 7.2% (n = 45) had quit more than 7 days ago (\u201clonger-term quitters\u201d).\nTable 3\nDemographic and smoking characteristics of study participants (N = 626)\nCharacteristic\nNumber of Participants (%)\nAge\n< 18\n5 (0.8)\n18-25\n117 (18.7)\n26-34\n232 (37.0)\n35-44\n161 (25.7)\n45-54\n87 (13.9)\n55-64\n20 (3.2)\n65 or older\n4 (0.6)\nGender\nMale\n243 (38.8)\nFemale\n383 (61.2)\nSmoking Status\nCurrent smoker\n472 (75.4)\nNot thinking of quitting\n1 (0.2)\nThinking of quitting in 6 months\n222 (35.5)\nThinking of quitting in 30 days\n249 (39.8)\nQuit \u2264 1 week\n109 (17.4)\nQuit > 1 week, \u2264 1 month\n43 (6.9)\nQuit > 1 month\n2 (0.3)\nThe majority of current smokers (52.8%, n = 249) planned to quit in the next 30 days, 47.0% (n = 222) planned to quit in the next 6 months, and one person (0.2%) was not thinking about quitting. Smokers had made an average of 5.1 quit attempts (SD = 14.7; median = 1) during the past year.\nInformation Preferences\nAs shown in Table 4, information preferences varied by smoking status. Current smokers were more likely than recent quitters and longer-term quitters to be interested in information about how to quit smoking (88.1%, 54.1%, and 40.0%, respectively; \u03a72\n2 = 104.7, P < .001) and medication usage (30.7%, 5.5%, and 4.4%, respectively; \u03a72\n2 = 41.0, P < .001). Not surprisingly, both recent quitters and longer-term quitters were more interested than current smokers in information about withdrawal (77.1%, 66.7%, and 59.7%, respectively; \u03a72\n2 = 11.7, P = .003).\nTable 4\nInformation sought by smoking status (N = 626)\nInformation\nCurrent Smoker (%)\n(n = 474)\nQuit \u2264 1 Week (%)\n(n = 109)\nQuit > 1 Week (%)\n(n = 45)\n\u03a722\nP value*\nHow to quit\n88.1\n54.1\n40.0\n104.7\n< .001\nMedications\n30.7\n5.5\n4.4\n41.0\n< .001\nAlternative methods\n57.6\n16.5\n17.8\n77.3\n< .001\nWithdrawal\n59.7\n77.1\n66.7\n11.7\n.003\n*Current smokers are the reference group.\nNote: Multiple responses were allowed, so total percentages within smoking category exceed 100%.\nPerceived Helpfulness of Cessation Services\nParticipants were also asked to rate the perceived helpfulness of various smoking cessation treatment interventions on a scale from 1 to 5, with 1 representing \u201cvery helpful\u201d and 5 representing \u201cnot helpful at all.\u201d As shown in Table 5, the three features that were rated most highly by all participants were (1) individually tailored information (mean = 1.90, SD = 1.18); (2) information on withdrawal (mean = 1.84, SD = 1.15); and (3) a meter that keeps track of personal data (mean = 2.14, SD = 1.37). The three features rated the lowest by all participants were (1) support from a telephone counselor (mean = 3.21, SD = 1.35); (2) email support (mean = 2.95, SD = 1.40); and (3) support from others (mean = 2.90, SD = 1.38). Ratings of perceived helpfulness varied according to smoking status. Current smokers rated information about medications, assistance in setting a quit date, and assistance in choosing a medication as more helpful than did recent quitters and ex-smokers. Support from others and information about withdrawal received higher ratings of perceived helpfulness from recent quitters and ex-smokers than from current smokers. As detailed in Table 6, information of withdrawal, individually tailored information, and tracking meters were rated as \u201chelpful\u201d or \u201cvery helpful\u201d by over half of the participants, while telephone counseling was thought to be helpful by less than 30% of participants.\nTable 5\nPerceived helpfulness of Internet features by smoking status\nFeature\nAll Participants, Mean (SD)\n(N = 626)\nCurrent Smokers, Mean (SD)\n(n = 472)\nQuit \u2264 1 Week\n(n = 109)\nQuit > 1 Week\n(n = 45)\nMean (SD)\nP value*\nMean (SD)\nP value*\nInformation on withdrawal\n1.84 (1.15)\n1.90 (1.17)\n1.67 (1.08)\n.06\n1.51 (0.75)\n.04\nIndividually tailored information\n1.90 (1.18)\n1.88 (1.18)\n2.00 (1.25)\n.36\n1.79 (0.95)\n.62\nA meter that keeps track of personal data\n2.14 (1.37)\n2.14 (1.37)\n2.14 (1.42)\n1.0\n2.15 (1.31)\n.97\nInformation on medication side effects\n2.59 (1.38)\n2.55 (1.38)\n2.79 (1.34)\n.11\n2.54 (1.43)\n.97\nAssistance in choosing a medication product\n2.72 (1.37)\n2.61 (1.36)\n2.97 (1.38)\n.02\n3.24 (1.24)\n.007\nInformation on medications\n2.72 (1.36)\n2.62 (1.36)\n2.97 (1.37)\n.02\n3.23 (1.23)\n.007\nOnline, personal help from a professional\n2.81 (1.38)\n2.79 (1.40)\n2.86 (1.29)\n.67\n2.88 (1.39)\n.70\nAbility to find buddies\n2.82 (1.37)\n2.87 (1.39)\n2.74 (1.29)\n.40\n2.59 (1.34)\n.22\nAssistance in setting a quit date\n2.83 (1.39)\n2.69 (1.37)\n3.25 (1.32)\n< .001\n3.39 (1.37)\n.003\nSupport via chat, forums, or email\n2.90 (1.38)\n2.98 (1.39)\n2.67 (1.35)\n.04\n2.57 (1.30)\n.07\nAdditional information that arrives by email\n2.95 (1.40)\n2.91 (1.43)\n3.06 (1.30)\n.34\n3.08 (1.26)\n.49\nTalking by phone with a professional counselor\n3.21 (1.35)\n3.17 (1.39)\n3.32 (1.22)\n.32\n3.46 (1.29)\n.20\n*\nP values compared to current smokers; P = ns for all comparisons between recent and long-term quitters.\nNote: 1 = very helpful; 2 = helpful; 3 = somewhat helpful; 4 = not very helpful; 5 = not helpful at all\nTable 6\nProportion of participants (N = 626) rating Internet cessation services as helpful or very helpful\nFeature Offered\nHelpful or Very Helpful\nn\n%\nInformation on withdrawal\n460\n73.5\nIndividually tailored information\n450\n71.9\nA meter that keeps track of personal data\n405\n64.7\nInformation on medication side effects\n303\n48.4\nInformation on medications\n275\n43.9\nAssistance in choosing a medication product\n273\n43.6\nOnline, personal help from a professional\n265\n42.3\nAbility to find buddies\n250\n39.9\nAssistance in setting a quit date\n248\n39.6\nSupport from others, via chat, forums, or email\n233\n37.2\nAdditional information that arrives by email\n223\n35.6\nTalking by phone with a professional counselor\n184\n29.4\nEstimating Incidence of Cessation Queries\nOver the course of 9 months, 541685 searches were extracted from MetaSpy, of which a total of 38 were smoking cessation related. Assuming a total search engine volume of 52 billion searches per year [14], this ratio yields an estimate of 3.6 million (99% CI = 2.5-4.8 million) cessation-related searches per year in the United States alone.\nDiscussion\nThe Internet holds great potential to impact population smoking prevalence by delivering evidence-based treatments to greater numbers of smokers who may never receive treatment through other modalities. This is the first study to characterize the population of individuals looking for cessation information online. Results suggest that the Internet may be an effective way to reach smokers who are younger, who search for cessation services during work hours, and who have recently quit on their own.\nThe relatively large proportion (17.4%) of recent quitters (within 7 days) in this study who are actively seeking assistance is of particular importance. The majority of self-quitters relapse within 8 days [15]. Over 16 million Americans try to quit on their own each year, but less than 5% maintain abstinence for 3 months [16]. Thus, more than 15 million smokers relapse. Until recently [17], this segment of the population of smokers received little attention once formal cessation treatments ended. Given the reach and 24\/7 availability of the Internet, effective relapse prevention interventions can and should be delivered to the thousands of smokers trying to maintain abstinence. An effective relapse prevention service for self-quitters with intensive support around the quit date could produce a significant impact on smoking prevalence and could be used in conjunction with any other cessation treatment.\nNew population-based strategies to identify and reach smokers with evidence-based cessation treatment are needed [3]. Currently, telephone quit lines are the primary public health delivery channel for low cost, effective tobacco treatment. Despite the obvious advantages of convenience and cost, uptake rates in states with quit lines have remained low despite aggressive promotion, with less than 2% of smokers participating [18]. Given that Internet searchers are more likely to prefer self-help treatment with lower efficacy rates, it is important to design interventions which capture initial interest that can successfully \u201cup-sell\u201d more intensive and effective treatment interventions such as telephone counseling and medication use. In this manner, the Internet may be able to provide a workable model for stepped care, where participants can be further triaged to receive telephone counseling; prescription medication; in-person, group, or individual counseling; or even inpatient treatment [19].\nLimitations\nSeveral limitations should be considered when interpreting results of this study. The relatively low response rate (29%) raises concern about the generalizability of findings. Survey respondents were more likely to go on to register with the site; this likely indicates that they were in a more advanced stage of change than nonrespondents. It may, however, also indicate that the survey itself acted as an incentive to proceed to registration. Furthermore, we worked from the assumption that individuals who clicked on the link to QuitNet in search engine results were representative of the entire population of searchers. Although consistent with utilization patterns of search engines, this assumption has never been tested for searches on smoking cessation, or the QuitNet site in particular. It is possible that less motivated searchers may find the query results unappealing and not click on any link at all, thus biasing our results toward individuals closer to quitting.\nA second potential limitation is the method we used to estimate the total number of people seeking smoking cessation information each year. This method does not take into account searches using other keywords or individuals using resources other than search engines to find information (eg, health Web portals, referrals from health professionals, direct-to-consumer advertising, or quit lines). In addition, individuals may search for information multiple times, making it difficult to estimate the actual number of unique individuals as opposed to the total number of searches. Finally, the dataset used to derive these estimates is of commercial nature and published online in a promotional context. It has not been peer-reviewed or made available in its raw form. The data for this study were collected from 2003-2004; it is possible that in the intervening time the demographics or search behavior of smokers has changed. However, given the limited changes in both search engine technology as well as the demographics of smokers in the United States, this seems unlikely. Despite these limitations, this study provides valuable information about people who search for smoking cessation information online, and it demonstrates a new methodology for validating this kind of survey data.\nConclusion\nThis study suggests that the potential public health impact that can be achieved through Internet-based smoking cessation programs is significant given the reach of the Internet\u2014should these interventions be proven effective. Given that individuals may conduct multiple searches, our estimate of 3.6 million active searches per year for smoking cessation information is consistent with the 2004 data that showed 7% (about 10 million) of Internet users in the United States had searched for information on quitting smoking [2]. With 1.25 billion smokers throughout the world [20], there is enormous potential to globally impact smoking prevalence.\nThe public health community has invested heavily over the past 15 years in successfully de-normalizing smoking and encouraging cessation. However, low uptake rates seen in clinical programs and telephone quit lines call for new population-based approaches. Even if Internet-assisted tobacco interventions prove to have limited efficacy, the Web may still serve as a point of entry to multi-modality treatment programs. These programs may serve to simply link online searchers to more traditional treatment programs (such as telephone counseling or local group sessions), provide pharmaceutical products, or, in more sophisticated settings, use the Web as a platform to integrate voice counseling, local groups, mailed pharmaceutical products, and other proven modalities. We anticipate that the consumer demand demonstrated in this report will ultimately drive increasing services that will reflect a mixture of these different evidence-based treatments.","keyphrases":["internet","smoking","cessation","search engine","query"],"prmu":["P","P","P","P","P"]}
{"id":"Cancer_Causes_Control-4-1-2323031","title":"Cancer screening in France: subjects\u2019 and physicians\u2019 attitudes\n","text":"Objective Since screening for cancer has been advocated, funded, and promoted in France, it is important to evaluate the attitudes of subjects in the general population and general practitioners (GPs) toward cancer screening strategies.\nIntroduction\nEarly detection of cancer can theoretically prolong overall survival of the screened subjects. Reductions in cancer-related mortality have been demonstrated for mammography for breast cancer (BC) and fecal occult blood test (FOBT) for colorectal cancer (CRC) [1, 2]. For these two cancers, mass-screening programs are implemented in France, in which targeted subjects are contacted by mail. The US Preventive Services Task Force guidelines rate CRC screening as \u201cA\u201d (strongly recommended), BC screening as \u201cB\u201d (recommended), and both prostate cancer (PC) and lung cancer (LC) screenings as \u201cI\u201d (evidence insufficient to come down in favor or against screening) [3]. The objectives of the nationwide EDIFICE opinion poll carried out in France were to collect data at the national level (the level of funding) about (1) subjects\u2019 individual access to cancer screening procedures (through organized programs or on subjects\u2019 own initiative) and (2) about general practitioners\u2019 (GPs) attitudes toward cancer screening. Four frequent cancer types were selected on the grounds of their different screening statuses: BC and CRC for evidence of efficacy of screening and existence of a national screening program and prostate and lung cancers for neither evidence of efficacy of screening nor official screening program.\nMethods\nGeneral population opinion poll\nThe population-based EDIFICE opinion poll was carried out by telephone from 18 January to 2 February, 2005 among a representative sample of 1,504 subjects living in France and between 40 and 75\u00a0years old (1,609 subjects minus 105 who had already been affected by cancer). Sample representativeness was assessed, in relation to the statistics of the French Employment Survey in year 2002 [4], based on the following criteria: sex, age (five categories), profession (eight categories), community size (five categories), and regional distribution (nine categories).\nGeneral practitioner opinion poll\nA nationwide opinion poll was carried out by telephone from 31 January to 18 February, 2005 among a representative sample of 600 GPs practicing in France. Sample representativeness was assessed based on the following criteria: age (four categories) and regional distribution (five categories).\nResults\nThe main results of EDIFICE opinion poll are summarized in Fig.\u00a01. Almost all (93%) interviewed women stated that they had undergone at least one mammography. In contrast, 25%, 36%, and 6% of the interviewed subjects stated that they had undergone screening tests for CRC, PC, and LC, respectively.\nFig.\u00a01Adherence to cancer screening strategies according to the French nationwide EDIFICE opinion poll (1,504 subjects, 600 general practitioners). In France, there are organized mass-screening programs for breast cancer by mammography (100% of the population covered at the time of the opinion poll) and for colorectal cancer using Hemoccult (almost 30% of the population covered). In contrast, there is no mass-screening program for prostate cancer and lung cancer. For subjects in the general population, figures indicate the rates of subjects stating that they had undergone at least one screening test. For general practitioners, figures indicate the rates of general practitioners stating that they systematically recommended cancer screening\nThe corresponding percentages of GPs who stated that they recommended cancer screening tests to their patients were consistent with the proportions of subjects who had undergone screening tests for each tumor type: 68%, 18%, 58%, and 4% of the interviewed GPs stated that they systematically recommended screening for BC, CRC, PC and LC, respectively.\nReasons given to explain why screening tests had not been performed\nPhysicians and subjects provided contrasting answers as to why cancer-screening tests had not been performed. Physicians mainly focused on subjects\u2019 fears while subjects denied this reason. Fear of results was cited by 44% of physicians as the main explanation why subjects did not undergo BC screening tests versus only 18% of subjects. For CRC screening, the figures were 16% and 3%, respectively. The corresponding odds ratios for fear being elicited by subjects versus GPs as the reason for not undergoing BC or CRC screening tests were 0.29 and 0.15, respectively (Table\u00a01). Furthermore, subjects focused on a lack of advice on cancer screening from their physicians whereas GPs rarely did so. Lack of advice from the physicians was cited by 11% of subjects as the main explicative factor for subjects not undergoing BC screening tests versus only 1% of GPs. For CRC screening, the figures were 16% and 9%, respectively. The corresponding odds ratios for lack of medical advice being elicited by subjects versus GPs as the reason for subjects not undergoing BC or CRC screening tests were 11.65 and 1.93, respectively (Table\u00a01).\nTable\u00a01Reasons cited by subjects in the general population and general practitioners why subjects did not undergo screening tests for breast cancer (BC) or colorectal cancer (CRC)Reasons citedFor not undergoing BC screening testsFor not undergoing CRC screening testsFemale subjects N\u00a0=\u00a038General practitioners N\u00a0=\u00a0600Subjects N\u00a0=\u00a0725General practitioners N\u00a0=\u00a0600Subjects fearing the result n (%)7 (18)264 (44)20 (3)96 (16)OR (CI95%)0.29 (0.12\u20130.66)0.15 (0.09\u20130.24)Lack of physician\u2019s advice n (%)4 (11)6 (1)116 (16)54 (9)OR (CI95%)11.65 (3.14\u201343.23)1.93 (1.37\u20132.71)OR indicates odds ratio; CI95% indicates confidence interval at the risk \u03b1\u00a0=\u00a00.05\nDiscussion\nIn the present study, we describe the adherence to different screening programs for four types of cancers (BC, CRC, PC, and LC) in France. It is important to point out that this opinion poll does not report an accurate incidence of subjects actually screened for cancer but indeed the proportion of subjects stating that they had undergone at least one screening test. It is also worth mentioning that the French national health insurance system currently makes the corresponding screening tests (FOBT, mammography, prostate specific antigen\u2014PSA\u2014testing and X-ray) available for free (or almost free) to all affiliates.\nThe major finding of this report is the obvious disagreement between evidence-based official recommendations and actual practice is both subjects in the general population and GPs in France. Despite a high level of evidence of reduction in cancer-related mortality of similar magnitude (\u223c15\u201320%) with both BC and CRC mass-screenings [1, 2], 93% of women undergo mammography screening whereas only 25% of subjects in the same age range have access to CRC screening tools. In contrast, 36% of men aged between 50 and 74\u00a0years have undergone a screening test for PC (mainly PSA testing) even though the benefit of PC screening remains unknown [5].\nEDIFICE showed, on the one hand, two \u201crational rates\u201d of screening\u2014high rate of women having undergone mammography and low rate of subjects having performed LC screening\u2014and, on the other hand, two \u201cinadequate rates\u201d of screening\u2014abnormally low rate of CRC screening (nationwide coverage by the on-going program is expected by the end of 2007) and abnormally high rate of PC screening. Even more striking, the French GPs\u2019 behavioral pattern of recommending individual cancer screening exhibited the same inconsistencies.\nThree reasons can be suggested to explain the observed relationships between subjects\u2019 and GPs\u2019 behaviors. First, subjects in the general population may be influenced by medical counseling. Alternatively, physicians may endorse their patients\u2019 views and agree \u201cunder pressure\u201d [6]. Lastly, both subjects and GPs are exposed to similar not evidence-supported recommendations\/information, with the \u201cmagic touch\u201d of blood analysis for PC screening being attractive to them. Appropriate information of subjects and physicians, including the possible benefits and risks of PC screening through PSA testing, could make these erroneous behaviors less frequent [7].\nWhereas GPs\u2019 and subjects\u2019 statements in EDIFICE appeared in rather good agreement with respect to the proportions of interviewed subjects having undergone cancer screening tests and of GPs recommending cancer screening to their patients, the reasons put forward why screening tests were not performed are different, indeed even opposite: based on their statements, GPs overestimated the negative impact of fear of the results on subjects\u2019 participation in cancer screening and they underestimated their own role. Nevertheless, both GPs and subjects interestingly seem to attach a more dreadful meaning to the result of BC screening than to the result of CRC screening. Indeed, fear of the result acting as a check upon carrying out cancer screening tests was cited by 18% of the subjects for BC screening whereas only 3% cited this reason for not undergoing CRC screening tests. Comparatively, 44% and 16% of GPs cited fear of the result as the reason for subjects not undergoing BC and CRC screening tests, respectively.\nIt thus appears that there is a need for more research in social science as well as in biology and public health to improve the effectiveness of cancer screening in the framework of a national health system.","keyphrases":["mass-screening","compliance","health care opinion poll","health services research","health services misuse"],"prmu":["P","U","M","R","M"]}
{"id":"J_Abnorm_Child_Psychol-4-1-2206247","title":"Trajectories of Delinquency and Parenting Styles\n","text":"We investigated trajectories of adolescent delinquent development using data from the Pittsburgh Youth Study and examined the extent to which these different trajectories are differentially predicted by childhood parenting styles. Based on self-reported and official delinquency seriousness, covering ages 10\u201319, we identified five distinct delinquency trajectories differing in both level and change in seriousness over time: a nondelinquent, minor persisting, moderate desisting, serious persisting, and serious desisting trajectory. More serious delinquents tended to more frequently engage in delinquency, and to report a higher proportion of theft. Proportionally, serious persistent delinquents were the most violent of all trajectory groups. Using cluster analysis we identified three parenting styles: authoritative, authoritarian (moderately supportive), and neglectful (punishing). Controlling for demographic characteristics and childhood delinquency, neglectful parenting was more frequent in moderate desisters, serious persisters, and serious desisters, suggesting that parenting styles differentiate non- or minor delinquents from more serious delinquents.\nDuring childhood the family environment constitutes the basic social ecology in which the child\u2019s behavior is manifested, learned, encouraged or suppressed (Dishion and Patterson 2006). Criminologists have long since acknowledged the association between parenting and delinquency (Loeber and Stouthamer-Loeber 1986) and various criminological theories have included parenting behaviors among their explanatory variables (e.g., Hirschi 1969). However, only with the advent of developmental criminology during the 1990s have criminological theories been proposed linking a variety of family factors and parenting practices to specific developmental trajectories of delinquency.\nIn this study we make use of data from the Pittsburgh Youth Study (PYS), a longitudinal study covering a period of over 14\u00a0years. Our aim is to test whether distinct developmental trajectories based on delinquency seriousness can be identified and whether parenting styles are differentially linked to membership of these trajectories.\nParenting and Delinquency\nA delinquent trajectory, the evolution of delinquency over age, can usefully be described by its level (intercept) and its rate of change over time (slope). Developmental criminological theories differ in the extent to which they consider between-individual variation on these two dimensions. Some theories account only for differences in the absolute level of delinquency, assuming, often implicitly, the shape of the delinquent trajectory to be relatively similar across individuals (e.g., Gottfredson and Hirschi 1990). Others explicitly recognize variation in both the intercept and slope of delinquent development, linking differently shaped trajectories to different etiological factors, including parenting practices (e.g., Moffitt 1993).\nA prominent example of a theory explaining only level differences in delinquent development is Gottfredson and Hirschi\u2019s (1990) \u2018General Theory\u2019. Their theory attributes delinquency to lack of self-control. While recognizing inherent individual differences, Gottfredson and Hirschi claim low levels of self-control to result from parents failing to monitor the child\u2019s behavior, to recognize deviant behavior when it occurs, and to punish such behavior (Gottfredson and Hirschi 1990). Other theories go beyond explaining only level differences in delinquency and examine how delinquency changes by age. Moffitt (1993), Patterson (e.g., Patterson and Yoerger 2002), and Lahey and Waldman (2003), for example, offer theories that try to explain why delinquent trajectories are differently shaped for different types of individuals. The basic premise of these models is that children differ, whether continuously (Lahey and Waldman) or discontinuously (Moffitt), in key temperamental and cognitive elements that make up antisocial propensity. According to these typologies difficult children negatively affect their parents\u2019 disciplinary strategies, resulting in harsher and inconsistent punishments and parents being less involved in the socialization process. These negative child\u2013parent transactions set a child off on a delinquent path that starts in the early teens, entails many delinquent acts and persists far into adulthood. In contrast, neuropsychologically healthy children with average temperamental profiles, raised in adaptive family environments, are unlikely to develop enduring and serious delinquency trajectories. These children tend to show minor, non-aggressive delinquent trajectories that peak in adolescence reflecting their desire to express autonomy from parental control (Moffitt 1993) or peer pressure to engage in delinquent acts (Lahey and Waldman 2003; Patterson and Yoerger 2002). Finally, children with extremely low risk profiles, experiencing both individual and structural barriers, will be impervious to these social influences and are expected to refrain from delinquency altogether.1\nFrom Parenting Dimensions to Parenting Styles\nThe vast majority of studies on the family-delinquency association have treated the family as a potential risk factor for delinquent behavior. Family risk factors include characteristics of parenting as well as other family-related issues such as marital discord, psycho-social problems of parents, and delinquency within the family (Loeber and Stouthamer-Loeber 1986). However, research adopting this risk factor approach is variable-centered, identifying differences among families on single dimensions, such as harsh parental discipline, supervision, and control, but not considering how these various dimensions coalesce within specific families. Several scholars have argued that adopting a typological approach is more suitable for studying a complex system such as a family (Bergman and Magnusson 1997). This approach combines aspects of variable- and case-centered approaches in which the whole functioning of the system is the unit of analysis by empirically organizing the variety of characteristics and dynamics of families (Henry et al. 2005; Mandara 2003; Mandara and Murray 2002).\nAn influential typology concerning the parenting context has been developed by Maccoby and Martin (1983). Elaborating on the work of Baumrind (1971), Maccoby and Martin proposed a typology, defining parenting styles according to a two-dimensional framework which consists of: (1) support, such as warmth, acceptance, affection, and responsiveness; and (2) control, which refers to punishment, restrictiveness, supervision, inductive parenting, and conformity demands. They identified four parenting styles: authoritarian (low support, high control), authoritative (high support and control), permissive (high support and low control), and neglecting (low support and control). Parenting styles are configurations of attitudes and behaviors of parents towards their child and create a context or a climate for the parent\u2019s behavior. A parenting style is not considered domain specific; that is, it is displayed across many different situations (Darling and Steinberg 1993). This multidimensional approach may consequently more fully cover the facets of child-rearing and may provide a more comprehensive understanding of the influence of patterns of parenting characteristics on the development of delinquency than single parenting characteristics commonly used as risk factors in predicting delinquency.\nPrior Research and Current Focus\nFindings from empirical research analyzing whether different delinquency trajectories are associated with different family factors are mixed. Some studies found at least partially different familial etiologies for different trajectories (Chung et al. 2002a, b; Fergusson et al. 2000; McDermott and Nagin 2001; Wiesner and Silbereisen 2003; Wiesner and Windle 2004), whereas other studies found no or very few differences (Nagin et al. 1995; White et al. 2001; Wiesner and Capaldi 2003). Although many theories attribute an important role to childhood parenting in the etiology of delinquency, most studies focused on family risk factors other than parenting, such as parental criminality, parental stress and family structure (Fergusson and Horwood 2002; Fergusson et al. 2000; McDermott and Nagin 2001) or examined only one or two single parenting dimensions in relation to delinquency trajectories (Nagin et al. 1995; White et al. 2001; Wiesner and Silbereisen 2003). To our knowledge, one study analyzed the link between family functioning patterns and offending trajectories. Gorman-Smith et al. (2000) found that struggling families (low in discipline, monitoring, structure, cohesion and beliefs) which may be comparable to the neglectful style, were found to be at increased risk for each type of offending, whereas exceptionally functioning families (high levels of positive parenting, adequate discipline, structure, and cohesion) were less likely to be involved in each of the offending patterns. Task-oriented families (high levels of structure, but low levels of warmth and beliefs about the family), which may be relatively similar to the authoritarian parenting style, appeared more likely to be involved in the serious chronic pattern of offending. Thus, although this study covered a relatively limited period of 4\u00a0years in middle adolescence, a concurrent link between patterns of family functioning and offending behavior was identified.\nThe present study builds on research on offending trajectories by analyzing the existence of distinct delinquency trajectories in a longitudinal sample of males who participated in the Pittsburgh Youth Study (PYS; Loeber et al. 1998). It adds to previous studies in at least four ways. First, the PYS covers a period of 14\u00a0years with 18 waves. We use data measured at ages 7 up to 19. Many previous studies applying trajectory analysis on self-report data had smaller numbers of assessments covering shorter periods (e.g., Chung et al. 2002a, b; Wiesner and Windle 2004). Second, whereas many previous studies conducted concurrent analyses measuring both risk factors and delinquency during adolescence (e.g., Gorman-Smith et al. 2000), in the present study risk factors were measured in childhood, thus before delinquency trajectory data was collected. Moreover, although most theories explaining delinquent behavior by family characteristics state that parenting and family influences are strongest during childhood (Gottfredson and Hirschi 1990; Moffitt and Caspi 2001), most previous studies concentrated on family factors measured during adolescence. Third, we further extend previous research by focusing on parenting styles instead of family factors in order to gain more insight in the influence of multidimensional styles of how parents interact with their children and whether these are linked to distinctive delinquency patterns across adolescence. Unlike previous studies our focus is on a broad range of parenting characteristics, including supportive and disciplining parenting behaviors and the quality of the relationship between parent and child. In addition, we control for risk factors known to be important, such as socioeconomic status and prior delinquent behavior (e.g., Farrington 2002). Fourth, parenting and all other risk factors were measured across six waves covering middle childhood rather than a snapshot of one point in time.\nIn sum, general theories of delinquency have argued that family risk factors discriminate between delinquents and non-delinquents. Indeed, there is extensive empirical evidence for family risk factors to explain level differences in delinquency. However, whether distinct delinquency trajectories are linked to different parenting styles is still ambiguous. Therefore, this paper addresses the following research questions: (1) which distinctive delinquency trajectories are empirically identifiable using self-reported and official delinquency from late childhood through late adolescence? (2) What are the delinquency characteristics of the trajectory groups? (3) Do parenting styles, which we consider to be composites of behaviors or relationships in which the parent and child are directly involved, differentiate between the offending trajectory groups, above and beyond prior delinquent behavior and demographic variables?\nMaterials and Methods\nSample and Procedure\nThe Pittsburgh Youth Study is a panel study that began in 1987 following boys from public schools in the inner city of Pittsburgh. The total sample consisted of three cohorts (grades 1, 4 and 7). Based on a screening of antisocial behavior during the first assessment, a risk score for antisocial or problem behavior was created with the most antisocial third of the sample (about 30%) considering the high risk group and the remaining two-thirds of the sample constituting the low risk group. About 500 boys per cohort, 250 from each risk group, were then randomly selected for further follow-up (for detailed information, see Loeber et al. 1998).\nFor this study we used data from the youngest cohort. The number of participants at the screening was 849 and at the first follow-up assessment 503 (256 high risk and 247 low risk). The average age was 6.5 at the screening and 6.9 at the first follow-up. The screening sample consisted of 56.4% African Americans and 57.3% of the first follow-up sample was African American. Many boys were living with their natural mother (94%) during the first follow-up but only a small percentage of them lived together with their natural father (38.5%). Demographic differences of the follow-up sample compared to the screening sample (normal population) are not large (see Loeber et al. 1998, p. 33, 36).\nAfter the screening, data have been collected by interviewing several informants: the youth, a parent (primary caretaker) and teacher. Also, official data, such as juvenile court records have been collected. Until 1990, follow-ups took place biannually with subsequent measurements conducted yearly until 2000. The youngest cohort has been followed up a total of 18 times until age 20. Attrition in the Pittsburgh Youth Study was quite low with 82% of the youngest cohort participating at the 18th assessment (Loeber et al. 2003).\nMeasures\nParenting variables We used the parenting data measured in childhood up to age 9.5 to identify parenting styles. Data on the relationship with primary caretaker, supervision, physical punishment, the quality of the caretaker\u2013child communication, and positive parenting strategies were used. Relationship with primary caretaker measured the parents\u2019 emotional closeness to the child and the ability to accurately read and understand the child\u2019s feelings and\/or needs (13 items reported by the boy; 16 items reported by the parent). Supervision measured to what extent the parent has knowledge about the adolescent\u2019s whereabouts and friends (four items). Physical punishment assessed to what degree the parent physically punished the child (one item). Communication measured the degree to which the caretaker and the boy communicate about emotions, disagreements, and problems (eight items). Reinforcement assessed the degree to which the parent expresses positive behaviors towards the boy (seven items reported by the boy; nine items reported by the parent). These measures have been described more extensively in Stouthamer-Loeber and Stallings (2007). Internal consistencies of the parenting scales were satisfactory with alphas ranging from 0.66 for supervision to 0.83 for relationship with primary caretaker, with the mean alpha 0.72. For each variable the informants were the primary caretaker, usually the mother (91.1%) and the boy, except for communication which is only reported by the primary caretaker.2 Mean scores of the caretaker and boy were used for analyses.\nDemographic variables Variables indicating low economic status and ethnicity (African American) were used as control variables in the multivariate analyses. Low socioeconomic status (SES) was measured using the Hollingshead (1975) index of social status. The scores were computed by multiplying the scale value for occupational prestige by a weight of five and the scale value for educational level by a weight of three. If a family had two parents the highest score was selected.\nDelinquency For the self-reported measurement of delinquency over the previous year, the Self-Reported Delinquency Scale (SRD) from Elliott et al. (1985) was used together with the Youth Self Report (Achenbach and Edelbrock 1987) items on stealing and fire setting. The informant of delinquent behaviors was the adolescent. The SRD questionnaire covered 22 delinquent acts ranging from petty theft to serious assault. The self-reported data was collected from age 7 up to age 19. In addition to the self-reported data, data were collected on officially registered convictions (45 different offences). Both self-reported data and official data are known to show biases, but in different ways. While serious offending is often underreported in self-report studies, minor offences are usually underreported in official data (Babinski et al. 2001; Maxfield et al. 2000). We therefore combined self-reported delinquency with official data on delinquency for the ages 10\u201319. This is especially important since earlier studies have pointed to the influence of the source of delinquency data on the parenting-delinquency link (e.g., Loeber and Stouthamer-Loeber 1986). The General Delinquency Seriousness Classification (Loeber et al. 1998) was used to classify self-reported and official delinquent behaviors (ages 10\u201319) as follows: No delinquency (level 0); Minor delinquency at home, including minor vandalism and stealing at home (level 1); Minor delinquency outside home, such as minor vandalism and fire setting with insignificant damage, shoplifting, and avoid paying (level 2); Moderately serious delinquency including pickpocketing, stealing from car, using illegal checks, and carrying weapons (level 3); Serious delinquency including murder, rape, robbery, and selling hard drugs (level 4), and Two or more serious level 4-offences (level 5). This classification places a boy in the category of the most serious delinquent act committed in the last year. The delinquency seriousness classification was based on work of Wolfgang et al. (1985)3Prior delinquency consisted of a summary measure of delinquency seriousness (self-reported) that took place before age 10 and was used as a control variable in the multivariate analyses. For descriptive purposes we also used data on types of delinquency, that is, the proportion of theft, violence, vandalism, fraud4, and other delinquent behaviors based on self-reported and official data between ages 10 and 19, total delinquency frequency, that is, the number of delinquent behaviors in the period of ages 10 up to 19, and total number of convictions between ages 10\u201319.The variables that were measured during middle childhood (parenting variables, demographic characteristic, and prior delinquency) had ordinal scales and were measured at six waves (ages 7\u20139.5). These variables were blocked by averaging the scores for each risk factor in order to improve the reliability of the data. For adding them as risk factors in the analyses the blocked constructs for low socioeconomic status and prior delinquency were dichotomized in the values 0 for the neutral part and 1 to indicate a risk effect. Percentiles at approximately 25 and 75% were used for cut-off scores. This cut-off point was based on previous analyses of the PYS data that resulted in a prevalence of about 25% serious delinquents in the risk population. All measures mentioned above have been described extensively in earlier publications (e.g., Loeber et al. 1998; Loeber et al. 2007).\nAnalytic Strategy\nWe checked for outliers within a parenting variable on the basis of standardized z-values larger than 3.29 or smaller than \u22123.29 (Tabachnick and Fidell 1989). Outliers were corrected to values that corresponded with 3.29 standard deviations below or above the mean. The self-reported frequency of delinquency which was used for descriptive purposes was corrected for outliers by transforming yearly counts above 365 into 365.\nTo identify parenting styles in the PYS data, we applied cluster analyses on the ordinal parenting variables measured up to age 9.5. To derive parenting styles multivariate methods are favored over bivariate approaches (Mandara 2003). Instead of defining parenting styles a priori based on subjective cut-off scores (e.g., Bronte Tinkew et al. 2006), in cluster analysis families are grouped according to their scores on a range of parenting characteristics (Henry et al. 2005). Cluster analysis also allows there to be more important dimensions than the two dimensions (i.e., support and control) on which most bivariate approach studies are based. Following the analytic strategy described in detail in Appendix\u00a0A we identified a three-cluster solution as the optimal solution.\nTo identify types of delinquency trajectories in the PYS data we used a semi-parametric model especially developed to study group based-developmental trajectories (Nagin 2005) using a SAS-based procedure (Jones et al. 2001). Conventional growth-curve models produce a mean estimated growth curve for the population and make the assumption that variation in the growth-curve parameters (level, shape) is normally distributed across individuals. Group-based models circumvent this normality assumption and approximate individual variation in trajectories by a number of discreet groups. The model used here links delinquency seriousness and age by a cubic function permitting parameters to vary freely across a finite number of groups. Consequently, the delinquency trajectory of each group may differ both in level and shape. We estimated models from one to eight groups.5 Based on the Bayesian Information Criterion (BIC) a five-group model proved to be the optimal solution for our data. The five-group model also performed well on the additional criteria to assess model fit reported by Nagin (2005). In addition to the trajectories themselves, the group-based model produces a probability of membership for each of the distinguished delinquency trajectory groups for each individual in the sample. In describing the different trajectories each boy was assigned to the trajectory group for which his posterior probability of group membership was highest. Indicators of model fit, parameter estimates and mean posterior probabilities are presented in Appendix\u00a0B.\nSubsequently, a multinomial logit model was used to sort out the redundancy among parenting styles and to control for risk factors, such as demographics, and prior delinquency. This model tested whether a parenting style affects the probability of group membership in each of the delinquency trajectory groups relative to the nondelinquency group. In addition, we conducted Wald tests to examine potential differences in risk factors between delinquency trajectories, that is, the trajectories excluding the nondelinquency trajectory.\nResults\nDescription of the Trajectories\nFive distinct delinquency trajectories were identified (Fig.\u00a01). Based on the overall level of the Delinquency Seriousness Classification (DSC) and the slope of development with age the five trajectories were labeled: (1) nondelinquent trajectory, which constituted 27.2% of the sample and consisted of boys reporting hardly any delinquent acts, (2) minor persisting trajectory (27.6%), comprised of boys more steadily reporting non-serious delinquency, (3) moderate desisting trajectory (6.8%) with boys showing more serious delinquency in their early teens, followed by a steep decline, (4) serious persisting trajectory (24.2%) with boys continually reporting serious delinquency throughout the follow-up period, and (5) serious desisting trajectory (14.3%) made up of boys showing high levels of serious delinquency, peaking in their mid-teens, but showing marked desistance from delinquency from ages 14 to 19.6Fig.\u00a01Predicted (pred) and observed (obs) mean delinquency seriousness for all trajectories\nAdditional Wald tests showed the serious persisting trajectory to be different both in level and shape from all other trajectories. Wald tests for the moderate and serious desister trajectories did not reject the hypothesis that these trajectories were similar, as did tests for the non- and minor delinquent trajectories. These groups did however show substantive differences on other offending characteristics reported below, which suggest that these trajectories should not be combined.7\nThe nondelinquent and minor persisting group differed most notably from the other trajectories in that their peak levels of delinquency seriousness never exceeded the DSC-level 1 (Table\u00a01). Estimated seriousness for the moderate desisting trajectory peaked at 1.7 and unlike the minor persisters, their trajectory showed a rapid decline with age. The serious persisting and serious desisting showed the highest levels of delinquency seriousness, with peak-level estimates of 2.4 and 3.0, respectively.\nTable\u00a01Delinquency characteristics of the offending trajectories\u00a0Nondelinquents (n\u2009=\u2009155)Minor persisting (n\u2009=\u2009124)Moderate desisting (n\u2009=\u200932)Serious persisting (n\u2009=\u2009111)Serious desisting (n\u2009=\u200981)General delinquency\u00a0Delinquency seriousness0.10.70.82.11.6\u00a0Peak level0.10.81.72.43.0\u00a0Peak age1919121514\u00a0Self-reported delinquency1.015.18.9109.266.6\u00a0Number of convictions0.11.40.15.54.0Crime mix (percentages)\u00a0Nondelinquent59.40.00.00.00.0\u00a0Theft11.346.137.448.854.0\u00a0Violence4.216.02.420.616.1\u00a0Vandalism3.213.113.315.513.1\u00a0Fraud3.915.128.214.111.7\u00a0Other18.19.718.80.95.0\u00a0Total100.0100.0100.0100.0100.0Figures are means per trajectory group, based on delinquency data measured from ages 10 up to 19. Self-reported delinquency and number of convictions are counts. Delinquency seriousness and crime mix percentages are based on self-reported and official data. The overall association between the seriousness and frequency of delinquency is significant (r\u2009=\u20090.53, p\u2009<\u20090.001, for self-reported and r\u2009=\u20090.59, p\u2009<\u20090.001, for official delinquency frequency).\nFrequency of delinquency based on self-reported or official data largely corresponded with the overall level of delinquency seriousness: the higher the overall level of delinquency seriousness the higher the average number of delinquent acts committed by boys on each of the five trajectories (Table\u00a01). A noteworthy exception is that both the number of self-reported and officially recorded delinquent acts was much higher in the minor persisting than in the moderate desisting trajectory, whereas their overall delinquency seriousness was relatively similar. Boys on the minor persisting trajectory thus engaged in minor acts of delinquency relatively often, while boys in the moderate desisting trajectory committed much less though more serious delinquency.\nAcross all groups the majority of the self-reported delinquent acts involved theft (Table\u00a01). The proportion of vandalism was relatively similar as well. Boys on the moderate desisting trajectory however diverged from boys on the other delinquent trajectories by showing markedly less violence (2.4%), and engaging most often in fraud (28.2%) such as using checks illegally and selling worthless goods, or other delinquency (18.8%). Furthermore, serious persisters were characterized by their high involvement in violent offences (20.6%), while the serious desisting trajectory showed relatively the most theft (54.0%) and the least fraud (11.7%), compared to the remaining delinquent trajectories.\nParenting Styles as Predictors\nThe cluster analysis identified three different parenting styles (see Appendix\u00a0A for details). Authoritative caretakers were characterized as highly supportive reflected in a relatively good relationship with their child, a high score on positive parenting (i.e., rewarding the child when he has done something good), and high communicative skills. They were also skilled in disciplining their child: they adequately supervised the child, while not using physical punishment as a discipline technique. Authoritarian caretakers (moderately supportive) scored relatively high on discipline characteristics, showing high levels of supervision and physically punishing their youngsters. Moreover, these parents had moderate scores on supportive variables such as relationship, communication, and positive parenting. Neglectful parents had the worst relationship with their children and their discipline techniques were also inadequate. They poorly supervised and physically punished their boys.\nIn order to assess whether parenting styles were linked to trajectory group membership, dummy indicators of parenting styles were added as risk factors to the trajectory model.8 The authoritative category was chosen as a control group since prior research clearly indicated that this style is related to positive child outcomes (e.g., Darling and Steinberg 1993; Maccoby and Martin 1983). Demographic variables (ethnicity and socioeconomic status) and an indicator of prior delinquency seriousness (before age 10) were also included as control variables. In all analyses the nondelinquent trajectory was used as a reference group, that is, the risk factor means for all the other trajectories were tested against the nondelinquent trajectory. We tested three multivariate models: (I) a model with only demographic variables, (II) a model with demographic variables and prior delinquency seriousness, and (IIII) the full model including demographic variables, prior delinquency seriousness, and parenting styles (authoritarian and neglectful styles). Results for Model I (Table\u00a02) showed a significantly larger proportion of African Americans on both serious delinquency trajectories (serious persisting and serious desisting). This difference disappeared when parenting styles were added to the model indicating the effects of ethnicity to largely result from differences in parenting style. Somewhat surprisingly, although nondelinquents have the lowest proportion of families with a low socioeconomic status (Table\u00a03), socioeconomic status does not differentiate between trajectories (Table\u00a02).\nTable 2Multivariate tests of differences in background, prior delinquency, and parenting style between offending trajectories\u00a0Nondelinquents (27.2%)Minor persisting (27.6%)Moderate desisting (6.8%)Serious persisting (24.2%)Serious desisting (14.3%)Model I\u00a0Demographics\u00a0\u00a0African Americans\u20130.60 (0.39)\u22120.04 (0.50)0.86 (0.30)**1.04 (0.41)*\u00a0\u00a0Low SES\u20130.50 (0.47)0.19 (0.69)0.50 (0.38)0.80 (0.45)****Model II\u00a0Demographics\u00a0\u00a0African Americans\u20130.66 (0.39)****0.10 (0.55)0.76 (0.38)*1.05 (0.44)*\u00a0\u00a0Low SES\u20130.96 (0.49)*0.38 (0.86)0.89 (0.49)****1.07 (1.52)*\u00a0Delinquency\u00a0Prior delinquency\u20130.98 (0.20)***1.25 (0.30)***1.58 (0.22)***1.21 (0.23)***Model III\u00a0Demographics\u00a0\u00a0African Americans\u20130.68 (0.37)****\u22120.26 (0.60)0.44 (0.43)0.98 (0.52)****\u00a0\u00a0Low SES\u20130.88 (0.47)****0.33 (0.81)0.50 (0.50)0.58 (0.59)\u00a0Delinquency\u00a0\u00a0Prior delinquency\u20130.87 (0.20)***1.20 (0.30)***0.25 (0.25)***1.03 (0.25)***\u00a0Parenting Style\u00a0\u00a0Authoritarian style\u20130.59 (0.40)1.70 (1.02)****0.48 (0.48)*0.28 (0.59)\u00a0\u00a0Neglectful Style\u20130.55 (0.72)2.44 (1.20)*0.71 (0.71)*1.96 (0.69)**Numbers are multinomial logit coefficients with standard errors given in parentheses. Trajectory 1 is used as a reference group. The BIC is \u22125,598.3 for model I, \u22125,447.9 for model II, and \u22125,459.9 for model III.*p\u2009<\u20090.05**p\u2009<\u20090.01***p\u2009<\u20090.001****p\u2009<\u20090.10Table\u00a03Means of background, prior delinquency, and parenting style for each offending trajectory based on the multinomal logit model (model III)\u00a0Nondelinquents (n\u2009=\u2009129)Minor persisting (n\u2009=\u2009161)Moderate desisting (n\u2009=\u200933)Serious persisting (n\u2009=\u2009101)Serious desisting (n\u2009=\u200959)Demographic variables\u00a0African Americans0.390.660.390.630.73\u00a0Low SES0.120.290.150.230.24Delinquency\u00a0Prior delinquency0.601.562.062.311.90Parenting styles\u00a0Authoritarian style0.320.500.580.510.25\u00a0Neglectful style0.050.140.330.310.47Prior delinquency is the delinquency seriousness classification during childhood (ages 7.5 up to 10) based on self-reports. The remaining means are proportions as the variables are dichotomous.\nFrom models II and III it became clear that prior delinquency seriousness was an important risk factor for later delinquency. However, although prior delinquency differentiated nondelinquents from delinquents, Wald tests showed that this risk factor did not differentiate the remaining four delinquent trajectories very well. Prior delinquency was only significantly different between the serious and minor persisting delinquency trajectories (\u03c72(1)\u2009=\u20099.09, p\u2009<\u20090.01) in model II. In the full model (model III), no significant differences in prior delinquency were found between the delinquent trajectories (i.e., all trajectories except nondelinquency).\nA neglectful parenting style was significantly linked to membership in the moderate desisting trajectory and the serious persisting and desisting trajectories compared to the nondelinquent trajectory. Furthermore, Wald tests revealed significant differences between serious persisting and serious desisting delinquents and minor persisters (\u03c72(1)\u2009=\u20093.89, p\u2009<\u20090.05 for serious persisting versus minor persisting; \u03c72(1)\u2009=\u20095.54, p\u2009<\u20090.05 for serious desisting versus minor persisting), suggesting that a neglectful parenting style primarily distinguished between moderate to serious trajectories and non- to minor trajectories (i.e., moderate desisting, serious persisting, and serious desisting versus nondelinquent and minor persisting). In addition to neglectful parenting being abundant, boys on the serious persisting trajectory also originated significantly more often from authoritarian families compared to the nondelinquents (see Table\u00a02, model III).\nDiscussion\nPrompted by recent theories distinguishing different developmental pathways and the role attributed by these theories to parenting in the etiology of these pathways, we set out to test whether there was evidence for adolescent boys to follow different trajectories of delinquency seriousness and whether these trajectories were linked to caretakers\u2019 parenting styles during childhood. Using person-centered methods and adopting a multidimensional perspective on parenting we identified five delinquency trajectories and three parenting styles. Parenting styles were differentially linked to delinquency with neglectful parenting linked to moderate desisting and serious persisting and desisting trajectories and authoritarian parenting linked to serious persistent delinquency.\nWhile the trajectories identified in this study resemble those postulated by developmental theories to some extent, there are also marked differences. Within the limits of our data range we identified a minor and serious \u2018persistent\u2019 group of boys continuously showing delinquent behavior resembling Moffitt\u2019s (2006) low chronic and life-course persistent trajectories both in level and age-pattern. Our serious desisting trajectory most closely resembled the adolescent-limited pathway: starting with minor delinquency in late childhood and rapidly escalating to relatively serious delinquency in early adolescence. This escalation process of delinquency seriousness has been described in the literature (e.g., Farrington 1997; Hawkins et al. 1998; Lipsey and Derzon 1998; Loeber et al. 2007). Yet, these boys constituted only 14% of the entire sample, while adolescence-limited delinquents were predicted to be common by the typology. Furthermore, over one in four of the boys reported no or hardly any delinquency during the 10\u00a0year-follow-up. Given the high risk nature of the Pittsburgh sample, this is at odds with the Moffitt typology which predicted abstainers to be a very rare phenomenon. Finally, we identified a moderate desisting trajectory with boys showing a marked decrease in delinquency seriousness from a peak at age 12 to virtually zero at age 19.\nGiven that most studies focus on delinquency frequency data (e.g., McDermott and Nagin 2001; Nagin et al. 1995; White et al. 2001; Wiesner and Silbereisen 2003), discrepancies between our and prior findings and theoretical predictions underscore the importance of considering other indicators of delinquency such as seriousness and offence type. To our knowledge, a group starting young with relatively serious delinquency levels but desisting early has not been identified previously. Notably, despite the fact that delinquency in these boys was relatively serious at a young age, the frequency of delinquency was rather low. Moreover, this group of boys reported very low levels of aggressive acts. Clearly, levels of delinquency seriousness are not necessarily related to the level of delinquency frequency.\nThis study aimed to analyze whether parenting styles were differentially associated with delinquency trajectories. A neglectful punishing style distinguished the moderate to serious trajectories from the nondelinquent and minor trajectories above and beyond childhood delinquency and demographic characteristics such as socioeconomic status and ethnicity. Even moderate desisters originated more often from neglectful families than nondelinquents. Notably, these youngsters had conviction rates that are comparable to nondelinquents, yet they committed relatively serious offences at younger ages. Our findings are in accordance with results from previous studies resulting in a link between a neglectful style and delinquency (Maccoby and Martin 1983; Steinberg et al. 1994). Furthermore, the neglectful style in our sample is similar to the concept of poor parenting described by Moffitt and Caspi (2001).\nGeneral theories state that high levels of poor parenting lead to high levels of delinquency, while typological theories argue that different types of delinquent trajectories stem from different etiological backgrounds. In the present study we found significant differences in parenting styles between delinquency trajectories, that is, differences between more serious and minor delinquency trajectories, yet parenting styles did not distinguish among the more serious trajectories. This finding is similar to that of White et al. (2001) who found the same family risk factors for adolescence-limited and life-course persistent delinquents. An explanation could be that parenting styles may not predict delinquency patterns later than early adolescence. While the boys following a moderate desisting and serious persisting pathways are similar during late childhood (i.e., at ages 10\u201311), with both displaying relatively high levels of delinquency seriousness at young age, the former experienced marked desistance as of age 11, while the latter did not. However, neither parenting styles nor prior delinquency (in middle childhood) could differentially predict membership for these groups. This suggests that neglectful parenting and delinquency during childhood can only partially predict development in delinquency over longer periods of time (i.e., after age 13). This is in accordance with previous research focusing on long-term association between parenting and delinquency (e.g., Farrington and Hawkins 1991; Hoeve et al. 2007; Sampson and Laub 1993).\nInterestingly, many of the theories that attribute an important role to childhood parenting in the etiology of delinquency conceive this role primarily as facilitating the development of some stable tendency towards delinquency (e.g., Gottfredson and Hirschi 1990; Moffitt 1993). Behavioral patterns originating in the family quickly become ingrained and increasingly hard to alter as children age. However, according to Sampson and Laub (2005) and others, changes in life circumstances are able to effectuate change in an individual\u2019s delinquent trajectory, notwithstanding the individual\u2019s rearing environment. Moreover, delinquency during adolescence is governed not only by bonds to the family, but also by bonds to peers, school and later work and romantic partners. Changes in any of these bonds continue to affect delinquent development. Since parenting styles and parenting dimensions have been found to be relatively stable over time (e.g., Holden and Miller 1999; Loeber et al. 2000; Steinberg et al. 1994), for the desisting groups bonds to school and peers may have become more salient in explaining delinquency during mid- and late adolescence than bonds to parents (Sampson and Laub 2005).9 The transition from primary to middle school and its changing opportunities for new friends and extracurricular activities may have set a group of boys off on a desisting pathway. Wiesner and Capaldi (2003) found that among adolescents with similar parenting experiences during childhood, low level offenders during adolescence were less involved with deviant peers than those youth who became high level offenders. Other life circumstances could have changed the delinquent pathway of some boys as well. For example, desisting youngsters may have participated in intervention programs.\nAmong the strengths of this study are the use of multiple informants, analyzing prospective relations between parenting and delinquency trajectories, examining a varied set of parenting characteristics to identify parenting styles, and besides assessing a broad range of delinquent acts combining self-reported and official delinquency data. However, several limitations should also be noted. First, a convicted boy may receive a custodial sentence which may influence the total time that a boy is actually at risk for committing delinquent acts. Prior research has shown that not controlling for exposure time may yield suppressed estimates of actual delinquency frequency (McCaffrey et al. 2007) and may account for much of the decline in delinquent trajectories past peak age (Piquero et al. 2001). Mathematical solutions offered to deal with this \u2018false\u2019 desistance, typically require data on incarceration. Unfortunately, official data on custodial sentences were not available for the PYS-sample. To gain some insight in the sensitivity of our findings to the possible bias of false desistance, we tested the five-group model on the subset of boys who were never convicted, and thus did not experience reduced risk of offending due to incarceration between ages 10 and 19. This resulted in trajectories with similar shapes and a classification of boys into similar groups (\u03ba\u2009=\u20090.93; N\u2009=\u2009324).10 A second limitation is the fact that the youngest sample has been followed up only until late adolescence and as a consequence we were not able to distinguish true life-course persistent offenders into adulthood. However, the PYS is an ongoing study and a follow-up is currently underway. A third limitation is that the sample consisted only of boys. Both delinquency trajectories as well as their associations to parenting styles could be different for girls. Future studies should focus on girls\u2019 delinquency trajectories and whether these relate to parenting.\nGiven that we found strong links between parenting styles and delinquency trajectories, we recommend that researchers include parenting styles or at least both elements of support and control measurements in their investigation. The typological approach offers an analytic strategy in which combinations of these parenting dimensions may more closely reflect the interactional nature of parenting dynamics and may have higher predictive value (Mandara 2003). Whereas many theories consider a particular parenting characteristic to be responsible for delinquent development (e.g., Hirschi 1969; Patterson 1982; Sampson and Laub 1993), our study shows that a neglectful parenting pattern consisting of a combination of low levels of warmth and support, inadequate discipline techniques, and harsh punishment predicts several serious delinquency trajectories.\nThese results have implications for family-oriented prevention strategies in that they provide information on which combinations of parenting dimensions are particularly relevant. Preventive actions should focus on neglectful families characterized by harsh punishment, inadequate discipline, and low levels of supportive parenting in their effort to reduce the risk of youngsters from these families setting off to a future of serious delinquency.","keyphrases":["parenting styles","development","delinquency trajectories","longitudinal"],"prmu":["P","P","P","P"]}
{"id":"J_Urban_Health-2-2-1705484","title":"Respondent-Driven Sampling in Participatory Research Contexts: Participant-Driven Recruitment\n","text":"This article reports on the use of respondent-driven sampling (RDS) in participatory and community-based research. Participant-driven recruitment (PDR) retains all of the analytic capabilities of RDS while enhancing the role of respondents in framing research questions, instrument development, data interpretation, and other aspects of the research process. Merging the capabilities of RDS with participatory research methods, PDR creates new opportunities for engaging community members in research addressing social issues and in utilizing research findings within community contexts. This article outlines PDR\u2019s synthesis of RDS and participatory research approaches, describes how PDR is implemented in community contexts, and provides two examples of the use of PDR, illustrating its process, potentials, and challenges.\nIntroduction\nThis article reports on the use of respondent-driven sampling (RDS) in participatory and community-based research. Participant-driven recruitment (PDR)1\u20133 retains the analytic capabilities of RDS (e.g., use of data on personal network sizes and recruiting patterns to control for selection bias and generate population estimates, sampling weights, and standard errors estimates) while enhancing the role of respondents in framing research questions, instrument development, data interpretation, and other aspects of the research process. PDR integrates two distinct research approaches\u2014the practices, analytic methods, and capacity to generate a statistically valid sample associated with RDS and the disciplines and techniques of participatory research including community-based participatory research (CBPR) and participatory action research (PAR).\nBy merging RDS with participatory research methods, PDR fosters new opportunities for engaging community members in research addressing social issues and in utilizing research findings within community contexts. In particular, it presents opportunities to integrate rigorous participatory methods with rigorous quantitative data analysis.\nThis article outlines PDR\u2019s synthesis of RDS and participatory research approaches, describes how PDR is implemented in community contexts, and provides two examples of the use of PDR illustrating its process, potentials, and associated challenges. The emphasis of the article is on field methodology, rather than on RDS data analysis.\nFoundations of PDR\nPDR developed through the integration of RDS with participatory research techniques. Key elements of RDS that distinguish it from other forms of chain-referral sampling include direct contact between recruiter and recruit (in contrast to referrals mediated through the researcher), presence of a pre-existing reciprocal relationship between the recruiter and recruit, documentation of recruitment patterns, regulation of the number of recruits per recruiter, and collection of data regarding participants\u2019 personal network sizes.4 Early in the development of RDS, as a way to engage injection drug users and other hidden populations in HIV prevention studies, researchers noted that peer-to-peer recruiting often led to informal peer education, resulting in a mild intervention effect.5 This paved the way for the development of a number of peer-driven interventions6 and suggested that RDS held potential as a sampling method in participatory research on social issues.\nParticipatory research projects have investigated international development issues,7 organizational change,8,9 community development and advocacy needs,10 health care and community health promotion,5,11,12 environmental health and urban design, and social issues affecting adolescent health and development.13\u201317\nParticipatory research approaches developed, in part, as a response to conditions of exclusion, oppression, and marginalization18 and, in part, as a strategy for addressing and compensating for some limitations of traditional research methods, enhancing the validity and utilization of research findings. Participatory approaches such as action research and empowerment evaluation are now practiced and discussed theoretically across a wide range of disciplines, including public health.11,12,19 Participatory research often engages those who are the focus of policies and programs to study the issues and conditions that affect their lives. Participatory methods also are used to research conditions within organizations, identifying issues and facilitating the development of strategies for resolving them.8 These approaches seek to elicit diverse perspectives, often using multiple research methodologies, including methods that promote discussion, deliberation, the development of critical thinking, and the exploration of social circumstances related to research questions. Participatory research seeks to generate knowledge that can be used to prompt collective action and change; its premise is that research should be useful to communities, organizations, programs and participants at the same time as contributing to the academic and disciplinary literatures.9\nOften, participatory research approaches seek to involve marginalized people in determining research questions and methods and in interpreting and utilizing research findings. Participatory research draws on local knowledge and emphasizes the involvement of non-academics. In particular, it seeks to mobilize and build upon the expertise of those whose lives are directly affected by the research issue.10 Ideally, stakeholders in participatory research stand positioned to act upon the new knowledge they have collectively generated. Action may occur at individual, interpersonal, organizational, and policy levels.13 If participants and communities involved in the research have access to data and findings, this may enable more nuanced data interpretation as well as higher commitment to utilizing results and to exploring emergent research questions. Research efforts that are viewed as sensitive to community concerns, locally engaged and sustained rather than extractive, may be better received by community members and potential respondents.11,20 Participatory research efforts often unfold over a relatively long period, and community-based participatory researchers have articulated crucial elements and stages that characterize effective collaborations.11\nParticipatory research faces a number of specific challenges: Careful negotiation of power dynamics between academic researchers and community members, as well as among community stakeholders, is crucial. Communication among partners is key and can be problematic (e.g., academic researchers may be ill-equipped to readily understand community concerns and priorities; community members may not readily endorse academic concerns about statistically valid samples). The creation of a \u201ccommon language\u201d and common standards of practice, including standards for sampling and data integrity, by the research team is a long, iterative, demanding process.\nThe synthesis of RDS with participatory research approaches contributes to resolving some of these challenges, in particular by providing a rigorous sampling technique that can present participatory researchers with a means to develop statistically credible samples without diminishing the quality of community engagement. RDS produces more reliable data than convenience or snowball samples through the use of data on personal network sizes and recruiting patterns to control for bias. In some cases, it generates more comprehensive data than either venue or institutional samples.21 Because RDS utilizes social networks and gives participants a central role in recruiting, it is more consistent with the values of participatory research than random sampling. Further, the involvement of participants in the sampling process opens up opportunities for dialogue and education regarding issues like data integrity, informed consent, and the overall aims of the research.\nAt first conceived as a sampling methodology, RDS worked to engage disenfranchised and stigmatized people in HIV prevention studies, giving them a central role in recruiting and building upon their capabilities and strengths.6 Nonetheless, the initial studies using RDS were not explicitly framed as participatory research efforts. We propose PDR as a careful synthesis of the two approaches. PDR provides a context for RDS that is specifically adapted to the framework and demands of participatory research, while at the same time maintaining the core elements of RDS.\nDescription of PDR\nAs in other participatory research approaches, studies using PDR may be initiated by either researchers or community stakeholders or may develop out of an on-going collaboration. In any of these cases, PDR involves a number of stages, shown in Figure\u00a01, that situate the peer recruiting effort within a participatory research process that seeks to ensure both the relevance of the research aims to community stakeholders and the utilization of research findings.\nFigure\u00a01Stages of a participatory research process utilizing PDR\nCommunity Engagement, Referral of \u201cSeeds\u201d and Pilot Activities\nIn most participatory research projects, researchers collaborate with informal and formal stakeholder organizations to set and refine the research questions and aims. In PDR, these organizations also identify a limited number of potential study participants with diverse perspectives21 to pilot, review, and assist in the revision and adaptation of research materials and questionnaires and to serve as \u201cseeds\u201d (the foundation of the peer recruitment process). The seeds in PDR have a pivotal role in the research process, involving several responsibilities and opportunities for engagement in addition to their roles as respondents and peer recruiters in RDS. Seeds take part in pilot groups to discuss the aims of the study and to assess and fine-tune research instruments, in some cases substantially co-designing the instruments in close collaboration with academic researchers.\nThe involvement of community members in the design and development of research projects has multiple benefits and challenges that have been well articulated in the community-based participatory research literature.20 Engaging seeds in discussions regarding the design and implementation of the study enhances their familiarity with the research process and contributes to the incremental development of a \u201ccommon language\u201d by diverse members of the research team. Increasing the commitment of seeds to the overall research project assists in addressing challenges regarding recruiting rates and productivity of seeds encountered in some RDS studies:22 The engagement and sense of ownership of the research by the seeds appears to lead to higher commitment to and capacity for peer recruitment and aids in developing recruiting momentum. It also helps to address challenges faced by participatory researchers: Peer recruitment enhances the level of engagement of participants in the overall research process by creating new opportunities for formal and informal dialogue about the research issues.\nPeer Recruitment\nAfter the pilot process has been completed, seeds recruit the first wave of participants, and peer recruitment continues through at least the number of waves required to obtain equilibrium.23 The recruiting process is generally similar to that in most studies using RDS, with the following differences: In most RDS studies, peer recruiters receive coupons with serial numbers and contact information for the research project, which they distribute to their recruits. In PDR, peer recruiters also receive written explanations of the research project, informed consent materials, and, in some cases, survey instruments and self-addressed stamped envelopes to convey to their recruits. In PDR studies that include group discussion or educational sessions, participants may have opportunities to discuss and\/or role-play recruiting techniques.\nIncentives\nParticipants receive small incentives for taking part in the study (e.g., $15 for survey completion) and for recruiting their peers (e.g., $10 for successfully recruiting an eligible participant). In addition to these extrinsic incentives, engagement with the overall development of the research project and the interpretation and utilization of findings may serve as an additional incentive, fostering intrinsic interest and motivation among participants. Evaluations of PDR indicate that, while the availability of extrinsic incentives initially attracted participants, they stayed engaged based on intrinsic motivation, the quality of participation, opportunities to discuss important issues with peers, or interest in the questions addressed by the research. In the words of one adolescent participant:At the beginning, I wanted the money, but now it\u2019s turned into something totally different.... It surprises me\u2014so much has changed. I\u2019ve been able to meet people that I know I wouldn\u2019t have met [otherwise]. I\u2019ve been able to expand and learn, you know, get more involved in things that I like to do and hope to do in the future.2\nLocation\nMost RDS studies have a research office or storefront location where recruits meet individually with research staff to assess their eligibility, complete informed consent documents, be interviewed, and, in some cases, receive an educational session. In PDR, self-administered surveys and educational or discussion sessions may take place in a wide range of community settings identified as accessible during the pilot process. While this may reduce costs associated with space rental and help to reduce the \u201cgeographic filtering\u201d24 of RDS samples, it demands extra care in terms of participant confidentiality and data integrity (see below).\nData Interpretation and Utilization\nPDR participants are invited to attend periodic updates on the research and data interpretation meetings. As preliminary findings are developed, community stakeholders as well as participants take part in further data interpretation meetings, discussion regarding the study\u2019s results, and activities leading to the dissemination and utilization of findings.\nConfidentiality and Management of Data Regarding Recruiting Patterns\nBecause PDR research is relatively decentralized, special care is taken to explain informed consent, confidentiality, and other human subjects considerations to participants and to recruiters; participants and other community members who are highly engaged in the research complete the same human subjects tutorial required of academic researchers. Participant referrals provide a tangible link between participants and their recruiters; participants describe their relationship with the person who recruited them in order to ensure that it is reciprocal.21 Contact forms facilitate follow-up and payment of incentives. Personally identifying information is kept separately from research data sets, which include unique identifiers for recruiters and recruits; all data is stored in secure locations at the university.\nExamples\nTwo studies conducted by research teams including the author illustrate the process and challenges of implementing PDR. The first involved dislocated workers in a study of social protections and employment trajectories; the second involved adolescents in community-based HIV prevention research. Instruments and procedures for both studies were approved by the Cornell University Committee on Human Subjects. Findings from both studies have been reported elsewhere2,3,25\u201330; the emphasis here is on the field methodology. The aims of the studies and the rationale for utilizing PDR in each of them are briefly summarized below. A chart comparing the use of PDR in the two studies is presented in Table\u00a01.\nTable\u00a01Comparison of the use of PDR in two studies\u00a0Plant closure studyRural adolescent HIV prevention studyEngagementCommunity organizations involved in framing research and in recruiting seedsWorkforce Improvement Board, Workers Rights Council, YWCAYouth Bureau, Catholic Charities, Aid to Victims of Violence, Jacobus Center (reproductive health clinic), YWCANumber of seeds involved in pilot process79Peer recruitmentDurationSummer 2000\u2013spring 2001Winter 2001\u2013spring 2002Projected sample size50125Actual sample size72128Number of waves57Distributed informed consent materialsYes (informed consent only)Yes (parental consent as well as informed consent\/assent)IncentivesFor completing survey$15$15For recruiting new participant$10$10Steering incentives$20 (for recruiting dislocated workers who had held part-time or contingent positions; for recruiting dislocated workers who had moved out of state to seek jobs)NoneLocationsWorkforce Improvement Board, Workers Rights Council, restaurants, private residences; data interpretation sessions held in meeting rooms at Fire Hall and YWCAYouth Bureau, YWCA, Rural Services meeting room, classrooms in rural schoolLocation sensitivityNone (surveys could be self-administered and mailed in; 11% of participants were living out-of-state at the time of the survey)Significant (in rural areas close to survey sites, participant pool was saturated, while rural areas distant from survey sites were under-represented)Data interpretationFormal meetings with participants to review preliminary dataYesYesFormal meetings with stakeholder organizations to review findingsYes (separate meetings with each agency)Yes (group presentation and meetings on-site with some agencies)Written report to participantsYesNoLocal media coverageYes (researcher, agencies and participants)NoPresentations and publications outside the local communityYes (researcher only)Yes (co-authored with participants)Individual or organizational actions taken by participants attributed to involvement in researchYesYes\nDislocated Workers\nPDR was developed in response to challenges encountered during a plant closure study conducted during 2000\u20132001. A non-unionized manufacturing plant employing nearly 500 people was shut down in early 1998 as a consequence of corporate restructuring. The plant had been the beneficiary of a variety of local economic development assistance packages in a community that had experienced the closure and downsizing of many other manufacturing firms. The study aimed to engage approximately 50 former workers in an extensive survey about their post-closure employment trajectories in order to inform ongoing social protection, planning and economic development efforts and to serve as a corrective to studies of the local impacts of the plant closure that neglected to gather data regarding dislocated workers.31 The former employees were in effect a hidden population; no comprehensive list could be obtained to serve as a sampling frame although human resource records provided precise aggregate demographic information on the displaced workers. Further, a proportion of the workers had moved out of state after the closure, either relocating to other plants owned by the original firm or traveling in search of employment opportunities. Peer recruitment exceeded the original participation objective, engaging 72 dislocated workers in the study. PDR had the capacity to build upon the social connections among former workers to locate study respondents while simultaneously providing a context for the participants to explore, reflect upon, and deliberate about economic development strategies, employment trajectories, and measures of job quality. RDS analysis of the data aided in estimating post-closure out-migration rates. By combining RDS and participatory research methods, the study enabled an economically vulnerable population to contribute to the development of recommendations regarding community social protection and economic development programs.\nRural Adolescents\nThe second illustration of PDR is drawn from an HIV prevention study of rural adolescents. Peer recruitment slightly exceeded the original sampling objective (128 youth participated; the study enrollment goal was 125). Although many approaches may be taken to sampling adolescents, peer recruiting produces empirical data about social networks that cannot be gathered in institutional or venue samples21; our study engaged homeless and out-of-school youth as well as youth enrolled in all local schools. RDS enables study of adolescents\u2019 social networks\u2014in particular, the relationship of risk and protective characteristics and behaviors to social networks. With empirical evidence of recruiting patterns, both demographic and non-demographic characteristics can be mapped in relation to recruiting networks. This enabled us to explore the roles that risk and protective characteristics play in the social network structures of adolescents.32,33 Further, RDS analysis contributes to identifying sub-groupings of adolescents, work that is crucial to the development of effective tailored interventions.34 PDR enabled not only the gathering of data on recruiting patterns needed for RDS analysis but also engaged youth in a participatory learning process that was particularly appropriate to their developmental needs.2,30,33\nChallenges and Lessons Learned\nThere are a number of challenges involved in implementing PDR, these include fully utilizing RDS analysis during data interpretation sessions, addressing location sensitivity so that sampling is not distorted by lack of access to study sites, and preventing the emergence of \u201csuper recruiters\u201d without dampening participants\u2019 engagement in the overall study.\nRDS Analysis\nBoth of the examples reported drew on early versions of RDS analysis software. This aided in identifying and addressing specific issues (e.g., the relative disengagement of contingent workers from permanent employees\u2019 social networks, the strong tendency of rural youth to recruit other rural youth). RDS analysis requires significant data set preparation before data can be imported and used with the current specialized software package (Respondent Driven Sampling Analysis Tool (RDSAT) can be downloaded at no cost from http:\/\/www.respondentdrivensampling.org). In the two studies cited, detailed RDS analyses were completed after recruitment ended and after most community-based discussions of the research had taken place. Deliberate procedures for incorporating RDS analyses into early data interpretation efforts are needed in order for community-based partnerships to benefit fully from the use of this sampling method.35\nLocation Sensitivity\nThe participatory processes that are well integrated into PDR may assist in addressing the location sensitivity and \u201cgeographic filtering\u201d24 of RDS-based samples. Useful practices include soliciting guidance from study participants on accessible times, locations, and methods of survey administration. For example, surveys can be administered in community locations recommended by participants as accessible sites rather than at a centrally located research office or storefront. Pilot studies may suggest a protocol in which respondents are provided with self-administered questionnaires and informed consent materials to give or mail to other potential participants, facilitating engagement of participants who might otherwise be missed by the study (e.g., dislocated workers who moved out of state after a plant closure).\n\u201cSuper Recruiters\u201d\nRegulating the number of participants that can be recruited by any one peer is a core element of RDS, generally accomplished by setting recruiting quotas and limiting the amount of recruiting materials (e.g., coupons) offered to each participant. In many studies, highly committed participants emerge who are eager to promote the study and to recruit more new participants than their quota allows. In PDR, potential \u201csuper recruiters\u201d can be engaged in elements of the participatory study other than recruiting, such as organizing data interpretation sessions or disseminating research findings within the community.\nConclusions\nPDR relies on the careful implementation of the core elements of RDS. Additional defining elements of PDR include recruitment of diverse seeds through formal or informal stakeholder organizations, involvement of seeds in piloting and co-designing study instruments and procedures, human subjects education for community research partners, involvement of participants and stakeholders in data interpretation sessions, and dissemination of findings to inform local actions. Some of these elements are specific to PDR; some are more general attributes of participatory or empowerment approaches.36\nPDR seeks to enable eligible participants to make informed judgments about their own level of engagement in the study of potentially sensitive social issues. PDR creates formal and informal contexts for civic discussion that are accessible to research participants who might not attend community meetings or utilize other traditional means of voicing their experiences and views. It creates a socially embedded research process in which discussion of the research questions may take place when peers meet informally (e.g., while shopping) as well as during formal data interpretation sessions. Being recruited by peers to take part in a research project serves for some as a bridge to civic engagement and fosters intrinsic interest in the questions and issues explored by the study. Thus, this approach to recruiting members of hidden, vulnerable, and marginalized populations to participate in research projects can contribute to more inclusive community dialogue about social issues and strengthen community participation in research efforts.","keyphrases":["respondent-driven sampling","participatory research","participant-driven recruitment","community-based participatory research","action research"],"prmu":["P","P","P","P","P"]}
{"id":"Bioinformation-1-4-1891668","title":"A database of 389 medicinal plants for diabetes\n","text":"Medicinal plants used to treat hypoglycemic and hyperglycemic conditions are of considerable interest to ethno-botanical community as they are recognized to contain valuable medicinal properties in different parts of the plant. The active principles of many plant species with desired properties are isolated to cure ailments such as diabetes type-1 and type-2, respectively. Here, we describe DiaMedBase, a database containing information of medicinal plants for diabetes.\nBackground\nSince olden days, plants are used to treat many ailments. India has about 45,000 plant species and several thousands have been claimed to possess medicinal \nproperties. [1] Medicinal plants used to treat hypoglycemic or hyperglycemic conditions are of \nconsiderable interest for ethno-botanical community as they are recognized to contain valuable medicinal properties in different parts of the plant and a \nnumber of plants have shown varying degree of hypoglycemic and anti-hyperglycemic activity. [1] The \nactive principles of many plant species are isolated for direct use as drugs, lead compounds or pharmacological agents. [3] Several species of medicinal plants are used in the treatment of diabetes mellitus, a disease affecting large number of people \nworld-wide. Traditional plant medicines or herbal formulations might offer a natural key to unlock diabetic complications. [2]\nDiabetes mellitus is the major endocrine disorder [4] responsible for renal failure, blindness or \ndiabetic cataract [5], poor metabolic control [6], \nincreased risk of cardiovascular disease including atherosclerosis and AGE (advanced glycation end) products. [7\n] Antioxidants play an important role to protect against damage by reactive oxygen species and their role in diabetes has been evaluated. Many plant \nextracts and products were shown to possess significant antioxidant activity. [8] Hence, we created \nDiaMedBase, a diabetes literature database of medicinal plants with abstract, plant parts, objective and a \u2018disease link\u2019 to diseases other \nthan diabetes for each medicinal plant.\nMethodology\nConstruction of DiaMedBase\nDiaMedBase is constructed using html and can be accessed at \nhttp:\/\/www.progenebio.in\/DMP\/DMP.htm. Data were collected from various literature sources such \nas, PubMed [9], ScienceDirect [10], Mary Ann \nLiebert [11], BlackWell Synergy [12], \nIngentaConnect [13], Scirus [14], Bentham \nPublishers [15], Wiley journals [16] and others. \nDiaMedBase includes 742 records, containing about 309 genus and 389 species of plants described to possess medicinal properties against diabetes. The complete \nlist can be found at \nhttp:\/\/www.progenebio.in\/DMP\/listz.htm. However, the list is not complete. They are provided alphabetically and the records are organized \nto simplify the task of finding relevant data for any plant. The database can be accessed alphabetically using genus name for information on specific plants.\nFeatures of DiaMedBase\nEach entry in DiaMedBase is provided with a unique accession number DMPXX001, where, XX represents the first letter of genus and species, respectively. \nDigits refer to the record number in the database. For example, the accession number of Aloe vera is DMPAv042, where, 042 represent record entry in the \nDiaMedBase. The Medicinal Plant row in some records display a list of medicinal plants and the representative plants are highlighted. DiaMedBase contains \n30 entries for genus Trigonella, 22 entries for Momordica, 19 for Gymnema, 13 entries for Opuntia and Panax, 11 for Allium species and 10 each for Aloe, \nTinospora, respectively. A screen-shot of the DiaMedBase is given in Figure 1. Of the collected data, 36% whole plant, \n26% leaves, 12% seeds, 10% roots, and 4% fruits. The characteristic feature of DiaMedBase is \u2018Disease Link\u2019, provided in each record displays \nthe list of diseases other than diabetes as a pop-up window indicating the importance of medicinal values of plants.\nUtility\nDiaMedBase emphasizes the importance of hypoglycemic and hyperglycemic properties possessed by medicinal plants. The database finds utility to the scientific \ncommunity for a quick review on the number of plants and plant parts for diabetes medicinal plant research.\nFuture development\nContinuous updates shall be released to include other plants of medicinal value periodically. The present access method shall be upgraded to browse records \non the studied plant parts. We plan to develop provisions to search the database to identify plants of interest using keywords.","keyphrases":["database","data","medicinal plants","diabetes","literature"],"prmu":["P","P","P","P","P"]}
{"id":"Exp_Brain_Res-3-1-2048826","title":"Depth cues, rather than perceived depth, govern vergence\n","text":"We studied the influence of perceived surface orientation on vergence accompanying a saccade while viewing an ambiguous stimulus. We used the slant rivalry stimulus, in which perspective foreshortening and disparity specified opposite surface orientations. This rivalrous configuration induces alternations of perceived surface orientation, while the slant cues remain constant. Subjects were able to voluntarily control their perceptual state while viewing the ambiguous stimulus. They were asked to make a saccade across the perceived slanted surface. Our data show that vergence responses closely approximated the vergence response predicted by the disparity cue, irrespective of voluntarily controlled perceived orientation. However, comparing the data obtained while viewing the ambiguous stimulus with data from an unambiguous stimulus condition (when disparity and perspective specified similar surface orientations) revealed an effect of perspective cues on vergence. Collectively our results show that depth cues rather than perceived depth govern vergence.\nIntroduction\nIn vision, binocular fusion is facilitated by vergence. Various different types of horizontal vergence have been distinguished based on inputs used by the vergence system, e.g. binocular disparity and accommodation. One of those, fusional vergence, uses binocular disparity as input. And another, proximal vergence, is associated with \u201cknowledge of nearness\u201d (Howard, 2002). Recently, the influence of depth perception on vergence has regained interest, because of the possibility to probe perception through vergence. In several studies, various depth cues were used to induce depth and the influence of perception on vergence was studied both without and with binocular disparity present. In this study, we investigate whether depth perception per se contributes to vergence.\nEnright (1987b) measured vergence movements corresponding to the depth relations implied by perspective foreshortening, under monocular viewing conditions. He concluded that the perceived depth suggested by perspective foreshortening could elicit vergence. Ringach et al. (1996) used the kinetic depth effect to induce perception of depth. They measured vergence, also under monocular viewing conditions, and found that vergence corresponded to depth perceived by the subject. This led them to conclude that perceived depth can elicit vergence.\nMore recently, several studies investigated the influence of perceived depth on vergence in the presence of disparity, i.e. under binocular viewing conditions. Sheliga and Miles (2003) used Ogle\u2019s induced effect in which vertical disparities give rise to depth. The authors reported that depending on the condition, a maximum of 41% of the vergence could be attributed to perceived depth. Both et\u00a0al. (2003) used Werner\u2019s illusion to induce depth. They reported slight vergence responses corresponding to perceived depth. These results led to the conclusion that perceived depth contributed to the vergence responses.1\nHowever, in all aforementioned studies, depth perception was correlated with stimulus properties, i.e. the depth cues giving rise to the perceived depth. Therefore, the distinction between an effect on vergence caused by perception and an effect due to depth cues could not be made. Thus, it may be possible that signals related to the depth cues contributed to vergence rather than perceived depth itself.\nTo investigate whether perceived depth itself is sufficient to influence vergence, a condition is required in which depth perception is not correlated with both monocular and binocular cues that give rise to the perceived depth (Allison et\u00a0al. 1998; Gillam 1968; Gillam and Ryan 1992; Gillam and Cook 2001). The slant rivalry stimulus offers the possibility to dissociate vergence effects due to depth cues from those caused by perceived depth (van Ee et\u00a0al. 2002). To create a slant rivalry stimulus, perspective foreshortening and binocular disparity are used to define slanted surfaces. If the cues specify opposite slant orientations, subjects report alternations between perceiving a slanted rectangle and perceiving an oppositely slanted trapezoid (van Ee et\u00a0al. 2002). Thus, perceived slant alternates, whereas the depth cues remain constant.\nUsing these slant stimuli, the contribution to vergence predicted by perception is different from that predicted by the depth cues. If perceived depth is sufficient to influence vergence, a difference in vergence should be observed while alternations occur between the two possibly perceived surface slants. However, if perceived slant does not influence vergence, vergence should remain stable regardless of the perceived surface slant orientation.\nTo study the effect of perceived surface slant on vergence, we analyzed saccades made under various stimulus and perceptual conditions. Binocular saccades usually contain a conjugate component (version) and a disjunctive component (vergence) (Erkelens et\u00a0al. 1989). Both components are preprogrammed, thus if perceived depth influences vergence, the disjunctive component should be related to the depth direction specified by the perceived slant. Hence, measured vergence at saccade offset can give insight into the input that drives the vergence system.\nMethods\nExperimental setup\nStimuli were displayed using a conventional Wheatstone stereoscope consisting of two TFT displays (20\u2033 LaCie Photon20Vision II, 1,600\u00a0\u00d7\u00a01,200, 75\u00a0Hz) and two small mirrors, see Fig.\u00a01. The mirrors were slanted about the vertical axis at an angle of 45\u00b0 with respect to the display. The virtual intersection point of the orthogonal mirrors was aligned with the center of the displays. Subjects were seated close to the mirrors so that the left eye could not see the right mirror and vice versa. The straight-ahead viewing distance (eye-mirror-display) was 57\u00a0cm. Subjects\u2019 heads were fixated using a bite-board.\nFig.\u00a01Wheatstone stereoscope. Subjects viewed one TFT display with the corresponding eye via one of the mirrors. The viewing distance (eye-mirror-display) was 57\u00a0cm. Note that the subjects were in reality much closer to the mirrors than depicted here and that there was no crossover, i.e. each eye could only see via one mirror\nEye movements were measured using the head-mounted Eyelink I system at 250\u00a0Hz. The cameras were positioned beneath the mirrors. The whole setup and experimental room were painted black matte and the room was darkened.\nStimuli\nA stimulus consisted of two images displayed, one on each of the displays. The displayed images were generated using custom Open GL based software. The images used were trapezoidal or rectangular shapes composed of lines and a sparse random dot texture, as shown in Fig.\u00a02. The images were surrounded by a fronto-parallel sparse random dot background (not shown).\nFig.\u00a02An image as shown on one display. Perspective foreshortening indicates a slant of 70\u00b0. The disparity gradient was produced by horizontally scaling the two eyes\u2019 half images. The red fixation cross is positioned in the center of the stimulus\nPerspective foreshortening of both images corresponded with a 2D projection (cyclopean viewpoint) of a rectangular surface slanted (\u00b1) 70\u00b0 about the vertical axis. A horizontal (binocular) disparity gradient defined a (\u2213) 50\u00b0 slant about the vertical axis, which was produced by scaling the two eyes\u2019 half images horizontally. After scaling, the horizontal angular width of the stimulus was 20\u00b0 for all stimuli independent of the imposed slant angles, see Fig.\u00a03a. The same figure also shows that a counter clockwise rotation (CCW) about the vertical axis was defined as a positive slant angle and a clockwise rotation (CW) as a negative slant angle.\nFig.\u00a03a The subtended angle from center to left or right side of the stimulus was 10\u00b0 (\u03b3) irrespective of the imposed surface slants defined by disparity (D) and perspective (P). A counter clockwise rotation of the surface about the vertical axis is defined as a positive surface slant angle (+). b Examples of different stimulus conditions. The surface slant angle as defined by disparity was \u00b150\u00b0 (\u03b1) and the perspective defined surface slant angle (\u03b2) was \u00b1 70\u00b0. In the unambiguous stimulus, disparity and perspective defined the same surface orientation yielding a stable perception of surface slant (1). Whereas in the ambiguous stimulus condition (2), the orientations of the defined surfaces were opposite, giving a bistable perception of surface slant\nStimulus parameters could define any of the following possibilities: (1) a pair of trapezoids in which the signs of slant defined by perspective foreshortening (P) and disparity (D) were equal [unambiguous stimulus, e.g. P\u00a0=\u00a070\u00b0 and D\u00a0=\u00a050\u00b0, see Fig.\u00a03b(1)] or (2) a pair of trapezoids in which the signs of slant defined by perspective foreshortening and disparity were opposite [ambiguous stimulus, e.g. P\u00a0=\u00a070\u00b0 and D\u00a0=\u00a0\u221250\u00b0, see Fig.\u00a03b(2)]. These physical parameters led to the following perceived slanted surfaces: ad (1) a slanted rectangle (stable slant) or ad (2) alternations of a slanted rectangle and an oppositely slanted trapezoid (bistable slant). Note that the slant angle defined by the monocular cues is larger than the angle defined by the binocular cues, to produce a sufficient degree of bistability for all subjects. The disparity gradient could not be diminished, as this would deteriorate the signal-to-noise ratio of vergence (see Sect.\u00a02.5 as well).\nFurthermore, a standing disparity was added to all stimuli, which made all surfaces appear to be positioned in front of the display. A fixation cross was presented in the center of the stimulus.\nProcedure and tasks\nExperimental trials consisted of a sequence of five different displayed items. Subjects were first presented with a fixation dot (used for offline drift correction) in the center of the display at display depth for 1.5\u00a0s (1). This dot was replaced by a fixation cross at the location and depth corresponding to the center of the stimulus (2). After 1.5\u00a0s, the stimulus images were added (3). A beep was sounded 2.0\u00a0s after stimulus onset. A monocular arrow appeared 2\u00a0s thereafter in the left eye\u2019s image (4). The screen was blanked after 4\u00a0s (5).\nDuring the trial, subjects were instructed to fixate the cross. When the monocular arrow had appeared, subjects were instructed to make one single saccade towards the side of the surface indicated by the arrow. The arrow always pointed towards the near side of the slanted surface defined by disparity. By doing this the vergence response (disjunctive part of saccade) was maximized, because some subjects showed considerably less vergence change when saccades were made towards the uncrossed disparity side of the stimulus. This is in agreement with the effect of version\u2013vergence ratio on the disjunctive part of the saccade as described by Enright (1984).\nSubjects had to report their percept prior to saccade onset. They were asked to report the perceived nearest side of the stimulus after the beep using a numerical keypad. By reporting which stimulus side they perceived as nearest, subjects implicitly indicated whether they perceived a slanted rectangle or a slanted trapezoid.\nWhen viewing a slant rivalry stimulus, subjects report perceiving spontaneous alternations of their percept. In addition, they are able to voluntarily control their perception as well (van Ee et\u00a0al. 2002, 2005b). To obtain equal sized data sets for each of both perceptual conditions, we used this ability to voluntarily control perception of the ambiguous stimulus. Thus, subjects were instructed to hold one of both possible perceived surfaces when viewing the bistable stimulus before the experiment started.\nOne experimental block consisted of a total of 16 trials, of which 8 were ambiguous stimuli conditions and the other 8 were unambiguous stimuli conditions. Both conditions were counter balanced for saccade direction and trials were randomized for conditions and saccade directions. Each experimental session contained three to four experimental blocks run consecutively. A calibration was performed, at the start of each block, i.e. every 5\u00a0min.\nData analysis\nRaw data provided by the Eyelink system was analyzed off line. We used a custom calibration procedure specifically designed to calibrate the raw data. This procedure consisted of a weighted least square error fit with a third order polynomial [35 measurement points, BFGS minimization technique (Press 1993)] (van Beers 2007). Offset correction was performed offline for each trial using the data obtained with the first fixation dot. On the calibrated data, saccade detection was performed based on version (mean gaze angle of left and right eye) velocity. A saccade was detected when the version velocity was higher than 120\u00b0\/s. The end of the saccade was defined when version velocity dropped below 20\u00b0\/s. Only saccades with an amplitude of at least 7\u00b0 in the indicated direction were analyzed. After the saccade, a slower vergence movement was usually present, vergence calculated as right eye gaze data\u2013left eye gaze data. This was assumed to be part of the initial preprogrammed vergence. We defined the preprogrammed vergence offset when vergence velocity was minimum within a timeframe of 100\u00a0ms after saccade offset and 5\u00b0\/s at the most. We used the data at the point of minimum vergence velocity for further analysis. As we were interested in the vergence difference between the onset and offset of the saccade, we present the data relative to the gaze direction before onset of the saccade. Vergence data were averaged [three nearest neighbor average (24\u00a0ms interval)] to remove high-frequency noise.\nSubjects\nSubjects (6 females and 9 males aged between 18 and 30) had normal or corrected to normal vision. Before taking part in the experiment, it was checked whether a subject was able to comply with the instructions of the experiment. Not all potential subjects could perceive the trapezoidal interpretation of the stimulus within the timeframe of the experiment (2\u00a0s), when disparity and perspective foreshortening were in conflict. These subjects either had poor stereovision or needed more than 2\u00a0s to elicit a voluntary controlled flip. In total eight subjects were excluded from further participation, because it would have been impossible to measure the effect of depth perception on vergence.\nResults\nSaccades had normal main sequence characteristics (amplitudes of about 9\u00b0, durations of about 56\u00a0ms, maximum velocities of about 310\u00b0\/s) (Collewijn et\u00a0al. 1988). Most saccades reached an amplitude of 80\u2013100% of the amplitude defined by the width of the stimulus. Sometimes a correction saccade was made after about 150\u00a0ms. Only a small fraction of saccades (less than 2%) was in the opposite direction as indicated by the monocular arrow presented.\nExperiment 1: Contribution of perception of depth to vergence\nTo investigate whether perceived surface orientation contributes to vergence, we analyzed the data obtained during the presentation of the ambiguous stimulus. As mentioned in Sect.\u00a02.3, we asked subjects to hold one of both possible orientations. In 52% of the 659 trials subjects reported perceiving a slanted rectangle and the slanted trapezoid was perceived in 48% of the trials. In 88% of all trials the percept was in agreement with the instruction given before the experiment started. The data of trials in which they failed (due to spontaneous or no (controlled) flips) are not included in the data presented here, but were not significantly different from the trials in which subjects were successful in obliging to the instruction.\nPredictions\nWhile the cues remained constant, perceived orientations alternated. Thus, we predict that if perception contributes to vergence, vergence should alternate accordingly. But, if not, the measured vergence changes should be independent of perceived slant orientation. Because we asked subjects to report only the sign of the orientation (and not the magnitude) of perceived slant, we can only predict the direction of the vergence changes, as illustrated in Fig.\u00a04a. When subjects perceived the trapezoid (disparity-based percept), the saccades were directed towards the near side of the perceived surface, whereas saccades were directed towards the far side when subjects perceived the rectangle (perspective-based percept). Consequently, the vergence changes predicted by perception are in the converging direction (blue area) when subjects perceived a trapezoid and in the diverging direction (red area) when they perceived the rectangle. The upper and lower limits of the predicted vergence regions in Fig.\u00a04a are defined by the vergence based on a zero depth change (grey line) and a vergence change based on the maximum slant defined by either depth cue: disparity (perception of trapezoid, blue line) or perspective (perception of rectangle, red line).\nFig.\u00a04a Predicted vergence changes based on perceived surface slant orientation. The transitions between vergence at saccade onset (t\u00a0=\u00a00\u00a0ms) and at saccade offset are described using a sigmoid function. The upper and lower limits of the predicted vergence regions are based on the vergence step corresponding with the depth defined by a single cue. The box indicates the range of the detailed graphs in b. b Vergence changes based on perceived surface slant orientation of subject S6 (top) and subject S1 (bottom). Mean traces for each condition with SE of every fourth data point are displayed. Saccade offset occurred at 55\u00a0ms for S6 and at 46\u00a0ms for S1 conform main sequence characteristics (saccade onset at t\u00a0=\u00a00\u00a0ms). These data show that (1) saccade offset does not coincide with offset of preprogrammed vergence per se, (2) that the \u201cperception of rectangle\u201d condition is more converging than the \u201cperception of trapezoid\u201d condition from S6, which is opposite to the traces of S1\nResults\nFigure\u00a04b shows representative vergence traces from the \u201cperception of trapezoid\u201d and \u201cperception of rectangle\u201d conditions. These vergence traces are means of 25 trials at the least, with standard error (SE) displayed at every fourth data point. Saccade onset (t\u00a0=\u00a00\u00a0ms) has been used to align the individual vergence traces for calculating the mean. Saccade offset occurred at t\u00a0=\u00a055\u00a0ms for S1 and at t\u00a0=\u00a046\u00a0ms for S6, which is in accordance with main sequence characteristics. Figure\u00a04b shows that at this point in time, the preprogrammed vergence movement has not ceased. Preprogrammed vergence offset was determined for individual traces (see Sect.\u00a02.4) and cannot be pinpointed exactly in these graphs. Preprogrammed vergence offset typically occurred at t\u00a0=\u00a0120\u00a0ms (\u00b130\u00a0ms), at which point the vergence velocity (mean of all subjects) was 0.2\u00b0\/s (SD\u00a0=\u00a00.15\u00b0\/s). The diverging movement present in the onset of the saccades is commonly observed in most conjugate saccades and caused by temporal\/nasal eye movement differences (Collewijn et\u00a0al. 1988).\nAs Fig.\u00a04b shows, the vergence changed in the converging direction for both perceptual conditions. Moreover, all subjects showed converging responses. Thus, if subjects perceived a slanted trapezoid, vergence was in the same direction as the perceived orientation of the surface. And, if they perceived a slanted rectangle, the direction of vergence was opposite to the perceived orientation of the surface. This indicates that the contribution of perceived orientation to vergence, if any, is small. When comparing the data traces with the predictions in Fig.\u00a04b, it is clear that the measured vergence changes are close to the value predicted by the disparity cue (blue dotted line). This result suggests that the largest contribution to vergence is related to the disparity cue.\nTo investigate the magnitude of the effect of perceived orientation on vergence, we further analyzed the data of all subjects, starting with selecting the end of the preprogrammed vergence movement as described in Sect.\u00a02.4. We then normalized the data, using the calculated prediction based on the disparity cue, thereby effectively removing the variability caused by the interocular distance.2 These normalized data are presented in Fig.\u00a05a. Because these data show individual biases (e.g. S4), we used the difference between the two conditions tested for calculations on group data (see Fig.\u00a05b). The data of leftward (bottom) and rightward (top) saccades are shown separately, because individual biases varied across these conditions (e.g. for S4). ANOVA statistics on individual and group data showed that there was no significant difference between the two investigated conditions (except for S1 leftward saccade, see Fig.\u00a05 caption), thereby rejecting the hypothesis that perceived orientation influences vergence. Thus, vergence is independent of perceived orientation.\nFig.\u00a05a Vergence (mean and SE selected as described in Sect.\u00a02.4) relative to predicted vergence based on the disparity cue (0 on vertical axis) from leftward saccade trials (bottom) and rightward saccade trials (top). These data show that (1) there are individual biases (e.g. S4), (2) the \u201cperception of rectangle\u201d (red circle) is not less convergent than the \u201cperception of trapezoid\u201d (blue pentagon) condition for all subjects (e.g. S2, S5) and both directions (e.g. S4, S6), rebutting the predictions based on perception. b Vergence (mean and SE selected as described in Sect.\u00a02.4) relative to \u201cperception of a trapezoid\u201d condition (0 on vertical axis). On the right side of these graphs the mean (and SE) of all subjects is shown. There is no systematic trend visible across subjects. All differences are nonsignificant except for S1, leftward saccade (F\u00a0=\u00a06.36, P\u00a0<\u00a00.05)\nExperiment 2: Contribution of depth cues other than disparity to vergence\nThe results from experiment 1 show that perceived orientation does not influence vergence. Still, as stated in the introduction, cues other than disparity may contribute to vergence. We investigated this hypothesis by analyzing the data from the ambiguous stimulus and the unambiguous stimulus presentations.\nPredictions\nFor both conditions the disparity defined slant was identical. In the unambiguous stimulus, the orientation of slant angle defined by perspective foreshortening was identical to the one defined by disparity, whereas it was opposite in the ambiguous stimulus. Thus, if perspective contributes to vergence, we predict that the vergence change of the ambiguous condition should be less convergent than that of the unambiguous condition. But, if not, vergence should be independent of the stimulus presented. Predictions of vergence changes based on slant cues are illustrated in Fig.\u00a06a. For both conditions, the disparity cue signaled a slant of 50\u00b0 in magnitude and as saccades were directed towards the crossed disparity side of the stimulus, the corresponding predicted vergence change converges (blue dotted line). The perspective cue signaled a slant of 70\u00b0 in magnitude in both conditions. In the unambiguous condition, the vergence change predicted by perspective converges (green dashed line) and consequently, the predicted vergence change in the ambiguous condition diverges (red line).\nFig.\u00a06a Predicted vergence changes based on the slant cues of the stimulus. The predicted values are based on the depth defined by a single cue. Details similar as in Fig.\u00a04. b Vergence changes from ambiguous and unambiguous stimulus trials of subject S6 (top) and subject S1 (bottom). Mean traces of each condition with SE of every fourth data point are displayed. Saccade offset occurred at 56\u00a0ms for S6 and 46\u00a0ms for S1 (saccade onset at t\u00a0=\u00a00\u00a0ms). These data show that the vergence step of the ambiguous stimulus condition is less convergent than the vergence step of the unambiguous stimulus condition for both subjects\nResults\nFigure\u00a06b shows representative traces of the unambiguous and ambiguous stimulus conditions. The vergence traces are means of 25 trials at the least with the SE displayed at every fourth data point. The characteristics of these vergence traces are similar to the traces in Fig.\u00a04b. The vergence traces in Fig.\u00a06b both converge towards the predicted value based on the disparity cue, see Fig.\u00a06a (blue dotted line). Thus, the contribution to vergence that can be related to disparity is relatively large, consistent with the results described before.\nWe further analyzed the data to investigate whether there was a significant effect of the perspective cue on vergence, using the same method as described in the previous subsection. Figure\u00a07a shows the normalized data, whereas Fig.\u00a07b displays the data from the ambiguous condition relative to the data from the unambiguous condition including the group means. Leftward (bottom) and rightward (top) saccades are shown separately. ANOVA statistics on group and individual data show that there is a significant difference between the ambiguous and unambiguous condition. The statistics for group data are: leftward saccades (lw) (difference\u00a0=\u00a00.14\u00b0, F\u00a0=\u00a013.04, P\u00a0<\u00a00.05) and rightward saccades (rw) (difference\u00a0=\u00a00.16\u00b0, F\u00a0=\u00a062.89, P\u00a0<\u00a00.001). And for individual subjects: S0 lw (F\u00a0=\u00a011.29, P\u00a0<\u00a00.01), S1 lw (F\u00a0=\u00a014.50, P\u00a0<\u00a00.001) and rw (F\u00a0=\u00a04.04, P\u00a0<\u00a00.05), S2 lw (F\u00a0=\u00a025.22, P\u00a0<\u00a00.0001) and rw (F\u00a0=\u00a024.14, P\u00a0<\u00a00.0001), S4 lw (F\u00a0=\u00a024.84, P\u00a0<\u00a00.0001), S6 lw (F\u00a0=\u00a05.22, P\u00a0<\u00a00.05) and rw (F\u00a0=\u00a011.02, P\u00a0<\u00a00.01).\nFig.\u00a07a Vergence (mean and SE selected as described in Sect.\u00a02.4) relative to predicted vergence based on the disparity cue (0 on vertical axis) from leftward saccade trials (bottom) and rightward saccade trials (top). Note that (1) there are some large individual biases, (2) that the ambiguous condition (purple triangle) is consistently less convergent than the unambiguous condition (cyan square), supporting the predictions based on slant cues. b Vergence (mean and SE) relative to unambiguous condition. On the right side of these graphs the mean of all subjects is shown. The systematic trend visible between and across subjects is significant for the whole group (leftward saccades: F\u00a0=\u00a013.04, P\u00a0<\u00a00.05 and rightward: F\u00a0=\u00a062.89, P\u00a0<\u00a00.001). All significant differences are denoted by a star (values are stated in text)\nThus, perspective, or signals related to it, significantly contributed to vergence.\nDiscussion\nOur findings show that depth cues rather than depth perception itself contribute to vergence accompanying saccadic movements. Perspective being congruent or incongruent with disparity caused a difference of about 14% in vergence changes predicted by disparity alone. Although our results show that vergence remained constant in the ambiguous stimulus condition, subjects perceived alternations of surface orientation. Collectively, these results show that perspective and disparity are each weighted differently for perception and vergence.\nBinocular studies\nOur result on the influence of perception on vergence refines the conclusions of previous studies relating perception and vergence (Sheliga and Miles 2003; Both et\u00a0al. 2003). These studies investigated the influence of perception on vergence under binocular viewing conditions, similar as in our study. However, contributions of perception of depth to vergence could not be dissociated from those of depth-inducing cues. This dissociation was possible in our study. We found that, perspective contributes to vergence, independent of its perceptual effect. This conclusion suggests that vertical disparities (Sheliga and Miles 2003), global horizontal disparities (Both et\u00a0al. 2003) and perspective (present study) contribute to vergence similarly as local horizontal disparities do, albeit to a lesser extent.\nMonocular studies\nThe perspective studies of Enright (1987a, b) show that vergence occurs corresponding to the depth implied by (linear) perspective under monocular viewing conditions. Enright concluded that the perceived depth contributed to vergence. However, in light of the present study, his results show that depth cues contribute to vergence under monocular viewing conditions as well. Moreover, without the dominantly contributing factor (disparity) being present, other depth cues become the main contributors to vergence.\nThe Necker cube study of Enright (1987a, b) and the ambiguous rotating sphere study of Ringach et\u00a0al. (1996) seem to provide similar stimulus conditions as our experiment. The single depth cue used induced alternations in depth perception. In both studies corresponding alternations in vergence were measured and both authors conclude that depth perception induced vergence.\nHowever, each depth cue used in these studies was inherently ambiguous. Thus, in these studies, the contribution of perception of depth to vergence could again not be dissociated from the contribution of the depth cues themselves. Taking the present study into consideration, there is evidence that their results reflect the alternations of the depth cue related signals and not those related to perception. In view of this conclusion, the fact that the vergence changes measured by Enright in the Necker cube experiment were much smaller than those in the linear perspective experiment may be explained by a degradation of the signals related to the depth cues due to the ambiguity herein.\nInfluence of perception on other visual phenomena\nOur results indicate that vergence is influenced by low-level (sensory) processes and not by high-level (voluntary control) processes. Studies on bistability (van Ee et\u00a0al. 2005b, a; Toppino 2003) show that high-level mechanisms can influence perception, whereas, as shown in the present study, they do not influence vergence. A recent study by Knapen and van Ee (2006) shows that slant adaptation is not influenced by perception or by voluntary control of perception, but is solely based on depth cues in the (slant rivalry) stimulus. Thus, both adaptation and vergence are influenced by low-level mechanisms, but not by high-level mechanisms.\nDifferent weighting of cues for vergence and perception\nTo explain the current results and those of Enright (1987a, b) and Ringach et\u00a0al. (1996), a single 3D surface representation used for perception and binocular eye movements as suggested by Ringach et\u00a0al. (1996) is inappropriate. The perceptual states and the alternations thereof are not reflected in the vergence responses, suggesting that two separate representations are used for perception and eye movements. Moreover, the present results suggest that the weights assigned to signals related to monocular and binocular depth cues are different for perception and vergence, resulting in either alternating or stable outcomes respectively, see Fig.\u00a08. These outcomes may construct two separate 3D space representations one used for perception and the other for vergence or, as an alternative, one 3D representation for perception whereas the signals for vergence are directly wired to the oculomotor system. However, in the latter situation the monocular signals must still be disambiguated before the motor command can be given to reorient the eyes.\nFig.\u00a08Perception and vergence are based on separate processing streams. Both monocular cues (such as relative motion, linear perspective, blur and looming) and binocular cues [such as horizontal disparity (global and local) and global vertical disparity] are used for perception as well as for vergence. The weights assigned to the individual cues might be different when used for perception than when used for vergence, resulting in different outcomes. In the present study, for example, the ambiguous stimulus yields a bistable perception of surface slant, whereas it yields a stable vergence angle. Cognitive factors, such as voluntary control, do not seem to exert influence on vergence, but they do influence perception\nPerception and action\nOur results constitute another example of a dissociation between perception and action systems as suggested by Goodale and Milner (1992). Since then evidence supporting a dissociation has been accumulating, for a review see Goodale and Westwood (2004), Carey (2001) and Bruno (2001). Haffenden and Goodale (2000) and Haffenden et\u00a0al. (2001) have shown that there are conditions in which perception has no influence on action, whereas other studies (Erkelens and Collewijn 1985; Masson et\u00a0al. 1997; Schreiber et\u00a0al. 2001; Banks et\u00a0al. 2001; van Ee and van Dam 2003) have shown that conditions exist in which eye movements have no influence on visual perception. On the other hand, some studies conclude that action is influenced by perception (Franz et\u00a0al. 2000, 2001; Franz 2001; Smeets et\u00a0al. 2002; Lopez-Moliner et\u00a0al. 2003; de\u00a0Grave et\u00a0al. 2006a, b; Bernardis et\u00a0al. 2005; Knox and Bruno 2007). As we have shown all depth cues are used by the vergence system. The use of pictorial depth cues by other motor systems could perhaps explain this incongruence.\nConclusion\nOur findings show that depth cues rather than perceived depth govern vergence that accompanies saccades. Perspective being congruent or incongruent with disparity caused a 14% difference in vergence change. These results combined with other studies show that in addition to local horizontal disparity, also global disparity, relative motion and perspective are used as input by the vergence system. Furthermore, our findings suggest that monocular and binocular cues are weighted differently for perception and vergence.","keyphrases":["vergence","ambiguous","disparity","depth perception"],"prmu":["P","P","P","P"]}
{"id":"Pediatr_Nephrol-3-1-1915619","title":"Linkage study of 14 candidate genes and loci in four large Dutch families with vesico-ureteral reflux\n","text":"Vesico-ureteral reflux (VUR) is a major contributing factor to end-stage renal disease in paediatric patients. Primary VUR is a familial disorder, but little is known about its genetic causes. To investigate the involvement of 12 functional candidate genes and two reported loci in VUR, we performed a linkage study in four large, Dutch, multi-generational families with multiple affected individuals. We were unable to detect linkage to any of the genes and loci and could exclude the GDNF, RET, SLIT2, SPRY1, PAX2, AGTR2, UPK1A and UPK3A genes and the 1p13 and 20p13 loci from linkage to VUR. Our results provide further evidence that there appears to be genetic heterogeneity in VUR.\nIntroduction\nVesico-ureteral reflux [VUR (MIM 193000)], the retrograde passage of urine from the bladder, is one of the most commonly detected congenital anomalies. With a prevalence of approximately 1% [1], VUR can be primary, due to an incompetent valve mechanism at the uretero-vesical junction, or secondary, due to a functional or anatomical urethral obstruction. VUR is often accompanied by non-neuropathic bladder\/sphincter dysfunction (NNBSD). This complex is a major cause of urinary tract infections in children [2] and the sometimes resulting reflux nephropathy is the cause of approximately 7% of end-stage renal disease in paediatric patients in the Netherlands [3]. Severe primary VUR can concur with congenital renal insufficiency based on hypoplasia\/dysplasia of one or both kidneys. Genetic factors play an important role in the aetiology of primary VUR, since siblings of affected children have a 32% risk of VUR [4], and since there is 80% concordance between monozygotic twins [5]. VUR may occur in isolation or as part of a syndrome, such as renal-coloboma syndrome. Apart from the recently published involvement of ROBO2 [6] little is known about the genetic causes of isolated primary VUR in humans. The aim of the present study was to confirm linkage to published candidate loci and genes. So far, only one genome-wide linkage study has been reported, which showed significant linkage to a 17\u00a0cM locus on chromosome 1p13 in five Caucasian families and suggestive linkage to chromosome 20p13 [7]. To date, these results have not been replicated [8]. Embryonal ectopic ureteral budding has been proposed to be a mechanism for the development of VUR [9, 10]. Defects of the RET and GDNF genes have been shown to cause ectopic ureteral budding [11, 12]. Hence, these and other genes involved in the RET\/GDNF pathway are obvious functional candidate genes for VUR. Genes involved in syndromal VUR and genes derived from mouse models with urinary tract abnormalities (such as AGTR2) are also attractive functional candidate genes for VUR. The aim of this study was to assess the 1p13 and 20p13 loci and appropriate candidate genes (Table\u00a01) for their role in the Dutch VUR population by performing linkage analysis in four large families.\nTable\u00a01Genes tested in linkage study of four large multi-generational VUR families (LOD logarithm of the odds, NPL non-parametric linkage, HLOD heterogeneity LOD, A ureteral budding, BRET\/GDNF pathway, C mouse and human phenotype, D linkage study, E in urothelial plaque with UPK3A (mouse model), F mouse model.)GeneRelevanceChromosomeLocation (cM)Multi-point LOD score at the gene locationNPLNPL p-valueAlphabHLODbReferenceGDNFA\/B554\u22122.030.700.220.100.01[13]RETA\/B1066\u22122.55\u22120.850.800.000.00[13]SLIT2A\/B434\u22122.150.350.320.150.80[14]SPRY1A\/B4126\u22123.25\u22120.980.860.000.00[14]PAX2A\/B10124\u22123.43\u22120.490.630.000.00[13]AGTR2A\/CX71\u22123.81\u22121.180.880.000.00[15]HLADRB1D646\u22121.840.250.350.200.16[16]UPK1AE1961\u22122.90\u22120.620.690.000.00[17, 18]UPK1BE31380.151.240.120.650.43[17, 18]UPK2E11115\u22121.50\u22120.220.520.000.00[17, 18]UPK3AA\/F2253\u22123.40a\u22121.200.800.000.00[17, 18]UPK3BE789\u22121.080.590.250.000.00[17, 18]aTwo-point analysis of marker D22S928; 0.5\u00a0cM away from UPK3A.bAlpha: estimated proportion of families linked to result in corresponding heterogeneity LOD (HLOD). HLOD analyses were performed, but did not contribute and are not discussed.\nMethods\nDNA of four unrelated Dutch VUR families was collected (Fig.\u00a01), which had been ascertained as part of a previous study [19]. Of a total of 51 samples there were 21 affected individuals. The families provided moderate power to detect linkage as calculated with SLINK (probability of obtaining LOD scores of at least 1.0, 2.0, or 3.0 was 74%, 49% and 18%, respectively). An affected phenotype for index patients was based on their having been treated for primary VUR, while for family members it was based on having a positive case history (of actual VUR, or multiple urinary tract infections with high fever as a child, or evidence of reflux nephropathy, such as requiring renal replacement therapy without obvious other causes) (see also Fig.\u00a01). All other family members were classified as \u201cunknown\u201d, despite negative imaging results at a young age in some of them. Dutch paediatric urologists consider the use of voiding cysto-urethrography (VCUG) in asymptomatic children just for research purposes inappropriate. Therefore, we could not classify family members as \u201cnot affected\u201d.\nFig.\u00a01VUR family pedigrees (21 affected individuals in 51 samples). Family\u00a01: 2 left kidney had to be removed at 5\u00a0years; 3, 4 end-stage renal disease (ESRD); 9 VUR and ESRD; 15 VUR and duplex collecting system; 19 VUR; 18 VUR and nephropathy. Family\u00a02: 68, 69, 73 not included in analyses; 25, 26, 30 and 31 VUR; 33 VUR and dysfunctional voiding. Family\u00a03: 39, 42, 43 VUR and dysfunctional voiding symptomatology; 47 VUR, dysfunctional voiding, meatal stenosis. Family\u00a04: 50 and 52 recurrent urinary tract infections (UTIs) as a child, duplex collecting system; 55 UTIs and urinary tract operation; 49 VUR; 51 VUR and dysfunctional voiding symptomatology\nSome of the candidate genes play roles in congenital anomalies of the kidney and urinary tract (CAKUT) phenotypes (such as VUR, duplex collecting system and renal hypoplasia in mice) [9, 15]. Therefore, families in which one or more patients had these kinds of phenotypes (and VUR) were not excluded. All participants gave their informed consent, and the Medical Ethics Committee of the University Medical Centre Utrecht approved the study.\nFor the 1p13 locus, we started out with the markers flanking the reported 1p13 linkage peak (D1S1653 and GATA176C01) [7]. Both markers now have different map locations if one is reviewing the most recent updates of the Ensembl (v38) and Marshfield databases. In fact, the telomeric marker GATA176C01 (D2S2972) even maps to a different chromosome (2q11). The centromeric marker D1S1653, which in our query result has roughly the same genetic position (164.09-166 cM) as previously published [7, 8], localizes on chromosome 1q23. Therefore, we tested both the entire 1p13\u20131q23 and 2q11 loci for linkage to VUR. A total of 11 short tandem repeat polymorphism (STRP) markers for 1p13\u20131q23 and seven STRP markers for 2q11 (with an average intermarker distance of 5\u00a0cM) were chosen to saturate the regions spanning 55.3\u00a0Mb on chromosome 1 and 46.3\u00a0Mb on chromosome 2. For 20p13, five STRP markers were selected, spanning 12.0\u00a0Mb (Supplementary Table\u00a01 Online).\nFor the candidate genes, we aimed to cover the specific location with an average intermarker distance of 2\u00a0cM (Supplementary Table\u00a01 Online).\nMarkers were genotyped as described elsewhere [20] in the 51 family members, together with three Centre d\u2019Etude Polymorphism Humaine (CEPH) reference samples and three negative controls. The polymerase chain reactions (PCRs) were carried out on a GeneAmp PCR system\u00a09700 machine (Applied Biosystems). The PCR products were separated on an ABI\u00a03730 DNA sequencer (Applied Biosystems). The output was analysed with Genemapper\u00a03.5 software (Applied Biosystems). Two investigators checked all the genotypes, and we verified the identity of the markers by comparing genotypes of the CEPH reference samples with the CEPH genotype database. A Mendelian inheritance check was performed with PedCheck\u00a01.1 software [21], and samples with Mendelian errors were excluded from the linkage analysis.\nMulti-point (both parametric and non-parametric) analyses were performed for all markers with GENEHUNTER (version\u00a02.1_r2 beta), or GENEHUNTER PLUS (for X-linked dominant calculations in AGTR2) [22]. We assumed an autosomal dominant model with reduced penetrance (0.8) for the parametric analyses, similar to the parameters previously described [7, 23]. This mode of inheritance agreed most with our pedigrees (Fig.\u00a01). The phenocopy rate was equal to the population frequency of VUR (0.01). Disease allele frequency was assumed to be 0.01. Regions with a parametric LOD score \u2264 \u22122 were defined as exclusion regions [24]. All significance levels applied in this study were based on previously proposed thresholds [24, 25].\nResults\nTwelve functional candidate genes were screened for linkage to VUR. The multi-point LOD score obtained for each of the 12 genes (at the genetic location of the gene) is shown in Table\u00a01, together with the non-parametric linkage (NPL) score and corresponding P value. Multi-point LOD scores with NPL score and corresponding P value for all markers are shown in Supplementary Table\u00a01 Online. Eight of the functional candidate genes (GDNF, RET, SLIT2, SPRY1, PAX2, AGTR2, UPK1A and UPK3A) were completely excluded. For the other four, the results were inconclusive, although linkage is highly unlikely.\nFor the reported linkage regions, no significant linkage was detected either. One of the markers reported to be on chromosome\u00a01 [7] appeared to reside on chromosome\u00a02 (see Methods section). Therefore, both the original locus on chromosome\u00a01p13 and the \u201cnew\u201d locus on chromosome\u00a02q11 were tested. The chromosome\u00a01 locus was completely excluded, as the multi-point LOD score was below \u22122 for the entire region. Sixty-one percent of the locus on chromosome\u00a02 could be totally excluded. The locus on chromosome\u00a020p13 was completely excluded (Fig.\u00a02).\nFig.\u00a02Multi-point LOD plots for the reported loci tested in the linkage study of four large multi-generational VUR families. Because one of the flanking markers of the 1p13 locus proved to actually map to chromosome\u00a02, we also tested the chromosome\u00a02q11 region. HLOD (heterogeneity LOD) analyses did not contribute. Positions in cM\nDiscussion\nWe performed a comprehensive screen of 12 functional candidate genes and two reported loci (which later proved to be three separate regions). All the genes, except HLADRB1, had, in some way, been proven to play a role (indirectly) in ectopic ureteral budding and were thought likely to play a role in human primary VUR. However, we did not detect linkage to any of them. We were able to exclude eight genes (GDNF, RET, SLIT2, SPRY1, PAX2, AGTR2, UPK1A and UPK3A) as major players in these Dutch VUR families. ROBO2, the receptor of SLIT2 [26], had already been ruled out, since it had been sequenced in the four probands in a parallel study; no mutations were detected [6], therefore it was not included in the present study. Nevertheless, these genes may still be involved in the mechanism causing VUR. The moderate power these pedigrees provided to reveal linkage means that it is possible that the genes that showed inconclusive results might have yielded positive results in a more highly powered study. Since the RET\/GDNF pathway plays such a central role in ureteral budding, it is plausible that these genes are, indeed, causative factors for VUR but that the pathogenetic variants lie in upstream or downstream regulatory elements. Other genes that are more or less directly involved in the RET\/GDNF pathway or in ureteral budding in general, such as EYA1, GATA3, WT1 or BMP4 [27], may also contribute to VUR.\nWe could not confirm linkage for any of the reported loci, and we were even able to exclude completely the 1p13 and 20p13 loci. This is the second non-replication of the 1p13 region [8]. Our data show that the 1p13 locus resides either on chromosome\u00a01p13 to 1q23 or on 2q11. We also excluded linkage to most of this 2q11 locus.\nWe realize that these families show some intra-familial and inter-familial heterogeneity. This heterogeneity, however, is similar to that described in VUR families by others [7]. In older generations (Fig.\u00a01) it is impossible to have more data than self-reported history, but the described phenotypes are very likely to be caused by VUR. Therefore, we did assign the affected status to these patients, but we are aware of the fact that this is one of the limitations of the study.\nOne of the major obstacles for linkage studies in VUR is the relative rarity of large pedigrees, which is due to many children growing out of the disorder, the reduced penetrance of the trait, and the locus heterogeneity [8]. Furthermore, when doing linkage studies in relatively few families, one assumes a large effect of one or few genes. Maybe the genes of interest do play a role, but their effect is too small to be picked up. Therefore, studies like ours and those published [7, 8] are useful to search for one or more major genes. For this reason, association studies with large sample sizes may offer a better approach for unravelling the genetics of VUR. Both a hypothesis-free (genome-wide) approach and a more elaborate candidate gene study would be interesting follow-up studies. Alternatively, it might be interesting to study the role of copy number variants (CNVs) in VUR, since, recently, such CNVs were proposed to be involved in the mechanism underlying a number of complex disorders [28, 29]. Apart from those in a recent study by Lu et al. [6], no genes have been published that appear to be directly involved in primary VUR in humans, and no replication of the linkage peak on 1p13 [7] has been reported. Our results provide further evidence for genetic heterogeneity in VUR. We hypothesize that several genes, which still have to be identified but which are likely to affect ureteral budding, will each play a role in the pathogenesis of VUR.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nSupplementary Table\u00a01 Online\nMarkers used in the linkage study of four large, Dutch, multi-generational VUR families. MPL multi-point LOD score (DOC 148 kb)","keyphrases":["vesico-ureteral reflux\/genetics","kidney diseases\/genetics","kidney diseases\/pathology","linkage (genetics)"],"prmu":["R","R","M","R"]}
{"id":"Int_J_Cardiovasc_Imaging-3-1-2048829","title":"A comparison of echocardiographic and electron beam computed tomographic assessment of aortic valve area in patients with valvular aortic stenosis\n","text":"The purpose of this study was to compare electron beam computed tomography (EBT) with transthoracic echocardiography (TTE) in determining aortic valve area (AVA). Thirty patients (9 females, 21 males) underwent a contrast-enhanced EBT scan (e-Speed, GE, San Francisco, CA, USA) and TTE within 17 \u00b1 12 days. In end-inspiratory breath hold, a prospectively ecg-triggered scan was acquired with a beam speed of 50\u2013100 ms, a collimation of 2 \u00d7 1.5 mm and an increment of 3.0 mm. The AVA was measured with planimetry. A complete TTE study was performed in all patients, and the AVA was computed using the continuity equation. There was close correlation between AVA measured with EBT and AVA assessed with TTE (r = 0.60, P < 0.01). The AVA measured with EBT was 0.51 \u00b1 0.46 cm2 larger than the AVA calculated with TTE measurements. EBT appeared to be a valuable non-invasive method to measure the AVA. EBT measures the anatomical AVA, while with TTE the functional AVA is calculated, which explains the difference in results between the methods.\nIntroduction\nAortic valve sclerosis is common in the elderly population, affecting about 25% of adults over 65\u00a0years of age [1, 2]. The presence of aortic sclerosis, with or without demonstrable haemodynamic obstruction, is associated with an increase of approximately 50% in the risk of cardiovascular death [3]. The timing of aortic valve replacement surgery has also an effect on mortality risk [4]. The indication for aortic valve replacement generally is based on haemodynamic variables, such as transvalvular pressure drop and the presence of symptoms. Significant reduction in the aortic valve area (AVA) is also an important indicator [4]. Hence, exact evaluation of the aortic valve and the AVA is necessary to make the best treatment decision.\nEvaluation of aortic valve stenosis by means of transthoracic echocardiography (TTE) has gained widespread acceptance in clinical routine and pressure gradients across the aortic valve have shown to correlate well with invasive measurements [5, 6]. Moreover, TTE is used to determine the functional AVA, using the continuity equation [5, 6]. However, the reliability of TTE measurements depends heavily on image quality, which is influenced by aortic valve calcification and adequacy of the ultrasound window [7\u20139]. The results also depend on the patient\u2019s haemoglobin level, heart rate and left ventricular function [7]. Thus, invasive confirmation is often still necessary preceding valve replacement surgery. For this purpose, cardiac catheterization evaluating AVA by applying the Gorlin formula is available [10]. However, this method may not give reliable results, depending on cardiac function and aortic regurgitation [11]. Aforementioned techniques rely on and are affected by physiological parameters. Moreover, catheterization is an invasive procedure that may be associated with serious complications [12]. Therefore, new techniques are necessary to evaluate the severity and progression of aortic sclerosis. These new techniques could measure the anatomical AVA, which measurement is independent of physiological parameters.\nContrast-enhanced computed tomography is a useful technique to evaluate cardiac structures, like coronary arteries and aortic valves [13]. EBT, a non-mechanical computed tomography scan with a high temporal resolution (50\u2013100\u00a0ms), is widely used to assess cardiac calcification, which is also an early marker of the aortic sclerotic process [1, 2]. To assess cardiac calcification, administration of contrast agent to the patient is not necessary. Several studies have shown the usefulness of assessing the degree of aortic valve calcification (AVC) with EBT as a measure of the severity of aortic valve sclerosis [14\u201317]. However, these studies did not use EBT to measure the AVA. MacMillan et al. were the first to use contrast-enhanced EBT to measure the AVA [18]. They were able to determine AVA in six patients with known aortic sclerosis. We wished to compare EBT and TTE in order to compare the assessment of aortic stenosis with both methods. We hypothesize that EBT is an useful method to evaluate aortic stenosis; both in terms of AVA as well as AVC.\nMethods\nBetween June 2004 and February 2006, patients with a known peak gradient (\u2265 30\u00a0mmHg) across their native aortic valve were invited to participate in the study during their control visit to the outpatient clinic of cardiology. All patients were under control for valvular aortic stenosis. The study was in accordance with principles that have their origin in the Declaration of Helsinki, and was approved by the local review board and all patients gave their informed consent. All patients were over 18\u00a0years of age, and had to be able to lie flat and hold their breath for 20\u00a0s. Patients with congenital heart defects, renal insufficiency (serum creatinin >120\u00a0\u03bcmol\/l), known contrast allergy or history of hyperthyroidism, thyroid cancer, Kahlers disease, myasthenia gravis, pheochromocytoma or mastocytosis were excluded. Patients with a body weight over 100\u00a0kg were also excluded because image quality of EBT is impaired in obese patients.\nElectron beam computed tomography\nScans were performed with an EBT scanner (e-Speed, GE, San Francisco, CA, USA), in the dual 1.5\u00a0mm slice mode with an image acquisition time of 50 or 100\u00a0ms depending on patient size. First, non-enhanced EBT data were collected during end-inspiratory breath hold. The non-enhanced scan was ecg-triggered at 42% of the R\u2013R interval. A set of 40 continuous axial coupes were obtained from under the trachea bifurcation to the heart. An Agatston calcification score of the aortic valve was obtained [19]. Area and volume of calcifications were calculated and summated. The localization and severity of AVC was graded using a scale of 1 to 6 according to Bahler et al. [20].\nSecond, contrast-enhanced EBT data were collected during end-inspiratory breath hold. To achieve optimal contrast enhancement, bolus time was determined for each patient individually before EBT scanning. Bolus timing, using 20\u00a0ml contrast agent, was done with the region of interest placed in the ascending aorta. The ecg-triggered multiphase scan was carried out with 120\u00a0ml contrast agent (Iomeron 400\u00ae, Bracco, Italy) followed by a saline bolus chaser. Contrast and saline bolus chaser were injected through a 18G venflon, placed in a cubital vein, with a flow rate of 4\u00a0ml\/s. Ecg-triggered multiphase scanning started at 5% of the R\u2013R interval, the first phase, and continued during systole till 50% of the R\u2013R interval, the last phase. Depending on the patient\u2019s heart rate and beam speed, measurements were performed during 7 to 10 phases.\nThe data were transferred to a workstation (GE Advanced Workstation, GE Medical, Waukesha, WI, USA) and reviewed by 1 observer (R D), blinded from echocardiographic results. An axial view of the aorta valve was created with a double oblique view. One oblique axis was put trough the aortic valve in the coronal view of the heart, see Fig.\u00a01(A). The second oblique axis was set in the created oblique view, see Fig.\u00a01(B). The AVA was than measured in six planes at several levels in the created axial view of the aortic valve, see Fig.\u00a01(B). The plane with the smallest AVA was selected and measured three times using planimetry, see Fig.\u00a01(C). The mean AVA was assessed in this way for each phase. The phase with the maximal mean AVA was selected and was considered to be the AVA of concern. This AVA was compared to TTE findings.\nFig.\u00a01All patients underwent electron beam computed tomography. (A) One oblique axis was put trough the aortic valve in the coronal view of the heart. Based on this orientation an oblique view was created. (B) The\u00a0second oblique axis was set in the created oblique view. Based on this orientation an axial view of the aortic valve was made. The aortic valve area (AVA) was than measured in six planes at several levels in the created axial\u00a0view of the aortic valve. (C) The AVA was measured three times at each level in each phase using planimetry. The mean AVA was assessed\u00a0for each phase. The phase with the maximal mean AVA was selected and was considered to be the AVA of concern\nTransthoracic echocardiography\nA complete TTE study was performed in all patients by an experienced sonographer, blinded for EBT, following a standard procedure (GE Vingmed Ultrasound Vivid Five, GE Medical Systems, Waukesha, WI). The diameter of the left ventricular outflow tract was measured from the parasternal long-axis view. Flow velocity in the left ventricular outflow tract was assessed by pulse-wave Doppler from the apical 4 chamber approach. Flow velocity across the aortic valve was measured in the apical 5 chamber view. The AVA was computed using the continuity equation [5]. The presence of aortic regurgitation was determined from standard images, and quantified as the short-axis area of the regurgitant jet as a percentage to the short-axis area of the left ventricular outflow tract, moreover standard images of the left ventricle were obtained to evaluate left ventricular function and hypertrophy. Left ventricular function was based on estimated ejection fraction and wall motion, and was graded as being normal if the ejection fraction \u226560%. Left ventricular dysfunction was mild if the ejection fraction was 45\u201360%, moderate if the ejection fraction 30\u201345%, or poor if the ejection fraction was <30%. To evaluate left ventricular hypertrophy, posterior wall and septal thickness were measured from the parasternal length axis.\nStatistical analysis\nFor the measurement of the AVA by EBT, 3 manual tracings made by 1 observer (R D) were averaged. Results of continuous normally distributed variables are expressed as mean\u00a0\u00b1\u00a0SD, results of continuous not normally distributed variables are expressed as median (range). The data were analyzed with the use of standard software (SPSS version 12.0.1, SPSS Institute, Chicago, IL, USA) on a PC. The Spearman\u2019s and Pearson\u2019s correlation coefficients (r) were used to establish the presence of linear relationships. To compare not normally distributed data, the Mann\u2013Whitney U test was used. A Bland\u2013Altman analysis was used to evaluate the agreement between the AVA assessed with EBT and TTE. A level of significance below 0,05 was defined as clinically significant.\nResults\nA total of 61 patients were screened for participation in the study. Nine females and 21 males (age 70.9\u00a0\u00b1\u00a010.0\u00a0years) were enrolled in the study. Eight patients were excluded because of renal insufficiency, 1 because of known contrast allergy, 1 because of body weight over 100\u00a0kg, and 4 patients declined to undergo an EBT. Another 17 patients were excluded because the AVA could not be assessed by TTE.\nIn the study group, the mean peak pressure gradient across the valve was 64\u00a0\u00b1\u00a021\u00a0mmHg. Ten patients had symptoms according to NYHA class I, 12 patients class II and 8 patients class III. Left ventricular end-diastolic diameter was 4.9\u00a0\u00b1\u00a00.6\u00a0cm, interventricular septal thickness was 1.2\u00a0\u00b1\u00a00.3\u00a0cm and posterior wall thickness was 1.1\u00a0\u00b1\u00a00.1\u00a0cm. Ninety percent of the patients had signs of left ventricular hypertrophy on TTE. Twenty two patients (73%) had normal left ventricular function, 6 patients (20%) had mild and 2 patients (7%) moderate left ventricular dysfunction. Mild aortic regurgitation was present in 8 patients (27%) and moderate aortic regurgitation in 2 patients (7%). The average time between TTE and EBT was 17\u00a0\u00b1\u00a012\u00a0days.\nAortic valve area\nMean AVA assessed with TTE was 0.99\u00a0\u00b1\u00a00.31\u00a0cm2. The median AVA measured with EBT was 1.34 (0.69\u20132.84) cm2. There was a significant correlation between AVA measured with EBT and AVA assessed with TTE (r\u00a0=\u00a00.60, P\u00a0<\u00a00.01), as shown in Fig.\u00a02. The AVA measured with EBT was on average 0.51\u00a0\u00b1\u00a00.46\u00a0cm2 larger than the AVA assessed with TTE, which was confirmed with Bland\u2013Altman analysis, see Fig.\u00a02. The difference in AVA between EBT and TTE showed a not statistically significant trend to be larger in patients with mild aortic regurgitation than without aortic regurgitation (0.62 (0.23\u20131.22) cm2 and 0.33 (\u22120.14\u20131.52) cm2, respectively, P\u00a0=\u00a00.08). The difference in AVA did not differ between patients with normal left ventricular function and left ventricular dysfunction (0.45 (\u22120.14\u20131.52) cm2 and 0.43 (\u22120.03\u20131.26) cm2, respectively, P\u00a0=\u00a00.78)\nFig.\u00a02By using electron beam computed tomography (EBT) planimetry the aortic valve area (AVA) can be measured. ( A) AVA measured with EBT correlate well with those assessed with transthoracic echocardiography (TTE) (r\u00a0=\u00a00.60, P\u00a0<\u00a00.01). (B) AVA measured with EBT is larger on average than AVA assessed with TTE, as is confirmed with Bland\u2013Altman analysis, exhibiting a mean difference of 0.51\u00a0\u00b1\u00a00.46\u00a0cm2. \u2013 \u2013 \u2013\u00a0=\u00a095% confidence interval\nAortic valve calcification\nThe median Agatston score of the aortic valve was 3363 (425\u201310230). There was a moderate, but significant, inverse correlation between the Agatston score of the aortic valve measured with EBT and AVA assessed with TTE (r\u00a0=\u00a0\u22120.38, P\u00a0=\u00a00.04), as shown in Fig.\u00a03. In 17 patients with an AVA <1.0\u00a0cm2 the aortic valve was more calcified than in 13 patients with an AVA\u00a0\u2265\u00a01.0\u00a0cm2 (Agatston score 3879 (1192\u201310230) and 2447 (425\u20136378), respectively, P\u00a0=\u00a00.04).\nFig.\u00a03Aortic valve calcification can be quantified with electron beam computed tomography using the Agatston score. The Agatston score correlates well with the aortic valve area (AVA) measured with transthoracic echocardiography (TTE)(r\u00a0=\u00a0\u22120.38, P\u00a0=\u00a00.04). \u2013 \u2013 \u2013\u00a0=\u00a095% confidence interval\nDiscussion\nEBT is an useful non-invasive method to evaluate aortic stenosis, both in terms of AVA and AVC. However, on average a larger AVA was measured with EBT than was assessed AVA with TTE. Indeed, this is not surprising since EBT determines the anatomical AVA, which is expected to be greater than the functional AVA assessed with TTE. Because of calcifications the valvular leaflets are less flexible, as a result the AVA will be irregularly shaped, hence the AVA becomes haemodynamically less efficient. This results in a discrepancy between the functional and anatomical AVA.\nTo our knowledge, the publication of MacMillan et al. is the first study investigating the usefulness of contrast-enhanced EBT to assess AVA [18]. In this study, 8 patients with known calcific aortic stenosis underwent EBT one day after cardiac catheterization. In six out of eight patients close agreement was found, within a 0.25\u00a0cm2 margin, between catheterization-derived AVA and AVA measured with EBT. In 1 patient the AVA could not be determined EBT and in 1 patient the AVA was overestimated by EBT with 0.60\u00a0cm2.\nThe results of our study are in line with those of MacMillan et al. Our study also showed that EBT measures larger AVA compared to TTE. The AVA is measured planimetrically with EBT, so it can be considered as the anatomical AVA. Our study as well as the study of MacMillan et al. compared this anatomical AVA to the functional AVA. This functional AVA is derived from haemodynamic characteristics of the aortic valve, (pressure changes and flow velocity changes) across the aortic valve. The functional AVA is supposed to yield the haemodynamic relevant AVA. However, using the continuity equation, the AVA is assumed to be circular, which does not reflect the actual morphology of the orifice of the stenotic valve, especially not in case of heavy calcification.\nTTE is the principal diagnostic tool in routine clinical practice to assess the AVA, next to cardiac catheterization. To determine the AVA with TTE, the continuity equation is used [5, 6], which yields the functional AVA. However, this technique has its limitations. First, TTE is not suitable in every patient with aortic valve sclerosis. Poor sonographic windows and calcified deposits in the aortic valve and cardiac wall hamper the determination of the left ventricular outflow diameter. A small fault in the measurement of this diameter can seriously influence the calculated AVA, because this diameter is squared in the continuity equation. Moreover, if the ultrasound beam is not directed parallel to the velocity jet, peak transvalvular velocity may be underestimated. Hence the peak pressure gradient and AVA may be underestimated. Second, additional cardiac dysfunction can influence the outcome of TTE measurements. The flow velocity can be decreased in case of poor left ventricular function, even though there is an anatomical small AVA. This leads to an underestimation of the severity of the aortic stenosis. Furthermore, the severity of aortic stenosis can be overestimated in cases with concomitant aortic regurgitation. Consequently, the aortic valve stenosis is overestimated [7\u20139]. Our study also showed a larger difference in AVA between EBT and TTE in patients with additional mild aortic regurgitation EBT is not influenced by aortic regurgitation, therefore EBT is a more reliable technique, in those patients, to conclude about the severity of aortic stenosis.\nThe larger AVA measured by EBT compared to TTE and cardiac catheterization may be related to slice orientation. In order to get an axial view of the aortic valve, the image has to be rotated in two planes by the observer. If the selected axial view is not situated perpendicular to the aortic valve, the AVA is overestimated. Moreover, from this axial view the plane with the smallest AVA has to be selected. Although the temporal resolution of EBT is high, it is possible that the smallest AVA is situated between 2 planes. Hence, the AVA can be overestimated. Nevertheless, we addressed this problem by acquiring several slices at different levels of the aortic valve. This is necessary to minimize the potential of AVA overestimation because of imprecise localization.\nIn addition to the measurement of AVA, AVC can be quantified with EBT. Many studies have made clear that EBT is also effective in the evaluation of AVC [14\u201317, 21, 22]. The findings of these studies suggest that elevated AVC scores are a marker for the presence of aortic stenosis; our study underlined these findings. A diagnostic threshold value of 1000 Agatson units has a sensitivity of 93% to detect severe aortic stenosis (AVA\u00a0<\u00a01.0\u00a0cm2) according to Messika-Zeitoun et al. [16]. Rosenhek et al. concluded that the extent of AVC was a strong independent predictor of outcome [23]. In a study population of 126 patients with asymptomatic severe aortic stenosis, four-year event-free survival was 75% in patients with mild AVC, compared to 20% in patients with moderate or severe AVC.\nAs mentioned above, the grade of AVC is a predictor of outcome in patients with aortic stenosis. Although the echocardiographic severity of aortic stenosis is correlated to AVC score, the AVA can not be predicted from the AVC score. So, to evaluate the severity of aortic valve stenosis and to indicate whether aortic valve replacement surgery is necessary it is inevitable to measure AVA. In our study, we showed a significant correlation between the AVA measured with EBT and the severity of aortic stenosis assessed with TTE. However, further research is needed to validate EBT as a technique to evaluate the AVA.\nNext to EBT, there are other non-invasive imaging techniques being evaluated for their value in AVA assessment. Alkadhi et al. compared 16 detector row computed tomography to tranesophageal echocardiography in the evaluation of AVA in 20 patients with and 20 patients without aortic stenosis [24]. They reported a mean difference of 0.06\u00a0cm2. Reant et al. performed a similar study using magnetic resonance imaging (MRI) [25]. They enrolled 39 patients with aortic stenosis and compared the AVA measured with MRI to tranesophageal echocardiography and cardiac angiography. Their results suggest good correlation between techniques; MRI and transesophageal echocardiography differed 0.01\u00a0cm2, MRI and cardiac angiography differed 0.06\u00a0cm2. Both studies show that non-invasive imaging techniques are a good possible alternative for the evaluation of aortic valve stenosis.\nConclusion\nEBT appears to be valuable as a non-invasive method to assess the severity of aortic valve sclerosis. EBT holds the qualifications for accurate assessment of the anatomy, morphology and physiology of the aortic valve. However, further investigations will have to be done to verify whether EBT is a suitable non-invasive imaging technique for evaluation of aortic valve disease.","keyphrases":["electron beam computed tomography","echocardiography","aortic valve stenosis"],"prmu":["P","P","P"]}
{"id":"Sci_Eng_Ethics-4-1-2413106","title":"To Recycle or Not to Recycle? An Intergenerational Approach to Nuclear Fuel Cycles\n","text":"This paper approaches the choice between the open and closed nuclear fuel cycles as a matter of intergenerational justice, by revealing the value conflicts in the production of nuclear energy. The closed fuel cycle improve sustainability in terms of the supply certainty of uranium and involves less long-term radiological risks and proliferation concerns. However, it compromises short-term public health and safety and security, due to the separation of plutonium. The trade-offs in nuclear energy are reducible to a chief trade-off between the present and the future. To what extent should we take care of our produced nuclear waste and to what extent should we accept additional risks to the present generation, in order to diminish the exposure of future generation to those risks? The advocates of the open fuel cycle should explain why they are willing to transfer all the risks for a very long period of time (200,000 years) to future generations. In addition, supporters of the closed fuel cycle should underpin their acceptance of additional risks to the present generation and make the actual reduction of risk to the future plausible.\nIntroduction\nThe worldwide need for energy is growing. The International Energy Agency foresees a 60% increase in energy need in the world between 2004 and 2030 and most of this expansion is expected to be met by fossil fuel [1]. Fossil fuels are not an attractive option, however, for reasons concerning the availability of resources and climate change. An increased need for alternative energy sources is therefore expected in the upcoming decades, e.g. wind energy, solar energy, but also nuclear energy. After being ruled out in many countries following the Chernobyl disaster in 1986, nuclear energy has recently made a serious comeback in the public and political debates about the future of energy. Many people consider nuclear energy at least as a serious alternative for the transition period between fossil fuels and sustainable energy sources. According to the World Nuclear Association, there were 435 operative nuclear reactors in January 2007; The United States, France, Japan and Russia together possess the vast majority of the operative reactors producing 370\u00a0GWe. As a whole, nuclear energy provides almost 16% of worldwide energy supply [2, 3].\nThe main advantage of nuclear energy\u2014compared to fossil fuels\u2014is its capability of producing a large amount of energy with relatively small amounts of fuel and a very small production of greenhouse gases. However, nuclear energy has serious drawbacks, such as accident risks, security concerns, proliferation threats, and nuclear waste. The waste problem is perhaps the Achilles\u2019 heel of nuclear energy as it remains radiotoxic for thousands of years [4].\nDiscussions about nuclear waste management must be related to the production of nuclear energy, as the most hazardous waste is produced during energy production. The question guiding this paper is whether spent fuel1 is to be disposed of directly or to be reused in the fuel cycle, referred to as the open and closed fuel cycle, respectively [5]. This issue is still topical after more than four decades of widely deployment of nuclear energy. In an open fuel cycle, uranium is irradiated once and the spent fuel is considered as waste to be disposed of directly. This waste remains radiotoxic for approximately 200,000\u00a0years; the period in which the radiotoxicity of spent fuel will equal that of the amount of natural uranium used to produce the fuel. Radiotoxicity is defined as the biological impact of radioactive nuclides on human health, in case they are digested or inhaled; these effects are indicated in sievert (Sv) or millisieverts (mSv). The closed fuel cycle reuses spent fuel after irradiation to produce energy and diminishes its toxicity and volume substantially. This fuel cycle has many long-term benefits, but it also creates extra short-term risks.\nThe question rises here how to deal with spent fuel in a proper way, taking the needs and interests of this generation and future generations into account. We should not foreclose options for future generations and should manage the waste in a such way that \u201cwill not impose undue burdens on future generations\u201d [6, 7]. In this paper we approach \u201cundue burdens\u201d in the light of fuel cycles and propose intergenerational justice as a framework in order to choose between the fuel cycle: are we willing to transfer all risks of spent fuel to future generations, or do we find it more just to diminish risks and hazards of our waste to the maximum extent and accept, consequently, some additional risks to the present generation. We discuss the idea of having right towards future generation and the concept of intergenerational justice. We further present the two fuel cycles and identify the associated risks with these fuel cycles. In the following chapter, we focus on conflicting values in choosing between them and reduce all trade-offs to a chief trade-off between the present and future generations. The next chapter provides a few underlying assumptions and possible counter-arguments.\nWhether nuclear energy is desirable or indispensable as an energy source in the future is a controversial issue, which is beyond the scope of this paper. At the same time, applying nuclear energy through different fuel cycles raises a number of ethical concerns and moral dilemmas; on those issues we focus here. Moreover, the existing spent fuel all around the world is an urgent problem that needs to be dealt with. 280,000\u00a0tons of spent fuel had been discharged globally by the end of 2004, of which one-third has been recycled, leaving 190,000\u00a0tons of spent fuel stored; the growth rate is estimated on 10,500\u00a0tons a year [8, 9]. The choice between the open and closed fuel cycle has significant influence on this growth. These intergenerational discussions are also crucial for the future of research investments on waste management issues. Partitioning and transmutation (P&T) is a new technology for further diminishing the waste radiotoxicity. P&T is still in its infancy and needs serious investments to be further developed [10, 11]; these investment are justified if and only if one chooses the closed fuel cycle, of which the P&T could be considered as an extension.\nFuture Rights, Present Obligations: Intergenerational Justice\nIncreasing concerns about depleting the Earth\u2019s resources and damaging the environment have invoked a new debate on justice across generations or intergenerational justice. This concept of justice was first introduced by John Rawls in 1971 as intergenerational distributive justice, which stands for an equal allocation of social benefits and burdens [12]. Justice for future implies that today\u2019s people have obligations towards their descendants [13, 14] and these obligations entail certain rights for the future [15\u201317]. These assumed rights have been challenged by some philosophers: \u201c\u2026the ascription of rights is probably to be made to actual persons\u2014not possible persons\u201d [18] and non-existing future people cannot be said to have rights, as our action and inaction define their composition and identity [19]; this is referred to as the Derek Parfit\u2019s \u2018non-identity-problem\u2019. Other objections against these alleged rights are expressed as the inability to predict future properly, the ignorance of the need and desire for future as well as the contingent nature of future. There have been a variety of arguments provided in the literature to these objections2 [20\u201323]: William Grey has proposed \u201cimpersonal principles subject to retroactive person-affecting constraints\u201d [24] and Wilfred Beckerman has argued that we should provide future people with the minimum opportunity for a \u201cdecent and civilised society\u201d [25].\nAlthough these fundamental discussions about right and obligation towards future people are very relevant, in this paper we will focus on the application of these assumed future rights to environmental policy and more specifically nuclear waste. In the last decades the climate change has given rise to serious concerns for the future [26, 27]. Do we have a duty to future generations [21, chap. 5] and if so what does this duty entail [28] and how should we realize it [29]?\nAnticipating technological progress in a rapidly developing world and being concerned about future generations, the World Commission of Environment and Development introduced the concept of sustainable development in 1987. This moment designates the introduction of intergenerational concerns in environmental policy. This Brundtland definition\u2014named after commission\u2019s chairperson\u2014states that the key to sustainable development is an equitable sharing of benefits and burdens between generations \u201c[\u2026] that meets the needs of the present without compromising the ability of future generations to meet their own needs\u201d [30]. The United Nations Conference on Environment and Development in Rio de Janeiro in 1992 (Earth Summit) not only endorsed this concept of sustainable development formally among 178 national governments, it also explicitly included the concept of equity in its principles [31, Principle 3].\nThe sustainability principle implies that there is a conflict of interest between the present and future generations. In an anthology edited by Andrew Dobson, the concept of sustainable development is evaluated in the light of intergenerational justice [32]. Wilfred Beckerman believes that the problems future people encounter have existed for millennia and states that our main obligation towards future people is \u201cmoving towards just institution and a \u2018decent\u2019 society\u201d, which encompasses future generations as well [33, p. 91]. Brian Barry investigates whether sustainability is a \u201cnecessary or a sufficient condition of intergenerational distributive justice\u201d. Barry emphasizes the obligations we have towards future generations and says that \u201cmeasures intended to improve the prospects of future generations [\u2026] do not represent optional benevolence on our part but are demanded by elementary considerations of justice\u201d [34, 35]3. Bryan Norton perceives of sustainability as \u201can obligation not to diminish the opportunity of future generations to achieve well-being at least equal to their predecessors.\u201d He further presents a model in order to compare well-being across time [36]. The \u201ccontested meaning of sustainability\u201d in technology is comprehensively discussed by Aidan Davison [37].\nWhat does the forgoing discussion about rights and obligations entail for nuclear fuel cycles, considering the fact that spent fuel life-time concerns a period between 1,000 and 200,000\u00a0years? The Nuclear Energy Agency (NEA4) introduces sustainability in one of its studies [11]. In this paper we adapt this definition both conceptually and practically and introduce intergenerational justice as a framework to choose between the fuel cycles. Intergenerational concerns have already been expressed about nuclear waste [38\u201340], but mainly with respect to the choice for final disposal of long living radioactive waste.\nNuclear Fuel Cycles: Open and Close\nThe characteristic difference in the fuel cycles is how spent fuel is dealt with after irradiation. Two main approaches to spent fuel outline the main dissimilarity between these cycles: (1) the direct isolation of the material from the environment for a long period of time in which it remains radiotoxic and (2) \u2018destroying\u2019 or converting the very long-lived radionuclides to shorter lived material [5]. The first approach represents the open fuel cycle in the production of energy. The closed fuel cycle is in accordance with the second approach. Here below we will elaborate on these two fuel cycles.\nOpen Fuel Cycle (OFC): Once-through Option\nIn the OFC, the lesser isotope of uranium (235U) is fissioned\u2014split\u2014in light water reactors (LWR) to produce energy; 90% of all operative nuclear reactors to produce energy are LWRs. Natural uranium contains two main isotopes, which constitute 235U and 238U. Only the first isotope (235U) is fissile and is used in LWRs as fuel, but it only constitutes 0.7% of natural uranium. This low concentration is not sufficient in nuclear reactors, the concentration of 235U is therefore deliberately enhanced to a minimum of 3% through a process called uranium enrichment [4].\nIrradiating uranium produces other materials, including plutonium (239Pu), which is a very long-lived radioactive isotope. Apart from plutonium-239, other fissile and non-fissile plutonium isotopes as well as minor actinides will be formed during irradiation. Actinides are elements with similar chemical properties: uranium and plutonium are the major constituents in spent fuel and are called major actinides; neptunium (Np), americium (Am), and curium (Cm) are produced in much smaller quantities and are called minor actinides. The presence of actinides in spent fuel defines the radiotoxicity and waste life-time. The OFC is also called the once-through strategy, as the spent fuel does not undergo any further treatment.\nThe spent nuclear fuel in an OFC will be disposed of underground for 200,000\u00a0years. This waste life-time in an OFC is dominated by plutonium. Neither minor actinides nor fission products have a significant influence on long-term radiotoxicity of waste in an OFC. Figure\u00a01 illustrates these radiotoxicities. The dashed line represents spent fuel in an OFC, decaying to the ore level in approximately 200,000\u00a0years. Fission products are a mixture of various radionuclides that will decay to the uranium ore level after approximately 300\u00a0years [41], indicated by the dotted line in Fig.\u00a01.\nFig.\u00a01Radiotoxicity of spent fuel, vitrified waste (HLW) and fission products, compared with regard to the radiotoxicity of uranium ore needed to manufacture the fuel\nClosed Fuel Cycle: Recycling Plutonium and Uranium\nAs stated above, less than 1% of the uranium ore consists of the fissile isotope 235U. The major isotope of uranium (238U) is non-fissile and needs to be converted to a fissile material for energy production: plutonium (239Pu). Spent fuel could undergo a chemical treatment to separate fissionable elements including Pu, this is referred to as reprocessing. During reprocessing, uranium and plutonium in the spent fuel are isolated and recovered. Recycled uranium could either be added to the front-end of the fuel cycle or used to produce mixed oxide fuel (MOX), a mixture of uranium-oxide and plutonium-oxide that can be applied in nuclear reactors as a fuel [42] (see Fig.\u00a02). Reprocessing is also called the \u201cwashing machine\u201d for nuclear fuel. The irradiated fuel is \u201cwashed and cleaned\u201d and \u201cclean\u201d materials (U\u00a0+\u00a0Pu) are reinserted into the fuel cycle to produce more energy, while the \u201cdirt\u201d is left behind (fission products and minor actinides) to be disposed of as high level waste (HLW) [4]. HLW contains fission products and minor actinides and will be put into a glass matrix in order to immobilize it and make it suitable for transportation, storage and disposal. This process is called conditioning of waste and results in so-called vitrified waste [6]. The ultimate radiotoxicity of vitrified waste will decrease to the uranium level in approximately 5,000\u00a0years [41], as illustrated by the dashed\u2013dotted line in Fig.\u00a01.\nFig.\u00a02An overview of the open and closed nuclear fuel cycle; the solid lines represent the OFC, the dashed lines the CFC\nAs uranium and plutonium are separated and reused, this fuel cycle is called the closed fuel cycle. The choice for a CFC is rightly associated with the choice to recycle spent fuel. Figure\u00a02 illustrates various steps in both nuclear fuel cycles and their different interpretations of spent fuel. As can be seen in Fig.\u00a02, the solid line representing the OFC is a once-through line. The CFC on the contrary is illustrated by separating plutonium and uranium and returning them to the fuel cycle, represented by the dashed lines. Nowadays, the main objective of reprocessing is to use uranium more efficiently and to reduce the waste volume and its toxicity considerably.\nIn the CFC, one can distinguish between two options with respect to nuclear reactors. In the first option, conventional LWRs are used, which are capable of using MOX as fuel. Reprocessed spent fuel is returned to the fuel cycle as MOX. Spent MOX fuel could again be reprocessed to separate uranium and plutonium. Further recycling of plutonium is only possible in another type of reactor capable of handling non-fissile plutonium: fast reactors, which constitute the second option. In the second option, the latter are basically used as energy producing reactors, in which MOX is the fuel. Due to the fast neutrons, fast reactors are capable of using the major isotope of uranium (238U) to the maximum extent via conversion to 239Pu [43].\nThe Future of the Closed Fuel Cycle; Maximal Recycling\nAs spent fuel is conceived of as the Achilles\u2019 heel of nuclear energy, there have been serious attempts to further reduce its radiotoxicity and volume. A new method is partitioning and transmutation (P&T), which could be considered as a recent supplementary method to reprocessing. Spent fuel comprises uranium and plutonium, minor actinides and fission products. Uranium and plutonium are separated during reprocessing in order to reuse; P&T focuses on \u201cdestroying\u201d minor actinides in spent fuel. If completely successful, P&T is expected to reduce the volume and radiotoxicity of spent fuel one hundred times (compared to OFC). After P&T, fuel radiotoxicity would decay to a non-hazardous level in 500 to 1,000\u00a0years [10]. The waste stream would then only consist of relatively short-lived fission products and curium isotopes. The latter will dominate the waste life-time and are considered to be too hazardous to be recycled at reasonable expenses and risks. P&T is merely available at the laboratory level at the moment; a considerable amount of R&D efforts is needed, before P&T could be utilized industrially [10, 11].\nWaste Management, Interim Storage, Long-term Storage and Repositories\nIrrespective of the fuel cycle choice, the remaining waste in a nuclear reactor after the (optional) treatments needs to be disposed. In waste management, a distinction is made between storage and disposal: storage means keeping the waste in engineered facilities aboveground or at some ten of meters depth underground, while disposal is the isolation and emplacement of the waste at significant depth (a few hundreds of meters) underground in engineered facilities, called \u2018geological repositories\u2019.\nUntil now, all the available storage facilities for spent fuel and high level waste have typically been above ground or at very shallow depth. Spent fuel is mostly stored under water for at least 3\u20135\u00a0years after removal from the reactor core; this stage is called interim storage. Water serves as radiation shielding and cooling fluid [39]. Bunn argued that interim storage for a period of 30\u201350\u00a0years has become an implicit consensus, as the world\u2019s reprocessing capacity is much less than globally spent fuel generation. In addition, there are no final repositories at our disposal yet. Interim storage of waste is also a crucial element in the safe management of radiotoxic waste since waste should be stored to allow radioactive decay to reduce the level of radiation and heat generation before final disposal. For the countries that favor reprocessing, spent fuel remains available for some decades to be reprocessed and there is no need to build up vast stockpiles of separated plutonium after reprocessing. For countries supporting direct disposal of spent fuel, interim storage allows more time to analyze and develop geological repositories appropriately [44].\nA commonly proposed alternative to geological disposal is the long term monitored storage on the surface. Spent fuel remains in this case retrievable in the future. However, the technical community appears largely to disregard this option and considers the surface storage only as an interim measure until the waste can be disposed of in geological repositories [5, 38, 39, 45]. Deep oceans and outer space are mentioned as possible locations for final disposal as well, but there are substantial political, ethical as well as technical impediments, mainly related to the safety of these locations [5].\nRisks and Associated Values\nIn this paper we distinguish moral values at play in the production of nuclear energy. Values are what one tries to achieve and strives for, as we consider them valuable; moral values refer to a good life and a good society. However, we should not confuse them with people\u2019s personal interest; moral values are general convictions and beliefs that people consider as worth striving for, in public interest [46]. We further identify dilemmas and moral problems rising from conflicting values: some trade-offs need to be made in order to choose a fuel cycle. The three main values we distinguished are as follows: sustainability, public health and safety and security. In the following sections we try to specify these values and, for the sake of comparison, relate them to risks and benefits of the open and closed fuel cycle.\nWe here distinguish between short-term and long-term effects, in which we consider the upcoming 50\u00a0years as short-term and after that as long-term. This period is chosen in view of comparisons in the literature between the fuel cycles: strong views about maintaining the OFC are mainly about the coming five decades [47] and in economic comparisons, short-term is defined as 50\u00a0years [48], probably based on estimations of reasonably assured uranium sources for the coming five to six decades in 2002 [49]. To conclude, 50\u00a0years is the period in which supply certainty of the OFC is assured. However, as will be shown later on, this period can be extended to 85\u00a0years or more without invalidating the arguments and conclusions of this paper.\nSustainability: Supply Certainty, Environmental Friendliness and Cost Affordability\nA comparative study of the Nuclear Energy Agency (NEA) on various P&T technologies introduces the following three axes in order to assess sustainability: (1) resource efficiency (2) environmental friendliness and (3) cost effectiveness [11]. In this paper we take these axes as a guideline for understanding sustainability with respect to nuclear energy and follow an adapted version in terms of concepts and terminology, with regard to the fuel cycles.\nSupply Certainty\nOn the first axis, sustainability refers to the continued availability of uranium: NEA uses the term resource efficiency for this. In this paper we apply the term supply certainty instead. Deploying resources efficiently means that we aspire to use as less as possible resources for the same purpose, while supply certainty refers to availability of resources in order to fulfill the needs. In energy discussion, certainty is a more significant concept than efficiency. Although this difference in designation has no consequences for the factual comparison in availability of uranium, we prefer the conceptually correct term.\nAs there are 50\u201360\u00a0years of reasonably assured uranium resources [49], there will be no significant short-term influences of the fuel cycle on the supply certainty. Later estimations of the NEA and the IAEA5 present approximately 85\u00a0years of reasonably assured resources (RAR) uranium are available for a once-through option in a LWR. These institutions estimate that this amount suffices for 2,500\u00a0years in a CFC, based on a pure fast reactor cycle, which is an improvement in supply certainty with a factor 30 [50]. Two later reports of the IAEA in 2006 adjust this period to 5,000\u20136,000\u00a0years, assuming that fast breeders allow essentially all non-fissile 238U to be bred to 239Pu in order to be used as fuel [8, 51]. It needs to be mentioned that these estimations are made under the explicit assumptions that fast breeders will be broadly deployed in the future.\nThe supply certainty benefits of the CFC will be relevant in the long run. Although there are no short-term significant differences between the fuel cycles, countries without natural fossil fuel, like Japan and France, tend to opt for reprocessing and recycling [52].\nEnvironmental Friendliness: Radiological Risks to the Environment\nThe second axis of the OECD approach in specifying sustainability concerns environmental friendliness. This value depends on the accompanying radiological risks to the environment. Radiological risks, as we perceive them in this paper, express the possibility or rather probability that spent fuel leaks to the biosphere and can harm both people and the environment.\nThe NEA proposes three stages to assess radiological risks: (1) mining and milling, (2) power production, and (3) reprocessing. They compare the radiological risks of the OFC with the (once) recycled and reused MOX fuel. In the power production phase, NEA argues, there is no difference between the cycles. The main difference lies in the two other steps: mining and milling and reprocessing. They further argue that deployment of reprocessing decreases the need for enriched uranium and, therefore, natural uranium, of which the mining and milling involve the same radiological risks as reprocessing and reusing plutonium as MOX fuel. In fact, NEA argues that under the described circumstances there are equal radiological risks for both fuel cycles [53]. This argument is probably sound in the long run, for large scale reprocessing enterprises and under ideal circumstances, but one can wonder whether the factual short-term consequences are such that radiological risks of both fuel cycles are quite similar. The question remains whether we should take comparisons under ideal circumstances or factual consequences into consideration (in moral discussions). Furthermore, NEA completely neglects the distribution of benefits and burdens: building a reprocessing plant in France will increase local risks to the surrounding area and will diminish the burdens in a uranium-exporting country, such as Canada.\nNEA further neglects the risks and hazards associated with the transport of waste in case of reprocessing: \u201c\u2026[R]adiological impacts of transportation are small compared to the total impact and to the dominant stage of the fuel cycle\u201d [53]. If we consider different aspects of public perception of risk, we cannot retain the idea that radiological risks of nuclear waste transportation are negligibly small [54]. Only a few reprocessing plants are currently available around the world and spent fuel needs to be transported to those plants and back to the country of origin. In Great-Britain, for instance, a serious debate is currently taking place about the possibilities to return Japanese reprocessed spent fuel to Japan.\nOne of the serious counterarguments against reprocessing is the large investments needed to build the plants; small countries with a few nuclear power plants and in favor of the CFC will probably not build a reprocessing plant and will keep transporting spent fuel to those countries capable of this technology. To illustrate, The Netherlands is one of the countries with favorable reprocessing policy: Dutch spent fuel is currently transported to La Hague (France). There is no real chance that The Netherlands will build its own reprocessing plant in the coming years. To conclude, we assume that reprocessing will result in more short-term radiological risks, both to the environment and to the public health and safety, as illustrated in Fig.\u00a03.\nFig.\u00a03Ethical values (first row) and their specification (second row) related to the OFC and CFC. A plus sign represent an improvement of the ethical value and has a positive connotation, a minus sign is a drawback of the value\nThe short-term and long-term effects mentioned above also pertain to environmental friendliness. Using the fuel to the maximum extent and maximally recycling the spent fuel could be considered as long-term \u2018environmentally friendly\u2019, as the environment is less exposed to potential radiological risks and radiotoxicity in the long run. One of the main arguments in favor of reprocessing\u2014along with enhanced resource efficiency\u2014is the vast reduction of waste volume and its toxicity and the accompanying advantages from a sustainability point of view. The volume of each ton of spent fuel containing approximately 1.5\u00a0m3 of HLW could be reduced through reprocessing three times [55]. The waste toxicity will decrease at least with a factor three [52].\nAffordability\nThe third axis the NEA proposes in its comparison is cost effectiveness. We adapt this axis here into affordability. We acknowledge the relevance of economic aspects for initiation and continuing a technological activity. Sustainability can be conceived of as durability, to that purpose. However, economic effectiveness goes much further than the question whether an activity is reasonably durable or affordable. Social security is, for instance, mostly ineffective economically but we consider that as a duty of the state with respect to its citizens; nevertheless, it is supposed to be neither economically effective nor profitable.\nIt is also arguable whether durability should be accepted as sustainability. This is an ongoing debate about different interpretations of the notion of sustainability. In a moral discussion, it is probably more just to separate economic considerations from other aspect of sustainability. However, for the sake of our analysis we follow here NEA\u2019s analysis and accept sustainability conceived as durability.\nIn 1994, a NEA study determined a slight cost difference between the reprocessing option and direct disposal. Based on best estimates and the uranium prices of that time, the cost of direct disposal was approximately 10% lower, which was considered to be insignificant, taking the cost uncertainties into account [56]. However, considering later uranium prices and resource estimations, there is a strong economic preference for the once-through strategy, even if a considerable growth of nuclear energy production is anticipated [52]. A MIT study in 2003 on \u2018The Future of Nuclear power\u2019 upholds the same view on economic aspects of reprocessing. Deutch et\u00a0al. conclude in this report that\u2014under certain assumptions and the US conditions\u2014the CFC will be four times as expensive as the OFC. The once-through option could only be competitive to recycling if the uranium prices increase [47]. These MIT researchers are not susceptible to the counterarguments that disposing of reprocessed HLW will be less expensive. They furthermore present a cost model in which reprocessing remains uneconomic, even if the cost of reprocessed HLW were zero [47]. Another international study compares reprocessing with the once-through option and concludes that\u2014even with substantial growth in nuclear power\u2014the open LWR fuel cycle is likely to remain significantly cheaper than recycling in either LWRs (as MOX) or fast breeders for at least the next 50\u00a0years [48].\nIn the previous reasoning we considered reprocessing as a broadly applied technology, which will create the need to build new reprocessing plants. Economic affordability appear totally different if we base our analysis on the existing reprocessing plants, as many small consumers of nuclear energy reprocess their spent fuel in France or Great-Britain. These countries do not have excessive initial expenditures for their CFC.\nPublic Health and Safety: Short-term and Long-term Radiological Risks\nThe second value is public health and safety. We again distinguish between short-term and long-term radiological risks, which cause hazards to public health and safety. Recycling of plutonium as MOX diminishes the eventual radiotoxicity of spent fuel with a factor three, assuming that spent MOX fuel is disposed of after one use (also called once-through recycling6). Theoretically, multiple recycling of plutonium in fast reactors can decrease the long term radiotoxicity of disposed waste by a factor 10. These scientific achievements could be brought into practice in several decades [52].\nRecycling spent fuel includes the separation and storage of plutonium. Along with security arguments which will be discussed later, plutonium contains serious potential risks to the public health due to its exceptional toxic nature. Plutonium needs especial isolation from humans, as it contains long-lived alpha emitters, which are very radiotoxic upon inhalation [57, p. 113)]. We included these risks in the short-term radiological risk for waste treatment. With respect to long-term radiological risks, the same reasoning as for the previously mentioned sustainability holds true: the short-term radiological risks associated with the CFC are significantly higher than the OFC.\nSecurity and Proliferation Hazards\nThe last, but certainly not least value at play in waste management is security as a result of production of plutonium during recycling. Concerns regarding nuclear weapon proliferation are extremely relevant given the current state of world security. Proliferation threats rise either by the use of enriched uranium (up to 70%) or by the production or separation of plutonium. To illustrate, eight kilograms of weapon grade plutonium (239Pu) are sufficient to produce a Nagasaki-type bomb [58].\nProliferation is also a potential hazard in countries capable of enriching uranium. One of the main tasks of the IAEA is to annually report to the United Nation\u2019s Security Council about nuclear energy possessing nations. Although both the OFC and the CFC need enriched uranium in the reactor, the short-term proliferation concerns of the CFC are considerably higher, due to the separation of plutonium during reprocessing.\nThe security concerns are double-edged: reprocessing increases proliferation concerns for the contemporary people, but at the same time it decreases those concerns for future generations, since the spent fuel residuals contain no plutonium any more. One can argue that the potential proliferation concerns of direct disposal of spent fuel in the OFC are negligible compared to the actual security concerns in case of reprocessing: disposed spent fuel cannot be retrieved unnoted, and expensive and inaccessible reprocessing plants are needed to separate plutonium from it for weapon manufacturing. Some scholars argue, on the other hand, that spent fuel in geological repositories becomes a better weapon-grade material as time goes by, due to the natural enrichment of 239Pu [10]. However, this effect will take place in several thousands of years. In sum, the CFC involves more short-term proliferation and security concerns but decreases those concerns in the long run, as illustrated in Fig.\u00a03.\nValue Conflicts in Fuel Cycles and Future Generations\nIn the preceding analysis, we formulated a number of values and aimed to translate risks and benefits of the fuel cycles into these values. In decision-making about the fuel cycles we are confronted with a number of value conflicts. It should be mentioned that the plus and minus signs in Fig.\u00a03 are merely approximations which enable us to make a comparison between the OFC and CFC, these signs are neither quantitative measures nor absolute entities. It should further be mentioned that plusses represent an improvement in terms of the three basic values, illustrated in squares on top of Fig.\u00a03; minuses are drawbacks of these values.\nValue Conflicts\nIn choosing between options, we have to accept certain trade-offs between these basic values. The CFC enhances sustainability in terms of supply certainty and creates less radiological risks to the environment. It also diminishes public health and safety concerns, as well as security concerns in the long run. At the same time, however, the CFC involves more short-term additional risks and, therefore, compromises public health and safety as well as security of contemporary people. It also deteriorates short-term sustainability, perceived as environmental friendliness. Trading off these conflicting values in a certain way can help one choose one of the fuel cycles. To illustrate, if one holds the cleanness of the environment we bequeath to our descendants as most important, she should be willing to accept some additional risks to the public in the present and, therefore, the CFC would appear the obvious choice. Short-term risks are traded off against the long-term benefits in the CFC. Another example: if one considers proliferation threats in the current security state of the world highly unacceptable, she trades off long-term benefits of the CFC against the short-term benefits of the OFC; the latter will be the outcome of this trade-off.\nIn the literature, implicit trade-offs are made. A MIT study in 2003 concluded unambiguously that the once-through fuel cycle is the best choice for the US for at least 50\u00a0years. MIT researchers asserted that the reprocessing plants in Europe, Russia, and Japan involve unwarranted proliferation risks and did not believe that benefits of the CFC outweigh the safety, environmental and security risks as well as economic costs [47]. Von Hippel upheld the same view on reprocessing: proliferation and economic costs of reprocessing are high and the environmental benefits are questionable. He maintained that direct storage of spent fuel after irradiation is cheaper, safer and more environmentally benign than reprocessing [59].\nProliferation of nuclear weapons is one of the main concerns in the discussions about recycling nuclear waste. IAEA director El-Baradei noted in 2004: \u201cWe should consider limitations on the production of new nuclear material through enrichment and reprocessing, possibly by agreeing to restrict these operations to being exclusively under multinational control\u201d [60]. Proliferation concerns with respect to reprocessing are the main reason why many countries prefer the OFC. The US, Sweden, Finland, and Canada have chosen the OFC to avoid plutonium separation. But unlike these countries, reprocessing occurs in many European countries such as Great-Britain and France as well as smaller nuclear energy consumers like The Netherlands, that reprocesses its nuclear waste in the French plants in La Hague. There are serious attempts to make reprocessing proliferation-resistant, including the US global nuclear energy partnership (GNEP) and the Russian Federation\u2019s global nuclear power infrastructure initiatives [8].\nIntergenerational Justice and Nuclear Waste Management\nOne of the key principles of radioactive waste management laid down by the IAEA in 1995 is that it should be managed in such a way that it \u201cwill not impose undue burdens on future generations\u201d [6]. This principle is founded on ethical consideration that the generation enjoying the benefits of an undertaking should manage the resulting waste. The NEA supported this definition in the same year in a collective opinion [7].\nAs illustrated, the CFC mostly has long-term benefits and compromises public health and safety as well as security of the contemporary people. Does the aim to avoid \u201cundue burdens on future generations\u201d mean that we are supposed to diminish waste radiotoxicity and its volume as much as possible? To what extent should we accept the increased risks and hazards to the present generation in order to accomplish the latter?\nThe questions how to interpret the \u201cundue burden\u201d can best be understood within the framework of intergenerational justice. Especially in fundamental policy decision-making, the question rises how one generation could equitably take the interest of future generations into account. Serious discussions about this issue started in the US [61] and are still ongoing in nuclear communities in choosing between options for final disposal of waste [38, 39]. Some scholars interpreted the NEA collective opinion in 1995 as a confirmation for the\u2014once and for all\u2014sealed underground repositories. Uncertainty in long-term safety and possible future needs to recover plutonium (from spent fuel) for its potential energy value are two serious objections to permanently closed repositories [62]; we are after all required not to deprive future generations of any significant option [6, 7]. Inequity of risks and benefits across generations are two other reasons opposing permanent disposal [63]. In other words, scholars argue that permanent disposal forecloses options to future generations to retrieve and reverse waste. Alternatives to permanent disposal are long-term continued surface storage or phased repositories, which remain open for an extended period of time. There seems to be consensus among nuclear experts that disposal in repositories should be given preference above surface storage, as it is believed to be a passively safe solution that does not require burden of care by future generations [38].\nIn a recent European study, Schneider et\u00a0al. argue that the main concerns in risk governance are the transfer of a whole waste management system, including a safety heritage, from the present to the future generations [64]. They approach various technical and societal issues, such as long-term responsibility, justice and democracy from the perspective of generations, both across generations and within one generation.\nIn this paper we propose to reduce the trade-offs in choosing the fuel cycle to a chief trade-off between the present and the future. Is it legitimate and just to transfer all the risks and hazards of nuclear waste to future generations? How can we arrange an equitable transfer of the whole waste management system\u2014as argued by Schneider et\u00a0al.\u2014to the future? Or is it more just and equitable to handle our waste as much as possible, in order to diminish its risks in the far future?\nThe OFC is to be associated with short-term benefits and the CFC primarily has long-term benefits7. In this reasoning, accepting the CFC means that we intend to diminish the risks and hazards to the future and accept some additional risks for the present generation. The OFC transfers the risks as much as possible to the future and avoid those risks in the present.\nUnderlying Assumptions and Possible Counter-arguments\nSo far we have argued that decision-making on the fuel cycles could best take place within the framework of intergenerational justice. This conclusion is based on the analysis in the foregoing chapter, in which we illustrated the choice between the OFC and the CFC mainly as a choice between the present and future generations. Obviously, there are a few assumptions at the basis of this analysis. Below, we will discuss some of these underlying assumptions and provide some possible counter-arguments and evaluate their validity.\nDefining Short-term as 50\u00a0years\nIn our analysis, we defined short-term as 50\u00a0years. Beyond half a century we considered as long-term. The question that rises here is whether 50\u00a0years constitute the real turning point in comparing the specified values, as we introduced in Fig.\u00a03. And more importantly, will other distinctions in time spans between short and long-term change our conclusion? As we mentioned earlier, the period of 50\u00a0years was taken from the comparisons we found in the literature. Most scholars preferring the OFC, pronounce their strong opinion for the coming five decades and economic comparisons are made for this period of time [47, 48]. Both mentioned studies based their strong opinion on estimations of reasonably assured uranium resources at the beginning of this century; NEA and IAEA considered this amount in 2001 enough for 50\u201360\u00a0years [49]. This period is, however, extended to 85\u00a0years in the 2005 estimations (of IAEA and NEA). It needs to be mentioned that the bulk of this increase is not due to discovering more resources, but it is a result of re-evaluation of previous resources in the light of the effects of higher uranium prices [50].\nLooking at the first columns in Fig.\u00a03 (supply certainty), the long-term benefits of the CFC will not change if we take 85\u00a0years as a turning point, the long-term benefits of supply certainty in the CFC will come into practice after this period. We should mention here that we founded our analysis on the identified resources. The total undiscovered resources of uranium are expected to be significantly higher [50]. If we base our analysis on the latter, the long-term benefits of the CFC will probably vanish entirely, even for a much longer period of time. However, an analysis based on undiscovered resources comprises such an amount of uncertainty that estimations are practically meaningless.\nWhether the column affordability will change, if we consider short-term as being 85\u00a0years, is not clear. We can state that high initial investments for the reprocessing plant might perhaps be affordable, if we consider a longer period of time. However, there have been no serious estimations based on the announced reasonably assured uranium resources in 2005.\nAll Released Pu will Eventually be \u2018Destroyed\u2019\nBeneficial long-term radiological risks of the CFC are based on the assumption that all plutonium is separated from spent fuel and \u201cdestroyed\u201d. As plutonium is the dominant element in indicating the waste life-time in spent fuel, its extraction from waste will diminishes waste radiotoxicity substantially. The mentioned period of radiotoxicity of vitrified waste after reprocessing of 5,000\u00a0years [41], includes the assumption of complete consumption of plutonium after separation. Less long-term proliferation hazards in the CFC are also based on the same assumption: extracted plutonium is ultimately fissioned. How realistic is this assumption if we consider the millions of kilograms weapon-grade plutonium and highly enriches uranium (to above 70%) discharged as a result of dismantlement of warheads after the Cold War?\nThese released materials could also either be considered as waste to be disposed of directly or as potential fuel for the production of energy. These different points of view mark the divergent approaches between the two superpowers in the Cold War. Americans believe that excess plutonium has no economic value, as it costs more to use as energy source than the energy is worth. However, since the other option of dealing with this hazardous material, i.e. its disposal, is costly as well, some plutonium is supposed to be used as reactor fuel (MOX), but only in a once-through scenario. This is perfectly in line with the American concerns about (civilian) plutonium which is not recycled and reused either. Russians hold a totally different view on this issue: they consider excess weapon plutonium as fuel having \u201csignificant energy potentials\u201d. Russia also acts in accordance with their CFC perspectives. However, they believe\u2014together with Americans\u2014that the potential value of these plutonium stockpiles cannot be cashed in the near future, as it needs substantial additional costs [58].\nPlutonium has already proven its benefit in the production of energy. Reprocessed plutonium from civil reactors is called civilian plutonium, a name that could mistakenly be understood as unfeasible weapon material (although it is very unfavorable as a weapon material). As reprocessing of plutonium has outpaced its use as fuel and due to technical and regulatory restrictions, no more than 30% of produced MOX could be fissioned in a reactor, which creates an imbalance between separated civil plutonium and reused MOX; in the beginning of this century an estimated amount of 200\u00a0tons of civilian plutonium was available in the stockpiles [52]. This amount is vastly growing and is believed to surpass the total amount of released weapon plutonium soon. Referring to the theft concern and concerns on excessive surpluses of plutonium, mainly in former Soviet Union countries, Bunn et\u00a0al. argue for an international phased-in moratorium on reprocessing [58, 65].\nIrrespective of Bunn\u2019s reasoning\u2019s validity regarding nuclear theft, we can easily state that separated plutonium for the purpose of reprocessing contains more proliferation concerns than plutonium \u2018embedded\u2019 in spent fuel. The latter needs advanced and very expensive technology to separate plutonium, which is not accessible outside the legal authorized and controlled way of the IAEA, which supports the argument that separated plutonium involves more security and proliferation concerns.\nA similar reasoning holds true for the toxic properties of plutonium. If we extract plutonium from spent fuel, under the assumption that it will eventually be fissioned and, consequently, prevent it of being disposed of underground, we create de facto more risks for the contemporary people. These risks were already included as more short-term radiological risks in Fig.\u00a03. However, if we fail to make it plausible that extracted plutonium will eventually be fissioned in reactors as MOX, we merely create more risks\u2014both short-term and long-term\u2014and that will substantially change our analysis. Considering the fact that one-third of separated plutonium is currently fissioned through reprocessing, the long-term benefits of the CFC will merely be meaningful under the assumption that MOX consumption will substantially expand.\nThe latter is possible under two scenarios: (1) broader deployment of MOX fuel and (2) less reprocessing, as produced MOX could first be consumed. Less deployment of reprocessing conflicts with the initial assumption. We were trying to give underpinnings for long-term benefits of the CFC, of which reprocessing is a crucial component. That leaves the first scenario open: less long-term risks of the CFC are plausible if and only if we take a wider deployment of MOX fuel for granted, either as a result of adapting existing reactors or due to a broader application of MOX in the planned reactors or reactors being built. According to the World Nuclear Association, there are 28 new reactors being built and 64 are ordered or planned worldwide. Furthermore, there are 158 reactors proposed and waiting for funding or approval [2]. These developments can give support to the long-term benefits of the CFC. Still, the protagonists need to make plausible that the stockpiles of civilian plutonium extracted through reprocessing will eventually be fissioned.\nHow Long does the \u2018Long-term\u2019 Last in Case of Radiological Risks?\nLet\u2019s go back to the first assumption discussed with respect to defined time spans in order to distinguish between the short-term and long-term. So far, we argued that the CFC has less long-term radiological risks, assuming that separated plutonium in reprocessing will eventually be fissioned. However, these radiological benefits will be noticeable only after 5,000\u00a0years, which represents the waste life-time of reprocessed waste (vitrified waste). After 50\u00a0years the CFC creates more additional risks to both public and the environment (at that moment), the more so since reprocessing will be an ongoing business in the CFC.\nThe question raises here whether this challenges our analysis. The trade-offs needs still to be made between the short-term and long-term radiological risks. The CFC is rightly associated with less long-term risks: perceived from now or after 50\u00a0years, there will be less long-term risks in remote future. The analysis is still valid, but these long-term benefits will reveal after a much longer period of time than the proposed 50\u00a0years for supply certainty. To sum up, 50\u00a0years is not applicable to all comparisons, but the line of analysis will not change as a consequence.\nThe CFC and the Transition Period\nLet\u2019s now take a look at the argument of nuclear energy being used in a transition period between conventional fuel resources (fossil fuel) and sustainable energy, from the perspective of the CFC. As we stated earlier, based on the 2004 nuclear energy consumption, the uranium resources are available for a period of approximately 85\u00a0years for a once-through option in a LWR [50]. There is also no economic reason for deployment of the CFC in the upcoming 50\u00a0years, as it remains uneconomic for this \u201cshort\u201d period of time and the high initial investments cannot be recovered, even if a considerable growth of nuclear energy is anticipated [47, 48, 52]. So far we argued that the benefits of the CFC will be revealed in the long run only, certainly in no less than 50\u00a0years. If this time exceeds the transition period, should those who believe in nuclear energy to bridge the transition period, be consequentially in favor of the OFC?\nThis transition period is not accurately defined in the literature; it concerns the transition of fossil fuel to sustainable energy sources. Nuclear energy is believed to play a significant part into this transition until 2020, due to its assured supply certainty and low emissions [66]. Which role nuclear energy will play after this period depends on developments in tackling safety, waste and proliferation issues. Most advocates of the transition-period argument do not exclude nuclear energy: they believe that nuclear energy is capable of being sustainable in the future, if the afore-mentioned concerns are being taken care of [66].\nIf we agree that the CFC is\u2014under some assumptions\u2014more environmentally benign in the long run and if the latter is the outcome of our trade-offs, we can argue that we should use the CFC for the transition period, no matter how short or how long this period is. The long-term burdens as a result of nuclear energy deployment will be there anyway, the CFC enables one to diminish those burdens to some extent. There are also no technical restrictions to deployment of the CFC in short periods of time, except the time needed to build a reprocessing plant. However, the argument we presented with respect to actually destroying plutonium holds stronger if one is in favor of applying nuclear energy to bridge a transition period: within that same period, all plutonium should then be destroyed.\nChoosing Between OFC and CFC. Is not that a False Dilemma?\nIn our analysis we presented two different methods in the production of nuclear energy. Prior to our analysis, we stated that the questions with respect to desirability of nuclear energy will be beyond the scope of our paper. We also listed the state-of-the-art in the production of nuclear energy, being responsible for 16% of world\u2019s energy production, and focused on existing moral conflicts. Under these assumptions, there are two methods to produce nuclear energy, namely the OFC and the CFC.\nThe question raises here whether there will be a third fundamentally different option, or in other words, whether the choice between the OFC and the CFC is a false dilemma? Future developments of nuclear energy mainly concern effort to reduce radiotoxicity of waste, such as the P&T presented in this paper. These options are to be considered as an extended CFC and are not essentially different. We still need to deal with the trade-offs as we described in this paper.\nOne can further argue that the framework of intergenerational justice can give rise to unacceptable risks in both scenarios. In other words, the intergenerational justice framework refutes both nuclear fuel cycles. Such reasoning challenge the assumptions we made with regard to nuclear energy rather than our analysis based on those assumptions.\nWhy do not we Talk about Justice Among Contemporaries?\nIn the preceding chapters we argued that the choice for a fuel cycle should be made within the framework of intergenerational justice. In other words, we should (also) take the needs and interest of future generations into consideration and make a trade-offs between the latter and the interest of contemporary people, in order to make a decision on the fuel cycle. The question rises here: is that a sufficient condition? Especially when we consider that the majority of nuclear plants is located in developed countries, while more than 30% of the world\u2019s uranium production is coming from developing countries [50]. Kazakhstan, Uzbekistan, Namibia, and Niger that are bearing the burdens of the front-end of the fuel cycle (i.e. milling, mining, etc.) do not have a power plant at all and will not be able to share the benefits of nuclear energy? Is not this a relevant question, perceived from distributive justice?\nThe authors fully acknowledge the relevance of evaluating justice among contemporaries in this discussion, which is referred to in the literature as intragenerational justice. However, intragenerational considerations are not decisive in the choice for the fuel cycle, they rather follow from the choice one makes. To illustrate, when a country decides to deploy the CFC, the question rises where the country is going to reprocess its waste; is it just that Dutch waste\u2014for instance\u2014goes to La Hague in France to be reprocessed? These intergenerational justice considerations are also relevant within a country: is it just that the Nevadans bear the burden of the whole American waste which probably will be disposed off under the Yucca Mountains in Nevada. Similar considerations are to be made in case of locating a nuclear power plant: people in the direct vicinity bear the burdens, while the whole nation enjoys the benefits.\nAs we briefly showed here, intragenerational considerations rather challenge the assumption we made in the beginning of this paper with regard to the deployment of nuclear energy, than to help us to make a choice between the fuel cycles. Intergenerational justice, however, offers a suitable framework for choosing the fuel cycle. Once this choice is made, intragenerational concerns are born.\nConclusions\nIn this paper we evaluate NEA\u2019s definition for sustainability [11] and adapt that definition both conceptually and practically: it is questionable\u2014from a moral standpoint\u2014whether sustainability can be related to economic issues and it is more correct to use economic affordability instead of cost effectiveness. We further argue that though sustainability\u2014as defined by NEA and adapted here\u2014is a crucial aspect in this discussion, it does not offer a proper basis to choose a fuel cycle: public health and safety as well as security concerns are at least as important to be included. By adding a time dimension to this comparison, we propose a new framework in order to choose the nuclear fuel cycle\u2014intergenerational justice\u2014and specify consequences of both fuel cycles within this new framework. To that purpose, we identify values at play and value conflicts one encounters in choosing between the fuel cycles: the CFC improves sustainability in terms of the availability of fuel and involves less radiological risks to the public and the environment in the long run, but it compromises public health and safety in the present. The CFC also poses serious security threats for the contemporary people, due to the production and the separation of plutonium. However, at the same time it diminishes those threats for future generations.\nThese trade-offs in nuclear energy are reducible to a chief trade-off between the present and the future. To what extent should we recycle our produced nuclear waste in order to avoid \u201cundue burdens\u201d on the future and to what extent should we accept additional risks for the present generation? These questions can be answered within the proposed framework of intergenerational justice. This concept of justice is often used in the nuclear discussions, mainly to tackle issues with respect to final waste disposal, waste retrievability in the future and, more recently, risk governance with regard to the question how we can equitably transfer a whole waste management system to the future.\nIn our analysis we used lots of estimations with regard to uranium resources, waste radiotoxicity and the radiological risks of the waste. How valid are these estimations if we include the uncertainties encompassing our analysis? Estimations and predictions are the key problems in dealing with the future, especially when we talk about the remote future. These uncertainties need to be further investigated in future studies in order to test the validity of provided analysis. It is also recommendable to quantify the probabilities of these risks in order to compare them in a more appropriate way. Do the decreased risks to the public and the environment in the remote future equal the increased risks to the present generation?\nIn this paper, we approach the choice between the fuel cycles perceived form the perspective of intergenerational justice. Advocates of he OFC should argue why they are willing to transfer all the risks for a very long period of time (200,000\u00a0years) to future generations and accept all the accompanying uncertainties for their descendants. Supporters of the CFC should underpin their acceptance of additional risks to the present generation. More importantly, they should make it plausible that separated plutonium during reprocessing is eventually \u201cdestroyed\u201d. Proliferation remains the leitmotiv in these discussions, as it is the main objection against the CFC.","keyphrases":["recycling","intergenerational justice","value conflicts","sustainability","future generations","nuclear waste management","reprocessing"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"Arch_Dermatol_Res-3-1-1800369","title":"Serologic and immunohistochemical prognostic biomarkers of cutaneous malignancies\n","text":"Biomarkers are important tools in clinical diagnosis and prognostic classification of various cutaneous malignancies. Besides clinical and histopathological aspects (e.g. anatomic site and type of the primary tumour, tumour size and invasion depth, ulceration, vascular invasion), an increasing variety of molecular markers have been identified, providing the possibility of a more detailed diagnostic and prognostic subgrouping of tumour entities, up to even changing existing classification systems. Recently published gene expression or proteomic profiling data relate to new marker molecules involved in skin cancer pathogenesis, which may, after validation by suitable studies, represent future prognostic or predictive biomarkers in cutaneous malignancies. We, here, give an overview on currently known serologic and newer immunohistochemical biomarker molecules in the most common cutaneous malignancies, malignant melanoma, squamous cell carcinoma and cutaneous lymphoma, particularly emphasizing their prognostic and predictive significance.\nIntroduction\nBiomarkers play an important role in the diagnosis and prognostic classification of various cancer entities and can moreover be useful in monitoring the patient\u2019s clinical course of disease and response to therapy. In general, biomarkers are proteins, less often carbohydrates or lipids, and their expression profiles are associated with malignant disease. In the majority of cases, the marker molecules are expressed by the tumour cells themselves or by cells of the tumour microenvironment. Thus, most biomarkers can primarily be found in malignant tissues, but after active secretion or passive release at tumour cell destruction also become detectable in body fluids like blood, lymph or urine.\nBesides morphological and histopathological biomarkers (anatomic site and type of the primary tumour, tumour size and invasion depth, ulceration, vascular invasion), an increasing variety of molecular markers have been identified, providing the possibility of a more detailed diagnostic and prognostic subgrouping of tumour entities, up to even changing existing classification systems. Recently published gene expression, as well as proteomic profiling data, indicates new candidate molecules involved in skin cancer pathogenesis, which may after further validation represent new markers superior to existing ones. This ongoing process of biomarker identification and validation would result in a rapidly changing molecular view and classification of skin cancers.\nMalignant melanoma\nSerologic markers of malignant melanoma\nDespite numerous therapeutic options, the prognosis of malignant melanoma, once metastasized, is still poor. Thus, the search for reliable methods to detect metastases as early as possible and to identify patients with high risk of disease progression is of major importance. The serological parameters most widely used for the early detection of a tumour relapse or metastasis in the follow-up of melanoma patients are the melanocyte lineage\/differentiation antigens S100-beta and melanoma inhibitory activity (MIA) (see Table\u00a01 and Fig.\u00a01). Both proteins are with high, but not exclusive, specificity expressed by melanoma cells and thus correlate with the patient\u2019s tumour load.\nTable\u00a01Serologic markers of malignant melanomaSerologic markerSelected literatureMelanocyte lineage\/differentiation antigensS100-beta[30, 71, 32, 33, 18, 43, 28]MIA (melanoma inhibitory activity)[9, 8, 10, 74, 28]Tyrosinase[2]5-S-Cysteinyldopa[91, 34]L-Dopa\/L-tyrosine[75]Proangiogenic factorsVEGF (vascular endothelial growth factor)[83, 56, 12]BFGF (basic fibroblast growth factor)[83, 12]IL-8 (Interleukin-8)[68, 83, 12]Molecules involved in cell adhesion and motilitysICAM-1 (soluble intracellular adhesion molecule 1)[34, 87, 94]sVCAM (soluble vascular cell adhesion molecule 1)[26, 87]Matrix metalloproteinases (MMP)-1 and 9[63, 53]Cytokines and cytokine receptorsIL-6 (Interleukin-6)[50, 73]IL-10 (Interleukin-10)[21, 51]sIL-2R (soluble interleukin-2-receptor)[11, 58]HLA moleculessHLA-DR (soluble HLA-DR)[62]sHLA-class-I (soluble HLA-class I)[89]OthersLDH (lactate dehydrogenase)[72, 18, 6]CRP (C-reactive protein) [19]Albumin[72]TuM2-PK (Tumour pyruvate kinase type M2)[81]sFas\/CD95 [84]YKL-40[69, 70]CYT-MAA (cytoplasmic melanoma-associated antigen)[85]HMW-MAA (high-molecular-weight melanoma-associated antigen)[85]Fig.\u00a01Schematic presentation of the cellular localization and function of the melanoma biomarkers S100-beta, tyrosinase and melanoma inhibitory activity (MIA)\nThe S100 protein is a 21-kDa thermo-labile acidic dimeric protein, which was originally isolated from the central nervous system (CNS) [49]. It consists of two subunits, alpha and beta, in the combinations alpha\/alpha, alpha\/beta and beta\/beta. S100 affects the assembly and disassembly of microtubules and also interacts in a calcium-dependent manner with the p53 tumour suppressor gene. The beta subunit is expressed in cells of the central nervous system as well as in cells of the melanocytic lineage. Therefore, S100-beta measured in the cerebrospinal fluid was known as a marker of CNS damage [59], years before S100-beta was shown to be a useful serum marker in melanoma [30]. MIA was originally detected in melanoma cell culture supernatant [9] and was shown to exert an important role in cell\u2013matrix interaction and metastasis [8].\nSerum S100-beta has been shown to be superior compared to MIA, as an early indicator of tumour progression, relapse or metastasis [18, 43], and its distribution as a serum biomarker of melanoma, therefore, is the broadest [36]. Both markers have been shown to be useful prognostic markers in melanoma patients with distant metastases (stage IV, classification system of the American Joint Committee on Cancer, AJCC, see Fig.\u00a02) [71, 33], but fail to provide prognostic significance in early stages of melanoma, especially in patients who are tumour-free after surgical procedures [74]. Because of the strong correlation of their serum concentrations with the patients\u2019 tumour load, S100-beta and MIA are useful markers in the monitoring of therapy response in advanced metastatic melanoma patients (AJCC stage IV) [32]. Again, none of both markers are suitable indicators of therapy response in tumour-free early-stage melanoma patients (AJCC stage II or III). Moreover, S100-beta has been shown to fail to identify patients with lymph node micrometastases detected by sentinel node procedure [1].\nFig.\u00a02Kaplan\u2013Meier survival curves of 65 patients with advanced metastatic melanoma (AJCC stage IV). Patients with elevated serum levels of S100-beta show a significantly reduced survival probability compared to patients with normal S100-beta serum concentrations (P\u00a0=\u00a00.003 by log rank test; detection system LIAISON Sangtec 100)\nThe strongest prognostic serum biomarker in advanced metastatic melanoma is lactate dehydrogenase (LDH), an unspecific marker indicating high tumour load in a variety of tumour entities, including melanoma. Studies comparing LDH, S100-beta and MIA using multivariate data analysis showed LDH to be the strongest independent prognostic factor in stage IV melanoma patients [18]. Due to its high prognostic significance, coupled with its easy, cost-efficient and widely distributed detection methodology, serum LDH is the only molecular marker that has been included in the current melanoma staging and classification system of the AJCC [6]. It, moreover, serves as a stratification parameter in most randomized therapy trials performed in stage-IV melanoma and may also be used to monitor therapy response in these patients.\nA variety of other molecules of peripheral blood have been described as markers of tumour load and disease progression in melanoma. These biomarkers are derived from different fields like melanocytic differentiation (e.g. tyrosinase; see Fig.\u00a01), tumour angiogenesis (e.g. VEGF, bFGF, IL-8), cell adhesion and motility (e.g. ICAM-1, MMPs), cytokines and their receptors (e.g. IL-6, IL-10), antigen presentation (e.g. HLA molecules), tumour cell metabolism (e.g. TuM2-PK), apoptosis (e.g. Fas\/CD95) and many others (see Table\u00a01). However, neither one of these markers could be confirmed to be superior to S100-beta or LDH in reflecting the prognosis of patients in advanced disease stages, nor could any marker be shown to be of strong prognostic relevance in early stage tumour-free patients.\nThe serum proteomic profiling is an innovative approach to identify new, potentially better serological biomarkers in melanoma. This methodology offers the possibility of screening the whole serum proteome for markers, which match different criteria like prognostic significance, prediction of therapy response, etc. Using this technology, marker proteins from thematic fields, different from the above-mentioned ones, might be found and thereafter validated for their clinical use. The first promising results have been obtained and are currently tested in large sets of serum samples [46].\nImmunohistochemical markers of malignant melanoma\nCutaneous malignant melanoma regularly develops from the radial to the vertical growth phase and thereafter to metastatic disease. The variability of this clinical course is only partially explained by morphological and histopathological parameters like primary tumour localization, patient gender and age, mitotic rate, tumour thickness and ulceration. There is a need to identify molecular variables, which help to assign patients to specific risk groups. The number of modalities for diagnosing and subclassifying malignant melanomas is rapidly increasing and includes immunohistochemistry of tissue sections and microarrays, gene expression profiling, comparative genomic hybridization and mutational analysis. These methodologies promise to improve our prognostic classification systems, as well as our diagnostic and therapeutic potential.\nFor diagnostic purposes, a small panel of melanocytic lineage markers, e.g. S100, MART-1\/MelanA and gp100\/HMB45, is sufficient to distinguish melanoma from non-melanocytic cancers. For the differentiation between benign and malignant melanocytic lesions, a review of immunohistochemical markers is given in ref. [44]. The present review focuses on newer markers with potential prognostic impact for the disease. For this purpose, the situation is more complex. The transformation from benign melanocytes to metastatic melanoma is the result of a compilation of genetic alterations crucial to cell division, differentiation, anti-apoptosis, invasion, angiogenesis and sustenance in a microenvironment distant from the point of origin of the cell. Several marker molecules involved in these genetic alterations have been identified, and their expression in primary melanoma has been studied and correlated with the prognosis. Table\u00a02 gives a current overview on already identified biomarkers, whose abnormal expression is associated with the patient\u2019s prognosis. It may be expected that the most detailed prognostic classification will result not from one, but rather from a panel of multiple biomarkers from this list.\nTable\u00a02Immunohistochemical markers of malignant melanoma associated with prognosisAssociation with poor prognosisSelected literatureMelanocyte lineage\/Differentiation antigens\u00a0gp100\/HMB45Increased expression[52]Tumour suppressors\/oncogenes\/signal transducers\u00a0p16 INK4ADecreased expression[47, 3]\u00a0PTENDecreased expression[48]\u00a0pRb (retinoblastoma protein)Inactivation due to protein phosphorylation[65]\u00a0EGFR (epidermal growth factor receptor)Increased expression[80]\u00a0p-Akt (activated serine-threonine protein kinase B)Increased expression[17]\u00a0c-KitExpression[35, 82]\u00a0c-mycIncreased expression[42]\u00a0AP-2 (activator protein-2alpha) transcription factorLoss of nuclear AP-2 expression[7]\u00a0HDM2 (human homologue of murine mdm2)Increased expression[61]bcl-6Expression[3]Cell cycle associated proteins\u00a0Ki67 (detected by Mib1)Increased expression[29, 3, 57]\u00a0Cyclin A, B, D, EIncreased expression[24, 25]\u00a0p21CIP1Decreased expression[3]\u00a0GemininIncreased expression[92]\u00a0PCNA (proliferating cell nuclear antigen)Increased expression[92]Regulators of apoptosis\u00a0bcl-2Increased expression[78]\u00a0bax Decreased expression[23]\u00a0BakDecreased expression[23]\u00a0APAF-1 (Apoptotic protease activating factor-1)Decreased expression[27]\u00a0SurvivingIncreased expression[78]Molecules involved in angiogenesis\u00a0LYVE-1 (lymphatic vascular endothelial hyaluronan receptor-1) Increased expression[16]\u00a0PTN (pleiotrophin)Increased expression[93]Molecules involved in cell adhesion and motility\u00a0P-CadherinStrong cytoplasmic expression[5]\u00a0E-CadherinDecreased expression[4]\u00a0Beta-cateninLoss of nuclear staining[5]\u00a0Integrins beta1 and beta3 Increased expression[66]\u00a0MMPs (matrix metalloproteinases)Increased expression[63]\u00a0DysadherinIncreased expression[54]\u00a0CEACAM1 (carcinoembryonic-antigen-related cell-adhesion molecule 1)Increased expression[79]\u00a0Osteonectin (also termed BM40 or SPARC (secreted protein, acidic and rich in cysteine))Increased expression[45]Others \u00a0TA (telomerase activity) Increased expression[13]\u00a0MelastatinDecreased expression[22]\u00a0ALCAM\/CD166 (Activated leukocyte cell adhesion molecule)Increased expression[77]\u00a0CXCR4 receptorIncreased expression[67]\u00a0MetallothioneinIncreased expression[88]\nIn a recent retrospective study, frozen tissue samples from primary melanomas with long-term clinical follow-up were analysed using a pangenomic oligonucleotide microarray [92]. The authors describe a signature of 174 genes, identifying patients at risk to develop distant metastasis. From these 174 genes, 141 were under-expressed and 33 were over-expressed in primary melanomas of patients who remained free of metastasis for 4\u00a0years. About 30 of these 174 genes have already been studied in melanoma and include genes involved in cell cycle (CKS2, CDC2, CCNB1, CENPF and DHFR), mitosis (HCAP-G and STK6), mitotic spindle checkpoint (BUB1), inhibition (BIRC5) or stimulation (GPR105) of apoptosis, DNA replication (TOP2A, RRM2, TYMS, PCNA, MCM4 and MCM6), stress response (GLRX2, DNAJA1, HSPA4, HSPA5, HSPD1 and TXNIP), ubiquitin cycle (SIP), actin and calmodulin binding (CNN3), intracellular signalling (STMN2), negative regulation of the Wnt signaling pathway (CTNNBIP1), inhibition of MITF expression (EMX2), regulation of proteolysis (TNA), testis cancer (CML66) and metastasis suppression (NME1). Winnepenninckx et\u00a0al. [92] recommend the use of a battery of antibodies that may improve the future determination of prognosis and treatment stratification. The determination of karyopherin-alpha2, MCMs (minichromosome maintenance proteins), geminin and PCNA over-expression could be used for screening in melanoma patients with a poor clinical outcome.\nSquamous cell carcinoma of the skin\nWhile primary cutaneous squamous cell carcinomas (SCC) are usually easily treatable, they have the potential to recur locally and even metastasize, then leading to a significant morbidity and mortality. Therefore, it is important to identify those tumours that are more aggressive and require closer follow-up and additional treatments, such as lymph adenectomy or radiation therapy. Established prognostic factors include anatomic site of primary, tumour size, depth of invasion, rapid growth, grade of differentiation, perineural invasion, history of previous treatment, host immunosuppression, and etiologic factors such as burn scars, radiation and chronic ulceration. The histological subtypes of SCC have also been considered as a factor in determining the prognosis [60].\nOnly few molecular markers are known to be associated with progression or prognosis of cutaneous SCC. In the following, we give an overview on some recently described proteins whose abnormal expression contributes to a malignant phenotype in this cancer entity. STAT3, a member of the signal transducer and activator of transcription (STAT) family of transcription factors is a known regulator of cell motility. The expression of phosphorylated STAT3 (p-STAT3) was described to be stronger in poorly differentiated than in well-differentiated SCCs. Moreover, the percentage of tumour cells expressing p-STAT3 correlated with the depth of tumour invasion and with metastasis formation [76].\nE-Cadherin is a Ca(2+)-dependent, intercellular adhesion molecule that is specifically expressed in epithelial cells and tissues and functions by maintaining intercellular connections. In some types of carcinomas, E-cadherin expression of tumour cells is decreased in association with metastasis. In cutaneous SCC, a decreased expression of E-cadherin in the primary lesion is correlated with the development of regional lymph node metastasis [41]. Additionally, a decreased expression is more often associated with well-differentiated than with poorly differentiated SCC. Therefore, E-cadherin might be useful as a marker for the metastatic potential of well-differentiated SCC [41]. The simultaneous detection of p-STAT3 and E-cadherin may contribute to the prediction of prognosis of cutaneous SCC patients [76].\nAnother marker to distinguish between well-differentiated and poorly differentiated SCC is Ets-1 [37]. Ets-1 is a transcription factor regulating the expression of various genes including matrix metalloproteinases (MMPs). Therefore, Ets-1 might be important in the pathogenesis of invasive SCC. MMP-12 was found to be expressed by tumour cells in squamous cell carcinoma of the vulva. Its expression correlates with invasiveness, while that of macrophages predict a better clinical outcome [38]. A cell surface marker, CD44, is a glycoprotein widely distributed in the extracellular matrix. CD44 isoforms, which arise from alternative mRNA splicing, were found to be implicated in the formation of tumour metastasis. In a study by Rodriguez-Rodriguez et\u00a0al., it was shown that lymph node metastases of cutaneous SCC of the vulva were immunoreactive for CD44\u20139v [64]. Also, CD44\u201310v expression was present in 78% of tumours compared to only 56% of normal epithelium. CD44\u201310v membrane expression, but not cytoplasmic expression, correlates with disease recurrence [64]. In ocular squamous cell carcinomas, over-expression of CD44\u20136v is correlated with tumour progression and metastasis [55].\nCutaneous T-cell lymphomas\nCutaneous T-cell lymphomas (CTCL) are a heterogeneous group of cutaneous non-Hodgkin\u2019s lymphomas. In this cutaneous malignancy, the tumour cells home to and persist in the skin, producing a broad spectrum of clinical entities. The prognosis of CTCL depends on histologic and molecular aspects. The new WHO\/EORTC classification for cutaneous lymphomas comprises mature T-cell and natural killer (NK)-cell lymphomas, mature B-cell lymphomas and immature haematopoietic malignancies. Marker proteins for the diagnosis of CTCL include, for example, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD16\/56, CD19, CD25, CD45, CD45RA and CD45R0 [90].\nThe probability of survival in CTCL can be accurately predicted by a formula based on the clinical CTCL-Severity-Index (CTCL-SI) [20], which evaluates the involvement of the skin, lymph nodes, blood and visceral organs [40]. Besides clinical and morphological parameters, several molecules have been investigated in CTCL that are involved in general cellular signalling processes, regulation of cellular proliferation and apoptosis, like Jun, Myc, c-myb, p53, STATs, bcl-2, Fas\/CD95 and SOCS-3, or contribute to the putative immunopathology of the disease such as expression of inhibitory MHC receptors (ILT2\/CD85j), NK receptors (p140\/KIR3DL2) and dendritic cell defects (CD40). The abnormal expression of these molecules could be relevant for the prognosis of CTCL, as it has been shown for other tumour entities [39].\nWith regard to serological biomarkers in CTCL, it has been shown that the serum concentrations of the soluble alpha-chain of the interleukin-2 receptor (sIL-2R) as well as lactate dehydrogenase (LDH) strongly correlate with lymph node size, but only sIL-2R significantly correlates with the severity of skin manifestations in erythrodermic patients [86]. Moreover, sIL-2R was demonstrated to be produced at a relatively low rate by tissue-based lymphoma cells, whereas large-cell transformation in CTCL results in a marked increase in the sIL-2R production in some patients [86]. In addition to sIL-2R, neopterin and beta2-microglobulin have been shown to be significantly elevated in the serum from patients with Sezary syndrome. Thus, sIL-2R seems to be the most sensitive marker, which is typically increased in Sezary syndrome. Concerning the outcome of the disease, in terms of disease progression versus non-progression, only neopterin showed a significant prognostic value in non-leukemic CTCL patients [31].\nConclusion and future directions\nTaken together, molecular markers provide additional and much more detailed information for the prognostic classification of cutaneous malignancies. Currently, this is particularly true for malignant melanoma, but will certainly also affect other entities in due time. In addition to the serological and immunhistochemical biomarkers discussed here, genetic abnormalities have recently been recognized to influence the prognosis of cancer patients in higher extents than previously assumed. With regard to malignant melanoma, a new classification system was proposed combining genetic aberrations with histomorphological changes, resulting in new insights into the pathogenesis of this malignancy [14, 15]. It may be expected that the rapidly increasing knowledge of molecular mechanisms would lead to mainly biomarker-based, rather than morphology-based, classification systems that might facilitate an individualized, molecular-driven cancer therapy.","keyphrases":["biomarker","skin cancer","prognosis","serum","immunohistochemistry"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_J_Appl_Physiol-3-1-1782100","title":"Oxygen kinetics and debt during recovery from expiratory flow-limited exercise in healthy humans\n","text":"In healthy subjects expiratory flow limitation (EFL) during exercise can lower O2 delivery to the working muscles. We hypothesized that if this affects exercise performance it should influence O2 kinetics at the end of exercise when the O2 debt is repaid. We performed an incremental exercise test on six healthy males with a Starling resistor in the expiratory line limiting expiratory flow to \u223c 1 l s\u22121 to determine maximal EFL exercise workload (Wmax). In two more square-wave exercise runs subjects exercised with and without EFL at Wmax for 6 min, while measuring arterial O2 saturation (% SaO2), end-tidal pressure of CO2 (PETCO2) and breath-by-breath O2 consumption taking into account changes in O2 stored in the lungs. Over the last minute of EFL exercise, mean PETCO2 (54.7 \u00b1 9.9 mmHg) was significantly higher (P < 0.05) compared to control (41.4 \u00b1 3.9 mmHg). At the end of EFL exercise %SaO2 fell significantly by 4 \u00b1 3%. When exercise stopped, EFL was removed, and we continued to measure During recovery, there was an immediate step increase in so that repayment of EFL O2 debt started at a higher than control. Recovery kinetics after EFL exercise was best characterized by a double-exponential function with fundamental and slow time constants of 27 \u00b1 11 and 1,020 \u00b1 305 s, compared to control values of 41 \u00b1 10 and 1,358 \u00b1 320 s, respectively. EFL O2 debt was 52 \u00b1 22% greater than control (2.19 \u00b1 0.58 vs. 1.49 \u00b1 0.38 l). We conclude that EFL exercise increases the O2 debt and leads to hypoxemia in part due to hypercapnia.\nIntroduction\nIn spite of the fact that dynamic hyperinflation (Calverley and Koulouris 2004; O\u2019Donnell et\u00a0al. 2001) and peripheral muscle deconditioning (Maltais et\u00a0al. 1998; Richardson et\u00a0al. 1999) are important factors limiting exercise in COPD, there is evidence that inadequate oxygen delivery to working locomotor and respiratory muscles may also play an important role (Aliverti and Macklem 2001; Aliverti et\u00a0al. 2005a; Iandelli et\u00a0al. 2002).\nThis situation has been modeled in normal humans by imposing expiratory flow-limitation (EFL) during exercise which limits exercise performance by intense dyspnea (Aliverti et\u00a0al. 2002; Iandelli et\u00a0al. 2002), while decreasing arterial oxygen saturation and cardiac output (Aliverti et\u00a0al. 2005a). This in turn leads to premature competition between respiratory and locomotor muscles for available energy supplies and the early onset of mixed respiratory and metabolic acidosis (Aliverti and Macklem 2001; Kayser et\u00a0al. 1997).\nAs a result of these previous experiments (Aliverti and Macklem 2001; Aliverti et\u00a0al. 2005a; Kayser et\u00a0al. 1997) we predicted that EFL exercise in normal subjects should increase the oxygen debt by decreasing systemic oxygen delivery to exercising muscles. Accordingly, to test this hypothesis, we measured O2 kinetics during repayment of the O2 debt after control and EFL exercise in young, normal, male subjects. Our results revealed that EFL increased O2 net debt by an average 52% and led to hypoxemia in part due to hypercapnia.\nMaterial and methods\nSubjects\nWe studied six healthy male individuals whose baseline characteristics are shown in Table\u00a01. All subjects gave signed\u2013informed consent and the protocol was approved by the University of Athens Ethics Committee, where the experiments were conducted.Table\u00a01Subjects\u2019 characteristicsSubjectAge (years)Height (m)Weight (kg)FEV1 (l)FEV1 (%predicted)Dyspnea (Borg scale)1381.76783.90971551.752221.73754.231001702.583191.69593.93971001.674221.94865.471121252.555221.88805.731091002.056341.66603.5296901.87Mean261.77734.461021232.16SD80.11110.917330.41FEV1, forced expiratory volume in 1\u00a0s in absolute and as percentage predicted; , maximal workload when EFL was imposed during the incremental cycling test;  maximal oxygen uptake when EFL was imposed during the incremental test\nPulmonary function tests\nSpirometry for the determination of forced expiratory volume in 1\u00a0s (FEV1) was performed by a Vmax 229 system (Sensor Medics, Anaheim, CA, USA) (Table\u00a01).\nExercise protocols\nThree exercise tests were performed for each subject on an electromagnetically braked cycle ergometer (Ergoline 800, Sensor Medics) with the subjects maintaining a pedaling frequency of 60\u00a0rpm. At the beginning an incremental test was performed to establish the individual subject\u2019s maximal workload when EFL was imposed  by the application of a Starling resistor in the expiratory line limiting flow to approximately 1\u00a0l\u00a0s\u22121. After 3\u00a0min of baseline measurements, followed by 3\u00a0min of unloaded pedaling, the work rate was increased every min by 20\u00a0W to the limit of tolerance (i.e. the point where the work rate could not be tolerated due to severe sensation of dyspnea, see Table\u00a01). On a separate day, two square-wave protocols were performed: the first without EFL (control) and the second with EFL. After 3\u00a0min of baseline measurements, followed by 3\u00a0min of unloaded pedaling, the work rate was set and maintained at  for 6\u00a0min in both protocols. At the end of exercise, EFL was removed and recovery was monitored for an additional 6\u00a0min (Fig.\u00a01).Fig.\u00a01Typical example of volume changes of the chest wall during 3\u00a0min of baseline measurements (QB quiet breathing), 3\u00a0min of unloaded cycling (W\/u warming up), 6\u00a0min of exercise at the pre-determined , and 6\u00a0min of recovery. Arrows indicate end of expiration and inspiration. Data acquisition was interrupted for 1\u00a0min every 2\u00a0min in order to allow data storage\nGas exchange and flow measurements\nInstantaneous inspired and expired O2 and CO2 concentrations, end-tidal values for O2 and CO2 (PETO2, PETCO2, respectively) and flow at the mouth were recorded breath-by-breath (Vmax 229, Sensor Medics) throughout both control and EFL exercise and recovery runs. Flow displaced at the mouth was also measured by a screen type pneumotachograph (3813 Hans Rudolph, Kansas City, MO, USA) and from these measurements the following parameters were obtained: minute ventilation,  tidal volume, (VT); breathing frequency, (fb); inspiratory (Ti), expiratory (Te), total respiratory (Ttot) cycle time; and duty cycle (Ti\/Tot). Cardiac frequency (fc) and percentage arterial oxygen saturation (% SaO2) were determined using the R\u2013R interval from a 12-lead on-line electrocardiogram (Marquette Max, Marquette Hellige GmbH, Germany) and a pulse oximeter (Nonin 8600, Nonin Medical, USA), respectively. The modified Borg Scale (Borg 1982) was used to rate the magnitude of dyspnea and leg discomfort every 2\u00a0min throughout exercise.\nOperational chest-wall volume measurements\nSimultaneously, throughout the square wave exercise and recovery protocols the volume of the chest wall was measured by optoelectronic plethysmography (OEP system, BTS, Milan, Italy) as previously described (Aliverti et\u00a0al. 1997; Cala et\u00a0al. 1996; Vogiatzis et\u00a0al. 2005). In brief, the motion of 89 retro-reflective markers placed front and back over the chest wall from clavicles to pubis was recorded. Each marker was tracked by six video cameras, three in front of the subject and three behind. Subjects grasped handles positioned at mid-sternum level, which lifted the arms away from the rib cage, so that lateral markers could be visualized. Dedicated software, which recognizes the markers on each camera in real time, reconstructed their 3D co-ordinates by stereophotogrammetry and using Gauss\u2019s theorem, calculated inspiratory and expiratory tidal volume variations. Thus, breathing pattern was measured both plethysmographically and by integration of flow. Data acquisition from the OEP system was interrupted for 1\u00a0min every 2\u00a0min (Fig.\u00a01) in order to allow data storage.\nCalculation of breath-by-breath oxygen consumption\nThe volume of O2 exchanged at the mouth differs from the volume of O2 taken up by pulmonary capillaries if the amount of O2 stored within the lung changes. This occurs if the volume inspired is different from the volume expired in a given respiratory cycle, and\/or if alveolar concentrations of O2 change (Capelli et\u00a0al. 2001). This is particularly important during on- and off-exercise transients (Cautero et\u00a0al. 2002). The changes in O2 stored within the lung must be subtracted from the volume of O2 exchanged at the mouth in order to obtain the amount of O2 exchanged at the alveolar level. This can be done by combining OEP with independent measures of the subdivisions of lung volume and continuously measuring changes in the absolute gas volume by OEP throughout all exercise tests. This method was recently described in detail (Aliverti et\u00a0al. 2004a, 2005a). Accordingly, in the present study, breath-by-breath oxygen consumption  was computed throughout using the data acquired by OEP, flow at the mouth and the continuous inspired and expired O2 and CO2 concentration recordings. Using this method corrections were made for (1) pneumotachograph integrator drift, (2) the sampling rates of the O2 and CO2 meters, (3) the time delay of the O2 analyzer and the flow through the O2 and CO2 sampling lines, (4) precise synchronization of O2 concentration and volume signals and (5) any changes in O2 stored within the lung. Thus, in contrast to commercial devices, we measured  at the level of the pulmonary capillaries, not at the mouth.\nCurve fitting\nTo characterize the kinetics of the  response during square-wave exercise and recovery single and double-exponential functions were applied, respectively, to the data using a non-linear least-squares fitting procedure (Figs.\u00a02, 3) without omitting the first 20\u00a0s of the response for either the on- or off-transient. The equations applied (Linnarsson 1974; Ozyener et\u00a0al. 2001) were\nwhere in Eq.\u00a02, the response is the sum of two components, each described by an exponential decay with two time constants: \u03c41\u00a0=\u00a01\/b (the fundamental  component) and \u03c42\u00a0=\u00a01\/d (the slow  component). In the same equation, a and c are the initial values of these two components and their sum (a\u00a0+\u00a0c) represents the initial value of the total response, i.e. the value of  at time zero. The adequacy of the fitting model was tested by calculating R2 values and residuals for single and double exponentials.Fig.\u00a02Typical example of breath-by-breath  during control (closed circles) and EFL exercise (open circles) and recovery. Upon completion of exercise the Starling resistor was removed. Note that time is aligned on the precise instant in which the recovery started (time zero)Fig.\u00a03Typical example of breath-by-breath  during recovery from control (closed circles) and EFL exercise (open circles). Data are fitted with double-exponential curves\nMeasurement of oxygen debt\nOxygen debt was calculated by integrating, for each subject, the fitting curves between time zero (start of recovery) and an arbitrary time of 400\u00a0s (Fig.\u00a03) Then, the value of mean  at baseline, multiplied by the same period of time, was subtracted (Aliverti et\u00a0al. 2005a).\nEstimation of cardiac output and arterio-venous oxygen difference\nTo estimate the cardiac output (CO) and the arterio-venous oxygen (a-v\u00a0O2) difference during EFL and control exercise the following equations (Stringer et\u00a0al. 1997) were used:\nStatistical analysis\nData are presented as mean\u00a0\u00b1\u00a0SD. Significant differences in recorded variables over the last minute of the square-wave exercise protocols between control and EFL tests were assessed by the Wilcoxon signed-rank test as were recovery data from control and EFL exercise tests. Linear regression analysis was performed using the least-squares method. A statistical significance of 0.05 was used for all analyses.\nResults\nSquare-wave protocols\nFigure\u00a02 displays a typical example of breath-by-breath  during control and EFL exercise protocols. Individual and mean values of  sustained during the two square-wave protocols are shown in Table\u00a01, whereas the gas exchange and ventilatory pattern parameters are shown in Table\u00a02.  tended to be lower (by 8\u00a0\u00b1\u00a02%) at the end of EFL compared to control exercise, albeit not significantly (Table\u00a02). However, EFL compared to control exercise was associated with significantly lower values for fb, and Ti\/Tot. Te at the end of EFL exercise was significantly higher compared to control (Table\u00a02). Over the last minute of EFL exercise, mean values for fc, PETCO2 (54.7\u00a0\u00b1\u00a09.9\u00a0mmHg) and dyspnea scores were significantly larger compared to control exercise (Fig.\u00a04), whereas mean PETO2 (87.0\u00a0\u00b1\u00a011.3\u00a0mmHg) was significantly lower (Fig.\u00a04). The mean fall (P\u00a0=\u00a00.044) in %SaO2 at the end of EFL exercise was 4\u00a0\u00b1\u00a03%. (Fig.\u00a04). Leg discomfort at the end of EFL exercise was not different to that at the end of control exercise (Fig.\u00a04). The on-transient data were best fitted with a mono-exponential function where the time constant of the fundamental  component was not significantly different during EFL exercise as compared to control exercise (Table\u00a03).Table\u00a02Exercise data at the end of the 6\u00a0min protocols with and without EFL\u00a0Control exerciseEFL exerciseWork rate (W)111\u00a0\u00b1\u00a036109\u00a0\u00b1\u00a0302.08\u00a0\u00b1\u00a00.311.93\u00a0\u00b1\u00a00.422.14\u00a0\u00b1\u00a00.381.88\u00a0\u00b1\u00a00.49*fc (beats\u00a0min\u22121\t)152\u00a0\u00b1\u00a019163\u00a0\u00b1\u00a019*59.8\u00a0\u00b1\u00a010.238.8\u00a0\u00b1\u00a015.1*VT (l)2.08\u00a0\u00b1\u00a00.482.05\u00a0\u00b1\u00a00.45fb (breaths\u00a0min\u22121)29\u00a0\u00b1\u00a0519\u00a0\u00b1\u00a03*Ti (s)1.00\u00a0\u00b1\u00a00.100.85\u00a0\u00b1\u00a00.15Te (s)1.15\u00a0\u00b1\u00a00.232.02\u00a0\u00b1\u00a00.43*Ti\/Tot (%)46\u00a0\u00b1\u00a0531\u00a0\u00b1\u00a04*Values are means\u00a0\u00b1\u00a0SD oxygen uptake;  carbon dioxide output; fc, cardiac frequency;  minute ventilation; VT, tidal volume; fb, breathing frequency; Ti, inspiratory time; Te, expiratory time; Ti\/Tot, duty cycle*Significant differences (P\u00a0<\u00a00.05) between EFL and control exerciseTable\u00a03Individual and mean data on the time constant of the fundamental and slow component of the  response for on- and off-transientsSubjectOn-transient \u03c41 (s)Off-transient \u03c41 (s)Off-transient \u03c42 (s)ControlEFLControlEFLControlEFL175.866.234.729.21,4291027.5250.562.932.123.11,428930.7349.054.633.932.8*976.1489.387.053.430.9*1605.8532.541.236.16.9909855.06m61.772.553.136.61,666727.7Mean59.864.140.626.61358.01020.5SD20.415.69.910.6319.5305.2P value0.2760.0280.068Fundamental (\u03c41) time constant of the on-transient  response during control and EFL exercise (first and second columns). Fundamental (\u03c41) and slow (\u03c42) time constants of the off-transient  response after control and EFL exercise (third to sixth column).*Values\u00a0>\u00a0106Fig.\u00a04Individual values for heart rate (fc, a), %SaO2 (b), PETO2 (c), PETCO2 (d), dyspnea (e) and leg discomfort (f) at the end of control and EFL exercise protocols\nRecovery oxygen kinetics and oxygen debt\nFigure\u00a03 displays a typical example of breath-by-breath  data fitted with a double-exponential function during recovery from control and EFL exercise square-wave tests. The R2 values of the fitted curves applied to the recovery breath-by-breath  data were not significantly different between EFL (0.89\u00a0\u00b1\u00a00.07) and control exercise (0.86\u00a0\u00b1\u00a00.09). Immediately after removing the Starling resistor at the end of EFL exercise there was a step increase in  so that repayment of O2 debt started at a higher  after EFL than control. Figure\u00a05 demonstrates individual values of the a, c, and a\u00a0+\u00a0c parameters obtained from Eq.\u00a02. All three parameters were significantly higher after EFL compared to control exercise (a: P\u00a0=\u00a00.04; c: P\u00a0=\u00a00.0002; a\u00a0+\u00a0c: P\u00a0=\u00a00.02). Also shown in Fig.\u00a05 is the total O2 debt which was 2.19\u00a0\u00b1\u00a00.58\u00a0l after EFL exercise compared to the control value of 1.49\u00a0\u00b1\u00a00.38\u00a0l, an increase of 52\u00a0\u00b1\u00a022% (P\u00a0=\u00a00.01) caused by EFL. In order to verify possible problems originated by the curve fitting, oxygen debt and deficit were also calculated directly from the original breath-by-breath data and did not differ significantly compared to the data obtained by the fitted curves (average difference 4.3\u00a0\u00b1\u00a03.1%).Fig.\u00a05Individual values of the parameters given in Eq.\u00a01: a (a), c (b), a\u00a0+\u00a0c (c) and calculated gross values for the oxygen debt (d) after the EFL and control exercise protocols\nIndividual and mean values for the off-transient time constants of the fundamental and slow  components are shown in Table\u00a03. The fundamental time constant was significantly (P\u00a0=\u00a00.028) shorter during recovery from EFL compared to control exercise (Table\u00a03). The off-transient slow component during recovery from EFL tended (P\u00a0=\u00a00.068) to be shorter compared to that after control exercise; however, in recovery from control exercise the slow component was not evident in subjects\u00a03 and 4 (Table\u00a03). Table\u00a04 reports the individual and mean data of the on and off  transients for EFL and control exercise.Table\u00a04Individual and mean data of the on and off  transientsSubjectOn-transientOff-transientAControl(l\u00a0min\u22121)aEFL(l\u00a0min\u22121)aControl(l\u00a0min\u22121)aEFL(l\u00a0min\u22121)cControl(l\u00a0min\u22121)cEFL(l\u00a0min\u22121)11.9441.4721.912.420.670.7821.7512.0592.334.930.720.9130.9931.0401.852.090.490.6041.8061.7421.872.620.410.6550.9811.7431.403.840.680.9461.5711.4541.291.890.500.67Mean1.5071.5851.782.970.570.76SD0.4210.3470.381.180.120.14P value0.6710.0380.001For the on-transient after control and EFL exercise the  value for a was obtained from Eq.\u00a01:  For the off-transient after control and EFL exercise the  values for a and c were obtained from Eq.\u00a02: \nEstimation of cardiac output and arterio-venous oxygen difference\nFrom Eq.\u00a03), COEFL\u00a0=\u00a0[1.9\u00a0l\u00a0min\u22121\/5.72 +\u00a0 (0.105 \u00d7\u00a0 95\u00a0\n1)]\u00a0=\u00a012.5\u00a0l\u00a0min\u22121.\nFrom Eq.\u00a04, a-v\u00a0O2EFL =\u00a0 5.72 +\u00a0 (0.105 \u00d7\u00a0 95)\u00a0= 15.2\u00a0ml O2 per 100\u00a0ml of blood. In addition, a-v\u00a0O2control\u00a0=\u00a05.72 +\u00a0 (0.10 \u00d7\u00a0 60\u00a0\n2)\u00a0=\u00a05.72 +\u00a05.9 =\u00a0 11.7\u00a0ml O2 per 100\u00a0ml of blood.\nBased on the above calculations, the mixed venous blood O2 content (CvO2) for EFL would be equivalent to 4.8\u00a0ml per 100\u00a0ml blood or 48\u00a0ml O2\u00a0l\u22121. For control, CvO2 would be equivalent to 8.3\u00a0ml O2 per 100\u00a0ml blood or 83\u00a0ml O2 \u00a0l\u22121.\nTaking into account that 70% of blood volume is located in the venous compartment at rest and during moderate exercise that figure falls to approximately 50% (Astrand and Rodahl 1986), then during EFL exercise with a cardiac output of 12.5\u00a0l\u00a0min\u22121 the CvO2 will be [(12.5\u00a0l\u00a0min\u22121 \u00d7\u00a0 0.5) \u00d7\u00a0 0.048\u00a0l\u00a0O2] =\u00a0 0.3\u00a0l or 300\u00a0ml\u00a0O2. For control exercise, the CvO2 will be [(13.75\u00a0l\u00a0min\u22121 \u00d7\u00a0 0.5) \u00d7\u00a0 0.083\u00a0lO2] =\u00a0 0.57\u00a0l or 570\u00a0ml\u00a0O2. Thus, the difference in CvO2 between EFL and control will be 570 \u2212 300 =\u00a0 270\u00a0ml. Hence from the 0.70\u00a0l difference in oxygen debt between EFL and control calculated in Table\u00a05 (2.19 \u2212 1.49 =\u00a0 0.70\u00a0l), 0.27 l (i.e. approximately one-third) would account for the replenishment of the blood oxygen stores.Table\u00a05Oxygen deficit and debt for control and EFL exercise\u00a0Control exerciseEFL exerciseO2 deficit (ml)1,448\u00a0\u00b1\u00a0 7391,692\u00a0\u00b1\u00a0575O2 debtfast component (ml)1,174\u00a0\u00b1\u00a02741,232\u00a0\u00b1\u00a0470O2 debtslow component (ml)3,470\u00a0\u00b1\u00a05514,201\u00a0\u00b1\u00a0683*O2 debttotal (ml)4,645\u00a0\u00b1\u00a04785,433\u00a0\u00b1\u00a0763* (ml)3,157\u00a0\u00b1\u00a04433,238\u00a0\u00b1\u00a0539O2 debtnet (ml)1,487\u00a0\u00b1\u00a03842,195\u00a0\u00b1\u00a0581*Values are means\u00a0\u00b1\u00a0SD oxygen uptake*Significant differences (P\u00a0<\u00a00.01) between EFL and control exercise\nDiscussion\nThere is growing evidence that an inadequate oxygen supply to meet demand may play a more dominant role in limiting exercise capacity in some patients with advanced COPD than impaired lung function (Aliverti et\u00a0al. 2004b, 2005b; Maltais et\u00a0al. 1998; Oelberg et\u00a0al. 1998; Potter et\u00a0al. 1971; Richardson et\u00a0al. 1999; Stark-Leyva et\u00a0al. 2004). In healthy subjects, limiting expiratory flow by a Starling resistor or increasing expiratory load by imposing a constant pressure at the mouth during expiration, have been shown to significantly reduce cardiac output during exercise (Aliverti et\u00a0al. 2005a; Stark-Leyva et\u00a0al. 2004). This results from the enforced decrease in the velocity of shortening of expiratory muscles and hypercapnia, both of which contribute to increased expiratory muscle force and decreased duty cycle so that expiration acts like a Valsalva maneuver with inadequate time to recover during inspiration (Aliverti et\u00a0al. 2002, 2005a; Iandelli et\u00a0al. 2002). Furthermore, the decreased duty cycle magnifies the effect of high expiratory pressures when averaged over the whole respiratory cycle. Our present results show that EFL increased O2 debt by an average 52% and led to hypoxemia in part due to hypercapnia. This supports our hypothesis that the reduction in cardiac output during EFL exercise decreases O2 supply to working locomotor and respiratory muscles resulting in an increased O2 debt. We conclude that the decreased cardiac output is important in limiting EFL exercise performance in healthy subjects.\nEFL as a model for COPD\nThe experimental model used in this study was designed to simulate, in part, the flow-limitation commonly experienced by COPD patients during exercise. The pros and cons of this model have been previously extensively discussed (Aliverti et\u00a0al. 2002; Iandelli et\u00a0al. 2002). The model has demonstrated that exercise with EFL induces intolerable dyspnea, CO2 retention, impaired exercise performance, expiratory muscle recruitment (Kayser et\u00a0al. 1997), blood shifts from trunk extremities, a reduced duty cycle, arterial desaturation, and a decrease in cardiac output (Aliverti et\u00a0al. 2005a).\nIn the present study, we confirmed many of these results and showed that the increased O2 debt was accompanied by a reduction in  by 8% and a fall in SaO2 by 4% at the end of EFL exercise, thus leading to a reduction in systemic O2 delivery of \u223c\u00a0\u00a012%, in close agreement with the 15% reduction reported by Aliverti et\u00a0al. (2005a). It is reasonable to assume that the reduction in systemic O2 delivery during EFL exercise would be associated with the progressive recruitment of fast-twitch fibers and hence the premature onset of the lactate threshold. The latter has been shown to be the case during exercise with EFL (Aliverti et\u00a0al. 2005a). When exercise workloads exceed the lactate threshold, energy supplies are inadequate to meet demands. The resulting competition between working locomotor and respiratory muscles for the available energy supplies, regulated by autonomic reflex mechanisms (Harms et\u00a0al. 1998), would be substantially worsened by the decrease in the available O2 and should further increase the lactate production. It is therefore likely that the muscle and blood lactate levels (not measured in the present study) would be appreciably higher during EFL exercise and consequently, the lactate related metabolic cost could significantly contribute to the repayment of the O2 debt in recovery from EFL exercise.\nIn addition to the reduction in systemic O2 delivery, the present study shows that application of EFL during exercise significantly decreased minute ventilation compared to control exercise, causing hypercapnia. This confirms earlier results that show that a vicious circle is induced whereby increasing central ventilatory drive increases expiratory pressure which further reduces alveolar ventilation and cardiac output (Aliverti et\u00a0al. 2002, 2005a; Iandelli et\u00a0al. 2002). Furthermore, the observed elevated heart rate during EFL exercise possibly reflects a reduction in stroke volume secondary to the decrease in venous return. In the study by Stark-Leyva et\u00a0al. (2004), expiratory loading during exercise increased heart rate in an attempt to minimize the effects of the reduced stroke volume on cardiac output. Thus, the reduced cardiac output secondary to the high expiratory pressures would also be expected to contribute importantly to the greater oxygen debt that was measured after EFL exercise.\nIn summary, EFL exercise in healthy subjects reproduces many features of COPD including acute respiratory failure, a condition resembling cor pulmonale, dyspnea, and impairment of exercise performance. Furthermore, it has led to testable predictions, one of which is the rationale for this study and which to date have been proven to be accurate (Aliverti et\u00a0al. 2004b, 2005a, b).\nOff-transient O2 kinetics\nThe increase in O2 debt by 52% that we found during EFL exercise impacted, as predicted, on O2 kinetics during recovery. Accordingly, the repayment of O2 debt after EFL exercise started at a higher  compared to control and the off-transient time constant of the fundamental and slow  components were shorter after EFL exercise. Collectively, the findings describing the EFL off-transient O2 kinetics reflect a more rapid replenishment of blood O2 stores (fundamental component) and a faster repayment of O2 tissue debt (slow component), both of which could result from the greater O2 deficit during EFL exercise as opposed to control exercise. Indeed, if we considered that cardiac output during EFL exercise was lower than control and  was not significantly different between EFL and control, then it would be reasonable to expect that during exercise with EFL, that is known to decrease pulmonary blood flow (Aliverti et\u00a0al. 2005a), the arterial to mixed venous blood O2 difference would be larger and the mixed venous blood O2 content would be lower. This notion was confirmed by our calculations of arterial to mixed venous blood O2. When at the end of exercise EFL was removed, the sudden increase in cardiac output and in blood perfusing the lungs brought about a refilling of the O2 stores of mixed venous blood; hence the sudden increase of  (marking the replenishment of the body\u2019s deprived oxygen stores), a faster  kinetics and a larger O2 debt in EFL (Table\u00a05). The larger depletion of the inner oxygen stores is also suggested by the longer time constants of the on- and off-transient  response during EFL exercise (Table\u00a03). Our calculations showed that approximately 30% of the O2 debt difference between EFL and control exercise was due to the blood O2 replenishment (Table\u00a05) caused by the reduced cardiac output during EFL, whereas the rest was due to other possible mechanisms described below.\nIn line with the results of Cunningham et\u00a0al. (2000) and Ozyener et\u00a0al. (2001) describing O2 kinetics after heavy exercise, the off-transient  data after EFL exercise were best fitted by a two component exponential function. The fundamental off-transient component after control exercise (40.6\u00a0s) was similar to that (\u223c\u00a0\u00a033\u00a0s) described by Cunningham et\u00a0al. (2000) and Ozyener et\u00a0al. (2001) following very heavy exercise, lasting as in the present study for 6\u00a0min. On the other hand, the time constant of the fundamental component after EFL exercise (26.6\u00a0s) was significantly shorter than that of control exercise, thus confirming replenishment of the body\u2019s deprived oxygen stores upon removal of EFL. In addition, the shorter time constant calculated after EFL is the result of fitting the data from higher starting points as evidenced by the significantly higher a\u00a0+\u00a0c values shown in Table\u00a04 and Fig.\u00a05c.\nFurthermore, the time constant of the slow component after EFL exercise (1,020\u00a0s) was twofold longer than the one (460\u00a0s) described previously for very heavy exercise (Ozyener et\u00a0al. 2001) possibly reflecting the additive effects of the EFL-induced reduction in systemic O2 delivery on the repayment of tissue O2 debt. Although at present the mechanism(s) of the  slow component is not fully understood, there are important factors that could influence the slow component after EFL exercise. These include the blood lactate concentration (Poole et\u00a0al. 1994), the influence of the metabolic acidosis on the HbO2 dissociation curve (Wasserman et\u00a0al. 1991), the increased respiratory and cardiac muscle energy requirement associated with EFL exercise (Aaron et\u00a0al. 1992; Harms et\u00a0al. 1998), the progressive recruitment of type-II fibers (Coyle et\u00a0al. 1992) and to a lesser extent the increased levels of circulating catecholamines (Gaesser et\u00a0al. 1994) associated with the greater cardiovascular response during EFL exercise. Importantly, excessive expiratory muscle recruitment has been shown in patients with airflow limitation to increase the oxygen cost of breathing threefold (Aliverti et\u00a0al. 2004b). Accordingly, it is reasonable to assume that the higher O2 cost of breathing during EFL exercise would significantly enhance the O2 dept as compared to exercise without EFL.\nIn the present study, we utilized a double-exponential function to describe the off-transient  kinetics not only for EFL exercise but also for control exercise [typically fitted by a mono-exponential function (Cunningham et\u00a0al. 2000; Ozyener et\u00a0al. 2001)], in order to allow adequate comparisons of relevant parameters of recovery for both exercise tests. Accordingly, the slow component observed after control exercise was not discernible in two subjects using the double-exponential function. This is in accordance with previously reported data by Cunnnigham et\u00a0al. (2000) who exercised healthy subjects at a similar work rate (100\u00a0W), yielding similar exercise  (1.7\u00a0l\u00a0min\u22121) as the one reached in the present study during control exercise. In conditions where exercise is sustained at a moderate intensity, as in the control test, the off-transient slow component is often not discernible such that  kinetics can retain first-order characteristics (Cunnnigham et\u00a0al. 2000; Gerbino et\u00a0al. 1996; Ozyener et\u00a0al. 2001).\nIn conclusion, the results of the present study provide further evidence that expiratory flow limitation during exercise reduces systemic O2 delivery, enhances the O2 debt and leads to hypoxemia in part due to hypercapnia.","keyphrases":["oxygen debt","exercise tolerance","copd models"],"prmu":["P","R","R"]}
{"id":"Arch_Orthop_Trauma_Surg-4-1-2413128","title":"Calcaneal nonunion: three cases and a review of the literature\n","text":"The long-term follow-up of intra-articular calcaneal fractures is often accompanied by complications. Frequently occurring are arthrosis, arthrofibrosis of the subtalar joint, and malunion. Uncommon is the calcaneal nonunion. A total of three cases is presented in this report, including a review of the literature. The occurrence of a nonunion appears to be more common after conservative treatment, but the pathophysiology remains unclear, however smoking may play a role.\nIntroduction\nDuring long-term follow-up after intra-articular calcaneal fractures complications frequently occur. In clinical practice, arthrosis and arthrofibrosis of the subtalar joint, malunion and nonunion are encountered [8, 12]. Nonunion is only rarely found, and studies concerning complications after intra-articular calcaneal fractures do not describe this complication [5, 7]. In only five studies, including nine patients, reports on nonunion are made [3, 6, 10, 11, 13]. Three patients with a pseudarthrosis after an intra-articular fracture of the calcaneus are presented in the current study.\nCases\nCase 1\nA male adult (age 49) fell from a height of 2\u00a0m fracturing his left calcaneus. According to the Essex\u2013Lopresti classification he sustained a joint-depression type, and a type IIA fracture according to the Sanders classification. There was no injury to the soft tissues. After 6\u00a0days the fracture was operated on, using percutaneous reduction and fixation according to the technique of Forgon and Zadravecz [9]. Post-operative treatment consisted of active range of motion exercises and 3\u00a0months non-weight bearing. Initial recovery was uncomplicated. Two years after the trauma, the patient however returned to our outpatient department with complaints of painful walking, and unable to work. A computed tomography (CT) scan showed a pseudarthrosis of the calcaneus and talocalcaneal joint surface incongruence (Fig.\u00a01). An injection of 10\u00a0cc lidocain in the subtalar joint temporarily reduced pain. A subtalar joint arthrodesis was performed, fusing the talus and calcaneus with the use of bone graft gained from the iliac crest. After 1\u00a0year the arthrodesis has fully consolidated, the patient is pain free, but has currently not yet returned to work.\nFig.\u00a01a Case 1: Axial CT-scan image 22\u00a0months after percutaneous reduction and fixation showing nonunion; b Sagittal view\nCase 2\nA female patient, aged 53, sustained a fracture of the right calcaneus after a fall from the stairs (Essex\u2013Lopresti joint depression type, Sanders type IIB). As patient was seen three weeks after the trauma, the initial treatment was conservatively, consisting of 1\u00a0week of plaster-of-Paris and 3\u00a0months non-weight bearing with active range of motion exercises. Two months later the patient was unable to bear weight at the fractured side due to pain. A CT showed a delayed union of the calcaneus (Fig.\u00a02). Peroperatively, 6\u00a0months after trauma, there was a nonunion of the fracture, without apparent signs of arthrosis of the talocalcaneal joint surface. Because of the smoking habits and diabetes in this patient, as independent causes of high wound complication rates, an early subtalar arthrodesis with bone graft harvested from the tibial tuberosity was performed instead of a correcting osteotomy. Three months after the salvage operation the arthrodesis had consolidated, the patient is walking pain free.\nFig.\u00a02a Case 2: Semi-coronal CT-scan image 5\u00a0months after conservative treatment showing delayed union; b Sagittal view\nCase 3\nA male adult, of 39-years-old, sustained a bilateral calcaneal fracture after jumping of a first storey balcony. The radiographs showed a comminuted intra-articular calcaneal fracture according to the Essex\u2013Lopresti classification at both sides and Sanders type IIC and IIIAC fractures on the left and right foot, respectively. Initial treatment was conservative, consisting of 1\u00a0week of plaster-of-Paris and 3\u00a0months non-weight bearing with active range of motion exercises. Thirteen months later the patient returned to the outpatient department with complaints of pain at the left heel. Additional radiographs and CT showed a nonunion of the fracture of the left calcaneus (Fig.\u00a03). A subtalar arthrodesis was performed, with bone graft from the tibial tuberosity. Two cannulated screws were inserted from the tuberosity of the calcaneus, bridging the primary fracture line. The after treatment was complicated with a superficial infection, which could be treated adequately with intravenous antibiotics. Six months after the secondary fusion the patient was able to walk pain free.\nFig.\u00a03a Case 3: Semi-coronal CT-scan image 13\u00a0months after conservative treatment showing nonunion; b Sagittal view\nDiscussion\nImproper healing after an intra-articular calcaneal fracture carries a high morbidity. Three groups: A, malunion; B, nonunion; and C, osteonecrosis, were suggested by Zwipp [13]. These groups are further divided according to the presence of joint incongruence, varus\/valgus, loss of height, translation of bones and luxation. Treatment of these complications after intra-articular calcaneal fractures depends upon the presence of these conditions [13].\nThe first written report on calcaneal fracture nonunion was by Thomas (1993). He presented a 36-year-old female patient, treated non-operatively, who developed a calcaneal nonunion after 6\u00a0months [11]. A correction of the displaced fragment was performed and stabilized using plate osteosynthesis and bone graft. The patient was able to fully bear weight 12\u00a0weeks after this procedure.\nGehr described a 38-year-old male patient with an intra-articular, comminuted fracture of the calcaneus [3]. This fracture was treated with open reduction and internal plate osteosynthesis. After removal of the plate at 18\u00a0months the patient returned to the clinic with local swelling and pain during walking. A nonunion was seen and a correction osteotomy was performed with bone graft and screw fixation. The fracture showed healing at 8\u00a0weeks.\nKarakurt et al. [6] presented one 42-year-old male patient with a nonunion of the calcaneus after conservative treatment. The patient sustained a comminuted, open calcaneal fracture which was treated in plastercast for 6\u00a0months. Eight months after trauma the patient was unable to walk without crutches because of severe pain of the heel. After removing fibrotic tissue the calcaneus was filled with bone graft. Eight months after the operation patient was able to walk and work without pain.\nZwipp and Rammelt [13] reported two patients with a nonunion of the calcaneus. A 61-year-old female patient, with a Sanders IIC fracture with a luxation of the tuberosity fragment, was treated conservatively. An arthrodesis of the subtalar and calcaneocuboid joint was performed 6\u00a0months after trauma. Postoperatively the American Orthopaedic foot and Ankle Society hindfoot score improved significantly, compared with pre-operative values. The second patient was a 45-year-old female polytrauma patient. After percutaneous reduction and fixation she was diagnosed with a nonunion 1\u00a0year after the trauma, for which a subtalar joint arthrodesis was performed. No data on outcome was provided in this case.\nFrom cases described in literature and in the current report no similarities that could indicate a risk factor of nonunion could be found. There are no apparent similarities besides the nonunion (Table\u00a01). There might be a trend of nonunion occurring after conservative treatment, suggesting that less rigid or no fixation may play a role in causing fracture nonunion in the calcaneus. However Howard et al. [5] saw no cases of nonunion in a group of 164 intra-articular calcaneal fractures treated nonoperative. Patient age varied between 36 and 61, both sexes were affected equally often and initial treatment differed between patients. Karakurt et al. [6] suggested that smoking could be the cause of the nonunion. All patients in this report were smokers. For tibial fractures strong evidence exists for delayed fracture healing in smokers [4]. And a significant lower union-rate was seen after subtalar arthrodesis in smokers versus non-smokers [2]. Assous et al. [1] however saw no difference in fracture healing between smokers and non-smokers in a small series of intra-articular calcaneal fractures treated operatively.\nTable\u00a01Summary of patients presented in the literature after a calcaneal nonunionStudyGenderAgeInitial treatmentSalvage procedureFollow-up*(months)Thomas and Wilson [11]Female36ConservativeOsteotomy, plate, bone graft3Thermann et al. [10]\u2013\u2013\u2013Subtalar arthrodesis62Gehr et al. [3]Male38ORIFOsteotomy, screws, bone graft2Karakurt et al. [6]Male42ConservativeBone graft8Zwipp and Rammelt [12]Female61ConservativeSubtalar arthrodesis, calcaneocuboid joint fusion\u2013Female45PercutaneousSubtalar arthrodesis\u2013Current studyMale49PercutaneousSubtalar arthrodesis14Female53ConservativeSubtalar arthrodesis3Male39ConservativeSubtalar arthrodesis6*\u00a0The follow-up period after the salvage procedure is given\nIn contrast to the infrequent occurrence of the calcaneal nonunion, stated in the case reports above, Thermann et al. [10] describe an in incidence of 10% nonunion (n\u00a0=\u00a04\/40) in a group of patients receiving a subtalar arthrodesis for persisting invalidating pain after an intra-articular calcaneal fracture. In these four patients the nonunion coincided with painful subtalar posttraumatic arthrosis, delineating the indication for a subtalar arthrodesis, instead of a correction osteotomy with internal fixation.\nCalcaneal nonunion has been reported on infrequently in the literature, but the number of patients seen by Thermann, and our own series, suggests that the incidence might be higher than expected.","keyphrases":["nonunion","fracture","pseudarthrosis","calcaneus"],"prmu":["P","P","P","P"]}
{"id":"J_Biol_Inorg_Chem-3-1-1915625","title":"Altered spin state equilibrium in the T309V mutant of cytochrome P450 2D6: a spectroscopic and computational study\n","text":"Cytochrome P450 2D6 (CYP2D6) is one of the most important cytochromes P450 in humans. Resonance Raman data from the T309V mutant of CYP2D6 show that the substitution of the conserved I-helix threonine situated in the enzyme\u2019s active site perturbs the heme spin equilibrium in favor of the six-coordinated low-spin species. A mechanistic hypothesis is introduced to explain the experimental observations, and its compatibility with the available structural and spectroscopic data is tested using quantum-mechanical density functional theory calculations on active-site models for both the CYP2D6 wild type and the T309V mutant.\nIntroduction\nCytochromes P450 (CYPs) are enzymes responsible for the oxidative biotransformation of a large variety of endogenous and exogenous substrates, like steroids, carcinogens and drugs, and have been found in virtually all organisms [1]. In human drug metabolism, one of the most important CYP isoforms is CYP2D6. This hepatic enzyme is involved in the phase I oxidative metabolism of about 30% of currently marketed drugs, including much prescribed antidepressants, \u03b2-blockers, opioids and antiarythmics [2]. The enzyme is known for its genetic polymorphisms and gene multiplicities, even increasing its clinical relevance [3]. Structural studies on CYP2D6 are of crucial importance to understand how this enzyme works and to be able to predict the metabolism of new (druglike) compounds [4].\nA common structural feature in all CYPs, including CYP2D6 [5], is the distal helix (I-helix) positioned above the heme plane, which constitutes an important part of the enzyme\u2019s active site (Fig.\u00a01a). Previously, we demonstrated the critical role of the I-helix residue T309 in the mechanism of oxidation by CYP2D6 [6]. Similar results were found after mutation of the homologous threonine residues T302 and T303 in rabbit CYP2B4 and CYP2E1, respectively [7, 8]. The I-helix threonine is highly conserved in CYPs and seems to be a key residue in the monooxygenation reaction. It has been studied in many CYPs with mutagenesis, spectroscopic and crystallographic techniques [9]: depending on the specific isoenzyme, the distal threonine has been attributed a variety of roles besides oxygen activation and proton delivery [9, 10], including electron transfer [11], substrate recognition [12] and control of heme spin state equilibrium [12\u201317]. This multiplicity of roles, as indicated by the diversity of results obtained from different CYPs, has been interpreted as a lack of a universal role of this residue, which might have instead distinct functions within the CYP superfamily [9, 14, 17]. Fig.\u00a01a The cytochrome P450 2D6 (CYP2D6) active site as obtained from crystallographic data (PDB 2F9Q). The heme (coordinated by C443) and part of the I-helix are shown in color with the heme and the residues A305 and T309 as ball-and-stick models. Hydrogen atoms are not modeled in the PDB file. b Optimized geometry of the CYP2D6 active site model, i.e., the A305\u2013G306\u2013M307\u2013V308\u2013T309 part of the I-helix and the iron porphyrin part of the heme catalytic center of CYP2D6 in its doublet (low-spin) electronic spin state, with a water molecule coordinated to the iron (position 1). c Geometry of the CYP2D6 active-site model corresponding to a second energy minimum (water in position 2). Carbon, nitrogen, oxygen and hydrogen atoms are colored green, blue, red and white, respectively. Iron and sulfur atoms are colored orange. Distances are in angstroms\nIn this study, spectroscopy is used to probe the active site of the wild type and the T309V mutant of CYP2D6 to establish whether the mutation influences the heme characteristics. Resonance Raman (RR), a vibrational spectroscopy technique based on inelastic scattering of laser radiation in resonance with an electronic transition of a chromophore, is used to obtain information about several properties of the heme prosthetic group [18, 19]. The heme-iron oxidation state, spin state and coordination state, as well as conformational changes of the vinyl and propionate side chains of the porphyrin macrocycle can be inferred from RR spectra [19]. Furthermore, the Fe\u2013CO stretching frequency of the ferrous CO\u2013heme complex is sensitive to variations in the electrostatic potential of the heme distal pocket induced by substrate-binding or mutated residues [20, 21]. RR was successfully employed in the past to study many CYPs [19] and the effects of mutations in their active site [22\u201327], including those of the conserved I-helix threonine [11, 28, 29].\nBesides spectroscopic data, a computational approach is employed to rationalize the experimental results. Although experimental data on threonine mutants are already available for many CYPs, theoretical descriptions have been only carried out at a classical force-field level for the T302A mutant of CYP2B4 [30] and at a density functional theory (DFT) level for the T252A mutant of CYP101 [31]. Quantum-mechanical DFT calculations can reliably predict the relative energies of the various electronic states of iron(II) and iron(III) porphyrins and have been previously used to study the resting state as well as further steps of the catalytic cycle of CYPs [31\u201335]. Therefore, this method, in absence of decisive experimental proof in favor of a specific model, is used in the present work to test the compatibility of a definite hypothesis with available structural and spectroscopic data on the CYP2D6 active site. In particular, through a computational analysis of the wild-type and the T309V-mutant active-site structures, interactions are investigated between residue T309, the heme moiety and a water molecule located above the heme in the resting state.\nMaterials and methods\nMaterials\nThe pSP19T7LT plasmid containing in tandem the complementary DNAs (cDNAs) of human CYP2D6 with a C-terminal His6-tag and the human NADPH-CYP reductase, was used as described before [6]. Escherichia coli JM109 was obtained from DSMZ (Braunschweig, Germany). Dextromethorphan hydrobromide (DX) was obtained from Sigma (St. Louis, MO, USA). All other chemicals were of analytical grade and were obtained from standard suppliers.\nExpression and purification of the enzymes\nThe pSP19T7LT plasmids containing the wild type or the T309V mutant of CYP2D6 cDNA were transformed into E. coli strain JM109. Expression and membrane isolation was carried out as described in [6]. Membranes were resuspended in 0.5% of the original culture volume of potassium phosphate\u2013glycerol buffer (50\u00a0mM potassium phosphate buffer, pH 7.4, 10% glycerol) and the enzymes were purified, using nickel affinity chromatography as described in [6]. CYP concentrations were determined by CO difference absorption spectra according to the procedure of Omura and Sato [36], with a Pharmacia Ultrospec 2000 spectrometer.\nRR spectroscopy\nSpectroscopic measurements were conducted using a homebuilt Raman microscope in a backscattering configuration. A Zeiss microscope (D-7082 with \u00d740 objective, numerical aperture 0.60, working distance 2\u00a0mm) was coupled to a single monochromator (Instruments, Metuchen, NJ, USA) with a mounted grating with 2,400\u00a0grooves per millimeter and a CCD camera (DV-420OE, Andor Technologies, Belfast, UK). The 413.1-nm line of a continuous-wave Kr ion laser (Coherent Innova 300c, Coherent, Santa Clara, CA, USA) and the 457.9-nm line of a continuous-wave Ar ion laser (Spectra Physics 2000\u2013336, Newport Corp., Mountain View, CA, USA) were used for excitation, and the Rayleigh scattered light was removed using third Millennium edge long pass filters (Omega Optical, Bratleboro, VT, USA). Laser powers of 5\u00a0mW (at 413.1\u00a0nm) and 1\u00a0mW (at 457.9\u00a0nm) at the sample were used throughout the experiments. The sloping background of the spectra was subtracted using a baseline fitted to the experimental data with the Andor CCD camera software. The fitting of experimental data with Lorentzian functions was performed with PeakFit 4.12 (SeaSolve Software, Richmond, CA, USA). For RR measurements, a 1-mm-diameter glass capillary was filled with 10\u00a0\u03bcl of various concentrations (1\u201350\u00a0\u03bcM) of CYP2D6 in potassium phosphate\u2013glycerol buffer and put in a spinning capillary holder under the microscope objective. To measure substrate binding, substrate concentrations of 5\u201310\u00a0mM were used, leading to a 500\u20131,000-fold excess of substrate with respect to the enzyme (saturating conditions, as indicated by the Ks reported for the substrates [6]). No further spectral changes were detected upon increasing substrate concentration, indicating that all the CYP2D6 in the sample was bound to the substrate [37]. To obtain the ferrous CO complexes, oxygen was removed from the CYP2D6 samples (with or without substrate) by stirring under a nitrogen atmosphere; then the samples were reduced by adding 5\u00a0\u03bcl of 25\u00a0g\u00a0l\u22121 sodium dithionite (in potassium phosphate\u2013glycerol buffer) to 20\u00a0\u03bcl of enzyme in the same buffer, and were exposed to CO for 2\u20133\u00a0min while stirring. During all measurements, the capillary containing the sample was kept spinning to minimize local heating and photodissociation of the iron-bound CO. Before and after every RR measurement, CYP integrity was monitored by CO difference absorption spectroscopy.\nComputational details\nModel\nThe crystal structure at 3.0-\u00c5 resolution of the substrate-free human CYP2D6 (PDB 2F9Q) [5] was used as the starting structure for the DFT calculations. The computational model includes only a small part of the distal I-helix from A305 to T309, the heme moiety and the proximal C443. In this model, the isopropyl amino acid side chain of V308 is replaced by a methyl substituent and the 2-(methylthio)ethyl side chain of M307 is replaced by a hydrogen. These alterations are applied to considerably reduce the computational cost and are usually justified by the fact that these amino acid side chains are not pointing toward the heme cavity [38]. Likewise, the heme substituents are replaced by hydrogens, and the C443 proximal heme ligand is mimicked by a thiolate anion (HS\u2212). Moreover, a water molecule is added above the heme iron to generate a six-coordinated (6c) resting state, as suggested by spectroscopic data (see \u201cResults\u201d and \u201cDiscussion\u201d). This results in an 89-atom model for the active site of the wild-type protein. The T309V mutant is approximated by replacing the alcohol moiety of the T309 by a methyl substituent to give V309 instead. These two models are used to provide a rationale for the differences between the observed relative amounts of high spin (HS) and low spin (LS) in the wild-type and T309V-mutant enzymes of CYP2D6.\nMethod\nDFT calculations were performed with the Amsterdam Density Functional program [39\u201341]. The atomic orbitals on the atoms of the alcohol moiety of T309, the carbonyl group of A305, the thiolate anion, iron and nitrogens of the heme moiety were described by an uncontracted triple zeta valence plus polarization (TZP) Slater-type orbital (STO) basis set. A double zeta valence plus polarization (DZP) STO basis set was used for the other atoms of the I-helix and the atoms of the periphery of the heme. The inner cores of carbon, nitrogen, and oxygen (1s2) and those of sulfur and iron (1s22s22p6) were kept frozen. The exchange\u2013correlation potential was based on the newly developed generalized gradient approximation exchange functional OPTX in combination with the nonempirical PBE correlation functional (OPBE) [42, 43]. The OPBE density functional was chosen because of its demonstrated superior performance in describing the spin states and electronic structures for iron complexes, which is of crucial importance in the present study [32]. The model was based on the geometry of 2F9Q [5] and is fully optimized for the doublet spin state (LS). This structure was used as a starting structure for the linear transit calculations (geometry scan) in which the water molecule was moved toward the threonine by decreasing the Owater\u2013OThr distance to yield the five-coordinated (5c) heme moiety. In these geometry optimizations, the positions of the \u03b1 carbons of A305 and T309 and two porphyrin meso carbons were fixed to mimic the rigidity of the protein tertiary structure (see supplementary material). Subsequently, analogous geometry optimizations of the LS geometries obtained were performed for the sextet spin states (HS).\nThe energy profiles for the mutant enzyme model were obtained by the same procedure for each structure of the wild-type profile in which the threonine hydroxyl moiety was replaced by a methyl substituent.\nResults\nEnzymes expression and purification, UV\u2013vis absorption spectroscopy\nRecombinant expression followed by affinity chromatography purification yielded approximately 60\u00a0nmol CYP2D6 per liter of cultured E. coli, in the case of the wild type and the T309V mutant of CYP2D6. The enzymes were pure, as judged by the observation of a single band of 55\u00a0kD on a Coomassie brilliant blue stained sodium dodecyl sulfate\u2013polyacrylamide gel. The absolute absorbance spectra of oxidized and reduced CO-bound, wild-type and T309V-mutant CYP2D6 are shown in Fig.\u00a02. Both enzymes show characteristic strong absorption bands at 418\u00a0and 448\u00a0nm, in their resting state and reduced CO-bound form, respectively. No significant differences are observed in the position or the relative intensity of absorption bands between the spectra of the wild type and the T309V mutant of CYP2D6, even by use of spectral subtraction (data not shown). An amount of P420 (inactive CYP, observed as a band at 420\u00a0nm in the reduced CO-bound form) was present in the both the wild-type and T309V-mutant samples, to approximately the same extent. Fig.\u00a02Electronic absorption spectra of 1\u00a0\u03bcM purified wild-type and T309V-mutant CYP2D6, in the oxidized resting state form (solid line) and the reduced CO-bound form (dashed line)\nRR spectroscopy\nThe RR spectra of the wild type and the T309V mutant of CYP2D6 in the resting state (i.e., without substrate) are at first sight rather similar (Fig.\u00a03). In both cases, the most intense band at 1,372\u00a0cm\u22121 is attributed to the \u03bd4 vibrational mode, indicative for an oxidized heme iron atom [19]. Fig.\u00a03Resonance Raman spectra of wild-type (WT) and mutant (T309V) CYP2D6. Spectra were acquired as described in \u201cMethods.\u201d Excitation wavelength 413.1\u00a0nm, laser power at the sample 5\u00a0mW, accumulation time 600\u00a0s\nThe high-frequency region of the RR spectrum of the wild-type enzyme (Fig.\u00a04) is indicative for a predominantly 6cLS oxidized heme, in agreement with a previous study [27]. The bands at 1,501, 1,582 and 1,635\u00a0cm\u22121 are attributed to the \u03bd3, \u03bd2 and \u03bd10 vibrational modes for a 6cLS heme, respectively [19]. However, the slight asymmetry of \u03bd3 suggests the presence of another band at lower wavenumbers, characteristic of a 5cHS species. A better fit of the spectroscopic data for wild-type CYP2D6 is obtained when including bands at 1,486, 1,567 and 1,623\u00a0cm\u22121 for, respectively, the \u03bd3, \u03bd2 and \u03bd10 vibrational modes of a 5cHS heme species in addition to the 6cLS bands. Fig.\u00a04Spin marker band region of the resonance Raman spectra of wild-type (WT) and mutant (T309V) CYP2D6, together with their difference spectrum. Excitation wavelength 413.1\u00a0nm, laser power at the sample 5\u00a0mW, accumulation time 600\u00a0s. The Lorentzian functions used to fit the spectrum of the wild-type enzyme (see \u201cMethods\u201d) are represented as solid lines (for high-spin marker bands) or dotted lines (low-spin marker bands). Frequencies of high-spin marker bands are reported in bold\nThe presence of 5cHS heme species in the RR spectrum of the wild type is more evident when compared with the spectrum of the T309V mutant (Fig.\u00a04). Although the two spectra seem similar, the difference spectrum of the two enzymes shows an intensity increase for the bands characteristic of a 6cLS species and a concomitant decrease of the \u03bd3, \u03bd2 and \u03bd10 vibrational modes that are typical for a 5cHS heme. Therefore, in the T309V mutant the content of 5cHS is decreased compared with that in the wild type, with a concomitant increase of the 6cLS fraction.\nIn the presence of saturating amounts of the substrate DX, the bands distinctive of the 5cHS species (\u03bd3 and \u03bd2 at 1,486 and 1,567\u00a0cm\u22121, respectively) increase in intensity, whereas the intensity of the corresponding bands for the 6cLS species (\u03bd3 and \u03bd2 at 1,501 and 1,582\u00a0cm\u22121, respectively) diminishes for both the wild type and the T309V mutant of CYP2D6 (Fig.\u00a05). The intensity ratio between the 6cLS and 5cHS \u03bd3 bands (at 1,501 and 1,486\u00a0cm\u22121, respectively) is higher in the T309V mutant than in the wild type for substrate-bound CYP2D6. Fig.\u00a05Spin marker bands region of the resonance Raman spectra of wild-type (WT) and mutant (T309V) CYP2D6 in the presence (solid lines) and in the absence (dotted lines) of the substrate dextromethorphan hydrobromide. The spectral difference between the enzyme in the presence and in the absence of substrate is shown for the spin marker band \u03bd3. Excitation wavelength 413.1\u00a0nm, laser power at the sample 5\u00a0mW, accumulation time 600\u00a0s\nThe T309V mutation does not alter the Fe\u2013CO stretching frequency (\u03bdFe\u2013CO) observed at 476\u00a0cm\u22121 in both the reduced CO-bound wild-type and the T309V-mutant CYP2D6 (Fig.\u00a06). As previously observed [27], in the RR spectrum of wild-type CYP2D6 \u03bdFe\u2013CO increases by 5\u00a0cm\u22121 upon addition of saturating amounts of DX. In the same experimental conditions, the \u03bdFe\u2013CO band of the T309V mutant broadens toward higher wavenumbers, indicating that a shift, although less evident, is taking place similarly to that in the wild type. Fig.\u00a06Low-frequency resonance Raman spectra of the reduced CO-bound CYP2D6 wild type (WT) and mutant (T309V), in presence and in absence of dextromethorphan hydrobromide (DX). Excitation 457.9\u00a0nm, laser power at the sample 1\u00a0mW, accumulation time 600\u00a0s\nDFT calculations\nThe optimized geometry of the enzyme\u2019s resting state was modeled by adding a water molecule above the heme using the crystal structure of the human CYP2D6 (Fig.\u00a01b). The root mean square deviation of the optimized backbone of the I-helix and the corresponding residues in the experimental structure is only 0.28\u00a0\u00c5, confirming that the modifications applied to the side chains of residues M307 and V308 in the model do influence the conformation of the I-helix in the active site. The water molecule is bound to the iron (water position 1 in Fig.\u00a01b, Fe\u2013O distance is approximately 2.2\u00a0\u00c5), yielding a 6cLS iron porphyrin complex that is energetically favored over the 6cHS state by 7.5\u00a0kcal\u00a0mol\u22121. The water molecule is hydrogen-bonded to the carbonyl oxygen atom of the A305-G306 peptide bond (approximately 1.9\u00a0\u00c5). The hydroxyl moiety of the threonine amino acid residue (T309) also forms a weak hydrogen bond to this amide oxygen (approximately 2.6\u00a0\u00c5). When the water molecule migrates from the iron to the threonine moiety, a hydrogen bond is formed (Fig.\u00a01c) in which the threonine alcohol group acts as a hydrogen-bond donor; the conformation with the OH group acting as a hydrogen-bond acceptor is 6.6\u00a0kcal\u00a0mol\u22121 less stable.\nThe energy profiles upon migration of the water molecule toward the threonine (i.e., from position 1 to 2) for both the LS and the HS states are shown as solid curves in Fig.\u00a07. This movement of the water molecule results in the increase of the relative energy of the LS state and a new local minimum (5cLS) is obtained, which is 1.4\u00a0kcal\u00a0mol\u22121 higher in energy with respect to the energy of the starting structure (6cLS). The relative energy of the HS state increases slightly upon shortening the threonine\u2013water distance and decreases again to reach a new minimum that is lower in energy. There is a spin crossover at a Owater\u2013OThr distance of approximately 3.1\u00a0\u00c5 and a global minimum for the HS state is obtained. In the corresponding structure, the water molecule is hydrogen-bonded to the T309 and occupies a second position in the active-site pocket, further referred to in the following sections as position 2 (Fig.\u00a01c). Fig.\u00a07The relative doublet (low spin, filled circles) and sextet (high spin, filled squares) energies for the active-site model of the wild-type CYP2D6 with respect to the distance between the oxygen atoms of the water molecule and the threonine alcohol side group. The dashed line represents the low-spin (open circles) and high-spin (open squares) energy profiles for the mutant model, obtained by replacing threonine with valine. LS low spin, HS high spin, 5c five-coordinated, 6c six-coordinated\nReplacement of the threonine with valine in each of the structures followed by partial geometry optimizations results in different energy profiles for the mutant (dashed curves in Fig.\u00a07). For both the HS and the LS mutant models, after a small initial decrease of approximately 2\u00a0kcal\u00a0mol\u22121 which yields minima at an Owater\u2013OThr distance of 3.9 and 3.5\u00a0\u00c5 for the LS and HS states, respectively, the relative energy increases steeply as the water approaches position 2, for which, in contrast to the wild type, no minima are found.\nThe interaction between residue T309 and the CO adduct was studied by full geometry optimization of the CO-bound iron porphyrin model species for both the wild type and the T309V mutant, showing that the bond distances, bond dissociation energies and bond orders of the Fe\u2013C\u2261O moiety are virtually identical (Table\u00a01). Population analysis of the CO adduct shows a substantial iron backdonation into the empty \u03c0* orbitals of CO, with occupation numbers of approximately 0.2 electrons for both the wild type and the mutant. Table\u00a01Selected bond distances, bond dissociation energies [\u0394E(Fe\u2013CO)], bond orders and \u03c0*(CO) orbital population of the CO\u2013heme model for the wild type and the T309V mutant of cytochrome P450 2D6Fe\u2013COFe\u2013C\u2261OWild typeMutantWild typeMutantDistance (\u00c5)1.8241.8511.1471.146\u0394E(Fe\u2013CO) (kcal\u00a0mol\u22121)\u221224.6\u221224.2\u2013\u2013Mayer bond order0.210.200.520.52\u03c0*(CO) population\u2013\u20130.19\u00a0e\u22120.18\u00a0e\u2212\nThe superimposed geometries of the fully optimized and experimental structures, of the partially optimized structures of the T309V mutant with water in positions 1 and 2, and of the fully optimized structures of the CO-bound species for both the wild type and the T309V mutant are available as supplementary material.\nDiscussion\nIn the resting state, the CYP heme iron atom is generally observed as oxidized (Fe3+), and mainly in the 6cLS state with a water molecule (or hydroxyl ion) as the sixth distal ligand trans to the endogenous cysteinate ligand [44]. Although no distal water ligand is reported in the structure determined by X-ray diffraction [5] (Fig.\u00a01a), both electronic absorption and RR spectroscopy indicate that wild-type CYP2D6 is predominantly 6cLS. The present DFT calculations show that the resting state is clearly a 6cLS state, in agreement with spectroscopic data (Fig.\u00a01b). In addition to experimental data, DFT calculations suggest a network of hydrogen bonds between the residue A305, the distal water ligand and the residue T309, in which T309 and the water ligand are too far to interact directly. The same structural pattern, where the distal water is hydrogen-bonded to an alanine (four positions before the conserved threonine in the primary sequence), which is in turn weakly hydrogen bonded to the threonine, is also present in the experimental structures of other bacterial and mammalian CYPs [45\u201347].\nMoreover, in agreement with the DFT calculations for CYP2D6, in all these structures the conserved threonine is too far to hydrogen-bond the distal water ligand. The absence of a direct interaction between the heme axial ligand and the threonine in CYP2D6 is experimentally corroborated by the absence of differences in the electronic absorption and RR spectra of the CO adducts of the wild type and T309V, where a CO ligand occupies the position of the water molecule in the resting state. In fact, hydrogen bonding or direct electrostatic interaction between T309 and the axial CO would alter the extent of backdonation of iron d\u03c0 electrons to the empty CO \u03c0* orbitals, shifting significantly both the Fe\u2013CO stretching frequency in RR spectra and the absorption maximum in the UV\u2013vis absorption spectra of the reduced CO-bound heme [48, 49]. A similar situation is observed for the T268A mutant of CYP102 A1, whose CO adduct has a UV\u2013vis absorption spectrum identical to that of the wild type [9, 14]. The absence of a significant interaction between the CO adduct and residue T309 is supported by DFT calculations on CO-bound CYP2D6, showing that the Fe\u2013CO bond energy, bond order, and the extent of Fe\u00a0\u2192\u00a0CO backdonation are virtually the same for both the wild type and the mutant (Table\u00a01).\nBesides the predominant 6cLS state, the 5cHS species is present as a minor component in RR spectra of CYP2D6 (Fig.\u00a04). The occurrence of the 5cHS species could not be detected in UV\u2013vis absorption spectra, probably because of its small amount. RR spectra have more, narrower bands which are usually better resolved than UV\u2013vis spectra at room temperature, making it easier to detect asymmetries or weak shoulders when using spectral subtraction or fitting. The presence of the 5cHS species in the resting state is not unusual and it has been reported in RR spectra of other bacterial and mammalian CYPs [50\u201353]. A shift of the spin equilibrium toward the 5cHS state, in which the heme is no longer ligated to an axial water molecule, is often observed when a substrate (like DX for CYP2D6; Fig.\u00a05) is present in the heme pocket, perturbing the iron-bound water molecule directly, or through inducing conformational changes in the enzyme [44]. However, this substrate-induced spin shift often does not lead to a pure 5cHS species, but rather to a mixture of the two species, with 6cLS still significantly present, like in the case of CYP2D6 with DX.\nThe decrease of 5cHS and the concomitant increase of the 6cLS content in the RR spectra of the T309V mutant of CYP2D6 indicate that the residue T309 is structurally involved in the spin equilibrium. This role is preserved in the presence of DX as the substrate itself perturbs the wild-type and mutant spin equilibria to the same extent, which is evident from the spectral subtraction of the \u03bd3 region of the resting state from the substrate-bound state (Fig.\u00a05). The same conclusion can be drawn from RR spectra of the CO adduct of the two enzymes in the presence of substrate. Moreover, the similar affinity of the two enzymes for DX (as was judged from optical titration experiments [6] as well as modeling studies [54]) does not indicate T309 to be involved in substrate binding in CYP2D6. This strongly suggests that the difference in the 6cLS-to-5cHS ratio in the RR spectra between the substrate-bound wild-type and mutant enzyme is not due to different binding modes of DX, but rather to a difference caused by the T309V mutation, as observed in the absence of a substrate.\nAn altered spin equilibrium upon mutation of the conserved threonine residue has been reported for many other CYPs [12, 14\u201317, 55, 56]. Notably in the T268A mutant of CYP102 A1, which share with the optimized CYP2D6 computational model the hydrogen-bonding pattern \u201cdistal water\u2013alanine\u2013threonine,\u201d the alteration is in favor of the 6cLS, like in CYP2D6 [12, 14].\nIn an effort to rationalize the experimental observations, we looked for an explanatory model, compatible with our spectroscopic data, which could be tested using available quantum-mechanical methods. The absence of direct interactions between T309 and the axial ligand indicates that T309 exerts its influence on the spin equilibria of CYP2D6 indirectly.\nIt might be argued that the increase of the 6cLS species in the T309V mutant is due to an increased solvent accessibility of the binding pocket caused by a mutation-induced conformational change. However, this hypothesis is incompatible with the spectroscopic data on the CO adduct, which rule out any significant change in polarity in the heme pocket due to the T309V mutation. A more plausible explanation is that T309 in CYP2D6 stabilizes, by means of hydrogen bonding, an alternative position of the water that is distant enough from the iron center to yield a 5cHS state. This model has also been proposed to account for spin equilibria in substrate-bound CYP102 A1 and its T268A mutant [13, 14]. Substitution of the threonine with a valine should eliminate the hydrogen-bonding interaction and perturb the equilibrium by destabilizing the 5cHS state in favor of the 6cLS species, as observed from spectroscopic data.\nDFT calculations clearly support this hypothesis, showing the destabilization of the 5cHS state upon the T309V mutation. In agreement with the experimental observation of a small amount of 5cHS in the resting state of the wild type, a spin transition from LS to HS occurs upon moving the water molecule from position 1 to 2, and a second energy minimum has been found for the water molecule hydrogen-bonded to T309 (Figs.\u00a01c, 7). Replacing the threonine with a valine results in a relative destabilization of the structure in which water is in position 2, most likely because of the absence of hydrophilic interactions between the water and the valine. This relative destabilization of the 5c heme with the water in position 2 will result in the occurrence of a higher relative amount of the 6cLS heme species in the T309V mutant of CYP2D6 with respect to the wild type.\nAlthough the computational model of the CYP2D6 active site used in the present study only includes a few amino acids and the iron\u2013porphyrin complex, and therefore does not take into account the rest of the protein, it provides a clear rationale for the observed experimental data. Unfortunately, an evident limit of this model is the inability to include the substrate and to study its influence on the spin equilibrium of the substrate-bound enzymes, for which extended models and methods have to be used. However, it should be stressed that experimental data indicate that the role of T309 in the CYP2D6 spin equilibrium is independent of that of DX; therefore, it is reasonable to assume that the implications of our model for the role of T309 would still be valid for the substrate-bound enzymes.\nConclusions\nOn the basis of RR data, it is concluded that the T309V mutant of CYP2D6 has an altered spin equilibrium with respect to the wild-type enzyme, with a relative higher amount of 6cLS species at the expense of the 5cHS species, in both the resting state and the substrate-bound forms. Apparently, there is no direct interaction between residue T309 and the heme sixth ligand, suggesting an indirect mechanism of action on the spin equilibrium. Spectroscopic data also indicate that the T309V mutation does not significantly alter the polarity of the heme environment, excluding an increased number of water molecules in the heme pocket as the reason for the altered spin state. DFT calculations show that a simple model, involving a water molecule alternatively occupying two positions inside the heme pocket corresponding to two different spin states, is able to explain the experimental data. In this model, the position corresponding to the 5cHS state is stabilized by a hydrogen bond with T309, and a T309V mutation will induce an increase of the 6cLS species, as experimentally observed.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material. \nSupplementary material (PDF 215 kb)","keyphrases":["t309v","cytochrome p450 2d6","resonance raman","spin equilibrium","threonine mutant"],"prmu":["P","P","P","P","P"]}
{"id":"Cancer_Metastasis_Rev-4-1-2362138","title":"The type 2C phosphatase Wip1: An oncogenic regulator of tumor suppressor and DNA damage response pathways\n","text":"The Wild-type p53-induced phosphatase 1, Wip1 (or PPM1D), is unusual in that it is a serine\/threonine phosphatase with oncogenic activity. A member of the type 2C phosphatases (PP2C\u03b4), Wip1 has been shown to be amplified and overexpressed in multiple human cancer types, including breast and ovarian carcinomas. In rodent primary fibroblast transformation assays, Wip1 cooperates with known oncogenes to induce transformed foci. The recent identification of target proteins that are dephosphorylated by Wip1 has provided mechanistic insights into its oncogenic functions. Wip1 acts as a homeostatic regulator of the DNA damage response by dephosphorylating proteins that are substrates of both ATM and ATR, important DNA damage sensor kinases. Wip1 also suppresses the activity of multiple tumor suppressors, including p53, ATM, p16INK4a and ARF. We present evidence that the suppression of p53, p38 MAP kinase, and ATM\/ATR signaling pathways by Wip1 are important components of its oncogenicity when it is amplified and overexpressed in human cancers.\nIntroduction\nCellular DNA is constantly exposed to various environmental and endogenous mutagenic insults. To maintain genomic integrity and prevent cancer in the face of these potentially mutagenic events, cells have evolved a sophisticated array of damage sensors, signaling molecules, and repair functions. Among the key sensors of DNA damage are the phosphoinositide-3-kinase-related kinase (PIKK) family, that include ATM (ataxia-telangiectasia mutated), ATR (ataxia-telangiectasia and Rad3-related), and DNA-PKcs (DNA dependent protein kinase catalytic subunit) [1, 2]. Most PIKKs are serine\/threonine kinases that are conserved from yeast to humans and phosphorylate key target proteins in various DNA damage response pathways [3, 4]. The direct importance of the ATM\/ATR-initiated damage response pathways in cancer prevention has recently been demonstrated by two groups [5, 6]. Human pre-neoplastic lesions from a variety of different human cancers were shown to express markers of an activated DNA damage response, including activated and phosphorylated ATM, Chk2, p53, and H2AX [5, 6]. Interestingly, late stage tumors often showed loss of these DNA damage response markers, suggesting that the disabling of DNA damage response pathways is an important prerequisite for cancer progression [5, 6].\nIn studies of the DNA damage response, most attention has been focused on the activation and execution of that response. Less attention has been given to the reversal of the response. Once cell division has been halted and the DNA damage has been repaired, how does the cell return to its normal pre-stress state and re-enter cell division? Since activation of the damage response often occurs through phosphorylation of key downstream targets of ATM\/ATR, phosphatases are obvious candidates as homeostatic regulators of the DNA damage response. In this review we will discuss the evidence that the Wild-type p53-induced phosphatase 1, or Wip1, is a major homeostatic regulator of the ATM\/ATR-initiated DNA damage response.\nIn addition to its homeostatic role in the DNA damage response, we will also describe how Wip1 downregulates important tumor suppressor molecules. Wip1 has been shown to inhibit p53 by multiple mechanisms and to downregulate p38 MAP kinase through dephosphorylation [5, 7\u201311]. Expression of p16INK4a and p14ARF have also been shown to be suppressed in some contexts by Wip1 [12]. The inhibition of these tumor suppressors is likely to be a major component of the oncogenic activity of this phosphatase.\nDiscovery and initial characterization of Wip1\nAppella and colleagues originally identified the human Wip1 gene by screening for genes induced in a p53-dependent manner in response to ionizing radiation (IR) in WMN Burkitt lymphoma cells [13]. Using mRNA differential display methodology, they identified a novel p53-induced gene. The Wip1 transcript was induced by ultraviolet (UV) and IR in a p53-dependent manner [13]. Tumor cell lines with wild-type p53 consistently showed IR-induced increases in Wip1 mRNA while p53-deficient cell lines showed little or no induction of Wip1 expression following radiation treatment. Cellular fractionation and indirect immunofluorescence indicated that the 61\u00a0kDa Wip1 protein localizes to the nucleus [13].\nThe 605 amino acid human Wip1 protein sequence can be subdivided into two major domains, a highly conserved N-terminal phosphatase domain from amino acids 1\u2013375, and a less conserved non-catalytic domain extending from amino acids 376\u2013605. This C-terminal domain of Wip1 may facilitate nuclear localization. However, although this domain contains two putative nuclear localization signals, mutation of these motifs failed to prevent nuclear localization [14]. In addition, the C-terminal domain shows high conservation among mammalian Wip1s and limited conservation with non-mammalian Wip1 molecules, but little or no similarity with other phosphatases (Fig. 1). The phosphatase domain of Wip1 shows the highest levels of similarity to the type 2C family of serine\/threonine protein phosphatases (PP2C), consistent with its observed biochemical activities (Fig. 1(b)) [13, 15].\nFig.\u00a01Protein sequence alignment of human Wip1 and human PP2C\u03b1. (a) The overall structures of Wip1 (top) and PP2C\u03b1 (bottom) show significant similarity. The conserved type 2C phosphatase domain is shaded in Wip1. The regions of more highly conserved sequences labeled I, II, and III are shown as black blocks. The C-terminal non-catalytic domain that is present only in Wip1 molecules (and is also well conserved among mammalian Wip1 orthologues) is indicated by the white block. A putative nuclear localization signal (NLS) is also indicated near the C-terminus of Wip1. (b) Primary amino acid sequence alignment between human Wip1 and human PP2C\u03b1 phosphatase domains is shown. Identical amino acids are highlighted with a black background while conservative amino acid substitutions are indicated with a gray background. The phosphatase domains of the two molecules show 30% identity and 45% similarity\nUsing the human Wip1 cDNA as a probe, our laboratory isolated the murine Wip1 gene and mapped it to mouse chromosome 11 [16]. The human Wip1 gene is located on chromosome 17q22\/q23 [17]. The murine Wip1 protein contains 598 amino acids and migrates at approximately 66\u00a0kDa on a SDS-PAGE gel [16]. The murine and human Wip1 protein sequences have an overall identity of 83% and an overall similarity of 86%. RT-PCR and Northern blot analyses revealed that the Wip1 mRNA is ubiquitously expressed in all mouse embryonic and adult tissues, with a very high level of expression in the testis. Wip1 mRNA levels fluctuate during development [16].\nWip1 is a type 2C phosphatase\nWip1 is a member of the magnesium-dependent serine\/threonine protein phosphatase (PPM) family [18, 19]. This is a large and varied family of protein phosphatases present in both prokaryotes and eukaryotes, whose defining member is PP2C\u03b1 [20, 21]. To date, 18 human PPM\/PP2C genes have been identified [19]. In prokaryotes and eukaryotes, the PPM\/PP2C family of phosphatases plays a role in regulating stress response pathways [18, 20, 21].\nLike other PPM\/PP2C family members, Wip1 is a monomeric enzyme that requires divalent cations, mainly Mg2+ or Mn2+, for catalytic efficacy and is insensitive to okadaic acid, a potent inhibitor of the PP1 and PP2A phosphatases [18, 20, 21]. Using a BLAST search of the sequence database, human Wip1 shows an overall identity of 30% and an overall similarity of 45% to human PP2C\u03b1 and PP2C\u03b2 (Fig. 1(b)). Like PP2C\u03b1 and PP2C\u03b2, Wip1 also negatively regulates the stress responsive p38 mitogen-activated protein kinase (MAPK) pathway by directly inactivating p38 through dephosphorylation of phosphothreonine 180 of the regulatory pTXpY motif found in the activation loop of the kinase [11, 22, 23].\nBased on the sequence homology between Wip1 and PP2C\u03b1, \u03b2, and \u03b3, Yamaguchi et al. [15] developed a structural model for the catalytic domain of Wip1. From these studies, Arg76 of human Wip1 (Arg69 of mouse Wip1) aligned with Arg33 of PP2C\u03b1, suggesting that Arg76 of human Wip1 performs the same role as the catalytic Arg33 of PP2C\u03b1. Substrate specificity studies indicated that peptides containing pSXpY inhibit Wip1, while pTXpY peptides are Wip1 substrates [24]. Sequences on either side of the pTXpY motif did not greatly affect Wip1 activity, but the residue (X) lying between the two conserved phospho-acceptors affected Wip1 affinity and correlated with selectivity for MAP kinases. From these studies a specific Wip1 inhibitor was developed, and using simulations with the proposed structural model of Wip1, the phospho-Ser of the inhibitor was shown to be in contact with the proposed catalytic Arg76, thus blocking its interaction with potential targets [24].\nWhile the specificity of Wip1 for pTXpY motifs is clear from biochemical and cell biology studies [11, 15, 24, 25], the recent identification of targets in which Wip1 dephosphorylates sites modified by ATM\/ATR indicates an additional specificity. ATM and ATR phosphorylate pS\/pTQ sites in over 700 proteins in the cell [4] and Wip1 has been shown to dephosphorylate pS\/pTQ sites in vitro and in vivo on at least five proteins, ATM, Chk1, Chk2, p53, and Mdm2 (see Fig. 2) [7\u20139, 26].\nIdentification of Wip1 targets reveals it to be a homeostatic regulator of the DNA damage response\nWip1 dephosphorylates DNA damage response\/repair proteins at TXY motifs\nOnce Wip1 was shown to be a serine\/threonine phosphatase, it was clear that its functional roles might best be understood by identifying Wip1 target proteins and dephosphorylation sites. Since the discovery of Wip1, at least seven Wip1 dephosphorylation targets have been definitively identified. These are listed in Table 1. Note that on the seven target proteins, two distinct motifs appear to be dephosphorylated by Wip1, pTXpY and pS\/pTQ. One key commonality is their importance in the cellular DNA damage\/repair response. Wip1 acts as an inhibitor or homeostatic regulator of the DNA damage response, facilitating the return of the cell to a normal pre-stress state following repair of the DNA damage. As indicated in Table 1, five of the seven identified dephosphorylation targets are phosphorylated by the PIKKs ATM and ATR, which are key sensor proteins that activate numerous components of the DNA damage response pathways in the cell. We hypothesize that Wip1 serves as a major off switch for the ATM\/ATR-initiated DNA damage signaling cascade [7, 26]. Note also that six of the seven Wip1 targets are important regulators of p53 function. The four kinases p38, Chk1, Chk2, and ATM all phosphorylate p53 and promote its activation [27\u201330]. Wip1 dephosphorylation of these p53 kinases decreases their intrinsic activity. Dephosphorylation of p53 at serine 15 by Wip1 also contributes to p53 degradation, as does dephosphorylation of Mdm2, which stabilizes Mdm2, an E3 ubiquitin ligase specific for p53 [31]. Thus, Wip1 appears to be a critical inhibitor of p53 function and such effects are likely to play a major role in Wip1 oncogenicity (see below). In this section, we will describe the proteins, which have been identified as targets for dephosphorylation by Wip1 and how Wip1 regulates the function(s) of these proteins.\nTable\u00a01Identified Wip1 dephosphorylation targetsProteinSiteaMotifKinaseProtein functionWip1 effectsp53 effect?Referencep38 MAPKT180TXYMKK3\/6Stress responseDec. kinase activityYes (dec.)Takekawa et al. [11]UNG2T6TXY?Base excision repairDec. uracil excisionNoLu et al. [25]Chk1S345S\/TQATRDNA damage responseDec. kinase activityYes (dec.)Lu et al. [10]p53S15S\/TQATMDNA damage responseDec. apoptosisYes (dec.)Lu et al. [10]Chk2T68bS\/T\/QATMDNA damage responseDec. kinase activityYes (dec.)Fujimoto et al. [9]ATMS1981S\/TQATMcDNA damage responseDec. kinase activityYes (dec.)Shreeram et al. [7]Mdm2S395S\/TQATMp53 regulationDec. p53 levelsYes (dec.)Lu et al. [8]aListed sites are from the human proteins; mouse sites (e.g. p53 S18 or ATM S1987) may be at different amino acid codonsbAlso sites Ser19, Ser33\/35, Thr68, and Thr432cActivated ATM autophosphorylates itself at S1981; S367 also dephosphorylated by Wip1\nTXY motif: p38\nThe first identified target of Wip1 was the p38 mitogen-activated protein kinase (p38 MAP kinase) [11]. Genotoxic stress such as UV radiation causes activation of p38 MAP kinase by dual phosphorylation on Thr180 and Tyr182 [32, 33]. The phosphorylated p38, in turn, phosphorylates p53 on Ser15, Ser33, Ser46 and Ser392 and increases p53 activities, including gene transcription and apoptosis [28, 34, 35]. p38 was shown to interact with Wip1 and to be dephosphorylated by Wip1 on its Thr180 residue [11]. Wip1 dephosphorylation of p38 was associated with reduced nuclear localization of p38 and reduced kinase activity towards Ser33 and Ser46 of p53 [11]. Dephosphorylation of Ser33 and Ser46 on p53 was accompanied by reduced p53 transcriptional activation activity and reduced p53-mediated apoptotic function following UV irradiation. Thus, Wip1 inhibits UV-induced phosphorylation of p53 on Ser33 and Ser46 via p38 downregulation, functioning as a mediator in a p53 negative feedback regulatory loop.\nTXY motif: UNG2\nUracil is a common DNA lesion formed by deamination of cytosine or misincorporation of dUMP, leading to transition mutations or generation of AP sites (apurinic\/apyrimidinic sites) in the genome. Such lesions are repaired by base excision repair (BER) that is initiated by a uracil DNA glycosylase [36]. At least four different mammalian uracil DNA glycosylases have been identified. Of these, the nuclear UNG2 encoded by the UNG gene is the major enzyme responsible for removing uracil and creating an apyrimidinic site for further repair processing [36].\nTo identify Wip1 interacting proteins, our laboratory performed bacterial two-hybrid assays with Wip1 bait constructs and we repeatedly pulled down UNG2 as a major interactor. In vitro and in vivo interaction studies by us and global human interactome screens by others confirmed that UNG2 is a Wip1 interacting protein [25, 37]. Analysis of the UNG2 sequence revealed two TXY motifs at Thr6 and Thr126, which show similarities to the Wip1 target site on p38 MAP kinase. We utilized UNG2 phosphothreonine 6 and 126 specific antibodies generated by the Appella laboratory to show that UV irradiation induced UNG2 phosphorylation at both Thr residues and that this enhances UNG2 enzymatic activity. Of these two target residues, only Thr6 was dephosphorylated by Wip1 in cells. Dephosphorylation of UNG2 by Wip1 resulted in reduced uracil-associated DNA incision activity, a critical step in BER [25]. Moreover, we were able to show that human cell lines overexpressing a Wip1 expression construct exhibited reduced global BER activity, while the same cells transfected with Wip1 siRNA exhibited enhanced global BER activity, indicating Wip1 inhibits base excision repair, in part by dephosphorylating UNG2 [25, 38].\nWip1 dephosphorylates DNA damage response proteins at S\/TQ motifs\nS\/TQ motif: Chk1, Chk2\nThe checkpoint kinases Chk1 and Chk2 are evolutionarily conserved kinases which play a crucial role in regulating DNA damage responses [39]. In response to DNA damage or replicative stress, Chk1 is phosphorylated on Ser317 and Ser345 mainly by the ATR kinase [40]. The phosphorylation on these two serine residues is a critical event for Chk1 activation in that mutants of Chk1 in which Ser317 and Ser345 residues were replaced with alanine showed poor kinase activity. Following activation, Chk1 phosphorylates and inactivates Cdc25 phosphatase family members to facilitate cell cycle arrest [41].\nChk2 activation is a multistep process initiated by phosphorylation on Thr68 mainly by ATM in response to DNA damage [42, 43]. Although Thr68 phosphorylation is not the only requirement for full activation of Chk2, the T68A mutation significantly reduced Chk2 kinase activity [44]. Activated Chk2 targets a variety of proteins involved in cell cycle control, DNA repair and apoptosis, including p53, BRCA1, PML, E2F-1, and the Cdc25 phosphatase family. Notably, ATR-Chk1 and ATM-Chk2 pathways are not strictly separated but rather highly connected and coordinated as evidenced by ATM-dependent phosphorylation of Chk1 in response to ionizing radiation [45], ATM-independent activation of Chk2 [46], and ATR activation regulated by ATM [47, 48].\nThe identification of Chk1 as a Wip1 target followed co-immunoprecipitation experiments in our laboratory that showed that Wip1 consistently bound Chk1 in cells [10]. In vitro phosphatase assays showed that Wip1 dephosphorylated Chk1 at Ser345, but not Ser317. In vivo assays demonstrated that overexpressed Wip1 suppressed UV-induced phosphorylation of Chk1 Ser345 while Wip1 siRNA enhanced Chk1 Ser345 phosphorylation compared to controls. Importantly, dephosphorylation of Chk1 at Ser345 resulted in reduced Chk1 kinase ability on Chk1 targets such as Cdc25C. Since Chk1 is an important cell cycle checkpoint protein, we also assessed the effects of either increasing or decreasing Wip1 expression on G2\/M and intra-S phase checkpoints. As expected, increased levels of Wip1 abrogated G2\/M and intra-S phase checkpoints, while decreased levels of Wip1 enhanced G2\/M and intra-S phase checkpoints induced by both UV and IR treatment of cells [10]. Because Chk1 phosphorylates and activates p53, the inhibition of Chk1 by Wip1 also places Wip1 in a negative feedback regulatory loop for p53 (Fig. 2) [26].\nFig.\u00a02Wip1 inhibits p53 activity by multiple mechanisms. When a cell is stressed by DNA damage, ATM, ATR, and p38 MAP kinase can phosphorylate p53 directly or through intermediary proteins such as Chk1 and Chk2. Phosphorylated p53 localizes to the nucleus and transactivates a battery of anti-proliferative genes. In addition, two p53 autoregulatory genes are activated, Mdm2 and Wip1. Mdm2 is an E3 ubiquitin ligase that promotes p53 degradation. However, early after the DNA damage response ATM (and possibly ATR) phosphorylate Mdm2 and this promotes Mdm2 degradation and prevents Mdm2 mediated p53 degradation. Activated p53 also upregulates Wip1 expression and after DNA damage is repaired, the accumulated Wip1 phosphatase inhibits a battery of proteins that activate p53. Wip1 dephosphorylates the upstream kinases that phosphorylate p53 (ATM, p38, Chk1, Chk2) and p53 itself (at Ser15). In addition, Wip1 dephosphorylates Mdm2 at Ser395 and this results in Mdm2 stabilization and Mdm2 mediated p53 degradation. Finally, increased Wip1 levels suppresses ARF which in turn results in increased Mdm2 activity and p53 proteolysis. The resulting destabilization of p53 helps return the normal cell to a pre-stress state after cellular damage is repaired. However, if Wip1 becomes amplified or overexpressed during tumor cell progression, this could result in chronic suppression of p53 activity and promote tumorigenesis. In the figure, proteins are indicated by circles or octagons and genes are indicated by rectangles. Small circles marked with P indicate phosphorylation sites. Black lines indicate early events in the DNA damage response and gray lines show later homeostatic events in the damage response\nChk2 was also shown to directly interact with Wip1 in vitro and in vivo by Fujimoto et al. [9]. These investigators showed that Thr68, which is phosphorylated by ATM after IR treatment, is dephosphorylated efficiently by Wip1. Moreover, Wip1 also dephosphorylated several other S\/TQ sites within Chk2. Overexpression of Wip1 was shown to suppress Chk2 kinase activity towards its substrate Cdc25C, while inhibition of Wip1 resulted in both increased and prolonged Chk2 kinase activity following IR. Interestingly, treatment of gamma-irradiated cells with Wip1 siRNA enhanced IR-induced apoptosis, suggesting that Wip1 negatively regulates irradiation-induced apoptosis by dephosphorylating and inactivating Chk2. Several laboratories have corroborated the inhibition of Chk2 activity by Wip1 and have shown that cancer cells with amplified Wip1 show reduced Chk2 activity [14, 49\u201351].\nS\/TQ motif: p53\nThe tumor suppressor p53 is a central node in the DNA damage response and mediates an array of responses, including the activation of multiple cell cycle checkpoints, DNA repair and apoptosis [52]. The importance of p53 in cancer prevention is supported by the observation that about half of all human cancer patients harbor p53 mutations [53]. Moreover, p53 deficient mice are highly susceptible to early onset spontaneous tumors [54]. In unstressed cells, p53 is maintained at low levels as a result of Mdm2-mediated ubiquitination and degradation [55]. When cells are confronted by genotoxic stress, ATM, ATR and Chk1\/2 kinases phosphorylate p53. This phosphorylation, along with modifications of other residues, blocks the p53-Mdm2 interaction, leading to p53 stabilization and an increase in p53 activity [56].\nWe investigated whether Wip1 dephosphorylated p53 at Ser15. In fact, both p53 phosphopeptides containing phosphoserine 15 and intact immunopurified p53 were efficiently dephosphorylated at Ser15 by purified Wip1 in vitro [10]. UV-irradiated cells exhibited reduced p53 Ser15 phosphorylation in the presence of overexpressed Wip1 and greatly augmented and sustained p53 Ser15 phosphorylation in the presence of Wip1 siRNA. As expected, increased Ser15 phosphorylation correlated with increased p53 protein levels in irradiated Wip1 siRNA treated cells.\nS\/TQ motif: Mdm2\nThe decreased stability of p53 in the presence of high Wip1 levels led us to investigate whether or not the effects of Wip1 on p53 stability are mediated by Mdm2. Mdm2 is an E3 ubiquitin ligase that specifically targets p53 for destruction [55]. Mdm2 binds to p53 and mediates its polyubiquitination as a prelude to its transport to the 26S proteosome and proteolytic degradation. Importantly, DNA damage results in ATM phosphorylation of Mdm2 at Ser395 and this phosphorylation is associated with Mdm2 destabilization [31]. Phosphorylation at this site also inhibits Mdm2 interactions with p53. Thus, p53 is stabilized.\nWe had noted that reduction of Wip1 levels corresponded with increased sustained p53 protein levels and increased p53 transcriptional activity after IR-induced DNA damage [8, 49]. One explanation for these observations was that Wip1 was affecting p53 stability through effects on Mdm2. To assess this possibility, we examined Wip1 interactions with Mdm2 and found that endogenous Wip1 and endogenous Mdm2 could form protein-protein interactions [8]. Moreover, Wip1 was shown to dephosphorylate Mdm2 at Ser395 both in vitro and in vivo. Dephosphorylation of Mdm2 by Wip1 was associated with decreased Mdm2 self-polyubiquitination and increased Mdm2 stability. Prevention of Mdm2 Ser395 dephosphorylation by Wip1 siRNA treatment destabilized Mdm2 following irradiation [8]. As expected, Wip1 overexpression increased Mdm2 interaction with p53 and increased p53 polyubiquitination, facilitating p53 proteolytic degradation. Thus, a primary role of Wip1 is to inhibit p53 stability in part through augmenting Mdm2 stability as a consequence of Mdm2 Ser395 dephosphorylation (Fig. 2).\nS\/TQ motif: ATM\nATM is a sensor kinase that is rapidly activated by DNA double strand breaks in part through autophosphorylation at Ser1981 (or Ser1987 in the mouse). Activated ATM then phosphorylates a diverse array of effector molecules that induce cell cycle arrest, DNA repair, DNA replication, and apoptosis. Activated ATM phosphorylates its targets at S\/TQ sites and it has recently been shown by Elledge and colleagues in a global proteomic screen that ATM\/ATR phosphorylates over 700 protein targets with widely different functions [4].\nBulavin and colleagues have demonstrated that Wip1 null mouse embryo fibroblasts (MEFs) treated with IR exhibit higher levels of Ser1987 phosphorylation, indicating higher levels of ATM activity [7]. Wip1 was shown to directly interact with ATM and its overexpression resulted in decreased levels of Ser1981 phosphorylation following IR treatment. Conversely, downregulation of Wip1 using siRNA resulted in increased Ser1981 phosphorylation. Moreover, immunopurified ATM was efficiently dephosphorylated at Ser1987 by purified Wip1, as was a phosphopeptide derived from this part of ATM. More recently, Shreeram et al. [57] have also shown that Wip1 dephosphorylates human ATM at Ser367 as well as Ser1981. The authors concluded that Wip1 was important in resetting ATM phosphorylation after repair of DNA damage.\nWip1 is an oncogene\nWhile a significant number of tyrosine phosphatases are associated with cancer initiation or development [58], few serine\/threonine phosphatases have been directly associated with oncogenesis. The only well characterized serine\/threonine phosphatase involved in oncogenic signaling is the phosphatase PP2A. PP2A has tumor suppressor activity and its inactivation has been associated with transformation of human primary cells [59]. It has also been shown to be either mutated or downregulated in some human cancers. Among the type 2C phosphatases, Wip1 appears to be the only one described so far with bona fide oncogenic function. The regulatory functions of Wip1 in the ATM-CHK1\/2-p53 and p38 MAPK-ARF\/p16INK4A signaling pathways would argue that this protein may possess major oncogenic potential.\nThe first evidence of an oncogenic role for Wip1 was published in a pair of papers by Bulavin et al. [17] and Li et al. [60]. Bulavin et al. used tissue microarray profiling to show that 37 of 326 primary breast tumors (11.3%) had Wip1 gene amplification. Similarly, Li et al., using DNA microarray analysis, showed that Wip1 was amplified at least 2.5 fold in 27 of 164 (16%) primary breast cancers. Both laboratories demonstrated that overexpression of Wip1 mRNA correlated well with Wip1 gene amplification. Interestingly, only one of eight tumors with Wip1 amplification examined by Bulavin et al. had a p53 mutation. The infrequent nature of p53 mutations in tumors with Wip1 amplification suggests that Wip1 may promote human tumors through its ability to inhibit p53, circumventing selective pressure to mutate p53 during tumor progression. Wip1 amplification in this context is reminiscent of tumors with Mdm2 amplification, where Mdm2 promotes degradation of p53 and few of these tumors exhibit p53 mutations [61]. Since these initial reports, other groups have confirmed amplification and overexpression of the Wip1 gene in breast cancers [62, 63]. Rauta et al. [62] showed Wip1 gene was amplified in 11% of breast cancers and this amplification was highly associated with ErbB2 amplification. Moreover, these investigators observed that breast cancers with Wip1 amplification had a significantly poorer prognosis than those without Wip1 amplification, though a breast cancer study by Yu et al. [63] failed to detect an effect of Wip1 overexpression on prognosis. In addition to breast cancers, Wip1 amplification and overexpression have been observed in ovarian clear cell adenocarcinomas [64], neuroblastomas [65], pancreatic adenocarcinomas [66], gastric carcinomas [51], and medulloblastomas [67\u201369] (Table 2). As shown in Table 2, where it was examined, tumors with Wip1 amplification rarely contained p53 mutations and often exhibited poorer prognosis than their counterparts with normal Wip1. Since only a handful of tumor types have been reported, it remains to be seen whether Wip1 amplification and overexpression occurs in most tumor types or only in a subset. Certainly, tumors with a low frequency of p53 mutations might circumvent p53 mutation or loss by Wip1 functional inactivation of p53 and these tumors would be good candidates for further investigation.\nTable\u00a02Human tumors with Wip1 gene amplification and\/or overexpressionOrgan\/TypeDNA\/RNA increasep53 mutationPrognosisaReferenceBreast adenocarcinoma(11% CNGb; ECGc)1\/8\u00a0Bulavin et al. [17]\u00a0(16% CNG; ECG)\u00a0\u00a0Li et al. [62]\u00a0(11% CNG; ECG)1\/10PoorerRauta et al. [64]\u00a0(35% Od)\u00a0\u00a0Yu et al. [65]Ovarian clear cell adenocarcinoma(40% CNG; ECG)\u00a0PoorerHirasawa et al. [66]Neuroblastoma(92% CNG; 28% O)2\/32PoorerSaito-Ohara et al. [67]Medulloblastoma(51% CNG; 88% O)\u00a0PoorerMendrzyk et al. [70]\u00a0(37% CNG; 27% O)\u00a0\u00a0Ehrbrecht et al. [69]Gastric carcinoma(74% O)\u00a0\u00a0Fuku et al. [53]Pancreatic adenocarcinoma(36% CNG)\u00a0PoorerLoukopoulos et al. [68]aPrognosis: \u201cPoorer\u201d indicates individuals with tumors with increased Wip1 copy number and\/or expression have significantly poorer prognosis than all individuals with that type of tumorbCNG: Wip1 DNA copy number gain (compared to DNA in normal tissues)cECG: increased Wip1 RNA expression significantly correlates with copy number gaindO: percentage of tumors with Wip1 RNA overexpression\nIn their papers describing the initial discovery of Wip1 amplification and overexpression in breast cancers, Bulavin et al. [17] and Li et al. [60] were also able to show that Wip1 can collaborate with known oncogenes such as Ras, Myc and Neu to transform rodent wild type primary fibroblasts and induce anchorage-independent growth in soft agar. It was also shown by both groups that overexpression of Wip1 could abrogate Ras-induced senescence of primary cells and could prevent apoptosis induced by serum starvation. Interestingly, transformation assays on p53 null MEFs showed that while Ras, Neu and Myc oncogenes could individually transform these cells, Wip1 could not [17]. These results argued that Wip1 is primarily oncogenic as a result of its ability to inhibit p53 signaling. In later studies, our laboratory was able to show that Wip1 transformation of primary rat embryo fibroblasts, in conjunction with the adenoviral E1A oncogene, was dependent on the phosphatase activity of Wip1 [49]. In contrast to wild-type Wip1, the phosphatase-dead mutant Wip1 (D307A) failed to transform primary fibroblasts.\nTo further demonstrate that Wip1 is oncogenic in an in vivo context, Demidov et al. [70] overexpressed Wip1 as a transgene in the mouse mammary gland. While the Wip1 transgenic mice did not develop spontaneous mammary tumors, the appearance of mammary tumors was accelerated when these animals were crossed with mammary tumor susceptible ErbB2 transgenic mice. Interestingly, the tumor promoting effects of the Wip1 transgene in the ErbB2 transgenics could be lost by further crossing in a constitutively activated MKK6 transgene [70]. MKK6 activates p38 MAP kinase and thus nullifies the effects of Wip1 dephosphorylation of p38, demonstrating the importance of Wip1 in regulating p38 signaling as well as p53 signaling in this particular model.\nMechanisms of Wip1 oncogenicity\nWip1 overexpression in MEFs and in transgenic mice promotes cell transformation and accelerated cancer progression [17, 49, 60, 70]. Moreover, a number of human cancers contain amplified and overexpressed Wip1 (Table 2). Generally, these tumors do not contain p53 mutations, suggesting that overexpressed Wip1 inhibits p53 during tumor progression, consistent with the fact that Wip1 suppresses p53 activity in the normal cellular context (Fig. 2). As an alternative approach to assess Wip1 function in promoting tumorigenesis, Wip1-deficient mice were generated in our laboratory. Wip1 null mice are viable but show some postnatal abnormalities, including variable male runting, male reproductive organ atrophy, reduced male fertility, and reduced male longevity [71]. The Wip1 null mice also showed increased susceptibility to pathogens and diminished T- and B-cell function.\nFibroblasts derived from Wip1 null embryos showed reduced growth rates, reduced colony forming ability, and premature senescence compared to their wild-type counterparts [12, 71]. In addition, Wip1 null MEFs exhibited an enhanced G1 checkpoint response to ionizing radiation. Bulavin et al. [12] showed that Wip1 null fibroblasts were significantly more resistant to transformation by various combinations of oncogenes compared to wild-type MEFs. Hras1 plus the adenoviral E1A oncogene transformed Wip1\u2212\/\u2212 MEFs displayed increased expression of p53 and cyclin dependent kinase inhibitors p21, p16INK4A, and p19ARF compared to Wip1+\/+ MEFs. In addition, the p38 MAP kinase showed increased phosphorylation in Wip1\u2212\/\u2212 MEFs [12]. Increases in p53, p21, and phosphorylated p38 MAP kinase in Wip1 null MEFs are supported by the fact that Wip1 directly dephosphorylates p38 and p53 and regulators of p53 (ATM, Chk1, Chk2). Since p21 is transcriptionally upregulated by p53, the increase in its levels is attributable to p53 activation.\nOne interesting result in these transformed Wip1 null MEF studies was the increase in p19ARF and p16INK4a levels. Wip1 regulation of these cyclin dependent kinase inhibitors appears to be at the transcriptional level, as transformed Wip1 null MEFs showed a three- to fourfold enhancement of transfected p16 promoter driven luciferase expression and p19 promoter driven CAT expression when compared to wild-type MEFs. Bulavin et al. [12] further showed that oncogene transformed p53 null MEFs produce tumors when transplanted into nude mice, while transformed doubly null p53 and Wip1 MEFs were resistant to tumors. Thus, Wip1 must regulate other pathways in addition to p53 to promote tumorigenesis, at least in this MEF model system. In contrast to p53\u2212\/\u2212 Wip1\u2212\/\u2212 MEFs, MEFs from Cdkn2a\u2212\/\u2212 (null for both p16 and p19, encoded from the same locus) Wip1\u2212\/\u2212 mice readily formed nude mouse tumors after oncogene transformation. This indicated that p16 or p19 or both genes were responsible for the Wip1 null MEF resistance to tumors. Subsequent experiments showed that much (though not all) of the transformation resistance was provided by p19ARF [12].\nBulavin et al. [12] also tested the effects of Wip1 deficiency on tumorigenesis in an in vivo context. Three mammary tumor susceptible models, MMTV promoter driven ErbB2, Hras1, and Wnt1 transgenic mice were crossed to Wip1 deficient mice and oncogene driven mammary tumorigenesis was examined in the presence and absence of Wip1. Wip1 female null mice were considerably more resistant to mammary tumors in the presence of the MMTV-HRas1 and MMTV-ErbB2 transgenes than were their Wip1 wild-type counterparts. However, Wip1 female null mice with the MMTV-Wnt1 transgene developed mammary cancers at the same rate as transgenic Wip1 wild-type females. Interestingly, the Wip1 null MMTV-ErbB2 tumors displayed a reduced mitotic index and an increased apoptotic cell index compared to the Wip1 wild-type MMTV-ErbB2 tumors. Moreover, p16INK4a protein levels in the Wip1 null tumors were low or absent, suggesting that loss or inactivation of high p16 expression (probably by p16 promoter methylation) is a likely prerequisite for tumor progression in this model. It was also observed that these Wip1 null mammary tumors also contained high levels of activated p38 MAP kinase (as indicated by increased levels of phosphorylated p38). Inhibition of p38 in the MMTV-ErbB2 Wip1 null mice by repeated injection of the inhibitor SB203580 resulted in the accelerated development of mammary tumors compared to water injected mice of the same genotype. Thus, constitutive activation of p38 MAP kinase in the absence of Wip1 may contribute to tumor resistance in the Wip1\u2212\/\u2212 MMTV-ErbB2 mice.\nIn another transgenic mouse model Shreeram et al. [57] examined lymphoma incidence in Wip1+\/+, Wip1+\/\u2212 and Wip1\u2212\/\u2212 mice bearing the E\u03bc-myc transgene in which Myc overexpression is restricted to B lymphocytes. Wip1+\/\u2212 and Wip1\u2212\/\u2212 mice exhibited a significant delay in E\u03bc-Myc-induced B cell lymphoma incidence. The median lifespan of Wip1+\/+ E\u03bc-Myc mice was 77\u00a0days compared to 107\u00a0days for Wip1+\/\u2212 E\u03bc-Myc mice and 138\u00a0days for Wip1\u2212\/\u2212 E\u03bc-Myc mice [14]. Subsequent crosses of the Wip1-deficient E\u03bc-Myc transgenic mice to p53, p19ARF, and ATM-deficient mice showed that Wip1 deficiency suppressed E\u03bc-Myc-induced lymphomagenesis in an ATM and p53-dependent, but ARF-independent manner [57]. Thus, overexpression of Wip1 in an oncogenic context could contribute to tumor promotion by inhibiting both p53 and ATM functions.\nIn addition to delaying oncogene-induced tumors, we found that Wip1 absence also resulted in far fewer spontaneous cancers than in mice with normal levels of Wip1 [49]. Lifelong monitoring of Wip1+\/+ mice recorded a 45% incidence of spontaneous cancers, while Wip1+\/\u2212 and Wip1\u2212\/\u2212 mice had a cancer incidence of 25% and 10%, respectively. These differences in tumor incidence between the Wip1+\/+ and Wip1\u2212\/\u2212 mice were significant (P\u2009=\u20090.016). These results suggest that the absence of Wip1 confers a significant degree of resistance to cancer development over the lifespan of the mouse.\nPart of the cancer resistance phenotypes of the Wip1 null mice may have been due to an enhanced DNA damage response in Wip1\u2212\/\u2212 tissues. Following whole body irradiation with 5 Gy, Wip1\u2212\/\u2212 tissues often exhibited increased phosphorylation of the DNA damage response proteins p53 (Ser15), Chk1 (Ser345), Chk2 (Thr68), and p38 (Thr180) compared to similar Wip1+\/+ tissues [49]. These results suggested that an enhanced DNA damage response might be one mechanism for the cancer resistance of Wip1 null mice, consistent with recent findings that the DNA damage response may be an important early failsafe system in preventing cancer progression [5, 6].\nIn summary, the studies described in this section and the previous section provide compelling evidence that Wip1 is a human oncogene. Table 3 recapitulates some of the evidence provided above in support of Wip1 oncogenic function. Its amplification and overexpression in human tumors, its clear effects on transformation of cells in culture and effects on tumorigenesis in animal models, and its ability to inhibit the activity of multiple tumor suppressors clearly define it as an oncogene. Perhaps the most important tumor suppressor modulated by Wip1 is the p53 protein. As shown in Fig. 2, Wip1 inhibits upstream kinase activators of p53 (ATM, p38, Chk1, Chk2), dephosphorylates p53 itself at Ser15, stabilizes a key mediator of p53 degradation, Mdm2, and inhibits the ARF upstream activator of p53. Its inhibition of p16INK4a levels also suggests Wip1 activity in suppressing retinoblastoma (Rb) tumor suppressor regulated pathways.\nTable\u00a03Evidence that the Wip1 gene is an oncogeneEvidenceReferences1. Wip1 specifically inhibits p53 signaling by multiple mechanisms[8\u201312, 17]2. Wip1 inhibits the activity of other tumor suppressors (ARF, p16INK4A)[12]3. Wip1 abrogates DNA damage response pathways and cell cycle checkpoints[10, 25, 51]4. Wip1 can transform primary rodent fibroblasts in conjunction with other oncogenes[17, 51, 62]5. Wip1 accelerates tumorigenesis in a mammary tumor susceptible model[72]6. Wip1 is amplified and overexpressed in multiple types of human tumors[17, 62, 64\u201371]7. Wip1 amplification and overexpression is often associated with poorer prognosis[64, 66, 67, 68, 70]8. Wip1 null primary embryo fibroblasts are resistant to transformation by oncogenes[12]9. Wip1 null mice are resistant to spontaneous and oncogene-induced tumors[12, 51, 59]\nWip1 as a target for cancer chemotherapeutic approaches\nBecause Wip1 inhibits so many tumor suppressor molecules (p53, ATM, p16INK4A, p14\/p19ARF), targeting Wip1 function in cancer cells may be an effective way to enhance tumor suppressor function, resulting in enhanced cancer cell arrest and\/or apoptosis. A number of laboratories have begun to experiment with this approach by developing and characterizing inhibitors of Wip1 phosphatase activity. Yamaguchi et al. [24] have developed a series of substituted linear and circular phosphopeptides that variably inhibited Wip1 activity. Optimization experiments resulted in two thioether cyclic phosphopeptides with a pSIpY core motif with essentially 100% inhibition of recombinant Wip1 phosphatase activity. Molecular modeling experiments indicated close interaction of the cyclic inhibitor with the key postulated catalytic residues (R76 and K238) in Wip1. Moreover, the inhibitor was specific for Wip1 and did not inhibit PP2A or PP2C\u03b1 [24].\nAnother approach was taken by Bulavin and colleagues who screened a diversity set library of 1990 compounds and identified 14 that could completely inhibit Wip1 phosphatase activity [72]. Two of these compounds were highly effective at concentrations as low as 0.5\u00a0\u03bcM and most of these hits did not inhibit PP2C\u03b1 and PP2A. When transformed MEFs were incubated with each of the 14 Wip1 inhibitors and tested for p38 MAPK phosphorylation (the prototype Wip1 target), only 5 of the compounds increased phosphorylation of p38. The most effective of these (compound M) was tested on various breast cancer cell lines and found to reduce cell viability by 30\u201350% in some lines and could also potentiate the anti-proliferative effects of the anti-cancer drug doxorubicin. Finally, compound M was injected into both mammary tumor susceptible MMTV-Neu mice and mouse xenograft models and was effective in reducing both tumor cell proliferation and tumor volume [72].\nAnother large high throughput screen of 65,500 compounds by Rayter et al. [73] identified six compounds that demonstrated strong inhibition of Wip1 phosphatase activity. However, only two of these compounds showed growth inhibitory activity on cancer cell lines. Of these, one specifically inhibited the growth of cells overexpressing Wip1, an effect that was lost in the presence of the p38 MAP kinase inhibitor SB203580. This indicated that suppression of p38 signaling by Wip1 is likely to be an important component of tumor promotion in some Wip1 overexpressing human cancer cells. The identification of novel Wip1 small molecule inhibitors is an encouraging advance and suggests that this approach merits further consideration for testing in human cancer patients.\nOther Wip1 biological functions\nAside from its clear importance in DNA damage response signaling and oncogenesis, the phenotypes observed in Wip1 knockout mice revealed other key physiological roles for Wip1. The Wip1 knockout mice display a range of abnormalities, including variable male runting, male reproductive organ atrophy with reduced male fertility as well as modestly diminished male longevity [71]. The reproductive defects of the Wip1\u2212\/\u2212 mice are only seen in older males and are presumably related to the abnormal seminiferous tubules and epididymi with a small number of mature spermatozoa that arise in these animals. The mechanisms of runting and reduced longevity seen in the null males may be a result of hormone level imbalance or deficient steroid receptor activation. Proia et al. [74] did demonstrate a rather unexpected regulatory effect of Wip1 on the progesterone receptor. This study showed that overexpression of Wip1 stimulates steroid receptor activity, by enhancing the intrinsic activity of p160 coactivators such as steroid receptor coactivator-1. One result of this activation is that Wip1 positively regulates the activity of estrogen, progesterone, and androgen receptors. This function appears to be independent of p38 MAPK because SB202190 (a potent p38MAPK inhibitor) is unable to reverse the inhibition of the progesterone receptor activity elicited by reducing Wip1 expression in MCF-7 cells.\nMice lacking Wip1 also showed immunological defects. The Wip1 null mice occasionally exhibited ulcerated skin lesions, disorganized and hyperplastic lymphoid organs, and increased inflammation in normal organs. B and T lymphocytes from Wip1\u2212\/\u2212 mice displayed a variety of unbalanced and ineffective responses to antigenic and mitogenic stimulation [71]. Moreover, Wip1 null mice were more likely to die from influenza virus infection than their normal counterparts.\nSchito et al. [75] in a more extended analysis of immune defects in the Wip1 null mice, showed that Wip1 is vital during the double negative to double positive T cell transition. Young Wip1 null mice had fewer splenic T cells and their thymi were smaller with fewer double positive (DP) and single positive (SP) CD4+ and CD8+ T cells. A partial block in transition from double negative (DN) to double positive T cells was noted and this correlates with a peak in Wip1 mRNA expression in the late DN stages of T cell maturation. DP T cells that did mature were found to have cell cycle abnormalities and increased apoptotic rates. Importantly, when the Wip1 null mice were crossed into a p53 null strain, many of the thymic and T cell deficiencies were rescued, indicating that a major component of the Wip1\u2212\/\u2212 T cell phenotypes were likely to be due to increased p53 activity.\nConclusions, future prospects\nSince the discovery of Wip1 by the Appella laboratory in 1997 there have been many advances in our understanding of how this p53-induced phosphatase functions. Yet in the decade or so since its discovery, there have been fewer than 50 papers associated with the study of Wip1, indicating that we have only scratched the surface in terms of gaining a complete understanding of the biological role of this protein. We have learned that Wip1\/PPM1D is an oncogene that functions in part by suppressing major tumor suppressors that include p53. It also plays a homeostatic role in reversing the effects of the ATM\/ATR-initiated DNA damage response pathway. This homeostatic role is generally benign until somehow the Wip1 gene becomes amplified and upregulated. Why and how this abnormal Wip1 alteration occurs in tumors is unclear, but should be of much interest in future studies.\nMany important questions remain to be answered. The structure, catalytic activities and functional domains of Wip1 are still poorly understood. What does the C-terminal non-catalytic domain do? Are pTXpY and pS\/pTQ the only target motifs recognized by Wip1 or are there others? And what are the other target proteins? The best guess is that there are likely to be hundreds of targets, if not more. Does Wip1 have other undiscovered functions? And perhaps most importantly, from a disease perspective, how many human cancers exhibit Wip1 overexpression, what are the mechanisms for Wip1-mediated oncogenesis, and how can our knowledge of these mechanisms assist us in designing novel therapies to fight cancer? The answers to these questions in the coming years should result in increased interest in this heretofore little studied protein.","keyphrases":["wip1","p53","ppm1d","serine\/threonine phosphatase","pp2c","atm"],"prmu":["P","P","P","P","P","P"]}
{"id":"Diabetologia-3-1-1998885","title":"High prevalence of low plasma thiamine concentration in diabetes linked to a marker of vascular disease\n","text":"Aims\/hypothesis To assess thiamine status by analysis of plasma, erythrocytes and urine in type 1 and type 2 diabetic patients and links to markers of vascular dysfunction.\nIntroduction\nDiabetes is increasing in incidence in the UK and elsewhere to epidemic proportions. The major health concerns of diabetes are achieving good control of blood glucose, BP and lipids to suppress the development of associated vascular complications\u2014microvascular complications (nephropathy, retinopathy and peripheral neuropathy) and macrovascular complications (cardiovascular disease and stroke) [1, 2]. This is not always achievable because of limitations of current drug therapy, patient compliance and other factors linked to the development of vascular complications [3]. Secondarily to the global epidemic of diabetes, therefore, is a global burden of vascular complications [4]. The significant residual risk of development of microvascular and macrovascular complications in prospective clinical trials where best efforts have been made to optimise glycaemic control [5, 6], and the failure of major genetic susceptibility factors for diabetic complications to emerge from intensive studies [7] (although some may be found with further study [8, 9]), encourage the search for other metabolic and nutritional factors that may predispose to the development of vascular complications in diabetes.\nVascular complications develop progressively from 5\u201340\u00a0years after the onset of diabetes, although they may appear earlier in type 2 diabetes when associated with a period of undiagnosed diabetes and impaired glucose tolerance. A strategy to counter multiple pathways of biochemical dysfunction linked to the development of vascular complications emerged from recent advances in understanding of the cell biology of diabetic complications: high-dose therapy with thiamine and related derivatives such as benfotiamine. High-dose thiamine therapy prevented the development of microvascular complications in experimental diabetes without improvement of metabolic control, as reviewed in [10]. Experimental diabetes was also associated with tissue-specific thiamine deficiency characterised by a marked decrease of plasma thiamine concentration and decreased activity of the thiamine-dependent enzyme of transketolase (TK) and decreased levels of TK protein in renal glomeruli linked to a profound increase in renal clearance of thiamine (ClThiamine) [11]. Thiamine deficiency was not detected by the conventional indicator of thiamine status, the erythrocyte \u2018thiamine effect\u2019 (the percentage unsaturation of TK with thiamine pyrophosphate [TPP] cofactor) [12]. The prevalence of similar low plasma thiamine concentrations and impaired renal handling of thiamine in clinical diabetes has not been addressed previously.\nIn this study, we sought to establish whether there is a disturbance of thiamine homeostasis prevalent in type 1 and type 2 diabetic patients in the UK and if this is linked to incipient nephropathy and markers of vascular dysfunction and early stage renal dysfunction.\nMethods\nPatients and normal healthy volunteers Diabetic patients were recruited from patients attending the Diabetes Clinic at Colchester General Hospital, and normal healthy control volunteers from partners and friends of the patients and investigators. Inclusion criteria were: diabetic patients with normoalbuminuria (AER <30\u00a0mg\/24\u00a0h) and microalbuminuria (AER 30\u2013300\u00a0mg\/24\u00a0h); matched for age and sex (18\u201365\u00a0years); diabetes duration of \u22655\u00a0years; HbA1c <10%; and BMI 19\u201340\u00a0kg\/m2. Exclusion criteria were: patients with end-stage renal disease; severe excess alcohol consumption (>50\u00a0U per week; 1\u00a0U is 8\u00a0g alcohol); significant comorbidities; known allergy or intolerance to thiamine; use of thiamine supplements or goldenseal (Hydrastis canadensis, a multipurpose herbal remedy); participating in an intervention study within 30\u00a0days; recipients of renal and\/or pancreatic transplants; and women who were pregnant or breastfeeding or of child-bearing potential not using adequate contraceptive precautions. Twenty-four hour urine collections and blood samples (fasting) were taken with informed consent of the patients and volunteers. Plasma, erythrocyte and mononuclear leucocyte (MNL) fractions were prepared immediately and stored at \u221280\u00b0C until analysis. Ethical approval for the study was given by the local ethics committee (North and Mid-Essex Local Research Ethics Committee).\nAssay of thiamine and phosphorylated metabolites Thiamine status was assessed by measurement of thiamine, thiamine monophosphate (TMP) and TPP, determined by HPLC with fluorimetric detection (pre-column derivatisation to thiachromes) [13]. The retention times, limits of detection, interbatch CV values and recoveries for these metabolites were: thiamine 13.1\u00a0min, 36\u00a0fmol, 1.1 and 97%; TMP 6.0\u00a0min, 52\u00a0fmol, 2.1 and 92%; and TPP 4.5\u00a0min, 51\u00a0fmol, 2.9 and 94%. There was no interference from glucose in the assay and validations were not significantly different in analysis of samples from normal healthy volunteers and diabetic patients. Sample storage studies indicated that analyte content was stable for plasma, erythrocytes and urine stored at \u221280\u00b0C for at least 6\u00a0weeks and storage of plasma, erythrocyte haemolysate and urine and related de-proteinised extracts for 8\u00a0h. Stock solutions of thiamine, TMP and TPP were calibrated by spectrophotometry assuming molar extinction coefficients of \u025b233\u2009=\u200914.2, \u025b247\u2009=\u200915.3 and \u025b247\u2009=\u200913.0 (mmol\/l)\u22121 cm\u22121, respectively [14]. Shewhart analysis assessing the stability of analyte estimates in samples every day over a period of ten consecutive days indicated the analysis had acceptable quality control (all estimates within mean\u2009\u00b1\u20092\u00a0SD) [15]. The concentration of thiamine metabolites was determined in plasma, erythrocytes and urine; ClThiamine and fractional excretion of thiamine (FEThiamine) were deduced.\nOther biochemical measurements Erythrocyte TK activity and percentage unsaturation with TPP (thiamine effect) were determined. Erythrocyte membrane content of thiamine transporter-1 (THTR-1) [16] and reduced folate carrier-1 (RFC-1), a transporter of TMP [17], were determined by SDS-PAGE electrophoresis and western blotting, normalising band intensities to \u03b2-actin and preparing erythrocyte membrane protein extracts using the optimised procedure described in [18], and commercial antibodies to THTR-1 and RFC-1 (Alpha Diagnostics, San Antonio, CA, USA).Markers of metabolic control were determined: plasma glucose, HbA1c, total cholesterol, HDL-cholesterol, triacylglycerol, systolic and diastolic BP and GFR assessed by Cockcroft\u2013Gault-corrected creatinine clearance. Markers of vascular and metabolic dysfunction (plasma plasminogen activator inhibitor-1; von Willebrand factor and soluble vascular adhesion molecule-1 [sVCAM-1]; and MNL protein kinase C) were determined by commercial ELISAs (Technoclone, Dorking, UK; R&D Systems, Abingdon, UK; and Stressgen, Ann Arbor, MI, USA, respectively).\nStatistical analysis The significance of differences between mean and median analyte concentrations was determined using Student\u2019s t test and the Mann\u2013Whitney U test, respectively, and correlation analysis was performed by calculating Spearman\u2019s \u03c1 statistic, as indicated in the text. Data presented are mean\u00b1SD or median (minimum\u2013maximum).\nResults\nPatient characteristics The characteristics of type 1 and type 2 diabetic patients and normal healthy control volunteers recruited for this study are given in Table\u00a01. The diabetic patients had moderate glycaemic control; mean HbA1c was 8.6\u20138.7%. GFR was within the normal range, indicative of normal renal function for creatinine clearance. Fifty diabetic patients (17 type 1 and 33 type 2) had urinary albumin excretion within the normal range (median 11.5, 2.3\u201329.1\u00a0mg\/24\u00a0h) and 24 had urinary albumin excretion characteristic of microalbuminuria and incipient nephropathy (median 46.1, 30.6\u2013297.3\u00a0mg\/24\u00a0h). Diabetic patients with and without microalbuminuria were of similar age (60\u2009\u00b1\u200914 vs 56\u2009\u00b1\u200915\u00a0years), duration of diabetes (17\u2009\u00b1\u200911 vs 16\u2009\u00b1\u200912\u00a0years) and HbA1c (8.9\u2009\u00b1\u20091.9 vs 8.5\u2009\u00b1\u20091.5%). Nine type 1 diabetic patients and 33 type 2 diabetic patients were receiving therapy with ACE inhibitors or angiotensin receptor blockers (ARBs).\nTable\u00a01 Characteristics of normal control volunteers and diabetic patients recruited for this studyParticipant typenSex M\/FAge (years)Duration of diabetes (years)BMI (kg\/m2)GFR (ml\/min)Fasting plasma glucose (mmol\/l)HbA1c (%)Systolic BP (mmHg)Diastolic BP (mmHg)ACE inhibitor\/ARB therapyControl volunteers2010\/1053\u2009\u00b1\u200910\u201327\u2009\u00b1\u2009489\u2009\u00b1\u2009185.6\u2009\u00b1\u20090.85.0\u2009\u00b1\u20090.2NDND\u2013Type 1 diabetes2610\/1648\u2009\u00b1\u20091522.1\u2009\u00b1\u200913.328\u2009\u00b1\u2009593\u2009\u00b1\u2009289.2\u2009\u00b1\u20091.7*8.7\u2009\u00b1\u20091.2*131\u2009\u00b1\u20092173\u2009\u00b1\u2009119Type 2 diabetes4829\/1962\u2009\u00b1\u20091213.0\u2009\u00b1\u20098.9**31\u2009\u00b1\u2009692\u2009\u00b1\u2009309.1\u2009\u00b1\u20092.2*8.6\u2009\u00b1\u20091.8*141\u2009\u00b1\u20092277\u2009\u00b1\u2009933Data are mean\u00b1SD.ND Not determined*p\u2009<\u20090.001 with regard to normal volunteers**p\u2009<\u20090.01 with regard to type 1 diabetes (Mann\u2013Whitney U test)\nThiamine status of patients with type 1 and type 2 diabetes Surveying the thiamine status in the study patients and normal volunteer groups, we found the plasma concentration of thiamine was decreased 76% and 75% in type 1 and type 2 diabetic patients, respectively. The plasma concentration of thiamine (mean\u2009\u00b1\u2009SD) was: normal volunteers 64.1\u2009\u00b1\u200912.0\u00a0nmol\/l, type 1 diabetes 15.3\u2009\u00b1\u20099.6\u00a0nmol\/l and type 2 diabetes 16.3\u2009\u00b1\u200911.5\u00a0nmol\/l, p\u2009<\u20090.001 (Student\u2019s t test) (Fig.\u00a01a). There was no significant difference between plasma thiamine concentration of type 1 and type 2 diabetic patients or between diabetic patients with and without microalbuminuria (14.4\u2009\u00b1\u20098.9 vs 16.7\u2009\u00b1\u200911.6\u00a0nmol\/l, p\u2009>\u20090.05). The urinary excretion of thiamine was increased fourfold and threefold in type 1 and type 2 diabetic patients with respect to normal volunteers. The urinary excretion of thiamine (median, minimum\u2013maximum) was: normal volunteers 0.31 (0.23\u20132.45) \u03bcmol\/24\u00a0h; type 1 diabetes 1.36 (0.39\u20137.65) \u03bcmol\/24\u00a0h; and type 2 diabetes 1.04 (0.08\u20136.85) \u03bcmol\/24\u00a0h, p\u2009<\u20090.001 (Mann\u2013Whitney U test). The urinary excretion of thiamine was higher in type 1 than in type 2 diabetic patients (p\u2009<\u20090.05) but was not significantly different in diabetic patients with and without microalbuminuria [1.10 (0.08\u20137.65) vs 1.23 (0.33\u20136.85) \u03bcmol\/24\u00a0h, p\u2009>\u20090.05]. ClThiamine was increased 24-fold in type 1 diabetic patients and 16-fold in type 2 diabetic patients. ClThiamine (median, minimum\u2013maximum) was: normal volunteers 3.7 (2.6\u201326.2) ml\/min; type 1 diabetes 86.5 (12.8\u2013228.4) ml\/min; and type 2 diabetes 59.8 (1.4\u2013256.6) ml\/min, p\u2009<\u20090.001 (Mann\u2013Whitney U test). There was no significant difference in ClThiamine between diabetic patients with and without microalbuminuria (72.9 [1.4\u2013355.7] vs 66.3 [5.8\u2013245.3] ml\/min). FEThiamine was increased 25-fold in type 1 diabetic patients and 15-fold in type 2 diabetic patients. FEThiamine (median, minimum\u2013maximum) was: normal volunteers 2.8% (1.4\u201317.2%); type 1 diabetes 71.2% (7.6\u2013165.9%); and type 2 diabetes 41.6% (1.1\u2013228.9%), p\u2009<\u20090.001 (Mann\u2013Whitney U test). There was no significant difference in FEThiamine between diabetic patients with and without microalbuminuria (57.7% [8.0\u2013228.9%] vs 43.4% [1.1\u2013165.9%]). Plasma thiamine concentration correlated negatively with ClThiamine (r\u2009=\u2009\u22120.531, p\u2009<\u20090.001; Spearman) and FEThiamine (r\u2009=\u2009\u22120.616, p\u2009<\u20090.001; Spearman) (Fig.\u00a01b,c). There was no significant difference in these thiamine-related variables between diabetic patients with and without ACE or ARB therapy.\nFig.\u00a01Thiamine status of diabetic patients and link to sVCAM-1. a Frequency ribbon graph of plasma thiamine concentration. b Inverse relationship between plasma thiamine concentration and thiamine clearance. Nonlinear regression equation: [Thiamine]Plasma\u2009=\u200947.7\u2009\u00d7\u2009ClThiamine\u22120.274, p<0.001. c Inverse relationship between plasma thiamine concentration and FEThiamine. Nonlinear regression equation: [Thiamine]Plasma\u2009=\u200974.0\u2009\u00d7\u2009FEThiamine\u22120.448, p<0.001Thiamine deficiency is assessed conventionally by assay of TK activity of erythrocytes [12]. Erythrocyte activity of TK was not changed significantly in diabetic patients, with regard to normal volunteers. TK activity was: normal volunteers 1.09\u2009\u00b1\u20090.06\u00a0mU\/mg Hb; type 1 diabetes 1.18\u2009\u00b1\u20090.25\u00a0mU\/mg Hb; and type 2 diabetes 1.01\u2009\u00b1\u20090.17\u00a0mU\/mg Hb. Erythrocyte activity of TK was not significantly different in diabetic patients with and without microalbuminuria (1.03\u2009\u00b1\u20090.24 vs 1.08\u2009\u00b1\u20090.21\u00a0mU\/mg Hb), although it correlated negatively with urinary albumin excretion (r\u2009=\u2009\u22120.232, p\u2009<\u20090.05; Spearman). All patients were not thiamine deficient by the definition of the \u2018thiamine effect\u2019; they had a thiamine effect of <15%. The concentration of thiamine in erythrocytes was not changed significantly in type 1 and type 2 diabetic patients, with respect to normal volunteers. The concentration of thiamine in erythrocytes (mean\u2009\u00b1\u2009SD) was: normal volunteers 0.318\u2009\u00b1\u20090.028\u00a0pmol\/mg Hb; type 1 diabetes 0.345\u2009\u00b1\u20090.025\u00a0pmol\/mg Hb; and type 2 diabetes 0.328\u2009\u00b1\u20090.137\u00a0pmol\/mg Hb.\nMasking of the clinical thiamine deficiency in erythrocytes by increased levels of thiamine transporter proteins We sought evidence to understand how erythrocytes of diabetic patients maintained a normal concentration of thiamine whilst the plasma concentration of thiamine was severely decreased. The transport of thiamine and TMP from plasma to the interior of erythrocytes is mediated by the transporters THTR-1 and RFC-1, respectively (Fig.\u00a02a). Western blotting of erythrocyte membrane proteins from diabetic patients showed increased contents of these transporters compared with membrane-associated housekeeping protein \u03b2-actin (Fig.\u00a02b). Thiamine transporter: \u03b2-actin blot intensity ratios (median, minimum\u2013maximum) were: normal volunteers (n\u2009=\u200910), THTR-1 0.69 (0.12\u20131.43) and RFC-1 0.042 (0.012\u20130.093); type 1 diabetes (n\u2009=\u200912), THTR-1 1.06 (0.64\u20132.02) (+54%, p\u2009<\u20090.05) and RFC-1 0.146 (0.021\u20130.902) (+248%, p\u2009<\u20090.01); and type 2 diabetes (n\u2009=\u200912), THTR-1 1.20 (0.62\u20132.39) (+74%, p\u2009<\u20090.01) and RFC-1 0.206 (0.038\u20131.382) (+390%, p\u2009<\u20090.001) (Mann\u2013Whitney U tests).\nFig.\u00a02a Schematic diagram of thiamine membrane transport and metabolism in erythrocytes. b Representative Western blot of THTR-1, RFC-1 and housekeeping protein \u03b2-actin of erythrocyte membranes. Lanes 1\u20133, normal volunteers; lanes 4\u20136, type 1 diabetic patients; and lanes 7\u20139, type 2 diabetic patients. T, Thiamine\nLow plasma thiamine concentration and markers of metabolic control and vascular dysfunction We screened markers of metabolic control and vascular dysfunction in diabetes for linkage to the low plasma thiamine concentration in diabetes. The only significant correlations were of plasma thiamine concentration with plasma sVCAM-1 (r\u2009=\u2009\u22120.246, p\u2009<\u20090.05; Spearman) (Fig.\u00a03a) and urinary excretion of thiamine with plasma sVCAM-1 (r\u2009=\u2009\u22120.311, p\u2009<\u20090.01) (Fig.\u00a03b).\nFig.\u00a03Correlation of sVCAM-1 with plasma thiamine concentration (a) and urinary thiamine excretion (b). Solid horizontal line, reference mean of sVCAM-1 of normal volunteers; broken horizontal line, upper limit of sVCAM-1 of normal volunteers\nDiscussion\nIn this study we found clinical diabetes was associated with a marked deficiency of thiamine in blood plasma linked to increased ClThiamine and FEThiamine. The low plasma thiamine concentration in diabetes was linked inversely to plasma sVCAM-1.\nThe strengths of this study were the use of a validated assay for thiamine and phosphorylated metabolites, a comprehensive assessment of thiamine-related variables (including thiamine transporter content of erythrocyte membranes, urinary excretion, ClThiamine and FEThiamine) and investigation of the association of thiamine-related variables with markers of metabolic and vascular dysfunction. The decreased plasma thiamine concentration in clinical diabetes was probably not due to a deficiency of dietary input of thiamine. The urinary excretion of thiamine of diabetic patients and normal healthy volunteers was within the normal range (>0.20\u00a0\u03bcmol\/24\u00a0h) [19], except for one type 2 diabetic patient with a urinary thiamine excretion of 0.08\u00a0\u03bcmol\/24\u00a0h. Urinary excretion of thiamine accounts for a minor part of normal thiamine turnover: pyrimidine and thiazole degradation products of thiamine are also excreted in the urine and account for the major component of thiamine turnover [20]. Rather, low plasma thiamine concentration was linked to a profound increase in ClThiamine and FEThiamine. The molecular mass of thiamine is <500\u00a0Da and hence it is filtered from plasma in renal glomeruli [10]. Increased ClThiamine is probably due to decreased re-uptake of thiamine in renal proximal tubules. Thiamine clearance was dysfunctional in diabetic patients with normal GFR, as assessed by creatinine clearance. This study suggests renal mishandling of thiamine in diabetic patients is an early marker of renal dysfunction in diabetes and, linked to the locus of renal thiamine re-uptake, particularly relates to proximal tubule dysfunction.\nRe-uptake of thiamine occurs in the proximal tubules by thiamine transporters THTR-1 and THTR-2 via a sodium-independent, proton antiport mechanism with regulation by Ca2+\/calmodulin [21]. The expression of the genes encoding THTR-1 and THTR-2 (and also RFC-1) transporters are regulated via SP1 promoter elements [22\u201324]. SP1 signalling in the tubular epithelium is impaired in hyperglycaemia associated with diabetes by increased O-glycosylation of the SP1 via enhanced hexosamine pathway activity [25]. Re-uptake of thiamine by the tubular epithelium in experimental diabetes [26] and clinical diabetes (this work) may be impaired by hexosamine pathway-linked decreased expression of the genes encoding THTR-1 and THTR-2, inhibition of these thiamine transporters by dicarbonyl glycation [27] and acidification of the tubular lumen [28]. This deserves further investigation.\nA weakness of the study was the finding of no strong link to a recognised clinical endpoint such as incipient nephropathy judged by microalbuminuria, although there was a weak negative correlation of erythrocyte TK activity with urinary albumin excretion. It is becoming increasingly evident, however, that urinary albumin excretion may not be a reliable marker of early stage decline in renal function in diabetes [29]. Future studies would be probably better addressed to investigation of the link between changes in thiamine homeostasis and early decline in GFR assessed by cystatin C measurement [30].\nDecreased plasma thiamine concentration has been reported in diabetic patients previously in studies with small patient numbers [31, 32] but ClThiamine and FEThiamine, thiamine and TMP transporter content of erythrocyte membranes and plasma sVCAM-1 were not evaluated and hence links to these variables were not disclosed. Thiamine deficiency is conventionally assessed by measuring the percentage unsaturation of erythrocyte TK with TPP cofactor [12]. Herein we found, however, that plasma deficiency of thiamine in diabetes was masked in erythrocytes by increased levels of THTR-1 and RFC-1 transporter protein. As erythrocytes lack protein synthetic capability, increased protein levels of these transporters was produced by increased expression of the genes encoding THTR-1 and RFC-1 in the erythrocyte precursors, reticulocytes and erythroblasts. Indeed, increased levels of THTR-1 and RFC-1 transporter proteins were also found in MNLs in these diabetic patients (data not shown). Increased thiamine transporter gene expression and protein level is a response to thiamine deficiency [21, 33] and is consistent with the low plasma thiamine concentration found in diabetic patients herein. This explains why a low plasma thiamine concentration in clinical diabetes has hitherto gone unrecognised. This study indicates that the measurement of the \u2018thiamine effect\u2019 based on erythrocyte TK activities is an inadequate assessment of clinical thiamine status as it can be masked by changes in thiamine transporter protein levels.\nLow plasma thiamine concentration in diabetes may be of limited significance if tissues can upregulate the gene expression and protein levels of thiamine transporters and maintain normal TK activity. Although this occurs in the normoglycaemic state [21, 33], our recent studies of experimental diabetes indicated it does not occur in renal glomeruli in the diabetic state [26] where enhanced hexosamine signalling may block increased expression of the genes encoding THTR-1, THTR-2 and RFC-1 and their protein levels. In diabetic glomeruli, TK activity and level of TK protein was decreased 60% [26], and similar impairment of thiamine uptake and metabolism may occur in the diabetic retina and peripheral nerve [34, 35]. Indeed, RFC-1 has impaired gene expression and protein levels in the diabetic retina [35].\nDecreased availability of thiamine in vascular cells in diabetes exacerbates metabolic dysfunction in hyperglycaemia. Increased plasma sVCAM-1 is a marker of endothelial dysfunction [36] and increased risk of atherosclerosis [37]. sVCAM-1 was increased in diabetic patients with normal renal function [38] and was linked to microvascular and macrovascular complications in diabetes [39\u201341], although the link to glycaemic control is less certain [39, 42, 43]. Low plasma thiamine concentration may be a confounding factor linked to increased sVCAM-1 in diabetes.\nThis study indicates that type 1 and type 2 diabetic patients in the UK exhibit low plasma thiamine concentration. The conventional indicator of thiamine sufficiency, erythrocyte TK activity, is masked in clinical diabetes by increased protein levels of thiamine and TMP transporters, THTR-1 and RFC-1. The deficiency of thiamine in clinical diabetes may increase the fragility of vascular cells to the adverse effects of hyperglycaemia and thereby increase the risk of developing microvascular complications. Correction of the low plasma thiamine concentration with thiamine supplements may decrease the risk of microvascular complications in diabetes.\nImportant areas for future study are: (1) confirmation of low plasma thiamine concentrations in diabetic populations of other countries independent of local dietary and culinary practice; (2) the evaluation of thiamine and thiamine derivatives to correct low plasma thiamine concentration in diabetes, reverse vascular dysfunction and prevent vascular complications; and (3) investigation of the mechanism of increased ClThiamine in diabetes. These studies are either ongoing or in preparation.","keyphrases":["thiamine","type 2 diabetes","microalbuminuria","type 1 diabetes","adhesion molecules"],"prmu":["P","P","P","P","M"]}
{"id":"Eur_J_Pharmacol-2-1-2258315","title":"Fucoidan partly prevents CCl4-induced liver fibrosis\n","text":"Fucoidan, a sulfated polysaccharide extracted from brown algae, has a wide range of biological activities, including anti-inflammatory, anti-viral, and anti-tumor activities. In the present study, we investigated the effects of fucoidan on CCl4-induced liver fibrosis. Administration of fucoidan reduced CCl4-induced acute and chronic liver failure. Hepatic fibrosis induced by CCl4 was also attenuated by injection of fucoidan. Damage to hepatocytes and activation of hepatic stellate cells are key events in liver fibrosis, and, interestingly, treatment of hepatocytes with fucoidan prevented CCl4-induced cell death and inhibited the proliferation hepatic stellate cells. These results indicate that fucoidan might be a promising anti-fibrotic agent possessing dual functions, namely, protection of hepatocytes and inhibition of hepatic stellate cell proliferation.\n1\nIntroduction\nHepatic fibrosis results from chronic damage to the liver in conjunction with the progressive accumulation of fibrillar extracellular matrix proteins (Friedman, 1993; Gressner, 1995; Lieber, 1999). The main causes of liver fibrosis are infection with hepatitis B or C, alcohol abuse, and non-alcohol steatohepatitis. There are over 100 million people with hepatic fibrosis in the world.\nThe liver consists of parenchymal (hepatocytes) and non-parenchymal cells (Kupffer, stellate, and endothelial cells). Hepatic fibrosis is triggered by specific intercellular interactions among some of these cells. Kupffer cells are activated by membrane components from damaged hepatocytes and infiltrating inflammatory cells. The activated Kupffer cells release pro-fibrotic factors, such as transforming growth factor-\u03b2, reactive oxygen species, and other factors (Wu et al., 1998). These pro-fibrotic factors act on hepatic stellate cells, which are key players in hepatic fibrosis (Gressner, 1995).\nHepatic stellate cells are normally quiescent and produce only small amounts of extracellular matrix components, such as laminin and collagen type IV, during the formation of basement membrane (Maher and Bissell, 1993). Exposure of hepatic stellate cells to the pro-fibrotic factors activates hepatic stellate cells, leading to a changed to a myofibroblast phenotype and an increase in the production of collagen (Friedman, 1999; Geerts, 2001). Preventing the activation of hepatic stellate cells is thus a promising anti-fibrotic strategy. Indeed, administration of antioxidants has been shown to suppress the activation of hepatic stellate cells, thereby preventing liver fibrosis, and inhibition of hepatocyte cell death can reduce liver fibrosis (Houglum et al., 1997; Maher et al., 1997; Horie et al., 2003; Canbay et al., 2002; Song et al., 2003).\nBrown algae seaweeds contain both soluble dietary fiber polysaccharides, including alginates, fucans, and laminarans, and insoluble fibers, which are mostly composed of cellulose (Lahaye and Kaeffer, 1997; Kloareg and Quatrano, 1988). The fucans are cell wall polysaccharides that contain variable amounts of fucose, uronic acids, galactose, xylose, and sulfate. They are classified in three types according to their chemical composition: fucoidan, ascophyllan, and glucuronofuco-galactan sulfate (Lahaye and Kaeffer, 1997; Kloareg and Quatrano, 1988; Mabeau et al., 1990). Fucoidan is a complex sulfated polysaccharide derived from Fucus vesiculosus, and it has a variety of biological activities, including anti-inflammatory, anti-viral, anti-liver failure, and anti-tumor activities (Boisson-Vidal et al., 1995; Saito et al., 2006). Furthermore, fucoidan interacts with transforming growth factor-\u03b2 and has antioxidative properties (Xue et al., 2001; McCaffrey et al., 1994). These findings indicate that fucoidan may have anti-fibrotic activity, but whether fucoidan can attenuate hepatic fibrosis is unknown. Therefore, in the present study, we evaluated the effects of fucoidan on hepatic injury and fibrosis. We found that fucoidan may be useful as a novel type of anti-fibrotic agent.\n2\nMaterials and methods\n2.1\nReagents\nCCl4 was purchased from Wako Pure Chemicals, Co. Ltd. (Osaka, Japan). Fucoidan was obtained from Sigma Chemical, Co. Ltd. (St. Louis, MO). CCl4 and fucoidan were dissolved in olive oil and saline in animal experiment, respectively. In cellular experiments, fucoidan was dissolved in cultured medium for hepatocytes or hepatic stellate cells as described below. CCl4 was dissolved in dimethylsulfoxide at 100\u00a0mM.\n2.2\nAnimals and experimental protocols\nAll of the experimental protocols conformed to the ethics guidelines of the Graduate School of Pharmaceutical Sciences, Osaka University. Male Sprague\u2013Dawley rats (200\u2013250\u00a0g) and male ddy mice (6\u00a0weeks old) were obtained from SLC (Shizuoka, Japan). The mice were housed in an environmentally controlled room (lights on from 8:00 to 20:00; temperature, 23\u00a0\u00b11.5\u00a0\u00b0C). Animals had free access to water and commercial chow (Type MF, Oriental Yeast, Tokyo, Japan).\nIn the acute liver injury model, CCl4 was intraperitoneally administrated to mice at 0.3\u00a0ml\/kg body weight with or without intravenous injection of fucoidan (25 and 50\u00a0mg\/kg body weight). After 24\u00a0h of fucoidan injection, the mice were anesthetized, and the blood was recovered. In the chronic liver injury model, CCl4 (0.3\u00a0ml\/kg body weight) and fucoidan (50\u00a0mg\/kg body weight) were administered orally and intravenously, respectively, to mice twice a week for 8\u00a0weeks. The liver and blood were recovered from the mice under anesthesia. The serum was separated from the blood and stored at \u2212\u00a020\u00a0\u00b0C before assay.\n2.3\nAspartate aminotransferase (AST) and alanine aminotransferase (ALT) assays\nSerum AST and ALT levels were measured using commercially available kits (Mitsubishi Kagaku Iatron Inc., Tokyo, Japan) according to the manufacturer's instructions.\n2.4\nAnalysis of fibrosis\nLiver specimens were fixed with 10% formaldehyde and embedded in paraffin. Tissue sections were mounted on slides, and Azan staining was performed to analyze the extent of fibrosis. After establishing a background for each micrograph, the number of pixels showing a blue color (stained collagen fibers) was determined with Scion Image (National Institutes of Health, Bethesda, MD), and the percentage of fibrosis in the liver was calculated as the ratio of the blue-colored area to the total area of the liver.\n2.5\nIsolation of hepatocytes and assay of viability\nHepatocytes were isolated from rats by perfusion of the liver with collagenase (Seglen, 1976). The cells were cultured in William's medium E containing 1\u00a0nM insulin, 1\u00a0nM dexamethasone, and 10% fetal bovine serum. The cells were seeded onto a dish at 1\u00a0\u00d7\u00a0105 cells\/cm2 in the absence or presence of fucoidan at 0, 0.3, 0.5, and 1.0\u00a0mg\/ml. After 6\u00a0h of treatment with fucoidan, CCl4 was added to the cells at 1\u00a0mM. After 3\u00a0h, the viability of the cells was assayed by staining with Trypan blue.\n2.6\nIsolation of hepatic stellate cells and assay of viability\nHepatic stellate cells were isolated from rat livers by perfusion with collagenase and pronase, followed by centrifugation on a Nycodenz cushion as described previously (Kawada et al., 1993). The cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The cells were then seeded onto a dish at 5\u00a0\u00d7\u00a0105 cells\/cm2 and then treated with fucoidan at 0, 0.3, 0.5, and 1.0\u00a0mg\/ml for 24 and 48\u00a0h. The viability of the cells was determined by mitochondrial conversion of 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Mosmann, 1983). The viability of the cells was calculated compared to cells at 0\u00a0h (i.e., prior to treatment with fucoidan).\n3\nResults\n3.1\nEffect of fucoidan on acute liver injury\nWe first examined the effect of fucoidan on acute liver failure induced by single injection of CCl4. Intraperitoneal injection of mice with CCl4 increased biochemical markers of liver injury. Specifically, 24\u00a0h after injection of CCl4, serum AST and ALT had increased from 125 to 707 and from 21 to 752 karumen unit\/ml, respectively (Fig. 1). Intravenous administration of fucoidan attenuated this elevation of serum AST and ALT (304 and 214 karumen unit\/ml, respectively, at 25\u00a0mg\/kg fucoidan). Notably, injection of 50\u00a0mg\/kg fucoidan restored serum AST and ALT to normal levels (77 and 67 karumen unit\/ml, respectively). Together, these results indicate that fucoidan is a potent inhibitor of acute CCl4-induced liver injury.\n3.2\nEffect of fucoidan on chronic liver injury\nTo evaluate the effect of fucoidan on chronic liver injury, we continuously administered CCl4 to mice twice a week for 8\u00a0weeks. Serum AST and ALT levels increased from 149 to 433 and from 101 to 568 karumen unit\/ml, respectively (Fig. 2A). Azan staining revealed that collagen accumulated in the liver, indicating the onset of liver fibrosis (Fig. 2B). Indeed, the fibrotic area in the liver increased from 1.9% to 14.8% of the whole liver (Fig. 2C). The elevation of AST and ALT levels was significantly reduced by administration of fucoidan (from 433 to 265 and from 568 to 238 karumen unit\/ml, respectively; Fig. 2A). The increase in fibrotic area induced by CCl4 was also attenuated by fucoidan (from 14.8% to 4.3%; Fig. 2C). Together, these findings show that fucoidan may be useful for treatment of hepatic fibrosis in chronic liver injury.\n3.3\nEffect of fucoidan on hepatocytes and stellate cells\nAs described above, our preliminary results indicated that fucoidan may be useful for the treatment of hepatic fibrosis. During the initiation of hepatic fibrosis, hepatocytes release a paracrine factor that stimulates hepatic stellate cell growth, leading to the damage of hepatocyte membranes (Gressner, 1995; Gutierrez-Reyes et al., 2007). Therefore, to determine how fucoidan prevents liver injury, we evaluated its effects on hepatocytes and hepatic stellate cells. As shown in Fig. 3A, treatment of hepatocytes with CCl4 (1\u00a0mM) reduced their viability to 63.3% of control (untreated) cells. In addition, fucoidan dose-dependently reduced CCl4-induced cell death, with complete prevention of cell death at 1\u00a0mg\/ml. Alone, fucoidan did not show any cytotoxicity at 1.0\u00a0mg\/ml in hepatocytes (data not shown). CCl4-induced cell death was not inhibited by pre-incubation of CCl4 with fucoidan (data not shown), indicating that the protective effects of fucoidan on CCl4-induced cell death is not due to absorption of CCl4 by fucoidan. In contrast, the viability of hepatic stellate cells was reduced to 26.4% of the control by a 48-h treatment with 1.0\u00a0mg\/ml fucoidan (Fig. 3B). Thus, both the protective effects of fucoidan against CCl4-induced cell death in hepatocytes and its cytotoxicity to hepatic stellate cells might contribute to its anti-fibrotic activity.\n4\nDiscussion\nFucoidan, the sulfated polysaccharides of brown algae, contains l-fucose residues as the main sugar constituent along with sulfate esters. Although fucoidan is known to have many biological activities, including anti-coagulant, anti-thrombosis, anti-inflammatory, anti-liver failure, and anti-tumor activities (Boisson-Vidal et al., 1995; Berteau and Mulloy, 2003; Saito et al., 2006), this is the first investigation of its anti-fibrotic activity. Our results show for the first time that fucoidan can reduce hepatic fibrosis in an animal model.\nStudies in animal models of hepatic fibrosis show that extracellular matrix components accumulate in interstitial regions of the liver around central veins or in the portal tracts. Normally, hepatic stellate cells exist in a quiescent state, but they become activated following liver injury. These activated hepatic stellate cells are primarily responsible for the excess production of extracellular matrix (Senoo et al., 1998). Thus, reduction of extracellular matrix production by activated hepatic stellate cells is crucial for the prevention of fibrogenesis. Damage to hepatocytes is the primary and continuing factor leading to hepatic stellate cell activation. Components released from the damaged cells, including lipid peroxides and reactive oxygen species, activate Kupffer cells in the liver, leading to their secretion of transforming growth factor-\u03b2 (Tsukamoto, 1999). These pro-fibrogenic factors activate hepatic stellate cells, which results in liver fibrosis. Here, we showed that treatment of hepatocytes with fucoidan attenuates CCl4-induced cell death.\nFucoidan has been reported to interact with transforming growth factor-\u03b2 and to scavenge reactive oxygen species (Boisson-Vidal et al., 1995; McCaffrey et al., 1994; Xue et al., 2001). CCl4 elevates serum transforming growth factor-\u03b2 levels and acts as a hepatotoxin by inducing the production of reactive oxygen species (Weiler-Normann et al., 2007). In agreement with this, we have found that administration of fucoidan reduces CCl4-induced lipid peroxidation (data not shown). Taken together, our results suggest that the anti-fibrogenic activity of fucoidan is due, at least in part, to attenuation of hepatic stellate cell activation by inhibition of transforming growth factor-\u03b2 and\/or by scavenging of reactive oxygen species, which can suppress the cascade of events that leads to hepatic stellate cell activation.\nHepatocyte injury is thought to promote fibrosis, leading to the release of activators from Kupffer cells. In contrast, hepatic stellate cell apoptosis is thought to be essential for the resolution phase of fibrosis (Canbay et al., 2002; Song et al., 2003; Iredale, 2001; Iredale et al., 1998; Issa et al., 2001). Thus, a compound that can prevent hepatocyte injury and\/or induce the death of hepatic stellate cells should be useful for the treatment of hepatic fibrosis. In the current studies we showed that fucoidan reduces the growth of hepatic stellate cells and that it can protect hepatocytes from injury. We also found that the fucoidan-treated hepatic stellate cells were stained with annexin V, a marker of apoptosis (data not shown). Taken together, these findings suggest that fucoidan may be useful for treating hepatic fibrosis.\nMost studies on fucoidan have used a commercially available crude extract from F. vesiculosus, and we used the crude fucoidan in the current study. Fucoidan contains heteropolysaccharides of various kinds besides those consisting predominantly of sulfate and fucose (Nishino et al., 1994). Patel et al. found that crude commercial fucoidan was more active than the purified fucoidan at inhibiting the proliferation of vascular smooth muscle cells, and then they speculated that a specific structure in the crude fucoidan may mediate its biological activities (Patel et al., 2002). Indeed, the content of the sulfated groups in fucoidan determines its anti-proliferative and anti-coagulant activities in fibroblasts (Haroun-Bouhedja et al., 2000). The biological activities may differ among the various structures of pure fucoidan as well as the different components of crude fucoidan. Identification of the structures of fucoidan that protect hepatocytes from hepatotoxins and that inhibit hepatic stellate cell growth is needed for the development of fucoidan as an anti-fibrotic agent.\nIn summary, we found that fucoidan prevents hepatocyte cell death and induces the death of hepatic stellate cells in an animal model of hepatic fibrosis. Future studies will examine the molecular mechanisms of fucoidan in hepatocytes and hepatic stellate cells. This is the first report that fucoidan has anti-fibrotic activity and that it is a promising lead for the development of anti-fibrotic agents. Identification of the molecular target and the active structure of fucoidan may lead to the development of novel anti-fibrotic agents.","keyphrases":["fucoidan","liver fibrosis","hepatocyte","hepatic stellate cell"],"prmu":["P","P","P","P"]}
{"id":"In_Vitro_Cell_Dev_Biol_Anim-4-1-2277440","title":"Effects of temperature and doxorubicin exposure on keratinocyte damage in vitro\n","text":"Cancer chemotherapy treatment often leads to hair loss, which may be prevented by cooling the scalp during drug administration. The current hypothesis for the hair preservative effect of scalp cooling is that cooling of the scalp skin reduces blood flow (perfusion) and chemical reaction rates. Reduced perfusion leads to less drugs available for uptake, whereas the reduced temperature decreases uptake of and damage by chemotherapy. Altogether, less damage is exerted to the hair cells, and the hair is preserved. However, the two mechanisms in the hypothesis have not been quantified yet. To quantify the effect of reduced drug damage caused by falling temperatures, we investigated the effect of local drug concentration and local tissue temperature on hair cell damage using in vitro experiments on keratinocytes. Cells were exposed for 4 h to a wide range of doxorubicin concentrations. During exposure, cells were kept at different temperatures. Cell viability was determined after 3 d using a viability test. Control samples were used to establish a concentration\u2013viability curve. Results show that cell survival is significantly higher in cooled cells (T < 22\u00b0 C) than in non-cooled cells (T = 37\u00b0 C), but no significant differences are visible between T = 10\u00b0 C and T = 22\u00b0 C. Based on this result and previous work, we can conclude that there is an optimal temperature in scalp cooling. Further cooling will only result in unnecessary discomfort for the patient and should therefore be avoided.\nIntroduction\nChemotherapy-induced hair loss is a feared side effect of cancer treatment (Katsimbri et al. 2000). Scalp cooling during administration of chemotherapy prevents hair loss (e.g., Ridderheim et al. 2003). Cooling can be achieved by means of a cap that is pre-cooled in a freezer or that exchanges coolant with a reservoir. The hair preservative effect of scalp cooling is attributed both to reduced blood flow by vasoconstriction and to reduced reaction rates in the body at the level of the hair follicle. When the scalp is cooled, vasoconstriction is induced, and through this, blood flow to the hair follicle is decreased. This reduced blood flow results in a decrease of the total amount of cytotoxic drug available for uptake in the hair follicle. The effect of reduced perfusion was studied by Janssen et al. (2007), and it was found that cooling the scalp to 20\u00b0 C reduces local blood flow down to 20% of normal. A further decrease in temperature did not result in a further decrease in local blood flow. Based on this, it might be expected that there is a limit in temperature below which the effectiveness of scalp cooling will not increase anymore.\nHowever, the hair preservative effect of scalp cooling is also attributed to reduced cell metabolism. Because of reduced temperatures during scalp cooling, cellular drug uptake and damage are assumed to be lower, and with this, hair follicles are thought to be less susceptible to cytotoxic drugs. Decorti et al. (2003) showed that in vitro doxorubicin uptake in kidney cells at 37\u00b0 C was 4.5 times higher than drug uptake at 4\u00b0 C. In addition, Decorti also showed that doxorubicin uptake is dependent on extracellular concentration. Unfortunately, no studies have been performed to quantify these processes in the human hair follicle.\nFor a better understanding of the functioning of scalp cooling, it is important to quantify the contribution of reduced drug uptake and drug damage, if any, on the hair preservative effect of scalp cooling. To this end, we investigated experimentally the relationship between doxorubicin exposure and cell damage at different temperatures using normal human epidermal keratinocytes.\nChemotherapy disrupts the rapidly dividing keratinocytes (the cells that actually produce the hair shaft) in the hair follicle (Cotsarelis and Millar 2001). Therefore, the use of keratinocytes as an in vitro model is indicative for chemotherapy-induced damage to the hair follicle. Moreover, human keratinocytes are commercially available and they are easy to cultivate, which means that with this model, a wide range of boundary conditions can quickly be investigated.\nThe goal of the experiments was to assess the effects of temperature and chemotherapy on keratinocyte survival.\nMaterials and Methods\nPooled neonatal normal human epidermal keratinocytes (NHEK) were obtained from Cambrex Bio Science Verviers, Belgium (catalog number CC-2507). These cells have a doubling time of approximately 24\u00a0h. Cells were cultured in T-75 flasks at a seeding density of 3,500 cells cm2 under an atmosphere of 95% air and 5% CO2 at 37\u00b0 C using 15\u00a0ml of keratinocyte general medium (KGM2; Cambrex Bio Science Verviers, catalog number CC-3107). Medium was refreshed the day after plating, and afterwards every other day until cells reached 70\u201380% confluence. Then, cells were harvested using 6\u00a0ml of trypsin solution and subsequently cryopreserved in liquid nitrogen at a density of 1.2\u2009\u00d7\u2009106 cells per milliliter using 80% KGM2, 10% fetal bovine serum, and 10% dimethyl sulfoxide (DMSO).\nFor the experiments on the effect of temperature and chemotherapy on keratinocyte survival, third passage cells were plated in 24-well plates at a cell density of 6,000 cells per square centimeter. Cells were incubated at 37\u00b0 C for a period of 24\u00a0h to allow the cells to recover from handling. Concentrations of doxorubicin in KGM2 (0.01, 0.04, 0.1, 0.5, 1.0, 3.0, or 10.0\u00a0\u03bcg ml\u22121) were prepared. Cells were then exposed to 250\u00a0\u03bcl of a specific doxorubicin concentration. Plates were incubated for 4\u00a0h at either a low temperature (TL\u2009=\u200910\u00b0 C), a medium temperature (TM\u2009=\u200922\u00b0 C), or a high temperature (TH\u2009=\u200937\u00b0 C). For each combination of temperature and doxorubicin concentration, a sample size of eight was used. Cells receiving medium without any doxorubicin were used as a control group for each specific temperature. At the end of the exposure time, doxorubicin was removed, and cells were washed with 500\u00a0\u03bcl phosphate-buffered saline (PBS). Fresh medium (250\u00a0\u03bcl per well) was added to each well, and plates were then incubated at 37\u00b0 C for a post-exposure time of 72\u00a0h, after which, a viability assay was performed as described below.\nThe damage to the NHEK cells exposed to different temperatures and doxorubicin concentrations was determined by a colorimetric MTT (tetrazolium) viability assay. The assay is based on the observation that viable cells have the ability to metabolize a water-soluble tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) into a product termed purple formazan (Mosmann 1983; Edmondson et al., 1988; Sgouras and Duncan, 1990). The purple formazan can be solubilized, and the optical density of the solute can be determined using a spectrophotometric technique. The resulting absorbance is directly proportional to the number of cells, and this linearity extends over a wide range of cell numbers (Mosmann 1983). A standard MTT solution was prepared by dissolving 5\u00a0mg ml\u22121 of MTT (MTT formazan, Sigma Aldrich, Zwijndrecht, The Netherlands) in PBS. The standard MTT solution was added to complete medium (KGM2) in a ratio of 1 to 10 to obtain a final solution of 10% MTT solution and 90% KGM2. A volume of 220\u00a0\u03bcl of this solution was added to all test wells, and the plates were left to incubate at 37\u00b0 C and 5% CO2 for a period of 45\u00a0min. After this period, the MTT\/medium solution was removed, and 200\u00a0\u03bcl of a solution of 90% DMSO and 10% Triton X was added to extract the purple formazan salt from the cells. Plates were sealed and kept in dark for 30\u00a0min, after which, 100\u00a0\u03bcl of each well was transferred to a new well of a 96-well plate. The optical density (OD) was measured using an automated spectrophotometric plate reader (Synergy HT, BioTek Instruments Inc., Winooski, VT, USA) set to 570\u00a0nm and using a reference wavelength of 650\u00a0nm. Blanks (100\u00a0\u03bcl of DMSO\/Triton X solution only) were used as an extra control. For each well, the corrected optical density OD(570\u2013650\u00a0nm) was provided by the plate reader. Mean optical density and standard error of each temperature and concentration group [ODT,C\u2009\u00b1\u2009SE(ODT,C)] and their respective control group [ODT,0\u2009\u00b1\u2009SE(ODT,0)] were calculated. Based on these values, the viability (S) for each temperature and concentration group can be calculated as:\nAn analysis of variance (ANOVA) was performed using statistical software (SPSS version 15.0, SPSS Inc., Chicago, IL). The ANOVA test is used to investigate whether significant differences in mean values exist between two or more groups. In this study, we want to investigate what the effect of temperature on the one hand, and the effect of doxorubicin concentration on the other hand, is on cell survival. Using a general linear model, the viability is statistically modeled as:\nThis means that we investigated whether temperature, concentration, and an interaction term are significantly relevant for describing our data. Where appropriate, a post hoc analysis was used using Tamhane\u2019s T2 criterium (based on unequal variances) to test for differences between various doxorubicin concentrations and various exposure temperatures. A significance level of p\u2009<\u20090.05 was used in these statistical tests.\nResults\nMicroscopic photographs of cells at selected concentrations are shown in Fig.\u00a01. Here, we can see that the control groups of all temperatures show similar cell counts. With increasing concentration, the number of cells decreases. At 3\u00a0\u03bcg ml\u22121 (C4), a clear difference is visible between the high temperature group and both the medium and low temperature group. The difference between these groups at other concentrations is less well defined. It can be seen that there is a difference in cell morphology between 0.04\u00a0\u03bcg ml\u22121 (C2) and 0.5\u00a0\u03bcg ml\u22121 (C3). Cells in the latter group, and in groups with higher doxorubicin concentrations, show enlarged and flattened cell shape and increased granularity. These are characteristics of senescence or aging, which is known to be caused by doxorubicin (Roninson 2003). The results of the viability determination are shown in Fig.\u00a02. This figure shows the cell viability as a function of doxorubicin concentration for different doxorubicin exposure temperatures. In this figure, we can see that the viability of keratinocytes as a function of doxorubicin concentration shows a decreasing S-curve with increasing concentrations. Viability levels are slightly above 1 for low concentrations (0.01\u00a0\u03bcg ml\u22121), and with increasing concentrations, viability gradually drops towards zero for high concentrations (10\u00a0\u03bcg ml\u22121). For different temperature groups, cell viability at a specific concentration is always lowest for the high temperature group. In the mid-section of the concentration range (i.e., 0.1\u20131\u00a0\u03bcg ml\u22121), the differences between the high temperature group (TH) and the two lower temperature groups (TL and TM) are more pronounced. Cell viability for these lower temperature groups decreases more slowly than for the high temperature group, until at a concentration of 1\u00a0\u03bcg ml\u22121, a rapid drop in cell viability is visible. The difference between the individual temperature groups at low and high concentrations is therefore small.\nFigure\u00a01.Microscopic photographs of cells exposed to selected doxorubicin concentrations. Photographs are taken at a magnification of ten times. C1\u2009=\u20090.01\u00a0\u03bcg ml\u22121, C2\u2009=\u20090.04\u00a0\u03bcg ml\u22121, C3\u2009=\u20090.5\u00a0\u03bcg ml\u22121, C4\u2009=\u20093.0\u00a0\u03bcg ml\u22121, C5\u2009=\u200910\u00a0\u03bcg ml\u22121.Figure\u00a02.Cell viability as a function of doxorubicin concentration at different temperatures. Delta: TL (10\u00b0 C), filled square: TM (22\u00b0 C), and open circle: TH (37\u00b0 C). Data points show the mean and standard error of eight replicates. The results of a significance test are shown in Table\u00a01.\nFrom an ANOVA analysis, we found that the effect of doxorubicin concentration on NHEK viability was highly significant [F(6,161)\u2009=\u2009165.213, p\u2009<\u20090.001], as was the effect of temperature on NHEK viability [F(6,161)\u2009=\u200937.054, p\u2009<\u20090.001]. The effect size of doxorubicin (; partial eta squared) was higher than the effect size of temperature (\u2009=\u20090.88 compared to \u2009=\u20090.36).\nFor all temperature groups, increased doxorubicin concentration has a significant decreasing effect on cell viability. This effect was highly significant for the high temperature group [F(6,49)\u2009=\u2009119.400, p\u2009<\u20090.001], the intermediate temperature group [F(6,49)\u2009=\u200933.166, p\u2009<\u20090.001], and the lowest temperature group [F(6,49)\u2009=\u200968.225, p\u2009<\u20090.001] Based on these results, a post hoc analysis was performed for each temperature group to check where a significant transition in cell viability as function of doxorubicin concentration occurs. This transition point may be viewed as the inflection point of the S-curve. For the low and medium temperature group, we found a transition point between 1.0 and 3.0\u00a0\u03bcg ml\u22121. The highest temperature group has a transition point slightly below 0.5\u00a0\u03bcg ml\u22121.\nThe ANOVA analysis also revealed a significant interaction between doxorubicin and temperature on cell viability [F(6,161)\u2009=\u20092.754, p\u2009=\u20090.002], although the effect is smaller than that of temperature and doxorubicin (\u2009=\u20090.199). This significant interaction term shows that the effect of temperature on cell viability is not the same at the levels of different doxorubicin concentrations. For low and high doxorubicin concentrations, there is hardly any influence of a lower temperature on cell survival. In the mid-section of the graph this influence is far more substantial.\nThe results of the ANOVA analysis are summarized in Table\u00a01. It can be seen that significant differences exist between TL and TH for concentrations of 0.1\u00a0\u03bcg ml\u22121 and up, except for a concentration of 1\u00a0\u03bcg ml\u22121. At this concentration, the significance level is not high enough, but still, a trend is visible (p\u2009<\u20090.1). Between TM and TH, significant differences exist for concentrations of 0.5\u00a0\u03bcg ml\u22121 and up, except for a concentration of 1\u00a0\u03bcg ml\u22121 where no significant difference is visible. The only significant difference between TL and TM exists at a concentration of 10\u00a0\u03bcg ml\u22121, although a trend is visible at a concentration of 0.1\u00a0\u03bcg ml\u22121.\nTable\u00a01.Results of an ANOVA analysis for determining the significance between different temperature groups at a specific doxorubicin concentrationDoxorubicin (\u03bcg ml\u22121)F valuep valueTL vs THTM vs THTL vs TM0.01F(2,21)\u2009=\u20090.3150.733\u00a0\u00a0\u00a00.04F(2,21)\u2009=\u20091.9580.166\u00a0\u00a0\u00a00.1F(2,21)\u2009=\u200915.187<0.001p\u2009<\u20090.01\u00a0p\u2009<\u20090.1*0.5F(2,21)\u2009=\u200931.605<0.001p\u2009<\u20090.001p\u2009<\u20090.001\u00a01F(2,21)\u2009=\u20094.380<0.05p\u2009<\u20090.1*\u00a0\u00a03F(2,21)\u2009=\u200912.908<0.001p\u2009<\u20090.01p\u2009<\u20090.01\u00a010F(2,21)\u2009=\u200922.587<0.001p\u2009<\u20090.001p\u2009<\u20090.05p\u2009<\u20090.05p Values are shown with their respective significance level. Trends (p\u2009<\u20090.1) are marked with an asterisk. See text for more detail.\nDiscussion\nWe examined the role reduced metabolism has on the hair preservative effect of scalp cooling. For this, a controlled in vitro experiment was conducted to assess the damage exerted by doxorubicin on human epidermal keratinocytes for a range of chemotherapy concentrations and exposure temperatures. The effects of temperature and doxorubicin on cell damage were determined from cell viability measurements (MTT assay).\nWe found that increased doxorubicin concentrations have a significant decreasing effect on keratinocyte cell viability. Viability was close to 1 for low concentrations and close to 0 for high concentrations. In between, the relationship between doxorubicin concentration and cell viability shows an S-curve. We also found that reduced temperature has a significant increasing effect on keratinocyte cell viability. The two cooled temperature groups (10\u00b0 C and 22\u00b0 C) show a higher cell viability for each doxorubicin concentration than the 37\u00b0 C group. At the mid-range of doxorubicin concentrations (0.04\u20131\u00a0\u03bcg ml\u22121), the effect of reduced temperature is more pronounced than in the extreme values of doxorubicin concentration. This observation is confirmed by the fact that an interaction term of doxorubicin concentration and exposure temperature was significant. The increased effect of reduced temperature was significant for doxorubicin concentrations of 0.1\u00a0\u03bcg ml\u22121 and higher. We found no significant differences between the low (10\u00b0 C) and medium (22\u00b0 C) group.\nThe results of the experiment are in line with the hypothesis of scalp cooling. In this hypothesis, the reduced temperature caused by scalp cooling will result in lower perfusion and a lower cellular metabolism. With this, the supply, uptake and damage of a chemotherapeutic agent will be diminished.\nFor low doxorubicin concentrations (0.01 and 0.04\u00a0\u03bcg ml\u22121), the MTT assay for each temperature group showed a higher viability than the respective control group. Because of the fact that each specific temperature group has its own control group, it is unlikely that this effect is caused by a measurement error. This means that apparently, cells are metabolically more active when exposed to a small amount of doxorubicin. It is possible that cells in the control group are close to reaching confluence at the time that viability is determined. As cells reach confluence, there is not enough room for new cells. Therefore, the rate at which cells divide will become lower, and hence, their metabolic activity will decrease. Another possibility is that a small amount of doxorubicin will stimulate cells to repair damage exerted to the cells. Hence, it may actually result in larger cell growth compared to a control group receiving no doxorubicin. At this point, however, we have no indication for which explanation may be true.\nAnother remarkable result is that there is no significant difference between the medium and low temperature group. Thus, it seems that there is a limit in effect of reduced temperature. In a pilot study to determine optimal doxorubicin exposure temperature, we found that an exposure temperature of 26\u00b0 C also showed increased cell viability (data not shown), and this increase did not differ from other temperature groups. During scalp cooling, a skin temperature of approximately 20\u00b0 C is reached. Based on the results of our experiment, one might expect that the limit in effect of reduced temperature during scalp cooling may be reached for a temperature as high as 26\u00b0 C. This means that during scalp cooling, the effect of reduced metabolism is already at its maximum. However, care has to be taken when the results of an in vitro experiment are generalized. Therefore, further studies on the effect of reduced temperature on cell damage are needed to exactly define this limit. Ideally, an in vitro hair follicle model is used in these studies. Our current study confines the boundary conditions in doxorubicin concentration and exposure temperature that can be used in these further studies.\nRecently, it was shown that there is a limit in perfusion during scalp cooling (Janssen et al. 2007). Cooling the scalp to approximately 18\u00b0 C resulted in a decrease in perfusion to 20%. Further cooling did not result in any further decrease in perfusion. The findings of the current study indicate that there is no difference in cell survival between 22\u00b0 C and 10\u00b0 C. Based on these findings, we can conclude that there is an optimal temperature in scalp cooling. Further cooling will only result in unnecessary discomfort for the patient. Therefore, strong cooling during scalp cooling should be avoided.","keyphrases":["doxorubicin","keratinocytes","scalp cooling","viability","chemotherapy induced hair loss"],"prmu":["P","P","P","P","R"]}
{"id":"Knee_Surg_Sports_Traumatol_Arthrosc-3-1-2082652","title":"Embolism of the popliteal artery after anterior cruciate ligament reconstruction: a case report and literature review\n","text":"Arterial complications after anterior cruciate ligament reconstruction (ACLR) are rare. We present a case report of a 44-year-old male patient with a subtotal occlusion of the popliteal artery, with sensory loss in the foot, 17 days after ACLR. Embolectomy and anticoagulant therapy led to full recovery of the peripheral arterial circulation. The sensory loss of the foot also fully recovered. To our knowledge, this is the first case report of an embolus of the popliteal artery after ACLR without relation to graft fixation. A literature review on vascular complications after ACLR is presented.\nIntroduction\nVascular complications after anterior cruciate ligament reconstruction (ACLR) are rare. Few peer reviewed case reports have been reported with various techniques of reconstruction [1, 3\u20139]. Allum did not report vascular lesions as a complication after ACLR in a review article on this subject [2]. The origin of vascular lesions after ACLR may be venous or arterial [4]. We present a case of arterial embolism of the popliteal artery after ACLR. A literature review on vascular complications after ACLR is presented.\nCase report\nA 44-year-old male has a previous medical history of open medial and lateral ligament repair of the right knee 15\u00a0years previously (motor accident). Functional instability of the right knee due to ACL deficiency was the reason for referral to our service. There were no signs of posterior nor posterolateral instability. An ACLR was performed with a quadruple hamstring graft. The graft was fixed with a Bone Mulch Screw on the femoral side and a WasherLoc device in the tibia (Arthrotek, Inc. Warsaw, USA). The latter is a spiked washer with bicortical screw fixation. Total tourniquet time was 90\u00a0min. No complications were noted during or after surgery. Thromboprophylaxis for deep venous thrombosis was given by means of Low Molecular Weight Heparin (2500\u00a0IE daily) during hospital stay. The hospital recovery was uneventful.\nOn the 17th day of post surgery, he experienced pain and swelling in the popliteal fossa of the right knee. The complaints partially resolved with physiotherapy. Two days later, the fossa pain returned with alterations of skin color, sensory loss and an increasing cold right foot. He was referred to a vascular surgeon. Adequate dorsal pedal and posterior tibial pulses were noted. Duplex ultrasound examination showed no sign of venous thrombosis. Angiography revealed a subtotal occlusion of the popliteal artery at the level of the superior genicular artery (Fig.\u00a01). An embolectomy was performed using a Fogarty catheter inserted in the femoral artery. The pedal pulses were diminished after embolectomy and a second angiography was performed. The occlusion at the level of the popliteal artery was no longer detected. No further emboli were noted, however, the peripheral flow qualified as too slow and suspect of small distal occlusions. Anticoagulant therapy with intravenous heparin as well as epidural analgesia was administered until complete recovery of peripheral circulation. The patient developed a superficial infection of the groin wound, treated by antibiotics. He was mobilized and discharged after 8\u00a0days. Sensory loss of the foot slowly recovered after 4\u00a0months. Vascular analysis in rest and strenuous activity was performed at 4\u00a0months. He had no more complaints, symmetrical ankle-brachial index in both legs and intact pulses at the foot and ankle. Vascular analysis did not reveal any other possible cause for arterial emboli. The patient has full range of motion of the right knee with a Lachman and anterior drawer test of 0\u20132\u00a0mm (International Knee Documentation Committee) and absent pivot shift test.\nFig.\u00a01Angiography of the right knee showing subtotal occlusion of the popliteal artery at the level of the superior genicular artery (reproduced with permission from [4])\nDiscussion\nVascular complications after ACLR are rare. The origin of vascular lesions may be venous or arterial [4]. A case of fatal pulmonary embolism after ACLR with venous origin has been published recently [5]. Hypothesis of the cause was a hereditary coagulopathy. Arterial lesions of the popliteal artery after ACLR have been presented in few peer reviewed case reports even with an all-inside technique of arthroscopic ACL reconstruction and fixation as well as any type of graft [4].\nRoth et al. [8] described an occlusion of the proximal popliteal artery. A composite graft consisting of a polypropylene ligament augmentation and the middle third of the quadriceps-patellar tendon was fixed to the lateral femur with a staple. The artery was trapped between the graft and the femur. A saphenous bypass was performed 6\u00a0weeks post surgery.\nSpalding et al. [9] reported a case of unilateral claudication 8\u00a0years after ACL reconstruction with use of a Gore-Tex ligament. A cyst had formed around the femoral insertion of the ruptured synthetic ligament and was excised without vascular repair.\nEvans et al. [3] reported a pseudoaneurysm of the medial inferior genicular artery following ACL reconstruction using a central third patellar tendon graft fixed with interference screws. At 5\u00a0weeks, ligation of the artery and removal of the thrombus led to full recovery. The cause of the lesion was elevation of the periosteum on the medial side of the tibia for tibial tunnel preparation.\nAldridge et al. [1] described an avulsion of the middle genicular artery after a bone-patellar tendon-bone autograft fixed with interference screws. Surgical exploration at 4\u00a0weeks revealed a tear in the popliteal artery. There was no rupture of the posterior capsule. Probable cause for the avulsion of the middle genicular artery was the debridement of the femoral ACL remnant tissue.\nWe have previously published two cases of popliteal artery lesions caused by a drill for bicortical tibia fixation after quadruple hamstring ACLR [6, 7]. In the first case, the drill had caused an intimal lesion at the level of the infragenicular popliteal artery which led to the pseudoaneurysm. Vascular repair was performed 12\u00a0days after ACL reconstruction but sensory loss of the saphenous and medial plantar nerves was still present at 4\u00a0months follow-up [7]. The second case was a simultaneous traumatic pseudoaneurysm and thrombosis of the popliteal artery after ACLR. At surgical exploration, the thrombosis was in line with the drill hole for bicortical tibial fixation. There was no apparent relation of the femoral fixation device to the pseudoaneurysm of the supragenicular popliteal artery. This pseudoaneurysm was thought to be pre-existent. The pseudoaneurysm was ligated and a venous jump graft was performed to bypass the thrombosis located more distally in the popliteal artery [6].\nIn this review of the literature on arterial complications after ACLR, all cases are associated with direct damage to the popliteal artery at time of ACLR [1, 3, 6\u20139]. There was no apparent direct damage to the popliteal artery in the 44-year-old male patient presented in this case report. The popliteal artery occlusion was not in line with either the femoral nor tibial fixation device. Vascular analysis did not reveal any pre-existent vascular causes for arterial embolus formation proximal to the popliteal artery. Our hypothesis of the cause was the traumatic knee dislocation 15\u00a0years previously. Precursors could have been pre-existent intimal vascular damage or adhesions of the artery at the level of the superior genicular artery in combination with the use of the tourniquet and ACLR.\nConclusion\nAwareness of possible arterial complications after ACL reconstruction is essential for early diagnosis. Clinical symptoms of pain in the popliteal fossa and sensory deficits in lower leg and foot should prompt the physician to analyze possible injuries of the popliteal artery. The differential diagnosis should include compartment syndrome and deep venous thrombosis. Doppler examination as well as intact pedal arterial pulses are unreliable in diagnosing arterial lesions after ACL reconstruction. Contrast-, CT- or MRI-angiography are the diagnostic tools of choice. Immediate surgical exploration is indicated to limit limb ischemia and neurological damage [4].","keyphrases":["embolism","popliteal artery","anterior cruciate ligament reconstruction","literature review","embolectomy","vascular complication"],"prmu":["P","P","P","P","P","P"]}
{"id":"Int_J_Cardiovasc_Imaging-3-1-2121121","title":"Editorial to Echocardiographic assessment of left atrial ejection force and kinetic energy in chronic congestive heart failure\n","text":"It is well known, that the left atrium plays an important role in filling of the left ventricle: In first instance by being a reservoir of expansion during systole, than by being a conduit phase during diastole and finally by an active phase (if sinus rhythm is present) during late diastole [1]. This active phase has an important role in compensation of decreased left ventricular compliance in patients with heart failure. It is well known, that cardiac output decreases with 15\u201320% in patients who are in atrial fibrillation or are only paced in the ventricle [2]. This is not only of importance from pathophysiological point of view, but left atrial function is also an independent determinant of prognosis in patients with heart failure [3]. Stretch of the left atrium is generally controlled by intra-atrial pressure. The Frank\u2013Starling mechanism of the left atrium can be described by a biphasic increase of contraction force after increase of the stretch level.\nLeft atrial function has been investigated with echocardiography for a long time [4].\nOne can make simple measurements of left atrial size or left atrial volume but also try to quantify left atrial function with Doppler and tissue Doppler. During heart failure, atrial function is characterised by augmented reservoir and pump function. This can be measured with pulsed wave Doppler across the mitral valve, which is generally expressed by a decreased E\/A ratio. However, there is a marked heterogeneity among heart failure patients with pseudo normal patterns and restrictive filling patterns. This makes it difficult to use pulsed wave Doppler as a determinant of prognosis and guide therapy. The current paper of Tripokiadis et\u00a0al. [5], describes the measurement of left atrial ejection volume and left atrial ejection force in patients with heart failure. The method is not new and has been described in normal volunteers and patients with atrial fibrillation [6]. Although there were some limitations of the study in sample size, matching of age groups and gender, the authors were able to demonstrate, that left atrial function increases during heart failure and so compensated the decrease of left ventricular function. The model, described by Tripokiadis et\u00a0al. can be used to estimate atrial function in patients with systolic and diastolic left ventricular dysfunction but also to evaluate the influence of atrial contribution to ventricular filling after medical treatment. Doing so, individual treatment can be optimised not only taking into account the clinical status, renal status, ventricular remodelling and neurohormonal status (BNP), but also atrial contribution to left ventricular filling.","keyphrases":["heart failure","left atrial function","echocardiography"],"prmu":["P","P","P"]}
{"id":"Exp_Cell_Res-1-5-2131725","title":"Concerted action of Aurora B, Polo and NHK-1 kinases in centromere-specific histone 2A phosphorylation\n","text":"The spatial and temporal control of histone modifications is crucial for precise regulation of chromatin structure and function. Here we report that phosphorylation of H2A at threonine 119 (T119) is enriched at centromere regions in Drosophila mitosis. We found that the Aurora B kinase complex is essential for this phosphorylation at centromeres, while Polo kinase is required to down-regulate H2A phosphorylation on chromosome arms in mitosis. Cyclin B degradation triggers loss of centromeric H2A phosphorylation at anaphase onset. Epistasis analysis indicated that Polo functions upstream of the H2A kinase NHK-1 but parallel to Aurora B. Therefore, multiple mitotic kinases work together to specify the spatial and temporal pattern of H2A T119 phosphorylation.\nIntroduction\nIn eukaryotes, genomic DNA is first packaged into nucleosomes and then organised into higher-order chromatin structures. Chromatin organisation is locally or globally changed in response to external and internal signals. The changes are required for executing critical biological functions, most notably in regulated gene expression and chromosome segregation. Various post-translational modifications take place on histones, mostly in their tail domains, and play critical roles in the regulation of chromatin structure and function, either directly or indirectly through the recruitment of specific chromatin binding proteins [1]. The importance of histone modifications in gene expression is well appreciated and has led to the hypothesis of \u2018the histone code\u2019, which proposes that the combination of various histone modifications defines the pattern of gene expression [2].\nUpon entry into mitosis, chromatin undergoes dramatic morphological changes to form mitotic chromosomes. On mitotic chromosomes, centromeres form unique chromosomal domains which are crucial for chromosome segregation in two respects [3]. First, centromeres are sites which connect two sister chromatids through cohesins until anaphase. Second, they serve as the foundation for kinetochores which provide the sites for microtubule attachment. To execute these functions, centromeres need to adopt a specialised chromatin structure which also changes during the cell cycle, particularly at the entry into mitosis, at the metaphase\u2013anaphase transition and during exit from mitosis. Moreover, different regulation is also required for meiotic divisions to achieve a correct meiotic chromosome segregation pattern [4].\nRecently a novel phosphorylation site was identified at threonine 119 (T119) in the C-terminal tail of Drosophila H2A [5]. The site is conserved in H2A amongst eukaryotes (serine in yeasts), but not in H2A variants, such as H2Av and H2AX. Here we show H2A T119 phosphorylation is enriched at centromeres during Drosophila mitosis. The Aurora B complex is required for this phosphorylation in centromeric regions, while Polo kinase suppresses phosphorylation by NHK-1 on chromosome arms. Inactivation of Cdc2 kinase is required for loss of centromeric phosphorylation at the metaphase-anaphase transition. Therefore, these mitotic kinases together control the temporal and spatial pattern of H2A phosphorylation at centromeres.\nMaterials and methods\nMolecular and immunological techniques\nStandard immunological, DNA manipulation and protein techniques were followed throughout [6,7]. Mouse \u03b1-tubulin antibody DM1A (Sigma) was used as a loading control in western blots. For immunoblotting, peroxidase-conjugated secondary antibodies (Jackson Lab) were used and detected using an ECL kit (Amersham). Primary antibodies used in this study include antibodies against Histone H2A (Upstate), dH2A-pT119 [5], phospho-H3 (Ser10; Upstate), CID [8], \u03b1-tubulin (DM1A; Sigma), GFP (3E6; Molecular Probes) and Aurora B [9].\nImmunofluorescence microscopy\nCulture and RNAi of S2 cells were carried out as described [10,11]. Effective depletion of target proteins was monitored by immunoblots or appearance of predicted phenotypes. S2 cells were immunostained as described with the exception that cells were fixed with 4% paraformaldehyde in PBS for 5\u00a0min [10]. Larval central nervous systems were dissected from late third instar larvae and fixed with 11% formaldehyde in 0.7% NaCl as described [12]. Secondary antibodies conjugated with Cy3 or Alexa488 (Jackson Lab or Molecular Probes) were used at 1\/250\u20131\/1000 dilution. S2 cells were transfected using Effectene Transfection Reagent (Qiagen). Non-degradable cyclin B fused to GFP (pUASp-CBTPM-GFP [13]) was co-transfected with ubiquitin-GAL4 to induce expression. Transfected cells were identified by the presence of GFP. The presence of dH2A-pT119 on centromeres of segregated chromosomes (>\u00a050 cells) was scored.\nCultured cells were examined using a Plan-Apochromat objective lense (100\u00d7, 1.4NA; Zeiss) attached to an Axioplan2 (Zeiss). Images were captured by a CCD camera (Orca; Hamamatsu) using OpenLab2 (Improvision). Larval central nervous systems were taken using a Plan-Apochromat lense (63\u00d7, 1.4NA; Zeiss) attached to an Axiovert 200\u00a0M (Zeiss) with a confocal scan head (LSM510meta; Zeiss). Confocal images were presented as a maximum intensity projection of the Z-stacks. All digital images were imported to Photoshop (Adobe) and adjusted for brightness and contrast.\nPhosphatase treatment\nFor western blotting of phosphatase treated cell extract, cell extracts were obtained by resuspending S2 cells in lysis buffer (150\u00a0mM NaCl, 20\u00a0mM Tris, 5\u00a0mM EDTA, 1% NP-40) with or without phosphatase inhibitors (100\u00a0mM NaF, 2\u00a0\u03bcM okadaic acid, 100\u00a0mM \u03b2-glycero-phosphate, 15\u00a0mM p-nitrophenylphosphate) and incubating on ice for 10\u00a0min. Lambda phosphatase (NEB) was added to the cell extract without phosphatase inhibitors and both samples incubated for 30\u00a0min at 37\u00a0\u00b0C. 2\u00d7 SDS sample buffer was then added to the extracts and boiled for 3\u00a0min. Samples were then western blotted with anti-dH2A-pT119 to compare phospho-protein levels. In addition, cells immediately resuspended in 1\u00d7 SDS sample buffer were included for comparison.\nFor phosphatase treatment of fixed cells for immunofluorescence with the anti-dH2A-pT119 antibody, cells were fixed with 4% paraformaldehyde in PBS followed by incubation with lambda phosphatase for 1\u00a0h at 37\u00a0\u00b0C. Cells were then washed and immunostained as described above. Microscope images with the same exposure settings were taken of immunostained cells with and without phosphatase treatment. Average pixel intensity of dH2A-pT119 staining on the DNA was measured in interphase and mitotic cells (16 cells in 2 separate experiments).\nFly stocks\nStandard techniques for fly manipulation were followed [14]. All stocks were grown at 25\u00a0\u00b0C in standard cornmeal media. A null nhk-1 mutant (nhk-1E107) used in this study was previously described [15].\nResults\nH2A T119 phosphorylation is specific to centromeres in mitosis\nTo examine the spatial and temporal control of H2A T119 phosphorylation in cells, we immunostained Drosophila S2 cells using an antibody which specifically recognises this phosphorylated form of H2A (anti-dH2A-pT119 [5]). We found a dynamic change in the phosphorylation pattern of H2A during the cell cycle. In interphase, phosphorylation was present throughout the chromatin in the nucleus (Fig. 1A). Interestingly, in mitosis, as the chromosomes begin to condense, phosphorylation was no longer spread throughout the chromatin but produced a more punctate pattern (Fig. 1B). Co-staining with a centromeric marker CID (the CENP-A homologue; [8,16]) revealed that in prometaphase and metaphase, phosphorylation was enriched in regions between and surrounding CENP-A positive regions, which we refer to as the centromeric regions (Figs. 1C\u2013E). This phosphorylation became dramatically reduced at the onset of anaphase (Fig. 1F). Phosphorylation only returned on decondensed chromatin at the end of mitosis.\nSpecificity of the signal obtained by this phospho-H2A antibody was confirmed by treatment with lambda protein phosphatase. Lambda phosphatase treatment of S2 cell extracts eliminated a single band (which comigrates with H2A) recognised by the antibody on immunoblots (Supplementary Fig. 1). Furthermore, the immunofluorescent signals obtained by the phospho-H2A antibody were greatly reduced by lambda phosphatase treatment of fixed S2 cells (Supplementary Fig. 1).\nIn syncytial embryos and oocytes, entire mitotic\/meiotic chromosomes are stained with the anti-dH2ApT119 antibody [5,17]. To confirm that the phosphorylation pattern found in S2 cells is not specific to this cell line, we examined H2A phosphorylation in somatic cells of developing flies. The larval central nervous system (CNS) is the tissue most commonly used for the study of standard mitotic cell cycles, which have two gap phases and checkpoint regulation [18]. Immunostaining of larval CNSs revealed a similar temporal and spatial pattern of H2A T119 phosphorylation as found in S2 cells (Supplementary Fig. 2A).\nCentromeric H2A T119 phosphorylation depends on Aurora B kinase\nPreviously, the conserved protein kinase NHK-1 was identified as phosphorylating H2A T119 in vitro [5]. A female sterile mutation in NHK-1 greatly reduced phosphorylation at this site in oocytes, but not in follicle or nurse cells [17]. This indicated that NHK-1 is the major kinase responsible for this phosphorylation at least in the oocyte nucleus. To test whether NHK-1 is responsible for this phosphorylation in S2 cells, we examined whether depletion of this kinase by RNA interference (RNAi) affects the phosphorylation. Down-regulation of NHK-1 in S2 cells did not eliminate the signal of the phospho-H2A antibody in immunostaining (Supplementary Figs. 3 and 5). This result was further confirmed by immunostaining of larval CNSs from a null mutant of NHK-1 (nhk1E107\n[15]) (Supplementary Fig. 2B). These results indicated that either a residual amount of NHK-1 kinase is sufficient to phosphorylate this site or kinases other than NHK-1 can phosphorylate this site in the absence of NHK-1.\nTo identify the regulatory mechanism of this dynamic change in H2A T119 phosphorylation, we first examined the potential role of Aurora B kinase which localises to the same centromeric domain as the H2A phosphorylation [19]. After Aurora B was depleted by RNAi, S2 cells were immunostained with phospho-H2A antibody. In Aurora B-depleted cells, the intense centromeric staining in mitotic cells was reduced to levels equivalent to that on the chromosome arms (Fig. 2A). However, nuclear staining in interphase cells remained high (Supplementary Fig. 5), suggesting that the phosphorylation is regulated in interphase and mitosis by different mechanisms.\nAurora B kinase is part of at least two functionally distinct complexes [19], a core complex (containing INCENP) and a larger complex (additionally containing Survivin and Borealin\/DasraB). To understand which complex is required for the H2A phosphorylation, we tested the requirement of other subunits for the phosphorylation. Depletion of any one of INCENP, Survivin and Borealin by RNAi greatly reduced H2A phosphorylation in centromeric regions in mitosis (Supplementary Fig. 4). Interphase phosphorylation was not affected in any of the cases. These results indicated that the large AuroraB complex is required for centromeric phosphorylation of H2A at T119 in mitosis.\nPolo kinase down-regulates the H2A phosphorylation on chromosome arms\nTo further study the regulatory mechanism of the phosphorylation, we examined the role of the crucial mitotic regulator Polo kinase [20]. After Polo kinase was depleted by RNAi, S2 cells were immunostained with phospho-H2A antibody. Surprisingly, in Polo-depleted cells, H2A T119 phosphorylation was not restricted to centromeric regions in mitosis but remained at a high level on the entire chromosome arms (Fig. 2A). Quantitative analysis indicated that the fluorescent signal from the phospho-H2A antibody on chromosome arms was dramatically increased in the absence of Polo kinase (Fig. 2B). This result suggests that Polo kinase is directly or indirectly required for down-regulating H2A T119 phosphorylation on chromosome arms to enrich the phosphorylation at centromeric regions.\nPolo suppresses phosphorylation by the H2A kinase NHK-1\nTo identify the relationship between Aurora B and Polo actions, both of the kinases were depleted simultaneously. If a loss of Polo kinase misregulates Aurora B kinase, simultaneous depletion would suppress H2A T119 phosphorylation on chromosome arms. Immunostaining of cells depleted of both Aurora B and Polo showed a high level of phosphorylation on the entire chromosome arms (Fig. 2). This indicated that H2A T119 phosphorylation on chromosome arms induced by loss of Polo kinase was independent of Aurora B activity.\nNext we tested the relationship between Polo and the H2A kinase NHK-1 by co-depletion. We found that NHK-1 depletion suppresses H2A T119 phosphorylation on arms induced by a loss of Polo (Fig. 3). Quantitative analysis confirmed that the phospho-H2A signal on chromosome arms in Polo NHK-1 double depletions was decreased to a level comparable to that of the control or NHK-1 depletion.\nFinally we tested the phenotype of double depletion of Aurora B and NHK-1. Like Aurora B single depletion, H2A T119 phosphorylation was greatly reduced from centromeric regions of mitotic chromosomes (Supplementary Fig. 5).\nThese epistasis studies suggested that Polo functions upstream of NHK-1 to suppress H2A T119 phosphorylation, but is independent of Aurora B.\nCyclin B degradation triggers a loss of H2A phosphorylation at initiation of anaphase\nCentromeric H2A T119 phosphorylation becomes greatly reduced at the onset of anaphase indicating a change in its regulation at this time. After alignment of all chromosomes, APC\/Cdc20 triggers degradation of Cyclin B and securin, leading to inactivation of Cdc2 kinase and activation of separase which cleaves cohesin to initiate anaphase [21]. To separate Cyclin B degradation from securin degradation, we expressed non-degradable Cyclin B in S2 cells and examined H2A phosphorylation by immunostaining. As previously reported [22,23,13], expression of non-degradable Cyclin B did not inhibit the onset of anaphase but prevented exit from mitosis, resulting in an accumulation of anaphase cells with overcondensed chromosomes. In cells expressing non-degradable Cyclin B, H2A phosphorylation was still retained at centromeric regions in most anaphase cells (Figs. 4A, B). Therefore, we concluded that cyclin B degradation, not anaphase onset, is required for triggering loss of phosphorylation at the metaphase-anaphase transition.\nDiscussion\nIn this study, we found dynamic changes in H2A T119 phosphorylation during the Drosophila cell cycle. This phosphorylation is enriched at centromeric regions early in mitosis and lost at the onset of anaphase. In interphase, H2A T119 phosphorylation was found throughout chromatin. Furthermore, our evidence showed that the combined action of at least four conserved mitotic kinases is required for precise spatial and temporal regulation of H2A T119 phosphorylation (Fig. 4C). Aurora B kinase is required for the enrichment of phosphorylation at centromeric regions in mitosis. Polo kinase is required for suppressing H2A phosphorylation by NHK-1 on chromosome arms. Furthermore, inactivation of Cdc2 kinase induced by Cyclin B degradation is required for the loss of centromeric phosphorylation at the onset of anaphase.\nCurrently we do not know what the function of this H2A phosphorylation is in cells. In higher eukaryotes which have many copies of histone genes, the function of histone modifications has been studied only indirectly by down-regulating responsible modifying enzymes. Unfortunately this approach is not suitable for kinases as they are likely to have multiple substrates (for example, Cdc2 and Aurora B mediating H1 and H3 phosphorylation).\nCentromeric distribution and regulation by conserved mitotic kinases may tempt us to speculate possible involvement of H2A T119 phosphorylation in chromosome segregation in mitosis. The phosphorylation might be important for generating or sensing tension between sister chromatids, or modes of microtubule attachment to kinetochores through the formation of centromere-specific chromatin or recruitment of centromere proteins during mitosis. A loss or misregulation of the H2A phosphorylation may be responsible for a subset of the highly pleiotropic phenotypes observed after down-regulation of Aurora B or Polo [19,20]. It would be a future challenge to define the precise roles of this H2A phosphorylation.","keyphrases":["aurora","polo","nhk-1","kinase","centromere","histone","drosophila","cyclin"],"prmu":["P","P","P","P","P","P","P","P"]}
{"id":"J_Gastrointest_Surg-3-1-1852379","title":"Treatment of Gastric Adenocarcinoma May Differ Among Hospital Types in the United States, a Report from the National Cancer Data Base\n","text":"The concept that complex surgical procedures should be performed at high-volume centers to improve surgical morbidity and mortality is becoming widely accepted. We wanted to determine if there were differences in the treatment of patients with gastric cancer between community cancer centers and teaching hospitals in the United States. Data from the 2001 Gastric Cancer Patient Care Evaluation Study of the National Cancer Data Base comprising 6,047 patients with gastric adenocarcinoma treated at 691 hospitals were assessed. The mean number of patients treated was larger at teaching hospitals (14\/year) when compared to community centers (5\u20139\/year) (p < 0.05). The utilization of laparoscopy and endoscopic ultrasonography were significantly more common at teaching centers (p < 0.01). Pathologic assessment of greater than 15 nodes was documented in 31% of specimen at community hospitals and 38% at teaching hospitals (p < 0.01). Adjusted for cancer stage, chemotherapy and radiation therapy were utilized with equal frequency at all types of treatment centers. The 30-day postoperative mortality was lowest at teaching hospitals (5.5%) and highest at community hospitals (9.9%) (p < 0.01). These data support previous publications demonstrating that patients with diseases requiring specialized treatment have lower operative mortality when treated at high-volume centers.\nIntroduction\nApproximately 22,000 patients in the United States (US) will be diagnosed with gastric carcinoma in 2006, a number that pales in comparison to more common malignancies such as colorectal (172,000 new cases) and breast cancer (211,000 new cases).1 Because of the relative infrequency of gastric cancer, most individual hospitals evaluate and treat a limited number of patients with stomach cancer, impairing the ability to develop expertise at many institutions. A lack of expertise may contribute to the dismal survival of gastric cancer in the US. In an earlier report from the National Cancer Data Base (NCDB), Hundahl et al.2 observed a 5-year survival rate of 78% for stage IA, 58% for stage IB, 34% for stage II, 20% for stage IIIA, 8% for stage IIIB, and 7% for stage IV disease.\nA recent report from the World Health Organization3 shows a decline in the mortality of gastric cancer worldwide. However, the observed rate of decline in mortality is less in the US than what is observed for Japan. Factors that might contribute to the improved Japanese survival includes greater operative experience leading to more skilled surgeons, earlier diagnosis, different biologies of gastric cancer between countries, improved pathologic staging with stage migration (Will Rogers effect),4 and the frequency of neoadjuvant or adjuvant chemoradiation therapy use.\nThese data highlight the need for improved treatment of gastric cancer to increase patient survival. A controversial way to accomplish this might be to limit gastric cancer care to high-volume centers, if the results were better at such sites. Evidence-based hospital referral has been adopted by some insurance companies and consortiums of large health care purchasers, such as the Leapfrog group, based on studies showing better outcomes for surgical services at high-volume centers.5\u20137 Birkmeyer et al.8 reported that Medicare patients had a lower operative mortality if they had cancer-related procedures (gastrectomy, esophagectomy, colectomy, pancreatectomy, cystectomy, nephrectomy, and pulmonary resection) or cardiovascular disease at a high-volume hospital. The Japanese have also found a similar pattern of improved survival after the treatment of gastric carcinoma at high-volume centers. Nomura et al.9 reported 5-year survival rates to be significantly higher at centers with high surgical volumes (96\u2013205\/year), when compared to very-low-volume hospitals (1\u201328 cases\/year).\nTo determine if patients treated for gastric cancer at high-volume and specialized centers in the US had better postoperative outcomes, we analyzed the preoperative evaluation and surgical treatment of gastric adenocarcinoma at three categories of hospitals, defined by the Commission on Cancer (CoC), namely, the Community Hospital Cancer Program (CHCP), Community Hospital Comprehensive Cancer Program (COMP), and Teaching Hospital Cancer Program (THCP). Data collected for the 2001 Gastric Cancer Patient Care Evaluation (PCE) by the NCDB were utilized.\nMaterial and Methods\nNCDB, Data Source, Case Selection, and Data Handling\nThe NCDB is a project of the American College of Surgeons (ACS) CoC. The NCDB was established in 1989 to serve as a comprehensive clinical surveillance resource for all forms of cancer diagnosed in the US and its operations have been supported in part by the American Cancer Society. In 2001, the database captured 73% of all newly diagnosed cancer cases in the US.\nData were submitted electronically in accordance with specified North American Association of Central Cancer Registries data transmission standards.10 Hospital cancer registrars abstracted each case according to a standardized set of data elements and definitions as described in the CoC\u2019s Registry Operations and Data Standards, volume II.11 The NCDB elements include patient characteristics: sex, age or date of birth, and race\/ethnicity; tumor characteristics: primary site, histology, behavior, grade, and American Joint Committee on Cancer (AJCC), fifth edition stage groups; and first course of treatment: surgery, radiation, chemotherapy, and others. In addition to the annual call for data, a call for participation in a 2001 PCE was issued and 711 of the 1,423 CoC-approved institutions in the US responded. Cancer registrars were also asked to provide information describing additional diagnostic and treatment information, 30-day mortality, and patient comorbidities. Data quality checks were conducted at the local and the depository level.\nPatient Population\nThe 2001 Gastric PCE included data submitted from 711 CoC-approved institutions in the US. Participating institutions submitted data for consecutive hospital admissions and clinic visits between January 1, 2001 and December 31, 2001. Patients eligible for participation had a \u201cmicroscopically confirmed neoplasm of the stomach that was either diagnosed or initially treated at the reporting facility.\u201d Of 7,084 total patients, 6,099 (86%) were diagnosed with gastric adenocarcinoma. For this study, 52 patients were excluded because they were treated at nine institutions without a specialized hospital type, leaving 6,047 patients at 691 cancer program types for evaluation. This group comprises the study population.\nCancer Program Categories\nCancer programs were characterized as CHCP, COMP, or THCP. Community Hospital Cancer Programs diagnose and\/or treat 100\u2013650 cancer patients every year and will commonly refer patients to other institutions for diagnostic evaluation or treatment. A CHCP has neither a medical school affiliation nor residency programs but does posses a medical oncology unit or functional equivalent and infrequently participates in cancer research. Community Hospital Comprehensive Cancer Programs diagnose and\/or treat more than 650 cancer cases per year, but are not associated with a medical school. A COMP may make outside patient referrals, has a medical oncology unit, and participates in cancer research. Teaching Hospital Cancer Programs are defined as facilities associated with a medical school that participates in the training of residents in at least four fields, two of which are medicine and surgery. A THCP offers a full range of diagnostic and therapeutic services on site and has an in-patient medical oncology unit. A THCP hospital also participates in cancer-related clinical research and has board-certified medical oncologists.\nStatistical Analysis\nAll analyses were performed using the SPSS statistical software (SPSS for Windows, version 14.0; SPSS Inc, Chicago, IL, USA). Frequency distributions were calculated to get the mean number of cases by hospital category type. The chi-square (\u03c72) test was used for comparisons of proportions across levels of categorical variables. When the overall test was significant, pairwise comparisons among the three hospital categories were also calculated to asses which hospitals differed in rates. The p values reported were adjusted for multiple comparisons using the Bonferroni adjustments.12 Results were based on two-sided tests with a p\u2009=\u20090.01 significance level, except where indicated. Where specific values were unknown, these cases were excluded from the analysis when appropriate.\nA forward stepwise binary logistic regression model was used to evaluate the impact of age, stage, and comorbid burden on determination of type of treatment, i.e., the odds of \u201csurgery, with or without other treatment\u201d compared to the odds of nonsurgical treatment (radiation and\/or chemotherapy). The Wald statistic was used to test significance. Exponentiated estimates of the beta coefficients were interpreted as the estimates for the effect (odds ratio) of a particular variable, controlling for the other variables in the equation. A receiver operating characteristic (ROC) curve was created to examine the prediction results. The true-positive probability was calculated to define the sensitivity of the classification rule and the true-negative probability was calculated to determine the specificity to summarize how well the model performed.\nConfidentiality\nData reported to the NCDB are retrospective in nature. No patient or physician identifiers were collected as part of the study. Case identification information (facility identification number and local registry accession number) was collected for administrative purposes only. Analyses were reported only at the aggregate level to assist hospital cancer programs with quality assurance, rather than used to make decisions about individuals and their care.\nThe ACS has executed a business associate agreement that includes a data-use agreement, with each of its CoC-approved hospitals. Results reported in this study were in compliance with the privacy requirements of the Health Insurance Portability and Accountability Act of 1996 as reported in the Standards for Privacy of Individually Identifiable Health Information; Final Rule (45 CFR Parts 160 and 164).\nResults\nTreatment Volumes\nA total of 691 cancer programs were included in the study: 258 CHCP sites, 267 COMP sites, and 166 THCP sites. Although 37.3% of the programs were CHCPs, only 22% (n\u2009=\u20091329) of the cases were treated in this setting; 40.8% (n\u2009=\u20092,468) of the cases came from COMPs; and 37.2% (n\u2009=\u20092,250) of the patients came from THCPs. Community Hospital Cancer Programs saw on average of 5.2 cases\/year; COMPs, 9.2; and THCPs 13.6. The THCPs treated more surgical patient on average (7.6 cases) than either the COMPs (5.3 cases) or the CHCPs (2.9 cases) (Table\u00a01). \nTable\u00a01Number and Percent of Cancer Programs, Number and Percent of Patients by Cancer Program, Mean Number and Range of All Cases by Cancer Program Type, Number and Percent of Surgically Treated Cases, and Mean Number of Surgical Cases and Range by Cancer Program Type\u00a0Community Cancer CentersComprehensive Community Cancer CentersTeaching\/Research HospitalsTotalNumber (%) of cancer programs258 (37.3)267 (38.6)166 (24.0)691Number (%) of patients in study1,329 (22.0)2,468 (40.8)2,250 (37.2)6,047Mean number and range of all cases5.2 (1\u201339)9.2 (1\u201349)13.6 (1\u201355)Number (%) of surgical cases673 (20.5)1,369 (41.8)1,235 (37.7)3,277Mean number and range of surgical cases2.9 (1\u201316)5.3 (1\u201337)7.6 (1\u201340)\nPatient Demographics\nMen, 3,751(62%), and women, 2,296 (38%), were equally distributed across hospital types. The mean age in years was 69.3 and the median age 71 (18\u2013103\u00a0years). Significantly more 70 and older patients were seen in CHCPs when compared to COMPs or THCPs (p\u2009<\u20090.01). Significantly more 50 and younger patients were seen at THCPs when compared with CHCPs and COMPs (p\u2009<\u20090.01) (Table\u00a02). \nTable\u00a02Patient Characteristics by Type Cancer Program\u00a0Community Cancer CenterComprehensive Cancer CenterTeaching\/ResearchTotaln (%)n (%)n (%)n (%)GenderMale805 (60.6)1,502 (60.9)1,444 (64.2)3,751 (62.0)Female524 (39.4)966 (39.1)806 (35.8)2,296 (38.0)Total1,3292,4682,2506,047Age<50106 (8.0)207 (8.4)264 (11.7)577 (9.5)50\u201369402 (30.2)865 (35.0)836 (37.2)2,103 (34.8)70 and older821 (61.8)1,396 (56.6)1,150 (51.1)3,367 (55.7)Total1,3292,4682,2506,047Race\/EthnicityCaucasian966 (72.7)1,723 (69.8)1,387 (61.6)4,076 (67.4)African American143 (10.8)278 (11.3)406 (18.0)827 (13.7)Hispanic120 (9.0)206 (8.3)207 (9.2)533 (8.8)Asian78 (5.9)218 (8.8)176 (7.8)472 (7.8)Other22 (1.7)43 (1.7)74 (3.3)139 (2.3)Total1,3292,4682,2506,047AJCC stageO2 (0.2)8 (0.3)2 (0.1)12 (0.2)I13 (1.0)28 (1.1)26 (1.2)67 (1.1)IA105 (7.9)210 (8.5)182 (8.1)497 (8.2)IB118 (8.9)218 (8.8)178 (7.9)514 (8.5)II160 (12.0)286 (11.6)242 (10.8)688 (11.4)III6 (0.3)12 (0.5)12 (0.5)30 (0.5)IIIA156 (11.7)347 (14.1)337 (15.0)840 (13.9)IIIB47 (3.5)96 (3.9)97 (4.3)240 (4.0)IV427 (32.1)856 (34.7)835 (37.1)2,118 (35.0)Unknown295 (22.2)407 (16.5)339 (15.1)1,041 (17.2)Total1,3292,4682,2506,047\nThe study population included 4,076 (67.4%) Caucasians, 827 (13.7%) African Americans, 533 (8.8%) Hispanic, 472 (7.8%) Asians, and 139 (2.3%) patients of other or unknown racial or ethnic background. Significantly fewer Caucasians were seen in THCPs when compared to CHCPs and COMPs (p\u2009<\u20090.01). THCPs saw significantly more African Americans than the other two types of cancer programs (p\u2009<\u20090.01). Significantly more Asians were seen at COMP hospitals when compared to CHCP hospitals (p\u2009<\u20090.01), but there was no significant difference when comparing the proportion of Asians in COMPs to the proportion found in THCPs.\nA large percentage of this patient population did not have a documented AJCC Stage reported (n\u2009=\u20091,041, 17.2%). Of those patients with a documented stage, more than 64.5% had advanced stage (stage III or IV) at presentation. The largest subgroup had stage IV disease (n\u2009=\u20092,118, 35.0%). There were no significant differences in the stage at presentation between the different types of medical institutions. When specified, the most common location of the primary cancer was the cardia 27.4% (n\u2009=\u20091,656), followed by the antrum 18.3% (n\u2009=\u20091,107) and stomach, not otherwise specified (NOS) 17.9% (1,080) (Fig.\u00a01). The distribution of tumor location was similar among all these categories of treatment institution.\nFigure\u00a01Location of cancer at presentation by cancer program types: A Community Hospital Cancer Program (CHCP), B Community Hospital Comprehensive Cancer Program (COMP), and C Teaching Hospital Cancer Program (THCP). Location of lesions: cardia, funds, body, antrum, pyloric, lesser curve, and greater curve by percent and others indicated by asterisk (C168, overlapping lesions and C169, stomach, NOS).\nDiagnostic Testing\nDiagnostic evaluation included computed tomography (CT) of the abdomen (n\u2009=\u20094,417, 73%) and pelvis (46.9%) over all hospital types. The abdominal CT suggested a diagnosis of cancer in 63.9% of patients at CHCP, 63.0% at COMP, and 68.2% at THCP. The CT of the abdomen was more likely to suggest cancer at the THCP when compared to the COMP (p\u2009<\u20090.01), but no significant difference was noted between the THCP and the CHCP. Computed tomography of the pelvis was suggestive for cancer in 36.9, 38.1, and 42.6% at CHCP, COMP, and THCP, respectively. The differences across cancer programs were not significant (p\u2009>\u20090.01). Only 15.6% (n\u2009=\u2009946) of these cases were evaluated with endoscopic ultrasonography. That procedure was used more often at THCPs (33.5%) when compared to both CHCP (13.8%) and COMP (17.6%) (p\u2009<\u20090.01). 2-18F-Fluoro-2-deoxy-d-glucose positron emission tomography (F18-FDG-PET) was rarely used at any type of hospital (4.6%). When done, THCPs were more likely to use the F18-FDG-PET than CHCP for diagnostic testing (p\u2009<\u20090.01). No significant differences were noted between THCP and COMP use.\nIntraoperative Assessment\nStaging laparoscopy was performed significantly more often at THCPs (18.9%) than at COMPs (13.6%) or at CHCPs (10.5%) (p\u2009<\u20090.01). No significant differences were seen between COMPs and CHCPs. Peritoneal lavage with cytology for the assessment of occult peritoneal disease was rarely used at THCPs, COMPs, and CHCPs with rates of 5.7, 4.3, and 2.7%, respectively. Significantly fewer peritoneal lavages occurred at CHCPs when compared to THCPs (p\u2009<\u20090.01). No significant differences were found between THCPs and COMPs.\nSurgical Treatment\nSurgical intervention was undertaken for 54.2% (n\u2009=\u20093,277) of the all patients in the study. At CHCP hospitals, 673 out of 1,329 (50.6%) patients were surgically treated, 1,369 out of 2,468 (55.5%) were surgical patients at COMP, and 1,235 out of 2,250 (54.9%) patients underwent surgery at THCP. When surgeries were grouped as less extensive or more extensive, significantly more extensive surgeries were performed at THCPs than at CHCPs (p\u2009<\u20090.01); however, no significant difference was observed between COMPs and THCPs (p\u2009>\u20090.01). More specifically, the most frequently recorded type of surgical resection was gastrectomy, NOS (overall, 21.7%; CHCP, 26.0%; COMP, 20.5%; and THCP, 20.6%) followed by distal gastrectomy (overall, 16.2%; CHCP, 15.6%; COMP, 17.3%; and THCP, 15.2%), and partial or subtotal gastrectomy (overall, 13.3%; CHCP, 12.2%; COMP, 12.7%; and THCP, 14.6%). A near total or total gastrectomy was uncommonly performed (overall, 5.7%; CHCP, 4.3%; COMP 5.7%; and THCP, 6.4%). After pathologic analysis, 83.5% of the entire surgical patient population had a resection with curative intent (R0 resection), whereas 16.5% had a palliative resection (R1 resection). The frequencies of R0 and R1 resections were similar (CHCP, 81.2\/18.8%, COMP, 84.6\/15.4%, and THCP, 83.6\/16.4%) among all institutions and not statistically different between different types of institution (p\u2009=\u20090.25).\nLymph node staging, a highly significant predictor of outcome, was evaluated using the number of lymph nodes examined after surgical resection. Analysis of at least 15 lymph nodes is required by the AJCC for accurate staging and exclusion of nodal metastases. Only 31.1% of patients at CHCPs, 31.0% at COMPs, and 38.4% at THCPs had more than 15 nodes examined (p\u2009<\u20090.01). A large number of patients from each hospital type had an unknown number of lymph nodes pathologically evaluated (26.6% CHCP, 22.6% COMP, and 15.4% THCP), but THCPs had significantly better documentation of lymph node evaluation (p\u2009<\u20090.05) (Fig.\u00a02). A D1 lymphadenectomy with removal of only perigastric nodes was noted in 56.7% of surgically managed patients. Some patients had removal of some lymph nodes that are included in D2 or D3 nodes but most across all hospital types did not have a formal extended lymphadenectomy (Table\u00a03).\nFigure\u00a02Percent of lymph nodes sampled at each hospital. CHCP = Community Cancer Center Program, COMP = Community Hospital Comprehensive Cancer Program, THCP = Teaching Hospital Cancer Program. p\u2009<\u20090.05 (asterisk). Error bars: 95% CI.Table\u00a03Lymph Nodes Sampled During Surgical ResectionResectedCommunity Cancer CenterComprehensive Cancer CenterTeaching\/Researchn (%)n (%)n (%)PerigastricaYes367 (71.1)775 (68.9)715 (70.4)No149 (28.9)350 (31.1)301 (29.6)HepaticbYes21 (4.2)68 (6.5)70 (7.5)No478 (95.8)979 (93.5)867 (92.5)CeliaccYes40 (7.9)100 (9.5)96 (10.1)No465 (92.1)952 (90.5)854 (89.9)SplenicdYes14 (2.8)49 (4.7)52 (5.5)No481 (97.2)988 (95.3)887 (94.5)ParaaorticeYes94 (18.8)170 (16.8)189 (20.2)No405 (81.2)840 (83.2)745 (79.8)Comparison of column proportions did not include the unknown values in calculations.aPerigastric unknown, n\u2009=\u2009620bHepatic unknown, n\u2009=\u2009794cCeliac unknown, n\u2009=\u2009770dSplenic unknown, n\u2009=\u2009806eParaaortic unknown, n\u2009=\u2009834\nMorbidity and Mortality\nThe prevalence of operative morbidity, specifically for postoperative hemorrhage, wound infection sepsis, and an anastomotic leak, was similar across all hospital categories (Table\u00a04). Operative mortality, defined by 30-day mortality, was different among the institution types. Teaching Hospital Cancer Programs had the lowest 30-day mortality at 5.5%, compared to 7.9% at COMPs and 9.9% at CHCPs (Table\u00a04). The proportional difference between THCPs and COMPs was not significant (p\u2009>\u20090.01), whereas, when compared to CHCPs, THCPs had significantly fewer deaths within 30\u00a0days of surgery (p\u2009<\u20090.01). For those that died within that 30-day period, no significant differences were found across all hospital types for stage (p\u2009>\u20090.01) or age (p\u2009>\u20090.01). In the logistic regression model stage was the most significant predictor of 30-day postoperative death (p\u2009=\u20090.0001), followed by age (p\u2009=\u20090.0001), and category of hospital (p\u2009=\u20090.004). Stage IV patients were 2.6 times (99% confidence interval [CI]\u2009=\u20091.6\u20134.3) more likely to die within 30\u00a0days of surgery; no other stage was significant (p\u2009>\u20090.01). Patients in CHCPs were almost twice as likely to die within 30\u00a0days when compared to THCPs (p\u2009=\u20090.001). Race and extent of surgery were not significant factors. The area under the ROC curve defined by the logistic model was 0.69 (99% CI\u2009=\u20090.64\u20130.74), where 0.5 represents a nondiscriminatory result. Long-term 5-year survival data are unavailable for this patient population and will not be until the fall of 2007. \nTable\u00a04Postoperative Mortality and Complications\u00a0Community Cancer CenterComprehensive Cancer CenterTeaching\/Researchn (%)n (%)n (%)30-Day mortalityaYes55 (9.9)93 (7.9)59 (5.5)No501 (90.1)1,080 (92.1)1,020 (94.5)BleedingbYes20 (4.2)40 (3.9)47 (5.1)No451 (95.8)993 (96.1)881 (94.9)Wound infectioncYes35 (7.4)54 (5.2)61 (6.6)No440 (92.6)981 (94.8)868 (93.4)SepsisdYes34 (7.1)62 (6.0)43 (4.7)No443 (92.9)973 (94.0)881 (95.3)Anastomotic leakeYes32 (6.7)68 (6.5)64 (6.6)No447 (93.3)979 (93.5)884 (93.2)Comparison of column proportions did not include the unknown values in calculations.a30-Day mortality unknown, n\u2009=\u2009469bBleeding unknown, n\u2009=\u2009845cWound infection unknown, n\u2009=\u2009840dSepsis unknown, n\u2009=\u2009841eAnastomotic Leak unknown, n\u2009=\u2009803\nNeoadjuvant and Adjuvant Therapy\nApproximately 38.9% of the surgical patients received chemotherapy; 30.5% received radiation therapy. Neither neoadjuvant radiation therapy nor chemotherapy was frequently provided at all hospital types, but highest, although not significant (p\u2009>\u20090.01) at the THCPs when compared to CHCPs and COMPs (Table\u00a05). Patients more frequently received adjuvant chemotherapy and radiation than neoadjuvant chemotherapy or radiation therapy at all the categories of hospitals. There were no statistically significant differences in the frequency of treatment with chemotherapy or radiation therapy by tumor stage between the hospital categories. \nTable\u00a05Radiation and Chemotherapy\/Surgery Sequence\u00a0Community Cancer CenterComprehensive Cancer CenterTeaching\/Research# (%)# (%)# (%)RadiationaNeoadjuvant23 (12.1)53 (13.0)68 (19.1)Adjuvant167 (87.9)356 (87.0)288 (80.9)ChemotherapybNeoadjuvant36 (14.6)71 (15.0)87 (20.3)Adjuvant210 (85.4)401 (85)342 (79.7)Comparison of column proportions did not include the unknown values in calculations.aRadiation sequence unknown, n\u2009=\u200943bChemotherapy sequence unknown, n\u2009=\u2009128\nDiscussion\nWhen examining the current status of gastric cancer patient care in the US and seeking ways to improve survival, looking to the East for guidance is a reasonable strategy. The standards of gastric cancer therapy have largely been set by the practices of Japanese physicians and surgeons, in a large part, because of their large experience with this disease. In Japan, there are 104,000 new cases annually in a population of 128\u2009\u00d7\u2009106,13 compared to the 22,000 cases seen annually in the US in a population of 296\u2009\u00d7\u2009106,1 an incidence that is more than 10 times greater than that of the US.\nThe high incidence seen in Japan has allowed the Japanese to develop surgical and medical strategies to improve mortality. However, several of the surgical principles practiced in Japan are difficult to incorporate into Western practices. Early detection programs in Japan have led to a significant decrease in mortality but this has not been replicated in the US because of the high cost to benefit ratio associated with the much lower incidence of gastric cancer in the US. A more controversial standard Japanese practice is the extended D2, D3, or even D4 lymphadenectomy performed for gastric cancer. Whereas extended lymphadenectomy has been associated with improved survival in Japan, with retrospective analysis, the value of this technique has not been proven in the West. Wanebo et al.,14 in a retrospective study from the US, as well as Bonenkamp et al.15 and Cuschieri et al.16, in randomized controlled trials from The Netherlands and Great Britain, respectively, have reported a lack of survival benefit with D2 lymphadenectomy. Both Bokenkamp et al.15 and Cuschieri et al.16 observed significantly higher mortality rates among those patients that had a D2 resection of 10 vs 4% and 13 vs 6.5%, respectively. The operative mortality after a D2 resection is much lower in Japan with reported rates below 2%.17,18 Kodera et al. from Japan reported an operative mortality rate of 0.8% from 523 patients in a D2 vs D3 study.19\nStudies examining large national databases have found improved surgical mortality after gastrectomy for gastric cancer at high-volume centers. A study examined using the National Inpatient Sample reported on 23,690 hospitalized patients with a hospital discharge code of gastric cancer and any gastrectomy noted significant differences in mortality among hospital types. Very-low-volume hospitals (<4 cases\/year) had a mortality rate of 8.9% compared with a rate of 6.4% seen at high-volume hospitals (>9 cases\/year).20 In a study by Birkmeyer et al.21 of the Medicare population, the observed mortality rate was 13% at very-low-volume centers (<5 cases\/year) compared to 8.7% at very-high-volume centers (>21 cases\/year). Improved survival among high-volume hospitals was also reported in Swedish22 hospitals that treated >20 surgical patients a year and German23 hospitals that treated >50 patients\/year.\nThe Japanese have also noted an association of lower patient survival rates among gastric cancer patients treated at low-volume centers when compared to high-volume centers. Nomura et al.9 analyzed a database of more than 55,000 patients and grouped them into the following time periods: 1975\u20131979, 1980\u20131984, 1985\u20131989, and 1990\u20131994. He found \u201cpositive relationships between hospital volume and 5-year survival\u201d but over time the survival benefit seen at high-volume centers decreased and persisted in comparison with the very-low-volume centers.\nNot all authors, however, have observed improved survival at high-volume centers. A Dutch study evaluated the impact of patient volume on operative mortality and found no differences. This study analyzed 1,987 gastric cancer patients treated at 22 hospitals between 1987 and 1997.24 A limitation of this study is the fact that only 1 of the 22 hospitals was a university hospital and the others were \u201cgeneral hospitals.\u201d Many of the participating hospitals were considered to be low-volume centers.\nInterpretation of the Results\nOur study had similar results to the aforementioned American, Japanese, and European studies. In this study, there was a marked improvement (>50%) in operative mortality at higher volume centers (\u226514 cases\/year) when compared to lowest volume institutions (\u22645 cases\/year). The average 30-day postoperative mortality at the low-volume community centers was almost 10%. The annual volume of gastric surgeries performed seems to be inversely related to 30-day postoperative mortality. On average 2.9, 5.5, and 7.6 gastric surgeries were reported from CHCPs, COMPs, and THCPs, respectively. Corresponding 30-day mortality was 9.9, 7.9, and 5.5%, respectively. There were no significant differences seen in postoperative morbidities such as wound infection and hemorrhage by hospital category. The logistical model revealed three predictors of perioperative mortality: stage IV disease, advanced age, and institution type. Patients that had surgery at a CHCP were twice as likely to die postoperatively compared to patients treated surgically at THCPs. Among those that died, there were no significant differences of stage or age at the different institutions.\nBecause the absolute differences in surgical case number among the hospital types were not vastly different, this observed difference in outcomes may be a reflection of the infrastructure of the institution rather than individualized surgeon skill. Centers affiliated with a medical school might have more experience with caring for the critically ill in the form of larger and better equipped intensive care units, resident and fellow coverage, newer technology, and more subspecialized physicians to help manage patient care. Unfortunately, we could not analyze the infrastructure for each hospital type nor look at surgeon-specific experience in this study as this was not a part of the study.\nClinical staging is affected by the sensitivity and specificity of the diagnostic studies performed during the preoperative evaluation. An assessment of staging at the different institutions revealed some major differences. Teaching hospitals were more likely to detect malignancy on a CT of the abdomen and pelvis than the other two hospital types. This might be a reflection on the quality of CT scans obtained and the experience of the radiologists. Preoperative utilization of endoscopic ultrasound was higher at teaching hospitals, a predictable finding given the recent adaptation of this technology and the expertise required to interpret these images.\nIn terms of pathologic staging, Karpeh et al.25 previously reported that evaluation of more than 15 lymph nodes allows a better estimate of patient survival. In fact, a study by Smith et al.26 found that overall survival was largely dependent on the number of nodes examined and found an increase in survival when up to 40 lymph nodes was assessed. We found that teaching hospitals did a significantly better job meeting this recommendation; however, even at THCPs only 38.4% of patients had greater than 15 nodes assessed. The D1 lymphadenectomy was the most common operation for of nodal dissection, probably as a result of controlled Western surgical trials showing no survival benefit from a D2 dissection. Only a limited number of patients had any D2\u2013D4 nodes resected and usually without a standardized extended lymphadenectomy. A notable observation was that the percentage of D2\u2013D4 lymphadenectomy performed at THCPs was not higher than what was observed at COMPs or CHCPs.\nTo improve and obtain accurate surgical staging of gastric cancer patients, current practice will have to improve and will be dependent upon both the excision of nodes by the surgeon and their retrieval from the specimen by the pathologist. Given so few patients had 15 or more nodes removed at the time of surgery, regardless of hospital type, this practice could be improved by surgeons and pathologists working together to achieve the goal of identifying at least 15 nodes followed by the proper documentation of the microscopic evaluation of these nodes in the pathology report. If intraoperative pathologic evaluation of the surgical specimen is possible and the lymph node sampling is inadequate, the surgeon should excise additional lymph nodes.\nA strength of this study is the large number of patients and hospitals included in the study. This broad sampling of hospitals leads to a close approximation of the current practice of gastric cancer treatment in the US. The main shortcoming is that many of the critical data categories had at least 20% \u201cunknown\u201d responses, and may have biased the reported results of this study. In addition, analysis of hospital infrastructure or surgeon volume was limited by lack data availability in the database. Another limitation is that the 5-year survival information has not yet been documented; these data will provide long-term outcome by type of treatment center.\nConclusion\nData from the Gastric PCE project suggests that there is significant room for improvement in the surgical management of gastric cancer in the US. Most hospitals, regardless of category, do not document the evaluation at least 15 lymph nodes necessary to meet AJCC standards. With a little over one third of all patients having more than 15 lymph nodes examined, pathologic staging is less accurate and results of surgical and adjuvant therapy are likely to be worse. Significantly more patients had appropriate staging with the recommended number of nodes included in the lymphadenectomy at teaching centers than at nonacademic centers. Thirty-day mortality rates after gastric cancer resection were significantly lower at teaching centers, further establishing the recommendation that complex oncologic operations should be performed at high-volume centers to obtain better patient outcomes, corroborating the results of several previous studies. Long-term survival data will provide more information on effectiveness of treatment at each of the different institution types.","keyphrases":["gastric cancer","operative mortality","survival","ncdb","hospital volume"],"prmu":["P","P","P","P","P"]}
{"id":"J_Mol_Med-3-1-1820750","title":"Genomic expression profiling of human inflammatory cardiomyopathy (DCMi) suggests novel therapeutic targets\n","text":"The clinical phenotype of human dilated cardiomyopathy (DCM) encompasses a broad spectrum of etiologically distinct disorders. As targeting of etiology-related pathogenic pathways may be more efficient than current standard heart failure treatment, we obtained the genomic expression profile of a DCM subtype characterized by cardiac inflammation to identify possible new therapeutic targets in humans. In this inflammatory cardiomyopathy (DCMi), a distinctive cardiac expression pattern not described in any previous study of cardiac disorders was observed. Two significantly altered gene networks of particular interest and possible interdependence centered around the cysteine-rich angiogenic inducer 61 (CYR61) and adiponectin (APN) gene. CYR61 overexpression, as in human DCMi hearts in situ, was similarly induced by inflammatory cytokines in vascular endothelial cells in vitro. APN was strongly downregulated in DCMi hearts and completely abolished cytokine-dependent CYR61 induction in vitro. Dysbalance between the CYR61 and APN networks may play a pathogenic role in DCMi and contain novel therapeutic targets. Multiple immune cell-associated genes were also deregulated (e.g., chemokine ligand 14, interleukin-17D, nuclear factors of activated T cells). In contrast to previous investigations in patients with advanced or end-stage DCM where etiology-related pathomechanisms are overwhelmed by unspecific processes, the deregulations detected in this study occurred at a far less severe and most probably fully reversible disease stage.\nIntroduction\nThe term dilated cardiomyopathy (DCM) describes a cardiac phenotype characterized by dilation and dysfunction of the ventricles. From an etiologic perspective, DCM encompasses a spectrum of disease entities, some of which are due to monogenic defects in cardiac expressed genes [1], others triggered by exogenous factors including cardiotropic viruses [2]. Targeting of etiology-related pathogenic pathways in etiologically distinct DCM subtypes may be more efficient than current standard treatment protocols employed in heart failure of any origin. One important DCM subtype characterized by cardiac inflammation is designated as inflammatory cardiomyopathy (DCMi), often found in conjunction with cardiac viral infections. Spontaneous elimination of cardiac viruses was associated with significant hemodynamic improvement for various viruses [3], and interferon-\u03b2 (IFN-\u03b2) therapy was useful to promote virus elimination and improve cardiac function and clinical status [4]. A search for new therapeutic targets in DCMi seems worthwhile due to the high prevalence of this condition and its still unsatisfactory long-term outcome. The current study combined in vivo and in vitro methods to identify possible DCMi-related gene regulatory networks that might be exploited to expand the therapeutic repertoire beyond the standard heart failure drugs currently used. The study does not address individual gene deregulations, but searches for disturbances of regulatory gene networks reflected by anomalous cardiac expression patterns. Genomic screening strategies require precise phenotyping of the study groups, as this will minimize interindividual differences and increase the likelihood of revealing distinctive pathomechanisms. Because, in advanced stages of DCM, such processes are most likely overwhelmed by unspecific reactions of the failing heart long after the initiating process has burnt out, we investigated patients with moderately severe disease in contrast to most previous microarray work in the field [5]. The standard clinical characterization was refined by endomyocardial biopsy (EMB)-based immunohistological and molecular virological analyses. In contrast to multiple fields of cancer research, restrictive disease phenotyping of this kind is rarely employed in cardiovascular research, mostly due to the difficult access to affected cardiovascular tissues in humans in vivo. The microarray part of our study was entirely based on RNA isolated from EMBs thus providing an approach suitable for follow-up investigations of clinical courses in relation to cardiac expression profiles. We report on a distinctive cardiac expression pattern in DCMi and two significantly altered gene networks centered around the cysteine-rich angiogenic inducer 61 (CYR61) and adiponectin (APN) gene.\nPatients and methods\nCharacterization of study groups\nThe patients investigated by microarray analysis of cardiac gene expression displayed the distinct DCM subtype of DCMi as defined by the World Health Organization and characterized by its association with cardiac inflammation. The DCMi group of this study was derived from a large series of patients initially submitted to our clinic with symptoms and signs of DCM; in all of whom, EMBs from the right ventricular septum were obtained by standard procedure for histological, immunohistological, and molecular virological analyses. All patients had given written informed consent. After standard clinical, morphological, and functional patient characterization, their EMBs were assessed for myocardial inflammation by histological and immunohistological methods. Their cardiac virological status was assessed by polymerase chain reaction (PCR)\/real-time polymerase chain reaction (RT-PCR) for the following viral genomes: enteroviruses, adenoviruses (ADV), Epstein\u2013Barr virus, human herpesvirus 6 (HHV-6), and parvovirus B19 (PVB19). Eight DCMi patients with cardiac inflammation in association with cardiac PVB19 were selected for the microarray study (DCMi group, Table\u00a01). This group was compared to a control group of four individuals submitted to our clinic and diagnosed to clarify a clinically suspected cardiomyopathy, in whom the full differential diagnostic workup finally revealed that their complaints were noncardiac in origin (Cont group, Table\u00a01). They had normal cardiac morphology and function, were negative for cardiac inflammation or viral genomes, and were subjected to microarray analysis, too. The DCMi group patients were on standard heart failure medication and in New York Heart Association functional class II (Table\u00a01). \nTable\u00a01Characterization of study groupsCodeGroupAge (years)EF (%)LVEDD (mm)INFLAMPVB19EVAdVHHV6EBVGA-1DCMi315671++\u2212\u2212\u2212\u2212HI-2DCMi393161++\u2212\u2212\u2212\u2212KA-3DCMi693660++\u2212\u2212\u2212\u2212PS-4DCMi603476++\u2212\u2212\u2212\u2212BA-5DCMi674359++\u2212\u2212\u2212\u2212DE-6DCMi315257++\u2212\u2212\u2212\u2212CH-7DCMi625173++\u2212\u2212\u2212\u2212GH-8DCMi453462++\u2212\u2212\u2212\u221252.5\u2009\u00b1\u200917 (31\u201369)42.0\u2009\u00b1\u20099.9 (31\u201356)62.8\u2009\u00b1\u20097.8 (57\u201373)RS-14Cont366847\u2212\u2212\u2212\u2212\u2212\u2212SM-15Cont466149\u2212\u2212\u2212\u2212\u2212\u2212KE-16Cont267447\u2212\u2212\u2212\u2212\u2212\u2212TU-17Cont366450\u2212\u2212\u2212\u2212\u2212\u221236.0\u2009\u00b1\u20098 (26\u201346)66.8\u2009\u00b1\u20095.6 (61\u201374)48.3\u2009\u00b1\u20091.2 (47\u201350)EF ejection fraction; LVEDD left ventricular end-diastolic diameter; INFLAM cardiac inflammation; PVB19 parvovirus B19; EV enterovirus; AdV adenovirus; HHV6 human herpesvirus 6; EBV Epstein\u2013Barr virus\nHigh density microarray studies of human cardiac biopsies\ncDNA was synthesized from 200\u00a0ng of total RNA isolated from EMBs. The minimal amount of total RNA routinely available from human EMBs was 200\u00a0ng. cRNA targets were generated using the small sample protocol of Affymetrix. Briefly, in vitro transcribed unlabeled cRNA from the first round of amplification was subjected to a second round of amplification. cRNA was transcribed into cDNA using random primers. After RNAse H-mediated removal of surplus cRNA, a second strand was synthesized using a T7 primer. cRNA amount was determined by ultraviolet spectroscopy, and distribution of cRNA fragment sizes of both cRNA and fragmentation products was checked on a BioAnalyzer. Each cRNA sample was hybridized for 16\u00a0h at 45\u00b0C to an Affymetrix Human Genome U133 Plus 2.0 GeneChip Array. Chips were washed and stained with streptavidin\u2013phycoerythrin using a fluidics station according to Affymetrix protocols. Probe arrays were scanned at 3-\/1.56-\u03bcm resolution using the Affymetrix GeneChip System Confocal Scanner 3000. Raw data were analyzed with Affymetrix GeneChip Operating Software. The detection p value of a transcript determines the detection call indicating if the transcript is reliably detected (p\u2009<\u20090.05; present) or not detected (absent). To enable the comparison between chips, data were normalized to a global intensity of 500. The expression data can be obtained from the Gene Expression Omnibus (GEO) website http:\/\/www.ncbi.nlm.nih.gov\/projects\/geo\/, GEO experimental series number: GSE4172. For TaqMan experiments to verify candidate genes, see next chapter.\nQuantitative real-time PCR analysis of human cardiac biopsies\nTotal RNA was extracted using TRIzol reagent (Invitrogen). Quality and quantity was checked on a Agilent 2100 Bioanalyzer. A minimal amount of only 200\u00a0ng of total RNA was routinely available from human EMBs for cDNA synthesis to become reverse transcribed using the High Capacity cDNA Archive Kit (Applied) according to the manufacturers instructions. For determination of gene expression, quantitative PCR assays were performed on an ABI PRISM 7900 Sequence Detector (Applied) with TaqMan Universal PCR Master Mix and TaqMan Gene expression assays (Applied) consisting of a FAM\u2122 dye-labeled TaqMan MGB probe and two PCR primers. The following oligonucleotide sets purchased from Applied Biosystems were used: human genes encoding APN (Hs00605917_m1), CYR61 (Hs00155479_m1), chemokine ligand 14 (CXCL14; Hs00171135_m1), Ras-responsive element protein 1 (RREB1; Hs00171486_m1), GTP binding protein overexpressed in skeletal muscle (GEM; Hs00170633_m1), membrane protein, palmitoylated 7 (MPP7; Hs00299584_m1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Hs99999905_m1), and hypoxanthine phosphoribosyltransferase 1 (HPRT1; Hs99999909_m1). Quantitative RT-PCR reactions were performed using standard cycle parameters determined by the company. Normalization and fold change were calculated with the \u0394\u0394Ct method with GAPDH and HPRT1 as reference mRNA species according to ABI Prism 7900 manufacturer\u2019s instructions [6]. The values given in Supplementary Fig. 2 are the means\u2009\u00b1\u2009SEM of two independent quantitative TaqMan PCRs performed on RNA isolated from the same EMB that was also used for the microarray analysis.\nCardiovascular cell cultures\nPrimary culture of neonatal rat cardiomyocytes (NRCMC): Cells were prepared from ventricular tissue of 1- to 3-day-old Wistar rat pups (Charles River) as described previously [7]. Briefly, the removed ventricles of 50\u201360 animals were placed into ice-cold calcium ion-free phosphate-buffered cell isolation medium (CIM) containing 120\u00a0mM NaCl, 4.56\u00a0mM KCl, 0.44\u00a0mM KH2PO4, 0.42\u00a0mM Na2HPO4, 25\u00a0mM NaHCO3, and 5.55\u00a0mM glucose, pH\u00a07.5, as well as 0.5\u00a0mg\/ml streptomycin and 5,000\u00a0IE\/ml penicillin G (Biochrom). Ventricular tissue was transferred to a Petri dish and minced into pieces of approximately 1\u00a0mm3 in size using two sterile scalpels. Stepwise disaggregation for 15\u00a0min each of tissue pieces into single cells was performed at 37\u00b0C under continuous mixing with a magnetic stirrer at 150\u00a0rpm in a 50\u00a0ml Erlenmeyer flask containing 15-ml CIM supplemented with 0.12% porcine trypsin 1:250 (Belger). The first tissue digest was discarded. The following three to eight supernatants obtained after each 15-min digestion period were poured into single sterile glass centrifuge tubes each containing 4\u00a0ml of ice-cold, heat-inactivated fetal calf serum (FCS; Biochrom). Cells were gently sedimented at 400\u00a0g for 10\u00a0min. The sedimented cells were resuspended in cell growth medium CMRL 1415-ATM (CMRL-ATM, Connaught Medical Research Laboratories-atmospheric) that was supplemented with 10% FCS, 10% horse serum (HS), and 0.02\u00a0mg\/ml gentamicin (all from Biochrom) and adjusted to pH\u00a07.4 using 1\u00a0N NaOH. For enrichment of cardiomyocytes, 20\u00a0ml of this suspension containing approximately 1.5\u2009\u00d7\u2009108 cardiac cells were incubated in 175\u00a0cm2 plastic dishes, Corning culture flask in a water-saturated atmosphere for 90\u00a0min at 37\u00b0C. During this incubation, most of the nonmuscle cells comprising 30\u201340% of total cell number attached to the bottom surface of the culture flask. The cardiomyocyte-enriched supernatant was carefully removed and pooled in an Erlenmeyer flask. The number of cells was counted using a light microscope and a hemocytometer. The cell density was adjusted with additional growth medium to 1.2\u2009\u00d7\u2009106 cells\/ml and the cells seeded to 6-well Nunc culture plates and incubated in a water-saturated atmosphere at 37\u00b0C. After 24\u00a0h of incubation, the FCS\/HS-supplemented growth medium was replaced either by a 10% FCS-supplemented CMRL 1415-ATM or serum-free, hormone-supplemented CMRL 1415-ATM medium containing 2-\u03bcM fluorodeoxfyuridine (Sigma) for inhibition of proliferation of contaminating nonmuscle cells. Vascular endothelial cell line: The endothelial cell line EA.hy926 (a hybrid line derived from human umbilical vein endothelial cells and A549 cells) was cultured in Dulbecco\u2019s modified Eagle\u2019s medium supplemented with hypoxanthine\u2013aminopterin\u2013thymidine, 10% FCS, and 1% penicillin and streptomycin.\nQuantitative real-time PCR analysis of activated cell cultures\nTo study regulatory factors of CYR61 and APN in cultured cardiovascular cells (Fig.\u00a02a,b), NRCMCs were grown in 6-well and EA.hy926 cells in 12-well dishes for 48\u00a0h. Thereafter, they were incubated for 24\u00a0h with the following cytokines (concentrations): IFN-\u03b21b (30\u00a0ng\/ml), IFN-\u03b21a (30\u00a0ng\/ml), IFN-\u03b3 (30\u00a0ng\/ml), IL-1\u03b2 (30\u00a0ng\/ml), IL-6 (100\u00a0ng\/ml), tumor necrosis factor (TNF)-\u03b1 (20\u00a0ng\/ml), and TNF-\u03b2 (50\u00a0ng\/ml). In some experiments (Fig.\u00a02a), EA.hy926 cells were preincubated for 1\u00a0h with APN (10\u00a0\u03bcg\/ml) before the addition of the above cytokines. This APN concentration was chosen because the APN plasma range in humans is 3\u201330\u00a0\u03bcg\/ml. Cells were harvested 24\u00a0h after cytokine addition. Of the total RNA, 1 to 3\u00a0\u03bcg were isolated from cell cultures to become reverse transcribed using the High Capacity cDNA Archive Kit, and TaqMan PCRs were then carried out employing the following primer systems (Applied) for the rat genes encoding APN (Acdc, Rn00595250_m1), CYR61 (Rn00580055_m1), and GAPD (Rn99999916_s1). mRNA amounts were standardized against the housekeeping gene GAPDH. The values given in Fig.\u00a02a,b are the means\u00b1SEM of two independent experiments each performed in triplicate.\nNetwork analysis\nThe networks shown in Fig.\u00a01a,c,e were derived by the Ingenuity Pathways Analysis (IPA; http:\/\/www.ingenuity.com) software. The microarray dataset containing gene identifiers and corresponding expression values was uploaded as an Excel spreadsheet using the template provided in the application. Each gene identifier was mapped to its corresponding gene object in the Ingenuity Pathways Knowledge Base (IPKB). A cutoff of fold change \u22651.5 was set to identify genes whose expression was significantly differentially regulated; these were then used as the starting point for generating regulatory networks. Biological functions were assigned to each network or the overall analysis, respectively, by using the findings that have been extracted from the scientific literature and stored in the IPKB. The biological functions assigned to each network or the overall analysis, respectively, are ranked according to the significance of that biological function to the network or the overall analysis, respectively. A Fischer\u2019s exact test is used to calculate a p value determining the probability that the biological function assigned to that network is explained by chance alone. The networks are displayed graphically as nodes (genes\/gene products) and edges (the biological relationships between the nodes). Human, mouse, and rat orthologs of a gene are stored as separate objects in the knowledge base but are represented as a single node in the network. The intensity of the node color indicates the degree of up-(red) or down-(green) regulation. Nodes are displayed using various shapes that represent the functional class of the gene product. Edges are displayed with various labels that describe the nature of the relationship between the nodes (e.g., B for binding, T for transcription).\nFig.\u00a01Altered gene expression profile and regulatory networks in DCMi. a, c, e The networks altered in DCMi are displayed graphically as nodes (genes\/gene products) and edges (biological relationships between the nodes). Human, mouse, and rat orthologs of a gene are stored as separate objects in the knowledge base but are represented as a single node in the network. The intensity of the node color indicates the degree of up- (red) or down- (green) regulation. Nodes are displayed using various shapes that represent the functional class of the gene product. Continuous edge lines indicate protein\u2013protein interactions, broken edge lines changes at the transcriptional level. The software further displays various labels that describe the nature of the relationship between the nodes (e.g., B for binding, T for transcription). b, d, f Show cluster analyses of the genes within the networks in (a), (c), (e), respectively. Here, analogous to the networks, red color denotes upregulation, green color denotes downregulation of a gene, whereas the brightness of the color indicates the extent of deregulation\nStatistical analysis\nFor statistical analysis of microarray data, we used the significance analysis of microarrays (SAM). SAM statistics identifies significant changes in gene expression by performing a set of gene-specific t-tests. For each gene, a score is calculated on the basis of its change of expression relative to the standard deviation of repeated measurements for that gene. Genes with scores greater than a given threshold \u0394 are defined as significantly deregulated. Manual adjustment of this threshold \u0394 allows the identification of smaller or larger cohorts of genes. In addition, a false discovery rate can be estimated based on random permutations of all measurements. With respect to quantitative TaqMan PCRs, Student\u2019s t-test was applied to demonstrate statistical significance. A value of p\u2009<\u20090.05 was considered statistically significant.\nResults\nBecause, in advanced or end-stage DCM, any etiology-related pathomechanisms are most likely overwhelmed by unspecific reactions of the terminally failing myocardium long after the initiating process has burnt out, we studied patients with moderately severe disease (Table\u00a01). The standard clinical characterization was refined by EMB-based immunohistological (Supplementary Fig. 1) and molecular virological analyses. The DCMi group was characterized by myocardial inflammation in association with PVB19. A distinctive expression profile was detected in the DCMi group by microarray analysis of the EMBs. The complete dataset is available from the website http:\/\/www.ncbi.nlm.nih.gov\/projects\/geo\/. Genes significantly (q value\u2009<\u20095.0) and \u22651.5-fold deregulated are listed in Supplementary Table 1. Subsequently, a network analysis of the complete dataset with 380 genes \u22651.5-fold down- and 1,038 genes \u22651.5-fold up-regulated was performed as shown in Fig.\u00a01a\u2013f. Several of these genes were validated by quantitative TaqMan PCR (Table\u00a02, Supplementary Fig. 2). Whereas confirmatory PCR analysis was desirable for a major number of DCMi-associated genes, the limited mRNA quantity available from human EMBs confined the number of such analyses. The minimal amount of total RNA available from EMBs for both microarrays and confirmatory PCR was only 200\u00a0ng. The genes selected for confirmatory PCRs are shown in Supplementary Fig. 2. They include CYR61, APN, CXCL14, and GEM as novel deregulated genes associated with DCMi, natriuretic peptide precursor A (NPPA), and natriuretic peptide precursor B (NPPB) as standard heart failure-associated genes and confirmed the microarray data in all cases. The microarray q values for each of the genes in Supplementary Fig. 2, the corresponding p values in the confirmatory TaqMan PCRs, and similar regulatory factors are proof of deregulation of these genes of particular interest.\nTable\u00a02Partial cardiac gene expression profile of human DCMi (further data at http:\/\/www.ncbi.nlm.nih.gov\/projects\/geo\/ and in Supplementary Table 1)NetworkSymbolRegulationFactorq valueGene NameAPNAPNaDown6.80.605Adiponectin14.4CYRCXCL14Down5.91.943Chemokine (C\u2013X\u2013C motif) ligand 144.4AKTTDGF1Down4.70.605Teratocarcinoma-derived growth factor 1CYRPDGFADown3.60.371Platelet-derived growth factor (PDGF)-\u03b1 polypeptideAPNSOS1Down3.10.371Son of sevenless homolog 1 (Drosophila)CYRTMSB10Down2.60.371Thymosin-\u03b210APNINSRDown2.50.371Insulin receptorCYREGFRDown2.52.226Epidermal growth factor (EGF) receptorAPNPIK3R1Down2.50.991Phosphoinositide-3-kinase regulatory subunit 1 (p85\u03b1)CYRLAMA4Down2.40.371Laminin \u03b14APNLMO4Down2.33.868Lin-11, Isl-1, Mec-3 (LIM) domain only 4CYRHSPG2Down2.12.226Heparan sulfate proteoglycan 2 CYRSOX9Down2.10.371Sex determining region Y (SRY)-box 9APNLIFRDown2.01.632Leukemia inhibitory factor (LIF) receptorCYRTNXBDown2.00.371Tenascin XBAKTTGFBR3Down2.00.895TGF-\u03b2 receptor III (betaglycan)AKTEIF4EBP1Down2.02.345Eukaryotic translation initiation factor 4E binding protein 1CYRSLIT2Down2.01.667Slit homolog 2 (Drosophila)CYRIGFBP5Down1.90.991Insulin-like growth factor binding protein 5CYRCYR61aUp3.61.943Cysteine-rich, angiogenic inducer, 613.3CYRTHBS1Up3.43.868Thrombospondin 1APNSTAT1Up3.01.222Signal transducer and activator of transcription 1AKTANKRD1Up3.02.226Ankyrin repeat domain 1 (cardiac muscle)CYRITGB1Up2.51.994Integrin \u03b21\u2009=\u2009\u03b2-subunit of fibronectin receptor (CD29)AKTBDNFUp2.43.641Brain-derived neurotrophic factorAKTMATR3Up2.40.895Matrin 3APNJAK2Up2.33.469Janus kinase 2APNSLC16A1Up2.31.943Solute carrier family 16 member 1APNHBEFGUp2.23.179Heparin-binding EGF-like growth factorCYRCD47Up2.14.108Integrin-associated signal transducerCYRFGF2Up2.12.868Fibroblast growth factor (basic)APNSORT1Up2.11.632SortilinAPNEPHA4Up2.12.345EPH receptor A4AKTSTX16Up2.12.226Syntaxin 16CYRNESUp2.02.487NestinCYRPDGFCUp2.03.469Platelet derived growth factor CCYRPTK2Up2.01.571Protein tyrosine kinase 2APNPTPN11Up2.01.632Protein tyrosine phosphatase non-receptor type 11 AKTGOSR2Up2.01.667Golgi SNAP receptor complex member 2APNNFAT4aUp1.92.345Nuclear factor of activated T cells 4CYRBIRC4Up1.91.943Baculoviral IAP repeat-containing 4CYRHSXIAPA1Up1.92.487XIAP associated factor-1AKTPPP2R1BUp1.93.641Protein phosphatase 2 regulatory subunit A \u03b2APNSMARCA2Up1.92.226SWI\/SNF related matrix associated regulator of chromatin AKTMID1Up1.93.175Midline 1AKTPARVAUp1.82.621Parvin-\u03b1APNLPLUp1.82.487Lipoprotein lipaseCYRLAMA2Up1.81.994Laminin-\u03b12AKTGSK3BUp1.80.991Glycogen synthase kinase 3\u03b2AKTPPP2R2AUp1.81.222Protein phosphatase 2 regulatory subunit B \u03b1APNSYNCRIPUp1.81.994Synaptotagmin binding RNA interacting proteinAKTPPP2R2AUp1.81.222Protein phosphatase 2 regulatory subunit B \u03b1AKTUPF3AUp1.83.173UPF3 regulator of nonsense transcripts A APNADIPOR1Up1.72.868Adiponectin receptor 1CYRTFPIUp1.73.868Tissue factor pathway inhibitorAPNSNX1Up1.74.362Sorting nexin 1APNSNX2Up1.74.467Sorting nexin 2APNRBBP4Up1.72.345Retinoblastoma binding protein 4AKTFBXO32Up1.72.487F-box protein 32AKTVPS33AUp1.72.226Vacuolar protein sorting 33AAPNNFAT1aUp1.65.091Nuclear factor of activated T cells 1AKTNFAT3aUp1.64.108Nuclear factor of activated T cells 3APNJAK1Up1.61.994Janus kinase 1APNSP3Up1.62.226Sp3 transcription factorAKTFRAP1Up1.64.362FK506 binding protein 12AKTSTX6Up1.61.994Syntaxin 6CYRPRELPUp1.64.757Proline arginine-rich end leucine-rich repeat proteinAPNRALBUp1.63.173v-ral simian leukemia viral oncogene homolog BAPNFGF13Up1.64.757Fibroblast growth factor 13APNTGFBR1Up1.64.108Transforming growth factor-\u03b2 receptor IAKTFTSUp1.63.469Fused toes homologAKTAKT1Up1.54.757v-akt murine thymoma viral oncogene homolog 1AKTPPP2CAUp1.54.362Protein phosphatase 2 catalytic subunit \u03b1All factors of deregulations are derived from the Affymetrix microarray analysis. For certain genes additional TaqMan analyses were conducted, which are then given in italics below the respective Affymetrix values (see also Supplementary Fig. 2).aPlease note the synonyma APN\u2009=\u2009ADIPOQ, CYR61\u2009=\u2009CCN1, NFAT1\u2009=\u2009NFATC2IP, NFAT3\u2009=\u2009NFATc4, NFAT4\u2009=\u2009NFATc3\nThe network analysis of the microarray data [8] was conducted by the IPA software employing the genes significantly (q value\u2009<\u20095.0) and \u22651.5-fold deregulated. IPA is based on modeled biological networks derived from information of peer-reviewed scientific publications. Analysis of our data revealed several strongly altered regulatory networks. The gene networks shown in Fig.\u00a01a,c,e have not been previously described in former microarray studies of DCM (details in discussion). One of these networks centered around the CYR61 or CCN1 gene (Fig.\u00a01a,b), another one around the APN gene (Fig.\u00a01c,d). These networks are related to cell-to-cell signaling and cellular compromise. Another network around the AKT1 gene (Fig.\u00a01e,f) also deserves consideration because AKT1 is involved in the regulation of both CYR61 [9] and APN [10] and because APN strongly influences CYR61 expression in vitro (Fig.\u00a02b). Table\u00a02 summarizes all member genes of these three networks with full gene names, q values, and fold changes. Those two genes (CYR61, APN) that were among the most strongly deregulated and, at the same time, key components of complex deregulated networks were further investigated in cardiovascular cell cultures in vitro (Fig.\u00a02a,b).\nFig.\u00a02Cytokines regulating of APN and CYR61 in cardiovascular cells. We searched for possible triggers of cardiac APN and CYR61 deregulation in DCMi in both endothelial cells and cardiomyocytes. a, b Summarize real-time quantitative TaqMan PCR data on the regulatory potential of inflammatory cytokines in these cell cultures investigated as first approximation to the far more complex in vivo situation with multiple resident and infiltrating cell types (compare Fig.\u00a03). a Endothelial cells expressed CYR61 and both APN receptors but no detectable amount of APN mRNA (data not shown). The inflammatory cytokines TNF-\u03b1\u00a0and IFN-\u03b2 induced CYR61 expression in these cells, whereas others showed no effect (IFN-\u03b3) or even reduced CYR61 (TNF-\u03b2). To reveal possible interactions between the APN and CYR61 regulatory networks, CYR61 induction experiments were conducted in the presence (asterisksin shaded bars) vs the absence of APN. The effect of APN on CYR61 induction by cytokines was unexpectedly strong: induction of CYR61 by TNF-\u03b1 or IFN-\u03b2 was not only abolished but reverted resulting in a net reduction of CYR61 expression below that in untreated controls. The APN concentration at which effect was observed is in the range 3\u201330\u00a0\u03bcg\/ml as observed in human plasma [14]. Error bars indicate the SEM from two independent experiments each performed in triplicate. b In contrast to endothelial cells, cardiomyocytes expressed APN and its receptors as well as CYR61. Several inflammatory cytokines (TNF-\u03b1, IFN-\u03b2,\u03b3) significantly reduced APN expression in cardiomyocytes, whereas\u00a0CYR61 expression was not significantly altered in this cell type (not shown). Error bars as in panel (a)\nThe APN regulatory network contains significantly deregulated (q value\u2009\u2264\u20095.0, \u22651.5-fold change) genes not previously reported in microarray studies of DCM (full gene names in Table\u00a01): STAT1 3.0-fold up; JAK2 2.3-fold up; HBEGF 2.2-fold up; SORT1 2.1-fold up; PTPN11 2.0-fold up; LPL 1.8-fold up; JAK1 1.6-fold up; SP3 1.6-fold up; APN 6.8-fold down; INSR 2.5-fold down; PIK3R1 2.5-fold down; LIFR 2.0-fold down; TNXB 2.0-fold down; and TGFBR3 2.0-fold down. Three genes encoding the nuclear factors of activated T cells (NFAT), NFAT1\u2009=\u2009NFATC2IP (1.6-fold up), NFAT3\u2009=\u2009NFATC4 (1.6-fold up), NFAT4\u2009=\u2009NFATC3 (1.9-fold up), are also linked to the APN network as negative regulators of APN expression [11]. Similarly, the ADIPOR1 gene (1.7-fold up) encoding the APN receptor 1 gene is part of the APN signal transduction system.\nA second regulatory network around CYR61 contains further deregulated genes with q value\u2009\u2264\u20095.0 and \u22651.5-fold change (full gene names in Table\u00a01): CYR61\u2009=\u2009CCN1 3.6-fold up; THBS1 3.4-fold up; ITGB1 2.5-fold up; CD47 2.1-fold up; FGF2 2.1-fold up; NES 2.0-fold up; PDGFC 2.0-fold up; PTK2 2.0-fold up; BIRC4 1.9-fold up; HSXIAPA1 1.9-fold up; LAMA2 1.8-fold up; TFPI 1.7-fold up; CXCL14 5.9-fold down; PDGFA\u00a03.6-fold down; TMSB10 2.6-fold down; EGFR 2.1-fold down; LAMA4 2.4-fold down; HSPG2 2.1-fold down; TNXB 2.0-fold; and IGFBP5 1.9-fold down. A recent publication also links CYR61\u2009=\u2009CCN1 to Wnt signaling pathways [13] and several Wnt signal pathway genes were deregulated in DCMi: STX16 2.1-fold up; GCLM 2.0-fold up; CSNK1A1 1.9-fold up; PPP2R1B 1.9-fold up; GSK3B 1.8-fold up; INSR 2.5-fold down; FBXO40 2.1-fold down; and CLIC5 2.0-fold down.\nIn addition to the multifunctional CYR61 and APN genes, several other deregulated genes are known to be involved in immunological and inflammatory processes: The CXCL14 gene was 5.9-fold and the neurturin (NRTN) gene 5.8-fold downregulated in DCMi. The interleukin-17D (IL-17D) gene, a member of IL-17A to F gene family, was 2.6-fold induced. IL-17 serum levels were elevated in DCMi patients (not shown). Altered intracardiac immune regulation is further suggested by upregulation of the immediate early response 3 (IER3, 2.6-fold) and 5 (IER5, 2.3-fold) genes, ubiquitin-specific protease 39 (USP39, 3.4-fold), phospholipase A2-activating protein (PLAA, 2.6-fold), and IFN-related developmental regulator 1 (IFRD1, 2.4-fold). Induction of the neural cell adhesion molecule (NCAM1, 2.0-fold) gene [14] and the coxsackie virus\u2013ADV-receptor (CXADR, 1.7-fold) gene [15] already known from immunohistochemical studies of DCMi hearts were recognized by the microarrays, too.\nAs a first step to elucidate the molecular mechanisms that deregulate CYR61 and APN expression in human DCMi, cell culture studies were conducted. Both genes were significantly deregulated in DCMi as components of complex gene networks. Figure\u00a02a shows that certain inflammatory cytokines induced CYR61 expression in vascular endothelial cell cultures similarly as observed in the DCMi hearts. This CYR61 induction by TNF-\u03b1 and IFN-\u03b2 was completely abolished in the presence of APN (Fig.\u00a02a). The APN plasma range in humans is 3\u201330\u00a0\u03bcg\/ml [16], and the concentration therefore chosen for the cell culture investigations was 10\u00a0\u03bcg\/ml. At this concentration, APN generally suppressed the CYR61 expression level in this cell type. In cardiomyocytes, TNF-\u03b1, IFN-\u03b2, IFN-\u03b3, and IL-1\u03b2 all significantly reduced the expression of APN (Fig.\u00a02b), whereas CYR61 expression was not affected by these cytokines in this cell type. Several other deregulated genes of interest known to be involved in immunological and inflammatory processes were neither expressed in endothelial cells nor in cardiomyocytes, and therefore could not be studied in these cell biological models.\nDiscussion\nIdentification of DCM subtypes\nDCM is the term used to describe any clinical phenotype characterized by dilation and dysfunction of the cardiac ventricles. From an etiologic perspective, however, DCM encompasses a diverse spectrum of disease entities, some of which are due to monogenic defects in cardiac expressed genes [1], others triggered by exogenous factors including cardiotropic viruses [2]. Therapies directed at etiology-related pathogenic pathways in these etiologically distinct DCM subtypes may be more efficient than current standard treatment protocols employed in heart failure of any origin. However, etiology-related early pathogenic pathways must be assumed to prevail only in the beginnings of the respective DCM subtype, whereas common unspecific molecular pathomechanisms will predominate in advanced heart failure irrespective of etiology. One important DCM subtype as defined by cardiac inflammation was investigated in the current study to further define it at the molecular level. Whereas previous studies focused on advanced or end-stage DCM [5] where specific pathomechanisms are overwhelmed by unspecific processes, we probed an earlier and restrictively defined phenotype (DCMi) to identify early and etiology-related disease processes. Importantly, our patients were not terminally ill nor on maximum supportive therapy as in previous studies of advanced DCM. Similarly, controls had no severe underlying condition as, e.g., heart transplantation donor hearts often used as controls for expression profiles but were free of cardiac disease after comprehensive exclusion of multiple cardiac differential diagnoses. Further, any search for etiology-related pathomechanisms is more likely successful if the study groups are highly homogenous. An excellent previous study has also defined its patient groups by clinical, morphological, and hemodynamic phenotyping [17] and derived interesting data on common expression profiles in disorders linked by common hemodynamic anomalies. We likewise defined a highly homogeneous study group by sequential stratification of a large DCM patient cohort, first, by standard clinical, morphological, and hemodynamic criteria, second, by biopsy-based detection of cardiac inflammation in conjunction with a cardiotropic virus. Cardiac inflammation in our patients was associated with the presence of PVB19 genomes in the myocardium. We think, however, that the observed anomalous expression pattern probably reflects a general inflammatory pathomechanism that may be triggered by diverse proinflammatory stimuli. After the current pilot study all the key genes of this anomalous pattern need to be further investigated in major patient groups with varying degrees of cardiac inflammation and hemodynamic impairment, to address issues of specificity and dynamic. In vivo biopsies on which the current study was entirely based further provide the perspective for follow-up studies of the clinical courses that would then add a third stratification criterion for patient selection to be used in the future.\nCardiac expression profile of DCMi\nThe anomalous cardiac expression pattern observed for human DCMi was not described in previous studies of human cardiac diseases or heart failure of any origin [5, 18\u201327]. Most network genes listed in Table\u00a02 and, in particular, CYR61 and APN have, to our knowledge, not been found to be deregulated in human microarray studies and also not in animal models of DCM. A comprehensive recent review [5] summarizes that the major molecular pathway changes in human DCM, and other human cardiomyopathies encompass sarcomeric, cytoskeletal, extracellular matrix, Ca2+ homeostasis, apoptosis, and energy metabolism genes. Major deregulations of inflammation- and immune system-associated genes as in DCMi were not included. Because we investigated a DCM subtype associated with intracardiac inflammation, a recent microarray study of two giant cell myocarditis patients [28] is of specific interest for comparison. That study addressed an immunohistologically defined inflammatory cardiac phenotype clearly different from that in our study and found deregulated T cell activation genes, including IL-10 receptor-\u03b1, \u03b22-integrin, chemokine receptor 4, and chemokine ligands 5, 9, 13, and 18. Remarkably, none of the latter genes was deregulated in DCMi but only CXCL14. Conversely, NFAT1 and NFAT4 gene upregulations and APN gene downregulation as observed in DCMi were not observed in the giant cell myocarditis study. This appears to reflect distinct immunological pathomechanisms. \nThe CYR61 gene and its associated network showed particularly strong deregulation in DCMi. CYR61\u2009=\u2009CCN1 is a member of the CCN gene family [29] (also including connective tissue growth factor [CTGF]\u2009=\u2009CCN2) and has already attracted attention as a multifunctional, secreted protein able to modulate multiple cell functions. It induces  apoptosis in fibroblasts [30] and mediates cell death in neurons [31] and in virus-infected cells [32]. From a therapeutic perspective, it is interesting that RNA interference-mediated silencing of the CYR61 gene protected cells against cell death, whereas CYR61 overexpression enhanced it [32]. Moreover, CYR61 coordinates genes that control inflammation (IL-1\u03b2), extracellular matrix remodeling (e.g., MMP1,3), cell\u2013extracellular matrix interactions, and angiogenesis [33, 34]. CYR61 acts in a cell type- and context-dependent manner through direct binding to at least five distinct integrins, including \u03b16\u03b21 and heparan sulfate proteoglycans as co-receptors [35], integrins \u03b1v\u03b23 and \u03b1v\u03b25 [36], and integrin \u03b1M\u03b22 on blood monocytes [37]. CYR61 is involved in integrin-linked kinase-mediated AKT and \u03b2-catenin-T cell factor\/Lef signaling [38]. It is also regulated by canonical Wnt signaling and involved in mesenchymal stem cell differentiation [13]. In contrast to this wealth of data on in vitro actions of CYR61, data on its physiological functions and pathogenic potential in humans are sparse. CYR61 upregulation has only recently been described in end-stage ischemic cardiomyopathy [39]. The current work shows, for the first time, CYR61 induction in an inflammatory cardiac disorder with far less severe clinical phenotype, where, most likely, no irreversible cardiac damage has yet occurred. Cardiac CYR61 expression is apparently induced by multiple stimuli [39], including inflammation in vivo and inflammatory cytokines in vitro (this study). Whereas transient CYR61 induction may be a temporarily beneficial response of the heart to various stress factors, chronic stimulation of the auto\/paracrine signaling pathways activated by CYR61 may be maladaptive and pathogenic in itself by, e.g., sustaining exuberant tissue inflammatory and remodeling processes [33]. Our observation of CYR61 overexpression in DCMi suggests to investigate further, if modulation of CYR61-dependent auto\/paracrine pathways might have therapeutic potential in cardiac diseases associated with local inflammation.\nIn the current study, we show, for the first time, that APN is synthesized by human hearts in situ, not only by adipose tissues as previously assumed, and that this local synthesis is strongly downregulated in a human cardiac disease. Cultured cardiomyocytes were already known to express APN and both of its receptors and secrete APN [42]. APN has encountered high interest in the cardiovascular field due to pioneering work in APN knockout mice, proving important functions of APN in the heart [40, 41]. These studies assumed, however, that cardioprotective effects were exerted by APN synthesized by adipose tissue and acting via the circulation. APN inhibits apoptosis and TNF-\u03b1 production in cardiomyocytes, modulates hypertrophic signals in the heart [40], and protects it from ischemic injury in a mouse model [41]. APN regulation has not been previously investigated in human hearts in situ. Previous studies [40, 41] and the present work suggest that cardiac APN is influenced by multiple factors acting on components of the APN gene network. AKT1 is involved in the regulation of both CYR61 [9] and APN [10]. Interestingly, APN abolished CYR61 induction by inflammatory cytokines in endothelial cells (Fig.\u00a02a). Because multiple other endothelial activation markers are induced in DCMi [54, 55], it is of interest to know the whole spectrum controlled by APN, but this is beyond the scope of the current study. In synopsis with a previous report that APN inhibits IL-8 synthesis by endothelial cells [10], these data suggest that local cardiac APN synthesis is required for adequate control of local inflammatory processes. If so, depression of cardiac APN synthesis by various mechanism may render the heart vulnerable to inflammatory challenges such as imposed by infiltrating immune cells in DCMi or under other conditions associated with enhanced cardiac inflammatory cytokine production [43, 44]. Modulation of the local cardiac APN system may therefore have therapeutic potential under certain conditions. Due to the complexities of the biological functions of both CYR61 and APN, it is impossible to predict the effects of their modulation in vivo. Synopsis of previous work and the current study suggests to investigate, however, if modulation of the balance between the CYR61 and APN networks may have therapeutic potential in cardiac diseases associated with local inflammation. One obvious approach would be cardiac APN gene therapy, another one RNA interference-mediated CYR61 suppression by cardiac shRNA expression.\nIn addition to the multifunctional CYR61 and APN proteins and interacting gene products such as the NFATs, several other deregulated genes are also directly involved in immunological and inflammatory processes. Two genes encoding proteins described as endothelial activation markers in DCMi [14, 15] were upregulated 2.0-fold (NCAM1) and 1.7-fold CXADR. CXCL14 (a component of the CYR61 network) belongs to a family of dendritic cell- and monocyte-attracting chemokine-like proteins [45, 46]. Loss of CXCL14 in tumors was associated with low infiltration by dendritic cells, whereas its restoration caused dendritic cell attraction in vitro and in vivo [47]. The downregulated NRTN gene belongs to the same family as glial cell line-derived neurotrophic factor (GDNF). GDNF and NRTN signaling is mediated by a two-component receptor: the signal-transducing component RET shared by both ligands and the ligand-specific binding components GFR-\u03b11 (GDNF) or GFR-\u03b12 (NRTN). Human T and B cells and monocytes produce RET and NRTN. GFR-\u03b11 and GFR-\u03b12 are transcribed in all immune cell subsets, with GFR-\u03b12 being the dominant ligand-binding chain in T cells, B cells, and monocytes [48]. Therefore, NRTN downregulation may alter immune cell communication, whereas little is known about its functions in other cell types. IL-17D belongs to a family of secreted proteins that play an active role in inflammatory and autoimmune diseases [49]. Because IL-17D stimulates production of IL-6, IL-8, and granulocyte\/macrophage colony-stimulating factor (GM-CSF) in human endothelial cells [50], its upregulation may enhance the activation of the endothelium, the primary attachment site for migrating immune cells. IL-17 functionally cooperates with TNF-\u03b1 [51], enhances growth of vascular endothelial cells [52], and stimulates matrix metallo-proteinase-9 expression [53]. The upregulated PLAA activates mammalian PLA2s involved in inflammatory responses [54]. Thus, PLA2-IB is upregulated during inflammation, stimulates CXCL8 production via extracellular signal-regulated kinase (ERK) and nuclear factor-\u03baB in neutrophils, and modulates inflammatory responses via the PLA2-IB receptor [55].\nWorking model of local cardiac inflammation control\nA working model of a novel inflammation-associated cardiac pathomechanism is outlined in Fig.\u00a03. The model proposes the existence and pathogenic relevance of a local inflammation control system in human hearts acting by local synthesis of anti-inflammatory proteins, a representative of which is APN. If local cardiac APN synthesis by cardiomyocytes is absent, as in knockout mice [40, 41], or suppressed, as in DCMi patients, inflammatory challenges, such as imposed by cardiac immune cell infiltrations (Supplementary Fig. 1), may result in grave damage to vascular endothelia and myocardium. The model assumes that cardiac inflammation control is predominantly mediated by auto\/paracrine actions of locally synthesized APN, whereas, in the context of cardiac ischemic injury in APN knockout mice, a complementary systemic model of APN action has been proposed, in which APN secreted by adipose tissue acts hormone-like to protect the heart [41] and the endothelia via the circulation [10]. APN acts directly upon immune cells, such as natural killer (NK) cells [12], and on nonimmune cells, such as vascular endothelia [10]. One new target controlled by APN is CYR61 that is locally upregulated in DCMi hearts in situ and whose endothelial induction by cytokines is abolished by APN (Fig.\u00a02a). Although CYR61 induces transient local inflammation and matrix remodeling during healing processes [33], its activity needs to be controlled so that it does not cause chronic tissue damage. In addition to such direct anti-inflammatory actions of APN upon non-immune targets (endothelial cells, cardiomyocytes), a recent study [12] shows that APN is also a potent negative regulator of IL-2-induced NK cell activation. IFN-\u03b3 production was suppressed by APN, accompanied by downregulation of the IFN-\u03b3-inducible TNF-related apoptosis inducing ligand and Fas ligands. APN and cellular immunity are further linked by the fact that NFAT transcription factors are key regulators of T cell function [56] and also negative regulators of APN expression [11]. All local cardiac functions of CYR61, APN, and other factors in the model are thought to be mediated via auto\/paracrine loops unrelated to the respective circulating plasma levels (3\u201330\u00a0\u03bcg\/ml for APN in humans, unknown for CYR61).\nFig.\u00a03Working model of local cardiac inflammation control. The model proposes the existence of a inflammation control system in human hearts acting by local synthesis of anti-inflammatory proteins, a representative of which is APN. If local cardiac APN synthesis by cardiomyocytes is genetically absent [38, 39] or suppressed (this study), inflammatory challenges may result in aggravated damage to endothelia and myocardium. This local model assumes that inflammation control and other regulatory functions are mediated by auto\/paracrine actions of APN synthesized locally by cardiomyocytes, whereas in the context of APN knockout mice [38, 39], a complementary systemic model of APN action was proposed in which APN secreted by adipose tissue acts hormone-like to protect heart and endothelia via the circulation. APN (downregulated in DCMi) acts directly upon immune cells [10] (right side of figure) and also on nonimmune cells such as vascular endothelia [8] (left side). One new target for control by APN is CYR61 that is locally upregulated in DCMi (center) and whose endothelial induction by cytokines is suppressed by APN (see Fig.\u00a02a). In addition to such direct anti-inflammatory action of APN on vascular endothelia (left side), APN is a potent negative regulator of IL-2-induced NK cell activation (right side) [10]. APN and cellular immunity are further linked by NFATs (upregulated in DCMi), which are key regulators of T cell function [54], and negative regulators of APN expression [9]. The functions of CYR61, APN, and other factors deregulated in DCMi in this local model are thought to be mediated via auto\/paracrine loops unrelated to the respective circulating plasma levels. CYR61-associated CXCL14 downregulation in DCMi (right side) may alter immune cell attraction to the heart. IL-17D induction in DCMi (left side) may stimulate IL-6, IL-8, and GM-CSF production by endothelial cells and thus activation of the endothelium, the primary attachment site for migrating immune cells\nLimitations and perspective of the study\nGain\/loss-of-function experiments are required to clarify which of the gene deregulations in DCMi are primary and causative or secondary and maladaptive. Conventional gain\/loss-of-function experiments in cell cultures may contribute to solve these questions but are rather artificial as compared to the complex physiological situation in intact tissues composed of multiple interacting cell types. Cardiac overexpression of deregulated genes and silencing of these genes by shRNA-mediated RNA interference in vivo has recently become feasible, however, by intravenous injection of cardiotropic AAV8 and AAV9 vectors in mice [57, 58]. These systems have considerable potential for gene therapy [59]. Beyond the classical knockout technologies, this novel approach may also help to clarify pathogenic cause\u2013effect relationships arising from genomic and proteomic screening studies in humans such as the current one based on EMBs of human hearts in situ.\nConclusions\nGenomic expression profiling evolves as a useful tool to identify novel pathomechanisms in human cardiac disorders [5, 8] because most animal models, if available at all, display multiple crucial differences to the respective human disease. Restrictive disease phenotyping significantly improves the chance of genomic approaches to identify such mechanisms and therapeutic targets even in complex human disorders. The current work shows, for the first time, local APN expression in human hearts in situ and its downregulation in a human cardiac disease. It further demonstrates that APN efficiently inhibits the endothelial expression of CYR61 that is strongly induced in DCMi. Synopsis of previous work and these data suggests to investigate further, if modulation of the balance between the CYR61 and APN network may have therapeutic potential in cardiac diseases associated with inflammation.\nElectronic Supplementary Material\nBelow is the link to the electronic supplementary material.\nFig.\u00a0S1\nThe disease phenotyping of the DCMi patient group (Table 1 and Patients and methods) that were investigated by genomewide Affymetrix microarray expression profiling included (in addition to clinical and molecular virological characterization) an immunohistochemical characterization of cardiac immune cell infiltration and vascular endothelial activation. The immunohistochemical studies were done on EMBs obtained by the same standard procedure as those used for the Affymetrix microarray expression profiling and for the TaqMan quantitative RT-PCRs (264.6 KB) (226.1 KB) (159.6 KB)\nFig.\u00a0S2\nReal-time quantitative TaqMan PCR data on shifts in gene expression in the DCMi patients versus the control group. TaqMan data on the above novel DCMi-associated genes were obtained by analysis of residual RNA extracted from the same EMB as already used for the genomic Affymetrix microarrays and confirmed the respective data from the microarray study reported in detail in Table\u00a02, Fig.\u00a02 (panels a\u2013f), and http:\/\/www.ncbi.nlm.nih.gov\/projects\/geo\/ in all cases. Sharp sign denotes p\u2009<\u20090.05 between the study groups. In addition to these new genes, the standard heart failure-associated genes NPPA and NPPB were significantly (p\u2009<\u20090.05) upregulated (18.7-fold and 39.0-fold, respectively) in the DCMi group as assessed by TaqMan PCR, confirming a trend observed in the microarrays (7.5-fold at q\u2009=\u20096.400 and 20.5-fold at q\u2009=\u20095.722; (PDF 22 kb).\nTable S1\nExtension of Table\u00a02 with all shifts in cardiac gene expression down to 1.5-fold change for DCMi patient group vs Cont control group, as assessed by Affymetrix microarray analysis of RNA extracted from EMB. For further details of the DCMi and Cont groups, see Table\u00a01 and Patients and methods. The complete Affymetrix dataset is available from the GEO website http:\/\/www.ncbi.nlm.nih.gov\/ projects\/geo\/. GEO experimental series number was assigned as GSE4172 (PDF 1 mb).","keyphrases":["genomic expression profiling","human inflammatory cardiomyopathy","dilated cardiomyopathy","heart failure","molecular pathomechanisms","molecular therapeutic targets"],"prmu":["P","P","P","P","P","R"]}
{"id":"Hum_Reprod_Update-1-1-2423221","title":"Cytogenetic determinants of male fertility\n","text":"BACKGROUND Cytogenetic abnormalities have been known to be important causes of male infertility for decades.\nIntroduction\nInfertility is a significant problem, affecting up to 15% of couples of reproductive age (de Kretser, 1997). For many years, it was assumed that most reproductive problems could be attributed to the female partner but research in recent years has demonstrated that \u223c30\u201350% of infertility is caused by a male factor (Lipshultz and Howards, 1997). Treatment of male-factor infertility by in vitro fertilization (IVF) was largely unsuccessful. However, the development of intracytoplasmic sperm injection (ICSI) (Palermo et al., 1992) revolutionized the treatment of male infertility, providing many men the chance to father their own children. ICSI also greatly stimulated research into the causes of male infertility, and our knowledge has increased tremendously in the past decade.\nThe development of new techniques has provided a wealth of information on the cytogenetics of human sperm. The human sperm\u2013hamster oocyte fusion system allowed the first analysis of human sperm chromosomes (Rudak et al., 1978; Martin et al., 1983; Brandriff et al., 1985; Mikamo et al., 1990; Templado et al., 1996). This technique provides precise karyotypes in which numerical and structural abnormalities can be assessed for each chromosome (Fig.\u00a01). Unfortunately, the technique is very difficult with only a handful of laboratories achieving success; also the data yield, though of great significance, is low.\nFigure 1:\nQ-banded karyotype of a normal 23,X sperm.\nFluorescence in situ hybridization (FISH) analysis with chromosome-specific DNA probes was developed in the 1990s, providing a faster, cheaper, easier alternative for detecting aneuploidy in human sperm (Martin et al., 1993; Robbins et al., 1993; Wyrobek et al., 1994; Martin et al., 1996). Also, sperm hampered by abnormalities in motility or other aspects of fertilization can be assessed using FISH analysis (Downie et al., 1997; Egozcue et al., 1997; Robbins et al., 1997; Martin et al., 1999a; Hristova et al., 2002). This is a simple technique that has been embraced by many laboratories, but it must be remembered that it is indirect: fluorescent signals, rather than chromosomes, are counted (Fig. 2).\nFigure 2:\nDisomic XY sperm, with green X signal, red Y signal and a single blue chromosome 1 signal (an internal control to distinguish disomy from diploidy).\nSingle sperm polymerase chain reaction (PCR) or single sperm typing is a difficult, time-consuming technique but it can be used in a very powerful manner for specific studies on recombination in delimited areas of the genome (Shi et al., 2001, 2002; Tiemann-Boege et al., 2006).\nThe latest development to shed light on the causes of chromosomal abnormalities and infertility is meiotic analysis of the synaptonemal complex (SC). Various important meiotic structures can be identified by the use of immunofluorescence. Antibodies against SCP1 (transverse elements) or SCP3 (lateral elements) can be used to visualize the SCs (the proteinaceous structure linking homologous chromosomes in prophase of meiosis I). The centromere can be localized with CREST antisera. Most importantly, recent studies have demonstrated that antibodies against the DNA mismatch repair protein MLH1 identify the sites of meiotic exchange in SCs in both mouse (Baker et al., 1996; Anderson et al., 1999) and human spermatocytes and oocytes (Barlow and Hult\u00e9n, 1998; Lynn et al., 2002; Tease et al., 2002).\nThe use of a multicentromeric FISH technique in conjunction with SC meiotic analysis is a powerful tool to study recombination and the fidelity of pairing in individual identified chromosomes (Sun et al., 2004b, 2006b, 2007a; Oliver-Bonet et al., 2006) (Fig.\u00a03). These studies have provided some important information on the meiotic abnormalities in infertile men and the association between meiotic recombination and aneuploidy (Gonsalves et al., 2004; Sun et al., 2004a, 2006a, 2007b; Codina-Pascual et al., 2005; Martin, 2005).\nFigure 3:\n(upper) Human pachytene spermatocyte with SCs shown in red, centromeres in blue and MLH1 foci in yellow. (lower) Subsequent cenM-FISH analysis permits identification of individual chromosomes so that recombination (MLH1) foci can be analyzed for each SC.\nIt is clear that cytogenetic abnormalities (both somatic and meiotic) are a major cause of male infertility. This review will summarize our knowledge of somatic chromosome abnormalities and their effects on sperm, susceptibility to sperm chromosome anomalies in chromosomally normal infertile men, and meiotic errors in translocation carriers and infertile men with a normal chromosome constitution.\nMaterials and Methods\nResearch publications from 1978 to 2008, from PubMed and the author's personal library, were reviewed. Methods for selecting and synthesizing the data were based on personal experience.\nSomatic chromosomal abnormalities\nSomatic chromosomal abnormalities are relatively common in humans. These can be numerical chromosomal abnormalities, such as an extra chromosome, or structural abnormalities such as a translocation. It has long been recognized that somatic chromosomal abnormalities are associated with infertility, an increased probability of pregnancy loss and the birth of handicapped children. The frequency of somatic chromosomal abnormalities in infertile men varies from 3% to 19%: 3% in the cases of mild infertility and 19% in men with non-obstructive azoospermia (NOA) (Yoshida et al., 1997). Thus, it is imperative that chromosome karyotyping be performed in all infertile men so that they can be apprised of their risks. Somatic chromosomal abnormalities have definite consequences on the cytogenetic abnormalities observed in sperm and consequently those observed in newborns.\nSex chromosomal abnormalities\n47,XYY\nMen with a 47,XYY karyotype are generally fertile, but they are seen more frequently in infertile populations. Many 47,XYY men produce normal children, but there has been no systematic analysis of children born to these men. Theoretically, 50% of the sperm cells should be abnormal. In a study of 75 sperm karyotypes from a 47,XYY male, we found no sperm disomic for a sex chromosome (Benet and Martin, 1988). Our results supported the hypothesis that the extra sex chromosome is eliminated during spermatogenesis. FISH analysis on the same male with 10 000 sperm studied demonstrated a small but significant increase for XY disomy to 0.6% (Martin et al., 1999b). Other laboratories have demonstrated increased frequencies of sperm aneuploidy for the sex chromosomes ranging from 0.3% to 15% (Mercier et al., 1996; Chevret et al., 1997; Shi and Martin, 2000b).\nThe majority of the more-stringent three-color FISH studies have demonstrated a low risk of 24,YY or 24,XY sperm of \u223c1%. However, a recent study of two 47,XYY men with severe oligozoospermia demonstrated a higher frequency of aneuploid sperm of 37\u201338%, with approximately one-half of the abnormalities caused by sex chromosomal aneuploidy (Gonzalez-Merino et al., 2007). This group found that the frequency of sperm aneuploidy was concordant with the frequency of aneuploidy in preimplantation embryos (32%).\nSince many 47,XYY men have normal semen parameters, the severe oligozoospermia observed in these men may indicate more perturbations during meiotic pairing, subsequent loss of germ cells and the production of aneuploid sperm.\n47,XXY\nPatients with Klinefelter syndrome (47,XXY) or mosaic variants of Klinefelter syndrome have greatly impaired spermatogenesis, with severe oligozoospermia or azoospermia. Nevertheless, these men are candidates for ICSI, particularly with the new methods used for recovering testicular spermatozoa. Studies on sperm chromosomes from men with Klinefelter syndrome have also demonstrated that the extra sex chromosome appears to be eliminated during spermatogenesis. FISH analysis has demonstrated that the frequency of aneuploidy for the sex chromosomes varies from 1.5% (Lim et al., 1999) to 7% (Kruse et al., 1998) in sperm from Klinefelter mosaics, and 2% (Rives et al., 2000) to 45% (Estop et al., 1998) in the sperm of men who appear to have a non-mosaic 47,XXY karyotype. The majority of babies born to 47,XXY men have been normal although chromosomally abnormal fetuses have been reported (Ron-el et al., 2000; Friedler et al., 2001). Staessen et al. (2003) studied 113 embryos by preimplantation genetic diagnosis (PGD) and found a significantly increased frequency of autosomal and sex chromosomal abnormalities. Thus, there appears to be a small increased risk for these men.\nStructural aberrations\nTranslocations\nRobertsonian translocations: Robertsonian translocation carriers have a fusion of the long arms of two acrocentric chromosomes. The fused short arms (containing redundant DNA) are generally lost so that the carrier has a balanced chromosomal constitution with 45 chromosomes. When the chromosomes pair during meiosis, they do so as a trivalent, and the resulting gametes can be chromosomally normal or aneuploid with an extra or missing chromosome q arm. This can cause translocation Down's syndrome or Patau's syndrome, e.g. sperm karyotyping studies in our laboratory, and others have demonstrated that the actual frequency of unbalanced sperm in seven men is lower than theoretically expected, with 3\u201327% of sperm being unbalanced because of the translocation (Martin, 1995; Ogawa et al., 2000). Similarly, FISH studies in 71 Robertsonian translocation heterozygotes have demonstrated that 7% to 40% are unbalanced, with a mean of 15% unbalanced (Rousseaux et al., 1995; Blanco et al., 2000; Escudero et al., 2000; Honda et al., 2000; Vegetti et al., 2000; Frydman et al., 2001; Morel et al., 2001; Pellestor et al., 2001; Acar et al., 2002; Baccetti et al., 2002; Anton et al., 2004; Anahory et al., 2005; Douet-Guilbert et al., 2005; Machev et al., 2005; Brugnon et al., 2006; Hatakeyama et al., 2006; Moradkhani et al., 2006a,b; Ogur et al., 2006; Chen et al., 2007; Nishikawa et al., 2008). Thus, all Robertsonian translocations have relatively similar segregation behaviors despite the participation of different acrocentric chromosomes. The risks of chromosomal imbalance at prenatal diagnosis are even lower, with generally only 1\u20132% of paternally derived Robertsonian translocations being unbalanced (Bou\u00e9 and Gallano, 1984). Even though the risks are low, prospective parents deserve to be informed of them as the abnormalities can be devastating, and trisomy 13 fetuses have been detected after ICSI using sperm from a father with a Robertsonian translocation (In't Veld et al., 1997a). Also, a special category of Robertsonian translocation, if found, would nullify the usefulness of ICSI: a Robertsonian translocation between the same two chromosomes, e.g. two chromosomes 13, would produce only abnormal embryos, trisomy 13 or monosomy 13, with no hope of long-term survival. A case with this particular problem was discovered in the Netherlands after three unsuccessful ICSI attempts (In't Veld et al., 1997b).Reciprocal translocations: reciprocal translocations occur when there are exchanges of chromosome material between any chromosomes. During meiosis, four chromosomes must pair in reciprocal translocation heterozygotes and the resulting segregations have a higher frequency of unbalanced chromosomes than Robertsonian translocations. Sperm karyotyping studies of 37 reciprocal translocation heterozygotes have shown that 19\u201377% of spermatozoa are unbalanced (Estop et al., 1995; Martin and Spriggs, 1995; Cifuentes et al., 1999). FISH analyses of chromosome segregations in 99 reciprocal translocation heterozygotes have also shown a large range in the frequency of unbalanced sperm, from 37% to 91% (Goldman and Hult\u00e9n, 1993; Spriggs and Martin, 1994; Rousseaux and Chevret, 1995; Estop et al., 1997, 1998, 1999, 2000; Van Hummelen et al., 1997; Blanco et al., 1998a, 2000; Martini et al., 1998; Mercier et al., 1998; Cifuentes et al., 1999; Giltay et al., 1999; Honda et al., 1999; Vegetti et al., 2000; Oliver-Bonet et al., 2001, 2002, 2004; Pellestor et al., 2001; Cora et al., 2002; Geneix et al., 2002; Trappe et al., 2002; Baccetti et al., 2003; Escudero et al., 2003; Lim et al., 2003; Anton et al., 2004; Morel et al., 2004; Douet-Guilbert et al., 2005; Machev et al., 2005; Brugnon et al., 2006; Midro et al., 2006; Yakut et al., 2006; Perrin et al., 2007; Wiland et al., 2007; Nishikawa et al., 2008; Vozdova et al., 2008). In one study, four male family members of a kindred segregating a chromosome 15;17 translocation were studied by FISH analysis (Cora et al., 2002; Vozdova et al., 2008). The segregation patterns were very similar in all four men, with \u223c50% of sperm chromosomally unbalanced. Also, Morel et al. (2004) found similar frequencies of imbalance of 37% and 43% in two brothers heterozygous for a chromosome 7;8 translocation. These studies demonstrate that the risk of meiotic imbalance is primarily determined by the characteristics of the chromosomes involved, and the break-point positions. They also demonstrate the reproducibility of the method. In both karyotyping and FISH studies, the mean frequency of sperm with unbalanced chromosomes is \u223c50% in reciprocal translocation carriers. Many of these imbalances are not compatible with survival, and the average frequency of paternally derived translocation imbalances at prenatal diagnosis is 12% (Bou\u00e9 and Gallano, 1984). However, some translocations have higher risks of imbalance and survival, and all have serious consequences of mental and physical handicaps. A number of fetuses with unbalanced segregations of reciprocal translocations have been reported after ICSI (Baschat et al., 1996; Meschede et al., 1997). Because the frequency of chromosome abnormality is very high, some men carrying reciprocal translocations have undergone PGD to implant only chromosomally normal or balanced embryos. Studies comparing the frequency of chromosome abnormalities in sperm and embryos from reciprocal translocation carriers show a close agreement in the abnormality frequencies (Escudero et al., 2003).\nInversions\nInversions occur when two chromosome breaks occur in the same chromosome and the breaks heal in an inverted order. All the genes are present in the correct number but difficulties arise during pairing of homologous chromosomes during meiosis. If a single crossover occurs in the inverted region of the paired chromosomes, offspring with chromosomal duplications and deficiencies can result from recombinant chromosomes.\nParacentric inversions: paracentric inversions occur when both break points are in one chromosome arm. If a single crossover occurs within the inversion pairing loop, one-half of the gametes are normal, one-quarter are acentric (and would be lost) and one-quarter are dicentric (which could lead to a break between the centromeres and chromosome imbalance). Paracentric inversions are rarely reported, since they can only be detected by the use of banding procedures (Pettenati et al., 1995). Some investigators have suggested that paracentric inversions in man are generally harmless (Madan, 1995); however, recombinant chromosomes have been observed in newborns, and the risk of viable recombinants has been estimated to be 3.8% (Pettenati et al., 1995). Only two men with paracentric inversions have been studied by sperm karyotyping (Martin, 1986, 1999). Neither showed any recombinant chromosomes in sperm, suggesting that either an inversion loop was not formed or that crossing over was suppressed within the loop. Similarly, one case has been studied by FISH analysis and 1% of sperm were recombinant, with both dicentric and acentric chromosomes observed (Devine et al., 2000). Thus, the risk for paracentric inversions appears to be small.Pericentric inversions: pericentric inversions occur when the chromosome breaks occur in both chromosome arms and include the centromere in the inversion. Sperm karyotyping studies have been performed in seven men heterozygous for pericentric inversions (Martin, 1999). Four men had no recombinant chromosomes and three men had frequencies of imbalance varying from 11% to 31%. FISH studies have been performed in 24 pericentric inversion carriers with the frequency of recombinant chromosomes varying from 0% to 54% (Anton et al., 2007; Chantot-Bastaraud et al., 2007; Morel et al., 2007). The inversions that produce recombinant chromosomes are, in general, large inversions encompassing more than half of the chromosome length. An overall risk at prenatal diagnosis has been estimated to be 10\u201315% (Daniel et al., 1989), but it is clear that the risks are dependent on the individual inversion.\nInterchromosomal effects\nA number of researchers have suggested that there is an increased frequency of chromosomal abnormalities unrelated to the structural abnormality and have termed this an \u2018interchromosomal effect\u2019 (Aurias et al., 1978), e.g. an increased frequency of trisomy 21 children born to Robertsonian translocation carriers. Sperm karyotype studies have not shown any support for an interchromosomal effect in translocation (Martin and Spriggs, 1995) or inversion carriers (Martin, 1999), despite the fact that all chromosomes can be analyzed. However, the number of sperm karyotypes is limited with a maximum of 548 sperm cells analyzed in one translocation carrier (Spriggs et al., 1992). Thus, a small interchromosomal effect would be missed by these studies.\nStudies employing FISH analysis have the advantage of much larger sample sizes, with a few hundred to several thousand sperm assessed. There is some suggestion for an interchromosomal effect in 58% (21\/36 studied for an interchromosomal effect) of Robertsonian translocations and 64% (35\/55) of reciprocal translocations studied for the segregation of other chromosomes by FISH analysis, since an increased frequency of numerical abnormalities was observed in at least one of the chromosomes evaluated. For inversions, only one of seven cases demonstrated a possible interchromosomal effect (Amiel et al., 2001). However, it is doubtful whether this is a true interchromosomal effect, since carriers of translocations and inversions are often infertile, with altered sperm profiles. In fact, almost all the Robertsonian translocation carriers studied for an interchromosomal effect had abnormal semen profiles, with only three normospermic men (none of whom demonstrated an interchromosomal effect). In reciprocal translocation heterozygotes, abnormal semen profiles were much more common among the cases demonstrating an interchromosomal effect (67%) compared with those that did not (11%). Infertile men with oligozoospermia, asthenozoospermia or teratozoospermia are known to have an increased frequency of chromosome abnormalities in their sperm (Moosani et al., 1995; Martin, 1996; Aran et al., 1999; Pang et al., 1999; Nishikawa et al., 2000). Therefore, it is possible that the increased frequency of numerical chromosomal abnormalities in some carriers of structural rearrangements may be related to infertility factors rather than the rearrangement. On the other hand, clear examples of translocations with asynaptic segments pairing with sex chromosomes or autosomes suggest that this type of interchromosomal effect may be causing the infertility (Oliver-Bonet et al., 2005a).\nPGD studies have demonstrated that embryos of translocation carriers have a high frequency of genetic imbalance. Some studies have suggested an interchromosomal effect for Robertsonian translocation carriers, since there appears to be a higher frequency of aneuploidy (Conn et al., 1998, 1999; Gianaroli et al., 2002), whereas others have not found a significant increase (Scriven et al., 2001; Munn\u00e9 et al., 2005). In summary, an interchromosomal effect may be a reality for some translocations, especially in infertile patients, and sperm chromosome studies may be useful to determine the level of risk.\nY chromosome microdeletions\nTiepolo and Zuffardi (1976) first recognized that six azoospermic men had deletions of the long arm of the Y chromosome, large enough to be recognized by light microscopy. Since that time, it has been determined that the majority are Yq microdeletions and therefore require analysis by molecular means. Most studies demonstrate that 4\u201314% of azoospermic or severely oligozoospermic men have an Yq microdeletion, making this a major contribution to male infertility (Foresta et al., 2001). Of significance is the fact that all sons are expected to be infertile.\nThree regions have been delineated as azoospermic factor (AZF) a, b and c with AZFc holding the best prognosis for viable testicular sperm retrieval (Foresta et al., 2001; Vogt, 2004). An interesting aspect of Y chromosome microdeletions is the instability of the chromosome suggested by recent studies. Patsalis et al. (2002) studied 12 mosaic 46,X\/46,XY patients with Turner syndrome trails or sexual ambiguities and 4\/12 had Y chromosome microdeletions. Ferlin et al. (2007) performed sperm FISH studies in 11 men with AZFc deletions and found only 33% Y-bearing sperm compared with 49% in controls, and a significant increase in disomic XY sperm and sperm nullisomic for the sex chromosomes. More research is clearly indicated, but this suggests that men with a Y chromosome microdeletion may have an increased risk of 45,X and 47,XXY offspring as well as mosaic offspring because of loss of the Y chromosome.\nGerminal mosaics\nMen with a normal somatic karyotype may still have an abnormal cell line in their testes. These men are called \u2018germinal mosaics\u2019, and it is difficult to discover them without a testicular biopsy. Studies have discovered that 1\u201317% of infertile men are germinal mosaics (Chandley et al., 1976; Hendry et al., 1976), so this is still a risk after a normal karyotype result, but the risks for abnormal offspring would be lower than those for non-mosaic individuals.\nInfertile men with a normal karyotype\nWith the advent of ICSI, it has become clear that infertile men with a normal somatic karyotype also have an increased risk of chromosomally abnormal sperm. Moosani et al. (1995) were the first to report that infertile men with a normal 46,XY karyotype have an increased risk for autosomal and sex chromosomal abnormalities in their sperm. More than 30 FISH studies have confirmed this association of increased sperm aneuploidy frequencies in 46,XY infertile men (Aran et al., 1999; Pang et al., 1999; Nishikawa et al., 2000). Most studies have reported the increase of sperm chromosome abnormalities in infertile men to be about three times higher than in control donors (Moosani et al., 1995; Lahdetie et al., 1999; Acar et al., 2000). Reports based on prenatal diagnosis of ICSI pregnancies (Van Steirteghem et al., 2002) and newborns (Aboulghar et al., 2001) have indicated the risk of de novo chromosome abnormalities to be \u223c2% to 3%, which is 3-fold higher than that in normal pregnancies. Thus, the increased frequency of chromosome abnormalities in ICSI pregnancies and newborns mirrors the increased frequency observed in the sperm of infertile ICSI patients. Furthermore, studies have indicated that these chromosome abnormalities are of paternal origin (Van Opstal et al., 1997), underscoring the fact that chromosomally abnormal sperm in ICSI patients become chromosomally abnormal fetuses and children.\nThe first studies of sperm chromosomal abnormalities in infertile men tended to lump all types in infertility together. However, it is possible that some subsets of infertility have an elevated risk of sperm chromosomal abnormalities whereas others do not. We have studied men with asthenozoospermia (motility defects; Hristova et al., 2002), teratozoospermia (abnormalities of form; Templado et al., 2002), various degrees of oligozoospermia (low concentration; Martin et al., 2003b) and azoospermia (no sperm in the ejaculate; Martin et al., 2003a). To our surprise, we found that men with any type of infertility had an increased frequency of sperm chromosomal abnormalities, varying from 2 to 10 times higher than that of control donors. Thus, it seems that any perturbation of spermatogenesis confers an increased risk of aneuploid sperm.\nOne rare type of teratozoospermia appears to confer a very high risk is men with a high percentage of macrocephalic, multinucleated, multiflagellate sperm. A number of studies have reported very high frequencies of aneuploidy and polyploidy in these men (50\u2013100%) (Benzacken et al., 2001; Devillard et al., 2002; Lewis-Jones et al., 2003). It has also been suggested that abnormalities of the centrosome may exist in surgically retrieved sperm, a problem that may lead to increased mitotic non-disjunction and mosaicism in resulting embryos (Silber et al., 2003).\nUtility of aneuploidy assessment in sperm\nThere have been a number of studies which have found a high frequency of sperm aneuploidy in men who subsequently fathered chromosomally abnormal children (Blanco et al., 1998b; Martinez-Pasarell et al., 1999a,b; Moosani et al., 1999; Soares et al., 2001; Nagvenkar et al., 2005). For example, in our original study of infertile men, one male had a frequency of 24,XY sperm that was 9-fold higher than controls (Martin, 1986). This man subsequently had ICSI and fathered a pregnancy that resulted in a 47,XXY fetus (Moosani et al., 1999). Gianaroli et al. (2005) studied sperm aneuploidy and correlated it to results in blastomeres after PGD in couples. They found a higher incidence of monosomies and trisomies in embryos from microepididymal sperm aspiration and testicular sperm extraction sperm, and aneuploidy for the sex chromosomes increased proportionally to the severity of the male-factor condition. These authors suggested that it is important to include sperm FISH analysis in preliminary tests given to infertile couples, especially in the case of repeated IVF failures. These preliminary studies suggest a correlation between sperm aneuploidy frequencies and ICSI outcome. However, unpublished results from our research laboratory demonstrated that only 10% of infertile men with a normal karyotype have a 24,XY sperm frequency that is \u22655 times that of controls, and only 3% have a frequency \u226510 times that of controls. Thus, it appears that a high frequency of sperm aneuploidy is relatively rare in 46,XY infertile men.\nRecombination analysis by single sperm PCR\nOur studies, and those of others, have shown that the frequency of aneuploidy in sperm is elevated for the sex chromosomes compared with the autosomes, for both fertile (Martin et al., 1991; Spriggs et al., 1996; Scarpato et al., 1998) and infertile men (Shi and Martin, 2000a; Hristova et al., 2002). The XY bivalent normally has only one crossover in the pseudoautosomal region during meiosis. If recombination is reduced or absent for these chromosomes, they may be particularly susceptible to non-disjunction. Indeed, it has been shown that 47,XXY of paternal origin is associated with a decreased recombination frequency (Hassold et al., 1991; Lorda-Sanchez et al., 1992). For a direct test of whether recombination is associated with non-disjunction in human sperm, we performed single sperm PCR analysis for a sex specific locus (STS\/STS pseudogene) and a pseudoautosomal locus (DXYS15) (Shi and Martin, 2001). Individual unisomic sperm (23,X or Y) were isolated using a FACStarPlus flow cytometer into PCR wells. To identify disomic 24,XY sperm, 3-color FISH analysis was performed with probes for chromosomes X, Y and 1. The 24,XY cells were identified using fluorescence microscopy, each disomic sperm was scraped off the slide using a glass needle attached to a micromanipulator and then put into a PCR well. Hemi-nested PCR analysis of the two markers was performed to determine the frequency of recombination. The frequency of recombination between the two DNA markers was 38% for the normal unisomic sperm compared with 25% for the 24,XY disomic sperm that had undergone non-disjunction. This difference was highly significant, and demonstrates that lack of recombination in the pseudoautosomal region is associated with XY non-disjunction and the production of aneuploid sperm.\nSC analysis\nThe discovery that lack of recombination is associated with non-disjunction is significant because it provides a definite molecular correlate with aneuploidy. The next logical step would be to assess recombination in other chromosomes. However, this is extremely difficult and time-consuming by single sperm PCR analysis. Luckily, new immunocytogenetic techniques allow assessment of recombination and chromosome pairing by visualization of the SC in early meiosis (Barlow and Hult\u00e9n, 1998; Lynn et al., 2002; Sun et al., 2004a, 2006b, 2007b). The SC can be analyzed throughout the stages of prophase (leptotene, zygotene, pachytene, diplotene) to assess the progress of meiosis and the fidelity of chromosome pairing and synapsis. The frequency and location of recombination sites on individual chromosomes can also be analyzed by use of antibodies to the mismatch repair protein MLH1 (Baker et al., 1996; Marcon and Moens, 2003; Sun et al., 2004b) combined with cenM-FISH to identify individual chromosomes (Fig.\u00a03). This technique provided the first recombination maps for every autosome (Sun et al., 2004b, 2006b).\nStudies in a number of laboratories have demonstrated that normal healthy men have \u223c50 recombination foci per pachytene cell, with a wide range in the mean number of recombination sites per cell and in the number of sites in individual pachytene cells (Barlow and Hult\u00e9n, 1998; Lynn et al., 2002; Sun et al., 2004b, 2006b). This number of recombination sites is very similar to chiasma counts at diakinesis (Hult\u00e9n, 1974; Laurie, 1985; Laurie and Hult\u00e9n, 1985) and also to the corresponding genetic length obtained from linkage data (Kong et al., 2002).\nSC analysis in translocation carriers\nReciprocal translocations are known to generate meiotic disturbances that affect both quantitative and qualitative sperm production. The chromosomes involved in a reciprocal translocation must pair as a quadrivalent, which is clearly visible at the SC level during pachytene (Oliver-Bonet et al., 2005a; Sun et al., 2005b). Meiotic studies of infertile men carrying chromosomal rearrangements have shown that quadrivalent configurations have different degrees of asynapsis around the break points. It has also been observed that these regions sometimes interact with the sex body. Associations between an autosome and the sex body can be visualized as continuous proteinaceous filaments that connect the two together. This association has been suggested to be the cause of infertility in carriers (Gabriel-Robez et al., 1986). It has been hypothesized that this may be due to the activation of X-linked genes, or a spreading of inactivation to the autosomes. Study of the SC permits analysis of these two hypotheses.\nThe X and Y chromosomes pair in only a small region, with the rest of the chromosomes remaining unpaired. Meiotic sex chromosomes are inactivated with many proteins locating to the sex body (Oliver-Bonet et al., 2005b). It has been hypothesized that the association of an autosome with the sex body causes reversal of the sex body inactivation and allows expression of some genes, with lethal results for the cell (Lifschytz and Lindsley, 1972). Others have suggested a spreading of sex body inactivation toward the autosomes connected to the sex body (Jaafar et al., 1993). Recent studies have shown normal timing and progression of condensation through the pachytene stage for the sex body in a carrier of a t(Y;1) translocation (Sun et al., 2005b) and for the sex body associated with the quadrivalent in a t(13;20) carrier (Oliver-Bonet et al., 2005b). These results, together with the fact that there is a strong relationship between sex body inactivation and XY condensation (Fernandez-Capetillo et al., 2003), do not support the model of gene activation on the X chromosomes. In addition, the observation that autosome arms invading the sex body show gradual heterochromatinization, mimicking the behavior of the sex chromosomes, suggests that spreading of inactivation to the autosome is taking place.\nIt has been suggested that transcriptional repression might not be an exclusive mechanism of the sex chromosomes, but rather a general mechanism that acts to silence any asynapsed region in the cell (Baarends and Grootegoed, 2003). Such a general mechanism has been described in Neurospora crassa (Shiu et al., 2001), and in the mouse (Turner et al., 2005). It is possible that a similar mechanism is operative in humans and that the spreading of sex body inactivation toward the translocated chromosome is a consequence of asynapsed regions within the quadrivalent attaching to the sex body. In this case, genes important in meiosis might be repressed with resultant destruction of the cell.\nThe meiotic process in two translocation carriers with different fertility outcomes, one normozoospermic and the other azoospermic, have been compared with interesting results (Oliver-Bonet et al., 2005a). A significant number of quadrivalents were attached to the sex body in the azoospermic carrier, whereas such an association was never detected in the normozoospermic carrier. In addition, this normozoospermic patient displayed heterologous synapsis within the quadrivalent. Thus, it appears that unpaired regions within the quadrivalent are likely to be detected by the pachytene checkpoint, so asynapsed regions seek each other out and try to pair, in order to escape the checkpoint and avoid apoptosis of the cell. The other option to shelter unsynapsed regions from the checkpoint is association with the sex body, but this situation may lead to anomalies disrupting the proper segregation of the chromosomes.\nSC analysis in infertile men\nStudies of infertile men have demonstrated a number of meiotic errors. Most of these meiotic studies have been performed on men with NOA, but some men with obstructive azoospermia and oligoasthenoteratozoospermia have also been studied. Approximately one-half of the men with NOA have no meiotic cells (Gonsalves et al., 2004; Sun et al., 2005a; Topping et al., 2006, 2007b). In those with meiotic cells, the progression of meiosis is altered, with significantly more cells observed in the early stages of prophase (leptotene and zygotene; Gonsalves et al., 2004; leptotene and zygotene; Ferguson et al., 2007; Sun et al., 2007b). This suggests problems in the development of the lateral and transverse elements of the SC, leading to difficulties in pairing and synapsis of homologous chromosomes.\nSun et al. (2005a,c, 2007b) determined that NOA men have a significant increase in the proportion of pachytene cells with unsynapsed regions. Also, a number of studies have demonstrated that NOA males have a significant reduction in the frequency of MLH1 foci compared with controls (Gonsalves et al., 2004; Sun et al., 2005a, 2007b), although two studies have not observed this reduction (Codina-Pascual et al., 2005; Topping et al., 2006). Achiasmate bivalents (with no crossovers) have been observed with a significantly elevated frequency in NOA males compared with controls (leptotene and zygotene; Gonsalves et al., 2004; Sun et al., 2005a, 2007b; Ferguson et al., 2007). For example, Sun et al. (2007b) observed that 29% of pachytene cells had at least one bivalent with no recombination foci, compared with 5% in controls. This is a dangerous situation, since without a crossover there is no mechanism to ensure orderly chromosome segregation at metaphase I. This could lead to engagement of the pachytene checkpoint and meiotic arrest or sperm aneuploidy.\nA minimum number of recombination sites (crossovers) for correct alignment and segregation of the chromosomes is one per chromosome arm (except for the short arms of the acrocentric chromosomes, which rarely recombine). Achiasmate chromosomes (those without a recombination site) are rare in humans, except for the sex chromosome pair, which has a visible recombination site only 56\u201393% of the time (Codina-Pascual et al., 2005; Sun et al., 2006a; Ferguson et al., 2007). However, when achiasmate chromosomes are observed in autosomes, they occur most frequently for chromosomes 21 and 22 (Codina-Pascual et al., 2006; Sun et al., 2006a). This is interesting, since it parallels data on aneuploidy in human sperm. Both studies on human sperm karyotypes (Martin and Rademaker, 1990) and FISH analysis (Williams et al., 1993; Spriggs et al., 1996; Blanco et al., 1998b; Scarpato et al., 1998) have demonstrated that chromosomes 21 and 22, and the sex chromosomes have the highest frequency of aneuploidy. This strengthens the association between lack of recombination and aneuploidy. However, these studies on meiotic recombination and sperm aneuploidy have not been performed on the same men. A more robust test of the association between meiotic recombination and aneuploidy would be to correlate both analyses in the same men.\nWhen we performed just such a study in vasectomy reversal patients, we found no correlation between meiotic recombination in individual chromosomes and sperm aneuploidy for the same chromosome (Sun et al., 2008). We hypothesized that our population of fertile men may not have reached the threshold of meiotic abnormalities necessary to demonstrate the relationship between recombination and aneuploidy, since achiasmate bivalents were rare in this group of men. Ferguson et al. (2007) found a significant inverse correlation between meiotic recombination for the sex chromosomes and XY disomy in sperm. They also found a significant relationship between the frequency of achiasmate chromosome 21 and sperm disomy 21. This analysis was performed in a mixed group of control donors (vasectomy reversals) and infertile men. When we studied men with NOA, we also demonstrated that a low frequency of meiotic recombination in the sex bivalent was significantly correlated with a high frequency of aneuploidy for the sex chromosomes (unpublished results). Thus, it is possible that men with more dramatic meiotic abnormalities are infertile because of loss of meiotic cells at the pachytene checkpoint, and also face an increased risk of aneuploid sperm because of non-recombinant cells that escape the checkpoint but are still susceptible to errors of chromosome segregation. Egozcue's group in Barcelona has performed elegant research in this area. They suggest that the best candidates for a meiotic study would be: infertile males with a normal karyotype and unexplained infertility, and among them, infertile males with normozoospermia and long-term sterility, or IVF failures (embryonic factor, no fertilization, repeated IVF failure), or infertile males with a severe oligozoospermia (<5 \u00d7 106 sperm\/ml) or a severe oligoasthenozoospermia (<1.5 \u00d7 106 motile sperm\/ml) (Egozcue et al., 2005).\nFunding\nR.H.M. holds the Canada Research Chair in Genetics and her research is funded by the Canadian Institutes of Health Research. Funding to pay the Open Access publication charges for this article was provided by the Canadian Institutes of Health Research.","keyphrases":["male infertility","meiosis","sperm aneuploidy","sperm chromosome abnormalities"],"prmu":["P","P","P","P"]}
{"id":"J_Mol_Biol-1-5-2082129","title":"A Switch in the Mechanism of Communication between the Two DNA-Binding Sites in the SfiI Restriction Endonuclease\n","text":"While many Type II restriction enzymes are dimers with a single DNA-binding cleft between the subunits, SfiI is a tetramer of identical subunits. Two of its subunits (a dimeric unit) create one DNA-binding cleft, and the other two create a second cleft on the opposite side of the protein. The two clefts bind specific DNA cooperatively to give a complex of SfiI with two recognition sites. This complex is responsible for essentially all of the DNA-cleavage reactions by SfiI: virtually none is due to the complex with one site. The communication between the DNA-binding clefts was examined by disrupting one of the very few polar interactions in the otherwise hydrophobic interface between the dimeric units: a tyrosine hydroxyl was removed by mutation to phenylalanine. The mutant protein remained tetrameric in solution and could bind two DNA sites. But instead of being activated by binding two sites, like wild-type SfiI, it showed maximal activity when bound to a single site and had a lower activity when bound to two sites. This interaction across the dimer interface thus enforces in wild-type SfiI a cooperative transition between inactive and active states in both dimers, but without this interaction as in the mutant protein, a single dimer can undergo the transition to give a stable intermediate with one inactive dimer and one active dimer.\nIntroduction\nType II restriction endonucleases recognise specific sequences in DNA, typically palindromic sites 4 to 8\u00a0bp long, and cut the DNA at specified positions within or close to the site.1 Their reactions usually (but not always2) require Mg2+ as a cofactor.3 Some Type II endonucleases are dimers of identical subunits that interact symmetrically with their palindromic sites.4,5 These have a single DNA-binding cleft at the subunit interface, and they act at individual copies of their target sites.6\u20138 The best-studied restriction enzymes, such as EcoRV and BamHI,8\u201314 and those most widely used in vitro as tools for molecular biology,15 all function in this manner. Consequently, these are often considered as \u201cstandard\u201d restriction enzymes. However, many Type II endonucleases differ from the standard in that they are fully active only after interacting with two copies of their recognition site.16\u201320 The Type II enzymes that need two sites fall into two subtypes, IIE or IIF.21 The Type IIE restriction enzymes bind two (or more22) copies of their recognition sequence yet cleave only one.23 They contain two DNA-binding clefts, catalytic and allosteric, but the catalytic cleft is inactive unless cognate DNA is also bound at the allosteric site.17,22 In contrast, the Type IIF enzymes form complexes with two DNA sites, at equivalent loci in the protein,24,25 and then cut both sites in a concerted reaction.26\u201330\nThe first restriction enzyme found to act concertedly at two DNA sites was the SfiI endonuclease.26,27 SfiI recognises the sequence GGCCNNNN\u2193NGGCC (where N is any base and \u2193 is the point of cleavage),31 but it cleaves substrates with two copies of this sequence more rapidly than DNA with a single copy. Moreover, in steady-state reactions at low enzyme concentrations, SfiI converts DNA with two sites directly to the final product cut at both sites, without liberating intermediates cut at one site:26,27 the intermediates remain bound to the enzyme until it has cut both strands at both sites.32 On DNA with two sites, SfiI loops out the DNA between the sites,33\u201335 but it can also bind simultaneously to two separate DNA molecules that each have one copy of the recognition sequence.36,37 The rates of their reactions on one-site substrates increase sigmoidally with DNA concentration, indicative of positive cooperativity.36 The interaction with two sites is obligatory, as virtually no DNA is cleaved by any complex of SfiI with a single recognition site.36,38 Many genetic events\u2014such as DNA replication and recombination, and the regulation of gene expression\u2014often depend on proteins interacting with two sites at separate locations in the DNA, and the SfiI restriction enzyme has become one of the principal test systems for analysing the mechanisms of long-range communications between distant DNA sites.39\nMany restriction enzymes are now known to belong to the Type IIF family: examples include Cfr10I,28 NgoMIV,24 Bse643I,40\u201342 BspMI,29 SgrAI,43,44 Mly113I and BbeI,19 and almost all of the Type II enzymes that cut DNA bilaterally on either side of their recognition sites, such as BcgI and AloI.20 All of these enzymes need to interact with two recognition sites for full activity. Proteins that interact with two DNA sites generally prefer sites in cis, on the same molecule of DNA, to sites in trans, on separate DNA molecules, simply because two sites in cis will almost always be in closer proximity than sites in trans.39,45 Consequently, all of these enzymes are capable of cleaving DNA with two sites more rapidly than DNA with one site.\nMost Type IIF enzymes, including SfiI, are known from analytical ultracentrifugation (AUC) studies to exist in solution as tetramers of identical subunits.26,28,29,34 [SgrAI is an exception, but while it is a dimer in solution, two dimers bound to separate sites associate to form a tetramer before cutting DNA.44] In the crystal structures of the tetrameric enzymes, two of the subunits (a primary dimer) constitute one DNA-binding cleft, and the other two subunits constitute a second identical cleft.24,25,28,40 The two primary dimers are arranged back-to-back, so that their DNA-binding clefts are on the opposite sides of the protein (Figure 1(a) and (b)). The primary dimers in SfiI are comparable to the dimeric restriction enzyme BglI,46 an enzyme whose recognition site, GCCNNNN\u2193NGGC, is a truncated SfiI site. The structures of the individual subunits of SfiI and the arrangement of the two subunits in its primary dimer are similar to those in BglI, likewise its motifs for DNA sequence recognition and catalysis.47 The subunit interface within the dimer has, however, a much smaller area in SfiI than in BglI. In addition, SfiI and BglI have very different surfaces opposite the DNA-binding cleft: polar and solvent-exposed in the case of dimeric BglI,46 but almost completely hydrophobic and buried in tetrameric SfiI.25\nIn previous studies, the mode of communication between the two DNA-binding clefts in another tetrameric Type IIF enzyme, Bse634I, was examined by mutating selected amino acids at the dimer\u2013dimer interface.41,42 A mutation that converted the tetramer into a dimer yielded an enzyme with much the same properties as a standard dimeric restriction enzyme: while wild-type (wt) Bse634I cleaves DNA with two target sites at a rapid rate and DNA with one site at a slow rate, the dimeric mutant cleaved both substrates at equally rapid rates.41 Another mutant at the dimer interface of Bse634I cleaved both one-site and two-site substrates at diminished rates, while a third cleaved both substrates at elevated rates, but in both cases the two-site DNA was still cleaved more rapidly than the one-site DNA.42 In this study, we report on the effect of a single amino-acid substitution at the dimer\u2013dimer interface of the SfiI endonuclease, a conservative change from a tyrosine to a phenylalanine that removes just one hydroxyl group per subunit. In contrast to the previous mutants of Bse634I, this mutation yields an enzyme that cleaves DNA with one cognate site more rapidly than DNA with two sites. It thus switches SfiI from an enzyme that is activated by binding two DNA sites to one that shows its maximal activity on binding a single site.\nResults\nSubunit communications in SfiI\nIn the crystal structure of SfiI bound to two copies of its recognition sequence, two subunits (a primary dimer) bind one duplex, with each monomer contacting one of the two GGCC elements of the sequence.25 The other two subunits bind the second duplex on the opposite side of the tetramer in a back-to-back arrangement (Figure 1(a) and (b)). The interface between the two monomers within each dimer has a surface area (1650\u00a0\u00c52) that is not only smaller than that for the related dimeric enzyme BglI (3500\u00a0\u00c52)46 but which is also smaller than those for other tetrameric restriction enzymes (viz. 3100\u00a0\u00c52 for Bse634I).40 Many of the amino acids at the interface within each dimer are polar in nature and interact with the opposite monomer.25\nIn contrast, the subunit interactions at the interface between the primary dimers have a larger contact area in SfiI than in the other tetramers (3450 \u00c52 compared to 1700\u00a0\u00c52 for Bse634I). In SfiI, the inter-dimer interface is composed almost entirely of nonpolar residues:25 the amino acids in one subunit that lie within 5\u00a0\u00c5 of the opposite subunit across the dimer interface (Figure 1(c)) coincide with a patch on this surface that is devoid of either positively or negatively charged side chains48 (Figure 1(d)). The subunit interactions across the inter-dimer interface are thus mainly van der Waals contacts between hydrophobic side chains, although they also include some contacts between peptidyl main-chain carbonyl and amino groups. However, there appear to be only two side-chain-to-side-chain hydrogen bonds across the inter-dimer interface: between Gln3 in one subunit and Gln26 in the opposite partner, likewise between Tyr68 and Gln30 (Figure 1(c)). Gln3 is located at the outside edge of the inter-dimer interface (Figure 1(c)) while Tyr68 is positioned near the centre of the contact area (Figure 1(c) and (d)). Moreover, the aromatic side chain of Tyr68 protrudes from the surface of each subunit into a pocket in the opposite subunit (Figure 1(b)). Given the paucity of directional interactions across this interface, it seemed plausible that Tyr68 might play a pivotal role in the communication between the two DNA-binding clefts. To test this possibility, the hydroxyl moiety from the Tyr68 side chain was removed by using site-directed mutagenesis to replace the tyrosine with phenylalanine to yield the mutant Y68F.\nEnzyme stability and quaternary structure\nDNA-cleavage reactions of the Y68F mutant were initially carried out at 50\u00a0\u00b0C (the standard temperature for SfiI assays) in the same way as for wt SfiI: the enzyme was diluted into dilution buffer (Materials and Methods), incubated at 50\u00a0\u00b0C and then added to a solution of DNA and MgCl2.26,32 However, Y68F lost activity rapidly between dilution and subsequent addition to the DNA: after 2 and 5\u00a0min in dilution buffer at 50\u00a0\u00b0C, its activity had fallen by factors of 10 and 1000, respectively, while the wt enzyme retained full activity (data not shown). However, when the Y68F enzyme was mixed with the DNA substrate before initiating the reaction with MgCl2, it retained full activity. For wt SfiI, no differences were observed between reactions initiated by adding diluted enzyme to the mix of DNA and MgCl2 and reactions initiated by adding MgCl2 to premixed enzyme and DNA. All subsequent analyses of Y68F were therefore carried out in the presence of cognate DNA, and all DNA-cleavage reactions were initiated by adding Mg2+ to mixtures of enzyme and DNA.\nSince the Y68F protein is unstable in the absence of DNA, its oligomeric state was determined with DNA present. The DNA was a 21-bp duplex with the recognition sequence for SfiI, HEX-21 (Table 1). This duplex carries a chromophoric label, hexachlorofluorescein (HEX), attached to the 5\u2032 end of the \u201ctop\u201d strand. HEX-21 has an absorbance peak at 539\u00a0nm, a wavelength at which neither protein nor nucleotides have any intrinsic absorbance.\nThe sedimentation of HEX-21 to equilibrium was measured by recording in the AUC the absorbance of the samples at 539\u00a0nm as a function of centrifugal radius. The samples comprised HEX-21 alone or HEX-21 with either Y68F or wt SfiI (the DNA was present at half the concentration of DNA-binding sites in the enzyme). In all three cases, the increase in absorbance with centrifugal radius was fitted to the equation for a single homogenous species49 to yield apparent Mr (relative molecular mass) values. By itself, HEX-21 gave an Mr of about 16,000, close to that expected for a HEX-labelled DNA of 21\u00a0bp (data not shown). The best fits to the data in the presence of wt SfiI or Y68F gave Mr values of 154,203 and 156,274, respectively (Figure 2), which are both close to that predicted for the SfiI tetramer bound to two duplexes (151,367).\nThe removal of the hydroxyl group from the side chain of Tyr68, by replacing it with Phe, diminished the stability of the protein: upon dilution to low protein concentrations, the mutant lost activity, possibly due to subunit dissociation at low concentrations. Nevertheless, as with wt SfiI, the Mr of Y68F bound to a 21-bp DNA matched that expected for the tetramer bound to two duplexes.\nDNA-binding studies\nGel retardation was used to compare the DNA\u2013protein complexes formed by Y68F and by wt SfiI (Figure 3). As in previous studies,36 two duplexes of different lengths were employed: HEX-21, the 21-bp DNA used in the AUC, and an elongated version, HEX-35 (Table 1). The complexes of enzyme bound to either duplex, or to both, were separated from each other, and from the free duplexes, by electrophoresis through polyacrylamide. The DNA was detected by HEX fluorescence.\nThe two duplexes were added, at a constant total concentration, to fixed amounts of either Y68F or wt SfiI, to give mixtures that contained twice the molarity of duplex over enzyme tetramer. The binding buffer36,37 contained Ca2+: this ion can promote specific binding by Mg2+-dependent enzymes without supporting DNA cleavage.10\u201312 The addition of HEX-21 alone to either protein gave a single retarded complex, as did the addition of HEX-35 alone (Figure 3, left-hand and right-hand lanes, respectively). The complex with HEX-21 had a faster electrophoretic mobility than that with HEX-35. When both duplexes were added, three complexes were observed: one with the same mobility as that with HEX-21, one equal to the complex with HEX-35 and a third with an intermediate mobility (Figure 3, central lanes). The yields of the three complexes varied with the ratio of the two duplexes in a binomial manner, with the 1:2:1 distribution occurring when the ratio of the concentrations of HEX-21 to HEX-35 was close to 1:1. The same pattern was observed with Y68F.\nThe intermediate complex, with a mobility in between that with HEX-21 alone and that with HEX-35 alone, must contain one molecule of HEX-21 and one molecule of HEX-35. The complexes with the highest and the lowest mobilities therefore correspond, respectively, to the SfiI tetramer bound to two molecules of HEX-21 and to two molecules of HEX-35. Both Y68F and wt SfiI can thus form complexes that contain two DNA duplexes bound to a tetrameric protein. However, both proteins were saturated at the DNA concentrations used here, so it remains to be determined whether there are any differences in affinity and\/or cooperativity between wt and mutant proteins.\nCleavage of two-site plasmid\nThe optimal substrates for SfiI are supercoiled (SC) plasmids that have two copies of its recognition sequence.27 A single tetramer of SfiI binds to two sites in cis and traps the intervening DNA in a loop.33\u201335 It then cleaves all four of its target phosphodiester bonds before dissociating from the DNA: the initial product liberated from the enzyme is the final product cut in both strands at both sites, to the virtual exclusion of intermediates cut at one, two or three bonds.32 To find out if the Y68F mutant acts like wt SfiI in this respect, the reactions of both enzymes were examined on a plasmid with two SfiI sites, pGB1.26 The reactions were initially conducted under steady-state conditions, with [E0]\u00a0<\u00a0[S] (Figure 4(a) and (b)), to be able to observe directly the nature of the DNA liberated from the enzyme, rather than the enzyme-bound intermediates generated during the reaction.\nUnder these conditions, wt SfiI converted almost all of the SC plasmid directly to the two linear products (L1 and L2) cut in both strands at both sites: none of the open circle (OC) form of the DNA, cut in one strand, and only a small amount of the full-length linear (LIN) form of the DNA, cut in both strands at one site, were released from the enzyme during the reaction (Figure 4(a)). To allow comparisons between substrates with one SfiI site and substrates with two SfiI sites, all steady-state rates are recorded here in terms of moles of SfiI sites cleaved per mole of enzyme tetramer per minute (mol\/mol\/min). The velocity of wt SfiI on this two-site plasmid was 2.2\u00a0mol\/mol\/min (Figure 4(a)), the Vmax for this reaction.26,27 The Y68F mutant also cleaved the two-site plasmid in a highly concerted manner: it liberated very little of either the nicked OC form or the LIN product cut at one site. Instead, it converted this DNA directly to the final products cut at both sites, L1 and L2 (Figure 4(b)). However, the rate at which Y68F cleaved pGB1 (0.11\u00a0mol\/mol\/min) was 20 times slower than wt SfiI.\nIt is unlikely that the 20-fold difference is due to a difference in Km as the gel-shift experiments (Figure 3) had shown that the DNA concentrations used here were sufficient to saturate either enzyme. However, the rate-limiting step for the complete reaction pathway for wt SfiI (the process that determines its steady-state velocity) is the final dissociation of the products cut at both sites: all of the preceding stages, including the four DNA-cleavage steps, are relatively rapid.32 The reduction in reaction velocity could thus be due to either product release from Y68F being 20-fold slower than from wt, or the rates of the DNA-cleavage steps in its reaction pathway being reduced to values below that for product release by wt SfiI. These possibilities can be distinguished by determining whether Y68F generates a pre-steady state burst of product formation. If the DNA cleavage steps are faster than product release, the enzyme will create a burst of enzyme-bound product, to a concentration equal to that of the enzyme, prior to a slow steady-state phase, the rate of which is limited by product release.50 On the other hand, if the DNA-cleavage steps are rate-limiting for Y68F, there will be no burst, and the reaction will proceed instead at the linear steady-state rate from time zero.\nThe reactions of both wt and mutant enzymes were studied under conditions where the presence of a burst phase is readily detected: with an enzyme concentration (2\u00a0nM) approaching that of the DNA (5\u00a0nM) so that the enzyme-bound product can constitute a significant fraction of the total product, and at a reaction temperature (30\u00a0\u00b0C) where SfiI has a very slow turnover rate32, thus allowing the reactions at elevated enzyme concentrations to still be monitored. Under these conditions, both wt and Y68F enzymes cleaved a fraction of the two-site substrate rapidly before entering a slower phase during which the concentration of the substrate declined linearly with time: the decline was more rapid with wt SfiI than with Y68F (Figure 4(c)). Hence, on a DNA with two sites, both enzymes are rate-limited by product release, but Y68F releases the doubly cut product more slowly than wt SfiI. [For both enzymes, the amount of substrate consumed in the initial burst phase was about 70% of the enzyme concentration rather than 100%: this was as expected,32 as the addition of SfiI to a two-site substrate leads not only to DNA with one SfiI tetramer bridging the two sites in cis but also to DNA carrying a tetramer at each site, which resists cleavage.33,35]\nCleavage of one-site plasmid\nThe turnover rate of wt SfiI on a plasmid with one site is typically about 10 times slower than on a plasmid with two sites.26,32,33 To see if the Y68F mutant behaved like wt, both enzymes were tested against pGB1\/S1, a plasmid that carries one of the two SfiI sites from pGB1.26 During these reactions, the SC substrate was cleaved directly to the final product, LIN DNA, with a double-strand break at the SfiI site, without liberating the nicked OC form (data not shown): only the decline in the substrate concentration is shown here (Figure 5).\nThe first tests employed the same enzyme concentration (0.5\u00a0nM; Figure 5(b)) as that used for the steady-state reactions on the two-site plasmid (Figure 4(a) and (b)). For wt SfiI, most of the reaction proceeded with a linear decline in the concentration of the substrate with time and, as expected, its reaction velocity was 10-fold lower than that on the two-site plasmid (0.21 compared to 2.2\u00a0mol\/mol\/min). However, the same concentration of Y68F yielded markedly biphasic kinetics: a rapid phase that could be fitted to a single exponential to give a rate constant of 0.7\u00a0min\u2212\u00a01, followed by a slower linear phase with a velocity of 0.10\u00a0mol\/mol\/min (Figure 5(b)). The amount of substrate consumed during the fast phase (about 1.5\u00a0nM) was larger than the amount of enzyme in this reaction (0.5\u00a0nM), so the fast phase cannot be due to a pre-steady-state burst of product formation stoichiometric with the enzyme. Instead, the fast phase must reflect multiple turnovers of an active form of the enzyme that decays exponentially to a less active form, the latter being responsible for the slow linear phase of the reaction. The initial rate for the utilisation of the one-site plasmid by Y68F, in the fast phase, was about sevenfold faster than its reaction on the two-site plasmid. Moreover, even during the slow linear phase, Y68F gave virtually the same rates on the one-site and two-site plasmids (0.11 and 0.10\u00a0mol\/mol\/min, respectively).\nTo see whether the biphasicity varied with enzyme concentrations, both Y68F and wt SfiI were tested against the one-site plasmid at lower (0.05\u00a0nM; Figure 5(a)) and at higher (1.5\u00a0nM; Figure 5(c)) concentrations. At each level of wt SfiI, the majority of the SC substrate was utilised at a single zero-order rate: the rates increased in direct proportion to the enzyme concentration. Low concentrations of Y68F also gave, for most of the reaction, a linear decline in the concentration of the one-site substrate with time, at a velocity (0.15\u00a0mol\/mol\/min; Figure 5(a)) that was similar to the slow phase of the reaction at 0.5\u00a0nM Y68F (0.10\u00a0mol\/mol\/min; Figure 5(b)). In contrast, at high levels of Y68F (Figure 5(c)), virtually all of the one-site substrate was consumed rapidly, at a similar rate to the fast phase from the reaction with 0.5\u00a0nM protein.\nThe active form of Y68F that gives rise to the rapid phase of cutting the DNA with one site is possibly the tetrameric enzyme bound to a single copy of its recognition sequence. This then may decay to a less active form upon binding a second copy to give the synaptic complex, the tetramer bound to two recognition sites. The synaptic complex will form more readily on a two-site DNA than on a one-site DNA,39 so both wt and mutant enzymes are likely to cleave DNA with two sites by forming a synaptic complex bridging two sites in cis. However, despite the difficulty of synapsing sites in trans, wt SfiI cleaves DNA with one site mainly by forming synaptic complexes between sites in separate DNA molecules and not by acting at individual sites.36,38 The complex of wt SfiI bound to one site is therefore either inactive or never present at a significant level. The Y68F mutant may differ from wt in one or both of these aspects.\nReactions in trans\nIf a restriction enzyme cleaves a plasmid with a single target by acting at that target alone, then the addition of an oligoduplex carrying the recognition sequence will reduce the rate at which that enzyme cleaves the plasmid, as the duplex will act as a competitive inhibitor.20,44 On the other hand, a restriction enzyme that cleaves plasmids with a single site by spanning two sites in trans will be activated by adding an appropriate concentration of the specific duplex, as the synaptic complex can then be formed more readily with one molecule of plasmid and one molecule of duplex rather than with two molecules of plasmid.17,26,28,30 [Higher concentrations of duplex will, however, lead to synaptic complexes with two molecules of the duplex and thus inhibit plasmid cleavage.] Hence, if the fast phase of the reaction of Y68F is due to its reaction at an individual site, it should be inhibited by adding even low concentrations of cognate duplex. But if it is due to a reaction in trans spanning separate molecules of DNA, it should be activated by low levels of the duplex.\nPrevious experiments had shown that 25\u00a0nM duplex activated wt SfiI against 5\u00a0nM one-site plasmid.26 This amount of duplex was therefore added to the reaction of Y68F on a one-site plasmid and, for comparison, the wt reaction (Figure 6). HEX-21 (Table 1) was again used as the duplex. Relatively high enzyme concentrations were employed so that Y68F cleaved almost all of the DNA in the fast phase (Figure 5(c)). The plasmid with one site was cleaved by wt SfiI more rapidly in the presence of HEX-21 than in its absence (Figure 6(a)). In marked contrast, the fast phase of the reaction of Y68F on the one-site plasmid was much slower in the presence of HEX-21 than in its absence (Figure 6(b)). In further experiments at this high concentration of Y68F, activated cleavage of the one-site plasmid was not observed at any level of duplex tested (from 1\u00a0nM to 1\u00a0\u03bcM). However, at 0.05\u00a0nM Y68F, an enzyme concentration that results in most of the one-site plasmid being cleaved in the slow phase (Figure 5(a)), the addition of HEX-21 enhanced the reaction rate (data not shown).\nIt thus seems likely that the fast phase of the reaction of Y68F on the plasmid with one SfiI site is indeed due to the tetrameric protein bound to an individual site, while the slow phase of the Y68F reaction and the monophasic reaction of wt SfiI on the one-site DNA are both due to a tetramer bound to two sites in trans. If so, the Y68F mutant bound to one SfiI site gives a much higher reaction velocity than wt SfiI at a solitary site. However, if a reaction in free solution is carried out by adding a one-site DNA to a protein that can bind two sites, then, as there exists no physical means to prevent the protein from binding two separate molecules of DNA, one can never completely exclude the possibility that the observed reaction involves the protein bridging sites in trans.41,42\nReactions at individual sites\nTo determine unequivocally whether a protein capable of binding two DNA sites is active after binding just one, the protein must be barred from contacting two DNA molecules at the same time. This can be achieved by immobilising the DNA on a solid surface at a low density, so as to hold the DNA molecules separate from each other.41,42 This strategy was applied to SfiI by using a 30-bp duplex with a single cognate site, BIO-30 (Table 1). The site in this duplex is the same as that in the one-site plasmid pGB1\/S1 with respect to spacer and flanking sequences. The duplex carried a biotin tag on the 5\u2032 end of one strand, and this was used to attach it to a streptavidin-coated bead. The ratio of streptavidin on the bead to biotinylated DNA was set at 125:1, conditions that result in the mean distance between adjacent DNA molecules on the surface of the bead being considerably longer than that between the two DNA-binding clefts in the SfiI tetramer. This leaves the individual DNA chains too far apart to allow SfiI to form a synaptic complex with two duplexes. The BIO-30 duplex also contained a 32P label at the 5\u2032 end of the other strand, so that cleavage of this strand could be monitored. These experiments employed enzyme concentrations in excess of immobilised DNA and thus are, effectively, single-turnover reactions. The observed rates of DNA cleavage thus reflect the phosphodiester hydrolysis step in the reaction pathway.\nEven though wt SfiI generally acts on DNA substrates with a single site by bridging two DNA molecules in trans (Figures 3(a) and 6(a)), it was still able to cleave a one-site DNA even when the individual molecules of that DNA were isolated from each other by immobilisation on the bead (Figure 7). The extent of cleavage of the immobilised substrate by wt SfiI was fitted to a single exponential to give an apparent rate constant of 0.1\u00a0min\u2212\u00a01. This rate constant for phosphodiester hydrolysis by wt SfiI at a single site is very much slower than measured previously for the same process in its synaptic complex with two sites (0.1\u00a0s\u2212\u00a01).32 Even though the immobilised duplex used here differs from the plasmids used previously for reactions in bulk solution, this 60-fold difference is consistent with other studies that have shown that the activity of wt SfiI bound to two sites is \u2265\u00a030 times higher than that at a single site.36\nThe single-turnover reaction of Y68F on the immobilised DNA with one SfiI site also gave an exponential decline in substrate concentration, with an apparent rate constant of 0.7\u00a0min\u2212\u00a01 (Figure 7), much faster than wt SfiI. Hence, the Y68F mutant bound to a single site cleaves that site more rapidly than wt SfiI at an individual site (Figure 7). Moreover, the rate constant for phosphodiester hydrolysis by Y68F bound to the immobilised DNA equals that from the fast phase of the reaction of Y68F on the one-site plasmid in bulk solution (Figure 5(b)). Although these experiments employed different types of DNA substrates\u2014in one case an oligoduplex attached to a coated bead, and in the other case a plasmid in free solution\u2014they still support the view that the fast phase from the reaction of Y68F on the one-site plasmid is due to the enzyme bound to a single site.\nDiscussion\nThe tetrameric SfiI restriction endonuclease26,34 is now one of the principal experimental systems used for the analysis of long-range interactions between distant DNA sites.39 Two of its subunits (a primary dimer)24,28 form one DNA-binding cleft, and the other two form a second cleft on the opposite side of the protein: the clefts are separated by about 80\u00a0\u00c5.25 SfiI recognises a palindromic DNA sequence31 and the two subunits in each primary dimer interact symmetrically with the cognate site, in much the same way as a standard dimeric restriction enzyme with a single DNA-binding cleft. However, virtually no DNA is cleaved by the complex of wt SfiI with one recognition site: instead, almost all of its reactions are due to the complex with two sites.33 Moreover, it has to be the scissile site in both clefts: a complex carrying the recognition sequence in one cleft and, in the other cleft, either a noncognate sequence 1\u00a0bp different or a phosphorothioate derivative of the correct sequence, fails to cleave either DNA.36,38 Hence, information about the occupancy of each DNA-binding cleft, and the susceptibility of the sequence in each cleft, must somehow be transmitted through the protein to the other cleft 80\u00a0\u00c5 away.\nThe transfer of information between the two DNA-binding clefts in a tetrameric restriction enzyme must occur through the interface between the two primary dimers.41,42 Inspection of the crystal structure of SfiI bound to two cognate duplexes suggests that Tyr68 may play a pivotal role at this interface (Figure 1). The aromatic ring of this tyrosine sits in a hydrophobic pocket in the opposite subunit, and its hydroxyl group forms an inter-subunit hydrogen bond across the dimer interface.25 The removal of the hydroxyl function from Tyr68, by the conservative mutation to phenylalanine, resulted in a protein, Y68F, that retains the tetrameric structure of wt SfiI and its ability to bind two DNA sites at the same time, to cleave two sites in cis concertedly (Figures 2, 3 and 4). In these respects, Y68F is like wt SfiI, though its turnover rate on the two-site plasmid is about 20-fold lower than wt, on account of its slow dissociation from the doubly-cut product (Figure 4(c)). However, Y68F differs markedly from wt on DNA with one SfiI site (Figure 5). The wt enzyme acts more slowly on one-site than on two-site DNA because it acts on the one-site DNA in trans, bridging two separate molecules of the DNA, and on the two-site DNA in cis, looping out the intervening DNA. In contrast, the steady-state reaction of Y68F on the plasmid with one SfiI site gave biphasic kinetics\u2014a fast phase that was considerably faster than its reaction on the two-site plasmid, but which then declined exponentially to a slower phase whose rate matched that on the two-site DNA. Both phases involve multiple turnovers of the enzyme. Further experiments showed that the fast phase is due to Y68F acting at individual sites and that the slower phase is due to its synaptic complex with two DNA sites (Figures 6 and 7). Y68F can thus cleave one-site DNA without the need to bridge two separate molecules of the DNA (Figure 6). The net effect of this Tyr\u2192Phe mutation is thus to switch SfiI from an enzyme showing high activity at two sites and low activity at one site to the exact opposite\u2014low activity at two sites and high activity at one site. It inverts the ratio of its turnover rates on two-site versus one-site substrates from 10:1 for wt SfiI to approximately 1:10 for Y68F (as measured from its fast phase on the one-site DNA).\nKinetic model for SfiI cooperativity\nCooperative action by an oligomeric protein is generally accounted for by either the Monod\u2013Wyman\u2013Changeux (MWC) or the Koshland\u2013Nemethy\u2013Filmer (KNF) scheme.51,52 In both schemes, each subunit of the oligomer exists in either an inactive low-affinity tense (T) state or an active high-affinity relaxed (R) state. In the following, the terms T and R apply to a primary dimer of SfiI, a single DNA-binding unit, so that the tetramer is noted as T\/T, T\/R or R\/R (Figure 8(a)): the subscript \u201cS\u201d will denote bound substrate. In the MWC model, the T\u2192R transition occurs in all or none of the subunits so that the protein never contains both T and R subunits at the same time. On the other hand, the KNF model proposes sequential T\u2192R transitions upon ligand binding to each subunit, allowing hybrid oligomers containing both T and RS subunits. [To keep the Y68F protein active, the reactions reported here were carried out by adding Mg2+ to mixtures of enzyme and DNA and thus might appear to start from an enzyme\u2013DNA complex rather than from free enzyme. However, these reactions had the same kinetics as those started by adding enzyme to the DNA\u2013Mg2+ mix, so the dissociation of any DNA bound in the pre-equilibrium must be rapid compared to the subsequent steps. Hence, these reactions can be considered as starting from the free enzyme. In the absence of metal ions as in the pre-equilibria, SfiI binds weakly and nonspecifically to DNA.36]\nThe properties of wt SfiI, its cooperative binding to two DNA duplexes and its enhanced activity on two-site over one-site substrates, can be accounted for readily on either the MWC or the KNF model. The wt enzyme almost always needs to bind two copies of its recognition site before it can cleave DNA; thus, on DNA with one site, it acts primarily in trans, bridging two DNA molecules. The wt reaction on a one-site DNA thus proceeds largely via the RS\/RS state, to the virtual exclusion of T\/RS or R\/RS forms. On a DNA with two sites, the wt enzyme binds both sites in cis to again yield the RS\/RS state, but in this case the transition from the free T\/T form to the doubly-liganded RS\/RS structure will occur much more readily than on a one-site DNA due to the local concentration effect for sites in cis.39,45 In contrast, the Y68F mutant of SfiI cleaves DNA readily after binding just one site, so that a substrate-induced conformational change in one primary dimer, to give the hybrid T\/RS state, is sufficient for Y68F activity. The presence of hybrid species is allowed by the KNF scheme but not by the MWC scheme; thus, if both Y68F and wt SfiI follow the same mechanism, it has to be a KNF scheme.\nA simplified three-state version of the KNF model for cooperative action by a dimer, as opposed to the full nine-state version, is shown in Figure 8(a): in this scheme, the substrate-binding and the T\u2192R steps always occur together so that the unliganded T\/R and R\/R states, the partially liganded T\/TS and R\/RS states and the fully liganded TS\/TS and TS\/RS forms are all excluded, leaving only T\/T, T\/RS and RS\/RS. It also coalesces the DNA-cleavage and product-release steps.\nTo examine whether the scheme in Figure 8(a) can describe the reactions of both wt and Y68F enzymes on both one-site and two-site substrates, the differential equations for the change in concentration of each species with time were solved by numerical integration, using a range of different values for each of the six rate constants: k1 and k\u2212\u00a01 for the first DNA-binding event; k2 and k\u2212\u00a02 for the second DNA-binding event; and k3 and k4 for DNA cleavage\/product release from the singly- and doubly-liganded protein. Numerous trials were undertaken, with different values, until a match between model and experiment had been obtained. The values used for Y68F and for wt SfiI differed from each other, but in both cases, the same sets of rate constants were used for their reactions on the one-site and two-site substrates. The distinction between one and two sites was modelled instead by multiplying k2[S], the association rate for the second DNA, by a factor \u03a9 to account for the local concentration of one DNA site in the vicinity of another being higher for sites in cis than for sites in trans: by definition, \u03a9\u00a0=\u00a01 for sites in trans and, by selection, \u03a9\u00a0=\u00a0100 for sites in cis. Even though the latter value was selected arbitrarily, it reflects experimental measurements45 and large (10-fold) variations in this figure made essentially no difference to these calculations (data not shown).\nFor both enzymes, a set of rate constants was found that yielded progress curves that closely matched the experimental data for that enzyme on both one-site and two-site substrates (Figure 8(c)) for wt; Figure 8(d) for Y68F: the corresponding experimental data are shown in Figures 4 and 5. To match the experimental data, it was necessary to stipulate that both rate constants leading to the formation of T\/RS (k1 and k\u2212\u00a02) were smaller for wt SfiI than for Y68F and that, conversely, both rate constants leading away from T\/RS (k\u2212\u00a01 and k2) had to be smaller for Y68F. In particular, for Y68F, k2 had to be held at a low value, comparable to that for the DNA-cleavage\/product-release steps from the T\/RS intermediate (k3), in order to simulate the biphasic reaction profile on the one-site DNA (Figure 5(b)). [If k2 was set either much smaller or much larger than k3, the substrate concentration declined linearly with time. In addition, in order to account for the slower turnover rate of Y68F on the two-site plasmid, k4 had to be fixed at a lower value than for wt SfiI, but this concurs with the experimental data: k4 encompasses both DNA cleavage by RS\/RS and product release, and the release of the doubly-cut product is slower from Y68F than from wt SfiI (Figure 4(c)).] However, the models used the same values for k3, the rate constant for DNA cleavage by the complex with one DNA (the T\/RS state). The fact that the T\/RS state leads to some DNA cleavage by Y68F, but virtually none by wt SfiI, is due to the differing extents to which this state is populated during their reactions.\nIn this scheme (Figure 8(a)), the principal effect of replacing Tyr68 with Phe is to reduce the differences in free energy between the T\/RS state and either the T\/T or the RS\/RS structures. With wt SfiI, the T\/RS state is very strongly disfavoured relative to either T\/T or RS\/RS in thermodynamic terms, and it also has a short lifetime so that wt SfiI cleaves DNA with one site by forming the RS\/RS complex with two sites in trans. Even when a high value is allotted to the wt enzyme for k3 (the rate constant for DNA cleavage by the T\/RS state), virtually all of the DNA is still cleaved by wt via its RS\/RS state. In contrast, the T\/RS state for Y68F is more stable thermodynamically than that for the wt enzyme and it also has a longer lifetime, due to a low value for k2, the rate constant for the T\/RS\u2192RS\/RS step. Hence, with Y68F, the one-site DNA is cleaved initially via the T\/RS state, but because the T\/RS\u2192RS\/RS transition still has a negative \u0394G\u00b0, it will eventually form the RS\/RS state with two DNA molecules in trans.\nStructural model for SfiI cooperativity\nHow does this Tyr\u2192Phe change stabilise the T\/RS intermediate? At present, a crystal structure is available for wt SfiI bound to two cognate duplexes, but not for the free protein without DNA. Given the scheme in Figure 8(a), the solved structure is presumably akin to the RS\/RS state, although it may denote a precursor to the catalytically active form.25 Nevertheless, the cartoon of RS\/RS (Figure 8(a)) relates to the known crystal structure (Figure 1(a)). In this structure, the two subunits that make up the primary dimers are in close proximity at the DNA-binding surface but lie distant from each other at the dimer interface.\nIn almost every case to date where crystal structures are available for a restriction enzyme in both DNA-bound and free forms,1,4,5 the DNA-binding cleft has an open configuration in the absence of DNA, but it closes up around the DNA in the complex. Hence, it is possible that the two DNA-binding clefts in SfiI also have open configurations in the absence of DNA but become narrower upon binding cognate DNA. If so, then the movement of two subunits towards each other across the DNA-binding cleft might result, given rigid body motion, in these subunits moving away from each other at the interface with the other primary dimer (as illustrated by the T\/T\u2192 RS\/RS transition in Figure 8(a)). The interface between the dimers is relatively flat and is almost entirely hydrophobic in nature,25 which might permit each subunit to slide or rotate past the opposite subunit in the other dimer. A similar scheme for intersubunit motion has been proposed for the subunit rotation step in site-specific recombination by \u03b3\u03b4 resolvase.53 However, in the crystal structure of wt SfiI bound to two duplexes, the hydroxyl groups of Tyr68 in all four subunits make direct hydrogen bonds to Gln30 in their partner subunits across the dimer interface. In order to satisfy the hydrogen-bonding potentials of both Tyr68 and Gln30, these interactions are likely to be present not only in the RS\/RS state, as observed in the crystal structure, but also in the free protein, the T\/T state, even though the free protein may have a different geometry at the dimer interface. On the other hand, when only one of the two primary dimers changes conformation from T to R upon DNA binding, the Tyr68 and Gln30 residues in the liganded RS dimer will no longer be in register with those in the unliganded T dimer (Figure 8(a)). This dislocation may destabilise the T\/RS state, as neither Tyr68 nor Gln30 will have a suitably positioned hydrogen-bonding partner.\nIn the above scheme, one function of Tyr68 in wt SfiI is to ensure that whenever one of the two dimers binds DNA and undergoes the T-to-RS transition, the other dimer is forced to follow suit and to undergo immediately its own switch from T to R, so as not to accumulate the highly unstable T\/RS state. However, without the Tyr68\u2013Gln30 interaction, as in the Y68F mutant, the T-to-RS transition in one dimer would no longer compel the other dimer to undergo straightaway its own T-to-R switch. The hybrid T\/RS form is more stable and has a longer lifetime in the reaction of Y68F compared to wt, so it accumulates sufficiently and exists long enough to allow for some DNA cleavage events while in this state.\nMaterials and Methods\nMutagenesis\nPlasmids containing the genes coding for the SfiI restriction endonuclease, pRRS-SfiIR+, and the SfiI modification methyltransferase, pSYX33-SfiIM+, were provided by Ira Schildkraut (New England Biolabs) and were used to transform Escherichia coli ER2353, first with pSYX33-SfiIM+ and then with pRRS-SfiIR+. The mixture of plasmids isolated from this strain was employed for site-directed mutagenesis of the SfiI restriction gene by the QuikChange method (Stratagene). The resultant PCR products were used to transform E. coli ER2353 (pSYX33-SfiIM+). The plasmids were isolated from the transformants, and the derivatives of pRRS-SfiIR+ were sequenced across the entire gene for the mutant SfiI endonuclease (University of Dundee Sequencing Service): only the Y68F mutation had been introduced.\nProteins and DNA\nWt SfiI and Y68F were purified from E. coli ER2238 cells that had been transformed successively with pSYX33-SfiIM+ and pRRS-SfiIR+ (or a derivative) as described previously.26 The purified enzymes were stored at \u2212\u00a020\u00a0\u00b0C. Concentrations of wt SfiI and the Y68F mutant were assessed by absorbance at 280\u00a0nm using an extinction coefficient of 123,200\u00a0M\u2212\u00a01 cm\u2212\u00a01, where M refers to the tetramer.34 All SfiI molarities are thus cited for the tetramer with Mr\u00a0=\u00a0124,176. Protein structures were analysed with INSIGHT II v. 2005 (Accelrys, San Diego), and surface charge was evaluated with GRASP v. 1.2.48\nThe plasmids pGB1\/S1 and pGB1,26 which contain one or two SfiI sites, respectively, were used to transform E. coli ER2267, and the resulting transformants were grown in M9 minimal media containing 37\u00a0MBq\/l [methyl-3H]thymidine (GE Healthcare). The covalently closed form of the plasmid was purified by density gradient centrifugations.32\u201335 The preparations contained mostly the SC form of the monomeric plasmid, with generally <\u00a010% as either dimer or OC. DNA concentrations were assessed by absorbance at 260\u00a0nm.\nAll oligonucleotides were obtained from Sigma Genosys as HPLC-purified samples. Oligonucleotides were annealed to give the duplexes shown in Table 1 by heating a mixture of two oligonucleotides with complementary sequences to 95\u00a0\u00b0C and then slowly cooling overnight to room temperature. The mixtures generally contained more of the \u201cbottom\u201d strand than the \u201ctop\u201d strand to ensure that all of the top strand was incorporated into the duplex. The bottom strand of the BIO-30 duplex (Table 1) was phosphorylated at its 5\u2032 end by using T4 polynucleotide kinase (Roche) and [\u03b3-32P]ATP (GE Healthcare), as described before.30,36\nAnalytical ultracentrifugation\nSedimentation to equilibrium was done at 20\u00a0\u00b0C in a Beckman XL-A analytical ultracentrifuge using an An60-Ti rotor with six channel centrepieces.44 The three sample channels contained 100\u00a0\u03bcl of HEX-21 or HEX-21 with either Y68F or wt SfiI in AUC buffer [10\u00a0mM Tris\u2013HCl, 50\u00a0mM NaCl, 10\u00a0mM CaCl2 and 1\u00a0mM dithiothreitol (DTT); pH 7.9]. The proteins and the DNA, both at 2.5\u00a0\u03bcM, had been dialysed previously against AUC buffer, and the reference channels contained 110\u00a0\u03bcl of the dialysate. Centrifugation was carried out sequentially at 10,000, 15,000 and 25,000\u00a0rpm. After 16 and 20\u00a0h at each speed, the differences in absorbance at 539\u00a0nm between samples and references were recorded as a function of centrifugal radius (r). At all speeds, the profiles at 16 and 20\u00a0h were identical, showing that equilibrium had been reached. Centrifugation was then continued for 8\u00a0h at 40,000\u00a0rpm to obtain the baseline offset. For each sample, plots of A539 against centrifugal radius at varied speeds were fitted globally to the equation for a single homogenous species,49to give values for the molecular mass (M, given here as Mr values): Ar and A0 are the absorbances at r and at the reference r0, respectively; \u03bd\u00af is the partial specific volume; \u03c1 is the buffer density; \u03c9 is the angular velocity; and B is the baseline offset. Values for \u03bd\u00af and \u03c1 were obtained as before.44\nDNA-binding studies\nBinding reactions were performed by adding aliquots of wt SfiI or Y68F in dilution buffer to either HEX-21 or HEX-35, or to mixtures of the two duplexes, to give solutions that had, in 20\u00a0\u03bcl of binding buffer, 5\u00a0nM SfiI tetramer and a total duplex concentration of 10\u00a0nM. Dilution buffer is composed of 20\u00a0mM Tris\u2013HCl, 10\u00a0mM \u03b2-mercaptoethanol, 0.1\u00a0mM EDTA, 10% vol\/vol glycerol, 1\u00a0mM spermine and 0.2% vol\/vol Triton X-100 (pH 7.5). Binding buffer is composed of 20\u00a0mM Tris\u2013HCl, 25\u00a0mM NaCl, 2\u00a0mM CaCl2, 5\u00a0mM \u03b2-mercaptoethanol and 100\u00a0\u03bcg\/ml bovine serum albumin (pH 7.5). After 30\u00a0min at room temperature, the samples were mixed with 10\u00a0\u03bcl of binding buffer containing 4% (wt\/vol) Ficoll 400 and applied to 8% polyacrylamide gels in 45\u00a0mM Tris\u2013borate (pH 8.3) and 2\u00a0mM CaCl2, as described previously.36 After electrophoresis, the gels were scanned in a Molecular Dynamics PhosphorImager with illumination at 550\u00a0nm.\nEnzyme assays\nFor kinetic experiments, the final reaction mixtures contained 5\u00a0nM 3H-labelled DNA, either pGB1\/S1 or pGB1, and various concentrations of either wt SfiI or the Y68F mutant in 200\u00a0\u03bcl of reaction buffer at 50\u00a0\u00b0C. Reaction buffer is composed of 10\u00a0mM Tris\u2013HCl, 50\u00a0mM NaCl, 10\u00a0mM MgCl2, 1\u00a0mM DTT and 100\u00a0mg\/ml bovine serum albumin (pH 7.9). The reactions were carried out by adding 10\u00a0\u03bcl of MgCl2 (200\u00a0mM) to 190\u00a0\u03bcl of enzyme and DNA in reaction buffer lacking MgCl2 (apart from some trial reactions initiated by adding enzyme to DNA in reaction buffer). Aliquots (15\u00a0\u03bcl) were removed from the reactions at various times after adding MgCl2 (one was removed before adding MgCl2 to serve as a zero time point) and vortexed immediately with 10\u00a0\u03bcl of an EDTA stop mix.18\u201320 The samples were analysed by electrophoresis through agarose under conditions that separate the various products from each other and from the SC substrate. The concentrations of each form at each time point were determined by scintillation counting.32\u201336 All values given here are presented as the means from three independent experiments, with error bars to denote standard errors. Zero-order rates were evaluated using GRAFIT (Erithacus Software) to fit the initial phases of substrate utilisation and\/or product formation to linear plots. For some reactions (viz. Figure 5(b)), the concentration of DNA substrate was fitted to an initial exponential decline followed by a zero-order phase. For the various reaction mechanisms considered here (Figure 8), extents of substrate utilisation were modelled by solving the differential equations for the time-dependent changes in the concentrations of each species during the course of the reaction by numerical integration in BERKELEY MADONNA.\nSome reactions on pGB1\/S1 also contained the oligoduplex HEX-21. These typically contained, in 200\u00a0\u03bcl of reaction buffer at 50\u00a0\u00b0C, 1.5\u00a0nM enzyme (either Y68F or wt SfiI), 5\u00a0nM pGB1\/S1 (3H-labelled) and 25\u00a0nM HEX-21: samples were taken at varied times after initiating the reaction with MgCl2 and analysed as described above to determine the amount of pGB1\/S1 that had been converted to its linear form.\nImmobilised oligonucleotides\nStreptavidin-coated magnetic beads from Promega (10\u00a0pmol of streptavidin) were mixed with 32P-labelled BIO-30 (0.08\u00a0pmol) in 80\u00a0\u03bcl of SSC,41 washed in SSC and resuspended in 200\u00a0\u03bcl of reaction buffer without MgCl2. The requisite concentrations of either wt SfiI or the Y68F mutant were then added before initiating the reactions with MgCl2 (final concentration, 10\u00a0mM). Aliquots (15\u00a0\u03bcl) were taken from the reactions at varied times and quenched with loading dye (10\u00a0mM NaOH, 100\u00a0mM EDTA, 95% formamide, 0.05% bromophenol blue and 0.05% xylene cyanol). After incubating first at 95\u00a0\u00b0C for 10\u00a0min and then on ice for 15\u00a0min, the samples were analysed by denaturing gel electrophoresis through a 12% polyacrylamide gel in TBE buffer [45\u00a0mM Tris\u2013borate and 1\u00a0mM EDTA (pH 8.3)] at \u223c\u00a040\u00a0V\/cm and 55\u00a0\u00b0C. The gels were fixed in 20% (vol\/vol) acetic acid and 20% (vol\/vol) methanol, dried, exposed overnight and scanned in a PhosphorImager. The amounts of intact and cleaved 32P strands of the duplex were quantified using ImageQuant (Molecular Dynamics), and the decline in substrate concentration with time fitted to a single exponential in GRAFIT.","keyphrases":["restriction enzyme","cooperativity","auc, analytical ultracentrifugation","dtt, dithiothreitol","hex, hexachlorofluorescein","knf, koshland\u2013nemethy\u2013filmer","lin, linear","mr, relative molecular mass","mwc, monod\u2013wyman\u2013changeux","oc, open circle","sc, supercoiled","wt, wild type","allostery","dna\u2013protein interaction","protein\u2013protein interaction"],"prmu":["P","P","R","R","R","R","R","R","R","R","R","M","U","R","M"]}
{"id":"Extremophiles-4-1-2175526","title":"Tetraether membrane lipids of Candidatus \u201cAciduliprofundum boonei\u201d, a cultivated obligate thermoacidophilic euryarchaeote from deep-sea hydrothermal vents\n","text":"The lipid composition of Candidatus \u201cAciduliprofundum boonei\u201d, the only cultivated representative of archaea falling in the DHVE2 phylogenetic cluster, a group of microorganisms ubiquitously occurring at hydrothermal vents, was studied. The predominant core membrane lipids in this thermophilic euryarchaeote were found to be composed of glycerol dibiphytanyl glycerol tetraethers (GDGTs) containing 0\u20134 cyclopentyl moieties. In addition, GDGTs with an additional covalent bond between the isoprenoid hydrocarbon chains, so-called H-shaped GDGTs, were present. The latter core lipids have been rarely reported previously. Intact polar lipid analysis revealed that they predominantly consist of GDGTs with a phospho-glycerol headgroup.\nIntroduction\nDeep-sea hydrothermal vents are unique environments, which are thought to represent models for both the origin of life on Earth and exploration of life on other planets. These vents contain ecosystems, which are predominantly fueled by geochemical energy and are host to many newly described free-living microbes, which are often associated with actively venting porous deep-sea vent deposits or \u201cchimneys\u201d. The steep chemical and thermal gradients within the walls of these deposits provide a wide range of microhabitats for microorganisms with suitable conditions for aerobic and anaerobic thermophiles and mesophiles (e.g. McCollom and Schock 1997). Indeed, both culture-dependent and -independent approaches have exposed a vast diversity of Bacteria and Archaea associated with deep-sea vent deposits (e.g. Reysenbach and Shock 2002; Schrenk et al. 2003). Numerous Archaea have been isolated from these deposits but few of these are found in environmental 16S rRNA gene clone libraries and most detected environmental clones in these environments have no representatives available in pure culture.\nIn particular, one archaeal lineage is widespread at deep-sea vents, namely the \u201cdeep-sea hydrothermal vent euryarchaeotic\u201d lineage DHVE2, and is frequently associated with actively venting sulphide deposits (e.g. Nercessian et al. 2003; Hoek et al. 2003; Reysenbach and Shock 2002; Takai et al. 1999, 2001). In some cases it has been reported as the most dominant clone type in archaeal clone libraries (Hoek et al. 2003), yet the physiology of these organisms was unclear. Recently, Reysenbach et al. (2006) isolated and cultivated a member of the DHVE2 phylogenetic DHVE2 cluster, Candidatus \u201cAciduliprofundum boonei\u201d, and showed it to be an obligate thermoacidophilic sulphur and iron reducing heterotroph capable of growing from pH\u00a03.3 to 5.8 and between 60 and 75\u00b0C. This provided the first evidence that thermoacidophiles may be key players in sulphur and iron cycling at deep-sea vents.\nArchaea are not only unique in their 16S rRNA phylogenetic position in the tree of life, but also synthesize specific membrane lipids. Analysis of cultivated hyperthermophilic Archaea showed that their membrane is predominantly composed of isoprenoid glycerol dibiphytanyl glycerol tetraethers (GDGTs) with additional cyclopentyl moieties (e.g. Structures I\u2013VI in Fig.\u00a01). The structural differences from diacyl membrane lipids of non-thermophilic Eukarya and Bacteria, i.e. ether bonds and the formation of a monolayer rather than a bilayer, have been suggested to contribute to the stability of membranes of hyperthermophiles at high temperatures and low pH (e.g. De Rosa and Gambacorta 1988; van den Vossenberg et al. 1998; Macalady et al. 2004). This suggests that members of the DHVE2 cluster may be also synthesizing GDGT membrane lipids. Therefore, in this study we analyzed the lipid composition of the only cultivated representative of DHVE2, Aciduliprofundum boonei, and examined both the core GDGT lipid composition as well as its intact polar lipid composition.\nFig.\u00a01HPLC\/APCI\/MS base peak chromatogram of GDGT core lipids in extract released after base hydrolysis (using KOH\/methanol mixture) of the cell material of Candidatus \u201cAciduliprofundum boonei\u201d. Inset shows the atmospheric pressure chemical ionization mass spectrum of GDGT VII. Note that the position of the covalent bond between the isoprenoid hydrocarbon chains in GDGTs VII\u2013XI is tentative (Morii et al. 1998)\nMaterials and methods\nCulture conditions\nAciduliprofundum boonei was grown as previously described (Reysenbach et al. 2006). Cells were harvested at late log phase of growth. Cell pellets were freeze-dried and stored frozen until analysis.\nCore lipid analysis\nCell material (74\u00a0mg dry weight) of A. boonei was hydrolyzed by refluxing in ca. 4\u00a0ml 1\u00a0M KOH in methanol for 1\u00a0h. The pH of the hydrolyzed extract was adjusted to pH\u00a03 using 2\u00a0M HCl\/methanol (MeOH) 1\/1 (v\/v) and transferred to a separatory funnel. The residual cell material was washed subsequently with 2\u00a0ml MeOH\/water 1\/1 (v\/v), MeOH and dichloromethane (DCM, three times). The washings and 6\u00a0ml bidistilled water were added to the separatory funnel, which was shaken vigorously. The DCM layer was removed and the residual water\/MeOH layer was re-extracted two times using DCM. The combined DCM layers were dried using a sodium sulfate column to yield the hydrolyzed extract. The extract released upon hydrolysis was condensed by rotary evaporation. Elemental sulphur was removed with activated Cu. The extract was methylated by diazomethane and filtered over a silicagel column using ethyl acetate. One aliquot of the eluate was silylated using BSTFA (with 1% TMCS) in pyridine (1\/1, v\/v) for 20\u00a0min at 60\u00b0C and subsequently analyzed by gas chromatography (GC) and GC\/mass spectrometry (GC\/MS). Another aliquot of the eluate was dissolved in hexane\/isopropanol (99:1, v\/v), ultrasonicated and filtered using a PTFE 0.45-\u03bcm filter prior to analysis for GDGTs on the high performance liquid chromatography (HPLC)\/MS.\nPart of the GDGT fraction was hydrogenated by dissolving it in ethyl acetate, adding a small amount of PtO2 and a drop of acetic acid and bubbling hydrogen for 1\u00a0h. Subsequently, the solution was stirred overnight and filtered over a small pipette column containing Na2CO3 (top) and MgSO4 (bottom). Ethyl acetate was evaporated and the hydrogenated GDGT fraction was dissolved in hexane\/isopropanol (99:1, v\/v), ultrasonicated and analyzed using HPLC\/MS.\nA second aliquot of the GDGT fraction was separated using HPLC with a preparative device (Foxy jr) and the fractions collected were analyzed by flow injection analysis mass spectrometry (Smittenberg et al. 2002). The fractions containing the unusual GDGT (see text below) were combined and treated with HI and LiAlH4 to release the ether bound hydrocarbon skeletons (Schouten et al. 1998). This fraction was analyzed by GC and GC\/MS.\nThe total extract and the GDGT fractions treated with HI\/LiAlH4 were analyzed by GC\/MS using a Finnigan Trace GC ultra coupled to a Finnigan Trace DSQ mass spectrometer. A fused silica capillary column (25\u00a0m\u00a0\u00d7\u00a00.32\u00a0mm) coated with CP-Sil 5 (film thickness 0.12\u00a0\u03bcm) was used with helium as carrier gas. The oven was programmed at a starting (injection) temperature of 70\u00b0C, which rose to 130\u00b0C at 20\u00b0C\/min and then to 320\u00b0C at 4\u00b0C\/min, at which it was maintained for 20\u00a0min. The column was directly inserted into the electron impact ion source of the DSQ quadrupole mass spectrometer, scanning a mass range of m\/z 50\u2013800 at three scans per second and with an ionization energy of 70\u00a0eV.\nHPLC\/MS analyses were performed according to Hopmans et al. (2000). Analyses were performed using an Agilent (Palo-Alto, CA, USA) 1100 series LC-MSD SL equipped with an auto-injector and Chemstation chromatography manager software. Separation was achieved on a Prevail Cyano column (2.1\u00a0\u00d7\u00a0150\u00a0mm, 3\u00a0\u03bcm; Alltech, Deerfield, IL, USA), maintained at 30\u00b0C. Injection volumes were 10\u00a0\u03bcl. GDGTs were eluted isocratically with 99% A and 1% B for 5\u00a0min, followed by a linear gradient to 1.8% B in 45\u00a0min, where A\u00a0=\u00a0hexane and B\u00a0=\u00a0isopropanol. Flow rate was 0.2\u00a0ml\/min. After each analysis the column was cleaned by back-flushing hexane\/isopropanol (90:10, v\/v) at 0.2\u00a0ml\/min for 10\u00a0min. Detection was achieved using atmospheric pressure positive ion chemical ionization mass spectrometry (APCI-MS) of the eluent. Conditions for the HP 1100 APCI-MSD SL were as follows: nebulizer pressure 60\u00a0psi, vaporizer temperature 400\u00b0C, drying gas (N2) flow 6\u00a0l\/min and temperature 200\u00b0C, capillary voltage \u22123\u00a0kV, corona 5\u00a0\u03bcA (\u223c3.2\u00a0kV). GDGTs were detected by mass scanning from m\/z 950\u20131,450.\nIntact polar lipid analysis\nFor analysis of the intact polar lipids, cell material of A. boonei were extracted using a modified Bligh\u2013Dyer procedure (Bligh and Dyer 1959). To an aliquot of the cell material, a solvent mixture of phosphate-buffer (0.05\u00a0M, pH\u00a07.4)\/MeOH\/DCM 0.8\/2\/1 (v\/v) was added. The mixture was sonicated for 10\u00a0min after which DCM and phosphate buffer were added to a volume ratio of 0.9\/l\/1. After centrifuging (2,500\u00a0rpm, 5\u00a0min) the DCM layer was collected. The residue was re-extracted twice following the same procedure. The combined DCM layers were concentrated by evaporating solvent under a N2 stream, dried with Na2SO4 and the solvent was removed under a N2 stream. The residue was dissolved in a mixture of hexane\/isopropanol (79:21, v\/v), ultrasonicated and filtered using a RC 0.45-\u03bcm filter prior to analysis on the HPLC\/electrospray ionization (ESI)\u2013MS.\nIntact core lipids were analyzed according to Sturt et al. (2004) with some modifications (Boumann et al. 2006). An Agilent 1100 series LC (Agilent, San Jose, CA, USA) was used, equipped with thermostatted autoinjector and column oven, coupled to a Thermo TSQ Quantum Ultra EM triple quadrupole mass spectrometer equipped with an Ion Max source with ESI probe (Thermo Electron Corporation, Waltham, MA, USA). Separation was achieved on an Inertsil diol column (250\u00a0mm\u00a0\u00d7\u00a02.1\u00a0mm, 5\u00a0\u03bcm particles; Alltech Associates Inc., Deerfield, IL, USA) maintained at 30\u00b0C. The following linear gradient was used with a flow rate of 0.2\u00a0mL\u00a0min\u22121: 100\u201335% A:65% B over 45\u00a0min, maintained for 20\u00a0min, then back to 100% A for 20\u00a0min to re-equilibrate the column, where A\u00a0=\u00a0hexane\/2-propanol\/formic acid\/14.8\u00a0M NH3aq ratios 79:20:0.12:0.04 (v\/v\/v\/v) and B\u00a0=\u00a02-propanol\/water\/formic acid\/14.8\u00a0M NH3aq ratios 88:10:0.12:0.04 (v\/v\/v\/v). For MS detection, source parameters were optimized using loop injections of standard phospholipids (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine, 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-glycerol, 1,2-dipalmitoyl-sn-glycero-3-phosphate, and soya l-\u03b1-phosphatidylinositol, Avanti Polar Lipids, Alabaster, AL, USA) into a stream of 0.2\u00a0ml\/min of eluent A. ESI settings were as follows: capillary temperature 250\u00b0C, sheath gas (N2) pressure 49 (arbitrary units, auxiliary gas) (N2) pressure 21 (arbitrary units), spray voltage 4.2\u00a0kV, and source CID-14\u00a0V. Mass detection was achieved on the first MS scanning from m\/z 1,000 to 2,000.\nResults and discussion\nCore lipids\nGC\/MS analysis of the lipid extract of hydrolyzed cell material of A. boonei did not reveal substantial amounts of dialkyl glycerol ethers, such as archaeol, suggesting that it does not accumulate these compounds in substantial amounts. By contrast, HPLC\/APCI-MS analysis of the fraction revealed the abundant presence of a range of GDGTs (Fig.\u00a01) suggesting that archaeol is effectively transformed into GDGTs. The main compound was GDGT II, containing no cyclopentyl moieties, with lower amounts of GDGTs III\u2013VI, containing 1\u20134 cyclopentyl moieties, and small amounts of GDGT I. These GDGTs are well known compounds of thermoacidophilic Archaea in both the euryarchaeotal and crenarchaeotal groups (cf. De Rosa and Gambacorta 1988; Koga and Morii 2005). Cultivated species of the Order Thermoplasmatales, the closest phylogenetic relatives to A. boonei (Reysenbach et al. 2006), also contain GDGTs II\u2013VI as their dominant membrane lipids (DeRosa and Gambacorta 1988; Uda et al. 2004; Macalady et al. 2004).\nBesides GDGTs I\u2013VI, another group of later-eluting compounds were present in the HPLC\/MS chromatogram with mass spectra (insert Fig.\u00a01) characteristic of GDGTs, i.e. a base peak likely representing the [M\u00a0+\u00a0H]+ ions and fragment ions with losses of 18\u00a0Da (hydroxyl group as water) and 74\u00a0Da (part of the glycerol moiety) (Hopmans et al. 2000). The main compound had a base peak ion of 1,300 with more minor compounds having base peak ions of 1,298, 1,296, 1,294 and 1,292, respectively. Remarkably, the distribution of these compounds is similar to that of GDGTs II\u2013VI and their base peak ions are all 2\u00a0Da lower than their GDGT counterparts. This suggests that these compounds are GDGTs with an additional double bond or an additional cyclic moiety. To investigate the first hypothesis, we hydrogenated the polar fraction to remove any double bonds. However, subsequent analysis by HPLC\/MS revealed no changes in GDGT composition, suggesting that these compounds did not contain any double bonds. The unknown GDGT was subsequently isolated by preparative HPLC and treated with HI\/LiAlH4 to release the hydrocarbon skeletons (cf. Schouten et al. 1998). GC\/MS analysis revealed, however, no distinct peaks suggesting that the hydrocarbon skeletons of the unknown GDGT do not consist of regular C40 isoprenoids. An explanation for these results can be that the hydrocarbon skeletons are covalently linked to each other, thereby forming a C80 hydrocarbon after ether-cleavage. The molecular weight of such a component is too high to elute over a regular GC column. Examples of such GDGTs have been previously reported, i.e. the so-called H-shaped GDGT (Morii et al. 1998) that contains a covalent bond between the two isoprenoid chains. We, therefore, analyzed a pure GDGT VII standard obtained from Methanothermus fervidus (a kind gift of Dr. T. Eguchi) by HPLC\/MS. The mass spectrum of this standard was identical to that of the major unknown GDGT in A. boonei and co-injection revealed that they have identical retention times, thus unambiguously identifying this compound as GDGT VII. This suggests that the other later eluting GDGTs are also H-shaped isoprenoid GDGTs but with additional cyclopentyl moieties, i.e., GDGTs VII\u2013XI. It must be noted that the exact position of the covalent bond in GDGT VII is tentative (Morii et al. 1998) and, thus, also the position of the covalent bonds in GDGTs VII-XI. Lutneas et al. (2007) recently determined the structure of structurally similar isoprenoid tetracids in which the covalent bond was between two methyl groups rather than between a methyl group and the aliphatic chain as suggested by Morii et al. (1998). Hence, future structural analyses are needed to unambiguously establish the position of the covalent bond.\nH-shaped isoprenoid GDGTs have been only rarely reported in cultivated archaea. GDGT VII was first identified in the hyperthermophilic methanogen Methanothermus fervidus (Morii et al. 1998). They were further identified by fast atom bombardment-MS in 4 out of 17 species of the Order Thermococcales (Sugai et al. 2004). Until now, they have only been identified in hyperthermophilic euryarchaeota growing at neutral pH with optimal growth temperatures >80\u00b0C. The identification of H-shaped GDGTs in A. boonei now shows that these compounds are also synthesized at lower growth temperatures (70\u00b0C) and more acidic pH\u00a0(4.5). The functional role of these GDGTs is not clear. Possibly, the introduction of a covalent cross-link between the alkyl chains will reinforce the strength of the monolayer membrane to protect the cell against membrane lysis at these high temperatures. The similar relative distribution of GDGTs II\u2013VI compared to GDGTs VII\u2013XI in A. boonei tentatively suggests a possible biosynthetic link, i.e., the latter GDGTs may be synthesized from GDGTs II\u2013VI.\nIntact polar lipids\nHPLC\/ESI-MS analysis of the Bligh\u2013Dyer extract of cell material of A. boonei revealed one major peak and one minor peak (Fig.\u00a02). The mass spectrum of the major peak is dominated by an ion at m\/z 1,456 with minor ions 15 and 22 Da higher (insert Fig.\u00a02). These latter ions are typical product of ammonia and sodium adductions, respectively, to protonated molecules in the ESI interface and suggests that the ion at m\/z 1,456 represents the [M\u00a0+\u00a0H]+ ion (cf. Sturt et al. 2004). As the major core lipid is GDGT II with a [M\u00a0+\u00a0H]+ ion of 1,302, it suggests that the head group has a mass of 154\u00a0Da in agreement with a phospho-glycerol (PG) moiety (Sturt et al. 2004). The mass spectrum of the minor peak eluting slightly later is similar to that of the major peak except that all ions are shifted 2\u00a0Da lower. In view of the results obtained with the core lipids it suggests that this peak represents H-shaped GDGTs with a PG head group. PG moieties are not uncommon as head groups of GDGTs in hyperthermophilic Archaea, though they are usually accompanied by a hexose head group attached to the other glycerol moiety (Koga and Morii 2005 and references cited therein). However, these compounds were not detected in A. boonei although we cannot exclude that they might be present in minor amounts.\nFig.\u00a02HPLC\/ESI\/MS base peak chromatogram of intact polar lipids in Bligh Dyer extract of cell material of Candidatus \u201cAciduliprofundum boonei\u201d. Inset shows the electrospray ionization mass spectrum of the major peak in the chromatogram with tentative structural identification\nConclusions\nOur analysis of lipid composition of Candidatus \u201cAciduliprofundum boonei\u201d, the only cultivated representative of archaea falling in the DHVE2 phylogenetic cluster, showed it to be composed of GDGTs containing 0\u20134 cyclopentyl moieties. Surprisingly, GDGTs with an additional covalent bond between the isoprenoid hydrocarbon chains, the so-called H-shaped GDGTs were also present in substantial amounts. These GDGTs mainly occurred with a phospho-glycerol headgroup in the membrane of A. boonei.","keyphrases":["aciduliprofundum boonei","thermoacidophile","dhve2 cluster","glycerol dialkyl glycerol tetraethers"],"prmu":["P","P","P","R"]}
{"id":"J_Gastrointest_Surg-3-1-1852391","title":"Predicting Stricture in Morbidly Obese Patients Undergoing Laparoscopic Roux-en-Y Gastric Bypass: A Logistic Regression Analysis\n","text":"Gastrojejunostomy stricture after Roux-en-Y gastric bypass occurs in 3 to 27% of morbidly obese patients in the USA. We questioned whether preoperative patient characteristics, including demographic attributes and comorbid disease, might be significant factors in the etiology of stricture. In this study from November 2001 to February 2006 (51 months), at a high-volume bariatric center, of the 1,351 patients who underwent laparoscopic gastric bypass, 92 developed stricture (6.8%). All but two were treated successfully by endoscopic dilation. All patients stopped nonsteroidal anti-inflammatory medications 2 weeks prior to surgery and did not restart them. The operative procedure included the use of a 21-mm transoral circular stapler to create the gastrojejunostomy; the Roux limb was brought retrogastric, retrocolic. In an effort to reduce our center\u2019s stricture rate, late in the study, U-clips used at the gastrojejunostomy were replaced by absorbable sutures, and postoperative H2 antagonists were added to the treatment protocol. The change to absorbable polyglactin suture proved to be significant, resulting in a lower stricture rate. The addition of H2 antagonists showed no significant effect. Following the retrospective review of the prospective database, univariate and multivariate logistic regression analyses identified factors associated with the development of stricture. Gastroesophageal reflux disease and age were each shown to be statistically significant independent predictors of stricture following laparoscopic gastric bypass.\nIntroduction\nBariatric surgery, particularly Roux-en-Y gastric bypass (RYGB), has been proven safe1,2 and effective in achieving long-term weight loss,3,4 comorbidity reduction,3,5 and enhancement of quality of life.6 Since its introduction by Wittgrove et al. in 1994,7 laparoscopic Roux-en-Y gastric bypass (LRYGB) has been shown to be as reliable as open RYGB8\u201310 and less traumatic, requiring a briefer recovery time and shorter duration of stay.11 It is effective in achieving comparable weight loss at 1\u00a0year3,12\u201315 and is successful in reducing the comorbidities of morbid obesity.3,9,13 Laparoscopic RYGB has surpassed other weight-loss methods for the morbidly obese in frequency of use in the USA.14\nLaparoscopic RYGB is a technically demanding procedure with a lengthy learning curve of approximately 100 cases.16,17 Gastrojejunostomy stricture following RYGB has been reported variously in 3 to 27% of patients.12,16\u201319 Actual incidence of this complication is a function of the accuracy of its diagnosis and reporting19 and has been shown to be relative to operative technique and surgeon experience.20 Other known etiologies of stricture following LRYGB are tension on the anastomosis, foreign body reaction, technical error in creation of the anastomosis, marginal ulcer, and leak with associated scarring.19\nIn 2003, Perugini and colleagues published an analysis of preoperative predictors of complications.20 They reported a 14.4% rate of gastrojejunostomy stricture (N\u2009=\u200927\/188), greater than half of all complications. With multivariate analysis by stepwise logistic regression, they found that surgeon experience was the most significant predictor of complications in general (not of stricture specifically) and that sleep apnea and hypertension were also significant independent predictors of complications.20 Perugini et al. cautioned that the factors they identified (experience, sleep apnea, hypertension) as predictive of complications in general might, in fact, \u201c be specific for the complication of gastrojejunal stenosis\u201d.20 Our study took up this question and, by univariate and multivariate logistic regression, analyzed preoperative predictors specifically for stricture.\nPatients and Methods\nPatients\nA consecutive series of morbidly obese patients underwent LRYGB between November 12, 2001, and February 28, 2006 (51\u00a0months), in a dedicated, community-based, bariatric program awarded Center-of-Excellence status by the Surgical Review Corporation in 2005.\nInstitutional review board approval and informed consent were obtained prior to prospective data collection. In a retrospective analysis, preoperative patient characteristics, including whether the patient was taking any nonsteroidal anti-inflammatory medications on a chronic basis prior to surgery, were compared for two groups: those who developed gastrojejunostomy stricture (N\u2009=\u200917\/92, 6.8%) and those who did not (N\u2009=\u2009311\/1,259, 93.2%). No patients were excluded from the study.\nPatients where tested for Helicobacter pylori and treated if positive. Anti-inflammatory medications were withheld 2\u00a0weeks preoperatively and not resumed postoperatively. Procedures were performed by four bariatric surgeons. Stenosis, or stricture, indicated by progressive dysphagia, nausea, and vomiting, was diagnosed by endoscopy.\nSurgical Technique\nThe procedure was performed using the transoral end-to-end anastomotic (EEA) technique similar to the Wittgrove technique.10 Using a Veress needle to an intra-abdominal pressure of 15\u00a0mm Hg, abdominal insufflation was performed. The patient was placed supine, in the reverse Trendelenburg position, and six abdominal trocars were introduced. The angle of His was dissected, and a 15-ml balloon was passed transorally and placed at the esophagogastric junction. The lesser sac was entered just below the balloon on the lesser curve of the stomach. A 15- to 20-ml gastric pouch was created with the Endo GIA 45-mm stapler (Ethicon Endo-Surgery, Cincinnati, OH, USA), using an average of three firings. A guidewire was introduced through the anterior abdominal wall and an endoscope was passed transorally. Cautery was used to pass the snare through the pouch and grasp the guidewire. The anvil of the 21-mm EEA Stealth stapler (Ethicon Endo-Surgery) was attached to the guidewire and brought down through the mouth. The ligament of Treitz was identified, and, approximately 20\u00a0cm distally, the jejunum was divided. A 100-cm Roux limb was created for patients with BMI\u2009<\u200955\u00a0kg\/m2, and a 150-cm Roux limb was created for patients with BMI\u2009\u2265\u200955\u00a0kg\/m2. The jejunojejunostomy was stapled side to side, and closed with the Endo GIA stapler. The Roux limb was brought retrocolic, retrogastric through the lesser curve of the stomach. The EEA circular stapler was introduced through the abdominal wall and the gastrojejunostomy was stapled end to end. The posterior wall of the gastrojejuostomy lays on soft tissue that holds the left gastric and vagus nerve and does not need reinforcement. All techniques for reinforcing the gastrojejunostomy were instituted only in the anterior wall of the anastamosis as a second layer. The initial technique was to sew a continuous second layer of the gastrojejunostomy anteriorly with 3\u20130 polyglactin absorbable suture. The angle is often technically demanding. In October 2005, the procedure was modified to one in which the gastrojejunostomy was oversewn with two Nitinol \u201cU\u201d clips that revert to preformed circular memory when placed, without additional suture. In October of 2005, these U-clips were replaced with two interrupted 3\u20130 polyglactin absorbable sutures. Finally, in January 2006, the addition of H2 antagonists for 90\u00a0days to the postoperative regimen was made. These technical changes were instituted in an attempt to lessen the stricture rate, which is followed monthly in our database.\nPatients who experienced persistent postoperative nausea, vomiting, and intolerance to solid food were referred for upper gastrointestinal studies, followed by referral to the gastroenterologist for upper endoscopy. Patients with stricture were treated endoscopically with balloon dilation to a maximum of 15\u00a0mm.\nStatistical Methods\nPatient data were collected prospectively and included preoperative patient demographics and comorbidities, as well as postoperative complications extracted to identify potential influences on stricture rates. Variables included in the univariate logistic regression analysis are listed in Table\u00a01. \nTable\u00a01Univariate Analysis to Identify Preoperative Patient Characteristics Associated with StrictureVariableStricture Group [N (%)]Nonstricture Group [N (%)]P ValueNo. patients (N\u2009=\u20091,351)921,259Age, mean (SD)41.4 (11.6)44.4 (10.4)0.039aCardiac disease6 (6.5)107 (8.5)0.885Chronic depression21 (22.8)278 (22.1)0.634Chronic respiratory disease22 (23.9)318 (25.3)0.766Chronic venous insufficiency53 (57.6)692 (55.0)0.347Degenerative joint disease89 (96.7)1,209 (96.0)0.893Diabetes, type 226 (28.3)307 (24.4)0.184Ethnicity, N (%)\u2013\u20130.145\u00a0Caucasian76 (82.6)1,122 (89.1)\u2013\u00a0Other16 (17.4)137 (10.9)\u2013GERD66 (71.7)715 (56.8)0.035aSex, N (%)\u2013\u20130.208\u00a0Female81 (88.0)1,042 (82.8)\u2013\u00a0Male11 (12.0)217 (17.2)\u2013Hypercholesterolemia\/hyperlipidemia40 (43.5)624 (49.6)0.958Hypertension41 (44.6)652 (51.8)0.838Infertility11 (12.0)96 (7.6)0.732Nonsteroidal anti-inflammatory medications17 (18.5)294 (23.4)0.284No. preoperative comorbidities5.2 (2.0)5.1 (1.9)0.259No. preoperative medications4.6 (3.6)4.2 (3.5)0.088Obstructive sleep apnea43 (46.7)564 (44.8)0.415BMI48.5 (8.5)49.2 (8.3)0.202Fasting blood sugar111.5 (45.4)108.9 (39.6)0.779HbA1C6.2 (1.8)6.1 (1.2)0.150Previous abdominal surgeries1.2 (1.1)1.2 (1.3)0.754Urinary stress incontinence52 (56.5)698 (55.4)0.322aSignificance at the 0.05 level\nThe SPSS\u2122 software package (version 14.0, SPSS, Chicago, IL, USA) was used to perform all statistical analyses. Statistical significance was set at P\u2009<\u20090.05. Binary logistic regression analysis was used in both univariate and multivariate modeling to identify independent preoperative variables associated with the development of gastrojejunostomy stricture following LRYGB surgery. Univariate analysis using logistic regression was applied to identify significant associations with the dichotomous outcome variable (stricture). For comparison, Pearson chi-square test for categorical variables and Student\u2019s t test for continuous variables were used. A priori preoperative factors of research interest (P\u2009<\u20090.21), as well as those found to be significantly associated with stricture (P\u2009<\u20090.05), were entered into multivariate analysis using forward stepwise logistic regression. Likelihood ratio tests were used for variable selection.\nIn the initial analysis of preoperative characteristics by univariate logistic regression, age, number of comorbidities, number of medications, BMI, fasting blood sugar, HbA1C, and previous abdominal surgeries were classified as continuous variables. With the exception of sex and ethnicity (dichotomized to male\/female and Caucasian\/non-Caucasian, respectively), all remaining variables were dichotomized (yes\/no). In the final multivariate forward stepwise logistic regression model, both age and BMI were redefined as categorical variables: age was coded to reflect four levels (\u226435, 36 to 45, 46 to 55, and \u226555\u00a0years) and BMI was coded to reflect two levels (<50 and \u226550).\nResults\nFrom November 12, 2001, to February 28, 2006 (51\u00a0months), 1,351 patients underwent LRYGB at a single institution. Mean preoperative patient age was 44\u00a0years, mean BMI was 49\u00a0kg\/m2, 83% of patients were female, and mean preoperative weight was 305\u00a0lb. Patients were predominantly Caucasian (89%); 7% were Hispanic; 3% were African American; and 1% were of Native American, Asian\/Pacific, and other ethnicities.\nOperative technique was the same in all procedures, namely, a retrocolic, retrogastric, transoral, 21-mm, circular stapled gastrojejunostomy. Mean intraoperative time was 96\u00a0min and length of stay averaged 2.8\u00a0days; 97.6% of operations were accomplished laparoscopically and 2.4% were conversions to open procedures. There were two reoperations with revision of the gastrojejunostomy in the \u201cstricture group.\u201d The first was in a patient who developed a leak immediately postoperatively and returned to the OR for definitive management. The patient developed a stricture that was not amenable to dilation and, at 7\u00a0months, underwent a revision of her gastrojejunostomy. The second patient had a stricture that was dilated to 12\u201315\u00a0mm. Subsequent endoscopy showed no recurrent stricture. She continued to eat very large portions in multiple meals, developing recurrent bezoars that had to be cleared endoscopically. The pouch dilated over time, and even though the gastrojejunostomy was shown to be only large enough to pass an endoscope, we were obliged to revise the pouch to a smaller size. Since that time she has had no further problem with bezoars.\nThere were no deaths in the stricture group. In the \u201cnonstricture group,\u201d early mortality was 0.2% (two deaths <31\u00a0days postoperatively); intermediate mortality, 0.3% (four deaths between 31 and 90\u00a0days postoperatively); and late mortality, 0.4% (five deaths >90\u00a0days postoperatively). Ninety-two patients (6.8%) were diagnosed with stricture and underwent subsequent endoscopic dilation. Seventeen of 92 stricture patients (18.5%) were on nonsteroidal anti-inflammatory medications prior to surgery, whereas 294 of the 1,259 (23.4%) nonstricture patients were on nonsteroidal anti-inflammatory medications prior to surgery (Table\u00a01). The difference in stricture rates between these two groups was not significant.\nOur rate of stricture (92\/1,351, 6.8%) was in the range of rates previously reported by Higa et al. (5.3%), Schauer et al. (4.7%), and DeMaria et al. (6.6%), as compiled by Perugini20, at the lower end of the national range of stricture prevalence. Other complications with the highest incidence were cholecystectomy (6.5% stricture group vs 3.0% nonstricture group), small bowel obstruction secondary to internal hernia (4.4 vs 1.9%), and intra-abdominal abscess (3.3 vs 2.0%). In the stricture and nonstricture groups, respectively, leakage occurred in 1.1 and 1.0% of patients, and port site infection occurred in 2.2 and 3.6% of patients. Peripheral neuropathy was 5.4% in the stricture group and 0.5% in the nonstricture group (Table\u00a02). \nTable\u00a02Forward Stepwise Multivariate Logistic Regression Model for Complications of StrictureVariableRate, N (%)P ValueOdds Ratio (95% Confidence Interval)Stricture GroupNonstricture Group30-day readmission27 (29.35%)109 (8.66%)0.000a4.381 (2.666\u20137.197)Cholecystectomy6 (6.52%)38 (3.02%)0.181NSInternal hernia1 (1.09%)35 (2.78%)0.337NSSmall bowel obstruction4 (4.35%)24 (1.91%)0.500NSIntra-abdominal abscess3 (3.26%)25 (1.99%)0.691NSPeripheral neuropathy5 (5.43%)6 (0.48%)0.000a11.979 (3.423\u201341.929)Infection2 (2.17%)2 (3.57%)0.151NSaSignificance at the 0.05 level\nMean percent excess weight loss at 1\u00a0year following surgery was the same for both groups (\u2212103.1\u00a0lb, 82.6% stricture group vs \u2212115.8\u00a0lb, 82.0% nonstricture group). Mean numbers of medications were reduced from 4.6 to 1.6 and from 4.2 to 1.5 in the stricture and nonstricture groups, respectively, at the most recent postoperative examination.\nBy univariate logistic regression analysis, two preoperative characteristics were identified as factors associated significantly (P\u2009<\u20090.05 level) with stricture: gastroesophageal reflux disease (GERD) (P\u2009=\u20090.035) and age (P\u2009=\u20090.039) (Table\u00a01). In addition to GERD and age, BMI (P\u2009=\u20090.202) and gender (P\u2009=\u20090.208) were incorporated into the final multivariate analysis. In the first multivariate model (Table\u00a03), with age defined as a continuous variable, forward stepwise multivariate logistic regression confirmed GERD (P\u2009=\u20090.006, OR\u2009=\u20091.917, CI\u2009=\u20091.200\u20133.062) and age (P\u2009=\u20090.010, OR\u2009=\u20090.973, CI\u2009=\u20090.953\u20130.993) to be independent factors associated with stricture. Seventy-two percent of all stricture patients were diagnosed with GERD prior to surgery, and the mean age was 41\u00a0years, whereas, in the preoperative nonstricture group, 57% were diagnosed with GERD and the mean age was 44\u00a0years. \nTable\u00a03Significance and Odds Ratios for Age, GERD, Gender, and BMI in Multivariate Models to Predict StrictureVariableP ValueOdds Ratio (95% Confidence Interval)Multivariate model #1a\u00a0Age0.010b0.973 (0.953\u20130.993) \u00a0GERD0.006b1.917 (1.200\u20133.062)\u00a0Gender0.324NS\u00a0Preoperative BMI0.086NSMultivariate model #2c\u00a0Age overall0.015\u00a0\u00a0Group 1: \u226435\u00a0years (reference category)1\u00a0\u00a0Group 2: 36\u201345\u00a0years0.0150.508 (0.294\u20130.877)\u00a0\u00a0Group 3: 46\u201355\u00a0years0.0040.431 (0.244\u20130.760)\u00a0\u00a0Group 4: >55\u00a0years0.1260.596 (0.308\u20131.156)\u00a0GERD0.0051.963 (1.227\u20133.141)\u00a0Gender0.353NS\u00a0Preoperative BMI0.100NSaAge classified as a continuous variablebSignificant at the 0.05 levelcAge classified as a categorical variable\nIn the second multivariate model (Table\u00a03), age was classified as a categorical variable to refine our understanding of the impact of age on stricture. Using age group 1 (\u226435\u00a0years) as the reference category, age groups 2 (36\u201345\u00a0years) and 3 (46\u201355\u00a0years) were significant at the 0.05 level [group 2, P\u2009=\u20090.015 (OR\u2009=\u20090.508, CI\u2009=\u20090.294\u20130.877); group 3, P\u2009=\u20090.004 (OR\u2009=\u20090.431, CI\u2009=\u20090.244\u20130.760)]. GERD remained significant (P\u2009=\u20090.005; OR\u2009=\u20091.963, CI\u2009=\u20091.227\u20133.141).\nThree postoperative complications were found to be associated significantly with the presence of stricture by univariate logistic regression, including readmissions within 30\u00a0days of surgery (P\u2009=\u20090.000), peripheral neuropathy (P\u2009=\u20090.000), and pneumonia (P\u2009=\u20090.031). The postoperative multivariate model determined readmissions within 30\u00a0days of surgery (P\u2009=\u20090.000, OR\u2009=\u20094.381, CI\u2009=\u20092.666\u20137.197) and peripheral neuropathy (P\u2009=\u20090.000, OR\u2009=\u200911.979, CI\u2009=\u20093.423\u201341.929) to be statistically significant covariates of stricture.\nDecreasing our rate of stricture by a protocol change to oversewing the gastrojejunostomy with polyglactin was shown, by chi square analysis, to be a significant decrease, with the rate of 6.8% diminishing to 2.8% (P\u2009=\u20090.028, OR\u2009=\u20090.358, CI\u2009=\u20090.143\u20130.895). The second protocol adjustment, administration of an H2 antagonist (prevacid) postoperatively, had no effect.\nDiscussion\nStrictures contribute to almost half of all readmissions in the early perioperative period and require instrumentation and rehydration. They can also be associated with the development of a more serious complication, peripheral neuropathy, as is demonstrated by these data.\nSpecific intraoperative factors (e.g., surgeon experience) associated with stricture have been identified as causal by prior studies. Isolating preoperative factors that may be causally related to stricture may aid in minimizing this complication. In their study, Perugini et al. showed preoperative hypertension and obstructive sleep apnea to be predictive of complications in general; yet, they cautioned that their findings might have been confounded by their high rate of stricture.20 By logistic regression analysis, our study found the variables of GERD and age to be significantly associated with a complication with one of the highest incidences\u2014stricture. Our results are supportive of Perugini\u2019s findings, in that we did not find the same preoperative predictors of complications (hypertension, obstructive sleep apnea) significantly associated with stricture and the overall rate was significantly less.\nThe odds of developing stricture in patients with preoperative GERD were found to be nearly two times higher than those for patients without GERD. GERD is a known inflammatory condition of the upper gastrointestinal tract that specifically affects the lower esophagus and fundus. If present, this inflammation may predispose patients to more vigorous scarring of the gastrojejunostomy. During the period of the study, we began testing patients preoperatively for H. pylori. Patients who tested positive were treated preoperatively. In general, the micropouch used to create the gastrojejunostomy has been assumed to be almost devoid of acid-producing cells, and the rate of ulceration at the anastomosis at the time of endoscopy is low. It is clear, though, that adding H2 antagonists for 30\u00a0days following surgery helps to decrease inflammation, and in addition, the polyglactin stitch may be less inflammatory in nature. Data were insufficient at study summary to demonstrate the statistical significance of administering H2 antagonists; however, we are encouraged by the trend of fewer strictures.\nFinding that younger rather than older age was associated with the development of stricture seems clinically counterintuitive. One reason for this is that age affects the small vessels\u2019 ability to provide necessary oxygenation to the anastomosis, and older patients are more likely overall to have diminished capability to oxygenate. In our center, maintenance on oxygen the night of surgery is standard; patients who require continuous positive airway pressure are started in the anesthesia recovery unit and are kept on supplemental oxygen until they can oxygenate in ambulating and resting modes at greater than 92%. However, as stated, in our first multivariate model, older age was shown to be slightly protective with respect to stricture. In the second multivariate model, designed to refine the interpretation of age effects on stricture, we found that the odds of developing stricture in those 35\u00a0years or younger were two times greater than those aged 36\u201345\u00a0years, and 2.32 times greater than those aged 46\u201355\u00a0years. The trend of age being protective against stricture leveled off and was not significant in those older than 55\u00a0years. More study of this finding is indicated.\nOur original assumption that higher rates of central fat distribution in males might place greater tension on the anastomosis predisposing male patients, particularly those with higher BMIs, toward stricture, may be in error. The effect of increased central fat on the anastomosis may not be injurious if meticulous technique is used to ensure a tension-free anastomosis. Also, the ischemic effects of diabetes, sleep apnea, and chronic respiratory disease at the gastrojejunostomy site may be insufficient, in themselves, to cause stricture.\nIt has been well documented that operative technique can contribute to increased stricture rates. Previous studies have reported fewer strictures with hand-sewn anastomoses than with stapled ones. Some authors have demonstrated that the 25-mm EEA stapler may be associated with fewer strictures that the 21-mm EEA stapler; we have a 6.8% stricture rate with the 21-mm EEA stapler. Further studies of stapling methodologies are warranted.\nIt has been proposed that stricture rates may be lessened by the creation of a larger gastrojejunostomy anastomosis. The effect on the restrictive element of the bypass and subsequent effect on weight loss long term has not been evaluated. We avoided excessive dilation to allow the patient to maintain as much restriction as possible. Using a protocol of minimal dilation to between 12.5 and 15\u00a0mm resulted in only one perforation and acceptable weight loss. In our study and those of others,21,22 weight loss at 1\u00a0year is not hindered by dilation.\nA limitation of this study is that is was retrospective. A prospective trial in which comorbidities were not controlled would not be feasible, although a prospective trial of H2 antagonists may be of value. It is difficult to discern the effect of absorbable suture on stricture rates vs the effect of H2 antagonists as their introduction to the protocol occurred in close proximity. The finding that readmissions within 30\u00a0days of surgery and postoperative peripheral neuropathy were statistically significant covariates of stricture most likely relates to the frequency of readmissions for peripheral neuropathy secondary to vitamin deficiencies following bariatric surgery.\nThe objective of this study was to reach back in the causal continuum, prior to the intraoperative effects of technique and surgeon experience, to identify other potentially salient contributors to postoperative stricture. This analysis of a large series of patients undergoing RYGB identified GERD and age as factors associated with gastrojejunostomy stricture.\nConclusions\nUsing absorbable suture at the gastrojejunostomy anastomosis appears to decrease stricture rates. As identified via multivariate logistic regression analysis, GERD and age are independent predictors of gastrojejunostomy stricture.","keyphrases":["stricture","obesity","gastric bypass","gastrojejunostomy","bariatric surgery"],"prmu":["P","P","P","P","P"]}
{"id":"Purinergic_Signal-3-4-2072920","title":"Modification of neuropathic pain sensation through microglial ATP receptors\n","text":"Neuropathic pain that typically develops when peripheral nerves are damaged through surgery, bone compression in cancer, diabetes, or infection is a major factor causing impaired quality of life in millions of people worldwide. Recently, there has been a rapidly growing body of evidence indicating that spinal glia play a critical role in the pathogenesis of neuropathic pain. Accumulating findings also indicate that nucleotides play an important role in neuron-glia communication through P2 purinoceptors. Damaged neurons release or leak nucleotides including ATP and UTP to stimulate microglia through P2 purinoceptors expressing on microglia. It was shown in an animal model of neuropathic pain that microglial P2X4 and P2X7 receptors are crucial in pain signaling after peripheral nerve lesion. In this review, we describe the modification of neuropathic pain sensation through microglial P2X4 and P2X7, with the possibility of P2Y6 and P2Y12 involvement.\nIntroduction\nThere is a type of pain that does not go away even though the tissue has already healed. One type of this pain is called neuropathic pain that typically develops when peripheral nerves are damaged such as through surgery, bone compression in cancer, diabetes, or infection. Neuropathic pain is a major factor causing impaired quality of life in millions of people worldwide and is frequently resistant to all known analgesic drugs. Over the last decade, accumulating evidence concerning how peripheral nerve injury creates neuropathic pain has suggested that nerve injury produces molecular and cellular alterations that result in multiple forms of neuronal plasticity and anatomical reorganization in the dorsal horn of the spinal cord. These alterations have been proposed to be crucial in the pathogenesis of neuropathic pain [1, 2]. While the dominant theme in research on neuropathic pain has been to understand the roles of neurons in the peripheral nervous system and the dorsal horn, there is a rapidly growing body of evidence indicating that spinal glial cells play a critical role in the pathogenesis of neuropathic pain.\nRecently, growing evidence has indicated that neuron-glia interaction is a key idea to understand functions of the central nervous system (CNS). Especially glia play important roles in pathophysiological situations of the CNS including psychiatric disorders, physical trauma, and infections [3]. Glia consist of three members: astrocytes, oligodendrocytes, and microglia. Accumulating findings also indicate that nucleotides play an important role in neuron-glia communication through P2 purinoceptors, even though ATP is recognized primarily to be a source of free energy and nucleotides are key molecules in cells. Microglia, which are thought to be residential macrophages in the CNS, express P2 purinoceptors, mainly P2X4 and P2X7 as well as P2Y2, P2Y6, and P2Y12 [4]. Damaged neurons release or leak nucleotides including ATP and UTP to stimulate microglia [5, 6]. It is clear that these nucleotides trigger the release of various neurotoxic and neuroprotective cytokines and growth factors via different purine receptors [4, 7] or induce P2Y12-dependent chemotaxis in cultured cells [8, 9]. It was shown in an animal model of neuropathic pain that microglial P2X4 and P2X7 receptors are crucial in pain signaling after peripheral nerve lesion [10\u201312]. Other purine receptors are upregulated in microglia in response to neuronal injury, as was recently demonstrated for P2Y6 [6]. The P2Y6 receptor triggers microglial phagocytosis [6]. In this paper, we review the modification of pain sensation through microglial P2X4 and P2X7, and also point out the possibility of P2Y6 and P2Y12 involvement in pain signaling.\nP2X4 in neuropathic pain\nWe found that the marked tactile allodynia that develops following nerve injury was reversed by acutely administering TNP-ATP intrathecally but was unaffected by administering PPADS [10]. From the pharmacological profiles of TNP-ATP (blocking P2X4 at high concentration) and PPADS (not blocking P2X4), it was suggested that tactile allodynia depends upon P2X4 in the spinal cord. The expression of P2X4 protein, normally low in the na\u00efve spinal cord, progressively increased in the days following nerve injury with a time course parallel to that of the development of tactile allodynia. Double immunolabeling analysis demonstrated that not neurons or astrocytes but activated microglia in the dorsal horn were intensely positive for P2X4 protein [10]. Moreover, intrathecally administered antisense oligodeoxynucleotide for P2X4R reduced the expression of P2X4 protein in spinal microglia and prevented the development of the nerve injury-induced tactile allodynia. In na\u00efve rats, intrathecal administration of cultured microglia that were preincubated with ATP to activate P2X4 produced tactile allodynia over the 3\u20135\u00a0h after the administration [10].\nSince it was already reported that the nerve injury-induced tactile allodynia depends on a depolarizing shift in the Eanion of spinal lamina I (LI) neurons in the dorsal spinal cord, resulting in converting the GABAA receptor- and glycine receptor-mediated inhibition to excitation [13], it was considered that microglia may affect Eanion in LI neurons. To investigate this possibility, microglia were administered to the lumbar spinal level of na\u00efve rats by an intrathecal catheter as described [11]. Administering microglia stimulated with ATP caused a progressive tactile allodynia over the 5\u00a0h after injection. Eanion in LI neurons from rats administered ATP-stimulated microglia was shifted to \u221261.6\u00a0mV from \u221268.3\u00a0mV of normal rats. In addition, we found that GABA response switched from hyperpolarizing in control rats to depolarizing in microglia-treated rats. Activated microglia secrete various biologically active molecules, one of which, BDNF, was implicated in the hypersensitivity of dorsal horn neurons that follows sensitization and inflammation [14\u201316] and in anion gradient shifts in the hippocampus [17]. Indeed, intrathecal administration of recombinant BDNF produced tactile allodynia comparable to that produced by ATP-stimulated microglia [11]. Eanion of LI neurons in slices treated with BDNF (>90\u00a0min, in vitro) was significantly less negative than that of LI neurons from control slices. The rise in [Ca2+]i was prevented by the GABAA receptor blocker bicuculline, confirming that the effect was mediated by GABAA receptors. Thus, acute administration of BDNF in slices caused a depolarizing shift in Eanion and caused GABA to produce net excitation [11]. Moreover, a function-blocking antibody against the TrkB receptor (anti-TrkB) and a BDNF-sequestering fusion protein (TrkB-Fc) acutely inhibited the allodynia and the shift of Eanion of LI neurons [11]. The administration of ATP-stimulated microglia with either anti-TrkB or TrkB-Fc did not develop tactile allodynia. After pretreatment of microglia with double-stranded short interfering RNA directed against BDNF (BDNF siRNA), the ATP-stimulated microglia injected intrathecally into normal rats did not cause the allodynia [11]. Anti-TrkB and BDNF siRNA prevented the shift in Eanion induced by ATP-stimulated microglia. ATP stimulation caused release of BDNF from microglia in culture. This effect of ATP was blocked by treating the cultures with the P2X receptor blocker TNP-ATP. In addition, pretreatment of the microglia with BDNF siRNA prevented release of BDNF by ATP stimulation. By bath application of TNP-ATP to spinal slices taken from allodynic rats 2\u00a0weeks after nerve injury, Eanion of LI neurons was returned to normal value [11]. These findings indicate that P2X4-dependent release of BDNF from microglia is necessary to sustain both the tactile allodynia and the depolarizing shift in Eanion in LI neurons that result from nerve injury (Fig.\u00a01).\nFig.\u00a01Schematic illustration of potential mechanisms by which P2X\/Y receptors in activated microglia modulate neuropathic pain signaling in the dorsal horn. Activated microglia in the spinal cord after nerve injury express ionotropic ATP receptors [e.g., P2X4 receptor (P2X4R) or P2X7R]. P2X4R or P2X7R activation leads to the release of bioactive diffusible factors such as BDNF and other proinflammatory factors (cytokines and chemokines). BDNF causes a collapse of transmembrane anion gradient in dorsal horn lamina I neurons presumably through the downregulation of KCC2, which in turn renders GABA and glycine effects depolarizing, rather than hyperpolarizing, in these neurons. Microglial factors may also interact with excitatory synapses of neighboring dorsal horn neurons and enhance the excitability in dorsal horn neurons. The net hyperexcitability in the dorsal horn pain network by these factors from activated microglia may be responsible for neuropathic pain. Microglia also express G protein-coupled ATP receptors [e.g., P2Y6 receptor (P2Y6R) and P2Y12R]. Activating P2Y12R and P2Y6R leads to chemotaxis and phagocytosis, respectively, but their functional relevance to neuropathic pain remained to be determined\nP2X7 in neuropathic pain\nP2X7 purinoceptors are a ligand-gated nonselective cationic channel and are expressed predominantly on immune cells [18]. Stimulation of the P2X7 receptors on microglia is associated with release of cytokines including tumor necrosis factor-\u03b1 (TNF-\u03b1) [5, 19], interleukin-6 (IL-6) [20], and interleukin-1\u03b2 (IL-1\u03b2) [21\u201323]. Several cytokines such as IL-1\u03b2, IL-6, and TNF-\u03b1 in the dorsal horn are increased after nerve lesion [24\u201326] and have been implicated in contributing to neuropathic pain [24\u201327]. Recent evidence indicates the relationship between TNF-\u03b1 and neuropathic pain [28\u201332], and TNF-\u03b1 released after injury is proposed as an initiator of abnormal pain sensation. TNF-\u03b1 is upregulated after nerve injury in both dorsal root ganglion (DRG) and spinal cord [33]. The inhibition of TNF-\u03b1 reduces the hyperalgesia in neuropathic pain models [34]. Recent evidence indicates the relationship between inflammatory cytokines including IL-1\u03b2 and neuropathic pain [25, 26, 28]. The expression of IL-1\u03b2 is upregulated in the spinal cord of several rat neuropathy models [24\u201326]. These findings further support a role for central IL-1\u03b2 in the development and maintenance of neuropathic pain through induction of a proinflammatory cytokine cascade (Fig.\u00a01).\nRecently, it was reported that in mice lacking P2X7 inflammatory and neuropathic hypersensitivity is completely absent to both mechanical and thermal stimuli, whilst normal nociceptive processing is preserved [12]. Contribution of P2X7 receptor to neuropathic pain is also demonstrated by using the recently developed selective antagonist for P2X7 receptor A-740003 [35]. The knockout animals were unimpaired in their ability to produce mRNA for pro-IL-1\u03b2, and cytometric analysis of paw and systemic cytokines from knockout and wild-type animals following adjuvant insult suggested a selective effect of the gene deletion on release of IL-1\u03b2 and IL-10, with systemic reductions in adjuvant-induced increases in IL-6 and MCP-1. In addition, P2X7 receptors were upregulated in human dorsal root ganglia and injured nerves obtained from chronic neuropathic pain patients [12]. It was hypothesized that the P2X7 receptor plays a common upstream transductional role in the development of pain of neuropathic and inflammatory origin via regulation of mature IL-1\u03b2 production.\nIn addition, a recent study has shown that activation of P2X7 receptors expressed on satellite glia that enwrap each DRG neuron leads to a release of TNF-\u03b1, which in turn increases the excitability of DRG neurons [36]. P2X7 receptors are upregulated in surrounding satellite glial cells in the DRG in humans [12]. Implication of TNF-\u03b1 has been reported in neuropathic pain [28\u201332]. Therefore, the contribution of P2X7 receptor to neuropathic pain might be related not only to the activation of microglial P2X7 receptors, but also to P2X7 receptors on other cell types such as satellite glia.\nP2Y6 and P2Y12 in neuropathic pain?\nWhen neurons are injured or dead, microglia are activated, resulting in their interaction with immune cells, active migration to the site of injury, release of proinflammatory substances, and the phagocytosis of damaged cells or debris. For such activation of microglial motilities, extracellular nucleotides have a central role. Extracellular ATP functions as a chemoattractant. Microglial chemotaxis by ATP via P2Y12 receptors was originally found by Honda et al. [8] and has recently been confirmed in vivo in P2Y12 receptor knockout animals [37]. Neuronal injury results in the release or leakage of ATP that appears to be a \u201cfind-me\u201d signal from damaged neurons to microglia to cause chemotaxis. In addition to microglial migration by ATP, another nucleotide, UDP, an endogenous agonist of the P2Y6 receptor, greatly activates the motility of microglia and orders microglia to eat damaged neurons. UDP does not cause chemotaxis, but instead causes phagocytosis by microglia [6]. Phagocytosis, a specialized form of endocytosis, is the uptake by the cell of relatively large particles (>1.0\u00a0\u03bcm) into vacuoles and has a central role in tissue remodeling, inflammation, and the defense against infectious agents [38]. Phagocytosis is initiated by the activation of various cell surface phagocytosis receptors, including Fc receptors, complement receptors, integrins, endotoxin receptors (CD18, CD14), mannose receptors, and scavenger receptors [39], which are activated by corresponding extracellular ligands. In the CNS, a full innate immune system, i.e., Fc receptors, complement system, scavenger receptors, and Toll-like receptors etc., has been described, and microglia reveal related roles as dedicated phagocytes. Since recognition is the first and the most important step for phagocytosis, extensive studies on phagocytosis receptors have been reported. It is well-known that dying cells express so-called eat-me signals such as phosphatidylserine on their surface membrane [39], by which microglia recognize the apoptotic cells to catch and remove them [39]. We first found that exogenously applied UDP caused microglial phagocytosis in a concentration-dependent manner, which was P2Y6 receptor-dependent. We found that neuronal injury caused by kainic acid (KA) upregulated P2Y6 receptors in microglia, the KA-evoked neuronal injury resulted in an increase in extracellular UTP, which was immediately metabolized into UDP in vivo and in vitro. Moreover, UDP leaked from injured neurons caused P2Y6 receptor-dependent phagocytosis in vivo and in vitro. These results suggest that UDP could be a molecule that signals the crisis of damaged neurons to microglia, triggering phagocytosis. It should be noted that nucleotides could be both \u201cfind-me\u201d and \u201ceat-me\u201d signals. Cells release ATP, and we also found that KA caused an increase in extracellular UTP\/UDP. Therefore microglia might be attracted by ATP\/ADP [8, 40, 41] and subsequently recognize UDP, leading to the removal of the dying cells and their debris.\nAre P2Y6 and P2Y12 receptors involved in neuropathic pain? For this question, there is a report indicating that the UDP-sensitive P2Y6 receptor produces inhibitory effects on spinal pain transmission in a neuropathic pain model [42]. In the neuropathic pain model, in which the sciatic nerves of rats were partially ligated, UDP (30 and 100\u00a0nmol\/rat) produced significant antiallodynic effects. UDP (100 nmol\/rat) caused no motor deficit in the inclined plane test [42]. The mechanism of this UDP-evoked inhibitory effect on neuropathic pain is unknown. There is no evidence to show the site (neuron or glia) of action of UDP. We are now investigating the involvement of P2Y12 in pain signaling and have obtained evidence suggesting the role of P2Y12 in the neuropathic pain state.\nConclusion\nNeuropathic pain is a major factor causing impaired quality of life in millions of people worldwide. We have to try to reveal the mechanism of this pain in order to develop effective drugs against the pain. Recently, there has been a rapidly growing body of evidence indicating that spinal glia, especially microglia, play a critical role in the pathogenesis of neuropathic pain and that nucleotides play an important role in neuron-glia communication through P2 purinoceptors. We described the modification of neuropathic pain signaling through microglial P2X4 and P2X7, with the possibility of P2Y6 and P2Y12 involvement (Fig.\u00a01). It was shown in an animal model of neuropathic pain that microglial P2X4 and P2X7 receptors are crucial in pain signaling after peripheral nerve lesion. Microglial P2Y6 and P2Y12 play very interesting roles for phagocytosis and chemotaxis, respectively. Besides, there is a possibility that these receptors are involved in pain sensation. Additional experiments are needed to clarify the possibility.","keyphrases":["neuropathic pain","atp","microglia","p2x4","spinal cord","allodynia"],"prmu":["P","P","P","P","P","P"]}
{"id":"J_Mol_Evol-3-1-1915622","title":"Positively Selected Codons in Immune-Exposed Loops of the Vaccine Candidate OMP-P1 of Haemophilus influenzae\n","text":"The high levels of variation in surface epitopes can be considered as an evolutionary hallmark of immune selection. New computational tools enable analysis of this variation by identifying codons that exhibit high rates of amino acid changes relative to the synonymous substitution rate. In the outer membrane protein P1 of Haemophilus influenzae, a vaccine candidate for nontypeable strains, we identified four codons with this attribute in domains that did not correspond to known or assumed B- and T-cell epitopes of OMP-P1. These codons flank hypervariable domains and do not appear to be false positives as judged from parsimony and maximum likelihood analyses. Some closely spaced positively selected codons have been previously considered part of a transmembrane domain, which would render this region unsuited for inclusion in a vaccine. Secondary structure analysis, three-dimensional structural database searches, and homology modeling using FadL of E. coli as a structural homologue, however, revealed that all positively selected codons are located in or near extracellular looping domains. The spacing and level of diversity of these positively selected and exposed codons in OMP-P1 suggest that vaccine targets based on these and conserved flanking residues may provide broad coverage in H. influenzae.\nIntroduction\nVaccination with Haemophilus influenzae type b polysaccharide (in the form of Hib conjugate vaccines) has proven to be highly effective in preventing invasive H. influenzae disease and has effectively reduced serotype b in many parts of the world (Black et al. 1992). Nontypeable H. influenzae strains, however, remain a serious problem (Zeckel et al. 1992). These strains are generally less invasive than their encapsulated type b counterpart but are a major cause of ear infections (otitis media) and sinusitis in children. They are also associated with respiratory tract infections such as pneumonia in infants, children, and adults. Ongoing efforts to develop a vaccine against nontypeable H. influenzae have mainly focused on immunogenic surface-exposed proteins (Bolduc et al. 2000). This approach clearly has potential, as immune responses against surface proteins have been shown to aid the recovery from otitis media (Shurin et al. 1980) and vaccination with surface-exposed domains of a major outer membrane protein (OMP-P1) provides protection in animal models (Bolduc et al. 2000). However, this effect is strain-specific (Gonzales et al. 1987) and, hence, not sufficient to provide broad protection. This problem may be overcome by the inclusion of less variable immunogenic protein regions in the vaccine.\nIdentification of candidate vaccine antigens is classically achieved via immunization studies with isolated or recombinant antigen and via epitope mapping, often in combination with analysis of correlates of protection. Vaccine development is often hampered by antigenic diversity within the surface-exposed regions. This limits a broad cross-reactivity of the elicited immune response. This holds also for proteins such as OMP-P1 in which large conserved domains separate relatively small variable domains (Munson and Grass 1988; Chong et al. 1995; Bolduc et al. 2000). On the other hand, the genetic diversity of vaccine candidates can be used to choose OMP-P1 variants for use in immunological assays (Bolduc et al. 2000). Nowadays, instead of an arbitrary selection of vaccine candidates, novel and rigorous computational approaches can be used to identify codons whose diversity is driven by the immune system. These codons are characterized by a higher rate of nonsynonymous substitutions (dN) relative to the synonymous substitution rate (dS) (Yang et al. 2003; Fitzpatrick and McInerney 2005). The codons are referred to as positively selected, although they may typically reflect immune selection (Fitzpatrick and McInerney 2005). To avoid confusion with the results of immunological studies, we refer here to codons with a higher rate of nonsynonymous substitution than synonymous substitution (dN > dS) as positively selected.\nKnowledge of the position of positively selected codons in a protein would constitute an excellent starting point for immunization studies and epitope mapping, not only because of their biological function, but also because the number of variable sites of proteins that are candidates for inclusion in a vaccine can be reduced. In the present study, we identified codons that evolved more rapidly through nonsynonymous than through synonymous substitutions in a sample of 36 OMP-P1 sequences. We compared the location of these codons with the location of peptides that were used in epitope mapping and with B- and T-cell OMP-P1-specific antigens to examine the congruence among these techniques and to identify regions that could be important for vaccine design. Finally, we localized stretches with positively selected codons in secondary structures and three-dimensional (3D) models of OMP-P1. Our computational approach led to the identification of several novel domains with positive selected codons within the OMP-P1 protein that may be attractive targets in future vaccine design.\nMaterials and Methods\nEvolutionary Analysis of the Selection Pressure on OMP-P1\nWe used the nr database for BLASTP searches under standard settings to collect closely related OMP-P1 sequences of H. influenzae using accession 9716616 as a query. The GenBank accession numbers and the data set of OMP-P1 sequences are provided as Supplementary Information. We aligned the sequences using the standard alignment parameters using Clustalx (Thompson et al. 1997) and checked the amino acid alignment with DNASP version 4.0 (Rozas et al. 2003). Codons comprised in insertions and deletions and in extremely variable stretches were removed from the alignment and are referred to in this work as hypervariable domains. A phylogenetic tree was reconstructed using PAUP* (Swofford 2003) with the maximum likelihood algorithm (100 random additions of taxa, TBR branch swapping) under the optimal nucleotide substitution model based on modeltest version 3.06 (Posada and Crandall 1998). Bootstrapping based on maximum likelihood was used to assess support for internodes using 100 random additions, SPR branch swapping, steepest descent, chuckscore = 0.1, and nchuck = 1. We used the alignment and tree topology of one of the maximum likelihood (ML) trees as input for PAML (Yang 1997). This program implements the currently most widely used algorithms to detect selection on codons, which take into account the transition\u2013transversion ratio and codon usage bias and allow identification of codons that accumulate nonsynonymous mutations more rapidly than synonymous ones. To detect such sites, the rates of nonsynonymous and synonymous substitutions are determined through ML analyses using the model of Goldman and Yang (1994). For immune-exposed proteins of pathogens, a dN\/dS ratio >1 indicates that nonsynonymous substitutions are driven by selection pressures exerted by the immune system (Derrick et al. 1999; Jiggins et al. 2002; Fitzpatrick and McInerney 2005). Because it is most likely that only a subset of codons in any protein-coding gene evolves under positive selection, several distributions of dN\/dS that reflect neutral (M1, M7) and selection (M2, M8) models can be fitted to the data using PAML (Yang 1997). The neutral models assume a single estimate of \u03c9 (0<\u03c9<1; M1) and a discrete beta distribution of \u03c9 with 10 categories (0<\u03c9<1; M7). The dN\/dS ratio under the neutral models is constrained to values \u22641 (Yang et al. 2003; Yang and Swanson 2002). The selection models M2 and M8 have an additional class of \u03c9 values that exceed unity. This latter codon category is believed to comprise codons that code for amino acids that are affected by the immune system (Yang et al. 2000) and that, consequently, evolve rapidly at the amino acid level. The cross-category \u03c9\u2019s under M7 and M8 were estimated by averaging the \u03c9 estimates of individual \u03c9 classes weighted by their frequency. We used multiple starting values for the selection models M2 and M8, because these models may have multiple local optima (e.g., Wong et al. 2004). The fit of M1 and M7 to the data was compared with selection models (M2 and M8, respectively). The ratio of the fit of neutral and selection models mentioned above follows a chi-square distribution with two degrees of freedom, i.e., for significance at the 1% significance level, two times the difference of the likelihood of neutral and selection models should be >9.21. We examined the evidence for positive selection in the entire alignment (excluding codons with ambiguity codes and gaps; see above), in a data set comprising 30 nontypeable isolates and in a data set of 36 sequences from which five variable domains were deleted. The latter data set enables the detection of positively selected codons in highly conserved domains. The selection models may give false-positive results in the case of a poor fit of the beta distribution to the true distribution of \u03c9 in the interval between 0 and 1 (Swanson et al. 2003). The test advocated by Swanson et al. (2003) to guard against such false positives of the ML methods implemented in PAML involves the comparison of M8 (\u03c92 > 1) and M8A (\u03c92 = 1). This likelihood ratio test (LRT) is asymptotically distributed as a 50:50 mixture of a point mass at 0 and a chi-square with one degree of freedom, which corresponds to 2.71 units difference between the likelihoods of M8A and M8 at the 1% level. If the fit of the selection models is significantly better than that of the corresponding neutral models, the selection models can be used to identify individual codons under positive selection (Yang et al. 2005). For inferring which sites are under positive selection, the Bayes theorem was used (Yang et al. 2005), which assumes that given the proportion of sites across \u03c9 classes as estimated from the ML analyses, assigns priors to model parameters to integrate over their uncertainties. The Bayes Empirical Bayes (BEB) approach used here was specifically advocated for small data sets, because it takes into account sampling errors of the proportions and \u03c9 ratios of site classes (Yang et al. 2005).\nThe rigorous identification of codons under positive selection is an important step when linking evolutionary and structural data, and when testing individual codons, p-values should ideally be confirmed by independent tests and corrected for multiple tests. To these ends, we applied the conservative tests of positive selection based on parsimony (Suzuki and Gojobori 1999) implemented as the Single Likelihood Ancestor Counting (SLAC) analysis in the datamonkey web server (Kosakovsky Pond and Frost 2005). In contrast to the original version of the Suzuki-Gojobori test, the modified version allows selection of a nucleotide substitution model. We also used a new ML method developed by Massingham and Goldman (2005), which does not assume an underlying distribution of \u03c9. Instead, their sitewise likelihood ratio (SLR) procedure, which uses the same codon substitution model as in Goldman and Yang (1994), performs a LRT on a sitewise basis, testing the null model (neutrality, \u03c9 = 1) against an alternative model (\u03c9 \u2260 1). In the SLR method, tree topology, branch lengths, equilibrium codon frequencies, and transition\/transversion rate ratio are assumed to be common to all sites in an alignment. Under the null model, all parameters except \u03c9 are allowed to vary freely. Under the alternative model, all parameters vary freely. The SLR method applies Hochberg\u2019s step-up procedure to correct for multiple tests (Massingham and Goldman 2005). Both the parsimony method and the SLR method also allow the identification of negatively selected sites.\nSecondary Structure of OMP-P1\nFollowing the localization of positively selected codons in the primary sequence, we determined the higher-order structure of amino acid stretches in OMP-P1 using TMHMM version 2.0 (Krogh et al. 2001) to determine whether stretches with positively selected codons take secondary structures typical of transmembrane domains. The exposition of regions of OMP-P1 was assessed by means of the solvent accessibility algorithm as implemented in Jpred (Cuff et al. 1998). We identified exposed residues using the secondary structure algorithm implemented in SSPRO 4.5 (Cheng et al. 2005) and Jnet5 (Cuff and Barton 1999). These attributes may suggest whether regions with positively selected codons are exposed to the immune system.\nTertiary Structure of OMP-P1\nWe examined the 3D structure of OMP-P1 using x-ray crystallography or NMR data on structurally related proteins. We used the precompiled Vector Alignment Search Tool (VAST) and NCBI\u2019s structural database to identify closely related structural variants that could serve as a template for homology modeling of OMP-P1. The entries in VAST contain experimentally verified information on secondary structure elements such as type, relative orientation, and connectivity. Structurally related proteins can be fitted to each other on a residue-by-residue basis using VAST\u2019s transformation matrices and a Gibbs sampling algorithm. In addition, alternative alignments can be generated which take into account the information in secondary structure, thereby increasing the confidence one can have in amino acid alignments of relatively divergent proteins. The VAST program was used for one randomly chosen H. influenzae OMP-P1 sequence (GenBank accession no. 9716616 of the nontypeable strain 1512A). Superimposed models of template and target protein were constructed to illustrate the position of gaps and insertions, hypervariable domains, and positively selected codons.\nAs an alternative to the identification of structural neighbors as implemented in VAST, homology modeling can be used. This always starts by looking for close relatives using standard PSI-BLAST or other similarity searches. We used homology modeling as implemented in GENO3D (Combet et al. 2002) and SWISS-MODEL (Peitsch 1996; Schwede et al. 2003) to assess the congruence with the VAST searches and to collect additional data on liability of portions of the 3D models of OMP-P1 and its structurally closest neighbors. The 3D models of OMP-P1 were visualized using Cn3d version 4.1, a free 3D model viewing and selection program. To judge the quality of the 3D reconstruction, we collected the percentage identity between template and target, the VAST P-value, the score reported by this program, and the squared root of mean square deviations (RMSD) of the backbone atoms between the template and the target proteins in angstroms (\u00c5) using a sliding window approach. To assess whether hypervariable domains were robustly assigned to their exact coordinates in the 3D structures, we also examined the reports of loop reconstructions by the CSP (Constrained Space Programming) routine as implemented in Swiss-Model (Schwede et al. 2003).\nTo further quantify the degree of similarity in \u03b2-strands between OMP-P1 and a structural template, we compared the distribution of amino acids maintaining \u03b2-barrels of transmembrane proteins using the TBB-PRED program (Liu et al. 2003), which uses atomic data to assign the most likely amino acid stretches that contribute to \u03b2-barrels. We used the Support Vector Machines (SVM) algorithm to determine the location of \u03b2-barrels.\nResults\nAlignment of OMP-P1 Sequences\nBLASTP searches used cutoffs of 0 (E-value) and 727 (score) to identify closely related OMP-P1 sequences. Thirty-six hits were obtained, including 33 protein-coding sequences (27 nontypeable, 1 a, 3 b, 1 e, and 1 f) that have previously been analyzed as part of a population genetics study of OMP-P1 (Bolduc et al. 2000; six sequences were left out because they were not among the top hits of the BLASTP search). The average pairwise amino acid similarity in the entire data set was 94%. The sequences of the nontypeable and typeable OMP-P1 protein variants comprised three hypervariable portions with many insertions and deletions (nucleotides 262\u2013282, 610\u2013621, plus 625\u2013633 and 1288\u20131302 plus 1309\u20131317) and a few codons with ambiguity codes or gaps (646\u2013651, 730\u2013732, 850\u2013852, 877\u2013879, and 1360\u20131362). In addition, 22 codons missed from the 5\u2019 end of the OMP-P1 gene of isolate 88591, leading to a total of 50 codons that were deleted to ensure a correct alignment. The amino acid and the nucleotide alignments of the five most variable domains that remained after removal of the codons mentioned above can be viewed as Supplementary Information. The nucleotide alignment revealed that many of the underlying codons in the variable domain frequently shared nucleotides at two of three positions, indicating that the alignment of these regions was correct.\nIdentification of Positively Selected Codons\nThe computational approaches implemented in PAML version 3.14 (Yang 1997) determine which, if any, codons may have evolved under positive selection. On the basis of a single phylogenetic estimate using the preferred nucleotide substitution model derived from MODELTEST (the transversion model TVM, which has variable base frequencies, variable transversion rates, and equal transition rates) with a proportion of invariant sites (0.66) and gamma distributed substitution rates (shape parameter, 0.51), we found 14 ML trees, one of which is shown in Fig.\u00a01 (ML \u20135104.27). Bootstrapping indicated that four branches received 100% support (Fig.\u00a01). Support for the other branches was <95% (not shown in Fig.\u00a01). Comparison of neutral and selection models using the OMP-P1 data set and the tree in Fig.\u00a01 indicated that both selection models M2 (likelihood, \u20135169.38) and M8 (\u20135177.30) fitted the data significantly better than the neutral models M1 (\u20135260.86) and M7 (\u20135270.13), respectively (Table\u00a01). Different starting values of \u03c9 did not lead to different parameter estimates after optimization (not shown). Both comparisons M1\u2013M2 and M7\u2013M8 exceeded by far the difference required for significance at the 1% level (see Materials and Methods). This suggested that some codons had a higher nonsynonymous than synonymous substitution rate. Similarly, the Swanson test indicated significant evidence for the occurrence of positive selection under M8 (Table\u00a01). Both selection models M2 and M8 suggested that the same codons were under positive selection, i.e., codons 93, 94, 97, 99, 105, 198, 222, 284, and 329, and possibly also codon 96 (under model M8). The OMP-P1 alignment (Supplementary Information) further showed that in this sample of OMP-P1 sequences, positively selected codons mostly flank hypervariable domains. Limitation of the analyses to the 30 nontypeable strains resulted in very similar findings. In these analyses, both M2 (likelihood, \u20134802.93) and M8 (\u20134808.87) fitted the data significantly better than M1 (\u20134883.86) and M7 (\u20134895.19), respectively (not shown). Thus, both model comparisons were again significant. This held also true for the positively selected codons. For M2, relative to the analyses in Table\u00a01, one additional positively selected codon (225) was found. For M8, no additional codons were found. After deletion of the five variable domains, no evidence for positive selection was left (M1, \u20133107.88; M2, \u20133107.77; M7, \u20133108.18; M8, \u20133106.35).\nFig.\u00a01.Maximum likelihood tree based on the TVM model of nucleotide substitution with a proportion of invariant sites and rate heterogeneity (see text). The error bar indicates the number of substitutions per nucleotide. Asterisks indicate branches with 100% bootstrap support.Table\u00a01.Positively selected codons in OMP P1 Heamophilus influenza according to neutral models (M1 and M7) and selection models (M2 and M8)LikelihoodTree lengthKappa \u03baParametersCodonAmino acidProbability positive selection (BEB)dN\/dS codon \u00b1 SEM1\u22125260.861.911.90\u03c90 = 0.02, \u03c91 = 1, p0 = 0.84, p1 = 0.16\u2014 \u2014M2\u22125169.382.172.13\u03c90 = 0.02, \u03c91 = 1, \u03c92 = 6.42, p0 = 0.83, p1 = 0.14, p2 = 0.0393A1.006.523 \u00b1 \u22120.83394S1.006.523 \u00b1 0.83396K0.573.979 \u00b1 2.63297I1.006.523 \u00b1 0.83399R1.006.523 \u00b1 0.834105Q1.006.523 \u00b1 0.833198A1.006.522 \u00b1 0.835222K1.006.523 \u00b1 0.834225T0.795.293 \u00b1 2.349284K1.006.519 \u00b1 0.844329H1.006.493 \u00b1 0.919M7\u22125270.132.071.91B(p = 2.14, q = 0.04)\u2014\u2014M8A\u22125260.961.921.91B(p = 2.14, q = 99), p0 = 0.84, p1 = 0.16, \u03c92 = 1M8\u22125177.302.172.12B(p = 0.07, q = 0.38), p0 = 0.97, p1 = 0.38, \u03c92 = 5.7993A1.005.445 \u00b1 0.58594S1.005.445 \u00b1 0.58595V0.844.661 \u00b1 1.72696K0.955.214 \u00b1 1.09997I1.005.445 \u00b1 0.58599R0.744.191 \u00b12.015100N1.005.445 \u00b1 0.585105Q1.005.445 \u00b1 0.585151I0.904.929 \u00b1 1.480198A1.005.445 \u00b1 0.585222K1.005.445 \u00b1 0.585225T0.965.238 \u00b11.098284K1.005.445 \u00b1 0.586329H1.005.442 \u00b1 0.596371Y0.764.279 \u00b1 2.030The likelihood indicates the fit of the data to the models of Yang (1997). Tree length is measured as the number of mutations per codon. Kappa is the transversion \u2013 transition ratio. pi denotes the proportion of sites falling in site class \u03c9i. The parameters p and q are the shape parameters of the beta distribution which underlies M8. The probability that codons were under positive selection was determined using Bayes Empirical Bayes (Yang et al. 2005), with the proportion, \u03c9, and standard error indicated per codon. Model 8A refers to the model of Swanson et al. (2003), in which \u03c92 is fixed at one under M8. The reference sequence is 9716616.\nThe modified Suzuki-Gojobori method indicated that four sites evolved under positive selection (substitution model 012010; codons 93 [P= 0.01], 94 [P= 0.01], 97 [P= 0.03], and 105 [P= 0.03]; likelihood, \u20135548.10; tree length per site, 0.59, dN\/dS over all codons = 0.32; middle row in Fig.\u00a02). Using the SLR method, we found eight positively selected codons. Relative to the results of PAML (top row in Fig.\u00a02), all but one positively selected codon found by the SLR method remained significant upon correction for multiple tests (except for codon 329, P= 0.14; bottom row in Fig.\u00a02 and Table\u00a01). Overall, the strongest evidence for positive selection based on the parsimony and ML methods was found for residues 93, 94, 97, and 105.\nFig.\u00a02.Codons under positive selection as inferred from the site models in PAML (top panel), the modified Suzuki-Gojobori method implemented as the SLAC procedure in the datamonkey server (middle panel), and the sitewise likelihood ratio test (bottom panel) of 36 OMP-P1 sequences of H. influenzae. Codons with significant evidence for positive selection have probabilities >0.95 (PAML and SLR methods) or <0.05 (modified Suzuki-Gojobori method). The spacing between codons is adjusted for the insertions and deletions in OMP-P1.\nTopographic Localization of the Amino Acids Encoded by Putatively Positively Selected Codons and Their Relation to Immunological Data of OMP-P1\nA priori, positively selected codons in B-cell and T-helper determinants in the OMP-P1 protein are suitable vaccination targets, because these sites can be expected to be highly immunogenic and involved in the clearance of OMP-P1 variants. The most promising broadly protective regions of OMP-P1 based on epitope mapping studies are mainly located outside the stretch of closely spaced positively selected codons (Fig.\u00a03). Peptide-specific antisera directed against these regions did not invoke protective immunity, although strong IgG and T-cell responses were obtained (Proulx et al. 1991; Chong et al. 1995). Only in rare cases was targeting of the conserved regions associated with protection (one epitope located on peptide HIBP1-4 and recognized by MAb 7C8 was protective [Panezutti et al. 1993]). Furthermore, two of five positively selected sites are present in a synthetic P1 peptide (HIBP1-12) that has been used in immunization studies (Proulx et al. 1992; Panezutti et al. 1993; Chong et al. 1995). The peptide, which was not identified as a conserved B-cell epitope across typeable and nontypeable H. influenzae isolates, did not carry an immunodominant epitope in animal models. Another peptide that was used for epitope mapping and immunological assays (Proulx et al. 1992; Chong et al. 1995) fully overlaps with the stretch with positively selected amino acids. This HIBP1-2 (Chong et al. 1995) or 2H (Proulx et al. 1992) peptide covers amino acids 60\u201388 of 1H in H. influenzae serotype b strain MinnA (Munson and Grass 1988), which correspond to residues 82\u2013108 in the reference sequence 9716616. However, anti-P1 antisera isolated from rabbits, guinea pigs, and humans did not react with HIBP1-2 (Chong et al. 1995), and vice versa, anti-HIBP1-2 specific sera did not recognize the intact P1 protein. Despite these negative immunological data, our computational analysis suggests that the stretches with positively selected codons are likely under immune selection in vivo. It may be worthwhile to study these stretches in more detail at the immunological level.\nFig.\u00a03.Diagram of different immunological and genetic attributes of OMP-P1 of Haemophilus influenzae. From top to bottom: linear B-cell epitopes in different hosts (murine, guinea pig, and rabbit), B-cell epitopes conserved among H. influenzae strains, murine T-helper determinants, positively selected codons, distribution of amino acids with a potentially extended (black) or a helical configuration (gray; Jpred) typical of \u03b1-helices and \u03b2-sheets, respectively, and distribution of amino acids with <5% solvent accessibility (Jnet5), location of hypervariable domains, and location of 13 synthetic peptides against OMP-P1, which were used for immunological studies (Proulx et al. 1992; Chong et al. 1995). The approximate locations of peptides HIBP1-2 and HIBP1-12 are boxed and dotted, respectively (see text).\nFive of the nine codons for which significant evidence for positive selection was obtained in both PAML selection models M2 and M8 cluster tightly in the primary sequence of OMP-P1 (amino acids 93\u2013105) and four of these are confirmed as positively selected sites using the other types of analyses of positive selection (Fig.\u00a02). Chong et al. (1995) found that some of these codons were part of a transmembrane domain based on secondary structure analysis, hydrophobicity, and reactivity with monoclonal antibodies. The nature of the variability of the first hypervariable domain has also been questioned on the basis of a lack of hydrophilic residues (Munson et al. 1992). TMHMM version 2.0 was used to determine whether stretches with positively selected codons involved a transmembrane domain or adopted secondary structures typical of such domains (Krogh et al. 2001). No secondary structures such as \u03b1-helices and \u03b2-sheets typically associated with transmembrane domains were found in this stretch (not shown). Investigation of the exposition of this region using the solvent accessibility algorithm as implemented in Jpred (Cuff et al. 1998) classified this portion of OMP-P1 as an exposed stretch with high solvent accessibility (Fig.\u00a03), which exceeded 25% for the entire stretch with the five positively selected codons. Thus, the positively selected codons are likely to be exposed. The above findings deviate from previous suggestions that these amino acids are part of a putative transmembrane domain (Chong et al. 1995), which would render this stretch unsuitable for vaccine purposes.\nLocalization of the Amino Acids Encoded by Putative Positively Selected Codons as Assessed by 3D Modeling\nThe apparent ambiguity about the putative transmembrane domain of OMP-P1 led us to examine the 3D topology of the protein. BLASTP searches identified one 3D structure with high similarity to OMP-P1 of H. influenzae; the FadL outer membrane protein of E. coli (39% similar in primary amino acid sequence). This protein has been analyzed at a resolution of 2.60 and 2.80 \u00c5 using two x-ray crystallography methods (van den Berg et al. 2004). Apart from its role in the transport of long-chain fatty acids across the outer membrane, FadL is also a receptor for the bacteriophage T2. OMP-P1 and FadL are both classified in PFAM03349.10, which also includes TodX from Pseudomonas putida F1 and TbuX from Ralstonia pickettii PKO1. The latter are membrane proteins of uncertain function that are involved in toluene catabolism. The VAST database identified the same structural neighbors (1T1A-B, 1T16A-B) as the BLASTP search and the homology modeling programs GENO3D and SWISS-MODEL (not shown).\nThere are many indications that the reconstructed model of OMP-P1 (Fig.\u00a04) is reliable. The percentage identity of the OMP-P1 sequences to the target based on secondary elements was approximately 43%, which, in principle, may allow confident 3D models as evidenced by the lower boundary for which homology modeling is used (25%; http:\/\/www.expasy.org\/swissmod\/SWISS-MODEL.html). Although VAST provides no easily interpretable confidence parameters (the score of this structure pair was 48.5 using an alignment length of 391 residues and the VAST P-value was 10\u221243), the level of similarity and the root-mean-square deviation (RSMD) of the fit across OMP-P1 and 1T16A indicate a high degree of structural similarity (RMSD < 3 \u00c5; Fig.\u00a05) (T. Madej, NCBI, personal communication). A large number of secondary structures such as helices, strands, and loops are shared between OMP-P1 and FadL as evident from VAST searches (Figs.\u00a04A and B). This interpretation is supported by the congruence among models of secondary structure to assign coiled regions that are exposed as determined through the use of SSPRO version 4.5 (not shown) and by the agreement of regions with a \u03b2-barrel structure between FadL and OMP-P1 as judged from the results of the TBB-PRED server based on atomic data of \u03b2-barrel proteins (Fig.\u00a05). Clearly, the fit of the overall structure of the reconstructed 3D model of OMP-P1 does not imply that the model is stable and robust for every region of the protein. The hypervariable domains introduce many differences in exposed portions of the 3D models between model and target in the superimposed models (Fig.\u00a04, bottom row). Further, it is important to note that only a single OMP-P1 sequence was used to model the 3D structure based on FadL. Slightly different loops may be obtained when using different OMP-P1 sequences. Not surprisingly, the CSP algorithm implemented in SWISS-MODEL had considerable difficulties in finding and optimally position loops (not shown). Many of the loops examined by the program could not be confidently reconstructed, and in many cases, the accepted loops (by the Spare Part procedure) were found only after shorter ones had not been accepted. These regions in many instances corresponded to hypervariable and looping regions (Fig.\u00a04A) that had much higher RMSD values than other regions of the OMP-P1 molecule. However, as judged from the secondary structure analyses these regions likely do not contribute much to the overall stability of the \u03b2-barrel structure of OMP-P1 (Fig.\u00a05).\nFig.\u00a04.Three-dimensional model of OMP-P1 of H. influenzae (A), FadL of E. coli (B), and superimposed models of both proteins (C). Brown arrows and threads mark strands, yellow threads mark regions with positively selected codons, green regions mark helical regions, and blue regions mark coiled regions. E, extracellular side; M, membrane; P, periplasm. In the superimposed models, a splitting of the two models indicates gaps or insertions.Fig.\u00a05.Alignment and structural analysis of OMP P1 9716616 of H. influenzae and FadL of E. coli based on the VAST 3D structural database. The deviation (\u00c5) of the 3D model of OMP P1 relative to the template FadL is shown for windows of five amino acids (above alignment). The contribution of individual residues to the \u03b2-barrel structure of OMP P1 as analyzed with TBB-PRED is marked by \u201cb\u201d (barrel) and \u201cn\u201d (nonbarrel). Asterisks above the alignment mark conserved amino acid positions; asterisks below the alignment mark positively selected amino acid positions. The alignment of OMP P1 9716616 starts at residue 23.\nThe most relevant conclusion of the 3D reconstruction was that the regions that comprised positively selected codons (yellow in Fig.\u00a04) were always on the extracellular side of the membrane, indicating that the positively selected codons are potential candidates for vaccine development. The positions of insertions and deletions between FadL and OMP-P1 that are longer than a few amino acids occurred\u2014in agreement with expectation\u2014mostly in these exposed regions of OMP-P1. Looping regions with insertions and deletions are indicated by the splitting of the two molecules in superimposed 3D models (Fig.\u00a04C). Overall, the superimposed 3D models agree with the models based on individual 3D structures in that, as expected for a \u03b2-barrel protein, particularly the main chain hydrogen-bonding patterns characteristic of strands contribute to the structural maintenance of OMP-P1.\nDiscussion\nPositive Selection on OMP-P1\nIdentification of positive selection on OMPs is generally hampered by extremely high levels of sequence divergence and the frequent occurrence of insertions and deletions in hot spots. These domains are, however, potentially interesting for vaccine studies, as they may be the targets of immune selection (Smith et al. 1995). To enable identification of positively selected codons, we focused on codons directly adjacent to hypervariable and unalignable regions (cf. Supplementary Information). The hypervariable regions were excluded, as it is simply not possible to identify homologous nucleotide positions for these domains. Our strategy to detect positively selected codons is still feasible, as the inference of positive selection is not very sensitive to the use of slightly different trees resulting from, for example, the deletion of hypervariable codons, if sufficient phylogenetic signal remains (Derrick et al. 1999). Our results indeed indicate that after deletion of the hypervariable regions, a substantial number of substitutions remains for OMP-P1 (see Supplementary Information), which allows the identification of positively selected codons.\nAnother important issue to consider in this type of study is how recombination affects the inferences of positive selection. Recombination generates different tree topologies when based on different regions of a gene, and it has been shown that\u2014depending on the level of recombination\u2014the LRT may be affected (Anisimova et al. 2003). In general, recombination is considered most problematic for analysis of selection pressures using divergent sequence data (cf. viral sequence data [Anisimova and Yang 2004]), but it has generally been difficult to determine its quantitative importance. Simulation studies provide information on the circumstances in which recombination may affect evolutionary analysis of selection pressures. For example, in spite of the congruent results between M2 and M8 in this study, these models are not equally affected by recombination (Anisimova et al. 2003). In addition, the Bayesian analysis that was used to identify positively selected residues is less affected by the presence of recombination than the LRTs, presumably because it does not depend strongly on the entire gene tree topology but, instead, reconstructs the numbers of synonymous and nonsynonymous mutations of individual codons. Finally, the proportion of correctly classified sites for the BEB procedure in simulations under positive selection and recombination increases with selection pressure (Anisimova et al. 2003). The higher \u03c9 values of positively selected codons in this study (Table\u00a01) relative to those of the simulation study mentioned above (\u03c92 = 2.55) and the large number of positively selected sites in OMP-P1 suggest that the inference of positive selection and sites under positive selection is valid.\nUse and Value of 3D Reconstruction of OMP-P1\nThe alternation of relatively conserved and hypervariable stretches in OMP-P1 and other OMPs also impacts the 3D reconstruction. Hypervariable amino acid stretches may render a 3D structure unstable, despite the fact that more conserved parts may share considerable similarity between target and template. Thus, although the level of congruence between the experimental 3D structure of FadL and the computational 3D structure of OMP-P1 may vary considerably, the most probable and approximate location of stretches with positively selected codons can be determined if the conserved portions of OMP-P1 can be confidently reconstructed. With this goal in mind, the identification of \u03b2-barrel regions and the degree of fit of stranded regions between model and template are most important. These attributes leave little doubt that the fit between FadL and OMP-P1 is sufficient to pinpoint the approximate localization of positively selected codons (Fig.\u00a05). This is not surprising, as more divergent structural templates and targets such as OmpF and PhoE of E. coli have successfully been used for the reconstruction of 3D models of porins in Neisseria (Derrick et al. 1999). In sum, there is every reason to believe that the basic barrel structure is not profoundly affected by hypervariable loops, even if these experience frequent insertions and deletions that are potentially also under positive selection (Smith et al. 1995; Derrick et al. 1999).\nAre Positively Selected Sites Useful for Vaccine Formulations?\nThe question arises why the peptides that partially (HIBP1-12) and fully (HIBP1-2) overlapped with the stretch of positively selected sites did not include immunodominant epitopes (Proulx et al. 1992; Panezutti et al. 1993; Chong et al. 1995). The stretches, however, were very long (29 and 27 amino acids, respectively) and they extended well into the interior of OMP-P1 (not shown). The potentially most interesting peptide (HIBP1-2) comprises the entire hypervariable domain of the Eagan strain (Chong et al. 1995), including residue 105 (SASQRNV is the C-terminus; Supplementary Information). Our analyses suggest that relatively short peptides starting at the C-terminal end of the first hypervariable domain would be suited for immunological experiments. This portion of OMP-P1 has not yet been explored.\nInterestingly, one of the positively selected residues in OMP-P1 that flank the first hypervariable domain allows examination of the impact and prospect of positively selected residues for vaccine formulation in a considerable fraction of the H. influenzae population. The positively selected codons 93, 94, and 97 are flanked on one side by the first hypervariable region and are separated from codon 105 by a conserved amino acid stretch (see Supplementary Information). The latter residue, in turn, is flanked by a long region at the C-terminal side that is conserved in the collection of H. influenzae strains examined by Bolduc et al. (2000). In our study sample, only three amino acids are encoded by residue 105, and this number increases only marginally in the larger set of H. influenzae isolates (Bolduc et al. 2000). Consequently, judging from the spacing of positively selected and conserved residues, there are opportunities to combine sites under purifying and positive selection in immunological experiments of OMP-P1. If it can be demonstrated by, for example, epitope mapping that these positively selected and exposed residues of OMP-P1 are located in immunologically relevant domains, a large portion of the H. influenzae population can be targeted by short peptides from this region of OMP-P1. Relative to traditional approaches to vaccine development, this strategy may allow a much broader coverage of the H. influenzae population.\nAnother question is to what extent the isolates studied here reflect the evolutionary diversity of H. influenzae. The isolates analyzed by Bolduc et al. (2000) were selected for OMP-P1 sequencing, because they were phylogenetically diverse in a collection of more than 500 H. influenzae isolates. Given the increase in multilocus sequence typing (MLST) of human pathogenic bacteria, it is also of interest to compare the diversity of the strains of Bolduc et al. (2000) with the phylogenetic relationships based on housekeeping genes in the MLST database of H. influenzae. In the case of H. influenzae, seven housekeeping genes are widely used to characterize phylogenetic relationships. In a housekeeping tree of 131 isolates (Meats et al. 2003), most of the nontypeable isolates were comprised of two highly divergent clusters, and in contrast to the housekeeping genes of encapsulated isolates, the congruence among trees based on single housekeeping genes of nontypeable isolates was only marginal. The current view of H. influenzae evolution is that nontypeable isolates are genetically distinct from encapsulated variants, and that nontypeable isolates form distinct and diverse populations. Among the five strains that were used by both Bolduc et al. (2000) and Meats et al. (2003), the shared isolates (B-RM7109, d-Rd, a-7416, nt-667, and e-6181) were scattered over the MLST tree. Apart from issues of sampling, the congruence of gene tree topologies based on different housekeeping genes and OMP-P1 hinges on the level of recombination between genes. It is currently unknown how important recombination is in H. influenzae. Although information on the clinical symptoms caused by the isolates with OMP-P1 sequences is extremely limited, strains 667 (9716582), BCH-1 (9716566), BCH-2 (9716610), and BCH-3 (9716568) caused middle ear infection, pneumonia, and nasopharyngeal infection in children. In spite of the limited amount of information on the nontypeable isolates with OMP-P1 sequences, the intermingling of nontypeable and typeable isolates on the housekeeping tree and the clinical symptoms of some of the nontypeable isolates suggest that, in principle, the OMP-P1 diversity studied here might be a suited starting point for vaccine development with the potential of providing broad coverage.\nRecent technological progress in vaccinology offers hope that epitopes based on moderately rapidly evolving portions of OMPs may turn out to be realistic vaccine material. Although it cannot be excluded that some epitopes will not be stable or immunogenic, or may not constitute stable constructs, extracellular loops in OMPs have been demonstrated to be immunodominant (Easton et al. 2005). Also, the inclusion of multiple OMPs in vaccine formulations is possible by using outer membrane vesicles or whole-cell vaccines based on recombinant bacteria (de Jonge et al. 2004) and the delivery of antigens based on OMPs of nontypeable H. influenzae based on fusion proteins (Riedmann et al. 2003) offer opportunities to target interesting vaccine candidate regions of OMPs. Finally, whole-cell vaccines of LPS mutants with strongly reduced toxicity are increasingly suited for vaccination purposes (Fisseha et al. 2005), thereby reducing the adverse effects of immune responses against these types of vaccines.\nImpact of Computational Analyses on Vaccine Development\nPositive selection on codons of exposed outer membrane proteins of pathogenic viruses and bacteria is common (Urwin et al. 2002; Yang et al. Yang 2003; Fitzpatrick and McInerney 2005). The lack of correspondence between interesting sites in OMP-P1 of H. influenzae in immunological and genetic diversity studies highlights the difficulty of finding the most promising epitopes, even among closely related sequences. The a priori identification of positively selected codons will normally greatly reduce the number of peptides that need to be screened using epitope mapping or immunization. Because the incorporation of antigenic diversity in the experimental setup of immunological and protection studies is a major bottleneck in terms of time and costs, the identification of a limited number of positively selected codons suggests that a multidisciplinary approach comprising evolutionary and structural data and tools can greatly improve the focus of ensuing empirical studies.\nElectronic Supplementary Material\nSupplementary material","keyphrases":["vaccine","haemophilus influenzae","epitope","immune selection","outer membrane proteini","structural information"],"prmu":["P","P","P","P","M","R"]}
{"id":"Ann_Surg_Oncol-4-1-2244700","title":"Perimembrane Aurora-A Expression is a Significant Prognostic Factor in Correlation with Proliferative Activity in Non-Small-Cell Lung Cancer (NSCLC)\n","text":"Purpose Aurora-A, also known as STK15\/BTAK, is a member of the protein serine\/threonine kinase family, and experimental studies have revealed that Aurora-A plays critical roles in cell mitosis and in carcinogenesis. However, no clinical studies on Aurora-A expression in non-small-cell lung cancer (NSCLC) have been reported. Thus, the present study was conducted to assess the clinical significance of Aurora-A status.\nPrimary lung cancer is the leading cause of cancer deaths in most industrialized countries, and non-small-cell lung cancer (NSCLC) accounts for 75\u201380% of primary lung cancer. Surgery is the most effective therapeutic modality for the cure, but the postoperative prognosis remains poor.1\u20133 To improve the prognosis, establishment of biological markers other than tumor-node-metastasis (TNM) factors that determine prognosis and response to a particular treatment is essential. Although many possible biological markers such as mutations of p53 and k-ras genes have been reported, none of them has been established as a marker in decision-making of the treatment of NSCLC. In addition, most NSCLC patients present with advanced stages and are appropriate candidates for surgery. For inoperable patients, chemotherapy with or without radiotherapy may be given. Recently, tyrosine kinase inhibitors targeting epidermal growth factor receptor (EGFR), gefitinib and erlotinib, have been introduced into the therapy of NSCLC, and experimental and clinical studies have revealed that these agents induce dramatic antitumor effects for tumors with activating mutations within the EGFR kinase domain.4,5 Thus, other molecular abnormalities in NSCLC should be revealed in the development of useful prognostic markers and in the development of effective molecular targeting agents.\nAurora-A, also known as BTAK, STK15, Aurora-2, ARKI, or AIKI, is a member of the serine\/threonine kinase family. As Aurora-A is involved in proper centrosome functions and chromosome segregation during normal cell mitosis,6\u20138 its abnormalities such as overexpression as well as gene amplification may contribute to development and progression of malignant tumors.9\u201311 In clinical studies, Aurora-A abnormalities have been reported in a variety of malignant tumor including glioma,12 medullobrastoma,13 gastric cancer,14 esophageal cancer,15,16 pancreatic cancer,17 ovarian cancer,18 breast cancer,19,20 bladder cancer,21 and hepatocellular carcinoma.22 In all these clinical studies, Aurora-A gene was amplified or Aurora-A expression was upregulated in tumor tissues compared with normal tissues, which also support experimental results suggesting that Aurora-A abnormalities are involved in carcinogenesis. In addition, many studies showed that Aurora-A abnormalities were positively correlated with aggressive tumor behavior such as poorly differentiated tumor grade, invasion, and nodal metastasis,15,16,19,20\u201322 but some studies showed no correlation12,17 or an inverse correlation.18 Some studies also assessed a prognostic significance of Aurora-A status; most studies showed that Aurora-A abnormalities were correlated with a significant poor prognosis,13,16,21,22 but one study failed to show.20 These results may suggest that clinical significant of Aurora-A status in malignant tumors remains controversial. Moreover, no clinical study on Aurora-A status in NSCLC has been reported. Thus, we conducted a clinical study on Aurora-A expression in resected NSCLC and assessed its clinical significance in correlation with other biomarkers including proliferative activity.\nPatients and Methods\nPatients\nWe retrospectively reviewed a total of 191 consecutive patients with pathologic (p)-stage I\u2013IIIA NSCLC who underwent complete tumor resection and nodal dissection without any preoperative therapy at the Department of Thoracic Surgery, Kyoto University, between January 1, 1985 and December 31, 1990. The p-stage was reevaluated and determined according to the current tumor-node-metastasis (TNM) classification as revised in 1997.1 Histological type was also redetermined according to the classification by the World Health Organization (WHO) as revised in 2004. Two patients who experienced operation-related death were excluded from the study. Thus, a total of 189 patients were finally evaluated in the present study. For all these patients, the inpatient medical records, chest x-ray films, whole-body computed tomography (CT) films, bone and gallium scanning data, and records of surgery were reviewed without knowledge of the results of immunohistochemical staining (IHS) or the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining.\nDetails of postoperative adjuvant therapy were described in previous studies.23,24 In brief, cisplatin-based chemotherapy, radiation, and oral administration of tegafur (a fluorouracil derivative drug) were prescribed postoperatively for 47, 30, and 49 patients, respectively. Intraoperative therapy was not performed on any patient.\nThe day of thoracotomy was considered the starting day for counting postoperative survival days. The median and mean follow-up periods were 1697 and 1983 days, respectively (range, 50\u20136381 days). This study has been approved by the Ethics Committee of Faculty of Medicine, Kyoto University.\nTissue Preparation\nAll tumor specimens were fixed immediately in 10\u00a0vol% formalin, and then embedded in paraffin. Serial 4-\u03bcm sections were prepared from each sample and used for routine hematoxylin and eosin (HE) staining, the TUNEL staining to detect apoptotic cells, and IHS to determine Aurora-A expression status, p53 status, intratumoral microvessel density (IMVD), and proliferative index (PI).\nImmunohistochemistry\nAurora-A expression was evaluated using a standard streptavidin-biotinylated horseradish peroxidase detection system. Dewaxed sections were incubated overnight with a rabbit polyclonal antibody (KR051, Transgenic Co. Ltd., Kumamoto, Japan; it had been produced and its reliability had been confirmed by western blotting on a commercial basis) diluted at 1:100. Diaminobenzidine-tetrahydrochloride (0.03%) containing 0.1% hydrogen peroxide was used as a chromogen, and sections were counterstained with hematoxylin. For negative control slides, rabbit immunoglobulin G was used instead of the primary antibody. Colon cancer sections that are known to express Aurora-A were used as positive control slides.\nAurora-A expression on tumor cells was first classified into negative or positive expression; a slide was judged to be Aurora-A positive expression, regardless of the staining intensity. For slides with positive Aurora-A expression, the staining pattern was, then, classified as follows: diffuse cytoplasmic staining (Fig.\u00a01B), perimembrane staining (Fig.\u00a01C). Slides were evaluated by two pathologists (E.O. and Y.T.) independently, without knowledge of any patient characteristic.\nFig.\u00a01.Aurora-A expression in non-small-cell carcinoma (NSCLC) as detected with imuunohistochemical staining. A Negative staining. B diffuse cytoplasmic staining. C Peri-membrane staining.\nPI, IMVD, and p53 status were also determined with IHS as described previously.23,25 Briefly, an anti-PCNA monoclonal antibody (PC-10, mouse immunogloblin G [IgG] 2a, kappa; DAKO Japan, Kyoto, Japan) diluted at 1:50 was used as the primary antibody to determine PI;23 PI was defined as the percentage of PCNA-positive staining tumor cells. IMVD was determined with an anti-CD34 monoclonal antibody QBEnd10 (mouse IgG1, kappa; DAKO Japan) diluted at 1:50; the 10 most vascular areas within a section were selected for evaluation of angiogenesis, and the average counts of CD34-positive vessels were recorded as IMVD in each case.25 An antihuman p53 monoclonal antibody DO-7 (mouse IgG2b, kappa, 250\u00a0\u03bcg\/mL, DAKO Japan) was used to determine p53 status. When the percentage of positive-staining cells exceeded 5%, the slide was judged to exhibit aberrant expression of p53.23\nDetection of Apoptosis\nThe TUNEL staining was performed using the In Situ Death Detection Kit, POD (Boehringer Manheim, Manheim, Germany) following the manufacture\u2019s protocol as described previously.23 The specificity of the TUNEL staining of apoptotic cells was confirmed by making the negative and the positive control slides at every staining. As negative control slides, sections incubated with the TUNEL reaction mixture without TdT were used. Sections treated with DNase I (Stratagene, La Jolla, CA) before the TUNEL reaction were used as positive control slides. Apoptotic cells were determined with careful observation of TUNEL-staining sections and serial HE-staining sections, and TUNEL-positive staining cells, if they represented the histological features of necrosis in HE-staining sections, were not considered to be apoptotic cells. In each case, a total of 10,000 tumor cells, consisting of 1000 tumor cells each in 10 different fields, were evaluated at high magnification (400\u00d7). The apoptotic index (AI) was defined as the number of apoptotic cells per 1000 tumor cells.\nStatistical Analysis\nCounts were compared by the chi-square test. Continuous data were compared using the t test if the distribution of the samples was normal, or using the Mann-Whitney U test if the sample distribution was asymmetrical. The postoperative survival rate was analyzed by the Kaplan-Meier method, and differences in the survival rates were assessed by the log-rank test. Death from any cause was included in calculation of postoperative survival. Multivariate analysis of the prognostic factors was performed using Cox\u2019s proportional hazard model. Differences were considered significant when the P value was less than .05. All statistical manipulations were performed using the SPSS ver.10 for the Windows software system (SPSS Inc., Chicago, IL).\nResults\nExpression of Aurora-A in NSCLC\nExpression of Aurora-A was not observed in normal adjacent lung tissues in any patient. Expression of Aurora-A in tumor cells was negative in 31 patients (16.4%) and positive in the other158 patients. Among Aurora-A positive patients, 124 patients showed diffuse cytoplasmic staining, 6 patients showed pure perimembrane staining, and 28 patients showed both diffuse cytoplasmic staining and perimembrane staining (mixed staining). Because the number of patients showing pure perimembrane staining was very small (n\u00a0=\u00a06), both patients with pure perimembrane staining and patients with mixed staining were classified into perimembrane patients. Thus, there were 31 negative patients (16.4%), 124 diffuse cytoplasmic patients (65.6%), and 34 perimembrane patients (18.0%) (Table\u00a01).\nTable\u00a01.Characteristics of patients and Aurora expressionNo. of patients (%)MAGE-D4 expressionP valueNegativePositiveDiffuse cytoplasmicPerimembraneAll patients189 (100)31 (16.4%)124 (65.6%)34 (18.0%)Age (mean\u00a0\u00b1\u00a0SD) 62.4\u00a0\u00b1\u00a09.660.5\u00a0\u00b1\u00a010.262.3\u00a0\u00b1\u00a09.264.3\u00a0\u00b1\u00a09.10.256Sex.137\u00a0\u00a0\u00a0\u00a0Male133 (70.4)26 (19.5%)86 (64.7%)21 (15.8%)\u00a0\u00a0\u00a0\u00a0Female56 (29.6)5 (8.9%)38 (67.9%)13 (23.2%)Performance status (PS).353\u00a0\u00a0\u00a0\u00a00164 (86.8)28 (17.1%)109 (66.5%)27 (16.5%)\u00a0\u00a0\u00a0\u00a01\u2013225 (13.2)3 (12.0%)15 (60.0%)7 (28.0%)Histologic type.302 (Sq vs Ad)\u00a0\u00a0\u00a0\u00a0Squamous cell (Sq)64 (33.9)8 (12.5%)41 (64.1%)15 (23.4%)\u00a0\u00a0\u00a0\u00a0Adeno (Ad)108 (57.1)19 (17.6%)73 (67.6%)16 (14.8%)\u00a0\u00a0\u00a0\u00a0Large cell (La)10 (5.3)4 (40.0%)4 (40.0%)2 (20.0%)\u00a0\u00a0\u00a0\u00a0Others7 (3.7)0 (0.0%)5 (85.7%)1 (14.3%)Tumor differentiation*.393\u00a0\u00a0\u00a0\u00a0Well68 (37.4)9 (13.2%)49 (72.1%)10 (14.7%)\u00a0\u00a0\u00a0\u00a0Moderately67 (36.8)13 (19.4%)43 (64.2%)11 (16.4%)\u00a0\u00a0\u00a0\u00a0Poorly47 (25.8)9 (19.1%)26 (55.3%)12 (25.5%)Pathologic (p-) stage.007 (for all p-stage)\u00a0\u00a0\u00a0\u00a0I104 (55.0)12 (11.5%)80 (76.9%)12 (11.5%)\u00a0\u00a0\u00a0\u00a0II22 (11.6)4 (18.2%)11 (50.0%)7 (31.8%)\u00a0\u00a0\u00a0\u00a0IIIA63 (33.3)15 (23.8%)33 (52.4%)15 (23.8%)*\u00a0Other histologic types were excluded in the analyses.\nThe incidence of perimembrane staining was significantly higher in higher p-stages; perimembrane staining was seen in only 11.5% (12 of 104) of p-stage I patients, but seen in as high as 25.8% (22 of 85) of p-stage II\u2013IIIA patients (Table\u00a01). There was no significant correlation between Aurora-A expression status and any other patients\u2019 characteristic (Table\u00a01).\nThe mean PI for perimembrane Aurora-A expression patients high tumor was significantly higher than that for Aurora-A negative or that for diffuse cytoplasmic Aurora-A expression patients (PIs: 63.5, 49.2, and 41.7, respectively; P\u00a0<\u00a0.001) (Table\u00a02). There was no significant difference in the mean IMVD or in the mean AI according to the status of Aurora-A expression; there was no significant correlation between Aurora-A expression status and p53 status (Table\u00a02).\nTable\u00a02.Correlation between Aurora expression and other biomarkersAurora expressionP valueNegativePositiveDiffuse cytoplasmicPerimembraneIntratumoral microvessel density (IMVD)*191.2\u00a0\u00b1\u00a016.3174.7\u00a0\u00b1\u00a08.9167.6\u00a0\u00b1\u00a014.6.584Proliferative index (PI) (%)49.2\u00a0\u00b1\u00a04.841.7\u00a0\u00b1\u00a02.563.5\u00a0\u00b1\u00a03.8<.001Apoptotic index (AI) (\/1000 tumor cells)13.5\u00a0\u00b1\u00a03.220.5\u00a0\u00b1\u00a02.015.9\u00a0\u00b1\u00a02.5.154p53-status: Aberrant expression (+)12\/31 (38.7%)55\/124 (44.4%)15\/34 (44.1%).848Values were shown as the mean\u00a0\u00b1\u00a0standard error (SE).*\u00a0Evaluated with an anti-CD34 antibody.\nExpression of Aurora-A and Postoperative Survival\nFor all 189 patients, 5-year survival rates of Aurora-A negative patients, diffuse cytoplasmic Aurora-A patients, and perimembrane Aurora-A patients were 67.8%, 66.7%, and 47.6%, respectively; perimembrane Aurora-A patients showed the poorest postoperative survival (P\u00a0=\u00a0.033) (Table\u00a03 and Fig.\u00a02).\nTable\u00a03.Aurora expression and postoperative survival in non-small-cell lung cancer (NSCLC) patients5-year survival rate (%)P valueAURORA expressionNegativePositiveDiffuse cytoplasmicPerimembraneAll patients67.8%66.7%47.6%.033Subset analysis, histologic type\u00a0\u00a0\u00a0\u00a0Squamous cell (Sq)75.0%74.2%34.5%.007\u00a0\u00a0\u00a0\u00a0Adeno (Ad)64.2%60.8%62.5%.894Pathologic (p-)stage\u00a0\u00a0\u00a0\u00a0I91.7%72.9%58.3%.098\u00a0\u00a0\u00a0\u00a0II75.0%68.6%57.1%.579\u00a0\u00a0\u00a0\u00a0IIIA50.4%50.9%28.9%.088Fig.\u00a02.Survival curves of patients who underwent complete resection for pathologic (p-) stage I\u2013IIIA non-small-cell lung cancer. Comparison according to Aurora-A expression status.\nSubset analyses revealed that perimembrane Aurora-A expression was a significant factor to predict a poor prognosis in squamous cell carcinoma patients, not in adenocarcinoma patients (P\u00a0=\u00a0.007 and P\u00a0=\u00a0.894, respectively) (Table\u00a03).\nA multivariate analysis of prognosis factors using a Cox proportional hazard model confirmed that perimembrane Aurora-A expression was an independent and significant factor to predict a poor prognosis in NSCLC (Table\u00a04).\nTable\u00a04Multivariate analysis of prognostic factors (Cox\u2019s proportional hazard model)FactorsBetaP valueHazard ratio (95% confidence interval)Gender (male\/female)\u22120.539.0960.583 (0.309\u20131.101)Age0.006.7141.006 (0.977\u20131.035)Performance status (0\/1\/2)0.183.5711.201 (0.637\u20132.264)Histologic type (Nonadenocarcinoma\/adenocarcinoma)\u22120.050.1620.951 (0.887\u20131.020)Pathologic stage (I\/II\/IIIa)0.551.0011.734 (1.311\u20132.295)Perimembrane Aurora expression (No\/Yes)\u22120.362.0320.343 (0.169\u20130.699)\nDiscussion\nThe present study is the first clinical study on Aurora-A expression in NSCLC and has revealed the clinical significance as follows: (1) positive Aurora-A expression is exclusively observed in tumor cells, not in normal cells; (2) there are two Aurora-A expression patterns, diffuse cytoplasmic staining and perimembrane staining; (3) perimembrane staining is significantly correlated to higher p-stage and higher proliferative activity; and (4) perimembrane staining is a significant and independent factor to predict a poor prognosis, especially in squamous cell carcinoma patients. As mentioned in the introduction section, previous experimental and clinical studies have demonstrated that Aurora-A is overexpressed and upregulated in tumor cells and\/or tissues,8\u201320,22,26 which is also true in NSCLC as demonstrated in the present study. The centrosomes play important roles in the maintenance of genomic instability by establishing bipolar spindles during mitosis and by ensuring equal segregation of replicated chromosome into two daughter cells.27 Aurora-A is involved in normal centrosome functions, and its abnormalities such as gene amplification and overexpression may lead to centrosome function disorder, chromosomal instability, and carcinogenesis.26Aurora-A gene amplification usually contributes to overexpression of Aurora-A gene and Aurora-A protein, but also common is overexpression without gene amplification.22 These results may suggest that Aurora-A expression is regulated not only by gene amplification but also by other mechanisms.22 In the present study, only Aurora-A protein expression was assessed immunohistochemically, and comparative study of overexpression and amplification should be conducted in future.\nIn previous clinical studies on Aurora-A expression assessed with IHS, Aurora-A expression was classified simply into negative or positive, and its clinical significance was examined.13,15\u201318,20 Some studies showed a significantly positive correlation between Aurora-A overexpression and aggressive tumor behavior such as poor differentiation and nodal metastasis,15,16,20 but others showed no17 or an inverse correlation.18 In addition, among three studies where prognostic significance of Aurora-A overexpression was assessed, two studies showed a significantly poor prognosis in patients with Aurora-A overexpression13,16 and one failed to show.20 These conflicting results on clinical significance of Aurora-A expression status might be partly due to the evaluation system. In fact, there was no difference in the proliferative activity as represented as PI or in the prognosis between negative Aurora-A tumors and diffuse cytoplasmic Aurora-A tumors in the present study (Tables\u00a02 and 3); when perimembrane Aurora-A expression was separated and analyzed, tumors with perimembrane Aurora-A expression showed an aggressive tumor phenotype correlated with a higher PI and a poor prognosis. The biochemical implication of perimembrane Aurora-A expression is unknown, staining patterns should be taken into consideration in evaluation of Aurora-A expression status in clinical samples.\nAnother reason for conflicting results of clinical significance of Aurora-A expression status might be difference in the histologic type. Two clinical studies on esophageal squamous cell carcinoma showed that Aurora-A overexpression was significantly correlated with aggressive tumor behavior.15,16 In contrast, two clinical studies on adenocarcinoma, one on pancreatic cancer17 and one on breast cancer,20 failed to show a significant correlation between Aurora-A status and tumor differentiation or prognosis. In fact, a subset analysis according to histologic types in the present study showed that perimembrane Aurora-A expression was a significant factor to predict a poor prognosis in lung squamous cell carcinoma, but not in lung adenocarcinoma (Table\u00a03). These results may suggest that clinical impact of Aurora-A expression status is enhanced in squamous cell carcinoma and reduced in adenocarcinoma. Further clinical studies on a variety of malignant tumors will give some answers to the issue.\nWe analyzed correlations between Aurora-A status and other biomarkers such as proliferative activity and p53 status. There has been no clinical study where Aurora-A status is assessed in correlation with proliferative activity. Considering experimental results that Aurora-A is a centrosome-associated kinase that contributes to regulation of cell mitosis,8,26 Aurora-A expression status may be correlated with proliferative activity in clinical samples as shown in the present study (Table\u00a02). However, it remains unknown why a specific Aurora-A expression pattern, the perimembrane expression, is correlated with higher proliferative activity. Experimental studies have also suggested an interaction between Aurora-A and p53,28\u201330 suggesting that the p53 inhibit oncogenic activity of Aurora-A28 and that Aurora-A, especially when overexpressed, inactivate tumor-suppressor activity of p53.29,30 One clinical study also documented that Aurora-A overexpression was significantly correlated with p53 mutation in hepatocellular carcinoma.22 These results strongly suggest that Aurora-A in concert with p53 contributes to the development and progression of malignant tumors. In the present study, we failed to show a significant correlation between Aurora-A expression status and p53 status as assessed with IHS. In a future study, p53 gene mutation status in correlation with Aurora-A expression should be examined.\nMore recently, increasing studies on Aurora-A gene polymorphisms and risk of malignant tumors have been reported.31\u201336 Two common single nucleotide polymorphisms in the coding regions are 91T->A transversion and 169G->A transversion, which result in phenylalanine\/isoleucine substitution at amino acid 31 (F31I) and valine\/isoleucine substitution at amino acid 57 (V57I), respectively. Several studies have shown such polymorphisms are associated with increased risk of occurrence of a variety of malignant tumors including esophageal,31,35 ovarian,32 and breast33,34 cancers. In future, correlation between Aurora-A polymorphism and risk of development of primary lung cancer should be investigated. In conclusion, Aurora-A expression status was a significant prognostic factor in correlation with proliferative activity in NSCLC. To confirm the clinical significance, future prospective studies should be conducted.","keyphrases":["aurora-a","lung cancer","surgery","prognosis","proliferation"],"prmu":["P","P","P","P","U"]}
{"id":"Dev_Biol-2-1-2098691","title":"Regulation of the Xenopus Xsox17\u03b11 promoter by co-operating VegT and Sox17 sites\n","text":"The gene encoding the Sox F-group transcription factor Xsox17\u03b11 is specifically expressed throughout the entire region of the Xenopus blastula fated to become endoderm, and is important in controlling endodermal development. Xsox17\u03b11 is a direct target of the maternal endodermal determinant VegT and of Sox17 itself. We have analysed the promoter of the Xenopus laevis Xsox17\u03b11 gene by transgenesis, and have identified two important control elements which reside about 9 kb upstream at the start of transcription. These elements individually drive transgenic endodermal expression in the blastula and gastrula. One contains functional, cooperating VegT and Sox-binding consensus sites. The Sox sites in this region are occupied in vivo. The other responds to TGF-\u03b2 signals like Activin or Nodals that act through Smad2\/3. We propose that these two regions co-operate in regulating the early endodermal expression of the Xsox17\u03b11 gene.\nIntroduction\nThe endoderm of Xenopus embryo arises in two successive phases, involving firstly cell-autonomous gene action, followed by dependence on cell signalling. The cell-autonomous phase is directly initiated by the maternal T-box transcription factor VegT, but then some key genes associated with endodermal differentiation become dependent on signalling by the group of TGF-\u03b2 family members that signal through Smad2\/3 These include the Nodal-related proteins or Xnrs, Derri\u00e8re, Vg1 and Activin. In this second phase, cells are sensitised to TGF-\u03b2 signalling by the maternal VegT that they inherit (Clements et al., 2001; Clements and Woodland, 2003; Engleka et al., 2001; Hudson et al., 1997; Yasuo and Lemaire, 1999). Since the essential TGF-\u03b2 signalling molecules are themselves induced by VegT, and VegT is indispensable for endoderm development (Xanthos et al., 2001), the overall effect is that only a critical mass of VegT-containing cells can generate sufficient signalling to sustain expression of these key endodermal genes. Scattered VegT containing cells will fail to become endoderm and conform to their surroundings. In contrast, some other genes (e.g. Xnr4), which are directly induced by VegT do not become signal-dependent. Finally, in the gastrula, endodermal gene expression becomes independent of cell signalling (Yasuo and Lemaire, 1999). This interpretation of endoderm initiation, establishment and maintenance is heavily based on studying the expression of the VegT target Xsox17, an HMG-box transcription factor, although Mix.1 and Mixer behave in a similar fashion.\nThere are three Sox17 genes in Xenopus laevis, Xsox17\u03b11, \u03b12 and \u03b2, but since no differences in their activity have so far been detected, for most purposes we refer to them collectively as Xsox17. The transcription of these Sox F group genes is activated prior to the mid-blastula transition (MBT), when very low levels are found ubiquitously, but Xsox17 transcription in the vegetal pole is enormously upregulated at MBT, precisely marking out the territory of the future endoderm through late blastula, gastrula and neurula stages (Hudson et al., 1997; Zorn et al., 1999). The Xsox17 genes have a key role in endoderm formation. Their ectopic expression induces endodermal gene expression, as well as changing the fate of cells. Interfering with their expression with a dominant negative Engrailed Xsox17 fusion construct has the converse effect, inhibiting endodermal gene expression and shifting cells out of an endodermal fate in intact embryos (Clements and Woodland, 2000; Hudson et al., 1997). The use of antisense morpholino oligos shows that the individual genes have non-redundant late roles in the developing mid- and hindgut, but that together they are needed for the completion of gastrulation (Clements et al., 2003). This correlates well with the essential role of the single-murine Sox17 gene for early development of endoderm fated to become mid- and hindgut, although in the absence of Sox17 there is also later loss of foregut cells (Kanai-Azuma et al., 2002). In zebrafish, two related Sox genes, Casanova and Sox17, are important in forming the endoderm; the former is more upstream and its mutants indicate that it has a vital role in endoderm development (Aoki et al., 2002). While mutations in the zebrafish Sox17 gene have not been described, it is likely that in the zebrafish endodermal gene network the combined action of Casanova and Xsox17 are roughly equivalent to the overall action of Xsox17 in Xenopus, particularly allowing for the fact that Casanova expression in the yolk syncytial layer is Nodal-independent, allowing a parallel with the Xenopus cell autonomous phase to be drawn (Kikuchi et al., 2001; Woodland and Clements, 2003). In Xenopus, ablation of Xsox17 expression with morpholinos halts gastrulation at an early stage. However, the immediate effects on gene expression were initially reported to be modest and were restricted to the direct Xsox17 targets Endodermin and Hnf-1\u03b2 (Clements et al., 2003). However, much wider effects have now been observed using microarrays (Sinner et al., 2006).\nSince Xsox17 is a crucial gene in the early endoderm, and its expression defines the endodermal territory, we have analysed the regulatory elements in the Xsox17\u03b11 promoter approximately 9\u00a0kb upstream of transcriptional initiation. We have identified two small elements, which can confer endodermal expression on a reporter gene in the early embryo. We have analysed one of these in detail and show that its activity depends on co-operating VegT and Sox17-binding sites, whereas the other regulatory region responds to Activin.\nMaterials and methods\nBiological materials\nEggs of X. laevis were obtained, fertilised, dejellied and cultured by standard methods, as described previously (Wilson et al., 1986). Oocytes, complete with their follicle, were manually removed using watchmakers forceps in modified Barths' saline (MBS; 110\u00a0mM NaCl, 1\u00a0mM KCl, 2.4\u00a0mM NaHCO3, 0.33\u00a0mM Ca(NO3)2, 0.41\u00a0mM CaCl2, 0.8\u00a0mM MgSO4, 15\u00a0mM Tris\u2013HCl, pH\u00a07.5).\nTransgenic methods\nThese followed the procedure of Kroll and Amaya (1996), except that a reduced amount of egg extract was used in the incubation of stored frozen sperm nuclei with DNA (2\u00a0\u03bcl in a 25\u00a0\u03bcl reaction). The reaction typically contained 150\u2013200\u00a0ng of linearised plasmid DNA and 0.5\u00a0\u03bcl of a 1:200 dilution of restriction enzyme (2\u00a0units\/ml). Injections of dejellied unfertilised eggs were performed in 6% Ficoll, 0.4\u00d7 Marc's modified Ringer (MMR) or 0.4\u00d7 MBS in polyheme-coated plastic dishes. Correctly cleaving eggs were sorted at the 2- to 4-cell stage and incubated in 6% Ficoll, 0.1\u00d7 modified Barth's medium. GFP fluorescence was monitored using a Leica MZFLIII dissecting microscope.\nTransient transgenesis in embryos and oocytes\nFor transient expression in embryos, DNA constructs were linearised and purified with the Qiagen gel extraction kit. 50\u00a0pg DNA together with 5\u00a0pg control Renilla luciferase reporter in 10\u00a0nl water were injected bilaterally, with or without mRNA, into the animal or vegetal poles of 2-cell embryos, cultured in 6% Ficoll, 0.4\u00d7 MBS. Embryos were analysed at gastrula or neurula stages using the Promega Dual Luciferase Reporter Assay System. Triplicate batches of 10 embryos were homogenised in 600\u00a0\u03bcl passive lysis buffer and incubated on ice for 10\u00a0min. Samples were centrifuged at 13,000\u00a0rpm for 2\u00a0min at 4\u00a0\u00b0C, and supernatants removed. They were equilibrated to room temperature for 30\u00a0min, and 60\u00a0\u03bcl assayed for bioluminescence after addition of 50\u00a0\u03bcl luciferin stock using the Luminoskan RS apparatus (Labsystems). Normalisation of the experimental reporter was achieved by quenching of the firefly luciferase reaction and measurement of Renilla luciferase luminescence.\nOocyte nuclei were microinjected with 18\u00a0nl water containing 300\u2013500\u00a0pg circular firefly test plasmid, plus one third this amount of control Renilla luciferase plasmid, with or without transcription factor mRNA. After culturing overnight in MBS, they were processed for luciferase activity as detailed above.\nCloning and characterisation of the Xsox17\u03b11 gene\nThe Xsox17\u03b11 gene was isolated by screening a X. laevis gilli PAC library (RZPD) with an Xsox17\u03b11 cDNA probe. One clone (BUMSP710J2012Q3) reacted strongly with this probe. A positive Not 1 fragment was subcloned into Bluescript and this sequence encompassed all 12\u00a0kb of 5\u2032 upstream sequence present in the PAC, the transcribed region itself and 3\u00a0kb downstream of the 3\u2032 UTR.\nTransgenic constructs and mutagenesis\nMutant promoter constructs were created by hybrid overlap extension PCR, using a series of external and overlapping internal primers. Primer sequences were as follows: B1 ext up 5\u2032CAACACTCACATTC 3\u2032. B1 T-box ext down 5\u2032CTTGAGAATGGGACTGTGTTAACAAACAATGATGATCAGAACTCTGG 3\u2032, Sox A int up 5\u2032 CTTGGGAACTAGTTGTGGATC 3\u2032, B1 Sox A int down 5\u2032GATCCACAACTAGTTCCCAAG 3\u2032, B1 Sox B int down 5\u2032 CTTGAGAATGGGACTGTGTTAACAACCATGGATGGTGTGAACTCTGG 3\u2032, B1 Sox B\u00a0+\u00a0T-box ext down 5\u2032CTTGAGAATGGGACTGTTTTAACAACCATGGATGATCAGAACTCTGG 3\u2032. Underlined text denotes mutated sequence. External primers had 5\u2032 Sac1 and 3\u2032 Kpn1 extension sequences. Amplification was performed over 20 cycles with 55\u00a0\u00b0C annealing temperature. Products were Qiagen column purified, cut with restriction enzyme, and cloned into pGL3basic-act-luc (transient assay) or pGL3basic-act-mgfp5 (transgenic assay).\nElectrophoretic band-shifts (EMSAs)\nEMSAs were performed using VegT protein synthesised in vitro in the rabbit reticulocyte system. 10\u00a0pmol single-stranded DNA oligo was 5\u2032 end-labelled with 20\u00a0\u03bcCi 32P \u03b3-ATP using T4 polynucleotide kinase. Forward and unlabelled reverse strands were annealed by heating to 90\u00a0\u00b0C for 5\u00a0min, followed by slow cooling overnight. DNA was recovered by ethanol precipitation and resuspended in TE buffer. 15\u00a0\u03bcl binding reactions contained 1\u00a0\u03bcl in vitro translated protein, 2\u00a0\u03bcg [poly dI.dC].[poly dI.dC], 3\u00a0\u03bcl MDB buffer (20\u00a0mM HEPES, pH\u00a07.9, 100\u00a0mM KCl, 12.5\u00a0mM MgCl2, 0.1\u00a0mM EDTA, 17% glycerol w\/v, 2\u00a0mM DDT). The probe was added following 10-min incubation at 30\u00a0\u00b0C, followed by a further 10-min incubation. Control binding reactions included a 50-fold excess of unlabelled specific competitor probe. Samples were analysed on a 15% polyacrylamide gel at 4\u00a0\u00b0C (200\u00a0V, 2\u00a0h). Gels were dried and autoradiographed or analysed with a phosphoimager. Probe sequences used were: B1 5\u2032-TGTCCAGAGTTCACACCATCATTGTTTGTTA-3\u2032, T-box mutant 5\u2032-TCTCCAGAGTACGCACATTCATTGTTTGTTA-3\u2032, T-box consensus 5\u2032-TCTCCAGAGTTCACACCTTCATTGTTTGTTA-3\u2032. Underlined text denotes T-box half site and variations thereof.\nChromatin immunoprecipitation (ChIP)\n30\u00a0gastrula stage embryos were fixed in 1% formaldehyde for 10\u00a0min at room temperature, and fixation reversed by addition of 125\u00a0mM glycine for 30\u00a0min. Embryos were washed in MMR (25\u00a0mM NaCl, 0.5\u00a0mM KCl, 0.25\u00a0mM MgCl2, 0.25\u00a0mM CaCl2, 1.25\u00a0mM HEPES, pH\u00a07.5), and homogenised in 500\u00a0\u03bcl low salt extraction buffer (25\u00a0mM Tris\u2013Cl, pH\u00a07.5, 70\u00a0mM KCl, 1\u00a0mM EDTA, 20% glycerol, 5\u00a0mM DTT\u00a0+\u00a0protease inhibitors). Shearing was performed by sonication at full power for 6\u00a0\u00d7\u00a010\u00a0s with 2-min breaks on ice. Shearing efficiency was assessed by agarose gel electrophoresis following reversal of cross links by the addition of 200\u00a0mM NaCl and incubation at 65\u00a0\u00b0C for 5\u00a0h, followed by proteinase K treatment, phenol extraction and ethanol precipitation. This preparation also yielded input DNA for quantification of PCR reactions.\n100\u00a0\u03bcl sheared chromatin preparation was diluted with 100\u00a0\u03bcl IP buffer (50\u00a0mM Tris\u2013Cl, pH\u00a08, 100\u00a0mM NaCl, 2\u00a0mM EDTA, 1\u00a0mM DTT, 1% NP-40\u00a0+\u00a0protease inhibitors) and pre-cleared with 40\u00a0\u03bcl protein A agarose (Sigma) for 2\u00a0hrs at 4\u00a0\u00b0C. Supernatant was incubated with 4\u00a0\u03bcl serum for 2\u00a0hrs at 4\u00a0\u00b0C. A no antibody control was also included. 40\u00a0\u03bcl 50% slurry protein A agarose pre-saturated with 1\u00a0mg\/ml BSA and 0.3\u00a0mg\/ml salmon sperm DNA in IP buffer was added, and reactions incubated overnight at 4\u00a0\u00b0C. Beads were washed successively in IP buffer plus 0.1% sodium deoxycholate, IP buffer with 500\u00a0mM NaCl, IP buffer with 250\u00a0mM LiCl, and TE (10\u00a0mM Tris, pH\u00a08, 200\u00a0mM NaCl). Following a pulse spin, 250\u00a0\u03bcl elution buffer (1% SDS, 0.1\u00a0M NaHCO3) was added to the beads and elution repeated until a 500\u00a0\u03bcl volume was obtained. Cross-links were reversed and DNA recovered as described above.\nPCR was performed as described previously. 32\u00a0cycles of amplification were used. Primer sequences are as detailed below:C3B1 F5\u2032 GCCAATAGACACCTTTCTAG 3\u2032C3B1 R5\u2032 GAGAATGGGACTGTGTTAAC 3\u2032Xsox17\u03b1 ORF F5\u2032 GGACGAGTGCCAGATGATG 3\u2032Xsox17\u03b1 ORF R5\u2032 CTGGCAAGTACATGTGTCC 3\u2032Xom Promoter F5\u2032 TGTTGGCTGAGTAGGAATGAGAGG 3\u2032Xom Promoter R5\u2032 AGGCAGAGATCAGTACCACCT 3\u2032\nThe Xom primers are from Messenger et al. (2005).\nResults\nStructure of the Xsox17\u03b11 gene\nThe Xsox17\u03b11 gene was isolated from a X. laevis gilli PAC library (RZPD) using an Xsox17\u03b11 cDNA probe. A Not 1 fragment that encompassed all 12\u00a0kb of 5\u2032 upstream sequence present in the PAC and 3\u00a0kb downstream of the 3\u2032 UTR was subcloned into Bluescript. The transcribed sequence contains a single intron of 705\u00a0bp, starting in codon 119. The upstream region was sequenced up to \u2212\u00a09.5\u00a0kb, although sequence from approximately \u2212\u00a07.7 to \u2212\u00a09.2\u00a0kb was highly repetitive and proved unsequenceable. The transcriptional start site was determined by primer extension (data not shown).\nIn parallel, an Xsox17\u03b2 phage \u03bb clone with 6\u00a0kb of 5\u2032 upstream sequence was isolated and all of its 5\u2032 regions sequenced. Since the alpha and beta genes have identical early expression patterns, a comparison was made of the proximal 5\u2032 promoter regions (Supplementary Fig. 1). There are many conserved features, including motifs for binding several transcription factors known to be relevant to endoderm development (Homeodomain, SMAD, T-box and Sox proteins), as well as a GA-rich region at about \u2212\u00a01860\u00a0bp. No other Xsox17\u03b2 or Xsox17\u03b12 clones were identified in any library.\nTransgenic analysis of the promoter of the Xsox17\u03b11 gene\nInitially the entire 12\u00a0kb upstream of the Xsox17\u03b11 gene was fused to a GFP expression cassette to give MR19. A second construct was made which also contained 3\u00a0kb downstream of the transcribed sequence. This was placed downstream of the GFP cassette (MR21) (Fig. 1, top panel). When the method of Kroll and Amaya (1996) was used to make transgenics containing these sequences, GFP expression was observed throughout the pre-endoderm of the gastrula, including the superficial region surrounding the blastopore lip, which is known as the involuting or extra-blastoporal pre-endoderm (Figs. 1A, B). Expression was screened by GFP fluorescence, which under-reports in the vegetal region (Ahmed et al., 2004), and it was confirmed by in situ hybridisation to GFP mRNA (C) which also under-reports in vegetal tissues, but to a much lesser degree if suitable protocols are used (Sasai et al., 1996). Bearing this in mind, Fig. 1 shows that the pattern at early and late gastrula was similar to the in situ hybridisation pattern of expression of the endogenous gene (E, F). Comparison of MR19 and MR21 transgenics indicated that they were similar in the gastrula (H), but that the 3\u2032 region of MR21 reduced expression outside the endoderm at later stages. This sequence has not been investigated further. The 260\u00a0bp minimal promoter was negative in terms of endodermal expression (I), but was expressed elsewhere. Typical numerical results for the transgenics are shown in Supplementary Tables 1 and 2. Constructs either consistently gave endodermal expression in the gastrula, or consistently failed to do so.\nThere was later expression of GFP in the pharynx and hindgut of the tailbud tadpole (Supplementary Fig. 2). This corresponds to regions where the endogenous gene is expressed, as judged by RT\u2013PCR (Clements et al., 2003), but there was no expression in the region of the developing gall bladder, where the gene is strongly expressed at this stage (Hudson et al., 1997; Zorn and Mason, 2001). Later there was robust expression in the foregut and proctodeum, as well as in the pancreas (Supplementary Fig. 2). Zorn and Mason (2001) did not report expression in the pancreas at these stages, so this may represent either incorrect regulation or persistent, stable GFP expression. There was also expression in lateral line organs and in the brain (fore\/mid brain boundary, cranial nerves). It is not known if Xsox17\u03b11 is expressed in all these regions. However there was GFP expression in the lens, and RT\u2013PCR shows that Xsox17 is expressed there, as are other Sox genes (data not shown) (Schlosser and Ahrens, 2004; Zygar et al., 1998). There was also expression in the olfactory organs, which has been shown for the endogenous genes by in situ hybridisation (Zorn, personal communication), as well as in skeletal elements of the developing limbs.\nWe have focused on blastula to early gastrula stage expression of the promoter because this is the time that the endodermal domain, marked by Xsox17 expression, is mapped out. To define the regions responsible, we first showed that there were no sequences promoting endodermal expression in the single intron or the 3\u00a0kb downstream of the cDNA sequence (not shown). We then showed that although the 260\u00a0bp proximal region contains sequences conserved in the Xsox17\u03b2 promoter, including T-box sites (Supplementary Fig. 1), it drove expression everywhere except the future endoderm. It is thus expressed only where Xsox17\u03b11 is not. However, later it gave expression in the pharynx, duodenum, pancreas, stomach and proctodeum, as illustrated for longer promoters in Supplementary Fig. 2. The 6\u00a0kb upstream of Xsox17\u03b2 also did not drive gastrula endodermal expression (not shown) and we could not identify any Xsox17\u03b12 clone. Thus a comparison of endodermal elements in the three Xsox17 genes could not be made. While it is possible to compare sequences with those in Xenopus tropicalis, any interpretation would be dubious since the detailed regulation of X. tropicalis has not been studied in this species.\nA wider screen for regulatory elements in Xsox17\u03b11 was made with a deletion series (Fig. 2). Early endodermal expression required regions about 9\u00a0kb from the start of transcription. This defined a 1.1\u00a0kb \u201cEndodermal element\u201d, which alone drove strong GFP expression in the early vegetal\/endoderm region. Internal deletions show that the E-element can drive strong endodermal expression when placed on the first 1.7\u00a0kb 5\u2032 to the start of transcription, which itself only gave low GFP expression in non-endodermal regions of gastrulae and later stages (Fig. 2J). Later experiments, described below, investigated sub-fragments of this region fused to a basal promoter from a Xenopus cytoskeletal actin gene that is expressed in embryonic striated muscle (Ahmed et al., 2004; Latinkic et al., 2002), showing that the E-element does not require sequences in the proximal region of the promoter for endoderm-specific expression. Thus, in the context of the sequences studied, the E-element is the only region both necessary and sufficient to direct expression in the progenitor of the endoderm and in the early gastrula.\nDissection of the E-element\nThe E-element was divided into three sub-fragments (A\u2013C), which were coupled to the basal cytoskeletal actin promoter and tested in transgenics (Fig. 3A). Only B and C gave vegetal expression. Each was further divided into two approximately equal fragments. For B, only B1 drove vegetal\/endodermal expression, but neither C sub-fragment did. However a sub-fragment from the central region of C gave vegetal expression, albeit with relatively high animal expression (Fig. 3A). Since the C1 and C2 fragments were made with an overlapping 18\u00a0bp region to avoid disrupting a regulatory sequence, there clearly are sequences within C1 and C2 which must co-operate to give the endodermal expression of C3.\nThus two independent Xsox17\u03b11 control regions, B1 and C3, are capable of driving expression in the presumptive endoderm of the gastrula. The expression of B1 (Figs. 3A, C) is notably similar to the endogenous Xsox17 genes (Figs. 1E, F). The GFP expression in the vegetal regions is detectable by early stage 10, the onset of gastrulation. Given that GFP requires an hour or more to mature and fluoresce, vegetal expression of the transgene must begin in the blastula, during the initiation and\/or establishment phases of Xsox17 regulation (Clements et al., 1999; Yasuo and Lemaire, 1999). As discussed earlier, GFP under-reports in the vegetal pole (Ahmed et al., 2004), so any detectable vegetal fluorescence indicates strong actual expression.\nTransgenic analysis of the T-box and Sox sites in element B1\nB1 contains several consensus binding sites for known endodermal regulators (Fig. 3B; Supplementary Fig. 3), including a variant T-box binding site (core CACCA rather than CACCT) and two canonical Sox sites (core CATTG). To test if the variant T-box binds VegT, we performed electrophoretic gel mobility shift assays (EMSA), comparing the binding of VegT to this sequence and the consensus sequence identified in the Derri\u00e8re promoter (White et al., 2002). Fig. 3D shows that the binding of the two is similar. As a control, no binding was observed to a sequence containing a mutant site (core CACAT).\nThe T-box and the two Sox sites were mutated and their efficiency in transgenics assessed (Fig. 3C). The numbers of transgenic embryos in a typical experiment are shown in Supplementary Table 2. The double Sox site mutant gave vegetal pole expression which was somewhat less intense than that produced by the wild-type B1. There was also raised relative expression in the animal pole, suggesting that these sites may play a possible repressive role in the non-endodermal region. The T-box mutant also gave vegetal expression, although again it appeared to be at level lower than from an unmutated B1 element, although this is difficult to quantify. When the T-box site was mutated in conjunction with the Sox sites (Fig. 3C, triple mutant), expression of GFP was excluded from the endodermal domain.\nTransient transgenic analysis of B1 and C3\nAlthough in transgenics the constructs either gave endodermal expression, or they did not, the results are not quantitative and the variability in the absolute level of expression from embryo to embryo makes it difficult to compare constructs objectively. Transient transgenesis using the quantifiable reporter luciferase circumvents this problem. DNA may be introduced into different parts of the embryo, or injected it into a region where Xsox17 is not expressed (i.e. the animal hemisphere) with or without mRNAs encoding specific regulatory molecules, like VegT. To enable this, promoter elements, together with the cytoskeletal actin basal promoter, were cloned into pGL3basic (Promega), which lacks eukaryotic promoter and enhancer sequences. The DNA was linearised for injection because sometimes this is necessary for expression (Wilson et al., 1986).\nFirst constructs containing B1 and C3 elements were injected into vegetal and animal hemispheres, to establish whether the transient expression mirrors the natural high expression of Xsox17 in the vegetal compared to the animal pole. Fig. 4A shows that B1 is much more active in the vegetal than the animal hemisphere, in agreement with the transgenics. This is also true, to a reduced degree, of the C3B1 combined fragment. Surprisingly, C3 showed reduced expression in the vegetal relative to the animal hemispheres. This may partly be because these results are expressed as a ratio of animal to vegetal expression. High expression in the animal hemisphere was also seen in GFP transgenics where we simply scored for vegetal expression. Presumably inhibitory elements are lacking.\nB1 contains T-box and Sox-binding sites that were essential for high endodermal expression in transgenics. We tested the functionality of the T-box site by co-injecting B1 DNA plus VegT mRNA into the animal pole, where VegT mRNA is normally present only at a low level. The basal promoter did not respond to VegT in these assays (not shown). VegT stimulates B1 activity by an amount comparable to that produced by vegetal compared to animal hemisphere injections (Fig. 4B). In contrast, C3 is inhibited slightly, even though it contains a consensus T-box site core sequence (CACCT). Flanking nucleotides of this site are divergent (AGACACCT; consensus TCACACCT), and White et al. found that this sequence bound VegT only weakly in EMSAs (White et al., 2002). The C3B1 fragment produced an intermediate result. Thus B1, but not C3, responds positively to VegT and the responses of B1 and C3 to VegT mirror their activity in vegetal versus animal poles.\nSince VegT, directly or indirectly, induces expression of many transcription factors which are part of the mesendodermal gene network, including other T-box proteins (Eomes and Xbra), we sought to confirm that VegT directly interacts with B1 in two ways. It is of course known that the endogenous Xsox17 genes are direct VegT targets from experiments with inducible VegT fusions (Clements et al., 1999; Clements and Woodland, 2003; Yasuo and Lemaire, 1999). Firstly we mutated the T-box site, which greatly reduced, but did not eliminate the stimulation by VegT (Fig. 4C, lane 3). This implicates a T-box protein in the regulation. Secondly, we examined expression in oocytes in response to co-injected VegT (White et al., 2002). The amount of transcription seen when DNA is injected into an oocyte nucleus is comparable to an embryo, this being supported by stores of chromosomal and transcriptional proteins. However there is only one nucleus in an oocyte, so the amount of downstream nuclear gene expression that the oocyte can support over the time course of our experiments is negligible, even over an 18-h time course (White et al., 2002). One hour of transcription in the late blastula would be equivalent to 10,000\u00a0h for the oocyte, equating to well over a year. This suggests that any effects of VegT on the co-injected DNA are directly dependent on VegT itself. Fig. 4C (last three lanes) shows that VegT stimulates B1 transcription in oocytes and that this is absent for the T-box mutant. The stimulation is less in oocytes than in embryos, presumably because cooperating genes are not significantly induced by VegT in oocytes. For example endogenous Xsox17 expression is dependent both on VegT itself and on the TGF-\u03b2 signalling that VegT induces (Clements et al., 1999; Clements and Woodland, 2003; Xanthos et al., 2001). To test if the B1 element responds to TGF-\u03b2s downstream of VegT (directly or indirectly), we blocked TGF-\u03b2 family signalling by co-expressing VegT and a truncated Activin receptor (tXAR) in animal hemispheres (Hemmati-Brivanlou and Melton, 1992). Fig. 4C, lane 4, shows that the stimulation of B1 by VegT is reduced, but not eliminated. These results are consistent with the proposal that B1 responds to VegT directly via its T-box site, but that co-operating molecules downstream of VegT-induced TGF-\u03b2 signalling are also necessary for the full VegT induction in an embryo. Consistent with this interpretation, in oocytes there is no VegT stimulation of the B1 element with a mutated T-box because VegT cannot induce downstream genes such as TGF-\u03b2 signals (Fig. 4C, last lane).\nTransgenesis also showed that B1 contained functional Sox-binding sites and it was already known that the endogenous Xsox17\u03b1 genes are direct Xsox17 targets (Sinner et al., 2004). Fig. 4D shows that in the animal hemisphere B1 responds strongly to Xsox17 and expression in the oocyte supports the view that this is a direct effect. Xsox17\u03b1 and \u03b2 induce themselves and each other (Sinner et al., 2004), but the autoinduction is quite slow, as shown in Fig. 4E. The luciferase inductions fitted this because there was no significant induction by Xsox17 at stage 10.5 (not shown), but it was clear by stage 17 (8\u00a0h later at 23\u00a0\u00b0C). Xsox17 probably normally co-operates with other factors that are maternal or downstream of VegT on certain promoters. However, this only applies to a subset of promoters, whereas other genes like Hnf-1\u03b2 do not show this delay, presumably because any co-operating factors are already present in the animal hemisphere. It is likely that when Xsox17 alone is expressed ectopically, it slowly induces the co-operating factors needed for auto-induction, eventually establishing a partial endodermal gene network. Normally the cooperating molecules are maternal or are induced by VegT. In contrast to B1, C3 did not respond to Xsox17, as expected from its lack of consensus Sox sites.\nWhile C3 was activated by neither VegT nor Xsox17, it contains several consensus Smad-binding sites coupled to those for co-operating FoxH1 (Fast1), suggesting that it might respond to TGF-\u03b2s like Activin. Fig. 4F shows that this is indeed the case, suggesting that it might mediate the TGF-\u03b2 response of Xsox17\u03b11. In oocytes, C3 is induced by constituently active Smad2, indicating that the induction by Activin is direct (Fig. 4G). In contrast, B1 shows very little response to Activin in embryos. This is surprising since many gene products that induce Xsox17\u03b11 are downstream of Activin, including the Xsox17s themselves. While in this experiment the luciferase was assayed before Xsox17 would have exerted its slow effect in animal caps (see above), there is little effect even at later time points (not shown), which suggests that the level of Xsox17 induced by Activin is too low to produce a strong induction without co-operating VegT. We show below that VegT and Xsox17 co-operate in regulating B1, and synergy between VegT and Activin was previously noted (Clements and Woodland, 2003).\nMutational analysis of B1 shows that one Sox site is most important and it co-operates with the VegT site\nTo assess the roles of the two Sox sites in B1, the response of a series of single, double and triple mutants of Sox and T-box sites was assessed by injection into animal hemispheres. Most of the response to VegT was dependent on the T-box site and the neighbouring B Sox site, whereas removal of the more distant A Sox site had much less effect (Fig. 5B). When all three sites were removed, there was no induction by VegT, and no expression in the vegetal pole. Since removal individually of either the B Sox or the T-box sites, but not the A Sox site, removed much of the response of B1, maximal induction clearly requires the presence of both B Sox and T-box sites. This suggests that there is synergy between the VegT and Xsox17 bound to these sites. The role of the two Sox sites in responses to Sox17 is examined in Fig. 5C. This confirms that Sox site B has the main activity and the T-box site is unimportant in induction by Sox17 alone.\nIt is possible that in the endoderm itself, the B1 Sox sites respond to Sox proteins other than Xsox17. To test this B1 DNA was injected into the vegetal pole, followed by injection of the three Xsox17 antisense morpholino oligos (MOs) that block translation of the three X. laevis Xsox17 mRNAs (Clements et al., 2003). The enhanced activity of B1 in vegetal poles, as compared to animal, was eliminated, whereas a control MO had no effect (Fig. 5D). This confirms that the endogenous Xsox17s are responsible for the high activity of the B1 element in the developing endoderm.\nThe B1 element is bound to Xsox17 in vivo\nTo confirm that the Sox sites in B1 are functional we needed to show that they are occupied by Xsox17 in the living embryo. We therefore performed a chromatin immunoprecipitation (ChIP) on mid-gastrulae stage embryos. Chromatin was isolated, sheared to DNA fragments of about 500\u00a0bp and immunoprecipitated with an Xsox17\u03b2 anti-serum (Clements et al., 2003). Fig. 6A shows PCRs from several regions in the resulting genomic DNA. The Xsox17 B1 plus C3 element was clearly precipitated by the anti-serum, but not pre-immune serum. Conversely, the Xsox17\u03b11 ORF was not precipitated, as expected since it is about 9\u00a0kb distant from the E-element. A second control was to probe for an enhancer previously used for immunoprecipitation with an Xbra anti-serum (Messenger et al., 2005). This ventrally expressed gene should not be regulated by Xsox17 and indeed the promoter element fails to precipitate with Xsox17\u03b2 antiserum. Therefore, as expected from the transgenic data, the E-element is bound to Xsox17\u03b2 in vivo.\nDiscussion\nExpression of the Xenopus Xsox17 genes defines the endodermal territory in the mid-blastula, when cells start rapid transcription and make the initial decisions to become endoderm. For this reason, we have focused on the regulation of one of these genes, Xsox17\u03b11, in the blastula and early gastrula. We have been able to identify two regulatory elements at approximately minus 9\u00a0kb that drive transcription in the future endoderm at blastula and gastrula stages. Study of the endogenous genes has suggested that the regulation of the Xsox17 genes is very dynamic, passing rapidly through initiation, establishment and maintenance phases (see Introduction). In the initiation phase, Xsox17\u03b11 is a direct target of the localised maternal transcription factor VegT, in the establishment phase it becomes signal-dependent and eventually maintenance is cell-autonomous. One would hope to find the basis of these regulatory processes in the Xsox17\u03b11 promoter.\nActivity of the C3 endodermal element\nOf the two adjacent elements capable of driving endodermal expression, B1 responds strongly to VegT and Xsox17, and C3 to Activin, but not to VegT. We have principally focused on B1, but we have made a preliminary analysis of C3. This 89-bp region must contain cooperating elements since two fragments, each containing half of C3 plus an 18-bp overlapping region, were negative in terms of endodermal expression. The Activin responsiveness of C3 correlates with its possession of three separate pairs of closely associated FoxH1\/Fast1 and Smad sites, which are known direct effectors of Activin\/Nodal signalling (Chen et al., 1997; Germain et al., 2000). However, Activin induces the expression of many downstream molecules, which could then be responsible for indirect induction of this element. To prove the effect was direct we showed that C3 is stimulated by activated Smad2 in the oocyte. This indicates a direct effect, since the single nucleus of this cell has 10,000-fold less transcriptional capacity to induce downstream effectors of Activin\/Smad action than does the early gastrula.\nMaternal depletion of FoxH1\/Fast1 by Kofron et al. (2004) showed that it was not essential for Xsox17\u03b1 expression. However, as they point out, Xenopus embryos also express XFast-3, which binds to the same target sequence (Howell et al., 2002), and could therefore redundantly regulate Xsox17 through the same promoter elements.\nC3 responds strongly to Activin, but with respect to vegetal expression and response to VegT, its behaviour is paradoxical. It is surprising that it does not respond to VegT in the embryo, both because C3 contains a consensus T-box core sequence and because VegT induces the expression of TGF-\u03b2s that, like Activin, act through Smads 2\/3; indeed VegT depends on this signalling for its overall biological effect. The T-box site in C3 entirely overlaps a Fast1\/Smad pair of sites, which may have a bearing on the fact that VegT actually inhibits C3 basal expression, just as its expression is inhibited in the vegetal pole compared to the animal. This is consistent with the observation that removal of the T-box site removes the inhibition (data not shown). C3 was identified by its ability to direct expression in the vegetal pole of transgenics, however here we simply scored expression in the vegetal pole itself, disregarding animal regions. Conversely, the luciferase measurements are of the ratio either of vegetal to animal expression or of VegT stimulated to control expression in the animal region. Therefore a high level of animal expression would mask vegetal activity of the promoter. However, it is important to note that both the VegT induction and vegetal expression are consistent. We believe that the response to TGF-\u03b2s is the important property of C3 and other problems are introduced by looking at small regulatory regions in isolation, where synergising and inhibitory effects are absent.\nPrevious work has shown that Xsox17 expression switches from direct VegT response to TGF-\u03b2 signal dependence. C3 is a therefore a good candidate for controlling the signal-dependent process. It is conceivable that the inhibitory effects we notice bear on the hypothetical inhibitor that make Xsox17 signal dependent in the establishment phase of endoderm development.\nThe activity of the B1 endodermal element\nThe activity of B1 is more straightforward. It is highly expressed in the presumptive endoderm in transgenics and it is much more highly expressed in the vegetal than the animal hemisphere in transient assays. It is also strongly stimulated by VegT. It contains a divergent T-box site which binds VegT in vitro and which mutation shows is partially responsible for the VegT stimulation in the embryo. Expression in oocytes shows that B1 responds directly to VegT through this site, although the stimulation is less than in embryos. This correlates with the fact that blocking TGF-\u03b2 signalling in embryos with a truncated Activin receptor reduces VegT stimulation to about the oocyte level. Thus B1 responds directly to VegT and synergistically to other molecules that are downstream of the VegT-induced TGF-\u03b2s, which would principally be Nodal-related signals. These synergistic molecules are the Xsox17 proteins themselves in an autoregulatory loop.\nB1 responds directly to Xsox17. Other molecules, such as Gata 4\u20136, are downstream of Xsox17 and could in principle regulate B1. Although B1 contains a possible variant Gata-binding site, mutation of the two consensus Sox sites removes much of the activity of B1, and the triple Sox\/VegT mutant is unresponsive both to VegT and vegetal pole expression. Further, mutation of the possible Gata site has no effect on the response of B1 to VegT (not shown). The direct action of Xsox17 on B1 was confirmed by transcriptional assays in oocytes. These observations fit with the fact that Xsox17 auto-induces itself (Sinner et al., 2004), but endogenous Xsox17 genes are very slow to be induced when Xsox17 is expressed ectopically in the animal cap, and this is equally true of the B1 regulatory region. Previously we have found the same for a direct Sox inductive site in the Endodermin promoter (Ahmed et al., 2004). In fact, we believe that the Sox sites in B1 are already needed at blastula stages because they are required for full vegetal expression of the GFP transgene as early as the onset to gastrulation, judged by GFP fluorescence. Since GFP takes several hours to mature (Davis et al., 1995), there must have been an earlier requirement for the Sox factors. We have argued previously, in connection with the Endodermin promoter, that Xsox17 must normally co-operate with other molecules found in the vegetal pole. In the animal cap Xsox17 presumably establishes the endodermal gene network more slowly than its normal establishment by multiple maternal inputs (Sinner et al., 2006). Since B1 is rapidly induced by VegT, and this depends largely on the Sox sites and Xsox17 expression (as shown by Sox site mutants and blocking Xsox17 action with combined Xsox17 group morpholinos), it seems that VegT is able to co-operate with Xsox17 to produce more rapid Sox action.\nThe kinetics of reporter expression suggest that the VegT and the T-box site are not simply needed at the very onset of endodermal gene expression in the initiation phase, but that this continues into gastrulation during the establishment phase. It is provocative that the Sox site of principal importance in B1 is very near to the VegT site, suggesting a direct interaction between VegT and Xsox17, although we have been unable to detect this by immunological co-precipitation.\nThe role of Xsox17 auto-induction in the endodermal network\nThe idea that a key endodermal gene, Xsox17, induces itself is attractive because it would give stability to Xsox17 expression and hence to the endodermal network. The requirement for signalling also adds a fail-safe for cells that become misplaced from the endodermal domain, but still initiate direct VegT-dependent endodermal gene expression. These conform to the fate of their neighbours (Clements et al., 1999; Clements and Woodland, 2003; Wardle and Smith, 2004; Wylie et al., 1987; Yasuo and Lemaire, 1999). However, blocking Xsox17 action with morpholino oligos has no effect on Xsox17 expression, even though other Xsox17 targets, including HNF1-\u03b2, Endodermin and Gata5, are down-regulated (Clements et al., 2003; Sinner et al., 2006). The most obvious reason for this paradox is redundancy. For example Xsox17 can be induced by Gata4, 5 and 6 and the first two are only partially and the latter not at all affected by Xsox17 morpholinos (Clements et al., 2003, and unpublished data). Xsox17 is a direct Gata6 target (Afouda et al., 2005), but while B1 and C3 elements contain possible divergent Gata sites, mutating these in B1 does not prevent its response to Gata4\u20136. Furthermore, only the Sox and T-box sites of B1 are essential for expression in the endoderm. While other Xsox17-inducing factors, like Mix\/Bix proteins, could be relevant, another candidate is maternal Xsox7, which is localised in the vegetal pole (Zhang et al., 2005). Indeed, we have shown that overexpression of Xsox7 in the animal hemisphere results in the induction of Xsox17, although again this is slow, as with Xsox17 itself (data not shown).\nNevertheless, the case for Xsox17 auto-regulation is compelling, now being based on three independent lines of evidence. Firstly, Xsox17 induces itself in ectopic expression experiments, and this is direct (Sinner et al., 2004); secondly, there are essential Sox sites in a promoter element that drives endodermal gene expression in transient and true transgenics; thirdly ChIP analysis shows that the endodermal element is bound to Xsox17 in vivo. Based on the activity of a glucocorticoid derivative of VegT in the absence of protein synthesis, Xsox17 is a direct target of VegT (Clements and Woodland, 2003), and our results show that it co-operates with Xsox17 (and possibly other transcription factors) to establish and amplify Xsox17 gene expression. The fact that Xsox17 expression is ultimately dependent on TGF-\u03b2 signalling makes this amplification and stabilisation of its own expression signal-sensitive and hence subject to a community effect (Gurdon et al., 1993a,b). We propose that this involves the C3 element. A simplified version of this network is shown in Fig. 6. An important point to note is that other members of the endodermal network, like the Mix\/Bix genes, are similar to Xsox17 with respect to VegT induction and signal dependence.\nThere are still many issues to be resolved relating to the regulatory elements. A VegT-induced inhibitor of its own action in inducing Xsox17 was postulated to explain the switch to signal dependence of VegT action, and this is still unknown. It may relate to the repressive effect of VegT and vegetal position on C3, a repression that is lifted by TGF-\u03b2 signalling. There also appear to be inhibitors of expression in other parts of the embryo. For example, the loss of the Sox sites appears to increase the background expression in the animal pole. This could involve the action of the inhibitor Xsox3 (Zhang et al., 2003, 2004). At later stages. there is also expression of most transgenes in regions like the axis, where Xsox17 is not expressed. This was not seen in a construct including 3\u00a0kb of 3\u2032 sequence, suggesting that late inhibitory sites are present in this region.\nThe regulatory interactions described here are elements within the core endodermal network (Sinner et al., 2006). The form of the regulation, where VegT induces several factors, such as TGF-\u03b2s and Xsox17, and co-operates with them to regulate an important control gene, is an important recurring regulatory motif, the feed-forward motif (Mangan and Alon, 2003). In this case, it also brings about autoregulation. Autoregulatory effects of proteins in transcription factor networks are thought to have special importance. Mochizuki (2005) found that the number of possible stable states of gene networks depended only on the number of autoregulated components. Since it is reasonable to equate stable states with differentiated states, the autoregulation of a key endodermal transcription factor would be most significant in endodermal differentiation.","keyphrases":["xenopus","sox17","promoter","vegt","endoderm","tgf-\u03b2","transgenic embryo"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"J_Med_Internet_Res-7-3-1550662","title":"The Role of User Input in Shaping Online Information From the National Cancer Institute\n","text":"The National Cancer Institute (NCI) was among the first federal agencies to recognize the potential of the Internet for disseminating health-related information. The evolution and refinement of NCI's online cancer information has been substantially \u201cuser driven\u201d\u2014from the launch of CancerNet in 1995 to the recent redesign of its award-winning successor, the NCI website. This article presents an overview of NCI's multi-pronged approach to gathering input about its online information products, including stakeholder meetings, focus groups, standard and customized online user surveys, usability testing, heuristic reviews, and search log analysis. Also highlighted are some of the many enhancements that have been made to NCI's online cancer information products based on user input.\nIntroduction\nThe National Cancer Institute (NCI) was among the first federal agencies to recognize the potential of the Internet for disseminating health-related information, and it launched its CancerNet website in 1995. This site was a natural extension of NCI's information dissemination efforts, which have been carried out in response to mandates from Congress in the National Cancer Act of 1971 [1] and subsequent legislation. Table 1 outlines major milestones in the development of NCI's Web presence.\nTable 1\nMilestones in the development of NCI's website\nYear\nMilestone\n1995\nCancerNet website is launched.\n1999\ncancerTrials website is launched.\n1999\nCancerNet website is redesigned.\n2002\nNCI's overarching website [2] is redesigned; CancerNet and cancerTrials websites are subsumed into the redesigned site.\n2004\nNCI website is redesigned.\nA large part of NCI's pre-1995 information dissemination efforts was targeted at health professionals through the Physician Data Query (PDQ\u00ae) cancer information database, which contains information summaries on numerous cancer-related topics and a cancer clinical trials registry. PDQ was available to medical librarians, physicians, oncology nurses, and other professionals through the National Library of Medicine's online information system [3-6].\nThere was, however, a new dynamic in the development of the Web. Cancer patients were coming online in large numbers, seeking to be informed decision makers in their own care. Simultaneously, the patient advocacy community was becoming more vocal in requesting that NCI provide products geared to patients. NCI responded to this growing audience by organizing the CancerNet website by audience type, with entry points for patients, health professionals, and researchers, and with information categorized accordingly.\nThe evolution and refinement of NCI's online cancer information has been notably \u201cuser driven.\u201d NCI has adopted a multifaceted approach to gathering feedback and other information about how its information products are used. This has included pre- and post-design tests in usability labs, heuristic or expert review, informal user feedback, standard online user surveys, focus groups, analysis of site usage and search logs, and special user survey projects. Each generation of NCI's Web presence has been informed by user feedback. NCI staff members were crucial leaders in developing usability guidelines and standards that are now widely accepted in the industry, and NCI was one of the first federal agencies to conduct systematic usability testing with its CancerNet website.\nThis article presents an overview of the methods NCI has used to gather input about its online information products and services. It is not the result of research projects that set out to test specific hypotheses about the impact of specific user-driven enhancements. Rather, it presents an approach to information architecture and design of a website that uses a variety of methods to gauge user behaviors and preferences. It highlights some of the many enhancements that have been implemented in response to user data and feedback. While NCI's website contains a wealth of additional information about cancer research opportunities, funding, NCI programs\/initiatives, cancer statistics, and information for the news media, space limitations prevent a discussion of the role of user input in the design and implementation of these areas. The focus of this article will be enhancements to patient-oriented cancer information and information about clinical trials.\nHow NCI Gathers User Input and Feedback\nStakeholder Input\nNCI solicits user input prior to any major online system design or redesign. For example, in response to a growing need for clinical trials information, and prior to a major redesign of the backend database and the user interface of its CancerNet website, NCI organized the Clinical Trials Information System meeting in Chantilly, Virginia, USA, in 1998.\nApproximately 200 patients, advocates, clinicians, oncology nurses, clinical investigators, and health information providers representing the core users of NCI's online information resources came together to brainstorm the design of a clinical trials information system.\nFigure 1Before and after screen shots of the CancerNet home page, showing a shift in focus from audience to topic with the 1999 redesign\nSome of the key recommendations of the meeting were the following: (1) that the NCI website avoid segmenting information pathways according to type of user (patient, physician, researcher); (2) that information be customized to provide varying degrees of technical detail, complexity, and reading level; and (3) that users be able to easily move between these levels. It was also recommended that the NCI website integrate clinical trials information with the full spectrum of cancer information; include information about clinical trials, patient rights, and the informed consent process; and include a feature covering news topics related to clinical trials.\nOne of the outcomes of this meeting was the development of a new NCI website, cancerTrials, to provide an educational context for the PDQ clinical trials registry that was offered on CancerNet. The cancerTrials website was launched in 1999. In addition to guidance on how to search the PDQ registry, visitors to the new site were offered original articles explaining what cancer clinical trials were, how they worked, and where to find them. They also were offered brief summaries of recently announced cancer trial results and other timely news related to the US clinical trials system.\nThe subsequent redesign of CancerNet in 1999 [7] carried out the Chantilly recommendation to abandon the partition of the site by audience (Figure 1). Now, the site gave all users information organized around a standard set of topics. Information was presented at varying levels for most of the common cancers\u2014including the \u201cWhat You Need To Know\u201d series for the most basic introduction, patient and health professional versions of the PDQ cancer information summaries, and abstracts (summaries) of clinical trial protocols written for patients and health professionals. The new design also made it easy for users to switch between the different information levels. Input obtained at the Chantilly meeting continues to influence the development of NCI's cancer information products and their presentation to users.\nOngoing Feedback from CIS Information Specialists\nInformation specialists at NCI's Cancer Information Service (CIS) are the front line of NCI's interactions with the cancer community, particularly the public [8]. Through the CIS toll-free telephone service (1-800-4-CANCER) and \u201cLiveHelp\u201d online chat sessions, information specialists help individuals who are seeking cancer information. As needed, they can assist callers and website visitors with NCI online tools and resources. As \u201cpower users\u201d of the NCI website, they often help test new features. Regular feedback from the CIS to website staff helps drive website improvements.\nUser Surveys\nA critical factor in achieving continuous improvement of NCI's Web resources is soliciting user feedback to learn what works, what doesn't, and where gaps in information or functionality exist. In preparation for the 1999 redesign of CancerNet, an online survey asked users to identify the information they were seeking (Table 2), difficulties they encountered on the site, features they found useful, and additional information or features that were needed. Users were also asked about their general Web usage and basic demographics.\nTable 2\nType of information users were seeking on CancerNet (1999)\nPercentage\nof Respondents\n(N = 780)\nInformation Sought\n22.8\nInformation on a specific type of cancer\n18.6\nTreatment information (general and specific)\n11.3\nClinical trial information (specific trials, general information, trial results)\n8.1\nSymptoms of cancer, causes, risk factors, detection, diagnosis, prevention\n6.8\nSpecific term (type of tumor or other term\u2014not by name of cancer)\n4.7\nCancer literature\/articles\n4.5\nNew treatments, news, recent findings, current research projects\n3.5\nPatient support (pain relief, diet\/nutrition, survivorship, exercise, follow-up, questions to ask doctor)\n3.1\nSide effects\n2.9\nStatistics (incidence rates, survival rates, mortality rates)\n2.8\nDrug information\n2.8\nAccess to other cancer resources (treatment facilities, physician names\/specialties, national tumor registry, cost information, insurance coverage, patient support group)\n2.2\nCaregiver information (how to help patient, what to expect as disease progresses, how to talk to patient, etc)\n2.1\nHistory of cancer research, information for reports\/projects\n1.8\nNCI publications (ordering information)\n0.8\nAlternative treatments\n0.6\nGenetic information (general and specific)\n0.4\nSearch engine for the site\n0.3\nInformation about oncology professions\nFigure 2The \u201cTypes of Cancer\u201d page on CancerNet\nFeedback from the online survey, along with input from the Chantilly meeting, guided the redesign of CancerNet in 1999. On the redesigned site, users could start with the \u201cTypes of Cancer\u201d page (Figure 2), which enabled users to quickly find information about specific cancers. They could then choose a cancer-specific home page (Figure 3), where information related to the cancer was organized by topics such as \u201cIntroductory Overview,\u201d \u201cStatistics,\u201d \u201cTreatment,\u201d and \u201cClinical Trials.\u201d\nFigure 3The \u201cBreast Cancer\u201d page on CancerNet, an example of a cancer-specific home page\nIn 2002, NCI's overarching website was redesigned, and the CancerNet and cancerTrials websites became the Cancer Information and Clinical Trials portal areas of the redesigned site. In 2004, the NCI site underwent another redesign, once again guided by extensive evaluation and user input.\nNCI's early decision to provide information tailored for patients and their families continues to be supported by surveys conducted during the past five years. Data from 1999 showed that 44% of visitors to the site described themselves as cancer patients or family members or friends of a cancer patient. Data from the American Customer Satisfaction Index (ACSI) survey posted on the NCI website in 2004 showed that more than 50% of respondents identified themselves as cancer patients or family or friends. The next largest audience in 2004 was health care providers, about 13%. NCI continues to keep the patient at the center of many of its online resources\u2014PDQ's cancer information summaries and clinical trial abstracts, clinical trial results summaries, fact sheets and other information products, and the website's dictionary are all written for lay audiences.\nAmerican Customer Satisfaction Index (ACSI Survey)\nBoth before and after the 2004 redesign, the website displayed the ACSI survey [9]. This survey gathers input from users at points within the website. The ACSI survey can be utilized site-wide or for a certain URL. One version of the survey can be posted to appear randomly on all pages of the site (Appendix 1), and another can be set to appear on a group of related pages to collect in-depth data on a particular subject.\nThe ACSI methodology provides continuous online feedback and is a uniform, national, cross-industry measure of customer satisfaction. A core set of ACSI questions measures overall satisfaction, and customized questions can be added regarding individual websites or pages.\nData from the survey are helpful in supporting or dispelling impressions of who uses a site and what their information needs are. For example, data from the 2004 ACSI survey showed that approximately 57% of NCI's website visitors are first-time users of the site. This underscores the need for intuitive site structure and navigation tools that can be easily grasped by users with no prior knowledge of the site. Multiple paths to core information, such as cancer-specific home pages and clinical trial search tools, were created in 2004 to help new users easily find the most sought-after information. While we cannot make a direct correlation between these enhancements and increased customer satisfaction, the ACSI survey results published in December 2004 named the NCI website the \u201cbest in customer satisfaction\u201d in the portal\/department main site category [10]. Overall satisfaction among visitors to major government online portals was 72.1, on a scale of 0 to 100. The NCI website led the category for government sites with an overall satisfaction score of 80. In the first quarter of 2005, the NCI website was again the highest scoring government portal site, with a score of 80. NCI expects to further analyze ACSI data to inform additional improvements to the website.\nUsability Testing and Expert Review\nUsability testing helps ensure that products and services address the needs and interests of website visitors [11,12]. In lab sessions with representative users, testers pose scenarios (see Appendix 2) and solicit comments to gauge the effectiveness of page designs, functions, navigation paths, labels and terminology, and other elements. Data from iterative testing inform the refinement of key pages and the development of new features. NCI also consults with experts on user-centered design to help ensure that its information products keep pace with current standards and trends. For example, prior to the launch of the redesigned NCI website in 2004, a panel of experts was involved in heuristic reviews, and their recommendations led to additional refinements prior to the launch.\nSearch Log Analysis\nSearch log analysis played an important role in the 2004 redesign of the NCI website. Each year, users enter approximately 2.5 million free-text searches in the basic search box on the site. More than 50% of searches are for types of cancer or specific body systems or locations. To give visitors immediate access to information on the most common cancers, prominent links for each of these cancers were added to the site's home page, along with multiple links to an A to Z list of cancers to enable easy information retrieval (Figure 4). The same selection of links to common cancers and the A to Z list was also placed on the site's Cancer Topics portal page (which replaced the Cancer Information portal page introduced in the 2002 redesign). (For more information about search log analysis, see \u201cBest Bets on the Website\u201d below.)\nFigure 4The home page of the current NCI website, prominently featuring \u201cTypes of Cancer\u201d and links to \u201cCommon Cancer Types\u201d\nSelected User-Driven Enhancements\nBest Bets on the Website\nWhen the NCI website was redesigned in 2002, the site's search tool was supplemented with a \u201cBest Bets\u201d feature that gives users a concise list of editorially selected NCI sites and pages that are displayed above the full set of search results. Whereas the full set of search results, which are generated by a free-text search of NCI's Web content, can number thousands of documents for a given search term, the Best Bets offer an average of two links, with a range of one to 18. There are currently 677 Best Bets categories (eg, lung cancer, mammography, cancer diagnosis program) with selected Spanish-language categories included.\nTo populate Best Bets initially, a team of information experts identified cancer-related information categories, selected the most relevant NCI sites and pages for each, and created a table of related terms for each category name. When a search term is entered in the search box on the site and a category name or related term matches the term or any part of the term, the associated list of Best Bets is displayed.\nSearch log analysis after the launch of the Best Bets feature in 2002 validated the choice of category names and related terms, the majority of which proved to be among the more popular search terms. Since 2002, the Best Bets database has been edited by NCI staff as needed, on the basis of periodic analysis of search logs and knowledge of new and changing NCI Web content. Log analysis has prompted a considerable expansion of the Best Bets database by suggesting new category names and related terms. In addition, there have been a few instances in which the large number of searches on a topic indicated the need for new content. These findings have already led to the creation of two important pieces of content (which, in turn, were classified as Best Bets), namely a fact sheet about cancer staging and a substantial resource on the NCI website called the \u201cTobacco and Cancer\u201d home page. Best Bets categories, related terms, and links have also been added in response to comments from users.\nClinical Trials Portal Redesign\nIn the summer and fall of 2002, NCI initiated a multi-pronged review of the Clinical Trials portal of its website (Figure 5) to determine whether the portal was meeting the needs of its users. Particular attention was given to the Clinical Trial Results section of the portal; articles in the section are also referred to as \u201cnews summaries\u201d [13].\nFigure 5The Clinical Trials portal home page on NCI's website (2002)\nEvaluation Methods\nThe 2002 evaluation used six qualitative and quantitative methods:\nThe initial phase of the evaluation involved key informant interviews with NCI staff integral to the development of the portal.\nA diary activity was conducted to capture feedback from users who were representative of three of the portal's target audiences, including patient advocates, oncology nurses, and CIS information specialists. Participants were asked to complete a written, formatted diary entry for each visit they made to the portal in the course of their regular activities over a period of one month.\nFocus groups and in-depth interviews were later conducted to gain more feedback.\nTwo online surveys were posted in the Clinical Trials portal of NCI's website. A general survey was presented to each user who visited any page of the portal except for news summaries. A news summary survey was presented to users who visited news summaries. Session cookies were used to recognize possible repeat visitors and to serve up the survey once per visitor during a 30-minute time period (to minimize both the burden on the public and duplicate responses). Once duplicate responses were eliminated, the adjusted survey sample contained 1589 general survey responses and 207 news summary survey responses.\nUsability testing was conducted to determine whether users could easily find and understand the news summaries. Perceived usefulness of the news summaries was also explored in usability testing with six participants.\nServer log file entries were analyzed using WebTrends log analysis software to collect the following usage statistics: unique visitors, visitors who visited once, visitors who visited more than once, sessions, median visit length, page views, and visits from referring sites.\nKey Findings\nSeveral key findings emerged from these evaluation methods [14]. The top three categories of information that visitors were looking for were (1) specific cancer clinical trials (ie, they wanted to search the PDQ registry); (2) recent research results about a specific cancer treatment, test, or prevention; and (3) recent research about a specific type of cancer.\nWith regard to the Clinical Trials portal, most users found the information they needed, were able to understand it, and found it useful. However, they had difficulty finding their way to the Clinical Trial Results news summaries, even though this type of information was among the top three categories of information desired. When directed there (or when identified as having been there via the pop-up exit survey), users found the summaries to be useful, understandable, and well organized.\nInformed Changes\nThese findings were used to inform changes to the content and design of the Clinical Trials portal over the course of 2003 and again during the 2004 redesign of the NCI website (Figure 6). For example, to make it quicker and easier for users to search the PDQ clinical trials registry, the website's basic search form for clinical trials was added to the Clinical Trials portal home page [15]. Links to this form are also located throughout the pages of the Clinical Trials portal and elsewhere on the site.\nFigure 6The Clinical Trials portal home page on the 2004 redesigned NCI website\nTo further help visitors locate specific trials in which they might be interested, a new section was created called Featured Clinical Trials [16]. This section is updated on a weekly basis and includes brief profiles of key NCI-sponsored clinical trials, with links to more detailed information about the trial. Both the Featured Clinical Trials and the Clinical Trial Results sections were redesigned to allow users to browse by type of cancer and to search the collections by keyword from anywhere in the sections.\nIn addition, links to the Clinical Trial Results pages organized by type of cancer are more prominently displayed on the Clinical Trials portal home page, and teasers (brief description and link) for the two most recently posted Results articles are prominently displayed on the Clinical Trials portal home page.\nImproved Searching for Clinical Trials\nThe PDQ clinical trials registry has been a key component of NCI's online cancer information services from its inception in the 1980s [3]. Originally designed for health professionals, the registry is now also widely used by patients and is one of the most popular features of NCI's website. Since January 2003, more than 50000 visitors per month, on average, have searched for clinical trials. Designing a search application that works equally well for patients, caregivers, health professionals, and researchers has been a major challenge, and NCI has relied on feedback from users as well as insight from experts to guide each version of the clinical trials search form.\nSince its appearance on the Web on CancerNet, the complexity of the clinical trials search form has been a topic of discussion within NCI. The PDQ clinical trials registry began as part of a DOS-based, menu-driven system used almost exclusively by health professionals, medical librarians, and cancer information specialists. Developers were wary of transplanting the sophisticated search functionality of this system to NCI's website because many of the site's visitors had little familiarity with clinical trials, cancer staging, treatment choices, and other elements in the original system. Initial methods of clinical trial searching on NCI's CancerNet website included a form with limited search options and clinical trial descriptions written in technical language, a legacy from the original system. Simply written, patient-friendly descriptions of clinical trials were introduced in 1997.\nTwo-Step Search Form\nThe second-generation search form that was launched in 1999 was based on recommendations from the Chantilly meeting, data from an online feedback form on the website, analysis of the search form, and personal interviews. In addition, a prototype of the form was developed through iterative rounds of usability testing. A two-step search form was designed to allow users to search by common search parameters, such as type of cancer, type of trial, and geographic location. Users could then review their search results or choose to narrow their search with other parameters, such as stage of cancer, drug (including brand and generic names), type of treatment, and trial sponsor. Users were also given the option of viewing two descriptions of each clinical trial, one for patients and one for health professionals.\nOther changes to the search form based on usability testing included a user's guide for less experienced users, annotated labels for search parameters with links to more detailed explanations, and explanations of how to select multiple items per field (eg, selecting several stages of breast cancer).\nAudience-Focused Search Form\nIn the 2002 redesign of the NCI website, the clinical trials search form was included in the Clinical Trials portal of the site, giving users a more integrated information pathway that grouped information such as patient safety, informed consent, and insurance issues with the listing of clinical trials.\nA major consideration in this redesign was the addition of a specific new group of users\u2014information specialists from the CIS, whose duties include assisting patients, their families, and health professionals in identifying clinical trials of interest. Information specialists had previously used the DOS-based, menu-driven PDQ search system that allowed them to perform complex searches, review results, refine as needed, and then prepare an \u201cinformation packet\u201d that could be emailed or mailed to callers. Web designers visited a CIS regional office to understand the needs of this group of users and did extensive usability testing with them prior to launching the revised form.\nGiven the diversity of users, it became clear that a single search form was not ideal. Some users found the detailed choices on the form confusing and beyond what they needed. An interactive search form that guided users through the search process was considered, but such a form would require JavaScript, which does not meet Web accessibility requirements for federal government websites. It was determined that the best approach was to develop two search forms with different levels of complexity. Web accessibility requirements could be satisfied by creating one form without JavaScript, and a more complex, interactive form could be created with JavaScript.\nThe basic search form, designed for the patient, caregiver, or busy health professional, provided three search options\u2014cancer type, type of trial, and zip code proximity. The results were also presented in a format more suited to the casual Web user, who was accustomed to clicking on a search result link to go to a page that contained more information. Usability testing had also indicated that users did not normally click on the check boxes that were provided with the search results in order to prepare a \u201cpackage\u201d for viewing or printing as a batch.\nThe advanced search form [17] was JavaScript enabled with key enhancements that included (1) dynamic population of the cancer subtype\/stage search options based on cancer type selection, and (2) expanded trial site and location searching, including searches by zip code proximity and hospital. In addition, browse lists for drugs, hospitals, and investigators were added to support more precise searching. Users could search for a character string and find appropriate values to add to the search form, or they could browse data-generated pick-lists alphabetically for drug, hospital, or investigator.\nIn addition, for the CIS users, the search results display was developed to enable information specialists to read a preliminary result set, so they could identify the most appropriate trials for their callers and prepare an \u201cinformation packet.\u201d\nBetter Visibility for Search Forms\nThe 2004 redesign of the NCI website saw further changes in clinical trials searching. Based on user input, the ability to narrow a search to subtype or stage of cancer was added to the basic search form. User feedback also indicated that physicians preferred trials to be listed by phase rather than by title, so the default display of search results was changed to a listing by phase, with phase IV and phase III trials appearing before phase II and phase I trials. The most substantial change, as a result of the Clinical Trials portal review, was adding the basic search form to the top of the Clinical Trials portal home page to give more ready access to the form [15]. With continued feedback from users, the search forms will be improved further to allow more precise clinical trial searching\u2014for example, an interactive format may be developed to help identify trials with eligibility criteria that match patient characteristics.\nPatient-Oriented Clinical Trial Abstracts\nIn the summer of 1996, NCI collaborated with the National Alliance of Breast Cancer Organizations (NABCO) to develop patient-oriented abstracts (summaries) of clinical trial protocols for breast cancer trials. By October 1996, these clinical trial abstracts were available on the NABCO and CancerNet websites in a one-paragraph format. After seeking input from many advocacy organizations, the patient-oriented clinical trial abstract format was redesigned, writing guidelines were developed, and the project was expanded to include all cancer types. By September 1998, patient-oriented abstracts for all active clinical trials were available on CancerNet. Since that time, clinical trial abstracts have been written according to the original guidelines.\nIn November 2001, selected patient-oriented and corresponding health professional clinical trial abstracts were evaluated. As a result of this evaluation, several problems were identified in the guidelines for writing the patient-oriented abstracts, including a lack of specificity in some respects and inconsistent application and interpretation of the guidelines. These findings led to the recommendation that the guidelines be redefined and expanded. Consequently, a qualitative and quantitative evaluation of the needs and preferences of users of the patient-oriented abstracts was undertaken. This evaluation included the following two elements: (1) a written survey of advocacy organizations, members of NCI's Consumer Advocates in Research and Related Activities (CARRA) Program, members of the NCI Director's Consumer Liaison Group (DCLG), comprehensive cancer center directors and administrators, cancer cooperative group chairs\/administrators, and oncology nurses; and (2) in-depth interviews with CIS information specialists.\nWritten Survey\nA 10-question survey was mailed to nearly 400 organizations and individuals, with a 43% return rate. A key question focused on whether or not users could understand and act on the information provided in the clinical trial abstracts for patients. Results showed that 82% of users could explain the rationale or purpose of the clinical trial, 93% could determine if basic eligibility requirements were met, and 73% could understand the treatment plan.\nThe organization and layout of the clinical trial abstracts were rated \u201cexcellent\u201d or \u201cgood\u201d by 72% of the respondents. Three samples of text written at 5th-, 8th-, and 12th-grade reading levels were included with the survey. The different reading levels were preferred by 37%, 42%, and 20% of the respondents, respectively.\nThe results of the survey were better understood when viewed in the context of comments from individual respondents. Taken as a whole, the respondents' comments were varied and, at times, contradictory. Several themes, however, emerged related to language and readability, access to other resources, and pursuing participation in a clinical trial. Although 27% of the respondents indicated they could not understand the treatment plan, few specific suggestions were offered for improvement.\nInterviews with CIS Information Specialists\nStructured interviews were conducted with staff in six CIS offices in different geographic areas of the United States in order to obtain their perceptions of users' needs, preferences, and comprehension of the standard elements (title, rationale, purpose, eligibility criteria, treatment, and study contacts) of the patient-oriented clinical trial abstracts. The CIS information specialists interact directly with users of the abstracts by answering their questions and by guiding their use of the abstracts online during a phone call or through LiveHelp. The information specialists emphasized the need to use consumer-oriented language and the fact that users \u201cskip\u201d disclaimer-type information.\nBased on these findings and on published principles [18], improvements to the patient-oriented abstracts were implemented as part of the 2004 redesign of the NCI website (Table 3). An example of the current abstract format [19] can be viewed online.\nTable 3\nSelected improvements to the patient-oriented clinical trial abstracts\nCriteria\nImprovement\nUse of Language\nProvide both simplified and health professional versions of the title. Avoid technical terms if a more common term is available (eg, \u201cremoved in surgery\u201d instead of \u201cresected\u201d). Aim for an 8th-grade reading level or lower, except for drug names and medical or scientific terms defined in the website's dictionary (terms are linked to dictionary definitions).\nReadability\nWrite sentences that are as short as the content will allow. Divide lengthy treatment descriptions into smaller paragraphs. Use bullets to separate information about different treatments.\nContent Display\nEmphasize how users who are interested in participating in a clinical trial can seek further information. Incorporate disclaimer information into the eligibility and trial contact information sections. Provide a boxed sidebar containing links to complementary information about clinical trials and drug information in the National Library of Medicine's MedlinePlus. Keywords in the title should not be linked to dictionary definitions. They should be linked from the purpose or treatment sections rather than the title.\nWeb-Friendly Cancer Information Summaries for Patients\nThe PDQ cancer information summaries are descriptions of the latest cancer information on treatment, supportive care, screening, prevention, genetics, and complementary and alternative medicine that are reviewed and updated monthly by cancer experts. Most of the summaries are available in two versions: one written for health professionals and a corresponding patient version written in lay language. (A small number of the summaries are available only in the health professional version.) In 2000, in response to the Chantilly meeting, work was initiated to reformat the patient-oriented information summaries. The goal was to present the information in a format and style of language that was easier to read and understand, to provide more detailed information, and to take advantage of features afforded by new Web technology.\nBased on design concepts that enhance readability, as well as on strategies used in information mapping, the process of reformatting and reorganizing the patient-oriented summaries was begun. \u201cKey Points\u201d boxes that highlighted critical concepts and linked to explanatory information in the body text were added. Links to pop-up definitions from the website's dictionary and to clinical trials information were included. For users who wished to print documents, a printer-friendly version was added that included dictionary terms and their definitions as an appended glossary.\nUsability testing was done to assess the ease of learning, efficiency in information gathering, and recall of information from the online documents. Based on testing results and Web design and usability guidelines [18], the template for the patient-oriented summaries was further refined, and the redesign has been well received by users. An example of the current summary format [20] can be viewed online.\nConclusion\nNCI's website is a leading resource for cancer information on the Web, consistently appearing high on the list of retrievals using search engines such as Google, Yahoo, MSN, and AltaVista. It has been awarded the Freddie Award in the website category of the 2004 International Health and Medical Media Awards, and it placed first or as an honorable mention in seven out of eight categories in the 2005 Medicine on the Net Web Excellence Awards. Its success can be at least partly attributed to NCI's efforts to make the site highly responsive to the needs of its users.\nThe large volume of traffic that the site receives offers tremendous opportunities to study user patterns, gather feedback, and test new ideas and designs. Online surveys are an efficient way to solicit opinions from users, and analysis of website logs provides insight into user needs. NCI's relationships with members of the cancer research and advocacy communities also facilitate the gathering of advice, suggestions, and other feedback related to NCI information products. The growing body of Web-design literature and advice from usability experts are important to the development of new Web features, but input from the site's wide range of users promises to have the greatest impact on shaping online information from the National Cancer Institute.","keyphrases":["online information","internet","cancer information","website","usability"],"prmu":["P","P","P","P","P"]}
{"id":"Exp_Appl_Acarol-3-1-2039800","title":"Plant structural changes due to herbivory: Do changes in Aceria-infested coconut fruits allow predatory mites to move under the perianth?\n","text":"Being minute in size, eriophyoid mites can reach places that are small enough to be inaccessible to their predators. The coconut mite, Aceria guerreronis, is a typical example; it finds partial refuge under the perianth of the coconut fruit. However, some predators can move under the perianth of the coconut fruits and attack the coconut mite. In Sri Lanka, the phytoseiid mite Neoseiulus baraki, is the most common predatory mite found in association with the coconut mite. The cross-diameter of this predatory mite is c. 3 times larger than that of the coconut mite. Nevertheless, taking this predator\u2019s flat body and elongated idiosoma into account, it is\u2014relative to many other phytoseiid mites\u2014better able to reach the narrow space under the perianth of infested coconut fruits. On uninfested coconut fruits, however, they are hardly ever observed under the perianth. Prompted by earlier work on the accessibility of tulip bulbs to another eriophyoid mite and its predators, we hypothesized that the structure of the coconut fruit perianth is changed in response to damage by eriophyoid mites and as a result predatory mites are better able to enter under the perianth of infested coconut fruits. This was tested in an experiment where we measured the gap between the rim of the perianth and the coconut fruit surface in three cultivars (\u2018Sri Lanka Tall\u2019, \u2018Sri Lanka Dwarf Green\u2019 and \u2018Sri Lanka Dwarf Green \u00d7 Sri Lanka Tall\u2019 hybrid) that are cultivated extensively in Sri Lanka. It was found that the perianth-fruit gap in uninfested coconut fruits was significantly different between cultivars: the cultivar \u2018Sri Lanka Dwarf Green\u2019 with its smaller and more elongated coconut fruits had a larger perianth-fruit gap. In the uninfested coconut fruits this gap was large enough for the coconut mite to creep under the perianth, yet too small for its predator N. baraki. However, when the coconut fruits were infested by coconut mites, the perianth-rim-fruit gap was not different among cultivars and had increased to such an extent that the space under the perianth became accessible to the predatory mites.\nIntroduction\nTo reduce the predation risk, some organisms show behavioural or morphological changes that are induced by their predators (Wiackowski and Staro\u0144ska 1999; Buskirk and McCollum 2000; Oku et\u00a0al. 2003). Some have adapted to find refuge in such a way that predators cannot reach them. Eriophyoid mites have a worm-like body with a very small cross-section diameter (40\u2013100\u03bcm) that allows them to reach concealed plant parts or to live in self-induced, small plant galls where they find protection from biotic and abiotic stresses (Sabelis and Bruin 1996). The coconut mite, Aceria guerreronis Keifer (Acari: Eriophyidae), feeds on the meristematic tissue beneath the perianth covering the base of the coconut fruit. Here it not only profits from the relatively nutritious value of this tissue, but also from the reduced risk of predation due to the perianth. Among the predatory mites that have been reported to be associated with the coconut mite in Sri Lanka, Neoseiulus baraki Athias-Henriot (Acari: Phytoseiidae) is the most frequently found species (Fernando et\u00a0al. 2003; Moraes et\u00a0al. 2004). This species was previously referred to as N. aff. paspalivorus (Fernando et\u00a0al. 2003), but was later confirmed as N. baraki (Moraes et\u00a0al. 2004). It has a flat and elongated idiosoma (Moraes et\u00a0al. 2004) and we suggest this might make this predator\u2014relative to other phytoseiid mites\u2014better suited to creep into narrow spaces.\nWhen the coconut mites are outside the perianth they are exposed and vulnerable to predators, but under the perianth of the coconut fruit they face less risk of being eaten. In the absence of natural enemies, coconut mite populations may grow exponentially and, consequently, the development of the coconut fruit will be impaired. Therefore, we expect the coconut palms to defend themselves directly against the coconut mites and\/or indirectly by promoting the efficiency of predators against these herbivores (e.g. Sabelis et\u00a0al. 2007). In this article, we investigate whether coconut fruits exhibit a mode of indirect defence that is similar to that observed in tulip bulbs by Lesna et\u00a0al. (2004). These authors have found that, when tulip bulbs are attacked from within by the eriophyoid mite Aceria tulipae Keifer (Acari: Eriophyidae), bulbs increase the gap between scales to such an extent that predatory mites can enter the interior of the bulbs. This prompted us to hypothesize that mite-infested coconut fruits undergo a change in perianth structure with the effect that predatory mites have better access to the space underneath the perianth and thereby to the coconut mites. To test this hypothesis we measured the gap between the perianth and the surface of coconut fruit (\u201cperianth-fruit gap\u201d) when uninfested and when infested by coconut mites, and compared the size of the gap with the size of the predatory mite N. baraki.\nThe perianth functions as a protective cover to the female flower and the tender meristematic zone of the growing coconut fruit. In young coconut fruits (i.e. 1\u20132\u00a0months after fertilization) the perianth is tightly appressed to the surface of the coconut fruit (Howard and Abreu-Rodriguez 1991), but, as the coconut fruit grows, the perianth-fruit gap increases slightly, but apparently just sufficient for the coconut mites to move under the perianth and feed on the meristematic zone of the coconut fruit. Tightness of the perianth (Howard and Abreu-Rodriguez 1991), bract arrangement (Moore 1986) and shape (Mariau 1986) of the coconut fruit have been shown to affect the susceptibility of coconut fruit to the coconut mites. Thus, perianth structure affects the probability of coconut mite infestation, but the extent of the effect depends on the growth phase of the coconut and on the palm cultivar. Coconut mites usually do not infest the meristematic zone of unfertilized coconut flowers (Mariau and Julia 1970; Hall and Espinosa-Becerril 1981; Moore and Alexander 1987). After fertilization, coconut fruits of all stages are susceptible to mite attack but in general, peak populations occur in 3- to 7-months-old coconut fruits (Moore and Alexander 1987; Ramaraju et\u00a0al. 2002; Fernando et\u00a0al. 2003). For our experiments we used 4-month-old coconut fruits of three cultivars, commonly grown in Sri Lanka. We measured the perianth-fruit gap for each of these cultivars when uninfested and when infested by coconut mites. Finally, we compared the size of the gap with that of the predatory mite, N. baraki, to make inferences on accessibility of the space under the perianth to the predators of coconut mites.\nMaterials and methods\nPerianth-fruit gap measurement\nFour-month-old coconut fruits (i.e. 4\u00a0months after fertilization) were collected from palms of the three cultivars: (1) cultivar \u2018Sri Lanka Dwarf Green\u2019 (DG), which has usually small, elongated coconut fruits, (2) cultivar \u2018Sri Lanka Tall\u2019 (SLT) with larger and more round-shaped coconut fruits, and (3) a hybrid \u2018Sri Lanka Dwarf Green\u00a0\u00d7\u00a0Sri Lanka Tall\u2019 (DGT). After bringing the coconut fruits to the laboratory they were first split transversely into two halves to remove nut water. This made it easier to dissect the coconut fruit into four longitudinal sections across the perianth (Fig.\u00a01). Dissected coconut fruits with disturbed perianth structure and loosened fibres at the coconut fruit surface were discarded from the measurements.\nFig.\u00a01Bract arrangement of the perianth on a coconut fruit. Longitudinal sections were taken along line A and B. OB-Outer bracts of the perianth, IB-Inner bracts of the perianth\nAfter splitting coconut fruits into four sections, the perianth-fruit gap was measured at two different places on each section (Fig.\u00a02) using a stereomicroscope with a graticule. The first measurement (L1) was made at the rim of the perianth where it touches the coconut fruit surface (Fig.\u00a02). The edge of each bract of the perianth has two different positions: (1) the edge that directly touches the surface of the coconut fruit, (2) the edge that overlaps (or is overlapped by) another bract. Measurements were not taken at the latter position, as it was difficult to dissect the coconut fruits along this position of the bract without disturbing the structure of the perianth. The second measurement (L2) was taken 1\u00a0cm away from position L1, higher up along the surface of the coconut fruit (Fig.\u00a02). These two perianth-fruit gaps were measured in 157 infested (61 from SLT, 39 from DG and 57 from DGT) and 114 uninfested (43 from SLT, 28 from DG and 43 from DGT) coconut fruits.\nFig.\u00a02Longitudinal section of a coconut fruit showing position L1 at the edge of the bract touching the coconut fruit and position L2, 1\u00a0cm away from L1 along the surface of the coconut fruit\nMite census\nAfter measuring the gap between the surface of the coconut fruit and the perianth, bracts of each coconut fruit were removed to count the number of mites on the lower surface of each of them as well as on the underlying surface of the fruit. Counts of total number of mobile stages were done under a stereomicroscope. Total number of N. baraki was counted, whereas the population level of A. guerreronis was estimated by counting the total number of mites from six randomly selected circular (1\u00a0cm \u00d8) patches, three on the lower side of the perianth and three on the surface of the coconut fruit under the perianth.\nSize of the predatory mites\nFor c. 30\u00a0min predatory mites were kept in a Petri dish on wet cotton wool, placed on ice to lower the temperature (to 3\u00b0C), thereby reducing mite activity. Thickness of the soma was taken as a measure of size. This was assessed for 12 female deutonymphs, just before their last moult, and for 20 adult females, ten of which were 1-day-old since their last moult and the other ten were more-than-5-days-old. Measurements in each stage of predatory mites were replicated four times. Because the migratory population of N. baraki mainly consisted of adult females (Kumara, unpublished data) and they are more likely to be the first invaders under the perianth of infested coconut fruits, sizes of the larval and protonymphal stages of the predatory mites were not used in our analysis. The other predatory mite that is commonly found under the perianth is N. paspalivorus which occurs mainly in the wet zone of Sri Lanka (Fernando, unpublished data). We did not measure the size of N. paspalivorus in our study since it does not occur in the dry-intermediate zone where we collected the coconuts.\nStatistical analysis\nThe perianth-fruit gap (L1) appeared to vary with the position along the perianth rim. The mean thickness of adult female N. baraki exceeded the mean value of the measurements taken at four positions along the perianth rim in most coconut fruits. Yet, the soma was less thick than the widest perianth-fruit gap on each coconut fruit. We hypothesized that the predatory mites can find the entrance to the interior of the perianth if there are places with a sufficiently large gap between fruit and perianth. Thus, the mean value of the perianth-rim-fruit gap (L1) is less relevant if it concerns perianth accessibility to the predatory mite. Therefore, it was assumed that the widestgap observed from data obtained from four sites on each coconut fruit was the most relevant variable to be taken into account in the data analysis. Generalized Linear Models (GLM) were used to test differences in the perianth-fruit gap between main factors (category of coconut fruits i.e. infested and uninfested coconut fruits and cultivar) and to assess the interactions between the main factors.\nUsing only the data on infested coconut fruits it was further investigated whether the perianth-fruit gap is a predictor of predator\/herbivore mite density under the perianth. Regression analyses were performed to assess the relation between the widest perianth-fruit gap and the per nut density of coconut mites under the perianth and the per nut density of predatory mites under the perianth. The difference in the density of coconut mites and predatory mites among cultivars were analyzed using one-way ANOVA on log-transformed data. All analyses were carried out using Minitab\u00ae, Version 11.\nResults\nThe mean of the widest perianth-fruit gaps at L1 and L2 in infested and uninfested coconut fruits in three cultivars are shown in Fig.\u00a03. In uninfested coconut fruits these were 41, 68 and 40\u00a0\u03bcm at L1 and 39, 78 and 45\u00a0\u03bcm at L2 in SLT, DG and DGT, respectively, whereas in infested coconut fruits these were 80, 75 and 99\u00a0\u03bcm at L1 and 84, 107 and 94 at L2 in SLT, DG and DGT, respectively. Thus, the perianth-fruit gap at L1 and L2 was significantly higher in infested coconut fruits than in uninfested coconut fruits (Table\u00a01; see also Figs.\u00a03 and 4). No significant difference was observed in the perianth-fruit gap among cultivars at L1. However, the perianth-fruit gap was significantly affected by the cultivar at L2 (Table\u00a01). The interaction between category of the coconut fruit (infested and uninfested) and cultivar was significant at L1, but not at L2 (Table\u00a01). This significant category-cultivar interaction at L1 arises because the increase in perianth-fruit gap due to coconut mite infestation is significant in two cultivars (SLT, DGT), but not in the third cultivar (DG). It should be noted that the perianth-fruit gap of uninfested fruits from the latter cultivar (DG) is very similar to the gap size of infested fruits in the two former cultivars (SLT, DGT) (Fig.\u00a03).\nFig.\u00a03Mean (\u00b1SE) of the widest gap at L1 and L2 in infested and uninfested coconut fruits of three cultivars: SLT\u00a0=\u00a0\u2018Sri Lanka Tall\u2019 (white rectangles), DG\u00a0=\u00a0\u2018Sri Lanka Dwarf Green\u2019 (black rectangles), DGT\u00a0=\u00a0\u2018Sri Lanka Dwarf Green\u00a0\u00d7\u00a0Sri Lanka Tall\u2019 hybrid (grey rectangles)Table\u00a01Analysis of variance of the gap (L1 and L2) between the perianth and the surface of the coconut fruitSources of variancedfMSFPAt L1Category1254,67033.88<0.001Cultivar25,9080.790.457Category\u00a0\u00d7\u00a0Cultivar240,2875.36<0.01Residual2657,516At L2Category1582,23549.27<0.001Cultivar256,5866.870.001Category\u00a0\u00d7\u00a0Cultivar211,8311.440.239Residual2658,232Category refers to classification into infested and uninfested coconuts; df\u00a0=\u00a0Degrees of freedom; MS\u00a0=\u00a0Mean of squares; P\u00a0=\u00a0Critical levelFig.\u00a04Frequency distribution of coconut fruits over classes of widest L1 gaps in infested (black rectangles) and uninfested (white rectangles) coconut fruits (all three cultivars together)\nTo test whether the widest perianth-fruit gap (L1) is a predictor of mite density under the perianth regression analysis was carried out. Because most infested coconut fruits had predatory mites under their perianth, it was not possible to obtain a sufficiently large sample of coconut fruits that had coconut mites only. A scatter plot of the few data points did not show any conspicious trend. Regression of the number of predatory mites under the perianth on L1 measured on the same coconut fruit did not reveal a significant relation (R2\u00a0=\u00a00.04, slope\u00a0=\u00a00.011, P\u00a0=\u00a00.436). This means that among the infested coconut fruits the perianth-fruit gap is not a reliable predictor of the density of predatory mites under the perianth. The absence of a correlation is not unexpected because the density records are snapshots of a dynamic process involving interactions between predator and prey. Sampling randomly over the time course of such an interaction is bound to show no correlation.\nThe frequency distribution of coconut fruits over different gap classes showed different patterns in infested and uninfested coconut fruits (Fig.\u00a04). At L1, 79% of the uninfested coconut fruits had a widest perianth-fruit gap less than 100\u00a0\u03bcm whereas 68% of the infested coconut fruits had a highest perianth-fruit gap exceeding 100 \u03bcm. These percentages, i.e. 21% for uninfested fruits and 68% for infested fruits, are significantly different according to Chi-square analysis of the 2\u00a0\u00d7\u00a02 frequency table that can be constructed from the table below Fig.\u00a04 (X2\u00a0=\u00a058.63\u00a0>>\u00a010.83 at \u03b1\u00a0=\u00a00.001). The frequency distribution of perianth-fruit gap at L2 followed the same pattern as that at L1. In conclusion, the gap between perianth and surface of the coconut fruits increased dramatically when infested by coconut mites.\nThe mean thickness (\u00b1SE) of the female deutonymphs of N. baraki was estimated to be 95\u00a0\u00b1\u00a04\u00a0\u03bcm (n\u00a0=\u00a012). In adult female predatory mites, the mean thickness was 100\u00a0\u00b1\u00a04\u00a0\u03bcm in mites 1-day-old after their last moult (n\u00a0=\u00a010) and 110\u00a0\u00b1\u00a07\u00a0\u03bcm in mites older than 5\u00a0days after their last moult (n\u00a0=\u00a010). However, the sizes of deutonymphs and two age classes of adult females were not statistically different from each other.\nThe number of A. guerreronis varied from 0 to 6491 with means of 1,178, 1,302 and 1,093 in six circular patches of 1\u00a0cm diameter in SLT, DG and DGT, respectively (Fig.\u00a05). Their density (per six circular patches of 1\u00a0cm diameter) was not significantly different among the infested cultivars (P\u00a0=\u00a00.322, df\u00a0=\u00a02 and F\u00a0=\u00a01.14 for log-transformed data). The number of N. baraki ranged from 0 to 55 with means of 9, 13 and 8 mites per coconut fruit in SLT, DG and DGT, respectively (Fig.\u00a05). There was a significant difference in the density of N. baraki among infested cultivars (P\u00a0=\u00a00.024, df\u00a0=\u00a02 and F\u00a0=\u00a03.82 for log transformed data): cultivar DG had a significantly higher density of predatory mites than the other two cultivars. We can therefore not exclude the possibility that the densities of coconut mites are blurred by effects of predatory mites. Unfortunately, sample sizes of coconut fruits \u2018with coconut mites but no predatory mites\u2019 were far too small to warrant further analysis (e.g. in DG there were only five coconut fruits of this type).\nFig.\u00a05Mean (\u00b1SE) of the number of coconut mites (white rectangles) and predatory mites (black rectangles) per coconut fruit from three coconut cultivars (SLT, DG, DGT; see legend of Fig.\u00a03)\nDiscussion\nWe found that the perianth-fruit gap was different among cultivars SLT, DG and DGT of the coconut palm. Except at L1 in infested coconut fruits, among the three cultivars used in our study, cultivar DG had the widest mean gap. In general DG bears smaller and more elongated coconut fruits than the other two cultivars. As observed by Mariau (1977), varieties with small coconut fruits are more susceptible to coconut mites than varieties with large coconut fruits. This may well be because the perianth of the smaller coconut fruits is less firmly attached to the coconut fruit, giving mites better access to the space under the perianth. In our study the highest mean number of coconut mites was also found on DG. However, the density of coconut mites did not significantly differ among cultivars. Therefore, the results of our study did not firmly support the idea that the coconut fruits with larger gap between perianth and the surface of the coconut fruit (in other words fruits with loosely attached perianth) are more susceptible to the coconut mite. It should be noted that the mean widest gap between the perianth and the surface of the uninfested coconut fruits exceeded 40\u00a0\u03bcm in all three cultivars, which is larger than the cross-diameter of adult female coconut mites (36\u201352\u00a0\u03bcm; Keifer 1965). Hence, the perianth-fruit gap in uninfested coconut fruits of any of the three cultivars is large enough for mature coconut mites to enter the space under the perianth. Hence, we expect the sub-perianth space of the three cultivars in our study to be equally accessible to mature coconut mites.\nThe widest perianth-fruit gap per uninfested coconut fruit was much less than 100\u00a0\u03bcm (see Fig.\u00a03) in 79% of the coconut fruits we observed in our study (Fig.\u00a04). However, the mean cross-diameter of the soma of adult female N. baraki was c. 110\u00a0\u03bcm. Therefore, it is clear that the perianth-fruit gap of most uninfested coconut fruits is not large enough for adult females of N. baraki to enter the space under the perianth. This largely applies to the deutonymphs of N. baraki as well, since their mean soma cross-diameter is close to 100\u00a0\u03bcm. Possibly, larvae and protonymphs of N. baraki have a soma small enough to pass the perianth-fruit gap of uninfested coconut fruits. However, juvenile predatory mites are unlikely to be the first invaders under the perianth, because they are much less mobile than adults and because female predatory mites can only lay eggs when there is a sufficient supply of prey. Adult females are therefore more likely to be the first to colonize a coconut, but they cannot enter sub-perianth space unless coconut mites have established. Hence, initially coconut mites experience reduced risk of being eaten by predatory mites when feeding on the tissue beneath the perianth.\nInterestingly, we found that the gap in coconut fruits infested by coconut mites was significantly wider than that in uninfested coconut fruits (Fig.\u00a04). Of the infested coconut fruits in our study, 68% had a perianth-fruit gap above 100\u00a0\u03bcm at L1 which is large enough for N. baraki to creep under the perianth. This suggests the following mechanistic hypothesis to be tested by future experimental analysis: when infested by coconut mites, the coconut fruits increase the gap between the perianth and the surface of the coconut fruit to such an extent that the predators can reach their otherwise-concealed prey. Our results on coconuts are consistent with observations by Lesna et\u00a0al. (2004) on tulip bulbs. When infested by the rust mite, A. tulipae, tulip bulbs become attractive to the predatory mite, Neoseiulus cucumeris Oudemans (Acari: Phytoseiidae) (Aratchige et\u00a0al. 2004). Having a small cross-sectional area, rust mites can easily move into the spaces in between bulbs scales where they are in refuge because the bulb scales in the apex (so called \u2018nose\u2019) of the bulbs are too tightly packed for predators to move in. However, in response to damage by rust mites, bulbs start to produce ethylene which triggers the bulbs to widen the gap between scales in the \u2018nose\u2019 of the bulb, apparently to an extent that is just enough to allow the predatory mites to enter the inside of the bulb (Lesna et\u00a0al. 2004). More importantly, treating the tulip bulbs with an ethylene blocker causes the space between bulb scales to be more narrow: tight enough to prevent access by predatory mites, but definitely not tight enough for the rust mite to enter the inside. Thus, it is not just the feeding damage to the bulb scale tissue that causes widening of the space between bulb scales (Lesna et al. unpublished data). Whether the structural changes in the perianth of coconut fruits in response to coconut mite attack is herbivore-induced plant response or a by-product of necrosis and suberization of the coconut fruit surface due to herbivory, remains to be elucidated, however. The \u2018by-product\u2019 hypothesis is not supported by the observation that the perianth-fruit gap of uninfested fruits from cultivar DG is very similar to the gap size of infested fruits from the same cultivar, as well as the two other cultivars (SLT, DGT) under study. Thus, coconut mite infestation has no significant effect on perianth-fruit gap in cultivar DG and the space beneath the perianth is always accessible to the predatory mite N. baraki, irrespective of coconut mite infestation. Whereas this shows that increased gap size is not a simple consequence of necrosis and suberization of the meristimatic tissue beneath the perianth, it does not disprove this hypothesis (e.g. feeding effects on perianth structure may depend on the structure itself) and\u2014as yet\u2014there is no evidence for the \u2018induction\u2019 hypothesis.\nDetermining whether or not the changes in perianth structure are induced or not, has wider implications. An induced response indicates a net benefit to the coconut palm and hence a reason why it may have been favoured by natural selection. Such an evolutionary response of the plant requires that the association between the eriophyids and the coconut palm has existed for a long enough time and that eriophyids have represented a selective factor of importance. This may not be true for the Americas because Cocos nucifera was introduced some 500\u00a0years ago, whereas A. guerreronis, while native to the Americas, has been first described and discovered as a pest in 1965 (Navia et\u00a0al. 2005). Moreover, there is insufficient data to make any inference on mite-palm associations for the Asian region. Thus, any speculation on the evolution of induced plant responses in perianth structure rests on the assumption of a sufficiently long association with some eriophyoid species or other herbivore (e.g. tarsonemid mites; see Lawson-Balagbo et\u00a0al. 2007) small enough to reach the space beneath the perianth. Alternatively, plants ancestral to coconut palms have been selected to induce a generalized \u2018open-the-gap\u2019 response to attack by any herbivore small enough to enter and feed in otherwise tight folds of plant tissue. Another crucial assumption is that there are predators small enough to enter sub-perianth space and capable of controlling coconut mites. This may hold for the predatory mite N. baraki since it is frequently found associated with coconut mites (Fernando et\u00a0al. 2003), but critical tests to prove its biocontrol capacity are still to be done.\nFor herbivore-induced increase of the perianth-fruit gap to be a plant defense strategy favoured by selection, the benefits should exceed the costs. Clearly, when the perianth-fruit gap is increased, coconut fruits may not only get assistance from predatory mites to combat coconut mites, but they would also become more vulnerable to herbivores that are somewhat larger than eriophyoid mites. This cost may be offset by providing sub-perianth access to a generalist predatory mite, like N. baraki, that can feed on (juvenile stages of) slightly larger herbivorous arthropods as well. However, the increased gap width may also provide intraguild predators, such as Proctolaelaps bickleyi (Acari: Ascidae), with access beneath the perianth, as recently shown in an extensive survey in Brazil (Lawson-Balagbo et\u00a0al. 2007). Most intraguild predators, including P. bickleyi, are larger than their intraguild prey, and this may either lead to partitioning of refuge space under the perianth depending on predator and refuge size or even to exclusion from the sub-perianth space (Lawson-Balagbo et\u00a0al. 2007). We therefore hypothesize that the maximum perianth-fruit gap created by the coconut palm represents a compromise between benefits in terms of protection by predatory mites and costs in terms of desiccation, increased diversity of herbivores and impact of intraguild predators of the effective predators of coconut mites.","keyphrases":["coconut","predatory mites","perianth","coconut mites","eriophyidae","phytoseiidae","induced response","indirect plant defence"],"prmu":["P","P","P","P","P","P","P","R"]}
{"id":"Acta_Neuropathol_(Berl)-3-1-1781098","title":"Subtypes of oligodendroglioma defined by 1p,19q deletions, differ in the proportion of apoptotic cells but not in replication-licensed non-proliferating cells\n","text":"Oligodendrogliomas may be divided into those with deletion of chromosomes 1p and 19q (Del+), and those without (Del\u2212). Del+ tumours show better survival and chemoresponsiveness but the reason for this difference is unknown. We have investigated whether these subgroups differ in (a) apoptotic index, (b) the proportion of cells licensed for DNA replication but not in-cycle, and (c) the relative length of G1-phase. Fluorescence in situ hybridisation with probes to 1p and 19q was used to determine the deletion status of 54 oligodendrogliomas, including WHO grades II and III. The apoptotic index was determined using counts of apoptotic bodies. Replication-licensed non-proliferating cells were determined from the Mcm2 minus Ki67 labelling index, whilst the geminin to Ki67 ratio was used as a measure of the relative length of G1. Del+ oligodendrogliomas showed a higher apoptotic index than Del\u2212 tumours (P = 0.037); this was not accounted for by differences in tumour grade or in proliferation. There were no differences in the Mcm2 \u2212 Ki67 index or in the geminin\/Ki67 ratio between the subgroups, but grade III tumours showed a higher proportion of licensed non-proliferating cells than grade II tumours (P = 0.001). An increased susceptibility to apoptosis in oligodendrogliomas with 1p \u00b1 19q deletion may be important in their improved clinical outcome compared to Del\u2212 tumours.\nIntroduction\nOligodendrogliomas are tumours composed of cells that morphologically resemble oligodendrocytes and that range in spectrum from well differentiated to anaplastic tumours. The current WHO classification of CNS tumours recognises two histological grades, WHO grades II and III, and grading is a significant predictor of survival [23]. Oligodendrogliomas have generally been considered to have a better prognosis than the corresponding grades of diffuse astrocytoma, and recent data continues to support this [21]. However, oligodendrogliomas, like astrocytomas, are diffusely infiltrating tumours, precluding complete surgical resection, and they may show malignant progression over time. There is therefore a role for adjuvant radiotherapy and\/or chemotherapy, particularly for anaplastic tumours.\nApproximately two-thirds of oligodendrogliomas show allelic losses from chromosomes 1p and 19q [24, 25], a genetic profile that appears to be associated with clinical outcome. Some anaplastic oligodendrogliomas respond to combination chemotherapy [2, 3], and better progression free survival and chemo-responsiveness appear to be linked to the presence of these deletions [4, 34]. Results from two recent large-scale trials provide support for the view that the presence of 1p 19q deletion identifies a subgroup with better clinical outcome [1, 41]. 1p 19q deletion may also identify better prognosis in low-grade oligodendrogliomas [13].\nThere may be some histopathological differences between the two genetic subgroups. Classical oligodendroglioma features, such as perinuclear haloes (in >50% of cells), uniform rounded nuclei, chickenwire vascular pattern and calcification, are associated with 1p 19q loss [14, 27, 42], suggesting that classical features, such as perinuclear haloes associated with tumour cellularity, can predict genotype [14]. However, there are cases of histologically typical oligodendrogliomas without 1p 19q deletion and more astrocytic appearing tumours with deletion. For these cases clinical behaviour appears to be better predicted by genotype than phenotype, so that 1p 19q deletion provides additional information to conventional histopathological analysis [27]. Some studies suggest that oligodendrogliomas with 1p 19q deletion have lower activity of O6-methylguanine DNA methyltransferase (MGMT) due to promoter hypermethylation and decreased expression. MGMT is a DNA repair enzyme associated with resistance to alkylating agents, so this may contribute to chemosensitivity [18], although other studies have not found a clear association [42]. At present, therefore, the biological basis for the intrinsically better prognosis of the 1p 19q deletion subgroup and the possible basis of a different interaction with adjuvant therapies remain poorly understood.\nIn this study we hypothesise that the difference in behaviour of tumours with and without these deletions is due to differences in kinetic properties of the tumours related to proliferation and apoptosis. Apoptotic bodies are often a conspicuous feature in oligodendrogliomas and levels of apoptosis increase with grade [43]. Tumour growth depends on the net effects of cell proliferation and loss [35] so that a subgroup of tumours with a greater propensity to apoptosis versus proliferation may have slower growth. It is thus possible that increased susceptibility of tumour cells to undergo apoptosis might underlie both a better intrinsic biological behaviour and a greater sensitivity to adjuvant therapy. We hypothesise therefore that tumours with 1p,19q deletions show a greater tendency to apoptosis that is reflected in increased basal levels of apoptosis in the tumours.\nDifferences in kinetic parameters related to DNA replication licensing are also candidates for relevance to growth capacity and chemosensitivity. The initiation of chromosomal replication is a crucial decision point in cell proliferation that lies at the point of convergence of all oncogenic signalling and transduction pathways that trigger proliferation. DNA replication initiation is precisely controlled through a core set of licensing factors (Orc1\u20136, Cdc6, Cdt1, Mcm2\u20137) that sequentially assemble into pre-replicative complexes (pre-RCs) at \u223c30,000 replication origins scattered along the chromosomes, resulting in chromatin being \u2018licensed\u2019 for replication in the subsequent S phase. Licensed replication origins are activated by the concerted action of S phase promoting kinases (Cdk2) and the ASK-dependent Cdc7 kinase in a process commonly known as \u2018origin firing\u2019. Origin firing results in local melting of the DNA helix at replication origins and the recruitment of DNA polymerases (reviewed in [40]). Notably the Mcm2\u20137 replication licensing factors, which have been shown to act as a replicative DNA helicase, are present within the nucleus in cells in-cycle, but are down-regulated in out-of-cycle states [10, 38].\nWe have previously shown that higher grade oligodendrogliomas show a greater proportion of cells that are licensed for DNA replication, as determined by the Mcm2 labelling index, which is of prognostic significance [44]. Certain tissues contain a population of licensed but non-proliferating cells [10, 38] and higher labelling indices for Mcm2 than for the conventional proliferation marker Ki67 (which marks all four phases of the cell-cycle) suggest that this may be true for oligodendrogliomas [44]. A population of licensed but non-proliferating cells might conceivably resist cell-cycle dependent cytotoxicity and form a viable cell cache for tumour re-population. We have used the difference between the Mcm2 and Ki67 labelling indices (Mcm2\u00a0\u2212\u00a0Ki67 LI) as an assay of this population.\nIt is essential for faithful propagation of the genome that origins are fired once and only once in each cell cycle to avoid re-replication events, which would lead to gene amplification. Geminin, a 23.5\u00a0kDa nuclear protein, is an endogenous regulator of chromosomal replication, absent during G1 phase but allowed to accumulate during S, G2 and M phase [17]. Geminin acts to prevent a second, inappropriate firing of replication origins by competitively binding to Cdt1 and thereby blocking recruitment of the Mcm2\u20137 helicase to replication origins [39, 48]. Thus pre-RC re-assembly and firing are prevented. At the metaphase to anaphase transition in mitosis, the anaphase promoting complex (APC\/C) becomes activated and geminin is targeted for polyubiquitination and degradation via the 26S proteosome pathway, lowering levels to the point where the protein no longer interferes with assembly of pre-RCs during the following G1 phase [17]. Since geminin is present in S\u2013G2\u2013M phase, the ratio of geminin to Ki67 may provide an index of the relative length of G1-phase. We have previously shown that this ratio decreases in higher-grade oligodendrogliomas, which may be a reflection of a shortened G1 in anaplastic tumours [45]. The length of G1-phase may be relevant to chemotherapeutic agents targeting the cell cycle machinery. We have therefore determined whether cytogenetic subgroups of oligodendrogliomas differ in their geminin\/Ki67 ratio.\nMaterials and methods\nA total of 55 cases of oligodendroglioma were identified and paraffin blocks retrieved from the archives of the Histopathology Department of the Royal Hallamshire Hospital, Sheffield, from a 12\u00a0year period from 1985 to 1997. As previously described [45], 25 cases were graded as WHO grade II (mean age 37.4\u00a0years) and 30 as grade III (mean age 44.6\u00a0years). The series comprised 22 female and 33 male patients. All of the cases were resection specimens; stereotactic and other small biopsies were excluded. Of these cases, 47 were first resections. Eight cases were recurrent resections that fell within the study period; the initial resections on these cases were prior to the study period and not included. All of the cases were used for the labelling index analyses but only the 47 first resections were used for survival analysis. Clinical follow-up data and data on radiotherapy and chemotherapy were obtained from the Clinical Oncology Department at Western Park Hospital, Sheffield. Approval for the study was granted by the Local Research Ethics Committee.\nImmunohistochemistry\nImmunohistochemistry was performed on serial sections from a representative paraffin block from each case using a standard ABC method and the signal visualised using diaminobenzidine. Immunostaining was performed to Ki67 (Novocastra), Mcm2 (BD Transduction Laboratories) and geminin (G95, rabbit polyclonal) on this series of tumours as previously described [45]. Apoptotic bodies were counted on haematoxylin and eosin stained sections. Areas of highest cellularity were used for quantitation. The percentage of labelled cells, derived from a count of at least 1000 cells, was determined for each marker using an eyepiece graticule [44, 45].\nFluorescence in situ hybridisation\nFluorescence in situ hybridisation (FISH) was performed on 4\u00a0\u03bc thick sections using the technique previously described [7]. The Vysis dual colour probe sets LSI 1p36\/LSI 1q25 and LSI 19p13\/LSI 19q13 were used according manufacturers instructions; probes were hybridised overnight using a PTC Peltier Thermal Cycler. Analysis was performed independently by two scientists, both scoring 30 cells for each probe set.\nStatistical analyses\nStatistical analyses were performed using the statistical package for the social sciences (SPSS v10.1). Comparison of age was made using an unpaired t test. Data on labelling indices was either not normally distributed and\/or did not satisfy criteria for equality of variance between groups (Levene\u2019s test). Therefore comparisons of labelling indices between groups were performed using the non-parametric Mann\u2013Whitney U test. Fisher\u2019s exact test was used for assessment of distribution between groups. For comparison of survival between groups, Kaplan Meier plots were constructed and the log rank test used. Multivariable linear regression analysis was used to examine the relationship of apoptotic index (AI) to proliferation and deletion status. Ki67 labelling index (LI) was used as measure of the former, rather than Mcm2 LI, as the latter also includes replication licensed but non-proliferating cells. To model a difference in the slope of the relationship between AI and Ki67 LI in the two subgroups, an interactive term between Ki67 LI and deletion status was included [Model: AI\u00a0=\u00a0\u03b20\u00a0+\u00a0(\u03b21\u00a0\u00d7\u00a0Ki67 LI)\u00a0+\u00a0(\u03b22\u00a0\u00d7\u00a0deletion status)\u00a0+\u00a0(\u03b23\u00a0\u00d7\u00a0Ki67 LI\u00a0\u00d7\u00a0deletion status)].\nResults\nCharacteristics of the deletion sub-groups\nFISH analysis for 1p and 19q deletion was successfully performed in 54 out of 55 cases (Fig.\u00a01). Cases were assigned to the Del+ group if they had deletion of both 1p and 19q, or 1p alone. Cases were assigned to the Del\u2212 group if they had no deletion or deletion of 19q only. There were 34 Del+ cases and 20 Del\u2212 (Table\u00a01). The Del+ group had an older mean age than the Del\u2212 group (Table\u00a02) (P\u00a0=\u00a00.002). There was no difference in the distribution of histological grade between the two groups (P\u00a0=\u00a01).\nFig.\u00a01Representative FISH images using the VYSIS dual colour probe sets; a relative loss of 1p (red) to 1q (green) with probable increase of chromosome number, b simple deletion of 1p (red), c relative loss of 19q (red) to 19p (green) with probable increase of chromosome number, d simple deletion of 19q (red)Table\u00a01Results of FISH analysisGroupDeletionNumberNo. in Del group% of casesDel+1p 19q3434631p 0Del\u2212None17203719q3Table\u00a02Charateristics of cytogenetic sub-groupsGroupDel+Del\u2212Mean age, SD46.0, 11.5 33.1, 17.9Grade II169Grade III1811% of grade II47.145.0\nSurvival analysis\nSurvival for the 47 cases included in the survival analysis was taken from the date of the surgical resection. The eight (recurrent) cases excluded from this analysis were distributed evenly between Del+ and Del\u2212 groups. The Del+ group showed a trend to better survival (Fig.\u00a02) but this did not reach significance (P\u00a0=\u00a00.092), probably due to the number of censored cases. Further survival analysis to include multivariate parameters was therefore not attempted.\nFig.\u00a02Kaplan Meier survival curves for Del+ (solid line) and Del\u2212 (dashed line) oligodendroglioma subgroups. Censored cases shown as cross marks\nComparison of indices between cytogenetic subgroups\nThe labelling index for apoptotic bodies (AI), assessed from H&E stained sections was used as a measure of apoptosis. Higher levels of apoptosis were seen in the Del+ group, carrying either deletions of 1p and 19q, or 1p alone (P\u00a0=\u00a00.037) (Table\u00a03, Fig.\u00a03). To further analyse the difference in apoptosis between the two cytogenetic subgroups, we considered whether higher levels of apoptosis in the Del+ group were a reflection of higher cell turnover. However, there was no difference in replication-licensed or proliferating cells between the two subgroups as assessed by Mcm2 (P\u00a0=\u00a00.87) or Ki67 (P\u00a0=\u00a00.77), respectively. Inspection of scatterplots of AI against Ki67 labelling index for the two subgroups suggested that with increasing tumour proliferation, apoptosis shows a greater increase in the Del+ subgroup (Fig.\u00a04). Because this suggested a steeper relationship of AI to proliferation in the Del+ subgroup, multivariable regression analysis was carried out with a model, incorporating Ki67 LI, deletion status and a term for interaction between these two variables as predictors of apoptosis index. This gave an overall significance of P\u00a0=\u00a00.002 for the model. The interaction between Ki67 LI and deletion status was significant (P\u00a0=\u00a00.031) and AI rose more steeply against Ki67 LI in the Del+ group (AI \u03b1 0.58xKi67LI) than in the Del\u2212 group (AI \u03b1 0.13 Ki67 LI).\nTable\u00a03Descriptive data for kinetic measuresSubgroupMeasureAIMcm2\u00a0\u2212\u00a0Ki67Gem\/Ki67Del\u2212Mean (SD)0.72 (0.38)8.31 (22.21)0.39 (0.25)Median (IQR)0.60 (0.48)6.65 (23.18)0.38 (0.44)Del+Mean (SD)1.10 (0.69)12.51 (15.11)0.29 (0.15)Median (IQR)0.95 (1.00)7.15 (21.88)0.26 (0.22)AI apoptotic index, Mcm2\u00a0\u2212\u00a0Ki67 Mcm2 minus Ki67 labelling index, Gem\/Ki67 ratio of geminin to Ki67 indices, SD standard deviation, IQR interquartile rangeFig.\u00a03Boxplot showing apoptotic indices in Del\u2212 and Del+ subgroupsFig.\u00a04Scatterplots of apoptosis index (AI) versus Ki67 labelling index for Del\u2212 (left) and Del+ (right) cases\nThe difference between Mcm2 and Ki67 labelling indices (Mcm2\u00a0\u2212\u00a0Ki67 LI) was used as a measure of the fraction of tumour cells that were licensed but not proliferating. In general tumours showed a higher labelling index for Mcm2 than Ki67, indicating the presence of a population of licensed cells that are not actively replicating. The Mcm2\u00a0\u2212\u00a0Ki67 LI was higher in grade III than in grade II tumours (P\u00a0=\u00a00.001, Fig.\u00a05). However, we demonstrated no difference in the proportion of licensed, non-cycling cells by this method between the two cytogenetic subgroups of oligodendrogliomas (P\u00a0=\u00a00.46). We also demonstrated no difference in the geminin\/Ki67 ratio (P\u00a0=\u00a00.22).\nFig.\u00a05Boxplot of Mcm2 minus Ki67 labelling index according to histological grade\nDiscussion\nThe basis for the difference in clinical behaviour between oligodendrogliomas with deletions of 1p and 19q (Del+) and those without (Del\u2212) remains unclear, but its elucidation may yield important new avenues for improved therapeutic approaches. In this study we have investigated whether there are differences in parameters related to tumour cell proliferation and death in these two cytogenetic subgroups of tumours.\nThe tumours were typed using FISH with probes to 1p and 19q. In our series, 63% of cases showed deletions in 1p and 19q, or 1p alone, a similar proportion to that reported in series of oligodendrogliomas in the literature where figures for combined loss from approximately 50 to 70% of cases have been described (sometimes higher in classical appearing WHO grade II tumours) [13, 19, 24, 25, 33]. Del+ and Del\u2212 had a similar distribution of histological grades whilst the older average age in the Del+ group is also unlikely to be of relevance to our findings as age is in general associated with a worse clinical behaviour, and opposite to the effects here. A trend towards better overall survival in Del+ cases in our series did not reach significance, probably due to the number of total and censored cases.\nAnalysis of the FISH probes in this cohort of cases revealed a pattern of multiple signals in some cases, indicating that increased copy number of chromosomes 1 and 19 were present, but that there was still effective loss of heterozygosity of short arm chromosome 1 and long arm chromosome 19. (Fig.\u00a01) These cases were interpreted Del+. One other subgroup was identified in the cohort: three cases in which there was 19q deletion present without concomitant 1p deletion. There was no significant association of these subgroups in our series with an obvious pathological feature, but more cases should be studied to determine any clinico-pathological correlation. The multiple signal cases, which would indicate complex aneuploid karyotypes, usually found in tumours with more malignant status, may be of particular interest. One other group has identified the single 19q deletion status cases and have suggested that 19q deletion alone may also predict a favourable prognosis [22].\nWe have previously reported values for apoptosis, Mcm2, Ki67 and geminin in this series of tumours [45], but we now define indices and relate these to cytogenetic subtype. We have investigated three selected parameters that we hypothesise may affect survival and therapeutic response. Apoptosis is an important process in tumour biology as defective apoptosis induction may contribute to cancer development and the susceptibility of tumour cells to apoptosis may affect rate of growth. Apoptosis may be induced by adjuvant therapies and is likely to become an increasingly important molecular therapeutic target as specific pathways are defined (reviewed in [26]). Apoptosis is variable between tumour types and a number of factors may contribute to apoptosis induction in a tumour. The apoptotic index often appears to be a function of the proliferation rate, with higher levels in more rapidly cycling tumours. In oligodendrogliomas, we and others have previously shown that apoptosis tends to increase with proliferation and with tumour grade [29, 43], though it has not been shown to be an independent prognostic factor. A similar relationship of apoptosis to histological grade and proliferation has also been demonstrated in the diffuse astrocytomas [8]. Although quantification of apoptosis, and so comparison between tumour types and studies, is beset with methodological difficulties, there is evidence that oligodendrogliomas show higher levels of apoptosis than other brain tumours of comparative WHO grade [15, 28]. Alterations in regulatory factors related to molecular genetic changes are likely to be important in setting the susceptibility to apoptosis in a given tumour type, whilst other factors such as ischaemia and the influence of tumoural inflammatory cells may also contribute to apoptosis induction. Thus variation in apoptosis is a candidate to explain differences in clinical behaviour of tumour subtypes.\nIn this series of oligodendrogliomas we have assessed apoptosis using counts of apoptotic bodies. This method has been used for other tumour types, including astrocytomas [8]. In a previous oligodendroglioma series we showed that the apoptotic index correlates with an assessment of apoptosis using the TUNEL method [43], but it is a much simpler method. In addition, the TUNEL technique can be capricious and lacks specificity as it may detect DNA damage in cells from processes other than apoptosis, including necrosis [12]. Immunohistochemical detection of caspase 3 has also been used to detect apoptotic cells in histological sections [5]. We have previously carried out caspase 3 immunohistochemistry on this series [45], but this showed weaker relationships to grade and proliferation than apoptotic index using counts of apoptotic bodies. Therefore, for this study, apoptotic index based on counts of apoptotic bodies was selected a priori for analysis.\nWe have demonstrated that Del+ oligodendrogliomas show a higher apoptotic index than Del- tumours, supporting the hypothesis that tumour cells from Del+ cases may have a greater susceptibility to undergo apoptosis. We have not investigated the molecular pathways that might lead to this difference. However, it is not simply a reflection of increased cell turnover as Del+ cases show no increase in either Mcm2 or Ki67 labelling indices compared to Del- cases (although a difference in cell-cycle length is not formally excluded). The greater rate of increase in apoptotic index against Ki67 labelling index for Del+ than Del- tumours suggests that oligodendrogliomas with 1p, 19q deletion have a greater tendency to the induction of apoptosis at higher rates of proliferation.\nImmunohistochemical demonstration and quantification of components of the DNA replication licensing machinery have recently been used for prognostic [16, 30, 32, 44] and surveillance [37, 46, 47] studies in human tumours. Theoretically demonstration of MCM proteins may be considered superior to Ki67 as it identifies not only cycling cells, but also those non-cycling cells with proliferative potential [10, 38], and this superiority is born out in many of these studies. Combined assessment of these markers with other cell cycle related markers may be used to obtain information on the relative length of cell cycle phases in archival biopsy material [9, 20, 31]. In this study, which adds to these applications, we have used a variable generated from the difference between the Mcm2 and Ki67 labelling indices (Mcm2-Ki67 LI) for each case to assess the proportion of tumour cells that are licensed to replicate but not actually in cycle. This index has been previously used in renal cell cancer, where it increases with tumour grade [6]. We now also demonstrate that this index increases with tumour grade in oligodendrogliomas, suggesting that the pool of cells that are licensed but not proliferating is larger in more anaplastic tumours, evidence for greater dysregulation of the origin licensing pathway with anaplasia. Such a licensed, non-proliferating fraction might conceivably act as a treatment-resistant population that could re-grow after adjuvant therapy. There is increasing evidence for tumour stem cells in brain tumours (reviewed in [11]) and it is intriguing to speculate whether a stem cell population, responsible for perpetuating tumour growth, might reside in this fraction. Its assay is therefore of potential interest and we hypothesised that the size of this fraction might be greater in Del- tumours. However, we did not demonstrate any difference in Mcm2\u00a0\u2212\u00a0Ki67 LI between Del+ and Del\u2212 tumours, suggesting that this is not important in determining their clinical differences.\nWe have previously shown that the ratio of geminin to Ki67 labelling indices is increased in grade III versus grade II oligodendrogliomas [45], implying a shortened G1-phase in higher grade tumours (assuming that other cell cycle phases are not substantially altered in duration). In this study, however, we have not demonstrated evidence for a differing G1-phase duration between Del+ and Del\u2212 tumours.\nMolecular differences between these two cytogenetic subtypes of oligodendroglioma are beginning to be defined, which will allow therapeutically relevant molecular classification [24]. Some of these genetic differences likely relate to the regulation of apoptosis, and may differ in the two subgroups. This study has shown that those oligodendrogliomas with 1p\u00a0\u00b1\u00a019q deletions have higher apoptotic indices than those without. Differences in cell death susceptibility may affect prognosis through a treatment independent effect on intrinsic growth rate. But apoptosis may also be induced by adjuvant therapies, and amongst the gliomas this may be particularly important for oligodendroglial tumours [36]. This study does not allow distinction between intrinsic growth rate and treatment response contributions of apoptosis. It has also been limited to histological methods and has not provided functional data on susceptibility or defined its molecular basis. However, the findings support the hypothesis that differences in clinical outcomes between these cytogenetic subgroups is related to differing susceptibility to apoptosis induction and suggest that detailed comparative studies of apoptotic pathways in molecular subgroups of oligodendrogliomas are warranted.","keyphrases":["oligodendroglioma","replication","apoptosis","dna replication licensing","cytogenetic"],"prmu":["P","P","P","P","P"]}
{"id":"Psychopharmacologia-2-2-1705495","title":"3,4-Methylenedioxymethamphetamine (MDMA) neurotoxicity in rats: a reappraisal of past and present findings\n","text":"Rationale 3,4-Methylenedioxymethamphetamine (MDMA) is a widely abused illicit drug. In animals, high-dose administration of MDMA produces deficits in serotonin (5-HT) neurons (e.g., depletion of forebrain 5-HT) that have been interpreted as neurotoxicity. Whether such 5-HT deficits reflect neuronal damage is a matter of ongoing debate.\nIntroduction\n3,4-Methylenedioxymethamphetamine (MDMA or Ecstasy) is an illicit drug used by young adults in the US, Europe, and elsewhere. The appeal of MDMA is related to its unique profile of psychotropic actions, which includes amphetamine-like stimulant effects, coupled with feelings of increased emotional sensitivity and closeness to others (Liechti and Vollenweider 2001; Vollenweider et al. 1998). MDMA misuse among children and adolescents is widespread in the US (Landry 2002; Yacoubian 2003); a recent sampling of high school students found 10% of 12th graders reported using MDMA at least once (Banken 2004). MDMA-related medical complications have risen more than 20-fold in recent years, consistent with increasing popularity of the drug. Serious adverse effects of MDMA intoxication include cardiac arrhythmias, hypertension, hyperthermia, serotonin (5-HT) syndrome, hyponatremia, liver problems, seizures, coma, and, in rare cases, death (Schifano 2004). Accumulating evidence indicates that long-term MDMA abuse is associated with cognitive impairments and mood disturbances, which can last for months after cessation of drug intake (Morgan 2000; Parrott 2002). Despite the risks of illicit MDMA use, some clinicians believe the drug may have therapeutic potential in the treatment of psychiatric disorders, such as post-traumatic stress disorder, and clinical studies with MDMA are underway (Doblin 2002). It is worth noting that MDMA has been administered to human subjects in controlled research settings, and few side effects are observed under these circumstances (Harris et al. 2002; Mas et al. 1999).\nThese considerations provide compelling reasons to evaluate the pharmacology and toxicology of MDMA and related compounds. In this review, we will examine four topics related to the hypothesis of MDMA-induced 5-HT neurotoxicity in rats: (1) the effects of MDMA on monoamine neurons, (2) the use of interspecies scaling to extrapolate doses of MDMA across species, (3) the effects of MDMA on established markers of neurotoxic damage, and (4) the functional impairments associated with MDMA-induced 5-HT depletions. The review will focus on data obtained from rats since most preclinical MDMA research has been carried out in this animal model. Previously published and new data from our laboratory at the National Institute on Drug Abuse (NIDA) will be included to supplement literature reports. Clinical findings will be mentioned in specific instances to note comparisons between rats and humans. All experiments in our laboratory utilized male Sprague\u2013Dawley rats (Wilmington, MA, USA) weighing 300\u2013350\u00a0g. Rats were maintained in facilities accredited by the American Association of the Accreditation of Laboratory Animal Care, and procedures were carried out in accordance with the Animal Care and Use Committee of the NIDA Intramural Research Program (IRP). Data from mice will not be considered here because this animal species displays the unusual characteristic of long-term DA depletions (i.e., DA neurotoxicity) in response to MDMA, rather than long-term 5-HT depletions observed in rats, nonhuman primates, and most other animals [reviewed by Colado et al. (2004)]. Finally, the present paper will not address possible molecular mechanisms underlying MDMA-induced 5-HT deficits, as several excellent reviews have covered this subject (Lyles and Cadet 2003; Monks et al. 2004; Sprague et al. 1998).\nEffects of MDMA on monoamine neurons\nTo address the topic of MDMA-induced 5-HT neurotoxicity, the pharmacology of MDMA must be briefly reviewed. Figure\u00a01 shows that MDMA is a ring-substituted analog of methamphetamine. Ecstasy tablets ingested by humans contain a racemic mixture of (+) and (\u2212) isomers of MDMA, and both stereoisomers are known to be bioactive (Johnson et al. 1986; Schmidt et al. 1987). Ecstasy tablets often contain other psychoactive substances such as substituted amphetamines, caffeine, or ketamine, which can contribute to the overall effects of the ingested preparation (Parrott 2004). Upon systemic administration, N-demethylation of MDMA occurs via first-pass metabolism to yield the ring-substituted amphetamine analog 3,4-methylenedioxyamphetamine (MDA) (de la Torre et al. 2004). Initial studies carried out in the 1980s showed that MDMA and MDA stimulate efflux of preloaded [3H]5-HT, and to a lesser extent [3H]DA, in nervous tissue (Johnson et al. 1986; Nichols et al. 1982; Schmidt et al. 1987). Subsequent findings revealed that MDMA interacts with monoamine transporter proteins to stimulate non-exocytotic release of 5-HT, DA and norepinephrine (NE) in rat brain (Berger et al. 1992; Crespi et al. 1997; Fitzgerald and Reid 1993).\nFig.\u00a01Chemical structures of MDMA and related compounds\nTable\u00a01 summarizes previously published data from our laboratory showing structure\u2013activity relationships for stereoisomers of MDMA, MDA, and related drugs as monoamine releasers in rat brain synaptosomes (Partilla et al. 2000; Rothman et al. 2001; Setola et al. 2003). Like other substrate-type releasers, MDMA and MDA bind to plasma membrane monoamine transporters and are translocated into the cytoplasm. The ensuing transmitter release occurs by a two-pronged mechanism: (1) transmitter molecules exit the cell along their concentration gradients via reversal of normal transporter function, and (2) cytoplasmic concentrations of transmitter are increased due to drug-induced disruption of vesicular storage [reviewed by Rothman and Baumann (2002) and Rudnick and Clark (1993)]. As shown in Table\u00a01, stereoisomers of MDMA and MDA are substrates for 5-HT transporters (SERT), NE transporters (NET) and DA transporters (DAT), with (+) isomers exhibiting greater potency as releasers. In particular, (+) isomers of MDMA and MDA are much more effective DA releasers than their corresponding (\u2212) isomers. It is noteworthy that (+) isomers of MDMA and MDA are rather nonselective in their ability to stimulate monoamine release in vitro. When compared to amphetamine and methamphetamine, the major effect of methylenedioxy ring substitution is enhanced potency for 5-HT release and reduced potency for DA release. For example, (+)-MDMA releases 5-HT (EC50=70.8\u00a0nM) about ten times more potently than (+)-methamphetamine (EC50=736\u00a0nM), whereas (+)-MDMA releases DA (EC50=142\u00a0nM) about six times less potently than (+)-methamphetamine (EC50=24\u00a0nM). \nTable\u00a01Profile of MDMA and related compounds as monoamine transporter substrates in rat brain synaptosomesDrug5-HT release EC50 (nM\u00b1SD)NE release EC50 (nM\u00b1SD)DA release EC50 (nM\u00b1SD)(+)-Methamphetamine736\u00b14512\u00b10.724\u00b12(\u2212)-Methamphetamine4,640\u00b124029\u00b13416\u00b120(\u00b1)-MDMA74.3\u00b15.6136\u00b117278\u00b112(+)-MDMA70.8\u00b15.2110\u00b116142\u00b16(\u2212)-MDMA337\u00b134564\u00b1603,682\u00b1178(+)-Amphetamine1,765\u00b1947.1\u00b11.025\u00b14(\u00b1)-MDA159\u00b112108\u00b112290\u00b110(+)-MDA99.6\u00b17.498.5\u00b16.150.0\u00b18.0(\u2212)-MDA313\u00b121287\u00b123900\u00b149The data are taken from Partilla et al. 2000, Rothman et al. 2001, and Setola et al. 2003. Details concerning in vitro methods can be found in these papers. Substrate activity at SERT, NET, and DAT is reflected as release efficacy for the corresponding transmitter\nConsistent with in vitro results, in vivo microdialysis experiments demonstrate that MDMA increases extracellular 5-HT and DA in rat brain, with effects on 5-HT being greater in magnitude (Baumann et al. 2005; Gudelsky and Nash 1996; Kankaanpaa et al. 1998; Yamamoto et al. 1995). Figure\u00a02 depicts new data from our laboratory showing the stimulatory effects of MDMA and MDA on extracellular 5-HT and DA in rat nucleus accumbens. In these experiments, i.v. injections of saline (0 dose) or drug were administered to conscious male rats undergoing in vivo microdialysis. Doses of 0.3 and 1.0\u00a0mg\/kg were chosen because these doses of MDMA are self-administered by rats (Ratzenboeck et al. 2001; Schenk et al. 2003). Dialysate samples were collected every 20\u00a0min beginning 2\u00a0h before injections until 2\u00a0h thereafter; samples were assayed for 5-HT and DA by high-performance liquid chromatography coupled to electrochemical detection (HPLC\u2013ECD) as described elsewhere (Baumann and Rutter 2003). Neurochemical data were converted to percentage of control values based on three preinjection control samples. The effects of MDMA and MDA are depicted as peak effects, which were observed in the first 20\u00a0min after injection. Peak effect data were analyzed using a one-way ANOVA (drug dose) followed by Duncan\u2019s post hoc test. MDMA caused significant increases in dialysate 5-HT [F2,15=19.47, P<0.001] and DA [F2,15=14.46, P<0.01]. MDA caused similar increases in 5-HT [F2,15=17.95; P<0.001] and DA [F2,15=7.69, P<0.01], but appeared to be slightly more potent at releasing DA. Both drugs produced elevations in 5-HT that were greater than the corresponding effects on DA. For example, the 1\u00a0mg\/kg dose of MDMA produced a tenfold rise in 5-HT and a twofold rise in DA.\nFig.\u00a02Effects of (\u00b1)-MDMA and (\u00b1)-MDA on extracellular levels of 5-HT (top panel) and DA (bottom panel) in rat nucleus accumbens. Male rats undergoing in vivo microdialysis received i.v. injections of saline (0 dose) or drug, and dialysate levels of 5-HT and DA were assayed by HPLC\u2013ECD (Baumann and Rutter 2003). Data are expressed as the percentage of three pre-injection baseline samples; each bar represents the mean\u00b1SEM peak effect measured 20\u00a0min posttreatment, N=6 rats\/group. Baseline levels of 5-HT and DA were 0.17\u00b10.01 and 1.31\u00b10.05\u00a0pg\/5\u00a0\u03bcl, respectively. Asterisk denotes significance with respect to zero dose control (P<0.05 Duncan\u2019s)\nAcute central nervous system (CNS) effects of MDMA are mediated by the release of monoamine transmitters, with the subsequent activation of presynaptic and postsynaptic receptors [reviewed by Cole and Sumnall (2003) and Green et al. (2003)]. As specific examples in rats, MDMA suppresses 5-HT cell firing, evokes neuroendocrine secretion, and stimulates locomotor activity. MDMA-induced suppression of 5-HT cell firing in the dorsal and median raphe involves activation of presynaptic 5-HT1A autoreceptors by endogenous 5-HT (Gartside et al. 1997; Sprouse et al. 1989). Neuroendocrine effects of MDMA include secretion of prolactin from the anterior pituitary and corticosterone from the adrenal glands (Nash et al. 1988). Evidence indicates that these MDMA-induced hormonal effects are mediated via postsynaptic 5-HT2 receptors in the hypothalamus, which are activated by released 5-HT. MDMA elicits a unique profile of locomotor effects, characterized by forward locomotion and elements of the 5-HT behavioral syndrome such as forepaw treading, flattened body posture, and head weaving (Gold et al. 1988; Slikker et al. 1989; Spanos and Yamamoto 1989). The complex motor effects of MDMA are dependent upon monoamine release followed by activation of multiple 5-HT and DA receptor subtypes in the brain [reviewed by Bankson and Cunningham (2001); Geyer (1996)].\nAdverse effects of acute MDMA administration, including cardiovascular stimulation and elevated body temperature, are thought to involve monoamine release from sympathetic nerves in the periphery or nerve terminals in the CNS. MDMA increases heart rate and mean arterial pressure in conscious rats (O\u2019Cain et al. 2000); this cardiovascular stimulation is likely mediated by MDMA-induced release of peripheral NE stores, similar to the effects of amphetamine (Fitzgerald and Reid 1994). MDMA has weak agonist actions at \u03b12-adrenoreceptors and 5-HT2 receptors, which might influence its cardiac and pressor effects (Battaglia and De Souza 1989; Lavelle et al. 1999; Lyon et al. 1986). Moreover, MDA is a potent 5-HT2B agonist and this property could contribute to adverse cardiovascular effects (Setola et al. 2003). The ability of MDMA to elevate body temperature is well-characterized in rats (Dafters 1995; Dafters and Lynch 1998; Nash et al. 1988), and this response has been considered a 5-HT-mediated process. However, a recent study by Mechan et al. (2002) provides convincing evidence that MDMA-induced hyperthermia involves the activation of postsynaptic D1 receptors by released DA.\nThe long-term adverse effects of MDMA on 5-HT systems have attracted substantial interest because studies in rats and nonhuman primates show that high-dose MDMA administration produces persistent reductions in markers of 5-HT nerve terminal integrity [reviewed by Lyles and Cadet (2003); Sprague et al. (1998)]. Table\u00a02 summarizes findings of investigators who first demonstrated that MDMA causes long-term (>1\u00a0week) inactivation of tryptophan hydroxylase activity, depletions of brain tissue 5-HT, and reductions in SERT binding and function (Battaglia et al. 1987; Commins et al. 1987; Schmidt 1987; Stone et al. 1987). These serotonergic deficits are observed in various regions of rat forebrain, including frontal cortex, striatum, hippocampus, and hypothalamus. Immunohistochemical analysis of 5-HT in cortical and subcortical areas reveals an apparent loss of 5-HT axons and terminals in MDMA-treated rats, especially the fine-diameter projections arising from the dorsal raphe nucleus (O\u2019Hearn et al. 1988). Moreover, 5-HT axons and terminals remaining after MDMA treatment appear swollen and fragmented, suggesting structural damage. \nTable\u00a02Long-term effects of (\u00b1)-MDMA on 5-HT neuronal markers in rats5-HT deficitDoseSurvival intervalReferenceDepletions of 5-HT in cortex, as measured by HPLC\u2013ECD10\u00a0mg\/kg, s.c., single dose1\u00a0weekSchmidt (1987)Depletions of 5-HT in forebrain regions as measured by HPLC\u2013ECD10\u201340\u00a0mg\/kg, s.c., twice daily, 4\u00a0days2\u00a0weeksCommins et al. (1987)Reductions in tryptophan hydroxylase activity in forebrain regions10\u00a0mg\/kg, s.c., single dose2\u00a0weeksStone et al. (1987)Loss of [3H]-paroxetine-labeled SERT binding sites in forebrain regions20\u00a0mg\/kg, s.c., twice daily, 4\u00a0days2\u00a0weeksBattaglia et al. (1987)Deceased immunoreactive 5-HT in fine axons and terminals in forebrain regions20\u00a0mg\/kg, s.c., twice daily, 4\u00a0days2\u00a0weeksO\u2019Hearn et al. (1988)\nTime-course studies indicate that MDMA-induced 5-HT depletion occurs in a biphasic manner, with a rapid acute phase followed by a delayed long-term phase (Schmidt 1987; Stone et al. 1987). In the acute phase, which lasts for the first few hours after drug administration, massive depletion of brain tissue 5-HT is accompanied by inactivation of tryptophan hydroxylase. Twenty-four hours later, tissue 5-HT recovers to normal levels but hydroxylase activity remains diminished. In the long-term phase, which begins within 1\u00a0week and lasts for months, marked depletion of 5-HT is accompanied by sustained inactivation of tryptophan hydroxylase and loss of SERT binding and function (Battaglia et al. 1988; Scanzello et al. 1993). The findings in Table\u00a02 have been replicated by many investigators, and the spectrum of decrements is typically described as 5-HT neurotoxicity. Most of the studies designed to examine MDMA neurotoxicity in rats have employed i.p. or s.c. injections of 10\u00a0mg\/kg or higher, either as single or repeated treatments. These MDMA dosing regimens are known to produce significant hyperthermia, which can exacerbate 5-HT depletions caused by the drug (Green et al. 2004; Malberg and Seiden 1998). All of these experiments have involved administration of MDMA that is not contingent on a specific behavior and this factor could significantly influence effects of the drug.\nThere are caveats to the hypothesis that MDMA produces 5-HT neurotoxicity. O\u2019Hearn et al. (1988) showed that high-dose MDMA administration has no effect on 5-HT cell bodies in the dorsal raphe, despite profound loss of 5-HT in forebrain projection areas. Thus, the effects of MDMA on 5-HT neurons are sometimes referred to as \u201caxotomy,\u201d to account for the fact that perikarya are not damaged (Molliver et al. 1990; O\u2019Hearn et al. 1988). MDMA-induced reductions in 5-HT levels and SERT binding eventually recover (Battaglia et al. 1988; Scanzello et al. 1993), suggesting the possibility that 5-HT terminals are not destroyed. Many drugs used clinically produce effects that are similar to those produced by MDMA. For instance, reserpine causes sustained depletions of brain tissue 5-HT, yet reserpine is not considered a neurotoxin (Carlsson 1976). Chronic administration of 5-HT selective reuptake inhibitors (SSRIs), like paroxetine and sertraline, leads to a marked loss of SERT binding and function comparable to MDMA, but these agents are therapeutic drugs rather than neurotoxins (Benmansour et al. 1999; Frazer and Benmansour 2002). Finally, high-dose administration of SSRIs produces swollen, fragmented, and abnormal 5-HT terminals, which are indistinguishable from the effects of MDMA and other substituted amphetamines (Kalia et al. 2000).\nThe caveats mentioned above raise a number of questions with respect to MDMA neurotoxicity. Of course, the most important question is whether MDMA abuse causes neurotoxic damage in humans. This complex issue is a matter of ongoing debate, which has been addressed by a number of recent papers (Gouzoulis-Mayfrank et al. 2002; Kish 2002; Reneman 2003). Clinical studies designed to critically evaluate the long-term effects of MDMA are hampered by a number of factors, including comorbid psychopathology and polydrug abuse among MDMA users. Animal models afford the unique opportunity to evaluate the effects of MDMA without many of these complicating factors, and the main focus here will be to review the evidence pertaining to MDMA-induced 5-HT neurotoxicity in rats.\n\u201cInterspecies scaling\u201d and MDMA dosing regimens\nA major point of controversy relates to the relevance of MDMA doses administered to rats when compared to doses taken by humans [see (Cole and Sumnall (2003)]. As noted above, MDMA regimens that produce 5-HT depletions in rats involve administration of single or multiple injections of 10\u201320\u00a0mg\/kg, whereas the typical amount of MDMA abused by humans is one or two tablets of 80\u2013100\u00a0mg or 1\u20133\u00a0mg\/kg administered orally (Green et al. 2003; Schifano 2004). Based on principles of \u201cinterspecies scaling,\u201d some investigators have proposed that neurotoxic doses of MDMA in rats correspond to recreational doses in humans (Ricaurte et al. 2000). To critically evaluate this claim, a brief discussion of interspecies scaling is warranted.\nThe concept of interspecies scaling is based upon shared biochemical mechanisms among eukaryotic cells (e.g., aerobic respiration), and it was initially developed to describe variations in basal metabolic rate (BMR) between animal species of different sizes [reviewed by White and Seymour (2005)]. In the 1930s, Kleiber (1932) derived what is now called the \u201callometric equation\u201d to describe the relationship between body mass and BMR. The generic form of the allometric equation is: , where Y is the variable of interest, W is body weight, a is the allometric coefficient, and b is the allometric exponent. In the case where Y is BMR, b is accepted to be 0.75. West et al. (2002) have shown that most biological phenomena scale according to a universal quarter\u2013power law, as illustrated by the space-filling fractal networks of branching tubes used by the circulatory system.\nGiven that the allometric equation is grounded in fundamental commonalities across organisms, it is not surprising this equation can describe the relationship between body mass and physiological variables, such as BMR, heart rate, and circulation time [e.g., Noujaim et al. (2004)]. Because circulation time and organ blood flow strongly influence drug pharmacokinetics, the allometric equation has been used in the medication development process to \u201cscale-up\u201d dosages from animal models to man [reviewed by Mahmood (1999)]. In general, smaller animals have faster heart rates and circulation times, leading to faster clearance of exogenous drugs. However, this relationship does not hold true for all classes of drugs, especially those that are extensively metabolized (Lin 1998).\nThe most important variable to consider when examining therapeutic or adverse effects of any drug is the concentration of bioactive compound reaching target tissues. Tissue drug concentrations are governed by drug absorption, distribution, metabolism, and elimination (ADME) in a complex manner. Many factors such as dose, route, species, strain, age, and gender can affect the ADME profile of a given drug, thereby affecting tissue concentrations. Campbell (1995) has described numerous limitations of interspecies scaling to predict pharmacokinetic parameters in different species. In particular, the allometric equation does not account for species-specific variability in tissue uptake of substituted amphetamines, as illustrated by the fact that brain-to-plasma ratios of the 5-HT releaser fenfluramine are 30\u201350 for the rat, yet <10 for humans. No studies have compared the brain tissue uptake of MDMA in various animal models. Perhaps the most problematic issue in extrapolating doses across species is the unpredictable nature of drug metabolism, which occurs chiefly in the liver [see Campbell (1996)]. Marked species differences in hepatic drug metabolism are due to variations in expression and activity of cytochrome P450 enzymes, which catalyze biotransformation reactions (Lin 1995).\nMDMA is extensively metabolized in humans, as depicted in Fig.\u00a03, and the major pathway of biotransformation involves: (1) O-demethylenation catalyzed by cytochrome P450 2D6 (CYP2D6) and (2) O-methylation catalyzed by catechol-O-methyltransferase (COMT) [reviewed by de la Torre et al. (2004)]. CYP2D6 and COMT are both polymorphic in humans, and differential expression of CYP2D6 isoforms leads to interindividual variations in the metabolism of serotonergic medications (e.g., SSRIs) (Charlier et al. 2003). Interestingly, CYP2D6 is not present in rats, which express a homologous but functionally distinct cytochrome P450 2D1 (Malpass et al. 1999; Maurer et al. 2000). A minor pathway of MDMA biotransformation in humans involves N-demethylation of MDMA to form MDA, which is subsequently O-demethylenated and O-methylated. N-demethylation of MDMA represents a more important pathway for rats when compared to humans (de la Torre and Farre 2004). The metabolism of MDMA and MDA generates a number of metabolites, some of which may be active [e.g., Escobedo et al. (2005); Forsling et al. (2002)]. Determining the potential neurotoxic properties of the various metabolites of MDMA is an important area of research [reviewed by Baumgarten and Lachenmayer (2004); Monks et al. (2004)].\nFig.\u00a03Metabolism of MDMA in man. CYP2D6 Cytochrome P450 2D6, CYP3A4 cytochrome P450 3A4, COMT catechol-O-methyltransferase. This is adapted from de la Torre et al. (2004)\nTo complicate matters further, de la Torre et al. (2000) have shown that MDMA displays nonlinear kinetics in humans such that administration of increasing doses, or multiple doses, leads to unexpectedly high plasma levels of the drug. Enhanced plasma and tissue levels of MDMA are most likely related to auto-inhibition of MDMA metabolism, mediated via formation of a metabolite\u2013enzyme complex that irreversibly inactivates CYP2D6 (Wu et al. 1997). Because MDMA displays nonlinear kinetics, repeated drug dosing could produce serious adverse consequences due to unusually high blood and tissue levels of the drug (Parrott 2002; Schifano 2004). The existing database of MDMA pharmacokinetic studies represents a curious situation where clinical findings are well-documented, while preclinical data are lacking. Specifically, few studies in animals have assessed the relationship between pharmacodynamic effects and pharmacokinetics of MDMA after administration of single or repeated doses [but see Chu et al. (1996)]. No studies have systematically characterized the nonlinear kinetics of MDMA in animal models. Collectively, the available data demonstrate that potential species differences in tissue drug uptake, variations in metabolic enzymes and their activities, and the phenomenon of nonlinear kinetics, preclude the use of interspecies scaling to extrapolate MDMA doses between animals and humans [reviewed by de la Torre and Farre (2004)].\nThe uncertainties and limitations of allometric scaling led us to investigate the method of \u201ceffect scaling\u201d as an alternative strategy for matching equivalent doses of MDMA in rats and humans [see Winneke and Lilienthal (1992)]. In this approach, the lowest dose of drug that produces a specific pharmacological response is determined for rats and humans, and subsequent dosing regimens in rats are calculated with reference to the predetermined threshold dose. In the case of MDMA, this strategy is simplified because CNS drug effects, such as neuroendocrine and behavioral changes, have already been investigated in different species. Theoretically, equivalent drug effects in vivo should reflect similar drug concentrations reaching active sites in tissue, suggesting that the method of effect scaling can account for differences in ADME across species (at least for low drug doses). Table\u00a03 shows the doses of MDMA that produce comparable CNS effects in rats and humans. Remarkably, the findings reveal that doses of MDMA in the range of 1\u20132\u00a0mg\/kg produce pharmacological effects that are equivalent in both species. It is noteworthy that MDMA is typically administered to rats via the i.p. or s.c. route, whereas humans take the drug orally. Given the similar effects of MDMA in rats and humans at the same doses, it appears that drug bioavailability is comparable after i.p., s.c., or oral administration [e.g., Finnegan et al. (1988)], but verification of this hypothesis awaits further investigation. \nTable\u00a03Comparative neurobiological effects of (\u00b1)-MDMA administration in rats and humansCNS effectDose in ratsDose in humansIn vivo release of 5-HT and DA2.5\u00a0mg\/kg, i.p. (Gudelsky and Nash 1996); 1\u00a0mg\/kg, s.c. (Kankaanpaa et al. 1998)1.5\u00a0mg\/kg p.o.a (Liechti et al. 2000; Liechti and Vollenweider 2001)Secretion of prolactin and glucocorticoids1\u20133\u00a0mg\/kg, i.p. (Nash et al. 1988)1.67\u00a0mg\/kg, p.o. (Mas et al. 1999);1.5\u00a0mg\/kg, p.o. (Harris et al. 2002)Drug discrimination1.5\u00a0mg\/kg, i.p. (Oberlender and Nichols 1988; Schechter 1988).1.5\u00a0mg\/kg, p.o. (Johanson et al. 2006)Drug reinforcement1\u00a0mg\/kg, i.v. (Wakonigg et al. 2003).1\u20132\u00a0mg\/kg, p.o.b (Tancer and Johanson 2003)\naSubjective effects were attenuated by 5-HT uptake blockers, suggesting the involvement of transporter-mediated 5-HT release\nbReinforcing effects were determined based on a multiple choice procedure\nAdministration of MDMA at i.p. doses of 1\u20133\u00a0mg\/kg causes marked elevations in extracellular 5-HT and DA in rat brain, as determined by in vivo microdialysis (Baumann et al. 2005; Gudelsky and Nash 1996; Kankaanpaa et al. 1998). The data from Fig.\u00a02 illustrate that doses of MDMA as low as 0.3\u00a0mg\/kg i.v. stimulate a significant rise in extracellular 5-HT in rat nucleus accumbens. Although it is impossible to directly measure 5-HT and DA release in living human brain, clinical studies indicate that subjective effects of recreational doses of MDMA (1.5\u00a0mg\/kg, p.o.) involve transporter-mediated release of 5-HT (Liechti et al. 2000; Liechti and Vollenweider 2001). Nash et al. (1988) showed that i.p. injections of 1\u20133\u00a0mg\/kg of MDMA stimulate prolactin and corticosterone secretion in rats, and similar oral doses increase plasma prolactin and cortisol in human drug users (Harris et al. 2002; Mas et al. 1999). The dose of MDMA discriminated by rats and humans is identical: 1.5\u00a0mg\/kg, i.p. for rats (Glennon and Higgs 1992; Oberlender and Nichols 1988; Schechter 1988) and 1.5\u00a0mg\/kg, p.o. for humans (Johanson et al. 2006). A few studies have shown that rats will self-administer MDMA at doses ranging from 0.25\u20131.0\u00a0mg\/kg i.v., indicating these doses possess reinforcing efficacy (Ratzenboeck et al. 2001; Schenk et al. 2003). Wakonigg et al. (2003) demonstrated that a single i.v. injection of 1\u00a0mg\/kg MDMA serves a powerful reinforcer in an operant runway procedure, and MDMA displays similar reinforcing potency in Sprague\u2013Dawley and Long\u2013Evans rat strains. Tancer and Johanson (2003) reported that 1 and 2\u00a0mg\/kg of MDMA have reinforcing properties in humans that resemble those of (+)-amphetamine. The findings summarized in Table\u00a03 suggest that there is no scientific justification for using interspecies scaling to \u201cadjust\u201d MDMA doses between rats and humans.\nBased on this analysis, we devised an MDMA dosing regimen in rats, which attempts to mimic binge use of MDMA in humans. Male Sprague\u2013Dawley rats weighing 300\u2013350\u00a0g were double-housed in plastic cages, under conditions of constant ambient temperature (22\u00b0C) and humidity (70%) in a vivarium. In our initial studies, three i.p. injections of 1.5 or 7.5\u00a0mg\/kg MDMA were administered, one dose every 2\u00a0h, to yield cumulative doses of 4.5 or 22.5\u00a0mg\/kg. Control rats received saline vehicle according to the same schedule. Rats were removed from their cages to receive i.p. injections but were otherwise confined to their home cages. The 1.5-mg\/kg dose was used as a low \u201cbehavioral\u201d dose, whereas the 7.5-mg\/kg dose was used as a high \u201cnoxious\u201d dose (i.e., a dose fivefold greater than threshold). Our repeated dosing regimen was designed to account for the common practice of sequential dosing (i.e., \u201cbumping\u201d) used by human subjects during rave parties (Parrott 2002). During the binge dosing procedure, body temperatures were measured by insertion of a thermometer probe into the rectum, and 5-HT-mediated behaviors were scored every hour. Rats were decapitated 2\u00a0weeks after dosing, brain regions were dissected, and tissue levels of 5-HT and DA were determined by HPLC\u2013ECD as described previously (Baumann et al. 2001). Neurochemical data were normalized to percentage of saline control values for each brain region examined. Data were analyzed using a one-way ANOVA (MDMA dose), followed by Duncan\u2019s post hoc test.\nFigure\u00a04 illustrates new data showing that our binge MDMA dosing regimen increases core body temperature in rats (F2,48=40.44, P<0.001). Specifically, repeated i.p. doses of 7.5\u00a0mg\/kg MDMA elicited persistent hyperthermia on the day of treatment, whereas doses of 1.5\u00a0mg\/kg did not. The 7.5-mg\/kg dose caused temperature increases that were about 2\u00b0C greater than control treatment. The data in Fig.\u00a05 demonstrate that binge MDMA treatment significantly decreases tissue 5-HT levels in the frontal cortex (F2,12=42.96, P<0.0001), striatum (F2,12=11.46, P<0.001), and olfactory tubercles (F2,12=21.27, P<0.0001) when assessed 2\u00a0weeks later. Post hoc tests revealed that high-dose MDMA produced long-term depletions of tissue 5-HT (\u223c50% reductions) in all three regions examined, but the low-dose group had 5-HT concentrations similar to saline controls. Transmitter depletion was selective for 5-HT neurons since tissue DA levels were unaffected. The magnitude of 5-HT depletions depicted in Fig.\u00a05 is similar to that observed by others (Battaglia et al. 1987; Schmidt 1987; Stone et al. 1987). Our findings demonstrate that repeated treatment with behaviorally relevant doses of MDMA does not cause acute hyperthermia or long-term 5-HT depletions. In contrast, repeated administration of MDMA at a dose that is fivefold higher than the behavioral dose causes both of these adverse effects. The data are consistent with those of O\u2019Shea et al. (1998), who reported that high-dose MDMA (10 or 15\u00a0mg\/kg, i.p.) but not low-dose MDMA (4\u00a0mg\/kg, i.p.) causes acute hyperthermia and long-term 5-HT depletion in Dark Agouti rats.\nFig.\u00a04Acute effects of (\u00b1)-MDMA on core body temperature in rats. Male rats received three i.p. injections of 1.5 or 7.5\u00a0mg\/kg MDMA, one dose every 2\u00a0h (i.e., injections at 0, 2, and 4\u00a0h). Saline was administered on the same schedule. Core temperature was recorded via insertion of a rectal thermometer probe every 2\u00a0h. Data are mean\u00b1SEM for N=5 rats\/group. Asterisk denotes significance with respect to saline-injected control at each time point (P<0.05 Duncan\u2019s)Fig.\u00a05Long-term effects of (\u00b1)-MDMA on tissue levels of 5-HT (top panel) and DA (bottom panel) in brain regions. Male rats received three i.p. injections of 1.5 or 7.5\u00a0mg\/kg MDMA, one dose every 2\u00a0h. Saline was administered on the same schedule. Rats were killed 2\u00a0weeks after injections; brain regions were dissected, and tissue 5-HT and DA were assayed by HPLC\u2013ECD (Baumann et al. 2001). Data are mean\u00b1SEM expressed as the percentage of saline-treated control values for each region, N=5 rats\/group. Control values of 5-HT and DA were 557\u00b124 and 28\u00b14\u00a0pg\/mg tissue for frontal cortex (CTX), 429\u00b136 and 10,755\u00b1780\u00a0pg\/mg tissue for striatum (STR), and 1,174\u00b1114 and 4,545\u00b1426\u00a0pg\/mg tissue for olfactory tubercle (OT). Asterisk denotes significance compared to saline-injected control for each region (P<0.05 Duncan\u2019s)\nEffects of MDMA on established markers of neurotoxicity\nHallmark indicators of neurotoxicity include cell death, silver positive staining (i.e., argyrophilia), and glial cell hypertrophy (Baumgarten and Lachenmayer 2004; O\u2019Callaghan and Sriram 2005; Switzer 2000). Despite the abundance of evidence showing that MDMA causes 5-HT depletions, few studies have examined the effects of MDMA on established markers of neurotoxic damage. Indeed, some investigators have argued that based on 5-HT abnormalities alone, one cannot infer the presence of neurotoxicity (O\u2019Callaghan and Miller 1993; Wang et al. 2004). Support for the hypothesis of MDMA-induced axotomy relies heavily upon immunohistochemical analysis of 5-HT levels, and the use of semiquantitative methods could produce misleading results if not validated by other means. For example, the MDMA-induced disappearance of immunoreactive 5-HT in specific brain regions could reflect depletion of transmitter to a low level (i.e., below the level of detection) in intact axons and nerve terminals. Thus, an alternative hypothesis consistent with available data is that MDMA-induced deficits in 5-HT systems are due to persistent adaptive changes in gene expression or protein function, and these changes reflect a state of metabolic quiescence or exhaustion, rather than neurotoxic damage. Table\u00a04 summarizes the findings from studies that have examined the effects of MDMA on established indicators of neurotoxic damage. All of the studies employed large doses of MDMA, either as single or repeated injections, which cause hyperthermia and its associated complications. \nTable\u00a04Effects of (\u00b1)-MDMA administration on markers of neuronal degeneration in ratsCNS markerDosing regimenSurvival intervalReferenceIncreased silver-positive staining in degenerating neurons80\u00a0mg\/kg, s.c., twice daily, 4\u00a0days15\u201348\u00a0hCommins et al. (1987)25\u2013150\u00a0mg\/kg, s.c., twice daily, 2\u00a0days2\u00a0daysJensen et al. (1993)Increased Fluoro-Jade B staining of degenerating neurons 20\u201340\u00a0mg\/kg, i.p., single dose2\u00a0daysSchmued (2003)Reactive astrogliosis as measured by elevations in GFAP75\u2013150\u00a0mg\/kg, s.c., twice daily, 2\u00a0days2\u00a0daysO\u2019Callaghan and Miller (1993)a\n20\u00a0mg\/kg, i.p., single dose1\u00a0weekAguirre et al. (1999)20\u00a0mg\/kg, s.c., twice daily, 4 days 3\u00a0days, 1\u00a0weekPubill et al. (2003)a\n7.5\u00a0mg\/kg, i.p., three doses2\u00a0weeksWang et al. (2004)a\naThese investigators found no effect of MDMA on GFAP at doses which significantly depleted 5-HT levels in brain tissue\nSilver staining methods are commonly utilized to identify degenerating neuronal elements in the CNS [reviewed by Switzer (2000)]. In particular, silver stains are sensitive indicators of damage caused by neurotoxic chemicals. Commins et al. (1987) examined the neurotoxic potential of MDMA by using the Fink\u2013Heimer procedure to detect argyrophilic neuronal structures. In their study, male Sprague\u2013Dawley rats received single or multiple s.c. injections of 80\u00a0mg\/kg MDMA and were killed 15 to 48\u00a0h later. Single doses of MDMA increased silver-positive staining only in the frontoparietal cortex. Multiple doses caused more extensive damage; degenerating nerve terminals were found in the striatum, while degenerating terminals, axons, and cell bodies were observed throughout layers III and IV of the parietal cortex. In a comparable study, Jensen et al. (1993) used the de Olmos cupric\u2013silver procedure to delineate areas of the brain damaged by MDMA. Male Long\u2013Evans rats received twice daily s.c. injections of 25\u2013150\u00a0mg\/kg MDMA for 2\u00a0days and were sacrificed 48\u00a0h thereafter. In this case, MDMA produced dose-dependent increases in silver-positive staining in the parietal cortex, with some irregular staining in the striatum and thalamus at higher doses. Most of the staining in the cortex was associated with degenerating axons and terminals, but cell bodies were also stained. The collective results show that administration of MDMA at sufficient doses (i.e., >25\u00a0mg\/kg) can cause neuronal damage, as indicated by sensitive silver staining methods.\nThere are several key factors to consider when interpreting the effects of MDMA on silver staining in rat brain. Most importantly, both of the cited studies employed massive cumulative doses of MDMA ranging from 80 to 600\u00a0mg\/kg. Based on the concept of effect scaling described previously (see Table\u00a03), the single injections of MDMA administered by Commins et al. and Jensen et al. are 16\u2013100 times higher than a behaviorally relevant dose of 1.5\u00a0mg\/kg. At such extraordinary doses of MDMA, excessive sympathetic activation and hyperthermia could cause physiological dysregulation sufficient to elicit nonspecific neuropathy. The idea that high-dose MDMA causes nonspecific neuronal damage is supported by the observation that increases in silver staining are not confined to 5-HT cells. Both studies noted the presence of argyrophilic cell bodies in the cortex of MDMA-treated rats, yet 5-HT cell bodies are not present in the cortex (Steinbusch 1981). Furthermore, the pattern of MDMA-induced silver staining, which is largely confined to the frontoparietal cortex, does not correspond to the pattern of 5-HT innervation or the pattern of 5-HT depletions. Given these findings, it seems feasible that increases in silver-positive staining produced by high-dose MDMA do not reflect 5-HT neurotoxicity per se.\nSchmued (2003) used a novel histological stain, Fluoro-Jade B, to examine neuronal damage produced by MDMA administration in rats. Fluoro-Jade B is an anionic fluorescein derivative that selectively stains degenerating terminals, axons, and cell bodies (Schmued and Hopkins 2000). Male Sprague\u2013Dawley rats received single i.p. injections of 10\u201340\u00a0mg\/kg MDMA and were killed 48\u00a0h later. After 10\u00a0mg\/kg MDMA, only one of four rats exhibited Fluoro-Jade staining in the cortex, and this rat experienced significant hyperthermia at the time of treatment. At higher doses of MDMA, most rats displayed Fluoro-Jade staining in a variety of brain areas. For example, degenerating pyramidal and nonpyramidal cells were stained in the parietal cortex, while degenerating multipolar cells were stained in the ventral thalamus. The findings with Fluoro-Jade B indicate that sufficient doses of MDMA (i.e., >20\u00a0mg\/kg) can produce neuronal degeneration. However, analogous to the findings with silver staining methods, many of the damaged cells identified by Fluoro-Jade B are nonserotonergic, and the pattern of staining does not overlap with the pattern of 5-HT deficits. Additionally, neuronal degeneration was only found in rats that experienced hyperthermia of >41\u00b0C, again suggesting an important link between elevated body temperature and adverse effects.\nA universal reaction to damage in the CNS is hypertrophy of astrocytes, or \u201creactive gliosis\u201d [reviewed by O\u2019Callaghan and Sriram (2005)]. This response is accompanied by enhanced expression of glial-specific structural proteins such as, glial fibrillary acidic protein (GFAP). O\u2019Callaghan et al. (1995) have verified that a wide range of neurotoxic chemicals increase the levels of GFAP in rodent CNS, indicating this protein can be used as a sensitive marker of neuronal damage. These investigators (O\u2019Callaghan and Miller 1993) carried out extensive studies to evaluate the effects of MDMA administration on GFAP expression in rat brain. Male Long\u2013Evans rats were treated twice daily with s.c. injections of 10\u201330\u00a0mg\/kg MDMA for 7\u00a0days and were sacrificed 48\u00a0h after the last dose. This high-dose regimen of MDMA did not alter the expression of GFAP, even though 5-HT levels were markedly reduced in the cortex, hippocampus, and striatum. A separate group of rats received twice daily s.c. injections of 75\u2013150\u00a0mg\/kg MDMA for 2\u00a0days, and these rats were killed 48\u00a0h later. In response to these massive doses of MDMA, levels of GFAP were significantly elevated in various brain regions but these increases did not correlate with the degree of 5-HT depletions. Finally, the effects of MDMA were compared to the effects of the 5-HT neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT). At doses of MDMA and 5,7-DHT which caused comparable 5-HT depletions, only 5,7-DHT increased GFAP.\nIn a more recent study, Pubill et al. (2003) compared the effects of MDMA and methamphetamine on SERT binding and a number of glial-specific markers. Male Sprague\u2013Dawley rats received twice daily s.c. injections of 20\u00a0mg\/kg MDMA for 4\u00a0days, and were killed at 3 and 7\u00a0days posttreatment. MDMA caused marked decreases in [3H]-paroxetine-labeled SERT binding but had no effect on any measures of reactive gliosis. In particular, MDMA treatment failed to induce astroglial activation as indicated by the lack of changes in immunoreactive GFAP. MDMA had no effect on microglial markers such as peripheral benzodiazepine receptors and OX-6 protein. Interestingly, methamphetamine produced significant increases in astroglial and microglial markers in this study, indicating an important distinction between methamphetamine and MDMA. Consistent with the findings of Pubill and colleagues, Bai et al. (2001) found that 10\u00a0mg\/kg s.c. MDA failed to increase GFAP expression in Sprague\u2013Dawley rats. In contrast, Aguirre et al. (1999) reported that a single i.p. dose of 20\u00a0mg\/kg MDMA enhanced histochemical staining for GFAP in the hippocampus of Wistar rats. The reasons for the discrepancies between the findings of Aguirre et al. vs the findings of others are not readily apparent, but could be due to differences in experimental methods across studies (i.e., different rat strains, different brain regions examined, etc).\nThe majority of data indicate that doses of MDMA causing significant 5-HT depletions (i.e., single or repeated doses of 10\u201320\u00a0mg\/kg) do not reliably induce silver staining or glial activation. Recent results from our laboratory support this assertion (Rothman et al. 2003; Wang et al. 2004). We examined the possible neurotoxic effects of MDMA and two other substituted amphetamines, (+)-fenfluramine and p-chloroamphetamine (PCA). Specifically, the effects of drug treatments on the expression of SERT and GFAP were evaluated using Western blot methods. In the first study, male Sprague\u2013Dawley rats received four i.p. injections of 6\u00a0mg\/kg (+)-fenfluramine or one i.p. injection of 5\u00a0mg\/kg PCA, and rats were killed 2\u00a0days or 2\u00a0weeks thereafter. (+)-Fenfluramine and PCA decreased cortical 5-HT levels (\u223c50\u201360%) but neither drug caused long-term changes in the expression of SERT or GFAP (Rothman et al. 2003). Similar results were found when three i.p. injections of 7.5\u00a0mg\/kg MDMA were administered to rats according to the binge dosing regimen described above (see Figs.\u00a04 and 5). MDMA did not alter levels of immunoreactive SERT or GFAP when examined 2\u00a0weeks after treatment, even though the same rats displayed substantial depletions of 5-HT in the cortex, striatum, and hippocampus (Wang et al. 2004). Thus, doses of MDMA that deplete tissue 5-HT do not alter expression of SERT or GFAP. The SERT data are especially surprising given that MDMA administration is known to reduce the number of SERT binding sites, thereby decreasing [3H]5-HT uptake capability (Battaglia et al. 1987; Commins et al. 1987; Schmidt 1987).\nTo explore this phenomenon further, we compared the effects of MDMA and 5,7-DHT on the expression of SERT, GFAP, and other markers of gliosis (Wang et al. 2005). In all cases, proteins were measured by Western blot analysis using sensitive polyclonal antibodies directed against the protein of interest. Sprague\u2013Dawley rats received three i.p. injections of 7.5\u00a0mg\/kg MDMA or saline vehicle, and were killed at 3\u00a0days and 2\u00a0weeks later. A separate group of rats received single intracerebroventricular (i.c.v.) infusions of 150\u00a0\u03bcg 5,7-DHT or 0.1% ascorbic acid vehicle. Both MDMA and 5,7-DHT caused 50\u201370% depletions of 5-HT in the frontal cortex at 3\u00a0days and 2\u00a0weeks after treatments. Figure\u00a06 depicts previously published data from our laboratory showing that MDMA does not affect tissue levels of SERT at 2\u00a0weeks posttreatment, whereas 5,7-DHT causes significant decreases in this protein (Wang et al. 2005). The data in Fig.\u00a07 reveal that binge MDMA treatment does not affect GFAP, while 5,7-DHT increases levels of this protein. It should be mentioned that treatment with 5,7-DHT, but not MDMA, produces persistent increases in another astrocyte marker, heat shock protein 32. On-going studies in our laboratory indicate that MDMA does not alter the intracellular trafficking of SERT proteins from endosomes to the plasma membrane (Wang et al. 2005). Rather, it seems that MDMA renders SERT proteins nonfunctional, though the mechanism responsible for this effect is not known. Our findings provide evidence that 5,7-DHT decreases SERT and increases GFAP, whereas MDMA does not alter expression of either protein. Accordingly, at the doses tested in these studies, 5,7-DHT displays the profile of a 5-HT neurotoxin, whereas MDMA does not.\nFig.\u00a06Comparative effects of (\u00b1)-MDMA (top panel) and 5,7-DHT (bottom panel) on SERT protein expression in dissected brain regions. One group of rats received three i.p. injections of saline or 7.5\u00a0mg\/kg MDMA, one dose every 2\u00a0h. Another group received single i.c.v. infusions of 5,7-DHT or vehicle. Rats were killed 2\u00a0weeks later and brain regions were dissected. Western blot analysis of SERT immunoreactivity in the frontal cortex (CTX), striatum (STR), and hippocampus (HIPP) was carried out as described (Wang et al. 2005). Blots were digitized and quantified using densitometry (NIH IMAGE software). Changes in immunoreactivity are expressed relative to their corresponding control (defined as 100% value). Each value is the mean\u00b1SEM for N=4 rats\/group. Asteriskdenotes significance with respect to control in each region (P<0.01 Student\u2019s t test). Data taken from Wang et al. 2005\nFig.\u00a07Comparative effects of (\u00b1)-MDMA (top panel) and 5,7-DHT (bottom panel) on GFAP expression in dissected brain regions. One group of rats received three i.p. injections of saline or 7.5\u00a0mg\/kg MDMA, one dose every 2\u00a0h. Another group received single i.c.v. infusions of 5,7-DHT or vehicle. Rats were killed 2\u00a0weeks later and brain regions were dissected. Western blot analysis of GFAP immunoreactivity in the frontal cortex (CTX), striatum (STR), and hippocampus (HIPP) was carried out as described (Wang et al. 2005). Blots were digitized and quantified using densitometry (NIH IMAGE software). Changes in immunoreactivity are expressed relative to their corresponding control (defined as 100% value). Each value is the mean\u00b1SEM for N=4 rats\/group. Asterisk denotes significance with respect to control in each region (P<0.01 Student\u2019s t test). Data were taken from Wang et al. 2005\nFunctional consequences of MDMA-induced 5-HT depletion\nAny operational definition of \u201cneurotoxicity\u201d must include the precept that functional impairments accompany neuronal damage (Moser 2000; Winneke and Lilienthal 1992). As noted previously, high-dose MDMA causes persistent inactivation of tryptophan hydroxylase, which leads to inhibition of 5-HT synthesis and loss of 5-HT (O\u2019Hearn et al. 1988; Stone et al. 1987). Moreover, MDMA-induced reduction in the density of SERT binding sites leads to decreased capacity for 5-HT uptake in nervous tissue (Battaglia et al. 1987; Schmidt 1987). Regardless of whether these deficits reflect neurotoxic damage or long-term adaptation, such changes would be expected to have discernible in vivo correlates. Many investigators have examined functional consequences of high-dose MDMA administration, and a comprehensive review of this subject is beyond the scope of the present paper [reviewed by Cole and Sumnall 2003; Green et al. (2003)]. The following discussion will consider long-term effects of MDMA (i.e., >1\u00a0week) on in vivo indicators of 5-HT function in rats, as measured by electrophysiological recording, microdialysis sampling, neuroendocrine secretion, and specific aspects of behavior. A number of key findings are summarized in Table\u00a05. In general, few published studies have been able to relate the magnitude of MDMA-induced 5-HT depletion to the degree of specific functional impairment. Furthermore, MDMA administration rarely causes persistent changes in baseline measures of neural function, and deficits are most readily demonstrated by provocation of the 5-HT system by pharmacological (e.g., drug challenge) or physiological means (e.g., environmental stress). \nTable\u00a05Effects of (\u00b1)-MDMA administration on functional indices of 5-HT transmission in ratsCNS effectDosing regimenSurvival intervalReferenceNo change in 5-HT cell firing20\u00a0mg\/kg, s.c., twice daily, 4\u00a0days2\u00a0weeksGartside et al. (1996)Reductions in evoked 5-HT release in vivo20\u00a0mg\/kg, s.c., twice daily, 4\u00a0days2\u00a0weeksSeries et al. (1994)10\u00a0mg\/kg, i.p., twice daily, 4\u00a0days1\u00a0weekShankaran and Gudelsky (1999)Changes in corticosterone and prolactin secretion20\u00a0mg\/kg, s.c., single dose2\u00a0weeksPoland et al. (1997)20\u00a0mg\/kg, s.c. twice daily, 4 days;4, 8, and 12\u00a0monthsIncreased anxiety-like behaviors5\u00a0mg\/kg, s.c., 1 or four injections, 2\u00a0days3\u00a0monthsMcGregor et al. (2003)a and Morley et al. (2001)7.5\u00a0mg\/kg, sc, twice daily, 3\u00a0days2\u00a0weeksFone et al. (2002)a\naThese investigators noted marked increases in anxiogenic behaviors in the absence of significant MDMA-induced 5-HT depletion in brain\n5-HT projections innervating the rat forebrain have cell bodies residing in the raphe nuclei (Steinbusch 1981). These neurons exhibit pacemaker-like electrical activity, which can be recorded using electrophysiological techniques (Aghajanian et al. 1978; Sprouse et al. 1989). Gartside et al. (1996) used extracellular recording methods to examine 5-HT cell firing in the dorsal raphe of rats previously treated with MDMA. Rats received two daily injections of 20\u00a0mg\/kg, s.c. MDMA for 4\u00a0days and were tested under chloral hydrate anesthesia 2\u00a0weeks later. MDMA pretreatment had no effect on the number of classical or burst-firing 5-HT cells encountered during recording. Additionally, 5-HT cell firing rates and action potential characteristics were not different between MDMA- and saline-pretreated groups. These data show that 5-HT neurons and their firing properties are not altered after MDMA administration, and this agrees with immunohistochemical evidence demonstrating that MDMA does not destroy 5-HT perikarya. The electrophysiological data from MDMA-pretreated rats differ from the findings reported with 5,7-DHT. In rats treated with i.c.v. 5,7-DHT, the number of classical and burst-firing 5-HT neurons is dramatically decreased in the dorsal raphe, in conjunction with a loss of 5-HT fluorescence (Aghajanian et al. 1978; Hajos and Sharp 1996). Thus, 5,7-DHT produces reductions in 5-HT cell firing that are attributable to cell death, but MDMA does not.\nIn vivo microdialysis allows continuous sampling of extracellular fluid from intact brain, and this method has been used to evaluate persistent neurochemical consequences of MDMA exposure (Gartside et al. 1996; Matuszewich et al. 2002; Series et al. 1994; Shankaran and Gudelsky 1999). Series et al. 1994 carried out microdialysis in rat frontal cortex to examine long-term effects of MDMA administration. Male Sprague\u2013Dawley rats received twice daily s.c. injections of 20\u00a0mg\/kg MDMA for 4\u00a0days, and were tested under chloral hydrate anesthesia 2\u00a0weeks later. Prior MDMA exposure did not affect baseline extracellular levels of 5-HT, but levels of the 5-HT metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were decreased to \u223c30% of control. Moreover, the ability of (+)-fenfluramine (10\u00a0mg\/kg, i.p.) to evoke 5-HT release was markedly blunted in MDMA-pretreated rats when compared to saline-pretreated controls. In an analogous investigation, Shankaran and Gudelsky (1999) assessed neurochemical effects of acute MDMA challenge in rats that had previously received four i.p. injections of 10\u00a0mg\/kg MDMA. In vivo microdialysis was performed in the striatum of conscious rats 1\u00a0week after high-dose MDMA treatment. Baseline levels of dialysate 5-HT were not altered by prior MDMA exposure, even though tissue levels of 5-HT in striatum were depleted by 50%. The ability of MDMA to evoke 5-HT release was severely impaired in MDMA-pretreated rats, while the concurrent DA response was normal. In this same study, effects of MDMA on body temperature and 5-HT syndrome were attenuated in MDMA-pretreated rats, suggesting the development of tolerance. Other investigations using in vivo microdialysis methods have shown that 5-HT release in response to physiological or stressful stimuli is impaired in rats pretreated with high-dose MDMA (Gartside et al. 1996; Matuszewich et al. 2002).\nTaken together, the microdialysis studies reveal important long-term consequences of MDMA administration: (1) baseline levels of dialysate 5-HT are not altered despite depletions of tissue transmitter, (2) baseline levels of dialysate 5-HIAA are consistently decreased, and (3) stimulated release of 5-HT is blunted in response to pharmacological or physiological provocation. The microdialysis findings with MDMA resemble those obtained from rats treated with 5,7-DHT, in which lesioned rats display normal baseline dialysate 5-HT but decreased 5-HIAA (Hall et al. 1999; Kirby et al. 1995; Romero et al. 1998). In a representative study, Kirby et al. (1995) used microdialysis in rat striatum to evaluate the long-term neurochemical effects of i.c.v. 5,7-DHT; these investigators showed that reductions in dialysate 5-HIAA and impairments in stimulated 5-HT release are highly correlated with loss of tissue 5-HT, whereas baseline dialysate 5-HT is not. In fact, depletions of brain tissue 5-HT up to 90% did not affect baseline levels of dialysate 5-HT (Kirby et al. 1995). It seems that adaptive mechanisms serve to maintain normal concentrations of synaptic 5-HT, even under conditions of severe transmitter depletion. A comparable situation exists after lesions of the nigrostriatal DA system, where baseline levels of extracellular DA are maintained in the physiological range despite substantial loss of tissue DA [see Zigmond et al. (1990)]. In the case of high-dose MDMA treatment, it seems feasible that reductions in 5-HT uptake (e.g., less functional SERT protein) and metabolism (e.g., decreased monoamine oxidase activity) compensate for 5-HT depletions to keep optimal concentrations of 5-HT bathing nerve cells. On the other hand, deficits in 5-HT release are readily demonstrated in MDMA-pretreated rats when 5-HT systems are taxed by drug challenge or environmental stressors.\n5-HT neurons projecting to the hypothalamus provide stimulatory input for the secretion of adrenocorticotropin (ACTH) and prolactin from the anterior pituitary (Van de Kar 1991). Accordingly, 5-HT releasers (e.g., fenfluramine) and 5-HT receptor agonists increase plasma levels of these hormones in rats and humans [reviewed by Levy et al. (1994)]. Neuroendocrine challenge experiments have been used to demonstrate changes in serotonergic responsiveness in rats treated with MDMA (Poland 1990; Poland et al. 1997; Series et al. 1995). In the most comprehensive study, Poland et al. (1997) examined effects of high-dose MDMA on hormone responses elicited by acute fenfluramine challenge. Male Sprague\u2013Dawley rats received single s.c. injections of 20\u00a0mg\/kg MDMA and were tested 2\u00a0weeks later. Prior MDMA exposure did not alter baseline levels of circulating ACTH or prolactin. However, in MDMA-pretreated rats, fenfluramine-induced ACTH secretion was reduced, while prolactin secretion was enhanced. The MDMA dosing regimen caused significant depletions of tissue 5-HT in various brain regions, including hypothalamus. In a follow-up time-course study, rats received twice daily s.c. injections of 20\u00a0mg\/kg MDMA for 4\u00a0days, and were challenged with fenfluramine (6\u00a0mg\/kg, s.c.) at 4, 8, and 12\u00a0months thereafter. As observed in the single dose MDMA study, rats exposed to multiple MDMA doses displayed blunted ACTH responses and augmented prolactin responses to fenfluramine. Interestingly, the impaired ACTH response persisted for 12\u00a0months in MDMA-pretreated rats, even though tissue levels of 5-HT were not depleted at this time point. The data show that high-dose MDMA can cause functional abnormalities for up to 1\u00a0year, and changes in 5-HT responsiveness do not necessarily parallel the extent of recovery from 5-HT depletions in brain.\nIn our laboratory, we wished to further explore the long-term neuroendocrine consequences of MDMA administration. Utilizing the binge MDMA dosing regimen described previously (see Figs.\u00a04 and 5), male Sprague\u2013Dawley rats received three i.p. injections of 1.5 or 7.5\u00a0mg\/kg MDMA, one dose every 2\u00a0h. Control rats received saline vehicle according to the same schedule. One week after MDMA treatment, rats were fitted with indwelling jugular catheters under pentobarbital anesthesia. After 1\u00a0week of recovery from surgery (i.e., 2\u00a0weeks after MDMA or saline), rats were brought into the testing room and i.v. catheters were connected to extension tubes. Each rat received an i.v. injection of 1\u00a0mg\/kg MDMA at time zero, followed by an injection of 3\u00a0mg\/kg MDMA 60\u00a0min later. Blood samples were withdrawn via the catheters immediately before and 30\u00a0min after each dose of MDMA. Plasma levels of corticosterone and prolactin were measured by radioimmunoassay methods (Baumann et al. 1998). Hormone data were normalized to percentage of preinjection control and analyzed using a two-way ANOVA (pretreatment \u00d7 acute treatment), followed by Duncan\u2019s post hoc test. Figure\u00a08 shows new data from our laboratory demonstrating the effects of binge MDMA pretreatment on hormone responses examined 2\u00a0weeks later. MDMA pretreatment did not alter baseline levels of either hormone. Acute administration of MDMA elicited dose-related elevations in circulating corticosterone (F2,63=62.03, P<0.0001) and prolactin (F2,63=45.41, P<0.0001) as shown by others (Nash et al. 1988). Importantly, MDMA pretreatment significantly attenuated MDMA-induced corticosterone (F2,63=6.89, P<0.01) and prolactin (F2,63=8.30, P<0.001) responses. Post hoc tests revealed that rats exposed to high-dose MDMA pretreatment displayed reductions in corticosterone and prolactin secretion in response to acute MDMA challenge, whereas hormone responses in the low-dose MDMA rats were indistinguishable from controls.\nFig.\u00a08Effects of (\u00b1)-MDMA pretreatment on secretion of corticosterone (top panel) and prolactin (bottom panel) evoked by acute (\u00b1)-MDMA challenge. Male rats received three i.p. injections of 1.5 or 7.5\u00a0mg\/kg MDMA, one dose every 2\u00a0h. Saline was administered on the same schedule. Two weeks later, each rat received a single i.v. injection of 1\u00a0mg\/kg MDMA at time zero, followed by a single injection of 3\u00a0mg\/kg MDMA 60\u00a0min later. Blood samples were drawn via indwelling catheters immediately before injections and 30\u00a0min after each injection; plasma corticosterone and prolactin were measured by RIA (Baumann et al. 1998). Data are mean\u00b1SEM expressed as the percentage of baseline hormone levels for N=8 rats\/group. Baseline corticosterone and prolactin levels were 73\u00b118 and 2.4\u00b10.6\u00a0ng\/ml of plasma, respectively. Asterisk denotes significance compared to saline-pretreated control group (P<0.05 Duncan\u2019s)\nOur neuroendocrine results are consistent with the development of tolerance to hormonal effects of MDMA. These findings do not agree completely with the data of Poland et al. (1997) discussed above; however, our findings do agree with previous data showing blunted hormonal responses to fenfluramine in rats with fenfluramine-induced 5-HT depletions (Baumann et al. 1998). Perhaps more importantly, the data shown in Fig.\u00a08 are strikingly similar to clinical findings in which cortisol and prolactin responses to acute (+)-fenfluramine administration are reduced in human MDMA users (Gerra et al. 1998, 2000; Gouzoulis-Mayfrank et al. 2002). Indeed, Gerra et al. (2000) reported that (+)-fenfluramine-induced prolactin secretion is blunted in abstinent MDMA users for up to 1\u00a0year after cessation of drug use. The mechanism(s) underlying reduced sensitivity to (+)-fenfluramine and MDMA are not known, but it is tempting to speculate that MDMA-induced impairments in evoked 5-HT release are involved, as shown by in vivo microdialysis studies. While some investigators have cited neuroendocrine changes in human MDMA users as evidence for 5-HT neurotoxicity, Gouzoulis-Mayfrank et al. (2002) provide a compelling argument that endocrine abnormalities in MDMA users could be related to cannabis use rather than MDMA. Further experiments will be required to resolve the precise nature of neuroendocrine changes in MDMA users.\nOne of the more serious and disturbing clinical findings is that MDMA causes persistent cognitive deficits in some users (Morgan 2000; Reneman 2003). Numerous research groups have examined the effects of MDMA treatment on learning and memory processes in rats, yet most studies failed to identify persistent impairments, even when extensive 5-HT depletions were present (Byrne et al. 2000; McNamara et al. 1995; Ricaurte et al. 1993; Robinson et al. 1993; Seiden et al. 1993; Slikker et al. 1989). While an exhaustive review of this literature is not possible here, representative findings will be mentioned. In an extensive series of experiments, Seiden et al. (1993) evaluated the effects of high-dose MDMA on a battery of tests including open-field behavior, schedule-controlled behavior, one-way avoidance, discriminated two-way avoidance, forced swim, and radial maze performance. Male Sprague\u2013Dawley rats received twice daily s.c. injections of 10\u201340 mg\/kg MDMA for 4\u00a0days and were tested beginning 2\u00a0weeks after treatment. Despite large depletions of brain tissue 5-HT, MDMA-pretreated rats exhibited normal behaviors in all paradigms. Likewise, Robinson et al. (1993) found that MDMA-induced depletion of cortical 5-HT up to 70% did not alter spatial navigation, skilled forelimb use, or foraging behavior in rats. On the other hand, Marston et al. (1999) reported that MDMA administration produces persistent deficits in a delayed nonmatch to performance (DNMTP) procedure when long delay intervals are employed (i.e., 30\u00a0s). Specifically, saline-pretreated rats exhibited progressive improvement in task performance over successive days of testing, whereas MDMA-pretreated rats did not. The authors theorized that delay-dependent impairments in the DNMTP procedure reflect MDMA-induced deficits in short-term memory, possibly attributable to 5-HT depletion.\nWith the exception of the findings of Marston et al., the collective behavioral data in rats indicate that MDMA-induced depletions of brain 5-HT have little effect on cognitive processes. There are several potential explanations for this apparent paradox. First, high-dose MDMA administration produces only partial depletion of 5-HT in the range of 40\u201360% in most brain areas. This level of 5-HT loss may not be sufficient to elicit behavioral alterations, as compensatory adaptations in 5-HT neurons could maintain normal physiological function. Second, MDMA appears to selectively affect fine diameter fibers arising from the dorsal raphe, and it is possible that these 5-HT circuits may not subserve the behaviors being monitored. Third, the behavioral tests utilized in rat studies might not be sensitive enough to detect subtle changes in learning and memory processes. Finally, the functional reserve capacity in the CNS might be sufficient to compensate for even large depletions of a single transmitter.\nWhile MDMA appears to have few long-term effects on cognition in rats, a growing body of evidence demonstrates that MDMA administration can cause persistent anxiety-like behaviors in this species (Fone et al. 2002; Gurtman et al. 2002; Morley et al. 2001). Morley et al. (2001) first reported that MDMA exposure induces long-term anxiety in rats. These investigators gave male Wistar rats one or four i.p. injections of 5\u00a0mg\/kg MDMA for two consecutive days. Subjects were tested 3\u00a0months later in a battery of anxiety-related paradigms, including elevated plus maze, emergence, and social interaction tests. Rats receiving either single or multiple MDMA injections displayed marked increases in anxiogenic behaviors in all three tests when compared to control rats. Because no 5-HT endpoints were examined, it was impossible to relate changes in behavior to changes in 5-HT transmission. In a follow-up study, Gurtman et al. (2002) replicated the original findings of Morley et al. using rats pretreated with four i.p. injections of 5\u00a0mg\/kg MDMA for 2\u00a0days; persistent anxiogenic effects of MDMA were associated with depletions of 5-HT in the amygdala, hippocampus, and striatum. Interestingly, Fone et al. (2002) showed that twice daily injections of 7.5\u00a0mg\/kg MDMA for 3\u00a0days caused impairments in social interaction in adolescent Lister rats, even in the absence of 5-HT depletions or reductions in [3H]-paroxetine-labeled SERT binding sites. These data suggested the possibility that MDMA-induced anxiety does not require 5-HT deficits.\nIn an attempt to determine potential mechanisms underlying MDMA-induced anxiety, McGregor et al. (2003) evaluated effects of the drug on anxiety-related behaviors and a number of postmortem parameters including autoradiography for SERT and 5-HT receptor subtypes. Rats received moderate (5\u00a0mg\/kg, i.p., 2\u00a0days) or high (5\u00a0mg\/kg, i.p., four injections, 2\u00a0days) doses of MDMA, and tests were conducted 10\u00a0weeks later. This study confirmed that moderate doses of MDMA could cause protracted increases in anxiety-like behaviors without significant 5-HT depletions. Furthermore, the autoradiographic analysis revealed that anxiogenic effects of MDMA may involve long-term reductions in 5-HT2A\/2C receptors, rather than reductions in SERT binding. Additional work by Bull et al. (2003, 2004) suggests that decreases in the sensitivity of 5-HT2A receptors, but not 5-HT2C receptors, could underlie MDMA-associated anxiety. Clearly, more investigation into this important area of research is warranted.\nSummary\nThe findings reviewed in this paper allow a number of conclusions to be drawn with regard to MDMA-induced neurotoxicity. First, there is little doubt that MDMA targets monoamine transporters, and transporter-mediated release of 5-HT, DA, and NE underlies pharmacological effects of the drug. While MDMA has been considered a predominately serotonergic agent, certain adverse effects including cardiovascular stimulation and hyperthermia likely involve NE and DA mechanisms, respectively. There seems to be no scientific rationale for using allometric scaling to adjust doses of MDMA between rats and humans because the pharmacologically relevant doses are similar in both species (e.g., 1\u20132\u00a0mg\/kg). Nonetheless, the complex metabolism of MDMA needs to be examined in various animal species to permit comparison with clinical literature and to validate appropriate preclinical models. With regard to MDMA-induced neurotoxicity, it seems that 5-HT deficits are not always synonymous with axonal death because doses of MDMA which cause marked depletions of brain tissue 5-HT in rats (e.g., 10\u201320\u00a0mg\/kg) are not associated with silver-positive staining, reactive gliosis, or loss of SERT protein. Like many other psychotropic drugs, MDMA is capable of producing bona fide neurotoxicity at sufficient doses (e.g., >25\u00a0mg\/kg), and damage is not confined to 5-HT neurons. Many aspects of 5-HT function appear to be normal in MDMA-pretreated rats despite significant loss of brain 5-HT, perhaps illustrating the profound adaptive capability of the CNS. On the other hand, MDMA-induced 5-HT depletions are accompanied by impairments in evoked 5-HT release and neuroendocrine secretion that suggest tolerance development. The clinical relevance of preclinical findings is often uncertain, but the fact that MDMA can produce persistent increases in anxiety-like behaviors without measurable 5-HT deficits suggests that even moderate doses may pose risks.","keyphrases":["mdma","neurotoxicity","serotonin","depletion","release","hormones","behavior"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"J_Comp_Physiol_A_Neuroethol_Sens_Neural_Behav_Physiol-3-1-1915583","title":"Olfactory receptors on the maxillary palps of small ermine moth larvae: evolutionary history of benzaldehyde sensitivity\n","text":"In lepidopterous larvae the maxillary palps contain a large portion of the sensory equipment of the insect. Yet, knowledge about the sensitivity of these cells is limited. In this paper a morphological, behavioral, and electrophysiological investigation of the maxillary palps of Yponomeuta cagnagellus (Lepidoptera: Yponomeutidae) is presented. In addition to thermoreceptors, CO2 receptors, and gustatory receptors, evidence is reported for the existence of two groups of receptor cells sensitive to plant volatiles. Cells that are mainly sensitive to (E)-2-hexenal and hexanal or to (Z)-3-hexen-1-ol and 1-hexanol were found. Interestingly, a high sensitivity for benzaldehyde was also found. This compound is not known to be present in Euonymus europaeus, the host plant of the monophagous Yponomeuta cagnagellus, but it is a prominent compound in Rosaceae, the presumed hosts of the ancestors of Y. cagnagellus. To elucidate the evolutionary history of this sensitivity, and its possible role in host shifts, feeding responses of three Yponomeuta species to benzaldehyde were investigated. The results confirm the hypothesis that the sensitivity to benzaldehyde evolved during the ancestral shift from Celastraceae to Rosaceae and can be considered an evolutionary relict, retained in the recently backshifted Celastraceae-specialist Y. cagnagellus.\nIntroduction\nRecently, progress has been made in our understanding of speciation in herbivorous insects, and mathematical models demonstrate that adaptive speciation driven by selection on ecological traits is theoretically plausible (Kondrashov and Kondrashov 1999; Dickman and Doebeli 1999; Doebeli 2005). However, as yet not much work has been done on the actual nature of these traits. The theoretical results make clear that host shifts and concomitant changes in host acceptance behaviour are important factors in adaptive speciation, but the underlying changes in the neural system are poorly studied and understood. Only recently work addressing this type of question is starting to appear (cf. Olsson et al. 2005a, b) .\nAlthough host acceptance in phytophagous insects is usually viewed as solely governed by the adult (\u201cmother knows best\u201d), it should be noted that in many species larval host acceptance is equally important. Lepidopterous larvae in particular often display striking food preferences, which are based on a small set of chemoreceptors (Schoonhoven and van Loon 2002), thus making caterpillars promising models to study the neural basis of host plant recognition, host shifts, and the evolution of insect\u2013plant relations. However, such an approach requires detailed knowledge about the responses of the involved receptors, information that is almost completely lacking.\nExternal chemoreceptors in caterpillars are located on the antennae, on the maxillae, and in the epipharynx. The gustatory sensilla styloconica on the galea are important in host acceptance behaviour, and a large body of data is available about their sensitivity (see Schoonhoven and van Loon 2002; Schoonhoven et al. 2005 for review). The other sets of receptors, the epipharyngeal organs on the inside of the labrum, and the sensilla on the antennae and the maxillary palps, are more difficult to study and as a consequence less well known. The maxillary palp receptors in particular have received very little attention. Except for the pioneering studies of Schoonhoven and Dethier (1966), recordings from the maxillary nerve of the silkworm by Hirao et al. (1976, and a recent paper on gustatory receptors in the palp of Choristoneura fumiferana (Albert 2003), no electrophysiological data on the maxillary palps of Lepidoptera appears to be available. However, behavioural work indicates that the influence of the palps on feeding can be as significant as that of the taste sensilla on the galea (Hanson and Dethier 1973; Glendinning et al. 1998; de Boer 1993, 2006).\nThe small ermine moth genus Yponomeuta Latreille (Lepidoptera: Yponomeutidae) consists of some 75 species (Gershenson and Ulenberg 1998). Most are specialised on Celastraceae, and phylogenetic analysis (Menken 1996; Turner H, Lieshout N, van Ginkel W, Menken SBJ, submitted; Ulenberg, personal communication) strongly suggest that this association is the ancestral state in the genus. Moreover, mapping host use upon the phylogeny indicates an intriguing evolutionary scenario: the genus most likely originated in the Far East and dispersed to the western Palearctic concomitant with a (probably single) shift to Rosaceae and further to Salicaceae (Menken 1996). Interestingly, one lineage within this clade, Y. cagnagellus (H\u00fcbner), shifted back to the ancestral host, so this species is secondarily associated with Celastraceae. Yponomeuta plumbellus on the other hand is present in Western Europe, but this species is more closely related to the clades in the Far East, and retained the ancestral relation with Celastraceae.\nThe feeding of the Western European Yponomeuta species, and in particular Y. cagnagellus, is relatively well studied. Both the behaviour (Gerrits-Heybroek et al. 1978; van Drongelen 1980; Kooi and van de Water 1988; Peterson et al. 1990) and the underlying sensory physiology (van Drongelen 1979; Roessingh et al. 1999) have been addressed. The results demonstrate a clade of predominantly monophagous species with well-adapted sensory systems displaying high sensitivity for sugar alcohols characteristic for the host\u2013plant families, as well as reduced sensitivities for host-specific deterrents. However, this whole body of work is focused on gustatory responses of styloconic sensilla on the galea that may provide only part of the relevant sensory inputs (de Boer 2006).\nHere we present a morphological, behavioural, and electrophysiological survey of chemoreceptors on the maxillary palps in the larvae of Yponomeuta cagnagellus. In addition, we performed a comparative study into the effects of benzaldehyde on feeding behaviour of three related Yponomeuta species. Benzaldehyde strongly stimulated sensory cells in the maxillary palp of Y. cagnagellus, but its role in feeding behaviour is unknown. This compound is of particular interest because it is prominent in Rosaceae such as Prunus spinosa (Humpf and Schreier 1992), the host of Y. padellus (L), but absent from Euonymus europaeus, the host of Y. cagnagellus and Y. plumbellus (Denis and Schifferm\u00fcller) (Fig.\u00a01). In this comparison we take advantage of the fact that the phylogenetic tree indicates that Y. plumbellus, although present in Western Europe, belongs to a clade that still displays the original ancestral association with Celastraceae. Yponomeuta padellus is in the middle of the clade that shifted to the benzaldehyde containing Rosaceae. Yponomeuta cagnagellus on the other hand is now feeding on the Celastraceous E. europaeus, that does not contain benzaldehyde, but this species most likely originated from an ancestor that was feeding on Rosaceae. An analysis of the phylogenetic pattern of the response to benzaldehyde in these three species will therefore contribute to our understanding of the flexibility of sensory systems and the role of sensory stimuli in host shifts and adaptive speciation.\nFig.\u00a01Phylogeny of Yponomeuta, based on the internal transcribed spacer region (ITS-1) and on 16S rDNA (16S) and cytochrome oxidase (COII) mitochondrial genes, using maximum parsimony (Turner H, Lieshout N, van Ginkel W, Menken SBJ, submitted). Host plant use is plotted on the tree and the presence of benzaldehyde in the host and feeding responses to this compound are indicated for the three tested species\nMaterials and methods\nInsects\nEgg batches of Y. cagnagellus and Y. padellus were collected in the field in The Netherlands from their host plants: Y. cagnagellus from E. europaeus and Y. padellus from P. spinosa and Crataegus spp. Yponomeuta plumbellus larvae were collected as fourth instars. Egg batches were kept at 4\u00b0C and 80% relative humidity until needed. Larvae were reared in 10\u00a0cm wide Petri dishes (at 25\u00b0C, ambient humidity, L18:D6\u00a0h photoperiod) on host foliage from potted plants grown in the greenhouse (E. europaeus for Y. cagnagellus and Y. plumbellus; P. spinosa for Y. padellus). During the rearing period larvae were fed ad libitum. For the electrophysiological experiments. 1- to 4-day-old fifth instars were used that were starved for 2\u20133\u00a0h. In feeding experiments the caterpillars were used during the whole duration of the fifth stadium. Between feeding experiments, caterpillars were kept at 4\u00b0C.\nMorphology\nThe morphology of the palps was studied using scanning EM. Fixation and drying often induce shrinking and cause collapse of soft tissue parts. We avoided these problems by using Cryo-SEM. Larvae were used directly after moulting into the fifth stadium. The preparation was stabilized by quick immersion in nitrogen slush (60\u00a0K), coated with a 200\u00a0\u00c5 thick layer of gold and viewed in the frozen state using a Philips 535 SEM equipped with a Hexland CT1000\/CP2000 and a Jeol 35C SEM equipped with an EMSCOPE SP2000A cryo preparation assembly.\nHost plant odour composition\nOne of the problems in determining the function of unknown sensory organs is the choice of the stimuli to be tested. To identify a set of relevant substances, the chemical composition of the headspace of E. europaeus, the host of Y. cagnagellus, was characterized. Branches (30\u00a0cm long) of E. europaeus growing in the field in Wageningen, the Netherlands, were cut with sharp garden scissors and transferred to the lab. About 2\u00a0kg of branches was placed in a 20-l stainless steel vessel within 30\u00a0min after cutting. Care was taken not to damage the leaves. With a membrane pump air was passed through KOH pellets, molecular sieves 5A and 3\u00d7, active charcoal, and a tenax pre-trap. This cleaned air flowed for 7\u201315\u00a0h at a rate of 70\u00a0ml\/min through the stainless steel vessel and subsequently through the tenax-TA sample trap. The collected volatiles were released from the tenax by the TCT technique (Thermo-desorption cold trap; Dicke et al. 1990). The TCT unit (Chrompack, Middelburg, The Netherlands) was heated (250\u00b0C) for 3\u00a0min and desorbed compounds were collected on a Sil5CB coated, fused silica capillary at \u2212100\u00b0C. Flash heating of the cold trap provided sharp injection of the compounds into the GC\/MS unit (Chrompack Sil19CB column, 25\u00a0m\u00a0\u00d7\u00a00.25\u00a0mm, df 0.25\u00a0\u03bcm). Electron impact ionisation was carried out at 70\u00a0eV on a VG MM 7070F mass spectrometer.\nElectrophysiological recording and odour stimuli\nA diagram of the recording set-up is presented in Fig.\u00a02. An isolated caterpillar head was mounted on a silver wire loop and connected via a 10-mm connector to the input terminal of a laboratory-built amplifier (Ri\u00a0>\u00a0109\u00a0\u2126, Ci\u00a0<\u00a01\u00a0pF, Ib\u00a0<\u00a01\u00a0pA). The sensory cells of the sensilla on the maxillary palp are located below the base of the terminal segment (Schoonhoven and Dethier 1966; Albert 1980). To gain access to these cells, the connecting membrane between the two most distal segments was pierced with a sharpened tungsten electrode. The actual single cell recordings were made using a glass capillary electrode filled with 3\u00a0M\u00a0KCl (impedance about 40\u00a0M\u2126) inserted through the hole and connected to ground (so actually serving as the indifferent electrode). The position of the glass electrode was carefully adjusted until action potentials could be recorded. The inside of the palp is probed with the tip until activity is picked up, analogous to the way in which recordings from the CNS are made. It should be noted that although this procedure has the advantage of allowing the recoding from single cells with good S\/N ratios, and easy identification of separate cells, the drawback is that the position of the tip (and therefore the identity of the recorded cell) is not known, and not reproducible between recordings.\nFig.\u00a02Schematic diagram of the electrophysiological set-up used to measure responses to both gustatory and olfactory stimuli. Open arrows indicate airflow, black arrows electrical signals. Note that in contrast to standard tip recording the stimulus pipette is electrically isolated\nThe preparation was placed in a continuous stream of clean, moistened air (80\u00a0ml\/s, 25\u00a0cm\/s). A second (control) air stream (2\u00a0ml\/s) was injected in the main stream through an empty pasteur pipette. Odour stimuli were delivered by switching this second stream for 1\u00a0s through a pasteur pipette containing the stimulus. For stimulation with CO2, a single injected stream of commercial available bottled CO2 (Hoekloos, the Netherlands) was switched on. This injected stream could be adjusted to produce CO2 concentrations in the main stream between 0.1 and 10%. Olfactory stimuli were diluted in paraffin oil (10\u22123, 10\u22122, and 10\u22121 v\/v). Twenty-five microlitres of these solutions was applied to filter paper strips (6.0\u00a0\u00d7\u00a00.5\u00a0cm) and was placed in pasteur pipettes. The following compounds (99% pure) were used: 1-hexanol and benzaldehyde (Fluka, Switzerland); (Z)-3-hexenyl acetate and (E)-2-hexenal (Z)-3-hexen-1-ol (Roth, West Germany); hexanal (98% pure) (Merck, USA); limonene, citral (97% pure), and geraniol (98% pure) (Aldrich, Germany). All stimuli were applied in random order with at least 30\u00a0s between them. Longer intervals were used after strong stimuli. Preparations were discarded after 60\u00a0min and were changed after a successful recording. Odour stimuli were stored at 4\u00b0C between recording sessions, and replaced every few days.\nThe AC amplified action potentials from the sensory cells were recorded on tape (Racal FM taperecorder). To facilitate computer-assisted analysis, the valve signal and a pre-pulse 1\u00a0s earlier were recorded on an additional channel.\nAnalysis of recordings\nThe recordings contained activity from one to four sensory cells but usually not more than two. Recordings with more than three cells were discarded. Multi-cellular responses were analysed with the help of an interactive computer program based on the methods described in Roessingh et al. (1992). Spikes were characterized primarily by amplitude. Amplitude boundaries for different cells were taken from an amplitude versus time plot. The separation of the cells is an interactive process in which spike amplitude and spike interval distributions are used as criteria to judge the separation. After the separation, responses for each cell were calculated as the difference in action potential frequency in the last second before the onset of stimulation and the first reaction second. A cell was assumed to respond to a stimulus if an increase of at least ten spikes could be evoked. Cells with weaker reactions were assumed to be sensitive to untested stimuli and were not further analysed. To test the significance of observed differences in spike frequency, analysis of variance was used on log transformed response data.\nNo-choice feeding assay\nA new no-choice feeding assay was designed to investigate the effect of benzaldehyde odour on larval feeding. The experimental setup is illustrated in Fig.\u00a03. A clean air stream was split into two branches, with airflow meters to control the flow. Each branch was connected to a cluster of 18 Petri dishes, each dish containing a feeding caterpillar. In the control group, clean air was passed over the feeding larvae. The air for the experimental group was passed through a small bottle of benzaldehyde diluted in paraffin oil. A bypass with a needle valve allowed mixing in clear air and fine-tuning of the concentration of benzaldehyde (i.e. 50% flow through the bypass reduces the final concentration with a factor 2. Six different dilutions (v\/v) of benzaldehyde in paraffin oil were used in the experiments, viz. 0.5\u00a0\u00d7\u00a010\u22124, 0.5\u00a0\u00d7\u00a010\u22123, 0.2\u00a0\u00d7\u00a010\u22122, 0.5\u00a0\u00d7\u00a010\u22122, 1\u00a0\u00d7\u00a010\u22122, and 0.5\u00a0\u00d7\u00a010\u22121. The total airflow was approximately 50\u00a0ml\/s, resulting in a flow of approximately 1.4\u00a0ml\/s through each Petri dish.\nFig.\u00a03Feeding assay air flow diagram. Both the control group and treatment group consisted of 18 Petri dishes\nA leaf disc with a diameter of 11\u00a0mm made from fresh host leaves was placed in each Petri dish on top of a piece of filter paper slightly wetted with distilled water. To reduce baseline palatability and detect feeding stimulation and\/or inhibition, the leaf discs were extracted in alcohol and then washed in distilled water for 10\u00a0min. The duration of extraction needed to induce a weak feeding response depending upon both the host plants and leaf quality. For Y. cagnagellus, E. europaeus leaf discs were soaked in 96% EtOH for 24\u00a0h. Prunus spinosa leaf discs for Y. padellus were soaked for 2\u00a0h in 70% EtOH. Yponomeuta plumbellus appears later in the season than the other two species and feeds on slightly older E. europaeus foliage. For these older leaves extraction of leaf discs for 1\u00a0h in EtOH was sufficient to reduce palatability. The feeding experiments lasted 4\u20135\u00a0h. Usually 1 or 2\u00a0h after the start of the experiment, caterpillars started eating. The experiment was terminated after at least two third of the insects had been eating for more than 3\u00a0h.\nTo quantify the amount of feeding, the remainders of the leaf discs were scanned with an EPSON perfection 1250 scanner. The surface area was measured on a Macintosh computer using the open source program NIH Image version 1.62 (developed at the U.S. National Institutes of Health and available on the Internet at http:\/\/www.rsb.info.nih.gov\/nih-image\/). The differences in consumption between treatment and control groups were evaluated using a Mann\u2013Whitney test in SPSS (SPSS Inc. 2001). For graphical display a feeding index (C\u00a0\u2212\u00a0T)\/(C\u00a0+\u00a0T)\u00a0\u00d7\u00a0100% (ranging from \u2212100 to 100%), was calculated, in which C and T denote, respectively, the mean of the remaining leaf surface in the control group and in the treatment group.\nResults\nMorphology\nThe palps of Y. cagnagellus are more elongated than in most other species of Lepidoptera, however, the sensory equipment does conform to the general pattern of ditrysian species as outlined by Grimes and Neunzig (1986). These authors divide the eight sensilla on the apex in a medial group (M1, M2), an apical group (A1, A2, A3), and a lateral group (L1, L2, L3). These groups can also be recognised in Y. cagnagellus, although the position of the sensilla is more linear than in the generalised pattern (4c).\nOn the apex seven sensilla basiconica and one sensillum styloconicum can be distinguished. On the side of the segment, a digitiform sensillum and at least one campaniform sensillum are visible (4b). With the exception of the styloconic sensillum, which has a prominent base, the sensilla on the apex are simple blunt cones. The lateral and medial sensilla, for which a gustatory function has been suggested, seem to be slightly more tapered (Fig.\u00a04e, f) but the difference is only slight. Structural details like grooved surfaces or apical pores were not observed.\nFig.\u00a04Cryo-SEM micrographs of Yponomeuta cagnagellus.a Ventral view of the head. b Maxillary palp showing the digitiform sensillum (leftmost arrow) and a large campaniform sensillum (rightmost arrow). c Overview of the tip of the most distal segment. Note the linear arrangement of the sensilla. d Detail of the digitiform sensillum. e On the apex of the palp, seven sensilla basiconica and one sensillum styloconicum are present. From left to right, L1, L2, L3, A1, a styloconic A2, A3, a partly hidden M1, and M2 can be seen (terminology after Grimes and Neunzig 1986). f High magnification of the lateral side of the apex. The two leftmost sensilla belong to the lateral group. The slightly more blunt sensillum on the right is A1. Note the absence of surface structures\nHost plant odour composition\nThe compounds used for olfactory stimulation were chosen on the basis of the headspace analysis of E. europaeus. Whole undamaged plants release only limited amounts of volatiles (Buttery et al. 1985; van Poecke and Dicke 2004), and indeed small quantities were found in the present study. The only compounds that could be detected consistently were C6-based \u201cgreen odours\u201d (Visser and Av\u00e9 1978). Other compounds (limonene, beta-ocimene, alpha-copaene, beta-bourbonene, benzaldehyde) could not be detected reliably. Based on this result (Z)-3-hexenyl acetate (E)-2-hexenal, 1-hexanal (Z)-3-hexen-1-ol, hexanol, limonene, citral, geraniol, and benzaldehyde were chosen as olfactory stimuli. Benzaldehyde is not known from E. europaeus, but was nevertheless included in the analysis because it is a major compound in the Rosaceous hosts of some other Yponomeuta species that are closely related to Y. cagnagellus.\nElectrophysiology\nIn experiments on 69 preparations, a total of 85 single cells were analysed. Twenty-four cells did not respond to any of the applied stimuli. Of the remaining receptors, 22 cells showed no response to odours, but responded to a 100\u00a0mM KCl solution. However, these gustatory responses were only weak (21\u00a0\u00b1\u00a04 spikes maximum) so the adequate stimulus was most likely not KCl. Gustatory responses will therefore not be considered here any further. Twenty-two other cells responded strongly to C6 compounds and to benzaldehyde (Fig.\u00a05). For 19 of those cells more or less complete response spectra could be obtained (Fig.\u00a06). At least two groups could be distinguished. Cells one to five responded to the aldehydes (E)-2-hexenal and hexanal. The other cells respond to the leaf alcohol (Z)-3-hexen-1-ol, to 1-hexanol, and to benzaldehyde. No cells were found that responded strongly to (Z)-3-hexen-1-yl acetate, or to the terpenoids limonene (6 cells tested), citral (3 cells tested) and geraniol (5 cells tested).\nFig.\u00a05I Responses of a (E)-2 hexenal cell in the maxillary palp of Yponomeuta cagnagellus to plant odour compounds. A Hexanal 10\u22121, B Hexanal 10\u22122. C (Z)-3-hexen-1-ol 10\u22121. D (Z)-3-hexen-1-ol 10\u22122. The stimulus period (1\u00a0s) is indicated by the horizontal bar. II Responses of a (Z)-3-hexen-1-ol best cell in the maxillary palp of Yponomeuta cagnagellus to plant odour compounds. A (Z)-3-hexen-1-ol 10\u22121. B (Z)-3-hexen-1-ol 10\u22122, C (Z)-3-hexen-1-ol 10\u22123. D (E)-2-hexenal 10\u22122, E hexanal 10\u22122, F benzaldehyde 10\u22122. Note in D and E the presence of an aldehyde-sensitive cell in the noise (arrow). The stimulus duration (1\u00a0s) is indicated by the horizontal barFig.\u00a06Response spectra for 19 cells sensitive to plant odour compounds in the maxillary palps of Yponomeuta cagnagellus at a concentration of 10\u22122. Response intensity is indicated by the diameter of the circles. Empty spots are missing observations\nDose response relations for green odours in the two main cell groups are given in Fig.\u00a07. In Fig.\u00a07a, the responses of the (E)-2-hexenal sensitive cells to the other green odours are plotted. The same is done in Fig.\u00a07b for the (Z)-3-hexen-1-ol cells. In the alcohol-sensitive group, several cells responded to both (Z)-3-hexen-1-ol and 1-hexanol (cells 6\u201312) but cells that reacted mainly to one of the compounds were also found (cells 13\u201319).\nFig.\u00a07Dose\u2013response relations of olfactory receptor cells in the cells of Yponomeuta cagnagellus. a (E)-2-hexenal sensitive cells (data from cell 1 to 5 in Fig.\u00a06, plus three additional cells). b (Z)-3-hexen-1-ol sensitive cells (data from cell 6 to 19 in Fig.\u00a06). c. benzaldehyde sensitive cells (data from cell 6 to 16 in Fig.\u00a06). The number of sensory cells actually recorded for each datapoint is given between brackets, vertical bars indicate the SE\nThe (Z)-3-hexen-1-ol and 1-hexanol cells were often also highly sensitive to benzaldehyde (Fig.\u00a07c). Evidence indicating that this sensitivity could be attributed to another cell (e.g. superpositions or irregular spike interval histograms) was not observed. The \u201caldehyde\u201d cells in our sample did not show this sensitivity and only two cells responded weakly to benzaldehyde.\nAlthough the sensitivity to benzaldehyde was often associated with that for the leaf alcohols, examples of both benzaldehyde insensitive cells (cell 14\u201315) and apparent benzaldehyde specialists (cells 18\u201319) were found.\nFourteen cells responded selectively to CO2 (Fig.\u00a08). The dose\u2013response curve (Fig.\u00a09) is almost linear from 0.2 to 2.5% CO2. No significant response to other stimuli was found for these cells. All tested C6 compounds sometimes inhibited spike activity in CO2 cells, but only at high concentrations (10\u22121 v\/v). The terpenoids limonene, citral, and geraniol never inhibited the CO2 cells (n\u00a0=\u00a014).\nFig.\u00a08Response of a CO2 sensitive cell in the maxillary palp of Yponomeuta cagnagellus. a to e are responses to, respectively, 0.26, 1.0, 1.5, 2.5, and 10% CO2. The time marker indicates 1\u00a0sFig.\u00a09Dose\u2013response relationship of 12 CO2 sensitive cells in the palp of Yponomeuta cagnagellus stimulated with CO2 or clean air. Vertical bars indicate 95% confidence interval\nThree cells did not react to chemical stimuli but responded to changes in temperature induced by a hot or cold metal rod in the vicinity of the palp. A drop in temperature evoked a strong response. An increase in temperature inhibited the cells (Fig.\u00a010), i.e. these cells functioned as cold receptors.\nFig.\u00a010Response of a cold receptor in the maxillary palp of Yponomeuta cagnagellus. a Stimulation with a cold metal rod at an upwind distance of about 10\u00a0mm. b Stimulation with a warm metal rod at an upwind distance of about 10\u00a0mm. Stimulus bar 2.5\u00a0s\nFeeding behaviour modulated by benzaldehyde\nFeeding assays were conducted for Y. cagnagellus at 5 different concentrations of benzaldehyde on 72 insects per treatment (Fig.\u00a011). Benzaldehyde had a positive feeding index at concentrations of 0.5\u00a0\u00d7\u00a010\u22123, 0.2\u00a0\u00d7\u00a010\u22122, and 0.5\u00a0\u00d7\u00a010\u22122. However, the stimulating effect of benzaldehyde was significant only at the concentration of 0.2\u00a0\u00d7\u00a010\u22122\u00a0M, with a feeding index of about 12% (percentage leaf disk left on benzaldehyde and control was, respectively, 60.9\u00a0\u00b1\u00a05.3 and 76.8\u00a0\u00b1\u00a04.4%, Mann\u2013Whitney U\u00a0=\u00a0427, P\u00a0=\u00a00.013). At higher concentrations, benzaldehyde tended to inhibit the feeding of Y. cagnagellus larvae. For example, the feeding index was \u22123% at the concentration of 0.5\u00a0\u00d7\u00a010\u22121 but feeding inhibition was not significant compared to the control group.\nFig.\u00a011Feeding index of three Yponomeuta species. FI\u00a0=\u00a0(C\u00a0\u2212\u00a0T)\/(C\u00a0+\u00a0T) (ranging from \u2212100 to 100%). C and T denote, respectively, the mean of the remaining leaf surface in the control group and in the treatment (benzaldehyde) group. Feeding indexes with P\u00a0<\u00a00.05 are indicated with an asterisk\nThe feeding response of Y. padellus was tested at four concentrations, i.e. 0.5\u00a0\u00d7\u00a010\u22124, 0.5\u00a0\u00d7\u00a010\u22122, 1\u00a0\u00d7\u00a010\u22122, and 0.5\u00a0\u00d7\u00a010\u22121 (Fig.\u00a011). Benzaldehyde significantly stimulated the feeding of caterpillars at all concentrations except at the lowest concentration (percentage leaf disk left on benzaldehyde and control at 0.5\u00a0\u00d7\u00a010\u22122 was, respectively, 76.5\u00a0\u00b1\u00a03.7 and 84.5\u00a0\u00b1\u00a03.4%, Mann\u2013Whitney U 375, P\u00a0=\u00a00.003, At 1\u00a0\u00d7\u00a010\u22122 76.6\u00a0\u00b1\u00a03.8 and 90.4\u00a0\u00b1\u00a01.7%, Mann\u2013Whitney U\u00a0=\u00a0321, P\u00a0=\u00a00.001 and at 0.5\u00a0\u00d7\u00a010\u22121 70\u00a0\u00b1\u00a03.8% and 82.8\u00a0\u00b1\u00a03.3% Mann\u2013Whitney U\u00a0=\u00a0408, P\u00a0<\u00a00.007). In Y. plumbellus, no effect of benzaldehyde on food intake was found, and the insects appeared to be fully indifferent to the compound (Fig.\u00a011).\nDiscussion\nMorphology\nA description of the external structure of the maxillary palps of ditrysian larvae is given by Grimes and Neunzig (1986). From their results it is clear that location, size, and shape of palpal sensilla are constant in lepidopterous larvae. Eight sensilla are always found on the apex. In addition, a digitiform sensillum and up to four campaniform sensilla or pore plates are present in the wall of the most distal segment. Grimes and Neunzig (1986) make a distinction between the more primitive, endophagous feeders, and larger, exophagous feeders. In endophagous species, the walls of the A sensilla are smooth, and A2 is often two-tiered (styloconic). In the more advanced, exophagous groups A2 is basiconic and the A sensilla are wrinkled or pockmarked. The Yponomeutidae are regarded as relatively primitive Ditrysia (Kristensen and Skalski 1999), and indeed a styloconic A2 and smooth A sensilla are found (Fig.\u00a04; see also van Drongelen 1979). On morphological grounds a gustatory function for L and M sensilla has been suggested (Grimes and Neunzig 1986), and for Choristoneura fumiferana this was confirmed by recordings (Albert 2003). In Helicoverpa armigera, however, some sensilla show ultrastructural features characteristic for both olfaction and taste (Keil 1996) and it is therefore difficult to assign a function based on mophology alone. Although we recorded responses to gustatory as well as olfacory stimuli, we stimulated all eight sensilla simultaneously and this method precludes assignment of the observed responses to particular sensilla. The wall of the most distal segment of the palp bears at least one campaniform sensillum. This sensillum is probably equivalent to the large plate sensillum described in C. fumiferana (Albert 2003), Euxoa messoria (Devitt and Smith 1982), and H. amigera (Keil 1996). The plate sensilla are multiporous, multiple innervated, and possess branching dendrites. If this is also true of Y. cagnagellus, these sensilla are presumably olfactory organs where some of the neural responses reported in this paper could originate.\nIn the wall of the most distal segment, a digitiform sensillum is present which is normally innervated by one neuron (Schoonhoven and Dethier 1966; Albert 1980; Devitt and Smith 1982; Keil 1996). In E. messoria and Heliotis amigera this sensillum appears to possess a laminated outer dendritic segment. Such laminated dendrites have been associated with cold receptors (see Altner and Loftus 1985), as well as with CO2 receptors (Chu-wang et al. 1975; McIver and Siemicki 1984; Lee et al. 1985; Bogner et al. 1986). In Y. cagnagellus both cold receptors and CO2 receptors were found. The digitiform sensillum of Y. cagnagellus might therefore house a CO2 receptor as suggested by Keil (1996) but without further electrophysiological evidence cold reception cannot be excluded.\nCarbon dioxide reception\nThe ecological function of the CO2 receptor in Y. cagnagellus is not clear. CO2 reception is well known in soil dwelling organisms that must locate plant roots (Johnson and Gregory 2006). CO2 sensitivity is also common in adult lepidoptera (Bogner 1990; Stange and Stowe 1999). Helicoverpa armigera for instance is in principle capable of detecting modulation of CO2 concentrations normally occurring in the environment (Stange 1992). The CO2 receptor of Y. cagnagellus and other lepidopterous larvae is clearly less sensitive than that of adult Lepidoptera, however, CO2 receptors with a similar low sensitivity range have been described in the tick Amblyomma varigatum (Steullet and Guerin 1992). During the day, CO2 levels near the surface of green leaves will be 0.038% or lower, while this level may rise to a maximum of 1.2% during nocturnal respiration (see Bogner 1990 for references). Although the latter concentration is just within the range of the larval receptor, it seems unlikely that the CO2 receptor is used to assess subtle changes in the respiratory status of the leaf (as a messure of leaf quality). Alternative hypotheses are that the receptor is used to monitor CO2 levels in the communal nest during periods of tight clustering, or that it facilitates aggregation behaviour. However, as yet experimental data to support these ideas is lacking.\nTemperature perception\nOnly qualitative data was collected from the three observed cold receptors. The actual drop in temperature induced by the hot and cold metal rods used, as stimulus is not known. The cold receptors might be used for monitoring the microclimate (Schoonhoven 1967) and in principle could be used to determine smaller temperature variations associated with leaf quality (Dethier and Schoonhoven 1968).\nPlant odour responses\nStimulation with plant odours yielded cells of at least two spectral types: aldehyde-sensitive cells and alcohol-sensitive cells. The two groups are clearly separated from each other. However, within each group responses to several chemically very different stimuli were found. A single receptor cell that reacts to different types of compounds used to be interpreted as a reflection of the presence of several receptor \u201csites\u201d on the membrane (Kaissling 1976; Selzer 1984; Hansen 1978; Wiezoreck and Wolff 1989). With the identification of the actual receptor genes and advanced labeling techniques it was suggested that normally only a single receptor protein is expressed in a particular sensory cell, the \u201cone receptor\u2013one neuron\u201d rule (see Gaillard et al. 2004 for review). Recently however, it was shown that in Drosophila olfactory organs this rule does not always hold, and coexpression of different receptor proteins was observed (Goldman et al. 2005). In C. elegans coexpression must also occur, simply because there are too many receptor genes to express for the limited number of sensory neurons. We observed single cells that responded to structurally different compounds such as (Z)-3-hexen-1-ol and benzaldehyde, but also cells that responded to only one of these two compounds. These observations can in each case still be explained by the expression of a single receptor protein per cell, with a different binding specificity (i.e. a cell that responds to several compounds expresses a receptor with a broad specificity, while a cell that responds to only a single compound expresses a narrowly tuned receptor protein). Alternatively, cells might be broadly tuned because they express an assembly of several narrowly tuned receptor proteins with different sensitivity spectra. In either case, following this line of reasoning a simple quantitative shift in the numbers of receptor proteins could in principle result in a qualitative change in host-acceptance behaviour and host shifts, processes that were postulated as major factors in the speciation process in Yponomeuta species (Menken et al. 1992; Menken and Roessingh 1998).\nFrom Fig.\u00a06 is clear that although at least two groups of cells with comparable spectral responses can be distinguished (cells 1\u20135, cells 6\u201319), however, there are no sharp boundaries between these classes (cf. cell 5), and considerable variation does exist. Since care was taken to record only from cells that were reliable and reproducibly responding, it is likely that this variation does reflect natural variability in the receptor cell population. The separation in aldehyde- and alcohol sensitive cells should therefore at present merely be considered as a convenient way to summarise the data. Until more recordings are made, it cannot be excluded that a continuum from more specialised to, more generalised cells exists.\nIn the present study, no cells with a primary response to (Z)-3-hexenyl acetate were found in the maxillary palps of Y. cagnagellus, but receptors sensitive to this compound have been found on the antennae of this species (Roessingh, unpublished results) as well as on the antennae of Pieris brassicae larvae (Visser and de Jong 1988). In this respect it is interesting to note that in Manduca sexta fibers from the maxillary nerve travel into a core of neuropile in the suboesophageal ganglion that also receives antennal axons (Kent and Hildebrand 1987).\nThe maxillary palps of caterpillars contain 15\u201330 chemoreceptors (Schoonhoven and Dethier 1966; Albert 1980; Devitt and Smith 1982). If the 85 cells in the present study represent a random sample from the population in the palps, then the proportion of neurons sensitive to each of the tested stimuli could be estimated. However, it must be stressed that the position of the electrode was not reproducable between recordings. As a consequence, the recordings by no means represent a random sample. In spite of this limitation it can be concluded that receptors for the tested terpenoids and for (Z)-3-hexen-1-yl acetate are relatively scarce, since no cells primarily sensitive to these stimuli were encountered.\nThe role of benzaldehyde in feeding behaviour\nBecause benzaldehyde is absent from E. europaeus, Euonymus feeders such as Y. plumbellus and Y. cagnagellus are not expected to be sensitive to this compound, or use it as a repellent. The electrophysiological results clearly show that benzaldehyde is stimulating the sensory cells in the maxillary palp of Y. cagnagellus; the straightforward interpretation would be that in Y. cagnagellus benzaldehyde signals a non-host compound, and therefore low acceptability. On the other hand, as benzaldehyde is prominently present in Rosaceae, it can be expected to be a feeding stimulant for Rosaceae feeders like Y. padellus, a species that is closely related to Y. cagnagellus.\nFrom a phylogenetic perspective a different hypothesis can thus be formulated. Yponomeuta cagnagellus is located within the European clade that feeds mostly on Rosaceae, which do contain benzaldehyde. Considering that the most recent common ancestor of this clade was presumably a Rosaceae feeder, the sensitivity of Y. cagnagellus to benzaldehyde could be viewed as a maintained sensitivity to a feeding stimulant from the ancestral host.\nUnder this hypothesis both Y. cagnagellus and Y. padellus are expected to be stimulated by benzaldehyde. Yponomeuta plumbellus, a Celastraceae feeder outside the European clade, is expected to be unresponsive because it has never been associated with Rosaceae nor have its ancestors. The results from the feeding experiment obtained from the three species are consistent with the latter scenario. Benzaldehyde stimulated feeding in Y. cagnagellus and Y. padellus and increased food intake by 6\u201312%. Yponomeuta plumbellus did not respond to benzaldehyde at all. The sensory response to benzaldehyde in Y. cagnagellus is therefore probably an evolutionary relict of the association with Rosaceae of the ancestral lineage of this species. In a previous study (van Drongelen 1979), dulcitol sensitivity was found in Y. evonymellus and Y. padellus, species feeding on Rosaceae that do contain very little or nodulcitol. In this case it was similarly concluded that the dulcitol sensitivity might be an evolutionary relict from the ancestral host association with Celastraceae.\nAlthough feeding in both Y. cagnagellus and Y. padellus was stimulated by benzaldehyde, Y. padellus (associated with a benzaldehyde-containing hosts) responded over a much larger concentration range than Y. cagnagellus (a species that is using a benzaldehyde-free host). It should be noted that care must be taken when interpreting these results. It is not clear what the natural concentrations of host plant odours that are experienced by the larvae, since a steep gradient in the boundary layer exists, and concentrations near the leaf surface can be much higher than those obtained from headspace samples (Schoonhoven et al. 2005). However, since the sensory responses show good dose\u2013response relations, and the behaviour yields a clear optimum, we feel that the used concentrations are covering the natural range. It will be interesting to investigate the actual genetics underlying the observed sensitivity differences between the species.","keyphrases":["yponomeuta cagnagellus","lepidoptera","adaptive speciation","sensory physiology","plant odours"],"prmu":["P","P","P","P","P"]}
{"id":"Plant_Cell_Rep-3-1-1824786","title":"Overexpression of defense response genes in transgenic wheat enhances resistance to Fusarium head blight\n","text":"Fusarium head blight (FHB) of wheat, caused by Fusarium graminearum and other Fusarium species, is a major disease problem for wheat production worldwide. To combat this problem, large-scale breeding efforts have been established. Although progress has been made through standard breeding approaches, the level of resistance attained is insufficient to withstand epidemic conditions. Genetic engineering provides an alternative approach to enhance the level of resistance. Many defense response genes are induced in wheat during F. graminearum infection and may play a role in reducing FHB. The objectives of this study were (1) to develop transgenic wheat overexpressing the defense response genes \u03b1-1-purothionin, thaumatin-like protein 1 (tlp-1), and \u03b2-1,3-glucanase; and (2) to test the resultant transgenic wheat lines against F. graminearum infection under greenhouse and field conditions. Using the wheat cultivar Bobwhite, we developed one, two, and four lines carrying the \u03b1-1-purothionin, tlp-1, and \u03b2-1,3-glucanase transgenes, respectively, that had statistically significant reductions in FHB severity in greenhouse evaluations. We tested these seven transgenic lines under field conditions for percent FHB disease severity, deoxynivalenol (DON) mycotoxin accumulation, and percent visually scabby kernels (VSK). Six of the seven lines differed from the nontransgenic parental Bobwhite line for at least one of the disease traits. A \u03b2-1,3-glucanase transgenic line had enhanced resistance, showing lower FHB severity, DON concentration, and percent VSK compared to Bobwhite. Taken together, the results showed that overexpression of defense response genes in wheat could enhance the FHB resistance in both greenhouse and field conditions.\nIntroduction\nFusarium head blight (FHB; scab), principally caused by Fusarium graminearum Schwabe (teleomorph Gibberella zeae (Schwein.) Petch), is a devastating disease of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) throughout the world (Sutton 1982; McMullen et\u00a0al. 1997). Between 1993 and 2001, in United States, an estimated US$ 8 billion loss was incurred from FHB (Nganje et\u00a0al. 2004). The disease is favored by warm conditions with frequent rainfall and high humidity during flowering. Yield reductions result from reduction in the kernel number and the presence of dry, shriveled kernels, often referred to as \u2018tombstone kernels\u2019. Additionally, reductions in grain quality are caused by Fusarium trichothecene mycotoxins such as deoxynivalenol (DON; Bai and Shaner 1994; Sutton, 1982; Tuite et\u00a0al. 1990).\nThe most economical and efficient way to protect wheat from FHB is to develop genetically-resistant varieties. Wheat breeding programs are utilizing resistance to initial infection (Type I) and resistance to spread of the disease (Type II) as the primary forms of resistance (Rudd et\u00a0al. 2001). However, these forms of resistance are partial (Kolb et\u00a0al. 2001), and the level of genetic resistance provided is generally insufficient to withstand a FHB epidemic. Therefore, novel sources of resistance are required, and genetic engineering is one approach to develop novel resistance in wheat.\nSeveral classes of genes have the potential to provide genetically-engineered resistance to FHB in wheat. One group of genes, referred to as pathogenesis-related (PR) or defense response genes, encode proteins such as \u03b2-1,3-glucanases, chitinases, thaumatin-like proteins (tlps) and thionins whose expression often increase as part of the plant host defense response to pathogen attack (Linthorst 1991). Many defense response genes were shown to be induced in wheat (Pritsch et\u00a0al. 2000, 2001; Li et\u00a0al. 2001; Kang and Buchenauer 2002; Kong et\u00a0al. 2005; Han et\u00a0al. 2005; Zhou et\u00a0al. 2005; Bernardo et\u00a0al. 2006) and barley (Boddu et\u00a0al. 2006) spikes during F. graminearum infection. In particular, PR1, PR-2 (\u03b2-1,3-glucanase), PR-3 (chitinase), PR-4, and PR-5 (tlp-1) transcripts accumulated in wheat spikes during F. graminearum infection (Pritsch et\u00a0al. 2000, 2001). In addition, polyphenol oxidase activities were detected in resistant wheat genotypes (Mohammadi and Kazemi 2002). Furthermore, Kang and Buchenauer (2003) showed accumulation of thionin proteins in F. culmorum-infected wheat tissues. These findings demonstrated that wheat and barley mount an induced defense response to Fusarium infection that involves many defense response genes.\nOverexpression of defense response genes in transgenic plants has provided enhanced resistance to a variety of fungal pathogens (Muehlbauer and Bushnell 2003). For example, transgenic wheat lines carrying a barley-seed class II chitinase exhibited enhanced resistance to powdery mildew (Bliffeld et\u00a0al. 1999; Oldach et\u00a0al. 2001). Varying amounts of resistance towards powdery mildew were observed in transgenic wheat lines carrying a barley chitinase or a barley \u03b2-1,3-glucanase (Bieri et\u00a0al. 2003). With respect to FHB, a transgenic wheat line carrying a rice tlp and a line carrying a combination of a wheat \u03b2-1,3-glucanase and chitinase exhibited delayed symptoms of FHB in greenhouse trials (Chen et\u00a0al. 1999; Anand et\u00a0al. 2003). However, neither transgenic wheat line exhibited any resistance to FHB under field conditions (Anand et\u00a0al. 2003). In addition, transgenic Arabidopsis carrying an overexpressed Arabidopsis thionin showed increased resistance to F. oxysporum (Epple et\u00a0al. 1997). Recently, transgenic wheat expressing the Arabidopsis NPR1 gene, a gene that regulates defense responses, was shown to exhibit a high level of resistance to FHB in greenhouse evaluations (Makandar et\u00a0al. 2006).\nAs part of our effort to increase variation for genetic resistance to FHB and to understand the relationship between defense response gene expression and FHB resistance, we produced wheat plants carrying a wheat \u03b1-1-purothionin, a barley tlp-1, or a barley \u03b2-1,3-glucanase transgene. We evaluated these plants against FHB under greenhouse and field conditions. Our results show that the overexpression of \u03b1-1-purothionin, tlp-1, or \u03b2-1,3-glucanase in wheat results in enhanced resistance to FHB.\nMaterials and methods\nPlant materials\nThe spring wheat cultivars \u2018Wheaton\u2019, \u2018Roblin\u2019, \u2018Alsen\u2019, \u20182375\u2019, \u2018Sumai 3\u2019, and \u2018Bobwhite\u2019 were used as checks for FHB responses. Wheaton and Roblin are hard red spring wheat cultivars that are highly susceptible to FHB; Bobwhite is a cultivar from CIMMYT that is susceptible to FHB; 2375 is moderately susceptible to FHB; Alsen is moderately resistant to FHB with resistance derived from Sumai 3; and Sumai 3 is a Chinese cultivar known for resistance to spread of disease in the spike (Type II resistance; Bai and Shaner 1996). The cultivar Bobwhite was used as parental material for transformations.\nPlant transformation plasmids\npAHC25\nThe pAHC25 plasmid (Fig.\u00a01; Christensen et\u00a0al. 1992), containing the uidA and bar genes under the control of the maize ubiquitin promoter, was kindly donated by Dr. Peter Quail of the Plant Gene Expression Center, University of California at Berkeley. The uidA gene encodes \u03b2-glucuronidase and the bar gene encodes the enzyme phosphinothricin acetyltransferase which confers resistance to the phosphinothricin-containing herbicides.\nFig. 1Plasmids used for wheat transformation. Plasmids containing the wheat \u03b1-1-purothionin (pKM1), barley tlp-1 transgene (pAHCBarPR5), and barley \u03b2-1,3-glucanase (pUBKBarGluc-3) were co-bombarded with pAHC25 to develop transgenic wheat plants. The ubiquitin 1 promoter and intron is from the maize ubiquitin gene, and the T nos termination sequence is from the nopaline synthase gene from Agrobacterium tumefaciens. The uidA gene encodes \u03b2-glucuronidase and is from Escherichia coli and the bar gene encodes the enzyme phosphinothricin acetyltransferase and is from Streptomyces hygroscopicus\npKM1\nA plasmid containing a 460 bp wheat \u03b1-1-purothionin gene (GenBank accession number X70665.1) under the control of the maize ubiquitin promoter was kindly provided by Dr. Ann Blechl (USDA-ARS, Albany, CA). The \u03b1-1-purothionin gene was cloned into the BamHI\/BglII site (replacement of the bar gene) of pUBK BglII\u2212. The pUBK BglII\u2212 vector, kindly provided by Drs. Ann Blechl, Pat Okubara, and Kent McCue (USDA-ARS, Albany, CA), is a derivative of the pAHC20 vector (Christensen et\u00a0al. 1992). pUBK BglII\u2212 contains the ubiquitin promoter, with the BglII site removed, driving the bar gene.\npAHCBarPR5\nBarley tlp-1 cDNA (GenBank accession number AM403331) was removed from the parent plasmid (kindly provided by Dr. David Collinge, Department of Plant Biology, Royal Veterinary and Agricultural University, Denmark) by XhoI digestion, blunt-end repaired and cloned into the blunt-end repaired BamHI site of pAHC17. The expression cassette in the pAHC17 vector (Christensen et\u00a0al. 1992) contains the maize ubiquitin promoter and the NOS terminator element. The pAHC17 vector was kindly provided by Dr. Peter Quail of the Plant Gene Expression Center, University of California at Berkeley.\npUBKBarGluc-3\nThe 1234 bp barley class-II \u03b2-1,3-glucanase cDNA (GenBank accession number M62907.1; Leah et\u00a0al. 1991) was removed from the parent plasmid (kindly provided by Dr. John Mundy, Carlsberg Research Laboratory, Copenhagen, Denmark) by EcoRI digestion, blunt-end repaired and ligated into the blunt-end repaired BglII\/BamHI site (replacement of bar gene) of the expression vector pUBK BglII\u2212.\nWheat transformation\nSpring wheat (cv. Bobwhite) was used for all transformations. Particle gun bombardment of embryos, selection, and regeneration were carried out as described by Mackintosh et\u00a0al. (2006). We conducted cotransformation of pAHC25 with pKM1, pAHCBarPR5, or pUBKBarGluc-3.\nRNA isolation and transcript analysis\nRNA was isolated from leaf tissue using the Trizol reagent (Invitrogen, Carlsbad, CA) as per manufacturers\u2019 instructions. RNA was subjected to RT-PCR based on the protocol accompanying the Calypso RT-PCR kit (GenSys Ltd., Farnborough, UK) using primers synthesized by Integrated DNA Technologies Inc. (Coralville, IA). The 5\u2032 sense primer was a maize ubiquitin promoter sequence (5\u2032-GATGCATATACATGATGGCATATGCAG-3\u2032) and the 3\u2032 antisense primers were oligonucleotides that corresponded to defense response gene coding sequences for \u03b1-1-purothionin (5\u2032-GTTACAGAAATTGACACAAGCATCGCC-3\u2032), tlp-1 (5\u2032-GACAGAAGGTGATCTGGTAGTTATTATT-3\u2032) and \u03b2-1,3-glucanase (5\u2032-GATGTTCACGGCAGGGTAGT-3\u2032 and 5\u2032-GCCACGTCCGTCATGTAGGCGTTC-3\u2032). A wheat actin gene (GenBank accession number AB181991) with the primer sequences 5\u2032-GCCACACTGTTCCAATCTATGA-3\u2032 and 5\u2032-TGATGGAATTGTATGTCGCTTC-3\u2019 was used as a positive control. Sizes for the amplified products from the \u03b1-1-purothionin and tlp-1 transgenes were 600 and 805, respectively. Sizes for the two primers for the \u03b2-1,3-glucanase transgene were 577 and 777 bp. Size for the wheat actin gene was 369 bp.\nGreenhouse evaluation of transgenic lines against Fusarium graminearum infection\nSeeds of each transgenic line were planted in the greenhouse. At anthesis, one spikelet at the central node of the main spike of each plant was inoculated with 10 \u03bcl of a macro-conidial spore suspension (100,000 conidia\/ml) of Fusarium graminearum isolate Butte 86ADA-11 (Evans et\u00a0al. 2000; NRRL 38661). Plants were placed in a dew chamber for 72\u00a0h following inoculation and then returned to the greenhouse. Disease severity was assessed at 20 days after inoculation by counting the number of infected spikelets and expressing the infection level as a percentage of the total number of spikelets for each spike. Bobwhite, Wheaton, and Sumai 3 were used as checks in each greenhouse test.\nField evaluation of transgenic lines against Fusarium graminearum infection\nTransgenic wheat lines were examined for their reaction to FHB in the field. RT-PCR positive plants of each of the lines were selected as the source of seed for the field plantings. In addition to the test lines, Bobwhite was included as the untransformed control. Two experiments were conducted during the summers of 2004 and 2005 at the University of Minnesota Agricultural Experiment Station, Crookston, Minnesota. T6 and T7 of the transgenic lines were used for the 2004 and 2005 field tests, respectively. The field tests were each a randomized complete block design with four replications. Entries were established in two-row plots; rows were 2.4 m (8 ft) long and were spaced 0.3\u00a0m (1 ft) apart. Within rows, seed was planted at a rate of 3.3\u00a0g of seed\/m. Alsen, 2375, Roblin and Wheaton were also included in the experiment as disease response checks. Additional plantings of noninoculated Wheaton were included in the field trial to determine the level of disease.\nInoculum consisted of a mixture of 12 isolates of F. graminearum. These came from naturally infected samples of grain from commercial fields of wheat and barley in Minnesota from 2002 to 2004. Plots were inoculated twice; the first time at anthesis and then 3 days later. Inoculum (1\u00a0\u00d7\u00a0105 macroconidia\/ml) was applied at a rate of 33\u00a0ml\/m of row using a CO2-powered backpack sprayer, at a pressure of 276\u00a0kPa and fitted with a flat-fan spray tip (TeeJet SS8003, Spraying Systems Co., Wheaton, IL).\nFHB incidence and severity were evaluated visually 21 days after the initial inoculation. Incidence was recorded as the percentage of spikes with symptomatic spikelets and severity as the percentage of symptomatic spikelets in 20 spikes of primary tillers arbitrarily selected per plot.\nPlots were harvested with a Wintersteiger classic combine (Wintersteiger, Ried, Austria) at maturity. The percentage of visually scabby kernels (VSK) was assessed on a hand-cleaned 50\u00a0g sample by comparison to standards with a known percentage of scabby kernels according to the procedure of Jones and Mirocha (1999).\nFollowing VSK analysis, the samples were ground for 2\u00a0min with a Stein Laboratory Mill (model M-2, Stein Laboratories, Atchison, KS) and analyzed for deoxynivalenol (DON) concentration using gas chromatography and mass spectrometry according to the procedures of Mirocha et\u00a0al. (1998) with the following modifications. DON was extracted from 4 g of the ground wheat placed in a 50\u00a0ml centrifuge tube to which 16\u00a0ml of acetonitrile:water (84:16 v\/v) was added. Samples were derivatized using 100\u00a0\u03bcl of the silylating reagent (TMSI\/TMCS, 100:1), 1\u00a0ml of isooctane and 1\u00a0ml of distilled water.\nWestern blot analysis\nSpike tissue was ground using liquid nitrogen and protein was extracted by vortexing the tissue at 4\u00b0C for 10\u00a0min in extraction buffer (50\u00a0mM NaH2PO4, pH 6.8, 100\u00a0mM PMSF). After micro-centrifugation at 4\u00b0C, full speed, for 5\u00a0min, supernatant protein measurements were conducted using Biorad reagent (Biorad) with bovine serum albumin as a standard. Extracts containing 10\u00a0\u03bcg protein were used to determine the amount of transgenic protein present in transgenic lines using Western blotting.\nSamples were subjected to SDS-PAGE using 12% gels, transferred to PVDF-PLUS transfer membrane (Micron Separations Inc., Westborough, MA) and cross reacted with an affinity-purified polyclonal antibody (1:1000 dilution of supplied material). The tlp-1 and \u03b2-1,3-glucanase antibodies were provided by Quality Controlled Biochemicals Inc., Hopkinton, MA. For tlp-1, two peptides (QAYQHPNDVATHAC and CINVPAG TQAGRIWAR) were used to raise the antibody. For \u03b2-1,3-glucanase, one peptide (CGLFNPDKSPAYNIQF) was used to raise the antibody. Protein was visualized using an ECF Western Blotting Reagent Pack (rabbit) (Amersham Biosciences, Piscataway, NJ), and fluorescence detection was carried out using a Storm 840 (Molecular Dynamics, Sunnyvale, CA). Specificity of \u03b2-1,3-glucanase and tlp-1 antibodies was confirmed through cross-reacting the antibodies with the peptides on Western blots.\nSouthern blot analysis\nDNA isolation, gel electrophoresis, gel blotting, hybridization, and washing were conducted according to de la Pe\u00f1a et\u00a0al. (1996). Radio-labeled probes for tlp-1, \u03b2-1,3-glucanase, and \u03b1-1-purothionin were used in the hybridization reactions. The subsequent banding patterns were visualized using autoradiography.\nStatistical analysis\nFor the greenhouse evaluation data, t-tests were used to compare each transgenic line to the parental Bobwhite controls. For the field evaluation data from 2004 and 2005, all analyses were performed with SAS\u00ae Version 9.1 (SAS Institute, Cary, NC). Analyses of variance were performed using PROC MIXED procedure. The statistical model included genotype, year and genotype-by-year interactions as fixed factors, and replication nested within year as a random factor was used as an error term for testing year effect. For each experiment, homogeneity of variances among genotypes was checked for each trait using PROC UNIVARIATE. For each trait, when variances were found to be more than four times different from each other A REPEATED\/GROUP\u00a0=\u00a0statement of PROC MIXED was used to account for the heterogeneity of variances. Least square means and pairwise comparisons between means were obtained using LSMEANS and PDIFF options.\nResults\nGeneration of transgenic wheat plants\nThe wheat cultivar Bobwhite was used as parental material for all transformation experiments. The pAHC25 plasmid and either the pKM1 (wheat \u03b1-1-purothionin), pAHCBarPR5 (barley tlp-1), or pUBKBarGluc-3 (barley \u03b2-1,3-glucanase) plasmids were used in cotransformation experiments. Figure\u00a01 shows a schematic of each plasmid. The correct orientation within the vector and open reading frame integrity of the inserted cDNA in pKM1, pAHCBarPR-5, and pUBKBarGluc-3 was confirmed by DNA sequence analysis. pAHC25 carries the uidA gene for visual scoring of \u03b2-glucuronidase (GUS) activity and the bar gene which confers tolerance to the herbicide phosphinothricin for selection. Both the uidA and bar genes were driven by the promoter from the maize ubiquitin gene. Selection and regeneration of plants was conducted as described in Mackintosh et\u00a0al. (2006).\nTo identify transgenic wheat plants carrying the \u03b1-1-purothionin, barley tlp-1 and barley \u03b2-1,3-glucanase transgenes, we conducted RT-PCR analysis on the T0 plants. We identified 25, 25, and 31 transgenic wheat lines carrying expressed wheat \u03b1-1-purothionin, barley tlp-1, and barley \u03b2-1,3-glucanase, respectively. Table\u00a01 shows the number of embryos bombarded for each plasmid, and the percent transformed plants carrying the expressed transgene of interest. Our efficiency for recovering transgenic wheat plants expressing the transgenes of interest ranged from 1.4 to 3%.\nTable 1Production of transgenic wheat plantsTransgeneNumber of embryos bombardedNumber of plants expressing transgeneaTransformation (%)Wheat \u03b1-1-purothionin1787251.4Barley thaumatin-like protein-1825253.0Barley \u03b2-1,3-glucanase1079312.9aExpression based on RT-PCR of each transgene.\nTo obtain T2 lines for further characterization, we grew five T1 seeds from each T0 plant. Each T1 plant was tested by RT-PCR for expression of the appropriate transgene, and T2 seed was collected from plants expressing each transgene.\nGreenhouse evaluation of transgenic plants for response to Fusarium head blight\nTo identify transgenic lines with enhanced resistance to FHB and to eliminate susceptible lines, we conducted two greenhouse evaluations for FHB resistance. Of the 81 transgenic wheat lines developed, 70 (18 of 25 wheat \u03b1-1-purothionin, 23 of 25 barley tlp-1 and 29 of 31 barley \u03b2-1,3-glucanase) lines produced enough T2 seed for FHB evaluations. Sixteen to 20 seeds were planted for each line and inoculated with F. graminearum. We assayed the spread of the disease following point inoculation, and analyzed the results as the percent disease severity at 20 days after inoculation. In addition, each plant in the \u03b1-1-purothionin lines was assayed for transgene expression using RT-PCR. Only those plants expressing the \u03b1-1-purothionin transgene were used to evaluate the efficacy of \u03b1-1-purothionin against FHB. The plants carrying the tlp-1 and \u03b2-1,3-glucanase transgenes were not assayed for transgene expression in the initial T2 FHB screen. For the lines carrying the tlp-1 and \u03b2-1,3-glucanase transgenes, data from all plants assayed for FHB resistance, which would have included transgene null together with transgene homozygous and hemizygous plants, were used to calculate the percent FHB severity. We compared FHB severity in the transgenic lines against the nontransformed Bobwhite parent.\nBased on this initial experiment, we eliminated the most susceptible lines (>50% disease severity) and reevaluated 6, 13, and 16 T2 lines carrying the \u03b1-1-purothionin, tlp-1, and \u03b2-1,3-glucanase transgenes, respectively. We also evaluated the T3 lines of the same 6 lines carrying the \u03b1-1-purothionin transgene, and 13 lines carrying the tlp-1 transgene. Again, we planted 16\u201320 plants per line, and evaluated the lines against FHB. In this screen, all plants were assayed for transgene expression. Only those plants expressing the transgene were used to calculate disease severity for comparison against the nontransformed Bobwhite.\nFrom the initial disease screens on the 70 transgenic lines, we identified seven lines with enhanced FHB resistance. One line had the \u03b1-1-purothionin transgene and is referred to as CM 17, two lines carried the tlp-1 transgene and are referred to as CM21 and CM23, and four lines had barley \u03b2-1,3-glucanase transgene and are referred to as CM27, CM28, CM30, and CM33. The results for these initial two screens of the resistant transgenic lines are shown in Table\u00a02. The resistant transgenic lines were evaluated in further FHB disease screens in the greenhouse (Table\u00a02). For these additional greenhouse evaluations, only plants expressing the transgene, based on RT-PCR assays, were used to calculate the percent FHB severity. One line carrying the \u03b1-1-purothionin transgene (CM17) significantly reduced FHB severity in four of five screens (P\u00a0<\u00a00.05), and had an overall average reduction of 34%. The tlp-1 transgenic CM23 and CM21 lines significantly reduced FHB severity when compared to the Bobwhite control in three of four or five screens, respectively (P\u00a0<\u00a00.05). Taking the average disease severity over all screen replicates, CM21 and CM23 reduced disease severity compared to Bobwhite by 30% and 36%, respectively. The \u00df-1,3-glucanase transgenic CM27, CM28, CM30 and CM33 lines significantly reduced FHB severity compared to the Bobwhite control in two to three screens (P\u00a0<\u00a00.05). The average reduction in disease severity compared to Bobwhite for CM27, CM28, CM30, and CM33 was 40, 49, 47, and 38%, respectively. While all seven transgenic lines had similar levels of enhanced disease resistance, the lines with the \u03b2-1,3-glucanase transgene had slightly better disease control.\nTable 2Percent Fusarium head blight severity in greenhouse evaluations of seven wheat lines carrying wheat \u03b1-1-purothionin, barley thaumatin-like protein 1, or barley \u03b2-1,3-glucanase that were selected in initial tests and three wheat varieties used as disease checksGeneration testedaGenotypebT2T2T2T3T3T3T3T4CM1738*(9)44**(8)\u2013c38**(10)74 (14)--28* (22)CM2139*(12)38 (4)26*** (11)89 (11)\u2013\u201357* (22)\u2013CM2337*(14)55 (14)44** (18)41*(12)\u2013\u2013\u2013\u2013CM2738*(12)34**(12)\u2013\u201355* (17)\u2013\u2013\u2013CM2826**(10)37*(10)\u2013\u201341** (12)\u2013\u2013\u2013CM3019***(8)36*(11)\u2013\u201351 (4)\u2013\u2013\u2013CM3333 (8)48*(11)44* (7)\u2013\u201340* (12)\u2013\u2013Bobwhite63 (31)71 (28)78 (28)71 (28)78 (28)64 (18)73 (33)54 (36)Wheaton70 (33)85 (45)\u201385 (45)\u201391 (21)94 (57)99 (60)Sumai 326 (22)9 (25)10 (78)9 (25)10 (78)21 (16)16 (46)7 (61)Numbers in parenthesis represent the number of plants in the screen.aIndicates the generation that was evaluated. T2 and T3 lines that were evaluated in the initial FHB disease screens are in bold. Each column, except for the second T2 screen and the first T3 screen, represent individual experiments where lines were evaluated simultaneously.bCM17 is a transgenic wheat line carrying the wheat \u03b1-1-purothionin, CM21 and CM23 are transgenic wheat lines carrying barley thaumatin-like protein 1, and CM27, CM28, CM30, and CM33 are the transgenic wheat lines carrying barley \u03b2-1,3-glucanase transgene. Bobwhite is the variety transformed and susceptible check, Wheaton is a susceptible check, and Sumai 3 is a resistant check. It is not known whether the transgenic lines were homozygous for the transgene or segregating.cIndicates that this line was not examined in this screen.*Significance at the 0.05 compared to Bobwhite.**Significance at the 0.01 compared to Bobwhite.***Significance at the 0.001 level compared to Bobwhite.\nMolecular characterization of transgenic plants\nTo verify that one, two, and four lines transformed with \u03b1-1-purothionin, tlp-1, and \u03b2-1,3-glucanase transgenes, respectively, were transgenic, we conducted Southern blot analysis. Our Southern blots also provided the opportunity to determine if the two tlp-1 lines and four \u03b2-1,3-glucanase lines were independent events. Genomic DNA was isolated from each line, digested with the appropriate restriction enzyme, blotted, and hybridized with a radio-labeled probe from the \u03b1-1-purothionin, tlp-1, or \u03b2-1,3-glucanase transgenes (Fig.\u00a02). Each transgenic line contained at least one unique band compared to the nontransformed Bobwhite, indicating that each of the lines were transgenic for the appropriate transgene. In addition, the banding patterns of the two tlp-1 lines and the four \u03b2-1,3-glucanase lines were distinct, indicating that the two tlp-1 lines were independent events, and the four \u03b2-1,3-glucanase lines were also independent events.\nFig. 2a\u2013c Southern blot analysis of transgenic wheat plants. aEcoRI-digested genomic DNA from untransformed Bobwhite (BW), and pUBKBarGluc-3 transgenic CM27, CM28, CM30, and CM33 plants hybridized with a probe designed to bridge the ubiquitin promoter and the \u03b2-1,3-glucanase transgene junction. bHindIII-digested genomic DNA from untransformed Bobwhite (BW), and pAHCBarPR5 transgenic CM21 and CM23 plants and hybridized with a probe designed to bridge the ubiquitin promoter and the tlp-1 transgene junction. cXhoI-digested genomic DNA from untransformed Bobwhite (BW), and pKM1 transgenic CM17 plants hybridized with a probe designed to bridge the ubiquitin promoter and the \u03b1-1-purothionin transgene junction\nTo confirm transgene expression, we conducted RT-PCR and Western blot analyses. As stated earlier, RT-PCR was conducted on each plant used in the greenhouse disease evaluations, except where indicated. Figure\u00a03 shows an example of the RT-PCR analysis of the lines carrying each transgene. We also conducted Western blot analysis on plants carrying the tlp-1, and \u03b2-1,3-glucanase transgenes. We isolated protein from spikes, blotted the protein, and cross-reacted the blots with antibodies specific for tlp-1 and \u03b2-1,3-glucanase proteins. Our results showed that the transgenic lines exhibited an increase in their appropriate transgene protein compared to the nontransgenic Bobwhite control (Fig.\u00a04).\nFig. 3RT-PCR analysis of transgenic wheat lines carrying the wheat \u03b1-1-purothionin (CM17), barley tlp-1 (CM21 and CM 23), and barley \u03b2-1,3-glucanase (CM27, CM28, CM30, and CM33) transgenes. The fragment sizes for the \u03b1-1-purothionin, barley tlp-1, and barley \u03b2-1,3-glucanase amplified the expected products of 600, 805, and 577 bp, respectively. The wheat actin gene was used as a positive control and it exhibited the expected size of 369 bpFig. 4a\u2013b Western blot analysis of transgenic wheat lines. a  Protein extracted from spikes of transgenic lines carrying barley \u03b2-1,3-glucanase (CM27, CM28, CM30, and CM33) transgene was subjected to SDS-PAGE using a 12% polyacrylamide gel. Our barley \u03b2-1,3-glucanase antibody does not distinguish the transgenic barley protein from the endogenous wheat protein. The transgene-specific protein band in line CM33 appears to exhibit a higher molecular weight. Molecular markers indicated the protein to be the expected 35.2\u00a0kDa size. b Protein extracted from spikes of lines carrying barley tlp-1 (CM21 and CM23) transgene was subjected to SDS-PAGE using a 10% polyacrylamide gel. Molecular markers indicated the protein to be the expected 17.5 kDa size\nField evaluation of transgenic plants for response to Fusarium head blight\nTo further examine the level of effect on FHB, we conducted field tests of these seven lines in the summers of 2004 and 2005. Seed for each of the seven lines was derived from plants expressing the transgene based on RT-PCR analysis. We scored the lines for percent FHB severity, DON concentration, and percent visually scabby kernels (VSK) and compared the lines to the parental cultivar Bobwhite (Table\u00a03). All transgenic lines tested in the field except for CM28 showed a significant reduction in at least one FHB disease measure in comparison with Bobwhite. Four, four, and two lines exhibited significant reductions in percent FHB severity, DON concentration, and percent VSK, respectively. Although CM30 showed a significant reduction in percent FHB severity, it showed a significant increase in DON concentration. CM27 was the only transgenic line that exhibited a significant reduction in all three disease measures.\nTable 3Percent Fusarium head blight (FHB) severity, deoxynivalenol (DON) concentration, and percent visual scabby kernels (VSK) in transgenic wheat plants carrying wheat \u03b1-1-purothionin, barley thaumatin-like protein l, and barley \u03b2-1,3-glucanase and check wheat varieties evaluated in the field in 2004 and 2005GenotypeaFHB severity (%)DON concentration (ppm)bVSK (%)Bobwhite65.116.329.6Alsen15.4***3.7***5.4***Wheaton81.2***26.2***51.9***Wheaton (noninoculated)64.317.832.2Roblin70.918.842.2*237546.2**8.3***11.6***CM1752.7*15.724.7CM2155.111.4***19.8*CM2357.213.4*21.0CM2746.5***9.9***17.7*CM2858.217.625.8CM3048.3***22.8**34.3CM3349.2***14.320.3aCM17 is a transgenic wheat line carrying the wheat \u03b1-1-purothionin, CM21 and CM23 are the transgenic wheat lines carrying barley thaumatin-like protein 1, and CM27, CM28, CM30, and CM33 are the transgenic wheat lines carrying barley \u03b2-1,3-glucanase transgene. T6 and T7 were used for the 2004 and 2005 field screens, respectively. Bobwhite is the variety transformed and susceptible check, Wheaton and Roblin are the susceptible checks, 2375 is a moderately resistant check, and Alsen is a resistant check.bParts per million deoxynivalenol concentration.*Significance at the 0.05 level compared to Bobwhite.**Significance at the 0.01 level compared to Bobwhite.***Significance at the 0.001 level compared to Bobwhite.\nDiscussion\nLarge-scale wheat breeding efforts have not resulted in the development of highly resistant varieties to FHB. This is due to the fact that resistance in wheat is partial and quantitative. That is, multiple loci in wheat explain just a portion of the variation for FHB resistance (e.g., Kolb et\u00a0al. 2001). Single genes conferring a high degree of resistance to FHB have not been found despite extensive searches of wheat germplasm resources (Leonard and Bushnell 2003). One characteristic of the wheat response to F. graminearum infection is the induction of defense response genes such as \u03b2-1,3-glucanase, tlp-1, and thionin genes (Chen et\u00a0al. 1999; Pritsch et\u00a0al. 2000, 2001; Li et\u00a0al. 2001; Kang and Buchenauer 2002; Han et\u00a0al. 2005; Zhou et\u00a0al. 2005; Bernardo et\u00a0al. 2006). These genes are thought to provide basal resistance during infection because they encode proteins with differing modes of action against fungal pathogens. Thionins and tlps damage fungal cell membranes by making them permeable (Bohlmann et\u00a0al. 1988; Yun et\u00a0al. 1998), whereas \u03b2-1,3-glucanases degrade cell wall polysaccharide linkages (Leah et\u00a0al. 1991). In this study, we produced transgenic wheat lines overexpressing either \u03b1-1-purothionin, a tlp-1, or a \u03b2-1,3-glucanase to test their efficacy against FHB.\nNumerous studies reveal that over-expression of defense response genes in transgenic plants results in enhanced resistance to various fungal pathogens (reviewed in Muehlbauer and Bushnell 2003). In general, these studies show that partial resistance can be achieved from over-expressing defense response genes in plants. In this study, we defined enhanced resistance as exhibiting a reduction in any of the three disease parameters. To date, there are no reports of commercially practical levels of fungal resistance derived from over-expressing defense response genes.\nFrom our initial 70 transgenic lines, there were seven lines carrying either \u03b1-1-purothionin, tlp-1, or \u03b2-1,3-glucanase transgenes that resulted in enhanced FHB resistance in the greenhouse. Enhanced resistance was not detected in each of these seven lines in every greenhouse screen conducted (Table\u00a02). These results are likely due to the high variability inherent in FHB disease screens. However, over multiple disease screens, the transgenic lines provided a level of resistance above that present in the nontransgenic control cultivar Bobwhite. In particular, we identified CM27, a line carrying a \u03b2-1,3-glucanase transgene that exhibited low FHB severity, low DON concentration, and low percent VSK in the field. Interestingly, in the field screens we observed lines, such as CM30, with significantly lower FHB severity and a high DON level. As seen in the greenhouse screens, these results are likely due to the variation in FHB disease screens. Variation in FHB readings from field grown plants can be difficult to control (Campbell and Lipps 1998).\nConsistent with our results, Chen et\u00a0al. (1999) and Anand et\u00a0al. (2003) showed that overexpression of tlp-1 and a combination of \u03b2-1,3-glucanase and chitinase transgenes in wheat resulted in enhanced FHB disease in the greenhouse. Interestingly, these authors only detected enhanced resistance during early stages of disease progression. They interpreted the action of these transgenes as delaying the development of FHB. Unfortunately, field disease screens of their lines lacked resistance (Anand et\u00a0al. 2003). For their field study, these authors used inoculated corn kernels, which provided a continuous source of inoculum, whereas in our field study, we sprayed fungal spores on the spikes twice. Thus, there is a distinct difference in the inoculation methodology between the two studies, which could lead to different disease reactions. Developmental differences such as the timing of flowering have resulted in different disease reactions to FHB. When inoculated grain is used for the inoculum, early heading plants can exhibit greater susceptibility than late heading plants as they are exposed to the inoculum for a longer period of time. In our study, we controlled the timing of inoculation through spraying the spikes, and we did not observe any obvious developmental differences in our transgenic lines. Thus, our results demonstrate that enhanced resistance to FHB can be obtained through overexpressing defense response genes.\nTo date, there are no wheat germplasm sources that exhibit immunity to FHB. The best available lines, such as Sumai 3 and Alsen, exhibit resistance to initial infection and spread of the disease but this resistance is partial and plants may become severely diseased when conditions are highly favorable for disease development. The transgenic lines described in this study may provide a potential wheat germplasm source for enhanced FHB resistance. Although the level of transgene resistance is not high enough to alone provide useful protection to FHB, our transgenic lines may extend and enhance FHB resistance germplasm when combined with other resistance sources. To increase the level of resistance, we crossed our \u03b2-1,3-glucanase and tlp-1 transgenic lines and combined the transgenes into a common background because developing lines with multiple transgenes in tobacco increased resistance to a fungal pathogen (Jach et\u00a0al. 1995). We have also initiated crosses of our transgenic lines with the moderately resistant genotype, Alsen. Alsen contains the chromosome 3BS QTL for FHB resistance (Waldron et\u00a0al. 1999). Our goal is to develop populations containing the 3BS QTL in combination with each of the three transgenes. Our expectation is that these combinations may result in enhanced resistance to FHB over the levels present in Alsen.","keyphrases":["transgenic wheat","wheat","fusarium head blight","fusarium graminearum","triticum aestivum"],"prmu":["P","P","P","P","P"]}
{"id":"Behav_Genet-4-1-2292426","title":"Genetic and Environmental Influences on Individual Differences in Attitudes Toward Homosexuality: An Australian Twin Study\n","text":"Previous research has shown that many heterosexuals hold negative attitudes toward homosexuals and homosexuality (homophobia). Although a great deal of research has focused on the profile of homophobic individuals, this research provides little theoretical insight into the aetiology of homophobia. To examine genetic and environmental influences on variation in attitudes toward homophobia, we analysed data from 4,688 twins who completed a questionnaire concerning sexual behaviour and attitudes, including attitudes toward homosexuality. Results show that, in accordance with literature, males have significantly more negative attitudes toward homosexuality than females and non-heterosexuals are less homophobic than heterosexuals. In contrast with some earlier findings, age had no significant effect on the homophobia scores in this study. Genetic modelling showed that variation in homophobia scores could be explained by additive genetic (36%), shared environmental (18%) and unique environmental factors (46%). However, corrections based on previous findings show that the shared environmental estimate may be almost entirely accounted for as extra additive genetic variance arising from assortative mating for homophobic attitudes. The results suggest that variation in attitudes toward homosexuality is substantially inherited, and that social environmental influences are relatively minor.\nIntroduction\nHomophobia can be defined as \u2018the fear of, aversion to, or discrimination against homosexuality or homosexuals\u2019 (Merriam\u2013Webster\u2019s Medical Dictionary 2007). Although the social and cultural status of homosexuality has improved, Kite and Whitley (1996) concluded from their meta-analyses of 112 studies that many heterosexuals still hold very negative attitudes toward homosexuals and their sexual behaviour. A great deal of research has focused on the personality and demographic features of homophobic individuals. Numerous studies indicated that men tend to hold more negative attitudes toward homosexuals than women, especially toward gay men (e.g. Davies 2004; Herek 1988; Herek and Gonzalez-Rivera 2006; Kite and Whitley 1996; LaMar and Kite 1998; Lingiardi et\u00a0al. 2005; Steffens and Wagner 2004). The most common explanation for this sex difference in homophobic attitudes is that men tend to hold more traditional gender beliefs, which encourage them to be more negative toward homosexuality, particularly toward gay men (Kite and Whitley 1996). Furthermore, homophobic individuals in general seem to be higher in authoritarianism, are more religious, hold traditional attitudes toward family and gender roles, have relatively low levels of education and have had fewer interactions with homosexuals (Herek 1988, 2002; Herek and Gonzalez-Rivera 2006). As Kite and Whitley (1996) stated, these data offer some insight into the profile of homophobic individuals, but the existing research is only descriptive in nature and hence provides little theoretical insight into prejudices regarding sexual orientation.\nThere is considerable support for the existence of generalized prejudice (see Ekehammar et\u00a0al. 2004). Different types of prejudice (e.g. racism, sexism and prejudice toward homosexuals) have been shown to be highly correlated (e.g. Bierly 1985; Ekehammar and Akrami 2003), implying that people who reject one out-group will also tend to reject others. As Ekehammar et\u00a0al. (2004) describe in their article, two major theories have risen to explain why some individuals are more prejudiced than others. In the first theory, individual differences in prejudice are considered to be due to stable factors within the individual (their personality characteristics); in this case prejudice is seen as an expression of personality (Ekehammar and Akrami 2003; Heaven and St. Quintin 2003). The second theory implies that individual differences in prejudice are caused by factors linked to the outside world, like intergroup relationships and social life (see e.g. Guimond et\u00a0al. 2003; Guimond 2000; Reynolds et\u00a0al. 2001).\nEarlier twin studies have demonstrated that individual differences in personality are substantially heritable (e.g. Jang et\u00a0al. 1996; Loehlin et\u00a0al. 1998), and therefore, it is possible that there are genetic influences on homophobia as well. On the other hand, a study concerning personality and social attitudes (including general conservatism and attitude to sex and politics) in a large sample revealed that, besides genetic effects, family (shared) environment also has a considerable contribution to social attitudes (Eaves et\u00a0al. 1999). Consequently, shared environmental influences on attitudes toward homosexuality can also be expected. Such results would fit the second theory that emphasizes social processes.\nAccordingly, the aim of the present study is to determine the contribution of both genes and shared environment to individual differences in attitudes to homosexuality, using data from a large twin sample. Findings will result in further insight into the aetiology of variation in homophobia.\nMethodology\nParticipants\nAll participants were drawn from the Australian National Health and Medical Research Council Twin Registry (ATR). Subjects for this study were recruited in two phases from a large twin-family study of alcohol use and abuse.\nIn phase one, all twin pairs (N\u00a0=\u00a04,269, aged between 18 and 25\u00a0years) participating in an extensive Health and Lifestyle Questionnaire (HLQ) were asked whether they were willing to participate in an anonymous study about sexual behaviour and attitudes. All those who agreed were mailed the sex questionnaire between July 1991 and October 1992. To ensure anonymity, twins were not asked about their name or date of birth, but to make up a 10 digit number. Both twins of a pair had to use the same number, which made it possible to connect the responses of both twins. Additionally, informed consent was obtained separate from the questionnaires.\nIn the second phase, an additional group of twin pairs in an older cohort (aged between 27 and 52\u00a0years old) was asked to participate in the sexual behaviour and attitudes study. Procedures for this data collection were the same as for the younger cohort, as described above. Twins who expressed willingness to participate were mailed the questionnaire between April and August 1992. For a more extensive description of the data collection procedure, the study sample, and the pairing of the returned questionnaires (see Kirk et\u00a0al. 2000).\nIn total, 1,907 complete pairs and 1,090 twins from incomplete pairs completed the questionnaire (1,824 male and 3,080 female individuals). However, due to missing data and ambiguous zygosities the effective study sample used for the homophobia analyses is lower than 4,904, as described in the result section.\nZygosity\nThe zygosity of the same-sex twins was determined during completion of the HLQ, based on their response to standard items about physical similarity and being mistaken for each other. Ambiguous replies were clarified by telephone call. According to Ooki et\u00a0al. (1990) and Martin and Martin (1975), concurrence on zygosity between discriminant analyses of questionnaire scores and DNA typing is at least 95% and telephone clarification will have increased accuracy. In the present study the zygosity of the twin pairs was premarked on their questionnaire and the twins were asked whether they did or did not agreed with the assessment.\nMeasures\nData used in this study were 10 items from a larger part of the sexual behaviour and attitudes questionnaire called \u2018Your Present Personality\u2019. This part of the questionnaire contained 47 items, selected from three former instruments, namely: Inventory of Attitudes to Sex (30 items; Eysenck 1976), Attitudes to Homosexuality (10 items; Klassen et\u00a0al. 1989) and Adult Gender Nonconformity (seven items; Finn 1987, The structure of masculinity and femininity self ratings, Unpublished manuscript). A principal components analysis of these 47 items (performed in SPSS-13.0 for Windows) revealed nine factors for males and 10 for females. The most apparent factor for both sexes, explaining 16% of the total variance for both sexes, was a factor regarding attitudes to homosexuality, comprising all 10 items of the Attitudes to Homosexuality scale created by Klassen et\u00a0al. (1989). Each item showed a high factor loading for both males and females (between 0.55 and 0.87).\nAccordingly, in the present study homophobia was measured using the 10 items of this Attitudes to Homosexuality scale, which comprised statements like \u2018Homosexuality is obscene and vulgar\u2019, and \u2018Homosexual men should be allowed to work as schoolteachers\u2019 (see Appendix\u00a0A for the total scale). Twins were asked to either tick YES if they agreed with the statement or NO if they disagreed. If a participant filled out both answers on an item (both YES and NO), the item was coded as missing. To enhance unbiased answering, a preamble to the section explained that there are no right or wrong answers and no trick questions and they were encouraged to work quickly and not to think too long about the exact meaning of questions.\nTo maximise the number of twin pairs available for genetic analysis, missing item responses were imputed where possible by using the imputation methods in PRELIS 2.30 (J\u00f6reskog and S\u00f6rbom 1999). Missing values were substituted with values from other cases with similar response patterns, but without missing data. Note that it was only possible to impute items in PRELIS if participants had only one missing value and that imputation depended on the response patterns from other cases. A total of 109 responses were imputed (0.2% of the total item responses), increasing the available twins for structural equation modelling from 4,442 to 4,551, a gain of 2.5% in the total available sample size.\nA total homophobia score was calculated by summing the answers on all 10 statements, where a homophobic response was counted as one and a non-homophobic response as zero. To further maximise the number of twin pairs available for genetic analysis, participants missing one (N\u00a0=\u00a0116) or two (N\u00a0=\u00a0101) responses were included in further analyses. Their homophobia score was corrected by adding their mean item score for each missing item. This resulted in another gain of 4.8% of the available sample size.\nAs the distribution of the homophobia data showed significant skewness and kurtosis, scores were grouped into four ordinal categories with roughly equal sample sizes. Individuals scoring 0 on the continuous scale were given an ordinal score of 0, while individuals scoring 1 or 2 were given score 1, individuals scoring 3\u20136 score 2, and individuals scoring 7\u201310 score 3.\nTo determine the participants\u2019 sexual orientation, twins were asked the following question in another part of the questionnaire, called \u2018Sexual Feelings and Behaviour\u2019: Do you consider yourself to be heterosexual (straight), bisexual or homosexual (lesbian or gay)? For the present study, sexual orientation was only subdivided into heterosexual and non-heterosexual. The latter includes both bisexual and homosexual individuals.\nData analysis\nAs the homophobia scale analysed in this study is an ordinal scale, the data were analysed using a threshold model (Falconer 1989). For these analyses it is assumed that there is an underlying continuum of liability which is normally distributed in the population. Upon this normal distribution three thresholds are placed to delimit the four categories. Variation in liability can be modelled as a function of genetic and environmental influences.\nDescriptive statistics for the homophobia scores are obtained using SPSS-13.0 for Windows. Subsequently, Structural Equation Modelling (SEM) is applied to estimate thresholds, polychoric correlations between twins and components of variance. First, the effects of gender, age and sexual orientation on the thresholds is tested. Next, heterogeneity of polychoric twin pair correlations was assessed as a function of sex and zygosity, and finally the importance of different sources of familial aggregation (i.e. shared genes and shared environment) was tested. SEM is conducted using Mx 1.65b, a matrix algebra calculator that fits structural equation models to observed data (Neale et\u00a0al. 2003). All analyses were conducted using maximum-likelihood estimation for raw ordinal data in Mx. Models were compared with a likelihood ratio \u03c72 test.\nTo determine the effects of age, sexual orientation and sex on the thresholds, different (nested) submodels are tested against a full model, in which all parameters were estimated. By looking for differences in model fit after constraining these effects to zero in a stepwise manner, the significance of parameters that contribute to explaining differences in thresholds can be determined.\nHeterogeneity of twin pair correlations is tested by determining the change in model fit when twin pair correlations (for sex and\/or zygosity) were equated.\nGenetic model fitting of twin data allows for decomposition of the observed variance in homophobia scores into its genetic and environmental components. Additive genetic variance (A) denotes the variance resulting from the sum of allelic effects across multiple genes, while D symbolizes non-additive genetic effects including dominance and epistasis. Common environmental variance (C) is that resulting from environmental influences shared within twin pairs, whereas unique environmental variance (E) results from non-shared environmental influences and includes measurement error. Estimates of A, C, D and E can be derived from the resemblance between monozygotic (MZ) twins who are genetically identical and dizygotic (DZ) twins who share, on average, 50% of their segregating genes. Accordingly, if MZ twins resemble each other more than DZ twins on a particular trait, this is an indicator that it is partly influenced by genetic effects. Further, if the DZ within-pair correlation is higher than half the MZ within-pair correlation, this implies that common environmental effects contribute to individual differences in the homophobia scores. By means of SEM, different combinations of A, C, D, and E can be evaluated to obtain the most parsimonious explanation for the observed pattern of MZ and DZ twin pair correlations. Note, however, that C and D cannot be estimated simultaneously (Martin et al. 1978) and that the choice for an ACE or ADE model depends on the pattern of MZ and DZ correlations.\nResults\nDescriptive statistics\nOf the 4,904 respondents to the questionnaire, 136 were omitted as they had more than two missing values on the 10 homophobia items and an additional 80 single twins were deleted due to ambiguous zygosity. Hence the total number of individuals utilised for the analyses is 4,688, comprising 2,846 twin families (1,822 pairs and 1,044 singletons). Complete twin pairs included 295 MZ males, 182 DZ males, 634 MZ females, 354 DZ females and 357 DZ opposite sex pairs. The age range of the study sample was 19\u201352\u00a0years for both sexes, with a mean age for males of 30.6 (S.D.\u00a0=\u00a08.3) and for females of 31.1 (S.D.\u00a0=\u00a08.5). Descriptive statistics for each homophobia statement for all individuals are presented in Table\u00a01.\nTable\u00a01Percentage of agreement (yes), disagreement (no) and missing answers on the 10 homophobia statements for males and females separatelyMales (N\u00a0=\u00a01,758)Females (N\u00a0=\u00a02,930)YesNoMissingYesNoMissingHomosexuality is not immoral50.948.20.964.035.11.0Allow as school teachers53.046.90.169.430.50.0Allow as court judges65.234.70.178.421.40.2Allow as ministers56.943.00.167.432.20.4Allow as medical doctors58.041.90.170.129.80.1Allow as government officials70.829.10.181.818.10.1Dangerous as teachersa34.463.42.220.377.81.9Homosexuality is obscenea48.051.30.730.368.61.1Homosexuality is social corruptiona29.269.31.519.977.72.4Allow to dance with each other in public49.149.21.762.235.22.6aNote that agreement on these statements indicate a homophobic attitude\nUsing the imputed data set, item scores were added together and subsequently recoded into a four-point ordinal scale ranging from 0 (not homophobic at all) to 3 (highly homophobic). Table\u00a02 shows the number and percentage of individuals within each homophobia category.\nTable\u00a02Number and percentage of males and females, heterosexuals and non-heterosexuals, for each category of the ordinal homophobia scale (0 is least, 3 is most homophobic)Homophobia category0123Males, heterosexual (N\u00a0=\u00a01,627)368 (22.6%)314 (19.3%)392 (24.1%)553 (34.0%)Males, non-heterosexual (N\u00a0=\u00a0111)77 (69.4%)18 (16.2%)13 (11.7%)3 (2.7%)Males (N\u00a0=\u00a01758)a450 (25.6%)338 (19.2%)408 (23.2%)562 (32.0%)Females, heterosexual (N\u00a0=\u00a02802)1,011 (36.1%)692 (24.7%)580 (20.7%)519 (18.5%)Females, non-heterosexual (N\u00a0=\u00a0100)78 (78.0%)9 (9.0%)10 (10.0%)3 (3%)Females (N\u00a0=\u00a02930)a1,098 (37.5%)705 (24.1%)600 (20.5%)527 (18.0%)aNote that for 20 males and 28 females self-reported sexual orientation is missing\nTo determine whether sexual orientation influences homophobic attitudes, individuals were asked which sexual orientation they considered themselves. Of the 1,758 male participants, 1,627 (92.5%) rated themselves as heterosexual, while 111 (6.3%) considered themselves to be non-heterosexual [20 responses (1.1%) were missing]. Of the 2,930 females, 2,802 (96.6%) rated themselves to be heterosexual, while 100 (3.4%) considered themselves to be non-heterosexual [28 responses (1.0%) were missing]. In Table\u00a02 the number and percentage of heterosexuals and non-heterosexuals within each homophobia category is shown.\nHypotheses concerning differences in threshold parsimony\nWithin sexes, thresholds for MZ and DZ twins could be equated without significant deterioration in model fit (\u03c7122\u00a0=\u00a08.60, P\u00a0=\u00a00.74), indicating no evidence for social interaction between twins. However, results show a significant difference in the distribution of attitudes toward homosexuality between sexes, such that males were more homophobic than females (\u03c732\u00a0=\u00a080.41, P\u00a0<\u00a00.001). Furthermore, a significant difference in distribution of thresholds was found between heterosexuals and non-heterosexuals (\u03c712\u00a0=\u00a0192.51, P\u00a0<\u00a00.001). On average, heterosexuals were more homophobic than non-heterosexuals.\nFinally, it was tested whether age had a significant influence on the distribution of the thresholds. Removing the effect of age from the model did not lead to a significantly worse model fit (\u03c712\u00a0=\u00a01.83, P\u00a0=\u00a00.18), implying that age has no appreciable influence on individual differences in attitudes toward homosexuality.\nHeterogeneity of twin pair correlations\nPolychoric twin pair correlations for each zygosity group, shown in Table\u00a03, were estimated in Mx by maximum-likelihood. Although the twin pair correlation for MZ males is higher than for MZ females, they were not significantly different (\u03c712\u00a0=\u00a03.18, P\u00a0=\u00a00.08).\nTable\u00a03Polychoric twin pair correlations (95% confidence intervals) for the homophobia score for each zygosity group, estimated in MxMZM 295 pairsDZM 182 pairsMZF 634 pairsDZF 354 pairsDZOS 357 pairsCorrelation0.61 (0.52\u20130.69)0.42 (0.37\u20130.55)0.51 (0.43\u20130.57)0.39 (0.28\u20130.50)0.29 (0.17\u20130.41)\nFurthermore, as the twin pair correlation for DZ opposite sex twin pairs is lower than for the DZ same sex twin pairs, qualitative differences in sources of familial aggregation between sexes could be expected. However, the difference in twin pair correlation between the opposite and the same sex DZ twin pairs was not significant (\u03c722\u00a0=\u00a02.46, P\u00a0=\u00a00.29).\nThe inability to find significant differences between the MZ twin pair correlations for males and females, and between the DZ same sex and opposite sex twin pair correlations may, however, be due to a lack of power to detect qualitative and quantitative differences between the groups.\nThe MZ twin pair correlations are significant higher than the DZ twin pair correlations (\u03c712\u00a0=\u00a015.64, P\u00a0<\u00a00.001), suggesting that genetic effects are a source of familial aggregation in attitudes toward homosexuality.\nGenetic modelling\nResults of the univariate SEM for the homophobia scores are shown in Table\u00a04. As the DZ twin pair correlations are more than half the MZ twin pair correlations, a model is fitted incorporating additive genetic (A), shared environmental (C) and unique environmental (E) effects. Since the DZ opposite sex correlation is lower (although not significantly) than the DZ same sex correlations, a general sex-limitation model is fitted, which allows for qualitative and quantitative differences in the sources of variation in attitudes toward homosexuality between sexes (Neale and Cardon 1992). To model the qualitative differences in genetic effects between sexes, the genetic correlation for DZ opposite sex twins (H) is estimated in the model instead of fixed at 0.5 as it is for same sex DZ twin pairs.\nTable\u00a04Genetic model fitting: Comparison of different genetic modelsModel\u22122lld.f.Vs\u0394\u03c72\u0394d.f.P-valueHDZOS1General sex-limitation model12,199.9346720.092Common effects sex-limitation model12,201.36467311.4310.230.503General ACE model12,205.88467524.5220.100.50\nSubsequently, a common effects sex-limitation model is fitted, by fixing the genetic correlation for DZ opposite sex twins at 0.5. Results show no significant deterioration of model fitting (\u03c712\u00a0=\u00a01.43, P\u00a0=\u00a00.23), consistent with the same sets of genes influencing homophobia scores for males and females.\nThird, the fit of a general ACE model, in which proportions of A and C are constrained equal in males and females, is compared with the fit of the common effects sex-limitation model. Results show no significant deterioration of model fit (\u03c722\u00a0=\u00a04.52, P\u00a0=\u00a00.10), indicating there are no magnitude differences in effects of A, C and E on variance in homophobia scores between sexes.\nUnder the general ACE model, additive genetic and common environmental effects together account for between 48% and 59% of the variance in homophobia (see Table\u00a05). It is impossible to distinguish between AE and CE models, as removing the effects of either A or C causes significant deterioration in model fit.\nTable\u00a05Proportions of variance (95% confidence intervals) attributable to A, C and E effects for both sexes for the general sex-limitation model, the common effects sex-limitation model and the general ACE modelAmalesCmalesEmalesAfemalesCfemalesEfemalesHDZOSGeneral sex-limitation model0.38 (0.08\u20130.68)0.23 (0.00\u20130.49)0.39 (0.31\u20130.44)0.23 (0.00\u20130.49)0.28 (0.04\u20130.49)0.49 (0.43\u20130.57)0.09 (0.00\u20131.00)Common effects sex-limitation model0.55 (0.16\u20130.69)0.07 (0.00\u20130.47)0.38 (0.30\u20130.47)0.20 (0.00\u20130.54)0.30 (0.00\u20130.51)0.50 (0.43\u20130.58)0.50General ACE model0.36 (0.19\u20130.54)0.18 (0.02\u20130.32)0.46 (0.41\u20130.52)0.36 (0.19\u20130.54)0.18 (0.02\u20130.32)0.46 (0.41\u20130.52)0.50\nAccordingly, the most parsimonious explanation for the observed pattern of MZ and DZ twin pair correlations is the general ACE model, where there are no qualitative and quantitative differences in sources of variance between sexes. The twin pair correlations, however, point to possible differences in the sources of variance between sexes. The inability to detect these qualitative and quantitative differences between sexes might be due to insufficient power from using a threshold model, necessitated by the extreme skewness of the raw data (Neale et\u00a0al. 1994).\nBased on the general ACE model, estimates of the influence of A, C and E on the variance in homophobia scores for both sexes are 36%, 18% and 46% (see Table\u00a05). In Fig.\u00a01, the graphical illustration of the general ACE model (a) and the general sex-limitation model (b), including the path coefficients, are presented.\nFig.\u00a01Graphical presentation of the general ACE model (a) and the general sex-limitation model (b) along with the standardised path coefficients. In the general sex-limitation model path coefficients for males are presented on the left and for females on the right side. Note that in the general sex-limitation model the correlation between opposite sex twins for additive genetic effects \nis estimated in the model\nDiscussion\nAnalyses of the homophobia scale showed that, in accordance with former literature (e.g. Kite and Whitley 1996), males have more negative attitudes toward homosexuality than females and heterosexuals are more homophobic than non-heterosexuals (Jellison et\u00a0al. 2004). In this study, age had no effect on attitudes toward homosexuality, whereas some others have found that younger individuals have more favourable attitudes regarding homosexuality than older persons (Herek 2002; Steffens and Wagner 2004). It should be noted however that the distribution of age in this study was highly skewed, with the majority of participants (70.9%) in age group 19\u201335 and only 29.1% of all participants aged between 35 and 52\u00a0years.\nThe aim of the present study was to determine the contribution of genes and environment to individual differences in attitudes toward homosexuality. Based on the general ACE model, estimates of the influence of A, C and E on the variance in homophobia scores are: 36%, 18% and 46%. However, although no significant qualitative and quantitative differences in sources of variation between males and females were found, twin pair correlations do suggest possible differences between the sexes. Under a general sex-limitation model, estimates of the influence of A and C on variation in homophobia scores are 38% and 23% for males and 23% and 28% for females, respectively. The additive genetic correlation for the DZ opposite sex twins is estimated at 0.09, considerably lower than the 0.5 assumed for DZ same sex twins. This finding would suggest that genes influencing homophobia in males are to a large extent different from those in females. However, since both A and C effects are present in both sexes, the parameterisation of sex limitation as genetic in origin is arbitrary. If it were parameterised as a lower common environmental correlation between males and females, the same model fit would be obtained. Moreover, there could be sex limitation of both A and C influences, but since there is only one degree of freedom to estimate them\u2014obtained from the DZ opposite sex twin pair correlation\u2014the two are completely confounded. In either case, differences in saliency of homosexuality between males and females make it unsurprising that there are distinct causes of variation in homophobia in the two sexes.\nA limitation to our confidence in the variance components estimated above is that strong assortative mating has been repeatedly found for social attitudes (e.g. Eaves et\u00a0al. 1999; Hatemi et\u00a0al. 2007; Martin et\u00a0al. 1986). This means that the relative genetic variance may have been underestimated. If spouses are correlated for homophobia through the assortment process, then both MZ and DZ twin correlations will be raised to the same degree. Mimicking the effect of shared environment this extra resemblance would yield higher estimates of \u2018C\u2019. As our sample does not include spouses, we rely on previous estimates of assortative mating to determine its effect on our estimates. Recently, Hatemi et\u00a0al. (2007) investigated spouse correlations on social attitudes, including an item on gay rights, for which they found a spouse correlation of 0.58. Assuming that this value is a reasonable estimate for assortative mating in our sample, it is possible to correct our C estimate for assortative mating by using a procedure described by Martin (1978).1\nAfter correction, the contribution of shared environment on individual differences in homophobia scores falls from 0.18 to 0.03, while the genetic estimate increases to about 0.51. This finding suggests that variation in attitudes toward homosexuality is substantially inherited, while social environmental influences are relatively minor.\nWhen applying the assortative mating correction to the results of the general sex-limitation model, the contribution of shared environment on individual differences in homophobia scores for males falls from 0.23 to 0.04 and for females from 0.28 to 0.24, while the genetic estimate increases to about 0.57 for males and 0.27 for females. As such, based on the general sex-limitation model familial aggregation for homophobia scores for males might be almost totally accounted for by genetic effects, while for females both genes and shared environment seem to have about equal effects. If this would be the case, and there is little or no C influence on homophobia scores for males, sex limitation for homophobia scores can only have a genetic origin.\nAnother important consideration is that the genetic contribution to homophobia might partly reflect genetic influences on related traits such as intelligence, personality, or social class. In our data, the polychoric correlation of the homophobia measure with educational level was \u22120.27 (P\u00a0<\u00a00.01) and with social class was \u22120.14 (P\u00a0<\u00a00.01), so these traits would only account for a modest amount of variance.\nWhile individuals\u2019 attitudes toward homosexuality are partly influenced by genes, shared environmental factors also contribute a modest proportion of variation in homophobic attitudes. This finding suggests that shared environmental influences, such as norms and values taught by parents, are aspects in forming positive or negative attitudes toward homosexuals, and possibly also toward other out-groups. The main source of variance is unique environment (46% under the general ACE model). While an unknown fraction of this will be due to measurement error, the number suggest a great influence on attitudes to homosexuality is the unique experiences of the individual shaping his or her attitudes independent of family influences. This study shows that one\u2019s own sexual orientation is one such influence, although previous research has shown that sexual orientation itself is substantially genetically influenced (e.g. Kendler et\u00a0al. 2000; Kirk et\u00a0al. 2000). Although the effect of sexual orientation on homophobia is large at the individual level, the percentage of non-heterosexuals is so low, it accounts for only a trivial proportion of population variance in attitudes to homosexuality.\nThere are a few important methodological limitations to be considered for this study. As already mentioned, the most important limitation of this study was a lack of statistical power. Although the sample size is fairly large, due to the fact that the homophobia scale had to be analysed as ordinal data, it is still insufficient. Neale et\u00a0al. (2004) showed that for a threshold study at least three times the sample size needed for equivalent power to a study using continuous data is required.\nAnother important limitation of this study is that no distinction has been made between attitudes toward male versus female homosexuals. Five out of the ten questions are asked in gender neutral terms, while the other five statements are about homosexual men. The term \u2018homosexuals\u2019 will by most individuals be interpreted as referring to gay males and not to females (Haddock et\u00a0al. 1993), so the items will predominantly measure attitudes toward gay men. As previously mentioned, heterosexuals tend to have quite different attitudes toward the two, with lesbians generally being viewed less negatively (e.g. Herek 2002; Kite and Whitley 1996). Hence, to better understand individual differences in attitudes toward homosexuality, researchers should distinguish between male and female homosexuals in their questions (Herek and Capitanio 1999; LaMar and Kite 1998; Kite and Whitley 1996).\nA further consideration is whether participation biases influenced the results of the present study, especially as this survey queried participants about very personal information. Dunne et\u00a0al. (1997) have examined this question for the survey used in the current study by comparing the social, psychological and behavioural features of the twins who explicitly consented to join the present study (52%) with those who either explicitly refused (27%) or initially agreed, but subsequently did not return the consent forms (19%). Results indicate that those individuals who explicitly consented had less conservative sexual attitudes and were more likely to agree with gay rights. However, the effect sizes of these findings were small, indicating the participation bias probably did not influence the results to a great extent.\nAnother limitation to consider is the possibility of socially desirable responding. Although it is not possible to exclude social desirability effects, twins were encouraged to be honest by guaranteeing anonymity and by asking them to fill out the questionnaire while alone with sufficient privacy.\nIn summary, this study concerning the aetiology of homophobic attitudes revealed that familial aggregation in attitudes toward homosexuality is accounted for by genes as well as by shared environmental factors. However, when the plausible effect of assortative mating on our estimates is taken into account, familial aggregation for homophobia scores might be almost totally accounted for by genetic effects. More research is necessary to further unravel the sources of variance in homophobia and to determine whether these differ between sexes. This future research will need greater sample sizes\u2014preferably including spouses\u2014and should analyse attitudes toward male and female homosexuals separately. It will also be of interest to compare prejudice toward homosexuals with prejudice toward other targets (like racism and sexism), to see to what extent similar results will be obtained and whether the same genetic and environmental sources explain individual differences in prejudice toward different out-groups.","keyphrases":["genetics","attitudes","homosexuality","twin study","homophobia","heritability"],"prmu":["P","P","P","P","P","P"]}
{"id":"Bioinformation-1-3-1891659","title":"SSRscanner: a program for reporting distribution and exact location of simple sequence repeats\n","text":"Simple sequence repeats (SSRs) have become important molecular markers for a broad range of applications, such as genome mapping and characterization, phenotype mapping, marker assisted selection of crop plants and a range of molecular ecology and diversity studies. These repeated DNA sequences are found in both prokaryotes and eukaryotes. They are distributed almost at random throughout the genome, ranging from mononucleotide to trinucleotide repeats. They are also found at longer lengths (> 6 repeating units) of tracts. Most of the computer programs that find SSRs do not report its exact position. A computer program SSRscanner was written to find out distribution, frequency and exact location of each SSR in the genome. SSRscanner is user friendly. It can search repeats of any length and produce outputs with their exact position on chromosome and their frequency of occurrence in the sequence.\nBackground\nSSRs (simple sequence repeats) or microsatellites are the genetic loci where one or few bases are tandemly repeated for varying \nnumbers of times. Such repetitions occur primarily due to slipped-strand mis-pairing and subsequent error(s) during DNA \nreplication, repair, or recombination. [1] SSRs comprising 1\u00ad6 bp long, \noccur frequently and are ubiquitously interspersed in many genomes. [2\u2013\n3] The biological importance of SSR tracts has been clearly delineated. Microsatellite \nloci show extensive length polymorphism, and hence they are widely used in DNA fingerprinting and diversity studies. They are also \nconsidered as ideal genetic markers for the construction of high-density linkage maps. [4\n\u20135] In spite of its high significance, a bioinformatics tool for the \nanalysis of these regions is not available. \nAvailable algorithms directly or indirectly detect tandem repeats. However, there are many limitations with these algorithms. \nThe drawbacks are high computational time required by the algorithm and their inability to predict the positions of SSRs in the \ngenome. The program Tandem Repeats Finder [6] locates repeats with motifs \nof any size and type, including repeats with insertions and deletions. The program Sputnik [\n7] (unpublished) uses recursion to search for both exact and approximate tandem repeats. Repeating unit \nlengths of 2 to 5 are sought, and a score is used to determine location. Tandem Repeat Occurrence Locator (TROLL) [8], uses a keyword tree adapted from bibliographic searching techniques and attempts to \nmatch the keywords exactly but it does not specify the positions of repeats. In this work, we describe a program called SSRscanner \n(Simple sequence repeat scanner) that uses dictionary approach to find simple sequence repeats of pre-selected motifs. \nSSRscanner is a PERL script developed for scanning genomes to find repeats of any length, their exact position on chromosome \nand frequency of occurrence. It is fast and requires a standard Personal Computer (PC) and PERL to operate. SSRscanner can accept \nlarge sequences as input and large number of motifs can be searched simultaneously. Thus, the running time of the program is \ngreatly reduced. To demonstrate the use of SSRscanner, Arabidopsis thaliana genome was analyzed for finding out \ndistribution, \nfrequency and specific position of SSRs in the genome. [9] The advantages \nover many other programs developed for SSR identification includes its ability to search motifs of any length repeated for a \nnumber of times and to give the exact position of the motif in the genome.\nMethodology\nProgram Input\nSSRscanner (implemented in PERL) accepts two text files (.txt). Upon execution of the program, it prompts to enter the file \nname containing the DNA sequence data. It also prompts for a file containing motifs of different repeat types. It then prompts for \nthe number of times for the motifs to be repeated (for example for searching tri-nucleotide repeats the user should enter 3) \n(Figure 1B). \nInput sequence and motifs are parsed to SSRscanner that extracts the SSRs giving their distribution\/frequency and \nspecific location. The results from SSRscanner are appended into result files (Figure 1A).\nProgram output\nSSRscanner gives two files in output (Figure 1A). They are (1) Motifposition.txt \n(gives the frequency of each repeat provided \nin the motif file) and (2) Motifresult.exe (gives the specific location of each repeat). The data obtained can then be arranged \ninto desired formats.\nCaveats and Future Development\nSSRscanner is a PERL script and it requires PERL to be installed on a PC before running the program. We are developing a web \nbased CGI for SSRscanner.","keyphrases":["ssr","scanner","repeats","microsatellite","script"],"prmu":["P","P","P","P","P"]}
{"id":"Photosynth_Res-3-1-1779624","title":"The metabolic significance of octulose phosphates in the photosynthetic carbon reduction cycle in spinach\n","text":"14C-Labelled octulose phosphates were formed during photosynthetic 14CO2 fixation and were measured in spinach leaves and chloroplasts. Because mono- and bisphosphates of d-glycero-d-ido-octulose are the active 8-carbon ketosugar intermediates of the L-type pentose pathway, it was proposed that they may also be reactants in a modified Calvin\u2013Benson\u2013Bassham pathway reaction scheme. This investigation therefore initially focussed only on the ido-epimer of the octulose phosphates even though 14C-labelled d-glycero-d-altro-octulose mono- and bisphosphates were also identified in chloroplasts and leaves. 14CO2 predominantly labelled positions 5 and 6 of d-glycero-d-ido-octulose 1,8-P2 consistent with labelling predictions of the modified scheme. The kinetics of 14CO2 incorporation into ido-octulose was similar to its incorporation into some traditional intermediates of the path of carbon, while subsequent exposure to 12CO2 rapidly displaced the 14C isotope label from octulose with the same kinetics of label loss as some of the confirmed Calvin pathway intermediates. This is consistent with octulose phosphates having the role of cyclic intermediates rather than synthesized storage products. (Storage products don\u2019t rapidly exchange isotopically labelled carbons with unlabelled CO2.)\nIntroduction\nThe formulation of the Calvin\u2013Benson\u2013Bassham pathway, hereafter called Calvin cycle, of photosynthesis (PS) was heavily dependent on the elucidation of a reaction sequence for the pentose pathway (PP) of glucose metabolism in the biochemistry of tissues. A reaction sequence for the PP was proposed from the results of experiments in which liver, pea leaf and pea root enzyme preparations were used to catalyse the conversion of [1-14C] Ribose 5-P (Rib 5-P) to14C-labelled hexose 6-P and unlabelled glyceraldehyde 3-P (Gap), which were formed in vitro over a 17-h period (Horecker et\u00a0al. 1954). For many years Williams and co-workers investigated and published results of research on PP in liver, some neoplasms, adipose tissue, heart, colonocytes and photosynthetic tissues (see Williams et\u00a0al. 1987; Williams 2004 for published listings). These investigations showed that the conclusion drawn from the results of the fundamental experiments on which the PP reaction sequence is based (Horecker et\u00a0al. 1954; Gibbs and Horecker 1954) was erroneous and because of its 17-h duration, the results were incapable of being interpreted due to the random scattering of 14C isotope in the glucose 6-P (Glc 6-P) product. The 14C labelling pattern of Glc 6-P was the result of a prediction labelling experiment that was used to hypothesize a reaction sequence for the PP. The unpredictable and randomized isotope scattering of 14C in Glc 6-P was finally shown to be due to the activity of the extensive 14C-exchange rates versus the slower rates of mass transfer reactions catalysed by the group transferring enzymes, aldolase (EC 4.1.2.13) (Ald), transketolase (EC 2.2.1.1) (TK) and transaldolase (EC 2.2.1.2) (TA) (Flanigan et\u00a0al. 1993). Williams et\u00a0al. (1978a) demonstrated that the reversible interconversions of Rib 5-P and hexose and triose phosphates by liver and the other tissues mentioned above, also involved ido- and altro-octulose mono- and bisphosphates, sedoheptulose 1,7-P2 (Seh 1,7-P2) and a low concentration of arabinose 5-P (Ara 5-P) as intermediates, together with a proposal for the inclusion of three new enzyme activities in the PP reaction sequence (see Fig.\u00a01).Fig.\u00a01The L-Type PP of glucose metabolism. The pathway features reactions of arabinose 5-phosphate, d-glycero-d-ido-octulose and sedoheptulose mono- and bisphosphates together with new enzymes to catalyse their reactions (Williams and Clark 1971; Williams et\u00a0al. 1978a; Williams 2004)\nCalvin and colleagues based the reaction sequence which regenerated pentose phosphates (especially Ru 1,5-P2) from hexose and triose phosphates in PS, on a selection of the reactions of a reverse-acting classical PP (Calvin 1956), thereby providing important credibility at the time for the PP which still carried an author-imposed tentative caveat. It was later noted (Clark et\u00a0al. 1974) that the omission of the above new PP intermediates and enzymes from the Calvin scheme provided further explanations of some of the early criticisms and anomalies of the Calvin pathway scheme (Clark et\u00a0al. 1974; Stiller 1962; Kandler and Gibbs 1956; Beck and Hopf 1982).\nThe investigations described here were made in order to (a) search for evidence that d-g-d-i-oct phosphates may be formed by carbon fixation reactions in spinach during PS and (b) quantitatively measure any formation of octulose phosphates and investigate whether their proposed path of synthesis conformed with a theoretical extension of the photosynthetic reaction scheme shown in Fig.\u00a02. This paper reports the results of studies on the formation and functions of octulose phosphates, with a particular initial focus on d-glycero-d-ido-octulose 1,8-P2 and the 8-monophosphate, together with other novel intermediates in spinach leaves and chloroplasts.Fig.\u00a02The initial hypothesis for a revised reaction scheme for the reductive PP of photosynthetic carbon reduction in C-3 plants. The hypothesis differs from the Calvin Pathway by the inclusion of new enzymes and reactions by octulose and arabinose phosphates and is tested by the results reported in this paper. The figure shows the distributions (for one cycle) of the coloured carbon of CO2 into traditional and proposed new intermediates of a modified Calvin Pathway\nThe initial singular attention on the ido- epimer rather than an inquiry that also included d-glycero-d-altro-octulose phosphates, rested on the exclusive roles assigned to d-g-d-i-oct phosphates in the L-type PP (Fig.\u00a01). However, a final conclusion drawn from the study suggests that both epimeric forms of octulose phosphates occur and that both may have roles in the path of carbon in PS. The investigation used the same 14C tracer and other methodologies that were pioneered by Calvin and his team in the unravelling of a path of carbon in PS. The accompanying paper reports the results of a subsequent mass spectrometric investigation of the incorporation of 13CO2 into C4 to C8 sugar phosphates in spinach chloroplasts during light-driven PS.\nMaterials and methods\nGrowth of plant material\nSpinach (Spinacia oleracea, L.,var. Yates 102) was grown by water culture in a glasshouse under natural lighting according to established methods (Anderson and Boardman 1966). Depending on the season, leaves were harvested five or eight weeks after sowing the seeds.\nChemical and chromatographic materials\nEnzymes and cofactors for analysis and synthesis of sugar phosphates were obtained from either Sigma Chemical Co., St. Louis, MO 63178, USA or from Boehringer-Mannheim Corp., D-6800 Mannheim, Germany. 14C- and 32P-labelled compounds were from Amersham (Bucks, England). Other analytical reagent grade chemicals were obtained from Ajax Chemicals, Sydney, Australia, BDH Poole, BH12 4NN, UK, or E. Merck, D-6100 Darmstad, Germany. Chromatographic media were obtained from the following sources: Ion exchange resins from BioRad, Richmond, CA 94804, U.S.A. Phenylboronate agarose gel (Matrex PBA 60) from Amicon Corp., Danvers, MA 01923, USA. Thin layer chromatography plates from E.Merck, 3Mm paper from Whatman, Maidstone, UK, Sephadex G10 and Sephadex A25 from Pharmacia, Uppsala, Sweden. Chromatographic columns were from Pharmacia or LKB, Bromma, Sweden. Ultrafiltration apparatus and Diaflow PM10 membranes were obtained from Amicon, Lexington, MA, USA.\nExtraction and analysis of photosynthetic tissue metabolites by gas-liquid chromatography\nYoung spinach leaves (6\u20138\u00a0weeks) were detached from plants, which had been exposed to full sunlight for 8\u00a0h, and then placed in liquid nitrogen. The tissue was ground to a fine powder using a stainless steel mortar and pestle and 6% perchloric acid was added. The temperature of this mixture was raised to 4\u00b0C and the solid was blended with the acid using a Potter-Elvejhem homogenizer. The liquid was separated from insoluble matter by centrifugation at 10,000g for 10\u00a0min. The pellet was re-extracted with perchloric acid and the two extracts combined. The solution was neutralized with 2\u00a0M KOH and stored at 2\u00b0C for several hours to complete the precipitation of potassium perchlorate which was then removed by centrifugation at 15,000g for 10\u00a0min. The supernatant fluid was treated with acid-washed activated carbon (Norite) to remove nucleotides and aromatic compounds and was lyophilized. The extract was dissolved in 5\u00a0ml of 0.2\u00a0M acetate buffer, pH\u00a05.0 and 20 units of acid phosphatase (EC 3.1.3.2 from potato) added. Mannitol (2\u00a0\u03bcmol) and erythritol (0.5\u00a0\u03bcmol) were added as internal standards. The mixture was incubated at 30\u00b0C to achieve complete sugar phosphate hydrolysis. The free sugars were recovered by thermally denaturing the protein, followed by centrifugation and treatment of the supernatant fraction with a three-fold excess (calculated from the amount of buffer used) of mixed bed resin (Dowex 50\u00a0\u00d7\u00a04, 100\u2013200 mesh, H+ form; Dowex 1\u00a0\u00d7\u00a04, 100\u2013200 mesh, HCO3\u2212 form). The completeness of deonization was established using a Radiometer conductivity meter. The solution was lyophilized and stored at \u221220\u00b0C. Lyophilized extracts were converted to their TMS ethers immediately before analysis using the procedure of Williams et\u00a0al. (1984). Dimethylformamide and bis-trimethylsilyltrifluoroacetamide (50\u00a0\u03bcl of each) were added and the mixture stirred for 45\u00a0min at 55\u00b0C. The analyses were carried out using a Pye-Unicam gas chromatograph (series 204), fitted with two glass columns (180\u00a0cm\u00a0\u00d7\u00a00.4\u00a0cm) packed with 3% SP-2250 on 100\/120 mesh Supelco, Bellefonte, PA; USA. FID responses were recorded on a computing integrator (Spectra-Physics, model 4100).\nPhotosynthesis experiments with whole spinach leaves\n14C isotope tracer and pulse\u2013chase investigations with whole spinach leaves were conducted using an exact application of the methods and apparatus described by Hatch and Slack (1966). This procedure and the method for the ethanol extraction, preparation of the soluble fraction and isolation of sugar phosphates was described by Bartlett et\u00a0al. (1989). Sugar phosphates were separated by formate anion-exchange chromatography and further purified using two-dimensional TLC or paper chromatography and the GW3\/GW3-phenylboronate solvents (Kapuscinski et\u00a0al. 1985). The insoluble residue remaining after ethanol extraction of all sugar phosphates was extensively washed with water, ethanol and acetone. The amount of 14C radioactivity in the insoluble glucan was determined using liquid scintillation counting (Arora et\u00a0al. 1987).\nPreparation of whole leaf homogenate\nApproximately 50\u00a0g of fresh spinach leaves were washed briefly in distilled water, de-ribbed and cut into small segments to facilitate maceration. The material was homogenized in 100\u00a0ml of 50\u00a0mM Tris\u2013HCl buffer (pH\u00a07.4) containing 1\u00a0mM EDTA, and strained through two layers of Miracloth (Calbiochem). After centrifugation at 10,000\u00a0\u00d7 g for 15\u00a0min, the preparation was concentrated four-fold by ultrafiltration using a 10,000\u00a0MW cut-off membrane (PM10 Amicon). The concentrated solution was dialysed for 10\u00a0h against six changes of the homogenization buffer. All operations were carried out at 4\u00b0C.\nChloroplast isolation\nThe procedure for the isolation of intact chloroplasts is based on a method which involves a two-step gradient of the silica-sol, Percoll (Pharmacia Biotech, Sweden) (Robinson 1983). Five- to six-weeks-old spinach leaves were harvested in the early morning in order to avoid the accumulation of starch granules, which can rupture the chloroplast envelope during centrifugation. The amount of leaf material required to produce a satisfactory yield of chloroplasts was not critical but the yield depended on the quality of the leaves and increased with the amount of material used. The harvested leaves were de-ribbed and floated in basins of water. Immediately before isolating the chloroplasts, the leaves were illuminated for 30\u00a0min with incandescent light at an intensity of 800\u00a0\u03bc\u00a0E\u00a0m\u22122\u00a0s\u22121. During this period, crushed ice was added to the water to maintain the temperature at 15\u00b0C. Typically, 55\u00a0g of coarsely chopped leaves were homogenized in 300\u00a0ml of medium for 1.5\u20132\u00a0s using a Polytron blender (setting 7) fitted with a PT35K probe. The homogenizing medium at pH\u00a06.5 was chilled to a semi-frozen consistency before use and contained 330\u00a0mM sorbitol, 10\u00a0mM Na4P2O7\u00b710H2O, 5\u00a0mM MgCl2, 2\u00a0mM isoascorbic acid and 0.1% bovine serum album (BSA) (Jensen and Bassham 1966). The brei was filtered through a double layer of Miracloth (Calbiochem). The filtrate was centrifuged at 1,200\u00a0\u00d7 g for 1\u00a0min in a Sorvall refrigerated centrifuge (DuPont Medical Products, Newtown, CT O647-5509 USA) using either an SS34 or SA600 rotor. The supernatant fluid was discarded and the sedimented material was gently resuspended, using a fine soft brush, in 6\u00a0ml of a medium at pH\u00a07.6 containing 330\u00a0mM sorbitol, 50\u00a0mM HEPES\u2013KOH, 2\u00a0mM EDTA, 1\u00a0mM MgCl2 and 1\u00a0mM MnCl2 (Jensen and Bassham 1966). An aliquot (3\u00a0ml) of the suspension was then placed into each of two 40-ml Corex tubes and carefully underlaid with 4\u00a0ml of Percoll medium made to 40% with resuspension medium and centrifuged. Finally, the chloroplasts were suspended in the resuspension medium (3\u00a0ml) and stored on ice. The above procedures were carried out on ice using previously chilled equipment. All parameters (density, yield, photosynthetic activity etc.) describing chloroplast properties were related to the Chl content measured according to the following procedure. Twenty five to 65\u00a0g of leaves produced between 3\u00a0mg and 8\u00a0mg of Chl. Provided the quality of leaf material was satisfactory and the Percoll was fresh, the method unfailingly produced a maximum yield of highly active chloroplasts which were better than 95% intact. Typical examples of preparations had the following mean values and S.D.: Activity 113.7\u00a0\u00b1\u00a08.6 (n\u00a0=\u00a010)\u00a0\u03bcmol CO2\u00a0h\u22121\u00a0mg\u22121 Chl; Intactness 95.75%\u00a0\u00b1 3.25% (n\u00a0=\u00a010).\nChlorophyll assay\nThe Chl content of isolated chloroplast suspensions was estimated by the method of Arnon (1949). In whole leaf experiments Chl was estimated using the method of Vernon (1960).\nMeasurements of chloroplast polarographic activity and intactness\nThe activity of the isolated chloroplast suspensions was determined polarographically using a Clark oxygen electrode. The reaction mixture (2\u00a0ml) contained, 0.33\u00a0mM sorbitol, 2\u00a0mM EDTA, 1\u00a0mM MgCl2, 1\u00a0mM MnCl2, 50\u00a0mM HEPES, 0.5\u00a0mM Pi, 5\u00a0mM pyrophosphate, 500\u00a0units of catalase (EC 1.11.1.6), 10\u00a0mM NaHCO3 and chloroplast suspension equivalent to 40\u00a0\u03bcg of Chl. The CO2-dependent O2 evolution of isolated chloroplast suspensions illuminated in an assay medium at 20\u00b0C was recorded using a Goerz Metrawatt SE120 chart recorder and the rate of oxygen evolution was calculated from the linear portion of the trace. The chloroplasts were illuminated at an intensity of 1,200\u00a0\u03bcE\u00a0m\u22122\u00a0s\u22121 using the quartz-halogen light from a slide projector. In the experiments described here, the range of acceptable activity of chloroplast preparations is 100\u2013120\u00a0\u03bcmol\u00a0h\u22121\u00a0mg\u22121 Chl. Light measurements were made using a Hansatech Quantum Sensor 3\/2897 (Hansatech Instruments, King\u2019s Lynn, Norfolk PE321, JL.UK).\nThe intactness of chloroplasts was measured by the ferricyanide reduction method (Lilley et\u00a0al. 1975).\nIsotopic tracer studies with isolated chloroplasts\nExperiments were carried out in the oxygen electrode (unless stated otherwise) by the procedure described for the assay of chloroplast activity. NaH14CO3 (100\u00a0\u03bcCi) was introduced when the rate of CO2-dependent oxygen evolution reached a linear rate. The reaction was terminated by rapid transfer of the mixture to boiling ethanol (80% w\/v). The insoluble material was removed by centrifugation and washed once with a small volume of water. The ethanol was evaporated from the combined supernatant solutions in a rotary evaporator. After passage through a cation-exchange column (Bartlett et\u00a0al. 1989), the sugar phosphates were separated and isolated by anion-exchange column chromatography and further purified by two-dimensional TLC or paper chromatography (Kapuscinski et\u00a0al. 1985).\nThe isolation of stromal metabolites used the method of Heldt (1980) which relied on rapid separation of intact chloroplasts from a reaction medium using the following silicon oil method. Fifty\u00a0\u03bcl of 10\u00a0M formic acid and 50\u00a0\u03bcl of silicone oil mixture (AR100 and AR150 mixed in a ratio of 3:1) were placed in a 400-\u03bcl microfuge tube followed by 200\u00a0\u03bcl of a chloroplast suspension that had been preincubated with 32P-orthophosphate (specific radioactivity 50\u00a0mCi\u00a0mmol\u22121). The extracts of the pellets containing the aqueous fractions that were pooled from four tubes were lyophilized and the sugar phosphates therein were isolated by anion-exchange column chromatography as described in Methods. Sugar phosphate peaks isolated in the column eluates were identified and quantitatively measured using the specific enzymatic and colourimetric procedures of Williams et\u00a0al. (1978b) and Kapuscinski et\u00a0al. (1985).\nPreparation of stromal enzyme extracts (SEP) from spinach chloroplasts\nThe soluble (stromal) enzyme fraction was prepared essentially as described by Furbank and Lilley (1981) for pea chloroplasts. Intact spinach chloroplasts were isolated with assayed rates of CO2-dependent O2 evolution in excess of 100\u00a0\u03bcmol\u00a0h\u22121\u00a0mg\u22121 Chl. Typically a chloroplast suspension equivalent to 2\u00a0mg of Chl was pelleted by centrifugation at 1,000\u00a0\u00d7 g for 1\u00a0min in an SS34 Sorvall rotor at 4\u00b0C. The supernatant solution was removed and 10\u00a0ml of chilled 10\u00a0mM HEPES buffer, pH\u00a08.0 containing 3\u00a0mM DTT was added. The pellet was resuspended by gentle shaking. The centrifuge tube was kept on ice for approximately 10\u00a0min after which it was centrifuged for 15\u00a0min at 9,000\u00a0\u00d7 g in a refrigerated Sorvall centrifuge RC-5 to separate the soluble proteins from the membrane fraction. The clear supernatant fluid, representing the stromal fraction of the chloroplasts, was concentrated to 4\u00a0ml in an Amicon ultrafiltration cell, using a PM10 membrane (nominal MW cut-off 10,000). Finally, dialysis was carried out against 3\u00a0\u00d7 500\u00a0ml of dialysis buffer containing 10\u00a0mM HEPES\u2013KOH (pH\u00a08.0), 3\u00a0mM DTT, 15\u00a0mM MgCl2 and 10\u00a0mM KCl. This last step was completed in 4\u00a0h. The pelleted membranes were retained for Chl analysis. The activity of the extracts was stable for 4\u20136\u00a0h from completion of the dialysis step. Some experiments used an alternative stromal isolation procedure based on the method of Kaiser and Bassham (1979). The chloroplast suspension was pelleted as described above but lysis of organelles was achieved with 25\u00a0mM HEPES\u2013NaOH, 2\u00a0mM MgCl2, 1\u00a0mM EDTA (pH\u00a07.6) and the same buffer was used for dialysis.\nAssay of the reductive PP using stromal enzyme preparation (SEP)\nThe activity of the reductive PP in the stromal extracts was determined by the method of Furbank and Lilley (1981). The progress of the reaction was followed spectrophotometrically using 0.2-cm light path cuvettes. The assay mixture contained 40\u00a0mM HEPES\u2013KOH buffer (pH\u00a08.0), 20\u00a0mM MgCl2, 8\u00a0mM KCl, 1\u00a0mM NADH, 0.8\u00a0mM EDTA, 10\u00a0mM DTT, 4\u00a0mM ATP, 10\u00a0mM creatine phosphate, 10\u00a0mM NaHCO3, 2 units of creatine kinase (EC 2.7.3.2), 1.25 units of glyceraldehyde-phosphate dehydrogenase (EC 1.2.1.12), 1.7 units of phosphoglycerate kinase (EC 2.7.2.3). SEP equivalent to 5\u201350\u00a0\u03bcg of Chl was added to make a final volume of 500\u00a0\u03bcl. SEP was preincubated for 5\u00a0min at 25\u00b0C in 20\u00a0mM MgCl2, 10\u00a0mM DTT, 10\u00a0mM NaHCO3 before addition of the test sugar phosphate substrate. Usually the following test substrates were individually used at 2\u00a0mM concentration in the reaction mixture: Rib 5-P, dihydroxyacetonephosphate (DHAP), or Seh 1,7-P2.\nMaximum catalytic capacity of the non-oxidative segment of the PP\nThis was determined by following the conversion of ribose 5-P to hexose 6-P in a coupled enzyme assay as described by Williams et\u00a0al. (1984). The reaction mixture contained 80\u00a0mM TEA\u2013HCl buffer, pH\u00a07.4, 0.4\u00a0mM NADP+, 10\u00a0mM MgCl2, 1.5 units of phosphoglucose isomerase (EC 5.3.1.9) (PGI), 2 units of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) (Glc 6-PDH), 0.4\u00a0mM ribose 5-P and stromal extract equivalent to 50\u00a0\u03bcg of Chl in a total volume of 1.0\u00a0ml.\nSynthesis of isotopically labelled compounds\n[U-14C] Arabinose 5-phosphate, [8-14C] d-g-d-a-oct 8-P and [8-14C] d-g-d-i-oct 8-P were prepared by the method of Arora et\u00a0al. (1987). [1-32P]-Sedoheptulose 1,7-bisphosphate was prepared by phosphorylation of sedoheptulose 7-P (altro-heptulose) with [\u03b3-32P] ATP using rabbit muscle phosphofructokinase (EC 2.7.1.11) (PFK). The reaction mixture (0.50\u00a0ml) contained: 20\u00a0\u03bcmol Tris\u2013HCl buffer (pH\u00a07.6), 2.5\u00a0\u03bcmol of MgCl2, 0.2\u00a0\u03bcmol of [\u03b3-32P] ATP (100\u00a0\u03bcCi), 2.5\u00a0\u03bcmol of Seh 7-P and 6 units of PFK. The mixture was incubated at 25\u00b0C and was 95% complete in 1\u00a0h. The title compound was purified using formate-anion exchange chromatography and TLC as described above and was shown by enzymatic analysis to be free of Fru 1,6-P2. Unlabelled d-glycero-d-ido- and d-glycero-d-altro-octulose 8-P and 1,8-P2 esters were prepared as described in Kapuscinski et\u00a0al. (1985).\nMeasurements of phosphotransferase activity\nArora et\u00a0al. (1985) published three assay procedures for the minimum estimate of the activity of d-glycero-d-ido-octulose 1,8-P2 or d-glycero-d-altro-octulose 1,8-P2: d-altro-heptulose 7-phosphotransferase (PT). Two of these assays measured the reaction for the non-oxidative PP (Fig.\u00a01) in the direction of octulose 8-P formation. The third procedure, and the one detailed here, measured the activity in the flux direction of the RPP (see Fig.\u00a02 and Eqs. (1) and (2)).\nOnly the third method was effective when SEP was the crude source of the putative enzyme activity. It is a radiochromatographic discontinuous-stop assay and is applied as follows. The reaction mixture in a volume of 0.50\u00a0ml contained 1\u00a0\u03bcmol of [8-14C]-octulose 8-P (0.8\u00a0\u03bcCi\u00a0\u03bcmol\u22121), 3\u00a0\u03bcmol Seh 1,7-P2, 20\u00a0\u03bcmol of TEA\u2013HCl buffer (pH\u00a07.4) and 16\u00a0\u03bcmol of KCl. The mixture was incubated for 30\u00a0min at 30\u00b0C and was initiated by the addition of SEP equivalent to 50\u00a0\u03bcg of Chl. Aliquots (0.1\u00a0ml) were sampled at 5.0\u00a0min intervals during the time-course of the reaction and injected into an equal volume of 6% perchloric acid. Denatured protein was removed by centrifugation, the pH of the supernatant fluid adjusted to 6.8 with KOH and precipitated KClO4 separated by centrifugation. The activity of the enzyme was estimated by separating the labelled octulose mono- and bis-phosphates by ion-exchange chromatography as follows: A precisely known volume of the above supernatant fraction was applied onto a column (5\u00a0mm\u00a0\u00d7\u00a05\u00a0mm) of ion-exchange resin (AG 1 X8, 200\u2013400 mesh, formate form) followed by 2\u00a0ml of deionized water. The sugar phosphates were eluted by successive washes with 2\u00a0M HCOOH (approx. 12\u00a0ml), which quantitatively removed monophosphate esters, and 5\u00a0ml of 4\u00a0M HCOOH\u20131\u00a0M HCOONH4 solution which eluted sugar bisphosphates. Fractions (2\u00a0ml) of the eluted sugar phosphates at each time point were analysed for radioactivity and the amount of octulose bisphosphate formed was calculated from the integrated peaks of radioactivity. Confirmation of the identity and amount of radioactivity in the 14C-labelled octulose phosphates used two-dimensional radiochromatography of each of the above fractions and the GW3\/GW3-phenylboronate solvents (Kapuscinski et\u00a0al. 1985). Control experiments using only a single substrate accompanied each activity measurement.\nArabinose 5-P activity with SEP\nThe reaction mixture (2.0\u00a0ml) containing 80\u00a0\u03bcmol TEA\u2013HCl buffer (pH\u00a07.4), 3.6\u00a0\u03bcmol Rib 5-P, 0.4\u00a0\u03bcmol of [U-14C]-Ara 5-P (0.2\u00a0\u03bcCi) and SEP equivalent to 200\u00a0\u03bcg of Chl was incubated for 1\u00a0h at 30\u00b0C. The reaction was terminated by heating and all sugar phosphates were isolated with greater than 90% recovery of 14C isotope. An identical control reaction using boiled SEP accompanied all incubations. Processed samples were chromatographed on paper using either the GW3 or GW3\u20132% PBA solvents and the chromatograms exposed to X-ray film (Kodak X-O mat RP), (Bleakley et\u00a0al. 1984).\nPositional isotopic analysis of sugar phosphates\nFollowing PS experiments with leaves or chloroplasts, 14C-labelled Glc 6-P, Rib 5-P, d-g-d-i-oct 1,8-P2 and PGA were isolated, resolved by ion-exchange chromatography and purified by two-dimensional TLC or paper chromatography. The fraction containing Glc 6-P was concentrated and chromatographed twice on paper (3MM, Whatman) using the GW3 solvent of Wood (1968) followed by solvent A of Bandurski and Axelrod (1951). The 14C-labelled Glc 6-P was located by autoradiography and comparison with a stained standard marker compound (Rosenberg 1959) and was then eluted from the paper with deionized water. The above regime ensured that the isolated 14C-labelled Glc 6-P was free of any other hexose phosphates. The phosphate group of Glc 6-P was removed by enzymatic hydrolysis as follows: Solid Glc 6-P was dissolved in 5\u00a0ml of 50\u00a0mM glycine\u2013NaOH buffer, pH\u00a010.4 and 5 units of Alkaline phosphatase (EC 3.1.3.1) from Escherichia coli was added and the mixture incubated at 37\u00b0C. The progress of the reaction was monitored by assaying the release of inorganic phosphate using the method of Tashima and Yoshimura (1975).\nWhen the reaction was completed the mixture was poured into a suspension of mixed-bed resin (1\u00a0meq of Dowex 50W, H+ form and 1\u00a0meq of Bio AG1 x\u00a04, HCO3\u2212 form) which was stirred for approx. 60\u00a0min. The solution was filtered through a sintered glass funnel (porosity 3) and the residual cake of resin thoroughly washed with deionized water. The 14C-labelled glucose filtrate was freeze-dried and stored at 4\u00b0C until used. The percentage distribution of 14C isotope in the individual carbons of glucose was determined as 14CO2 using a combination of microbiological and chemical methods (Williams et\u00a0al. 1971).\n14C-Labelled octulose phosphates from spinach leaves or chloroplast extracts were isolated and purified as above and degraded using the methods of Williams et\u00a0al. (1985). Following isolation by anion-exchange and TLC chromatography, 14C-labelled Rib- and Ara 5-phosphates were dephosphorylated (Williams et\u00a0al. 1984) and Ara and Rib were chemically degraded using the periodate method (Genovese et\u00a0al. 1970). [14C]-3-Phosphoglyceric acid derived from DHAP was chemically degraded by the procedure of Andrews et\u00a0al. (1965).\nRecovery of 14CO2 as BaCO3 and measurement of radioactivity\nDiscs (2\u00a0cm) of Whatman No. 42 filter paper were used to collect barium carbonate precipitates. The discs were washed with water, absolute ethanol and diethyl ether, dried and weighed before use. BaCO3 was deposited on the papers using gentle suction, then washed with CO2-free water, alcohol and ether. The discs were dried under vacuum over silica and equilibrated at room temperature before weighing on a semi-micro balance. Approx. 20\u00a0mg of dried BaCO3 was exactly weighed, placed into a vial with 10\u00a0ml of scintillation cocktail and counted for radioactivity as a suspension in the \u201cCab-O-Sil\u201d (Godfrey L. Cabot Inc.) scintillant of Cluley (1962).\nReplication and statistical analysis\nData presented are the mean values (\u00b1S.D.) from results of five or more experiments or mean values (\u00b1P.E.) (Probable Error) for all experiments conducted on different days.\nResults and discussion\nGLC analysis of perchloric acid extracts of spinach leaves\nPrincipal aims of the experiments were an investigation of the occurrence, identity and quantitative levels of the ido- and altro-octuloses in fresh photosynthetic tissue and the assessment of their hypothesized functions (especially that of d-g-d-i-oct 1,8-P2) in PS. It was noted that Charlson and Richtmyer (1959) and Begbie and Richtmyer (1966) had isolated and characterized octulose from avocado and Primula officinalis, respectively. In a remarkable finding Howarth et\u00a0al. (1996) showed that d-glycero-d-ido-octulose represented 90% of the total carbohydrates in fully hydrated leaves and 50% of dried leaf matter of the resurrection plant Craterostigma plantagineum. Heath (1984) also showed evidence for the L-type, but not the F-type, PP in Chlorella sorokiniana. Since the mono- and bisphosphate esters of d-g-d-i-oct are a unique feature of the L-type PP (Fig.\u00a01), the need to identify and measure these esters in fresh spinach leaf and chloroplasts is obvious. Different methods were used to establish whether d-glycero-d-ido-octulose 1,8-P2 occurred in spinach leaves. The first approach used the simple and sensitive GLC method. A gas chromatogram of the pertrimethylsilyl (TMSi) derivatives of the dephosphorylated sugar phosphates extracted from spinach leaves is shown in Fig.\u00a03.Fig.\u00a03Gas liquid chromatogram of dephosphorylated TMSi-derivatized sugars extracted from young spinach leaves during light-driven carbon fixation. Octuloses and arabinose feature in the chromatogram. For details see Materials and methods section\nThe high molecular weight region was well resolved and allowed tentative identification (by retention times and co-chromatography with authentic compounds) of d-g-d-i-octulose and its 5\u2032-altro-epimer, d-g-d-a-oct. Estimation of the amounts of these compounds from the known response factors of the gas chromatograph gave values of 11.8\u00a0\u00b1\u00a00.98\u00a0nmol\u00a0mg\u22121 Chl for ido-octulose and 18.2\u00a0\u00b1\u00a00.78\u00a0nmol\u00a0mg\u22121 Chl for altro-octulose. These values are tentative because other compounds may co-elute with the octuloses, e.g. the TMS ethers of heptitols have similar retention times to those of eight carbon sugars. In addition, the GLC method used in this study cannot differentiate between free and phosphorylated sugars. Different approaches were therefore adopted to determine the levels of specifically phosphorylated octuloses present in photosynthesizing tissue.\n14C-d-g-d-i-oct 1,8-P2 isolation from spinach leaves: 14CO2 fixation and pulse\u2013chase studies\nThe second approach used the combination of formate ion-exchange chromatography followed by phenylboronic acid and two-dimensional TLC, to achieve isolation, identification and complete separation of d-g-d-i-oct 1,8-P2 from all other sugar phosphates (see Methods). This methodology both confirmed and extended the GLC findings and unequivocally demonstrated that d-g-d-i-oct 1,8-P2 occurred in spinach leaf extracts.\nThe conditions adopted for the study of the time-course of 14CO2 fixation into the products of PS involved an exact application of the method of Hatch and Slack (1966). This provided steady-state conditions for PS in intact spinach leaves at a CO2 concentration of 470\u00a0ppm that followed from a 50\u00a0ml injection of 0.80\u00a0mCi of 14CO2 and light intensity of 50% direct sunlight (1,200\u00a0\u03bcE\u00a0m\u22122\u00a0s\u22121). The rate of 14CO2 incorporation into the soluble leaf fraction (an index of the total 14CO2 fixed) was approximately linear (1.9\u00a0\u00d7 106\u00a0cpm\u00a0s\u22121\u00a0mg\u22121 Chl) during a 120-s interval (Fig.\u00a04, panel A). The 14C labelling of PGA was marginally more rapid and extensive than that of Glc 6-P which reached 14C isotope saturation in the interval 75\u2013120\u00a0s.Fig.\u00a04Shows the kinetics of 14C isotope incorporation from 14CO2 into a selection of sugar phosphates in whole spinach leaves during light-driven PS for 120\u00a0s. The time course of the total 14C fixed in the soluble fraction of the leaf extract is also shown. Labelled sugar phosphates were resolved by anion-exchange chromatography and were then individually isolated and further purified by two-dimensional paper or TLC chromatography as described in the Materials and methods Section. The 14C-radioactivity incorporated in glucose 6-P and PGA (panel A) was an order of magnitude greater than that incorporated in the sugar phosphates shown in panel B. Panel A: Total 14C isotope fixed, \u2022; radioactivity incorporated in Glc 6-P, \u25a0 and PGA, \u25b4. Panel B. Rib 5-P, \u25a1; other pentose 5-P, \u25cb; Seh 1,7-P2, \u0394 and d-g-d-i-oct 1,8-P2, \u25ca\nGlc 6-P was labelled at an approx rate of 2.8\u00a0\u00d7\u00a0105\u00a0cpm\u00a0s\u22121\u00a0mg\u22121 Chl (Fig.\u00a04, Panel A). The radioactivity in PGA and Glc 6-P was an order of magnitude greater than that fixed in all other sugar phosphates investigated (see Fig.\u00a04, panel B). The fast rate of 14C incorporation into Rib 5-P was initially inclined to linear for 75\u00a0s (2.0\u00a0\u00d7\u00a0104\u00a0cpm\u00a0\u22121\u00a0mg\u22121 Chl). The bisphosphates Seh 1,7-P2 and d-g-d-i-oct 1,8-P2 were more slowly labelled at 7.3\u00a0\u00d7\u00a0103 and 2.4\u00a0\u00d7\u00a0103\u00a0cpm\u00a0s\u22121\u00a0mg\u22121 Chl, respectively. The kinetics and relative degree of 14C isotope incorporation into individual PS intermediates shown at Fig.\u00a04 are very similar to the results for 14CO2 incorporation in Scenedesmus (Benson et\u00a0al. 1952). Moreover, d-g-d-i-oct 1,8-P2 was labelled early and was saturated with 14C isotope at 75\u00a0s, at which point its concentration in the leaf was 1.6\u00a0nmol\u00a0mg\u22121 Chl and the rate of label incorporation was 0.13% of that fixed in the soluble fraction. When the above experiments were conducted in the dark the distribution of 14C in the leaf soluble fraction was uneven and at 120\u00a0s was only 0.01% of that measured in the light. None of the sugar phosphates shown at the A and B panels of Fig.\u00a04 were dark labelled.\nHatch and Slack (1966) described an apparatus for measurements of the kinetics of 14CO2 fixation and 12CO2 exchange by pulse\u2013chase in bulk samples of intact leaves undergoing PS. Their method was able to delineate Calvin Cycle intermediates, which rapidly exchanged 14C for 12C during and following a short pulse with 12CO2, thereby identifying rapidly cycling compounds. These are clearly distinguished by their delabelling kinetics (Figs.\u00a05 and 6) from the cytoplasmic storage compounds sucrose, starch and cationic compounds that retained or increased their levels of 14CO2 following the pulse phase (Fig.\u00a05).Fig.\u00a05Shows the results of pulse\u2013chase experiments where spinach leaves were confined in 14CO2 for 20\u00a0s (the pulse) and were then exposed to non-radioactive air (the chase). See methods. The figure shows the time-course of the partition of 14C isotope into various fractional groups of compounds in spinach leaves following their exposure to the \u2018chase\u2019 with unlabelled CO2 for 240\u00a0s and illuminated to the equivalent of 50% direct sunlight. It is of note that starch and cationic compounds retained and continued to show increasing 14C radioactivity while the radioactivity in the total soluble fraction and the pool of anionic compounds declined. The time-course of changes in 14C radioactivity are shown for starch, \u2022; neutral and anionic compounds, \u25a0; the cationic fraction, \u25a1 and total soluble radioactivity, \u0394Fig.\u00a06Shows the change in the 14C-radioactivity of individual sugar phosphates and sucrose in spinach leaves during the time-course of the \u2018chase\u2019 with unlabelled CO2. Sugar phosphates from leaves that had been subjected to the \u2018pulse-chase\u2019 experiments (See Fig.\u00a04b results) were separated by anion-exchange chromatography and individually purified by two dimensional TLC or paper chromatography. See Materials and methods for details. Panel A shows the kinetics over 240\u00a0s of the decline in radioactivity of Rib 5-P, \u25a0; Fru 1,6-P2, \u25b4; DHAP, \u25a1; and d-g-d-i-oct 1,8-P2, \u2022. Panel B shows the time-course over 80\u00a0s of the decline in radioactivity of PGA, \u25b4; Glc 6-P, \u25a1; Fru 6-P, \u25cb; and the initial increase in radioactivity in sucrose, \u2022\nTaken together the results of Figs.\u00a04 and 6 showed that d-g-d-i-oct 1,8-P2 was rapidly labelled and saturated with isotope during 14CO2 fixation. It was delabelled on exposure to 12CO2 during a comparative study which showed that it exhibited the same kinetics of 14C loss as other cycling bisphosphate intermediates of the Calvin Pathway. It was therefore unambiguously distinguished from slow 14C-release carbon storage and other cytoplasmic end products of PS by the above kinetics.\nd-glycero-d-ido-octulose 1,8-P2 in spinach chloroplasts\nThe above results suggested that d-g-d-i-oct 1,8-P2 may have a chloroplast origin. In order to test this proposal, PS experiments were carried out with isolated spinach chloroplasts. If d-g-d-i-oct 1,8-P2 is synthesized by enzymes of the reductive PP, it should become rapidly labelled following the metabolism of 32P-inorganic phosphate (Pi) or 14C-bicarbonate. It was noted above that d-g-d-i-oct 1,8-P2 incorporated 14C isotope during PS by isolated chloroplasts in the presence of NaH14CO3 (see Methods). These observations were further tested using 32P-orthophosphate as the isotopic marker (see Methods section) in order to investigate whether d-g-d-i-oct 1,8-P2 was exclusively present in the organelles and was not the result of external reactions catalysed by enzymes released from any ruptured chloroplasts. Intact chloroplasts were rapidly filtered through a layer of silicone oil (Heldt 1980) and the 32P-labelled metabolites were separated by ion-exchange chromatography (see Methods section). The unambiguous identification of the [32P]-d-g-d-i-oct 1,8 P2 was made by rechromatographing the recovered octulose phosphate fractions on paper using the GW3 and GW3\/PBA solvents. From the known specific radioactivity of inorganic phosphate in the reaction mixture and the absolute amount of 32P-isotope in the purified d-g-d-i-oct 1,8-P2, the concentration of the sugar phosphate in the stroma was estimated.\nUsing the value of the sorbitol impermeable space of 26\u00a0\u03bcl\u00a0mg\u22121 Chl (Schafer et\u00a0al. 1977), the concentration of d-g-d-i-oct 1,8-P2 was estimated to be 1.3\u00a0nmol\u00a0mg\u22121 Chl \u00b10.50 (50\u00a0\u03bcM, three determinations). This value is less than that measured for ido-oct in the whole leaf PS study, but is in the same concentration range as that of some other Calvin Cycle metabolites in spinach chloroplasts (Portis et\u00a0al. 1977; Heldt et\u00a0al. 1978, 1980; Stitt et\u00a0al. 1980; Petterson and Ryde-Petterson 1988). Measurement of the amount of the altro-epimer of the octulose bisphosphate in chloroplasts was not made in this study (see the accompanying paper for the results of a mass spectrometric study of 13C labelled sugar phosphates formed in spinach chloroplasts during PS). A carry-through of the external medium may affect the estimation of the stromal volume as a sorbitol impermeable space and consequently the determination of the molar concentration of metabolites contained in the stroma. However, the amount of d-g-d-i-oct 1,8-P2 in the stroma is quoted per mg of Chl and is thus independent of the volume of external medium that may be carried through silicon oil with the organelles. The results of control experiments, using 14CO2, showed that following centrifugation, the external medium contained less than 10% of the 14C-labelled d-g-d-i-oct 1,8-P2 found in the pelleted chloroplast fraction. Thus it is concluded that d-g-d-i-oct 1,8-P2 is probably synthesized and may react exclusively in the stromal compartment of spinach chloroplasts. Its concentration is estimated to be 50\u00a0\u03bcM using the above data and the following equation:\nOrigin of d-g-d-i-oct 1,8-P2 and distributions of 14C isotope in a selection of sugar phosphates isolated from spinach leaves and chloroplasts assimilating 14CO2 in photosynthesis\nThe most likely biosynthetic routes leading to d-g-d-i-oct phosphate formation involve catalysis by the carbon\u2013carbon cleavage and group-transferring enzymes of sugar phosphate metabolism, namely transketolase (Eq. (3)), aldolase (Eq. (4)) and transaldolase (Eq. (5)).\nThe short-term PS experiments described in the Methods and Results sections were used to identify which of the above three enzymes may have operated for the synthesis of d-g-d-i-oct phosphate in spinach leaves and chloroplasts. However, it is of note that the initial hypothesis (Fig.\u00a02) for the modified reductive PP (Clark et\u00a0al. 1974; Williams et\u00a0al. 1987), proposed that d-g-d-i-oct-8-P was formed by the reaction of (Eq. (3)) and d-g-d-i-oct-1,8-P2 by the PT reaction of (Eq. (6)) (Arora et\u00a0al. 1985).\nFigure\u00a02 shows that in one cycle of 14CO2 fixation, d-g-d-i-oct 1,8-P2 is labelled in carbons 5 and 6. In order to test this aspect of the mechanism of the pathway, the distributions of 14C isotope into the individual carbons of Glc-6P, phosphoglyceric acid (PGA), Rib-5-P and d-g-d-i-oct 1,8-P2 were determined in samples isolated from spinach leaves and chloroplasts during light-driven PS in 14CO2. The procedures used (see Methods section) allowed each of the carbons of Glc 6-P and PGA to be isolated for the determination of the degree of positional carbon isotopic labelling. However, only carbons 1, 2, 3, 4 and 8 of d-g-d-i-oct 1,8-P2 were able to be individually isolated for 14C measurement and carbons 5, 6 and 7 were collectively measured as a group (Williams et\u00a0al. 1985). Only the 14C isotope levels of carbons 1 and 5 of Rib-5-P were uniquely determined by the methods applied in this study, with carbons 2, 3 and 4 being estimated as a group 14C-measurement. The data of Table\u00a01 show the results of these experiments using spinach leaves photosynthetically assimilating 14CO2 for 10, 45, 75 and 120\u00a0s, respectively (see Methods section). The data of Table\u00a02 show similar 14C isotope distributions in PGA, Glc 6-P and d-g-d-i-oct 1,8-P2 isolated from intact spinach chloroplasts after 60\u00a0s of PS in 14CO2.Table\u00a01Distribution of 14C isotope in sugar phosphates isolated from spinach leaves following specified intervals of PS in 14CO2aCarbon10\u00a0s of 14CO2 fixation45\u00a0s75\u00a0s120\u00a0sGlc 6-Pd-g-d-i-oct 1,8-P2Rib 5-PPGAGlc 6-Pd-g-d-i-oct 1,8-P2Glc 6-Pd-g-d-i-oct 1,8-P2Glc 6-P\u00a0\u00a0FoundPredicted\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a013.235.6(33.3)23.484.912.720.4(38.7)15.129.9(37.3)18.523.225.3(33.3)}7.27.421.1(22.6)10.330.6(25.4)11.8337.239.1(33.3)71.67.924.658.5(38.7)22.039.5(37.3)17.2450.521.2(3.1)38.113.1(8.5)33.413.3(12.1)25.052.5}}5.07.0}}9.1}}10.763.4(60.7)(93.2)10.279.2(79.8)10.163.7(76.0)17.17818.1(3.5)7.7(11.7)23.0(11.9)% recovery of 14C-isotope8891b76c8610110682b75c9385b89c90aThe conduct of the experiments is described in the Methods section. Sugar phosphates were isolated and purified by ion-exchange and paper chromatographic procedures. Results are mean values of duplicate degradations except for all of the compounds isolated after 10\u00a0s of 14CO2 fixation, which were degraded in triplicate. b, c% 14C recovered in the top 3 and bottom 5 carbons of d-g-d-i-oct 1,8-P2Table\u00a02Distribution of 14C isotope in sugar phosphates isolated from intact spinach chloroplasts following carbon fixation by light-driven PSaCarbonPercent specific radioactivity40\u00a0s60\u00a0sPGADHAPGlc 6-PPGAGlc 6-Pd-g-d-i-oct 1,8-P2\u00a0Found\u00a0(Theory)155.159.412.546.613.656.7(32.5)218.417.27.329.214.428.4(35.0)326.523.423.024.218.314.9(32.5)439.133.618.4(17.0)58.59.2}}69.610.964.0(70.0)7817.6(13.0)% 14C recovered9810189979588b, 94ca\u00a0The 14C-labelling experiments were carried in an oxygen electrode apparatus as described in the Methods section. Illuminated chloroplast suspensions (100\u00a0\u03bcg\u00a0Chl\u00a0ml\u22121) were incubated with 10\u00a0mM NaHCO3 until a linear rate of CO2-dependent O2 evolution was attained. One hundred\u00a0\u03bcCi of NaH14CO3 was added to adjust the specific radioactivity of the substrate to 5\u00a0\u03bcCi\u00a0\u03bcmol\u22121. The subsequent steps are detailed in the Methods section. The specific radioactivity of individual carbons of sugar phosphates are the mean value of triplicate determinations. b,c The percent recovery of 14C isotope from the top 3 and bottom 5 carbon atoms of d-g-d-i-oct 1,8-P2, respectively. The method for calculating the theoritical bracketed values in Tables 1 and 2 is given in the Appendix\nDegradative data for PGA, DHAP and Glc 6-P following 40\u00a0s of PS are also listed. Both sets of results implicate a TK-catalysed reaction (Eq. (3)) as the most likely route for the biosynthesis of d-g-d-i-oct 8-P leading to the bisphosphate by the reaction of Eq. (6).\nIn order to compare the above TK path of synthesis with the 14C distribution pattern that may be imposed by the reactions of Eqs. (4) and (5), catalysed by either Aldolase (ALD) or TA, theoretical distributions of 14C isotope in d-g-d-i-oct 1,8-P2 are also presented as bracketed values in Tables\u00a01 and 2. The Appendix shows the method for calculating the theoretical values. These distributions were calculated using the experimentally measured isotope levels in Glc 6-P (Table\u00a01) and an assumption that Fru 6-P formed in the reductive pathway is in chemical and isotopic equilibrium with Glc 6-P formed by the action of PGI. It is also assumed that the 14C distributions in the mono- and bisphosphates of the d-g-d-i-octuloses of Eqs. (3) and (6) are the same.\nEquation (3a) shows detail of the spread of 14C in each carbon of d-g-d-i-oct 8-P at 10\u00a0s (see data Table\u00a01) using the mean values of triplicate estimations of 14C isotope in each of the six carbons of Glc 6-P and the assumptions described above. The individual distributions measured in the top three carbons of the d-g-d-i-oct 1,8-P2 sampled at all times support the proposal that d-g-d-i-oct 8-P in spinach leaves (Table\u00a01) and chloroplasts (Table\u00a02) may be formed by the TK reaction of Eq. (3). However, the leaf concentrations of 14C in C-4 and C-8 of d-g-d-i-oct 1,8-P2 in the 45-s and 75-s samples and the 60-s chloroplast sample are greater than expected from contributions of C-1 and C-6 of hexose phosphates to these carbons of oct 8-P formed by the reaction of Eq. (3). The level of 14C in position 8 of some ido-octulose 1,8-P2 samples was significant and may be the result of a second cycle of 14C labelling or action by a phenomenon that was first noted by Van Sumere and Shu (1957). Their results showed an inversion of 14C-labelling of [1-14C] Rib and production of [5-14C]-Xlu via the intermediacy of 14C-d-arabinitol formed by d-arabinitol: NAD+ 4-oxidoreductase (EC 1.1.1.11). Rephosphorylation and isomerization to form the isotopically inverted aldopentose 5-P and its Aldx catalysed exchange with d-g-d-i-oct 1,8-P2 will specifically label position 8. Contamination, isolation, recovery and analytical errors may be ruled out since authentically labelled d-g-d-i-oct 1,8-P2 samples subject to the above procedures showed that the error in 14C analysis of C-8 was <\u00a08%. However, it is important to note that neither the results of the Calvin group, the reactions of the Calvin pathway, the scheme of Fig.\u00a02, nor the results of Table\u00a01 show pentose phosphates labelled at positions other than carbons 1, 2 and 3. The scrambling of 14C into carbons 2 and 3 of 3-PGA (see Table\u00a02 for 60-s chloroplast data) by recycling in the path of carbon in PS is predicted to lead to some enrichment of 14C isotope into positions 5 and 6 of d-g-d-i-oct 1,8-P2 (via the prior labelling of Fru 6-P from PGA). However, only heavily labelled [1-14C] PGA was found after 10\u00a0s PS in spinach leaves (Table\u00a01). After 120\u00a0s of 14CO2 exposure there was an almost uniform 14C distribution in all carbons of Glc 6-P consistent with extensive recycling of the isotope in the carbon path of PS.\nThe results of the partial degradations of d-g-d-i-oct 1,8-P2 samples do not support exclusive paths of synthesis by the reactions of Eqs. (4) and (5), since both of these reactions will very heavily label carbon 3 of octulose phosphate. Because [1-14C]-PGA is the earliest and most heavily labelled intermediate detected during 14C fixation in C-3 plant PS and is the precursor of [1-14C]-DHAP, then it follows that the aldolase reaction of Eq. (4) forms a predominantly labelled [3-14C]-d-g-d-i-oct 1,8-P2. The TA reaction of Eq. (5) uses Fru-6-P which will be most heavily, but unequally, labelled (Gibbs Effect) in positions 3 and 4 in short time-course experiments (Tables\u00a01 and 2). Thus the transfer of the [3-14C]-DHA-TA moiety from [3,4-14C]-Fru 6-P to Ara 5-P in the reaction of Eq. (5) forms d-g-d-i-oct 8-P which would have C-3 notably labelled. These predictions cannot be made in longer term experiments (much beyond 30\u00a0s in leaves) because of the PS-induced spread of 14C label in PGA and hexose 6-P (Tables\u00a01 and 2). The most supportive evidence for the TK reaction in the synthesis of d-g-d-i-oct phosphate is best shown in the 10\u00a0s fixation in spinach leaves and 60\u00a0s in spinach chloroplasts. It is of note that the inability of the degradative procedures (see Methods section) to provide quantitative individual 14C distributions for all eight carbons of the octulose phosphates was addressed using the mass spectrometric method (MacLeod et\u00a0al. 2001), the results of which are reported in the accompanying paper. The above quantitative 14C- labelling of positions 5 and 6 of d-g-d-i-oct 1,8-P2 was qualitatively confirmed by Bartlett et\u00a0al. (1989), who used two-dimensional NMR Spectrometry and 60\u00a0s of 13CO2 fixation by PS with intact spinach leaves (Hatch and Slack 1966) to demonstrate formation of [5,6-13C]-d-g-d-i-oct 1,8-P2. Their results also included the first demonstration of Aldx catalysis by showing that the [5,6-13C]-d-g-d-i-oct 1,8-P2 and [1,2,3-13C]-Rib 5-P, both products of PS, interconverted with [4,5,6-13C]-d-g-d-a-oct 1,8-P2 and [2,3-13C]-Ara 5-P.\nWhile the above results and the scheme of Fig.\u00a02 support the proposal that the TK reaction leading to Oct 8-P involved Glc and Fru 6-phosphates, it should be noted that TK can accept various other substrates (Williams et\u00a0al. 1987). Using the theoretical analysis method dealt with earlier in this section, it can be shown that Seh 7-P serving as a two-carbon fragment donor will give the same labelling result as that imposed by Fru 6-P. On the other hand, reaction between Glc 6-P and Xlu 5-P results in a different pattern, with C-1 and C-2 of the resulting d-g-d-i-oct 8-P having much heavier labelling than C-3 and thus this reaction possibility is excluded. Finally, another potential TK donor substrate is \u03b2-hydroxypyruvate. Daley and Bidwell (1977) showed that in its phosphorylated form, 3-phosphohydroxypyruvate together with phosphoserine, accounted for a substantial portion (35%) of the 14C fixed by Phaseolus vulgaris leaves during the first minute of exposure to 14CO2 in PS. If P-hydroxypyruvate is synthesized by a carboxylation process, as the above authors imply, and is converted to hydroxypyruvate, then reaction of the latter with Glc 6-P would produce d-g-d-i-oct 8-P labelled in the same fashion as that derived from Glc 6-P and Fru 6-P, since the distribution of 14C in hydroxypyruvate resembles that in carbons 1\u20133 in hexose monophosphate. However, lack of data prevents our further speculation on the possible role of hydroxypyruvate as a path-of-carbon intermediate in the synthesis of octulose phosphates in spinach during PS.\nThis section on octulose phosphate synthesis and reactivity in PS would be incomplete without mention of the 5\u2032-epimerization of octulose phosphates (Williams et\u00a0al. 1978a). In such a mechanism d-g-d-a-oct 8-P or the corresponding bisphosphate may be reversibly generated from the ido-epimeric phosphate esters. The manner of this epimerization and its significance in the proposed modification of the path of carbon in PS is treated in the last section of this Discussion.\nStromal enzyme preparation and photosynthesis\nIn 1981, Furbank and Lilley showed that reaction sequences of a complete photosynthetic carbon reduction pathway could be demonstrated using a SEP (see Methods section) from the chloroplasts of peas. By supplying SEP with ATP, NAD(P)H, Mg2+ and dithiothreitol (DTT) and operating at pH\u00a08.0, the need for light was eliminated. When SEP was provided with a small priming level of 3-PGA, there was an immediate and rapid oxidation of NAD(P)H which was spectrophotometrically monitored at 340\u00a0nm. The amount of NAD(P)H oxidized was equated with the number of moles of 3-PGA added. During the period of rapid NAD(P)H oxidation it was shown, in parallel experiments using 14CO2, that no carbon fixation took place. However, after this initial rapid period there followed an interval of further NAD(P)H oxidation at a slower rate, which was linear for at least 15\u00a0min and which was accompanied by CO2 fixation. It was assumed that CO2 was fixed by Ru 1,5-P2 production for the ribulosebisphosphate carboxylase (EC 4.1.1.39) reaction which was supported by the test sugar-P added to the SEP reaction mixture (Table\u00a03). Furbank and Lilley hypothesized that, in spite of a 500-fold dilution of the intact enzyme complement of chloroplasts and the loss of any attendant structural regulation, the pathway catalysed by SEP is mechanistically similar to the overall carbon fixation rate that occurs in intact organelles, although we suggest that it may not be quantitatively equivalent for all intermediates. Thus it was of interest to prepare SEP from spinach chloroplasts and investigate its comparative ability to react octulose and arabinose phosphates, and adopt its use for measurement of catalytic activity of the phosphotransferase enzyme, and the rates of the oxidative and reductive PPs (see Methods).\nDetermination of CO2 fixation using SEP and activity of the reductive PP with various sugar phosphate substrates\nThe relative capacity of SEP to fix CO2 (see Methods) depended primarily on the nature of the sugar-P primer in the incubation. In Table\u00a03, the rates of spinach SEP-catalysed NADH oxidation in the presence of various sugar phosphates are compared with the data reported by Furbank and Lilley (1981) using SEP from peas.Table\u00a03Rates of reductive PP sequences, measured by NADH oxidation, in reactions catalysed by spinach SEP with a variety of sugar phosphates as substratesSubstrateConcentration (mM)Rate of NADH oxidation (\u03bcmol\u00a0h\u22121\u00a0mg\u22121 Chl)SpinachaPeabRu 1,5-P22.0592\u00a0\u00b1\u00a079 (9)546Rib 5-P2.0743\u00a0\u00b1\u00a092 (10)453Rib 5-P0.2366\u00a0\u00b1\u00a039 (10)\u2013Seh 7-P2.0183\u00a0\u00b1\u00a032 (12)184Seh 1,7-P22.076\u00a0\u00b1\u00a014 (10)112Fru 1,6-P22.010.1\u00a0\u00b1\u00a02.7 (10)74.1Fru 1,6-P21.018.2\u00a0\u00b1\u00a02.9 (11)\u2013Fru 6-P2.01.0c\u00a0\u00b1\u00a00.3 (13)77.6Fru 6-P + DHAP2.0\/0.201.8\u00a0\u00b1\u00a00.4 (10)142Fru 6-P + PGA2.0\/0.202.9\u00a0\u00b1\u00a00.4 (8)\u2013DHAP2.013.1\u00a0\u00b1\u00a03.1 (10)53DHAP0.205.0\u00a0\u00b1\u00a01.4 (11)22.6Ara 5-P2.01.1 (9)d\u2013d-g-d-i-oct 1,8-P22.03.7\u00a0\u00b1 1.7 (5)\u2013d-g-d-a-oct 1,8-P22.09.3\u00a0\u00b1 1.5 (6)\u2013d-g-d-i-oct 8-P2.06.3\u00a0\u00b1 2.4 (5)\u2013d-g-d-a-oct 8-P2.08.2\u00a0\u00b1 3.0 (5)\u2013a\u00a0Shows the results of this study. Results are mean values\u00a0\u00b1\u00a0standard deviation. The number of determinations using different batches of SEP are shown in bracketsb\u00a0Shows the data of Furbank and Lilley (1981) for peasc\u00a0When preparations were made using sonicated spinach chloroplast suspensions and Fru 6-P as substrate, varying (non-reproducible) rates up to 5\u00a0\u03bcmol\u00a0h\u22121\u00a0mg\u22121 Chl were recordedd\u00a0Results were variable with different SEP batches, three of the SEP preparations with Ara 5-P did not support any NADH oxidation\nIn general, metabolites which immediately precede the carboxylation reaction in the scheme of the reductive PP, namely Rib 5-P, Ru 5-P, Ru 1,5-P2, Seh 7-P, Seh 1,7-P2, gave very high rates of CO2 fixation as indicated by the oxidation rate of NADH, consistent with their role in autocatalysis (Walker and Lilley 1974). However, triose, arabinose, fructose, ido- and altro-octulose phosphates exhibited low rates that were notably below the maximum rate of CO2 fixation in intact chloroplasts. Triose-P and hexose-P rates using pea SEP were also below the physiological PS rate. In particular Fru 6-P and Glc 6-P (results not shown) were very inefficient substrates for spinach SEP giving much lower values than those reported by Furbank and Lilley (Table\u00a03) for pea preparations, results which we were able to confirm.\nLilley and Walker (1979) also reported that substrate concentrations of Fru 6-P and a reconstituted chloroplast system yielded very low rates for the enablement or support of Calvin Cycle regenerative activity by this substrate.\nA number of modifications of the original procedure were introduced in an attempt to obtain higher rates of CO2 fixation with several sugar phosphates, Fru 6-P in particular. These modifications included variations in the composition of the low osmolality buffer used for the disruption of the organelles, freeze\u2013thawing and sonication of chloroplasts, the inclusion of various components such as ThPP or glycerol in the dialysis medium and the omission of selected steps from the procedure. Notably better reproducibility, in rates of NADH oxidation, were obtained using 10\u00a0mM HEPES buffer (pH\u00a08.0) with 3\u00a0mM DTT. Consequently, this method of SEP preparation was used for all reported results. The low rates obtained with hexose and the octulose phosphates were initially thought to be due to depressed activity of a key group-transferring enzyme, particularly transketolase. This was shown to be unlikely since the maximum catalytic activity of TK, as well as that of several other enzymes of the reductive PP in SEP, was sufficient to support rates of CO2 fixation in excess of 100\u00a0\u03bcmol\u00a0h\u22121\u00a0mg\u22121 Chl (Table\u00a04). The enzyme activities were measured between 4 and 8\u00a0h after chloroplasts were disrupted and are representative of their activities in SEP during progressive or serial assays of NADH oxidation.Table\u00a04Maximum catalytic activity of selected enzymes of the reductive PP in Spinach SEPEnzymeCatalytic activity (\u03bcmol\u00a0h\u22121\u00a0mg\u22121 Chl)This studyData of Latzko and Gibbs (1969) for Spinach leaf extractPhosphoglycerate kinasea19902423Glyceraldehyde 3-phosphate dehydrogenase (NADP+)b207269Transketolasec199i; 103j194Fructose-bisphosphate aldolased113102Fructose-bisphosphatasee5346Sedoheptulose-bisphosphatasef35k3.8Phosphotransferaseg2.7l; 13.6m\u2013Transaldolaseh2.47.7Phosphotransferaseg in Pea leaf extract25.3nPhosphotransferaseg in Chlorella fusca extract20.9nPhosphotransferaseg in Spinach leaf extractNDn; 19.3mEnzymes were assayed using the methods of: a, b\u00a0Latzko and Gibbs (1969); c\u00a0Brin (1974); d\u00a0Bergmeyer and Bernt (1974); e\u00a0Latzko and Gibbs (1974); f\u00a0Woodrow and Walker (1982); g\u00a0Arora et\u00a0al. (1985); h\u00a0Brand (1974).i\u00a0Assayed using Ery 4-P and Xlu 5-P as substratesj\u00a0Assayed using Rib 5-P and Xlu 5-P as substratek\u00a0Assayed using the Pi liberation methodl\u00a0Assayed using [8-14C]-d-g-d-i-oct 8-P and Seh 1,7-P2 as substratesm\u00a0Assayed using [8-14C]-d-g-d-a-oct 8-P and Seh 1,7-P2 as substratesn\u00a0Assayed using d-g-d-i-oct 1,8-P2 and Seh 7-P as substratesND: not detected\nBoth the mono- and bisphosphates of d-g-d-a-oct were marginally more active substrates for the support of CO2 fixation (Table\u00a03) than the ido-oct phosphate esters. d-g-d-i-oct 1,8-P2 was least effective and has not been assigned a substrate role in a new reaction sequence for PS presented as Scheme 1 and is a conclusion of this paper. This is a curious finality to this study of the octuloses in PS since d-g-d-i-oct 1,8-P2, because of its identification in the L-type PP, was the initial substrate of choice to test the hypothesis of this study (see Introduction). Although d-g-d-i-oct 1,8-P2 has a degree of 14C isotopic equilibrium with all the octulose phosphates listed in Table\u00a03, we find no other evidence or reason to now support its inclusion in the new PS scheme. d-g-d-a-oct 1,8-P2 is the most reactive octulose substrate in both reductive CO2 fixation and in its formation from the monophosphate by PT catalysis in the reductive direction of PS (see later discussion on PT). However, d-g-d-a-oct 1,8-P2 forms Rib 5-P if it is cleaved by the ALD activity of SEP, so the higher CO2 fixation by d-g-d-a-oct 1,8-P2 (Table\u00a03) may in part be attributed to some Rib 5-P release. d-g-d-i-oct 8-P is also more effective in the support of CO2 fixation (Table\u00a03) than its bisphosphate and it is also independent, in the new PS scheme, of any need to transact the PT step at reaction XII of the new scheme (see later section). It is also noted that the Furbank and Lilley (1981) assay for CO2 fixation identified slow reacting intermediates in the reduction assay (Table\u00a03). These intermediates may accumulate and thus be confined to a linear, non-autocatalytic (Walker and Lilley 1974) expression of PS in\u00a0vitro with resultant slow synthesis of Ru 1,5-P2.\nDetermination of the activity of the non-oxidative segment of the PP\nThe capacity to convert Rib 5-P to hexose 6-P is the measure of the activity of the enzymes comprising the non-oxidative segment of the PP (Williams et\u00a0al. 1978b, 1987; Fig.\u00a01). When the products of this conversion are removed by NADP+ reduction, using an excess of PGI and Glc 6-PDH, the reduction rate of the pyridine nucleotide is the measure of the maximum catalytic capacity of the preparation (see Methods). The average max rate of Rib 5-P to hexose 6-P conversion by SEP was 2.29\u00a0\u03bcmol\u00a0h\u22121\u00a0mg\u22121 Chl (S.D.\u00a0\u00b1\u00a00.41, n\u00a0=\u00a017). The ability of SEP from spinach chloroplasts (Furbank and Lilley 1981) to catalyse the above process (half-life 24\u00a0h) was greater than its stability in measurements of the reductive PP (half-life 6\u00a0h). When SEP was prepared by the method of Kaiser and Bassham (1979), it catalysed the non-oxidative segment reactions of the PP at the rate of 3.22\u00a0\u03bcmol\u00a0h\u22121\u00a0mg\u22121 Chl and this was enhanced, at all test times, by 30% over a 48-h stability time course by the inclusion of ThPP (0.1\u00a0mM). This suggests that TK may have been susceptible to inactivation by the assay system (Murphy and Walker 1982). However, comparison of these data with the above results for the reductive PP show that the maximum catalytic capacity of the non-oxidative segment of PP in SEP, using Rib 5-P as substrate, is only 0.6% of Rib 5-P carboxylating activity in the reductive PP (Table\u00a03). The above finding thus discounts the possibility that the complete network of the PP (Fig.\u00a01) in SEP can make any significant overlapping contribution to the flux of the reductive PP of PS.\nPhosphotransferase activity\nThe proposed alternative mechanism for the regenerative phase of the path of carbon in PS (Fig.\u00a02) presents a number of features in common with the L-type PP in animal tissues (Fig.\u00a01) (Williams et\u00a0al. 1978a, 1987; Williams 1980). In particular, both pathways require the participation of a specific phosphotransferase, PT (see Methods). The presence and an excess of PT activity to support the maximum flux of Rib 5-P conversion to hexose and triose-P in\u00a0vitro was reported for a number of animal and plant tissues (Arora et\u00a0al. 1985). PT catalysed the reaction of Eq. (6), using the same rat liver enzyme catalyst that generated results used for the initial hypothesis of the classical (F-type) PP (Horecker et\u00a0al. 1954; Horecker 2002; Williams et\u00a0al. 1987). PT was also detected in French Press extracts of green algae (Chlorella fusca) and enzyme extracts of pea leaf (Arora et\u00a0al. 1985) (see Table\u00a04). The PT enzyme has not previously been investigated for any putative role in the metabolism of plants.\nThe detection of 13C and 14C-labelled d-g-d-i-oct 1,8-P2 in spinach leaves (Bartlett et\u00a0al. 1989) and isolated chloroplasts following short periods of 14CO2 assimilation (this paper), raised the question of the origin of this bisphosphate compound. The reaction of Eq. (3) shows the formation of d-g-d-i-oct 8-P and the data reported here examine the activity and mechanism of phosphate donor and transfer for the formation of d-g-d-i-oct 1,8-P2. In particular the phosphotransferase proposed by Williams and Clark (1971), Clark et\u00a0al. (1974) and Arora et\u00a0al. (1985) is featured. The L-type PP (Fig.\u00a01) and the hypothesized path of carbon in PS (Fig.\u00a02) proposed a phosphotransferase which catalysed a reversible phosphate transfer between octulose and sedoheptulose phosphates (Eq. (6)). The investigations reported on this reaction were made using spinach leaf extracts and SEP preparations from spinach chloroplasts. PT activity was not detected using either spinach leaf extract or SEP to catalyse the reaction of Eq. (6) when the measurements were performed in the oxidative direction of d-g-d-i-oct 8-P formation (Fig.\u00a02). A study of SEP-catalysed phosphate transfer from Seh 1,7-P2 to [8-14C]-d-g-d-i-oct 8-P (Eq. (1) in the reductive direction of the PS pathway), using the radiochromatographic stop assay procedure (see Methods), showed PT activity was 2.7\u00a0\u03bcmol\u00a0h\u22121\u00a0mg\u22121 Chl (S.D.\u00a0\u00b1\u00a00.80; n\u00a0=\u00a07) (Table\u00a04). This value exceeded the maximum rate of SEP-catalysed conversion of Rib 5-P to hexose phosphates but was only 16% of the chloroplast CO2 fixation rate required by the PT reaction step (See New Scheme) and 15% of the degree of carboxylation required for the ab\u00a0initio synthesis of octulose (120\/8) by PS. 14C-labelled d-g-d-a-oct 8-P was a more effective substrate in this PT assay with activity of 13.6\u00a0\u03bcmol\u00a0h\u22121\u00a0mg\u22121 Chl (P.E.\u00a0\u00b1\u00a03.0; n\u00a0=\u00a04; see Table\u00a04), which is 80% of the required chloroplast CO2 fixation rate at the PT step of the revised RPP scheme and 91% of the activity necessary for octulose formation. Significant PT activity was found in spinach leaf extract (19.3\u00a0\u03bcmol\u00a0h\u22121\u00a0mg\u22121 Chl, see Table\u00a04) using the above radiochromatographic method and the [8-14C]-d-g-d-a-oct 8-P substrate (Eq. (2)). No activity was detected in the leaf extract using d-g-d-i-oct 1,8-P2 and oxidative direction assays (see Table\u00a04). However, it is not concluded that positive PT measurement in the reductive PS mode is evidence for an exclusive unidirectional flux mechanism. Rather the failure in the application of the oxidative-mode methods (Arora et\u00a0al. 1985) is attributed inter\u00a0alia to subversively high levels of Mg+ and DTT in SEP.\nA test of the absolute specificity of the PT reaction was made using [1-32P]-Seh 1,7-P2 substrate and d-g-d-i-oct 8-P. The reaction was carried out using SEP and exactly the same conditions as the above radiochromatographic stop assay. All 32P-labelled products of the reaction were resolved using formate ion-exchange chromatography (see Methods) and their radioactivity measured. 32P-d-g-d-i-oct 1,8-P2, which coincided with the elution peak of the authentic compound, was recovered and its identity further confirmed by rechromatography with DEAE-Sephadex A25 (borate form) (Kapuscinski et\u00a0al. 1985). Aldolase cleavage of the purified Oct 1,8-P2 and isolation of 32P-DHAP using short column (0.9\u00a0\u00d7\u00a015\u00a0cm2) formate ion-exchange chromatography, showed that >90% of the incorporated 32P was in this compound. The results of this experiment indicated that qualitatively SEP has enzymatic activity capable of specifically phosphorylating d-g-d-i-oct 8-P at position 1 using Seh 1,7-P2 as the phosphate donor. There was also an extensive 12% conversion of the [1-32P]-Seh 1,7-P2 to 32Pi and 30% formation of [32P]-Fru 1,6-P2. Only 25% of the [1-32P]-Seh 1,7-P2 substrate remained after 30\u00a0min of reaction time. When 10\u00a0mM Mg2+ and 10\u00a0mM DTT were included in the above reaction mixture, 93% of the 32P label of [1-32P]-Seh 1,7-P2 was converted to 32Pi and only 0.08% to the isotopically labelled d-g-d-i-oct 1,8-P2. It is suggested that high concentrations of Mg2+ in SEP may have activated chloroplastic bisphosphatases and account in part or completely for an inability to demonstrate significantly higher activities of PT. Inclusion of DTT and the endogenous levels of aldolase in SEP and spinach leaf extracts possibly acted to further diminish the full expression of the activity of the PT enzyme. Hence the minimum qualification for the measurements of the crude activity of PT.\nDuring the conduct of the above investigations and measurements of PT activity, the various SEP preparations were also used to test the possibility that other intermediary compounds and enzymes may act to phosphorylate d-g-d-i-oct 8-P. Among the test substrates were [\u03b3-32P]-ATP, 1,3-diphosphoglycerate, inorganic pyrophosphate, Fru 1,6-P2 and Ru 1,5-P2. The following purified enzymes and enzyme preparations were also investigated; PFK from rabbit muscle, phosphoribulokinase (EC 2.7.1.19) from spinach, the endogenous kinases of SEP using [\u03b3-32P]-ATP as substrate and sedoheptulose 1,7-bisphosphatase. In summary, none of these enzymes or substrates was effective except for muscle PFK which catalysed the formation of [1-32P]-d-g-d-i-oct 1,8-P2 from the reaction of [\u03b3-32P]-ATP and d-g-d-i-oct 8-P. Notwithstanding this, when SEP was used to catalyse the same reaction only a miniscule rate of labelled d-g-d-i-oct 1,8-P2 formation was measured (0.3\u00a0\u03bcmol\u00a0h\u22121\u00a0mg\u22121 Chl). PFK accepts Fru 6-P and Seh 7-P as substrates (Karadsheh et\u00a0al. 1973) and on the basis of the above finding d-g-d-i-oct 8-P may be added to the list. However, the maximum catalytic activity of PFK in spinach chloroplasts is only 2.5\u00a0\u03bcmol\u00a0h\u22121\u00a0mg\u22121 Chl (Kelly and Latzko 1977) and it is therefore unlikely to be a significant contributor to d-g-d-i-oct 1,8-P2 formation in RPP reactions in PS. The results of this section (Table\u00a04) showed that minimum estimates of PT were compromised when SEP is the source of activity. The results also showed that d-g-d-a-oct phosphate was the most active substrate and that PT may be the rate-limiting enzyme for octulose involvement in path of carbon reactions. This limitation in minimum estimates of PT activity in SEP (Table\u00a03) may account for altro-octuloses being restricted to the support of only 11% of the rate of CO2 fixation by intact spinach chloroplasts and\/or 80% of the maximum CO2 rate required at the PT reaction step (See later discussion of the new reactions in Scheme \n1).\nArabinose 5-phosphate reactions with spinach stromal enzyme preparation\nIn the L-type PP (Fig.\u00a01), the prior formation of Ara 5-P, and its use by aldolase is the key step for the formation of d-g-d-i-oct 1,8-P2. In contrast, the hypothetical mode of d-g-d-i-oct 8-P synthesis and reaction in the PS scheme of Fig.\u00a02 results in the later formation of Ara 5-P and its contribution to Rib 5-P (via Ru 5-P). Thus it was anticipated that Ara 5-P by its initial assignment of an intermediary role in the flux of the pathway of Fig.\u00a02, should support CO2 fixation in the SEP assay system. The result (Table\u00a03) of the test of this proposition was negative. Arabinose 5-P, as a single test substrate, had negligible ability to support CO2 fixation in SEP-catalysed reductive PP reactions in PS. A second question raised by the hypothetical scheme of Fig.\u00a02 is whether SEP is able to catalyse Ara 5-P dissimilation and interconvert it into other intermediates of either the Calvin Pathway or octulose phosphates. SEP catalysed the incorporation of radioactivity from [U-14C]-Ara 5-P into the carbons of the following sugar phosphate intermediates of the reductive PP; triose-P, ketopentose 5-phosphates, hexose and heptulose mono- and bisphosphates (Fig.\u00a07).\nFig.\u00a07Radioautogram showing the spinach SEP-catalysed conversion of [ U-14C]-Ara 5-P, in the presence of a nine-fold excess of unlabelled Rib 5-P, to other radioactively labelled sugar phosphates. [ U-14C]-Ara 5-P and Rib 5-P were incubated for 1\u00a0h with spinach SEP as detailed in Materials and methods. The products of the reaction were separated by descending paper chromatography using the GW3-PBA solvent. Lanes 1 and 3 of the chromatogram show the mixture of 14C-labelled sugar phosphates formed from Ara 5-P dissimilation. Lane 2 shows the radioautograph of products in the absence of Rib 5-P. Lane 4 shows an absence of product formation when boiled SEP was used. The identity, by visualization, of the 14C-labelled sugar phosphates at Lanes 1 and 3 was established by comparison with the positions (not shown in Fig.\u00a07) of the following sugar phosphate marker standards that accompanied the four lanes of the chromatogram, Fru 1, 6-P2 (FBP), Seh 1,7-P2, Seh-7-P, Glc 6-P, Fru 6-P, Ara 5-P, DHAP, Ru 5-P, Xlu 5-P. The one-dimensional chromatography system used does not permit the identification of octulose phosphates (see Kapuscinski et\u00a0al. 1985 for a list of Rp-i values for some of the above sugar phosphates resolved by the GW3-PBA solvent). The visualization and tentative identification of the labelled products was distinct except for the band running just ahead of Ara 5-P which may have involved ketopentose 5-phosphates and DHAP\nHowever, 14C-incorporation into these products only occurred in the presence of an excess (nine-fold) of unlabelled Rib 5-P. The process only faintly labelled Rib 5-P. When 2\u00a0mM [U-14C]-Ara 5-P was the sole substrate, its conversion to other identified 14C-labelled sugar phosphates was only 2% and there was clear incorporation into an unresolved band of phosphorylated compounds with an Rf value coincident with the ketopentose phosphates and also close to DHAP (Fig.\u00a07). It was imperative to ensure that 14C-incorporation into the sugar phosphates was not the result of non-enzymatic conversions occurring during sample processing. This possibility was eliminated on the basis of several control experiments which included different ways of terminating the reaction (boiling and perchloric acid treatment) and incubations with SEP boiled before the addition of [U-14C]-Ara 5-P.\nWhen the incubations were carried out with SEP at pH\u00a08.0 in the presence of 10\u00a0mM MgCl2 and 10\u00a0mM DTT (conditions favouring activation of some key regulatory enzymes of the reductive PP), there was no incorporation of 14C isotope from [U-14C]-Ara 5-P (and excess Rib 5-P) into any other metabolites. Since the experiments used SEP that had provided approx 10% 14C incorporation from [U-14C]-Ara 5-P (in the presence of a nine-fold excess of Rib 5-P) into hexose 6-P and by visual judgement of the chromatogram, incorporations into Triose-P, Ru 5-P, Fru- and Seh-bisphosphates, it was concluded that Mg2+ or the reductant may be inhibitory. These latter observations were not pursued further. The above is the first report of Ara 5-P reaction in a chloroplast preparation.\nArabinose 5-P is a competitive inhibitor of TA-catalysed reactions, with a Ki of 70\u00a0\u03bcM (Williams et\u00a0al. 1978a). d-manno-Heptulose 7-P, a product of TK catalysis using a ketosugar-P donor and Ara 5-P, is a competitive inhibitor of TK-catalysed reactions involving Ery 4-P (Arora 1984). Moreover, in all animal and neoplastic tissues investigated (Williams et\u00a0al. 1987), Ara 5-P acted as a powerful competitive inhibitor (Ki: 40\u00a0\u03bcM, Arora 1984) when the tissue enzyme preparations converted Rib 5-P to hexose and triose phosphates using standard assay conditions for the non-oxidative PP (Methods section). Thus it was reasoned that enzyme inhibition may be responsible for the failure to find uncontentious evidence that Ara 5-P is a utilized substrate in reactions catalysed by spinach SEP. This proposition was tested with SEP using [Ara 5-P]:[Rib 5-P] ratios ranging from 0.25 to 1.0. Some inhibition was found but it was notably less than that uniformly encountered with animal and neoplastic tissue enzyme preparations. Even a 1:1 mixture of Ara 5-P and Rib 5-P with SEP exhibited 75% of the control rate measured with Rib 5-P alone. This small degree of inhibition is therefore not an explanation for the inability of SEP to catalyse Ara 5-P as a substrate in carboxylation reactions. In experiments that involved incubation of SEP with unlabelled Ara 5-P and NaH14CO3, slow 14C fixation occurred (1\u00a0\u03bcmol\u00a0h\u22121\u00a0mg\u22121 Chl.).\nReplacement of Ara 5-P with Rib 5-P resulted in a rapid rate of formation of acid stable 14C-labelled product consistent with the peak activity of Rib 5-P in reductive PP reactions, as shown by the data of Table\u00a03. Moreover, using the polarographic method for measuring CO2-dependent O2 evolution (Methods section) and intact spinach chloroplasts, it was found that the inclusion of 0.2\u00a0mM Rib 5-P stimulated the rate of O2 evolution by 27% and the same concentration of Ara 5-P stimulated the rate by 7.5%. Schafer et\u00a0al. (1977) found that free Ara and Rib were transported into intact chloroplasts at equal rates and the above data suggest that spinach chloroplasts may be differentially permeable to the phosphates, with Rib 5-P being 3.6\u00a0times more accessible and effective than Ara 5-P. There is also controversy about the substrate role of d-Ara 5-P in higher organisms (a brief statement of its intermediary metabolic history is given in Williams et\u00a0al. 1987). Interconversion of Ara 5-P with other pentose phosphates (via isomerization to Ru 5-P) by microorganisms has been known for a considerable time (Volk 1960) but the direct evidence for such a process in rat liver was first demonstrated by Bleakley et\u00a0al. (1984). The enzyme equilibrating Ara 5-P with other pentose phosphates was originally proposed to be of the epimerase type (Williams and Clark 1971), however, work with pig liver suggested that utilization and conversion of Ara 5-P to other metabolites may proceed via Ru 5-P (Williams unpublished results). The essence of the controversy concerning the metabolism of Ara 5-P is not so much its formation and presence in plant and other tissues but its comparatively low rate of substrate utilization by tissue enzyme preparations in\u00a0vitro, usually only at 2\u201310% of the rate of Rib 5-P dissimilation. This is the case reported here where SEP catalysed utilization of Ara 5-P was extremely low. This controversy was settled when Bartlett et\u00a0al. (1989) and Flanagan et\u00a0al. (1993) demonstrated the role that catalytic quantities of Ara 5-P and Rib 5-P were able to play in TA and ALD exchange reactions. These exchange reactions affected 5\u2032-epimerase interconversions of d-g-d-i-oct 8-P and d-g-d-a-oct 8-P and their bisphosphates. It was shown that Ara 5-P was reversibly exchanged into positions 4\u20138 of the octulose phosphates with exchange rates that were greater than the mass transfer flux rates catalysed by the maximum catalytic capacities of each of the group transferring enzymes TA and ALD acting in the reactions of the following equations.\nThe roles of Eqs. (7) and (8) are shown at reactions IX and X in the following new reaction sequence (Scheme 1) for the path of carbon in PS. Taken together these reactions catalyse the epimerization of the ido- and altro-octulose monophosphates. Equation (11) shows the interconversion of ido- and altro-octulose bisphosphates by the aldolase exchange reaction (Bartlett et\u00a0al. 1989; Flanagan et\u00a0al. 1993). It is proposed that the reaction of 11 assists in the prevention of an accumulation of the much slower reacting d-g-d-i-oct 1,8-P2 by rapidly channelling its carbon into the more reactive 5\u2032-epimeric form, d-g-d-a-oct 1,8-P2 (see XII and XIII in Scheme I involving octulose phosphates in PS). The experimental results of this paper and the reactions of IX, XII and XIII in the reaction scheme suggest that d-g-d-a-oct mono- and bisphosphates become the reactive forms of ido-octulose phosphates by the reactions of (7) and (11) using Rib 5-P and Ara 5-P as cycling catalytic co-factors in reactions catalysed by TAx and Aldx. We now discount any role for d-glycero-d-ido-octulose 1,8-P2 as was initially proposed for the Fig.\u00a02 hypothesis. The revised scheme only differs significantly from Fig.\u00a02 at reaction IX, where 5\u2032-epimerization of d-g-d-i-oct 8-P to d-g-d-a-oct 8-P occurs. From that point ido-octulose phosphates are omitted from any further involvement in the completion of the cycle. Using PT activity data with d-g-d-a-oct 8-P as substrate (Table\u00a04), it is calculated that the revised sequence may make a contribution that ranges from 11% (13.6\/120), based on the rate of CO2 fixation (120\u00a0\u03bcmol\u00a0h\u22121\u00a0mg\u22121 Chl) in intact chloroplasts, to a maximum of 80% (13.6\/17) based on the required CO2 fixation rate (17\u00a0\u03bcmol\u00a0h\u22121\u00a0mg\u22121 Chl) at the PT step (reaction XIII) of Scheme \n1 for the RPP.\nScheme\u00a01\u00a0\nFinally, it is concluded that metabolic function has directed a distinct and confining intermediary metabolism on ido- and altro-octulose phosphates. In the L-type PP (Fig.\u00a01) selection of d-glycero-d-ido-octulose phosphates realised the formation of the metabolically active products Fru 6-P and Glc 6-P. d-glycero-d-altro-Octulose phosphates in the pathway of Fig.\u00a01 will only lead to the formation of Fru 6-P and the dead-end product d-allose 6-P. In Scheme 1 formulated here, the formation of d-g-d-a-oct 1,8-P2 leads to the production of Rib 5-P, the most active intermediate for the support of CO2 fixation (Table\u00a03). Formation and further utilization of d-g-d-i-oct 1,8-P2 by the reaction scheme would produce stoichiometric amounts of Ara 5-P which, as a metabolic product, has no ability to fix CO2 (Table3).","keyphrases":["octulose phosphates","chloroplasts","l-type pentose pathway","calvin cycle","stromal enzymes","phosphotransferase","spinach leaf","arabinose 5- phosphate","14c-carbon dioxide","14c-labelled novel compounds","keto-group exchange","octulose-p epimerization","revised rpp in ps"],"prmu":["P","P","P","P","P","P","P","M","U","R","M","M","R"]}
{"id":"J_Gastrointest_Surg-3-1-1852389","title":"An Innovative Option for Venous Reconstruction After Pancreaticoduodenectomy: the Left Renal Vein\n","text":"Introduction Pancreatic ductal adenocarcinoma has a high mortality rate with limited treatment options. One option is pancreaticoduodenectomy, although complete resection may require venous resection. Pancreaticoduodenectomy with venous resection and reconstruction is becoming a more common practice with many choices for venous reconstruction. We describe the technique of using the left renal vein as a conduit for venous reconstruction during pancreaticoduodenectomy.\nIntroduction\nPancreatic ductal adenocarcinoma has a high mortality rate1, which approaches the incidence, and treatment options remain limited. For those patients diagnosed with pancreatic ductal adenocarcinoma, resection continues to offer the only chance for cure. Historically, involvement of local vasculature was considered a contraindication to pancreaticoduodenectomy (PD), with early experience associated with prohibitively high morbidity and mortality rates.2 As surgeon experience has grown, morbidity and mortality rates have decreased, and at high-volume pancreatic surgery centers, invasion of local mesenteric venous structures is no longer a contraindication to resection. Venous resection occurs in up to 25% of patients undergoing PD at several centers.3,4 Several techniques are described for reconstruction of the venous system after PD when necessary: primary lateral venorrhaphy, primary end-to-end anastomosis, and interposition grafting.5,6 Both synthetic grafts and autologous vein grafts have been used, with several donor sites available.3,7\nThe goal of this report is to describe the use of the left renal vein as a conduit for venous reconstruction after PD with venous resection. Historically, the left renal vein has been used to repair the portal vein, hepatic vein, or inferior vena cava during resection of hepatic hilar carcinomas,8\u201311 and its use offers distinct advantages for venous reconstruction during PD. Importantly, previous studies have demonstrated the safety of left renal vein ligation, specifically in relation to renal function.12 We describe the technique of mesenteric venous reconstruction after PD with venous resection using a left renal vein graft and report on a group of patients that have undergone this repair.\nPatient Selection and Technique\nContrast-enhanced computed tomography (CT) is used to evaluate a pancreatic head mass and its relation to vascular structures. CT accurately diagnoses mesenteric vein involvement, aiding in operative planning. The CT is also used to assess the length and caliber of the left renal vein, the status of the kidneys bilaterally, and the presence of the left gonadal and adrenal veins, which serve as collateral venous drainage. Additional imaging of the pancreas with endoscopic ultrasound (EUS) is frequently used to further evaluate the location and extent of any venous involvement.\nAfter initiation of the operation, a Kocher maneuver allows assessment of tumor location and its relationship to the superior mesenteric artery (SMA), further assessing resectability. The lesser sac is entered by mobilizing the greater omentum off of the transverse colon through an avascular plane. The middle colic vein is followed centrally to identify the superior mesenteric vein (SMV) and the gastrocolic venous trunk. The gastrocolic venous trunk is routinely divided. (Fig.\u00a01a) If venous resection is anticipated, to increase mobility, the middle colic vein and several other tributaries are ligated and divided. (Fig.\u00a01b)\nFigure\u00a01a Division of gastrocolic venous trunk and middle colic vein. b Mobilization of superior mesenteric vein in preparation of venous reconstruction.\nThe superior border of the pancreas is approached by incising the gastrohepatic ligament. The right gastric and gastroduodenal arteries are routinely ligated and divided. Retraction of the common hepatic artery cephalad allows dissection of the portal vein (PV), thereby, allowing completion of the plane between the pancreas and the PV-SMV.\nThe gallbladder is dissected from the liver, and the hepatic duct is encircled near the cystic duct junction. If a pylorus-preserving PD is planned, the superior and inferior aspects of the duodenum are skeletonized, individually ligating the right gastroepiploic vessels. The ligament of Treitz is mobilized, and the proximal jejunum is divided. Sequential ligation and division of the bowel mesentery exposes the uncinate process. The mobilized duodenum and jejunum are passed beneath the superior mesenteric vessels into the right upper quadrant.\nMobilization is now complete and transection begins at the duodenum approximately 3\u00a0cm distal to the pylorus. The bile duct is transected, and the margin is evaluated for malignancy. Stay sutures are placed on the superior and inferior borders of the pancreas to aid in retraction and hemostasis. The neck of the pancreas is transected over a clamp to protect the portal vein. (Fig.\u00a02a) Reflection laterally allows visualization and ligation of venous tributaries. (Fig.\u00a02b)\nFigure\u00a02a The neck of the pancreas is transected over a clamp, thereby, protecting the portal vein from injury. b Venous tributaries to the portal vein are individually ligated while mobilizing the head and uncinate process of the pancreas.\nOften, venous invasion is not discovered until this juncture, and although some resections can be limited to tangential excision and primary lateral venorrhaphy, there are oncologic and vascular considerations that make other options, including segmental resection with primary end-to-end anastomosis or interposition grafting with autologous or synthetic material, advantageous. Early in our experience, we completed venous resection before division of the arterial branches and soft tissue along the right lateral aspect of the superior mesenteric artery (SMA) (Fig.\u00a03a). More recently, we have altered our technique by performing the dissection of the retroperitoneal margin before venous resection. The advantages of this are to avoid the need for venous anastomosis before removal of the specimen, minimize venous occlusion time, and allow preservation of the splenic vein. This is accomplished by performance of a generous Kocher maneuver and isolation of the superior mesenteric artery both at its origin and caudad to the uncinate process. The Kocher maneuver orients the superior mesenteric artery posterior to the PV-SMV and allows access for completion of the retroperitoneal dissection (Fig.\u00a03b). Arterial branches coursing into the uncinate are sequentially clamped, divided, and ligated, thereby, completely freeing the pancreas from the SMA. The pancreatic head is then rotated back to its normal anatomic orientation, and it is at this juncture that a decision is made for primary end-to-end venous reconstruction or renal vein interposition grafting.\nFigure\u00a03Dissection of the superior mesenteric artery proximal and distal to the area of venous invasion will limit total venous occlusion time after resection is performed. This can be approached anteriorly (a) or posteriorly (b) as necessary. Inflow occlusion of the SMA during posterior dissection is used selectively if maintenance of hemostasis is problematic.\nMobilization of the portal vein superior to the pancreas and the peritoneum along the root of the small bowel mesentery may provide length for the SMV or PV segment. This is accomplished by ligating and dividing small branches to the SMV, PV, and splenic vein (SV). Although primary end-to-end anastomosis is preferred, if interposition grafting is necessary, autologous vein and specifically the left renal vein is utilized for two reasons. First, the vein may be exposed within the same operative field, thereby, eliminating a second operative field and dissection. Second, the caliber and wall thickness of the vein is similar to the portal vein in most instances, providing good handling and suturing properties. Harvest of the left renal vein is undertaken after the retroperitoneal dissection when the specimen remains attached to the portal vein segment only (before venous resection). This allows the best assessment of the need for interposition grafting and minimizes the amount of clamp time by harvesting the graft before SMA and venous occlusion.\nThe left renal vein is optimally exposed by extending the Kocher maneuver to the left and elevating the head of the pancreas. (Fig.\u00a04a) The vein is divided at the junction of the left gonadal and left adrenal veins, always preserving these vessels as collateral venous outflow for the left kidney. The vein is divided again flush with the inferior vena cava.(Fig.\u00a04b) This typically provides a 3- to 4-cm venous segment. For venous division, we prefer a linear stapling device. The vein segment is placed in heparinized saline and then can be used as an interposition graft. (Fig.\u00a04c)\nFigure\u00a04a The Kocher maneuver is extended to the left and elevation of the pancreatic head allows exposure of the entire left renal vein and the left adrenal and gonadal veins. b The vein is transected with a linear stapling device distal to the insertion of the left adrenal and gonadal veins and again flush with the inferior vena cava. c The left renal vein is used as an interposition graft to restore continuity to the mesenteric venous system.\nVascular control includes the SMA with placement of a Rummel tourniquet (in addition to venous occlusion proximal and distal) and is obtained immediately before resection. This allows inflow occlusion during the resection and reconstruction, decreasing the amount of intestinal engorgement, thereby, facilitating an easier pancreaticojejunostomy. The patient is not systemically heparinized. Venous resection is done sharply to obtain a margin.(Fig.\u00a05) If the specimen has not been entirely freed from the SMA, the remaining branches are now ligated. Communication between the surgical and pathological teams is crucial to fully evaluate the specimen. Of specific importance is the venous segment for margin status and invasion and the retroperitoneal (uncinate) margin. (Fig.\u00a06) Frozen section analysis allows additional margin to be obtained if necessary before reconstruction. After completion of both anastomoses, intraoperative ultrasound is routinely utilized to evaluate the reconstruction for patency. After satisfactory venous reconstruction, the remainder of the gastrointestinal reconstruction is completed. (Fig.\u00a07)\nFigure\u00a05Vascular clamps are used to control the superior mesenteric vein, splenic vein, and portal vein before sharp dissection and resection of the involved venous segment. Inflow occlusion of the superior mesenteric artery during reconstruction reduces bowel engorgement.Figure\u00a06The specimen is carefully marked for all margins, including the venous segment margin. The portal vein groove and the retroperitoneal margin should be inked, and the venous segment should be evaluated histologically for malignant invasion (posterior view).Figure\u00a07In the example shown, reconstruction of the PV-SMV confluence with an interposition graft utilizing left renal vein was performed initially, followed by reimplantation of the splenic vein into the graft (end-to-side) Gastrointestinal reconstruction is performed in a standard fashion after completion of venous reconstruction.\nResults\nNine patients have undergone reconstruction of the SMV-PV during PD with an autologous left renal vein graft. There were seven men and two women with a mean age of 57\u00a0years (range, 31\u201377). Preoperative abdominal CT had suggested mesenteric vein involvement in seven of the nine patients. EUS was completed in three patients. In one patient, EUS suggested there was no vein involvement, while in the remaining two patients, EUS did suggest involvement. Preoperative serum creatinine levels ranged from 0.8 to 1.1\u00a0mg\/dl in these patients (normal values 0.9\u20131.4\u00a0mg\/dl ). Mean follow-up was 6.8\u00a0months.\nThe procedure consisted of three standard PD and six pylorus-preserving PD. Venous reconstruction consisted of eight interposition grafts and one patch graft. The patch graft was located on the lateral edge of the SMV and PV. Five of the interposition grafts were placed in the SMV, inferior to the confluence. One interposition graft was placed between the SMV and PV with reimplantation of the splenic vein; an additional was placed between the SMV and PV without reimplantation of the splenic vein, and the final graft was in the portal vein. The mean operating time was 7.8\u00a0hours (range, 6.5\u20139.5). The mean tumor size was 3.4\u00a0cm (range, 2.2\u20135). The mean estimated blood loss was 1,300\u00a0ml (range, 350\u20132,500). Eight patients were found to have node-positive disease with six of these patients noted to have histological involvement of the venous segment, while one additional patient had pathologically negative lymph nodes and no evidence of malignant invasion of the vein. In two patients, the uncinate margin was microscopically positive.\nOne patient was monitored overnight in the intensive care unit. There were no operative mortalities, and reoperation was not required in any of the patients. The mean length of hospitalization was 14\u00a0days (range, 9\u201329). Immediate perioperative morbidity included a superficial wound infection in one patient, delayed gastric emptying in one patient, and postoperative gastrointestinal bleeding in one patient. None of the patients experienced a pancreatic leak. No hematuria was noted. One patient was diagnosed with ascites and stenosis of the left renal vein interposition graft anastomosis 1\u00a0month after the operation. This patient had a congenitally cystic (nonfunctioning) left kidney. The left renal vein was reduced in caliber, but felt to be adequate for grafting at the time of the original operation. The patient underwent stenting of the graft by interventional radiology with resolution of her symptoms. Eight patients underwent adjuvant treatment, which included radiation therapy in six patients. None of the six patients receiving radiation therapy experienced a decrement in renal function after radiation therapy. Two patients had died 8.3 and 18.2\u00a0months after the operation of recurrent disease. Median survival has not been reached.\nAfter discharge, all patients were evaluated with contrast-enhanced CT. All grafts were patent, and both kidneys were perfused well. Renal function was monitored by following serum creatinine levels. After the operation, creatinine levels transiently increased, but normalized by discharge. (Table\u00a01) Creatinine values were available for a mean of 6.8\u00a0months postoperatively, with all levels within the normal range. One patient was anticoagulated with clopidogrel, while subsequent patients were treated with aspirin. Our current protocol is to treat all patients with daily aspirin if no clot is noted on the postoperative imaging, and heparin transitioned to coumadin if clot is noted. \nTable\u00a01A Comparison of Creatinine LevelsSerum Creatinine Concentrations (mg\/dL)PatientPreoperativePeakTime of Discharge11.11.11.121.11.41.1311.5140.91.31.1511.3160.91.31.370.80.90.980.81.10.990.80.80.7\nDiscussion\nVenous resection and reconstruction is not uncommon during PD at high volume centers.3,4 Involvement of the vein by malignancy is not always suggested preoperatively by imaging and is often discovered at a time during the operation after commitment has already been made to resection. This fact necessitates that the surgeon has a plan for completing the resection and reconstruction of the venous system.13 Whereas many such cases can be completed with segmental resection and primary end-to-end anastomosis, a number will require either patch repair or interposition grafting. Other groups routinely use the internal jugular vein3 or superficial femoral vein14 as a conduit. While these groups have demonstrated good results with these conduits, the use of the left renal vein for autologous grafting offers some significant advantages and avoids the handling difficulties that can be encountered with the internal jugular vein and the risk of lymphedema or venous thrombosis that can be encountered with use of the superficial femoral vein.\nThe left renal vein provides a graft with good length, good caliber, and is easily accessible. The left renal vein typically provides a graft of 3\u20134\u00a0cm in length when harvested from the junction of the left gonadal and left adrenal vein proximally and the inferior vena cava distally, although some reports have indicated lengths up to 6\u00a0cm.9 The caliber of the graft allows for excellent flow, as demonstrated by CT and Doppler ultrasound. The ease of harvesting the graft also is an important consideration. Exposure of the left renal vein can be accomplished through a standard PD incision, without requiring any further prepping, an additional incision, or the need for an additional operating team. Furthermore, use of the left renal vein leaves the patient with all possible routes of central venous access.\nImportantly, the operation is tolerated well from a renal standpoint. Previous work demonstrated that good collateral flow and functional capacity of the left kidney is preserved despite ligation of the left renal vein. McCullough and colleagues reported that after a right nephrectomy and ligation of the left renal vein for malignancy, only one of three patients experienced transient renal insufficiency.12 In our series, serum creatinine levels transiently increased after operation, but all normalized before discharge. Creatinine levels remained normal throughout follow-up.\nConclusions\nResection offers the only chance at cure for patients with pancreatic cancer, and potentially curative resection may require venous resection. When reconstruction of the venous system necessitates the use of interposition grafting, autologous vein interposition grafts are preferred. The left renal vein provides an additional choice for an autologous graft, and its use is distinguished by ease of harvest and maintenance of renal function. The use of the left renal vein for interposition grafting and patch repair should be considered by surgeons experienced in SMV-PV reconstruction during PD.","keyphrases":["pancreaticoduodenectomy","left renal vein","venous resection","portal vein","superior mesenteric vein","pancreatic cancer"],"prmu":["P","P","P","P","P","P"]}
{"id":"Int_J_Cardiovasc_Imaging-3-1-2121120","title":"The additional value of first pass myocardial perfusion imaging during peak dose of dobutamine stress cardiac MRI for the detection of myocardial ischemia\n","text":"Purpose of this study was to assess the additional value of first pass myocardial perfusion imaging during peak dose of dobutamine stress Cardiac-MR (CMR). Dobutamine Stress CMR was performed in 115 patients with an inconclusive diagnosis of myocardial ischemia on a 1.5 T system (Magnetom Avanto, Siemens Medical Systems). Three short-axis cine and grid series were acquired during rest and at increasing doses of dobutamine (maximum 40 \u03bcg\/kg\/min). On peak dose dobutamine followed immediately by a first pass myocardial perfusion imaging sequence. Images were graded according to the sixteen-segment model, on a four point scale. Ninety-seven patients showed no New (Induced) Wall Motion Abnormalities (NWMA). Perfusion imaging showed absence of perfusion deficits in 67 of these patients (69%). Perfusion deficits attributable to known previous myocardial infarction were found in 30 patients (31%). Eighteen patients had NWMA, indicative for myocardial ischemia, of which 14 (78%) could be confirmed by a corresponding perfusion deficit. Four patients (22%) with NWMA did not have perfusion deficits. In these four patients NWMA were caused by a Left Bundle Branch Block (LBBB). They were free from cardiac events during the follow-up period (median 13.5 months; range 6\u201320). Addition of first-pass myocardial perfusion imaging during peak-dose dobutamine stress CMR can help to decide whether a NWMA is caused by myocardial ischemia or is due to an (inducible) LBBB, hereby preventing a false positive wall motion interpretation.\nIntroduction\nDobutamine stress Cardiac MRI (CMR) is used to detect myocardial ischemia of the left ventricle by means of wall motion analysis during the infusion of high-dose dobutamine [1\u20134]. Previous studies have reported a broad range of sensitivity (83\u201391%) and specificity (80\u201386%) of dobutamine stress CMR for the detection of myocardial ischemia [1\u20133, 5]. It has been proven to be more accurate than dobutamine stress echocardiography [2]. The addition of myocardial tagging even further increased the sensitivity (96%) of dobutamine stress CMR [4]. False positive dobutamine stress CMR\u2019s were described for left bundle branch block (LBBB) or an incidental low interpretability [4]. Means to overcome for these false-positive dobutamine stress CMR\u2019s could increase specificity even further.\nAssessment of myocardial perfusion is used to provide information on the hemodynamic significance of a coronary artery stenosis. Segments with New Wall Motion Abnormalities (NWMA) detected with dobutamine stress CMR should also show perfusion deficits, since perfusion abnormalities precede wall motion abnormalities in the ischemic cascade [6].\nNormal stress perfusion SPECT-results predict a less than 1% annual risk of cardiac death or myocardial infarction, thereby yielding a high negative predictive value [7\u201310]. High negative predictive values are also reported for MR perfusion imaging combined with MRI cine-angiography (100%) in a small study of 15 patients [11].\nThe addition of a perfusion sequence on peak-dose dobutamine may further enhance the interpretation of dobutamine stress CMR, by ruling out false positive findings, through the combined strength of both methods.\nThe purpose of this study is to assess whether the addition of perfusion imaging to dobutamine stress CMR at peak-dose dobutamine reduces the number of false-positive dobutamine stress CMR examinations.\nMaterials and methods\nPatient population\nBetween September 2004 and April 2006, 124 consecutive patients were referred from the department of Cardiology for a dobutamine stress CMR. The study was approved by the local ethical committee. Informed consent was obtained prior to the study, after the nature of the procedure had been explained. All patients had chest pain and an inconclusive diagnosis of coronary artery disease by means of history, ECG recordings at rest and, if performed, during a bicycle exercise test. Patients with an acute coronary syndrome, atrial fibrillation, severe arterial hypertension (>220\/120), CMR-incompatible metallic implants or known claustrophobia were not eligible.\nProtocol for dobutamine stress CMR with myocardial perfusion on peak-dose dobutamine\nTo ensure cardiac response to dobutamine, all anti-anginal medication was stopped 4\u00a0days before the dobutamine stress CMR examination. After the patient was positioned on the scanning table, intravenous access was established via an anticubital vein. ECG monitoring leads, a phased-array surface coil covering the heart, and a brachial blood pressure cuff were applied. A single lead ECG was continuously monitored on the MRI-console. Systolic and diastolic blood pressures were recorded using an automatic device (Welch-Allyn, Emro-medical) at baseline and every 3\u00a0min throughout the procedure. Blood pressure and heart rate were recorded. The imaging methodology of dobutamine stress CMR has been described in detail previously [4]. Dobutamine stress CMR was performed on a 1.5 T system (Magnetom Avanto, Siemens Medical Systems, Erlangen, Germany). Three short-axis cine breath-hold CMR images of the left ventricle, with and without myocardial tagging, were acquired at rest and during incremental dosage of dobutamine up to 40\u00a0\u03bcg\/kg\/min. An ECG-triggered segmental gradient-echo pulse sequence was used: TrueFisp: TR 57.64, ms; TE, 1.1\u00a0ms; \u03b1, 59\u00b0; FOV, 284\u00a0\u00d7\u00a0350\u00a0mm; slice-thickness 6\u00a0mm; and matrix 125\u00a0\u00d7\u00a0192; iPAT 2. Tagging was performed with a standard FLASH grid-sequence: TR, 46\u00a0ms; TE, 3.8\u00a0ms; \u03b1, 14\u00b0; FOV, 284\u00a0\u00d7\u00a0350\u00a0mm; slice-thickness 6\u00a0mm and matrix 141\u00a0\u00d7\u00a0256. The basal plane was taken 1.5\u00a0cm below the mitral valves. The midventricular and apical short-axis views were divided equally over the remaining part of the left ventricle. When a wall motion abnormality (WMA) was detected at baseline, infusion was started at 5\u00a0\u03bcg\/kg\/min, after which the dose of dobutamine was increased to 10, 20, 30 and 40\u00a0\u03bcg\/kg\/min. Starting dose of dobutamine was 10\u00a0\u03bcg\/kg\/min when no WMA was detected at baseline. Imaging started 6\u00a0min after each dose increase and required 3\u00a0min per dose increase.\nTermination criteria for dobutamine stress CMR were the development of new wall motion abnormalities (NWMA) or worsening WMA, a fall of systolic blood pressure of more than 40\u00a0mmHg, marked hypertension above 240\/120\u00a0mmHg, severe chest pain, ventricular arrhythmias and intolerable side effects. NWMA are indicative of myocardial ischemia.\nOn peak-dose dobutamine a bolus injection of 0.1\u00a0mmol\/kg gadolinium-DTPA (Omniscan\u00ae) was given and a perfusion sequence was started: TrueFisp: TR, 150.5 or 163.1\u00a0ms; TE 1.03\u00a0ms; TI 100\/103\u00a0ms; \u03b1 45\/50\u00b0; FOV 300\u00a0\u00d7\u00a0300; slice-thickness 6\u00a0mm; matrix 76\u00a0\u00d7\u00a0128; iPAT 2. The same three slices as the cine and tagging series were acquired.\nDuring the examination a radiologist present in the MR suite to monitor the condition of the patient and to visually evaluate the images. When NWMA\u2019s with a corresponding perfusion deficit were seen, a coronary angiography (CAG) was performed within 3\u00a0weeks. Patients with NWMA\u2019s and a normal first pass perfusion on peak stress entered follow-up.\nImage analysis\nWall motion was scored on six segments of the basal plane, six on the midventricular plane and four on the apical plane. Segmental wall motion was qualitatively graded as 1\u00a0=\u00a0normal or hyperkinesis; 2\u00a0=\u00a0hypokinesis; 3\u00a0=\u00a0akinesis; and, 4\u00a0=\u00a0dyskinesis. Myocardial ischemia was defined as a new (induced) or worsening WMA in at least two segments at consecutive planes of the left ventricle during infusion of dobutamine. Analysis was performed using both cine and tagging images as described in detail previously [4].\nWall Motion Score Index (WMSI) was derived as the mean score of all segments of all short-axis images. WMSI data from the combined analysis of the cine and tagging images were determined from baseline and peak stress images.\nThe first pass perfusion images on peak-dose dobutamine were visually analyzed by an experienced radiologist and cardiologist in a consensus reading after the wall motion analysis by the same physicians. A perfusion abnormality, corresponding to the coronary artery distribution areas, in at least two segments at consecutive short-axis slices or one segment of the most apical slice of the left ventricle was defined as myocardial ischemia.\nFollow-up\nFollow-up data were obtained in September 2006. The present status of the patient was determined by review of the hospital records or contacting the patient\u2019s general physician. The date of the last review was used to calculate follow-up time.\nEvaluated end points were non-fatal myocardial infarction (angina of >30\u00a0min duration and either 2\u00a0mm ST segment elevation in two consecutive ECG leads or a rise in creatine kinase level and its myoglobine fraction two times the upper limit of normal), cardiac death (death in the presence of acute myocardial infarction, significant cardiac arrhythmias or refractory congestive heart failure) and coronary revascularization.\nResults\nPatient population\nFrom the 124 consecutively included patients, in nine patients the examination could not be completed due to: intolerable side effects (nausea, vomiting) in four patients, claustrophobia in three patients. Two patients were excluded due to insufficient image quality, one of whom had an irregular rhythm with triggering difficulties, and the other patient was unable to sustain breath holds.\nTherefore, 115 patients all with good image quality were analysed (93%) with a mean age 61\u00a0\u00b1\u00a011\u00a0years, 20 women (30%). Demographic and hemodynamic data are listed in Table\u00a01.\nTable\u00a01Demographic and hemodynamic dataVariableMean or %Age, years61\u00a0\u00b1\u00a011Female, %29.6Previous myocardial infarction, %38.0Revascularization, %31.0Rest wall motion abnormalities (RWMA), %40.9Body weight, kg78\u00a0\u00b1\u00a012Resting diastolic blood pressure, mmHg87\u00a0\u00b1 11Peak diastolic blood pressure, mmHg78 \u00b1 12Resting systolic blood pressure, mmHg152\u00a0\u00b1\u00a026Peak systolic blood pressure, mmHg151\u00a0\u00b1\u00a031Resting heart rate, bpm79\u00a0\u00b1\u00a015Peak heart rate, bpm119\u00a0\u00b1\u00a021Rate-pressure producta at rest12,030\u00a0\u00b1\u00a03,545Rate-pressure product at peak stress17,935\u00a0\u00b1\u00a04,807Wall Motion Score Index (WMSI) at baseline1.18\u00a0\u00b1\u00a00,32Wall Motion Score Index (WMSI) at peak dose1.21\u00a0\u00b1\u00a00.34Values are expressed as mean\u00a0\u00b1\u00a0SD or percentageaRate-pressure product\u00a0=\u00a0(heart rate)\u00a0\u00d7\u00a0(systolic blood pressure)RWMA\u00a0=\u00a0Rest Wall Motion Abnormality; WMSI\u00a0=\u00a0Wall Motion Score Index\nDobutamine stress CMR with myocardial perfusion on peak-dose dobutamine\nEighteen of the 115 patients (16%) had NWMA of whom 14 (78%) showed perfusion deficits on peak-dose dobutamine in the corresponding segments. Four patients (22%) with NWMA did not have a perfusion deficit. In these four patients, NWMA were attributable to a LBBB as could be confirmed with an independent (stress) ECG. Two of these were inducible LBBB, not known prior to the examination. CAG was positive for the corresponding segments in the 14 patients (100%) with NWMA and a corresponding perfusion deficit.\nNinety-seven patients (84%) had no NWMA. The perfusion images on peak-dose dobutamine showed absence of perfusion deficits in 67 of these 97 patients (69%) and perfusion deficts in 30 (31%). Of these patients 29 (97%) were attributable to a known previous myocardial infarction in the patients history combined with the presence of RWMA. One patient (3%) had a small perfusion deficit inferior which could not be assigned to a known previous event in the patients history. Figure\u00a01 illustrates the course and outcome of the study. In Fig.\u00a02 the additional value of cine or grid tagging images combined with stress first-pass perfusion images is illustrated.\nFig.\u00a01Flow chart illustrating course of the study and outcome NWMA\u00a0=\u00a0New Wall Motion Abnormality; LBBB\u00a0=\u00a0Left Bundle Branch Block; CAG\u00a0=\u00a0Coronary AngiogramFig.\u00a02Short axis views at peak-dose dobutamine. Cine image illustrating a NWMA inferior. (A) Perfusion abnormality in the corresponding segment. (B) Dyskinetic septal wall in another patient. (C) No perfusion abnormalities in the corresponding segment. (D) Dyskinetic septal wall in C was due to a LBBB, this differentiation could be made by a perfusion sequence on peak-dose dobutamine. Arrows indicate the wall motion abnormality or perfusion abnormality NWMA\u00a0=\u00a0New Wall Motion Abnormality; LBBB\u00a0=\u00a0Left Bundle Branch Block\nFollow-up results (median 13.5\u00a0months, range 6\u201320\u00a0months) were obtained from all patients with NWMA\u2019s and a normal first-pass perfusion. None of these patients had cardiac events or revascularizations at follow-up.\nDiscussion\nThis study demonstrates that adding first-pass myocardial perfusion imaging during peak-dose dobutamine has direct clinical relevance for the interpretation of dobutamine stress CMR examinations, by increasing the interpretation of possible wall motion abnormalities (NWMA) of the left ventricle. The addition of first pass perfusion relies on the conceptual use of the ischemic cascade. Ischemic wall motion abnormalities are preceded by perfusion abnormalities, therefore visualized NWMA due to myocardial ischemia should also show perfusion abnormalities. A normal perfusion study is used to identify wall motion abnormalities not due to myocardial ischemia, and an abnormal corresponding perfusion deficit is used to confirm NWMA, indicative for myocardial ischemia.\nThis is the first study to assess the additional value of a first pass myocardial perfusion imaging sequence on peak-dose dobutamine during a dobutamine stress CMR. Previous studies have reported a broad range of sensitivity and specificity for dobutamine stress CMR in the detection of myocardial ischemia [4]. In this protocol we have chosen for a prolonged infusion time of dobutamine from 3 to 6\u00a0min without atropine and the use of the target heart rate rule, as described before [4, 12\u201317].\nThe use of the target heart rate rule from a physical exercise based concept can not be generalized to a pharmacological stress setting and has been questioned in several reports [12\u201314, 18]. It has also been shown that the target heart rate rule can not be extrapolated to a pharmacological stress examination [19]. In a recent overview of published dobutamine stress CMR examinations by Strach et\u00a0al. [20], the approach we used [4] showed to provide the highest diagnostic accuracy for significant coronary artery disease defined by a >50% luminal stenosis on a coronary angiogram. Specificity, although already high, was lowered, according to our opinion, in part due to LBBB. This could be overcome with a perfusion sequence, taking into account the high negative predictive value of normal myocardial perfusion imaging. The additional value of myocardial perfusion MRI could be used for this purpose and may add significant diagnostic information.\nIn this study we added a perfusion sequence on peak-dose dobutamine in all patients. The results show a good agreement between the absence of NWMA and myocardial perfusion. The main purpose of this study was to assess the additional value of first-pass perfusion imaging in the presence of NWMA. Our recommendation for future clinical use is to add a first pass perfusion sequence on indication, namely if there is doubt on whether a NWMA is due to myocardial ischemia. In this way, the specificity of dobutamine stress CMR can be even further increased. By combining dobutamine stress CMR with myocardial perfusion, one still has the opportunity to examine for viability, which seems even superior to scar quantification [21].\nAbsolute specificity values cannot be given with this study, because a CAG was not performed in case of a negative dobutamine stress examination. The outcome of the dobutamine stress CMR and myocardial perfusion on peak-dose dobutamine was used as a direct arbiter for subsequent clinical follow-up. In this respect follow-up was considered the reference standard. None of the patients with NWMA\u2019s and a normal myocardial perfusion had an adverse outcome at follow-up. Therefore, it is reasonable to state that adding first pass myocardial perfusion imaging on peak-dose dobutamine increases the specificity of dobutamine stress CMR in this patient group.\nOur data could have been influenced by a referral bias, but in light of previous results, we found it not justifiable to perform a CAG in case of a negative examination [12]. Furthermore, the high negative predictive value of dobutamine stress CMR examination without perfusion imaging has been proven before [12, 22]. Mahrholdt et\u00a0al. [23]. examined 139 patients with a LBBB. All 139 patients had fixed perfusion defects with SPECT, 19 could not be confirmed with CAG. Rest wall motion analysis and myocardial perfusion (between 5 and 30\u00a0min after contrast administration) was performed on these 19 patients using MRI. All 19 patients showed septal wall motion abnormalities, but none showed subendocardial or transmural contrast enhancement. This also indicates that contrast enhanced imaging can help differentiate between WMA on the basis of coronary artery disease or a LBBB.\nLong term follow-up will provide information about the case in which a small perfusion abnormality was seen inferior without NWMA. Whether this abnormality is a \u201ctrue\u201d abnormality or an artefact is unclear. This patient did not have any adverse cardiac event at 14\u00a0months follow-up.\nAnother limitation of our study is the fact that images were analysed semi-quantitatively. Quantitative wall motion analysis could possibly provide additional information, but due to the time-consuming nature this is not yet feasible in clinical practice. Furthermore, no decisions can be made during the examination, which we believe is crucial, since overstressing may lead to serious complications [13].\nVisual analysis of wall motion and perfusion images was performed in a consensus reading by an experienced radiologist and cardiologist. This can be regarded as a limitation since the observers are not blinded for the previous wall motion images. However, this does represent routine clinical practice and is fundamental to the underlying concept of using first-pass perfusion imaging in the presence of NWMA. In recent studies the inter-observer agreement of dobutamine stress CMR was investigated and good agreement was found [24, 25].\nIn a clinical post-infarct setting, delayed contrast enhancement imaging may provide valuable diagnostic information. To our opinion, this would in our study not have provided additional information regarding the fact whether a NWMA was caused by myocardial ischemia or a non-ischemic cause, and guide the necessity for an invasive coronary angiogram. Cine and grid tagging images were acquired at rest to look for rest wall motion abnormalities. New wall motion abnormalities in this setting would represent myocardial ischemia (whether or not in the presence of a previous myocardial infarction). Delayed contrast enhancement imaging was therefore not performed in this protocol.\nIn this protocol we only acquired stress first-pass perfusion images. Perfusion imaging at rest may be very useful in stress-rest perfusion imaging studies, but the main focus in this protocol is on a normal perfusion in case of NWMA. A rest perfusion exam would in this context not provide additional information (the rest perfusion exam will also be normal). In this way the protocol is not unnecessarily prolonged and a second bolus injection of gadolinium-DTPA can be omitted.\nConclusion\nGood agreement exists between the absence of NWMA and a normal myocardial perfusion. Furthermore, a perfusion sequence on peak-dose dobutamine can help decide whether a NWMA is caused by ischemia or is due to a LBBB. First pass myocardial perfusion during peak-dose dobutamine can be used as an additional tool to reduce the number of false-positive NWMA\u2019s, to improve the detection of myocardial ischemia.","keyphrases":["stress","mri","ischemia","heart","myocardium"],"prmu":["P","P","P","P","U"]}
{"id":"Theory_Biosci-4-1-2254468","title":"Tree thinking cannot taken for granted: challenges for teaching phylogenetics\n","text":"Tree thinking is an integral part of modern evolutionary biology, and a necessary precondition for phylogenetics and comparative analyses. Tree thinking has during the 20th century largely replaced group thinking, developmental thinking and anthropocentricism in biology. Unfortunately, however, this does not imply that tree thinking can be taken for granted. The findings reported here indicate that tree thinking is very much an acquired ability which needs extensive training. I tested a sample of undergraduate and graduate students of biology by means of questionnaires. Not a single student was able to correctly interpret a simple tree drawing. Several other findings demonstrate that tree thinking is virtually absent in students unless they are explicitly taught how to read evolutionary trees. Possible causes and implications of this mental bias are discussed. It seems that biological textbooks can be an important source of confusion for students. While group and developmental thinking have disappeared from most textual representations of evolution, they have survived in the evolutionary tree drawings of many textbooks. It is quite common for students to encounter anthropocentric trees and even trees containing stem groups and paraphyla. While these biases originate from the unconscious philosophical assumptions made by authors, the findings suggest that presenting unbiased evolutionary trees in biological publications is not merely a philosophical virtue but has also clear practical implications.\nIntroduction\nModern evolutionary biology relies on population thinking and tree thinking (O\u2019Hara 1997). Both ways of thinking have replaced earlier philosophies that turned out to be incompatible with the empirical basis (Hull 1965; Simberloff 1980; Sober 1980). The transition from essentialism to population thinking has been described by Ernst Mayr (1988, 1996; but see Levit and Meister 2006; Winsor 2003). This transition followed directly from the Darwinian revolution (Darwin 1859): it is organisms with unique trait combinations that make up populations, that constitute biodiversity, and that are the basis of evolutionary change. The earlier paradigm, which viewed organisms as blueprints of unchangeable essences, was incompatible with these observations.\nEqually important to modern biology is tree thinking. Each and every species occupies one specific place in the tree of life. Consequently all species have shared certain parts of their history with all other living and extinct species. The degree to which this history has been shared, allows us to predict the extent and way in which species differ from and resemble each other. A description of biodiversity is impossible or at least incomplete without taking this knowledge into account. It is necessary both for the idiographic activity of reconstructing evolutionary relationships between species, and for the nomothetic activity of testing hypotheses about biological laws of nature (Ghiselin 1997; Hull 1999). Phylogenetic reconstruction (Felsenstein 2004) and the comparative method (Martins and Hansen 1996) are thus entirely unthinkable without tree thinking.\nTree thinking became widely accepted somewhat later than population thinking, as it can be said to have originated with phylogenetic systematics (Hennig 1950). However, this does not mean that tree thinking has superseded population thinking (O\u2019Hara 1997). Both perspectives are compatible, and both are necessary in biology today. As Robert O\u2019Hara (1997) has shown, what has been replaced by tree thinking, are the earlier paradigms of group thinking and developmental thinking in evolutionary biology. Group thinking had equated systematics with classification, i.e. it perceived taxonomic groups as units that could be defined rather than entities that had to be discovered. Group thinking thus represented essentialism in systematics, because it failed to understand that taxa are evolving entities and thus, ontologically speaking, individuals (Ghiselin 1974, 1997; Hull 1976). Developmental thinking, on the other hand, had assumed that organic evolution was a teleological process (Ghiselin 2002; Hull 1974; Mayr 1988). It was thus based upon the ancient idea that change in nature has an ultimate goal. This is incompatible with the irregular and divergent nature of evolution.\nOne might presume that the replacement of group and developmental thinking by tree thinking is of merely historical interest\u2014a subject to be dealt with by historians of science, just as the replacement of essentialism by population thinking. To the contrary, I here argue that the transition of both group thinking and developmental thinking to tree thinking is still happening, and that it deserves more attention by those who teach phylogenetics and who communicate phylogenies. The transition is still happening in the sense that students of biology have to accomplish it in the course of their \u201cacademic ontogenies\u201d: tree thinking is an ability that has to be acquired and cannot be taken for granted by teachers. Sadly, many text books indirectly and implicitly promote developmental thinking. This further aggravates the problem. Given the importance (even necessity) of tree thinking in order to fully appreciate the meaning of evolutionary results, this problem deserves attention.\nMaterials and methods\nI devised a short questionnaire which was handed out during the first lesson in several classes of a university course in systematics and evolution (at Universitetet i Troms\u00f8, Norway). The course was intended for students who had studied biology for several terms, but this was not a formal requirement. The students who followed normal progression had been taught evolutionary principles and processes in earlier courses. The same questionnaire was used in a similar course at a regional university college (H\u00f8gskolen i Bod\u00f8, Norway). The students were asked to answer the questions, using the time they needed, and were assured anonymity. They did not have access to any books or notes.\nThe questions (and correct answers) were:\u201cGiven the following evolutionary tree [reproduced in Fig.\u00a01], where letters symbolise species: which species is (are) most closely related to species B?\u201d1(The correct answer was \u201cC, D, E and F\u201d.)\u201cHow many classes of vertebrates and angiosperms are there? Fill in the scientifically correct answer:...\u201c(There is no scientifically correct answer to this question.)\u201cWhat are the differences between species and genera, or between orders and classes? Give one or more keywords that describe these differences. (a) Differences between species and genera:...(b) Differences between orders and classes:...\u201c(The correct answer was that species differ from genera [and orders and classes], for instance by being populations within which gene flow can occur. The difference between orders and classes is merely one of definition, none of nature.)\u201cWhat does the \u2018tree of life\u2019 look like? Please draw the evolutionary tree which describes the relationships between groups of organisms. Include as many groups as you are able to recall\u201d.Fig.\u00a01Example phylogeny, where letters symbolise species. Not a single student (n\u00a0=\u00a031) was able to give the correct answer to the question \u201cwhich species is\/are most closely related to species B\u201d\nResults\nTwenty one university students returned questionnaires that were at least partly answered. On average they had previously taken biology courses for 1.6\u00a0years of full time study load (median 1.5, range 0.3\u20134.0\u00a0years). Four were graduate students, the remainder were undergraduates. A further ten students of the regional university college returned the questionnaire.\nNot a single correct answer was given to question 1. In most cases, species A (cf. Fig.\u00a01) was the whole answer or part of the answer (17 of 20 university students, and 8 of 9 college students).\nOf 20 answers to question 2, 18 consisted of numbers. One student entered strokes, and 1 left the answers blank.\nIn response to question 3, 5 of 18 respondents answered that species were fundamentally different from genera, while there was no such difference for orders and classes. The remainder answered that there was no important differences at all (n\u00a0=\u00a08) or that there were important differences between both species and genera, and between orders and classes (n\u00a0=\u00a05).\nAs an answer to question 4, 20 university students produced a drawing, however, 7 of them did not enter any names, and 1 entered one name only. The remainder provided between 4 and 23 taxon names. See Fig.\u00a02 for an example.\nFig.\u00a02Phylogenetic tree drawn by a university student. The question posed was \u201cWhat does the \u2018tree of life\u2019 look like? Please draw the evolutionary tree which describes the relationships between groups of organisms. Include as many groups as you remember\u201d. Note the prominent position of mammals and the unresolved \u201cside branches\u201d. Taxon names provided by the student were translated verbatim into English, the drawing itself is unchanged\nSeven of 13 students depicted 1 to 9 extant taxa as stem groups in their drawings. A total of 14% of all names provided were drawn as stem taxa. 11 of 13 students drew 1 to 11 branches which they named with paraphyletic groups. A total of 31% of all names provided referred to paraphyla.\nAs regards the placement of our own species, or the taxon containing Homo sapiens, the placement of our taxon seemed arbitrary in four drawings, and in two cases no names provided referred to taxa that contained our species. Our taxon was placed at top of the drawing in five cases, and at the outermost position in two cases. This means that 7 of 13 respondents gave a special position to our taxon. Counting the number of nodes at which the path to H. sapiens leads to the uttermost or uppermost position, a continuous \u201chuman utterness score\u201d can be calculated (Sandvik 2008). Combined for the eleven drawings to which it was applicable, the average score was 0.90 (43 of 48 nodes), which is highly significantly biased in favour of man (P\u00a0<\u00a010\u22128).\nThe resolution of the tree also favoured our species. I calculated scores of the relative attention the tree drawings devoted to three species, viz. H. sapiens (Mammalia), Vanessa atalanta (Lepidoptera) and Veronica fruticans (Lamiales), in the following manner: a score of 1 was assigned if the species was named, of 2 if at least the \u201corder\u201d was named, of 3 if at least the \u201cclass\u201d was named, of 4 if at least the \u201cphylum\u201d was named, of 5 if a higher taxon was named, and of 6 if no named branch contained the species. The average attention score was 3.6\u00a0\u00b1\u00a01.5 (SD) for man, 4.8\u00a0\u00b1\u00a01.2 for the butterfly, and 4.6\u00a0\u00b1\u00a01.3 for the flower. A one-tailed Wilcoxon two-sample tests showed that man received more attention than the other two species (W\u00a0=\u00a0155.5, P\u00a0<\u00a010\u22125). If using the \u201chuman attention score\u201d defined elsewhere (Sandvik 2008), a continuous measure that expresses the degree to which tree resolution favours our own species, the average for the 13 tree drawings was 0.60, which is highly significantly biased in favour of man (P\u00a0<\u00a010\u22124).\nDiscussion\nO\u2019Hara (1997:327) remarked that \u201cbeginning students in biology should be taught how to read trees [...] just as beginning students in geography need to be taught how to read maps\u201d. My findings drastically support this opinion. None of the undergraduate or graduate students in my (admittedly small) sample was able to correctly interpret a simple cladogram (Fig.\u00a01). The cladogram was certainly not the first evolutionary tree that the students had met, since most had studied biology for several terms, including introductory courses in evolutionary biology.\nIt does not require many sentences to explain how a cladogram should be read. The concept of evolutionary relatedness is not hard to grasp once it is explained. If it is not, however, erroneous conceptions seem to be able to survive for several years during prospective biologists\u2019 educational careers. At the end of my course all university students were able to give correct interpretations of cladograms. Matters differed for the students of the regional university college. These students were asked which of the small cladograms in Fig.\u00a03b\u2013i were compatible with the large cladogram (Fig.\u00a03a). This latter question was posed after the meaning of cladograms had been explained and after a considerable amount of time had been spent practicing the reading of cladograms. Still, only one answer out of fourteen correctly identified all of the three compatible cladograms. The remainder failed to identify at least one compatible cladogram and\/or indicated at least one incompatible tree as compatible. This sobering result might be related to the fact that many of the college students simply needed some biology courses in order to study, e.g. fishery. They might thus not have been genuinely interested in biology per se, so that these findings would need to be replicated with university students. Still, it is independent evidence that reading cladograms is very much an acquired ability rather than one that can be presupposed.\nFig.\u00a03Nine phylogenetic trees over Mollusca with different topologies. a was used as a reference phylogeny. Students were asked which of the eight small trees were compatible with the reference tree. The eight topologies b\u2013i differ from a in either the phylogenetic relationships displayed (making them incompatible with a), resolution of some of the branches, placement of taxa along the left\u2013right axis, or a combination of those (only c, g and h are compatible with a. In the tree distributed to students, all names were given as unabbreviated Norwegian vernacular names)\nIn their answers, the students proved to pay more attention to how the taxon names at the tree tips were ordered along the left\u2013right axis, than to the topology of the cladogram. Given that the information of cladograms is conveyed in the branching order of taxa, while the left\u2013right ordering is arbitrary, this wrong focus necessarily leads to wrong conclusions.\nThe main problem is presumably that students simply have not been taught how to read cladograms. Teachers may often assume that cladograms are self-explicatory graphic devices to illustrate phylogenetic relationships, when in fact their interpretation is not trivial. As Halstead (1978:760) put it: \u201cCladograms are difficult enough for experts in the field to comprehend fully\u201d. Ironically, what he was advocating as the obvious alternative was phylograms\u2014which were meant to not only express phylogenetic relationships, but, in addition, phenotypic similarity, distribution through geologic time and species number. As such, phylograms are not only harder to interpret for untrained readers than cladograms. They may also convey incorrect information on any one of the aspects because they represent a two-dimensional compromise of several multidimensional measures.\nRegrettably, the presentation of systematics in text books has been lagging behind the cutting edge of systematic research by several decades. It is thus only quite recently that most biological text books have replaced phylograms with cladograms. A less appreciated fact is that even displaying cladograms does not guarantee that the information provided is unbiased, as many cladograms are drawn in an anthropocentric fashion (Sandvik 2008). It might thus well be that one source of confusion for students is the ambiguity of evolutionary tree drawings in text books. Drawings of evolutionary trees cannot be clearer than their authors\u2019 thoughts about evolutionary processes. And the latter have long been rather muddled, as can be exemplified by the occurrence of \u201cstem groups\u201d in many text book trees (e.g. Villee et al. 1984: Fig. 19.6; Willmer 1990: Fig. 14.2, which has even been reprinted by a number of other text books). However, stem groups have never existed in nature. A taxon above the species level cannot possibly give rise to other taxa\u2014nor to anything else (Ghiselin 1997). One might say, therefore, that stem groups are just another way of expressing (or, worse still, trying to hide) ignorance. That some taxonomists have chosen to even give scientific names to their ignorance (e.g. \u201cProcoelomata\u201d; Bergstr\u00f6m 1989), does not really make the matter more transparent for biologists under education. No wonder, then, that stem groups occurred in 54% of the student drawings.\nOne problem is thus that many tree drawings which students meet during their studies are biased and thereby distort the evolutionary understanding of their readers. Another is that this distorted understanding even impinges on the interpretation of correctly drawn phylogenetic trees. In other words, both problems re-enforce each other. I have demonstrated elsewhere that even cladograms in phylogenetic text books are biased in an anthropocentric way (Sandvik 2008). In terms of ordering of taxa and differential resolution of branches, otherwise correct cladograms may distort the understanding of evolution. These findings make the evidence presented here even more alarming: If taxa are ordered in an anthropocentric manner in most cladograms, and if students rely more on ordering than topology, this affects the interpretation of relationships. The taxon containing our own species is most often placed in the rightmost position in cladograms (Sandvik 2008). This can be illustrated with Fig.\u00a04, which displays the phylogeny of Osteognathostomata in an anthropocentric manner. The topology of the cladogram is the same as in Fig.\u00a01. Accordingly, many untrained student of biology can be expected to conclude that coelacanths are more closely related to ray-finned fishes than to mammals.\nFig.\u00a04Cladogram depicting the phylogeny of bony vertebrates (Osteognathostomata). The cladogram is anthropocentric in that both the resolution of its branches and the ordering of taxon names are biased in favour of mammals. Note that the tree topology is identical to Fig.\u00a01. See text for further details\nThe students\u2019 inability to answer question 1 (Fig.\u00a01) might be argued to have other reasons. An obvious alternative explanation would be that the error was caused by a simple misunderstanding of the word \u201crelationship\u201d, and that it is sufficient to tell students that relationship, as used in evolutionary biology, is a technical term meaning \u201cthe relative recency of common ancestry\u201d (Mayr 1974). However, the question to the students was posed in Norwegian, were the word \u201cslektskap\u201d only has the latter meaning. In other words, while knowledge of the precise meaning of \u201crelationship\u201d is necessary (especially in English), it is not sufficient for students to understand cladograms.\nIn passing I would like to mention a speculation on the reason why phylogenetic systematics and cladistic methodology was rather quickly accepted in Germany (Ax 1977; Remane 1956; Schlee 1969), but provoked intense debates in English-language journals (verifiable with almost any issue of Systematic Zoology from the 1970s). I suspect that part of the problem was semantic. The German word for \u201crelationship\u201d is \u201cVerwandtschaft\u201d, but while the English word has all kind of abstract and symbolic connotations, including overall similarity, the German term is reserved for true, genealogical bonds (as is the Norwegian \u201cslekt\u201d, see footnote 1). The statement that for instance the lungfish is more closely \u201cverwandt\u201d to the cow than to the salmon is quite uncontroversial in German. On the other hand, the statement that the lungfish is more closely related to the cow than to the salmon, was able to create a heated discussion\u2014which was only peripherally concerned with the actual phylogeny of the groups concerned (Gardiner et al. 1979; Halstead 1978; Halstead et al. 1979).\nA final observation concerns the importance attributed to Linnean categories (i.e. labels such as \u201cfamily\u201d, \u201corder\u201d, etc.) by students. It is well-established that Linnean categories above the species level do not carry information, that they are not comparable across taxa, and that it is entirely arbitrary to which taxa they are assigned in the first place (de Queiroz and Gauthier 1992; Donoghue 2001; Ereshefsky 1994, 2001, 2002). Many taxonomists have abandoned categories altogether, simply referring to taxa by their names. However, few students are aware of the arbitrariness of Linnean categories. In many undergraduate text books, the enumeration (!) of animal \u201cphyla\u201d or insect \u201corders\u201d seems to be more important than the discussion of interrelationships between the taxa concerned (e.g. Barnes et al. 1998). This leaves students with the impression that categories must express something. Unless told otherwise, they tend to make up their own explanations. As my results indicate, only a tiny proportion of students seems to question the existence and reality of Linnean categories. Of course, blank answers to question 2 do not necessarily indicate the deliberate rejection of categories\u2014the two students may also have wished to give a number, but were unable to \u201crecall the correct one\u201d. On the other hand, question 2 was clearly a leading (not to say, loaded) question, which may have biased the respondents into thinking that a number was the only acceptable answer. Still, findings from question 3 show that at least 27% of the students perceive the distinction between \u201corders\u201d and \u201cclasses\u201d to be a reflection of real differences between natural levels of organisation.\nThe findings presented here re-enforce earlier reports that reading cladograms is an ability that has to be practiced (O\u2019Hara 1992, 1997). Even though the transition from developmental thinking to tree thinking is more or less completed in the science of systematics at the collective level, it has to be accomplished anew by every generation of biology students on the individual level. This must not be forgotten when teaching phylogenetics or writing or illustrating text books. Other topics that need to be addressed explicitly are the artificiality of Linnean categories, the non-existence of stem taxa, and the arbitrariness of paraphyla. It does not seem to be sufficient to \u201cget the facts right\u201d in teaching and text books. Students tend to fall back to group and developmental thinking unless explicitly told otherwise.","keyphrases":["group thinking","evolutionary tree","stem group","essentialism","cladogram","biological education"],"prmu":["P","P","P","P","P","R"]}
{"id":"Virchows_Arch-3-1-1888717","title":"Assessment of epidermal growth factor receptor (EGFR) expression in primary colorectal carcinomas and their related metastases on tissue sections and tissue microarray\n","text":"Metastatic colorectal carcinomas (CRC) resistant to chemotherapy may benefit from targeting monoclonal therapy cetuximab when they express the epidermal growth factor receptor (EGFR). Because of its clinical implications, we studied EGFR expression by immunohistochemistry on tissue sections of primary CRC (n=32) and their related metastases (n=53). A tissue microarray (TMA) was generated from the same paraffin blocks to determine whether this technique could be used for EGFR screening in CRC. On tissue sections, 84% of the primary CRC and 94% of the metastases were EGFR-positive. When matched, they showed a concordant EGFR-positive status in 78% of the cases. Moreover, staining intensity and extent of EGFR-positive cells in the primary CRC correlated with those observed in the synchronous metastases. On TMA, 65% of the primary CRC, 66% of the metastases, and 43% of the matched primary CRC metastases were EGFR-positive. There was no concordant EGFR status between the primary and the metastatic sites. A strong discrepancy of EGFR status was noted between TMA and tissue sections. In conclusion, EGFR expression measured in tissue sections from primary CRC and their related metastases was found to be similar and frequent, but it was significantly underestimated by the TMA technique.\nIntroduction\nEpidermal growth factor receptor (EGFR), also called HER1, is a member of the transmembrane tyrosine kinase receptor family. In normal and malignant cells, the activation of EGFR receptor cascades has multiple consequences, such as cell growth, differentiation, and proliferation [10]. The EGFR signaling pathway may also promote malignant transformation, angiogenesis, and metastatic dissemination [10]. To block the activation of this receptor, targeted therapies have been developed, representing a new and promising strategy for cancer management [15]. Thus, antibodies directed against EGFR have recently been integrated into the treatment of metastatic colorectal carcinomas (CRC) resistant to chemotherapy [3, 23]. Such a strategy requires an EGFR immunohistochemical assessment by the pathologist to ensure that the targeted receptor is present in the tumor. Cancer patients included in these protocols are not always treated in the same institute for their primary disease and for their metastatic disease. Moreover, metastases are not always available for histology. It is thus possible that EGFR expression be evaluated in only one of the tumor locations (i.e., primary or metastatic). However, few and controversial data are available concerning the EGFR status in its primary site and the related metastatic sites [6, 11, 13, 24].\nThe first goal of our study was to analyze whether EFGR expression in colorectal primary tumors and their metastatic sites was similar or not. To answer this question, formalin-fixed and paraffin-embedded primary CRC from 32 patients and their related synchronous metastases (n=45) were examined using a standardized EGFR immunohistochemical procedure. Additionally, metachronous metastases (n=8) from four out of 32 patients were also processed for EGFR immunohistochemistry.\nThe second goal of this study was to evaluate EGFR expression on a tissue microarray (TMA) generated from the same paraffin blocks and to compare these results with those obtained by using the whole tissue sections. This comparison was carried out to determine whether the increasingly used TMA technology is a reliable tool for high-throughput EGFR screening in CRC [17, 26].\nMaterials and methods\nPatient characteristics\nClinical and histopathological characteristics of the patients are reported in Table\u00a01. Thirty-two patients, having undergone surgical resection of both the primary tumor and the corresponding metastatic sites in our institution between 2000 and 2004, were selected from a pathological database of colorectal cancer cases. There were 15 men (47%) and 17 women (53%). Median age at surgery was 60\u00a0years (ranging from 45 to 81\u00a0years). Twenty-one patients (66%) had colon cancer and 11 (34%) had rectal cancer. The most common histological type was adenocarcinoma (91%), the remaining three cases (9%) being mucinous carcinomas. According to the latest TNM classification [28], all the patients had stage-IV disease because of synchronous metastases at the time of diagnosis. Among them, four patients received neoadjuvant radiotherapy (n=2) or radiochemotherapy (n=2) for rectal carcinomas and six others received neoadjuvant chemotherapy before surgical resection of their metastases. Eighty-five paraffin-embedded tumor samples from CRC (n=32) and liver, pulmonary, and ovarian metastases (n=51, 1, and 1, respectively) were available. Synchronous metastases (n=45) were unique in 23 patients and multiple (n=22) in nine patients, consisting of two, three, or four locations in the liver (Table\u00a01). Metachronous metastases (n=8) from four patients were also collected and assessed for EGFR immunohistochemistry. They consisted of five multiple liver metastases for one patient, one single liver metastasis for two patients, and one pulmonary metastasis for one patient. Synchronous and metachronous metastases were obtained by metastasectomy. All resected samples were received fresh, then immediately fixed in 10% pH neutral formalin for 48\u00a0h and embedded in paraffin before processing. \nTable\u00a01Clinical data and EGFR status among primary CRC and their related metastasesClinical dataEGFR statusa on tissue sectionsCase no.SexAge (years)pTNMLocationNeoadjuvant treatmentPrimary siteSynchronous metastases (location number)Metachronous metastases (location number)1234123451M60T3N1M1C+++++2M60T3N2M1RRCT++++3M66T2N0M1R++++4M49T3N2M1RCT++++++++5M70T3N1M1C+++6F74T3N1M1RRCT+++7F59T3N2M1RCT+++\u22128M71T3N2M1C+++9F55T3N2M1R\u2212+\u221210F61T4N1M1CCT+++11F68T3N1M1CCT+++12M75T3N1M1R++13M72T3N1M1C++14M54T3N0M1C++15F55T4N1M1C++16F57T4N2M1C++17F52T3N1M1R++18M53T3N0M1C++19M62T4N2M1C++20F77T3N2M1C++21F49T3N1M1C++22F51T3N2M1C++23M62T3N2M1RRT++24F65T3N1M1CCT++25M56T4N1M1CCT++26F47T3N1M1RRT++27F59T3N1M1C+\u221228F45T3N2M1C+\u221229F81T3N1M1R\u2212+30M58T2N0M1C\u2212+31M69T3N1M1C\u2212+32F68T3N2M1C\u2212+Liver metastases in all cases except for a metachronous pulmonary metastasis and a synchronous ovarian metastasis in patients 7 and 15, respectivelyC colon, R rectum, RCT radio-chemotherapy, CT chemotherapy before surgical resection of metastasis, RT radiotherapyaPositive if >1% of tumor cells expressed EGFR\nEGFR immunohistochemistry\nEGFR immunohistochemistry was carried out on freshly sectioned tissue slides which has been shown to be critical for optimal EGFR immunoreactivity [1]. Immunohistochemistry of the tumors was performed by using the Dako autostainer and the EGFR pharmDx\u2122 kit (K1494, Dako, Glostrup, Denmark), according to the manufacturer\u2019s instructions. Briefly, 4-\u03bcm-thick sections were mounted on silanized slides and allowed to dry overnight at 37\u00b0C. After deparaffinization and rehydratation, slides were incubated with 3% hydrogen peroxide for 5\u00a0min. After washing with the supplied buffer, tissue sections were covered for 5\u00a0min with a proteinase K solution. The slides were then incubated for 30\u00a0min with the primary mouse anti-EGFR monoclonal antibody (clone 2-18C9), which binds to a formalin-resistant epitope near the ligand-binding site on the extracellular domain of EGFR. After two rinses in buffer, the slides were incubated with a horseradish peroxidase-labeled polymer coupled to secondary antibodies for 30\u00a0min. Tissue staining was visualized with diaminobenzidine as substrate-chromogen solution. Slides were counterstained with hematoxylin, dehydrated, and mounted. Negative control sections were processed without the primary antibody but with an irrelevant murine IgG1 supplied with the kit. Negative and positive control cell slides (CAMA-1 and HT29 cell lines, respectively), provided with the EGFR pharmDx\u2122 kit, were also used to ensure that each assay run was performed appropriately and according to protocol specifications. Furthermore, perineurium and normal mucosae on primary CRC and hepatocytes surrounding the metastases were considered as positive internal controls on tumor slides.\nTissue microarray\nTissue blocks appearing to have enough material upon gross inspection were initially selected and hematoxylin\u2013eosin-stained sections were evaluated by two observers (F. Bibeau and F. Boissi\u00e8re-Michot) for the presence of carcinoma. The areas to be used for the construction of the TMAs were marked on the slide and the donor block. Particular attention was made to select invasive cells near the tumor front. The tissues corresponding to selected areas were sampled using a manual arraying instrument (Manual Tissue Arrayer 1, Beecher Instruments, Sun Prairie, WI, USA). Two TMAs were constructed using 0.6-mm tissue cores. The sampling consisted of three malignant cores from different areas of the tumor from a single case of invasive colorectal cancer, placed at specified coordinates. When possible, normal mucosa was also sampled as internal control. After the arraying was completed, TMA blocks were sectioned at a thickness of 4\u00a0\u03bcm. One section was stained with hematoxylin\u2013eosin, and EGFR immunostaining using the immunohistochemical system kit EGFR pharmDx\u2122 was performed on the adjacent section. Among the 32 cases sampled, 31 primary CRC, 29 metastatic sites, and 28 matched sites were assessable with this technology.\nEGFR immunohistochemical staining scoring system\nEGFR assessment was realized according to the EGFR pharmDx\u2122 scoring guidelines. EGFR expression was evaluated on sections including the deepest region of tumor invasion as this region was shown to contain the greatest density of EGFR-positive cells [6]. Results were reported as positive when a complete or incomplete circumferential membrane staining was observed in at least 1% of the tumor cells. Staining was defined as immunostaining of tumor cell membranes above background level and scored as follows: 1+=weak, 2+=moderate, and 3+=strong. The absence of membrane staining or cytoplasmic staining was reported as negative. The percentage of stained cells was assessed as follows: 1\u201310, 10\u201350, and >50%. Slides were scored by two pathologists (F. Bibeau and J.C. Sabourin), who were blinded to the patients\u2019 characteristics. In the case of disagreement (three out of 91 specimens), the EGFR status was determined by consensus after simultaneous dual reexamination.\nIn addition to these standardized criteria, a semiquantitative analysis was performed taking into account both the staining intensity and the percentage of positive tumor cells.\nStatistical analysis\nThe cut-off value for negative\/positive expression of EGFR was 1% of tumor cells displaying a membranous staining, as specified by the EGFR pharmDx\u2122 scoring guidelines. Data were summarized by frequency and percentage for categorical variables and by means, standard deviations, median, and range for continuous variables. For TMA analysis, the percentage of positive cells was calculated from the mean of triple cores.\nThe correlation between the percentage of positive cells observed on primary tumors and matched metastases was evaluated with the Spearman correlation coefficient. Associations between categorical variables were examined using McNemar\u2019s chi2 test. A P value of less than 0.05 was considered statistically significant. Due to the small number of patients with metachronous metastases, only the correlation between the matched primary CRC and the synchronous metastatic sites was made.\nResults\nEGFR expression of primary CRC and their related metastases on tissue sections\nEGFR reactivity was not homogeneous throughout the tumors. EGFR immunostaining was mainly observed in the deepest region of the primary CRC or at the periphery of metastases, especially in the liver. Of note, EGFR immunoreactivity was often greater in isolated tumor cells and in small clusters of tumor cells.\nEighty-four percent of primary CRC (27 out of 32) and 94% of the synchronous metastases (30 out of 32) displayed EGFR reactivity in more than 1% of the tumor cells (Table\u00a01). Overall, all patients displayed an EGFR-positive status considering EGFR-expression in the primary tumor and\/or the metastatic sites. EGFR expression was positive at both sites in most of the cases (25 out of 32; 78%) and rarely in only one site, i.e., primitive (two out of 32; 6%) or metastastic (five out of 32; 16%). However, the discordant cases (i.e., positive primary sites with negative metastasis and conversely) were not statistically significant (P=0.453).\nNine (28%), 10 (31%), and eight (25%) of the primary CRC and seven (22%), 14 (44%), and nine (28%) of the synchronous metastases displayed a weak, moderate, and strong positivity, respectively. According to the extent of EGFR immunoreactivity, five (16%) primary CRC had no reactivity, 12 (37.5%) had <10% reactive cells, eight (25%) had >10\u201350% positive cells, and seven (22%) had >50% labeled cells. In the synchronous metastases, two cases (6%) were negative, 13 (41%) had <10% reactive cells, 11 (34%) had >10\u201350% positive cells, and six (19%) had >50% labeled cells.\nInterestingly, the percentage of EGFR-positive tumor cells in the primary site was significantly correlated with that observed in the synchronous metastases (r2=0.616, P=0.0002). This correlation was also demonstrated when combining EGFR scoring (labeling intensity) with the percentage of EGFR-positive tumor cells (r2=0.559, P=0.0009). One example of the pattern of EGFR labeling in both sites is shown in Fig.\u00a01. EGFR expression was positive among the multiple synchronous liver metastases in eight out of nine patients (i.e., 21 out of 22 samples, Table\u00a01). The related primary site of the discordant case (two synchronous metastases: one was EGFR-positive, the other was EGFR-negative) displayed an EGFR-negative status (case number 9, Table\u00a01).\nFig.\u00a01Example of EGFR expression in tissue sections in a primary colorectal carcinoma (a) and the matched metastasis (b). Strong membranous staining of numerous cells in both sites (immunoperoxidase; bar 50\u00a0\u03bcm)\nRegarding the analysis of EGFR expression in metachronous metastases, we observed an analogous EGFR-positive status between all the available liver metastases (i.e., five metastases for one patient, one metastasis for two patients) and their related primary sites and synchronous metastases (case numbers 4, 10, and 11, Table\u00a01). The only metachronous pulmonary metastasis available for analysis did not show any EGFR immunoreactivity, whereas both the corresponding hepatic synchronous metastases and the primary site expressed EGFR (case number 7, Table\u00a01).\nEGFR expression of primary CRC and their related metastases on TMA\nOn the first TMA block, the EGFR status was found to be positive in 18 out of the 31 (58%) assessable primary tumors and 18 out of 29 metastases (62%) (Table\u00a02). Only nine out of 28 (32%) assessable matched tumors were positive for both the primary and the metastatic sites, whereas four out of 28 (14%) were negative. Fifteen out of 28 (54%) cases were discordant (i.e., seven positive primary and negative metastases, and eight, conversely). \nTable\u00a02Comparison of EGFR expression in primary CRC and their synchronous metastatic sites on whole tissue sections and TMA\u00a0Primary siteMetastasesMatched sitesTissue sections27\/32 (84%)30\/32 (94%)25\/32 (78%)First TMA18\/31 (58%)18\/29 (62%)9\/28 (32%)Second TMA14\/31 (45%)16\/29 (55%)8\/28 (29%)Both TMAs20\/31 (65%)19\/29 (66%)12\/28 (43%)Data are expressed as the number of positive cases\/total number of  specimens\nOn the second TMA block, 14 out of 31 (45%) primary tumors and 16 out of 29 (55%) metastases were EGFR-positive (Table\u00a02). Eight out of 28 (29%) assessable matched tumors were positive for both sites, whereas nine (32%) were negative. Eleven out of 28 cases (39%) were discordant (i.e., four positive primary sites and negative metastases, and seven, conversely).\nWhen combining the two TMAs (six tissue cores), the number of positive cases increased, to reach 20 out of 31 of the primary tumors (65%) and 19 out of 29 of the metastases (66%) (Table\u00a02). Twelve out of 28 (43%) assessable matched tumors were positive for both sites, whereas three (11%) were negative. Nevertheless, 13 cases (46%) remained discordant (i.e., six positive primary and negative metastases, and seven, conversely). Moreover, using the TMA technology and whatever the number of cores (three or six), we failed to demonstrate any correlation of EGFR expression between primitive sites and matched metastases.\nComparison of EGFR expression between tissue sections and TMA\nThe EGFR status on the triple core-tissue array was significantly underestimated at both sites when compared with tissue sections (P=0.011 for both primitive and metastatic sites for the first TMA; P=0.0005 and P=0.0023 for primitive and metastatic sites, respectively, for the second TMA). When combining both TMAs (six tissue cores), although the number of positive cases increased, we still observed a significant discrepancy between both techniques for primitive sites (P=0.034) and metastases (P=0.0001).\nDiscussion\nEGFR is a 170-kDa transmembrane cell surface receptor encoded by the human HER1 gene, which promotes malignant cell proliferation and cancer progression [15]. Interfering with EGFR cell signaling by targeting strategies represents a novel approach to the treatment of solid tumors [15]. Among them, cetuximab (Erbitux\u00ae), a chimeric monoclonal human\u2013mouse antibody, is used for the treatment of EGFR-expressing metastatic CRC that have progressed following chemotherapy [3, 23]. To identify patients eligible for this treatment, an immunohistochemical evaluation of EGFR expression is required. Although numerous studies have demonstrated EGFR expression in 70\u201397% of CRC [4, 6, 29], only few and controversial data are available on the EGFR status in distant metastases [6, 11, 13, 24]. Consequently, knowledge of EGFR metastastic status could be of potential value for therapeutic decisions, particularly if it differs from the primary tumors.\nTo answer the question as to whether EGFR expression is similar in different tumor sites, we compared the EGFR status of a set of primary CRC and their corresponding synchronous distant metastases, using a standardized immunohistochemistry test (EGFR pharmDx\u2122, Dako). We also analyzed EGFR expression in metachronous metastases, when available, to study possible changes of EGFR expression during the metastatic process of CRC.\nOn whole tissue sections, our results demonstrated that EGFR expression was frequent and similar in both the primary and the metastatic sites. In our study, immunoreactivity was observed in 84 and 94% of the primary tumors and the metastases, respectively (Table\u00a01). The 32 matched primary and synchronous metastatic CRC expressed a concordant EGFR status in 78% of the cases, all immunopositive, and the discordant cases were not statistically significant (P=0.453). Our data are in agreement with three recent studies that showed a high frequency and concordance of EGFR expression in matched primary tumors and distant metastatic lesions of CRC [8, 11, 13]. To our knowledge, only one study reported no correlation of EGFR expression on primary CRC and related metastases [24].\nIn addition to the concordant EGFR status between primary and metastatic locations, our study showed that both the staining intensity and the percentage of EGFR-positive cells in the primary site were significantly correlated with those observed in synchronous metastases (Fig.\u00a01). Our work also emphasized the EGFR-positive status among multiple synchronous and metachronous metastatic sites in most of the patients (Table\u00a01). Furthermore, we noticed an analogous EGFR-positive status between all metachronous metastases but one, their related synchronous metastases, and their primary CRC (Table\u00a01). Such data, to our knowledge, have never been reported in clinical samples and seem to agree with the preclinical results, suggesting that EGFR expression is required for the tumor to acquire metastatic potential [19, 22, 27]. Moreover, in our cohort, all the patients displayed an EGFR-positive status. This would mean that most of the patients are potentially eligible for therapy with EGFR-targeted monoclonal antibodies, given the results of the immunohistochemistry.\nThe clinical relevance of EGFR immunohistochemical detection is today a matter of debate because EGFR-negative patients might respond to cetuximab and EGFR-positive patients might not [2, 3, 5, 23]. One reason proposed to explain this lack of correlation is a possible difference in the EGFR status between the primary tumor and the metastatic sites [24]. Our results do not support this hypothesis. The lack of response in EGFR-positive patients could more likely be explained by alterations in the critical downstream signals, activated via other receptors or other pathways. It is also plausible that the lack of correlation between EGFR expression and antitumor activity could be due to the potential for cetuximab to induce antibody-dependent cell-mediated cytotoxicity and to the absence of discrimination between the high- and low-affinity receptors with the commonly used antibodies [2, 14, 16]. Moreover, negative immunohistochemical results may be linked to the heterogeneity of EGFR expression and could, in part, represent false negative cases that might respond to EGFR-targeted therapy.\nThe second goal of our study was to perform the same analysis on TMA generated with the same paraffin blocks used for EGFR assessment on the whole tissue sections. TMA has been employed to study the molecular profile of different cancer types and to test hypotheses regarding colorectal carcinogenesis and prognosis [9, 12, 20]. However, EGFR expression in primary CRC and their related metastases has rarely been studied with this approach and the few published results are controversial [8, 25]. In our work, TMA was carried on multiple (three) cores, selected from the deepest region of tumor invasion. Nevertheless, the results obtained were systematically lower than those observed on the whole tissue sections. Even when combining the two TMAs, EGFR positivity only reached 65 and 66% in primary CRC and their related metastases, respectively (Table\u00a02). In contrast to what we observed on tissue sections, we failed to demonstrate a correlation of EGFR expression between both sites using the TMA technology. The discordant results between the two technologies may be explained by cases containing rare stained cells (i.e., <10%) or small invasive clusters, which may be not selected when TMA is performed even using three or six cores. These small tumor clusters, also called tumor \u201cbudding,\u201d belong to a phenomenon which might be involved in metastatic spreading and seem to represent a poor prognostic factor [18, 21, 30]. Such data are of importance because TMA has been recognized as a useful and powerful tool for screening immunohistochemical markers for prognostic purposes and for carcinogenesis studies, as well.\nIn conclusion, we demonstrated that EGFR expression on whole tissue sections is frequent and similar in primary CRC and their related metastases. Our results also indicated that the EGFR status of the primary is concordant with those of metastases, whatever the number of sites and the time of occurrence, i.e., synchronous or metachronous. This may have clinical implications because patients are not always treated in the same institute for their primary disease and for their metastatic disease. In our experience, TMA does not represent an appropriate technique for EGFR screening in CRC because it significantly underestimates EGFR expression and does not provide data similar to those obtained with whole tissue sections. Undoubtedly, this technology is a reliable tool for the high-throughput assessment of homogeneously distributed proteins [7], but it appears to generate unreliable information with very heterogeneous markers such as EGFR.","keyphrases":["epidermal growth factor receptor","colorectal carcinoma","metastases","tissue microarray"],"prmu":["P","P","P","P"]}
{"id":"Cardiovasc_Intervent_Radiol-4-1-2367387","title":"Evaluation of the Occupational Doses of Interventional Radiologists\n","text":"The aim of the present study was to determine whether there is a linear relation between the doses measured above and those measured under the lead apron of the radiologists performing interventional procedures. To monitor radiation exposure the International Commission of Radiological Protection (ICRP) recommends the use of a single dosimeter under the protective apron. To determine the exposure more accurately an additional dosimeter is recommended above the protective apron. The exposure of eight radiologists was monitored with two personal dosimeters during 3 consecutive years. To measure the doses uniformly the two dosimeters were worn in a special holder attached to the lead apron. The two personal dosimeters were replaced every 4 weeks on the same day. The doses above and under the protective aprons of seven radiologists did not differ significantly. A significant lower dose above and under the protective apron was measured for one of the radiologists. During a 4-week period the average dose measured above the lead apron was 3.44 mSv (median, 3.05 mSv), while that under the 0.25-mm lead apron was 0.12 mSv (median, 0.1 mSv). The coefficients of the regression line result in the equation Y = 0.036X \u2212 0.004, with Y as the dose under the lead apron and X as the dose above the lead apron. The statistical analysis of the data established a linear relation between the doses above and those under the lead apron (R2 = 0.59). Before the special holder was introduced it was not possible to derive a relation between the doses above and those under the lead apron, as the doses were measured at varying places above and under the lead apron. There is no evidence that the effective dose can be estimated more accurately when an additional dosimeter is used. The present study revealed a threshold before doses under the lead apron were measured. Due to the threshold it can be concluded that the doses under the lead apron will not be underestimated easily when doses above the lead apron are used to calculate them. This is not the case when the doses above the lead apron are calculated for the doses under the lead apron.\nIntroduction\nLike many other hospitals the Academic Medical Centre (AMC) in Amsterdam uses personal dosimeters to monitor the occupational doses from x-ray procedures to clinical staff. The occupational doses of the majority of the clinical staff do not exceed 1\u00a0mSv in a single year. However, for clinical staff involved in fluoroscopically guided interventional procedures, significant doses are measured. As a consequence of the radiation exposure, the risk of fatal cancer for the radiologists performing interventional procedures increases. This stochastic risk can increase to a value of about 0.1% for radiologists performing these procedures during many years [1]. In recent years the number of fluoroscopically guided interventional procedures at the AMC has increased, as has the number of radiologists performing the procedures. Moreover, the discipline to wear personal dosimeters has improved. As a result, the number of radiologists with doses exceeding 20\u00a0mSv in a single year has increased. Whenever the dose exceeds 20\u00a0mSv in a single year a report has to be sent to the Dutch labor inspection, even when the doses are measured above the lead apron. In the report evidence has to be given that the limit for stochastic effects is not exceeded. In order to prove that the doses of 20\u00a0mSv measured above lead aprons do not exceed the stochastic dose limit, it was decided to provide additional personal dosimeters to the radiologists performing interventional procedures at the AMC. These additional personal dosimeters were meant to monitor the occupational doses under the lead apron.\nWhen in 2004 it was decided to provide the radiologists with an additional dosimeter, the AMC had already substantial data of doses monitored on both sides of the lead apron. These data were collected at the Department of Cardiology, where additional dosimeters were introduced in 1998. Until 2004 the data collected at the Department of Cardiology established no relation between the doses measured above and those measured under the lead apron. This was thought to be caused by the fact that the two dosimeters were often transposed, that the dosimeters were not always returned at the same time, and that unmistakable instruction regarding the position of the two personal dosimeters was missing. To rule out the influence of these factors on the dose measured, it was decided to develop a special holder for the two dosimeters at the time the radiologists were provided with additional dosimeters. Moreover, it was decided to replace all personal dosimeters in the study on the same day by one person, to preclude the influence of differences in measuring periods and problems with transposed dosimeters.\nFor this department the doses measured under the lead apron were considered to be a substitute for the effective dose as recommended by the ICRP [2], while the doses measured above the lead apron were used to estimate the effective dose more accurately.\nThe aim of the present study was to determine whether a relation exists between the doses measured above and those measured under the lead aprons of radiologists performing interventional procedures and whether two personal dosimeters provide additional information compared to a single dosimeter.\nMaterials and Methods\nFrom 2004 onward the occupational doses of eight radiologists involved in interventional fluoroscopically guided procedures were monitored by means of two personal dosimeters. The personal dosimeters were used to measure the doses above and under the lead aprons of the radiologists. To receive consistent measurements the two dosimeters were worn in a specially designed holder that was fixed to the lead apron (Figs.\u00a01 and 2). The holder with the two personal dosimeters was worn breast-high. The two dosimeters were replaced every 4\u00a0weeks on the same day, while the holder remained fixed to the lead apron during the whole study.\nFig.\u00a01The specially designed holder fixed to the lead apron breast-highFig.\u00a02The specially designed holder with the two personal dosimeters\nThe doses were measured during 39 periods of 4\u00a0weeks in 3 consecutive years.\nAt the start of the study six of the radiologists were provided with two personal dosimeters, while throughout the study another two radiologists were provided with two dosimeters. Three radiologists left the AMC before the end of the study.\nThe personal dosimeters were provided by the Nuclear Research and Consultant Group (NRG, Arnhem, the Netherlands). The personal dosimeters were read by the NRG and reported to the AMC. The dosimeters contain LiF:Ti, Mg (TLD100) detectors behind 2\u00a0mm of aluminum for the measurements at a depth of 10\u00a0mm. In the dose reports the doses were presented by means of the personal dose equivalent at a depth of 10\u00a0mm, the Hp(10). The doses were reported in multiples of 0.01\u00a0mSv. Doses below 0.005\u00a0mSv were reported as <0.01\u00a0mSv [3]. \nAt the Department of Radiology the interventional procedures were performed with two Philips Integris Allura machines (Philips Medical Systems, Best, the Netherlands) with the same potential range of 50\u2212133\u00a0kVp. During all interventional procedures the radiologists used wrap-around lead aprons (skirts and vests; Scanflex Medical AB, T\u00e4by, Sweden) with an overall lead equivalence of 0.25\u00a0mm at 100\u00a0kV. The wrap-around apron has an overlap due to which the actual thickness of the protective apron in the front is 0.5\u00a0mm lead equivalent. The doses were monitored above and under a layer of 0.25\u00a0mm lead equivalence.\nThe doses measured above and under the lead apron were statistically analyzed using SPSS software (release 11.5.1 for Windows). Under the lead apron doses <0.01\u00a0mSv were treated as 0\u00a0mSv for the statistical analysis. Above the lead apron doses <0.01\u00a0mSv were entered as missing values. For the statistical analysis the doses under the lead apron were entered as dependent variables, while the doses above the lead apron and the eight radiologists were independent variables. Statistical significant differences were set at a level of 5% (p\u00a0=\u00a00.05).\nResults\nDuring the study 392 dosimeters were provided to eight radiologists. All dosimeters were returned to the NRG. The doses were read and reported in 13 4-weekly dose reports. In total 196 doses were recorded above the lead apron, ranging from <0.01 to 16.78\u00a0mSv. Under the lead apron the doses ranged from <0.01 to 0.83\u00a0mSv. Five readings were excluded from the study, as the doses measured above and under the lead apron were reported to be <0.01\u00a0mSv. In 27 cases the doses measured under the lead apron were <0.01\u00a0mSv. These doses were treated as 0\u00a0mSv in the statistical analysis.\nIn Tables\u00a01 and 2 and in Figs.\u00a03 and 4, the mean dose, median, and quartiles above and under the lead apron are presented for the eight radiologists. A Kolmogorov-Smirnov test showed that the doses of the eight radiologists were normally distributed (p\u00a0>\u00a00.05). The doses measured above and under the lead apron differed significantly among the eight radiologists (one-way ANOVA, p < 0.05). The Levenes test established that the variance of the doses measured above and under the lead apron of seven radiologists was equal (p\u00a0>\u00a00.05). The doses of these seven radiologists did not differ significantly (one-way ANOVA, p\u00a0>\u00a00.05). The average dose of the seven radiologists was 3.85\u00a0mSv in 4\u00a0weeks above the lead apron and 0.13\u00a0mSv in 4\u00a0weeks under the lead apron. The average dose of radiologist 5 was 1.79\u00a0mSv in 4\u00a0weeks above the lead apron and 0.07\u00a0mSv under the lead apron. The dose above the lead apron of radiologist 5 differed significantly from the doses of radiologists 6, 7, and 8 (Tamhane post hoc analysis, p\u00a0<\u00a00.05). Under the lead apron the dose of radiologist 5 differed significantly from the dose of radiologist 7 (Tamhane post hoc analysis, p\u00a0<\u00a00.05).\nTable\u00a01The number of measurements, mean dose, standard deviation, and percentiles (25th, 50th [median], and 75th) of the doses measured above the lead aprons of radiologists 1 to 8Radiologist no.NMean (mSv)SD25th (mSv)50th (mSv)75th (mSv)Minimum (mSv)Maximum (mSv)143.411.222.163.584.481.884.582343.673.211.323.155.260.0316.78392.331.241.382.173.590.284.00493.652.611.763.564.720.359.245a381.791.090.881.702.290.145.406b393.682.372.253.264.180.1311.527b213.661.712.493.084.570.666.948b374.752.313.104.876.131.0513.23Total1913.442.411.743.054.610.0316.78Note. Doses followed by a superscript a differ significantly from doses followed by a superscript b (p\u00a0<\u00a00.05).Table\u00a02The number of measurements, mean dose, standard deviation, and percentiles (25th, 50th [median], and 75th) of the doses measured under the lead aprons of radiologists 1 to 8Radiologist no.NMean (mSv)SD25th (mSv)50th (mSv)75th (mSv)Minimum (mSv)Maximum (mSv)140.180.090.100.170.270.090.292340.130.150.030.110.170.000.83390.060.060.010.070.110.000.15490.140.090.060.150.210.000.245a380.070.090.000.060.090.000.496390.120.080.060.110.190.000.377a210.200.120.120.180.270.020.508370.130.120.050.120.180.000.66Total1910.120.110.050.10.170.000.83aThese doses differ significantly (p\u00a0<\u00a00.05). Fig.\u00a03Boxplot of the doses (mSv) measured above the lead apron. The black line in the red box marks the median, the box demarks the 25th and 75th percentiles, and the whiskers mark the minimum and maximum observed values that are not statistical outliers. Extreme values are marked with an asterisk. The extreme values are more than three box lengths from the upper edge of the box. The box length is the interquartile rangeFig.\u00a04Boxplot of the doses (mSv) measured under the lead apron. The black line in the red box marks the median, the box demarks the 25th and 75th percentiles, and the whiskers mark the minimum and maximum observed values that are not statistical outliers. Extreme values are marked by an asterisk. The extreme values are more than three box lengths from the upper edge of the box. The box length is the interquartile range\nIn Fig.\u00a05 the doses measured under the lead apron are plotted against the doses above the lead apron. The statistical analysis of the data established a linear relation between the doses above and those under the lead apron (ANOVA, p\u00a0<\u00a00.05, R2\u00a0=\u00a00.59). The coefficients of the regression line representing the relation between the doses measured above and those measured under the leaded apron were derived, resulting in the equation Y\u00a0=\u00a00.036X \u2212 0.004, with Y as the dependent variable (dose under the lead apron) and X as the predictor value (dose above the lead apron). The 95% confidence interval for the coefficients ranged from 0.034 to 0.038. The standard error of the models tested was 0.07. The standard error of the model was lower than the standard deviation of the average dose under the lead apron (0.11). The residuals were normally distributed (p\u00a0>\u00a00.05) (Fig.\u00a06).\nFig.\u00a05Plot of doses (mSv) measured above the lead apron (X-axis) and under the lead apron (Y-axis). The line represents the equation for the regressionFig.\u00a06Distribution of the residuals (mean\u00a0=\u00a00, standard deviation\u00a0=\u00a01)\nThe linear relation was not influenced by the doses measured for radiologist 5. The equation remained the same. With the equation and the lowest detectable dose under the lead apron (<0.005\u00a0mSv), the corresponding dose above the lead apron was calculated: <0.25\u00a0mSv. Above this threshold the relation between doses above and those under the lead apron was determined another time. The statistical analysis once again established a linear relation (ANOVA, p < 0.05). The coefficients of the regression line remained the same. Only the strength of the model was lower (R2\u00a0=\u00a00.57).\nDiscussion\nThe dose measurements above and under the lead apron established that the occupational exposure of the radiologists performing interventional procedures in the AMC is comparable. There was only one exception: radiologist 5. As radiologist 5 performed fewer procedures than his colleagues, his exposure was expected to be lower. It was, however, unknown what reduction in doses was to be expected, as data on the number and the length of procedures were not collected for the study. Despite this, it is likely that the lower exposure of radiologist 5 was caused by a lower workload, as the doses above and under the lead apron decreased proportionally compared to the other radiologists\u2019: 46% and 50%.\nResults of doses measured above and under the lead apron of physicians performing interventional procedures have been published by several authors. Moreover, measurements for cardiologists performing interventional procedures were carried out. Renaud et al. [4] reported measurements above and under the lead aprons of in-room personnel during cardiac catheterization. Vano et al. [5] published measurements of cardiologists during a 15-year follow-up. However, differences in the way radiologists and cardiologists perform interventional procedures make it difficult to compare the doses of cardiologists and radiologists. A limited number of clinical studies on doses above and under the lead apron have been carried out among radiologists. Williams et al. [6] reported average monthly doses above the lead aprons of radiologists that ranged from 1.08 to 6.55\u00a0mSv. The highest average dose in this study was found for a radiologist who performed all interventional procedures in patients with liver disease. The average dose of the other radiologists in the study was 1.56\u00a0mSv. The findings of Williams et al. are comparable to the average dose above the lead apron in the present study, where the doses varied between 1.09 and 3.21\u00a0mSv.\nNiklason et al. [7] published annual radiation doses above the lead aprons of 28 radiologists. The average annual dose in their study was 48\u00a0mSv. The average annual dose in the study by Niklason et al. was higher than the average annual dose in the present study: 45\u00a0mSv. However, the average annual dose found by Niklason et al. was lower than the average annual dose when radiologist 5 was excluded. The average annual dose of the seven remaining radiologists was 50\u00a0mSv.\nIn the studies by Williams et al. and Nicklason et al. the doses under the lead apron were also measured. Williams et al. found that the average monthly dose under the lead apron ranged from 0 to 0.48\u00a0mSv. As for the results above the lead apron, Williams et al. reported that the highest average dose was caused by the differences in the nature of work of one of the radiologists. The average monthly dose under the lead apron of all other radiologists (the radiologist with the highest dose was excluded) was 0.08\u00a0mSv. In the present study the doses under the lead apron varied between 0.06 and 0.2\u00a0mSv in a 4-week period. The average dose, 0.12\u00a0mSv, was higher than the average dose reported by Williams et al. The differences between the two studies are possibly caused by the differences in thicknesses of the lead aprons between them. Williams et al. reported that the doses were measured under lead aprons of 0.35 and 0.5\u00a0mm, while in the present study doses were measured under a layer of 0.25\u00a0mm.\nNiklason et al. measured doses under lead aprons of 0.25 and 0.5\u00a0mm. As for the doses measured above the lead apron, the doses under the lead apron were annual doses. The average annual dose under the lead apron reported by Niklason et al. was 0.88\u00a0mSv, while in the present study the average annual dose was 1.6\u00a0mSv (median, 1.3\u00a0mSv). As for the results reported by Williams et al., the differences in doses were probably caused by the variation in thickness of the lead aprons worn by the radiologists.\nIn the study reported by Williams et al., the radiologists ensured that the personal dosimeters were worn in all cases. However, the personal dosimeters under the lead aprons were worn not only during interventional procedures but also during other radiology procedures. The authors described that the dedication of the radiologists to wearing additional dosimeters under the lead apron was probably less consistent than for the other dosimeters. In the present study the dedication of the radiologists was not tested, as the dosimeters were simultaneously worn in the special holder. Moreover, the dosimeters in this study were worn exclusively during interventional procedures.\nThe duration of the measurements in the study by Niklason et al. was approximately 2\u00a0months, while in the present study the doses of the majority of the radiologists were measured during 3 consecutive years.\nThe regression analysis revealed a linear relationship between the doses measured above and those measured under the lead apron. The relation was described by the formula Y\u00a0=\u00a00.036X \u2212 0.004. The slope of the line (0.036) is a measure of the transmission of the lead aprons. In the present study a transmission of 3.6% was found for lead aprons of 0.25\u00a0mm. This transmission was derived breast-high, at the height of the lead collar of radiologists. According to Kharrati et al. [8], the energy of the scattered radiation at the height of the lead collar of radiologists is \u223c70\u00a0kVp. The transmission fraction in the present study (3.6%) is in accordance with the data on transmission factors of different primary beam energies found by other authors. Murphy et al. [9] found a transmission factor of 3% for lead aprons of 0.3\u00a0mm in the primary beam of 80\u00a0kVp. Vano et al. [5] found a fraction transmitted through 0.25-mm lead aprons of between 3.3% at 70\u00a0kVp and 5.7% at 80\u00a0kVp. Christodoulou et al. [10] reported about transmission fraction through lead aprons of 0.25\u00a0mm at a tube potential of 70\u00a0kVp. The transmission fraction ranged from 4.3% up to 10%.\nFrom the formula Y\u00a0=\u00a00.036X \u2212 0.004 the intercept of the regression line can be calculated. The intercept with the X-axis lies at 0.11\u00a0mSv. This value theoretically represents the threshold of the doses above the lead apron before doses under the lead apron. This means that for the doses above the lead apron of \u22640.11\u00a0mSv, only doses below the diction limit are expected. The doses will be reported as <0.01\u00a0mSv. As doses up to 0.005\u00a0mSv are below the detection limit of the dosimeters and are reported as doses <0.01\u00a0mSv, the theoretical threshold is even higher: 0.25\u00a0mSv, versus 0.11\u00a0mSv for the doses above the lead apron. As a matter of fact, in the present study for all measurements above the lead apron <0.25\u00a0mSv, the corresponding doses under the lead apron were reported to be below the detection limit of 0.01\u00a0mSv. The lowest dose in this study above the lead apron for which a dose under the lead apron of 0.01\u00a0mSv reported was 0.59\u00a0mSv.\nThe minimum thickness recommended by the IAEA [11] for lead aprons of radiologists performing interventional procedures is 0.35\u00a0mm. In the present study the doses were measured at 0.25\u00a0mm. The doses were measured under one layer of wrap-around lead aprons. Due to the overlap of these aprons, the thickness of the lead apron in the front of the radiologists was 0.5\u00a0mm. For practical reasons it was decided to measure the doses under one layer of 0.25\u00a0mm despite the fact that the doses measured were higher than the occupational doses to the body of the radiologists. The occupational doses of the radiologists under the lead apron in this study are therefore overestimated.\nThe present study showed that additional monitoring under the lead apron is not necessary to estimate the occupational exposure more accurately. This result is in agreement with the conclusions of Schultz et al. [12], based on dose calculations with Monte Carlo simulations. A transmission of 3.6% for lead aprons \u2265 0.25 mm does not underestimate the exposure for radiologists.\nIn the present study the doses under the lead apron were determined almost at the same place as the doses above the lead apron. This was possible as a special holder was used. Before the special holder was introduced it was not possible to derive a relation between the doses above and those under the lead apron.\nConclusion\nThe aim of the present study was to determine whether a relation exists between the doses measured above the lead apron and those measured under the lead apron of radiologists performing interventional procedures and whether two personal dosimeters provide additional information compared to a single dosimeter. It might be concluded that a linear relation between the measurements above and those under the lead apron is proved in this study. With this, there is no evidence for a more accurate estimation of the effective dose when an additional dosimeter is used.\nAs the results of this study were derived from measurements by radiologists performing interventional procedures, it is not known whether a factor of 0.036 is applicable for other specialists performing interventional procedures, such as interventional cardiologists and electrophysiologists. For this reason, more investigation is needed to derive correction factors for other physicians performing interventional procedures.","keyphrases":["lead apron","effective dose","dosimetry","fluoroscopy","radiation protection"],"prmu":["P","P","U","U","R"]}
{"id":"Environ_Manage-4-1-2233706","title":"Analyzing the Impacts of Dams on Riparian Ecosystems: A Review of Research Strategies and Their Relevance to the Snake River Through Hells Canyon\n","text":"River damming provides a dominant human impact on river environments worldwide, and while local impacts of reservoir flooding are immediate, subsequent ecological impacts downstream can be extensive. In this article, we assess seven research strategies for analyzing the impacts of dams and river flow regulation on riparian ecosystems. These include spatial comparisons of (1) upstream versus downstream reaches, (2) progressive downstream patterns, or (3) the dammed river versus an adjacent free-flowing or differently regulated river(s). Temporal comparisons consider (4) pre- versus post-dam, or (5) sequential post-dam conditions. However, spatial comparisons are complicated by the fact that dams are not randomly located, and temporal comparisons are commonly limited by sparse historic information. As a result, comparative approaches are often correlative and vulnerable to confounding factors. To complement these analyses, (6) flow or sediment modifications can be implemented to test causal associations. Finally, (7) process-based modeling represents a predictive approach incorporating hydrogeomorphic processes and their biological consequences. In a case study of Hells Canyon, the upstream versus downstream comparison is confounded by a dramatic geomorphic transition. Comparison of the multiple reaches below the dams should be useful, and the comparison of Snake River with the adjacent free-flowing Salmon River may provide the strongest spatial comparison. A pre- versus post-dam comparison would provide the most direct study approach, but pre-dam information is limited to historic reports and archival photographs. We conclude that multiple study approaches are essential to provide confident interpretations of ecological impacts downstream from dams, and propose a comprehensive study for Hells Canyon that integrates multiple research strategies.\nIntroduction\nRivers have been dammed and diverted for millennia and river damming is one of the most prominent human impacts on fresh water ecosystems (Graf 1999; Naiman and others 2005; Nilsson and others 2005). With damming, the flooded zone upstream undergoes an abrupt, dramatic transition from river valley to reservoir (Nilsson and Berggren 2000; Naiman and others 2005). In addition, river damming and flow regulation also impact downstream ecosystems and these downstream impacts often influence longer river reaches than the segments that are inundated (Williams and Wolman 1984; Johnson 1998; Rood and others 2005).\nDownstream ecological impacts often follow from three environmental alterations (Rood and others 2005): changes to the quantity and timing of downstream water flow (Williams and Wolman 1984; Rood and Mahoney 1990; Magilligan and others 2003), reduced passage of alluvial materials and particularly suspended sediments (Ligon and others 1995; Kondolf 1997), and the fragmentation of the river corridor, with interruptions in downstream and even upstream passage of biota (Ward and Stanford 1995a, 1995b; Jansson and others 2000). Alterations to the water flow regime are determined by dam operations, while sediment trapping and interruption to corridor connectivity are largely unavoidable consequences of major dams and reservoirs.\nMany prior studies have investigated the ecological impacts downstream from specific dams and there have also been integrative reviews of some environmental impacts by Petts (1984), Williams and Wolman (1984), Ligon and others (1995), Friedman and others (1998), Grant and others (2003) and others. Conversely, there are few analyses of the research strategies to investigate downstream impacts. A systematic analysis of prospective research strategies is of interest for three reasons. First, relative to the broader understanding of ecosystem function, river systems are especially dynamic (Poff and others 1997; Naiman and others 2005; Schumm 2005) and this provides conceptual and practical challenges in resolving human impacts that are superimposed on natural spatial and temporal variation (Underwood 1994; Stewart-Oaten and Bence 2001). Second, relative to an understanding of fundamental river processes, each dam represents a major perturbation and the investigation of the physical and biological consequences can provide insight into the functioning of aquatic (instream) and riparian (streamside) ecosystems (Williams and Wolman 1984; Nilsson and Berggren 2000). Third, there is a need to develop rigorous study designs for environmental analyses of new dams that are being implemented, especially in China and India, and for relicensing applications of existing dams, particularly in North America and Europe (Johnson and others 1995; Trush and others 2000; Hughes and Rood 2003). Prior dams were generally implemented without comprehensive environmental assessment and the relicensing phase provides the opportunity to analyze and even mitigate some environmental impacts (Richter and Richter 2000; Rood and others 2005).\nConsequently, in this study we reviewed and categorized the different research strategies and, as an illustrative case-study, we considered how these approaches might apply in the analysis of environmental impacts downstream from a sequence of three major dams and reservoirs along the Snake River, the largest tributary of the Columbia River (Palmer 1991). The dams are located at the upstream end of Hells Canyon, a spectacular river reach that provides the border between Oregon and Washington, and Idaho (Fig.\u00a01). Brownlee, Oxbow and Hells Canyon dams were completed in 1958, 1961, and 1967, respectively, and are collectively referred to as the Hells Canyon Complex (HCC).\nFig.\u00a01Map of the study region showing rivers and dams of the Hells Canyon Complex\nMethods\nThe study commenced with a literature survey of reports analyzing ecological impacts downstream from dams and especially studies investigating riparian zones along bedrock-dominated river canyons of western North America (Table\u00a01). We categorized the research strategies relative to their conceptual approach and study design, and considered scientific strengths and weaknesses, including confounding factors. The relevant studies often involved multidisciplinary approaches, with integrative analyses of river hydrology, fluvial geomorphology, and riparian ecology.\nTable\u00a01Recent studies that include multiple comparisons for investigating ecological impacts from dams above river canyons of western North America (north to south) and the Snake RiverAuthor (year)River(s)Location1. Upstream versus downstream2. Progressive downstream3. Dammed versus free-flowing4. Pre- versus post-dam5. Sequential post-damming6. Flow modification7. Process-based modelingRood and others (1995); Rood and Mahoney (2000); Rood and others (2005)aSt. Mary River through Box CanyonAlbertaXXXXXXAndrews 1986; Schmidt and others (1995); Andersen & Cooper (2000); Merritt & Cooper (2000); Grams & Schmidt (2002); Cooper and others (2003); Adair and others (2004)aGreen and Yampa rivers through Dinosaur MonumentColoradoXXXXSchmidt and others (1995, 2001); Stevens and others (1995); Webb (1996); Collier and others (1997); Patten and others (2001)aGrand Canyon of the Colorado RiverArizonaXXXXXXXJohnson and others (1995); Dixon & Johnson (1999) Snake River, upstream of Hells CanyonIdahoXXSchmidt and others (1995)Snake River, Hells CanyonIdaho, OregonXXProposed composite studySnake River, Hells CanyonIdaho, OregonXXXXX?Xaadditional relevant articles are cited within the articles in this group\nFollowing categorization, we considered the prospective applicability of the research strategies for the Hells Canyon reach of the Snake River. The suitability of spatial comparisons was considered by observing biophysical conditions along (1) the 93 km Hells Canyon reach of the Snake River versus (2) the 35 km upstream Weiser reach of the Snake River, (3) the adjacent 107 km reach of the Lower Salmon River Gorge below White Bird, Idaho, and (4) the 32 km reach of the Snake River below the Salmon River confluence to the Grande Ronde River (Fig.\u00a01).\nRiver channel and valley characteristics were assessed by field visits and float trips along all of these reaches and from 1:24,000 scale USGS quadrangle topographic maps. From these maps, channel widths were measured at 1.6 km intervals from the left to right bank and incorporated islands if present. Longitudinal profiles were determined from elevational contours and calculated as both river and valley gradients. Historic hydrologic data were accessed from regional USGS gauges along the Snake and Salmon Rivers. Statistical comparisons of the river channel characteristics were undertaken with Kruskal-Wallis nonparametric comparisons with JMP 5.0 (SAS Institute Inc., Cary, North Carolina).\nFollowing from the consideration of different research strategies, we compared the different spatial and temporal study approaches relative to the similarity of the proximal, or local, rivers and landscapes, and the distal, headwater environments. We considered: (1) climate and hydrology and especially the seasonal river flow pattern; (2) aspects of fluvial geomorphology, with the geomorphic context for the local comparison, and sediment inputs for the distal comparison; and (3) biological aspects and especially the composition of the riparian vegetation communities. We applied a quartile scaling with: 0\u00a0=\u00a0very different; 0.25\u00a0=\u00a0different; 0.5\u00a0=\u00a0somewhat different; 0.75\u00a0=\u00a0very similar; or 1.0\u00a0=\u00a0same. The local environment was considered most critical for riparian vegetation and, consequently, we doubled the weighting of this component and subsequently added the proximal and distal scores to produce the comparability index that could range from 0 (entirely different) to 4 (identical).\nResults\nPrevious studies investigating ecological impacts downstream from major dams in western North America (Table\u00a01) have applied research strategies that may be broadly classified into three categories: (1) comparative studies, (2) manipulative experiments, and (3) process-based biophysical modeling. Comparative studies have been most common and are generally correlative in nature. These can be divided into studies involving spatial comparisons or temporal comparisons and we have sub-divided these into five comparative research strategies (Table\u00a02). For each of these, we present the conceptual approach, followed by consideration of its applicability to Hells Canyon.\nTable\u00a02Research strategies to analyze ecological impacts downstream of dams and their suitability for the Snake River through Hells CanyonResearch strategyStudy designConsideration \/ complexitySuitability for Hells CanyonI. Comparative studiesA. Spatial comparison1. Upstream versus downstreamPaired comparison: upstream\u00a0=\u00a0control, downstream\u00a0=\u00a0treatmentDams are not randomly situated and are often at geomorphic transitionsLow - an extreme geomorphic transition exists2. Progressive downstreamQuantitative comparison suitable for pattern analysisInflowing tributaries can mitigate impacts of damming but also introduce system complexityMedium - the inflow of the Salmon River provides considerable system recovery3. Dammed versus free-flowingPaired or multiple comparison: free-flowing\u00a0=\u00a0control or reference, dammed\u00a0=\u00a0treatmentAlthough regional rivers have biophysical similarities, each river is somewhat uniqueHigh - in biophysical condition, the Salmon River Gorge is very similar to Hells CanyonB. Temporal comparison4. Pre- versus post-damPaired comparison: pre-dam\u00a0=\u00a0control, post-dam\u00a0=\u00a0treatmentComprehensive biophysical inventories were seldom undertaken prior to dammingMedium - historic descriptions and archival photographs are available5. Sequential post-dammingMultiple comparison that may be suitable for pattern analysisOther coincidental changes complicate interpretationMedium - sequential aerial photographs permit comparison of large-scale featuresII. Manipulative experiments6. Flow or sediment modification or dam removalPre-determined paired or multiple comparisonA specialized post-damming comparison that may test causal associationUncertain - following recent relicensing , there may be slight, but probably not major, changes in dam operationsIII. Biophysical modeling7. Process-based modelingQuantitative modeling with hydrogeomorphic foundation (i.e., hydrology & geomorphology \u2794 vegetation \u2794 wildlife)Requires defined and deterministic relationships but some processes remain poorly understoodHigh - the physically rigid landscape simplifies hydrogeomorphic modeling and riparian vegetation is limited\nSpatial Comparisons\nSpatial comparisons involve assessments of different reaches along a particular river or comparisons with nearby reaches of different rivers. Scientific interpretation is based on the general assumption that regional river reaches will demonstrate ecological similarities because they often share hydrologic and geomorphic contexts, have similar climatic regimes, and at least prior to damming, share some ecosystem communities. Correspondingly, the strength of comparison within or across rivers relies on environmental similarity without damming.\nUpstream Versus Downstream\nConcept \u2013 Probably the most obvious spatial comparison contrasts river reaches upstream versus downstream from a dam and reservoir (Fig.\u00a02, Tables\u00a01 and 2). This comparison is based on the expectation that sequential reaches along a river will experience similar but gradually changing ecological conditions and processes, an expectation consistent with the river continuum concept (Vannote and others 1980). The dam and reservoir separate the upstream versus downstream reaches, which subsequently experience different physical and biological influences (Ward and Stanford 1995a, 1995b). Although upstream and downstream reaches are both impacted by some alterations such as fragmentation of the river corridor (Jansson and others 2000), the upstream reach is unaltered relative to the fundamental fluvial processes of hydrology and sediment flux. There is, consequently, an expectation that the upstream reach will continue to function in a natural manner, similar to the condition without the dam. In contrast, the downstream reach is impacted by aspects such as sediment depletion (Kondolf 1997) and hydrologic changes that reflect the pattern of dam operation. Thus, the upstream versus downstream comparison represents a paired comparison whereby the upstream reach provides the control and the downstream reach represents the treatment condition (Table\u00a02).\nFig.\u00a02Schematic showing general spatial comparisons to analyze environmental impacts downstream from dams. Triangles represent dams and reservoirs\nWhile upstream versus downstream comparison is a common study approach there is frequently a confounding factor. Dams are not randomly situated but are instead positioned at strategic locations that may involve valley narrowing. These sites are often at geomorphic transitions that are naturally associated with ecological change. Consequently, natural differences in river reaches commonly exist upstream versus downstream from dam sites. In addition, the upstream environments can also be altered by damming. For example, dams can interrupt the upstream movement of marine-derived nutrients contributed by carcasses of anadromous salmonids (Naiman and others 2005).\nApplication \u2013 The reaches of the Snake River upstream versus downstream from the HCC differ dramatically in their natural geomorphic setting (Fig.\u00a03). The upstream reach near Weiser, Idaho flows through a broad, 3 km wide river valley (Fig.\u00a04) with extensive floodplain zones and terraces dominated by agricultural development. The river channel averages about 300 m in width (Fig.\u00a04), with frequent islands. The longitudinal gradient is shallow, averaging about 0.27 m\/km (Fig.\u00a04) and consequently, stream velocities are typically slow. The river banks and floodplain consist of alluvial deposits without bedrock exposure or confinement. Thus, the channel would be relatively dynamic over the time period of decades or a few centuries that correspond to the life spans of native riparian shrubs and trees such as the locally abundant sandbar willow (Salix exigua L.) and black cottonwood (Populus trichocarpa Torrey & Gray) that require a dynamic floodplain environment (Polzin and Rood 2006).\nFig.\u00a03Typical views of the Snake River upstream (top, near Weiser, July 1997) and downstream (bottom, below Hells Canyon Dam, July 1997) from the Hells Canyon Complex of three dams and reservoirsFig.\u00a04Comparisons of channel slopes (longitudinal gradients) and channel and valley widths for the Weiser reach of the Snake River upstream of the Hells Canyon Complex (n\u00a0=\u00a022), for the Hells Canyon reach downstream of the dams (n\u00a0=\u00a058), and for the adjacent lower gorge of the Salmon River (n\u00a0=\u00a067). Different letters indicate significantly different (p\u00a0<\u00a00.05) widths\nIn contrast to the alluvial reach upstream, Hells Canyon below the HCC is an exceptionally erosion-resistant, bedrock-dominated canyon landscape (Fig.\u00a03). The Snake River has probably been flowing through Hells Canyon for 2 to 6 million years and the deep canyon was considerably scoured during the draining of Lake Idaho about 2 million years ago (Vallier 1998). Progressive river incision has compensated for gradual uplifting of the mountainous region to create the present, exceptionally deep (reaching 2100 m) V-shaped valley canyon. While slight changes in specific channel configuration have occurred, the general river valley form has probably been only slightly altered over the past 100,000 years (Malde 1991; Vallier 1998).\nThrough Hells Canyon below the HCC, the Snake River has a typical width of about 75 m, about one-quarter of the channel width of the upstream Weiser reach (Fig.\u00a04). Basalt bedrock exists as a dominant surface material in the riparian zones, along with large, jagged colluvial boulders that have fallen from the hill-slope bedrock due to physical weathering (Fig.\u00a03). Given the prominence of erosion-resistant bedrock and massive boulders, the position of the river channel and banks would be almost static over the life span of riparian plants.\nWith the steep cross-section of the canyon extending down to the valley floor and into the river, alluvial floodplain development is minimal along the Hells Canyon reach. The typical river valley width is about 130 m, only 55 m wider than the river channel and about 1\/25th of that of the upstream Weiser reach (Fig.\u00a04). The longitudinal gradient of the river channel through Hells Canyon is 6.7\u2013fold steeper than the gradient along the upstream Weiser reach. As a result, the upstream versus downstream reaches differ significantly, complicating this spatial comparison (Table\u00a02).\nProgressive Downstream\nConcept \u2013 The progressive downstream research strategy investigates biophysical conditions along consecutive reaches of the dammed river, and thus represents another within-river longitudinal comparison (Fig.\u00a02, Tables\u00a01 and 2). Instead of an upstream control reach, observations are made along the segments downstream from a dam to investigate progressive change. The longitudinal patterns provide insight into the nature of the environmental influences and may resolve impacts due to water flow regulation versus sediment change (Rood and others 2005). Impacts from sediment trapping would be most severe in the tail-water zone directly below the dam and would initially be less severe downstream (Williams and Wolman 1984; Kondolf 1997). In contrast, ecological consequences of flow alteration could be more uniform along the downstream reach. For both water- and sediment-associated impacts there is some recovery with tributary inflows that contribute water, sediments, and other materials (Andrews 1986; Cooper and others 1999).\nWhile the upstream versus downstream comparison represents a paired comparison, the progressive downstream or synoptic comparison involves a sequence of river segments to reveal quantitative patterns that are suitable for regression or other trend analyses. The progressive downstream approach also overlaps with temporal comparison because impacts such as sediment depletion may extend downstream over time.\nApplication \u2013 The Hells Canyon reach of the Snake River is fairly uniform with respect to riparian vegetation and over the past half-century the zone of sediment depletion has extended through the full reach downstream to the Salmon River (Schmidt and others 1995). However, there are numerous dams above the HCC, including those along the Boise and Payette rivers that formerly provided extensive sands originating from the Idaho Batholiths, and it is thus difficult to differentiate some of the physical impacts of the HCC from impacts due to the upstream dams (Parkinson and others 2003).\nWithin Hells Canyon, tributary inflows are quite minor, except for the free-flowing Imnaha River shortly upstream from the Salmon River junction (Fig.\u00a01). There is little evidence of sediment or vegetation response due to the Imnaha inflow because the downstream Snake River segment is in a severely confined canyon zone dominated by steep bedrock walls rising directly from the river. In contrast, accompanying the inflow of the Salmon River there is an abrupt change in the riverine environment. Along the Lower Hells Canyon reach (downstream from the Salmon River inflow), sandy beaches are abundant as are interstitial sands sifted between alluvial cobbles and colluvial boulders. In contrast to the Hells Canyon reach, sandbar willow is prolific below the confluence of the Snake and Salmon Rivers, particularly at the fringes of sandbars and in other zones with interstitial sand.\nHowever, the Snake River valley also widens with the inflow of the Salmon River and valley wall slopes are shallower. Further, the Salmon River drains a geologically different catchment, dominated by the Idaho Batholiths that provide extensive sand sources. Consequently, the change in riparian conditions along the Snake River below the Salmon River partly reflects a natural transition in the physical landscape. Despite this transition, the general river valley landscape is quite similar through Hells Canyon above and below the Salmon River inflow, and the riparian vegetation communities are very similar. Consequently, comparison between reaches above and below the Salmon River should provide a useful study approach (Fig.\u00a02, Table\u00a02).\nDammed Versus Free-Flowing Rivers\nConcept \u2013 Another commonly applied spatial comparison involves the assessment of a river reach downstream from a dam versus a reach(es) along a nearby river(s) that is free-flowing or has experienced a different history of damming and flow regulation (Fig.\u00a02, Tables\u00a01 and 2). This scientific comparison is based on the expectation that adjacent rivers will experience similar climates and regional-scale geologic and geomorphic conditions. As a result, adjacent rivers often support similar aquatic and riparian ecosystems. Consequently, impacts due to damming and flow regulation could result in differences between the flow-regulated versus free-flowing river reaches. The free-flowing river thus provides the study control or reference reach and the dammed river reach provides the treatment condition.\nHowever, each river is somewhat unique and while there are similarities across regional rivers, there are also some differences in hydrology, geomorphology, and aquatic and riparian biology (Naiman and others 2005). Processes along river reaches also reflect impacts and characteristics of the upstream watershed that also vary across rivers. Thus, an effective comparison of a dammed versus an adjacent free-flowing reach must consider watershed influences as well as the local conditions along the comparative study reaches (Hewlett and others 1969).\nApplication \u2013 As previously noted, Hells Canyon is a particularly distinctive landscape with a large, steep-gradient river in a deep, bedrock-dominated V-shaped valley. The Lower Salmon River Gorge has a biophysical context very similar to Hells Canyon. Both river canyons are deeply incised, producing narrow, V-shaped valley canyons with minimal floodplains (Fig.\u00a05). Exposed bedrock and angular colluvia dominate both landscapes and steep bedrock walls flank the valleys of both river reaches that share a common climate and support sparse vegetation.\nFig.\u00a05Schematic showing the proposed spatial comparisons to analyze environmental impacts downstream from the Hells Canyon Complex of dams\nThe physical similarities of Hells Canyon and the Lower Salmon River Gorge are confirmed by channel and valley characteristics (Fig.\u00a04). River channel and valley widths are almost identical and the longitudinal gradients are also very similar (Fig.\u00a04). While their channel geometry is very similar, the associated hydrology differs considerably (Table\u00a03). The annual discharge of the Snake River is about twice that of the Salmon River but the Salmon River has about 1\/3 greater peak flows. The Salmon River has a much smaller watershed that receives greater precipitation and provides fairly synchronous snowmelt-dominated seasonal flow.\nTable\u00a03Hydrological characteristics of river reaches in the Hells Canyon regionRiver gaugeaYears of recordDrainage area (km2)Mean annual discharge (m3\/s)Annual peak discharge (m3\/s)AveMaxMinAveMaxMinSnake River at Murphy1914\u20131998108,5213145431917061339306Major tributaries above Weiserb1914\u2013199870,707198488485931053116Snake R. at Weiser1914\u20131998179,228513103123912992393422Snake R. at Hells Canyon Dama1965\u20131997189,847585103527613562777578Salmon R. at Whitebird1914\u2013199835,09531650616517983681617aHells Canyon Dam data were provided by Idaho Power Corp. and data for other gauges were derived from USGS gauging stationsbOwyhee, Boise, Malheur, Payette, and Weiser rivers. The associated peak flow values are estimates based on data from Weiser and Murphy gauges\nAlthough these adjacent river reaches are very similar, their headwater reaches vary substantially with respect to both natural and human influences. The Snake River originates in western Wyoming and then flows in a wide arc across the Snake River Plain of southern Idaho, a region with a combination of erosion-resistant lava beds with minimal soil cover and limited sediment input and agricultural landscapes with greater sediment inputs. Immediately upstream of the Hells Canyon reservoirs, five major tributaries (Boise, Malheur, Owyhee, Payette, and Weiser rivers) double the drainage area of the Snake River (Fig.\u00a01).\nThe Snake and Salmon rivers also vary considerably in accessibility and the extent of human impact. The Snake River corridor has been the focus for agricultural development and human settlement in Idaho. In contrast, much of the Salmon River flows through the Frank Church River of No Return Wilderness Area, one of the least developed areas of the contiguous United States. Only about 150 km of the Salmon River flows through lands with developed agriculture and these areas are minor compared to agricultural developments along the Snake River. Because virtually all land uses within a watershed impact hydrology, sediment and nutrient fluxes, the different human histories of the watersheds would result in different inputs into the Snake versus Salmon River systems.\nAlso related to human history, the Snake and Salmon Rivers represent an extreme contrast with respect to river damming. The Salmon River is nondammed, although it was briefly impounded by the small Sunbeam Dam in its headwaters near Stanley, Idaho. The Salmon River is one of the last large free-flowing rivers in the contiguous United States, whereas the Snake River is one of the most extensively dammed and diverted rivers in North America (Palmer 1991). Damming commences in the headwater region of Grand Teton National Park with Jackson Lake Dam that elevates a natural lake. It is followed by Palisades Dam, a sequence of weirs near Idaho Falls, and substantial dams at American Falls, Minidoka, Milner, Shoshone Falls, Twin Falls, Upper Salmon, Lower Salmon, Bliss, CJ Strike, and Swan Falls, upstream of the HCC. There are also 41 major dams along the tributaries of the Snake River upstream from Hells Canyon. These dams and reservoirs would considerably modify the flow regime and trap sediment above the HCC.\nThese assessments provide mixed conclusions relative to comparing the Hells Canyon reach of the Snake River with the Lower Salmon River Gorge. The local river valley landscapes and riparian vegetation communities are very similar, but there are considerable differences in the watersheds and major differences in upstream damming. Analyses of the Hells Canyon reach of the Snake River versus the Lower Salmon River Gorge should thus provide a useful but somewhat confounded spatial comparison (Table\u00a02).\nTemporal Comparisons\nTemporal comparisons involve sequential analyses of the same river reach(es) and may involve comparative field measurements at different time periods, consideration of indirect records, such as ground or aerial photographs, or the analyses of ecological elements that provide chronosequences, such as progressive arcuate bands of vegetation or tree rings (studies cited in Table\u00a01). The focus of temporal comparisons is on the river reach downstream from a dam, but simultaneous study of other reaches along the same river or an adjacent river(s) would reveal broader regional patterns upon which the impacts of damming are superimposed (Table\u00a01).\nTemporal comparisons are common for ecological analyses following environmental disturbance (Underwood 1994; Stewart-Oaten and Bence 2001), and are based on the assumption that a particular region should demonstrate ecological consistency over time. Consequently, observed changes following damming may be interpreted to reveal impacts of damming and flow regulation (Williams and Wolman 1984; Ligon and others 1995; Friedman and others 1998). However, river systems are naturally dynamic, with considerable seasonal and interannual variations in hydrology, including periodic disturbance, particularly from floods, that can produce major ecological change (Junk and others 1989; Naiman and others 2005; Rood and others 2007). Thus, the scientific challenge in interpreting temporal patterns is to resolve the impacts due to damming and flow regulation from the natural variations of these physically-dynamic fluvial systems.\nPre- Versus Post-Dam\nConcept \u2013 The analysis of sequential change along a particular river reach following damming may provide the most direct approach for analyzing ecological impacts downstream from dams. However, rigorous temporal comparisons are hindered due to the history of river damming projects and the nature of research funding. Relative to project history, many dams were implemented in the twentieth century following geotechnical and hydrologic studies but with minimal pre-project biophysical study. Neither the values nor the vulnerabilities of river ecosystems were generally appreciated and many of the major dams in western North America were situated in remote locations and were implemented with limited public interest in environmental consequences. Because these dams were undertaken before comprehensive environmental impact analyses were required, ecological attributes were often neglected and pre-dam ecological conditions were rarely inventoried.\nThe second impediment is the nature of research funding. Because funding is generally limited in duration, it is more practical to seek funding for a limited-term project with a short-term \u201cdeliverable\u201d than for a long-term study, potentially with an uncertain duration and outcome. The nature of academic study also favors a shorter-term comparison because research projects often involve two or three-year intervals to suit graduate student and post-doctoral projects.\nDue to these practical limitations, temporal comparisons have often relied upon archival materials such as ground-level and aerial photographs (studies cited in Table\u00a01). Ground-level photographs were seldom based on pre-determined sampling strategies for ecological investigation but were instead generally taken for human interest. The sites of historic photographs are often biased towards locations with ease of access or for atypical landscape features that are dramatic or scenic. Aerial photographs provide more systematic coverage, but are insufficient to reveal small-scale features such as plant species and community types. Comparisons involving both ground-level and aerial photographs are often complementary because the two approaches partially compensate for the prospective weaknesses.\nApplication \u2013 The pre- versus post-dam comparison has considerable merit relative to Hells Canyon but pre-dam information particularly regarding riparian vegetation is sparse. Hells Canyon is remote and sparsely inhabited and very few photographs exist from the pre-dam period. In contrast, Hells Canyon is now one of the world\u2019s most highly regarded recreational river trip destinations. The spectacular landscape provides a prime attraction and consequently the river valley has been extensively, but not systematically, photographed in recent decades.\nSequential Post-Damming\nConcept \u2013 Similar to pre- versus post-dam comparisons, the sequential post-damming comparison provides a temporal approach that focuses on the specific river reach below a dam. It involves two or more observation or sampling periods after the dam is implemented and, especially with multiple observations, it can reveal quantitative patterns that may enable future forecasting (Dixon and Johnson 1999). This approach may also be more practical than pre- versus post-damming comparisons due to the deficiency of pre-dam inventory. Additionally, remote sensing inventories have become more common through the twentieth century and aspects such as aerial photographs and more recently, digital multispectral imagery, are now available with repetitive coverage for many landscapes (Lorang and others 2005).\nFor sequential temporal comparison, an appropriate time-frame must be considered relative to the dam project and the environmental components of interest. Some responses occur within a few years, while others require decades or even centuries for change (Williams and Wolman 1984; Church 1995). As a composite study, the combination of pre-project inventory followed by sequential post-dam study can strengthen the analysis since this overlaps the two temporal study approaches.\nApplication \u2013 For the Hells Canyon reach of the Snake River, the sequential post-damming comparison is enabled by periodic aerial photographs. These commenced when the first dam was under construction and have been repeated at about one to two decade intervals thereafter. Early photographs were black and white and more recent photographs are often in true-color or false-color, infra-red. The resolution of aerial photographs limits the scale of landscape feature that can be assessed and those for Hells Canyon are only suitable for large physical features such as river channel position and the extent of major sand bars. Through the interpretation of sequential aerial photographs, Schmidt and others (1995) previously interpreted post-damming depletion in sand bars along the Hells Canyon reach of the Snake River, particularly in the first two decades after damming.\nLong-lived woody plants also enable investigations of riparian landscape chronology. In Hells Canyon netleaf hackberry and sandbar willow are abundant small trees and shrubs that are appropriate to investigate distribution and population age structure and hence, prospective impacts of damming and flow regulation on recruitment, expansion and mortality (Rood and others 1995).\nFlow Modification\nConcept \u2013 Comparative study approaches yield correlative data, including abundances in ecological attributes, such as woodland groves or sandbars, that may change following damming. This reveals correlative pattern but not causal association. The deliberate modification of flow or sediment regime provides an experimental manipulation that can confirm causal association (Rood and Mahoney 2000; Patten and others 2001; Schmidt and others 2001; Rood and others 2003b). Flow modification may follow comparative investigations and enable testing of hypotheses arising from observed responses. Although deliberate flow modification may provide the most definitive study approach, its implementation is restricted by practical considerations. There have been relatively few instances in which dam operations have been deliberately altered in response to ecological considerations but following some initial successes (Rood and others 2005) modifications may increase in future.\nApplication \u2013 The HCC has recently undergone an environmental review in association with the Federal Energy Regulatory Commission relicensing process. Following from that review it is possible that dam operations will remain relatively unchanged. However, if dam operations are altered, appropriate ecological investigations should be undertaken to assess the environmental consequences and to capitalize on the research opportunities.\nProcess-Based Biophysical Modeling\nConcept \u2013 Process-based modeling relies on systematic relationships between underlying physical components of hydrology and geomorphology, and subsequent biological responses, such as the establishment, survival, and growth of riparian plants (Auble and others 1994; Johnson and others 1995; Springer and others 1999; Mahoney and Rood 1998; Richter and Richter 2000). This modeling considers stochastic patterns and assumes deterministic relationships that are predictably quantitative and represents a relatively new approach for analyzing ecological impacts downstream from dams.\nRelative to riparian ecology, modeling requires an understanding of the life history strategies of different riparian plants, including both native and nonnative species (Shafroth and others 2002; Karrenberg and others 2002; Rood and others 2003a). Life history defines the phenology (timing) of seed release and other developmental events, as well as aspects of the physiological water relations that underlie flood and drought tolerance (Tyree and others 1994; Mahoney and Rood 1998; Nilsson and Svedmark 2002). Modeling involves hydrologic analysis of river stage in conjunction with discharge patterns since it is the water elevation that determines the moistening or inundation of riparian zones critical to seedling (or clonal) colonization (Auble and others 1994; Scott and others 1996; Rood and others 2003b). The modeling requires analyses of riparian substrate and particularly sediment textures since this influences erosion resistance and moisture retention that also contributes to seedling survival (Mahoney and Rood 1990; Polzin and Rood 2006). The modeling involves multiple year simulations to account for the natural variation in inflows and the multiple-year life cycle of perennial plants (Auble and others 1994; Scott and others 1996). The modeling may emphasize the large, woody plants that are especially important for wildlife habitat as these provide \u201cstructure\u201d through vertical development of woodland groves (Rood and others 2003b).\nApplication \u2013 The Hells Canyon reach of the Snake River is well-suited for process-based modeling. The bedrock dominated landscape is static relative to the longevity of the riparian plants, reducing the need to account for the dynamic channel changes along alluvial river reaches. The hydrology may also be simpler than along other rivers because the major inflow originates from dam release and only small tributaries occur along the Hells Canyon reach. With a very dry regional climate in the valley bottom, local precipitation and ground-water contribution are also limited, strengthening the linkage between river regulation and riparian soil moisture. With the prominent bedrock and minimal floodplain zones, associated vegetation are relatively limited in both extent and species diversity, thus reducing the range of plants needed to be considered in hydrogeomorphic modeling. Additionally, with a xeric upland landscape, adjacent vegetation is naturally sparse and this would reduce some complexity due to competition and other biological interactions. On the basis of these considerations, the Hells Canyon reach of the Snake River could provide an ideal study system for the development or refinement of process-based hydrogeomorphic models (Auble and others 1994; Mahoney and Rood 1998) similar to those implemented for the Middle Snake River (Johnson and others 1995).\nComparative Validity Across Study Approaches\nThe semi-quantitative comparison of the different comparative strategies indicated that for the Hells Canyon reach: (1) pre- versus post-dam comparison would provide a valid study approach (Table\u00a04), (2) spatial comparison of the Hells Canyon reach versus the Lower Salmon River Gorge would also be useful, particularly due to the biophysical similarities of the adjacent canyon environments, and (3) an upstream versus downstream comparison is complicated by the natural geomorphic transition. A combination of all three comparisons would provide the most comprehensive approach since this could account for natural and anthropogenic differences in both the proximal (local) and distal (watershed) landscapes.\nTable\u00a04Assessment of different comparison studies for analyzing impacts of damming and flow regulation on the Snake River through Hells CanyonProximal score (P) local landscape (weight\u00a0=\u00a02)Distal score (D) watershed conditions (weight\u00a0=\u00a01)Comparability index\u00a0=\u00a0P + DUpstream vs. downstream (Weiser vs. Hells Canyon)Different (0.5)(almost the) Same (1)1.5Dammed vs. free-flowing (Hells Canyon vs. Salmon)Very similar (1.5)Different (0.25)1.75Pre- vs. post-dam (Hells Canyon)Same (2)Very similar (0.75)2.75We applied a quartile scaling of: 0\u00a0=\u00a0very different; 0.25\u00a0=\u00a0different; 0.5\u00a0=\u00a0somewhat different; 0.75\u00a0=\u00a0very similar; or 1.0\u00a0=\u00a0same; and multiplied this value by the weight to produce the P and D score\nDiscussion\nIn this study, we reviewed and categorized various research strategies that researchers have used to analyze ecological impacts in riparian zones downstream from dams in western North America (Table\u00a01). Similar research strategies have been used for dams in other regions worldwide and many of the fundamental considerations are universal (Petts 1984; Williams and Wolman 1984; Ligon and others 1995; Lytle and Poff 2004). We also provided qualitative analyses of the suitability of these approaches for Hells Canyon (Table\u00a02) and provided a semi-quantitative consideration of validity (Table\u00a04). However, these research strategies are prone to a number of potentially confounding factors (Table\u00a02). With respect to comparative studies, responses are correlative in nature and some effects may not be caused by the dam or the associated alteration to downstream flows. As a result, the following factors should be considered: natural variation, coincidental influences, cumulative and sequential impacts, threshold effects, and latent effects.\nNatural Variation\nRiparian zones are naturally extremely dynamic reflecting river flows that vary seasonally across years (Trush and others 2000; Naiman and others 2005; White and others 2005). Occasional floods provide powerful agents of erosion and deposition and can immediately have dramatic impacts on aquatic and riparian zones. Floods often enable bursts of recruitment by riparian plants and some other biota and thus, the initial \u201cdestruction\u201d may be followed by ecosystem rejuvenation, a sequence of events consistent with the flood pulse concept (Junk and others 1989; Scott and others 1996).\nIn contrast to natural floods, droughts lead to natural declines in some riverine biota such as riparian vegetation, due to drought-induced mortality (Tyree and others 1994). Following particular wet or dry years or multiple-year wet and dry cycles, aquatic and riparian populations naturally experience episodes of decline and recovery. These natural cycles provide a variable baseline upon which impacts of damming and flow-regulation are superimposed.\nCoincidental Influences\nAquatic and riparian ecosystems are influenced by many natural and anthropogenic factors (Naiman and others 2005), which may temporally or spatially coincide with river damming. For example, exotic weeds have progressively migrated through the western United States and in some areas their expansion coincides with periods of extensive river damming. Within remote landscapes, such as Hells Canyon, major dam projects introduce roads and utility corridors that facilitate many human uses and impacts that further alter riverine environments.\nCumulative and Sequential Impacts\nLike many other rivers in North America and worldwide (Graf 1999; Nilsson and others 2005), the Snake River is extensively dammed and diverted (Palmer 1991). The combined impacts from the various water resource projects make it very difficult to isolate those effects specifically related to the Hells Canyon Complex. With respect to river environments, we suggest that cumulative impacts be viewed as those effects that accumulate spatially along the longitudinal corridor whereas sequential impacts are those that accumulate over time. Some of these impacts may be additive while other response functions may be more complex and more difficult to resolve or model.\nThreshold Effects\nThreshold effects are related to cumulative and sequential impacts. For threshold effects, the ecosystem or component may remain relatively unaltered up to a point at which a substantial response occurs. Threshold effects are particularly relevant to physiological stresses that are tolerable within a specific range of environmental conditions. For example, cold water fish may be unaffected until aquatic conditions exceed particular thresholds in temperature and oxygen levels (Ruckelshaus and others 2002). Similarly for riparian plants, water stress due to instream flow reduction may have minor impact until the xylem cavitation threshold is reached which can lead to abrupt mortality (Tyree and others 1994). Thus, threshold effects reflect nonlinear ecosystem dynamics that confound analyses such as instream flow needs (IFN) calculations.\nLatent Effects\nLatent effects are those in which the timing of a response is delayed, thus complicating temporal comparisons. For example, an alteration in stream flow pattern may eliminate fish spawning or vegetation recruitment, but if monitoring is focused on the population of mature fish or trees, the impact may not be revealed until a substantial fraction of the life cycle passes. Alternately, higher-order members of a riverine ecosystem may not be affected by a negative impact until the lower-order prey base is substantially diminished (Power and others 1995).\nMultiple Comparisons\nDue to the limitations of individual study approaches and the range of confounding factors, simple comparisons involving specific spatial or temporal comparison are vulnerable (Stewart-Oaten and Bence 2001). Conversely, integrative comparisons among multiple river reaches over several time intervals (Table\u00a02) would enhance data interpretation and subsequent study conclusions. In the ideal case, several comparative approaches would be used although this is rarely practical (Table\u00a01).\nFor Hells Canyon, the Schmidt and others (1995) interpretation of aerial photographs provided a form of sequential post-damming comparison. This study would be complemented by further research to reveal the range of dam-related impacts on the riparian ecosystem along the Hells Canyon corridor (Table\u00a01).\nA Composite Study Strategy for Hells Canyon\nThe analysis of different comparisons (Table\u00a04) suggests that the pre- versus post-dam analysis would provide a particularly valid single study approach for the Hells Canyon reach of the Snake River. However, pre-dam information is limited to a few historic descriptions and archival photographs with limited spatial coverage. Comparisons of the historic descriptions and photographic views with contemporary conditions would be useful and may especially reveal changes in vegetation that would complement Schmidt and others\u2019 (1995) study of sand bars. Subsequently, current vegetation and sediment conditions could be more thoroughly investigated through a composite spatial analysis (Fig.\u00a05). Further, vegetation analyses could also be linked to habitat studies to assess prospective influences on wildlife (Blair and others 2002), and reveal the extent of invasive weeds which are increasingly problematic in riparian zones (Naiman and others 2005). When coupled with detailed analyses of hydrology, these data could provide a confident foundation for hydrogeomorphic modeling of the plant species and communities.\nThis composite study design would thus involve overlapping all three spatial comparisons (Fig.\u00a05) to compliment and calibrate a process-based modeling approach (Tables\u00a01 and 2). This study design would also provide a dam operator with information to assist in the management of large dams for multiple benefits, including environmental conservation along the downstream river reach (Richter and Richter 2000; Rood and others 2005). The development and implementation of this comprehensive study design would also provide an informative case study that would be relevant for other rivers impacted by dams and regulated flows.\nAlthough the upstream versus downstream comparison is confounded by a natural geomorphic transition, we propose to initiate this composite study along the Weiser reach upstream from the HCC (Fig.\u00a05). Sampling could continue along each of the three sequential reservoirs (Fig.\u00a01), with more intensive sampling downstream from Hells Canyon Dam. Sequential sampling would also extend below confluence of the Salmon River (Fig.\u00a05). Complementary sampling along the Lower Salmon River Gorge would enable spatial comparisons between a free-flowing river and the dammed Hells Canyon reach of the Snake River. To complement longitudinal (downstream) sampling, transverse (upslope from the river) patterns in vegetation and substrate would also be inventoried to provide yet another form of spatial analysis, revealing correspondence between water-levels, substrate, and vegetation.\nAs a final component of the composite study design, we propose the extension and implementation of process-based modeling (Johnson and others 1995). The proposed field sampling strategy would be extensive and as indicated, we consider that the exceptionally static, bedrock-dominated Hells Canyon landscape may be particularly well suited for hydrogeomorphic model development. In contrast to alluvial rivers with frequently shifting channels and banks, changes in river channel position along Hells Canyon are minimal within a time frame corresponding to the life cycle of riparian plants. Additionally, the hot and dry climate restricts the number of local plant species further simplifying this system. Thus, despite the vast scale and remote situation of this dramatic landscape, we consider that Hells Canyon presents an ideal opportunity to advance process-based models to analyze functional interactions in this riparian ecosystem and to refine research strategies used to analyze ecological impacts downstream of dams.","keyphrases":["river damming","riparian ecology","environmental impact analysis"],"prmu":["P","P","R"]}
{"id":"Exp_Brain_Res-4-1-2190788","title":"No effect of auditory\u2013visual spatial disparity on temporal recalibration\n","text":"It is known that the brain adaptively recalibrates itself to small (\u223c100 ms) auditory\u2013visual (AV) temporal asynchronies so as to maintain intersensory temporal coherence. Here we explored whether spatial disparity between a sound and light affects AV temporal recalibration. Participants were exposed to a train of asynchronous AV stimulus pairs (sound-first or light-first) with sounds and lights emanating from either the same or a different location. Following a short exposure phase, participants were tested on an AV temporal order judgement (TOJ) task. Temporal recalibration manifested itself as a shift of subjective simultaneity in the direction of the adapted audiovisual lag. The shift was equally big when exposure and test stimuli were presented from the same or different locations. These results provide strong evidence for the idea that spatial co-localisation is not a necessary constraint for intersensory pairing to occur.\nIntroduction\nIn many circumstances people experience external events by a number of different sensory modalities. For example, when someone is talking, there is auditory and visual information that is initially processed by specialized neural pathways. Ultimately, though, the different sensory signals are integrated into a coherent multimodal percept of the speaker. Many behavioural and neurophysiological studies have emphasized the importance of spatial co-localisation and temporal synchrony for intersensory pairing to occur (e.g., Welch and Warren 1980; Bedford 1989; Stein and Meredith 1993; Radeau 1994; Bertelson 1999; Welch 1999). However, there is accumulating evidence that some intersensory phenomena may not require spatial alignment (Welch et al. 1986; Scheier et al. 1999; Morein-Zamir et al. 2003; Murray et al. 2004; Teder-Salejarvi et al. 2005; Vroomen and Keetels 2006; Keetels et al. 2007). In the present study, we explored the importance of spatial alignment for audio\u2013visual (AV) temporal recalibration.\nTemporal recalibration refers to the phenomenon that the brain adapts itself to (small) temporal asynchronies. In a multi-modal percept, it usually appears that information from different senses arrive at the same time. This occurs, despite the fact that there are natural asynchronies between the senses caused by differences in signal transduction time through air and differences in neural transmission time. At least two options are available to handle these asynchronies: one is concerned with immediate corrections, the other is important for adaptation on a longer time scale. As concerns the immediate effect, several studies have shown that the brain corrects for small AV temporal asynchronies by shifting one or both modalities on the time scale so that the temporal discordance is reduced. For example, when a sound and a light are presented at slightly different onset times (usually in the order of \u223c100\u00a0ms), the temporal asynchrony is reduced by a capturing effect of the light by the sound; a phenomenon called temporal ventriloquism (Scheier et al. 1999; Fendrich and Corballis 2001; Morein-Zamir et al. 2003; Vroomen and de Gelder 2004; Stekelenburg and Vroomen 2005; Vroomen and Keetels 2006). Temporal ventriloquism can, for example, be demonstrated by the use of a visual temporal order judgment (TOJ) task in which participants are presented two lights at various stimulus onset asynchronies (SOAs) and judge which light came first. By presenting a sound before the first and after the second light, the just noticeable difference (JND) improves (i.e. participants become more sensitive), presumably because the two sounds attract the temporal occurrence of the two lights, and thus effectively pull the lights further apart in time (Scheier et al. 1999; Morein-Zamir et al. 2003; Vroomen and Keetels 2006).\nThere are also long-term effects reflecting an adaptive change to AV asynchrony, a phenomenon called temporal recalibration (Fujisaki et al. 2004; Vroomen et al. 2004). For example, Vroomen et al. studied temporal recalibration by exposing participants to 3\u00a0min of sound and light flashes with a constant time lag, after which an AV TOJ or AV simultaneity task was performed. Following exposure, observers were given AV test stimuli and judged whether the sound or the light came first, or whether the sound and light were simultaneous or successive. The results showed that the point of subjective simultaneity (PSS), the point of perceived temporal alignment between the sound and the light, was shifted in the direction of the exposure lag. So, following exposure to a train of sound-first stimulus pairs, participants perceived sound-first trials as more simultaneous than after light-first exposure. Fujisaki et al. (2004) demonstrated similar findings and also provided somewhat mixed evidence that temporal recalibration may generalize to different test stimuli than the ones presented during exposure. The authors adapted participants to asynchronous tone-flash stimulus pairs and later tested them on the \u201cbounce\u201d illusion (Sekuler et al. 1997). In this illusion, two visual targets that move across each other can be perceived either to bounce off or to stream through each other. A brief sound presented at the moment that the visual targets coincide generally biases visual perception in favour of a bouncing motion, while without sound observers tend to report a streaming percept. Following exposure to asynchronous sound\u2013light pairs, the optimal delay for obtaining the bounce illusion was shifted in the same direction, but in other conditions, the magnitude of the after-effect was smaller for some of the cross-adaptation conditions.\nTemporal recalibration may also occur between other modalities than AV. For example, Navarra et al. (2006) demonstrated audio\u2013tactile temporal recalibration by exposing participants to streams of brief auditory and tactile stimuli presented in synchrony, or else with the auditory stimulus leading by 75\u00a0ms. Rather than a shift in the PSS, they observed that the JND to resolve audio\u2013tactile temporal order was larger after exposure to the desynchronized streams than after exposure to the synchronous streams. The authors argued that the temporal window for integration was widened due to audio\u2013tactile asynchrony.\nThe goal of the present study was to explore whether spatial disparity between a sound and light affects temporal recalibration. According to the \u201ccommon notion\u201d of intersensory pairing, intersensory effects should be bigger when the individual components of a multisensory stimulus come from the same location (e.g. Welch and Warren 1980; Bedford 1989; Stein and Meredith 1993; Radeau 1994; Bertelson 1999; Welch 1999). However, Vroomen and Keetels (2006) demonstrated that, at least for temporal ventriloquism, spatial correspondence between sound and light is not important. In their study, a visual TOJ task was used with a sound presented before the first and after the second light. Temporal ventriloquism manifested itself as an improvement in the JNDs but, crucially, the improvement was unaffected by whether the sounds came from the same or a different position as the lights, whether the sounds were static or moved, or whether the sounds and lights came from the same or opposite sides of fixation. Keetels et al. (2007) further examined how principles of auditory grouping (Bregman 1990) relate to intersensory pairing. They embedded two sounds that normally enhance sensitivity on the visual temporal order judgement task in a sequence of flanker sounds, which either had the same or different frequency, rhythm, or location. In all experiments, temporal ventriloquism only occurred when the two capture sounds differed from the flankers, thus demonstrating that intramodal grouping of the sounds in the auditory stream took priority over intersensory pairing. By combining principles of auditory grouping with intersensory pairing, they also demonstrated that the capture sounds could, counter-intuitively, be more effective when their locations differed from that of the lights rather than when they came from the same position, thus demonstrating that sound location mattered for auditory grouping, but not intersensory pairing.\nHere we examined whether, like in temporal ventriloquism, spatial disparity is ignored when temporal recalibration is at stake. Participants were exposed for 3\u00a0min to a train of asynchronous sounds and lights that came either from the same or a different location. Following exposure, participants performed an AV TOJ task with sounds and lights from either the same or different location. This design allowed us to address two questions. First, we could test whether temporal recalibration is affected by spatial disparity between the sounds and lights. Recalibration is usually considered to be a low-level perceptual learning phenomenon necessary for re-alignment of the senses (Bertelson and de Gelder 2004). Observing an after-effect following exposure to spatially disparate sound\u2013light pairs would provide strong evidence that spatial co-occurrence is, even at this early stage, not necessary for intersensory pairing to occur. Secondly, the use of an exposure\u2013test design allowed us to introduce a change between the exposure and test stimulus so that we could test whether after-effects generalize to different test stimuli. Here we tested whether spatial similarity between the exposure and test sound affects after-effects. If spatial co-location plays no role in intersensory pairing, one would expect stimulus generalization across space to be complete.\nMethod\nParticipants\nThirty students from Tilburg University received course credits for their participation. All reported normal hearing and normal or corrected-to-normal vision. They were tested individually and were unaware of the purpose of the experiment. The study was carried out along the principles laid down in the Helsinki Declaration and informed consent from the participants was obtained.\nStimuli\nParticipants sat at a table in a dimly lit and sound-proof booth. Head movements were precluded by a chin-rest. Visual stimuli were presented by a green LED, positioned at central location, at 70\u00a0cm from the subject\u2019s eyes (diameter of 0.5\u00a0cm, luminance of 40\u00a0cd\/m2). Auditory stimuli were 88\u00a0dB sound bursts presented by one of two loudspeakers; one directly behind the green LED and the other placed laterally at 70\u00a0cm distance on either the far left or the far right of the subject (i.e., 90\u00a0degrees of spatial separation between the sound and light). See Fig.\u00a01 for a schematic view of the experimental set-up. The sounds and lights each had a duration of 10\u00a0ms. A small red LED, placed 2\u00a0cm below the green LED, was constantly lit during the experiment and served as fixation point.\nFig.\u00a01Schematic illustration of the experimental conditions. In the exposure phase, the subject was exposed to a sound\u2013light pair with 100\u00a0ms temporal offset (either sound-first or light first). During exposure, sounds were either presented from central (a, c) or lateral location (b, d). In the test phase, sound\u2013light pairs were presented with a particular SOA ranging between \u2212240 and 240\u00a0ms, with negative values indicating that the sound was presented first. Sounds of the test-stimulus pair either came from central (a, b) or lateral location (c, d )\nDesign\nThree within-subjects factors were used: exposure lag during the exposure phase (\u2212100 and +100\u00a0ms, with negative values indicating that the sound was presented first), location of the sound during exposure (exposure-sound central or lateral) and SOA between the sound and light of the test stimuli (\u2212240, \u2212120, \u221290, \u221260, \u221230, 0, +30, +60, +90, +120, and +240\u00a0ms, with negative values indicating that the sound came first). The location of the test sound (central or lateral) was a between-subjects variable. Half of the participants were tested with central test sounds, the other with lateral test sounds. These factors yielded 44 equi-probable conditions for each location of the test sound (2\u00a0\u00d7\u00a02\u00a0\u00d7\u00a011), each presented 12\u00a0times for a total of 528 trials. Trials were presented in eight blocks of 66 trials each. The exposure lag and the location of the exposure sound were constant within a block, while the SOA between sound and light varied randomly. The order of the blocks was counterbalanced across participants. In half of the blocks with a lateral exposure sound, the sound came from the left, in the other half from the right. The lateral test sounds were presented from the same side as during exposure.\nProcedure\nEach block started with an exposure phase consisting of 240 repetitions (\u223c3\u00a0min) of a sound\u2013light stimulus pair (ISI\u00a0=\u00a0750\u00a0ms) with a constant lag (\u2212100 or +100\u00a0ms) between the sound and the light. After a 2,500\u00a0ms delay, the first test trial then started. To ensure that participants were fixating the light during exposure, they had to detect the occasional occurrence of the offset (150\u00a0ms) of the fixation light (i.e., a catch trial). Participants then pushed a special button.\nThe test phase consisted of two parts: a short AV re-exposure phase followed by three AV test trials of which the temporal order of the sound and light had to be judged. The re-exposure phase consisted of a train of ten sound-light pairs with the same lag, ISI, and sound location as used during the immediately preceding exposure phase. After 1\u00a0s, the three AV test trials were presented with a variable SOA between the sounds and lights. The participant\u2019s task was to judge whether the sound or the light of the test stimulus was presented first. An unspeeded response was made by pressing one of two designated keys on a response box. The next test stimulus was presented 500\u00a0ms after a response, and the re-exposure phase of the next trial started 1,000\u00a0ms after the response on the third test stimulus.\nTo acquaint participants with the TOJ task, experimental blocks were preceded by four practice blocks in which no exposure preceded the test trials. The first two practice blocks were to acquaint participants with the response buttons, and consisted of 16 trials in which only the largest SOAs were presented (\u00b1240 and \u00b1120). During this part, participants received verbal feedback (\u201ccorrect\u201d or \u201cwrong\u201d) about whether they gave the correct response or not. The next two practice blocks consisted of 66 trials in which all SOAs were presented 6\u00a0times randomly without verbal feedback. Total testing lasted approximately 2.5\u00a0h.\nResults\nTrials of the practice session were excluded from analyses. The proportion of \u201clight-first\u201d responses was for each participant calculated for each combination of exposure lag (\u2212100, +100\u00a0ms), location of the exposure sound (central, or lateral), location of the test sound (central, or lateral) and SOA (ranging from \u2212240 to +240\u00a0ms). Performance on catch trials was flawless, indicating that participants were indeed looking at the fixation light during exposure. For each combination of exposure lag, location of the exposure sound and location of the test sound, an individually determined psychometric function was calculated over the SOAs by fitting a cumulative normal distribution using maximum likelihood estimation. The mean of the resulting distribution (the interpolated 50% crossover point) is the point of subjective simultaneity (henceforth the PSS), and the slope is a measure of the sharpness with which stimuli are distinguished from one another. The slope is inversely related to the just noticeable difference (JND) and represents the interval (absolute SOA) at which 25 and 75% visual-first responses were given.\nThe PSS and the JND data are shown in Fig.\u00a02 and Table\u00a01. Temporal recalibration was expected to manifest itself as a shift of the PSS in the direction of the exposure lag. The temporal recalibration effect (TRE) was therefore computed by subtracting the PSS following auditory-first exposure from visual-first exposure.\nFig.\u00a02The proportions of visual-first responses (V-first) for each exposure lag (\u2212100\u00a0ms sound-first, 100\u00a0ms light-first) for each combination of location of exposure sound (central, lateral) and location of the test sound (central, lateral)Table\u00a01Mean points of subjective simultaneity (PSSs) in ms, and mean just noticeable differences (JND) in parenthesesLocation test sound Location of the exposure soundCentral LateralAV-lag (ms)PSS (JND)TREPSS (JND)TRECentral\u2212100\u221212.5 (39.3)14.5\u22129.9 (37.8)6.41002.0 (40.8)\u22123.5 (38.6)Lateral\u22121006.1 (38.2)14.2\u221214.3 (36.4)16.810020.3 (36.1)2.5 (42.0)Exposure stimulus pairs were presented with an auditory\u2013visual Lag (AV-lag) of \u2212100 and +100\u00a0ms with sounds either central or lateral; the location of the test stimulus sound was either central or lateral. The temporal recalibration effect (TRE) reflects the difference in PSSs between the \u2212100 and +100\u00a0ms audio\u2013visual lags\nAn overall 2\u00a0\u00d7\u00a02\u00a0\u00d7\u00a02 ANOVA with as within-subjects factors exposure lag, location of the exposure sound and as between-subjects factor location of the test sound was run on the JNDs. None of the effects was significant (all P\u00a0>\u00a00.08), except for a second-order interaction between exposure lag, exposure location and test location, F(1,28)\u00a0=\u00a04.6, P\u00a0=\u00a00.041. Inspection of Table\u00a01 shows that the differences between the JNDs (on average 38.7\u00a0ms) were rather small and unsystematic.\nThe ANOVA on the TREs only showed a significant effect of exposure lag, F(1,28)\u00a0=\u00a023.0, P\u00a0<\u00a00.001, demonstrating, as predicted, that the exposure phase shifted the PSS such that there were more visual-first responses after sound-first exposure than after light-first exposure (i.e. the TRE). The average TRE was 12.9\u00a0ms or 6.5% of the exposure lag. The overall size of this effect corresponds well with previous reports (Fujisaki et al. obtained an average TRE of 12.5%; Vroomen et al. an average TRE of 6.7%). There were, furthermore, no main effects of location of the exposure and test sound, and the crucial interaction between the location of the exposure and test sound was non-significant (all F\u00a0<\u00a01). Temporal recalibration thus manifested itself no matter whether exposure sounds came from central or lateral location, and whether the location of the exposure and test sounds was changed or not.\nDiscussion\nThe goal of the present study was to address whether spatially co-located AV asynchronous stimulus pairs induce temporal recalibration as much as spatially dislocated stimuli do, and whether spatial correspondence between the exposure and test sound affects the size of this effect. Results showed that in all cases, there were clear temporal recalibration effects as subjective simultaneity was shifted in the direction of the adapted audiovisual lag. The shift was equally big for spatially separated and spatially co-located exposure stimuli. Apparently, spatial separation between the sound and light did not hinder temporal realignment. Stimulus generalization across space was also complete, as the shift in temporal alignment was equally big for when the exposure and test sound came from the same or different positions. The results therefore support the notion that spatial alignment between the senses is unimportant for AV pairing in the temporal domain. The results are also in line with previous reports on temporal ventriloquism (Keetels et al. 2007; Vroomen and Keetels 2006) where it was shown that spatial separation does not affect the capturing effect of a light by a sound. Taken together, these findings provide strong evidence that spatial co-occurrence is, even at early perceptual stages, not a necessary constraint for intersensory pairing.\nOne might object, though, that spatial ventriloquism has diminished the potential effects of spatial discordance. It is well-known that the apparent location of a sound can be shifted towards a visual stimulus that is presented at approximately the same time (Howard and Templeton 1966; Radeau and Bertelson 1978; Welch 1978; Bertelson and Radeau 1981; Bertelson 1994, 1999; Radeau 1994). Could it be, then, that the AV spatial discordance in our set-up was diminished, if not became unnoticeable due to spatial ventriloquism? If so, one may not observe an effect of spatial separation on temporal recalibration. This argument, though, seems highly unlikely because it is known that spatial ventriloquism dramatically declines whenever spatial separation exceeds approximately 15\u00a0degrees (Slutsky and Recanzone 2001; Godfroy et al. 2003). Given that we maximized the spatial separation between the sound and light (i.e., at 90\u00a0degrees azimuth), and that informal testing indeed confirmed that spatial separation was clearly noticeable, it seems safe to assume that spatial ventriloquism did not diminish the effect of spatial discordance.\nOne might also ask whether the visual task as used during the exposure phase (i.e., detection of the offset of visual fixation) resulted in an attentional shift towards the visual modality. According to the \u201claw of prior entry\u201d (Titchener 1908), attending to one sensory modality speeds up the perception of stimuli in that modality, resulting in a change in the PSS (see also Shore et al. 2001, 2005; Spence et al. 2001; Schneider and Bavelier 2003; Zampini et al. 2005b). Our visual task might thus result in a shift of the PSS towards more \u201cvisual-first\u201d responses. However, this shift should be uniform for all conditions, and given that temporal recalibration is expressed as a difference in the PSS between exposure lags, the possible role of attention will be subtracted out.\nA remarkable aspect of the data is that previous studies have demonstrated that AV temporal order judgements become more sensitive (i.e. smaller JND) when the sound and light of the test stimuli are spatially separated (see also Bertelson and Aschersleben 2003; Spence et al. 2003; Zampini et al. 2003a, b, 2005a; Keetels and Vroomen 2005). Here, there was a small trend in this direction (average JND of 39.1 vs. 38.2\u00a0ms, for spatially co-located vs. separated test stimuli, respectively), but the effect was non-significant. Possibly, we might have picked up this difference if the effect were measured as a within-subjects factor. For the current purpose, though, this was considered to be unpractical because it would have doubled individual testing time. Despite that we did not observe an effect of AV spatial separation on the JNDs, the data speak on the interpretation on this effect. At least two explanations have been brought up for the improved temporal sensitivity when the locations of test sound and light differ. One is that there is more intersensory integration with as a consequence that the temporal discordance is fused; the other is that there are extra spatial cues that help TOJ performance (Spence et al. 2003). Given that our results show that intersensory pairing occurs independent of a spatial mismatch (see also Vroomen and Keetels 2006; Keetels et al. 2007), it seems more likely that the previously observed effects of spatial separation on temporal sensitivity were induced by the availability of redundant spatial cues rather than fusion per se.\nTo conclude, our results provide strong evidence for the claim that commonality in space between a sound and light is not relevant for AV pairing in the temporal domain. This may, at first sight, seem unlikely, because after all, most natural multisensory events are spatially and temporally aligned. However, a critical assumption that underlies the idea of spatial correspondence for cross-modal pairing is that space has the same function in vision and audition. This notion, though is arguable, as it has been proposed that the role of space in hearing is to steer vision (Heffner and Heffner 1992), while in vision it is an indispensable attribute (Kubovy and Van Valkenburg 2001). If one accepts that auditory spatial perception evolved for steering vision, but not for deciding whether sound and light belong together, there is no reason why cross-modal interactions would require spatial co-localization. Our results therefore have also important implications for designing multimodal devices or creating virtual reality environments, as they show that the brain can ignore cross-modal discordance in space.","keyphrases":["auditory\u2013visual","spatial disparity","temporal recalibration","temporal order judgment","intersensory perception"],"prmu":["P","P","P","P","R"]}
{"id":"Surg_Endosc-3-1-2077356","title":"Duhamel procedure: a comparative retrospective study between an open and a laparoscopic technique\n","text":"Background Few studies are available comparing open with laparoscopic treatment of Hirschsprung\u2019s disease. This study compares a laparoscopic series of 30 patients with a historical open series of 25 patients.\nThe treatment of Hirschprung\u2019s disease has become less invasive over the years. For a long time the surgical treatment was performed in three tempi: the creation of a proximal diverting ostomy, resection of the aganglionic bowel segment, and restoration of bowel continuity. Since the 1990s the operation is usually performed in one session, without ostomy [1\u20133]. In several centers the operation is now undertaken shortly after birth, eliminating the need for bowel washouts in the period between diagnosis and definitive surgical treatment.\nMore recently minimal access techniques have been introduced for taking biopsies to determine the proximal extension of the disease and for dissection of the aganglionic part of the bowel to be removed [4\u20136]. Transanal resection of the aganglionic colon has also been performed without taking biopsies at the beginning of the operation in cases with presumed classic extension of the disease [7].\nThe treatment for Hirschsprung\u2019s disease in our institution has been the Duhamel technique for a long time. With the introduction of minimal access techniques in the early 1990s, we developed a laparoscopic variant of the open technique and published preliminary results in 1995 [4]. There is little doubt that the laparoscopic variant of the open Duhamel leads to less pain, a quicker recovery, and better cosmesis, but the question arises whether the operation is as safe as the open one and whether the functional results are comparable. In this study we compare a series of laparoscopic Duhamel procedures with a historical series of open Duhamel procedures.\nMaterials and methods\nThe charts of all patients who underwent a Duhamel procedure in the period from June 1987 through July 2003 were retrospectively analyzed. From June 1987 through March 1994 the Duhamel procedure was performed in an open way (OD), and from March 1994 through July 2003 it was performed laparoscopically (LD).\nTo obtain a relatively homogeneous group of patients, patients were excluded from the study for the following reasons: preoperative colostomy, extended aganglionosis, trisomy 21, Waardenburg syndrome, or other associated malformations.\nPreoperative bowel preparation consisted of antegrade washout until the effluent became clear [8].\nThe laparoscopic technique was described earlier [4]. In short, the extension of the aganglionosis was diagnosed by frozen section examination of seromuscular biopsies taken during surgery. Originally the dissection of the rectum was performed circumferentially up to the pelvic floor, allowing the rectal stump to be closed transanally. The latter was difficult and required considerable traction on the stapled side-to-side anastomosis in order to reach the upper part of the stump [4]. The technique was therefore modified. The anterior dissection was limited to just below the peritoneal reflexion, while posterior dissection was done up to the pelvic floor. Dissection of the bowel in the proximal direction was close to the bowel wall up to a good location for a biopsy. Either the rectum was amputated at the level of the peritoneal reflexion after placing a proximal ligature around the rectum to prevent leakage or the mobilized colon was everted through the anus. The everted rectum was then transected under traction close to the anus, while the bowel was further exteriorized up to the good biopsy location. The everted bowel was then amputated and closed. The everted bowel was reintroduced into the abdomen as was the remaining rectum.\nAbout 0.5 cm above the dentate line a transverse incision was made in the posterior rectal wall and the retrorectal space was entered to meet the space dissected from above. The proximal end of the bowel was grasped, pulled through, and anastomosed circumferentially with the created opening in the posterior rectum. A side-to-side anastomosis was then made between the anterior aganglionic rectum and the posterior pulled-through ganglionic bowel using an EndoGIA (blue cartridge 3.5cm long; Tyco Heathcare) under laparoscopic control. Usually two cartridges needed to be fired. Finally, the upper rectum was closed laparoscopically with a running 2 \u00d7 0 Vicryl suture. Outcome measures included postoperative complications, hospital stay, and long-term outcome such as constipation, enterocolitis, fecal incontinence, enuresis, stenosis, and adhesive obstruction.\nThe data were statistically analyzed by using SPSS v9.0 (SPSS Inc., Chicago, IL). A p value of less than 0.05 was considered significant. For comparing weight and age between the two groups the t test was used, whereas for hospital stay and start of oral feeding the Mann-Whitney U test was used. Fisher\u2019s exact test was used for comparing the postoperative complications and additional surgical procedures. Data are given as mean (range) unless stated otherwise.\nResults\nIn total 117 patients were operated on during the study period. Sixty-two patients underwent an open Duhamel in the period from June 1987 through March 1994, and 55 had a laparoscopic Duhamel in the period from March 1994 through July 2003. After applying the exclusion criteria 25 patients (21 males and 4 females) who underwent an open Duhamel remained and 30 patients (23 males and 7 females) who had a laparoscopic Duhamel remained. Patient characteristics are shown in Table\u00a01. There were no statistically significant differences between the groups with respect to gender or age at the time of operation.\nTable\u00a01.Patient characteristicsOpen DuhamelLaparoscopic DuhamelGender (m,f)21, 423, 7Age at diagnosis (months)4.2 (0.13\u201372.4)4.6 (0.7\u201367.8)Age at operation (months)6.8 (1.2\u201374.9)8 (0.9\u201372)Weight at operation (kg)7.1 (4.5\u201318.5)8 (3.2\u201322)Operation time (h)not available4.5 (2.3\u20139)Data given as median (range)\nNo intraoperative complications were recorded in both groups. The operative time for the open group could not be traced anymore. In the LD group the mean operative time was 4.8 h (range = 2.3\u20139 h).\nPostoperative events are given in Table\u00a02. Two patients in the LD group underwent a second operation in the early postoperative period: One because of leakage of the rectal stump, which was treated by laparotomy and ileostomy, and the second had laparoscopy because of suspicion of leakage that could not be confirmed. There was a statistically significant difference between the groups with respect to length of hospital stay (p < 0.001) and time of first oral intake (p < 0.001).\nTable\u00a02.Postoperative eventsOpen Duhamel (n = 25)Laparoscopic Duhamel (n = 30)Postoperative fever 31Leakage of rectum stump01Abscesses00Start of oral feeding (days)5.1 (4\u20138)3.4 (2\u20139) Hospital stay (days)7.8 (6\u201313)6 (3\u201315)\nBecause of the study design the period of follow-up is different between the two groups. Two patients in the LD group were readmitted for dilatation of the anorectum for stenosis. In one of these patients a rectal spur needed to be transected; this was performed under laparoscopic control. In this patient initially only one cartridge had been used. Reoperation was carried out in one OD patient and in two LD patients.\nAlthough statistically not significant, there was a higher incidence of admission for enterocolitis in the LD group. There were more children with adhesive obstruction and with enuresis in the OD group, but again the differences was not statistically significant (Table\u00a03).\nTable\u00a03.Results at follow-upOpen Duhamel (n = 25)Laparoscopic Duhamel (n = 30)Follow-up (months)87.8 (7\u2013211)39.5 (\u2013113)Reoperation (Duhamel)12Stenosis02Obstructive ileus30Admission for enterocolitis39Admission for constipation75Incontinence00Enuresis 30\nDiscussion\nHirschsprung\u2019s disease is basically incurable. Even when the proximal transection plane of the bowel shows a normal plexus at pathologic examination, there is no guarantee of a good outcome because the distal rectum is and remains abnormal. The best that surgery can achieve is a delicate balance between constipation and incontinence. More often than not the balance tips in one direction. End points are difficult to set and final results are therefore difficult to evaluate.\nHirschsprung\u2019s disease is a relatively rare disease and is heterogeneous in the sense that its extension varies and that it may be part of a syndrome. To obtain a group of patients that was as homogeneous as possible, many variables in the present study were excluded. The disadvantage of that is that the subgroups become smaller which makes it harder to obtain significant differences between the groups. With these limitations in mind, we can conclude that there was no difference in intraoperative complication rate. Although the operative times for patients in the OD group were not available, the operative times for patients in the LD group were long, which emphasizes that the laparoscopic procedure is not simple. The difference in starting time of oral feeding and in postoperative hospital stay were significantly longer in the OD group. The first seems to be the result of bias as the postoperative feeding protocol changed over the years toward faster introduction of feeding. The same may apply to the shorter postoperative hospital stay in the LD group although this is less obvious. There were no significant differences in postoperative complications or in the follow-up end points. However, relatively more patients in the LD group were readmitted for enterocolitis, maybe more rectum is left behind with LD. Better functional results have been reported when a short rectal pouch is left [8]. Although not statistically different, adhesive bowel obstruction was seen in the OD group only and the same applies for enuresis. The cosmetic results were definitely better in the LD group.\nConclusion\nThe laparoscopic variant of the Duhamel procedure is not simple, as reflected by its long operation time. There seems to be no essential difference between the open and the laparoscopic procedure with respect to postoperative complications or functional results at follow-up. Although there is a tendency for a higher enterocolitis rate in the LD group, lower adhesive obstruction and enuresis rates were encountered. There is no doubt that the LD is cosmetically superior. Despite the fact that the transanal approach is becoming more popular in recent years, there certainly remains a place for the laparoscopic Duhamel\u2013Martin procedure, particularly when extended Hirschsprung\u2019s disease is present.","keyphrases":["duhamel","hirschsprung","aganglionosis","laparoscopy","child"],"prmu":["P","P","P","P","U"]}
{"id":"Dig_Dis_Sci-4-1-2292501","title":"Lack of Relationship Between Chronic Upper Abdominal Symptoms and Gastric Function in Functional Dyspepsia\n","text":"To determine the relationship between gastric function and upper abdominal sensations we studied sixty FD patients (43 female). All patients underwent three gastric function tests: 13C octanoic gastric emptying test, three-dimensional ultrasonography (proximal and distal gastric volume), and the nutrient drink test. Upper abdominal sensations experienced in daily life were scored using questionnaires. Impaired proximal gastric relaxation (23%) and a delayed gastric emptying (33%) are highly prevalent in FD patients; however, only a small overlap exists between the two pathophysiologic disorders (5%). No relationship was found between chronic upper abdominal symptoms and gastric function (proximal gastric relaxation, gastric emptying rate, or drinking capacity) (all P > 0.01). Proximal gastric relaxation or gastric emptying rate had no effect on maximum drinking capacity (P > 0.01). The lack of relationship between chronic upper abdominal sensations and gastric function questions the role of these pathophysiologic mechanisms in the generation of symptoms.\nIntroduction\nFunctional dyspepsia (FD) is a common disorder seen in daily clinical practice, characterized by the presence of pain or discomfort in the upper abdomen in the absence of organic, systemic, or metabolic disease [1]. Functional dyspeptic patients complain about a variety of symptoms, which are frequently intermittent, and mostly related to food intake [2]. For that reason, a subdivision of patients has been proposed, in order to clarify the heterogeneity of this disorder and to direct treatment options [3]. Attempts have been made to subdivide patients according to their symptoms; however, a large overlap of symptoms exists and many patients do not fit into one of the subgroups [1].\nCurrently, many efforts are being made to subdivide patients according to gastric (dys)function, and to find new ways of treating these proposed pathophysiologic disorders [4]. Three pathophysiologic mechanisms have been described as possible etiologic factors: (1) a delayed gastric emptying, (2) impaired proximal gastric accommodation, and (3) visceral hypersensitivity. Delayed gastric emptying is present in approximately 30% of FD patients, and may be one of the underlying mechanisms for symptoms (vomiting and postprandial fullness) [5]. Impaired proximal gastric relaxation may be an important etiologic factor in the pathophysiology of functional dyspepsia, considering the high prevalence of approximately 40% in FD patients, and a possible association with early satiety, weight loss, and fullness [6, 7]. Finally, an increased visceral sensitivity is highly frequent in FD patients, and even though difficult to measure, an important etiologic factor in the pathophysiology of FD [8]. However, when targeting specific pathophysiologic mechanisms by the use of pharmacologic agents, the effect on symptoms is questionable [6, 9, 10]. In other words, the relationship between specific upper abdominal sensations and the above-mentioned mechanisms remains to be matter of debate.\nWe conducted a study to assess the relationship between gastric function and upper abdominal sensations. Gastric emptying rate, proximal gastric relaxation, and maximum drinking capacity were assessed in FD patients. The symptoms experienced in daily life (chronic upper abdominal symptoms) were assessed and related to the primary outcome parameters of the gastric function tests. We hypothesised that chronic upper abdominal symptoms and specific pathophysiologic mechanisms have no correlation in functional dyspepsia.\nMaterials and methods\nPatients\nAll patients visiting the outpatient clinics at our hospital, fulfilling the Rome II criteria for functional dyspepsia [1], were subjected to three non-invasive functional tests of the stomach. A total of 60 functional dyspeptic patients were prospectively evaluated; 43 female (median age 40\u00a0years; range 18\u201365) and 17 male (median age 37\u00a0years; range 21\u201364).\nThe inclusion criteria were (a) the presence of dyspeptic symptoms, assessed using the questionnaire described below; (b) no evidence of macroscopic inflammation of the esophageal mucosa or focal lesions of the esophagogastroduodenal mucosa at upper gastrointestinal endoscopy (performed within 1-year prior to inclusion); (c) no abnormalities seen during upper abdominal ultrasonography (performed within 1\u00a0year prior to inclusion); (d) absence of serious concomitant illness; and (e) the absence of major gastrointestinal surgery (excluding appendectomy).\nThe protocol was approved by the ethics committee of the University Medical Center Utrecht. All patients gave written informed consent for inclusion in the trial.\nChronic dyspeptic symptoms questionnaire\nEach patient completed a reproducible dyspepsia questionnaire [5, 6, 11]. Patients were asked to score six different symptoms (pain or discomfort centered in the upper abdomen, early satiety, bloating in the upper abdomen, fullness, nausea, and vomiting) from 0 to 5 (0\u00a0=\u00a0none, 1\u00a0=\u00a0very mild; awareness of symptoms but easily tolerated, 2\u00a0=\u00a0mild; tolerated without interference with usual activity, 3\u00a0=\u00a0moderate; enough to cause some interference with usual activity, 4\u00a0=\u00a0severe; enough to cause significant interference with usual activity, 5\u00a0=\u00a0very severe; incapacitating with inability to work or do usual activity). For inclusion, two of these symptoms had to be scored as moderate, severe or very severe and these symptoms needed to be present for at least 12\u00a0weeks, not necessary consecutive, in the preceding 12\u00a0months.\nStudy protocol\nAll patients underwent three functional tests of the stomach on three separate days; the \u00b9\u00b3C-octanoic breath test, three-dimensional ultrasonography of the stomach, and a nutrient drink test. The order of the three study days was arbitrary. Each of the study days started at 08:00\u00a0h after an overnight fast of at least 10\u00a0h. The time interval between the tests was three days to 2\u00a0weeks. Patients were asked to discontinue any medication known to influence gastrointestinal motility or sensitivity for at least 7\u00a0days prior to the study, including PPI therapy. The use of narcotics, anticholinergic medication, serotonergic medication (including selective serotonin reuptake inhibitors), and antidepressants was considered an exclusion criterion. None of the patients were on NSAID therapy.\n13C-octanoic breath test\nThe rate of gastric emptying was assessed using the 13C-octanoic breath test. The test meal consisted of two fried eggs, one slice of bread, 5\u00a0g margarine and 150\u00a0ml water (total caloric value of 294\u00a0kcal and a nutrient composition of 16\u00a0g protein, 16\u00a0g carbohydrate, 18\u00a0g fat) [12]. The egg yolk of one egg was labelled with 100\u00a0mg 13C-sodium-octanoic acid (598\u00a0\u03bcmol; Campro Scientific, Veenendaal, The Netherlands), dissolved in 1\u00a0ml distilled water. Breath samples were taken at baseline, before the meal and from start of ingestion of the meal every 2\u00a0min the first 30\u00a0min, every 5\u00a0min for the next 30\u00a0min and every 15\u00a0min thereafter up to 4\u00a0h.\nThree-dimensional ultrasonography\nTotal-, proximal-, and distal gastric volumes were assessed before and after ingestion of a nutrient drink using 3D-US [13\u201316]. Ultrasonographic data were acquired in a sitting position, while fasting and at 5, 15, 30, 45, and 60\u00a0min after ingestion of a nutrient drink. The nutrient drink (500\u00a0ml) consisted of 200\u00a0ml lactose- and fiber-free milk drink, containing 12.0\u00a0g proteins, 11.6\u00a0g fat and 36.8\u00a0g carbohydrate (300\u00a0kcal) (Nutridrink, Nutricia, Zoetermeer, The Netherlands) mixed with 300\u00a0ml of water, and was ingested within 3\u00a0min.\nThe 3D imaging system consisted of an ultrasound scanner with a 3.5\u00a0MHz curved probe and a tracking system (Esaote-Pie Medical, Maastricht, The Netherlands). The tracking system consisted of a transmitter generating a spatially varying magnetic field and a small receiver, firmly attached to the ultrasound probe, containing three orthogonal coils to sense the magnetic field strength [17]. A standardized ultrasound-scanning pattern was used, starting at the left lateral subcostal margin and then moving distally towards the pylorus having the probe in a vertical position [14]. The 2D sagittal planes were used to draw the region of interest, corresponding to the inner layer of the stomach wall (the interface between the outer profile of the gastric wall mucosa and the liquid nutrition). A 3D reconstructed image of the stomach and the gastric volume was obtained using software with rendering and volume estimation capability (In Vivo ScanNT, Medcom GmbH, Darmstadt, Germany).\nThe proximal gastric volume was defined as the gastric volume between the diaphragm and a dividing plane 10\u00a0cm below the point where the fundic top reaches the diaphragm. Similarly, a distal part was separated, defined as the gastric region between the antral area (the sagittal ultrasound plane in which the antrum, the left liver lobe, the superior mesenteric vein and the abdominal aorta are seen simultaneously) and the gastroduodenal junction [18]. At every time point, we subtracted fasting total or partial gastric volume leaving the change in total or partial gastric volume. Proximal and distal gastric volume ratios were calculated by dividing proximal or distal gastric volume by total gastric volume. Recently, we defined impaired proximal relaxation as the average of the proximal gastric volume ratios of 5 and 15\u00a0min smaller than the lower limit of the 95% confidence interval (0.32\u20130.57) of healthy controls; 35 healthy controls (16 male; mean age 31 (18\u201353) years) [7]. All measurements were made by a single investigator (N.v.L.) who was blinded for the results of the gastric emptying test and the drink test.\nNutrient drink test\nThe nutrient drink test was used to measure the drinking capacity and the symptoms evoked by a nutrient drink [19, 20]. Patients were asked to ingest a nutrient drink (Nutridrink; 1.5\u00a0kcal\/ml) at a constant rate of 15\u00a0ml\/min. At 5-min intervals, they scored satiety using a graphic rating scale that combines verbal descriptors on a scale graded 0\u20135 (0\u00a0=\u00a0no satiety, 5\u00a0=\u00a0maximum satiety). The test ends when the subject reaches maximum satiety. In healthy controls, maximum satiety occurs after ingestion of 1005\u00a0ml (1508\u00a0kcal). The lower limit of normal was 653\u00a0ml (979\u00a0kcal) [19]. In the same study, FD patients ingested 361\u00a0ml (542\u00a0kcal) before reaching maximum satiety.\nStatistical analysis\nThe main focus of our analysis was (a) to analyse a possible relationship between the rate of gastric emptying, total or partial gastric volumes after meal ingestion, and drinking capacity, and (b) to compare the outcome of the 13C-octanoic breath test, the 3D-US test, and the drink test with the symptoms of patients experienced during daily life (chronic symptoms).\nThe primary end points of the gastric function tests are dichotomous and continuous. The relationship between the outcomes of the three gastric function tests was studied using a Pearson\u2019s correlation between continuous variables (half-emptying time, retentions after 120\u00a0min, fasting gastric volume, proximal gastric volume ratio, distal gastric volume ratio, and maximum drinking capacity). Secondly, patients were subdivided in two groups, according to postprandial proximal gastric relaxation (normal or impaired), and according to the rate of gastric emptying (normal or delayed), in order to compare multiple variables between the sub-groups using the Students\u2019 t-test.\nChronic upper abdominal symptoms (ordinal variables) were compared between FD patients with normal- or delayed gastric emptying or a normal or impaired proximal gastric relaxation (dichotomous variable), using the \u03c72 test. Secondly, the relationship between chronic upper abdominal symptoms and half-emptying time, retention after 120\u00a0min, fasting gastric volume, proximal gastric volume ratio, distal gastric volume ratio, and maximum tolerated volume (continuous variable) was analysed using one-way analysis of variance (ANOVA).\nWe have analysed the effect of age, height, weight, BMI, and sex on the outcome of the gastric function tests and the chronic symptoms using a regression analysis. All variables were tested in single models and using multivariable analysis.\nDue to the high number of comparisons made, we considered a P value of <0.01 as statistically significant. All statistical analysis was performed using commercially available software (SPSS 11.0 for Microsoft windows). For the \u03c72 test and the Students\u2019 t-test, on the basis of a Cohen\u2019s effect size of 0.8, a power of 0.80 was obtained with 60 subjects (considering \u03b1\u00a0=\u00a00.01) [21]. With the same number of subjects, there was good power (0.80) to detect an R value of 0.40 with an \u03b1\u00a0=\u00a00.01.\nResults\nCombined assessment of the 13C-octanoic breath test, 3D-US, and the nutrient drink test was performed in 60 FD patients. The frequency of upper abdominal sensations scored as moderate or higher was  upper abdominal pain (80%), early satiety (59%), bloating (77%), fullness (71%), and nausea (51%). Vomiting was present in 13% of patients (Table\u00a01).\nTable\u00a01Frequency of severity grading for each of six dyspeptic symptoms in 60 dyspeptic patients (chronic symptoms)0 (None)1\u20132 (Very mild\u2013mild)3 (Moderate)4\u20135 (Severe\u2013very severe)Upper abdominal pain1 (2)11 (18)18 (30)30 (50)Early satiety7 (12)18 (30)19 (32)16 (27)Bloating4 (7)10 (17)27 (45)19 (32)Fullness2 (3)15 (25)26 (43)17 (28)Nausea10 (17)19 (32)14 (23)17 (28)Vomiting46 (77)6 (10)2 (3)6 (10)Numbers in parentheses represent row percentages\nGastric emptying and proximal gastric relaxation\nFigure\u00a01 shows the frequency of a delayed gastric emptying rate, defined as a half emptying time \u2265120\u00a0min and\/or a retention after 120\u00a0min \u226540%, and impaired proximal gastric relaxation, defined as an average proximal gastric volume ratio of 5 and 15\u00a0min postprandially \u2264 0.32 (95% CI healthy controls) in the patient group [7]. A delayed gastric emptying with a normal proximal gastric relaxation was found in 33% of patients. Impaired proximal gastric relaxation with a normal gastric emptying rate was observed in 23% of patients. In 38% of all patients, none of both pathophysiologic disorders were found. Finally, only a small overlap exists between the two pathophysiologic disorders (5% of patients). These numbers are in concordance with literature [5, 6].\nFig.\u00a01Gastric emptying and proximal gastric relaxation in 60 FD patients: 33% of patients have a delayed gastric emptying and normal proximal gastric relaxation (black), 23% of patients have impaired proximal gastric relaxation and a normal gastric emptying rate (striped), 38% of patients have none of the pathophysiologic disorders (white), and 5% of patients have both disorders (grey)\nNutrient drink test\nThe average amount of nutridrink ingested before reaching maximum satiety was 399.8 (344\u2013447)\u00a0ml (600\u00a0kcal). Table\u00a02 summarizes the results of the nutrient drink test. No effect of age or BMI on MTV and upper abdominal sensations was observed (all P\u00a0>\u00a00.01). Male patients had a maximum tolerated volume (MTV) of 500 (381\u2013618), and female patients of 359 (301\u2013417) (P\u00a0=\u00a00.017).\nTable\u00a02The effect of age, BMI, and sex on maximum tolerated volume (MTV) and the change in upper abdominal sensations after the nutrient drink testAgeBMISex\u03b20\u03b21\u03b20\u03b21FemaleMaleMTV329 (160\u2013499)1.7 (\u22122.2\u20135.6)363 (3\u2013725)1.6 (\u221214 \u201318)359 (301\u2013417)500 (381\u2013618)Delta symptoms \u00a0\u00a0\u00a0\u00a0Pain11 (0\u201335)0.02 (\u22120.5\u20130.6)24 (0\u201374)-0.5 (\u22123\u20132)10 (0\u201320)14 (0\u201328)\u00a0\u00a0\u00a0\u00a0Fullness49 (23\u201376)-0.1 (\u22120.7\u20130.5)49 (0\u2013100)-0.1 (\u22122\u20132)48 (37\u201359)42 (29\u201355)\u00a0\u00a0\u00a0\u00a0Nausea46 (20\u201372)-0.5 (\u22121\u20130.1)28 (0\u201385)-0.2 (\u22123\u20132)21 (11\u201331)31 (12\u201350)\u00a0\u00a0\u00a0\u00a0Hunger\u221228 (\u221250\u20130)0.09 (\u22120.4\u20130.6)\u22127 (\u221252\u201340)\u22120.8 (\u22123\u20131.2)\u221224 (\u221233  to  \u221215)\u221225 (\u221237 to \u221214)\u03b20: intercept of the model. \u03b21: slope of the corresponding variable. Numbers in parenthesis represent the 95% confidence interval of \u03b2I. MTV and delta symptoms in female and male patients are presented as mean (95% confidence interval for mean). No effect of age, BMI, or sex on MTV or the change in upper abdominal sensations after the nutrient drink test was observed (all P\u00a0>\u00a00.01)\nRelationship between gastric emptying, total and partial gastric volume, and drinking capacity\nTable\u00a03 displays some of the patient characteristics in FD patients with normal or impaired proximal gastric relaxation and with normal or delayed gastric emptying. Age or BMI did not influence proximal gastric relaxation or the rate of gastric emptying. Patients with a normal gastric emptying rate had an MTV of 439\u00a0ml (359\u2013519) whereas patients with a delayed gastric emptying had a MTV of 334\u00a0ml (279\u2013389) (P\u00a0=\u00a00.032). MTV in patients with normal or impaired proximal relaxation was very similar (404 and 384\u00a0ml respectively). In patients with normal proximal gastric relaxation, an average fasting gastric volume of 50\u00a0ml (41\u201359) was determined, opposed to 34\u00a0ml (24\u201344) in patients with impaired proximal relaxation (P\u00a0=\u00a00.029). The fasting gastric volume in patients with normal or delayed gastric emptying was 44 and 47\u00a0ml, respectively.\nTable\u00a03Characteristics of FD patients, subdivided according to the extent of proximal gastric relaxation or the rate of gastric emptying (n\u00a0=\u00a060)Proximal gastric relaxationGastric emptyingNormal (n\u00a0=\u00a043)Impaired (n\u00a0=\u00a017)Normal (n\u00a0=\u00a037)Delayed (n\u00a0=\u00a023)Age41 (36\u201345)42 (35\u201349)40.9 (36\u201345)41 (35\u201348)BMI22 (21\u201323)23 (21\u201325)22 (21\u201323)22 (21\u201323)MTV (ml)404 (346\u2013461)384 (237\u2013530)439 (359\u2013519)334 (279\u2013389)Fasting gastric volume (ml)50 (41\u201359)34 (24\u201344)44 (33\u201355)47 (37\u201356)Data are presented as mean (95% confidence interval for mean). No effect of age and BMI on proximal gastric relaxation or gastric emptying was observed\nTwenty out of 43 female patients had a delayed gastric emptying (47%) and 18% of all male patients had a delayed gastric emptying (P\u00a0=\u00a00.038). The prevalence of impaired proximal gastric relaxation in male and female patients was 21% and 35% respectively (not shown in the table).\nA positive correlation was observed between the proximal gastric volume ratio (3D-US) and half emptying time (r\u00a0=\u00a00.32, P\u00a0=\u00a00.015) and the retention after 120\u00a0min (r\u00a0=\u00a00.30, P\u00a0=\u00a00.024) (13C breath test). No correlation was found between the distal gastric volume ratio and the main outcome parameters of the gastric emptying test.\nRelationship between chronic symptoms and gastric function\nNo effect of age, sex, or BMI on any of the chronic upper abdominal sensations was observed, except that patients with a higher BMI or a higher weight scored lower on symptoms of early satiety (both P\u00a0=\u00a00.007). Figure\u00a02 shows the chronic upper abdominal symptoms in FD patients with normal or delayed gastric emptying and normal or impaired proximal gastric relaxation. No differences were observed in the percentage of patients who scored any of the upper abdominal symptoms as moderate or higher between these groups (all P\u00a0>\u00a00.01). We did not find any correlation between half emptying time, retention after 120\u00a0min, proximal gastric volume ratio, distal gastric volume ratio, fasting gastric volume, and any of the chronic upper abdominal symptoms (all P\u00a0>\u00a00.01).\nFig.\u00a02Chronic upper abdominal symptoms scored from 0 (nonexistent) to 5 (very severe; incapacitating with inability to work or do usual activity). The figure shows the number of the patients who scored three (moderate) or higher on the questionnaire (expressed as a percentage of the total) in subgroups with; (Fig. 1A) normal- (\u25a1) or delayed (\u25a0) gastric emptying, and (Fig. 1B) normal- (\u25a1) or impaired (\u25a0) proximal relaxation\nFigure\u00a02 depicts all FD patients categorized according to their chronic complaints; mild (1\u20132), moderate (3), and severe (4\u20135). No difference in maximum drinking capacity was observed between the three groups (all P\u00a0>\u00a00.01) (Fig.\u00a03). Interestingly, patients who reported early satiety as moderate or higher have a comparable drinking capacity with patients who do not experience this symptom in daily life.\nFig.\u00a03Nutrient drink test: maximum tolerated volume in FD patients, categorized according to the chronic symptoms. No difference in drinking capacity was observed between patients experiencing mild (1\u20132), moderate (3), or severe (4\u20135) pain, early satiety, fullness, or nausea in daily life (all P\u00a0>\u00a00.01)\nDiscussion\nThe following were the most important findings of this study: (1) no relationship was found between chronic upper abdominal symptoms and gastric function (proximal gastric relaxation, gastric emptying rate, or drinking capacity), (2) a third of all FD patients had a normal gastric emptying rate and a normal proximal gastric relaxation, and only a small overlap existed between the two pathophysiologic disorders (7%), and (3) there was an absence of any relationship between maximum drinking capacity and proximal gastric relaxation or gastric emptying rate.\nThe observation that approximately 40% of FD patients had a delayed gastric emptying, and approximately 30% of FD patients had impaired proximal gastric relaxation, is confirmatory of previous studies [5, 6]. The relative small overlap between the two pathophysiologic mechanisms may suggest that one abnormality excludes the other. However, no significant positive correlation between the proximal gastric volume ratio and half emptying time or retention after 120\u00a0min was found. The rate of gastric emptying is most likely dependent on many factors, including fundal, antral, pyloric, and duodenal motility [22]. For that reason, gastric emptying and postprandial gastric relaxation should be considered as two separate mechanisms [23].\nFor assessment of partial gastric volumes we used 3D-US as a noninvasive alternative for the barostat technique. Recently, a head-to-head comparison between the barostat and 3D-US was performed, in which it was shown that 3D-US was able to identify almost all patients with impaired accommodation assessed by barostat. It was, however, emphasized that the two techniques are not interchangeable, which is likely to be due to the difference in invasiveness of both techniques. Since there is no absolute concordance between the two techniques, we must preserve some reservations in generalizing the results of the current study.\nThe nutrient drink test has been suggested as a tool to measure meal-induced satiety, and as a non-invasive alternative for the detection of normal- or impaired accommodation of the stomach [6, 19]. Furthermore, a positive relationship between the rate of gastric emptying and the amount of Kcal ingested during the nutrient drink test has been described, thereby suggesting that the maximum tolerated volume is not only influenced by gastric accommodation [24]. However, many studies have shown conflicting results, displaying no relationship between drinking capacity and barostat or SPECT findings, [25, 26] and a negative relationship between gastric emptying rate and maximum tolerated volume [27].\nSince the results from different studies do not correspond, it remains a mystery what it is we are testing with the nutrient drink test. The suggestion that the nutrient drink test can be used to discriminate between FD patients with normal or impaired visceral sensitivity is disputable [9]. In the current study, we did not observe any relationship between MTV and chronic upper abdominal symptoms or between MTV and proximal gastric relaxation or gastric emptying rate. Most studies do agree that the drink test differentiates between FD patients and healthy controls, as we have found in the present study. Notably, the average amount of nutridrink ingested until maximum satiety, was very similar to what others have found (approximately 360\u00a0ml) [19], which is below the 95% confidence interval of healthy controls. No effect of age or BMI on MTV was observed in the current study, however we did observe a modest effect of gender, although this did not reach statistical significance (P\u00a0=\u00a00.017) [20].\nThe nutrient drink test is also being used in pharmacological trials, and a resemblance between symptoms evoked by the meal challenge and symptoms experienced in daily life has been observed [27]. Recently, we have performed a double blind, placebo-controlled, crossover trial, in which the activity of a new drug was tested, using the outcome of the nutrient drink test as one of the end points in the study [28]. FD patients who participated in the pharmacological trial drank significantly more compared to the FD patients in the current study; 569\u00a0\u00b1\u00a090\u00a0ml and 360\u00a0\u00b1\u00a030\u00a0ml respectively (P\u00a0<\u00a00.001). A strong placebo effect and cognitive influences like motivation should therefore be considered as confounding factors. No differences in age, sex, BMI, chronic symptoms, or upper abdominal sensations experienced during the drink test were observed between the patients who participated in the pharmacological trial and those who did not.\nIn summary, the maximum drinking capacity of FD patients, seen at a tertiary referral practice, is not influenced by gastric emptying rate or proximal gastric relaxation. The question is raised what usefulness this test has, in terms of diagnosis or treatment options, in FD patients and as a tool to analyze gastric function or upper abdominal sensations. Since we did not find any relationship between upper abdominal symptoms and MTV, the nutrient drink test cannot be regarded as an alternative for measuring visceral perception, as can be done by gastric barostat. In our opinion, many subjective factors, like motivation, probably play an important disturbing factor in the outcome of the test.\nIn conclusion, in spite of a high prevalence of impaired proximal accommodation and delayed gastric emptying in FD patients, the lack of correlation between chronic upper abdominal sensations and gastric function questions the role of these pathophysiologic mechanisms in the generation of symptoms. Consequently, gastric function does not serve as a clear marker for the symptoms experienced by FD patients in daily life, and limited effect on symptoms may be expected when targeting these specific mechanisms. Finally, despite many efforts, no (measurable) motoric disorder can be appointed as a possible pathophysiologic mechanism underlying the presence of upper abdominal symptoms. Most likely, other factors like visceral perception play a vital role in functional dyspepsia.","keyphrases":["upper abdominal symptoms","functional dyspepsia","gastric emptying","nutrient drink test","stomach","3d-ultrasonography"],"prmu":["P","P","P","P","P","U"]}
{"id":"J_Med_Internet_Res-8-4-1794003","title":"Age-Specific Search Strategies for Medline\n","text":"Background Many clinicians and researchers are interested in patients of a specific age (childhood, geriatrics, and so on). Searching for age-specific publications in large bibliographic databases such as Medline is problematic because of inconsistencies in indexing, overlapping age categories, and the spread of the relevant literature over many journals. To our knowledge, no empirically tested age-specific search strategies exist for Medline.\nIntroduction\nClinicians and researchers seeking research reports for specific age categories, including generalists and those who are engaged in clinical specialties such as adult medicine, geriatric medicine, pediatric medicine, neonatal medicine, or obstetrics, need to target their literature searches so that the information they retrieve is relevant to their patient population. Difficulty in finding pertinent evidence contributes to the challenges\thealth professionals have in keeping up-to-date and practising evidence-based medicine [1-7].\nFinding age-specific evidence in Medline is a difficult task for several key reasons. In large bibliographic databases such as Medline, optimal search retrieval for individual topics is hampered by the overwhelming amount of available information that is not pertinent to the question. When users search in Medline they have the potential to retrieve articles from any of the approximately 4800 journals that are currently indexed in the database. The size of this general purpose biomedical database coupled with imperfections in indexing [1-3] lead to a high risk of missing articles that are relevant to the topic of the search while at the same time retrieving many articles that are off target. Effective ways to refine the search may be helpful for those wanting to keep up-to-date and for those looking for an answer to a specific patient care question.\nSearching in Medline for a specific patient population by selecting \"age-specific\" journals will not help because studies relevant to any age group are scattered through a wide range of journals, including general journals that cater to no particular age group. Moreover, in Medline, the indexing practices used to identify the ages of those involved in a study are so liberal that they create a very imprecise representation of the age categories of the participants within the study. Medline indexers apply all relevant age-specific index terms to an article regardless of how many participants fall within that category. Thus, if just one patient or participant in the study falls into a particular age category, that age-specific medical subject heading (MeSH) term will be applied. For example, if a researcher was interested in intercultural communication in family medicine around issues of newborn care, the study by Harmsen and colleagues [8] might be retrieved using the following index terms: infant, newborn; ethnic groups; communication; and family practice. However, looking at the patient population studied, only 0.9% of the participants were children under the age of 12 years\u2014likely very few of these were newborns. The study included participants from many age categories, resulting in eight age-specific index terms being assigned to this article (infant, newborn; infant; child, preschool; child; adolescent; adult; middle aged; and aged). For searchers who are interested in communication around newborn issues, this article is likely not useful even though the indexing indicates that it is potentially relevant. These age-classification problems are compounded by the less than optimal search strategies used by clinicians, including their lack of knowledge about how to narrow searches without missing relevant information, and their uncertainty about when to stop searching [9,10].\nTo assist clinicians searching for studies on age-specific patient populations, we have developed and tested Medline search strategies for detecting studies for specific age categories as well as tested age-specific search terms pertinent to five age-related clinical specialties. In this paper, we report on the evaluation of the retrieval performance of age-specific search strategies in Medline compared with a manual review (the \"gold standard\" search) of each article in every issue of 161 journals in the year 2000.\nSearch strategies are useful tools when searching in large electronic databases. We previously developed search strategies for use in Medline to detect clinically relevant scientifically sound articles in the areas of causation, prognosis, treatment, and diagnosis [11-15]. After publishing our initial work on search strategy development [15], we were approached by neonatologists and gerontologists to develop age-specific search strategies because they expressed frustration with the inefficiency of the current system for finding content specific to their patient population. Using only the age-related MeSH terms when searching can be time-consuming because retrievals can be very large and imprecise. To our knowledge, no empirically developed age-specific search strategies have been previously reported for Medline.\nMethods\nThe study compared the retrieval performance of age-specific search terms and phrases in Medline (accessed using Ovid) with a manual review of each article in every issue of 161 journal titles for the year 2000. The 161 journals were chosen over several years in an iterative process based on a hand search review of over 400 journals. The journals were recommended by clinicians, librarians, editors, and publishers and were chosen based on Science Citation Index impact factors and ongoing assessment of their yield of studies and reviews of scientific merit and clinical relevance [16] in the production of 4 evidence-based medicine secondary journals (ACP Journal Club, Evidence-Based Medicine, Evidence-Based Nursing, and Evidence-Based Mental Health). The 161 journals include content for the disciplines of internal medicine (eg, Annals of Internal Medicine), general medical practice (eg, BMJ, JAMA, and Lancet), mental health (eg, Archives of General Psychiatry, British Journal of Psychiatry), and general nursing practice (eg, Nursing Research).\nSix research assistants hand searched the 161 journals for the year 2000 and collected data on age of the study participants according to our hand search categories defined in Table 1. This data collection was part of a larger study in which the research assistants applied methodological criteria to each article in each issue to determine if the article was methodologically sound for seven purpose categories (eg, treatment and diagnosis). All purpose category definitions and corresponding methodological rigor have been outlined in previous papers [4,17]. Research staff were rigorously calibrated for applying all these criteria, including the age classification of study participants, and interrater agreement for application of all criteria exceeded 80% beyond chance \u03ba = 0.81; 95% CI = 0.79-0.84) [4].\nTable 1\nComparison of hand searching and Medline MeSH classification of age categories\nHand Search Category\nOur Definition\nMedline MeSH Term Category\nMeSH Definition\nFetus\nFetus\n-\n-\nNewborn\nBirth to 1 month\nInfant, newborn\nBirth to 1 month\nInfant\n> 1 month to < 24 months\nInfant\n1 to 23 months\nPreschool\n2 years to < 6 years\nChild, preschool\n2 to 5 years\nChild\n6 years to < 13 years\nChild\n6 to 12 years\nAdolescent\n13 years to < 19 years\nAdolescent\n13 to 18 years\nAdult\n19 years to < 45 years\nAdult\n19 to 44 years\nMiddle age\n45 years to < 65 years\nMiddle aged\n45 to 64 years\nAged\n65 years to < 80 years\nAged\n65 to 79 years\nAged 80\n\u2265 80 years\nAged, 80 and over\n80 years and over\nND\nNondiscernible\n-\n-\nMeSH terms and textwords related to age (eg, infant, child, adult) were downloaded from Medline and were treated as \"diagnostic tests\" for detecting studies with an age-specific population as determined by a hand search of the literature from 161 journals (the gold standard). The hand search data were obtained by reading each issue completely. The downloaded Medline data from the 161 journals included the retrieval sets for each of the individual terms. After these two data sources were obtained (ie, the Medline downloads and the hand search review), a database was created that included the matched merged content from these two sources. These Ovid retrieval sets were then manipulated by our own set of programs to calculate our outcome measures\u2014the operating characteristics of each age-specific searching term (eg, sensitivities, specificities, and precision) for individual terms and for combinations of terms. When we merged the two data sets (Medline and hand search), we determined the match. If Medline included an item that was not indexed, we went back to the journal and scored it. If we had scored an item that was not in Medline, we removed it from the merged database. Therefore, the final merged database included only items that had hand search scores and Medline indexing. This merged database was used to develop the age-specific search strategies [17].\nBorrowing from the concepts of diagnostic test evaluation and library science, we determined the sensitivity, specificity, precision, and accuracy of single- and multiple-term Medline searches. We considered these operating characteristics as the indicators of search term performance. Sensitivity for a given age-specific topic is defined as the proportion of relevant articles (ie, articles with the desired age-specific content) that are retrieved; specificity is the proportion of nonrelevant articles (ie, articles that are outside the desired age-specific content) not retrieved; precision is the proportion of retrieved articles that are relevant (a library science term that is equivalent to \"positive predictive value\" in diagnostic test evaluation); and accuracy is the proportion of all articles that are correctly classified (ie, overall proportion of relevant articles retrieved and nonrelevant articles not retrieved). Our hand search of the 161 journals indexed in Medline led to the classification of all articles in these journals for age-related content. Search terms were then tested to determine their performance in retrieving age-relevant articles while eliminating those that were nonrelevant. An automated process (which we developed and implemented using a computer program) was used to calculate the operating characteristics (performance) for each single and combination term in Medline. Formulae for calculating the operating characteristics (ie, sensitivity, specificity, precision, and accuracy) of searches are shown in Table 2.\nTable 2\nFormulae for calculating the sensitivity, specificity, precision, and accuracy of searches for detecting age-specific articles*\nHand Search\nDetection of Search Terms\nMeets Criteria\nDoes Not Meet Criteria\nDetected\na\nb\nNot detected\nc\nd\na+c\nb+d\n*Sensitivity = a\/(a + c); precision = a\/(a + b); specificity = d\/(b + d); accuracy = (a + d)\/(a +b + c + d). All articles classified during the manual review of the literature, n = (a + b + c + d).\nIndividual search terms with sensitivity > 25% and specificity > 75% for a given age category were incorporated into the development of search strategies that included two or more terms. All combinations of terms used the Boolean \"OR.\" For the development of multiple-term search strategies to either optimize sensitivity or specificity, we tested all two-term search strategies with sensitivity of at least 75% and specificity at least 50%.\nTo construct a comprehensive set of search terms, a list of MeSH terms and textwords was initially generated, and input was sought from clinicians and librarians in the United States and Canada through interviews with known searchers, requests at meetings and conferences, and requests to the National Library of Medicine. These experts were asked which terms or phrases they used when searching for age-specific studies, as well when searching for studies in specific purpose categories. Search terms could be MeSH terms, including publication types and subheadings, or textwords specific to age in titles and abstracts of articles. Various truncations were also applied to the textwords, phrases, and MeSH terms. We compiled a list of 543 age-specific terms (Multimedia Appendix). All terms were tested in Medline using the Ovid Technologies searching system.\nAge categories for the hand search were modeled from the MeSH terms used to index age content. A comparison of hand search categories and MeSH term definitions is shown in Table 1. The major difference between the hand search age categories and the MeSH terms is in how they were applied. During the hand search, we classified the age of study participants in primary studies or review articles in the following way: select one age category, if possible, or up to three to represent where \u2265 50% of participants fell. This procedure is intended to more accurately represent the focus of age-category research of clinical relevance than the comprehensive indexing of all participants' ages provided by the Medline index terms (which may be more pertinent for nonclinical purposes).\nWe defined five age-specific specialty areas by collapsing our hand search age categories (see Table 1) and through discussions with clinicians from each specialty area about which definition most appropriately reflected the age of their patients in clinical practice: geriatric medicine (\u2265 65 years of age), adult medicine (19 to < 65 years of age), pediatric medicine (> 1 month to < 19 years of age), neonatal medicine (birth to 1 month), and obstetrics (fetus).\nResults\nTables 3 to 7 show the operating characteristics of top-performing combinations of terms with best sensitivity, best specificity, and best optimization of sensitivity and specificity while minimizing the difference between the two, for detecting studies on geriatric medicine, adult medicine, pediatric medicine, neonatal medicine, and obstetrics in Medline in 2000. Search strategies are reported using Ovid's search engine syntax for Medline (mp = multiple posting\u2014term appears in title, abstract, or subject heading; sh = subject heading [MeSH term]; tw = textword\u2014word or phrase appears in title or abstract; : = truncation; pt = publication type; exp = explode\u2014a search term that automatically includes closely related MeSH terms; tu = therapeutic use as a subheading; xs = exploded subheading).\nGeriatric Medicine\nThe single term \"exp adult\" yielded the best sensitivity (96.4%) with a specificity of 55.9% for retrieving articles about geriatric medicine. However, by using the next best sensitivity combination, \"aged.sh. OR age:.tw.\", a small sacrifice in sensitivity (1% absolute decrease) resulted in a much better specificity compared with the most sensitive term (absolute increase 14.4%) and improved precision (absolute increase 5.2%) and accuracy (absolute increase 13.3%). As expected, precision improved slightly when specificity was maximized (absolute increase 8.6%). The term that yielded the best optimization of sensitivity and specificity, \"aged.sh.\", resulted in 93.6% sensitivity and 82.7% specificity.\nTable 3\nCombination of terms with the best sensitivity, best specificity, and best optimization of sensitivity and specificity for detecting studies about geriatric medicine (\u2265 65 years) in Medline in 2000\nSearch Strategy*\nOperating Characteristics\u2020\nSensitivity, %\n(95% CI)\n(n = 3309)\nSpecificity, %\n(95% CI)\n(n = 45719)\nPrecision, %\u2021\n(95% CI)\nAccuracy, %\n(95% CI)\n(n = 49028)\nBest sensitivity(exp adult)\n96.4(95.8-97.1)\n55.9(55.5-56.4)\n13.7(13.2-14.1)\n58.7(58.2-59.1)\nNext best sensitivity(aged.sh. OR age:.tw.)\n95.4(94.7-96.1)\n70.3(69.8-70.7)\n18.9(18.2-19.4)\n72.0(71.6-72.3)\nBest specificity(aged, 80 and over.sh. OR of age.tw.)\n63.3(61.7-65.0)\n84.0(83.7-84.4)\n22.3(21.5-23.1)\n82.6(82.3-83.0)\nNext best specificity(aged.sh.)\n93.6(92.8-94.5)\n82.7(82.4-83.1)\n28.2(27.3-29.0)\n83.5(83.1-83.8)\nBest optimization of sensitivity and specificity(aged.sh.)\n93.6(92.8-94.5)\n82.7(82.4-83.1)\n28.2(27.3-29.0)\n83.5(83.1-83.8)\n*Search strategies are reported using Ovid's search engine syntax for Medline (if a single search term is shown, this term outperformed two- and three-term combinations). Best sensitivity while keeping specificity \u2265 50%; Best specificity while keeping sensitivity \u2265 50%; Best Optimization of Sensitivity and Specificity is based on lowest possible absolute difference between sensitivity and specificity; exp = explode, a search term that automatically includes closely related indexing terms; sh = subject heading; : = truncation; tw = textword (word or phrase appears in title or abstract).\n\u2020Total database has 49028 articles, of which 3309 articles are relevant to geriatric medicine and 45719 are irrelevant to geriatric medicine.\n\u2021n varies by row.\nAdult Medicine\nThe three-term strategy \"adult.mp. OR middle aged.sh. OR age:.tw.\" yielded the best sensitivity (94.9%) and had a specificity of 64.5% for retrieving articles about adult medicine. When specificity was maximized (85.2%) with the single term \"middle aged.sh.\", sensitivity lowered to 72.3%, but precision improved to 62.1% (absolute increase 14.8%) and accuracy improved as well (absolute increase 9.8%). The best optimization of sensitivity and specificity occurred with the combined terms \"middle aged.sh. OR of age.tw.\", with values approaching 79%.\nTable 4\nCombination of terms with the best sensitivity, best specificity, and best optimization of sensitivity and specificity for detecting studies about adult medicine (19 to < 65 years) in Medline in 2000\nSearch Strategy*\nOperating Characteristics\n\u2020\nSensitivity, %\n(95% CI)\n(n = 12307)\nSpecificity, %\n(95% CI)\n(n = 39721)\nPrecision, %\u2021\n(95% CI)\nAccuracy, %\n(95% CI)\n(n = 49028)\nBest sensitivity(adult.mp. OR middle aged.sh. OR age:.tw.)\n94.9(94.5-95.3)\n64.5(64.4-64.9)\n47.3(46.6-47.8)\n72.1(71.7-72.5)\nBest specificity(middle aged.sh.)Next best specificity(adult.sh.)\n72.3(71.5-73.1)75.3(74.6-76.1)\n85.2(84.8-85.5)81.4(81.0-81.8)\n62.1(61.3-62.8)57.6(56.8-58.4)\n81.9(81.6-82.3)80.0(79.5-80.3)\nBest optimization of sensitivity and specificity(middle aged.sh. OR of age.tw.)\n78.7(78.0-79.4)\n77.9(77.4-78.3)\n54.4(53.7-55.1)\n78.1(77.7-78.5)\n*Search strategies are reported using Ovid's search engine syntax for Medline (if a single search term is shown, this term outperformed two- and three-term combinations). Best sensitivity while keeping specificity \u2265 50%; Best specificity while keeping sensitivity \u2265 50%; Best Optimization of Sensitivity and Specificity is based on lowest possible absolute difference between sensitivity and specificity; mp = multiple posting\u2014term appears in title, abstract, or subject heading; sh = subject heading; : = truncation; tw = textword (word or phrase appears in title or abstract).\n\u2020Total database has 49028 articles, of which 12307 articles are relevant to adult medicine and 39721 are irrelevant to adult medicine.\n\u2021n varies by row.\nPediatric Medicine\nThe three-term strategy \"child:.mp. OR adolescent.mp. OR infan:.mp.\" yielded the best sensitivity of 98.0% with a specificity of 81.2% for retrieving articles about pediatric medicine. When specificity was maximized (97.1%) with the single term \"children.tw.\", a striking trade-off in sensitivity occurred as it was lowered to 58.2% (absolute decrease 39.8%). Yet, as expected, precision improved (absolute increase 30.9%). The three-term strategy \"adolescent.tw. OR children.tw. OR child, preschool.sh.\" yielded the best optimization of sensitivity and specificity (89.3% and 87.3%, respectively).\nTable 5\nCombination of terms with the best sensitivity, best specificity, and best optimization of sensitivity and specificity for detecting studies about pediatric medicine (> 1 month to < 19 years) in Medline in 2000\nSearch Strategy*\nOperating Characteristics\u2020\nSensitivity, %\n(95% CI)\n(n = 2845)\nSpecificity, %\n(95% CI)\n(n = 46183)\nPrecision, %\u2021\n(95% CI)\nAccuracy, %\n(95% CI)\n(n = 49028)\nBest sensitivity(child:.mp. OR adolescent.mp. OR infan:.mp.)\n98.0(97.4-98.5)\n81.2(81.1-81.4)\n24.6(23.8-25.4)\n82.4(82.1-82.8)\nBest specificity(children.tw.)\n58.2(56.4-60.0)\n97.1(97.0-97.3)\n55.5(53.7-57.2)\n94.9(94.7-95.1)\nBest optimization of sensitivity and specificity(adolescent.tw. OR children.tw. OR child, preschool.sh.)\n89.3(88.1-90.4)\n87.3(87.0-87.6)\n30.3(29.3-31.3)\n87.4(87.1-87.7)\n*Search strategies are reported using Ovid's search engine syntax for Medline (if a single search term is shown, this term outperformed two- and three-term combinations). Best sensitivity while keeping specificity \u2265 50%; Best specificity while keeping sensitivity \u2265 50%; Best Optimization of Sensitivity and Specificity is based on lowest possible absolute difference between sensitivity and specificity; mp = multiple posting\u2014term appears in title, abstract, or subject heading; : = truncation; tw = textword (word or phrase appears in title or abstract); sh = subject heading.\n\u2020Total database has 49028 articles, of which 2845 articles are relevant to pediatric medicine and 46183 are irrelevant to pediatric medicine.\n\u2021n varies by row.\nNeonatal Medicine\nBest sensitivity (95.3%) was achieved by the three-term strategy \"infan:.mp. OR child:.mp. OR gestation:.tw.\", with a specificity of 83.6% for retrieving articles about neonatal medicine. An expected trade-off occurred in sensitivity (absolute decrease 41.7%) with the most specific term, \"infants.tw.\" (98.7%). However, precision increased to 38.2% (absolute increase 30.8%) and accuracy reached 98.2%. The three-term strategy \"infan:.mp. OR gestation:.tw. OR neonatal.tw.\" yielded the best optimization of sensitivity and specificity, reaching values of 93% (which were the highest among all five specialties).\nTable 6\nCombination of terms with the best sensitivity, best specificity, and best optimization of sensitivity and specificity for detecting studies about neonatal medicine (birth to 1 month) in Medline in 2000\nSearch Strategy*\nOperating Characteristics\u2020\nSensitivity, %\n(95% CI)\n(n = 663)\nSpecificity, %\n(95% CI)\n(n = 48365)\nPrecision, %\u2021\n(95% CI)\nAccuracy, %\n(95% CI)\n(n = 49028)\nBest sensitivity(infan:.mp. OR child:.mp. OR gestation:.tw.)\n95.3(93.7-96.9)\n83.6(83.3-83.9)\n7.4(6.8-7.9)\n83.8(83.4-84.1)\nBest specificity(infants.tw.)Next best specificity(infants.tw. OR neonatal.tw.)\n53.6(52.6-60.2)67.7(64.0-71.1)\n98.7(98.6-98.8)98.2(98.0-98.3)\n38.2(34.9-41.0)33.7(31.0-36.0)\n98.2(98.0-98.3)97.8(97.6-97.9)\nBest optimization of sensitivity and specificity(infan:.mp. OR gestation:.tw. OR neonatal.tw.)\n92.5(90.5-94.5)\n92.6(92.4-92.8)\n14.7(13.6-15.7)\n92.6(92.4-92.8)\n*Search strategies are reported using Ovid's search engine syntax for Medline (if a single search term is shown, this term outperformed two- and three-term combinations). Best sensitivity while keeping specificity \u2265 50%; Best specificity while keeping sensitivity \u2265 50%; Best Optimization of Sensitivity and Specificity is based on lowest possible absolute difference between sensitivity and specificity; mp = multiple posting\u2014term appears in title, abstract, or subject heading; : = truncation; tw = textword (word or phrase appears in title or abstract).\n\u2020Total database has 49028 articles, of which 663 articles are relevant to neonatal medicine and 48365 are irrelevant to neonatal medicine.\n\u2021n varies by row.\nObstetrics\nThe combination of terms \"gestation:.tw. OR fetal.tw. OR pregnancy.tw.\" produced the best sensitivity of 82.0%, with a very high specificity of 97.1% for retrieving articles about obstetrics. The maximization of specificity (reaching almost 99%) with the single term \"gestation:.tw.\" yielded a 1.8% increase in specificity but with a marked trade-off in sensitivity, which decreased to 52.0% (absolute decrease 30%).\nTable 7\nCombination of terms with the best sensitivity, best specificity, and best optimization of sensitivity and specificity for detecting studies about obstetrics (fetus) in Medline in 2000\nSearch Strategy*\nOperating Characteristics\u2020\nSensitivity, %\n(95% CI)\n(n = 516)\nSpecificity, %\n(95% CI)\n(n = 48512)\nPrecision, %\u2021\n(95% CI)\nAccuracy, %\n(95% CI)\n(n = 49028)\nBest sensitivity(gestation:.tw. OR fetal.tw. OR pregnancy.tw.)\n82.0(78.7-85.3)\n97.1(97.0-97.3)\n23.4(21.4-25.3)\n97.0(96.9-97.1)\nBest specificity(gestation:.tw.)\n52.0(47.6-56.3)\n98.9(98.8-99.0)\n33.6(30.2-36.7)\n98.4(98.3-98.5)\nBest optimization of sensitivity and specificity(pregnancy.tw. OR fetal.tw. OR age:.tw.)\n80.7(77.2-84.0)\n79.3(78.9-79.7)\n4.0(3.6-4.4)\n79.3(79.0-79.7)\n*Search strategies are reported using Ovid's search engine syntax for Medline (if a single search term is shown, this term outperformed two- and three-term combinations). Best sensitivity while keeping specificity \u2265 50%; Best specificity while keeping sensitivity \u2265 50%; Best Optimization of Sensitivity and Specificity is based on lowest possible absolute difference between sensitivity and specificity; : = truncation; tw = textword (word or phrase appears in title or abstract).\n\u2020Total database has 49028 articles, of which 516 articles are relevant to obstetrics and 48512 are irrelevant to obstetrics.\n\u2021n varies by row.\nDiscussion\nOur study shows that selected age-specific search strategies can achieve high retrieval of studies for age-specific populations. Our age-specific search strategies performed differently among the five specialties we investigated. The highest sensitivity and specificity were achieved for pediatric medicine (98% and 81.2%, respectively) and neonatal medicine (95.3% and 83.6%, respectively). This finding may be a result of these age groups being more precisely defined and that studies tend to be narrowly focused on them. Search strategies within obstetrics yielded a higher specificity (97.1%) than sensitivity (82%), indicating that this strategy was better at filtering out nonrelevant age-specific articles than retrieving them. The best performing strategy for optimizing sensitivity and specificity was achieved within neonatal medicine (92.5% and 92.6%, respectively). In all cases, precision was low, a consequence of searching in large multi-purpose databases. Future research is focusing on potential ways to improve precision without compromising sensitivity, for example, by searching in journal subsets.\nA possible limitation to our study is the generalizability of our findings to other publication years as our data was collected in the year 2000. We believe, however, that our search strategies are robust because no major changes have been made to age-specific MeSH terms since the year 2000. Moreover, we have previously shown that search strategies developed in 1990 were robust when searching in 2000 [18]. Another potential limitation of our study is that our interrater agreement for classifying age content did not reach 100%. However, exceeding the level of agreement achieved in our study (> 80% beyond chance) is rarely done in diagnostic studies. The scope of journals investigated in our journal subset could be a limitation, but we have no indication that these search strategies would perform differently in other journal subsets aside from the precision values reported. Precision is affected by the prevalence of on-target articles within the database. Thus, our precision figures are presented as estimates of search strategy performance.\nThe utility of age-specific filters will vary according to the needs of clinicians and researchers who must weigh the consequences of using a sensitive or specific search. Although a sensitive search will not miss many relevant articles, such searches are less precise and entail time-consuming sorting through irrelevant articles. The narrower yield of a specific search will capture many relevant articles and take less weeding, but it has greater potential for missing key articles.\nSearch Examples\nTo illustrate the use of age-specific search strategies, if a geriatrician was looking for information about current treatment strategies for Huntington disease, she might begin her search by entering the content term \"Huntington disease\" in Medline, which would yield 5907 articles (Table 8).\nTable 8\nExample: best sensitivity (keeping specificity \u2265 50%) search strategies for detecting treatment studies in geriatric medicine (patients \u2265 65 years of age) in Medline (1996 to July week 3, 2005)\nSearch Strategy*\nContent Term\nBoolean Operator\nBest Sensitivity Combination Strategy for Treatment Studies\nBoolean Operator\nBest Sensitivity Combination Strategy for Geriatric Medicine\nNumber of Articles\nHuntington disease\n-\n-\n-\n-\n5907\nHuntington disease\nAND\nclinical trial.mp. OR clinical trial.pt. OR random:.mp. OR tu.xs.\n-\n-\n901\nHuntington disease\nAND\nclinical trial.mp. OR clinical trial.pt. OR random:.mp. OR tu.xs.\nAND\nexp adult\u2020\n483\n*Search strategies are reported using Ovid's search engine syntax for Medline. mp = multiple posting\u2014term appears in title, abstract, or subject heading; : = truncation; pt = publication type; tu = therapeutic use as a subheading; xs = exploded subheading; exp = explode\u2014a search term that automatically includes closely related indexing terms.\n\u2020Outperformed two- and three-term combinations.\nHowever, sifting through such a large number of articles would be time-consuming and many of these articles would not be relevant to treatment studies in geriatric medicine. By combining the content term \"Huntington disease\" with the most sensitive combination of terms for treatment studies (clinical trial.mp. OR clinical trial.pt. OR random:.mp. OR tu.xs.), the search can be narrowed to 901 articles. Further, by adding the most sensitive strategy for geriatric medicine (exp adult) to this search string with the Boolean operator AND, the search is refined to 483 articles, which is much more manageable than the original 5907 articles retrieved from searching the content term only. A sensitive search such as this would be an efficient beginning for researchers interested in conducting systematic reviews.\nA more specific approach may be especially useful for physicians who do not have time to process an exhaustive search. In the above example, by combining the content word \"Huntington disease\" with the most specific search strategy for treatment studies [12], \"randomized controlled trial.mp. OR randomized controlled trial.pt.\", and the most specific search strategy for geriatric medicine, \"aged, 80 and over.sh. OR age.tw.\", the search yields five articles (Table 9). This is a dramatic reduction in the number of articles retrieved by searching the content term alone (5907 articles), but key articles can be missed.\nTable 9\nExample: best specificity (keeping sensitivity \u2265 50%) search strategies for detecting treatment studies in geriatric medicine (patients \u2265 65 years of age) in Medline (1996 to July week 3, 2005)\nSearch Strategy*\nContent Term\nBoolean Operator\nBest Specificity Combination Strategy for Treatment Studies\nBoolean Operator\nBest Specificity Combination Strategy for Geriatric Medicine\nNumber of Articles\nHuntington disease\n-\n-\n-\n-\n5907\nHuntington disease\nAND\nRandomized controlled trial.mp. OR randomized controlled trial.pt.\n-\n-\n46\nHuntington disease\nAND\nRandomized controlled trial.mp. OR randomized controlled trial.pt.\nAND\naged, 80 and over.sh. OR of age.tw.\n5\n*Search strategies are reported using Ovid's search engine syntax for Medline. mp = multiple posting\u2014term appears in title, abstract, or subject heading; pt = publication type; sh = subject heading; tw = textword (word or phrase appears in title or abstract).\nConclusion\nSelected age-specific search strategies can enhance the retrieval of studies for clinicians and researchers who need information relevant for a well-defined age-category patient population. The optimal trade-off between sensitivity and specificity should be determined according to the needs of the searcher.","keyphrases":["medline","medical subject headings","information storage and retrieval"],"prmu":["P","P","M"]}
{"id":"J_Neurooncol-4-1-2174521","title":"Medulloblastomas overexpress the p53-inactivating oncogene WIP1\/PPM1D\n","text":"Medulloblastoma is the most common malignant brain tumor of childhood. Despite numerous advances, clinical challenges range from recurrent and progressive disease to long-term toxicities in survivors. The lack of more effective, less toxic therapies results from our limited understanding of medulloblastoma growth. Although TP53 is the most commonly altered gene in cancers, it is rarely mutated in medulloblastoma. Accumulating evidence, however, indicates that TP53 pathways are disrupted in medulloblastoma. Wild-typep53-induced phosphatase 1 (WIP1 or PPM1D) encodes a negative regulator of p53. WIP1 amplification (17q22-q23) and its overexpression have been reported in diverse cancer types. We examined primary medulloblastoma specimens and cell lines, and detected WIP1 copy gain and amplification prevalent among but not exclusively in the tumors with 17q gain and isochromosome 17q (i17q), which are among the most common cytogenetic lesions in medulloblastoma. WIP1 RNA levels were significantly higher in the tumors with 17q gain or i17q. Immunoblots confirmed significant WIP1 protein in primary tumors, generally higher in those with 17q gain or i17q. Under basal growth conditions and in response to the chemotherapeutic agent, etoposide, WIP1 antagonized p53-mediated apoptosis in medulloblastoma cell lines. These results indicate that medulloblastoma express significant levels of WIP1 that modulate genotoxic responsiveness by negatively regulating p53.\nIntroduction\nMedulloblastoma is the most common malignant brain tumor of childhood [1, 2]. Treatment with surgery, radiation, and chemotherapy successfully cures many patients, but survivors can suffer significant long-term toxicities affecting their neurocognitive and growth potential [3]. Despite clinical advances, up to 30% of children with medulloblastoma experience tumor progression or recurrence, for which no curative therapy exists. The lack of more effective, less toxic therapies stems from our imperfect understanding of the molecular processes that underlie medulloblastoma growth.\nAmong the most common cytogenetic lesions affecting medulloblastoma are the gain of the long arm of chromosome 17 (17q) and isochromosome 17q (i17q), consisting of 17p deletion with duplication of 17q, in approximately one-third of cases [4, 5]. Because i17q has been described as the sole cytogenetic lesion in certain medulloblastomas, it may represent a primary event rather than an alteration associated with clonal evolution [4]. Investigators have long sought oncogenes on 17q and tumor suppressor genes on 17p, but with limited success [6].\nAlthough the TP53 tumor suppressor gene (17p13.1) is mutated in approximately half of human malignancies, it is rarely mutated in medulloblastoma [7\u20139]. However, several lines of evidence suggest that the p53 pathway is perturbed in medulloblastoma. Frank et\u00a0al. have described activation of the p53-p14ARF pathway in the large cell\/anaplastic variant of medulloblastoma [10, 11]. Our collaborators have noted significant nuclear p53 immunopositivity consistent with its activation in approximately 50% of primary human medulloblastoma examined (Adekunle Adesina, personal communication). Deletion of the murine homolog, Trp53, in the Patched haploinsufficient (Ptch+\/\u2212) mouse model increases the incidence of spontaneous medulloblastoma from approximately 15 to 100% [12]. Individuals with Li\u2013Fraumeni syndrome who carry germline TP53 mutations are at increased risk for developing medulloblastoma, but fewer than 10% of sporadic medulloblastoma display TP53 mutations [4, 5].\nThe activity of p53 may be abrogated by alternate mechanisms as observed in other cancers with wild-typeTP53 [7]. One mechanism by which p53 function is limited is through amplification or overexpression of MDM2. MDM2 encodes an E3 ubiquitin ligase that normally functions in a negative feedback loop to down-regulate p53 expression, and is amplified in approximately 7% of human malignancies that lack mutation of TP53 [13]. However, Adesina et\u00a0al. and Batra et\u00a0al. have documented the absence of MDM2 amplification in medullobastoma [14, 15]. Although MDM2 overexpression has been detected in adult patients [16], there is no functional data to implicate MDM2 in p53 inactivation in medulloblastoma, suggesting alternative mechanisms.\nThe p53-induced proto-oncogene, WIP1 (wild-typep53-induced phosphatase 1 or protein phosphatase, magnesium-dependent 1, delta, PPM1D) maps to 17q22-q23. Amplification of 17q22-q23 has been described in several cell lines and malignancies including neuroblastoma, breast and ovarian carcinomas, most of which are wild-type for TP53 [17\u201319]. WIP1 displays p53-dependent oncogenic properties by inactivating p53, p38MAPK, and ATM pathways [17\u201321]. Previous reports of genomic analysis of medulloblastoma have implicated the gain of the WIP1 locus in its overexpression in a subset of tumors [22, 23].\nWe present data that confirm prior observations and also define the effects of WIP1 on medulloblastoma cell growth. We have quantitated WIP1 overexpression in primary medulloblastoma specimens and cell lines previously analyzed by comparative genomic hybridization (CGH) [24]. Using fluorescence in\u00a0situ hybridization (FISH), we determined that WIP1 copy gain and amplification is prevalent among but not exclusively found in the tumors with 17q gain and i17q. We surveyed 33 primary medulloblastoma specimens for WIP1 expression and determined that RNA levels were significantly higher in 16 tumors with 17q gain or i17q. Similarly, our Western immunoblot analysis confirmed significant levels of WIP1 protein expression in primary tumors, generally higher in those with 17q gain or i17q. Genotoxic stress induces p53 phosphorylation and WIP1 expression in D283Med and Daoy medulloblastoma cell lines. Transient transfections indicate that WIP1 antagonizes p53-mediated cell cycle arrest and apoptosis in response to the chemotherapeutic agent, etoposide. These results support the hypothesis that medulloblastoma express significant levels of WIP1 that modulate genotoxic responsiveness by negatively regulating p53.\nMaterials and methods\nMedulloblastoma tissue and cell lines\nMedulloblastoma samples (n\u00a0=\u00a033) were obtained from children diagnosed at Texas Children\u2019s Hospital, Houston, TX, between 1996 and 2004, following informed consents. Tissues were snap-frozen and stored in liquid nitrogen until processed. All specimens were obtained at the time of diagnosis prior to radiation or chemotherapy and subjected to histopathologic review according to WHO criteria [1]. Seven tumors displayed classic histology, 11 were uniformly desmoplastic, nodular, or well-differentiated, seven were predominantly anaplastic or large cell, an additional seven displayed some degree of anaplasia or large cell histology, and one was regarded as none of the above.\nTen patients were female and 23 were male. The mean age at diagnosis was 82\u00a0months (median 79\u00a0months) with four patients less than 36\u00a0months old (range: 12\u2013216\u00a0months). The median and mean time of follow-up was approximately 42\u00a0months from diagnosis. The majority of patients were M0 (non-metastatic), while one was M2 and eight were M3 stage at diagnosis. All studies were performed according to the institutional guidelines of Texas Children\u2019s Hospital, and with the approval of the Institutional Review Board of Baylor College of Medicine.\nThe breast carcinoma cell line MCF-7 with known high-level WIP1 amplification, the un-amplified breast epithelial cell line MCF-10A, and the medulloblastoma cell lines, Daoy and D283Med (American Type Culture Collection, Manassas, VA) were maintained as previously described in Dulbecco\u2019s modified Eagle\u2019s medium (Invitrogen, Carlsbad, CA) with high glucose (6\u00a0g\/l), 2\u00a0mM l-glutamine and 10% (vol\/vol) heat-inactivated fetal calf serum (Invitrogen, Carlsbad, CA) at 37\u00b0C in 5% CO2. Etoposide (VP-16) and cisplatin (CDDP) were applied to cell cultures as media-diluted stock solutions in PBS and DMSO, respectively (Sigma, St. Louis, MO). Ultraviolet (UV) irradiation was performed by exposing cell cultures using a GS Gene Linker UV Chamber (Bio-Rad Laboratories, Hercules, CA).\nFluorescence in\u00a0situ hybridization (FISH) analysis\nPrimary medulloblastoma cells were harvested from first passage cultures. Chromosomes were prepared according to standard methods, including confirmatory karyotyping with G-banding [25]. Eleven first or second passage cultures of primary tumors were harvested when cultures were still sub-confluent. The BAC containing the WIP1 locus at chromosome 17q23 (RP11-67D12) and a plasmid containing chromosome 17 centromeric sequences (pZ17-14, generously provided by Dr. Mariano Rocchi) were fluorescently labeled with Spectrum Green and Spectrum Orange, respectively (Vysis, Downers Grove, IL), by nick translation and hybridized to metaphase or interphase spreads of cell lines (MCF-7, Daoy, D283) and primary tumor specimens [26]. We confirmed the map position of WIP1 on normal human metaphase spreads by FISH, as described previously [27]. The slides were counterstained with DAPI, and the images were captured using the Quips Pathvysion System (Applied Imaging, Santa Clara, CA). To determine the WIP1 copy number status, we analyzed 100\u2013200 individual interphase nuclei for each case. The presence of copy gain or amplification was indicated if more than 10% of tumor nuclei displayed an increased copy number relative to the chromosome 17 centromeric probe signals and\/or tumor ploidy. In cases in which there was a discrepancy between the number of centromeric signals and ploidy, the ploidy number was used to estimate the relative copy number.\nQuantitative real-time RT-PCR (qRT-RTPCR) analysis\nTotal cellular RNA was prepared from frozen tumor tissue with either Trizol (Invitrogen) or an RNeasy kit (Qiagen, Valencia, CA) depending upon relative abundance, according to the manufacturers\u2019 recommendations. Integrity and concentration of RNA were verified on the Baylor College of Medicine, Houston, TX Microarray Core Facility Bioanalyzer (Agilent Technologies, Santa Clara, CA). Total cellular RNA was reverse transcribed with SuperScript First-Strand Synthesis (Invitrogen) primed with oligo-(dT)12. PCR reactions were performed in a Bio-Rad iCycler using iQ Syber Green Supermix (Bio-Rad Laboratories), and primers listed below. Each PCR reaction was performed for 40 cycles, and performed in triplicate. Primer sequences included: WIP1 sense, 5\u2032-ATCCGCAAAGGCTTTCTCGCTT-3\u2032 and antisense, 5\u2032-TTGGCCATTCCGCCAGTTTCTT-3\u2032; TP53 sense, 5\u2032-CCATCTACAAGCAGTCACAGC-3\u2032 and antisense, 5\u2032-GAGTCTTCCAGTGTGAGATG-3\u2032; GAPDH sense, 5\u2032-AAGGTGAAGGTCGGAGTCAA-3\u2032 and antisense, 5\u2032-AATGAAGGGGTCATTGATGG-3\u2032. Expression of target RNA was internally normalized to expression of GAPDH gene and expressed relative to target gene expression in a human fetal brain RNA pool (Stratagene, La Jolla, CA). Human adult cerebellar (Stratagene) and human fetal cerebellar RNA samples (BioChain, Hayward, CA) were also assayed to provide tissue controls. Amplification products were verified by analyzing melting curves, agarose gel electrophoresis, and with direct sequencing of PCR products.\nWestern immunoblot analysis\nProtein was extracted from frozen tissue by solubilization in boiling lysis buffer (0.5% SDS, 50\u00a0mM Tris\u2013Cl, pH 8, 5\u00a0mM Na2EDTA, 2% \u03b2-mercaptoethanol). Lysates were stored at \u221280\u00b0C and separated by SDS-PAGE, transferred to nitrocellulose membranes (Pall Life Sciences, Ann Arbor, MI), and immunoblotted using standard methods. Primary antibodies included: WIP1 monoclonal antibody (Trevigen, Gaithersburg, MD), Phospho-p53 serine-15 (Ser15)-specific monoclonal antibody (Cell Signaling Technology, Danvers, MA), p53 monoclonal antibody (DO-1, Santa Cruz Biotechnology, Santa Cruz, CA), and \u03b2-Actin monoclonal antibody (Sigma). Staining with secondary antibody (Alexa Fluor 680-nm-conjugated goat anti-mouse IgG, Invitrogen) was visualized using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, Nebraska). Intensity of immunostaining was quantitated using ImageQuant 5.2 (Molecular Dynamics, Piscataway, NJ). Expression of target proteins in each sample was internally normalized to \u03b2-actin (determined on re-probed blots), relative to expression in the MCF-10A cell line.\nTransfections and luciferase reporter assays\nCell lines were transiently transfected with expression and reporter plasmids using Lipofectin according to manufacturer\u2019s recommendations (Invitrogen). Expression plasmids included: the human cytomegalovirus (CMV) immediate-early (IE) promoter-driven pP53-EGFP plasmid, encoding a p53-enhanced green fluorescent protein (GFP) fusion protein (Clontech, Mountainview, CA). Reporter plasmids included the pP21-luciferase construct to detect p53-activated transcription [28] and the actin promoter-driven Renilla luciferase plasmid, pB-Actin-RL, to provide transfection controls [29]. In each transfection, the expression and reporter plasmids were supplemented by empty vector plasmid (pcDNA3.1, Invitrogen) to achieve an equivalent total DNA concentration.\nFor flow cytometry, the EGFP-expressing pMaxGFP plasmid (Amaxa, Gaithersburg, MD) provided transfection controls. For functional assessment of in\u00a0vitro p53 activation, cells transfected with luciferase-encoding reporter plasmids were harvested and processed for dual color luciferase assay according to manufacturer\u2019s recommendations (Promega, Madison, WI), and quantitated using a BenchMark Plus plate spectrophotometer (Bio-Rad).\nFlow cytometric and terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick end-labeling (TUNEL) analysis\nWe monitored DNA-indices for cell cycle analysis by multi-parametric flow cytometry using standard methods. Analyses were performed using a Becton Dickinson FACScan flow cytometer (BD Biosciences, San Jose, CA) for the detection of cells stained with propidium iodide (PI) and a 488\u00a0nm laser with filter combination for fluorescein isothiocyanate (FITC) and GFP. Single cell suspensions were isolated from culture, fixed in methanol, and stained with PI (100\u00a0\u03bcg\/ml in PBS). Each histogram represents 10,000\u2013100,000\u00a0cells for measuring DNA-index and cell cycle. Histogram analysis was performed with the CellQuest program (BD Biosciences). We calculated the sub-G0\/G1 peak at a position in the hypodiploid area below a DNA-index of one (2n). Because the nucleus becomes fragmented during apoptosis and numerous individual chromatin fragments may be present in a single cell, the percentage of objects with a fractional DNA-content is represented by the sub-G0\/G1 peak. Apoptotic nuclei were identified as a sub-diploid DNA peak, and were distinguished from cell debris on the basis of forward light scatter and PI fluorescence. The significance of differences in apoptotic populations was determined using paired Student t-test.\nTo detect apoptotic nuclei in sub-G0\/G1 peaks, a subset of unstained cells was analyzed for DNA-fragmentation using the Frag-EL DNA fragmentation detection kit, according to manufacturer\u2019s recommendations (Oncogene Research Products, Cambridge, MA). Cells were also counter-stained with PI for DNA quantification. FITC signal was detected on one channel in the logarithmic mode, while UV fluorescence (PI) was recorded in the linear mode on a separate channel. For each measurement, at least 10,000\u00a0cells were analyzed. We used CellQuest software for multi-parametric calculations and analyses. Cutoff negative and positive cells resulted from FITC-fluorescence isotype control measurements for each sample.\nResults\nWIP1 DNA copy number is increased in medulloblastomas with 17q gain\nWe employed FISH to analyze copy numbers of the WIP1 gene in primary medulloblastoma specimens and cell lines previously analyzed by CGH (Fig.\u00a01). Compared to the centromeric chromosome 17 probe, which consistently displayed 2\u20134 signals in each metaphase or interphase nucleus, the 17q23 BAC probe (RP11-67D12) containing the WIP1 locus revealed a wider range of copy numbers. By FISH, we identified high-level amplification of the WIP1 gene (>50\u00a0copies\/cell) in all of the MCF-7 cells analyzed (Fig.\u00a01A). This finding is consistent with previously identified amplification of the 17q23 chromosomal region in MCF-7 cells. The D283Med (D283) medulloblastoma cell line, with i17q, displayed low-level amplification of the WIP1 locus with four to nine copies in each interphase spread examined (Fig.\u00a01B). In contrast, the Daoy medulloblastoma cell line with trisomy 17 displayed an average of three copies of WIP1 (Fig.\u00a01C).\nFig.\u00a01WIP1 copy number is increased in medulloblastomas with gain of chromosome 17q. High power micrographs of interphase nuclei of cell lines and primary human medulloblastoma cells hybridized to fluorescently-labeled WIP1 BAC (green) and centromeric chromosome 17 probes (red): (A) MCF-7 nucleus showing numerous WIP1 amplicons; (B) D283Med (D283) nucleus exhibiting low-level WIP1 amplification; (C) Daoy nucleus showing gain of 17; (D\u2013L) interphase nuclei of medulloblastoma cells from nine patients, illustrating examples of (D\u2013H) diploid and near-diploid nuclei, and (I\u2013L) those with low-level amplification of WIP1\nAmong the 71 tumors analyzed by CGH, 11 primary medulloblastomas were examined for WIP1 amplification by FISH [24]. Six tumors displayed i17q, two showed trisomy 17, one had loss of 17p12-p13 with gain of 17p11.2-q25, and two had no change in chromosome 17 (Table\u00a01). We detected low-level amplification of the WIP1 locus in 7 of 11 primary medulloblastoma specimens, which also had gain of 17q, including four with i17q by G-banding.\nTable\u00a01WIP1 amplification is associated with gain of chromosome 17q or i17q in primary medulloblastoma specimensSpecimenCopy number (FISH)Amplification*CNA (CGH)CEP 17WIP1WIP1Chr 17 statusD2832\u201344\u20139Yi17qDaoy43N+17MCF-74>50Y**MB1326\u20137Y\u221217p12-p13, +17p11.2-q25MB12\u201345\u20138Yi17qMB32\u201343\u20137Yi17qMB142\u201342\u20137Y+17MB152\u201342\u20136Y+17MB526Yi17qMB23\u201346\u201311Yi17qMB624Ni17qMB2822NN\/CMB2722NN\/CMB42\u201332\u20133Ni17qFISH analysis for WIP1 and chromosome 17 centromere reveals low-level amplification of WIP1 in 7 of 11 primary medulloblastoma samples. WIP1 copy gain is significantly associated with gain of 17q (P\u00a0=\u00a00.00057)*Amplification is defined as >4 copies as determined by FISH**Reportedly amplified, but not determined in the present studyAbbreviations: MB, medulloblastoma; +17, gain of chromosome 17; \u221217, loss of chromosome 17; N\/C, no change in chromosome 17; CNA, copy number aberration; CEP 17, centromeric chromosome 17 probe\nFour tumors were diploid or near-diploid overall (Fig.\u00a01D\u2013G), and seven displayed copy number gain of the WIP1 locus (Fig.\u00a01H\u2013L). Our previously reported CGH analysis of 71 medulloblastoma revealed that 46.8% had gain of 17q, including 30% with i17q and 2.6% with amplifications involving 17q, consistent with previous reports [4]. Comparing CGH results with our FISH analysis reveals that WIP1 DNA copy number correlates with gain of 17q (including i17q) by CGH (P\u00a0<\u00a00.001, Student\u2019s t-test). All the tumors with copy number gain of WIP1 by FISH also had gain of the WIP1 locus on chromosome 17q22-q23 by CGH. WIP1 amplification in medulloblastoma was low-level compared to the high-level amplification observed in MCF-7 cells. These cytogenetic results indicate that low-level amplification of the WIP1 locus are prevalent in a significant proportion of medulloblastoma.\nMedulloblastomas with gain of 17q, including i17q, overexpress WIP1 RNA\nUsing qRT-RTPCR, we determined the expression of WIP1 RNA in cell lines and a larger series of primary medulloblastoma samples. Human fetal cerebellum specimens displayed 2.1-fold greater WIP1 expression (\u00b10.2, SEM) compared to fetal whole brain. Human adult cerebellum expresses 4.0-fold more WIP1 RNA (\u00b11.0) than fetal total brain. WIP1-amplified MCF-7 cells had 14-fold higher levels of WIP1 RNA (\u00b12.6), relative to human fetal brain (Fig.\u00a02A). Daoy cells expressed 0.23-fold WIP1 RNA (\u00b10.054, SEM), compared to fetal brain (Fig.\u00a02A). In contrast, i17q-positive D283 cells displayed 9.8-fold (\u00b11.8) higher expression of WIP1 RNA than human fetal brain.\nFig.\u00a02Medulloblastomas with gain of 17q express significantly higher WIP1 RNA levels. (A) WIP1 RNA expression, by qRT-RTPCR analysis, is significantly higher in the D283 cell line and in primary medulloblastoma specimens with gain of 17q or i17q (+17q\/i17q MB, n\u00a0=\u00a016) by conventional karyotyping and CGH, compared to Daoy, and primary tumors without i17q or gain of 17q (non-17q gain MB, n\u00a0=\u00a017). For reference, expression in the WIP1-amplified MCF-7 breast cancer cell line is also shown. Columns, mean expression of at least three experiments; error bars, SEM Y-axis, RNA expression normalized to GAPDH expression and relative to human fetal brain. (B) Relative WIP1 RNA expression in individual medulloblastoma specimens (n\u00a0=\u00a033) assayed by qRT-RTPCR, and listed with their associated chromosome 17 status. (C) Relative WIP1 RNA expression in medulloblastomas with WIP1 amplification (open diamond) is significantly higher than non-amplified tumors (open circle). Abbreviations: ALL MB, mean (\u00b1SEM) of all primary human tumors; i17q, isochromosome 17q; +17, gain of chromosome 17; \u221217, loss of chromosome 17; NC, no change in chromosome 17\nWe surveyed 33 primary medulloblastoma specimens for WIP1 expression and determined that RNA levels were significantly higher in 16 tumors with 17q gain or i17q (Fig.\u00a02, Table\u00a02). Of the primary medulloblastomas examined by FISH, WIP1-amplified tumors (n\u00a0=\u00a07) displayed significantly higher RNA levels than non-amplified tumors (n\u00a0=\u00a04) (27.7-fold (\u00b11.6) vs. 14.1-fold (\u00b14.5) more WIP1 RNA than fetal control; P\u00a0=\u00a00.050) (Fig.\u00a02C, Table\u00a02). Among the larger set of analyzed specimens, primary medulloblastoma samples displayed 13.0-fold higher levels of WIP1 RNA (\u00b12.1) than human fetal brain (Fig.\u00a02A\u2013B, Table\u00a02). Analyzed separately, tumors with gain of 17q, including i17q, (n\u00a0=\u00a016) expressed 20.3-fold more WIP1 RNA (\u00b12.1), compared to those without gain of 17q or i17q (n\u00a0=\u00a017) with only 5.9-fold more WIP1 RNA (\u00b11.0), relative to human fetal brain (Fig.\u00a02A\u2013B, Table\u00a02). In other words, those tumors with gain of 17q, including i17q, expressed 3.4-fold more WIP1 RNA (\u00b10.86) than those without such cytogenetic lesions. The association between WIP1 RNA expression and the presence or absence of 17q gain (including i17q) was statistically significant (P\u00a0<\u00a00.00001) (Table\u00a02).\nTable\u00a02WIP1 overexpression is associated with gain of chromosome 17q or i17q in primary medulloblastoma specimensTumors grouped by CNAnWIP1 RNAP value+17q MB328.30.02*i17q MB625.4<0.0001*+17q or i17q MB926.3<0.00001*Non-17q gain MB26.5\u2013WIP1 gain\/amplification727.70.05**Normal WIP1 copy number414.1\u2013Tumors analyzed by FISH (subtotal)11+17q MB520.20.0533*i17q MB1120.3<0.0001*+17q or i17q MB1620.3<0.00001*Non-17q gain MB175.9\u2013All tumors analyzed by CGH (total)3313.0\u2013WIP1 amplification by FISH was associated with increased WIP1 RNA expression by qRT-RTPCR (P\u00a0=\u00a00.05). Relative WIP1 RNA level was significantly associated with gain 17q, including i17q (P\u00a0=\u00a00.000001)*P value: Student t-test of tumors with specified CNA versus tumors without that CNA**P value: Student t-test of WIP1-ampllified versus non-amplified tumorsAbbreviations: MB, medulloblastoma; +17, gain of chromosome 17; \u221217, loss of chromosome 17; CNA, copy number aberration\nMedulloblastomas with gain of 17q overexpress WIP1 protein\nA subset of representative tumors with sufficient tissue was analyzed for WIP1 protein expression. Western immunoblot analysis confirmed significant protein expression in lysates from medulloblastoma cell lines and tumor samples, internally normalized to \u03b2-actin expression and relative to expression in the MCF-10A cell line (Fig.\u00a03A\u2013B). The medulloblastoma cell line, Daoy, with trisomy 17, expressed 3.1-fold more WIP1 protein (\u00b10.5, SEM) than MCF-10A cells (Fig.\u00a03A\u2013B). The D283 cell line, with i17q, displayed 15.4-fold higher WIP1 levels (\u00b12.0) than MCF-10A (Fig.\u00a03A\u2013B). Since UV irradiation activates p53 by phosphorylation at serine 15 and induces WIP1 expression [30], we used lysates prepared from irradiated D283 cells to provide control specimens with induced p53 and WIP1 proteins. In UV-irradiated D283 cells, p53 levels stabilized with increased phosphorylation at Ser15 (Fig.\u00a03A). The WIP1-amplified breast cancer line, MCF-7, expressed 4.5-fold more WIP1 (\u00b10.4) than MCF-10A cells (Fig.\u00a03A\u2013B).\nFig.\u00a03Medulloblastomas express significant WIP1 protein. (A) Western blots of cell lines and four primary medulloblastoma specimens reveal significant WIP1 and p53 protein expression. UV irradiation (30\u00a0J\/m2) of D283 increases WIP1 and p53 expression and induces p53 phosphorylation detected as phospho-p53 (Ser15). Breast carcinoma cell lines, WIP1-amplified MCF-7 and non-amplified MCF-10A are shown for reference. Associated chromosome 17 lesions are also labeled. (B) Western analysis of WIP1 protein expression among primary medulloblastoma specimens (n\u00a0=\u00a010), normalized to internal reference \u03b2-actin and relative to expression in the MCF-10A cell line. Abbreviations: all MB, mean (\u00b1SEM) of all primary human tumors (n\u00a0=\u00a010); i17q, isochromosome 17q; +17, gain of chromosome 17; \u221217, loss of chromosome 17; CNA, copy number aberration\nOf the ten primary medulloblastoma specimens analyzed, five displayed gain of 17q or i17q, one had trisomy 17, another had loss of 17p, and three showed no change in chromosome 17 by CGH. Overall, mean WIP1 protein expression was 6.1-fold (\u00b11.1) greater than expression in MCF-10A cells, internally normalized to \u03b2-actin (Fig.\u00a03B). As a group, primary tumors exhibited even more WIP1 than the MCF-7 cell line, which is widely employed as an example of WIP1-amplification. Analyzed separately, primary medulloblastomas with 17q or i17q (n\u00a0=\u00a06) displayed 7.1-fold (\u00b11.5) more WIP1 protein than MCF-10A, compared to non-17q gain tumors (n\u00a0=\u00a04) with only 4.6-fold (\u00b11.3) levels (Fig.\u00a03B). We also analyzed primary medulloblastoma lysates for expression of total p53 protein and p53 phosphorylation at serine 15 (Ser15) (Fig.\u00a03). Total p53 and phospho-p53 (Ser15) were readily detectable on Western blots, and suggest functional significance. Overall, Western analysis indicated significant levels of WIP1 protein expression in primary tumors, generally higher in those with 17q gain or i17q, which might be responsible for antagonizing p53 activation.\nGenotoxic stress induces p53 phosphorylation and WIP1 expression in medulloblastoma cell lines\nTo determine the functional significance of WIP1 overexpression, we transiently transfected established medulloblastoma cell lines and assayed changes in cell cycle and apoptosis in response to altered levels of WIP1. The D283 and Daoy cell lines differ in their basal WIP1 expression and their TP53 status, providing cell systems for gain-of-function transfection studies. Genotoxic stress in the form of exposure to UV irradiation or the chemotherapeutic agent VP-16 stabilized p53 levels and induced phosphorylation of p53 in D283 and Daoy cells, respectively (Fig.\u00a04A). We examined the effects of increased constitutive WIP1 by transient transfection with a flag-tagged expression plasmid. Flag-tagged WIP1 could be readily distinguished from endogenous WIP1 on Western blots and WIP1 transfection was accompanied by decreased phospho-p53 (Ser15) in treated D283 and Daoy cells that are wild-type and mutant for TP53, respectively (Fig.\u00a04A).\nFig.\u00a04WIP1 antagonizes p53 activation in medulloblastoma cell lines. (A) Western blots of D283 and Daoy medulloblastoma cell lines: Treatment with UV irradiation (UV, 30\u00a0J\/m2) or etoposide (VP-16, 1\u00a0\u03bcM) stabilizes wild-type p53 protein and induces phosphorylation of p53 at Ser15. Transfection with the WIP1-flag expression plasmid detected as higher molecular weight band using anti-WIP1 or anti-flag antibodies. Overexpressed WIP1 reduces p53 phosphorylation at serine 15 (Ser15). (B) Increased TP53 and WIP1 RNA in D283 and Daoy cell lines transfected with TP53-GFP and WIP1-flag (WIP1) expression plasmids compared to the MaxGFP control plasmid, as measured by qRT-RTPCR. (C) Luciferase assays reveal that co-transfection of WIP1-flag (WIP1) partially abrogates p53-dependent transcription from the p21-luciferase reporter (relative to constitutively expressed control Renilla-luciferase reporter) in TP53-GFP transfected D283 and Daoy cells (error bars, SEM). (D) TUNEL assay and flow cytometric methods confirm VP-16-induced apoptosis in Daoy and D283 cell lines\nDetermination of RNA levels with qRT-RTPCR confirmed significant expression of TP53 and WIP1 by transfection with WIP1-flag and TP53-GFP expression plasmids (Fig.\u00a04B). To confirm the transcriptional effects of WIP1- and TP53-expression plasmids, we co-transfected cell lines with the p53-responsive p21-luciferase reporter plasmid and an actin promoter-driven Renilla luciferase plasmid to control for transfection efficiency. Transfected WIP1 partially abrogated p53-dependent luminescence indicating anti-p53 activity in both cell lines in\u00a0vitro. Presumably because of endogenous wild-type TP53, the relative response in D283 cells was less dramatic than in Daoy cells (Fig.\u00a04C).\nVP-16 and constitutively expressed p53 induce apoptosis\nExpression from epitope-tagged plasmids enabled us to employ flow cytometric methods to analyze the cell cycle distribution of fluorescently immunolabeled WIP1- and p53-overexpressing transfectants. To stimulate genotoxic responses in\u00a0vitro, we employed minimally toxic dose concentrations of etoposide (VP-16), a chemotherapeutic topoisomerase II-inhibitor used to treat medulloblastoma clinically. VP-16-induced changes in apoptosis were also determined by flow cytometric quantitation of cells in sub-G0\/G1 fractions, which were confirmed by TUNEL analysis (Fig.\u00a04D). Similar results were obtained with micromolar concentrations of another chemotherapeutic agent, cisplatin (data not shown).\nWhen exposed to low concentrations of VP-16, the D283 medulloblastoma cell line underwent increased apoptosis indicated by an increased fraction of cells in sub-G0\/G1 by flow cytometric analysis, from 4.5 to 17.2% (P\u00a0=\u00a00.009) (Fig.\u00a05F). These results indicate that VP-16 induces apoptosis, as described for other cell types with wild-type p53.\nFig.\u00a05WIP1 antagonizes p53-mediated apoptosis and arrest in medulloblastoma cell lines. Flow cytometric analysis of cell cycle distributions of: (A) untreated D283 controls, illustrating the cell populations with 2n DNA content in G0\/G1 phase, 4n DNA content in G2\/M phase, intermediate DNA content in S phase, and the sub-G0\/G1 peak representing apoptotic nuclei; (B) WIP1-flag transfected D283, (C) p53-GFP transfected D283, (D) WIP1-flag and p53-GFP (WIP1\u00a0+\u00a0p53) doubly transfected D283, and (E) etoposide (VP-16)-treated D283 (Y-axis, cell number; X-axis, DNA content). Histograms summarizing flow cytometric analyses of transfected cultures: (F) apoptotic D283 and (G) apoptotic Daoy in Sub-G0\/G1, with plasmids and treatment with V-16, as indicated (Y-axis, %; X-axis, transfected plasmid(s) and culture conditions). The following annotation symbols refer to statistically significant differences in sub-G0\/G1 cell populations, as determined by paired Student t-test: * D283 cells transfected with p53-GFP plasmid versus mock-transfected D283 cells (P\u00a0=\u00a00.033). ** D283 cells co-transfected with both p53-GFP and Flag-WIP1 plasmids versus D283 cells transfected with p53-GFP alone (P\u00a0=\u00a00.026). *** D283 cells transfected with p53-GFP plasmid and treated with VP-16 vs. VP-16-treated, mock-transfected D283 cells (P\u00a0=\u00a00.019). # D283 cells transfected with Flag-WIP1 plasmid versus untreated, max-GFP control-transfected D283 cells (P\u00a0=\u00a00.013). ## Daoy cells transfected with p53-GFP plasmid and treated with VP-16 vs. VP-16-treated, mock-transfected Daoy cells (P\u00a0=\u00a00.044)\nIncreasing p53 in D283 cells with a GFP-tagged p53 expression plasmid also increased apoptosis (16.9% in sub-G0\/G1) compared to mock-transfected controls (4.5%, P\u00a0=\u00a00.033) (Fig.\u00a05F). VP-16 treatment of p53-transfected D283 cells further increased the sub-G0\/G1 population to 25.6% (P\u00a0=\u00a00.019 compared to mock-transfected VP-16-treated controls) (Fig.\u00a05F). These results suggest that additional constitutive p53 expression in transfected D283 cells can overcome negative regulatory pathways including endogenous MDM2 and WIP1.\nWe also transfected the TP53-mutant Daoy medulloblastoma cell line. Like wild-typeTP53 D283 cells, control Daoy cells treated with VP-16 increased their apoptotic sub-G0\/G1 population to 20.4 vs. 6% in untreated controls, suggesting that VP-16 induces apoptosis even in the absence of wild-type p53 (Fig.\u00a05G). Constitutive expression of transfected wild-type p53 in TP53-mutant Daoy cells enhanced VP-16-induced apoptosis to 33.5% (sub-G0\/G1), compared to mock-transfected, VP-16-treated controls (14.6%, P\u00a0=\u00a00.044) (Fig.\u00a05G). These data suggest that transfected p53 expression partially rescues TP53 mutation in Daoy cells.\nWIP1 overexpression limits VP-16- and p53-induced apoptosis\nWe examined the effects of constitutive WIP1 expression in wild-typeTP53 D283 cells. VP-16 exposure increased apoptosis to 18.5 from 5.7% in untreated WIP1-transfected D283 (Fig.\u00a05F). Despite limited transfection efficiency, extra WIP1 expression appeared to antagonize p53 activity even in the absence of genotoxic stress. When D283 cells were co-transfected to constitutively overexpress both WIP1 and TP53, they displayed fewer cells in sub-G0\/G1 than p53-transfectants (10 vs. 16.9%, respectively; P\u00a0=\u00a00.026) (Fig.\u00a05F). These data suggest that additional WIP1 limits exogenous p53-mediated apoptosis. In double WIP1- or TP53-transfected D283 cells, VP-16 increased apoptosis (15%) (Fig.\u00a05F). Compared to VP-16-treated p53-transfected D283, double transfectants displayed a trend toward less apoptosis and fewer cells in G0\/G1 phase (Fig.\u00a05F). These data are consistent with anti-p53-induced apoptosis, presumably due to WIP1 overexpression, despite genotoxic stimulation.\nWIP1-transfected Daoy cells lacking wild-type p53 did not differ in their cell cycle profile from control transfectants under basal conditions, demonstrating the p53-dependence of WIP1 effects. However, VP-16-treated WIP1-transfectants underwent more apoptosis (23.6%) than untreated counterparts (4.1%) (Fig.\u00a05G). These data suggest a minimal effect of constitutively expressed WIP1 in the absence of wild-type p53. Overexpressed WIP1 also limited p53-mediated apoptosis in doubly co-transfected Daoy cells. As in controls, VP-16 treatment of double WIP1\/TP53 Daoy transfectants increased cells in sub-G0\/G1 (21.8%) (Fig.\u00a05G). However, VP-16-treated double WIP1- or TP53-transfectants displayed fewer apoptotic cells compared to VP-16-treated single p53-transfectants. Thus, excess WIP1 also appears to protect VP-16-treated Daoy cells from p53-induced arrest and apoptosis.\nDiscussion\nRegulation of the responses of medulloblastoma to genotoxic therapies is not well understood. Because TP53 has been implicated but rarely mutated or deleted in medulloblastoma, we focused attention on the candidate oncogene, WIP1. Recently published studies employed genomic methods to associate WIP1 with gain of 17q23 in medulloblastomas. Mendrzyk et\u00a0al. used array-CGH to identify gain of chromosomal region 17q23.2-qter, a locus containing WIP1, in 24 of 47 (51%) medulloblastomas [22]. WIP1 mRNA was moderately overexpressed with respect to normal cerebellum control in seven of nine tumors. Immunostaining of a large series of tissue micro-arrays revealed strong nuclear expression in 148 of 168 (88%) tumor samples. Ehrbrecht et\u00a0al. used conventional and array-CGH to identify amplification of 17q23 in three (19%) tumors and copy number gain of 17q23 in another 3 out of 16 medulloblastomas [23]. In 3 of 11 (27%) tumors, WIP1 mRNA was expressed less than fivefold in excess of expression in normal cerebellum control. Using additional approaches, we have confirmed gain of the WIP1 locus in medulloblastomas and quantitated its relative overexpression in our larger series of tumors. These results raise the possibility that overexpressed WIP1 functions as an oncoprotein in medulloblastoma.\nWIP1 is a p53-dependent gene induced by genotoxic stress [20, 31]. As a PP2C serine\u2013threonine phosphatase family member, Wip1 can counteract the tumor suppressive activity of p53 through several distinct mechanisms. WIP1 dephosphorylates p53 that has been activated in response to genotoxic stress [21]. WIP1 also dephosphorylates and stabilizes MDM2, promoting p53 degradation (Xiongbin Lu, manuscript submitted). This provides another feedback loop that returns the cell to a homeostatic state following DNA repair. Wip1 can complement different oncogenes in their transformation of mouse embryo fibroblasts. Wip1 overexpression also phenocopies the anti-apoptotic effects of TP53 mutation in\u00a0vitro and increases tumorgenicity in murine models in\u00a0vivo [20, 32].\nAmplification or overexpression of WIP1 in breast and ovarian cancers may phenocopy the loss of p53 function, thereby contributing to growth and resistance to genotoxic therapies [17, 18]. In breast carcinomas with amplified and overexpressed WIP1, the TP53 locus is rarely mutated, suggesting that WIP1 amplification inhibits p53 activity and reduces selection for p53 mutations during tumor progression [17]. Although WIP1 expression reportedly correlates with clinical outcomes in patients with breast and ovarian carcinomas [33, 34], conclusive multivariate analysis of additional factors (such as the concurrent deletion of a TP53 allele and loss of 17p seen with i17q) awaits future analysis of greater tumor numbers.\nAnalysis of our series of primary medulloblastomas revealed low-level amplification of WIP1 as well as increased expression of WIP1 mRNA transcript and protein, especially in tumors with 17q gain and i17q. The connection between WIP1 amplification and these common cytogenetic lesions suggests a selective advantage. Since a few tumors with i17q did not display WIP1 amplification, other factors probably contribute to the emergence of this characteristic cytogenetic lesion. Conversely, one or more tumors without 17q gain displayed WIP1 overexpression. Although extra WIP1 copy numbers may explain relative overexpression via gene dosage effects, regulation at the post-transcriptional and -translational levels may also affect WIP1 protein levels. Consistent with our FISH results, qRT-RTPCR and Western blot analyses demonstrated increased levels of WIP1 mRNA and protein; both significantly associated with 17q gain and i17q. The degree of WIP1 RNA overexpression relative to normal cerebellar tissue corresponded to earlier reports [22, 23]. The apparent discrepancy between WIP1 expression at the RNA and protein levels suggests that post-translational modifications also contribute to WIP1 overexpression. The positive and negative regulatory networks appear to involve p38MAPK, MDM2, CHK1, and other DNA damage response factors [21, 30, 33].\nOf note, our analysis was limited to those tumor specimens with available fresh tissue for molecular studies and displays an unintended selection bias for mixed tumors with anaplastic features. However, none of our findings appeared to correlate with histologic subtype or clinical features. Having confirmed the gain of WIP1 copy numbers and quantitated its relative overexpression in our medulloblastoma series, we hypothesized that overexpressed WIP1 contributes to treatment resistance by interfering with p53 function in medulloblastoma.\nBecause the tumor specimens were obtained before genotoxic treatment, their WIP1 and p53 levels do not reflect their activity. Therefore, we employed established cell lines to analyze the effects of overexpression of WIP1. Despite their phenotypic differences, D283 and Daoy cells provide readily manipulable systems for comparing the cell cycle effects of WIP1 in the setting of wild-type versus mutant p53. Our results indicate that chemotherapy-induced genotoxic stress or p53 overexpression alone can induce apoptosis in D283 cells. The constitutive overexpression of p53 overcame endogenous negative regulation in D283 cells, and rescued TP53-mutation in VP-16-treated Daoy cells. These results are consistent with studies of other p53 wild-type cell types, including the neuroepithelial lineage, indicating that VP-16 induces apoptosis in a p53-related manner [35\u201337].\nMedulloblastoma cell lines displayed predicted effects of WIP1 overexpression. In addition to high basal WIP1 levels in D283 cells, additional transfected WIP1 counteracted the apoptotic effects of endogenous p53. The anti-p53 and chemoprotective effects of overexpressed WIP1 were also evident upon VP-16 treatment. These results support the hypothesis that WIP1 overexpression modulates genotoxic responsiveness by negatively regulating p53 in medulloblastoma cells. WIP1 overexpression appears to antagonize p53 activation, and provides medulloblastoma cells the ability to resist arrest and apoptosis due to genotoxic stress.\nThe frequency of WIP1 amplification and the degree of overexpression in primary medulloblastoma strongly suggests the acquisition of selective advantages that may explain the prevalence of 17q gain and i17q. Based on evidence from medulloblastoma cell lines , we hypothesize that WIP1 regulates p53 function, which contributes to tumor growth and genotoxic treatment resistance. The recent development and characterization of agents that inhibit WIP1 raise the possibility of therapeutic intervention in those medulloblastomas with wild-type TP53 [38, 39].","keyphrases":["medulloblastoma","p53","wip1\/ppm1d","isochromosome 17q","apoptosis","fish"],"prmu":["P","P","P","P","P","P"]}
{"id":"Apoptosis-3-1-1914234","title":"Homocysteine affects cardiomyocyte viability: concentration-dependent effects on reversible flip-flop, apoptosis and necrosis\n","text":"Background Hyperhomocysteinaemia (HHC) is thought to be a risk factor for cardiovascular disease including heart failure. While numerous studies have analyzed the role of homocysteine (Hcy) in the vasculature, only a few studies investigated the role of Hcy in the heart. Therefore we have analyzed the effects of Hcy on isolated cardiomyocytes.\nIntroduction\nHyperhomocysteinaemia (HHC) reflects an impairment of the methionine\u2013homocysteine metabolism. Normally homocysteine (Hcy) is converted to cysteine via transsulfuration or to methionine by addition of a methyl group donated by folate [1]. However, when these pathways are deranged because of genetic mutations of enzymes involved or relative deficiencies of folate, vitamin B6 or vitamin B12, the serum concentration of Hcy increases [2, 3].\nIn 1969 Mc Cully et al. reported elevated concentrations of Hcy in young patients with severe cardiovascular disease [4]. They hypothesized the existence of a pathogenic link between HHC and atherogenesis. Since then other studies have also suggested that HHC is an independent risk factor for cardiovascular complications [5\u20138]. It is now assumed that even mild HHC constitutes an independent risk factor for cardiovascular disease [9] with equal predictive strength as hypercholesterolemia and smoking [10]. Whether Hcy itself or a Hcy-related substance such as S-adenosyl-homocysteine (SAH) is the culprit, and what the exact pathogenic mechanism of the cardiovascular complications induced by HHC could be, remains to be elucidated. In addition, large-scale intervention trails using high-dose-B-vitamins to reduce plasma Hcy levels in cardiovascular patients have as yet not shown an overall clinical benefit [11\u201313]. This raises further questions about the mechanisms underlying the association between HHC and cardiovascular disease, and about the possible adverse effects of high-dose-B-vitamins as a means of reducing Hcy levels [14].\nStudies on the effects of Hcy on the vascular wall, especially on endothelial and smooth muscle cells, indicate that increased Hcy leads to cellular stress and also to cell damage [15\u201317]. Hcy primes human neutrophils for an increased production of reactive oxygen species (ROS) and induces ROS production in endothelial cells [15, 18]. ROS production has also been found in the vascular wall of coronary arteries during HHC [19] and a role of NADPH oxidases in this process has been suggested.\nFrom the Framingham Heart Study it is known that increased plasma homocysteine concentrations, next to their effect on the vasculature, also form an independent risk factor for the development of heart failure [20]. Several other studies have also shown a link between HHC and heart failure [21, 22]. However, so far no studies have addressed possible effects of Hcy on cardiomyocytes. Therefore, we performed the present study to analyze the effects of Hcy on isolated cardiomyocytes.\nMaterials and methods\nCell cultures\nRat cardiomyoblasts (H9c2 (2\u20131) cells; ATCC, Manassas, VA, USA) were cultured in Dulbecco\u2019s Modified Eagles Medium (DMEM; BioWhittaker, Verviers, Belgium) supplemented with 10% heat-inactivated fetal calf serum (FCS; BioWhittaker), 2\u00a0mM l-glutamine (GIBCO, Paisley, UK), 100 IU\/ml penicillin (Yamanouchi Europe BV, Leiderdorp, Netherlands) and 100\u00a0\u03bcg\/ml streptomycin (Radiopharma-Fisiopharma, Palomonte, Italy), and grown at 37\u00b0C in a humidified 5% CO2\/95% air atmosphere. Experiments were performed with cells grown to a confluency of 60\u201380%. Adult rat cardiomyocytes were isolated as described before [23].\nAntibodies and chemicals\nMonoclonal Antibody 48 against NOX2 IgG 1 (dilution 1:28) was obtained from Sanquin Research at CLB, Amsterdam, The Netherlands [24]. Cy3-labeled goat-anti-rabbit-Ig (1:75) (Alexa Fluor; Leiden, Netherlands) was used as a secondary antibody. Polyclonal \u03b1-Nitrotyrosine IgG (1:50) (Molecular Probes Inc, Eugene, OR, USA) was used as an antibody to measure nitrotyrosin residues which is an indicator for ROS production. In this case, Cy5-labeled goat-anti-rabbit-Ig (1:50) (Jackson Immuno Research, West Grove, PA, USA) was used as a secondary antibody. Isotype controls and PBS were used to determine aspecific binding. Monoclonal antibody 1501R against pan-actin (1:6000; Chemicon International, Inc, Temecula, CA, USA) was used as a loading control for the western blot analysis.\nCells were incubated with several concentrations (0.1\u00a0mM\u20132.7\u00a0mM) of d,l-homocysteine (Hcy) (Sigma, St. Louis, MO, USA) in growth medium for 24\u00a0h at 37\u00b0C in a humidified 5% CO2\/95% air atmosphere.\nMeasuring Hcy concentration in growth medium\nThe H9c2 cells were incubated with different concentrations of d,l-Hcy. Several studies have suggested that only the l form is bioactive [25, 26]. Next to d,l-Hcy concentration we therefore also measured the l-form. d,l-Hcy was measured by HPLC using fluorescence detection [27]. Intra- and interassay coefficients of variation (CVs; std dev\/average *100%) were 1.8 and 3.5%, respectively. l-Hcy was measured by the Abbott IMx fluorescence polarization immunoassay (IMx; Abbott Laboratories, Abbott Park, IL, USA). Intra- and interassay CVs were less than 2 and 4%, respectively. The concentration of Hcy in culture medium was measured before incubation (t\u00a0=\u00a00) and after 24\u00a0h (final timepoint for the rest of the experiments) of incubation with H9c2 cells to determine the amount of putative degradation and\/or uptake of the Hcy during this incubation period.\nDetermination of intracellular S-Adenosylmethionine (SAM) and S-Adenosylhomocysteine (SAH)\nSince previous studies suggested that an increase in SAH is the culprit in cellular damage due to elevated levels of Hcy, we also determined the intracellular concentration of SAM and SAH in H9c2 cells after 24\u00a0h of incubation with d,l-Hcy. Tandem mass spectrometry (MS\/MS) was used for the determination of SAM\/SAH concentrations as previously described [28].\nFlow cytometry\nPhosphatidylserine exposure was assessed with FITC-labeled human recombinant annexin-V (Bender Med Systems, Vienna, Austria), while propidium iodide (PI; Bender Med Systems) was used to determine the permeability of the cellular membrane. Experiments were performed in the presence or absence of Z-Asp-Glu-Val-Asp-fluoromethylketone (Z-VAD FMK, 25\u00a0\u03bcM, Alexis Biochemicals, Lausen, Switzerland), a general caspase inhibitor, to differentiate between reversible flip-flop of plasma membrane phospholipids, apoptosis and necrosis [23]. After treatment with Hcy, the cells were trypsinized and centrifuged at 400g for 5\u00a0min at room temperature. Cells were then washed with serum-free DMEM, and resuspended in serum free DMEM containing annexin V (1:40) for 30\u00a0min in the dark at 37\u00b0C in a humidified 5% CO2\/95% air atmosphere. Cells were subsequently washed and resuspended in serum free DMEM containing PI (1:40). Cells were measured with a FACSCalibur (Becton Dickinson, San Jose, CA, USA). Results were analyzed by Cell Quest Pro software (Becton Dickinson).\nDetection of caspase-3 activity\nCells were grown in a 96\u2212wells plate (20,000 cells\/well). After treatment with Hcy and\/or Z-VAD FMK, cells were lysed and incubated with DEVD-rhodamine 110 substrate (Roche, Mannheim, Germany) for 1\u00a0h at 37\u00b0C. Subsequently the amount of free rhodamine was determined at a microplate fluorescence reader (TECAN spectrafluor, Switzerland). The developed fluorochrome was proportional to the concentration of activated caspase-3 and could be quantified by a calibration curve of diluted free rhodamine. Each condition was measured in triplo per measurement (total of three measurements).\nImmunofluoresence microscopy\nTo measure the expression of NOX2 and the putative formation of nitrotyrosin, cells were incubated with or without Hcy for 24\u00a0h in the 4-well chamber slides (Nalge Nunc International, Naperville, IL, USA). Cells were washed with PBS and fixated with 4% formaldehyde for 10\u00a0min at 37\u00b0C. Cells were subsequently washed with PBS, permeabilized with acetone\u2013methanol (70%\u201330%) for 10\u00a0min at \u221220\u00b0C, and then washed again with PBS\/Tween-20 (0.05% (v\/v) Tween-20 in PBS). Subsequently cells were incubated with primary antibodies against NOX2 and nitrotyrosin for 60\u00a0min at room temperature followed by incubation overnight at 4\u00b0C. PBS and isotype controls were also tested to determine nonspecific binding of the antibodies and background signal. The following day the cells were washed with PBS\/Tween and incubated with the secondary antibodies for 30\u00a0min at room temperature. After subsequent washes in PBS\/Tween and PBS, the slides were covered in mounting medium containing DAPI (Vector Laboratories Inc, Burlingame, CA, USA) to visualize nuclei. Thereafter the slides were covered with coverslips.\nSubsequently, cells were analyzed by means of a 3I MarianasTM digital imaging microscopy workstation (Zeiss Axiovert 200\u00a0M inverted microscope; Carl Zeiss, Sliedrecht, Netherlands), equipped with a nanostepper motor (Z-axis increaments; 10\u00a0nm) and a cooled CCD camera (Cooke Sensicam, 1280\u00a0\u00d7\u00a01024\u00a0pixels; Cooke Co, Tonawanda, NY, USA). Visualization of NOX2 and nitrotyrosine was performed with a 40\u00d7 air lens. The microscope, camera and data viewing process were controlled by SlideBookTM software (version 4.0.8.1; Intelligent Imaging Innovations, Denver, CO, USA).\nLive cell analysis of \u0394\u03a8m\nTo visualize mitochondrial membrane potential (\u0394\u03a8m) via Life Cell Imaging, cells were loaded for 30\u00a0min at 37\u00b0C with 0.5\u00a0\u03bcmol\/l of 5,5\u2032,6,6\u2032-tetrachloro-1,1\u2032,3,3\u2032-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Cell Technology Inc, Minneapolis, USA). In non-apoptotic cells, JC-1 exists as a monomer and is visible on the green channel (FITC). In healthy cells these monomers accumulate as aggregates in the mitochondria due to existing \u0394\u03a8m. These aggregates are visible on the red channel (CY3) when viewed with a fluorescence microscope. Cells were washed with PBS before viewing and subsequently analyzed by use of a 3I MarianasTM digital imaging microscopy workstation as described above.\nWestern blot analysis\nAfter treatment H9c2 cells were harvested into modified ELB lysis buffer (250\u00a0mM NaCl, 0.1% Nonidet P-40, 50\u00a0mM HEPES pH 7.0, 5\u00a0mM EDTA, 0.5\u00a0mM DTT) wherein protease inhibitor cocktail (PIC, 1:40; Sigma) was added, and the cell suspension was mixed rigorously and then incubated for 30\u00a0min on ice. After determination of the protein concentration of the samples with the BCA protein assay kit (Pierce, Rockford, IL, USA), reducing sample loading buffer (0.25\u00a0M TRIS pH 6.8, sodium dodecyl sulfate (SDS), glycerol, 2-mercaptoethanol, bromophenol blue) was added and the samples were mixed and heated at 95\u00b0C for 10\u00a0min. A total of 50\u00a0\u03bcg protein of each sample was then subjected to SDS-PAGE, transferred to nitrocellulose membranes and analyzed for NOX2 expression with monoclonal antibody 48 (1:250 dilution) and for pan-actin with monoclonal antibody 1501R as a loading control (1:6000), followed by horseradish\u2013peroxidase\u2013conjugated rabbit\u2013anti-mouse immunoglobulins (R\u03b1M-HRP; 1:1000 dilution; DakoCytomation, Glostrup, Denmark). The blots were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences AB; Uppsala; Sweden). Staining was quantified with a charge-coupled device camera (Fuji Science Imaging Systems; D\u00fcsseldorf, Germany) in combination with AIDA Image Analyzer software (Isotopenmessger\u00e4te; Staubenhardt, Germany).\nATP measurement via Luciferase\u2013Luciferin assay\nAfter treatment cells were collected by trypsinization and centrifugation at 400g for 5\u00a0min. Cells were then counted and equal amounts were taken per condition. After centrifugation for 2\u00a0min (400g) the supernatant was discarded. The pellet was resuspended in 150\u00a0\u03bcl of ice-cold perchloric acid (PCA) 0.4\u00a0M. Cells were left on ice for 30\u00a0min and then centrifuged again for 5\u00a0min (2000g) at 4\u00b0C. To the isolated supernatant 7.5\u00a0\u03bcl of K2CO3 5\u00a0M was added to neutralize pH. Samples were subsequently stored at \u221280\u00b0C until measuring. The amount of ATP in the samples was determined with a Luciferase\u2013Luciferin assay (Biaffin GmbH & Co KG, Kassel, Germany) according to the manufacturers\u2019 protocol. Samples were measured in a FluoroNunc MaxiSorp plate (Nalge Nunc International, Rochester, NY). Luminescence was measured by using a Tecan GENios Plus reader (Tecan Benelux, Mechelen, Belgium).\nStatistics\nStatistics were performed with the SPSS statistics program (windows version 9.0). To evaluate whether observed differences were significant, One Way ANOVA with post hoc Bonferroni tests were used. All values are expressed as mean\u00a0\u00b1\u00a0standard error of the mean (SEM). A P value (two sided) of 0.05 or less was considered to be significant.\nResults\nMeasurement of the concentration of d,l-homocysteine (d,l-Hcy), l-homocysteine (l-Hcy), S-adenosyl methionine (SAM) and S-adenosyl homocysteine (SAH)\nWe tested the effects of d,l-Hcy at concentrations of 0.1\u00a0mM, 1.1\u00a0mM and 2.7\u00a0mM, in accordance with previous studies in isolated vascular cells [15, 17, 26, 29\u201331]. We quantified the exact concentration of d,l-Hcy in medium added to the cells, via HPLC. As depicted in Table\u00a01, the concentration of d,l-Hcy was according to the concentration added to the growth medium.\nTable\u00a01Concentration of d,l-Hcy and l-Hcy in culture medium on t\u00a0=\u00a00, l-Hcy after 24\u00a0h incubation, and intra-cellular concentration of SAM\/SAH after 24 h incubationmM Hcy addedmM Hcy measured[SAM] (nM) t\u00a0=\u00a024[SAH] (nM) t\u00a0=\u00a024[d,l-Hcy] [l-Hcy] t\u00a0=\u00a00[l-Hcy] t\u00a0=\u00a0240.0 00.0007 (+\/\u22120.00009)0.002 (+\/\u2212\u00a00.00004)464.9 (+\/\u2212\u00a036.6)6.3 (+\/\u2212\u00a00.7)0.1 0.14 (+\/\u2212\u00a00.03)0.06 (+\/\u2212\u00a00.01) 0.06 (+\/\u2212\u00a00.01)468.3 (+\/\u2212\u00a077.2)7.0 (+\/\u2212\u00a02.9)1.1 1.08 (+\/\u2212\u00a00.04)0.46 (+\/\u2212\u00a00.03)0.32 (+\/\u2212\u00a00.01)*319.6 (+\/\u2212\u00a056.2)16.9 (+\/\u2212\u00a06.8)2.7 2.73 (+\/\u2212\u00a00.06)1.18 (+\/\u2212\u00a00.05)0.84 (+\/\u2212\u00a00.03)*204.4 (+\/\u2212\u00a074.5)**164.8 (+\/\u2212\u00a034.6)***All mediators were measured in n\u00a0=\u00a03. A significant decrease of l-Homocysteine (l-Hcy) can be seen at 1.1\u00a0mM and 2.7\u00a0mM after 24\u00a0h (*\u00a0P\u00a0<\u00a00.001). At 2.7\u00a0mM a significant decrease of S-adenosylmethionine (SAM,** P\u00a0=\u00a00.006); but an increase in S-adenosylhomocysteine (SAH, ***\u00a0P\u00a0<\u00a00.001) was detected, compared to all other concentrations\nSince previous studies have shown that only the l form of Hcy is bioactive [25, 26], we also determined the l-Hcy concentration in medium by IMx. We found that 42.7%, 42.5% and 43.3% of the total d,l-Hcy amount 0.1\u00a0mM, 1.1\u00a0mM and 2.7\u00a0mM, respectively, in fact was l-Hcy at t\u00a0=\u00a00. Furthermore, we determined the concentrations of l-Hcy at 24\u00a0h, the final time point of incubation, and found a significant decrease in the concentrations of l-Hcy, namely for 1.1\u00a0mM a significant decrease of 0.14\u00a0mM l-Hcy; +\/\u22120.011 (P\u00a0<\u00a00.001) and for 2.7\u00a0mM a significant decrease of 0.34\u00a0mM l-Hcy; +\/\u22120.0301 (P\u00a0<\u00a00.001).\nIt has been well documented that increased levels of SAH inhibit several important methylation reactions [32\u201334]. Therefore we also determined the intracellular concentrations of both SAH and SAM, being one of the primary methyl donors (Table\u00a01). Measuring intracellular concentrations of SAM and SAH in H9c2 cells incubated with and without d,l-Hcy showed a significant depletion of SAM at 2.7\u00a0mM d,l-Hcy compared to untreated cells and 0.1\u00a0mM d,l-Hcy treated cells (P\u00a0=\u00a00.006; n\u00a0=\u00a03). While the concentration of SAH increased significantly at this same concentration compared to all other conditions (P\u00a0<\u00a00.001; n\u00a0=\u00a03). Therefore we also examined the effects of the higher, non-physiological concentrations of Hcy.\nEffect of Hcy on cell viability\nWe analyzed the effect of Hcy on H9c2 cell viability by measuring annexin V and\/or PI positivity using flow-cytometry. Incubation with 0.1\u00a0mM d,l-Hcy had no effect on single-annexin-V-positivity (Fig.\u00a01A), double-annexin-V, PI positivity (Fig.\u00a01B) or single-PI positivity (not shown, approximately 3% for each condition). In contrast, incubation with 1.1\u00a0mM d,l-Hcy resulted in a significant increase in single- annexin-V-positive cells (P\u00a0<\u00a00.002) (Fig.\u00a01A). This increase in single-annexin-V-positive cells was only partly, but not significantly inhibited by Z-VAD FMK (31.6% decrease).1.1\u00a0mM d,l-Hcy had no significant effect on the percentage of double-annexin-V\/PI-positive cells (Fig.\u00a01B) or on the percentage of single-PI-positive cells (not shown). Incubation with 2.7\u00a0mM d,l-Hcy, however, resulted in a significant increase of single-annexin-V positive cells (P\u00a0<\u00a00.001) and double-annexin-V\/PI-positive cells (P\u00a0<\u00a00.044) that could not be inhibited significantly by Z-VAD FMK. Again we did not see any significant increase in single-PI-positive cells (not shown). Thus, depending on the concentration of Hcy, either reversibly flip-flopped cardiomyocytes and\/or late apoptotic\/necrotic cells can be found.\nFig.\u00a01Effect of Hcy on cell-viability via flow-cytometry: Flow cytometry analysis of H9c2 cells incubated with different concentrations of d,l-Hcy during 24\u00a0h, with or without 25\u00a0\u03bcM Z-VAD FMK, a pan caspase inhibitor. A total of 10,000 cells were measured (n\u00a0=\u00a03). (A) Percentage of single annexin V positive, PI-negative labeled cells, which is a marker of flip flop of the plasma membrane phospholipids. (B) Percentage of double-annexin-V\/PI-positive cells depicting late apoptotic and\/or necrotic cells. Data represent mean percentages and standard error of the mean (SEM)\nCaspase-3 activity\nTo determine whether the population of double-annexin-V\/PI-positive cells consists of late apoptotic cells or necrotic cells, we performed an assay of caspase-3 activity. Incubation with 0.1\u00a0mM and 1.1\u00a0mM d,l-Hcy did result in a small, but not significant increase in caspase-3 activity that was completely inhibited when Z-VAD FMK was added. Incubation with 2.7\u00a0mM d,l-Hcy for 24\u00a0h, however, induced a significant increase of caspase-3 activity (P\u00a0<\u00a00.001) that was also completely inhibited with Z-VAD FMK (Fig.\u00a02). Caspase-3 activity measured at earlier time points (6 and 16\u00a0h) of Hcy incubation was not significantly increased (results not shown). Thus, significant apoptosis induction of H9c2 cells was only present at 2.7\u00a0mM d,l-Hcy.\nFig.\u00a02Effect of Hcy on caspase-3 activity: H9c2 cells were incubated during 24\u00a0h with different concentrations of d,l-Hcy in the presence and absence of Z-VAD FMK (n\u00a0=\u00a09). Caspase-3 activity was measured as indicated under Methods. Data represent mean and SEM\nEffect of Hcy on NOX2 expression and nitrosylation\nIn a previous study we have found that ischemia of cardiomyocytes induced nuclear NOX2 expression coinciding with nuclear nitrotyrosine residues, resulting in subsequent apoptosis of these cells [35]. Given the pro-apoptotic effect of 2.7\u00a0mM d,l-Hcy, we analyzed by Western blotting the effect of Hcy on NOX2 expression. We found that NOX2 was constitutively present in whole cell lysates of H9c2 cells (Fig.\u00a03). NOX2 expression was slightly increased at 0.1\u00a0mM d,l-Hcy and increased even more at concentrations of 1.1\u00a0mM and 2.7\u00a0mM of d,l-Hcy. It is noteworthy that although we found this increase in NOX2 expression, we were not able to detect apoptosis at the lower concentrations of Hcy. Therefore we subsequently analyzed the intracellular localization of NOX2 with digital imaging. In the cytosol, we found diffuse positivity for NOX2 at all concentrations of Hcy (data not shown). Incubation with 0.1\u00a0mM and 1.1\u00a0mM, but especially with 2.7\u00a0mM d,l-Hcy resulted in a significant increase in NOX2 expression in the nucleus (P\u00a0<\u00a00.001; P\u00a0=\u00a00.047; P\u00a0<\u00a00.001, respectively) (Fig.\u00a04A). These results appear in contrast with the caspase-3 measurements. We therefore also analyzed the presence of nitrotyrosine residues as an indicator for ROS production. We found that neither 0.1\u00a0mM nor 1.1\u00a0mM d,l-Hcy resulted in significant nitrotyrosine formation in the nucleus. In contrast, 2.7\u00a0mM d,l-Hcy did induce a significant formation of nitrotyrosine residues in the nucleus (P\u00a0<\u00a00.001) (Fig.\u00a04B). These results underline that not only nuclear NOX2 expression but also nuclear ROS production is necessary for the induction of apoptosis. It has to be noticed that especially at 2.7\u00a0mM d,l-Hcy a significant loss of approximately 50% of the cells was found (P\u00a0<\u00a00.001, results not shown). As these cells theoretically are predominantly double-annexin-V\/PI or single-PI-positive, we conclude that the effect of incubation with 2.7\u00a0mM d,l-Hcy on the amount of double-annexin-V\/PI or single-PI-positive cells (see Fig.\u00a01) in all probability is underestimated.\nFig.\u00a03Effect of Hcy on NOX2 expression: (A) Western Blot analysis for NOX2 expression of whole-cell lysates from H9c2 cells incubated with or without Hcy for 24\u00a0h. Data represent mean and SEM for three independent experiments. (B) Western Blot analysis of the same blots for pan-actin as a loading controlFig.\u00a04Effect of Hcy on intracellular localization of NOX2 and nitrosylation in H9c2 cells: Analysis of the effect of different concentrations of D,L-Hcy at 24\u00a0h incubation on the nuclear localization of NOX2 expression (A) and nitrotyrosine residues (B) measured by means of digital imaging microscopy. The arbitrary units are defined as sum intensity per nucleus and depicted as % increase versus control where control was set to 0 (n\u00a0=\u00a06). Data represent mean and SEM\nWe next wanted to corroborate the findings of NOX2-mediated nitrotyrosine formation in adult rat cardiomyocytes. Because the Hcy concentrations analyzed in H9c2 cells were lethally toxic for adult rat cardiomyocytes, we used lower concentrations for these cells. Incubation with 0.07\u00a0mM d,l-Hcy induced significant NOX2 expression in the nucleus (P\u00a0<\u00a00.022; Fig.\u00a05D) but no significant nitrotyrosine formation. Incubation with 0.1\u00a0mM d,l-Hcy led to significant nuclear NOX2 expression and presence of nitrotyrosine residues (P\u00a0<\u00a00.01; Fig.\u00a05A\u2013E). As with H9c2 cells, we found only little NOX2 positivity in the cytosol under all Hcy concentrations analyzed (arrow in Fig.\u00a05A).\nFig.\u00a05Effect of Hcy on intracellular localization of NOX2 and ROS production in adult rat cardiomyocytes: Digital Imaging microscopy pictures of adult rat cardiomyocytes incubated with 0.1\u00a0mM d,l-Hcy during 24\u00a0h. Red color (A) indicates NOX2 expression which is visible in the nucleus and in the cytosol (arrow). Green color (B) represents nitrotyrosine formation. The merge image (C) also shows blue DAPI staining for DNA which shows co-localization of NOX2 expression and nitrotyrosine formation in the nucleus. (D) Amount of nuclear NOX2 expression in adult rat cardiomyocyt in control, 0.07\u00a0mM and 0.1\u00a0mM d,l-Hcy samples (n\u00a0=\u00a03). (E) Amount of nitrotyrosine formation in adult rat cardiomyocyt in control, 0.07\u00a0mM and 0.1\u00a0mM d,l-Hcy sample (n\u00a0=\u00a03). The arbitrary units are defined as sum intensity per nucleus and depicted as % increase versus control where control was set to 0. Data represent mean and SEM\nATP depletion\nAn important determinant in the process of cell death is ATP, as it is for instance necessary to complete the process of apoptosis. We therefore measured ATP concentrations in H9c2 cells (Fig.\u00a06). Incubation with 0.1\u00a0mM d,l-Hcy resulted in a significant increase in ATP concentration (P\u00a0=\u00a00.05). In contrast, incubation with 1.1\u00a0mM d,l-Hcy led to a decrease in ATP concentration that was not significant compared to control cells, but was significant compared to ATP concentration found in cells that were incubated with 0.1\u00a0mM d,l-Hcy (P\u00a0=\u00a00.024). Incubation with 2.7\u00a0mM d,l-Hcy induced an more pronounced decrease of ATP concentration that was significant also when compared to control cells (P\u00a0=\u00a00.036).\nFig.\u00a06Effect of Hcy on ATP levels: Increase or decrease in ATP levels (as compared to control levels) in H9c2 cells after incubation with different concentrations of d,l-Hcy during 24\u00a0h. Data represent mean and SEM of four independent experiments\n\u0394\u03a8m\nTo explain the contrasting effects of different Hcy concentrations on ATP concentrations, we analyzed the mitochondrial membrane potential (\u0394\u03a8m) with JC-1 staining in H9c2 cells. We found an increase in red signal (P\u00a0=\u00a00.006) when the H9c2 cells were incubated with 0.1\u00a0mM d,l-Hcy compared to control cells (Fig.\u00a07A\/B) which is indicative for more actively respiring mitochondria. \u0394\u03a8m after incubation with 1.1\u00a0mM and 2.7\u00a0mM d,l-Hcy did not differ significantly from that in control cells (Fig.\u00a07A\u2013E). A difference in the morphology of mitochondria was also visible. In control cells a heterozygous mitochondria population is visible consisting of round and elongated mitochondria. Incubation with 0.1\u00a0mM d,l-Hcy showed mostly elongated mitochondria, whereas incubation with 1.1\u00a0mM and 2.7\u00a0mM d,l-Hcy resulted in a more rounded morphology of the mitochondria, the so called thread-to-grain transition which is seen as an early marker for apoptosis [36].\nFig.\u00a07Effect of Hcy on mitochondrial membrane potential: H9c2 cells were incubated with or without Hcy for 24\u00a0h and analyzed with digital imaging microscopy. Green indicates the JC-1 monomers. Red indicates JC-1 aggregates, which are formed in the mitochondria when a sufficiently high membrane potential is reached. (A) H9c2 cells incubated in growth medium alone. Normal heterozygous population of elongated and round mitochondria have a red fluorescence signal. (B) H9c2 cells incubated with 0.1\u00a0mM d,l-Hcy. An increase in actively respiring mitochondria (red) is visible, and a mostly elongated morphology can be seen. (C) 1.1\u00a0mM d,l-Hcy shows a similar amount of functioning mitochondria in the cells that did not receive Hcy; only the morphology of the mitochondria is mostly round, a phenomenon which is known as thread-to-grain transition. (D) H9c2 cells incubated with 2.7\u00a0mM d,l-Hcy. Some cells have no functioning mitochondria left, but overall there is no significant loss of red signal. The thread-to-grain transition of the mitochondrial reticulum is complete. (E) Analysis of sum intensity in Cy3 signal (red) per condition as an indicator for \u0394\u03a8m. Data represent mean and SEM (n\u00a0=\u00a03)\nDiscussion\nIn the present study we examined the effects of Hcy on cell viability of cardiomyocytes and found different effects, depending on Hcy concentration (Fig.\u00a08).\nFig.\u00a08Effects of different concentrations of Hcy on cardiomyocyte viability Scheme of the proposed events caused by Hcy, which ultimately lead to cell death: (A) control cells. (B) 0.1\u00a0mM d,l-Hcy results in increase of \u0394\u03a8m and ATP levels, and translocation of NOX2 to the nucleus. (C) 1.1\u00a0mM d,l-Hcy results in flip-flop of the plasma membrane (bended arrows in plasma membrane indicate flip-flop of phophatidyl serine to the outer leaflet of the membrane), coinciding with localization of NOX2 in the nucleus. (D) 2.7\u00a0mM d,l-Hcy next to flip-flop and NOX2 localization in the nucleus, nuclear ROS production is found. ATP is then decreased, as well as \u0394\u03a8m. Caspase-3 is activated and PI can enter the cell indicating cell death\nThe lower concentration of 0.1\u00a0mM Hcy resulted in mitochondrial hyperpolarization, an increase of ATP and translocation of NOX2 to the nucleus but without local nitrotyrosin formation. Mitochondrial hyperpolarization and an increase in ATP concentration have been described as first signs of mitochondrial disturbance on the pathway to apoptosis [36, 37]. On the other hand, they have also been described as part of a cell-protective mechanism [38]. Therefore, the effect of this lower Hcy concentration may be a cellular state characterized by early activity of both pro- and anti-apoptotic pathways, which does not necessarily result in cell death.\nIn contrast, the intermediate concentration of 1.1\u00a0mM Hcy led to rounding of mitochondria, a slight, non significant decrease of \u0394\u03a8m with a visible thread-to-grain transition of the mitochondrial reticulum, a slight decrease in ATP concentrations, a translocation of NOX2 to the nucleus, again without presence of nitrotyrosin residues, and a flip-flop of the cell membrane phospholipids as indicated by single annexin v positivity. We did not find caspase-3 activation at this concentration. Although annexin V positivity of cells has generally been considered as a marker for apoptosis, in a previous study however, we have demonstrated that this is not absolute as such and that membrane phospholipid flip-flop can be reversible [23], which is supported in this study by the observation that complete inhibition of caspases by Z-VAD-FMK did not affect this phenotype. The caspase-dependent pathway of apoptosis was, therefore, not yet activated at this concentration of Hcy, probably related to the lack of nuclear nitrotyrosin residues.\nThe highest concentration of 2.7\u00a0mM Hcy entailed a completion of the thread-to-grain transition of the mitochondrial reticulum, a further decrease of ATP, translocation of NOX2 to the nucleus and nuclear nitrotyrosin formation, caspase-3 activation, and also a clear increase in both single-annexin-V and annexin-V\/PI-positive cells. These changes represent a cellular phenotype irreversibly committed to cell death, in which nuclear ROS production apparently plays a deciding role as indicated by the presence of nitrotyrosin residues. Whether cell death then follows the process of apoptosis or necrosis depends on the remaining concentration of ATP. Apoptosis is energy consuming, and therefore depends on minimal residual ATP concentrations, whereas necrosis is the default route in the absence of ATP [39].\nInduction of apoptosis generally follows one of two pathways. The first pathway, the death receptor or extrinsic pathway, is activated by ligand binding of cell-surface-expressed death receptors. The second pathway, the mitochondrial or intrinsic pathway, is promoted by physical or chemical stress agents [40]. Intracellular accumulation of Hcy and its metabolites constitutes considerable intracellular stress [15, 17, 41]. In line with this notion, several studies in endothelial cells have suggested that Hcy-induced apoptosis follows the mitochondrial pathway [42,43]. Our study in cardiomyocytes has shown that \u0394\u03a8m is modified in a concentration-dependent manner, and thereby supports the concept that Hcy-induced apoptosis mainly follows the mitochondrial pathway, also in cardiomyocytes. However, in addition we found that translocation of NOX2 to the nucleus coinciding with the presence of nitrotyrosin residues indicative for ROS production is even more important in the effector phase of the process of apoptosis induction in cardiomyocytes.\nSince we only found a significant decrease in L-Hcy at the highest concentrations, but not with 0.1\u00a0mM d,l-Hcy we also determined SAH concentrations. Several studies provided further evidence that SAH could be the culprit in Hcy-mediated damage since it is a potent inhibitor of various methylation reactions [32\u201334]. In this study we found that only the concentration of 2.7\u00a0mM d,l-Hcy resulted in a significant increase in SAH, which was the concentration that resulted in cell death of cardiomyocytes. Therefore our study underlines that SAH is an important mediator in the induction of cell death of cardiomyocytes by Hcy.\nA limitation of the present study is the use of relatively high concentrations of Hcy. However, it should be noted that cells in culture usually up-regulate their Hcy-metabolizing enzymes, in particular cystathionine beta-synthase (CBS) [44]. Furthermore short-term exposure to high Hcy concentrations may reflect a prolonged exposure to moderately elevated Hcy concentrations as occur life-long in patients. Therefore, our data may provide, at least in part, an explanation why HHC contributes to heart failure.\nConclusion\nHcy is increasingly recognized as an independent cardiovascular risk factor [5\u20138] and an extensive literature exists that links Hcy-induced changes in endothelial [15, 26, 45\u201348] and smooth muscle cells [16, 17, 49\u201352] to atherosclerotic disease. The Framingham Heart Study also showed that increased plasma Hcy levels are a risk factor for the development of heart failure [20]. To our knowledge, no reports have yet been published on the effect of Hcy on cardiomyocytes specifically. Our study now clearly indicates that Hcy, and possible SAH, indeed affects also cardiomyocytes, primarily by inducing apoptosis\/necrosis in a concentration-dependent manner, in which nuclear ROS production, related to nuclear NOX2 expression, is crucial.","keyphrases":["cardiomyocytes","apoptosis","hyperhomocysteinaemia","heart failure","oxidant stress"],"prmu":["P","P","P","P","M"]}
{"id":"J_Med_Internet_Res-5-4-1550575","title":"Improving Web Searches: Case Study of Quit-Smoking Web Sites for Teenagers\n","text":"Background The Web has become an important and influential source of health information. With the vast number of Web sites on the Internet, users often resort to popular search sites when searching for information. However, little is known about the characteristics of Web sites returned by simple Web searches for information about smoking cessation for teenagers.\nIntroduction\nThe World Wide Web, with over 3 million public Web sites and over 1.4 billion Web pages [1], has become an important and influential source of health information [2]. In September 2002, there were an estimated 605 million people online worldwide [3]. In the United States, 90% (48 million) of the children and adolescents between the ages of 5 and 17 use computers, and 75% of the 14 to 17 year olds use the Internet [4]. With the vast amount and dynamic nature of information on the World Wide Web, it is not surprising to find that over 75% of those online use search sites to navigate the Web [5]. However, the amount of results returned from a search is often overwhelming. For example, 115000 results were found with the search terms \"teen quit smoking\" in Google.\nOf the several thousand search sites or directories [6], only a few are of high popularity as indicated by their audience reach and time spent on them [7]. Although Google will provide up to a thousand results from a query, few users are likely to examine them all. In an observational study on 16 adult subjects, only 9 participants ever looked beyond the first search pages and only 5 of them ever clicked a link on those pages [8]. A survey done in 2002 on 1403 e-mail participants showed that only 23% of the users went beyond the second page [9]. Another pilot study of 12 teenagers found they looked past the fourth page of results less than 5% of the time [10]. Thus, position ranking in Web-search results, especially on the first few pages, is an important determinant of information accessibility by users.\nSeveral studies have reported substantial variability in health-related Web-site content [11- 14]. While guidelines for evaluating the quality of health information on the Web are available [15- 19], the correlation between these guidelines and accuracy of health information is debated [20- 22]. Position ranking in search results was not associated with content quality [23]. Using the search term \"breast cancer,\" Meric et al [24] reported that popularity of Web sites was associated with type rather than quality of content. In a sample of 75 Web sites that provided information on urinary incontinence, the Internet popularity indexes\u2014as measured by the number of links to the main incontinence page of each Web site and by the number of links to all pages of each Web site divided by the number of pages of the site\u2014were not correlated with a quality score based on Silberg et al [16] and the HONcode principles [25].\nThe aim of this study was: (a) to identify the characteristics of Web sites with information on smoking cessation for teenagers that ranked in the top 30 positions in a typical Web search on popular search sites and (b) to evaluate the association between those characteristics and the position ranking for sites that are of direct relevance to smoking cessation for teenagers. The findings are relevant for improving consumer access to health information.\nMethods\nThis study was carried out from May 2003 through June 2003. Web sites with information on smoking cessation for teenagers were identified with 4 popular search sites using a specific search term. The characteristics of the identified sites were collected with a Web-site characteristic checklist; 2 raters evaluated each Web site independently (details below).\nSearch Protocol\nFour popular search sites (Table 1) were used in this study. Users spend over 5 million search hours per month at each site. A search hour equals the number of visitors to a site multiplied by the average number of hours each visitor is estimated to have spent at the site.\nTable 1\nPopular search sites in the United States*\nSearch Site\nURL\nTotal Search Hours (Millions of Hours) in January 2003\nMainUnderlying SearchEngine\nGoogle\nwww.google.com\n18.7\nGoogle\nAOL\nsearch.aol.com\n15.5\nGoogle\nYahoo!\nwww.yahoo.com\n7.1\nGoogle and Overture (for paid listings)\nMSN\nsearch.msn.com\n5.4\nLookSmart, Inktomi, Microsoft proprietary editor, and Overture (for paid listings)\n* Source: Search Engine Watch [26].\nThe search term on smoking cessation for teenagers was selected based on information from the Overture Search Term Suggestion Tool [27] and the 7search Keyword Suggestion Tool [28]. These sites provide a count of the search terms that were submitted to their search engines. Overture provides their search results to various popular search sites including Yahoo, MSN, AltaVista, Lycos, HotBot, and AllTheWeb [29]. For example, in April 2003 there were 40036 searches submitted to Overture with \"quit smoking,\" 27812 with \"stop smoking,\" and 9001 with \"smoking cessation.\" Various other combinations of \"teen,\" \"youth,\" \"adolescent,\" \"quit smoking,\" \"stop smoking,\" and \"smoking cessation\" were compared. Based on the frequency of searches performed on the Web as recorded by the Overture database, the search terms \"teen quit smoking\" were submitted to the 4 search sites to locate sites with information on smoking cessation for teenagers.\nTo mimic the search behavior of Web users, only the top 30 search results were included in the study. Sites ranking below the top 30 results are likely to be found only by more-persistent searchers [30]. Thirty results are equivalent to 3 pages (2 clicks) of the default number of results per page in Google and AOL, 2 such pages (1 click) in MSN, and one and a half such pages (one click) in Yahoo. The results from the 4 search sites were combined into one list to provide an overall picture of the search activity on the Web. The sites were reranked by first grouping the sites into 4 groups by the number of search sites that included them (1 to 4 search sites) and then by the position ranking provided by the search results within each group. The top 30 reranked sites formed the sample for the analysis.\nSince the rankings of Web sites within search-site results change frequently, the search results were captured in spreadsheet format using the Google API Search Tool [31]. The Web pages of sites identified by search results were captured using Offline Explorer software [32] to facilitate the recall of the exact page content when necessary and to provide consistency for the 2 raters.\nTable 2\nSite characteristics and correlation with search ranking for 14 sites relevant to teenagers who are seeking information on smoking cessation\nSite Characteristic\nInter-Rater Reliability (Kappa)\nCharacteristics\nNumber (%)\nKendall Rank Correlation, Tau (P value)\nEssential\nSearch feature in the site\n0.86\nPresent\n8 (57)\n0.15 (.52)\nAbsent\n6 (43)\nSite navigation system on page\n0.76\nPresent\n11 (79)\n-0.16 (.42)\nAbsent\n3 (21)\nPrivacy statement\n0.57\nPresent\n8 (57)\n0.18 (.41)\nAbsent\n6 (43)\nDisclaimer\n0.69\nPresent\n9 (64)\n0.11 (.70)\nAbsent\n5 (36)\nReadability grade level\nNA*\n< 8.0\n5 (36)\n-0.39 (.05)\n\u2265 8.0\n9 (64)\nBroken links on page\nNA\nPresent\n7 (50)\n0.08 (.75)\nAbsent\n7 (50)\nEnhancement\nIndication of sponsorship\n0.19\nPresent\n11 (79)\n0.09 (.66)\nAbsent\n3 (21)\nPop-up advertisements or banner advertisements\n0.59\nPresent\n4 (29)\n0.18 (.17)\nAbsent\n10 (71)\nContact e-mail address\n0.51\nPresent\n10 (71)\n-0.46 (.01)\nAbsent\n4 (29)\nPhone number or mailing address\n1.00\nPresent\n4 (29)\n0 (1.00)\nAbsent\n10 (71)\nContent on cessation method: behavioral approach\n0.43\nPresent\n11 (79)\n0.31 (.10)\nAbsent\n3 (21)\nContent on cessation method: medication approach\n0.84\nPresent\n10 (71)\n-0.43 (.02)\nAbsent\n4 (29)\nContent on cessation method: alternative approach\n0.51\nPresent\n5 (36)\n-0.42 (.02)\nAbsent\n9 (64)\nAnnotated external hyperlinks\n0.72\nPresent\n5 (36)\n-0.39 (.04)\nAbsent\n9 (64)\nInteractive component (quiz, game, or bulletin board)\n0.53\nPresent\n8 (57)\n-0.18 (.44)\nAbsent\n6 (43)\nMaterial in video or audio format\n1.00\nPresent\n1 (7)\n-0.20 (.31)\nAbsent\n13 (93)\nTechnical\nPage size (kilobyte)\nNA\n< 35\n6 (43)\n-0.39 (.04)\n\u2265 35\n8 (57)\nMeta description tag\nNA\nPresent\n8 (57)\n-0.48 (.002)\nAbsent\n6 (43)\nMeta keywords tag\nNA\nPresent\n11 (79)\n-0.31 (.13)\nAbsent\n3 (21)\nPersistent cookies\nNA\nPresent\n3 (21)\n-0.34 (.06)\nAbsent\n11 (79)\nPart of a larger Web site\n0.72\nYes\n6 (43)\n0.03 (.90)\nNo\n8 (57)\nLink density (reverse links)\nNA\n1\n6 (43)\n-0.58 (.02)\n2-100\n4 (29)\n> 100\n4 (29)\n* NA = Not applicable. Kappa values for these characteristics were not available because they were analyzed by the WebXact Watchfire Page Analysis [35], except for readability grade level which was evaluated by only 1 rater.\nChecklist of Web-Site Characteristics\nA checklist was uses to evaluate the characteristics of the Web sites (see Table 2 for checklist items). The readability was estimated by the Flesch-Kincaid grade-level score [33]. (The Flesch-Kincaid grade-level score rates text on a United States grade-school level. For example, a score of 8.0 means that an eighth grader can understand the document.) Sample passages from the Web pages with information pertaining to smoking cessation of the identified sites were pasted into Microsoft Word XP for Windows to obtain the score. The results were recorded in a spreadsheet and subsequently imported into SPSS [34] for analysis. The number of broken links, page size, presence of meta tags, and presence of persistent cookies were obtained from WebXact Watchfire Page Analysis [35]. (Meta tags are HTML [hypertext markup language] tags that provide information about the content of a Web page for indexing by search engines but do not affect how a Web page is displayed by a browser.) Link density was obtained by using a reverse-lookup query (link:siteURL, where siteURL is replaced by the Web site's URL) in Google. The link density of a site is the number of external sites that have a link to that site. A site with a higher link density is generally more likely to be found by visitors because they may find it through the external sites.\nStatistical Analysis\nCorrelations between position ranking and the Web-site characteristics were calculated using the Kendall rank correlation. The value of the coefficient (tau) ranges from -1 to 1. A value of zero indicates no correlation, values near 1 indicate a strong direct correlation, and values near -1 indicate a strong inverse correlation. Interobserver reliability between the 2 raters was calculated using Kappa statistics on all variables except readability, link density, and those returned by WebXact Watchfire Page Analysis. We regarded P\u00a3 .05 as statistically significant.\nResults\nOf the top 30 sites identified by the 4 search sites using the search terms \"teen quit smoking,\" only 14 were relevant to teenagers who are seeking information on smoking cessation. We also evaluated the search results from Google by using other similar search terms. The number of relevant sites ranged from 5 to 17 (Table 3). Although we used only 1 search site to illustrate the effect of search terms on the type of Web sites found, the result should be similar at other search sites.\nCharacteristics of the 14 Relevant Web Sites\nThe characteristics of the 14 sites are summarized in 3 categories (Table 2).\nEssential-Characteristic Category\nThe essential-characteristic category contains those characteristics that contribute to user dissatisfaction if absent or inadequately provided. The presence of a privacy statement and disclaimer, although it appears not to be required for the functioning of a Web site, wasreported to be essential in a Web-user interface study [36].\nThe correlation between the 2 raters ranged from 1.00 for 2 characteristics (presence of phone number or mailing address and presence of material in video or audio format) to 0.19 for indication of sponsorship. The median correlation was 0.69 for the 15 characteristics evaluated by both raters.\nIn the essential category, 8 sites (57%) contained a site-search feature and 11 sites (79%) contained links for navigation in the site. However, 2 sites contained neither of the features. Over half of the sites contained either a privacy statement (57%) or a disclaimer (64%) but only a third of the sites contained both. About one-third of the sites have readability below eighth-grade school level and they ranked significantly higher (tau = -0.39, P= .05) than those that have readability above or equal to that level. The median grade level was 8.5. Half the sites contained one or more broken internal or external hyperlinks.\nEnhancement-Characteristic Category\nIn the enhancement-characteristic category, 11 sites (79%) indicated their sponsorship. Apparently because most of the sites were sponsored by organizations, government bodies, or educational institutions, only 4 sites (29%) had either pop-up advertisements or in-page banner advertisements. E-mail address (71%) was the most-common contact information available while phone number or mailing address was present in 29% of the sites. Sites that ranked higher were significantly associated with the presence of e-mail address for contact (tau = -0.46, P= .01). Eleven sites (79%) had information on behavioral approach as a method of smoking cessation. Ten sites (71%) had information on a medication (nicotine replacement) approach, and 5 sites (36%) had information on alternative approaches such as acupuncture, hypnosis, laser therapy, and herbal cigarettes. Both the presence of medication (tau = -0.43, P= .02) and alternative approaches (tau = -0.42, P= .02) were significantly associated with a higher search ranking. Five sites provided annotated hyperlinks to external sites and their presence was significantly associated with a higher search ranking (tau = -0.39, P= .04). Eight sites contained interactive components such as quizzes, games, or bulletin boards. Only 1 site provided material in video or audio format.\nTable 3\nType of Web sites found with different search terms using Google search site\nType of Web Site\nSearch Terms Used\nteen quit smoking\nteen stop smoking\nteen smoking cessation\nyouth quit smoking\nadolescent quit smoking\nSite with information to help teenagers quit smoking\n14\n5\n5\n17\n5\nPage with hyperlinks to Web sites with information to help teenagers quit smoking\n3\n1\n5\n3\n4\nNews or press release\n4\n5\n3\n3\n5\nReport of study results or proceedings from conferences\n1\n2\n5\n2\n9\nRecruitment of study subjects\n2\n1\n1\n0\n0\nCommercial site\n3 (2 were redirects*)\n4 (1 was a redirect)\n0\n0\n2 (both redirects)\nSite for teenagers but not on smoking\n1\n2\n0\n0\n0\nResources on teenager smoking cessation for parents or health professionals\n1\n4\n5\n3\n2\nHealth organizations or community centers\n0\n1\n3\n1\n1\nPage not found\n1\n4\n2\n1\n0\nOther\n0\n1 (alt.support.stop-smoking Usenet archive)\n1 (porno-graphic Web site)\n0\n2 (mental health Web site)\n* The visitor was automatically sent to a page other than the page listed in the search results (see Discussion for details).\nTechnical-Characteristic Category\nIn the technical-characteristic category, the largest file size of the landing page (the page reached when clicking on the search-site result) was 134 kilobytes, which is equivalent to approximately 19 seconds of download time on a 56 Kbps modem. Sites that were equal to or larger than 35 kilobytes (57%) were ranked significantly higher (tau = -0.39, P= .04) by the search sites. Eight (57%) and 11 (79%) of the sites had meta description and meta keywords tags, respectively. The presence of a meta description tag was significantly associated with a higher search rank (tau = -0.48, P= .002). Although 5 sites used cookies (small files sent to the browser along with a Web page for tracking a visit), only 3 of them used a persistent cookie that is stored on the user's hard disk and 4 used a session cookie that is automatically deleted from the browser's cache when the browseris closed. Six (43%) sites were just part of larger Web sites containing information other than smoking. The median link density of the 14 Web pages was 6 and the maximum was 735. A higher link density was significantly associated with a higher search rank (tau = -0.58, P= .02).\nDiscussion\nThe key finding of this study was that using simple search terms on popular search sites to look for information on smoking cessation for teenagers, less than half (14 of 30) of the sites found were of direct relevance. The remaining sites were study reports, news, and hyperlinks.\nWe did not include all information retrieved from Web searches, as has been done in studies on other topics [37], since users tend not to go beyond the first few pages of search results [9,10]. Instead, we evaluated only the top 30 search results to mimic typical Web search behavior.\nSearching with the terms \"teen quit smoking\" on 7 popular search sites, Edwards et al [38] also reported that only 40% of the 140 potential hits were focused on cessation. In our study, 1 site of pornographic nature was found when using the search terms \"teen smoking cessation\" but no such sites were found when using the search terms \"teen quit smoking\" in contrast to a previous report [39] where 7 out of the top 20 sites were teen pornography sites.\nOf public health concern was the finding that 3 sites were commercial sites and 2 of them were linked back to a single online drug store using a page-redirect spamming technique. With page redirection, an optimized page with unique and specific terms is submitted to search sites with the single purpose of ranking high on a specific topic. However, anyone clicking the link to this page is automatically sent to a real destination page, which often contains material unrelated to the initial search terms. For example, one site used \"what-happens-to-your-body-when-you-quit-smoking.htm\" as the name of its Web page. However, this page contains no information on smoking cessation. Instead, it is a page with a JavaScript that immediately redirects visitors to an online drug store.\nSeveral important associations were found between Web-site characteristics and position ranking in the top 30 search results. These results can be used for optimizing site development in future smoking-cessation Web sites.\nEssential-Characteristic Category\nAs an example of how these results can be used, of the 6 items in the essential-characteristic category, readability (lower grade level) was associated with higher position ranking. The lack of search box, navigational menu, privacy statement, or disclaimer, or the presence of broken links, was not uncommon, but their absence was not associated with lower position ranking.\nEnhancement-Characteristic Category\nIn the enhancement-characteristic category, presence of contact e-mail address, medication-cessation information, alternative-approach information, and annotated external links were associated with higher position ranking. It is surprising to find that only 1 site displayed a HONcode insignia which, along with the associated membership, is an indication that a site complies with an 8-point code of conduct put forth by Health on the Net [18]. Although 73% of young people said that knowing who produced health information is very important to them, only 29% of those who looked up health information online checked the source the last time they conducted a search [5] and it is likely that fewer will check for the authenticity (for example, verify the membership status of a site at the HON Web site) of any indications of external recognition even if they are present [8].\nTechnical-Characteristic Category\nIn the technical-characteristic category, page size that was larger than 35 kilobytes, presence of a meta description tag, and a high link density were associated with higher ranking. The strong association between site description meta tag and ranking (tau = -0.48, P= .002) suggests that such information is relevant to the ranking algorithms of the search-engines used. Including a concise description tag is likely to be more effective in improving search-engine visibility than just a comprehensive keywords list. In fact, due to high rate of keyword repetition and spam, search sites such as Google and AltaVista do not give consideration to the keywords meta tag in their ranking [40,41]. As expected, link density is strongly associated with ranking (tau = -0.58, P= .02). Search engines generally use the number of incoming links (link density) in their ranking algorithm. However, Google's PageRank algorithm also takes into account the number of outgoing links on the page of each of the incoming links [42].Therefore, to achieve a high ranking a Web site should try to get listed on as many sites as possible and, in particular, on those sites that have as few external links as possible. Since search engines assign higher ranking to sites with incoming links that originate from pages containing fewer external links, and sites with annotated external links tend to have fewer links than those sites without annotated external links, this may explain the association between the presence of annotated external links and higher ranking (tau = -0.39, P= .04).\nTo improve search efficiency, users may want to supplement results from search sites with those from subject-based Web directories that are created and maintained by people, rather than by algorithms, such as Yahoo! Directory, which has a teen-smoking section [43]. Using the Yahoo! directory, we found 25 sites listed, of which only 4 were found using our search terms at the 4 popular search sites. In addition, users may want to learn and apply the specific syntax of their favorite search sites when searching for information. For example, quit-smoking Web sites of the commercial (.com) domain can be eliminated from the search results by entering \"quit smoking -site:.com\" in the search box in Google.","keyphrases":["teens","teenagers","internet","smoking cessation","search engines","web page analysis"],"prmu":["P","P","P","P","P","R"]}
{"id":"Doc_Ophthalmol-3-1-1820752","title":"ISCEV Standard for Clinical Electro-oculography (EOG) 2006\n","text":"The Clinical Electro-oculogram (EOG) is an electrophysiological test of function of the outer retina and retinal pigment epithelium (RPE) in which the change in the electrical potential between the cornea and the ocular fundus is recorded during successive periods of dark and light adaptation. This document sets out a Standard Method for performance of the test, and also gives detailed guidance on technical and practical issues, and on reporting test results. The main object of the Standard is to promote consistent quality of testing and reporting within and between centres. This 2006 Standard, from the International Society for Clinical Electrophysiology of Vision (ISCEV: www.iscev.org ), is a revision of the previous Standard published in 1993, and reviewed and re-issued in 1998.\nPurpose and use of this Standard\nThis Standard is one of a series of Standards and Guidelines for electrophysiology of vision [1\u20138] available for download from www.iscev.org, and is a revision of the ISCEV Standard for Clinical Electro-oculography (first issued 1993 \u2013 re-approved 1998) [1, 2], which provides a Standard Method for measurement of the electro-oculogram (EOG). Also included is advice on possible variations on the Standard Method, additional tests, and comment on the procedures recommended. The major change in this current standard compared with the earlier EOG standards is that it recommends one single standard testing protocol and measurement strategy.\nClinical and research users of the clinical EOG are encouraged to use the current Standard Method where possible, to achieve consistency of results within and between test centres. Reports of EOG recordings performed to the Standard Method given here should cite this 2006 Standard. Where a method is used which deviates from the Standard Method, the deviations should be stated, together with any normative or reference data. Where the method used conforms to a previous EOG Standard, this may be cited instead.\nEOG origins, pathological effects, and principles of measurement\nElectrophysiology of the RPE in dark and light adaptation\nThe eye has a standing electrical potential between front and back, sometimes called the corneo-fundal potential. The potential is mainly derived from the retinal pigment epithelium (RPE), and it changes in response to retinal illumination. The potential decreases for 8\u201310\u00a0min in darkness. Subsequent retinal illumination causes an initial fall in the standing potential over 60\u201375\u00a0s (the fast oscillation (FO)), followed by a slow rise for 7\u201314\u00a0min (the light response). These phenomena arise from ion permeability changes across the basal RPE membrane. The clinical electro-oculogram (EOG) makes an indirect measurement of the minimum amplitude of the standing potential in the dark and then again at its peak after the light rise. This is usually expressed as a ratio of \u2018light peak to dark trough\u2019 and referred to as the Arden ratio. The behaviour of the corneo-fundal potential in the normal eye is predictable in defined conditions, such as those described in this Standard, but changing from dark to light actually initiates a triggered response extending for about 2\u00a0h in the form of a diminishing sinusoidal oscillation.\nDiseases affecting the light response of the EOG\nThe light response is affected in diffuse disorders of the RPE and the photoreceptor layer of the retina including some characterised by rod dysfunction, or chorio-retinal atrophic and inflammatory diseases. In most of these there is correlation with the electroretinogram (ERG), except notably in the case of Best\u2019s vitelliform maculopathy, in which the clinical EOG is usually highly abnormal in the presence of a normal ERG.\nMeasurement of the clinical EOG\nThe potential across the RPE causes the front of the eye to be electrically positive compared to the back. As a result, potentials measured between two electrodes placed on the skin at each side of an eye will change as the eye turns from left to right. The EOG method is used widely to record eye movements, on the assumption of unchanging corneo-fundal potentials. In the clinical EOG described here, we use defined eye movements to monitor the changes in corneo-fundal potential. If the test subject looks alternately at targets a fixed angle apart, the potential recorded from the skin resembles a square wave whose amplitude will be a fixed proportion of the corneo-fundal potential. During a light\/dark cycle, this indirectly measured potential will change in the same way as the source potentials, so that the Arden ratios (and timing of peaks etc.) will be a close approximation to the average changes occurring across the RPE.\nThe Standard Method\nThis section outlines the Standard Clinical EOG method, definitions, explanations, and instrument specifications. Further notes regarding testing strategies are given in later sections.\nPupils Apply dilating drops before any other action to allow pupil dilation to be relatively complete by the start of the test (see notes below). Their size should be checked at the start and end of the test and recorded at the end of the test.\nElectrodes After suitable skin preparation, place small recording electrodes, close to the canthi of each eye as in Fig.\u00a01. Connect the electrodes from each eye to separate channels of a differential amplifier. The \u2018ground\u2019 electrode can be placed on the forehead. The impedance between any pair of electrodes should not exceed 5\u00a0k\u03a9. The electrodes, amplifier and impedance meter must be approved for medical use.\nFig.\u00a01(a and b). Recording electrode positions\nAmplifier This should have a band pass of either 0 (d.c.) to 30\u00a0Hz, or 0.1 to 30\u00a0Hz, to provide recordings of the saccades which appear as square waves.\nFull field (Ganzfeld) stimulator This should have a comfortable head\/chin rest, and two red fixation lights 15 degrees left and right of centre. The fixation lights should be bright when the light adapting background is on, and as dim as practical in the dark.\nPre-adaptation The test subject should be in stable indoor lighting for as long as possible before the test, and should not be exposed to any large changes in lighting (lighter\/darker) during this period, such as indirect ophthalmoscopy. As near as is practical, the pre-test light exposure should be the same for all test subjects.\nPreparing the test subject Explain the procedure including, \u2018chin\/head on rest\/restraint in stimulator, 15\u00a0min dark, 15\u00a0min light, fixation lights alternate in simple rhythmic manner, for 10\u00a0s each minute, when lights change, move eyes in single sweep to next one, do not turn head, do not anticipate the changes\u2019. Practice the procedure with the recording system on and coach the subject if there is head movement, overshoot, stepping, or anticipation.\nDark phase In total darkness for 15\u00a0min, except for the dim fixation lights, alternate the fixation lights every 1\u00a0s for 10\u00a0s every 1\u00a0min, and record the resulting EOG potentials. The test subject should remain looking into the stimulator the whole time if possible, and should be warned of the start of each measurement sequence to ensure attention. The operator must have an instant view of the recordings to check for patient compliance, and errors such as overshoot.\nLight phase Bring on the ganzfeld background light of 100 photopic cd\/m2 . If necessary, bring this on gradually over a short period (e.g. 20\u00a0s) for patient comfort, especially in cases of photophobia. Continue recording every 1\u00a0min as above. Keep the test subject forward in the stimulator bowl for the whole time and with eyes open.\nThis completes the procedure for the test subject.\nMeasure the amplitude of the EOG Taking care to remove the effects of overshoot (see Fig.\u00a02 and notes below) and stepped saccades, measure the EOG amplitude in \u03bcV, either manually or by a computer algorithm. Calculate the average of the amplitudes in each 10\u00a0s trial.\nFig.\u00a02Idealised saccadic recording with d.c. amplifier (top) and example a.c. coupled amplifier with high pass filter at 0.5\u00a0Hz and 0.1\u00a0Hz. Overshoot is hard to recognise using 0.5\u00a0Hz\nPlot the change in the responses Plot the average amplitude of the EOG in \u03bcV on a graph representing the 30\u00a0min of the test. Ignore any points known to be incorrect (e.g. subject did not follow the fixation lights properly), and identify the underlying curve (see notes) as in Fig.\u00a03 (lower panel).\nFig.\u00a03Idealised (underlying) EOG response (top) and practical response with noise and trial\/trial variability. Arrows show the dark trough (DT) and light peak (LP). The underlying curve must be estimated before recording the Arden ratio (LP\/DT)\nCalculate the Arden ratio This is the ratio between the peak and trough of the underlying curve, not the maximum and minimum recorded values.\nReport As a basic factual report state the Arden ratio, the first (dark) trough amplitude in \u03bcV, the time from start of the light phase to the peak (if present), the pupil size, and the type of adapting light source. The report should also state any difficulties which were encountered during the test session which may affect confidence in the results, such as the test subject\u2019s difficulty in performing the test.\nDeviation from the Standard\nThis Standard represents a basic or core procedure. If a laboratory chooses a procedure which varies from the Standard Method above, it is critical to cite this Standard, but specify the deviations from the Standard Method, such as different luminance level for the adapting light. If a statistical report is given, then this must be supported by reference data obtained under the same conditions.\nAdditional tests\nSome centres measure the \u2018Fast Oscillation\u2019 (FO), often in conjunction with the Clinical EOG.\nThe fast oscillations:\nThe fast oscillations (FOs) have the opposite polarity to the light rise of the Standard Clinical EOG. At light onset there is a decrease in the standing potential that recovers 30\u201340\u00a0s after light onset. This decrease, the \u2018light-trough\u2019 (LT), is caused by a decrease in intracellular Cl- that results in a decrease of Cl- transport across the basolateral membrane resulting in a hyperpolarisation of the RPE basal membrane.\nThe FO is recorded using the same parameters as for the clinical EOG (amplification, electrode placement and stimulus light intensity). However, recordings should be made continuously as the subject executes regular horizontal saccades at 1\/s. Alternating light and dark for 60\u00a0s each induces the FOs, which have a near sinusoidal appearance. During the light interval a light trough (LT) develops and begins to rise again after 30\u201340\u00a0s. The subsequent interval of darkness results in a dark rise (DR) at 30\u201340\u00a0s following the onset of darkness. The next interval of light induces another light trough. The total number of light-dark intervals should be at least 4 with 60\u00a0s periods of light and dark, making a total test time of 8\u00a0min. Pre-adaptation does not affect the FO, and so this test can be performed either independently or in conjunction with the clinical EOG, provided pre-adaptation conditions for the latter are consistent within the lab.\nFigure\u00a04 shows a schematic idealised representation of the FO cycle, from which the ratio of DP:LT should be reported. The normal amplitude ratio is typically between 1.05 and 1.30. However, at present each laboratory should obtain and use their own reference data. It should be noted that high blood glucose levels increase the FO amplitudes.\nFig.\u00a04Idealised representation of fast oscillations (FO). In the dark intervals (black bars) the standing potential increases to a dark rise maximum (DR). Following light onset the standing potential falls to a light trough (LT). The FO ratio of the DR:LT standing potentials should be recorded\nPractical notes, instruments and definitions (alphabetical)\nAmplifiers For a 30 degree saccade, the typical EOG amplitudes are between 250 and 1000\u00a0\u03bcV with essential frequency content of 0 to 30\u00a0Hz. With such signal amplitudes, no special shielding nor interference protection should be necessary. Medical grade amplifiers intended for human physiological recording are required to be \u2018electrically isolated\u2019 to prevent any risk of electric shock to the patient via the recording electrodes. Laboratory grade amplifiers may not have this protection. To obtain recordings of saccadic movements as a \u2018square wave\u2019 (the ideal) requires a d.c. amplifier (i.e. no high pass filter) but there are operational penalties in terms of base line shift due to electrode polarisation and movement, which can be partially eliminated by the use a high pass filter (e.g. 0.1\u00a0Hz). If a higher frequency is used (e.g. 0.5\u00a0Hz) there is considerable \u2018droop\u2019 of the square wave, making identification of overshoot and stepped saccades more difficult (see Fig.\u00a02). For the low pass filter, 30\u00a0Hz is sufficient. Including higher frequencies adds noise to the response (e.g. EMG) and since most automatic cursor placement regimes find the lowest and highest parts of the wave, this exaggerates the recorded amplitudes. Since both the peak and the trough amplitudes will be equally exaggerated the Arden ratio will be underestimated. For guidance on verifying amplifier performance, see reference [3].\nAmplifier saturation EOG potentials measured during saccadic eye movements as in the Standard Clinical Method can vary by about 5:1 in amplitude between subjects, which, with the light rise, may mean a total amplitude range of up to 15:1. Thus, the operator must be able to see the recordings of the saccades to ensure saturation does not occur, and to adjust the amplifier gain settings accordingly.\nArden ratio Using the Standard clinical EOG method described here, the Arden ratio is the peak EOG amplitude occurring in the light phase, divided by the minimum amplitude during the dark phase. The peak and trough values must be measured from the \u2018underlying curve\u2019 as shown on Fig.\u00a03, not from individual measurements.\nCompliance of the patient Test subjects will have difficulty performing the saccadic movements if they do not have good central vision, or suffer from diplopia or dysconjugate eye movements, have nystagmus, are very young, or are infirm and are unable to take up the physical positions required for the duration of the tests. Some patients also suffer claustrophobia or fear of the dark, and so the testing must be performed in such a way as to minimise these fears. Real-time view of the EOG potentials, and infrared camera view of the patient\u2019s eyes, are very useful in ensuring full compliance. In most cases, coaching under observation can remedy poor co-operation. A common issue is the patient closing eyes during the light phase, which can be seen by a camera.\nThe cost of unreliable performance is that the margins of error are widened. This does not mean that no information can be gleaned. If the suspected disorder is one with a clear test outcome, it may be possible to make the diagnosis on one or two reliable trials performed near the time of the trough and the peak, to ensure that there is indeed a light rise of some amplitude, or not.\nDiplopia Test subjects often find the fixation lights too near for them to achieve convergence. The subject may be advised to look between the pair of images in cases of intractable diplopia. Subjects with ocular motility problems are likely to give unreliable results. If the suspected retinal disorder is bilateral, it is acceptable to patch one eye to perform the tests (see the notes on interaction between eyes below).\nElectrodes Recording the EOG is relatively undemanding as regards the electrodes. These should be relatively non-polarisable such as standard medical EEG or ECG electrodes, of a size appropriate for attachment to the side of the nose. If baseline drift is excessive, less polarisable electrodes may be necessary.\nFixation targets These should be small, red, and adjustable in brightness so that they can be seen clearly by all subjects above the background illumination, and to be dim (just visible to the test subject) in darkness.\nFull field (Ganzfeld) stimulator This should be as large as practicable to allow adequate distance from eye to fixation lights. It should have a chin rest and forehead bar to ensure stable head position, and it should have fixation lights 15 degree each side of centre. The background intensity should be stable and \u2018visibly white\u2019 (see notes on Light below).\nInteraction between eyes The EOG potentials from one eye will contaminate the response from the other. This is approximately 15% with electrodes placed on each side of the nose close to the inner canthi, rising to about 40% as they come close together and then touch (e.g. becoming a common central electrode placed on the bridge of the nose). This can give misleading results in cases of an electrically inactive (e.g. total retinal detachment), or absent eye, in which case the defective eye appears to have the same Arden ratio as for the fellow eye, albeit on a much smaller apparent standing potential. More confusing is when the eyes have similar standing potentials but different Arden ratios. In these cases, the measured Arden ratio from the better eye is enhanced at the expense of that from the weaker eye, which can in fact appear to have an Arden ratio of less than 1.0, solely due to interaction.\nLight\nLuminance For the light phase the luminance of the full field adapting light should be 100 photopic cd\/m2 as measured with a photometer (photopic filter) with a traceable calibration (see also notes on pupil dilation below). The calibration of the ganzfeld stimulator should be carried out periodically, e.g. once a year, and corrective action applied (see ref [3] for guidance). Modest room lighting may be turned on during the light phase when the impact on the effective luminance in the bowl should be negligible with the test subject looking into the bowl.\nDarkness The dark phase should take place in total darkness, and the fixation lights should be dimmed to the minimum necessary to enable fixation.\nColour There are several possible sources of adapting light such as tungsten, halogen, LED, and fluorescent. For a commercial recording system, the type will be stated in the manufacturer\u2019s literature. Spectrally these are quite different, and the spectra for tungsten and halogen also change with brightness. This issue makes the definition of a single Standard Method for recording the clinical EOG difficult at present, and therefore all reports should state the type of light source used. If the user has additional data, such as colour temperature, or scotopic luminance, then this should be stated.\nNormative data\/reference range At present there is no standard international reference range for the clinical EOG. With the introduction of this Standard (The Standard Clinical EOG Method 2006) there is a greater possibility of developing and comparing normative data (see notes on reporting).\nPhotophobia The EOG \u2018light rise\u2019 is a \u2018triggered\u2019 response, and is assumed to start from the onset of the light phase. Some test subjects will find the light too bright and it may be necessary to bring the adapting light up gradually over, say, a 20-second period. If so, this variation should be stated. If the adapting light is brought on very slowly (e.g. minutes), the response is changed.\nPlotting The average EOG amplitude calculated from each 10-second trial should be plotted (see Fig.\u00a03). The first critical step is that the underlying physiologic curve is recognised and drawn for derivation of the trough and peak. This may require a curve fitting algorithm or a traditional \u2018flexicurve\u2019 (curve fitting ruler) on paper. Only then should the Arden ratio be calculated. It is helpful if any uncertain values have been identified and marked at the time of recording, so that they can be ignored when identifying the underlying curve.\nPupil dilation Having dilated pupils means less variability in the light entering the eye. There may be occasions when full pupil dilation is impossible or undesirable. If pupils are not artificially dilated, then the report should state this. An attempt should be made to adjust the adapting luminance to accommodate smaller or variable pupils, and this luminance should be quoted (it is usual to try to achieve the same value in Trolands, as for dilated pupils). In many cases useful diagnostic information can be obtained without dilated pupils, especially with extreme results (no light rise, strong light rise). Intermediate results for the Arden ratio will obviously be of less value with undilated and therefore variable pupil sizes.\nReporting\nBasic factual report This should include the Arden ratio, the first (dark) trough amplitude in \u03bcV, the time from the start of the light phase to the light peak (if present), the pupil size at the end of the test, and the type of adapting light source.\nStatistical report This should show how the calculated Arden ratio relates to a quoted reference range. If the reference range was obtained with any different test parameters, this should be explained.\nNo fully authenticated normal reference data is available currently for the Standard Clinical EOG method given above. However, from a review of existing published data, Arden ratios <1.5 are reported as being abnormally low, and those >2.0 are reported as probably normal, and in between as borderline. This guidance may be useful in the absence of local normative data, but the values do not constitute validated diagnostic criteria.\nSaccade measurement Use a scale or electronic calliper to measure the change in EOG potential resulting from each saccade, and calculate an average for each 10 second trial. The average should include only those measurements judged to be reliable. Alternative measurement methods may be used, including computer algorithms.\nIf a computer algorithm is used there is a need to ensure that the values returned properly represent the true EOG amplitudes. Each algorithm is likely to make some mistakes. Fortunately, the final dark\/light response curve will form an \u2018average of averages\u2019 in which the influence of a few errors should be swamped by the remaining data.\nParticular causes of unreliability are overshoot (see Fig.\u00a02), stepped saccades, missing saccades, inverse saccades (eyes go opposite way to fixation lights), and eccentric fixation in which the saccade length switches between two or more values. When measuring manually, these difficulties are fairly obvious.\nStanding potentials This Standard recommends the reporting of the minimum standing potential (dark trough minimum), taken from the underlying response curve, not the minimum recorded value. This value is not often used in diagnosis at present, but if the value is abnormally low it may indicate an inactive retina (e.g. total retinal detachment), and the calculated Arden ratio may be unreliable because of the low value of the divisor in the ratio. Normal values for the minimum standing potential should be established, but as a guide, minimum standing potentials less than 150\u00a0\u03bcV should be treated as low, and more likely to produce unreliable Arden ratios.\nWarning of start of each trial There should be a warning, verbal or automatic, of the impending start of each trial, to ensure readiness of both test subject and operator. Some users have sounds (beeps) to co-incide with the alternations of the fixation lights. However, it may be better to rely solely on the visual stimuli, to avoid the possibility of alternation without actual fixation.\nHistory and acknowledgements\nThis Standard forms part of series of Standards, Recommendations and Guidelines prepared by the International Society for Clinical Electrophysiology of Vision (ISCEV):\nParticipants in the review: ISCEV EOG Standard Revision Committee 2006; Malcolm Brown (Chairman), Royal Liverpool University Hospital, Liverpool UK\nMichael Marmor, Stanford University, California USA; Vaegan (ISCEV Board Member-at-Large) University of New South Wales, Australia, Eberhart Zrenner University Eye Hospital, Tuebingen, Germany\nEOG Standard Advisory Panel 2006 includes the members above plus: Michael Bach, (ISCEV President), Universit\u00e4t-Augenklinik, Freiburg, Germany: Mitchell Brigell, (ISCEV Director of Standard), Pfizer Global. R&D, Ann Arbor, Michigan USA: Graham Holder, (ISCEV Director of Education), Moorfields Eye Hospital, London UK\nPaul Constable, City University, London UK: Masao Yoshikawa, Mayo Corporation, Aichi, Japan; Carol Westall, Hospital for Sick Children, Toronto Canada; Geoffrey Arden, (Honorary ISCEV member) City University, London UK","keyphrases":["electro-oculogram","retinal pigment epithelium","light adaptation","arden ratio","clinical standards"],"prmu":["P","P","P","P","R"]}
{"id":"J_Membr_Biol-3-1-1784061","title":"Pharmacology and Surface Electrostatics of the K Channel Outer Pore Vestibule\n","text":"In spite of a generally well-conserved outer vestibule and pore structure, there is considerable diversity in the pharmacology of K channels. We have investigated the role of specific outer vestibule charged residues in the pharmacology of K channels using tetraethylammonium (TEA) and a trivalent TEA analog, gallamine. Similar to Shaker K channels, gallamine block of Kv3.1 channels was more sensitive to solution ionic strength than was TEA block, a result consistent with a contribution from an electrostatic potential near the blocking site. In contrast, TEA block of another type of K channel (Kv2.1) was insensitive to solution ionic strength and these channels were resistant to block by gallamine. Neutralizing either of two lysine residues in the outer vestibule of these Kv2.1 channels conferred ionic strength sensitivity to TEA block. Kv2.1 channels with both lysines neutralized were sensitive to block by gallamine, and the ionic strength dependence of this block was greater than that for TEA. These results demonstrate that Kv3.1 (like Shaker) channels contain negatively charged residues in the outer vestibule of the pore that influence quaternary ammonium pharmacology. The presence of specific lysine residues in wild-type Kv2.1 channels produces an outer vestibule with little or no net charge, with important consequences for quaternary ammonium block. Neutralizing these key lysines results in a negatively charged vestibule with pharmacological properties approaching those of other types of K channels.\nIntroduction\nMore than 80 mammalian genes code for K channel protein subunits, so it is not surprising that the physiological roles of K channels include such diverse actions as controlling action potential duration, action potential firing patterns and salt and water movement (Hille, 2001). Given the important, complex physiological processes controlled by K channels, it is not surprising that considerable effort has been expended on understanding their pharmacological properties (Tamargo et al., 2004; Yost, 1999). K channels are blocked by many chemical agents, including many types of complex peptides, especially peptide toxins, and by simple amine compounds, including the quaternary amine tetraethyl ammonium (TEA).\nThe study of the pharmacology of K channels has been greatly aided by mutational analysis of cloned K channels and the solution of the crystal structures of several bacterial K channels (Doyle et al., 1998; Jiang et al., 2002, 2003) and a mammalian voltage-gated flavor (Long, Campbell & MacKinnon, 2005). This work has revealed a remarkable conservation of the general structure of the outer vestibule and selectivity filter of K channels. In spite of this structural conservation, there are considerable differences in the pharmacological properties of K channels.\nCompared to the peptide toxins and other agents that inhibit K channels, the simple quaternary amine TEA ought to be the easiest to understand. Early mutational analysis revealed a \u201chot spot\u201d for external TEA block: the affinity for this compound changes over more than two orders of magnitude depending on the amino acid at this position (Kavanaugh et al., 1991; MacKinnon & Yellen, 1990). Additional studies revealed that an aromatic amino acid side chain at this hot spot on all four subunits of the homotetrameric channels contributes to high-affinity TEA binding (Heginbotham & MacKinnon, 1992; Kavanaugh et al., 1992). This amino acid (position 449 of the prototypical voltage-gated K channel, Shaker) is located just outside the entrance to the pore selectivity filter, and all the data appeared consistent with the idea that TEA was stabilized in its blocking site through cation \u03c0-electron interaction with this aromatic side chain.\nMore recent studies, however, have suggested that the mechanism of TEA and other quaternary ion block of K channels is more complex. Analysis of the structure-activity relations for K channel block of quaternary amines revealed contributions from hydrophobic interactions as well as a role for dehydration of the blocking ions (Jarolimek et al., 1995, 1996). In addition, amino acids other than that at position 449 are involved in TEA binding (Pascual et al., 1995). A molecular dynamics study (Crouzy, Berneche & Roux, 2001) showed that K channel proteins do not have the three-dimensional geometry necessary for TEA: \u03c0-electron interaction. Finally, studies of the ability of TEA to alter the chemical modification of introduced cysteine residues indicate that TEA likely blocks at a location more external than position 449 (Andalib et al., 2004).\nOne factor that has received little attention in the study of the quaternary ammonium block in K channels is the involvement of electrostatic charges on the channel surface, which can certainly affect the interaction of charged blockers like quaternary amines. Indeed, TEA block of frog node K channels is increased in low-ionic strength solutions or in solutions with reduced Ca2+ concentration (Mozhayeva & Naumov, 1972). TEA block of a Ca2+-activated K channel is sensitive to solution ionic strength, and a chemically induced neutralization of negative surface charges reduces this ionic strength dependence (MacKinnon, Latorre & Miller, 1989). The pH-dependent TEA block of Kv1.1 channels is sensitive to changes in bulk solution ionic strength (Bretschneider et al., 1999). Finally, we have shown that TEA block of Shaker K channels is sensitive to bulk solution ionic strength and that block by a trivalent TEA analog (gallamine) is even more sensitive (Quinn & Begenisich, 2001). The results of these various studies suggest a role for surface electrostatics in quaternary ammonium block of K channels.\nKv2.1 channels are rather unique among K channels in that they have a relatively low affinity for TEA in spite of possessing an aromatic amino acid at the TEA hot spot that confers high-affinity block to other K channels. They are unique also in having two lysine residues in the outer vestibule that are not conserved in other K channels. These lysine residues have a powerful effect on TEA block of these channels (Immke & Korn, 2000; Immke et al., 1999). We have investigated the mechanism by which the positively charged side chains of these lysines affect TEA block. We tested the ionic strength dependence of block by TEA and the trivalent TEA analog gallamine as a way to probe for surface electrostatic effects. Kv3.1 channels have a high affinity for TEA and do not contain the Kv2.1 lysine residues noted above (Grissmer et al., 1994; Taglialatela et al., 1991). We found that, like Shaker K channels, quaternary amine block of Kv3.1 channels was ionic strength-dependent but block of wild-type Kv2.1 channels with the lysine residues was not. We also found that Kv2.1 channels were insensitive to gallamine. Neutralization of one or the other of the nonconserved lysine residues in Kv2.1 channels conferred ionic strength sensitivity to TEA block of Kv2.1 channels. Neutralization of both lysine side chains produced channels that were blocked by gallamine, and this block was more sensitive to solution ionic strength than block by TEA. Thus, surface electrostatics appears to play a role in TEA block of some, but not all, K channels depending on the location of some particular charged residues.\nMaterials and Methods\nK CHANNEL CONSTRUCTS\nSeveral K channel constructs were used in this study, including wild-type Kv2.1 and Kv3.1 channels (Chan Test Inc., Cleveland, OH). Three mutant Kv2.1 channels were also investigated: K356F, K382Q and the double mutant K356F\/K382Q. The replacement amino acids were introduced into the Kv2.1 clone using a two-step polymerase chain reaction (PCR) protocol and the resulting mutants analyzed by DNA sequencing.\nOOCYTE ISOLATION AND MICROINJECTION\nXenopus laevis oocytes were maintained as described by Goldin (1992). Isolated ovarian lobes were rinsed with Ca2+-free OR-2 solution (in mM): 82.5 NaCl, 2.5 KCl, 1\u00a0MgCl2 and 5 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; pH 7.6, NaOH) and then defolliculated by incubation for 60\u201390 min with 2 mg\/ml collagenase type 1A (Sigma, St. Louis, MO). Cleaned oocytes were transferred and maintained for 2 h in ND-96 solution (in mM): 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES and 2.5 Na-pyruvate (pH 7.6, NaOH) before injection of mRNA coding for the channel construct of interest. Injected oocytes were transferred to multiwell tissue culture plates and incubated at 18\u00b0C in ND-96 solution supplemented with 100 U\/ml penicillin and 100\u00a0\u03bcg\/ml streptomycin. Animal (frog) handling and care utilized methods consistent with the National Institutes of Health\u2019s Guide for the Care and Use of Laboratory Animals.\nELECTROPHYSIOLOGICAL RECORDINGS\nK channel currents were recorded 1\u20135 days after mRNA injection. Recordings were made at room temperature (20\u201322\u00b0C) using the cut-open oocyte voltage-clamp apparatus (model CA-1B; Dagan, Minneapolis, MN). Connections to the different compartments were made with glass capillaries containing 75\u00a0\u03bcm platinum wires and filled with a 1 M NaCl, 3% agar solution. The experimental chamber (ELV-1, Dagan) was modified to include a low-volume (80\u00a0\u03bcl) chamber insert. The low chamber volume and high flow rates (\u223c1 ml\/min) produced efficient solution exchange. The measured difference in junction potential for the two experimental solutions (see below) was <2 mV, so no correction to membrane voltages was made.\nRecording electrodes were made either of 1 BBL glass with filament (1.5 mm outer diameter; World Precision Instruments, Sarasota, Fl) or of GC-150F glass (Warner Instrument, Hamden, CT). Electrodes had tip diameters of \u223c2\u00a0\u03bcm and were filled with a 3 M KCl solution. Data acquisition was performed using a 12-bit analog\/digital converter controlled by a personal computer. Current records were filtered at 5 kHz. Series resistance compensation was used: a mean value of 0.74 \u00b1 0.05 (standard error of them mean, SEM) k\u03a9 in normal-ionic strength solutions and 2.2 \u00b1 0.11 k\u03a9 in reduced-ionic strength solutions (n = 50).\nElectrical access to the internal compartment was achieved by 0.125% saponin treatment in a solution of (in mM) 100 KCl and 10 HEPES (pH 7.4, N-methyl-D-glucamine [NMDG]). The external solution was (in mM) 140 NaCl, 2 CaCl2, 10 KCl, 10 HEPES (pH 7.4, NMDG). An external solution of reduced ionic strength was used that consisted of (in mM) 35 NaCl, 0.5 CaCl2, 10 KCl, 10 HEPES (pH 7.4, NMDG). The osmolarity of this solution was matched to that of normal ionic strength by addition of 216 mM glucose. In order to test for possible effects of glucose beyond simply maintaining solution osmolarity, we also used mannitol and sucrose. We found that the ionic strength sensitivity of both TEA and gallamine block was the same regardless of which agent was used for osmotic balance. Osmolarity of all solutions was verified using a vapor pressure osmometer (Wescor, Logan, UT). Addition of TEA-Cl was made by substitution with NaCl, which maintained solution ionic strength. Gallamine-I3 (1,2,3-tris[2-triethylammonium ethoxy]benzene triiodide; Sigma) was added to the external solution with no compensation for changes in solution ionic strength.\nOocytes were clamped at a holding potential of \u221280 mV, and 80-ms test pulses were applied to elicit channel current. Channel block by TEA or gallamine was computed by calculating the steady-state current recorded at the end of each pulse as a fraction of the average current recorded before application and following washout of the blocker. Only those results with at least 90% recovery from block were considered. Concentration-response relationships were constructed by plotting the mean fraction of current blocked at a test potential of +40 mV. These were fit by the standard binding isotherm:\nwhere [B] is the concentration of TEA or gallamine, Bmax is the maximal fraction of current blocked and Kapp is the concentration that blocks half the maximal current.\nWe used the crystal structure of the KcsA K channel (RCSB Protein Data Bank entry 1J95) as a template for the Kv2.1 channel (Doyle et al., 1998). The KcsA residues in the outer vestibule were mutated to their Kv2.1 counterparts with the DeepView\/Swiss-PdbViewer, version 3.7 (http:\/\/www.ca.expasy.org\/spdbv\/). The molecular views of the K channel structures were generated with ViewerLite 4.2 for Windows (Accelrys, San Diego, CA; http:\/\/www.accelrys.com\/). The electrostatic surface view was also generated with ViewerLite using Gasteiger charges.\nResults\nCHARGED RESIDUESINTHE OUTER VESTIBULE OF K CHANNELS\nFigure\u00a01 illustrates two views of the outer vestibule of K channels. Shown are schematics based on the crystal structure of the bacterial K channel KcsA, but all crystallized K channels, including the mammalian voltage-gated K channel Kv1.2, share a common structure for this outer part of the channel (Long et al., 2005). On the left is a view from above the channel, and the right part of the figure illustrates a side view with two of the subunits removed. Shown below the structures is an alignment of the amino acids in the outer vestibule and selectivity filter of Shaker, Kv2.1 and Kv3.1 channels. As noted in the Introduction, an aromatic residue at Shaker position 449 confers high-affinity TEA block. Replacement of the native threonine at position 449 in Shaker channels to a tyrosine renders the channel highly sensitive to TEA (Kavanaugh et al., 1991; MacKinnon & Yellen, 1990). Both Kv2.1 and Kv3.1 channels have a tyrosine at this position (shown in green).\nFigure\u00a01.Sequence and structure of the outer vestibule of K channels. Bottom, Amino acid sequence alignment of three K channels in the region that forms much of the channel pore outer vestibule. In blue are the two lysine residues (K356 and K382) in Kv2.1 channels that are a major focus of this study. Kv2.1 and Kv3.1 channels have a tyrosine amino acid (shown in green) at the position that is important for high-affinity TEA block (see text). In red are several amino acids (aspartate and glutamate) in the vestibule area that have negatively charged side chains. Top left, Structures of gallamine and TEA. Bottom left, Top-view structure of KcsA. The fourfold symmetry of the channel (in ribbon form) is apparent in this view. The Kv2.1 K356 and K382 lysine residues are shown with space-filling atoms (without hydrogens). The tyrosine important for TEA binding is shown with \u201cball and stick\u201d atoms in green. Right, Side view of the channel with two subunits removed for clarity. Apparent are the outer vestibule and narrow selectivity filter. As in the top view, the relevant lysines and tyrosines are shown with space-filling and ball-and-stick atoms, respectively.\nAll three channels contain several negative amino acids in this vestibule region (indicated in red in the alignment). Kv2.1 channels are unique in having two positively charged amino acids (K382 and K356) in the outer vestibule (blue in the alignment and illustrated as space-filling atoms in the structural diagrams). The influence of these lysine residues on TEA block is an important part of the present study.\nIONIC STRENGTH-DEPENDENT BLOCK OF KV3.1 CHANNELS BY TEA+ AND GALLAMINE3+\nAs described above, TEA block of Shaker K channels is sensitive to solution ionic strength, and block by a trivalent TEA analog, gallamine, is even more sensitive (Quinn & Begenisich, 2001). These results demonstrate that negative charges on the surface of the channel contribute to an electrostatic potential that influences block by these charged molecules. As a first step toward identifying the amino acids that could contribute to this type of surface electrostatics, we tested the ionic strength dependence of TEA and gallamine block of two other K channels: Kv3.1 and Kv2.1.\nShown in the upper part of Figure\u00a02A are raw current traces from an oocyte expressing Kv3.1 channels. TEA (50\u00a0\u03bcM) produced a small (near 25%) block of Kv3.1 channels in a normal-ionic strength solution (see Materials and Methods) but almost a 50% block when applied in a solution of low ionic strength. The lower panel illustrates the concentration dependence of current block, measured at +40 mV, at normal and low ionic strength. The lines are fits of equation 1 to the data with a Kapp value of 0.14 mM for TEA block in the normal-ionic strength solution, well within the 0.09\u20130.2 mM range previously reported for TEA block of this K channel (Grissmer et al., 1994; Jarolimek et al., 1995; Taglialatela et al., 1991). TEA was more effective in the low-ionic strength solution with a Kapp value of 0.055 mM, a 2.5-fold increase in apparent TEA affinity in the low-ionic strength solution.\nFigure\u00a02.Ionic strength-dependent TEA and gallamine block of Kv3.1 channels. (A) Top, Raw currents as indicated in response to step depolarizations of 80 ms to \u221220, 0, +20 and +40 mV from a \u201380-mV holding voltage. Calibration, 1\u00a0\u03bcA, 20 ms. Bottom, Dose-response relation for TEA block of current at +40 mV in normal- (\u25a0) and low- (\u25cb) ionic strength solutions. Mean values from three to five measurements and SEM limits (if larger than symbol) are plotted. Lines are fits of equation 1 to the data with Kapp values of 0.14 and 0.055 mM for normal- and low-ionic strength conditions, respectively. Bmax values of 0.91 and 0.89 for normal- and low-ionic strength conditions, respectively. (B) Top, Raw currents as indicated in response to step depolarizations of 80 ms to \u221220, 0, +20 and +40 mV from a \u201380-mV holding voltage. Calibration, 0.5\u00a0\u03bcA, 20 ms. Bottom, Dose-response relation for gallamine block of current at +40 mV in normal- (\u25a0) and low- (\u25cb) ionic strength solutions. Mean values from three to five measurements and SEM limits are plotted. Lines are fits of equation 1 to the data with Kapp values of 0.066 and 0.017 mM for normal- and low-ionic strength conditions, respectively. Bmax values of 0.91 and 0.94 for normal- and low ionic-strength conditions, respectively.\nThe upper part of Figure\u00a02B contains raw currents illustrating block by 20\u00a0\u03bcM gallamine at normal and low ionic strength. This concentration of gallamine blocked very little current at normal ionic strength (<20%), but more than half the current was blocked when the gallamine was applied in the low-ionic strength solution. The lower part of Figure\u00a02B shows the concentration dependence of block by gallamine, measured at +40 mV, at normal and low ionic strength. The lines are fits of equation 1 to the data with Kapp values of 0.066 mM at normal and 0.017 mM at low ionic strength, a 3.9-fold increase in apparent gallamine affinity in the low-ionic strength solution. Similar results have been observed for gallamine block of Shaker K channels (Quinn & Begenisich, 2001).\nThus, quaternary amine block of Kv3.1 channels, like Shaker K channels, was sensitive to solution ionic strength and this sensitivity was greater for trivalent than monovalent blocking ions. These results demonstrate that Kv3.1, like Shaker channels, contain charged residues that contribute to a negative electrostatic potential that influences block by these charged molecules. Our further investigations indicated that not all channels share this property, as demonstrated in Figure\u00a03.\nFigure\u00a03.Ionic strength-dependent TEA block of Kv2.1 channels. Top, Raw currents as indicated in response to step depolarizations of 80 ms to \u221220, 0, +20 and +40 mV from a \u221280-mV holding voltage. Calibration, 2\u00a0\u03bcA, 20 ms. Bottom, Dose-response relation for TEA block of current at +40 mV in normal- (\u25a0) and low- (\u25cb) ionic strength solutions. Mean values from 4\u201310 measurements and SEM limits (if larger than symbols) are plotted. Line is a fit of equation 1 to the pooled normal- and low-ionic strength data with Kapp and Bmax values of 5.1 mM and 0.83, respectively.\nTEA+ BLOCK OF KV2.1 CHANNELS WAS NOT SENSITIVE TO IONIC STRENGTH\nThe upper part of Figure\u00a03 illustrates TEA block of currents recorded from Kv2.1 channels. The raw current records show that, in this oocyte, block by 5 mM TEA was about 35%, independent of the solution ionic strength. The lower part of the figure demonstrates the dose-response relation for block of the ionic current at +40 mV from many oocytes in the normal- and low-ionic strength solutions. Clearly, TEA block of Kv2.1 channels was not ionic strength-dependent and well described by a Kapp value of 5.1 mM. This Kapp value is quite similar to the 4.2\u20134.5 mM values previously reported for these channels (Immke et al., 1999; Taglialatela et al., 1991) and illustrates the fact that these Kv2.1 channels are almost 40-fold less sensitive to TEA block than other K channels in spite of the fact that they contain the aromatic amino acids normally associated with high-affinity TEA block. In addition, unlike Shaker and Kv3.1 channels, TEA block of Kv2.1 channels was not sensitive to solution ionic strength.\nKv2.1 channels also differed from Shaker (T449Y) and Kv3.1 channels with respect to block by gallamine. We could detect no significant block of Kv2.1 channels by this trivalent TEA analog. In the normal-ionic strength solution, there was an average 3.5% increase in current in the presence of 5 mM gallamine, but this was not significantly different from zero (n = 3). Similar results showing no significant block were obtained with 10 mM gallamine and with the low-ionic strength solution.\nOur low-ionic strength solution contained one-fourth the normal Na+ and Ca2+ concentrations than the normal-ionic strength solution. In addition to changes in ionic strength induced by alterations in the monovalent ion concentrations, changes in Ca2+ concentrations are known to shift the voltage dependence of K channel gating but do not block these channels (e.g., Begenisich, 1975; Frankenhaeuser & Hodgkin, 1957; Mozhayeva & Naumov, 1972). It is because of this effect of alterations in Na+ and Ca2+ concentrations that we measured TEA and gallamine block at +40 mV, a voltage at which there are minimal gating changes. Nevertheless, it is worthwhile to demonstrate that these gating shifts do not affect our measurement of TEA block in the normal- and low-ionic strength solutions. Thus, in a separate set of experiments, we measured TEA block of Kv3.1 and Kv2.1 channels at several voltages in the two ionic strength solutions. As can be seen in Figure\u00a04, there is a slight voltage dependence to the TEA block of Kv3.1 channels and little or no voltage dependence to block of Kv2.1 channels, consistent with previous observations on these channels (Hartmann et al., 1991; Taglialatela et al., 1991). Importantly, these results show that the ionic strength dependence of TEA block was independent of test potential, so our use of +40 mV introduced no bias into the analysis.\nFigure\u00a04.Voltage dependence of TEA block of Kv3.1 and Kv2.1 channels. (A) Fraction of Kv3.1 channel current blocked by 100\u00a0\u03bcM TEA at the indicated membrane potentials in normal- (\u25a0) and low- (\u25cb) ionic strength solutions. (B) Fraction of Kv2.1 channel current blocked by 5 mM TEA at the indicated membrane potentials in normal- (\u25a0) and low- (\u25cb) ionic strength solutions.\nAs noted in Introduction, the low Kv2.1 sensitivity to TEA is surprising since, like Kv3.1, this channel contains the tyrosine residue at the \u201chot spot\u201d necessary for high-affinity TEA binding (see Fig.\u00a01). This issue has been discussed previously (Taglialatela et al., 1991), and one possibility that those authors suggested is that the positively charged side chain of the nearby K382 (see Fig.\u00a01) could \u201crepulse TEA\u201d and so reduce Kv2.1 TEA sensitivity. If there were a significant through-space electrostatic repulsion of TEA by the charged side chain of K382, then that repulsion should increase in a low-ionic strength solution. The data presented in Figure\u00a03, however, show that this did not occur.\nWe suggest that there is little or no net surface charge on wild-type Kv2.1 channels in a position to influence TEA block, so ionic strength should have no effect, as the data demonstrated. If this is correct, then removing some of the positive charge should shift the balance to a net negative surface charge and confer ionic strength dependence to TEA block. Thus, we mutated K382 in Kv2.1 to the neutral glutamine that occupies the analogous location in Kv3.1 channels. The action of TEA on these mutant channels is illustrated in Figure\u00a05A.\nFigure\u00a05.Ionic strength-dependent TEA block of mutant Kv2.1 channels. (A) Dose-response relation for TEA block of current (at +40 mV) from Kv2.1 K382Q channels in normal- (\u25a0) and low- (\u25cb) ionic strength solutions. Mean values from three measurements and SEM limits (if larger than symbols) are plotted. Line is a fit of equation 1 to the data with Kapp values of 4.9 and 2.1 mM for normal- and low-ionic strength conditions, respectively. Bmax values of 0.96 and 0.91 for normal- and low-ionic strength conditions, respectively. (B) Dose-response relation for TEA block of current (at +40 mV) from Kv2.1 K356F channels in normal- (\u25a0) and low- (\u25cb) ionic strength solutions. Mean values from four to six measurements and SEM limits are plotted. Line is a fit of equation 1 to the data with Kapp values of 6.1 and 2.8 mM for normal- and low- ionic strength conditions, respectively. Bmax values of 0.73 and 0.80 for normal- and low-ionic strength conditions, respectively.\nPOSITIVE CHARGE NEUTRALIZATION IN KV2.1 CHANNELS\nAs illustrated in Figure\u00a05A, TEA blocked K382Q Kv2.1 channels with an apparent Kapp value near 5 mM in the normal-ionic strength solution. TEA was more effective when applied in the low-ionic strength solution: block was described by a Kapp value near 2 mM. Thus, as predicted, removal of positive charge altered the electrostatic environment in the vestibule such that TEA block became sensitive to solution ionic strength. These K382Q channels remained resistant to block by 5 mM gallamine (not significantly different from zero, n\u00a0=\u00a03).\nInspection of the likely topology of the outer pore of Kv2.1 channels (Fig.\u00a01) suggests that the other positively charged amino acid unique to Kv2.1 (K356) may also be in a position to electrostatically effect TEA block. Thus, we tested TEA block of Kv2.1 channels with the lysine at position 356 mutated to the equivalent residue in Shaker (phenylalanine). Figure\u00a05B shows TEA block of these K356F channels. As can be seen, replacement of the charged K356 residue, like neutralization of K382, resulted in a channel with ionic strength-dependent TEA block: Kapp values of 6.1 and 2.8 mM in the normal- and low-ionic strength solutions, respectively. This construct was also quite insensitive to block by gallamine: 5 mM inhibited only 4.4 \u00b1 0.5% of the current at +40 mV.\nWe also tested the ionic strength dependence of block of channels with both charged lysine residues neutralized. Figure\u00a06A shows that TEA block of the double mutant K356F\/K382Q channels was sensitive to solution ionic strength. Figure\u00a06B demonstrates that these double mutant channels, unlike wild-type channels or channels with only a single neutralization, could be blocked by millimolar concentrations of gallamine. In normal-ionic strength solution, the Kapp for gallamine block was about 1.2 mM; in reduced-ionic strength solution, it was about 0.13 mM. This almost 10-fold increase in gallamine block with ionic strength change was even larger than that seen in Kv3.1 channels (Fig.\u00a02B).\nFigure\u00a06.Ionic strength-dependent TEA and gallamine block of K356F\/K382Q Kv2.1 channels. (A) Dose-response relation for TEA block of current (at +40 mV) in normal- (\u25a0) and low- (\u25cb) ionic strength solutions. Mean values from three to six measurements and SEM limits are plotted. Line is a fit of equation 1 to the data with Kapp values of 5.6 and 2.8 mM for normal- and low-ionic strength conditions, respectively. Bmax values of 0.80 and 0.91 for normal- and low-ionic strength conditions, respectively. (B) Dose-response relation for gallamine block of current (at +40 mV) in normal- (\u25a0) and low- (\u25cb) ionic strength solutions. Mean values from three to six measurements and SEM limits (if larger than symbols) are plotted. Line is a fit of equation 1 to the data with Kapp values of 1.2 and 0.13 mM for normal- and low-ionic strength conditions, respectively. Bmax values of 0.47 and 0.56 for normal- and low-ionic strength conditions, respectively.\nDiscussion\nTEA block of some K channels is sensitive to ionic strength changes (MacKinnon et al., 1989; Quinn & Begenisich, 2001), indicating that surface electrostatics plays a role in the permeation and pharmacological properties of such channels. We showed here that this property is shared by Kv3.1 channels. In contrast, TEA block of wild-type Kv2.1 channels was insensitive to solution ionic strength. Kv2.1 channels are also unusual in having a relatively low TEA affinity in spite of having the type of amino acid at the critical position that normally confers high TEA affinity (Kavanaugh et al., 1991; MacKinnon & Yellen, 1990). We showed that Kv2.1 channels were also unusual in being insensitive to the trivalent TEA analog gallamine.\nInspection of the crystal structures of the outer vestibule of K channels and the specific amino acid composition of Shaker, Kv2.1 and Kv3.1 channels (see Fig.\u00a01) reveals that the noted pharmacological differences of Kv2.1 channels could result from the presence of charged lysine residues not represented in Kv3.1 or Shaker channels. Neutralization of either K356 or K382 in Kv2.1 conferred an ionic strength-sensitive TEA block to these channels (Fig.\u00a05), but these mutant channels remained very insensitive to block by gallamine. Kv2.1 channels with both positively charged lysines neutralized attained a reasonable sensitivity to gallamine block, which was particularly sensitive to solution ionic strength (Fig.\u00a06).\nThe lack of ionic strength sensitivity of TEA block of Kv2.1 channels indicates that these channels, in contrast to Shaker and Kv3.1 channels, have little or no net charge on the outer vestibule in a position to affect TEA block. If so, then neutralization of the charged lysines not represented in Shaker and Kv3.1 channels should result in a net negative surface charge and thus confer ionic strength sensitivity to TEA block on the mutant Kv2.1 channels. This was the result that was obtained (Fig.\u00a04). Figure\u00a07 illustrates the outer entrance of the KcsA channel pore which has been \u201cmutated\u201d to contain Kv2.1 amino acids. Shown is the electrostatic potential arising from the charged amino acids as it is mapped onto the protein. Negative potentials are in red and positive potentials are in blue. The most prominent charged side chains are identified, and it is clear that K356 and K382 sit quite close to the pore entrance but that several negatively charged amino acids are also nearby. The lack of ionic strength dependence of TEA block of Kv2.1 indicates a negligible surface potential in a position to affect TEA block. Thus, it is likely that that the two lysine residues near the pore (K382 and K356) and the one farther away (K350) are sufficient to balance the electric field from the four negatively charged amino acids in the vicinity (D351, E352, D353, D354). According to this picture, mutating either or both of K356\/K382 would cause the near zero electrostatic potential of the wild-type channel to shift negative, accounting for the ionic strength dependence of the TEA block of these mutant channels.\nFigure\u00a07.Electrostatic potential map of the outer vestibule of Kv2.1 channels. Top view of the channel with the pore in the middle; the fourfold symmetry of these K channels is apparent. Electrostatic potentials computed as described in Materials and Methods and mapped onto the surface of the Kv2.1 channel. Negatively charged amino acid side chains are red, and blue represents positive charges. The two lysine residues that are the focus of this work (K336 and K382) as well as the other exposed charged amino acids are indicated in one of the four channel subunits.\nThe \u201cuncovering\u201d of negative charges caused by replacing the lysines with uncharged amino acids would be expected not only to confer ionic strength dependence on TEA block (as observed) but also to increase the apparent affinity for TEA block. While these charge-mutant channels did exhibit an ionic strength-dependent TEA block, their TEA sensitivities were essentially the same as the wild-type channel. It is possible that the mutations also produced some minor structural alteration in the outer vestibule that decreased the TEA affinity, masking the apparent increase expected with a more negatively charged vestibule. This view is supported by the fact that replacement of these lysines with neutral amino acids different from those used here (K382V, K356G) does, in fact, increase TEA affinity (Immke et al., 1999).\nOur data also suggest that at least one component of the gallamine sensitivity of K channels may be the net charge in the outer vestibule. The intrinsic gallamine affinity for K channels may be relatively low, but the negatively charged vestibules intrinsic to, for example, Shaker and Kv3.1 channels would increase the local gallamine concentration and thus overcome the intrinsic low affinity. The essentially neutral vestibule of wild-type Kv2.1 channels cannot be occupied by gallamine at millimolar levels. Even the mild negativity of K356F and K382Q Kv2.1 channels is not sufficient to allow reasonable block by gallamine. However, removal of both positively charged lysines (a total of eight positive charges on these tetrameric channels) apparently produces a sufficiently negative vestibule to provide for gallamine block. Consistent with this strongly negatively charged vestibule is the very large ionic strength dependence of gallamine block of these double mutant channels (Fig.\u00a05B).\nAnother feature of the gallamine block of the double mutant Kv2.1 channels was the apparent saturation of block at levels well below 100% (Fig.\u00a06B). We do not know the reason for this effect, but it is worth noting that, under some circumstances, TEA block of Kv2.1 channels displays this phenomenon (Hartmann et al., 1991; Immke & Korn, 2000; Immke et al., 1999). Such a result would be observed if the channels had both high and very low affinity for these blocking ions. The outer vestibule of KcsA channels exists in two conformations controlled by K+ levels (Zhou et al., 2001), and Shaker K channels exhibit both high- and low-affinity TEA block configurations also controlled by the K+ levels (Thompson & Begenisich, 2005). Future experiments will be required to test if Kv2.1 channels (or mutant Kv2.1 channels) exhibit such behavior with respect to gallamine block.\nFinally, it should be noted that electrostatic potentials contribute to the block of other types of ion channels. For example, snail toxin peptide block of voltage-gated Na channels, like scorpion toxin block of K channels (MacKinnon et al., 1989), exhibits strong charge-dependent effects (Hui et al., 2002). While the details of block are different in these two examples, these and the results of the present study demonstrate the widespread importance of electrostatic potentials in the outer vestibule of ion channels.\nIn summary, the results reported here indicate that surface electrostatics may play a significant role in the pharmacology of K channels. Shaker and Kv3.1 channels have negative charges in the outer vestibule that contribute to TEA and gallamine block. Wild-type Kv2.1 channels have a lower TEA affinity than Kv3.1 and other channels even though these channels possess the aromatic amino acid necessary for high-affinity TEA block. Our results suggest that at least one reason for this is that Kv2.1 channels do not have the exposed negative charges that contribute to TEA block in Kv3.1 and Shaker channels, likely because these negative charges are rendered ineffective by the nearby positively charged side chains of lysines at positions 356 and 382. Gallamine cannot block Kv2.1 channels unless both lysines are neutralized, likely because this TEA analog has an intrinsically low affinity but is particularly sensitive to electrostatic potentials owing to its trivalent nature. A full understanding of the pharmacology of the outer vestibule of K channels will have to take into account the electrostatic charges in this structural region.","keyphrases":["k channel","tetraethylammonium","gallamine","channel block","surface potential","quaternary ammonium ion"],"prmu":["P","P","P","P","P","R"]}
{"id":"Clin_Oral_Investig-4-1-2238777","title":"Erosion\u2014diagnosis and risk factors\n","text":"Dental erosion is a multifactorial condition: The interplay of chemical, biological and behavioural factors is crucial and helps explain why some individuals exhibit more erosion than others. The erosive potential of erosive agents like acidic drinks or foodstuffs depends on chemical factors, e.g. pH, titratable acidity, mineral content, clearance on tooth surface and on its calcium-chelation properties. Biological factors such as saliva, acquired pellicle, tooth structure and positioning in relation to soft tissues and tongue are related to the pathogenesis of dental erosion. Furthermore, behavioural factors like eating and drinking habits, regular exercise with dehydration and decrease of salivary flow, excessive oral hygiene and, on the other side, an unhealthy lifestyle, e.g. chronic alcoholism, are predisposing factors for dental erosion. There is some evidence that dental erosion is growing steadily. To prevent further progression, it is important to detect this condition as early as possible. Dentists have to know the clinical appearance and possible signs of progression of erosive lesions and their causes such that adequate preventive and, if necessary, therapeutic measures can be initiated. The clinical examination has to be done systematically, and a comprehensive case history should be undertaken such that all risk factors will be revealed.\nIntroduction\nErosion has, for many years, been a condition of little interest to clinicians and researchers. This has changed during the last years, and there is some evidence that the presence of dental erosion is growing steadily. In the UK, the prevalence of erosion was shown to have increased from the time of the children\u2019s dental health survey in year 1993 compared to 1996\/1997 [82]. In another UK study, the progression of erosion was investigated: 1,308 children were examined at the age of 12\u00a0years and 2\u00a0years later. In this study, 4.9% of the subjects at baseline and 13.1% 2\u00a0years later had deep-enamel or dentine lesions. Approximately 12% of erosion-free children at 12\u00a0years developed the condition over the subsequent 2\u00a0years. New or more advanced lesions were seen in 27% of the children over the study period [20]. The progression of erosion seems to be greater in older adults (52\u201356\u00a0years) compared to younger (32\u201336\u00a0years) and has a skewed distribution [64]. In this study, the group with high-erosion progression was found to have four or more dietary acid intakes per day, a low buffering capacity of stimulated saliva and used a hard-bristle toothbrush. Intake frequency of the same magnitude was also associated with an increased risk for erosion in children. In this investigation, the erosion group ate fruits significantly more frequently and had different drinking habits, such as swishing, sucking or holding drinks in their mouths [83].\nDental erosion is a multifactorial condition. To prevent further progression, it is important to detect this condition as early as possible. It is fundamental to diagnose the possible risk factors such that preventive measures can be initiated. This overview is aimed to give some basic aspects about the diagnosis and the risk factors of erosion. A more detailed description can be found elsewhere [67].\nDiagnosis\nDiagnosis of early forms of erosion is difficult, as it is accompanied by few signs and fewer if any symptoms. There is no device available in routine dental practice for the specific detection of dental erosion and its progression. Therefore, clinical appearance is the most important feature for dental professionals to diagnose this condition. This is of particular importance in the early stage of dental erosion [68]. At a more advanced stage, it can be very difficult to determine if dentine is exposed or not [31]. It is possible to use disclosing agents to render dentine involvement visible.\nThe appearance of smooth silky-glazed, sometimes dull, enamel surface with the absence of perikymata and intact enamel along the gingival margin are some typical signs of enamel erosion on facial and oral sites. It has been hypothesized that the preserved enamel band along the oral and facial gingival margin could be due to some plaque remnants, which could act as a diffusion barrier for acids. This phenomenon could also be due to an acid-neutralizing effect of the sulcular fluid [66]. In the more advanced stages, further changes in the morphology can be found. These changes result in developing a concavity in enamel, the width of which clearly exceeds its depth. Facial erosion should be distinguished from wedge-shaped defects which are located at or apical to the enamel\u2013cementum junction. The coronal part of wedge-shaped defects ideally has a sharp margin and cuts at right angle into the enamel surface, whereas the apical part bottoms out to the root surface. Thereby, the depth of the defect exceeds its width.\nThe initial features of erosion on occlusal and incisal surfaces are the same as described above. Further progression of occlusal erosion leads to a rounding of the cusps and restorations rising above the level of the adjacent tooth surfaces. In severe cases, the whole occlusal morphology disappears. Erosive lesions have to be distinguished from attrition and abrasion. The latter are often flat, have glossy areas with distinct margins and corresponding features at the antagonistic teeth. It is sometimes challenging to distinguish between the influences of erosion, attrition or abrasion during a clinical examination. Indeed, they may occur simultaneously with sometimes similar shape. The most commonly reported areas with this condition are occlusal surfaces [6]. Figures 1, 2, 3, and 4 show typical pattern of dental erosion process.\nFig.\u00a01Facial erosion: The intact enamel border along the gingival margin of tooth 12 and some plaque remnants are clearly visible. Note the smooth silky-glazed appearance and the absence of perikymata on the enamel surfaceFig.\u00a02Advanced facial erosion of teeth 43, 44 and 45 with dentinal involvement. The width of the lesions exceeds its depthFig.\u00a03Erosion with involvement of dentine on the oral surface of tooth 13, 12 and 11. Intact enamel borders along the gingival marginFig.\u00a04a\u2013c Typical pattern of advanced occlusal erosion of teeth 45 and 46 of three different patients: The whole occlusal morphology disappears, and extensive exposed dentinal areas are visible\nThe clinical examination should be done systematically using a simple but accurate index. This is a difficult task to achieve as an index with a too-fine grading shows a small inter- and intraexaminer reliability [60]. For a dental practitioner, the most important part is to recognize the condition and to describe its dimension and severity. It is important to search for a general pattern and not to over-interpret one single sign. For epidemiological purpose, an index with high detection capability and reliability is most important. Whenever possible, the clinical examination should be accomplished by a thorough history taking with respect to general health, diet and habits and by the assessment of saliva flow rates and buffer capacity. People who show signs and symptoms of erosion are often not aware of this condition. Only when a comprehensive case history is undertaken will all the risk factors be revealed.\nIt is difficult to judge the activity and progression of dental erosion. One tool is the comparison of clinical photographs of tooth surfaces to estimate the possible substance loss over time. Thereby, the discoloration of the lesions and their sensitivity state may give some information about the activity of the tooth surface. Further, study casts as well as the examination of dental radiography, especially bitewings longitudinally taken, can provide information about the substance loss over time. For research purpose, computed controlled mapping [13] or profilometric measurements using acid-resistant markers [6, 92] are tools to monitor progression.\nRisk factors\nWhen an acidic solution comes in contact with enamel, it has to diffuse first through the acquired pellicle, and only thereafter can it interact with enamel. The acquired pellicle is an organic film, free of bacteria, covering oral hard and soft tissues. It is composed of mucins, glycoproteins and proteins, including several enzymes [41]. On the surface of enamel, the hydrogen ion component of the acid will start to dissolve the enamel crystal. First, the prism sheath area and then the prism core are dissolved, leaving the well-known honey comb appearance [74]. Thereafter, fresh, unionized acid will eventually diffuse into the interprismatic areas of enamel and dissolve further mineral in the region underneath the surface [23, 27, 65]. This will lead to an outflow of ions (dissolution) and subsequently to a local pH rise in the tooth substance immediately below and in the liquid surface layer adjacent to the enamel surface [65]. The events in dentine are, in principle, the same but are even more complex.\nThere are different predisposing factors and aetiologies of the erosive condition. The interplay of chemical, biological and behavioural factors is crucial and helps explain why some individuals exhibit more erosion than others, even if they are exposed to the same acid challenge in their diets. Comprehensive knowledge of the different risk factors is a prerequisite to initiate adequate preventive (non-interventive) and, if necessary, therapeutic (interventive) measures. When a restoration becomes inevitable, in all situations, the preparations have to follow the principles of minimally invasive treatment.\nFigure 5 is an attempt to reveal the multifactorial predisposing factors and aetiologies of the erosive condition.\nFig.\u00a05Interactions of the different factors for the development of dental erosion (adapted from [68])\nChemical factors\nSeveral in vitro and in situ studies show that the erosive potential of an acidic drink or foodstuff is not exclusively dependent on its pH value but is also strongly influenced by its mineral content, its titratable acidity (\u2018the buffering capacity\u2019) and by the calcium-chelation properties [7, 8, 37, 44, 61, 72, 73, 76, 77, 80, 86, 88, 94, 95, 96]. The pH value, calcium, phosphate and fluoride content of a drink or foodstuff determine the degree of saturation with respect to the tooth mineral, which is the driving force for dissolution. Solutions oversaturated with respect to dental hard tissue will not dissolve it. A low degree of undersaturation with respect to enamel or dentine leads to a very initial surface demineralization which is followed by a local rise in pH and increased mineral content in the liquid surface layer adjacent to the tooth surface. This layer will then become saturated with respect to enamel (or dentine) and will not demineralize further.\nAcids such as citric acid exist in water as a mixture of hydrogen ions, acid anions (e.g. citrate) and undissociated acid molecules, with the amounts of each determined by the acid-dissociation constant and the pH of the solution. The hydrogen ion directly attacks the crystal surface. Over and above the effect of the hydrogen ion, the citrate anion may complex with calcium, also removing it from the crystal surface. Each acid anion has a different strength of calcium complexation dependent on the structure of the molecule and how easily it can attract the calcium ion [28]. Consequently, acids such as citric acids have double actions and may be very damaging to the tooth surface. Up to 32% of the calcium in saliva can be complexed by citrate at concentrations common in fruit juices, thus reducing the super-saturation of saliva and increasing the driving force for dissolution with respect to tooth minerals [75].\nThe dissolution with water of drinks containing organic acids with high buffering capacity will hardly reduce the pH but will reduce the titratable acidity. This is of some importance, as the greater the buffering capacity of the drink, the longer it will take for saliva to neutralize the acid. But dilution will also reduce concentrations of Ca and P (if present), which have a protective effect [10, 61, 62].\nThe calcium and phosphate content of a foodstuff or beverage are important factors for the erosive potential as they influence the concentration gradient within the local environment of the tooth surface. The addition of calcium (and phosphate) salts to erosive drinks showed promising results. Addition of calcium to a low-pH blackcurrant juice drink has been shown to reduce the erosive effect of the drink [48]. In a follow-up study, a blackcurrant drink with added calcium was compared to a conventional orange drink in situ. Servings of 250\u00a0ml of each drink were consumed four times per day during 20 working days. Measurements of enamel loss were made by profilometry on enamel samples for up to 20\u00a0days. The experimental carbonated blackcurrant drink supplemented with calcium caused significantly less enamel loss than the conventional carbonated orange drink at all time points measured [98]. When Ca was added to a sports drink, a reduction of the erosive potential was found [46]. Today, several Ca-enriched orange juices and sports drinks are on the market which hardly soften the enamel surface. Yoghurt is another example of food with a low pH (~4.0), yet it has hardly any erosive effect due to its high calcium and phosphate content, which makes it supersaturated with respect to apatite. A yoghurt or another milk-based food may have an erosive potential when it has a low content of Ca and\/or P and a low pH. It has to be kept in mind that, with the added mineral, enamel dissolution could not always be completely prevented. But the progression can be retarded which has some implications for the patient and the clinician.\nTheoretically, fluoride has some protective effect in a drink with a pH higher than that indicated by the saturation curve of fluorapatite at given Ca and P concentrations. Lussi et al. [61, 62] and Mahoney et al. [71] found an inverse correlation of the erosive potential of different beverages with their fluoride content. It is unlikely that fluoride at the concentration present in beverages alone has any great beneficial effect on erosion because the challenge is high. However, it is possible that, under conditions in which the other erosive factors are not excessive, fluoride in solution may exert some protective effect [71]. It appears that topical fluoride application can positively affect the tooth-wear process. The influence of immersion in fluoride solutions on brushing abrasion of eroded dentine slabs was investigated by Attin et al. [3]. Bovine dentine specimens were alternately stored in a demineralising and in a remineralising solution and brushed five times. Before each immersion in the remineralising solution, the specimens were not treated, treated with 250\u00a0ppm or treated with 2,000\u00a0ppm neutral sodium fluoride solution for 1\u00a0min. The least wear was found in the uneroded controls, whereas the greatest wear was found in the group without fluoride treatment. Significantly greater wear resistance was found as fluoride concentration increased. The same group evaluated the abrasion resistance of eroded enamel brushed with an acidified fluoride gel [4]. They found that treatment with a slightly acidic amine\/sodium fluoride gel increased the abrasion resistance of bovine enamel compared to an unfluoridated gel. They speculated that the fluoride is incorporated into and deposited on the enamel during treatment with the acidic gel, which could be one reason for the higher resistance.\nThe adhesiveness and displacement of the liquid are other factors to be considered in the erosive process. There appear to be differences in the ability of beverages to adhere to enamel based on their thermodynamic properties, e.g. the thermodynamic work of adhesion [50].\nIn summary, the two very often-cited parameters, the pH and the titratable acidity, do not readily explain the erosive potential of food and drink. The mineral content is also an important parameter, as is the ability of any of the components to complex calcium and to remove it from the mineral surface.\nBiological factors\nBiological factors such as saliva, acquired pellicle, tooth structure and positioning in relation to soft tissues and tongue are related to dental erosion development.\nA very important biological parameter is saliva. Several salivary protective mechanisms come into play during an erosive challenge: dilution and clearance of an erosive agent from the mouth, neutralisation and buffering of acids, and slowing down the rate of enamel dissolution through the common ion effect by salivary calcium and phosphate [104]. Practical experience demonstrates the importance of saliva in patients suffering from salivary-flow impairment. Studies have shown that erosion may be associated with low salivary flow or\/and low buffering capacity [53, 64, 89]. The dry mouth condition is usually related to aging [18, 81, 84], even though some other studies have not found this correlation [9, 43]. It is well established that patients taking medication can also present decreased saliva output [101], as well as those who have received radiation therapy for neck and head cancer [19]. Tests of the stimulated and unstimulated flow rate as well as of the buffer capacity of saliva may provide some information about the susceptibility of an individual to dental erosion. However, it has to be kept in mind that these parameters are two of a multifactorial condition. Sialometric evaluations should be carried at a fixed time point or in a limited time interval in the morning, avoiding intra-individual variations due to the circadian cycle [29]. Studies have shown that sour foodstuff has a strong influence on the anticipatory salivary flow [15, 55], which can be significantly increased when compared to the normal unstimulated flow rate [25]. Hypersalivation also occurs in advance of vomiting as a response from the \u2018vomiting center\u2019 of the brain [56], as frequently seen in individuals suffering from anorexia and bulimia nervosa, rumination or chronic alcoholism. It is suggested that this could minimize the erosion caused by acids of gastric origin. On the other hand, patients with symptoms of gastro-esophageal reflux disease (GERD) should not expect the salivary output to increase before the gastric juice regurgitation because this is an involuntary response not co-coordinated by the autonomic nervous system [42, 90]. Therefore, there may be insufficient time for saliva to act before erosion occurs. The influence of saliva on the remineralisation\/rehardening of erosive damaged dental hard tissue is discussed controversially. There is evidence that acid-softened enamel can reharden after exposure to saliva or remineralisation solution and that dietary products and fluoride can enhance the rehardening process [2, 26, 33]. Other investigations could not find a significant rehardening effect of saliva in situ [16, 32, 34, 58]. Some limited increase of the abrasion resistance of softened enamel was found after intraoral exposure to saliva [5, 51]. It seems that in vitro, some rehardening could be expected when supersaturated solution or saliva with no protein added are used [23, 24], whereas in situ, this is only the case to a very small amount.\nThe salivary acquired pellicle is a protein-based layer which is rapidly formed on dental surfaces after its removal by tooth brushing with dentifrice, other prophylaxis, measures or chemical dissolution. This organic layer becomes detectable on dental surfaces after few minutes of exposure to the oral environment [39, 93]. Enzymatic activity is also detectable at early stages of pellicle formation [40, 41]. It is suggested that it grows until reaching an equilibrium between protein adsorption and de-sorption within 2\u00a0h [57].\nThe acquired pellicle may protect against erosion by acting as a diffusion barrier or a perm-selective membrane preventing the direct contact between the acids and the tooth surface [39, 102, 103], reducing the dissolution rate of hydroxyapatite [57].\nMillward et al. [78] monitored the pH at the surface of teeth of healthy volunteers after drinking 1% citric acid. They observed that the pH recovered to above pH\u00a05.5 within 2\u00a0min from a site adjacent to the palatal surface of the upper central incisor and within 4\u20135\u00a0min from another palatal surface of the upper first molar. Other observations have revealed a longer clearance time on upper incisors for patients with active erosions and normal saliva values compared to patients with no erosion (Lussi, unpublished data). These differences could be due to the anatomy of the teeth and soft tissues that may influence the retention\/clearance pattern of erosive agents. Furthermore, soft tissue movements by the tongue and buccal mucosa and swallowing pattern can influence clearance rate. The importance of the tongue in modifying the tooth-wear process has long been the subject of speculation. Holst and Lange [45] considered mechanical abrasion caused by tongue to be a contributing factor in erosion caused by vomiting. Observations from animal studies also provide support in that beverages produced erosion mainly on the lingual surfaces of rat molar teeth in areas where the tongue contacts the teeth [96]. Dugmore and Rock [21] found that orthodontic anomaly appeared to confer a protective effect.\nBehavioural factors\nDuring and after an erosive challenge, behavioural factors play a role in modifying the extent of tooth wear. The manner that dietary acids are introduced into the mouth will affect which teeth are contacted by the erosive challenge and possibly the clearance pattern. As lifestyles have changed through the decades, the total amount and frequency of consumption of acidic foods and drinks have also changed. Soft drink consumption in the USA increased by 300% in 20\u00a0years [11], and serving sizes increased from 185\u00a0g (6.6\u00a0oz) in the 1950s to 340\u00a0g (12\u00a0oz) in the 1960s and to 570\u00a0g (20\u00a0oz) in the late 1990s. Around the year 1995, between 56 and 85% of children at school in the USA consumed at least one soft drink daily, with the highest amounts ingested by adolescent males. Of this group, 20% consumed four or more servings daily [36]. Studies in children and adults have shown that this number of servings per day is associated with the presence and the progression of erosion when other risk factors exist [64, 83].\nHigh erosion was associated with a method of drinking whereby the drink was kept in the mouth for a longer period [54]. One study investigated a randomly selected group of Swiss adults and diagnosed the cause of tooth wear. It showed that the consumption of erosive drinks and foodstuffs was strongly associated with erosion on facial and occlusal surfaces. Severe palatal erosions were scarce and highly associated with chronic vomiting [60]. The presence of calculus (odds ratio 0.48) or eating fruit other than apples or citrus fruit (0.48) reduced the chances of erosion. High consumption of carbonated drinks increased the odds of erosion being present at 12\u00a0years by 252% and was a strong predictor of the amount of erosion found at age 14 [21]. In a sample of 987 children (2 to 5\u00a0year-olds), consumption of vitamin C supplements, carbonated drinks and fruit syrup from a feeding bottle at bed- or naptime were related to erosion [1]. Considerable risk of erosion was found when citrus fruits were eaten more than twice a day and soft drinks were drunk daily [53].\nExcessive consumption of acidic candies combined with a low salivary buffering capacity may aggravate erosive lesions [17, 63]. The high intake of herbal teas, widely perceived as a healthy drink, may have an erosive potential exceeding that of orange juice [85]. An increase in agitation (e.g. when a patient is swishing his\/her drink in the mouth) will enhance the dissolution process because the solution on the surface layer adjacent to tooth mineral will be readily renewed. Further, the amount of the drink in the mouth in relation to the amount of saliva present will modify the dissolution process [69]. Several authors have suggested that using a straw is beneficial, since the straw directs drinks past the anterior teeth and towards the pharynx [22, 38, 49]. However, the placing of a straw labial to the anterior teeth can be destructive [70]. Nighttime exposure to erosive agents may be particularly destructive because of the absence of salivary flow. Two examples of this are bedtime baby bottle-feeding with acidic beverages and gastroesophageal reflux with regurgitation during sleeping.\nHealthier lifestyle paradoxically can lead to dental health problems in the form of dental erosion, as it often involves regular exercise and what is considered healthy diets with more fruits and vegetables. A lactovegetarian diet, which includes the consumption of acidic foods, has been associated with a higher prevalence of dental erosion [30]. The benefits of exercise are well-proven; however, exercise increases the loss of body fluids and may lead to dehydration and decreased salivary flow. A few case reports and studies have reported an association between sports activities and dental erosion. The cause could be direct acid exposure or strenuous exercise which may increase gastroesophageal reflux. Risk groups are swimmers exercising in water with low pH and athletes consuming frequently erosive sport drinks. In a study with 25 swimmers and 20 cyclists, the latter showed significantly more tooth wear into dentine. However, no association between erosion and sports drink consumption was found [79]. Professional swimmers train several hours in the water, which should have proper pH regulation. The main disinfection techniques used are gas chlorination and sodium hypochlorite. In the Netherlands, where the sodium hypochlorite method is used, only 0.14% of the tested pools were found in the year 2001 to have low pH values [59]. Another case report confirmed these findings [91]. In a review by Geurtsen [35], an increased prevalence of dental erosion among intensive swimmers due to low-pH gas-chlorinated pool water was described. The recommended pH for swimming pools is between pH\u00a07.2 and 8.0. Swimming activities in pH-adjusted pools do not harm the teeth [100]. However, erosion among competitive swimmers was found in 39% of swim-team members who trained in a pool with a pH of 2.7 which is a H concentration 100,000 times higher than that recommended for swimming pools [12]. Sports drinks are often erosive [46, 47, 95, 97], and when consumed during strenuous activity when the person is in a state of some dehydration, the possible destructive effects may be enhanced further.\nHealth-conscious individuals also tend to have better than average oral hygiene. While good oral hygiene is of proven value in the prevention of periodontal disease and dental caries, frequent tooth brushing with abrasive oral hygiene products may enhance dental erosion.\nAt the other end of the spectrum, an unhealthy lifestyle may also be associated with dental erosion [105]. Alcoholics may be at particular risk for dental erosion and tooth wear. Robb and Smith [87] reported significantly more tooth wear in 37 alcoholic patients than in age- and sex-matched controls. Tooth wear was most pronounced in men and those with frequent alcohol consumption.\nWine has properties such as low pH and low content of P and Ca, which renders it to have an erosive potential. Professional wine tasting is very common all over the world. In some countries (e.g. Sweden, Finland), wine tasters are employed by the state to support their state-owned wine shops. Full-time Swedish wine tasters test on average 20\u201350 different wines nearly 5\u00a0days a week. Wiktorsson et al. [99] investigated the prevalence and severity of tooth erosion in 19 qualified wine tasters in relation to number of years of wine tasting, salivary flow rate and buffer capacity. Salivary flow rate and buffer capacity of unstimulated and stimulated saliva were measured. Data on occupational background and dental and medical histories were collected. Fourteen subjects had tooth erosion mainly on the labio-cervical surfaces of maxillary incisors and canines. The severity of the erosion tended to increase with years of occupational exposure. Caries activity in all subjects was low. It was concluded that full-time wine tasting is an occupation associated with an increased risk for tooth erosion. In a cross-sectional comparative study, the prevalence and severity of tooth-surface loss between winemakers (exposed) and their spouses (non-exposed) was examined. There was a difference in the prevalence and severity of tooth surface loss between the two groups [14]. On the other hand, many other studies were not able to find an association between dental erosion and behavioural factors [30, 52], or they found only a weak association [82]. One can only speculate about the reasons. A possible explanation is the mode of questioning the persons (orally vs written questionnaire), the statistics employed (multivariate vs univariate) and the population group under study (selected vs randomly).\nAlthough no detrimental effects were described on a population level, one has to keep in mind that factors like sports-drink consumption and occupation can be, for some patients, a cofactor in the development of or in the increase in dental erosion when other factors are present. It is unlikely that one or two isolated factors (e.g. sports drink, dehydration) will be responsible for a multifactorial condition like erosion.\nConclusion\nThis overview shows the importance of early diagnosis of dental erosion and of accurate detection of possible risk factors and their interplay. These facts are prerequisites to initiate adequate preventive (and therapeutic) measures.","keyphrases":["erosion","chemical","clinical appearance","biological and behavioural risk factors"],"prmu":["P","P","P","R"]}
{"id":"Eur_J_Pediatr-3-1-1914295","title":"Stimulation programs for pediatric drug research \u2013 do children really benefit?\n","text":"Most drugs that are currently prescribed in pediatrics have not been tested in children. Pediatric drug studies are stimulated in the USA by the pediatric exclusivity provision under the Food and Drug Administration Modernization Act (FDAMA) that grants patent extensions when pediatric labeling is provided. We investigated the effectiveness of these programs in stimulating drug research in children, thereby increasing the evidence for safe and effective drug use in the pediatric population. All drugs granted pediatric exclusivity under the FDAMA were analyzed by studying the relevant summaries of medical and clinical pharmacology reviews of the pediatric studies or, if these were unavailable, the labeling information as provided by the manufacturer. A systematic search of the literature was performed to identify drug utilization patterns in children. From July 1998 to August 2006, 135 drug entities were granted pediatric exclusivity. Most frequent drug groups were anti-depressants and mood stabilizers, ACE inhibitors, lipid-lowering preparations, HIV antivirals, and non-steroidal anti-inflammatory and anti-rheumatic drugs. The distribution of the different drugs closely matched the distribution of these drugs over the adult market, and not the drug utilization by children.\nIntroduction\nMost drugs prescribed in pediatrics have not been tested in children. A recent review demonstrates that up to 80% of prescriptions for children in hospital and in general practice are either unlicensed (without a license for children) or used off-label (outside the product license) [15]. Of commercially available drugs in Europe, only 35% are authorized for use in children [4]. Although there are reasons why children do not often participate in clinical trials, including ethical, scientific and commercial considerations [2, 25], it is considered unacceptable to treat children with drugs that have not been studied properly.\nIn 1997, the Food and Drug Administration (FDA) and the Congress introduced the Food and Drug Administration Modernization Act (FDAMA), and this was followed by the Best Pharmaceuticals for Children Act. Closely linked to this legislation is the Pediatric Rule (1998), which requires the industry to perform research in the pediatric population. In the European Union (EU), final legislation on this topic has been approved and will come into effect at the beginning of 2007.\nIn both continents, the measures taken to address the problems broadly follow the same pattern. The first incentive is aimed at new medicines and intended for products covered by a patent or a supplementary protection certificate (SPC). For these drugs, a 6-month extension of market exclusivity is granted if a pediatric study is performed. The second incentive has the objective of increasing the knowledge on drugs that are no longer patent protected. In order to obtain the data necessary to establish safety, quality and efficacy specifically in children, either funding for the studies (USA) or market exclusivity (a so-called Pediatric Use Marketing Authorization\u2014PUMA) can be given. Central to the second incentive is that experts are involved in determining for which drugs the greatest medical need exists and in ensuring that these drugs will be given priority. In the USA, the FDA plays this central role, and in the EU, a Pediatric Committee will be established within the European Medicines Agency (EMEA) which will be given a similar role.\nThe EU Commission apparently follows the approach in the USA with regard to patent-protected drugs because, according to the EU Commission, \u201cthe pediatric exclusivity provision has been extremely successful in the USA in stimulating the development of medicinal products for pediatric use\u201d [1].\nAt the brink of implementing new programs (EU) or decisions on continuation of existing programs (USA), we questioned what the influence of the pediatric exclusivity regulation has been on pediatric drug development. We evaluated the drugs that are granted pediatric exclusivity in the USA by studying research that has been performed as a consequence of the exclusivity provision, and by comparing the drugs granted pediatric exclusivity with medicines actually used by children.\nMaterials and methods\nThe drugs granted pediatric exclusivity since the introduction of the FDAMA in July 1998 until August 2006 were retrieved from the FDA website [17]. All drugs were classified into subgroups according to the Anatomical Therapeutical Classification (ATC) system of the European Pharmaceutical Marketing Research Association. The data submitted to the FDA that resulted in granting the exclusivity were examined. For each drug, the publicly available summaries of medical and clinical pharmacology reviews of the pediatric studies were scrutinized. These summaries contain information on the pediatric research performed to obtain pediatric exclusivity. If these summaries were unavailable, the labeling information of the drug with pediatric exclusivity was studied to retrieve information about the pediatric studies performed. Each drug label contains a pediatrics section stating whether the drug is tested in children and, if so, information on the performed studies is given [6, 17, 26]. Data were extracted about the type of study, number and age of participants, whether long-term follow-up (defined as >1\u00a0year with specific attention for long term effects on growth and development) was undertaken and whether the study led to a pediatric indication being included on the label. Participants were divided into pediatric age categories according to the International Conference on Harmonization (ICH) guidelines: neonates (birth\u201327\u00a0days), infants (28\u00a0days\u201323\u00a0months), children (2\u201311\u00a0years) and adolescents (12\u201318\u00a0years).\nTo obtain data about drug use in children, recently published surveys of drug prescribing in hospitals and general practice were reviewed by a systematic search of the literature on drugs used by children. Details on the literature search and selection criteria are provided in box 1. Drug use in adults was assessed by using the sales figures from public databases and publications thereof [7, 8]. The data were categorized according to the ATC system as mentioned above.\nLiterature search and selection strategy\nStep 1: literature searches of the PubMed and Embase bibliographic databases for papers published in English between 1990 and July 2005 combining the following search terms (keywords and appropriate medical subject headings): \nChild, preschool (or) child (or) infant (or) infant, newborn (or) adolescent (or) pediatric (or) paediatric (or) paediatrics.Pharmaceutical preparations (or) drugs, non-prescription (or) drugs, generic (or) drug, therapy (or) prescriptions, drug (or) medicine (or) medication.Drug utilization (or) pharmacoepidemiology (or) drug utilization review.\nStep 2: the references contained in articles identified in step 1 were examined to identify further relevant studies.\nStep 3: based on the titles and abstracts of the papers, we next identified and then located full-text copies of 35 potentially relevant studies for closer examination.\nStep 4: selection of articles by two independent reviewers (I.B., R.S.), using the following selection criteria: \nStudy performed in the industrialized world, defined as in Europe, North America, Australia and New Zealand.Study population of children from 0 to at least 14 years, to include all age groups (neonates, infants, children and adolescents).Sufficient information in the paper about drug utilization to classify 90% of the drugs in an ATC drug groups.Avoidance of selection bias by exclusion of voluntary surveys.\nStep 5: inclusion of nine papers meeting all selection criteria.\nResults\nPediatric exclusivity provision\nAccording to the FDA, 135 drugs (130 active moieties) were granted pediatric exclusivity from July 1998 to August 2006. Most frequent drug groups were central nervous system drugs (19%), such as anti-depressants and psycholeptics, cardiovascular drugs (16%), mainly ACE inhibitors and lipid-lowering preparations, systemic anti-infectives (12%), among which largely HIV antivirals, cytostatics (11%), and alimentary tract medication (12%), among which proton pump inhibitors and oral antihyperglycemic medication (Table\u00a01). \nTable\u00a01Active moieties granted pediatric exclusivity according to ATC groupDrug categoryNumber of drugs (% of total)Most frequent drug classesNumber of drugsCentral nervous system24 (19%)Anti-depressants8Psychostimulants, agents used for ADHD and nootropics4Anti-epileptics3General anaesthetics3Anti-migraine preparations2Cardiovascular system21 (16%)ACE inhibitors, plain6HMG-CoA reductase inhibitors5Beta-blocking agents4Alimentary tract and metabolism16 (12%)Drugs for peptic ulcer and gastro-oesophageal reflux disease5Oral blood glucose lowering drugs4Anti-infectives for systemic use15 (12%)Direct acting antivirals10Antibacterials for systemic use4Antineoplastic and immunomodulating agents14 (11%)Other antineoplastic agents5Antimetabolites3Alkylating agents2Musculo-skeletal system7 (5%)Anti-inflammatory and anti-rheumatic products, non-steroids6Drugs affecting bone structure and mineralization1Respiratory system10 (8%)Antihistamines for systemic use4Drugs for obstructive airway disease3Decongestants and other nasal preparations for topical use2Sensory organs10 (8%)Antiglaucoma preparations and miotics4Decongestants and anti-allergics3Dermatologicals6 (5%)Corticosteroids, potent (group 3)3Genito-urinary system and sex-hormones3 (3%)Other urologicals, including antispasmodics2Blood and blood forming organs2 (2%)Anti-thrombotic agents1Systemic hormonal preparations1 (1%)Hypothalamic hormones1Parasitology1 (1%)Anti-malarials1Total130 (100%)\nFrom 118 drugs (91%), information about studies performed in children to obtain pediatric exclusivity could be retrieved from the FDA summaries of medical and clinical pharmacology reviews (n\u2009=\u200961) of the pediatric studies or the prescribing information as provided by the manufacturer (n\u2009=\u200957). No information could be found on 12 of the drug entities, mainly including over the counter drugs. The product label of 13 drugs merely stated that safety and\/or efficacy in the pediatric population had not been established. For the remaining 105 drugs, in total, 326 studies were performed for the approval of pediatric exclusivity. At least 40,075 pediatric patients participated in these 326 studies (the number of participants was not noted for 27 of these studies). In the majority of the applications, children over a wide age range were included. Information on the age of participants was not provided for 54 of the 326 studies. Children participated in 224 of the remaining 272 studies (82%), adolescents in 177 (65%), infants were included in 105 (39%) of the trials, and neonates in 38 trials (14%). Only 1 (0.4%) study included preterm infants. The objective of most (62%) of the 326 studies was to determine the safety and efficacy of a drug. Pharmacokinetics and\/or pharmacodynamics were studied in 147 (45%) trials, efficacy-only in 13 (4%) and safety-only in 38 (12%). From the 299 studies in which information about the number of participants was given, on average 134 children participated per study. This varied from 8 children in a safety and efficacy study of a HIV antiviral in neonates, to 994 children participating in a safety database of an antibiotic. Long-term (>1\u00a0year) follow-up was either done or planned for 25 (21%) of the 118 drugs.\nFor 42% of the drugs granted pediatric exclusivity and for which information was available (50 out of 118), the information obtained from the pediatric studies led to an approved pediatric indication.\nThe 130 active moieties granted pediatric exclusivity were categorized according to the ATC system. The results are shown in Table\u00a01.\nDrug utilization by children and adults\nThe search strategy retrieved nine papers addressing drug utilization in children (Table\u00a02). The data were summarized by calculating a weighted average for each drug category with weights proportional to the number of prescriptions (Table\u00a03). Drugs most often used by children are respiratory drugs, anti-infectives for systemic use and dermatologicals. \nTable\u00a02Characteristics of included studies on pediatric drug utilizationAuthor (year)SettingPopulation sizeAge range (y)Number of prescriptionsNiclasen [14] (1995)Pharmacy dispensing records in-and outpatients1,7040\u2013145,876Rokstad [21] (1997)Prescriptions of general practitionersa0\u2013198,215Thrane [28] (1999)Pharmacoepidemiological prescription database outpatients48,0910\u201315154,189Schirmb [24] (2000)Pharmacy dispensing records outpatients15,0010\u201316373,925Lewis [12] (2001)Prescriptions pediatric outpatients12,6280\u20131633,140Pandolfini [16] (2002)Prescriptions general pediatric hospitals1,3250\u2013144,265Schirm [23] (2003)Pharmacy dispensing records outpatients18,9430\u20131666,222Ufer [30] (2003)Pharmacy dispensing records outpatients357,7840\u201316644,817Sanz [22] (2004)Prescriptions outpatients by GPs and pediatricians12,2640\u20131427,486a Not statedb Not included in weighted average due to presentation of data in paper (percentage of children using a drug category).Table\u00a03Drug utilization pattern in childrenDrug category% of prescriptionsFrequent used drug classesRespiratory system30 %Drugs for obstructive airway diseaseNasal preparationsCough and cold preparationsAntihistamines for systemic useGeneral anti-infectives, systemic28%Antibacterials for systemic useDermatologicals12%Antifungals for dermatological useEmollients and protectivesCorticosteroids, dermatological preparationsSensory organs7%OphtalmologicalsOtologicalsCentral nervous system4%Analgesics\/ antipyreticsPsychostimulants, agents used for ADHD and nootropicsAnti-epilepticsHormones3%Corticosteroids, for systemic useBlood and blood forming organs3%Vitamin KAlimentary tract and metabolism2%Drugs for peptic ulcer and gastro-oesophageal reflux diseaseInsulins and analoguesMusculo-skeletal system2%Anti-inflammatory and anti-rheumatic products, non-steroidsGenito-urinary system and sex-hormones2%Hormonal contraceptives for systemic useCardiovascular system1%DiureticsOther<1%Total105% (exceeds 100% due to rounding)\nThe pattern of drug use in adults based on sales over the last 12\u00a0months to May 2005 (Table\u00a04) shows that drugs used for central nervous system, cardiovascular, alimentary tract and metabolism disorders make up over 50% of the market. \nTable\u00a04Drug utilization patterns in adults, according to the drug sales in North America over the period May 2004\u2013May 2005Drug categoryAdult prescriptionsCentral nervous system23%Cardiovascular system19%Alimentary tract and metabolism14%Respiratory system9%General anti-infectives, systemic8%Musculo-skeletal system6%Genito-urinary system and sex-hormones6%Other2%Antineoplastic and immunomodulating agents4%Dermatologicals3%Blood and blood forming organs3%Sensory organs2%Total100%\nDiscussion\nThe introduction of the pediatric exclusivity laws in the USA has led to pediatric drug trials for 135 drugs tested in the past eight\u00a0years. Over 300 studies were performed with over 40,000 pediatric patients participating. Based on these data, the FDA claims \u201cthe pediatric exclusivity provision has done more to generate clinical studies and useful prescribing information for the pediatric population than any other regulatory or legislative process to date\u201d [20]. The EU Commission and other policy makers see a rapid adaptation of similar legislation in Europe essential to boost pediatric drug research in the EU as soon as possible [1, 3, 19]. Although it is true that more pediatric studies have been done, we question the content of the research.\nA major discrepancy is apparent between the drug prescription pattern in children and the drugs granted pediatric exclusivity. The majority of drugs granted pediatric exclusivity are rarely used by children, and drugs that are frequently used by children are underrepresented in the pediatric studies to obtain exclusivity. This difference is not accounted for by the licensing status of the used drugs. For instance, in general practice, 37% of the prescriptions of respiratory drugs are still used in an unlicensed or off-label manner [27]. This suggests that the pediatric studies did not address the real needs in pediatric drug development. Whilst the pediatric exclusivity scheme has stimulated pediatric research quantitatively, the nature of the scheme has led to priorities for the type of research that are largely driven by the adult market for medicines rather than by the needs of the pediatric population. An important built-in motive in the stimulation program for performing pediatric studies is the financial benefit gained from the exclusivity provision. The drugs granted pediatric exclusivity include 5 out of the \u201cTop 10\u201d prescription drugs with the highest sales figures in North America in 2005: atorvastatin (Lipitor), simvastatin (Zocor), omeprazole (Nexium), lansoprazol (Prevacid), and sertraline (Zoloft). Sales of these 5 drugs amounted to $24.1 billion US in 2005 [9]. Extension of the SPC on the basis of pediatric exclusivity has also been granted to other adult best-selling drugs, such as pravastatine, enalapril, metformin, amlodipine, paroxetine, fluoxetine, and rofecoxib (currently withdrawn). It is thus clear that the patent extension has an enormous financial benefit for the pharmaceutical companies, which easily outweighs the cost of the average pediatric trial [1]. This is not only the case for best-selling drugs but also for drugs with a smaller market share. A Tufts University report indicates that an estimated $35 million US in undiscounted profits per drug can be earned by obtaining pediatric exclusivity based on median 2004 sales for all pediatric exclusivity drugs after accounting for costs and market protection extensions [29]. Although the increased profit was intended by the legislator, we doubt if the other intention, which aimed at providing data to reduce the volume of drugs taken by children that are off-label or unlicensed, has also been met.\nOur approach to define essential drugs for children by volume and by number of children that need a certain medicine is incomplete, as indications and severity of the conditions for which the drug is prescribed also play an important role [10]. Secondly, the method employed in our literature search may have introduced some bias toward outpatient drug consumption, thus underestimating the use of, for example, anesthetic or cardiovascular drugs. Clearly, hypertension or lipid disorders occur in children, and knowledge about these drugs in this population is important. However, these conditions are certainly not major causes of morbidity, and subsequent drug use in children and the research priorities lie elsewehere.\nFurthermore, there are specific issues in pediatric clinical pharmacology that have remained unaddressed by the current product-related approach. First, the age distribution of the children participating in the trials does not reflect the actual medical needs. Drug use in children follows a pattern, with a relatively high percentage of children using a prescription drug in the first year of life [24] while the proportion of off-label and unlicensed drug use is highest (\u223c90%) in the vulnerable group of neonatal ICU patients [5]. These data are not reflected in the participation in the trials to obtain pediatric exclusivity, with neonates and infants being included in, respectively, 14 and 39% of the study groups. In fact, it is surprising to see that the majority of applications included children of a wide age range, thereby ignoring the delineation of age groups as defined in the ICH guidelines. This is problematic, as in the pediatric population significant age-related differences may exist in the pharmacokinetics and the effects of drugs [11]. Secondly, the effects of drugs (that are intended for chronic use) on development and maturation of children are largely unknown, but this issue has hardly been addressed. Third, off-label drugs are hardly or not studied. Only ten drugs in this category are under consideration for study by pharmaceutical companies (FDA website). From these ten drugs, six already have been refused by industry for studying, and have been referred to the National Institutes of Health to obtain the necessary information for rational use in pediatric populations. These clinical trials are at the moment awaiting funding [13, 18]. Surprisingly, in the same Tufts study that reports on a profit of $35 million US per drug granted exclusivity, it is also suggested that the current incentives for studying non-patent protected drugs are too low [29].\nFinally, methodological research has remained underfunded by this approach. Any expansion of research will require specialized techniques that allow samples and data to be obtained in children with minimal discomfort and risk. For instance, most pharmacokinetic assays require an amount of blood that is too large for the average neonate, so highly sensitive assays need to be developed. Accurate assessment of drug effects on neuro-development and behavior also require further development, independently of studies with particular compounds.\nThe recently approved EU guidelines contain differences from the USA that may remedy some of the deficiencies indicated. It contains a section for the stimulation of off-patent drugs that can be granted a special label (PUMA), and data protection if data necessary to establish safety, quality and efficacy in children are submitted. The \u2018Medicines Investigation for the Children of Europe\u2019 program, funded by the EU, will be created to stimulate research of off-patent drugs. The proposed establishment of a Pediatric Committee, operating within the European Medicines Agency, will guard study of significant drugs for children and avoid unnecessary studies, provide free advice to industry, and stimulate long-term pharmacovigilance. It will also play a role in the implementation of the requirement for industry to submit data they already hold on use of their medicines in children. New drugs will not be granted Marketing Authorisation unless the need for pediatric research has been waived by the Pediatric Committee, or deferral of initiation or completion of an already approved Pediatric Investigation Plan has been agreed to by this committee. In addition, an EU network of investigators and trial centers will be formed [1]. Although elements of this network are now beginning to form, their funding is unclear and certainly not at the level provided to the patent holders by a patent extension. Also, it is not immediately clear why the EU measures will deliver, when this was not achieved with the formation of Pediatric Pharmacology Research Units and the oversight of research priorities by the FDA in the USA.\nIn conclusion, the schemes implemented in the US generated new knowledge and led to the rapid development of an infrastructure to carry out pediatric drug trials. Although these are certainly positive developments, we do believe that the findings of this survey warrant additional efforts to stimulate research on drugs used more frequently by children, and generally applicable methodological research, as at least the short-term effect of these initiatives seems to have drawn the focus of industry-sponsored research to the most profitable part of the market. The funding of the research we propose should not be problematic. The pediatric exclusivity schemes generate a flow of public money to the sponsoring drug companies because generic replacement and price reductions are postponed. There is no particular reason why these public funds could not be at least partly applied in an alternative manner.","keyphrases":["pediatrics","patents","clinical trials","pharmaceutical preparations","licensure"],"prmu":["P","P","P","P","U"]}
{"id":"Int_J_Cardiovasc_Imaging-3-1-2048828","title":"Value of assessment of tricuspid annulus: real-time three-dimensional echocardiography and magnetic resonance imaging\n","text":"Aim To detect the accuracy of real-time three-dimensional echocardiography (RT3DE) and two-dimensional echocardiography (2DE) for tricuspid annulus (TA) assessment compared with magnetic resonance imaging (MRI).\nIntroduction\nEvaluation of tricuspid annulus (TA) continues to be a major problem in the surgical decision-making process due to its complex three-dimensional shape [1\u20133]. Accurate assessment of TA has many values in clinical application. For example, the decision of tricuspid valve repair may change due to discrepancy between pre-operative measurement of TA diameter (TAD) by two-dimensional transthoracic (or transesophageal) echocardiography (2DE) and direct surgical visualization [4]. Several studies described a strong correlation between TA motion and right ventricular (RV) function [5\u20138]. Analysis of TA velocity by tissue Doppler imaging has been found useful for RV functional assessment [9, 10]. In these studies, the assessment of TA motion relied on 2DE, M-mode and tissue Doppler recording. Since Real-time three-dimensional echocardiography (RT3DE) has become available for clinical practice, it is now possible to examine the TV more completely [11]. This study aimed to use RT3DE in evaluation of TA morphology, size and cyclic changes during the cardiac cycle. It also aimed to correlate TA function with RV function that was assessed by magnetic resonance imaging (MRI).\nSubjects and methods\nThe study included 30 patients (mean age 34\u00a0\u00b1\u00a013\u00a0years, 60% were males) who were scheduled for routine MRI examination for evaluation of right ventricular function (10 patients with congenital heart disease, five with chronic pulmonary disease, five with multivalvular affection, and 10 normals). 2DE and RT3DE were performed at the same day of MRI examination after their informed consent for assessment of TA. The inclusion criteria for selection were good 2D image quality, sinus rhythm and mild to moderate tricuspid regurgitation.\nMRI studies were performed with a 1.5 T MRI (General Electric, Milwaukee WI; Signa 1.5 T MRI) equipped with a four-element torso coil. A cardiac-triggered, steady-state, free-precession sequence (FIESTA; temporal resolution and repetition time of echo of 3.5 and 1.3\u00a0ms, respectively, flip angle of 45\u00a0degrees) was used for quantitative analysis. 10 cine short axis slices were acquired (slice thickness 10\u00a0mm, gap 0\u00a0mm) during a breath-hold period. Additional imaging parameters were a field of view of 320\u2013380\u00a0mm and a matrix of 160\u00a0\u00d7\u00a0128. These series of high quality images encompassing the right ventricle (RV) produced a three-dimensional data set with sharp edge between the blood pool and myocardium. Quantitative analysis was performed using standardized software (MassPlus, Medis Inc., Leiden, NL). By this software, manual tracing was done for TA and RV endocardial border on all images (end-diastolic and end-systolic). The following measures were obtained at end-diastolic and end-systolic frame: (1) TA area (TAAMRI), (2) TADMRI, (3) RV volume, (4) RV ejection fraction (RV-EF) was calculated as (RV end-diastolic volume- RV end-systolic volume\/RV end-diastolic volume)\u00a0\u00d7\u00a0100%, (5) TA fractional shortening (TAFSMRI) defined as (end-diastolic TADMRI\u2013end-systolic TADMRI)\/end-diastolic TADMRI\u00a0\u00d7\u00a0100% and (6) TA fractional area changes (TAFACMRI) defined as (end-diastolic TAAMRI\u2013end-systolic TAAMRI)\/end-diastolic TAAMRI\u00a0\u00d7\u00a0100%.\n2DE was done with a Sonos 7500 ultrasound system attached to a S3 transducer (Philips, Best, The Netherlands). The TV was imaged from apical 4-chamber view with the patient in the left lateral decubitus position. The following measures were obtained: (1) TAD2D was defined as the distance between the insertion sites of septal and anterior TV leaflets and obtained at an end-diastolic and end-systolic still frame and (2) TA fraction shortening (TAFS2D) defined as (end-diastolic TAD2D\u2013end-systolic TAD2D)\/end-diastolic TAD2D\u00a0\u00d7\u00a0100%\nRT3DE was done with the same ultrasound system attached to X4 matrix array transducer capable of providing real-time B-mode images. A full volume 3D data set was collected within approximately 5\u201310\u00a0s of breath holding in full volume mode from an apical window. The 3D data set was transferred for off-line analysis with TomTec software (Unterschleissheim, Munich, Germany). Data were stored digitally and subsequently evaluated by two blinded observers (AMA, OIIS). Data analysis of 3D images was based on a 2D approach relying on images obtained initially from the apical view. The TA was sliced between two narrow lines to exclude other tissues on the 2D image leading to clarification of annulus by a 3D image. TA was viewed and traced manually from the atrial aspect and once this is completed the surface area was automatically calculated and could be visualized from different points of views. Manual modification was made to correct any inconsistence. The following RT3DE variables were obtained from both an end-diastolic and end-systolic still frame: (1) TA area (TAA3D), and (2) TAD3D defined as the widest TAD (see Fig.\u00a01), Subsequently, TAFS3D and TA fractional area changes (TAFAC3D) (%) were calculated by the same formula used in 2DE.\nFig.\u00a01Tricuspid annulus oval shape as seen by graphic representation (A), real-time three-dimensional echocardiography (B), magnetic resonance imaging (C), and tricuspid annulus with 2 lines inside, the larger is the TAD by real-time three-dimensional echocardiography and the smaller is the TAD by two-dimensional echocardiography with large difference in measurements\nStatistical analysis\nAll data obtained by MRI, 2DE and RT3DE are presented as mean\u00a0\u00b1\u00a0SD. A paired t-test and was performed for comparing means of variables. The level of significance was set to P\u00a0<\u00a00.05. A SPSS statistical package was used (SPSS, version 12.1, SPSS Inc, Chicago). Pearson\u2019s coefficient was used for correlation between RT3DE and MRI data. Interobserver agreement for RT3DE measurements was assessed according to the Bland and Altman principle [12].\nResults\nAcquisition of RT3DE data was performed in all patients in a reasonable time (approximately 1\u00a0min for acquisition and 5\u00a0min for data analysis). The TA was clearly delineated in all patients. An oval-shaped TA (not circular) was visualized by RT3DE and MRI (Fig.\u00a01). TAD3D was obtained with very good interobserver agreement (mean difference \u22120.4\u00a0\u00b1\u00a01.5\u00a0mm, agreement \u22123.4\u20132.6). There was a good correlation between TADMRI and TAD3D (R\u00a0=\u00a00.75, P\u00a0=\u00a00.001), while TAD2D was fairly correlated with TAD3D and TADMRI (R\u00a0=\u00a00.5, P\u00a0=\u00a00.01 for both). There were no significant differences between RT3DE and MRI in TAD, TAA, TAFS, and TAFAC measurements, while TAD2D and TAFS2D were significantly underestimated (P\u00a0<\u00a00.001) (Table\u00a01). TAFS2D was not correlated with RV-EF, while TAFS3D and TAFAC3D were fairly correlated with RV-EF (r\u00a0=\u00a00.49, P\u00a0=\u00a00.01, and r\u00a0=\u00a00.47, P\u00a0=\u00a00.02 respectively).\nTable\u00a01Comparison between measurements of TA size and function by 2DE, RT3DE and MRI2DERT3DEMRIEnd-diastolic TAD (mm)33.0\u00a0\u00b1\u00a08.5 43.2\u00a0\u00b1\u00a010.044.1\u00a0\u00b1\u00a09.2End-diastolic TAA (mm2)\u2014-1835\u00a0\u00b1\u00a04251869\u00a0\u00b1\u00a0392End-systolic TAD (mm)27.0\u00a0\u00b1\u00a07.832.0\u00a0\u00b1\u00a08.732.5\u00a0\u00b1\u00a08.2End-systolic TAA (mm2)\u20141120\u00a0\u00b1\u00a03071137\u00a0\u00b1\u00a0288TAFS (%) 18.7\u00a0\u00b1\u00a04.026.3\u00a0\u00b1\u00a05.926.7\u00a0\u00b1\u00a05.7TAFAC (%)\u201439.3\u00a0\u00b1\u00a04.939.5\u00a0\u00b1\u00a04.7TAD: Tricuspid annular diameter, TAA: Tricuspid annular area, TAFS: Tricuspid annulus fractional shortening and TAFAC: tricuspid annular fractional area change\nDiscussion\nIn the present study, the morphological and functional aspects of TA were assessed by MRI and RT3DE. The main findings of our study are (1) TA shape was not circular but oval, (2) TAD measurement by RT3DE is more accurate than by 2DE, and (3) TA function was fairly correlated with RV function.\nTAD measurements have an important role in the TV surgical decision-making process not only for the selection of patients undergoing surgery, but also the type of surgical technique (valve plication or ring placement) [13\u201315]. Although 2DE is helpful to assess TV function and to detect TR severity it has important limitations in describing TV morphological details, such as TAD [14, 15]. RT3DE may yield more detailed anatomical description of TA morphology and function [16, 17]. In the present study, the TA was visualized well in all subjects allowing even measurements of its area. This is in accordance with Schnabel et al. [11] who reported well or at least sufficient TA visualization in over 90% of patients. The measurements of TAD by RT3DE and MRI showed good correlation without significant difference between both techniques. When TAD2D measurements were compared with the TAD3D and TADMRI measurements, 2D measurements were significantly underestimated. Analysis of TA motion by M-mode, 2DE and tissue Doppler imaging has been studied as a feasible marker for RV function reflecting the longitudinal RV shortening and lengthening. All these analyses described the physiological behaviour of TA plane systolic motion towards the apex along the RV long axis [5\u201310]. The TA plane systolic displacement is not influenced by its complex structure and asymmetrical shape [18] In the present study TA circumferential and horizontal motion along the RV short axis was assessed by RT3DE. TA motion along the RV short axis, which was fairly correlated with RV function that was assessed by MRI as a standard method for assessing RV ejection fraction. Fair and not good correlation may be explained by the meridional motion of the muscles and other structures of the RV inflow region. Furthermore, the myocytes are disposed longitudinally in the inflow region and more sensitive to meridional stress [19]. Despite this fair correlation, the summation of TA motion along both RV long and short axes increases the accuracy and correlation values for estimation of RV function [20].\nStudy limitation\nThe main limitation of this study is that RT3DE images more critically depend on image quality than 2DE images and the value of RT3DE should be assessed in a more non-selected (image quality). Due to the high cost of MRI, a small number of patients were included.\nConclusions\nRT3DE helps in accurate assessment of TA comparable to MRI, while 2DE could not be relied on due to underestimation. This may have important implications in the TV surgical decision-making processes. RT3DE analysis of TA function could be used as a marker of RV function.","keyphrases":["tricuspid annulus","real-time three-dimensional echocardiography","magnetic resonance imaging"],"prmu":["P","P","P"]}
{"id":"J_Med_Internet_Res-7-1-1550637","title":"What Is eHealth (4): A Scoping Exercise to Map the Field\n","text":"Background Lack of consensus on the meaning of eHealth has led to uncertainty among academics, policymakers, providers and consumers. This project was commissioned in light of the rising profile of eHealth on the international policy agenda and the emerging UK National Programme for Information Technology (now called Connecting for Health) and related developments in the UK National Health Service.\nIntroduction\nThe application of information and communications technology (ICT) in health care has grown exponentially over the last 15 years and its potential to improve effectiveness and efficiency has been recognized by governments worldwide [1]. National strategies aimed at developing health information infrastructures and \u201cinfostructures\u201d are emerging across North America, Australia, Europe and elsewhere [2-5]. These are united by a vision to improve the safety, quality and efficiency of patient care by enabling access to electronic health records and by supporting clinical practice, service management, research and policy though availability of appropriate evidence and data. In addition, these strategies emphasize the importance of standards and policies for ensuring interoperability and data security, and many incorporate a commitment to facilitate consumer empowerment and patient self-care through provision of electronic information and\/or telemedicine facilities. In the United Kingdom, these principles are reflected in the National Information Strategy for Health and are being addressed via the UK National Programme for Information Technology (NPfIT, now called Connecting for Health) and related initiatives [6,7].\nWhile such initiatives have been taking place, the focus of health care information technology (IT) has been changing, from an emphasis on hardware, systems architectures and databases, to innovative uses of technology for facilitating communication and decision making, coupled with a growing recognition of the importance of human and organizational factors. At the same time, Internet technologies have become increasingly pervasive. In parallel, the language of health care IT has been changing, and references to the concept of eHealth have proliferated in international health policy, management and research arenas. Despite the clear interest in and apparent marketability of eHealth, it was not evident, at the time this research was commissioned, what exactly was meant by the term. It had been variously used as a synonym for health informatics, telemedicine, consumer health informatics and e-business, as well as more specific technological applications, but no consensus existed on its conceptual scope and it was unclear whether it indeed represented a new concept, or simply a linguistic change. An international call for definitions of eHealth posted in 2001 failed to generate any published responses and the call was updated in June 2004, suggesting that this is still a grey area [8,9].\nIn view of these uncertainties, it was considered important by the UK National Health Service (NHS) Research and Development Programme to define eHealth and to assess its scope and value for the future of health care, in particular to synthesize the available evidence relating to its potential impact, likely trajectory, and implications for service development and organization. The current paper reports descriptive work to profile and define the field, which was conducted independently of, but complements, the systematic review of definitions of eHealth provided elsewhere in this volume [10]. This work produced a framework for locating evidence on the effectiveness, promise and challenges of eHealth, as well as recommendations for future research, which are reported elsewhere [11].\nPotential areas of eHealth considered at the outset of the project are shown in Table 1. This was derived by group discussion among the research team, utilizing team members' a priori knowledge of topics and issues in medical informatics (drawing on backgrounds in health care research, practice, policy, and computing), key eHealth discussion papers, and the results of a preliminary Medline search suggesting that eHealth is closer to the emerging area of health informatics than to medical informatics as a whole. While it was established that eHealth is about the use of information technology to facilitate patient and citizen health care or service delivery, rather than technology per se, uncertainty remained about what specific topics or issues, among those shown, fall within the scope of, or have relevance to, the concept.\nIt was recognized that in order to fully explore the area, multiple sources of information would need to be examined. While identifying the scope of eHealth research was a crucial objective, the published research literature presents a filtered record of activity and thinking and, given the fast-moving pace of the field and its importance beyond academia, nonresearch sources are likely to yield rich information about the current status of eHealth and future trends. For this reason we conducted two parallel, large scale reviews\u2014one focusing on the medical and related scientific literature and the other drawing on alternative sources available via the World Wide Web, including independent scoping exercises (of which there have been several), policy documents and technology reports. The results of these exercises were converged in order to derive a conceptual map and are considered together in this report.\nTable 1\nPotential eHealth areas and issues considered at the outset of the project\nWhat\nissues currently dominate eHealth?\nWhat is going on in eHealth?\nWhat emerging technologies are likely to impact on health care?\nHow does research inform eHealth?\nHow do developments in eHealth inform research?\nProfessional Clinical Informatics- Decision aids for practitioners (eg, prompts, reminders, care pathways, guidelines)- Clinical management tools (eg, electronic health records [EHRs\/EPRs], audit tools)- Educational aids (guidelines, medical teaching)- Electronic clinical communications tools (eg, e-referral, e-booking, e-discharge correspondence, clinical email\/second opinion, laboratory test requesting\/results reporting, e-shared care)- Electronic networks (NHS-Net and disease-specific clinical networking systems)- Discipline\/disease-specific tools (eg, diabetes informatics)- Telemedicine applications (for interprofessional communication, patient communication and remote consultation)- Subfields eg, nursing & primary care informatics) \nElectronic Patient\/Health Records (EPR, EHR)- Electronic medical records. Record linkage. The Universal Patient Indicator. Databases and population registers.- Achieving multiprofessional access. Technical and ethical issues.- Data protection\/security issues- Patient access and control- Integration with other services (eg, social work, police)- Clinical coding issues (terminologies, etc)\u00a0Healthcare Business Management- Billing and tracking systems- Audit & quality assessment systems\nConsumer Health Informatics- Decision aids for patients facing difficult choices (eg, genetic screening)- Information on the web and\/or digital TV (public information and educational tools for specific clinical groups)- Clinician-patient communication tools:1. Remote: Clinical email and web-based messaging systems for consultation, disease monitoring, service-oriented tasks (eg, appointment booking, prescription reordering).2. Proximal: Shared decision making tools, informed consent aids3. Mixed: On-line screening tools (eg, for depression) and therapeutic interventions (eg, cognitive behaviour therapy)- Access and equity issues (data protection issues, the Digital Divide)- Quality issues for health information on the net- \u201cvirtual\u201d health communities\nNew Technologies- Satellite communications (eg, for remote medicine )- Wireless networks (eg, within hospitals, across geographical areas)- Palmtop technologies (for information, for records)- New mobile telephones- Digital TV (for disseminating health information & communicating with patients)- The WWW and it's applications for health (issues: quality control, confidentiality, access) NHS-Direct etc.- Virtual reality (eg, remote\/transcontinental surgery)- Nanotechnology- Intersection of bioinformatics and health informatics.\nResearch Input- Development -Need for user involvement in product conception, design and testing. Iterative development. Needs assessment, accessibility and usability research. Multi-faceted expertise required.- Implementation \u2013 Understanding people and organizational factors eg, system acceptability, resistance to change etc. Use of tailored implementation strategies.- Innovative methods for mapping functional and technology needs eg, place of systems in the organization - Knowledge management, systems approaches, communication networks models, organizational development to map pathways.- EvaluationFormative, as above, also:Outcome assessment to establish impact of new systems on clinical outcomes, processes and costs. )\nResearch Outcomes- Potential of electronic databases such as population registers for epidemiological research.- Research into the impact or use of informatics tools suggests appropriate and cost-effective priorities for policymakers.- Areas of cross-over (eg, bioinformatics)\nMethods\nAssessing the Taxonomic Structure of Research Databases and the Presence of eHealth\nIn the formative stage of the project, we explored the subject taxonomies, or thesauri, of multiple databases of abstracts in order to identify high-level subject headings which could be used to profile the volume and content of the medical informatics literature and to construct searches for pertinent evidence. In the case of Medline the thesaurus containing a hierarchical controlled vocabulary is referred to as Medical Subject Headings, or MeSH (see below). As part of this we sought to assess whether eHealth was explicitly represented within these thesauri. A further objective was to determine the ontological structure of the databases in relation to medical informatics and eHealth and the implied relationships between alternative subfields.\nThe databases examined were Medline (PubMed), the Cumulative Index of Nursing and Allied Health Literature (CINAHL), the Science Citation Index (SCI), the Social Science Citation Index (SSCI), the Cochrane Library Database (including Dare, Central, NHS Economic Evaluation Database [NHS EED], Health Technology Assessment [HTA] database, NHS EED bibliographic) and Index to Scientific and Technical Proceedings (ISTP, now known as ISI proceedings), all of which predate the targeted search period.\nExploring the Composition of the Medical Informatics Literature Using the Existing MeSH Thesaurus\nMeSH has been developed (and is constantly updated by) the US National Library of Medicine. It consists of sets of terms naming descriptors in a hierarchical structure that permits searching at various levels of specificity. At the most general level of the hierarchical structure are very broad headings such as Anatomy or Information Science. More specific headings are found at more narrow levels of the eleven-level hierarchy, such as Ankle or Medical Informatics. There are 22568 descriptors in MeSH.\nHistorical trends in the literature indexed by the individual Medline MeSH terms subsumed within the broad Medical Informatics category were assessed for the period 1987 to 2003, and part way through 2004. Individual MeSH definitions were examined to assess the range and nature of the topics covered and to clarify which are most clearly related to common conceptions of eHealth (eg, specific applications of information technology (IT) to health care versus technical issues). The number of publications in Medline was profiled by year, as was the type of publication, subject to the limitations of the Medline categorization scheme (Randomized Controlled Trial\/Controlled Trial\/Meta-analysis\/Review). In addition, the MesH tree was compared with an expert-derived taxonomy from the International Medical Informatics Association (IMIA) in order to assess its coverage of key areas and its merits as a means of identifying appropriate literature.\nUsing eHealth as a Search Term\nApplying eHealth as free-text search term to multiple databases offered a \u201cgrounded\u201d method of defining the field, as represented in the research literature. In order to identify publications specificially relating to eHealth and to place the concept within the wider medical informatics literature, all the databases described previously were searched for the presence of the word eHealth or its variants in the title or abstract for the period January 1, 1997 to December 31, 2003 (search string: Ehealth OR e-health OR e*health). Results were organized to show the number of articles arising each year, the journals in which they appeared, and the range of topics covered.\nProfiling the Literature From Wider Web-Based Sources\nMixed methods were used to (a) identify current commentary and analysis relating to the emergence, nature, scope and potential of eHealth, and (b) locate evidence and opinions on general trends in technology and technology adoption with direct or indirect relevance to eHealth now or in the future. Relevant terms (including e health, e-health, ehealth, healthcare information technology and healthcare computing) were applied, singly and in combinations, to the Google search engine, which indexes over 8 billion URLs and ranks results by relevance and link popularity. In addition, websites previously identified as being likely to contain information relevant to eHealth were visited directly and scrutinized for pertinent information. In some cases, this was guided by the results of preliminary Google searches or by following up leads suggested in documents found earlier on, while in others it was guided by the existing knowledge of team members. As the searches were predominantly opportunistic and iterative in nature, it is inappropriate to try to document them exhaustively; however, the following types of information were targeted:\nprevious exercises to map, scope or define eHealth;\nwhite papers, technical reports, predictions and early research reports on aspects of technology in health care, eHealth related policy, evaluation and trends, from the United Kingdom, Europe and beyond;\nfunding programmes for eHealth- and\/or health-and-technology - focused research and development;\nrelevant articles from computing and information science-focused academic publications;\neHealth and health technology-focused websites, web logs and online journals, online ehealth news feeds, email discussion groups and email newsletters;\nonline sources with a focus on human-computer interaction, usability and accessibility, with specific attention on health care issues;\ntechnology-oriented news websites profiling general and health-related trends and developments;\nonline studies, reports and statistical surveys relating to general technology take-up; consumer purchasing trends; attitudes and strategies of consumers and clinicians towards adoption of technology in general and for health care-focused tasks in particular; evaluation of the effectiveness of technological innovation, in the health care sector and beyond.\nGiven the increasing online availability of refereed academic literature there was inevitably some overlap between the information identified by the two searches.\nAggregating and Analyzing Definitions of eHealth\nScientific abstracts identified using the key word search were examined in order to assess the presence of definitions. While hand searching of full text articles was not a primary objective, this was done where easy Web-based access to this information was available. In the case of Web-based reports or commentary the definition was extracted from the page in which it appeared or was quoted. In both cases the initial extraction was performed by one research fellow and the results checked for inclusion eligibility by a second investigator. Our aim was not to perform an exhaustive and systematic review of definitions (because of time constraints) but to aggregate those appearing most easily and commonly in the research and wider arenas, as a means of supplementing our wider scoping study. The aggregated definitions were then analyzed thematically in order to assess the applications, stakeholders, contexts and theoretical perspectives targeted, so that the heterogeneity of conceptualizations could be determined. They were also considered with reference to the perspectives of the defining individual or organization and associated clarifications within the source document.\nResults\nAssessing the Taxonomic Structure of Research Databases and the Presence of eHealth\nOf the databases of scientific abstracts consulted, only Medline has a comprehensive hierarchical taxonomy of descriptors for the broad field of medical informatics. This part of the MeSH tree is shown in Figure 1. Medical informatics is also represented on CINAHL; however the subtree is relatively shallow and undifferentiated, forming only a small branch of the higher Information Science category, with many potentially relevant areas subsumed within other branches.\nThat eHealth has yet to be explicitly included among these thesauri, indicates the relative youth of the topic and the lack of an agreed conceptual definition. The literature relevant to eHealth is thus distributed among a range of existing MeSH fields.\nThe Medline MeSH structure for Medical Informatics contains 3 main subbranches: Public Health Informatics, Medical Informatics Computing, and Medical Informatics Applications. Examining the definitions of these and their lower order MeSH descriptors indicates that the Medical Informatics Applications tree encompasses the greatest number of component categories relevant to eHealth, taken broadly as the use of information and communication technologies to facilitate health care. For example, it subsumes the lower-order categories of Decision Making, Computer Assisted (which subsumes Computer Assisted Therapy and Diagnosis, among others); Information Systems (electronic information systems, networks, clinical decision support) and Information Storage and Retrieval (databases, laboratory information systems, etc). In contrast, Medical Informatics Computing is mainly characterized by an emphasis on systems and hardware, although it does contain MeSH descriptors relevant to eHealth \u2014 most importantly Internet, which may appear in eHealth publications as a specific technology or an application of technology. Public Health Informatics is concerned with the application of information and computer sciences to public health practice, research, and learning. Although this potentially encompasses eHealth-relevant research (for example, use of information and communications technologies for population health surveillance), the term was only recently introduced and has yet to contain any subcodes, limiting its usefulness at the present time. While the broader taxonomic categories each have their own character, there is clearly overlap between them. For example, decision support systems appear within both Medical Informatics Applications and Medical Informatics Computing, and electronic databases are a common feature in medical informatics applications, as well as representing a type of system.\nComparison of the MeSH tree with an expert-derived conceptual map endorsed by the International Medical Informatics Association (IMIA) revealed interesting differences in terms of the breadth of included concepts and their structural relationships (Table 2) [12]. For example, human and organizational factors appear to be underrepresented within Medline, while applications for consumers do not have a specific MeSH term (however, the IMIA taxonomy also appears to underrepresent consumer issues). This reflects the historical evolution of the MeSH hierarchy, which has been added to as the need arose by elaborating upon existing structures. Nonetheless, all the main areas apparently relevant to eHealth were encompassed by the MeSH tree and we are confident that using it as the basis of our search enabled the majority of pertinent literature to be identified.\nFigure 1Hierarchy of MeSH descriptors found below the Medical Informatics descriptor in the MeSH tree\nTable 2\nMedical informatics scientific content map endorsed by the International Medical Informatics Association (IMIA) [12]\nApplied Technology\nInformation Technology Infrastructure\nData-Infrastructure Related\nApplications and Products\nHuman-Organizational\nEducation and Knowledge\nAlgorithms\nBioinformatics\nBiosignal processing\nBoolean logic\nCryptology\nHuman genome related\nHuman interfaces\nImage processing\nMathematical models in medicine\nPattern recognition\nArchival-repository systems for medical records- EPR-CPR-EMR\nAuthentication\nChip cards in health care\nDistributed systems\nHealth professional workstation\nInterfaces\nKnowledge based systems\nNetworks\nNeural networks\nPen based\nSecurity\nSpeech recognition\nStandards\nSystems architecture\nTelehealth\nUser interfaces\nClassification\nCoding systems\nConcept representation-preservation\nData acquisition-data capture\nData analysis-extraction tools\nData entry\nData policies\nData protection\nDatabase design\nIndexing\nSyntax\nLanguage representation\nLexicons\nLinguistics\nModeling\nNomenclatures\nStandards\nTerminology-vocabulary\nThesaurus tools\nBiostatistics\nClinical trials\nComputer-supported surgery\nDecision support\nDiagnosis related\nDisease management\nEPR-CPR-EMR\nEpidemiological research Hosp IS\nEvent-based systems\nEvidence based guidelines\nExpert systems\nHealth services research\nHealth Information Systems management\nKnowledge-based systems\nLaboratory data\nImage processing\nOperations\/resource management\nOutcomes research and measurement\nQuality management\nPatient identification\nPatient monitoring\nMinimum data sets\nSupply chain\nTelematics\nTelemedicine\nAssessment\nCompliance\nCognitive tasks\nCollaboration\nCommunication\nEconomics of IT\nEthics\nImplementation-deployment\nDiffusion of IT\nEvaluation\nHuman Factors\nLegal issues, implementing national laws\nManagement\nManaging change\nNeeds assessment\nOrganizational redesign processes\nOrganizational transformation\nPlanning\nPolicy issues\nPrivacy\nProject management\nSecurity\nStrategic plans\nUnique identifiers\nUser-computer interface\nBibliographic\nCognitive learning\nComputer aided instruction\nComputer-supported training\nConsumer education\nContinuing education\nDigital libraries\nE-Business\nHealth\/medical informatics education\nInformation management- dissemination\nKnowledge bases\nKnowledge management\nLearning models\nOnline\/distance education\nClinical Disciplines: Anesthesia, Behavioral, Cardio\/Thoracic, Cardiovascular, Dentistry, Dermatology, Emergency Medicine, Environmental Health, Gastroenterology, Human Genetics, Internal Medicine, Neurosurgery, Nursing, Obstetrics & Gynecology, Ophthalmology, Orthopedics, Pathology, Pediatrics, Pharmacy, Primary Care, Psychiatry, Radiology, Surgery, Urology\nExploring the Composition of the Medical Informatics Literature Using Existing Taxonomic Systems\nFigure 2 describes trends in the volume and nature of the literature indexed by the Medical Informatics MeSH descriptor (note that searching for MeSH terms in PubMed automatically includes the more specific MeSH terms in a search). There has been a steady growth in the volume of medical informatics research literature. The annual number of publications increased from 1987 to 2003 five-fold.\nFigure 2Number of publications over time indexed with the MeSH descriptor Medical Informatics\nPublications indexed with MeSH keywords from each of the 3 main medical informatics MeSH subtrees (medical informatics computing, medical informatics applications, public health informatics) all follow this steady upwards trend, as do most narrower MeSH (eg, Information Systems; Therapy, Computer Assisted). However, the frequency of publications concerned with Clinical Laboratory Information Systems (Figure 3), appears to be decreasing, while research concerned with computer-assisted diagnosis increased rapidly in 2003 (Figure 4).\nA breakdown of Medical Informatics MeSH, including definition, year of introduction, number and type of publications is supplied in Multimedia Appendix 1.\nFigure 3Number of publications over time indexed with the MeSH descriptor Clinical Laboratory Information Systems\nFigure 4Number of publications over time indexed with the MeSH descriptor Diagnosis, Computer Assisted\nUsing eHealth as a Search Term\nAs mentioned previously, there are currently no MeSH or equivalent coding categories in any of the databases searched which explicitly incorporate the term eHealth or its variants in their thesauri. This suggests that articles making reference to eHealth are being absorbed within existing classification schemes, such as Medline's Medical Informatics taxonomy.\nWhen duplicates across databases were discarded we identified a total of 392 publications which explicitly referred to eHealth in the title, abstract, or journal title. Of these, most were represented in Medline. Appearing only in the Medline database were 283 (72%) articles, 54 (14%) only on the CINAHL database, and 55 (14%) only on the SCI, SSCI and ISTP databases.\nFigure 5 illustrates trends in the volume of eHealth publications appearing across databases over time. This shows that the term did not start to be used in the research literature until 2000. References to eHealth showed a dramatic rise in 2000 to 2001 and, despite a small dip in 2002 a general upward trend persists. Note that we also retrieved publications from the Journal of Telemedicine and E-health which were picked up due to the journal name, not necessarily because they dealt with eHealth.\nFigure 5Number of publications found using the search term eHealth (or variants) in 5 research databases by year.\nIn Which Journals Do Publications Using the Term eHealth Appear?\nIn our study, publications containing the term eHealth were found in 154 different journals. A research fellow classified these by type, using a scheme agreed by the research team. The number of articles appearing within each journal were documented. Of the 387 publications found across multiple databases (after eliminating 5 that were clearly irrelevant), 77 appeared in clinical journals, 61 in health-services - related journals, 7 in finance-related journals, 4 in legal journals, 3 in journals related to medical education, and 28 in other journals not easily categorized. The journal titles with the most articles containing the term eHealth (n=9 for each journal) were the Journal of Medical Internet Research, Managed Care Interface, and Journal of AHIMA \/ American Health Information Management Association. The majority of publications were IT-related (207): however, among these, 116 articles were published in the Journal of Telemedicine and E-health, which were mainly picked up due to the journal name: only 4 articles actually contained the term eHealthin the abstract or title. Further details are provided in Table 3 and a detailed breakdown of journal titles is given in Multimedia Appendix 2.\nTable 3\nTopical areas of journal titles containing articles using the term eHealth\nMain Topic Area\nMore Specific Topics\nNumber of Publications (%)\nInformation Technology\nTelemedicine\n124* (32%)\nMedical Informatics\n35 (9%)\nInternet\n23 (6%)\nMedical Computing\n6 (1.5%)\nBiotechnology\n2 (0.5%)\nOthers\n17 (4 %)\nSub total\n207 (53%)\nClinical\nSpecialist Medical\n30 (8%)\nGeneralist Medical\n16 (4%)\nNursing\n13 (3%)\nOthers\n18 (4%)\nSub total\n77 (19%)\nHealth Services\nManagement\n30 (8%)\nCase Management\n16 (4%)\nOthers\n15 (4%)\nSub total\n61 (16%)\nFinance\nSub total\n7 (2%)\nLegal\nSub total\n4 (1.5%)\nEducation\nSub total\n3 (1.5%)\nOthers\nSub total\n28 (7%)\nTotal\n387 (100%)\n* Of the 124 publications listed under telemedicine, 116 articles were published in the Journal of Telemedicine and E-health, of which only 4 articles actually contained the term e-health\nWhat Topics are Covered in the Literature Using the Term eHealth?\nIn our study, in order to identify the topics dealt with in papers explicitly referring to eHealth, article titles and abstracts were examined by a research fellow and classified using narrative descriptors. This indicated that the most common topics are related to telemedicine (25% of publications) or the Internet (13%), while some (6%) are concerned with issues such as the scope of eHealth, future trends, or progress and challenges in the field. Note that this view is possibly biased towards the telemedicine field, as all articles published in the Journal of Telemedicine and E-health were retrieved, even if they did not mention eHealth specifically. Other topics are distributed across a range of diffuse areas such as antiterrorism and medical errors, none of which is represented by more than 4 papers (hence relevant percentages have not been calculated). A heuristic summary is provided in Figure 6, which highlights the key topics and subtopics identified. These results are based on preliminary analysis; further validation work is underway. \nFigure 6Map of topics in published articles using the term eHealth\nDefinitions of eHealth\nWe identified 36 definitions of eHealth [13-52] appearing in published scientific abstracts and Web-based information sources (Table 4). As stated previously, our aim was not to perform an exhaustive and systematic review of definitions (which would have necessitated hand searching of full-text articles and reference lists), but to aggregate the most salient and easily accessible examples. Since many research databases are Internet accessible, there was some overlap between the definitions obtained by the two methods; however, they did yield largely unique results. In total, 36 definitions were identified. Definitions 1 to 15 were accessed via the research literature and 16 to 36 via the independent online searches, while 1, 5, 6, 7, 15 and 28 emerged from both searches.\nDefinitions were analyzed thematically in order to highlight specific technologies, applications or stakeholders referred to, and other theoretical concepts addressed, as detailed in Table 4. Analysis was initially performed by one investigator and the results checked by two others, thereby establishing agreement.\nOur analysis suggests that there is significant variability in the scope and focus of existing definitions of eHealth both within the research literature and relevant sources on the World Wide Web. In terms of its functional scope, most definitions conceptualize eHealth as a broad range of medical informatics applications for facilitating the management and delivery of health care. Purported applications include dissemination of health-related information, storage and exchange of clinical data, interprofessional communication, computer-based support, patient-provider interaction and service delivery, education, health service management, health communities, and telemedicine, among others. A few narrow the concept down to specific applications, such as telemedicine or e-business, but these are the exceptions. While the range of applications is broad, a general theme relates to communication. One example is \u201cE-health is connectivity; it is transactional; it is clinical. It is informational, interactive and interventional.\u201d[43]\nThe majority of definitions (n=24) specify the use of networked information and communications technologies, primarily the Internet, and digital data, thus differentiating eHealth from the broader field of medical informatics, which incorporates \u201charder\u201d technologies, such as scanning equipment, and bioinformatics research which tends to take place in isolation and is less directly applicable to health care service delivery. It is acknowledged that the Internet \u201c\u2026has the reach, the infrastructure, and the acceptance to achieve widespread change\u201d [17] and it is envisaged that \u201cInternet technology may rank with antibiotics, genetics and computers as among the most important changes for medical care delivery.\u201d[16] Only 1 definition makes specific reference to harder technologies such as nanotechnology, robotics and laboratory tools [27], although another refers to Internet-compatible ICTs such as digital TV [40]. Of the 36 definitions identified, a sizable proportion make reference to telemedicine or telecare, either explicitly (7 examples) or in terms commonly used to describe these areas, such as delivery of care over distances. In most cases this is presented as part of a wider sphere of applications, although the definition from NHS Wales clearly identifies eHealth with telemedicine and telecare [45]. We identified 6 definitions that make explicit reference to business or e-business, although others contain related ideas such as the online trading of goods and services. In the majority of cases, such commercial applications are presented as merely one expression of eHealth.\nIn terms of the stakeholders considered to be the users or targets of eHealth, many definitions emphasize applications for providers and organizations\u2013particularly those stressing electronic data exchange for clinical and administrative purposes. Others emphasize provision of information, education and services to consumers, including patients and \u201ccitizens\u201d, with a small number clearly identifying eHealth with consumer health informatics [14, 46, 50]. Nevertheless the majority appear to encompass applications for all stakeholder groups, whether specified or implied by the breadth of the definition.\nThere is also variation in the degree to which alternative definitions consider wider theoretical issues, such as the influence of eHealth on society or on professional behaviour. Several highlight the changing cultural environment of health care; particularly growing patient empowerment (access to information and ability to use it), and point to the potential of eHealth to facilitate doctor-patient communication, partnership and shared decision making. Others emphasize the changes required to ensure that eHealth reaches its full potential, recognising that it requires new ways of working and attitudes and must take account of human and organizational influences affecting technology adoption and change. More broadly, eHealth is said to require a fundamental rethinking of health care processes and a commitment for networked global thinking to improve health care [22]. Overall, the definitions suggest a general excitement and optimism about the potential of this rapidly evolving field to improve health care processes and patient outcomes, and many clearly identify projected benefits such as improved clinical decision making, efficiency and safety.\nTable 4\nDefinitions of eHealth identified from searching databases of scientific abstracts and wider Web-based information sources\nDefinition\nSource\nDate\nTechnologies Specified\nApplications Specified\nStakeholder Focus\n(and Other Concepts)\n1) \u201ce-Health is a consumer-centred model of health care where stakeholders collaborate, utilizing ICTs, including Internet technologies to manage health, arrange, deliver and account for care, and manage the health care system\u201d\nAlvarez [13], based on Ontario Hospital e-health Council [14]\n2002(2002)\nICTs including Internet\nGeneral: manage health, arrange, deliver and account for care, and manage the health care system\nConsumer centered but also emphasizes collaboration with providers\n2) \u201cHealthcare delivery is being transformed by advances in e-health and by the empowered, computer-literate public. Ready to become partners in their own health and to take advantage of online processes, health portals, and physician web pages and e-mail, this new breed of consumer is slowly redefining the physician\/patient relationship. Such changes can effect positive results like improved clinical decision-making, increased efficiency, and strengthened communication between physicians and patients.\u201d\nBall and Lillis [15]\n2001\nInternetonline processes, health portals, physician en-pages, email.\nGeneral: healthcare delivery\nConsumers (Change. Citizen empowerment. Physician\/patient relationship\/ communication. Improved clinical decision making, efficiency)\n3) \u201cThe \"e-health\" era is nothing less than the digital transformation of the practice of medicine, as well as the business side of the health industry\u2026. The Internet is the next frontier of health care. Health care consumers are flooding into cyberspace, and an Internet-based industry of health information providers is springing up to serve them. Internet technology may rank with antibiotics, genetics, and computers as among the most important changes for medical care delivery.\u201d\nCoile [16]\n2000\nInternet\nThe practice of medicine as well as the business side of the health industry\nConsumers and providers(Change. New frontiers. Transformation of medical practice.)\n4) \u201cE-health\u2014any electronic exchange of healthcare data or information across organizations\u2014reflects an industry in transition\u2026. The Internet clearly drives the development and adoption of e-health applications; standing alone, it has the reach, the infrastructure, and the acceptance to achieve widespread change.\u201d\nDeLuca and Enmark [17]\n2000\nInternet\nElectronic exchange of healthcare data or information across organizations\nNot specified. Implies focus on professional & organizational levels(Change)\n5) \"a new term needed to describe the combined use of electronic communication and information technology in the health sector... the use in the health sector of digital data - transmitted, stored and retrieved electronically - for clinical, educational and administrative purposes, both at the local site and at distance\"\nDella Mea [18], based on Mitchell [19]\n2001[1999]\nCombined use of electronic communication in and IT in the health sector. Digital data transfer\nTransmission of digital data locally and across distances, for clinical, educational and administrative purposes\nProfessionals and organizations\n6) \u201ce-health is the use of emerging information and communication technology, especially the Internet, to improve or enable health and healthcare.\u201d\nEng [20], based on Eng [21]\n2004[2001]\nEmerging ICTs, especially the Internet\nGeneral: To improve or enable health and health care\nNot specified but implies consumers and providers\n7) \u201ce-health is an emerging field in the intersection of medical informatics, public health and business, referring to health services and information delivered or enhanced through the Internet and related technologies. In a broader sense, the term characterizes not only a technical development, but also a state-of-mind, a way of thinking, an attitude, and a commitment for networked, global thinking, to improve health care locally, regionally, and worldwide by using information and communication technology.\u201d\nEysenbach [22]\n2001\nBroad definition encompassing many aspects of health informatics but focusing on the Internet and related technologies\nDelivery of health services and information\nNot specified. Implies consumers and providers.(\u201ca state of mind, a way of thinking, an attitude and commitment for networked, global thinking to improve healthcare\u2026\u201d)\n8) \u201cMany of the major forces of change impacting health care today have technological underpinnings, and many of the less desirable impacts may have technological solutions. Two related technological forces are transacting business, online (e-business) and delivering health care online (e-health).\u201d\nEllis and Schonfeld [23]\n2001\nInternet\nGeneral: Delivering healthcare\n\u201cDelivering\u201d implies focus on professionals(Change.Relationship between eHealth and eBusiness)\n9) \u201cehealth includes use of the internet or other electronic media to disseminate health related information or services.\u201d\nGustafson and Wyatt [24]\n2004\nInternet or other electronic media\nDissemination of health related information or services\nImplies consumers\n10) \u201cAs a special expression of e-business in the health service the sphere of e-health has developed in recent years which increasingly manifests itself in the internet via health portals. Next to the transmitting of medical contents, the offer of community functions and the trading with goods from the medical sector, these health portals now increasingly provide advisory services for citizens by medical experts.\u201d\nKhorrami [25]\n2002\nIncreasingly manifests itself in the Internet via health portals.\ne-businessHeath advice. Information exchange. Community functions. Advisory services for citizens\nConsumersHealthcare organizations\n11) \u201ce-Health (use of interactive communication and information technologies to engage in health-related activities) includes not only telehealth-related media and telecommunications but also a wide array of consumer and healthcare provider activities that use the Internet.\u201d\nMaddox [26]\n2002\nInteractive ICT, telehealth, internet etc\nGeneral: health-related activities\nConsumer and healthcare provider\n12) \u201c \u2026technologies with practical applications that have the potential to improve both quality of and access to healthcare\u2026.Telemedicine, Health Information Systems, Databases, Genomics, Biotechnology, eLearning, Continuing Professional Development, Nanotechnology, Drug Treatment Technologies, Decision Making Tools, Diagnostic Aids, eLibraries, Laboratory tools, and Robotics are all innovative or 'disruptive' technologies that promise a better health for our children.\u201d\nMcConnell [27]\n2002\nWide range of digital technologies\u00a0\nWide range of informatics applications that may contribute to improved quality of and access to healthcare\nProviders and patients(Quality. Access. \u201cDisruptive technologies\u201d)\n13) \u201ce-Health offers the rich potential of supplementing traditional delivery of services and channels of communication in ways that extend the healthcare organization's ability to meet the needs of its patients. Benefits include enhanced access to information and resources, empowerment of patients to make informed healthcare decisions, streamlined organizational processes and transactions, and improved quality, value, and patient satisfaction.\u201d\nNazi [28]\n2003\nNot specified\nDelivery of services Communication. Access to information and resources.\nPatients (empowerment, satisfaction)Organization (efficiency and quality)\u00a0\n14) \u201cthe use of the Internet for health purposes\u201d\nProvost et al [29]\n2003\nInternet\nGeneral: \u201cHealth purposes\u201d\nAny\n15) \"a means of applying new low cost electronic technologies, such as 'web enabled' transactions, advanced networks and new design approaches, to healthcare delivery. In practice, it implies not only the application of new technologies, but also a fundamental re-thinking of healthcare processes based on using electronic communication and computer-based support at all levels and for all functions both within the healthcare service itself and in its dealings with outside suppliers. eHealth is a term which implies a way of working rather than a specific technology or application\".\nRichardson [30], based on Silicon Bridge [31]\n2003\u00a0[2001]\nInternetNew low-cost electronic technologies such as \u201dweb enabled\u201d transactions and advanced networks\u201d\u00a0\nGeneral: \u201cHealthcare delivery\u201d\u00a0Electronic communication and computer-based support at all levels and for all functions\n\u2018Healthcare delivery [and] processes' implies organizational\/ professional level(\u201c\u2026a way of working\u201d)\n16) \u201cThe healthcare industry's component of business over the internet.\u201d\nBlutt [32]\u00a0\u00a0\n2001\nInternet\nBusiness\nImplies organizations\n17) \"The application of the Internet and other related technologies in the healthcare industry to improve the access, efficiency, effectiveness, and quality of clinical and business processes utilized by healthcare organizations, practitioners, patients, and consumers to improve the health status of patients.\"\nBroderick and Smaltz [33]\n2003\nInternet and related technologies\nImprovement of access, efficiency, effectiveness and quality of clinical and business processes\nOrganizations, practitioners, patients, consumers\n18) \u201ceHealth includes the development, application and implementation of technology to improve effectiveness in healthcare. But it also includes getting it out there wherever it's needed in the service and making it happen across the service. It includes the use of telemedicine and clinical systems used for diagnosis and care pathways. We also apply the term to the policies and protocols that assure the confidentiality and security of sensitive data. Most of all it includes those aspects that support major change of working practice - training, support and Organisational Development.\u201d\nChisholm [34]\n2003\nTechnology\nTelemedicineClinical systems for diagnosis and care pathwaysPolicies and protocols\nNot specified, but implies organizational\/professional focus\u00a0(Importance of organizational and professional behaviour change recognized. Also confidentiality and security issues.)\n19) \u201c\u2026using Information and Communications Technologies to ensure the right treatment to each patient, specialised to each individual's context and situation, and to deliver healthcare where patients and providers need not be in the same place at the same time.\nCSIRO [35]\nUn-dated\nICTs\nDelivery of personalized patient care. Telemedicine implied\nNot specified. Implies provider focus but also interaction with patients\n20) \"Put simply, e-health is a wide-ranging area of social policy that uses new media technologies to deliver both new and existing health outcomes. In the UK, it incorporates everything from NHS Direct online to Internet pharmacies to webcast operations involving consultants in another country\u2026At the moment, the main focus of e-health is on patient empowerment and self-care. As the area develops, e-health could expand to include online long-term disease management, personalised health checks, and more efficient primary care services due to informed patients accessing the healthcare system at the most appropriate point.\"\nGJW Government Relations Ltd [36]\n2000\nNew media technologies\nOn-line health informationLong-term disease management and patient self-careTelemedicine\nPatients and professionals(Patients emphasized)\n21) \u201csomething to do with computers, people, and health\u201d(Centre for Global e-Health Innovation, 2003)\nGustafson [37]\n2003\nComputers in general\nVery broad \u2013 computers, people and health\nImplies all stakeholders\n22) \u201cthe application of information and communication technologies (ICT) across the whole range of functions which, one way or another, affect the health of citizens and patients.\u201d\nEuropean Commission [38]\n2003\nICTs\nBroad \u2013 the whole range of functions which, in one way or another, affect the health of citizens and patients\nAll stakeholders. Providers, patients, citizens.\n23) \u201cthe emerging world of e-health can be defined as the application of information, communication and video technologies to the delivery of timely, professional and safe healthcare.\u201d\nEuropean Health Telematics Association [39]\n2004\nICT and video technologies\nBroad \u2013 delivery of timely, professional and safe care\nNot specified. Implies professional perspective.\n24) \u201cthe use of emerging interactive technologies (i.e., Internet, interactive TV, interactive voice response systems, kiosks, personal digital assistants, CD-ROMs, DVD-ROMs) to enable health improvement and health care services. For this Initiative, these technologies should focus primarily on health behavior change and chronic disease management for consumers\/patients.\u201d\nHealth e-Technol-ogies Initiative [40]\n2002\nEmerging interactive technologies (Internet, interactive TV, interactive voice response systems, kiosks, personal digital assistants, CD-ROMs, DVD)\nEnabling health improvement and health care services,chronic disease management, health behaviour change\nConsumers, patients\n25) \u201cthe use of ICT to support and improve healthcare\u201d\nHoving et al [41]\n2002\nICT\nGeneral: support and improve health care\nNot specified.\n26) \"eHealth means taking the most recent developments in computer and networking technology, and applying it to the problems facing the healthcare community in all its forms - eHealth is the endeavour to produce reliable, easy-to-use, highly-automated, accurate systems, so that health care professionals can spend less time and resources on finalising the paperwork, and more time doing what they do best - taking care of people's health!\"\nIBA eHealth [42]\nUn-dated\nRecent developments in computer and networking technology\nGeneral: Applying it to the problems facing the healthcare community in all its formsSpecific: administrative and clinical information to improve efficiency\nProfessionals(improved efficiency)\n27) \u201cThe \"e\" is for electronic. Placed before the word health, it implies all things transmitted and technological in health care, which help improve the flow of information and the process of health care delivery. \"E\" networks integrate isolated towers of information and create new knowledge through the creation of relational databases. The spectrum of \"E\" is broad and goes beyond the use of a computer as a box on the desktop. It includes wireless communication using hand-held devices and the storage and function by the microchip which is revolutionizing health care, as it is inserted into everything we use to diagnose, treat, record, sort, analyze, and conclude. It also incorporates electronic forms of care delivery, such as telemedicine, providing health care over a distance, communicating by sound and image transmission. E-health is connectivity; it is transactional; it is clinical. It is informational, interactive and interventional.\u201d\nMarcus and Fabius [43]\nUn-dated\nElectronic networks, relational databases. Wireless communication.\nAll things transmitted and technological in health care, which help improve the flow of information and the process of health care deliveryElectronic care delivery (telemedicine)Sound and image transmission\nNot specifiedConnectivity; communication, interactivity, intervention\n28) \"the health services organisation and societal approach to health and health services which result from the introduction of, and increasing access to, new digital technologies: including the Internet, other computerised networks and tele- or distant health care facilitated by new digital technologies\".\nNHS SDO Programme [44]\n2002\nNew digital technologies InternetOther computerized networks Telemedicine\nHealth service organization\u201cSocietal functions\u201d\nOrganizationsSociety (citizens)\n29) \u201cMore commonly known as \u201ceHealth\u201d, the headings of Telemedicine and Telecare are themselves subsumed under the framework category of \"health informatics\", which basically means the delivery of healthcare and medical knowledge through the application of advanced information and computer technologies.\u201d\nNHS Wales [45]\n2003\nAdvanced information and computer technologies\nTelemedicine and Telecare.\nNot specified.(Identified eHealth with telemedicine)\n30) \u201cThe big difference between yesterday's knowledge-based patient care and that of tomorrow is a fundamental premise that patients will explore the web world with a desire to learn more about their condition, including its treatment and prognosis. This has evolved into the concept of e-health\u201d\nPodichetty and Biscup [46]\n2003\nInternet\nPatient information and decision support\nPatients(Cultural shift to patient participation\/ empowerment in health care)\n31) \u201ceHealth signifies a concerted effort undertaken by some leaders in healthcare and hi-tech industries to harness the benefits available through convergence of the internet and healthcare\u2026\u201d\nRx2000 Institute [47]\nUn-dated\nInternet\nNone specified\nNot specified. Implies organizations(Harnessing benefits of converging internet and healthcare)\n32) \u201ceHealth describes the application of information and communications technologies (ICT) across the whole range of functions that help health. It is the means to deliver responsive healthcare tailored to the needs of the citizen.\u201d\nSilber [48]\n2003\nICTs\nBroad \u2013 the whole range of functions that help health\nCitizens (consumers, patients, public)\n33) \u201cE-health is a new term used to describe the combined use of electronic communication and information technology in the health sector OR is the use, in the health sector, of digital data-transmitted, stored and retrieved electronically-for clinical, educational and administrative purposes, both at the local site and at a distance.\u201d\nWHO [49]\nUn-dated\nICTsDigital data\nClinical, educational and administrative purposes, at the local site and at a distance\nOrganizations\/professionals\n34) \u201cUsing the internet and other electronic media to disseminate or provide access to health & lifestyle information or services\u201d\nWyatt [50]\n2003\nInternet and other electronic media\nAccess to health and lifestyle information or services\nPatients, public\n35) \u201ce-Health refers to all forms of electronic healthcare delivered over the Internet, ranging from informational, educational and commercial \"products\" to direct services offered by professionals, non-professionals, businesses or consumers themselves. e-Health includes a wide variety of the clinical activities that have traditionally characterized telehealth, but delivered through the Internet. Simply stated, e-Health is making health care more efficient, while allowing patients and professionals to do the previously impossible.\u201d\nWysocki [51]\n2001\nInternet\nDelivery of informational, educational and commercial \"products\"Direct delivery of servicesClinical activities traditionally characterized telehealth\nProfessionals, consumers, businesses(Making health care more efficient, while allowing patients and professionals to do the previously impossible)\n36) \u201cE-health is a very broad term that encompasses many different activities related to the use of the Internet for healthcare. Many of these activities have focused on administrative functions such as claims processing or records storage. However, there is an increasing use of e-health related to patient and clinical care.\u201d\nAmerican Telemed-icine Association [52]\n2001\nInternet\nAdministrative functions, patient and clinical care\nNot specified. Implies organizational and professional focus(increasing use of eHealth for patient and clinical care)\nDiscussion\nWe have established that eHealth is a new term which has yet to be formally represented in bibliographic research taxonomies but is part of the wider field of medical or health informatics. The Medical Informatics MeSH tree encompasses most topics likely to be classed as eHealth and is broadly compatible with an expert-derived taxonomy endorsed by IMIA. Since eHealth cuts across a range of health informatics topics a new MeSH term may neither be necessary nor appropriate at the present time. Topics related to eHealth are distributed across all component MeSH trees within the broader field, although most are represented by the Medical Informatics Applications tree, which emphasizes functions of technologies, rather than technologies themselves, and prioritizes delivery of clinical information, care or services. The medical informatics literature has grown steadily over the last 15 years although research on some topics, such as clinical laboratory information systems, is becoming less prevalent, while that on others, such as computer-assisted diagnosis, has recently increased rapidly, reflecting a change in emphasis from systems and database architectures to supportive applications.\nResearch articles explicitly referring to eHealth or its variants begun to appear in 2000 and are accumulating rapidly. The majority of such articles are indexed by Medline, although others appear in alternative databases. Such articles are published in a wide range of journals, spanning information science to law, but they are most commonly represented in journals related to health care information technology and telemedicine. A vast array of topics is covered by research articles referring to eHealth, highlighting the diffuse nature of the field and the lack of an agreed conceptual definition.\nDefinitions of eHealth demonstrate variation in the breadth and focus of alternative conceptualizations. At the extremes these range from the highly vague and diffuse, eg, \u201csomething to do with computers, people and health\u201d [37] to the highly specific, eg, \u201cthe healthcare industry's component of business over the internet.\u201d [32] Nevertheless, most conceptualize eHealth as a broad range of medical informatics applications for facilitating the management and delivery of health care, including dissemination of health-related information, storage and exchange of clinical data, interprofessional communication, computer-based support, patient-provider interaction, education, health service management, health communities and telemedicine, among other functions. A general theme relates to electronic communication, which is supported by the fact that most definitions specify the use of networked digital information and communications technologies, primarily the Internet. This differentiates eHealth from its parent field of medical informatics, which encompasses fixed technologies, such as X-Ray equipment, and pure bioinformatics research. While Internet technologies represent the prevailing theme, there is sufficient reference to applications that may be enabled by other interactive ICTs to suggest caution before identifying eHealth exclusively with this medium. This is supported by the high profile of decision support as a generic topic within the health informatics literature, which may, for example, take the form of clinical decision support systems or patient decision aids available via CD-ROM. Nevertheless, rapid increases in bandwidth and desktop computing capability make it likely that most such tools will soon be accessible using digital networked systems.\nMany conceptualizations of eHealth incorporate telemedicine and although most do so as part of a wider sphere of applications, some authors use the terms synonymously [45]. We suggest that the latter is more likely due to a misuse of the term than, as some have speculated, \u201cthe death of telemedicine\u201d in favour of eHealth [19] (cited in [18]). While telemedicine is certainly a theme in the eHealth literature, and the ICTs used in this area are common to many eHealth functions, it clearly represents only one domain of the broader field. Similarly, while several definitions extend to e-business, primarily meaning online transactions between suppliers and purchasers (2% of eHealth-related articles appear in journals of finance), most of these portray it as merely one application of eHealth for service management or care delivery.\nMost definitions appear to encompass applications for all stakeholder groups, although many emphasize support for providers and organizations and a few see eHealth as an application of consumer health informatics or, even narrower, as the use of \u201cinternet and other electronic media to disseminate or provide access to health & lifestyle information or services.\u201d[50] Our review of eHealth topics in the research and Web-based literature also indicates that the concept extends across stakeholder groups, including providers, patients, citizens, organizations, managers, academics and policymakers. A tendency has been noted for an inclusive model to predominate in Europe and a narrower consumer-focused one in the USA, possibly reflecting top-down versus bottom-up health systems and cultures [53]. However our results indicate that there is currently more overlap than difference between conceptualizations emanating from either side of the Atlantic, with the inclusive view predominating (also the case for Australia). Even of those conceptualizations tending toward the consumer informatics model, most emphasize interaction with professionals rather than simply passive delivery or provision of information to citizens or patients, thus drawing in the professional stakeholder. While there may be a valid argument for narrowing eHealth down to consumer health informatics in the future, namely to circumscribe the field and thereby make it more manageable, analysis of the existing eHealth landscape suggests that the concept is currently more inclusive.\nExisting conceptualizations also vary in the extent to which they consider broader issues relating to the place, function or promise of eHealth in the modern world, such as its ability to promote patient self-care and communication, and the implications of this for the doctor-patient relationship. Many see eHealth as facilitating the transition of decision making control and responsibility from the professional to the empowered consumer, consistent with conceptions of the information age flipping over the \u201cpower pyramid\u201d of health care [54]. The human and organizational changes required to effect new ways of working and attitudes also represent a strong theme. This is reflected in the relatively large number of publications, identified by the keyword search, that are concerned with issues such as challenges to implementation, as opposed to specific technologies or applications. We therefore agree that the concept incorporates \u201ca state-of-mind, a way of thinking, an attitude.\u201d [22] Such human and organizational factors appear to be underrepresented in the MeSH Medical Informatics taxonomy at present, suggesting that a review may be warranted to bring it into line with expert-derived ontologies such as that endorsed by IMIA. More broadly, eHealth is said to require a fundamental rethinking of healthcare processes\u201d [31] and a \u201ccommitment for networked global thinking to improve healthcare\u201d [22], but there is clearly a general optimism surrounding the potential benefits of this rapidly evolving field for health care processes and patient outcomes.\nOf course, definitions do not exist in isolation and the source documents for those reviewed provide further elaboration. For example, Eng provides a \u201c5 C's model\u201d of functions and capabilities of eHealth (content, connectivity, community, commerce, care) [21]; Eysenbach lists \u201c10 essential E's\u201d in eHealth (efficiency, enhancing quality of care, evidence-based, empowerment of consumers, etc) [22], and Richardson proposes a \u201c4-pillar model\u201d (under the headings of clinical applications, healthcare professional continuing education, public health information, and education and lifetime health plan) [30]. Yet others have attempted to define eHealth in terms of its potential role during a patient's care pathway [55] or with reference to the settings in which it may be useful [48]. Nonetheless, most authors have successfully distilled their concepts within the definitions they provide. Converging these with the other information sources documented in this report provides a fairly comprehensive overview of the concept and enables us to draw broad conclusions about its nature and scope.\nIn an editorial on the website, Health Informatics Europe, Ahmad Risk posed the question: \u201cSo, is this it? \u2026 Does 'eHealth' mean 'web health informatics'?\u201d[9] Based on our results, our conclusion is largely \u201cYes\u201d, or \u201cIt soon will be\u201d, recognising that the parameters of the field currently extend to other interactive ICTs which, with increasing computing power, bandwidth and wireless capability, may rapidly be accommodated by Internet technologies. Based on our analysis of the place of eHealth within the wider informatics field and the nature of research activity and general commentary on the topic, we conclude that it is well represented by the global definitions suggested by Eng and Eysenbach early in the emergence of the field, with a minor change to the latter, as indicated below:\ne-health is the use of emerging information and communications technology, especially the Internet, to improve or enable health and healthcare. [21]\ne-health is an emerging field of medical informatics, referring to the organization and delivery of health services and information using the Internet and related technologies. In a broader sense, the term characterizes not only a technical development, but also a new way of working, an attitude, and a commitment for networked, global thinking, to improve health care locally, regionally, and worldwide by using information and communication technology. (adapted from Eysenbach [22])","keyphrases":["ehealth","telemedicine","internet","medical informatics"],"prmu":["P","P","P","P"]}
{"id":"Pathol_Oncol_Res-4-1-2413131","title":"EGFR Inhibitor Enhances Cisplatin Sensitivity of Oral Squamous Cell Carcinoma Cell Lines\n","text":"Epidermal growth factor receptor (EGFR) is involved in multiple aspects of cancer cell biology. EGFR has already been identified as an important target for cancer therapy, with various kinds of EGFR inhibitors currently used in treatment of several human cancers. Recently, EGFR and its downstream signaling pathways were identified as being associated with cisplatin sensitivity. In addition, EGFR inhibitors have shown significant promise for patients who failed cisplatin-based therapy. In this study, we investigated whether treatment with an EGFR inhibitor improves cisplatin sensitivity in oral squamous cell carcinoma (OSCC) cell lines. The effects of a combination of AG1478, a specific EGFR tyrosine kinase inhibitor, with cisplatin were evaluated in cultured OSCC cell lines and cisplatin-resistant sublines. Higher expression of EGFR and p-EGFR was found in the two cisplatin-resistant cell lines compared with the corresponding parental cell lines. In addition, augmented inhibition of OSCC cell growth by the combination of AG1478 with cisplatin was found in both cell lines. These results suggest that the combination of an EGFR inhibitor and cisplatin may be useful as a rational strategy for the treatment of patients with oral cancer with acquired cisplatin resistance.\nIntroduction\nEpidermal growth factor receptor (EGFR) is expressed at high levels in a variety of solid tumors including oral cancers [1, 2]. EGFR and its downstream signaling pathways are involved in multiple aspects of cancer cell biology, including tumor cell proliferation, inhibition of apoptosis, invasion, metastasis, and angiogenesis [1\u20134]. EGFR has already been identified as an important target for cancer therapy, and various kinds of EGFR inhibitors are currently used in the treatment of several human cancers [5\u201310].\nCisplatin-based combination chemotherapy displays significant anti-tumor activity against solid tumors of oral squamous cell carcinoma (OSCC). However, the effectiveness of cisplatin in the treatment of recurrent\/metastatic tumors is limited because of acquired or intrinsic resistance. EGFR and its signaling pathways are involved in the mechanism of cisplatin resistance. Cells that are resistant to cisplatin have an altered response to the EGF ligand and enhanced activation of the protein kinase [11]. In addition, several studies have suggested that enhanced expression of EGFR may be associated with cisplatin resistance in a variety of solid tumors including oral cancers [12, 13]. Increased EGFR expression may be a survival response by some tumors exposed to chemotherapeutic agents [14].\nIncreased availability of EGFR inhibitors in cisplatin-resistant cells has also been reported previously [13]. EGFR inhibitors have shown significant activity in cases failing cisplatin-based therapy [15, 16]. Therefore, EGFR blockade may be a useful therapeutic tool in the treatment of patients with acquired cisplatin resistance.\nIn this study, we established a cisplatin-resistant cell line from an OCSS-derived cell line and investigated the differential EGFR and phosphorylated EGFR (p-EGFR) expression between OSCC cell lines and the cisplatin-resistant sublines. In addition, we examined the effect of combination therapy with an EGFR inhibitor and cisplatin on the growth of OSCC cells.\nMaterials and Methods\nCell Lines\nTwo human OSCC cell lines have been established at Wakayama Medical University, Wakayama, Japan. The H-1 line was derived from a biopsy specimen of moderately differentiated OSCC in the lower gingiva. The Sa-3 line was derived from a biopsy specimen of well-differentiated OSCC in the upper gingiva. Both cell lines were cultured in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM; Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Equitech-Bio, Kerrville, TX, USA), 100 units\/ml penicillin, and 100\u00a0\u03bcg\/ml streptomycin (Gibco BRL, Grand Island, NY, USA) in a highly humidified atmosphere of 5% CO2 at 37\u00b0C.\nIn accordance with previously described methods [17, 18], the cisplatin (CDDP)-resistant sublines H-1\/CDDP and Sa-3\/CDDP were established by repeated subculture in the presence of increasing concentrations of cisplatin (Nippon Kayaku Corporation, Tokyo, Japan), from 0.1\u00a0\u03bcg\/ml until cells became fully resistant to cisplatin and could grow exponentially; in each case the final cisplatin concentration was 0.5\u00a0\u03bcg\/ml. The drug-resistant cell lines were passed in drug-free medium, and there was no loss of resistance during the two-month testing period.\nCell Growth Analysis with MTT Assay\nCells were seeded in 96-well plates at 2000 cells per well in DMEM containing 10% FBS. After 24\u00a0h, cells were exposed to one of nine concentrations (0.05, 0.1, 0.25, 0.5, 1, 1.25, 2.5, 5 and 10\u00a0\u03bcg\/ml) of cisplatin or five concentrations (1, 5, 10, 20 and 30\u00a0\u03bcM) of AG1478 (Calbiochem San Diego CA, USA). After cells were incubated with cisplatin or AG1478 for 24\u00a0h, medium was changed to drug-free DMEM and cells were incubated for an additional 72\u00a0h. Thereafter, the number of cells per well was quantified with a MTT cell growth assay kit (Funakoshi, Tokyo, Japan) according to the manufacturer\u2019s instructions. Briefly, after 10\u00a0\u03bcl MTT solution was added to each well, the well was incubated for 4\u00a0h and scanned at 550\u2013630\u00a0nm by a MTP-300 microplate reader (Corona, Tokyo, Japan). Six wells were used for each drug concentration, and the experiment was repeated three times. The 50% inhibitory concentration (IC50) was calculated from the survival curve. In another experiment, to test whether the combination of cisplatin and AG1478 would achieve higher growth inhibition than the single agent at a concentration lower than the IC50, fixed concentrations of each drug were then tested in combination treatment of OSCC cell lines. All cells were exposed to AG1478 for 1\u00a0h before cisplatin. The statistical significance of differences was analyzed by applying Mann\u2013Whitney U-test. The level of significance was set at p\u2009<\u20090.05.\nWestern Blot Analysis\nSubconfluent cells were scraped from culture dishes, washed twice with phosphate buffered saline (PBS), and suspended in 700\u00a0\u03bcl Western blot lysis buffer containing 62.5\u00a0mM Tris\u2013HCl (ph\u00a06.8), 25% glycerol, and 2% sodium dodecyl sulfate (SDS). Samples were centrifuged at 15,000\u00a0rpm for 20\u00a0min at 4\u00b0C, after which supernatants were collected. After heating at 95\u00b0C for 5\u00a0min, equal amounts of proteins were separated with the use of 10% SDS-PAGE. After electrophoresis, proteins were transferred to a PVDF membrane in Tris-glycine buffer containing 20% methanol. The membrane was blocked with 3% skim milk containing 0.01% polyoxyethlene sorbitan monolaurate for 60\u00a0min, and incubated overnight with the corresponding primary antibodies {a 1:750 dilution of anti-rabbit polyclonal EGFR antibody [Santa Cruz Biotechnology, Santa Cruz, CA, USA], and a 1:750 dilution of anti-goat polyclonal p-EGFR (Tyr 1173) antibody [Santa Cruz Biotechnology]} at 4\u00b0C. The membrane was washed three times for 5\u00a0min each with PBS containing 0.05% polyoxyethlene sorbitan monolaurate and horseradish peroxidase-conjugated anti-rabbit or anti-goat antibody for 1\u00a0h at room temperature, respectively. Protein signals were visualized by enhanced chemiluminescence using ECL Western blotting detection reagents (Amersham, Arlington Heights, IL, USA) for 1\u00a0min and exposed to Kodak Biomax XAR film.\nResults\nSingle Agent Effects\nThe sensitivities of all cell lines to various concentrations of cisplatin and AG1478 were determined by MTT assay and plotted on a survival curve (Fig.\u00a01). The IC50 values of cisplatin and AG1478 are shown in Table\u00a01. The IC50 values of the H-1\/CDDP and Sa-3\/CDDP lines to cisplatin were about 10-fold greater than the values for the parental H-1 and Sa-3 lines. Resistance of all cell lines to AG1478 was similar, but the IC50 values for parental cell lines tended to be slightly higher.\nFig.\u00a01Inhibitory effects of cisplatin and AG1478 on growth of OSCC cell lines. The four lines, H-1, H-1\/CDDP, Sa-3, and Sa-3\/CDDP, were treated with cisplatin (0.05\u201310\u00a0\u03bcg\/ml; a) or AG1478 (1\u201330\u00a0\u03bcM; b) for 24\u00a0h as described in the Materials and methods section. Cell viability was measured by MTT assay and is expressed as a percentage relative to control cells. The results showed that cisplatin and AG1478 inhibited OSCC cell growth in a dose-dependent mannerTable\u00a01Cisplatin and AG1478 sensitivity of OSCC cell linesCell lineIC50IC50Cisplatin (\u03bcg\/ml)AG1478 (\u03bcM)H-10.4925.3Sa-30.4328.5H-1\/CDDP6.523.3Sa-3\/CDDP5.921.2\nCombination Effects\nAll cell lines were treated with AG1478 (10\u00a0\u03bcM) for 1 hour before cisplatin (0.25\u00a0\u03bcg\/ml) for 24\u00a0h. The combination of AG1478 with cisplatin inhibited cell growth >60% in parental cell lines > 50% in cisplatin-resistant cell lines (Fig.\u00a02). In both cell lines, we showed augmented effects of inhibition and showed a large effect of augmentation in cisplatin-resistant cell lines.\nFig.\u00a02Effect of a combination of AG1478 with cisplatin on growth of OSCC cell lines. The four lines, H-1, H-1\/CDDP, Sa-3, and Sa-3\/CDDP, were treated with AG1478 (10\u00a0\u03bcM) for 1\u00a0h before addition of cisplatin (0.25\u00a0\u03bcg\/ml) for 24\u00a0h. Cell viability was measured by MTT assay and is expressed as a percentage relative to control cells. In both cell lines, we showed augmented effects of inhibition and showed large-effect augmentation in cisplatin-resistant cell lines. *p\u2009<\u20090.05, compared with cisplatin-treated cells\nExpression of EGFR and p-EGFR\nTo investigate the molecular basis for the increased sensitivity of chemoresistant tumors to cisplatin, we used Western blot analysis to determine the level of EGFR and p-EGFR expression in parental and cisplatin-resistant cell lines. Higher EGFR and p-EGFR expression was found in cisplatin-resistant cell lines compared with the corresponding parental cell lines (Fig.\u00a03).\nFig.\u00a03Total expression of epidermal growth factor receptor (EGFR) and phosphorylated receptor (p-EGFR). Total expression of EGFR and p-EGFR was determined by Western blot analysis. Ten micrograms of total cellular lysates prepared from parental and cisplatin (CDDP)-resistant cells were resolved by electrophoresis, transferred to a PVDF membrane, and blotted with anti-EGFR and anti-p-EGFR antibodies as described in the Materials and methods section\nDiscussion\nOur studies suggest that changes in the expression of EGFR play an important role in regulating the drug-resistant phenotype of cells in response to cisplatin. Cisplatin is widely used for chemotherapy of many malignancies, especially of oral squamous cell carcinoma (OSCC). However, the effectiveness of cisplatin in the treatment of recurrent\/metastatic tumors is limited because of acquired or intrinsic resistance. Increased EGFR expression may be a survival response by some tumors exposed to chemotherapeutic agents [14]. In our study, higher EGFR expression was found in two cisplatin-resistant cell lines compared with the corresponding parental cell lines (Fig.\u00a03).\nEGF receptors initiate cytoplasmic signaling through autophosphorylation of their intracellular domains [19]. Phosphorylated EGFR (p-EGFR) cytoplasmic tyrosine residues initiate the activated protein kinase pathway [20]. This mitogen-activated protein kinase (MAPK) pathway is involved in the mechanism of cisplatin resistance [21\u201323]. Activation of JNK and p38 by cisplatin has been shown to promote apoptotic cell death [22, 23]. In addition, activation of the ERK pathway by cisplatin was reported to promote induction of cell death [21]. In our study, higher p-EGFR expression was found in two cisplatin-resistant cell lines compared with the corresponding parental cell lines. These findings offer further evidence that activation of EGFR downstream signaling pathways is involved in cisplatin sensitivity.\nThe effect of combining EGFR inhibitors with cytotoxic agents has been studied in various types of cancer [16, 24]. The quinazoline derivative AG1478 is a specific reversible inhibitor of EGFR. The cancer cell growth depression effect of AG1478 has been reported in various carcinomas [24\u201326]. In our study, the combination of AG1478 and cisplatin showed augmented effects of inhibition in both cell lines and showed large-effect augmentation in cisplatin-resistant cell lines. These results suggest that acquired resistance to cisplatin is associated with enhanced sensitivity to an EGFR tyrosine kinase inhibitor, which correlates with increased EGFR and p-EGFR expression.\nAugmented effects of inhibition of OSCC cell growth through the combination of AG1478 and cisplatin provide a potential and novel strategy for patients with oral cancer with acquired cisplatin resistance. In addition, it would be very advantageous if an equal chemotherapeutic effect could be obtained with a smaller dosage of cisplatin. The combination of AG1478 and cisplatin against OSCC definitely deserves additional in vivo and clinical studies.","keyphrases":["egfr inhibitor","epidermal growth factor receptor (egfr)","oral squamous cell carcinoma (oscc)","cisplatin-resistant oscc cell line","cisplatin sensitivity and resistance"],"prmu":["P","P","P","R","R"]}
{"id":"Clin_Rheumatol-3-1-2071969","title":"The role of PET\/CT in Cogan\u2019s syndrome\n","text":"We report on the case of a 60-year-old woman with complaints of fatigue, coughing, anorexia, atypical chest pain, recurrent fever, and also ear pain and hearing loss. A test for anti-neutrophil cytoplasmic antibody (ANCA) was myeloperoxidase positive with p-ANCA specificity. Laboratory acute phase parameters were increased. A 2-deoxy-2-[18F]fluoro-d-glucose positron emission tomography\/computed tomography investigation showed pathological uptake in the aorta ascendens, with no other involvement of the large vessels. After therapy with methylprednisolon intravenously and later prednisolon orally with methothrexate, her general condition and hearing loss improved both subjectively and objectively. \u201cAtypical\u201d Cogan\u2019s syndrome was diagnosed on the basis of sensorineural deafness with improvement on steroids and large-vessel vasculitis of the aortic arch.\nIntroduction\nThe association of nonsyphilitic interstitial keratitis and audiovestibular involvement was first reported in 1934 by Mogan and Baumgartner. Their description was extended several years later by Cogan [1].\nIn \u201ctypical\u201d Cogan\u2019s syndrome, the presence of interstitial keratitis is necessary, the term \u201catypical\u201d Cogan\u2019s syndrome is used when other types of inflammatory eye disease, including conjunctivitis, uveitis, scleritis, and choroiditis are associated with the vestibuloauditory abnormalities [2]. In many cases, the symptomatology is not only restricted to the eyes and the ears but also other organs, thus resembling systemic vasculitis in one third of the patients. The most common symptoms are cardiovascular, musculoskeletal, neurological, gastrointestinal, and mucocutaneous [3].\nPositron emission tomography (PET) with 2-deoxy-2-[18F]fluoro-d-glucose (FDG) is becoming increasingly important in diagnosis, staging, and therapy monitoring in clinical oncology and has recently been used in the diagnosis of infectious diseases with elevated intracellular glucose metabolism. Activated inflammatory cells have been shown to overexpress glucose transporters and to accumulate increased amounts of glucose and structurally related substances such as F18-FDG [4, 5].\nTherefore FDG-PET is also introduced as a diagnostic means to assess involvement in large vessel vasculitis [6]. In this report, we report the use of FDG-PET\/computed tomography (CT) scanning in the diagnosis of Cogan\u2019s syndrome.\nCase report\nA 60-year-old Caucasian woman was admitted to the hospital with a 4-month history of excessive fatigue, coughing, anorexia and weight loss, night sweats, and atypical chest pain. She also experienced short periods of fever. She experienced headaches and ear pain and hearing loss for over the last month, mainly on the left side, and felt sometimes dizzy. No blurred vision complaints or eye problems were noted. She was not known with any allergies.\nFor her hypothyroidism (multinodular goiter), she used Thyrax (l-thyroxine)150\u00a0mcg once a day. She did not smoke and consumed alcohol only moderately.\nThe family history revealed a daughter with systemic lupus erythematosus.\nPhysical examination revealed a pulse of 104, and bloodpressure was 125\/85\u00a0mmHg and the temperature 37.1\u00b0C. Heart sounds were normal, and the lungs were clear. The outer ears were normal. No lymphadenopathy was detected and no scalp tenderness or decreased pulsation at the temporal arteries was noted.\nLaboratory tests revealed an erythrocyte sedimentation rate (ESR) of 51\u00a0mm\/h and C-reactive protein (CRP) of 53\u00a0mg\/L. Test for rheumatoid factor was 42\u00a0kU\/L (<10), and tests for antinuclear facor and double-stranded DNA antibodies were negative.\nA test for anti-neutrophil cytoplasmic antibody (ANCA) appeared to be myeloperoxidase positive with p-ANCA specificity.\nSerum electrolytes and creatinine were normal. Her differential blood count, alkaline phosphatase, and transaminases were normal. The urine gave a negative test for protein, and the sediment contained no white cells, red cells, or casts in the urine.\nInitially arteritis temporalis was suspected, but Doppler ultrasonography of the temporal arteries showed no abnormalities. A chest X-ray revealed no interstitial or focal abnormalities. Subsequently, a PET\/CT was performed, which showed pathological uptake in the wall of the aortic arch. More intens pathological uptake was seen at the beginning of the aorta descendens in the lateral wall, most likely a sign of perivascular inflammation. Maximum standard uptake value (SUV max) measured 11.9. No other involvement of large vessels was noted (Fig.\u00a01). A magnetic resonance imaging (MRI) scan of the cerebrum showed abnormalities suspicious for bilateral mastoiditis, possibly as a consequence of bilateral otitis media.\nFig.\u00a01\na Transverse fused PET\/CT slice showing pathological uptake in the wall of the aortic arch and particularly in the lateral wall and perivascular space adjacent to the truncus pulmonalis (SUV max 11.9). b Follow-up PET\/CT 3\u00a0weeks later, after treatment with methylprednisolon i.v. and prednisolon orally, showing clearly decreased uptake in the aortic arch (SUV max 4.3). c Second follow-up PET\/CT 6\u00a0months later, while patient was in a stable condition with methotrexate and low-dose prednisone. Again, high pathological uptake in the aortic arch with higher intensity in the lateral wall and perivascular space adjacent to the truncus pulmonalis (SUV max 12.9)\nAfter placing inner ear tubes, her hearing loss improved only little. Culture of the ear secretion was negative for pathogenic microorganisms. Audiograms confirmed sensorineural hearing loss, particularly in the left ear.\nInvestigation of the eyes revealed no abnormalities, especially no keratitis.\nTherapy was started with three cycles of 1,000\u00a0mg methylprednisolon intravenously and later 60\u00a0mg prednisolon daily orally. Her general condition and hearing loss improved subjectively. Audiograms 6\u00a0weeks later showed also objective improvement of hearing. A control CT scan showed improvement of the abnormalites in both mastoid regions.\nAs a consequence of the high doses of steroids, risedronate and calcium supplementation were started, and also methothrexate was added for the reason of its steroid-sparing effect. \u201cAtypical\u201d Cogan\u2019s syndrome was diagnosed on the basis of sensorineural deafness with improvement on steroids and large-vessel vasculitis of the aortic arch.\nA follow-up PET\/CT investigation 3\u00a0weeks later showed clearly decreased uptake in the aortic arch, especially a dramatic decrease at the location of the focal uptake in the lateral wall, compared to the first PET\/CT. The SUV max was calculated 4.3. ESR and CRP after 2\u00a0weeks of therapy were, respectively, 11\u00a0mm\/h and <2\u00a0mg\/L. White cell count was still elevated (16.9.109\u00a0L).\nSix months later, she was in a stable condition with methotrexate and low-dose prednisone, and her ESR, CRP, and white cell count were now, respectively, 24\u00a0mm\/h, 14\u00a0mg\/L, and 13.7.109\u00a0L.\nA third PET\/CT showed higher pathological activity, compared to the first PET\/CT, in the wall of the aortic arch and in the perivascular space adjacent to the truncus pulmonalis (the SUV max was12.9 compared to the SUV max of 11.9 of the first pretherapy PET\/CT investigation). Consequently, the dose of methothrexate and prednisone were both increased to 20\u00a0mg\/day.\nDiscussion\nThe etiology of Cogan\u2019s syndrome is unknown; a minority of patients have rheumatoid factor, antinuclear bodies, and diminished complement levels. Histology of biopsies shows often vasculitis and perivascular inflammation. Giant cells may be present [7]. Specific involvement of large arteries has been reported in Cogan\u2019s syndrome [8]. Aortitis in Cogan\u2019s syndrome is indistinguishable from Takayasu\u2019s arteritis. Cardiac involvement during the course of Cogan\u2019s syndrome is, above all, aortic insufficiency. It is a severe complication that may require valve replacement, without which left ventricle involvement insufficiency develops, which can be fatal [9, 10].\nStandard diagnostic modalities such as biopsy, angiography, ultrasound, and MRI are commonly unable to demonstrate the full extent of vascular involvement in large-vessel vasculitis. PET investigations might play an important role here as large-vessel F18-FDG uptake is positively correlated with the level of acute phase reactant markers in patients with large vessel vasculitis. In a study of 18 patients with Takayasu\u2019s arteritis, the F18-FDG-PET examination showed a sensitivity of 92% and a specificity of 100% [11].\nThis is, to the best of our knowledge, the first report of Cogan\u2019s syndrome diagnosed by the use of FDG-PET\/CT scanning. This case report is also supportive in the hypothesis that with PET, the inflammatory activity of large-vessel vasculitis is more accurately assessed compared to laboratory acute phase parameters [12]. The CT component of PET\/CT is useful in the precise anatomical localization of the PET abnormalities and may provide information about changes in the wall structure or luminal flow [13].\nThe conclusion seems justified that F18-FDG-PET\/CT is helpful in risk assessment of large-vessel vasculitis, as it provides intrinsically fused morphologic and functional data in a single examination.","keyphrases":["cogan\u2019s syndrome","hearing loss","large-vessel vasculitis","f18-fdg pet\/ct"],"prmu":["P","P","P","R"]}
{"id":"Ann_Surg_Oncol-4-1-2190337","title":"High-Volume versus Low-Volume for Esophageal Resections for Cancer: The Essential Role of Case-Mix Adjustments based on Clinical Data\n","text":"Background Most studies addressing the volume\u2013outcome relationship in complex surgical procedures use hospital mortality as the sole outcome measure and are rarely based on detailed clinical data. The lack of reliable information about comorbidities and tumor stages makes the conclusions of these studies debatable. The purpose of this study was to compare outcomes for esophageal resections for cancer in low- versus high-volume hospitals, using an extensive set of variables concerning case-mix and outcome measures, including long-term survival.\nSince Luft published his study about the inverse relationship between surgical volume and hospital mortality in 1979, a plethora of studies has demonstrated an improvement of clinical outcome with increased hospital volume.1 Most of these studies use hospital mortality as the sole outcome measure. Often, data are obtained from insurance company\u2019s databases, and few studies use clinical data for risk-adjustment.2\nThe surgical treatment of esophageal cancer is often mentioned as one of the procedures for which concentration in high-volume centers might improve outcome.3,4 Nevertheless, a clear volume cut-off point at which a cancer center is justified to perform esophageal resections can hardly be defined.5,6 Also, the volume\u2013outcome literature for esophageal resections is limited to postoperative mortality as the sole determinant of outcome.\nConsidering the growing evidence for this volume\u2013outcome relationship for esophageal cancer surgery, we decided to investigate the outcome of these procedures in our region from 1990 until 1999. During this study period none of the 11 hospitals affiliated with the Comprehensive Cancer Center Leiden (CCCL) in the Netherlands performed more than seven esophageal resections a year; all are considered low-volume hospitals (LVH).\nIn contrast to most volume\u2013outcome studies, we decided to use clinical data obtained from the original patients\u2019 files. We retrieved information about comorbid diseases, tumor characteristics, treatment, and outcome. Next to hospital mortality, several determinants of outcome were examined, such as the number of tumor-free margins and complication rates. Assuming that survival is an essential indicator for quality in cancer surgery, we included a 5-year follow-up. To put our data in the right perspective, we compared these outcomes to the results of the topographically nearest high-volume referral center (HVH).\nPATIENTS AND METHODS\nAll surgically treated esophageal carcinomas in the period 1990\u20131999 were retrospectively identified through the Leiden Cancer Registry (LCR) of the Comprehensive Cancer Center Leiden (CCCL), in which all cancer patients treated in the midwestern part of the Netherlands are registered (1.7 million inhabitants). All 11 hospitals gave consent to participate in this audit and were visited by two investigators to retrieve the original patient files. Patient demographics, pathological notes, data on the surgical and (neo)adjuvant treatments, comorbidity as well as postoperative morbidity, mortality, length of stay, radicality of the resection, and long-term survival could all be retrieved from the patient\u2019s files.\nAll tumors were staged according to the UICC TNM classification of 1997. This was done by two independent researchers. The obtained pTNM stages were checked with the pTNM stages registered in the LCR. Any discrepancies were discussed between the researchers and a trained data manager from the CCCL. If consensus could not be reached, the pTNM stage was registered as \u201cunknown.\u201d\nTo make a comparison with the outcomes of the nearest high-volume center, data were categorized according to the database of this center. In this hospital, data of patients operated on for an esophageal carcinoma are prospectively collected by a trained data manager.\nDifferences in patient, tumor, and treatment characteristics as well as outcome measurements were assessed using the Kruskal\u2013Wallis test for continuous variables and the chi-square test for categorical variables. Logistic regression was used to determine prognostic factors of in-hospital mortality. Variables were entered in the multivariate model as a prognostic factor when P values <.10.\nSurvival was calculated as the difference between date of surgery and either the date of death or the date of last patient follow-up. For both groups, follow-up of the patients was completed until December 31,, 2005. Observed survival rates were estimated by using the Kaplan\u2013Meier method. The log-rank test was used to assess differences in survival between patients who were operated in LVHs and the HVH. All analyses were conducted using SPSS software (version 12.0; SPSS Inc., Chicago. IL).\nRESULTS\nHospital Volume\nIn 1990\u20131999 the evaluation and treatment of patients with an esophageal carcinoma were performed in 11 hospitals in the region of the CCCL (one university hospital, five teaching hospitals, and five general hospitals). In 342 patients the tumor was resected with curative intent. Figures\u00a01A and B illustrate the distribution of surgical procedures within the studied time period and between the different hospitals. None of the CCCL hospitals performed more than seven esophageal resections a year, which makes them low-volume hospitals (LVHs).7 In the same period, 561 esophageal resections were performed in the nearest high-volume referral center (HVH); a mean volume of 56 resections a year.\nFIG.\u00a01. (A) Number of esophageal resections per year in HVH versus LVH group (1990\u20131999). (B) Total number of esophageal resections per hospital for HVH and LVHs (1990\u20131999).\nPatient, Tumor, and Treatment Characteristics\nTable\u00a01 shows the patient, tumor, and treatment characteristics of both groups. More patients from the HVH had a squamous cell carcinoma and an advanced stage of the disease. Operative strategy as well as adjuvant or neoadjuvant treatment varied widely between the groups. The vast majority of resections in the HVH was performed according to the transhiatal technique, with a gastric tube reconstruction and anastomosis to the cervical remnant esophagus. In the LVH group a substantial number of anastomoses were located in the thoracic cavity, after a (partial) gastroesophagectomy with either a gastric tube reconstruction or esophagojejunostomy. In the pathology, clear surgical margins (R0) were reported in 72 % and 67 %, respectively for the LVHs and the HVH group.\nTABLE\u00a01.Patient, tumor, and treatment characteristics of esophageal resections in LVHa and HVHbCharacteristicsLVHHVHP valueNo. of patients%No. of patients%Age (years)6564.240\u00a0\u00a0Range (years)33\u20138731\u201383Gender.072\u00a0\u00a0Male2497343878\u00a0\u00a0Female932712322Comorbidity.078\u00a0\u00a0No1424227349\u00a0\u00a01 organ system1113217932\u00a0\u00a02 organ systems51158014\u00a0\u00a0\u22653 organ systems113275\u00a0\u00a0Unknown27820Histology.039\u00a0\u00a0Adenocarcinoma2386934762\u00a0\u00a0Squamous962819334\u00a0\u00a0Barrett\u2019s dysplasia4161\u00a0\u00a0Other21143\u00a0\u00a0Unknown2110Tumor localization.740\u00a0\u00a0Cervical esophagus72143\u00a0\u00a0Mid esophagus53158615\u00a0\u00a0Distal esophagus1143320436\u00a0\u00a0Gastroesophageal junction1664925145\u00a0\u00a0Unknown2161Stage (pTNM)<.001\u00a0\u00a00 and I43126111\u00a0\u00a0II1624721438\u00a0\u00a0III1073118633\u00a0\u00a0IV2169417 Unknown9361(Neo)-adjuvant treatment<.001\u00a0\u00a0None3169246483\u00a0\u00a0Chemotherapy1759317\u00a0\u00a0Radiotherapy0020\u00a0\u00a0Chemoradiation4100 Unknown5210Surgical approach<.001\u00a0\u00a0Abdomino-cervical1504446683\u00a0\u00a0Thoraco-abdominal97286011\u00a0\u00a0Abdomino-thoraco-cervical4313173\u00a0\u00a0Abdominal5215183Anastomoses<.001\u00a0\u00a0Cervical1955754196\u00a0\u00a0Thoracic912782\u00a0\u00a0Abdominal561682\u00a0\u00a0Unknown0040Total No. of patients342561a LVH, low-volume hospitals.b HVH, high-volume hospital.\nMorbidity and Mortality\nA significantly higher postoperative morbidity rate was found in the LVH group, which probably is also reflected by the longer hospital stay (Table\u00a02). The clinical anastomotic leakage rate differed between both groups: LVHs 17% versus HVH 5%. The mortality rate was almost three times higher for patients treated in the LVHs than those who had their operation in the HVH: 13% vs 5%, respectively (P < .001). None of the LVHs had a mortality rate lower than the 5% of the HVH (Table\u00a03). Univariate analysis showed that hospital volume, age, and comorbidity are prognostic factors for mortality (Table\u00a04). The mortality risk increased with higher age and the number of organ systems affected. Especially cardiac (OR 3.22, CI 1.91\u20135.44), vascular (OR 2.49, CI 1.45\u20134.27), and respiratory (OR 1.90 CI 1.09\u20133.33) comorbidity were risk factors for postoperative mortality.\nTABLE\u00a02.Outcome after resection of esophagus for cancer in LVHa and HVHbOutcomeLVHHVHP valueNo. of patients%No. of patients%Margins.93\u00a0\u00a0R02487237767\u00a0\u00a0R1551616128\u00a0\u00a0R23511214\u00a0\u00a0Unknown4121Complications\u00a0\u00a0Surgical complications1444220737.01\u00a0\u00a0General complications1915620737<.001\u00a0\u00a0No complications892624744<.001Hospital stayMedian (days)2114<.001In-hospital \u00a0\u00a0Mortality4513285<.001\u00a0\u00a0Survival\u00a0\u00a0Median (months)2122.90\u00a0\u00a0Range (months)(1\u2013171)(1\u2013158)Total No. of patients342561a LVH, low-volume hospitals.b HVH, high-volume hospital.TABLE\u00a03.Mortality after resection of esophagus for cancer in LVHa and HVHbHospitalsmIn-hospital mortalityNo. of patientsNo. of deaths%HVH561285.0LVH 116212.5LVH 219210.5LVH 32827.1LVH 425312.0LVH 51417.1LVH 62827.1LVH 73425.9LVH 8641218.7LVH 9441022.7LVH 106233.3LVH 1164710.9Total No. of patients903738a LVH, low-volume hospitals.b HVH, high-volume hospital.TABLE\u00a04.Univariate analysis of in-hospital mortalityUnivariate analysisOR95% CIP valueRegion<.001\u00a0\u00a0HVH1.00Refa\u00a0\u00a0LVH2.881.76\u20134.72Age (years).01\u00a0\u00a0<500.190.04\u20130.79\u00a0\u00a050\u2013590.510.25\u20131.04\u00a0\u00a060\u2013691.00Refa\u00a0\u00a0>701.200.70\u20132.04Gender.20\u00a0\u00a0Male1.00Refa\u00a0\u00a0Female0.670.36\u20131.24Comorbidity<.001\u00a0\u00a0No1.00Refa\u00a0\u00a01 organ system2.021.06\u20133.86\u00a0\u00a02 organ systems4.512.30\u20138.85\u00a0\u00a0\u22653 organ systems4.971.92\u201312.83Histology.97\u00a0\u00a0Adenocarcinoma1.00Refa\u00a0\u00a0Squamous0.990.60\u20131.65Stage.24\u00a0\u00a0I1.00Refa\u00a0\u00a0II0.500.24\u20131.04\u00a0\u00a0III0.800.39\u20131.63\u00a0\u00a0IV0.650.26\u20131.61Tumor localization.33\u00a0\u00a0Cervical\/mid esophagus1.00Refa\u00a0\u00a0Distal esophagus\/gastroesophageal junction1.410.71\u20132.80Neoadjuvant treatment.14\u00a0\u00a0No1.00Refa\u00a0\u00a0Yes0.490.20\u20131.25Surgical approach.31\u00a0\u00a0Transhiatal1.00Refa\u00a0\u00a0Transthoracic1.510.90\u20132.54.12Anastomosis.46\u00a0\u00a0Cervical1.00Refa\u00a0\u00a0Thoracic1.520.77\u20133.01\u00a0\u00a0Abdominal1.260.52\u20133.04a Ref, reference category.\nMultivariate analysis showed that both hospital volume and comorbidity were independent prognostic factors for hospital mortality (Table\u00a05).\nTABLE\u00a05.Multivariate analysis of in-hospital mortalityMultivariate analysisOR95% CIP valueRegion<.001\u00a0\u00a0HVH1.00Refa\u00a0\u00a0LVHs3.051.82\u20135.11Age (years).10\u00a0\u00a0<500.220.05\u20130.96\u00a0\u00a050\u2013590.600.29\u20131.25\u00a0\u00a060\u2013691.00Refa\u00a0\u00a0>701.070.61\u20131.88Comorbidity.004\u00a0\u00a0No1.00Refa\u00a0\u00a0Yes2.341.30\u20134.19a Ref, Reference category.\nSurvival\nFigure\u00a02 shows the crude 10-year overall survival rate of all patients, in which an esophageal resection for cancer was performed. Survival rates for patients treated in the HVH are significantly better (P = .01). This survival benefit loses its statistical significance, after exclusion of patients who died postoperatively of complications of the surgical procedure (Fig.\u00a03). Only, when we select patients with stage I and II disease do we see a better survival in the HVH (Fig.\u00a04), meaning that its overall results are worsened by the poor survival in the higher stages of the disease, stage III and IV. This can be explained by the unfavorable tumor mix, with significantly more stage IV disease treated in the HVH, than in the LVHs (16.7 vs 6.1%).\nFIG.\u00a02.Overall survival after esophagus resection for cancer: LVHs vs HVH (in-hospital mortality included).FIG.\u00a03.Overall survival after esophagus resection for cancer: LVHs vs HVH (in-hospital mortality excluded).FIG.\u00a04.Overall survival after esophagus resection for stage I and II carcinoma: LVHs vs HVH (in-hospital mortality excluded).\nDISCUSSION\nCurrently, there is extensive interest in comparing outcome of complex surgical procedures between high- and low-volume providers. Most of the studies are registry-based or relatively small. Our series offers additional proof to the volume\u2013outcome relationship, because it is based on clinical data, retrieved from the original patient files. This allows us to make reliable comparisons for comorbidities and tumor stage, which proved to be important prognostic factors for in-hospital mortality and survival.\nA review of the evidence for a volume\u2013outcome relationship was published by Dudley in 20008 and Halm in 2002.2 In the latter publication 135 studies were reviewed, of which only five were not from the United States or Canada. The majority of reports were based on state or national hospital discharge databases, where only a few studies used clinical data for risk adjustment. The outcome measure was \u201cdeath\u201d in 79% of the studies, without analyzing other dimensions of \u201coutcome,\u201d such as morbidity, length of hospital stay, reoperations, et cetera. For cancer-related procedures, long-term survival was not mentioned. Higher-level methodological issues were rarely addressed. Only five studies concerning cancer treatment adjusted for (neo)-adjuvant therapies or the type of surgical resection, but without any adjustment for tumor stage.\nSince 2002, more extensive studies on hospital or surgeon volume appeared in the international literature. Birkmeyer reported a total number of 2.5 million operations concerning 14 different surgical procedures derived from the MEDICARE database.9 Mortality was the only outcome measure. Even after risk adjustment, which decreased the outcome differences between high- and low-volume hospitals, the differences in results for esophageal and pancreatic resections were highly significant, favoring surgery in a high-volume center. Two more recently published reviews of the volume\u2013outcome relationship for esophagectomies came up with 12 papers addressing this subject.4,5 Only two of these studies were based on clinical data. Although both showed a decrease in mortality, they failed to show a statistically significant relationship of operative mortality with hospital volume.10,11 In our own review of the literature we identified another study from the United Kingdom using clinical data, in which hospital case volume independently predicted operative mortality2 (Table\u00a06).\nTABLE\u00a06.Volume\u2013outcome articles for in-hospital mortality after esophagectomy 1998\u20132006AuthorJournal\/YearDataVolume \u201ccut-off\u201dConclusionDimick et al.32Ann Thorac Surg 2005Adm<6>SUrbach and Baxter33BMJ 2004Adm<9>NSMcCulloch et al.12BMJ 2003Clin<10\u201320>SChristian et al.34Ann Surg 2003Adm<22>SFinlayson et al.35Arch Surg 2003Adm<4\u20139>SUrbach et al.36CMAJ 2003AdmaSDimick et al.37Surgery 2003Adm<7>SBirkmeyer et al.9N Engl J Med 2002Adm<2\u20134\u20137\u201319>SGillison et al.11Br J Surg 2002Clin<19>NSBachmann et al.10Br J Surg 2002ClinaNSDimick et al.38Ann Thorac Surg 2001Adm<4\u201315>Svan Lanschot et al.7Cancer 2001Adm<10\u201320>SKuo et al.39Ann Thorac Surg 2001Adm<6>SSwisher et al.40J Thorac Cardiovasc Surg 2000Adm<5>SGordon et al.41J Am Coll Surg 1999Adm<10\u201320\u201350>SBegg et al.42JAMA 1998Mixed<5\u201310>SPatti et al.43J Gastrointest Surg 1998Adm<1\u20132\u20134\u20136>Sa Urbach and Bachmann used equally sized groups and reported only median volumes of these groups.Adm denotes administrative data; Clin denotes clinical data; S denotes significant; NS denotes not significant.\nIn the present study, independent data managers collected data retrospectively from the patient files. Not only the (in-hospital) mortality rate was obtained, but also a range of other outcome data, such as complication rates, resection margins, length of stay, and long-term survival. In our opinion the latter is an important performance indicator in surgical oncology, surprisingly sporadically mentioned in the volume\u2013outcome literature.\nThe results of patients treated in 11 low-volume hospitals were compared with the results of patients treated in the nearest high-volume referral center. Significant differences in outcome could be revealed. In-hospital mortality was significantly higher in the low-volume hospitals. The retrieved information about comorbidity and stage of the disease made an extensive preoperative risk and tumor load comparison possible. Risk adjustment is an important issue in outcome research, because patients with severe comorbidity may be unequally distributed between (groups of) hospitals. Especially, when only administrative data are used to assess hospital performances, a selection-bias could lead to inadvertently penalizing those surgeons who provide excellent care to patients with more severe comorbid disease.7,13 Administrative data sets were never designed to predict risk and should probably not be used as such.14 Therefore, the validity of studies that fail to make case-mix adjustments based on clinical data, has to be questioned.\nNevertheless, a multivariate analysis of our data shows hospital volume to be an independent prognostic factor for in-hospital mortality. Although differences in surgical technique could be detected, with more transthoracic esophagectomies and intrathoracic anastomoses in the low-volume group, these factors are not significantly related to mortality. These findings are confirmed by earlier reports.15\u201318 Also, there is little evidence for a beneficial role of neoadjuvant therapies.19\u201322 However, above all, choices made concerning diagnostic strategy, neoadjuvant treatments, and surgical technique are related to the knowledge, experience, and judgment of the (team of) specialists.\nAfter exclusion of in-hospital mortality, the survival of patients in the HVH was equal to those treated in the LVHs. However, the results of the HVH were negatively influenced by its case-mix. More patients with stage IV disease were treated in the HVH, corresponding with its status as a tertiary referral center. The very poor survival in this group of patients influences the overall results significantly. Only when we are informed about differences in tumor stage, we are able to detect real differences in survival between patients treated in different hospitals. Although in this study, all pathology reports were reviewed, and the number of lymph nodes resected was equal for both groups, we still have to be cautious suggesting a survival benefit for high-volume surgery. Only when a uniform pathologic evaluation is guaranteed, can we be sure that observed differences in tumor stages are truly characteristic for patient groups. This could be the reason that few studies have attempted to examine the influence of hospital volume on long-term survival in cancer surgery, only one of them concerning esophagectomies.23\u201327 A recent study from the Netherlands failed to show a survival benefit in high-volume hospitals (>20 resections a year), but did show an improved survival for esophagectomies performed in university compared to non-university hospitals.28 On the other hand, for pancreatectomies and hepatectomies registered in the MEDICARE-database, Fong showed a significantly better survival for procedures performed in high-volume centers.25 In his study, administrative data about age, gender, comorbidity, and extent of the resection were included in a univariate and multivariate analysis, but stages of the disease, radicality, and intent of the resection (palliative or curative) were not reported.\nIn conclusion, our study shows that hospital volume is an important determinant of perioperative morbidity and mortality in esophageal cancer surgery. Nevertheless, volume in itself is no guarantee for high quality of surgical care in a specific institution. Selecting (only) favorable patients can be the basis of superior results. Therefore, case-mix adjustments are essential in the assessment of surgical performance of different institutions.","keyphrases":["case-mix","comorbidity","high-volume hospitals","esophageal cancer\u2013esophagectomy","surgical outcomes"],"prmu":["P","P","P","M","R"]}
{"id":"Nutr_J-2-_-293472","title":"Effects of isoflavones (soy phyto-estrogens) on serum lipids: a meta-analysis of randomized controlled trials\n","text":"Objectives To determine the effects of isoflavones (soy phyto-estrogens) on serum total cholesterol (TC), low density lipoprotein cholesterol (LDL), high density lipoprotein cholesterol (HDL) and triglyceride (TG).\nIntroduction\nIn recent years, phytoestrogens have attracted a great deal of interests in the medical and scientific literature. It also appears in the lay press for its effects on cardiovascular and menopausal health, and even cancer prevention. These compounds are present in large quantities in soybeans, clover and some legumes. Because of resemblances to human estrogen and the observations that Asian populations who consume more isoflavones compared with women in western countries have less menopausal symptoms, isoflavones are postulated as natural products that may be beneficial to postmenopausal women in cardiovascular health. Commercial products containing different quantities and mixtures of isoflavones are now widely available. A meta-analysis of soy protein in 1995 found significant cholesterols lowering effects when compared with animal proteins [1]. The authors suggest that isoflavones may be the principal physiologically active components responsible for the lipid lowering effects. However, it is still controversial [5]. Many reviews and editorials [2,3] have discussed the relevance of phytoestrogens on cardiovascular health and hypocholesterolemic effects but there was no meta-analysis up to our searching.\nMethods\nSearching strategy\nWe searched the databases from ACP Journal Club 1991 to Oct 2002, Cochrane Controlled Trials Register 3rd Quarter 2002, Cochrane Database of Systematic Reviews 4th Quarter 2002, Database of Abstracts of Reviews of Effectiveness 4th Quarter 2002, British Nursing Index (BNI) 1994 to Oct 2002, CANCERLIT 1975 to Oct 2002, CINAHL 1982 to Oct Week 4 2002, CSA \u2013 Life Sciences Collection 1982 to Oct 2002, EMBASE 1980 to 2002 Week 45, International Pharmaceutical Abstracts 1970 to Oct 2002, PREMEDLINE Oct 27, 2002, MEDLINE 1996 to Oct Week 4 2002. We searched the keywords with Ovid software version rel6.2.0: 'soy', 'soy protein', 'soybean', 'tofu', 'phytoestrogen', 'isoflavone', 'genistein', 'daidzein', 'formononectin' and 'biochanin A' by the method described by Dickersin 1994 [4]. We did not restrict any languages during the searching. Hand searching was made by retrieving relevant articles from the obtained studies and unpublished data were obtained through contacting experts. We identified on-going trials by searching ClinicalTrials.gov, the UK National Research Register and Meta-register of controlled trials on the internet.\nSelection of eligible trials\nWe included both single and double blind randomized controlled trials with baseline and after treatment values for synthesizing risk (mean) differences. The outcome measures were differences of serum total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C) and triglycerides (TG) between post-randomization baselines and after treatments. Eligible interventions were isoflavones tablets of single isolated component or any mixtures of genisteins, daidzein, formononectin and biochanin A. Isoflavones interventions in forms of soy protein supplements or diets were also included as long as they compared isoflavones containing treatments with isoflavones depleting controls. Treatments with soy polysaccharides, fiber, and phytosterols as their primary interventions were excluded.\nValidity assessment and data abstraction\nTwo independent investigators reviewed the articles obtained without masking. There were no scorings to the included trials. Data were entered twice to reduce input errors. Inter-rater reliability was not performed. Data were abstracted with a designed form before analysis. Duplicated trials or studies with the same population were counted once to reduce the duplicated publication bias. Data disagreement between the two reviewers was resolved by discussion.\nStudy characteristics\nThe characteristics of the obtained studies were tabulated with subtypes of isoflavones interventions, subjects' serum lipids status, dosages of isoflavones and lengths of treatment. Sub-group analysis was performed with different forms of isoflavones interventions, such as isolated genistein or mixtures of isoflavones tablets versus placebo, isoflavones containing versus depleting soy protein diets. Sensitivity analysis was also made across different population characteristics and lengths of treatment. Funnel plots were used to detect possible publication bias or treatment heterogeneity across sample sizes.\nQuantitative data synthesis\nWe obtained the risk differences (RD) from the post-randomization baselines and after-treatment values in each trial and calculated the pooled standard deviation of the RD as:\nwhere n1 and SD1 were sample size and standard deviation from treatment and n2 and SD2 were from control. Inverse variance method was used to pool all trial results [6] with software 'Review Manager 4.2 [31]. Fixed-effects model was used as the method of combination and it was supplemented with random-effects model if necessary. Since log odds ratios were not available in the trials, funnel plots were plotted with standard errors against risk (mean) differences.\nResults\nTrial flow\nThe trial flow chart was illustrated in Figure 1. Seventeen studies [7-23] (21 comparisons) with 853 subjects were included in this meta-analysis. Reasons for exclusion were non-randomization, lack of control [24-27], insufficient original data and baseline values [28,29].\nFigure 1\nTrial flow chart of including isoflavones studies. Databases generated 3542 potential eligible randomized controlled trials (RCT). After careful review, seventeen RCT (reference [7-23]) were included in this meta-analysis.\nStudy characteristics\nThe characteristics of the trials included were shown in Table 1. The populations being studied were adults of age between 18 and 73. Soy phytoestrogens interventions varied from isoflavones tablets to isoflavones containing soy diets. Isoflavones tablets were introduced in 11 trials, soy capsule in 1 trial, isolated soy protein (ISP) in 3 trials, and soy foods in 2 trials. The average intake of isoflavones was 73 mg per day (ranging from 28.5 to 150 mg) and length of treatment was 10 weeks (ranging from 2 to 26 weeks). Three trials recruited hyperlipidemic subjects while 8 included participants with normolipidemia. Thirteen studies recruited female, two recruited male and two recruited subjects with both genders.\nTable 1\nCharacteristics of the 17 included studies. There were 9 crossover and 8 parallel studies. The isoflavones tablets introduced were mainly in aglycone form. The abbreviations were listed below table.\nStudy\nTreatment (Rx) \u2020\nIsoflavones content in Rx (mg\/d) \u2021\nControl\nIsoflavones content in control (mg\/d)\nNo. of subjects\nLipid status \u00a7\nSubject gender ||\nLength of treatment (weeks)\n*Calvert 1981 [7]\nsoy biscuits\nNA\nsoy biscuits\n0\n10\nH\nmale\n4\nClifton-Bligh 2001(I) [8]\nT\n57\nT\nClifton-Bligh 2001(II) [8]\nT\n85.5\n28.5\n46\nB\nfemale\n26\nDewell 2002 [9]\nT\n150 G = 40; D = 50; glycosides = 60\nplacebo\n0\n36\nH\nfemale\n24\nHale 2002 [10]\nT\n80 G = 40; D = 40\nplacebo\n0\n29\nNS\npost\n2\nHan 2002 [11]\nsoy protein capsule\n100 G = 70; D = 18; glycitein = 12\nsoy protein capsule\n0\n78\nN\nperi\n16\nHodgson 1998 [12]\nT\n55 G = 30; D = 1; B = 16; F = 8\nplacebo\n0\n59\nN\nmale & female\n8\nHowes 2000 [13]\nT\n40 G = 1; D = 0.5; B = 26; F = 16\nplacebo\n0\n75\nNS\npost\n5\nMackey 2000 [14]\nISP\n65\nISP\n< 4\n49\nH\npost\n12\n*Merz-Demlow 2000(I) [15]\nISP\n113\u2013144\nISP\n*Merz-Demlow 2000(II) [15]\nISP\n55\u201374\n9\u201311\n13\nN\nfemale\n3\n*Nestel 1997 [16]\nT\n40\u201380 G= 22\u201343; D = 17\u201333; glycitein= 1\u20133\nplacebo\n0\n21\nB\nfemale\n5\n*Nestel 1999(I) [17]\nT\n80 G = 8; D = 7; B = 49; F = 16\nplacebo\n*Nestel 1999(II) [17]\nT\n40 G = 4; D = 3.5; B = 24.5; F = 8\n0\n13\nB\nfemale\n5\n*Samman 1999 [18]\nT\n86 G = 8.6; D = 7.4; B = 51.4; F = 18.6\nplacebo\n0\n14\nN\npre\n16\n*Sanders 2002 [19]\nsoy burger\n56 G = 34.8; D = 21.2\nsoy burger\n2\n22\nN\nmale & female\n2\n*Simons 2000 [20]\nT\n80\nplacebo\n0\n20\nN\npost\n8\nSquadrito 2002 [21]\nT\n54 G= 54\nplacebo\n0\n60\nN\nfemale\n24\n*Urban 2001 [22]\nT\n70 G = 42; D = 27\nISP\n3\n28\nN\nelderly men\n6\n*Wangen 2001(I) [23]\nISP\n110\u2013154\nISP\n*Wangen 2001(II) [23]\nISP\n54\u201376\n6\u20138.2\n18\nNS\npost\n4\n* crossover design \u2020 'T' = isoflavone tablet; 'ISP' = isolated soy protein \u2021 'G' = Genistein; 'D' = Daidzein; 'B' = Biochanin A; 'F' = Formononectin \u00a7'H' = hyperlipidemic subjects, 'N' = normolipidemic subjects; 'B' = both hyper- and normolipidemic subjects; 'NS' = not specified || 'pre' = premenopausal women; 'peri' = perimenopausal women; 'post' = postmenopausal women\nQuantitative data synthesis\nWe found that isoflavones tablets insignificantly increased serum TC by 0.01 mmol\/L (95% CI = -0.17 to 0.18; heterogeneity p = 1.0) (Figure 2); LDL by 0.00 mmol\/L (95% CI = -0.14 to 0.15; heterogeneity p = 0.9); HDL by 0.01 mmol\/L (95% CI = -0.05 to 0.06; heterogeneity p = 1.0); and triglyceride by 0.03 mmol\/L (95% CI = -0.06 to 0.12; heterogeneity p = 0.9). Isoflavones containing soy biscuits or burger had insignificant effects on serum TC with reference to isoflavones depleting soy biscuits or burger. Only isoflavones containing isolated soy protein (ISP) or soy protein (SP) capsule reduced serum TC when compared with isoflavones depleting ISP or SP capsule (Table 2). There was only one trial [11] introduced SP capsule as treatment. Mixtures of the four soy phytoestrogens (genistein, daidzein, formononectin and biochanin A) or isolated genistein did not have significant effects on serum TC. The significant result from intervening the mixtures of genistein and daidzein had shown to be statistically heterogeneous to combine (heterogeneity p = 0.06) as genistein and daidzein tablets were introduced among 3 studies [9,10,16] while Sanders [19] chose genistein and daidzein soy burger and Urban [22] used genistein and daidzein ISP. In subgroup analysis, the effect of genistein and daidzein tablets on serum TC from the 3 studies was shown to be insignificant, 0.1 mmol\/L (95% CI = -0.25 to 0.45; heterogeneity p = 0.87). Dose-response effect was not found in any forms of interventions in this meta-analysis. The results remained insignificant when the length of treatment increased. Although one trial [22] (without washout period) produced significant effect, it seemed that study design had no influence on the serum TC. There were insignificant decreases of serum TC among pre- and postmenopausal women. Interestingly, cholesterol lowering effects could only be found among normolipidemic subjects but not participants with hyperlipidemia and men were shown to be benefited with isoflavones treatment. Isoflavones in the forms of tablets and ISP were all insignificant on serum low density lipoprotein cholesterol (LDL), high density lipoprotein cholesterol (HDL) and triglycerides (TG). These results were shown in Table 3. Funnel plots of isoflavones tablets were asymmetrical and the results of the published trials tended to favor treatment.\nFigure 2\nFunnel plot of risk difference of isoflavones tablets versus placebo on serum total cholesterol. Total treatment size was 274 and total control was 208. The pooled risk difference of isoflavones tablets versus placebo was 0.01 with 95% confidence interval between -0.17 and 0.18. Heterogeneity p-value was 1.00. Note: 'N' = sample size; 'SD' = standard deviation; 'RD' = risk difference; '95% CI' = 95% confidence interval\nTable 2\nSubgroup analysis of the effects of isoflavones on serum total cholesterol. Isoflavones were shown to have significant benefits when given in the form of ISP or soy protein capsule. Regardless the forms of prescription, isoflavones decreased serum total cholesterol among normolipidemic but not hyperlipidemic subjects.\nSubgroup outcome\nNo. of comparisons\nNo. of subjects ||\nTreatment effect on TC (mmol\/L) \u00b6\nHeterogeneity p-value \u00a7\nForm of intervention \u2020:\n\u2003a.) isoflavones tablets\n12 [8-10,12,13,16-18,20,21]\n482\n0.01 (-0.17, 0.18)\n1.00\n\u2003b.) ISP(+)\n8 [14,15,22,23]\n229\n-0.11 (-0.21, -0.01)*\n0.34\n\u2003c.) soy protein capsule\n1 [11]\n78\n-0.69 (-1.19, -0.19)*\nNA\n\u2003d.) soy biscuit\n1 [7]\n20\n-0.05 (-0.47, 0.37)\nNA\n\u2003e.) soy burger\n1 [19]\n44\n0.09 (-0.38, 0.56)\nNA\n\u2003f.) Overall\n21 [7-23]\n853\n-0.09 (-0.18, -0.01)*\n0.77\nIsoflavones mixture \u2021:\n\u2003G&D&B&F\n5 [12,13,17,18]\n214\n-0.06 (-0.34, 0.21)\n0.96\n\u2003G&D\n6 [9-11,16,19,22]\n285\n-0.22 (-0.41, -0.03)*\n0.06\n\u2003G\n1 [21]\n60\n0.10 (-0.35, 0.55)\nNA\nIsoflavones intake (mg per day):\n\u2003< 50\n3 [8,13,17]\n132\n-0.12 (-0.54, 0.29)\n0.99\n\u200351\u2013100\n12 [8,10,12,14,15,17-23]\n484\n-0.10 (-0.21,0.01)\n0.74\n\u2003101 \u2013 150\n4 [9,11,15,23]\n176\n-0.10 (-0.24, 0.05)\n0.09\nDesign :\n\u2003parallel crossover\n9 [8-14,21]\n447\n-0.08 (-0.27, 0.10)\n0.48\n\u2003\u2003a.) no washout\n1 [22]\n56\n-0.43 (-0.73, -0.13)*\nNA\n\u2003\u2003b.) washout mentioned\n6 [15,19,20,23]\n208\n-0.07 (-0.17, 0.03)\n0.91\nGender:\n\u2003male\n2 [7,22]\n76\n-0.30 (-0.54, -0.05)*\n0.15\n\u2003female\n17 [8-11,13-18,20,21,23]\n674\n-0.07 (-0.17, 0.02)\n0.89\nTreatment length:\n\u20032\u201310 weeks\n14[7,10,12,13,15-17,19,20,22,23]\n541\n-0.09 (-0.18, 0.00)\n0.84\n\u200311\u201320 weeks\n3 [11,14,18]\n155\n-0.22 (-0.52, 0.07)\n0.07\n\u200321\u201330 weeks\n4 [8,9,21]\n157\n0.02 (-0.28, 0.31)\n0.96\nMenopausal status:\n\u2003pre-menopausal\n3 [15,18]\n80\n-0.06 (-0.17, 0.06)\n0.58\n\u2003peri-menopausal\n1 [11]\n78\n-0.69 (-1.19, -0.19)*\nNA\n\u2003post-menopausal\n13 [8-10,13,14,16,17,20,21,23]\n516\n-0.05 (-0.20, 0.10)\n1.00\nSubjects with:\n\u2003normolipidemia\n8 [11,12,15,18,19,21,22]\n377\n-0.11 (-0.21, -0.01)*\n0.08\n\u2003hyperlipidemia\n3 [7,9,14]\n105\n-0.05 (-0.36, 0.26)\n1.00\n* statistically significant \u2020 'ISP (+)' = isoflavone containing isolated soy protein; 'ISP (-)' = isoflavone depleting isolated soy protein \u2021 'G' = Genistein; 'D' = Daidzein; 'B' = Biochanin A; 'F' = Formononectin \u00a7 NA = not applicable || Since subjects acted as their own controls in a crossover trial, the 'calculated' total number was thus doubled in a single pair comparison. \u00b6 95% confidence interval in parenthesis\nTable 3\nEffects of isoflavones on serum LDL-cholesterol, HDL-cholesterol and triglycerides levels. Isoflavones in the forms of tablets or isolated soy protein (ISP) did not show significant benefits over serum LDL-cholesterol, HDL-cholesterol and triglycerides levels. The results were not heterogeneous to combine.\nType of intervention*\nNo. of trials\nNo. of comparisons\nNo. of subjects\nTreatment effect (mmol\/L) \u2020\nHeterogeneity p-value\n1.) Isoflavones tablets\n\u2003LDL\n9\n11[8,10,12,13,16-18,20,21]\n446\n0.00 (-0.14, 0.15)\n0.94\n\u2003HDL\n10\n12[8-10,12,13,16-18,20,21]\n482\n0.01 (-0.05, 0.06)\n0.98\n\u2003TG\n9\n12[8-10,12,13,16,17,20,21]\n482\n0.03 (-0.06, 0.12)\n0.93\n2.) ISP (+)\n\u2003LDL\n3\n5 [14,15,23]\n173\n-0.06 (-0.16, 0.03)\n0.84\n\u2003HDL\n3\n5 [14,15,23]\n173\n-0.01 (-0.07, 0.05)\n0.97\n\u2003TG\n3\n5 [14,15,23]\n173\n0.02 (-0.05, 0.09)\n1.00\n* 'ISP (+)' = isoflavones containing isolated soy protein; 'LDL' = Low density lipoprotein cholesterol; 'HDL' = High density lipoprotein cholesterol; 'TG' = Triglycerides \u2020 95% confidence interval in parenthesis\nDiscussion\nMain findings\nPhytoestrogens with either isolated genistein or isoflavones mixtures of genistein, daidzein, formononectin and biochanin A were found to be statistically insignificant in lowering serum total cholesterol (Table 2). Resolving the heterogeneity from pooling the mixtures of genistein and daidzein, we found that tablets prescription with these two isoflavones [9,10,16] yielded insignificant benefits over serum TC, 0.1 mmol\/L (95% CI = -0.25 to 0.45; heterogeneity p-value = 0.87). One trial [19], introducing soy burger as the prescription of these 2 isoflavones, found insignificant benefits. Another trial [22], prescribing them in the form of isolated soy protein (ISP), had shown a decrease in serum TC by 0.43 mmol\/L (95% CI = -0.73 to -0.13). It may be a result of bias as this trial lacked a washout period. When dose-response effect and treatment length were taken into account, phytoestrogens seemed to have insignificant effects over serum cholesterol (Table 2). Isoflavones in the forms of tablets, soy burger or biscuits seemed to have insignificant benefits over serum TC. Only Han [11] reported a significant benefit for isoflavones in the form of soy protein capsule. Isoflavones in the forms of tablets or ISP could not be shown to have significant benefits over serum low density lipoprotein (LDL) cholesterol, high density lipoprotein (HDL) cholesterol and triglycerides (TG) (Table 3).\nWe could not demonstrate significant benefits with all types of prescriptions when the dosages or treatment lengths increased. No dose-response effect was detected nor benefits on serum cholesterol with any forms of prescription among pre- and post-menopausal women and hyperlipidemic subjects. The benefit over normolipidemic subjects was heterogeneous (p = 0.08). It may be again the result of prescription diversity. Separated analysis found that isoflavones tablets [12,18,21] had insignificant effect on serum TC among normolipidemic subjects, 0.05 mmol\/L (95% CI = -0.23 to 0.33; heterogeneity p = 0.9). Only isoflavones in the form of soy protein capsule [11] or ISP [22] reported benefits on participants with normolidemia. In fact, some suggest that the beneficial effects of soy protein require synergistic reactions between isoflavones and other soy components [32]. This hypothesis was supported by some animal experiments [33-35]. It seemed that isoflavones in the form of tablets, up to 150 mg per day (genistein about 50 mg, daidzein about 50 mg), had no significant benefits on serum cholesterols in this meta-analysis. Phytoestrogens treatments in forms of soy protein supplements or soy protein capsule may need further investigations, especially interactions between other chemically active components inside prescriptions.\nValidity and limitation\nIn subgroup analysis, the sample sizes were inevitably reduced and there might be insufficient power to detect small significant differences. Isoflavones treatments with dosages beyond 150 mg per day or treatment lengths beyond 26 weeks could not be shown in this study. The effects on serum cholesterols among subjects with hypertension, diabetes mellitus or other cardiovascular risk factors were also beyond this meta-analysis. Generalizability over other phytoestrogens such as coumestans, lignans and resveratrol may need further trials to clarify the questions. No funnel plots were shown in this study because the choice of risk differences as the horizontal axes may lead to funnel plot asymmetry [36]. Publication bias was still possible although vigorous searching had been attempted.\nBiological plausibility\nIt was suggested that isoflavones readily bind estrogen receptors \u2013 beta (ER-beta) which are an important receptors in both the central nervous and cardiovascular systems [37-39]. Isoflavones may also have anti-oxidant effects on blood vessels [40-42]. However, compared with estradiol, isoflavones bind estrogen receptors with 100 and 1,000 times less affinity [30]. Both conjugated and aglycone forms of daidzein and genistein are rapidly cleared from the plasma following a single dose of isoflavones [43] and urinary excretion is virtually nil within 48 hours after ingestion [44,45]. Biological effects of soy phytoestrogens are still controversial. The hypothesis from an influential meta-analysis [1] stating soy estrogens may be responsible for the lipid reduction effects was criticized for the fact that the trials involved largely isoflavone-free soy protein products [46]. Apart from phytoestrogens, in fact, some other components such as phytic acid, saponins and fibers were potential candidates responsible for the hypocholesterolemic effects [32,48,49].\nSuggestion to future trials\nTablets of single isolated isoflavone with considerable dosages may be important in future randomized trials. Intervention with isoflavones mixtures may be helpful but interactions between components should be handled with extra cares. Introduction of phytoestrogens in forms of soy diets is not suggested because we found much heterogeneity inside the diets, ranging from soy biscuits to burger, which may be difficult to combine and analyze. Confounding factors such as fiber, fatty acids, amino acids and energy intake are especially hard to control. Complications associated with hyperlipidemia, such as coronary heart disease (CHD) or cardiovascular accident (CVA), may be selected as other endpoints in future trials. It may help constructing convincing funnel plots with less heterogeneity as trials accumulate.\nConclusions\nIsoflavones tablets, up to 150 mg per day, had insignificant effects in lowering serum total cholesterol, LDL-cholesterol and triglyceride. There was also insignificant benefit over serum HDL-cholesterol. The results were consistent when tablets were introduced as isolated genistein, mixture of genistein and daidzein, or mixture of genistein, daidzein, formononectin and biochanin A. No significant effects were found among participants with normo- or hyperlipidemia and women with pre- or postmenopausal status. Isoflavones interventions in the forms of soy proteins, such as isolated soy protein (ISP), soy diets or soy protein capsule, were inconclusive due to inadequate sample size, heterogeneity and presence of potentially uncontrolled confounders.\nList of abbreviations\n'ISP' = isolated soy protein; 'TC' = total cholesterol; 'LDL' = Low density lipoprotein cholesterol; 'HDL' = High density lipoprotein cholesterol; 'TG' = Triglycerides\nCompeting interests\nNone declared.\nAuthors' contributions\nJY participated in the design of this manuscript. JY and YTF participated in abstracted the data and performed statistical analysis. All authors read and approved the final manuscript.","keyphrases":["isoflavones","soy","lipids","meta-analysis","phytoestrogens"],"prmu":["P","P","P","P","P"]}
{"id":"Intensive_Care_Med-2-2-1315314","title":"SAPS 3\u2014From evaluation of the patient to evaluation of the intensive care unit. Part 1: Objectives, methods and cohort description\n","text":"Objective Risk adjustment systems now in use were developed more than a decade ago and lack prognostic performance. Objective of the SAPS 3 study was to collect data about risk factors and outcomes in a heterogeneous cohort of intensive care unit (ICU) patients, in order to develop a new, improved model for risk adjustment.\nIntroduction\nFollowing the publication in the early 1980s of the Acute Physiology and Chronic Health Evaluation Score (APACHE [1]), Simplified Acute Physiology Score (SAPS [2]), and-some years later\u2014APACHE II [3] systems, outcome prediction became an important topic among European intensivists. Ten years later, a new generation of these instruments was published: APACHE III [4], SAPS II [5], and Mortality Probability Model (MPM) II [6]. All of these newer systems were developed by using sophisticated statistical techniques in large multinational databases, and were found to perform better than their predecessors [7, 8].\nThe availability of such sophisticated methods for risk adjustment facilitated outcome research in critically ill patients, which became increasingly important over time. Risk adjustment systems now have a fixed place in critical care research for various purposes. At the patient level, the reporting of severity of illness and the use of risk-adjusted mortality rates to draw inferences from their results are a prerequisite for any study to be published. At the intensive care unit (ICU) level, observed-to-expected mortality ratios (or the use of direct standardisation techniques based on severity scores) have become standard for assessing the impact of ICU-related factors on outcome, such as the effects of organisation and management [9, 10].\nHowever, a series of studies assessing the performance of risk adjustment systems unveiled a lack of prognostic performance of these systems: In most cases, lack of calibration was evident over several subgroups of patients, often accompanied by an underestimation of mortality in low-risk patients and an overestimation in high-risk patients. This pattern was observed for all published outcome prediction models in several countries [11, 12, 13, 14, 15, 16, 17, 18] and seemed to be worsening over time [19].\nFor this reason, several researchers tried to improve the prognostic performance of various systems through recalibration, using one of two possible approaches. A level 1 customization requires calculation of a new equation for the prediction of hospital mortality (without changing the weights of the constituent variables). A level 2 customization involves a reweighting of each variable contained in the model. Although recalibration was able to improve prognostic accuracy in some cases [13, 14], it generally did not solve the various problems inherent in the models.\nThese problems can be classified as either user-, patient-, or model-dependent. User-dependent problems include differences in the definitions and application criteria [20, 21]. Patient-dependent problems are mainly shifts in the baseline characteristics of the populations over time [22]: age distribution, distribution of illnesses, and the development of new treatments, all of which affect prognosis. Model-dependent problems have many different causes, such as the lack of important prognostic variables (e.g., diagnostic information [4, 23]) or the presence, location and aetiology of infection [24, 25, 26]. Confounding variables and statistically wrong assumptions [9, 27] also distort performance results.\nIf recalibration is not sufficient to improve the performance of the prognostic model, the only alternative is to develop a new model that takes into account the results of studies done since the original model was developed. This means incorporating missing variables that have been shown to affect outcome, minimizing problems with the application of the model, and reducing the possibility of other confounders.\nThe objective of the SAPS 3 project was to cope with the above-stated problems by developing a new model for improved risk adjustment in critically ill patients. Another important goal was to make the new model available free of charge for use in the scientific community.\nIn the SAPS 3 study (which took place at the end of 2002), data about risk factors and outcomes in an international multicentric cohort of critically ill patients were prospectively collected so that a high-quality database would be available for further analysis of the associations between risks and outcomes in our patients.\nMaterials and methods\nProject Organization\nThe SAPS 3 project was conducted by the SAPS 3 Outcomes Research Group. The project was endorsed by the European Society for Intensive Care Medicine (ESICM, http:\/\/www.esicm.org) and conducted in cooperation with the Section on Health Services Research and Outcome of the ESICM. The SAPS 3 Outcomes Research Group consists of a project coordinator and a steering group. The steering group was responsible for the scientific conduct and consistency of the project. An additional advisory board integrated further scientists with special expertise who were asked for comments on the scientific content and for help in conducting the project. The complete board lists can be found in Appendix D of the Electronic Supplementary Material (ESM).\nDuring the data collection phase, a coordination and communications centre (CCC) was installed. The CCC was responsible for the management and control of the project. This included the administration of all project tasks and implementation of actions and activities as necessary; communication between project partners (e.g., centres, researchers and institutions) through sampling and distribution of necessary information; and pooling and administration of the data provided by project participants. In addition, the CCC provided almost around-the-clock service to answer urgent questions and resolve problems during the phase of data collection.\nIn each country, a country coordinator was responsible for operational management and direct communication with the participating ICUs in that country, including giving specific help when necessary. The country coordinator was responsible for ensuring completion of the various tasks required of the participating ICUs. The list of country coordinators can be found in Appendix E of the ESM.\nAt the ICU level, an ICU coordinator was responsible for local activities, such as obtaining approval from the local ethics or data-protection committees where applicable. In addition, the ICU coordinator was responsible for supervising the daily data collection, problem management, controlling the completeness of the data, data quality control, training medical and nonmedical staff for data collection, management of the data, and transmission of the data to the CCC or country coordinator. The list of ICU coordinators can be found in Appendix F of the ESM.\nData collection\nPatient data were recorded by using either online data collection software (provided by iMDsoft, Tel Aviv, Israel) or the SAPS 3 stand-alone database system (provided by the CCC). The latter software used a Microsoft Access database (Microsoft Corporation, Redmond, WA, USA) for data storage and needed no Internet connection for data entry. Both systems maintained a variety of plausibility controls to ensure the quality of the recorded data. Each variable was precisely defined before the start of data collection (see Appendix C of the ESM). Detailed definitions of the variables were available to participants in both paper and electronic form. To facilitate plausibility checking, each variable was assigned a probability range, encompassing the range of probable values for that variable. In addition, a range of possible values (storage range) for that variable was defined (e.g., for FiO2, no values <21% or >100% could be accepted). Thus, formal plausibility controls in the software systems were used wherever possible and ensured the maximum of data quality checking during data collection.\nParticipants who could not use one of the two software options were allowed to record the data on paper forms and submit them to the CCC (n=26 ICUs). Patient data were then entered into the SAPS 3 stand-alone software system and thus checked for plausibility. In cases of uncertainty, ICU coordinators were contacted for clarification.\nIn addition, each ICU received a questionnaire with detailed questions about ICU structures and about resources available in other areas of the hospital.\nData were collected at ICU admission, on days 1, 2 and 3, and on the last day of the ICU stay. Data from the day of admission (aside from sociodemographic data such as age and sex) were categorized into different levels: (i) data about the condition of the patient before ICU admission, such as chronic conditions and medical diseases; (ii) data about the patient\u2019s condition at ICU admission, such as the reason for admission, infection at admission, and surgical status; and (iii) data about the patient\u2019s physiologic derangement at ICU admission. These data were collected within an hour before or after ICU admission.\nOn the following days of the ICU stay, further information was collected: severity of illness, as measured by the SAPS II [5]; number and severity of organ dysfunction, as measured by the Sequential Organ Failure Assessment (SOFA) [28]; length of ICU and hospital stay; and outcome data, including vital status at ICU and hospital discharge. All patients were subjected to mandatory follow-up until hospital discharge, but not longer than 90\u00a0days after ICU admission. Patients still remaining in the hospital at 90\u00a0days were at that time classified as being \u201cstill in the hospital\u201d.\nTo record diagnoses, a three-level system was used. (i) An acute medical disease was recorded for all patients, independent of surgical status, i.e., the acute (or acute on chronic) disease that best explained the ICU admission. If the reason for ICU admission was infectious disease, then this was recorded. (ii) Surgical status at admission and the anatomic site of surgery were recorded for all patients undergoing surgery during the hospital stay before ICU admission. (iii) A concrete reason for admission had to be selected. At least one reason for admission was required, but several selections were possible (one within each organ system). If no other reason was present, at least \u201cbasic and observational care\u201d had to be selected.\nAll participants received detailed documentation of patient- and ICU-based data items as well as a detailed description of the data collection process. Moreover, specific forms to check the completeness of the patient-based documentation were provided. Additionally, a training session for ICU coordinators was organised at the 15th Annual Congress of the ESICM in Barcelona, Spain, before the start of data collection. Throughout the project, the project website (http:\/\/www.saps3.org) provided all necessary information. In addition, the CCC was available to answer questions by email, fax and phone. Data were to be collected from all consecutively admitted patients between 14 October and 15 December 2002. ICUs with a high number of beds (and thus also admissions) could stop patient enrolment after contributing 100 patients.\nDatabase\nData were collected and pooled by the CCC. The final database file was then imported into the SAS system, Version 8e (SAS Institute Inc., Cary, USA). Data cleaning was accomplished through the application of a variety of additional plausibility controls and cross-checking of information between redundant data fields.\nA total of 22,791 admissions were recorded in the 309 participating ICUs during the study period. For patients who were admitted more than once (n=1,455), only the first admission was included, giving 21,336 admitted patients. Patients who were <16\u00a0years of age (n=628), those without ICU admission or discharge data (n=1,074), and those with records that lacked an entry in the field \u201cICU outcome\u201d (n=57) were excluded. The Basic SAPS 3 Cohort thus comprises 19,577 patients from 307 ICUs.\nFor the development of a predictive model for hospital mortality as outcome, patients with a missing entry in the field of \u201cvital status at hospital discharge\u201d (n=2,540) or an entry of \u201cstill in the hospital\u201d at the end of the follow-up period (n=253) were further excluded. The SAPS 3 Hospital Outcome Cohort thus comprises 16,784 patients from 303 ICUs.\nBecause the study was an observational study and no additional interventions were performed, the need for informed consent was waived by the institutional review board. Each ICU, however, was made responsible for obtaining local permissions as necessary.\nData quality\nRecorded data were evaluated for completeness of the documentation and reliability. Interrater quality control was performed through rescoring of the data from day 0 (the day of ICU admission) for three randomly selected patients in each ICU. From the rescored data, kappa coefficients and intra-class correlation coefficients were calculated, as appropriate. Availability of the variables necessary to calculate the SAPS II was used as an indicator for the completeness of the data.\nStatistical analysis\nStatistical analysis was performed using the SAS system, version 8e (SAS Institute Inc., Cary, NC, USA). A P value of <0.05 was considered significant. Unless otherwise specified, results are expressed as median and interquartile ranges (quartile). Observed-to-expected (O\/E) mortality ratios were calculated by dividing the number of observed deaths per group by the number of expected deaths per group (as predicted by the SAPS II). To test for statistical significance, we calculated 95% confidence intervals (CI) according to the method described by Hosmer and Lemeshow [29]. The Hosmer-Lemeshow goodness-of-fit \u0124-statistic and \u0108-statistic [30] were used to evaluate the calibration of the SAPS II. Discrimination was tested by measuring the area under the receiver operating characteristic (aROC) curve, as described by Hanley and McNeil [31]. Reliability of data collection was analysed using K-statistics or intra-class correlation coefficients, as appropriate. Statistical methods used for the development of the predictive model are described in Part 2 of this report.\nResults\nData quality\nFour hundred eighty-three rescored patients could be identified and linked to their original counterparts (2.5% of admitted patients). Data quality was found to be excellent, with the majority of coefficients being >0.85. Only two of the more than 50 tested variables had coefficients <0.80 (body weight, 0.79; positive end-expiratory pressure, 0.72), and only one was <0.70 (leukocytes [maximum], 0.57). For a detailed list of coefficients see Table E1 in the ESM. Data completeness was also found to be satisfactory, with 1 [0\u20133] SAPS II parameter missing per patient.\nDescription of ICUs\nThe Basic SAPS 3 cohort includes 307 ICUs from 35 countries. On average each ICU contributed 50 (27\u201378) patients to the cohort. To assess heterogeneity of results between different geographic regions, seven regions were defined: Australasia, Central and South America, Central and Western Europe, Eastern Europe, North America, Northern Europe, and Southern Europe and Mediterranean countries. The allocation of countries to these regions can be seen from Table E10 of the ESM.\nSeventy percent of the participating ICUs identified themselves as mixed medical-surgical (Table E2, ESM). Roughly half of the ICUs (46%) were located in university-affiliated or teaching hospitals. Eighty-four percent of ICUs (n=258) reported having a full-time medical director, and 272 (88.6%) reported having a full-time nursing director. On weekdays, 76.6% of ICUs reported having an intensivist available on the ICU 24\u00a0hours per day, whereas 6.2% had an intensivist available in the hospital. In 12.1% of ICUs, the intensivist was at home, on-call, during the daytime. During weekends, this proportion did not change much (74.3%, 5.5%, and 15.0% on the ICU, in the hospital, and on-call, respectively). None of the participating ICUs reported having no intensivists available during night or weekend shifts.\nDescription of patients\nThe Basic SAPS 3 Cohort comprises 19,577 patients admitted to participating ICUs during the study period. More than 70% of patients were admitted from the same hospital as the ICU, with operating rooms, emergency departments and normal wards contributing most of the patients (Table\u00a01). Almost two thirds of the admissions were classified as unplanned. The mean age of patients was 60.0\u00b117.7\u00a0years (Fig.\u00a01), and 39.2% were female. Comorbidities were recorded in 65% of admitted patients, with arterial hypertension, chronic obstructive pulmonary disease, and chronic heart failure being the most frequent (Table E3, ESM).Table\u00a01 ICU admission data for the two cohorts (Basic cohort: SAPS 3 basic cohort; HO cohort: SAPS 3 Hospital Outcome Cohort; n: number of patients)Basic cohortHO cohortn%n%Number of patients19,57716,784100.0Gender\u00a0\u00a0Female7,67839.26,61039.4\u00a0\u00a0Male11,88160.710,16160.5 \u00a0\u00a0Missing180.1130.1Age, years (median, quartiles)6349-746449-74Origin\u00a0\u00a0Home2,81014.42,34314.0\u00a0\u00a0Same hospital13,92671.112,06371.9\u00a0\u00a0Chronic care facility740.4640.4\u00a0\u00a0Public place5192.74322.6\u00a0\u00a0Other hospital2,12510.91,79110.7\u00a0\u00a0Other800.4590.4\u00a0\u00a0Missing430.2320.2Intra-hospital location before ICU admission\u00a0\u00a0Emergency room5,41927.74,63027.6\u00a0\u00a0Intermediate care unit\/High dependency unit5622.94752.8\u00a0\u00a0Operating room7,53738.56,44938.4\u00a0\u00a0Other5522.84132.5\u00a0\u00a0Other ICU6983.66113.6\u00a0\u00a0Recovery room4822.54002.4\u00a0\u00a0Ward3,41117.43,03618.1\u00a0\u00a0Missing9164.77704.6ICU admission status\u00a0\u00a0Planned admission6,75034.55,59833.4\u00a0\u00a0Unplanned admission12,33863.010,80164.4\u00a0\u00a0Missing4892.53852.3Acute Infection at ICU admission\u00a0\u00a0No infection15,25477.912,96877.3\u00a0\u00a0Clinically improbable\/colonization3421.72981.8\u00a0\u00a0Clinically probable\/documented2,76114.12,42214.4\u00a0\u00a0Microbiologically documented12066.21,0836.5\u00a0\u00a0Missing130.1130.1Surgical status\u00a0\u00a0No surgical procedure8,43743.17,30543.5\u00a0\u00a0Scheduled surgery6,80034.75,70034.0\u00a0\u00a0Emergency surgery3,32117.02,93017.5\u00a0\u00a0Missing1,0195.28495.1Fig.\u00a01 Age distribution and associated mortality. The figure shows the age distribution of the Basic SAPS 3 Cohort (n=19,577) and the corresponding ICU mortality rates for each stratum. Columns: Number of patients as percentages of the whole cohort; squares: ICU mortality rates for the corresponding stratum\nCardiovascular, respiratory and neurologic diseases were the most frequent organ-specific reasons for admission (Table E4, ESM). The acute medical diseases necessitating ICU admission included a broad spectrum of diagnoses (Table E5, ESM). Approximately one half of the patients underwent surgery before ICU admission, with abdominal, cardiac and vascular surgery being the most frequent procedures (Table E6, ESM).\nRegarding discharge details (Table\u00a02), it is notable that a high percentage of patients were discharged unplanned (8.15%), i.e., without at least a 12-hour planning window. 15.2% of patients from the SAPS 3 Basic cohort died within the ICU. As can be seen from Table\u00a03, patient cohorts differed significantly between regions. Both, ICU and hospital mortality rates exhibited a broad spectrum between ICUs: hospital mortality was on average 28% (17\u201342%) in the SAPS 3 Hospital outcome cohort.Table\u00a02 ICU discharge and outcome data for the two cohorts (Basic cohort: SAPS 3 basic cohort; HO cohort: SAPS 3 Hospital Outcome Cohort; n: number of patients;\nICU LOS: ICU length of stay; IMCU\/HDU: intermediate care unit\/high dependency unit; Q1, Q3: lower and upper interquartile range, respectively)Basic cohortHO cohortn%n%Number of patients19,577100.016,784100.0ICU LOS, days (median, quartiles)21\u2013621\u20136ICU discharge\u2014destination\u00a0\u00a0Home4382.23612.2\u00a0\u00a0Same hospital14,94676.312,47774.3\u00a0\u00a0Other hospital1,0295.38525.1\u00a0\u00a0Missing3,16416.23,09418.4Intrahospital discharge\u00a0\u00a0Emergency room580.3500.3\u00a0\u00a0IMCU\/HDU2,22211.41,87311.2\u00a0\u00a0Other3031.52571.5\u00a0\u00a0Other ICU5833.04792.9\u00a0\u00a0Recovery room3061.62181.3\u00a0\u00a0Ward12,25062.610,29161.3\u00a0\u00a0Missing3,85519.73,61621.5ICU discharge\u2014status\u00a0\u00a0Planned discharge14,87276.012,26273.1\u00a0\u00a0Unplanned discharge1,5958.11,4678.7\u00a0\u00a0Missing3,11015.93,05518.2Risk adjustment\u00a0\u00a0SAPS II score (median, Q1\u2013Q3)3020\u2013423121\u201343\u00a0\u00a0SOFA score (median, Q1\u2013Q3)96\u20131196\u201311Outcome\u00a0\u00a0ICU mortality (%)15.217.7Table\u00a03 ICU admission and discharge data for the seven defined geographic regions (SAPS 3 Basic Cohort; n=19,577)AustralasiaCentral & South AmericaEastern EuropeCentral and Western EuropeNorthern EuropeSouthern Europe and Mediterranean countriesNorth AmericaNumber of patients2,235\u00a02,540\u00a01,084\u00a04,712\u00a0355\u00a07,854\u00a0797\u00a0Females, %38.0\u00a044.6\u00a042.2\u00a040.1\u00a042.5\u00a036.9\u00a038.0\u00a0Age, years (median, quartiles)5945\u2013713146\u2013746248\u2013726552\u2013756651\u2013766459\u2013746449\u201375SAPS II score (median, Q1-Q3)2819\u2013403020\u2013422718\u2013432921\u2013403525\u2013463222\u2013442920\u201339SOFA score (median, Q1-Q3)86\u20131086\u20131197\u20131185\u20131197\u20131197\u20131196\u201311ICU mortality, %12.7\u00a017.4\u00a016.9\u00a010.8\u00a020.6\u00a018.1\u00a08.5\u00a0\nPerformance of the SAPS II\nThe performance of the original SAPS II model [5] (using data from the first 24\u00a0hours) was tested in the SAPS 3 Hospital Outcome Cohort (n=16,784). Discrimination was good with an aROC of 0.83 (95% CI: 0.824\u20130.838). SAPS II showed underestimation of hospital mortality: The O\/E ratio of the overall cohort was 1.08 (1.06\u20131.10). O\/E ratios significantly differed between regions: from 0.86 (0.81\u20130.91) for Central and Western Europe to 1.32 (1.25\u20131.38) for Central and South America, with four out of the seven defined regions exhibited O\/E ratios significantly different from 1 (Table E7, ESM). Calibration, as assessed by the Hosmer-Lemeshow \u0124 + \u0108 statistics, was poor for the overall cohort: \u0124 227.21, \u0108 184.70; both p<0.0001; This lack of calibration was present for all tested subgroups except for the region of North America (see Table\u00a0E7, ESM).\nDiscussion\nTo the best of our knowledge, the SAPS 3 study is the largest prospective epidemiologic multicentre, multinational study conducted in health services and outcomes research in intensive care medicine to date.\nThe project was first intended to focus on Europe because it was believed such a strategy would produce a more homogeneous cohort of patients, which would in turn provide a more stable reference line for further comparisons. This idea was discussed during several investigator meetings and finally abandoned\u2014first, because interest from outside Europe was enormous: 39% of ICUs that registered for the project were located outside Europe. The SAPS 3 board members thus agreed that such a high level of interest should not be ignored. Second, some investigators questioned whether a concentration on European ICUs would be successful in reducing heterogeneity anyway. Provision of intensive care in Europe is extremely variable, with enormous differences in severity of illness, provision of treatments and mortality from north to south and from west to east [32, 33].\nFor these reasons ICUs from regions outside Europe were invited to participate. Our results prove that we were right in our assumptions: First, one can easily see that the four European regions (as defined in our study) are hardly comparable: severity of illness as measured by the SAPS II varied from 27 to 35 points, and ICU mortality ranged from 10.8 to 20.6%\u2014almost a doubling of mortality figures (Table\u00a03). Second, almost a third of the patient cohort (28.5%) was contributed from regions outside Europe.\nAlthough the decision to accept ICUs worldwide probably increased the heterogeneity of our sample, it also allowed the SAPS 3 database to better reflect important differences in patients\u2019 and health care systems\u2019 baseline characteristics that are known to affect outcome. These include, for example, different genetic makeups, different styles of living or a heterogeneous distribution of major diseases within different regions, as well as issues such as access to the health care system in general and to intensive care in particular, or differences in availability and use of major diagnostic and therapeutic measures within the ICUs [32, 34]. Although the integration of ICUs outside Europe and the U.S. surely increased it\u2019s representativeness, it must be acknowledged, that the extent to which the SAPS 3 database reflects case-mix on ICUs worldwide cannot be determined yet.\nIt should additionally be noted that allocation of countries to regions does not always follow geographic borders (Table\u00a03; see also Table E10 in the ESM). Partitioning of the sample was done to adjust for some of the above-stated differences between different populations and to develop a system that uses several different reference lines to compare ICUs on a similar level. Thus, patients are not necessarily representative of their respective regions.\nTo minimize possible seasonal influences, we chose late fall in the Northern Hemisphere for data collection. Thus, participants in both late fall\/winter (Northern Hemisphere) and spring\/summer (Southern Hemisphere) are represented in our cohort. A recent study [35] showed, moreover, that differences in seasonal mortality rates, at least in a sample of ICUs in the United Kingdom, were related to variations in case mix rather than to a specific impact of season on outcome.\nPerformance of the SAPS II was, not surprisingly, found to be similar to that in previous studies: acceptable discrimination but lack of calibration. Possible reasons for this have already been alluded to in the Introduction. In contrast to previous studies, however, we found an underestimation of hospital mortality, which contradicts the rationale that the shifting in calibration is due only to the development of new and possibly better therapies and thus to better ICU performance [19].\nAnalyzing the various geographic regions provides evidence that the underestimation of hospital mortality by the SAPS II might be partially attributable to the composition of the cohort: SAPS 3 is the first large international study on severity of illness systems to include patients from all continents. South America, for example, where provision of intensive care is much more limited than it is in Europe or North America, contributed extensively to the patient cohort. High O\/E ratios have already been reported for this continent [36] and are probably linked to the limited availability of resources.\nData quality was one of our major concerns. Completeness of the documentation was found to be satisfactory: The amount of missing data is in fact smaller than reported from previous cohort studies on severity of illness systems [11, 12, 16]. With respect to reliability, intraclass-correlation coefficients and kappa coefficients were generally similar to or even better than those found in previous studies, showing a high degree of interrater agreement (see Table E1 in ESM) [37, 38].\nWe did, however, experience problems with the cohort of rescored patients: First, we had to exclude all rescored patients for whom the original counterpart was also excluded due to the application of any of the exclusion criteria. Second, in some cases the original patient identification was either missing or documented in such a way that a unique allocation was not possible. Both of these exclusions reduced the number of rescored patients available for analysis.\nTwo strategies to build up a cohort are available: first, to recruit only patients who meet well-documented inclusion criteria (such as documented vital status at hospital discharge) or, second, to document all patients and then exclude patients based on a predefined set of exclusion criteria. For the SAPS 3 study we chose the second option\u2014to form two different cohorts\u2014because we needed to provide a basic cohort for all further analyses of the SAPS 3 database. Since some studies will focus on different outcomes (e.g., ICU outcome rather than hospital outcome), we decided to use missing ICU outcome (and not hospital outcome) as an exclusion criterion for the basic cohort.\nA possible limitation of the SAPS 3 database is that vital status at hospital discharge was not available for all admitted patients. Despite several efforts from the CCC and sufficient time to allow for a close follow-up, we did not succeed to receive all hospital outcomes documented. Recording of hospital outcome (or later outcomes) still poses major problems for ICUs in European and non-European hospitals, either because of technical problems or possibly because of data security algorithms in the hospitals. Exclusion of these patients did, however, not affect major criteria, such as geographic representation, ICU admission or discharge data, co-morbidities, or the distribution of the reasons for admission (Tables 1 and 2).\nWe conclude that the SAPS 3 database is within the above discussed limits of high quality and reflects the heterogeneity of current intensive care provision. As such, it provides an excellent basis for the development of a new risk adjustment system.\nElectronic Supplementary Material\n(PDF 794 KB)","keyphrases":["intensive care unit","risk adjustment","severity of illness","hospital mortality","icu mortality"],"prmu":["P","P","P","P","P"]}
{"id":"Arch_Gynecol_Obstet-2-2-1705528","title":"Single-day therapy: an expert opinion on a recent development for the episodic treatment of recurrent genital herpes\n","text":"One common method for treating recurrent genital herpes outbreaks is 3\u20135 day episodic therapy with nucleoside analogues. However, since maximum viral replication occurs within 24 h after the onset of symptoms, short-term patient-initiated episodic therapy started at prodromal onset or at the first appearance of lesions in patients without a prodrome may represent an important option. In a recent randomized trial, single-day famciclovir treatment decreased lesion healing time and the duration of pain and other symptoms by approximately 2 days compared to placebo, and prevented progression to a full outbreak in almost one in four patients. Because single-day treatment is more convenient than traditional therapies, it may lead to improved patient compliance and better overall management of recurrent genital herpes outbreaks.\nIntroduction\nRecurrent genital herpes outbreaks are predominantly caused by herpes simplex virus type 2 (HSV-2), although the incidence of genital herpes caused by herpes simplex virus type 1 (HSV-1) is rising due to changing attitudes about oral-genital sexual behavior [10]. Approximately one in four adults in the United States is seropositive for HSV-2 [7], an incurable infection that initially enters the body through mucous membranes or abraded skin. Once primary infection has occurred, the virus remains dormant in the dorsal root ganglia, where it can reactivate to cause uncomfortable, recurrent outbreaks [20].\nHSV-2 infection is somewhat more prevalent in women than in men [7], and can have a profound psychological impact as well as physical repercussions, resulting in a decrease in measures of quality of life. The fear of spreading the infection can result in lowered self-esteem or sexual desire and, less commonly, social phobia and depression [6]. In addition, patients with genital herpes have a significantly increased risk of acquiring human immunodeficiency virus (HIV) [8].\nEpisodic treatment for genital herpes\nAlthough there is no cure for HSV-2 infection, patients have two treatment options: treating outbreaks as they occur (episodic therapy), or attempting to prevent future outbreaks (suppressive therapy). Suppression with daily oral antivirals may be most appropriate for patients with frequent or uncomfortable recurrences and to reduce genital herpes transmission to sexual partners [4]. Although suppressive therapy is effective, one quarter to one half of patients on suppressive therapy have at least one recurrence per year [5, 14].\nAlthough episodic treatment has not been proven to reduce the risk of transmission, it still remains an important option for patients who do not want to take daily suppressive therapy, are not concerned about the frequency of recurrences, or are not sexually active. The best method for administering episodic therapy is to make medication readily available to patients to self-administer at the first onset of symptoms or lesions [16]. This type of patient-initiated episodic therapy has been successfully used in previous clinical trials of recurrent genital herpes [13, 15]. The ability to treat recurrences sooner may also help decrease the duration of an outbreak or halt progression to a full outbreak [9, 13, 15, 19]. This expert opinion will examine the results from a recently published clinical trial about patient-initiated, single-day oral antiviral therapy (famciclovir) for recurrent genital herpes [2] and compare its efficacy with data from currently available therapies.\nTreatment of recurrent genital herpes with oral antivirals\nPatients with recurrent episodes of genital herpes often experience such prodromal symptoms as itching, tingling, burning, and pain, with papules and vesicles typically forming 12\u201324\u00a0h after onset of these symptoms [3]. Maximum HSV-2 concentration and lesion pain usually occur within the first 24\u00a0h of onset of these prodromal symptoms [3]. Under the assumption that one can extrapolate from the pathogenesis of herpes labialis [17], it is thought that the most effective way to treat recurrent genital herpes with antiviral therapy would be within the first 24\u00a0h after the onset of clinical symptoms, when viral replication is highest [16]. Oral antivirals, acting as nucleoside analogues enter infected cells and bind to viral thymidine kinase, resulting in their phosphorylation. After the antivirals are further phosphorylated by cellular enzymes, they compete with nucleosides to bind to the viral DNA polymerase, resulting in polymerase inactivation and a decrease in viral replication [16].\nAlthough topical acyclovir was initially used to treat recurrent genital herpes, it has since been proven ineffective and is no longer recommended [1, 12]. Today, the most commonly prescribed drugs are the oral antivirals acyclovir, valacyclovir, and famciclovir. Acyclovir is approved for 5-day episodic treatment, although a 2-day regimen has been shown to be effective [13, 19]. Acyclovir is effective, but its poor bioavailability requires more frequent dosing. Valacyclovir, a prodrug of acyclovir, has a higher bioavailability than acyclovir and has been approved for 3-day episodic treatment [9, 14]. Famciclovir, a prodrug of penciclovir, is currently indicated for 5-day treatment [11, 15]. The high bioavailability of famciclovir (77%) and the rapid onset of viral replication in recurrent genital herpes suggested that this medication would be efficacious in an even shorter single-day treatment regimen.\nData from single-day famciclovir clinical trial\nA clinical trial was performed to assess whether single-day famciclovir was effective in treating recurrent genital herpes in immunocompetent individuals [2]. The 329 study participants experiencing a recurrence of genital herpes were predominately female (71%) and 48% had had more than six recurrences in the preceding year. Participants were given a single day of famciclovir 1,000\u00a0mg bid or matching placebo and were instructed to take the medication within 6\u00a0h of the onset of prodromal symptoms and\/or genital herpes lesions during their next recurrence. The trial results showed that a single-day regimen of famciclovir significantly decreased lesion-healing time and significantly reduced time to resolution of all symptoms by 2\u00a0days, as compared to placebo. Famciclovir also halted the progression to a full genital herpes outbreak in almost one in four patients. Adverse events were of mild-to-moderate intensity and were similar to the adverse events experienced by the placebo group.\nAlthough no head-to-head studies have been conducted between single-day famciclovir and other oral antivirals, patients who received single-day famciclovir experienced effects similar to those seen in previous clinical trials of traditional longer-term therapies (Table\u00a01) [2, 9, 13, 15, 18, 19]. Single-day therapy appears to inhibit viral replication enough to significantly reduce both symptoms and the tissue damage characteristic of a full outbreak, preventing progression to a full recurrence in some cases.\nTable\u00a01Effectiveness of oral antivirals in clinical trials of episodic treatment for genital herpesDrugTreatment regimenMedian time (days) to lesion healing (treatment vs control)Percent of aborted episodes (treatment vs control)Acyclovir [13, 19]800\u00a0mg tid \u00d7 2\u00a0days versus placebo200\u00a0mg 5 times daily \u00d7 5\u00a0days versus placebo4.0 versus 6.0 (P\u00a0=\u00a0.001) 5.7 versus 7.2a (P\u00a0\u2264\u00a0.001)27.0 versus 10.6% (P\u00a0=\u00a0.029)    Was not performedValacyclovir [9, 18]500\u00a0mg bid \u00d7 3\u00a0days versus 500\u00a0mg bid \u00d7 5\u00a0days1,000\u00a0mg bid \u00d7 5\u00a0days versus 200\u00a0mg acyclovir 5 times daily \u00d7 5\u00a0days versus placebo4.4 versus 4.7 (P\u00a0=\u00a0NS)4.8 versus 4.8 versus 6.0 (P\u00a0<\u00a0.001)b25.4 versus 26.6% (P\u00a0=\u00a0NS)25.9 versus 24.8 versus 19.8% (P\u00a0=\u00a0NS)Famciclovir [2, 15]1,000\u00a0mg bid \u00d7 1\u00a0day versus placebo125\u00a0mg bid \u00d7 5\u00a0days versus placebo4.3 versus 6.1 (P\u00a0<\u00a0.001)3.8 versus 4.8 (P\u00a0<\u00a0.001)23.3 versus 12.7% (P\u00a0=\u00a0.003)Was not performedaMean time to healingbP values correspond to both valacyclovir versus placebo and acyclovir versus placeboNS\u00a0=\u00a0Not significant\nConclusion\nThe rapid onset of viral replication in recurrent genital herpes suggests that a shorter course of therapy would be as efficacious as traditional treatments. Patient-initiated single-day therapy could prove helpful for obstetricians\/gynecologists who treat patients with genital herpes, as it may provide a more convenient option for treating the disease, and could help to promote patient compliance. High bioavailability, ease of use, and the added benefit of preventing progression to a full genital herpes outbreak in some patients make famciclovir an excellent candidate for single-day patient-initiated episodic therapy.","keyphrases":["genital herpes","patient-initiated episodic therapy","single-day famciclovir"],"prmu":["P","P","P"]}
{"id":"Transgenic_Res-4-1-2268725","title":"Mast cell-specific Cre\/loxP-mediated recombination in vivo\n","text":"Mast cells are important effectors of type I allergy but also essential regulators of innate and adaptive immune responses. The aim of this study was to develop a Cre recombinase-expressing mouse line that allows mast cell-specific inactivation of genes in vivo. Following a BAC transgenic approach, Cre was expressed under the control of the mast cell protease (Mcpt) 5 promoter. Mcpt5-Cre transgenic mice were crossed to the ROSA26-EYFP Cre excision reporter strain. Efficient Cre-mediated recombination was observed in mast cells from the peritoneal cavity and the skin while only minimal reporter gene expression was detected outside the mast cell compartment. Our results show that the Mcpt5 promoter can drive Cre expression in a mast cell-specific fashion. We expect that our Mcpt5-Cre mice will be a useful tool for the investigation of mast cell biology.\nIntroduction\nMast cells are central effector cells in type I allergy (Kawakami and Galli 2002; Metcalfe et\u00a0al. 1997), but have recently been shown to be important initiators and effectors also of innate immunity as well as modulators of adaptive immune responses (Galli et\u00a0al. 2005a, b; Marshall and Jawdat 2004). Many reports demonstrated a role of mast cells in host defence against pathogens (Dawicki and Marshall 2007; Echtenacher et\u00a0al. 1996; Malaviya et\u00a0al. 1996) and implicated a function of these cells in wound healing, tissue remodeling and transplant tolerance (Lu et\u00a0al. 2006; Trautmann et\u00a0al. 2000; Weller et\u00a0al. 2006). A pathogenic role of mast cells was discussed in rheumatoid arthritis, systemic sclerosis, multiple sclerosis and atherosclerosis (Bachelet et\u00a0al. 2006; Irani et\u00a0al. 1992; Lee et\u00a0al. 2002; Secor et\u00a0al. 2000; Sun et\u00a0al. 2007).\nInvestigation of mast cell biology was severely hampered by the scarcity of these cells in tissues. Experimental systems that have widely been used are in\u00a0vitro analysis of mast cell lines or mast cells differentiated in\u00a0vitro from bone marrow (BMMC). In\u00a0vivo analysis of the function of genes in mast cells until today relied on the reconstitution of mast cell-deficient rodents with mast cells derived from bone marrow or hematopoietic cells of gene-deficient animals or from gene-deficient embryonic stem cells (Kitamura et\u00a0al. 1978; Nakano et\u00a0al. 1985; Tsai et\u00a0al. 2002, 2000). These systems yielded valuable information, but suffer important limitations. In the mouse lines kitW\/W-v and kitW-sh\/W-sh, which are commonly used for reconstitution experiments, spontaneous mutations of the kit gene coding region or its regulatory elements result in reduced activity of the kit receptor that controls development and survival of mast cells (Galli et\u00a0al. 2005b; Grimbaldeston et\u00a0al. 2005). In addition to mast cell-deficiency, kitW\/W-vanimals are characterized by sterility, anemia, absence of melanocytes and interstitial cells of Cajal, a high incidence of dermatitis, papillomas of the forestomach, gastric ulcers and dilation of the duodenum (Galli et\u00a0al. 2005b). kitW-sh animals, which lately are being used instead of kitW\/W-v mice, seem to feature a more narrow defect with deficiency for mast cells, melanocytes, and interstitial cells of Cajal (Grimbaldeston et\u00a0al. 2005). The transfer of in\u00a0vitro cultivated mast cells into mast cell-deficient animals by intradermal, intraperitoneal or intravenous injections can result in mast cell numbers, which, dependent on the route of cell transfer and the anatomical site, can approach normal mast cell density, but often remain considerably lower with significant variability (Grimbaldeston et\u00a0al. 2005; Tsai et\u00a0al. 2005).\nThe Cre\/loxP recombination system represents a powerful tool that allows for conditional, e.g. cell type-specific, inactivation of genes in the mouse (Rajewsky et\u00a0al. 1996). Since the first description of in\u00a0vivo mutagenesis using the Cre\/loxP system (Gu et\u00a0al. 1994), an impressive number of floxed mouse lines has been generated. Herein, we report the first Cre transgenic line allowing mast cell-specific gene inactivation in\u00a0vivo.\nMaterial and methods\nConstruction of the transgene and generation of transgenic mice\nThe construct was based on a bacterial artificial chromosome (BAC) encompassing the entire Mcpt5 gene. The BAC clone RP23-284A14 from the mouse BAC library RPCI-23 (Osoegawa et\u00a0al. 2000) was obtained from RZPD (Deutsches Ressourcenzentrum f\u00fcr Genomforschung GmbH, Berlin, Germany). All modifications were done by homologous recombination in E. coli (Zhang et\u00a0al. 1998) and sequences of all oligonucleotides used for construction by homologous recombination are listed in Table\u00a01. Neighbouring genes contained in the BAC were excluded by replacing several kb of DNA at both ends of the insert by resistance genes (Fig.\u00a01). In a next step, a modified iCre (improved Cre, Shimshek et\u00a0al. 2002) gene cassette (Testa et\u00a0al. unpublished) was inserted into the Mcpt5 gene. The Cre cassette was first assembled with a loxm2-flanked neomycin-resistance cassette (neo) and flanked by homology regions (HR) for recombination into the Mcpt5 gene. This was done by subcloning the Cre cDNA into a minimal vector consisting of the origin of replication and a chloramphenicol resistance gene amplified from pACYC184 (New England Biolabs, Frankfurt, Germany). The primers used for the amplification contain the HRs for both subcloning of Cre and insertion into the Mcpt5 gene (see Table\u00a01). In a next step, the neomycin cassette was inserted 3\u2032 of Cre and 5\u2032 of the Mcpt5 HR. The HR-Cre-Neo-HR cassette was released from the minimal vector by NotI digestion and recombined into the BAC replacing the coding part of exon 1 of the Mcpt5 gene followed by Cre-mediated deletion of neo. The construct was purified for pronucleus injection as described by Sparwasser et\u00a0al. (2004) with modifications. Briefly, the construct was separated from the BAC backbone by NotI digest and purified by gel electrophoresis and electroelution. Pronucleus injection was done by R. Naumann, MPI of Molecular Cell Biology and Genetics, Dresden, Germany. Genotyping of Mcpt5-Cre transgenic mice was performed by PCR (primers: Mcpt5-CreFor 5\u2032ACAGTGGTATTCCCGGGGAGTGT, Mcpt5-CreRev 5\u2032 GTCAGTGCGTTCAAAGGCCA). Founder mice were bred to the ROSA26-EYFP reporter line (Srinivas et\u00a0al. 2001) and five to 15\u00a0week old mice were analyzed for reporter gene expression. Animals were housed under SPF conditions and all experiments were done according to institutional guidelines.\nTable\u00a01Primers used for transgene construction by homologous recombinationOligonucleotides used for transgene construction by homologous recombination. The respective 50\u00a0bp homology regions (HR) were included into the primers used for the amplification of selection markers or the minimal vectorFig.\u00a01Strategy of transgene construction. The BAC RP23-284A14 was shortened by replacing 51.3\u00a0kb of insert DNA at the T7 end of the vector backbone and 42.4\u00a0kb at the SP6 end by an ampicillin (Amp) and zeocin (Zeo) resistance cassette, respectively, resulting in a residual insert length of 129\u00a0kb. A NotI restriction site was inserted along with each of the resistance genes. The coding part of the first exon of Mcpt5 was replaced by a cassette comprising the Cre cassette and a loxm2-flanked neomycin resistance cassette (Neo) by homologous recombination and subsequent deletion of neo by Cre-mediated deletion in E. coli. (NotI, N, EcoRI, I, arrows represent open reading frames, shaded boxes represent untranslated regions)\nFlow cytometric analysis\nTotal splenocyte suspensions were separated into lymphocyte and non-lymphocyte fractions by magnetic cell sorting (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany) using anti-CD3-Biotin (clone 145-2C11, BD Pharmingen), anti-Biotin MicroBeads, anti-CD19 MicroBeads and LS MACS columns (Miltenyi Biotec). Skin cell suspensions were prepared from shaved abdominal skin plus one ear pinna by mincing followed by an incubation with 2\u00a0mg\/ml collagenase IV (Worthington Biochem. Corporation, Lakewood, NJ, USA) at 37\u00b0C. Cells in peritoneal lavage as well as single cell suspensions from skin and spleen were stained with phycoerythrin (PE)-conjugated anti-CD3 (clone 145-2C11), anti-CD19 (clone 1D3); anti-CD11c (clone HL3), anti-CD45 (clone 30-F11) and anti-Siglec-F (clone E50-2440) (all from BD Pharmingen) or anti-Fc\u03b5RI\u03b1-PE (clone MAR-1), allophycocyanin (APC)-conjugated anti-CD117 (clone 2B8) and anti-CD49b (clone D\u00d75), F4\/80-Biotin (clone BM8) and streptavidin-PE (all from eBiosciences, SanDiego, CA) or anti-Gr-1-PE (clone RB6-8C5) (Miltenyi Biotech). The cells were analyzed on a FACSCalibur (BD Biosciences).\nResults\nTransgene construction and screening of transgenic mice for Cre expression\nIn order to drive Cre expression in mast cells, we used a BAC clone containing the entire Mcpt5 gene along with abundant upstream and downstream flanking DNA (Fig.\u00a01). Neighboring genes were excluded by shortening the BAC on both ends to avoid possible effects of altered gene dosage in the transgenic mice. In a next step, the coding part of the first exon of Mcpt5 was replaced by the Cre cassette (see Methods and Fig.\u00a01). The construct was released from the BAC backbone by NotI digest (Fig.\u00a01) and injected into the pronuclei of C57BL\/6 oocytes. Six Mcpt5-Cre transgenic founder animals were obtained and mated to a ROSA26 reporter line (Srinivas et\u00a0al. 2001) which expresses enhanced yellow fluorescent protein (EYFP) under the control of the ubiquitous ROSA26 promoter following Cre-mediated deletion of a loxP-flanked stop element. Mcpt5-CreROSA26-EYFP double transgenic animals were analyzed for EYFP expression in peritoneal and skin mast cells by FACS as described below (Fig.\u00a02). Reporter gene expression was detected in mast cells from three of the six founder lines. One of these lines showed EYFP expression in only 80% of peritoneal mast cells and was not investigated further. Close to 100% of peritoneal mast cells were EYFP positive in two lines, which, because of autosomal inheritance of the transgene in one line and X-linked inheritance in the other, were called \u201cA-Mcpt5-Cre\u201d and \u201cX-Mcpt5-Cre\u201d, respectively.\nFig.\u00a02Efficiency of Cre-mediated reporter gene activation in mast cells. EYFP expression is demonstrated in cells from peritoneal lavage fluid and single cell suspensions of skin of Mcpt5-Cre ROSA26-EYFP double transgenic mice. Peritoneal mast cells were stained for CD117 and Fc\u03b5RI\u03b1 (n\u00a0=\u00a07) and skin mast cells for CD117 and CD45 (n\u00a0=\u00a04). Mast cell populations were gated and their EYFP fluorescence displayed in histogram plots. The black graph represents Mcpt5-Cre ROSA26-EYFP double transgenic mice, the shaded graph represents the Cre-negative but ROSA26-EYFP-positive control (littermates in most instances)\nEfficient and mast cell-specific Cre-mediated recombination in the lines A-Mcpt5-Cre and X-Mcpt5-Cre\nHighly efficient Cre-mediated activation of reporter gene expression was uniformly observed in peritoneal CD117+Fc\u03b5RI\u03b1+ mast cells of seven A-Mcpt5-Cre ROSA26-EYFP double transgenic animals (98.7\u201399.6%, on average 99.1%, Fig.\u00a02). In skin cell suspensions mast cells were detected in extremely low numbers, on average 0.32%. (Skin mast cells were defined as CD117+CD45+ cells. A staining of Fc\u03b5RI\u03b1 on skin mast cells was not reliable in our hands, likely due to loss of epitopes after enzymatic tissue digestion). In the five animals for which this mast cell population was investigated, the majority of the few CD117+CD45+ cells gated was EYFP positive (43\/47, 54\/64, 32\/39, 23\/27 and 77\/88 gated events, Fig.\u00a02). Given that the mast cell gate will inevitably collect also some non-mast cells upon analysis of high total cell numbers (40,000 events), the percentage of skin mast cells expressing EYFP is probably close to 100%. A similar efficiency of Cre-mediated activation of the reporter gene was observed in three male X-Mcpt5-Cre mice (not shown) whereas the two female animals analyzed showed EYFP fluorescence in only 61% and 32% of peritoneal mast cells consistent with random X-chromosome inactivation (not shown).\nIn both lines, Cre-mediated activation of EYFP expression was specific for mast cells. In seven A-Mcpt5-Cre and three male X-Mcpt5-Cre animals we detected no or only minimal numbers (i.e. below 1%) of EYFP positive cells (below 3% in one A-Mcpt5-Cre animal) in hematopoietic populations other than mast cells (Fig.\u00a03) or in non-hematopoietic cells of the skin cell suspensions (primarily keratinocytes and fibroblasts, not shown). As a control, we used mice with germline deletion of the stop element of the ROSA26-EYFP Cre excision reporter demonstrating that all non-mast cell populations analysed for reporter expression in Mcpt5-Cre mice can, in principle, express EYFP (not shown). In contrast to the seven A-Mcpt5-Cre mice (described above) showing mast cell-specific Cre-mediated recombination, one A-Mcpt5-Cre mouse displayed non-specific reporter gene expression in 15\u201325% of all cell types analyzed including non-hematopoietic cells of the skin (not shown). This finding likely reflects accidental early activity of the transgenic Mcpt5 promoter.\nFig.\u00a03Absence of EYFP reporter gene expression in hematopoietic cells other than mast cells. EYFP expression was analyzed in hematopoietic cells from peritoneal lavage and spleen cell suspensions of Mcpt5-Cre ROSA26-EYFP double transgenic mice. In order to enrich granulocytes, splenocytes were separated into a lymphocyte and non-lymphocyte fraction by MACS. Basophils were double stained for CD49b and Fc\u03b5RI\u03b1. Macrophages, dendritic cells, neutrophils, eosinophils, B cells and T cells were stained for F4\/80, CD11c, Gr-1, Siglec-F, CD19 and CD3\u03b5, respectively. The respective populations were gated and displayed in histogram plots. The black graph represents Mcpt5-Cre ROSA26-EYFP double transgenic mice, the shaded graph represents the Cre-negative but ROSA26-EYFP-positive control (littermates in most instances)\nAbsence of significant Cre-mediated genotoxicity in mast cells of Mcpt5-Cre animals\nIn order to exclude a reduced proliferative potential of mast cells in Mcpt5-Cre transgenic mice due to Cre-mediated genotoxic effects, we compared the percentage of mast cells in peritoneal lavage and the proliferation of peritoneal mast cells from Mcpt5-Cre ROSA26-EYFP double transgenic or wild-type mice in\u00a0vitro. Cell suspensions obtained by peritoneal lavage contained similar numbers of mast cells in wild-type and double transgenic mice (Fig.\u00a04a). Total peritoneal lavage cells were cultured in the presence of stem cell factor. After 23\u00a0days, these cultures contained 98% mast cells as judged by expression of CD117 and Fc\u025bRI\u03b1 (not shown). By this time, absolute cell numbers had increased more than 200-fold in both cultures (Fig.\u00a04b). Importantly, the mast cells grown from the Mcpt5-Cre ROSA26-EYFP positive mouse uniformly expressed EYFP (not shown) ruling out an overgrowth of the culture by a (hypothetical) minor population of mast cells, which do not express Cre. These results show that Mcpt5-Cre positive mast cells are not growth retarded and therefore do not seem to be significantly affected by Cre-mediated genotoxicity.\nFig.\u00a04Numbers and proliferative potential of peritoneal mast cells in Mcpt5-Cre transgenic mice. (a) Mast cells were quantified in peritoneal lavage fluid from control (n\u00a0=\u00a09) and Mcpt5-Cre (7 A-Mcpt5-Cre and 3 male X-Mcpt5-Cre) mice. Mast cells were detected as CD117+Fc\u03b5RI\u03b1+ cells by FACS. (b) Total cells from peritoneal lavage fluid from one control and one A-Mcpt5Cre ROSA26-EYFP double transgenic mouse were incubated in complete Opti-MEM supplemented with 4% supernatant from CHO transfectants secreting murine SCF (Malbec et\u00a0al. 2007). The CHO transfectants were generated by S. Lyman, Immunex, Seattle and were kindly provided by P. Dubreuil. Cell numbers were quantified at each passage every 3\u20134\u00a0days by counting trypan blue-excluding cells\nDiscussion\nHere, we describe a novel Cre transgenic mouse allowing mast cell-specific knock out of genes in\u00a0vivo. We employed BAC transgene technology to express a Cre cassette under the control of the Mcpt5 promoter. To our knowledge, this is the first report of mast cell-specific Cre-mediated recombination in the mouse.\nTo date, important insights into functions of mast cells have been gained by reconstitution of mast cell-deficient mice with mast cells differentiated in\u00a0vitro from gene deficient bone marrow or embryonic and hematopoietic stem cells (Galli et\u00a0al. 2005b; Grimbaldeston et\u00a0al. 2005; Kitamura et\u00a0al. 1978; Tsai et\u00a0al. 2005). While this model yielded important information, inherent technical problems limit experimentation and interpretation of data in this system (Tsai et\u00a0al. 2005). We expect that mast cell-specific knock out of genes using our new Cre transgenic line will greatly facilitate investigation of mast cell biology.\nThe two Cre transgenic lines demonstrate that the Mcpt5-promoter allows efficient and cell type-specific expression of a Cre transgene inserted into the first exon. One animal of the line A-Mcpt5-Cre (characterized by autosomal transgene inheritance) showed non-specific Cre-mediated recombination in non-mast cells while seven animals of this line did not. This finding likely reflects accidental early activity of the transgenic Mcpt5 promoter, e.g. in one stem cell during the embryonic four cell stage. Future analysis will define the frequency of such events more precisely. In females of the X-Mcpt5-Cre line (X-chromosomal inheritance), Cre-mediated recombination occurred in only about half of the mast cells due to inactivation of the transgene-carrying X-chromosome. Incomplete inactivation of particular genes may be advantageous if the attenuation of a severe phenotype is desired.\nCre recombinase has been shown to mediate genotoxicity in some Cre transgenic or Cre knock in mouse strains resulting in a reduced proliferative potential or even death of Cre expressing cells (Schmidt-Supprian and Rajewsky 2007). In Mcpt5-Cre mice, however, we found no difference in mast cell numbers or proliferation of mast cells ex vivo in response to stem cell factor indicating that Cre-mediated genome damage is not a prominent feature of these mice.\nWe are currently refining our system further to achieve also inducible mast cell-specific gene inactivation in adult mice. In addition, we are breeding the new lines to iDTR mice (Buch et\u00a0al. 2005) aiming at diphtheria toxin-inducible ablation of the mast cell lineage in adult animals.","keyphrases":["mast cells","cre\/loxp-mediated recombination","conditional gene targeting","cre transgenic mice"],"prmu":["P","P","M","R"]}
{"id":"Brain_Struct_Funct-4-1-2226080","title":"The impact of maternal separation on adult mouse behaviour and on the total neuron number in the mouse hippocampus\n","text":"The maternal separation paradigm has been applied to C57BL\/6J mice as an animal developmental model for understanding structural deficits leading to abnormal behaviour. A maternal separation (MS) model was used on postnatal day (PND) 9, where the pups were removed from their mother for 24 h (MS24). When the pups were 10 weeks old, the level of anxiety and fear was measured with two behavioural tests; an open field test and an elevated plus maze test. The Barnes platform maze was used to test spatial learning, and memory by using acquisition trials followed by reverse trial sessions. The MS24 mice spent more time in the open arms of the elevated plus maze compared to controls, but no other treatment differences were found in the emotional behavioural tests. However, in the reverse trial for the Barnes maze test there was a significant difference in the frequency of visits to the old goal, the number of errors made by the MS24 mice compared to controls and in total distance moved. The mice were subsequently sacrificed and the total number of neurons estimated in the hippocampus using the optical fractionator. We found a significant loss of neurons in the dentate gyrus in MS mice compared to controls. Apparently a single maternal separation can impact the number of neurons in mouse hippocampus either by a decrease of neurogenesis or as an increase in neuron apoptosis. This study is the first to assess the result of maternal separation combining behaviour and stereology.\nIntroduction\nDeveloping an animal model of mental disorders is controversial due to the human nature of the symptoms such as hallucinations, delusions and poverty of speech. These symptoms can only be adequately assessed by psychological assessments and therefore cannot be modulated in animals. A way to try to circumvent these problems is studying certain psychological or psychophysiological aspects of mental disorders such as latent inhibition, prepulse inhibition or P50 gating (Ellenbroek and Cools 1990, 1995; Geyer and Markou 1995; Ellenbroek et al. 2004). However, it is still unclear how (and if) these abnormalities are linked to the symptoms of mental illness. In rodent models prepulse inhibition (PPI) of the acoustic startle response is a model of sensorimotor gating mechanisms in the brain, while an equivalent reaction in humans is eye blinking (Braff et al. 1978; Ellenbroek et al. 1998). It has been shown that rat pups that underwent 24\u00a0h maternal separation on postnatal day (PND) 6 or 9 expressed reduced prepulse inhibition (PPI) on postnatal day 69 and had hyperactivity of the dopamineric system involving the dopamine neurotransmitter system via the hypothalamic\u2013pituitary\u2013adrenal (HPA) axis (Ellenbroek and Cools 1995). Further, the PPI deficits could be reversed with typical antipsychotic drugs like haloperidol and were not detected prior to puberty (Ellenbroek et al. 1998). Due to the changes seen in the HPA axis, the dopamine system, hippocampus and long-term behavioural effects modelling deficits seen in mental patients has lead Ellenbroek and co-workers to hypothesis the 24-h maternal deprivation model to be a \u201cschizophrenia-like\u201d neurodevelopmental animal model (Ellenbroek and Cools 1998, 2002; Ellenbroek et al. 1998, 2004, 2005).\nOne of the striking characteristics of the developing neuroendocrine stress system in the mouse and rat is a period of reduced stress-responsiveness, the so-called stress hypo-responsive period (SHRP) (Schapiro et al. 1962; Cirulli et al. 1994; Schmidt et al. 2002). From about postnatal day (PND) 4\u201314 in the rat, and PND 1\u201312 in the mouse, the animals neuroendocrine system is characterized by a low basal corticosterone level and by the inability of a mild stressor, e.g. exposure to novelty, to induce a corticosterone response (Cirulli et al. 1994; Schmidt et al. 2003). In this study, we used the C57BL\/6 inbred mouse. Due to a similar postnatal neurodevelopmental course in the mouse and rat (Clancy et al. 2001), we used PND 9 as the day of separation equivalent to Ellenbroek and co-workers day of choice in the rat. We tested if a 24-h maternal separation on PND 9 can cause an adult phenotype characterized by altered levels of activity and anxiety, learning and memory dysfunction, deficits in behavioural flexibility (reversal deficits) as well as changes in number of neurons in the hippocampus and its subregions in the mouse brain.\nMaterial and methods\nAnimals\nThe offspring of 4 male and 8 female C57BL\/6J mice (8\u00a0weeks old) obtained from Taconic Europe (Taconic Farms Inc., DK) were used in this study. The animals were acclimatized to the animal facility for 1\u00a0week. Multiparous females were used, since there is a higher rate of offspring survival. The animals were housed under a 12:12\u00a0h light\/dark cycle (lights on at 6 a.m.) with constant temperature (21\u00a0\u00b1\u00a02\u00b0C) and humidity (52\u00a0\u00b1\u00a02%) in Macrolon type III cages with environmental enrichment in the form of wood splints bedding (aspen 4HV), wood shavings, 1 piece of Aspen Corner 15, 1 standard mouse house made of recycled cardboard, 1 aspen chewing stick size medium, and pads of nesting material (all obtained from Brogaarden, DK). Food (Altromin pills NR 1324) and tap water were available ad libitum. Two females were placed in a male\u2019s cage for a period of 1\u00a0week to ensure conception, followed by separation of the two females to their own cages.\nMaternal separation\nPregnant females were checked for litters daily at 09:00 a.m. If litters were found, the day of birth was defined as PND 0 for that litter. On PND 0, litters were randomly assigned to maternal separation (MS) (N\u00a0=\u00a016, 9 males and 7 females), or to standard facility rearing (SFR). For the behavioural testing, 16 MS animals (4 males and 6 females) and 5 SFR animals (2 males and 3 females) were included, while 12 of the MS animals (2 males and 10 females) and 7 control animals (2 males and 5 females) were used for cell counting. Litters were not culled or sexed at birth to minimize the handling of the pups, but male and female pups were separated at weaning (PND 28) and group housed with their siblings, which resulted in 2\u20135 mice per cage. The environmental enrichment applied to the mothers was also applied to the MS and control animals.\nThe 24-h deprivation was carried out on PND 9 starting at 8 a.m. The pups remained in the home cage but were placed in a separate room with the same temperature, humidity and lighting conditions as the home stable. The cage was placed on a heating pad, which had a constant temperature of 31\u00b0C. No food or water was available during the separation. The dam was placed in a cage with similar facilities as the home cage in the home stable. The pups were checked every 3\u00a0h, using a red light during the night. Body weights were recorded before and after separation. Immediately after 24\u00a0h the dams were returned to the home cage and reunited with the pups.\nTest for anxiety and fear related behaviour\nWhen the pups reached 10\u00a0weeks of age they were subjected once to the open field test (Hall 1934) and the elevated plus maze test, which is based on the procedure used by Montgomery (1955) and later validated by Pellow et al. (1985). Behaviour was analysed using EthoVision (Noldus, Groeningen, The Netherlands). All behavioural testing took place between 10 a.m. and 3 p.m.\nOpen field test (OFT)\nThe open field consisted of a circular wooden platform (diameter 90\u00a0cm) surrounded by a 43\u00a0cm high wall with a camera mounted directly above. A central circle of 31\u00a0cm diameter was defined in the behaviour analysis software. Three 60-W light bulbs illuminated the arena. On the day of testing each animal was transported in a cardboard box to the centre of the open field and behaviour was recorded for 10\u00a0min. After the trial the maze was cleaned with a solution of acetic acid and soap water and faecal boli were counted.\nThe following parameters were calculated; total distance moved (cm), time spent in central circle and time spent in peripheral zone (expressed as % of session duration).\nElevated plus maze\nThe plus maze was elevated 50\u00a0cm above the ground and consisted of two opposing open arms (21\u00a0cm\u00a0\u00d7\u00a08\u00a0cm) connected by a central square (8\u00a0cm\u00a0\u00d7\u00a08\u00a0cm) to two opposing enclosed arms of the same size with 32\u00a0cm high walls. A video camera placed above the maze recorded the animals\u2019 behaviour. On the day of testing, the animal was placed in the centre of the maze and behaviour was recorded for 10\u00a0min. Between trials the maze was cleaned as described for the OFT. The following parameters were calculated; total duration (s) in open and closed arms, the number of entries into the open and closed arms and the total distance moved (cm). From these parameters the ratio of entries into the open arms to the total number of arm entries, and the ratio of time spent in the open arms to time spent in both open and closed arms was calculated.\nTest for spatial memory; Barnes maze\nThe Barnes maze (Barnes 1975) consisted of a circular, white-coated platform 90\u00a0cm in diameter and elevated 50\u00a0cm over the ground. Sixteen 5\u00a0cm wide holes were evenly distributed around the perimeter, 2.5\u00a0cm from the edge. A pair of rails was placed under two opposing holes to hold the hidden escape box. The escape box was a dark plastic storage box with a 5\u00a0cm diameter hole in the lid. A dark cylindrical cardboard tube (7.5\u00a0cm\u00a0\u00d7\u00a07\u00a0cm high) with a lid was used as the transport and start chamber.\nThree 60-W bulbs illuminating the maze and high irregular rock and techno music played from a computer in a random manner provided the aversive stimuli. As with the previous test, a digital video camera mounted above the maze recorded animal\u2019s behaviours.\nShaping\nFor 2\u00a0days before testing commenced, the animals were trained to enter the hidden escape box. Using the transport cylinder, the mouse was placed near the edge of the target hole with the hidden escape box underneath. Two cardboard walls blocked entry to adjacent holes. Only dim lightning and no noise was used during this phase of the experiment. The animal was allowed 5\u00a0min to enter the escape box and if this failed, it was placed manually inside. When the animal entered the box it was quickly carried to the home cage.\nAcquisition trials\nSix consecutive trials were given, one per day. The animal was placed in the transport cylinder, oriented in a random direction, in the centre of the maze. The aversive stimuli were turned on and the cylinder removed. Recording in the behaviour-observation software began immediately after the experimenter had left the room. The trial ended after 5\u00a0min or when the animal entered the hidden escape box. If the animal failed to enter the box or re-entered the maze after recording was stopped, the aversive stimuli were turned back on and the animal was allowed 5\u00a0min more to enter the escape box. If that also failed, the animal was manually placed in the box. After completion of each trial, the box was placed in the home cage.\nReversal trials\nThree days after acquisition trials, the escape box was placed underneath the hole opposite to the hole that had been the target during acquisition training. Reversal training was conducted for seven consecutive days as described above.\nParameters\nEach of the 16 escape holes in the Barnes maze was defined as a separate zone-of-interest in the behaviour analysis software. The following parameters were analysed: latency to target [time from start of the trial to first entry into the target hole zone (s)], total distance moved (cm) and error frequency (number of visits to other holes than the target hole). During reversal training two additional parameters were analysed: the mean number of visits to the old target hole over the seven trials (the hole where the escape box was located during acquisition training) and the mean number of visits to the two holes adjacent to the old target hole.\nThree different search strategies could be distinguished and were scored manually. The random search strategy is characterized by a non-systematic exploration of the maze with many centre maze crossings and some perseverations. Perseverations were defined as repeatedly searching the same hole or two adjacent holes (Bach et al. 1995). Secondly, there is the serial search strategy, defined as systematic consecutive hole searching in a clockwise or counter clockwise manner and finally a spatial search strategy, defined as searching less than three holes from the location of the goal (Barnes 1979; Bach et al. 1995; Inman-Wood et al. 2000; Zhang et al. 2002; Raber et al. 2004) (Fig.\u00a01).\nFig.\u00a01Three different search strategies could be applied: Random, a pattern that include many hole examinations in a random manner; Serial, a relative systematic search either clockwise or counterclockwise; Spatial, a pattern where the target box is found within relative short time and with high accuracy\nFixation and embedding\nApproximately 1\u00a0week after all tests were concluded, the animals were scarified by CO2 asphyxiation and within 1\u00a0h the brains were dissected out and placed in a 37% formalin solution (Fixatin). The brains were split through corpus callosum, separating the two hemispheres. After systematic random assignment of left or right hemisphere they were coloured on the outer surface to preserve a coded sequence and embedded in paraffin with an automatic vacuum tissue processor (Leica ASP300). The hemispheres were then mounted horizontally balancing on needles and embedded in a paraffin block with up to four hemispheres in one block\nSectioning\nThe whole hemisphere was cut horizontally with a Leica (model SM 2400) microtome with a microtome setting of 40\u00a0\u03bcm thickness. All sections were mounted on silicone-coated glass (Frost +). After a minimum of 24\u00a0h in a 40\u00b0C heating cupboard, the sections were sampled systematic uniformly randomly (SURS). The first section was randomly selected from a random number table, hereafter every 6th section was sampled systematically for staining and counting. This provided a total of 8\u201310 sections containing the hippocampus per specimen. To be able to account for block advance (BA) the block height was measured for every 100th section. The BA determines the hitting probability of the particles within the block (see later for calculation). Furthermore, the paraffin shrinkage effect was calculated and amounted to about 70%. The sections were stained with a modified Vogt\u2019s Cresyl violet acetate (Armed forces).\nOptical design equipment\nThe optical design equipment consisted of an Olympus BH-2 microscope with an oil immersion 100\u00d7 objective of high numerical aperture (NA\u00a0=\u00a01.40), which allows focusing in a thin focal plane inside a thick section. A camera transmits the image to a computer screen where a counting frame is superimposed using the computer-assisted stereological (CAST)-Grid software (Visiopharm, H\u00f8rsholm, DK). A motorized automatic stage was used to control movement in the x,y- plane via a connected joystick. Movement in the z-axis was controlled manually with the focus button on the microscope and the distance between the upper and lower surfaces of the sections was measured with a Heidenhein microcator (Heidenhain, Germany) with a precision of 0.5\u00a0\u03bcm.\nDefinitions and divisions of the hippocampus\nThe hippocampus was subdivided into five regions; the dentate gyrus (DG), the hilus of the dentate gyrus (CA4), regio inferior (CA3\/2), region superior (CA1) and subiculum.\nNeurons were counted manually in the sub-sampled sections containing the hippocampus, using the optical disector design. The layers comprising the hippocampus were defined consistently in all sections using the terminology of Blackstad (1956). It was not considered too difficult to differentiate the neuronal layers in horizontal sections (Fig.\u00a02). The granule cell layer and the pyramidal cell layers also contain the cell bodies of basket cells and glial cells. The glial cells can be easily identified and counted separately, but the basket cells are so similar in appearance to the granule and pyramidal cells that they are included in the estimates. It was previously shown that they comprise less than 1% of the neurons in these layers (review West et al. 1991).\nFig.\u00a02A schematic drawing of the hippocampus with the five subregions identified in this study. Dg, the granule cell layer of the dentate gyrus; h\/CA4, hilus of the dentate gyrus; ri, regio inferior; CA3\/2, rs, region superior; CA1, s, subiculum\nStereological equations\nEstimation of the total neuron number, N\nFor the estimation of total neuron numbers, neurons were counted in optical disectors and sampled according to the so-called fractionator principles. In a fractionator, cells are counted directly in a known fraction of the different hippocampal subdivisions and the total neuron number, N, is estimated by multiplying the number of particles counted with the reciprocal sampling fractions: where ssf is the section sampling fractions, asf the area sampling fraction, and hsf the height sampling fraction. The bilateral cell number is estimated by multiplying the unilateral number \u2211Q\u2212 by 2. This is admissible when the right or left hippocampus is sampled systematically randomly.\nThe asf is known as the area of the counting frame of the disector relative to the area associated with each x,y-step movement of the disector: After having ascertained that the cell density was constant within the disector height, the height sampling fraction was defined as: is the q\u2212 weighted mean section thickness (Gundersen et al. 1988; Dorph-Petersen 2001).\nEstimation of total volume\u2014the Cavalieri estimator\nBesides estimating total neuron numbers, it was also decided to estimate total volume of the different compartments of the hippocampus although the main purpose was estimation of cell numbers. Estimates of volume were obtained according to the principles of Cavalieri\u2019s basic estimator (Gundersen and Jensen 1987) and corrected for shrinkage. Notice that these volumes were not used for the estimation of total neuron number. The estimates of total neuron number obtained by the fractionator design are independent of the containing volume. Further, when total number and volume is estimated, the density, NV (cells\/mm3) can be obtained as well without extra work.\nError predictions\nThe precision of the two different estimates (neuron number and volume) can be expressed by the coefficient of error, CE. The CE for the Cavalieri estimation of volume was first formulated and described by Gundersen and Jensen (1987) revised in Gundersen et al. (1999). Table\u00a01 gives an example of how CE for both volume and neuron estimates are calculated in this study (see also Gundersen et al. 1999).\nTable\u00a01Example of how the CE is calculated for neuron number and volume respectivelySectionQj\u2212A:Qi\u00a0\u00a0\u00d7\u00a0QiB:Qi\u00a0\u00d7\u00a0Qi\u00a0+\u00a01C:Qi\u00a0\u00a0\u00d7\u00a0Qi\u00a0+\u00a02SectionPj\u2212A:Pi\u00a0\u00d7\u00a0PiB:Pi\u00a0\u00d7\u00a0Pi\u00a0+\u00a01C:Pi\u00a0\u00a0\u00d7\u00a0Pi\u00a0+\u00a021111216162311242282563,1361,1761,0642111214433321441399378341612124193613425743993518324541985393963933486113371112117616573996816256240352839615915225330909241081022484132\u20131052520\u201311636\u2013\u201311416\u2013\u2013Sum1985,5143,4982,583Sum4122313897\nWhen an appropriate number of sections have been chosen (10 or a little less), it is the number of points counted (the noise) which decides the precision of the estimate. To count about 200 points per sample is usually enough to obtain a CE around 5\u20138%, unless the object is very irregular (Gundersen and Jensen 1987; Gundersen et al. 1988; West and Gundersen 1990). When CE is estimated for the cell counting, Noise is equal to \u2211Q\u2212.\nStatistical analyses\nAll data were analysed with the statistical software packages SPSS (Statistical Package for the Social Sciences, version 14.00) or SigmaStat (version 2.0). If Levene\u2019s test for equality of variances did not fail, a t test was applied to test for differences between the two experimental groups. For data where normality tests failed, the non-parametric tests Mann\u2013Whitney U test and Kruskal\u2013Wallis test were applied. The animals performance over the six acquisition trials and the seven reversal trials in the Barnes maze was analysed by mixed-model analysis of variance with trial as within-subject factor and experimental group as between-subjects factor. Homogeneity of the variance-difference scores was determined by Mauchly\u2019s test of sphericity [SPSS Inc. Chicago, IL, USA. SPSS Base version 11.5 User Manual; 2004]. When the assumption of sphericity was violated, degrees of freedom were adjusted with the Huynh\u2013Feldt correction. Group differences in strategy selection in the Barnes maze were analysed with the likelihood-ratio chi-square test for each trial separately. Group differences were considered significant when P\u00a0<\u00a00.05.\nResults\nOnly significant behavioural results are depicted and statistically elaborated.\nBody weights\nA significant weight loss was found in the pups after separation (P\u00a0=\u00a00.019, paired t test). However, 8\u00a0weeks after separation the body weights of MS and SFR animals were similar (P\u00a0=\u00a00.61, unpaired t test).\nOpen field test\nBoth experimental groups spent more time in the periphery of the open field than the centre (P\u00a0<\u00a00.001, t test). There was no significant difference between MS and SFR groups in either distance moved or frequency of visits to and time spent in the centre or periphery of the open field (data not shown).\nElevated plus maze\nThe MS animals spent more time in the open arms than the SFR animals and allotted a greater percentage of time spent in both open and closed arms to the open arms (P\u00a0=\u00a00.035 and P\u00a0=\u00a00.046, t test, respectively; Fig.\u00a03).\nFig.\u00a03The mean (+SEM) time spent in both the open and closed arms. *There was a significant difference between MS 24 (N\u00a0=\u00a016) and SFR 24 (N\u00a0=\u00a05) in time spent in open arms (P\u00a0<\u00a00.05, ANOVA). ** Time spent in closed arms compared with time spent in open arms (P\u00a0<\u00a00.001, t test)\nIn trial progress there was a significant difference in both frequency and duration in closed and open arms [Closed arms frequency: (F(9,171)\u00a0=\u00a03.544, P\u00a0<\u00a00.0001) and duration: (Wilk\u2019s \u03bb F(9,11)\u00a0=\u00a06.045, P\u00a0=\u00a00.004), Open arms frequency: (Wilk\u2019s \u03bb F(9,11)\u00a0=\u00a08.086, P\u00a0=\u00a00.001) and duration: (F(9,171)\u00a0=\u00a06.253, P\u00a0<\u00a00.0001)].\nThere was no significant difference in any of the other parameters analysed.\nBarnes maze\nAcquisition trials\nLatency to reach the target hole, total distance moved on the maze, and error frequency all decreased significantly over the course of the six acquisition trials (F(4.3,77.4)\u00a0=\u00a03.88, P\u00a0=\u00a00.005; F(5,90)\u00a0=\u00a02.56, P\u00a0=\u00a00.033; F(3.9,70.1)\u00a0=\u00a02.49, P\u00a0=\u00a00.05; respectively), indicating that the animals indeed learned the task. There was, however, no significant difference between the two experimental groups.\nReversal trials\nLatency to reach the new target hole (Fig.\u00a04 left) total distance moved on the maze (Fig.\u00a05), and error frequency (Fig.\u00a06) all decreased significantly over the course of the seven reversal trials (F(3.1;55.95)\u00a0=\u00a03.08, P\u00a0<\u00a00.05; F(1.85;33.41)\u00a0=\u00a016.24, P\u00a0<\u00a00.001; F(3.43,61.68)\u00a0=\u00a018.79, P\u00a0<\u00a00.001, respectively). The MS24 animals made significantly more errors over the course of reversal training (F(1, 18)\u00a0=\u00a04.79, P\u00a0<\u00a00.05; Fig.\u00a06) and travelled longer distances (Fig.\u00a05; P\u00a0=\u00a00.008, Mann\u2013Whitney U-test). This might indicate that the MS animals did not learn the reversal task as fast as the controls.\nFig.\u00a04Left: Mean (\u00b1SEM) latency to \u201cGoal\u201d. There was a significant difference in Trial progress and a significant Trial x Group interaction (P\u00a0<\u00a00.05). Right: Mean (\u00b1SEM) frequency of visits to \u201cOld Goal\u201d. There was * a significant difference between the two groups (P\u00a0<\u00a00.05, Mann\u2013Whitney U test) and a significant decrease over time (P\u00a0<\u00a00.05, RM ANOVA). MS 24 (N\u00a0=\u00a016) and SFR (N\u00a0=\u00a05)Fig.\u00a05Mean (\u00b1SEM) distance moved in reverse trials. There was a significant decrease over time (P\u00a0<\u00a00.05, RM ANOVA) and * a significant difference between the two treatment groups (P\u00a0<\u00a00.05, Mann\u2013Whitney U test) but no significant Trial x Group interaction. MS 24 (N\u00a0=\u00a016) and SFR (N\u00a0=\u00a05)Fig.\u00a06Mean (\u00b1SEM) frequency of errors in MS 24 (N\u00a0=\u00a016) and SFR (N\u00a0=\u00a05) * there was a significant difference between the two treatment groups and a significant decrease in Trial progress (P\u00a0<\u00a00.05). MS 24 (N\u00a0=\u00a016) and SFR (N\u00a0=\u00a05)\nThe MS24 animals made significantly more visits to the old target hole (Fig.\u00a04 right) and the two adjacent holes over the course of the seven reversal trials (t(17.5)\u00a0=\u00a03.72, P\u00a0<\u00a00.01; t(17.5)\u00a0=\u00a04.34, P\u00a0<\u00a00.001).\nThis tendency to venture to the old goal and the adjacent holes could be an indication of perseveration, a behavioural pattern also found in other animal models and in schizophrenic patients (Bleuler 1950; Bach et al. 1995; review Crider 1997)\nSearch strategies\nThe animal\u2019s search pattern evolved over time (Fig.\u00a07). The usual behaviour sequence was a progression from a random to a serial, and finally to a spatial search strategy. This sequence indicates that the animals became more accurate and more efficient in locating the correct hole. Likelihood-ratio chi-square tests showed that the experimental groups differed in their choice of search strategy on days 2 and 3 of acquisition training (\u03c7(2)\u00a0=\u00a010.5, P\u00a0<\u00a00.01; \u03c7(2)\u00a0=\u00a06.6, P\u00a0<\u00a00.05, respectively). Already at this early time the SFR animals adopted a serial search strategy while the MS animals maintained a random search strategy. On all other acquisition and reversal training days the two experimental groups did not differ significantly with respect to their choice of search strategy.\nFig.\u00a07Overview of mean (%) search strategies applied in the acquisition and reverse trial sessions. The search pattern evolved over time from a random to a serial, and finally to a spatial search strategy. From acquisition days 2 and 3, the SFR animals adopted a serial search strategy, while the MS animals maintained a random search strategy. On all other acquisition and reversal training days the two experimental groups did not differ significantly with respect to their choice of search strategy. SFR (N\u00a0=\u00a05) animals to the left and MS (N\u00a0=\u00a016) animals to the right\nNeuron number\nThe stereological sampling is shown in Table\u00a02.\nTable\u00a02Overview of the stereological sampling used in this studySubiculumCA4CA1CA3DGMS (maternal separated)Area (frame) (\u03bcm2)770\u20131,160710\u20131,100259259259Z depth (\u03bcm)2020202020X and Y step (\u03bcm)17580100100170\u2211Q\u2212208\u00a0\u00b1\u00a036.5140\u00a0\u00b1\u00a023.8254\u00a0\u00b1\u00a015.7193\u00a0\u00b1\u00a010.3205\u00a0\u00b1\u00a015.4Mean section thickness, tq\u2212 (\u03bcm)39.5\u00a0\u00b1\u00a00.240.0\u00a0\u00b1\u00a00.3039.0\u00a0\u00b1\u00a00.338.9\u00a0\u00b1\u00a00.3039.3\u00a0\u00b1\u00a00.30Height sampling fraction, hsf1.97\u00a0\u00b1\u00a00.012.00\u00a0\u00b1\u00a00.011.95\u00a0\u00b1\u00a00.021.95\u00a0\u00b1\u00a00.011.96\u00a0\u00b1\u00a00.02Area sampling fraction, asf57.4\u00a0\u00b1\u00a011.516.8\u00a0\u00b1\u00a04.0638.7\u00a0\u00b1\u00a00.0030.7\u00a0\u00b1\u00a00.01111\u00a0\u00b1\u00a00.00Section sampling fraction (k), ssf66666Guard zone, (\u03bcm)5.5\u201375.5\u201375.5\u201375.5\u201375.5\u20137SFR (Control)Area (frame) (\u03bcm2)1,222\u00a0\u00b1\u00a061.61,388\u00a0\u00b1\u00a034.7197\u00a0\u00b1\u00a00.1240\u00a0\u00b1\u00a06.2197\u00a0\u00b1\u00a00.1Z depth (\u03bcm)2020202020X and Y step (\u03bcm)20095100100170\u2211Q\u2212245\u00a0\u00b1\u00a014.7190\u00a0\u00b1\u00a014.4215\u00a0\u00b1\u00a013.6188\u00a0\u00b1\u00a011.8201\u00a0\u00b1\u00a07.5Mean section thickness, tq\u2212 (\u03bcm)39.0\u00a0\u00b1\u00a00.6039.1\u00a0\u00b1\u00a00.538.5\u00a0\u00b1\u00a00.638.2\u00a0\u00b1\u00a00.438.6\u00a0\u00b1\u00a00.4Height sampling fraction, hsf1.95\u00a0\u00b1\u00a00.031.95\u00a0\u00b1\u00a00.021.93\u00a0\u00b1\u00a00.031.91\u00a0\u00b1\u00a00.021.93\u00a0\u00b1\u00a00.02Area sampling fraction, asf33.3\u00a0\u00b1\u00a01.626.46\u00a0\u00b1\u00a00.2950.9\u00a0\u00b1\u00a00.0342.0\u00a0\u00b1\u00a01.28147\u00a0\u00b1\u00a00.07Section sampling fraction (k), ssf66666Guard zone, (\u03bcm)5.5\u201375.5\u201375.5\u201375.5\u201375.5\u20137\nThere was no significant difference in the total bilateral neuron number in hippocampus (Table\u00a03). However, in the five subregions of hippocampus, there was a significant difference in the dentate gyrus (P\u00a0=\u00a00.029, Students t test), but none in the four other regions (see Fig.\u00a08; Table\u00a03). The neuron loss in the dentate gyrus is equivalent to a 20% reduction in the maternal separated animals compared with controls. The results should be interpreted with caution due to the low number of subjects.\nTable\u00a03Total estimated neuron number in five subregions of the hippocampusMaternally separated (MS) N\u00a0=\u00a012SexSubiculumCA4CA1CA3DGTotalSubjectNeurons (103)CENeurons (103)CENeurons (103)CE Neurons (103)CE Neurons (103)CENeurons (103)CEFemale3310.0842.90.132920.061900.076720.071.5290.09Female2640.1034.80.132430.061890.075760.071.3070.09Female3710.0818.60.183030.062260.064740.081.3930.09Female1070.0821.20.081420.081120.093340.107160.09Female1480.0723.10.072130.071440.083540.108820.08Female1320.0720.60.082620.061740.076650.071.2540.07Female1380.0720.20.071550.081630.085520.071.0280.07Female1890.1267.90.132020.071340.084600.081.0530.10Female1440.0523.10.072300.071710.075970.071.1660.07Female1970.0520.90.072290.072030.074840.091.1350.07Male2370.1067.20.122570.061910.074870.091.2390.09Male2420.1037.40.132240.061860.077880.061.4880.09Mean2080.0833.20.112290.071740.075380.081.1820.08CVcontrol (SFR) N\u00a0=\u00a070.500.360.280.210.260.25Female1860.0726.30.072850.071750.088040.071.4760.07Female1760.0730.20.092400.071910.086580.081.2960.08Female1690.0731.50.072730.072050.076550.081.3330.07Female1940.0525.30.091820.081860.076170.061.2040.08Female1620.0834.10.092780.071990.087250.071.3970.05Male1570.0820.30.092060.081150.096130.091.1120.07Male2210.0628.00.072850.071910.076440.091.3680.07Mean1810.0727.90.082500.071800.086740.081.3120.08CV0.070.120.170.060.110.08Student\u2019s t testP\u00a0=\u00a00.41P\u00a0=\u00a00.46P\u00a0=\u00a00.37P\u00a0=\u00a00.65P\u00a0=\u00a00.02*P\u00a0=\u00a00.20*\u00a0P < 0.05Fig.\u00a08The total neuron number for the two treatment groups in five sub-regions of the mouse hippocampus for the pooled data in MS (N\u00a0=\u00a012) and SFR (N\u00a0=\u00a07). * MS\u00a0\u2260\u00a0SFR, P\u00a0<\u00a00.05, Student\u00b4s t test in the dentate gyrus. filled circle MS; open circle SFR; Sub Subiculum, CA 4 Hilus, DG Dentate Gyrus\nVolume\nAfter Cavalieri estimation of volume and correction for shrinkage (67.7%) a Student\u2019s t test did not show any significant difference in either the total hippocampal volume or in the five subregions of the hippocampus between groups (Table\u00a04).\nTable\u00a04Total estimated volume in five subregions of the hippocampusMaternally separated (MS) N\u00a0=\u00a012SexSubiculumCA4CA1CA3DGTotalSubjectVolume mm2CEVolume mm2CEVolume mm2CEVolume mm2CEVolume mm2CEVolume mm2CEFemale4.330.050.690.081.460.051.520.051.800.039.790.05Female3.380.050.690.061.140.061.390.051.230.047.840.05Female4.250.050.590.071.540.051.720.041.180.049.290.05Female1.660.120.600.070.520.091.000.061.060.044.840.08Female2.400.090.860.060.790.081.260.051.290.046.590.07Female1.730.120.650.071.070.061.390.051.330.036.170.07Female2.260.100.690.060.680.091.420.051.220.046.260.07Female2.290.071.040.081.000.061.260.051.120.066.710.06Female2.150.070.790.060.910.071.450.051.130.036.430.06Female3.000.060.740.060.980.071.470.051.130.067.320.06Male2.980.061.010.061.070.061.390.051.150.057.000.06Male3.310.050.910.051.220.061.630.042.180.039.250.05Mean2.810.080.770.071.030.071.410.051.320.047.340.06CVContol(SFR) N\u00a0=\u00a070.320.200.290.130.250.20Female2.970.080.780.071.260.061.690.041.560.038.250.06Female2.290.090.910.071.010.061.220.051.450.036.880.07Female2.510.090.870.061.160.061.880.041.380.047.820.06Female2.550.090.520.090.850.071.300.050.730.046.050.07Female2.100.110.860.070.980.071.480.051.300.046.730.07Male2.320.100.520.101.030.071.190.051.730.056.780.08Male2.830.080.700.071.170.051.460.051.580.047.720.06 Mean2.510.090.750.081.070.051.460.051.390.047.180.07CV0.120.190.130.170.230.11Student\u2019s t testP\u00a0=\u00a00.40P\u00a0=\u00a00.79P\u00a0=\u00a00.76P\u00a0=\u00a00.62P\u00a0=\u00a00.66P\u00a0=\u00a00.79\nDiscussion\nIn the present study, 24\u00a0h maternal separation on PND 9 in mice pups resulted in a 20% neuron decrease in the dentate gyrus and behavioural perseverations in the Barnes maze. The anxiety and thus stress related behavioural tests conducted did not show any indications of an elevated anxiety level. On the contrary, the MS mice showed a reduced sign of anxiety, since they ventured more often onto the open arms than the equivalent control group. This could indicate that the SHRP was not repressed, in spite of our expectations. The results were thus surprising and contradicted some but not all other findings (Pihoker et al. 1993; Plotsky and Meany 1993; Cirulli et al. 1994; Wigger and Neuman 1999; Boccia and Pedersen 2001; Parfitt et al. 2004). A study by Lehman et al. (1999) found that MS 24 at PND 9 did not result in the anxiety\/fear response predicted by the group (Lehmann et al. 1999). Others have also found that effects of a manipulation of the HPA axis cannot always be used to predict effects of the same manipulation of fear\/anxiety expression at the behavioural level (see Lehmann et al. 1999; Parfitt et al. 2004). Furthermore, Parfitt et al. (2004) reported that maternally separated C57BL\/6 male mice had a prolonged increase in plasma CORT after an acute stressor, but in adulthood showed no increased fear\/anxiety behavioural response (Parfitt et al. 2004). The lack of fear response does evidently not necessarily mean that the corticosterone plasma level is not elevated in MS animals but since the corticosterone level was not measured this question remains unanswered. Finally, a study by Francis et al. (2002) concluded that MS rats subjected to an enriched environment could reverse the effect from MS on the HPA function and anxiety behaviour (Francis et al. 2002). In conclusion, we found that the MS mice in our study did not show behavioural indications on a repressed SHRP, which might be explained by the enriched environment the mice were kept in. The corticosterone plasma level was not measured.\nIn the data presented for the Barnes maze, there were no significant differences between the two treatment groups during the acquisition period. Latency to reach the target hole, total distance moved on the maze, and error frequency all decreased significantly over the course of the six acquisition trials and seven reversal trials, indicating that the animals were able to learn the task with time, which is in agreement with other studies (e.g. Pompl et al. 1999).\nOne consequence of hippocampal dysfunction is perseveration (Devenport et al. 1988). For the MS animals the group differences in the number of errors made, the goal parameter and the distance moved were therefore of interest. The differences found in the parameter \u201cClose to Old Goal\u201d the significantly higher frequency of \u201cErrors\u201d and visits to \u201cOld Goal\u201d by the MS 24 animals indicate that the MS animals were more perseverant which could point to a hippocampal lesion (Devenport et al. 1988; Bach et al. 1995). However, since perseveration is also a feature of prefrontal cortical dysfunction and there was no hippocampal dysfunction in the acquisition trials, we can only conclude that the behaviour in reversal trial may be related to hippocampus, but we cannot exclude that it is was caused by, e.g. a prefrontal cortical deficit.\nOne of the advantages of the Barnes maze is its ability to reveal the search strategies applied by the mice. The search strategies can be an indication of how well the cognitive abilities in the mice are due to the use of extra-maze cues (Barnes 1979; Holmes et al. 2002). In this study, an overall search pattern evolved with time. On initial trials the mice tended to use a random pattern and explore many incorrect holes, often returning to the centre after investigating the edge of the platform. With more experience the number of centre crossings decreased and a more systematic search was applied. Thus in later trials many mice went directly to the correct hole or one or two holes away before locating the hidden box. The mice learned the task better after a previous introduction to the platform and used the \u201cSpatial\u201d search strategy earlier. Furthermore, the SFR mice used the search strategy \u201cSerial\u201d significantly faster than the MS mice in the acquisition trials, but this was not true in the reversal trials. The serial search strategy requires the mouse to use the multiple relationships among extra-maze stimuli to find the escape tunnel (Barnes 1979; Bach et al. 1995). So apparently the MS mice needed more time to\u00a0acquire\u00a0to the spatial search strategy when learning the task, which could indicate a learning disability. However, since the MS mice did not show difficulties in using the spatial search strategy in the reverse trials, no firm conclusions can be made on this point.\nOver 85% of granule cell neurogenesis are previously described to occur postnatally in the rodent with peak neurogenesis between PND 5 and 7 and total cell number increasing throughout the first year and continuing throughout life (Altman and Das 1965; Schlessinger et al. 1975; Bayer et al. 1982; Kuhn et al. 1996; Kempermann et al. 1998). Much research has focused on the neurogenesis in both the adult and newborn hippocampus of mammals (Kempermann et al. 1997; Gould et al. 1997; Eriksson et al. 1998; Tanapat et al. 1998, 2001; Gould et al. 1999; Lemaire et al. 2000; Malberg et al. 2000; Raber et al. 2004; Mirescu et al. 2004; Greisen et al. 2005) but only a few of these studies have used modified stereological methods (Kempermann et al. 1997; Eriksson et al. 1998; Gould et al. 1999; Lemaire et al. 2000; Malberg et al. 2000; Greisen et al. 2005). None of these previous studies have quantified the total cell numbers in the hippocampus using stereology in early trauma animal models or in humans. The model can apparently induce a neuron change in the hippocampus but whether the lower neuron number is a decrease due to a neuron loss or because neurogenesis has been affected in the peak period could not be determined in this study. Secondly, the impact of the neuron loss is not immediately linked to a possible memory deficit. Even though the MS animals had a reduced emotional responds in the elevated plus maze and showed perseverance in the Barnes maze, the cognitive deficits could just as well be due to a prefrontal damage and thus not a result of the decreased neuron numbers in DG.\nWe tested if a 24-h maternal separation on PND 9 can cause an adult phenotype characterized by altered levels of activity and anxiety, learning and memory dysfunction, deficits in behavioural flexibility (reversal deficits) as well as changes in number of neurons in the hippocampus and its subregions in the mouse brain. We found that a single 24\u00a0h maternal separation on PND 9 could elicit a reduced stress response in the elevated plus maze and induce perseveration behaviour in the Barnes maze. Further, a 20% reduction in total neuron numbers was found in the dentate gyrus of the hippocampus.\nEthics\nThe experiment was carried out in accordance with the European Communities Council Directive of 24 November 1986 (86\/609\/EEC) and the Danish legislation regulating animal experiments (Animal care and housing BEK nr 687 from 25\/07\/2003). The Danish Animal Experiments Inspectorate approved the protocols (Journal No. 2003\/561\u2013781).","keyphrases":["maternal separation","behaviour","hippocampus","stereology"],"prmu":["P","P","P","P"]}
{"id":"Neuroradiology-3-1-2082066","title":"Multislice CT angiography in the selection of patients with ruptured intracranial aneurysms suitable for clipping or coiling\n","text":"Introduction We sought to establish whether CT angiography (CTA) can be applied to the planning and performance of clipping or coiling in ruptured intracranial aneurysms without recourse to intraarterial digital subtraction angiography (IA-DSA).\nIntroduction\nSubarachnoid haemorrhage (SAH) is caused by aneurysmal rupture in 70\u201385% of patients [1, 2]. In a systematic review, Hop et al. found fatality rates ranging from 32% to 67% [3]. Furthermore, 10\u201320% of patients remained functionally dependent after SAH. Rapid diagnostic evaluation and treatment are crucial for the patient\u2019s outcome.\nIntraarterial digital subtraction angiography (IA-DSA) has been the main technique for detecting and characterizing intracranial aneurysms and remains the gold standard. However, IA-DSA is invasive and time consuming, and carries a risk of neurological complications of 0.5\u20131.8% with permanent deficit in 0.09\u20130.5% [4\u20136]. Serious non-neurological complications, which occur in 0.6% of patients, include groin hematoma, peripheral thromboembolism, transient hypotension and arteriovenous fistulas [4]. Furthermore, IA-DSA may increase the risk of rebleeding [7, 8]. It has been demonstrated that three-dimensional CT angiography (3D-CTA) can reliably detect intracranial aneurysms [9\u201314]. Only after replacement of IA-DSA by CTA can the advantages of CTA be fully realized in the clinical setting. We report here our clinical experience with both 16- and 64-detector row CTA as the first and intended only diagnostic and treatment decision-making study for intracranial aneurysms in patients with acute SAH.\nMaterials and methods\nSubjects\nBetween April 2003 and January 2006 all patients presenting with a SAH to the University Medical Centre Groningen consecutively underwent CTA as the first diagnostic study. Based on the CTA findings, patients were selected for surgical clipping or endovascular coiling of a ruptured intracranial aneurysm.\nSAH was suspected on clinical grounds and confirmed by unenhanced CT or by blood pigments on lumbar puncture.\nImaging protocols\nThe CT examinations were performed on a 16- or 64-multidetector row spiral CT machine (Somatom Sensation 16 or 64; Siemens Medical Systems, Erlangen, Germany), based on a standard protocol. The 64-multisclice CT was implemented in the Emergency Department in December 2004.\nParameters for 16-slice CT for diagnosis of aneurysm: via an intravenous cannula in the antecubital fossa, 80\u00a0ml of contrast agent (Visipaque 320) was injected with a power injector at a rate of 4\u00a0ml\/s. Injection of contrast agent was followed by a flush of 50\u00a0ml 0.9% saline (Stellant; NaCl Neck Angio) injected at the same rate. A manual fluoroscopic bolus-triggered system, with the internal carotid arteries as reference point and a delay of 4\u00a0s, determined the optimal timing. The CTA protocol parameters were as follows: spiral mode, rotation time 0.5\u00a0s, reconstruction interval 0.75\u00a0mm at Kernel H20, 120\u00a0kV\/200\u00a0mAs, acquisition time 10\u00a0s, scan range from the C1 vertebral body to the vertex parallel to the orbitomeatal line.\nParameters for 16-slice CT for diagnosis of SAH: gantry un-angled, spiral mode, rotation time 0.75\u00a0s, 16-detector rows at 0.75-mm intervals, table speed 6\u00a0mm\/rotation, reconstruction interval 3\u00a0mm at Kernel H30 and acquisition parameters 120\u00a0kV\/200\u00a0mAs. The actual acquisition time was approximately 15\u00a0s.\nParameters for 64-slice CTA for diagnosis of aneurysm: rotation time 1\u00a0s, table speed 15.4\u00a0mm\/rotation, reconstruction interval 0.6\u00a0mm at Kernel H20, 120\u00a0kV\/260\u00a0mAs, acquisition time 9\u00a0s and scan range extending from the C1 vertebral body to the vertex parallel to the orbitomeatal line. The protocol parameters for contrast agent injection remained unchanged.\nParameters for 64-slice CT for diagnosis of SAH: gantry un-angled, spiral mode, rotation time 1\u00a0s, 64 detector rows at 0.6-mm intervals, table speed 9.6\u00a0mm\/rotation, reconstruction interval 2\u00a0mm at Kernel H30, acquisition parameters 120\u00a0kV\/260\u00a0mAs and acquisition time 14\u00a0s.\nPostprocessing of CTA\nSource images were transferred to a remote computer workstation (Odelft Benelux diagnostic imaging) for viewing. Initial careful review of axial images was considered imperative. During this review any areas of concern could be noted. Two-dimensional maximum intensity projection (MIP) views and three-dimensional (3-D) surface-rendered and volume-rendered reconstructions were reformatted from the raw image date on a Vitrea computer workstation by one of the neuroradiologists.\nParameters for IA-DSA and postprocessing\nFrom April 2003 until April 2004 the IA-DSA studies were produced on a digital angiographic unit (Siemens Multiskop with InfiMed image processing) with a 512\u00d7512 pixel matrix. From April 2004 onwards the studies were performed on a Siemens Axiom Artis angiographic unit with a 1024\u00d71024 pixel matrix. Selective four- or six-vessel angiography using a standard projection format was performed initially and additional views were obtained if required to identify the parent vessel and aneurysm neck more clearly. The amount of contrast medium (Visipaque 270) used was 8\u00a0ml for the internal carotid artery and 6\u00a0ml for the external carotid artery, and the injection rate was 6\u00a0ml\/s when the tip of the catheter was in the internal carotid artery and 3\u20134\u00a0ml\/s when the tip of the catheter was in the external carotid artery. The rate of injection into the vertebrobasilar system was 6\u20138\u00a0ml\/s to a total amount of 8\u00a0ml.\nIn certain situations, rotational 3-D angiography was performed to better delineate the anatomic details of an aneurysm. Rotational 3-D angiography was performed on a Siemens Axiom Artis angiographic unit. The C-arm rotates in a continuous 200\u00b0 arc around the patient\u2019s head during a prolonged intraarterial catheter injection of contrast medium (28\u00a0ml Visipaque, injection rate 4\u00a0ml\/s). The raw date images were transferred to a Leonardo workstation (AX Applications) from which 3-D volume-rendered reconstructions were reformatted.\nImage review and data analysis\nThe presence of an aneurysm, its size and morphology, its parent and feeding vessels and the collateral circulation at the circle of Willis were determined by one of the diagnostic or interventional neuroradiologists. If multiple aneurysms were detected, the usual criteria were applied to decide which aneurysm was responsible for the haemorrhage. These criteria included the unenhanced CT findings (distribution of blood) and the size and irregularity of the aneurysm.\nAll diagnostic findings were discussed with the neurosurgeons. The CTA results were categorized into proven ruptured aneurysm, inconclusive or negative. Patients with a proven ruptured aneurysm were selected subsequently for coiling or clipping. The surgical and endovascular findings were compared to the CTA findings. In general, ruptured aneurysms in the anterior circulation were selected for either coiling or clipping. Ruptured aneurysms located in the posterior circulation were preferably coiled. Giant intracranial aneurysms were preferably treated surgically. A ruptured aneurysm in association with an intraparenchymatous haemorrhage was most often selected for clipping of the aneurysm and surgical evacuation of the haematoma.\nPatients categorized as inconclusive or negative underwent IA-DSA. In patients with a perimesencephalic blood distribution, one IA-DSA examination was performed. In patients with a nonperimesencephalic blood distribution a second IA-DSA was performed if the first one was negative. IA-DSA was considered the gold standard. CTA was considered false-negative when IA-DSA revealed a ruptured aneurysm or when rebleeding occurred.\nThe positive predictive value, negative predictive value, sensitivity, specificity and accuracy of CTA per patient were calculated. The chi-squared test was used to compare the performance of 16-slice CTA and 64-slice CTA for the identification of intracranial aneurysms. Differences with a P value less than 0.05 were considered significant.\nThe IA-DSA findings in patients in the inconclusive category were compared with the CTA findings to assess whether IA-DSA actually provided any additional information.\nResults\nPatient population\nFrom April 2003 until January 2006 292 patients with SAH underwent CTA. Excluded from the study were 68 patients, of whom 24 were excluded because of a nonaneurysmal cause of the SAH including trauma (n\u2009=\u200917), arteriovenous malformation (n\u2009=\u20096) and anticoagulant therapy (n\u2009=\u20091), 3 because of hypertension and intracerebral haematoma, 4 because of comorbidity or advanced age, 2 were excluded because of poor clinical condition and 1 because if poor clinical grading and advanced age, and 31 died from the initial effect of SAH, rebleed or vasospasm with ischemia. Two patients refused therapy and one patient was referred to another hospital for treatment.\nThe study included 224 patients, 89 men and 135 women with a mean (\u00b1SD) age of 52.7\u2009\u00b1\u200910.7\u00a0years (range 22\u201379\u00a0years). Their clinical condition just before treatment was classified according to the original Hunt and Hess grading system: 99 patients were classified as grade I, 45 as grade II, 58 as grade III, 20 as grade IV, and 2 as grade V [15].\nDetection of intracranial aneurysms\nOf the 224 patients, 140 underwent 16-slice CTA and 84 underwent 64-slice CTA. The CTA results were categorized as proven ruptured intracranial aneurysm (133 patients, 59%), inconclusive (31 patients, 14%), or negative for aneurysm (60 patients, 27%).\nPositive CTA result\nIn this category 133 ruptured aneurysms were diagnosed in 133 patients. In 25 patients 32 associated unruptured aneurysms were diagnosed. The distributions of the locations and sizes of the aneurysms are shown in Tables\u00a01, 2, 3 and 4. An overview of the results in this subgroup is presented in Fig.\u00a01.\nTable\u00a01Location of symptomatic intracranial aneurysms in 224 patientsAneurysm locationCTA-positive (n\u2009=\u2009133)CTA-inconclusive (n\u2009=\u200931)aCTA-negative (n\u2009=\u200960)bCoiling (n\u2009=\u200978)Clipping (n\u2009=\u200955)Coiling (n\u2009=\u20098)Clipping (n\u2009=\u200912)Anterior circulationAnterior communicating artery3425551Pericallosal artery22Middle cerebral artery318161Internal carotid artery3111Posterior communicating artery1781Anterior choroideal artery1Posterior circulationBasilar tip11Vertebral junction1Posterior cerebral artery1Posterior inferior cerebellar artery511Superior cerebellar artery1a11 patients had no proven aneurysm.bFive patients had false-negative CTA, in four of whom a ruptured aneurysm was diagnosed on repeat angiography.Table\u00a02Size distribution of symptomatic intracranial aneurysms in 224 patientsSize (mm)CTA-positive (n\u2009=\u2009133)CTA-inconclusive (n\u2009=\u200931)aCTA-negative (n\u2009=\u200960)b\u2009<\u20095471245\u2013970410\u20131414315\u20132411\u2265251a11 patients had no proven aneurysm.bFive patients had false-negative CTA, in four of whom a ruptured aneurysm was diagnosed on repeat angiography.Table\u00a03Location of asymptomatic intracranial aneurysms in 224 patientsAneurysm locationCTA-positiveCTA inconclusiveAnterior circulationAnterior communicating artery6a3Pericallosal artery2Middle cerebral artery11b4Internal carotid artery9aPosterior communicating artery6aPosterior circulationBasilar tip1Junction of vertebral artery1Posterior inferior cerebellar artery1aOne false-negative on CTA.bTwo false-negatives on CTA.Table\u00a04Size distribution of asymptomatic intracranial aneurysms in 224 patients\u00a0Size (mm)CTA-positiveCTA-inconclusive\u2009<\u2009530a45\u201396310\u2013141aFive false-negative on CTA.Fig.\u00a01Flow chart of CTA results\nThe majority of patients were treated within 3\u00a0days of SAH (n\u2009=\u200999, 75%). Of the 133 CTA-positive patients, 78 (59%) were coiled and 55 (41%) were clipped. Treatment conversion was needed in three patients, in two because of difficult aneurysm morphology and in one because of rebleeding during surgery (dura not yet opened). In two patients treatment conversion was necessary because of incorrect treatment selection based on CTA (Fig.\u00a02).\nFig.\u00a02CTA and IA-DSA results in a 66-year-old woman with SAH Hunt and Hess grade III. CTA showed four aneurysms: an aneurysm of the anterior communicating artery (AComA) and two bilateral aneurysms of the middle cerebral artery and one aneurysm of the pericallosal artery. The aneurysm of the AComA was regarded as symptomatic at the time of initial SAH and its CTA-proven morphology showed both coiling and clipping to be a difficult challenge. Coiling of the AComA aneurysm was tried on the 2nd day. The session was aborted because the neck of the aneurysm was broad and the aneurysm incorporated both A2 segments. Unfortunately, rebleeding occurred after coiling. The morphology of the aneurysm excluded complete occlusion by clipping on the 25th day. In the postoperative course again two rebleedings occurred and the patient died. At autopsy a ruptured pericallosal aneurysm was seen more distal to the clipped aneurysm. a Coronal MIP CTA; b volume-rendered CTA; c AP view DSA, selective catheterization of left internal carotid artery; d volume-rendered IA-DSA; e, f autopsy (red arrow anterior communicating artery aneurysm, yellow arrow middle cerebral aneurysm, black arrow anterior cerebral artery (A2 segment), blue arrow pericallosal artery aneurysm\nAll ruptured intracranial aneurysms were confirmed by surgery or endovascular treatment. In two patients IA-DSA was performed after surgical treatment for evaluation of coiling of asymptomatic aneurysms.\nIn four patients (3%) a fatal rebleeding occurred during follow-up, in one patient soon after complete occlusion of the aneurysm with coiling, in one patient on the 5th day after clipping, in one patient 2\u00a0weeks after incomplete occlusion of the aneurysm with coiling and in one patient almost 3\u00a0years after clipping. In all patients the blood distribution of the rebleeding was the same as that of the primary SAH. Although permission was not granted for an autopsy in two patients, it was considered most probable that the rebleeding was caused by the treated aneurysm. In two patients an autopsy was performed. In one a ruptured pericallosal aneurysm was seen, 1.5\u00a0cm more distal from the clipped anterior communicating artery aneurysm which was regarded as symptomatic at the time of initial SAH (Fig.\u00a02), and in the other a haematoma surrounding a prepontine cavernous haemangioma and an endovascular treated dissecting aneurysm of the basilar artery were seen.\nThe presence of CT-diagnosed additional asymptomatic aneurysms was checked in 22 patients. These patients had 29 aneurysms. Five aneurysms were confirmed at surgery and subsequently clipped, 5 aneurysms were checked with IA-DSA and subsequently coiled and 19 aneurysms were confirmed with IA-DSA. Three aneurysms in three patients were not verified. In four patients five asymptomatic aneurysms were false-negative on CTA. All were smaller than 5\u00a0mm. Four aneurysms were diagnosed with IA-DSA during an embolization session, one of them was also embolized. Another aneurysm was considered a vessel loop of the middle cerebral artery on CTA. However, an aneurysm of the middle cerebral artery was seen during surgery of a ruptured aneurysm of the anterior communicating artery. Clipping of the aneurysm of the middle cerebral artery was also performed.\nInconclusive CTA result\nIn 31 patients IA-DSA was performed because of inconclusive CTA results. The indications for IA-DSA examination are presented in Table\u00a05. An overview of the results in patients in this category is presented in Fig.\u00a01.\nTable\u00a05Indications for IA-DSA examination in 31 patientsIndicationNo. of patientsMore information required regarding location and orientationSymptomatic aneurysm10Asymptomatic aneurysm2More information required regarding presence of intraaneurysmal thrombus in symptomatic giant aneurysm2Differentiation between asymptomatic and symptomatic aneurysm1Differentiation between infundibulum, vessel loop and aneurysm5Fisher grade IV SAH1Arterial vasospasm3Discrepancy between diagnosed intracranial aneurysm and distribution of blood1Incomplete angiography of circle of Willis3Overprojection of venous structures1Variance of normal intracranial vessel anatomy1Amalgam artefacts1\nIn 11 patients (35%) IA-DSA confirmed the results of CTA. In 17 patients (55%) IA-DSA was able to give further diagnostic information required for a correct patient selection for therapy. In two patients (6%) no additional diagnostic information could was obtained from IA-DSA. In both patients vasospasm of a vertebral artery resulted in an inconclusive CTA, but also excluded selective catheterization with IA-DSA. A second CTA was negative in both patients. In one patient (3%) treatment selection was based on a false-positive IA-DSA. CTA was inconclusive because of amalgam artefacts in the region of the right posterior inferior cerebellar artery (PICA). An aneurysm of the right PICA was diagnosed on the first IA-DSA. A second IA-DSA was performed with the intention of coiling. However, with additional views the aneurysm turned out to be a vessel loop.\nNo ruptured aneurysms were found in ten patients (four nonperimesencephalic SAH, four with perimesencephalic SAH and two negative on unenhanced CT). In 20 patients 20 ruptured intracranial aneurysms and 6 additional unruptured aneurysms were found. In one patient only an asymptomatic aneurysm was diagnosed (Tables\u00a01, 2, 3 and 4). Of the ruptured aneurysms, 12 were clipped and 6 were coiled. Two ruptured aneurysms were clipped after failure of endovascular treatment. Two asymptomatic aneurysms were clipped.\nNegative CTA result\nIn 60 patients CTA was considered negative. An overview of the results in this category of patients is presented in Fig.\u00a01. Of these 60 patients, 13 (22%) had negative findings on unenhanced CT, and 47 (78%) had positive findings on unenhanced CT, and of the latter 30 had a perimesencephalic blood distribution and 17 had a nonperimesencephalic SAH [16]. In 11 (85%) of those with negative findings on unenhanced CT, one IA-DSA was done. In one patient a second IA-DSA was performed and in one patient a second CTA was performed. No rebleedings occurred. CTA was true-negative in all these patients.\nIn 21 patients (70%) with perimesencephalic SAH, IA-DSA was performed once, and in one of them CTA was repeated once and in one CTA was repeated twice. In eight patients IA-DSA was repeated once and in one patient IA-DSA was repeated twice. In this category CTA was true-negative in all these patients. No rebleedings occurred.\nIn nine patients (53%) with nonperimesencephalic SAH IA-DSA was performed once, and in two of them a follow-up MRA was done and in one CTA was repeated. In seven patients a second IA-DSA was done. In one patient IA-DSA was repeated twice. In five patients (29%) with nonperimesencephalic SAH, CTA was false-negative (Tables\u00a01 and 2). In one of these patients only IA-DSA was able to detect a 3-mm ruptured aneurysm of the anterior communicating artery, and in the other four a rebleeding occurred despite an initially negative CTA and IA-DSA. Repeat angiography was performed in three patients: a ruptured aneurysm was shown by CTA in two and by IA-DSA in one. One patient died before repeat angiography. Two patients were treated successfully. One patient died before treatment due to the direct effect of the rebleeding. The explanations for false-negative results were interpretation mistakes (two aneurysms recognized retrospectively on CTA and IA-DSA), and haematoma demonstrated on CTA surrounding and compressing the aneurysm leading to interpretation error on both CTA and IA-DSA in one patient and on only CTA in another (IA-DSA showed the aneurysm); the findings were uncertain in one patient.\nStatistical analysis\nThe diagnostic value of both 16- and 64-slice CTA are outlined in Tables\u00a06 and 7. A comparison of the results of 16-and 64-slice CTA is presented in Table\u00a08. No statistically significant differences were found.\nTable\u00a06Diagnostic value of CTA in ruptured aneurysmsDiagnostic valueTrue positive132 patientsFalse positive1 patientTrue negative55 patientsFalse negative6 patientsPositive predictive value99%Negative predictive value90%Sensitivity96%Specificity98%Accuracy96%Table\u00a07Diagnostic value of CTA in additional aneurysmsDiagnostic valueTotal number detected with CTA25 patients (32 aneurysms)Presence checked22 patients (29 aneurysms)True positive22 patients (29 aneurysms)False positive0True negative120 patientsaFalse negative4 patients (5 aneurysms)Positive predictive value100%Negative predictive value97%Sensitivity85%Specificity100%Accuracy97%aIncluding 60 CTA-negative patients and 60 CTA-positive patients. Of the CTA-positive patients, 44 were not examined with IA-DSA as standard control.Table\u00a08Comparison of results of 16- and 64-slice CTA for detection of intracranial aneurysms\u00a016-slice (n\u2009=\u2009140 patients)64-slice (n\u2009=\u200984 patients)CTA resultPositive7459Negative4515Inconclusivea2110Ruptured aneurysmsTrue positive7359False positive10True negative4213False negative4b2Positive predictive value (%)99100Negative predictive value (%)9187Sensitivity (%)9597Specificity (%)98100Accuracy (%)9697Unruptured aneurysmsTotal number on CTA12 (15 aneurysms)13 (17 aneurysms)Presence checked11 (14 aneurysms)11 (15 aneurysms)True positive11 (14 aneurysms)11 (15 aneurysms)False positive00True negative75c45dFalse negative1 (1 aneurysm)3 (4 aneurysms)Positive predictive value (%)100100Negative predictive value (%)9994Sensitivity (%)9279Specificity (%)100100Accuracy (%)9995aPatients with an inconclusive result were not included in the statistical analysis.bIncluding one CTA-positive patient.cIncluding 45 CTA-negative patients and 30 CTA-positive patients. Of the CTA-positive patients, 31 were not examined with IA-DSA as standard control.dIncluding 15 CTA-negative patients and 30 CTA-positive patients. Of the CTA-positive patients, 13 were not examined with IA\u2013DSA.\nDiscussion\nOur primary aim was to assess whether CTA is useful clinically in planning and performing clipping or coiling, especially in the acute phase in ruptured intracranial aneurysms, without recourse to IA-DSA. We demonstrated that it was possible to treat more than half of all patients with a ruptured intracranial aneurysm using only CTA. By avoiding conventional angiography, it was possible to streamline the management of ruptured aneurysm during the acute phase. Further, 3D-CTA was able to help in deciding whether to clip or to coil; in only two patients was treatment conversion needed due to incorrect treatment selection based on CTA.\nWe found 3D-CTA to be a simple, reliable, quick and minimally invasive imaging modality that reduces the risk of complications caused by conventional angiography and reduces the delay between the patient\u2019s arrival at the hospital and treatment, leading to diminished rebleeding. Matsumoto et al. analyzed the rate of rebleeding of ruptured aneurysms during CTA and conventional angiography, and found 0% (none of 160 patients) for CTA and 1.5% (5 of 317 patients) for conventional angiography [17]. In patients with a ruptured aneurysm and intracerebral haemorrhage CTA saves time when aiming for a fast clot removal. Another advantage is that the radiation dosage is low compared to IA-DSA (1.0\u00a0mSv at 200\u00a0mAs with the CTA Siemens Sensation 16 and 1.8\u00a0mSv at 380\u00a0mAs with the CTA Siemens Sensation 64 compared with 3.5\u20136.5\u00a0mSv with conventional angiography). Furthermore, the cost of CTA is one-fourth that of conventional angiography.\nSeveral other studies assessing whether CTA may serve as the sole imaging method for the preoperative work-up of patients with ruptured intracranial aneurysms have been published [17\u201326]. An overview of these previous studies is presented in Table\u00a09. There is a wide variation in the percentage of patients who have had their symptomatic aneurysms treated based on CTA. This may be influenced positively by the very high aneurysm prevalence and the subsequent very low negative rates of CTA in some studies [22, 23, 25]. In other studies patients with a negative CTA were not enrolled at all [17, 20]. In general, a mean of 15\u201320% negative angiographies after SAH is accepted [27]. The present study showed a high negative rate for CTA. This may reflect the good awareness of the diagnosis SAH in first-line and second-line health-care and the good access to CTA when the diagnosis SAH is considered. Furthermore, the wide variation in CTA-based treatment may be partially explained by differences in hardware and software used by each group, the rate of technical failures in performing CTA, scanning parameters set for screening the circle of Willis and more peripheral vessels, the experience and scrutiny of the neuroradiologist evaluating each CTA and the willingness of the neurosurgeon and neurointerventional radiologist to rely on CTA alone in each individual case.\nTable\u00a09Presentation of previous studies and present studyStudyNo. of patientsCTA-positiveCTA-negativeCTA inconclusive or no CTA-based treatmentTotal patientsCTA-based treatmentTrue-positive CTATotal patientsTrue-negative CTATotal patients218746 (55%)44 (96%)44 (100%)15 (17%)6 (60%)26 (30%)2210988 (81%)87 (99%)87 (100%)5 (5%)5 (100%)16 (15%)238462 (74%)62 (100%)62 (100%)7 (8%)0 (0%)15 (18%)199045 (100%)45 (100%)45 (100%)\u2013\u201345 (50%)1815061 (41%)61 (100%)60 (98%)a24 (16%)24 (100%)65 (43%)2512040 (27%)40 (100%)40 (100%)13 (11%)13 (100%)67 (56%)247827 (35%)27 (100%)27 (100%)20 (26%)20 (100%)b31 (40%)1710093 (93%)93 (100%)93 (100%)\u2013\u20137 (7%)209687 (91%)87 (100%)86 (99%)2\u2013\u20139 (9%)266144 (72%)44 (100%)44 (100%)15 (25%)14 (93%)2 (3%)Present study224133 (59%)133 (100%)132 (99%)260 (27%)55 (92%)31 (14%)aOne false-negative and one false-positive ruptured aneurysm in one patient.bIn five patients with perimesencephalic SAH, IA-DSA as the gold standard control was not performed.\nIn the present study CTA was false-negative in 8% of patients. The risk of rebleeding after a negative initial CTA was 7%. All false-negatives were in patients with a nonperimesencephalic blood distribution, giving a false-negative rate of 29% and a risk of rebleeding of 24%. It seems unlikely that the false-negative rate of initial CTA and the risk of rebleeding despite a negative initial CTA in patients with a nonperimesencephalic SAH might be influenced negatively by the use of CTA as the first diagnostic tool. Firstly, in all patients with a rebleeding, repeat IA-DSA was also false-negative. Secondly, repeat angiography with CTA performed after a rebleeding still demonstrated an aneurysm.\nFurthermore, the findings of other studies using IA-DSA as the first diagnostic tool were similar. In the study by Urbach et al. in 67 patients with a negative initial angiogram after SAH, four ruptured aneurysms were revealed by repeat angiography [28]. Three patients presented with a nonperimesencephalic SAH and one presented with a perimesencephalic SAH. In the study by Bradac et al., 60 of the 440 patients presenting with spontaneous SAH had a negative angiogram [29]. A second angiogram performed 1\u20134\u00a0weeks later revealed a ruptured aneurysm in 5 of the 40 patients. Of these patients, 3 had a second SAH. In all patients a nonperimesencephalic blood distribution was seen on CT.\nBecause in the present study some aneurysms could be correctly identified retrospectively, we suggest that if, under strong clinical suspicion of a ruptured aneurysm, the CTA is reported as normal, the study should be reviewed by a second neuroradiologist before proceeding to repeat angiography. It is essential to perform a review of axial raw source images. Next, we recommend repeat CTA or IA-DSA when the initial CTA is negative in patients with a nonperimesencephalic SAH. There is no consensus about the time interval for repeat angiography. In practice, the guideline is to repeat angiography after several days to months. The substantial risk of rebleeding in patients with an aneurysmal pattern of haemorrhage in the present study indicates that some cerebral aneurysms are occult on initial CTA. Several factors may explain this finding. Most importantly, there is a learning curve in assessing aneurysms on CTA. Pedersen et al. reported an increase in sensitivity from 88% to 94% after 1\u00a0year\u2019s experience [30]. Small aneurysms can be missed when using CTA. CTA had a sensitivity of 50% for aneurysms <2\u00a0mm in the study of Wintermark et al. [13]. Distal pericallosal and PICA aneurysms can be missed when restricting the area of coverage to the proximal circle of Willis [31\u201334]. Thrombosis of the neck of the aneurysm or of the entire sac is another possible reason [23]. Perianeurysmal blood or haematoma may reduce lesion conspicuity [34]. Aneurysms may be mistaken for vascular infundibula (persistent fetal nonaneurysmal dilatation of the proximal vessel) of the posterior communicating or anterior choroidal artery origins if a vessel cannot be identified arising from them [35]. Aneurysms may masquerade as tight vascular loops if the MIP thickness is wide (>3\u00a0mm) [34]. In patients with multiple intracranial aneurysms large aneurysms may obscure smaller ones on the CT reconstruction [33]. Aneurysms close to bone (e.g. carotid siphon, ophthalmic and posterior communicating artery) may be overlooked when relying on surface-rendering and volume-rendering techniques or using MIP with bone editing [32, 34, 36\u201338]. Aneurysms located within or close to the cavernous sinuses are easy to overlook unless thin-section axial and coronal MIP images are reviewed on a slightly wider window width [9].\nIn patients with a perimesencephalic SAH the chance of finding a posterior fossa aneurysm is low: 2.5\u20135% [39, 40]. Nonaneurysmal perimesencephalic haemorrhage carries no risk of vasospasm and rebleeding and has been shown to follow a benign course with an excellent prognosis [41]. The chance of finding an aneurysm in 5% of patients has to be weighed against the risk of complications from angiography imposed upon the remaining 95% of patients. CTA has a high accuracy for diagnosis of vertebrobasilar aneurysms and of intracranial aneurysms in general [9\u201313, 42]. In the present study, in patients with a perimesencephalic SAH and a negative initial CTA, no rebleedings occurred and CTA was true-negative in all. Similarly, in the prospective study of Huttner et al., 69 patients with a perimesencephalic SAH had a negative initial CTA and IA-DSA [43]. A repeat IA-DSA was performed in 38 patients (55%). None of the repeat IA-DSAs showed any additional distinctive features with respect to the first IA-DSA. It therefore seems practical and safe to perform CTA as the first diagnostic tool and to omit repeat angiography if CTA is negative. A formal decision analysis based on these observations confirmed that a strategy where CTA is performed and not followed by conventional angiography, if negative, results in a better utility than a strategy of CTA followed by conventional angiography or of conventional angiography as primary investigation [44].\nAccording to the results of the present study, it seems important to distinguish the two patterns of SAH on CT. The CT criteria of perimesencephalic bleeding have been defined [40]. Different data show that experienced radiologists can accurately discriminate between a perimesencephalic and nonperimesencephalic SAH [12, 40, 45]. Early CT within 3\u00a0days is necessary for reliable assessment of the pattern of haemorrhage [12, 40, 46].\nA criticism of this study might be that patients treated with endovascular coiling underwent IA-DSA as part of the endovascular procedure and thus should not be counted in the analysis of efficacy of the prospective protocol. However, a shift in management of ruptured intracranial aneurysm from surgery to endovascular treatment has appeared [47]. Endovascular treatment is replacing clipping. The use of CTA as the initial investigation for cerebral aneurysms may offset some of this increased workload whilst also improving workflow.\nIn conclusion, in this evaluation of the use of 16-row and 64-row multislice CTA in the management of ruptured intracranial aneurysms, we demonstrated that CTA can be used as the first-line diagnostic modality for the management of SAH patients. In CTA-negative patients IA-DSA provided no or marginal added value. IA-DSA is not needed in patients with negative CTA and classic perimesencephalic SAH. Repeat IA-DSA or CTA should still be performed in patients with a nonperimesencephalic SAH, due to false-negative CTAs and IA-DSAs in this patient group. The remaining true indication for IA-DSA was in patients with an inconclusive CTA result. In more than half of those IA-DSA provided relevant new diagnostic information.","keyphrases":["intracranial","aneurysm","cta","subarachnoid haemorrhage"],"prmu":["P","P","P","P"]}
{"id":"Mar_Biotechnol_(NY)-2-2-1475948","title":"Characterization of Promoter Activities of Four Different Japanese Flounder Promoters in Transgenic Zebrafish\n","text":"An important consideration in transgenic research is the choice of promoter for regulating the expression of a foreign gene. In this study several tissue-specific and inducible promoters derived from Japanese flounder Paralichthys olivaceus were identified, and their promoter activity was examined in transgenic zebrafish. The 5\u2032 flanking regions of the Japanese flounder complement component C3, gelatinase B, keratin, and tumor necrosis factor (TNF) genes were linked to green fluorescence protein (GFP) as a reporter gene. The promoter regulatory constructs were introduced into fertilized zebrafish eggs. As a result we obtained several stable transgenic zebrafish that displayed green fluorescence in different tissues. Complement component C3 promoter regulated GFP expression in liver, and gelatinase B promoter regulated it in the pectoral fin and gills. Keratin promoter regulated GFP expression in skin and liver. TNF gene promoter regulated GFP expression in the pharynx and heart. TNF promoter had lipoplysaccharide-inducible activity, such that when transgenic embryos were immersed lipopolysaccharide, GFP expression increased in the epithelial tissues. These 4 promoters regulated the expression of GFP in different patterns in transgenic zebrafish.\nIntroduction\nTransgenic technology is a powerful tool not only for analyzing molecular biological functions but also for obtaining commercially important traits for aquaculture. In the last decade fish transgenesis has become common and has progressed considerably (Chen et al., 1996; Hackett and Alvarez, 2000). Various transgenic fish that possess commercially important traits, such as growth enhancement (Zhang et al., 1990) or disease resistance (Sarmasik et al., 2002), have been generated. One of the most critical factors in transgenic research is the regulation of foreign gene expression. Early fish transgenic research was conducted using housekeeping gene promoters such as \u03b2-actin (Liu et al., 1990; Higashijima et al., 1997; Hwang et al., 2003; Noh et al., 2003) and elongation factor (EF) (Gao et al., 1997; Kinoshita et al., 2000). These promoters resulted in excess expression in various tissues. However, tissue-specific or inducible promoters are needed to regulate foreign genes in different situations. The activities of tissue-specific promoters were assessed in zebrafish using green fluorescence protein (GFP) as a reporter gene (Udvadia and Linney, 2003). This technique has provided a powerful tool for analyzing the regulation of gene expression in living fish. The pattern of expression of GFP in these transgenic zebrafish was the same as that of the gene from which the promoter was derived. However, promoters derived from most marine aquaculture species have not been described. To generate transgenic aquaculture fish, tissue-specific and stress-inducible promoters from these species need to be identified.\nJapanese flounder Paralichthys olivaceus is widely cultured and one of the most important food fish in East Asia. Because this industry is seriously affected by disease, the development of disease-resistant strains is a high priority. Previous studies by our laboratory have used expressed sequence tags (ESTs) (Aoki et al., 1999, 2000; Nam et al., 2000, 2003) and molecular genetic analyses, especially of immune-related genes in Japanese flounder (Hirono et al., 2003). In these reports we focused on cDNA clones that displayed tissue-specific expression or inducible expression, and 4 Japanese flounder genes were chosen for this study: complement component C3, gelatinase B, keratin, and tumor necrosis factor (TNF) genes. These genes were chosen because they were revealed to be expressed in specific tissues of Japanese flounder by EST analysis. We assumed that the promoter region of these genes would show tissue-specific or inducible activities.\nIt is possible to generate an effective transgenic fish using tissue-specific or inducible promoters. Indeed, such promoters can regulate foreign gene expression faithfully. In contrast, use of a housekeeping gene promoter would result in nonspecific expression (i.e., overexpression) of foreign genes that may be harmful to the host fish. In this study we developed several Japanese flounder promoters that showed tissue-specific or inducible gene expression with the aim of using these in Japanese flounder. Although we have succeeded in introducing a foreign gene into Japanese flounder fertilized eggs and shown transient expression in Japanese flounder embryos, it has been difficult to conduct promoter assays in vivo because the technology has not been sufficiently developed to produce transgenic Japanese flounder with the necessary efficiency. However, the transgenic zebrafish model system has the advantages of shorter generation time, better optical transparency, and easier treatment schemes when compared with Japanese flounder. In this study, promoter regions of the complement component C3, gelatinase B, keratin and TNF genes were isolated and their promoter activities were characterized in transgenic zebrafish.\nMaterials and Methods\nmRNA Expression in Japanese Flounder Tissues\nTotal RNA was extracted from spleen, fin, liver, head kidney, post kidney, skin, blood, heart, intestine, ovary, gill, leukocyte, and peripheral blood leukocytes (PBLs) stimulated with lipopolysaccharide (LPS) using TRIzol (Invitrogen). The purified total RNA (10 \u03bcg) was reverse transcribed into cDNA using an avian myeloblastosis virus (AMV) transcriptase First-strand cDNA synthesis kit (Amersham Bioscience). The reverse-transcribed sample (1 \u03bcl) was used in 50 \u03bcl of PCR mixture. The PCR primers for Japanese flounder complement component C3 mRNA detection were (sense) 5\u2032-GCTGGAGAAAG TCGTCTTGG-3\u2032 and (anti) 5\u2032-GGATACCTCTCAACTCTG CC-3\u2032, gelatinase B mRNA detection, (sense) 5\u2032-GCA GGAGCCACCAGTCAAAA-3\u2032 and (anti) 5\u2032-GGTCCAGTG TTCATCATCGT-3\u2032, keratin mRNA detection, (sense) 5\u2032-ACTCCGTCGCACAATGCAGA-3\u2032 and (anti) 5\u2032-CTGCA ATTTCCATCTCCAGC-3\u2032, and TNF mRNA detection, (sense) 5\u2032-CCCTATGAACTGTAACAGTTTG-3\u2032 and (anti) 5\u2032-GTCAGGTACTTAACCCTCAT-3\u2032. The \u00df-actin primer set for an internal control was (sense) 5\u2032-TTTCCCT CCATTGTTGGTCG-3\u2032 and (anti) 5\u2032-GCGACTCTCAGCTC GTTGTA-3\u2032. The PCR was performed with an initial denaturation step of 2 minutes at 95\u00b0C and then 20 cycles were run as follows: 30 seconds of denaturation at 95\u00b0C, 30 seconds of annealing at 55\u00b0C, and 1 minute of extension at 72\u00b0C. The reaction products were electrophoresed in a 2.0% agarose gel.\nIdentification of 5\u2032 Flanking Region Sequences\nThe 5\u2032 flanking regions of 4 genes were isolated from a Japanese flounder genomic BAC library (Katagiri et al., 2000) using cDNA clones of complement component C3 (AB021653), gelatinase B (AU091035), keratin (AB079729), and TNF (AB040448), as probes for gene screening. To determine the transcription start site, the primer extention method was performed according to a modified version of that in Hikima et al. (2001). A 20mer synthetic oligonucleotide corresponding to the complementary sequence of the first exon was labeled with fluorochrome. The fluorochrome-labeled primer was hybridized to 10 \u03bcg of kidney total RNA (extracted using Trizol, Invitrogen) and then extended using an AMV reverse transcriptase first-strand cDNA synthesis kit (Amersham Bioscience). Subcloned plasmid DNA, containing the first exon of each gene, was adopted as a template for the sequencing using the same fluorochrome-labeled primer. The sequence products were analyzed with primer-extended cDNA fragment by the cycle sequencing method.\nConstruction of Promoter-EGFP Plasmid\nThe reporter gene vector pEGFP-1 (Clontech) was used for the construct. Four different Japanese flounder promoters including 5\u2032 untranslated region (UTR) were amplified by specific PCR primers and inserted into the multiple cloning site of pEGFP-1. GFP reporter gene was the endogenous initiation codon directly replaced with that of GFP. To confirm the sequence and direction of insert, the recombinant plasmid was sequenced using a Thermo Sequenase II kit and automated DNA sequencer 373A with a primer designed for pEGFP-1.\nProduction of Transgenic Zebrafish\nAdult zebrafish (Danio rerio) and embryos were maintained at 28.5\u00b0C on a 14-hour light and 10-hour dark cycle. The recombinant plasmid was propagated in Escherichia coli JM109 and purified using a GFX Micro Plasmid prep kit (Amersham Biosciences). Purified plasmid DNA was adjusted to 50 ng\/\u03bcl in distilled water and microinjected into one-celle-stage zebrafish embryos. Initially 400 to 500 embryos were injected with plasmid DNA in each promoter-GFP construct. Living embryos were examined under an SZX12 fluorescence microscope (Olympus), and GFP-positive zebrafish embryos were selected. After maturation the GFP-positive fish were mated with wild types to select fish for germline transmission. GFP expression in F1 embryos was also examined under the fluorescence microscope in each developmental stage.\nLPS Treatment of TNF-GFP Transgenic Zebrafish Embryo\nF2 generations of transgenic zebrafish injected with TNF-GFP at 30 hours postfertilization (hpf) were stimulated with LPS (Sigma) at 140 \u03bcg\/ml for 30 minutes and then washed in sterilized water. GFP expression in F2 embryos was examined under the fluorescence microscope at 48 hpf.\nDetection of GFP Gene Expression in Adult Transgenic Zebrafish by RT-PCR\nTotal RNA extraction and PCR was performed as described above. Total RNA was extracted from fin, skin, liver, kidney, muscle, and brain of adult transgenic zebrafish. The following PCR primers were designed for the detection of EGFP gene: GFP-F, 5\u2032-GGTCG AGCTGGACGGCGACG-3\u2032, and GFP-R, 5\u2032-ACGAACTCC AGCAGGACCAT-3\u2032. The zebrafish \u00df-actin primer set for internal control was zactin-F, 5\u2032-TTTCCCTCCATT GTTGGTCG-3\u2032, and zactin-R, 5\u2032-GCGACTCTCAGCTCG TTGTA-3\u2032.\nResults and Discussion\nThe expression of complement component C3, gelatinase B, keratin, TNF and \u03b2-actin genes in Japanese flounder was detected by reverse transcriptase polymerase chain reaction (RT-PCR). The complement component C3 gene was expressed only in the liver. The gelatinase B gene was expressed in the kidney, blood, and leukocytes. The keratin gene was expressed in various tissues. The TNF gene was expressed only in PBLs when stimulated with LPS. The \u03b2-actin gene used as an internal control was expressed in all tissues (Figure\u00a01). Thus the expression patterns of these genes were different in different Japanese flounder tissues. We assumed that the tissue-specific and inducible expression of these genes was regulated by their promoter region.Fig.\u00a01.Expression of Japanese flounder tissue-specific genes in different tissues. Messenger RNA expression of complement component C3, gelatinase B, keratin, and TNF genes in spleen, fin, liver, head kidney, post kidney, skin, blood, heart, intestine, ovary, gills, leukocyte, and PBLs stimulated with LPS from Japanese flounder were detected by RT-PCR.\nThe 5\u2032 flanking regions of the genes for complement component C3 (1381 bp), gelatinase B (1420 bp), keratin (1288 bp), and TNF (2351 bp) were isolated, and the respective transcriptional initiation sites were determined (Figure\u00a02). There were some predicted transcriptional factor binding sites, such as TATA box, CAAT box or NF-\u03baB binding site, in the 5\u2032 flanking region. These sites were highly homologous with mammalian transcriptional factor binding sites. It is not certain that these predicted transcriptional factor binding sites can be applied to Japanese flounder promoters as there are few reports on fish transcriptional factor binding protein. It should be interesting to determine if these transcriptional factor binding sites are common to mammal and fish. These promoter regions were cloned into pEGFP-1 plasmid vector. These promoter regulatory constructs were designated C3-GFP, gelatinase-GFP, keratin-GFP, and TNF-GFP, respectively.Fig.\u00a02.The 5\u2032 flanking region sequence of the Japanese flounder gene: complement component C3 (A); gelatinase B (B); keratin (C); and TNF gene (D). We isolated 1381 bp of the 5\u2032 flanking regions from transcription initiation codon for complement component C3; 1420 bp for gelatinase B; 1288 bp for keratin; and 2351 bp for TNF. The first exon is indicated in capital letters. The coding region and deduced amino acids are indicated in boldface capital letters. The transcription initiation site is shown by an asterisk. The predictive transcriptional factor binding site is underlined.\nWe obtained F1 transgenic individuals using each promoter regulatory constructs. An F2 strain was established with the C3-GFP, gelatinase-GFP, and keratin-GFP construcst, and two F2 strains from two different founders were established with the TNF-GFP construct. Similar GFP expression patterns were shown in F1 individuals and their F2 strains. GFP expression controlled by the complement component C3 promoter was observed in the yolk sac from the gastrula stage, in F2 transgenic zebrafish strains. In 10-day embryos strong GFP expression was observed only in the liver (Figure\u00a03). GFP expression could not be observed in adults. The RT-PCR analysis showed that complement component C3 promoter also induced GFP expression in the adult liver (see Figure\u00a07). Fish complement component C3 gene has the same function as the mammalian gene, and is expressed mainly in the liver (Abelseth et al., 2003). Japanese flounder complement component C3 was also expressed mainly in the liver (Figure\u00a01). This was similar to the promoter activity observed in transgenic zebrafish. This suggests that Japanese flounder complement component C3 promoter could also induce expression of genes in the same tissues in zebrafish.Fig.\u00a03.GFP expression in C3-GFP transgenic zebrafish embryos. The larval embryos at 24 hpf (A) and liver of the 10-day embryos (B) of F2 transgenic strain. Arrowheads indicate bladder and eye.\nGFP expression regulated by the gelatinase B promoter was observed after the one-cell stage, and was expressed strongly in the whole body until 48 hpf. However, GFP expression in the whole body decreased in the course of development. In 5-day embryos, strong GFP expression was detected only in the pectoral fins and gills (Figure\u00a04). In adult transgenic fish gelatinase B promoter regulated GFP expression in the fin (see Figure\u00a07). This result agrees with the GFP expression at embryonic stage. The GFP expression under the control of the Japanese flounder gelatinase B gene promoter in the transgenic zebrafish was not identical with the expression of the gelatinase B gene in Japanese flounder, in which it was expressed mainly in hematopoietic tissues (Figure\u00a01). This promoter may lack a region essential for tissue-specific expression.Fig.\u00a04.GFP expression of gelatinase B-GFP transgenic zebrafish. GFP expression at one-cell stage (A), 7 hpf (B), 12 hpf (C), 48 hpf (D), and 5-day embryo, lateral view (E) and dorsal view (F).\nThe keratin promoter regulated strong GFP expression from the embryo stage to the adult stage. In the embryo strong GFP expression was observed in the epithelial tissues and liver. In adult fish GFP expression was detected in skin, gills, fins, and liver (Figure\u00a05). This expression pattern was similar to that of the Japanese flounder keratin gene (Figure\u00a01). The zebrafish keratin 8 promoter was found to induce expression only in the skin (Gong et al., 2002). These results indicate that the promoter of the Japanese flounder keratin gene induces gene expression in the same tissues in zebrafish as in Japanese flounder. However, the sequences of the keratin promoter region of zebrafish and Japanese flounder are not the same (data not shown). This may be because some transcription factors that enhance skin-specific expression in zebrafish can bind and enhance the promoter activity of keratin promoter derived from Japanese flounder in transgenic zebrafish embryo.Fig.\u00a05.GFP expression in keratin-GFP F2 transgenic zebrafish under the fluorescence microscope. A: The 4-day embryo. B: Adult fish. C: Adult fish with operculum removed and abdominal muscle surgically opened. Arrowheads indicate gill and liver.\nThe TNF promoter regulated GFP expression only in the heart and pharynx in 5-day embryos. GFP expression was strongly induced in the epithelial tissues in 2-day transgenic embryos after LPS treatment (Figure\u00a06). GFP expression was not detected in adult transgnic zebrafish by RT-PCR. This result suggested that TNF promoter controlled GFP expression in transgenic zebrafish under normal conditions (Figure\u00a07). The Japanese flounder TNF promoter has inducible activity in zebrafish similar to that of Japanese flounder TNF gene expression in LPS-stimulated PBLs (Hirono et al., 2000). These results suggest that the Japanese flounder TNF promoter used in this study is sufficient to regulate the expression of a downstream gene.Fig.\u00a06.GFP expression in TNF-GFP transgenic zebrafish under the fluorescence microscope. A: The 5-day embryo of wild type. B: F2 transgenic strain, 5-day embryo. Arrowheads indicate heart and pharynx. C: Nontreated 3-day transgenic zebrafish embryo. D: LPS-treated 3-day transgenic zebrafish embryo.Fig.\u00a07.Detection of mRNA expression of GFP gene in fin, skin, liver, kidney, muscle, and brain from adult transgenic zebrafish by RT-PCR. GFP expression regulated by TNF promoter was not detected. N.C. indicates negative control.\nIn this study we showed that heterologous promoters derived from Japanese flounder worked in transgenic zebrafish. However, the expression patterns different from endogenous Japanese flounder expression patterns. Notably, the keratin and TNF promoters seemed to work faithfully in zebrafish embryos, but the gelatinase B promoter result differed from the results predicted from Japanese flounder. This may have been due to the short 5\u2032 flanking region used in this study. Furthermore, we only observed the GFP expression pattern in a single transgenic line. Whether this GFP expression pattern is representative of gelatinase B promoter activity needs to confirmed in future studies by establishing multiple transgenic lines. Similarly, the fate of these promoters in Japanese flounder needs to be determined by production of transgenic lines of Japanese flounder.\nThe Japanese flounder promoters developed in this study can be adapted for transgenic fish in a variety of situations. Keratin promoter induced expression in epithelial tissues, where it acts as a first line of defense against bacterial infection. It is possible to generate disease-resistant transgenic fish expressing an antibacterial or antiviral peptide only in epithelial tissues. Moreover, using the Japanese flounder TNF promoter, it is possible to generate transgenic fish expressing an antibacterial or antiviral peptide only in cases of infection with pathogens. Thus these promoters should be useful for production of disease-resistant transgenic fish. The overexpression of a foreign gene, especially the gene for an antimicrobial peptide, may suppress growth or maturity of the host fish. In addition, these transgenic fish are safe for human consumption, as the foreign gene is not expressed in the muscle. The Japanese flounder TNF gene promoter can also be used to monitor bacterial infection in live fish using GFP as an indicator. Furthermore, these transgenic zebrafish were suitable for developmental analysis of specific tissues or organs. It is possible to observe development of liver, gills, and skin in living embryos using GFP expression.\nWe also developed a method for introducing a gene into Japanese flounder fertilized eggs by particle gun bombardment (Yazawa et al., 2005). The development of tissue-specific, inducible promoters in this study will support the establishment of transgenic technology for Japanese flounder and should contribute to a better understanding of the biology and culture of this fish species.","keyphrases":["japanese flounder","transgenic zebrafish","inducible promoter","green fluorescence protein (gfp)","tissue-specific promoter"],"prmu":["P","P","P","P","P"]}
{"id":"Breast_Cancer_Res_Treat-4-1-2217620","title":"An overview of prognostic factors for long-term survivors of breast cancer\n","text":"Background Numerous studies have examined prognostic factors for survival of breast cancer patients, but relatively few have dealt specifically with 10+-year survivors.\nIntroduction\nBreast cancer (BC) is the most common cancer among women, with a lifetime risk of up to 12% and a risk of death of up to 5% [1]. Its incidence has been increasing but after a period of continuous rise in many industrialized countries BC mortality has been stable or has even decreased in the last 10\u201315\u00a0years [2, 3]. The introduction of mass mammographic screening programmes also resulted in earlier detection and diagnosis of small and less aggressive tumours. This, in combination with therapeutic improvements, has led to a substantial increase in BC survivors over the last few decades (Fig.\u00a01). A long-term survivor is commonly defined as a person who is still alive 5\u00a0years after cancer diagnosis [4]. For BC, the relative survival at 5 and 10\u00a0years after diagnosis is 88% and 77%, respectively, both substantially higher than the 5-year relative survival of all cancers together (64%) [4]. Thus, it seems logical to consider factors known to play an important role in predicting 5-year survival of BC patients and to question their importance in survival 10\u00a0years after diagnosis and even longer. Furthermore, in recent years major advances in the prognostic value of several molecular markers have been achieved, hence the need to incorporate this data into our current knowledge. Therefore, we have summarized available knowledge on the determinants of survival 10\u00a0years or more after breast cancer diagnosis. We supported our analyses and considerations with data from the population-based, long-standing Eindhoven cancer registry in the Netherlands.Fig.\u00a01Proportion of breast cancer patients (3-year moving average) diagnosed between 1973 and 1993 who survived 10\u00a0years or longer in Southeastern Netherlands\nMethods\nWe initially searched PubMed, using the search MESH term for \u2018breast neoplasms\u2019 AND \u2018prognoses\u2019 AND \u2018long-term\u2019. Only papers published in English between 1995 and 2006 (September) which researched female adults (19+\u00a0years) were included. We retrieved 528 articles and studied the abstracts (sometimes also the methods section). We selected only articles that assess or show the results for those surviving 10\u00a0years or longer with cohorts having a mean\/median follow-up of 10\u00a0years or longer. If mean\/median follow-up time was not reported, we examined the proportion of patients who survived 10\u00a0years after diagnosis, and this ought to be larger than 50%. If, for a specific topic of interest, no relevant studies with a follow-up of at least 10\u00a0years were found (such as BRCA mutation or gene profiling, which have been studied only during the last decade), then studies with the longest available follow-up were chosen. Furthermore, the following inclusion criteria were used: overall and\/or BC-specific survival was reported; relative risk or hazard rate and statistical probability values were given; at least 250 BC patients included at the beginning of study. We also searched the reference lists collected by this search strategy and selected those that were relevant to both our study question and inclusion criteria. Reviews and books that gave general overviews were also included in the reference list.\nWe present data from the Eindhoven Cancer Registry (ECR) to illustrate the role of factors such as age, tumour size, lymph node involvement and time since diagnosis. Within the Netherlands, ECR is unique because it has collected follow-up data since 1970, including clinical aspects of cancer patients. This is a population-based cancer registry covering a population of almost 2.4\u00a0million people in 2004 [5]. Cumulative survival proportion was calculated using the Kaplan Meier method. Relative survival was calculated by comparing the survival of BC patients to the general population.\nThroughout the text the term long-term and\/or survival will frequently be mentioned; this corresponds to at least 10-year survival unless otherwise indicated.\nResults and discussions\nDeterminants of survival BC 10\u00a0years or longer\nPatient characteristics\nAge at diagnosis\nVery young women, i.e. younger than 30\/35\u00a0years [6, 7], exhibited a particularly poor survival as do those older than 70 (Fig.\u00a02) [8, 9]. Young BC patients were more likely to have a more negative clinical presentation, such as affected lymph nodes, negative for oestrogen receptors, and have large tumour with a high fraction of p53 nuclei and overexpression of c-erb-2 oncoprotein [6, 10, 11]. However, current adjuvant treatment seems to diminish the poor prognostic value of young age [6]; young women who did not receive adjuvant treatment had a significantly increased risk of dying; those diagnosed at 35\u201339\u00a0years and <35\u00a0years had a 1.4 and 2.2\u00a0higher risk of death, respectively, compared to those of 45\u201349\u00a0years [6]. Older patients exhibited higher mortality rates [12], probably because of less extensive treatment (either related to advanced age itself or the presence of serious concomitant diseases (comorbidity)) [13].Fig.\u00a02Relative survival of breast cancer patients (n: 13,279) diagnosed in 1990\u20132002 and followed until 2004, according to age at diagnosis in southeastern Netherlands\nComorbidity\nConcurrent health conditions (comorbidity) at the time of BC diagnosis have a significant impact on early [13] as well as long-term survival of BC patients [12]. The most prevalent conditions were cardiovascular disease (7%), previous cancer (7%) and diabetes mellitus (6%), all becoming more common with increasing age [13]. Compared to those without comorbidity whose 5-year relative survival was 87%, those with diabetes mellitus or cardiovascular disease represented 78% and 83% of the respective survival estimates [13]. Patients with severe comorbidity exhibited a 2.7\u20133.4 higher risk of death in 10\u00a0years compared to those without comorbidity [12, 14].\nPeriod of diagnosis\nAccess to care and treatment of BC has improved over time in most industrialized countries, which is reflected in the higher long-term survival of BC cases across all age groups and the tumour characteristics of those diagnosed more recently [15\u201318]. In Finland, relative survival 10\u00a0years after diagnosis among patients younger than 50\u00a0years increased from 49% for those diagnosed in 1953\u20131959 to 68% for the 1983\u20131989 cohort [15]. Furthermore, 60% of node-positive BC patients diagnosed in 1978\u20131979 in Italy survived 10\u00a0years or longer compared to the 50% probability 10-year survival for those diagnosed in 1968\u20131969 [17]. In addition, changes in BC diagnosis, e.g. screening[19, 20] and better staging [17], may partly be responsible for the observed increase in the proportion of survivors.\nTime after diagnosis\nThe longer a woman survives BC the more the prognosis improves, illustrated by conditional survival [16, 21]. Probably the subgroup of patients who survived longer had less aggressive tumours due to a different genetic make-up or better life-style. In Australia, 79% of women with localized BC survived 10\u00a0years after diagnosis, yet among those still alive 5\u00a0years after diagnosis 84% had a 10-year survival [16]. The respective values for regional vs. advanced BC were 53% and 68% [16]. Unlike other cancers, relative conditional survival remained stable below 100% after 12\u00a0years of survival and decreased again after about 19\u00a0years (Fig.\u00a03) [5]. This may be a consequence of late recurrences and metastases, second cancers or late side-effects of treatment [23].Fig.\u00a03Conditional 5-year relative survival (calculated using period analysis [22] of breast cancer patients diagnosed in southern Netherlands in 1985\u20132002 and followed until 2004, according to age. (Dashed line): diagnosed at 25\u201349\u00a0years, (solid line): diagnosed at 50\u201374\u00a0years\nSocioeconomic status (SES) and race\nA population-based study of BC patients diagnosed in 1968\u20131999 in France showed a diminishing role of SES on excess mortality among women with BC over these periods [24]. Long-term follow-up studies reported that women with BC from low social classes had a 20\u201350% poorer survival compared to patients from higher social classes [25, 26], although others contradicted this [27]. Low SES patients were more likely to be diagnosed at a later stage, had more aggressive tumour characteristics and might have received sub-optimal treatment. However, differences in these prognostic factors did not fully explain the variation in survival according to social class [25]. This is also the case when breast cancer survival is studied according to race\/ethnicity. Ten years after treatment 58% of African Americans were still alive compared to 66% of the white Americans. After adjusting for other prognostic factors, 41% excess mortality from all causes was still observed among African Americans compared to caucasians [28]. This suggests other residual factors such as lifestyle (higher body weight was observed among African Americans), comorbidity [14], genetics or variation in the delivery of treatment, which influence outcome beyond variation in tumour aggressiveness [29].\nTumour-related characteristics\nTumour size\nTumour size is one of the strongest prognostic indicators (Fig.\u00a04) [7, 30], even after 20\u00a0years of follow-up [8, 31]. A larger tumour has been related to more positive lymph nodes [32], thus their interaction further influences the survival from BC. Nonetheless, the independence of survival by node status is shown by the lower 10-year overall survival rate found for node-negative patients with a tumour of 2\u20135\u00a0cm compared to those with a tumour smaller than 1\u00a0cm, 66% vs. 79%, respectively [33].Fig.\u00a04Cumulative survival proportion of breast cancer patients diagnosed in southern Netherlands in 1970\u20131994 and followed until 2004, according to tumor size (based on pathological diagnosis). \u25a0 tumor size: <2\u00a0cm (n: 3263) \u2022 tumor size: 2\u20135\u00a0cm (n: 3420) \u25b4 tumor size: >5\u00a0cm (n: 474) x tumor size: involvement of skin (n: 1133) and unknown\/not applicable tumor size: 1410\nHistological type\nThe prognostic value of histological type can be grouped into four: excellent, good, poor and very poor prognosis [34]. BC with an excellent prognosis, such as invasive cribriform, tubular [35], tubulo-lobular and mucinous [36, 37] showed >80% survival at 10\u00a0years [9]. Tubular mixed, mixed ductal with special type, atypical medullary [38] and alveolar lobular carcinoma have a good prognosis with a 60\u201380% 10-year survival. Those with invasive papillary, classic lobular and medullary cancers have a worse prognosis. Finally, 10-year survival among those with ductal, solid lobular, mixed ductal and lobular carcinoma is below 50% [34]. In most populations infiltrating ductal carcinoma covers about 70% of all diagnoses [36, 39]. Inflammatory BC has a particularly poor prognosis: about 30% survived 10\u00a0years [40].\nHistological grade\nThe most widely used grading systems are Scarff-Bloom-Richardson classification, Fisher grading nuclear system and Nottingham Combined Histologic Grade (NCHG) [41]. The validity of grading has been subjected to inter-observer reproducibility and subjectivity [42]. However, higher grades have been quite consistently associated with lower long-term survival [7, 8, 31, 43\u201345]. Depending on other prognostic factors, such as nodal status or tumour size [46, 47], cumulative survival among patients with the lowest score was 90\u201394% 10\u00a0years after diagnosis and 30\u201378% among those with the highest score [37, 48].\nRegional lymph node involvement\nLymph node involvement is a valuable indicator of long-term survival (Fig.\u00a05) [8, 32]. Node- positive patients have about a 4\u20138 times higher mortality than those without nodal involvement [8, 9, 49]. The more nodes involved the worse the prognosis. Prognosis for patients with 10 or more involved axillary nodes showed 70% more deaths at 10\u00a0years than for those with 1\u20133 involved nodes [32]. The survival of node-positive patients improved due to better staging procedures and application of systemic treatment [7, 31, 50].Fig.\u00a05Cumulative survival proportion of breast cancer patients diagnosed in southern Netherlands in 1970\u20131994 and followed until 2004, according to nodal status (based on pathological diagnosis). \u25a0 node negative (n: 4452) \u2022 node status: 1\u20133 positive nodes (n: 3266) \u25b4 node status: 4\u20139 positive nodes (n: 255) x node status: 10+ positive nodes (n: 189), unknown\/not applicable node status: 1538\nLymphovascular invasion (LVI) and molecular markers of tumours angiogenesis\nAt the St. Gallen meeting in 2005, LVI was added to the prognostics for node-negative patients [51]. Compared to patients having no LVI, a 60% higher BC mortality was observed for node-negative BC patients having positive LVI [52, 53], although others did not observe the independent role of LVI [46, 50]. In this line of research, studies have also focused on the value of microvessel density [44], blood invasion (BVI) [54] and markers of angiogenesis (VEGFR (vascular endothelial growth factor receptor), CD105, Tie-2) [55, 56] in predicting long-term survival of BC patients, although the results are still conflicting.\nGrouped prognostic factors\nSome of the prognostic factors have been combined into a prognostic index, such as the TNM classification and also the more current Nottingham Prognostic Index (NPI), both highly predictive for estimating long-term survival [41]. TNM staging consists of information on primary tumour size, involvement of the regional lymph node and the presence of distant metastasis. Only 53% of patients with regional or locally advanced BC had survived 10\u00a0years after diagnosis compared to 79% of those with localised BC [16]. Patients with metastasis (stage: M1) at diagnosis exhibited very poor 10-year survival (3.4%) [57].Fig.\u00a06Cumulative survival of breast cancer patients diagnosed in southern Netherlands in 1970\u20131994 and followed-up until 2004, according to second cancer. Follow-up for patients with second cancer begins at the date of second cancer diagnosis. \u25a0 no second cancer (n: 8137) \u2022 second breast cancer (n: 744) \u25b4 second non-breast cancer (n: 819)\nTumour size, grade and lymph node status make up the NPI [11, 46, 49]. In a large series of 2879 BC patients, 10-year survival proportion was 85% for those with the lowest NPI score and 19% for those with the highest score [11].\nRecurrence, metastasis and second cancer\nPatients with recurrent, metastasized or second cancer generally exhibited lower long-term survival than those without [9, 21, 58\u201361]. Ten years after surgery, the probability for survival for another 10\u00a0years, thus 20\u00a0years after diagnosis, for node-negative patients aged \u226545\u00a0years, tumour \u22641\u00a0cm, grade 1 and without a recurrence or metastasis was 0.89. If a recurrence occurred, the probability of being alive at 20\u00a0years dropped to 0.72. If a metastasis was observed the probability of survival was only 0.18 [21]. The prognosis decreases with larger primary tumour size, nodal involvement [62], higher grade,[21] early recurrence (within 5\u00a0years of surgery)[63], location of recurrence (regional rather than local ipsilateral) [59] and inadequate primary cancer treatment [9, 64]. In the dataset of the ECR, overall survival was better for women without second primary tumours than for women who developed a new primary cancer (Fig.\u00a06). Only 68% of early BC patients with second malignancies had survived 10\u00a0years of follow-up compared to 78% of those without multiple cancers [65]. Younger BC patients are reported to have poorer survival and a higher risk of second cancer [59]. Corrected for race and grade, women in the 20-29\u00a0year old category who had a second BC had a probability of 10-year survival probability of only 23% compared to 57% for those without multiple cancers.\nOther tumour markers\nHormone receptors\nThe presence of hormone receptors such as oestrogen (ER) and progesterone (PR) receptors predicts the long-term outcome of hormonal therapy [66], thus they have been more commonly used as a predictive marker rather than as a prognostic marker. Thus given a particular treatment, e.g. tamoxifen, ER-positive patients have a considerably better prognosis than ER-negative patients. The prognostic value is weak [30, 43] or negligible [37], particularly in the early years after diagnosis [67].\nHER-2 expression\nNode-positive patients with BC cells showing amplification of the gene for human epidermal growth factor receptor type 2 (HER2), and\/or overexpression of its product had a lower 10-year overall survival proportion, 50% versus 65% for those without HER2 amplification [17, 68]. After 10\u00a0years the difference in survival persisted, although it became somewhat smaller[17]. Tumours that overexpress HER2 are more likely to contain p53 abnormalities, to be hormone receptor- and bcl-2-negative and to have lymphoid infiltration and a high mitotic index, all known to be markers of poor prognosis for BC [17, 69, 70]. As for patients with node- negative tumours, HER2 did not seem to affect long-term survival significantly [17, 37, 69]. HER-2 expression has been valuable in predicting treatment responses to trastuzumab, certain endocrine therapies and chemotherapy, adding to it\u2019s role as a predictive marker [68].\nMitotic Activity Index (MAI)\nMAI is an indicator of tumour proliferative activity that represents the mitotic activity in a given area of the tumour. Combined with another prognostic factor (NCHG), MAI has proven to be an accurate tool for assessment of long-term survival [48]. In a population-based study women with node-negative tumours <5\u00a0cm and a MAI \u226510 exhibited 80% survival at 10\u00a0years compared to 90% for an MAI <10 [71].\nGene expression profile\nA very promising new finding is the microarrays method, in which a set of intrinsic genes is clustered and segregated into major subgroups; BC with a good and poor prognosis profile is correlated to the probability of distant metastases [72] or a tumour with basal or luminal characteristics which are strongly associated with ER status [73]. In a study of 295 patients diagnosed with stage I or II breast cancer, those classified as having a good prognosis profile had a 95% overall 10-year survival rate compared to 55% for those with a poor profile [74]. This classification predicted outcome regardless of the nodal status, implying that more accurate criteria have become available for administering adjuvant systemic treatment.\nVarious molecular markers\nBRCA1 & 2 mutations were first identified in 1994 and are BC risk factors for some specific groups [75]. Their role as prognostic indicator for long-term (more than 10-year) survival has not yet been established. A study of 496 women (median follow-up: 116\u00a0months), 56 of whom (11%) carried a BRCA1\/BRCA2 mutation, showed worse BC-specific survival for women with BRCA1 mutations than for those without (62% at 10\u00a0years versus 86%; P\u00a0<\u00a00.0001), but not for women with the BRCA2 mutation [76]. However, another study which compared patients from BRCA1, BRCA2 and non-BRCA1\/2 families as well as sporadic cases did not confirm the prognostic role of BRCA1\/2 [77].\nLong-term follow-up studies have not demonstrated an independent effect of p53 mutations on long-term survival. The P53 mutation was related to a poor clinical profile for patients, hence in multivariate analysis its role on survival diminished [10, 69, 78, 79].\nA high level of tissue urokinase-type plasminogen activator (uPA) and its inhibitors has been correlated with poor outcome for node-negative and node-positive patients. Those having the highest level of uPA have a five times greater risk of dying from BC compared to those with the lowest level [69]. Other factors such as Ki67 (MIB-1), cathepsin-D, DNA ploidy and S-phase have been suggested as prognosticators of survival, with conflicting results, particularly among long-term survivors. Their use in general clinical settings is therefore not recommended [80, 81].\nMiscellaneous\nLifestyle\nGenerally, increased death rates due to BC (13\u201320%), other causes (49\u201386%) and all causes (14\u201370%) have been observed among obese patients [82\u201385]. Normal body weight tended be more beneficial in death from other causes than from BC: [83, 84] 9.5% of obese patients died from non-BC causes compared to 6.4% and 5.8%, respectively, of the normal or intermediate groups [82]. Obesity was also related to a 2-fold increased risk of postmenopausal contralateral BC and a 60% higher occurrence of second other cancers [84]. Therefore, normal weight may reduce the risk of second post-menopausal BC, second other cancers and overall mortality [83, 84, 86].\nCompared with women who engaged in less than 9 metabolic equivalent task (MET)-hours per week of activity, women who engaged in 9 or more MET-hours per week had a 40% lower risk of death from all causes, translating into a 6% absolute (unadjusted) reduction in mortality [87], which emphasizes the need to advise physical activity.\nSo far, although studies have not convincingly shown the positive influence of eating fruit, vegetables and soy bean on long-term BC survival [85, 88], diets high in fruits, vegetables, legumes, poultry, and fish and a low intake of red meat, desserts and high fat dairy products are likely to protect against mortality from non-BC causes [89].\nModification of BC\u2019s prognostic factors\nVarious studies have questioned the role of BC risk factors in determining the biological tumour features as mentioned above. Indeed, BC risk factors seem to differ according to histological type, grade, size, nodal status and ER\/PR receptor status [90\u201393]. For example, excessive alcohol intake and obesity increased the risk for the development of ER-positive tumours [92, 93]. As for late age at first full-term birth and obesity are related to an increased risk of large tumours [91]. Hence, risk factors for BC may also affect breast biology and clinical behaviour, thus also BC prognosis.\nChanging importance of prognostic factors over time after diagnosis\nCommonly, the value of prognostic factors decreases depending on the length of the follow-up period [31, 94]. Survival curves according to prognostic factors usually show a large drop in survival for all stages during the first 5\u00a0years; afterwards the curve stabilizes. Studies agreed on the long-lasting influence of tumour size at diagnosis on survival, albeit attenuating over time [31, 94, 95]. Grade, nodal status and metastases were also valuable in predicting survival up to 20\u00a0years after diagnosis [31, 95]. Although, others have reported that 10\u00a0years after diagnosis only tumour size [94] or nodal status [8] or old age [8] remained as an independent predictor of long-term survival. Similarly, ER\/PR status and MAI only had a significant prognostic role in the first 5\u201310\u00a0years after diagnosis [67, 71, 96]. Because even 10\u00a0years after BC diagnosis the probability of survival for BC patients does not seem to reach that of the general population, the role of other prognostic factors in determining survival for long-term survivors still needs to be determined.\nThe role of early detection\nIncreased awareness among women and improvement in diagnostic procedures have enabled earlier and better detection of BC. Trials on population screening have reported 21\u201329% reduction in BC mortality for women invited for screening within 14\u201316\u00a0years of follow-up [19, 97]. Screening identified tumours at an early stage consequently, survival improved [98, 99]. Screening also identified patients with slowly growing tumours who might receive unnecessarily aggressive cancer treatment. Thus, Joensuu et\u00a0al. [100] examined recurrence rates among patients detected by screening compared to those detected outside screening. After adjusting for tumour aggressiveness (tumour size, nodal status, grade, age, treatment, PR status, HER-2), hence eliminating bias towards detection of indolent cancers (length bias), the benefit of screening for the prognosis for BC patients remained evident.[100] This suggests that other factors explain the indolent behaviour of BC detected by screening. Hence, until this factor is established, detection mode should probably be considered as a prognostic factor and thus be taken into account in patient management.\nThe role of treatment\nImprovement in BC treatment has undoubtedly also increased the long-term survival of BC patients [101], as reflected by the improved overall survival across all BC stages [16]. Using historical data from population-based studies in periods when effective treatment was not available, it was estimated that without treatment only 4% of BC patients would survive 10\u00a0years or longer [102]. BC treatment guidelines have been modified continuously in the last 28\u00a0years, tailored to most of the prognosticators mentioned earlier [51]. Effectiveness of various treatment modalities has been summarized by others who conclude that radiation, chemotherapy and hormonal therapy may reduce long-term mortality by up to 57% [66, 103\u2013105]. Emerging new therapeutic approaches using a monoclonal antibody directed against HER-2 have yielded improved short-term survival for advanced stage [106] as well as operable BC patients [107]. Quality of treatment as indicated by loco-regional failure [108], surgeon workload [109] or hospital volume [110], may affect survival although its role on long-term survival still needs confirmation. In conclusion, on the one hand we have observed a shift in stage towards less aggressive cancers; on the other hand, better and more (systemic) treatment has become available, leading to improved survival for BC patients.\nConclusion\nThe prognosis of BC has become relatively good, with current 10-year relative survival about 70% in most western populations [16, 111], especially if up-to-date statistical method such as the period analyses is used [111] (Table\u00a01). Even better, the longer patients survive their BC the higher their survival chance [16]. Our review shows conventional prognostic factors of survival, such as tumour size, lymph node status and grade, remain the most important determinants of 10-year survival for BC patients (Table\u00a02). Most studies agreed on the value of MAI and LVI for prediction of long-term survival. The influence of host factors including age, race\/ethnicity or socio-economic factors and tumour-related factors such as histological type and angiogenesis diminishes after correction for other factors. For most recent markers such as Her2, gene profiling, p53 mutation and uPA level longer follow-up is needed. Recurrence, metastases and a second cancer double the burden of disease thus increase risk of mortality. Similarly, co-occurrence with other diseases is in no doubt decrease survival.Table\u00a01Overview of studies reporting long-term prognostic factors for breast cancer (BC) patientsNo.Author, yearNo. of patientsMean\/median follow-up (yrs)Univariate (UV) analysis significantMultivariate (UV) analysis significantNot significantRemarks1Haerslev and Jacobsen 1995 [70]a49010.6MS, T, N, Htyp, MI, G, PR, Her2All patients: N & PR In N+: MI & PR In N-: MS & GHer2Overall survival was measured. P53 was related to absence of tubular formation, high G, ER-negative, high PCNA (proliferating cell nuclear antigen) score2Pietilainen 1995 [10]39211.1P53, N, T, Htyp, tubular formation, intraductal growth, margin formation, necrosis, DNA ploidy, S-phase fractionAll patients: N, T, MI In N+: T, MI In N\u2212: T, p53, GOverall survival was measured. P53 is related to younger age, MI, AI, G, nuclear pleomorphism3Haerslev 1995 [79]a49011PCNAT, G, PRHer2 & PCNA (Proliferating cell nuclear antigen)Overall survival was measured. PR was only an independent factor in N-positive pts. Her2 & PCNA were related to more positive N, higher G, ER-\/PR-negative4Gamel 1996 [39]163,808NR. Range 1mos-19\u00a0yrsHistological type by stage (localized & regional BC)Breast cancer specific survival was measured5West 1996 [12]1196NR. Diagnosed: 1973\u20131986. End FU: 1994.ComorbidityLevel of comorbidityAdjusted for age, race, stage, N, therapy. Values for these factors were not shownOverall survival was measured. Charlson comorbidity index was used. There is no difference in the significance of comorbidity on survival of Caucasians and African American (AA)6Haerslev 1996 [112]a487>10Ki-67, PCNAT, G, PRKi-67 & PCNA.Overall survival was measured. PR only an independent factor for N+ patients. Ki-67 was related to T, MI, G7Northridge 1997 [36]Mucinous BC: 4082. Infiltrating duct BC: 139,154NR. Diagnosed: 1973\u20131990HTyp, period of diagnosis, Stage, GAgeBreast cancer specific survival was measured8Kollias 1997 [11]a2879 age\u00a0\u226470, T <5cm>10Age <35, NPIT, G, NAgeOverall survival was measured. Younger than 35\u00a0yrs had higher grade, more LVI and worse NPI group. After 10yrs NPI did not change OS9Zahl and Tretli 1997 [95]8802 age <70Diagnosed: 1965\u201374. End FU: 1991Survival categorized by age, stage and follow-up timeExcess hazard from breast cancer was measured. After 8\u00a0yrs being younger than 35\u00a0does not influence survival. Stage was an important prognosticator up to 20\u00a0yrs10Pinder 1998 [47]46512N by grade, Treatment by gradeOverall survival was measured. The study aimed to confirm value of Nottingham grading system for survival. N+G3 patients benefited from prolonged chemotherapy11Gaffney 1998 [113]BRCA1: 30BRCA2: 20 Control: 18278 BC ptsBRCA1: 9.8 BRCA2: 7.5 Control: NRBRCA1 vs. BRCA2 vs. controlOverall survival was measured Case and control were matched for date of birth, date of diagnosis and tumour size. Patients with BRCA+ were younger. Patients with BRCA1 had higher grade12Wojcik 1998 [28]6577 patients Whites: 5879 African American (AA): 698At 10\u00a0yrs 59\u201367% patients were aliveRace, G, N, T, stage, waiting time, smoking, being a widow, having other family as dependentRace, age, stageUV: alcohol, family historyOverall survival was measured. AA is more likely to be younger at diagnosis, have larger tumour, higher stage and more lymph nodes13Mansi 1999 [114]35012.5Bone marrow micrometastasesN, TBone marrow micrometastases, LVIOverall and breast cancer-specific survival was measured. Bone marrow metastases may be useful as prognostic factor for BC pts without information on T and N14Kollias 1999 [46]a319 T \u22641cm>10G, N, LVI, NPIG, NLVIOverall survival was measured15Tabar 1999 [45]2468NR. Diagnosed: 1977\u201385. End FU: 1996T, N, G, detection mode, HTypTXN, age*N, Htyp*N, T*N*GOverall survival was measured. Screening arrests disease progression. Tumour progression is more rapid in BC patients <50\u00a0yrs. OS of T1a(1\u20135\u00a0mm) vs. T1b(6\u201310\u00a0mm) NS.16Holmes 1999 [85]198213.1BMI \u2265303rd  to 5th quintile of protein protein intake after diagnosis, N, T, GBMI, protein intake prior diagnosis, alcohol intakeAll cause mortality was measured. MV for BMI was corrected for age, diet interval, oral contraceptive use, hormone replacement therapy, MS, age at menarche, aget at birth and parity, smoking, T, G, N, ER, PR BMI <21 and 1st quintile of protein intake were reference. Significant trend of higher mortality from lowest to highest quintiles of fibre, lutein and zeaxanthin, calcium and protein intake, with 13\u201335% lowest mortality in the lowest quintile17Nomura 1999 [58]1857 <80\u00a0yrs stage I-III12Second cancer and recurrenceAge, ER, N, recurrence, second cancerOverall survival was measuredRecurrence is related to higher stage, younger age at diagnosis, Htyp, and therapy. Second cancer is related to younger age. Death related to recurrence and second cancer is increased 12 yrs after diagnosis.18Reed 1999 [37]613 T1-2N015.5Age >50, T, GG, T, treatmentUV: treatment, ER, PR, Her2, P53Overall survival was measured. Her2 was related to PR-, ER-negative, P53, G. P53 was related to PR-. Treatment was ovarian & locoregional irradiation that had lower mortality rate19Aebi 2000 [7]3700 pre- & perimenopausal12Age <35\u00a0vs. \u226535N, T, G, age <35*ER+Age, EROverall survival was measured. Younger patients with ER+ who were not amenorrhoea had a significantly shorter survival20Ferrero 2000 [78]297 N-11T, ER, P53T, ERAge, PR, GBreast cancer-specific survival was measured. P53 was related to grade, T, ER-negative. P53 was continuous variable21Kroman 2000 [6]10,356 age <50NR. Diagnosed: 1978\u201396.Age, T, N, GPeriod of treatment and surgeryRelative survival was measured for excess mortality due to BC. When chemotherapy was given BC at young age does have worse prognosis22Ferrero-Pous 2000 [69]48810ER, uPA, G, N, PR, P53 by Her2All patients: uPA, N, T, Her2, age In N\u2212: uPA, T In N+: N: uPA, T, age, Her2Overall survival was measured. For patients who received chemotherapy uPA, T & N determined OS. For patients who received hormonal therapy uPA, Her2 & N determined OS23Kato 2001 [44]a37710T, N, GAMC, T, N, GNecrosisOverall survival was measured. AMC is a good prognostic factor for N- and T2-3 patients24Liu 2001 [96]79116.3T, N, G, ER, Her2, p53, MIB-1, MAI, AIAll patients: N, T, G, ER, Her-2 In N\u2212: G In N+: N, age, ER, Her2UV: age. MV All patients: AI, MI, MIB-1, ER, G MV in N- & N+: AI, MI, ER, G.Breast cancer-specific mortality was measured. When patient FU was truncated at 5\u00a0yrs, MI was prognostic factor for N+ and N-25Page 2001 [115]311 no adjuvant therapy.11.6High risk group (ER- or T\u22653cm) vs. low risk (ER+ and T <2\u00a0cm)T, risk group (high vs. low)G, MIOverall survival was measured. MI was only significant when FU was truncated at 5\u00a0yrs. Grade was significant prognostic factor for short- and long-term survival26Frkovic-Grazio and Bracko 2001 [48]270 T1N0M012.5G, Tubular score, MITubular score and MIBreast cancer-specific survival was measured. This study confirmed the use of Nottingham grading system in their cohort27D\u2019Eredita 2001 [49]402\u226516T, N, Htyp, G, LVI, NPIT, N, GUV: Age, MS, ER, type of surgery MV: LVI & HtypOverall survival was measured. NPI gives similar survival prognosis as T, N, G28Thomson 2001 [26]23786At 10\u00a0yrs about 50% patients were aliveAge stratified by SESIntermediate vs. high SES group corrected for age, ER, N, T, stageDeprived vs. high SES group corrected for age, ER, N, T, stageDeprived women have more ER- tumours. ER distribution and treatment method accounted for 20% of disparities in survival29Vorgias 2001 [30]269 stage II12NRT, N, age, ER\/PRMS, therapyOverall survival was measured30Vincent-Salomon 2001 [43]685 T\u22643\u00a0cm10.8G, N, ER, necrosisN, necrosis, GUV: Vascular density, LVI, age, PROverall survival was measured. Intratumoral vascular density was related to larger tumour size and higher grade31Eerola 2001 [77]Familial BC: 359Sporadic BC: 59517NR Diagnosed: 1953\u20131995 End FU: 1997Stage, age, period of BC diagnosis, FU time (after 2 and 3\u00a0yrs of diagnosis)BRCA1, BRCA25-year relative survival was measured for excess mortality due to BC32Kitchen 2001 [35]952012Tubular BC type vs. other type, by nodal status and chemotherapyOverall survival was measured. Tubular BC type had better prognosis than other type. This type was more likely to have low G & ER+33Kato 2002 [50]a42210P53, MI, necrosis, T, N, LVIMI, T, NUV: AIOverall survival was measuredIn MV P53 & MI were independent prognostic factors for N-patients only. P53 was related to MI, AI, necrosis, G, T, N, ER\/PR34Kato 2002 [54]a39810BVI, T, N, G, chemotherapyBVI, T, N, G, chemotherapyUV: necrosisOverall survival was measured35Costa 2002 [67]67011.4N, T, age, ER\/PRN, T, ageMS, ER\/PRBreast cancer-specific survival was measured. After 5\u00a0yrs of FU ER and PR were not independent prognostic factors36Menard 2002 [17]1928Diagnosed in 1968\u201369 and 1978\u201379Her2, N, T, MS, lymphoid infiltration, PR-G, T, N, lymphoid infiltrationOverall survival was measured. HER-2 was related to large tumours, higher G, lymphoid infiltration, higher mitotic index, PR-37Van de Vijver 2002 [74]295 age <53, stage I-II6.7Gene profile (Good vs. bad prognosis) for all patients, N+, N\u2212Gene profile, T, N, chemotherapyVI, G, age, hormonal therapyOverall survival was measured38Van\u2019t Veer 2002 [72]117 age <55NRBetter classification of patients with high risk of metastasis and in need of chemotherapy39Hatteville 2002 [21]318015.8OS <5\u00a0year: N, G, recurrence or metastasis OS\u22655\u00a0yr: G and recurrence or metastasisAge, TIf patient remains without recurrence or metastasis, effect of prognostic factors decreases over time. With metastases, this effect increases40Sotiriou 2003 [73]996.1Gene profile (luminal 1\u20133 vs. basal 1\u20132 & Her2 type)Luminal-like 1\u20133 was predominantly ER+. Basal-like 1\u20132 and Her2 was predominantly ER-41Dignam 2003 [83]3385 N\u2212, ER+13.8BMI <18.5 and BMI \u226530 higher total mortality and other deaths.BMI on deaths after BC events.Total mortality, death after BC events and other deaths as well as recurrence rate and occurrence of a second cancer was were measured. MV was adjusted for treatment, age, MS, race, T, ER and PR. Reference group was BMI 18.5\u201324.9.42Olivotto 2003 [57]620 stage IIIB-M1>20Supraclavicular BC, Stage IIIB and M1Overall and breast cancer specific survival were measured. Patients with supraclavicular metastases had significantly better survival than patients with M1. Survival of these patients resembles that of BC stage IIB. (FU for living patients 20 yrs, for all patients 4.5\u00a0yrs)43Weiss 2003 [32]905 N+ Chemotherapy+22.6N+ (N1\u20133 vs. N4\u20139 vs. N >10), also by treatment and follow-up timeN, T, MSMV: NXT, MSXT, additional vincristine and prednisonOverall survival was measured. N was related to T. MS was related to receptor status44Taylor 2003 [16]54,228At 10\u00a0yrs 65% patients were alivePeriod of diagnosis, stage by age, FU time by stageRelative survival was measured for excess mortality due to BCThe longer the survival the better the prognosis. Improvement in relative survival for all patients and all stages since 197245Dales 2004 [56]905 aged 25\u20138111.7In N- : CD105+ vessels. In all pts: CD31, Tie-2\/TekIn all pts: G, CD105 vessels, ER In N\u2212: G, CD105 vessels, PRIn all pts: T, Htyp, CD31, PR, age. In N\u2212: T, CD31 vessels, ER, ageOverall survival was measured. MV: Tie-2\/Tek showed significant role for predicting OS in all patients and N- patients46Brenner and Hakulinen 2004 [15]18,578 age <50NR. Diagnosed: 1953\u20131999.Period of diagnosis, stage, stage*period, time after diagnosisImprovement of prognosis for BC patients younger than 50 over the past decades. Relative survival remains lowered even 40 yrs after diagnosis47Robson 2004 [76]584 Ashkenazi Jewish116BRCA1, T, N, ER, age, chemotherapyBRCA1, T, N, AgeTamoksifen, BRCA2Breast cancer-specific survival was measured. No effect of BRCA on non-BC death. BRCA1 only predicted BC death in patients without chemotherapy48Chia 2004 [33]1187 LVI-, N-, Adjuvant systemic therapy-10.4T, GTXGOverall and breast cancer specific survival were measured. Patients with higher grade and size have greater chance to die from other & those with low risk disease greater chance of death from BC49Yoshimoto 2004 [18]15,416NR. Diagnosed 1946\u20132001.Period of diagnosisOver the decades, there were less extensive surgery and lymph node examination, less radiotherapy, more chemo- and hormonal therapy50Houterman 2004 [116]527 age \u2265404.7Comorbidity, N, Therapy, age\u226570, comorbidity*NIn age <70: comorbidity, N In age \u226570: comorbidity, ageIn age <70: therapy In pts age\u226570: N, therapyRelative survival was measured for excess mortality due to BC Older patients with comorbidity were not treated differently but had a worse prognosis51Schoppmann 2004 [53]37422.4LVI, G, N, TherapyLVI, G, NLMVD (Lymphatic Microvessel Density), T, Htyp, ER, age, MSOverall survival was measured. LVI is related to young premenopausal BC, lower G, N+52Warwick 2004 [31]2299>10G, N, T, MetastasesG, N, T, MetastasesBreast cancer specific survival was measured. All studied factors predicted long-term survival, but their value decreased over time53Berclaz 2004 [82]679214BMI 25\u201329, BMI \u226530 lower overall survivalBMI \u226530BMI 25\u201329Overall survival and also disease free survival were measured. Reference group was BMI \u2264 24.9. MV adjusted for ER, T, N, MS, treatment, chemotherapy, hormonal- in combination with chemotherapy54Dignam 2005 [84]4077 N-, ER-NRBMI \u226535 and AA had higher overall mortality and non- BC deathBMI and race on death after BC eventsTotal mortality, death after BC events and other deaths as well as recurrence rate and occurrence of a second cancer were measured. MV was adjusted for treatment, age, MS, race, T, ER and PR Reference group was BMI \u226424.955Holmes 2005 [87]298796\u00a0monthsPhysical activity after diagnosis (MET \u22659) on BC and total mortalityBreast cancer and total mortality were measured. MV corrected for age, interval between diagnosis and physical activity assessment, smoking, BMI, MS, hormone therapy use, age at first birth, parity, energy intake, stage and treatment. MET: metabolic equivalent task hours per week. Patients with BMI \u226525 and more physical activity before diagnosis there was a significant trend for less breast cancer death56Robsahm and Tretli 2005 [27]5042NR. Diagnosed: 1964\u201392. End FU: 1992NRLocation of home, age at first child, physical activity at workMV corrected for: age, period of diagnosis, birth cohort, educational levelBreast cancer-specific survival was measured. Incidence of BC increases with higher educational level, and case fatality decreases by increasing education level57Vu-Nishino 2005 [38]1490 received breast- conserving treatment13.9Medullary BC vs. other BC typeOverall survival was measured. Medullary BC type had better prognosis than other type. This type was more likely to have ER+, PR+ & less BRCA1\/2 mutation. Medullary type was only a prognostic factor for the first 5\u00a0yrs58Galper 2005 [63]2102 stage I-II, 314 with local recurrence (LR)13.1NRNo LR treatment, Invasive LR, time (yrs) to local recurrence, age at initial BC diagnosisT, detection method, number of nodes sampled, ER\/PR, histological type, G, LVI, marginsMeasure of survival: distant failure, second malignancy, or death Patients with a longer time to recurrence have prolonged survival59Voogd 2005 [62]266 BC with LR11.2 after LR for living ptsNRLocation of LR, size of LR, skin involvement of LR, N+ for primary tumourOverall survival was measured. Early detection of local recurrence may improve the treatment outcome60Louwman 2005 [13]8966Diagnosed 1995\u20132001. End FU: 20042 or more comorbidities, diabetes mellitus and previous cancerPrevious cancer, CVD, DM, cerebrovascular disease, dementia, 2 or more comorbidities, stage, treatment (RT, ST, age)Overall as well as relative survival was measured for excess mortality due to BC Primary treatment of BC patients with serious comorbidity was less extensive than treatment of those without comorbidity61Tammemagi 2005 [14]90610Number of severe comorbidities, race, type of comorbidityAll patients: 3 or more comorbidities adjusted for stage, age, ER, surgery, chemotherapy, radiotherapyOverall survival was measured. AA had more diabetes and hypertension. After adjustment for these 2 comorbidities disparity disappeared62Meunier-Carpentier 2005 [55]909\/918 age: 25\u20138111.3Tie2\u2013UV: VEGFR-2, VEGFR-2Overall survival was measured. VEGFR-1 and Tie2 were reported as independent prognostic factors corrected for T, G, Htyp, in all patients and N-63Tai 2005 [40]6184 Inflammatory BCNR. Diagnosed 1973\u20131995. End FU: 2000Period of diagnosisBreast cancer-specific survival was measured. Prognosis has improved over the decades due to more aggressive therapy64Louwman 2005 [71]492 T1\u20132 N0>10\u00a0yrsMAIOS: age, T BCS: MAIOS: HTyp, therapy, period of diagnosis. BCS: therapy, period of diagnosis, age, T, HtypOverall (OS) as well as relative survival (BCS) was measured for excess mortality due to BC Higher MAI was a significant prognostic factor for N\u2212 and N+, but only during the first 10\u00a0yrs of FU65Arrigada 2006 [8]2410 T \u22647\u00a0cm N1\u2013219T, skin fixation, muscle fixation, G, N, ageTotal FU: T, N, G, age <35, age\u226555 FU 0\u20135\u00a0yrs: T, N, G, age \u226555 FU 5\u201310\u00a0yrs: N, G, age <35, age\u00a0\u2265\u00a055 FU 10\u201315\u00a0yrs: N >10, age >55. FU 15\u201320yrs: age\u226565Overall survival was measured. Long-term effect of prognostic factors vanishing66Newman 2006 [29]90,124. White American: 76,111. AA: 14,013Age, stage, SESMeta-analysis. African American is an independent predictor of poor outcome for overall survival and breast cancer specific mortality67Menvielle 2006 [24]407,435 women followed for BC death (N:1408)Women who died of BC in 1968\u201396Level of education by period of diagnosisBreast cancer death among women with the highest education compared to women with the lowest education in 1968\u201374 was 0.43; and in 1990\u201396: 1.17 (NS)68Bouchardy 2006 [25]3920 age <70NR. Diagnosed in 1980\u20132000SESSES corrected for age, period of diagnosis, marital status, country of birth, Htyp, ER, detection method, stage, sector of care, therapyOverall survival was measured. Lowest SES had less frequently screen-detected cancers, less stage I, less lobular BC, less BCT, less lymph node dissection69Siegelmann-Danieli 2006 [117]992, age \u2265706.9Being in wheelchair, renal insufficiency, dementia, CHF, cardiac arrhythmia, DM, IHD, osteoporosis, PVD, cerebrovascular disease, COPD, Parkinson\u2019s disease, valvular heart diseaseIn stage 1A-2A: age, CHF, DM, PVD, stage, cardiac arrhythmia, Parkinson\u2019s disease, renal insufficiency In stage 2B-4: G, stage, N, wheelchair-bound, renal insufficiency, COPD, age, DMSystemic therapyOverall survival was measured. CHF: Cardiac Heart Failure. DM: Diabetes Mellitus. IHD: Ischemic Heart Disease. PVD: Peripheral Heart Disease. COPD: Chronic Obstructive Pulmonary Disease. Role of comorbidity varies by age70Pritchard 2006 [68]639 premenopausal N+10Her2 amplificationHer2 corrected for age, N, ER, type of surgeryOverall survival was measured. Those with amplified Her2 have improved survival with CEF71Lee 2006 [52](A) Adjuvant therapy \u2212 : 990. (B) Adjuvant treatment +: 1765Group A: 13 Group B: 6.8.LVIGroup A: T, G, LVI, HtypGroup B: T, G, LVI, chemotherapy, hormonal therapyB: ER, age, HtypBreast cancer-specific survival was measuredFor patients without adjuvant treatment, role of G in survival was higher in the first 5\u00a0yrs. Role of Htyp was not significant for the first 5\u00a0yrs of FUaindicates the overlapping patients used by the same author to answer another research question; yrs: years; UV: Univariate analysis. MV: Multivariate analysis. MS: Menopausal Status; T: Tumour size; N: Nodal involvement; Htyp: Histological type; MI: Mitotic Index; G: Grade; PR: Progesterone Receptor status; ER: Oestrogen Receptor status; PCNA: proliferating cell nuclear antigen; mos: months; NR: Not Reported; AA: African American; age: is in year and indicate age at primary breast cancer unless otherwise state; NPI: Nottingham Prognostic Index; LVI: Lymphovascular Invasion; (Prognostic factor)*(Prognostic factor): interaction between two factors; BMI: Body Mass Index; AMC: Average Microvessel Count; MAI: Mitotic Activity Index; AI: Apoptosis Index; FU: Follow-up; SES: Socioeconomic Status; BVI: Blood vessel Invasion; LMVD: Lymphatic Microvessel Density; LR: local recurrence; CVD: Cardiovascular Disease; DM: Diabetes Mellitus; RT: radiotherapy; ST: Systemic therapy; VEGFR: Vascular Endothelial Growth Factor Receptor; OS: Overall survival; BCS: Breast Cancer Specific Survival; NS: not significant; CHF: Cardiac Heart Failure; IHD: Ischemic Heart Disease. PVD: Peripheral Heart Disease. COPD: Chronic Obstructive Pulmonary. CEF: cyclophosphamide, epirubicin and fluorouracilTable\u00a02Selected prognostic factors for long-term overall mortality of breast cancer (BC) patientsPatient groups basedHazard ratio (HR) for overall follow-up or survival probability (S) 10\u00a0years after diagnosisMorphology basedHazard ratio (HR) for overall follow-up or survival probability (S) 10\u00a0years after diagnosisMolecular basedHazard ratio (HR) for overall follow-up or survival probability (S) 10\u00a0years after diagnosisAge at diagnosis [8]HR:Lymph node status [31]HR:HER2[69]HR:\u2003 \u00a0\u00a0\u00a0\u00a0\u00a0<35\u00a0vs. 35\u2013441.4 (P: 0.07)\u2003 N\u22651 vs. N02.4 (1.9\u20132.9)\u2003 >500 vs. \u22645001.82 (1.1\u20132.9)\u2003\u00a0\u00a0\u00a0\u00a0\u00a045\u201354 vs. 35\u2013441.1 (ns)Metastases vs. N022.73 (16.1\u201332.2)Only in node-positive\u2003 \u00a0\u00a0\u00a0\u00a0\u00a055\u201364 vs. 35\u2013442.0 (P: 0.000)\u2003\u00a0\u00a0\u00a0\u00a0\u00a065\u201375 vs. 35\u2013442.5 (P: 0.000)Period of diagnosis [16]Relative survivalb:Tumour size (mm)[31]HR:Cell proliferation indexHR:\u2003\u00a0\u00a0\u00a0\u00a0\u00a01972\u2013197659%\u2003 T10\u201314 vs. T1\u201391.2 (0.8\u20131.9)(MAI)\u2003\u00a0\u00a0\u00a0\u00a0\u00a01977\u2013198664%\u2003 T15\u201319 vs. T1\u201391.7 (1.1\u20132.6)\u2003 >10 vs. \u226410 [10]1.02 (1.00\u20131.03)\u2003\u00a0\u00a0\u00a0\u00a0\u00a01987\u2013199170%\u2003 T20\u201329 vs. T1\u201392.5 (1.6\u20133.9)Only in node-positive patients\u2003 T30\u201349 vs. T1\u201393.8 (2.4\u20136.0)\u2003 T\u226550 vs. T1\u201394.6 (2.9\u20137.6)Time after diagnosis [16]Relative survival:Tumour grade [44]HR:Gene expression profile [74]S:\u2003\u00a0\u00a0\u00a00 vs. 5 yrs after diagnosis\u2003 II vs. I2.5 (1.0\u20136.1)55% vs. 95%\u2003\u2003\u00a0\u00a0\u00a0\u00a0\u00a0Regional BC79% vs. 84%\u2003 III vs. I5.7(2.6\u201312.4)\u2003 Poor vs. good signaturef\u2003\u2003\u00a0\u00a0\u00a0\u00a0\u00a0Locally advanced BC53% vs. 68%Socioeconomic status [26]HR:Tumour type[34]S:ER\/PR status [30]HR:\u2003\u2003\u00a0\u00a0\u00a0\u00a0\u00a0Intermediate vs. affluent1.2 (1.0\u20131.4)\u2003 Poor vs. excellent d,e<50% vs. >80%\u2003 Positive vs. negative0.38 (0.02\u20131.06)\u2003\u2003\u00a0\u00a0\u00a0\u00a0\u00a0Deprived vs. affluent1.2 (0.99\u20131.53)LifestyleHR:\u2003\u2003\u00a0\u00a0\u00a0\u00a0\u00a0Body Mass Index\u2003\u2003\u2003\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0<21 vs. 29+ kg\/m21.4 (0.97\u20132.00) [85] c\u2003\u2003\u00a0\u00a0\u00a0\u00a0\u00a0Physical activity\u2003\u2003\u2003\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0<\u00a03 vs. 23.9 MET-h\/wka0.56 (0.4\u20130.8) [87]HR: Hazard ratio calculated within multivariate analysis of breast cancer patients followed for a median\/mean of 10\u00a0years or longer a Metabolic equivalent task hours per week; b Estimates taken from graph; c Higher alcohol intake no significant effect on mortality. Significant trend of higher mortality for lowest compared to highest quintiles of fibre, lutein and zeaxanthin, calcium & protein intake; d Becomes larger as numbers of involved lymph nodes increases [8]; e Excellent prognosis: tubular, invasive cribriform, mucinous, tubulolobular. Poor prognosis: mixed lobular, solid lobular, ductal and mixed ductal lobular; f unadjusted estimates\nHealthier lifestyle generally increases long-term survival. Modifiable risk factors (such as alcohol consumption and obesity) not only affect incidence but also tumour\u2019 clinical behaviour and thus survival.\nAlthough a lot is known about the prognosis for BC patients, effect of traditional prognostic factors appears to attenuate over time, leaving room for studies on the role of other and newer factors for long-term survival.","keyphrases":["prognostic factors","long-term","survival","breast cancer"],"prmu":["P","P","P","P"]}
{"id":"FEBS_Lett-1-5-1942070","title":"Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NF\u03baB activity in vertebrate B-cells\n","text":"To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569\u20131577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NF\u03baB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology.\n1\nIntroduction\nThe protein kinase D (PKD) serine\/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLC\u03b3 and the subsequent production of diacylglycerol (DAG) which stimulates classical and\/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3\u20136]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8\u201312].\nPKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13\u201316], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NF\u03baB transcription factor and in regulating cell survival during oxidative stress [17,21\u201323]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26\u201328].\nTo investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NF\u03baB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems.\n2\nMaterials and methods\n2.1\nCell culture, transient transfections and cell stimulation\nThe generation, culture and activation of PKD1\u2212\/\u2212, PKD3\u2212\/\u2212 and PKD1\/3\u2212\/\u2212 knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29].\n2.2\nsIgM staining\nDT40 B cells (2\u00a0\u00d7\u00a0106 cells per point) were resuspended in 200\u00a0\u03bcl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20\u00a0min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry.\nAll results shown are representative of at two to four independent experiments unless otherwise indicated.\n3\nResults\n3.1\nLoss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases\nDT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1]. In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3\u2212\/\u2212 cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter. Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1\/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A).\nPreviously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1\/3\u2212\/\u2212 B cells and can restored upon re-expression of a single PKD isoform [1]. The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence. We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3. As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells. BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B). However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C). Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C). In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D). Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases.\n3.2\nCellular proliferation and survival in DT40 B cells lacking expression of PKD family kinases\nPKD enzymes have previously been linked to the regulation of cell proliferation and survival (reviewed in [20]). To investigate the effect that loss of PKD kinases had on B cell survival and\/or proliferation we cultured wild-type and PKD-null cells in the presence (PKD1\/3\u2212\/\u2212: Flag-PKD3+ve) or absence (PKD1\/3\u2212\/\u2212) of doxycycline and monitored exponential growth. As shown in Fig. 2A, PKD1\/3\u2212\/\u2212 cells proliferated exponentially and re-expression of Flag-PKD3 in these cells had no impact on the rate of proliferation. Furthermore, the viability of PKD1\/3\u2212\/\u2212 B cells during routine culturing was not significantly different from that of wild-type B cells (data not shown). It was noted that the population doubling time of PKD1\/3\u2212\/\u2212 cells was slightly slower than that of wild type DT40 cells (12.7\u00a0\u00b1\u00a02.8\u00a0h versus 10.2\u00a0\u00b1\u00a00.4\u00a0h) but the failure of PKD3 re-expression to modify the proliferation rate of PKD1\/3\u2212\/\u2212 cells suggests that these small differences were most likely the result of clonal variation and were not caused specifically by loss of PKD enzymes. Thus, PKD family enzymes are not essential for regulating basal survival and proliferation of DT40 B cells.\nPKD enzymes, specifically PKD1 and PKD2, have previously been linked to a protective role against oxidative stress-induced injury in 3T3 fibroblast, HeLa and epithelial cell lines [17,30\u201332]. We therefore addressed the role of PKD family kinases in regulating B cell survival in response to oxidative stress and other stress stimuli. As shown in Fig. 2B, loss of PKD1\/3 expression had no significant impact on the survival of DT40 B cells in response to mitochondrial stress stimuli (H2O2 or serum deprivation); DNA damaging agents (etoposide or doxorubicin); ER pathway stress due to calcium overload (thapsigargin) or following prolonged treatment with phorbol esters or Trichostatin A, an inhibitor of class I\/II HDACs. Thus, PKD kinases do not play an essential role in regulating B cell survival in response to a range of different stress stimuli.\n3.3\nAntigen receptor regulated signalling pathways in PKD-null DT40 B cells\nTo further explore the contribution of PKD kinases to DT40 B cell biology we investigated whether specific BCR-regulated signalling events were defective in the PKD-null B cells. Initial experiments revealed that surface expression of the BCR was reduced in PKD1\/3\u2212\/\u2212 (and in PKD1\/3\u2212\/\u2212:Flag-PKD3+ve) cells compared to wild-type DT40 B cells (Fig. 3A and data not shown). Nevertheless, BCR-crosslinking of PKD1\/3\u2212\/\u2212 cells was sufficient to induce the activation of a number of signalling cascades, similar to that observed in wild-type cells (Fig. 3B). Hence, BCR-induced activation of the Akt, mTOR\/p70 S6 kinase (as shown by S6 ribosomal protein phosphorylation) and MAPK signalling pathways was clearly detectable in PKD1\/3-null B cells (Fig. 3B). Furthermore, enhanced tyrosine phosphorylation of multiple cellular proteins as well as an increase in intracellular calcium levels was also observed following BCR stimulation of PKD1\/3-null B cells (data not shown). We did observe that the strength of BCR (but not phorbol ester)-induced regulation of the Erk1-RSK1 signalling pathway was reduced in PKD1\/3\u2212\/\u2212 B cells compared to wild-type B cells (Fig. 3B). One interpretation of this data is that PKD enzymes may modulate Erk activation. Indeed, PKD enzymes have previously been linked to the growth factor-regulated Erk signalling in fibroblast and endothelial cell lines [33\u201335]. However, BCR-induced Erk phosphorylation was also reduced in PKD1\/3\u2212\/\u2212-Flag-PKD3+ B cells (data not shown) suggesting that reduced BCR levels on the surface of PKD1\/3\u2212\/\u2212 (and PKD1\/3\u2212\/\u2212-Flag-PKD3+) B cells may itself impact on the strength of activation of this specific intracellular signalling pathway.\nTo search for other potential PKD targets that may show defective regulation in PKD1\/3\u2212\/\u2212 DT40 B cells, we used a PKD substrate phospho-antibody that recognises consensus phosphorylation sequences targeted by PKD enzymes (LxRxxpS\/T) [36]. As shown in Fig. 3C, phorbol ester- and BCR-induced phosphorylation of cellular substrates detected by this phospho-antibody was similar in wild-type and PKD1\/3\u2212\/\u2212 cells and is therefore independent of PKD enzymes. However, pretreatment of both wild-type and PKD1\/3\u2212\/\u2212 DT40 B cells with GF109203X, a bisindoylmaleimide derivative that inhibits PKCs prevented the induction of proteins that contain phosphorylated LxRxxS\/T motifs. Thus loss of PKD1\/3 enzymes does not globally disrupt the phosphorylation of cellular proteins that contain LxRxxpS\/T motifs. This result is perhaps not surprising as LxRxxS\/T motifs also act as good substrates for other serine\/threonine kinases such as MAPKAPK2. However these experiments do provide further evidence that phosphospecific antisera are not sufficiently selective to be designated kinase specific substrate antisera.\nBCR-induced signalling pathways culminate in the activation of gene transcription events that control B cell survival, proliferation and function. In this context, it has been proposed that PKD family members control of gene transcription through activation of the NF\u03baB transcription factor. Thus, PKD-mediated activation of NF\u03baB occurs downstream of a variety of different signals, including mROS\/oxidative stress, lysophosphatidic acid and the Bcr-Abl oncogene [17,21,23,30,37]. Furthermore, expression of an activated PKD1 mutant enhances HPK1-mediated NF\u03baB activation [38]. In B cells, NF\u03baB is known to be regulated via DAG and PKC\u03b2 [39,40] but whether PKDs are key intermediaries for NF\u03baB regulation has not been explored.\nThe data (Fig. 4A) show that NF\u03baB transcriptional activity was strongly induced in both wild-type and PKD1\/3\u2212\/\u2212 DT40 B cells in response to either phorbol ester or BCR stimulation. In contrast, BCR and phorbol ester-induced NF\u03baB transcriptional activity was abolished in PKC\u03b2\u2212\/\u2212 DT40 B cells (Fig. 4A), although strong activation of PKD kinases (as assessed by autophosphorylation of PKD1 at S916) was observed in the PKC\u03b2\u2212\/\u2212 cells (Fig. 4B). Thus, PKD kinases are neither essential nor sufficient to mediate BCR-induced NF\u03baB activation in DT40 B cells and hence do not participate in DAG\/PKC mediated control of NF\u03baB.\n4\nDiscussion\nProtein kinase D serine kinases have been proposed to regulate diverse cellular functions including the phosphorylation and nuclear localisation of class II HDACs and the phosphorylation of HSP27. It has also been suggested that PKDs act as mitochondrial sensors for oxidative stress and play a role in regulating NF\u03baB transcription factors [41]. Most of the data about the function of PKDs has come from experiments that ectopically express active or inhibitory PKD mutants or that use RNAi to reduce PKD expression. We have used gene targeting to specifically delete PKD alleles in DT40 chicken B cells and can thus use PKD-null DT40 cells to assess the relative contribution of individual PKD isoforms in class II HDAC control versus oxidative stress responses and NF\u03baB regulation in lymphocytes. We have previously used these PKD-null DT40 cells to define an essential role for PKDs in regulation of class II HDACs, the present report now describes an indispensable role for PKDs in regulating the phosphorylation of HSP27 on serine 82, a site previously identified as a target for the p38-MAPKAPK2 signalling cascade [42]. However, studies of PKD-null DT40 cells reveal that PKD family kinases are not essential for oxidative stress survival responses nor are they required for activation of NF\u03baB transcription factors. These latter findings are in striking contrast to previous observations in HeLa and epithelial cell lines where overexpression\/RNAi approaches have implicated PKD1\/2 in the control of proliferation, survival and NF\u03baB activation [20,23]. Hence, the present report shows that the proposed roles for PKDs as key sensors that modulate survival pathways in response to oxidative stress and regulate cell survival and proliferation are not ubiquitous and may be restricted to certain cell lineages. Taken together, these data indicate that loss of expression of PKD family members does not globally impact on early BCR-regulated signalling pathways.","keyphrases":["protein kinase d","proliferation","survival","nf\u03bab","pkd","hsp27","pkd, protein kinase d","pkc, protein kinase c","bcr, b cell antigen receptor"],"prmu":["P","P","P","P","P","P","R","M","R"]}
{"id":"Abdom_Imaging-4-1-2386533","title":"Detection of inflammatory bowel disease: diagnostic performance of cross-sectional imaging modalities\n","text":"Different cross-sectional imaging techniques can be used as a diagnostic tool for the evaluation of inflammatory bowel disease (IBD). In this report the diagnostic performances of ultrasonography, magnetic resonance imaging and computed tomography in the detection of IBD and the evaluation of known IBD are described, together with a short update on patient preparation and imaging technique of the respective modalities discussed.\nIntroduction\nChronic inflammatory bowel disease (IBD) consists of two main subtypes, i.e., Crohn\u2019s disease (CD) and ulcerative colitis (UC). During the last decades, the incidence of CD has continued to increase worldwide, reaching incidence rates ranging from 3.1 to 14.6\/100,000 in North America and from 0.7 to 9.8\/100,000 in Europe [1]. Incidence rates of UC differ greatly between studies and regions, varying from 1.5 to 24.5 per 100,000 person-years [1, 2].\nCrohn\u2019s disease can be localized in any part of the gastrointestinal tract, although the location of predilection is the terminal ileum, involvement of the terminal ileum is observed in 90% of the patients with small-intestinal CD, who in turn constitute 30\u201340% of all CD patients. In 40\u201355% of the patients both ileum and colon are affected, while in a minority (15\u201325%) only a colonic localization is observed.\nThe earliest change caused by CD occurs in the mucosa and submucosa and consists of hyperemia and edema. Tiny aphthoid or superficial ulcerations can be seen when disease progresses. In more severe disease, the disease extends transmurally with sometimes serosal involvement. In this stage, mucosal ulcerations merge to form deep longitudinal and transverse ulcerations while bowel wall thickening and narrowing of the bowel lumen can be observed due to significant mucosal edema and associated bowel spasms. In long-standing disease, chronic obstruction can develop due to scarring, luminal narrowing, and stricture formation. Extramural manifestations of CD are fistulas, abscesses, adhesions, creeping fat, and enlargement of lymph nodes.\nUlcerative colitis exclusively affects the colon with a predictable way of spreading from distal to proximal in a continuous manner; the rectum is often involved, but rectal sparing can be observed. In previous cases, small superficial erosions can be seen, whereas in more severe disease these ulcerations can be quite large. However, only in very severe disease they penetrate the muscularis layer. The mucosa is thickened because of round-cell infiltration in the lamina propria. In chronic UC, a marked hypertrophy of muscularis mucosae can be seen, causing contraction, shortening, and narrowing of the involved colon. The submucosa becomes thickened because of the deposition of fat or, in acute or subacute cases, edema [3, 4].\nDiagnostic modalities\nThe gold standard examination for the small bowel traditionally has been small bowel barium examination (SBE), either by using an enteroclysis technique or by using small-bowel follow-through [5, 6]. SBE is invasive and burdensome, and requires an extensive bowel preparation (dietary restrictions, use of laxatives). Moreover, in the young population of CD patients, the ionizing radiation required for SBE limits the use of this technique for follow-up of disease.\nThe advent of video capsule endoscopy (VCE) and double-balloon endoscopy (DBE) has increased the diagnostic possibilities. For VCE a capsule is swallowed after a fasting period of up to 12\u00a0h and is propelled through the bowel by peristalsis. Thus, the mucosal surface of the small bowel can be depicted in detail (Fig.\u00a01). However, with VCE there is no facility to increase visibility by insufflation of air or by tissue rinsing. Moreover, tissue sampling and therapeutic interventions are not possible. The use of VCE is contraindicated in patients with (suspicion of) obstruction due to the risk of capsule retention.\nFig.\u00a01.VCE image of a 14-year-old male patient with known CD. VCE was performed as small-bowel disease was suspected. Image shows severe inflammation of the small bowel with a stenosis.\nFor DBE, a high-resolution video-endoscope with a flexible overtube is used. By alternately inflating and deflating two balloons attached to the overtube and endoscope the small bowel is threaded on the overtube. Both an oral and an anal approach are possible; for the oral approach no specific preparation is required, although patients are asked to fast for at least 6\u00a0h before the procedure. If the anal approach is used, bowel cleansing such as is employed for traditional colonoscopy is used. By using both the anal and oral approach, DBE allows visualization of the entire small bowel, with the possibility of obtaining tissue for analysis and the added advantage of the possibility of endoscopic therapy (e.g. dilatation of a stricture, cauterization of a bleeding site). For DBE conscious sedation is a necessity.\nTraditionally, ileocolonoscopy (CS) with tissue sampling is considered to be the most valuable tool for diagnosis and follow-up of disease in the colon and terminal ileum [5, 7]. As UC solely affects the mucosa of the colon, CS would suffice for diagnosis of disease and evaluation of disease activity and extent. However, when strictures are present as a complication of disease, these might hamper execution of a complete examination, while in severe attacks of UC CS is relatively contra-indicated due to the increased risk of bleeding or perforation. For ileocolonic localizations of CD CS would suffice as well, although inspection of the terminal ileum is reported to fail in up to 27.8% of examinations [8, 9].\nCross-sectional imaging modalities\nThe trans- and extramural extent of IBD cannot be visualized with any of the abovementioned techniques. Much research has been directed toward the potential of cross-sectional imaging modalities for the diagnosis and evaluation of IBD as with these techniques the bowel lumen, the bowel wall and the extra-intestinal abdomen including the visceral fat, the lymph nodes and the vascular structures feeding and draining the bowel can be visualized. An added advantage of these techniques is the fact that they are limitedly to non-invasive.\nUltrasonography (US), computed tomography (CT) and magnetic resonance imaging (MRI) are often used for the evaluation of the abdomen. While in the USA the technique of choice is CT, in Europe the focus is more on MRI and US. This inclination is reflected by the majority of CT studies on IBD patients originating from the USA, while the majority of published studies on MRI and US has been conducted in Europe.\nUltrasonography\nPatient preparation and US technique\nPatients are usually asked to fast for several hours before the scan to diminish peristaltic movements and the amount of intraluminal air; in the available literature the minimum fasting time described is 4\u00a0h, whereas overnight fasting is also sometimes employed. Usually, no additional dietary or cleansing measures are taken. Due to the limited patient preparation necessary and the non-invasive nature of this examination, US can be considered to be a relatively patient-friendly and straightforward examination.\nUltrasonography is mostly performed without the use of enteral contrast medium. In two recent studies, the effect of enteral contrast medium for diagnosis of IBD of the small bowel was investigated; higher sensitivity values were obtained when enteral contrast medium had been administered [10, 11]. In both studies the additional value of enteral contrast medium permitted detection of jejunal lesions that had escaped detection with conventional US. For colonic IBD, use of enteral contrast medium also increased accuracy [12, 13].\nHigh-frequency transducers are preferred, such as 7.5\u00a0MHz [13\u201316].\nThe use of Doppler-US might provide helpful additional information on IBD, particularly on the degree of disease activity. Using Doppler-US the vascularity of the bowel wall can be assessed according to the intensity of color signals and\/or by the analysis of Doppler curves (measurement of resistive index) obtained from vessels detected within the bowel wall. Measurement of flow parameters of the superior and inferior mesenteric arteries can also be performed.\nImaging criteria\nThe criterion that is most extensively used for the diagnosis of CD is bowel wall thickening. In most studies, the bowel wall is considered to be thickened when thickness exceeds 3\u00a0mm (Fig.\u00a02). In a meta-analysis conducted by Fraquelli et al. [17] concerning US in CD the respective diagnostic accuracies of different cut-off values were compared; sensitivity for diagnosis of CD decreased using a cut-off value of 4\u00a0mm instead of 3\u00a0mm (from 88% to 75%), but specificity increased slightly (from 93% to 97%). Other features that are considered characteristic of CD on US are the presence of a stiff bowel wall, modification or disappearance of bowel wall stratification, presence of deep ulcers (seen as interruption of the submucosal hyperechoic rim by a hypoechoic tract), a reduction of peristalsis and loss of haustration in the colon. Extramural findings are fibrofatty proliferation, enlarged lymph nodes, and\/or the presence of an abscess or fistula.\nFig.\u00a02.A 14-year female patient with known CD and intermittent abdominal pain. US image shows the thickened wall of the terminal ileum (arrowheads) with some infiltration of the perivisceral fat.\nBowel wall thickening is considered to be a characteristic feature of UC as well. Mural stratification is preserved in most UC patients due to the superficial pattern of inflammation. This feature can be used to differentiate between CD and UC, although this was not regarded sufficient for differentiation in all studies on this topic [12, 13]. In long-standing UC a tubular appearance of the colon and loss of haustration can be seen.\nDiagnostic accuracy of US\nMost studies regarding diagnostic accuracy of US for diagnosis and follow-up of IBD have been conducted in the last decade. Although reported sensitivity and specificity values are high, with the state-of-the-art equipment diagnostic accuracy could possibly be higher than that previously reported.\nIn the hands of an experienced radiologist, US can be very accurate for the detection of IBD. Reported sensitivity values of US for the detection of IBD in patients with suspected disease vary from 76% to 92% [15, 18, 19]; specificity values are also high. In patients with proven IBD, reported sensitivity values for US are higher, probably reflecting a higher index of suspicion [10, 20]. Reported segmental sensitivity values are lower; these are below 78%, even if gray-scale US is combined with power Doppler [21, 22]. Regarding the detection of extramural complications, fistulas and abscesses can be identified accurately on US [23, 24] (Fig.\u00a03).\nFig.\u00a03.A 25-year-old female patient with known CD of the terminal ileum. A US image shows a large abscess (arrowheads) that was located ventrally and cranially of the bladder. B US image shows a fistula (arrowheads) that originated from the abscess.\nAlthough many US studies have been carried out, most concerned either CD patients or both CD and UC patients, and reported accuracy values are usually applicable for both subgroups of disease. Although in the study by Limberg and Osswald [13] separate accuracy values are provided for US and CD, it is not clear from these data if accuracy values were obtained from post hoc separation of data. To our knowledge, as of yet no prospective study has been performed with predetermined imaging parameters to differentiate between CD and UC.\nAlthough US can be used for the assessment of both small bowel and colon, disease localized in the duodenum and jejunum is often missed [18, 19, 25]. Moreover, the rectum and distal sigmoid cannot be visualized accurately due to their pelvic location. This makes US less suitable for the assessment of UC.\nDoppler US has been proved useful in assessing whether IBD is in an active phase or in remission; significant correlations were found between Doppler parameters and disease activity, both in UC [26] and in CD [27]. However, the only distinction made was between active and inactive disease, meaning no conclusions can be drawn about the severity of active disease from these data. As of yet, there is no standardized scale to determine the degree of disease activity on US, neither for CD nor for UC.\nThe spatial resolution of US is not high enough to permit the detection of superficial pathology, making this modality less suitable for the diagnosis of early diseases when compared with SBE. Although SBE can reportedly be highly accurate when performed by skilled radiologists [28, 29], compared with VCE or DBE its sensitivity is low [30]. In this regard, comparison between US and VCE and\/or DBE might be very interesting in order to determine the accuracy of US for small lesions and accuracy for bowel segments that are difficult to access. To our knowledge, no comparative studies have been performed as of yet.\nComputed tomography\nPatient preparation\nPatients are usually asked to fast for several hours before the scan to diminish peristaltic movements [31\u201333]. In addition, in some institutions a bowel-cleansing regimen is applied, as a rule consisting of mild laxatives. Dietary restrictions are also often applicable. Although with this bowel preparation residual feces are usually present to some degree, the mural presentation of disease enables the identification of disease even if the bowel wall is partly obscured.\nThere is consensus as to the indispensability of enteral contrast medium for an abdominal CT examination for IBD. The contrast medium of choice should be neutral (meaning an attenuation value comparable with water), as a neutral contrast medium allows optimal distinction between bowel wall and lumen. While in some institutions enteral contrast medium is administered orally (CT enterography), in other institutions controlled distention is achieved by inflow of contrast medium through a nasojejunal catheter (CT-enteroclysis). Although by some authors CT-enteroclysis is propagated as the controlled infusion provides a more consistent distention of the small bowel than CT-enterography, especially of the jejunum [34], others report that with the right choice of contrast medium and correct timing of intake excellent distention of all small bowel loops can be obtained after the oral administration of contrast medium [31, 33]. In only one small study, CT enteroclysis and CT enterography were compared, but both the degree of luminal distention and the diagnostic accuracy did not show significant differences [35].\nImaging technique\nTechnical developments have allowed the widespread use of multi-slice scanners. With these scanners volumes can be scanned in a very short breath hold, allowing the acquisition of isotropic voxels for multiplanar reformatting. Thin slices should be used to permit the detection of subtle pathology.\nBefore CT enterography sometimes metoclopramide is given to increase gastric emptying and peristaltic movements of the small bowel [31]. The use of antiperistaltic drugs is not standard. In a recently published update on CT enteroclysis, Rajesh and Maglinte [32] report that in their institution all patients undergoing CT enteroclysis receive conscious sedation. Although this most probably decreases patient discomfort to a large degree, this can considerably increase the in-hospital time for patients as they will have to stay in a recovery unit after the procedure until the anesthetic effects have worn off.\nIntravenous contrast medium should be administered for a comprehensive CT examination of the bowel. In a recent study, the optimal timing of scanning after the administration of intravenous contrast medium was determined; Schindera et al. [36] found that peak mural enhancement of normal small-bowel wall occurs on average about 50\u00a0s after contrast administration or 14\u00a0s after peak aortic enhancement.\nImaging criteria\nThe main feature considered indicative of both CD and UC is bowel wall thickening. Whereas in earlier studies cut-off values for pathological bowel wall thickening have varied between 2.5\u00a0mm\u00a0and 10\u00a0mm, in recent publications of experts in the field bowel wall thickness exceeding 3\u00a0mm is described as pathological [31, 33] (Fig.\u00a04). Mural thickening is not seen in early superficial disease, precluding CT from being a first-line examination for (suspicion of) superficial disease.\nFig.\u00a04.A 60-year old female patient who underwent CT enterography for suspected bowel obstruction. Just 1\u00a0month earlier at ileocolonoscopy CD of the terminal ileum was discovered; the terminal ileum was not intubated because of the stenosis. Axial image shows the severely thickened bowel wall of the ileum (arrowheads) with only a pinpoint bowel lumen remaining.\nIncreased bowel wall enhancement after the administration of intravenous contrast medium is also considered as an indicative of active disease [37\u201339]. Whereas in earlier studies the only distinction regarding enhancement was between pathological enhancement (i.e., hyper-enhancement) and normally enhancing bowel walls, in a recent publication the degree of mural enhancement was found to correlate with disease activity [38].\nBowel wall enhancement can be transmural, but also layered. This layered enhancement pattern, which is represented by a thickened intestinal wall with a middle layer of low attenuation surrounded on each side by layers of higher attenuation, has been termed the target sign; this is due to the presence of edema or the deposition of fat in the submucosa.\nDiagnostic accuracy of CT\nThe accuracy of CT has mainly been investigated for small-bowel disease. In suspected CD sensitivity was 83% when compared with SBE [40]. When compared with ileoscopy sensitivity values vary from 80% to 88% [34, 35, 41]. Segmental sensitivity of CT was somewhat lower (71.8%) in a study by Molnar et al. [42], comparing CT with SBE and CS.\nSuperficial lesions (such as aphthoid lesions) are not accurately visualized on CT, making CT less suitable as a first-line examination for the suspicion of mild disease. This was already evident from studies comparing CT with CS and\/or SBE, but in a recent meta-analysis comparing CT with VCE was shown that the yield of CT compared with the yield of CE was 30% vs. 69% [43]. No comparative studies have been published regarding CT vs. DBE.\nExtramural complications are well shown on CT, although the lower contrast resolution of CT makes this modality less suitable for the detection of fistulas and abscesses than MRI in patients with CD [44] (Fig.\u00a05). In a recent study, the relationship between increased attenuation of perivisceral fat and disease activity was determined; one of the most specific markers of active disease was increased attenuation in the perivisceral fat [37].\nFig.\u00a05.A 25-year-old female patient with known CD of the terminal ileum (same patient as pictured in Fig.\u00a03). A CT-scan was performed to determine involvement of the small bowel. A Coronal image shows the abscess (arrowheads). B Axial image again shows the abscess again (arrowheads). C Coronal image shows the fistula (arrowhead).\nHardly any studies have focused on the accuracy of CT colonography for the detection of ileocolonic IBD. This is possibly partly due to the fact that for the rectal administration of contrast medium the rectum is obscured by the rectal catheter, precluding diagnosis of rectal IBD, specifically UC. It does seem clear that CTC is unable to detect ulcerative lesions; even diffuse inflammation with large ulcerations can be missed. CTC might however be useful in patients with colonic stenosis or narrowing [45, 46].\nMagnetic resonance imaging\nPatient preparation\nWhile in some studies on MRI a period of several hours of fasting was deemed sufficient, in others full bowel cleansing was performed, as the reference standard, (i.e., CS) was performed on the same day. There is no consensus yet as to what constitutes the optimal bowel preparation for MRI. However, as a limited bowel preparation does not seem to negatively affect accuracy, it might be sufficient to limit the bowel preparation to a fasting period taking into account the patient-friendliness of the respective preparations.\nLuminal distention by means of use of enteral contrast medium is indispensable for an adequate evaluation of the bowel as collapsed bowel can hide or mimic disease. As was the case with CT, for MRI of the small bowel contrast medium is either administrated by mouth or by enteroclysis. An advantage of MR enteroclysis over MR enterography is the fact that it allows fluoroscopic monitoring of the inflow of contrast medium and thereby provides functional information on bowel distensibility. An advantage of MR enterography is the fact that it can be considered more patient-friendly and also that no ionizing radiation is necessary. To our knowledge, only one study has been carried out in which both methods of contrast medium administration were compared. In this study by Schreyer et al. [47] all patients (n\u00a0=\u00a021) underwent both MRI enterography and MRI enteroclysis; no difference in accuracy compared with SBE was noted by the investigators (Fig.\u00a06).\nFig.\u00a06.A A 38-year-old female with complaints of vomiting and an iron-deficiency anemia who was suspected of CD and underwent MRI-enteroclysis to evaluate the small bowel. Coronal TrueFISP image shows good distention of jejunal bowel loops after controlled infusion of contrast medium. B A 12-year-old male patient with known CD who underwent MR enterography for the evaluation of the small bowel. Coronal TrueFISP image shows good distention of jejunal bowel loops after oral administration of contrast medium.\nWhereas for MR enteroclysis mostly a methylcellulose suspension is used, for MR enterography many different contrast media have been tested. The main subdivision is between positive, negative, and biphasic contrast media. A biphasic contrast medium performs best for the identification of pathology on both T2-and T1-weighted sequences as adequate delineation between hypointense bowel wall and hyperintense lumen is seen on T2-weighted sequences while on T1-weighted images the enhancing bowel wall can be easily discriminated from the hypointense lumen. An artificial sugar-solution (mannitol or sorbitol) has been shown to cause good distention of small bowel loops with negligible side effects [48, 49].\nImaging technique\nMostly, both T2-weighted and T1-weighted sequences are used for the MRI evaluation of the bowel. On T2-weighted images the bowel wall can be appreciated and bowel wall stratification\u2014if present\u2014can be well apprehended. As feces can show bright signal intensity on T1-weighted sequences, it is important to perform a pre-contrast T1-weighted sequence in order to be able to determine whether high signal intensity was already present before intravenous contrast administration, indicating the presence of stool.\nAnother sequence that is propagated by many authors is the TrueFISp sequence, a sequence that is insensitive to motion and breathing artifacts. This sequence, that makes use of a T2\/T1 ratio, adequately delineates the bowel wall and the mesentery, allowing adequate assessment of disease (Fig.\u00a07). When combining a T2-weighted sequence or TrueFISP sequence and a T1-weighted sequence, a comprehensive MRI examination can be carried out in less than 30\u00a0min.\nFig.\u00a07.A 18-year-old female patient with known CD. MRI-enterography was performed for suspicion of active CD of the neoterminal ileum. A Coronal TrueFISp image shows enlarged mesenteric lymph nodes (arrowheads). B Coronal TrueFISP image shows thickened bowel wall of the neoterminal ileum (arrowheads).\nImaging criteria\nA bowel wall thickness exceeding 3\u00a0mm should be considered as an indicative of disease. Besides bowel wall thickening the most important criterion indicative of active IBD is pathological bowel wall enhancement after the administration of intravenous gadolinium. Bowel wall enhancement can always be seen as the bowel is a highly vascularized structure. However, in active IBD increased enhancement can be observed, due to the increased vascularization and the increased capillary leakage of the affected tissue (Fig.\u00a08). In CD it has been hypothesized that the degree of enhancement correlates with the degree of disease severity, but this statement has not been extensively corroborated [50\u201353].\nFig.\u00a08.A 12-year-old male patient with known CD who underwent MR-enterography for the evaluation of the small bowel (same patient depicted in Fig.\u00a06b). Axial T1-weighted image shows pathological enhancement of thickened small-bowel loops after administration of intravenous contrast medium (arrowheads). Approximately, 1\u00a0m of small bowel (terminal jejunum, proximal ileum) was shown to be affected.\nBowel wall stratification can be observed on T2-weighted images as a bright line within the two dark stripes of the mucosal and muscularis propria layers, likely related to the presence of fat or edema in the submucosal layer. On fat-suppressed T2-weighted images it is possible to determine the nature of the bright signal as a persistent bright signal suggests the presence of edema, whereas complete suppression of the submucosal signal suggests fat infiltration and quiescent disease [54].\nExtramural manifestations of disease that can be identified on MRI are fistulas, abscesses, fibrofatty proliferation, and enlarged lymph nodes.\nDiagnostic accuracy\nThe accuracy of MRI of the small bowel has been extensively investigated. In many European institutions, conventional enteroclysis is increasingly being replaced by MRI enteroclysis or MRI enterography as MRI has proved to be highly accurate in both the detection of disease in patients with known IBD as in patients in whom IBD of the small bowel was suspected [52, 55, 56]. However, the studies that have been performed were mostly small and concerned selected populations with either a high suspicion of disease or known CD of the small bowel. Larger studies including the full spectrum of disease activity should be conducted.\nAs was the case with CT and US, MRI is not suitable for the detection of superficial disease due to the limited spatial resolution. This finding is corroborated by a study comparing MRI and VCE in patients with CD [57]. Significantly more inflammatory lesions were detected with VCE in the jejunum and partly in the ileum of patients with CD. However, these findings had no effect on the therapeutic approach of the individual patients. The accuracy of MRI has not been compared with DBE as of yet.\nAs mentioned before, MRI can be used for the evaluation of extramural disease. Due to the high contrast resolution abscesses are very conspicuous on T1-weighed fat suppressed images after the administration of intravenous Gadolinium. MRI is also very sensitive for the detection of fistulas [47, 52, 58] (Fig.\u00a09).\nFig.\u00a09.A 25-year-old female patient with known CD of the terminal ileum (same patient as pictured in Figs.\u00a03 and 5). A Coronal T1-weighted image clearly shows the abscess (arrowheads) that was also depicted on US and CT. B Coronal T1-weighted image showing a fistula\nIn recent years, the accuracy of MRI for the detection of ileocolonic IBD has been investigated by means of MRI colonography. After administration of rectal contrast medium the colon (and sometimes the terminal ileum) was assessed for disease. Conflicting results were reported: while in one study high accuracy values were reported [59], in others segmental sensitivity values were around 32% [60, 61].\nRegarding the accuracy of MRI in differentiation between CD and UC conflicting results have been reported; while some authors report that based on the location of inflammatory changes, the degree of involvement, the continuity or discontinuity of disease, and the presence of complications it was possible to differentiate between CD and UC [51, 62], others report a limited value in differentiation of disease [63].\nTheoretically, a whole-bowel examination would be possible with MRI, by the administration of contrast medium orally and rectally. This has been attempted [64, 65] and was deemed feasible. More research is needed to establish the diagnostic value of this combined approach. At the moment, MRI colonography does not seem to be able to replace CS.\nDiscussion\nCompared with conventional imaging methods, CT, US, and MRI are accurate methods for the detection of IBD of the small bowel, both in patients suspected of disease as in patients with known IBD. Although subtle lesions cannot be depicted with any of these modalities, clinically more relevant findings can be accurately depicted. Therefore, cross-sectional imaging should be incorporated in a comprehensive clinical evaluation of suspected IBD and for follow-up of CD. The exact role cross-sectional imaging techniques can play for follow-up in UC should be more extensively studied.\nAs US is easily accessible, widely available, and inexpensive, it is recommended to use abdominal US as first-line modality in patients with suspected IBD of the small bowel. MR enterography would be a good alternative, especially as the assessment of the degree of disease activity can be better performed on MRI than on US. Although CT enterography is a very accurate technique and is used in many institutions, its role in IBD is limited by the ionizing radiation needed, especially due to the repetitive use for follow-up in often young individuals. If possible, it might be advisable to reserve this technique for patients in whom imaging is needed at very short notice as CT enterography can be performed very fast and is readily available.\nAlthough VCE has shown to be more accurate in depicting subtle lesions in the small bowel than MRI or CT, its role should be limited as of yet as the true benefit of VCE is not clear yet. As there are presently no standardized criteria for the diagnosis of CD with VCE, definitions with regard to what constitutes a positive finding might differ between studies. Moreover, the clinical significance of finding a single mucosal break or a few superficial aphthous lesions is not clear yet. Also, specificity and positive predictive values for VCE have not been established. At this time, it might be good to reserve VCE as a second-line modality if cross-sectional imaging has not shown abnormalities but the suspicion of disease remains standing despite these negative findings.","keyphrases":["inflammatory bowel disease","diagnostic performance","ultrasonography","magnetic resonance imaging","computed tomography"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_J_Epidemiol-4-1-2190782","title":"Short- and long-term mortality after acute myocardial infarction: comparison of patients with and without diabetes mellitus\n","text":"Aims To compare short- and long-term mortality after a first acute myocardial infarction (AMI) in patients with and without diabetes mellitus. Methods and results A nationwide cohort of 2,018 diabetic and 19,547 nondiabetic patients with a first hospitalized AMI in 1995 was identified through linkage of the national hospital discharge register and the population register. Follow-up for mortality lasted until the end of 2000. At 28 days and 5 years respectively, absolute mortality risks were 18 and 53% in diabetic men, 12 and 31% in nondiabetic men, 22 and 58% in diabetic women, and 19 and 42% in nondiabetic women. Crude mortality was significantly higher in diabetic patients than in nondiabetic patients in both men (28-day hazard ratio (HR) 1.55; 95% confidence interval (CI) 1.32\u20131.81, 5-year HR 2.01; 95% CI 1.84\u20132.21) and women (28-day HR 1.19; 95% CI 1.03\u20131.37, 5-year HR 1.53; 95% CI 1.40\u20131.67). After multivariate adjustment, risk differences became nonsignificant at 28 days, but diabetes was still associated with a significantly higher long-term mortality in both men (28-day HR 1.16; 95% CI 0.99\u20131.36, 5-year HR 1.49; 95% CI 1.36\u20131.64) and women (28-day HR 1.12; 95% CI 0.97\u20131.28, 5-year HR 1.39; 95% CI 1.27\u20131.52). The interaction between diabetes mellitus and gender did not reach significance in the analyses. Conclusion Our findings in an unselected cohort covering a complete nation show a significantly higher long-term mortality after a first acute myocardial infarction in diabetic patients. Yet, short-term mortality is not significantly higher in diabetic patients. Risks appear to be equally elevated in men and women.\nIntroduction\nIt is well established that patients with diabetes mellitus (DM) have a higher short-term mortality after acute myocardial infarction (AMI) than patients without DM. Several recent studies have found that hospital, 28-day and 1-year mortality after AMI was approximately 1.5\u20132\u00a0times higher in diabetic patients [1\u20133]. As it is known that most patients survive beyond this period, it is important to study the difference in long-term survival after an AMI between diabetic and nondiabetic patients as well. Survival after an AMI may be particularly worsened beyond the poorer prognosis already conferred by the presence of DM itself, as diabetic patients with an AMI may experience more severe coronary heart disease and more complications (left ventricular dysfunction and heart failure, significant ventricular arrhythmias) than nondiabetic patients with an AMI [2, 3]. There are relatively few recent studies that address differences in long-term prognosis after an AMI between diabetic and nondiabetic patients with follow-up lasting through the second half of the 1990s [4\u20138]. Moreover, data largely originate from clinical trials or single centers [4, 5, 7]. These studies reported that DM is independently associated with long-term mortality [4\u20138].\nThe aim of the present study was to compare short- and long-term mortality after a first hospitalized AMI in patients with and without DM encompassing the entire country by using data from linked national registers.\nMethods\nDesign\nThe study methods, including enrollment of the study population, have recently been described in detail [9]. In brief, cases of first hospitalized AMI in the Netherlands in 1995 were identified by linkage of the Dutch national hospital discharge register and the Dutch population register. Thus, the study included a total of 21,565 patients hospitalized for a first AMI in 1995. These patients had not been previously hospitalized for AMI from 1991 to 1995. Information about demographic characteristics, medical history and hospital outcomes was obtained from the hospital register and the population register. All general and academic hospitals and most specialty hospitals participate in the hospital register. For each hospital admission a new record is created in the hospital register, including the following information: date of birth, gender, numeric part of postal code, hospital-specific patient identification code, type of hospital, admission date and principal and secondary diagnoses of the admission. The principal diagnosis is determined at discharge and is in retrospect the main reason for admission. The diagnoses are coded using the ninth revision and clinical modification of the International Classification of Diseases (ICD-9-CM) [10]. The population register contains information on all registered persons living in the Netherlands, including date of birth, gender, current address, postal code, nationality, native country (both of registered person and his\/her parents), date of death and date of emigration. Patients whose parents were both born in the Netherlands were classified as native Dutch. Patients were classified as having a history of cardiovascular disease or having DM when cardiovascular disease (ICD-9-CM [10] codes 390\u2013459 and subcategories, principal diagnoses) or DM (ICD-9-CM [10] code 250 and subcategories, principal and secondary diagnoses) was registered during previous hospital admissions in 1991\u20131995 and in case of DM also during the index admission in 1995. Data on mortality of the patients were derived by linking their records with the population register and the cause of death certificates. In the population register, it is recorded when a registered person has died. Linkage of the population register with the cause of death certificates yielded information on primary causes of death.\nData analysis\nSurvival time was calculated as the time from the initial AMI admission date in 1995 to the date of death from any cause, or a patient was censored at the date of loss to follow-up in the population register (4% of patients, e.g. in case of emigration) or the end of study at December 31, 2000, whichever came first. Crude short-term (28-day), 1-year and long-term (5-year) mortality risks were computed for diabetic and nondiabetic patients. Cox proportional hazard analyses were used to examine the association between DM and overall survival in men and women. DM (yes\/no) and other predictors, including age (continuous), previous cardiovascular disease (yes\/no) and ethnic origin (native or non-native Dutch) were included in the models. Furthermore, Cox proportional hazards models were used to determine whether gender, age, previous cardiovascular disease and ethnic origin were independently associated with overall survival in patients with DM and patients without DM. Interaction terms were included in the models to assess the interaction of gender with DM, age with DM and age with gender.\nResults\nThe characteristics of the diabetic and nondiabetic patients with a first AMI are presented in Table\u00a01. Most of the diabetic patients were women (52%), whereas most of the nondiabetic patients were men (69%). In both diabetic and nondiabetic patients, women were older and had a longer duration of hospital stay than men. Diabetic patients were older and more likely to have previous cardiovascular disease (excluding AMI) compared to nondiabetic patients.\nTable\u00a01Characteristics of first acute myocardial infarction patients with and without diabetes mellitusPatients with diabetes mellitusPatients without diabetes mellitusMenWomenMenWomenNumber of patients9691,04913,4946,053Age at admission (years)\u00a0\u00a0\u00a0\u00a0Mean (standard deviation)68.7 (11.0)73.2 (10.3)64.0 (12.3)71.6 (12.0)Prior admission for CVD (%)31.731.016.816.3Type of hospital (%)\u00a0\u00a0\u00a0\u00a0Academic6.76.16.25.7\u00a0\u00a0\u00a0\u00a0General93.393.993.894.3Length of stay (days)\u00a0\u00a0\u00a0\u00a0Mean (standard deviation)11.1 (8.5)12.5 (11.8)9.7 (7.4)10.6 (9.7)\u00a0\u00a0\u00a0\u00a0Median10.010.09.09.0\u00a0\u00a0\u00a0\u00a0P25\u2013P75a7.0\u201313.06.0\u201316.06.0\u201312.06.0\u201313.0Native ethnic origin (%)87.088.090.090.0CVD\u00a0=\u00a0cardiovascular disease, excluding acute myocardial infarctiona25th and 75th percentile\nDuring admission, 18% of the diabetic patients (men 16%, women 20%) and 13% of the nondiabetic patients (men 11%, women 18%) died. In both diabetic and nondiabetic patients, AMI was the most frequent cause of death (during hospitalization 71 and 82%, respectively, and during 5\u00a0years of follow-up 39 and 45%, respectively).\nTable\u00a02 shows the higher absolute short- and long-term mortality risks for diabetic patients compared to nondiabetic patients in men and women.\nTable\u00a02Short- and long-term mortality in patients with a first hospitalized acute myocardial infarction (AMI) in the Netherlands in 1995 stratified by gender and presence of diabetes mellitusMenDiabetic patients (n\u00a0=\u00a0969)Non-diabetic patients (n\u00a0=\u00a013,494)Deaths (n)Deaths (%)Deaths (n)Deaths (%)At 28\u00a0days17017.5 (15.1\u201319.9)1,55511.5 (11.0\u201312.1)At 1\u00a0year28929.8 (26.9\u201332.7)2,31617.2 (16.5\u201317.8)At 5\u00a0years51653.3 (50.1\u201356.4)4,11630.5 (29.7\u201331.3)WomenDiabetic patients (n\u00a0=\u00a01,049)Non-diabetic patients (n\u00a0=\u00a06,053)Deaths (n)Deaths (%)Deaths (n)Deaths (%)At 28\u00a0days23522.4 (19.9\u201324.9)1,15219.0 (18.0\u201320.0)At 1\u00a0year36835.1 (32.2\u201338.0)1,62626.9 (25.7\u201328.0)At 5\u00a0years61158.2 (55.3\u201361.2)2,53841.9 (40.7\u201343.2)Figures are numbers and percentages of deaths (95% confidence intervals in brackets) based on actuarial life table method\nCrude short- and long-term mortality was significantly higher in diabetic patients than in nondiabetic patients in both men (28-day hazard ratio (HR) 1.55; 95% confidence interval (CI) 1.32\u20131.81, 5-year HR 2.01; 95% CI 1.84\u20132.21) and women (28-day HR 1.19; 95% CI 1.03\u20131.37, 5-year HR 1.53; 95% CI 1.40\u20131.67). When differences in age and other covariates between diabetic and nondiabetic patients were taken into account, risk differences became nonsignificant at 28\u00a0days, but DM was still associated with a significantly higher long-term mortality in both men and women (Table\u00a03). The interaction between gender and DM for 28-day, 1-year and 5-year mortality did not reach significance in the analyses, indicating that risks are equally elevated among men and women.\nTable\u00a03Multivariate analysis of the association between diabetes mellitus and short- and long-term mortality in first acute myocardial infarction patients (n\u00a0=\u00a021,565) by gender and ageAge (years)Follow-up duration28\u00a0days1\u00a0year5\u00a0yearsMen<601.41 (0.79\u20132.53)1.38 (0.86\u20132.23)1.83 (1.34\u20132.51)60\u2013691.46 (1.02\u20132.08) 1.73 (1.31\u20132.28)1.80 (1.46\u20132.21)70\u2013791.04 (0.80\u20131.36)1.16 (0.94\u20131.42)1.42 (1.23\u20131.65)\u2265801.11 (0.82\u20131.50)1.32 (1.04\u20131.66)1.37 (1.14\u20131.65)All ages1.16 (0.99\u20131.36)1.33 (1.17\u20131.50)1.49 (1.36\u20131.64)Women<602.06 (1.09\u20133.88)2.16 (1.25\u20133.75)2.13 (1.40\u20133.25)60\u2013691.38 (0.92\u20132.06)1.65 (1.19\u20132.29)1.99 (1.57\u20132.54)70\u2013790.94 (0.72\u20131.22)1.12 (0.92\u20131.37)1.38 (1.19\u20131.60)\u2265801.15 (0.94\u20131.42)1.18 (0.99\u20131.40)1.25 (1.09\u20131.44)All ages1.12 (0.97\u20131.28)1.23 (1.09\u20131.37)1.39 (1.27\u20131.52)TotalAll ages1.13 (1.02\u20131.26)1.27 (1.17\u20131.38)1.44 (1.35\u20131.53)Figures are hazard ratios (95% confidence intervals in brackets) with nondiabetic patients representing the reference groupResults from Cox proportional hazards analyses with diabetes mellitus, previous cardiovascular disease and ethnic origin (age- and gender-specific hazard ratios) and age (gender-specific overall hazard ratios) and gender (overall hazard ratios) included in the model\nIn the multivariate analyses, gender differences in mortality in nondiabetic patients varied over time with a higher mortality in women at 28\u00a0days and a higher mortality in men at 5\u00a0years (28-day HR 1.11; 95% CI 1.03\u20131.20, 5-year HR 0.93; 95% CI 0.88\u20130.98). Largely comparable, yet not significant, gender differences in mortality were found in diabetic patients (28-day HR 1.09; 95% CI 0.89\u20131.34, 5-year HR 0.93; 95% CI 0.82\u20131.04).\nDiscussion\nOur study provides nationwide estimates of the difference in mortality after a first hospitalized AMI between diabetic and nondiabetic patients. Long-term mortality was significantly higher in diabetic patients than in nondiabetic patients. Yet, there were no significant differences in short-term mortality. Risks appeared to be equally elevated in men and women.\nSome aspects of the study need to be addressed. In our study, the presence of DM was merely based on information from hospital admissions and on a retrospective period of maximal 5\u00a0years. As a result, the estimate of the effect of DM is likely an overestimate due to selection of the more severe cases (those hospitalized with or due to DM), while a fairly limited number of people with less severe stages of DM (not requiring hospital care) will be diluted in the large group of people who truly did not have DM.\nIn our study, we were able to jointly evaluate a number of predictors of mortality using information from the hospital register. However, we were unable to examine the contribution of other predictors on our outcomes, including other risk factors, comorbidity and treatment, as registration in the hospital register is limited. Yet, this does not invalidate the finding that DM is related to a worse long-term prognosis in patients with a first AMI, as confounding is not an issue in descriptive (prognostic) research [11]. However, if cofactors such as the presence of poorly treated hypertension or dyslipidemia become the focus of investigation, the lack of more detailed information from the hospital register renders such analyses impossible.\nIn our study, the percentage patients with a cardiovascular history (other than AMI) was about 2\u00a0times higher in diabetic patients than in nondiabetic patients. To be sure that the worse prognosis of diabetic patients compared to nondiabetic patients was real and not merely a reflection of the difference in cardiovascular history, we repeated the analyses in diabetic and nondiabetic patients without a cardiovascular history. These analyses yielded similar findings.\nThe present study involved hospitalized first AMI patients. Out-of-hospital deaths from first AMI were not included. A substantial and possibly selective mortality with regard to DM might have preceded the baseline of our study, e.g. if DM were more prone to out-of-hospital death [2] then this may result in less clear differences over the rest of the first 28\u00a0days, because the early deaths were not recorded. Therefore, the study population does not represent (the diabetes distribution of) all first AMI patients in the Netherlands, but specifically hospitalized first AMI patients, and results might differ from population-based studies, which usually manage to include the out-of-hospital deaths.\nAs data on patients without an AMI were not available, we were not able to compare the effect of DM on mortality in patients without an AMI with the effect in patients with an AMI, i.e. whether survival after an AMI is particularly worsened beyond the poorer prognosis already conferred by the presence of DM itself.\nThe strength of our study lies in the validity of the registries and linkage methods, the large size and lack of selection of the cohort and the long duration of follow-up. Recently, it was shown that 99% of the personal, admission and discharge data and 84% of the principal diagnoses (validated through medical record review by medical specialists) were correctly registered in a random sample of all hospital admissions registered in the hospital register [12]. Furthermore, over 97% of the uniquely linked hospital admissions resulting from linkage of the hospital register with the population register were shown to be correctly linked [13].\nResults from different studies regarding differences in mortality between diabetic and nondiabetic patients are conflicting. Consistent with our findings, it has been reported that DM has no independent predictive value on short-term mortality [14]. Yet, several other recent studies showed that short-term (<1\u00a0year) mortality after AMI was significantly higher (approximately 1.5 to 2\u00a0times) in diabetic patients compared with nondiabetic patients [1\u20133, 15]. Weitzman et\u00a0al. [16] found a significant higher 1-year mortality in diabetic men (odds ratio (OR) 1.5; 95% CI 1.2\u20131.9), but no significant association in women. Recent studies regarding differences in long-term mortality (follow-up ranging from 4 to 10\u00a0years) after AMI showed an approximately 1.5\u20132.5 higher risk of dying in diabetic patients [4\u20138, 17]. Ishihara et\u00a0al. [18] showed that DM was an independent predictor of 10-year mortality in patients with single vessel disease (OR 1.81; 95% CI 1.27\u20132.54), but in patients with multivessel disease the influence of DM was nonsignificant (OR 1.17; 95% CI 0.85\u20131.60). Melchior et\u00a0al. [5] reported that the difference in mortality after AMI between diabetic and nondiabetic patients increased with time (relative risk ranging from 1.03 [95% CI 0.81\u20131.31] at 30\u00a0days to 1.43 [95% 1.24\u20131.66] at 2\u00a0months-3\u00a0years and 1.74 [95% CI 1.36\u20132.23] at 7\u20139\u00a0years in patients admitted between 1990 and 1992).\nWe found no significant differences in short- and long-term mortality after a first AMI between men and women in diabetic patients. A few other studies examined gender differences in mortality after an AMI in diabetic patients. Crowley et\u00a0al. [19] found that women with DM were at increased risk of hospital death compared to men (OR 1.37; 95% CI 1.08\u20131.75), but no significant gender difference in 1-year mortality (HR 1.25; 95% CI 0.99\u20131.58) and 10-year mortality (hazard ratio 1.00; 95% 0.99\u20131.58) was found. Two other studies showed a higher short-term (hospital or 28-day) mortality in women [2, 20].\nThe higher risk of long-term mortality in first AMI patients with DM reinforces the importance of vigorous preventive measures by lifestyle advice and drugs in these patients. Currently, achievement of lifestyle and risk factor goals for reducing mortality in diabetic patients with AMI is poor, as illustrated by Pyorala et\u00a0al. [21] who reported that 20% of diabetic patients continued to smoke, 43% were obese, 57% had hypertension and 55% had hypercholesterolemia at least 6\u00a0months after hospitalization for coronary heart disease. A long-term, intensive approach consisting of behavior modification and pharmacologic therapy aimed at multiple risk factors is necessary, as it has been shown that this results in an impressive reduction in cardiovascular complications in patients with DM [22].\nIn conclusion, our findings in an unselected cohort covering a complete nation show that diabetic patients are at an increased risk of long-term mortality after a first acute myocardial infarction. Yet, there are no significant differences in short-term mortality. Risks appear to be equally elevated in men and women. These results stress the importance of secondary prevention by lifestyle advice and drugs in diabetic patients after a first AMI.","keyphrases":["mortality","myocardial infarction","diabetes mellitus","survival","prognosis","registries","epidemiology"],"prmu":["P","P","P","P","P","P","U"]}
{"id":"Eur_J_Nucl_Med_Mol_Imaging-3-1-1998889","title":"Serial O-(2-[18F]fluoroethyl)-L-tyrosine PET for monitoring the effects of intracavitary radioimmunotherapy in patients with malignant glioma\n","text":"Purpose Intracavitary radioimmunotherapy (RIT) offers an effective adjuvant therapeutic approach in patients with malignant gliomas. Since differentiation between recurrence and reactive changes following RIT has a critical impact on patient management, the aim of this study was to analyse the value of serial O-(2-[18F]fluoroethyl)-l-tyrosine (FET) PET scans in monitoring the effects of this locoregional treatment.\nIntroduction\nThe life expectancy of patients with high-grade gliomas, in particular glioblastoma multiforme, is still very poor. Standard treatment including surgery, radiation therapy and, if suitable, systemic chemotherapy results in median survival times ranging between 1 year for glioblastoma and about 3 years for anaplastic astrocytoma [1, 2]. Standard treatment is not able to control tumour progression for a longer period since in most cases microscopic tumour cell clusters located in the peritumoural brain tissue are left and become the starting point for (usually) early tumour recurrence [3, 4]: complete surgical resection or eradication by subsequent conventional radiation therapy is usually impossible. To overcome these limitations, more specific approaches for brain tumour treatment have been introduced, such as additional targeted radiotherapy, e.g. in form of a locoregional radioimmunotherapy (RIT) [5\u20139]. The infusion of radiolabelled monoclonal antibodies directly into the postsurgical resection cavity after surgery has enabled the delivery of high radiation doses to the affected area without harming the surrounding normal brain tissue or distant organs.\nTreatment efficacy can be evaluated by different neuro-imaging modalities. Conventional magnetic resonance imaging (MRI) and computed tomography (CT) assess morphological parameters such as changes in tumour size, oedema or contrast enhancement. However, these changes and alterations in blood-brain barrier properties are not always related to tumour regrowth; rather, they can also represent non-specific inflammatory treatment effects and radiation or tumour necrosis, especially after locoregional administration of high-dose radiation [10\u201312]. Functional imaging methods have been proposed as an alternative for monitoring treatment and are claimed to be more specific than structural imaging methods.\nPositron emission tomography (PET) using radiolabelled amino acid analogues, particularly [methyl-11C]-l-methionine (MET) and, in recent years, O-(2-[18F]fluoroethyl)-l-tyrosine (FET), has been employed in numerous metabolic studies of malignant brain tumours [13\u201321]. Since amino acid uptake has been shown to be increased relative to normal brain tissue in most low- and high-grade tumours, it has gained an important role in the diagnostic work-up of brain tumours [21\u201324]. PET using MET or FET has also been shown to differentiate reliably between tumour recurrence and post-therapeutic benign lesions after standard treatment modalities like surgery, external radiation therapy or chemotherapy [15, 16, 20, 25\u201328]. In a recent study, FET PET was evaluated for the first time in a novel locoregional therapeutic approach, for the purpose of monitoring the effects of convection-enhanced delivery of paclitaxel in patients with recurrent glioblastoma. Preliminary data in a small patient group suggested that FET PET seems to fulfil the clinical requirement for a method that is sensitive enough to assess therapeutic effects early and to distinguish between harmless post-therapeutic changes and tumour regrowth after this aggressive locoregional approach [29].\nThe aim of the present study was to evaluate the diagnostic value of FET PET in another locoregional approach, namely in monitoring the therapeutic effects of locoregional RIT following intracavitary administration of 131I- or 188Re-labelled monoclonal anti-tenascin antibodies (TN-mAb).\nMaterials and methods\nPatients\nTwenty-four patients (9 females, 15 males) with a mean age of 49\u00b114 years and histopathologically proven tenascin expression of malignant gliomas (5 anaplastic astrocytomas, 19 glioblastomas) were included in the study. All patients had undergone primary surgery on the tumour or re-operation of tumour recurrence with implantation of an Ommaya reservoir into the resection cavity. Primary surgery had been followed in 23 of the 24 patients by external radiation therapy and in six patients by additional systemic chemotherapy.\nIn 7 of the 24 patients, small residual tumours with an average diameter of less than 1 cm had to be left because of their location in high-risk and functionally important areas. In 17 patients no obvious tumour was seen on baseline MRI examinations. Patients underwent one to five cycles of intracavitary RIT with 131I-labelled (n=19) or 188Re-labelled (n=5) murine TN-mAb (BC4, which is directed against the EGF-like repeats of human TN) at 6- to 8-week intervals (maximal cumulative activity 5,740\u00a0MBq for 131I and 470\u00a0MBq for 188Re). In 20 patients, serial FET PET and MRI scans were performed preceding every RIT cycle and at 3-month intervals after the end of RIT in order to detect tumour recurrence and to monitor the therapeutic effects. In the remaining four patients, follow-up with serial FET PET scans was not started until 10, 12, 17 and 33 months after the end of RIT. Altogether, a total of 119 PET scans were performed.\nPET studies\nFET PET scans were obtained with a Siemens ECAT EXACT HR+ scanner. To obtain standardised metabolic conditions, patients fasted for a minimum of 6\u00a0h before performing the PET scan. The scanner acquires 63 contiguous transaxial planes, simultaneously covering 15.5\u00a0cm of axial field of view. After a 15-min transmission scan (68Ge sources), 180\u00a0MBq [18F]FET was injected intravenously. Since FET accumulates and reaches a peak in malignant gliomas within 15\u201320\u00a0min after injection and only slowly decreases afterwards [14], a static PET study from 20 to 50\u00a0min post injection (three added frames of 10\u00a0min each, 128\u00d7128 matrix, 3D acquisition) was acquired to obtain adequate count rate statistics. Images were reconstructed by filtered backprojection using a Hann filter with a cutoff frequency of 0.5 Nyquist and corrected for scatter and attenuation. For semiquantitative evaluation, data were transferred to a HERMES work station (Hermes Medical Solutions, Sweden) and the slice with the highest FET uptake around the former resection cavity was evaluated. For this slice, maximal uptake (maximal counts within the tumour: TUmax) was determined and the ratio to the background (BG) was calculated. For background information, the mean uptake within a 70% isocontour threshold ROI mirrored to the opposite non-tumour-bearing hemisphere was determined.\nStatistical analysis\nThe diagnostic performance in differentiating between recurrence and tumour-free status was assessed by receiver operating characteristic (ROC) analyses [30, 31]. For this purpose, data of serial PET scans were separately categorised into true- and false-positive and true- and false-negative findings using clinical follow-up or the results of histopathology as the gold standard. Then sensitivity (SN) and specificity (SP) pairs as a function of the respective decision thresholds were plotted as ROC curves to describe the inherent discrimination capacity of the diagnostic system and to define the optimal threshold for decision making. The threshold was considered optimal when the sum of paired values for SN and SP reached a maximum in two evaluations: the analysis of all performed scans (n=119) and the analysis of only that scan of each patient which presented with the highest uptake value during the entire follow-up period.\nIn addition, SN and SP values are given for a visual analysis using nodular versus non-nodular FET uptake as the criterion to distinguish between patients with tumour recurrence and tumour-free patients.\nThe duration of progression-free survival after the first RIT cycle was computed according to the Kaplan-Meier method using the SPSS for Windows (SPSS, Version 13.0, Chicago, IL) statistics package. Two groups were compared, those with maximal individual TUmax\/BG ratios above \u22652.4 and those with ratios below <2.4, which was considered the threshold value for best differentiation between recurrent tumour and reactive post-therapeutic changes. Statistical differences between the two curves were tested using log rank and Breslow tests.\nResults\nOur clinical experience with RIT has been reported previously. We observed substantially prolonged survival times for patients who received RIT (n=37: a larger number than in this study) compared with a similarly treated group without additional RIT [5]. Therefore, readers are referred to this previous article for information on the clinical findings associated with this form of therapy; here we shall focus on the role of FET PET during follow-up only.\nDuring the study period, 17 of the 24 patients ultimately presented with tumour progression while seven were considered tumour free. For the latter group, Table\u00a01 summarises patient characteristics, number and time points of RIT cycles and cumulative activity, number of serial PET scans with FET uptake ratios at baseline and last follow-up, and patient survival after initial surgery. The tumour-free status was documented by biopsy in two patients (these patients presented with contrast enhancement on MRI which was highly suspicious for recurrence) and by stable clinical follow-up for between 23 and 87 months after initial surgery in the other five. Six of these seven patients demonstrated slightly increasing homogeneous FET uptake surrounding the resection cavity without any signs of additional nodular uptake; this uptake presented with a peak (baseline TUmax\/BG 1.78\u00b10.30, peak TUmax\/BG 2.07\u00b10.25, mean\u00b1SD) up to 18 months after RIT, followed by stable or decreasing values (last follow-up TUmax\/BG 1.63\u00b10.22; representative example in Fig.\u00a01). One of these six patients unfortunately decided to discontinue the follow-up with FET PET 4 months after RIT. However, this patient was clinically followed and showed a stable course over 27 months after RIT and 35 months after initial surgery. In one of the seven patients with slightly increased stable and homogeneous FET uptake, RIT had been completed about 3 years prior to the beginning of FET PET monitoring; therefore the post-therapeutic phase of increasing FET uptake and its peak were probably missed. The peak values of TUmax\/BG observed in tumour-free patients corresponded to the threshold value of 2.4 for TUmax\/BG ratio as assessed by ROC analyses which allowed best differentiation between post-therapeutic reactive changes and tumour recurrence. In this small patient group, the extent and changes of FET uptake over time following RIT seemed to be independent of the delivered radiation dose since the peak TUmax\/BG ratio reached similar levels in all patients. Furthermore, the time point of the peak uptake, which ranged between 2 and 18 months, also showed no obvious relation with the dose administered. \nTable\u00a01Patient and tumour characteristics and therapeutic details of tumour-free patientsAge (yrs)SexWHOSurgeryRIT cyclesCum. activity (MBq)\/nuclideRITNo. of PET scansTUmax\/BG: baselineTUmax\/BG: peak TUmax\/BG: last follow-up Survival (mo.) post surgerySerial FET PET monitoring started after the end of RIT36fIII05\/9922,460\/131I10\/99\u201311\/999\u20132.4 (02\/01)1.5 (01\/05)77 (l.c.)39mIII10\/9855,740\/131I05\/99\u201301\/008\u20132.3 (09\/01)1.5 (10\/05)85 (l.c.)37fIV03\/0011,270\/131I11\/0012\u20132.1 (04\/02)1.5 (02\/05)66 (l.c.)54fIV08\/9833,490\/131I04\/99\u201308\/995\u2013peak missed1.6 (05\/05)87 (l.c.)Serial FET PET monitoring during and after RIT27fIV08\/0111,280\/131I03\/0232.0 (02\/02)2.2 (04\/02)2.0 (06\/02)35 (l.c.)22fIV01\/0433,560\/131I10\/04\u201302\/0561.4 (09\/04)2.1 (04\/05)1.8 (09\/05)21 (l.c.)40fIV11\/031380\/188Re05\/0471.4 (04\/04)1.7 (03\/05)1.4 (09\/05)23 (l.c.)l.c. last contactFig.\u00a01Serial MRI and FET PET scans of a 36-year-old woman after surgery for an anaplastic astrocytoma WHO III and following two RIT cycles (10\/99 and 11\/99 with 2,460\u00a0MBq 131I-TN-mAb). The patient was clinically tumour free at follow-up for up to 77 months after surgery. Slight linear contrast enhancement surrounding the small tumour cavity in the right central area is observed on MRI. On FET PET there is slightly increasing homogeneous FET uptake surrounding the cavity (which in this particular case mimics a more focal appearance owing to the small lesion size in combination with the limited resolution of the PET scanner), peaking 15 months after RIT (02\/01) and decreasing during further follow-up. The respective TUmax\/BG ratios are given below the PET images\nAmong the 17 of 24 patients who ultimately presented with tumour progression, ten experienced tumour recurrence and seven, regrowth of residual tumour. These diagnoses were histopathologically proven by stereotactic biopsy in five patients and by re-operation in three patients. In the remaining nine patients, suspected tumour regrowth or recurrence was assumed because of concordantly increasing contrast enhancement on MRI and clinical deterioration. Table\u00a02 summarises the characteristics and results of patients with progressive disease. The ten patients without obvious tumour at baseline initially presented with normal ratios (TUmax\/BG 1.64\u00b10.26) but showed focally increasing FET uptake values, resulting in pathological ratios during follow-up (TUmax\/BG 2.67\u00b10.43). A representative example is shown in Fig.\u00a02. In seven patients with known residual tumour on the baseline scan, further FET PET investigations demonstrated pathological ratios which were stable or increased over time (baseline TUmax\/BG 2.49\u00b10.42; last follow-up TUmax\/BG 3.04\u00b10.49). Six of these seven patients showed pathological contrast enhancement on baseline MRI as well. Most interestingly, in one of the seven patients (with anaplastic astrocytoma WHO grade III), MRI was rated as normal owing to the lack of contrast enhancement, whereas the concomitant PET scan showed pathological focal FET uptake ventral of the cavity, clearly indicating recurrence. This was proven by stereotactic biopsy. This patient underwent re-operation of this area, but unfortunately developed another tumour manifestation distant from the primary location that showed contrast enhancement on MRI as well (Fig.\u00a03). \nTable\u00a02Patient and tumour characteristics and therapeutic details of patients with tumour recurrence or regrowth of residual tumour during follow-upAge (yrs)SexWHOSurgeryRIT cyclesCum. activity (MBq)\/nuclideRITNo. of PET scansTUmax\/BG: baselineTUmax\/BG: last follow-up Survival (mo.) post surgeryPatients without obvious tumour at baseline followed by tumour recurrence40mIII10\/0034,510\/131I05\/02\u201310\/0261.5 (12\/01)2.5 (03\/03)36 (\u2020)53mIV08\/0111,690\/131I03\/0221.7 (01\/02)3.1 (04\/02)19 (\u2020)46fIV11\/0134,550\/131I07\/02\u201301\/0361.5 (07\/02)2.0 (09\/03)26 (\u2020)70mIV04\/0323,020\/131I02\/04\u201306\/0441.8 (02\/04)2.7 (09\/04)22 (\u2020)69mIV05\/042470\/188Re07\/04\u201310\/0442.1 (07\/04)3.3 (03\/05)13 (l.c.)55mIV07\/0233,060\/131I11\/03\u201303\/0452.0 (09\/03)2.1 (09\/04)34 (\u2020)61mIV12\/0211,770\/131I10\/0321.6 (08\/03)2.8 (12\/03)17 (l.c.)56mIV04\/0311,850\/131I10\/0371.6 (10\/03)2.5 (02\/05)24 (l.c.)37fIII04\/0134,130\/131I01\/05\u201305\/0551.2 (12\/04)2.4 (07\/05)54 (l.c.)39fIV03\/0421,420\/131I07\/04\u201310\/0461.6 (07\/04)2.8 (07\/05)19 (l.c.)Patients with small residual tumour at baseline followed by tumour progression30mIII02\/0111,870\/131I07\/0242.6 (07\/02)4.0 (12\/02)25 (\u2020)61mIV04\/0034,250\/131I09\/01\u201312\/0133.3 (09\/01)3.6 (12\/01)25 (\u2020)60mIV02\/0122,670\/131I12\/01\u201301\/0232.5 (12\/01)2.6 (03\/02)28 (\u2020)59mIV05\/031380\/188Re03\/0432.1 (03\/04)2.9 (07\/04)24 (l.c.)68mIV10\/021370\/188Re05\/0422.5 (05\/04)3.1 (06\/04)21 (l.c.)50mIV10\/0311,110\/131I01\/0422.1 (01\/04)2.2 (03\/04)12 (l.c.)68mIV08\/0224,090\/131I02\/05\u201307\/0552.3 (11\/04)2.9 (09\/05)38 (l.c.)\u2020 death, l.c. last contactFig.\u00a02Serial MRI and FET PET images of a 69-year-old man after surgery and radiation therapy of a glioblastoma located in the left occipital lobe during and after two RIT cycles (07\/04 and 10\/04 with 470\u00a0MBq 188Re-TN-mAb). Slightly increased homogeneous FET uptake is evident surrounding the tumour cavity (07\/04\u201312\/04). Development of tumour recurrence lateral to the resection cavity is indicated by additional nodular FET uptake as shown in the last scan (03\/05). The respective TUmax\/BG ratios are given below the PET imagesFig.\u00a03Serial MRI and FET PET scans of a 30-year-old man after surgery and radiation therapy of an anaplastic astrocytoma WHO III located in the right parietal lobe before and after 1 RIT cycle (07\/02 with 1870\u00a0MBq 131I-TN-mAk). a At baseline and 3 months after therapy, slightly increased homogeneous FET uptake at the cavity borders with additional nodular FET uptake ventral of the cavity indicating recurrence. MRI shows no suspicious contrast enhancement. b After reoperation development of another tumour manifestation developed distant the primary location with focal FET uptake and contrast enhancement on MRI as well. The respective TUmax\/BG ratios are given below the PET images\nIn total, 119 PET scans were performed, 34 in the presence of recurrent tumours and 85 while the patient had a tumour-free status. ROC analyses based on two approaches\u2014(a) including all performed PET scans (Fig.\u00a04a) and (b) considering only the PET scan with the highest individual uptake in each patient during follow-up (n=24) (Fig.\u00a04b)\u2014demonstrated the highest discrimination capacity between patients with tumour recurrence and tumour-free patients at a threshold value of 2.4 for the TUmax\/BG ratio.\nFig.\u00a04ROC curves illustrating the diagnostic performance of FET PET when varying the decision thresholds. Analyses were performed twice: for all available PET scans (n=119, including 85 scans performed while patients had a tumour-free status and 34 scans in patients with recurrent tumours) (a) and for only that scan presenting with the highest individual uptake (n=24) (b). The sensitivity and specificity pairs for the threshold values between 2.0 and 2.5 are given in the boxes\nComparison of patients with ratios below and above this threshold using Kaplan-Meier curves for progression-free survival (Fig.\u00a05) demonstrated that survival times were significantly longer (p<0.05) in patients presenting with values below this threshold.\nFig.\u00a05Kaplan-Meier curves showing progression-free survival in patients with ratios below (top) and above (bottom) the suggested optimal threshold value (TUmax\/BG=2.4)\nVisual analysis using a nodular versus a non-nodular uptake pattern as the criterion to distinguish between tumour recurrence and tumour-free status led to correct detection of recurrence in 32 of 34 scans (sensitivity 94%). In 80 of 85 scans performed in a tumour-free status, no additional nodular uptake pattern was present (specificity 94%). In five scans, uptake appeared somewhat focally accentuated owing to circumscribed folding of the cavity wall, and thus mimicked nodular uptake. Restricting visual analysis only to the one scan per patient presenting with the highest FET uptake resulted in correct detection of tumour recurrence in 16 of 17 patients (sensitivity 94%) and of a tumour-free status in five of seven (specificity 71%). In two of seven tumour-free patients, FET uptake that appeared nodular was misleading for reasons mentioned above.\nDiscussion\nRecent studies on locoregional RIT have demonstrated that local administration of radiolabelled monoclonal antibodies may significantly prolong survival times in patients with malignant gliomas [5\u20139]. The high locally achieved radiation dose, however, may cause inflammatory infiltrates and radiation necrosis more frequently than do standard treatment modalities, rendering differential diagnosis between benign reactive post-therapeutic effects and tumour recurrence more difficult. In particular, abnormal contrast enhancement in necrotic tissue often mimics tumour recurrence and, therefore, neither MRI nor CT allows reliable distinction between tumour and reactive changes in contrast-enhancing lesions [10\u201312]. Due to the poor prognosis of patients with glioma, however, knowledge of whether RIT has been successful or not is required so that a decision can be made to re-operate or to change to alternative aggressive treatment modalities when necessary.\nSo far, to our knowledge only one study has reported on results of functional imaging following RIT of glioma. Using PET with FDG as the metabolic marker, Marriott et al. [32] reported in a limited number of ten patients that a metabolic rim of FDG was observed around the tumour cavity after intracavitary administration of 131I-labelled monoclonal antibodies. Based on their findings they suggested that the development of such a rim may be dose dependent but independent of malignant disease. Histologically, the metabolic rim was associated with an inflammatory infiltrate consisting of a relatively increased number of macrophages and fibroblasts. Furthermore, malignant recurrence was suggested in the event of development of new nodularity in the non-malignant FDG-accumulating rim.\nTo our knowledge, there have been no previous reports of experience with PET and amino acid tracers as metabolic markers in the special setting of locoregional RIT. A recent study that monitored a limited number of patients with FET PET after another locoregional treatment of glioma, namely convection-enhanced delivery of paclitaxel, suggested that FET PET may be more reliable than MRI in differentiating reactive post-therapeutic changes in stable disease from tumour progression [29]. These results strongly encourage the evaluation of FET PET for monitoring of other forms of locoregional glioma therapy.\nThis is the first study to investigate the use of an amino acid tracer to monitor the effects of locoregional administration of radiolabelled monoclonal antibodies. Twenty-four patients with malignant gliomas were followed up with serial FET PET scans during and\/or after intracavitary RIT. Similar to the results reported by Marriott et al. [32], in all cases, even those considered tumour free, PET demonstrated slight homogeneous FET uptake surrounding the resection cavity, which was considered to be a consequence of therapy. However, the results of in vitro and animal studies on the uptake of FET in soft tissue infection [33] or cerebral radiation injury [34] suggest that FET uptake is probably to be explained by a breakdown in the blood-brain barrier rather than by active uptake in macrophages or inflammatory infiltrates, as in the case of FDG. This is supported by the following four observations: Breakdown of the blood-brain barrier following RIT was documented in all patients by a linear contrast-enhancing rim surrounding the resection cavity on MRI scans. Kaim et al. demonstrated that FET uptake was not increased in activated white blood cells of experimental soft tissue infection [33]. In rats, comparison of tracer accumulation in cryolesions characterised by the absence of inflammatory cells and radiation injury demonstrated that slight FET uptake was most likely due to a disruption of the blood-brain barrier and not additional trapping by macrophages. Uptake in radiation injury has generally been reported to be lower than uptake in tumours, suggesting that FET may be superior to FDG for distinguishing radiation necrosis from tumour recurrence [34].\nIn general, the homogeneous FET uptake around the resection cavity after RIT was somewhat higher than the unspecific uptake observed in a larger patient group after surgery and conventional external radiation therapy [20]. For these standard treatment modalities, the preliminary threshold value for best differentiation between post-therapeutic effects and tumour recurrence has been considered to be 2.0 for the TUmax\/BG ratio, whereas in the present study following RIT the threshold value was determined to be 2.4. The somewhat higher unspecific FET uptake after RIT may be explained by a more severe breakdown of the blood-brain barrier, probably induced by the higher local radiation dose. However, based on the limited experience so far, we were not able to document a relationship between extent and time course of FET uptake with the dose administered.\nIn addition to these general considerations regarding the rim uptake, the time course of FET uptake in relation to the RIT cycles also has to be taken into account. In the seven patients without clinical signs of tumour recurrence, PET demonstrated slightly increasing values initially, with a peak at up to 18 months after RIT, and stable or even decreasing values during further follow-up. In these patients, FET uptake was homogeneous and the TUmax\/BG ratio reached the threshold value of 2.4 in only one patient and then in only one (peak) of nine serial scans. In contrast, in the 17 patients presenting with tumour progression during follow-up, PET showed a more focally configured FET uptake in addition to the rim uptake. Semiquantitative evaluation of the slice with the highest uptake showed either stably increased values (in patients with residual tumour) or increasing values, starting from normal ratios and progressing to pathological ratios(in patients who developed recurrence).\nUsing the threshold value of 2.4 (as determined by ROC analyses) and regardless of the uptake pattern, FET PET was able to distinguish with high discriminatory power between tumour progression during or after RIT and therapy-induced benign changes: in 21\/24 patients there was accurate assessment in all serial PET scans, while in the remaining three patients there was false negative assessment, with TUmax\/BG ratios of 2.0, 2.1 and 2.2 respectively. However, in all three cases the nodular configuration of FET uptake led to the correct diagnosis. Even in one patient without focal contrast enhancement on MRI, PET showed focally increased FET uptake clearly indicating tumour regrowth, which was later proven by biopsy. Incidentally, this case supports the hypothesis that tumoural uptake independent of blood-brain barrier disruption is mainly mediated by active transport via the transport system L [35\u201337]. Visual assessment based solely on the FET uptake pattern seemed to provide slightly more sensitive results than those obtained by the ratio method, but at a lower specificity. Both the TUmax\/BG ratio and the configuration of FET uptake should be taken into account in order to differentiate optimally between recurrence and benign post-therapeutic effects. If findings are then still considered equivocal, a shorter time interval between follow-up scans (e.g. 6 weeks) offers another option to achieve reliable diagnostic assessment as early as possible.\nEven though it was not the aim of this study to assess the predictive value of FET PET for clinical outcome, FET uptake values following RIT could have been used to compare Kaplan-Meier-overall survival curves of patients with ratios below and above the suggested threshold value (TUmax\/BG<2.4). This approach, however, was not followed in our study owing to the heterogeneous patient group (patients with and without residual tumour at baseline, various intervals between initial surgery and radioimmunotherapy, various starting points of FET PET monitoring). Instead, a Kaplan-Meier analysis for progression-free survival following RIT was performed, showing significantly longer progression-free survival in patients in whom the maximal individual uptake was below the suggested threshold value, compared to those presenting with values above this threshold.\nOne possible limitation of the present study was the lack of histological confirmation in 15\/24 patients, and especially in five of the seven patients who were considered tumour free. Histopathological confirmation would have been ethically hard to justify in these patients, but its lack gives rise to the question of possible false negative findings. Nevertheless, the unsuspicious long-term clinical follow-up in the patients in question and the fact that the study population consisted only of patients with high-grade gliomas, and in particular glioblastomas (19\/24 patients), strongly support the PET results.\nAnother possible limitation is that in four of the 24 patients, follow-up with serial FET PET scans did not start until at least 10 months after the end of RIT, with the consequence that some earlier changes in FET uptake following RIT might have been missed. However, since in three of those four patients FET uptake was still increasing during the initial PET scans, followed by a peak and subsequent decrease, it is unlikely that crucial information was missed. Only in one patient in whom RIT had been completed as long as 33 months prior to the beginning of FET PET monitoring were the post-therapeutic phase of increasing FET uptake and its peak probably missed.\nFinally, in the present study we abstained from systematically correlating FET PET results with MRI data because the primary goal was to evaluate the value of serial FET PET scans for monitoring the effects of intracavitary RIT. Direct correlations between FET PET and MRI in a similar series of patients following multimodal systemic and\/or locoregional treatment have been published recently [38], showing that MRI is insufficient to distinguish between benign side-effects of therapy and tumour recurrence and that FET PET is a powerful tool to improve the differential diagnosis in these patients. Therefore, FET PET might be a method of choice for monitoring locoregional forms of treatment, like RIT, which frequently cause treatment-related inflammatory reactions or tumour necrosis.\nConclusion\nThis study indicates that FET PET is a sensitive tool for monitoring the effects of high local radiation doses given by intracavitary RIT. Homogeneous, slightly increasing FET uptake around the tumour cavity with a peak up to 18 months after RIT, followed by stable or decreasing FET uptake, is indicative of benign, therapy-related changes. These findings are independent of tumour recurrence and must not be misinterpreted as progressive disease. In contrast, focally increased FET uptake is an early and reliable indicator of tumour progression.\nSince both radiation necrosis and active tumour growth can present with clinical deterioration and are often impossible to distinguish by means of structural brain imaging, FET PET may be a powerful tool for planning further patient management. While radiation necrosis may be treated by steroids or, in extensive cases, by debulking surgery, recurrent tumour requires change of ineffective treatment or palliative care only.","keyphrases":["intracavitary radioimmunotherapy","malignant glioma","serial fet pet","therapy monitoring"],"prmu":["P","P","P","R"]}
{"id":"Sci_Eng_Ethics-4-1-2225997","title":"\u2018No Time to be Lost!\u2019\n","text":"Severe Traumatic Brain Injury (TBI) remains a major cause of death and disability afflicting mostly young adult males and elderly people, resulting in high economic costs to society. Therapeutic approaches focus on reducing the risk on secondary brain injury. Specific ethical issues pertaining in clinical testing of pharmacological neuroprotective agents in TBI include the emergency nature of the research, the incapacity of the patients to informed consent before inclusion, short therapeutic time windows, and a risk-benefit ratio based on concept that in relation to the severity of the trauma, significant adverse side effects may be acceptable for possible beneficial treatments. Randomized controlled phase III trials investigating the safety and efficacy of agents in TBI with promising benefit, conducted in acute emergency situations with short therapeutic time windows, should allow randomization under deferred consent or waiver of consent. Making progress in knowledge of treatment in acute neurological and other intensive care conditions is only possible if national regulations and legislations allow waiver of consent or deferred consent for clinical trials.\nIntroduction\nSevere Traumatic Brain Injury (TBI) remains a major cause of death and disability afflicting mostly young adult males and elderly people, resulting in high economic costs to society [1]. Road traffic accidents, domestic and work-related falls and assaults are the main causes of TBI in Europe. The fatality rate for severe TBI is about 30% and a significant disability in 35\u201340% in unselected series. The primary injury initiates a complex sequence of events resulting in secondary brain damage, which can be exacerbated by systemic insults, such as hypotension and hypoxia. Therapeutic approaches focus on reducing the risk on secondary brain injury. Pharmacological neuroprotective agents aim to limit secondary brain damage after the primary acute injury and aim to improve overall outcome. Various neuroprotective agents, mainly targeting specific pathophysiologic mechanisms, have been tested in TBI, but convincing benefit has not been shown [2]. These data signify an ethical imperative to develop and test new therapeutic strategies and neuroprotective pharmacological agents in the field of TBI.\nThe most important ethical issues pertaining to clinical pharmacological trials in severe TBI are: emergency nature of the researchincapacity of the patients to consentshort therapeutic time windowsrisk\/benefit ratio based on the concept that in relation to the seriousness of the injury, significant adverse side effects may be acceptable for treatments with possible benefit.\nThe importance of implications of these issues is not fully recognized outside, and even within, the expert field of treatment of severe TBI.\nTherapeutic trials to evaluate the efficacy and safety of pharmacological agents are subject to the ethical and juridical principles of Good Clinical Practice, national legislation and European and international regulations. The guiding ethical principles underlying these investigations of treatment are respect for autonomy of the subjects, protection against discomfort, harm, risk and exploitation and the prospect of benefit. The prospect of benefit is almost always complicated by the equipoise underpinning the statistical null hypothesis of pharmacological trials: the hope that an individual patient will benefit, but that this is not more certain than the chance of non benefit.\nCountries in the European Union are amending legislation to comply with the European Union Directive 2001\/20\/EC [3]. In this European legislation, emergency research under deferred or waiver of consent is however not permitted. This will impede or even obviate emergency research phase III trials in TBI in the European countries [4\u20136].\nThe European Clinical trial Directive 2001\/20\/EC was originally aimed as a European-wide harmonization of the provisions concerning clinical pharmacological trials, with a focus at the facilitation of multi-national clinical research. Since the publication in 2001, several articles drew attention to the serious threat to the development of evidence-based critical care and emergency research within the European Union (EU) posed by the Directive 2001\/20\/EC which requires prior informed written consent before subjects can be recruited to clinical trials of medicinal products [7\u201319].\nThe Directive made no direct exception for emergency and critical care situations, and therefore threatened to prevent all emergency trials involving patients with acute catastrophic illness causing loss of decision-making capacity and facing (very) short therapeutic time windows, such as severe shock, circulatory arrest, acute myocardial infarction, severe stroke and other acute neurological conditions, and moderate and severe traumatic brain injury.\nImplementation by all EU countries was required by May 2004. The wording of the Directive permitted some flexibility so that variations were expected that might impact on emergency research [20]. Lemaire et\u00a0al. [9] have described the variations in national legislative responses to the Directive within Europe; they called on legislators to permit waivers of informed consent for emergency and critical care research, to clarify terms and definitions, and to remove the artificial distinction between interventional and observational research. Concerning practice in The Netherlands, the requirements as described in the Directive have been transposed into the revision of the Medical Research in Human Subjects Act (WMO) and Medicine law (WOG) [21]. The amended WMO will change the rules governing drugs studies in The Netherlands. There will be little, if any, change to non-drug research. The Dutch Parliament has accepted the amended WMO for the implementation (amended WMO) at November 22, 2005 and the revised Act became effective in The Netherlands on March 1, 2006.\nThe Directive was conceived in part to ensure that participants enrolled in research projects are given adequate information about the nature of the trials and the associated risks. Legislation to protect the interests of patients was necessary and timely. The research community welcomed most of the Articles in the Directive; they offer guidance and will help to maintain confidence in the probity of medical research. Unfortunately, however, neither those responsible for the Directive, nor many who drafted enabling legislation within Member States, considered the special problems relating to research in emergency nor critical care situations, where consent cannot be obtained from subjects and where the need for emergency treatment does not allow time for contact with relatives or other legal representatives. Moreover, in the United States, in 1996, the FDA had published a waiver of informed consent for certain types of emergency and critical care research after earlier strict provisions had brought to a halt important progress in some critical clinical situations.\nThis shortcoming and the variable response within European Member States to the requirements of the Directive, prompted an expert meeting to be convened in Vienna, Austria on 30 May 2005 (\u2018Vienna Initiative to save European Research\u2019 [VISEAR]). A final report was presented in December 2005 and full reports appeared in the Wiener Klinische Wochenschrift in 2006. The initiative to the meeting was supported by the Department for Ethics in Medical Research of the Vienna Medical University, in cooperation with the European Forum for Good Clinical Practice (EFGCP), the European Clinical Research Infrastructures Network (ECRIN), and the Vienna School of Clinical Research. One of the six working groups aimed at \u2018clinical trials including patients who are not able to consent; the concept of individual direct benefit from research and informed consent in case of the temporarily incapacitated patient\u2019 Their recommendations were published in 2006 [6].\nConsent Procedures\nInformed consent in TBI victims can, due to the severity of the brain injury, never be obtained from the patients. Proxies or an independent physician must give consent for inclusion in research, or consent must be deferred or waived. Most ethical committees in European countries consider consent by legal representatives (proxy consent) valid. The moral basis for proxy consent is restricted to the substituted judgment about the inclusion into the trial. The proxy is supposed to act as the patient, if competent, would have decided. The question remains if the patient wants to be represented by relatives for inclusion in a trial. Roupie et\u00a0al. [22] found that only 40.6% of 1,089 patients would want their spouse\/partner to be their surrogate, 28% want to be represented by the physician in charge of their care.\nCoppolino and Ackerson [23] concluded that surrogate decision makers for critical care research resulted in false-positive consent rates in up to 20%. In the study by Sulmasy et\u00a0al. [24], agreement between patients and proxies varied between 57% and 81%, depending on whether previous discussions had taken place on similar situations. It is very unlikely that such existential discussions occur frequently in the target population prone to TBI (young adult males), resulting in lack of evidence as to what their relative would have wanted in case of severe TBI. Most proxies seem to make decisions in emergency situations based on what they hope that will happen (survival of the patient), rather than what is likely to happen (possible death or [severe] disability); this will bias decision-making towards possible therapeutic benefit, however small the chance will be [12].\nIn some European countries consent for randomization may be given by an independent physician. Different perceptives on consent by a physician are reflected in conflicting reports. In one study, 84% of patients with myocardial infarction felt that the physician could independently decide on inclusion, if the patient was unable to consent for himself [25]. In the field of neonatology only 11% of parents believe that physicians should decide regarding research participation [26].\nWith deferred (proxy) consent patients are included into the research without prior consent. After inclusion, the patient (deferred consent) or his\/her representatives (deferred proxy consent) should be informed as soon as possible and subsequent informed consent should be requested.\nWith waiver of consent, all consent is waived. Emergency research without prior consent (deferred consent or waiver of consent) can morally be accepted on the principles of fairness, justice and beneficence [27].\nAs severe TBI mostly occur outside the domestic situation (road traffic accidents), proxies are rarely available during the first hours after TBI [28]. This prompted investigators to use deferred (proxy) consent and waiver of consent in emergency research facing very short therapeutic time windows. In the National Acute Brain Injury Study: Hypothermia (NABIS-H) resulted the adoption of waiver of consent in a higher enrolment and reduced the time between injury and treatment by approximately 45\u00a0min [27]. In this study, relatives of only 11 out of 113 patients arrived within 6\u00a0h of the injury. In a septic shock trial the investigators could not contact the proxies within the inclusion time in 74% of the cases, and these were included under waiver of consent [19]. In the CRASH trial, mean time to randomization was significantly longer in those hospitals where consent was required compared with those it was not (4.4\u00a0h [SE\u00a0=\u00a00.21] vs. 3.2\u00a0h [SE\u00a0=\u00a00.16]), the difference in the mean time to randomization was 1.2\u00a0h [95% CI 0.7 to 1.8\u00a0h] [29]. In our series in the dexanabinol trial only 174 out of 6,303 (2.7%) were excluded for reason that proxy consent could not be obtained within 6\u00a0h after injury [30].\nEven when proxies are available, many do not know what the patient\u2019s wishes are [31]. Surrogate decision makers for critical-care research resulted in false-positive consent rates of 16\u201320.3% [23]. The emotional nature of an emergency situation limited the reliability of proxy consent for clinical research [26, 31, 32]. Only 48% of 79 representatives of European Brain Injury Consortium (EBIC) associated neuro-trauma centers in 19 European countries feel that relatives can make a balanced decision in an emergency situation, 72% believed that a consent procedure forms a significant factor causing decrease in enrolment rate in a TBI study, and 83% believed that prior consent is a significant factor causing delay in initiation of study treatment [12]. Under emergency circumstances, proxy consent does not seem to secure proper patient\/subject protection. To our experience the validity of informed consent and proxy consent given in an emergency situation is at least troubling. When consent for clinical research is sought during an emergency situation, comprehension is generally less than optimal [33\u201335]. A small minority realized that pharmacological trials are designed to assess not only efficacy but safety as well [36]. One study searching for public views on emergency exception to informed consent found that most (88%) of 530 people believed that research subjects should be informed prior to being enrolled, while 49% believed enrolling patients without prior consent in an emergency situation would be acceptable and 70% (369) would not object to being entered into such a study without providing prospective informed consent [37]. In another study 11 of 12 stroke patients stated that, if the patient of family was not able to consent, then the treating physician should make the decision for inclusion in an emergency trial [38].\nThe requirement for all patients to give written informed (proxy) consent before enrolment can result in major selection biases, such that registry patients were not representative of the typical patient [39].\nThe Emergency Nature of Research in TBI\nTraumatic Brain Injury is by definition an acute condition. The emergency nature of pharmacological research in TBI is reflected by the fact that experimental and clinical studies have shown that patho-physiological cascades are initiated within minutes to hours following primary injury. Time windows for treatment modalities are therefore considered to be short. Experimental studies have shown the efficacy of many neuroprotective agents, if these were administered before, or within 15\u00a0min after injury; others have shown a window of efficacy of 3\u20136\u00a0h.\nIn the most recent international pharmacological trial in TBI, a phase III randomized, placebo-controlled clinical trial investigating the efficacy and safety of a single dose Dexanabinol [40], the experimental data have consistently shown better protection the sooner the agent is administered after TBI [41]. In the animal model for TBI, this agent given up to 3\u00a0h after TBI was protective against breakdown of the blood\u2013brain barrier and reduced formation of edema and resulted in less severe neurological symptoms [42]. Administered between 4\u00a0h and 6\u00a0h after injury, no significant reduction of cerebral edema was noticed, nevertheless neurological symptoms improved. Based on these findings, it may be concluded that in the experimental model the patho-physiologic endpoint can be determined at 3\u00a0h. If this time window also form the clinical therapeutic border in patients with severe TBI remains however uncertain. Time windows as applied to clinical trials in TBI have rarely been based on experimental evidence, but were rather determined by organizational and logistical considerations as to the time window within which investigators expected that a considerable number of patients could be enrolled. This was also the case in the recent Dexanabinol trial. One of the inclusion criteria in this trial was \u2018sustained TBI within the past 6\u00a0h [40] Informed consent could, seen the severity of the brain injury, not be obtained from the patients. Proxy consent was accepted in all participating countries. Deferred patient or proxy consent was only allowed in Australia, Austria, Finland, France and Germany and consent by an independent physician was allowed in Israel, Italy, Spain and the United Kingdom. In all cases of deferred consent, subsequent written assent by patient or proxy was obtained.\nAs coordinating quality control and assurance center for this trial, we had the opportunity to study time windows in more detail [43]. We defined for this analysis four different time windows: the time between injury and admission in a neuro-trauma center;the time between admission in a neuro-trauma center and first head CT scan;the time between the first head CT scan and proxy consent for inclusion in the trial;the time between proxy consent and study drug administration.\nFor analysis of these four time windows we selected 631 patients. The only selection criterion was that the study drug was administered after written proxy consent. Patients included in the trial under deferred consent were excluded from our analysis. Furthermore, we only included patients from Europe and Israel, excluding patients from Australia and the United States for other reasons [44]. The time between injury and admission at the neuro-trauma center was for all selected patients between 1\u00a0h, 16\u00a0h and 2.35\u00a0h (Table\u00a01, Fig.\u00a01). 501 (79.4%) patients were directly admitted to the neuro-trauma center, 130 cases (20.6%) concerns secondary referrals. In all patients the window between admission and the first diagnostic CT scan remains within 1\u00a0h. With exception of France, in all countries the median time between injury and completion of the CT scan remained within the 3\u00a0h (Table\u00a01). The longest time window was found between the first diagnostic CT scan and obtaining the required proxy consent (between 1.71\u00a0h and 2.74\u00a0h). The median time between injury and obtained proxy consent was between 3.75\u00a0h and 5.00\u00a0h (IQR 2.75\u20135.38\u00a0h) (Table\u00a01). After proxy consent was given, almost all patients subsequently received the study drug within one hour (Table\u00a01, Fig.\u00a01). In 85.3% of all cases the time between injury and study drug administration was longer than 4\u00a0h, in 60% of the cases even longer than 5\u00a0h.\nTable\u00a01Time windows per country (median\u00a0+\u00a0IQR)Country (N)Hours between injury and admission NTC median (IQR)Hours between injury and CT scan median (IQR)Hours between injury and obtained consent median (IQR)Hours between injury and SDA median (IQR) Belgium (23)0.93 (0.65\u20131.27)1.80 (1.28\u20132.27)3.75 (2.75\u20134.75)4.60 (3.98\u20135.42)Netherlands (73)1.00 (0.75\u20131.33)1.65 (1.32\u20132.00)4.53 (3.95\u20135.05)5.53 (5.07\u20135.75)Israel (116)0.93 (0.72\u20131.40)1.91 (1.58\u20132.47) 4.01 (3.20\u20134.83)4.67 (4.00\u20135.33)Spain (75)1.33 (0.97\u20131.67) 2.07 (1.65\u20132.53)4.17 (3.33\u20135.00)5.17 (4.30\u20135.58)Germany (109)1.20 (0.88\u20132.00)1.65 (1.30\u20132.13)4.08 (3.42\u20134.98)5.25 (4.25\u20135.67)Italy (146)1.25 (0.83\u20132.60)1.77 (1.40\u20132.35)4.92 (4.08\u20135.28)5.50 (4.98\u20135.75)France (34)2.17 (1.42\u20133.00)3.08 (1.97\u20133.53)5.00 (4.50\u20135.38)5.75 (5.17\u20135.83)Other countries* (55) 1.47 (1.00\u20132.67)1.82 (1.33\u20132.50)4.00 (3.08\u20134.75)5.25 (4.33\u20135.75)*Countries with small patient populations (United Kingdom, Denmark, Austria, Poland, and Turkey) were combinedFig.\u00a01Time between injury and admission neurotrauma center, time between admission and first CT scan, time between first CT scan and informed consent for inclusion in trial and time between consent and start study drug admission (from Ref. 43)\nDexanabinol was one of the promising new pharmaceuticals in the treatment of TBI, but it shown to be safe but not effective in the treatment of severe TBI [40]. Nevertheless, one can conclude that chances of efficacy increase if treatment is provided earlier. Fact is that in almost all of the studied cases the time between injury and completion of the primary diagnostic CT scan remains within 3\u00a0h post injury, which is shown to be the therapeutic time window in the animal model. In 60% of the cases the time between injury and study drug administration was however longer than 5\u00a0h, and in 85.3% of all cases longer than 4\u00a0h (Fig.\u00a01). Our data provide the empirical proof for considering deferred consent or waiver of consent in trials with a very short therapeutic time window.\nRisk-Benefit Ratio\nTo my opinion the balance between risk and benefit should be the guiding principle in emergency research in severe TBI. This also applies to the nature and the type of consent procedures. The ethical principle of respect for the autonomy of the patient underpinning the informed consent procedures is not valid for acutely incapacitated patients as TBI victims. Significant concerns has been raised on the validity and ethics of proxy consent in acute emergency situations, and the required written consent cause a significant delay in treatment initiation, as we have shown with our analysis of the time windows. The possible therapeutic benefit, as has been shown in experimental models, form the moral justification for randomizing patients under deferred consent or waiver of consent within a sufficient period of time. The risks should however be acceptable in relation to the severity of the disease or injury. For trials under deferred consent or waiver of consent in acute emergency situations we would constrain the institution of an independent safety committee, under the auspices of regulatory authorities. The obligation to such a committee is based on the experience of a dramatically harmful outcome in some trials under waiver of consent in other fields of medicine [45, 46].\nConclusions\nSpecific ethical issues pertaining in clinical testing of pharmacological neuroprotective agents in TBI include the emergency nature of the research, the incapacity of the patients to informed consent before inclusion, short therapeutic time windows, and a risk-benefit ratio based on concept that in relation to the severity of the trauma, significant adverse side effects may be acceptable for treatments with possible benefit.\nTime windows as applied to randomized controlled clinical trials in TBI have rarely been based on experimental evidence, but were rather determined by organizational and logistical considerations as to a time window within which investigators expected that a considerable number of patients could be enrolled [12]. The main determinant is now formed by the informed (proxy) consent procedures, as also has been compelled in the new European Union Directive 2001\/20\/EC [3, 6]. These requirements assume that relatives are available in emergency situations, and that these relatives can be fully informed and given sufficient time to make a balanced decision in a relatively short time period. The conflict between the desire for early initiation of experimental treatment versus the time required for following consent procedures and the conflict between the desire for following consent procedures requiring prior consent and the doubts about the validity of proxy consent in acute situations are the most problematic aspects of emergency research in TBI.\nClinical research in emergency situations without prospective informed or proxy consent is ethically challenging. Severe TBI is without doubt an emergent and life-threatening condition and existing therapy is unsatisfactory seen the high morbidity and mortality in a mostly young group of patients. This should qualify severe TBI for emergency exception form informed consent for randomized clinical controlled trials with pharmacological agents with promising therapeutic benefit facing short therapeutic time windows. Randomized controlled investigations are necessary to determine the safety and effectiveness of new developed agents in these conditions. We have proved that the requirement of previous written proxy consent causes a significant delay till study drug administration in a trial with a neuroprotective agent in TBI. With waiver of consent or deferred (proxy) consent the first dose of the experimental drug can be administered directly after completion of the first diagnostic CT scan, which is very close to the experimental therapeutic time window. Randomized controlled phase III trials investigating the safety and efficacy of agents with promising benefit, conducted in acute emergency situations with short therapeutic time windows, should allow randomization under deferred (proxy) consent or waiver of consent. Making progress in knowledge of treatment in acute neurological and other intensive care conditions is only possible if national regulations and legislations allow waiver of consent or deferred (proxy) consent for clinical trials [15]. As two of us have said before: \u2018treat first, ask later\u2019 seems ethically defendable in acute care research [4].","keyphrases":["traumatic brain injury","deferred consent","waiver of consent","pharmacological trial","proxy consent","clinical ethics"],"prmu":["P","P","P","P","P","R"]}
{"id":"Pflugers_Arch-3-1-1915652","title":"Nitric oxide differentially regulates renal ATP-binding cassette transporters during endotoxemia\n","text":"Nitric oxide (NO) is an important regulator of renal transport processes. In the present study, we investigated the role of NO, produced by inducible NO synthase (iNOS), in the regulation of renal ATP-binding cassette (ABC) transporters in vivo during endotoxemia. Wistar\u2013Hannover rats were injected with lipopolysaccharide (LPS+) alone or in combination with the iNOS inhibitor, aminoguanidine. Controls received detoxified LPS (LPS\u2212). After LPS+, proximal tubular damage and a reduction in renal function were observed. Furthermore, iNOS mRNA and protein, and the amount of NO metabolites in plasma and urine, increased compared to the LPS\u2212 group. Coadministration with aminoguanidine resulted in an attenuation of iNOS induction and reduction of renal damage. Gene expression of 20 ABC transporters was determined. After LPS+, a clear up-regulation in Abca1, Abcb1\/P-glycoprotein (P-gp), Abcb11\/bile salt export pump (Bsep), and Abcc2\/multidrug resistance protein (Mrp2) was found, whereas Abcc8 was down-regulated. Up-regulation of Abcc2\/Mrp2 was accompanied by enhanced calcein excretion. Aminoguanidine attenuated the effects on transporter expression. Our data indicate that NO, produced locally by renal iNOS, regulates the expression of ABC transporters in vivo. Furthermore, we showed, for the first time, expression and subcellular localization of Abcb11\/Bsep in rat kidney.\nIntroduction\nThe free radical nitric oxide (NO) has been shown to play an important role in various physiological processes in the kidney, including salt and fluid reabsorption, renal hemodynamics, renin secretion, and tubuloglomerular feedback [1]. Endogenous NO is enzymatically produced from conversion of the amino acid l-arginine to l-citrulline, a reaction that is catalyzed by the enzyme NO synthase (NOS). This enzyme exists in three isoforms; the neuronal (nNOS) and endothelial, both of which are Ca2+- and calmodulin-dependent constitutive isoforms, and an inducible (iNOS) isoform [2]. Mediators associated with sepsis, such as endotoxin and the proinflammatory cytokines, interleukin-1\u03b2 (IL-1\u03b2), IL-2, and tumor necrosis factor-\u03b1 (TNF-\u03b1), have been shown to induce iNOS, thereby increasing production of NO. Sustained, high-output generation of NO by iNOS can cause cell death and tissue damage through lipid peroxidation, DNA damage, and proapoptotic effects [3]. This is the result of NO itself and the oxidant peroxynitrite, which is generated after reaction of NO with superoxide anions [4].\nIn contrast to many other organs, iNOS is constitutively expressed in the kidney, in particular in the medulla and in proximal tubules [5]. Many in vivo animal studies showed the importance of iNOS as a contributor to the pathophysiology of acute kidney injury (AKI). For example, it has been shown that selective chemical inhibitors of iNOS [6, 7], NO scavengers [8], iNOS gene knockout mice [9], or antisense oligodeoxynucleotides to iNOS [10] resulted in less renal tubular injury and improved structural and functional outcome. This renoprotective effect was partly due to the rescue of tubular epithelial cells from injury by NO [3].\nUsing killifish renal proximal tubules, we found previously that NO has a regulatory role in the transport activity of the ATP-binding cassette (ABC) transporter multidrug resistance protein 2 (Abcc2\/Mrp2) via an intracellular signaling pathway in response to the action of several nephrotoxic chemicals in vitro [11]. This pathway involved at least endothelin (ET) release, binding to the basolateral ETB receptor, and activation of NOS, soluble guanylyl cyclase, and protein kinase C [12]. A similar regulatory pathway was found for the ABC transporter P-glycoprotein (Abcb1\/P-gp) in rat brain capillaries [13]. Both studies indicate that the activation of NOS is most likely Ca2+-independent and we concluded that the production of NO is a result of the activation of iNOS.\nThe proximal tubule is responsible for the excretory transport of xenobiotics, xenobiotic metabolites, and waste products of metabolism from blood into urine. Consequently, the proximal tubule is also an important target for toxic effects and is often the first site of damage in AKI [14]. Luminal transport proteins, like the ABC transporters ABCB1\/P-gp, ABCC2\/MRP2, and ABCC4\/MRP4, are important efflux transporters in the kidney [15] and, therefore, play a role in the detoxification of tubule cells. The observed effects of NO on Abcc2\/Mrp2 in killifish proximal tubule may be part of the normal progression of cellular events that occur during AKI.\nIn the present study, we hypothesized that iNOS has a role in the regulation of renal ABC transporters during nephrotoxicity in vivo. An endotoxemia model was used in which rats were exposed to lipopolysaccharide (LPS), and the gene expression of 20 ABC transporters was evaluated. In addition, we examined the effect of the iNOS inhibitor, aminoguanidine, to determine whether the change in expression of the renal efflux transporters is caused directly by NO generated by iNOS.\nMaterials and methods\nAnimals and experimental design\nSpecified pathogen-free male Wistar\u2013Hannover rats (240\u2013295\u00a0g; purchased from Harlan, Zeist, The Netherlands) were kept under routine laboratory conditions at the Central Animal Laboratory of the Radboud University Nijmegen Medical Centre. This strain was used because they do not develop hydronephrosis spontaneously like normal Wistar rats, which in turn could have a down-regulating effect on iNOS expression [16]. The local Animal Care Committee approved all experimental procedures.\nDifferent pilot studies have been conducted to determine the route of administration, intravenously or intraperitoneally (IP), concentration of the endotoxin LPS (LPS+, Escherichia coli 0127:B8, Sigma-Aldrich, Zwijndrecht, The Netherlands) and aminoguanidine (Sigma-Aldrich), and the time point of coadministration in this strain. LPS and aminoguanidine were dissolved in sterile Hank\u2019s balanced salt solution (HBSS, Gibco, Paisly, UK) supplemented with 10\u00a0mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES, Gibco), and set to pH\u00a07.4 with NaOH.\nThirty rats were, stratified to body weight, injected IP with 5\u00a0mg\/kg LPS+ and were killed after 3, 6, 12, 24, or 48\u00a0h (n\u2009=\u20096 for each experimental group). Fifteen rats served as controls and received 10\u00a0mg\/kg detoxified LPS (LPS\u2212, n\u2009=\u20093 per group), in which the biologically active component, lipid A, essential for the activation of NOS, is lacking [17]. Although the control group is small, standard errors appeared to be satisfactory to allow a reduction and refinement of animals in this group. Twelve rats were injected IP with both 5\u00a0mg\/kg LPS+ and, after 1\u00a0h, 100\u00a0mg\/kg of the iNOS inhibitor aminoguanidine (Sigma-Aldrich), and were killed 6 and 12\u00a0h after LPS+ (n\u2009=\u20096 per group). For the isolated perfused kidney experiments, six rats received LPS+, four received LPS\u2212, and four were untreated and served as controls, and were killed after 12\u00a0h as described under the \u201cIsolated kidney perfusion\u201d section.\nTo collect urine before and after the injection, rats were housed in metabolic cages, washed with 2% chlorohexidine (Genfarma, Zaandam, The Netherlands) to prevent bacterial growth. Urine volumes were recorded and samples were snap-frozen in liquid nitrogen and stored at \u221280\u00b0C until assayed. During the experiment, body weight and body temperature were determined. At indicated time points after LPS injection, the rats were anesthetized with 5% isoflurane and heparin (125\u00a0IU\/100\u00a0g) was injected via the penile vein to minimize blood clotting. The left kidney was removed, divided in four pieces, and snap-frozen in liquid nitrogen for immunohistology or fixed in Bouin\u2019s solution for routine histology. Arterial blood was obtained by aortic puncture and the rats were killed by exsanguination. The right renal vein was punctured and, subsequently, the kidney was perfused with HBSS\u2013HEPES for 2\u00a0min at 7.5\u00a0ml\/min (Minipuls 2, Gilson, Villiers-le-Bel, France). After perfusion, the kidney was removed, divided, and snap-frozen in liquid nitrogen for quantitative real-time polymerase chain reaction (RQ-PCR) and Western blotting. Thrombocyte and leukocyte counts were determined in blood as inflammatory parameters, and the concentrations of creatinine, urea, protein, glucose, and sodium were analyzed in blood and urine by routine clinical chemistry. The activity of alkaline phosphatase, a proximal tubule injury marker, was determined in 26-fold diluted urine samples, as described before [18].\nDetermination of mRNA expression\nRNA was isolated using a Mikro-dismembrator U (Sartorius B. Braun Biotech, Melsungen, Germany). To prevent RNA degradation, we washed the metal cylinders with 0.5\u00a0M NaOH. Perfused kidneys were pulverized (2,000\u00a0rpm, 30\u00a0s) and transferred in ice-cold TRIzol reagent (Invitrogen, Breda, The Netherlands), and then RNA was isolated as described previously [19]. RQ-PCR on cDNA was performed according to the TaqMan\u00ae protocol in optical tubes using either the ABI PRISM 7700 single reporter Sequence Detection System (n\u2009=\u20096, Applied Biosystems, Zwijndrecht, The Netherlands) or the ABI\/PRISM 7900HT Gene Expression Micro Fluidic Card Sequence Detection System (three pooled samples from nine different rats, Applied Biosystems) according to the manufacturer\u2019s instructions. All experiments were performed in triplicate. Different rat genes were amplified with a predeveloped Gene Expression Assay, provided by Applied Biosystems.\nWestern blotting\nTotal membrane and cytosolic fractions from perfused kidneys were isolated using a Mikro-dismembrator U (Sartorius BBI Systems GmbH, Melsungen, Germany; 2,000\u00a0rpm for 30\u00a0s) and subjected to Western blotting as described previously [20]. Protein concentration was determined with the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA) using bovine serum albumin (BSA) as standard. Samples were separated on a 6 or 12% sodium dodecylsulfate polyacrylamide gel and transferred to Hybond-C pure nitrocellulose membrane (Amersham, Buckinghamshire, UK). The membrane was incubated overnight at 4\u00b0C with antibodies against NOSII\/iNOS (1:1,000, according to Vos et al. [21]), ABCB1\/P-gp (1:200, C219, DakoCytomation, Dako, Denmark), Abcc2\/Mrp2 (1:250, M2III-6, Alexis Biochemicals, San Diego, CA, USA), ABCC4\/MRP4 (1:1,000, as described by van Aubel et al. [22]), Abcb11\/bile salt export pump (Bsep) (1:1,000, kindly provided by Dr. Bruno Stieger, University Hospital Z\u00fcrich, Switzerland), or \u03b2-actin (1:10,000, Sigma) in Tris-buffered saline supplemented with 0.1% Tween-20 and 1% nonfat dried milk.\nNitrate and nitrite determination\nThe total amount of the stable NO metabolites (NOx), nitrate and nitrite, were determined as a measure of NO radicals production, using the Griess reaction, according to Moshage et al. [23]. Heparinized plasma was diluted 1:4 and urine samples were diluted 1:40 or 1:80 with distilled water. Each sample was determined in triplicate. Calibration curves were made with sodium nitrite and sodium nitrate in distilled water. The nitrate recovery was 90\u2013100% over the entire range tested (0\u201350\u00a0\u03bcmol\/l). The coefficient of variation between the different experiments was less than 5%.\nHistology and immunohistology\nKidneys fixed in Bouin\u2019s solution were dehydrated, embedded in paraplast (Amsterstad, Amsterdam, The Netherlands), and cut into 3-\u03bcm sections, which were stained with periodic acid-Schiff for routine histology. For immunohistologic studies, serial tissue sections of 1.5\u00a0\u03bcm were cut from frozen kidneys using a cryostat as described previously [24]. The slices were incubated for 60\u00a0min at room temperature with antibodies against either NOSII\/iNOS (1:200, according to Vos et al. [21]) or ABCB1\/P-gp (1:50, C219, DakoCytomation) in PBS supplemented with 1% BSA. For the antibodies peroxynitrite (1:500, Sigma) and Abcb11\/Bsep (1:1,000, as described by Huls et al. [24]), cryostat slices of 4\u00a0\u03bcm were used, fixed in acetone for 5\u00a0min. Endogenous peroxidase was blocked by incubation with 5% H2O2 for 5\u00a0min, and nonspecific protein binding was blocked with 10% normal goat serum (Vector, Burlingame, CA, USA) in PBS for 10\u00a0min at room temperature. Slices were incubated with the primary antibody in PBS 1% BSA overnight at 4\u00b0C. Primary antibody was detected using the Vectastain Elite peroxidase ABC kit (Vector) with 3,3\u2032-diaminobenzidine as substrate. Finally, slices were counterstained with hematoxylin, dehydrated, and mounted in mounting medium. Sections were examined with a confocal laser-scanning microscope (Leica lasertechnik GmbH, Heidelberg, Germany).\nIsolated kidney perfusion\nTwelve hours after the administration of LPS+ (n\u2009=\u20096) or LPS\u2212 (n\u2009=\u20094), the transport activity of Abcc2\/Mrp2 was determined by monitoring the urinary excretion of calcein in an isolated perfused rat kidney, as described in detail previously [25]. Briefly, rats were anesthetized IP with pentobarbital (6\u00a0mg\/100\u00a0g), and furosemide was injected IP (1\u00a0mg\/100\u00a0g) to prevent deterioration of the distal nephron. Heparin (125\u00a0IU\/100\u00a0g) was injected in the spleen. The kidney was excised and placed in a fluid bath with a constant temperature of 37.5\u00b0C and rats were killed by exsanguination. Vitamin B12 was used for the determination of the glomerular filtration rate (GFR) and calcein-acetoxymethylester was used as a source for the fluorescent substrate calcein [25].\nStatistical analysis\nValues are given as mean \u00b1 SE. Differences between the experimental groups were tested using one-way ANOVA with Bonferroni\u2019s correction or two-way ANOVA corrected for repeated measurements. A two-sided P value\u2009<\u20090.05 was considered significant.\nResults\nSigns of inflammation\nA few hours after LPS+ administration, inflammatory symptoms appeared, like diarrhea and piloerection. Body temperature was elevated after 24\u00a0h and the loss in body weight increased from \u22123% (t\u2009=\u20093\u00a0h), due to normal circadian rhythm, to 12% (t\u2009=\u200948\u00a0h), compared to the controls (Table a in the supplementary material\u00a01). Furthermore, administration of LPS+ resulted in the expected leucopenia (t\u2009=\u20093\u00a0h) followed by leukocytosis (t\u2009=\u200948\u00a0h) and thrombocytopenia, with a maximum at 48\u00a0h. Coadministration of LPS+ with aminoguanidine did not affect the inflammatory parameters, except for a greater loss in body weight 12\u00a0h after LPS administration.\niNOS induction after LPS treatment\nRQ-PCR was used to determine the iNOS mRNA levels in kidney samples. The relative expression of iNOS in LPS\u2212 rats was normalized for the average cycle threshold (CT) value for the housekeeping gene, GAPDH (CT\u2009=\u200916.06\u2009\u00b1\u20090.04, n\u2009=\u200915), and set to 1. GAPDH expression remained stable during endotoxemia (CT\u2009=\u200916.22\u2009\u00b1\u20090.09, n\u2009=\u200942). Furthermore, no circadian rhythm for the different genes was detected in control animals.\nAnalysis of three separate experiments revealed a more than 40,000 times induction in iNOS mRNA 3\u00a0h after LPS+ compared to controls (Fig.\u00a01a), which decreased with time, but was still 100 times higher than control values 48\u00a0h after LPS+. Treatment with both LPS+ and aminoguanidine slightly, but not significantly, reversed iNOS mRNA induction compared to LPS+ alone (Fig.\u00a01a). Maximal iNOS protein expression was observed in cytosolic fractions 6\u00a0h after LPS+, and returned to LPS\u2212 level at 48\u00a0h (Fig.\u00a01b). In contrast to its effect on iNOS mRNA expression, aminoguanidine reversed the induction in protein expression (Fig.\u00a01c).\nFig.\u00a01iNOS mRNA and protein. a iNOS mRNA expression in rat kidney at different time points after LPS+ was determined with RQ-PCR without (closed bars, n\u2009=\u20096) or with coadministration of aminoguanidine (gray bars, n\u2009=\u20096). The iNOS mRNA expression was normalized for the GAPDH CT value (16.06\u2009\u00b1\u20090.04, n\u2009=\u200915), and \u0394CT values for the LPS\u2212 were set to 1. Data are expressed as mean\u00b1SE. Significantly different compared to the LPS\u2212 (double asterisksP\u2009<\u20090.01, triple asterisksP\u2009<\u20090.001, quadruple asterisksP\u2009<\u20090.0001). Cytosolic fractions of rat kidney were isolated and expression of iNOS was determined by Western blotting. Representative images (n\u2009=\u20094) show maximal iNOS protein expression 6\u00a0h after LPS+ administration (b), and coadministration with aminoguanidine reversed this induction (c)\nNOx levels in plasma and urine were measured using the Griess assay. An increase in NOx concentration in plasma was already visible 3\u00a0h after LPS+, with a peak concentration at 12\u00a0h. Coadministration with aminoguanidine reversed the increase in NOx significantly at both 6 and 12\u00a0h (Fig.\u00a02a). The urinary excretion of NOx increased 12\u00a0h after LPS+, and was still elevated 48\u00a0h later. Aminoguanidine returned this increase to baseline levels at both time points (Fig.\u00a02b).\nFig.\u00a02NOx in plasma and urine. NOx levels in plasma and urine were measured at various times after LPS\u2212 (open bars, n\u2009=\u20093) or LPS+ (closed bars, n\u2009=\u20096) treatment or LPS+ together with aminoguanidine (gray bars, n\u2009=\u20096). a Blood samples show a peak concentration at 12\u00a0h and coadministration with aminoguanidine reversed this increase at both 6 and 12\u00a0h. b The urinary excretion of NOx increased 12\u00a0h after LPS+ treatment, and aminoguanidine returned this increase to baseline levels. Data are expressed as mean\u00b1SE. Significantly different compared to the LPS\u2212 (single asterisksP\u2009<\u20090.05, double asterisksP\u2009<\u20090.01, triple asterisksP\u2009<\u20090.001) or LPS+ (double sharp signsP\u2009<\u20090.01, triple sharp signsP\u2009<\u20090.001)\nRenal localization of iNOS and formation of 3-nitrotyrosine-protein adducts, a reliable biomarker of peroxynitrite generation [26], were determined by immunohistology. There was no iNOS staining visible in kidney sections of rats treated with LPS\u2212. Positive iNOS staining was seen in both experimental groups after LPS+, in inflammatory cells, and in proximal tubule cells. Forty-eight hours after LPS+, iNOS staining was no longer detectable. Figure\u00a03 shows representative immunohistochemical images with iNOS expression in all segments of the proximal tubule (3A+C). After colocalization with Abcb1\/P-gp (3B), iNOS expression in the vicinity of the apical membrane was visible (3A+C). 3-Nitrotyrosine-protein adducts were predominantly present in the cortex in peritubulary capillaries and proximal tubules after LPS+ (Fig.\u00a03d,e). Coadministration with aminoguanidine attenuated the formation of adducts in the cortex (Fig.\u00a03f,g).\nFig.\u00a03Immunofluorescence microscopic analysis of iNOS and peroxynitrite in the kidney. a\u2013c Representative immunohistochemical images show that iNOS (green staining) is expressed in the vicinity of the apical membrane (P-gp counterstaining, red) in all segments of the proximal tubule 12\u00a0h after LPS+ treatment. d, e Formation of 3-nitrotyrosine-protein adducts, a reliable biomarker of peroxynitrite generation, were visible in the cortex in peritubulary capillaries and proximal tubules 12\u00a0h after LPS+ treatment. f, g Coadministration with aminoguanidine resulted in attenuated formation of 3-nitrotyrosine-protein adducts at 12\u00a0h. G glomerulus, asterisks peritubular staining, P proximal tubule, D distal tubule. Original magnification d, f \u00d7200 and a\u2013c, e, g \u00d7400\nAminoguanidine restores the slightly impaired kidney function and loss of brush border membrane\nKidney function was slightly impaired after LPS+ as indicated by increased blood urea nitrogen (BUN), an increase in plasma creatinine, and an increased urinary glucose excretion (Table\u00a01). Furthermore, the urine\/plasma creatinine ratio was decreased (data not shown) as was the creatinine clearance, although not significantly. \nTable\u00a01Renal functional parametersKidney functionTime (h)Controls (LPS\u2212)LPS+LPS+ and aminoguanidineTotal urine volume (ml)31.6\u2009\u00b1\u20090.241.7\u2009\u00b1\u20090.2762.4\u2009\u00b1\u20090.654.45\u2009\u00b1\u20090.987.3\u2009\u00b1\u20090.76123.2\u2009\u00b1\u20090.5710.0\u2009\u00b1\u20090.70a9.4\u2009\u00b1\u20090.65a2411.5\u2009\u00b1\u20090.2513.7\u2009\u00b1\u20092.114823.5\u2009\u00b1\u20093.4718.0\u2009\u00b1\u20091.57BUN (mmol\/l)36.6\u2009\u00b1\u20090.209.9\u2009\u00b1\u20090.7464.6\u2009\u00b1\u20090.7214.0\u2009\u00b1\u20091.48b12.1\u2009\u00b1\u20090.82b126.3\u2009\u00b1\u20090.2412.3\u2009\u00b1\u20091.38a15.4\u2009\u00b1\u20091.27b247.0\u2009\u00b1\u20090.5810.6\u2009\u00b1\u20090.58486.5\u2009\u00b1\u20090.109.1\u2009\u00b1\u20090.38Blood creatinine (mmol\/l)30.035\u2009\u00b1\u20090.0020.049\u2009\u00b1\u20090.002b60.039\u2009\u00b1\u20090.0020.053\u2009\u00b1\u20090.002b0.043\u2009\u00b1\u20090.019d120.037\u2009\u00b1\u20090.0020.048\u2009\u00b1\u20090.002c0.036\u2009\u00b1\u20090.032e240.038\u2009\u00b1\u20090.0010.051\u2009\u00b1\u20090.001a480.042\u2009\u00b1\u20090.0010.052\u2009\u00b1\u20090.001Glucose excretion (\u03bcmol\/h)30.290\u2009\u00b1\u20090.0450.481\u2009\u00b1\u20090.34160.333\u2009\u00b1\u20090.0860.420\u2009\u00b1\u20090.1000.478\u2009\u00b1\u20090.051120.425\u2009\u00b1\u20090.1060.537\u2009\u00b1\u20090.0540.332\u2009\u00b1\u20090.080240.254\u2009\u00b1\u20090.0060.984\u2009\u00b1\u20090.039c480.325\u2009\u00b1\u20090.0370.606\u2009\u00b1\u20090.128Creatinine clearance (ml\/min)31.33\u2009\u00b1\u20090.120.78\u2009\u00b1\u20090.1961.06\u2009\u00b1\u20090.150.87\u2009\u00b1\u20090.090.93\u2009\u00b1\u20090.35121.01\u2009\u00b1\u20090.050.90\u2009\u00b1\u20090.031.01\u2009\u00b1\u20090.13241.19\u2009\u00b1\u20090.020.91\u2009\u00b1\u20090.20481.00\u2009\u00b1\u20090.071.08\u2009\u00b1\u20090.37FENa (%)30.816\u2009\u00b1\u20090.0040.365\u2009\u00b1\u20090.07361.046\u2009\u00b1\u20090.1450.358\u2009\u00b1\u20090.094b1.423\u2009\u00b1\u20090.310e121.094\u2009\u00b1\u20090.0970.458\u2009\u00b1\u20090.075a1.610\u2009\u00b1\u20090.282e241.070\u2009\u00b1\u20090.0240.429\u2009\u00b1\u20090.065c481.033\u2009\u00b1\u20090.0770.121\u2009\u00b1\u20090.024aData are expressed as mean\u00b1SE, n\u2009=\u20096 for each time point for both LPS+ and LPS+ +aminoguanidine and n\u2009=\u20093 for LPS\u2212 FENa fractional sodium excretionaSignificantly different compared to the control group; P\u2009<\u20090.01bSignificantly different compared to the control group; P\u2009<\u20090.001cSignificantly different compared to the control group; P\u2009<\u20090.05dSignificantly different compared to the LPS+ group; P\u2009<\u20090.01eSignificantly different compared to the LPS+ group; P\u2009<\u20090.001\nHistological examination after LPS+ revealed damage to the proximal tubules, compared to LPS\u2212 (Fig.\u00a04a,b). Loss of brush border membranes and formation of vacuoles were observed. Aminoguanidine reduced LPS+-induced damage to the proximal tubule (Fig.\u00a04c). The activity of alkaline phosphatase in urine samples was assayed to get an indication of proximal tubule injury. Twelve hours after LPS+, the activity of alkaline phosphatase was increased in urine samples and was still significantly increased after 48\u00a0h (Fig.\u00a04d). Furthermore, the expression of kidney injury molecule 1 (Kim-1 [27]) was strongly up-regulated, 400 times more than control values, 6 and 12\u00a0h after LPS+ (Table b in the supplementary material 1). Except for BUN, aminoguanidine improved all parameters of renal function, as shown in Table\u00a01 and Fig.\u00a04. No differences in urinary protein excretion were found and, remarkably, the fractional excretion of filtered sodium was decreased (Table\u00a01).\nFig.\u00a04Proximal tubular damage during endotoxemia. Histological examination after LPS+ revealed damage to the proximal tubules compared to LPS\u2212. a LPS\u2212-treated rat with intact brush border membrane in proximal tubules. b Twelve hours after LPS+, damage to the proximal tubules is observed with loss of brush border membranes and formation of vacuoles. c Treatment with both LPS+ and aminoguanidine resulted in less damage to the proximal tubule. Kidneys of LPS+-treated rats killed after 24 or 48\u00a0h also showed signs of proximal tubule damage (data not shown). Proximal tubules are indicated by arrows. Original magnifications (a\u2013c) \u00d7400. d Histological data are supported by an increase in the activity of alkaline phosphatase, marker for proximal tubule damage, in urine samples 12\u00a0h after LPS+ administration (closed bars, n\u2009=\u20096), compared to controls (open bars, n\u2009=\u20093). Coadministration with aminoguanidine (gray bars, n\u2009=\u20096) reduced this proximal tubule damage. Data are expressed as mean\u00b1SE. Significantly different compared to the LPS\u2212 (double asterisksP\u2009<\u20090.01) or LPS+ (sharp signP\u2009<\u20090.05)\nFurthermore, the mRNA expression of different biotransformation enzymes [28], enzymes involved in the NO pathway [2] or known to be activated during oxidative stress [29, 30] was determined. Twelve and 24\u00a0h after LPS+ treatment, expression of the metabolizing enzymes glutathione S-transferases (GST-\u03b12 and GST-\u03b15) and uridine diphosphate-glucuronosyltransferases (UDPGT1a6) was down-regulated (Table b in the supplementary material\u00a01, at least a 2 times lower expression). The arginine metabolic enzymes, argininosuccinate synthetase and arginase 2, were not changed after LPS+. In contrast, arginase 1 mRNA expression was reduced. The gene expression of the ETB receptor, which gets activated as part of the NO-dependent signaling pathway regulating Abcb1\/P-gp and Abcc2\/Mrp2 [13, 31], did not change during endotoxemia. Three and 6\u00a0h after LPS+ the mRNA expressions of super oxide dismutase 2, heam-oxygenase 1, and nuclear factor \u03baB (NF-\u03baB) were up-regulated (i.e., at least a 2 times higher expression). Coadministration of aminoguanidine had no effect on the gene expression of all these proteins. In contrast, aminoguanidine reversed the down-regulation of nNOS gene expression during endotoxemia (Table b in the supplementary material\u00a01).\nLPS+ treatment affects ABC drug transporter expression in the kidney\nThe mRNA expression of 20 renal ABC transporters was profiled. Abcb1a\/P-gp, Abcb1b\/P-gp, Abcc2\/Mrp2 and Abcc4\/Mrp4 expression was determined with the ABI PRISM 7700 single reporter sequence detection system. In rodents, Abcb1\/P-gp exists as two functional isoforms, which are encoded by two different genes: Abcb1a and Abcb1b [32]. The other ABC transporters were assessed with the ABI\/PRISM 7900HT Gene Expression Micro Fluidic Card (Table\u00a02). Their expression was normalized for the average CT value for GAPDH, as described for iNOS. Other housekeeping genes, like glucuronidase and hypoxanthine guanine phosphoribosyl transferase 1, gave comparable values to GAPDH (data not shown). Abcb1b\/Pg-p and Abcc2\/Mrp2 were clearly up-regulated 3\u00a0h after LPS+ and were still elevated 12 and 24\u00a0h later (Fig.\u00a05b,c). Furthermore, Abcb1a\/P-gp and Abcc4\/Mrp4 were slightly, but not significantly, increased 12\u00a0h after LPS+ treatment (Fig.\u00a05a,d). Table\u00a02 presents a list of the expression of other transporter genes during endotoxemia. Abca1 showed a slight up-regulation 3\u00a0h after LPS+, but returned to basal levels within 12\u00a0h. Furthermore, Abcb11 was up-regulated, but only significantly after 48\u00a0h. A down-regulation was observed for Abcb4 and Abcc8 (at least a 2 times lower expression). The expression of the Abcg genes was not affected by endotoxemia. \nTable 2Gene expression of efflux transporters during endotoxemiaGene symbolProtein synonymAssay IDsDelta CT valuesaLPS\u2212 (controls)LPS+t\u2009=\u20093 hLPS+t\u2009=\u20096 hLPS+t\u2009=\u200912 hLPS+t\u2009=\u200924 hLPS+t\u2009=\u200948 hLPS+ + amino t\u2009=\u20096 hLPS+ + amino t\u2009=\u200912 hAbca1Abca 1Rn00710172_m17.1\u2009\u00b1\u20090.315.1\u2009\u00b1\u20090.165.8\u2009\u00b1\u20090.227.3\u2009\u00b1\u20090.557.3\u2009\u00b1\u20090.356.8\u2009\u00b1\u20090.236.1\u2009\u00b1\u20090.238.0\u2009\u00b1\u20090.12Abca2Abca 2Rn00577821_m13.5\u2009\u00b1\u20090.403.5\u2009\u00b1\u20090.354.1\u2009\u00b1\u20090.154.5\u2009\u00b1\u20090.384.3\u2009\u00b1\u20090.353.6\u2009\u00b1\u20090.064.4\u2009\u00b1\u20090.384.4\u2009\u00b1\u20090.12Abca5Abca 5Rn00597824_m19.8\u2009\u00b1\u20090.069.8\u2009\u00b1\u20090.359.8\u2009\u00b1\u20090.109.5\u2009\u00b1\u20090.1210.0\u2009\u00b1\u20090.2310.2\u2009\u00b1\u20090.219.5\u2009\u00b1\u20090.259.7\u2009\u00b1\u20090.35Abcb1aP_gpRn00591394_m18.4\u2009\u00b1\u20090.01Fig. 5aAbcb1bP_gpRn00561753_m110.8\u2009\u00b1\u20090.04Fig. 5bAbcb4Abcb 4Rn00562185_m110.7\u2009\u00b1\u20090.6212.3\u2009\u00b1\u20090.1612.5\u2009\u00b1\u20090.2112.8\u2009\u00b1\u20090.1510.9\u2009\u00b1\u20090.157.3\u2009\u00b1\u20090.4213.2\u2009\u00b1\u20090.4612.4\u2009\u00b1\u20090.24Abcb6Abcb 6Rn00589801_m15.5\u2009\u00b1\u20090.355.9\u2009\u00b1\u20090.266.4\u2009\u00b1\u20090.256.5\u2009\u00b1\u20090.525.9\u2009\u00b1\u20090.385.4\u2009\u00b1\u20090.146.4\u2009\u00b1\u20090.256.2\u2009\u00b1\u20090.32Abcb9Abcb 9Rn00573757_m18.1\u2009\u00b1\u20090.208.5\u2009\u00b1\u20090.389.3\u2009\u00b1\u20090.298.8\u2009\u00b1\u20090.178.6\u2009\u00b1\u20090.108.7\u2009\u00b1\u20090.179.2\u2009\u00b1\u20090.428.9\u2009\u00b1\u20090.15Abcb11BsepRn00582179_m110.5\u2009\u00b1\u20090.408.6\u2009\u00b1\u20090.3710.3\u2009\u00b1\u20090.2411.9\u2009\u00b1\u20090.218.7\u2009\u00b1\u20090.175.4\u2009\u00b1\u20090.499.9\u2009\u00b1\u20090.219.7\u2009\u00b1\u20090.21Abcc1Mrp1Rn00574093_m15.9\u2009\u00b1\u20090.256.2\u2009\u00b1\u20090.266.1\u2009\u00b1\u20090.256.1\u2009\u00b1\u20090.256.3\u2009\u00b1\u20090.215.9\u2009\u00b1\u20090.236.4\u2009\u00b1\u20090.406.2\u2009\u00b1\u20090.15Abcc2Mrp2Rn00563231_m16.7\u2009\u00b1\u20090.31Fig. 5cAbcc3Mrp3Rn00589786_m18.4\u2009\u00b1\u20090.158.7\u2009\u00b1\u20090.128.8\u2009\u00b1\u20090.5088.6\u2009\u00b1\u20090.068.0\u2009\u00b1\u20090.358.2\u2009\u00b1\u20090.128.8\u2009\u00b1\u20090.328.7\u2009\u00b1\u20090.23Abcc4Mrp4Rn01465699_q16.6\u2009\u00b1\u20090.06Fig. 5dAbcc5Mrp5Rn00588341_m17.1\u2009\u00b1\u20090.017.9\u2009\u00b1\u20090.037.6\u2009\u00b1\u20090.156.4\u2009\u00b1\u20090.066.5\u2009\u00b1\u20090.506.5\u2009\u00b1\u20090.387.6\u2009\u00b1\u20090.456.4\u2009\u00b1\u20090.36Abcc6Mrp6Rn00578778_m17.0\u2009\u00b1\u20090.147.0\u2009\u00b1\u20090.018.4\u2009\u00b1\u20090.218.4\u2009\u00b1\u20090.127.8\u2009\u00b1\u20090.056.8\u2009\u00b1\u20090.358.6\u2009\u00b1\u20090.078.3\u2009\u00b1\u20090.14Abcc8Sur1Rn00564778_m111.5\u2009\u00b1\u20090.4011.4\u2009\u00b1\u20090.2111.4\u2009\u00b1\u20090.2613.4\u2009\u00b1\u20090.1514.8\u2009\u00b1\u20090.1513.2\u2009\u00b1\u20090.2511.7\u2009\u00b1\u20090.2313.7\u2009\u00b1\u20090.31Abcc9Sur2Rn00564842_m16.8\u2009\u00b1\u20090.017.7\u2009\u00b1\u20090.268.4\u2009\u00b1\u20090.318.1\u2009\u00b1\u20090.157.2\u2009\u00b1\u20090.326.8\u2009\u00b1\u20090.408.0\u2009\u00b1\u20090.358.2\u2009\u00b1\u20090.32Abcg1Abcg1Rn00585262_m16.8\u2009\u00b1\u20090.157.8\u2009\u00b1\u20090.256.8\u2009\u00b1\u20090.257.1\u2009\u00b1\u20090.406.8\u2009\u00b1\u20090.316.7\u2009\u00b1\u20090.216.4\u2009\u00b1\u20090.317.3\u2009\u00b1\u20090.36Abcg2Abcg2Rn00710585_m14.1\u2009\u00b1\u20090.244.5\u2009\u00b1\u20090.074.6\u2009\u00b1\u20090.215.0\u2009\u00b1\u20090.064.5\u2009\u00b1\u20090.144.5\u2009\u00b1\u20090.074.9\u2009\u00b1\u20090.425.2\u2009\u00b1\u20090.07Abcg5Abcg5Rn00587092_m118.8\u2009\u00b1\u20090.29baData of nine pooled samples, measured in triplicate, are expressed as mean \u00b1 SDbLow expressed transport protein: CT value is not considered as validFig.\u00a05mRNA expression of different renal ABC transporters. Gene expression of Abcb1a\/P-gp (a) and Abcb1b\/P-gp (b), Abcc2\/Mrp2 (c), and Abcc4\/Mrp4 (d) were determined in rat kidney at different time points after LPS\u2212 (open bars, n\u2009=\u20093) or LPS+ (closed bars, n\u2009=\u20096) treatment or LPS+ together with aminoguanidine (gray bars, n\u2009=\u20096). \u0394CT values for the LPS\u2212, given in Table\u00a02, were set to 1. Data are expressed as mean\u00b1SE. Significantly different compared to the LPS\u2212 (single asterisks P\u2009<\u20090.05, double asterisks P\u2009<\u20090.01, triple asterisks P\u2009<\u20090.001) or LPS+ (sharp signs P\u2009<\u20090.05)\nCoadministration with aminoguanidine blocked the induction in mRNA expression of both Abcb1b\/P-gp and Abcc2\/Mrp2 at 6 and 12\u00a0h, respectively (Fig.\u00a05b,c). Furthermore, aminoguanidine reduced the increased protein expression of both transporters 12\u00a0h after LPS+ treatment (Fig.\u00a06b,d), indicating a regulatory role for NO in the expression of these efflux transporters. Abcc4\/Mrp4 protein levels, on the other hand, decreased even more after treatment with both LPS+ and aminoguanidine. Gene expression levels of other transporters were not affected by aminoguanidine (Table\u00a02).\nFig.\u00a06Protein expression of different renal ABC transporters. Total membrane fractions of rat kidney were isolated and expression of Abcb1\/P-gp (a, b), Abcb11\/Bsep (c), Abcc2\/Mrp2 (d, e), and Abcc4\/Mrp4 (f, g) was determined by Western blotting. Representative images (n\u2009=\u20094) show maximal Abcb1\/P-gp protein expression 12 and 24\u00a0h after LPS+ (a), and coadministration with aminoguanidine reversed this induction (b). The protein expression of Abcb11\/Bsep was up-regulated 12\u00a0h after LPS+ treatment, which was reversed by aminoguanidine (c). Maximal Abcc2\/Mrp2 protein expression was seen 12\u00a0h after LPS+ (d) and aminoguanidine reversed this induction (e). Abcc4\/Mrp4 protein expression was down-regulated during endotoxemia (f), which was intensified by aminoguanidine (g)\nThe expression patterns of Abcb1\/P-gp, Abcb11, Abcc2\/Mrp2, and Abcc4\/Mrp4 were investigated further. Using total membrane fractions, we found an up-regulation of Abcb1\/P-gp 6\u00a0h after LPS+, with maximal protein expression 12 and 24\u00a0h after LPS+ (Fig.\u00a06a). Abcb11 is the major liver canalicular bile salt export pump (Bsep), but surprisingly, this transporter is also expressed at the apical membrane of mice renal proximal tubule, as we discovered recently [24]. In agreement with an increased mRNA expression, Abcb11\/Bsep protein was up-regulated after LPS+ (Fig.\u00a06c). Although we are not completely certain whether the C219 antibody against Abcb1\/P-gp also detects Abcb11\/Bsep [33], our Western blot results showed expression of Abcb1\/P-gp, with a molecular weight of 130\u00a0kDa, and it does not seem to cross-react with Bsep at 160\u00a0kDa.\nBy using immunohistochemistry, the expression and localization of Abcb11\/Bsep was investigated in endotoxemic rat kidneys. In agreement with mouse kidney, positive staining was observed in the apical membranes of the proximal tubule 12\u00a0h after LPS+ treatment (Fig.\u00a07). In addition, we observed staining in rat glomerular endothelium and peritubular capillaries.\nFig.\u00a07Distribution of Abcb11\/Bsep in rat kidney cortex. a, b Representative immunohistochemical images show apical staining in proximal tubules (indicated by arrows). In addition, glomerular and peritubular staining of Bsep in the cortex was found. G glomerulus, asterisks peritubular staining, P proximal tubule, D distal tubule. Original magnification in a \u00d7200, and in b \u00d7400\nFurthermore, a clear increase in Abcc2\/Mrp2 protein was observed after LPS+ (Fig.\u00a06d), but a down-regulation in Abcc4\/Mrp4 was found (Fig.\u00a06f). We measured the function of Abcc2\/Mrp2 by monitoring the urinary excretion of calcein in an isolated perfused rat kidney 12\u00a0h after LPS+ or LPS\u2212. A 16\u2009\u00b1\u20093% increase in calcein excretion was observed after LPS+, as shown in Fig.\u00a08. We do not expect calcein to be a Bsep substrate because Mrp2-deficient (TR\u2212) rats clearly showed that calcein excretion was highly reduced [25], despite the presence of Bsep in these strains (unpublished data). Twenty-four hours after LPS+, Abcc2\/Mrp2 activity was still elevated, although not significantly. In accordance, endotoxemia affected renal function as observed by diminished GFR and fractional reabsorption of water in isolated perfused rat kidneys (Table\u00a03).\nFig.\u00a08Renal calcein excretion in isolated perfused rat kidneys. Secretion of the fluorescent calcein into the urine was measured after 12\u00a0h in untreated (closed squares control; n\u2009=\u20094), LPS\u2212 (open squaresn\u2009=\u20094), or LPS+ (trianglesn\u2009=\u20096) rats. Urinary excretion was corrected for GFR. Significantly different compared to the LPS\u2212 (asterisk P\u2009<\u20090.05)Table\u00a03Functional parameters of isolated perfused rat kidneys in control rats (no additional treatment) and after exposure to LPS\u2212 or LPS+ for 12\u00a0hParameterControls (n\u2009=\u20094)LPS\u2212 (n\u2009=\u20094)LPS+ (n\u2009=\u20096)GFR (\u03bcl\/min)350\u2009\u00b1\u200925380\u2009\u00b1\u200917290\u2009\u00b1\u200920aDiuresis (\u03bcl\/min)18.6\u2009\u00b1\u20091.821.1\u2009\u00b1\u20091.317.4\u2009\u00b1\u20091.0FRwater (%)94.7\u2009\u00b1\u20090.294.5\u2009\u00b1\u20090.392.7\u2009\u00b1\u20090.6aRPP (mmHg)b88.4\u2009\u00b1\u20091.584.5\u2009\u00b1\u20091.788.7\u2009\u00b1\u20091.0Data are expressed as mean \u00b1 SE of four to six perfused kidney experiments over the period 25-115\u00a0min. Untreated rats served as controls for this model. Kidneys were perfused for 150\u00a0min as described in the \u201cMaterials and methods\u201d sectionaSignificantly different from perfused control kidney (P\u2009<\u20090.05)bConstant flow during the experiments was 18\u2009\u00b1\u20090.5\u00a0ml\/minFR fractional reabsorption, RPP renal perfusion pressure\nDiscussion\nIn the present study, we investigated the role of NO, produced by iNOS, in the differential expression of renal ABC transporters in rats in vivo. A clear up-regulation of Abca1, Abcb1\/P-gp, Abcb11\/Bsep, and Abcc2\/Mrp2 expression was found after LPS+. In addition, we observed a functional up-regulation of Abcc2\/Mrp2 in perfused kidneys. This supports a detoxifying role of the efflux pump, which is in accordance with the up-regulation of Abcc2\/Mrp2 protein in killifish renal tubules [34, 35], in rat kidney, and in MDCKII cells overexpressing human ABCC2\/MRP2 after long-term exposure to the nephrotoxicant gentamicin [20], and of abcb1\/P-gp in mouse kidneys after ischemic reperfusion injury [24]. Indeed, blocking Abcb1\/P-gp during endotoxemia leads to further renal damage (Heemskerk, Wouterse, Russel, and Masereeuw, preliminary findings). The renal drug transporters are likely to be under direct influence of NO produced by iNOS, as coadministration of the iNOS inhibitor, aminoguanidine, attenuated the LPS-induced effects on transporter expression. Aminoguanidine treatment results in decreased NO production and, subsequently, reduced oxidative stress by decelerating the formation of peroxynitrite. Under these conditions, less toxic metabolites may be produced, explaining the restoration in transporter expression and reversal of proximal tubular damage.\nIn contrast to our findings, Cherrington et al. [36] reported that coadministration of aminoguanidine failed to change the expression of different hepatic drug transporters during endotoxemia. This discrepancy might be due to organ differences and the fact that Cherrington et al. pretreated the animals with the NOS inhibitor, whereas we gave aminoguanidine 1\u00a0h after LPS+. The lack of an effect of aminoguanidine on the expression of different biotransformation enzymes, enzymes involved in the iNOS pathway and during oxidative stress, supports a direct function of NO itself in ABC transporter regulation.\nThe role of two up-regulated efflux pumps, Abca1 and Abcb11\/Bsep, in the kidney is unknown. In humans, ABCA1 is widely expressed and found to be the causative gene in Tangier disease, a disorder of cholesterol transport between tissues and the liver, mediated by binding of the cholesterol onto high-density lipoprotein particles [37]. ABCA1 controls the extrusion of membrane phospholipid and cholesterol toward specific extracellular acceptors; however, the exact role of the protein in this process is not known. Moreover, its expression and function in the kidney requires further investigation. The presence of Abcb11\/Bsep, the major canalicular bile salt export pump, was recently demonstrated by us in mouse kidney [24], and here we show a clear apical expression in rat kidney proximal tubules for the first time as well. Hence, the function of Abcb11\/Bsep in the kidney still needs to be determined. In addition to their up-regulation in the kidney, we found a down-regulation of Abcb1\/P-gp, Abcc2\/Mrp2, and Abcb11\/Bsep in the liver (data not shown). This is in agreement with previous findings for the hepatic expression of the transporters during inflammation [36, 38\u201340], obstructive jaundice [41], and cholestasis [42]. An induction of the transporters in the kidney during liver failure may function as a compensatory elimination mechanism to reduce enhanced levels of circulating bile salts caused by endotoxin-induced cholestasis [39] and\/or to protect proximal tubular cells against the higher exposure via primary urine.\nA differential regulation was found for Abcc4. Although this transporter is expressed in the same nephron segments as Abcb1\/P-gp and Abcc2\/Mrp2, the apically expressed efflux pump clearly has a distinct regulation. In killifish, we previously found renal proximal tubules exposed to nephrotoxicants or ET-1 that Abcc2\/Mrp2-mediated transport and Mrp2 expression in the luminal membrane were increased after 24\u00a0h [34], whereas the expression of Abcc4\/Mrp4 remained unchanged [35]. Furthermore, Abcc4\/Mrp4 protein levels in rat kidney were decreased during cholestasis in contrast to the Abcc2\/Mrp2 levels [43]. No changes in Abcc4\/Mrp4 mRNA were found, suggesting a posttranscriptional regulation of transporter expression, including phosphorylation and protein routing. A down-regulation of ABC transporters, as observed for Abcc8\/Sur1 and initially also for Abcb4, may be explained by the different functions of these ABC members in the kidney.\nLPS itself may affect transporter expression after binding to the Toll-like receptor 4 and a second, not yet identified, factor through activation of NOS-dependent pathways, as was found for Abcb1\/P-gp in rat brain capillaries [44]. Furthermore, systemic TNF-\u03b1, released during endotoxemia, may regulate efflux pumps, including ABC transporters [45, 46]. This cytokine might act through NOS activation [44], but also through a pathway that does not involve iNOS-derived NO during endotoxin-associated renal failure [47]. Furthermore, regulation of iNOS after exposure to LPS occurs at the level of gene transcription and may require activation of the NF-\u03baB pathway [48]. It has been shown previously that an up-regulation of Abcb1\/P-gp in renal failure, i.e., after ischemic\u2013reperfusion damage [24] or exposure to cadmium [49], might be signaled through an NF-\u03baB-dependent pathway. Consistent with these findings, we observed a clear induction (sevenfold) of NF-\u03baB expression 3\u00a0h after LPS+ as well.\nSelective inhibition of renal iNOS has important implications for the treatment of septic acute kidney disease. Changes in the expression levels of renal drug transporters alters the pharmacokinetics of many clinically important drugs [50] and has implications for the prediction of overall drug disposition during sepsis. For example, glucocorticoids are substrates for Abcb1\/Pg-p and are often prescribed for their anti-inflammatory properties in septic patients. An up-regulation of Abcb1\/Pg-p, as observed in our study, might have consequences for therapeutic levels of these substrates [51].\nIn conclusion, after LPS+, a clear up-regulation in Abca1, Abcb1\/P-gp, Abcb11\/Bsep, and Abcc2\/Mrp2 was found, whereas Abcc8\/Sur1 was down-regulated. Abcb4 and Abcc4\/Mrp4 show a differential pattern during the course of endotoxemia. The renal drug transporters are likely to be under direct influence of NO produced by iNOS, as coadministration of the iNOS inhibitor, aminoguanidine, attenuated the LPS-induced effects on their expression. Apparently, an up-regulation of important efflux pumps diminishes the renal accumulation of toxic compounds and attenuates further proximal tubular damage during endotoxemia.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material. \nSupplementary data (DOC 47.6 kb)","keyphrases":["inos","lipopolysaccharide","aminoguanidine","nephrotoxicity","renal drug transport"],"prmu":["P","P","P","P","P"]}
{"id":"Cell_Tissue_Res-4-1-2295254","title":"Accumulation of fibronectin in the heart after myocardial infarction: a putative stimulator of adhesion and proliferation of adipose-derived stem cells\n","text":"Stem cell therapy is a promising treatment after myocardial infarction (MI). A major problem in stem cell therapy, however, is that only a small proportion of stem cells applied to the heart can survive and differentiate into cardiomyocytes. We hypothesized that fibronectin in the heart after MI might positively affect stem cell adhesion and proliferation at the site of injury. Therefore, we investigated the kinetics of attachment and proliferation of adipose-tissue-derived stem cells (ASC) on fibronectin and analysed the time frame and localization of fibronectin accumulation in the human heart after MI. ASCs were seeded onto fibronectin-coated and uncoated culture wells. The numbers of adhering ASC were quantified after various incubation periods (5\u201330 min) by using DNA quantification assays. The proliferation of ASC was quantified after culturing ASC for various periods (0\u20139 days) by using DNA assays. Fibronectin accumulation after MI was quantified by immunohistochemical staining of heart sections from 35 patients, after different infarction periods (0\u201314 days old). We found that ASC attachment and proliferation on fibronectin-coated culture wells was significantly higher than on uncoated wells. Fibronectin deposition was significantly increased from 12 h to 14 days post-infarction, both in the infarction area and in the border-zone, compared with the uninfarcted heart. Our results suggest that a positive effect of fibronectin on stem cells in the heart can only be achieved when stem cell therapy is applied at least 12 h after MI, when the accumulation of fibronectin occurs in the infarcted heart.\nIntroduction\nCardiovascular diseases are a leading cause of morbidity and mortality in the western world (Wang and Sjoquist 2006), with heart failure attributable to myocardial infarction (MI) being the most common aetiology (Lu et al. 2004). Heart failure partly is caused by the loss of cardiomyocytes and the limited capacity of spontaneous regeneration of the heart. This results in the replacement of injured myocardium by scar tissue, which adversely affects cardiac function (Smits et al. 2005; Wang and Sjoquist 2006).\nTo reduce heart failure after MI, current therapeutic strategies focus on the restoration of the blood flow to the myocardium and on reducing cardiac remodelling by using drug therapy (inhibitors of angiotensin-converting enzyme and beta blockers; Fraser et al. 2004). Although these strategies significantly reduce mortality (Khalil et al. 2001; Lange and Hillis 2002), they do not replace the lost cardiomyocytes.\nA promising tool to restore contractile function after myocardial infarction is stem cell therapy. Stem cells not only restore neovascularization, but also replace lost cardiomyocytes, since they have the capacity to differentiate into cardiomyocytes (Orlic et al. 2001; Strauer et al. 2002; Wang and Sjoquist 2006). However, to restore contractile function of the heart effectively after MI, sufficient stem cells need to attach to the infarcted area and subsequently proliferate and differentiate into cardiomyocytes, thereby replacing a substantial fraction of the jeopardized cardiomyocytes. Unfortunately, recent studies suggest that only a small proportion of stem cells applied to the heart invade the infarcted area and finally differentiate into cardiomyocytes. Furthermore, most of the applied stem cells die within the first week after transplantation (Wang and Sjoquist 2006; Zhang et al. 2001).\nThis high stem cell death rate is probably caused by the inferior environment that is found in the heart after myocardial infarction and into which the cells are transplanted; this poor environment is caused by the presence of inflammatory mediators and ischemia (Azarnoush et al. 2005; Wang and Sjoquist 2006; Zhang et al. 2001). Therefore, stem cell therapy has to be applied at that moment after infarction when the environment is most favourable for stem cell adhesion and cardiomyocyte formation. However, to the best of our knowledge, this ideal time frame has yet not been determined (Mishra 2005). In most animal studies, investigators apply stem cells during the same operational procedure as the infarct induction, whereas recent studies have shown that later time points might be more favourable for the survival of the stem cells (Bermejo et al. 2006; Lu et al. 2004; Wang and Sjoquist 2006).\nHoming, growth and differentiation of stem cells after myocardial infarction is known to depend on several environmental factors, including the presence of adhesion factors at the site of injury (Chastain et al. 2006; Lu et al. 2004; Malek et al. 2006; Salasznyk et al. 2004; Wang and Sjoquist 2006). Fibronectin has been demonstrated to enhance the attachment of bone-marrow-derived stem cells in vitro, compared with several other extracellular matrix (ECM) molecules (Cool and Nurcombe 2005; Ogura et al. 2004). Fibronectin is a large molecular weight glycoprotein, present at low levels in the ECM of the healthy heart (Heng et al. 2004; Willems et al. 1996). In addition to the influence of fibronectin on cell attachment, the presence of fibronectin can also affect stem cell behaviour such as migration and proliferation, both in vitro and in vivo (Tate et al. 2002; van der Loo et al. 1998; Voermans et al. 1999; Wijelath et al. 2004). However, the effect of fibronectin on adipose-tissue-derived stem cells (ASC) has as yet not been investigated. Therefore, we have examined whether fibronectin stimulates the attachment of ASC and have compared this with the attachment of ASC to other ECM proteins that are normally expressed in the heart and that are upregulated after acute MI (AMI), namely collagen type 1 and laminin (Deten et al. 2001; Morishita et al. 1996). We have also investigated the effect of fibronectin on the proliferation of ASC in vitro.\nIn vivo, fibronectin is strongly upregulated in the heart after MI (Froen and Larsen 1995; Knowlton et al. 1992; Willems et al. 1996). We have hypothesized that fibronectin serves as an attractant for ASCs, thereby improving stem cell homing, attachment and proliferation. Therefore, we have studied the kinetics and the localization of fibronectin accumulation in the human heart after MI in detail, in heart sections from 35 patients, representing various infarction periods.\nMaterials and methods\nAdipose tissue donors\nHuman subcutaneous adipose tissue samples were obtained as waste material after elective surgery and donated upon informed consent of the patients from various clinics in Amsterdam, The Netherlands. Adipose tissue was harvested from the abdomen or hip and thigh region by using either resection or tumescent liposuction. Five donors (age-range: 27\u201349\u00a0years) were included in this study.\nIsolation of the stromal vascular fraction of adipose tissue\nAdipose tissue was stored in sterile phosphate-buffered saline (PBS) at 4\u00b0C and processed within 24\u00a0h after surgery as described previously (Oedayrajsingh-Varma et al. 2006). In brief, resected material was minced by using a surgical scalp before processing. After extensive washes with PBS, the ECM was enzymatically digested with 0.1% collagenase A (Roche Diagnostics, Mannheim, Germany) in PBS containing 1% bovine serum albumin (BSA; Roche Diagnostics) under intermittent shaking for 45\u00a0min at 37\u00b0C. Then the material was washed with Dulbecco\u2019s modified Eagle\u2019s medium (DMEM-glucose; BioWhittaker, Cambrex, Verviers, Belgium) containing 10% fetal bovine serum (FBS) and centrifuged for 10\u00a0min at 600g. The cell pellet was resuspended in PBS and passed through a 200-\u03bcm mesh (Braun\/Beldico, Marche-en-Famenne, Belgium) to obtain a single cell suspension. To remove contaminating erythrocytes, the cells were subjected to Ficoll density centrifugation (lymphoprep, \u03c1=1.077\u00a0g\/ml, osmolarity: 280\u00b115\u00a0mOsm; Axis-Shield, Oslo, Norway). The cell-containing interface was harvested and washed with DMEM containing 10% FBS. Cells were frozen under \u201ccontrolled rate\u201d conditions and stored in liquid nitrogen until needed in experiments.\nCell culture\nCells from the stromal vascular fraction were seeded at 1\u00d7105 cells\/cm2 and cultured for several passages in DMEM supplemented with 10% FBS, 100\u00a0U\/ml penicillin, 100\u00a0\u03bcg\/ml streptomycin, 2\u00a0mM L-glutamine (all from Gibco, Invitrogen, Calif., USA; normal culture medium), in a humidified atmosphere of 5% CO2 at 37\u00b0C. Media were changed twice a week. When reaching 80%\u201390% confluency, cells were detached with 0.5\u00a0mM EDTA\/0.05% trypsin (Gibco, Invitrogen) for 5\u00a0min at 37\u00b0C and replated.\nAttachment of ASC\nTo investigate attachment of ASC to different coatings, culture-expanded cells (passages 3\u20136; p3-6) were seeded in a 96-well culture plate at a density of 25,000 cells per well. Wells were either uncoated or coated with fibronectin (5 or 0.5\u00a0\u03bcg\/cm2; Roche Diagnostics), laminin (0.12\u00a0\u03bcg\/cm2; Roche Diagnostics) or collagen (2.5\u00a0\u03bcg\/cm2, rat tail collagen type I; BD Biosciences, Bedford, Mass., USA). Cells were then allowed to attach undisturbed in a humidified incubator (5% CO2, 37\u00b0C) for different incubation times (5\u00a0min, 10\u00a0min, 30\u00a0min), after which unattached cells were removed by washing with PBS. To standardize the conditions, all the conditions at the same timepoint were added simultaneously to one microplate. The number of attached cells was quantified by assaying for DNA with a CyQUANT Cell Proliferation Assay Kit (Invitrogen) according to the manufacturer\u2019s protocol.\nTo achieve an in vitro situation more comparable to the in vivo situation after AMI, attachment of ASC to cardiac muscle cells and to fibronectin-coated cardiac muscle cells was investigated. Cardiomyoblasts (H9c2, p18; ATCC, Manassas, Va., USA) were seeded into 24-well culture dishes, at a density of 10,000 cells per well, and when wells were 70% confluent, cells were either coated with fibronectin (0.5 or 5.0\u00a0\u03bcg\/cm2) or remained uncoated. Then culture-expanded cells (p3\u2013p7) from four donors were seeded into the culture dishes, at a density of 40,000 cells per well. Cells were allowed to attach undisturbed in a humidified incubator (5% CO2, 37\u00b0C) for various incubation times (5\u00a0min, 10\u00a0min, 30\u00a0min), after which the unattached cells were removed by washing with PBS. The cells were subsequently trypsinized, washed, centrifuged at 600g for 5 min, resuspended in 50\u00a0\u03bcl PBS and incubated with anti-CD90 phycoerythrin-labelled antibody (1:20; BD Biosciences Pharmingen, San Diego, Calif., USA) for 30 min on ice. The percentage of CD90-positive cells (ASC) was quantified by using fluorescence-activated cell sorting (FACS) analysis (FACScaliber, Becton Dickinson, San Jose, Calif., USA).\nProliferation of ASC\nTo assess ASC proliferation on fibronectin-coated culture dishes, culture-expanded cells (p3\u2013p6) from three donors were plated into 6-well culture plates, at a density of 25,000 cells per well, either coated with 5\u00a0\u03bcg\/cm2 fibronectin or uncoated, in normal culture medium. After a 1-day incubation in a humidified incubator (5% CO2, 37\u00b0C), unattached cells were removed by washing with PBS. Cells were then allowed to proliferate for various incubation times (0\u00a0days, 3\u00a0days, 6\u00a0days, 9\u00a0days). Cells were washed with PBS to remove dead cells. The amount of living cells removed by this washing was negligible (<0.5%). The number of attached cells was quantified by assaying for DNA with a CyQUANT Cell Proliferation Assay Kit (Invitrogen) according to the manufacturer\u2019s protocol.\nMyocardial tissue from infarction\nMyocardial tissue from 27 autopsied patients who had died from AMI and from eight autopsied patients with no cardiac lesions of any kind was obtained from the Department of Pathology for authopsy, with approval of the ethics committee of the VU Medical Centre, Amsterdam. The use of left-over material after the pathological examination is completed, is part of the standard patient contract in our hospital. Heart tissue was sampled as soon as possible, within 24 h of death.\nCharacterization of infarction phase\nRecent AMI was defined by using macroscopic (lactate dehydrogenase decolouration) and microscopic criteria to estimate infarct duration and the viability of cardiomyocytes in all myocardial tissue specimens (paraffin-wax-embedded sections stained with haematoxylin and eosin). Clinical data with respect to the duration of the infarction corresponded to the time intervals of the various morphological phases of infarction. Jeopardized myocardium was characterized by the intensity of eosinophilic staining of involved myofibres, cell condensation, loss of nuclei and cross striation and was visualized by complement (C3d) staining. We defined heart tissue sections without microscopic changes but with macroscopic lactate dehydrogenase decolourization as a phase 1 infarct (3\u2013 to 12-h-old AMI), infiltration of polymorphonuclear leucocytes as a phase 2 infarct (12-h to 5-day-old AMI) and infiltration of lymphocytes and macrophages and fibrosis as a phase 3 infarct (5\u2013 to 15-day-old AMI; Krijnen et al. 2003). Table\u00a01 shows the distribution of the various infarct phases among the different patients.\nTable\u00a01Characterization of the different infarct phases used in the immunohistochemical study (LDH lactate dehydrogenase)InfarctionphaseNumberof patientsInfarct durationDefinition08No infarctionDoes not apply1113\u201312 hLDH decolourization,without extravascularneutrophilic granulocytes.2912 h\u20135\u00a0daysExtravascular neutrophilicgranulocytes.375\u201314\u00a0daysGranulation tissueformation\nImmunohistochemistry\nMyocardial tissue was fixed with 4% buffered formaldehyde and embedded in paraffin. Paraffin sections (3\u20134\u00a0\u03bcm) were mounted on glass slides, dried, deparaffinized, rehydrated, and then used for immunohistochemistry. Sections were stained for fibronectin. Serial sections were stained for complement factor C3d to define the microscopical infarction area (Krijnen et al. 2003). All antibodies used were from DakoCytomation (Glostrup, Denmark).\nFor C3d staining, antigen retrieval was performed by boiling sections in 10\u00a0mM sodium citrate buffer, pH 6.0, for 10\u00a0min in a microwave oven. Sections were pre-incubated with normal swine serum (1:10; DakoCytomation) for 10 min at room temperature, followed by an incubation with rabbit antibodies against human C3d (1:1,000) for 1 h at room temperature. The sections were then washed with PBS and incubated with biotin-conjugated swine-anti-rabbit antibodies (1:300) for 30\u00a0min, washed in PBS and incubated with streptavidin-biotin complex\/horseradish peroxidase (1:200) for 1 h. Staining was visualized by using 3,3\u2032-diaminobenzidine (DAB; 0.1\u00a0mg\/ml, 0.02% H2O2).\nFor fibronectin staining, antigen retrieval was performed by incubating the sections with 0.1% pepsine-HCl for 30\u00a0min at 37\u00b0C. Sections were incubated with rabbit antibodies against human fibronectin (1:18,000) for 1\u00a0h at room temperature, followed by incubation with 100\u00a0\u03bcl envision (ready for use kit, anti-Mouse IgG and anti-Rabbit IgG) for 30\u00a0min at room temperature. Staining was visualized with envision DAB (DakoCytomation). Finally, the sections were counterstained with haematoxylin, dehydrated and cover-slipped. Control sections were incubated with PBS instead of the primary antibody.\nAll slides were judged and scored for infarct age and anatomical localization of fibronectin and C3d, as visualized by immunohistochemical staining. The border-zone of the infarct was defined as the area surrounding the microscopic infarction area, as characterized by C3d staining. Since only the fibronectin on the plasma membrane and in the ECM are available for ASC attachment, fibronectin deposition was scored separately for the ECM, plasma membrane and intracellular components (nucleus and cytoplasm). The extent of the fibronectin deposits was determined by quantifying the mean surface area occupied by cardiomyocytes positive for fibronectin and expressed as a percentage of the total surface area of the sections in the infarcted region, border-zone and non-infarcted areas. Areas were scored as negative (0), 1%\u20135% of the cells positive (1), 6%\u201325% positive (2), 26%\u201350% positive (3), 51%\u201375% positive (4) or 76%\u2013100% positive (5). Since positivity of fibronectin in the ECM is difficult to quantify, fibronectin staining in ECM was related to its intensity score as follows: negative (0), minor positive (1), intermediate positive (2) or strongly positive (3).\nStatistics\nStatistics were performed with the SPSS statistics program (Windows version 9.0) and Graphpad Prism (version 4). Attachment and proliferation data were analysed by using Repeated Measures. Fibronectin accumulation was analysed by the Kruskal Wallis and the Dunn\u2019s Multiple Comparison Test. A P-value of less than 0.05 was considered to be significant.\nResults\nAttachment and proliferation of ASCs on fibronectin\nTo investigate the attachment of ASC to various ECM molecules, the percentage of ASCs adhering to laminin, fibronectin, collagen-1 and uncoated culture wells was analysed. We monitored the number of ASCs in the culture wells and compared this with the numbers of cells in uncoated culture wells. As shown in Fig.\u00a01, after 5 and 10\u00a0min of incubation, significantly higher numbers of ASC were detected on fibronectin-coated culture wells as compared with the uncoated culture wells (P<0.05). This effect was dose-dependent, since a lower concentration of fibronectin (0.5\u00a0\u03bcg\/cm2 instead of 5.0\u00a0\u03bcg\/cm2) did not increase ASC attachment. Laminin and collagen-1 did not significantly affect ASC attachment, although collagen-1 showed a trend towards increased attachment of ASC. After 30 min, a plateau level seemed to be reached for all conditions. This suggested that ASC adhered more rapidly to fibronectin than to laminin, collagen or uncoated culture wells.\nFig.\u00a01ASC adherence to laminin-coated, collagen-1 (col-1)-coated and fibronectin (fibro)-coated (5 and 0.5\u00a0\u03bcg\/cm2) and uncoated culture wells monitored with time. Numbers of adhered ASCs were quantified after 5 (a), 10 (b) and 30 (c) min of attachment. Results are expressed as the percentage of applied cells. Error bars represent means\u00b1SEM (n=4, *P<0.05, Repeated measures)\nTo investigate the kinetics of ASC attachment in a setting more comparable to the in vivo situation after AMI, the percentage of ASCs adhering to fibronectin-coated and uncoated cardiac muscle cells was monitored with time. Numbers of adhered ASCs were quantified after 5, 10 and 30 min of attachment and are shown as the percentage of total cells applied in Fig.\u00a02. ASC rapidly adhered to the fibronectin-coated cardiac muscle cells. This rapid attachment was most prominent after 5 min of incubation, when 5 times more ASC attached to the fibronectin-coated cells than to the uncoated cells (P<0.05). This effect was independent of the dose of fibronectin, since the ASC also attached rapidly to the cardiac muscle cells coated with a lower concentration of fibronectin (0.5\u00a0\u03bcg\/cm2 instead of 5.0\u00a0\u03bcg\/cm2). On the uncoated cardiac muscle cells, however, attachment was limited when compared with the fibronectin-coated cells and increased only gradually the first 10 min. For all conditions, a plateau level was reached after 30 min of attachment. These findings suggested that ASC attached more rapidly on fibronectin-coated cardiac muscle cells than to uncoated cardiac muscle cells.\nFig.\u00a02Kinetics of ASC adherence to fibronectin-coated (5 and 0.5\u00a0\u03bcg\/cm2) and uncoated cardiac muscle cells monitored with time. Numbers of adhered ASCs were quantified after 5 (a), 10 (b) and 30 (c) min of attachment. Results are expressed as the percentage of applied cells. Error bars represent means\u00b1SEM (n=4, *P<0.05, Repeated measures)\nTo study the proliferation of ASCs on fibronectin, ASCs were seeded in fibronectin-coated culture wells. Unattached cells were washed away after 24 h (day 0). We monitored the number of ASCs in the culture wells with time (days 0, 3, 6, 9) and compared this with the numbers of cells in uncoated culture wells. Significantly higher numbers of ASC were detected on fibronectin-coated culture wells compared with the uncoated culture wells during the 9-day culture period (P=0.012, Repeated measures, n=3; Fig.\u00a03).\nFig.\u00a03Kinetics of ASC proliferation on fibronectin-coated (5\u00a0\u03bcg\/cm2) and uncoated culture wells. Numbers of adhered ASCs were quantified after 0, 3, 6 and 9\u00a0days of culture by using the CyQUANT cell proliferation assay. Results are expressed as percentage of cells, compared to day 0, both for the fibronectin-coated and uncoated wells. Error bars represent means\u2009\u00b1\u2009SEM. (n\u2009=\u20093, p\u2009=\u20090,012, Repeated measures)\nFibronectin accumulation after infarction\nFibronectin accumulation after myocardial infarction was investigated in heart sections from 35 patients, representing various infarction periods (no infarction, infarction of 3\u201312\u00a0h old, 12\u00a0h\u20135\u00a0days old, and 5\u201314\u00a0days old). To define jeopardized cardiomyocytes and thus the microscopical infarction area, we stained serial sections for complement factor C3d (Fig.\u00a04c). Fibronectin was found not only in cardiomyocytes (plasma membrane, cytoplasm and nucleus; Fig.\u00a04a), but also in the ECM (Fig.\u00a04b). Fibronectin co-localized with complement factor C3d but fibronectin deposition was also found in the border-zone adjacent to the infarction area (complement-negative). To analyse fibronectin depositions in the human heart after myocardial infarction, we scored all sections for the extent and the anatomical localization of fibronectin depositions. Notably, in AMI phase 1, the border-zone and infarcted area could not be analysed as such with the slides from the macroscopical infarction area, since it was not possible to identify a microscopical infarction area clearly at this early phase, as complement was then negative. As shown, the intensity of fibronectin depositions in the ECM (Fig. 5) was significantly increased at the site of myocardial infarction of AMI phases 2 (P<0.01) and 3 (P<0.05) compared with the uninfarcted heart. The extent of fibronectin deposition was also increased in the border-zone of phases 2 and 3; however, this increase was not statistically significant. In phase 1 infarction and in the uninfarcted areas, no increase in ECM fibronectin intensity score was found, when compared with uninfarcted hearts (not shown).\nFig.\u00a04Localization of fibronectin in myocardial infarction of the human heart. Fibronectin accumulation after myocardial infarction was investigated in heart sections representing different infarction periods. a High power view of fibronectin deposition in infarct phase 2. Fibronectin depositions were found on the plasma membrane (arrow I), in the nucleus (arrow II) and in the cytoplasm (arrow III) of jeopardized cardiomyocytes. b Fibronectin depositions in infarct phase 3 in the extracellular matrix (ECM; arrow IV). c C3d deposition in infarct phase 2 (+ jeopardized cardiomyocytes, i.e. complement-positive cells, \u2212 complement-negative cells). d Fibronectin colocalization within C3d-positive areas in a serial section from the same patient as in c, in infarct phase 2. As a control, AMI sections were incubated with PBS instead of mAb and gave no positive staining (not shown)Fig.\u00a05Fibronectin deposition in the ECM of the human heart after myocardial infarction. Intensity of the fibronectin deposition in the ECM was scored and compared with that of the non-infarcted heart (stadium 0). Areas were scored negative (0), minor positive (1), intermediate positive (2) or strongly positive (3). Error bars represent means\u00b1SEM (*P<0.05, **P<0.01)\nWe subsequently scored fibronectin positivity in the cardiomyocytes. The extent of fibronectin deposition was significantly increased in the infarcted area of AMI phase 2, compared with controls, at the plasma membrane (P<0.05), in the cytoplasm (P<0.001) and in the nucleus (P<0.05). Fibronectin depositions were increased not only in the infarcted area, but also in the border-zone of phase 2; however, this increase was only statistically significant in the nucleus when compared with the non-infarcted heart. In AMI phase 3, no cardiomyocytes in the infarction area could be analysed since this area no longer contains cardiomyocytes in this stadium, but only granulation tissue. The extent of fibronectin deposition was still significantly increased in the border-zone of AMI phase 3 in the nucleus and cytoplasm but was not positive on the plasma membrane (Fig.\u00a06a). Neither in AMI phase 1 (Fig.\u00a06) nor in non-infarcted areas (not shown) was an increase in fibronectin positivity found in the cardiomyocytes when compared with controls (Fig.\u00a06).\nFig.\u00a06Fibronectin deposition in cardiomyocytes in the human heart after myocardial infarction (IH immunohistochemical). a Area of fibronectin deposition in the plasma membrane. b Area of fibronectin deposition in the cytoplasm. c Area of fibronectin deposition in the nucleus. Areas are scored from 0 for no fibronectin-positive cells up until 5 for 75%\u2013100% fibronectin-positive cells. Error bars represent means\u00b1SE (*P<0.05, **P<0.01) (Phase 0: n=8, phase 1: n=11, phase 2 border: n=6, phase 2 infarct: n=9, phase 3 border: n=7)\nDiscussion\nWe hypothesized that accumulation of fibronectin in the heart after MI might improve the fate of ASC after transplantation in the heart. Therefore, we have studied the effect of this protein on the attachment and proliferation of ASC in vitro. In addition, we have investigated the time course and the localization of the accumulation of fibronectin after myocardial infarction in the human infarcted heart. We have found that ASC attachment and proliferation on fibronectin-coated plates is significantly increased compared with that on uncoated plates (early attachment: P<0.05, proliferation: P=0.009, Repeated measures, n=4) and that attachment of ASC to cardiac muscle cells is significantly increased when cells are coated with fibronectin (0.5\u00a0\u03bcg\/cm2 and 5.0\u00a0\u03bcg\/cm2), after 5 min of attachment. In the ECM of the human heart, fibronectin depositions are significantly increased at AMI phase 2 and phase 3 in the infarction area, whereas fibronectin deposition in the cytoplasm, at the plasma membrane and in the nucleus of cardiomyocytes is significantly increased at AMI phase 2, but not in AMI phase 3, except for depositions in the nucleus.\nWhen culture wells are coated with fibronectin, we have found an increased attachment of ASC when compared with uncoated plates, even though ASC are mesenchymal stem cells that are defined by their strong attachment to plastic culture wells (Wagner et al. 2005). We have also found that neither laminin nor collagen-1 significantly increases the attachment of ASC, unlike fibronectin, which does cause an increase. These findings are comparable with studies of Ogura et al. (2004) and Cool and Nurcombe (2005), in which an increased attachment of human bone-marrow-derived MSC to fibronectin-coated culture plates has been found when compared with albumin-coated or other ECM-coated culture plates. Ogura et al. (2004) have also reported that attachment to fibronectin is most prominent within the first 20 min of incubation. We now have shown that fibronectin also improves the attachment of ASC.\nIn an experiment mimicking the in vivo situation after a myocardial infarction, we have demonstrated that fibronectin also increases the attachment of ASC to cardiac muscle cells, even when a relatively low concentration of fibronectin is used. These results are in agreement with our hypothesis that fibronectin depositions will also increase stem cell attachment in vivo, independent of fibronectin concentration, underlining the importance of fibronectin in stem cell transplantation.\nIn this study, we have additionally demonstrated a significant increase in the proliferation of ASC cultured in fibronectin-coated wells, compared with proliferation in uncoated culture wells (P=0.012). This effect is most prominent during the first 5\u00a0days of culture; thereafter, the cells grow confluently and the effect diminishes. Cool and Nurcombe (2005) have found no significant effect of fibronectin on proliferation, although a trend is visible; this might be caused by the timepoint of their study, since they have only measured the difference in cell numbers at day 7, or could be related to the source of stem cells.\nSince the presence of fibronectin promotes ASC attachment and proliferation in vitro in our study, we have hypothesized that the presence of fibronectin in the heart after myocardial infarction might positively affect stem cell adhesion and proliferation at the site of injury. As such, stem cell therapy would be most efficient when fibronectin depositions are high. We have found an increase in the extent of fibronectin depositions in cardiomyocytes during AMI phase 2, whereas fibronectin deposition in the ECM is significantly increased in the infarction area during AMI phase 2 and phase 3, when compared with the uninfarcted heart.\nThese results are comparable with the findings of several animal studies, in which increased fibronectin mRNA expression has been demonstrated, together with increased fibronectin deposition in the infarcted area as early as the first day after infarct induction (Carlyle et al. 1997; Froen and Larsen 1995; Knowlton et al. 1992; Shekhonin et al. 1990; Ulrich et al. 1997). Moreover, several studies have shown increased deposition of fibronectin in the infarction area after MI in human (Hu et al. 1996; Ishikawa et al. 2000, 2003; Ortmann et al. 2000; Willems et al. 1996). However, none of these studies have scored the fibronectin depositions as accurately as we have during the different infarction phases, i.e. by using complement to define the microscopic infarction area, by differentiating between infarction area and border-zone and by separately scoring the ECM and the various anatomical locations in the cells.\nWe have studied the various anatomical locations of fibronectin depositions separately, since theoretically only fibronectin on the plasma membrane and in the ECM should play a direct role in stem cell attachment. Although fibronectin has been detected on the ECM and the plasma membrane of cardiomyocytes, depositions in the cytoplasm and the nuclei are more widespread than on the plasma membrane. These strong depositions in the nucleus and cytoplasm have also been reported by Ishikawa et al. (2000); however, they have only described these nuclear depositions and not scored them.\nNotably, we have found that the extent of fibronectin depositions in the heart is only increased in the infarcted area and the border-zone, and not in the non-infarcted areas. This is an important observation, since stem cell attachment and differentiation is only desirable in the infarcted area and the area surrounding it for stem cell therapy. In accordance with this finding, Tran et al. (2006) have indeed shown that rat bone marrow MSC are retained better in the infarcted area, when compared with the intact myocardium.\nSeveral other studies have also investigated the time frame after MI with respect to when best to apply stem cell therapy, both in animal models and human stem cell transplantation studies, by investigating molecular events and stem cell retention at the site of infarction (Bermejo et al. 2006; Lu et al. 2004; Ma et al. 2005). These studies suggest that stem cell therapy should neither be applied during the acute inflammation phase (within 1\u00a0day after infarction), nor after a period longer than 2\u00a0weeks when scar tissue has been formed. In agreement with this suggestion, our results indicate that a putative effect of fibronectin on stem cell therapy can only be achieved when stem cell therapy is applied more than 12 h but within 14\u00a0days after infarction.\nIn conclusion, we have shown, for the first time, that fibronectin improves ASC attachment and proliferation. To achieve a positive effect of fibronectin on stem cell therapy after AMI in the heart, this therapy should be applied when fibronectin depositions in the heart are high. We have accurately scored fibronectin deposition in the human heart after AMI and found that the fibronectin intensity score significantly increases from 12\u00a0h after MI in both the infarction area and the border-zone.","keyphrases":["heart","infarction","adhesion","stem cell","human","adipose tissue"],"prmu":["P","P","P","P","P","P"]}
{"id":"Pediatr_Radiol-4-1-2292495","title":"Malfunctioning central venous catheters in children: a diagnostic approach\n","text":"Central venous access is increasingly becoming the domain of the radiologist, both in terms of the insertion of central venous catheters (CVCs) and in the subsequent management of these lines. This article seeks to provide an overview of the CVC types available for paediatric patients and a more detailed explanation of the spectrum of complications that may lead to catheter malfunction. A standard catheter contrast study or \u2018linogram\u2019 technique is described. The normal appearances of such a study and a detailed pictorial review of abnormal catheter studies are provided, together with a brief overview of how information from catheter investigations can guide the management of catheter complications.\nIntroduction\nCentral venous access forms a vital part of the management of many paediatric conditions. For many years, tunnelled central venous catheters were largely reserved for patients on chemotherapy regimens or long-term total parenteral nutrition (TPN), but more recently, reliable central venous access has proved central to the management of an extensive range of conditions such as those requiring long-term antibiotic therapy, chemotherapy or haemodialysis. The emergence of image-guided vascular access techniques has brought central venous access increasingly into the domain of interventional radiology (IR), with perceived advantages of shorter operating times, an increased likelihood of achieving access in difficult cases, fewer procedural complications, lower operating costs, and a probable improvement in long-term venous patency rates [1\u201310]. This shift from general surgery to radiology has, however, brought with it an increased demand on radiology departments to maintain and manage indwelling central venous catheters (CVCs). In children, meticulous attention to catheter position is vital to ensure that CVCs are kept functioning for as long as possible in order to minimize the number of catheter replacements a child may have to undergo. In some practices, malfunctioning catheters are removed and replaced without investigation; in other centres, including our institution, it is recognized that there are a number of interventions available to salvage blocked or displaced catheters, allowing alternative access sites to be preserved for future use. Plain radiographs and fluoroscopic contrast examinations are central to the radiological investigation of malfunctioning catheters [11, 12]. Many radiologists, however, may be perplexed by the increasingly wide variety of venous access devices available and unaware of some of the potential CVC-related complications that develop in children or the catheter-salvage techniques available.\nThis article gives a brief overview of the central venous access devices available and the potential causes of catheter malfunction. A standard catheter contrast study or \u2018linogram\u2019 technique is described. The normal appearances of such a study and a detailed pictorial review of abnormal catheter studies are provided, together with a brief overview of how information from catheter investigations can guide the management of catheter complications.\nBackground\nCentral venous access is required for the safe delivery of a number of drugs and other intravenous therapies into large-calibre vessels, to allow adequate dilution of the drug and avoid vessel irritation. Here the term \u2018central\u2019 is taken to encompass the brachiocephalic veins, the superior vena cava (SVC), the suprahepatic inferior vena cava (IVC) and the right atrium (RA). A centrally placed catheter with a reasonable-size lumen also allows regular noninvasive blood sampling, which has significant benefits for both the clinical team and the patient.\nThere is much debate surrounding the issue of the ideal CVC tip position and a detailed presentation of this argument is beyond the remit of this article. Suffice to say that the argument that a catheter tip position at the level of the RA is unsafe due to the risk of myocardial perforation and subsequent tamponade is increasingly unsupported in modern practice with the advent of softer catheter tips, with the probable exception of 2F catheters, traditionally inserted by the cot-side as neonatal long lines, which have a slightly stiffer tip. There is much in the literature regarding cardiac perforation by 2F neonatal long lines in low-birth-weight infants and it seems generally accepted that these catheters are safer when positioned outside the pericardial reflections [13\u201320]. This paper, however, deals specifically with radiologically or surgically inserted Silastic (silicone elastomer) catheters of 4F calibre and above. It is now recognized that these larger-calibre catheters are significantly less likely to develop tip thrombus or a fibrin sheath when left to move freely within fast-flowing blood at the level of the right atrium and are likely to function for a significantly longer period of time in this position [8, 21\u201325]. The position of the RA is best estimated at a level 1.5 vertebral bodies below the carina on a chest radiograph [26, 27]. It is worth remembering, however, that the position of a catheter tip will vary widely with patient positioning, particularly in children, and should be evaluated with caution on any image [8, 22, 28\u201330].\nCatheter types\nThere is a vast array of central venous access devices available for paediatric use; particular devices may be preferred in different centres. In general, however, catheter subtypes can be defined according to their site of insertion, whether they are placed via a subcutaneous tunnel or, with particular reference to haemodialysis catheters, according to function. To achieve a central tip position, a catheter can be inserted using a tunnelled or a nontunnelled technique. Tunnelled devices are usually inserted via access to a neck vein and are tunnelled to that site through the adjacent subcutaneous tissues, exiting the skin at an appropriate position on the chest wall. The tunnelled, extravascular portion of the catheter is considered to provide a barrier to ascending infection and with time will adhere to the subcutaneous tissues, decreasing the likelihood of catheter displacement. This adherence is encouraged by the presence of a short Dacron cuff attached to the catheter, which causes a focal region of fibrosis or ingrowth in the track and thus provides further anchorage for the catheter (Fig.\u00a01). Insertion of such catheters almost always requires general anaesthesia in children to allow safe venous puncture. The vein puncture itself can be performed using a percutaneous, US-guided technique or a surgical open venotomy. Removal of a cuffed catheter that has been in situ for longer than a week or so generally also requires a general anaesthetic in children, as removal or dissection of the cuff can be traumatic. Hence, tunnelled CVCs are best suited for medium- to long-term venous access requirements such as chemotherapy and TPN regimens. Tunnelled catheters range in calibre from 2.7F to 12F and have a single or double lumen. Haemodialysis catheters are very similar to other double-lumen catheter devices. The lumens are generally of a larger calibre, however, to allow high flow rates for efficient dialysis and the end holes or tips may be staggered (Fig.\u00a02).\nFig.\u00a01A 4F single-lumen Hickman (Bard Access Systems, Salt Lake City, Utah) catheter for tunnelled central venous access. Inset illustrates the Dacron ingrowth cuffFig.\u00a02A permanent (tunnelled) haemodialysis catheter with staggered tip for optimal flow rates (12.5F Vas-Cath Soft Cell; Bard Access Systems, Salt Lake City, Utah)\nAn alternative to a traditional tunnelled CVC is a totally implanted CVC or implanted venous access device (iVAD), commonly known as a portacath. This comprises a reservoir attached to a standard Silastic catheter (Fig.\u00a03). The catheter is inserted using a standard tunnelled catheter technique. The reservoir is buried within the subcutaneous tissues of the chest wall, so that the device is completely implanted. When required, the reservoir is accessed percutaneously via a specialised noncoring needle. The reservoir can be accessed up to 1,000 times before replacement is needed. Such buried devices have a lower infection rate than externalized catheters and allow greater freedom for social activities such as swimming, but require patients to undergo repeated needle access procedures. They are best suited for intermittent therapies such as enzyme infusions in metabolic disorders, factor infusions in haemophilia or recurrent antibiotic requirements for conditions such as cystic fibrosis.\nFig.\u00a03A totally implantable port (Bard Access Systems, Salt Lake City, Utah) with reservoir and 6.6F catheter. Inset illustrates the specialized noncoring port access needle\nNontunnelled CVCs are placed directly into a vein via a skin incision overlying the vein and are simply sutured to the adjacent skin (Fig.\u00a04). Access sites include the neck and the extremities. Nontunnelled neck lines are commonly placed for complex anaesthesia cases or in the intensive care setting. The neck veins can also be used for short-term dialysis catheters in patients who do not require a longer-term tunnelled device. For longer-term indications for a nontunnelled line, operators usually prefer to access venous sites in the extremities. Small-calibre (4\u20135F) peripherally inserted central catheters (PICCs) are usually placed via the veins of the upper arm (Fig.\u00a05). In a cooperative child they can often be placed via a peripheral venous cannula, without sedation or general anaesthesia, and are most useful for providing reliable access for medium-term therapies in children. The catheter is advanced to the level of the central vessels using fluoroscopic guidance in the radiology department or a non-image-guided \u2018best-estimate\u2019 technique at the bedside. These catheters provide reliable venous access for indications such as extended antibiotic treatment in patients with osteomyelitis or infected neurosurgical shunts and for repeated blood-sampling in needle-phobic patients, or children in whom peripheral access is becoming technically difficult. PICCs are also increasingly used for longer-term therapies such as chemotherapy, pulmonary hypertension and TPN, and can function for a year or more. Single-lumen peripherally inserted CVCs or PICCs are commonly 4F in calibre; this allows more reliable function than the 2F neonatal long lines, particularly for blood sampling (aspiration via a catheter requires a faster flow rate in the vessel being sampled, and is therefore more sensitive to lumen calibre when compared with infusion through the same catheter). Double-lumen 5F catheters are also widely used, although each lumen is small and hence these catheters, although useful for multidrug therapies, are less reliable for blood sampling. Some types of PICC are now approved for CT contrast medium infusions via a pump injector.\nFig.\u00a04A triple-lumen 7F catheter (Arrow-Howes; Arrow International, Reading, Pa.) for short-term nontunnelled central venous accessFig.\u00a05A double-lumen 5F PICC (Bard Access Systems, Salt Lake City, Utah)\nStrategies in managing catheter malfunction\nCVC malfunction can be approached by the clinical team and the radiologist in a number of ways. In some centres a policy of removing all malfunctioning catheters without investigating the cause of the malfunction is maintained. In most institutions, however, protocols are available for attempted thrombolysis of blocked catheters and in some units revision of displaced or incorrectly positioned lines is attempted. In our opinion, an attempt should always be made to diagnose the cause of a malfunctioning catheter, with the aim of possible catheter salvage and, therefore, a potential reduction in the number of both repeated anaesthesia and venous access attempts that a patient may have to undergo. An institutional policy regarding the management of malfunctioning CVCs is helpful and should be made with the facilities of that institution in mind. In particular, contrast studies involving radiation exposure should only be undertaken if the results of the study would alter the subsequent management of the line. In general, an IR department is more likely to be able to offer salvage procedures than a surgically managed service, due to differences in both the imaging and interventional techniques available within each speciality [3, 10, 31].\nInitial examination of the catheter\nIt is important to examine both the catheter and its skin exit site prior to performing any radiographic investigation, so that a simple cause of catheter malfunction, such as a break in the catheter itself or wear at the clamp site, can be established. It is not uncommon to find that the skin suture of a newly placed catheter is too tight and is restricting flow.\nFluoroscopic or plain radiographic views\nPlain radiographic views of the catheter should be acquired prior to any contrast-enhanced study. These can be acquired as a chest radiograph or by using low-dose fluoroscopy at the time of a planned contrast investigation. This allows misplaced catheters (Fig.\u00a06) and catheters of an incorrect length to be identified (Fig.\u00a07). The entire catheter must be imaged. Studies limited to the intravascular portion of the catheter will frequently miss abnormalities such as kinking of the catheter within the skin, a finding that is particularly common with portacaths (Fig.\u00a08), and looping or twisting of a catheter in the neck (Fig.\u00a09). More than one view may be required to confirm a kink in the catheter of a portacath; angulation of the catheter most commonly occurs at the origin of the subcutaneous tunnel, adjacent to the reservoir (Fig.\u00a010). Occasionally, a portacath reservoir can become detached from its catheter, and children who are compulsive \u2018port fiddlers\u2019 can encourage a catheter to loop around the port chamber so that over time the intravascular portion is shortened. A catheter may be accidentally pulled back or may migrate backwards into the soft tissues of the neck, so that the tip lies outside the vein entirely. This second complication seems to occur most commonly in chubby infants with redundant tissue in the neck (Fig.\u00a011). Despite accurate initial placement, catheter tips can flick into an incorrect position with changes in posture or intrathoracic pressure; this occurs more commonly with catheter tips placed at the level of the SVC rather than the RA. The commonest site for a catheter tip to migrate to is the ipsilateral internal jugular vein or the contralateral brachiocephalic vein (Fig.\u00a012). Observing the catheter tip during fluoroscopic screening, even before injecting contrast medium, allows the operator to determine whether the tip is moving freely or is wedged against a vessel or chamber wall. Exaggerated movement of the catheter when in an intracardiac position is strongly suggestive of malposition of the catheter tip within the right ventricle; most operators would agree that such a line should be shortened or replaced in such a situation, even if the child is asymptomatic, due to the risk of catheter-induced arrhythmias. Fluoroscopic screening should be performed using a low pulse rate (three pulses per second) and where available, the image-grab facility should be used to minimize radiation exposure.\nFig.\u00a06Misplaced catheter. a Fluoroscopic image of a 10-month-old male with a portacath in situ, the tip of which has been placed into the coronary sinus. b The position of the tip is confirmed by a contrast medium studyFig.\u00a07A chest radiograph in a 13-month-old male with a permanent haemodialysis catheter, the tip of which lies at or beyond the level of the tricuspid valve causing intermittently poor flow ratesFig.\u00a08Kinking of the catheter. a Chest radiograph of a 6-year-old male with a portacath in situ. The catheter is kinked just beyond the reservoir (arrow). b Fluoroscopic image of a femoral temporary dialysis catheter in a 15-year-old male. The catheter is kinked at the vessel entry site, causing poor flow ratesFig.\u00a09Fluoroscopic image of a tunnelled catheter in a 2-year-old child. The catheter has twisted in the soft tissues of the neck, causing the catheter tip to be partially withdrawn (arrow)Fig.\u00a010Kinking of the catheter. a, b AP (a) and lateral (b) views of a portacath in a 2-year-old female demonstrating sharp angulation of the proximal catheter, limiting infusion flow rates. The kink is not clearly seen on the AP viewFig.\u00a011Chest radiograph of a 9-month-old infant with a tunnelled CVC in situ. The catheter has migrated back into the redundant soft tissues of the neck and chest wallFig.\u00a012Migration of the catheter tip. a Chest radiograph of a 4-year-old male with a portacath in situ. The catheter tip lies in the right internal jugular vein (short arrow). Note that the proximal catheter is also kinked (long arrow). b Chest radiograph of a 2-year-old female with a tunnelled CVC in situ. The catheter tip lies in the left brachiocephalic vein\nPerforming a contrast study\nIf the initial examination of the line is unremarkable, a contrast-enhanced study (\u2018linogram\u2019) should be performed. The catheter should be accessed using a sterile technique and, where possible, any heparin solution dwelling in the line should be aspirated and discarded. Inability to aspirate from the catheter should be documented, as this narrows the list of potential causes of catheter malfunction, as discussed below. If only one lumen of a multilumen catheter is malfunctioning, imaging should first be performed through the functioning lumen as this may demonstrate the underlying problem and obviate the need for access of the second lumen. Water-soluble nonionic contrast medium should be used and the operator should aim to minimize the dose of contrast medium that the child receives.\nThe initial contrast medium injection should be performed slowly while the entire catheter is screened so that complications anywhere along the length of the line are readily identified (Fig.\u00a013). The operator should look for signs of pooling of contrast medium in the soft tissues surrounding the catheter. Extravasation and pooling of contrast medium in the neck are usually painful; a slow gentle injection will minimize both the patient\u2019s discomfort and the risk of loss of the child\u2019s confidence in the operator. When examining a malfunctioning portacath device, tangential and en face views of the port reservoir should be obtained, to ensure that it fills uniformly and does not contain thrombus (Fig.\u00a014). If the initial slow injection of contrast medium appears normal, a controlled but forceful hand injection should then be performed to study in more detail the flow of contrast medium away from the catheter tip. Digital subtraction angiography (DSA) should be used during image acquisition where possible, to achieve a diagnostic study with one injection, minimizing both contrast agent and radiation dose. Cooperative patients should be encouraged to breath-hold. If the study is performed in an imaging suite without DSA capability, screening should be focused on the catheter tip and the fluoroscopic pulse rate may have to be increased to either 15 pulses per second or \u2018continuous\u2019 screening to obtain adequate images. Diagnostic imaging of the contrast medium jet is hardest in small patients with rapid breathing and significant cardiac motion.\nFig.\u00a013Fluoroscopic imaging of a tunnelled catheter in a 4-month-old female. The entire catheter is imaged during slow instillation of contrast mediumFig.\u00a014Normal portacath. a, b Tangential (a) and en face (b) views of a portacath reservoir in a 7-year-old male. Note that in both views there is normal opacification of the reservoir during contrast medium instillation\nThe contrast medium jet should be of the same diameter as the lumen of the catheter and should exit the catheter in a linear forward direction, rapidly moving away from the catheter tip rather than pooling at the tip. The jet should fan out appropriately to fill the lumen of the vessel or chamber (Fig.\u00a015). The operator should be aware of the configuration of the catheter, so that a catheter with a side hole, such as the Groshong PICC (Bard Access Systems, Salt Lake City, Utah) is not misinterpreted as a fractured or partially occluded line (Fig.\u00a016).\nFig.\u00a015Fluoroscopic image of contrast medium flowing freely from the catheter tip in an 8-year-old male. The contrast medium immediately fans out to fill the right atriumFig.\u00a016Normal appearance of the contrast medium jet exiting the side hole of a PICC\nIt is important to remember to study both lumens of a double-lumen catheter if injection of the first lumen is normal. In patients with intermittent catheter malfunction, attempts should be made to recreate the situation in which problems occur; often, catheter function is position-dependent and placing the child in the troublesome position may demonstrate the cause of catheter malfunction. This may include a significant change in tip position following a change in posture or intermittent occlusion of the catheter lumen at the level of the clavicle due to catheter compression between the clavicle and the first rib (\u2018pinch-off\u2019 syndrome).\nIf the child presents with symptoms of venous occlusion, such as localized pain and swelling over the venous access site or sudden swelling of the face or extremity, a formal venogram via a peripheral cannula can be performed to determine the presence of catheter-related venous thrombosis (see below).\nOnce the study is completed, the catheter should always be flushed and, where required, \u2018locked\u2019 with heparin solution of an appropriate concentration. This should be documented in the clinical notes as part of the examination. If the catheter is deemed unsafe for use, this too should be clearly documented.\nInterpreting an abnormal contrast study\nOnce contrast medium has been instilled, a careful examination of the entire length of the catheter should be performed. Abnormal findings may be subtle, such as narrowing of the lumen at the site of the skin suture or clamp (Fig.\u00a017). Contrast medium will leak into the soft tissues at the site of any break in the catheter wall (Fig.\u00a018). In such cases the point of extravasation can often be clinically predicted by the patient\u2019s clinical symptoms (focal swelling within the subcutaneous tunnel or leak of infusate from the catheter exit site) and the contrast agent dose therefore minimized.\nFig.\u00a017Catheter narrowing. a Image acquired during contrast injection through a double-lumen catheter in a 7-year-old male. The catheter is damaged at the site of previous kinks or catheter clamps (arrows). b Focal narrowing of the opacified lumen of a PICC (arrow) in a 3-year-old male where the skin suture device has been attached to the catheter too tightlyFig.\u00a018Extravasation. a Extravasation of contrast medium into the soft tissues at the site of a catheter break. b Contrast medium is seen leaking from a break in the connection between the portacath reservoir and the catheter\nInability to aspirate from a catheter that otherwise flushes well indicates one of three possible causes of catheter malfunction: the presence of a fibrin sheath, catheter tip thrombus, or position of the catheter tip against a vessel or chamber wall. All three causes create a valve effect at the catheter tip, so that the obstruction is temporarily alleviated by forward pressure during infusion but recurs with negative pressure on attempted aspiration (Fig.\u00a019).\nFig.\u00a019Line drawing demonstrating the effect of a fibrin sheath. a Flushing the catheter displaces the sheath or thrombus from the catheter tip and allows saline to exit the lumen. b Attempted aspiration sucks the sheath back onto the catheter tip, causing withdrawal occlusion\nGentle contrast medium injection through a catheter that is resting against a vessel or chamber wall will demonstrate rapid but asymmetric or oblique flow of contrast agent away from the catheter tip (Fig.\u00a020). This appearance should be distinguished from an irregular jet due to tip thrombus (see below). It is vital to identify this problem during an initial slow instillation of contrast medium as a forceful injection may damage the vessel wall.\nFig.\u00a020Fluoroscopic contrast study of a haemodialysis catheter in a 9-year-old female. The lumen with a more distal tip has been opacified. The catheter is too long and the distal tip lies up against the inferior wall of the right atrium, causing the contrast jet to be deflected\nA fibrin sheath or sleeve thrombus is caused by build-up of fibrin material around the intravascular portion of the catheter. This phenomenon may occur in the majority of catheters, as early as 24\u00a0h after catheter insertion [8, 32, 33]. Fibrin sheaths cause malfunction only when the sheath extends around or over the tip of the catheter. A complete sheath prevents any contrast medium from flowing away from the catheter tip; instead it tracks retrogradely along the intravascular portion of the catheter, between the catheter and the fibrin sheath, and then spills into the soft tissues at the point of venous access (Fig.\u00a021). In such cases, the patient will present with symptomatic swelling at the venous access site, usually the neck, during attempted catheter flushes. It is essential to identify initial contrast medium pooling at the catheter tip in the early phase of the injection followed by tracking of contrast medium back along the length of the catheter on later images, so that this is not misinterpreted as extravasation due to catheter fracture. To ensure a correct diagnosis in such cases, sequential images should be acquired or \u2018grabbed\u2019 during a very slow injection. In some cases, the fibrin sheath may be very thin and it is broken or fenestrated during flushing of the catheter. A circumferential but incomplete fibrin sheath will cause narrowing of the contrast jet as it exits the catheter, with delayed fanning out of contrast medium. This is a subtle, but classic, sign of a fibrin sheath (Fig.\u00a022). The operator must ensure that the contrast agent jet is of a similar diameter to that of the catheter lumen, so that a circumferential thrombus or fibrin sheath causing partial occlusion of the end hole is not missed.\nFig.\u00a021Fibrin sheaths. a, b Contrast medium is instilled into a tunnelled catheter in a 4-month-old child. The catheter has been partially pulled back and the tip lies within the left brachiocephalic vein. A fibrin sheath occludes the catheter. Instead of flowing away from the catheter tip, contrast medium tracks back along the side of the catheter (aarrow) and extravasates into the tissues of the neck (b). c Contrast study performed in a 2-year-old child with a catheter occluded by a fibrin sheath. Contrast medium tracks back along the catheter and into the subcutaneous tunnel, outlining the cuff, as shown diagrammatically in the line drawingFig.\u00a022Fibrin sheaths. a Catheter contrast study in a 5-year-old male. Contrast medium exits the catheter tip in a linear manner, but the contrast jet is significantly narrower than the lumen of the catheter (arrow), implying the presence of a circumferential, nonocclusive fibrin sheath. The effect of the fibrin sheath is shown diagrammatically. b Contrast medium exits the tip of a portacath in an 11-year-old male. The jet fails to fan away from the tip (arrow) of the catheter, instead simply filling the lumen of the sheath. c A similar appearance is seen during a contrast study of a PICC in another patient. An arrow marks the catheter tip. Note the apparent irregular calibre of the catheter itself, a sign that contrast medium is tracking back along the catheter as shown in Fig.\u00a021c\nCatheter thrombus is most likely to occur in catheters that are inadequately or infrequently flushed, or that are sited in small vessels with a low volume and rate of blood flow around the tip, as has occurred in the case shown in Fig.\u00a021. Thrombus within the catheter lumen itself is seen as a filling defect in the catheter during injection (Fig.\u00a023). Tip thrombus causes irregularity of the jet of contrast medium as it exits the catheter and may deflect the jet away from the long axis of the catheter (Fig.\u00a024). Thrombus may extend into the vessel itself, which will cause irregular filling of the vessel beyond the catheter tip (Fig.\u00a025).\nFig.\u00a023Image acquired during contrast medium injection through one lumen of a double-lumen catheter in a 2-year-old female. There is a filling defect within the catheter (arrow) at the site of a focal intraluminal thrombusFig.\u00a024Thrombus. a Fluoroscopic image acquired during a catheter contrast study in a 3-year-old female. The contrast medium jet is deflected away from the long axis of the catheter, implying the presence of partially occlusive thrombus at the catheter tip. The effect of the tip thrombus is shown diagrammatically. b Thrombus is adherent to the tip of the distal lumen of a haemodialysis catheter in a 7-year-old male. Contrast medium exits the lumen eccentrically and outlines the thrombusFig.\u00a025Contrast study of a left femoral catheter in a 7-month-old child. The short white arrow shows the tip of the catheter. Contrast medium outlines the common iliac veins and IVC. There are several filling defects in the IVC (long white arrows) consistent with venous thrombus beyond the catheter tip. Note that contrast medium also refluxes into ascending lumbar veins on the left (black arrows)\nCatheter-induced venous thrombosis is influenced by catheter dwell time, the size of the catheter relative to the vessel, the access vein used, infusate type, and the presence of any prior venous interventions or systemic comorbidities in the patient [34\u201338]. It is likely to be a relatively common occurrence [39], although evidence for this in the literature is skewed by variations in the diagnostic techniques and pathological definitions used in various studies. Many cases of pericatheter venous thrombosis are asymptomatic and the catheter continues to function, so this complication is likely to be under-reported. Where they occur, symptoms include localized pain and swelling overlying the accessed vein and sudden facial or extremity swelling. It is important that the clinical team rule out septic thrombophlebitis in symptomatic patients. Isolated facial or extremity swelling is, however, strong evidence for acute venous thrombosis [34]. Contrast-enhanced catheter studies will demonstrate irregular occlusion of the involved vein (see Fig.\u00a025). In well-established cases, drainage may occur only via tortuous collateral vessels. It may be appropriate to perform a venogram of the affected vessel via a peripheral cannula rather than the catheter at this stage, to assess drainage of the extremity (Fig.\u00a026). Some operators may prefer to perform a venogram immediately prior to placement of a new catheter, to allow time for possible recanalization of the vessel or formation of substantial collaterals in the interim period (Fig.\u00a027).\nFig.\u00a026Left arm venography in a 1-year-old female with a malfunctioning tunnelled catheter in situ. Contrast medium fails to fill the occluded SVC and there are irregular filling defects within the left brachiocephalic vein, consistent with mural thrombi. Small collaterals are forming in the left supraclavicular regionFig.\u00a027Right arm venogram in an 8-year-old boy performed prior to placement of a new catheter. The study demonstrates multiple irregular venous collaterals draining the arm following occlusion of the subclavian and brachiocephalic veins\nImplications for catheter salvage techniques\nDiagnostic catheter studies are central to the management of malfunctioning catheters. A detailed discussion of catheter salvage techniques is beyond the remit of this review; a number of the techniques outlined below are dependent on the services of a proactive IR department. Most institutions at least have a ward-based thrombolysis protocol for occluded CVCs and these techniques play a significant role in extending the life of a malfunctioning catheter [10, 40\u201345].\nCatheters that are found to be too long can be shortened by a variety of techniques. The simplest involves withdrawal of the intravascular portion of the catheter at the venous access site while the tunnelled portion is left in situ. The catheter is then re-measured and shortened, prior to re-puncture of the venous access site and replacement of the intravascular portion of the catheter [46, 47]. If there is sufficient excess length, the catheter can be cut when it is partially withdrawn, and the tip wired to maintain access, avoiding the need for re-puncture.\nKinks in the subcutaneous tunnel of a CVC can often be straightened by accessing either the venous access site or the skin exit site of the catheter. Intervention at the venous access site may result in an increase in the intravascular length of the catheter, a result that is beneficial in catheters that have migrated back into the tunnel or the neck.\nCatheter tips that have flicked out of position can occasionally be repositioned using one of several techniques. Often a forceful injection of saline through the displaced catheter will encourage the tip to return to its original position, in the direction of flowing blood [10]. Obstinate catheters may respond to placement of a guidewire through the catheter lumen, which stiffens the catheter sufficiently to encourage repositioning of the catheter [10, 48]. An alternative technique involves venous access via a femoral approach, allowing the displaced catheter to be snared and pulled back into the appropriate vessel [10, 49\u201351].\nCatheter tip thrombus can be dislodged by inserting a tip deflecting guidewire through the catheter and rotating the wire once it is just beyond the tip of the catheter [9, 10]. Such techniques, although effective, are rarely long lasting.\nFibrin sheath stripping techniques can be attempted in patients in whom replacement of a malfunctioning catheter is undesirable, such as those with a high risk for general anaesthesia or limited alternative access sites. Again, femoral venous access is obtained and the catheter is ensnared using a standard vascular snare device (Fig.\u00a028). Gentle manipulation of the snare along the length of the catheter is performed in an attempt to dislodge and retrieve the fibrin sheath [9, 10, 51\u201355]. Symptomatic pulmonary embolism is unlikely, although the procedure is contraindicated in subjects with a right to left shunt or severe cardiopulmonary disease that will not tolerate a pulmonary embolus [32, 55].\nFig.\u00a028The distal portion of a malfunctioning haemodialysis catheter has been snared from a femoral approach in an attempt to strip the fibrin sheath from the catheter\nImplications for catheter replacement techniques\nThe results of catheter contrast medium studies are often valuable in planning catheter replacement. Operators are likely to alter their approach to venous access in the presence of a documented fibrin sheath or venous occlusion. Resiting a catheter into the lumen of a fibrin sheath is highly likely to result in a rapid reestablishment of the sheath and subsequent catheter occlusion. In cases where reaccess of a vessel containing a fibrin sheath is necessary, it is sensible to attempt an US-guided puncture parallel to the fibrin sheath, rather than into it (Fig.\u00a029). Alternatively, the sheath can be deliberately cannulated so that a guidewire is placed through the sheath followed by a standard angioplasty balloon that, when inflated, encourages break-up of the sheath or incorporation of the sheath into the vessel wall [9, 53].\nFig.\u00a029US image of the right internal jugular vein in a 12-year-old female. A linear filling defect is attached to the vessel wall (arrow), likely to represent a previous fibrin sheath in this patient with a history of multiple previous access catheters\nDocumentation of complete venous occlusion is vital prior to replacement of a CVC. This allows a more considered approach to catheter placement and appropriate discussion with the family prior to a difficult procedure. Recanalization and venous stenting techniques or use of unusual access sites should be considered in children who would otherwise require open surgical procedures to maintain reliable venous access.\nConclusion\nReliable central venous access is key to the management of many paediatric conditions. The insertion of central venous access devices is increasingly becoming the remit of the interventional radiologist, a paradigm shift that will require general radiologists to play some role in the subsequent management of these catheters. In institutions where the specific cause of catheter malfunction may alter subsequent management, radiologists need to be familiar with the catheter types used and their imaging appearances, both on plain radiography and on contrast investigations. Accurate diagnosis of CVC malfunction can significantly alter outcome for patients in whom repeated venous access procedures can often become a source of greater morbidity than their primary condition.","keyphrases":["central venous catheter","children","complications"],"prmu":["P","P","P"]}
{"id":"Angiogenesis-4-1-2268723","title":"Judah Folkman, a pioneer in the study of angiogenesis\n","text":"More than 30 years ago, Judah Folkman found a revolutionary new way to think about cancer. He postulated that in order to survive and grow, tumors require blood vessels, and that by cutting off that blood supply, a cancer could be starved into remission. What began as a revolutionary approach to cancer has evolved into one of the most exciting areas of scientific inquiry today. Over the years, Folkman and a growing team of researchers have isolated the proteins and unraveled the processes that regulate angiogenesis. Meanwhile, a new generation of angiogenesis research has emerged as well, widening the field into new areas of human disease and deepening it to examine the underlying biological processes responsible for those diseases.\nEarly evidence of tumor cells releasing specific growth factor for blood vessels\nIn 1939, Ide et\u00a0al. [1] were the first to suggest that tumors release specific factors capable of stimulating the growth of blood vessels. In 1945, Algire and Chalkley [2] were the first to appreciate that growing malignancies could continuously elicit new capillary growth from the host. They used a transparent chamber implanted in a cat\u2019s skin to study the vasoproliferative reaction secondary to a wound or implantation of normal or neoplastic tissues. They showed that the vasoproliferative response induced by tumor tissues was more substantial and earlier than that induced by normal tissues or following a wound. They concluded that the growth of a tumor is closely connected to the development of an intrinsic vascular network.\nIn 1956, Melwin and Algire [3] found that the vasoproliferative response of normal or neoplastic tissues transplanted into muscle was not significantly different with respect to the time of onset of new blood vessels, though it was stronger when the implantation was performed in a resection area. In addition, while normal tissues induced a vasoproliferative response confined to the host, tumor tissues induced the formation of neovessels that pierced the implant. Lastly, the intensity of the response seemed to be influenced by the distance between the implant and the host\u2019s vessels: normal tissue was unable to induce a response if placed more than 50\u00a0\u03bcm away, whereas tumor tissue had a longer activity range.\nIn 1968, Greenblatt and Shubik [4] implanted Millipore chambers (pore size 0.45\u00a0\u03bcm) into a hamster\u2019s cheek pouch and placed some tumor fragments around them. In a few days, the growing tumor mass engulfed the whole chamber, whose pores were permeable to the tumor interstitial fluid, but not to the tumor cells. New blood vessels, however, were formed in any case very likely through the release of a diffusible factor that could pass through the pores. Ehrman and Knoth [5] confirmed these data with tumor fragments laid on Millipore filters planted on the chick embryo chorioallantoic membrane (CAM).\nTumors in isolated perfused organs: absence of angiogenesis\nIn 1963, Folkman and Becker studied hemoglobin solutions as potential substitutes for blood transfusion. To test which solution was optimal for tissue survival, they perfused these solutions through the vasculature of canine thyroid glands, by using an apparatus with a silicone rubber oxygenator. The glands survived for about 2\u00a0weeks. They could distinguish different hemoglobin preparations by histologic analysis of the thyroid glands after a week or more of continuous arterial perfusion. To determine if these isolated organs could support growth, they injected them with adult mouse melanoma cells. Tiny tumors developed but stopped growing at 1- to 2-mm diameter and never became vascularized [6]. Endothelial cells swelled and could not proliferate in the presence of free hemoglobin solutions lacking platelets [7]. However, the tumors were not dead. When they were transplanted to their host mice, they rapidly vascularized and grew to more than 1\u00a0cm3.\nFolkman et\u00a0al. observed that when tumor cells were inoculated into isolated perfused organs, tumors were limited in size to 1\u20132\u00a0mm3 [6]. Subsequently, they found that neovascularization does not occur in isolated perfused organs and that tumors transplanted from these organs to syngeneic mice became vascularized and grew rapidly to 1\u20132\u00a0mm3. This was the first evidence that the absence of neovascularization correlated with severe restriction of tumor growth.\nThe data were consistent with work from Harry Green, who had shown long before that growth of rabbit tumors transplanted into the anterior chamber of the guinea pig coincided with the growth of new blood vessels. Tumors that remained viable, but did not grow, had no visible blood vessels [8].\nHypothesis: tumor growth is angiogenesis dependent\nIn 1971, Folkman published in the \u201cNew England Journal of Medicine\u201d a hypothesis that tumor growth is angiogenesis dependent and that inhibition of angiogenesis could be therapeutic [9]. This article also introduced the term anti-angiogenesis to mean the prevention of new vessel sprout from being recruited by a tumor. The hypothesis predicted that tumors would be enable to grow beyond a microscopic size of 1\u20132\u00a0mm3 without continuous recruitment of new capillary blood vessels.\nThis concept is now widely accepted because of supporting data from experimental studies and clinical observations carried out over the intervening years.\nEvidence that tumors are angiogenesis dependent\nFolkman and collaborators provide evidence for the dependence of tumor growth on neovascularization: Tumor growth in the avascular cornea proceeds slowly at a linear rate, but after vascularization, tumor growth is exponential [10].Tumors suspended in the aqueous fluid of the anterior chamber of the rabbit eye and observed for a period up to 6\u00a0weeks remain viable, avascular, and of limited size (less than 1\u00a0mm3) and contain a population of viable and mitotically active tumor cells. These tumors induce neovascularization of the iris vessels but are too remote from these vessels to be invaded by them. After implantation contiguous to the iris, which had abundant blood vessels, the tumors induced neovascularization and grow rapidly, reaching 16,000 times the original size within 2\u00a0weeks [11]. This experiment introduced the concept of tumor dormancy brought about by prevention of neovascularization. In a parallel study, tumors were suspended in the aqueous humor of the anterior chamber, placed at various distances from the iris vessels, and compared with tumors implanted directly on the iris and with those implanted in the cornea [12]. Moving the distant, dormant tumors closer to the iris jump started their growth. This suggested that this type of tumor dormancy was caused not by cell cycle arrest or immune control, but by a lack of blood supply.B-16 mouse melanoma, V-79 Chinese hamster lung cells, and L-5178 Y murine leukemia cells were plated in soft agar [13]. After 6\u20137\u00a0days of incubation, spheroid colonies of 0.1\u00a0mm were visible. All spheroids first enlarged exponentially for a few days and then continued on a linear growth curve for 5\u201323\u00a0weeks before reaching a diameter beyond which there was no further expansion. This was termed the dormant phase. After the dormant diameter was reached, these spheroids remained viable for 3\u20135\u00a0months, or as long as they were frequently transferred to new medium. Cells in the periphery of the spheroid incorporated 3H-labeled thymidine while cells in the center died. This is a form of population dormancy in which the proliferating cells near the surface of the spheroid just balance those dying cells deep in the center of the spheroid.Tumors implanted on the CAM of the chick embryo do not exceed a mean diameter of 0.93\u00a0\u00b1\u00a00.29\u00a0mm during the prevascular phase (approximately 72\u00a0h). Rapid growth begins, however, within 24\u00a0h after vascularization, and tumors reach a mean diameter of 8.0\u00a0\u00b1\u00a02.5\u00a0mm by 7\u00a0days [14].Tumors grown in the vitreous of the rabbit eye remain viable but attain diameters of less than 0.50\u00a0mm for as long as 100\u00a0days. Once such a tumor reaches the retinal surface, it becomes neovascularized and within 2\u00a0weeks can undergo a 19,000-fold increase in volume over the avascular tumor [15].The CAM appears at day\u00a05 during development of the chick embryo. The 3H-thymidine labeling index of its vascular endothelium decreases with age, with an abrupt reduction at day\u00a011 [16]. Prior to day\u00a011, labeling index is approximately 23%; during 11\u00a0days, the labeling index decreases to 2.8%, and subsequently, the cells begin to acquire the structural characteristics of matured, differentiated endothelium. One-millimeter fragments of fresh Walker 256 carcinoma were implanted on the CAM from day\u00a03 to day\u00a016 [14]. The size of the tumors was measured daily, and the onset of vascularization of each tumor was determined in\u00a0vivo with a stereomicroscope and confirmed with histological sections. Proliferation of chick capillaries occurred in the neighborhood of the tumor graft by 24\u00a0h after implantation, but capillary sprouts did not penetrate the tumor graft until approximately 72\u00a0h. During the avascular phase, tumor diameter did not exceed 1\u00a0mm. Small tumor implants of 0.5\u00a0mm or less grew to 1\u00a0mm and stopped expanding. Larger tumor implants of 2- or 3-mm shrank until they reached 1-mm diameter. During the first 24\u00a0h after penetration by capillaries, there was a rapid tumor growth. Neovascularization was not grossly observable with the stereomicroscope until after day\u00a010 or 11. Tumors implanted on the CAM after day\u00a011 grow at slower rate in parallel with the reduced rates of endothelial growth.When tumor grafts of increasing size (from 1 to 4\u00a0mm) are implanted on the 9-day CAM, grafts larger than 1\u00a0mm undergo necrosis and autolysis during the 72-h prevascular phase. They shrink rapidly until the onset of neovascularization, when rapid tumor growth resumes [14]. In another study [17], the behavior of tumor grafts on the CAM was compared to grafts of normal adult and embryonic tissues. In tumor tissue, preexisting blood vessels within the tumor graft disintegrated by 24\u00a0h after implantation. Neovascularization did not occur until after at least 3\u00a0days, and only by penetration of proliferating host vessels into the tumor tissue. There was marked neovascularization of host vessels in the neighborhood of the tumor graft. By contrast, in embryonic graft, preexisting vessels did not disintegrated. They reattached by anastomosis to the host vessels within 1\u20132\u00a0days, but with minimal or almost no neovascularization on the part of the host vessels. In adult tissues, the preexisting graft vessels disintegrated; there was no reattachment of their circulation with the host, and adult tissues did not stimulate capillary proliferation. These studies suggest that only tumor grafts are capable of stimulating formation of new blood vessels in the host.In transgenic mice that develop carcinomas of the \u03b2 cells in the pancreatic islets, large tumors arise only from a subset of preneoplastic hyperplastic islets that have become vascularized [18].\nIsolation of the first angiogenic tumor factor\nUntil the early 1970s, it was widely assumed that tumors did not produce specific angiogenic proteins. The conventional widsom was that tumor vasculature was an inflammatory reaction to dying or necrotic tumor cells.\nPrevious studies had shown that tumor-stimulated vessel growth did not require direct contact between tumor and host tissue [4, 5]. This made sense to Folkman, who reasoned that a soluble factor would be more likely to reach nearly than distant blood vessels. He and his colleagues isolated an angiogenic factor in 1971 [19]. The homogenate of a Walker 256 carcinoma\u2014a breast tumor of Sprague-Dawley rats\u2014was fractionated by gel-filtration on Sephadex G-100. The fraction that exhibited the strongest angiogenic activity had a molecular weight of about 10,000\u00a0daltons and consisted of 25% RNA, 10% proteins, and 58% carbohydrates, plus a possible lipid residue. It was inactivated by digestion with pancreatic ribonuclease, or by heating at 56\u00b0C for 1\u00a0h, and was not modified when kept at 4\u00b0C for 3\u00a0months, nor when treated with trypsin for more than 3\u00a0days. This active fraction was subsequently called \u201ctumor angiogenesis factor\u201d (TAF) [19]. Both the cytoplasmic and the nuclear fractions of tumor cells stimulated angiogenesis. In the nuclear fraction, this was found to be associated with nonhistonic proteins [20]. Tumor angiogenesis factor has since been nondestructively extracted from several tumor cell lines, and several low molecular weight angiogenic factors have been isolated, again from the Walker 256 carcinoma. These factors induced a vasoproliferative response in\u00a0vivo when tested on rabbit cornea or chick CAM, and in\u00a0vitro on cultured endothelial cells [21\u201323].\nFirst evidence of the existence of the avascular and vascular phases of solid tumor growth\nThe earliest evidence of the existence of the two phases was obtained by Folkman and collaborators in 1963, who perfused the lobe of a thyroid gland with plasma and inoculated a suspension of melanoma B16 tumor cells through the perfusion fluid. These cells grew into small, clearly visible black nodules. The nodules did not exceed 1\u00a0mm in diameter and did not connect with the host\u2019s vascular network. Their outer third generally remained vital, while the interior portion underwent necrosis. Reimplanted nodules, on the other hand, equipped themselves with a vascular network and grew very rapidly. The conclusion was thus drawn that the absence of vascularization limits the growth of solid tumors.\nFurther research by Folkman\u2019s group resulted in an experimental system in which the tumor, or its extracts, could be separated from the vascular bed [24, 25]. This system was based on subcutaneous insufflation to lift the skin of a rat and form a poorly vascularized region below it. Millipore filters containing Walker 256 cancer cells or their cytoplasmic or nuclear extracts (TAF) were implanted into the fascial floor of the dorsal air sac. At intervals thereafter, 3H-labeled thymidine was injected into the air sac, and the tissues were examined by autoradiography and electron microscopy. Autoradiographs showed thymidine-3H labeling in endothelial cells of small vessels, 1\u20133\u00a0mm from the site of implantation, as early as 6\u20138\u00a0h after exposure to tumor cells. DNA synthesis by endothelium subsequently increased, and within 48\u00a0h new blood vessels formation was detected. The presence of labeled endothelial nuclei, endothelial mitosis, and regenerating endothelium was confirmed by electron microscopy. Tumor angiogenesis factor also induced neovascularization and endothelial DNA synthesis after 48\u00a0h. Further ultrastructural autoradiographic studies were carried out with the same model [25]. It was apparent that by 48\u00a0h there was ultrastructural evidence of regenerating endothelium, including marked increase in ribosomes and endoplasmic reticulum, scarce or absent pinocytotic vesicles, and discontinuous basement membrane. Labeled endothelial cells were seen along newly formed sprouts as well as in parent vessels. Furthermore, pericytes were also shown to synthesize DNA.\nIn another series of experiments, 1-mm fragments from Brown-Pearce and V2 carcinomas were implanted into the avascular stroma of a rabbit cornea 1\u20136\u00a0mm away from the limbic vessels, and observed the tumor growth daily with a stereomicroscope [10]. After 1\u00a0week, new blood vessels had invaded the cornea starting from the edge closer to the site of implantation and developed in that direction at 0.2\u00a0mm and then about 1\u00a0mm\/day. Once the vessels reached the tumor, it grew very rapidly to permeate the entire globe within 4\u00a0weeks.\nDormancy of micrometastases may be governed by angiogenesis\nFolkman and collaborators found that metastases were suppressed when a primary tumor was implanted and allowed to grow in nude mice, whereas they underwent neovascularization and became clinically evident when primary neoplasm was removed. In the absence of angiogenesis, micrometastases rarely exceeded 0.2-mm diameter and contained many proliferating tumor cells balanced by many apoptotic cells. When they were allowed to become angiogenic, they grew rapidly. Dormancy may be generalizable to a variety of tumors in which blocked angiogenesis results in balanced tumor cell proliferation and apoptosis [26].\nPrognostic significance of tumor vascularity\nIn 1972, Brem in the Folkman laboratory reported the first quantitative method for histologic grading of tumor angiogenesis. He correlated neovascularization in human brain tumors with tumor grade [27]. In the early 1990s, Weidner and collaborators [28\u201330] showed that measurement of microvascular density within isolated regions of high vessel concentration (i.e., hotspots) was a prognostic indicator for human breast and prostate carcinomas.\nMicrovascular density counting protocols have become the morphological gold standard to assess the neovasculature in human tumors. This method requires the use of specific markers to vascular endothelium and of immunohistochemical procedures to visualize microvessels. Microvascular density determined in primary tumors is significantly associated with metastasis and prognosis in several solid and hematological tumors.\nAntiangiogenesis\nThe existence of specific angiogenesis inhibitors was first postulated by Folkman in 1971 in an editorial. No angiogenesis inhibitors existed before 1980, and few scientists thought at that time that such molecules would ever be found.\nFrom 1980 to 2005, Folkman\u2019s laboratory reported the discovery of 12 angiogenesis inhibitors (Table\u00a01). The first angiogenesis inhibitor was found in cartilage, an avascular tissue that resists invasion by many tumors [31]. Brem and Folkman demonstrated that tumor-induced vessels were inhibited by a diffusible factor from neonatal rabbit cartilage [32]. The partially purified inhibitor suppressed tumor growth when it was infused into the vascular bed of murine and rabbit tumors [33]. 2-methoxyestradiol was first reported by Fotsis et\u00a0al. [34], and the article reporting 2-methoxyoestradiol and its molecular mechanism as an inhibitor of tubulin polymerization by acting at the colchicine site was published a month later [35].\nTable\u00a01Angiogenesis inhibitors discovered in Folkman\u2019s laboratory from 1980 to 20051980. Interferon alpha-beta1982. Platelet factor 4\/protamine1985. Angiostatic steroids1990. Fumagillin1994. Angiostatin1994. Thalidomide1994. 2-methoxyestradiol1997. Endostatin1999. Cleaved antithrombin III2002. 3-Aminotholidomide2003. DBF-maf2005. Caplostatin\nInterferon alpha\nInterferon alpha was first shown to inhibit endothelial cell migration in a dose-dependent and reversible manner in 1980 by Zetter in Folkman\u2019s laboratory [36]. Since 1988, interferon alpha has been used successfully to cause complete and durable regression of life-threatening pulmonary hemangiomatosis, hemangiomatosis of the brain, airway and liver in infants, recurrent high-grade giant cell tumors refractory to conventional therapy, and angioblastomas [37\u201339].\nA 12-year-old boy with fatal pulmonary hemangiomatosis had a complete remission and recovered completely after 7\u00a0months of therapy with interferon alpha. Therapy was continued for 7\u00a0years. This led to the successful use of low-dose daily interferon alpha therapy administered subcutaneously to infants with sight-threatening or life-threatening hemangiomas and hemangioendotheliomas of the heart, airway, and liver [37\u201339].\nPlatelet factor 4\/protamine\nProtamine was shown to be an angiogenesis inhibitor [40], but cumulative toxicity from prolonged administration and a narrow window of angiostatic efficacy prevented its consideration for clinical use.\nPlatelet factor 4 was first tested for antiangiogenic activity because its method of binding and neutralizing heparin is similar to that of protamine [40]. Recombinant human platelet factor 4 (rHuPF4) has been produced [41]. It specifically inhibited endothelial proliferation and migration in\u00a0vitro [42]. The inhibitory activities are associated with the carboxy-terminal region of the molecule. The growth of human colon carcinoma in athymic mice, as well as the growth of murine melanoma, was markedly inhibited by intralesional injections, whereas tumor cells were completely insensitive to rHuPF4 in\u00a0vitro at levels that inhibited normal endothelial cell proliferation. Systemic administration of rHuPF4 has so far been ineffective against tumor growth, perhaps because of rapid inactivation or clearance of the peptide.\nAngiostatic steroids\nFolkman had begun to use the CAM of the chick embryo to detect angiogenic activity in fractions being purified from tumor extracts. The addition of heparin increased the speed of development of the angiogenic reaction so that it could be read 1\u20132\u00a0days later [40]. But one problem with this assay is that occasionally eggshell dust falls on the CAM and causes background inflammation. Folkman guessed that adding cortisone to the CAM might eliminate the irritation from the shell dust but not abolish the tumor-angiogenic reaction. As expected, cortisone alone prevented shell dust inflammation without interfering with angiogenesis induced by tumor extracts. The surprise was that when heparin and cortisone were added together, tumor angiogenesis was inhibited [43]. Furthermore, when this combination of heparin and steroid was suspended in a methylcellulose disk and implanted on the young (6-day) CAM, growing capillaries regressed leaving in their place, 48\u00a0h later, an avascular zone. The antiangiogenic effect was specific for growing capillaries. Mature nongrowing capillaries in older membranes were unaffected. Nonanticoagulant heparin had the same effect. A hexasaccharide fragment with a molecular weight of approximately 16,000 was found to be the most potent inhibitor of angiogenesis (in the presence of a corticosteroid). The combination of the heparin hexasaccharide fragment and cortisone also inhibited tumor-induced angiogenesis in the rabbit cornea.\nThe regression of a growing vessel exposed to heparin-steroid combinations begins with endothelial cell rounding and is followed by cessation of endothelial proliferation, desquamation of endothelial cells, and retraction of the capillary sprout [44]. These events occur as 24\u201348\u00a0h and are accompanied by dissolution of the basement membrane of the new capillary vessels.\nFumagillin\nFumagillin was found by Ingber in the Folkman laboratory to inhibit endothelial cell proliferation without causing endothelial cell apoptosis, when a tissue culture plate of endothelial cells became contaminated with a fungus Aspergillus fumigatus fresenius [45]. A conditioned medium from fungal cultures contained an inhibitor of endothelial cell proliferation and angiogenesis, which, upon purification, was found to be fumagillin, a polyene macrolide. When capillary endothelial cells were stimulated by fibroblast growth factor-2 (FGF-2), half-maximal inhibition was observed with fumagillin at 100\u00a0pg\/ml. This antiproliferative effect appeared to be relatively specific for endothelial cells because inhibition of nonendothelial cells, including tumor cells, was observed at up to 1,000-fold higher concentrations.\nScientists at Takeda Chemical Industries (Osaka, Japan) made a synthetic analogue of fumagillin, called TNP-470, which inhibits endothelial proliferation in\u00a0vitro at a concentration 3\u00a0logs lower than the concentration necessary to inhibit fibroblasts, and tumor cells.\nAngiostatin and endostatin\nThey were discovered by M. O\u2019Reilly in Folkman laboratory based on Folkman\u2019s hypothesis of a mechanism to explain the phenomenon that surgical removal of certain tumors leads to rapid growth of remote metastases. This hypothesis said that if tumors produce both stimulators and inhibitors of angiogenesis, an excess of inhibitors could accumulate within an angiogenic tumor. In the circulation however, the ratio would be reversed. Angiogenesis inhibitors would increase relative to stimulators, because of rapid clearance of stimulators from the blood. Folkman formulated this hypothesis after reading Bouck\u2019s first report in 1989 that the emergence of tumor angiogenesis was the result of a shift in balance between positive and negative regulators of angiogenesis in a tumor [46]. Bouck reported that the switch to angiogenesis during tumorigenesis of transformed hamster cells was associated with downregulation of an inhibitor of angiogenesis, thrombospondin. She suggested that the switch to the angiogenic phenotype could be the result of a shift in the net balance of positive and negative regulators of angiogenesis.\nIn 1991, O\u2019Reilly began to screen a variety of transplantable murine tumors for their ability to suppress metastases. A Lewis lung carcinoma was the most efficient. When the metastasis-suppressing primary tumor was present in the dorsal subcutaneous position, microscopic lung metastases remained dormant at a diameter of less than 200\u00a0\u03bcm surrounding a preexisting microvessel, but revealed no new vessels. Within 5\u00a0days after surgical removal of the primary tumor, lung metastases became highly angiogenic and grew rapidly, killing their host by 15\u00a0days [47]. This striking evidence that primary tumor could suppress angiogenesis in its secondary metastases by a circulating inhibitor was further supported by the demonstration that a primary tumor could also suppress corneal angiogenesis by an implanted pellet of FGF-2. O\u2019Reilly then succeeded in purifying this inhibitor from the serum and urine of tumor-bearing animals. It was a 38-kD internal fragment identical in amino acid sequence to the first four kringle structures of plasminogen and it was named angiostatin. Angiostatin specifically inhibited the proliferation of growing vascular endothelial cells and had no effect on resting confluent endothelial cells or on other cell types, including smooth muscle cells, epithelial cells, fibroblasts and tumor cells. It also inhibited growth of primary tumors by up to 98% [48] and was able to induce regression of large tumors (1\u20132% of body weight) and maintain them at a microscopic dormant size.\nBased on the same rationale and strategy, O\u2019Reilly isolated and purified another angiogenesis inhibitor from a murine hemangioendothelioma. This inhibitor, called endostatin [49], is a 20-kD protein with an N-terminal amino acid sequence identical to the carboxyterminus of collagen XVIII. It was purified directly from tumor cell-conditioned medium. Endostatin is also a specific inhibitor of endothelial proliferation and has no effects on resting endothelial cells or on other cell types. It is slightly more potent than angiostatin and also causes regression of large tumors to a microscopic size.\nThalidomide\nIn 1994, D\u2019Amato in the Folkman laboratory reported that thalidomide is an angiogenic inhibitor [50]. Corneal neovascularization in rabbits induced by FGF-2 or VEGF was blocked by orally administered thalidomide. This activity of thalidomide was mainly the result of its direct effect on inhibiting new blood vessel formation and not on suppression of infiltrating host inflammatory cells. Histologic sections of the pretreated neovascularized corneas were virtually free of inflammatory cells. Thalidomide also inhibited corneal neovascularization in mice, but it was necessary to give the drug by the intraperitoneal route and at high doses, because mice do not metabolize thalidomide effectively.\n2-Methoxyestradiol\nIn 1994, D\u2019Amato in Folkman laboratory demonstrated that a metabolite of estradiol, 2-methoxyestradiol, inhibited angiogenesis in the chick CAM [35]. Moreover, since 2-methoxyestradiol causes mitotic perturbations, they examined its interactions with tubulin and showed that 2-methoxyestradiol bound to colchicine site of tubulin and, depending on reaction conditions, either inhibited assembly or seems to be incorporated into a polymer with altered stability properties.\nCleaved antithrombin III\nA human small-cell lung carcinoma suppressed angiogenesis and tumor growth at remote sites in immunodeficient mice. These cells generated an enzyme in\u00a0vitro that converted the 58-kD conformation of circulating antithrombin III to a 53-kD form of the protein [51] in which the externally configured stressed loop of antithrombin was retracted into the body of the molecule. The 53-kD form is a specific endothelial inhibitor and a potent angiogenesis inhibitor and has no thrombin binding activity.\nCaplostatin\nCaplostatin is a nontoxic synthetic analogue of fumagillin conjugated to a water-soluble-N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer [52, 53]. Caplostatin has a similar broad antitumor spectrum of TNP-470 without any toxicity. In addition to its antiangiogenic activity, caplostatin is the most potent known inhibitor of vascular permeability [53]. Caplostatin prevents vascular leakage induced by VEGF, bradykinin, histamine, and platelet-activating factor and prevents pulmonary edema induced by interleukin-2.\nAntiangiogenic chemotherapy\nBrowder in the Folkman laboratory was the first to demonstrate the concept that by optimizing the dosing schedule of conventional cytotoxic chemotherapy to achieve more sustained apoptosis of endothelial cells in the vascular bed of a tumor, it is possible to achieve more effective control of tumor growth in mice, even if the tumor cells are drug resistant [54]. Conventional chemotherapy is administered at maximum tolerated doses followed by off-therapy intervals of 2\u20133\u00a0weeks to allow the bone marrow and gastrointestinal tract to recover. In contrast, antiangiogenic chemotherapy is administered more frequently at lower doses, without long interruptions in therapy and with little or no toxicity. During antiangiogenic chemotherapy, endothelial cell apoptosis and capillary dropout precede the death of tumor cells that surround each capillary.\nConcluding remarks\nCurrently, several compounds with angiostatic activity are approved for clinical use, and many are in late-stage clinical development. However, the results from clinical trials have not shown the antitumor effects which were expected following preclinical studies. It appears that clinical applications of antiangiogenic therapy are more complex than originally thought.\nThe main problem in the development of antiangiogenic agents is that multiple angiogenic molecules may be produced by tumors, and tumors at different stages of development may depend on different angiogenic factors for their blood supply. Therefore, blocking a single angiogenic molecule was expected to have little or no impact on tumor growth. Current development of targeted antiangiogenic agents include their use in adjuvant settings and the combination of different antiangiogenic inhibitors to take a more comprehensive approach in blocking tumor angiogenesis.\nAdvancing insights into fundamental mechanisms will be necessary in the development of novel anticancer strategies based on inhibition of angiogenesis.","keyphrases":["angiogenesis","antiangiogenesis","history of medicine","tumor progression"],"prmu":["P","P","M","M"]}
{"id":"Auton_Neurosci-2-1-2428072","title":"Immunohistochemical detection of connexin36 in sympathetic preganglionic and somatic motoneurons in the adult rat\n","text":"Gap junctional communication in the adult CNS plays an important role in the synchronization of neuronal activities. In vitro studies have shown evidence of electrotonic coupling through gap junctions between sympathetic preganglionic motoneurons and between somatic motoneurons in the neonatal and adult rat spinal cord. Electrotonic transmission of membrane oscillations might be an important mechanism for recruitment of neurons and result in the generation of rhythmic sympathetic and somato-motor activity at the population level. Gap junctions in the adult spinal cord are constituted principally by connexin36 (Cx36). However, the distribution of Cx36 in specific neuronal populations of the spinal cord is unknown. Here, we identify Cx36-like immunoreactivity in sympathetic preganglionic and somatic motoneurons in thoracic spinal cord segments of the adult rat. For this purpose, double immunostaining against Cx36 and choline acetyltransferase (ChAT) was performed on transverse sections (20 \u03bcm) taken from spinal segments T6\u2013T8. Cx36 punctate immunostaining was detected in the majority of ChAT-immunoreactive (-ir) neurons from lamina VII [intermediolateral cell column (IML) and intercalated cell group (IC)], lamina X [central autonomic nucleus (CA)] and in ventral horn neurons from laminae VIII and IX. Cx36 puncta were distributed in the neuronal somata and along dendritic processes. The presence of Cx36 in ChAT-ir neurons is consistent with electrical coupling between sympathetic preganglionic motoneurons and between somatic motoneurons through gap junctions in the adult spinal cord.\n1\nIntroduction\nGap junctions are specialized cell\u2013cell contacts that provide direct intercellular communication. They often occur as plaques that can contain thousands of individual gap junction channels. Each channel is formed by the association of 12 connexin (Cx) proteins. Eleven of the 20 members connexin family have been identified so far in the mammalian central nervous system (Nagy et al., 2004), but only Cx36 is expressed almost exclusively by neural cells (Belluardo et al., 2000; Condorelli et al., 2000; Rash et al., 2000). Previous studies have shown that gap junctions play an important role in the synchronization of neuronal activity in various areas of the neonatal and adult rodent brain, including the cortex (Deans et al., 2001), hippocampus (Hormuzdi et al., 2001; Buhl et al., 2003), thalamic reticular nucleus (Landisman et al., 2002), inferior olive (Long et al., 2002; De Zeeuw et al., 2003; Leznik and Llinas, 2005) and olfactory bulb glomeruli (Christie et al., 2005). Such junctions enable the intercellular flow of electric current (electrotonic transmission) which can result in the generation of coordinated activity within populations of neurons. In the rat spinal cord, electrotonic and dye coupling have been shown to occur between somatic motoneurons (Gogan et al., 1977; Walton and Navarrete, 1991; Chang et al., 1999; Mentis et al., 2002) and between sympathetic preganglionic motoneurons (Logan et al., 1996; Nolan et al., 1999). It has been suggested that synchronization of electrical activity through gap junctions plays an important role in the generation of somatomotor and sympathetic motor rhythms induced by NMDA and\/or 5-HT (Marina et al., 2006; Tresch and Kiehn, 2000). In the spinal cord of adult rats, gap junctions are composed mainly by Cx36 and are distributed in the white matter and the grey matter (Rash et al., 2000; Lee et al., 2005). However, the expression of Cx36 protein in specific neural populations of the spinal cord remains unknown. In this study we used immunofluorescence to identify Cx36-like immunoreactivity in ChAT-positive putative sympathetic preganglionic and somatic motoneurons within thoracic spinal segments.\n2\nMaterials and methods\n2.1\nTissue preparation\nExperiments were performed in accordance with the UK Animals (Scientific Procedures) Act 1986 and associated guidelines. Six male Sprague\u2013Dawley rats (210\u2013250\u00a0g, bred in-house at UCL) were anaesthetized deeply with 20% urethane (1.6\u00a0g kg\u2212\u00a01 i.p.) and perfused through the ascending aorta with 100\u00a0ml of 0.9% saline at room temperature, followed by 500\u00a0ml of 4% paraformaldehyde in 0.1\u00a0M phosphate buffered saline (PBS, pH\u00a07.4) at 4\u00a0\u00b0C. The thoracic spinal cord and the brain stem were removed, postfixed for 1\u00a0h and cryoprotected in 10% sucrose in 0.1\u00a0M PBS overnight at 4\u00a0\u00b0C. Transverse slices (20\u00a0\u03bcm thick) from spinal segments T6\u2013T8 and caudal medulla were collected with a freezing microtome and stored in cryo-protectant (30% ethylene glycol and 20% glycerol in 0.05\u00a0M PBS) at \u221220\u00a0\u00b0C.\nCaudal medulla slices were taken to assay specificity of the Cx36 antibody and to determine optimal antibody concentration using the inferior olive nucleus (ION) as a positive control. Cx36-immunostaining was analyzed in spinal segments T6\u2013T8 because previous studies have shown evidence of electrotonic coupling among sympathetic preganglionic motoneurons and expression of Cx36 mRNA in somatic motoneurons from thoracic spinal cord slices (Logan et al., 1996; Parenti et al., 2000).\n2.2\nImmunohistochemistry\nAll series of tissue slices from all animals (20\u201330 slices\/animal) were processed together in the same assay to ensure uniformity of immunostaining. After slices were washed in 0.1\u00a0M PBS with 0.1% Triton X (PBS-T), non-specific binding was blocked with 10% normal donkey serum (Sigma-Aldrich) in PBS-T (blocking medium) for 60\u00a0min. Tissue was then incubated in Cx36 antibody (1:100 dilution in blocking medium) at 4\u00a0\u00b0C overnight. The antibody was obtained by immunizing rabbits with a synthetic peptide corresponding to amino acids LQNTETTSKETEPDC of the murine Cx36 protein. The peptide sequence was compared to protein sequence databases from the Basic Local Alignment Search Tool (BLAST 2.0, NCBI, National Institutes of Health, USA), and showed local alignments with murine Cx36 protein only. The specificity of the Cx36 antibody was shown in Western blots and in preabsorption control experiments (Bittman et al., 2002). The slices were then washed again with PBS-T and incubated with fluorescent secondary antibody (Alexa Fluor 488 donkey anti-rabbit IgG, Molecular Probes, 1:500 dilution in blocking medium) for 60\u00a0min at room temperature, and washed again with PBS.\nCholine acetyltransferase (ChAT) immunodetection was employed to identify putative sympathetic preganglionic and somatic motoneurons (Marina et al., 2002). Following staining for Cx36, slices were incubated in goat anti-ChAT affinity purified polyclonal antibody (Chemicon, 1:100 dilution in 1% normal donkey serum) overnight at 4\u00a0\u00b0C. Tissue was washed and incubated with Alexa Fluor 568 donkey anti-goat IgG antibody (Molecular Probes, 1:500 dilution in 1% normal donkey serum) for 60\u00a0min at room temperature, washed again in PBS and cover-slipped with Vectashield hard set mounting medium with the fluorescent nuclear dye DAPI (Agar Scientific, Stansted, Essex).\n2.3\nImage analysis\nThe anatomical distribution of Cx36 immunostaining was determined by visual inspection through 63\u00d7 and 100\u00d7 objectives with an Olympus BH2-RFCA epifluorescence microscope equipped with light filters (exciter 380\u2013490\u00a0nm, emitter 519\u00a0nm for the green channel). ChAT-Immunoreactive (ir) neurons were considered positive for Cx36 if punctate staining was observed within the cell body or along the dendritic processes.\nThe cellular distribution of Cx36 in sympathetic preganglionic and somatic motoneurons was analyzed using a Leica TCS NT SP laser scanning confocal microscope with argon, krypton, and helium-neon lasers. Green and red channels were each scanned sequentially through a 63\u00d7 objective. Alexa Fluor 488 labelling was observed by exciting at 488\u00a0nm and collecting at 515\u2013550\u00a0nm. Alexa Fluor 568 labelling was visualized by using excitation at 568\u00a0nm and collecting at 590\u2013626\u00a0nm. Serial optical sections were taken at intervals of 0.5\u00a0\u03bcm starting at the upper cell surface, i.e., the first optical section where cytoplasmic ChAT staining became visible, and all the way down through the cell to the lower cell surface, i.e., where ChAT staining was no longer detected. Composite figures were created for each optical section by merging both channels using Image J 1.37c (National Institutes of Health, USA). Colocalization of Cx36 (green) and cytoplasmic ChAT immunofluorescence (red) appeared as yellow. Thus, green puncta on the cell surface, i.e., surrounding ChAT-ir cytoplasm, suggested the presence of Cx36 on the plasma membrane; yellow intracellular puncta in ChAT-positive regions were indicative of cytoplasmic Cx36. Serial sections were projected as a single image to create a 2D reconstruction of 3D data set from the sections of the cell bodies and dendritic processes, using Leica Confocal Software (Leica Microsystems Heidelberg GmbH). Both the individual and projected images were saved directly to a computer as TIFF files. Images were minimally adjusted for brightness and contrast with Adobe Photoshop\u00ae 6 running on a Dell Pentium 4 PC. Images were then imported into Adobe illustrator\u00ae CS2 where groups of images were assembled and labelled.\n3\nResults\n3.1\nConnexin 36 immunostaining in the inferior olivary nucleus\nCaudal medulla slices were immunostained for Cx36 to be used as a positive control. Cx36-immunostaining could only be detected using high magnification lenses (at least 63\u00d7) and appeared as minuscule round immunofluorescent puncta distributed in restricted regions within the inferior olivary nucleus (Fig. 1A). Cx36 expression in other medullary nuclei was extremely low, as illustrated in Fig. 1B, where Cx36-like-ir puncta are virtually undetectable in the trigeminal spinal nucleus. No staining was observed in negative controls where the primary antibody was omitted (data not shown).\n3.2\nConnexin36 immunostaining in the spinal cord\nUnder low magnification (40\u00d7 or less), Cx36 immunostaining is very hard to detect as immunofluorescent puncta are small and sparsely distributed in the ventral horn (Rash et al., 2000; Lee et al., 2005). Under high magnification (63\u00d7 and higher), several Cx36-like-ir puncta were revealed within the soma of spinal neurons (Fig. 2).\n3.3\nCholine acetyltransferase immunostaining in the spinal cord\nSympathetic preganglionic and somatic motoneurons were identified by immunohistochemical staining of ChAT in transverse spinal cord slices from thoracic spinal segments T6\u2013T8. Intracellular ChAT immunostaining (red immunofluorescence) was detected extranuclearly in the cytoplasm and in the dendritic projections (Figs. 2 and 3). ChAT-ir neurons located in lamina VII (IML and IC) and lamina X (CA) were considered as sympathetic preganglionic motoneurons, according to the spatial arrangement described by Cabot (1990; see Fig. 2A). Lateral and medial ChAT-ir projections were easily identified in all sympathetic preganglionic motoneurons within the IML; dorsal and ventral dendritic projections were not identified in transverse spinal slices stained for ChAT. On the other hand, ChAT-ir neurons and their respective dendritic projections located in laminae VIII and IX were considered as somatic motoneurons, according to the anatomical boundaries described by Paxinos and Watson (1998; see Fig. 3).\n3.4\nConnexin 36 immunostaining in sympathetic preganglionic motoneurons\nThe presence of Cx36-like-ir puncta in transverse spinal cord slices taken from T6\u2013T8 spinal segments immunostained for ChAT was analyzed with wide field epifluorescence microscopy. The cellular distribution of Cx36-like-ir puncta in putative sympathetic preganglionic and somatic motoneurons was analyzed in individual optical sections scanned sequentially along the cell body (z series) under high magnification. Optical sections were stacked to show a three-dimensional reconstruction of the neurons. Figs. 2 and 3 show examples of somal and dendritic labelling in ChAT-ir neurons taken from a representative spinal slice from animal number 5. Co-expression of Cx36 (green) and ChAT (red) immunofluorescence appears as yellow. Cx36 immunostaining in ChAT-ir neurons was similar in all the slices taken from all six animals used in this study. All ChAT-ir neurons located in the different sympathetic nuclei, i.e., IML, IC and CA, showed Cx36-like-ir puncta (See Fig. 2A and A1). Both, somal and dendritic puncta were observed in putative sympathetic preganglionic motoneurons. A detailed analysis of each optical section revealed that membrane labelling was very scarce; this was indicated by the small amount of green puncta surrounding ChAT-ir cytoplasm (Figs. 2A1). In contrast, moderate intracellular punctate staining was detected in ChAT-ir regions, i.e., perinuclearly, where the Cx36 is generated in the Golgi-ER complex, suggesting a substantial cytoplasmic production of Cx36 protein. On the other hand, Cx36 staining was consistently detected in ChAT-ir dendrites projected from sympathetic preganglionic neurons within the IML, i.e., lateral and medial projections. Dendritic labelling was characterized by fine puncta distributed along the primary dendrites and thin filaments. Robust Cx36-like staining was often observed in medially projecting bundled dendrites (Fig. 2B1). In some cases, fine puncta were also detected in areas where fine dendrites were seen in close proximity to a cell body (Fig. 2B1). As mentioned before, dorsal dendritic projections were not detected in transverse spinal slices stained for ChAT, and therefore, the presence of Cx36-like-ir puncta in dorsal dendrites could not be determined.\n3.5\nConnexin 36 immunostaining in somatic motoneurons\nCx36-immunoreactive puncta were consistently observed in somatic motoneurons from laminae VIII and IX. Cx36 labelling in somatic motoneurons was characterized by moderate punctate staining distributed perinuclearly in the cytoplasm and to a lesser extent on the plasma membrane. Punctate staining was also distributed along the dendritic arbour and it was often observed in dendro-dendritic and somato-dendritic sites of close apposition (Fig. 3).\n4\nDiscussion\n4.1\nMethodological considerations\nIn the present study, connexin 36-immunoreactivity was identified in putative sympathetic preganglionic motoneurons and somatic motoneurons from the lower thoracic spinal cord of adult rats. The staining pattern obtained was similar to that described previously in the spinal cord and in the ION (Rash et al., 2000; Placantonakis et al., 2006). Even though the peptide used to immunize the rabbits showed local alignments with murine Cx36 protein only, it is important to mention that the specificity of the antibody has only been partially tested in preabsorption control experiments and in Western blots (Bittman et al., 2002). Further experimental evidence is required to fully determine the specificity of this antibody using tissue from Cx36 knock-out animals.\nIt is important to consider that connexin expression can only be estimated at the subcellular level using high magnification objectives. Cx 36-ir puncta are quite small, with an average diameter of 0.61\u00a0\u03bc (Placantonakis et al., 2006) and Cx36 protein levels in the adult spinal cord are distinctly low. Therefore, visualization at low magnification can be extremely difficult; this point can also be illustrated by the apparent difference in the expression of Cx36mRNA levels in the adult spinal cord described in previous studies. Showing a panoramic photograph of the spinal cord at low magnification (judged by the scale bars), Lee et al. (2005) showed that Cx36 mRNA is strong in the neonatal rat spinal cord but in the adult spinal cord, expression is very low and barely above background. In contrast, Cx36 mRNA expression in the adult rat spinal cord can be clearly seen in higher magnification images by Chang et al. (2000). In the present study, it was also difficult to identify Cx36-like-ir puncta in panoramic images of the spinal cord (see Fig. 1A). However, a closer inspection revealed the presence of Cx36 within ChAT-ir neurons.\n4.2\nCellular distribution of Cx36 immunostaining in ChAT-ir neurons\nThis study shows that ChAT-ir neurons from the adult rat thoracic spinal cord express the neuron-specific gap junction protein Cx36. Such labelling was detected both in the IML, IC and CA and in laminae VIII and IX and it was distributed in neuronal somata and along dendritic processes. Somal labelling was mainly characterized by moderate cytoplasmic punctate staining with scarce labelling on the plasma membrane and cell nucleus. Like all membrane proteins, connexins are synthesized by ribosomes and are transported in small vesicles that follow the cell's secretory pathway from the endoplasmic reticulum to the plasma membrane (Yeager et al., 1998; Martin and Evans, 2004). Removal of gap junctions occurs through the formation of annular gap junctions by phagocytosis and lysosomal degradation (Yeager et al., 1998). Therefore, the cytoplasmic labelling observed in putative sympathetic preganglionic motoneurons and in somatic motoneurons may represent the trafficking pathways of connexins leading to the formation and\/or degradation of gap junction channels. Our results therefore support the contention that Cx36 is actively synthesized by the adult spinal cord. This is the first documentation of connexin 36-like immunostaining in ChAT-ir (putative sympathetic preganglionic) neurons in sympathetic nuclei of the spinal cord. Furthermore, our results are consistent with several studies, which have demonstrated that Cx36 mRNA is widely expressed in somatic motoneurons during the embryonic and neonatal life, and continues to be expressed in the adulthood (Chang et al., 1999; Chang et al., 2000; Parenti et al., 2000).\nCx36 labelling was scarce on the somatic plasma membrane of ChAT-ir neurons and it was never found in sites of close apposition between neighbouring cell bodies. However, abundant Cx36 labelling was identified in and along dendritic processes. In fact, Cx36-like-ir puncta were commonly detected in tightly bundled dendrites and in areas where fine dendrites were in close proximity with other cell bodies. The presence of Cx36 in these areas might represent a potential site for gap junctional coupling between dendrites and between the somata and the dendrites of the neighbouring sympathetic preganglionic and somatic motoneurons. This is consistent with in vitro studies using slice preparations, which have identified putative sites of coupling between dendrites and\/or cell bodies in dye-coupled sympathetic preganglionic and somatic motoneurons (Becker and Navarrete, 1990; Shen and Dun, 1990; Mentis et al., 2002). This has also been confirmed at the ultrastructural level, where dendro-dendritic gap junctions have been identified in somatic motoneurons (van der Want et al., 1998). In fact, gap junctions in somatic motoneurons are distributed predominantly in dendro-dendritic and dendro-somatic sites and to a much lesser extent, in somato-somatic points (Matsumoto et al., 1988). Further ultrastructural studies are required to determine whether dendritic Cx36 protein is involved in the formation of homotypic gap junctional plaques among sympathetic preganglionic motoneurons or even heterotypic gap junctions between sympathetic preganglionic motoneurons and interneurons or astrocytes. For instance, previous studies have shown the presence of heterotypic gap junctions in the lumbar spinal cord, composed of Cx32 in superficial dorsal horn neurons on one side, and Cx43 in astrocytic processes on the other (Qin et al., 2005). Interestingly, neuronal tracing studies have proposed the presence of dendro-dendritic connections (presumably through gap junctions) between lamina V neurons from the dorsal horn and sympathetic preganglionic motoneurons from the IML (Cabot et al., 1994). It will be interesting to determine whether Cx36 in sympathetic preganglionic motoneurons is involved in the formation of heterotypic gap junctions with lamina V neurons.\n4.3\nPhysiological role of gap junctional communication between sympathetic preganglionic motoneurons\nPaired cell recordings have shown evidence of synchronization of action potential firing and subthreshold membrane potential oscillations between electrotonically coupled sympathetic preganglionic motoneurons (Logan et al., 1996; Nolan et al., 1999). Since action potential discharge in one of the sympathetic preganglionic motoneurons was almost simultaneously registered as a spikelet in the second sympathetic preganglionic neuron, it was considered that gap junctions mediating electrotonic coupling are located near the site of action potential generation (Logan et al., 1996). These data are consistent with the abundance of the gap junction protein Cx36 that we found between primary dendrites in sympathetic preganglionic motoneurons and supports the idea that electrical synapses might be located in dendro-dendritic sites of close apposition. Electrical synapses in sympathetic preganglionic motoneurons behave in a similar way to a low-pass filter, allowing electrotonic transmission of sub- and suprathreshold activity (Nolan et al., 1999). Therefore, it has been recently proposed that by increasing the degree of synchrony of discharges in the population of sympathetic preganglionic motoneurons, gap junctional communication might play a significant role in the generation of relatively low frequency coherent sympathetic motor rhythms induced by 5-HT in the adult rat (Marina et al., 2006).\n4.4\nPhysiological role of gap junctional communication between somatic motoneurons\nSeveral studies have shown evidence of electrical and dye coupling between somatic motoneurons in the neonatal spinal cord (Gogan et al., 1977; Becker and Navarrete, 1990; Walton and Navarrete, 1991; Chang et al., 1999; Mentis et al., 2002). Experiments using gap junction blockers and NMDA receptor antagonists have shown that coherent somato-motor rhythms result from the interaction between membrane properties at the single cell level and the transmission of this activity through gap junctions (see Kiehn and Tresch, 2002). It has been suggested that gap junction coupling is a very important factor that contributes to the synchronization of individual somatic motoneurons to produce a coordinated rhythmic motor output.\nThe dye transfer properties between neonatal somatic motoneurons change as development progresses, moving from Lucifer yellow to neurobiotin coupling (Becker and Navarrete 1990; Mentis et al., 2002). The number and intensity of labelling of dye-coupled somatic motoneurons are substantially reduced and becomes almost absent shortly after birth (Gogan et al., 1977; Becker and Navarrete, 1990; Walton and Navarrete, 1991; Chang et al., 1999; Mentis et al., 2002). Electrotonic coupling between somatic motoneurons in adult animals has only been observed in pathological conditions such as nerve injury (Chang et al., 1999). However, Cx36 mRNA expression in axotomized somatic motoneurons remains unaffected (Chang et al., 1999). In a similar way, recent evidence has shown that spinal cord injury is associated with a strong up-regulation of Cx43 in astrocytes, but in contrast, neural Cx36 expression remains unaffected (Lee et al., 2005). This suggests that an increased synthesis of Cx36 protein is not involved in the reappearance of coupling in adult injured animals (Chang et al., 1999). Therefore, it might be possible that the reappearance of coupling between adult somatic motoneurons following injury might be produced by different mechanisms, such as the activation of gap junctions that contain different connexins other than Cx36 or by the release of factors that affect either connexin assembly into functional gap junctions, or the open state of the channels. In fact, several studies have shown that gap junction coupling can be modified by the activation of multiple G protein-coupled receptors, by pH and intracellular calcium levels (see Bennett, 1997; Bennett and Zukin 2004). For instance, early postnatal blockade of NMDA receptors has been shown to prevent the decrease in electrotonic and dye coupling observed during the first postnatal week (Mentis et al., 2002). These data show the importance of gap junction coupling regulation by neuromodulators and suggest the possibility that electrotonic coupling between somatic motoneurons could also reappear in the adult animal under different kinds of pathological states, or even in physiological conditions. To date, this possibility has not been explored.\n5\nConclusions\nOur results confirm and expand previous observations regarding Cx36 expression in the spinal cord (Rash et al., 2000; Lee et al., 2005) and provide anatomical evidence that suggests that gap junctions composed by Cx36 may mediate electrotonic coupling between sympathetic preganglionic motoneurons and between somatic motoneurons. Further work using different approaches, such as blockers for Cx36 gap junction channels, Cx36-knock-out animals, transfection of sympathetic preganglionic motoneurons and somatic motoneurons with dominant-negative constructs of Cx36 and Cx36-specific antisense oligodeoxynucleotides, will reveal the precise role of Cx36 in the generation of coherent sympathetic and somato-motor rhythmic activity in the spinal cord of adult animals.","keyphrases":["sympathetic","motoneurons","gap junctions","spinal cord","rhythmic"],"prmu":["P","P","P","P","P"]}
{"id":"Angiogenesis-4-1-2268731","title":"NF-\u03baB: a new player in angiostatic therapy\n","text":"Angiogenesis is considered a promising target in the treatment of cancer. Most of the angiogenesis inhibitors in late-stage clinical testing or approved for the treatment of cancer act indirectly on endothelial cells. They either neutralize angiogenic growth factors from the circulation or block the signaling pathways activated by these growth factors. Another group of angiogenesis inhibitors are the direct angiostatic compounds. These agents have a direct effect on the endothelium, affecting cellular regulatory pathways, independently of the tumor cells. The reason that this category of agents is lagging behind regarding their translation to the clinic may be the lack of sufficient knowledge on the mechanism of action of these compounds. The transcription factor NF-\u03baB has been recently connected with multiple aspects of angiogenesis. In addition, several recent studies report that angiogenesis inhibition is associated to NF-\u03baB activation. This is of special interest since in tumor cells NF-\u03baB activation has been associated to inhibition of apoptosis and currently novel treatment strategies are being developed based on inhibition of NF-\u03baB. The paradigm that systemic NF-\u03baB inhibition can serve as an anti-cancer strategy, therefore, might need to be re-evaluated. Based on recent data, it might be speculated that NF-\u03baB activation, when performed specifically in endothelial cells, could be an efficient strategy for the treatment of cancer.\nIntroduction\nThe NF-\u03baB\/Rel proteins are a family of transcription factors that include five proteins, p50, p52, p65 or RelA, RelB, and c-Rel, that exist as homo- and hetero-dimers. The most common NF-\u03baB heterodimer is composed of p50 and p65. In resting cells, NF-\u03baB is mainly sequestered in the cytoplasm by its association with proteins belonging to the I\u03baB inhibitor family. Stimuli such as the proinflammatory cytokines tumor necrosis factor (TNF)-\u03b1, and interleukin-1 (IL-1), or bacterial products such as lipopolysaccharide (LPS) can activate NF-\u03baB. In the canonical pathway, these stimuli activate I\u03baB kinases (IKKs), which in turn phosphorylate the main NF-\u03baB inhibitor, I\u03baB\u03b1. This phosphorylation step leads to the ubiquitination and subsequent degradation by the proteasome of I\u03baB\u03b1. The NF-\u03baB complex translocates to the nucleus where it binds to \u03baB enhancers present in the regulatory regions of various genes and where it activates transcription [1]. The NF-\u03baB target genes are involved in a wide range of biological functions including proliferation, survival, and inflammation (Fig.\u00a01).\nFig.\u00a01Schematic NF-\u03baB pathway. In the canonical activation pathway, NF-\u03baB (often the dimer composed of p50\/p65) is sequestered by its main inhibitor I\u03baB-\u03b1. Upon stimulation, cell surface receptors activate IKK complex, which then phosphorylate I\u03baB-\u03b1. These phosphorylations lead to its degradation by the proteasome and the entry of NF-\u03baB in the nucleus, which turns on target genes\nNF-\u03baB activation has been connected with multiple aspects of oncogenesis, including the control of tumor cell proliferation, migration, cell cycle progression, and inhibition of apoptosis [2\u20134]. Indeed, NF-\u03baB is constitutively activated in several types of cancer cells and it is generally regarded as an anti-apoptotic and pro-oncogenic signal. The most studied and well-established functions of NF-\u03baB in promoting oncogenesis are its ability to (i) induce growth promoting genes such as cyclin D1 and c-myc and (ii) induce anti-apoptotic genes such as c-IAP-1, c-IAP-2, or XIAP [3]. Therefore, activation of NF-\u03baB in cancer cells by chemotherapy or radiation therapy is often associated with the acquisition of resistance to apoptosis. This has emerged as a significant impediment to effective cancer treatment. In combination with chemotherapy, inhibitors of the NF-\u03baB pathway (e.g., proteasome inhibitors) were recently used with success as treatment against cancer [5]. Next to this direct effect, it has also been reported that NF-\u03baB activity can be tumorigenic by activation of pro-angiogenesis genes, such as VEGF, IL-8, and MMP-9 [6].\nIn contrast to the negative effects of NF-\u03baB activation, recent reports suggest that in certain situations NF-\u03baB can promote apoptosis and may be viewed as a tumor suppressor gene. For example, blockade of NF-\u03baB predisposes murine skin to squamous cell carcinoma [7]. This observation could be explained by the fact that in normal human epidermal cells, NF-\u03baB activation induces cell cycle arrest [8]. In addition, Ryan et\u00a0al. describe the role of NF-\u03baB in p53-mediated programmed cell death. The tumor suppressor p53 inhibits cell growth through activation of apoptosis and cell cycle arrest. Using a p53-inducible Saos-2 cell line, it was demonstrated that induction of p53 causes activation of NF-\u03baB. Furthermore, inhibition of NF-\u03baB abrogated p53-induced apoptosis demonstrating that inhibition of NF-\u03baB in tumors that retain wild-type p53 may reduce a therapeutic response [9]. Loss of p65 can also cause resistance to different agents that induce apoptosis through p53 [10]. Independently, it was demonstrated that activation of NF-\u03baB is essential for the cytotoxic effect of doxorubicin and its analogs [11]. Many hypotheses have been put forward to explain this dual activity. The overall conclusion that is emerging is that the final outcome of NF-\u03baB activation depends on cell type, the stimulus, and the context of activation [12\u201314]. The dual activity of NF-\u03baB complicates the systemic use of broad spectrum NF-\u03baB inhibitors for the treatment of cancer and it has been suggested to design better therapeutics that specifically unleash the pro-apoptotic activity of NF-\u03baB [15, 16].\nNF-\u03baB in ongoing angiogenesis\nNF-\u03baB signaling has been found to regulate endothelial cell integrity and vascular homeostasis in\u00a0vivo. Treatment of zebrafish embryos with NF-\u03baB inhibitors provokes vascular leakage and alters vessel morphology [17]. The role of NF-\u03baB signaling in tumor angiogenesis has also been recently investigated. Inoculated tumors grow faster in transgenic mice expressing mutated I\u03baB\u03b1, under control of the Tie-2 promoter, resulting in endothelial repression of NF-\u03baB [18]. Histological analysis revealed a striking increase in tumor vascularization in these mice. This study highlighted, for the first time, the in\u00a0vivo role of NF-\u03baB in tumor angiogenesis, indicating an inhibitory role for NF-\u03baB in tumor angiogenesis. Based on this study, NF-\u03baB activation in endothelial cells appears to be a way to block angiogenesis. However, it is unknown how specific activation of NF-\u03baB in endothelial cells can be realized and through which mechanisms NF-\u03baB activation leads to inhibition of angiogenesis.\nAngiogenesis occurs in a coordinated series of steps, which can be divided into a destabilization, a proliferation, and a maturation phase. Whereas inhibition of angiogenesis can prevent diseases with excessive vessel growth such as cancer, diabetes retinopathy, and arthritis, stimulation of angiogenesis would be beneficial in the treatment of diseases such as coronary artery disease and critical limb ischemia in diabetes [19]. One of earliest events in angiogenesis is the degradation of the vascular basement membrane and the remodeling of the extracellular matrix (ECM). The role of NF-\u03baB in the regulation of these systems is well documented. In line with a pro-oncogenic activity, NF-\u03baB promotes expression of several matrix metalloproteinases (MMPs), including MMP-2, -3, and -9 [20\u201322]. However, NF-\u03baB could also inhibit endothelial cell migration via the up-regulation of tissue inhibitors of metalloproteinase-1 (TIMP-1) as described in astrocytes [23]. Next to the MMPs, plasmin is a broad-spectrum protease that also hydrolyzes many extracellular proteins, the most notable of which is fibrin. Plasmin is produced from an inactive precursor called plasminogen. uPA (urokinase plaminogen activator) and tPA (tissue-type plasminogen activator) are two proteases with high affinity for plasminogen. The activation of plasminogen into plasmin could be negatively regulated by the physiological inhibitors, namely plasminogen activator inhibitor (PAI)-1 and -2 [24]. In endothelial cells, it has been described for both reactive oxygen species as well as for TNF-\u03b1 induced expression of PAI-1 via NF-\u03baB [25]. In addition, activation of NF-\u03baB by TNF-\u03b1 can also lead to the inhibition of the tPA expression [26]. These data suggest that NF-\u03baB activation could impair angiogenesis via a decrease in ECM degradation capacity.\nIntegrins are the principle adhesion receptors used by endothelial cells to interact with the extracellular environment and are necessary for cell migration, proliferation, and survival [27]. It has recently been demonstrated that the interaction of \u03b1V\u03b23-integrin with the ECM activates NF-\u03baB by activation of the IKK complex and degradation of I\u03baB-\u03b1. This activation triggers a pro-survival signal for example in rat aortic endothelial cells [28].\nMany soluble molecules control the balance between cell proliferation and cell death. While angiogenic factors such as VEGF and bFGF are mitogenic and act as survival factors, angiostatic agents induce cell cycle arrest and promote endothelial cell death [29]. Activation of NF-\u03baB in endothelial cell leads to the expression of angiogenic and angiostatic factors. VEGF expression is up-regulated by hypoxia-induced mitogenic factor through activation of the NF-\u03baB pathway [30]. On the contrary, vascular endothelial growth inhibitor (VEGI, reported to inhibit endothelial cell proliferation) has also been found to be induced by NF-\u03baB [31]. In addition, the promoters of thrombospondin-1 and -2, which are among the first naturally occurring angiostatic agents discovered, contain NF-\u03baB binding sites [32].\nThe role of NF-\u03baB in the cell cycle progression has been also investigated [33]. NF-\u03baB induces expression of activators of the cell cycle such as cyclin D or -E [34] as well as expression of inhibitors such as p21\/cip1 [35] demonstrating that the overall effect of NF-\u03baB on cell proliferation is difficult to predict. To our knowledge, there are no reports on a direct relationship between NF-\u03baB activation and proliferation in endothelial cells.\nProgrammed cell death or apoptosis occurs mainly by two connected pathways. The extrinsic pathway involves activation of caspase-8 by cell surface death receptors, while the intrinsic pathway, involves cytochrome-c release from mitochondria and subsequent caspase-9 activation. As previously described for tumor cells, diverse activities have been observed in endothelial cells. Silibinin, a cancer chemopreventive agent, was found to induce apoptosis in endothelial cell line by inhibiting NF-\u03baB [36] and a report describes that pro-survival effect of VEGF is mediated by NF-\u03baB activation [37]. However, a large body of evidence exists that NF-\u03baB activation plays a pro-apoptotic role in endothelial cells. A high concentration of glucose activates the production of reactive oxygen species and induces caspase-3 activation in endothelial cells [38]. Recently, it has been demonstrated that this is mediated via NF-\u03baB and subsequent c-Jun N-terminal protein kinase activation [39]. In human cardiac microvascular endothelial cells, IL-18 induces activation of both the intrinsic and extrinsic apoptotic pathways via NF-\u03baB activation [40]. Angiopoietin-1 inhibits endothelial cell apoptosis induced by growth factor deprivation. This effect is mediated via the activation of an endogenous inhibitor of NF-\u03baB, namely A20 binding inhibitor of NF-\u03baB (ABIN-2) [41]. In addition, in endothelial cells, it was observed that A20 inhibition and subsequent activation of NF-\u03baB, effectively leads to reduced tube formation in a matrigel assay [42].\nA role for NF-\u03baB in angiostatic therapy?\nThe observations described above indicate that NF-\u03baB is involved in the regulation of migration and\/or proliferation\/survival of endothelial cells and suggest a strong link between NF-\u03baB in angiostatic agent signaling. The following section will highlight the role of NF-\u03baB in the angiostatic agent signaling. Indeed, several angiostatic compounds, already described to block tumor growth, have been reported to act on endothelial cells via NF-\u03baB activation [29].\nPlatelet factor-4 (PF4) is an \u03b1-chemokine naturally secreted by platelets and is known to inhibit angiogenesis [43]. PF4 promotes the expression of E-Selectin in HUVEC. Data provide direct evidence that the NF-\u03baB\u2013binding site is required for PF4-mediated activation of the E-selectin promoter. In addition, EMSA experiments demonstrate that PF4 treatment of HUVECs results in binding of NF-\u03baB to the E-Selectin promoter already after 1\u00a0hour of stimulation [44]. The angiostatic properties of angiostatin, a cleavage product of plasminogen, have also been linked with NF-\u03baB. Chen et\u00a0al. have analyzed the global action of angiostatin in endothelial cells. By microarray screening, they have found an altered expression of 189 genes after treatment with angiostatin. These genes are mainly involved not only in growth, apoptosis, migration but also in inflammation [45]. Even though no direct evidence points a role of NF-\u03baB, angiostatin promotes mRNA expression of RelB as well as many NF-\u03baB target genes, namely E-selectin, intracellular adhesion molecule-1 (ICAM-1), Cyclin D1, p21\/cip1, and FasL (for the complete list of altered gene expression, see reference [45]). Based on these data, there is a strong suggestion that NF-\u03baB is also activated by angiostatin. The 16\u00a0kDa N-terminal fragment of prolactin (16\u00a0K PRL) is a potent angiostatic agent in various in\u00a0vivo models and has been shown to inhibit endothelial cell migration and proliferation [46\u201348]. We have demonstrated that NF-\u03baB activation is required for 16\u00a0K hPRL-induced caspase-8 and -9 activation and subsequent apoptosis [49]. In addition, it is interesting to note that NF-\u03baB activation appears to be a very proximal event. The angiostatic agent Neovastat, which is currently in phase III clinical studies, inhibits angiogenesis through an increase in tPA activity and it has been shown that this induction is NF-\u03baB dependent [50]. Finally, administration of statins has been shown to decrease tumor growth and angiogenesis [51]. Statins up-regulate the expression of endothelial and inducible nitric oxide synthase through NF-\u03baB activation [52].\nBased on all these reports, it is suggested that the activity of angiostatic compounds is dependent on activation of NF-\u03baB. Therefore, activation of NF-\u03baB specifically in endothelial cells might be an attractive therapy. TNF-\u03b1 is one of the most potent NF-\u03baB activators. The clinical use of TNF-\u03b1 as an anti-cancer drug is limited to local treatment (e.g., isolated limb perfusion) because of its systemic toxicity [53]. To circumvent this problem, targeted delivery of TNF-alpha to tumor vessels was achieved by coupling this cytokine with cyclic CNGRC peptide, an aminopeptidase N (CD13) ligand that targets the tumor neovasculature. Administration of this compound leads to a reduced toxicity, a marked endothelial cell apoptosis, destruction of blood vessels, and improvement of the anti-tumor activity of doxorubicin [54]. These studies indicate that NF-\u03baB activation, specifically in endothelial cells, can be an efficient strategy for the treatment of cancer.\nIn conclusion, while in tumor cells NF-\u03baB is mostly described as an oncogenic factor, up-regulation of NF-\u03baB in endothelial cells is associated with angiostatic activity. It might therefore be warranted to revisit anti-cancer therapies based on inhibition of NF-\u03baB activity for effects on angiogenesis.\nAn indirect anti-tumor activity of NF-\u03baB through circumvention of endothelial cell anergy\nNext to a direct anti-tumor activity of NF-\u03baB through inhibition of tumor angiogenesis, the activation of NF-\u03baB could also be connected with an indirect anti-tumor activity through reversal of endothelial unresponsiveness to inflammatory signals, a process called endothelial cell anergy. The latter is defined as the inability of tumor endothelial cells to express adhesion molecules such as ICAM-1\/-2, vascular endothelial cell adhesion molecule-1 (VCAM-1) or E-selectin, in response to inflammatory cytokines such as TNF-\u03b1, interferon-\u03b3 and interleukin-1. These adhesion molecules mediate leukocyte rolling along, adhering to, and diapedesis through the vessel wall, and thus have an important role in the selection of an inflammatory infiltrate [55].\nThe observation of a reduced number of infiltrated leukocytes in the tumor [56] has been correlated with the fact that tumor endothelial cells display a reduced expression of adhesion molecules (ICAM-1 and ICAM-2) as compared with normal endothelial cells [57, 58]. In\u00a0vitro and in\u00a0vivo studies on endothelial cell anergy have demonstrated that this reduced expression is caused by exposure to angiogenic growth factors such as VEGF and bFGF [59]. It has been recently described that bFGF down-regulates ICAM-1 expression via NF-\u03baB inhibition [60]. Furthermore, we have demonstrated that suppressed leukocyte-vessel wall interactions in tumor vessels can be normalized by angiostatic compounds, such as endostatin, angiostatin, anginex, and 16\u00a0K hPRL, as well as by treatment with chemotherapeutic agents [61, 62]. This normalization has been correlated with up-regulation of ICAM-1, VCAM-1, and E-selectin in endothelial cells [44, 63]. Therefore, activation of NF-\u03baB by angiostatic therapy does not directly affect angiogenesis but also has an indirect effect via expression of adhesion molecules and subsequent reversal of endothelial cell anergy. Therefore, such activation of NF-\u03baB resulting in stimulation of anti-tumor immunity but also in inhibition of angiogenesis clearly results in an anti-tumor outcome.\nWhere to go from here?\nInhibition of angiogenesis is a promising therapeutic approach to fight cancer. From several recent findings it is likely that activation of NF-\u03baB is a common mechanism of angiostatic agents, resulting in both inhibition of angiogenesis and stimulation of anti-tumor immunity. While these data raised a cautionary note about the pharmaceutical agents that block NF-\u03baB, they also suggest that a targeted activation of NF-\u03baB, specifically in endothelial cells, could represent a new and promising strategy in cancer treatment (Fig.\u00a02). Further studies remain necessary to fully understand the molecular mechanisms induced by angiostatic agents and the role of NF-\u03baB in endothelial cells.\nFig.\u00a02Model of the dual role of NF-\u03baB in tumorigenesis. In tumor cells, activation of NF-\u03baB leads to tumor growth by both direct and indirect mechanisms. The direct mechanisms involve expression of anti-apoptotic and pro-proliferative molecules while the indirect mechanism involves promotion of angiogenesis. In endothelial cells, activation of NF-\u03baB can block tumor progression by both, angiostatic activity, via the production of pro-apoptotic molecules and by improvement of immune response via expression of adhesion molecules","keyphrases":["nf-\u03bab","angiostatic"],"prmu":["P","P"]}
{"id":"Anal_Bioanal_Chem-4-1-2175028","title":"Problems in obtaining precise and accurate Sr isotope analysis from geological materials using laser ablation MC-ICPMS\n","text":"This paper reviews the problems encountered in eleven studies of Sr isotope analysis using laser ablation multicollector inductively coupled plasma mass spectrometry (LA-MC-ICPMS) in the period 1995\u20132006. This technique has been shown to have great potential, but the accuracy and precision are limited by: (1) large instrumental mass discrimination, (2) laser-induced isotopic and elemental fractionations and (3) molecular interferences. The most important isobaric interferences are Kr and Rb, whereas Ca dimer\/argides and doubly charged rare earth elements (REE) are limited to sample materials which contain substantial amounts of these elements. With modern laser (193 nm) and MC-ICPMS equipment, minerals with >500 ppm Sr content can be analysed with a precision of better than 100 ppm and a spatial resolution (spot size) of approximately 100 \u03bcm. The LA MC-ICPMS analysis of 87Sr\/86Sr of both carbonate material and plagioclase is successful in all reported studies, although the higher 84Sr\/86Sr ratios do suggest in some cases an influence of Ca dimer and\/or argides. High Rb\/Sr (>0.01) materials have been successfully analysed by carefully measuring the 85Rb\/87Rb in standard material and by applying the standard-sample bracketing method for accurate Rb corrections. However, published LA-MC-ICPMS data on clinopyroxene, apatite and sphene records differences when compared with 87Sr\/86Sr measured by thermal ionisation mass spectrometry (TIMS) and solution MC-ICPMS. This suggests that further studies are required to ensure that the most optimal correction methods are applied for all isobaric interferences.\nIntroduction\nThe radioactive beta (\u03b2\u2212) decay of 87Rb to 87Sr is an important isotope system that has been widely applied for geochronological purposes. More importantly the isotopic system is extensively used to constrain the rates and fluxes involved in a wide range of geological processes operating from within the hydrosphere of the Earth to the deep mantle. In addition, recently Sr isotopes have become widely applied as provenance tracers in many different scientific disciplines (e.g. biology, nutrition, medical, forensic and art history) [1\u20137]. Sr isotope ratios have always been difficult to analyse to high precision (better than 20\u00a0ppm), because there are large differences in the abundances of the isotopes 84Sr, 86Sr, 87Sr and 88Sr. In many materials the low abundance of Rb and their relatively young age leads to small isotopic variations (e.g. 0.05% in mantle rocks and biogenic carbonates). This range is considerably smaller than the combined U-Th-Pb isotope system for example. Recent technical improvements in thermal ionisation mass spectrometry (TIMS) design, most notably in terms of Faraday collectors and amplifier electronics, now allow analytical precisions better than 0.0005% (5\u00a0ppm, 1\u00a0SE). Despite this significant improvement in precision, the classical TIMS technique requires time-consuming liquid chromatographic techniques to remove matrix and interfering elements (87Rb), inhibiting any possibility for an \u201conline\u201d in situ analytical technique.\nThe arrival of the first commercial multicollector inductively coupled plasma mass spectrometry (MC-ICPMS) instruments in the first half of the 1990s, coupled with laser ablation (LA) facilities promised an enormous new potential for in situ analytical techniques in petrology, marine sciences and many other applications (e.g. see [8, 9]). The study of small-scale variations of Sr isotopes in geological materials has provided very important constraints on the rates of volcanic processes and the fluxes that operate in diverse geological environments (e.g. see [2, 10]). However, these studies are extremely time consuming, typically averaging more than 10\u00a0hours per sample because they require careful microdrilling techniques, low-blank liquid chromatographic separation techniques and TIMS analyses. The MC-ICPMS coupled with a laser ablation system should be able to perform a Sr isotope analyses within minutes, while importantly maintaining the spatial resolution and avoiding the extensive wet chemistry and warm up times on the TIMS instrument.\nDespite the great potential of LA-MC-ICPMS analyses for Sr isotopes and its availability for more than a decade, the technique has not become routine. Eight out of eleven papers published in the period 1995\u20132006 that reported in situ analyses of Sr isotopes by LA-MC-ICPMS are mainly focussed on technique development [11\u201321]. There is clearly a huge potential and a demand for the application of in situ Sr isotope analysis. Therefore the goals of this paper are to: (1) describe the different approaches that have been followed to date, (2) summarize the problems encountered, (3) discuss the solutions attempted to solve the encountered problems and (4) discuss studies which are needed in the near future to improve the LA-MC-ICPMS technique so that \u201croutine\u201d Sr isotope analysis becomes possible.\nDescription of instrument setups\nAll LA-MC-ICPMS instrument setups have the same basic geometry of LA system, pulsed laser focussed onto ablation chamber, sample transfer-inlet system, differential vacuum pumping system, mass-energy filter and signal detection (Fig.\u00a01). It is beyond the scope of this review to discuss the actual performance of the different mass spectrometers, so we concentrate upon the sample handling and data interpretation aspects of the LA-MC-ICPMS technique.\nFig.\u00a01Typical LA-MC-ICPMS setup with He as carrier gas. ESA electrostatic filter analyser\nLaser ablation systems\nLaser ablation systems have been tremendously improved in the last decade (see [22] for a review). This development has mainly focussed on how to obtain maximum sensitivity in both sample ablation and transfer of the ablated sample to the ion optics of the ICP-MS and a minimized fractionation of elemental ratios, for example Th\/U (e.g. see [23, 24]). The early studies of Christensen et al. [11] and Davidson et al. [12] used a pulsed 266-nm quadrupled Nd:YAG laser, whereas more recent studies have tended to use shorter wavelengths, e.g. pulsed 213-nm and Excimer 193-nm lasers (Table\u00a01). Owing to the more efficient absorption of shorter wavelength light by most materials, the reported interelement fractionation is less with lower wavelength lasers (e.g. see [25, 26]). Consequently, the application of lower wavelength lasers tends to produce more accurate Sr isotope results. However, it is difficult to judge from the published papers which laser system has the highest sensitivity, as this is highly dependent on the repetition rate of the laser, the pit size or raster technique used and the energy density of the laser system. Moreover, ICP-MS inlet design and instrument sensitivity have improved by several orders of magnitude in the last decade, so direct comparison between the sensitivity of systems of different age is perhaps pointless.\nTable\u00a01Instrument setups and materials analysed in eleven publications on Sr isotope analyses by LA-MC-ICPMSReferenceLaser typePit sizes (\u03bcm)Materials (Sr concentration)InstrumentSensitivity\/blank levels[11]Nd:YAG, 266\u00a0nm, 8\u00a0Hz, 20\u201330\u00a0mJ\u00a0pulse\u22121, carrier gas ArSpot 20\u201340 and 150\u2013300Carbonate (2,000\u00a0ppm)VG P54LA 2,000\u00a0ppm, sample material gives >3\u00a0V total SrFeldspar (2,000\u00a0ppm)No blank reported[12]Nd:YAG, 266\u00a0nm, 5\u201320\u00a0Hz, 1.2\u00a0mJ, carrier gas not specifiedSpot 100\u2013300, rasterPlagioclase (1,200\u20132,100\u00a0ppm)IsoProbeNo blank reported[13]Nd:YAG, 266\u00a0nm, 10\u00a0Hz, 0.66\u00a0mJ, carrier gas not specifiedSpot 70, rasterCarbonate, fresh water otolith (ca. 500\u00a0ppm)VG P54No blank reported[14]Nd:YAG, 266\u00a0nm and 213\u00a0nm, 20\u00a0Hz, 4\u00a0mJ, carrier gas ArSpot 10\u2013200Plagioclase, apatite sphene, clinopyroxene otolithVG Axiomca. 25\u00a0V\u00a0ppm\u2212188Sr (solution work); blank 0.5\u20133\u00a0mV 85Rb and 88Sr[15]Excimer, 193\u00a0nm, 1\u20132\u00a0Hz, 4\u20135\u00a0J\u00a0cm\u22122, carrier gas HeSpot 150\u2013330Apatite (>3,000\u00a0ppm)IsoProbeNot reportedCarbonate (>3,000\u00a0ppm)[16]Excimer, 193\u00a0nm, (see [15])Spot 330Clinopyroxene (100\u2013400\u00a0ppm), plagioclase, carbonateIsoProbeNot reported[17]Nd:YAG, 213\u00a0nm, 10\u00a0Hz, 7\u201310\u00a0J cm\u22122, carrier gas HeSpot 80, raster 160\u2009\u00d7\u2009500Carbonate (1,000\u00a0ppm)ThermoFinnigan Neptune88Sr blank <5\u00a0mVPlagioclase (900\u00a0ppm)Clinopyroxene (50\u00a0ppm)Basaltic groundmass (400\u00a0ppm)[18]Excimer, 193\u00a0nm, 5\u00a0Hz, 10\u00a0J\u00a0cm\u22122, carrier gas HeSpot 10\u2013350, rasterCarbonate otolithNu PlasmaNot reported[19]Nd:YAG, 213\u00a0nmRaster 60\u2013500, 80 deepCarbonate, fresh water otolith (ca. 300\u2013800\u00a0ppm)ThermoFinnigan NeptuneNot reported[20]Excimer, 193\u00a0nm, 5\u00a0Hz, 50\u00a0mJ, carrier gas HeRaster, 80 wideCarbonate, otolithsThermoFinnigan NeptuneNot reported[21]Nd:YAG, 213\u00a0nm, 20\u00a0HzSpot 120, rasterMelt inclusionsThermoFinnigan Neptuneca. 40\u00a0V\u00a0ppm\u2212188Sr (solution work)Blank not reported\nHe (0.5\u20131.0\u00a0L\u00a0min\u22121) is used as the transport gas through the ablation cell in nearly all studies (see Table\u00a01) following the publication of [27], which demonstrated that He gives a higher sensitivity and less inter-element fractionation (Rb\/Sr) compared with other potential carrier gases (e.g. Ar). The He flow is mixed with Ar flow before it enters the plasma in a ratio between 0.5 and 1.0 (see Fig.\u00a01).\nWith laser ablation analyses there are essentially two possible ablation methods: spot analysis and rastering. A spot analysis leaves the laser beam in the same position where the laser beam progressively ablates material from deeper levels of the ablation pit. There are two advantages of spot analysis: first, the optimal spatial resolution is obtained, typically between 100\u2013300\u00a0\u03bcm for Sr isotopes; second, this approach limits the influence of surface contamination. A significant disadvantage of the single spot analysis is that on many laser ablation systems the laser will go out of focus if the depth to diameter ratio of the ablation pits is larger than one [28]. This will result in lower beam intensities due to lower ablation rates coupled with less efficient sample fragmentation that also reduces ionisation efficiency in the plasma. Furthermore, if the depth to diameter ratio becomes larger than 6, significant fractionation occurs between elements [28], which could make the 87Rb correction on 87Sr less accurate. For a 50-\u03bcm spot analyses the maximum ablation time is 1\u00a0min (assuming 1\u00a0\u03bcm\u00a0s\u22121 ablation rate, see Fig.\u00a02).\nFig.\u00a02Signal intensity obtained by ablating different spot sizes (50 and 120\u00a0\u03bcm) and rastering (120-\u03bcm spot, moving at 5\u00a0\u03bcm\u00a0s\u22121) as a function of Sr abundance in a mineral (modified from Fig. 2 in [12]). The ablation rate is 1\u00a0\u03bcm\u00a0s\u22121, and the typical efficiency is 0.05% (e.g. 1 in 2,000 ions that get ablated are counted by the detector). This diagram illustrates that with a 50-\u03bcm spot size, only the minerals with >2,000\u00a0ppm can be analysed with sufficient precision. Rastering results in slightly better 88Sr signals, but its greatest advantage is that the beam does not run out of focus during the analysis, and the 88Sr ion current does not decay as with a spot analysis\nThe rastering technique involves the movement of the sample with a low speed (typically 1\u20135\u00a0\u03bcm\u00a0s\u22121, e.g. see [18]). The advantage is that the ion beam size is more stable over time, and the 88Sr signal is larger (see Fig.\u00a02). Disadvantages are that the surface contamination of the sample could be a problem and the advantage of high spatial resolution of LA is diminished. The surface contamination problem can be overcome by \u201cpre-cleaning\u201d the ablating area with the same raster area but with a lower ablation rate.\nMC-ICPMS instruments\nFive different MC-ICPMS instruments have been used to obtain Sr isotopes LA-MC-ICPMS (see Table\u00a01). Although different in configuration, the type of MC-ICPMS instrument does not seem to be very important. All MC-ICPMS instruments can collect the Sr isotope masses and the additional masses required to perform the isobaric corrections on the Sr isotope masses. There are probably differences in the sensitivity, whereby the older generation instruments (P54, Isoprobe and Axiom) have a lower sensitivity than more recently designed instruments (ThermoFinnigan Neptune and Nu Plasma instruments). The collision cell arrangement is not beneficial for Sr isotope analysis (maybe for Fe interferences, see below), nor is the high-resolution capability of the Nu Plasma HR, Nu Plasma 1700 and the ThermoFinnigan Neptune instruments because isobaric interferences, such as 87Rb on 87Sr, can only be resolved with a mass resolution of >10,000, significantly beyond the capabilities of all MC-ICP-MS instruments.\nMaterials ablated\nTable\u00a01 lists the different types of geologically relevant materials which have been analysed. Except for the melt inclusion work reported by Jackson and Hart [21], most of the tested materials are characterized by low Rb\/Sr (<0.002) ratios. The majority of studied materials are marine carbonate and magmatic plagioclase. Most studies have used present-day marine carbonate as an \u201cin-house\u201d standard, assuming that it has the 87Sr\/86Sr composition of present-day seawater: 0.709172 [29]. Marine carbonate has in the order of 2,000\u00a0ppm Sr. Plagioclase is another suitable material, since it has also low Rb\/Sr ratios and high Sr contents (600\u20131,800\u00a0ppm). Although these minerals have low Rb\/Sr ratios, the Ca\/Sr ratios are high, which could result in potential interferences from Ca (see below).\nOther materials that have been ablated and analysed successfully are clinopyroxene, magmatic carbonates (Sr-rich >3,000\u00a0ppm and Rb-poor <1\u00a0ppm, [15]), apatite and melt inclusions from relatively Sr-rich alkaline melts. These materials generate several types of isobaric interferences which will be discussed below.\nThe highest Rb\/Sr ratios materials were analysed by Jackson and Hart [21]. In their study they analysed basaltic melt inclusions with Rb\/Sr ratios of up to 0.14, although the reported external reproducibility of the 87Sr\/86Sr is about 5 times worse than that of a melt inclusion with a Rb\/Sr of 0.04.\nFactors influencing the data quality of Sr isotope analysis by LA-MC-ICPMS\nThe following factors influence the quality of data which can be obtained by LA-MC-ICPMS: (1) counting statistics, (2) blank levels, (3) instrumental mass discrimination and laser-induced elemental and isotopic fractionation and (4) molecular interferences.\nCounting statistics\nThe precision which can be obtained by Sr isotope analyses by LA-MC-ICPMS depends upon the number of ions counted. The number of ions that can be collected by the Faraday cups depends on (1) the amount of Sr in the sample (see Fig.\u00a02), (2) ablation spot size, ablation rate (energy density and pulse rate), (3) the laser ablation efficiency (e.g. the particle size distribution of ablated material), (4) transport from sample chamber to plasma and (5) tuning conditions of the plasma and mass spectrometer.\nThe amount of Sr brought to the plasma is unclear in most publications. Davidson [12] reported that approximately 10\u00a0ng of Sr is needed for a 3-V beam of 88Sr. The theoretical best possible precision for such beam intensity is then 23\u00a0ppm.\nBackground (blank levels)\nThe instrumental background levels for 88Sr and 85Rb that have been reported are 0.5\u20135.0\u00a0mV ([14, 17], see Table\u00a01). Since running samples spiked with 87Rb and 84Sr resulted in non-natural background isotopic composition of these isotopes, Waight et al. [14] concluded that the Sr and Rb background is an accumulation of material from samples within the introduction system. It is important to note that these backgrounds are significantly higher than those reported for Pb and Hf isotopes, and that careful monitoring of the background is clearly vital to obtain accurate results. In nearly all studies the blank is measured by collecting data with conditions as during the analysis, except for not firing the laser. Only in two studies [13, 21] are baselines measured \u201coff-peak\u201d as with a standard TIMS analysis.\nInstrumental mass discrimination and laser-induced elemental and isotopic fractionation\nBoth the laser system and the MC-ICPMS induce isotopic fractionations. The isotope fractionation in the MC-ICPMS is generally referred to as instrument mass discrimination. In this case the measured elemental or isotope ratio are different from the \u201ctrue\u201d ratio due to differences in ionisation potentials, space charge effects, effects of the matrix of the sample, preferential transmission of one type of ion, and reactions in the ICP (e.g. oxide formation). The instrument mass discrimination is generally corrected during solution MC-ICPMS analyses by using a stable isotope pair, which can be corrected by an exponentional law (similar to TIMS mass fractionation correction), or when there is no stable isotope pair available (e.g. Pb isotopes), corrected using a standard-sample bracketing method [30].\nIn addition to the instrument mass discrimination, it is now well established that during the ablation process of a sample, both isotopic and elemental fractionations are induced. The best studied elemental fractionations have been reported for U\/Pb, for which an increase of the U\/Pb ratio by a factor 2 during a single ablation has been reported (e.g. see [24, 31]). Laser-induced isotopic fractionation has also been reported, e.g. for Fe isotopes [32].\nBoth the laser-induced isotopic fractionation and the instrument mass discrimination can be corrected for during Sr isotope analyses by using the stable 86Sr\/88Sr ratio and an exponential correction [30]. All 11 LA ablation studies have done so, except one [13] in which no mass bias correction was applied to the data.\nElemental fractionation induced by the laser is another important factor. The Sr isotope ratios can be internally normalized to 86Sr\/88Sr, but correcting for the 87Rb interference on the 87Sr (see below) is complicated by the fractionation of the Rb\/Sr ratio. In seven of the published studies it is assumed that the laser-induced isotope fractionation and instrument mass discrimination are the same for both Sr and Rb. This is possibly not the case, as suggested by the study of Jackson and Hart [21] who employed a standard-sample bracketing method to establish the total Rb laser-induced fractionation and instrument mass discrimination, in order to cope with large Rb corrections (see below).\nSimilarly, most authors make the Kr corrections on the Sr masses by assuming that the laser-induced isotope fractionation and instrument discrimination are the same for Sr and Kr. For a good Kr correction, the mass bias has to be established in an independent way. We will use the term mass bias to describe both the laser-induced isotope fractionation and the instrument mass discrimination.\nMolecular interferences\nMeasurement and correction of the interferences of elements and molecules on the Sr masses (Table\u00a02) is perhaps the most challenging aspect of Sr isotope analyses by MC-ICPMS. This is especially true when comparing LA analysis with solution work, where some of the interfering elements (e.g. Ca, Rb and the REE) can be removed by chromatographic purification of the sample or \u201cburnt off\u201d by heating at temperatures below that required for Sr ionization on a TIMS instrument. Unfortunately, for LA-MC-ICPMS Sr isotope analysis, large corrections for interfering elements need to be made. Since these interferences can be more than half of the signal intensity on the Sr isotope mass, these corrections are large and the correction procedures need to be extremely rigorous if accurate and precise Sr isotope ratios are to be obtained.\nTable\u00a02Sr isotope masses and possible interferences in the mass region 82\u201389Source of interferenceMass8283848586878889Sr84Sr86Sr87Sr88SrKr82Kr83Kr84Kr86KrRb85Rb87RbREEY89YYb2+168Yb2+170Yb2+172Yb2+174Yb2+176Yb2+Er2+166Er2+168Er2+170Er2+Lu2+176Lu2+Hf2+174Hf2+176Hf2+Fe\/Zn\/Ga oxides54Fe54Fe16O254Fe16O17O54Fe16O18O54Fe17O256Fe56Fe16O266Zn66Zn17O67Zn67Zn16O68Zn68Zn16O68Zn17O68Zn18O70Zn70Zn16O70Zn17O70Zn18O69Ga69Ga17O69Ga18O71Ga71Ga16O71Ga17OCa dimers40Ca43Ca40Ca44Ca42Ca43Ca40Ca46Ca40Ca48Ca42Ca44Ca42Ca46Ca43Ca243Ca44Ca44Ca2Ca argides43Ca40Ar48Ca36Ar46Ca38Ar48Ca38Ar44Ca40Ar46Ca40Ar48Ca40ArCa-P40Ca31P16O\nThere are many potential interfering elements and molecules on the Sr isotope spectrum, most of which have been extensively discussed by Ramos et al. [17]. This study concluded that careful monitoring and correcting for the interferences can produce Sr isotope data with a precision suitable for use in most geological applications. In the next section we discuss the main interferences and how they can compromise Sr isotope data, leading to errors in excess of those that can be tolerated for everyday applications.\nRubidium\nRb is a well-known interference on 87Sr during TIMS analyses (e.g. see [33]). The presence of a significant Rb signal can hamper TIMS and solution- and LA-MC-ICPMS analyses of Sr isotopes. Whereas with TIMS and solution MC-ICPMS work, the Rb can be separated from the Sr by chromatographic techniques, this is obviously not possible during LA analysis.\nAll LA-MC-ICPMS studies (see Table\u00a01) use the peak stripping method for the Rb correction, by measuring the 85Rb signal on mass 85 and subtracting the 87Rb signal from 87Sr using the 85Rb\/87Rb value of 2.58745 [33]. An additional problem with LA-MC-ICPMS analysis is the unknown mass bias of Rb during the analysis. Most studies (see Table\u00a02) assume that the mass bias of Rb and Sr (derived from the 88Sr\/86Sr ratio) is constant and correct the Rb interference using the Sr mass bias. However, Jackson and Hart [21] deployed a different strategy to correct for high Rb\/Sr alkaline melt inclusions. The mass bias of Rb was measured by sample-standard bracketing natural basalt glasses with sample material. The measured 85Rb\/87Rb was than used to correct the mass bias of the melt inclusions measured between successive glass standards. By determining a more accurate Rb mass bias they were probably in a better position to measure high Rb\/Sr material, although the reproducibility of their isotopic data deteriorated with increasing Rb\/Sr. A similar approach was followed by McCulloch et al. [20].\nUnfortunately, Jackson and Hart [21] did not report measurement of standards of known Rb and Sr isotopic composition, so it is difficult to fully assess the accuracy of their method. There is no consensus as to how high the Rb\/Sr ratio of materials can be analysed to still produce a reliable LA analysis. Ramos et al. [17] proposed that the materials should have Rb\/Sr < 0.002, whereas Jackson and Hart [21] and Davidson et al. [12] report successful corrections of materials with Rb\/Sr of 0.14 and 0.2, respectively.\nKrypton\nThe noble gas krypton interferes with masses 84Sr and 86Sr and is therefore an important interference that needs to be corrected. The source of the krypton is the argon (and helium) gas used to transport the sample into the plasma, and to generate the argon plasma (see Fig.\u00a01). The amount of krypton in the argon gas is supplier dependent. Studies that report the magnitude of the Kr interference indicates a total Kr contribution between 20\u00a0mV [18] and 40\u201350\u00a0mV ([14, 17, 21]). Woodhead et al. [18] observed that Ar derived from compressed gas cylinders gives a more stable Kr signal than those from liquid Ar supplies. They further noted that the Kr abundances in the Ar supply vary largely between different batches. It is important to note that Jackson and Hart [21] suggest that there are also isobaric interferences of unknown origin on the 83Kr peak, making a good Kr correction extremely difficult (see Table\u00a02). In addition, as with Rb it is impossible to monitor directly the mass bias of the Kr isotope ratios during an analysis if there are isobaric interferences on 82Kr or 83Kr, so sample-standard bracketing may be required.\nFour different correction methods have been deployed to correct for the Kr interference:\n\u201cGas blank\u201d or \u201con peak zero\u201d [11, 12, 14\u201320]. This method measures the Kr levels as a blank measurement, with the He flow going through the sample chamber, but without ablating the sample (laser shutter closed, or no laser firing). The advantage of this method is that there is no mass bias correction involved. The gas blank also corrects for minor amounts of Sr and Rb on masses 84, 85, 86, 87 and 88 which are thought to be derived from the sample introduction system (see above).Peak stripping. In this case the 83Kr (or 82Kr) signal is used to calculate the 84Kr and 86Kr corrections on mass 84Sr and 86Sr, respectively. Christensen et al. [11] used this method to correct for the 86Sr\/88Sr and 87Sr\/86Sr ratios. However, they do not report how they performed the mass bias correction on this ratio, but instead used a 86Kr\/83Kr ratio of 1.53, which is significantly higher than the natural ratio of 1.5 [34]. To date there is no report in the literature of a method to calculate the mass bias of Kr from the 82Kr\/83Kr ratio, which should be possible provided there are no isobaric interferences on 82Kr or 83Kr.Waight et al. [14] used the on-peak-zeros to correct for the Kr interference, and subsequently correct any remaining Kr interference by the peak stripping technique by monitoring the 83Kr signal.Jackson and Hart [21] monitored both the 82Kr and the 83Kr and observed deviations of up to a factor of 2 in the 83Kr\/82Kr (ca. 1). Therefore, these authors deployed another technique to correct for Kr on the Sr masses which uses the most abundant Kr isotope at mass 84 (abundance 57%) instead of 82,83Kr. This mass has a major 84Sr \u201cinterference\u201d that can reach 35\u201375% of the signal. Correction is made by subtracting 84Kr from the 84 signal until the internationally accepted ratio of 0.00675476 is obtained for the 84Sr\/88Sr ratio [34]. Iterations are necessary for the mass bias correction (e.g. substitute the Kr number in the 86Sr\/88Sr ratio, and repeat the calculations until there is no change in the calculated isotope ratios). This technique will only work if there are no other interferences involved on masses 84, 86 and 88. A significant disadvantage of this Kr correction technique is that the 84Sr\/86Sr ratio cannot be used to check if the interference corrections on the 87Sr\/86Sr ratio were successful by providing an independent check that the 84Sr\/86Sr obtained from the analysis is the same as the internationally accepted value of 0.056500.\nIn summary: there are several ways to correct for Kr on masses 86 and 84. The most widely applied technique is the on peak zero, which seems to be successful for low Rb\/Sr (<0.1) ratios. The method published by Jackson and Hart [21] is the only one reporting Sr isotope analyses on high Rb\/Sr (>0.1) samples.\nCalcium dimers and argides\nWhen ablating sample material with high Ca\/Sr ratios (e.g. marine carbonate, ca. 500 and plagioclase, ca. 50\u2013200) calcium dimer and calcium argides can be formed. Ca dimers have been reported to be interfering molecules during SIMS analyses [35]. Waight et al. [14] and Bizarro et al. [15] were the first to suggest that Ca argides (e.g. 44Ca40Ar) could interfere with the Sr isotope masses for materials with high Ca\/Sr ratios. The published studies on Sr isotopes are confusing regarding Ca dimer and argides. Ramos et al. [17] conducted solution work using NIST SRM-987 doped with Ca in such a way that the Ca\/Sr ratios varied between 50 and 550. They did not observe a change of the 87Sr\/86Sr outside the reported error. In addition, Jackson and Hart [21] did not find a change in the 87Sr\/86Sr ratio outside analytical error in alkali basalts with Ca\/Sr ratios of ca. 150. In contrast, Woodhead et al. [18] reported Ca argide and Ca dimer signals of approximately 100\u00a0mV for all Sr masses during the ablation of carbonates. This study also reports that the influence on the accuracy of the 87Sr\/86Sr ratio is beyond the levels of within-run precision. In addition, Woodhead et al. [18] also reported that the 84Sr\/86Sr ratio can be significantly modified by Ca dimer and argides when ablating carbonates, increasing the ratio from 0.0565 to 0.0575. They successfully corrected the interferences on mass 84, 86 and 88 by monitoring the 42Ca40Ar\/42Ca40Ca peak on mass 82. Peak stripping resulted in the correct 84Sr\/86Sr ratio, because the relative isotopic abundances of these argide and dimer molecules are very similar. Therefore, it is not necessary to know the correct argide\/dimer ratio. It is important to note that Jackson and Hart [21] reported significant variations in the 82Kr\/83Kr which they attributed to interferences. Ca argides and dimer appear a probable source of such interferences.\nREE\nThe rare earth elements (REE) can interfere on the Sr isotope masses and on interfering element masses as doubly charged ions (see Table\u00a02). The formation of doubly charged REE in an Ar plasma depends on the operating conditions (e.g. RF power, gas flows), but is generally in the order of 1\u20132% (e.g. see [17]). The REE interferences of course depend on the material analysed. For recent marine carbonates with low absolute REE abundances (<10\u00a0ppm total REE) the effect can be neglected, but for clinopyroxene, with significant REE abundances (Dy ca. 3\u00a0ppm, Yb ca. 2\u00a0ppm), it could be a potential problem (e.g. see [17]).\nWaight et al. [14] and Ramos et al. [17] were the first to study the interferences of Er and Yb in detail. For Sr isotope analyses 168Er2+ and 170Er2+ are relevant, because these ions interfere with 84Sr and 85Rb, respectively (Table\u00a02). With an increasing Er interference on mass 85 the 87Sr\/86Sr ratio will decrease due to an incorrect Rb correction [17]. The net effect is that the amount of 85Rb on mass 85 is overestimated, and too much 87Rb will be subtracted from the 87 ion current, resulting in a low 87Sr\/86Sr ratio. In a similar way, an increasing interference of Er will result in a higher 84Sr\/86Sr ratio, because the Kr correction on the 86 mass is not correct. The signal on mass 84 is assumed to be 84Sr, and therefore the measured 84Sr\/86Sr ratios will be too high due to the presence of 168Er2+.\nAnother potential interfering REE on the Sr isotope masses is Yb (see Table\u00a02). Yb has five isotopes which appear on masses 84, 85, 86, 87 and 88 as doubly charged ions. The 87Sr\/86Sr and 86Sr\/88Sr ratios will increase with increasing Yb content of the ablated material, whereas the 84Sr\/86Sr ratio will decrease [17]. The correction for these interfering doubly charged REE can be done by monitoring half masses. Erbium can be accounted for by measuring 167Er2+ at mass 83.5 during an analysis. Peak stripping with mass 168Er2+ (mass 84) can then be done, using a natural Er solution prior to the Sr LA measurement to establish the Er mass bias. The mass bias can also be established by measuring the 171Yb2+\/173Yb2+ ratio on masses 85.5 and 86.5 and assume that the mass bias for Yb is equal to that of Er [17]. The Yb corrections can be deployed by using the 171Yb2+ abundance and mass bias from the 171Yb\/173Yb ratio. Ramos et al. [17] also examined if Hf (e.g. 176Hf2+) interferes with the Sr isotope masses, but did not find a significant contribution.\nZn, Ga and Fe\nSchmidberger et al. [16] did not observe correlations between the FeO content and Sr isotope ratios of ablated clinopyroxenes. Iron can potentially generate two isobaric interferences (54Fe32O2\u2009=\u200986, 56Fe32O2\u2009=\u200988), which are according to Schmidberger et al. [16] eliminated by the collision cell in the Isoprobe instrument. Ramos et al. [17] only reported a limited effect of Fe oxide on the Sr isotope ratios, and did not correct for Fe oxide interferences.\nGa and Zn oxides (e.g. 71Ga16O+ and 68Zn16O+) could also interfere with the Sr isotope masses. However, Ramos et al. [17] did not find significant deviations in solution work with Sr\/Ga (10\u201320) and Sr\/Zn (1\u20135) ratios typical of geological materials, and decided not to correct for these potential interferences during laser ablation work.\nCalcium phosphates\nDue to the large interference of 40Ca31P16O on 87Sr during LA-MC-ICPMS (see Table\u00a02), it has become apparent that in situ analysis of biogenic phosphates and apatite is extremely challenging and currently does not yield accurate results (e.g. see [36, 37]).\nProposed solutions to correct for isobaric interferences\nFrom the above discussions of the interferences on the strontium masses (see Table\u00a02) it is clear that the order in which the interference corrections are applied is very important. Nearly all interference corrections interact with other corrections. For example the Rb correction is important for the 87Sr, but is also influenced by the Yb correction. In some cases iterations of calculations are necessary to minimize the errors on the calculations. In Fig.\u00a03. the order of corrections is given for the eleven published Sr LA-MC-ICPMS procedures. The correction routines can be divided in two groups: (I) Kr (gas blank), Rb, Sr mass bias correction and (II) the Jackson and Hart [21] method with Kr correction using 84Kr and Rb mass bias correction by sample standard bracketing.\nFig.\u00a03Order of interference corrections in the eleven publications concerning Sr isotope analysis by LA-MC-ICPMS. **Not mentioned in publication, but inferred from published isotope ratios. See text for discussion\nThe group I correction method in its most simple form has been deployed by Christensen et al. [11] and involves Kr correction by 83Kr peak stripping, 85Rb peak stripping and subsequently normalization to 86Sr\/88Sr ratio of 0.1194 (see Fig.\u00a03). The more elaborate correction methods of group I [17, 18] include corrections for the tail, REE [17] and Ca argide\/dimers [18]. The group I data reduction method does work for the relatively simple matrix of carbonate material and plagioclase. The group II method from Jackson and Hart [21] is unique, and has as the major advantage that the Rb correction is done very precisely, resulting in the possibility to analyse higher Rb\/Sr materials (see above), but more standards should be analysed to fully validate this approach.\nPrecision and accuracy of Sr isotopes by laser ablation\nFrom the above discussion it is obvious that LA-MC-ICPMS will probably never obtain the 5- to 10-ppm precision and accuracy which are possible with state-of-the-art TIMS (e.g. see [38]). Therefore, LA-MC-ICPMS will not replace microdrilling of sample material, dissolution and chromatographic processing in a clean laboratory environment and measurement by TIMS when high-precision analysis are required in sample materials with limited variation in Sr isotopes (see [10]). However, the microdrilling\/TIMS technique is very time consuming, and requires a significant skill from the operator. Measurement of Sr isotopes by LA-MC-ICPMS, on the other hand, has great potential if the precision and accuracy required is in the order of 50\u00a0ppm, with the additional advantage of reduced analysis time (average of <5\u00a0min, including wash out and standard analysis, compared with >100\u00a0min for the TIMS analysis).\nIn Fig.\u00a04 the precision and accuracy are reported for the eleven Sr isotope studies by LA-MC-ICPMS for which data were available. The precision is controlled by the counting statistics that are mainly controlled by the concentration of Sr in the ablated material. From Fig.\u00a04 it is obvious that due to their high Sr contents, calcium carbonate and plagioclase provide the best precision. The precision for carbonate material is better than 150\u00a0ppm for six laboratories that have published data on marine carbonate. The precision for other materials can be significantly worse, for example for many clinopyroxenes the precision is between 300 and 1,000\u00a0ppm. Plagioclase also gives good results with a precision better than 250\u00a0ppm in four studies.\nFig.\u00a04Difference between laser ablation and TIMS in ppm (87Sr\/86SrLA\u221287Sr\/86SrTIMS)\/87Sr\/86SrTIMS\u00d7106) for 87Sr\/86Sr (a) and 84Sr\/86Sr (c) and the precision (2sd\/average)\u00d7106 for laser ablation analysis (in ppm) for 87Sr\/86Sr (b) and 84Sr\/86Sr (d). Publications 1\u201311=[11\u201321], respectively. Typical TIMS precision (in ppm) is represented by the grey shaded area (e.g. see [38])\nThe accuracy of the data is more important than the precision. The accuracy, expressed in ppm deviations of 87Sr\/86Sr from reported values, is given in Fig.\u00a04. The accuracy is calculated for samples where LA data and chromatographically cleaned samples measured by TIMS or solution MC-ICPMS were given. Some interesting conclusions can be made:\nCarbonate material gives the most accurate results: data are within 150\u00a0ppm of the solution MC-ICPMS and TIMS values. The good results for carbonate material are to be expected, since corrections for the REE and Rb are minimal in this material. The good accuracy also suggests that the Ca dimer and argides are probably not a significant problem for carbonate material, since a correction for these interferences was actually performed in only one study [18]. However, this is a question that needs to be explored in greater detail in future studies. Another observation is that all the LA-MC-ICPMS carbonate data are shifted towards higher 87Sr\/86Sr compared with TIMS and solution MC-ICPMS data (Fig.\u00a04a).Plagioclase tends to result in 87Sr\/86Sr ratios that are higher than TIMS or solution MC-ICP-MS values. The plagioclase samples of Ramos et al. [17] and Christensen et al. [11] are closest to the TIMS\/solution-MC-ICPMS value, whereas those of Waight et al. [14] and Davidson [12] are 150\u2013250\u00a0ppm too high. The most likely cause for this discrepancy is the significant Rb correction for this material (especially those of Davidson [12], since these samples are artificially enriched in Rb).In contrast, clinopyroxene results tend to be lower than the published TIMS\/solution MC-ICPMS results. This is true in the work reported by Waight et al. [14] and Ramos et al. [17], but the clinopyroxene 87Sr\/86Sr accuracy published by Schmidberger et al. [16] is highly variable (accuracy varies between \u2212408 and 382\u00a0ppm). The large range in the accuracy of clinopyroxene is probably partly caused by the significantly lower Sr content of this material compared with plagioclase and carbonate material (see Fig.\u00a04b\u2013d). The variation in precision is up to 900\u00a0ppm, a range that is also observed in the accuracy (ca. 800\u00a0ppm). In addition, clinopyroxene needs significant corrections for Rb and the REE, hampering both the precision and accuracy.Sr isotopes by laser ablation analysis of other geological materials also looks promising (e.g. groundmass), whereas apatite and sphene require such large corrections that accurate results will be difficult to obtain.\nUnfortunately, not all studies published so far have reported the 84Sr\/86Sr ratio. From the five studies who did measure this ratio, the results from Woodhead et al. [18] are only presented in a figure. A summary of the data from the other four studies is shown in Fig.\u00a04c and d. As can be expected from the significant Kr corrections, there are large deviations from the \u201ctrue\u201d value of 0.0565 (e.g. see [38]). Results from clinopyroxene display a large variation of ca. 14,000\u00a0ppm, but the same is true for carbonate material (ca. 12,000\u00a0ppm). Sphene again has the largest deviation from the true 84Sr\/86Sr value, suggesting problems with corrections of Ca dimer and\/or argides. We recommend that 84Sr\/86Sr ratios are routinely reported as an indicator for the quality of the Sr isotope analysis and that greater effort is made to account for Ca dimer and argides.\nFuture directions\nAll published studies so far have demonstrated the potential of Sr isotopes by LA-MC-ICPMS. As discussed above the precision and accuracy of Sr by LA-MC-ICPMS is clearly limited by the corrections involved with this technique. Some of these are technique dependent (such as the Kr corrections), whereas others depend on the sample material analysed (e.g. Rb, REE and Ca dimer). Some of the potential developments for this technique are evaluated below:\nLaser systems. In the past decade there has been a tremendous effort to improve laser design and shorter wavelength lasers are now being used [25]. The net effect of using shorter wavelength lasers is the more efficient volatilization of the sample which produces smaller aerosol particles in the plasma that are more efficiently ionized (e.g. see [26]). This development was partly driven by the effects of trace-element fractionation during laser ablation of materials where the measurement of elements with different volatility was hampered if large aerosol particles were produced, for example U\/Pb dating of zircons [24]. Since corrections for isobaric interferences on Sr isotope masses are so large (compared with TIMS) the assumption that Rb instrumental discrimination and laser-induced elemental fractionation are the same as for Sr is probably not true. Lasers with low wavelength (e.g. 193\u00a0nm) will reduce this elemental and isotopic fractionation. However, the mass-bias contributions by instrumental mass discrimination and laser-induced elemental fractionation need to be determined independently.Careful determination of the Rb instrumental discrimination by introducing a Rb standard aerosol through a desolvating nebulizer to correct for instrumental discrimination may help. However, probably the best solution would be that followed by Jackson and Hart [21] by measuring Rb-bearing standards with the laser in a standard-sample bracketing method.The Kr interference can be reduced by using clean sources of (liquid) Ar. It could be useful to experiment with Kr removal techniques, in a similar manner as suggested by Zuzel et al. [39].The interferences of the REE are probably only important for materials that contain significant amounts of REE. Experimenting with optimal plasma conditions, which reduce the creation of doubly charged REE, is important.The situation with Ca argides and dimer is unclear. More experimental work is needed, especially by ablating Ca-rich materials, which do not contain Sr.\nConclusions\nThe eleven publications reporting on strontium isotope analyses by LA-MC-ICPMS all conclude that it is a very powerful technique which is feasible, although it does have significant limitations, depending mainly on the material ablated. So far low Rb\/Sr ratios have proved essential for a good analysis. Successful corrections for Rb have been reported for values up to 0.05. This suggests that carbonate and plagioclase represent reliable materials for analysis, but the analysis of high Rb\/Sr minerals such as mica and biotite are not currently possible and will remain so unless there is a significant improvement in the way that the Rb correction on 87Sr is performed. Higher Rb\/Sr materials have been ablated using a standard-sample bracketing technique for correct Rb corrections with, to date, limited success.\nCorrection of Kr interferences on Sr masses was reported not to be a major problem in the eleven studies published to date; however, the accuracy and precision of the data reported so far do not fully substantiate this conclusion. Different strategies are followed to correct for Kr interference, in which gas blank subtraction seems to be the most preferable. Further work is required to determine the instrumental mass discrimination of Kr to ensure the correct 87Sr\/86Sr and 84Sr\/86Sr ratios are obtained.\nDoubly charged REE interferences are only a problem in materials where significant REE contents are present, such as clinopyroxene. However, successful correction is possible by collecting data at half masses.\nThe influence of Ca dimer and Ca argides, is unclear, and needs more investigation. It appears that the effect is limited on the 87Sr\/86Sr ratio, but for 84Sr\/86Sr corrections are necessary. Given that monitoring 84Sr\/86Sr is a good way to establish the veracity of the data, these corrections should be performed routinely.","keyphrases":["sr isotopes","laser ablation","mc-icpms","interferences","in situ analysis"],"prmu":["P","P","P","P","P"]}
{"id":"J_Med_Internet_Res-2-2-1761853","title":"e-Health Code of Ethics (May 24)\n","text":"The Internet is changing how people receive health information and health care. All who use the Internet for health-related purposes must join together to create an environment of trusted relationships to assure high quality information and services; protect privacy; and enhance the value of the Internet for both consumers and providers of health information, products, and services. The goal of the e-Health Code of Ethics is to ensure that people worldwide can confidently and with full understanding of known risks realise the potential of the Internet in managing their own health and the health of those in their care. The final e-Health Code of Ethics, presented in this paper, has been prepared as a result of the \"e-Health Ethics Summit,\" which convened in Washington DC on 31 January 2000 - 2 February 2000. The summit, organized by the Internet Healthcare Coalition and hosted by the World Health Organisation\/Pan-American Health Organisation (WHO\/PAHO), was attended by a panel of about 50 invited experts from all over the world and produced the foundation for a draft code, which was released 18 February [1] for an online public consultation period which ended on 14 April 2000. The final Washington e-Health Code of Ethics sets forth guiding principles under eight main headings: candor; honesty; quality; informed consent; privacy; professionalism in online health care; responsible partnering; and accountability.\nVision Statement\nThe goal of the e-Health Code of Ethics is to ensure that people worldwide can confidently and with full understanding of known risks realise the potential of the Internet in managing their own health and the health of those in their care.\nIntroduction\nThe Internet is changing how people give and receive health information and health care. All people who use the Internet for health-related purposes-patients, health care professionals and administrators, researchers, those who create or sell health products or services, and other stakeholders-must join together to create a safe environment and enhance the value of the Internet for meeting health care needs.\nBecause health information, products, and services have the potential both to improve health and to do harm, organisations and individuals that provide health information on the Internet have obligations to be trustworthy, provide high quality content, protect users' privacy, and adhere to standards of best practices for online commerce and online professional services in health care.\nPeople who use Internet health sites and services share a responsibility to help assure the value and integrity of the health Internet by exercising judgment in using sites, products, and services, and by providing meaningful feedback about online health information, products, and services.\nDefinitions\nHealth information includes information for staying well, preventing and managing disease, and making other decisions related to health and health care.\nIt includes information for making decisions about health products and health services.\nIt may be in the form of data, text, audio, and\/or video.\nIt may involve enhancements through programming and interactivity.\nHealth products include drugs, medical devices, and other goods used to diagnose and treat illnesses or injuries or to maintain health. Health products include both drugs and medical devices subject to regulatory approval by agencies such as the U.S. Food and Drug Administration or U.K. Medicines Control Agency and vitamin, herbal, or other nutritional supplements and other products not subject to such regulatory oversight.\nHealth services include specific, personal medical care or advice; management of medical records; communication between health care providers and\/or patients and health plans or insurers, or health care facilities regarding treatment decisions, claims, billing for services, etc.; and other services provided to support health care.\nHealth services also include listserves, bulletin boards, chat rooms, and other online venues for the exchange of health information.\nLike health information, health services may be in the form of data, text, audio, and\/or video, and may involve enhancements through programming and interactivity.\nAnyone who uses the Internet for health-related reasons has a right to expect that organisations and individuals who provide health information, products or services online will uphold the following guiding principles:\nGuiding Principles\nTable 1\nGuiding Principles\n1. Disclose information that if known by consumers would likely affect consumers' understanding or use of the site or purchase or use of a product or service.\nCandorPeople who use the Internet for health-related purposes need to be able to judge for themselves that the sites they visit and services they use are credible and trustworthy. Sites should clearly indicate who owns or has a significant financial interest in the site or servicewhat the purpose of the site or service isFor example, whether it is solely educational, sells health products or services, or offers personal medical care or adviceany relationship (financial, professional, personal, or other) that a reasonable person would believe would likely influence his or her perception of the information, products, or services offered by the siteFor example, if the site has commercial sponsors or partners, who those sponsors\/partners are and whether they provide content for the site\n2. Be truthful and not deceptive\nHonestyPeople who seek health information on the Internet need to know that products or services are described truthfully and that information they receive is not presented in a misleading way. Sites should be forthright in all content used to promote the sale of health products or servicesin any claims about the efficacy, performance, or benefits of products or servicesThey should clearly distinguish content intended to promote or sell a product, service, or organisation from educational or scientific content.\n3. Provide health information that is accurate, easy to understand, and up to date.\nQualityTo make wise decisions about their health care, people need and have the right to expect that sites will provide accurate, well-supported information and products and services of high quality. \nTo assure that the health information they provide is accurate, e-Health sites and services should make good faith efforts to evaluate information rigorously and fairly, including information used to describe products or servicesprovide information that is consistent with the best available evidenceassure that when personalized medical care or advice is provided that care or advice is given by a qualified practitionerindicate clearly whether information is based on scientific studies, expert consensus, or professional or personal experience or opinionacknowledge that some issues are controversial and when that is the case make good faith efforts to present all reasonable sides in a fair and balanced wayFor example, advise users that there are alternative treatments for a particular health condition, such as surgery or radiation for prostate cancerInformation and services must be easy for consumers to understand and use. Sites should present information and describe products or services in language that is clear, easy to read, and appropriate for intended usersFor example, in culturally appropriate ways in the primary language (or languages) of the site's expected audiencein a way that accommodates special needs users may haveFor example, in large type or through audio channels for users whose vision is impairedSites that provide information primarily for educational or scientific purposes should guarantee the independence of their editorial policy and practices by assuring that only the site's content editors determine editorial content and have the authority to reject advertising that they believe is inappropriate. Consumers have a right to expect that the information they receive is up to date. Sites should clearly indicate when the site published the information it provides (and what version of the information users are seeing if it has been revised since it was first published)when the site most recently reviewed the informationwhether the site has made substantive changes in the information and if so, when the information was most recently updated\nand\nProvide the information users need to make their own judgments about the health information, products, or services provided by the site.\nIndividuals need to be able to judge for themselves the quality of the health information they find on the Internet. Sites should describe clearly and accurately how content is developed for the site by telling userswhat sources the site or content provider has used, with references or links to those sourceshow the site evaluates content and what criteria are used to evaluate content, including on what basis the site decides to provide specific links to other sites or servicesFor example, by describing the site's editorial board and policiesWhen health products or services are subject to government regulation, sites should tell users whether those products (such as drugs or medical devices) have been approved by appropriate regulatory agencies, such as the U.S. Food and Drug Administration or U.K. Medicines Control Agency\n4. Respect users' right to determine whether or how their personal data may be collected, used, or shared.\nInformed ConsentPeople who use the Internet for health-related reasons have the right to be informed that personal data may be gathered, and to choose whether they will allow their personal data to be collected and whether they will allow it to be used or shared. And they have a right to be able to choose, consent, and control when and how they actively engage in a commercial relationship.\nSites should clearly disclose that there are potential risks to users' privacy on the InternetFor example, that other organisations or individuals may be able to collect personal data when someone visits a site, without that site's knowledge; or that some jurisdictions (such as the European Union) protect privacy more stringently than othersSites should not collect, use, or share personal data without the user's specific affirmative consent. To assure that users understand and make informed decisions about providing personal data, sites should indicate clearly and accurately what data is being collected when users visit the siteFor example, data about which parts of the site the user visited, or the user's name and email address, or specific data about the user's health or online purchaseswho is collecting that dataFor example, the site itself, or a third partyhow the site will use that dataFor example, to help the site provide better services to users, as part of a scientific study, or to provide personalised medical care or advicewhether the site knowingly shares data with other organisations or individuals and if so, what data it shareswhich organisations or individuals the site shares data with and how it expects its affiliates to use that dataFor example, whether the site will share users' personal data with other organisations or individuals and for what purposes, and note when personal data will be shared with organizations or individuals in other countriesobtain users affirmative consent to collect, use, or share personal data in the ways describedFor example, to collect and use the visitor's personal data in scientific research, or for commercial reasons such as sending information about new products or services to the user, or to share his or her personal data with other organisations or individualswhat consequences there may be when a visitor refuses to give personal dataFor example, that the site may not be able to tailor the information it provides to the visitor's particular needs, or that the visitor may not have access to all areas of the site\"E-commerce\" sites have an obligation to make clear to users when they are about to engage in a commercial transaction and to obtain users' specific affirmative consent to participate in that commercial transaction.\n5. Respect the obligation to protect users' privacy.\nPrivacyPeople who use the Internet for health-related reasons have the right to expect that personal data they provide will be kept confidential. Personal health data in particular may be very sensitive, and the consequences of inappropriate disclosure can be grave. To protect users, sites that collect personal data should take reasonable steps to prevent unauthorised access to or use of personal dataFor example, by \"encrypting\" data, protecting files with passwords, or using appropriate security software for all transactions involving users' personal medical or financial datamake it easy for users to review personal data they have given and to update it or correct it when appropriateadopt reasonable mechanisms to trace how personal data is usedFor example, by using \"audit trails\" that show who viewed the data and whentell how the site stores users' personal data and for how long it stores that dataassure that when personal data is \"de-identified\" (that is, when the user's name, email address, or other data that might identify him or her has been removed from the file) it cannot be linked back to the user\n6.\nRespect fundamental ethical obligations to patients and clients.\nProfessionalism in Online Health CarePhysicians, nurses, pharmacists, therapists, and all other health care professionals who provide specific, personal medical care or advice online should abide by the ethical codes that govern their professions as practitioners in face-to-face relationshipsdo no harmput patients' and clients' interests firstprotect patients' confidentialityclearly disclose any sponsorships, financial incentives, or other information that would likely affect the patient's or client's perception of professional's role or the services offeredclearly disclose what fees, if any, will be charged for the online consultation and how payment for services is to be madeobey the laws and regulations of relevant jurisdiction(s), including applicable laws governing professional licensing and prescribing\nand\nInform and educate patients and clients about the limitations of online health care.\nThe Internet can be a powerful tool for helping to meet patients' health care needs, but users need to understand that it also has limitations. Health care professionals who practice on the Internet should clearly and accurately identify themselves and tell patients or clients where they practice and what their professional credentials aredescribe the terms and conditions of the particular online interactionFor example, whether the health care professional will provide general advice about a particular health condition or will make specific recommendations and or referrals for the patient or client, or whether the health care professional can and will or cannot and will not prescribe medications in the particular situationmake good faith efforts to understand the patient's or client's particular circumstances and to help him or her identify health care resources that are available locallyFor example, to help the patient or client determine whether particular treatment is available in his or her home community or only from providers outside his or her communitygive clear instructions for follow-up care when appropriate or necessaryHealth care professionals who offer personal medical services or advice online should clearly and accurately describe the constraints of online diagnosis and treatment recommendationsFor example, providers should stress that because the online health care professional cannot examine the patient, it is important for patients to describe their health care needs as clearly they canhelp \"e-patients\" understand when online consultation can and when it cannot and should not take the place of a face-to-face interaction with a health care provider\n7. Ensure that organisations and sites with which they affiliate are trustworthy.\nResponsible PartneringPeople need to be confident that organisations and individuals who operate on the Internet undertake to partner only with trustworthy individuals or organisations. Whether they are for-profit or nonprofit, sites should make reasonable efforts to ensure that sponsors, partners, or other affiliates abide by applicable law and uphold the same ethical standards as the sites themselvesinsist that current or prospective sponsors not influence the way search results are displayed for specific information on key words or topicsAnd they should indicate clearly to users whether links to other sites are provided for information only or are endorsements of those other siteswhen they are leaving the siteFor example, by use of transition screens\n8. Provide meaningful opportunity for users to give feedback to the site.\nAccountabilityPeople need to be confident that organisations and individuals that provide health information, products, or services on the Internet take users' concerns seriously and that sites make good faith efforts to ensure that their practices are ethically sound. e-Health sites should indicate clearly to users how they can contact the owner of the site or service and\/or the party responsible for managing the site or serviceFor example, how to contact specific manager(s) or customer service representatives with authority to address problemsprovide easy-to-use tools for visitors to give feedback about the site and the quality of its information, products, or servicesreview complaints from users promptly and respond in a timely and appropriate mannerSites should encourage users to notify the site's manager(s) or customer service representatives if they believe that a site's commercial or noncommercial partners or affiliates, including sites to which links are provided, may violate law or ethical principles.\nand\nMonitor their compliance with the e-Health Code of Ethics.\ne-Health sites should describe their policies for self-monitoring clearly for users, and should encourage creative problem solving among site staff and affiliates.","keyphrases":["ethics","internet","quality of health care"],"prmu":["P","P","R"]}
{"id":"Ann_Biomed_Eng-2-2-1705519","title":"A Modeling Study of Notch Noise Responses of Type III Units in the Gerbil Dorsal Cochlear Nucleus\n","text":"A computational model of the neural circuitry of the gerbil dorsal cochlear nucleus (DCN), based on the MacGregor\u2019s neuromime model, was used to simulate type III unit (P-cell) responses to notch noise stimuli. The DCN patch model is based on a previous computational model of the cat DCN [Hancock, K. E., and H. F. Voigt. Ann. Biomed. Eng. 27:73\u201387, 1999]. According to the experimental study of Parsons et al. [Ann. Biomed. Eng. 29:887\u2013896, 2001], the responses of gerbil DCN type III units to notch noise stimuli are similar to those of cat DCN type IV units, which are thought to be spectral notch detectors. This suggests that type III units in the gerbil DCN may serve as spectral notch detectors. In this modeling study, a simplified notch noise response plot\u2014spike discharge rate vs. notch cutoff frequency plot\u2014was used to compare model responses to the experimental results. Parameter estimation and sensitivity analysis of three connection parameters within the DCN patch have been studied and shows the model is robust, providing reasonable fits to the experimental data from 14 of 15 type III units examined [work supported by a grant from NIDCD, Boston University\u2019s Biomedical Engineering department and Hearing Research Center].\nINTRODUCTION\nThe cochlear nucleus (CN) is the sole target of auditory nerve fibers and is the first brainstem nucleus in the ascending auditory pathway. The dorsal part of the cochlear nucleus (DCN) is one of its three sub-nuclei and has initial integration functions. The DCN\u2019s role in audition appears, in part, to be involved in sound localization in the median plane equidistant from the two ears where the traditional sound source location cues, interaural time and level differences, are absent. For broadband signals, the pinna attenuates energy in a narrow band whose center frequency can be correlated with sound source elevation. The pinnae-induced attenuation is called a spectral notch. These notches are seen in the head-related transfer functions (HRTFs), which are the Fourier transforms of the impulse responses from free space sound sources to the tympanic membrane. These spectral features are found in human, cat and gerbil HRTFs.12,13 Principal cells of the DCN, type III units in the gerbil15 and type IV units in the cat,22 exhibit sharp sensitivity to notch noise signals.14,18 This suggests that the DCN is a spectral notch detector across different species.\nThe previous study of Hancock and Voigt6 used a computational model of the DCN to model the responses of three type IV units to notch noises and presented close fits to the physiological data recorded by Spirou and Young.18 The gerbil DCN has fewer type IV units than the cat DCN (11% type IV units in gerbil vs. 31% in cat)2,17 and is comprised primarily of type III units (62%).2 As the principal units in gerbil DCN, type III units undoubtedly play an important role in gerbil sound localization. In this study, we used the same computational model to fit the physiological data obtained from the gerbil DCN by Parsons et\u00a0al.15 Despite the fact that type III units have quite different responses to tones than type IV units, this study shows that by modifying the connectivities within the neuron groups we can simulate both type III and type IV units as notch detectors. Also in this work we have good fits for 14 out of 15 type III units in gerbil; this is substantially more modeling data than previously presented and points to the robustness of the DCN model.\nRESPONSE MAP SCHEME AND RATE VS. CUTOFF FREQUENCY PLOTS\nA response map (RM) is a plot of excitatory and inhibitory responses to tonal stimuli shown in a stimulus frequency vs. sound pressure level plane. It is a popular method to categorize the neurons in the CN of unanethetized, decerebrate animals.4,17,22,23 Type III units are characterized by their RMs (Fig.\u00a01A), which show a center V-shape excitation region flanked by side-band inhibition. The tip of the excitation region points to the unit\u2019s best frequency (BF), the frequency to which the unit was most sensitive. In Fig.\u00a01A, there are seven discharge-rate vs. frequency curves shown for seven sound pressure levels presented. RMs of type IV units typically show an excitatory region over BF and an inhibitory area above that ranging over a broad frequency range; sometimes a narrow excitatory area appears off BF (see Fig.\u00a01 of Ref. 6).FIGURE 1.Physiology of a type III unit: (A) Response map composed of rate vs. frequency curves of unit L021199 1.01 in Parsons et\u00a0al.15. Excitatory responses are shaded black while inhibitory responses left blank. (B) Rate-level curves when given wideband noise and notch noise stimuli. Only four notch noise responses with notch (nw) widths 1.0 kHz, 2.0 kHz, 4.0 kHz, and 8.0\u00a0kHz are shown. (C) Rate vs. Cutoff frequency plot. Here notch widths varies systematically from 0.5 to 8.0\u00a0kHz in step of 0.5\u00a0kHz. The average rates are calculated from the range of 46\u201354\u00a0dB SPL as shown by black bar in (B).\nResponses to broadband noise, in addition to tones, are used to classify units in the DCN. We also used the same notch noise (NN) stimuli in the model simulations as in the physiological experiments.15 These have band reject notches of 30\u00a0dB centered at the BF with varying widths. In Fig.\u00a01B, discharge-rate vs. level curves are plotted for broadband noise and four NN stimuli with widths of 1, 2, 4, and 8\u00a0kHz. These responses were taken from a series of responses to BF-centered NN stimuli whose widths systematically varied from 0.5 to 8.5\u00a0kHz with a 0.5\u00a0kHz step. For Unit L021199 1.01 in Fig.\u00a01 there were 16 NN stimuli applied with widths from 0.5 to 8.0\u00a0kHz. Only four NN responses are plotted for clarity.\nTo summarize the information in these rate-level curves, Spirou and Young18 further characterized NN responses with rate vs. cutoff frequency plots (Fig.\u00a01C), where the firing rates to each NN were averaged over a range of sound levels from 46 to 54\u00a0dB SPL, where the inhibition reached the maximum, as indicated with black bar on the abscissa in Fig.\u00a01B. The cutoff frequencies included both the notch noise\u2019s lowpass and the highpass cutoff frequencies, thus the average rate of each notch noise response is plotted twice. In such plots, 11 of 15 units in Parsons et\u00a0al.'s experimental data showed a specific \u201cMexican Hat\u201d shape, indicating that when increasing the notch width, the average response rate dropped sharply from excitatory to inhibitory and then showed some degree of recovery. The unit in the figure was excited by the broad-band noise and NN with narrow-notch width (0.5\u00a0kHz, not shown in Fig.\u00a01B but shown in Fig.\u00a01C), but was inhibited by NN with wider widths (from 1.0 to 6.0\u00a0kHz, also shown in Fig.\u00a01C). Also it showed some degree of recovery to a less inhibitory stage when NN with an even wider notch was applied (from 6.5 to 8.0\u00a0kHz, shown in Fig.\u00a01C). This recovery was seen in 11 of 15 units in the paper of Parsons et\u00a0al.15 Similar responses to NN have been found in type IV units in cat by Spirou and Young.18\nThis demonstrated the sensitivity of type III units in gerbils to the NNs. Thus, type III units in gerbils are capable of detecting spectral notches as do type IV units in cats.\nFIGURE 2.Conceptual model of gerbil dorsal cochlear nucleus circuitry. Only inhibitory connections are shown. The size of the triangles indicates connection strength. The horizontal axis represents the input from the auditory nerve fibers with increasing frequency, which target\u00a0all cells. Modified from Davis et\u00a0al.2\nCONCEPTUAL MODEL OF DCN CIRCUITRY\nTo illustrate the functional role of type III units in the gerbil DCN, the conceptual model from Davis et\u00a0al.2 was chosen (see Fig.\u00a02). The gerbil DCN conceptual model is more complex than the cat model since the principal cells in gerbils can be either type III units or type IV units, including subtypes of type IV units: type IV-i and type IV-T units. In addition, besides type II units and the wide band inhibitor (WBI) units, type III-i units were included in the circuitry to provide necessary inhibitory input to type IV-i units. In this paper, however, we only discuss the properties and responses of type III units, thus only type II units and WBI units are taken as inhibitors.\nAuditory nerve (AN) fibers provide excitatory inputs to type II units, type III units and WBI units. However, DCN units show spontaneous activity even after destruction of the cochlea.9 Thus non-specific afferents (NSA) were added to the principal cells in our computational model to provide noncochlear inputs to the DCN (Fig.\u00a03). In order not to confuse physiologically defined unit types and model unit types, we use different names to distinguish them (see Table\u00a01).FIGURE 3.A patch of DCN model. Five cell populations are organized in a tonotopic manner with frequency step equal to 0.005 octaves. The excitatory and inhibitory connections are shown in a single frequency slice. P cells, representing type III units in gerbil and type IV units in cat; I2 cells, type II units; W cells, providing wide band inhibition; AN fibers, the auditory nerve fiber input. Modified From Hancock and Voigt, 1999.6\nFusiform cells in gerbil DCN are mostly type III units in intracellular recording and marking experiments by Hancock and Voigt7 whereas type II units arise from vertical cells in the DCN21 and WBI units are thought to project from the posteroventral CN.20 In addition, WBI units send inhibitory connections to both type IV units (principal units in cat) and type II units in cat DCN. We are assuming that these connections to the gerbil principal units remain intact.TABLE 1.Physiological unit types with corresponding model unit types.Physiological unit typeModel unit typeType III unitsP-cellsType II unitsI2-cellsWideband inhibitorsW-cellsAuditory nerve fibersAN-fibers\nMETHODS\nOrganization of the DCN Model\nThe gerbil model is based on a previous computational model of the cat DCN6 (see Fig.\u00a03). The model contains five cell groups that are arranged into 800 isofrequency slices centered at 5\u00a0kHz with two octaves below and above in intervals of 0.005 octaves. Figure\u00a03 shows the connections among the model cells within a single frequency slice, where P-cells represent the principal cells, I2-cells represent interneuron inhibitors with type II responses and W-cells represent wide band inhibitors. AN-fibers excite P-cells, I2-cells and W-cells. W-cells inhibit P-cells and I2-cells. I2-cells inhibit P-cells. P-cells also are excited by non-specific afferents.\nThe relationship among the model cells can be discussed in terms of the connection parameters as shown in Fig.\u00a04. For a given population of model cells (e.g., P-cells), the target cell \u201cB\u201d will receive N inputs from the \u201cA\u201d source cells, which are chosen from a band of \u201cA\u201d source cells with bandwidth BW, offset from the BF of B by C. The subscript  indicates the source unit, A to the target unit, B. In the simulations shown here, all the P-cells\u2019 BFs will be 5\u00a0kHz.FIGURE 4.Group connection parameters. B is the target cell and a group of source cells A connect to it.  and  are the step increase and time constant in response to the input spikes. Here B cell receives N inputs from A cells, which are randomly chosen from a band of bandwidth and center frequency offset specified cells.TABLE 2.Intrinsic parameters.Population\u03c4m (ms)\u03b8 (mV)bk\u03c4k (ms)Ek (mV)Eex (mV)Ein (mV)W-cells5.04.02.001.0\u221210+70\u221210I2-cells6.014.51.751.0\u221210+70\u221210P-cells10.07.52.001.0\u221210+70\u221210\nNeuron Model\nThe neuron model (Fig.\u00a05) is based on the MacGregor neuromime model,10 which is a parallel circuit model containing the membrane capacitance, leakage conductance, a potassium channel branch and the excitatory\/inhibitory connection branches. Each excitatory or inhibitory input will add a branch, with the variable conductance controlled by the parameters, step \u03c3 and time constant \u03c4.FIGURE 5.(A) Vm is the membrane potential relative to rest; Cm is membrane capacitance; G is resting conductance; Gk and Ek are variable conductance and reversal potential of potassium; Gex\/in and Eex\/in are excitatory\/inhibitory synaptic conductance and reversal potential. (B) Expression of connection synapse conductance. Target cell will undergo a step increase and exponential decay, time constant in response to input spikes.\nEvent time is recorded when the membrane potential exceeds its threshold, \u03b8, and the potassium conductance is activated to induce the cell\u2019s refractory period. Thus, the computation is reduced by leaving out the details of action potential generation. The following equations describe the neuronmime model:\nwhere gk = Gk\/G, gex = Gex\/G, and gin = Gin\/G; G, Gk, Gex, and Gin represent respectively the conductances of neuron membrane, potassium channel, excitatory synaptic inputs and inhibitory synaptic inputs. The latter three are normalized by G to simplify the equations. Vm is the membrane potential. Ek, Eex, and Ein represent the reversal potentials. \u03c4m and \u03c4k are the membrane time constant and the refractory time constant. Equation (1) states the current fluxes across the neuronal membrane. Equation (2) states the change of the potassium conductance, where bk represents the sensitivity to potassium conductance. S is the spiking variable with two alternative values: 1 indicates a cell is firing.TABLE 3.Connection parameters.Connection  (octave) (octave) (ms)0.0Varies1400.06100.00.4480.55100.00.448Varies100.00.1151.40100\/0.20.115Varies10\u22120.2\/00.6210.11n\/an\/a150.153\nFor target cell B, the variable conductance that represents synapses from source cells A is described by\nwhere SA are the input spikes.\nParameter Values\nIn the model, we used four intrinsic (\u03c4m, \u03b8, bk, and \u03c4k) and five connection parameters (, , , , and ). The intrinsic parameter set describes the membrane properties of the cell, which determines the steady firing rate and the threshold sound pressure level of the single cell. The connection parameter set describes the connections between two groups of cells; this is used to specify the connectivity within the neuronal circuitry of the model. We used the intrinsic values in the former work of Hancock and Voigt6 (Table\u00a02), as they have little or no effect on the spectral response characteristics.\nFIGURE 6.Best fit of Unit L041499 13.01 and parameter sensitivity analysis. (A) Best fit of the model to physiological data. The best frequency of physiological data and the center frequency of the model data have been lined up through simply translation of the physiological data rate vs. cutoff frequency plot. The circles represents the physiological data and the solid line represents the best fit of the model. The dashed line represents the average spontaneous rate of the physiological data. The minimum error \u0424min and the values of the three important parameters of the best fit are shown in the upper left. (B)\u2013(D) Contours of equal \u0424\/\u0424min (1.5, 2, 5, 10, and 15, from inside to outside) showing the sensitivity of the fit to the parameter values. , , and  are held to be the value in the best fit corresponsively in plots (B), (C), and (D).FIGURE 7.Best fit of Unit L110199 2.01 and parameter analysis. Organized as in Fig. 6 except that in the contours plots (B\u2013D) equal \u03a6\/\u03a6min of 1.1 was added to show the center.FIGURE 8.Best fit of Unit L122299 4.01 and parameter analysis. Organized as in Fig. 6.\nMacGregor11 pointed out that the steady state effect of one population on its target is proportional only to the product , which enabled us to set only one parameter from these three to be a free parameter; here we chose , the strength from cell A to B. Thus, only three connection parameters, , , and  were taken into account in the simulations. The values of the parameters are listed in Table\u00a03.\nAs in Table\u00a03, three parameters were set to be main parameters that vary: , , and . Center offset  and  were set to two possible values 0\/\u22120.2 octaves and 0\/0.2 octaves respectively according to the physiological data in cross-correlation studies.  was set to 0.1 to obtain type III RM properties. Non-specific afferent firing rate was set to 80, 90, or 120\u00a0spikes\/s according to the various spontaneous rates of type III units.\nAuditory Nerve Model\nAN-fibers were based on the model described by Carney.1 The input to the AN-fiber is a sound pressure signal and the output is a spike train. Each fiber consists of a gammatone filter that provides the frequency selectivity, a nonlinear process that generates an inner hair cell potential and a compartmental model of neurotransmitter release. This in turn creates an instantaneous firing probability. AN-fiber thresholds and spontaneous activity rates were randomly assigned from physiological distributions of these parameters.5\nIn order to save time, all stimuli were processed by the auditory nerve filter bank once and the spike time responses were saved for use in subsequent simulations of the DCN circuitry.\nSimulation Protocol and Data Analysis\nAll simulations were carried out on an IBM pSeries 655, which is a 48-processor system composed of six nodes. Each p655 node consists of eight Power4 processors running at 1.1\u00a0GHz and sharing 16\u00a0GB of memory. There are three levels of cache on this machine. Each processor has a 32\u00a0KB L1 cache and then each pair of processors share a 1.41\u00a0MB L2 cache, and each p655 node shares a 128\u00a0MB L3 cache.\nTo create RMs, the model was stimulated by 50-ms tone bursts presented every 250\u00a0ms with a 32-ms delay while sound pressure levels varied from 0 to 60\u00a0dB SPL in 2\u00a0dB SPL steps and the frequency varied in 0.1 octave steps within a three octave band above and below 5\u00a0kHz. Thus, there are 31 frequency slices in one RM simulation. The spikes of the last 40\u00a0ms of each trial were used to compute the spontaneous rate and the spikes of the last 40\u00a0ms of each tone burst were used to compute the driven rate. In this paper, RMs composed of rate vs. frequency curves for seven levels were plotted (0\u201360\u00a0dB SPL in 10\u00a0dB SPL steps). This is because the physiological data were taken only to 60\u00a0dB SPL due to increasing recording artifacts above these levels. At each level, the horizontal line represents the unit\u2019s spontaneous discharge rate; excitation and inhibition are shown as rates above and below this line, respectively. The excitatory region was filled black and the inhibitory region left blank.\nWe used the same notch noises and broadband noise stimuli to obtain rate-level data as in Hancock and Voigt.6 In the simulation the sound level was varied from 0 to 90\u00a0dB SPL in 2\u00a0dB SPL steps and the noise bursts were presented for 200\u00a0ms in 1000-ms trials.\nTo obtain rate vs. notch cutoff frequency plots, the firing rates from 46 to 54\u00a0dB SPL were averaged to capture the most inhibitory area, consistent with Hancock and Voigt.6 These plots are sometimes referred to as \u201cMexican Hats.\u201d\nTABLE 4.Data fits for all 15 type III units.In RefUnitBFSR\u03a6minNSA1Fig. 3AL122299 3.014.3383.490003.25a0.250.902Fig. 3BL021199 1.014.7773.81200.203.25a0.451.803Fig. 3CL110199 2.012.6132.480003.000.120.254Fig. 4AL111799 9.014.992.8800.2\u22120.22.000.120.255Fig. 4BL122299 4.0111.0144.7800\u22120.21.250.240.906Fig. 4CL110199 11.024.21211.080001.00a0.150.05a7Fig. 4DL102599 7.0110.2265.190001.500.250.708Fig. 4EL041499 16.013.8308.1800.2\u22120.22.500.120.209Fig. 4FL021199 4.017.1152.880003.25a0.180.2510Fig. 4GL102599 8.016.4225.680001.500.120.4011Fig. 4HL041499 13.011.2352.480001.250.240.8012Fig. 4IL041499 1.021.399.8800.203.25a0.060.1513Fig. 4JL041499 1.011.2495.8800\u22120.21.00a0.240.9014Fig. 4KL040700 3.0111.4201.3800.203.000.240.2515Fig. 4LL040700 7.0115.1240.4120002.750.452.00aNote. Ref. refers to Parsons et\u00a0al.15; BF in kHz, SR in spikes\/s, NSA in spikes\/s.aThe parameter value reached the parameter limit.\nRESULTS\nFit of the Model to Physiological Data and Parameter Sensitivity Analysis\nDischarge rate versus cutoff frequency plots were used to evaluate the effectiveness of our DCN model quantitatively when fit to the physiological data obtained by Parsons et\u00a0al.15 The physiological discharge rate versus cutoff frequency plots were translated along the frequency axis to align the BF of the unit to that of the model at 5\u00a0kHz without shape change in logarithmic frequency plots. This operation was done mathematically by scaling the cutoff frequencies by CF\/BF, where CF is the center frequency of model and BF is the best frequency of physiological data. This operation also made the after-translation physiological data look like that obtained by notch noise stimuli centered at 5\u00a0kHz while notch widths scaled by the factor CF\/BF. Then linear interpolation was used to obtain \u201cnew\u201d physiological data corresponding to model data cutoff frequencies and this made comparison of fits among different units possible. Here we used an objective function, \u03a6, to demonstrate quantitatively the fit of one unit, or for comparisons across the units as done by Hancock and Voigt6:\nwhere yd and ym are the mean firing rates in the physiological data and model results respectively, and N is the total number of cutoff frequencies used in the comparison. The value of the objective function is scaled by 500 to make it more readable, consistent with Hancock and Voigt.6\nThe three parameters , , and , were systematically varied to get the \u201cbest fit\u201d (minimum \u03a6, \u03a6min) and also to show contours of equal goodness or quality of fit. Center offsets  and , were set to 0 or \u22120.2 octaves and 0 or 0.2 octaves respectively, and the fitting results were only slightly different with similar \u03a6min when best fits were obtained. Non-specific afferent (NSA) firing rate was set to 80, 90, or 120\u00a0spikes\/s to adjust to the various spontaneous rates of type III units and this improved the fits, especially for some low-spontaneous rate units. An alternative way to adjust spontaneous rate was to vary the connection strength of NSA to P-cells as in Hancock and Voigt.6 These two methods show no difference from each other since P-cells are the only targets of the NSA and the different firing rates of NSA or the strength of the connection between NSA and P-cells will only affect the total NSA excitatory inputs to P-cells.\nFigures\u00a06\u20138A show the best fits to three physiological units labeled Unit L041499 13.01, Unit L110199 2.01 and Unit L122299 4.01. Figures\u00a06\u20138B\u2013D are contours showing parameter sensitivity analyses of these parameters. As shown in the figures,  varied from 1.0 to 3.25 octaves in steps of 0.25 octaves;  varied from 0.05 to 0.5 or from 0.03 to 0.3; and  varied from 0.1 to 1.0 or from 0.05 to 0.5\nVery good fits are obtained for these three units. Not only are the values of \u03a6min small, but the centers of the contours are shown clearly and the contours themselves resemble ellipses.FIGURE 9.Response maps and rate-level plots for notch noise stimuli. (A, B) Response map composed of rate vs. frequency curves plot of Unit L041499 13.01 and the model using the set of best fit parameters in Fig. 6. The best frequency of this unit is 1.2\u00a0kHz and has been moved to 5\u00a0kHz with the same method in \u201cMexican Hat\u201d plots for easier comparison to model RM. (C, D) Rate-level curves of broad-band noise and notch noise responses of the unit and the model data.FIGURE 10.Response maps and rate-level plots responding to notch noise stimuli. Unit L110199 2.01. The best frequency of this unit is 2.6\u00a0kHz. Picture is organized as in Fig. 9.\nTable\u00a04 shows the results for all 15 units. Of the 15 units from Parsons et\u00a0al., 15 eight units had best fits within closed ellipses. Another six units also gave good fits, although the  parameter in these cases was actually at its limit (four reaching the upper limit and one reaching the lower limit of the parameter range, one reaching the upper limit of ). These 14 units had values of \u03a6min < 9.8, which shows that the model is robust. The final unit was difficult to fit because the rate vs. notch noise cutoff frequency plot showed additional features (see Parsons et\u00a0al., Fig.\u00a04C).15\nComparisons Between Physiological Data and Model Data: Response Maps and Notch Noise Rate-Level Curves\nSince the rate vs. notch noise cutoff frequency curves are the functions that were modeled, to what extent do the model RMs and notch noise rate-level curves resemble their physiological counterparts? Figures\u00a09\u201311A and B show the physiological RMs together with the model RMs for the units of Figures\u00a06\u20138. The model RMs were simulated using the model parameters set found when \u03a6 = \u03a6min. For two sets of RMs we found that the model RMs show insufficient excitatory regions across the frequency axis when high-level tone bursts are given. This occurs in another five units where the physiological data show wide excitatory regions and indicates that our model parameters may need adjustment to get better tone responses for these units. All three model RMs, however, show nice center excitatory regions and sideband inhibitory regions, which is characteristic of type III units.\nFigures\u00a09\u201311C and D show the rate-level curves corresponding to the BBN and NN stimuli. For Unit L041499 13.01 and Unit L110199 2.01 (Figures\u00a09 and 10), the maximum notch widths could not reach 8.0\u00a0kHz since the units had low BFs (1.2 and 2.6\u00a0kHz). Unit L122299 4.01 has a higher BF at 11.0\u00a0kHz and the notch width was varied from 0.5 to 8.5\u00a0kHz as in Parsons et\u00a0al.15 In general, the responses to BBN are greatest in both model and physiological data, but there are great differences in shape, maximum rate, saturation rates and thresholds. The model\u2019s curves for the NN stimuli are qualitatively similar to the physiology, but again several differences are observed. The model seems to be providing more inhibition than that found in the physiology.\nDISCUSSION\nSpirou and Young18 showed that type IV units in the cat DCN (projection neurons) are notch detectors. Hancock and Voigt6 used a computational model of cat DCN circuitry, whose P-cells had type IV response properties to show that these also have notch noise sensitivity and that the variability seen in the units\u2019 responses to notch noise are accounted for by parameter variations rather than different circuit arrangements. The gerbil DCN shows far fewer type IV units than the cat and the projection neurons in gerbil DCN are predominately type III units. Parsons et\u00a0al.15 showed that gerbil type III units are sensitive to notch noises in much the same way as cat DCN type IV units. Thus, it appears that DCN projection neurons from both species are doing the same analysis on notch noise and thus may play similar roles in sound source detection in the median plane.\nThis study demonstrates that the same computational model used to model the notch noise behavior in cat type IV units can be used to model type III unit notch noise sensitivity in gerbil DCN. The fact that we can use the same neural circuit to model the DCN in both species suggests that perhaps this aspect of the DCN neural circuit is invariant across species.\nThe Parsons et\u00a0al.15 data set of 15 type III units provided a rich database to test our model. In all, the model provided reasonable fits to 14 type III units. Prior to this, notch noise responses from only three type IV units from cat were available for modeling.6,18 The objective function used here is the same one used in Hancock and Voigt6; this allowed direct comparisons to that study. In this study, the RMs of the model P-cells with the best-fit parameters were compared to the physiological units\u2019 RMs. The model RMs had excitatory center regions with inhibitory surrounds. These are similar to the physiological RMs and correspond to type III units. The model rate-level curves to broadband noise were similar to the physiological data, but the rate-level curves to notch noise showed many differences. Of course, these data are not part of the objective function used and so we would expect differences. It is possible to modify the objective function to include the rate-level curves, the RMs, or both, and this may be done in the future.FIGURE 11.Response maps and rate-level plots responding to notch noise stimuli. Unit L122299 4.01. The best frequency of this unit is 11.0\u00a0kHz. Picture is organized as in Fig. 9.\nOur model also shows reduced sensitivity to  in half of the units. Four units reached the upper limit of  octave and two units reached the lower limit of  octave, which indicates that better fits might be obtained by extending the range of . This is not possible in the present version of the model, which is limited to a four octave frequency range.\nNon-specific afferent (NSA) firing rate affected the \u03a6min in a significant way for some units. This was not true, however, for most of the units, where NSA firing rates of 80, 90, and 120\u00a0spikes\/s produced similar \u03a6min. The best-fit parameter values and sensitivity plots, however, would change significantly when different rates applied. In general, the parameters of the best fits showed no specific relations to unit BF or spontaneous rates.\nThe DCN computational models for cat and gerbil are identical in form and differ only in connectivity. The model is still incomplete, however, in that it lacks model cartwheel cells, granule cells and other cell types known to exist. The CN also receives input from somatosensory neurons that subserve tactile and kinesthetic sensations from the trigeminal ganglion16 the interpolar and caudal spinal trigeminal ganglion,8,25 cuneate nucleus19 as well as from other locations. These indicate that in addition to auditory function, the CN is involved in sensory integration at a very early stage in the brain. These are not currently in the model, but provide rich directions to explore in the future.","keyphrases":["parameter estimation","sensitivity analysis","hearing","sound localization"],"prmu":["P","P","P","P"]}
{"id":"Pediatr_Radiol-3-1-1891645","title":"Multidetector row CT for imaging the paediatric tracheobronchial tree\n","text":"The introduction of multidetector row computed tomography (MDCT) scanners has altered the approach to imaging the paediatric thorax. In an environment where the rapid acquisition of CT data allows general hospitals to image children instead of referring them to specialist paediatric centres, it is vital that general radiologists have access to protocols appropriate for paediatric applications. Thus a dramatic reduction in the delivered radiation dose is ensured with optimal contrast bolus delivery and timing, and inappropriate repetition of the scans is avoided. This article focuses on the main principles of volumetric CT imaging that apply generically to all MDCT scanners. We describe the reconstruction techniques for imaging the paediatric thorax and the low-dose protocols used in our institution on a 16-slice detector CT scanner. Examples of the commonest clinical applications are also given.\nIntroduction\nImaging of the airways with subsequent 2-D and 3-D reconstructions has significantly improved with the advent of the multidetector row computed tomography (MDCT) scanners. These scanners allow volumetric acquisition of isotropic datasets. As a result, the scanning time is significantly reduced, and large anatomical regions are covered within seconds with optimal intravenous contrast material enhancement. MDCT scanners have significantly improved the image quality as the artefacts resulting from partial volume averaging and motion have been dramatically reduced. Additionally, their fast and user-friendly workstations allow postprocessing of impressive multiplanar 2-D and 3-D reconstructions, which are desirable to clinicians as they can be used for comprehensive presurgical planning [1, 2]. In chest applications, the whole of the tracheobronchial tree can be adequately depicted with a single-breath-hold volume acquisition within 4\u20136\u00a0s; normal or complex anatomical structures, pathological processes and their anatomical relationships can be precisely imaged [3, 4]. Finally, on the 16-slice MDCT scanner, by applying the \u201cCombiscan\u201d protocol (0.75-mm collimated scans after administration of intravenous contrast medium), postprocessing of the original data pool can additionally generate high-resolution images of the chest.\nTechnicalities for MDCT of the paediatric airways\nImaging of the airways and thoracic vessels in infants and children has been significantly affected by the wide spectrum of applications of 16-slice MDCT. The key achievement with MDCT is the increased volume coverage per time unit at high axial resolution, which results in improved temporal resolution. With MDCT, scanning techniques may vary when different collimation is selected: the narrowest collimation is applied when partial volume effect needs to be minimal and the 3-D postprocessing images of optimal quality, e.g. CT angiography (CTA) and cardiac CT [3].\nPreparation of the child: is sedation always needed?\nDue to the faster scanning times possible with MDCT scanners, routine sedation is no longer required. In the neonate, recent feeding usually provides tranquillity while children over 3\u00a0years of age tend to cooperate after explanation of the procedure through play therapy and verbal reassurance. As a result, CT waiting lists have shortened.\nWhich protocol should be applied?\nPlanning the examination protocol is essential as this will provide the optimal imaging results that will lead to an accurate diagnosis. Suggested paediatric protocols applied in our institution are displayed in Tables\u00a01, 2, 3, 4 and 5.\nTable\u00a01Imaging protocol for the paediatric chest using a 16-row MDCT scanner\u00a0Routine scanCombiscan\/CTAIndicationStrictureCardiovascular anomaliesTumourSmall tracheobronchial stenosesTracheomalaciaPeripheral airways diseaseAnatomical areaThoracic inlet to diaphragmTube collimation (mm)1.50.75Slice width \u2013 reconstructed (mm)53Table feed (mm\/rotation)2412Exposure factors100\u00a0kVp100\u00a0kVp20\u201375 effective mAs (dependent on patient weight)20\u201375 effective mAs (dependent on patient weight)0.5\u00a0s scan time0.5\u00a0s scan timeRespirationSuspended inspiration; single breath-hold where possibleThree to five expiratory scans for tracheomalacia\/small airways diseaseContrast mediumNoneYesTriggering for CTAAlgorithmSoft tissueSoft tissue plus reconstruction on bony algorithm for high-resolution CT of the lungsTable\u00a02Suggested delay times from the injection of contrast medium\u00a0Manual injectionPressure injectorScan initiationtime delay10\u00a0s from termination of injection25\u00a0s from start of injectionFlow rate2\u00a0ml\/sAge rangeAll age groupsAll age groupsTable\u00a03Volumetric CT chest scanning parameters according to child\u2019s weight when routine and Combiscan protocols are performed\u00a0<15\u00a0kg15\u201324\u00a0kg25\u201334\u00a0kg35\u201344\u00a0kg45\u201355\u00a0kgVolumeCombiVolumeCombiVolumeCombiVolumeCombiVolumeCombikVp100100100100100100100100100100Effective mAs20202525353555557575Collimation (mm)1.50.751.50.751.50.751.50.751.50.75Scan slice width (mm)5555555588Table feed (mm)24122412241224122412Scan time (s)0.50.50.50.50.50.50.50.50.50.5Calculated Effective Dose (mSv)\u2013CT EXPO0.91.01.131.311.581.752.482.753.383.75Table\u00a04High-resolution CT chest scanning parameters according to child\u2019s weight\u00a0<15 kg15\u201330 kg>30 kgkVp100100100Effective mAs203055Collimation (mm)111Scan slice width (mm)111Table feed (mm)101010Scan time (s)0.360.750.75Calculated Effective Dose (mSv)\u2013CT EXPO0.210.320.59Table\u00a05CT angiography scanning parameters according to child\u2019s weight\u00a0<15 kg15\u201324 kg25\u201334 kg35\u201344 kg45\u201355 kgkVp100100100100100Effective mAs2025354050Collimation (mm)0.750.750.750.750.75Scan slice width (mm)11111Table feed (mm)1515151515Scan time (s)0.750.750.750.750.75Calculated Effective Dose (mSv)\u2013CT EXPO1.01.261.762.02.5\nRadiation dose\nWhen dealing with children, the issue of performing a low-dose examination is crucial and of the utmost importance. Radiation dose is a significant issue in paediatrics as it is well established that the life-time cancer mortality risk attributable to CT examinations is considerably higher than in adults [1, 2]. As proposed by the ALARA principle, the selection of appropriate scanning parameters focuses on the optimization of the image quality whilst delivering the lowest possible radiation dose and shifting the risk\u2013benefit balance towards benefit [1, 2, 5].\nTechnical parameters that need to be selected for any scan include: thickness of collimation, tube current, and kilovoltage. The thickness of collimation is the minimum section thickness that can be acquired once the scan is finished and in a 16-row MDCT scanner is usually 1.5\u00a0mm. Thinner collimation (0.75\u00a0mm) increases the radiation dose by approximately 30% with our in-house reduced protocol and is applied only in selected cases of vascular abnormalities, visualization of small structures and in cardiac CT (Tables\u00a01 and 6). The axial images are reconstructed at 5-mm thickness and archived to the PACS system within our hospital. In recent years we have made efforts to standardize low-dose protocols for the children scanned in our institution, and the currently applied parameters are summarized in the Tables\u00a01, 3, 4 and 5. Methods adopted to minimize radiation dose in MDCT include: \nApplying a dose modulation function, where the system samples the patient thickness and adjusts (i.e. reduces) the exposure accordingly when the tube is in the AP\/PA position, as patients are narrower in the frontal than in the side-to-side orientation.Reduction of the kilovoltage to 100\u00a0kVp when imaging the thorax. Further reduction to 80\u00a0kVp is possible for CTA, but as resolution of the parenchyma is not ideal this is applied only if lung pathology is unlikely.Selecting tube collimation of 1.5\u00a0mm. The 0.75-mm collimation improves spatial resolution but, as already mentioned, increases the radiation dose, and is therefore reserved for CTA or where thin-slice reconstruction is indicated.Selecting appropriate mAs selection dependent on the patient\u2019s weight or cross-sectional diameter.Table\u00a06Dose comparison for different scanning protocols in a phantom study in our institutionWeight range (kg)Effective dose (mSv)Volume (1.5\u00a0mm)Combi (0.75\u00a0mm)High-resolution CTCTAChest radiographyMaleFemaleMaleFemaleMaleFemaleMaleFemaleAPLateral<150.770.900.91.050.360.421.301.510.004870.0079915\u2013240.931.091.131.310.360.421.621.890.008740.0108625\u2013341.341.561.581.840.540.632.242.620.011630.0096835\u2013442.112.462.482.891.001.172.573.00.017690.01452\nUnlike the single-slice scanner, an increase or decrease in table feed time on the MDCT scanner only affects the overall scanning time. An increase in table speed results in a concomitant increase in mA and this has no effect on the dose delivered. The tube current is automatically compensated to ensure that the preset effective and total mAs is delivered, i.e. a fast table movement results in an automatic increase in the mA keeping the mAs constant.\nAnatomical coverage\nFor imaging of the paediatric thorax, regular coverage extends from the thoracic inlet to the diaphragm. Greater coverage may be warranted in certain clinical cases, such as an extralobar pulmonary sequestration that may derive its blood supply from the upper abdomen.\nIn order to increase spatial resolution, the field of view (FOV) should closely approximate the cross-sectional area of the body part being studied. A large FOV would result in waste of matrix space and partial volume averaging would generate poor quality images [2].\nBreathing during scan\nIdeally, scans should be performed during suspended inspiration at total lung capacity, but this is not often possible in younger children. Experience has proved quiet breathing to be more successful than the confusion created over attempts to acquire the scans during suspended inspiration [6]. When subtle air-trapping due to small airways disease is suspected, a few supplementary expiratory sections may be obtained. If the child is too young to breath-hold, decubitus scans may replace expiratory scans, the dependent lung behaving as the \u201cexpiratory\u201d lung and the nondependent lung behaving as the \u201cinspiratory\u201d lung [6]. The acquisition of prone scans is rarely required in older children who may demonstrate hypoventilation changes in the dependent areas of the lungs. General anaesthesia is therefore applied only in a limited number of cases.\nContrast medium\nWhen imaging the airways, intravenous nonionic contrast material is only administered if paratracheal abnormalities, such as vascular rings, anomalous origin of the pulmonary artery, and mediastinal masses are suspected [2]. \u201cCombiscans\u201d provide the potential to image equally well the lung parenchyma by using the high-resolution techniques (HR) for reconstruction algorithms and the mediastinal structures by injecting contrast medium. However, since 0.75-mm fine collimation is required, there is an inevitable increase in the radiation dose (Table\u00a06) and its application is limited to cases where lung parenchymal detail is of high importance.\nAn intravenous cannula should preferably be placed in situ on the ward so that the child will not associate CT with venepuncture, especially in those in whom subsequent scans may be required. It is ideally inserted via a peripheral vein of the upper or lower limb. Central lines are used for injection in conjunction with clinical agreement and are more desirable as the experience is less unpleasant for the child than having to undergo repeated cannulation. Nonionic contrast medium is administered at a dose of 2.0\u00a0ml\/kg up to a maximum of 50\u00a0ml via a 20\/22G cannula using either a power injector or hand injection (when the cannula is placed in the hand or the wrist). The power injector has the advantage of instilling contrast medium at a constant delivery rate. A flow-rate of 2.0\u00a0ml\/s is used rising to 2.5\u00a0ml\/s for cardiac studies. The delay time between the start of injection and the start of the scan can be empirical and bolus tracking is routinely used in cardiac CT studies (Table\u00a02).\nIn children with complex vascular anatomy, the radiologist should be present for the examination. In those children, low-dose planning scans are performed at a predetermined anatomical level and the injection of contrast medium is triggered by the radiologist. The volumetric scan begins when the contrast medium has reached the relevant preselected vascular structure.\nPostprocessing\nThere are four reconstruction displays available for postprocessing of the volumetric data, which are applied accordingly: multiplanar reformation or reconstruction (MPR), 3-D shaded-surface display (SSD), multiprojective volume reconstruction (MPVR) and 3-D volume rendering (VR). In fact, the axial images include all the information about the anatomy of the airways that is provided with 2-D and 3-D reformats. However, postprocessing gives added value to imaging since the axial scans that need to be studied are usually numerous, and oblique structures as well as interfaces and surfaces parallel to the axial plane are poorly demonstrated and sometimes occult.\nMultiplanar reformations\nThese provide additional diagnostic information in different planes and are as accurate as the axial scans due to the nature of the isovolumetric acquisition of the data [3]. MPRs are one-voxel-thick 2-D tomographic sections that can be displayed in coronal, sagittal, or parasagittal planes or in a single tomographic \u201ccurved\u201d plane, along the axis of a structure of interest, e.g. a bronchus or a feeding vessel (Figs.\u00a01, 2, 3, 4, 5, 6, 7, 8, 9 and 10) [2, 3]. They are real-time easy-to-reconstruct images, producible as soon as the axial sections are completed. They generally improve our perception of images and give information that although contained in transverse images, is sometimes less effectively displayed. Their diagnostic value is substantial in demonstrating and documenting the presence of small focal lesions, defining the vertical extent of a bronchial stenosis, which may go undiagnosed from the axial source CT images (Figs.\u00a04, 5 and 6), and are invaluable prior to surgical remodelling of vascular rings and the tracheobronchial tree [3]. However, to avoid misinterpretations due to partial volume effect, e.g. overestimation of the degree of a stenosis, overlapping and thinner cuts should be applied when processing the raw data. Likewise, when processing curved MPRs, the trace should be centred within the lumen of interest to avoid anatomical distortion.\nFig.\u00a01Coronal oblique MPR in 4.5-month-old boy with trisomy 21. There is right lung hypoplasia and diaphragmatic eventration. An incidental finding is an aberrant right subclavian artery (arrow)Fig.\u00a02Coronal MPR of the chest in a child with a history of recurrent infections due to a congenital pulmonary airway malformation. A thin-walled multicystic lesion in the right lower lobe is shownFig.\u00a03Combiscan in an 11-month-old child with a congenital pulmonary airway malformation in the right lower lobe. a Coronal MPR in lung window setting shows the air-filled cystic component of the lesion. b, c Axial CT slice (b) and coronal MPR (c) showing a large arterial feeder (arrow). This is, however, visualized and appreciated better with an oblique MIP along the axis of the vessel (d) and with coronal anterior and posterior 3-D VR (e). These images additionally demonstrate the pulmonary venous drainage of the lesion, i.e. intralobar sequestration (curved arrows)Fig.\u00a04Congenital tracheal stenosis in a 3-year-old girl with trisomy 21. a Series of axial slices demonstrating stenosis in the central third of the trachea. b This is more sensitively demonstrated on coronal MPR, which correlates well with the bronchographic appearances(c) (arrows). d Virtual bronchoscopy also demonstrates mild stenosis of the supracarinal portion of the trachea (arrowheads)Fig.\u00a05Chest CT (1.5\u00a0mm collimation following intravenous injection of contrast medium) in 10-month-old boy with recurrent wheezing due to congenital tracheal stenosis treated previously with tracheoplasty. There is mild stenosis of the trachea and the origin of the right main bronchus, which is appreciated with difficulty on the axial scans(a), but is nicely shown in coronal MPR (arrow) (b). Virtual bronchoscopy in this case was unnecessary. Bronchography (c) and bronchoscopy confirmed stenosis in the right main bronchus (arrow) that was caused by granulation tissueFig.\u00a06Cardiac CT (0.75 collimation after intravenous injection of contrast medium) in a 6-month-old girl with absent pulmonary valve syndrome. a Axial contrast-enhanced CT shows massive dilatation of the main and the left pulmonary arteries with possible compression of the left main bronchus. This is also appreciated on serial oblique MIPs (b), but becomes more apparent on the oblique MIP along the axis of the left main bronchus (c) (black arrow) and on the 3-D VR (d) (white arrow)Fig.\u00a07Combiscan of the chest in 15-year-old girl with laryngeal\/pulmonary papillomatosis. a There is irregular thickening and narrowing of the proximal tracheal wall that is well demonstrated on axial imaging. b, c Coronal MPRs additionally demonstrate intrapulmonary cavitating lesions. Same findings are impressively demonstrated on 3-D VR (d)Fig.\u00a08Myofibroelastic tumour in a child with a history of cough and haemoptysis. Axial imaging (a) and coronal (b) MPR demonstrate infiltration of the right main bronchus from a heterogeneous soft-tissue massFig.\u00a09Coronal MPR shows in detail the extent of the pneumomediastinum with associated surgical emphysema and right pneumothorax in a child with severe combined immune deficiency (SCID) and Pneumocystis carinii pneumoniaFig.\u00a010Coronal MPR in lung window setting demonstrates left lower lobe bronchiectasis (arrow) in a boy with a history of chronic aspiration\n3-D imaging\nThis is a diagnostic tool only in certain cases as it usually requires more time and postprocessing skills to provide information already included and demonstrated in the axial images and the MPRs. There is no doubt, however, that the 3-D reformatted images may further increase the diagnostic confidence which eventually affects patient management, particularly pertinent in presurgical assessment. Communication with the referring clinicians is simplified as the images portray the spatial relationships of important anatomical structures.\n3-D shaded-surface display techniques\nThese are only applied in the imaging of the central airways and they are usually more visually impressive than clinically useful. Their generation from original data is time-consuming and they carry the risk of loss of density information due to problems with thresholding.\nMultiplanar volume reconstructions\nThese are \u201cselective\u201d 3-D images that resemble the 2-D MPRs and depict peripheral airways better than individual sections. With this technique, a combination of the spatial resolution of thin sections and the anatomical display of thicker slices is achieved, and all the information acquired in the raw data set is used. The routine CT images are combined in multiples to create an image thicker in voxels, the volume \u201cslab\u201d, which constitutes an interactive sum of axial, coronal and sagittal reconstructed sections [2, 3]. By using different algorithms and setting thresholds, maximum or minimum intensity voxels can be highlighted within the slab. For the evaluation of the airways, minimum intensity projections within the slab are usually applied (Fig.\u00a011) [2]. Slabs are useful in detecting and localizing micronodular or microtubular patterns and in analyzing mild forms of uneven attenuation of the lungs [7]. However, they are time-consuming and should be reserved for cases of complex morphology, to give the clinicians a comprehensive multilevel roadmap 3-D image.\nFig.\u00a011Complete vascular ring around the trachea and the oesophagus formed by a double aortic arch. a Diagrammatic representation of the abnormality. b Corresponding contrast-enhanced axial image at the level of the double aortic arch, the right part being the dominant. c 3-D VR image of the double aortic arch from a left anterior oblique view. Volume slab (d) and coronal MPR (e) additionally demonstrate the resulting significant narrowing of the trachea\n3-D volume rendering\nThese represent the main technique for 3-D reformatting of the airways and the vascular structures of the mediastinum. The VR technique is particularly useful for displaying structures that course parallel or oblique to the transverse plane and those that develop or extend into multiple planes [2, 3]. However, preliminary editing is still necessary and can be time consuming, altering work patterns in CT. In VR, different anatomical tissues are represented by proportional values that are assigned to every voxel and depend on the range of tissue attenuation values in the original data set. Voxels are selected by the probability of belonging to the object of interest; thus they are displayed in different colours or several shades of grey, different transparency or opacity. Transition from the reconstructed surface to the surrounding media is gradual and the depiction of interfaces, like the inner tracheal lining, is supposed to approach their true appearances. Although this 3-D segmentation technique is better and more complicated than the others previously mentioned, some information is lost during processing, so that the axial images are still indispensable for the radiologist to assess extraluminal disease and identify artefacts. Three-dimensional VR-reconstructed images are attractive and appealing to the clinicians as they may better illustrate short focal areas of narrowing, the craniocaudal length of a tracheobronchial stenosis, and complex congenital cardiovascular and tracheobronchial anomalies (Figs.\u00a02, 3, 6, 7, 11, 12).\nFig.\u00a012a Contrast enhanced 16-slice MCDT (0.75-mm collimation) showing marked tracheal narrowing and an aberrant origin of the left pulmonary artery from the right pulmonary artery (pulmonary sling). b 3D VR demonstrating an elongated and stenotic trachea with identification of a separate bronchus for the right upper lobe\nVirtual bronchoscopy\nThe inner surface of the air-containing tracheobronchial tree can be displayed with virtual bronchoscopy (VB), which is performed with either polygonal SSD or direct VR. VB is a noninvasive and accurate technique that can provide \u201cbronchoscopic\u201d views of the central and the peripheral airways (Figs.\u00a04 and 13). This technique, like fibreoptic bronchoscopy, is considered supplementary to CT and referral to the axial sections is again valuable in recognizing artefacts and in gaining perception of orientation within the virtual airways. VB uses perspective surface rendering, which takes advantage of the natural contrast between the airway and the surrounding tissues [1]. The point for viewing is located intraluminally so that external structures do not overlap and editing takes shorter time periods. Sub-millimetre (0.75\u00a0mm) slice thickness allows deeper penetration and visualization of the bronchial surface down to bronchial diameters of less than 5\u00a0mm; it is, however, associated with an inevitable increase in radiation dose [4].\nFig.\u00a013Accessory bronchus. a Bronchography and b volume rendered 16-slice VB demonstrating an accessory bronchus (curved arrows). On the MDCT image at the level of the carina, a more cephalad origin of the apical accessory bronchus isnoted\nAgain, VB practically never really adds anything new to the established diagnosis. However, pulmonologists may prefer and relate to these images in conjunction with the axial or the reformatted ones [2]. In practice, VB is of limited value, reserved for cases where bronchoscopy is not applicable (children in whom traditional bronchoscopy presents a risk) or precise navigation is not possible to guide airway interventional procedures, and in emergencies such as infant airway (tracheal) stenosis which cannot be otherwise evaluated [4]. The images produced resemble the actual bronchoscopic ones and can additionally \u201cadvance\u201d distal to an airway obstruction, where the real endoscope cannot penetrate [3, 8]. However, when compared to fibreoptic bronchoscopy, VB does not allow any therapeutic manoeuvring, is incapable of detecting endoluminal lesions smaller than 2\u20133\u00a0mm that may go undetected due to the partial volume effect, and provides limited information about mucosal detail (morphology, vascularity and colour), making differentiation between pathological processes and retained secretions difficult [1\u20134, 9, 10]. There is consensus that measurements should be performed only on 2-D sections as they may otherwise be inaccurate [3, 8]. The technique is additionally affected by the partial volume effect, which may lead to misinterpretation of severe stenoses as occlusions, and the threshold level is therefore of importance for displaying accurate simulations [8].\nDynamic and functional inspiratory and expiratory scanning\nIn conjunction with single-breath-hold MDCT, dynamic and functional inspiratory and expiratory scanning helps identify strictures, areas of air-trapping and tracheobronchomalacia, but is rarely applied in paediatric practice because of the associated radiation burden [2].\nPeripheral airways\nModern volumetric techniques have partially overcome the difficulty in demonstrating 3-D images of the peripheral airways. Data are acquired and reconstructed at a later time, allowing thin-slice high-resolution images at any level, so that a whole affected bronchiectatic segment can be displayed on a single MPR\/VR oblique section (Fig.\u00a010) with images that resemble conventional bronchography and may influence the approach for tissue sampling [3, 7]. If there is suspicion of small-airways disease, some additional expiratory slices may be useful. Whatever the application we perform, all the techniques are considered as an adjunct to conventional HRCT evaluation of the peripheral airways that allows accurate and precise assessment of diffuse lung disease at very low doses equivalent to approximately ten chest radiographs [3, 6].\nClinical applications of volumetric imaging of the airways in children\nSelection of the most appropriate CT protocol for each individual case is paramount in imaging of the airways in children. Ultimately, the best protocol is the one that provides the most relevant information at the lowest radiation burden possible. The indications include: (1) congenital bronchial anomalies (e.g. accessory bronchi, bronchial hypoplasia and atresia, and bronchopulmonary foregut malformations), (2) tracheomalacia, (3) tracheobronchial strictures (congenital and acquired) or tumours, and (4) peripheral (small) airways disease.\nTracheobronchial anomalies\nApproximately 1\u201312% of adult patients who undergo bronchoscopy demonstrate some form of congenital variant [1, 11]. Tracheobronchial anomalies may be associated with recurrent episodes of pulmonary infection and airway obstruction and usually go undiagnosed with conventional imaging [1, 12].\nTracheal bronchus\nTracheal bronchus, with an incidence of 0.1\u20135%, represents an aberrant bronchus that usually arises from the right tracheal wall above the carina and ventilates the apical segment of the upper lobe (Fig.\u00a013) [1, 13]. If the right upper-lobe bronchus has a normal trifurcation then the tracheal bronchus is supernumerary and may end blindly; it is also called a tracheal diverticulum. Left tracheal bronchus has also been reported [13]. Its presence may be associated in some patients with other abnormalities of the lung or within the spectrum of the VACTREL (V vertebral, A anorectal atresia, C cardiac, T tracheobronchial, R renal, E esophageal, L lumbosacral\/limb abnormalities) association. Diagnosis of tracheal bronchus should be considered early in the clinical course of intubated patients with recurrent right upper-lobe complications [11]. Coronal MPRs are particularly helpful in demonstrating the presence of a tracheal bronchus, which may go undetected on axial images. Findings can be additionally supported with VB.\nBronchial atresia\nBronchial atresia may present with a variety of findings depending on the child\u2019s age (water-density mass in newborns, bronchocele and air-trapping during childhood, and solitary pulmonary nodule or area of emphysema in adults). In doubtful cases, MPRs help identify mucoid impaction and bronchocele as a branching structure radiating from the hilum [13].\nBronchopulmonary foregut malformations\nThese are anomalies of pulmonary development that are due to abnormal budding of the embryonic foregut and tracheobronchial tree. They include duplication cysts characterized by an isolated portion of lung tissue communicating with the upper gastrointestinal tract or the central nervous system such as bronchogenic cysts, enteric cysts, and neurenteric cysts. Symptoms are usually provoked by the size and location of the cysts, which may cause compression of the trachea or bronchi leading to distal collapse and air trapping. Infection is less commonly encountered.\nPulmonary underdevelopment\nThe spectrum of pulmonary underdevelopment includes agenesis of the bronchus and lung (pulmonary agenesis), presence of a rudimentary blind-ending bronchus without lung tissue (pulmonary aplasia) and bronchial hypoplasia with reduction of lung tissue of variable degree (pulmonary hypoplasia) (Fig.\u00a01) [13].\nScimitar syndrome\nThe scimitar syndrome, also called venolobar syndrome and hypogenetic lung syndrome, is a rare congenital cardiovascular anomaly involving the right lung. It consists of ipsilateral anomalous pulmonary drainage of part of the right lung into the inferior vena cava, hypoplasia of the right lung, dextrorotation of the heart, hypoplasia of the right pulmonary artery, and anomalous systemic arterial supply to the lower lobe of the right lung from the subdiaphragmatic aorta or its main branches. It may be associated with abnormal bronchial anatomy, abnormal diaphragm, hemivertebrae, and anomalies of the genitourinary tract. CT with MPRs shows the size of the right hemithorax, the associated bronchial anomalies, and the anomalous pulmonary (scimitar) vein [13].\nSequestration spectrum\nThe sequestration spectrum includes congenital pulmonary airway malformations (CPAMs), anomalies previously termed congenital cystic adenomatoid malformations, the pulmonary sequestrations and also hybrid lesions that histologically consist of both entities (Figs.\u00a02 and 3). CT defines their location, extent and anatomical relationships, and evaluates their density. MPRs and VRs can impressively demonstrate the arterial feeder from a subphrenic aortic branch and detect the venous drainage into the systemic or pulmonary circulation for the extra- and intralobar sequestrations, respectively (Fig.\u00a03). Enhancement of the cystic wall implies infection.\nTracheomalacia\nTracheomalacia is a condition that refers to softening of the tracheal wall. This is caused from an abnormality of the cartilaginous ring and hypotonia of the myoelastic elements. There is dynamic collapse of the trachea during expiration which leads to airway obstruction with development of wheeze, cough, stridor, dyspnoea, cyanosis and recurrent respiratory infections. Primary tracheomalacia is thought to be caused by congenital immaturity of the tracheal cartilage and may be associated with other developmental defects, such as a vascular ring\/compression or tracheo-oesophageal fistula. The natural history shows that it improves by the end of the first year. In secondary tracheomalacia, previously normal cartilage undergoes degeneration [13].\nTracheobronchial strictures\nCongenital tracheal stenosis\nCongenital tracheal stenosis is a rare disorder characterized by a fixed tracheal narrowing caused by complete tracheal cartilage rings (Fig.\u00a04). Approximately 50% of congenital tracheal stenoses are focal, located usually in the lower third of the trachea, 30% are generalized and 20% are funnel-shaped [13]. The bronchi are usually of normal size. This abnormality which may be associated with other anomalies, more commonly pulmonary artery sling, should be suspected when a distal tracheal stenosis is recognized in an infant [13].\nCongenital lobar emphysema\/overinflation\nAreas of malacia or stenosis of the bronchial cartilage are thought to be the aetiological factors in the development of congenital lobar emphysema\/overinflation through a check-valve mechanism, which is characterized by progressive distension\/hyperinflation of a lobe, most commonly the left upper lobe. CT will typically show hyperinflation of the affected lobe without destruction of the alveolar walls and will help to differentiate this condition from others (Fig.\u00a014).\nFig.\u00a014Axial CT of an infant with mediastinal shift shown on a chest radiograph showing hyperinflation of the left upper lobe without destruction of the alveolar walls, consistent with congenital lobar emphysema\nAcquired tracheobronchial stenoses\nAcquired tracheobronchial stenoses may result from prolonged intubation (post-intubation webs) or at the anastomosis sites following surgical intervention\/lung transplantation. In patients in whom the axis of the stenosis is vertical or slightly oblique it is usually difficult to perceive on the axial images alone, and MPRs\/VRs are useful adjunctive tools (Fig.\u00a05).\nCompression of the airways of cardiovascular origin\nThis is a relatively common complication of cardiovascular diseases that may go unrecognized for some time. Compression is caused either because of an underlying anomalous anatomical relationship between the tracheobronchial tree and the vascular structures, e.g. a double aortic arch, or is extrinsic, caused by enlarged cardiac or pulmonary vascular structures, e.g. congenital heart disease, dilated pulmonary arteries (Fig.\u00a06). In children with unexplained respiratory distress, stridor, wheezing, dysphagia and apnoea, a high index of suspicion is required to identify these lesions that cause mechanical airway compression [14, 15].\nA vascular ring is an aortic arch anomaly in which the trachea and the oesophagus are completely surrounded by vascular structures. They are formed because components of the aortic arch complex persist or regress abnormally. If the airway is incompletely encircled then a vascular sling is produced [13\u201315].\nThe most common and clinically serious type of vascular ring is the double aortic arch, which represents a persistence of both right and left embryonic fourth arches joining the aortic portion of the truncoaortic sac to their respective dorsal aortae [14]. It typically consists of a right and a left arch that encircle the trachea and oesophagus in a tight ring, joining distally to form a common descending aorta (Fig.\u00a011). Variations of this anomaly have been reported, e.g. hypoplasia of one arch [13].\nAnother vascular sling can be produced by an abnormal origin of the left pulmonary artery from the right pulmonary artery (Fig.\u00a012). As the former crosses to the left, it encircles and compresses the right main bronchus and the trachea and occasionally the left main bronchus. This condition is usually associated with other congenital anomalies [14].\nForeign body aspiration\nThis is one of the most common and potentially life threatening events in children, accounting for 7% of lethal accidents in infants aged 1\u20133\u00a0years [16]. MDCT of the chest for the detection of radiolucent foreign bodies is not the investigation of first choice and not routinely used unless a pulmonary infiltrate that fails to resolve within the normal recovery period of 10\u201314\u00a0days is present [16]. It is vital to remember that actual bronchoscopy should be performed even in cases of low clinical suspicion and no direct history since the risk of overlooking foreign body aspiration may be lethal and may cause long-term pulmonary damage.\nLocal extension of neoplasms\nMPRs\/VRs with coevaluation of the axial images are the method of choice for staging of hilar or mediastinal neoplasms. They demonstrate interfaces accurately, e.g. the relationship of the lesions to the bronchovascular walls and the presence of mediastinal lymph nodes, since they maintain voxel density values (Figs.\u00a07 and 8). More complex rendering techniques are not usually required [3].\nPeribronchial air collections\nMPRs\/VRs are helpful in demonstrating small extraluminal air collections and leakages of surgical anastomoses, especially in orthotopic lung or heart\/lung transplant patients (Fig.\u00a09). They can impressively show fistulous paths that may be indiscernible on axial images and affect patient management [3]. Additionally, by correlating with VB the precise location of the leak can be traced prior to actual bronchoscopy.\nPeripheral airways\nMPRs\/VRs using MDCT technology have achieved more accurate visualization of the distal airways. The VR technique is applied in thin, overlapping, transverse reconstructed sections using a high-resolution algorithm. This is particularly helpful in the search for bronchiectasis where a single oblique MPR\/VR section may include the whole portion of the affected bronchi (Fig.\u00a010). Additionally, when planning the best approach for tissue sampling the relation between peripheral nodules and the afferent bronchus is better appreciated using the MIP\/VR sections. Although the applications of conventional HRCT for the evaluation of the peripheral airways are beyond the scope of this review article, the value of HRCT should not be overlooked. When performed individually, HRCT is considerably less of a radiation burden than MDCT (Table\u00a06), but with the standard HRCT protocol, limited slices are performed and the imaging of the mediastinum is insufficient. On the contrary, the Combiscan MDCT protocol with the possibility of HR reconstructions allows detailed parenchymal evaluation of the whole of the chest and simultaneous imaging of the mediastinum; however, there is the disadvantage of extra radiation burden (Table\u00a06).\nConclusion\nMDCT with 2-D and 3-D reconstructed imaging has enhanced the applications of CT in imaging of the chest in children. Obtaining high-quality scans should always be attempted at the lowest radiation dose possible. CT and bronchoscopy are supplementary examinations in the diagnostic work-up of children with tracheobronchial pathology. Although 2-D and 3-D rendering techniques are not the first-line diagnostic tools, they significantly reinforce the confidence in a diagnosis even in complex cases. In specific scenarios, they may supply information that is more easily interpreted among the different specialties than conventional axial scans. In the future, virtual tracheobronchial endoscopy is anticipated to be applied for interactive virtual-reality guidance in surgical procedures of the airways.","keyphrases":["tracheobronchial tree","children","volumetric","multidetector ct"],"prmu":["P","P","P","R"]}
{"id":"J_Interv_Card_Electrophysiol-4-1-2262917","title":"Experience of robotic catheter ablation in humans using a novel remotely steerable catheter sheath\n","text":"Background A novel remotely controlled steerable guide catheter has been developed to enable precise manipulation and stable positioning of any eight French (Fr) or smaller electrophysiological catheter within the heart for the purposes of mapping and ablation.\nIntroduction\nMapping and catheter ablation of cardiac arrhythmias can be technically complex and challenging. Currently the majority of such procedures are performed using manually deflectable catheters. These catheters have limited range and flexibility and rely on operator skill to manoeuvre the catheter tip and maintain stability at target sites within the heart.\nA novel electromechanical master\/slave system that can remotely control a steerable guide catheter (Hansen Medical, Inc., Mountain View, CA, USA) has been developed to enable precise positioning and manipulation of any type of electrophysiological catheter within the heart for the purposes of mapping and ablation. The system comprises three linked components: the physician\u2019s workstation (Sensei\u2122 robotic control system), remote catheter manipulator (RCM) and steerable guide catheter (Artisan\u2122 Control Catheter) (Fig.\u00a01).\nFig.\u00a01The remote catheter manipulator (RCM) (left) is attached to the steerable guide catheter (Artisan\u2122) through which a mapping catheter can be positioned within the heart. The physician\u2019s workstation (right) navigates the steerable guide catheter remotely\nThe physician\u2019s workstation\nThe physician\u2019s workstation consists of display screens for contact intracardiac electrophysiology data and 3D mapping systems as well as a central fluoroscopic control view also displaying a superimposed icon of the Artisan Control Catheter (Fig.\u00a02). Integral to the workstation and its control of the Artisan is the Instinctive Motion Controller (IMC); effectively a 3-dimensional hand operated joystick. The position of the IMC handle is calculated from internal sensors, and this position is fed to control computers in a separate electronics rack which in turn guides the remote catheter manipulator (RCM).\nFig.\u00a02The Instinctive Motion Controller (top left) used with a control panel on the physician\u2019s workstation (bottom left) remotely guides the steerable guide catheter which can be seen on the central \u201ccontrol view\u201d display (right). Real-time data on catheter orientation, catheter-tip pressure, fluoroscopic views as well as intracardiac echocardiography are shown\nRemote catheter manipulator\nThe RCM is a robot designed to accept the Artisan catheter. The RCM receives catheter position commands from the control computers as issued by the IMC. Acting on these commands, the RCM uses servo motors to control its motions. These motions transfer to the Artisan catheter\u2019s pull wires, ultimately determining the position of the catheter tip.\nSteerable guide catheter (Artisan)\nThe Artisan is a single use sterile guide catheter that is composed of two parts: a steerable inner guide within a steerable outer guide (Fig.\u00a03). The outer guide catheter (outer size 14\u00a0Fr, inner size 11\u00a0Fr), controlled by two pull wires 180\u00b0 apart, provides a stable base for the inner guide (11\u00a0Fr outer, 8.5\u00a0Fr inner). Four orthogonal pull wires deflect the inner guide catheter in x and y direction so it can reach anywhere within a roughly toroidal workspace defined by bend of up to 270\u00b0 and 10\u00a0cm extension. Both sheath movements are controlled by the operator. Combined with the RCM\u2019s ability to insert the entire Artisan forward and rotate it, the tip has a versatile reach. Conventional 8\u00a0Fr or smaller mapping and ablation catheters are inserted through the inner guide catheter and locked in place at the proximal end of the Artisan. The deflection mechanisms of these catheters are not required and remain within the Artisan. The only part of the catheters to protrude from the tip of the Artisan control catheter are the distal bipole and occasionally the proximal bipole, as determined by the operator on insertion of the conventional catheter into the Artisan.\nFig.\u00a03The Artisan control catheter and its components. SIG Steerable inner guide, SOG steerable outer guide\nWe report our initial experience in testing the hypothesis that robotic remote catheter ablation in humans is feasible and safe using existing catheters and mapping systems. This study was approved by St. Mary\u2019s Hospital local ethics committee.\nMaterials and methods\nConsecutive patients meeting inclusion criteria and without exclusion criteria (Table\u00a01) were recruited. As part of the initial safety protocol, ten patients underwent remote mapping only and these patients subsequently underwent conventional ablation after the steerable guide catheter was removed. A further ten patients underwent both remote mapping and ablation using the Artisan, the results of which are presented in this paper. Informed written consent was obtained from all patients prior to the procedure. All were studied in the fasted state with or without intravenous sedation.\nTable\u00a01Inclusion and exclusion criteriaInclusion criteriaExclusion criteriaSuitable for catheter mapping\/ablationSevere cerebrovascular disease18\u201385\u00a0years of ageSerum creatinine > 2.5Body Mass Index < 40Active gastrointestinal bleedingSigned informed consentActive infection or feverShort life expectancy <1\u00a0yearSignificant anemiaSevere electrolyte imbalanceAllergy to contrastCongestive heart failure (NYHA Class IV), ejection fraction <30%Unstable angina requiring emergent percutaneous interventionRecent myocardial infarction within 2\u00a0weeksBleeding or clotting disordersUncontrolled diabetesInability to receive IV anticoagulants\nVarious diagnostic catheters were inserted and manipulated manually through the left femoral vein for initial arrhythmia mapping. These included Josephson\u2122 quadripolar catheters in combination with Cardima\u2122, Halo\u2122 and Lasso\u2122 catheters for mapping a left lateral accessory pathway, two atrial flutter circuits and for seven atrial fibrillation ablation procedures respectively.\nTranseptal access was performed to treat the patients with an accessory pathway and atrial fibrillation. In the cases of atrial fibrillation ablation, a single transeptal puncture was made from the left femoral vein, the needle was removed, the sheath withdrawn into the right atrium and an .035\u00a0J-wire left across the puncture site. The Artisan containing the ablation catheter was then guided from the right femoral vein through the puncture site into the left atrium for ablation. It is useful to note that although the outer guide is able to cross the inter-atrial septum, for the majority of cases, this remains on the right atrial side of the septum and catheter navigation within the left atrium was done mainly using the inner sheath. The pulmonary veins were mapped manually with a Lasso\u2122 placed via the left femoral transeptal sheath. For left-sided procedures titrated intravenous heparin boluses of up to 10,000 units were given every 30\u00a0min to maintain an activated clotting time of at least 300\u00a0s.\nThe Artisan control catheter was inserted into the right femoral vein through a short non-irrigated 14\u00a0Fr sheath. Various conventional 4 and 8\u00a0mm non-irrigated tip (EPT Blazer II\u2122) and irrigated-tip (Navistar\u2122 Thermocool\u2122) catheters were deployed through the lumen of the Artisan for ablation. Continuous heparinised saline flushing was maintained through the side ports of the inner and outer sheaths of the Artisan.\nConventional mapping was complemented with 3D non-fluoroscopic mapping technologies as required, including NavX\u2122 (St. Jude Medical, St. Paul, MN, USA), CARTO\u2122 and CARTOMERGE\u2122 (Biosense Webster Inc., Diamond Bar, CA, USA).\nAll 3D maps, computed tomography scan registration, mapping and ablation were performed remotely. In the atrial fibrillation cases some ablation points were \u2018drag\u2019 lesions used to construct ablation lines.\nConventional endpoints of loss of accessory pathway function, bidirectional cavotricuspid isthmus block and four pulmonary vein isolation were used to determine immediate procedural success.\nAt the end of the procedure the Artisan was removed from the 14\u00a0Fr venous sheath manually. If heparin had been given during the procedure, intravenous protamine was administered and removal of the 14\u00a0Fr sheath was done under manual pressure once the activated clotting time was less than 150\u00a0s.\nResults\nTwenty patients were studied. In ten, only mapping to specific anatomical sites was performed using the Hansen system for regulatory purposes without any procedural complication related to using the system. The other ten patients underwent remote catheter ablation and are the subjects of this report. All mapping and ablation endpoints were achieved using only the Sensei robotic control system, without manual manipulation of the ablation catheter in these ten patients.\nTable\u00a02 shows the procedures undertaken, equipment used, radiation exposure, procedural and ablation times. (Note that ablation times are taken from the first application to the last application of radiofrequency energy. All RF applications were given for up to 1\u00a0min at a time). Figures 4 and 5 show examples of the use of the steerable catheter in combination with each mapping system.\nFig.\u00a04Examples of the use of the remotely controlled catheter sheath in conventional electrophysiological procedures. LAO projection of a left free wall accessory pathway ablation (left) and PA projection of segmental right upper pulmonary vein isolation (right). (SGC steerable guide catheter, Abl ablation catheter tip)Fig.\u00a05Non-fluoroscopic mapping system images created using the remotely controlled steerable guide catheter. Anteroposterior view of the left and right atrial geometry using NavX\u2122 (top left) and posteroanterior view of the left atrium during wide area circumferential ablation using CARTO\u2122 (top right). Posteroanterior view of the left atrium (bottom left) and cross-sectional view of the left sided pulmonary veins (bottom right) during segmental pulmonary vein isolation using CARTOMERGE\u2122. (Ablation lesions in red)Table\u00a02Robotic ablation procedural dataPatientSexAgeDiagnosisProcedureMapping systemCatheter tipNumber of RFsRF timeTotal procedure timeFlouro timePatient DAPWorkstation DAP1F76Atrial flutterCTIconventional4\u00a0mm irrigated3810314044.58,38302M55Permanent atrial fibrillationPVI, left atrial mazeNavX\u21224\u00a0mm irrigated6319125911624,66703M50Paroxysmal atrial fibrillationPVINavX\u21224\u00a0mm irrigated421111705211,21604M41Accessory pathwayPathway ablationconventional4\u00a0mm non-irrigated33110613.477905F71Atrial flutterCTIconventional8\u00a0mm non-irrigated15337019.91,04206M53Persistent atrial fibrillationPVI roof line, MINavX\u21224\u00a0mm irrigated3912316555.26,57707M73Persistent atrial fibrillationPVI and roof lineNavX\u21224\u00a0mm irrigated4614316664.24,73908F63Paroxysmal atrial fibrillationPVI and roof lineNavX\u21224\u00a0mm irrigated4811815742.87,27109F46Atrial ectopy\/paroxysmal atrial fibrillationPVICARTOMERGE\u21224\u00a0mm irrigated3114312441.72,262010M54Persistent atrial fibrillationPVI and roof lineCARTO\u21224\u00a0mm irrigated6815522343.45,8780[CTI cavotricuspid isthmus line, PVI pulmonary vein isolation, MI mitral isthmus, line, RF radiofrequency ablation. RF time \u2013 time from first RF to the end of the last RF. All times are in minutes. Radiation exposures are expressed as dose area product DAP (cGycm2).]\nNo major complication occurred. One patient (accessory pathway) had a small <1\u00a0cm pericardial effusion measured on echocardiogram 24\u00a0h post-procedure. The patient was not on post-procedural anticoagulation and was discharged without any clinical sequalae. There were no femoral haematomas associated with the use of the system.\nA radiation dosimeter (Mydose Mini X, Aloka Co. Ltd., Japan) positioned at the physician\u2019s workstation approximately 2.7\u00a0m away from the X-ray source demonstrated negligible exposure to the operator despite a mean cumulative dose area product of 7,281.4\u00a0cGycm2 for all ten ablation procedures.\nDiscussion\nComplex ablation procedures to treat cardiac arrhythmias can be time consuming and technically challenging. There is always a need to improve procedural success, reduce procedure times and minimize fluoroscopy screening times. These issues are largely governed by the skill and efficiency of the operator using manually controlled catheters that may be limited in their flexibility and manoeuvrability. Computer assisted remote catheter ablation systems may minimize some of these difficulties by reducing the manual skill required, potentially reducing the operator\u2019s learning curves.\nThe potential advantages of remote ablation include improved catheter manoeuvrability, precision and especially stability in areas within the heart which may be difficult to reach manually and the ability to reproducibly and accurately return to sites of interest during a procedure. Another remotely controlled catheter technology in current clinical use involves magnetic tipped catheters that are directed within a magnetic field. Its efficacy has already been demonstrated in mapping and ablation of accessory pathways, atrial, nodal and ventricular arrhythmias [1\u20135].\nElectromechanical systems are an alternative means of remote catheter ablation. An in-depth comparison of the advantages and disadvantages of both systems is beyond the remit of this paper; particularly as robotic catheter navigation has only just been developed whereas magnetic navigation has already established itself in clinical trials. However, one can see the advantages of being able to use a remote navigation system that is portable; does not exclude patients that have metal implants or devices and is compatible with other mapping systems and catheters. It remains to be seen whether robotic navigation can achieve the long-term efficacy and safety of magnetic navigation. In particular, whilst catheter-tip stability is improved, the amount of energy applied and the duration needed to achieve successful ablation without increasing the risk of \u201cpops\u201d and resultant perforation needs to be determined.\nRobotic systems have already been used in surgical procedures, demonstrating improved precision, stability and dexterity [6]. Our study has shown that the Sensei robotic system in combination with the Artisan control catheter is safe, feasible and effective in achieving conventional endpoints in mapping and ablation within the human heart.\nSafety\nPrevious studies with porcine models as well as a small human study have shown its safety in vivo [7\u20139]. A potential problem with a remote catheter control system is the lack of mechanical feedback that one would receive from manually controlling a catheter. This is important in assessing how much force is being applied in moving and maintaining catheter tip position. Knowledge of this force is important in avoiding damage to or perforation of cardiac and vascular structures. A system called Intellisense\u2122 Fine Force Technology uses two force sensors that grip the shaft of the working catheter as it protrudes from the Artisan catheter. The working catheter is pulsed a short distance (<1.5\u00a0mm) in and out of the Artisan four times each second and with each pulse, coaxial force data are collected and compared. Forces applied to the working catheter tip are measured and displayed on the main screen as visual feedback of force (Fig.\u00a06). This does not provide feedback on all the multidirectional forces applied to all parts of the catheter and, whilst it would be ideal to receive mechanical feedback through the instinctive motion controller, the visual data provided on the most important part of the catheter helps the operator to overcome this potential problem.\nFig.\u00a06The Intellisense system display (close-up edited view). Coaxial forces sensed at the catheter tip are displayed as a realtime waveform strip chart and gauge. On the strip chart there is a \u201cwarning level\u201d threshold which is set by the operator. When forces exceed this level, the tip of the animated catheter changes colour (from white to yellow), and the strip chart plot line changes colour as well\nIn the authors\u2019 personal experience using this system, although visual representation of the forces applied at the catheter tip is better than none at all, it does not replace the tactile responses that experienced operators use to prevent damage or perforation of cardiac structures. The Intellisense system allows the operator to predetermine what pressure level is deemed to be \u2018excessive\u2019 and if pressures exceed this arbitrary threshold the tip of the virtual catheter and the pressure waveform changes colour as a warning but there is no auditory alarm, requiring the operator to be constantly vigilant.\nAnother potential problem is the potential for thrombus to form within the Artisan\u2019s sheaths. This was avoided by high flow continuous flushing and appropriate heparinisation. No thrombotic complication occurred in this study.\nFluoroscopy and radiation exposure\nComplex ablation procedures such as for atrial fibrillation can be lengthy and radiation exposure to both patients and operators remains a concern. The present study does not fully represent the potential for a robotic system to reduce X-ray exposure to patients. For the operator, during conventional ablation procedures, despite adequate radiation protection, areas such as the face and extremities are still subject to scattered radiation exposure which impacts on cumulative dose [10, 11]. Our study has shown that negligible operator radiation exposure can already be achieved from being less than 3\u00a0m away from the radiation source whilst still being within the laboratory, potentially reducing the long-term risks to operators from radiation. Once the learning curve is overcome and procedure times improve, when used fully in combination with non-fluoroscopic mapping systems, robotic ablation could potentially reduce patient radiation exposure as well.\nVersatility\nThis study has demonstrated the system\u2019s compatibility with various irrigated and non-irrigated tipped ablation catheters as well as its use with existing non-fluoroscopic mapping systems such as CARTO\u2122 and NavX\u2122. There was no technical difficulty in setting up and performing mapping and ablation in any of the cases. In the case using a Thermocool Navistar 8\u00a0mm tip catheter, insertion into the Artisan was not problematic, though the fit prevented the Intellisense force feedback system from moving the catheter tip. Mapping and ablation was performed unhindered but with the Intellisense system switched off.\nLimitations\nThis is a small feasibility study therefore conclusions with regards to overall efficacy and efficiency of the system for each type of arrhythmia cannot be made. For comparison, averaged data from conventional procedures (n\u2009=\u200910 for each) undertaken at our laboratory are shown in Table\u00a03. It should be noted that this data represents the work of several operators of varying skill and experience. The robotic atrial fibrillation data (RF time 140\u2009\u00b1\u200927\u00a0min, procedure time 181\u2009\u00b1\u200945\u00a0min, n\u2009=\u20097) show that times are longer on average compared to conventional procedures. This is not surprising as, with any new device, there is a learning curve to its use.\nTable\u00a03Conventional ablation procedural dataAblation procedureNumber RFsRF timeTotal procedure timeFlouro timeDAPAtrial fibrillation\u2013111\u2009\u00b1\u200951 (44\u2013171)143\u2009\u00b1\u200952 (45\u2013204)61.4\u2009\u00b1\u200931 (24\u2013121)6,636\u2009\u00b1\u20095,867 (1,776\u201319,489)Atrial flutter7\u2009\u00b1\u20095 (2\u201318)21\u2009\u00b1\u200916 (4\u201356)62\u2009\u00b1\u200924 (25\u201394)23\u2009\u00b1\u200913.3 (7\u201349)1,369\u2009\u00b1\u20091,108 (189\u20133,596)Accessory pathway3\u2009\u00b1\u20093 (1\u201311)14\u2009\u00b1\u200925 (1\u201383)97\u2009\u00b1\u200953 (36\u2013186)22.9\u2009\u00b1\u200912.2 (9\u201344)2,899\u2009\u00b1\u20093,224 (219\u201311,055)Figures represents data taken from conventional procedures (n\u2009=\u200910) for each arrhythmia type. Means are given with standard deviations and ranges. Times are in minutes. RF\u2014radiofrequency energy lesion. Dose area product (DAP) in cGycm2.\nThe operators themselves underwent an intensive two day training course to familiarise themselves with the set-up and use of the equipment in animals prior to the study being undertaken. During the animal case the physician is instructed in how to drive to certain places in the right atrium, how to cross the septum and drive to defined points in the left atrium and also how to create a 3D atrial geometry. It is understandable that great caution was employed with its use in the first human subjects. Despite this, the data are sufficient to conclude that the system can work safely and effectively in humans and can achieve conventional endpoints of ablation.\nConclusions\nThe Sensei robotic control system in combination with the Artisan control catheter is compatible with current mapping and ablation technologies, enabling clinically effective remote navigation of ablation catheters. Remotely controlled catheter ablation for cardiac arrhythmias using this system reduces operator radiation exposure.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nESM 1\n(MOV 4.26\u00a0MB)","keyphrases":["catheter ablation","remote navigation","robotic navigation"],"prmu":["P","P","P"]}
{"id":"J_Gastrointest_Surg-3-1-1852382","title":"Acquiring Tetanus After Hemorrhoid Banding and Other Gastrointestinal Procedures\n","text":"Tetanus after hemorrhoidal banding is an extremely rare but serious complication of the procedure. We describe the second reported case of this complication and review the literature concerning tetanus after different gastrointestinal procedures. Although a rare complication, practicing physicians need to be aware of the clinical presentation of this deadly disease when encountered in at-risk patient populations. Such cases also reemphasize the importance of primary tetanus immunization and follow-up boosters for all vulnerable patients.\nIntroduction\nTetanus is currently a rare disease in the United States and other developed countries where active immunization programs provide a considerable degree of immunity to the infection in the general population. The incidence of tetanus has dropped dramatically in the United States and other developed countries due to a successful vaccination program instituted in the 1940s.1 During the period from 1998 to 2000, the Centers for Disease Control (CDC) reported the average annual incidence of tetanus to be 43 new cases in the United States per year. This is equivalent to 0.16 cases\/million people in the US.2 The worldwide incidence of tetanus, however, is much higher and estimated to be around 500,000 to 1 million per year.3 In developed countries, tetanus is most commonly seen in immigrant patients who have never received a complete primary immunization series. Another at-risk group is the elderly people who have not received recent booster doses. Such cases in the older, previously vaccinated adult reflects waning immunity if a booster shot is not received every 10\u00a0years.\nTetanus is caused by the toxin-producing anaerobic bacterium Closteridium tetani. C. tetani is a spore-forming bacteria that once inside the body of susceptible host, produces a potent toxin named tetanospasmin. Tetanospasmin binds to the central nervous system causing diffuse muscle spasms and autonomic instability that characterizes tetanus. Tetanus is usually seen in association with soil contamination of a cutaneous wound; however, on rare occasions it may occur after a surgical procedure such as gastrointestinal surgery. In this paper, we describe the second reported case of tetanus after banding ligation of internal hemorrhoids and will review the other reported cases of tetanus after gastrointestinal procedures.\nCase Report\nA 63-year-old female with no significant past medical history presented with chief complaint of 5\u00a0days of anal discomfort due to hemorrhoids. Physical exam showed a 1.5\u00a0cm soft and inflamed external hemorrhoid. She was initially treated with pramoxine suppositories and a week later was scheduled for elective hemorrhoid banding. At the time of surgery, she was noted to have a small noninflamed external hemorrhoid as well as a grade III internal hemorrhoid at the eight o\u2019clock position; there was no evidence of infection. The patient underwent rubber band ligation of the internal hemorrhoid with no immediate complications. After the procedure, the patient was instructed to take daily Sitz baths and given a prescription for docusate (oral) and psyllium to prevent constipation. Five days after the surgery, the patient returned to the hospital with a 1-day history of sore throat, pain upon swallowing, headache, and inability to open her mouth. She was noted to have a hoarse voice and stated that she had difficulty sticking out her tongue. Initial vital signs showed blood pressure of 150\/70\u00a0mmHg, pulse of 95, temperature of 37.1\u00b0C, and respiratory rate of 18 per minute. Neurological examination was remarkable for inability to completely open the mouth; the rest of the neurological examination showed normal findings including muscle tone, bulk, and strength. A neck x-ray was obtained and it was normal. Patient\u2019s inability to open mouth prompted further evaluation and the diagnosis of tetanus was entertained. Further history revealed that patient had never been immunized against tetanus. She was subsequently admitted to the intensive care unit and initially treated with 1,000 units of intervenous (IV) human tetanus immune globulin, 2\u00a0g of IV ceftriaxone every 24\u00a0h, IV diazepam, and methylprednisolone. On the next day, the patient had severe pain on attempting to open her mouth and diffuse muscular spasms of jaw, neck, abdominal musculature consistent with trismus and generalized tetanus. A rectal exam at the time showed normal postoperative changes; there were no masses, bleeding, signs of infection, or hemmorhoids. She subsequently developed respiratory difficulties due to laryngeal spasms and required intubation and paralysis. In the next days, she developed a number of complications related to hospitalization and the underlying tetanus. The patient had autonomic lability with associated hypertension (systolic blood pressure up to 220\u00a0mmHg) and tachycardia (pulse of 110s); she developed an episode of chest paint and was found to have an acute anterior wall ST-elevation myocardial infarction confirmed with EKG abnormalities and elevated troponins. Additional complications included a small pneumothorax after placement of a Swann\u2013Ganz catheter, left lung collapse due to mucous plugging, and nosocomial pneumonia due to Acinetobacter baumanii. Attempts to wean her during endotracheal intubation were unsuccessful and the patient required a tracheostomy. After about 2\u00a0months of hospitalization, she gradually improved and was weaned from the respirator. She had upper and lower muscle atrophy, global muscle weakness, and ankle contractures bilaterally. She had no obvious sensory, cognitive, or language deficits. She was able to roll herself in bed and feed herself; however, she was unable to stand, walk, or place herself on bedpan. She was subsequently transferred to an inpatient rehabilitation facility for intensive physical and occupational therapy. At the completion of a month-long inpatient rehabilitation program, she showed some improvement but still had some lower extremity weakness (grade 3\u20134 out of 5 of muscle strength bilaterally) and residual ankle contractures; she could walk with assistance using a forearm crutch. The patient was discharged from the hospital with a continuing outpatient rehabilitation program.\nReview of Literature\nMethods\nWe searched the English language articles from 1966 to January 2005 in the MeSH system of PubMed for relevant case reports and articles. MeSH keywords identified included tetanus, combined with digestive system surgical procedures, surgery, surgical procedures operative, and hemorrhoids. All the relevant articles were reviewed, and their reference list examined for other relevant articles. Other articles were obtained and reviewed from these reference lists. Postoperative tetanus cases after obstetric\u2013gynecology procedures in which the appendix was removed were not included; one such paper is included in the reference list however.4\nResults\nWe found 14 case reports of tetanus after gastrointestinal procedures. Findings and case summaries are outlined in the tables below (see Tables\u00a01 and 2). \nTable\u00a01Reported Cases of Gastrointestinal Procedures Complicated by TetanusCasesReferencesOpen cholecystectomy5\u20137Cholecystectomy with exploration of bile duct8Resection for a gangrenous perforated small intestine9,10Rubber band ligation of hemorrhoids11Cryosurgery for internal hemorrhoids12Drainage of anorectal abscess13Sigmoidoscopic polypectomy14Gastrectomy, Billroth II, and transverse colectomy, (for large cell lymphoma)15Exploratory laparotomy (for carcinoma in omentum and liver)5Table\u00a02Summary of Presentations for the Reported CasesAuthorYearAge (Year)Gender (Male\/Female)Time to Initial Symptoms. (Days Postprocedure)Initial SymptomsLater SymptomsOnset of Later Symptoms (Days Postprocedure)Tetanus ImmunizationOutcomeOpen cholecystectomy: (four cases) 5\u20137Parker& Mandal 5198447Female10No mentionTrismus10uncertainAliveParker& Mandal 5198459Female17Spastic gaitNo mention22No mentionDeadO\u2019Riordain, Buckley, & Kirwan 6199146Male11Abdominal spasm, pain, fever, mild trisumusTrismus12NoDeadCrokaert, Glupczynski, Fastrez, Alle, & Yourassowsky 7198444No mention6Neck and face pain, stiffnessOpisthotonus6No mentionNo mentionCholecystectomy with exploration of bile duct: (two cases) 8Lennard, Gunn, Sellers, & Stoddart 8198449Female12Sub-costal pain, abdominal distentionOpisthotonusNo mentionNoAliveLennard et al. 8198466Female16Abdominal painTrismus19NoAliveResection for a gangrenous perforated small intestine: (two cases) 9,10Furui et al. 9199875Male1Jerking, limb rigidityOpisthotonus1NoAliveClay & Bolton 10196461Male2JerkingOpisthotonus4No mentionAliveRubber band ligation of hemorrhoids: (two cases) 11Murphy 11197833Female7Dysphagia, neck painTrismus9NoAlivePresent case199763Female4DysphagiaTrismus6NoAliveCryosurgery for internal hemorrhoids: (one case) 12Singh, Chhina, & Kaul 12199242Male14Fever, DysphagiaTrismus19NoAliveDrainage of anorectal abscess: (one case) 13Myers et al. 13198462Male10Restlessness, limb rigidityTrismus12NoAliveSigmoidoscopic polypectomy: (one case) 14Segel & Shaff 14196955Female10Fatigue, weaknessTrismus19NoAliveGstrecotmy, Billroth II, and transverse colectomy, for \u201clarge cell lymphoma\u201d: (one case) 15Fleshner, Hunter, & Rudick 15198848Male21Fever, abdomen, back pain, dysphagiaTrismus and opisthotonus26NoAliveExploratory laporatomy (for carcinoma in omentum and liver): (one case) 5Parker & Mandal 5198465Female12No mentionNo mention12NoDead\nDiscussion\nThis is the second case of tetanus after hemorrhoid banding that we were able to find in the literature. As noted in Table\u00a01, tetanus can occur after a wide variety of gastrointestinal surgical procedures including major intraabdominal surgery (e.g., cholecystectomy for cholecystitis; exploratory laparotomy for intestinal perforation), as well as relatively \u201ctrivial\u201d procedures such as hemorrhoid surgery and sigmoidoscopic polypectomy. Tetanus occurs after germination of the spores and subsequent production of toxin by the organism. Clostridium tetani will not grow in healthy tissue, therefore a number of factors need to be present for germination to occur including ischemia, devitalized tissue, coinfection, and injury from penetration or foreign body.1 In gastrointestinal surgical procedures, presence of ischemic or devitalized tissues permits proliferation of C. tetani and subsequent toxin production. In our case, the devitalized tissue from banded hemorrhoid was the likely entry point for the organism with subclinical C. tetani infection occurring at the site. C. tetani may be isolated from stool flora in asymptomatic individuals; in farming regions where individuals are in constant contact with soilor domestic animals (e.g., horses, cattle), up to 1\/3 of individuals may have C. tetani in their stools.16 Given standard sterilization procedures, tetanus associated with infected surgical instruments is extremely uncommon in industrialized countries\u2014our patient most likely developed the condition after contamination of the wound with organisms from the stool or external environment. In our experience with patients from Latin America, it has come to our attention that some of them use herbal products such as out-door plants known as aloe vera (\u201cbuena herba\u201d in Spanish) for treatment of local wounds. Although we are not aware of any documented case report of this practice causing transmission of tetanus, we wonder if application of contaminated outdoor plants might play a role in some of the postoperative cases. Our patient did not provide history of such practice and the exact mode of contamination will not change the clinical presentation or management. Once suspicion arises for presence of tetanus, identification depends upon recognition of the characteristic clinical syndrome. Although isolation of C. tetani from a wound is supportive of the diagnosis, many patients have negative wound cultures and presence of the characteristic clinical syndrome is adequate evidence to support the diagnosis.1\nAlthough rare, it is prudent for practitioners to be mindful of signs that suggest the possibility of postoperative tetanus. Patients with tetanus usually have painful spasms and contractions of their skeletal muscles; this can present as stiff neck, trismus, or opisthotonus. Lockjaw, also known as trismus, leads to inability to open the mouth and the characteristic sardonic smile (risus sardonicus)\u2014it is a sign of spasm of muscles of mastication. Trismus, along with back pain and diffuse muscle spasms, is one of the most frequent findings in tetanus.17 Opisthotonus is a sign of generalized tetanus and is characterized by painful involuntary bending of spine and extremities. It leads to forward convexity of the body, with patient\u2019s torso arching upward and body supported on head and heels. There can also be periods of apnea due to thoracic, glottal, pharyngeal muscle contractions often requiring intubation and respiratory support. Irritability, restlessness, sweating, labile vitals signs, or even a myocardial infarction can occur due to autonomic instability. Presence of these signs or symptoms could be due to underlying tetanus and should alert the practitioner of possibility of the condition. Despite the case reports in Table\u00a01, it should be emphasized that tetanus is a highly unusual complication of gastrointestinal surgical procedures. Given the extremely rare incidence of this complication, it does not appear to be practical to require evidence of full tetanus immunization before a procedure. Nevertheless, our case reemphasizes the importance of routine tetanus immunization, especially in patient populations that were not immunized in childhood or failed to receive a complete series of vaccinations. As part of their general health maintenance, adults should continue to receive periodic booster shots every 10\u00a0years as recommended by public health authorities.18 Furthermore, despite its rarity, practitioners need to be aware of the clinical presentation of tetanus and consider the diagnosis in at-risk patients who present with characteristic symptoms such as trismus and muscle rigidity.\nConclusion\nPostoperative cases of tetanus have been reported after a number of different gastrointestinal procedures. Tetanus can occur after relatively minor procedures such as hemorrhoidal banding. Practicing physicians need to be aware of the clinical presentation of this deadly disease when encountered in vulnerable patient populations.","keyphrases":["tetanus","hemorrhoids","digestive system surgical procedures","surgical procedures operative","postoperative complications"],"prmu":["P","P","P","P","R"]}
{"id":"Cell_Tissue_Res-4-1-2386751","title":"Analysis of obstetric complications and uterine connective tissue in tenascin-X-deficient humans and mice\n","text":"Tenascin-X (TNX) is a large, multi-domain, extracellular matrix glycoprotein. Complete deficiency of TNX in humans leads to a recessive form of Ehlers-Danlos syndrome (EDS), and TNX haploinsufficiency is a cause of hypermobility type EDS. EDS patients appear to have a higher risk of several complications during pregnancy, such as pelvic instability, premature rupture of membranes, and postpartum hemorrhage. Here, we present a study of genitourinary and obstetric complications in TNX-deficient women of reproductive age. We have found complications, such as uterus prolapses, that are in agreement with previous findings in other EDS types. In TNX knockout (KO) mice, we have observed mild pregnancy-related abnormalities. Morphological and immunohistological analysis of uterine tissues has not revealed obvious quantitative or spatial differences between TNX KO and wildtype mice with respect to collagen types I, III, V, and XII or elastic fibers. We conclude that TNX-deficient women are at risk of obstetric complications, but that TNX KO mice show only a mild phenotype. Furthermore, we show that TNX is involved in the stability of elastic fibers rather than in their initial deposition.\nIntroduction\nTenascin-X (TNX) is a large, multi-domain, extracellular matrix (ECM) glycoprotein composed of epidermal growth factor (EGF) like-repeats, fibronectin type III repeats, and a C-terminal fibrinogen domain (Bristow et al. 1993; Lethias et al. 1996; Elefteriou et al. 1997; Ikuta et al. 1998; Tucker et al. 2006). Complete deficiency of TNX in humans leads to a rare recessive form of Ehlers-Danlos Syndrome (EDS), and TNX haploinsufficiency is a cause of hypermobility type EDS. Patients of both EDS types exhibit mild to severe joint hypermobility. The skin of TNX-deficient patients is markedly lax with poor recoil properties and shows easy bruising. The collagen density appears reduced in the dermis of these patients, and the elastic fibers are abnormal (Burch et al. 1997; Schalkwijk et al. 2001; Zweers et al. 2003; Peeters et al. 2004; Lindor and Bristow 2005). Most of the literature concerning pregnancy in EDS uses the old classification of nine subgroups. In our study, we use the revised classification of six subtypes of EDS (Beighton et al. 1998). Tissue fragility is a hallmark of EDS and is present in all the different subtypes. EDS patients appear to have a higher risk of several complications during pregnancy, such as pelvic instability, premature rupture of membranes, and postpartum hemorrhage. Pregnancy in classical and hypermobility type EDS has a relative favorable maternal and neonatal outcome. Pregnancy in vascular type EDS patients is associated with severe complications including maternal mortality (Pepin et al. 2000; Carley and Schaffer 2000; Roop and Brost 1999; Wegrowski et al. 1999; Ramos-e-Silva et al. 2006; Lind and Wallenburg 2002; Kuczkowski 2005; Parry and Strauss 1998). The effect of TNX deficiency on pregnancy has not been studied so far, and only one case report of pregnancy in a TNX-deficient woman has been reported in the literature (Lindor and Bristow 2005). Here, we present a study of all known pregnancies and genito-urinary (GU) abnormalities in TNX-deficient woman of reproductive age identified in our clinic (Schalkwijk et al. 2001) and in the literature (Lindor and Bristow 2005; Schalkwijk et al. 2001). Furthermore, we have extended our studies to a TNX knockout (KO) mouse model in which we have investigated uterine development during pregnancy. We have previously established that TNX is essential for the development of tissue strength in the skin and is able to bind to elastin, a major component of elastic fibers, and to collagens type I, III, V, and XII (Egging et al. 2006a, b; Veit et al. 2006). We have investigated the expression of these molecules at the protein level.\nMaterials and methods\nTNX-deficient patients\nWe investigated pregnancies and GU abnormalities in all currently identified female tenascin-X deficient patients of reproductive age identified in our clinic and in the literature. The study protocol was approved by the local medical ethics committee, and written informed consent was obtained from the patients.\nExperimental animals\nTNX knockout (KO) mice were obtained as described previously (Mao et al. 2002). For all studies, we used TNX KO mice that were crossed back with six generations of C57BL\/6N mice. Wildtype C57BL\/6 (WT) mice were used as a control. All mice investigated for pregnancy abnormalities and morphological changes in uterus tissue were between 2 and 6\u00a0months of age, reflecting a normal age distribution of a breeding mice population. Skin samples were taken from 2-month-old and 9-month-old mice, and aortic tissue was obtained from 9-month-old mice. The experimental design was approved by the Animal Use Committee of Radboud University, Nijmegen.\nBreeding\nBreeding pairs were kept in a 5:1 female:male ratio. Females were inspected daily for vaginal plugs, and those with plugs were isolated and inspected each day for abnormalities and progression of pregnancy. Litter size was determined after birth. Neonatal survival is defined as the difference in litter size after partus and 2\u00a0weeks postpartum.\nProcessing of samples for (immuno)histochemistry\nMice were killed in a sealed compartment by exposure to a mixture of carbogen gas and increasing concentrations of CO2. Skin samples for frozen sections were embedded in Tissue-Tek O.C.T. compound (Sakura Finetek Europe, The Netherlands) and snap frozen in liquid nitrogen. Skin samples for paraffin sections were fixed in 4% buffered formalin for 4\u00a0h.\nAffinity-purified TNX antibody production\nTNX FNIII repeats 27\u201332 were amplified by polymerase chain reaction (PCR; forward primer: 5\u2019-GGAATTCGAGCTACCTCCCCAC-3\u2019, reverse primer: 5\u2019-CAGGTCGACTCAGGTGAAAGAGGTGGA-3\u2019) by using a previously described 2.7-kb human TNX cDNA as a template (Morel et al. 1989). The PCR product was ligated into the pCR2.1TOPO vector (Invitrogen, Breda, The Netherlands) according to the manufacturer\u2019s instructions for easy digestion with restriction enzymes. The pCR2.1 TOPO vector with insert was digested with EcoRI and SalI. The region coding for FNIII27-32 was inserted into the EcoRI\/SalI site of the pET28(a)+ plasmid (Brunschwig Chemie, Amsterdam, The Netherlands). The sequence of the TNX domains was verified by dideoxy sequencing with a 3730 DNA analyzer (Applied Biosystems, Nieuwekerk a\/d IJssel, The Netherlands). TNX FNIII27-32 protein was expressed and purified according to the manufacturer\u2019s instructions (Brunschwig Chemie). Purified TNX FNIII27-32 protein was used for immunization of a rabbit. Aliquots of 500 \u03bcg TNX FNIII27-32 in 500\u00a0\u03bcl phosphate-buffered saline were mixed with an equal volume of Freund\u2019s complete adjuvant for the first injection and Freund\u2019s incomplete adjuvant for boosters. Three injections were administered subcutaneously at 3-week intervals. Polyclonal antibodies against TNX FNIII27-32 were purified by affinity chromatography on a column with the antigen coupled to CNBr-activated sepharose 4B (GE Healthcare Life Sciences, Diegem, Belgium).\nHistopathology, histochemistry, and immunohistochemistry\nStaining of elastic fibers was performed by modified Hart\u2019s stain on paraffin sections (7 \u03bcm; Starcher et al. 2005). Paraffin sections were also stained with hematoxylin and eosin (H&E) stain to assess uterus morphology and pathology, in collaboration with a professional pathologist. Frozen sections (7 \u03bcm) were stained with antibodies against collagen types I, III, and V (SouthernBiotech, USA) and elastin (Elastin Products, USA) as previously described by Egging et al. (2006b). Collagen XII was stained as previously described by Veit et al. (2006). TNX was stained with affinity-purified antibodies directed against FNIII27-32 of TNX. Detection was performed with antibodies labeled with fluorescein isothiocyanate (FITC, green; DakoCytomation, Denmark) and Alexa Fluor 594 (AF594, red; Molecular Probes, The Netherlands). Cell nuclei were made visible with 4,6-diamidino-2-phenylindole (DAPI, blue; Molecular Probes) counterstaining. Assesment of immunostaining was performed as previously described (Egging et al. 2007, 2006b). Images were captured and processed with Axiovision software (Carl Zeiss, The Netherlands).\nElectron microscopy\nFor electron microscopy, WT and TNX KO samples from uterine wall at 3\u00a0weeks post-partum, skin from 2-month-old and 9-month-old mice, and aortas from 9-month-old mice were fixed in 3% glutaraldehyde in 0.1\u00a0M sodium cacodylate overnight and washed in 0.1\u00a0M sodium cacodylate buffer. The tissues were then sequentially treated with osmium tetroxide, tannic acid, and uranyl acetate and then dehydrated and embedded in Epon as previously described (Davis 1993). Thin sections (60\u00a0nm) were counterstained with 7% uranyl acetate in absolute methanol and lead citrate and examined in a Tecnai 12 transmission electron microscope, at 120\u00a0kV.\nStatistics\nFor statistical analyses, Fisher\u2019s exact or Chi-square tests were used where appropriate for proportional data. Student\u2019s t-test was used to compare mean litter size after partus and neonatal survival (difference in litter size after partus and 2\u00a0weeks postpartum). A P-value of <0.05 was considered statistically significant.\nResults\nPregnancy in TNX-deficient patients\nWe investigated pregnancy and GU abnormalities in TNX-deficient woman of reproductive age. General reproduction characteristics and GU abnormalities of female TNX-deficient patients are presented in Table\u00a01. Maternal and neonatal outcome was generally normal; one out of 13 pregnancies resulted in an intrauterine death of the fetus. Tissue laxity of TNX-deficient patients was demonstrated by the occurrence of vaginal, uterine, and rectal prolapses, even at a relative young age. One case of postpartum hemorrhage was observed in 12 childbirths. None of our patients had urinary incontinence symptoms. TNX-deficient EDS is an autosomal recessive disorder; therefore, all offspring of a TNX-deficient patient and an unaffected individual are obligatory heterozygotes. We have previously found that approximately 60% of adult females that are haploinsufficient for TNX suffer from hypermobility type EDS or benign joint hypermobility syndrome (Zweers et al. 2003). We found no obvious abnormalities (e.g. floppy infant syndrome, premature birth) in the neonates (obligatory heterozygotes) of our TNX-deficient population; however, some TNX-deficient patients themselves were born prematurely (Table\u00a01; Lindor and Bristow 2005). Four out of six of the TNX-deficient women had postpartum complications (one of the patients had no pregnancies). This was in agreement with the findings in the literature of an increase in maternal GU complications in EDS-deficient women compared with the control population (Ainsworth and Aulicino 1993; Carley and Schaffer 2000; Kuczkowski 2005; Lind and Wallenburg 2002; Ramos-e-Silva et al. 2006).\nTable\u00a01Pregnancy in TNX-deficient woman (VUE vaginal uterine extirpation, IUD intra-uterine death, CAH congenital adrenal hyperplasia). Age represents the age of the patient at the anamnesisPatient IDAge (years)Gravida number\/Para numberGenito-urinary complicationsSpecification150G1P1NoneNone2 (sister of patient\u00a01)60G2P2 VUE after uterine prolapse (at age 49)None Vaginal prolapse 2 weeks after VUENone346G0P0NoneWas born prematurely (week 35) with shoulder luxation, rectal prolapse at age 1 4 (sister of patient 3)51G3P3 One incident of hemorrhage postpartum (>1000 ml blood loss)None538G0P0NoneCAH 651G3P2 IUD (24\u00a0weeks) with relatively large blood lossDeceased at age 51, severe co-morbidity including cardiomyopathy, arrhythmia, arteriosclerosis, and amputation of left leg (Schalkwijk et al. 2001). Undefined prolapse (at age 36)757*G4P4 Precipitious second stage at term for all birthsLindor and Bristow 2005 Uterine prolapse (at age 20), also recurrent rectal prolapses\nPregnancy in TNX KO mice\nWe investigated GU and pregnancy-related abnormalities in TNX KO and WT mice ranging between 2\u20136\u00a0months of age. Litter size (TNX KO: 7\u00b12 pups, WT: 7\u00b13 pups) and neonatal survival (TNX KO: 74%\u00b136%, WT: 54%\u00b140%) did not differ significantly (Student\u2019s t-test). GU abnormalities of the TNX KO and WT mice are presented in Table\u00a02. Overall, we noted relatively few pregnancy-related abnormalities. Obstruction of the uterine or vaginal canals during partus resulting in a failure to deliver pups was noted more often in WT mice compared with TNX KO mice, but the difference was not significant (Chi-square test). The length of term did not differ significantly for the TNX KO mice (mean: 19.3\u00a0days) compared with WT mice (mean: 19.6\u00a0days, Fisher\u2019s exact test). A striking difference, however, between TNX KO and WT was found for the location of the vaginal plug that was present after mating. Generally, the vaginal plug in TNX KO mice was located much deeper in the vaginal canal than in WT mice. This made identification of vaginal plugs in TNX KO mice markedly more difficult, resulting in a significant increase in pregnancies with unknown length of pregnancy (Fig.\u00a01a, Chi-square test). No uterine or rectal prolapses were observed in this particular study (67\u00a0pregnancies of KO and WT mice). We did, however, observe rectal prolapses in the breeding colony of our TNX KO mice population (shown in Fig.\u00a01b), albeit at a low frequency (<1%). We did not observe rectal prolapses in our WT mice population.\nTable\u00a02Pregnancy in TNX-deficient miceGenito-urinary complicationsTNX KO miceWT miceNumber of miceNumber of miceMaternal death during pregnancy10Miscarriage20Obstruction during partus13Excessive bleeding of vagina (as seen at vaginal plug check)20Cannibalism after partus11Total number of evaluated pregnancies3631Fig.\u00a01TNX KO mice abnormalities. Vaginal plugs, which are present after mating, are much more difficult to asses in TNX KO mice than in WT mice. This resulted in a significant increase of pregnancies in which the date of impregnation was unknown (a, *P<0.025, Chi-square test). Total evaluations: TNX KO mice, n=32; WT mice, n=28. In this study, no uterine prolapses were observed, although some TNX KO mice suffered from a rectal prolapse (b). Rectal prolapses were also observed in TNX-deficient patients, although the incidence in TNX KO mouse was lower (<1% in mice)\nStructure of mouse uterus\nThe mouse uterus consists of three layers: an outer layer, which includes the single-cell layered perimetrium and a thin layer of connective tissue; a muscular layer, the myometrium, composed of two oppositely orientated layers of muscle; and an endometrium, which undergoes remodeling during the menstrual cycle and pregnancy. The endometrium contains luminal and glandular epithelial cells, stromal cells, resident immune cells of various types, and endothelial cells. No differences in the overall morphology of the uterine tissues were noticeable between TNX KO and WT mice (Fig.\u00a02; uteri on 1\u00a0day postpartum) as assessed by light microscopy of H&E-stained sections. As we mainly focused on the ECM compartment of the uterus, we did not attempt to make quantitative analyses of the cell type distribution.\nFig.\u00a02Structure of TNX KO and WT uterus. H&E-stained sections of the uterus of WT mice (a) and of TNX KO mice (b). No differences in the structure of the uteri were noticeable between TNX KO and WT mice. The mouse uterus consists of a thin outer layer, the perimetrium (P). A muscle layer, the myometrium (M), consists of two oppositely orientated layers of muscle, although this is difficult to distinguish in H&E-stained slides (E endometrium, L lumen). Bars 0.01\u00a0mm\nTNX distribution in mouse uterus\nTNX was present throughout the uterus of virgin mice and in the uterus during and after pregnancy (Fig.\u00a03a-c). Immunostaining for TNX was discontinuous in the perimetrium (Fig.\u00a03a-c,f). TNX was detected in the endometrium, although the epithelium lining the endometrium was negative for TNX (Fig.\u00a03d). Furthermore, TNX was present in the layers of connective tissue ensheathing the muscle bundles of the myometrium (Fig.\u00a03f). Overall, no change in the localization of TNX immunostaining was observed during and after pregnancy. The specificity of our TNX antibody was demonstrated by the complete absence of positive staining in the tissues of the TNX KO mice (Fig.\u00a03e,g).\nFig.\u00a03Immunostaining of TNX in the uterus (P perimetrium, M myometrium showing longitudinal muscle bundles, M* myometrium showing transverse muscle bundles, E endometrium, Epi epithelium of the lumen, L lumen). TNX (green) is present throughout the uterus of virgin mice (a) and in the uterus during and after pregnancy, shown for uteri at 13\u00a0days pregnant (b) and 3\u00a0weeks postpartum (c). Cell nuclei are stained with DAPI (blue). TNX is present in the endometrium (d) and the layers of connective tissue ensheathing muscle bundles of the myometrium (f). TNX immunostaining of the perimetrium is relatively weak (a-c, f). The epithelium of the lumen is negative for TNX (d). The specificity of the TNX antibody is demonstrated in e, g. d\u2013g Uteri at 3\u00a0weeks postpartum. Bars 50\u00a0\u03bcm\nCollagen distribution in mouse uterus\nWe have previously demonstrated the binding and the colocalization of TNX with collagen types I, II and V, in the skin (Egging et al. 2006a, b). In the uterus, TNX also colocalizes with major fibrillar collagens types I, III, and V, as is shown for uteri 3\u00a0weeks postpartum (Fig.\u00a04). The collagens are present in the perimetrium, the endometrium, and the layers of the connective tissue ensheathing the muscle bundles of the myometrium. Similar results have been obtained for uteri from virgin, 13-days pregnant, and 1-day postpartum mice. No differences in collagen type I, III, and V immunostaining are found between WT and TNX KO mice (data not shown), which is in agreement with our observations in skin (Egging et al. 2006b). TNX does not exclusively colocalize with collagens, however, because TNX also cololalizes with other ECM components such as (tropo)elastin. Collagen type XII, an interaction partner of TNX (Veit et al. 2006), is present throughout the uterus (Fig.\u00a05). Collagen type XII occurs in the transverse muscle bundles of the myometrium around blood vessels and around the lumen, although the signal intensity is not completely continuous around the lumen and blood vessels. TNX and collagen type XII are both present throughout the uterus. No differences in the collagen type XII localization in the uterus between WT (Fig.\u00a05a,c,e,g) and TNX KO (Fig.\u00a05b,d,f,h) mice have been observed.\nFig.\u00a04TNX colocalizes with major fibrillar collagens types I, III, and V (P perimetrium, M myometrium with longitudinal muscle bundles, M* myometrium with transverse muscle bundles, E endometrium, Epi epithelium of the lumen, L lumen). TNX (b, e, h, green) and collagen types I (a), III (d), and V (g, red) colocalize (c, f, i, yellow, orange, magenta) in uteri 3\u00a0weeks postpartum. Similar results have been obtained for uteri from virgin, 13-days pregnant, and 1-day postpartum mice. No differences in collagen type I, III, and V immunostaining are apparent between WT and TNX KO mice (data not shown). In k, l, m, higher magnification images of the myometrium are shown to demonstrate the colocalization of the fibrillar collagens with TNX (collagen type III is used as an example). Bars 50\u00a0\u03bcmFig.\u00a05Immunostaining of collagen type XII in uterus (P perimetrium, M myometrium with longitudinal muscle bundles, E endometrium). Collagen XII (red) is present throughout the uterus. Immunostaining appears to be the strongest in the transverse muscle bundles of the myometrium (M*), around blood vessels (BV), and around the lumen (L), although the signal intensity is not always completely continuous. No differences in the collagen type XII localization in the uterus between WT (a, c, e, g) and TNX KO (b, d, f, h) mice are observed. Bars 50\u00a0\u03bcm\nElastic fibers in mouse tissues\nElastic fibers in the skin of TNX-deficient patients are known to be abnormally shaped (Zweers et al. 2004), and thus we have investigated their structure in TNX KO mouse tissues. Elastic fibers in the uterus are mostly located in the myometrium and perimetrium (Fig.\u00a06a,b). The endometrium contains few elastic fibers as shown for WT (Fig.\u00a06a) and TNX KO (Fig.\u00a06b) mice. In the myometrium and perimetrium, positive staining for elastin is seen predominantly in the layers of connective tissue ensheathing the transverse muscle bundles (Fig.\u00a06c,d). Despite the relative low abundance of mature elastic fibers in the endometrium, the presence of elastin can be detected by immunohistochemistry, possibly a consequence of the sensitivity of the detection methods (Fig.\u00a06c-d). Elastin localization is similar for WT (Fig.\u00a06c) and TNX KO (Fig.\u00a06d) mice. We have previously demonstrated binding of TNX with elastin and the colocalization of these two proteins in skin (Egging et al. 2006a, b). Consistent with these findings, elastin also (partially) colocalizes with TNX in the uterus, as shown in the myometrium (Fig.\u00a06e). Colocalization of TNX with elastin is not complete, since TNX also colocalizes with fibrillar collagens; however, all elastin-reactive material colocalizes with TNX. In previous work, we have showed an increase in elastin-positive material in the skin of aging TNX KO mice (Egging et al. 2006a, b). Although, by light microscopy, the elastic fibers in the TNX KO mouse skin do not appear to be abnormally shaped, an increase in staining might have resulted from an increase in less mature or less organized elastin fibers, an increase in soluble tropoelastin within the matrix, and\/or the consequence of more immunoreactive epitopes being available because of an increased susceptibility for degradation. These results are consistent with our past observations of elastic fibers in the skin of TNX-deficient patients in whom irregular and immature elastic fibers and fibers devoid of microfibrils can be seen at the ultrastructural level (Egging et al. 2006b; Zweers et al. 2004). Thus, to expand these findings further in skin and to investigate the elastic fibers in the uterus, we have performed an ultrastructural analysis of the elastic fibers in several tissues. Elastic fibers in WT (Fig.\u00a07a) and TNX KO mice (Fig.\u00a07b) uteri do not appear to differ in shape or size (shown for uterus 3\u00a0weeks postpartum). Similarly, in aorta, the elastic laminae of WT (Fig.\u00a07c) and TNX KO mice (Fig.\u00a07d) are similar in shape and number (aorta of 9-month-old mice). In skin of older TNX KO mice (as shown for 9-month-old mice), however, a difference in elastic fiber ultrastructure is apparent compared with WT skin. In addition to normal-appearing elastic fibers, irregular elastin aggregates, as seen in the skin of TNX-deficient patients (Zweers et al. 2004), can be observed (Fig.\u00a07e,f). These elastin aggregates are not found in the skin of 2-month-old TNX KO mice or in 9-month-old WT mice. Interestingly, elastic fibers in the skin of 9-month-old TNX KO mice often appear larger than those of WT mice of the same age (compare Fig.\u00a07g,h).\nFig.\u00a06Elastin and elastic fibers in the uterus (L  lumen). Elastic fibers (purple, modified Hart\u2019s staining) are mostly located in the myometrium (M, M*) and perimetrium (P), whereas the endometrium (E) appears to contain fewer elastic fibers in WT (a) and TNX KO (b) mice. No elastic fiber abnormalities are found in the TNX KO mice. Elastin immunostaining (red) is observed in the myometrium, predominantly in the transverse bundles (M* in c, d). The layers of connective tissue ensheathing the muscle bundles of the myometrium (M) and perimetrium (P) are stained positively for elastin (red). Strong elastin staining is also seen in the endometrium (E). Elastin immunoreactivity is similar for WT (c) and TNX KO (d) mice. Elastin (red) colocalizes (orange) with TNX (green) in the myometrium (M* in e). Not all TNX colocalizes with elastin as TNX also colocalizes with different collagen types (Fig. 4). Bars 50\u00a0\u03bcm Fig.\u00a07Ultrastructural evaluation of elastic fibers. Elastic fibers in WT (a) and TNX KO (b) mouse uterus do not appear to differ in shape or size. The dark structures (arrows) are elastic fibers (shown for uterus at 3\u00a0weeks postpartum). The elastic laminae (arrows) in the aorta of WT (c) and TNX KO (d) mice are similar in shape and number (aorta of 9-month-old mice). Skin of older TNX KO mice (9\u00a0months old) shows differences in elastic fibers from those of WT skin. Irregular elastin aggregates can be observed in the TNX KO mouse skin (e, near a sebaceous gland). A higher magnification of an elastin aggregate (arrow) is shown in f. These aggregates were not found in skin of 2-month-old TNX KO mice or in 9-month-old WT mice. No irregularities in the shape of elastic fibers are observed in 9-month-old TNX KO mice skin; however, larger elastic fibers than in WT mice (g) are often observed in the TNX KO mice (h, arrows elastic fibers). Bars 0.5\u00a0\u03bcm (a, b), 10\u00a0\u03bcm (c, d), 2\u00a0\u03bcm (e, g, h), 1\u00a0\u03bcm (f)\nDiscussion\nGU complications occur frequently in patients with various types of EDS (Carley and Schaffer 2000; Ramos-e-Silva et al. 2006; Lind and Wallenburg 2002; Kuczkowski 2005; Parry and Strauss 1998; Pepin et al. 2000). In classical and hypermobility types of EDS, which are the most common, the outcome of pregnancy is generally favorable. Maternal complications however, such as postpartum hemorrhage and pelvic instability, are more common than in the general population (Ramos-e-Silva et al. 2006; Kuczkowski 2005; Parry and Strauss 1998). Furthermore, EDS patients appear to have an elevated risk of uterine prolapse (Carley and Schaffer 2000; Lind and Wallenburg 2002). Pregnancy in patients with vascular type EDS may even lead to maternal death through uterus or vessel rupture (Pepin et al. 2000). We have investigated pregnancies and GU abnormalities in TNX-deficient woman of reproductive age. Generally, pregnancy is without major complications in TNX-deficient patients, apart from one noted incident of postpartum hemorrhage. However, uterine and vaginal prolapse regularly occur in TNX-deficient women, even at a young age, suggesting laxity of GU tissues. Premature rupture of fetal membranes is a risk in pregnancy with EDS affected fetuses (Lind and Wallenburg 2002; Parry and Strauss 1998). No premature births have been observed in the offspring of the TNX-deficient patients; however, some TNX-deficient patients had been born prematurely. Obviously, some caution must be taken in making conclusions and extrapolating data from such a small group of TNX-deficient woman.\nComplete TNX deficiency in humans is a rare condition, and so far, only a few patients have been identified. We have therefore investigated pregnancy and uterine tissue structure in our TNX KO mouse model. No gross significant differences have been found with regard to abnormalities during pregnancy or reproduction between the TNX KO and WT mice. A trend toward a reduction in the length of pregnancy in TNX KO mice. The only significant difference has been observed in the location of the vaginal plugs, which suggests laxity in the vaginal wall.\nIn a previous study, we have not found any differences in cutaneous collagen deposition between TNX KO and WT mice (Egging et al. 2006b), as has been observed in the skin of TNX-deficient patients (Schalkwijk et al. 2001; Zweers et al. 2004). Alterations in collagen deposition in the skin of TNX KO mice is, however, a matter of debate (Egging et al. 2006b; Mao et al. 2002; Minamitani et al. 2004). In the present study, we have found elastin aggregates and enlarged elastic fibers in 9-month-old skin of TNX KO mice at the ultrastructural level; this is in accordance with our previously published data concerning increased elastin staining in aging TNX KO mouse skin (Egging et al. 2006b). We have detected no differences in elastic fibers in the uterus or elastic laminae of the abdominal aorta between TNX KO and WT mice suggesting a specific role for TNX in the maturation or maintenance of elastic fibers in skin. However, abnormal elastic fibers might not be present in the uterus, since the ECM is constantly being remodeled during the menstrual cycle and pregnancy.\nAlthough the TNX KO mice appear to have a much milder phenotype compared with TNX-deficient humans, the skin of TNX KO mice is significantly weaker and more lax compared with WT mice (Bristow et al. 2005; Burch et al. 1997; Egging et al. 2006b; Lindor and Bristow 2005; Mao et al. 2002; Matsumoto et al. 2001; Minamitani et al. 2004; Schalkwijk et al. 2001; Zweers et al. 2004). Our data suggest a more lax vaginal canal in TNX-deficient patients and to a certain degree in TNX KO mice. An attractive speculation, considering the observations from our studies in mice, is that TNX is essential for tissue integrity, independent of the quantity of collagen deposition or elastic fiber structure. In conclusion, we would advise obstetricians and gynecologists to monitor TNX-deficient patients closely, as they are likely to have weaker GU connective tissue and could therefore be at risk of GU complications.","keyphrases":["tenascin-x","human","ehlers-danlos syndrome","pregnancy","collagen","elastin","mouse (tnx knockout; c57bl\/6)"],"prmu":["P","P","P","P","P","P","R"]}
{"id":"Eur_Radiol-3-1-2077917","title":"Feasibility study of computed tomography colonography using limited bowel preparation at normal and low-dose levels study\n","text":"The purpose was to evaluate low-dose CT colonography without cathartic cleansing in terms of image quality, polyp visualization and patient acceptance. Sixty-one patients scheduled for colonoscopy started a low-fiber diet, lactulose and amidotrizoic-acid for fecal tagging 2 days prior to the CT scan (standard dose, 5.8\u20138.2 mSv). The original raw data of 51 patients were modified and reconstructed at simulated 2.3 and 0.7 mSv levels. Two observers evaluated the standard dose scan regarding image quality and polyps. A third evaluated the presence of polyps at all three mSv levels in a blinded prospective way. All observers were blinded to the reference standard: colonoscopy. At three times patients were given questionnaires relating to their experiences and preference. Image quality was sufficient in all patients, but significantly lower in the cecum, sigmoid and rectum. The two observers correctly identified respectively 10\/15 (67%) and 9\/15 (60%) polyps \u226510 mm, with 5 and 8 false-positive lesions (standard dose scan). Dose reduction down to 0.7 mSv was not associated with significant changes in diagnostic value (polyps \u226510 mm). Eighty percent of patients preferred CT colonography and 13% preferred colonoscopy (P<0.001). CT colonography without cleansing is preferred to colonoscopy and shows sufficient image quality and moderate sensitivity, without impaired diagnostic value at dose-levels as low as 0.7 mSv.\nIntroduction\nSince computed tomography colonography (CTC) has shown good results in detecting clinically relevant polyps [1\u20136], studies have been performed focusing on increasing patient acceptance by reducing the bowel preparation. This can be done by adding an oral contrast agent to meals (fecal tagging) [7\u201312] in combination with a low-fiber diet, and sometimes lactulose for stool softening, thereby obviating extensive cleansing.\nDifferent studies have been performed using barium, iodine or a combination of both as tagging material. However, only a limited number of these studies on CTC have used limited bowel preparation. Few have evaluated the diagnostic value and patient acceptance of CTC. In parallel with the development of limited bowel preparation strategies, (ultra) low-dose protocols have been evaluated [13\u201315].\nTo our knowledge, to date no studies have evaluated the effect of dose reduction on sensitivity and specificity in patients with a limited bowel preparation. Only one study [7] has evaluated limited bowel preparation at a relatively low dose level (140\u00a0kVp, 10\u00a0mAs). This study has shown very good results; however, these data have not been reproduced yet. Moreover, as only one dose-level was studied, the effect of dose reduction remains unknown.\nThe purpose of this study was to evaluate limited bowel-preparation CTC using an oral contrast agent (amidotrizoic acid) in terms of image quality, patient acceptance and polyp visualization using conventional colonoscopy (CC) as a reference standard. A second objective was to determine the effect of substantially reducing the radiation dose levels on the diagnostic accuracy of limited bowel preparation CTC, again using CC as the reference standard.\nMaterials and methods\nStudy population\nPatients of the Onze Lieve Vrouwe Gasthuis at increased risk for colorectal cancer (personal or family history of colorectal polyps or cancer) [16] who were scheduled to undergo CC between April 2002 and August 2003 were invited to participate in the study. Exclusion criteria were: impossibility to understand the information\/informed consent, age below 18\u00a0years, pregnancy and inflammatory bowel disease. The study was approved by the institutional review board of the Onze Lieve Vrouwe Gasthuis and Academic Medical Center. All patients gave written informed consent.\nCTC bowel preparation and scanning protocol\nFour weeks prior to the CC patients were asked to ingest amidotrizoic acid (20\u00a0mg\/ml, made by the hospital pharmacy, 11.7\u00a0mg I\/ml; corresponding to approximately 30 times diluted Gastrografin370\u00ae) three times a day (100\u00a0ml at breakfast and lunch, 300\u00a0ml at dinner) with a low-fiber diet (well cooked vegetables and meat, no fibrous fruit, no whole-wheat products, no nuts) starting 2\u00a0days prior to CTC. Lactulose (12 g, lactulose CF powder 6\u00a0g\/sachet, Centrafarm, Etten-Leur, The Netherlands) was taken in the morning for 3\u00a0days prior to CTC for stool softening. Twenty mg of butylscopolaminebromide (Buscopan; Boehringer-Ingelheim, Ingelheim, Germany), when contraindicated, 1 mg of glucagon hydrochloride (Glucagen; Novo-Nordisk, Bagsvaerd, Denmark) was administered intravenously. The colon was insufflated with a CO2-air mixture (13.2% vol.) using a flexible catheter until patients experienced discomfort (\u00b12\u20133\u00a0l). Patients were scanned in prone and supine position with a four-slice MX8000 (Philips Medical Systems, Best, The Netherlands) CT scanner (120\u00a0kV, rotation time 0.75\u00a0s, pitch 1.25, collimation 4*2.5\u00a0mm, section thickness 3.2\u00a0mm, and reconstruction interval 1.6\u00a0mm, 50 or 70 milliampere-second (mAs); 70\u00a0mAs if the abdominal circumference was >102.5\u00a0cm, scan time 20\u201325\u00a0s).\nColonoscopy\nPrior to CC, each patient was instructed to ingest 4\u00a0l of a macrogol solution (Colofort macrogol 4000 sachets, Ipsen, Hoofddorp, The Netherlands), starting on the evening prior to the CC. CC was performed by one of three experienced gastroenterologists of the Onze Lieve Vrouwe Gasthuis (experience as gastroenterologist: 23, 12 and 4\u00a0years) and recorded on videotape. Patients received sedatives [5\u00a0mg midazolam (Dormicum; Roche, Basel, Switzerland)] and analgesics [0.05\u00a0mg fentanyl (Janssen Pharmaceuticals, Beerse, Belgium)] at the request of the patient. If polyps were present, the location was determined and size was estimated, based on comparison with an open biopsy forceps prior to removal. A polyp was considered flat if its height was less than one-half of the diameter of the lesion [17]. Only segments visualized at CC were used for analysis.\nSimulation of low-dose CT colonography\nTo study polyp detection at lower dose, CTC examinations were simulated with a lower dose\/mAs-value using an established simulation technique [18, 19] that has been applied in two earlier CTC studies [13, 14]. This simulation method consists of the controlled increase of noise in the raw CT transmission data prior to reconstruction of the images. In this way, scans were simulated at 20\u00a0mAs (in the order of 2.3\u00a0mSv for two scans: supine and prone) and 6\u00a0mAs (\u00b10.7\u00a0mSv). A previous simulation study has demonstrated that the radiation dose of CTC after extensive bowel cleansing can be reduced down to 0.2\u00a0mSv [13]. Since the contrast between tissue and tagged fecal material in limited bowel preparation colons is lower than that between air and tissue, we chose 0.7\u00a0mSv as the lowest simulated dose-level next to 2.3\u00a0mSv.\nCTC evaluation\nAn unblinded research fellow (J.F., experience: evaluated 300 CTC) matched findings at CC (polyps \u22656\u00a0mm and colorectal cancer) with the CTC data to provide a frame of reference for true positive findings at CTC. Only exact matches in shape, size and location were scored as a positive match. All images were then independently evaluated using a primary 2D display mode by three blinded observers. The first observer (research fellow, R.v.G., experience: compared >300 CTCs with CC videos) evaluated the original CTC scan for image quality and for polyps. Scores were assigned (per patient\/segment, Table\u00a01) for overall image quality, luminal distension, homogeneity and presence of stool. The second observer evaluated the original CTC for polyps and marked those examinations that were not diagnostic. If both observers rated a CTC as not diagnostic, this patient was excluded from analysis. The third observer first evaluated the simulated 0.7\u00a0mSv scans for presence of polyps at least 4 weeks after the simulated 2.3\u00a0mSv scans, and again at least 4 weeks later the original CTC scans. All lesions were measured and screenshots were taken. The second observer (V.v.d.H., experience: 8\u00a0years of clinical CT experience as abdominal radiologist) and the third observer (A.v.R., a research fellow in radiology with no prior experience with CTC) had had a learning curve in evaluating 50 CTCs with CC feedback. \nTable\u00a01Scales used by observer 1 to rate image quality (upper part) and scales used by the patients to rate experience and preferenceObserver 1:ScaleImage quality (patient, segment)*1: poor, not diagnostic; 2: moderate, diagnostic with limitations; 3: good, diagnostic with minor limitations; 4: excellent, no limitationsDistension (segment)1: collapsed; 2: poorly distended; 3: only moderately distended but segment is distended over its full length; 4: good; 5: very goodHomogeneity (segment)1: poor; 2: moderate; 3: good; 4: very goodPresence of stool (segmemt)1: large amount of stool, segment fully filled; 2: moderate amount of stool, ~50% of lumen filled; 3: small amount of stool; 4: only contrast layer on the wall; 5: no stool at allPatients:Most burdensome aspectCTC preparation: diet, lactulose, contrast agentCTC: iv puncture, catheter placing, insufflation, breathholds, prone positionCC: iv puncture, moving of scope, air insufflation, monitoring after CCHow burdensome\/painfulNot, mild, moderate, severe, extremePreferenceDefinitely CTC, probably CTC, possibly CTC, indifferent, possibly CC, probably CC, definitely CCMost reluctant factorCC, bowel preparation prior to the CC, CTC, the limited bowel preparation prior to the CTC*The items were scored per patient and per segment.\nPolyp detection and image quality\nA polyp detected at CTC was labeled as true positive if three criteria were met: segmental location and location within the segment corresponded with CC (when situated near the borders of the segment, localization in the adjacent segment was also accepted), the polyp size as estimated by the endoscopist (open forceps) corresponded with size as measured on CTC (50% margin based on the CC size was allowed), and appearance (morphology) closely resembled that of the corresponding polyp at videotaped CC. The unblinded research fellow determined the nature of false-positive findings \u226510\u00a0mm by reviewing the videotaped CC and CTC. If the polyp was possibly missed at CC, a repeat CC was called for.\nPatient questionnaires\nPatient experience and preference were evaluated by six questionnaires (scales are shown in Table\u00a01): 2\u00a0weeks prior to the CTC, directly after the CTC bowel preparation, after the CTC, after the CC preparation, after the CC and 5\u00a0weeks later. The first and the last questionnaires were sent by mail. After the CTC bowel preparation, patients were asked how burdensome they rated the CTC bowel preparation, and the most burdensome aspect. After CTC they rated how much pain they had experienced and how burdensome the CTC had been. Prior to CC patients were asked how burdensome the extensive bowel preparation had been. After the CC they were asked how much pain they had experienced and how burdensome the CC had been. After the CC and 5\u00a0weeks later, patients were asked for their preference for either CTC or CC assuming that both techniques were equally accurate, while in 20% of CTC examinations clinically relevant lesions would be shown, necessitating a therapeutic CC. Two weeks prior to the CTC, directly after the CC and 5\u00a0weeks later, patients were asked what they were most reluctant to undergo.\nStatistical analysis\nDifferences in quality between CTC images of different segments were assessed using ordinal regression analysis. For this analysis we first determined the best segment and used this segment as reference segment. The regression coefficient of each segment estimates the change in the log transformed diagnostic odds ratio (DOR) compared to the reference segment. It can be interpreted, after antilogarithm transformation as relative diagnostic odds ratio (RDOR). A lower RDOR implies inferior image quality of the respective segment compared to the reference segment. Differences in polyp detection rates between CC and lower dose CTC were assessed with the McNemar test for paired proportions. The Wilcoxon signed rank test statistic was used to evaluate differences in patient experience between CTC and CC and to evaluate differences in preference between the questionnaire filled out directly after the procedures and 5\u00a0weeks later. The data were first dichotomized as preference for CTC versus preference for CC. These differences were tested using the chi-square test statistic. All P-values <0.05 were considered to indicate statistical significance.\nResults\nSixty-three eligible patients (53%) gave written informed consent. Two patients were excluded. One patient was excluded since he did not undergo CC for unknown reasons. In another patient with a 20-mm carcinoma opposite the ileocecal valve at CC, the CTC was rated as of no diagnostic value by both observers (parts of the large bowel not in the field of view, stool not tagged sufficiently, and breathing movement artifacts). Therefore, 61 patients were analyzed (Table\u00a02); 51 of these 61 patients could be reviewed regarding dose reduction. \nTable\u00a02Baseline characteristics of the study populationIncluded61Male\/female40\/21Age in years: mean\u2009\u00b1\u2009sd\u2020 (range)61\u2009\u00b1\u200912 (27\u201381)Indication:H\/O\u2020 colorectal polyps38H\/O colorectal carcinoma9F\/H\u2020 of colorectal polyps or cancer14Coexistent complains: Abdominal pain\/hematochezia\/altered bowel habits12\/4\/3Colonoscopy: number of polyps\/patients with polyps:Any size94\/38\u22656\u00a0mm28\/20\u226510\u00a0mm15\/12\u226510\u00a0mm initial colonoscopy13\/11Morphology of polyps \u22656\u00a0mm (sessile\/stalked\/flat\/CRC)12\/7\/7\/2Morphology of polyps \u226510\u00a0mm (sessile\/stalked\/flat\/CRC)3\/6\/4\/2Colonoscopy: no. of patients receiving Sed+analg\/sed\/analg\/none29\/7\/3\/22Stool consistency prior to CTC (diarrhea\/soft\/normal)15\/13\/33Abdominal pain prior to CTC (major\/minor)1\/7Flatulence prior to CTC (major\/minor)3\/27Spasmolitycs during CTC (Buscopan\/Glucagon\/neither)47\/12\/2\u2020sd: standard deviation; H\/O: history of; F\/H: family history, sed: sedatives. Analg: analgesics\nIn five patients it was not possible to fully inspect the colon endoscopically. In one patient a 14-mm polyp was seen at CTC in a segment not inspected at CC and confirmed at surgery (not included in the analysis). In two patients, repeat CC showed a 30-mm polyp and a 30-mm carcinoma (included in the analyses), both missed at the initial CC.\nImage quality\nOverall image quality was rated as excellent in 2 patients, good in 41 and moderate in 18. One patient who received a score of poor\/not diagnostic was excluded. Image quality was significantly lower (Fig.\u00a01) in the cecum (both positions), sigmoid (prone position) and rectum (supine position). This was mainly due to inferior homogeneity in the cecum and rectum and inferior distension in the sigmoid and rectum.\nFig.\u00a01Figure showing RDOR with confidence intervals of all six segments of the colorectum in both prone and supine position regarding overall image quality, distension, presence of stool and homogeneity. All segments are compared to the best segment (DOR by definition 1). Confidence intervals not reaching 1 indicate significantly inferior results\nDiagnostic value\nIncluding the findings at repeat CC, 12 patients had 15 polyps \u226510\u00a0mm (including 2 carcinomas of 35\u00a0mm and 30\u00a0mm) while 20 patients had in total 28 polyps \u22656\u00a0mm (Fig.\u00a02). Twenty-three of 61 patients did not have any polyps at colonoscopy. The initial CC detected 13 of the 15 large polyps (sensitivity 87%). The per-patient sensitivity for the initial CC was 92% (11\/12). The unblinded research fellow was able to match 13\/15 (87%) large polyps (in 10\/12 patients: 83%), while 2 large flat lesions were not visible in retrospect. For polyps \u22656\u00a0mm he matched 23\/28 (87%) polyps (3 additional flat lesions not visible in a patient with already a large flat lesion) in 18\/20 (90%) patients. Observers 1 and 2 correctly identified 10\/15 (67%) and 9\/15 (60%) polyps \u226510\u00a0mm, with respectively 5 and 8 false-positive lesions (Table\u00a03). All but two were explained as stool.\nFig.\u00a02A 76-year-old woman with a 7-mm stalked polyp (arrow) submerged in a layer of tagged stool at CTC (original dose; a: supine position, b prone position) and at colonoscopy (c)Table\u00a03Performance characteristics per observer per size categoryVariablePolyps \u226510\u00a0mmPolyps \u22656\u00a0mmObserver:Observer:1212Analysis according to polypSensitivity10\/15 (67%)9\/15 (60%)17\/28 (61%)15\/28 (54%)FP582028PPV10\/16 (63%)9\/17 (53%)17\/38 (45%)15\/43 (35%)Analysis according to patientSensitivity8\/12 (67%)8\/12 (67%)13\/20 (65%)13\/20 (65%)Specificity45\/49 (92%)41\/49 (84%)30\/41 (73%)25\/41 (73%)PPV8\/12 (67%)8\/16 (50%)13\/24 (54%)13\/29 (45%)NPV45\/49 (92%)41\/45 (91%)30\/37 (81%)25\/32 (78%)PPV: positive predictive value; NPV: negative predictive value; FP: false positives; CI: confidence interval\nPolyp detection at lower dose-levels\nIn 51 of 61 patients, we were able to simulate and reconstruct data at lower dose levels. In 10 of 61 patients this was not possible due to missing original raw data. Dose reduction down to 0.7\u00a0mSv was not associated with significant changes in diagnostic value for patients with lesions \u226510\u00a0mm (Table\u00a04, Figs.\u00a03 and 4). Only focusing on the data of these 51 patients, reader 3 showed diagnostic values comparable to or even better than reviewer 1 and 2: sensitivity for reviewer 1 and 2 both 5 out of 10 large polyps with respectively 5 and 8 false-positive lesions \u226510\u00a0mm and respectively 10 and 9 out of 20 polyps \u22656\u00a0mm with respectively 20 and 25 false-positives \u22656\u00a0mm. Specificity for patients without lesions \u22656 mm was significantly lower (P=0.003) at 6\u00a0mAs when compared to the original dose. \nTable\u00a04Performance characteristics of observer 3 per dose levelVariablePolyps \u226510\u00a0mmPolyps \u22656\u00a0mmDose levelDose levelOriginal2.3\u00a0mSv0.7\u00a0mSvOriginal2.3\u00a0mSv0.7\u00a0mSvAnalysis according to polypSensitivity5\/10 (50%)7\/10 (70%)4 \/10 (40%)11\/20 (55%)13\/20 (65%)10\/20 (50%)FP22451120Analysis according to patientSensitivity5\/9 (56%)7\/9 (78%)4 \/9 (44%)10\/15 (67%)12\/15 (60%)9\/15 (45%)Specificity40\/42 (95%)40\/42 (95%)39\/42 (93%)33\/36 (92%)26\/36 (72%)23\/36SS (64%)SS: statistically significant. FP: false positivesStatistical analysis (only performed on the per patient data) showed that only the specificity for patients without lesions \u22656\u00a0mm was significantly lower (P\u2009=\u20090.003) at 0.7\u00a0mSv when compared to the original doseFig.\u00a03Figure showing a large 12-mm false-positive lesion (arrow) in the transverse colon at three different dose levels in a 65-year-old male patient (a: 0.7\u00a0mSv, b: 2.3\u00a0mSv, c: original). At lower dose levels it is more difficult to differentiate this lesion from a true-positive lesion (polyp) when fecal material is insufficiently tagged, this especially applied to smaller lesions. Well-tagged material is clearly differentiated from colon wall, even at 0.7\u00a0mSv (arrowhead)Fig.\u00a04Figure showing a large 10-mm stalked polyp (arrowhead) in the sigmoid at three different dose levels in a 57-year-old male patient (a: 0.7\u00a0mSv, b: 2.3\u00a0mSv, c: original)\nPatient experience and preference\nThe 6 questionnaires were filled out by respectively 58 (95%), 61 (100%), 61 (100%), 56 (92%), 57 (93%) and 57 (93%) patients. Two weeks prior to the CTC patients were most reluctant to undergo CC, then the CC bowel preparation, thirdly the limited CTC bowel preparation. The CTC was feared least. After both procedures (fifth questionnaire), the order was the same. Five weeks later the bowel preparation prior to the CC was regarded to be the most burdensome, followed by the CC, then the CTC preparation and the CTC. None of the three aspects of the bowel preparation for CTC (the diet, lactulose and contrast agent) was rated significantly worse than the others (Fig.\u00a05). Patients rated the limited bowel preparation (prior to CTC) less burdensome than the bowel preparation prior to CC (Fig.\u00a06, P<0.001). Patients also experienced more pain during CC than during CTC (Fig.\u00a06, P<0.001). The most burdening aspect of the CTC was insufflation of air (58%). The most burdening aspect of CC was the movement of the scope (59%). Directly after the CC (fifth questionnaire) 80% (43\/54) of the patients preferred CTC for their next examination, 13% (7) preferred CC (P<0.001) and 7% (4) were indifferent (Fig.\u00a06). Five weeks after the procedures, 71% (39\/55) preferred CTC, 13% (7) preferred CC (P<0.001) and 18% (10) were indifferent. After 5\u00a0weeks there was no change (P=0.12) in preference of CTC as the next screening test.\nFig.\u00a05Graph showing how patients rated the three different components of CTC with limited bowel preparation. How burdensome were the diet, lactulose and the contrast agent?Fig.\u00a06Graphs show patients\u2019 experience of the bowel preparation (upper left) and the CTC examination (upper right) and patient preference for one of the two modalities (CTC with limited bowel preparation versus CTC with standard bowel preparation, lower). How burdensome was the limited bowel preparation prior to CTC (grey) as compared to cleansing prior to CC (black)? How burdensome were the CTC (grey) and CC (black) examinations (upper right graphs)? What did participants prefer for their next examination (lower graph) directly after both examinations and in the questionnaire sent at home 5\u00a0weeks later?\nDiscussion\nThis study demonstrates that CT colonography without cleansing is preferred to colonoscopy and shows moderate sensitivity (60\u201367%) for polyps \u226510\u00a0mm without impaired diagnostic value at mSv levels as low as 0.7\u00a0mSv.\nImage quality was good on average. Nevertheless, the cecum, sigmoid and rectum showed overall reduced image quality. Although this can be caused by the pelvis causing more noise, reduced imaged quality must probably be attributed to inferior homogeneity in the cecum and rectum and inferior distension in the sigmoid and rectum in the prone position. In contrast to the known problems in CTC with distension, which are solved by dual positioning, inferior homogeneity in the cecum and rectum are typical for the limited bowel preparation protocol. Insufficient homogeneity in the cecum is probably caused by the fact that patients ate food after the last amount of contrast agent was taken, resulting in inadequately tagged stool. The inhomogeneous stool in the rectum probably was caused by the fact that in patients with a long transit time stool was already shaped before the fecal tagging was started.\nWe assume that two adaptations are necessary to improve image quality. First, contrast must be taken as long as the patients are eating. Second, stool should be made softer, for example by replacing lactulose by a stronger osmotic laxative (e.g., low-dose magnesium salts), thereby also reducing the transit time and reducing the amount of non-tagged stool in the rectum. Although these adaptations increase the burden of the bowel preparation, they are slight and are required to improve image quality and thereby maybe the sensitivity and specificity.\nOur study showed an average sensitivity of 63% for large polyps (with a limited number of large false-positive lesions). This is lower than the 100% average sensitivity described by Iannaccone et al. [7] using a more or less comparable bowel preparation; no fiber-rich food and 200\u00a0ml of 370\u00a0mg\/ml iodinated contrast agent in 48 h (which is 6.3 times the amount given in our study), but no lactulose. In addition to the differences in bowel preparation, differences in experience of the readers or reader performance, difference in study groups (polyp prevalence and spectrum) might explain these discrepant results. A recent study [20] has shown inferior results regarding diagnostic value of CTC in populations at increased risk, as is used in the current study, possibly due to a relatively high number of hard-to-see polyps. When comparing our study with three large studies on CTC with extensive bowel preparation [21\u201323] (showing per polyp sensitivities of respectively 52%, 32\u201373% and 53% for polyps \u226510\u00a0mm), our results are comparable or slightly better.\nSince two of five (observer 1) and two of six (observer 2) missed polyps were not seen in retrospect by the unblinded observer, the problem of missing large polyps can be regarded as predominantly an interpretation problem rather than a visualization problem. Future developments, such as better homogeneity, better distension, better learning curve, intuitive display modes, electronic cleansing and computer-aided detection can help in reducing the number of missed polyps.\nSensitivity and specificity were impaired at lower dose levels for patients with lesions \u226510\u00a0mm. This is especially of interest in screening protocols. Regarding lesions \u22656\u00a0mm, specificity was significantly lower at the simulated 0.7\u00a0mSv low-dose scans. Increased noise levels made it impossible to see the tiny air bubbles inside stool. These results may seem to be in contrast with those in an earlier report on low radiation dose CTC [13] showing fewer false-positive lesions. However, these studies in patients with extensively cleansed colons used a three-dimensional display mode and filters to smooth the bowel wall. By using these smoothing filters, small artifacts and small amounts of residual stool were masked when evaluating the colon three dimensionally. Making stool softer and scanning with thinner slices possibly allows for better visualization of the air bubbles, thereby reducing the number of false positives.\nPatient acceptance was found to be very good in the current study. Patient preference was convincingly in favor of CTC; 71% of patients preferred CTC with limited bowel preparation to CC for their next examination. Other studies have shown discrepant results on patient acceptance of CTC versus CC, some favoring CTC [24\u201327], others CC [28, 29]. In a study where patients first underwent extensive cleansing and consecutively CTC and CC [24] fewer patients (71% directly after the CC) favored CTC when compared to our study (80% directly after CC). This difference cannot be attributed to the bowel preparation because of a different study setup.\nSeveral potential limitations must be considered. The number of patients included is relatively low, especially for determining the detection parameters at the 2.3 and 0.7\u00a0mSv levels.\nIn the current study no segmental unblinding during CC was performed. Although repeat CC showed that two large false-positive lesions were missed at the initial CC, segmental unblinding has the advantage of evaluating all false positives.\nSince no electronic cleansing was used and many polyps were submerged, 3D evaluation of these lesions was not of additive value. However, 3D evaluation with electronic cleansing might have given better results, especially since most missed polyps were visible in retrospect.\nAlthough the two readers that had no previous experience in CTC underwent a learning curve of 50 patients, some studies have shown that this may not be sufficient for optimal polyp detection [30, 31]. For CTC using limited bowel preparation, the learning curve is possibly even longer.\nWhen filling out the questionnaires, sensitivity for CC and CTC were assumed equal. If patients knew that the sensitivity of CTC was lower than for CC, this most likely would have influenced the preference for CTC negatively.\nIn conclusion, these results show that CTC with the limited bowel preparation protocol used in this study is feasible, even when using doses as low as 0.7\u00a0mSv levels. Although sensitivity was not as high as previously reported, this is most likely not due to the limited bowel preparation, but to interpretation problems. Technical developments will probably increase the sensitivity, while minor adjustments in bowel preparation may reduce the number of false positives, especially when using low-dose protocols. Since patient acceptance was very good, this technique can be regarded as promising in screening patients of populations with a low prevalence of polyps.","keyphrases":["ct colonography","colonoscopy","fecal tagging","colonic neoplasm"],"prmu":["P","P","P","M"]}
{"id":"Eur_Spine_J-2-2-1602197","title":"Instrumented fusion of thoracolumbar fracture with type I mineralized collagen matrix combined with autogenous bone marrow as a bone graft substitute: a four-case report\n","text":"In order to avoid the morbidity from autogenous bone harvesting, bone graft substitutes are being used more frequently in spinal surgery. There is indirect radiological evidence that bone graft substitutes are efficacious in humans. The purpose of this four-case study was to visually, manually, and histologically assess the quality of a fusion mass produced by a collagen hydroxyapatite scaffold impregnated with autologous bone marrow aspirate for posterolateral fusion. Four patients sustained an acute thoracolumbar fracture and were treated by short posterior segment fusion using the AO fixateur interne. Autologous bone marrow (iliac crest) impregnated hydroxyapatite-collagen scaffold was laid on the decorticated posterior elements. Routine implant removal was performed after a mean of 15.3 months (12\u201320). During this second surgery, fusion mass was assessed visually and manually. A bone biopsy was sent for histological analysis of all four cases. Fusion was confirmed in all four patients intraoperatively and sagittal stress testing confirmed mechanical adequacy of the fusion mass. Three out of the four (cases 2\u20134) had their implants removed between 12 and 15 months after the index surgery. All their histological cuts showed evidence of newly formed bone and presence of active membranous and\/or enchondral ossification foci. The last patient (case 1) underwent implant removal at 20 months and his histological cuts showed mature bone, but no active ossification foci. This four-case report suggests that the fusion mass produced by a mineralized collagen matrix graft soaked in aspirated bone marrow is histologically and mechanically adequate in a thoracolumbar fracture model. A larger patient series and\/or randomized controlled studies are warranted to confirm these initial results.\nIntroduction\nEffectiveness of a bone graft can be described as having three core properties: osteoinductivity, osteoconductivity and osteogenicity [25, 28]. Autogenous bone graft is the only biological structure simultaneously possessing all three properties. However, the long-term morbidity due to autogenous bone harvesting can be as high as 30%, although improved technique has probably lowered the incidence [1, 2, 10, 26]. Allograft bone is the primary alternative to autograft for a number of spinal fusion procedures. However, allograft bone is a poor posterior onlay graft with rates of fusion reported to be consistently lower than autograft except in cases of pediatric deformity [4, 12, 17, 18]. Therefore, there has been an increasing shift in the past decade to the use of bone graft substitutes for spinal fusion.\nOne commercially available bone graft substitute comprised a Type I mineralized collagen matrix (MCM), coated with hydroxyapatite (Healos\u00ae Bone Graft Substitute, DePuy Spine, Inc, Raynham, MA, USA). Kraiwattanapong et al. [21] reported a 0% fusion rate for posterolateral fusion with MCM and bone marrow in rabbits. However, in that study, bone marrow was harvested from the iliac crest which is not a rich source of osteoprogenitor cells in rabbits. Despite these unique findings, the efficacy of this bone graft substitute has been demonstrated in a previous rabbit study of posterolateral fusion preformed by Tay et al. [29] harvesting bone marrow from rabbit tibiae, with a fusion rate of 100%. Autogenous bone marrow aspirate has been successfully used in the treatment of congenital and post-traumatic pseudarthrosis [7, 11, 28]. The effectiveness of this method has been further enhanced by the use of a three-dimensional scaffold which avoids wash out of osteogenic bone marrow cells by blood circulation. In humans, Kitchel has recently reported two lumbar posterolateral fusion studies [19, 20] demonstrating equivalent rates of fusion for MCM to autograft using CT scans for assessment.\nThe current understanding of fracture healing is primarily based on animal histological and radiological studies. The majority of human studies discussing fusion rates are based on radiological fusion criteria which are not completely reliable, even when CT scans are utilized [6]. In a 1993 study involving spinal implant removal following fusion, Blumenthal et al. [3] found an overall agreement between preoperative radiographs and surgical findings in only 69% of the cases. Brodsky et al. [5] found a similar overall agreement in their study published in 1991. The ultimate method to assess the quality of a fusion is perioperative manual palpation and histological analysis of a bone biopsy following implant removal.\nThere are no reports in the literature of the histological behavior of this bone graft substitute in humans. We present here our findings of radiological and histological results on four patients that had surgery performed for an acute thoracolumbar fracture without neurologic deficit.\nMaterials and methods\nFive consecutive patients underwent surgical management for an acute vertebral fracture of the thoracolumbar spine without neurologic deficit. There were three females and two male patients, and the mean age was 47\u00a0years (range 30\u201364). Three patients sustained their fracture after a fall from a horse, one patient after a fall from a scaffold, the last after a fall from a tree. All patients were initially admitted to our institution, which is a level one trauma facility. Initial management was applied according to Advanced Trauma Life Support (ATLS\u00ae) guidelines. Four out of the five patients were diagnosed with no other lesion than the vertebral fracture. The patient who fell from a scaffold was also diagnosed with pulmonary contusions and severe tibial pilon fractures, open type IIIA according to Gustilo\u2019s classification [13, 14]. All patients were worked-up under standard hospital protocol which included plain anteroposterior and lateral radiographs as well as a CT scan of the spine.\nThe AO classification was used to characterize the type of fracture in each patient [23]. Three patients sustained an L1 fracture: two patients had a type A2.3 (Burst-split), one patient had a type B1.2 fracture (flexion-distraction). One patient sustained a type C2.1 fracture (flexion-distraction with rotation) of T12. The fifth patient sustained a type A3.1 fracture of L2 (lateral burst).\nAll patients were conscious and given the choice between non-surgical and surgical management of their fracture after discussing with each of them the advantages and disadvantages of each treatment method, and alternatives. All patients signed an informed written consent to be included in this pilot study. All surgeries were performed by two fellowship trained surgeons (AAF and AJK).\nSurgical technique\nThe short segment fusion-stabilization technique as described originally by Lindsey and Dick [9, 22] was utilized. The spine was approached posteriorly with a midline incision. Musculature was detached subperiosteally and the fracture was exposed posteriorly, both inferiorly and superiorly, to immediately adjacent vertebrae, taking care to preserve the facet joints of the intact segment. Following this, pedicle screws were placed in one vertebra above and one vertebra below the fracture under fluoroscopic control. Whenever possible, the authors attempted to stabilize (and fuse) only one segment if pedicle screws could be placed in the fractured vertebra, typically hemiburst fractures (AO classification type A3.1) where the inferior part of the vertebral body is primarily intact. This was not possible in either of the patients with type A fractures in this study. Following pedicle screw placement, careful decortication of the facet joints and posterior arch of the fractured vertebra was performed. Typically, transverse processes are not decorticated unless a laminectomy to achieve decompression is necessary.\nPercutaneous aspiration of 10\u00a0cc of autogenous bone marrow from the posterior iliac crest was performed. Two 5\u00a0cc rectangles of the MCM graft were saturated with the bone marrow aspirate and laid bilaterally on the posterior elements previously decorticated. No other bone, bone graft extender, or substitute was placed as part of the graft. In the patient who sustained a lateral burst fracture, we performed a transpedicular bone grafting of the vertebral body, but no other graft substitute other than Healos\u00ae was used for the posterior fusion stabilization. Rods were connected to the pedicle screws and fracture reduction in the sagittal plane, and when necessary coronal plane, was achieved by means of ligamentotaxis through the \u201cfixateur interne\u201d (USS\u00ae: Universal Spine System, Synthes Spine, Paoli, PA, USA) as described by Walter Dick [9, 22].\nStandard procedure with this instrumentation is to remove it at 12\u00a0months following surgery when radiological and clinical healing is evident. There are two major reasons for this: (1) implant removal allows the fixed but non-fused segment to recover some mobility which may protect the adjacent segment from accelerated degeneration; (2) in general, after the fracture is radiologically healed, patients experience some discomfort when trying to increase their physical activity. Patients clearly differentiate this discomfort from the pain they had from the fracture and during the healing period. The USS system is in fact a very bulky implant, which the authors believe, is eventually responsible for this discomfort.\nDue to surgeon and patient availability, the time to instrumentation removal is often variable, but always performed at a minimum of 12\u00a0months following the index surgery at our institution. When radiological and clinical healing was achieved, implant removal was performed in four patients. The fifth patient declined implant removal following healing of the fracture because she did not experience any discomfort. Technically, the second surgery required a very short general anesthesia and lasted approximately 30\u00a0min from skin incision to skin suture.\nDuring the second surgery, the mechanical stability of the fused segment was tested using thin curettes introduced in the pedicle screw tracts and manual stress was applied sagittally. A bone biopsy was performed in the heart of the fusion mass which was clearly distinguishable from the lamina and sent for histological analysis. Biopsies were sent in formalin to the institution\u2019s pathology department. They were then decalcified in formic acid, embedded in paraffin and cut in 3-\u03bcm slices with a microtome. They were further colored with hematoxylin\u2013eosin and analyzed under a microscope at magnifications ranging from 20\u00d7 to 400\u00d7.\nRadiological assessment\nAnteroposterior and lateral radiographs were taken in the supine position immediately after surgery, in the standing position a few days after surgery, then at approximately 6\u00a0weeks, 3, 6, and 12\u00a0months following the index surgery, as well as following implant removal. Assessment of healing radiographically was based only on the remodeling of the vertebral body. Overall fusion assessment was based on radiographic healing of the vertebral body as well as patient pain, function, and activity level. Fusion of the posterior elements could not be assessed radiologically. CT scan was not used because it would not have been possible to assess fusion status of the posterior elements due to metallic artifacts from implants. When smoothening of bony edges and some sclerosis of the fractured vertebral body became visible, the fracture was considered to be healed. This was also based on the patient\u2019s subjective appreciation. When fracture and surgery pain regressed and the patient felt he or she could go back to almost all previous all day activity, the fracture was considered clinically healed. No specific clinical outcome scale was used in this limited patient sample.\nResults\nThe four patients who had the secondary surgery underwent implant removal after a mean period of 15.3\u00a0months from the index surgery (12\u201320). All patients had returned to their previous activity at latest follow-up, except one who was involved in worker\u2019s compensation litigation.\nDuring the second surgery, we visually confirmed that the resorbable matrix had been replaced by new bone (Fig.\u00a01) and that this new bone had formed at the location where the MCM graft had been laid. Manual palpation and sagittal stress through curettes placed in the pedicle tracts demonstrated that no movement could be detected visually in any of the four patients.\nFig.\u00a01This 30-year-old female sustained an AO type C2.1 fracture of T12 after a fall from a horse. Initial work-up in the ER did not show any other lesion. She was neurologically intact. We performed a T11-L1 posterior short segment fusion as previously described. Implant removal was performed 14\u00a0months following the index surgery. The biopsy demonstrated irregular bony trabeculae, foci of enchondral ossification and numerous osteoblasts, all characteristic elements of ongoing bone remodeling. Several aggregates of lymphoplasmocytes were observed and have been attributed to a foreign body inflammatory type of response. a Preoperative sagittal CT. b Preoperative lateral radiograph. c Lateral radiograph at 5\u00a0months postop. d Perioperative image of extensive new bone growth during the secondary surgery. e AP radiograph 15\u00a0months following the index surgery after implant removal. f Histology at 100\u00d7 magnification. g Histology at 400\u00d7 magnification\nHistological analysis of these four cases showed clear evidence of newly formed bone. Patients 1, 3, and 4 had implant removal performed between 12 and 15\u00a0months follow-up. Their histological cuts also showed several foci of active membranous and\/or enchondral ossification. Interestingly, patient 2 had implant removal performed at 20\u00a0months of follow-up and though his histology cuts showed newly formed bone, he had no visible active ossification focus. Patient 3 had implant removal performed at 14\u00a0months. His histology showed newly formed bone and active ossification foci.\nDiscussion\nAutogenous graft harvesting site morbidity is a significant issue, and the incidence may be underestimated [15]. Other substitutes such as hydroxyapatite [30\u201333] and other ceramics have had variable results when used for spinal fusion or other orthopedic applications. Disease transmission through allograft though remote, still remains a possibility. The effectiveness of bone morphogenetic proteins (BMPs) has been shown in human clinical trials. But high cost, unknown potential long-term biological effects, and issues with carriers and dosing may alter widespread clinical use, at least in the short term [16, 27].\nAutogenous bone marrow aspirate has been successfully used in the treatment of congenital and post-traumatic pseudarthrosis [7, 8, 11, 25, 28]. In 1997, Muschler et al. [24] studied the prevalence and concentration of osteoblastic progenitors in marrow aspirates from the anterior iliac crest of 32 patients without systemic disease. They determined the number of alkaline phosphatase-positive colony-forming units that grew after placing the bone-marrow derived cells into tissue-culture medium. Three important conclusions have been drawn from this study: (1) the authors estimated that 80% of the cells found in the first 2\u00a0ml of aspirated bone marrow have a prevalence of alkaline phosphatase-positive colony-forming of one for 35,000 nucleated cells; (2) the bone marrow derived cells concentration decreases with the volume of aspirate due to dilution; (3) the cellularity of bone marrow and the prevalence of osteoblastic progenitor cells significantly differ between humans. The prevalence of osteogenic precursors cells, can be increased by centrifugation [7] or using a three-dimensional structure to which these cells can attach [25]. MCM is such a three-dimensional matrix.\nAs described previously, implant removal was performed usually as soon as there was radiological evidence of vertebral body fracture healing and the patient could return to all-day activity, but not earlier than 12\u00a0months after surgery. Patient 2, who suffered from a polytrauma, underwent repeat surgery for his open pilon fractures and was bound to a wheelchair for a long period. This is the reason why implant removal has been done later than in other patients, almost 2\u00a0years after index surgery (20\u00a0months).\nConclusions\nWe conclude that this study shows some evidence that this MCM graft may be a valid bone substitute for posterior or posterolateral spinal fusion under favorable conditions: presence of bone marrow cells, presence of healthy bleeding bone and mechanical stability (provided by the AO fixateur interne in these cases). A larger patient series and\/or randomized controlled studies are warranted to confirm these initial results.","keyphrases":["fusion","fracture","bone graft substitute","thoracolumbar spine"],"prmu":["P","P","P","P"]}
{"id":"Cancer_Causes_Control-3-1-2039842","title":"Dietary fat and risk of colon and rectal cancer with aberrant MLH1 expression, APC or KRAS genes\n","text":"Objective To investigate baseline fat intake and the risk of colon and rectal tumors lacking MLH1 (mutL homolog 1, colon cancer, nonpolyposis type 2) repair gene expression and harboring mutations in the APC (adenomatous polyposis coli) tumor suppressor gene and in the KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) oncogene.\nIntroduction\nAlthough dietary fat has been implicated in the etiology of colorectal cancer [1], results from epidemiological studies are inconsistent [2, 3] and often do not support an association, as observed recently in the Women\u2019s Health Study [4]. Fortunately, current molecular techniques to detect DNA alterations on a large scale allow studying molecular endpoints for colorectal cancer, characterized by acquired (epi) genetic defects in tumor DNA [5]. This approach may improve our ability to observe associations between dietary factors and cancer that may otherwise remain undetected.\nA multistep model linking sporadic colorectal carcinogenesis to molecular aberrations has been proposed [6\u20138], with DNA repair genes, tumor suppressor genes and oncogenes, operating in multiple genetic pathways. About 10\u201320% of sporadic colon carcinomas are characterized by microsatellite instability, predominantly due to promoter methylation of the MLH1 (mutL homolog 1, colon cancer, nonpolyposis type 2) DNA mismatch repair gene, which prevents expression of the enzyme [9]. Up to 90% of colon and rectum carcinomas are chromosomally instable [10, 11] and are associated with mutations in the APC (adenomatous polyposis coli) and TP53 (tumor protein 53) tumor suppressor genes and in the KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) oncogene [12]. However, simultaneous occurrence of mutations in these three genes is rare suggesting that, even within this group of chromosomally instable tumors, different genetic pathways to colorectal cancer exist [13, 14]. Mutations in the APC gene are found to occur relatively early in colorectal tumorigenesis and are observed in up to 80% of both adenomas and carcinomas [8, 15]. Mutations in the KRAS gene are observed in approximately 10\u201320% of small adenomas and 40\u201350% of larger adenomas and carcinomas, suggesting it to be an important event in the progression of adenoma to carcinoma [15]. Mutations in the TP53 gene are postulated to affect relatively late stages of colorectal carcinogenesis [15].\nBreivik et\u00a0al. proposed that the type of genetic instability in cancer cells reflects the selection pressures exerted by specific carcinogens [16]. Bardelli et al. subsequently tested this hypothesis in immortal genetically stable human cells and concluded that exposure to specific carcinogens can indeed select for tumor cells with distinct forms of genetic instability and vice versa [17]. Therefore, DNA adducts derived from dietary fat metabolism could also be associated with colorectal tumors exhibiting chromosomal instability. This is supported by the observations that malondialdehyde (MDA), generated during lipid peroxidation and arachidonic acid metabolism, can form DNA adducts and induce G\u2192T transversions and G\u2192A transitions in DNA [18, 19]. In addition, higher levels of MDA-DNA adducts have been observed in colorectal tissue of adenoma patients than in tissue of controls [20]. MDA levels are modulated by dietary factors, with polyunsaturated fatty acids, and specifically \u03c9-6 polyunsaturated fatty acids, presumably increasing MDA levels [21]. This is in line with our previous report of a significant association between the intake of linoleic acid, the most abundant \u03c9-6 polyunsaturated fatty acid in the diet, and increased risk of colon carcinomas with a mutated KRAS gene within the Netherlands Cohort Study (NLCS) on diet and cancer [22].\nThese observations and hypotheses prompted us to investigate the associations between the intake of total fat and different types of fat and the risk of colon and rectal tumors lacking MLH1 expression and with and without APC gene mutations, two early events in colorectal tumorigenesis, independent of tumors harboring KRAS gene mutations.\nMaterials and methods\nStudy population\nThe prospective NLCS was initiated in The Netherlands in September 1986. The study design has been described in detail elsewhere [23]. Briefly, at baseline a total of 58,279 men and 62,573 women, between the ages of 55\u00a0and 69\u00a0years, completed a self-administered food frequency and lifestyle questionnaire. Incident cancer cases are identified by monitoring of the entire cohort for cancer occurrence through annual record linkage to the National Cancer Registry (NCR), consisting of nine regional cancer registries throughout The Netherlands, and to PALGA, a nationwide network and registry of histo- and cytopathology [24]. The NCR and PALGA together provide a near 100% coverage of the 204 municipalities included in the NLCS.\nAccumulation of person-time in the cohort was estimated through biennial vital status follow-up of a subcohort of 3,500 men and women who were randomly selected after baseline exposure measurement [24]. Cases with prevalent cancer other than non-melanoma skin cancer were excluded from the subcohort, which left 3,346 men and women for analysis next to all colorectal cancer cases from the entire cohort. No subcohort members were lost to follow-up. A flow diagram of subcohort members and patients on whom the analyses are based is given in Fig.\u00a01.\nFig.\u00a01Flow diagram of the number of subjects on whom the final statistical analyses were based. aNetherlands Cancer Registry. bPathologisch Anatomisch Landelijk Geautomatiseerd Archief. cPatients with rectosigmoid tumors were not included in the analyses. dmutL homolog 1, colon cancer, nonpolyposis type 2. eAdenomatous polyposis coli. fMutation cluster region. gv-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog. hPatients with rectal tumors were not included in the analysis according to MLH1 expression\nThe first 2.3 years of follow up were excluded because of possible preclinical disease affecting exposure status and because of incomplete nationwide coverage of PALGA alone (i.e., not in combination with the NCR) in some of the municipalities included in the NLCS. Within this period, 83 subcohort members deceased or were diagnosed with cancer other than non-melanoma skin cancer, leaving 3,263 subcohort members for analysis. From 1989 to 1994, 929 incident cases with histologically confirmed colorectal cancer were identified within the entire cohort, of whom 819 could also be linked to a PALGA report of the lesion. The PALGA reports were used to identify and locate tumor tissues from eligible colorectal cancer patients in 54 pathology laboratories throughout the Netherlands. Cancers were classified according to site as follows, colon: cecum through sigmoid colon (ICD-O codes 153:0, 153.1, 153.2, 153.3, 153.4, 153.5, 153.6, 153.7), proximal colon (ICD-O codes 153.0, 153.1, 153.4, 153.5, 153.6), distal colon (ICD-O codes 153.2, 153,3, `53.7), rectosigmoid (ICD-O code 154.0), and rectum (ICD-O code 154.1).\nTissue samples\nApproval for collection of archival tissue samples from colorectal cancer patients was obtained from the Ethical Review Board of University Maastricht, the NCR and PALGA. The tissue specimen collection started in August 1999 and was completed in December of 2001. For five percent of patients, tissue samples could not be retrieved (44\/819) due to administrative inconsistencies. Of 775 available tissue samples, 737 (95%) contained sufficient tumor material for molecular analyses of MLH1 expression and mutations in APC and KRAS genes.\nSince the rectosigmoid can be considered as a clinically applied term rather than an anatomically defined transitional zone between the colon and rectum, the 85 patients with a rectosigmoid tumor were excluded from data analysis. Moreover, the group of patients with a rectosigmoid tumor was too small for adequate stratified analysis.\nMLH1 expression analysis\nFormalin-fixed, paraffin-embedded tissues were sectioned at 4\u00a0\u03bcm and contained tumor tissue and normal adjacent mucosa. Endogeneous peroxidase activity was blocked with 3% H2O2. Slides were submitted to microwave antigen retrieval in 1mM EDTA buffer (pH 8.0) and incubated with 10% normal horse serum for 10\u00a0min at room temperature. Then, sections were incubated overnight at 4\u00b0C with mouse monoclonal antibodies against MLH1 protein (clone G168\u201315, PharMingen, San Diego, CA) at a 1:100 dilution. Antibody binding was detected by incubating the sections at room temperature with the peroxidase-labeled DAKO Envision System (DAKO, Carpinteris, CA) and using DAB as a chromogen. Sections were counterstained with haematoxylin.\nLesions were considered to lack MLH1 protein expression when unequivocal absence of nuclear staining of the tumor epithelial cells was observed. Nuclear staining of normal epithelial and stromal cells or lymphocytes served as internal positive control. Two investigators reviewed the immunohistochemical staining independently and discrepancies were re-examined and discussed with a pathologist until consensus was reached. MLH1 expression status was determined successfully in 98% of samples, i.e., 468 colon tumors and 173 rectum tumors (Fig.\u00a01).\nAPC mutation analysis\nThe majority of somatic mutations in APC occur within the mutation cluster region. Mutation analysis of the mutation cluster region (codons 1,286\u20131,520), was performed on archival adenocarcinoma specimens, using macrodissection followed by extraction of tumor DNA. Then nested PCR was used to amplify the mutation cluster region in four overlapping DNA fragments and the purified fragments were sequenced. This procedure has been described in detail elsewhere [25]. In brief, in a first round of PCR, two overlapping fragments were generated, that served as templates for a second round of PCR to amplify four overlapping biotin-labeled PCR fragments that were subsequently used for direct sequencing. The sequence profile was analyzed on ALFexpress DNA Analysis System using ALFwin software (Amersham Biosciences, Roosendaal, The Netherlands). Evaluation of the sequence patterns and data entry were independently performed by two observers. Sensitivity and specificity was assessed by analyzing the mutational status of APC in six colorectal cancer cell lines. Both sensitivity and specificity were regarded to be satisfactory since specific mutations in the mutation cluster region of APC were confirmed in CaCo2 cells, SW480 cells and LOVO cells, as previously described [25, 26], and wild type sequences were confirmed in HCT116, Colo205 and HT29, for the mutation cluster region of APC [25]. In addition, the detection limit was 5% of mutated DNA [25]. Reproducibility of mutation analysis was regarded to be satisfactory since 85% of duplicate analyses, from flank PCR of genomic DNA to sequencing of the four fragments (i.e., 61 out of 72 fragments), revealed identical mutation status of APC [25].\nFrom 47 colon cancer patients and 25 rectum cancer patients, one or more fragments of the APC gene mutation cluster region could not be amplified and these patients were not included in this study, leaving 429 colon and 151 rectum cancer cases with successful analysis of the mutation cluster region of the APC gene (Fig.\u00a01).\nKRAS mutation analysis\nMutation analysis of the exon1 fragment of the KRAS oncogene, spanning codons 8\u201329, was performed on archival adenocarcinoma specimens, using nested PCR, followed by direct sequencing of purified fragments [27]. The detection limit was 5% mutated DNA. Reproducibility was regarded to be satisfactory, since 88% of duplicate analyses, from tissue sectioning to DNA sequencing (i.e., 28 out of 32), revealed identical mutation status of KRAS [27]. Mutation analysis was performed with success on 476 colon and 176 rectum cancer cases (Fig.\u00a01).\nExposure assessment\nThe 150\u2013item semi-quantitative food frequency questionnaire concentrated on habitual consumption of food and beverages during the year preceding the start of the study. Mean individual nutrient intakes per day were computed using the computerized Dutch food composition table of 1986 [28]. The questionnaire was validated against a 9-day diet record [29]. Crude and energy-gender-adjusted (in parentheses) correlation coefficients were 0.72 (0.52) for total fat, 0.73 (0.58) for saturated fat and 0.73 (0.75) for polyunsaturated fat [29]. For energy intake the correlation coefficient was 0.74. On average, the questionnaire covered 91% of the energy intake assessed by the record intake. Questionnaire data were key-entered twice and processed for all incident cases in the cohort and for all subcohort members in a manner blinded with respect to case\/subcohort status.\nFor 7% of subjects (either cases or subcohort members), dietary data were incomplete or inconsistent, and they were excluded from the analyses. Questionnaires were considered incomplete when either: (1) more than 60 items were left blank and less than 35 items were eaten at least once a month; or (2) one or more item blocks (groups of items, e.g., beverages) were left blank. Additional details are given elsewhere [29]. This resulted in the availability of 3,048 subcohort members, 441 colon cancer cases for whom MLH1 expression status was known, and 414 colon and 136 rectal cancer cases for whom APC and KRAS mutation status was known. No data-analyses were conducted for lack of MLH1 expression in rectal cancer cases since there were only two such cases in the cohort (Fig.\u00a01).\nIntake of specific fatty acids was based on a food composition database with specific fatty acids derived from the TRANSFAIR study [30]. For this database, the hundred foods that contributed most to fat intake in the Dutch dietary pattern were sampled and analyzed as methyl esters of the fatty acids present in the foods. In the database, total fat includes triglycerides and other lipids such as phospholipids and sterols. The percentage of triglycerides in total fat is assumed to be 93% on average, but varies across food sources. Daily intakes of total fat (g\/day), saturated fat (g\/day), monounsaturated fat (g\/day), polyunsaturated fat (g\/day), and linoleic acid (C18:2, C9, 12) (g\/day) and linolenic acid (C18:3, C9, 12, 15) (g\/day) as the main constituents of polyunsaturated fat, were used as exposure variables. Linoleic and linolenic acid were used as the most abundant sources of \u03c9-6 polyunsaturated fatty acids and \u03c9-3 polyunsaturated fatty acids in the diet. In all analyses, the values for fat intake variables are adjusted for energy intake by the residual method [31]. For data analyses, quartiles of the intake of fat and different types of fats were computed based on the distribution of subcohort members. Daily intake of dietary fiber (g\/day), alcohol (g\/day), fruit (g\/day), vegetables (g\/day) and total energy (kJ\/day) and age at baseline (years), sex (men\/women), body mass index (kg\/m2), non-occupational physical activity (<30\u00a0min\/day, 30\u201360 min\/day, 60\u201390\u00a0min\/day,\u00a0>90\u00a0min\/day), family history of colorectal cancer (yes\/no) and smoking status (never\/ex\/current) were regarded as potential confounders.\nStatistical analysis\nData analyses were based on study participants for whom data on fat intake and confounding variables were complete, i.e., 2,948 subcohort members, 428 colon cancer patients for whom MLH1 expression status was known, and 401 colon cancer and 130 rectal cancer patients for whom APC and KRAS mutation status was known (Fig.\u00a01).\nData analyses were conducted separately for overall colon and rectal cancer, colon cancer lacking MLH1 expression, colon and rectal cancer with or without a truncating APC mutation, described here as APC+ and APC\u2212 tumors respectively. Truncating APC mutations lead to the introduction of a stop codon and result in a truncated and therefore, inactive APC protein. The analyses with truncating APC mutations were also conducted separately for the most common point mutations resulting in the introduction of a stop codon, i.e., C:G \u2192 T:A or G:C \u2192 T:A point mutations. As indicated previously, associations between fat intake and KRAS mutated tumors have been described in this population previously, and a positive association between the intake of linoleic acid and KRAS mutated colon tumors was observed [22]. Therefore, when (borderline) significant associations were observed with any of the colon tumor endpoints, analyses were repeated excluding tumors harboring mutations in KRAS.\nSince tumors may harbor multiple mutations it is difficult to assess whether observed associations are specific for tumors with a particular gene defect. We therefore, conducted additional analyses when (borderline) significant associations were observed. In these analyses subgroups of tumors were formed characterized by either the absence of the three gene defects, or by defects in a single gene, i.e., either only lack of MLH1 expression, only a truncating APC mutation or only an activating KRAS mutations. Activating KRAS mutations are defined as mutations in codons 12 and 13 of the KRAS gene leading to an altered amino acid.\nMean values of the intake of fat variables (g\/day), and possible confounding variables including age at baseline (years), dietary fiber (g\/day), alcohol (g\/day), intake of fruit (g\/day), vegetable (g\/day), energy (kJ\/day), and BMI (kg\/m2), as well as distributions of the variables sex, family history of colorectal cancer (yes\/no), smoking status (never\/ex\/current smoker) and physical activity in leisure time (<30, 30\u201360, 60\u201390,\u00a0>90\u00a0min\/day) were evaluated for subcohort members, colon and rectal cancer patients with or without a truncating APC mutation and colon cancer patients lacking MLH1 expression. Differences in mean values of the continuous variables between patients with or without truncating nonsense or frameshift mutations in the mutation cluster region of the APC gene, and between patients with or without MLH1 expression, were tested using the Mann\u2013Whitney-U-test since the variables were not normally distributed among cases. The distributions of the categorical variables between patients with and without truncating APC mutations were tested with the \u03c72\u2013test.\nIncidence rate ratios (RR) and corresponding 95% confidence intervals (CI) for colon and rectal cancer patients were estimated according to quartiles of intake of fat variables, and one standard deviation increment of intake, using Cox proportional hazards regression models. In addition, associations were estimated for specific molecular endpoints of the tumors.\nStandard errors were estimated using the robust Huber\u2013White sandwich estimator to account for additional variance introduced by sampling the subcohort from the entire cohort [32, 33]. The proportional hazards assumption was tested using the scaled Schoenfeld residuals [34]. Tests for dose response trends over the different quartiles and categories of fat intake were estimated by fitting the ordinal exposure variables as continuous variables and evaluated using the Wald test.\nThe covariates included in the multivariate analyses were those found to significantly (p\u00a0<\u00a00.05) contribute to the multivariate model for colon and\/or rectal cancer (age at baseline, sex, body mass index, family history of colorectal cancer, and smoking status) or to influence the RR by more than ten percent, as well as energy intake.\nResults\nLack of expression in MLH1 was observed in 13% (54 out of 428) of tumors from colon cancer patients (Table\u00a01). APC truncating mutations were observed in tumors from 32% of colon cancer patients (127 out of 401) and 44% of rectal cancer patients (57 out of 130) (Table\u00a01). C:G\u2192T:A transitions or G:C\u2192T:A transversions that would result in a stop codon were observed in 10% and 5% of colon cancer patients and 12% and 5% of rectal cancer patients, respectively. These figures are similar to the percentages reported for the total group of colon and rectal cancer patients for whom APC mutation status was available, but for whom dietary intake data were not always complete [25].\nTable\u00a01Baseline dietary intake and other characteristics of the subcohort and colon and rectum cancer patients from The Netherlands cohort studySubcohortColon cancer (n\u00a0=\u00a0428)p-valuebColon cancer (n\u00a0=\u00a0401)p-valuebRectal cancer (n\u00a0=\u00a0130)p-valuebMLH1 expressionaNo MLH1 expressionaAPC\u00a0\u2212\u00a0cAPC\u00a0+cAPC\u00a0\u2212cAPC\u00a0 +cN2,948374542741277357\u00a0\u00a0\u00a0\u00a0Sex (%men)4856410.0452560.4964670.79\u00a0\u00a0\u00a0\u00a0Age (years)61.3\u00a0\u00b1\u00a04.263.0\u00a0\u00b1\u00a04.062.8\u00a0\u00b1\u00a04.50.9163.1\u00a0\u00b1\u00a04.062.7\u00a0\u00b1\u00a04.00.3462.2\u00a0\u00b1\u00a04.462.6\u00a0\u00b1\u00a03.50.63Fat variables (g\/day)d\u00a0\u00a0\u00a0\u00a0Total fat83.8\u00a0\u00b1\u00a015.885.1\u00a0\u00b1\u00a014.784.1\u00a0\u00b1\u00a017.50.5785.2\u00a0\u00b1\u00a015.084.8\u00a0\u00b1\u00a014.90.8085.1\u00a0\u00b1\u00a014.286.7 \u00b1\u00a014.80.52\u00a0\u00a0\u00a0\u00a0Saturated fat33.2\u00a0\u00b1\u00a07.533.5\u00a0\u00b1\u00a06.633.3\u00a0\u00b1\u00a07.60.6433.3\u00a0\u00b1\u00a06.733.5\u00a0\u00b1\u00a07.00.9233.0\u00a0\u00b1\u00a05.835.5\u00a0\u00b1\u00a07.70.05\u00a0\u00a0\u00a0\u00a0MUFAe31.4\u00a0\u00b1\u00a07.031.9\u00a0\u00b1\u00a06.531.1\u00a0\u00b1\u00a07.50.4531.8\u00a0\u00b1\u00a06.631.9\u00a0\u00b1\u00a06.60.9831.8\u00a0\u00b1\u00a06.132.8\u00a0\u00b1\u00a06.20.37\u00a0\u00a0\u00a0\u00a0PUFAf17.3\u00a0\u00b1\u00a07.517.9\u00a0\u00b1\u00a07.517.4\u00a0\u00b1\u00a06.80.8518.2\u00a0\u00b1\u00a07.317.8\u00a0\u00b1\u00a07.60.4418.6\u00a0\u00b1\u00a08.716.5 \u00b1\u00a06.90.27\u00a0\u00a0\u00a0\u00a0Linoleic acid16.0\u00a0\u00b1\u00a07.516.7\u00a0\u00b1\u00a07.516.6\u00a0\u00b1\u00a07.00.7617.0\u00a0\u00b1\u00a07.416.6\u00a0\u00b1\u00a07.70.3617.5\u00a0\u00b1\u00a08.815.2 \u00b1\u00a07.20.15\u00a0\u00a0\u00a0\u00a0Linolenic acid1.3\u00a0\u00b1\u00a00.61.2\u00a0\u00b1\u00a00.51.3\u00a0\u00b1\u00a00.60.981.2\u00a0\u00b1\u00a00.51.3\u00a0\u00b1\u00a00.50.571.3\u00a0\u00b1\u00a00.61.3\u00a0\u00b1\u00a00.60.79Other dietary factors\u00a0\u00a0\u00a0\u00a0Fibre (g\/day)27.0\u00a0\u00b1\u00a08.224.5\u00a0\u00b1\u00a08.125.2\u00a0\u00b1\u00a07.10.1326.7\u00a0\u00b1\u00a07.927.9\u00a0\u00b1\u00a08.10.2028.1\u00a0\u00b1\u00a07.028.2\u00a0\u00b1\u00a09.00.78\u00a0\u00a0\u00a0\u00a0Alcohol (g\/day)g10.1\u00a0\u00b1\u00a014.111.0\u00a0\u00b1\u00a014.810.7\u00a0\u00b1\u00a017.00.4911.0\u00a0\u00b1\u00a014.911.3\u00a0\u00b1\u00a016.50.8912.2\u00a0\u00b1\u00a014.914.5 \u00b1\u00a018.10.53\u00a0\u00a0\u00a0\u00a0Fruit (g\/day)177.0\u00a0\u00b1\u00a0118.0178.8\u00a0\u00b1\u00a0122.1160.5\u00a0\u00b1\u00a0137.70.07169.7\u00a0\u00b1\u00a0121.9187.5\u00a0\u00b1\u00a0132.20.11197.4\u00a0\u00b1\u00a0155.1205.0 \u00b1\u00a0118.30.21\u00a0\u00a0\u00a0\u00a0Vegetables (g\/day)193.8\u00a0\u00b1\u00a082.2191.3\u00a0\u00b1\u00a082.3185.4\u00a0\u00b1\u00a073.80.80187.9\u00a0\u00b1\u00a080.7192.7\u00a0\u00b1\u00a085.20.71186.3\u00a0\u00b1\u00a068.5169.0 \u00b1\u00a0122.20.69\u00a0\u00a0\u00a0\u00a0Energy (kj\/day)8,028\u00a0\u00b1\u00a02,1648,080\u00a0\u00b1\u00a02,0597,505\u00a0\u00b1\u00a01,7180.077,845\u00a0\u00b1\u00a01,8998,335\u00a0\u00b1\u00a02,3060.098,433\u00a0\u00b1\u00a01,9248,449 \u00b1\u00a01,6160.92Other characteristics\u00a0\u00a0\u00a0\u00a0BMIh (kg\/m2)25.1\u00a0\u00b1\u00a03.125.6\u00a0\u00b1\u00a03.225.6\u00a0\u00b1\u00a03.50.5725.5\u00a0\u00b1\u00a03.2825.8\u00a0\u00b1\u00a03.10.3025.3\u00a0\u00b1\u00a03.125.1 \u00b1\u00a02.80.92\u00a0\u00a0\u00a0\u00a0Family history of CRCi (% yes)61390.4911110.9810110.86Smoker (%)\u00a0\u00a0\u00a0\u00a0Never37373336382633\u00a0\u00a0\u00a0\u00a0Ex-smoker35453746434739\u00a0\u00a0\u00a0\u00a0Current smoker2818300.1218190.8827280.59Physical activity (%)g\u00a0\u00a0\u00a0\u00a0<30 min\/day21212219261726\u00a0\u00a0\u00a0\u00a030-60 min\/day32332233282832\u00a0\u00a0\u00a0\u00a060-90 min\/day21211922182418\u00a0\u00a0\u00a0\u00a0>90 min\/day2725370.2026270.3132250.43aFor rectal cancer there were only two patients without MLH1 expression, these are not shown separately in this tablebp-value for the difference between cancer patients with and without MLH1 expression and colon and rectal cancer patients with and without a mutation leading to the introduction of a stop codon in APCcAPC \u2212: cancer patients without a mutation in the MCR of the APC gene leading to a stop codon; APC\u00a0+\u00a0: cancer patients with a mutation in the MCR of the APC gene leading to a stop codondAdjusted for energy intakeeMonounsaturated fatfPolyunsaturated fatgFor alcohol intake and physical activity the mean levels in the subcohort are based on 2,862 and 2,915 subjects respectively. Four and six colon cancer cases had missing values for alcohol intake and physical activity respectively. Two and one rectal cancer case had missing values for alcohol intake and physical activity respectivelyhBMI: body mass indexiCRC: colorectal cancer\nColon and rectal cancer patients were generally older and more frequently men than subcohort members (Table\u00a01). Colon cancer patients lacking MLH1 expression in their tumor were significantly less often men than patients with expression of the gene (41% vs. 56%). There were no striking differences in fat intake between patients and subcohort members or between patients with or without MLH1 expression or APC mutations in their tumors. Only rectal cancer patients with a tumor harboring a truncating APC mutation had a higher intake of saturated fat than rectal cancer patients without a truncating APC mutation (p\u00a0=\u00a00.05).\nNeither total fat nor different types of fat appeared to be associated with overall colon cancer risk in this population (Table\u00a02). For different subgroups of colon cancer based on absence of MLH1 expression or absence or presence of APC truncating mutations in their tumors, total fat intake and most of the specific types of fat intake variables were also not associated with risk. However, polyunsaturated fat intake, and especially linoleic acid intake, appeared to be associated with an increased risk of colon tumors without MLH1 expression and with colon tumors without APC truncating mutations, but not with colon tumors with APC truncating mutations (Table\u00a02). For colon tumors without MLH1 expression, the RR according to the quartiles of linoleic acid intake were increased, though not significantly, for all the categories of intake above the reference (lowest quartile of intake), i.e., 1.66 (95% CI 0.69\u20133.98), 2.14 (95% CI 0.91\u20135.00) and 2.02 (95% CI 0.86\u20134.76) for the second through the fourth quartiles respectively, and the test for linear trend was borderline significant (p\u00a0=\u00a00.08). A similar trend was observed for the risk of colon tumors without APC truncating mutations (RR over the quartiles of linoleic acid intake: 1.50 (95% CI 1.02\u20132.21), 1.68 (95% CI 1.15\u20132.45) and 1.44 (95% CI 0.99\u20132.11) respectively, p-trend\u00a0=\u00a00.05 (Table\u00a02). Additional analyses for subgroups of colon tumors with specific truncating point mutations in APC did not show any associations with the intake of fat or different types of fat (results not shown).\nTable\u00a02Adjusted incidence rate ratios and 95% confidence intervals for colon cancer patients overall, without MLH1 expression, and with and without an APC mutation leading to a stop codon, according to the intake of fat variables (The Netherlands Cohort Study)Dietary fat intakeMedian intake (g\/day)Person yearsbColon cancerOverallNo MLH1 expressionAPC \u2013aAPC +aMenWomenNumber of patientsRRc(95% CI)cNumber of patientsRRc(95% CI)cNumber of patientsRRc(95% CI)cNumber of patientsRRc(95% CI)cTotal fat\u00a0\u00a0\u00a0\u00a0Q178.063.03,5491101.00(reference)111.00(reference)611.00(reference)401.00(reference)\u00a0\u00a0\u00a0\u00a0Q290.271.43,5561251.09(0.82\u20131.44)171.47(0.69\u20133.14)871.34(0.94\u20131.90)320.80(0.49\u20131.28)\u00a0\u00a0\u00a0\u00a0Q398.577.63,579890.75(0.55\u20131.01)110.94(0.40\u20132.17)550.82(0.56\u20131.21)240.57(0.33\u20130.96)\u00a0\u00a0\u00a0\u00a0Q4108.885.33,5881100.96(0.72\u20131.28)151.30(0.59\u20132.86)711.11(0.77\u20131.59)310.74(0.46\u20131.20)\u00a0\u00a0\u00a0\u00a0p-valued0.290.820.710.13\u00a0\u00a0\u00a0\u00a01SD incremente1.00(0.88\u20131.13)1.21(0.82\u20131.77)1.04(0.89\u20131.22)0.93(0.76\u20131.14)Saturated fat\u00a0\u00a0\u00a0\u00a0Q128.923.93,567991.00(reference)91.00(reference)591.00(reference)331.00(reference)\u00a0\u00a0\u00a0\u00a0Q233.727.83,5501201.20(0.89\u20131.61)141.48(0.63\u20133.49)841.38(0.97\u20131.97)300.94(0.55\u20131.61)\u00a0\u00a0\u00a0\u00a0Q338.330.93,5831151.11(0.82\u20131.49)191.93(0.86\u20134.32)731.15(0.79\u20131.66)331.01(0.60\u20131.68)\u00a0\u00a0\u00a0\u00a0Q445.836.63,5711000.94(0.69\u20131.27)121.25(0.52\u20133.00)580.90(0.62\u20131.32)310.89(0.53\u20131.48)\u00a0\u00a0\u00a0\u00a0p-valued0.540.480.350.72\u00a0\u00a0\u00a0\u00a01SD incremente0.97(0.87\u20131.08)1.12(0.82\u20131.51)0.95(0.83\u20131.09)0.95(0.79\u20131.14)MUFAf\u00a0\u00a0\u00a0\u00a0Q128.222.43,546981.00(reference)111.00(reference)561.00(reference)311.00(reference)\u00a0\u00a0\u00a0\u00a0Q233.226.03,5461221.20(0.89\u20131.61)141.15(0.52\u20132.53)791.33(0.92\u20131.91)361.18(0.72\u20131.95)\u00a0\u00a0\u00a0\u00a0Q336.928.93,5801131.12(0.83\u20131.52)141.15(0.52\u20132.54)761.30(0.89\u20131.89)311.03(0.60\u20131.77)\u00a0\u00a0\u00a0\u00a0Q442.533.13,6001010.99(0.73\u20131.34)151.25(0.58\u20132.71)631.08(0.74\u20131.57)290.90(0.54\u20131.52)\u00a0\u00a0\u00a0\u00a0p-valued0.790.590.800.57\u00a0\u00a0\u00a0\u00a01SD incremente0.99(0.88\u20131.12)1.04(0.73\u20131.46)1.01(0.87\u20131.17)0.98(0.81\u20131.18)PUFAg\u00a0\u00a0\u00a0\u00a0Q111.68.83,507911.00(reference)81.00(reference)491.00(reference)321.00(reference)\u00a0\u00a0\u00a0\u00a0Q216.012.43,5621181.37(1.02\u20131.86)162.03(0.86\u20134.81)731.55(1.06\u20132.28)331.13(0.68\u20131.89)\u00a0\u00a0\u00a0\u00a0Q320.916.23,6181131.24(0.91\u20131.68)162.00(0.84\u20134.76)781.57(1.07\u20132.31)300.96(0.57\u20131.62)\u00a0\u00a0\u00a0\u00a0Q429.322.53,5801121.21(0.89\u20131.63)141.75(0.72\u20134.24)741.47(1.01\u20132.16)320.98(0.59\u20131.63)\u00a0\u00a0\u00a0\u00a0p-valued0.380.260.060.79\u00a0\u00a0\u00a0\u00a01SD incremente1.03(0.94\u20131.14)1.07(0.82\u20131.38)1.09(0.97\u20131.23)1.00(0.84\u20131.19)Linoleic acid\u00a0\u00a0\u00a0\u00a0Q110.07.53,509861.00(reference)81.00(reference)491.00(reference)261.00(reference)\u00a0\u00a0\u00a0\u00a0Q214.811.23,5861221.49(1.10\u20132.02)131.66(0.69\u20133.98)711.50(1.02\u20132.21)401.65(0.99\u20132.76)\u00a0\u00a0\u00a0\u00a0Q319.514.93,5991121.32(0.97\u20131.79)172.14(0.91\u20135.00)821.68(1.15\u20132.45)251.00(0.57\u20131.76)\u00a0\u00a0\u00a0\u00a0Q428.021.23,5741141.30(0.96\u20131.77)162.02(0.86\u20134.76)721.44(0.99\u20132.11)361.35(0.80\u20132.28)\u00a0\u00a0\u00a0\u00a0p-valued0.200.080.050.65\u00a0\u00a0\u00a0\u00a01SD incremente1.06(0.96\u20131.17)1.15(0.90\u20131.48)1.12(0.99\u20131.26)1.02(0.86\u20131.21)Linolenic acid\u00a0\u00a0\u00a0\u00a0Q10.80.63,5181071.00(reference)111.00(reference)681.00(reference)311.00(reference)\u00a0\u00a0\u00a0\u00a0Q21.20.93,5741040.95(0.70\u20131.30)171.32(0.60\u20132.89)720.99(0.68\u20131.43)240.84(0.47\u20131.49)\u00a0\u00a0\u00a0\u00a0Q31.51.23,5711171.10(0.82\u20131.48)110.91(0.40\u20132.10)620.89(0.62\u20131.29)431.48(0.91\u20132.40)\u00a0\u00a0\u00a0\u00a0Q42.01.63,6041061.01(0.76\u20131.36)151.32(0.61\u20132.84)721.08(0.76\u20131.53)290.97(0.57\u20131.65)\u00a0\u00a0\u00a0\u00a0p-valued0.680.730.820.52\u00a0\u00a0\u00a0\u00a01SD incremente0.98(0.89\u20131.09)1.07(0.79\u20131.44)0.97(0.85\u20131.10)1.01(0.86\u20131.19)aAPC \u2212: cancer patients without a mutation in the MCR of the APC gene leading to a stop codon; APC +: cancer patients with a mutation in the MCR of the APC gene leading to a stop codonbPerson years at risk are estimated from the subcohortcIncidence rate ratios (RR) and 95% confidence intervals (95% CI) are adjusted for age, sex, body mass index, smoking, energy intake and family history of colorectal cancerdp-value for trend over the quartiles of intake of fat variableseFor 1 standard deviation of intake of fat in the subcohort (see Table\u00a01); i.e., 15.8\u00a0g\/day for total fat, 7.5\u00a0g\/day for saturated fat, 7.0\u00a0g\/day for monounsaturated fat, 7.5\u00a0g\/day for polyunsaturated fat, 7.5\u00a0g\/day for linoleic acid and 0.6\u00a0g\/day for linolenic acidfMonounsaturated fatgPolyunsaturated fat\nFor overall rectal cancer, associations with the intake of total fat or different types of fat were not observed (Table\u00a03). Also after taking account of truncating APC mutations in rectal tumors, none of the fat intake variables were significantly associated with risk of rectal cancer. Only the intake of saturated fat appeared to be inversely associated with rectal tumors without APC truncating mutations (RR for the highest versus the lowest quartile of intake: 0.46 (95% CI 0.22\u20130.97), p-trend\u00a0=\u00a00.07) (Table\u00a03). For rectal tumors with specific types of APC truncating mutations no associations were observed with any of the fat intake variables (results not shown).\nTable\u00a03Adjusted incidence rate ratios and 95% confidence intervals for rectal cancer patients overall, and with and without an APC mutation leading to a stop codon, according to the intake of fat variables. (The Netherlands Cohort Study)Dietary fat intakeMedian intake (g\/day)Person yearsbRectal cancerOverallAPC \u2212aAPC +aMenWomenNumber of patientsRRc(95% CI)cNumber of patientsRRc(95% CI)cNumber of patientsRRc(95% CI)cTotal fat\u00a0\u00a0\u00a0\u00a0Q178.063.03,549431.00(reference)241.00(reference)151.00(reference)\u00a0\u00a0\u00a0\u00a0Q290.271.43,556390.89(0.56\u20131.39)170.70(0.37\u20131.34)120.79(0.36\u20131.70)\u00a0\u00a0\u00a0\u00a0Q398.577.63,579330.74(0.46\u20131.19)160.65(0.33\u20131.26)140.91(0.43\u20131.90)\u00a0\u00a0\u00a0\u00a0Q4108.885.33,588380.87(0.55\u20131.36)150.60(0.31\u20131.16)161.06(0.51\u20132.19)\u00a0\u00a0\u00a0\u00a0p-valued0.420.130.80\u00a0\u00a0\u00a0\u00a01SD incremente0.91(0.76\u20131.09)0.84(0.65\u20131.08)0.94(0.69\u20131.28)Saturated fat\u00a0\u00a0\u00a0\u00a0Q128.923.93,567431.00(reference)231.00(reference)141.00(reference)\u00a0\u00a0\u00a0\u00a0Q233.727.83,550340.80(0.49\u20131.29)180.81(0.42\u20131.56)120.87(0.39\u20131.91)\u00a0\u00a0\u00a0\u00a0Q338.330.93,583441.02(0.66\u20131.59)210.92(0.49\u20131.73)120.86(0.40\u20131.88)\u00a0\u00a0\u00a0\u00a0Q445.836.63,571330.74(0.46\u20131.18)110.46(0.22\u20130.97)191.30(0.65\u20132.63)\u00a0\u00a0\u00a0\u00a0p-valued0.380.070.47\u00a0\u00a0\u00a0\u00a01SD incremente0.95(0.81\u20131.12)0.81(0.65\u20131.02)1.16(0.89\u20131.50)MUFAf\u00a0\u00a0\u00a0\u00a0Q128.222.43,546441.00(reference)231.00(reference)151.00(reference)\u00a0\u00a0\u00a0\u00a0Q233.226.03,546310.70(0.44\u20131.13)180.79(0.41\u20131.49)90.62(0.27\u20131.43)\u00a0\u00a0\u00a0\u00a0Q336.928.93,580400.91(0.58\u20131.44)150.67(0.34\u20131.33)201.36(0.67\u20132.78)\u00a0\u00a0\u00a0\u00a0Q442.533.13,600390.88(0.56\u20131.37)170.72(0.38\u20131.36)130.88(0.40\u20131.90)\u00a0\u00a0\u00a0\u00a0p-valued0.820.280.76\u00a0\u00a0\u00a0\u00a01SD incremente0.94(0.78\u20131.12)0.85(0.67\u20131.08)1.00(0.76\u20131.33)PUFAg\u00a0\u00a0\u00a0\u00a0Q111.68.83,507451.00(reference)171.00(reference)211.00(reference)\u00a0\u00a0\u00a0\u00a0Q216.012.43,562320.74(0.46\u20131.18)191.17(0.60\u20132.28)100.49(0.23\u20131.07)\u00a0\u00a0\u00a0\u00a0Q320.916.23,618390.86(0.55\u20131.34)160.94(0.47\u20131.88)160.76(0.39\u20131.48)\u00a0\u00a0\u00a0\u00a0Q429.322.53,580380.82(0.53\u20131.29)211.20(0.63\u20132.29)100.47(0.22\u20131.00)\u00a0\u00a0\u00a0\u00a0p-valued0.530.730.11\u00a0\u00a0\u00a0\u00a01SD incremente0.98(0.83\u20131.16)1.06(0.84\u20131.34)0.78(0.60\u20131.03)Linoleic acid\u00a0\u00a0\u00a0\u00a0Q110.07.53,509391.00(reference)181.00(reference)181.00(reference)\u00a0\u00a0\u00a0\u00a0Q214.811.23,586391.03(0.65\u20131.64)170.98(0.50\u20131.92)140.80(0.40\u20131.63)\u00a0\u00a0\u00a0\u00a0Q319.514.93,599350.90(0.56\u20131.44)160.90(0.45\u20131.79)150.84(0.42\u20131.68)\u00a0\u00a0\u00a0\u00a0Q428.021.23,574411.03(0.65\u20131.62)221.19(0.63\u20132.24)100.54(0.25\u20131.18)\u00a0\u00a0\u00a0\u00a0p-valued0.950.650.15\u00a0\u00a0\u00a0\u00a01SD incremente0.99(0.84\u20131.17)1.09(0.86\u20131.38)0.79(0.60\u20131.04)Linolenic acid\u00a0\u00a0\u00a0\u00a0Q10.80.63,518421.00(reference)181.00(reference)181.00(reference)\u00a0\u00a0\u00a0\u00a0Q21.20.93,574380.92(0.57\u20131.48)201.16(0.58\u20132.31)100.55(0.25\u20131.24)\u00a0\u00a0\u00a0\u00a0Q31.51.23,571360.87(0.55\u20131.38)150.87(0.43\u20131.75)130.72(0.35\u20131.48)\u00a0\u00a0\u00a0\u00a0Q42.01.63604380.92(0.58\u20131.44)201.13(0.60\u20132.14)160.90(0.45\u20131.80)\u00a0\u00a0\u00a0\u00a0p-valued0.680.930.91\u00a0\u00a0\u00a0\u00a01SD incremente0.95(0.81\u20131.12)0.98(0.77\u20131.24)1.03(0.81\u20131.31)aAPC \u2212: cancer patients without a mutation in the MCR of the APC gene leading to a stop codon; APC +: cancer patients with a mutation in the MCR of the APC gene leading to a stop codonbPerson years at risk are estimated from the subcohortcIncidence rate ratios (RR) and 95% confidence intervals (95% CI) are adjusted for age, sex, body mass index, smoking, energy intake and family history of colorectal cancerdp-value for trend over the quartiles of intake of fat variableseFor 1 standard deviation of intake of fat in the subcohort (see Table\u00a01); i.e., 15.8\u00a0g\/day for total fat, 7.5\u00a0g\/day for saturated fat, 7.0\u00a0g\/day for monounsaturated fat, 7.5\u00a0g\/day for polyunsaturated fat, 7.5\u00a0g\/day for linoleic acid and 0.6\u00a0g\/day for linolenic acidfMonounsaturated fatgPolyunsaturated fat\nAdditional analyses were conducted to assess whether the observed associations of polyunsaturated fat intake, and especially linoleic acid intake, with the increased risk of colon tumors lacking MLH1 expression and with the increased risk of colon tumors without APC truncating mutations, were observed because of an underlying association with colon tumors harboring a KRAS mutation, as previously observed [22]. Excluding tumors with a KRAS mutation resulted in the absence of a statistically significant association of polyunsaturated fat intake and linoleic acid intake with colon tumors lacking MLH1 expression (p-trend\u00a0=\u00a00.34 and 0.12, respectively) and those lacking APC tuncating mutations (p-trend\u00a0=\u00a00.77 and 0.99, respectively). Intake of polyunsaturated fat or linoleic acid was neither associated with the risk of colon cancer without any of the three gene defects, nor with the risk of colon cancer only lacking MLH1 expression, nor with the risk of colon cancer with only truncating APC mutations (Table\u00a04). With increasing intake of polyunsaturated fat and of linoleic acid, a strongly increased risk of colon cancer with only activating KRAS mutations was observed (Table\u00a04) (p-trend\u00a0\u2264\u00a00.001 for both polyunsaturated fat and linoleic acid intake). The RRs for one standard deviation increase in intake were 1.40 (95% CI 1.17\u20131.68) and 1.41 (95% CI 1.18\u20131.69), respectively. The RRs for polyunsaturated fat (not shown) and linoleic acid intake were of similar size when estimated separately for men (1.41, 95% CI 1.15\u20131.72 for 1 standard deviation increase in linoleic acid intake) and women (1.42, 95% CI 0.96\u20132.10), and were elevated for proximal (1.23, 95% CI 0.99\u20131.53) and distal colon cancer (1.53, 95% CI 1.21\u20131.95). Likewise, a positive association was observed for all colorectal cancers (1.24, 95% CI 1.06\u20131.47 and p-trend\u00a0=\u00a00.01), based on a total of 87 cases (i.e., including the rectosigmoid).\nTable\u00a04Adjusted incidence rate ratios and 95% confidence intervals for colon cancer patients without any of the gene defects or with only a single gene defect, i.e., either lack of MLH1 expression, a truncating APC gene mutation or an activating KRAS gene mutation, according to the intake of polyunsaturated fat and linoleic acid intake (The Netherlands Cohort Study)Dietary fat intakePerson yearseColon cancerNo gene defectsaOnly lack of MLH1 expressionbOnly truncating APC mutationscOnly activating KRAS mutationsdNumber of patientsRRf(95% CI)fNumber of patientsRRf(95% CI)fNumber of patientsRRf(95% CI)fNumber of patientsRRf(95% CI)fPUFAgQ13,507391.00(reference)61.00(reference)181.00(reference)41.00(reference)Q23,562441.15(0.73\u20131.81)142.48(0.94\u20136.54)211.31(0.68\u20132.53)143.76(1.21\u201311.69)Q33,618451.10(0.70\u20131.73)111.89(0.69\u20135.16)150.86(0.43\u20131.74)195.00(1.67\u201314.99)Q43,580350.86(0.54\u20131.38)101.72(0.61\u20134.81)130.70(0.34\u20131.45)286.74(2.36\u201319.51)p-valueh0.510.480.19\u22640.0011SD incrementi0.96(0.82\u20131.13)1.01(0.72\u20131.40)0.90(0.71\u20131.14)1.40(1.17\u20131.68)Linoleic acidQ13,509381.00(reference)71.00(reference)141.00(reference)41.00(reference)Q23,586451.20(0.77\u20131.90)101.51(0.58\u20130.93)262.03(1.03\u20133.99)154.06(1.32\u201312.46)Q33,599451.15(0.73\u20131.81)142.08(0.84\u20135.19)130.99(0.46\u20132.13)215.61(1.88\u201316.68)Q43,574350.90(0.56\u20131.44)101.49(0.56\u20133.97)140.97(0.46\u20132.06)256.02(2.09\u201317.41)p-valueh0.620.300.38\u22640.0011SD incrementi0.98(0.84\u20131.15)1.08(0.79\u20131.49)0.91(0.73\u20131.14)1.41(1.18\u20131.69)aColon cancer patients with MLH1 expression and without truncating APC or activating KRAS gene mutationsbColon cancer patients lacking MLH1 expression but without truncating APC or activating KRAS gene mutationscColon cancer patients with a truncating APC gene mutation but with MLH1 expression and without activating KRAS gene mutationsdColon cancer patients with an activating KRAS gene mutations but with MLH1 expression and without truncating APC gene mutationsePerson years at risk are estimated from the subcohort.fIncidence rate ratios (RR) and 95% confidence intervals (95% CI) are adjusted for age, sex, body mass index, smoking, energy intake and family history of colorectal cancer.gPolyunsaturated fathp-value for trend over the quartiles of intake of fat variablesiFor 1standard deviation if intake of fat in the subcohort (see Table\u00a01); i.e., 7.5\u00a0g\/day for polyunsaturated fat and 7.5\u00a0g\/day for linoleic acid\nLikewise, additional analyses were conducted for saturated fat intake in relation to risk of rectal cancer without APC truncating mutations, also excluding individuals with a KRAS mutation and lack of MLH1 expression. The association did not change substantially. Again, only the highest level of intake showed a significant reduced risk of cancer compared to the reference category (RR 0.40 95% CI 0.14\u20131.15, p-trend\u00a0=\u00a00.09).\nDiscussion\nIn this prospective study, we observed that the intake of total, saturated and monounsaturated fat was not associated with the risk of colon cancer, rectal cancer, or the different molecular subgroups of cancer based on lack of MLH1 expression or truncating mutations in the APC gene. This was also found for polyunsaturated fat intake and rectal cancer. However, linoleic acid showed an association with increased risk of colon tumors with only an activated KRAS mutation and no additional truncating APC mutation or lack of MLH1 expression.\nNone of the other epidemiological studies report on specific fatty acids and the risk of molecular surrogate end-points for colon or rectal cancer or adenomas [35\u201343]. Some of these studies report on various types of fat depending on saturation level, but the results are inconsistent across the studies [35, 38, 41, 42] including the current study.\nDiergaarde et al. observed unsaturated fat intake to be associated with increased colon carcinomas with a truncating APC mutation [38]. No distinction was made between mono- and polyunsaturated fats. In our study, we did not observe any association between various types of fat intake and risk of colon or rectal cancer with or without truncating APC mutations after patients also harboring a KRAS mutation in their tumor were excluded from the analyses. We observed a possible inverse association between saturated fat intake and risk of rectal tumors without a truncating APC mutation. However, the association was weak, did not increase gradually according to the quartiles of intake and was only a result of the reduced risk in the highest category of intake. Furthermore, in absence of a biological explanation for this finding and considering the large number of associations investigated, the observation may best be attributed to chance.\nSlattery et\u00a0al. observed saturated and monounsaturated fats, but not polyunsaturated fat, to be associated with increased risk of colon tumors with specific KRAS mutations, i.e., a G\u2192T transversion at codon 12 [41]. No distinction was made between \u03c9-6 and \u03c9-3 fatty acids. We observed an association between polyunsaturated fat intake, especially linoleic acid, and increased risk of colon tumors with a KRAS mutation, regardless of the type of mutations [22].\nFinally, Bautista et\u00a0al. observed an inverse association between monounsaturated fats, mostly derived from olive oil in the Spanish diet, and risk of colon cancer without KRAS mutations [35]. Since olive oil was rarely consumed by this elderly Dutch population in the years preceding 1986 (the cohort baseline), this could explain the lack of association for this type of fat in our study. However, a recent Dutch case\u2013control study on risk factors for colorectal adenomas showed a significant positive association between monounsaturated fats and adenoma risk [42].\nSeveral factors hamper comparisons between our findings and those of other epidemiological studies and may in part explain observed inconsistencies. First, our study is the first large prospective cohort study incorporating molecular end-points for colon and rectal cancer. One of the other studies was a cross-sectional case\u2013case comparison study [40], and the others were case\u2013control studies of varying size (ranging from 108 to 1,510 cases) [35\u201339, 41\u201343]. Second, varying end-points were considered. Three of the other studies focused on adenomas instead of carcinomas [37, 40, 42], and three studies also incorporated rectal tumors but did not distinguish between colon and rectum [35, 37, 42].\nPreviously, we reported the association between linoleic acid intake and increased risk of colon tumors with KRAS mutations (adjusted RR for one standard deviation of increase 1.22 (95% CI 1.05\u20131.42)) [22]. Now, we report that the association appears to be confined to those colon tumors with activating KRAS mutations and an otherwise intact APC gene and with MLH1 expression. In addition, the association appears to be robust since RRs clearly increase over the quartiles of linoleic acid intake and the RRs for one standard deviation increase in linoleic acid intake are similar for men and women, and is increased for proximal and distal colon cancer as well as for overall colorectal cancer, including the rectosigmoid. The activating KRAS mutations at codons 12 and 13 are predominantly G\u2192T and G\u2192A mutations [27] which could be a result of MDA DNA adduct formation [18, 19] associated with increased \u03c9-6 polyunsaturated fat intake [21]. Therefore, even though chance cannot be ruled out and verification by others is warranted, the association seems quite plausible.\nStill several issues remain puzzling. First, why is the observed association for linoleic acid confined to colon cancer and not rectum cancer with only a KRAS mutation? The multistep model for molecular aberrations underlying colorectal carcinogenesis is likely to apply equally for both tumor subsites [7, 8, 15]. However, lack of MLH1 expression in our study is rare among rectum cancer patients and there is growing evidence for differences in the etiology of colon and rectal tumors [33].\nSecond, why is the association with linoleic acid observed for KRAS and not for truncating APC gene mutations? It is unlikely that adduct formation selectively occurs in one gene but not in the other. However, KRAS is an oncogene requiring only one mutation for the gene to be activated, whereas APC is a tumor suppressor gene requiring an additional aberration in the other allele for loss of function. In addition, more than half of the patients with an APC mutation had multiple mutations in this gene [25], complicating data analyses and interpretation. Additional analyses for subgroups of colon tumors with specific truncating point mutations in APC did not show any associations with the intake of fat or different types of fat.\nThis still does not satisfy our third query, i.e., why are the associations specifically confined to this subgroup of colon cancer patients whose tumors are characterized by activating KRAS mutations, and not truncating APC mutations or lack of MLH1 expression? It is speculative, but plausible, that when KRAS is the only one of the three genes affected, the mutation may more likely be the result of exogenous exposure, for example a relatively high linoleic acid intake. In contrast, when a KRAS mutation co-occurs with a mutation in APC or, although more rarely, in addition to a defective MLH1, these other early gene defects also had a role in tumor formation and may have resulted in a mutator phenotype leading to mutations in other genes (such as the KRAS gene) irrespective of exogenous factors. Since there is no information on the timing of genetic aberrations in this type of human studies, we cannot verify this with our data. Aberrations in other genes, not available for this study, but possibly involved in early tumorigenesis of colorectal cancer, could not be accounted for in analyses and may have influenced results. However, a recent systematic sequence analysis of 13,023 exons in individual colorectal cancers showed that the prevalence of mutations other than in APC, KRAS and TP53 is rather low [44], and mutations in TP53 is not an early event in colorectal carcinogenesis. Finally, results are based on relatively small numbers of patients, especially in the reference group of polyunsaturated fat or linoleic acid intake (four patients, see Table\u00a04), and point estimates of RRs for quartiles of intake of polyunstaturated fat or linoleic acid should therefore, be interpreted cautiously. Nevertheless, as discussed previously, the association appears to be robust when regarding the results for one standard deviation increase in linoleic acid intake (based on a total of 65 patients). Therefore, Breivik and Glaudernack\u2019s hypothesis for distinct carcinogens to exert their effect on two proposed types of genetic instability, i.e., microsatellite instability and chromosomal instability [16], may be extended to the potential effect of carcinogens on more specific genetic pathways to colorectal tumorigenesis, as for example the KRAS mutated pathway.\nThe data from this large prospective cohort study suggest that linoleic acid intake is strongly associated with colon tumors with an aberrant KRAS gene, but an intact APC gene and MLH1 expression. Verification in other studies is warranted. Possibly, tumors revealing the involvement of distinct genetic pathways on the basis of specific genetic aberrations, may have a unique etiology.","keyphrases":["dietary fats","epidemiology","molecular","colorectal neoplasms"],"prmu":["P","P","P","M"]}
{"id":"Diabetologia-3-1-2039826","title":"Metabolic and vascular determinants of impaired cognitive performance and abnormalities on brain magnetic resonance imaging in patients with type 2 diabetes\n","text":"Aims\/hypothesis The determinants of cerebral complications of type 2 diabetes are unclear. The present study aimed to identify metabolic and vascular factors that are associated with impaired cognitive performance and abnormalities on brain MRI in patients with type 2 diabetes.\nIntroduction\nDiabetes mellitus is associated with slowly progressive changes in the brain [1]. Neuropsychological studies show that patients with type 2 diabetes mellitus have mild to moderate impairments in attention and executive functioning, information processing speed and memory (for reviews see [2, 3]). Patients with type 2 diabetes also show changes on brain magnetic resonance imaging (MRI), such as cortical and hippocampal atrophy [4, 5]. We have recently shown that cognitive dysfunction in patients with type 2 diabetes was associated with white matter lesions (WML), (silent) brain infarcts and to a lesser extent with atrophy [6].\nThe determinants of changes in cognition and abnormalities on brain MRI of patients with type 2 diabetes are uncertain [2]. Some studies report associations with hypertension [3, 4, 7, 8], but this was not supported by others [5, 9, 10]. Associations between impaired cognition and chronic hyperglycaemia have also been noted [9]. Studies on other diabetic complications may provide leads for potentially relevant determinants. Complications like nephropathy, retinopathy and neuropathy are thought to be due to hyperglycaemia-induced microangiopathy [11, 12], with additional involvement of hypertension and macrovascular disease [13\u201315]. Since atherosclerosis and hypertension are established risk factors for age-related cognitive decline and brain MRI changes in the general population [16\u201319], we hypothesised that the combined effects of atherosclerotic macrovascular disease, chronic hyperglycaemia and hypertension are involved in the development of cognitive impairments in patients with type 2 diabetes.\nThe aim of the present study was to identify possible metabolic and vascular determinants of cognitive dysfunction and changes on brain MRI in patients with type 2 diabetes. Given the uncertainty about these determinants, an exploratory design was chosen. A detailed neuropsychological examination and brain MRI were obtained from a large cross-sectional sample of type 2 diabetes patients and related to different measures of glucose metabolism, vascular risk factors, microvascular complications and macrovascular disease.\nMethods\nParticipants The Utrecht Diabetic Encephalopathy Study aims to identify determinants of cognitive impairment in patients with diabetes [6]. Therefore, patients were not selected for the presence or absence of diabetic complications, co-morbid conditions (e.g. hypertension) or exposure to other risk factors (e.g. smoking). For inclusion patients had to be 55 to 80\u00a0years of age, functionally independent and speakers of Dutch, with a minimal diabetes duration of 1\u00a0year. Exclusion criteria for all participants were: a psychiatric or neurological disorder that could influence cognitive functioning; a history of alcohol or substance abuse and dementia; and, for the control group, a fasting blood glucose \u22657.0\u00a0mmol\/l [20]. Participants with a history of stroke who were still fully functionally independent were classified as eligible. To increase statistical power for within-group analyses in the type 2 diabetes group, twice as many patients as controls were enrolled.Overall, 122 patients with type 2 diabetes (age 56\u201380\u00a0years) and 56 controls (age 57\u201378\u00a0years) were included in the present study. Patients were recruited through their general practitioners; controls were spouses or acquaintances of the patients. Groups were comparable for age, sex and educational level. The study was approved by the local medical ethics committee and each participant signed an informed consent form. All participants underwent a 2\u00a0day protocol, which included brain MRI, a neurological and neuropsychological examination, retinal photography and ultrasonography of the carotid arteries. Fasting blood and urine samples were collected and blood pressure was recorded. In one control person and two type 2 diabetes patients it was not possible to perform the neuropsychological examination. Similarly, MRIs could not be obtained in five controls and nine type 2 diabetes patients, mostly due to MRI contraindications (claustrophobia, pacemaker).\nNeuropsychological examination The neuropsychological examination tapped the major cognitive domains in verbal and non-verbal ways. Eleven tasks were administered in a fixed order, taking about 90 min to complete. These tasks were divided into five cognitive domains, as described previously [6]: (1) attention and executive functioning; (2) information processing speed; (3) memory; (4) abstract reasoning; and (5) visuoconstruction. For analysis the test scores were standardised into z scores for each of the five domains, based on the means of the whole group. The mean performance from each participant across the domains is expressed as the composite cognitive z score.Premorbid IQ was assessed with the Dutch version of the National Adult Reading Test. To control for possible effects of mood disturbances or affective disorders a Beck depression inventory [21] was performed.\nBrain MRI The MRI investigation (1.5 T; Philips Medical Systems, Best, the Netherlands) consisted of an axial T1-weighted and an axial T2 and T2 fluid-attenuating inverse recovery (FLAIR) scan (TR\/TE\/TI: 6000\/100\/2000, field of view 230\u00a0mm, matrix 180\u2009\u00d7\u2009256, slice thickness 4.0\u00a0mm, contiguous, 38 slices).WML were rated according to the Scheltens scale [22] with slight modifications [6]. Periventricular WML (PWML) were rated on a severity scale (0\u20132) at the frontal and occipital horns and the body of the lateral ventricle on both sides (sum score 0\u201312). For the rating of deep (subcortical) WML (DWML) the brain was divided into six regions: frontal, parietal, occipital, temporal, basal ganglia and infra-tentorial. Per region the size and number of WML were rated on a scale ranging from 0 to 6. The total score thus ranged from 0 to 36.Cortical atrophy was evaluated by the frontal interhemispheric fissure ratio and the Sylvian fissure ratio [23]. Subcortical atrophy was evaluated by the bifrontal ratio and by the bicaudate ratio [23]. These ratios were converted to z scores: a cortical atrophy z score (mean of z frontal fissure ratio and z Sylvian fissure ratio) and a subcortical atrophy z score (mean of z bicaudate ratio and z bifrontal ratio).All MRI scans were rated by two investigators (S. M. Manschot and G. J. Biessels) blinded for presence or absence of diabetes or other characteristics. In case of disagreement of more than 1 point on the WML scales in a particular region or more than 5\u00a0mm (actual size) on any of the atrophy measurements (2\u00a0mm for fissure widths), a consensus reading was held (0% of PWML, 4% of DWML and 4% of atrophy ratio readings were thus affected). In all other cases the readings of both raters were averaged.\nDiabetes characteristics and glucose metabolism A standardised questionnaire addressed medical history, medication use, diabetes duration and the life-time occurrence of severe hypoglycaemic episodes (defined as episode of hypoglycaemia severe enough to require the assistance of another person, hospitalisation or emergency room visit). BMI was calculated as weight divided by height square.Blood was drawn by venepuncture to assess HbA1c, fasting glucose and insulin levels. Insulin resistance was estimated with the homeostasis model assessment of insulin resistance (HOMA-IR). The HOMA-IR is calculated as fasting glucose (mmol\/l) \u00d7 fasting insulin (mU\/l)\/22.5 [24]. Because insulin was expressed in pmol\/l we used the formula fasting glucose (mmol\/l) \u00d7 fasting insulin (pmol\/l)\/(22.5\u2009\u00d7\u20096.945) [24].\nVascular risk factors Blood pressure was measured at home at nine fixed time points during the day with an automatic blood pressure machine (705CP; Omron, Mannheim, Germany). These measurements were averaged. In the primary analysis hypertension was defined as a mean systolic blood pressure >160\u00a0\u00a0mmHg or a mean diastolic pressure >95\u00a0mmHg or the use of antihypertensive medication. In a second analysis cut-off values for systolic and diastolic blood pressure of 140 and 90\u00a0mmHg were used.Smoking habits were classified as \u2018current\u2019 and \u2018past or never\u2019. Total cholesterol, HDL-cholesterol, LDL-cholesterol and triacylglycerol were assessed in a fasting venous blood sample.\nMicrovascular disease Following mydriasis with phenylephrine and tropicamide, single-field photographs were taken of both eyes with a 50-degree retinal camera (Zeiss FF 450, Carl Zeiss B.V., Sliedrecht, the Netherlands), centred on the macula. Retinopathy was rated on slides, according to the diabetic retinopathy severity scale (grades 1\u20137) as used in the Wisconsin Epidemiologic Study of Diabetic Retinopathy [25]. Photocoagulated eyes were rated at grade 5 or higher (severe non-proliferative diabetic retinopathy). Ratings were performed by two investigators, blinded to patient characteristics. In case of disagreement (2%), a third investigator was involved and a consensus was made. Retinopathy was defined as a grade of 1.5 or higher.Neuropathy was rated with the Toronto Clinical Neuropathy Scoring System [26], with a slight modification. A sensory test for temperature was not performed, so that the maximum score was 18 points (severe polyneuropathy) instead of 19. A score of 0\u20135 indicated no neuropathy, 6\u20138 indicated mild neuropathy, 9\u201311 moderate neuropathy and \u226512 severe neuropathy. Neuropathy was defined as a score of \u22656.Urine was collected overnight. Albuminuria was defined as microalbuminuria (albumin 0.03\u20130.25\u00a0g\/l) or macroalbuminuria (albumin 0.25\u00a0g\/l or positive protein dipstick test).\nMacrovascular disease Several composite measures of macrovascular disease were defined. \u2018Any peripheral arterial disease\u2019 was defined as current complaints of intermittent claudication (assessed with the Rose questionnaire [27]) or a history of surgery or endovascular treatment for arterial disease of the legs or the abdominal aorta. \u2018Ischaemic heart disease\u2019 was defined as a history of myocardial infarction or surgery or endovascular treatment for coronary artery disease. \u2018Any vascular event\u2019 was defined as a history of myocardial infarction or stroke, or a history of operative or endovascular treatment for coronary, carotid or peripheral (legs, abdominal aorta) artery disease.Brain infarcts were rated on brain MRI, by location (cortical and subcortical), size (lacunar [<1.5\u00a0cm] or large) and number. A lesion was considered an infarct if it was hypo-intense on T1 and FLAIR images and if its appearance was unlike a perivascular space.Carotid intima-media thickness (CIMT) was measured in both common carotid arteries as described previously [28] with an ATL Ultramark 9 (Advanced Technology Laboratories, Bothell, WA, USA) equipped with a 10-MHz linear-array transducer. Scanning was performed at three different longitudinal projections (anterior-oblique, lateral and posterior-oblique). The CIMT was measured in a 1\u00a0cm section proximal to the beginning of the dilatation of the carotid bulb in all three projections, in both carotid arteries. CIMT was calculated as the average of these six measurements. CIMT readings were not available in six type 2 diabetes patients and one person in the control group.\nStatistical analysis The differences between patients and the control group were examined with t test for means, Mann\u2013Whitney U was used for non-parametric data and \u03c72 test for proportions. In the text and tables, data are shown as mean \u00b1 SD or proportions, unless stated otherwise.Within the type 2 diabetes population, cognition (five domains) and brain MRI findings (cortical and subcortical atrophy z scores, PWML, DMWL and infarcts) were related to the different measures of glucose, insulin and lipid metabolism, and to microvascular complications and macrovascular disease by linear or logistic regression analyses, adjusting for age, sex and estimated IQ. In order to limit the number of analyses the \u2018composite cognitive z score\u2019 was used as the primary cognitive outcome measure in the regression analyses. For significant associations, post hoc tests were performed per domain. Secondary analyses were performed with information processing speed, the domain most markedly affected by type 2 diabetes. The results were essentially the same as for the composite cognitive z score (data not shown).In the regression analyses, B values >0 indicate that a variable is associated with more severe MR abnormalities; for cognition B values <0 indicate that a variable is associated with more pronounced performance impairments. For the between and within-group analyses, p\u2009<\u20090.05 was considered statistically significant. All variables that reached a significance level of p\u2009\u2264\u20090.1 in the adjusted univariate risk factor analyses were included in a multivariate model that also included age, sex and estimated IQ.\nResults\nParticipant characteristics The age, sex, level of education and estimated IQ in the groups were comparable (Table\u00a01).\nTable\u00a01Participant characteristicsCharacteristicType 2 diabetes groupControl groupParticipants (n)12256Sex (male\/female)62\/6025\/31Age (years)66.0\u2009\u00b1\u20095.865.1\u2009\u00b1\u20095.2Level of education (1\u20137)a4 (3\u20135)4 (3\u20135)Estimated premorbid IQ99\u2009\u00b1\u200915101\u2009\u00b1\u200914Diabetes duration (years)b8.7\u2009\u00b1\u20096.1Diabetes treatment (%)\u00a0Diet10\u00a0Oral medication alone61\u00a0Insulinb29HbA1c (%)b6.9\u2009\u00b1\u20091.2**, d5.5\u2009\u00b1\u20090.3Fasting glucose levels (mmol\/l)b8.6\u2009\u00b1\u20092.9**5.5\u2009\u00b1\u20090.6Fasting insulin levels (pmol\/l)b,c120\u2009\u00b1\u2009110**, e (n\u2009=\u200982)76\u2009\u00b1\u200950 (n\u2009=\u200954)HOMA-IRb,c6.6\u2009\u00b1\u20096.4**, f2.6\u2009\u00b1\u20091.8BMI (kg\/m2)b28.1\u2009\u00b1\u20094.427.3\u2009\u00b1\u20095.3Data are given as number or percentage (as indicated), mean \u00b1 SD or ranges**p\u2009<\u20090.01 for type 2 diabetes vs control groupaLevel of education was expressed in seven categories [49]bEntered as explanatory variable in the regression analyses within the type 2 diabetes groupcOnly from participants who were not treated with insulin and did not have antibodies against insulinStatistically significant associations within the type 2 diabetes group: with composite cognitive z scores\u2014dp\u2009<\u20090.05; with MRI abnormalities\u2014ep\u2009<\u20090.01, fp\u2009<\u20090.05Detailed neuropsychological and MRI data have been reported previously [6]. In short, performance of patients with type 2 diabetes was worse than that of the control group across all five cognitive domains, with statistically significant differences on attention and executive functioning (difference mean z scores 0.23 [95% CI 0.03, 0.43]; p\u2009=\u20090.02), information processing speed (0.40 [0.17, 0.63]; p\u2009=\u20090.001) and memory (0.20 [0.05, 0.36]; p\u2009=\u20090.01). Patients with type 2 diabetes had more pronounced cortical atrophy (difference mean z scores 0.62 [95% CI 0.33, 0.91]; p\u2009<\u20090.001) and subcortical atrophy (0.38 [0.07, 0.68]; p\u2009=\u20090.01). They also had more severe DWML (controls, median [range]: 5 [0, 18]; type 2 diabetes: 7 [0.5, 27.5]; p\u2009=\u20090.02), but PWML severity in the two groups was similar (control: 6 [4, 10]; type 2 diabetes: 6 [3, 12]; p\u2009=\u20090.13). Patients with type 2 diabetes also had more (silent) cerebral infarcts than controls (type 2 diabetes 22\/113, control 4\/54; p\u2009=\u20090.06).\nGlucose metabolism HbA1c, fasting glucose and insulin levels were higher (all p\u2009<\u20090.01) in patients with type 2 diabetes than in the control group. BMI was similar in both groups. Only a small proportion (6%) of type 2 diabetes patients had ever experienced a severe hypoglycaemic event (Table\u00a01).In the regression analyses within the type 2 diabetes group, HbA1c levels were significantly related to cognition (composite z score: B [per % HbA1c]: \u22120.07 [\u22120.14, 0] p\u2009=\u20090.047; post hoc per domain: information processing speed: B [per % HbA1c]: \u22120.15 [95% CI: \u22120.27, \u22120.2], p\u2009=\u20090.02; abstract reasoning: B: \u22120.15 [\u22120.29, \u22120.01], p\u2009=\u20090.04). Elevated fasting insulin levels and HOMA-IR were related to increased DWML severity (B [per 10\u00a0pmol\/l insulin]: 0.14 [0.04, 0.26], p\u2009=\u20090.009; B [HOMA-IR]: 0.21 [0.04, 0.39], p\u2009=\u20090.02).\nVascular risk factors Table\u00a02 shows that patients with type 2 diabetes had higher systolic blood pressure (p\u2009<\u20090.01) and pulse pressure (p\u2009<\u20090.05) than controls. They also had hypertension more often (p\u2009<\u20090.01). Total cholesterol was lower in the type 2 diabetes group (p\u2009<\u20090.01), but the proportion of individuals taking lipid-lowering drugs was higher in that group (p\u2009<\u20090.01). There were no statistically significant differences between type 2 diabetic patients and the control group in the proportion of participants who smoked or had dyslipidaemia (Table\u00a02).\nTable\u00a02Vascular risk factorsCharacteristicType 2 diabetes group (n\u2009=\u2009122)Control group (n\u2009=\u200956)Mean arterial pressure (mmHg)a103\u2009\u00b1\u200911*,b98\u2009\u00b1\u200910Pulse pressure (mmHg)a65\u2009\u00b1\u200915*59\u2009\u00b1\u200916Hypertension (%)a73**34Antihypertensive drugs (%)70**32Current smoking (%)a2214Total cholesterol (mmol\/l)a5.0\u2009\u00b1\u20090.9**5.8\u2009\u00b1\u20091.1HDL-cholesterol (mmol\/l)a4.3\u2009\u00b1\u20091.24.4\u2009\u00b1\u20091.5Triacylglycerol (mmol\/l)a1.9\u2009\u00b1\u20091.01.6\u2009\u00b1\u20091.1Lipid-lowering drugs (%)a54**,c21Data are given as percentage or mean \u00b1 SD*p\u2009<\u20090.05, **p\u2009<\u20090.01 for type 2 diabetes vs control groupaEntered as explanatory variable in the regression analyses within the type 2 diabetes groupbp\u2009<\u20090.05 for association with more severe MRI abnormalitiescp\u2009<\u20090.05 for reverse associationIn the regression analyses within the type 2 diabetes group there were no statistically significant associations with the composite cognitive z score. Non-significant trends (p\u2009\u2264\u20090.10) were observed for associations between both hypertension and current smoking and impaired cognitive performance (hypertension B: \u22120.19 [\u22120.38, 0], p\u2009=\u20090.053; smoking B: \u22120.21 [\u22120.43, 0], p\u2009=\u20090.051), and between the use of lipid-lowering drugs and better performance (B: 0.15 [\u22120.03, 0.32], p\u2009=\u20090.10). Reanalysis with cut-off values for hypertension of 140\/90\u00a0mmHg made the association with the composite cognitive z score less strong. Mean arterial pressure was associated with more severe PWML (B [per 10\u00a0mmHg]: 0.28 [0.03, 0.53], p\u2009=\u20090.03). The use of lipid-lowering drugs (statins in all but one patient) was associated with less severe MRI abnormalities (PWML: B: \u22120.68 [\u22121.25, \u22120.12], p\u2009=\u20090.02; cortical atrophy: B: \u22120.36 [\u22120.69, \u22120.03], p\u2009=\u20090.03). These effects were not affected by additional adjustment for the actual cholesterol levels (Table\u00a02).\nMicrovascular disease In 20 patients with type 2 diabetes and 8 control individuals it was not possible to perform retinal photographs. Mostly due to logistical reasons, overnight urine samples could not be obtained from 21 diabetic patients and 13 controls. Patients with type 2 diabetes had more retinopathy and neuropathy than the control group (both p\u2009<\u20090.01). Although albuminuria was more common in the type 2 diabetic than in the control group, this difference was not statistically significant (Table\u00a03).\nTable\u00a03Microvascular diseaseCharacteristicType 2 diabetes group (n\u2009=\u2009122)Control group (n\u2009=\u200956)Retinopathy (diabetes n\u2009=\u2009112; control n\u2009=\u200948)a37 (33)**,b1 (2)\u00a0Background 33 1\u00a0Severe non-proliferative4 Neuropathya47 (39)**7 (13)\u00a0Mild neuropathy25 7 \u00a0Moderate neuropathy18 0\u00a0Severe neuropathy4 0Albuminuria (diabetes n\u2009=\u2009101; control n\u2009=\u200943)a16 (16)3 (7)\u00a0Microalbuminuria93\u00a0Macroalbuminuria7 0Any microvascular diseasec72 (59)**11 (20)Data are given as values (percentage)**p\u2009<\u20090.01 for type 2 diabetes vs control groupaEntered as explanatory variable in the regression analyses within the type 2 diabetes groupbp\u2009<\u20090.05 for association with MRI abnormalitiescIncludes all patients with albuminuria, neuropathy or retinopathyIn the regression analyses within the type 2 diabetes group, there were no statistically significant associations with the composite cognitive z score. Retinopathy was associated with more pronounced cortical atrophy (B: 0.48 [0.11, 0.85], p\u2009=\u20090.01) (Table\u00a03).\nMacrovascular disease Patients with type 2 diabetes were more likely to have had intermittent claudication (p\u2009<\u20090.01) or a history of ischaemic heart disease (p\u2009<\u20090.01). There was no difference between the two groups in the CIMT (Table\u00a04).\nTable\u00a04Macrovascular diseaseCharacteristicType 2 diabetes group (n\u2009=\u2009122)Control group (n\u2009=\u200956)Any peripheral arterial diseasea18 (15)** 0\u00a0Claudicatio intermittens14 (11)**0\u00a0Vascular surgery femoral artery4 (3)0\u00a0Vascular surgery (AAA)3 (3)0Ischaemic heart diseasea23 (19)**,b2 (4)\u00a0Myocardial infarction15 (12)*1 (2)\u00a0CABG13 (11)*1 (2)Brain infarct on MRIa22 (20)*,b,c4 (8)History of brain infarcta7 (6)2 (4)Carotid surgerya2 (2)1 (2)Any vascular event a33 (27)**,b,c4 (7)CIMTa0.093\u2009\u00b1\u20090.0180.092\u2009\u00b1\u20090.023Data are given as value (percentage) or mean \u00b1 SDAAA, abdominal aortic aneurysm; CABG, coronary artery bypass grafting*p\u2009<\u20090.05, **p\u2009<\u20090.01 for type 2 diabetes vs control groupaEntered as explanatory variable in the regression analyses within the type 2 diabetes groupStatistically significant associations: with MRI abnormalities bp\u2009<\u20090.05; with impaired cognition cP\u2009<\u20090.05In the regression analyses within the type 2 diabetes group, a history of \u2018any vascular event\u2019 and the presence of brain infarcts on MRI were associated with an impaired composite cognitive z score as follows: (1) vascular event: composite z score B: \u22120.25 (\u22120.44, \u22120.05), p\u2009=\u20090.01; post hoc per domain: information processing speed B: \u22120.46 (\u22120.80, \u22120.12), p\u2009=\u20090.008; and memory B: \u22120.23 (\u22120.41, \u22120.06), p\u2009=\u20090.01; (2) infarct on MRI: composite z score B: \u22120.28 (\u22120.50, \u22120.06), p\u2009=\u20090.01; post hoc per domain: information processing speed B: \u22120.77 (\u22121.14, \u22120.39), p\u2009<\u20090.001; and abstract reasoning B: \u22120.41 (\u22120.82, 0.01), p\u2009=\u20090.06. A history of \u2018any vascular event\u2019 was also associated with more pronounced DWML (B: 2.0 [0, 4.1], p\u2009=\u20090.05) and with an increased occurrence of infarcts on MRI (odds ratio: 2.9 [1.1, 7.9], p\u2009=\u20090.04). Patients with a (silent) infarct on MRI tended to have more pronounced PWML (B: 0.7 [0, 1.4], p\u2009=\u20090.06) and cortical atrophy (B: 0.51 [0.10, 0.92], p\u2009=\u20090.02) relative to type 2 diabetes patients without infarcts on MRI (Table\u00a04).Exclusion of patients with a history of stroke attenuated the association between \u2018any vascular event\u2019 and the cognitive z score, but not that between \u2018any vascular event\u2019 and DWML.\nMultivariate analysis For the majority of risk factors the values of the standardised regression coefficients \u00df were similar in the single risk factor and multivariate models, indicating that interaction between factors was limited. Values for \u00df for individual factors varied between 0.15 and 0.30, indicative of modest associations (Table\u00a05).\nTable\u00a05Multivariate risk factor analyses within the type 2 diabetes group\u00a0Single factors \u03b2 (95% CI)Full model \u03b2 (95% CI)Final model \u03b2 (95% CI)Cognition\u00a0Age\u22120.30 (\u22120.44, \u22120.16)***\u22120.29 (\u22120.43, \u22120.14)***\u00a0Sex\u22120.12 (\u22120.24, 0.02)\u00a0Estimated IQ0.65 (0.50, 0.79)***0.65 (0.50, 0.80)***\u00a0HbA1c\u22120.14 (\u22120.28, \u22120.002)*\u22120.11 (\u22120.25, 0.02)\u00a0Hypertension\u22120.14 (\u22120.26, 0.002)\u22120.12 (\u22120.26, 0.01)\u22120.14 (\u22120.27, \u22120.001)*\u00a0Current smoking\u22120.14 (\u22120.29, 0.001)\u22120.15 (\u22120.29, \u22120.02)*\u22120.14 (\u22120.27, 0.001)\u00a0Lipid-lowering drugs0.12 (\u22120.02, 0.26)0.17 (0.03, 0.31)*0.17 (0.03, 0.31)*\u00a0Any vascular event\u22120.18 (\u22120.32, \u22120.03)*\u22120.18 (\u22120.32, \u22120.03)*\u22120.19 (\u22120.34, \u22120.05)**\u00a0Brain infarct on MRI a\u22120.18 (\u22120.32, \u22120.04)*Cortical atrophy\u00a0Sex\u22120.005 (\u22120.23, 0.22)\u00a0Age0.35 (0.18, 0.52)***0.34 (0.18, 0.51)***\u00a0Estimated IQ\u22120.07 (\u22120.24, 0.10)\u00a0Lipid-lowering drugs\u22120.20 (\u22120.37, \u22120.02)*\u22120.19 (\u22120.36, \u22120.03)*\u22120.18 (\u22120.35, \u22120.02)*\u00a0Retinopathy0.24 (0.05, 0.43)*0.21 (0.04, 0.39)*0.22 (0.06, 0.39)**\u00a0Brain infarct on MRI0.22 (0.04, 0.39)*0.23 (0.06, 0.40)**0.23 (0.06, 0.40)**DWML\u00a0Sex0.13 (\u22120.05, 0.30)\u00a0Age0.20 (0.02, 0.35)*0.22 (0.04, 0.39)*\u00a0Estimated IQ\u22120.14 (\u22120.31, 0.04)\u00a0Insulin level0.29 (0.08, 0.51)**0.26 (0.04, 0.47)*0.24 (0.03, 0.45)*\u00a0Any vascular event0.19 (\u22120.002, 0.37)0.15 (\u22120.03, 0.34)PWML\u00a0Sex0.16 (\u22120.02, 0.34)0.16 (\u22120.02, 0.34)\u00a0Age0.25 (0.07, 0.43)**0.24 (0.07, 0.42)**\u00a0Estimated IQ\u22120.12 (\u22120.30, 0.06)\u00a0Mean arterial pressure0.20 (0.02, 0.38)*0.15 (\u22120.03, 0.33)0.17 (\u22120.004, 0.35)\u00a0Lipid-lowering drugs\u22120.22 (\u22120.35, \u22120.001)*\u22120.20 (\u22120.38, \u22120.02)*\u22120.18 (\u22120.35, 0.001)*\u00a0Brain infarct on MRI0.17 (\u22120.006, 0.35)0.18 (0.01, 0.36)*0.18 (0.009, 0.36)*\u03b2 is the standardised regression coefficient B. The first column contains the values of \u03b2 for all variables that reached a significance level of a least p\u2009=\u20090.10 in the univariate risk factor analyses within the type 2 diabetes patient group, adjusted for age, sex and estimated IQ. These variables were included in a multivariate model that also included age, sex and estimated IQ (second column, full model). The final model (third column) is based on backward elimination of non-significant factors. This final model shows which variables that were related to the outcome measures in the univariate model remained significantly associated with the outcome measures independently of the other factors in the full modelFor cognition \u03b2 values <0 indicate that a variable is associated with more pronounced performance impairments; for MRI \u03b2 values >0 indicate that a variable is associated with more severe MRI abnormalities*p\u2009<\u20090.05**p\u2009<\u20090.01***p\u2009<\u20090.001aBecause \u2018infarct on MRI\u2019 and \u2018history of any vascular event\u2019 were interrelated in the univariate analyses (see Macrovascular disease section) only \u2018history of any vascular event\u2019 was entered in the multivariate modelIn the multivariate model, hypertension and a history of vascular events were associated with worse cognitive performance and statin use with better performance. Retinopathy and brain infarction on MRI were associated with more severe cortical atrophy and statin use with less atrophy. A higher insulin level was associated with more DWML, brain infarction on MRI with more PWML and statin use with less PWML. Overall, macrovascular disease (history of macrovascular events or infarct on MRI) were most consistently associated with the different outcome measures (Table\u00a05).\nDiscussion\nPatients with type 2 diabetes had more cortical and subcortical atrophy and more DWML than control participants and their overall performance in the five cognitive domains was worse. As expected, patients with type 2 diabetes had more microvascular complications, more macrovascular (atherosclerotic) disease and more hypertension than the control group. In multivariate regression analyses within the type 2 diabetes group, hypertension and a history of vascular events were associated with worse cognitive performance, while statin use was associated with better performance. Retinopathy and brain infarcts on MRI were associated with more severe cortical atrophy and statin use with less atrophy. Insulin level and brain infarcts were associated with more severe WML and statin use with less severe WML.\nCognitive function in patients with type 2 diabetes has been studied extensively (for reviews see [2, 3]). Performance in the domains verbal memory and information processing speed, and probably also executive functioning and non-verbal memory, is moderately impaired. Our results are in keeping with these findings. Thus far, relatively few studies have specifically addressed brain MRI abnormalities in patients with type 2 diabetes. In agreement with our observations, modest cortical and subcortical atrophy and symptomatic or asymptomatic infarcts have been found more often in type 2 diabetes patients than in control individuals [4, 5, 29]. Results of previous studies on the association between type 2 diabetes and WMLs are less consistent [30]. This might be due to the study populations involved and the use of relatively insensitive WML rating scales [30].\nChronic hyperglycaemia might be a determinant of cerebral changes in patients with type 2 diabetes. In the present study, HbA1c levels were related to the composite cognitive z score, but only in de univariate analysis. Moreover, retinopathy, which is generally considered to be a consequence of chronic exposure to hyperglycaemia [11], was related to cortical atrophy. Previous studies on cognition in patients with type 2 diabetes have also reported an association with HbA1c levels [2, 9, 31]. The relation with fasting blood glucose or duration of diabetes is, however, inconsistent [31, 32]. No previous studies have provided detailed data on the association between glycaemic control and MRI changes in type 2 diabetes. Studies in type 1 diabetes mellitus, however, have shown an association between diabetic retinopathy (as a proxy of chronic hyperglycaemia) and both brain atrophy [33, 34] and cognitive functioning [35]. There are no previous studies on the relation between insulin levels and cerebral complications in type 2 diabetes. The association with WML severity, observed by us in the present study, is of particular interest in the light of recent studies in the general population, which link insulin to vascular abnormalities and degenerative changes in the brain [36, 37].\nPrevious studies in the general population indicate that risk factors for vascular disease, such as hypertension, dyslipidaemia, increased BMI and smoking, are associated with an increased risk of cognitive decline and dementia and with brain MRI changes, including WML (e.g. [38\u201341]). Previous studies on the modulating effect of hypertension on cognitive function in type 2 diabetes show conflicting results [7, 9, 42, 43]. In the present study, hypertension was related with impaired cognitive performance and mean arterial pressure with PWML severity. To our knowledge, the relation between other vascular risk factors and both cognition and brain MRI in patients with type 2 diabetes has not been examined previously. The reverse association between the use of statins and both cognition and MRI findings is intriguing. Nevertheless, this observation cannot be regarded as proof of a possible treatment effect. It should be noted that the association between statin use and both cognition and age-related brain MRI changes in the general population is still being debated [44]. The present findings will need to be confirmed by further studies.\nMacrovascular atherosclerotic disease appeared to be the most consistent determinant of impaired cognition and brain MRI abnormalities in the type 2 diabetes patients in the present study. We have not found any previous studies that presented detailed data on the relation between macrovascular disease and cerebral changes in people with type 2 diabetes. In the general population, however, several studies have shown that macrovascular atherosclerotic disease is associated with age-related cognitive impairment and changes in brain MRI. In a large cross-sectional study, for example, previous vascular events, presence of plaques in the carotid arteries and presence of peripheral arterial atherosclerotic disease were negatively associated with cognitive performance [17]. In another study, the association between the number of cardiovascular disease conditions and cognitive impairment appeared to show a \u2018dose\u2013response\u2019 relationship [18]. With regard to brain MRI changes, a history of stroke or myocardial infarction has been associated with the presence of WML [19] and plaques in the carotid artery with PWML [16, 45].\nThe strength of our study is that we combined detailed data on cognitive function and brain MRI with detailed data on metabolic and vascular risk factor clusters, thus allowing an accurate assessment of the relation between these factors. Possible limitations include patient selection, the cross-sectional design and the large number of explanatory variables addressed. With regard to patient selection, we aimed to obtain a representative sample of functionally independent patients with type 2 diabetes from the general population. Although the rather demanding testing protocol may have deterred patients with relatively severe mental or physical limitations, the prevalence of microvascular and macrovascular disease, hypertension and smoking habits, as well as the level of metabolic control in our study sample is comparable with those found in other population-based studies in the Netherlands [46\u201348]. To minimise the effects of lifestyle and socioeconomic factors, control participants were recruited from the direct environment of the type 2 diabetic patients. Consently, the prevalence of risk factors such as hypertension and high BMI was higher than would be expected in the general population in the Netherlands. If anything, this would have decreased the differences in cognition and MRI ratings between the groups. The cross-sectional design of our study precludes inferences about causal relationships. Moreover, the cognitive and imaging outcome measures were probably influenced by a large number of factors, some of which are specific to type 2 diabetes mellitus (e.g. chronic hyperglycaemia, diabetes treatment) and some not (e.g. age, hypertension, atherosclerosis). Our exploratory analysis included a large number of explanatory variables, which has certain drawbacks. First, different explanatory variables might be interrelated. The relatively small regression coefficients and effect sizes affect the evaluation of these interrelations and limit statistical power. This may also explain why some of the variables that reached statistical significance in the univariate analyses dropped out of the multivariate model. Nevertheless, the multivariate analysis as presented in Table\u00a05 does indicate which variables were the strongest independent determinants of cognition and MRI abnormalities in the model used. The second drawback is that the large number of regression analyses can lead to type I errors. Nevertheless, we feel that this first detailed study of cognition and brain MRI in type 2 diabetes patients in relation to metabolic and vascular risk factors does provide important leads that could be further evaluated in future studies. Such studies should: (1) preferably have a longitudinal design; (2) include assessment of cognition and brain MRI in relation to chronic hyperglycaemia and atherosclerotic vascular disease; and (3) allow the assessment of potential confounders (e.g. hypertension).\nType 2 diabetes is associated with modest impairments in cognition, as well as with atrophy and vascular lesions on MRI. This \u2018diabetic encephalopathy\u2019 is a multifactorial condition, for which atherosclerotic (macroangiopathic) vascular disease is an important determinant. Chronic hyperglycaemia, hypertension and hyperinsulinaemia may play additional roles.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nESM\nThe Utrecht Diabetic Encephalopathy Study Group (PDF 12.8 kb)","keyphrases":["cognitive performance","cerebral complications","brain mri","type 2 diabetes mellitus","white matter lesions","vascular risk factors","diabetic encephalopathy","brain atrophy","metabolic risk factors","neuropsychological investigation"],"prmu":["P","P","P","P","P","P","P","P","R","R"]}
{"id":"Behav_Genet-4-1-2257994","title":"Environmental Factors in Obsessive-Compulsive Behavior: Evidence from Discordant and Concordant Monozygotic Twins\n","text":"To investigate environmental factors that protect against or exacerbate obsessive-compulsive (OC) symptoms, we selected 25 monozygotic (MZ) twin pairs discordant, 17 MZ twin pairs concordant high and 34 MZ pairs concordant low on OC symptoms from a large longitudinal Dutch sample of adult twin pairs and their family members, applying stringent criteria for OC symptomatology. Data were collected on psychopathology, family structure, health, lifestyle, birth complications and life events. Unique environmental factors were studied using within-discordant MZ pair comparisons, whereas between-concordant MZ pair comparisons were used to study environmental factors that are shared by the twins of an MZ pair. The high-scoring MZ twins of the discordant group reported more life events (especially sexual abuse) than their low-scoring twin-siblings. The between-pair comparisons showed lower birth weight in the discordant MZ pairs than in the concordant MZ pairs. Further, the concordant high MZ pairs as well as their spouses had a lower educational level than the two other groups. On scale scores of anxious-depression, neuroticism, and somatic complaints, concordant high MZ pairs showed highest scores, and the discordant MZ pairs scored intermediate, except for neuroticism, on which the high-scoring twins of discordant MZ pairs were equal to the concordant high pairs. Discordance on psychological scale scores between the concordant MZ pairs was evident from 1991 onward, and within the discordant MZ pairs from 1997 onward, confirming previous reports of an association of early-onset OC symptoms with higher genetic load. Parent scores of OC symptoms and anxious-depression suggested intermediate genetic load in the discordant MZ group. In conclusion, this study reports on both unique and shared environmental factors associated with OC symptomatology. Whether these factors operate in addition to or in interaction with genetic disposition is to be elucidated in future studies.\nIntroduction\nObsessive-compulsive disorder (OCD) is characterized by repetitive distressing and anxiety-provoking intrusive thoughts, mostly in combination with time-consuming repetitive actions designed to reduce tension or anxiety caused by the disturbing thoughts (American Psychiatric Association 1994). OCD can run, especially if untreated, a chronic and disabling course (Nestadt et\u00a0al. 1998). Family studies have quite convincingly shown that early-onset OCD is familial (Pauls et\u00a0al. 1995; Nestadt et\u00a0al. 2000). Studies in 7- to 12-year-old twins have indicated that between 47 and 58% of the variance in obsessive-compulsive (OC) behavior is explained by additive genetic factors (Hudziak et\u00a0al. 2004). The remaining variance is almost entirely explained by unique environment, with a small contribution of shared environmental factors (16%) at age 12. In adults, twin studies have indicated a more modest contribution of genetic factors (van Grootheest 2005 and others). One twin study in women suggested heritability of 33 and 26% respectively for obsessions and compulsions (Jonnal et\u00a0al. 2000). Further, a recent twin study in 5,893 mono- and dizygotic twins, and 1,304 additional siblings from the population-based Netherlands Twin Register (NTR; Boomsma et\u00a0al. 2002), indicated heritability estimates of 47% for both men and women (van Grootheest et\u00a0al. 2007a, b).\nThe course of OCD is moderately stable: longitudinal twin studies as well as epidemiological and clinical studies have indicated that on average 50% of cases remit over time (van Grootheest et\u00a0al. 2007a, b; Angst et\u00a0al. 2004; Skoog and Skoog 1999). Environmental factors explain about half of persistence in boys and two-third of persistence in girls. Thus, environmental factors are of substantial importance in the likelihood to obtain and persist or remit with respect to OC symptomatology.\nTo date, only few studies have addressed the specific nature of these environmental factors in OC phenomenology. Which environmental influences can be detected from the literature? Family studies have revealed that parents of children with OCD suffer from poorer mental health and have fewer coping strategies than parents of healthy children (Derisley et\u00a0al. 2005). A-specific risk factors for (the persistence of) OCD include: earlier age at onset, presence of co-morbid conditions and low socio-economic status (Skoog and Skoog 1999; Stewart et\u00a0al. 2004, 2006; Angst et\u00a0al. 2004). Further, OCD patients report more often than healthy controls to have been overprotected or emotionally neglected by their parents (Cavedo and Parker 1994). Patients with the hoarding subtype of OCD in particular, report a lack of parental emotional warmth (Alonso et\u00a0al. 2004). Perinatal risk factors, such as prolonged labor and edema during pregnancy, have been reported to increase the risk of later OCD (Vasconcelos et\u00a0al. 2007). Childhood sexual abuse appears to be an important mediator for later OCD, especially in women (Lochner et\u00a0al. 2002). The relationship between religiosity and OCD is unclear. Some authors find increased frequencies of religious obsessions and hand washing among highly religious protestants in comparison with less or non-religious subjects (Abramowitz et\u00a0al. 2004), while others find no relation between religiosity and an increase in OC symptoms (Assarian et\u00a0al. 2006), and argue that religiosity is merely a form in which OC symptoms can be displayed (religious obsessions) (Tek and Ulug 2001). Finally, \u03b2-hemolytic streptococcal infections have been reported to be associated with OC symptom exacerbation (March et\u00a0al. 1990).\nThe comparison of monozygotic (MZ) twins who score high on a trait with their low-scoring co-twins, comprises a powerful method to identify environmental factors involved in a disorder (Martin et\u00a0al. 1997). MZ twins have identical genomes and are born and raised at the same time in the same family, thus sharing a very similar family environment. Consequently, discordance on the trait is mostly explained by differences in the non-shared (i.e., unique) environment that act either directly on the phenotype, or by epigenetic mechanisms (Fraga et\u00a0al. 2005). Environmental factors that are shared by both members of a twin pair (such as maternal smoking during pregnancy, or parental divorce) can be studied by comparing MZ twins who are concordant high on the trait with MZ twins who are concordant low.\nComparisons within discordant MZ pairs or between concordant MZ pairs have not been employed to study environmental factors involved in OC phenomenology. In other psychiatric disorders, such as schizophrenia and ADHD (Stabenau and Pollin 1993; Lehn et\u00a0al. 2007), as well as in somatic disorders such as diabetes mellitus (Bo et\u00a0al. 2000), this method has been successfully used. One twin study on a disorder related to OCD, i.e., Gilles de la Tourettes\u2019 Syndrome, has studied basal ganglia D2-receptorbinding in five MZ twins who were discordant on tic severity, and found that caudate nucleus D2 receptor binding increased by up to 17% in the more severely affected twins when compared with their less severely affected twin siblings (Wolf et\u00a0al. 1996). This within-MZ twin discordance reflects unique environmental influences on D2-caudate receptor density.\nIn this study, we used prospective data of adult twins from the NTR, who have been followed between 1991 and 2002, and about whom information on a wide range of variables was collected every 2\u20133\u00a0years (Boomsma et\u00a0al. 2000). Differences between the MZ concordant and discordant groups were described using measures of anxiety and depression co-occurring with OC behavior. The aim of this explorative study was to replicate and extend the information from previous studies on both unique and shared environmental influences that might protect against or exacerbate OC behavior. Unique environmental factors were studied using within-discordant MZ twin pair comparisons. To study environmental factors shared by both twins of a pair, between-MZ pair comparisons were used. Parent data on level of education and on drinking and smoking behavior were used to compare the groups of twin pairs on these common environment influences. Further, measures of anxiety, depression and personality were compared between the parents of the concordant and discordant twin pairs, with the following reasoning: concordance between MZ twin pairs on OC behavior most likely results from genetic similarity between the twins of a pair. Thus, the contrasts between twin pairs who are concordant high and low reflect differences in genetic vulnerability to OC behavior. As a consequence, the parent scores on OC symptoms, on anxious depression and on neuroticism (the latter characteristics are known to be related to OC symptoms) are expected to reflect these differences in genetic vulnerability and therefore to be highest in the parents of the concordant high MZ pairs, to be intermediate in the parents of the discordant MZ pairs and to be low in the parents of the concordant low MZ pairs.\nFinally, longitudinal measures of psychopathology were studied to investigate age at onset of OC symptoms, anxiety and depressive symptoms in the concordant and discordant groups. Family studies have suggested that lower age at onset is associated with higher familiarity, possibly reflecting higher genetic load (Delorme 2005). We hypothesized that the concordant high MZ twin pairs, in whom the OC symptoms are theoretically more genetically determined, would show lower age at onset than the high-scoring twins of the discordant group in whom unique environmental factors might be more important.\nMethod\nSample selection\nThe data of this study originate from a longitudinal study in twin families registered with the NTR (Boomsma et\u00a0al. 2002). Since 1991, twins and their families received a survey by mail every 2\u20133\u00a0years containing questionnaires about health, personality, life events, perinatal circumstances and lifestyle. The 2002 survey formed the starting point of this study. The Padua Inventory Abbreviated (PI ABBR) was added to the 2002 wave of data collection and was derived from the Padua Inventory-Revised version (PI-R), a widely used self-report inventory on OC symptoms (Sanavio 1988; van Oppen 1992). The PI-R is a 41-item self-report instrument that measures OC symptoms on a 0\u20134 scale, and contains five subscales: washing, checking, rumination, precision and impulses (van Oppen et\u00a0al. 1995). It has been validated in the Netherlands, shows good psychometric qualities, and moderately correlates with the Y-BOCS symptom checklist, a clinician-derived checklist on OC symptoms (Denys et\u00a0al. 2004). For the purpose of this epidemiological twin study, the PI-R was reduced to 12 items. Item choice was based on two items of each subscale with highest factor loadings in a previous validation study (van Oppen et\u00a0al. 1995), and with one additional item for each of the more equivocal obsession subscales: rumination and impulses. The PI-R ABBR is shown in Table\u00a01. To investigate its psychometric qualities psychometric analyses have been conducted in three groups derived from an earlier study by van Oppen et\u00a0al. (1995). These groups encompassed a population-based control group (n\u00a0=\u00a0428), a psychiatric control group (n\u00a0=\u00a0272) and a clinical OCD group (n\u00a0=\u00a0120); for an extensive description of the study groups (see van Oppen et\u00a0al. 1995). Cronbachs\u2019 \u03b1 of the scale was 0.73, which is an indication of good internal consistency. Analyses of variance (ANOVAs) of PI-R ABBR scores within the three groups revealed a significant main between-group effect (p\u00a0<\u00a00.0001). Post-hoc t-tests showed that the mean PI-R ABBR OC score for the OCD group (20.7\u00a0\u00b1\u00a08.1) was significantly higher than scores of the psychiatric control group (12.4\u00a0\u00b1\u00a07.4) as well as the population control group (6.6\u00a0\u00b1\u00a05.6; p\u00a0<\u00a00.0001 in both comparisons). To investigate whether the PI-R ABBR can accurately screen for OCD, and to establish cut-points of OC behavior, receiver operating characteristic (ROC) analyses were carried out. ROC analyses use the association between sensitivity and specificity to derive an area under the curve (AUC), which indicates how well a measure distinguishes between case positives (i.e., OCD group) and case negatives (i.e., psychiatric controls or population controls) irrespective of the base rate. A value of 0.50 of the AUC indicates chance level and 1.0 indicates a perfect diagnostic tool (Swets 1996; McFall and Treat 1999). The AUC for the PI-R ABBR when compared with clinical controls was 0.78 (95% CI\u00a0=\u00a00.73\u20130.83). When compared with the population controls, the AUC was 0.93 (95% CI\u00a0=\u00a00.90\u20130.95). At the best cut-off point of 16 (i.e., maximum difference between sensitivity and 1-specificity), the sensitivity was 0.74 with a specificity of 0.72, when compared with clinical controls. (A detailed description of the ROC analyses on the PI-R ABBR is available upon request through the first author.)\nTable\u00a01The Padua Inventory-Revised abbreviated (PI-R ABBR)PI-R ABBROriginal factor1In certain situations, I am afraid of losing my self-control and doing embarrassing thingsImpulses2I check and recheck gas and water taps and light switches after turning them offChecking3I feel obliged to follow a particular order in dressing, undressing and washing myselfPrecision4When I see a train approaching I sometimes think I could throw myself under its wheelsImpulses5I return home to check doors, windows, drawers etc., to make sure they are properly shutChecking6When I start thinking of certain things, I become obsessed with themRumination7I feel I have to repeat certain numbers for no reasonPrecision8Unpleasant thoughts come into my mind against my will and I cannot get rid of themRumination9My thoughts constantly go astray, therefore I find it difficult to attend to what is happening around meRumination10I sometimes have to wash or clean myself dimply because I think I may be dirty or \u2018contaminated\u2019Washing11I get upset and worried at the sight of knives, daggers and other pointed objectsImpulses12If I touch something which I think is \u2018contaminated\u2019, I immediately have to wash or clean myselfWashingFig.\u00a01The 2002 NTR wave using the PI-R abbreviated\nOf the adult twins, 2,672 pairs, their family members and\u2013in some instances\u2014their spouses (a total of 9,950 individuals) returned the survey. Monozygotic twin pairs were selected on the basis of high or low scores on the PI-R ABBR. Using the stringent criteria derived from the analyses described above, discordant, concordant high and concordant low MZ twin pairs were selected. Twin pairs were considered to be discordant when one twin scored >17 (in the clinical range), and his\/her MZ twin sibling scored <7 (population control range). Pairs were considered to be concordant high when both twins scored >17, and concordant low when both twins scored <7. Information on zygosity from DNA polymorphisms was available in 19 MZ twin pairs (25%) of the final sample. When DNA polymorphisms were not available zygosity was determined from questions about physical similarity of the twins and confusion of the twins by family members, friends and strangers. Overall, agreement between zygosity diagnoses based on questionnaire and DNA data is 97% (Willemsen et\u00a0al. 2005). After exclusion of incomplete pairs, 25 MZ discordant pairs, 17 MZ concordant high pairs and 521 MZ concordant low pairs were identified. Concordant low pairs were matched on age and sex with concordant high pairs and oversampled, so that 34 concordant low pairs were finally retained (Fig.\u00a01).\nOf the final 76\u00a0MZ twin pairs selected for this study, 18 pairs participated in wave 2002 only, 28 pairs participated in two waves, 4 pairs in three, 8 pairs in four, 13 pairs in five, and 4 pairs in all six waves.\nMeasures and instruments\nThe NTR survey contains a broad range of longitudinal measurements taken at six time points between 1991 and 2002, as well as cross-sectional measurements. Information is obtained on life events, perinatal adversities, physical and mental health, lifestyle factors such as physical activity, religiosity, drinking, smoking and drug behavior, and on demographic variables such as relationships, number of children, level of education, living situation, and work status. Since this is an exploratory study, all available information was taken into account.\nReligiosity was assessed by asking whether the respondent had had a religious upbringing (yes\/no), the person\u2019s current religion, and whether the respondent currently was an active church member.\nOn alcohol and smoking behavior, respondents were questioned about their consumption ever, in the past year and past month, as well as the number of cigarettes or glasses of alcohol per week. Alcohol dependence was assessed by the CAGE (four questions) (Bush et\u00a0al. 1987).\nThe occurrence of negative life events throughout the lifespan was measured in the 2002 survey, using an adapted version of the Dutch life event scale (Schokverwerkings Inventarisatie Lijst\u00a0=\u00a0SchIL) (van der Velden et\u00a0al. 1992). This scale gathers information on: death of a spouse, father, mother, child, sibling or significant other; serious illness or injury of self or a significant other; divorce\/break-up of a relationship; traffic accident; violent and sexual assault or rape, and robbery. Response categories are: never experienced; 0\u20136\u00a0months ago; 6\u201312\u00a0months ago; 1\u20135\u00a0years ago, and more than 5\u00a0years ago.\nData on anxiety and depression were available at most time points between wave 1 and 6, and were assessed with: the Spielberger State-Trait Anxiety Inventory, trait scale (Spielberger et\u00a0al. 1970; van der Ploeg 1979), and the young adult self-report (YASR), anxious-depressed subscale (Achenbach 2000; Verhulst et\u00a0al. 1997). Questionnaires between waves 1 and 4 (between 1991 and 1997) also contained the 8-item OC symptom subscale of the YASR (Nelson et\u00a0al. 2001; Geller et\u00a0al. 2006). Neuroticism, somatic complaints and extraversion were measured with the Amsterdamse Biografische Vragenlijst (ABV; Amsterdam Biographical Questionnaire) (Wilde 1970). The ABV neuroticism and extraversion scales are very similar to the Eysenck Personality Questionnaire neuroticism and extraversion subscales (Eysenck and Eysenck 1964), and contain answer categories: yes\/no\/don\u2019t know. The satisfaction with life scale and the subjective happiness scale (Lewis and Joseph 1995), a combined 10-item scale with scoring possibilities between 1 and 7 were taken, and the Rosenberg self-efficacy scale, a 10-item scale scoring between 1 and 4 (Rosenberg 1965; Helbing 1982).\nSocio Economic Status (SES) in 2002 was assessed using a full description of the occupation of the twins according to the descriptions provided by the Central Office for Statistics in the Netherlands. The work level was coded into three levels based on the mental complexity of the work, ranging from low skilled (1) to academic work (3). Living situation was coded between 1 and 3 (1\u00a0=\u00a0with parents; 2\u00a0=\u00a0alone; 3\u00a0=\u00a0with partner).\nFinally, we used child-derived information on their parents\u2019 level of education. Direct parent information was used to collect information on their smoking and drinking behavior and on their scores of OC behavior, anxiety and depression.\nStatistical analyses\nWithin-pair analyses\nWithin-discordant pair differences between the high and low-scoring twins on the PI-R ABBR were calculated using paired t-tests (t-tests for two related samples) for continuous data, Wilcoxon signed- rank tests for ordinal data and McNemar \u03c72 tests of matched pairs for nominal data.\nBetween-pair analyses\nVariables that measure psychological health, as well as measures of environmental influences on OC symptoms were compared between the concordant high, the concordant low and the discordant MZ twin pairs, using one-way ANOVAs for continuous data, Kruskal\u2013Wallis tests for ordinal data and \u03c72 tests for nominal data. Post hoc comparisons were conducted using post hoc Scheff\u00e9\u2019s (continuous data) and Mann\u2013Whitney U-tests (ordinal data). Post hoc Scheff\u00e9 testing, although more liberal than Bonferroni correction, provide some correction of type I error. Two-tailed probabilities were used in all analyses, since we had no clear expectation of the direction of the findings.\nTo adjust for correlated error in the between-group comparisons of common environment variables, separate regression analyses (multiple regression for continuous measures and logistic regression for categorical measures) were conducted in STATA 9.2 for these variables (StatCorp, College Station, TX, USA). The robust cluster option was used to account for nonindependence of the twin pairs on the variables that reflected common environmental influences (i.e., caesarean section, birth weight, and religious upbringing of the twin; parental death and divorce, death of a sibling, and level of education, alcohol use and smoking behavior of the parents). Alpha was set at 0.05.\nResults\nWithin-pair analyses of discordant pairs\nTwenty-five MZ twin pairs discordant on OC behavior were included, of whom 18 pairs were female. Their mean age was 29.6\u00a0years (SD 6.8\u00a0years). Mean PI-R ABBR OC scores in the high-scoring twins of the discordant pairs were 21.4 (SD 5.9), in the low scoring twins 4.5 (SD 2.0).\nHealth and lifestyle characteristics\nHigh-scoring twins of the discordant pairs experienced lower general health (p\u00a0=\u00a00.03) and more impediments in physical activity (p\u00a0<\u00a00.001) than the low-scoring co-twins. The duration of the current relationship of the high-scoring twins tended to be shorter (p\u00a0=\u00a00.06), and they tended to have fewer children (p\u00a0=\u00a00.07). They were less satisfied with life (p\u00a0=\u00a00.02), less happy (p\u00a0=\u00a00.001) and had lower self-efficacy scores (p\u00a0=\u00a00.006). They showed no differences with respect to birth weight, birth order, church participation, drinking or smoking behavior, nor on level of education, work status, living situation, or number of (mental) health contacts. The high-scoring twins of the discordant pairs scored significantly higher on the YASR anxious-depressed subscale (p\u00a0<\u00a00.001), on the neuroticism subscale (p\u00a0<\u00a00.001), on the ABV subscale of somatic complaints (p\u00a0<\u00a00.001), and on STAI-trait (p\u00a0<\u00a00.001). On the ABV extraversion scale, no within-pair differences were found (Table\u00a02).\nTable\u00a022002 wave of data collection: within-discordant twin pair characteristicsLow risk twin, mean (SD)High risk twin, mean (SD)Test statistic*p-ValueBirth weight (g)2,189 (806)2,028 (667)0.9n.s.Birth order (first born)n\u00a0=\u00a010n\u00a0=\u00a0141.7n.s.General health (1\u20135)4.1 (0.7)3.8 (0.6)2.30.03Mental health contacts ever yesn\u00a0=\u00a07n\u00a0=\u00a090.5n.s.Sumscore impediments physical activity43.5 (12.5)53.9 (12.4)\u22124.2<0.001Number of persons drinking evern\u00a0=\u00a023n\u00a0=\u00a0230n.s.Number of drinks per week (past 12 months)11.1 (1.4)13.4 (0.7)0.19n.s.CAGE score alcohol dependence4.0 (0.2)4.3 (0.7)\u22121.4n.s.Duration current relation (years)5.9 (7.4)3.0 (0)1.97n.s.Number of children1.1 (1.2)0.7 (0.6)1.87n.s.Education level self (1\u201313)7.9 (3.0)8.3 (2.6)\u22120.8n.s.Education level partner (1\u201313)8.3 (3.6)8.1 (3.1)\u22121.2n.s.Living situation (1\u20134)2.8 (0.8)2.7 (0.8)\u22121.3n.s.PI-R ABBR OC scale4.5 (2.0)21.4 (5.9)\u221213.7<0.001YASR anxious depression scale4.3 (2.8)11.6 (4.3)\u22126.4<0.001ABV extraversion51.0 (16.7)45.6 (12.5)1.1n.s.ABV neuroticism48.6 (23.1)85.3 (27.4)\u22125.8<0.001ABV somatic complaints16.6 (5.1)24.4 (10.3)\u22124.0<0.001STAI-trait31.6 (4.7)46.4 (10.8)\u22126.5<0.001Satisfaction with life scale scores27.4 (4.1)23.8 (7.0)2.30.02Happiness scores22.7 (3.9)17.9 (5.8)230.001Self-efficacy scores31.3 (4.0)27.5 (4.7)2.90.006PI-R ABBR, Padua Inventory-Revised Abbreviated; OC, obsessive-compulsive; YASR, young adult self-report; ABV, Amsterdamse Biografische vragenlijst; STAI, State trait Anxiety Inventory; SBL, Spannings behoefte Lijst (sensation seeking list); CI, confidence interval; n.s., not significant\nUnique environment influences\nThe only within-pair difference found on unique life events, was the tendency of the high-scoring twins of the discordant pairs to have experienced more sexual assault than the low-scoring twins (p\u00a0=\u00a00.08). All persons who had experienced sexual assault were women. Two low-scoring twins of the discordant pairs reported on sexual assault, versus five high-scoring twins. The low-scoring twins and four of the five high-scoring twins of the discordant pairs reported to have experienced the assault more than 5\u00a0years ago, versus one twin who had experienced sexual abuse between 1 and 5\u00a0years ago (Table\u00a03).\nTable\u00a03Within-discordant twin comparisons\u2014unique life eventsLow OC twin, mean (SD)High OC twin, mean (SD)p-ValueBirth weight (g)2,189 (806)2,028 (667)n.s.Disease self (0\u20132)0.08 (0.4)0.26 (0.6)n.s.Disease child (0\u20132)0.09 (0.4)0.6 (0.1)n.s.Disease partner (0\u20132)0.09 (0.4)0n.s.Disease significant other (0\u20132)0.82 (0.9)0.82 (0.9)n.s.Death child (0\u20132)00n.s.Death partner (0\u20132)00n.s.Death significant other (0\u20132)1.1 (0.9)1.0 (0.9)n.s.Sexual abuse* (0\u20132)0.17 (0.5)0.43 (0.8)0.08Violence (0\u20132)0.17 (0.6)0.17 (0.6)n.s.Relationship termination (0\u20132)0.52 (0.8)0.82 (0.9)n.s.Theft (0\u20132)0.47 (0.8)0.56 (0.8)n.s.Traffic accident (0\u20132)0.52 (0.8)0.38 (0.7)n.s.Dismissal (0\u20132)0.30 (0.7)0.48 (0.7)n.s.Total score life events2.35 (1.9)2.76 (1.7)n.s.\nLongitudinal data\nYoung adult self-report OC subscale scores, taken in 1991, 1995 and 1997, revealed significant differences between high and low-scoring twins of the discordant pairs in 1997 (p\u00a0=\u00a00.007). Further, scale scores between 1991 and 2002 revealed significant within-pair differences on the YASR anxious-depressed subscale from 1997 onward (p\u00a0=\u00a00.001), on the neuroticism subscale from 1993 onward (p-value between 0.01 and <0.001 at wave 2\u20135), on the ABV subscale of somatic complaints from 1997 onward (p-value between 0.015 and <0.001), and on STAI-trait scores from 1997 onward (p-values between 0.007 and <0.001).\nBetween-pair analyses of concordant and discordant pairs\nSeventeen MZ twin pairs were included who were concordant high on OC behavior, of whom 14 pairs were female. Their mean age was 30.0\u00a0years (SD 11.2\u00a0years), mean PI-R ABBR OC scores were 23.7 (SD 6.7). Thirty-four MZ twin pairs were included who were concordant low on OC behavior, of whom 28 pairs were female. Their mean age was 30.0\u00a0years (SD 11.3\u00a0years), mean PI-R ABBR OC scores were 3.8 (SD 2.2).\nHealth and lifestyle characteristics\nThe concordant low group generally experienced the best health, with the discordant group scoring intermediate between high and low concordant groups. Members of the discordant group more often had a spouse than the concordant high group, and were living with a spouse more often than both concordant groups. No between-group differences were found for smoking behavior. For drinking behavior, the concordant high group showed the highest scores on alcohol dependence (p\u00a0=\u00a00.02 and 0.04 in comparison with the concordant low and discordant group), although they scored intermediate between the low and discordant groups on current number of drinks per week. On religious upbringing, there were no significant differences between the study groups. Interestingly, the concordant low MZ twin pairs, as well as their spouses, reported to have a higher level of education than the concordant high and discordant twin pairs (p-values 0.02 in both comparisons) (Table\u00a04).\nTable\u00a042002 wave of data collection: between concordant and discordant twin pair health and lifestyle characteristicsConcordant Low twin pairs, mean (SD)Concordant high twin pairs, mean (SD)Discordant twin pairs, mean (SD)Low\u2013high p-valueLow\u2013discordant p-valueHigh\u2013discordant p-valueGeneral health (1\u20135)4.4 (0.7)3.6 (1.2)4.1 (0.7)<0.0010.007n.s.Mental health contacts ever yesn\u00a0=\u00a08 (12%)n\u00a0=\u00a021 (61%)n\u00a0=\u00a016 (32%)<0.0010.0060.009Impediments physical activity41.3 (12.6)53.0 (17.1)43.5 (12.5)0.001n.s.0.03Specialized medical treatment ever yesn\u00a0=\u00a011 (16%) n\u00a0=\u00a015 (44%)n\u00a0=\u00a011 (22%)0.002n.s.0.04Currently active in church (1\u20133)0.8 (0.7)0.5 (0.7)0.9 (0.8)n.s.n.s.n.s.Number of persons drinking evern\u00a0=\u00a062 (91%)n\u00a0=\u00a019 (56%)n\u00a0=\u00a035 (70%)n.s.0.003n.s.Number of drinks per week (past 12 months)2.7 (1.4)2.3 (1.5)1.9 (1.2)n.s.0.01n.s.CAGE score alcohol dependence4.1 (0.5)4.5 (0.8)4.2 (0.5)0.02n.s.0.04Number of persons smoking evern\u00a0=\u00a020 (29%)n\u00a0=\u00a014 (41%)n\u00a0=\u00a018 (36%)n.s.n.s.n.s.Number of cigarettes per day (1\u20137)4.1 (1.1)4.7 (1.1)3.8 (1.0)n.sn.s.0.07Number of persons with partnern\u00a0=\u00a041 (60%)n\u00a0=\u00a017 (50%)n\u00a0=\u00a035 (70%)n.s.n.s.0.04Children yesn\u00a0=\u00a016 (23%)n\u00a0=\u00a08 (23%)n\u00a0=\u00a025 (50%)n.s.0.0030.015Education level self (1\u201313)9.4 (2.3)8.27 (2.8)8.24 (2.8)0.020.02n.s.Education level partner (1\u201313)9.1 (2.8)6.7 (3.7)8.20 (3.4)0.006n.s.n.s.Living situation (1\u20133)2.4 (0.8)2.2 (1.0)2.8 (0.8)n.s.0.040.01PI-R ABBR OC scale3.8 (2.2)23.7 (6.7)12.9 (9.5)<0.001<0.001<0.001YASR anxious depression scale2.9 (4.7)14.5 (19.3)8.7 (12.6)<0.001<0.001<0.001ABV extraversion62.8 (16.2)46.4 (17.7)48.3 (17.7)<0.001<0.0010.001ABV neuroticism36.2 (18.9)92.8 (19.7)66.6 (31.1)<0.001<0.001n.s.ABV somatic complaints15.7 (3.7)27.5 (8.3)20.3 (8.8)<0.0010.003<0.001STAI-trait29.4 (6.4)36.9 (7.9)37.6 (7.5)<0.001<0.001<0.001Satisfaction with life28.7 (4.0)19.8 (7.2)25.6 (5.9)<0.0010.010.001Happiness24.2 (3.2)16.2 (5.7)20.4 (5.4)<0.001<0.0010.01Self-efficacy33.7 (3.9)25.5 (3.9)29.4 (4.7)<0.001<0.001<0.001\nOn life events, the concordant high MZ twin pairs reported more often that they had been dismissed from work than the concordant low scoring pairs (p\u00a0=\u00a00.04), with the discordant pairs scoring between the concordant high and low pairs. Further, the discordant pairs reported more often to have been sexually assaulted in comparison with both the concordant low and high-scoring pairs; n\u00a0=\u00a07 individuals in the discordant group versus n\u00a0=\u00a00 and n\u00a0=\u00a01 individual in the concordant high and low groups (p\u00a0=\u00a00.02 and 0.03 respectively). Finally, the discordant pairs reported more traffic accidents than the other groups (p\u00a0=\u00a00.05 and 0.02 when compared with the concordant low and high pairs respectively).\nOn psychological scale scores, the concordant high group scored, as expected, overall higher on the PI-R-ABBR (p-values <0.001), the YASR anxious-depressed scale (p-values <0.001), ABV neuroticism (p\u00a0<\u00a00.001 in low\u2013high comparison; p\u00a0=\u00a0n.s. between high and discordant twin pairs), somatic complaints (p-values between <0.001 and 0.003), and STAI-trait anxiety (p-values <0.001). Further, the concordant high group had lower scores on ABV extraversion (p-values 0.001), satisfaction with life (p-values between 0.01 and 0.001), happiness (p-values between 0.01 and <0.001) and self-efficacy (p-values <0.001) than the concordant low and discordant groups.\nShared environment influences\nBetween-group analyses revealed that the discordant group had the lowest rate of caesarean sections (p-values of 0.005 and 0.006 in comparison with the concordant low and high groups), while there was no difference between the concordant groups. The discordant group had the lowest birth weight (p\u00a0=\u00a00.008 compared with the concordant low pairs and p\u00a0<\u00a00.001 compared with the concordant high pairs). There were no between-group differences on level of education of the parents (p-values\u00a0=\u00a0n.s. in all comparisons). There were no between-pair differences in the occurrence of parental death. The concordant low MZ pairs reported most on death of a sibling (p\u00a0=\u00a00.05 between concordant low and high pairs). There were no between-group differences with respect to relationship termination of the parents. On both drinking and smoking behavior of the parents, surprisingly the concordant low parents reported more drinking than the discordant parents, although alcohol consumption as well as number of cigarettes were low on average (Table\u00a05).\nTable\u00a05Between twin-pair comparisons: comparison of common environment characteristics (after correction for interrelatedness)Concordant low twin pairs, mean (SD)Concordant high twin pairs, mean (SD)Discordant twin pairs, mean (SD)Low\u2013high p-valueLow\u2013discordant p-valueHigh\u2013discordant p-valueCaesarean section (yes)n\u00a0=\u00a05 pairsn\u00a0=\u00a03 pairsn\u00a0=\u00a00 pairsn.s.0.008<0.001Birth weight (g)2,650 (876)2,685 (795)2,109 (736)n.s.0.0040.009Religious upbringing yesn\u00a0=\u00a045 (67%)n\u00a0=\u00a016 (47%)n\u00a0=\u00a034 (69%)n.s.n.s.n.s.Education level fathera (1\u201313)7.5 (4.0)5.5 (3.7)5.7 (3.5)n.s.n.s.n.s.Education level mothera (1\u201313)6.3 (3.7)5.2 (3.4)4.7 (3.0)n.s.n.s.n.s.Death mother (0\u20132) yesn\u00a0=\u00a02 (3%)n\u00a0=\u00a03 (10%)n\u00a0=\u00a04 (8%)n.s.n.s.n.s.Death father (0\u20132)n\u00a0=\u00a012 (19%)n\u00a0=\u00a07 (24%)n\u00a0=\u00a06 (12%)n.s.n.s.n.s.Death sibling (0\u20132)n\u00a0=\u00a06 (10%)n\u00a0=\u00a00n\u00a0=\u00a01 (2%)0.05n.s.n.s.Relationship termination parents (0\u20132)bn\u00a0=\u00a04 (14%)n\u00a0=\u00a02 (25%)n\u00a0=\u00a01 (7%)n.s.n.s.n.s.Number of parents drinking (ever; yes)b91%100%74%0.06n.s.0.04Number of drinks\/week parents (1\u20137)b3.5 (4 drinks\/week)2.7 (2\u20133 drinks\/week)2.3 (1\u20132 drinks\/week)n.s.0.03n.s.Number of parents smoking ever (yes)b71%89%48%n.s.0.060.07Number of cigarettes\/day parents (1\u20137)b4 (6\u201310 cig\/day)5(11\u201320 cig\/day)5 (11\u201320 cig\/day)n.sn.s.n.s.aReported by twin children and by parentsbDirect parent data\nLongitudinal data\nYoung adult self-report OC scale scores revealed significant differences between low and high-scoring twin pairs in the 1995 (p\u00a0=\u00a00.02) and 1997 wave (p\u00a0<\u00a00.001). YASR anxious-depressed scale scores revealed significant differences between the concordant low and high groups from 1991 on (p-values\u00a0<\u00a00.05 in all comparisons). ABV extraversion scores revealed significant between-group differences from 1993 onward (p-values between <0.001 and 0.008), whereas ABV neuroticism scores revealed significant between-group differences at all waves (p-values between 0.05 and <0.001). ABV somatic complaints showed significant between-group differences from 1997 on (p-values <0.001).\nParent data\nParent data were available for 66 persons; the 34 parents of concordant low twin pairs had a mean age of 53.6 years (SD 5.9), a PI-R ABBR mean score of 5.2 (SD 3.8); 9 parents of concordant high twin pairs had a mean age of 51.6 years (SD 2.5), and a PI-R ABBR mean score of 11.7 (SD 3.8); and 23 parents of discordant twin pairs had a mean age of 57.5 years (SD 6.9) and a PI-R ABBR mean score of 9.9 (SD 5.7). Between-group analyses of psychological scale scores showed that the parents of the discordant pairs scored between the parents of the concordant low and high pairs on anxious depression, satisfaction with life, happiness and self-efficacy scales. On somatic complaints and extraversion they showed higher scores than the other groups. On the PI-R ABBR, STAI trait and neuroticism they scored equal to the parents of the concordant high groups (Table\u00a06).\nTable\u00a062002 wave of data collection: between-parents comparisons of psychological scalesParent concordant low, mean (SD)Parent concordant high, mean (SD)Parent discordant, mean (SD)High\u2013low p-valueLow\u2013discordant p-valueHigh\u2013discordant p-valuePI-R ABBR OC scale5.2 (3.8)11.7 (3.8)9.9 (5.7)0.0020.002n.s.YASR anxious depression scale4.5 (3.5)11.7 (4.2)7.7 (4.3)<0.0010.020.04ABV extraversion53.4 (17.5)44.9 (12.962.4 (13.3)n.s.n.s.0.02ABV neuroticism36.3 (25.3)79.8 (26.5)62.7 (13.3)<0.001<0.001n.s.ABV somatic complaints16.3 (4.5)21.2 (6.6)26.9 (2.3)0.01<0.0010.005STAI-trait29.5 (6.7)44.1 (7.1)47.7 (4.1)<0.001<0.001n.s.Satisfaction with life28.1 (4.9)18.6 (8.5)25.5 (7.1)0.001n.s.0.03Happiness23.5 (3.5)16.3 (6.9)15.1 (3.3)<0.001<0.001n.s.Self-efficacy32.1 (3.8)28.0 (4.2)25.3 (2.6)0.01<0.001n.s.PI-R ABBR, Padua Invetroy-Revised Abbreviated scale; OC, obsessive-compulsive; YASR, young adult self-report; ABV, Amsterdamse Biografische vragenlijst; STAI, State trait Inventory\nDiscussion\nThe most important aim of this MZ twin study has been to explore unique and shared environmental factors involved in OC symptoms.\nUnique and shared environmental factors\nThe within-twin pair comparisons of the MZ discordant pairs were primarily used to study unique environmental factors associated with OC symptoms. Although the discordant pairs were genetically identical, were raised at the same time in the same family, and were selected from an epidemiological sample, the twins differed substantially on several measures across time. The twins who scored low on OC symptoms reported to feel healthier, to be more satisfied with life, happier and more self-efficient than their high scoring MZ twin siblings. They tended to have longer relationships and more children. Further, they had lower scores on anxious depression and on neuroticism, mostly from 1997 onward. The most striking unique environmental factor to explain these within-discordant pair differences was the relatively high frequency of sexual assault experienced by the high-scoring twins of the discordant pairs in comparison with their low-scoring twin siblings, which is in line with previous reports on this issue (Lochner et\u00a0al. 2004). However, two of the low-scoring twins of the discordant pairs reported on sexual assault as well, underscoring the complexity of presumed causality in the interplay between environmental and genetic factors in OCD. Interestingly, no sexual assault was reported by the concordant high-scoring MZ twin pairs. Thus, although the high-scoring respondents of the discordant pairs show similar OC symptomatology when compared with the concordant high MZ pairs, the pathways along which similar OC symptoms develop seem to differ between the high-scoring discordant twins on the one hand, and the high-scoring concordant pairs on the other. Although one can only speculate about causal relationships in this explorative study, the OC symptoms in the high-scoring twins of the discordant pairs seem to be associated more with environmental stressors (i.e., sexual assault) than are the OC symptoms in the concordant high-scoring pairs.\nThe between-twin pair comparisons to study environmental factors that are shared by the twins of a pair revealed low birth weight and low rates of caesarean section in the discordant pairs. We were unable to take the relationship between low birth weight and gestational age into account in the analyses, and were therefore unable to distinguish whether the study persons had been pre- or dysmature at birth. However, a recent twin study showed that low birth weight in itself resulted in an increase in problem behavior in later life. Children with low birth weight appeared to be more vulnerable to negative environmental factors than normal birth weight children (Wichers et\u00a0al. 2002), possibly in association with a negative interaction between genetic vulnerability for problem behavior and low birth weight. Low birth weight can be indicative of a range of prenatal adversities such as maternal psychological stress, alcohol, drug abuse, or smoking during pregnancy. These adversities cause immunological challenge, and lead through various mechanisms to a diversity of psychopathology, including anxiety and depression (Meyer et\u00a0al. 2006; Huizink et\u00a0al. 2004; Nigg and Breslau 2007). In this study, we did not find an indication of alcohol, smoking or drug abuse in the parents of the twin pairs, but other sources of prenatal stress can not be ruled out. Further, no discordant twin pairs were born through caesarean section, as opposed to eight concordant pairs. This is remarkable in light of the fact that in general, caesarean section is carried out more often in multiple pregnancies, especially when one suspects low birth weight in the fetuses (Colla et\u00a0al. 2001). Although it might be a chance finding, one can speculate that\u2014since caesarean section is intended to decrease perinatal adversities\u2014the discordant group of this sample has been \u2018under treated\u2019, providing an additional negative environmental factor to explain between-group differences.\nThere were no between-group differences in rates of parental death or death of a sibling, nor in frequency of relationship termination between the parents, life events that reflect shared environmental stressors. In general, rates of these life events were low in this relatively young twin group, possibly hampering detection of between-twin pair differences. Alcohol use by the mother (especially during pregnancy) as well as maternal smoking are considered to be common environmental risk factors for problem behavior such as ADHD (Smidts 2007). However, neither alcohol use nor smoking behavior of the parents was associated with OC symptoms in the concordant high or discordant groups of this study. Further, there was no association between OC symptoms and a religious upbringing in the study groups, which is in line with the literature on the lack of association between religiosity and OCD (Tek et\u00a0al. 2004), but deviates from reports of a protective effect of religion on other forms of psychopathology such as alcohol and drug abuse, depression and disruptive behavior (Kendler et\u00a0al. 1999). Apparently, different problem behavior is associated with different environmental risk factors.\nFinally, level of education of the parents (as a measure of socio-economic status, a risk factor reported in OCD) was not found to be associated with OC symptoms in this study, although the parents of the twins who were concordant low on OC symptoms tended to have a higher level of education than the other groups, a difference that may have failed to reach significance due to the small sample size.\nFinally, between-twin pair comparisons on unique life events revealed an elevated rate of dismissal in the concordant high-scoring twin pairs compared with the other pairs. Since dismissal typically represents a unique negative environmental influence on each twin of a pair, instead of being an environmental influence shared between the twins of a pair, its elevated rate among the high-scoring concordant MZ pairs is better explained as being the consequence of OC symptomatology rather than causing OC symptoms; elevated dismissal rates in these OC twin pairs might result from over-scrupulosity and slowness in work\u2014characteristics well known in OC symptomatology\u2014and subsequent dysfunction.\nHealth and lifestyle characteristics\nOverall, as expected, the concordant low pairs reported highest scores of health, fewest mental and medical health contacts, and lowest scores on OC symptoms, anxiety and depression, neuroticism, and somatic complaints compared with the other groups. Further, they reported to be more extravert, more satisfied with life, happier and more self-efficient, with the discordant pairs scoring in between the concordant low and high pairs. On alcohol use, the concordant high-scoring twin pairs scored in between the low and discordant twin pairs over the past 12\u00a0months, with the number of drinks per week well below the quantity required to fulfil criteria for alcohol abuse or dependence according to DSM-IV criteria. However, subjective reports of alcohol withdrawal and dependence (CAGE scores) were increased in the high-scoring twin pairs compared with the low-scoring and discordant pairs. This might reflect increased scrupulosity and feelings of guilt, a well-known phenomenon in persons with OC symptoms, (Olatunji et\u00a0al. 2006), related to alcohol use and its toxic effects rather than a verifiable alcohol problem in the concordant high-scoring MZ pairs.\nA protective effect of level of education on OC symptoms was suggested by the finding of a higher level of education in the concordant low-scoring twin pairs than in the concordant high and the discordant twin pairs. Not only the concordant high-scoring twin pairs but also their spouses had a lower level of education, which suggests that low level of education and OC symptomatology might share genetic vulnerability. Deficits in encoding complex information and subsequent memory impairments have been reported in OCD (Buhlmann et\u00a0al. 2006; Deckersbach et\u00a0al. 2000). These (genetically determined) impairments possibly mediate low educational level. On the other hand, low level of education in the concordant high-scoring group might be a consequence of the OC symptomatology in itself, a notion that is supported by the literature (Sorensen et\u00a0al. 2004).\nThe longitudinal data\nAs expected, the longitudinal data on OC symptoms, anxiety and depressive symptoms in the concordant and discordant groups revealed an earlier age at onset of OC and related symptoms in the concordant high group (from 1991 on) than in the discordant group (mostly from 1997 on). This confirms data from family-based studies where an earlier age at onset was associated with higher familial load (do Rosario-Campos et\u00a0al. 2005). Thus, assuming that OC symptoms in the concordant high-scoring twin pairs are more genetically mediated than in the discordant pairs, this study is in line with clinical studies indicating that age at onset might be an important phenotypic characteristic that reflects differences in genetic characteristics underlying OCD (Delorme et\u00a0al. 2005).\nThe parent data\nAs parent scores on OC symptoms and related psychopathology were expected to reflect genetic vulnerability, we expected scores to be highest in the concordant high parents, to be intermediate in the discordant parents and to be low in the concordant low parents. On most measures of psychopathology, this assumption was confirmed. Thus, the intermediate scores in the parents of the discordant twins on OC, anxious-depression and neuroticism scales may be the consequence of the intermediate amount of genetic vulnerability to OC symptoms in this group. Therefore, these parent data suggest that the symptoms in the high-scoring twins of the MZ discordant group are likely to be the consequence of a moderate genetic vulnerability to OC pathology in addition to or in interaction with environmental mediators.\nLimitations\nFirst, sample size is small; although we sampled from a large group of MZ twins, only a small sample was retained due to the use of rigorous criteria. Consequently, especially in the within-discordant pair comparisons, some of the negative outcomes might in fact be the result of lack of power to detect within-pair differences. Alternatively we could have relaxed the stringent selection criteria, with the disadvantage of including twin pairs not scoring in the clinical range of OCD, thus representing an unclear group of problem behavior.\nSecond, considering the large number of tests relative to the small sample size, we only mildly corrected for type I errors. However, considering the exploratory nature of this study, an increase in the odds of type II errors by correction of type I errors was undesirable. Therefore, we decided to compromise by only applying a mild correction of type I errors (Perneger 1998).\nFinally, the database used in this study was not primarily designed to specifically inquire about environmental factors, leaving some questions unanswered, especially with respect to protective environmental mediators of OC symptomatology.\nConclusion\nThis study has been a first attempt to identify characteristics of the environment associated with OC symptoms using a twin study design. Some important environmental factors involved in OC symptomatology have been identified. Two crucial questions to be addressed in future studies are: (1) what is the differential impact of the various environmental mediators on OC symptoms, and under which circumstances and at which age are they most harmful? (2) Along which lines do the environmental factors found in this study operate? Do they add to genetic risk factors, are they causal in themselves, or do they operate through gene\u2013environment interaction? Future studies are needed to study the differential effects of environment and genes on phenotypes (and endophenotypes), and to elucidate the nature of the interplay between genes and environment.","keyphrases":["environmental factors","discordant","concordant","monozygotic twins","life-events","obsessive-compulsive symptoms"],"prmu":["P","P","P","P","U","R"]}
{"id":"J_Mol_Model-4-1-2386531","title":"Application of the PM6 method to modeling the solid state\n","text":"The applicability of the recently developed PM6 method for modeling various properties of a wide range of organic and inorganic crystalline solids has been investigated. Although the geometries of most systems examined were reproduced with good accuracy, severe errors were found in the predicted structures of a small number of solids. The origin of these errors was investigated, and a strategy for improving the method proposed.\nIntroduction\nThe semiempirical method PM6 [1] was designed primarily for the investigation of molecular species of biochemical interest. That is, the objective of parameter optimization was to reproduce the properties of molecules. When other semiempirical methods, e.g., MNDO [2, 3], AM1 [4], and PM3 [5, 6], were developed, initial reports indicated that they were significantly more accurate than earlier methods. But later, when each new method was used for modeling species that were significantly different from those used in the training set, average errors typically increased quite significantly. This unfortunate result was a natural consequence of the way in which semiempirical method development was done: if, during a survey, a systematic fault was identified, then the training set would be modified in such a way as to correct the fault. The close relationship between the survey set and the training set meant that, by its nature, properties of species in the survey set were reproduced with a higher accuracy than those of species not in the survey set.\nDuring the development of PM6, efforts were made to minimize the potential for this increase in error. Among these were the construction and use of very large survey and training sets. In contrast to previous methods in which the training set was a subset of the survey set, during the development of PM6 the training set was a superset of the survey set.\nNo solids were used in either the training set or the survey set while PM6 was being developed because inclusion of even one solid in the parameter optimization would have made the whole process extremely slow, which in turn would have precluded optimization of the parameters in any reasonable time. Because of this, solids were excluded from the parameterization, and therefore they form an ideal, clearly defined set of systems for testing the applicability of PM6 to species that were not used in the development of the method.\nTheory\nThere are several problems associated with solid-state calculations that do not exist when discrete molecules are modeled, all of which are related to the fact that there are an infinite number of interacting atoms. The most obvious consequence of this is that the electric potential experienced by each of these atoms is the result of the contributions of an infinitely large number of electrostatic terms arising from the partial charges of all the other atoms. Another implication is that the number of one-electron wavefunctions contributing to the density matrix during the solution of the self-consistent field (SCF) equations is also infinite. Various techniques have been developed for solving these problems. Thus, in all solid-state methods, the assumption is made that the wavefunctions exhibit a perfect periodicity; this assumption is formalized in the Born\u2013von K\u00e1rm\u00e1n [7] periodic boundary conditions.\nThe electrostatic contribution or Madelung integral can be solved analytically using the Ewald sum [8]. In this procedure, an elegant mixture of real and reciprocal space contributions is used in the evaluation of the potential. To assist in the solution of the SCF equations, the near infinite number of occupied wavefunctions contributing to the density matrix is replaced by an integration over the Brillouin zone. In turn, this integration is approximated by a Simpson\u2019s rule technique involving a weighted sampling of points within the zone.\nSeveral complete procedures have been developed for modeling solids using semiempirical methods. One of these, the MOSOL program [9], used sampling of the Brillouin Zone but, because it used complex mathematics, it was impractical for application to anything more complicated than simple binary solids. If the unit cell used is sufficiently large, then, instead of sampling the Brillouin Zone using a regular mesh of points, only one point need be used and, if the point chosen is at the origin of k-space, i.e. the \u0393 point, then complex mathematics can be avoided entirely. This is the basis for the large unit cell [10] or cluster [11] approximation. More recently, Gale [12] has addressed the problem of solving the Ewald sum when neglect of diatomic differential overlap (NDDO) methods are used, and developed a technique that would allow the Madelung integral to be solved more rapidly. In turn, this has allowed the structures and energies of some crystalline oxides, such as corundum and some of the polymorphs of silica, and of ice, to be modeled.\nDuring the development of a procedure to allow PM6 to be applied to solids, various deficiencies and limitations were found in earlier procedures. Some of these, and the resulting modifications that had to be made in order to allow PM6 to be used for modeling solids, will now be described.\nNDDO error\nThe NDDO methods pioneered by Dewar and Thiel use the Dewar-Sabelli [13\u201315]-Klopman [16] (DSK) approximation, Eq.\u00a01, which is equivalent to the Ohno approximation [17], for the two-electron two center integral \u03b3AB involving atoms A and B separated by a distance RAB.\nThe DSK approximation has the correct behavior at the extremes. That is, it converges to the exact point-charge expression as the interatomic distance becomes very large, and also converges to the exact two-electron, one-center term, GA, when the interatomic separation becomes zero. Additionally, it has good behavior at chemical bonding distances. Over the past 40\u00a0years, the DSK approximation has proven very successful in NDDO models when applied to both discrete species and to polymers.\nSurprisingly, in its unmodified form, the DSK approximation gives rise to an infinite error when applied correctly to any non-elemental crystalline solid. This error arises from the fact that the one-center two-electron integrals differ from element to element. The origin of the error can be understood by considering the potential at an atom A in a simple binary solid, AB, arising from all atoms on the surface of a spherical shell of radius R; in such a solid, if the charge on atom A is Q, then the charge on atoms of type B would be \u2212Q. When R becomes large enough, the fraction of atoms of type A and B at that distance will be the same, and the resulting electric potential, V, at atom A could then be represented by Eq.\u00a02.\nA Taylor series expansion of this function shows that V is proportional to the reciprocal of the distance. For all values of R greater than about 10\u00a0\u00c5 this potential is clearly very small. In solids, however, the potential at an atom is the result of the summed electric fields of all such shells, out to infinity. For convenience, this sum can be replaced by an integral, Eq.\u00a03.\nThe value of this integral is infinity, which means that, if the DSK approximation is used and the integration is done correctly, the potential experienced by an atom of type A arising from the electrostatic contributions of all other atoms would then be either plus infinity or minus infinity, depending on the sign of its partial charge. This is an obviously unphysical result.\nThis catastrophe can readily be avoided by modifying the DSK approximation to ensure that it converges to the point charge expression for large values of R. The simplest modification would be to ensure that, as the interatomic separation increased, a smooth transition is made from the DSK equation to the exact point charge equation. Several trial functions were examined and, from these, a Gaussian function was selected as having the best characteristics; this function is shown in Eq.\u00a04. Below 5\u00a0\u00c5, the unmodified DSK equation would be used; at larger distances, Eq.\u00a04 would be used. This function is well-behaved in that it is single-valued and has finite first and second derivatives.\nElectrostatic interaction\nEvaluating the electrostatic potential at an atom in a crystal involves summing interactions from all surrounding atoms, and since there are, for all practical purposes, an infinite number of these, the direct sum must be replaced by a tractable alternative. The simplest and most efficient method of evaluating the electrostatic potential arising from an infinite lattice of point charges is the Ewald sum [8]. In this summation, the contribution to the potential is divided into two terms, a real-space and a reciprocal- or Fourier-space term. When an appropriate error function is used, the Ewald sum is both accurate and readily evaluated, and is the method of choice when the model used represents the electrostatic potential as the sum of contributions from classical point charges. Gale successfully applied a modified version of the Ewald sum [12] in evaluating the electrostatic potential used in MNDO, AM1, and PM3 solid state calculations.\nIn all NDDO [18, 19] methods, including PM6, the electrostatic contribution to the potential at an atom arising from the charges on distant atoms can be represented by the classical point charge equation, at small distances by the DSK, and at intermediate distances by the modified DSK approximation. Gale [12] noted that a modification must be made to the potential in order for the Ewald summation to be used in an NDDO method. This change requires the point-charge contribution to the potential of each atom that arises from all nearby atoms to be replaced by the exact NDDO contribution. Derivatives of the energy with respect to geometry require all potential functions to be continuous, but if corrections of the type just described were made, the resulting function would obviously be discontinuous, and further corrections would be needed. So, although the Ewald sum is aesthetically attractive, its practical implementation would necessarily involve aesthetically unattractive corrections.\nAn alternative to the Ewald sum would be to modify the way in which the electrostatic sum is evaluated. In this approach, use is made of the fact that an integer number of interacting unit cells are used in a solid state calculation. If the DSK equation, either unmodified or modified as in Eq.\u00a04, is used, then the potential at any given atom arising from the direct summation of the NDDO electrostatic terms from all the other atoms would contain artifacts reflecting the asymmetric environment. In other words, the presence of boundary effects introduces spurious terms into the potential. If these terms were not eliminated, they would have a perturbative effect on the optimized geometry that would severely compromise the validity of the results. A method for removing these spurious effects was developed that involves modifying the distance term in the DSK approximation.\nThe potential experienced by each atom in a solid that arises from the partial charges on other atoms falls off rapidly with increasing distance. This is a natural result of the fact that the net charge arising from all atoms in a spherical shell must rapidly converge to zero as the radius increases. An implication of this is that, for large radii, the precise value of the radius used in evaluating the potential is unimportant. Conversely, when the radius is small, and there are relatively few atoms, the potential arising from the associated partial charges is large. In that case, the value of the interatomic separation used is of great importance. This behavior can be used as the basis for modifying the electrostatic sum. At large distances, because the electrostatic effect of the distant atoms is small, the value of the interatomic distance used in calculating the potential can be different from the actual value, and, in fact, can be set to any arbitrary large fixed value. That is, all potentials arising from distant atoms can be treated as if their partial charges were moved in to the surface of a sphere of fixed radius. A result of this is that the gradient or force arising from a charge that was initially outside the sphere would be exactly zero: any potential motion of the central atom in response to the presence of a charge on the surface of the sphere would be accompanied by a simultaneous motion of that charge. A consequence of this is that the gradient of the potential arising from a charge on the surface of the sphere is precisely zero. This modification of the effective interatomic distance (EID) used in evaluating the electrostatic potential completely eliminates all directional effects, in particular all artifacts arising from the use of a finite number of interacting unit cells.\nIf no further modifications to the EID were made, then there would be a discontinuity in the gradient arising from the presence of the sphere. The gradient arising from a partial charge just inside the sphere would be finite, but if that charge were to move just outside the sphere, its gradient would now become zero, and there would be a discontinuity. The presence of such discontinuities would then preclude the gradients being used in subsequent operations such as geometry optimization and calculation of vibrational frequencies. To avoid them, the EID must be further modified to ensure that the gradient arising from an atom near the surface of the sphere drops smoothly to zero as the atom approaches the surface of the sphere. This is most simply accomplished by reducing the EID of an atom as it approaches the surface of the sphere.\nTo summarize: the value of the EID is set to a constant for all atoms separated by a large distance, is set less than the actual distance for intermediate distances, and is equal to the actual distance when the interatomic separation is small. A simple function that satisfies these criteria can be defined using three domains, as shown in Fig.\u00a01.\nFig.\u00a01Truncation approximation for Madelung integral. C\u2009=\u200930\u00a0\u00c5\nFor atoms that are at a distance less than some predefined value, 2\/3C, the exact DSK approximation is used. Between 2\/3C and 4\/3C, the EID to be used in Eq.\u00a04 would be reduced as shown in Eq.\u00a05. \n, and at distances greater than 4\/3C the value of the EID would be a constant C. The effect of these changes when applied to an example set of charges is illustrated in Fig.\u00a02, with the original charges shown in black, and the locations of the charges that would be used in evaluating the electrostatic potential shown in green.\nFig.\u00a02Effect of truncation on apparent position of charges. For a set of charges (black), the position used in evaluating the electrostatic interactions is shown in green\nProvided enough unit cells are used to ensure that all atoms within the sphere of radius 4\/3C are present, the effect of this modification is to remove any directional influence, specifically surface effects, arising from the presence or absence of distant atoms. As with the unmodified DSK equation, the potential arising from an atom at any distance is single-valued and its first derivative is finite. An integer number of unit cells is always used in the evaluation of the electrostatic potential; therefore, the net charge on the surface of the sphere of radius 4\/3C precisely counterbalances the sum of all the charges within the sphere, regardless of how many unit cells are outside the sphere. This is a natural and necessary consequence of the requirement that unit cells in a solid must have a zero charge.\nThe electrostatic potential is, of course, dependent on the value of C. With increasing values of C, the potential converges rapidly to a constant, but also as C increases the number of unit cells that need to be used increases rapidly. The value of C was set to 30\u00a0\u00c5, this being the best compromise between computational effort and numerical stability.\nUnlike the Ewald summation, this modified DSK approximation can be used directly in evaluating the electrostatic potential. The new approximation is relatively simple in that the use of error functions and reciprocal space terms are avoided.\nSolids with unpaired electrons\nMany solids, particularly those containing transition metals, have unpaired electrons. Of the two standard methods available for modeling such systems when only molecules are involved, unrestricted Hartree Fock (UHF) and restricted Hartree Fock followed by configuration interaction (RHF-CI), only UHF is suitable for modeling solids. The use of RHF-CI methods is precluded because of the very large active space involved. For example, consider the garnet uvarovite, calcium chromium silicate, Ca3CrIII2Si3O12.. Each chromium ion in this mineral has three unpaired d electrons. The unit cell contains eight formula units, so, if the RHF-CI procedure was used, at a minimum the active space would need to include all 80 molecular orbitals of predominantly d character. Even if the reasonable assumption was made that all the unpaired electrons had the same spin, the number of microstates involved would still be very large, , and evaluation of the gradients for the resulting non-degenerate state would be prohibitively slow.\nFor solids in which the ions with unpaired electrons are well separated, that is, where the ions are electronically isolated from each other, the assumption can be made that the spin-state of one ion will not interact significantly with the spin-state of any adjacent ion. In addition, if the atom in question is a transition metal ion, then the spin state can usually be inferred from its environment. In the case of uvarovite, each chromium ion is in an almost octahedral environment (the exact symmetry is S6), being surrounded by six oxygen atoms, so the three d electrons would be in a t2g manifold, and would therefore be unpaired. If the Hund\u2019s rule assumption is made that the spin state is a maximum, then each chromium atom would be in a local 4A2g state, and any Jahn-Teller tendency to geometric distortion to a lower symmetry would be avoided. This assumption can be formalized in the calculation when a UHF method is used by defining the difference between the number of electrons of \u03b1 and \u03b2 spin to correspond to the maximum possible spin state of the entire system. For uvarovite, the unit cell would then be defined as having a spin of Ms\u2009=\u200924, and therefore would have 48 more \u03b1 than \u03b2 electrons.\nApplications\nOrganic compounds\nData sets were constructed for each organic compound, with, in each case, the starting geometry being the X-ray structure: i.e., the observed geometry. In contrast to molecular calculations, where internal coordinates are normally used, in the work reported here Cartesian coordinates were used exclusively. An attempt was made initially to use internal coordinates, but the numerical instabilities associated with the geometric gradients at the interfaces of the unit cells rendered their use impractical; no such difficulties were encountered when Cartesian coordinates were used. Each cluster consisted of between 100 and 200 atoms, and geometries were converged until the gradient norm had dropped below 5\u00a0kcal\u00a0mol\u22121\u00a0\u00c5\u22121, this corresponding to an uncertainty in the optimized geometry of about 0.001\u00a0\u00c5. All unit cell parameters were optimized, as were the coordinates of all atoms within the unit cell. Unless indicated otherwise, symmetry was not used to accelerate the optimization. All calculations were done using MOPAC2007 [20] on a 3.6\u00a0GHz Pentium computer, and each geometry optimization took between 20 min and 1\u00a0day, with most taking about 1\u00a0h. No problems were encountered in any of the optimizations.\nWith the possible exception of polymers, crystalline organic compounds consist of discrete molecules held together by relatively weak forces. As PM6 has been shown [1] to reproduce bond lengths and angles of simple organic compounds with useful accuracy, in this work attention was focused on the prediction of the structures of entire molecules and on the forces and energies arising from intermolecular interactions. A useful measure of accuracy of prediction of molecular structure is the root-mean-square (RMS) difference between the calculated and reference geometries of a single molecule or ion in a crystal. This quantity differs from the geometric quantities reported earlier [1] in that it measures the accuracy of prediction of the overall structure of a molecule, not just the accuracy of prediction of individual bond lengths and angles. It is possible for only relatively small distortions to exist in individual angles and, at the same time, for the overall structure to be severely in error. The RMS error is therefore complementary to the errors in individual bond lengths and angles. In order to probe the suitability of PM6 for modeling organic solids, compounds were selected that illustrate a wide range of common intermolecular interactions, the most important of these being, in order of the energies involved: ionic, hydrogen bonding, and \u03c0-stacking.\nDensities\nAnother useful measure of the accuracy of prediction of organic and inorganic solids is the density. In most cases when the density is accurately reproduced the internal structure of the unit cell is also accurately predicted. This is not an infallible rule, in that it is possible for the density to be predicted with good accuracy and, at the same time, the unit cell structure to be significantly distorted. This rare occurrence can usually be detected by distortions of the unit cell parameters. No cases were found where the unit cell parameters were predicted with good accuracy and, at the same time, significant errors existed in the internal structure of the unit cell. A comparison of PM6 and X-ray unit cell parameters for 124 organic solids is presented in Table\u00a01. In this table, the unit cell used was often different from that reported in the literature, particularly so in hexagonal crystals, that is, crystals in which the interface angles are 90\u00b0, 90\u00b0, and 60\u00b0. Unit cells were chosen that would maximize the size of sphere that could be contained in a given cluster; to this end, most hexagonal unit cells were replaced by equivalent orthorhombic unit cells. Predicted densities were reproduced with good accuracy, the average unsigned error in density being 6.9%, with the bulk of this error arising from errors in the calculated intermolecular distances. Although most systems optimized with only small changes in the geometry, in three instances quantitative changes occurred.\nTable\u00a01Calculated and X-ray structural parameters for organic compounds\u00a0RMS errorPM6X-rayDensity (g\/cm3)dabc\u03b1\u03b2\u03b3abc\u03b1\u03b2\u03b3(Z Z)-N N\u2032-Dimethylurea (NIJHUJ)0.119.694.3611.3890.992.396.410.354.5711.4090.090.0102.71.23 (1.11)1, 4 Dioxocine 6-carboxylic acid chloride (CUWWIA)0.2914.6712.303.9089.691.789.215.1512.493.8190.087.790.01.63 (1.59)1, 3, 5-Triaminobenzene, 1, 3, 5-trinitrobenzene (NIBZAM)0.0514.696.8413.8277.590.389.915.086.9814.0676.590.090.01.65 (1.55)1, 3-bis((Pyrid-2-ylamino)carbonyl)adamantane \u03b1-ketoglutaric acid (RIZWUS)0.149.9713.6610.7292.0113.167.210.5313.7310.7391.8114.767.81.42 (1.35)1,4-diazabicyclo[2.2.2]octane azelaic acid (UNEGEZ)0.5030.368.335.9391.189.191.328.158.606.9890.093.090.01.33 (1.18)1-diazonia-4-azabicyclo[2.2.2]octane glutarate (UNEFIC)0.2910.5611.6710.3989.483.191.410.2812.4610.6890.066.190.01.42 (1.30)2-(2-(3-Carboxypyridyl))-4-isopropyl-4-methyl-5-oxo-imidazole (JAZCOC01)0.1016.0710.747.20188.786.585.516.3110.787.1785.690.090.01.40 (1.38)2, 4, 4, 6, 6-Pentachloro-2-(piperidyl)cyclotriphosphazene (POTKEO)0.238.1222.5017.2089.590.190.18.3222.0117.2690.090.090.01.68 (1.67)2, 4, 6-Tribromoaniline (BRANIL)0.094.2013.5912.9990.090.090.04.2614.6213.4490.090.090.02.96 (2.62)2, 4, 6-Trinitro-N-methyl-N-nitroaniline (Tetryl) (MTNANL)0.3011.377.2715.9690.472.390.410.617.3714.1390.084.990.01.52 (1.73)2-Pyridone (PYRIDO04)0.035.775.9312.7590.290.490.15.605.7913.5690.090.090.01.45 (1.43)4, 5-bis(Dimethylamino)-1,8-dihydroxynaphthalene (RISBIE)0.0610.3313.758.91107.790.390.110.6513.779.32107.090.090.01.36 (1.25)4-Aminobenzoic acid 4-nitroaniline (RILJEB)0.0530.908.664.8695.089.591.231.258.644.8793.790.090.01.41 (1.39)4-Fluoro-2-(phosphonomethyl)benzenesulfonic acid monohydrate (KIXQIR)0.178.499.717.60105.8108.699.48.499.257.92104.8110.097.41.74 (1.75)4-Hydroxybenzoic acid isonicotinamide (VAKTOR)5.4822.299.3790.390.091.96.0720.679.4090.090.095.31.51 (1.47)5H-Dibenz(b,f)azepine-5-carboxamide saccharin solvate (UNEZAO)0.1712.2311.467.1669.684.682.712.6810.457.5175.485.783.61.50 (1.46)5-Methyl-2-((2-nitrophenyl)amino)-3-thiophenecarbonitrile (QAXMEH)0.2318.5015.964.2488.789.688.918.6916.403.9586.290.090.01.38 (1.43)5-Methyl-2-((2-nitrophenyl)amino)-3-thiophenecarbonitrile (QAXMEH01)0.1116.428.328.5790.089.689.716.418.548.5088.290.090.01.47 (1.47)5-Methyl-2-((2-nitrophenyl)amino)-3-thiophenecarbonitrile (QAXMEH02)0.117.4811.907.91104.2117.078.67.4911.917.79104.5116.477.801.42 (1.44)5-Methyl-2-((2-nitrophenyl)amino)-3-thiophenecarbonitrile (QAXMEH03)0.178.0211.3613.6690.690.4106.57.9811.6813.3290.090.0104.81.44 (1.44)5-Methyl-2-((2-nitrophenyl)amino)-3-thiophenecarbonitrile (QAXMEH04)0.2010.9612.054.3789.690.573.011.2512.324.5990.291.871.21.56 (1.43)5-Methyl-2-((2-nitrophenyl)amino)-3-thiophenecarbonitrile (QAXMEH05)0.1213.2922.577.9190.690.190.513.1822.808.0290.090.090.01.45 (1.43)5-Methyl-2-((2-nitrophenyl)amino)-3-thiophenecarbonitrile (QAXMEH12)0.177.9312.1612.2590.890.9102.88.2312.3111.8290.090.0102.51.50 (1.47)6-(Pyran-4-one-2-yl)-1,4-dioxocine (CUWWEW)0.197.6910.9110.9690.774.490.67.9110.8011.5890.070.490.01.53 (1.46)9-(2-Hydroxyethyl)adenine (FABFUJ)0.117.188.3413.9690.290.089.47.348.2713.5790.090.090.01.42 (1.45)Acetophenone (ACETPH)0.048.718.459.7590.958.590.68.568.6810.2690.059.090.01.31 (1.22)Acetylacetone (LIWPIQ01)0.047.854.1115.4892.590.189.88.464.1516.0390.090.090.01.33 (1.18)Acetylcholine chloride (ACHOLC01)0.1715.295.949.6290.0 89.390.215.326.309.8990.090.090.01.38 (1.26)Adenosine-3\u2033-phosphate dihydrate (ADPOSD)0.355.9111.6210.5588.690.090.26.3411.909.9487.890.090.01.76 (1.70)Adenosine-5\u2033-diphosphate dihydrate (HMADPH)0.2918.506.768.8991.789.393.018.446.899.2090.087.590.01.75 (1.66)\u03b1 Glycine0.105.095.5010.7790.190.069.65.105.4611.9790.090.068.31.77 (1.61)\u03b1 Resorcinol0.055.609.5910.3889.789.990.15.609.5310.5390.090.090.01.31 (1.30)Anthracene (ANTCEN14)0.0210.367.326.8489.9109.489.811.176.028.5590.0124.690.01.21 (1.25)Aqua-tris(2-((dimethylamino)methyl)phenyl)borane (QEVYEV)0.1619.818.4114.9989.975.189.620.358.5715.2390.072.390.01.19 (1.13)Aspartic acid (LASPRT)0.205.136.387.7090.0106.290.05.146.987.6290.099.890.01.83 (1.64)Barium oxalate oxalic acid dihydrate (BAHOXH11)4.7510.6813.0968.990.190.15.4112.4614.4563.890.090.03.77 (2.67)Benzene (BENZEN)0.019.276.797.1590.190.289.89.556.927.4490.090.090.01.15 (1.06)Berberine sulfate (CISREB)0.1519.917.4327.5197.6117.390.720.377.4427.4397.7116.285.51.61 (1.56)\u03b2 Glycine0.085.346.095.1490.065.789.85.386.275.0890.066.890.01.64 (1.59)\u03b2 Resorcinol0.048.814.9912.3489.988.990.07.815.4312.6290.090.090.01.35 (1.37)bis(1,4-Diazoniabicyclo(2.2.2)octane) bis(1-aza-4-azoniabicyclo(2.2.2)octane) tetrakis(tribromide) dibromide[26] (DAHGUO)15.3920.6012.81103.789.990.016.1721.3713.23101.090.090.02.65 (2.33)bis(3,5-Dinitro-4-methylbenzoic acid) 1,2-bis(4-pyridyl)ethane (LAPTUS)0.1812.0116.117.2775.489.794.112.7715.777.2773.191.193.41.56 (1.51)bis(Pyridinium) oxalate oxalic acid (DEFCUM)4.1211.397.77102.199.891.04.0011.328.44102.097.388.61.60 (1.52)bis(Urea) oxalic acid (UROXAL01)12.347.005.1384.289.890.012.376.885.0583.690.090.01.58 (1.64)Bromo-tris(2-dimethylaminoethyl)amine-manganese(ii) (DAEAMN)0.2711.8511.8511.8390.290.389.712.2212.2212.2290.090.090.01.78 (1.62)Calcium acetate chloride pentahydrate (CALCLA)0.0413.2611.206.66119.189.788.613.7211.516.82116.790.090.01.73 (1.55)Camphor (UGAHUF)0.068.4526.777.3290.891.189.98.9327.047.3890.090.090.01.22 (1.14)Cholesteryl acetate (CHOLAD04)0.1216.989.2116.2887.971.788.417.629.2216.5290.072.890.01.18 (1.11)cis Copper Diglycinate (CUGLYM02)0.195.3312.6010.5489.990.189.95.1913.5510.6990.090.090.02.16 (2.03)Citric acid (CITRAC10)0.3111.925.3812.1889.5115.289.611.475.6212.8190.2111.590.01.81 (1.66)Coronene (CORONE)0.0316.024.799.8890.168.890.016.124.7010.1090.069.190.01.41 (1.40)Cyclohexane (CYCHEX)0.028.126.4111.2390.0107.190.08.206.4411.2390.0108.890.01.00 (1.00)Cyclotrimethylene-trinitramine (RDX)0.2211.2411.4814.3289.888.489.910.7111.5713.1890.090.090.01.60 (1.81)Cystine (LCYSTI10)0.425.3454.255.3788.459.989.25.4256.285.4290.060.090.01.78 (1.67)Cytosine (CYTOSM03)0.0410.217.097.7987.290.488.49.827.527.7379.590.090.01.52 (1.53)Dinicotinic acid (DINICA10)0.186.7111.099.6889.7111.990.16.5911.159.7090.0107.890.01.66 (1.64)Dipyridinium bis(hydrogen-oxalate) oxalic acid (DUVLUB)27.718.307.3590.083.890.026.848.917.4590.087.190.01.69 (1.60)Disodium adenosine-triphosphate trihydrate (ADENTP)0.766.2420.5831.3790.091.488.37.0720.8830.4590.090.090.02.00 (1.79)DL-2-Amino-4-phosphonobutyric acid monohydrate (CAXDIO01)0.118.114.8319.8987.6102.390.68.404.9520.6090.0100.490.01.76 (1.58)\u03b3 Glycine0.075.526.938.94114.450.990.75.487.048.92113.252.190.01.65 (1.59)Glucose (GLUCSA)0.1910.274.2015.1290.090.090.010.364.9714.8490.090.090.01.84 (1.57)Guanine0.063.6216.849.8888.285.996.23.5516.349.6990.090.090.01.68 (1.79)8-Azaguanine (AZGUAN01)0.063.6711.8116.3889.6102.789.73.5611.4416.4790.095.190.01.46 (1.51)Hydroquinone bis(aniline) (HIBFUT)5.038.2118.4276.890.090.05.398.2218.7477.590.090.01.33 (1.22)Isonicotinamide (EHOWIH)0.089.985.8010.1190.079.389.910.035.7310.1790.081.890.01.41 (1.40)Isonicotinamide 3-hydroxybenzoic acid (LUNMEM)20.705.1622.1989.996.290.020.875.1422.4290.097.790.01.47 (1.45)Keggin pentakis(Tetraethylammonium) bis(meso-tetraphenyl-porphyrinato-zinc) tetraconta-oxo-silicon-dodeca-molybdenum bromide(PIJFUJ)15.1432.0114.9890.190.390.115.2232.2815.2290.090.090.01.88 (1.83)l-Alanine0.065.9311.675.8690.089.989.95.7912.265.9390.090.090.01.46 (1.41)Leucine (LEUCIN02)0.1313.855.249.4489.282.490.214.525.309.5690.085.890.01.28 (1.19)Lithium acetate (Li(CH3COO) 2(H2O))0.0211.236.387.1598.394.488.210.886.626.8290.090.090.01.34 (1.38)l-Lysine monohydrochloride dihydrate (LYSCLH)0.1112.355.7614.3881.690.489.813.325.8814.9881.290.090.01.44 (1.25)Malonic acid bis(isonicotinamide) (ULAWEJ)14.6514.6812.2565.881.868.815.6814.8611.9467.385.665.71.55 (1.49)m-Aminophenol (MAMPOL02)0.028.246.1211.3390.089.589.98.286.1011.2390.090.090.01.27 (1.28)m-Aminopyridine (AMIPYR)0.036.305.6815.2390.189.5111.46.195.7115.3090.090.0110.51.23 (1.24)m-Cresol N,N,N\u2032,N\u2032-tetraisopropyloxamide (DUGRIG)6.6226.7211.8893.489.889.86.8627.1912.0896.190.289.81.15 (1.07)m-Diaminobenzene, 3,5-dinitro-1-cyanobenzene (REDDEJ)13.0710.399.8584.089.590.413.2610.459.8886.790.090.01.51 (1.46)Methionine (LMETON02)0.115.1816.409.3185.393.883.35.2014.839.4981.090.090.01.27 (1.37)Methyl (+)-(1\u03b1,2\u03b2,8\u03b1,9\u03b1,10\u03b2)-2-chloro-4-aza-3-oxatetracyclo(8.4.0.02,9.04,8) tetradecane-9-carboxylate oxalic acid monohydrate (HUZKOC)0.156.1918.358.1196.5101.681.76.6117.898.07102.399.883.21.47 (1.43)m-Hydroxybenzoic acid (BIDLOP)0.0323.804.975.0490.074.390.023.894.945.4990.074.390.01.60 (1.47)tetrakis(2-Carboxypyridinium) octacyano-molybdenum(iv) (PYCMOA) (4[C6H6NO2] [MoIV(CN)8]4-)0.0917.9610.469.7093.391.489.218.5110.159.1789.890.090.01.46 (1.54)m-Toluidine (FANDOO)0.089.135.7923.1291.780.989.48.765.8124.8790.079.990.01.18 (1.14)Natroxalate (Na2(C2O4))0.584.239.295.3890.190.286.53.4510.385.2490.090.090.02.11 (2.38)N, N\u2032-(6 6\u2033-dimethylbiphenyl-2 2\u2033-diyl) bis(methylamine) (ENIWIH)0.0710.6711.7710.1590.190.190.011.0912.0210.5790.090.090.01.25 (1.13)N, N-2 6-Tetramethyl-4-nitroaniline (FOCVOI)0.127.8516.177.6790.795.688.97.6418.087.6490.090.090.01.33 (1.22)N, N-Dimethylaniline (DMAFBZ01)0.087.0913.208.7182.4101.295.46.7812.338.2680.574.092.01.29 (1.57)Nicotinamide adenine dinucleotide tetrahydrate (CEVYEH11)0.258.898.2011.3383.269.7103.28.848.5911.1989.470.4103.91.66 (1.58)N-Methylurea (MEUREA)0.096.856.648.0890.191.290.36.926.988.4890.090.090.01.34 (1.20)N, N-Dimethylbenzamide (ODOTOQ)0.086.5116.197.4990.090.690.86.6316.307.6590.090.090.01.26 (1.20)N, N-Dimethylurea (WIFKEB)0.099.278.496.0690.489.6109.39.278.606.0490.090.0108.81.30 (1.29)o-Aminophenol (AMPHOM02)0.057.508.1019.7090.090.590.07.857.2519.7590.090.090.01.21 (1.29)o-Aminopyridine (AMPYRD)0.047.415.6511.6290.085.989.97.595.6711.7190.084.590.01.29 (1.25)o-Diaminobenzene (BAGFIY)0.047.727.579.9689.982.590.07.727.5410.3290.080.090.01.25 (1.21)o-Dimethoxybenzene (TUKGEL)0.0612.949.355.5890.189.889.913.359.925.5390.090.090.01.36 (1.25)Oxalic acid (OXALAC06)0.036.916.088.1689.989.990.06.566.097.8590.090.090.01.74 (1.91)Oxalic acid dihydrate (OXACDH26)0.044.9211.453.4489.790.0106.96.0911.933.4789.790.0106.92.26 (1.73)Oxytocin 1XY10001 [23]0.4422.068.8926.7390.699.188.423.049.0427.2790.0102.290.01.43 (1.33)p-Aminophenol (AMPHOL01)0.037.9512.904.7890.190.190.18.1812.955.2690.090.090.01.48 (1.30)p-Aminopyridine (AMPYRE)0.045.4811.927.1989.690.391.05.5712.127.3290.090.090.01.33 (1.26)p-Chloroaniline (CLANIC05)0.038.377.308.9890.089.790.28.597.249.1990.090.090.01.54 (1.48)p-Diaminobenzene (PENDAM)0.038.2923.085.9489.990.093.58.3722.955.9790.090.093.61.27 (1.26)Phenanthrene (PHENAN08)0.049.165.738.5690.280.989.99.446.148.4490.082.090.01.34 (1.22)Phenolphthalein (NIMDAO)0.0911.2414.5819.0190.089.889.511.3914.8219.2790.090.090.01.34 (1.28)Phloroglucinol (PHGLOL)0.0312.719.354.8789.889.689.912.569.374.8390.090.090.01.45 (1.47)p-Hydroxybenzoic acid (JOZZIH)0.046.2110.0518.4689.885.790.16.3410.4618.5190.086.890.01.60 (1.50)Picric acid (PICRAC)0.159.63 19.499.3888.990.489.89.7019.139.2590.090.090.01.73 (1.77)Potassium hydrogen acetate0.013.916.7725.0090.389.882.14.017.1923.8890.090.081.71.60 (1.54)1-Dimethylamino-8-dimethylammonionaphthalene saccharin dihydrate (AJOHUC)0.098.819.5424.5090.299.690.19.259.1724.9390.095.890.01.42 (1.37)Salicylaldoxime (SALOXM)0.0712.915.4510.0890.365.489.813.605.0810.4190.067.190.01.41 (1.38)Sodium acetate Na(CH3COO)\u00b73(H2O) (NAACET01)11.6511.3915.4088.978.891.710.4010.4712.3590.068.390.00.90 (1.45)Sodium hydrogen acetate16.3316.3316.3389.989.990.015.9215.9215.9290.090.090.01.30 (1.40)Sucrose (SUCROS01)0.2410.368.597.5090.0103.090.010.828.687.7290.0103.090.01.75 (1.61)Tetramethylammonium dihydrogen phosphate monohydrate (FIJHEL)0.028.018.0712.32 90.190.098.18.528.4412.9090.090.099.11.59 (1.37)Tetramethylurea (TIDBIR)0.168.696.0011.7392.193.191.69.976.2610.6390.093.090.01.27 (1.17)Thymine (THYMIN01)0.056.796.9611.7178.189.889.66.856.7812.8975.190.090.01.55 (1.45)trans Copper digycinate (IWUBEH)0.329.5327.275.3289.092.388.59.4427.935.0690.090.090.01.85 (1.91)Trinitrotoluene (ZZZMUC01)0.115.4322.6314.7290.191.490.16.0820.0214.9990.090.090.01.67 (1.66)tris(Acetylacetonato) titanium(iv) perchlorate (TIACPC)0.258.9711.739.7891.083.592.38.6811.749.8988.584.392.01.45 (1.47)Tryptophan (TRYPTC)0.1315.265.176.9089.378.388.914.675.307.4590.081.290.01.50 (1.40)Tyrosine (LTYROS10)6.8520.395.9089.990.090.06.91 21.125.8390.090.090.01.46 (1.41)Urea (UREAXX13)0.034.675.585.5890.090.090.04.695.575.5790.090.090.01.37 (1.37)Urea nitrate (UREANT02)7.818.209.7089.952.890.17.508.209.5490.055.890.01.65 (1.69)Weddellite (Ca(C2O4)\u00b72(H2O)7.2311.7211.7289.789.989.97.3612.3712.3790.090.090.02.20 (1.94)Whewellite (Ca(C2O4)\u00b7(H2O) (CALOXM03)6.049.7714.5789.990.5107.76.2910.1214.5890.090.0109.52.37 (2.22)tris(2,2,6,6-tetramethylheptane-3,5-dionato)-yttrium(iii) (HAHTOZ01)0.289.578.8918.9589.686.490.210.639.9817.8790.090.090.01.32 (1.12)dX-ray densities in parenthesisValues of a, b, c in \u00c5ngstroms; \u03b1, \u03b2, \u03b3 in degrees. Reference crystal structures from the Cambridge Structural Database [21]. RMS Root-mean-square\nIn the solid state, individual molecules of oxalic acid in oxalic acid dihydrate [Cambridge Structural Database [21] (CSD) entry OXACDH26] exist as the neutral species. PM6 incorrectly predicts them to be fully ionized, as oxalate, [C2O4]2-, plus two hydronium ions, [H3O]+. This change was accompanied by a very large increase in density, from 1.73 to 2.23\u00a0g\/cc, a direct consequence of going from a neutral to an ionic species.\nA related system is barium oxalate oxalic acid dihydrate (CSD entry BAHOXH11) in which there exist polymeric chains of oxalate groups connected by bridging protons. As with oxalic acid, in the optimized PM6 geometry the proton is abstracted by the water molecule to give oxalate groups and hydronium, resulting in an  increase in density of almost 40%.\nSodium acetate trihydrate is an ionic solid consisting of sodium ions surrounded by an acetate group and five water molecules, four of which form bridges between pairs of sodium ions. PM6 completely fails to predict the observed structure: the distance between the sodium ions increases considerably, effectively destroying any tendency of the water molecules to form bridges.\nWhen these three solids were removed from consideration, the average unsigned error (AUE) in density decreased to 6.1%. The most common intermolecular interaction is hydrogen bonding, which PM6 predicts to be too short by about 0.1\u00a0\u00c5, with the result that the average signed error in calculated densities of organic compounds is too high by 3.9%. When a systematic correction to the density was made, the AUE in density decreased still further to 4.8%.\nHeats of formation\nTable\u00a02 presents a comparison of experimental and calculated heats of formation of organic solids. The largest difference occurs with 2, 4, 6-tribromoaniline, which is predicted to be too stable by 44.5\u00a0kcal\/mol. The optimized structure revealed an unrealistically short intermolecular Br\u2013N distance of 2.14\u00a0\u00c5, indicating that the Br\u2013N core-core repulsion was severely underestimated. Examination of the values of the PM6 parameters for the Br\u2013N core-core interaction revealed that the Voityuk interaction would be negligible at chemical bonding distances. This error in PM6 can be attributed to the absence of appropriate reference data in the training set, a fault that could be readily corrected in future work.\nTable\u00a02Comparison of calculated and experimental heats of formation of organic compounds (kcal\/mol)PM6ReferenceaDifference(Z Z)-N, N\u2032-Dimethylurea (NIJHUJ)\u221262.0\u221276.314.32, 4, 6-Tribromoaniline (BRANIL)\u221230.713.8\u221244.5\u03b1 Glycine\u2212122.9\u2212126.13.2\u03b1 Resorcinol\u221279.3\u221288.08.7Anthracene26.430.0\u22123.6\u03b2 Glycine\u2212121.8\u223c\u2212126.1\u223c4.3Camphor\u221266.3\u221276.310.0Citric acid\u2212348.7\u2212369.020.3Cyclotrimethylene-trinitramine (RDX)\u22125.918.9\u221224.8Cystine\u2212237.9\u2212246.88.9\u03b3 Glycine\u2212120.8\u223c\u2212126.1\u223c5.3l-Alanine\u2212132.0\u2212134.12.1Leucine\u2212142.7\u2212152.39.6m-Aminophenol (MAMPOL02)\u221237.6\u221247.910.3m-Aminopyridine (AMIPYR)23.214.48.8m-hydroxybenzoic acid\u2212123.7\u2212142.018.4o-Aminophenol (AMPHOM02)\u221238.2\u221248.19.9o-Aminopyridine (AMPYRD)21.49.412.0o-Diaminobenzene (BAGFIY)12.99.33.6Oxalic acid\u2212175.5\u2212198.422.9p-Aminophenol (AMPHOL01)\u221240.7\u221246.45.7p-Aminopyridine (AMPYRE)19.010.09.0p-Chloroaniline (CLANIC05)2.5\u22128.010.5p-Diaminobenzene (PENDAM)5.110.1\u22125.1Phenanthrene24.126.2\u22122.1p-Hydroxybenzoic acid\u2212125.9\u2212145.019.2Picric acid\u221243.4\u221252.18.7Salicylaldoxime\u221227.0\u221243.916.9Sucrose\u2212535.8\u2212532.0\u22123.8Trinitrotoluene\u22125.7\u221215.19.4Tyrosine\u2212150.7\u2212163.713.0Urea\u221265.7\u221279.613.9aReference values taken from the CRC Handbook [32]\nHeats of sublimation\nThe heat of sublimation is a measure of the intermolecular interaction energy. In some cases sublimation is accompanied by large geometric and electronic changes. For example, the simple amino acids exist as the Zwitterion in the crystal phase, but in the gas phase they are unionized. Representative values for calculated and observed heats of sublimation are presented in Table\u00a03. Determining the accuracy of PM6 for the prediction of heats of sublimation is made difficult by the acknowledged unreliability of many experimental measurements. Thus in the reference compendium of sublimation enthalpies [22], the authors indicate that the reported value for aspartic acid, 22.9\u2009\u00b1\u20091.0\u00a0kcal\/mol, was likely unreliable.\nTable\u00a03Comparison of calculated and experimental heats of sublimation\u00a0Reference [22]PM6Difference(Z Z)-N, N\u2032-Dimethylurea (NIJHUJ)22.115.9\u22126.2\u03b1 Glycine32.629.7\u22122.9Anthracene23.833.19.3Aspartic acid22.937.814.9Benzene10.03.2\u22126.8Camphor12.46.5\u22125.9Cyclohexane9.02.1\u22127.0Guanine44.525.2\u221219.3l-Alanine31.732.60.9Leucine36.028.5\u22127.5m-Aminophenol (MAMPOL02)23.611.8\u221211.8m-Aminopyridine (AMIPYR)19.39.5\u22129.8Methionine32.027.2\u22124.8m-Hydroxybenzoic acid29.515.3\u221214.3N-Methylurea22.617.0\u22125.6N, N-Dimethylbenzamide21.410.0\u221211.4o-Aminophenol (AMPHOM02)22.317.6\u22124.7o-Aminopyridine (AMPYRD)18.39.2\u22129.1o-Diaminobenzene (BAGFIY)20.49.1\u221211.3Oxalic acid22.319.2\u22123.1p-Aminophenol (AMPHOL01)24.219.3\u22124.9p-Aminopyridine (AMPYRE)20.810.4\u221210.4p-Chloroaniline (CLANIC05)21.78.7\u221213.0p-Diaminobenzene (PENDAM)22.016.0\u22126.1Phenanthrene22.030.28.2p-Hydroxybenzoic acid29.514.5\u221215.1Picric acid25.114.6\u221210.5Trinitrotoluene26.914.7\u221212.2Tyrosine24.133.79.6Urea21.717.5\u22124.2\nBiomolecules\nThe primary objective in developing PM6 was to more accurately model systems of biochemical interest. The applicability of PM6 to the study of crystals of biochemical importance was therefore of interest.\nOligopeptides\nThe X-ray structures of many small polypeptides have been determined and are readily available in the Protein Data Bank [23]. One representative entry in this collection, 1XY1, is the nonapeptide deamino-oxytocin, Cys-Tyr-Ile-Glu-Asp-Cys-Pro-Leu-Gly. The structure of this hormone had been refined to a resolution of slightly better than 1.1\u00a0\u00c5 [24]. Each deamino-oxytocin molecule contains a disulfide bridge between atoms S1 and S6, and two strong hydrogen bonds between N2 and O5, and N5 and O2. The unit cell contains four deamino-oxytocin molecules related by a pseudo C2 operation, that is, there are two inequivalent polypeptide molecules and 26 water molecules. The coordinates of the hydrogen atoms in the peptide were given, but not those of the hydrogen atoms on the water molecules. That water of crystallization exists implies that peptide\u2013water hydrogen bonds also exist.\nBecause the positions of the hydrogen atoms on the water molecules were not given in the X-ray structure, an estimate of the locations of the 52 hydrogen atoms had to be made before the geometry could be optimized. For this operation, the \u201cice rules\u201d were used: each oxygen atom in a water molecule was involved in forming two hydrogen bonds and each hydrogen atom formed one hydrogen bond. Of necessity, some of these bonds involved atoms on the peptide.\nSeveral candidate structures, each of which satisfied these conditions, were constructed, and the positions of all hydrogen atoms optimized while those of the other atoms were held constant. An incidental benefit of this operation was that any potential errors in the X-ray positions of some hydrogen atoms, in particular the apparently faulty location reported for H147, were automatically corrected. After the positions of the hydrogen atoms were optimized, an unconstrained optimization on the unit cell was carried out. This involved the optimization of the Cartesian positions of all the atoms in the unit cell of formula (C43H65N11O12S2)4\u00b726(H2O), and the unit cell dimensions, i.e., the simultaneous optimization of 1,839 coordinates.\nEach initial geometry optimized to give a different final structure. That is, the optimized geometry was very sensitive to the choice of initial locations of the hydrogen atoms assigned to the water molecules. As a result, it was not possible to unambiguously define an optimized PM6 structure; however, all the fully optimized structures were within a few kcal\/mol of each other, so one structure was chosen arbitrarily and used in the following analysis.\nThe optimized PM6 unit cell dimensions are shown in Table\u00a01. The optimized PM6 structure of the entire unit cell had an RMS error of 0.61\u00a0\u00c5, and an RMS error of 0.44\u00a0\u00c5 for a single molecule of deamino-oxytocin. Deviations from the pseudo-C2 symmetry were small, and were very sensitive to the initial choice of hydrogen bonds; it is likely that the time-average would be exactly pseudo-C2. All the weak intra-chain bonds were preserved: PM6 predicted the disulfide bridges, S1\u2013S6 to be 2.06\u00a0\u00c5 and 2.05\u00a0\u00c5 compared with the X-ray values of 2.08\u00a0\u00c5 and 1.95\u00a0\u00c5, and the hydrogen bonds between N2 and O5 to be 1.98\u00a0\u00c5 versus the X-ray value of 1.93\u00a0\u00c5, and N5 and O2 1.98\u00a0\u00c5, compared to the X-ray, 1.90\u00a0\u00c5.\nAcetylcholine One of the simplest of the important biochemicals is the neurotransmitter acetylcholine, [CH3COOCH2CH2N(CH3)3]+ (ACh). In one form, this ion exists in the solid state as the chloride (CSD entry ACHOLC01). PM6 reproduced the structure of ACh in this salt with good accuracy, the RMS error for a single ACh being only 0.21\u00a0\u00c5. Part of this error can be attributed to the C\u2013H and N\u2013H bond-lengths from the X-ray structure being about 0.2\u00a0\u00c5 too small; when only the heavy atoms are used in the comparison, the RMS error decreased to 0.13\u00a0\u00c5. An estimate of the effect of the crystal-packing forces can be obtained by comparing the PM6 predicted structure of the gas-phase ion with that found in the crystal; when this was done the RMS difference increased to 0.54\u00a0\u00c5. This is unequivocal evidence that inclusion of crystal-packing forces is essential in order to reproduce the observed structure.\nAdenosine diphosphate Adenosine diphosphate (ADP) is an important intermediate in energy transfer in biochemistry. ADP contains an pyrophosphate group, P2O7, which, in the tris(hydroxymethyl)-methylammonium dihydrate salt (CSD entry HMADPH), is described as being doubly ionized, with the counterions being the adenine and the quaternary ammonium ion. Hydrogen atoms were added to the structure given in the CSD and, in a preliminary calculation, their positions were optimized using PM6, the rest of the geometry being fixed at the X-ray structure. One of the hydrogen atoms in the resulting geometry was located between an oxygen of the terminal PO4 group on one ADP ion and a nitrogen atom on an adenine in an adjacent ADP ion. Because these oxygen and nitrogen atoms were only 2.57\u00a0\u00c5 apart, it is likely that a bridging hydrogen bond exists between them in the crystal. The geometry of the entire system was then optimized. This resulted in an insignificant increase in the N\u2013O distance to 2.58\u00a0\u00c5, and gave an RMS error for a single ADP ion of 0.41\u00a0\u00c5. This suggests that a better description of the pyrophosphate moiety would be that it is singly ionized and that it is part of a two molecule ADP\u2013adenine system connected by a bridging hydrogen. As with acetylcholine, the structure of ADP depends strongly on its crystal environment: when the geometry of a single ATP ion was optimized using PM6, the structure distorted dramatically and the resulting RMS error relative to the X-ray structure increased to 2.08\u00a0\u00c5.\nAdenosine triphosphate The nucleotide adenosine-triphosphate (ATP) is a source of energy for many biochemical reactions and, as such, its structure and properties are of considerable interest. Only one simple compound of ATP was found in the CSD, disodium adenosine-triphosphate trihydrate, ADENTP. Its structure was very badly reproduced when PM6 was used, with the RMS error of the ATP molecule being 1.18\u00a0\u00c5. Examination of the unit cell revealed that the sodium ions had formed spurious weak bonds to nearby hydrogen atoms, and that this was partially responsible for the distortion of the ATP ion. This was confirmed when potassium was used in place of sodium and the RMS error dropped to 0.70\u00a0\u00c5. Because of this and other results, there is convincing evidence that PM6 sodium has severe problems when solid state systems are being modeled, and it is highly probable that part of the error in prediction of the structure of ADENTP can be attributed to faults in sodium parameters.\nNicotinamide adenine dinucleotide In contrast to ATP, the structure of nicotinamide adenine dinucleotide (NAD) tetrahydrate (CSD entry CEVYEH11) was reproduced with good accuracy, the RMS error being only 0.33\u00a0\u00c5. Like ATP, NAD contains a polyphosphate group.\nHydrogen bonding\nBecause of the importance of hydrogen bonding in biochemistry, a range of types of hydrogen bond were examined. Most of the important hydrogen bonds in biochemistry involve a proton positioned between either two oxygen atoms, two nitrogen atoms, or an oxygen and a nitrogen atom, the more exotic bonds, such as those involving halogen ions, while interesting, being of secondary importance.\nIndividual types of hydrogen bonds\nO\u2013H\u2013O A common example of hydrogen bonds is provided by simple organic compounds that contain hydroxyl groups. Examples of such systems are sucrose, aspartic acid, citric acid, glucose, oxalic acid, and the hydroxybenzoic acids. In all such simple hydrogen bonded systems, the geometry predicted by PM6 was similar to that observed. Of more interest are those hydrogen bonds that occur in systems in which relatively large charges are involved.The tendency for a proton to form a bridging structure between two oxygen atoms in organic acids was investigated. Such a situation occurs in the polymeric solid potassium hydrogen acetate, where a single proton is shared between two acetate groups, the whole assembly, two acetates plus the proton, having a formal charge of \u22121. In catena-((\u03bc4-acetato)-(\u03bc2-acetic acid)-potassium) (CSD entry KHACET02), this structure has the geometry O\u2013O\u2032: 2.49\u00a0\u00c5, O\u2013H: 1.09\u00a0\u00c5 and O\u2032\u2013H 1.40\u00a0\u00c5, the three atoms forming a straight line. For this solid, PM6 predicts the following: O\u2013O\u2032: 2.52\u00a0\u00c5, O\u2013H: 1.22\u00a0\u00c5 and O\u2032\u2013H 1.30\u00a0\u00c5, with the O\u2013H\u2013O angle being 178\u00b0.Solid 4-fluoro-2-(phosphonomethyl)benzenesulfonic acid monohydrate (CSD entry KIXQIR) exists as the Zwitterion. In this system, the sulfonic acid group donates a proton to the phosphono group, giving \u2013[SO3]\u2013 and its counterion \u2013[P(OH)3]. One of the hydrogen atoms of the phosphono group then forms a strong hydrogen bond with the nearby water molecule. PM6 incorrectly predicts this bridging hydrogen to be nearer to the water than to the phosphono group. Where the X-ray structure has the PO\u22efH distance of 1.05\u00a0\u00c5, PM6 predicts 1.48\u00a0\u00c5; the corresponding distances for the H\u22efOH2 are 1.37\u00a0\u00c5, observed, and 1.10\u00a0\u00c5, calculated.Another interesting hydrogen bond exists in crystalline acetylacetone (CSD entry LIWPIQ01). In this system the X-ray structure shows that the hydrogen atom involved in hydrogen bonding is disordered over two equivalent positions within a single molecule, with the result that the observed bond lengths and angles are symmetric about the central C\u2013H unit. As expected, when the geometry is optimized using PM6, the extra symmetry is destroyed. This is a consequence of the requirement that, in a quantum chemical calculation, each atom must be in a defined position. However, although the calculated structure was of lower symmetry, when the optimized geometries of the two halves of the molecule were averaged, the result was in good agreement with the X-ray structure: where PM6 predicted the C\u2013O bond to be 1.25\u00a0\u00c5 and 1.35\u00a0\u00c5, the X-ray structure gave 1.28\u00a0\u00c5, and where PM6 gave the C2\u2013C3 distance as 1.36\u00a0\u00c5 and 1.44\u00a0\u00c5, the observed value is 1.40\u00a0\u00c5. The structure of the hydrogen bond was also well reproduced, with the calculated O\u2013O distance being 2.62\u00a0\u00c5 compared with the observed 2.54\u00a0\u00c5, albeit the calculated O\u2013H bond length, 1.06\u00a0\u00c5 was much greater than the reported value of 0.92\u00a0\u00c5.\nN\u2013H\u2013N In 1-dimethylamino-8-dimethylammonionaphthalene saccharin dihydrate (CSD entry AJOHUC), saccharine donates a proton to the \u201cproton sponge\u201d 1,8-bis(dimethylamino)-naphthalene to form an ionic crystal. The reported structure has that proton asymmetrically positioned between the two nitrogen atoms: N\u2013N\u2032: 2.56, N\u2013H: 1.35, N\u2032\u2013H: 1.26\u00a0\u00c5. The optimized PM6 structure gives N\u2013N\u2032: 2.68, N\u2013H: 1.71, N\u2032\u2013H: 1.11. PM6 thus both underestimates the bridging power of the proton and exaggerates the asymmetry of the bond.A closely related species, 4,5-bis(dimethylamino)-1,8-dihydroxynaphthalene, exists as the Zwitterion in the solid (CSD entry RISBIE). In this system, the geometry of the N\u2013H\u2013N\u2032 structure is symmetric, N\u2013H: 1.27\u00a0\u00c5, N\u2013N\u2032: 2.57\u00a0\u00c5, and the O\u2013H\u2013O structure is unsymmetric, O\u2013H: 1.00\u00a0\u00c5, O\u2013O\u2032: 2.45\u00a0\u00c5. PM6 predicts both the N\u2013H\u2013N\u2032 (N\u2013H: 1.12\u00a0\u00c5, N\u2013N\u2032: 2.68\u00a0\u00c5) and the O\u2013H\u2013O\u2032 structures (O\u2013H: 1.09\u00a0\u00c5, O\u2013O\u2032: 2.49\u00a0\u00c5) to be unsymmetric. In the gas phase, 4,5-bis(dimethylamino)-1,8-dihydroxynaphthalene would most likely exist as the neutral species; a B3LYP\/6\u201331G(d) calculation predicts the energy of the Zwitterionic form to be 0.6\u00a0kcal\/mol above that of the neutral form; however, PM6 incorrectly predicts that the Zwitterion should be 15.8\u00a0kcal\/mol more stable than the neutral form.In 2005, an even stronger proton sponge, 1,8-bis(hexamethyltriaminophosphazenyl)naphthalene, HMPN, was reported [25]. The effect of steric crowding in HMPN arising from the \u2013N\u2009=\u2009P(N(Me2))3 groups distorts the naphthalene skeleton so that the reported C1\u2013C9\u2013C10\u2013C5 torsion angle, Fig.\u00a03, is 173.9\u00b0. The fully optimized PM6 crystal structure predicted this angle to be 170.3\u00b0. For the gas-phase structure, B3LYP\/6\u201331G* predicted the torsion angle to be 172.8\u00b0 [25] while PM6 gave an angle of 165.8\u00b0, indicating that the PM6 model was producing either a less rigid naphthalene structure or a greater steric repulsion energy.\nFig.\u00a031,8-bis(hexamethyltriaminophosphazenyl)naphthaleneAn additional example of the importance of crystal packing forces is provided by the accuracy of prediction of the structure of HMPN in the gas and solid state phases. Excluding hydrogen atoms, the RMS difference between the B3LYP gas-phase structure and the X-ray structure was 0.286\u00a0\u00c5, while for the PM6 crystal structure the equivalent difference was 0.267\u00a0\u00c5, and for the PM6 gas-phase structure the RMS difference was 0.677\u00a0\u00c5. That is, the B3LYP gas-phase structure was a significantly better fit to the observed crystal structure than that given by PM6, but when crystal forces were included in the PM6 calculation, the PM6 gave a slightly better fit than the B3LYP result.The energetics involved in the sublimation process for HMPN can be modeled in three stages. In the solid phase, PM6 gives a \u0394Hf of \u221247.8\u00a0kcal\/mol for HMPN. Using the optimized geometry for the crystal form, PM6 gives a \u0394Hf of \u221217.9\u00a0kcal\/mol for HMPN in the gas phase. When the geometry is allowed to relax, the \u0394Hf of the optimized gas-phase geometry decreased to \u221227.9\u00a0kcal\/mol. From this it follows that crystal packing forces distort the geometry of HMPN so that its energy increases by 10\u00a0kcal\/mol. This increase is, however, more than offset by the intermolecular stabilization energy of 29.9\u00a0kcal\/mol resulting in a net sublimation energy of 19.9\u00a0kcal\/mol.Because HMPN has a very high proton affinity, it is interesting to speculate about the minimum energy structure of gas-phase 4,5-bis(hexamethyltriaminophosphazenyl)-1,8-dihydroxynaphthalene. As with 4,5-bis(dimethylamino)-1,8-dihydroxynaphthalene, PM6 predicts this system to exist as the Zwitterion in the gas phase, yet, like 4,5-bis(dimethylamino)-1,8-dihydroxynaphthalene, a BLYP\/6\u201331G* calculation suggests that the neutral form is the more stable form, but only by 0.3\u00a0kcal\/mol. That is, the possibility exists that 4,5-bis(hexamethyltriaminophosphazenyl)-1,8-dihydroxynaphthalene might form a stable gas-phase Zwitterion, but the probability of this being the case is low.An unusual complex resulting from the reaction of elemental bromine and 1, 4-diazabicyclo[2.2.2]octane (DABCO), (DABCO)4\u22c5Br14 has been reported [26] to contain an almost linear structure of three DABCO groups and seven bromine atoms (Fig.\u00a04) with the bromine atoms split into three fragments of , , and Br\u2212, resulting in a formal charge of \u22123. From the X-ray structure (CSD entry DAHGUO), the authors concluded that partial protonation had occurred, that the complex of three DABCO units included four protons, and that the remaining isolated DABCO moiety was doubly protonated.\nFig.\u00a04Detail of bis(1,4-Diazoniabicyclo(2.2.2)octane) bis(1-aza-4-azoniabicyclo(2.2.2)octane) tetrakis(tribromide) dibromideWhen the structure suggested by the authors was optimized using PM6, only very small changes occurred in the geometry, as shown in Table\u00a04; all the essential features proposed by the original authors were reproduced, and are therefore confirmed. These included the conjectures that, in the set of three DABCO units, the central DABCO was doubly ionized, that the terminal DABCOs were singly ionized, and that the isolated DABCO was doubly charged. The two tribromide ions had charges of \u22120.98 and \u22121.00, with the central atom in each group carrying only an insignificant charge, in accordance with the postulated existence of a tribromide anion, and the isolated bromide ion had a partial charge of \u22120.78, again in accordance with the proposed structure.\nTable\u00a04Interatomic distances and charges in bis(1,4-Diazoniabicyclo(2.2.2)octane) bis(1-aza-4-azoniabicyclo(2.2.2)octane) tetrakis(tribromide) dibromideInteratomic distancesChargesX-rayPM6PM6N1-N22.692.70DABCO1+1.01N2-H1.16DABCO2+1.68N3-H1.16DABCO3+1.01N3-N42.662.70DABCO4+1.81N5-Br13.172.97Br1\u22120.78Br1-Br23.603.00Br2\u22120.54Br2-Br32.512.48Br3\u22120.05Br3-Br42.592.50Br4\u22120.39Br4-Br53.252.98Br5\u22120.45Br5-Br62.452.47Br6\u22120.09Br6-Br72.692.53Br7\u22120.47\nN\u2013H\u2013O The simple amino acids exist as Zwitterions in the solid state. In this form, an \u2013NH3+ group on one ion electrostatically interacts strongly with a \u2013COO\u2013 ion on a neighboring ion. In all cases examined, the structure of the resulting salt was reproduced with good accuracy. This included the structures of three polymorphs of glycine, \u03b1, \u03b2, and \u03b3, a set of systems in which any differences in heats of formation could arise only from the different intermolecular interactions. PM6 predicted all three polymorphs to have heats of formation within 2\u00a0kcal\/mol of each other, albeit not in the order observed experimentally. Of more interest are those cases where the energy of interaction is smaller and, consequently, the geometry of the N\u2013H\u2013O system would be more sensitive to the environment. A good example of such a system is methyl (+ \u2212)-(1\u03b1,2\u03b2,8\u03b1,9\u03b1,10\u03b2)-2-chloro-4-aza-3-oxatetracyclo(8.4.0.02,9.04,8) tetradecane-9-carboxylate oxalic acid monohydrate (CSD entry HUZKOC), in which a molecule of oxalic acid hydrogen bonds to a neutral nitrogen on the large organic fragment. In this system, PM6 predicts the N\u2013O distance to be slightly too large, 2.61\u00a0\u00c5 compared to the reported 2.57\u00a0\u00c5.The existence of a uniquely short heteroatom separation in a hydrogen-bonded compound, 2-(2-(3-carboxypyridyl))-4-isopropyl-4-methyl-5-oxo-imidazole, Fig.\u00a05, was reported in 1989 [27]. In this system (CSD entry JAZCOC01), the pyridine and imidazole rings are almost coplanar, a condition essential to the formation of the strong hydrogen bond. The optimized gas-phase geometry predicted by AM1 and PM3, and now PM6, all have large twist angles, 47, 37, and 40\u00b0, respectively. In the solid state, however, PM6 predicts the rings to be coplanar, and the N\u2013O distance to be 2.50\u00a0\u00c5, which is very close to the reported 2.47\u00a0\u00c5. In the observed crystal structure the hydrogen atom is asymmetrically positioned, with the O-H and N-H distances being 1.16\u00a0\u00c5 and 1.32\u00a0\u00c5, and the O\u2013H\u2013N angle being 170\u00b0. PM6 also predicts the hydrogen to be asymmetrically positioned, but in the opposite sense: the PM6 O\u2013H and N\u2013H distances being 1.46 and 1.12\u00a0\u00c5, and the O\u2013H\u2013N angle to be 150\u00b0.\nFig.\u00a052-(2-(3-Carboxypyridyl))-4-isopropyl-4-methyl-5-oxo-imidazoleThe tendency of PM6 to incorrectly favor the Zwitterionic form of a hydroxy\u2013amine over the neutral was investigated by modeling the three isomers of hydroxyaniline. All three isomers exist in the solid state as the neutral form; however, on optimizing the geometry using PM6, the ortho and para forms spontaneously transitioned to the Zwitterion. Meta-hydroxyaniline optimized to the neutral form, but when the calculation was repeated, starting with the Zwitterionic form, the structure optimized to the Zwitterionic form, and in that form was 1.1\u00a0kcal\/mol lower in energy than the neutral form. As expected, the density increased from 1.27 to 1.34 on going from the neutral to the Zwitterion. PM6 thus has a definite and demonstrable error in its exaggerated tendency to form Zwitterions.DABCO forms several very unusual hydrogen-bonded systems, among which are 1,4-diazabicyclo[2.2.2]octane azelaic acid (CSD entry UNEGEZ), where each DABCO forms two different types of bridging N\u2013H\u2013O bonds to adjacent azelaic acid molecules. PM6 predicts one of these to have the structure N\u2013O: 2.53(2.55), N\u2013H: 1.29(1.21), and O\u2013H: 1.27(1.40), (X-ray structures in parentheses) and the other to be N\u2013O: 2.75(2.61), N\u2013H: 1.75(1.50), and O\u2013H: 1.06(1.11). The X-ray structure shows the existence of a polymeric chain of alternating azelaic acid and DABCO units, from which it follows that the different geometries of the two N\u2013H\u2013O structures can only be attributed to crystal packing forces. A closely related compound is 1-diazonia-4-azabicyclo[2.2.2]octane glutarate (CSD entry UNEFIC), in which a similar structure exists but now with the bridging hydrogen being nearer to the nitrogen than to the oxygen. In UNECIF, the N\u2013H distance predicted by PM6 was 1.10 (0.95) \u00c5 and the O\u2013H 1.65 (1.76) \u00c5.\n\u03c0\u2212\u03c0 stacking\n\u03c0\u2212\u03c0 stacking occurs in the polycyclic aromatic hydrocarbons where it is the result of Van der Waals (VDW) interactions between the rings. In general, VDW interactions are weaker than hydrogen bonding interactions, and, historically, have been the hardest to model using semiempirical methods. Thus, when the default single determinant wavefunction is used, VDW terms are completely absent, and, in order to mimic the effects of VDW attraction, the normal procedure is for modifications to be made to the core\u2013core interaction. An estimate of how accurately PM6 can reproduce the VDW interaction is provided by anthracene (CSD entry ANTCEN14), benzene (CSD entry BENZEN), and coronene (CSD entry CORONE). In anthracene, the molecules are stacked in a staggered arrangement. While PM6 reproduces the density with good accuracy, the optimized structure predicts the parallel sheets of anthracene molecules to be separated by 5.37\u00a0\u00c5 rather than the observed 2.83\u00a0\u00c5, and pairs of anthracene molecules forming \u201cT\u201d structures rather than the observed \u201cV\u201d configuration. Conversely, both benzene and coronene crystallize with a perfect herringbone packing, and this structure is reproduced with very good accuracy by PM6, the calculated and observed inter-plane distance being essentially identical.\nVery weak interactions\nIn small saturated hydrocarbons the strongest intermolecular interaction energy arises from instantaneous correlation or VDW forces. A consequence of this is that such compounds are, in general, highly volatile and most have very low melting points. Another characteristic of compounds of this type is that intermolecular separations are typically very large: in cyclohexane the smallest intermolecular separation is about 2.6\u00a0\u00c5. When the structure of cyclohexane was optimized from the experimental structure using PM6, the final and X-ray structures agreed almost exactly. However, the heat of formation of each cyclohexane decreased from an initial +65.8\u00a0kcal\/mol for the starting X-ray structure to the final \u221229.6\u00a0kcal\/mol for the optimized PM6 structure, reflecting the relaxation of the X-ray C\u2013H bond lengths, which are normally too short. However, the PM6 \u0394Hf for isolated cyclohexane is predicted to be \u221227.5\u00a0kcal\/mol, from which it follows that the energy of interaction amounted to only 2.1\u00a0kcal\/mol, much less than the reported 9.0\u00a0kcal\/mol. The implication is that, although PM6 was able to reproduce the observed crystal structure, the magnitude of the VDW interaction was grossly underestimated.\nIn solid methionine (CSD entry LMETON02) the molecules form double layers with the hydrophobic end, \u2013CH2\u2013S\u2013CH3, on the outside. The X-ray structure shows that these layers are separated by about 2.21\u00a0\u00c5; PM6 predicts the inter-layer separation to be much larger, 2.83\u00a0\u00c5. Further investigation of the properties of sulfur predicted by PM6, see below, indicated the lack of any sulfur\u2013sulfur VDW attraction. This deficiency is likely responsible for the unrealistically large inter-layer separation.\nMost acetylacetonato transition metal complexes, such as tris(acetylacetonato) titanium(iv) perchlorate (CSD entry TIACPC), adopt an almost octahedral coordination of the oxygen atoms around the central metal ion. An exception is the hexamethyl acetylacetone complex of yttrium(iii), tris(2,2,6,6-tetramethylheptane-3,5-dionato)-yttrium(iii) (CSD entry HAHTOZ01), where the oxygen atoms form a trigonal prism. This structure was reproduced by PM6. The deviation from the conventional octahedral structure cannot be attributed to crystal packing forces\u2014when the geometry of the isolated complex ion was optimized using PM6, the trigonal prism structure was retained. However, when the geometry of the unsubstituted complex, tris(acetylacetonato) yttrium(iii), was optimized, the expected D3d structure resulted, suggesting that the likely driving force was steric crowding arising from the tertiary butyl groups.\nPolymorphs\n5-Methyl-2-((2-nitrophenyl)amino)-3-thiophenecarbonitrile is unique in the CSD in that there are seven distinct polymorphs [28]; because three of these are red, orange, and yellow, this chemical is commonly referred to as ROY. To test the suitability of PM6 for modeling polymorphs, the structure of each of the polymorphs of ROY was optimized using PM6, starting with the X-ray geometry. In every case, the optimized PM6 structure was qualitatively the same as the X-ray structure; that is, the crystal packing arrangement was preserved. Within each unit cell, the structures of the individual molecules were reproduced with good accuracy (Table\u00a01). Although the calculated heats of formation of the X-ray structures spanned a range of 31\u00a0kcal\/mol, the optimized PM6 structures all had similar heats of formation, spanning a range of 4.3\u00a0kcal\/mol, as would be expected for polymorphs.\nCo-crystals\nDesigning crystal structures for active pharmaceutical ingredients presents an important challenge to crystal engineers. One promising avenue of research involves designing co-crystals, so determining the suitability of PM6 as a tool for this work is of obvious interest. The structures of several co-crystals were optimized using PM6; all the resulting geometries were in good agreement with the structures found in the CSD. In all of the systems investigated the two components were held together by hydrogen bonds. Thus in the bis-urea\u2013oxalic acid co-crystal (Fig.\u00a06, CSD entry UROXAL01), each oxalic acid forms two strong O\u22efH\u22efO hydrogen bonds to the keto groups of the neighboring urea molecules, this resulting in a tri-molecular unit. In turn, these units pack together in the crystal using weaker N\u22efH\u22efO hydrogen bonds. PM6 predicts that the co-crystal would be significantly more stable than its two separate precursors: the predicted \u0394Hf of the co-crystal is \u2212313.4\u00a0kcal\/mol, while the sum of the \u0394Hf of the precursors, Table\u00a02, is \u2212306.9\u00a0kcal\/mol.\nFig.\u00a06Crystal structure of the co-crystal of urea and oxalic acid\nAnother example of such a co-crystal is provided by isonicotinamide 3-hydroxybenzoic acid, Fig.\u00a07 (CSD entry LUNMEM), in which each hydroxybenzoic acid forms hydrogen bonds to three adjacent isonicotinamide molecules in a complicated three-dimensional structure. As with the previous co-crystal, PM6 predicts LUNMEM to be more stable than its precursors but, in this case, only by 0.1\u00a0kcal\/mol.\nFig.\u00a07Co-crystal of isonicotinamide and 3-hydroxybenzoic acid showing PM6 hydrogen-bond lengths (X-ray in parenthesis)\nMetal-containing species\nMany organic compounds that contain elements of Groups I or II are ionic salts. A good example is calcium oxalate, which forms three well characterized solids: whewellite, (Ca(C2O4)\u00b7(H2O)), weddellite, (Ca(C2O4)\u00b72(H2O)), and caoxite, (Ca(C2O4)\u00b73(H2O)), at least one of which the author involuntarily prepared in vivo, the separation of which from the surrounding organic material was accompanied by severe algia. The structures of all three minerals are reproduced with good accuracy. Anhydrous disodium oxalate exists as the mineral natroxalate. In contrast to the calcium oxalates, the structure predicted by PM6 was completely different to that observed: within each oxalate dianion, one carboxylate group was rotated by \u223c90\u00b0 to give an approximately D2d structure. More seriously, some sodium\u2013sodium distances became unrealistically short, 1.23\u00a0\u00c5 instead of the observed 3.30\u00a0\u00c5. This specific error is attributable to the faulty PM6 values of the Na\u2013Na core\u2013core parameters.\nSolids containing metal complex ions can be regarded as salts: that is, as molecular metal complexes, cations or anions, plus counterions. An example is bromo-tris(2-dimethylaminoethyl)amine-manganese(ii) (CSD entry DAEAMN), where the metal complex consists of the neutral tris(2-dimethylaminoethyl)amine that chelates a manganese dication, the whole complex behaving like a large dication. Two bromide counterions are present for each such complex ion in order to maintain electroneutrality. PM6 predicts the single N\u2013Mn distance to be 2.09\u00a0\u00c5 versus 2.19\u00a0\u00c5 in the X-ray structure, and the three N\u2013Mn to be 1.96\u00a0\u00c5 versus 2.27\u00a0\u00c5 in the X-ray.\nInorganic compounds\nIn general, most inorganic solids differ from crystalline organic compounds in that they do not involve discrete molecules. Instead, they exist as extended covalently or ionically bound infinite systems. A consequence of this is that identification of simple structural units in inorganic solids is often either difficult or impossible.\nA more subtle consequence is that the band-structure of inorganic solids is usually more complicated than those of organic solids: for the same reciprocal distance, bands arising from inorganic solids generally have a much greater curvature than those for organic solids, this being a consequence of the strong bonds that extend throughout such solids. Conversely, in most organic solids there is an intermolecular gap that effectively confines molecular orbitals to individual molecules. This means that the band-structure of most organic compounds consists of relatively flat bands. In the cluster method, the \u0393 point represents the entire Brillouin zone so that a much larger cluster must be used when inorganic solids that are not composed of discrete molecules are modeled. In practice this means that the cluster used has to be large enough to contain a sphere of radius 10\u201312\u00a0\u00c5, in contrast to the 7\u20138\u00a0\u00c5 used in modeling organic solids.\nA survey of the applicability of PM6 to a wide range of inorganic solids was carried out, using structural reference data obtained from the American Mineralogist Crystal Structure Database [29]. As with organic crystals, the starting geometry was the X-ray structure. But, in contrast to most organic solids, many inorganic solids had to be modified before a PM6 calculation could be started. Semiempirical calculations require that a definite structure be used. However, some minerals, such as forsterite, (FeII,Mg)2SiO4, are of variable composition, with one or more sites occupied by the two types of metal atoms at random. Before a calculation on such a system can be performed, all variable atoms have to be replaced by definite atoms. In the case of forsterite, all iron atoms were replaced by magnesium atoms. In a few solids there was still disorder in the lattice although the formula was stoichiometric. In spinel, for example, the formula is MgAl2O4 but, in the observed crystal structure, a small fraction of the aluminum sites are replaced by magnesium atoms, and vice versa. Of necessity, the model used in the PM6 calculation was idealized so that sites that were 80% magnesium were made 100% magnesium, and sites that were 90% aluminum were made 100% aluminum. Although this idealized structure does not occur naturally it could be considered a good approximation to the observed structure.\nSome minerals that contain hydrogen atoms did not have the positions of these atoms reported. In those cases a preliminary calculation was carried out in which the hydrogen atoms were positioned in likely sites, and then the positions of those atoms optimized. During this operation, the positions all the other atoms were fixed at the experimental values.\nThe results can conveniently be partitioned according to the types of solids involved and sequenced in order of complexity. In most cases, the calculated structure was similar to the X-ray structure in that no bonds were made or broken as a result of geometry optimization. Unit cell parameters for those solids where the calculated PM6 structure inside the unit cell was substantially similar to the reference are presented in Table\u00a05 and Table\u00a06, and heats of formation are given in Table\u00a07.\nTable\u00a05Calculated and X-ray structural parameters for inorganic compounds. Values of a, b, c are in \u00c5ngstroms; \u03b1, \u03b2, \u03b3 in degreesPM6X-rayDensity (g\/cm3)dabc\u03b1\u03b2\u03b3abc\u03b1\u03b2\u03b3ElementsDiamond (C)3.593.593.5990.090.090.03.573.573.5790.090.090.03.44 (3.52)Glitter (C)e2.602.605.9690.190.190.02.602.605.9090.090.090.02.98 (3.08)Graphite (C)2.472.4787.1789.690.060.02.462.466.7090.090.060.02.12 (2.28)Silicon (Si)5.115.115.1190.090.090.05.435.435.4390.090.090.02.78 (2.33)Sulfur (S)11.7714.0126.8190.490.289.810.4312.8324.3690.090.090.01.54 (2.09)Group I HalidesCesium bromide (CsBr)4.354.354.3590.090.090.04.304.304.3090.090.090.04.29 (4.43)Cesium chloride (CsCl)4.174.184.1890.389.889.74.124.124.1290.090.090.03.84 (3.99)Cesium fluoride (CsF)6.026.026.0290.090.090.06.016.016.0190.090.090.04.62 (4.64)Cesium iodide (CsI)4.734.734.7390.090.090.04.574.574.5790.090.090.04.07 (4.51)Potassium bromide (KBr)6.796.756.7587.690.090.06.616.616.6190.090.090.02.55 (2.74)Potassium chloride (KCl)6.126.126.1290.090.090.06.296.296.2990.090.090.02.16 (1.99)Potassium fluoride (KF)5.035.035.0390.090.090.05.385.385.3890.090.090.03.02 (2.48)Potassium iodide (KI)7.177.177.1790.190.090.07.077.077.0790.090.090.02.99 (3.12)Lithium bromide (LiBr)5.495.485.4890.090.090.05.505.505.5090.090.090.03.50 (3.46)Lithium chloride (LiCl)5.135.135.1390.090.090.05.145.145.1490.090.090.02.09 (2.07)Lithium fluoride (LiF)4.034.044.0590.089.990.04.034.034.0390.090.090.02.62 (2.64)Lithium iodide (LiI)6.046.036.0490.090.090.06.036.036.0390.090.090.04.04 (4.06)Sodium bromide (NaBr)6.276.206.2085.990.090.06.046.046.0490.090.090.02.84 (3.10)Sodium chloride (NaCl)6.246.246.2490.090.090.05.605.605.6090.090.090.01.60 (2.21)Sodium fluoride (NaF)4.794.794.7990.290.189.84.614.614.6190.090.090.02.54 (2.85)Sodium iodide (NaI)7.027.027.0290.090.090.06.476.476.4790.090.090.02.87 (3.67)Rubidium bromide (RbBr)6.876.886.8890.390.090.06.906.906.9090.090.090.03.38 (3.35)Rubidium chloride (RbCl)6.706.686.6889.190.090.06.636.636.6390.090.090.02.69 (2.76)Rubidium fluoride (RbF)5.865.865.8690.090.090.06.016.016.0190.090.090.03.45 (3.20)Rubidium iodide (RbI)7.847.767.7683.390.090.07.357.357.3590.090.090.03.01 (3.55)HalidesCalcium iodide (CaI2)6.214.484.48119.990.090.16.964.484.48120.090.090.04.51 (4.04)Carnallite (KMg(H2O)6Cl3)15.8622.3110.2473.192.895.916.1222.479.5590.090.090.01.61 (1.60)Chloromagnesite (MgCl2)19.403.753.75119.989.190.917.593.603.60107.990.090.02.00 (2.41)Cryolite (Na3AlF6)5.678.035.6990.190.089.95.467.805.6190.090.090.22.69 (2.92)Fluorite (CaF2)5.495.455.4890.090.090.05.465.435.4690.090.090.03.17 (3.20)Frankdicksonite (BaF2)6.226.226.2390.090.090.06.206.206.2090.090.090.04.83 (4.89)Hydrophilite(CaCl2)4.286.426.3889.990.090.04.206.436.2490.090.090.02.10 (2.19)Magnesium dibromide (MgBr2)5.834.024.02119.191.688.36.263.813.81120.090.090.03.71 (3.88)Magnesium diiodide (MgI2)6.284.274.24118.290.689.66.884.144.14120.090.090.04.61 (4.52)Na3(TiCl6)10.237.357.0090.087.189.89.817.096.6890.089.790.02.08 (2.36)Neighborite (NaMgF3)8.148.138.1390.090.090.17.677.677.6791.490.090.02.57 (3.07)Sal ammoniac (NH4Cl)4.074.073.6890.089.973.53.863.863.8690.090.090.01.52 (1.54)Sellaite (MgF2)3.074.624.6290.090.090.03.054.634.6390.090.090.03.16 (3.17)Oxides\u03b1 Crystobalite (SiO2)5.365.367.4789.990.189.64.984.986.9590.090.090.01.86 (2.32)\u03b1 Quartz (SiO2)5.195.185.7490.090.0120.04.914.915.4190.090.0120.02.24 (2.65)Anatase (TiO2)10.293.923.9290.190.289.69.523.783.7890.090.090.03.36 (3.90)Arkelite (ZrO2)6.094.915.5090.089.990.15.135.135.1390.090.090.04.97 (6.06)Barium oxide (BaO)5.645.645.6489.890.090.05.635.635.6390.090.090.05.69 (5.72)\u03b2 Quartz (SiO2)5.195.185.7490.090.060.05.005.005.4690.090.060.02.24 (2.54)\u03b2 Tridymite (SiO2)5.345.348.7290.090.0119.95.055.058.2790.090.0120.01.86 (2.18)Brookite (TiO2)5.425.679.6190.090.490.05.145.459.1790.090.090.03.59 (4.13)Cassiterite (SnO2)4.944.963.4990.090.089.94.744.743.1990.090.090.05.86 (7.00)Chabazite (SiO2)9.559.569.5693.993.993.89.409.409.4094.394.394.31.38 (1.46)Coesite (SiO2)7.4312.857.4990.060.890.07.1712.377.1490.059.790.02.56 (2.92)Claudetite (As2O3)14.005.435.0689.885.881.812.874.545.2586.290.090.03.46 (4.29)Corundum (Al2O3) (a\u2009=\u2009b\u2009=\u2009c,\u03b1\u2009=\u2009\u03b2\u2009=\u2009\u03b3)5.135.125.1256.256.256.25.135.135.1355.355.355.33.91 (3.99)Corundum (Al2O3) (a\u2009=\u2009b\u2009\u2260\u2009c,\u03b1\u2009=\u2009\u03b2\u2009\u2260\u2009\u03b3)4.834.8312.9190.090.0120.04.764.7612.9990.090.0120.03.91 (3.99)Ice-I (Ice-Ih)7.454.367.0990.590.389.37.834.527.3890.090.090.01.04 (0.92)Ice-II7.2212.265.9189.9106.591.07.7812.986.2490.0105.590.01.43 (1.18)Ice-III6.696.506.4689.989.590.76.736.736.7390.090.090.01.28 (1.18)Ice-V7.029.448.8070.190.692.67.5310.359.2070.790.089.11.53 (1.24)Ice-VI5.385.945.9090.989.589.05.706.186.1890.090.090.01.59 (1.37)Ice-VIII6.444.364.3690.090.590.16.414.454.4590.090.090.01.96 (1.89)Ice-X (0Gpa)2.902.902.9090.090.090.02.732.732.7390.090.090.02.46 (2.96)Ice-X (62Gpa)2.722.722.7290.090.090.02.732.732.7390.090.090.02.98 (2.96)Ice-XI7.624.267.1590.190.289.97.834.527.3890.090.090.01.03 (0.92)Ice-XIII9.777.058.4690.569.590.610.297.479.2490.070.390.01.54 (1.25)Ice-XIV3.838.077.8490.390.090.34.088.358.1490.090.090.01.48 (1.29)Keggin Dodecatungstophosphoric acid hexahydrate H3(PO4@W12O36).6(H2O)13.0113.4313.0090.492.289.712.5112.5112.5190.090.090.04.37 (5.07)Lime (CaO)4.954.954.9590.090.190.04.824.824.8190.090.090.03.07 (3.34)Mordenite (SiO2)18.6821.007.7590.290.090.018.1320.497.5290.090.090.01.58 (1.71)Lead oxide (PbO)3.834.743.8389.990.089.93.985.023.9890.090.090.010.68 (9.34)Periclase (MgO)4.294.294.2990.090.090.04.224.224.2290.090.090.03.40 (3.57)Perovskite (CaTiO3)5.385.547.8489.789.990.15.385.447.6490.090.090.03.87 (4.04)Plattnerite (PbO2) (Rutile structure)3.504.664.7389.890.090.03.394.964.9690.090.090.010.31 (9.55)Rutile (TiO2)4.844.843.1290.090.090.04.594.592.9690.090.090.03.71 (4.25)Stishovite (SiO2)2.824.384.3890.090.090.02.674.184.1890.090.090.03.70 (4.28)Zincite (ZnO)3.333.335.2690.090.060.33.253.255.2090.090.060.05.33 (5.68)SpinelsChrysoberyl ((BeO)(Al2O3))4.589.585.7290.390.090.04.409.335.4490.090.090.03.36 (3.78)Spinel (MgAl2O4)8.3116.6316.6390.290.090.08.2116.4116.4190.090.090.03.29 (3.42)BoratesBoracite (MgO)5(MgCl2)(B2O3)78.548.5912.1190.090.090.08.558.5512.0990.090.090.02.93 (2.95)Borax (Na2B4O7\u00b710(H2O))12.3311.2716.20105.193.783.012.1910.7411.8990.073.490.01.18 (1.70)Parahilgardite (Ca2(B5O9)Cl(H2O))17.196.396.18119.699.884.617.506.496.31119.2100.484.02.84 (2.69)Ulexite (NaCaB5O9\u00b78(H2O))6.7713.628.8897.4108.2103.36.6812.878.82110.0109.190.41.82 (1.96)CarbonatesAragonite (CaCO3)4.557.806.0890.090.090.14.967.975.7490.090.090.03.08 (2.93)Azurite ((CuO)3(CO2)2(H2O))5.735.129.5091.090.190.05.855.0110.3492.490.090.04.10 (3.78)Calcite (CaCO3)6.326.326.3245.245.245.26.366.366.3646.146.146.12.87 (2.73)Cerussite (PbCO3)5.846.43 7.0989.391.489.35.186.148.4990.090.090.06.66 (6.57)Dawsonite ((Na2O)(Al2O3)(CO2)2(H2O)2)6.735.985.7590.090.290.06.715.215.5890.090.090.02.07 (2.45)Dolomite (CaMg(CO3)2)4.744.7415.3290.089.9120.04.814.8116.0290.090.0120.03.08 (2.86)Huntite (CaMg3 (CO3)4)9.409.357.6790.590.259.89.509.507.8290.090.060.03.02 (2.88)Ikaite ((CaO)(CO2)(H2O)6)8.727.8510.8191.371.390.38.878.2311.0290.069.890.01.97 (1.83)Kalicinite(KHCO3)19.634.164.1690.977.890.215.195.633.7190.075.590.02.00 (2.17)Magnesite (MgCO3)14.914.577.9590.090.290.015.024.638.0390.090.090.03.11 (3.01)Natrite (Na2CO3)9.936.815.6690.290.0102.08.916.045.2490.090.0101.31.88 (2.55)Thermonatrite (Na2CO3(H2O))5.8511.817.8190.089.391.65.2610.726.4790.090.090.01.53 (2.26)Otavite (CdCO3)5.564.7115.5990.088.990.94.934.2716.2790.090.090.04.21 (5.02)Smithsonite (ZnCO3)4.834.8314.4689.590.0120.04.654.6515.0390.090.0120.04.28 (4.44)Strontianite (SrCO3)6.598.655.0290.190.090.06.008.365.0990.090.090.03.42 (3.84)Witherite (BaCO3)4.997.268.4989.989.989.95.316.438.9090.090.090.04.26 (4.32)Zabuyelite (Li2CO3)5.268.806.2367.590.090.04.978.366.2065.290.090.01.84 (2.10)Group IVMoissanite (SiC)5.005.363.0990.090.090.05.055.333.0890.090.090.03.22 (3.22)Silicon carbide (SiC)4.394.394.3990.090.090.04.354.354.3590.090.090.03.16 (3.24)Groups III-VAluminum antimonide (AlSb)5.945.945.9490.090.090.06.146.146.1490.090.090.04.71 (4.28)Aluminum arsenide (AlAs)5.915.915.9190.090.090.05.665.665.6690.090.090.03.27 (3.73)Aluminum nitride (AlN)4.404.404.4090.090.090.04.374.374.3790.090.090.03.20 (3.25)Aluminum phosphide (AlP)5.495.495.4990.090.090.05.425.425.4290.190.190.12.33 (2.42)Boron Nitride (BN)3.643.643.6490.090.090.03.613.613.6190.090.090.03.42 (3.49)Gallium arsenide (GaAs)5.665.665.6690.090.090.05.655.655.6590.090.090.05.31 (5.32)Indium arsenide (InAs)5.975.975.9790.090.090.06.066.066.0690.090.090.05.93 (5.67)Group VICadmium telluride (CdTe)5.585.585.5890.090.090.06.366.366.3690.090.090.09.17 (6.20)Coloradoite (HgTe)6.616.616.6190.090.090.06.326.326.3290.090.090.07.56 (8.63)Lead selenide (PbSe)4.344.344.3590.090.090.06.176.176.1790.090.090.023.18 (8.10)Lead telluride (PbTe)7.877.877.8790.090.090.07.107.107.1090.090.090.04.56 (6.22)Stibnite (Sb2S3)10.674.0511.4090.087.990.011.293.8311.2190.090.090.04.58 (4.66)Stilleite (ZnSe)5.955.955.9590.090.090.05.545.545.5490.090.090.04.56 (5.65)HydridesBH3NH34.824.245.2594.490.190.14.994.895.3990.090.090.00.96 (0.78)HydroxidesBrucite (Mg(OH)2)3.203.214.3089.890.0120.13.143.144.7790.090.0120.02.53 (2.38)Diaspore (AlO(OH))4.179.753.0090.089.989.84.409.432.8590.090.090.03.26 (3.38)Gibbsite (Al(OH)3)9.0410.384.4590.190.090.09.7410.164.3490.085.590.02.48 (2.42)MnO(OH)7.006.2313.4590.288.489.85.295.8013.3189.789.690.02.99 (4.29)Orthoboric acid (B(OH)3)10.977.267.26120.183.4100.46.357.047.02119.878.587.50.84 (1.56)NitratesAmmonium nitrate (NH4NO3)8.8213.8510.7695.889.472.19.4912.2310.7690.089.991.31.71 (1.70)Niter (KNO3)8.965.386.5990.689.189.59.165.416.4390.090.090.02.11 (2.11)Nitratine (NaNO3)18.665.434.0890.196.290.516.835.074.3990.090.090.02.06 (2.26)PhosphatesBobierrite (Mg3(PO4)2(H2O)8)10.4626.994.5889.4104.393.010.0727.934.6790.0105.090.02.16 (2.13)Calcium phosphate (Ca3(PO4)2)5.5319.975.3488.756.990.35.2518.675.2590.060.090.03.13 (3.47)Fluorapatite (Ca5(PO4)3F)9.289.296.8789.890.059.69.379.376.8890.090.060.03.28 (3.20)Dorfmanite (Na2HPO4\u00b72(H2O))8.4911.7715.9089.990.290.16.6010.3616.8790.090.090.01.48 (2.05)Lithiophosphate (Li3PO4)5.496.7511.6290.090.190.14.936.1210.5390.090.090.01.79 (2.42)Newberyite (MgHPO4\u00b73(H2O))9.8710.0610.3290.090.090.410.0210.6810.2090.090.090.02.26 (2.12)Phosphammite ((NH4)2HPO4)7.756.7410.9189.966.490.18.036.7011.0490.066.690.01.68 (1.61)Potassium dihydrogen phosphate (KH2PO4)7.697.577.5988.091.088.76.977.457.4590.090.090.02.05 (2.33)Potassium oxovanadium(IV) phosphate(V) (KVPO5)7.667.718.8190.289.890.07.617.318.4190.090.090.02.56 (2.85)Sodalite (AlPO4)8.328.338.6089.490.590.49.049.049.0490.090.090.02.04 (1.64)Wagnerite (Mg2PO4F)12.1112.429.6590.074.690.011.9612.689.6490.071.790.03.09 (3.11)SulfatesAlum (KAl(SO4)2\u00b712(H2O))12.3112.3512.2789.389.890.512.1812.1812.1990.090.090.01.69 (1.74)Aluminite (Al2SO6 9(H2O))6.9015.3311.2990.2108.090.07.4415.5811.7090.0110.290.02.01 (1.80)Alunite (KAl3(SO4)2(OH)6)7.017.0017.3889.989.760.26.966.9617.3590.090.060.02.79 (2.84)Ammonium sulfate (2(NH4)SO4)10.995.757.2991.090.190.210.645.997.7890.090.090.01.90 (1.77)Anglesite (PbSO4)7.406.337.0092.491.495.16.968.485.4090.090.090.06.18 (6.32)Anhydrite (CaSO4)6.798.397.0987.190.090.26.996.257.0090.090.090.02.24 (2.96)Arcanite (K2SO4)7.559.626.3889.990.190.27.4810.075.7690.090.090.02.50 (2.67)Barite (BaSO4)7.445.379.3490.189.990.07.155.468.8890.090.090.04.15 (4.47)Celestine (SrSO4)8.435.676.9390.189.891.18.395.366.8990.090.090.03.68 (3.94)Epsomite (MgSO4 (H2O)7)11.8211.816.1989.990.090.111.8912.016.8690.090.090.01.89 (1.67)Glauberite (CaNaSO4)8.4110.5910.1850.590.190.18.318.5310.1376.790.090.02.64 (2.78)Gypsum (CaSO4\u00b72H2O)5.5814.426.7190.2106.190.25.6715.106.4990.0118.590.02.21 (2.34)Hanksite (KCl)2(Na2O)22(SO3)18(CO2)411.6511.7621.8788.090.5119.210.4710.4621.1990.090.0120.01.99 (2.59)Hexahydrite (MgSO4\u00b76(H2O))24.306.929.6589.878.290.024.417.2110.1190.081.790.01.91 (1.72)Langbeinite (K2Ca2(SO4)3)10.7010.3810.5889.389.090.210.4310.4310.4390.090.090.02.52 (2.62)Leonite (K2Mg(SO4)2\u00b74(H2O))10.738.9913.0489.995.690.09.859.4711.7890.084.790.01.95 (2.23)Mercallite (KHSO4)9.8517.858.71590.290.090.69.8018.968.4190.090.090.02.36 (2.32)Thenardite (Na2SO4)6.3413.2811.3190.291.190.25.8712.309.8390.090.090.01.98 (2.66)Zinkosite (ZnSO4)4.936.368.8590.186.690.14.776.758.6090.090.090.03.87 (3.87)SulfidesCinnabar (HgS)4.314.319.8790.090.060.04.154.159.5190.090.060.07.30 (8.17)Galena (PbS)5.875.875.8790.090.090.05.945.945.9490.090.090.07.86 (7.60)Greenockite (CdS)6.707.154.1390.089.989.96.757.164.1390.090.090.04.85 (4.80)Hawleyite (CdS)3.843.843.8490.090.090.05.835.835.8390.090.090.05.02 (4.84)Metacinnabar (HgS)6.296.296.2990.090.090.05.855.855.8590.090.090.06.21 (7.71)Niningerite (MgS)5.205.205.2090.090.090.05.205.205.2090.090.090.02.66 (2.66)Sphalerite (ZnS)5.415.415.4190.090.090.05.435.435.4390.090.090.04.08 (4.04)Wurtzite (ZnS)3.843.846.3090.090.090.03.813.816.2390.090.060.04.03 (4.12)Vanadates, tungstates, chromates, molybdatesDescloizite (PbZn(VO4)(OH))8.366.309.3594.788.990.87.606.079.4490.090.090.05.48 (6.17)Ferberite (FeWO4)5.005.354.6489.995.489.94.955.704.7390.090.090.08.17 (7.55)Phoenicochroite (Pb2CrO5)5.677.7415.6742.191.888.25.677.1414.0064.890.090.07.90 (7.07)Scheelite (CaWO4)5.455.7314.4391.087.892.45.245.2411.3890.090.090.04.25 (6.12)Tarapacaite (K2CrO4)10.576.057.7089.989.890.310.395.927.6690.090.090.02.62 (2.74)Vanadinite (Pb5(VO4)3Cl)7.7811.9716.7487.492.391.67.3410.3317.8990.090.090.06.04 (6.93)Wulfenite (PbMoO4)5.6212.495.5990.390.089.25.4712.185.4790.090.090.06.22 (6.69)Zincochromite (ZnCr2O4)8.408.408.4090.090.090.08.338.338.3390.090.090.05.24 (5.36)Zirconium tungstate (ZrW2O8)9.439.499.5090.289.789.89.189.189.1890.090.090.04.59 (5.04)dX-ray densities in parenthesiseSee [31]Table\u00a06Calculated and X-ray structural parameters for silicates. Values of a, b, c are in \u00c5ngstroms; \u03b1, \u03b2, \u03b3 in degreesPM6X-rayDensitydabc\u03b1\u03b2\u03b3abc\u03b1\u03b2\u03b3CyclosilicateBenitoite (BaTiSi3O9)6.7611.609.8290.090.390.06.6011.439.7190.090.090.03.57 (3.75)Beryl (Be3Al2Si6O18)19.0919.069.0290.090.0120.018.4318.439.2490.090.090.02.51 (2.63)Dravite (NaMgTi2Al6(BO3)3(Si6O18)O4)16.1416.137.3989.989.960.215.9515.957.2190.090.060.02.99 (3.14)InosilicateCummingtonite (Mg7Si8O23\u00b7(H2O))9.0618.385.6790.0100.090.09.5118.195.3390.0101.990.02.79 (2.87)Diopside (MgCaSi2O6)5.369.119.2290.076.390.05.258.909.7590.074.490.03.29 (3.28)Enstatite (MgSiO3)5.388.9617.6990.189.990.05.188.8218.2390.090.090.03.13 (3.20)Jadeite (NaAlSi2O6)8.689.085.7290.1106.290.19.478.615.2490.0107.690.03.10 (3.30)Johannsenite (CaMnSi2O6)9.795.269.7975.690.290.19.165.299.9874.590.090.03.36 (3.52)Kosmochlor (Si2CrNaO6)5.699.3910.0790.076.290.25.278.729.5890.072.690.02.89 (3.59)Spodumene (LiAlSi2O6)9.526.118.0464.086.287.48.395.229.4669.990.090.02.95 (3.18)Tremolite (Ca2Mg5Si8O22(OH)2)9.3018.835.5890.177.490.09.8418.055.2790.075.390.02.83 (2.98)Wollastonite (CaSiO3)9.1611.127.63100.0102.182.710.1211.077.3199.5100.583.43.10 (2.93)Nesosilicate\u00c5kermanite (Ca2MgSi2O7)7.917.734.7790.087.290.17.847.845.0190.090.090.03.11 (2.94)Andalusite (Al2SiO5)6.137.965.9097.185.987.95.567.907.8090.090.090.03.78 (3.14)Chromium silicate (Cr2SiO4)12.5211.385.7789.890.289.09.5911.175.7090.090.090.03.17 (4.26)Euclase ((BeO)2(Al2O3)(SiO2)2(H2O))4.8315.124.4190.099.990.04.7814.324.6390.0100.390.03.03 (3.09)Forsterite (Mg2SiO4)4.776.1010.2390.189.990.14.766.0010.2290.090.090.03.14 (3.20)Garnet (Al2O3(CaO)3(SiO2)3)11.7511.7511.7590.090.090.011.8611.8511.8590.090.090.03.69 (3.59)Kyanite (Al2SiO5)7.965.775.6582.184.978.37.857.135.5778.990.074.04.29 (3.67)Pyrope ((Al2O3)(MgO)3(SiO2)3)11.3611.3711.3689.889.789.811.5511.5511.5590.090.090.03.65 (3.48)Sillimanite (Al2SiO5)12.497.365.7389.890.090.011.557.687.4990.090.090.04.09 (3.24)Titanite (CaTiSiO5)7.447.008.4389.989.6113.97.076.578.7290.090.0113.93.24 (3.52)Topaz (Al2SiO4F2)7.114.188.9589.789.189.69.334.428.3990.090.090.04.60 (3.54)Uvarovite (Ca3Cr2Si3O12)12.0712.0612.0689.990.190.012.0012.0012.0090.090.090.03.79 (3.85)Willemite (Zn2SiO4)8.948.938.95108.8108.7108.78.628.628.63107.9107.9107.93.93 (4.26)Zircon (ZrSiO4)6.096.088.5390.690.190.36.616.615.9890.090.090.03.85 (4.66)PhyllosilicateApophyllite ((Na2O)(CaO)8(SiO2)16(H2O)15)9.559.8114.9192.185.989.08.978.9715.7790.090.090.02.08 (2.28)Chlorite ((MgO)10(Al2O3)3(SiO2)4(H2O)9)5.459.3914.1689.397.289.25.249.0714.2990.097.090.02.57 (2.64)Kanemite (Na2O)(SiO2)4(H2O)75.1420.707.6090.490.091.14.9520.507.2890.090.090.01.76 (1.93)Kaolinite (Al2Si2O5(OH)4)6.599.655.3487.577.888.67.408.945.1689.975.188.32.59 (2.60)Lithium disilicate (Li2Si2O5)5.3714.786.4690.090.090.04.7814.655.6890.090.090.01.95 (2.50)Prehnite (Ca2Al2Si3O10(OH)2)4.365.8818.4390.488.889.84.655.4818.4990.090.090.02.90 (2.91)Pyrophyllite (AlSi2O5(OH))9.435.6320.7099.690.190.08.905.1618.64100.890.090.02.21 (2.85)Talc (Mg3Si4O10(OH)2)5.579.619.7991.181.290.25.299.179.4690.481.389.92.43 (2.78)SorosilicateBertrandite ((BeO)4(SiO2)2(H2O)16.264.928.7590.190.090.015.274.578.7190.090.090.02.26 (2.60)Danburite (CaB2Si2O8)8.708.198.2490.090.090.08.048.757.7390.090.090.02.78 (3.00)Hardystonite (Ca2ZnSi2O7)8.048.044.8286.486.989.87.837.835.0190.090.090.03.36 (3.39)Hemimorphite (Zn4Si2O7)(OH)2\u00b7(H2O))5.4010.618.3290.189.990.05.1210.738.3790.090.090.03.36 (3.48)Lawsonite (CaAl2Si2O7(OH)2\u00b7(H2O))7.566.3313.3490.190.090.08.805.8513.1490.090.090.03.27 (3.09)Rosenhahnite (H2Ca3Si3O10)9.376.706.8288.479.2105.09.486.816.9682.984.1108.63.01 (2.90)Tanzanite (Ca2Al3(SiO4)(Si2O7)O(OH))15.615.529.9590.090.090.116.225.5410.0390.090.090.03.52 (3.35)Thortveitite (Sc2Si2O7)7.444.468.9889.989.773.46.654.698.6290.090.077.83.00 (3.27)TectosilicateAlbite (NaAlSi3O8)8.7612.697.0489.665.090.28.1512.877.1193.163.589.72.46 (2.62)Mordenite (NaAlSi11O24)18.6721.007.7590.290.090.018.1320.497.5290.090.090.01.58 (1.71)Nepheline (KNa3Al4Si4O16)10.1510.298.2690.090.061.110.0510.058.3890.090.060.02.57 (2.64)Paracelsian (SrGa2Si2O8)7.059.379.3890.190.290.18.409.489.0090.089.390.04.40 (3.81)Sodalite (Na4Al3Si3O12Cl)8.918.908.9090.190.190.08.888.888.8890.090.090.02.28 (2.30)Thomsonite (NaCa2Al5Si5O206H2O)13.6913.5712.7480.775.884.213.2513.0613.1090.090.090.02.37 (2.36)dX-ray densities in parenthesisTable\u00a07Comparison of calculated and experimental heats of formation of inorganic compounds (kcal\/mol)\u00a0PM6ReferenceaDifference\u03b1 Quartz (SiO2)\u2212189.4\u2212217.728.3\u03b1 Crystobalite (SiO2)\u2212193.2\u03b2 Tridymite (SiO2)\u2212193.2\u03b2 Quartz (SiO2)\u2212189.4Chabazite (SiO2)\u2212190.7Coesite (SiO2)\u2212187.2Mordenite (SiO2)\u2212191.2Stishovite (SiO2)\u2212160.7Aluminum nitride (AlN)\u221242.7\u221276.033.3Aluminum phosphide (AlP)\u221226.5\u221239.813.3Ammonium chloride (NH4Cl)\u221270.0\u221275.15.1Ammonium nitrate (NH4NO3)\u221288.0\u221287.4\u22120.6Ammonium sulfate ((NH4) 2SO4)\u2212251.5\u2212282.230.7Anatase (TiO2)\u2212229.3\u2212224.4\u22124.9Andalusite (Al2SiO5)\u2212665.2\u2212619.5\u221245.7Anglesite (PbSO4)\u2212221.9\u2212219.9\u22122.0Anhydrite (CaSO4)\u2212275.3\u2212342.967.6Aragonite (CaCO3)\u2212269.0\u2212288.619.6Arcanite (K2SO4)\u2212377.5\u2212343.6\u221233.9Arkelite (ZrO2)\u2212286.4\u2212263.0\u221223.4Barium oxide (BaO)\u2212182.2\u2212131.0\u221251.2Barite (BaSO4)\u2212383.8\u2212352.1\u221231.7Boron (B)\u22128.20.0\u22128.2Boron Nitride (BN)\u221275.5\u221260.8\u221214.7Brookite (TiO2)\u2212229.8Brucite (Mg(OH)2)\u2212176.6\u2212221.044.4Calcite (CaCO3)\u2212271.7\u2212288.616.9Calcium Iodide (CaI2)\u2212108.8\u2212127.518.7Cassiterite (SnO2)\u221259.8\u2212138.178.3Celestine (SrSO4)\u2212297.4\u2212291.6\u22125.8Chloromagnesite (MgCl2)\u2212153.9\u2212153.3\u22120.6Chrysoberyl ((BeO)(Al2O3))\u2212490.6\u2212549.959.3Cinnabar (HgS)\u221253.3\u221213.9\u221239.4Coloradoite (HgTe)\u221220.4\u221210.0\u221210.4Corundum (Al2O3)\u2212370.9\u2212400.529.6Cryolite (Na3AlF6)\u2212871.0\u2212792.8\u221278.2Enstatite (MgSiO3)\u2212324.2\u2212370.246.0Fluorite (CaF2)\u2212207.1\u2212293.085.9Forsterite (Mg2SiO4)\u2212452.9\u2212520.367.4Frankdicksonite (BaF2)\u2212305.3\u2212288.5\u221216.8Galena (PbS)\u221224.8\u221224.0\u22120.8Gallium arsenide (GaAs)\u221235.5\u221217.0\u221218.5Graphite (C)1.30.01.3Greenockite (CdS)\u221285.1\u221238.7\u221246.4Hawleyite (CdS)\u221285.0\u221238.7\u221246.3Hydrophilite(CaCl2)\u2212151.4\u2212190.138.7Indium arsenide (InAs)\u221217.6\u221214.0\u22123.6Lead selenide (PbSe)\u2212124.7\u221224.6\u2212100.1Lead telluride (PbTe)6.2\u221216.923.1Lime (CaO)\u2212116.2\u2212151.835.7Magnesite (MgCO3)\u2212224.8\u2212265.740.9Magnesium dibromide (MgBr2)\u2212116.9\u2212125.38.4Magnesium diiodide (MgI2)\u221267.2\u221287.019.8Sodium sulfate (Na2SO4)\u2212372.5\u2212331.5\u221241.0Orthoboric acid (B(OH)3)\u2212238.3\u2212261.723.4Lead oxide (PbO)\u221285.9\u221252.3\u221233.6Lead dioxide (PbO2) (Rutile structure)\u221221.6\u221266.344.7Periclase (MgO)\u221296.9\u2212143.746.8Rutile (TiO2)\u2212230.1\u2212225.6\u22124.5Sellaite (MgF2)\u2212234.9\u2212268.733.8Silicon (Si)\u221213.50.0\u221213.5Silicon carbide (SiC)\u221237.6\u221215.6\u221222.0Smithsonite (ZnCO3)\u2212175.7\u2212194.318.6Soda Niter (NaNO3)\u2212159.6\u2212111.8\u221247.8Sphalerite (ZnS)\u221240.4\u221249.28.8Spinel (MgAl2O4)\u2212493.7\u2212549.555.8Sulfur (S)1.50.01.5Willemite (Zn2SiO4)\u2212375.9\u2212391.215.3Wurtzite (ZnS)\u221239.4\u221246.06.6Zincite (ZnO)\u221284.6\u221283.8\u22120.8Zinkosite (ZnSO4)\u2212211.0\u2212234.924.0Zircon (ZrSiO4)\u2212461.0\u2212529.968.9aReference values taken from the CRC Handbook [32]\nElements\nThree allotropes of carbon were modeled: diamond, consisting of pure covalent sp3 bonds, graphite, with sp2 bonds in-plane and \u03c0-stacking or VDW forces between planes, and \u201cglitter\u201d, a hypothetical structure composed of 1,4 cyclohexadiene fragments [30]. The simple C\u2013C bond in diamond is reproduced with good accuracy, 1.57\u00a0\u00c5 versus 1.54\u00a0\u00c5 observed, as is the in-plane bond in graphite, 1.43\u00a0\u00c5 versus 1.42\u00a0\u00c5. This last result was unexpected in light of the zero band gap of graphite, in that severe difficulties in achieving a SCF are often encountered when systems with very small band-gaps are modeled; graphite appears to be a unique exception to this. Graphite layers are held together by weak VDW forces, which are poorly represented in all NDDO methods. One consequence of this is that the predicted interlayer distance, 3.56\u00a0\u00c5, is significantly larger than the observed 3.35\u00a0\u00c5. Intermediate between graphite and diamond is the hypothetical glitter, a tetragonal allotrope of carbon composed of single and double bonds. A PM6 calculation predicted that the structure would be essentially the same as that given by DFT methods [31].\nThe standard state of carbon is graphite, and therefore by definition the value of its \u0394Hf is 0.0\u00a0kcal\/mole-atom. PM6 predicts the \u0394Hf of graphite to be 1.29\u00a0kcal\/mole-atom, in good agreement with the reference value. The higher energy allotrope, diamond, has a reported heat of formation of 0.45\u00a0kcal\/mole-atom [32]; for diamond, PM6 predicts the \u0394Hf to be 1.34\u00a0kcal\/mole-atom, i.e., 0.05\u00a0kcal\/mole-atom above graphite. Glitter is a hypothetical allotrope, but is predicted by PM6 to be unlikely to be formed under equilibrium conditions: both PM6 and DFT predict the \u0394Hf to be large and positive +9.1\u00a0kcal\/mole-atom and +11.8\u00a0kcal\/mole-atom [33], respectively.\nSilicon also crystallizes in the diamond lattice, but the calculated silicon\u2013silicon distance, 2.22\u00a0\u00c5, is significantly less than the observed value of 2.35\u00a0\u00c5, and while the experimental \u0394Hf is, by definition, zero, the calculated heat of formation is \u221216.6\u00a0kcal\/mole-atom.\nSulfur forms eight-membered rings, with 16 rings per unit cell. Because the unit cell is so large, and because there is a distinct insulating gap between each ring, the approximation that \u0393 represents the entire Brillouin zone is valid even when only a single unit cell is used. Within each ring, the sulfur\u2013sulfur distance is 2.04\u00a0\u00c5, in perfect agreement with the 2.04\u00a0\u00c5 observed, but the inter-ring distance is badly predicted, resulting in a calculated density of 1.54\u00a0g\/cc, considerably less than the observed 2.06\u00a0g\/cc. This lack of inter-ring interaction is the likely cause of the calculated heat of formation being 1.45\u00a0kcal\/mole-atom, rather than being nearer to the reference 0.0\u00a0kcal\/mole-atom.\nHalides\nOne of the simplest sets of inorganic solids are the alkali metal halides. Most of these crystallize with the rock-salt lattice, the exceptions being cesium chloride, bromide, and iodide, which crystallize in the body-centered cubic or cesium chloride structure. Because of their high symmetry there is only one geometric variable, so for this group of solids, symmetry was used to accelerate the geometry optimization. Geometries were optimized for both the rock-salt and cesium chloride structures for each salt. With the exception of rubidium fluoride, the calculated heats of formation (Table\u00a08) of the isolated molecule and of the crystal are reproduced with good accuracy, although PM6 did a very poor job of predicting which of the two crystal forms was the more stable.\nTable\u00a08Comparison of calculated and experimental heats of formation of alkali metal halides (kcal\/mol)SaltMoleculeCrystalPM6Reference [1]PM6 (NaCl)PM6 (CsCl)Experimental [32]Lithium fluoride\u221281.5\u221286.0\u2212138.2\u2212139.7\u2212147.4Lithium chloride\u221246.8\u221253.9\u2212107.4\u2212108.2\u221297.6Lithium bromide\u221236.8\u221238.1\u221289.6\u221287.3\u221283.9Lithium iodide\u221219.4\u221216.1\u221272.0\u221273.2\u221264.6Sodium fluoride\u221269.6\u221264.0\u2212123.2\u2212125.7\u2212137.5Sodium chloride\u221243.4\u221249.4\u221287.7\u221286.8\u221298.3Sodium bromide\u221234.2\u221237.6\u221299.4\u2212101.7\u221286.4Sodium iodide\u221219.0\u221219.8\u221275.2\u221275.4\u221268.8Potassium fluoride\u221278.1\u221274.9\u2212185.7\u2212183.5\u2212135.9Potassium chloride\u221251.2\u221253.4\u2212112.0\u2212118.3\u2212104.4Potassium bromide\u221243.0\u221244.3\u221299.8\u2212113.2\u221294.1Potassium iodide\u221230.0\u221230.0\u221282.5\u221293.9\u221278.4Rubidium fluoride\u221279.2\u2212101.3\u2212152.8\u2212111.3\u2212133.3Rubidium chloride\u221254.7\u221261.3\u2212117.9\u2212108.2\u2212104.1Rubidium bromide\u221243.7\u221250.8\u2212107.7\u2212103.5\u221294.3Rubidium iodide\u221232.1\u221224.0\u221266.4\u221269.6\u221279.8Cesium fluoride\u221285.2\u221281.9\u2212155.9\u2212115.3\u2212132.6Cesium chloride\u221257.4\u221262.9\u2212128.4\u2212130.2\u2212105.8Cesium bromide\u221250.0\u221255.0\u2212106.0\u2212117.6\u221297.0Cesium iodide\u221236.3\u221243.5\u221286.0\u221298.4\u221282.8\nElements of Group II form two main groups of halides: those with 8 coordinate metal atoms, such as fluorite, CaF2, and frankdicksonite, BaF2, and the six-coordinate layer structures of the chlormagnesite, MgCl2, type. Other structures include the rutile lattice, as in sellaite, MgF2, and the distorted rutile lattice, hydrophilite, CaCl2. For all alkaline earth halides investigated, the calculated structure had the same lattice type as that in the starting geometry.\nA more complex halide is carnallite, KMgCl3\u00b76(H2O), composed of isolated potassium and chloride ions, and magnesium ions that are octahedrally coordinated by water molecules. For the X-ray structure, PM6 predicted the charge on the coordination complex ion [Mg(H2O)6]2+ to be +1.89, on the potassium ion, +0.74, and on the chloride, \u22120.87. Within the complex ion, the charge on magnesium was +0.79, and, on average, each water molecule had a charge of +0.183. The complex ion thus behaves like an extremely ionic Group II element; the net charge being much greater than that on any Group II metal ion in any halide.\nTrisodium hexachlorotitanate crystallizes in a cryolite-like structure [34]. Each titanium ion has one unpaired electron and although the Ti\u2013Cl bond is highly covalent, the smallest distance between the [TiCl6]3- complex ions is more than 3.6\u00a0\u00c5; therefore, from an electronic perspective, the unpaired electrons could be regarded as isolated. To verify the validity of this assumption, the geometry of solid Na3TiCl6 was optimized using the UHF Hamiltonian. A cluster of 16 formula units was used, this representing 8 unit cells. In one calculation, the magnetic component of spin, Ms, was set to zero, and in the other it was set to the maximum value for 16 unpaired electrons, i.e., Ms\u2009=\u20098. Both optimized geometries were essentially the same, geometric differences were negligible, and the two heats of formation were \u2212459.2\u00a0kcal\/mol and \u2212462.0\u00a0kcal\/mol, respectively. Therefore. the assumption that the unpaired electrons are electronically isolated, at least when the focus of interest is energetics and structure, is justified.\nOxides\nSiO2 PM6 predicts the Si\u2013O distance in silicon dioxide to be too large by 3\u20135%, with the result that the predicted densities of the polymorphs of silica were all underestimated. This is best seen in the very dense stishovite, where the predicted density, 3.70, is about 14% less than the observed 4.28. Stishovite has a rutile structure, so the positions of the atoms in the unit cell can be defined using two bond lengths and one angle. PM6 predicts the Si\u2013O\u2013Si angle correctly, 97.8\u00b0 compared to the observed 98.8\u00b0, but overestimates the Si\u2013O bond-lengths, 1.88\u00a0\u00c5 and 1.87\u00a0\u00c5 compared to the 1.76\u00a0\u00c5 and 1.81\u00a0\u00c5 observed. In \u03b1-quartz the predicted Si\u2013O distance, 1.65\u00a0\u00c5, is also larger than that observed, 1.61\u00a0\u00c5, but, in addition, the Si\u2013O\u2013Si angle opens from the observed 143.7\u00b0 to 157.2\u00b0. This additional decrease in density results in the predicted density of \u03b1-quartz being too low by 15.5%. The error in the Si\u2013O\u2013Si angle reaches its maximum in \u03b1-crystobalite where PM6 predicts it to be 180.0\u00b0 instead of the observed 146.8\u00b0. This results in a large error in density, of \u221220%. Linear Si\u2013O\u2013Si systems are not unknown in nature: in the high temperature form of tridymite, hexagonal \u03b2-tridymite [35] and in thortveitite, (Sc,Y)2Si2O7 [36], these atoms form a straight line, and, as expected, this angle is also precisely reproduced by PM6.In addition to the dense phases of silica there are open lattice structures which, of their nature, necessarily have very low densities. Two examples are mordenite and chabazite. Calculated and X-ray structures in these systems are shown in Figs.\u00a08 and \u00a09. Chabazite has the lowest density of any silica polymorph, 1.46. PM6 reproduces these systems but, as with the higher density polymorphs, it underestimates the density, but in this case by about 5%.\nFig.\u00a08Part of the unit cell of Mordenite, SiO2. Left X-ray structure, right optimized PM6 mirror image structureFig.\u00a09Chabazite, SiO2Left X-ray structure,  right optimized PM6 structure\nH2O Water forms several polymorphs. In Ice-I or Ice-Ih, the common polymorph of ice, oxygen atoms form a hexagonal lattice and each atom has two hydrogen atoms strongly covalently bound to it and two hydrogen atoms at a larger distance. The locations of the hydrogen atoms can be specified by application of the \u201cice rules\u201d. However, even when that is done correctly, the positions of the hydrogen atoms are not unambiguously defined. That is, although the X-ray structure shows that the oxygen atoms are located precisely on the vertices of the hexagonal lattice, there is statistical disorder in the positions of the hydrogen atoms\u2014between each pair of adjacent oxygen atoms there are two potential positions that a hydrogen atom can occupy, and in the observed structure each position has a 50% occupancy. This type of disorder must be resolved before a solid state calculation can be carried out, so in Ice-Ih, an arbitrary distribution of hydrogen atoms consistent with the ice rules was used.Geometry optimization of the Ice-Ih structure gave a recognizable hexagonal lattice, as expected, but as a result of the asymmetric distribution of hydrogen atoms each oxygen atom was slightly displaced relative to the ideal lattice.All the ices, except Ice-IV and Ice-VII, were modeled successfully. The simple unit cell of Ice-IV contains 16 water molecules but, in order to satisfy the ice rules and have the hydrogen atoms disordered, a large cluster would be needed. Assigning hydrogen atoms in such a cluster in order to achieve both randomness and compliance with the ice rules would be a non-trivial task. However, Ice-VII is the high-temperature (above 5\u00b0C) modification of Ice-VIII and differs from Ice-VIII only in that the positions of the hydrogen atoms are disordered. As such, Ice-VII is trivially different from Ice-VIII.With the exception of Ice-X, each polymorph optimized to the correct structure. That is, the structure of each calculated polymorph was qualitatively similar to that of the observed polymorph. Ice-X is unique in that the oxygen atoms form a body-centered cubic lattice with hydrogen atoms equidistant between half of the adjacent pairs of oxygen atoms: all oxygen atoms are symmetrically tetrahedrally coordinated by hydrogen atoms. Unconstrained optimization of this structure resulted in a disordered, essentially amorphous, solid. When symmetry constraints were imposed, the geometry of Ice-X optimized to give an O-H distance of 1.214\u00a0\u00c5. Ice-X is stable at pressures above 62\u00a0GPa and, at that pressure, PM6 predicts the O\u2013H distance to be 1.179\u00a0\u00c5, in good agreement with the X-ray value [37] of 1.181\u00a0\u00c5. At a much higher pressure, about 480\u00a0GPa, PM6 predicts that the antifluorite structure would become the preferred polymorph; in this structure each hydrogen bonds to four oxygen atoms and each oxygen bonds to eight hydrogen atoms.\nAl2O3 An attempt [12] by Gale to use the MNDO, AM1, and PM3 methods to predict the structure of corundum met with only limited success. The failure to reproduce the observed structure was attributed to deficiencies, mainly the lack of d-orbitals, in the basic semiempirical methods. To a large degree these deficiencies have been corrected in PM6, as can be seen by comparing the structures predicted by the various methods with the known X-ray structure (Table\u00a09).\nTable\u00a09Calculated and observed structure and \u0394Hf of \u03b1-corundumExperimentalMNDOaAM1aPM3aPM6a (\u00c5)4.764.854.695.284.83c (\u00c5)12.9913.1212.3315.4712.91\u0394Hf (kcal\/mol)\u2212400.5\u2212266.5\u2212241.9\u2212236.4\u2212370.9aResults for MNDO, AM1, and PM3 taken from Gale [12]\nTiO2 Within each of the polymorphs of titanium dioxide, all the titanium atoms are in the same approximately octahedral environment, surrounded by six oxygen atoms. This is illustrated in Fig.\u00a010 for rutile, the commonest polymorph. The structures of all three polymorphs were qualitatively reproduced by PM6, but the Ti\u2013O bond-length was over-estimated by 5%, as shown in Table\u00a010 for rutile, resulting in the predicted density being too low by about 16%.\nFig.\u00a010Unit cell of rutile (TiO2). Crossed-eyes stereo viewTable\u00a010Interatomic distances in rutileDistancePM6X-rayTi-O2.0221.981Ti-O\u20192.0801.948Ti-Ti3.1162.959\nB(OH)3 Orthoboric acid, B(OH)3, forms a layer structure in which individual molecules within each layer are held together by hydrogen bonds, and the layers are held together by VDW forces and very weak electrostatic forces. Although the layer structure is reproduced with good accuracy, the predicted inter-layer distance, 11.0\u00a0\u00c5, is much greater than the observed 6.35\u00a0\u00c5. The lack of any significant inter-layer interaction can be attributed to the absence of a VDW core\u2013core term in boron. It is likely that if such a term were added, the correct interlayer interaction would be reproduced.During the survey of organic solids, the structure of the very complicated organometallic pentakis(tetraethylammonium) bis(meso-tetraphenyl-porphyrinato-zinc) tetraconta-oxo-silicon-dodeca-molybdenum bromide (CSD entry PIJFUJ) was accurately reproduced. In contrast, problems had been encountered with various other complicated solids, particularly those involving protonated species. To determine the ability of PM6 to model complex ionic systems, a solid containing an archetypal proton ion, Zundel\u2019s cation [38], [H5O2]+, was used. This ion had been identified [39] in Keggin\u2019s [40] pentahydrate, dodecatungstophosphoric acid hexahydrate. X-ray analysis indicated that the [H5O2]+ ion has D2h symmetry and that the O\u2013O separation is 2.38\u00a0\u00c5. However, a PM6 calculation on the isolated ion predicted its structure to be C2h with an O\u2013O separation of 2.51\u00a0\u00c5. This was not unexpected: ab-initio calculations [41, 42] indicate that the ion should have C2 symmetry with the Cs structure being only about 0.4\u00a0kcal\/mol higher in energy, and that the D2h structure should be significantly higher in energy. When D2h symmetry was used, PM6 predicted that the separation would decrease to 2.45\u00a0\u00c5, and the energy would increase by 7.8\u00a0kcal\/mol. Disorder had been reported in Keggin\u2019s pentahydrate, with Zundel\u2019s ion assuming two different orientations, each having a 50% occupancy. After resolving the disorder, an unconstrained geometry optimization was performed. Examination of the optimized structure revealed that the Zundel ion had been destroyed and that the proton had migrated to the polyoxometalate. This phenomenon, the neutralization of a polyanion, had also been observed in tetrakis(2-carboxypyridinium) octacyano-molybdenum(iv), 4[C6H6NO2]+ [MoIV(CN)8]4-, CSD entry PYCMOA, where PM6 predicted that protons on the pyridinium ions would migrate to the metal complex.\nOther AB-type solids\nA number of solids of the type AB are formed from elements of Group III and V, while others involve elements of Group IV, and still more involve elements of Group VI and heavy elements. Some of these occur naturally, such as wurtzite and sphalerite (zinc sulfide), and coloradoite (mercury telluride), while others are formed synthetically, often by chemical vapor deposition methods. In most of these materials, each atom of type A is tetrahedrally coordinated to four atoms of type B. This results in two types of packing, best exemplified by the two polymorphs of zinc sulfide. For convenience all compounds of this type will be grouped together.\nMany of these compounds are semiconductors, and have small band-gaps between the occupied and virtual orbitals. A consequence of this is that the cluster used must be very large in order to minimize errors arising from the cluster approximation. In addition, it was anticipated that the narrow band-gap would give rise to difficulties in solving the self-consistent field equations\u2014such difficulties had frequently occurred when molecules that had small HOMO\u2013LUMO gaps were being studied. Surprisingly, the SCF equations were solved using default options, albeit more iterations than normal were needed.\nSymmetry was used to accelerate the geometry optimization of those solids that had the sphalerite structure; such solids have only one adjustable parameter. In most cases, the calculated density for the optimized structure was close to that expected, the exceptions being cadmium telluride, which PM6 predicts to be too dense, and coloradoite, where the density was predicted to be too low.\nCarbonates, nitrates, and borates\nOf the simple carbonate minerals that crystallize in the calcite lattice (Fig.\u00a011), PM6 correctly reproduces the structures of magnesite, MgCO3, smithsonite, ZnCO3, otavite, CdCO3, and calcite, CaCO3 itself (Table\u00a011). PM6 was unable to reproduce the structures of two other carbonates: rhodochrosite, MnCO3, and siderite, FeCO3. The UHF method was used in modeling both of these minerals because they contain transition metal ions that have unpaired electrons. In the case of rhodochrosite, each metal ion has five d-electrons in an approximately octahedral environment. Because the crystal field of the carbonate ions is too weak to cause a large separation of the t2g and eg levels, MnII would be in the high-spin configuration. This implies that the local electronic structure would be approximately 6A1g, and that there would be no angular terms arising from the d-electrons. That is, a MnII ion would be expected to behave like a Group II ion. The failure of PM6 to reproduce the calcite structure for rhodochrosite indicates a fault in the parameterization of manganese, most likely attributable to errors in the core-core terms.\nFig.\u00a011Unit cell of calcite. Crossed-eyes stereo viewTable\u00a011Interatomic distances in calciteDistancePM6X-rayCa-O2.2972.389C-O1.2891.210Ca-Ca3.9894.036\nThe other common polymorph of calcite is aragonite. Other minerals that have the aragonite structure are cerussite, PbCO3, strontianite, SrCO3, and witherite, BaCO3. Of these, PM6 predicts correctly the structures of aragonite, strontianite, and witherite, albeit some carbonate groups were rotated slightly about their C3 axis. In cerussite, the carbonate groups were tilted out of plane, giving rise to a herringbone-like structure.\nThe densities of all Group I carbonates are predicted by PM6 to be too low. This includes the simple carbonates natrite, Na2CO3, and zabuyelite, Li2CO3, potassium hydrogen carbonate, kalicinite, and sodium carbonate monohydrate, thermonatrite.\nNitrates\nPM6 correctly reproduces the structures of ammonium nitrate and potassium nitrate (niter), but the predicted structure of sodium nitrate, nitratine, is completely incorrect; as with other sodium compounds, the predicted Na\u2013Na distances are unrealistically small.\nBorates\nSeveral borates were examined, including the simple sodium salt, borax. Like other solids that contain a large amount of sodium, its optimized PM6 structure was severely in error. With that single exception, the structures of the borates were reproduced with good accuracy. Almost all minerals have some symmetry in their unit cell, the most well known exception being the naturally-occurring calcium chloroborate, parahilgardite, Ca2(B5O9)Cl(H2O). PM6 was able to reproduce the unit cell of this almost unique mineral with good accuracy, despite the fact that the unit cell was completely devoid of any symmetry (Fig.\u00a012). This system was unique among the solids studied in that, unlike all the organic compounds, there were no readily identifiable discrete molecular fragments, and, unlike the other inorganic solids, it is devoid of any elements of symmetry.\nFig.\u00a012Unit cell of parahilgardite. Crossed-eyes stereo view\nMolybdates, tungstates chromates vanadates, sulfates, and phosphates\nThis group of solids is characterized by the presence of tetrahedral oxyanions, e.g., [SO4]2-, [PO4]3-, [MoO4]2-, [WO4]2-, and [CrO4]2-, with counterions ranging from the highly ionic Group I and Group I-like ions, e.g., [NH4]+, to softer cations such as Pb2+ and Zn2+. With the exception of descloizite, PbZn(VO4)(OH), the internal structures of all systems modeled were quantitatively reproduced. In descloizite, the distance between lead and zinc ions was predicted to be unrealistically small, which, in turn, resulted in severe distortions to the positions of the other entities in the unit cell. There are diatomic core\u2013core repulsion parameters in PM6 for Pb and Zn, but clearly the magnitude of the term was too small. As with other errors of this type, this fault could readily be corrected by re-parameterizing the core\u2013core terms only.\nCharges on the ions are significantly reduced from the formal values. Thus, in fluorapatite (Fig.\u00a013, Table\u00a012), calcium ions have charges of +1.35 and 1.29, depending on environment, fluoride is \u22120.77, and each phosphate group has a net charge of \u22121.95, all indicating a high degree of covalence in the bonds.\nFig.\u00a013Unit cell of fluorapatite. Crossed-eyes stereo viewTable\u00a012Interatomic distances in fluorapatite. Ca1 Ca on a vertex, Ca2 Ca on an edge, Ca3 Ca attached to FDistancePM6X-rayCa1-Ca15.4155.408Ca1-Ca23.4503.456Ca1-O2.3052.338Ca3-O2.3862.375Ca3-F2.3302.361P-O1.5801.585\nStrong hydrogen bonding can occur in some sulfates and phosphates, which can be illustrated by salts in which the acid is partially neutralized, such as compounds of the type MHSO4, MH2PO4, and M2HPO4, where M is a Group I element. A common structure in systems of this type consists of a hydrogen atom positioned between two oxygen atoms on two different acid groups. The positions of the protons as determined by X-ray analysis are of limited reliability, so for the purposes of comparison the oxygen\u2013oxygen distances were used. In mercallite, potassium hydrogen sulfate (Fig.\u00a014), PM6 predicts the oxygen\u2013oxygen distance (Table\u00a013) in perfect agreement with the X-ray structure. PM6 also predicts the hydrogen atoms to be asymmetrically positioned between the two oxygen atoms, this also being in accord with the published X-ray structure. Potassium dihydrogen phosphate is more complicated in that each phosphate forms four hydrogen bonds to four different phosphate groups (Fig.\u00a015). Again, PM6 predicts (Table\u00a014) the oxygen\u2013oxygen distance with good accuracy, and predicts the hydrogen atoms to be asymmetrically positioned; however, in both the hydrogen sulfate and hydrogen phosphate PM6 underestimates the degree of asymmetry.\nFig.\u00a014Structure of hydrogen sulfate in KHSO4. Top X-ray structure, bottom PM6 structureFig.\u00a015Detail of structure of dihydrogen phosphate in KH2PO4 (upper pair) and in (CH3)4NH2PO4. Left X-ray structures on left, right PM6 structureTable\u00a013Interatomic distances in mercallite (KHSO4)DistanceX-rayPM6O(H) \u22efO2.632.63O\u2013H0.731.04S\u2013S4.404.60Table\u00a014Interatomic distances in hydrogen phosphate in KH2PO4DistanceX-rayPM6O(H)\u22efO2.492.53O\u2013H1.071.20O\u2013H1.431.33P\u2013P4.114.26\nThe potassium ion is relatively small in comparison to a phosphate ion, so in KH2PO4 the phosphate ions can form a three-dimensional lattice. If a larger cation, e.g., tetramethylammonium, is used, the three-dimensional phosphate lattice becomes energetically unfavorable relative to a one-dimensional chain of phosphates. This structure was also reproduced accurately.\nPM6 reproduces the structures of most of the phosphates, but, as expected, in the case of sodium phosphate the predicted structure was qualitatively incorrect.\nSilicates\nSilicates form the largest and most complicated group of minerals and exhibit a very wide range of properties. From a chemical perspective, however, they are relatively simple: most silicates involve SiO4 tetrahedra in various degrees of polymerization, and in turn these tetrahedra interact with fully oxidized metal ions. In addition, for any given empirical formula, naturally occurring minerals represent the most stable, or almost the most stable, structure. Some structures might be quite complicated; nevertheless, because they formed under equilibrium conditions, they are near or at the energy minimum, and, for any given empirical formula, an accurate computational model should not be able to generate a polymorph that is significantly more stable than any that occurs naturally. This is in contrast to organic chemistry, where high energy forms of the same empirical formula occur frequently: for example, dimethyl ether is the high energy isomer of C2H6O, with ethanol being the low energy form. For the purposes of this study, the silicates can be divided into families based on the degree of aggregation of the silicate units.\nIsolated SiO4 tetrahedra: nesosilicates The simplest of the silicates are the nesosilicates, which contain isolated SiO4 groups. With the exception of the three polymorphs of Al2SiO5, kyanite, andalusite, and sillimanite, the structures of the nesosilicates are reproduced with good accuracy. In the aluminum silicates there are two distinct anions, an [SiO4]4- ion and an isolated oxygen ion, [O]2-. PM6 incorrectly predicts these ions to coalesce to form a SiO5 moiety.\nDouble and triple tetrahedra: sorosilicates All the sorosilicates examined were reproduced with good accuracy. In most of the minerals that contained the [Si2O7]6- moiety the Si-O-Si system is bent. As expected, PM6 reproduced the bent structure. The only exception was thortveitite, an important source of scandium, which exists in nature as (Sc,Y)2Si2O7, but which, for the purposes of this work, was simplified to Sc2Si2O7. In the crystal structure of this mineral [36], the [Si2O7]6- moiety has C2h symmetry; consequently the Si\u2013O\u2013Si angle is precisely 180\u00b0. When the structure of thortveitite was optimized using PM6 the symmetry did not change: the predicted Si\u2013O\u2013Si angle was also 180\u00b0.\nChains: inosilicates With the exception of spodumene, where PM6 predicted the aluminum-silicon distances to be too short, the structures of all the chain silicates were reproduced with good accuracy. As with the aluminum silicates, the fault in spodumene can be attributed to errors in the Al\u2013Si core\u2013core parameters. In the case of cummingtonite, Mg7Si8O23\u00b7(H2O), hydrogen atoms were added before the structure was optimized.\nCyclosilicates Three cyclosilicates were examined: benitoite, BaTiSi3O9, beryl, Be3Al2Si6O18, and dravite, NaMgTi2Al6(BO3)3(Si6O18)O4. All were reproduced with good accuracy, as illustrated by the bond lengths and angles (Table\u00a015) in the simplest of these, beryl (Fig.\u00a016). Dravite has a particularly complicated structure, involving seven different elements, in which the positions of the cations Na+, Mg2+, Al3+, and Ti4+ and the anions [O]2-, [BO3]3- and [Si6O18]12- are determined by roughly equal contributions of covalent and ionic terms.\nFig.\u00a016Unit cell of beryl. Crossed-eyes stereo viewTable\u00a015Interatomic distances and angles in berylGeometric quantityX-rayPM6Al-Be2.7542.656Al-O1.9281.906Si-O(Al)1.6551.610Si-O(Si)1.6111.607Si-O-Si168.1165.3Al-O-Si128.6137.1\nSheets: phyllosilicates The structures of the phyllosilicates are reproduced with modest accuracy. In talc (Fig.\u00a017) for example, in-plane distances are reproduced well, but the inter-plane separation (Table\u00a016) is only poorly modeled. This fault can be attributed to the absence of strong inter-plane interactions: any errors in the weak long-range interatomic terms would give rise to disproportionately large errors in inter-plane separations.\nFig.\u00a017Unit cell of talc. Crossed-eyes stereo viewTable\u00a016Interatomic distances in talcDistancePM6X-rayMg-O(H)2.0602.062Mg-O(Si)2.0842.080Si-O(Mg)1.6291.624Si-O(Si)1.6741.622O\u2013Oa3.7623.095aDistance between layersThe mineral chlorite has a variable stoichiometry, (Fe, Mg, Al)6(Si, Al)4O10(OH)8, which does not allow a simple resolution into a fixed stoichiometry. In it, the presence of main-group metals of different oxidation states (MgII and AlIII) in the cationic part of the formula requires that, in order to maintain electrical neutrality, for any given ratio of magnesium to aluminum, there should be a corresponding composition in the anionic part. In nature, this condition would be satisfied by the random distribution of magnesium, aluminum, and silicon atoms in the lattice. Computationally, however, such a distribution is currently not possible, and fixed stoichiometries are required. In the extremes, these would be Mg6Si4O10(OH)8 and Mg2-Al8O10(OH)8. However, the lattice structures of these two extremes would likely be so different to that of chlorite that comparison between any computed result for one of these extremes and that of chlorite would be meaningless. In order to obtain a meaningful result, an unusually large cluster was used, in which the ratios of the elements was as near to that of chlorite as possible.\nFrameworks: tectosilicates With the exception of thomsonite, NaCa2Al5Si5O20\u00b76H2O, PM6 predicted the internal structures of the tectosilicates accurately. As with the other aluminosilicates, in thomsonite the predicted aluminum-silicon distance was unrealistically short.\nDiscussion\nWith a few exceptions, the geometries of individual organic molecules and ions and their packing arrangement in the crystal lattice were reproduced with good accuracy. In the original PM6 article it was shown that the average error in predicted bond-lengths in organic compounds was about 2\u20133%. The effect of intermolecular forces on the geometries of the component molecules is likely to be small, so by implication the accuracy of prediction of the geometries of those molecules would be similar to that reported in the earlier article. By far the largest structural error in organic solids involves intermolecular separations. The values of these are determined by several forces, from the weak VDW and \u03c0-stacking attractions, in cyclohexane and coronene, through simple hydrogen bonding of the type found in sucrose, to very strong intermolecular interactions, usually ionic, best typified by the Zwitterions, such as the simple amino acids. Errors in intermolecular separations could be determined by a direct comparison of calculated and reference structures. However, because errors in intermolecular separation have a direct impact on the unit cell dimensions, and consequently on the density, a convenient and reliable estimate of the accuracy of prediction of intermolecular separations of molecules in the unit cell can be obtained from a comparison of predicted and X-ray densities.\nThe internal structures of three organic solids were incorrectly predicted using PM6; for all other solids the internal packing was qualitatively correct. Two of the faulty solids involved the oxalate and water moieties. From analysis of other systems known to contain neutral species, but which PM6 predicts to be ionic, it appears that PM6 unrealistically favors the formation of the hydronium ion. The fault in sodium acetate trihydrate appears to arise from faults in sodium core\u2013core parameters, in that similar faults were found in several other inorganic sodium-containing compounds. Interestingly, the fault was not present in the closely related species sodium hydrogen acetate, a system chosen because of the presence of the hydrogen-bonded structure, [Ac2H]\u2013. When the three solids that were badly predicted were removed from consideration, the average error in density decreased to 6.1%, with most of this attributable to the 2% average error in intermolecular separations. This error was not evenly distributed within the organic solids in that when interaction energies were large, as in the ionic solids, in particular Zwitterions, PM6 reproduced the intermolecular separations with good accuracy, but when the interaction energies were small, as in species where only VDW forces are involved, such as methionine, errors in intermolecular separations were often quite large.\nThe structures of most of the inorganic solids were predicted with useful accuracy, with the highest accuracy being exhibited by minerals in which metal atoms interact with oxygen, and the oxygen then interacts with a main-group element. Most of the silicates, phosphates, and sulfates fall into this group. One reason for this can be attributed to the procedure used in developing PM6, in that a large quantity of reference data for systems that had metal-oxygen or main-group oxygen bonds was used during the parameterization. This naturally resulted in increased emphasis being placed on the structure and thermochemistry of oxygen-containing systems.\nOn the other hand, inorganic systems exhibit a much wider range of types of interaction than those found in organic chemistry, and this makes the general application of PM6 to inorganic solids more difficult. An implication of the fact that PM6 uses the Voityuk diatomic core-core term is that parameters must be present for each pair of elements in a system, unless the pair of elements is separated by a distance sufficiently large that there would be no significant core-core interaction. Only a limited number of types of interactions were surveyed in this work; even within that number, several instances were found in which the PM6 values of the Voityuk parameters were severely in error and gave rise to results that were nonsense. Because of this, great care should be exercised in determining the suitability of PM6 for modeling solids that include diatomic interactions of types not found in any of the species reported here.\nIn general, PM6 reproduced the X-ray densities of both organic and inorganic crystals with useful accuracy, Fig.\u00a018, the R2 value being 0.94 and the least squares fit was \u03c1PM6\u2009=\u20090.936\u03c1X-ray+0.121. The average unsigned error in the predicted densities of inorganic solids, 9.3%, is higher than that of organic solids, 6.9%, although the average signed error was \u22122.5% compared to the equivalent +3.9% for organic solids.\nFig.\u00a018Comparison of calculated and X-ray densities\nSystems that are badly predicted\nOf the systems whose properties were badly predicted, three types could be identified. In the first group, illustrated by lead selenide and 2, 4, 6-tribromoaniline (TBA), the origin of the error could be traced to the values of individual diatomic parameters used in PM6. These were either incorrect, as in lead selenide, where the PM6 value for Voityuk\u2019s lead\u2013selenium core\u2013core repulsion was much too small, and in TBA where the bromine\u2013nitrogen parameters were either physically unrealistic or absent. Errors of this type could be easily corrected by carrying out a small parameterization operation involving only the faulty parameters and using a training set consisting of examples of the two atoms in close proximity.\nThe second type of error was found in some solids for which no equivalent error was found in the isolated molecule. This is best illustrated by the Group I halides, where PM6 predicted the lowest energy structure to be either rock salt or cesium chloride, almost at random. During the development of PM6, only small representatives of I-VII species were used. Apparently, these systems were too small to allow the lattice properties to be accurately characterized. Errors of this type were therefore not immediately obvious, and only became apparent when full solid-state calculations were done. The implication is that future parameterizations should include solids in the training set. The best solids for use in parameterization would be those that were badly predicted by PM6. Addition of solids to the parameterization would be unlikely to cause a significant increase in error in the prediction of molecular properties. Although the use of solids in parameterization is impractical at present, it is likely that increases in computer power will make such calculations possible in the not-too-distant future.\nThe third type of error is specific to organic compounds, where PM6 predicts some ions to be too stable. Thus the aminophenols are predicted to exist in the solid state as the Zwitterions rather than as the neutral species. The hydrate of oxalic acid was predicted to exist as oxalate and hydronium ions, a prediction completely in variance with the X-ray structure. No instances were found where PM6 underestimated the stability of ions relative to their neutral counterparts. That is, the error was completely systematic and unequivocally indicated a fault in the parameterization. The origin of this error most likely lies in the set of atomic electronic parameters, rather than in the diatomic core\u2013core parameters, and as such could only be corrected by a re-parameterization. There was no evidence that any faults were due to an underlying defect in the set of approximations.\nAccuracy of geometry vs hardness\nIn general, the structures of solids that are mechanically extremely hard, seven or more on Moh\u2019s scale, are predicted with good accuracy, whereas the geometries of many softer solids, such as most organic species, the transition metal carbonates, and various layer silicates, particularly the micas, are predicted with significantly less accuracy. This inverse relationship of mechanical hardness and computational accuracy can be rationalized by consideration of the interatomic forces involved. In hard solids, all atoms are connected to adjacent atoms by strong covalent bonds, and, of their nature, those bonds have large force constants. In general, semiempirical methods predict such geometries with good accuracy. Any tendency to deviation from the expected geometry would result in a large energy penalty, therefore distortions in predicted geometries are likely to be small.\nConversely, in mechanically weak solids some atoms interact only weakly with adjacent atoms. Thus the layers in the micas and in boric acid are held together by low energy electrostatic and VDW forces. Much effort was expended during the development of PM6 in attempting to accurately model hydrogen bonds and other weak interactions because of their importance in biochemical systems. This work has apparently been of limited success, and, as a result, in solids that are mechanically weak very small changes in energy are often associated with large geometric distortions.\nThe nature of this inverse relationship can be used in future method development, where, currently, the use of solids in parameterization is precluded because of the heavy computational effort required. At the present time, however, solids can be used in surveys. There, the hardness-accuracy relationship can be used in the choice of solids to be used, in that potential errors in any new method are more likely to be detected in a survey of mechanically weak rather than very hard solids.\nCrystal packing automatically considered\nIn order to accurately reproduce molecular structures determined from X-ray analysis, theoretical methods must not only be able to accurately reproduce gas-phase geometries, but must also be able to include crystal packing forces. When these forces are small, the gas-phase structure is a good approximation to that of the solid, but the converse is also true, in that, in systems where there are large intermolecular interactions, particularly strong ionic forces, a solid state calculation is essential in order to accurately reproduce such structures. This was most evident in the case of HMPN-H(+), where the gas-phase B3LYP geometry is a significantly worse match to the observed crystal structure than that generated by the PM6 solid state calculation, despite the intrinsically higher accuracy of B3LYP. In this system, it is obvious that the packing forces arising from the electrostatic interaction of the [PF6]\u2013 ion with the HMPN-H(+) have significant geometric consequences.\nConclusions\nThe newly developed PM6 method has been demonstrated to reproduce the geometries and heats of formation of many solids, both organic and inorganic, with useful accuracy, although in a few cases the geometries predicted by PM6 were severely in error. Most of these errors can be attributed to incorrect values of the Voityuk core\u2013core parameters. Three examples of this type of error were identified: the Pb\u2013Sb, Br\u2013N, and Pb\u2013Zn interactions all had serious errors. Where diatomic parameters were either missing or improperly optimized, as a result of a fault in the training set used, a simple correction to the method can be made. This would involve only a re-optimization of the faulty diatomic parameters, and would not alter the performance of PM6 when applied to other systems. One general error was found in the treatment of organic compounds: many compounds that exist in the solid state as neutral species were incorrectly predicted by PM6 to be ionized. Examples include the aminophenols and compounds involving oxalic acid and water, such as oxalic acid dihydrate and barium oxalate oxalic acid dihydrate. In contrast to the errors in the values of the Voityuk parameters, errors of this type involve electronic properties and can only be corrected by a complete re-parameterization. The error was not detected during the development of PM6 because solids were not used. Many solids, particularly those that are mechanically weak, can be used as sensitive detectors of potential faults in new methods, and as such they should be used for quality control in future method development work.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nESM\u00a01\nArchive files of all systems, both organic and inorganic, described in this article are provided as separate files in a ZIP collection. Each file contains a complete Z-matrix of the geometry optimized using PM6, together with all keywords used in the calculation. The archive files are suitable for running using MOPAC2007 (PDF 32.4 MB)","keyphrases":["pm6","solid state","periodic boundary conditions","unit cell","minerals","hydrogen bonding"],"prmu":["P","P","P","P","P","P"]}
{"id":"Mar_Biotechnol_(NY)-4-1-2263118","title":"DNA Microarrays for Identifying Fishes\n","text":"In many cases marine organisms and especially their diverse developmental stages are difficult to identify by morphological characters. DNA-based identification methods offer an analytically powerful addition or even an alternative. In this study, a DNA microarray has been developed to be able to investigate its potential as a tool for the identification of fish species from European seas based on mitochondrial 16S rDNA sequences. Eleven commercially important fish species were selected for a first prototype. Oligonucleotide probes were designed based on the 16S rDNA sequences obtained from 230 individuals of 27 fish species. In addition, more than 1200 sequences of 380 species served as sequence background against which the specificity of the probes was tested in silico. Single target hybridisations with Cy5-labelled, PCR-amplified 16S rDNA fragments from each of the 11 species on microarrays containing the complete set of probes confirmed their suitability. True-positive, fluorescence signals obtained were at least one order of magnitude stronger than false-positive cross-hybridisations. Single nontarget hybridisations resulted in cross-hybridisation signals at approximately 27% of the cases tested, but all of them were at least one order of magnitude lower than true-positive signals. This study demonstrates that the 16S rDNA gene is suitable for designing oligonucleotide probes, which can be used to differentiate 11 fish species. These data are a solid basis for the second step to create a \u201cFish Chip\u201d for approximately 50 fish species relevant in marine environmental and fisheries research, as well as control of fisheries products.\nIntroduction\nCompared with terrestrial ecosystems, little is known about marine biodiversity and changes in species richness and ecosystem function. This is mainly because of sampling difficulties and problems in taxonomy. Many marine organisms and especially their diverse developmental stages, such as (1) eggs and larvae of fishes and invertebrates, (2) zoo- and phytoplankton, and (3) benthic invertebrates, are cumbersome and difficult to identify by morphological characters. Classical microscopy methods are time-consuming and require a high degree of taxonomic expertise, which is currently falling short. In many cases identifying a species is the major bottleneck in marine biodiversity and ecosystem research, hampering the necessary monitoring of marine biodiversity. As an example, a review of 138 studies on invertebrate diversity in European seas showed that approximately one-third of the specimens could not have been identified to species level (Schander and Willassen 2005).\nDNA-based identification methods are meanwhile established (Barlow and Tzotzos 1995) as powerful tools, exhibiting an unprecedented accuracy because of their inherently highest possible resolution, which can reach even the level of single base changes in a whole genome. Using these methods, the following marine animals have been investigated: eggs, larvae, and adults of fishes (Rocha-Olivares 1998; Noell et al. 2001; Fox et al. 2005; Ward et al. 2005), planktonic copepods (Bucklin et al. 1999), invertebrate larvae (Garland and Zimmer 2002; Barber and Boyce 2006), and prey in the gut content or feces of penguins, whales, and fishes (Jarman et al. 2002; Saitho et al. 2003).\nSequences of the small subunit of the rRNA gene are used as a standard method for identifying microbial organisms (Ludwig et al. 2004), and a fragment of the mitochondrial cytochrome oxidase I gene is in use as \u201cDNA-barcode\u201d for the identification of metazoans (Hebert et al. 2002; Ward et al. 2005). As shown above, a growing number of recently published studies are using molecular genetic identification methods. Nevertheless, their application is still restricted mainly because of methodical problems and because special knowledge and experience in molecular genetics is required. This is especially true if DNA-based identification is performed by using microarray platforms that are error-prone and difficult to quantify (Shi et al. 2006). Whereas most of the methods presently in use, such as PCR-based DNA amplification followed by sequencing techniques, allow to handle only single or a few species at the same time, DNA microarrays are believed to have the potential of identifying hundreds of species in parallel and to differentiate them against an even larger number of related species.\nCommonly, microarrays are glass microscope slides on which oligonucleotide probes are spotted that are complementary to the DNA target sequences to be analysed (Rel\u00f3gio et al. 2002, Pirrung 2002, Dufva 2005). The DNA target, which is usually fluorophore-labelled during PCR amplification, hybridises with the oligonucleotide probe on the microarray and can be detected after washing steps by its label. Whereas applying of DNA microarrays for gene expression has already reached the routine level of high throughput systems (Blohm and Guiseppi-Elie 2001, Hoheisel 2006), they have been only recently applied for the identification of organisms, such as microbes (Wang et al. 2002; Call et al. 2003; Korimbocus et al. 2005; Loy and Bodrossy 2005), animals (Pfunder et al. 2004), and plants (R\u00f8nning et al. 2005). In terms of identifying marine organisms, microarrays have been used for bacteria (Peplies et al. 2003; Peplies et al. 2004) and phytoplankton (Metfies and Medlin 2004; Metfies et al. 2005; Godhe et al. 2007) Other DNA-hybridisation methods for the identification of higher marine organisms, such as invertebrate larvae (Goffredi et al. 2006), copepods (Kiesling et al. 2002), and larvae of fishes (Rosel and Kocher 2002), have been applied, but microarrays have not been used for this kind of studies. Other applications of microarrays in research on marine organisms are gene expression analysis (Williams et al. 2003; Lidie et al. 2005; Wang et al. 2006; Cohen et al. 2007; Jenny et al. 2007) and genotyping in population genetics (Moriya et al. 2004; Moriya et al. 2007).\nOne of the methodical limitations for using microarrays is the design of species specific probes. On the one hand, oligonucleotide probes designed in silico do not always exhibit the experimental hybridisation properties they were selected for and must be empirically tested. On the other hand, the molecular marker must have highly selective characteristics, such as low intraspecific and a high interspecific variation. One of the most frequently used markers in phylogenetics of fishes is the mitochondrial 16S rRNA gene. This gene has a well-characterized secondary structure (Meyer 1993; Ort\u00ed et al. 1996) and especially the loop regions exhibit many insertions, deletions, and substitutions forming highly variable molecular features, which usually allow the design of highly specific probes. This is underlined by a study on lionfishes (Kochzius et al. 2003), which showed that individuals of one species exhibit identical 16S rDNA haplotypes even though they were sampled at sites thousands of kilometres apart, but clear differences could be detected between closely related lionfish species.\nIn this study, the development of a DNA microarray is described, demonstrating the suitability of the 16S rRNA gene for designing oligonucleotides as microarray probes to differentiate at least 11 fish species from European seas. Based on these data, a \u201cFish Chip\u201d for approximately 50 fish species is under construction to support the identification of eggs and larval stages from species that are otherwise difficult to identify, and of adult or processed fishes in fisheries industry.\nMaterial and Methods\nSampling and DNA Extraction\nTo consider possible intraspecies sequence variations, fishes were collected in five different regions of the European seas: North Sea, Bay of Biscay, and Western, Central, as well as Eastern Mediterranean (Fig.\u00a01, Table\u00a01). All together 267 individual samples from 79 fish species were investigated. In addition sequences from the EMBL sequence data base were included. Voucher specimens and tissue samples have been preserved in absolute ethanol and stored at 4\u00b0C or were frozen at -20\u00b0C. DNA was extracted from gill filaments with the Agowa mag midi DNA isolation kit (AGOWA, Berlin, Germany) or from muscle tissue with the DNeasy tissue kit (Qiagen, Hilden, Germany) according to the instructions of the manufacturer.\nFigure\u00a01Map with the sampling areas; NS North Sea; BB Bay of Biscay; WM Western Mediterranean; CM Central Mediterranean; EM Eastern Mediterranean.Table\u00a01Number of sequences per fish species and sampling regionSpeciesFamilyOrderNorth SeaBay of BiscayWestern MediterraneanCentral MediterraneanEastern MediterraneanEMBL sequence data base and other projectsTotalAccession number of EMBL data base sequencesTarget species\u00a0Boops boopsSparidaePerciformes3418AF247396\u00a0Engraulis encrasicolusEngraulidaeClupeiformes3216\u00a0Helicolenus dactylopterusSebastidaeScorpaeniformes316111AY538975\u00a0Lophius budegassaLophiidaeLophiiformes369\u00a0Pagellus acarneSparidaePerciformes3339\u00a0Scomber scombrusScombridaePerciformes3126AB120717, AF055615\u00a0Scophthalmus rhombusScophthalmidaePleuronectiformes33118AY359665\u00a0Serranus cabrillaSerranidaePerciformes347\u00a0Sparus aurataSparidaePerciformes3216AF247432\u00a0Trachurus trachurusCarangidaePerciformes125210AB108498, AB096007\u00a0Trigla lyraTriglidaeScorpaeniformes336Additional species\u00a0Chelidonichthys lucernusTriglidaeScorpaeniformes13610\u00a0Diplodus sargusSparidaePerciformes246\u00a0Gadus morhuaGadidaeGadiformes1236X99772, NC_002081, AY850363\u00a0Merluccius merlucciusMerlucciidaeGadiformes233614\u00a0Mullus surmuletusMullidaePerciformes252211\u00a0Mullus barbatusMullidaePerciformes257\u00a0Pagellus erythrinusSparidaePerciformes12710\u00a0Platichthys flesusPleuronectidaePleuronectiformes42410AY359670, AB125255, AY157320, AF113180\u00a0Pleuronectes platessaPleuronectidaePleuronectiformes426AY359673, AY157328\u00a0Psetta maximaScophthalmidaePleuronectiformes134\u00a0Sardina pilchardusClupeidaeClupeiformes33410\u00a0Scorpaena notataScorpaenidaeScorpaeniformes5611\u00a0Scorpaena porcusScorpaenidaeScorpaeniformes3317\u00a0Serranus hepatusSerranidaePerciformes2237\u00a0Solea soleaSoleidaePleuronectiformes332311AB125247, AF488442, AF112845\u00a0Zeus faberZeidaeZeiformes332614NC_003190, AF488474, AF221894-AF221896, AP002941\u00a0\u03a3 230Accession numbers are given for sequences obtained from EMBL sequence databaseTaxonomy according to FishBase (2007)\nPolymerase Chain Reaction and Sequencing\nA fragment of approximately 1380\u00a0bp length from the mitochondrial 16S rRNA gene was amplified with the primer 16fiF140 (5\u2032-CGY AAG GGA AHG CTG AAA-3\u2032), which has a single-base modification compared with Palumbi et al. (1991, unpublished manuscript) as well as with the newly designed primer 16fiR1524 (5\u2032-CCG GTC TGA ACT CAG ATC ACG TAG-3\u2032). Polymerase chain reaction (PCR) reactions with a total volume of 15\u00a0\u03bcl contained 1.5\u00a0\u03bcl 10 X reaction buffer, 1.5\u00a0\u03bcl dNTPs (10\u00a0mM), 0.05\u00a0\u03bcl of each primer (100\u00a0pmol\/\u03bcl), 5\u00a0\u03bcl DNA-extract, 0.3\u00a0\u03bcl Teg polymerase (3 U\/\u03bcl; Prokaria, Reykjavik, Iceland), and 6.6\u00a0\u03bcl deionized water. Thermal profile began at 94\u00b0C for 4\u00a0min, followed by 35 cycles of 94\u00b0C (30 s), 54\u00b0C (30 s), 72\u00b0C (90 s), with a final step of 7\u00a0min at 72\u00b0C.\nPCR products were purified by using the ExoSAP-IT for PCR clean-up (GE Healthcare, Uppsala, Sweden). The newly designed sequencing primer 16fiseq1463 (5\u2032-TGC ACC ATT AGG ATG TCC TGA TCC AAC-3\u2032) was used to sequence one strand of the amplified fragment using the BigDye Terminator Cycle Sequencing Kit (ver. 3.1, PE Biosystems, Foster City, USA). The sequencing reactions were run in an ABI Prism 3730 automated DNA Analyzer (Applied Biosystems, Foster City, USA) according to the manufacturer\u2019s instructions.\nSequence Analysis and Oligonucleotide Probe Design\nIn the framework of this project, 267 sequences have been acquired and 944 sequences have been obtained from EMBL sequence database and other projects, representing approximately 380 species of fish from European seas. Probes were designed based on 230 sequences obtained from 27 species (Table\u00a01). A multiple alignment of these 230 sequences was performed with the programme Clustal W (Thompson et al. 1994) as implemented in BioEdit (version 7.0.4.1; Hall 1999) to ensure that all sequences represent a homologous fragment of the 16S rDNA. Before probe design gaps have been removed from each sequence. A computer program developed by the bioinformatics group of the Centre for Applied Gene Sensor Technology (CAG) and the Zentrum f\u00fcr Technomathematik (ZeTeM), both at University of Bremen, was used to design species-specific oligonucleotide probes, which ideally cover all sequences of one species and do not match any other species (N\u00f6lte 2002). The following criteria have been considered: (1) length of 23 to 27\u00a0bp, (2) melting temperature (Tm) of 81 to 85\u00b0C based on the unified model (SantaLucia 1998), (3) GC content of 52% to 54%, (4) secondary structure of the oligonucleotides and the target sequence, (5) possible dimer formation, and (6) the energy content of a bond between the probe and the target sequence. Minimal free energy (mfe) structures are computed by using RNAfold (Hofacker et al. 1994). Probes exhibiting strong secondary structures or binding to a region of the target with such a strong secondary structure were not used. If more than one probe qualified for a species, the one with the highest binding energy between probe and target was chosen. Already during the design-phase the selected oligonucleotide probes were tested in silico against 1211 background sequences from approximately 380 fish species.\nPreparation of DNA Microarrays and Hybridisation Experiments\nAminosilane (3-aminopropyltrimethoxysilane)-coated glass slides were used with a PDITC-linker (1,4-phenylendiisothiocyanate) from Asper Biotech (Tartu, Estonia). Oligonucleotide probes (Thermo Hybaid, Ulm, Germany) with a 5\u2032-amino-C6-modification were spotted in 150\u00a0mM Na3PO4 buffer (pH 8.5) at a concentration of 30\u00a0\u03bcM using a spotting robot based on a modified version of the contactless TopSpot\u00ae technology. The spotted volume of this oligonucleotides solution was 200\u00a0pl, producing a spot diameter of approximately 220\u00a0\u03bcm. Each probe was spotted in four replicates per block. An array contained five blocks and three arrays were spotted on one microarray slide (Fig.\u00a02). After spotting, the microarrays were incubated for 16\u00a0h in a wet chamber to ensure efficient covalent binding of the oligonucleotides. Finally, the microarrays were shrink-wrapped under a nitrogen atmosphere and were storable at 4\u00b0C for up to 6\u00a0months.\nFig.\u00a02Layout of the microarray\nDNA for hybridisation experiments was amplified and labelled with 5\u2032-Cy5-modified primers. The primers 16sar-L (3\u2032-CGC CTG TTT AAC AAA AAC AT-3\u2032) and 16sbr-H (5\u2032-CCG GTT TGA ACT CAG ATC ACG T-3\u2032) amplify a fragment of approximately 600\u00a0bp length from the mitochondrial 16S rRNA gene (Palumbi et al. 1991, unpublished manuscript). PCR reactions with a volume of 100\u00a0\u03bcl contained 10\u00a0\u03bcl 10 X reaction buffer, 4\u00a0\u03bcl dNTPs (5\u00a0mM), 2\u00a0\u03bcl of each primer (10\u00a0\u03bcM), 2\u00a0\u03bcl DNA-extract, 0.4\u00a0\u03bcl Taq polymerase (5 U\/\u03bcl), 2\u00a0\u03bcl BSA (20\u00a0mg\/ml), and 77.6\u00a0\u03bcl deionized water. The PCR thermal profile began at 95\u00b0C for 2\u00a0min, followed by 35 cycles of 95\u00b0C (30 s), 54\u00b0C (45 s), 72\u00b0C (60 s), followed by a final step of 10\u00a0min at 72\u00b0C. The Cy5-labelled PCR amplified DNA was purified using the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany).\nHybridisation experiments were performed with 11 target and 14 nontarget fish species. As nontarget species fishes were chosen that are closely related to the target species and from which false-positive signals could be expected according to the in silico specificity tests performed during the design phase of the probes (Table\u00a02).\nTable\u00a02Nontarget species tested in hybridisation experimentsSpeciesFamilyOrderDentex dentexSparidaePerciformesDiplodus vulgarisSparidaePerciformesGadus morhuaGadidaeGadiformesMelanogrammus aeglefinusGadidaeGadiformesMerlangius merlangusGadidaeGadiformesMerluccius merlucciusGadidaeGadiformesMicromesistius poutassouGadidaeGadiformesMullus surmuletusMullidaePerciformesPollachius pollachiusGadidaeGadiformesPollachius virensGadidaeGadiformesPsetta maximaScophthalmidaePleuronectiformesSerranus hepatusSerranidaePerciformesTrachurus mediterraneusCarangidaePerciformesTrachuru picturatusCarangidaePerciformesTaxonomy according to FishBase (2007)\nA positive control at a concentration of 1\u00a0nM (5\u2032-CGT GTG AGT CGA TGG ATC ATA-3\u2032; 5\u2032-Cy3-labelled) and 10\u00a0nM of the purified Cy5-labelled PCR product were hybridized to the microarray in a volume of 65\u00a0\u03bcl using GeneFrames\u00ae (ABgene House, Epsom, UK), which were applied to the microarray slides according to the manufacturer\u2019s instruction (Fig.\u00a02). Hybridisation was conducted at 50\u00b0C in a hybridisation oven. After 2\u00a0h hybridisation time the GeneFrames\u00ae were removed and the microarrays were washed 5\u00a0minutes each with 2\u2009\u00d7\u2009SSC (sodium chloride trisodium citrate) buffer containing 0.05% SDS (sodium dodecyl sulphate), 1\u2009\u00d7\u2009SSC containing 0.05% SDS, and 1\u2009\u00d7\u2009SSC. Finally the microarrays were dried in a centrifuge at 2000\u00a0rpm for 3\u00a0minutes.\nMeasurement of Fluorescence Signals and Data Analysis\nHybridisation signals were measured using an Axon 4000B fluorescence microarray scanner at 635\u00a0nm (Cy5) as well as at 528\u00a0nm (Cy3). The fluorescence signal analysis was conducted with the software GenePix 4.1 (Axon, Union City, USA). The fluorescence signals of each probe were measured and the arithmetic mean was calculated. However, data were removed from the analysis if the spots showed artefacts caused during the spotting process (e.g., inhomogeneous spots documented by a monitoring camera during spotting) or experimental artefacts (e.g., air bubbles). Background noise was corrected by subtracting the arithmetic mean of the negative control measurement from the arithmetic mean of the spot measurements. Negative values were set to zero.\nResults\nProbes for 11 commercially important fish species have been designed (Table\u00a03). Positions of the oligonucleotide probes in the 16S rDNA fragment used for probe design are given in Fig.\u00a03. Following the nomenclature of Ort\u00ed et al. (1996), the binding sites of the microarray probes for Engraulis encrasicolus, Sparus aurata, and Trigla lyra are located in the variable region j, whereas the probes for Boops boops, Helicolenus dactylopterus, Lophius budegassa, Pagellus acarne, Scomber scombrus, Scophthalmus rhombus, Serranus cabrilla, and Trachurus trachurus bind to the variable region l.\nTable\u00a03Oligonucleotide probes for the identification of fish species from European seasSpecies nameProbe nameProbe sequence (5\u2032-3\u2032), 5\u2032-amino-C6-modifiedLength (bp)Tm (\u00b0C)GC (%)Oligo mfeDimer mfeSpecificity (in silico)Boops boopsBooboo_315GCACCACACTCCTAAACCCAAGA2382.6452\u22650\u22120.07speciesEngraulis encrasicolusEngenc_213CAAGTCCTAAATACCCGCAGCCT2382.4952\u22650\u22120.17speciesHelicolenus dactylopterusHeldac_317ACCCCTCCTACAATTAAGAGCCG2381.8452\u22650\u22120.22speciesLophius budegassaLopbud_312AACACCCTTCCTATCACCCAGAGCTAC2784.3952\u22650\u22120.2genusPagellus acarnePagaca_317TACTACACTCCCACATCCGAGAGC2482.7754\u22650\u22120.89speciesScomber scombrusScosco_321CAACTACTCCTACAGTCAAGAGCCACC2782.9152\u22650\u22120.43speciesScophthalmus rhombusScorho_322CCCCTTAACTCCTCGAAGCAAGA2381.8852\u22650\u22120.37speciesSerranus cabrillaSercab_313CCATTTTCCTACAACCCAGAGCGAC2582.7452\u22650\u22120.18speciesSparus aurataSpaaur_201AGAACAGCTCACGTCAAACACCC2383.0252\u22650\u22120.5speciesTrachurus trachurusTratra_333TTCCTCTCCTCCCACAAGCAAGA2383.6252\u22650\u22120.15genusTrigla lyraTrilyr_232AAGACCGAACCAAATGAGCCCTG2383.1652\u22650\u22120.17familyThe number in the probe name indicates the binding site in the 16S rDNA sequenceOligo mfe minimal free energy of the secondary structure of the oligonucleotide; Dimer mfe minimal free energy of the dimer of two identical oligonucleotide moleculesValues for mfe are given in kcal\/molFig.\u00a03Alignment (5\u2032\u2009>\u20093\u2032) of representative 16S rDNA sequences from the target species with binding sites (light grey) of probes (5\u2032\u2009>\u20093\u2032; probes hybridise to the reverse complementary target strand). Double stranded (dark grey) and single stranded regions (grey) of the secondary structure are indicated in the reference sequence of Pygoplites nattereri (Ort\u00ed et al. 1996; Accession number: U33590)\nAll single target hybridisations of the Cy5-labelled 16S rDNA fragment gave true-positive fluorescence signals for the corresponding probe (Fig.\u00a04), but the hybridisation efficiency is very different. The signal intensity of the weakest probe-target pair gave approximately 1,000 arbitrary units and the strongest almost 30,000 (Table\u00a04). Under the experimental conditions selected, single target hybridisations with B. boops, E. encrasicolus, H. dactylopterus, L. budegassa, S. rhombus, S. aurata, T. trachurus, and T. lyra did not show any false-positive signals. Eight probes gave under these conditions very weak false-positive signals with P. acarne, S. scombrus, and S. cabrilla, representing approximately 7% of all possible cross-hybridisation reactions. The values of these false-positive signals were several orders of magnitude lower than the true positive-signals.\nFigure\u00a04Signals (background subtracted from absolute signal) of single target and nontarget hybridisations. White bars represent true-positive signals; false-positive signals are shown as grey bars. Numbers at the basis of the bars indicate the amount of measured spots. The number of hybridisations is given in brackets after target and nontarget names. Replication and absolute signal intensities (\u00b1 standard deviation) of hybridized targets to the corresponding probe are given in Table\u00a04.Table\u00a04Target hybridisationsHybridized targetsNo. of hybridisationsMeasurements of specific probesMeasured probes\/absolute no. of probesMean absolute fluorescence signal in arbitrary unitsStandard deviationBoops boops221\/402991\u00b11491Engraulis encrasicolus220\/401659\u00b1962Helicolenus dactylopterus220\/403502\u00b1912Lophius budegassa240\/403450\u00b11515Pagellus acarne227\/403727\u00b11270Scophthalmus rhombus115\/201528\u00b1269Scomber scombrus240\/4027827\u00b15330Serranus cabrilla240\/4010814\u00b14396Sparus aurata240\/40963\u00b1227Trachurus trachurus120\/202015\u00b1880Trigla lyra235\/402343\u00b1560\nSingle nontarget hybridisations showed 48 very weak false-positive signals, representing 31% of 154 possible cross-hybridisations. These false-positive signals were at least one order of magnitude weaker than the true-positive signal. Merlangius merlangus and Merluccius merluccius did not gave any false-positive signal, whereas Dentex dentex, Diplodus vulgaris, Gadus morhua, Melanogrammus aeglefinus, Micromesistius poutassou, Mullus surmuletus, Pollachius pollachius, Pollachius virens, Psetta maxima, Serranus hepatus, Trachurus mediterraneus, and Trachurus picturatus showed two to eight cross-hybridisations. Considering all possible cross-hybridisations, only 18% showed usually very weak false-positive signals.\nDiscussion\nThe results show that the \u201cFish Chip\u201d described enables the identification of 11 commercially important fish species from European seas in certain experimental limits. These limits are primarily given by the fact that the fluorescence signal intensities of true-positive hybridisation signals were heterogeneous. This phenomenon is commonly encountered in DNA microarray experiments (Peplies et al. 2003; Warsen et al. 2004; Korimbocus et al. 2005; R\u00f8nning et al. 2005; Tobler et al. 2006) and can probably be overcome only by an extreme methodical effort (Shi et al. 2006). The problems encountered when single-colour microarray experiments need to be quantified are severe and in part not yet solved, because complex parameters are influencing the results. These are the sequence dependent hybridisation efficiency, specifically steric hindrance, secondary structures (Southern et al. 1999), and the relative position of the fluorescent label at the target (Zhang et al. 2005).\nSometimes the duplex formation can be favoured by using spacers, which obviously give the captures a greater degree of freedom from their neighbouring molecules and from the surface (Southern et al. 1999), leading to an enhancement of signal intensity with increasing spacer length (Peplies et al. 2003).\nAn important criterion of probe design was the base composition (Southern et al. 1999) and therefore all but one capture oligonucleotides were designed to have a GC content of 52% and 54% for the exception (Table\u00a03). The different hybridisation efficiencies of the captures and the varying sensitivity for the different fishes is still a severe disadvantage, because it can hamper the estimation of a small amount of fishes of one species in the presence of a larger number of individuals of another species in a mixed sample. These limits are presently under investigation with fish eggs and other biological material.\nBecause all oligonucleotide probes bind to the variable regions j and l of the 16S rRNA gene, which represent large single-stranded loops, the secondary structure is unlikely to be a factor contributing to the partially low sensitivity. Also, the POL effect, a phenomenon decreasing the fluorescence signal with increasing distance between the probe binding site and the label on the target (Zhang et al. 2005) seems not to be important in this case. Most of the probes used in this study hybridise to the variable region l of the 16S rDNA and their distance from the fluorescence label is more or less identical.\nAlthough cross-hybridisations occur, true-positive signals could clearly be differentiated from false-positive signals because of their generally higher signal. Most false-positive signals occurred when the 16S rDNA fragment of nontarget species was hybridised on the microarray.\nTesting of nontarget species is seemingly important in studies using DNA microarrays for the identification of organisms, because even if a comprehensive sequence background is utilised for probe design and even if extensive in silico testing has been performed, the specificity of oligonucleotide probes has to be evaluated with closely related species and specifically those that show cross-hybridisations in silico.\nThis study shows that the 16S rRNA gene of fishes is suitable to design oligonucleotide probes that are able to differentiate eleven fish species from European seas by single target hybridisation on a microarray. Such a \u201cFish Chip\u201d can hopefully be applied in marine environmental and fisheries research, as well as in fisheries and food control if the uneven hybridisation signal intensities of the different probe-target pairs can be improved or compensated.\nThe correct identification of fish eggs and larvae is crucial for fish stock assessment based on ichthyoplankton surveys. Genetic identification has shown that the majority of eggs in the Irish Sea, wrongly believed to be from cod, were actually from whiting, leading to an overestimation of cod stocks (Fox et al. 2005). A study on food fish in the United States revealed that three-quarters of fish sold as \u201cred snapper\u201d were mislabelled and belonged to other species (Marko et al. 2004), a situation that needs better analytical tools to be changed. The European Union (EU) also has strict regulations for seafood labelling, which must include, for example, the species name (EU Council Regulation No 104\/2000; EU Commission Regulation No 2065\/2001). Approximately 420 species of fish are sold in the German market alone, making a reliable identification method urgently necessary to protect the customer. DNA microarrays might have the potential to fulfill these requirements.\nRecent efforts in compiling sequences of fishes, such as the European projects \u201cFishTrace\u201d http:\/\/www.fishtrace.org) and \u201cFish & Chips\u201d (http:\/\/www.fish-and-chips.uni-bremen.de; Kochzius et al. 2007), as well as the international \u201cFish Barcode of Life Initiative\u201d http:\/\/www.fishbol.org; Ward et al. 2005), will provide the necessary sequence background for the design of species specific oligonucleotide probes for the development of DNA microarrays for the identification of fishes.","keyphrases":["capture oligonucleotide","dna chips","species identification","pisces"],"prmu":["P","R","R","U"]}
{"id":"Neurochem_Res-4-1-2295255","title":"Increased Nitric Oxide Production and GFAP Expression in the Brains of Influenza A\/NWS Virus Infected Mice\n","text":"The cause of influenza to the brain was investigated using the A\/NWS\/33 influenza virus infected BALB\/c mouse model. NOS-2 mRNA levels in the infected mouse brain was greater than in control mice in all brain regions examined, particularly in the olfactory bulb and hippocampus by 1 day p.i. On the contrary, no differences in NOS-1 or NOS-3 mRNA levels were found between infected and control mice. There was also a marked increase in the levels of metabolites of nitric oxide in the olfactory bulb and hippocampus. Immunohistochemistry showed positive staining for anti-NOS-2 primarily in the hippocampus of infected mice. Further, anti-NOS-2 and GFAP staining was mostly found around capillary blood vessels of the hippocampus starting early in the course of the disease. These results indicate that the NWS enhances the activation of astrocytes and NOS-2 expression which in turn enhances NO production and the expansion of capillary blood vessels.\nIntroduction\nInfluenza associated encephalopathy (IE) is one of the most severe neurological diseases amongst children, mostly under the age of 5. It is marked by rapid progression and the mortality rate is estimated to range from 27 to 44% [1]. Recently, we reported that production of the biological free radical gas, nitric oxide (NO), in the serum and cerebrospinal fluid (CSF) of patients with IE was increased in the initial stages and correlated with disease severity [2, 3].\nYokota et\u00a0al. hypothesized that the influenza virus activates the limbic system followed by glial cell activation which in turn produces NO and inflammatory cytokines which flood the brain and cause a cytokine storm [4]. Recent in\u00a0vitro studies by Imanishi et\u00a0al. showed that a mouse lung-adapted strain of influenza virus could induce inducible NO synthase (NOS-2) mRNA expression and NOS-2-mediated NO production in murine macrophage cell [5]. Moreover, Ino et\u00a0al. studied five autopsies cases of IE patients. They reported that four out of the five patients showed degenerated astrocytic spines in the cortex, by glial fibrillary acidic protein (GFAP) staining. They suspected that the IE was largely due to these altered glial cells (Ino et\u00a0al. unpublished).\nTaken together, it is likely that IE is related to both the influence of NO and glial cells changes. Nevertheless, there have been no reports on a relationship between NOS-2 and glial cells within the brain after infection with the influenza virus. Thus, the aim of these studies is to better understand the clinical state of IE by focusing on NO and astrocytes in the central nervous system (CNS) after influenza virus infection. By using the A\/NWS\/33 influenza virus infected BALB\/c model mouse, we were able to demonstrate that influenza virus infection leads to an up regulation of NOS-2 and astrocytes, mostly around capillary blood vessels of the hippocampus and olfactory bulb, starting at an early stage of the disease.\nAlthough IE has been reported for more than a decade, a therapy for IE has not yet been defined. We expect that our results will provide evidence which will aid in the development of a novel IE therapy.\nExperimental procedures\nVirus\nInfluenza A\/NWS virus, which is a mouse brain adapted type of human influenza A\/NWS\/33 virus was used. (A kind gift from Dr. K. Hayashi, Toyama University, Japan.)\nExperimental animals\nIn total, 82 male BALB\/c mice (5-week-old) were purchased from Japan Charles River Co. and given free access to food and water (Oriental Yeast, Co., Tokyo) and acclimatized for at least a week before the experiment.\nAnesthetics\nEach mouse was anesthetized by inhalation of ether before being inoculated intranasally with 50\u00a0\u03bcl NWS, 5.0\u00a0\u00d7\u00a0107 plaque-forming units (PFU)\/ml (5.0\u00a0\u00d7\u00a0105 PFU\/mouse). For negative control mice, the same amounts of 0.1\u00a0M phosphate-buffered saline (PBS) were intranasally inoculated. The mice were examined at 1, 3 and 6\u00a0days postinfection (p.i.).\nBody weight\nBody weight changes were monitored daily.\nEuthanasia\nAll mice were decapitated whilst under ether inhalational anesthesia. Each brain was divided into eight regions (frontal cortex, occipital cortex, cerebellum, medullar oblongata, hippocampus, corpus striatum, thalamus\/hypothalamus and olfactory bulb). Then each section was immediately frozen with liquid nitrogen.\nNO2\u2212 and NO3\u2212 measurements by HPLC-UV method\nBoth NO2 and NO3 (NOx) levels in each brain region were measured by HPLC-UV system (ENO-10, EICOM) which was reported by us [3]. Briefly, thawed brain regions were deproteinized with a ten times volume of methanol and then centrifuged (12,000 r.p.m. for 20\u00a0min at 4\u00b0C) to avoid an occlusion in the polystyrene polymer column used in this system, by protein or other macromolecules. Detection was measured at 540\u00a0nm (absorption) and the concentrations were calculated from the area under the curve (AUC) of NO2 and NO3 (Power Chrome, EICOM, Kyoto) by Griess reaction.\nRNA extraction\nRNA was extracted from each part of the brain by using a QIA amp RNA extraction kit (QIAGEN K.K., Tokyo), according to the protocol suggested by the manufacturer.\ncDNA synthesis\ncDNA was synthesized from purified RNA by using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Switzerland). Random hexamer primer was used and the protocol was as follows; after annealing for 10\u00a0min at 25\u00b0C, and incubating 30\u00a0min at 55\u00b0C, the reaction was heated to 85\u00b0C for 5\u00a0min and chilled on ice.\nReal time reverse transcriptase-polymerase chain reaction (RT-PCR)\nMessenger RNA levels of NOS-1, NOS-2 and NOS-3 were quantified by using a Light Cycler Fast Start DNA Master HybProbe (Roche). This was done according to the following protocol; Pre-incubation was 95\u00b0C for 30\u00a0s. This was followed by 40 cycles of denaturation at 95\u00b0C for 10\u00a0s, annealing at 62\u00b0C for 15\u00a0s, amplification at 72\u00b0C for 8\u00a0s for 45 cycles. Cycles were followed by 30\u00a0s 40\u00b0C cooling program. The primers and probes were designed by the Light Cycler primer and probe set (Roche Diagnostics Applied Science, Tokyo). NOS-2, NOS-3 and NOS-1 primer sequences were designed on GenBank database NM_0100927, NM_008713, and NM_008712 respectively. All data were normalized by dividing with the corresponding GAPDH mRNA from the same sample.\nImmunohistochemistry\nAnesthetized animals were perfused intracardially with 20\u00a0ml of 0.1\u00a0M PBS, followed by 30\u00a0ml of 4% paraformaldehyde in 0.1\u00a0M phosphate buffer. The brains were removed, postfixed overnight and cryoprotected in 30% sucrose, processed through graded alcohols and embedded in paraffin. Then, they were cut as 6\u00a0\u03bcm thick coronal sections. They were incubated with 0.3% H2O2 for 20\u00a0min. The sections were then rinsed with tris-buffered saline, pH 7.6 (TBS). To identify reactive astrocytes, we stained with Anti-GFAP (rabbit polyclonal, DAKO, Denmark). Sections were incubated over night at a 1:2,000 dilution. The NOS-2 stains were incubated for 4\u00a0h at a 1:1,500 dilution (Anti-iNOS\/NOSII, rabbit polyclonal IgG;Upstate Biotechnology, Lake Placid, NY). After the first staining, each section was incubated with the secondary antibody for 30\u00a0min (Nichirei Histofine Simple Stain, anti-rabbit IgG) at room temperature after TBS rinsing. Labeling was visualized by incubating with a solution containing 2%, 3,3\u2032-diaminobenzidine, 0.1\u00a0M imidazole and 30% H2O2.\nAll sections were counterstained with Mayer\u2019s hematoxylin (SIGMA, MI).\nStatistical analysis\nFisher\u2019s exact test was used when comparing body weights. All results obtained from the real time RT-PCR were subjected to one- or two-way analysis of variance (ANOVA) and differences among the means were analyzed by two-way ANOVA followed by a Newman\u2013Keuls range test at the 0.05 significance level. Results are expressed as mean\u00a0\u00b1\u00a0standard error.\nResults\nBody weight\nFigure\u00a01 shows the change in body weight of the non-infected (PBS) and infected (Flu) mice over day. The mice infected with virus showed a decrease in weight and ruffled hair from 3\u00a0days p.i. and by 6\u00a0days p.i. and there was a significant decrease in the body weight of the infected mice as compared to the non-infected mice (P\u00a0<\u00a00.05). Prominent neurological signs in the infected mice did not appear during the experimental period and none of the mice died during the experiment, although spontaneous locomotor activity was reduced in the infected mice.\nFig.\u00a01Changes in body weight of A\/NWS\/33 influenza virus infected BALB\/c model mice after virus infection. Each result shows the mean\u00a0\u00b1\u00a0SE value of eight experiments. () non-infected (PBS treatment) mice group, () infected (Flu) mice group\nViral titers\nAt one day p.i., 120\u00a0PFU\/50\u00a0mg virus was detected in the infected mouse brain and 136\u00a0\u00d7\u00a0104 PFU\/100\u00a0mg virus was detected in the infected mouse lung.\nReal-time RT-PCR for NOS-2, NOS-3 and NOS-1\nFigure\u00a02 shows the NOS-2\/GAPDH and NOS-3\/GAPDH ratios as measured in the olfactory bulb and hippocampus of infected mouse brains as compared to the non-infected mice on days 1, 3 and 6 p.i. Due to the scattered results obtained in both the infected and non-infected mice, data on the NOS-1\/GAPDH ratio is not reported. The increases in the NOS-2\/GAPDH ratio as seen in the olfactory bulb(OB) and hippocampus(HIP) at day 1 and 3 p.i., were obvious as shown in Fig.\u00a02a and b. However, no changes were seen in the levels of NOS-3\/GAPDH in the same regions (Fig.\u00a02a\u2019, b\u2019).\nFig.\u00a02NOS-2\/GAPDH and NOS-3\/GAPDH ratios in the olfactory bulb and hippocampus of non-infected and infected mice on days 1, 3 and 6 p.i. The open column shows the mean and S.E. value obtained from nine non-infected mice and the closed column shows the mean and S.E. value obtained from eight infected mice. *Significant difference between non-infected mice and infected mice. *P\u00a0<\u00a00.05. (a and a\u2019): olfactory bulb, (b and b\u2019): hippocampus\nChanges in brain NO levels between non-infected and infected mice\nFigure\u00a03 shows that NO levels in the eight brain regions examined, either with or without NWS virus infection, changes in a day-by-day manner after virus infection. The levels of NO2 and NO3 were measured separately, as shown in the Materials and methods. In the isolated brain regions, NO3 levels were more than 40-fold higher than NO2 levels, and the final data are shown as the total value (NO) of NO2 plus NO3. At day 1 p.i., the NO levels detected in olfactory bulb (OB), hippocampus (HIP) and frontal cortex (FC) of the infected mice (Flu) were significantly increased as compared to the levels in the same brain regions of the non-infected (PBS) mice (P\u00a0<\u00a00.05). Further, at day 6 p.i., the increased levels of NO in the olfactory bulb (OB) and the frontal cortex (FC) returned to the levels seen in non-infected mice. However, the levels of NO in the hippocampus (HIP) were significantly increased (P\u00a0<\u00a00.05) at all the time points examined.\nFig.\u00a03Day to day changes in NO levels in the eight brain regions either with or without NWS virus infection. The open column shows the mean and S.E. value obtained from eight non-infected mice and the closed column shows the mean and S.E. value obtained from seven infected mice. *Significant difference between non-infected mice and infected mice. *P\u00a0<\u00a00.05. OB: olfactory bulb, HIP: hippocampus, St: striatum, Hypo\/Thal: hypothalamus and thalamus, MO: medulla oblongata, CE: cerebellum, FC: frontal cortex, and OC: occipital cortex\nImmunohistochemistry\nFigure\u00a04 shows the results of anti-NOS-2 staining and GFAP staining in the hippocampus of non-infected (PBS) and infected mice (Flu) at 1 (Fig.\u00a04a) and 6 (Fig.\u00a04b) days p.i. in the same magnification each. The brown-yellow staining was recognized as positive staining for anti-NOS-2. We found positive staining in the hippocampus, especially in the area surrounding capillary blood vessels of the infected mice at 1\u00a0day p.i. (Fig.\u00a04a, Day 1, Infected). By 6\u00a0days p.i., positive staining had spread to the striatum, hippocampus and cortex of the infected mice and positive staining were seen in the areas surrounding capillary vessels (Fig.\u00a04b, Day 6, Infected). Add that infected mice showed an increase in GFAP staining on days 1 much more so than that seen in non-infected mice. Positive staining was most notable around hippocampal blood vessels in infected mice. Additionally, most of the blood vessels in hippocampi of infected mice were markedly expanded as compared to those seen in the non-infected mice.\nFig.\u00a04Anti-NOS-2 immunohistochemistry and GFAP immunohistochemistry in the hippocampus at 1 (Fig.\u00a04a) and 6 (Fig.\u00a04b) days p.i\nDiscussion\nSince our aim was to see the effect to the brain after influenza infection, this experiment was undertaken by using the A\/NWS\/33 virus because it is proven by Li et\u00a0al that the NWS\/33 strain can replicate in the brain of adult mice after intracerebral injection, add that the virus usually fails to spread beyond the respiratory epithelium after internasal instillation [6].\nDuring the experiment, we observed that the NWS virus infected mice lose significantly more body weight at 6\u00a0days p.i. (Fig.\u00a01) in agreement with previous studies [5, 7]. Since bodyweight changes are an indicator of the overall condition of the mice, this bear out that the mice was infected to the virus. More over, we have checked that RT-PCR for A\/NWS\/33 virus were positive in all brain regions expect medullar oblongata on day 1 p.i. (data not shown).\nThe reason why we determined the amounts of influenza virus in this experiment is as followed. During the past experiment, we had experienced that the mice die within 3\u00a0days p.i. when inoculating any more than the virus we used this time and when we use fewer viruses, there were hardly any difference in the non-infected and infected behavioral change.\nOur results show that an enhancement NO biosynthesis was induced in the NWS virus infected mouse brain. Over-production of NO was substantiated by determining an increase in the expression of NOS-2 mRNA as shown in Fig.\u00a02, and increased immuno-histochemical staining for anti-NOS-2 as shown in Fig.\u00a04. At 1\u00a0day p.i., the amount of NOS-2 mRNA within the infected mouse brain was greater than that seen in control mice in all brain regions studied. Above all, the olfactory bulb showed the greatest increase. On the basis of these results, we compared NOS-2 mRNA expression in the olfactory bulb on days 1, 3, and 6 p.i., respectively. This showed that the NOS-2 mRNA ratio was greater in the infected mouse brain at all experimental times studied (Fig.\u00a02). On the contrary, there was no difference in NOS-3 or NOS-1 mRNA levels between infected and control mice (data not shown). These results are important since these three isoforms of NOS, NOS-2 the inducible form of NOS, is capable of producing a continuous flux of NO and a large amount of NO generated by NOS-2 over a sustained period is implicated in the pathogenesis of various disorders and with the response to infectious organisms [8].\nIn regards to the immuno-histochemical analysis, positive staining for anti-NOS-2 was mostly detected in the hippocampus of infected mouse brains, whereas hardly any staining was seen in control brains (Fig.\u00a04). Furthermore, positive staining had spread to the hippocampus and striatum by 6\u00a0days p.i., which correlated with the transition of NO levels.\nNOS-2 has been shown to play a role in the defense against bacteria, parasites and viruses. However, it seems that NO induced by the influenza virus has a dual role. Rimmelzwaan et\u00a0al. reported an inhibitory effect of NO on virus growth [9], whereas Akaike et\u00a0al. reported that NO played a pathological role in the influenza virus-induced pneumonia in mice [10]. The fact that the influenza virus can induce NO has been reported previously [2, 3]. Yao et\u00a0al. reported that the mRNA\u2019s of both NOS-2 and NOS increased in the brain after the intranasal infection with influenza A virus of newborn mice [11]. Further, Imanishi et\u00a0al. pointed out that a mouse lung-adapted strain of influenza virus could induce NOS-2 mRNA expression and NOS-2-mediated NO production in murine macrophage cells [5]. Our study has demonstrated that the NWS virus can induce NOS-2 expression in the infected mouse brain, especially in the olfactory bulb and hippocampus. These results may relate to the fact that intranasally administered NWS virus can invade the brain through the olfactory pathway as has been reported previously [12\u201316]. Recently, Yossi et\u00a0al. reported that astrocytes are capable of rapid and massive NOS-2-dependent NO production in brains of normal mice in situ [17]. According to the report by Ino et\u00a0al. on the brain autopsy of patients who had died of IE, four out of five IE patients showed degenerated astrocytic spines in the cortex by GFAP staining. They suspected that the IE was largely due to altered glial cells (Ino et\u00a0al. unpublished). Takahashi et\u00a0al. also made a statement on the autopsied case of a 2-year-old girl with encephalopathy associated with influenza A virus infection and according to their report; reactive astrocytes were seen in the vicinity of blood vessel in the brain but astrocytosis was not seen anywhere, including the areas where virus antigen-positive neurons were abundant [18]. Additionally, Yokota et\u00a0al. hypothesized that the influenza virus activates the limbic system followed by the activation of glial cells which produce NO and other inflammatory cytokines which together flood the brain and cause a cytokine storm [4]. Astrocytes are the major glial cell in the CNS and have important physiological properties in terms of CNS homeostasis. Astrocytes also influence the formation and maintenance of the blood\u2013brain barrier (BBB). The importance of astrocytes is demonstrated by a study from Bush et\u00a0al. According to their report, an astrocyte ablation led to failure of BBB repair and vasogenic edema [19]. There have been several reports related to glial cells and NO [17, 20, 21]. However, there has not been a report considering the relationship between influenza virus infection, NOS-2 and astrocytes together. By determining an increase in GFAP staining as shown in Fig.\u00a04, our results show that astrocytes are activated in the infected mouse brain and especially in hippocampus. Interestingly, we noticed that both the increased NOS-2 and GFAP staining were mostly found around capillary blood vessels of the hippocampus starting at an early stage. Our results suggest that since NOS-2 and astrocytes became activated around capillary blood vessels after viral infection, this may lead to brain capillary blood vessels break down. However, we were unable to determine whether the NOS-2 measured is derived from vascular endothelial cells or macrophages which will be an issue for future experiments.\nAlthough IE has been mostly reported in Asian countries, influenza infection occurs in epidemics in both the northern and southern hemispheres and is a pandemic disease throughout the year. Recently, influenza virus which is resistant to anti-influenza drugs has been shown to emerge and there is real concern in regards to a new type of influenza pandemic [22]. However, no standardized therapy for the treatment of IE has yet been defined. In this paper, we suggest that the influenza virus causes the activation of astrocytes and NOS-2 in the brain. Thus it is likely that our results will add supportive evidence for the development of a novel IE therapy.","keyphrases":["balb\/c mouse","no","nos-2","olfactory bulb","hippocampus","astrocyte","influenza a\/nws\/33 virus","brain capillary blood vessels"],"prmu":["P","P","P","P","P","P","P","P"]}
{"id":"Photosynth_Res-3-1-2117334","title":"Time sequence of the damage to the acceptor and donor sides of photosystem II by UV-B radiation as evaluated by chlorophyll a fluorescence\n","text":"The effects of ultraviolet-B (UV-B) radiation on photosystem II (PS II) were studied in leaves of Chenopodium album. After the treatment with UV-B the damage was estimated using chlorophyll a fluorescence techniques. Measurements of modulated fluorescence using a pulse amplitude modulated fluorometer revealed that the efficiency of photosystem II decreased both with increasing time of UV-B radiation and with increasing intensity of the UV-B. Fluorescence induction rise curves were analyzed using a mechanistic model of energy trapping. It appears that the damage by UV-B radiation occurs first at the acceptor side of photosystem II, and only later at the donor side.\nIntroduction\nThe solar spectrum that reaches the earth\u2019s surface includes wavelengths in the visible or photosynthetically active range (PAR, 400\u2013700\u00a0nm), in the ultraviolet-A (UV-A, 320\u2013400\u00a0nm), ultraviolet-B (UV-B, 280\u2013320\u00a0nm) and in the ultraviolet-C (UV-C, 200\u2013280\u00a0nm) ranges. While UV-C is totally absorbed by the atmosphere, ozone is the principal atmospheric attenuator of UV-B radiation (Cockell and Horneck 2001). The depletion of ozone by the emission into the atmosphere of man-made chlorine- and bromine-containing compounds has been correlated with an increase in the background level of UV-B radiation.\nUV-B radiation can affect a multitude of physiological and morphological plant processes that ultimately can lead to inhibition of growth and reproduction (Jansen et\u00a0al. 1998). Potential molecular targets for direct UV-B damage via photomodification or photosensitization include nucleotides, proteins, lipids and pigments (Jordan 2002). Besides direct macromolecular damage, UV-B can also induce specific signal transduction pathways and changes in gene expression that stimulates acclimation and repair processes (Holl\u00f3sy 2002; Brosch\u00e9 and Strid 2003).\nThe photosynthetic process can be affected by UV-B radiation at different levels, including alterations in plant and leaf morphology that decreases light interception (Jansen 2002), changes in stomatal function that limit the availability of CO2, changes in photosynthetic pigments (Strid and Porra 1992), on the expression of photosynthetic genes and on enzymes of the carbon fixation pathway. However, it is the effects of UV-B radiation on light harvesting and primary photochemical reactions of photosynthetic membranes, particularly on the Photosystem II (PS II) reaction center, which has attracted much attention and study (Vass 1997; Vass et\u00a0al. 2005).\nPS II is one of the reaction centers that, together with the cytochrome b6f complex, the photosystem I (PS I) reaction center, and the ATP-synthase, forms the electron transport chain that drives energy transduction in the thylakoid membranes of oxygenic eukaryotes. The photochemical core of PS II is formed by the D1\/D2 heterodimer, where the redox electron carriers and cofactors of electron transport are bound. Light absorbed by the antenna system of PS II induces the excitation of a special reaction center chlorophyll (P680), which is photo-oxidized on the first electron transfer reaction of PS II, with a pheophytin molecule (Pheo) acting as the primary electron acceptor and the formation of a radical pair state (P680+Pheo\u2212). This radical pair state is \u201cstabilized\u201d at the reducing (acceptor) side of PS II by the electron transfer from Pheo\u2212 to a primary D2-bound quinone electron acceptor (QA) and then to the secondary D1-bound quinone acceptor (QB), which, upon accumulation of two reducing equivalents, and becoming protonated to plastoquinol, dissociates from the reaction center. At the oxidizing (donor) side of PS II, P680+ is reduced by a redox-active tyrosine residue of D1 (Yz) which acts as an electron transfer intermediate between P680+ and the oxygen-evolving complex (OEC), the metalloenzyme system composed of a cluster of four Mn ions and inorganic cofactors (Ca2+ and Cl\u2212) that oxidizes water into molecular oxygen. Accordingly, the consecutive photochemical formation of the primary radical pair P680+Pheo\u2212 by light absorption and the reduction of P680+ forming the radical YZ+ drives the sequential four-step oxidation of the OEC inducing the S-state transitions and ultimately the splitting of the water molecule. For detailed reviews on oxygen evolution and PS II structure and function see, Diner and Babcock (1996), Xiong et\u00a0al. (1998), and Van Rensen and Curwiel (2000).\nFrom the above brief description of the PS II components and the potential molecular targets of UV-B radiation, it is clear that several components of PS II could be directly affected, as actually has been observed (Vass et\u00a0al. 1996). Several studies, most of them in\u00a0vitro, have shown that UV-B can inhibit PS II electron transport by damage to the quinone electron acceptors\u2019 redox function (Rodrigues et\u00a0al. 2006), to Yz function, to the OEC, and to the D1 protein. It should be noted that the time-sequence of events that ultimately leads to inactivation of PS II function is not completely clear, as it might involve a direct destruction or impairment of the absorbing molecule, modification of the protein environment to which these redox components are bound as well as a process of energy transfer by the sensitizer species to the damaged site (Vass 1997).\nThe time sequence of damage to acceptor and donor sides of PS II, respectively has never been measured in a single experiment. In this work, we have studied the effect of UV-B radiation on the function of PS II in leaves of Chenopodium album using both steady-state (light-adapted) fluorescence and the time-resolved fast Chl-a fluorescence induction curve. For a review on Chl-a fluorescence, see Govindjee (1995). The results were analyzed based on a mechanistic model of energy trapping and electron transport of PS II (Vredenberg 2000). It appears that damage to the acceptor side of PS II occurs first, and that only later the donor side becomes affected.\nMaterials and methods\nPlant growth\nPlants of Chenopodium album L. were grown as described earlier (Rodrigues et\u00a0al. 2006). After around 28\u00a0days the plants were transferred to a growth cabinet with a constant temperature of 20\u00b0C, 70% relative humidity and a PAR level of 60\u00a0\u03bcmol\u00a0m\u22122\u00a0s\u22121. The plants were watered daily and after 10\u201314\u00a0days the sampling of leaves for the UV exposure treatments started. Several different batches of plants were used and no significant difference could be detected when comparing results from experiments replicated with plants with similar ages but from different batches.\nUV-B radiation treatments\nThe plant material used for the exposure to UV light was either fully developed detached leaves or leaf discs (0.6\u00a0cm2) floating on petri-dishes filled with demineralized water with the abaxial leaf surface exposed to the radiation. The control samples were either kept in the darkness or exposed to white light (PAR). In some experiments leaves were vacuum infiltrated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) in 0.5% ethanol before being exposed to UV-B, with the controls infiltrated with a 0.5% ethanol solution.\nUV-B irradiations were performed using a Vilber Lourmat lamp with filter (VL-115M, Marne-la-Vall\u00e9e, France). This lamp has a peak emission at 312\u00a0nm. UV-B irradiance was measured with an optometer (United Detector Technology Inc, Baltimore, USA) equipped with a probe specific for UV radiation.\nChlorophyll a fluorescence measurements\nMeasurements of Chl-a fluorescence were performed at room temperature on leaf samples that had been dark adapted for 1\u00a0h after the exposure to UV-B radiation, unless otherwise stated. Steady-state Chl-a fluorescence was measured with a pulse-amplitude fluorometer (PAM 101\u2013103, Walz Inc., Effeltrich, Germany). A weak modulated measuring light (1.6\u00a0kHz, 650\u00a0nm) was used for the determination of the minimal fluorescence level Fo, followed by a 1 s pulse of saturating light (5,500\u00a0\u03bcmol\u00a0m\u22122\u00a0s\u22121) for the measurement of the maximum fluorescence level Fm. The leaf was then irradiated with a fixed or increasing level of actinic PAR and, at each level, we measured the steady state light-adapted fluorescence (Fo\u2032), the maximum fluorescence of a light-adapted state (Fm\u2032) induced by a saturating light pulse superimposed on the actinic light, and after removing the actinic light and the application during 10 s of far-red light (6\u00a0\u03bcmol\u00a0m\u22122\u00a0s\u22121, 720\u00a0nm), the minimum fluorescence of a light-adapted state (Fo\u2032). During the saturating pulses the modulation of the measuring light was automatically changed to 100\u00a0kHz. Several fluorescence parameters were calculated from the measured fluorescence levels (Schreiber et\u00a0al. 1986; Genty et\u00a0al. 1989; Van Kooten and Snel 1990): the potential photochemical efficiency (quantum yield) of PS II, expressed as the ratio Fv\/Fm with Fv\u00a0=\u00a0Fm\u00a0\u2212\u00a0Fo; the coefficient of photochemical quenching, qP, given by (Fm\u2032\u00a0\u2212\u00a0Fs)\/Fv\u2032 with Fv\u2032\u00a0=\u00a0Fm\u2032\u00a0\u2212\u00a0Fo\u2032; the efficiency of excitation energy capture by open PS II reaction centers, Fv\u2032\/Fm\u2032; and non-photochemical quenching, qN, given by 1\u00a0\u2212\u00a0(Fm\u2032\u00a0\u2212\u00a0Fo\u2032)\/(Fm\u00a0\u2212\u00a0Fo) (Walters and Horton 1991).\nThe fast Chl-a fluorescence rise was measured with the Plant Efficiency Analyser (PEA, Hansatech Ltd, Norfolk, UK). Leaf samples were excited with a saturating pulse of red light (peak at 650\u00a0nm, 3,500\u00a0\u03bcmol\u00a0m\u22122\u00a0s\u22121) and the fluorescence signal recorded at an acquisition interval of 10\u00a0\u03bcs in the first 2\u00a0ms of excitation (for more details on this instrument see Strasser et\u00a0al. (1995, 2000): a model describing discrete steps of excitation energy and electron transfer associated with PS II was published by Zhu et\u00a0al. (2005). Due to the limiting response time of the fluorescence detector in the time region below 50\u00a0\u03bcs, the Fo level was taken as the model-calculated (see below) fluorescence level at 10\u00a0\u03bcs. This procedure gave similar results to the method of extrapolation as proposed by Vredenberg (2000). The fast Chl-a fluorescence rise is plotted in a log-time scale either as the absolute measured values or as the relative variable fluorescence (rFv), calculated as (Fm\u00a0\u2212\u00a0Ft)\/(Fm\u00a0\u2212\u00a0Fo), with Ft as the fluorescence level at any time.\nModeling and interpretation of fast Chlorophyll a fluorescence rise\nThe fast Chl-a fluorescence rise curve was modeled according to Vredenberg et\u00a0al. (2001). The mathematical formulation is based on a three-state model of energy trapping and fluorescence (Vredenberg 2000) and equates the polyphasic fluorescence rise upon a saturating pulse of light to a multi-state transfer pattern of the PS II reaction center.\nResults and discussion\nExposure of the leaves to UV-B radiation caused an increase of Fo and a decrease of Fm (Fig.\u00a01B), resulting in a decrease of the Fv\/Fm value (Fig.\u00a01A). Using a lower intensity of UV-B radiation on grape leaves, Pf\u00fcndel (2003) also found a decrease of Fm. However, in his experiments, the Fo first decreased with time, and only increased after about 3\u00a0h. The increase of the Fo indicates an increase in the amount of QB-nonreducing centers; the decrease of the Fv\/Fm value indicates that UV-B has a strong damaging effect on the functioning of PS II.\nFig.\u00a01Effect of UV-B radiation of variable duration on Fo, Fm, and the Fv\/Fm ratio in leaf discs. The UV-B radiation level was 720 (\u25a0), or 150 (\u25ef)\u00a0\u03bcmol\u00a0m\u22122\u00a0s\u22121, respectively (PAR level was zero); control samples were kept in darkness. Each value is the mean of four measurements and the bars indicate \u00b1S.D.; bars not shown are within the symbols\nIllumination with white light during 6\u00a0h decreased the photochemical quenching qP from 1.0 at the lowest intensity to a value of 0.58 at 656\u00a0\u03bcmol\u00a0m\u22122\u00a0s\u22121 (Fig.\u00a02). Curwiel et\u00a0al. (1993), using the same plant material, reported a decrease to about 0.80 after illumination at about 500\u00a0\u03bcmol\u00a0m\u22122\u00a0s\u22121. Up to 100\u00a0\u03bcmol\u00a0m\u22122\u00a0s\u22121, irradiation with UV-B has a stronger effect on qP than white light; at 200\u00a0\u03bcmol\u00a0m\u22122\u00a0s\u22121 and higher, the effect of UV-B is smaller. The efficiency of excitation energy capture by open PS II reaction centers, Fv\u2032\/Fm\u2032, was already decreased strongly after 6\u00a0h at 5.4\u00a0\u03bcmol\u00a0m\u22122\u00a0s\u22121 of UV-B, while the effect of white light is much smaller; comparable results were found for the quantum yield of PS II given by (Fm\u2032\u00a0\u2212\u00a0Fs)\/Fm\u2032. The non-photochemical qN increased from 0 at the lowest intensities to 0.51 (white light) and 0.41 (UV-B) at the highest irradiation levels. Curwiel et\u00a0al. (1993) reported a value of about 0.6 after illumination with white light at about 550\u00a0\u03bcmol\u00a0m\u22122\u00a0s\u22121.\nFig.\u00a02Effects of 6\u00a0h irradiation, at various levels, of detached leaves on some fluorescence parameters. CO is control; control is white light (PAR); UV-B is given by the Vilber lamp (zero PAR). Each value is the average of 4 measurements and the bars indicate \u00b1S.D.; bars not shown are within the symbols\nMeasurements of Chl-a fluorescence transients of intact leaves with the PEA fluorometer are presented in Fig.\u00a03. In Fig.\u00a03A the effects of 0, 1, 3, and 6\u00a0h irradiation with the Vilber UV-B lamp at 150\u00a0\u03bcmol\u00a0m\u22122\u00a0s\u22121 are illustrated. Also in these measurements, the most obvious effects of UV-B are an increase of the Fo level and a decrease in Fm. After infiltration with water (plus 0.5% ethanol) the effects of UV-B appear the same (compare in Fig.\u00a03B, \u25a1 with \u25a0). Compared with infiltration with water, infiltration with DCMU (\u25cf) causes a much higher Fo level, and a faster increase to the J-P level; irradiation with UV-B after DCMU-infiltration (\u25ef) causes a still higher Fo level and a slower increase to the J-P level. Comparable results were found using another PS II-inhibiting herbicide, atrazine (not shown). The higher Fo level indicates an increased amount of QB-nonreducing centers. Apart from the Fo level, the differences between the curves before and after UV-B treatment are much smaller in the presence of DCMU. This may be related with the finding by Jansen et\u00a0al. (1993) that PS II-inhibitors like DCMU, which replace QB from its niche on the D1 protein, inhibit UV-B-driven D1 degradation.\nFig.\u00a03Fluorescence induction curves after various treatments of leaves. (A) Fluorescence transients after 0, 60, 180, and 360\u00a0min irradiation with UV-B light at 150\u00a0\u03bcmol\u00a0m\u22122\u00a0s\u22121. ( PAR is zero). (B) \u25a0, After infiltration with 0.5% ethanol (CO is control), no UV-B; \u25cf, after infiltration with 10\u22125\u00a0M DCMU in 0.5% ethanol, no UV-B; \u25a1, after infiltration with 0.5% ethanol and 6\u00a0h irradiation with 150\u00a0\u03bcmol\u00a0m\u22122\u00a0s\u22121 UV-B light; \u25ef, after infiltration with 10\u22125\u00a0M DCMU in 0.5% ethanol and 6\u00a0h irradiation with UV-B. Each curve is the average of three measurements\nTo study the kinetics of the various parts of the transients, induction curves were normalized (Fig.\u00a04). While the level of the J transient increases a little after 60\u00a0min UV-B, it decreases clearly after 180 and 360\u00a0min UV-B; the kinetics of the O-J increase become lower after UV-B irradiation. In all cases the level of the I transient decreases and the kinetics of the J-(I)P rise decreases. From curves like those in Fig.\u00a04, rate constants of reactions involved in the photochemical trapping in the reaction center of PS II were calculated using the Three-State Trapping Model of PS II (Vredenberg 2000; Vredenberg et\u00a0al. 2001). The rate constants for QA\u2212 oxidation by QB (kAB1) decreased very fast, both at 150 and 720\u00a0\u03bcmol\u00a0m\u22122\u00a0s\u22121; it is down to about 50% of control already after 15\u00a0min UV-B irradiation (Fig.\u00a05A). The rate constant for QA\u2212 oxidation by QB\u2212 (kAB2) was down to 5% of control, already after 15\u00a0min UV-B (not shown). This indicates that UV-B irradiation has a fast action on the reducing side of PS II. The rate constants for Yz+ reduction by the OEC in S1 and S2 (k1 and k2 in Fig.\u00a05B and C, respectively), were also affected, but much slower. From this we conclude that the damage by UV-B starts at the reducing side, and affects the donor side later. In Fig.\u00a05D it is illustrated that kL (excitation rate) increases by UV-B irradiation. This indicates that UV-B radiation decreases the antenna size, or that a lower number of open reaction centers is available after UV-B treatment.\nFig.\u00a04Relative variable fluorescence measured after illumination of leaves during various times (T, in min) with UV-B light at 150 (A) or 720\u00a0\u03bcmol\u00a0m\u22122\u00a0s\u22121 (B); PAR is zero. CO is control. Each curve is the average of three (A) or four (B) measurementsFig.\u00a05Rate constants of some reactions involved in the photochemical trapping in the reaction center of PS II, calculated using the Three-State Trapping Model of PS II (Vredenberg 2000). Curves like those presented in Fig.\u00a05 were taken as a source for the calculations; these curves were measured after various times of irradiation with the Vilber lamp at 150\u00a0\u03bcmol\u00a0m\u22122\u00a0s\u22121 (\u25cb) or 720\u00a0\u03bcmol\u00a0m\u22122\u00a0s\u22121 (\u25a0); PAR was zero. Average of four measurements and the bars indicate \u00b1S.D.; bars not shown are within the symbols\nAs may be concluded from the results presented in Fig.\u00a05, UV-B radiation affects rate constants of reactions at both the acceptor and donor sides of PS II. With respect to the acceptor side, Rodrigues et\u00a0al. (2006) reported that QA\u2212 is a photosensitizer for UV-B radiation; absorption of UV-B by this semiquinone radical initiates reactions leading to damage to PS II. The group of Vass, most often using isolated plant preparations, has reported several times on the effect of UV-B radiation on the donor side of PS II. In a recent paper (Szilard et\u00a0al. 2006), they describe a model where UV-B-induced inhibition of water oxidation is caused either by direct absorption within the catalytic manganese cluster or by damaging intermediates of the water oxidation process.\nVass et\u00a0al. (1996) measured the effects of UV-B radiation on the OEC, the QAFe2+ complex, the redox-active tyrosines, and the D1 protein in isolated spinach PS II membrane particles. While all these functions were affected, the OEC appeared to be the most sensitive; the authors concluded that the primary damage by UV-B occurs at the OEC, and that modification and\/or inactivation of tyrosine and the quinone acceptor complex are subsequent events. Our work confirms that UV-B radiation affects both acceptor and donor sides of PS II. However, our analysis leads to the conclusion that the acceptor side of PS II is affected first and the donor side later. This different conclusion may be caused by the different material and methods of the two works. Vass et\u00a0al. (1996) used PS II particle membranes, while in our work intact leaves were measured. Furthermore, Vass et\u00a0al. (1996) measured the effect of UV-B separately on the various PS II functions, while we calculated the effects of UV-B from one single measurement.","keyphrases":["photosystem ii","uv-b radiation","chlorophyll a fluorescence","chenopodium album","photodamage","photosynthesis"],"prmu":["P","P","P","P","U","U"]}
{"id":"Tuberculosis_(Edinb)-1-5-1913930","title":"The role of GlnD in ammonia assimilation in Mycobacterium tuberculosis\n","text":"Summary\nIntroduction\nMycobacterium tuberculosis is responsible for the largest number of human deaths from a single bacterial agent. Nearly two million people die from tuberculosis each year and more than eight million are newly infected.1 A better understanding of the basic metabolism of this pathogen could lead to new strategies for eradication. Although progress has been made in understanding some of the nutritional requirements of this organism both in vitro and in vivo, in particular its carbon source acquisition,2,3 little is known about nitrogen metabolism.\nM. tuberculosis has four enzymes with glutamine synthetase (GS) activity (GlnA1-4).4,5 Of these, GlnA1, GlnA3 and GlnA4 synthesise l-glutamine, whereas GlnA2 synthesises the d-glutamine and d-isoglutamine required for cell wall biosynthesis.5 The major GS, GlnA1, is expressed to a high level and is exported.6 The role of the secreted enzyme is not clear, but it has been suggested that it may play a role in pH modulation, although the biosynthetic reaction does require a source of ATP.\nGS catalyses the production of glutamine from glutamate and ammonia. Since this reaction requires ATP, it needs to be strictly regulated in the presence of excess ammonia to conserve both energy and glutamate pools in the cell. GS activity can be controlled by several mechanisms, including feedback inhibition, transcriptional control of gene expression and by post-translational modification. In Escherichia coli a regulatory cascade of three proteins, GlnD, PII and GlnE, is involved in the latter mechanism (Fig. 1). GlnD is a uridylyl transferase which modifies the PII protein. PII in turn controls the activity of GlnE. GlnE is an adenylyl transferase which controls the interconversion of GS and GS-AMP. Transfer of the AMP moiety to the GS enzyme reduces its glutamine synthetic activity. GlnE is also able to deadenylylate GS with the predominant reaction being determined by interaction with the PII protein; PII promotes the adenylylation reaction, whereas PII-UMP promotes the deadenylylation reaction. In this way the cells can rapidly control GS biosynthetic activity in response to ammonia availability. An M. tuberculosis GlnA1 mutant is auxotrophic for glutamine, and attenuated in macrophages and guinea pigs,7 suggesting that the assimilation of ammonia via this pathway is required in vivo.\nWe have previously shown that, in contrast to other bacteria including closely related organisms such as Streptomyces coelicolor, GlnE is an essential gene in M. tuberculosis.8 Thus the control of GS activity seems critical to normal growth. Here we show that glnD is not essential and that although it has an effect on GS activity in the cells, it is not required for virulence.\nMaterials and methods\nCulture\nM. tuberculosis H37Rv (ATCC25618) was grown in Middlebrook 7H9 plus 10% v\/v OADC supplement (Becton Dickinson) and 0.05% w\/v Tween 80, Middlebrook 7H10 agar with 10% v\/v OADC supplement or TSM media (1.5\u00a0g\/L K2HPO4, 0.5\u00a0g\/L KH2PO4, 0.5\u00a0g\/L MgSO4, 0.5\u00a0mg\/L CaCl2, 0.1\u00a0mg\/L ZnSO4, 0.1\u00a0mg\/L CuSO4 and 50\u00a0mg\/L ferric chloride) supplemented with 10% v\/v OADC and 0.05% w\/v Tween 80.9 Working pH 7.2. For TSM-high ammonia, 30\u00a0mM (NH4)2SO4 was added; for TSM-low ammonia, 0.1\u00a0mM (NH4)2SO4 was added, l-amino acids (alanine asparagine, glutamine and glutamate) were added to 3\u00a0mM. Growth curves were obtained in 12\u00a0mm diameter borosilicate tubes with 4\u20135\u00a0ml media and stirring at 250\u00a0rpm with an 8\u00a0mm flea. Hygromycin was used at 100\u00a0\u03bcg\/ml and kanamycin at 20\u00a0\u03bcg\/ml.\nQuantitative RT-PCR\nProbes and primers were designed for quantitative PCR for sigA (endogenous control), amt, glnB and glnD using the software Primer Express (Table 1). cDNA was synthesised from RNA using RT and random hexamer primers using AMV reverse transcriptase. PCR was carried out in a Taqman 7900 using a standard PCR master mix. For sigA, amt, glnB and glnD, the primer pairs were SigA-R and SigA-F, Amt-R and Amt-F, GlnB-R and GlnB-F, and GlnD-F and GlnD-R, respectively, and the probes used were SigA-T, Amt-T, GlnB-T and GlnD-T. The primer and probe concentrations were first optimised. The optimal primer concentration was 300\u00a0nM for all four genes, the probe concentration was 100\u00a0nM for sigA and glnB, 125\u00a0nm for glnD and 200\u00a0nm for amt. In order to measure relative gene expression levels, standard curves for each primer-probe set were generated using genomic DNA. CT values were converted into the equivalent of ng using the standard curve. Control reactions without RT were used to confirm that there was no significant contaminating genomic DNA present. CT values for genomic DNA were converted to ng and subtracted from the plus RT values. In order to standardise the samples to ensure that equal amounts of cDNA were used, each value was standardised to sigA to generate unit-less values. At least three independent RNA samples were assayed in triplicate for each gene.\nConstruction of glnD mutant\nWe used our previous method for generating delivery vectors with a marker cassette.10 The delivery vector was constructed by amplifying two regions flanking glnD such that an in-frame deletion was engineered and cloning them into p1NIL. Primer pairs gap7 CACAACGGATACCACAAC and gap8 CGTCAATGCTGTTGCTGC, and gap5 CAAGACCTGGGGAGACGC and gap6 CAGTTTGTCGGTGCCCTC were used to amplify the upstream and downstream regions and the PCR products were cloned into pGEM EasyT (Promega). The two regions were then excised as KpnI-EcoRI and EcoRI\u2013HindIII fragments respectively and cloned into the KpnI-HindIII sites of p1NIL thereby deleting 1.7\u00a0kbp of the glnD gene. The marker cassette from pGOAL19 (hyg, lacZ, sacB) was introduced as a PacI fragment to generate the final delivery vector pKOD3.\nA deletion mutant was constructed according to our previous method.10 Briefly, we treated the vector DNA with UV and electroporated M. tuberculosis to generate a single cross-over strain.11 This strain was streaked out without antibiotics and double cross-overs selected and screened on 2% w\/v sucrose and 50\u00a0\u03bcg\/ml X-gal. White colonies were patch tested for hygromycin and kanamycin sensitivity and then screened by PCR for the deletion gene. Potential mutants were confirmed by Southern blotting using XhoI digested genomic DNA and hybridising to a probe derived from the upstream flanking region.\nGS assays\nCell-free extracts were generated using the MiniBeadBeater.12 Culture filtrates were prepared using 0.2\u00a0\u03bcM filters and the filtrates were concentrated using Centricon 20 units (Amicon). Total GS activity was assayed using the transferase assay.13 GS activity is given in nmol per minute per mg of total protein.\nVirulence assays\nTHP-1 cells were maintained in culture, treated with PMA to induce differentiation, washed and then infected as described.14 A total of 5\u00d7105 macrophages were infected at MOIs of 1:50 and 1:5 bacteria to macrophage. Extra-cellular bacteria were removed by washing several times. Determination of the initial inoculum was assessed by plating serial dilutions and the number of intra-cellular bacteria was monitored over 7 days. Mice were infected with approximately 106 viable mycobacteria in 200\u00a0\u03bcl of pyrogen-free saline via a lateral tail vein. Where appropriate, infected mice were killed by cervical dislocation in accordance with humane endpoint protocols under the Animals Scientific Procedures Act, 1986 (UK).\nResults\nExpression of glnD and nitrogen regulation\nWe are interested in the regulatory cascade that controls the activity of GS by post-translational modification. We previously demonstrated that GlnE, an adenylyl transferase which modifies GS, is essential in M. tuberculosis.8,15 The activity of GlnE is modified by the PII protein (encoded by GlnK or GlnB) and PII is controlled by the GlnD protein (Fig. 1). In order to gain a better understanding of this pathway, we extended our work to look at the other members of the cascade.\nThe glnD gene is arranged in an apparent operon with two other genes, amt and glnB (Fig. 3A). The start and stop codons of amt and glnB overlap, but there is a 60\u00a0bp gap between glnB and glnD which could theoretically contain a promoter. We wanted to determine if the genes in this region are controlled by ammonia availability. We used RT-quantitative PCR to look at the expression levels of the three genes relative to sigA (Fig. 2). Amt and glnB were up-regulated three-fold and glnD was up-regulated two-fold in low-ammonia medium as compared to high-ammonia medium. The lesser induction of glnD is not unexpected as it is often seen with genes that are at the 3\u2032 end of the operon. Alternatively, glnD could be independently expressed from a promoter located in the intergenic region.\nConstruction of glnD mutant\nIn order to characterise the role of GlnD in the regulatory cascade which controls GS activity, we constructed a deletion mutant. An in-frame deletion of the gene was made in the vector p1NIL and the gene cassette from pGOAL19 containing the hyg, lacZ, sacB genes was inserted. A two-step homologous recombination process was used to generate the mutant.10 The in-frame deletion and expected genotype was confirmed by Southern blotting (Fig. 3). Out of 32 double cross-overs screened, 12 were mutants. One strain (Tame 69) was selected for further study.\nWe analysed the ability of the glnD\u0394 strain to grow in various nitrogen sources, since it has been shown that mutations in the control of GS activity can lead to deleterious effects on growth. We measured growth of the deletion mutant Tame 69 with various nitrogen sources. The mutant was able to use all the nitrogen sources and there was no difference in the growth rates from the wild-type strain (data not shown).9 Thus the mutant is not compromised in its ability to utilise any of these substrates.\nControl of total GS activity\nIn other bacteria, GlnD is involved in the post-translational modification cascade which ultimately controls GS activity by modulating its adenylylation state. It has also been shown to play a role in regulation of nitrogen-controlled genes in Corynebacteria.16 Therefore, we investigated total GS activity of the mutant grown with ammonia or glutamate as nitrogen sources (Fig. 4) to determine if deletion of glnD affected the activity of GS. The majority of GS is found in the culture filtrate in M. tuberculosis6 and our data confirmed that we had higher activity in this fraction than in the cell-free extracts for the wild-type strain. Interestingly, we found that total GS activity was higher in ammonia-rich conditions (30\u00a0mM ammonium sulphate) as compared to either low ammonia (0.1\u00a0mM) or 7H9 (glutamate) in the wild-type strain. This was in agreement with RT-qPCR data for glnA1 which showed increased expression in high-ammonia medium (Pashley and Parish, unpublished data), although glnA2 expression was unchanged.9 There was no significant difference in the total GS activity between the mutant and wild-type in the cell-free extracts. However, in culture filtrates the situation was very different. We found a large reduction in total GS activity in the mutant strain grown in all media except low ammonia (where the level of activity was at its lowest). This was particularly pronounced in ammonia-rich conditions, where the wild-type activity was at its highest.\nVirulence in macrophages and SCID mice\nM. tuberculosis glnA1 mutants are glutamine auxotrophs and showed a reduced ability to multiply in macrophages and guinea pigs.7 Since extra-cellular GS activity was reduced in the glnD mutant, we determined whether GlnD played any role in intra-cellular survival. We used two measures of this: (1) the ability of the mutant to grow in macrophages and (2) its ability to cause disease in mice. For the macrophage infection assay we used the human macrophage-like THP-1 cell line. Infections were carried out at two different MOI (Fig. 5). The results showed that there was no difference between the wild-type and mutant strain, indicating that disruption of GlnD function had no effect on intra-cellular survival. We also tested virulence in the SCID mouse model to detect any strong attenuation profile (Fig. 5). Again, the mutant strain behaved just as the wild-type and was not attenuated.\nDiscussion\nWe have shown that expression of glnB and amt is controlled in response to ammonia levels. The up-regulation of these genes in low-ammonia conditions is in agreement with previous findings in other related bacteria. For example, in Corynebacterium glutamicum, these genes are transcribed as an operon under the control of nitrogen availability; the operon is switched off in nitrogen-rich media and is turned on during nitrogen starvation.17,18\nOur previous results indicate that at least one member of the GS regulatory cascade is essential, as we have been unable to construct GlnE mutants in M. tuberculosis.8,15 Our current hypothesis is that GlnE is required to inactivate GlnA1 because the latter is expressed at such a high level. If it were all enzymatically active, the intra-cellular levels of glutamate and\/or ATP would quickly be depleted. In agreement with this we have shown that the adenylylation function is essential, but the deadenylylation function is not (Pashley et al., unpublished). Deletion of GlnD would result in a lack of uridylyation of the PII protein. Since unmodified PII promotes the adenylylation activity of GlnE, inactivation of GS should still occur in the mutant and therefore deletion of GlnD is possible. However, our hypothesis predicts that PII is likely to be essential since it directly modulates GlnE activity. This concurs with our preliminary evidence that we are unable to make a glnB deletion mutant, in contrast to the relative ease of constructing a glnD mutant (Pashley and Parish, unpublished data).\nAlthough the glnD mutant was viable, there were substantial changes in the level of GS activity. Normally, a large amount of GS is exported from the cell during growth.6 Export of GS has been linked to high levels of expression, rather than any specific export mechanism.19 If glnD deletion leads to reduced expression of glnA1, this would explain why only extra-cellular levels are depleted. The observation that total GS activity in the culture filtrates was markedly reduced in the glnD mutant is an intriguing one. It is possible that overall expression of GlnA1 is markedly reduced and therefore little of it will be exported to the outside. Alternatively, GlnA1 production could be completely abolished. The enzyme assay we used does not distinguish between the four M. tuberculosis GS enzymes, so GS activity measured could arise from GlnA2-4. However, this seems unlikely, since it has already been demonstrated that deletion of the glnA1 gene leads to glutamine auxotrophy, implying that all the biosynthetic activity in the cell is GlnA1.7\nIt is likely that the GlnD regulatory cascade only controls activity of GlnA1, although we cannot rule out the possibility that GlnA3 activity would also be modulated. However, GlnA2 and GlnA4 will not be controlled by this cascade (since they cannot be modified by GlnE). Thus GS activity arising from enzymes other than GlnA1 cannot be rule out. Further work to determine the levels of expression of each GS would help to further elucidate the mechanism of control of GS activity in the whole cell. However, the fact that GlnA1 is the major GS in M. tuberculosis confirms the importance of this post-transcriptional regulatory control system.\nAlthough extra-cellular GS was markedly reduced in the glnD mutant, the strain was still fully virulent in macrophages and SCID mice. This was a surprising observation, since it has already been demonstrated that GlnA1 is required for virulence.7 Our data indicate that GS is not required at high levels for virulence in the SCID mouse and that the presence of the lower level of intra-cellular GS is sufficient for bacterial survival. It is possible that a defect may be seen in the glnD mutant under conditions in which intra-cellular multiplication is seen or in activated, rather than resting macrophages. However, since we have not measured extra-cellular GS directly during infection, we cannot exclude the possibility that extra-cellular GlnA1 is found in the glnD mutant in vivo. Therefore, the attenuation of GlnA1 mutants most likely results from their auxotrophic nature and the lack of availability of glutamine in the host environment.\nIn conclusion, we have shown that glnD is not an essential gene, but that it is required for the normal expression and activity of GS in culture filtrates of M. tuberculosis. Future work to address the question of how deletion of glnD results in altered expression of GS by determining the mechanism of regulation for ammonia-regulated genes is underway.","keyphrases":["nitrogen metabolism","glutamine synthetase","gene regulation"],"prmu":["P","P","R"]}
{"id":"Ann_Biomed_Eng-2-2-1705544","title":"Proteoglycan Breakdown of Meniscal Explants Following Dynamic Compression Using a Novel Bioreactor\n","text":"Motivated by our interest in examining meniscal mechanotransduction processes, we report on the validation of a new tissue engineering bioreactor. This paper describes the design and performance capabilities of a tissue engineering bioreactor for cyclic compression of meniscal explants. We showed that the system maintains a tissue culture environment equivalent to that provided by conventional incubators and that its strain output was uniform and reproducible. The system incorporates a linear actuator and load cell aligned together in a frame that is contained within an incubator and allows for large loads and small displacements. A plunger with six Teflon-filled Delrin compression rods is attached to the actuator compressing up to six tissue explants simultaneously and with even pressure. The bioreactor system was used to study proteoglycan (PG) breakdown in porcine meniscal explants following various input loading tests (0\u201320% strain, 0\u20130.1 MPa). The greatest PG breakdown was measured following 20% compressive strain. These strain and stress levels have been shown to correspond to partial meniscectomy. Thus, these data suggest that removing 30\u201360% of meniscal tissue will result in the breakdown of meniscal tissue proteoglycans.\nINTRODUCTION\nMechanical loading of the meniscus plays a crucial role in the metabolic activity of fibrochondrocytes.7,11,12,15 Previous studies have demonstrated that increased load on meniscal tissue leads to an increase in proteoglycan and collagen levels,16 whereas unloading of meniscal tissue causes a decrease in aggrecan expression, collagen formation and cell growth.2,3 It is not fully understood how biomechanical and biochemical events interact to produce changes in the extracellular matrix. This lack in knowledge is, in part, due to the difficulties associated with performing real time meniscal loading experiments in vivo.\nRecreating the physiological forces in vitro using tissue explants while measuring the biological response provides one method for observing the effect of mechanical stress on the meniscus;9,15 however, the majority of commercially available bioreactors may not be suitable for application to meniscal loading studies. The Biopress system (The Biopress system, Flexcell International, Hillsborough, NC, USA) uses air pressure applied to a flexible bottom under each well. The Biopress has been used to apply pressures of 0.1\u00a0MPa in previous studies on meniscal explants,7,15 noting strain levels of approximately 10%. Because pressures up to 10\u00a0MPa and strains ranging from 2% to 20% are seen in the meniscus13,18 this system may not generate high enough pressures to mimic the full range of meniscal strains thought to occur during active loading of healthy and damaged tissue in vivo. Stresses and strains are approximately 5\u201310%, and 3\u00a0MPa, respectively, in the normal healthy meniscus, but these strain levels increase with a partial meniscectomy to approximately 20% and 8\u00a0MPa.18 Another biaxial tissue-loading device, previously used to compress articular cartilage explants, is able to create a maximum 400\u00a0N axial force on as many as 12 explants at once, however, is limited to 100\u00a0\u03bcm of motion.8\nPrevious studies have shown that following knee trauma, such as ACL transection, prior to any articular cartilage damage, there are signs of meniscal tissue degeneration.10 We have previously shown that following partial meniscectomy, the remaining meniscal tissue is subjected to an altered loading state.18 This altered loading state may cause a direct breakdown of matrix components such as proteoglycans (PG), or indirectly affect matrix production by induction of catabolic biomolecules.\nThe objectives of this study were (1) to design and build a practical, cost-effective device for applying homogeneous strains to tissue explants, and (2) to utilize the system to overload and underload meniscal tissue and measure the biochemical output. In this communication, we show that the ensuing device is a simple biocompatible design that applies accurate and reproducible strains and is made of components that can be sterilized. We utilized the bioreactor to apply both load and displacement controlled dynamic compression tests. Dynamic strain compression tests showed PG breakdown following overloading of meniscal tissue. No significant changes in concentration of PG released to the conditioned media was measured following various levels of dynamic compressive stress. A comparison between stress controlled dynamic compression versus strain controlled dynamic compression was also made.\nMATERIALS AND METHODS\nDesign of Bioreactor\nThe frame consists of two 2.54\u00a0cm thick parallel aluminum plates separated by 2.54\u00a0cm diameter aluminum support rods (Fig.\u00a01). Centered on the bottom plate is a load cell that is attached to a six well dish. The system is driven by a belt-driven linear actuator utilizing a control package by Animatics (Smartmotor 1720, Ultramotion, Mattituck, NY, USA). The actuator has a maximum stroke length of 5\u00a0cm and can thrust to 2225\u00a0N. It also has a maximum speed of 50\u00a0cm\/s with bi-directional repeatability of \u00b10.00762\u00a0mm and a unidirectional repeatability of \u00b10.00254\u00a0mm. Motor control was achieved by using the SmartMotor Interface (SMI). This language allows the motion of the actuator to be controlled by the signal generated by the load cell or by the displacement of the actuator. Displacement resolution for the actuator is 0.4\u00a0\u03bcm.\nFIGURE\u00a01.A 2-dimensional view of the assembly of the plunger, dish, and cap. The test frame is composed of two aluminum plates supported by aluminum rods. The actuator is positioned in a centered hole in the top plate and tighten into alignment with an adjustable collar.\nA strain gage load cell (Model 1210AF, Interface, Scottsdale, AZ) with a load capacity of 1334\u00a0N (sensitivity of \u223c1.3\u00a0N) was utilized. For tests that require loads near or above 1334\u00a0N, a dimensionally identical load cell with 8896\u00a0N (sensitivity \u223c2.17\u00a0N) (Interface, Scottsdale, AZ, USA) capacity is interchangeable with the current load cell. This design feature of interchangeable load cells allows for a larger range of loads to be accurately measured. A 2100 series signal conditioner (Vishay Instruments, Raleigh, NC, USA) amplifies the load cell signal to produce a 5\u00a0V signal at the maximum load.\nThe load cell is centered on a 2.54\u00a0cm thick aluminum plate that is the base of the system frame. A threaded stud leaving the load cell connects to the aluminum dish via a quick disconnect pin. The dish has six 10\u00a0mm deep wells equally spaced in a circular orientation. Teflon-filled Delrin compression rods (diameter\u00a0=\u00a08\u00a0mm) for each well are press fit into a plunger which attaches to the actuator via a quick disconnect pin. To ensure only one plunger\/dish orientation and to keep the compression rods centered in each well, the plunger also features two press-fit aluminum pins that slide into matching holes in the dish. An aluminum cap rests on the shoulder of the dish and houses a linear bearing that is press fit into the cap. Along the resting edge of the cap, four shallow grooves were machined to allow carbon dioxide supply to the explants during testing. The linear bearing allows the plunger to move up and down within the cap and restricts the plunger to vertical motion (Fig.\u00a02). The entire frame is small enough that it can be placed in a commercially available CO2 incubator to maintain physiological conditions (Model 5015, VWR, West Chester, PA, USA).\nFIGURE\u00a02.A 2-dimensional drawing of the test frame shows the side and top view of the test system. The linear actuator is attached to the plunger using a quick-disconnect pin. The dish is attached to the load cell in the same manner. The cap improves alignment of the plunger by utilizing a linear bearing.\nAccuracy Evaluation of the System\nUltra-low pressure film (Sensor Products Inc., East Hanover, NJ, USA) was used to measure well pressure during compression. First, the repeatability of the pressure film was tested by loading the film (seven times) between flat platens in a tensile testing machine (Model 8872, Instron Corp., Canton, MA, USA) to a 70\u00a0N target load, corresponding to a pressure of 0.477\u00a0MPa for the given indentor size. Calibration of the pressure film was also done using the tensile testing machine and included loading pieces of pressure film ranging from 0.2 to 1.64\u00a0MPa. Pressure film analysis was completed using commercial software (Scion Image, National Institute of Standards and Technology, Gaithersburg, MD, USA) to measure the density of the pressure film samples. Film was compressed between the platens and a piece of rubber similar to the rubber used for testing well pressure.\nTo determine well pressure in the bioreactor, a machined plate was set on top of the dish with a 3\u00a0mm thick piece of uniform rubber. Pressure film was placed on top of the rubber and the plunger was lowered near the surface of the film. Four tests were conducted, each to the same pressure (0.477\u00a0MPa) to determine the repeatability of the bioreactor in load control. The film from the bioreactor was analyzed and density measured to determine the difference between each compression rod. The difference in film density and the maximum percentage error was determined to demonstrate the accuracy of the system.\nDetermination of Displacement Repeatability\nTo further investigate the accuracy of the system, the gap between the bottom of the compression rods and the bottom of the wells was measured while the system was assembled into the bioreactor. This was done by using the actuator to compress commercially available Fibre-Strand body filler (6371, The Matin Senour Comp., Cleveland, OH, USA) until the gap between the bottom of the compression rod and well bottom was filled. The actuator remained at this position until the body filler hardened completely. After plunger removal, a micrometer (2.54\u00a0\u03bcm resolution) was then used to measure the thickness of the body filler. This process was repeated three times with the same plunger and dish orientation.\nCompliance of the System\nThe compliance of the system was determined by placing a flat stainless steel plate over the wells and running a load-deformation test in the absence of menisci. The test was repeated three times and the load-deformation data recorded.\nApplication of the System\nPigs used in the experiment were 18\u00a0weeks old, sacrificed 24\u00a0h after death (received from Mayo Clinic, Rochester, MN, USA). The explants were collected from both the lateral and medial meniscus using sterile technique. The explants were 6\u00a0mm in diameter and cut using a biopsy punch, perpendicular to the superior surface to maximize the amount of superior tissue preserved. To ensure two parallel flat faces on the cylindrical explants, a specialized cutting device was utilized. Explants were held such that a fixture containing two razorblades, 5\u00a0mm apart, cut the tissue perpendicular to the long axis of the cylindrical explant. This ensured that each explant was the same height with parallel faces (Fig.\u00a03a).\nFIGURE\u00a03.a \u2013 Meniscus following biopsy, b \u2013 Maximum axial strain following different partial meniscectomy simulated by FE analysis. Various percentages (5%, 10%, 30% and 60%) removed from different portion of the medial meniscus (A \u2013 anterior, C \u2013 central, P \u2013 posterior). Knee loaded with 1200\u00a0N axial force.\nExplants were incubated at 37\u00b0C (5% CO2) for 48\u00a0h in growth media (89% DMEM\/F-12, 10% FBS, 1% Penn\/Strep) which was changed after 24\u00a0h. For mechanical testing, the explants were placed in the six well bioreactor filled with 400\u00a0\u03bcl of the test media (97% DMEM\/F-12, 2% FBS, 1% Penn\/Strep). The explants were loaded for 2\u00a0h at 1\u00a0Hz, at one of the following levels: 5%, 10%, 15% or 20% strain, 0.05 or 0.1\u00a0MPa unconfined dynamic compression. Each loading group consisted of six explants taken from six different animals (except 15% test \u2013 4 animals). Control explants were placed in wells, but not exposed to compression. Previous experiments (FE analyses) showed that under two times body weight the intact knee meniscus experiences about 10% maximum compressive strain. Removal of 5\u201310% of the meniscus minimally affects the maximum strain level, however, removal of 30\u201360% of the meniscal body increases the maximum strain to 15% or greater. Generally speaking, 0% strain is likely underloading the tissue, 5\u201310% is approximately physiological and 15\u201320% is considered overloading18 (Fig.\u00a03b). Following compression the explants were bisected into superior and deep zone, by cutting them in half (Fig.\u00a04), weighted and placed in 24-wells plates, in 1\u00a0ml of fresh test media. Samples were post-incubated at 37\u00b0C (5% CO2) for 24\u00a0h. Post-incubation media was collected and stored at \u221280\u00b0C for future analysis.FIGURE\u00a04.Cross-section of the meniscus with the direction of the cut and explants showing superior and deep zones.\nThe content of sulfated glycosaminoglycan (GAG) released to the conditioned media was assayed using dimethylmethylene blue (DMMB) dye solution.5,6 The standard curve was generated with a known concentration of shark cartilage chondroitin sulfate C. All samples were run in duplicates. The concentration was normalized to the wet weight of the explant. GAG release was then normalized to the no load control samples for each animal.\nData Analysis\nThe concentration of the GAG released to the media was calculated using a standard curve (R2\u00a0>\u00a00.85). The final value was an average from duplicates. All data is presented as mean\u00a0\u00b1\u00a0standard error. A one way ANOVA followed by Fisher\u2019s PLSD post-hoc testing was used to measure statistical differences (p\u00a0<\u00a00.05 was considered significant). Paired t-tests were used at each stress or strain level to determine differences between superficial and deep zone PG release to the media.\nRESULTS\nAccuracy Evaluation of the System\nThe repeatability test showed an average of 0.4773\u00a0\u00b1\u00a00.0003\u00a0MPa. The pressure film demonstrated equal pressure in each well for each load (Fig.\u00a05). There was less than a 1% error (Table\u00a01).\nFIGURE\u00a05.Pressure film impressions at 0.477\u00a0MPa under the six bioreactor compression rods.TABLE\u00a01.Results of pressure film verification.0.477\u00a0MPaPressure (MPa)Test #Rod 1Rod 2Rod 3Rod 4Rod 5Rod 6AverageStd. Dev.10.47730.47680.47710.47710.47680.47680.47700.000220.47680.47690.47680.47680.47690.47720.47690.000130.47730.47680.47690.47700.47690.47710.47700.000240.47730.47680.47690.47680.47690.47680.47690.0002\nDetermination of Displacement Repeatability\nThe micrometer measurements from the body filler showed that the bioreactor was extremely repeatable. Well 3 had the largest standard deviation in height, 3.4\u00a0\u00b1\u00a00.01\u00a0mm, whereas well 1 had the lowest standard deviation 3.4\u00a0\u00b1\u00a00.0015.\nCompliance of the System\nThe results of the compliance test indicate a linear load-deformation response (R2\u00a0>\u00a00.99). The slope of the load-deformation curve was 3465\u00a0\u00b1\u00a0200\u00a0N\/mm. Thus, for the range of strains and stresses seen in this study, the compliance is negligible.\nApplication of the System\nTo determine an appropriate post incubation time, preliminary tests were run. PG concentration in the media was monitored following 2, 4, and 6\u00a0h and 1, 2, and 3\u00a0days following 10% dynamic compression and for controls samples (Fig.\u00a06). The shortest time showing a strong signal was chosen to minimize the duration of the experiment (1\u00a0day).\nFIGURE\u00a06.PG concentration following different incubation time (n\u00a0=\u00a02 for controls (C), n\u00a0=\u00a03 for 10% compression test (10%)). Data presented is mean\u00a0\u00b1\u00a0standard error.\nThe highest break down of PG was measured following 20% compressive strain for both the superior and deep zones of the meniscal explants (Fig.\u00a07). There were significant differences between release of GAGs into the media for the overloaded condition (20%) versus physiological loading (10%) for both superior and deep zones. No significant differences were found between superficial and deep zones for any compression level. Explants exposed to pressures of either 0.05 or 0.1\u00a0MPa did not show any significance differences in either the superior or deep zone for PG breakdown (Fig.\u00a08).FIGURE\u00a07.PG concentration following different compression tests (n\u00a0=\u00a06 except 15% test (n\u00a0=\u00a04)). Data presented is mean\u00a0\u00b1\u00a0standard error. *Statistically different than 20% (p\u00a0<\u00a00.05).FIGURE\u00a08.PG concentration following different load level compression tests (n\u00a0=\u00a06). Data presented is mean\u00a0\u00b1\u00a0standard error.\nDisplacement controlled tests showed a rapid drop in load within the first 1000 cycles with little change in the following 6200 cycles (Fig.\u00a09). For displacement tests at or below 15% strain, loads settled just below 0.05\u00a0MPa while the 20% strain test remained above 0.1\u00a0MPa throughout the duration of the test. The change in pressure measured from the beginning of the test to the end is shown in Table\u00a02. Load controlled tests (Fig.\u00a010) showed a rapid increase in compressive displacement within the first 2000 cycles. The 0.05\u00a0MPa load level reached maximum displacement near 3000 cycles and remained at that level to the end of the test. The 0.1\u00a0MPa tests reached 18% strain near 3000 cycles but steadily increased to 20.7% strain by the final cycle. The differences in strain from the start to the end of the tests can be found in Table\u00a03.FIGURE\u00a09.Stress vs. time for four representative displacement control tests. Only peak values during each cycle are plotted. Stress was calculated by dividing the peak load by the initial cross-sectional area.Table\u00a02.Change in pressures over the duration of the stress-relaxation tests.StrainPressure (MPa)StartEnd5%0.166\u00a0\u00b1\u00a00.108 *#0.038\u00a0\u00b1\u00a00.010 #10%1.141\u00a0\u00b1\u00a00.103 #0.046\u00a0\u00b1\u00a00.010 #15%2.185\u00a0\u00b1\u00a00.8270.035\u00a0\u00b1\u00a00.026 #20%3.548\u00a0\u00b1\u00a00.4290.128\u00a0\u00b1\u00a00.020Data represents mean\u00a0\u00b1\u00a0standard deviation. n\u00a0=\u00a06 for all groups. * Significantly different than 15% (p\u00a0<\u00a00.05), # significantly different than 20% (p\u00a0<\u00a00.05).FIGURE\u00a010.Strain vs. time for two representative load control tests. Only peak strain values during each loading cycle are plotted. Strain was calculated by dividing the peak displacement by the original height of the explant.Table\u00a03.Change in strains over the duration of the creep tests.Pressure (MPa)Strain (%)StartEnd0.052.6\u00a0\u00b1\u00a00.5311.6\u00a0\u00b1\u00a01.360.13.0\u00a0\u00b1\u00a00.1220.7\u00a0\u00b1\u00a01.45*Data represents mean\u00a0\u00b1\u00a0standard deviation. n\u00a0=\u00a06 for all groups. * Significantly different than 0.05\u00a0MPa (p\u00a0<\u00a00.05).\nDISCUSSION\nThe explant compression system meets the criteria necessary to obtain a realistic representation of physiological forces present in the knee joint. This system is able to apply known pressures to six explants at once, which is important when trying to gather data for hypothesis testing. It is capable of applying physiological and supraphysiological levels of load and displacement, and has the ability to test in load or displacement control. SMI programming allows for flexibility in frequency, duration, amplitude, and waveform. The system is small enough to fit in a standard incubator and is made of materials that can endure autoclaving and alcohol. An important feature to this system is the ability to keep the explants and media sterile from the culture hood to the incubator. The plunger, dish, and cap form an enclosure that allows easy transport between the culture hood and incubator without allowing open air and bacteria to infect the sample. Since the cap incorporates a linear bearing it does not need to be removed for testing. Utilizing the system features and designing the correct protocol will help maintain a physiological loading sterile environment.\nThis bioreactor is capable of creating higher loads and greater displacements than previous systems used for compressing explants.7,8,15 The Biopress system (Flexcell International, Hillsborough, NC, USA) is capable of loads as high as 69\u00a0N, whereas the current systems actuator can thrust to 2225\u00a0N. An advantage of our system over the biaxial tissue-loading device presented in Frank et\u00a0al. (2000), is that it can create displacements over 10\u00a0mm with a resolution of 0.4\u00a0\u03bcm.8 Our actuator also has a bi-directional repeatability of \u00b17.62\u00a0\u03bcm compared to the \u00b125\u00a0\u03bcm used in Sah et\u00a0al. (2003).11 In addition, the present system is capable of 1\u00a0Hz cyclic compression in a sinusoidal wave using displacement or load control. The bioreactor can be used to compress any tissue that fits under an 8\u00a0mm compression rod and in a 10\u00a0mm deep well. All surfaces are machined to a smooth, frictionless finish, to ensure the sample is exposed to pure unconfined compression. Although we believe this bioreactor to be an improvement over others, we realize that there are some limitations that need to be compensated for. For example, this system can only perform unconfined compression currently. However, the system could be modified to run confined compression tests as well by outfitting a new plunger\/well assembly.\nSince the stress tests were conducted at 0.05 and 0.1\u00a0MPa, and resulted in strains between 2.6% and 20%, the compliance of the system was negligible. Similarly, for the 5% strain control tests, the loads were small enough that the compliance of the system was negligible. However, for the 10%, 15% and 20% strain control tests, stresses were between 3.548 and 0.035\u00a0MPa. Thus, at the larger stress levels, the compliance of the system was greater. For instance, during the 10% strain control test, initially stress levels of 1.1\u00a0MPa would lead to approximately 57\u00a0\u03bcm of compliance, and thus, for the first 150 of the 7200\u00a0s test, strains were closer to 8.8% instead of 10%. For the 15% strain test, strains were closer to 13% for the first 200\u00a0s of the test, and as the material relaxed, the last 1\u00a0h and 57\u00a0min of the test were at 15% strain.\nWe hypothesized that PG breakdown (as determined by GAG in the media) would be high for both the underloading and overloading condition. PG breakdown was significantly increased at 20% strain. Meniscal tissue is a mechanically sensitive, and mechanical loading has been shown to regulate gene expression.14 Hence, we expected that loading the tissue below normal physiological levels of magnitude (underloading) would result in PG breakdown. Lack of increased PG breakdown for control samples (underloading) is surprising and needs to be further explored. Perhaps the degraded PG is not being released into the media for the control samples, whereas the overloaded samples have mechanical compression to help move the broken down PG into the media. Future studies will measure PG breakdown in the tissue explants following compression to determine the integrity of the PG within the tissue. These data suggest that removing 30\u201360% of meniscal tissue, and thereby increasing tissue strains over 15% results in an increase in PG breakdown and tissue destruction. Thus, not only does meniscectomy affect the underlying articular cartilage but the remaining meniscal tissue appears to begin to breakdown, possibly leading to a change in meniscal material properties. This data is supported by previous work that showed following an ACL transection, degenerative changes were seen in the meniscus prior to any articular cartilage changes.10\nDiMicco et\u00a0al. showed an increase in GAG release from bovine cartilage that had been exposed to an injurious level single uniaxial, unconfined compression.1 Inhibitors of biosynthesis or degradative enzymes did not affect PG breakdown, suggesting that the breakdown was a mechanical consequence of compression. GAG release 1\u20137\u00a0days post injury was slowed by metalloprotease inhibitors. Shin et\u00a0al.12 showed that dynamic compression (0.1\u00a0MPa for 24\u00a0h at 0.5\u00a0Hz) increased both GAG synthesis as well as release to the media compared to unloaded controls. Our loading scheme was designed to simulate approximate physiological walking conditions (2\u00a0h, 1\u00a0Hz). It is difficult to compare our GAG data to a study of a single injurious insult, or dynamic compression for 24\u00a0h. We created an approximate daily physiological loading environment that might occur during walking, and simulated loads from unloaded to overloaded due to meniscectomy. In the future, we will investigate the mechanisms of GAG release, whether it is mechanical damage or activation of enzymatic activity.\nIt was surprising that explants tested at 0.05\u00a0MPa showed greater PG breakdown than explants tested at 0.1\u00a0MPa for the superficial zone. One possible reason for this result might be related to the cell viability. A compression of 0.1\u00a0MPa may induce more cell death than 0.05\u00a0MPa of compression and hence fewer cells may be available for production of metalloproteases that may contribute to the breakdown of PG. Current studies are underway to document the degree of cell death in the explants.\nIt is interesting to compare the load control results to the displacement control results. Based on Fig.\u00a09, the 20% strain test applied an initially very high load (\u223c3.5\u00a0MPa), but equilibrated at 0.1\u00a0MPa. Similarly, the 0.1\u00a0MPa test reached a steady state of 20% strain (Fig.\u00a010). Hence, we would expect to see similar levels of PG breakdown in the 20% strain test as well as the 0.1\u00a0MPa test. This is also true for the 10% strain test and the 0.05\u00a0MPa stress tests. For example, in the deep zone the 20% strain control test resulted in 3.7\u00a0\u00b1\u00a01.4\u00a0ug\/ml per gram of tissue, whereas the 0.1\u00a0MPa test resulted in 1.9\u00a0\u00b1\u00a00.5\u00a0ug\/ml per gram of tissue. This large difference could be due to either \u201clift-off\u201d (separation between loading platen and sample during a 1\u00a0Hz test)4 or the initially high stress that was reached initially in the 20% displacement control tests. In contrast to the differences seen above, the 10% strain test and 0.05\u00a0MPa stress test resulted in 1.3\u00a0\u00b1\u00a00.2 and 1.8\u00a0\u00b1\u00a00.5\u00a0ug\/ml per gram of tissue, respectively. Previous 3-D computational studies of an entire human knee joint have shown that mean contact pressures changed from 1.57\u00a0MPa for an intact healthy meniscus to 3.09\u00a0MPa following 60% meniscectomy.18 Maximum contact pressures on the superior surface of the meniscus changed from 4.7 to 7\u00a0MPa when 60% of the meniscus was removed.18 The differences between stress and strain control studies noted above make the data presented difficult to interpret due to the non-physiological nature of in vitro unconfined compression studies. Clearly, this indicates that meniscal tissue may respond differentially to stress versus strain, the duration of strain levels, or most likely the loading history. Before definitive clinical implications can be made regarding the effects of meniscectomy on meniscal tissue, we must first determine if the levels of PG breakdown noted in this study correspond to changes in the load-bearing capacity of the tissue and its function in the knee joint.\nOne of the limitations of this study is lack of investigation of differences between the medial and lateral menisci as well as specific location within the meniscus from which explants were harvested. This likely resulted in higher standard deviations. Future studies with a larger set of animals are proposed in which lateral versus medial, anterior versus posterior and inner radial versus outer radial differences could be studied. Previous researchers have shown a difference between inner and outer radial explants.14 Bisection of explants has previously been shown to release many growth factors.17 Since all samples in this study were bisected, the relative differences found are still significant. It should be noted that the measured response may be due not only to mechanical stimuli but also growth factors released due to cutting the samples. In addition to bisecting the samples, other factors, such as FBS concentration and post-incubation time may effect the absolute concentrations of GAG analyzed in this study. Therefore, only relative comparisons between treatment groups should be considered.\nIn summary, this simple and practical experimental system allows for reproducible application and quantification of homogeneous stresses or strains to explants tissues, thereby providing a systemic and quantitative method for correlating external mechanical stimuli to cellular and molecular mechanisms of mechanotransduction.","keyphrases":["mechanotransduction","meniscectomy","apparatus"],"prmu":["P","P","U"]}
{"id":"Graefes_Arch_Clin_Exp_Ophthalmol-4-1-2292470","title":"Do dissociated or associated phoria predict the comfortable prism?\n","text":"Background Dissociated and associated phoria are measures of latent strabismus under artificial viewing conditions. We examined to what extent dissociated and associated phoria predict the \u201ccomfortable prism\u201d, i.e. the prism that appears most comfortable under natural viewing conditions.\nIntroduction\nSince Ogle\u2019s work [14] dissociated phoria has been distinguished from associated phoria. Dissociated phoria is defined as a deviation from the orthovergence position that occurs when no fusionable contours are provided. Associated phoria is a deviation of the eyes that appears under prism correction of fixation disparity: associated phoria equals the \u201caligning prism\u201d [1] that nullifies fixation disparity. To avoid the technical requirements for measuring the eye position objectively, fixation disparity is commonly determined according to the observer\u2019s directional perception of monocular Nonius lines, embedded in binocular contours. Both dissociated [11, 18] and associated phoria [9, 19] have been recommended as indicators for prismatic correction in the case of asthenopia, but recently associated phoria has been preferred, because the stimulus for associated phoria appears to be more natural, in that both eyes are presented with a nearly identical configuration [1, 4, 5, 16, 20]. Moreover, Yekta et al. [20] found the correlation of asthenopic complaints to be significant only with associated, not with dissociated phoria.\nKromeier et al. [8], however, suggested that dissociated and associated phoria should be similar, provided the accommodative demand in the two procedures is equal. Their argument was based on the consideration that the feedback loop for fusion is opened under both conditions. Concerning dissociated phoria, it is obvious that the feedback loop cannot operate; fusionable contours are absent, hence there is no error signal. Concerning associated phoria, the argument is more complicated because of the fundamental difference between fixation disparity and associated phoria. When the observer looks at a test for fixation disparity, any tendency to deviate from orthoposition is largely kept in check, because the disparity of the binocular contours provides an error signal: the fusional feedback loop is functioning. In the test for associated phoria, however, prisms are added. Prompt and repeated adjustment of the prismatic power nullifies any disparity of fusionable contours. This continuous and artificial resetting of the error signal to zero means that the fusional feedback loop cannot fulfill its purpose, namely stabilisation of a certain vergence angle.\nThe experimental results of Kromeier et al. supported their theoretical consideration. These authors suggested that the discrepancies between dissociated and associated phoria encountered in previous studies [15, 20] might be due to different accommodative stimuli.\nTo clarify this issue, we compared dissociated and associated phoria using the same object for fixation, thus keeping the accommodative demand similar. We further investigated to what extent dissociated and\/or associated phoria predict the \u201ccomfortable prism\u201d. This term defines the prismatic power reached by the observers when they looked at a fully fusionable display, and tuned a pair of counterrotating prisms, called Herschel\u2019s or Risley\u2019s rotary prisms [17], such that the viewing of a fully fusionable object appeared most comfortable. Hence, the comfortable prism anticipated the real-life situation for which prismatic spectacles might be considered.\nMethods\nApparatus\nStimuli were generated by a PowerMacintosh G4 and presented at a distance of 4.0\u00a0m on a 19\u201d Philips GD403 CRT monitor with a resolution of 800\u2009\u00d7\u2009600 pixels. By means of liquid crystal shutter goggles (ELSA 3D Revelator), separate images were presented to the two eyes. The goggles were synchronized to the CRT refresh such that the frames were alternately presented to the right and left eyes. The refresh rate was 120\u00a0Hz, i.e. 60 Hz for each eye, just above the flicker fusion frequency. The phosphor persistence of the monitor, measured with a photoelectric cell, was down to 10% after 4.0\u00a0ms, i.e. within a shorter time than the frame time of 8\u00a0ms. An alternate cover test assured us that this technique provided a complete separation between the images of the two eyes. Herschel\u2019s counterrotating prisms, manufactured by Zeiss, were mounted in front of their right eye (Fig.\u00a01).\nFig.\u00a01Herschel\u2019s prisms mounted in front of the right eye. The observer, looking through shutter goggles, adjusts the prisms by means of a lever arm. A cogwheel belt transmits the prismatic power to a potentiometer\nThese prisms, equipped with a lever arm, allowed a variation of the prismatic power between 30\u00a0cm\/m base in and 30\u00a0cm\/m base out. A calibration performed with a laser beam showed that the readings on the scale of the prisms varied only about 2% around the true inflection. A cogwheel belt connected the prisms with a potentiometer that provided a signal linearly proportional to the prism power. The electric signal was directly transmitted to a computer.\nDuring the trials the room was dark; in the intervals the room was moderately illuminated so that the test person was able to see a coloured print (Henri Matisse \u201cFleurs et c\u00e9ramique\u201d, 35\u2009\u00d7\u200940\u00a0cm, mean luminance 13.8\u00a0cd\/m2) mounted directly above the monitor.\nTest figures\nDissociated phoria\nAs depicted in Fig.\u00a02a, the left eye was presented with a fixation cross, flanked by two O letters (\u201cO X O\u201d). Each of the three letters had a diameter of 10 arcmin and a line thickness of 1.5 arcmin. Above and below the cross were two Nonius lines, 20 arcmin high and 5 arcmin wide. The interval between the lines and the centre of the \u201cO X O\u201d was 10 arcmin. A bright circular background of 40 arcmin radius, embedded in a random dot pattern, surrounded these dark features. The right eye was presented with a bright dot of 4.3 arcmin diameter on a homogenous dark field. The luminance of the dark features was 0.2\u00a0cd\/m2, and that of the bright features 22.6 cd\/m2 (measured through the liquid crystal shutter goggles). The luminance outside the monitor was 0.1\u00a0cd\/m2.\nFig.\u00a02Test figures. a Test for dissociated phoria. All the elements contained in the Mallett figure, including the two Nonius lines, are displayed to the left eye. The right eye is presented with a white dot on a homogenous dark field. b Test for associated phoria, according to Mallett [9]. c Test for the comfortable prism. An identical figure is displayed to both eyes\nAssociated phoria\nAs depicted in Fig.\u00a02b, the configuration was similar to Mallett\u2019s test for fixation disparity [9]. The \u201cO X O\u201d served as a fusional stimulus. Vertical Nonius lines were used as monocular markers, in that the line above the fixation cross was shown to the right eye, and the line below the fixation cross to the left eye.\nComfortable prism\nBoth eyes were presented with all the Mallett elements including both Nonius lines (Fig.\u00a02c).\nProcedure and instructions\nThe observers were seated in a comfortable chair. During the trials, the observers had to rest their chin on a support, to lean their forehead against a bar, and to adjust Herschel\u2019s prisms with the lever arm. Before each trial, the experimenter set the prisms to \u00b10. To limit prism adaptation [12], the time for the adjustment was restricted to 30 seconds. In the interval between the single trials (about 60 to 120 s), the observers were encouraged to move their head freely and to look around in the room or to behold the Matisse painting.\nEach of the three conditions (dissociated phoria, associated phoria, comfortable prism) was tested in a block of eight trials. The conditions were presented in the following order:\nAssociated phoria. If the observers saw an offset between the Nonius lines, they had to align them with the least possible excursion of the lever arm so that they appeared as stable to each other as possible. This instruction resembles the suggestion of Karania and Evens [6] to observe whether one or both of the Nonius lines ever move. If an observer did not see an offset at the start of the trial, or the Nonius lines oscillated around zero, he or she was encouraged to \u201cplay\u201d a little with the lever arm and set Herschel\u2019s prisms such that the Nonius lines became aligned as stably as possible. The observers were repeatedly reminded to look at the centre of the \u201cO X O\u201d, not at the Nonius lines. Further, the observers were instructed to ignore if one Nonius line, or even if both of them seemed to disappear every now and then (fading). Most observers had such an experience. They had to adjust Herschel\u2019s prisms according to the percept when they saw both Nonius lines.Dissociated phoria. The observers were asked to align the white dot with the midline of the \u201cO X O\u201d, neglecting any vertical offset of the white dot. While doing so, they were urged to strictly look at the centre of the \u201cO X O\u201d, not at the white dot.Comfortable prism. The observers were asked to look at the centre of the \u201cO X O\u201d and to set the prisms such that viewing appeared most relaxing. They were encouraged to start with rather bold searching excursions and to refine the tuning gradually during the available 30\u00a0seconds.\nTo get used to the manoeuvring of Herschel\u2019s prisms, the experiment was preceded by a few trials in which the observers practised bringing the white dot to the midline of the \u201cO X O\u201d (test for dissociated phoria) and aligning the Nonius lines to each other (test for associated phoria).\nTo examine the reproducibility, we repeated the whole experiment for all observers in a second session after an interval of 24 to 130\u00a0days.\nObservers\nTwenty observers participated in the study (members of our department or recruits via a public advertisement, aged between 20 and 71\u00a0years, median 26.5\u00a0years). The observers were selected according to the following 4 criteria: (1) Visual acuity of each eye (with full spherical and cylindrical correction) at least 1.0decimal (= 6\/6Snellen), (2) absence of strabismus, ascertained with the unilateral cover test, (3) absence of a prism in the spectacles, and (4) presence of random dot stereopsis (Lang Test 1). The observers were refracted without dilating their pupils, using streak retinoscopy and crossed cylinders, taking particular care to avoid any uncorrected hyperopia. During the experiment, the observers wore full spherical and cylindrical spectacle corrections. Care was taken to ensure that the glasses remained centred throughout the study. We did not inquire whether the observers had asthenopic symptoms or not.\nWe explained to the observers that the study intended to optimise the comfort of seeing. Otherwise, the observers were kept naive as to the purpose of the study. Each observer provided informed written consent to participate in the experiments. The study followed the tenets of the Declaration of Helsinki and was approved by the institutional human review board.\nData acquisition and analysis\nThe prismatic power derived from the potentiometer attached to Herschel\u2019s prisms was recorded by PowerLab with a sampling rate of 100\u00a0Hz. To enable analysis of the dynamic behaviour of the adjustments we recorded the full 30 seconds of each trial. Offline examination was accomplished with Igor Pro\u00ae (Wavemetrics, Inc., Lake Oswego, OR, USA) and Statview\u00ae (Abacus Concepts, Inc., Berkeley, California, USA). Statistical analysis was performed with Statview\u00ae and SPSS\u00ae (SPSS Inc., Chicago, Illinois, USA). We used multifactorial ANOVA (including condition, session and trial) and paired comparisons. For post-hoc tests, Bonferroni adjustment was performed.\nResults\nInspection of curves\nThe curves obtained from the 20 observers varied considerably between the 8 trials. For example see Fig.\u00a03. There was no trend, e.g. with the early trials (thin lines) yielding smaller values than the late ones (progressively thicker lines), or vice versa (p\u2009=\u20090.34). Most observers adjusted Herschel\u2019s prisms stepwise, reaching the final value after about 20 seconds. Therefore, we limited the numerical evaluation to the remaining 10 seconds.\nFig.\u00a03Adjustment of Herschel\u2019s prisms by observer #10 for dissociated phoria, associated phoria, and the comfortable prism. First session. Positive values indicate base out prisms (eso deviation), negative values base in prisms (exo deviation). The upper panels show the original recordings of the eight single trials. The lower panels show the mean values (solid lines) with the standard error of the mean (SEM, dashed lines). Note that the scale of the ordinate for the comfortable prism differs from those in the two phoria conditions\nFor dissociated phoria, most observers saw an offset right at the beginning of each trial and promptly moved the lever arm accordingly.\nFor associated phoria, some observers initially did not see an offset between the Nonius lines. In this case, the observers followed the instruction to \u201cplay\u201d a little with the lever arm and find a position in which the lines appeared as stable as possible.\nFor the comfortable prism, most observers started with rather bold searching excursions and refined the tuning gradually (Fig.\u00a03, right panels). Only exceptionally, an observer ended up with a rather large range for the comfortable prism.\nStatistical analysis\nWe averaged the 1000 values recorded during the last 10 seconds in each of the eight trials, as most observers reached the final value after about 20 seconds. As stated above, there was no trend between the eight trials, e.g. with the early trials yielding smaller values than the late ones, or vice versa (p\u2009=\u20090.34). This allowed us to treat the 8 trials together and calculate an intertrial mean\u00b1SEM (standard error of the mean) for each observer (Fig.\u00a04). Among the 20 observers, the range of the intertrial mean was for dissociated phoria from +9.3\u00a0eso to \u22125.9\u00a0cm\/m exo deviation, for associated phoria from +11.2\u00a0eso to \u22123.3\u00a0cm\/m exo deviation, and for the comfortable prism from +4.8\u00a0eso to \u22124.1\u00a0cm\/m exo deviation.\nFig.\u00a04Mean of the last 10 seconds of eight trials \u00b1SEM for each of the 20 observers. The values obtained in the first session are represented in the left columns, those obtained in the second session in the right columns. DP = dissociated phoria, AP = associated phoria, CP = comfortable prism. The observers are identified with #1 to #20. Ordinate: positive values = eso deviation, negative values = exo deviation. Note that the ordinates in #7 and #19 are scaled down\nTo assess the intertrial variability over all 20 observers, we averaged the 95% confidence interval (\u00b11.96\u00a0SD) per session. The values were as follows. Dissociated phoria: first session \u00b11.3\u00a0cm\/m, second session \u00b11.0\u00a0cm\/m; associated phoria: first session \u00b11.6\u00a0cm\/m, second session \u00b11.6\u00a0cm\/m; comfortable prism: first session \u00b12.0\u00a0cm\/m, second session \u00b12.2\u00a0cm\/m. Hence, the variability was similar in the two sessions. Combining the two sessions resulted in a greater overall scatter (95% confidence interval\u2009=\u2009\u00b11.96\u00a0SD): \u00b15.8\u00a0cm\/m for dissociated phoria, \u00b15.4\u00a0cm\/m for associated phoria, and \u00b14.3\u00a0cm\/m for the comfortable prism. The marked increase in the overall scatter indicates that the values changed between the two sessions. In some cases the mean values of the second session were even outside the 95% confidence interval of the first session: 5\/20 for dissociated phoria, 9\/20 for associated phoria, and 6\/20 for the comfortable prism. The change was not always concordant in the three conditions. For example, in observer #13, the associated phoria changed from +0.1\u2009\u00b1\u2009SEM 0.4\u00a0cm\/m to +4.6\u2009\u00b1\u2009SEM 0.5\u00a0cm\/m, i.e. in the eso direction, and the comfortable prism changed from \u22120.1\u2009\u00b1\u2009SEM 0.4\u00a0cm\/m to \u22121.8\u2009\u00b1\u2009SEM 0.2\u00a0cm\/m, i.e. in the exo direction.\nAre these changes clinically relevant? As several practitioners consider prescribing prisms from a minimum of about 1.0\u00a0cm\/m onwards [13], we identified the observers in whom the change was greater than 1.0 cm\/m: for dissociated phoria 6\/20 (up to 3.2\u00a0cm\/m), for associated phoria 11\/20 (up to 4.5\u00a0cm\/m), and for the comfortable prism 12\/20 (up to 3.3\u00a0cm\/m). To find out whether the observers who changed their values by more than 1.0\u00a0cm\/m could be recognized at the first session, we compared the intertrial variability at the first session in two groups: the observers who changed their values more than 1.0\u00a0cm\/m with those who changed them less than 1.0\u00a0cm\/m. There was no statistically significant difference between the two groups (p\u2009=\u20090.67 for dissociated phoria, 0.11 for associated phoria, 0.52 for the comfortable prism).\nComparison between dissociated phoria, associated phoria, and the comfortable prism\nThe ANOVA over all trials of all 20 observers (n\u2009=\u20098\u2009\u00d7\u20092\u2009\u00d7\u200920\u2009=\u2009320) revealed that dissociated and associated phoria differed significantly (p\u2009<\u20090.01). In 11 observers, the dissociated phoria was lower, in four higher than the associated phoria; in five observers, dissociated and associated phoria were similar.\nFigure\u00a05 demonstrates that the majority of observers had an eso deviation for dissociated and associated phoria (13\/20 and 17\/20, respectively), but an exo deviation for the comfortable prism (17\/20). The tendency towards eso deviation in the two phoria conditions was also obvious in the average \u00b1SD over all trials of all 20 observers (n\u2009=\u20098\u2009\u00d7\u20092\u2009\u00d7\u200920\u2009=\u2009320): +1.0\u2009\u00b1\u20092.9\u00a0cm\/m eso deviation for dissociated phoria, and +2.0\u2009\u00b1\u20092.7\u00a0cm\/m eso deviation for associated phoria, versus \u22120.6\u2009\u00b1\u20092.2\u00a0cm\/m exo deviation for the comfortable prism. The difference between dissociated phoria and comfortable prism, on the one hand, and the difference between associated phoria and comfortable prism on the other were both significant (p\u2009<\u20090.05 and p\u2009<\u20090.001 respectively).\nFig.\u00a05Mean of the two sessions \u00b1SEM for associated phoria, dissociated phoria, and comfortable prism. The first column in each graph represents observer #1, followed by #2 etc\nTo investigate whether the tendency towards eso deviation in the two phoria conditions, as compared with the comfortable prism, was brought about by the sequence of tests, we made a spot check. We chose observer #10, because she had a marked eso deviation in the two phoria conditions, but an exo deviation in the comfortable prism. In an extra session, this observer determined the comfortable prism before the tests for dissociated and associated phoria. The values obtained were of the same magnitude as those in the two previous sessions (ANOVA p\u2009=\u20090.084 for tested difference).\nDiscussion\nWe compared three vergence parameters: dissociated phoria, associated phoria, and the comfortable prism. To avoid any (possibly prejudiced) influence of the experimenter, we recorded the prismatic power set by the observer with a potentiometer. The comfortable prism anticipates the real-life situation in which prismatic spectacles are worn. Therefore, the comfortable prism is very close to the endpoint of therapeutical considerations, i.e. prescribing prismatic spectacles. In contrast, dissociated and associated phoria, determined under artificial viewing conditions, are surrogate measures of the comfortable prism. We studied whether these surrogate measures predict the comfortable prism, assuming that dissociated phoria, associated phoria, and the comfortable prism might be similar, because all three parameters are determined by opening the feedback loop for fusional vergence. When measuring dissociated phoria, fusionable contours are absent; hence, there is no disparity error signal. When associated phoria is measured, any disparity of the (defective) fusional pattern, which initially constitutes an error signal, is promptly nullified by repeated prism adjustment. Concerning the comfortable prism, one has to realize that the fusional feedback loop, which uses disparity as its error signal, can stabilize the vergence angle only if the stimuli are presented to the two eyes in a fixed angle, e.g. through a certain unchanging prism. If, however, the observer is asked to adjust a variable prism according to his or her comfort, the fusional feedback loop is open and cannot stabilize the vergence angle. Instead, the fusional feedback loop is replaced by another feedback loop whose error signal is discomfort. Accordingly, the vergence position reached with the comfortable prism may also be described as the \u201cvergence position of rest when both eyes are exposed to identical pictures\u201d.\nTo our surprise, we found marked differences between the three vergence parameters: as indicated in Fig.\u00a05, the majority of observers had an eso deviation under the two phoria conditions (13\/20 for dissociated and 17\/20 for associated phoria), but preferred base-in for the comfortable prism, corresponding to an exo deviation (17\/20). The maximal difference occurred in observer #10 who showed in his second session an eso deviation for associated phoria of +4.9\u00a0cm\/m, and an exo deviation for the comfortable prism of \u22123.0\u00a0cm\/m.\nThe tendency towards eso deviation in the two phoria conditions was also evident in the significant difference between the averages \u00b1SD over all 20 observers: +1.0\u2009\u00b1\u20092.9\u00a0cm\/m eso deviation for dissociated phoria, +2.0\u2009\u00b1\u20092.7\u00a0cm\/m eso deviation for associated phoria, but \u22120.6\u2009\u00b1\u20092.2 cm\/m exo deviation for the comfortable prism. Although the test condition for associated phoria is commonly regarded as being more natural than that for dissociated phoria, the associated phoria was not closer to the comfortable prism than the dissociated phoria. This means that the difference in the overall luminance for the two eyes, present in our test for dissociated phoria, did not push the vergence away from the value obtained with the comfortable prism. This finding supports the finding of Kromeier et al. [7, 8] that unequal luminance of the images for the two eyes does not necessarily influence the vergence position.\nWhat is the reason for the tendency towards eso deviation in the two phoria conditions? A different accommodative demand can be excluded, because we used a similar target in all three conditions. Rather, we suggest that the shift towards eso deviation is brought about by the dissimilarity between the images of the two eyes. This suggestion pertains even to the associated phoria condition, in which ample fusionable contours are available, and the dissimilarity is limited to the monocular Nonius lines. Nevertheless, this dissimilarity was conspicuous for the observers: most of them reported that one or both Nonius lines seemed to disappear every now and then. Correspondingly, the observers reported occasional disappearance of the white dot when they determined their dissociated phoria, although this was less pronounced than the disappearance of the Nonius lines in the associated phoria condition. These observations raise the possibility that the irritating perception of binocular rivalry drives the eso shift in the two phoria conditions. Compatible with this idea is that the eso shift was stronger in the associated than in the dissociated phoria condition, i.e. in the condition with the more pronounced fading.\nConsidering the open feedback loop for fusional vergence in all three conditions, it is not surprising that the values obtained with all three methods (dissociated phoria, associated phoria, and comfortable prism) were rather variable. This was true in each of the two sessions.\nThe values changed in many observers from the first to the second session beyond the 95% confidence interval of the eight trials in the first session. Comparing the three conditions, they did not always change in the same direction (eso or exo). We do not see a specific reason for these changes, as there was no hint that the general health or attentiveness had changed between the two sessions. Rather, the variability might be understandable as there is no need for the ocular motor system to keep the vergence angle stable under open-loop conditions. Surely, adaptation maintains the open-loop vergence in a certain range [10, 12], but this range appears to be rather wide.\nWhat are the practical inferences of our study?\nAs mentioned in the introduction, both dissociated [11, 18] and associated phoria [9, 19] have been recommended as indicators for prismatic correction in the case of asthenopia. We purposely did not select our observers according to the presence or absence of asthenopic complaints. Rather, we avoided using the relief from asthenopic complaints as a criterion, because this approach would have required prolonged wearing of various test prisms, including placebo, in a controlled design, to face the following four problems. First, a causal relationship between heterophoria and asthenopia is hard to establish, because the prevalence of both conditions is high in the general population, so that an unrelated coincidence can easily occur. Second, wearing prisms for hours or days leads to adaptation, so that the patient may later value even prisms that he or she initially disliked. Third, most asthenopic symptoms are waxing and waning. It is highly likely that patients seek remedy at a time when their symptoms are relatively intense. Hence, a subsequent improvement may well be due to a regression to the mean, rather than to a prism [21]. Fourth, a relief from symptoms may be brought about by the supportive behaviour of the therapist.\nAlthough we did not use the relief from asthenopic complaints as a criterion, we think that our findings relate to the prescription of therapeutic prisms for such patients. The discrepancy between dissociated and associated phoria on the one hand and the comfortable prism on the other hand would probably also occur in patients with asthenopia, and it is plausible to assume that these patients would benefit more from the comfortable prism, chosen by themselves under natural viewing conditions, than from a prescription based on one of the phoria parameters, which carry artefacts due to the artificial test conditions.\nAnother practical aspect of our study is the finding that both phoria parameters and the comfortable prism can change from one session to the next. Are these changes clinically relevant? Several practitioners consider prescribing prisms from 1.0\u00a0cm\/m onwards [13]. On this background, our finding that 12 of the 20 observers changed their comfortable prism by more than 1.0\u00a0cm\/m (up to 3.3\u00a0cm\/m) within an interval of a few weeks is important. We examined whether patients tending to a long-term change of their open-loop vergence might be identifiable in the first session by a large scatter. Unfortunately, our results did not support this notion: the intertrial variability at the first session was not larger in the observers who changed their values more than 1.0\u00a0cm\/m, as compared with observers who changed their values less than 1.0\u00a0cm\/m. Hence, repeated determinations of the comfortable prism on different days appear advisable to identify patients in whom the values remain relatively stable. Only in these patients would a prescription of prisms be reasonable.\nWhat are the limitations of our study?\nTowards the end of our experiments, we considered that it might have been preferable to randomize the sequence of tests between the 20 observers. However, it is unlikely that such a randomization would have produced significantly different results, for the following two reasons: (1) an influence of training or fatigue is unlikely since there was no trend between the eight trials of each test, e.g. with the early trials yielding smaller values than the late ones, or vice versa (p\u2009=\u20090.34), and (2) a spot check in observer #10 with the reverse order of tests confirmed this observer\u2019s marked discrepancy between an eso deviation in the two phoria conditions, and an exo deviation in the comfortable prism (ANOVA p\u2009=\u20090.084 for tested difference).\nThe angles encountered in our 20 observers were relatively small: the dissociated phoria ranged from +9.3\u00a0cm\/m eso to \u22125.9\u00a0cm\/m exo deviation, the associated phoria from +11.2\u00a0cm\/m eso to \u22123.3\u00a0cm\/m exo deviation, and the comfortable prism from +4.8\u00a0cm\/m eso to \u22124.1\u00a0cm\/m exo deviation. An extrapolation of our conclusions to observers with larger angles may not be justified.\nOur tests corresponded to just one of many \u201cnatural\u201d viewing conditions: with respect to luminance, visual angle, and distance (4 meters), the conditions were similar to watching television. According to previous work [2, 3], it is likely that, at closer viewing distances, the values would be shifted in the exo direction. Hence, we suggest that the practitioner should determine the comfortable prism in the viewing distance, for which therapeutic prisms are being considered. This recommendation corresponds with the concept of Jaschinski [4], who suggested that observers should choose their comfortable viewing distance for computer screens in the real-life situation.","keyphrases":["fixation disparity","aligning prism","asthenopia","heterophoria","mallett unit"],"prmu":["P","P","P","P","M"]}
{"id":"Histochem_Cell_Biol-4-1-2413113","title":"Connexons and cell adhesion: a romantic phase\n","text":"Recent evidence indicates, that gap junction forming proteins do not only contribute to intercellular communication (Kanno and Saffitz in Cardiovasc Pathol 10:169\u2013177, 2001; Saez et al. in Physiol Rev 83:1359\u20131400, 2003), ion homeostasis and volume control (Goldberg et al. in J Biol Chem 277:36725\u201336730, 2002; Saez et al. in Physiol Rev 83:1359\u20131400, 2003). They also serve biological functions in a mechanical sense, supporting adherent connections between neighbouring cells of epithelial and non-epithelial tissues (Clair et al. in Exp Cell Res 314:1250\u20131265, 2008; Shaw et al. in Cell 128:547\u2013560, 2007), where they stabilize migratory pathways in the developing central nervous system (Elias et al. in Nature 448:901\u2013907, 2007; Malatesta et al. in Development 127:5253\u20135263, 2000; Noctor et al. in Nature 409:714\u2013720, 2001; Rakic in Brain Res 33:471\u2013476, 1971; J Comp Neurol 145:61\u201383 1972; Science 241:170\u2013176, 1988), or mediate polarized movements and directionality of neural crest cells during organogenesis (Kirby and Waldo in Circ Res 77:211\u2013215, 1995; Xu et al. in Development 133:3629\u20133639, 2006). Since, most data describing adhesive properties of gap junctions delt with connexin 43 (Cx43) (Beardslee et al. in Circ Res 83:629\u2013635, 1998), we will focus our brief review on this isoform.\nIntroduction\nFor those readers who are not familiar with the molecular composition of gap junctions, we will briefly recollect the substructure of this cell contact and refer to recent detailed reviews (Duffy et al. 2002; Gaietta et al. 2002; Goodenough and Paul 2003; Meier and Dermietzel 2006; Willecke et al. 2002).\nGap junctions are formed by hemichannels (connexons), which consist of an oligomer of six proteins (connexins). At present, at least 20 genes seem to be present in the human and rodent genome (Willecke et al. 2002), which may oligomerize in monomeric or heteromeric patterns to form a hemichannel. A complete gap junction channel is formed by two hemichannels in mirror symmetry (Fig.\u00a01). Heterotypic configurations of different isoforms are allowed for some connexins while others occur exclusively in homotypic configuration. In the history of gap junctions, the junctional plaque has always been considered to occur in a \u201cnaked\u201d form without cytoplasmic adjuncts like scaffolding proteins or cytoskeletal elements (Hirokawa and Heuser 1982). However, accruing evidence indicates that gap junctions are associated with a complex system of scaffolding and cytoskeletal proteins, which seem to assemble in cell specific patterns (Duffy et al. 2002 for recent review).Fig.\u00a01General structure of a gap junction plaque. Gap junctions are formed by paired hemichannels (connexons) of two adjacent cells. A single connexon is made by a polymer of six connexins. Only apposed connexons allow intercellular transfer of ions (ionic coupling) and small metabolites (metabolic coupling). Unapposed connexons seem to perform per se functions\nIn the following, we will put main emphasis on heart and brain tissues for which most of the data on gap junctions and cell adhesion have been reviewed.\nCardiomyocytes: directed trafficking of connexin43 involves the cytoskeleton and adhesion plaques\nIn the terminal intercalated discs between cardiomyocytes, Cx43 celebrates an example for integrating its hemichannel and cell coupling functions (Gros and Jongsma 1996; Shaw and Rudy 1997; van Veen et al. 2001). Asides the terminal intercalated discs, gap junctions are also localised in the lateral sarcolemma of the heart (Fig.\u00a02), and thus described to form site-to-site and end-to-end connections (Yao et al. 2003). Cardiomyocytes also represent unopposed connexons with hemichannel function in the lateral sarcolemma (Saez et al. 2003; Schulz and Heusch 2006; Yao et al. 2003).Fig.\u00a02a shows cultured cardiomyocytes immunolabelled with an anti-Cx43 antibody (red). Immunolabelling is prevalent in apposed cell membranes, but also in some unapposed domains. Nuclei are counterstained with Hoechst dye. b Immunolabelling of heart tissue with Cx43 antibody. Intercalated discs (red) are intensivley stained. Bar indicates 25\u00a0\u03bcm\nTo ensure its\u2019 trafficking and functional integration into a gap junction plaque or in form of unpaired connexons (hemichannels) into the plasma membrane, Cx43 has to interact with other proteins. In the intercalated discs of coupled cardiomyocytes for instance, gap junction plaques are embedded into adherens junctions, which are primarily formed by cadherins (Matsuda et al. 2006; Niessen 2007; Zuppinger et al. 2000). Whilst the adherens junction supports the mechanical coupling (Gutstein et al. 2003; Niessen 2007), the gap junction ensures the propagation of action potentials along the cardiomyocytes (Gros and Jongsma 1996; Shaw and Rudy 1997).\nMultiple models describe the pathway from connexon assembly to the initial gap junction formation and interaction with cadherins in the adherens junction. The most common examples are based on half-life time determined trafficking and junctional protein (cadherins) mediated activation of Cx43. The half-life time of Cx43 is restricted between 1 and 3\u00a0h, and implicates a dynamic process of assembly, insertion and replacement of connexons, and pairing of connexons to form gap junctions (Beardslee et al. 1998; Hofer and Dermietzel 1998; Laird et al. 1991). Cx43 synthesis was shown to be located on membrane bound ribosomes, where connexin proteins are rapidly oligomerized into homo or heteromeric connexons (Evans et al. 1999; Martin and Evans 2004). Final packing into hemichannel loaded vesicles occurs in the trans-Golgi network as shown by Musil and Goudenough (1993) followed by directed transport along microtubules to multiple insertion sites in the membrane (Akhmanova and Hoogenraad 2005; Jordan et al. 1999; Lauf et al. 2002; Mimori-Kiyosue et al. 2005; Shaw et al. 2007). Once arrived at the gap junction borders at the membrane, connexons are assumed to be inserted via flipping events into the membrane and to diffuse into the centre of the plaque, whilst elderly connexin proteins are shifted to the plaque periphery for subsequent disposal (Gaietta et al. 2002; Laird 2005; Segretain and Falk 2004).\nThe finding that mislocated Cx43 gap junction plaques in the ischemic myocardium are associated with similarly misplaced adherens junctions (Matsushita et al. 1999), underlines an interdependence between Cx43 and the adherens junction related cadherins (Angst et al. 1997; Li et al. 2005; Luo and Radice 2003; Matsushita et al. 1999). E-cadherin transfections into gap junction incompetent cells, allowed the transfectants to build out functional gap junctions (Matsushita et al. 1999). Furthermore, N-cadherin knockout mice (Luo and Radice 2003) and conditional knockdown of N-cadherin in the heart caused mislocalisation and compromized expression of gap junctions. Conditional knockdown of N-cadherin in the heart was additionally shown to lead to arrhythmogenic death (Li et al. 2005), which may involve aberrant regulation of gap junction function (for reviews see: Duffy et al. 2007)\nRecently, Shaw et al. (2007) described microtubule-mediated target-delivered transport of Cx43 via microtubule plus-end-tracking proteins (+TIPs) and interaction partners such as p150(GLUED) (Berrueta et al. 1999), a component of the dynein\/dynactin complex, which in turn is potent to tether microtubules at the adherens junctions (Chausovsky et al. 2000; Ligon et al. 2001). Studies implicating fluorescence recovery after photobleaching (FRAP) on Cx43-YFP transfected HeLa cells that do not endogenously express Cx43, revealed a rapid Cx43 delivery to gap junction plaques. Deconvolution clarified that microtubules extend directly to the gap junction plaques at the cell\u2019s border and total internal reflection fluorescence (TIRF) microscopy and time lapse imaging revealed the appearance of a preferential and prolonged association of microtubule plus ends with the plaques. Most strikingly, Shaw et al. (2007) were able to show that gap junction plaque formation was disrupted by siRNA knockdown of the dimeric +TIP EB1. EB1 associates directly with the plus ends of microtubules and provides, in turn, dual binding sites for adherens junction related proteins like p150(GLUED) and \u03b2-catenin. Furthermore, in this setting gap junction plaques could also be disrupted via Nocodazol and Taxol treatment, peptides, which compromise the homophilic cadherin-cadherin interaction in adherens junctions. This setting according to the recent paper by Shaw et al. (2007) is sketched in the cartoon (Fig.\u00a03). While Nocodazol interrupts formation of microtubules by depolimerization, Taxol lets microtubules remain stable, but interferes with their EB1 interaction partner (Nakata and Hirokawa 2003).Fig.\u00a03Model for microtubulus mediated delivery of vesicle-bound connexons to adherens junctions (adapted from Shaw et al. 2007). Microtubules bind via their +end to EB1. EB1 in turn binds to P150 (GLUED), a component of the dynein\/dynactin complex, which interacts with \u03b2-catenin through P120-catenin with the adherens junction. This interaction is understood to tether the microtubule to the junction and to serve as a gateway for connexon delivery\nIn this context, actin is discussed to act as an initial sensor of cell-cell interaction, driving the localisation of adherens junctions with assistance from Rho-GTPases (Noren et al. 2001, 2003).\nSpread of Shigella flexneri requires connexin43 hemichannels\nHow close cytoskeletal (re-)organization and intracellular connexin distribution are related is demonstrated for polarized intestinal cells during Shigella flexneri invasion (Clair et al. 2008). For invading the colonic mucosa (Labrec et al. 1964), the gram negative enteric bacillus requires RhoGTPases, Src and Abl\/Arg tyrosine kinases for actin polymerization and formation of cytoplasmic extensions of surrounding cells (Burton et al. 2003; Tran Van Nhieu et al. 2000). The invasion and dissemination of the bacteria causes intense inflammatory responses, and especially ATP-dependent paracrine signalling induced by Cx hemichannel opening (Tran Van Nhieu et al. 2000). E-cadherins were indispensable for the intercellular spreading of S. flexneri (Sansonetti et al. 1994). It is assumed that a cytoskeletal reorganization toward the formation zone of gap junctions is induced in this process to allow the spread of the bacteria (Clair et al. 2008; Tran Van Nhieu et al. 2000). On the epithelial level there is mounting evidence that connexin hemichannels regulate intercellular signalling (Stout et al. 2004), which might be of importance for incoming phagocytic cells during bacterial infection (Ferrari et al. 1997; Griffiths et al. 1995; John et al. 2001; Korcok et al. 2004).\nThe developing brain and hemichannel adhesion\nThe necessity of gap junction adhesion via regulation by its cytoskeleton interaction partners p120 catenin, integrin and actin has become well identified in the developing brain (Xu et al. 2001, 2006). Here, stem cells of the developing neocortex give rise to neurons (Malatesta et al. 2000; Noctor et al. 2001) and provide guidance of the developing neurons to the target zones of the cortical plate, where they are meant to become pyramidal cells of the adult cortex (Rakic 1971, 1972, 1988). Electron microscopy showed, that during the process of migration of neuronal precursors, gap junctions occur between radial fibres and migrating neurons and nestin\u2212 and nestin+ cells (Huang et al. 1998a). The most important gap junction protein isoforms are Cx43 and Cx26 in developing brain tissue (Dermietzel et al. 1989). Until now there was evidence that gap junctions between radial glia and migrating neurons served for chemical and electrical communication. Elias et al. (2007) recently found that Cx26 and Cx43 are expressed in \u03b2-III tubulin positive migrating neurons in the contacting regions close to vimentin positive radial glial fibres. Using a RNA knockdown of Cx43 and Cx26 by short hairpin RNA (shRNA) constructs in rat, the authors were able to demonstrate a reduced fractioning of neurons in the cortical plate. In addition, transplantation of Cx26 and Cx43 shRNA knocked down donor cells into E17 wildtype mice revealed an intact engrafting of the donor cells into the host brain, but no migration. Immunocytochemistry of the shRNA knocked down transplanted neurons in the recipient brains showed no compromised cell cycle exit and no alterations of differentiation. Furthermore, the expression of the adherens related proteins ZO-1, N-cadherin and \u03b2-integrin was not altered, indicating that gap junctions mediate glial-guided radial migration of developing neurons in the cortex. This migration was additionally demonstrated to rely on the adhesive and not on the channel properties of Cx26 or Cx43 (Elias et al. 2007). Dominant negative connexin mutants lacking channel properties were still able to form adhesive contacts. In reverse experiments, the authors demonstrated that channel, but no adhesion forming mutants, were unable to rescue the Cx43 shRNA induced migration defect. Finally, time lapse imaging of Cx43\/Cx26 shRNA expressing neurons affirmed their inability to stabilize their processes and to continue to extend along the radial glia.\nNeural crest cells and colonization\nGap junction regulated polarized cell movements and directional migration are not restricted to developmental processes within the central nervous system (Elias et al. 2007; Schaar and McConnell 2005). Studies focusing on Cx43 expression of cardiac neural crest cells indicated a clear relationship between their migratory properties and Cx43 expression (Huang et al. 1998a, b; Li et al. 2002; Lo et al. 1999; Reaume et al. 1995; Sullivan and Lo 1995; Xu et al. 2001, 2006). Neural crest cells are ectomesenchymal cells emerging from epithelial mesenchymal cell transformation in the dorsal neural tube from where they disperse throughout the embryo to generate a variety of tissues (Kirby and Waldo 1995; Xu et al. 2006). Neural crest cells from different axial levels of the neural tube use multiple migratory pathways to reach their terminal destinations. Cardiac neural crest cells (CNCs) have been shown to migrate along a circumpharyngeal pathway to reach the aortic arches and the heart (Kirby et al. 1983; Lumsden et al. 1991). This deployment has been shown to be modulated by Cx43 and cytoskeletal interaction partners with the extracellular matrix (Xu et al. 2006). The finding, that dynamic di- and reassembly of focal contacts is essential for polarized cell movements and directional cell migration moved the heterodimeric receptor group of integrins into the centre of related studies. Integrins cluster to form focal domains within the cell membrane, linking the extracellular matrix to the actin cytoskeleton. Since, it could be shown that neural crest cells express multiple integrins (Delannet et al. 1994; Monier-Gavelle and Duband 1997) and perturbation studies provide evidence that integrins modulate the migratory behaviour of neural crest cells, (Strachan and Condic 2003, 2004, 2008) the question arose whether integrin signalling might be affected in Cx43 expressing versus Cx43 knock out cells (Xu et al. 2006).\nIn neural tube explants of the post-otic hindbrain folds from E8.5 mice, underlying either an Cx43 knockout or Cx43 overexpression, neural crest cells were generated that emerge from the same axial level as CNCs, which migrate to the heart. In contrast to overexpressing CNCs, the Cx43 deficient CNC type was characterized by a severe loss of directionality and reduced adhesion whilst being cultured on a fibronectin matrix. Furthermore, an increase in the fibronectin matrix density leads to reductions in the migratory speed of Cx43 deficient CNCs, as shown via time lapse videomicroscopy (Xu et al. 2001). In fact double-immunostaining against \u03b21-integrin and vinculin as markers for focal adhesion, was significantly reduced in Cx43 deficient CNCs, indicating a reduction in the actin-cytoskeletal linkage for matrix adhesion. A modulatory influence of Cx43 on the actin cytoskeleton became evident in rhodamine-phalloidine stainings, where Cx43 knockout CNCs represented shorter stress fibre bundles (Xu et al. 2001). Additionally, these bundles exhibit no anchoring via vinculin to focal adhesions, as being observed for Cx43 overexpressing CNCs. Furthermore, adhesion and migration of Cx43 deficient CNCs on a fibronectin matrix could be inhibited by semaphorin application, which is described to act as a potent blocker of integrin activation (Brown et al. 2001). This approach also confirmed that Cx43 modulates the retraction of cellular processes. Immunoprecipitation, Western blot and immunocytochemistry pointed out that Cx43 in CNCs does not co-localise with \u03b21-integrin, but with vinculin and actin-filaments (Osborne et al. 2005; Pasterkamp and Kolodkin 2003; Serini et al. 2003; Xu et al. 2006). This finding is supported by the co-localisation of Cx43 with several actin binding proteins, such as ezrin, IQGAP, \u03b1-actinin and drebrin (Butkevich et al. 2004). No correlation between gap junctional coupling properties and the density of fibronectin matrix was found for Cx43 knockouts and Cx43 overexpressing CNCs in dye coupling experiments, although an upregulation of gap junction communication with altered integrin-matrix interactions has previously been described for other cell types (Czyz et al. 2005; Lampe et al. 1998; Shanker et al. 2005).\nIn summary, asides their function as gap junction forming elements, unpaired connexons have been shown to modulate the cells\u2019 migratory and adhesive functions whilst being in permanent crosstalk with an elaborate complex of cytoskeletal interaction partners.\nPerspectives on pathology\nHow important the connexin-cytoskeleton interaction is, becomes elucidated in case of pathology. For instance, cadherin-cadherin interactions might be critically affected during tumour formation. The interaction normally becomes active in cell sorting mechanisms during development (Wheelock and Johnson 2003). Since, Shaw et al. (2007) have shown that Cx43 can reach adherens junctions via microtubule directed delivery, it may be suggested that gap junctions are formed preferentially with cells, expressing the same type of cadherin (Wheelock and Johnson 2003). Additional studies provide evidence, that loss of E-cadherin or upregulation of N-cadherin can increase tumour invasiveness and Cx43 downregulation in malignant cells (Mesnil 2002). It is thought that gap junction channels and their interactions with molecules such as p120 catenin, integrin and the actin cytoskeleton are important for neural crest cell migration (Xu et al. 2001, 2006), and that glioblastoma invasion of the brain parenchyma requires functional gap junctions between tumour cells and astrocytes (Lin et al. 2002). Furthermore, the migration of lung and skin cancer cells has also been associated with gap junction expression, although no clear mechanism has been proposed so far (Ito et al. 2000; Lois et al. 2002).\nDevelopmental defects are also related to mutations of the Cx43 gene. As already indicated, Cx43 knockout mice reveal comprised conotruncal heart development, which is associated with a reduction in the number of cardiac neural crest cells targeted to the heart (Xu et al. 2006). Mutations in Cx43 which seem to influence cytoskeletal organization in a strong manner range from disease patterns like deafness, cataracts, germ cell developmental defects, ocludentodigital dysplasia to cardial outflow abnormalities (Polontchouk et al. 2002) and left ventricular remodelling (Kanno et al. 2003).\nAt its final extent, gap junction channels, in particular in form of hemichannels, constitute a new player in the complex interaction of cell adhesion and cytoskeletal activation, which underlies directed migration during development and in mature tissue. It is a romantic phase where anything may go, but time has to approve what will remain forever.","keyphrases":["cell adhesion","gap junction","hemichannel","heart","developing brain"],"prmu":["P","P","P","P","P"]}
{"id":"J_Neurooncol-3-1-1915654","title":"Hemangioblastomatosis in a patient with von Hippel-Lindau disease\n","text":"Case presentation\nA woman developed a cerebellar hemangioblastoma in 1991 at the age of 17 and von Hippel-Lindau disease was diagnosed. She underwent five craniotomies for recurrent hemangioblastoma. In 2001 she had a laminectomy for an intramedullar hemangioblastoma at C2-C3 level. In 2004 a renal cell carcinoma was found and a partial nephrectomy was performed. A few weeks after her last craniotomy in July 2005 she developed progressive right hemisensory disturbances and sensory ataxia. Spinal MRI showed leptomeningeal contrast enhancement around the entire spinal cord (see Figure). The sensory ataxia progressed and she got tetraparesis, most severe proximally in her arms, especially myotome C4. Although hemangioblastoma are not very radiosensitive, radiotherapy was started because of the rapid clinical deterioration. High-dose total neuraxis radiotherapy was considered to toxic. It was judged that most of her clinical complaints could be attributed to the cervical spine. Also the MRI showed the most severe lesions at the cervical region. Therefore she was treated from C1 to Th1 (30\u00a0\u00d7\u00a01.8\u00a0Gy) combined with dexamethasone (2\u00a0\u00d7\u00a08\u00a0mg). During radiotherapy she developed also symptoms outside the radiotherapy field (thoracal radiculopathy).\nAs the main problem in VHL is uncontrolled angiogenesis by overexpressing VEGF and VEGF receptors, Thalidomide (400\u00a0mg, later 600\u00a0mg) was added. Thalidomide inhibits angiogenesis induced by VEGF [1]. Despite the treatment she developed a total tetraplegia and died 3\u00a0months after diagnosis as a result of respiratory failure.\nThis syndrome appears to result from delayed growth of subarachnoid tumor cells disseminated by surgery. Hemangioblastomatosis is also seen in hemangioblastoma patients without von Hippel-Lindau disease and it is thought that additional genes are likely to be the source of this malignant behaviour [2].","keyphrases":["hemangioblastoma","von hippel-lindau disease"],"prmu":["P","P"]}
{"id":"Breast_Cancer_Res_Treat-3-1-2001216","title":"The discovery and mechanism of action of letrozole\n","text":"Because estrogen contributes to the promotion and progression of breast cancer, a greater understanding of the role of estrogen in breast cancer has led to therapeutic strategies targeting estrogen synthesis, the estrogen receptor, and intracellular signaling pathways. The enzyme aromatase catalyses the final step in estrogen biosynthesis and was identified as an attractive target for selective inhibition. Modern third-generation aromatase inhibitors (AIs) effectively block the production of estrogen without exerting effects on other steroidogenic pathways. The discovery of letrozole (Femara\u00ae) achieved the goal of discovering a highly potent and totally selective AI. Letrozole has greater potency than other AIs, including anastrozole, exemestane, formestane, and aminoglutethimide. Moreover, letrozole produces near complete inhibition of aromatase in peripheral tissues and is associated with greater suppression of estrogen than is achieved with other AIs. The potent anti-tumor effects of letrozole were demonstrated in several animal models. Studies with MCF-7Ca xenografts successfully predicted that letrozole would be clinically superior to the previous gold standard tamoxifen and also indicated that it may be more effective than other AIs. An extensive program of randomized clinical trials has demonstrated the clinical benefits of letrozole across the spectrum of hormone-responsive breast cancer in postmenopausal women.\nIntroduction\nStudies have consistently shown that lifetime exposure to estrogens increases the risk of breast cancer [1]. The degree of risk is increased by persistently elevated blood concentrations of estrogen [2]; clinical indicators of persistently elevated blood estrogen concentrations, for example, age at menarche, first live birth, menopause, alcohol consumption, and obesity [3\u20135]; and, although still controversial, exposure to exogenous estrogen, for example, some forms of hormone replacement therapy and oral contraceptives [6\u201312]. The presence of some of these factors also increases the risk of breast cancer being estrogen receptor (ER)-positive [13]. Studies have shown that higher levels of endogenous estrogen and testosterone (which is converted to estrogen by aromatase) increases breast cancer risk, regardless of predicted breast cancer risk [14\u201316]. These data indicate that estrogen is an important risk factor even in women considered at high risk of developing the disease, for example, those with a family history of breast cancer.\nEstrogen is thought to contribute to the initiation and contributes to the promotion and progression of breast cancer via two complementary mechanisms [1], the carcinogenic effects of estrogen metabolites, notably hydroxyl metabolites [3, 17, 18], and stimulation of ER signaling pathways, including those initiated by activation of epidermal growth factors, notably the mitogen-activated phosphoinositide 3 kinase pathway [19\u201330]. Greater understanding of the role of estrogen in breast cancer has led to therapeutic strategies targeting estrogen synthesis (aromatase inhibitors [AIs]) [31], the ER (selective ER modulators [SERMs], pure antagonists) [32], and intracellular signaling pathways (signal transduction inhibitors) [33].\nHormone receptor (HR)-positive tumors are defined as those with ER or progesterone receptor (PgR) expression detectable above a pre-set limit [34]. Patients whose ER or PgR expression is below this pre-set limit are considered HR\u2013. Approximately two thirds of breast cancer patients have HR+ tumors [13] and are candidates for treatment strategies designed to counteract the growth effects of estrogen. This review describes the rational development of the potent AI letrozole, which has therapeutic utility in HR+ tumors across the breast cancer continuum.\nMechanism of action of aromatase inhibitors\nAromatase\nAromatase (cytochrome P-450 [CYP] 19) catalyzes the rate-limiting step (conversion of steroidal C-19 androgens to C-18 estrogens) in estrogen biosynthesis [35\u201337]. Aromatization is the final step in steroid biosynthesis (Fig.\u00a01) [38]; and, therefore, aromatase is an attractive target for selective inhibition [39, 40]. Aromatase is expressed primarily in the ovary and also in central and peripheral tissues, fat, muscle, liver, and breast [41, 42]. With increasing age, as ovarian estrogen production declines [43], the contribution of peripheral production of estrogens increases [44], and in postmenopausal women, peripheral aromatization of androstenedione produced by the adrenal gland (Fig.\u00a01) [38] becomes the main source of endogenous estrogens [45\u201349]. Of note, normal and malignant breast tissue contributes to the peripheral synthesis of estrogens [14, 50\u201353]. Thus, expression of aromatase in breast tumors may contribute significantly to the degree of cellular exposure to estrogens [14]; therefore, it is important to target both intra-tumoral and peripheral aromatase [31].\nFig.\u00a01Aromatization of androgens to estrogens in postmenopausal women. A androstenedione, E1 estrone, E1S estrone sulfate, E2 estradiol, T testosterone. Reprinted from [38] with permission from the Society of Endocrinology\nThe presence of intracellular aromatase activity could explain why estrogen concentrations are 10\u201320\u00a0times higher in peripheral tissue than blood in postmenopausal but not pre-menopausal women [41, 54\u201358]. Moreover, estrogen concentrations are higher in tumors than in surrounding non-malignant tissue [41, 54\u201358]. Recent research has increased understanding of how aromatase is regulated by tissue-specific promoters [59] and how genetic variation may affect the pathophysiology of estrogen-dependent disease [60]. Pharmacogenomics may become an increasingly important tool for individualizing hormonal therapy for patients with breast cancer.\nAromatase inhibitors\nModern third-generation AIs effectively block the production of estrogen without exerting effects on other steroidogenic pathways and have been heralded as a \u201ctriumph of translational oncology\u201d [61]. The search for potent and selective inhibitors of aromatase started with the first-generation inhibitor aminoglutethimide [62]. However, aminoglutethimide lacked selectivity for aromatase [63] and inhibited biosynthesis of cortisol, aldosterone, and thyroid hormone [64] as well as aromatase; moreover, aminoglutethimide was also found to induce hepatic enzymes (Fig.\u00a02) [65, 66]. Second-generation AIs included the nonsteroidal inhibitor fadrozole and the steroidal inhibitor formestane (4-hydroxyandrostenedione). Fadrozole was superior to aminoglutethimide in terms of potency, selectivity, and safety [67], but its selectivity was not complete and clinical trials suggested that it was no more effective than tamoxifen [68, 69].\nFig.\u00a02The development of aromatase inhibitors (AIs) has culminated in agents with high specificity and potency for aromatase. Spectrum of action of first- through third-generation AIs: The third-generation AIs act exclusively on the aromatase enzyme and do not appear to exert additional effects. Potency of AIs determined by degree of inhibition of total body aromatase: 4-OHA 4-hydroxyandrostenedione. Reprinted from [66] with permission from the Society of Endocrinology\nTo improve on fadrozole, Novartis synthesized a series of new compounds. Structure-activity relationship studies were then performed to identify the most potent AI from a series of benzyl-azole derivatives of fadrozole [70]. The third-generation AI letrozole (Femara\u00ae) was the result of this structure-activity approach to drug design and achieved the research goal of creating a highly potent and totally selective AI [71]. These compounds were also used to design pioneering molecular modeling techniques used to map the active site of aromatase [70, 72]. Other third-generation AIs developed during this period were the nonsteroidal agents vorozole (since discontinued) and anastrozole [73] (Fig. 2) [66] and the steroidal agent exemestane [74]. AIs have been classified as steroidal (type I; for example, exemestane) or nonsteroidal (type II; for example, letrozole and anastrozole) [75]. A comprehensive review of AIs focuses on the pharmacology and clinical development of letrozole [76].\nLetrozole pharmacodynamics and pharmacokinetics\nPotency\nThe chemical structure of letrozole (4,4\u2032-[(1H-1,2,4-triazol-1-yl) methylene] bis-benzonitrile) is compared with other AIs in Fig.\u00a03 [77]. The nitrogen-containing structures like the imidazoles and the triazoles bind to the iron in the heme moiety of CYP-450, whereas the cyanobenzyl moiety present in the nonsteroidal AIs such as letrozole partially mimics the steroid backbone of the enzyme\u2019s natural substrate androstenedione. Furthermore, the triazole compound letrozole was found to be superior to other derivatives of fadrozole in terms of in vivo inhibition of aromatase [70].\nFig.\u00a03Comparison of the molecular structures of aromatase inhibitors. Reprinted from [77] with permission from Elsevier\nLetrozole is a highly potent inhibitor of aromatase in vitro, in vivo in animals, and in humans. The relative potencies of letrozole, anastrozole, and fadrozole were determined in a variety of model cellular endocrine and tumor systems containing aromatase (hamster ovarian tissue fragments, adipose tissue fibroblasts from normal human breast, the MCF-7Ca human breast cancer cell line transfected with the human aromatase gene, and the JEG-3 human choriocarcinoma cell line) [31]. These studies showed that although letrozole and anastrozole are approximately equipotent in a cell-free aromatase system (human placental microsomes), letrozole is 10\u201330 times more potent than anastrozole in inhibiting intracellular aromatase in intact rodent cells, normal human adipose fibroblasts, and human cancer cell lines (Fig.\u00a04) [31]. In several other studies, letrozole has consistently demonstrated greater potency compared with anastrozole, exemestane, formestane, and aminoglutethimide (Table\u00a01) [31, 71, 75, 78\u201382].\nFig.\u00a04Relative potencies with which letrozole, anastrozole, and fadrozole inhibit aromatase from non-cellular and intracellular sources. Reprinted from [31] with permission from ElsevierTable\u00a01Inhibitory concentrations of letrozole, anastrozole, exemestane, fadrozole, 4-hydroxyandrostenedione and aminoglutethimide against the aromatase enzyme derived from various cellular and non-cellular sources. Reprinted from [77] with permission from ElsevierAromatase inhibitorIC50 values (nM), (relative potency; letrozole\u00a0=\u00a01) Human placental microsomesParticulate fractions of human breast cancerRat ovarian microsomesMCF-7Ca cancer cellsJEG-3 cancer cellsCHO cellsHamster ovarian tissueHuman breastLetrozole2 (1)0.8 (1)Anastrozole8 (0.25)15 (0.053)Exemestane15 (0.13)5 (0.16)4-OHA30 (0.07)30 (0.027)AG20,000 (0.0001)10,000 (0.0008)Letrozole11 (1)0.07 (1)0.07 (1)20 (1)0.8 (1)Anastrozole23 (0.48)0.82 (0.085)0.99 (0.071)600 (0.033)14 (0.057)Fadrozole5 (2.2)0.05 (1.4)0.07 (1.0)30 (0.67)1 (0.80)4-OHA62 (0.18)AG1900 (0.0058)Letrozole1.02 (1)0.35 (1.0)0.45 (1)0.14 (1)Anastrozole5.35 (0.19)3.62 (0.097)5.66 (0.080)17.17 (0.0082)4-OHA0.59 (0.59)1.6 (0.28)0.72 (0.19)Letrozole7 (1)Anastrozole25 (0.28)Fadrozole7 (1)Letrozole1.4 (0)Anastrozole27 (0.052)4-OHA60 (0.023)AG5500 (0.00025)4-OHA 4-hydroxyandrostenedione, AG aminoglutethimideValues quoted are IC50 values representing the concentration needed to achieve 50% inhibition of aromatase activity. The relative potency of each inhibitor compared with letrozole is shown in parentheses\nThe degree of aromatase inhibition can be determined in vivo by measuring uterine weight after treatment with a standard dose of androstenedione in immature female rats [71]. Using this assay, it was found that the in vivo potency of letrozole is more than four orders of magnitude greater than aminoglutethimide (50% effective dose [ED50], 1\u20133\u00a0\u03bcg\/kg vs. 30\u00a0mg\/kg, respectively) [71]. It has also been shown that neoadjuvant letrozole profoundly inhibits in situ aromatase activity and reduces endogenous estrogens within the breast in postmenopausal women with large primary breast cancers [75].\nIn postmenopausal women, letrozole achieves significantly greater plasma estrogen suppression of estrogens and greater inhibition of in vivo aromatization than anastrozole [83]. In the study, levels of aromatase were detectable in 11 of 12 patients during treatment with anastrozole (mean percentage inhibition in the whole group, 97.3%) but in none of the 12 patients during treatment with letrozole (>99.1% suppression in all patients; Wilcoxon, P\u00a0=\u00a00.0022, comparing the two drug regimens). Suppression of estrone and estrone sulfate was found to be significantly greater during treatment with letrozole compared with anastrozole (P\u00a0=\u00a00.019 and 0.0037, respectively). Another study conducted in 54 postmenopausal women with invasive breast cancer showed that more complete inhibition of aromatase was achieved with 2.5\u00a0mg of letrozole than 1\u00a0mg of anastrozole, resulting in significantly greater suppression of estradiol (P\u00a0<\u00a00.0001), the most bioactive estrogen [84]. This recent study confirms previous observations showing that letrozole produces near complete inhibition of aromatase in peripheral tissues, associated with greater suppression of estrogen than achieved with other AIs [78, 85\u201390].\nSelectivity\nLetrozole is highly selective for aromatase and unlike first- and second-generation AIs does not significantly affect cortisol, aldosterone, or thyroxine [77]. In vitro studies showed that letrozole was more than three orders of magnitude more selective than aminoglutethimide in its effects on progesterone and corticosterone production, and more than 300-fold more selective against aldosterone than fadrozole [71, 78]. In vivo adrenocorticotrophic hormone (ACTH) stimulation tests in rats showed that letrozole had no significant effect on either aldosterone or corticosterone levels, even at a dose 1,000\u00a0times greater than that required for inhibition of aromatase [71].\nThe selectivity of letrozole has been demonstrated in clinical studies in postmenopausal women. These studies showed that letrozole has no effect on the plasma levels of 17\u03b1-OH progesterone, thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), or androstenedione and does not affect normal urine electrolyte excretion or thyroid function [86, 91\u201393]. Of note, the vast majority of patients treated with letrozole have a normal response to synthetic ACTH [86].\nAnti-tumor activity in vivo\nThe potent anti-tumor effects of letrozole have been demonstrated in several animal models [77, 78, 94]. Letrozole induced complete regression of estrogen-dependent, 9,10-dimethylbenz-a-anthracene (DMBA)-induced mammary tumors in adult female rats [95]. The ED50 for letrozole was determined to be 10\u201330\u00a0\u03bcg\/kg\/day.\nThe use of MCF-7 cells transfected with human aromatase gene (MCF-7Ca) and implanted into athymic nude mice has proved to be an effective in vivo model for predicting clinical results with AIs [61, 96, 97]. Using this model, it has been shown that letrozole produces dose-dependent inhibition of tumor growth, resulting in complete inhibition at a daily dose of 10 \u03bcg\/animal\/day [94, 98]. Comparative studies using the MCF-7Ca model have shown that letrozole is more effective at suppressing tumor growth than the pure anti-estrogen fulvestrant and the SERM tamoxifen [99]. While anastrozole was also better than fulvestrant and tamoxifen in suppressing tumor growth, only letrozole was shown to induce tumor regression [99].\nAnother study, also using the MCF-7Ca model, demonstrated that letrozole potently inhibits mammary tumor growth but does not have the estrogenic effects of tamoxifen, as measured by its uterotrophic effects [100]. The observation that tamoxifen has an agonist effect even when estrogen synthesis is inhibited by letrozole suggests that there may be a degree of antagonism between these compounds [100]. Interestingly, studies in the MCF-7Ca model showed that letrozole is more effective as monotherapy than when combined with tamoxifen [80, 101]. In the study reported by Long et al. [101] tumor volume doubling times were 3\u20134\u00a0weeks in controls, 16\u00a0weeks with tamoxifen alone, 18\u00a0weeks with tamoxifen plus letrozole, and 34\u00a0weeks with letrozole alone. First-line treatment with letrozole was shown to be significantly superior to treatment with tamoxifen alone or with the two drugs combined (at week 16, both P\u00a0<\u00a00.001). Tumors that progressed during treatment with tamoxifen remained sensitive to second-line letrozole therapy, whereas tumors that progressed on letrozole did not respond to second-line treatment with tamoxifen or fulvestrant. In another series of experiments conducted by the same group using the MCF-7Ca model, letrozole was even effective as third-line therapy for a limited period when administered after treatment with tamoxifen and exemestane [102]. The studies showed that although exemestane was more effective than tamoxifen in controlling tumor growth, letrozole as first-line therapy was the most effective treatment overall, both in terms of the degree of tumor suppression and the length of effectiveness of treatment [102].\nThe potential of letrozole as a chemopreventive agent was investigated in an in vivo model using aromatase-transgenic female mice [103]. The model provided evidence to show that aromatase overexpression is sufficient to induce and maintain early preneoplastic and neoplastic changes that can be completely abrogated by treatment with letrozole. Carcinogenicity studies have also found that letrozole decreases the incidence of spontaneous mammary tumors and granular cell tumors in rats [104].\nPharmacokinetics of letrozole\nClinical pharmacokinetic studies of letrozole have been conducted in healthy volunteers [105\u2013107] and in patients with breast cancer [108, 109]. Following oral administration, letrozole is rapidly and completely absorbed (mean absolute bioavailability of 99.9%) and extensively distributed to tissues. It has a large apparent volume of distribution at steady state (1.87 l\/kg [range, 1.47\u20133.24]), and approximately 60% is bound to plasma proteins, mainly to albumin (55%). The terminal half-life (T1\/2) of letrozole is 42\u00a0h. The terminal T1\/2 was observed to be longer and area under the curve (AUC) greater in patients with breast cancer than in healthy volunteers, possibly due to reduction in metabolic clearance [109]. The major route of elimination of letrozole is metabolism by CYP-450 isoenzymes (CYP 3A4 and CYP 2A6) into an inactive carbinol metabolite. Systemic exposure to metabolites is, therefore, low. Steady-state concentrations of letrozole are reached after 2\u20136\u00a0weeks and maintained for long periods with no evidence of drug accumulation.\nIn marked contrast to the first-generation AI aminoglutethimide, no significant drug interactions have been reported for letrozole; however, when combined with tamoxifen, letrozole plasma concentrations are reduced by between 35% and 40% [110]. Age does not have an effect on the pharmacokinetics of letrozole. Exposure to letrozole, measured by AUC, is increased in renally impaired subjects but remains in the range seen in subjects without impaired function. However, hepatic impairment can markedly increase the T1\/2 of letrozole, and caution is required in such patients.\nDifferences in pharmacokinetics, including uptake rates, elimination T1\/2, and metabolism and clearance exist between AIs and have been reviewed by L\u00f8nning et al. [111]. The clinical significance of such differences is not known.\nClinical development of letrozole\nLetrozole entered clinical trials on the basis of its high potency and selectivity for aromatase, the demonstration of unsurpassed anti-tumor effects in models of human breast cancer, and the development of a convenient oral formulation. Daily doses of 0.1\u20135\u00a0mg have been shown to suppress estradiol, estrone, and estrone sulfate plasma concentrations by 75\u201395% from baseline, while doses >0.5\u00a0mg suppress estrogens to below limit of detection [92, 112, 113]. Based on pharmacokinetic and pharmacodynamic studies, the recommended dose of letrozole is one 2.5\u00a0mg tablet once daily.\nPreclinical models [97, 101] successfully predicted that letrozole would be superior to tamoxifen, the previous gold standard in the treatment of breast cancer. An extensive program of clinical trials has been conducted with letrozole across the spectrum of hormone-responsive breast cancer in postmenopausal women. The first randomized controlled trials demonstrated consistent superiority for letrozole compared with megestrol acetate, aminoglutethimide, and tamoxifen in patients with advanced breast cancer [114\u2013118]. The clinical efficacy of letrozole in advanced breast cancer is described in a review by Dr. Mouridsen in this supplement.\nPreclinical MCF-7Ca models have also predicted that letrozole should be clinically more effective than other less potent third-generation AIs [99, 102]. Letrozole (2.5\u00a0mg\/day) and anastrozole (1\u00a0mg\/day) were directly compared in a randomized, open-label phase IIIb\/IV study involving 713 postmenopausal women with advanced breast cancer previously treated with an anti-estrogen [119]. While there was no difference between the treatment arms in the time to progression, letrozole produced a significantly higher overall response rate than anastrozole (19.1 vs. 12.3%, P\u00a0=\u00a00.013). Letrozole and anastrozole are currently being compared in a large randomized head-to-head trial in early breast cancer (ClinicalTrials.gov identifier NCT00248170) [120]. A review by O\u2019Shaughnessy in this supplement provides the rationale for this trial and a description of its design.\nThe clinical benefits of letrozole in early breast cancer have already been demonstrated in landmark randomized clinical trials. MA.17 was the first trial to show improved clinical outcomes with extended adjuvant hormone therapy [121]. In this trial, letrozole given after initial adjuvant therapy with tamoxifen significantly improved disease-free survival compared with placebo [121, 122]. Full details of this trial are provided in a review by Dr. Goss in this supplement.\nSubsequently, the Breast International Group 1-98 trial provided high-level evidence for the superiority of letrozole over tamoxifen as initial adjuvant therapy [123]. A detailed description of this ongoing trial, which will also help to define the optimal sequence for hormone therapies in hormone-responsive early breast cancer, is provided in a review by Dr. Th\u00fcrlimann in this supplement. Letrozole has also demonstrated superior efficacy compared with tamoxifen when used as neoadjuvant therapy [124]. This treatment setting is particularly interesting in terms of drug development because the effects of hormone therapy on breast tumors can be detected early and may be predictive of long-term outcome [125].\nConclusions\nLetrozole is a highly potent and selective AI that inhibits the enzyme activity of intracellular aromatase at the major sites where it is found, resulting in almost complete suppression of whole body aromatization. By effectively blocking estrogen synthesis, letrozole inhibits the growth or induces the regression of hormone-responsive breast tumors in vivo. Estrogen is implicated as a major risk factor in the majority of breast cancers; therefore, use of the most potent AI is a logical treatment strategy.\nStudies conducted using in vitro and in vivo models have demonstrated that letrozole is the most potent of the third-generation AIs. Preclinical data obtained from MCF-7Ca xenograft models suggest that the greater potency of letrozole compared with anastrozole and exemestane may translate into clinically meaningful differences in postmenopausal women with hormone-responsive breast cancer. These models accurately predicted that letrozole would be more effective than tamoxifen in the clinical setting. The superiority of letrozole over tamoxifen has been consistently demonstrated in advanced and early breast cancer [118, 123]. Outstanding clinical questions, including what is the most effective AI and what is the optimal sequence for adjuvant hormonal therapy, will be answered by the results of ongoing trials involving letrozole.\nIn conclusion, experimental data indicating that letrozole efficiently inhibits aromatase activity have been confirmed clinically, leading to approved indications across the spectrum of breast cancer. The broad range of indications for letrozole in unique clinical settings is reshaping the management of hormone-sensitive breast cancer.","keyphrases":["estrogen","breast cancer","aromatase","postmenopausal"],"prmu":["P","P","P","P"]}
{"id":"Breast_Cancer_Res_Treat-3-1-2092410","title":"BRCA1 and BRCA2 germline mutation analysis in the Indonesian population\n","text":"Specific mutations in BRCA1 and BRCA2 genes have been identified in specific populations and ethnic groups. However, little is known about the contribution of BRCA1 and BRCA2 mutations to breast cancers in the Indonesian population. One hundred-twenty moderate to high risk breast cancer patients were tested using PCR-DGGE, and any aberrant band was sequenced. Multiplex ligation-dependent probe amplification (MLPA) was performed on all samples to detect large deletions in the two genes. Twenty-three different mutations were detected in 30 individuals, ten were deleterious mutations and 20 were \u201cunclassified variants\u201d with uncertain clinical consequences. Three of seven (c.2784_2875insT, p.Leu1415X and del exon 13\u201315) and two of four (p.Glu2183X and p.Gln2894X) deleterious mutations that were found in BRCA1 and BRCA2 respectively, are novel. Several novel, pathogenic BRCA1 and BRCA2 germline mutations are found in early onset Indonesian breast cancer patients, these may therefore be specific for the Indonesian population.\nIntroduction\nBreast cancer is the most common cancer in women. In 5% to 10% of breast cancer cases, the disease results from a hereditary predisposition [1, 2], which can to a large extent be attributed to mutations in either of two tumour suppressor genes, BRCA1 (MIM# 113705) and BRCA2 (MIM# 600185) [3\u20135]. These genes are involved in DNA repair as well as transcriptional regulation [6, 7]. Women carrying pathogenic germline mutations in either of these genes tend to develop breast cancer at an early age [8, 9].\nThe BRCA1 and BRCA2 genes encode large proteins of 1,863 and 3,418 amino acids, respectively. Over 300 distinct mutations in BRCA1 and BRCA2 have been described [10, 11]. These mutations are widely scattered across both genes and most affect the structure and function of the gene. Nevertheless, a significant proportion (34% of BRCA1 and 38% of BRCA2 mutations) (http:\/\/www.nhgri.nih.gov\/Intramural_research\/Lab_transfer\/Bic) are missense mutations that alter one amino acid, but do not truncate the protein and are rare sequence variants of unknown functional consequence. Moreover, a number of base substitutions do not alter the amino acid sequence or result in amino acid changes not associated with disease (polymorphisms) [12]. Hence the biggest challenge in interpreting the mutation analysis of BRCA1 and BRCA2 genes is to distinguish between harmless polymorphisms and deleterious mutations associated with increased cancer risk.\nIn addition, mutations specific for certain populations and ethnic groups have been identified in both genes. For example, specific BRCA1 and BRCA2 mutations were reported for Ashkenazi Jews [13]. Other common BRCA1 mutations were especially found in Italian, Canadian, Belgian or Dutch breast cancer families [14\u201316]. In Indonesia, the contribution of the BRCA1\/BRCA2 mutations to the population incidence of early-onset breast cancer is largely unknown. In one pilot study, however, a new BRCA2 mutation was identified [17] indicating that it was worthwhile to more extensively study the Indonesian population, which was the aim of this study. The accumulating knowledge about the prevalence and nature of BRCA1 and BRCA2 mutations in specific populations may facilitate the interpretation of genetic analysis with regard to breast cancer risk of individual patients.\nMaterials and methods\nPatients\nA total of 120 unrelated breast cancer patients and 16 of their family members from three Indonesian cities (Jakarta and Jogjakarta on the Java island, Denpasar on the Bali island) were analyzed. Breast cancer patients at moderate to high-risk of a hereditary predisposition were selected according to the following criteria: (A) Breast cancer before the age of 41 (n\u00a0=\u00a0102); (B) Two cases of breast cancer in the same family before the age of 60 (n\u00a0=\u00a09); (C) Three or more cases of breast cancer in the same family (n\u00a0=\u00a02); (D) Bilateral breast cancer (n\u00a0=\u00a07). Subjects were asked to fill out questionnaires to evaluate their personal and family histories, and blood specimens were collected for determination of BRCA mutations. Informed consent was obtained from all the subjects in this study.\nDNA extraction and PCR amplification\nGenomic DNA was isolated by the saturated salt extraction procedure as described in [18]. All 22 coding exons of BRCA1 and 26 coding exons of BRCA2 were amplified using primer sequences developed by the University of Groningen, The Netherlands [19]. Primers for DGGE were obtained from Ingeny (Goes, The Netherlands). Genomic DNA was amplified using 100\u2013200\u00a0ng of template DNA, 10\u00a0pmol of the mixture of 40-mer primers, 30\u00a0mM of MgCl, 3\u00a0mM dNTPs (Invitrogen) and 0.7\u00a0unit of Platinum Taq (Invitrogen) in 9\u00a0\u03bcl PCR reactions. In order to speed up the test, the PCR reaction was placed in 384 well plates using a pipetting robot (TECAN Miniprep 75). PCR conditions were performed as previously described [17].\nDenaturing Gradient Gel Electrophoresis and DNA sequencing\nA 4\u20136\u00a0\u03bcl aliquot of each PCR product with relatively large melting temperature differences were pooled as previously described [17] with some modifications for optimal results. The fragment pool was designed based on melting profiles and sequence. Electrophoresis was performed in 0.5 TAE buffer at 58\u00b0C, 120\u00a0V for 16\u00a0h for BRCA1 gene, and 55\u00b0C, 100\u00a0V for 18\u00a0h for BRCA2. Gels were stained with ethidium bromide and photographed under a UV transilluminator. The aberrantly migrating samples were re-amplified using sequencing primers and sequencing was performed using Big Dye Cycle-sequencing kit according to the manufacturer\u2019s instructions. The reaction products were analyzed using an ABI 3100 DNA Sequencer (Applied Biosystems, Torrence, CA, USA) and sequence files were edited using the Bio Edit program. The classification of gene alterations was performed in accordance with the entries in the Breast Cancer Information Core (BIC, Bethesda, MD).\nMultiplex ligation-dependent probe amplification (MLPA)\nThe principle of the MLPA technique has been described elsewhere [20]. The MLPA test for BRCA1 (P02) and BRCA2 (P45) mutations were obtained from MRC-Holland, Amsterdam, The Netherlands. The fragments were analyzed on an ABI model 310 capillary sequencer (Applied Biosystems, Torrence, CA, USA) using Genescan-TAMRA 500 size standards (Applied Biosystems). Fragment analysis was performed with Genescan software.\nResults and discussions\nWe identified 120 incident Indonesian breast cancer cases diagnosed before the age of 41\u00a0years, or having family history of breast cancer, or harboring bilateral breast cancer during September 1999\u2013April 2005 (Jogjakarta) and during July 2004\u2013April 2005 (Jakarta and Denpasar). In addition, 16 of their family members were analyzed.\nThe entire coding regions and exon-intron junctions of BRCA1 and BRCA2 were screened in these 136 persons of breast cancer patients and their families using PCR-DGGE (Fig.\u00a01) followed by sequencing (Fig.\u00a02) for samples with aberrant migrating bands. To optimize the screening, MLPA, a relatively new technique, was also performed in all samples (Fig.\u00a03). Here, we report on 116\/120 women (96.7%) for whom BRCA1\/2 analysis were completed. The remaining four patients (all from group A) had to be excluded due to the small amount of extracted DNA that did not allow complete screening of the BRCA1 and BRCA2 genes.\nFig.\u00a01DGGE analysis of fragments 11.15\u00a0g, 11.4 and 11.10 of the BRCA2 gene in ten unrelated breast cancer patients. The arrows show altered band mobility compare to other patientsFig.\u00a02Sequence electropherogram of a normal individual showing (A) wild-type BRCA2 exon 11 sequence and (B) of breast cancer patient (B-3-5) showing c.2699_2704delTAAATG mutationFig.\u00a03MLPA analysis of BRCA1 gene of patient sample (blue) compare to the normal control (red). X and Y axis represent peak size and peak height respectively. There are reduced peaks in the patient sample compared to the normal control in exons 13, 14 and 15 (arrows) indicating deletions\nBRCA1 and BRCA2 pathogenic mutations\nThe analysis of 116 unrelated breast cancer patients with breast cancer revealed that nine patients (7.8%) carried pathogenic germline mutations especially the early onset patients: 3 within BRCA1 (2.6%) and 6 within BRCA2 (5.2%) which is comparable to previous studies [21]. We only found BRCA1 and BRCA2 mutations in groups A (\u201cearly onset\u201d, n\u00a0=\u00a07 out of 98, 7.1%) and B (two cases of breast cancer in the same family before the age of 60, n\u00a0=\u00a02 out of 9 (22.2%)) (Table\u00a01). There were twice as many BRCA2 mutations as BRCA1 mutations. Although the absolute numbers are low and no firm conclusions can therefore be drawn, this is comparable to other Asian regions [22\u201324] but seems to discern the Indonesian population from non-Asian ethnic groups where the reverse trend is seen.\nTable\u00a01BRCA1 or BRCA2 germline mutations in Indonesian women with early onset breast cancerPatientAgeageneExonMutationbmutation typePathogenic mutationBICcAE25BRCA111c.2784_2785insTframeshift+noB1031BRCA113p.Leu1415Xnonsense+noAA40BRCA113\u201315\u2212dlarge rearrangement+noAB34BRCA211c.3040_3043del4frameshift+1B566BRCA211p.Glu2183Xnonsense+noB665BRCA211p.Glu2183Xnonsense+noB-III-530BRCA211p.Leu824Xnonsense+noAZ40BRCA211p.Leu824Xnonsense+noW-II37BRCA221p.Gln2894Xnonsense+noQ-II40BRCA12c.101\u201310T>CIVS\u00b16P-III-1919BRCA19p.Val191IleMissense\u00b16J2232BRCA111p.Leu1209ValMissense?noAZ40BRCA116p.Met1652IleMissense\u00b135B1 24BRCA120c.5313\u201331A>GIVS?noB731BRCA124p.Arg1835GlnMissense?no21633BRCA124p.Thr1852IleMissense?noP-III-1919BRCA25p.Gln147ArgMissense\u00b16B324BRCA210p.Gln609GluMissense?noC-II-739BRCA211p.Met1149ValMissense\u00b15AO28BRCA211p.Met1149ValMissense\u00b15AQ44BRCA211p.Met1149ValMissense\u00b15BH38BRCA211p.Met1149ValMissense\u00b1517236BRCA211p.Gln699LeuMissense?noJ3229BRCA211p.Arg2108CysMissense\u00b116J633BRCA211p.Val950IleMissense?no20637BRCA225c.9485\u201316T>CIVS\u00b14BC35BRCA227p.Ile3412ValMissense\u00b110916633BRCA227p.Ile3412ValMissense\u00b1109J2435BRCA227p.Ile3412ValMissense\u00b110920637BRCA227p.Lys3326Xnonsense \u00b1289aAge at time of diagnosisbGen Bank Accession number, BRCA1: U14680, BRCA2: U43746cnumber of times reported in BICdnot determined, detected by MLPA\nSeven pathogenic mutations were found in nine probands: three in BRCA1 (c.2784_2785insT, pL1415X (c.4361_4362insT), del exon 13\u201315) and four in BRCA2 (c.3040_3043delGCAA, p.Glu2183X (c.6775G>T), p.Leu824X (c.2699_2704delTAAATG), p.Gln2894X (c.9008C>T)). All these mutations were classified as pathogenic as they are predicted to result in protein truncation. The three pathogenic mutations found in BRCA1 were not previously reported in the BIC database as well as two novel nonsense mutations (p.Glu2183X and p.Gln2894X) identified in BRCA2. The p.Glu2183X mutation was found in 2 related patients that had breast cancer above the age of 60.\nOne of seven pathogenic mutations found in BRCA1 and BRCA2 showed a significant clinical impact on the patient (Table\u00a02). Patient AE with a one nucleotide insertion (Thymine) between nucleotide 2784 and 2785 (c.2784_2785insT) in exon 11 of BRCA1 suffered from bilateral breast cancer at a relatively early age (25\u00a0years). The insertion leads to frameshift and creates a premature stop codon in exon 11. The mutation takes place in the sequence within BRCA1 encoding for aminoacids 758\u20131064 which interact with RAD51 protein that is required for homologous recombination (HR) repair of double strand breaks (DSBs) [25], which is one of the most important functions of the BRCA1 protein. This patient presented in a late stage (stage III for both breasts) and only survived for 9\u00a0weeks after treatment. Her mother did not carry this mutation. Although her father may be carrier, the mutation is probably de novo as there was no family history of breast or other cancers.\nTable\u00a02Clinicopathological features of Indonesian breast cancer patients with deleterious BRCA1 or BRCA2 germline mutationsPatientAgeaGene with germline mutationMutationbstageDiagnosisMenopausal statusfamily history of cancerSurvival statusAE25BRCA1c.2784_2785insTIIIB\/IIIAIDC, bilateralpreNoDOD 9 wB1031BRCA1p.Leu1415XIIDCpreNoDOD 57 wAA40BRCA1-cIIIBIDC N+preNoAWDAB34BRCA2c.3040_3043del4IIIBIDC N+preSister, IntDOD 17 wB563BRCA2p.Glu2183XIVTubularpostSister,BrAWDB665BRCA2p.Glu2183XIIIIDCpostBrother, BrAWDB-III-530BRCA2p.Leu824XIIDCpreNoAWDAZ40BRCA2p.Leu824XIVIDCpreSister, CvDOD 46 wW-II37BRCA2p.Gln2894XIIIAIDCpreNoDOD 107 waAge at time of diagnosisbGen Bank Accession number, BRCA1: U14680, BRCA2: U43746cnot determined, detected by MLPAIDC: invasive ductal carcinoma; DOD: dead of disease; bil: bilateral breast cancer; N+: with metastatic to lymph node; Int: intestinum cancer, Br: breast cancer; Cv: cervical cancer\nThe second pathogenic mutation with a significant clinical manifestation was a cytosine for thymine substitution on nucleotide 9008 of BRCA2 leading to a premature stop codon in position 2894, c9008C>T (p.Gln2894X). Patient W presented at age 37 in a late stage and survived for only 107\u00a0weeks after initial treatment. She had no family history of breast or other cancers. This mutation lies within exon 21 of BRCA2 which is the proposed site for interaction with the DSS1 protein that seems to have a fundamental role in enabling the BRCA2-RAD51 complex to associate with sites of DNA damage [26].\nThe c.2699_2704delTAAATG (p.Leu824X) in BRCA2 that has been reported previously by us in the Indonesian population [17], was found in one other patient in the present study (Table\u00a01). This mutation lies in exon 11 BRCA2, within the BRC repeats domain. The truncating mutation causes loss of three quarters of the protein leading to lack of interaction with the RAD51 protein. Different from BRCA1, the repair of DSBs by HR is the most important function of the BRCA2 protein [27]. Patient B-III-5 was diagnosed with early stage breast cancer at age 30 with no family history of breast or other cancers. Her sister carried the same mutation, but with no present clinical manifestation as yet. Patient AZ who was diagnosed at 40\u00a0years of age, presented in late stage, only survived 46\u00a0weeks after initial treatment. This patient also harbored a mutation in exon 16 of BRCA1, a G to A substitution in nucleotide 5075 (c.5075G>A), which leads to amino acid change from Methionine to Isoleucine, (p.Met16521Ile) which has to date been reported 35 times in BIC as a UV mutation. As the c.2699_2704delTAAATG mutation was found in two unrelated patients, this mutation could be a good candidate as a founder mutation.\nNone of the families with more than 3 cases of breast cancer and families with bilateral breast cancer showed pathogenic mutations in the BRCA1 and BRCA2 genes. Family U had four first-degree relatives that were affected by breast cancer. Two of four members had bilateral breast cancer. In spite of this high familial breast cancer incidence, no BRCA1\/2 mutations were found.\nBRCA1 and BRCA2 unclassified variants\nSixteen (7 BRCA1 and 9 BRCA2) rare mutations of so far unknown significance (\u201cunclassified variants\u201d, UVs) were detected in 18 patients: 13 missense changes and 3 intronic variants. Of these 16 UVs, 7 were novel, whereas the other UVs have been previously reported in the BIC database (Table\u00a01). From the 18 patients which carried UV mutations, two patients were detected in families from group D; one patient in a group B family and the other fifteen patients in families from group A.\nSeven UV were found in the BRCA1 gene, two mutations occurring in the intronic region between exons 1 and 2 (c.101\u201310T>C) and between exons 19 and 20 (c.5313\u201331A>G), and five missense mutations identified: p.Val191Ile (c.690G>A), p.Leu1209Val (c.3744T>G), p.Met1652Ile (c.5075G>A), p.Arg1835Gln (c.5623G>A) and p.Thr1852Ile (c.5674C>T).\nFour out of seven BRCA1 missense mutations; p.Leu1209Val (c.3744T>G), c.5313\u201331A>G, p.Arg1835Gln (c.5623G>A) and p.Thr1852Ile (c.5674C>T) were have not been described previously in the BIC. The p.Leu1209Val may not be a significant change as both Leucine and Valine belong to the same group of non polar amino acids. However, p.Arg1835Gln is possibly an important alteration since a positively charged Arginine is replaced by an uncharged Glutamine, which may have an effect on the structure and\/or function of the protein. Another potentially important alteration concerns p.Thr1852Ile, where the hydrophilic amino acid Threonine is replaced by a hydrophobic Isoleucine. The sites of mutation of both p.Arg1835Gln and p.Thr1852Ile also have to be considered as they lie within the site for the activation domain of the BRCA1 protein [28]. The intronic UV c.5313\u201331A>G also deserves further investigation as it may theoretically have an effect on splicing. However, according to splice site finder (http:\/\/www.genet.sickkids.on.ca\/\u223cali\/splicesitefinder.html), the splicing sites in the wild type and mutant alleles are similar, so therefore we can suggest that the c.5313\u201331A>G has no effect on splicing.\nNine different UVs of the BRCA2 gene were found in fourteen patients (Table1), and three of them were novel; p.Gln609Glu 9c.2053C>G), p.Gln699Leu (2324A>T) and p.Val950Ile (3076G>A). One truncating mutation near the C-terminal end of BRCA2, p.Lys3326X (c.10204A>T) is probably not pathogenic. Since the truncating mutation is at the very end of the protein, it is possible that protein functions are not affected. Most of the few entries in databanks describing nonsense mutations near the C terminus of BRCA2 between codon 3308 and 3408 are described as UVs. Thus, the effect of this truncating mutation on cancer predisposition remains unclear.\nThe p.Val950Ile may not be a significant change as both Valine and Isoleucine belong to the same group of non polar, hydrophobic amino acids. However, p Gln609Glu and p.Gln699Leu are potentially important alterations as for p.Gln609Glu, a non acidic, polar, hydrophilic Glutamine is replaced by a negatively charged Glutamic acid, whereas for p.Gln699Leu, an uncharged hydrophilic Glutamine is replaced by a hydrophobic Leucine. As it takes place within the BRC repeats of the BRCA2 protein, the p Gln699Leu alteration might affect protein structure and function.\nTo know more about the importance of amino acid substitutions for protein function, we compared the amino acid sequence of interest in seven other species, i.e. Mus musculus, Rattus rattus, Bos taurus, Gallus gallus, Canis familiaris, Macaca mullata and Monodelphis domestica. The missense mutation p.Leu1209Val lies in the conserved region of exon 11 of the BRCA1 gene as the sequence is maintained in seven other species, whereas p.Arg1835Gln and p.Thr1852Ile are only conserved in four and three other species (comparison of p.Arg1835Gln and p.Thr1852Ile with Bos taurus sequence is not possible because the BRCA1 gene is shorter). Therefore, even tough the Leucine to Valine changes may not give any effect on amino acid charge, its conservation in evolution is suggestive of a functional role. Interestingly, p.Gln609Glu and p.Gln699Leu of BRCA2 that result in a quite dramatic amino acid subtitution that might lead to protein structure changes, are only conserved in four and five species respectively. As for the p.Val950Ile, the conservation in evolution is quite low. Although p.Gln609Glu is less conserved, we still believe that Glutamine to Glutamic acid substitution may have an effect on protein conformation as two adjacent acidic amino acids will be formed as the result of the substitution.\nGlycosylation moiety of an amino acid also plays a role in protein function. Amino acid substitutions involving Serine, Threonine and Asparagine, should also be checked for their O-GlcNac potential and threshold. Here we have a Threonine to Isoleucine substitution (p.Thr1852Ile) that after checking with YinOYang (http:\/\/www.cbs.dtu.dk\/services\/YinOYang) showed no significant threshold changes between the wildtype and the mutant allele.\nThe possible effect of amino acid changes in proteins can also be assessed using similarity scores (based on Grantham table [29]), in which a value above 100 for an amino acid substitution indicates a higher chance of impact on protein function. Among seven novel UVs in the BRCA1 and BRCA2 genes found in the present study, only p.Gln699Leu in BRCA2 has a similarity score above 100, whereas p.Gln609Glu and p.Val905Ile in BRCA2 have the lowest score (Table\u00a03).\nTable\u00a03The amino acid properties of novel unclassified mutations in BRCA1 and BRCA2 within an Indonesian breast cancer populationGeneAmino acid changeChange of chargeChange of amino acid groupSimilarity scorea# species with conserved sequenceBRCA1Leu to ValNoneNo327a,b,c,d,e,f,gBRCA1Arg to GlnPos to no chargeYes434a,c,f,gBRCA1Thr to Ilepolar to non polarYes893a,c,gBRCA2Gln to GluNo charge to negYes294a,b,c,gBRCA2Gln to LeuPolar to non polarYes1135a,b,d,e,fBRCA2Val to IleNoneNo292f,gabased on Grantham table [Grantham et\u00a0al. [29], a score above 100 indicates significance changesa\u00a0=\u00a0Macaca mullata, b\u00a0=\u00a0Bos taurus, c\u00a0=\u00a0Canis familiaris, d\u00a0=\u00a0Rattus rattus, e\u00a0=\u00a0Mus musculus, f\u00a0=\u00a0Gallus gallus, h\u00a0=\u00a0Monodelphis domestica\nOverall, we propose that among the seven novel UVs, there are three mutations that are possibly pathogenic: p.Leu1209Val for its location in a conserved region, and p.Gln609Glu and the p.Gln699Leu because of two adjacent acidic amino acid being formed and a high similarity score, respectively.\nWhen comparing the three different Indonesian regions, the percentages of breast cancer patients with pathogenic BRCA1\/2 mutations was significantly higher in Denpasar (Bali island) than in Jogjakarta and Jakarta (Java island) ((25% (3\/12), 7.2% (6\/83) and 0% (0\/25) respectively (P\u00a0=\u00a00.0255, chi-square test)). The percentages of breast cancer patients with UV mutations in Jakarta, Jogjakarta, and Denpasar were 16% (4\/25), 12% (10\/83), and 25% (3\/12), respectively (n.s.). Although the number of patients is too small to draw firm conclusions, these data may point to geographic differences within Indonesia.\nIt was initially suggested that the BRCA1 and BRCA2 genes would be responsible for most cases of inherited breast cancer, but more recent studies suggest that they would account for a far smaller proportion, with considerable variation among different populations [30]. We found that the incidence of mutations in these genes varies, depending on the diagnostic group. In this sense, mutations were present in (22\/102) 21.6% of early onset patients (group A), 28.7% (2\/7) in patients with bilateral breast cancer (group D) and (2\/9) 22.2% of patients with two cases of breast cancer before the age of 60 (group B). The proportion of families affected by BRCA1\/2 mutations depends on the population analyzed and on the criteria used to select the patients. Family history of breast cancer was, however, absent or not suggestive of a hereditary predisposition in three-fourth of the deleterious mutations carriers and in more than 90% of UV carriers. This suggests that BRCA screening policies based on family history only would miss a considerable proportion of mutation carriers.\nIn conclusion, a relatively high percentage of early onset Indonesian breast cancer patients carry a germline mutation in either BRCA1 or BRCA2. Several novel, pathogenic BRCA1 and BRCA2 germline mutations have been found, as well as a variety of novel \u201cunclassified variant\u201d mutations that may therefore be specific for the Indonesian population. It is likely that some of the \u201cunclassified variant\u201d mutations may have a functional role in breast cancer development, which deserves to be explored further.","keyphrases":["brca1","brca2","mutation analysis","hereditary breast cancer"],"prmu":["P","P","P","R"]}
{"id":"Purinergic_Signal-4-1-2246001","title":"Remyelination after chronic spinal cord injury is associated with proliferation of endogenous adult progenitor cells after systemic administration of guanosine\n","text":"Axonal demyelination is a consistent pathological sequel to chronic brain and spinal cord injuries and disorders that slows or disrupts impulse conduction, causing further functional loss. Since oligodendroglial progenitors are present in the demyelinated areas, failure of remyelination may be due to lack of sufficient proliferation and differentiation of oligodendroglial progenitors. Guanosine stimulates proliferation and differentiation of many types of cells in vitro and exerts neuroprotective effects in the central nervous system (CNS). Five weeks after chronic traumatic spinal cord injury (SCI), when there is no ongoing recovery of function, intraperitoneal administration of guanosine daily for 2 weeks enhanced functional improvement correlated with the increase in myelination in the injured cord. Emphasis was placed on analysis of oligodendrocytes and NG2-positive (NG2+) cells, an endogenous cell population that may be involved in oligodendrocyte replacement. There was an increase in cell proliferation (measured by bromodeoxyuridine staining) that was attributable to an intensification in progenitor cells (NG2+ cells) associated with an increase in mature oligodendrocytes (determined by Rip+ staining). The numbers of astroglia increased at all test times after administration of guanosine whereas microglia only increased in the later stages (14 days). Injected guanosine and its breakdown product guanine accumulated in the spinal cords; there was more guanine than guanosine detected. We conclude that functional improvement and remyelination after systemic administration of guanosine is due to the effect of guanosine\/guanine on the proliferation of adult progenitor cells and their maturation into myelin-forming cells. This raises the possibility that administration of guanosine may be useful in the treatment of spinal cord injury or demyelinating diseases such as multiple sclerosis where quiescent oligodendroglial progenitors exist in demyelinated plaques.\nIntroduction\nDemyelination contributes to functional deficits in central nervous system (CNS) disorders [1\u20133]. Traumatic spinal cord injury (SCI) induces local inflammation and demyelination in the white matter around the lesion resulting in disrupted axonal conduction [4\u20137]. Remyelination is critical for recovery, but does not occur spontaneously [8\u201311]. Spinal cords contain endogenous progenitors that can proliferate and differentiate into mature oligodendroglia and remyelinate axons under certain circumstances [12\u201314]. After acute SCI in rats, oligodendrocyte precursors proliferate in the first 2\u00a0weeks but are \u201csilent\u201d thereafter [15], possibly because the oligodendroglial precursors cannot differentiate into mature oligodendroglia capable of remyelinating axons. Enhancement of proliferation of oligodendrocytes or progenitors may lead to repair and restoration of function after SCI.\nNeurotrophic factors restore physiological function by promoting oligodendrocyte proliferation and axonal sprouting [16\u201319], regulating the proliferation of oligodendrocyte progenitors [3] and modulating the differentiation [20\u201322] and the maturation of oligodendrocyte precursors into myelin-forming cells [23, 24].\nGuanosine has trophic effects on many cell types [25\u201328], and we have some evidence that guanine acts similarly. For example, it stimulates proliferation of a variety of cells in culture [25, 28] and promotes synthesis and release of several potentially neuroprotective trophic factors from a variety of cells, including neuron growth factor (NGF) from astrocytes as well as basic fibroblast growth factor (bFGF) and transforming growth factor beta (TGF beta) [25, 27]. Extracellular guanosine itself also enhances outgrowth of nerve processes from PC12 cells and from primary cultures of embryonic rat brain neurons [25]. As well, it acts synergistically with NGF to promote outgrowth of neurites [29, 30].\nOur preliminary data [31] showed that 35\u00a0days after moderate traumatic injury of the spinal cord of rats, when the initial spontaneous incomplete recovery of function had plateaued, systemic administration of guanosine (intraperitoneally 8\u00a0mg\/kg body weight) for 7\u00a0days enhanced locomotor functional recovery that continued to improve throughout the 7-day duration of the treatment and correlated with the increase in myelination. In the present study, we treated rats for up to 14\u00a0days to determine whether guanosine-induced functional improvement was due to triggering the endogenous adult progenitors. Meanwhile, we also examined the uptake and tissue distribution of systemically administered guanosine.\nMaterials and methods\nAll experiments were performed in compliance with the requirements of the Animals for Research Act of Ontario, Canada and the Guidelines of the Canadian Council on Animal Care, and had been approved by the Animal Research Ethics Board of McMaster University.\nSpinal cord injury Adult female Wistar rats (280\u2013300\u00a0g weight, Charles River) were anaesthetized with isoflurane (3\u20135%): O2 (1\u00a0l\/min). Buprenorphine (0.03\u00a0mg\/kg body weight, subcutaneously) was administered prior to surgery for pain relief. Moderate spinal cord injury was induced by crushing exposed cords with modified coverslip forceps [31\u201333]. The forceps were closed slowly over 2\u00a0s and the compression sustained for 15\u00a0s. The muscles and fat pad over the lesion were sutured and the skin was closed with stainless steel clips. Post-operatively, the rats were kept quiet and warm [31, 34, 35].\nBehavioural assessment and drug administration Rats were handled daily for 2\u00a0weeks pre-operatively to acclimatize them to the experimental procedures and behavioural test. After the spinal injury, locomotor recovery was assessed weekly for 35\u00a0days in an open field walking task (OFWT) before treatment. Cagemates (two animals) were placed in the centre of the open field, formed by a child\u2019s circular plastic swimming pool (1.3\u00a0m in diameter). They were observed for 5-min periods and scored for general locomotor ability using the Basso-Beattie-Bresnahan (BBB) locomotor rating scale [36, 37]. Rats were rated on a scale of 0 to 21, with 0 being no function and 21 being normal. If the animal stopped moving for a minute, it was placed again in the centre of the open field; otherwise it was left alone for the duration of the 5-min test period.On day 35 following the injury, rats were randomly assigned to three groups. One group of rats was used for baseline behavioural and histological analysis. The other rats received either daily intraperitoneal (i.p.) injections of 8\u00a0mg\/kg guanosine or the same volume of saline containing 0.001\u00a0N NaOH [31] for 2\u00a0weeks. The open field walking testing was performed once a day from days 35 to 48 post-surgery (0 to 14\u00a0days post-treatment). Groups of 9\u201312 rats were killed by perfusion\/fixation for immunohistochemical analysis at 1, 3, 7 or 14\u00a0days after treatment.\nBrdU administration Bromodeoxyuridine (BrdU) was used to label proliferating cells after treatment. BrdU (50\u00a0mg\/kg in sterile saline; Sigma, B-5002) was injected intraperitoneally daily for 1, 3, 7 or 14 consecutive days, beginning the day treatment was initiated, thereby reflecting cell proliferation over the entire treatment period.\nTissue processing and immunohistochemistry On post-operative days 35, 36, 38, 42 or 49 (representing baseline, 1, 3, 7 or 14\u00a0days treatment), rats were deeply anaesthetized with sodium pentobarbital (50\u201360\u00a0mg\/kg body weight, i.p.) immediately following the final locomotor testing and perfused transcardially with 100\u00a0ml 0.1\u00a0M phosphate-buffered saline (PBS) containing 0.1% heparin, followed by 300\u2013500\u00a0ml of 4% paraformaldehyde (PFA). T9 to L1 segments of the spinal cords were removed and incubated in the same fixative for 2\u00a0h at room temperature and then cryoprotected in 30% sucrose PBS solution. A segment of each cord, extending from 5\u00a0mm rostral to 5\u00a0mm caudal to the lesion site was embedded in medium (Tissue-Tek\u00ae O.C.T. compound, Sakura Finetek USA, Inc., Torrance, CA, USA). Serial transverse sections were cut at 15- to 20-\u03bcm intervals on a cryostat and mounted onto slides (ColorFrost\/Plus; Fisher, Pittsburgh, PA, USA). Every fifth section was stained with the lipophilic dye Luxol fast blue to stain myelin [38]. Adjacent sections were stained immunohistochemically with the various antibodies described below.For BrdU immunohistochemistry, 6% hydrogen peroxide was used to reduce endogenous peroxidase activity, followed by three 10-min PBS rinses and incubation in 1\u00a0N HCl at 37\u00b0C for 30\u00a0min. After rinsing, a blocking solution [5% bovine serum albumin (BSA) in 0.1% Triton-100] was applied for 1\u00a0h; sections were subsequently incubated in rat anti-BrdU (1:200 in 1% BSA in 0.1% Triton-100; Serotec, Raleigh, NC, USA) at 4\u00b0C overnight in a humid chamber. Sections were then rinsed and incubated with biotinylated horse anti-rat IgG secondary antibodies. Sections were then incubated with Elite ABC (Vector Laboratories, Burlingame, CA, USA) for 1\u00a0h, followed by 3,3\u2032-diaminobenzidine (DAB) substrate kit for peroxidase (Vector Laboratories, Burlingame, CA, USA) for 5\u201310\u00a0min. Sections were dehydrated, coverslipped and examined under a microscope.To determine the fate of newly born cells after SCI, double-fluorescent immunolabelling was performed, combining BrdU with one of the cell-specific phenotypic markers listed below. For double immunofluorescence, sections were pretreated with HCl as described above and incubated with BrdU antibody mixed with one of the following antibodies in PBS at 4\u00b0C overnight in a humid chamber: rabbit anti-MBP (myelin basic protein) polyclonal antibodies (1:50; Chemicon Int., Temecula, CA, USA), rabbit anti-NG2 polyclonal antibodies (chondroitin sulfate proteoglycan for oligodendroglial progenitors; 1:200; Chemicon Int. Temecula, CA, USA), mouse anti-Rip monoclonal antibodies (mature oligodendrocyte-specific marker: Reactive Immunology Protein; 1:200; Chemicon Int., Temecula, CA, USA; [39]), rabbit anti-glial fibrillary acidic protein (GFAP) polyclonal antibodies (1:600; Zymed\u00ae Lab-SA System Kit, San Francisco, CA, USA) and mouse anti-microglial-specific marker OX-42 monoclonal antibodies (1:200; CD11b, Research Diagnostics Inc., Flanders, NJ, USA). For Rip and OX-42 monoclonal antibodies single staining, sections were developed with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG. For BrdU and polyclonal antibodies (NG2 or MBP or GFAP) double immunolabelling, sections were developed using a mixture of FITC-conjugated goat anti-rat IgG and rhodamine-conjugated goat anti-rabbit IgG in 1% normal goat serum and 0.25% Triton X-100; 1:200 (Invitrogen, Carlsbad, CA, USA) for 2\u00a0h. To examine the contribution of Schwann cells from the peripheral nervous system (PNS) in the remyelination process, sections were immunolabelled with specific markers for Schwann cell myelin P0 (a rabbit polyclonal anti-P0 antibody was kindly donated by Prof. Marie T. Filbin, Department of Biology, CUNY, NY, USA; 1:200) and secondary antibody using rhodamine-conjugated goat anti-rabbit IgG. For detection of apoptotic cells, a terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick end labelling (TUNEL) stain was performed using the \u2018In situ Cell Death Detection Kit, Fluorescein\u2019 (Roche Molecular Biochemicals, Mannheim, Germany), according to the manufacturer\u2019s instructions.\nQuantification To quantify the amount of myelin present at the injury site before and after treatment, the Luxol fast blue-stained section from each cord (n\u2009=\u20097\u201310 for each group) that contained the greatest lesion area was selected together with the adjacent two caudal and two rostral Luxol blue-stained sections. Luxol fast blue staining was also done on cords of five additional unoperated normal animals for comparative purposes. Digital photographs were taken of the sections. The total area of each section and the area of the fast blue-stained portion were measured using a computerised Bioquant BQ-TCW98 image analysis program by an investigator who was blind to group assignment.For quantification of cell proliferation, five to seven sections taken from the penumbra of the lesion and spaced about 100\u00a0\u03bcm apart were analysed for each animal (n\u2009=\u20097\u20139 animals per group). Cellular proliferation was determined by counting the total number of BrdU+ nuclei in spinal cord cross sections. Low power sections were digitized and manually outlined using an image analysis system. Any cavities present in the sections were excluded from analysis. Positively labelled nuclei were counted automatically. The threshold was set such that only positively labelled profiles of the appropriate size were counted. Data are expressed as cells per section.The total number of NG2+ progenitor cells and the number of those that proliferated (double-labelled for NG2 and BrdU: NG2+\/BrdU+) was determined by manual counting. Cell counting was done conservatively, and a positive profile was counted as a cell only when clearly defined borders could be detected completely around and adjacent to the entire nucleus. Data are expressed as the number of immunostained cells per section.Mature oligodendrocytes in injured cords were identified with Rip, a specific marker for mature oligodendrocytes that recognises an unknown epitope [39] and has been used successfully in the CNS to label both oligodendrocyte processes and myelin sheaths. The number of mature oligodendrocyte (Rip+ cells) in the tissue sections was determined by manual counting.\nEvaluation of exogenous guanosine distribution A guanosine solution (8\u00a0mg\/kg) was administered intraperitoneally to rats with spinal cord injury induced 5\u00a0weeks previously. Samples of spinal cord were taken from the site of the lesion and immediately above or below the lesion just before the intraperitoneal injection of guanosine and at 7.5, 30 and 60\u00a0min after the guanosine injection. There were between three and six animals at each time point. Samples of spinal cord were placed in 0.5\u00a0ml of cold 0.4\u00a0M perchloric acid solution and immediately homogenized. Samples were then centrifuged (4\u00b0C, 10,000\u00a0g, 10\u00a0min) to remove proteins precipitated by perchloric acid and the supernates containing the soluble molecules were neutralized with KOH solution and centrifuged (4\u00b0C, 10,000\u00a0g, 5\u00a0min). Samples were filtered with 0.2-\u03bcm filters (Millipore, Billerica, MA, USA) and stored at \u221270\u00b0C before high-performance liquid chromatography (HPLC) [40] or capillary electrophoresis (CE) analysis.\nTissue concentrations of guanosine and guanine To evaluate the concentration of guanosine and its principal degradation product, guanine, in spinal cords before and after the intraperitoneal injection of guanosine, either HPLC or CE was used. There was no significant difference in the values obtained by these two methods so the data represent the mean of values obtained by both analyses. HPLC analysis was carried out using an Agilent 1100 series HPLC (Agilent Technologies, Waldbronn, Germany) and an ion-pair technique. Separation was carried out with a reverse-phase analytical column (LiChroCART 125-4 LiChrospher 100 RP-18 5\u00a0\u03bcm, Merck, Darmstadt, Germany) and the flow rate was 1.5\u00a0ml\/min. A 15-min linear gradient was applied from 100% buffer A (60\u00a0mM KH2PO4 and 5\u00a0mM tetrabutylammonium phosphate, pH 6) to 100% buffer B (30% methanol plus 70% buffer A). CE separations were carried out using an HP3DCE capillary electrophoresis system (Agilent Technologies, Waldbronn, Germany) equipped with a diode array detection system. Analysis was performed using a 80.5\u00a0cm \u00d7 50\u00a0\u03bcm ID \u00d7 375\u00a0\u03bcm OD uncoated fused silica capillary from Agilent Technologies (Waldbronn, Germany) with an effective length of 72\u00a0cm to the detector window. The capillary temperature was kept constant at 37\u00b0C and the running buffer was 10\u00a0mM borate, pH 9.3. The samples were injected by pressure (50\u00a0mb for 40\u00a0s) and the separation was conducted at 22\u00a0kV. Detection took place at 254\u00a0nm and purines were identified by their migration times and UV spectra using the diode array detector (Agilent Technologies, Waldbronn, Germany).\nStatistical analysis Data are expressed as mean\u2009\u00b1\u2009SEM. Behavioural scores were analysed by the Kruskal-Wallis non-parametric analysis of variance (ANOVA). Post hoc comparisons were made using the Dunnett\u2019s test. Correlation between behavioural and histological outcomes was analysed using regression analysis. Significance was set at p\u2009<\u20090.05.\nResults and discussion\nAfter SCI, motor function in the legs partially recovered over 3\u20134\u00a0weeks, after which there was no further improvement (Fig.\u00a01a). Daily guanosine for 14\u00a0days (8\u00a0mg\/kg intraperitoneally beginning 35\u00a0days after injury) produced improvement in locomotor performance over the next 2\u00a0weeks; control rats showed no improvement (Fig.\u00a01b). This treatment regimen was associated with significantly increased amounts of myelin in the penumbra of the cord lesions shown by Luxol fast blue staining (Fig.\u00a01c\u2013e). The extent of Luxol fast blue staining in the injured cords correlated with each animal\u2019s open field walking scores (r\u2009=\u20090.72; p\u2009<\u20090.05; Fig.\u00a01f), indicating that guanosine-induced functional recovery may be related to the increase in myelin. Immunostaining for CNS myelin basic protein (MBP; Fig.\u00a01g,h) and a specific marker for Schwann cell myelin, P0 (Fig.\u00a01i,j) showed that Schwann cells from the peripheral nervous system did not enter spinal cord and, therefore, did not contribute to the remyelination.\nFig.\u00a01Systemic treatment with guanosine improves locomotor function and correlates with increased myelination in rats with stable chronic spinal cord injury. a After spinal cord crush, locomotor function was assessed in an open field walking task (OFWT) and scored using a standard scale (\u201cMaterials and methods\u201d). No further improvement in locomotor performance occurred after 28\u00a0days. b On days 35\u201348 after injury (days 1 to 14 of treatment in the figure; the first test done immediately prior to the first treatment), animals were randomly divided into two groups. One group received guanosine (\u25b4), the other group received vehicle (\u2666). The locomotor function in the control group did not change significantly throughout the 14\u00a0days of vehicle administration. In contrast, rats that received guanosine showed a marked improvement in locomotor behaviour, which was significantly (p\u2009<\u20090.05) better than vehicle-treated control animals from the 5th day of treatment onward. c, d Luxol fast blue staining of cross sections of spinal cords from guanosine- (d) and vehicle- (c) treated animals at the lesion site 48\u00a0days after the initial lesion (after 14\u00a0days treatment) shows increased Luxol fast blue-stained material in d compared to the vehicle-treated control group in c. DC dorsal column at the lesion. Scale bar\u2009=\u200950\u00a0\u03bcm. e The total area of each section and of the Luxol fast blue-stained portion were measured following the protocol in \u201cMaterials and methods\u201d. The quantitative results show that the cords from guanosine-treated animals had significantly greater myelinated area (*p\u2009<\u20090.05), as estimated from the Luxol fast blue staining, than cords from vehicle-treated control animals, which in turn had significantly less myelinated area (**p\u2009<\u20090.001) than unoperated animals. f Correlation between behavioural and histological outcomes was analysed using regression analysis. The data show that the Luxol fast blue-stained area in the injured cords from animals treated with guanosine correlated with their behavioural recovery in open field walking scores, correlation coefficient of 0.72, indicating that the recovery of function may be related to the increase in myelin. Myelin immunofluorescent staining of cross sections of spinal cords from vehicle- (g) and guanosine- (h) treated animals at the lesion site 14\u00a0days after treatment shows that guanosine-treated cords (h) contained more myelin basic protein (MBP)-positive profiles from central nervous system at the injured site compared to the vehicle-treated control group (g). IS injured site. Whereas, in i (vehicle-treated cord) and j (guanosine-treated cord) immunolabelling with specific markers for Schwann cell myelin P0 indicated that Schwann cells from the peripheral nervous system did not contribute to guanosine-induced remyelination. Scale bar\u2009=\u200950\u00a0\u03bcm for all\nTo determine whether existing cells elaborated the myelin or whether it was a product of other cells that proliferated and subsequently elaborated myelin, the number of proliferating cells was determined by counting all BrdU+ nuclei in cross sections of the lesion penumbra at different times after treatment. In the first 3\u00a0days after treatment, cell proliferation at the injury site in both guanosine-treated and control animals was not different from baseline (immediately before treatment; Fig.\u00a02c), although at 3\u00a0days there was a trend toward more BrdU+ cells in guanosine-treated animals than controls (112\u2009\u00b1\u200925 vs 79\u2009\u00b1\u200916). At 7 and 14\u00a0days after guanosine treatment, the number of BrdU+ cells increased significantly; there were 382\u2009\u00b1\u200942 BrdU+ cells per section at 7\u00a0days and 477\u2009\u00b1\u200949 at 14\u00a0days compared with only 88\u2009\u00b1\u200920 at 7\u00a0days and 95\u2009\u00b1\u200922 at 14\u00a0days in controls (p\u2009<\u20090.05; Fig.\u00a02a\u2013c). There were few dividing cells beyond the lesion margins.\nFig.\u00a02Quantitative assessment of immunostaining shows that systemic treatment with guanosine stimulates endogenous oligodendroglial progenitors (NG2+ cells) to proliferate and mature in rats with stable chronic spinal cord injury. a, b Bromodeoxyuridine (BrdU) was used to label proliferating cells after treatment. BrdU immunostaining in cross sections from vehicle- (a) and guanosine- (b) treated animals at the lesion 14\u00a0days after treatment shows increased BrdU+ nuclei in b compared to the vehicle-treated control group in a. Scale bar\u2009=\u200950\u00a0\u03bcm. c Quantitative analysis shows that 7 or 14\u00a0days after guanosine treatment there was a significant increase in the number of BrdU+ cells compared with vehicle-treated controls. d, e Demonstrate examples of double-fluorescent immunostaining using antibodies against BrdU (in green) and a marker (NG2) for oligodendroglial progenitors (in red) in cross sections from vehicle- (d) and guanosine- (e) treated animals at the lesion 14\u00a0days after treatment. Scale bar\u2009=\u200950\u00a0\u03bcm. f\u2013h Show quantification of proliferating progenitors (NG2+\/BrdU+ double-labelled cells; f) and total number of NG2 cells (g). Data indicate that after 7 or 14\u00a0days administration guanosine significantly stimulates proliferation of oligodendroglial progenitors (f, g). Concurrently, guanosine also significantly increases the number of mature oligodendroglia shown in i and j compared to vehicle-treated controls (h, j) using fluorescent immunostaining with a specific marker (Rip) to label the mature oligodendroglia. Scale bar\u2009=\u200950\u00a0\u03bcm\nOligodendrocytes that survive the demyelinating insult are not thought to be able to contribute to remyelination [41, 42]. Instead, endogenous oligodendrocyte progenitor cells local to the lesion are believed to be the source of new myelinating cells [43]. Previous studies have illustrated that NG2+ cells are the major cycling cell within the resting adult CNS [14, 44] and express many characteristics of oligodendroglial lineage cells, not least their ability to differentiate into oligodendrocytes when isolated into culture. Their role in demyelination and remyelination has been extensively studied and there is evidence that they are the cells responsible for myelin repair [43, 45\u201348]. To determine whether the proliferating cells were related to NG2+ oligodendroglial progenitor cells, we counted the total number of NG2+ progenitor cells and the number that had proliferated (NG2+\/BrdU+) from 2\u00a0mm rostral to 2\u00a0mm caudal to the lesion site (Fig.\u00a02d,e). There was minimal turnover in the NG2+ cells in the spinal cord during the first 3\u00a0days in both guanosine-treated and control animals (13\u2009\u00b1\u20095 in the guanosine group vs 9\u2009\u00b1\u20094 cells per section in controls; Fig.\u00a02f). By 7\u00a0days, guanosine significantly stimulated progenitor proliferation (85\u2009\u00b1\u200918 vs 13\u2009\u00b1\u20094 NG2+\/BrdU+ cells\/section; Fig.\u00a02f); these cells were observed throughout the penumbra with the majority localised in the dorsal columns. Enhanced proliferation of the NG2+ progenitors after guanosine persisted, and by 14\u00a0days, double-labelled progenitors in the guanosine-treated group were eightfold more than in controls (108\u2009\u00b1\u200912 vs 13\u2009\u00b1\u20095 NG2+\/BrdU+ cells\/section; Fig.\u00a02f).\nThe total number of NG2+ cells was also determined. During the first 3\u00a0days, there was no significant difference in the numbers of NG2+ cells in cords at baseline after vehicle treatment or guanosine treatment (Fig.\u00a02g). By 7\u00a0days, guanosine treatment increased the total NG2+ cells from 96\u2009\u00b1\u200922 cells\/section at baseline to 266\u2009\u00b1\u200938 cells\/section\u2014more than 2 times higher than in the control group (110\u2009\u00b1\u200924 \/section; Fig.\u00a02g). By 14\u00a0days, the numbers of double-labelled progenitors in the cords of guanosine-treated animals were threefold higher than controls (347\u2009\u00b1\u200936 NG2+ cells\/section for guanosine-treated animals; 115\u2009\u00b1\u200924 cells\/section for controls; Fig.\u00a02g).\nMature oligodendrocytes were identified with Rip+ staining (Fig.\u00a02h,i). Guanosine had no effect on mature oligodendroglia 3\u00a0days after treatment; at 7 and 14\u00a0days, there was a significant increase in Rip+ cells in the penumbra of the cords of animals that had received guanosine compared to controls (7\u00a0days: 468\u2009\u00b1\u200940 vs 205\u2009\u00b1\u200925; and at 14\u00a0days: 492\u2009\u00b1\u200943 vs 219\u2009\u00b1\u200923 per section, p\u2009<\u20090.01; Fig.\u00a02j).\nGuanosine treatment stimulated astrocyte proliferation in the penumbra as early as 3\u00a0days and throughout the 14\u00a0days of treatment (Fig.\u00a03a,b) and was detected by antibodies to glial fibrillary acidic protein (GFAP). Astrocytes may stimulate oligodendroglial progenitor proliferation or induce their migration [49]. Guanosine did not alter the numbers of OX-42+ microglia\/macrophages until 14\u00a0days after treatment. At day 14, guanosine significantly increased OX-42+ cells compared to vehicle treatment (Fig.\u00a03c\u2013e; p\u2009<\u20090.05).\nFig.\u00a03Cross sections of the cords at the injury site staining with GFAP and OX-42 from guanosine-treated and vehicle-treated rats 14\u00a0days after treatment following chronic spinal cord injury. a, b Demonstrate examples of GFAP-positive astrocytes (in red) in cross sections from vehicle- (a) and guanosine- (b) treated animals at the injury site 14\u00a0days after treatment. c, d Show OX-42-immunofluorescent staining of cross sections of spinal cords from vehicle- (c) and guanosine- (d) treated animals at the lesion site 14\u00a0days after treatment. Quantification showed more OX-42-immunolabelled cells in cords of guanosine-treated rats (d, e) compared to cords of vehicle-treated rats (c, e; p\u2009<\u20090.05). Scale bar\u2009=\u200950\u00a0\u03bcm for all\nGuanosine inhibits apoptosis due to a number of stimuli [50\u201352]. After stroke and spinal cord injury, intrinsic stem cells and progenitors proliferate and differentiate but many die through apoptosis [15, 53\u201355]. In these experiments, guanosine did not prevent cell death (measured by TUNEL) (Fig.\u00a04), indicating that the protective effects of guanosine do not result from a reduction in precursor cell apoptosis.\nFig.\u00a04Guanosine treatment did not affect apoptosis in the cords with chronic, stable spinal cord injury. For detection of apoptotic cells, a terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick end labelling (TUNEL) stain was performed using the \u2018In situ Cell Death Detection Kit, Fluorescein\u2019. No obvious difference in number of TUNEL-positive cells was observed between vehicle- (a) or guanosine- (b) treated spinal cord sections. Scale bar\u2009=\u200950\u00a0\u03bcm for all\nBefore the intraperitoneal injection of guanosine, there were detectable amounts of guanosine and its metabolic product guanine in the spinal cords of injured rats (time 0, Fig.\u00a05a,b). There were no significant differences in guanosine or guanine in the samples of the spinal cords above, at or below the lesion. After guanosine injection, the amount of both guanosine and guanine increased, reaching a maximum by 30\u00a0min; the amounts of guanosine and guanine were about twofold and fivefold higher than those detected before the injection (Fig.\u00a05a,b). As with the basal levels (time 0), there was no difference in the amounts of guanosine above, at or below the lesion. Interestingly, guanine levels were always higher than those of guanosine at each of the times tested after injection (Fig.\u00a05a,b).\nFig.\u00a05Guanosine (a) and guanine (b) in spinal cord samples of rats with chronic spinal cord injury before and after intraperitoneal injection of guanosine. Guanosine (8\u00a0mg\/kg) was administered intraperitoneally to rats 5\u00a0weeks after the spinal cord injury. Before or at the indicated time points after the injection, samples of spinal cord from 3\u20136 animals were taken from the site of the lesion and immediately above and below the lesion. The tissue content of guanosine and its metabolic product guanine was measured by HPLC and CE analysis. Data are the mean\u2009\u00b1\u2009SEM\nNew oligodendrocytes in injured spinal cords come from a population of endogenous progenitor cells present in the CNS that can differentiate into mature cells capable of myelinating axons [3, 12, 13, 56]. The data from the present study showed that the increase in the number of NG2+ cells is associated with an increase in Rip+ cells. Our data indicated that guanosine treatment induced an increase of mature oligodendroglia (Rip+ cells), likely due to proliferation and differentiation of their progenitor (NG2+) cells. Therefore, one would expect that Rip+ cells increase after NG2+ cells. However, this was not observed. But guanosine may well stimulate the proliferation of oligodendroglial progenitor cells at several stages of development. This would account for the observed results. Additionally, we cannot exclude that other possible mechanisms are involved in the process; for example, mature oligodendroglia could migrate from the adjacent normal tissue to the injury site soon after guanosine treatment.\nWhether the effects on remyelination are due to guanosine, guanine or both is not clear. However, since a putative receptor for guanosine has been identified in the CNS, and since PNP (purine nucleoside phosphorylase) in CNS permits the interconversion of guanine and guanosine enabling guanine to act as a reservoir for guanosine, it is possible that guanosine may be responsible for causing the proliferation of the endogenous progenitor cells and the subsequent remyelination process, although this is by no means certain. But, since guanine itself is difficult to administer because of its poor solubility, administration of guanosine is the most effective delivery mechanism.\nThe mechanism by which remyelination is facilitated after chronic, traumatic SCI is unknown. Guanosine has been shown to stimulate the production and\/or release of a variety of trophic factors [25]; for example bFGF, NGF and NT-3 have been demonstrated to increase the proliferation and survival of oligodendrocyte progenitors [3, 16, 57], induce oligodendroglial genesis [58\u201361], stimulate the proliferation of oligodendrocyte precursors and enhance remyelination after spinal cord traumatic injury [18, 19] and after toxin-induced demyelination [62]. There are no published reports of the effects of guanine in the vivo systems. However, in cultured cells, guanosine stimulates cell division apparently through a pertussis toxin-sensitive process involving MAP kinase [63]. Moreover, there is evidence of a G protein-coupled receptor in brain membranes that might be a candidate for a guanosine receptor [64]. Further studies will permit us to distinguish amongst these possibilities.\nThe present findings are the first to show that systemic treatment with guanosine after chronic SCI induces an improvement in function that is accompanied by the formation of mature oligodendrocytes. This effect of guanosine\/guanine may be attributable to direct or indirect stimulation of endogenous oligodendrocyte lineage progenitors in the spinal cord and remyelination of axons at the injury site.\nThese findings raise the possibility that the administration of guanosine may be effective in treating spinal cord injuries and in the treatment of demyelinating diseases such as multiple sclerosis where quiescent progenitors exist in demyelinated plaques [65\u201370].","keyphrases":["myelination","spinal cord","guanosine","oligodendrocyte","cell proliferation","guanine","precursors","functional recovery","immunohistochemistry"],"prmu":["P","P","P","P","P","P","P","P","P"]}
{"id":"Naunyn_Schmiedebergs_Arch_Pharmacol-3-1-1915621","title":"Pharmacological characterisation of capsaicin-induced relaxations in human and porcine isolated arteries\n","text":"Capsaicin, a pungent constituent from red chilli peppers, activates sensory nerve fibres via transient receptor potential vanilloid receptors type 1 (TRPV1) to release neuropeptides like calcitonin gene-related peptide (CGRP) and substance P. Capsaicin-sensitive nerves are widely distributed in human and porcine vasculature. In this study, we examined the mechanism of capsaicin-induced relaxations, with special emphasis on the role of CGRP, using various pharmacological tools. Segments of human and porcine proximal and distal coronary arteries, as well as cranial arteries, were mounted in organ baths. Concentration response curves to capsaicin were constructed in the absence or presence of the CGRP receptor antagonist olcegepant (BIBN4096BS, 1 \u03bcM), the neurokinin NK1 receptor antagonist L-733060 (0.5 \u03bcM), the voltage-sensitive calcium channel blocker ruthenium red (100 \u03bcM), the TRPV1 receptor antagonist capsazepine (5 \u03bcM), the nitric oxide synthetase inhibitor N\u03c9-nitro-l-arginine methyl ester HCl (l-NAME; 100 \u03bcM), the gap junction blocker 18\u03b1-glycyrrhetinic acid (10 \u03bcM), as well as the RhoA kinase inhibitor Y-27632 (1 \u03bcM). Further, we also used the K+ channel inhibitors 4-aminopyridine (1 mM), charybdotoxin (0.5 \u03bcM) + apamin (0.1 \u03bcM) and iberiotoxin (0.5 \u03bcM) + apamin (0.1 \u03bcM). The role of the endothelium was assessed by endothelial denudation in distal coronary artery segments. In distal coronary artery segments, we also measured levels of cyclic adenosine monophosphate (cAMP) after exposure to capsaicin, and in human segments, we also assessed the amount of CGRP released in the organ bath fluid after exposure to capsaicin. Capsaicin evoked concentration-dependent relaxant responses in precontracted arteries, but none of the above-mentioned inhibitors did affect these relaxations. There was no increase in the cAMP levels after exposure to capsaicin, unlike after (exogenously administered) \u03b1-CGRP. Interestingly, there were significant increases in CGRP levels after exposure to vehicle (ethanol) as well as capsaicin, although this did not induce relaxant responses. In conclusion, the capsaicin-induced relaxations of the human and porcine distal coronary arteries are not mediated by CGRP, NK1, NO, vanilloid receptors, voltage-sensitive calcium channels, K+ channels or cAMP-mediated mechanisms. Therefore, these relaxant responses to capsaicin are likely to be attributed to a non-specific, CGRP-independent mechanism.\nIntroduction\nCapsaicin, a pungent constituent of red pepper, is known to activate sensory C-fibres via transient receptor potential vanilloid receptors type 1 (TRPV1; Caterina et al. 1997; Gunthorpe et al. 2002; Szallasi 2006), which are nonselective cation channels. Activation of these channels increases influx of mono- and divalent cations, which leads to an increase in the intracellular Ca2+ concentrations. Consequently, an array of neuropeptides like calcitonin gene-related peptide (CGRP), substance P and neurokinin A are released (Franco-Cereceda et al. 1988; Mitchell et al. 1995). These neuropeptides play a role in the regulation of normal vascular smooth muscle tone and are also implicated in several pathological conditions like ischemic preconditioning (Chai et al. 2006), preeclampsia (Dong et al. 2005) and migraine (Arulmani et al. 2004b). In the pathophysiology of migraine, vasodilatation of cranial blood vessels, especially extracerebral intracranial blood vessels, which are richly innervated by nerves containing a number of peptides such as CGRP, seems pivotal (Arulmani et al. 2006). Vasodilatation of these intracranial arteries leads to activation of nociceptors, which stimulate the pain centres in the brain (Goadsby et al. 2002). Involvement of CGRP in the pathophysiology of migraine is further strengthened by the observation that olcegepant (BIBN4096BS, Doods et al. 2000), a CGRP receptor antagonist, is effective in the acute treatment of migraine attacks (Olesen et al. 2004). Capsaicin has been widely used in various in vivo models of migraine to induce cranial vasodilatation, which is attributed to endogenous release of neuropeptides, especially CGRP (Akerman et al. 2003; Arulmani et al. 2004a; Gupta et al. 2006a). The involvement of CGRP is further substantiated in all these in vivo studies by the fact that the capsaicin-induced vascular responses are all amenable to blockade with CGRP receptor antagonists. Repeated capsaicin challenges are reported to deplete a number of neurotransmitters, including CGRP in various animal models (Tang et al. 1997; Tang et al. 1999; Zhou et al. 2002). In addition to the in vivo models, also in vitro vascular models involving meningeal (Gupta et al. 2006b) and coronary (Edvinsson et al. 2002; Gupta et al. 2006c) arteries have been used in migraine research, but vasorelaxation in these models was induced by exogenously administered CGRP. Several major vascular beds, including the meningeal and coronary (Franco-Cereceda 1988), are richly innervated with capsaicin-sensitive sensory fibres containing CGRP (Edvinsson et al. 1987). Meningeal blood vessels have been shown to be involved in the plasma protein extravasation in rat dura mater (Delepine and Aubineau 1997; Seabrook et al. 1996), a model of neurogenic inflammation, one of the putative mechanisms in migraine pathophysiology. In this model, capsaicin pre-treatment has been shown to block plasma protein extravasation, again by depleting neuropeptides (Delepine and Aubineau 1997). Therefore, it is of interest to investigate the release of endogenous CGRP in various blood vessels and capsaicin seems most appropriate for such studies.\nInterestingly, very few studies have investigated capsaicin-induced relaxations in human isolated blood vessels (Franco-Cereceda 1991a; Franco-Cereceda et al. 1987). Based on CGRP-like immunoreactivity, it was claimed that capsaicin-induced relaxations are mediated by CGRP (Franco-Cereceda 1991b), but no CGRP receptor antagonists were used to unequivocally demonstrate the role of CGRP in these responses. Therefore, we were interested in studying capsaicin-mediated relaxations in human and porcine isolated arteries as a model to study endogenous release of CGRP in view of its relevance in the pathophysiology of migraine. Remarkably, some recent studies indicate that capsaicin-induced relaxations are mediated by non-CGRP-mediated mechanisms in guinea pig ileum, rabbit coronary arteries and equine tracheal smooth muscle preparations (Fujimoto et al. 2006; Yeon et al. 2001; Zhu et al. 1997), which is contrary to the observation that capsaicin-induced relaxations are mediated principally by CGRP. In these studies, relaxant responses to capsaicin were mainly attributed to different Ca2+-activated K+ channels. Therefore, in the present study, we tried to characterise capsaicin-induced relaxant response in human and porcine vessels, with special emphasis on the role of CGRP, and also investigated other mechanisms, using different pharmacological tools.\nMaterials and methods\nTissue preparation\nThe study protocol was approved by the ethical committee of Erasmus MC. Human coronary artery was obtained from \u2018heart-beating\u2019 organ donors (13 men, 19 women; 13\u201365\u00a0years) who died due to non-cardiac disorders. The hearts were provided by the Heart Valve Bank, Rotterdam, The Netherlands, after donor mediation by Bio Implant Services Foundation\/Eurotransplant Foundation (Leiden, The Netherlands). The meningeal arteries were obtained from patients (four men, six women; 41\u201375\u00a0years) undergoing craniotomy at the neurosurgical unit (n\u2009=\u20096) or from the Department of Pathology (postmortem, n\u2009=\u20094) within 24\u00a0h of death, at Erasmus MC, Rotterdam. During the surgical procedure or autopsy, the dura mater together with a small piece of the meningeal artery was cut and placed in a plastic container filled with ice-cold (0\u20134\u00b0C), physiological salt solution. The artery segment was immediately transported to the laboratory and placed in cold oxygenated Krebs bicarbonate solution of the following composition (mM): NaCl 119, KCl 4.7, CaCl2 1.25, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25 and glucose 11.1; pH\u00a07.4. Excess tissue was removed. The meningeal artery (internal diameter of 350\u2013800\u00a0\u03bcm) was used the same day or stored overnight in cold oxygenated modified Krebs solution and used the following day. Porcine hearts and heads (pigs of either sex; 6\u201312\u00a0months of age) were collected from a local slaughterhouse. Porcine basilar (internal diameter of 150\u2013200\u00a0\u03bcm) as well as meningeal (internal diameter of 100\u2013250\u00a0\u03bcm) arteries were dissected out from the skull and were placed in a cold oxygenated Krebs solution as described earlier for human meningeal arteries. In the laboratory, both human and porcine proximal (internal diameter, 2\u20133.5\u00a0mm) and distal (internal diameter, 200\u2013600\u00a0\u03bcm) coronary arteries were dissected out of the right ventricle; distal coronary arteries were dissected with the aid of a microscope and stored in bicarbonate solution of following composition (mM): NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25 and glucose 11.1; pH\u00a07.4.\nFunctional studies\nBoth human and porcine proximal coronary artery segments (3\u20134\u00a0mm length) were suspended with the help of stainless steel hooks in 15-ml organ baths with a pretension of 15\u00a0mN (optimal tension shown in earlier experiments). Distal coronary and cranial artery segments were cut into ring segments of 1\u20132\u00a0mm length and were mounted in Mulvany myographs between two parallel titanium wires with a tension normalised to 90% of l100 (distance when transmural pressure equals 100\u00a0mmHg), thus achieving optimal conditions for active force development (Nyborg et al. 1987). Vessels were allowed to equilibrate for 30\u00a0min in Krebs solution at 37\u00b0C, a similar equilibration period was repeated after each physical or pharmacological challenge. In case of human arteries, the cyclo-oxygenase inhibitor indomethacin (0.1\u00a0\u03bcM) was added to prevent prostaglandin synthesis during the whole experimental protocol. Two successive challenges to KCl (30\u00a0mM) were performed to test the reproducibility of responses. Endothelial integrity was assessed by observing relaxation to substance P (10\u00a0nM) after precontraction with U46619 (9,11-dideoxy-11\u03b1, 9\u03b1-epoxymethano-prostaglandin F2\u03b1; 10\u00a0nM\u20131\u00a0\u03bcM), followed by washout of the agonists. KCl (18\u201330\u00a0mM, except where higher concentrations are indicated) or U46619 (10\u00a0nM\u20131\u00a0\u03bcM), in case of the K+ channel blockers, was used to obtain a stable contraction plateau of around 60\u201375% of the maximal contraction reached with KCl (100\u00a0mM). Subsequently, capsaicin was added in a cumulative manner in log steps. The different antagonists were used to antagonise capsaicin responses, and the concentration of each of these antagonists was decided on the basis of highest concentration in the literature unless mentioned otherwise. Concentration response curves were constructed in the absence or presence of the CGRP receptor antagonists olcegepant (1\u00a0\u03bcM; Gupta et al. 2006b,c) and CGRP8\u201337 (10\u00a0\u03bcM; Gupta et al. 2006b,c), the neurokinin NK1 receptor antagonist L-733060 (0.5\u00a0\u03bcM; Seabrook et al. 1996), the voltage-sensitive calcium channel blocker ruthenium red (100\u00a0\u03bcM, 45\u201390\u00a0mM KCl was required to get a similar precontraction as in the corresponding control segments; Franco-Cereceda and Rudehill 1989), the nitric oxide synthetase inhibitor N\u03c9-nitro-l-arginine methyl ester HCl (l-NAME; 100\u00a0\u03bcM; Batenburg et al. 2004a,b), as well as the vanilloid receptor antagonist capsazepine (5\u00a0\u03bcM; Fujimoto et al. 2006; Gazzieri et al. 2006). At still higher concentrations of capsazepine (>5\u00a0\u03bcM) and L-733006 (>0.5\u00a0\u03bcM), even higher concentrations of preconstricting agents could not produce a workable precontraction.\nWe also investigated the role of different K+ channels by using various inhibitors, 4-aminopyridine (1\u00a0mM; Fujimoto et al. 2006; Yeon et al. 2001), a voltage-dependent K+ channel (Kv) blocker and charybdotoxin (0.5\u00a0\u03bcM), a blocker of Ca2+-dependent K+ channels for large conductance (BKCa) and intermediate conductance (IKCa), in combination with apamin (0.1\u00a0\u03bcM), a blocker of small-conductance Ca2+-dependent K+ channels (SKCa; Batenburg et al. 2004b; Fujimoto et al. 2006; Zhu et al. 1997). The effect of the RhoA kinase inhibitor, Y-27632 (1\u00a0\u03bcM; Fujimoto et al. 2006) was investigated both in the absence or presence of 4-aminopyridine (1\u00a0mM). We also investigated the effect of the gap junction inhibitor 18-\u03b1-glycyrrhetinic acid (10\u00a0\u03bcM) on capsaicin-induced relaxations. Where indicated, the endothelium was removed with a human hair, and removal was confirmed by observing a relaxation of less than 10% of precontraction of U46619 after addition of substance P.\nFurther, in porcine distal coronary artery, we also studied the effect of repeated administration of capsaicin (50\u00a0\u03bcM, four times) to verify the reproducibility of the responses in view of possible depletion of endogenous peptide pools or other agents.\nMeasurements of cAMP\nHuman proximal and distal, as well as porcine distal, coronary artery segments were incubated in a medium containing isobutylmethylxanthine (0.5\u00a0mM) for 30\u00a0min in the absence or presence of olcegepant (1\u00a0\u03bcM). The arterial segments were exposed to KCl (30\u00a0mM), challenged with ethanol (vehicle of capsaicin, final concentration in the baths 0.56%), capsaicin (10\u00a0\u03bcM) or h-\u03b1CGRP (100\u00a0nM, serving as a positive control) for 5\u00a0min and then snap frozen. The samples were stored at \u221280\u00b0C until cyclic adenosine monophosphate (cAMP) assay. To determine cAMP, tissues were homogenised in 0.5\u00a0ml 0.1\u00a0M HCl using a stainless steel ultraturrax (Polytron, Staufen, Germany). Homogenates were centrifuged at 3,300\u00d7g, and cAMP was measured in 300\u00a0\u03bcl supernatant using the ELISA kit according to the instructions of the manufacturer (R&D Systems Europe, Abingdom, UK).\nMeasurements of CGRP in organ bath fluid\nHuman distal coronary artery segments were subjected to a similar protocol as during the functional studies, while bath fluids were collected after construction of the concentration response curve. The bath fluids were collected from the segments treated with vehicle and capsaicin (100\u00a0\u03bcM), and Krebs solution was used as a control. Bath fluids were stored in tubes containing aprotinin (0.6\u00a0TIU\/ml) and stored at \u221280\u00b0C. A competitive radioimmunoassay (Peninsula Lab INC., San Carlos, CA, USA) was used according to the instructions of the manufacturer to measure the CGRP concentrations in the bath fluid.\nData presentation and statistical analysis\nThe relaxant responses elicited by the agonists are expressed as percentage relaxation of the tone induced by 30\u00a0mM KCl or U46619 (in case of the K+ channel blockers). All data are presented as means\u00b1SEM, and n represents the number of blood vessel segments, all obtained from different subjects. The effect of all potential inhibitors of the relaxations to capsaicin was investigated in a paired parallel setup; that is, relaxations in segments with inhibitors were always compared to relaxations obtained in control segments from the same subject. The concentration response curves for all agonists were analysed using nonlinear regression analysis, and the potency of agonists was expressed as pEC50 using Graph Pad Prism 3.01 (Graph Pad Software, San Diego, CA, USA), setting the Emax of capsaicin in the presence of potential inhibitors to that in the respective control segment in case it was lower than the control Emax. The blocking potency of the antagonists was estimated by calculating median effective concentration (EC50) ratios, and apparent pKB values were calculated for the antagonists at each given concentration, with the slope set to unity. Statistical differences between concentration response curves to capsaicin in the absence and presence of potential inhibitors were determined using Student\u2019s paired t-test with \u03b1 set to 1.00\u20130.95(1\/n) (Motulsky 2003) to correct for multiple comparisons. For the measurements of CGRP in the organ bath fluids, we could not exclude a non-Gaussian distribution due to the large degree of variability in the data. Therefore, the levels of CGRP in bath fluids were analysed by the non-parametric Kruskal\u2013Wallis test, followed by Dunn\u2019s post hoc multiple comparison test. Significance was assumed at P\u2009\u2264\u20090.05.\nCompounds\nHuman \u03b1-CGRP and \u03b1-CGRP8\u201337 were obtained from Polypeptide, (Wolfenb\u00fcttel, Germany), olcegepant (BIBN4096BS, 1-piperidinecarboxamide, N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl] pentyl] amino]-1-[(3,5-dibromo-4-hydroxyphenyl) methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl)-, [R-(R*,S*)]-) was a gift from Boehringer Ingelheim Pharma (Biberach\/Riss, Germany); 4-aminopyridine was purchased from ICN Biomedicals (Aurora, OH, USA); L-733060 was purchased from Tocris (Bristol, UK); apamin, capsaicin, capsazepine, 3-isobutyl-1-methyl-xanthine, l-NAME, charybdotoxin, ruthenium red, substance P, U46619 and Y-27632 were purchased from Sigma-Aldrich (Zwijndrecht, The Netherlands), and KCl was obtained from Merck (Darmstadt, Germany). Capsaicin was dissolved in 70% ethanol, and the dilution series was also prepared in ethanol 70%. Capsazepine was dissolved in methanol; olcegepant was dissolved in a small amount of 1\u00a0N HCl and then diluted with distilled water. The other compounds were dissolved in distilled water, and all compounds were stored in aliquots at \u221280\u00b0C.\nResults\nFunctional studies\nHuman arteries\nSubstance P relaxed artery segments precontracted with U46619 (10\u00a0nM\u20131\u00a0\u03bcM); responses were equi-efficacious in distal coronary (80\u2009\u00b1\u20095% of contraction to U46619, n\u2009=\u200928) and meningeal (75\u2009\u00b1\u20098%, n\u2009=\u20098) artery and significantly less in proximal coronary artery (27\u2009\u00b1\u200915%, n\u2009=\u20094). Both in the meningeal and distal coronary arteries, capsaicin induced concentration-dependent relaxations. In human proximal coronary artery, relaxant responses were only observed at the highest concentration of 100\u00a0\u03bcM, and the maximum relaxant response (34\u2009\u00b1\u200914% of contraction to 30\u00a0mM KCl) was significantly less than that observed in the distal arteries (94\u2009\u00b1\u20091% of contraction to 18\u201330\u00a0mM KCl). In human meningeal arteries, there was no difference in capsaicin-induced responses between arteries obtained perioperatively or postmortem; therefore, these data were pooled for further analysis. Capsaicin was equipotent and equi-efficacious in human distal coronary and human meningeal artery. In human distal coronary artery segments, the lower concentrations of capsaicin (0.1\u00a0nM\u20131\u00a0\u03bcM) in some cases induced contractions, but in all cases, we uniformly only measured the relaxant responses. The relaxations to capsaicin in proximal and distal coronary as well as meningeal arterial segments were insensitive to blockade by the CGRP antagonist olcegepant (1\u00a0\u03bcM; Fig.\u00a01, Table\u00a01).\nFig.\u00a01Effect of capsaicin or its vehicle in the absence or presence of various pharmacological agents or interventions in precontracted human and porcine distal coronary arteriesTable\u00a01Effect of various antagonists\/interventions on capsaicin-induced relaxations in human isolated artery segmentsAntagonist or other intervention (n)Emax (%)\u0394 EmaxpEC50\u0394 pEC50Human distal coronary artery\u00a0(Control) (32)94\u2009\u00b1\u200915.27\u2009\u00b1\u20090.12Olcegepant (1\u00a0\u03bcM) (10)89\u2009\u00b1\u200948\u2009\u00b1\u200944.84\u2009\u00b1\u20090.090.33\u2009\u00b1\u20090.16CGRP8\u201337 (10\u00a0\u03bcM) (5)96\u2009\u00b1\u200933\u2009\u00b1\u200944.79\u2009\u00b1\u20090.060.16\u2009\u00b1\u20090.08Capsazepine (5\u00a0\u03bcM) (13)91\u2009\u00b1\u200931\u2009\u00b1\u200925.10\u2009\u00b1\u20090.130.07\u2009\u00b1\u20090.16Ruthenium red (0.1\u00a0mM) (9)92\u2009\u00b1\u200935\u2009\u00b1\u200935.01\u2009\u00b1\u20090.130.21\u2009\u00b1\u20090.22L-733060 (5\u00a0\u03bcM) (7)94\u2009\u00b1\u200922\u2009\u00b1\u200926.03\u2009\u00b1\u20090.78\u22120.54\u2009\u00b1\u20090.34Denuded endothelium (5)90\u2009\u00b1\u200969\u2009\u00b1\u200955.34\u2009\u00b1\u20090.48\u22120.21\u2009\u00b1\u20090.58l-NAME (0.1\u00a0mM) (7)94\u2009\u00b1\u200923\u2009\u00b1\u200925.23\u2009\u00b1\u20090.48\u22120.24\u2009\u00b1\u20090.4518-\u03b1-Glycyrrhetinic acid (10\u00a0\u03bcM) (3)96\u2009\u00b1\u200920\u2009\u00b1\u200925.08\u2009\u00b1\u20090.28\u22120.15\u2009\u00b1\u20090.38Olcegepant (1\u00a0\u03bcM)\u2009+\u2009L-733060 (5\u00a0\u03bcM) (3)90\u2009\u00b1\u200961\u2009\u00b1\u200936.04\u2009\u00b1\u20090.230.06\u2009\u00b1\u20090.21\u00a0(Control) (10)97\u2009\u00b1\u200915.91\u2009\u00b1\u20090.324-Aminopyridine (1\u00a0mM) (6)95\u2009\u00b1\u200922\u2009\u00b1\u200935.64\u2009\u00b1\u20090.380.94\u2009\u00b1\u20090.46Charybdotoxin (0.5\u00a0\u03bcM)\u2009+\u2009apamin (0.1\u00a0\u03bcM) (8)97\u2009\u00b1\u200910\u2009\u00b1\u200915.80\u2009\u00b1\u20090.370.23\u2009\u00b1\u20090.37Iberiotoxin (0.5\u00a0\u03bcM)\u2009+\u2009apamin (0.1\u00a0\u03bcM) (5)96\u2009\u00b1\u200920\u2009\u00b1\u200925.94\u2009\u00b1\u20090.650.15\u2009\u00b1\u20090.38Y-276323 (1\u00a0\u03bcM) (3)99\u2009\u00b1\u20091\u22121\u2009\u00b1\u200925.39\u2009\u00b1\u20090.21\u22120.58\u2009\u00b1\u20090.14Y-276323 (1\u00a0\u03bcM)\u2009+\u20094-Aminopyridine (1\u00a0mM) (3)100\u2009\u00b1\u20090\u22123\u2009\u00b1\u200945.12\u2009\u00b1\u20090.180.33\u2009\u00b1\u20090.28Human proximal coronary artery\u00a0(Control) (4)34\u2009\u00b1\u2009144.30\u2009\u00b1\u20090.14Olcegepant (1\u00a0\u03bcM) (4)36\u2009\u00b1\u2009164.40\u2009\u00b1\u20090.17Human meningeal artery\u00a0(Control) (10)91\u2009\u00b1\u20095 5.04\u2009\u00b1\u20090.09Olcegepant (1\u00a0\u03bcM) (10)96\u2009\u00b1\u20091\u22125\u2009\u00b1\u200945.03\u2009\u00b1\u20090.07\u22120.07\u2009\u00b1\u20090.08Capsazepine (5\u00a0\u03bcM) (4)81\u2009\u00b1\u200998\u2009\u00b1\u200974.90\u2009\u00b1\u20090.310.11\u2009\u00b1\u20090.31Ruthenium red (0.1\u00a0mM) (3)74\u2009\u00b1\u2009155\u2009\u00b1\u2009145.13\u2009\u00b1\u20090.420.02\u2009\u00b1\u20090.62L-733060 (5\u00a0\u03bcM) (4)79\u2009\u00b1\u2009128\u2009\u00b1\u2009154.80\u2009\u00b1\u20090.31\u22120.09\u2009\u00b1\u20090.31Emax is the maximum relaxant response, expressed as percentage of the respective precontraction; pEC50 is the \u2212logEC50, where EC50 is the concentration of agonist required to produce half the maximal response. The arteries were precontracted with KCl (18\u201330 mM) except where KCl (45\u201390 mM; bold) or U46619 (ital).\nAs the relaxant responses to capsaicin were small in human proximal coronary arteries and the availability of human meningeal arteries was very limited, further experiments were only carried out in human distal coronary artery. In this preparation, the CGRP receptor antagonist CGRP8\u201337 (10\u00a0\u03bcM), the TRPV1 receptor antagonist capsazepine (5\u00a0\u03bcM) and the NK1 receptor antagonist L-733006 (0.5\u00a0\u03bcM) also did not attenuate capsaicin-induced relaxations (Table\u00a01). Similarly, there was no significant difference in relaxant responses in endothelium-intact or endothelium-denuded segments (Table\u00a01). Also, in the absence or presence of the NO synthase inhibitor l-NAME (100\u00a0\u03bcM) or in the presence of the gap junction blocker 18-\u03b1-glycyrrhetinic acid, capsaicin caused equipotent relaxations compared to the respective control segments (Table\u00a01). Various K+ channel blockers, namely, 4-aminopyridine (1\u00a0mM), charybdotoxin (0.5\u00a0\u03bcM)\u2009+\u2009apamin (0.1\u00a0\u03bcM) and iberiotoxin (0.1\u00a0\u03bcM)\u2009+\u2009apamin (0.1\u00a0\u03bcM) were also unable to block the relaxant responses to capsaicin. The RhoA kinase inhibitor Y-27632 (1\u00a0\u03bcM) alone or in combination with 4-aminopyridine was also unable to block the relaxant responses to capsaicin. Ruthenium red (100\u00a0\u03bcM), a nonselective blocker of Ca2+ transport through membrane channels, also did not significantly block these responses (Table\u00a01). It is noteworthy that at higher concentrations, capsazepine and L-733060 significantly attenuated the responses to their respective preconstriction agents, and also in case of preincubation with Y-27632 and ruthenium red, 2- to 20-fold higher concentrations of precontracting agents were required to induce 60\u201375% of the maximal contraction reached with KCl (100\u00a0mM). At still higher concentrations of capsazepine (>5\u00a0\u03bcM) and L-733060 (>0.5\u00a0\u03bcM), even higher concentrations of preconstricting agents could not produce a workable precontraction. Additionally, the combination of olcegepant (1\u00a0\u03bcM)\u2009+\u2009L-733060 (0.5\u00a0\u03bcM) in human distal coronary artery or 4-aminopyridine (1\u00a0mM)\u2009+\u2009Y-27632 (1\u00a0\u03bcM) both in human and porcine distal coronary artery were also unable to block the responses to capsaicin. The vehicle of capsaicin (0.56% ethanol) did not induce any significant relaxations. It should be noted that the potency of capsaicin was higher on a precontraction with U46619 (10\u00a0nM\u20131\u00a0\u03bcM; pEC50 of capsaicin, 5.91\u2009\u00b1\u20090.32) than on a precontraction with KCl (18\u201330\u00a0mM; pEC50 of capsaicin, 5.27\u2009\u00b1\u20090.12; P\u2009=\u20090.029). However, irrespective of the precontracting agent used, the antagonists behaved in a similar fashion towards capsaicin. For example, the effects of 4-aminopyridine on capsaicin-induced relaxations were similar after precontraction with KCl (pEC50, 5.01\u2009\u00b1\u20090.05 and 5.12\u2009\u00b1\u20090.41; n\u2009=\u20097, in the absence and presence of 4-aminopyridine, respectively) and with U46619 (pEC50, 6.58\u2009\u00b1\u20090.47 and 5.64\u2009\u00b1\u20090.38; n\u2009=\u20096, in the absence and presence of 4-aminopyridine, respectively). In human meningeal artery, capsazepine, L-733006 and also ruthenium red did not block the responses to capsaicin (Table\u00a01).\nPorcine arteries\nIn porcine arteries, substance P induced relaxations in precontracted arteries to a varying degree in different vessels. The relaxations in distal coronary (64\u2009\u00b1\u20096% of contraction to U46619, 10\u00a0nM\u20131\u00a0\u03bcM, n\u2009=\u200926) basilar (44\u2009\u00b1\u20093%, n\u2009=\u20093) and meningeal (57\u2009\u00b1\u200915%, n\u2009=\u20093) artery were similar, whereas that in proximal coronary (8\u2009\u00b1\u20098%, n\u2009=\u20094) artery was significantly smaller. In porcine proximal and distal coronary arteries, capsaicin induced concentration-dependent relaxations. Unlike in human coronary arteries, the maximum response was not significantly different between porcine proximal and distal coronary arteries (Table\u00a02). In both the arteries, olcegepant (1\u00a0\u03bcM) did not block the responses to capsaicin. Further experiments were carried out in distal coronary arteries and, similar as in the human distal coronary artery, capsazepine (1\u00a0\u03bcM), ruthenium red (0.1\u00a0mM), L-73360 (0.5\u00a0\u03bcM), 4-aminopyridine (1\u00a0mM), l-NAME (0.1\u00a0mM), charybdotoxin (0.5\u00a0\u03bcM)\u2009+\u2009apamin (0.1\u00a0\u03bcM), iberiotoxin (0.5\u00a0\u03bcM)\u2009+\u2009apamin (0.1\u00a0\u03bcM), Y-27632 (1\u00a0\u03bcM), 18-\u03b1-glycyrrhetinic acid (10\u00a0\u03bcM) and endothelium denudation did not affect capsaicin-induced relaxations (Fig.\u00a01, Table\u00a02). Additionally, Y-27632 (1\u00a0\u03bcM), alone or combined with 4-aminopyridine (1\u00a0mM), was also unable to block the responses to capsaicin. Similar as in the other arteries, the vehicle did not induce a relaxation (Fig.\u00a01). Interestingly, in contrast to what we observed in the human distal coronary artery, precontracting the arteries with either KCl or U46619 did not change the relaxant responses to capsaicin. Four consecutive challenges to capsaicin (50\u00a0\u03bcM) did not significantly affect the magnitude of the responses (Fig.\u00a02). \nTable\u00a02Effect of various antagonists\/interventions on capsaicin-induced relaxations in porcine isolated artery segmentsAntagonist or other intervention (n)Emax (%)\u0394 EmaxpEC50\u0394 pEC50Porcine distal coronary artery\u00a0(Control) (56)96\u2009\u00b1\u200915.27\u2009\u00b1\u20090.09Olcegepant (1\u00a0\u03bcM) (10)92\u2009\u00b1\u200923\u2009\u00b1\u200925.26\u2009\u00b1\u20090.11\u22120.02\u2009\u00b1\u20090.11CGRP8\u201337 (10\u00a0\u03bcM) (5)97\u2009\u00b1\u20093\u22122\u2009\u00b1\u200954.78\u2009\u00b1\u20090.060.00\u2009\u00b1\u20090.03Capsazepine (5\u00a0\u03bcM) (7) 99\u2009\u00b1\u20091\u22123\u2009\u00b1\u200925.15\u2009\u00b1\u20090.260.26\u2009\u00b1\u20090.16Ruthenium red (0.1\u00a0mm) (12)88\u2009\u00b1\u200937\u2009\u00b1\u200944.89\u2009\u00b1\u20090.090.47\u2009\u00b1\u20090.23L-733060 (5\u00a0\u03bcM) (7)88\u2009\u00b1\u200934\u2009\u00b1\u200944.89\u2009\u00b1\u20090.09\u22120.04\u2009\u00b1\u20090.15Denuded endothelium (9)91\u2009\u00b1\u200942\u2009\u00b1\u200925.62\u2009\u00b1\u20090.490.21\u2009\u00b1\u20090.16l-NAME (0.1\u00a0mM) (6)90\u2009\u00b1\u200988\u2009\u00b1\u200984.84\u2009\u00b1\u20090.150.28\u2009\u00b1\u20090.31\u00a0(Control) (15)99\u2009\u00b1\u200905.08\u2009\u00b1\u20090.1518-\u03b1-Glycyrrhetinic acid (10\u00a0\u03bcM) (3)99\u2009\u00b1\u200910\u2009\u00b1\u200915.73\u2009\u00b1\u20090.75\u22120.59\u2009\u00b1\u20090.534-Aminopyridine (1\u00a0mM) (7)96\u2009\u00b1\u200923\u2009\u00b1\u200924.84\u2009\u00b1\u20090.15\u22120.46\u2009\u00b1\u20090.35Charybdotoxin (0.5\u00a0\u03bcM)\u2009+\u2009apamin (0.1\u00a0\u03bcM) (11)99\u2009\u00b1\u200910\u2009\u00b1\u200915.27\u2009\u00b1\u20090.15\u22120.04\u2009\u00b1\u20090.20Y-276323 (1\u00a0\u03bcM) (9)96\u2009\u00b1\u200923\u2009\u00b1\u200925.17\u2009\u00b1\u20090.190.04\u2009\u00b1\u20090.21Y-276323 (1\u00a0\u03bcM)\u2009+\u20094-Aminopyridine (1\u00a0mM) (8)96\u2009\u00b1\u200934\u2009\u00b1\u200935.22\u2009\u00b1\u20090.200.00\u2009\u00b1\u20090.11Porcine proximal coronary artery\u00a0(Control) (4)100\u2009\u00b1\u200905.33\u2009\u00b1\u20090.42Olcegepant (1\u00a0\u03bcM) (4)90\u2009\u00b1\u200965.79\u2009\u00b1\u20090.16Porcine basilar artery\u00a0(Control) (3)97\u2009\u00b1\u200914.70\u2009\u00b1\u20090.05Olcegepant (1\u00a0\u03bcM) (3)100\u2009\u00b1\u200904.97\u2009\u00b1\u20090.24Capsazepine (5\u00a0\u03bcM) (3) 100\u2009\u00b1\u200904.80\u2009\u00b1\u20090.01Porcine meningeal artery\u00a0(Control) (3)99\u2009\u00b1\u200914.82\u2009\u00b1\u20090.02Olcegepant 1\u00a0\u03bcM (3)99\u2009\u00b1\u200914.88\u2009\u00b1\u20090.04Emax is the maximum relaxant response, expressed as percentage of the respective precontraction; pEC50 is the \u2212logEC50, where EC50 is the concentration of agonist required to produce half the maximal response. The arteries were precontracted with KCl (18\u201330 mM) except where KCl (45\u201390 mM; bold) or U46619 (ital).Fig.\u00a02Effect of four consecutive challenges to capsaicin (50\u00a0\u03bcM) in porcine distal coronary arteries precontracted with KCl (30\u00a0mM)\nThere were no significant differences in efficacy or potency of capsaicin in porcine basilar (Emax, 97\u2009\u00b1\u20091%; pEC50, 4.70\u2009\u00b1\u20090.05; n\u2009=\u20093) and meningeal (Emax, 99\u2009\u00b1\u20091%; pEC50, 4.82\u2009\u00b1\u20090.02; n\u2009=\u20093) arteries as compared to porcine distal coronary arteries. In both these arteries, responses to capsaicin were not affected by olcegepant (1\u00a0\u03bcM).\nMeasurements of cAMP\nCapsaicin (10\u00a0\u03bcM) did not affect cAMP levels in comparison to its vehicle or the control, which was only exposed to 30\u00a0mM KCl. In contrast, \u03b1CGRP (100\u00a0nM), which was used as a positive control, significantly increased cAMP levels in both human and porcine distal coronary arteries, which was blocked after incubation with olcegepant (1\u00a0\u03bcM; Fig.\u00a03).\nFig.\u00a03Changes in cAMP levels in human (n\u2009=\u20094\u20137) and porcine (n\u2009=\u20094\u20139) distal coronary artery segments after exposure to various pharmacological agents. *Significantly different (P\u2009<\u20090.05) from KCl (30\u00a0mM)-treated segments\nMeasurements of CGRP levels in organ bath fluid\nCapsaicin and its vehicle induced a significant CGRP release from human distal coronary artery segments into the organ bath fluid (Fig.\u00a04) as compared to levels observed in Krebs solution. There was no significant difference between CGRP levels obtained after incubation with capsaicin or its vehicle.\nFig.\u00a04CGRP levels measured in bath fluids (Krebs buffer, control) after capsaicin or vehicle challenge in precontracted human distal coronary artery segments. *Significantly different (P\u2009<\u20090.05) from Krebs buffer\nDiscussion\nIn the present study, we investigated the role of CGRP in capsaicin-induced relaxations in human and porcine isolated arteries. In all arteries investigated, there does not seem to be any relevant role of CGRP in the relaxant responses to capsaicin. Further, the effects of capsaicin appear to be mediated by non-specific mechanisms.\nIn all arteries investigated, capsaicin induced concentration-dependent relaxations, although these had a limited potency and efficacy in human proximal coronary artery. The responses to capsaicin were resistant to blockade with olcegepant in all tissues studied, which suggests no involvement of CGRP receptors. The potency of capsaicin is in line with what has earlier been observed in human coronary arteries (Franco-Cereceda 1991a) and guinea pig ileum (Fujimoto et al. 2006). As expected in view of its resistance to olcegepant, CGRP8\u201337 (10\u00a0\u03bcM) also did not block capsaicin-induced relaxations in human distal coronary artery segments. This is contrary to the observations by Franco-Cereceda and Rudehill (1989), where the authors claim the relaxations to capsaicin are mediated by CGRP on the basis of the observation that the sustained relaxations in the human coronary induced by capsaicin are similar to those induced by exogenous CGRP, unlike the transient relaxations to substance P, which are followed by rapid tachyphylaxis. In another publication, these authors claim involvement of CGRP in responses to capsaicin in the human isolated coronary artery on the basis of increased CGRP-like immunoreactivity after exposure of the artery segments to capsaicin (Franco-Cereceda 1991b). It should be noted that in the above study, the authors show an increase in CGRP-like immunoreactivity in large arteries, where no functional studies were performed. Further, the classical prerequisite for demonstrating the involvement of a particular pharmacological agent, by using the corresponding antagonist to block functional responses, was lacking in this study. Admittedly, the same group did demonstrate that CGRP8\u201337 inhibited the responses to capsaicin in porcine coronary arteries (Franco-Cereceda 1991b), although we could not block these responses even at 10-\u03bcM concentration.\nAs CGRP-induced responses are mediated by increases in cAMP (Gupta et al. 2006c), we measured the levels of this second messenger after exposure to capsaicin and CGRP. We observed no increase in cAMP after addition of capsaicin, in contrast to the increased levels of cAMP after exposure of the vessel segments to (exogenous) \u03b1-CGRP. These increased levels were, as expected, blocked by the CGRP receptor antagonist olcegepant. Interestingly, after exposure to capsaicin, we observed CGRP release in the organ bath fluid, where the human distal coronary arteries were mounted in accordance with earlier observations (Franco-Cereceda 1991a). However, a similar increase was observed after administration of vehicle, while the control was not studied in the study of Franco-Cereceda et al. (1991a). Although capsaicin is known to activate TRPV1, there are reports that ethanol, also via activation of TRPV1, induces the release of CGRP as well (Trevisani et al. 2002, 2004). Moreover, as obvious from Fig.\u00a01, the vehicle did not induce any relaxations in the precontracted arteries, and hence, the released CGRP cannot account for relaxations induced by capsaicin. Interestingly, the concentration of CGRP detected in the bath fluid of about 3\u00a0pM should have been about 10,000 times higher in the vessel segments (\u223c0.5\u00a0mg tissue in 5-ml organ bath fluid). Thus, the concentration of CGRP in the vessel segments should have been in the nanomolar range, which is equal to or even higher than the pEC50 in human distal coronary artery under similar experimental conditions (Gupta et al. 2006c) and should, thus, have induced a detectable relaxation. Therefore, it is most likely that the radioimmunoassay displayed cross-reactivity to another ligand, not CGRP. Admittedly, our observation that ruthenium red, even at a very high concentration (100\u00a0\u03bcM), did not block the responses to capsaicin, is in contrast with the observations described by Franco-Cereceda (1991a), where ruthenium red completely blocked the responses to capsaicin. Additional evidence for the fact that CGRP and TRPV1 are not involved in relaxant responses to capsaicin is provided by the fact that capsazepine, a competitive antagonist of TRPV1 (Caterina et al. 1997), did not block responses to capsaicin in our study.\nIn view of reports of involvement of various K+ channels in the relaxant responses to capsaicin in various smooth muscle preparations (Yeon et al. 2001; Zhu et al. 1997), we also investigated various K+ channel inhibitors in similar or even higher concentrations, but none of these blocked the responses to capsaicin. 4-Aminopyridine, a blocker of delayed rectifier K+ channels, which is reported to block the responses to capsaicin in rabbit coronary artery (Yeon et al. 2001) and guinea pig ileum (Fujimoto et al. 2006), was ineffective in human as well as porcine distal coronary artery. Similarly, charybdotoxin, a BKCa and IKCa blocker, which blocked capsaicin responses in equine tracheal smooth muscle (Zhu et al. 1997), did not block these responses at similar concentrations and even in combination with apamin, a SKCa blocker. We also used the RhoA kinase inhibitor Y-27632, but unlike in guinea pig ileum, (Fujimoto et al. 2006) it also did not antagonise responses to capsaicin in the isolated preparations used in the present study. The capsaicin-mediated responses appear to be endothelium-independent, as denudation of the endothelium did not significantly change the capsaicin-induced responses. Further, the vehicle of capsaicin was without effect and, hence, cannot account for the relaxant responses to capsaicin. Moreover, repeated administration of capsaicin in porcine coronary artery did not significantly decrease the responses, suggesting that stored neuropeptides are not responsible for the relaxations, as these would most likely be depleted after repeated challenges to capsaicin (Sams-Nielsen et al. 2001). Unlike in the present study, relaxations in precontracted guinea pig isolated pulmonary artery could not be repeated with capsaicin indicating depletion of neurotransmitters (Maggi et al. 1990). Although we did not include a positive control for the various K+ blockers in the current study, in the same set up at our laboratory, relaxant responses to l-S-nitrosocysteine were blocked by the combination of charybdotoxin (100\u00a0nM) and apamin (100\u00a0nM) in porcine distal arteries (Batenburg et al. 2004b). Further, the concentrations of the various inhibitors that we used in our study were equal to (Yeon et al. 2001; Zhu et al. 1997) or even higher (Yeon et al. 2001) than those employed by others.\nTaken together, our observations in human and porcine distal coronary artery suggest that capsaicin-induced responses are not mediated by CGRP, substance P or TRPV1 receptors and also do not involve various Ca2+-activated K+ channels. The relaxations to capsaicin are mediated by a cAMP independent pathway. The major component of capsaicin-induced relaxations, therefore, appears to be mediated by non-specific actions of capsaicin, rather than from the release of neuropeptides like CGRP.","keyphrases":["capsaicin","cgrp","human coronary artery","human meningeal artery","porcine coronary artery"],"prmu":["P","P","P","P","P"]}
{"id":"Environ_Manage-3-1-2094719","title":"Using Soil and Water Conservation Contests for Extension: Experiences from the Bolivian Mountain Valleys\n","text":"Soil and water conservation (SWC) contests among farmer groups were organized in five rural villages in the Bolivian mountain valleys. The contests were aimed at quickly achieving widespread sustainable results. This article analyzes the effectiveness of these contests as an extension tool. Mixed results were obtained. In three villages, participation rates in the SWC activities introduced in the contests were still high even 2 years after project withdrawal. These were all villages where a solid foundation for sustainable development had been laid before the contests were held. Two years later, most families were still involved in maintenance of the SWC practices introduced in the contests, and many farmers had started to experiment with different soil management practices. However, replications of these SWC practices were not widespread, Conservation Leaders did not continue with their training activities, and the quality of maintenance of the practices was often not satisfactory. In order to become a more effective extension tool and achieve widespread impact, SWC contests must receive continued support by a catalyst agency. Moreover, other SWC contests should also be organized in which practices are not predefined. Given that SWC contests are a low-budget extension tool, local municipalities could become more actively involved.\nIntroduction\nAgricultural development programs and extension services, both in developed and developing countries, struggle with frustratingly low adoption rates of soil and water conservation (SWC) practices. In response, Savenije and Huijsman (1991) called for a \u201cmaking haste slowly\u201d approach that focuses more emphasis on developing and implementing solutions together with rural people. Numerous experiences, principally from Non-Governmental Organizations (NGOs), have proven that these more participatory (slow) approaches often work and that sustainable livelihoods can be established. Successes to date, however, consist of scattered small-scale projects; wide-scale impact (haste) has yet to be achieved. The major dilemma remains combining widespread and short term impact (Farrington 1998). In SWC extension in particular, participatory approaches are too limited in scope, while existing governmental extension services are often not participatory enough and not sustainable. Hence, strategies and accompanying methodologies and tools are needed that achieve sustainable results in a short time and on a widespread scale.\nDespite urgent calls to modify attitudes and develop strategies for the extension of SWC (Sombatpanit and others 1996), the agricultural extension service in Bolivia has completely disappeared, and a governmental strategy for tackling soil erosion does not exist. NGOs have partially filled this gap, and several small-scale successes were obtained with the participatory introduction of SWC practices. However, these successes have not been reproduced at a large enough scale to have an impact at national (or even departmental) level. Government officials are often not even aware of these experiences, and NGOs themselves generally do not have the capacity for wide-scale dissemination of technology options (Farrington 1998). As a consequence, land degradation during the past decades has increased (Kessler and Stroosnijder 2006), and rural poverty remains a serious threat to sustainable development in Bolivia.\nIn response, a project executed by the Japan Green Resources Corporation (JGRC) developed and validated a strategy to motivate poor Bolivian mountain farmers to engage in adequate natural resources management, the logical strategy for SWC (Kessler 2007a). Unlike other small-scale participatory experiences, the logical strategy not only aims at slowness but also at making haste (i.e., in finding participatory methodologies and tools that achieve sustainable widespread success in SWC). Scaling-up and integrating SWC in a future extension system at a national level is the ultimate objective. One of the logical strategy\u2019s most innovative tools is conducting SWC contests among organized groups of farmers. The objective of this article is to analyze the effectiveness of these SWC contests as an extension tool. This is principally done by monitoring and evaluating farmers\u2019 participation in maintaining and replicating SWC practices after execution of the contests. The results will be used to discuss possibilities of using SWC contests in an extension strategy at a national level.\nStudy Area\nThe study was conducted in the five research villages of the JGRC project. They are situated in the north-Chuquisaca region of the inter-Andean valleys of Bolivia (Fig.\u00a01), which is a semi-arid region located at an altitude of 2500 to 3100 m above sea level. The majority of the families in this region still manage a mixed farming system, with mainly subsistence agriculture and a flock of goats and sheep. Potatoes are still the main crop and are the only crop that receives small amounts of manure. Other important crops are maize, wheat, and barley. Animal traction is mostly used for land preparation. Chuquisaca is one of the least developed areas in Bolivia, with the second lowest Human Development Index (HDI) in Bolivia, equaling only 0.49 (United Nations Development Programme 1997). Similarly, a study by the JGRC project revealed that the Human Poverty Index (HPI) for the research villages is among the highest in the world (on average 45%), with lack of access to drinking water and health facilities being the principal constraints. Hence, the region is very poor and faces enormous socioeconomic and physical constraints.\nFig.\u00a01Location of the study area in the inter-Andean valleys of Bolivia\nIn three of the research villages, the \u201cexperimental villages\u201d (Tomoroco, Kaynakas, and Sirichaca), the logical strategy was developed and tried out over a period of 4 years. The other two villages were \u201cvalidation villages\u201d (Talahuanca and Patallajta); in these villages the strategy was validated for 2 years. The villages were carefully selected, in order to obtain a representative sample of villages for the north-Chuquisaca region. Therefore, geographical, climatic, cultural, and socioeconomic characteristics are different among the villages. Rainfall varies from 350 mm\/year (in Tomoroco) to 750 mm\/year (in Kaynakas). Sirichaca has more flat lands for potato production, whereas the other villages have steeper and more erosion-prone slopes.\nSWC is a barely developed activity in this region. Since the colonial era, when farmers started using a combination of Spanish and traditional agricultural practices to maintain adequate production levels, practices have not changed much. As a consequence, once cultivable land began to be used more intensively, traditional conservation practices were no longer able to control erosion and maintain soil fertility. Most of the SWC practices currently found in the research villages are still based on traditional knowledge, but given their ineffectiveness in conserving soil and water, farmers have become increasingly sceptical about prospects for sustainable agriculture in their villages. Therefore, migration rates are high in most of the villages. Attempts of development agencies to promote improved SWC practices have not been successful, mainly because of failing intervention strategies and the lack of adequate extension tools.\nResearch Methodology\nThe SWC contests were conducted in the \u201cexperimental villages\u201d in 2001, and in the \u201cvalidation villages\u201d in 2003. In each \u2018\u2018experimental village\u2019\u2019 only one SWC contest, which focused on all physical SWC practices, was conducted; in the \u201cvalidation villages\u201d two SWC contests were conducted, focusing on two SWC practices each. During the SWC contests, the number of labor days invested per family was monitored for each SWC practice. Moreover, the rate of participation in the contests (i.e., percentage of families actively involved in SWC activities) was evaluated in each village.\nThe most important data for this study were obtained in the ex-post evaluation in 2005, 2 years after the JGRC project\u2019s withdrawal from the villages. The ex-post evaluation was conducted in all villages, except Sirichaca. In this village an evaluation was not considered necessary, due to the disappointing results and low participation rates during the contests. In the other villages, a random sample of 30 families was taken for the ex-post evaluation. Although the diversity of farmers within each village is high, and farmers who are progress-driven and economically better-off tend to invest more in SWC practices than others (Kessler 2006), this was not considered a variable. Hence, large families, as well as, for example, widows were included in the sample. Similarly, farmers from the higher economic stratum (with more land) as well as very poor small farmers were considered.\nIn the ex-post evaluation, the percentage of families actively involved in both maintenance and replications of SWC practices was assessed by means of a field survey. If at least one type of SWC practice was properly maintained, the family was considered to be actively involved in maintenance. If at least one SWC practice was replicated on other fields after the project\u2019s withdrawal, the family was considered to be actively involved in replications. Similarly, the active usage of other (nonphysical) SWC practices was evaluated during the ex-post evaluation.\nFinally, in order to obtain data concerning the popularity of the executed SWC practices, quality of maintenance of these practices was evaluated within the same sample of families. For each family an average score was given for the quality of maintenance of each type of SWC practice, ranging from very bad (or abandoned) to very good. Based on these scores, and in order to be able to compare the villages, average scores for each village were calculated.\nThe Need for More Effective Extension Tools\nAlthough Bolivia has tried out different extension service models (Bojanic 2001), these were always technology-centered and top-down, with weak research-extension linkages. Most widely used was the Training & Visit extension system, in which knowledge trickled down from the research institutes to the extension worker and finally to the farmers. Farmer participation in this model was mainly functional. In 1991 a World Bank project strengthened the country\u2019s research capacity, but extension remained extremely weak, resulting in poor adoption rates of improved practices (Bojanic 2001). In contrast to other developing countries, where low adoption rates forced extension agencies to apply more people-centered extension approaches, the governmental extension service in Bolivia simply disappeared. The research and extension component remained an independent foundation only for the study of potatoes. Currently, a demand-driven research and extension system (SIBTA, the Bolivian Agricultural Technology System) is in place that promotes innovative technologies in support of productive chains. Given the system\u2019s focus on promoting regional cash-crops, (poor) subsistence farmers are largely excluded.\nIn Bolivia, as in many other Latin America countries, NGOs fulfilled the more intensive extension tasks. In contrast to the functional type of participation of public sector organizations, most NGOs aim at an empowering type of participation (Farrington 1998). Several NGO-initiated experiments with Farmer Participatory Research (FPR), were based on the farmer-first approach (Chambers and others 1989). Their pioneering work with a large set of participatory techniques and innovative extension approaches has led to the realization that community participation and integrated (multi-sector) approaches are essential elements for success. In Latin America, farmer-to-farmer extension has had a notable impact and has led to adequate natural resources management in Mexico (Ramos 1998), Honduras (Sherwood and Larrea 2001), and Nicaragua (Braun and Hocd\u00e9 2000), among other countries. Given that NGOs can spend considerable resources in a few villages, and often invest in time-demanding and costly face-to-face participation, they have achieved higher participation rates than governmental extension services. However, the principal constraint of these extension approaches is their limited scope: they are difficult to replicate on a wide scale in the absence of local support organizations (Farrington and others 2002). With the prime objective of agricultural extension being to reach all farmers, this is a crucial constraint.\nThe JGRC project\u2019s logical strategy for SWC is not the first attempt to develop effective strategies that combine farmer participation at the grassroots level with extension and scaling-up. Killough (2005), for example, proposes \u201cparticipatory extension through [the] accompaniment model.\u201d This extension approach is a middle road between the traditional extensionist-centered approaches and the more recent farmer-led approaches. In this model, professional extension workers help farmer-promoters conduct on-farm experiments and provide farmer-to-farmer training. Local empowerment (through the development of farmer leaders) and strengthening local institutions are primary goals of this holistic approach. It is therefore important that such extension approaches promote integrated human development. Evidence from Honduras shows that farmers would have likely abandoned SWC practices long ago, if the extension approach had not addressed essential aspects of the interaction between human development and agriculture (Sherwood and Larrea 2001). Farmers conceptualized this interaction and holistic approach as \u201cthe human farm.\u201d\nImplementing such an integrated and farmer-based extension approach is urgently needed in Bolivia; only then can a wide-scale impact be achieved. Although municipalities have considerable budgets for rural development, they do not have the human capacity to provide this service, and they regard extension as a state responsibility. Hence, there is a need for (1) profound changes at institutional level to give priority to extension, and (2) effective extension tools that can be used in a farmer-based extension approach. SWC contests are an example of such a tool.\nUsing SWC Contests Within a Farmer-Based Extension Approach\nOrganizations in several countries have experimented with farmer contests or competitions. The State Farm Bureaus in the United States regularly hold such contests, in particular for young farmers. Management, growth and progress in farming operations are major factors in judging the contests. The outstanding farmers receive financial awards, which are usually made available by sponsors. Farmer competitions in New Zealand and Australia are more focused on developing technical skills and knowledge, with conservation and land management practices being one of the several competition modules. In developing countries all kinds of farmer contests are held, but rarely focused on SWC. Chuma and Murwira (1999) report of farmer competitions in Zimbabwe organized to stimulate the process of experimentation and revival of farmer knowledge regarding best farming practices.\nThe best results with respect to SWC have been achieved in two rural development projects in Bolivia and Peru, where SWC contests among farmers were used to speed up execution of SWC practices. Both projects make use of the \u201clearning from the best\u201d principle (i.e., learning from the best families and villages) (Van Immerzeel and De Zutter 2005). Knowledge management (i.e., combining knowledge and capacities from different farmers to find sustainable and fast solutions) is essential in this approach. The contests are used as a catalyst tool to disseminate this knowledge and motivate farmers to learn from the best, to experiment and innovate, and to win prizes by improving on what they have learned (Van Immerzeel and De Zutter 2005).\nIn Peru, the rural development project, MARENASS (Management of Natural Resources in the Southern Highlands), uses farmer contests to promote new technological practices for improving natural resources management, agricultural production and living conditions. An important characteristic of the project is the transfer of decision-making and responsibility for planning and financial resources to the villages. Each participating village receives financial support to hire direct technical assistance (Posthumus 2005). These external service providers can be farmers with much experience (\u201cthe best farmers\u201d), consultants or technical staff members. When a contest is organized, the villages themselves select and contract these privatized services to provide training to a number of selected farmers. By means of farmer-to-farmer training, the trained farmers in turn teach the other villagers the new techniques they have learned. Contests are held both at village level, with farmers competing against each other, and on district level between villages (Posthumus 2005). Jury members are selected by the participants; the families, or villages that best apply the recommendations provided by technical staff earn a cash prize. The innovations of MARENASS have been successful, and the project is still in progress (International Fund for Agricultural Development 2005); in 360 villages about 60% of all households have been reached (De Zutter 2004). Nevertheless, since internal problems in some villages limit their participation in the project, the impact of activities on watershed level is quite low (Posthumus 2005).\nIn Bolivia, the SID (Strategies for International Development) project, Pachamama Urupa, approaches the dual problems of soil erosion and rural poverty with the understanding that neither can be resolved without simultaneously addressing both. Competitions among villages are organized to encourage participation in natural resources management, and to recognize the most successful farmers (Strategies for International Development 2005). SID hires farmers who are skilled in one or more of the land conservation and reclamation practices as part-time staff. These para-professional extension agents train selected farmers in about five villages, who in turn train all the other participants of the contest in their respective villages (Borda 2002). The competitions are flexible enough to permit farmers to find their own solutions to their problems (i.e., to experiment and innovate). All farmers participate in the judging, which stimulates the sharing of knowledge and the adoption and improvement of the practices. The winning villages and families receive farm tools, seeds, and animals as prizes. Participation rates in the competitions are about 80% to 90%. The project has recently won the World Bank\u2019s \u201cDevelopment Marketplace Competition\u201d for innovative ideas in international development.\nThe MARENASS and SID projects are quite similar; they aim at wide-scale adequate natural resources management through a farmer-based extension approach that builds on local knowledge and capacities. Massive participation in farmer contests and farmer-to-farmer training are crucial aspects of this approach. Moreover, progressive learning and improvement is stimulated through constant interchanges, the participatory judging process and the closing ceremonies. Two important differences between the projects are that:in Bolivia the practices being judged in a contest are specifically defined, while in Peru the contests have a more general character (e.g., soil conservation) and practices are not specified;in Bolivia money is not involved (and prizes are in the form of goods), while in Peru farmers are responsible for contracting the trainers and cash prizes can be won.Based on these experiences, SWC contests are undeniably a very promising tool for farmer-based extension strategies, and for achieving fast and widespread sustainable impact in natural resources management. In the following section we will focus on the SWC contests of the JGRC project.\nSWC Contests in the JGRC Project\nIn the logical strategy of the JGRC project, SWC practices are executed within a framework of integrated rural development. They are always accompanied by \u2013 and often integrated in \u2013 other activities that aim at improving local livelihoods. An important feature of this strategy is the laying of a solid foundation for sustainable development before starting a SWC contest, with activities focusing, for example, on better village organization, responsible participation and effective collaboration (Kessler 2007b). The project\u2019s strategy stresses the human dimension of sustainable development: genuine participation of stakeholders is essential (Kessler 2007a). The objective of the SWC contests is to train farmers in basic SWC practices. Moreover, the contests encourage farmers to experiment and to decide which practices best fit their specific conditions.\nReflection and dialog \u2013 two key features of Participatory Research & Extension (Percy 2005) \u2013 are constantly used. Participatory research with a selected group of farmers, as well as farmer-to-farmer training and knowledge transfer, are crucial before, during and after the SWC contests. Lessons learned through the farmer-to-farmer movement that began in the Guatemalan highlands (Bunch 1982) are, therefore, taken into account (e.g., to use small-scale experimentation) to start slowly and small, to achieve early recognizable success and to limit the introduction of technology. The essential multiplier effect is provided by the SWC contests and farmer-to-farmer training.\nIn the next section we explain the SWC contests in more detail. In all the activities the project\u2019s extension worker plays a crucial role. At the end of the section we also present the differences between the approach of the aforementioned two projects and the JGRC project.\nActivities Preceding the SWC Contests\nPreceding the SWC contests (i.e., during the laying of a solid foundation for sustainable development in a village), SWC activities start with a group of about 10 Conservation Leaders (CLs). CLs are chosen by the assembly, taking into account personal characteristics such as responsibility, honesty and willingness to innovate. They receive intensive training from the project\u2019s extension worker, aiming at the generation of a progress-driven attitude and at conducting experiments on their fields. Experimentation focuses both on physical SWC practices (stone lines, diversion ditches, bench terraces, etc.) and agronomical soil management practices (more efficient manure use, composting, green manure, etc.). CLs are also stimulated to establish some test-sites for comparing with and without cases, with the objective of obtaining more visible results and making on-site comparisons. This might convince visiting farmers that the positive effects are indeed a result of the practices, and not of different physical conditions between their farm and the CLs\u2019 farms. Apart from experimentation and providing demonstrations on their own farms, CLs also have many less tangible tasks such as mobilizing the villagers to become involved in development activities. They are thus both promoters and technicians. Finally, training of CLs in techniques for knowledge transfer is given before and during the farm visits.\nOnce a solid foundation is laid in the village and CLs are sufficiently trained, the group decides when the first SWC contest will be held and which practices will be executed. In the dry season, practices like stone lines and gully control measures are considered and in the wet season practices that require digging such as diversion ditches and bench terraces. The village is informed in the assembly and by distributing information leaflets. During a period of at least a few weeks, families have the opportunity to decide whether to participate in the contest. CLs have an active role in motivating their neighbors, and in starting to organize groups based on vicinity. Eventually, each CL should lead a group of five to eight families. During these weeks of group formation, possible conflicts must be resolved, especially between neighbors. Given that effective group collaboration is crucial for a successful SWC contest, group formation should be given the required time.\nExecution and Evaluation of the SWC Contests\nA contest generally deals with two SWC practices. In a later stage a certain contest can be repeated and\/or more integrated contests can be considered, combining different practices. The first contest is the most important one, because it serves as a selection tool for distinguishing the interested families (those with a progress-driven attitude) from the others. In this first contest a subsidy is given \u2013 as an incentive to all participants \u2013 for the purchase of a set of tools that are essential to conduct SWC practices. Each family pays 20% of the original cost. Families that start participating in a later stage have no access to the subsidized tools, and should themselves catch up with the already executed practices.\nEach SWC contest takes about one or two months. Twice a year a contest can be held; one in the dry season and the other one in the wet season. The criteria for evaluating the contests are clearly indicated before starting: (1) executed quantity, (2) quality of the work, (3) knowledge, and (4) group collaboration. Major emphasis is given to training and learning during the contests. An essential technique for conducting most of the SWC practices is the adequate handling of the A-frame, which is used for establishing the contour lines in a field. This is generally taught during the first contest.\nOnce the contest starts, each group \u2013 under the guidance of their CL \u2013 decides how the work will be done. Reciprocal group work (or \u201cayni\u201d in Quechua) is mostly used. In this system, farmers work on each others\u2019 farms on a rotation basis. It is especially useful for labor-intensive work, and hastens the pace of execution. The host family provides food; money is never used. Reciprocity in the Andes region is based on mutual trust. Torrico and others (1994) argue that it still contains many religious aspects, and that, therefore, quality of the work is never discussed. Similar to the \u201calayon\u201d, a traditional form of village cooperation in the Philippines (Moneva and others 2000), the ayni serves as a venue for group learning, problem solving and the promotion of equitability among farmers. SWC practices are mainly conducted on fields situated near the farmer\u2019s homestead; later they can be replicated on other fields. The project\u2019s extension worker regularly monitors group work, and assists the CLs in their training. In the beginning, CLs often encounter problems and feel uncertain; regular meetings help to solve this.\nOnce a contest has finished, each CL measures the quantity of practices executed by his or her group. The verification of this quantity, as well as the evaluation of quality and knowledge, is done by means of cross-visits with other CLs. Quality criteria are harmonized between CLs before starting the evaluation. Knowledge is evaluated by asking some practical questions. For these three criteria (quantity, quality and knowledge) scores of 1 (bad) to 3 (good) can be obtained. This is written down on evaluation sheets. Based on observations, the extension worker evaluates the fourth criterion: group collaboration and cohesion. All criteria are given the same weight-factor in the final calculation.\nDuring the final closing ceremony the groups receive a reward for their efforts. Recognition of efforts is important because it engenders a sense of pride and it increases self-confidence (Cinn\u00e9ide and Conghaile 1990). The most recommendable rewards are seeds for green manure and vegetables, which contribute to more sustainable agriculture. Additionally, the winning groups also receive, for instance, potato, maize, or barley seeds, all in small quantities. These prizes are useful products and their value is small enough to avoid participating in the SWC contests for the wrong reason. In this respect, money or food must never be used as prizes.\nActivities Succeeding the SWC Contests\nMaintenance of the newly constructed practices is the first priority, and complementary vegetative and soil management practices are essential in order to achieve impact on soil productivity. Vegetative conservation practices (grasses, bushes, or trees) also strengthen most SWC practices and make them more sustainable. They require, however, controlled grazing and strict rules at village level that are respected by all villagers. Hence, only when such regulations are collectively agreed upon will vegetative practices be viable and will SWC practices work.\nGiven the importance of vegetation, establishing tree nurseries in each village \u2013 preferably at family level \u2013 is part of the holistic approach of the logical strategy. By means of an Integrated Project (Kessler 2007a), a group of farmers interested in agroforestry is trained to become trainers in this topic. Through farmer-to-farmer training, technical knowledge regarding agroforestry practices can be spread to a large number of farmers. Similarly, other groups of farmers specialize in, for example, manure management (improved stables, manure storage and collection methods), composting or green manure practices.\nThe spreading and replication of SWC practices to all the other fields that need to be conserved is the responsibility of each family. Although, ideally, groups that participated in the SWC contest will continue to work in ayni, most families will have to do it on their own. The role of the CLs is to provide support whenever it is requested.\nDifferences with the MARENASS and SID Projects\nAlthough similar in many aspects, especially in considering SWC as being part of an integrated approach to sustainable rural development, the JGRC project conducted its SWC contests in a slightly different way than the MARENASS and SID projects. This concerned four crucial aspects:The emphasis on training in specific SWC practices during each contest instead of leaving more space for experimentation. The reason: the SWC contests are foremost an extension tool, i.e., they aim at providing farmers with basic practical knowledge of some simple SWC alternatives. After the contests farmers experiment with innovations and will adapt and improve the practices.The emphasis on laying a solid foundation for sustainable development before starting the SWC contests. The reason: only farmers with a progress-driven attitude will continue to experiment and innovate after the project\u2019s withdrawal.The use of contests between groups instead of families or villages. The reason: to stimulate group formation and collaboration within a village, i.e., to maintain or re-establish the traditional work in ayni, and to contribute to better internal relations and knowledge exchange.The subsidizing of tools to stimulate participation in the contests, but no (or insignificant) prizes for winning groups. The reason: not having the tools is often a major limitation for participating, but once farmers participate, they must become convinced by the result of their work and not by the prizes they can win.\nResults and Discussion\nTable\u00a01 shows that similar numbers of family labor days were invested during the SWC contests in all villages. Groups generally worked two aynis (or two complete labor days) on each group member\u2019s fields. Stone lines were the most popular practice executed during the contests, with labor accounting for 40% to 70% of total labor days invested. In Tomoroco and Kaynakas \u2013 with more steeply sloping land \u2013 considerable investments were made in bench terraces, while in Sirichaca and Patallajta gully control works (especially the smaller ones) and earth bunds (due to the absence of stones) were given more attention.\nTable\u00a01Average number of labour days invested per family for each SWC practice, during the SWC contestsSWC practicesExperimental villagesValidation villagesTomorocoKaynakasSirichacaTalahuancaPatallajtaStone lines 8.15.64.55.25.4Diversion ditches 0.80.81.22.82.8Gully control0.30.61.80.52.5Bench terraces 2.12.7\u2013\u2013\u2013Earth bunds \u20130.11.60.21.1Infiltration ditches 0.20.3\u2013\u2013\u2013Individual terraces\u20130.51.4\u2013\u2013Total 11.510.710.58.911.8Source: M&E data in 2001 (experimental villages) and 2003 (validation villages)\nTable\u00a02 shows that the percentage of families participating in the construction of practices during the SWC contests was lowest in Sirichaca (48%) and Patallajta (66%); these are both villages in which a solid foundation for sustainable development was never laid. In Tomoroco the participation rate was highest with 86%. These data show that in the villages where activities concerning organization, collaboration and environmental awareness raising had already been successfully executed, the SWC contests were able to mobilize more people.\nTable\u00a02Percentage of families actively involved in SWC activitiesSWC activityExperimental villagesValidation villagesTomorocoKaynakasSirichacaTalahuancaPatallajtaConstruction of SWC practices during the SWC contests86754884 66Maintenance of SWC practices two years after project withdrawal9169\u20138453Replications of SWC practices two years after project withdrawal78252030Source: M&E data in 2001 and 2003 (during the contests) and ex-post evaluation data in 2005\nHowever, initial motivation is easy; continued motivation is what really matters (Savenije and Huijsman 1991). The effectiveness of the SWC contests can only be properly measured by evaluating the continued use of SWC practices after the project\u2019s withdrawal. Table\u00a02 shows the results of the ex-post evaluation, in which the percentage of families that have maintained and replicated SWC practices was assessed.\nConcerning maintenance, in Tomoroco this percentage is highest and has even increased; presently, 91% of all families perform adequate maintenance of one or more SWC practices. Quality of maintenance was moderate to good in Tomoroco (Table\u00a03); stone lines and bench terraces were especially well maintained. In Kaynakas 6% fewer families are currently involved in SWC activities; some families have not maintained their practices or have even removed stone lines and gully control measures. In some cases stones from stone lines were used for fruit tree terraces; such farmers experimented with and adapted practices. Farmers often refine their practices under environmental pressure (Veihe 2000). They may, for example, consider stones more effective for terrace building than for stone bunds. This is supported by Table\u00a03: in Kaynakas the quality of maintenance is better for bench terraces than for stone lines. Bench terraces are popular and productive for vegetables.\nTable\u00a03Quality of maintenance of SWC practices two years after project withdrawalSWC practicesExperimental villagesValidation villagesTomorocoKaynakasTalahuancaPatallajtaStone lines++\/\u2212+++\/\u2212Diversion ditches+\/\u2212+\/\u2212+-Gully control+\/\u2212-+\/\u2212+\/\u2212Bench terraces++n.a.n.a.Earth bundsn.a.n.a.\u2013\u2013++ very good; + good; +\/\u2212 moderate; - bad; \u2013 very bad (abandoned); n.a. not applicable (not executed)Source: Ex-post evaluation data in 2005\nAbandonment of SWC practices is highest in Patallajta, where two consecutive severe drought years and lack of impact of SWC practices caused general disillusion among the villagers. Migration increased and fields were left unattended. Table\u00a03 shows that quality of maintenance is bad to moderate in this village; earth bunds, which were heavily damaged during a high-intensity rain storm, were all abandoned. In neighboring Talahuanca, however, despite severe drought and similar damage to earth bunds, all participating families in the SWC contests are still actively involved in maintenance, except for earth bunds. The presence in Talahuanca of a solid foundation for sustainable development before starting the SWC contests explains the differences between both \u2018validation\u2019 villages. Maintenance of gully control measures was generally given little attention in all of the villages, although most people are of the opinion that these practices work very well.\nConcerning replications, a field survey in 2003 revealed that in Tomoroco and Kaynakas wide-scale replications were conducted during the two years in which the JGRC project was in effect. All participating families in the SWC contests constructed new SWC practices. This was mainly attributed to the inclusion of SWC activities in other activities of the JGRC project (namely Integrated Projects, see Kessler 2007a). Stone lines and diversion ditches were mostly replicated and covered large areas of the agricultural land in both villages. The average investment in these villages in maintenance and replications of SWC practices was estimated to be 20 labor days per family during these two years (2001 to 2003).\nHowever, more important from a sustainability viewpoint is what happened after the project\u2019s withdrawal (starting in 2003). Table\u00a02 shows that in Tomoroco 78% of all families constructed replications of SWC practices in the subsequent two years; in the other villages this was much lower (about 25%). Stone lines, especially, were replicated, given their relatively low labor requirements. The differences between the villages are explained by the fact that Tomoroco has more potential land for constructing stone lines, while in Talahuanca and Kaynakas the most important fields had already been protected. Moreover, on the steeper slopes of Kaynakas, stone lines sometimes disturb land preparation. In this village bench terraces were found more useful, but their replication requires higher investments. Replications of stone lines were found in only one village outside the project area, near Tomoroco..\nHence, the SWC contests have achieved mixed results in the five villages. They were effective in three villages: Tomoroco, Kaynakas and Talahuanca. The most positive outcome is that in these villages (on average) more than 80% of the families are currently involved in one way or another in SWC activities, and this was achieved without using incentive schemes or cash prizes. Most villagers also consider the executed SWC practices useful, and more than half of the farmers plan to replicate more measures in the near future. The three villages have in common that they all have solid foundations for sustainable development, which were laid before the SWC contests were conducted (in the first Phase of the logical strategy). This has triggered a renewed interest in alternatives to improve living conditions, including better soil management. The most negative outcomes are that in two of these villages (Kaynakas and Talahuanca) replication rates are currently very low, and in all villages CLs are no longer active as trainers. It seems that the dynamics of the process came to a halt after the project\u2019s withdrawal. Some farmers cautiously experiment and replicate SWC practices, but most of them only maintain existing practices and wait for tangible results before investing in new ones. Similarly, the CLs find themselves in a vacuum; they are rarely asked for advice, and there is no common objective to keep the CLs\u2019 groups active. The catalyst of the process, the project, is no longer there. The CLs were expected to fulfill this motivating role after the project\u2019s withdrawal, but this has not happened.\nOn the other hand, in Tomoroco and Kaynakas the landscape has visibly changed due to the installed SWC practices, and internal regulations concerning controlled grazing are being complied with (Kessler 2007a). According to the respondents in the ex-post evaluation, SWC contests work; 80% of respondents are positive about the actual impact of the contests. Most importantly, for many farmers the experiences acquired during the contests with alternative techniques and practices have served as a basis for experimentation. Under the marginal conditions of poor farmers, adapting innovations is more important than adopting innovations (Van de Fliert and Braun 2002). The message that alternatives are available to improve productivity has come through; people are interested and have started to experiment with new techniques.\nIn this respect, it is interesting to observe the usage of other SWC practices that were not included in the contests, but are now practiced by interested farmer groups and CLs. Table\u00a04 shows that several of these practices are currently in use by a considerable number of families, especially improved traditional practices like crop rotations (including leguminous crops) and mixed cropping systems. Hence, knowledge transfer from farmer to farmer does also occur spontaneously. If these SWC practices would have been included in the SWC contests, their adoption rate could have been much higher. Therefore, a first recommendation of this paper is to also conduct SWC contests in which practices are not previously defined. The contests described in this paper filled an important knowledge gap and provided farmers with basic information. However, the failure of earth bunds and the removal of other practices by several families prove that only executing contests with some predefined practices is not enough. Stimulating people\u2019s creativity and having each individual family decide which management practices best fit their conditions is considered crucial in the MARENASS and SID projects (Van Immerzeel and De Zutter 2005). Local adaptations of existing practices will become available sooner if more space is left for farmers\u2019 initiatives during the contests. Hence, the JGRC-type of contests can be maintained as a training tool, but other (more general) SWC contests should be organized as a follow-up activity.\nTable\u00a04Percentage of families using other SWC practices two years after project withdrawalSWC practicesExperimental villagesValidation villagesObservationsTomorocoKaynakasTalahuancaPatallajtaManure use361650Improved traditional practiceGreen manure200100New practiceCrop rotations60283015Improved traditional practiceStrip cropping2412150New practiceAgroforestry28642545New practiceMixed cropping68487065Improved traditional practiceComposting4020\u201310New practiceSource: Ex-post evaluation data in 2005\nA second recommendation is to strengthen the role of local organizations in providing follow-up support to the SWC contests. The major weakness of the JGRC project was its incapacity to institutionalize the process: SWC activities continued at a high rate when the project fulfilled its catalyst function, but drastically decreased after the project\u2019s withdrawal. Of course, municipalities were always involved in the activities, but their genuine participation was not achieved. Longer-lasting institutional support in organizing and facilitating more SWC contests would have strengthened the CLs\u2019 role, and would have kept the dynamic process going. To some extent, farmers can respond to land degradation without external support, but they need continued provision of technical assistance and information in order to make progress (Paudel and Thapa 2001). Farmers have often lost the self-confidence and capacity to adapt and innovate (Reijntjes and others 1998); without external support they will continue to farm in the way they have in the past (Percy 2005). Given that SWC contests are a low-budget extension tool, they can be easily organized by municipalities and local NGOs, for example. However, despite the involvement of local leaders during the SWC contests, the active involvement of many extension workers is needed, especially when larger areas are to be covered in order to achieve a wide-scale impact. This can be a major limitation for local institutions; policies at the macro-level that enable the implementation of a farmer-based extension approach are therefore required. Earlier in this paper we already mentioned that in Bolivia profound changes at institutional level are required that give priority to extension. Only when such changes are made can SWC contests become an effective extension tool \u201cto make haste slowly.\u201d\nIn Bolivia, to date, governments have never committed themselves to extension; strategies were not clearly defined and the extension service constituted a large burden on the state budget (Bojanic 2001). Presently, many participatory approaches and tools are available (Chambers and others 1989) that have proven their effectiveness over the last decades. Based on case studies in Thailand and Laos, Connell (2000) concludes that there are several opportunities for the institutionalization of participatory approaches in mainstream extension, but that they all require significant political commitment. Particularly regarding environmental problems, different interest groups often pull in complementary and opposing directions (R\u00f6ling and Pretty 1997). The challenge ahead for effective extension is to combine efforts. This has been done in Chile, where the government has contracted private technology companies to cater for the larger commercial farmers, and NGOs for small subsistence-oriented farmers. Rivera and Qamar (2003) for example propose a mixture of funding and service delivery modalities; governments could provide funding and NGOs could deliver the extension services. If the political willingness is present, this could also present an interesting opportunity in Bolivia.\nConclusions\nThe challenge we face in SWC is to quickly achieve widespread sustainable results, i.e., \u201cto make haste slowly\u201d (Savenije and Huijsman 1991). Based on the success of SWC contests elsewhere in the Andes (Van Immerzeel and De Zutter 2005), the JGRC project used this innovative tool in five rural Bolivian villages to put \u201cmaking haste slowly\u201d into practice. Mixed results were achieved with SWC contests between farmer groups. On the one hand, in villages where a solid foundation for sustainable development had already been laid, participation rates in the SWC contests were high. Most farmers were still involved in SWC activities even two years after project withdrawal, without receiving any incentive. In these villages large areas are currently protected with physical SWC practices and farmers have also started to experiment with other soil management practices. On the other hand, in the same villages the renewed system of collaboration focused on SWC was lost when the project withdrew, and Conservation Leaders did not continue with their training activities. Despite the visible widespread impact of the contests, sustainability is thus not yet assured. Farmers easily become disillusioned and unmotivated in the absence of a catalyst to keep the process of SWC contests and farmer-to-farmer training going. Moreover, tangible results such as higher soil productivity take a long time to appear. Farmers are opportunistic; in poor regions like the Bolivian mountain valleys, especially, they will grasp any opportunity to increase income.\nTwo recommendations were given in this paper to make SWC contests more effective in an extension strategy. First, in addition to the contests described here, other SWC contests should be organized in which practices are not predefined; this will stimulate peoples\u2019 creativity in developing adaptations of existing practices. Second, commitment is required from local institutions to support SWC contests as an extension tool; only then can Conservation Leaders continue their activities, and can a widespread impact be achieved. Given the responsibility of Bolivian municipalities for rural development, they must be the first to become actively involved in extension. However, steering and support with adequate strategies from departmental and state institutions is indispensable; this will motivate municipalities to take natural resources conservation and rural development more seriously. Without such support, any attempt to spread SWC practices via participatory extension methodologies \u2013 such as farmer contests \u2013 will likely fail; no matter how logical and well-designed the strategy may be.","keyphrases":["soil and water conservation","bolivian mountain valleys","farmer-to-farmer training","farmer-based extension","farmer contests","sustainable rural development"],"prmu":["P","P","P","P","P","P"]}
{"id":"Purinergic_Signal-3-3-2096642","title":"Ecto-5\u2032-nucleotidase and intestinal ion secretion by enteropathogenic Escherichia coli\n","text":"Enteropathogenic Escherichia coli (EPEC) triggers a large release of adenosine triphosphate (ATP) from host intestinal cells and the extracellular ATP is broken down to adenosine diphosphate (ADP), AMP, and adenosine. Adenosine is a potent secretagogue in the small and large intestine. We suspected that ecto-5\u2032-nucleotidase (CD73, an intestinal enzyme) was a critical enzyme involved in the conversion of AMP to adenosine and in the pathogenesis of EPEC diarrhea. We developed a nonradioactive method for measuring ecto-5\u2032-nucleotidase in cultured T84 cell monolayers based on the detection of phosphate release from 5\u2032-AMP. EPEC infection triggered a release of ecto-5\u2032-nucleotidase from the cell surface into the supernatant medium. EPEC-induced 5\u2032-nucleotidase release was not correlated with host cell death but instead with activation of phosphatidylinositol-specific phospholipase C (PI-PLC). Ecto-5\u2032-nucleotidase was susceptible to inhibition by zinc acetate and by \u03b1,\u03b2-methylene-adenosine diphosphate (\u03b1,\u03b2-methylene-ADP). In the Ussing chamber, these inhibitors could reverse the chloride secretory responses triggered by 5\u2032-AMP. In addition, \u03b1,\u03b2-methylene-ADP and zinc blocked the ability of 5\u2032-AMP to stimulate EPEC growth under nutrient-limited conditions in vitro. Ecto-5\u2032-nucleotidase appears to be the major enzyme responsible for generation of adenosine from adenine nucleotides in the T84 cell line, and inhibitors of ecto-5\u2032-nucleotidase, such as \u03b1,\u03b2-methylene-ADP and zinc, might be useful for treatment of the watery diarrhea produced by EPEC infection.\nIntroduction\nEcto-5\u2032-nucleotidase (CD73, EC 3.1.3.5) is a key extracellular enzyme which catalyzes the hydrolysis of extracellular nucleoside monophosphates to their corresponding nucleosides, with 5\u2032-AMP being the preferred substrate. Ecto-5\u2032-nucleotidase is highly expressed in endothelium, liver, and intestinal mucosa and at varying levels in other tissues including lymphocytes, kidney, and certain cancers. Ecto-5\u2032-nucleotidase is tethered to the extracellular surface of mammalian cells by a glycosylphosphatidylinositol (GPI) lipid anchor. Most of the research on this enzyme has been in the cardiovascular system because of its role in the production of adenosine, which protects cells against ischemia by multiple mechanisms including vasodilation [1, 2].\nEnteropathogenic Escherichia coli (EPEC) is a common cause of watery diarrhea in children in developing countries. While some aspects of EPEC infection, such as adherence, are well studied, the way that EPEC triggers watery diarrhea has been obscure, since EPEC produces no toxins. We recently proposed a theory that release of adenine nucleotides from host intestinal cells, followed by breakdown to adenosine, could trigger watery diarrhea by activation of adenosine receptors on intestinal cells [3].\nIn the gastrointestinal tract, ecto-5\u2032-nucleotidase was found to be a key enzyme necessary for enterocyte responses to 5\u2032-AMP, the neutrophil-derived secretagogue [4\u20136]. At that time the significance of ecto-5\u2032-nucleotidase was felt to be in the setting of invasive, inflammatory pathogens because polymorphonuclear neutrophils (PMNs) release 5\u2032-AMP during the process of chemotaxis into areas of infection or inflammation. Since that time, however, we have discovered that a noninvasive pathogen, EPEC, triggers a large release of ATP directly from host intestinal cells without any requirement for PMNs to be present [3]. Other diarrheal pathogens not considered classically invasive, such as Aeromonas hydrophila, also cause ATP release from enterocytes [7]. Extracellular ATP is broken down to ADP, AMP, and then adenosine, where it triggers a vigorous fluid secretory response in intestinal epithelium. We suspected that ecto-5\u2032-nucleotidase also played a role in the diarrheal fluid secretion induced by EPEC and initiated this study to determine if it is the case, and, if so, whether inhibitors of ecto-5\u2032-nucleotidase could block fluid secretion triggered by adenine nucleotides and EPEC infection. We used the T84 colon carcinoma cell line as a model because we have used these cells to study EPEC infection, they express ecto-5\u2032-nucleotidase, and they can be studied in the Ussing chamber to measure chloride secretion. In order to facilitate our study we also developed a nonradioactive, non-high-pressure liquid chromatography (non-HPLC) method for measuring ecto-5\u2032-nucleotidase in living T84 cell monolayers.\nMaterials and methods\nBacterial strains used E. coli strains used included laboratory E. coli strain HB101 (O: rough), commensal strain HS (O9: H4), and classic human EPEC strains E2348\/69 (serotype O127: H6), B171-8 (O111: NM), and JCP88 (O119: B14) as described in several publications [8\u201312]. EPEC mutants included JPN15, an E2348 derivative which has lost the EPEC adherence factor (EAF) plasmid [13], UMD874, the espF mutant derived from E2348, which is deficient in host cell killing [3, 14], and SE1010, with a mutation in sepZ (also called espZ), which is defective in type III secretion [15]. Bacteria were added to yield a multiplicity of infection (MOI) of 100:1.\nMaterials The following reagents were obtained from Sigma-Aldrich Chemicals: \u03b1,\u03b2-methylene-ADP, adenosine, adenosine 5\u2032-monophosphate (AMP), tetramisole (also called levamisole), polymyxin B, neomycin, purified phosphatidylinositol-specific phospholipase C (PI-PLC, from Bacillus cereus), and zinc acetate. BIOMOL (Plymouth Meeting, PA, USA) was the source of the BIOMOL GREEN reagent used in the phosphate release assay for nucleotidase activity and of U73122, a PI-PLC inhibitor. U73122 is 1-(6-[17 beta-3-methoxyestra-1,3,5- (10) triene-17-yl] amino\/hexyl) 1H-pyrroledione. A cell permeant PI-PLC activator, 3M3-FBS, was from the Calbiochem Division of EMD Biosciences (La Jolla, CA, USA). 3M3-FBS is 2,4,6-trimethyl-N-(m-3-trifluoromethylphenyl)benzenesulfonamide. Phosphate-free DMEM medium was purchased from MP Biomedicals (formerly ICN Biomedicals, Aurora, OH, USA). UNIFILTER plates were from Whatman (Clifton, NJ, USA).\nBacterial culture E. coli strains were grown overnight in Luria-Bertani (LB) broth at 37\u00b0C with 300\u00a0rpm shaking, then subcultured for 2\u00a0h in serum-free DMEM\/F12 medium supplemented with 18\u00a0mM NaHCO3, 25\u00a0mM hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer, pH 7.4, and 1% D-mannose as previously described [3]. For experiments with bacteria in minimal medium, bacteria were subcultured at a dilution of 1:2,000 into minimal medium (M9 salts plus casamino acids supplemented with 2\u00a0mM glucose). For convenience we used M9-CA liquid broth packets (E. coli Fast Media, MBI-Fermentas, Hanover, MD, USA) and added 2\u00a0mM glucose before use.\nCell culture T84 colon cancer cells were grown in DMEM\/F12 medium supplemented with 7.5% fetal bovine serum (Gibco\/Invitrogen, Grand Island, NY, USA), 18\u00a0mM NaHCO3, 20\u00a0\u03bcg\/ml vancomycin, and 15\u00a0\u03bcg\/ml gentamicin as previously described [16]. Ussing chamber studies of secretion were performed on T84 cell monolayers grown in Snapwell inserts (Corning Costar, Corning, NY, USA). The Snapwell inserts, which had a 0.4\u00a0\u03bcm pore size, were coated with 32\u00a0\u03bcg collagen per well by applying 0.16\u00a0ml of 0.2\u00a0mg\/ml type III collagen (Sigma; dissolved in warm 0.2\u00a0M acetic acid) to the Snapwell and allowing it to dry in the tissue culture hood under UV light. T84 cells were seeded onto the Snapwell inserts at ~1.2\u2009\u00d7\u2009106 cells per well and allowed to grow to confluency for 7\u20139\u00a0days. At this time the monolayers had transepithelial electrical resistances (TER) of 400\u20131,000\u00a0\u03a9 \u00b7 cm2.\nAssay for ecto-5\u2032-nucleotidase by phosphate release An assay for ecto-5\u2032-nucleotidase activity in living cells was developed based on the ability to detect inorganic phosphate (Pi) released from 5\u2032-AMP. This method has been used to detect activity of protein phosphatases such as PTEN [17] and lipid phosphatases [18] and is based on sensitive detection of low levels of Pi using the BIOMOL GREEN reagent, an enhanced and stabilized formulation of malachite green. To carry out the assay a phosphate-free buffer was used consisting of (in mM): NaCl, 154; KCl, 2: MgCl2, 4; NaHCO3, 18: HEPES, pH 7.4, 25; and glucose, 10. This buffer is referred to as nucleotidase buffer. To measure ecto-5\u2032-nucleotidase activity, the cell monolayer was rinsed once with sterile normal saline, then the medium was replaced with warm nucleotidase buffer. For cells in a 48-well plate, 0.25\u00a0ml of nucleotidase buffer were added per well, and the cells were allowed to rewarm to 37\u00b0C in the CO2 incubator. During pipetting the multiwell plate was kept warm using a metal heating block set at 37\u00b0, and a stopwatch was used to time the AMP addition and to terminate the assay. The procedure used for measuring monolayer activity was slightly different from that used to measure 5\u2032-nucleotidase activity released into the supernatant, as described below.\nCell-bound or monolayer activity To measure cell-bound ecto-5\u2032-nucleotidase activity in cell monolayers, 5\u2032-AMP was added to yield a final concentration of 0.2\u00a0mM to quadruplicate wells. Two other wells were left without addition of AMP (the \u201cno AMP blank\u201d). After a 10-min incubation at 37\u00b0 an aliquot (usually 50\u00a0\u03bcl) was removed and quickly transferred to a well of a 96-well plate to terminate the reaction.\nE. coli-induced release of nucleotidase activity into the supernatant medium For nucleotidase release experiments, the cell monolayer was changed to warm, phosphate-free DMEM, then infected with an E. coli strain for 35\u00a0min to allow adherence, then the medium was changed to nucleotidase buffer and the infection was allowed to continue for 2 or 3\u00a0h. Note that in this procedure any nucleotidase activity that is released in the first 35\u00a0min is discarded and not detected by our method. However, this two-stage procedure with the medium change was necessary because EPEC bacteria did not adhere normally if they first encountered the host cell in nucleotidase buffer. After a period of infection, supernatant medium was collected with a multichannel pipettor and transferred to the wells of a Whatman UNIFILTER plate (a 96-well with 0.45-\u03bcm membrane for sterile filtration). Sterile filtrates were prepared by centrifugation with collection of the filtered medium into another 96-well plate placed beneath the UNIFILTER as previously described [19]. Once again, experimental conditions were usually done in groups of six, with two wells not receiving any AMP (no AMP blanks) and four wells receiving 0.2\u00a0mM AMP. Again, the usual assay condition was 10\u00a0min at 37\u00b0 before the reaction was stopped by addition of 10\u00a0\u03bcl of 1\u00a0M HCl (\u201cstop solution\u201d).\nBIOMOL GREEN detection of phosphate released from AMP Stopped samples in a 96-well plate were brought to 100 or 110\u00a0\u03bcl volume with water if necessary, then treated with 100\u00a0\u03bcl of BIOMOL GREEN reagent. A standard curve of inorganic phosphate was prepared and run with every experiment; standards and unknown samples were incubated at room temperature for 20\u00a0min to allow a green color to develop, then the 96-well plate was read on a multiwell plate spectrophotometer at 620\u00a0nm. Unknown values were calculated from the standard curve using a hyperbolic curve fit using GraphPad Prism software, version 4.0. Results of monolayer activity were expressed as nmol of Pi produced\/min per 106 cells. For experiments showing nucleotidase release, the results were often expressed as nmol Pi released\/ min per well since the assay was done on a cell-free filtrate and because we often noted some detachment of cells during the longer incubations of 2\u20133\u00a0h needed to observe release.Although we believed we were developing a new method for assay of ecto-5\u2032-nucleotidase by phosphate release, during the course of this work another group reported using a virtually identical method, also based on detection of phosphate released from 5\u2032-AMP [1].\nDetection of CD73 by Western immunoblot To prove that the released 5\u2032-nucleotidase activity we measured was of host cell rather than bacterial origin, we performed immunoblots on the supernatants of infected T84 cells with antibodies against CD73. Initial attempts at immunoblotting using a commercially available monoclonal anti-CD73 antibody (Abnova Corp., Taipei, Taiwan) were unsuccessful. Dr. Linda F. Thompson, Oklahoma Medical Research Foundation, kindly sent us mouse monoclonal antibodies against human CD73 which had been generated by Dr. Wolf Gutensohn several years earlier. Of these, the two antibodies that gave the best results were designated CD73.4 and CD73.6 by Dr. Gutensohn; both were of isotype IgG2b and were used at a concentration of 1\u00a0\u03bcg\/ml. After washings, the secondary antibody was goat anti-mouse IgG2b conjugated to peroxidase at a dilution of 1:3,000 (Roche Molecular Biochemicals, Indianapolis, IN, USA). Blots were developed by chemiluminescence as previously described [20].\nUssing chamber studies A Snapwell insert containing a monolayer of T84 cells was placed in the plexiglass \u201cslider\u201d and inserted into the Ussing chamber (Physiologic Instruments, San Diego, CA, USA) at 37\u00b0C and continuously short-circuited by a four electrode, automatic voltage-clamp apparatus which measured short-circuit current (Isc) and transepithelial resistance (TER); chamber fluid resistance was automatically subtracted. Transepithelial resistance was determined by passing 10-s 10-mV current pulses through the tissues. Short-circuit current was measured by passing sufficient current through the tissues via Ag\/AgCl electrodes to reduce the spontaneous transepithelial potential to zero. The composition of the tissue bathing solution was (in mM): 140 Na+, 124 Cl\u2212, 21 HCO3-, 5.4 K+, , 1.2 Mg2+, 1.2 Ca2+, and 10 glucose. Raw short-circuit current (Isc) values were converted to \u03bcA per cm2 by dividing by the area of the Snapwell monolayer (1.13\u00a0cm2). Other details of the Ussing chamber methods were exactly as described [16].\nProtein assay Protein assay was by the Coomassie blue dye binding assay method of Bradford, using a Bio-Rad kit [21].\nExpression of ecto-5\u2032-nucleotidase RNA by reverse transcription and real-time polymerase chain reaction (PCR) T84 cells grown in 24-well plates were infected with EPEC for 35\u00a0min, then the medium was changed to remove unbound bacteria. Three hours after the medium change, ciprofloxacin was added to 25\u00a0\u03bcg\/ml to kill EPEC and the incubation was continued 1 more hour. Old medium was removed, and the cell monolayer was lysed in extraction buffer with 10% \u03b2-mercaptoethanol (RNeasy Kits, Qiagen, Valencia, CA, USA). RNA was subjected to reverse transcription using Invitrogen Superscript III reverse transcriptase; 5\u00a0\u03bcl of purified RNA was used per 50\u00a0\u03bcl reaction, and gene-specific primers at 0.2\u00a0\u03bcM were used. Reverse transcription reaction was at 55\u00b0 for 1\u00a0h. Copy DNA from reverse transcription was diluted 100-fold, then analyzed by quantitative real-time PCR using the same oligonucleotide primers. For ecto-5\u2032-nucleotidase the primers used were 5\u2032-TTC CAC CCT GAA GAA GGC CTT TGA-3\u2032 (forward) and 5\u2032-ATA ACT GGG CAC TCG ACA CTT GGT-3\u2032 (reverse). As a normalizing gene we used glyceraldehyde phosphate dehydrogenase (GAPDH) as described by Khan et al. [22] except that we redesigned longer primers which were 5\u2032-TCG ACA GTC AGC CGC ATC TTC TTT-3\u2032 and 5\u2032-ACC AAA TCC GTT GAC TCC GACC CTT-3\u2032. PCR was carried out using a MyiQ Single-Color qRT-PCR machine from Bio-Rad (Hercules, CA, USA) using SYBR Green as the dye to monitor the amplification. Relative expression was calculated by the \u0394\u0394Ct (\u201cLivak\u201d) method as described [23], where Ct is the number of cycles to threshold. SYBR Green PCR reagents were from Bio-Rad and to reduce the cost, the PCR reaction volume was reduced to 25\u00a0\u03bcl. PCR was performed using a two-step protocol with an annealing temperature of 58.7\u00b0 and denaturation at 95\u00b0 for 30\u00a0s each (i.e, no extension step) for 35 cycles. Thermal melt curve analysis was performed at the end of the PCR amplification and showed a single sharp peak for the genes analyzed.\nData analysis and presentation All error bars shown in graphs and error values reported in the text are standard deviations. Significance was tested by one-way analysis of variance (ANOVA) with the Tukey-Kramer post-test for multiple comparisons, using InStat software for the Macintosh from GraphPad software (San Diego, CA, USA). Graphs were prepared using Prism 4.0 software, also from GraphPad. Asterisks shown on graphs indicate a p value of <\u20090.05.\nResults\nEcto-5\u2032-nucleotidase activity was readily measurable in T84 cell monolayers using the phosphate release assay method developed in this study. Figure\u00a01 shows the characteristics of the ectoenzyme in this system. Figure\u00a01a shows a curve of activity vs substrate concentration, demonstrating that the enzyme shows Michaelis-Menten kinetics. In four experiments similar to that shown in Fig.\u00a01a, the KM was 229\u2009\u00b1\u200930\u00a0\u03bcM and the Vmax was 2.7\u2009\u00b1\u20091.8\u00a0nmol\/min per 106 cells (mean \u00b1 SD of 4 experiments). While the KM was very consistent over time and from experiment to experiment, the Vmax was more variable as we compared experiments separated by months or years. We noted that highly T84 passaged cells (passage number of 70 or greater) showed lower levels of ecto-5\u2032-nucleotidase activity than less passaged cells from the same source (experiments not shown). The KM we observed in intact T84 cells was higher than that reported for the purified enzyme derived from a colon carcinoma cell line, BCS-TC2 cells [24]. Figure\u00a01b shows the raw data of Pi generated over time in the presence and absence of added AMP substrate and demonstrates that the assay was linear for 10\u00a0min; therefore, 10\u00a0min was chosen as the standard assay duration.\nFig.\u00a01Characterization of ecto-5\u2032-nucleotidase activity in T84 cell monolayers. 5\u2032-Nucleotidase activity was measured by the release of inorganic phosphate (Pi) from AMP in phosphate-free buffer using a colorimetric method as described in \u201cMaterials and methods.\u201d For a, c, and d, the spectrophotometric reading in the absence of AMP (\u201cno AMP blank\u201d) was subtracted from the total reading. In b the \u201cno AMP blank\u201d was not subtracted and is shown in b as labeled. a Michaelis-Menten curve of enzyme velocity vs concentration of AMP substrate. b Time course of Pi release vs time. c Lack of inhibition by tetramisole, an inhibitor of alkaline phosphatase. d pH dependence of 5\u2032-nucleotidase activity. e, f HPLC traces showing conversion of AMP (e) to adenosine (Ado, f) in T84 cell monolayers. g Sensitivity of the AMP-hydrolyzing activity to the inhibitor \u03b1,\u03b2-methylene-ADP, confirming it as ecto-5\u2032-nucleotidase\nAlkaline phosphatase is another abundant intestinal ectoenzyme which can hydrolyze 5\u2032-AMP [25]. Alkaline phosphatase can be distinguished from 5\u2032-nucleotidase by its alkaline pH optimum and sensitivity to inhibition by tetramisole. Figure\u00a01c shows that the ectonucleotidase we measured on T84 cells was not inhibited by tetramisole (also known as levamisole). Furthermore, its activity did not increase with increasing pH (Fig.\u00a01d) providing evidence that the activity being measured was not alkaline phosphatase. In addition, we used HPLC to confirm that 5\u2032-AMP was being converted to adenosine in the supernatant medium of T84 cells (Fig.\u00a01e and f). Morover, the 5\u2032-nucleotidase activity we measured by phosphate release was sensitive to inhibition by \u03b1,\u03b2-methylene-ADP, a known inhibitor of ecto-5\u2032-nucleotidase (Fig.\u00a01g and see also Fig.\u00a04). Lastly, after we initiated this study, other researchers also reported using phosphate release from 5\u2032-AMP as their assay for ecto-5\u2032-nucleotidase [1]. We have found that the increased throughput of the phosphate release assay increases its usefulness compared to the much slower HPLC methods for ecto-5\u2032-nucleotidase.\nFigure\u00a02 shows the effect of EPEC infection on ecto-5\u2032-nucleotidase activity released into the supernatant medium during an experimental EPEC infection of cultured T84 cells. Wild-type EPEC strains E2348\/69 and JCP88 triggered a release of 5\u2032-nucleotidase into the supernatant medium while nonpathogenic E. coli strains HB101 and HS did not. The supernatant medium was subjected to sterile filtration prior to assay to remove bacterial cells as well as any detached host cells, so that the activity reflects a soluble and not a cell-bound form. In experiments where we determined both the supernatant release and the monolayer activity in the same experiment, supernatant release by wild-type EPEC was 30\u201340% of monolayer activity 3\u00a0h after the medium change (Fig.\u00a02a and data not shown). To determine if the 5\u2032-nucleotidase could possibly be derived from the E. coli bacteria rather than from the host we also assayed sterile filtrates of E. coli for 5\u2032-nucleotidase activity and it was virtually absent from the culture filtrates of all E. coli strains tested, including EPEC (Fig.\u00a02b, light gray bars). Even after treatment with a concentration of polymyxin B sufficient to cause >\u200999% bacterial cell lysis the amount of bacterial nucleotidase-like activity was far less than the amounts we observed in the supernatant in Fig.\u00a02a. In addition, our protocol included a medium change step in order to remove unbound bacteria. Therefore we concluded that the 5\u2032-nucleotidase activity being released in Fig.\u00a02a was of host cell origin.\nFig.\u00a02Effect of E. coli infection on 5\u2032-nucleotidase activity in the supernatant medium. a Nonpathogenic E. coli strains (HB101 and HS) or EPEC strains E2348\/69 and JCP88 were subcultured in DMEM medium for 2\u00a0h, then used to infect T84 cells at a multiplicity of infection of 100:1 in phosphate-free DMEM. After 35\u00a0min to allow adherence, the medium was changed to nucleotidase buffer and aliquots were collected at various times after the medium change, filtered through a 0.45-\u03bcm filter to remove bacterial cells, then assayed for 5\u2032-nucleotidase activity. Since the activity was measured in the cell-free sterile filtrates the activity is expressed as nmol\/min per well. bE. coli suspensions were subjected to sterile filtration without treatment (light gray bars) or following treatment with 50\u00a0\u03bcg\/ml polymyxin B, a lytic antibiotic (dark gray bars). Then the nucleotidase activity of a 100-\u03bcl aliquot was measured. The 100\u00a0\u03bcl volume was chosen because this was the volume of inoculum needed to achieve an MOI of 100:1 for the slowest growing strain (E2348\/69) in a typical 48-well plate of T84 cells\nTo determine if this hypothesis was correct and to try to determine the mechanism of EPEC-induced 5\u2032-nucleotidase release, we compared the 5\u2032-nucleotidase release triggered by wild-type EPEC strain E2348\/69 with the plasmid-cured derivative, JPN15, and its espF mutant, UMD874 (Fig.\u00a03a). The espF mutant is defective in host cell killing and in inducing damage to monolayer integrity [14, 26]. Interestingly, the espF mutant was not attenuated in its ability to trigger 5\u2032-nucleotidase release, but instead it consistently outperformed the wild-type strain in this regard. In six experiments 5\u2032-nucleotidase release induced by the espF mutant exceeded that of the wild-type E2348\/69 by an average of 2.3\u2009\u00b1\u20090.14 fold (p\u2009=\u20090.03 by paired t-test), suggesting that host cell death or damage is not the mechanism of EPEC-induced nucleotidase release.\nFig.\u00a03a Effect of EPEC mutants and PI-PLC inhibitors and activators on induced 5\u2032-nucleotidase release into supernatant. Release of 5\u2032-nucleotidase activity into supernatant medium was measured as described in \u201cMaterials and methods\u201d and in the legend to Fig.\u00a02. Once again, activity in the cell-free sterile filtrates is expressed as nmol\/min per well. However, for purposes of comparison 48-well plates contain ~0.25\u2009\u00d7\u2009106 T84 cells per well at confluency. b U73122, an inhibitor of PI-PLC, was used at a concentration of 2\u00a0\u03bcm and was re-added after the medium change; *significantly decreased compared to the EPEC strain alone, p\u2009<\u20090.05. cm-3M3-FBS, a cell-permeant sulfonamide activator of PI-PLC, was added at the concentrations and for the times indicated. d Inhibitory effect of neomycin on PI-PLC-induced 5\u2032-nucleotidase release; this experiment was performed on cells grown in a 24-well plate, with ~0.8\u2009\u00d7\u2009106 cells per well; *significantly decreased compared to PI-PLC alone. e PI-PLC was again added to a final concentration of 0.1\u00a0U\/ml for the times shown; *significantly different from the corresponding control. This figure is a composite of experiments that were separated in time and with cells of different passage number; therefore the absolute amount of activity varies among the figure parts. f Effect of EPEC infection on expression of RNA encoding ecto-5\u2032-nucleotidase in T84 cells, by reverse transcription and real-time PCR. T84 cells were infected for 35\u00a0min, then the supernatant medium was changed to remove unattached bacteria, then the T84 cell monolayer harvested for RNA extraction 4\u00a0h after the medium change. Reverse transcription and PCR conditions were as described in \u201cMaterials and methods.\u201d Expression of ecto-5\u2032-nucleotidase was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). f Normalized expression of a single representative experiment (mean \u00b1 SD of 6 PCR wells). g Immunoblot analysis of the proteins released into the supernatant after 2\u00a0h of infection with EPEC or an EPEC mutant (SE1010, sepZ\/espZ), using monoclonal antibody against CD73. Lane 1, supernatant from uninfected, control T84 cells. Lanes 2 and 3, supernatant from cells infected with wild-type E2348\/69. Lanes 4 and 5, supernatants from cells infected with the sepZ mutant. GPI-linked CD73 has an apparent molecular size of 72\u00a0kDa (not seen in this blot) and the GPI-cleaved soluble portion runs at 51\u00a0kDa (heavy band in lanes 2 and 3)\nEcto-5\u2032-nucleotidase is tethered to the extracellular surface of the cell by a glycosylphosphatidylinositol (GPI) lipid anchor [24, 27]. GPI lipid anchors are cleaved by phosphatidylinositol-specific phospholipases C (PI-PLC), and EPEC has been shown to activate at least one type of PI-PLC in the host cell, PI-PLC-\u03b3 [28]. Therefore we considered whether PI-PLC activation by EPEC could be a nonlethal mechanism by which EPEC triggered 5\u2032-nucleotidase release from host intestinal cells.\nFigure\u00a03b shows an experiment in which we tested whether a PI-PLC inhibitor could block EPEC-induced nucleotidase release. Indeed, 2\u00a0\u03bcM U73122 did reverse the nucleotidase release triggered by wild-type EPEC strains. Conversely, a newly described cell-permeable activator of PI-PLC, called m-3M3-FBS [29], triggered nucleotidase release from uninfected cells in a time- and concentration-dependent manner (Fig.\u00a03c). Experiments to test if m-3M3-FBS would enhance EPEC-induced release were inconsistent, with some experiments showing a mild enhancement or additive effect and others showing no enhancement or mild antagonism (results not shown).\nIn addition we also tested whether purified PI-PLC added to the apical surface of the T84 cell monolayer induced 5\u2032-nucleotidase release and it did (Fig.\u00a03d and e). Figure\u00a03d also shows that 50\u00a0\u03bcg\/ml neomycin could partially block 5\u2032-nucleotidase release by purified PI-PLC. In addition to its antimicrobial effects, neomycin is an inhibitor of several types of PLC enzymes including PI-PLC. Figure\u00a03e shows that after a 2-h treatment with PI-PLC not only was 5\u2032-nucleotidase activity increased in the supernatant, but that monolayer activity was depleted as well (right portion of Fig.\u00a03e). In experiments similar to Fig.\u00a03e done with EPEC infection, we could not detect any decrease in monolayer activity 2\u20133\u00a0h after infection, despite the liberation of 5\u2032-nucleotidase activity into the supernatant. We considered the possibility that increased host cell synthesis of ecto-5\u2032-nucleotidase might be occurring in response to the cleavage of the enzyme from the cell surface. We analyzed the abundance of RNA encoding ecto-5\u2032-nucleotidase by reverse transcription and real-time quantitative PCR in response to EPEC infection. Figure\u00a03f and Table\u00a01 show that infection with wild-type EPEC and the EPEC espF mutant (UMD874) increased the abundance of ecto-5\u2032-nucleotidase RNA compared to uninfected control cells. The increase in expression of ecto-5\u2032-nucleotidase RNA by wild-type EPEC was 33% above control. While this is not a large increase, it does explain the maintenance of normal levels of ecto-5\u2032-nucleotidase on the surface of EPEC-infected cells, because under the most optimum conditions the release of enzyme activity into the supernatant is about 30\u201340% of monolayer activity (e.g., Fig.\u00a03e).\nTable\u00a01Effect of EPEC infection on expression of RNA encoding ecto-5\u2032-nucleotidase in T84 cells, by reverse transcription and real-time PCRConditionNormalized expression ratioaUninfected control1.0, by definition+1\u00a0U\/ml PI-PLC1.23\u2009\u00b1\u20090.26HS-infectedb0.66\u2009\u00b1\u20090.22E2348\/69-infectedb1.33\u2009\u00b1\u20090.21cUMD874 (espF)-infectedb1.40\u2009\u00b1\u20090.19caCells were infected for 35\u00a0min, then the supernatant medium was changed to remove unattached bacteria, then the T84 cell monolayer harvested for RNA extraction 4\u00a0h after the medium change. Expression of ecto-5\u2032-nucleotidase was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data shown are the means \u00b1 SD of 5 similar experimentsbBacteria were added with the intent of achieving a multiplicity of infection (MOI) of 100:1; actual MOIs in the 5 experiments ranged from 71 to 144cSignificantly increased compared to uninfected control by paired t-test (p\u2009=\u20090.04)\nThe 5\u2032-nucleotidase released into the supernatant appeared to be of host cell origin, because it could be triggered by chemical stimuli in the absence of EPEC infection (Fig.\u00a03c\u2013e). The host origin was also proven using a mouse monoclonal antibody against human CD73. Wild-type EPEC infection of T84 cells triggered the release into the supernatant of a 51\u00a0kDa protein recognized by a mAb against CD73 (Fig.\u00a03g, lanes 2 and 3). In contrast, the EPEC sepZ\/espZ mutant, which is defective in type III secretion, triggered the appearance of a fainter band which was of ~55\u00a0kDa in size. The origin of this faint band is unknown, but it is possible it is generated by proteolysis rather than PI-PLC activation. For example, E2348\/69 expresses EspC, an autotransporter protease whose export is independent of type III secretion.\nFigure\u00a04 shows that 5\u2032-nucleotidase activity in living T84 cells was susceptible to the same inhibitors as the purified enzyme studied by others. Figure\u00a04a shows that \u03b1,\u03b2-methylene-ADP inhibited ecto-5\u2032-nucleotidase activity and that its inhibition has the characteristics of a competitive inhibitor. In the presence of \u03b1,\u03b2-methylene-ADP the enzyme shows an apparent increase in KM but no increase in the Vmax, and the inhibition can be overcome at high concentrations of AMP substrate. In contrast in Fig.\u00a04b one observes that zinc acetate behaves as a noncompetitive inhibitor of the enzyme, with a decrease in the Vmax but no increase in the KM (in fact there is a small, paradoxical decrease in KM in the presence of zinc). Figures\u00a04c and d show the inhibitory dose-response curves at a single 0.2\u00a0mM concentration of AMP substrate. For both inhibitors, when concentration is expressed on a logarithmic scale, the curves are well fit by a sigmoidal, single-site model, allowing determination of the inhibitory concentration 50% (IC50) as shown in Figs.\u00a04c and d. In addition to \u03b1,\u03b2-methylene-ADP and zinc acetate, ecto-5\u2032-nucleotidase activity was also inhibited by 3\u201310\u00a0mM sodium fluoride as reported for the purified enzyme (data not shown). 5\u2032-Nucleotidase activity released into the supernatant was also sensitive to inhibition by \u03b1,\u03b2-methylene-ADP and zinc acetate with similar potencies (data not shown).\nFig.\u00a04Effect of the 5\u2032-nucleotidase inhibitors on enzyme activity in T84 cell monolayers. a, b Michaelis-Menten curves of enzyme activity vs substrate concentration in the absence (open symbols) and presence (closed symbols) of the inhibitor shown. Enzyme parameters were calculated by nonlinear curve fitting using GraphPad Prism and the fitted parameters of Vmax and KM are shown below the graphs. c, d Dose-response curves for inhibition of ecto-5\u2032-nucleotidase activity by \u03b1,\u03b2-methylene-ADP (c) and zinc acetate (d) using a 0.2\u00a0mM concentration of AMP substrate. In all parts of Fig. 4 the inhibitor was added before the AMP, and in c and d the curves were fitted to a sigmoidal inhibitory dose-response curve and the inhibitory concentration 50% (IC50) value derived from the curve fit is shown\nIn Fig.\u00a04 the inhibitor was always added to the cell monolayer prior to the AMP substrate. Using the Ussing chamber apparatus to measure chloride secretion, we realized we could monitor the effects of inhibition of 5\u2032-nucleotidase activity dynamically and assess the ability of inhibitors to reverse a secretory response already underway. In preliminary experiments we found that to reverse an ongoing secretory stimulus we had to add inhibitors at concentrations considerably above their IC50 values. The requirement for addition of inhibitors well above the IC50 is due to the fact that adenosine is a very potent secretagogue in intestinal tissues and a very high degree of inhibition of ecto-5\u2032-nucleotidase is needed to reduce the production of adenosine below the secretory threshold.\nFigure\u00a05a shows that secretion triggered by 10\u00a0\u03bcM AMP is reversed by theophylline, an adenosine receptor antagonist. Figure\u00a05a emphasizes the fact that 5\u2032-AMP has no secretory ability unless it is converted into adenosine. Adenosine then acts via apical adenosine A2b receptors to trigger chloride secretion toward the apical side of the monolayer. Figure\u00a05b shows that chloride secretion triggered by 1\u00a0\u03bcM AMP is partially antagonized by 2\u00a0mM zinc acetate even when the inhibitor is added after secretion has started. Addition of 1\u00a0\u03bcM adenosine restored the short-circuit current (Isc) to its previous trend, showing that zinc does not block adenosine receptors or \u201cpoison\u201d the secretory machinery. Under the same experimental conditions, 200\u00a0\u03bcM \u03b1,\u03b2-methylene-ADP produced a much larger degree of reversal of Isc than did zinc (Fig.\u00a05c). As with zinc, \u03b1,\u03b2-methylene-ADP did not prevent a subsequent, normal Isc response to addition of adenosine (Fig.\u00a05c, right-hand portion), showing that it is acting as a 5\u2032-nucleotidase inhibitor and not an adenosine antagonist. The efficacy of inhibition by \u03b1,\u03b2-methylene-ADP in this paradigm, however, was strongly dependent on the concentration of AMP used to trigger secretion. At higher concentrations of AMP (e.g., 20\u00a0\u03bcM), the degree of inhibition by \u03b1,\u03b2-methylene-ADP was markedly less (Fig.\u00a05d) than when 1\u00a0\u03bcM AMP was used as the stimulus. In contrast, zinc acetate produced the same, modest inhibition of Isc regardless of the concentration of AMP used. Figure\u00a05e summarizes the effectiveness of the two inhibitors, zinc and \u03b1,\u03b2-methylene-ADP, when tested against various different concentrations of AMP as the initial stimulus. Figure\u00a05e shows that the effectiveness of \u03b1,\u03b2-methylene-ADP declines significantly with increasing concentrations of AMP used, whereas zinc\u2019s inhibitory effects are fairly constant over a range of AMP concentrations. The results of Fig.\u00a05 (b\u2013e) vividly illustrate, at the level of of an electrophysiologic response, the difference between a competitive and a noncompetitive mode of action of an enzyme inhibitor (compare with Fig.\u00a04a and b).\nFig.\u00a05Effect of 5\u2032-nucleotidase inhibitors on the chloride secretory response to AMP in T84 cells in the Ussing chamber. Short-circuit current (Isc) was measured in T84 cells grown in Snapwell inserts as described in \u201cMaterials and methods\u201d; in this cell line and electrode configuration, a positive Isc (upward deflection on the graphs) represents chloride secretion in the basolateral to apical direction. All of the additions of agonists and inhibitors in these parts of the figure was on the apical (mucosal) side of the monolayers only. a Reversal of AMP-induced secretory response by theophylline, an adenosine receptor antagonist. b Partial inhibition of AMP-induced secretion by 2\u00a0mM zinc acetate, with restoration of the tracing to its previous trend by adenosine, demonstrating that adenosine receptors are not blocked by zinc. c Substantial reversal of Isc by \u03b1,\u03b2-methylene-ADP when a low concentration of AMP (1\u00a0\u03bcM) is used as the stimulus. Note that adenosine fully restores secretion to its previous level. d Lesser degree of reversal of Isc by \u03b1,\u03b2-methylene-ADP when a higher concentration of AMP (20\u00a0\u03bcM) is used as stimulus. e Summary of many experiments in which fixed concentrations of zinc and \u03b1,\u03b2-methylene-ADP were tested for inhibitory efficacy against varying concentrations of AMP as test stimulus. In Fig. 5e the slope of the \u03b1,\u03b2-methylene-ADP curve was \u22121.73\u2009\u00b1\u20090.5 by linear regression (95% confidence limits of the slope value \u22123.27 to \u22120.2, significantly different from zero, p\u2009<\u20090.04). In contrast the slope of the zinc curve was \u22120.04\u2009\u00b1\u20090.23 and did not differ significantly from zero\nSince the discovery of EPEC-induced ATP release we have hypothesized that EPEC bacteria may gain a nutritional advantage by this inflicting kind of damage on the host cell [3]. The lumen of the gastrointestinal tract is a purine-limited environment, as demonstrated by in vivo gene expression screens such as in vivo expression technology (IVET) [30] and signature-tagged mutagenesis (STM) [31]. Nucleotides and nucleosides enhance growth of EPEC strains in vitro, and these growth-promoting effects are most obvious when adenosine is added to minimal medium (Fig.\u00a06). Compared to minimal medium, addition of 30\u00a0\u03bcM adenosine markedly accelerated growth of EPEC strains such as E2348\/69 (Fig.\u00a06). Addition of 5\u2032-AMP also increased EPEC growth, although not as much as adenosine. Alpha-\u03b2-methylene-ADP alone has no effect, stimulatory or inhibitory, on EPEC growth, but it did completely reverse the growth-promoting effects of 5\u2032-AMP (Fig.\u00a06a and b, right-hand bars). Alpha-\u03b2-methylene-ADP did not block the enhanced growth seen with adenosine (not shown). Similarly, 100\u00a0\u03bcM zinc acetate did not affect growth in minimal medium (Fig.\u00a06c, left-hand bars) or the enhanced growth seen with adenosine (Fig.\u00a06c, two middle bars), but zinc did abolish the enhancement of growth by AMP. E. coli 5\u2032-nucleotidase is susceptible to inhibition by \u03b1,\u03b2-methylene-ADP and zinc; in fact, the crystal structure of the E. coli enzyme was solved with \u03b1,\u03b2-methylene-ADP bound in the catalytic pocket [32]. Bacterial utilization of extracellular nucleotides is usually by dephosphorylation followed by uptake of the nucleoside (in this case adenosine) and inorganic phosphate [33]. Although we began studying \u03b1,\u03b2-methylene-ADP and zinc as inhibitors of the host cell enzyme, CD73, a side effect (or side benefit) of these compounds may be inhibition of bacterial 5\u2032-nucleotidase activity as well, an effect that is able to block the nutritional benefits that E. coli bacteria derive from extracellular AMP (Fig.\u00a06).\nFig.\u00a06Effect of 5\u2032-nucleotidase inhibitors on growth of EPEC strain E2348\/69 in minimal medium supplemented with 30\u00a0\u03bcM adenosine or AMP. Minimal medium here refers to M9 salts, 2% casamino acids, and 2\u00a0mM glucose. Growth was at 37\u00b0C with 300\u00a0rpm shaking after a 1: 2,000 dilution from an overnight culture in LB medium. a, c Culture turbidity by spectrophotometric absorbance at 600\u00a0nm. b Bacterial protein content. a, b Growth-enhancing effects of AMP are blocked by \u03b1,\u03b2-methylene-ADP. c Growth-enhancing effects of AMP are blocked by zinc acetate; *significantly decreased compared to AMP alone (p\u2009<\u20090.05)\nDiscussion\nWe became interested in ecto-5\u2032-nucleotidase (CD73) because of its likely role in the generation of adenosine from adenine nucleotides released during EPEC infection. This host enzyme has not received a great deal of scrutiny in the context of microbial pathogenesis, and when it was studied its role was felt to be in the setting of a polymorphonuclear neutrophil (PMN) response, since activated PMNs release 5\u2032-AMP during migration into an infected area [4, 34]. EPEC behaves clinically as a noninvasive pathogen and does not trigger an influx of fecal leukocytes, but nevertheless triggers ATP release from the host. Therefore our results suggest that ecto-5\u2032-nucleotidase plays a role in the response to a broader range of pathogens, including noninvasive enteric pathogens, than was previously thought.\nOne important finding of our study is that EPEC infection triggers a release of ecto-5\u2032-nucleotidase from a cell-bound to a soluble, free form (Figs.\u00a02 and 3). The significance of this finding is not clear at present but there are several possibilities to be considered. First, studies by other investigators have shown that cleavage of ecto-5\u2032-nucleotidase from its GPI lipid anchor increases its activity significantly via an increase in the Vmax [27]. This could result in an increase in the activity of 5\u2032-nucleotidase present in an EPEC-infected portion of the intestine, which could be an advantage to EPEC (see Fig.\u00a06 and additional discussion below). The observed increase in transcription of ecto-5\u2032-nucleotidase in response to EPEC infection (Fig.\u00a03f and Table\u00a01), if accompanied by a similar increase in protein synthesis and surface expression of the enzyme, would replace ecto-5\u2032-nucleotidase lost due to cleavage of the lipid anchor and also contribute to an increase in total activity. Third, release of nucleotidase from its lipid anchor would change its distribution from a mucosal location to a soluble form in the intestinal lumen, where it could possibly be more accessible to substrate. This could be important in the human or animal EPEC infection because expression of ecto-5\u2032-nucleotidase is highest in the intestinal crypts [35], whereas EPEC adheres to villus tips as well as crypts. Last, after many days of infection the mucosa could be stripped of ecto-5\u2032-nucleotidase activity, leading to malabsorption of nucleotides in the diet. Indeed, malabsorption of electrolytes has been demonstrated after 7\u00a0days of infection in a rabbit model of EPEC infection [36]. In addition to ecto-5\u2032-nucleotidase, several other important digestive enzymes have GPI lipid anchors, including alkaline phosphatase and aminopeptidase N (CD13) and these also could be liberated by EPEC-induced activation of PI-PLC. Another GPI-linked protein of interest in the intestinal tract is decay-accelerating factor (CD55), the cellular receptor for adhesion by diffuse adherent E. coli [37, 38] and which is also involved in internalization of Salmonella into vacuoles.\nSeveral virulent pathogens have been found to secrete their own microbial ATPases or nucleotidases into the extracellular medium, including Entamoeba histolytica [39], Trichinella spiralis [40], and Vibrio cholerae [41]. Researchers have speculated that secreted microbial nucleotidases may serve in protection against killing by host immune cells, acquisition of nutrients from the host, or dampening of host immune responses. EPEC does not appear to secrete nucleotidase activity under any conditions we have encountered, but may achieve the same goal by triggering the release of 5\u2032-nucleotidase from the host cell.\nEcto-5\u2032-nucleotidase acts on nucleoside monophosphates, with 5\u2032-AMP as the preferred substrate, and is inactive toward ATP. Therefore, the generation of AMP from ATP requires another enzyme or enzymes. CD39, or ectonucleoside triphosphate diphosphohydrolase, is the major enzyme responsible for this conversion in the cardiovascular system and probably in the gastrointestinal tract as well [42, 43]. CD39 is also deserving of further study in the context of microbial pathogenesis.\nOne surprising finding of our study was that the espF EPEC mutant was not attenuated but in fact better than the wild-type EPEC in release of ecto-5\u2032-nucleotidase (Fig.\u00a03a). Although this mutant is defective in host cell killing, its phenotype has been instructive in many ways. For example, the ability of the espF mutant to trigger a robust ATP release led to us the discovery of a new pathway for ATP release from the host cell; this \u201csecond pathway\u201d is not dependent on cell death but is dependent on the cystic fibrosis transmembrane regulator (CFTR) for ATP efflux [19]. The competence of the espF mutant in release of ATP and of ecto-5\u2032-nucleotidase from the host cell may help explain why this mutant is only modestly attenuated compared to wild-type in some animal models of EPEC infection, such as Citrobacter rodentium in mice [44]. On the other hand, the sepZ\/espZ mutant is attenuated in its ability to trigger ecto-5\u2032-nucleotidase release (Fig.\u00a03f), showing that type III secretion is necessary for PI-PLC activation and release of the enzyme from its lipid anchor.\nThe role of ecto-5\u2032-nucleotidase in triggering EPEC-induced fluid secretion is highlighted in the Ussing chamber studies shown in Fig.\u00a05. Even after AMP has induced a chloride secretory response, addition of the nucleotidase inhibitor \u03b1,\u03b2-methylene-ADP is able to reverse the short-circuit current in a manner similar to that of an adenosine receptor antagonist, theophylline (compare Fig.\u00a05a and c). Tracings such as those in Fig.\u00a05 seem to indicate that to sustain a secretory response there must be on ongoing production of adenosine from AMP. In other words, there seems to be a \u201csink\u201d for depletion of adenosine from the extracellular medium, either by cellular reuptake or by adenosine deaminase. The rapid turnover of adenosine implied in these experiments suggested that inhibitors of ecto-5\u2032-nucleotidase might be able to block EPEC-induced fluid secretion in animal models of infection.\nIn discussions of zinc on ecto-5\u2032-nucleotidase it is worthwhile to note that zinc supplements have been shown to reduce the duration and severity of watery diarrhea in children in developing countries around the world [45\u201348]. In most of these trials the microbial etiology of the diarrhea was not carefully investigated, so it is unknown if zinc is particularly efficacious in diarrhea due to certain pathogens, such as EPEC, or if it beneficial \u201cacross the board\u201d for multiple agents. Likewise, the mechanism by which zinc exerts its beneficial effects has not been carefully studied in the field trials. In earlier reports it was assumed that zinc was acting by correcting a zinc deficiency, but this assumption has been called into question by the finding that zinc supplements have a beneficial effect on diarrhea even in children with normal serum zinc levels at the outset [49]. Kelleher et al. showed that zinc provided some added benefit beyond a probiotic (Lactobacillus rhamnosus) in rhesus monkeys infected with the human EPEC strain E2348\/69; the monkeys studied were not zinc deficient [50]. Inhibition of ecto-5\u2032-nucleotidase or K+ channels [51] by zinc could slow diarrhea but this should be considered a pharmacological effect of the mineral rather than repletion of a deficiency. Inhibition of 5\u2032-nucleotidase also blocks the growth-enhancing effects of AMP on EPEC bacteria (Fig.\u00a06); since this is a direct effect on bacterial cells this action of zinc would not depend on the nutritional status of the host. The effects of zinc on EPEC infection and on diarrheal diseases in non-zinc-deficient children deserve further study.","keyphrases":["adenosine","zinc","extracellular nucleotides","diarrheal disease"],"prmu":["P","P","P","P"]}
{"id":"J_Mol_Biol-1-5-2291451","title":"Plasticity Within the Obligatory Folding Nucleus of an Immunoglobulin-like Domain\n","text":"A number of \u03b2-sandwich immunoglobulin-like domains have been shown to fold using a set of structurally equivalent residues that form a folding nucleus deep within the core of the protein. Formation of this nucleus is sufficient to establish the complex Greek key topology of the native state. These nucleating residues are highly conserved within the immunoglobulin superfamily, but are less well conserved in the fibronectin type III (fnIII) superfamily, where the requirement is simply to have four interacting hydrophobic residues. However, there are rare examples where this nucleation pattern is absent. In this study, we have investigated the folding of a novel member of the fnIII superfamily whose nucleus appears to lack one of the four buried hydrophobic residues. We show that the folding mechanism is unaltered, but the folding nucleus has moved within the hydrophobic core.\nIntroduction\nStudies of structurally related proteins have clearly indicated that for\nmany proteins the folding mechanism is determined primarily by the native state\ntopology.1,2 This is evident from comparative \u03a6-value analyses of proteins\nthat share similar folds but are very different in sequence. Such studies\ninclude representatives from all protein classes; all-\u03b1 proteins,3\u20139 all-\u03b2 proteins,10\u201322 and mixed \u03b1\/\u03b2 proteins.23\u201328 The folding mechanisms of these proteins range from purely\nhierarchical, where secondary structural elements form before any tertiary\nstructure, to pure nucleation-condensation, where secondary and tertiary\nstructure form concomitantly.29 A study of representative members of the homeodomain superfamily\nfamily has suggested that their folding mechanisms are dependent on inherent\nsecondary structural propensity, and the authors propose that all folding\nmechanisms are in fact variations of the same theme:3 as the propensity for forming secondary structures decreases,\nthe folding mechanism shifts from pure hierarchical to polarized transition\nstates, and ultimately to the classical nucleation-condensation mechanism first\nshown for CI2.30 Separate investigations have suggested that a folding nucleus\nconsists of obligatory and critical components: the specific interactions\nnecessary to establish the correct topology form first, followed by a subset of\nsurrounding residues that provide the critical stabilising\ninteractions.31\u201333\nAs part of the \u201cfold approach\u201d we have studied the folding of a number of\nproteins with an Ig-like fold,16\u201318,34\u201336 all of which are composed of two anti-parallel \u03b2-sheets packed\nagainst each other. The deep hydrophobic core is always formed from the packing\nof the four central B, C, E and F \u03b2-strands,37 but the number and position of the edge strands varies between\nthe superfamilies.38 Although proteins in different superfamilies share the same\nfold, they are apparently unrelated in sequence and are found in proteins with a\nwide variety of functions. The stabilities of the Ig-like proteins studied to\ndate range from about 1\u00a0kcal mol\u2212\u00a01 to 9\u00a0kcal\nmol\u2212\u00a01, and the folding rate\nconstants vary by six orders of magnitude. However, there is a correlation\nbetween folding rate and thermodynamic stability, which suggests that the\ninteractions that are critical in stabilising the fold also govern the folding\nprocess.34\nAll members of the fold studied to date fold via a\nnucleation-condensation mechanism where the obligatory folding nucleus comprises\na set of structurally equivalent buried hydrophobic residues in the B, C, E and\nF-strands that form a \u201cring\u201d of interactions in the core (Figure 1). Early\npacking of the residues, which are distant in sequence, ensures formation of the\ncorrect native state topology.35,36 The critical nucleus surrounds this obligatory nucleus, but the\ndegree of structure formation varies between different proteins.\nThe residues that form the obligatory folding nucleus are highly conserved\nwithin immunoglobulin domains but are conserved only in terms of residue type in\nfibronectin type III (fnIII) domains. However, there are rare examples of\nproteins that appear to have a disparate nucleation pattern. Here, we have\nidentified a fnIII domain in which one of the hydrophobic residues in the\nconserved folding nucleus has been replaced by a surface polar residue, and we\nask how the folding mechanism has been affected. An extensive protein\nengineering \u03a6-value analysis reveals that the folding mechanism is unaltered,\nbut that a spatially different set of core residues is used to form the\nobligatory folding nucleus, where interactions within each sheet establish the\ncorrect hydrogen bond registry between the core \u03b2-strands. Subsequent\ninteractions between two such pairs are able to bring the \u03b2-sheets together and\nset up the complex Greek key topology.\nResults\nResidue conservation within the folding nucleus of fnIII\ndomains\nA non-redundant multiple sequence alignment from Pfam was used to\nanalyse the residue conservation in the putative folding nucleus of fnIII\ndomains.39 These nucleation positions were identified through\ncomparison with the third fnIII domain from human tenascin (TNfn3), which\nhas been studied extensively in our laboratory. In most fnIII domains (73%),\nall four residues in the proposed folding nucleus positions are hydrophobic.\nFurther analysis reveals that where there is a polar residue in one of these\nfolding positions, it is almost invariably in the C or E strand, and these\nhydrophilic residues are usually arginine or lysine. These can act as\nhydrophobic residues, since the long aliphatic side-chains can traverse the\ncore and allow the charged terminus to reside on the surface of the\nprotein.40 Small hydrophilic residues, such as asparagine or aspartate,\nare found very rarely (only in \u223c\u00a03% of all cases).\nMost fnIII domains (65%) have a single aromatic residue in the proposed\nfolding nucleus. Analysis of the distribution of aromatic residues in the\nfour obligatory folding nucleus positions shows clearly that aromatic amino\nacids are located preferably within the C\u2013F sheet (Figure 2).\nFurthermore, the type of aromatic residue present is affected by the solvent\naccessibility of the \u03b2-strand: the C-strand position is partly\nsolvent-accessible, and hence the majority of aromatic residues occurring\nwithin this strand are tyrosine (thereby allowing hydrogen bonding of the\nhydroxyl group with solvent molecules). In contrast, the F-strand position\nis deep within the core and phenylalanine is almost always the aromatic\nresidue of choice. Approximately 20% of the sequences have more than one\naromatic residue in the folding nucleus, and again these residues are almost\nexclusively located in the C\u2013F sheet (86% of all such sequences). This\nasymmetry is likely caused by the presence of an adjacent conserved\ntryptophan in the B-strand that is essential for stability but not involved\nin the folding nucleus.16,18 Interestingly, about 15% of the fnIII domains appear to\nfold without any aromatic residues at the supposed folding positions,\nsuggesting that a large side-chain is not crucial for the formation of the\nobligatory nucleus.\nSelection of CAfn2 as a candidate\nAll known fnIII structures were surveyed to find a candidate protein\nthat was missing a hydrophobic residue at one of the four putative folding\npositions. Only one candidate protein was identified, the second fnIII\ndomain in chitin A1 from Bacillus circulans (CAfn2),\nwhich has a surface-exposed asparagine in the putative nucleus position in\nthe C-strand. It has an aromatic residue in the F-strand folding position\n(Phe66).41\nIn this work we intended to compare the folding of CAfn2 with TNfn3, as\nthis is the most extensively studied \u201ctypical\u201d fnIII domain. The structure\nof CAfn2 was superimposed on the structure of TNfn3 to reveal a RMSD of only\n1.7\u00a0\u00c5 over all structurally equivalent positions (69 residues), even though\nthe two proteins have just 13% sequence identity (Supplementary Data Figure 1). The major differences are\nrestricted to the loops and turns, which are different in length in the two\nproteins. CAfn2 has the same Greek key topology as all other fnIII domains,\n(Figure 1), and possesses both\nthe highly conserved Trp residue in the B-strand and the conserved tyrosine\ncorner motif (Supplementary Figure\n1).42 A comparison of the structures of CAfn2 and TNfn3 reveals\nthat the lengths of the highly conserved EF-loop and the AB-turn are\nidentical in these proteins, whereas the rest of the loops show some\nvariation. Most notably the C and C\u2032-strands are shorter in CAfn2 and are\njoined by a short tight turn (Figures 1 and\n3). The packing\ninteractions are almost identical in the two proteins, with the majority of\nthe contacts being made within the same sheet, and the interactions between\nsheets occurring in \u201clayers\u201d.18 To help visualize the interactions that occur within the\nproteins, all residue positions are described according to both the \u03b2-strand\nand the core layer in which they reside (Figure\n3).\nImportantly, however, inspection of the supposed obligatory folding\nnucleus of CAfn2 clearly shows that the C-strand residue, N40, does not pack\nagainst the other putative nucleus residues (Figure 1). How, therefore, does this domain\nfold?\nCharacterization of wild-type CAfn2\nThe equilibrium stabilities of wild-type CAfn2 and all its mutant\nproteins were determined through the use of standard denaturation curves fit\nto a two-state equation.43 From a number of repeated measurements of wild-type CAfn2\nthe free energy of unfolding (\u0394GD\u2013N)\nwas estimated to be 6.7(\u00a0\u00b1\u00a00.3) kcal\nmol\u2212\u00a01 at pH 5.0 and 25\u00a0\u00b0C.\nKinetic studies reveal a single unfolding phase, but two refolding\nphases. Since the slower phase accounts for less than 15% of the total\namplitude, and is apparently independent of the concentration of denaturant,\nit was attributed to proline isomerisation. Both arms of the chevron plot\nare linear (Figure\n4), and the\n\u03b2T for wild-type CAfn2 is estimated to be 0.56. This is\nsimilar to that of TNfn3,44 indicating that the two transition state structures are of a\nsimilar compactness.\nEffect of mutations\nUsing the \u03a6-value analysis of TNfn3 as a basis, a total of 23\nnon-disruptive mutations were made throughout the CAfn2 protein. Where\npossible, all mutations were conservative deletions.45 Contacts lost upon mutation are given in Table 1. The\ndifference in free energy between wild-type and mutant\n(\u0394\u0394GD\u2013N) was calculated using an\naverage m-value,\n<m>, of 1.15(\u00b1\u00a00.02)\nkcal mol\u2212\u00a01 M\u2212\u00a01. It has been shown that m-values are\nhard to determine with accuracy (the range of\nm-values observed is typical for that observed in\nmost large-scale protein engineering studies) so that use of an average\nm-value reduces the error in\n\u0394\u0394G.46 Most mutations are destabilizing but\n\u0394\u0394GD\u2013N values range between\n\u2212\u00a01.5\u00a0kcal mol\u2212\u00a01\nand +\u00a06.0\u00a0kcal mol\u2212\u00a01.\nChevron plots for all mutants are shown in Figure 4, with the kinetic data given in Table 2.\nUsing these data, \u03a6-values were calculated from refolding data at 0\u00a0M\ndenaturant. Note that for three highly destabilized proteins (L22A, Y36L and\nL58A) there were too few data points in the refolding arm to determine the\ngradient (mkf) accurately. In these\ncases an average mkf (1.06\nM\u2212\u00a01) was used to fit the data.\nNote that this has no effect on the final \u03a6-value determined at 0\u00a0M\ndenaturant. Only one mutant, V38A, has a folding\nm-value that is significantly different from this\nmean value.\nSeveral mutant proteins exhibit roll-over in the unfolding arm, and\nsome mutants have increased mku\nvalues. Such behavior in unfolding has several possible explanations. It has\nbeen ascribed to \u201cHammond\u201d behaviour, where there is a broad transition\nstate barrier,47 or to population of a high-energy intermediate.48 We do not have sufficient data to distinguish these two\npossibilities and, since there is no roll-over in the wild-type protein, it\nis not possible to determine \u03a6-values for the \u201clate\u201d transition state using\nunfolding data. However, the model used has no effect on analysis of the\n\u201cearly\u201d transition state, at 0\u00a0M denaturant.49\nStructure of the transition state\nA number of positions in each \u03b2-strand of CAfn2 were probed using\n\u03a6-value analysis. \u03a6 is a measure of the extent of structure at a given\nresidue in the transition state (\u2021). A \u03a6-value of 1 indicates that the\ninteractions are fully formed in \u2021, whereas a \u03a6-value of 0 indicates that\nthe structure is as unfolded in \u2021 as in the denatured state. The precise\ninterpretation of fractional \u03a6-values is ambiguous but is usually taken to\nmean that the residue is partly structured in \u2021.50 However, it is generally accepted that, particularly when\ncomparing homologous proteins, the best approach is to look at patterns of\n\u03a6-values rather than considering the absolute values of individual\nresidues.2\nThe CAfn2 \u03a6-values range from 0 to 0.5, indicating that none of the\npositions analyzed is completely structured at the transition state\n(Table 2). In general, the\n\u03a6-values in the A and G-strands are close to 0, while those in the central\nB, C, C\u2032, E and F\u00a0\u03b2-strands are higher, and the \u03a6-values in these central\nstrands are higher in the central layers of the core than at the extremes\n(Figure 3), as observed in\nTNfn3,18 the tenth fnIII domain of fibronectin (FNfn10)16 and the titin immunoglobulin domain TI I27.17 The \u03a6-values were classified into low (\u03a6\u00a0\u2264\u00a00.2), medium (0.2\u00a0<\u00a0\u03a6\u00a0<\u00a00.4) and high (\u03a6\u00a0\u2265\u00a00.4)\nclasses. These \u03a6-values are mapped onto the CAfn2 structure in Figure 5.\nA and G-strands\nAll mutations in the A and G-strands gave low \u03a6-values, indicating\nvery little structure formation in these strands in the transition state\n(Table 2; Figures 3 and\n4). Both mutated sites in the\nA-strand, L10A(A3) and S12A(A2), pack onto the neighbouring B-strand,\nwhilst inter-sheet interactions are formed with residues from the F and\nG-strands (Table 1). The\n\u0394\u0394GD\u2013N for the S12A(A2)\nmutation is too low for a reliable \u03a6-value to be determined. The three\nresidues probed in the G-strand, S81(G4), V84(G3) and V86(G2), make\ninteractions mainly with residues from the A and F-strands\n(Table 1).\nStrands B, C, E and F\nAll core positions in CAfn2 were mutated, with the exception of W24\nin the B-strand. The distribution of \u03a6-values reveals that the\nhydrophobic core is only partially formed in the transition state\n(Table 2; Figures 3 and\n4). The highest values occur\nat positions V38(C4) and I55(E3), with moderate \u03a6-values at four other\npositions: I20(B2), L22(B3), V68(F4) and A70(F5).\nC\u2032-strand\nThe C\u2032-strand is connected by two short loops to the central C and\nE-strands. Three of the four mutations within this strand, L44A(C\u20323),\nT46A(C\u20324), and V48A(C\u20325), have high \u03a6-values (Table 2; Figures 3 and\n4). T46 and V48 interact\nmainly with the buried residues from the C and E-strands, whereas the\nL44 contacts are limited to residues within the C and C\u2032-strands\n(Table 1).\nDiscussion\nCAfn2 folds by a nucleation-condensation mechanism\nThere has been much discussion of folding mechanisms in recent years.\nThe two \u201cextremes\u201d are represented by the framework model, where local\nsecondary structure forms before tertiary structure, and\nnucleation-condensation, where secondary structure and tertiary structure\nform concomitantly. Such extremes have distinct patterns of \u03a6-values. In the\nframework model, \u03a6-values will fall into two groups, one set close to 1 and\nthe other close to 0. This has been termed a polarised transition state. In\na nucleation-condensation mechanism, the transition state structure will be\nmore diffuse, involving most of the protein, and \u03a6-values will all be\ngenerally between 1 and zero. Furthermore, in the nucleation condensation\nmechanism, the pattern of \u03a6-values is generally distinctive, with a subset\nof residues having slightly higher \u03a6-values, with \u03a6-values gradually\nbecoming lower as structure condenses around the early \u201cnucleus\u201d. The\npattern of \u03a6-values shows CAfn2 to have a diffuse nucleus, with two-thirds\nof the residues having \u03a6-values between 0.10 and 0.45. Moreover, these are\narranged in the structure as one would predict from a\nnucleation-condensation pattern, with higher \u03a6-values at the centre of the\ncore, becoming lower towards the edges of the molecule. This suggests\nstrongly that CAfn2 folds, as do other Ig-like proteins,\nvia a nucleation-condensation folding mechanism.\nHowever, again like other Ig-like proteins, there is a significant number of\nresidues, in the peripheral A and G-strands, and in loops that have \u03a6-values\nclose to 0, suggesting that in the final stage of folding these peripheral\nstrands and loops pack onto the central region of the protein.\nIdentification of the obligate (embryonic) folding nucleus\nOliveberg and co-workers have suggested that residues that constitute\nthe transition state for folding in a nucleation-condensation mechanism\nmight be divided into two sets.31,32 The first set of residues make up the \u201cembryonic\u201d or\nobligate folding nucleus, defined as the set of primary contacts that are\nobliged to form to establish the topology of the protein. The second set is\nthe residues that pack onto this embryonic nucleus forming the \u201ccritical\ncontact layer\u201d, providing sufficient interactions to drive the folding\nprocess downhill. Note that residues that form the embryonic nucleus have to\nform a network of contacts that establish the topology of the protein, but\nthat residues in the critical contact layer may contribute significantly\ntowards stabilising the transition state for folding. Identifying the most\nlikely obligate folding nucleus from a pattern of \u03a6-values is non-trivial,\nespecially for complex Greek key structures. This has been discussed in\ndetail.18 In summary, it is not possible simply to \u201cpick\u201d residues\nwith the highest \u03a6-values as being those that form the folding nucleus: one\nalso has to consider the packing of the residues. In TNfn3, as is observed\nhere for CAfn2, residues in the B-strand have generally low \u03a6-values\ncompared to the \u03a6-values in the other central \u03b2-strands. This does not\nnecessarily mean that the residues in the B-strand are less important for\nfolding. Residue L22(B3) in CAfn2, for example, forms about half of its\ninter-strand contacts with residues in the A and G-strands, which have\n\u03a6-values of \u223c\u00a00. Thus, the contacts with the C (V38), E\n(A53 and I55) and F (F66 and V68) strands must be more formed than the\nmoderate \u03a6-value would indicate. Similarly, the high \u03a6-values in the\nC\u2032-strand probably reflect the fact that the residues in this strand make\nthe vast majority of their tertiary contacts with residues in the C and\nE-strands, which are themselves partially formed. In TNfn3 it was suggested\nthat this C\u2032-strand is \u201cobliged\u201d to fold when the adjoining C and E-strands\npack together. Thus, we would now, following the nomenclature suggested by\nthe Oliveberg model, assign residues in the C\u2032-strand to the critical\ncontact layer and not to the obligatory embryonic nucleus.\nFor TNfn3, the residues with the highest \u03a6-value in the B, C, E and\nF-strands were initially chosen as putative nucleus residues. Examination of\nthe structure showed that these residues are all found in the same core\nlayer, and that they pack to form a \u201cring\u201d of interactions in the core of\nthe protein. It was suggested that this \u201cobligatory\u201d nucleus alone was\nsufficient to establish the topology of the native protein. This picture of\nthe folding transition state was confirmed by subsequent restrained\nmolecular dynamics simulations.36 A similar method has been used to identify the folding\nnucleus in a structurally related immunoglobulin domain.17,35\nUsing the same strategy, the \u03a6-value pattern of CAfn2 was investigated\nto identify the putative obligate folding nucleus, a set of residues with\nsignificant \u03a6-values that interact such that these interactions are\nsufficient to establish the topology of the protein. The layer that contains\nresidues with consistently high \u03a6-values in the B and E-strands is layer 3\n(I55 and L22), as in TNfn3. However, for the C and F-strands, the layer with\nthe highest \u03a6-values is layer 4 (V38 and V68). Examination of the structure\nof CAfn2 shows that although residues L22 and I55, and V38 and V68 sit in\ndifferent core layers, these four residues are still able to pack together\nin the centre of the core to form a ring of contacts (Figure 6). We\nsuggest that the residues surrounding the obligate nucleus, residues in the\nC\u2032-strand and more peripheral residues in the B, C E and F-strands, pack\nonto these obligate nucleus residues and, together, form the critical\ncontact layer required to stabilise the transition state structure\nsufficiently to drive folding.\nThere is a caveat we should make. We note that in CAfn2 it is more\ndifficult to select which residues are likely to form part of the obligate\nnucleus than it was in TNfn3. Consider the B-strand. I20(B2) exhibits a\n\u03a6-value that is slightly lower, but is within error of L22(B3). However, I20\nforms no contacts with the nucleating residues from the opposite sheet,\n(V38(C4) and V68(E4)), suggesting that it does not form part of the\nobligatory nucleus that establishes the topology of the molecule.\nFurthermore, as was the case in TNfn3, we were unable to determine a \u03a6-value\nfor the highly conserved Trp in the position B4. Simulations confirmed for\nTNfn3 that this Trp residue had a low \u03a6-value (as we had inferred from the\npattern of \u03a6-values surrounding the Trp residue). Trp 24 makes 150\nside-chain\u2013side-chain contacts in CAfn2, and 65% of these contacts are with\nresidues that have \u03a6-values that are (or are predicted to be) low (\u03a6\u00a0\u223c\u00a00.15, 52 contacts) or zero (46 contacts).\nLess than one-third of the contacts made by Trp24 are with residues in the\nputative obligatory nucleus (with L22, V38 and V68) and no contact is made\nwith I55. Thus, we tentatively propose that if Trp24 does have a role in the\nfolding nucleus, it is more likely to be in the critical layer than in the\ntopology-defining obligate nucleus. Also consider residue A70 in the\nF-strand in position F5. The \u03a6-value for this residue is very slightly\nhigher than that for V68 in layer F4. However, Ala to Gly mutation must be\nconsidered to be non-conservative and, furthermore, Ala70 makes no contact\nwithin the proposed obligate nucleus i.e. it cannot have a role in\nestablishing the Greek key topology.\nComparison of the transition states of CAfn2 and TNfn3\nThe \u03a6-values for TNfn3 are generally higher than those in CAfn2,\nranging from 0 to 0.6;18 however, the pattern of \u03a6-values is similar. For both\nproteins, the mutational results can be separated into two classes. The\nfirst group consists of residues in the central \u03b2-strands, which show\nsignificant formation of structure in the transition state. The second group\nconsists of mutations probing the terminal A and G-strands, and residues\nfrom the extremities of the central strands. These two populations are\nclearly observed in a Br\u00f8nsted plot (Figure\n7).\nNevertheless, there are important differences between the two domains,\nwhich are apparent in the pattern of \u03a6-values (Figure 8). In the\nC\u2013F-sheet, the highest \u03a6-values in TNfn3 are found in core layer 3 (V70,\n0.54; Y36, 0.53), with the \u03a6-values in layer 4 being significantly lower\n(L72, 0.29; L34, 0.35). However, in CAfn2 the \u03a6-values in layer 3 are very\nlow (F66, 0.07; N40, 0.01), whereas the \u03a6-values in layer 4 (V68, 0.25; V38,\n0.40) are significantly higher (Figures 3\nand 8). This indicates that the absence of a buried\nhydrophobic residue in position C3 has forced the obligate folding nucleus\nof CAfn2 to \u201cmigrate down\u201d one layer within the core (Figure 6). Perhaps unexpectedly, a\ncorresponding \u201cdownwards migration\u201d has not occurred in the B\u2013E-sheet; (even\nif Trp24 was important, the \u03a6-value for A53(E4) is unambiguously low\n(0.14)). Such migration is not necessary; analysis of the CAfn2 structure\nshows clearly that residues L22(B3) and I55(E3) form significant\ninteractions with V68(F4) and V38(C4) in the opposite sheet. Thus, these\ninter-sheet interactions would be sufficient to establish the Greek key\ntopology.\nFurther support for this migration hypothesis comes from \u03a6-values in\nthe EF-loop. TNfn3 exhibits moderate \u03a6-values in this loop, (Y68(F2), 0.42;\nL62(E2), 0.33), which indicates that it is significantly structured in the\ntransition state. It was argued that this loop is \u201cobliged\u201d to be structured\nin the transition state of TNfn3 to allow for formation of the adjacent\nfolding nucleus: the more distant BC-loop exhibits lower values. However, in\nCAfn2 the folding nucleus has shifted away from the EF-loop (Figure 6) and consequently it is less\nrestrained within the transition state (\u03a6-values for Y64(F2) and L58(E2) are\n0.04 and 0.16, respectively).\nBoth TNfn3 and CAfn2 display high \u03a6-values in the C\u2032-strand. We suggest\nthat these residues are not involved in the obligatory folding nucleus, but\nresult from short CE-loops that force the C\u2032-strand to pack as the nucleus\nforms;18 thus, these residues form part of the critical contact\nlayer. In CD2d1, an immunoglobulin Ig variable domain, the nucleating C and\nE-strands are joined by a much longer loop comprising three \u03b2-strands, C\u2032,\nC\u02ba and D. In this case these strands do not pack until late in\nfolding.18,19\nIn summary, for both proteins we observe the formation of a specific\nnucleus in the core of the protein involving formation of long-range\ntertiary contacts between a single residue from each of the B, C, E and\nF-strands. Formation of this \u201cobligate\u201d nucleus establishes the topology of\nthe protein. Other residues pack around this obligate nucleus to form the\ncritical contact layer until sufficient contacts have formed to surmount the\nfree-energy barrier. This is typical of a nucleation-condensation folding\nmechanism. The peripheral strands and the loops pack late, mainly after the\nrate-limiting step for folding.\nConclusion: plasticity within the obligatory folding nucleus\nin Ig-like domains\nUnlike other classes of proteins, such as the homeodomain proteins, all\nIg-like proteins appear to fold by the same, nucleation condensation\nmechanism. The obligate nucleus is defined by the interactions that are\nnecessary to establish the complex Greek key \u03b2-sheet topology of the native\nstate. Previous biophysical studies of members of the Ig-like fold have\nshown that this folding nucleus always comprises a ring of interacting\nresidues within the hydrophobic core: one residue from each of the B, C, E\nand F-strands. Whereas the obligatory nucleus in the immunoglobulin\nsuperfamily proteins is highly conserved and is based around the invariant\ntryptophan located within the C-strand, members of the fnIII superfamily\nshow more variability. Instead of restricting a particular structural\nposition to a specific amino acid, each position simply needs to possess a\nhydrophobic residue. Here, we have shown that the fnIII nucleus is more\nflexible still, and that when this pattern of residue conservation is lost\nupon mutation, fnIII proteins can \u201cmigrate\u201d the folding nucleus, thereby\nrevealing plasticity in the early stages of the folding process, while\nretaining the same folding mechanism.\nSuch plasticity in the folding of Ig-like proteins has been observed\npreviously; the Ig domain TI I27 has been shown to fold by alternative,\nparallel pathways.51 Although the wild-type protein folds only through one\npathway under physiological conditions, extremes of temperature and\ndenaturant, or mutations within its obligate folding nucleus also result in\na switch of folding pathway. Lindberg and Oliveberg have suggested recently\nthat a \u201cmalleable\u201d protein folding energy landscape will allow proteins to\nretain efficient folding during the course of evolution, even though the\nfiner details of the folding pathway are dependent on individual\nsequence.52 It is possible that the ability of these Ig-like domains to\nalter their folding pathway on mutation has contributed to the success of\nthis fold, and has contributed to its abundance in the proteome (over\n40,000\u00a0Ig-like domains are listed in the current Pfam database).\nMaterials and Methods\nProtein expression and purification\nThe fibronectin type III domain used in this work consists of 88\nresidues (SwissProt P20533, residues 559\u2013646, PDB 1K85) of the\nBacillus circulans chitinase A1. The synthetic\ngene was produced using overlapping primers and standard PCR techniques, and\nwas inserted into a modified version of pRSETA vector (Invitrogen)\ncontaining an N-terminal His-tag followed by a thrombin cleavage site.\nSite-directed mutagenesis was performed using the QuikChange Kit\n(Stratagene). The identity of wild-type and mutants was confirmed by DNA\nsequencing.\nProtein expression was carried out in Escherichia\ncoli C41 cells.53 Transformed cells were grown to an absorbance at 600\u00a0nm of\n0.6 at 37\u00a0\u00b0C before induction with IPTG and growth overnight at 28\u00a0\u00b0C. The\ncells were harvested and lysed by sonication. The soluble fraction was bound\nto Ni2+-agarose resin, washed several times to remove\nweakly bound proteins, and eluted from the Ni2+-agarose\nresin in a high concentration of imidazole. After dialysis to remove the\nimidazole, the proteins were cleaved overnight with thrombin. Uncleaved\nprotein and remaining His-tag were removed by using small amounts of\nNi2+-resin before further purification by\ngel-filtration chromatography using a Pharmacia Biotech Superdex 75 column.\nWhen not used immediately, proteins were flash-frozen and stored at \u2212\u00a080\u00a0\u00b0C.\nEquilibrium measurements\nThe stability of the CAfn2 wild-type and mutant proteins was determined\nby equilibrium urea denaturation in 50\u00a0mM sodium acetate buffer, pH 5.0\n(15\u00a0mM HOAc, 35\u00a0mM NaOAc) at 25\u00a0\u00b0C. The solutions were left to equilibrate\nat 25\u00a0\u00b0C for at least 2\u00a0h before measurements were recorded. All experiments\nwere carried out in thermostatted cuvettes at 25\u00a0\u00b0C. The experiments used an\nexcitation wavelength of 280\u00a0nm, and an emission wavelength of 360\u00a0nm. Data\nwere fit to an equation describing a two-state transition.43\nChange of free energy on mutation\nThe change of free energy on mutation,\n\u0394\u0394GD\u2013N, was determined using\nequation (1):54Where [urea]50% is the concentration of urea\nat which 50% of the protein is unfolded for wild-type (wt) and mutant (mut)\nproteins, and <m> is the mean\nm-value determined from all measurements on\nwild-type and mutant proteins.\nKinetic measurements\nAll kinetic experiments were done using an Applied Photophysics\nstopped-flow fluorimeter. The excitation wavelength was 280\u00a0nm and the\nemission was monitored at wavelengths >\u00a0320\u00a0nm. All\nexperiments were carried out in 50\u00a0mM sodium acetate buffer (pH 5.0) at\n25\u00a0\u00b0C. The final concentration of all proteins was 1\u00a0\u03bcM. Refolding rates at\n0\u00a0M denaturant were determined using CAfn2 unfolded at pH 12.4 as\ndescribed.18 Between three and five traces were averaged for each\nconcentration of denaturant. The refolding data were fit to an equation\nusing a single-exponential term. An average refolding\nm-value of 1.06\u00a0M\u2212\u00a01 was used for mutations L22A, Y36L and L58A. Fitting data to an\nequation with two exponentials did not improve the residuals. The unfolding\ndata were fit to an equation describing a single-exponential process with\ncurvature.\n\u03a6-Value analysis\nThe \u03a6-value for folding was determined using equation (2):54.where \u0394\u0394GD\u2013\u2021 is the\nchange in the difference in free energy between D and the transition state\n(\u2021) upon mutation and calculated from refolding data as follows:where kf and\nkf\u2032 are refolding rate constants\nfor wild-type and mutant proteins (at 0\u00a0M urea), respectively.","keyphrases":["folding nucleus","ig domain","protein folding","ig, immunoglobulin","fniii, fibronectin type iii","cafn2, the second fniii domain of chitin a1 from bacillus circulans","tnfn3, the third fniii domain of human fibronectin","phi-value analysis"],"prmu":["P","P","P","R","R","R","R","M"]}
{"id":"Eur_Spine_J-2-2-1602190","title":"Thoracic cord compression caused by disk herniation in Scheuermann\u2019s disease\n","text":"We present the case of a 14-year-old male with Scheuermann\u2019s disease and significant neurological deficit due to thoracic disk herniation at the apex of kyphosis. He was treated with an anterior decompression, anterior and posterior fusion in the same setting using plate, cage and a segmental instrumentation system. The patient had an excellent outcome with complete neurological recovery.\nIntroduction\nScheuermann\u2019s disease (juvenile kyphosis dorsalis) is a structural kyphosis of the thoracic spine initially described by Scheuermann in 1921 [19]. It occurs commonly in adolescents (0.4\u20138.3% of the general population) and in most cases is characterized by minimal deformity and few clinical symptoms. Scheuermann\u2019s disease is rarely associated with neurological complications [5, 20].\nThe purpose of the present paper is to present the case of a patient with Scheuermann\u2019s disease and severe neurological deficit due to a thoracic disc hernia at the level of kyphos, to review the literature and compare with previously published similar cases.\nCase report\nA 14-year-old male was admitted with a severe spastic paraparesis. He had gradually developed a gait disorder and bilateral numbness of lower limbs in the past 5\u00a0months. During that period his symptoms deteriorated rapidly. He was unable to walk and a week before his admission he developed spastic paraplegia. There was no history of trauma.\nClinical examination revealed spastic paraparesis below T9. The superficial sensation was diminished below the T9 dermatome. He had a muscle strength of grade II according to the Medical Research Council (MRC) scale. A barely detectable contraction could be seen below the knees by palpation over the muscles (M1 on the MRC scale). Plantar responses (Babinski sign) were present on both sides. Knee and ankle reflexes were brisk bilaterally, clonus was present in both ankles as well. Patient had hypalgesia and decreased temperature sensation. Light touch and pin prick sensation were diminished over both lower limbs and absent from the knees downward. Rectal tone and sensation while urinating were decreased.\nAll laboratory findings were normal including an investigation for systemic diseases. Plain radiographs of the spine in standing position revealed anterior wedging of more than 5\u00b0 of several adjacent vertebrae (T7:12\u00b0, T8:14\u00b0, T9:17\u00b0, T10:15\u00b0) at the apex of the kyphosis and vertebral endplate irregularities. The thoracic kyphosis from T5 to T12 measured 66\u00b0 (Fig.\u00a01). These findings are in agreement with the criteria for diagnosis of Scheuermann\u2019s disease. A magnetic resonance imaging scan revealed thoracic disc heniation with spinal cord compression at T8\u2013T9 level (apex of the kyphosis). The intervertebral spaces from T6 to T10 were also very narrow and the vertebral bodies had an anterior wedging (Figs.\u00a02, 3).\nFig.\u00a01a, b Anteroposterior and lateral radiographs of the thoracic spine in standing position, at the time of admission. The thoracic kyphosis from T5 to T12 is 66\u00b0Fig.\u00a02a, b Magnetic resonance imaging scan revealed thoracic disc herniation with spinal cord compression at T8\u2013T9 level (apex of the kyphosis)Fig.\u00a03Three-dimensional reconstruction computed tomography of the thoracic spine preoperatively\nSurgical treatment was decided based on the severity of the neurological deficit. A right seventh rib transthoracic approach to the spinal column was performed followed by decompression at T8\u2013T9 level. A disc fragment was found and removed behind the thoracic body of T9. We performed an anterior fusion using plate, screws, an interbody titanium cage and also a posterior fusion from T6 to T12 using a double-rod multihook and transpedicular screws segmental instrumentation system. Bone grafts were applied between T8 and T9 and intertransversaly from T7 to T10 (Fig.\u00a04).Fig.\u00a04a, b Postoperative anteroposterior and lateral radiographs of the thoracic spine. Note the anterior and posterior spinal fusion\nPostoperatively the patient had a surprisingly rapid improvement of the neurological deficit. Four weeks after the operation he could walk steadily and 2\u00a0months later his neurological examination was entirely normal.\nAt 2\u00a0years follow-up the patient had a normal gait, he was symptoms free and there was no increase of the spinal deformity.\nDiscussion\nScheuermann in 1921 [19] described a condition, which he called juvenile dorsal kyphosis, distinguishing it from the more common postural kyphosis. The etiology of Scheuermann\u2019s disease is unknown. Scheuermann proposed that the kyphosis resulted from avascular necrosis of the ring apophysis of the vertebral body [19]. Schmorl suggested that the vertebral wedging was caused by herniation of disc material into the vertebral body (Schmorl\u2019s nodes). According to Ippolito and Ponseti [10] a biochemical abnormality of the collagen and matrix of the vertebral endplate cartilage may be another contributing factor. Bradford et al. [6] claimed that osteoporosis may be responsible for the development of Scheuermann\u2019s disease. Mechanical factors and repetitive trauma have been also considered to play a significant role in the appearance of the disease [20]. Case reports in monozygotic twins support the theory that there is also a genetic contribution [9]. In conclusion, the etiology of Scheuermann\u2019s disease remains unclear and probably is multifactorial.\nThe criteria for the diagnosis of Scheuermann\u2019s disease are: (1) more than 5\u00b0 of wedging of at least three adjacent vertebrae at the apex of the kyphosis, (2) endplates irregularities and (3) a thoracic kyphosis of more than 45\u00b0 [5]. Our patient had typical roentgenografic features of Scheuermann\u2019s disease and his kyphosis was 66\u00b0 from T5 to T12.\nNeurological complications in Scheuermann\u2019s disease are rare [2\u20134, 7, 11, 15, 18, 21, 24]. Three different types of neural compression have been reported: (1) extradural spinal cyst, (2) compression of the cord at the apex of the kyphos and (3) disk hernia at the apex of the kyphos [4]. To the best of our knowledge only 20 cases fall in the last category [2\u20134, 8, 12, 14, 17, 21\u201324]. Data of all these cases are presented in Table\u00a01.\nTable\u00a01Cases of Scheuermann\u2019s disease causing spinal cord compression as the result of thoracic disc herniationReferencesLevelAge\/sexFindingsAppro achProcedureResults (follow up)Muller [14]T1040 MParaplegiaNot reportedNot complete recoveryVan Landingham [23]T7\u2013T8\u2013T917 MSpastic paraparesis, no sphincter dysfunctionPT7\u2013T9 laminectomyComplete recovery (4 months)Roth et al. [17]T9\u2013T1061 MSpastic paraparesisPT9\u2013T10 laminectomyParaplegiaTurinese and Raven [22]T5\u2013T7\u2013T816 MSpastic paraparesis, sphincter dysfunctionBed rest, Minerva jacketComplete recovery Bradford and Garcia [4]T7\u2013T816 MSpastic paraparesisPT7\u2013T9 laminectomyResidual hypereflexia(120\u00a0months) Ryan and Taylor [18]T8\u2013T918 MSpastic paraparesisTT8\u2013T10 partial vertebrectomy, anterior spinal fusionSpastic paraparesis(36\u00a0months)Yablon et al. [24]T7\u2013T829 MSpastic paraparesis, sphincter dysfunctionCT\/TT7\u2013T8 discectomy and fusionComplete recovery (14\u00a0months)Lesoin et al. [12] (six cases) T9\u2013T1049 FMonoparesis (left)PT8\u2013T10 laminectomyNot complete recovery (12\u00a0months)T8\u2013T955 FSpastic paraparesisTT8\u2013T9 discectomySmall motor deficit (8\u00a0months)T11\u2013T1261 MMonoparesis (right)PPosterior spinal fusionNot complete recovery (10\u00a0months)T8\u2013T927 MLower back pain, Babinski\u2019s sign (right)PLT8\u2013T9 discectomy, T7\u2013T10 posterior spinal fusionComplete recovery (3\u00a0months)T8\u2013T930 MLower back pain, slight motor deficit (left)PLT8\u2013T9 discectomy, T8\u2013T9 posterior spinal fusionComplete recovery (3\u00a0months)T8\u2013T935 MSpastic paraparesisPLT8\u2013T9 discectomy, T8\u2013T9 posterior spinal fusionSmall motor deficit (3\u00a0months)Bohlman and Zdeblick [3] ( two cases)T11\u2013T12T12\u2013L138 FMonoparesis. Back and leg pain (for 16\u00a0years)CTT11\u2013L1 discectomy and fusionComplete recovery (36\u00a0months)T8\u2013T925 FBack and leg pain (for 7\u00a0years)TT8\u2013T9 discectomy and fusionSignificant pain relief (26\u00a0months)Stambough et al. [21]T6\u2013T7\u2013T821 MSpastic paraparesisTT6\u2013T8 discectomy, anterior spinal fusion Complete recovery (24\u00a0months)Bhojraj and Dandawate [2] (three cases)T11\u2013T1216 MSpastic paraparesis, urinary hesitancyPT10\u2013T12 laminectomy, posterior interbody fusion Complete recovery (36\u00a0months)T12\u2013L124 MSpastic paraparesis, urinary hesitancyPT12\u2013L1 laminectomy, posterior interbody fusionComplete recovery (24\u00a0months)T11\u2013T1216 FSpastic paraparesisPT11\u2013T12 laminectomy, posterior interbody fusionTransient worsening postoperatively, subsequently complete recovery (15\u00a0months)Chiu and Luk [8]T11\u2013L235 FSpastic paraparesisTT11\u2013L2 discectomy, spinal fusion Small motor deficit (24\u00a0months)This report (2005)T8\u2013T914 MRapidly progressive spastic paraparesis, finally spastic paraplegiaTT8\u2013T9 discectomy, anterior fusion, T6\u2013T12 posterior fusionComplete recovery (25\u00a0months)M Male, F female, P posterior, T transthoracic, CT costotransversectomy, PL posterolateral\nDespite the fact that this condition has a uniform sex distribution [15, 20], only 6 of all 20 cases were female. Males with Scheuermann\u2019s disease are at greater risk for the development of spinal cord compression in the second decade of life [1, 15]. This is in agreement with our case (male, 14\u00a0years old). This difference between the sexes is probably due to the fact that males have more longitudinal spinal growth than females and their maximum growth in trunk length, as well as the maximum deformity of the kyphosis, occurs about 2\u00a0years later.\nIn all cases in which the degree of deformity was reported, the average angle was only 56.3\u00b0. The severity of the neurological complications had no obvious correlation with the magnitude of the deformity [11, 18]. In our case the deformity was 66\u00b0. Lonstein et al. [13] found that in 43 cases with neurological deficits secondary to spinal deformity (what ever the etiology) the average angle of kyphosis was 95\u00b0. He suggested that the greater the angle of kyphosis, the greater the risk of neurological impairment. Neurological complications are less likely to occur when the deformity is present over a large number of segments, instead of sharply angular deformities.\nDisc ruptures tend to occur at the apex of the thoracic kyphosis [13]. The majority of them have occurred at T7\u2013T8 or at T8\u2013T9. This is also confirmed by our case (at T8\u2013T9).\nIt is interesting that a relatively small disc herniation such as in our case produces such a major neurological deficit. Factors that may influence the onset of cord compromise are the tenuous vascularization and the relatively small canal diameter of the thoracic cord [16]. Additionally, in young patients the disc material is not degenerated and can act as a solid mass. Bradford and Garcia [4] suggested that the high incidence of Schmorl\u2019s nodes in Scheuermann\u2019s disease can prevent disc herniation by decompressing the disc space. In our case there were no Schmorl\u2019s nodes either at this level or at any other level.\nSurgical treatment is indicated in the cases of Scheuermann\u2019s disease with acute cord compression and generally combines anterior release with posterior instrumentation and fusion. A posterior decompression is ineffective because the compressive force is acting from the front [11, 13, 15, 18]. Although some authors have advocated posterior laminectomy and decompression, Patterson and Arbit [16] showed that 45% of patients undergoing laminectomies for thoracic disc herniations either had no relief or deteriorated.\nFor the 20 reported cases with thoracic disc herniation and Scheuermann\u2019s disease six patients were treated with anterior spinal fusion, four patients with laminectomy, four with posterior spinal fusion, three patients with interbody fusion and pedicular screw plate fixation and one patient was treated with discectomy without spinal fusion. Only one patient was treated conservatively with bed rest and Minerva jacket without any decompressive surgery [22]. In one case [14] the treatment was not reported. If a significant or progressive kyphosis, i.e. 55\u00b0 or more exists, a combined posterior spinal fusion and instrumentation should be considered [18, 21]. Due to the age of the described patient and to the degree of kyphosis we performed discectomy, decompression, anterior and also posterior fusion using a double-rod multihook and transpedicular screws segmental instrumentation system.\nThe majority of the reported cases had good results. In 10 out of 20 cases the patients had complete recovery while only in one case the final outcome was paraplegia [17]. Our patient had an excellent result with complete recovery in 3\u00a0months. At the final follow-up 2\u00a0years postoperatively, he is neurologically entirely normal and the spinal fusion is solid.\nThis case is interesting because of the severity of the symptoms. The patient had a progressive bilateral spastic paraparesis and finally a spastic paraplegia. Spastic paraparesis was the most common symptom in all cases but neurological symptoms were not so severe to result in paraplegia. Only one case had paraplegia, as the described patient [14]. It is impressive that despite the significant neurological deficit there was a complete recovery. It seems that when symptomatic compression of the spinal cord occurs, surgery is the best option.\nConclusion\nThoracic disc herniation is an extremely rare complication of Scheuermann\u2019s disease, resulting in cord compression and marked neurological deficit, most commonly in young patients. This rare form of spinal cord compression once demonstrated, should be treated as soon as possible with surgical decompression and stabilization of the spine anteroposteriorly.","keyphrases":["scheuermann\u2019s disease","spastic paraparesis","spinal cord compression","thoracic disc herniation","acute myelopathy"],"prmu":["P","P","P","P","M"]}
{"id":"Eur_Arch_Otorhinolaryngol-3-1-1914267","title":"Donor site morbidity of the fasciocutaneous radial forearm flap: what does the patient really bother?\n","text":"The objective of this study was the evaluation of donor site morbidity in head and neck cancer patients after reconstruction using a free vascularized radial forearm flap with emphasis on subjective complaints. Fifty patients who underwent at least 6 months before a reconstruction using a free vascularized radial forearm flap were asked to fill out two questionnaires regarding cosmetics and sensibility and forearm disabilities. Furthermore, a function test including movement extensions (flexion\u2013extension, ulnar\u2013radial deviation and pronation\u2013supination), strength (pinch and grip) and temperature (digiti I and V) of the donor and non-donor site were measured and compared. Thirty-five percent of the patients reported no complaints regarding cosmetics and sensibility and 75% mentioned no forearm disabilities. There was no difference in movement extensions, temperature and grip strength between donor and non-donor sites. The difference in pinch strength appeared to be significant (p < 0.001). The total score of the questionnaire on forearm disabilities correlated significantly with extension, pronation and grip strength of the donor arm. Donor site morbidity of the radial forearm flap measured by objective functional tests was limited but subjective self-ratings revealed complaints regarding cosmestics and sensibility and to a lesser extent regarding forearm disability. The present data may be used for solid patient counselling.\nIntroduction\nThe free radial forearm flap (FRFF), introduced by Yang et al. in 1981 [1], has established itself as a versatile, reliable and widely used method for reconstruction of defects in the head and neck region [2, 3]. The pliability and thinness of FRFF allow its use in complex reconstructions. The vascular pedicle of the FRFF provides adequate vessel diameter and length for microvascular anastomosis. A split skin graft is most frequently used for the forearm donor site reconstruction. Donor site morbidity includes functional and aesthetical outcome. Hand and wrist function is the most important issue in the assessment of morbidity of the FRFF. In a previous study we found that donor site morbidity of the radial forearm flap measured by objective functional tests is negligible but in the patient\u2019s perception this is substantial [4]. Some retrospective studies describe a reduced forearm and\/or wrist mobility and a reduced strength [5], stiffness of the wrist with movement disability, dysaesthesia, swelling of hand and\/or wrist and pain [6], reduced sensibility of the radial nerve area and a less aesthetic result [7, 8], while others describe rare or no movement disabilities after using the FRFF [9, 10].\nThe subjective morbidity after harvest of the FRFF is one of the reasons that some reconstructive surgeons shift their attention to other fasciocutaneous flaps for reconstruction of defects in the head and neck [11, 12]. The goal of the present study is to investigate these subjective complaints in more detail and in relation to the objective function tests in a larger group of patients.\nMethods\nPatients\nAll patients who visited the outpatient clinic of the department of otolaryngology\/head and neck surgery of the VU Medical Center during the period of March till July 2004 and had a reconstruction of the surgical defect by using a FRFF at least 6 months before were asked to participate in the study. Exclusion criteria were arthritis of the upper extremities or a recent trauma of the wrist and\/or lower arm.\nDuring this period 69 patients, who underwent such a operation, visited the outpatient clinic. Fifty patients (28 men and 22 women, aged between 26 and 77\u00a0years, mean 59.2\u00a0\u00b1\u00a011.1\u00a0years) met the inclusion criteria. Eight of these 50 patients had had a forearm or wrist injury before surgery (five patients had a wrist fracture, two had a Dupyutren\u2019s contracture and one had surgery because of a ganglion on dorsal side of the wrist), of which four patients at the donor side. None of these patients had pre-existing complaints and\/or range of motion disability of the hand or wrist. In 45 patients the left arm was used for the harvest of the FRFF and in five patients the right arm. In three patients the donor side was the side of the dominant hand.\nHarvest of the flap was done simultaneously with the ablative procedure whenever possible. All patients underwent a preoperative and intra-operative Allen\u2019s occlusion test to rule out inadequate blood supply from the ulnar artery. Fasciocutaneous flaps were raised under a tourniquet in a conventional subfascial or superfascial manner about 2\u00a0cm proximal to the wrist skin fold. The superficial radial nerve and branches of the lateral antecubital nerve were preserved. The cephalic vein was used as donor vein. The radial artery was not reconstructed in any patient. The donor defect was closed with a split skin graft (0.6\u00a0mm) taken from the upper thigh at the same site. In order to aid healing the arm a pressure dressing of a paraffin gauze and foam was placed over the skin and the arm was immobilized for 7\u00a0days in a dorsal hand-to-upper-arm splint.\nDonor site morbidity questionnaires\nSubjectively the donor site morbidity was measured by means of a questionnaire regarding cosmetics and sensibility (Table\u00a01) and a questionnaire regarding forearm disabilities of the operated arm (Table\u00a02). Response possibilities were yes, no, or not applicable. All themes relate to the last 24\u00a0h. A total score on each questionnaire was calculated by dividing all yes-scores by the number of questions answered yes or no. The total score ranged from 0 to 100 with 0 indicating no problems (no disability) and 100 indicating severe problems (severe forearm and\/or wrist disabilities).\nTable\u00a01Questionnaire regarding sensibility and cosmetics of the donor arm (n\u00a0=\u00a050)QuestionsNumber of positive answer (%)No complaints17 (35)Complaints\u00a01. Can you wear a wristwatch or bracelet?12 (24)\u00a02. Does the hand feel numb?11 (22)\u00a03. Does the scar itch?11 (22)\u00a04. Do you experience problems in the cold?4 (8)\u00a05. Does the appearance bother you?13 (27)In case of complaints, multiple answers were possibleTable\u00a02Questionnaire regarding forearm disabilities in the last 24\u00a0h (n\u00a0=\u00a050)Questions Number of positive answer (%)No forearm disabilities38 (75)Disabilities\u00a01. I wake up at night because of my forearm1 (2.0)\u00a02. I have complaints lying on my forearm 4 (8.0)\u00a03. I have complaints during daily life activities2 (4.0)\u00a04. I have complaints during movements of my wrist3 (6.0)\u00a05. I have complaints during leaning on my elbows or hands3 (6.0)\u00a06. I have complaints with writing (or typing)1 (2.0)\u00a07. I have complaints holding my wheel of my car or bike2 (4.0)\u00a08. I have complaints during lifting an object5 (10.0)\u00a09. I have complaints opening or closing a door2 (4.0)\u00a010. I rub my wrist or forearm more than once a day9 (18.0)\u00a011. I am irritable for people in my environment due to my forearm1 (2.0)\nDonor site morbidity tests\nThree tests were used for objective measurement of donor site morbidity. All measurements were performed at the operated and non-operated side. The first test covers movement extensions of the wrist. The angles of the maximal flexion and extension of the wrist, the ulnar and radial abduction and the lower arm pronation and supination were measured by the Mediclino Inclinometer (Bodybow-Holland, Nieuwegein, The Netherlands) with an accuracy of 2\u00b0 and a range of 0\u2013180\u00b0 (Fig.\u00a01).\nFig.\u00a01Inclinometer used for measuring of movements\nThe second test measures the strength of the hand. The grip strength (strength measured in the hand) was measured in kg\/m2 with a hydraulic dynamometer (Smith & Nephew Roylan, Germantown, WI, USA) with an accuracy of 2.0\u00a0kg and a range of 0\u201390.0\u00a0kg (Fig.\u00a02). The pinch-strength (strength of digiti I and V) was measured with a pinch gauge (B&L Engineering, Santa Fe Springs, CA, USA) with an accuracy of 0.5\u00a0kg and a range of 0\u201312.5\u00a0kg (Fig.\u00a03).\nFig.\u00a02Hydraulic dynamometer used for measuring grip strengthFig.\u00a03Pinch gauge used for measuring pinch strength\nThe third test measures the skin temperature of digiti I and V with a Tempcontrol MT 100 KC and probe (Tempcontrol Industrial Electronic Products, Voorburg, The Netherlands) on the skin surface with an accuracy of 0.1\u00b0C and a range of \u2212200 to 1350\u00b0C.\nStatistical analyses\nDescriptive statistics were generated for the range of outcome variables in the study. Student\u2019s t-tests were used to determine the differences in objective tests between the donor and non-donor site. Spearman correlation-coefficients were calculated to assess correlations between subjective and objective forearm disability.\nFor all tests, a two-sided p-value less than .05 was considered statistically significant.\nResults\nRegarding subjective evaluation of cosmetics and sensibility, 35% of the patients had no complaints. The other patients scored positively (thus indicating complaints) on several of the questions (Table\u00a01).\nRegarding forearm disabilities, 75% of the patients had no complaints (Table\u00a02). The other patients showed a variety of complaints, especially on rubbing and lifting an object. The mean total score on the forearm disability questionnaire was 4.6 (SD 12.6) with a range from 0 to 82.\nThe results of the objective tests revealed that no differences were measured between the donor and non-donor arm regarding movement, grip and temperature. Pinch values appeared to be significantly different (Table\u00a03).\nTable\u00a03Movement extensions, strength and temperature of donor and non-donor wrist and hand\u00a0Donor sideNon-donor sideMeanSDMeanSDExtension57.311.759.811.0Flexion71.18.070.29.2Ulnar abduction57.78.258.98.0Radial abduction15.38.414.85.1Pronation85.410.885.311.0Supination79.612.280.412.9Grip28.710.429.99.7Pinch7.42.48.02.4*Temperature digit I29.33.329.43.6Temperature digit V29.03.929.14.1*Significant difference (p-value\u00a0<\u00a0.01))\nThe total score on the forearm disabilities questionnaire appeared to be significantly related to extension (r\u00a0=\u00a00.29, p\u00a0<\u00a00.05), pronation (r\u00a0=\u00a00.30, p\u00a0<\u00a0.05) and grip strength (r\u00a0=\u00a00.30, p\u00a0<\u00a0.05).\nDiscussion\nThe most important goal of reconstructive surgery in head and neck cancer patients is the optimal restoration of function. The FRFF is a very reliable flap which can restore function in the head and neck function very well [13, 14, 15]. However, the donor site morbidity after harvesting the flap is another important issue. While the main postoperative concern was the reconstruction site, during follow-up the donor site become more important to patients [16].\nHand and wrist functions are important in the assessment of morbidity of the FRFF. The only objectively observed difference in our study was pinch strength between operated and non-operated sides. It must be emphasized that in 94% of the patients the FRFF was harvested from non-dominant side. Therefore, the pinch strength on the operated arm may be already less than on the non-operated side before surgery. All other functional tests revealed no statistically significant differences between operated and non-operated sides. Also Ho et al. [17] did not find any significant difference between the operated and the non-operated arm for strength, range of motion and dexterity.\nThe questionnaires on the other hand revealed donor site complaints related to the FRFF. This difference between objective and subjective findings was also reported by others [4, 7, 8, 9]. The questionnaire on forearm disabilities showed that the vast majority (75%) of the patients had no functional complaints at all. The most frequent positive answer was about rubbing the forearm, which may be more a sensibility than a functional problem. In a study of Toschka et al. [18] postoperative hand function received a subjective rating of 80\u2013100% of the preoperative function by 89% of their patients. Other studies report no subjective impairment of function in 63\u2013100% of patients [7, 10]. Ho et al [17] found using a questionnaire consisting of a list of activities of daily living (ADL) that required use of wrist and forearm, that postoperative function appears to be quite satisfactory.\nWhile the forearm disability questionnaire showed that functional problems are limited, the cosmetics and sensibility questionnaire revealed that only 39% of the patients had no cosmetic or sensible complaints. The most frequent (27%) complaint was on the appearance of the donor site. Other studies found complaints of poor aesthetic results in up to 28% of patients, particularly female patients [7, 9, 10, 16]. Bardsley et al. [10] examined the cosmetic result by a subjective assessment on a scale of 0\u201310. The cosmetic result was acceptable in men (mean score 1.5) but was less so in women (mean score 4). Ito et al. [16] scored the dissatisfaction about the FRFF donor site of 23 patients in five items: color (4%), scar width (0%), depression (30%), wrist mobility (0%) and sensation (4%). The majority of patients (61%) had no complaints at all. The mean number of items of which patients were dissatisfied was 0.39. On the other hand in the study of Toschka et al. [18] 94% of the patients rated the aesthetic outcome as fair or good. Lutz et al. [8] reported 98% of patients rating the aesthetic outcome as satisfactory.\nOnly extension, pronation and grip strength had a clear correlation with the score on the forearm disabilities questionnaire. For all other objective test no significant correlation with this score was found. Therefore, most objective test results may not be indicative for donor site complaints by the patient. This finding has been confirmed earlier by others [4, 7, 8, 9].\nTo diminish donor site morbidity several harvesting and donor site closure technique modifications have been proposed.\nThe technique of harvesting the flap may have some impact on donor site function. In the early days of this flap when radial bone was included for mandibular reconstruction, morbidity was substantial, even leading to wrist fractures [7]. Suprafascial dissection is claimed by some to lead to superior results from a standpoint of wound healing, but there are no comparative studies that confirm this [8, 18]. Wolff et al. [19] reported on a small series of prefabricated fascial-split-thickness skin flap: after a split-thickness skin graft is transplanted to the forearm fascia the flap can be raised with complete preservation of the forearm skin and microsurgically transplanted like a conventional radial flap. The prelaminated fasciomucosal radial forearm flap as described by Nehrer-Tairych et al. [20] claims to provide better cosmetic and functional donor site effects, although the series of patients they describe is rather small.\nAvery et al. [5] reported good aesthetic results of repair of the FRFF donor site by full-thickness skin graft from the inner upper arm. Ho et al. [17] compared the functional and aesthetic outcomes of FRFF donor sites reconstructed with full-thickness skin graft, split skin graft alone and split skin graft overlying an acellular dermal matrix and found that all three methods of reconstruction have comparable low morbidity, postoperative satisfactory aesthetic and functional outcomes. Negative pressure wound dressing has been used for rapid healing and decreased donor site complications [21]. Bardsley et al. [10] used a ulnar artery-based transposition flap for primary closure to reduce wound healing and improve cosmetic results. Hsieh et al. [22] reported good results of primary closure of the FRFF donor site with a bilobed flap based on the fasciocutaneous perforator of the ulnar artery. Several types of tissue expansion have been reported to allow the use of local tissues for primary closure [23]. The preferred technique of closure is difficult to assess, because of limited comparative studies, limited number of patients and lack of significant differences.\nThe presented data can be used for solid counselling patients who are scheduled for FRFF reconstruction. Elaborate pre-surgical counselling may reduce the impact of functional and cosmetic impairment at the FRFF donor site [24]. Moreover, these data may serve as benchmark for future studies that use other free fasciocutaneous flaps.\nConclusion\nDonor site morbidity of the radial forearm flap measured by objective functional tests is negligible but in the patient\u2019s perception this is substantial. Subjective measurements show especially problems in cosmesis and sensibility. The present data may be used for solid patient counselling.","keyphrases":["donor site morbidity","head and neck cancer","cosmetics","free radial forearm flap","functional results"],"prmu":["P","P","P","P","R"]}
{"id":"J_Antimicrob_Chemother-1-1-2386079","title":"Cellular pharmacokinetics of telavancin, a novel lipoglycopeptide antibiotic, and analysis of lysosomal changes in cultured eukaryotic cells (J774 mouse macrophages and rat embryonic fibroblasts)\n","text":"Background Telavancin is a lipoglycopeptide with multiple mechanisms of action that include membrane-destabilizing effects towards bacterial cells. It shows bactericidal activity against forms of Staphylococcus aureus (phagolysosomal infection) with different resistance phenotypes [methicillin-resistant S. aureus, vancomycin-intermediate S. aureus or vancomycin-resistant S. aureus]. We examine here the uptake, efflux and intracellular distribution of telavancin in eukaryotic cells as well as its potential to induce lysosomal changes (in comparison with vancomycin and oritavancin).\nIntroduction\nTelavancin is a novel lipoglycopeptide derivative of vancomycin1 with marked bactericidal activity against vancomycin-susceptible and vancomycin-resistant organisms2 due to a multifunctional mechanism of action that combines inhibition of cell wall synthesis and disruption of bacterial cell membrane permability.3 It shows a high penetration in tissues,4 including human alveolar macrophages.5 In a recent study, we showed that telavancin exerts time- and concentration-dependent bactericidal activity against intraphagocytic Staphylococcus aureus, disregarding their resistance phenotypes (methicillin-resistant S. aureus, vancomycin-intermediate S. aureus or vancomycin-resistant S. aureus).6\nThe present investigation was initiated to further document and rationalize this observation by examining the uptake and subcellular disposition of telavancin in eukaryotic cells. Because previous studies had disclosed marked lysosomal alterations in eukaryotic cells exposed to another lipoglycopeptide, oritavancin,7 we also undertook to assess the impact of telavancin in this context, using vancomycin as a comparator. The study was performed with both phagocytic (J774 macrophages) and non-phagocytic (fibroblasts) cells because this allowed us, in the past, to obtain a comprehensive picture of the behaviour of other antibiotics accumulating in cells and causing specific lysosomal alterations.8\u201311\nMaterials and methods\nCells, cell cultures and assessment of membrane integrity\nJ774 mouse macrophages and rat embryo fibroblasts were cultivated, as previously described,7 in RPMI 1640 or in Dulbecco's modified Eagle's medium, respectively, both supplemented with 10% fetal calf serum (unless stated otherwise). The integrity of the pericellular membrane upon exposure to the antibiotics was assessed by measuring the release of lactate dehydrogenase in the culture medium, as described previously.6\nDetermination of cellular antibiotic concentration\nCells incubated with 14C-labelled telavancin were washed three times in ice-cold 0.9% NaCl, collected by scraping in distilled water and used for radioactivity determination (liquid scintillation counting) and protein assay (a detailed description of the methodology has been published previously12). In pilot experiments, cell-associated antibiotic was also measured by a microbiological technique (with Micrococcus luteus ATCC 9341 as test organism and antibiotic medium 11).12 A correlation coefficient (R2) of 0.95 was found between the two methods for cells incubated 24 h at concentrations spanning from 10 to 100 mg\/L (n = 12).\nCell fractionation studies\nCells were collected by gentle scraping in 0.25 M sucrose\/1 mM EGTA\/3 mM imidazole pH 7.4, homogenized and fractionated, as described previously.12 In brief, homogenates were separated into an \u2018unbroken cells\/nuclei\u2019 fraction by a low-speed centrifugation. The resulting cytoplasmic extract was separated into a \u2018granules\/membranes\u2019 fraction and a final supernatant by high-speed centrifugation. The granule\/membrane fraction was then further analysed by isopycnic centrifugation in a sucrose gradient. Protein and [14C]telavancin contents were determined in the fractions in parallel with the activity of marker enzymes of the main organelles, namely, inosine 5\u2032-diphosphatase (E.C. 3.6.1.6.; plasma and endoplasmic reticulum membranes), cytochrome c oxidase (E.C. 1.9.3.1.; mitochondria) and N-acetyl-\u03b2-glucosaminidase (E.C. 3.2.1.30.; lysosomes). Results are expressed as the percentage of enzyme activity, protein or drug recovered in each fraction. For isopycnic centrifugation, distributions were standardized for equal density increments ranging from 1.08 to 1.21, as described previously.13\nBiochemical studies\nCell sheets were washed three times in ice-cold 0.9% NaCl, collected by scraping in distilled water and lysed by sonication. Total phospholipids and cholesterol were extracted and assayed, as described previously.7 Proteins were assayed by the Folin\u2013Ciocalteau\/biuret method.14\nElectron microscopy\nSample preparation was performed as described previously.15,16 Observations were made in a Zeiss electron microscope operated at 80 kV. Morphometric analysis was performed on pictures taken at random, using the Image J software available from the NIH web site (http:\/\/rsb.info.nih.gov\/ij\/) and examining a total cell surface of cell profiles of 1000\u20132000 \u00b5m2. For each cell profile, we manually selected the zones occupied by electron-dense material (for which a limiting membrane could not always be clearly recognized) or corresponding to large vesicles filled with heterogeneous material (see Figure\u00a03 for examples). Results were expressed as percentage of the whole cell surface profile, which is numerically identical to the percentage of cell volume.17\nFigure\u00a03\nSelected pictures illustrating typical changes observed in macrophages (a) and fibroblasts (b) after incubation with 90 mg\/L telavancin for 24 or 72 h, respectively. Both cells show markedly enlarged and ill-shaped lysosome-like bodies with a pleiotropic material that is highly osmiophilic (often organized in a distorted concentric fashion). (c) Example of loose material and (d) example of highly osmiophilic material organized in multiple separate bodies. Bars are 1 \u00b5m. The dotted lines show examples of profiles selected for morphometric analysis: i, a large profile showing mainly a moderately osmiophilic and pleiomorphic material; ii, a small profile showing mainly osmiophilic material [possibly a polar section of a lysosome with mixed content (as in iii), but cut as shown by the arrow].\nMaterials\nTelavancin hydrochloride (powder for microbiological evaluation and >90% purity) and [14C]telavancin trifluoroacetate (33.8 mCi\/mmol and radiochemical purity >91%) were supplied by Theravance, Inc. (South San Francisco, CA, USA). The labelled drug was mixed with unlabelled telavancin to obtain a specific activity of 5 mCi\/mmol. Stock solutions were prepared at a final concentration of 1\u20132 mg\/L by vigorous vortexing in distilled water (the use of acidified DMSO recommended by the manufacturer was avoided because preliminary experiments disclosed a decrease in cell viability). Oritavancin (supplied as diphosphate salt fully hydrated) was obtained from Intermune (Brisbane, CA, USA). Vancomycin was procured as VANCOCIN\u00ae from GlaxoSmithKline, Belgium. Cell culture or microbiology media were from Invitrogen (Paisley, Scotland, UK) and Difco (Sparks, MD, USA). Other reagents were of analytic grade and purchased from E. Merck AG (Darmstadt, Germany) or Sigma-Aldrich-Fluka (St Louis, MO, USA).\nResults\nInfluence of telavancin on pericellular membrane integrity\nIn preliminary experiments, we measured the release of lactate dehydrogenase, a cytosolic enzyme, from cells exposed to telavancin (90 mg\/L, corresponding to the human Cmax18), in comparison with vancomycin (50 mg\/L, corresponding to the human Cmax19) and oritavancin (25 mg\/L, corresponding to the human Cmax20). No difference in controls (5.0 \u00b1 0.2%) was seen with telavancin (4.9 \u00b1 1.0%) or vancomycin (5.0 \u00b1 1.5%) in J774 macrophages after 24 h of incubation, whereas oritavancin induced a small but significant increase in enzyme release (15.5 \u00b1 1.5%; P < 0.001; n = 3 for all conditions). No significant effect was seen for fibroblasts after 72 h of incubation between controls (13.4 \u00b1 3.7%) and cells incubated with telavancin (16.9 \u00b1 2.5%), vancomycin (17.1 \u00b1 1.3%) or oritavancin (18.2 \u00b1 3.0%; n = 3 for all testing conditions).\nKinetics of uptake and release of telavancin\nFigure 1(a and b) shows the kinetics of uptake of telavancin in J774 macrophages and fibroblasts incubated with an extracellular concentration of 90 mg\/L. The uptake proceeded linearly over time at a rate that was similar in both cell types (see figure captions for details). Figure 1(c and d) examines the uptake of telavancin at increasing extracellular concentrations and at fixed time points (24 h for macrophages and 72 h for fibroblasts). The uptake was linearly related to the extracellular concentration in both cell types, allowing us to calculate a clearance rate for all conditions shown in Figure 1 of ~10 \u00b5L\/mg of cell protein per h. For macrophages, we also examined the drug efflux (after an initial uptake of 12 h in the presence of 90 mg\/L telavancin), which occurred at an apparent rate \u223c5.7-fold lower than that of influx (Figure 1a).\nFigure 1\n(a and b) Kinetics of uptake of telavancin (filled symbols and continuous line) in J774 macrophages (a) or embryo fibroblasts (b) incubated for the indicated times with an extracellular concentration of 90 mg\/L at 37\u00b0C in medium supplemented with 10% fetal calf serum. (a) Kinetics of efflux of the drug from J774 macrophages exposed to telavancin (90 mg\/L) for 12 h and re-incubated in a drug-free medium for an additional 24 h (open symbols and broken line) are also shown. (c and d) Cellular concentration of telavancin in J774 macrophages (c) or embryo fibroblasts (d) incubated at 37\u00b0C for 24 h or 72 h, respectively, in the presence of telavancin at the extracellular concentrations indicated on the abscissa. Results are given as arithmetic means \u00b1 SD (n = 3) and analysed by linear regression to calculate the corresponding clearances (\u00b5L\/mg of protein\/h): J774 macrophages, 9.6 \u00b1 0.6 (R2 = 0.98; a) and 10.0 \u00b1 0.4 (R2 = 0.99; c); fibroblasts, 8.2 \u00b1 0.4 (R2 = 0.97; b) and 9.0 \u00b1 0.7 (R2 = 0.98; d).\nThe influence of serum on telavancin uptake was examined in J774 macrophages by performing experiments in culture medium not supplemented with calf serum. The incubation was limited to 5 h to ensure maintenance of cell viability. Telavancin uptake remained linear over the 10\u201390 mg\/L range of extracellular concentrations, but proceeded at a rate ~1.7-fold faster than in the presence of serum (data not shown).\nSubcellular distribution of telavancin\nThe subcellular distribution of cell-associated telavancin was examined in homogenates obtained from J774 macrophages incubated for 24 h with the drug at an extracellular concentration of 90 mg\/L. The distribution of cell-associated telavancin and N-acetyl-\u03b2-glucosaminidase (lysosome marker) among the unbroken cells\/nuclei, granules\/membranes and supernatant fractions was similar (35% and 27%, 55% and 67% and 10% and 6%, respectively). The granule\/membrane fraction was therefore subfractionated by isopycnic centrifugation. The density distribution of telavancin, in comparison with that of N-acetyl-\u03b2-glucosaminidase (lysosomes), cytochrome c oxidase (mitochondria) and inosine 5\u2032-diphosphatase (plasma\/endoplasmic reticulum membranes), is shown in Figure 2. The distribution pattern of telavancin was largely superimposable on that of the N-acetyl-\u03b2-glucosaminidase, clearly dissociated from that of cytochrome c oxidase and also distinct from that of inosine 5\u2032-diphosphatase.\nFigure 2\nDensity distribution of marker enzymes [N-acetyl-\u03b2-glucosaminidase (lysosomes), cytochrome c oxidase (mitochondria) and inosine 5\u2032-diphosphatase (plasma and endoplasmic reticulum membranes)] and of telavancin after isopycnic centrifugation in a linear sucrose gradient of a granule fraction prepared from homogenates of J774 cells that were incubated for 24 h with 90 mg\/L telavancin at 37\u00b0C in medium supplemented with 10% fetal calf serum. The ordinate shows the percentage of each constituent recovered in each fraction.\nUltrastructural alterations\nElectron microscopy was used to examine whether telavancin induces morphological alterations in the subcellular organelles of cells incubated in its presence. Figure 3 shows selected pictures of alterations that could be observed in J774 macrophages and fibroblasts (90 mg\/L; 24 and 72 h, respectively). There was an enlargement of lysosomes that were filled with a pleiotropic material, made of partly highly osmiophilic, concentric structures and partly of a more loose appearance material.\nTo gain quantitative information on these changes, a morphometric analysis was performed on pictures taken at random. Figure 4 shows the relative abundance of these abnormal lysosomal profiles in cells incubated with three different concentrations of telavancin, in comparison with control cells or cells exposed to 50 mg\/L vancomycin or 20 mg\/L oritavancin. In macrophages (24 h incubation) as well as in fibroblasts (72 h incubation), telavancin induced a concentration-dependent increase in the percentage of cell volume occupied by overloaded lysosomes. The morphometric analysis showed that: (i) changes induced in fibroblasts were more marked than those in macrophages (which may have been a consequence of the longer incubation time in fibroblasts); (ii) incubation of macrophages with 25 mg\/L telavancin or of fibroblasts with 90 mg\/L caused changes similar to those seen with 50 mg\/L vancomycin in either cell types and (iii) alterations induced by telavancin at the highest concentration tested (90 mg\/L) were considerably milder than in cells incubated with 20 mg\/L oritavancin in both cell types.\nFigure 4\nMorphometric analysis of the material accumulated in macrophages (left-hand panel) or fibroblasts (right-hand panel) after 24 and 72 h of incubation, respectively, in control conditions or in the presence of 5, 25 or 90 mg\/L telavancin (TLV), 50 mg\/L vancomycin (VAN) or 20 mg\/L oritavancin (ORI). Results are expressed as percentage of the cell surface occupied by the electron-dense material and\/or large vesicles filled with a material of undetermined nature. Surface analysed was ~2000 \u00b5m2 for macrophages and 1000 \u00b5m2 for fibroblasts.\nInfluence of telavancin on cell phospholipid and cholesterol contents\nIn view of the ultrastructural changes observed, we looked for changes in phospholipids and cholesterol content of cells exposed to 90 mg\/L telavancin in comparison with vancomycin (50 mg\/L) and oritavancin (25 mg\/L). Figure 5 illustrates the data obtained after 24 h in macrophages and 72 h in fibroblasts. Neither telavancin nor vancomycin caused detectable increase in the phospholipid content. In contrast, oritavancin induced significant increases in the phospholipid content in both cell types. Telavancin and vancomycin caused a slight but not significant increase in the cholesterol content, whereas oritavancin again induced a marked, statistically significant increase.\nFigure 5\nTotal phospholipid content (upper panels) or total cholesterol content (lower panels) of J774 mouse macrophages (left-hand panels) or rat embryo fibroblasts (right-hand panels) exposed for 24 and 72 h, respectively, to glycopeptides at their human Cmax (VAN, vancomycin 50 mg\/L; TLV, telavancin 90 mg\/L and ORI, oritavancin 25 mg\/L). Results are expressed as percentages of control values for total phospholipids and cholesterol. Values are arithmetic means \u00b1 SD (n = 6\u20138). Values for control macrophages and fibroblasts were, respectively, 204 \u00b1 10 and 295 \u00b1 8 nmol\/mg of protein for total phospholipids and 72 \u00b1 3 and 130 \u00b1 16 nmol\/mg of protein for total cholesterol. Statistical analysis (ANOVA): ***P < 0.001, **P < 0.01, *P < 0.05 when compared with the matching control. Other differences were not significant.\nDiscussion\nThe present study provides evidence that telavancin is taken up by cultured macrophages where it becomes associated with lysosomes, as assessed by cell fractionation studies. These results rationalize our previous data, showing that telavancin exerts a marked antibiotic activity against intraphagocytic S. aureus, which is known to develop in the phagolysosomes of infected macrophages.21,22\nThe accumulation level reached by telavancin in macrophages at 24 h (\u223c45-fold) is intermediate between that recorded previously in the same conditions for vancomycin (\u223c8-fold) and oritavancin (\u223c370-fold).12 Its uptake is not specific to phagocytic cells, as it is also observed with fibroblasts, both cell types taking up the drug at similar rates. The entry of drugs into lysosomes may occur through either diffusion\/segregation or pinocytosis,23 as illustrated by the behaviour of macrolides8 and aminoglycosides,24 respectively. Diffusion\u2013segregation seems unlikely in view of the slow efflux of telavancin, as this process is usually considered to be reversible (although binding of the drug to intracellular constituents could explain this slow efflux, as is observed for azithromycin in fibroblasts16). Pinocytosis, therefore, appears more plausible, especially if considering the size of the molecule that would prevent its fast diffusion through membranes. However, the clearance rates recorded here (\u223c10 \u00b5L\/mg of protein\/h) are ~15- to 30-fold higher than those reported for fluid-phase pinocytosis markers (\u223c0.7 \u00b5L\/mg of protein\/h in macrophages15 and \u223c0.3 \u00b5L\/mg of protein\/h in fibroblasts24). Telavancin uptake, therefore, should involve a process of adsorptive pinocytosis (through binding to cell surface; see discussion and modelling in Silverstein et al.25) However, the lack of saturation of telavancin uptake upon increase of its extracellular concentration is intriguing as this (i) is an hallmark of adsorptive pinocytosis25 and (ii) was observed for oritavancin.12 The initial uptake clearance rate of oritavancin, however, is considerably larger (\u223c150 \u00b5L\/mg of protein\/h),12 which indicates that binding of telavancin should be much weaker and may not actually show saturation in the concentration range investigated here. A weaker membrane binding of telavancin is actually consistent with its lower lipophilicity when compared with oritavancin (reported logP values of 0.6 for telavancin versus 4.1 for oritavancin) [Advanced Chemistry Development Software Solaris V4.67, Sci Finder Scholar 2006, American Chemical Society, Washington, DC, USA].\nBeyond its therapeutic interest, the lysosomal accumulation of telavancin may also be responsible for the morphological alterations observed in cells exposed to the drug. Yet, these changes appear quantitatively minor, as is also the case for vancomycin. Moreover, and in contrast to what is observed with oritavancin,7 telavancin did not significantly affect phospholipid or cholesterol cellular levels. This difference may be related to the lower uptake of telavancin, although we cannot exclude, at this stage, a true difference in the intrinsic capacity of the two molecules to interfere with lipid metabolism. Further studies are required to determine the exact nature of the accumulated material and to decipher the underlying molecular mechanisms. These may be more complex than those evidenced for aminoglycoside antibiotics, which mainly induce an accumulation of phospholipids.26 Interestingly, telavancin was without detectable effect on lactate dehydrogenase release. This suggests that the membrane-destabilizing properties exerted by telavancin towards bacterial membranes,3 which probably contribute to its marked bactericidal effect, involve constituents that are specific to or more abundant in prokaryotic cells.\nIndependent studies have shown that telavancin accumulates in human alveolar macrophages, reaching a cellular concentration of ~50 mg\/L within 8\u201324 h after systemic administration of therapeutic doses.5,27 In our experiments, macrophages exposed for 24 h at 90 mg\/L have an apparent cellular concentration of 4 mg\/mL [based on an estimated mean cellular volume of 5 \u00b5L\/mg of protein (see Van Bambeke et al.12 and references cited therein)]. The lower cellular uptake of telavancin reported in vivo may result from (i) its high protein binding (\u223c90% to 93%) and (ii) the fluctuation of its serum levels related to its once-daily administration and its half-life of 7.5 h.18 As extensively discussed in previous publications,6,7,12,22 our cellular model suffers from the limitation that it does not take into account these two important pharmacokinetic determinants. This may lead to an overestimation of the accumulation of telavancin versus that of vancomycin, for which the free fraction in vivo oscillates between 10% and 55%.19 Thus, assuming that only the free fraction of telavancin is available for the uptake by macrophages in vivo, its effective extracellular concentration will oscillate between \u223c10 and 0.5 mg\/L, which, in our model, would create a cellular concentration of \u223c0.24 \u00b5g\/mg of protein (i.e. \u223c48 mg\/L). At this concentration, which is of the same order of magnitude as that observed in vivo, telavancin does not cause any morphological change in our model.\nTaken together, our data further document the tissue-directed pharmacokinetics of lipoglycopeptides.28 As telavancin is clearly superior to vancomycin with respect to bactericidal activity towards both extracellular and intraphagolysosomal S. aureus6 while showing a similar cellular safety profile (as far as lipid metabolism and subcellular morphology are concerned), the data support its potential interest for the treatment of infections where intracellular foci are present.29 Further studies using in vivo models are, however, required to confirm the improved intracellular efficacy and cellular inocuity of telavancin.\nFunding\nF. V. B. is Ma\u00eetre de Recherches of the Belgian Fonds National de la Recherche Scientifique. Funding: Belgian Fonds de la Recherche Scientifique (FRS\u2013FNRS) and Belgian Fonds de la Recherche Scientifique M\u00e9dicale (FRSM; grant nos 1.5.223.05 F, 3.4.549.00 F and 3.4.639.04 F); Belgian Federal Science Policy Office (Research project P5\/33; research action P5); Fonds Sp\u00e9ciaux de Recherches and Actions de Recherches Concert\u00e9es of the Universit\u00e9 catholique de Louvain; Grant-in-Aid from Theravance, Inc. (for the pharmacokinetic studies with telavancin).\nTransparency declarations\nF. V. B. and P. M. T. are members of the European Advisory Board of Targanta Inc. (current owner of oritavancin). The remaining authors have none to declare.","keyphrases":["cellular pharmacokinetics","glycopeptides","membrane","vancomycin","oritavancin","lipids"],"prmu":["P","P","P","P","P","P"]}
{"id":"Eur_J_Nucl_Med_Mol_Imaging-3-1-1914271","title":"Cardiovascular molecular MR imaging\n","text":"Introduction Cardiovascular molecular imaging is a rapidly evolving field of research, aiming to image and quantify molecular and cellular targets in vivo. MR imaging has some inherent properties that make it very suitable for cardiovascular molecular imaging. Until now, only a limited number of studies have been published on cardiovascular molecular imaging using MR imaging.\nIntroduction\nConventional imaging modalities, including magnetic resonance (MR), are primarily based on anatomical, functional or metabolic properties to study (patho)physiology. Molecular imaging is a rapidly evolving field of research, aiming to image and quantify molecular and cellular targets in vivo. Molecular imaging can be applied to a wide range of scientific and clinical fields of interest. One of the most promising applications of molecular imaging is in the field of cardiovascular imaging. Imaging of cardiac anatomy, dimensions and function has some limitations concerning, for example, prediction of therapy outcome. Addition of specific information on, for instance, plaque composition and total plaque burden can be very helpful in guiding therapy.\nImaging of molecular processes is desirable because cardiovascular disease may be detected earlier, risk stratification may be more accurate, monitoring of innovative therapies may be improved, or a more accurate prognosis may be provided [1].\nMR imaging has some inherent properties that make it very suitable for cardiovascular molecular imaging. The interaction between inherent tissue properties and specific contrast agents may lead to more specific clinical conclusions and prediction of therapy outcome. Thereby, cardiovascular molecular MR imaging may help in diagnosing cardiovascular disease, and in deciding whether expected beneficial effects of (invasive) therapy counterbalance the risk of complications of therapy.\nA conventional approach to molecular MR imaging concerns MR spectroscopy. Furthermore, there are two main innovative contrast agents that may be used clinically soon: (1) iron oxide MR contrast agents and (2) fibrin-targeted MR contrast agents.\nMR spectroscopy\nMR spectroscopy (MRS) allows non-invasive characterisation of myocardial metabolism. In principle, MRS is a pure form of molecular \u2018imaging\u2019 technique. Clinically, several nuclei allow noninvasive MRS of the heart. Initially, human MRS research was focussed on the 31P nucleus to study high-energy phosphate metabolism. An example concerning the effects of diabetes type 2 on myocardial high-energy phosphate metabolism is shown in Fig.\u00a01 [2]. Another interesting new application of 31P-MRS was published by Smith et al. [3], who measured myocardial creatine kinase (CK) metabolite concentrations and adenosine triphosphate synthesis through CK, the primary energy reserve of the heart, to test the hypothesis that ATP flux through CK is impaired in patients with left ventricular hypertrophy (LVH) and chronic heart failure. It turned out that myocardial ATP levels were normal, but creatine phosphate levels were 35% lower in LVH patients than in normal subjects. Furthermore, the myocardial CK rate constant was normal in LVH, but halved in patients with LVH combined with chronic heart failure. Thereby, they could show that it is not the relative or absolute CK metabolite pool size but rather the kinetics of ATP turnover through CK that distinguishes failing from non-failing hypertrophic hearts. These observations support the hypothesis that a deficit in myofibrillar energy delivery contributes to chronic heart failure pathophysiology in human LVH. The same technique was applied by Weiss et al. to study ATP flux through CK in the normal, stressed and failing human heart [4]. The latter studies are elegant examples of the capability of MR to measure non-invasively the concentration of metabolites and even the rate constant of enzyme turnover.\nFig.\u00a01Left panel: planscan of the volume of interest on transverse and sagittal spin-echo MR images. Right panel: phosphorus-31 magnetic resonance spectroscopy (31P-MRS) obtained at rest from the anterior left ventricular wall in a patient with diabetes type 2 (left) and a healthy subject (right). Note the decreased signal amplitude of phosphocreatine in the type 2 diabetes patient. Courtesy of Diamant et al. [2]\nAnother interesting application of metabolic imaging is 23Na MR imaging, as show by Jansen et al. They applied this innovative spectroscopic imaging technique as a diagnostic modality for early detection of myocardial ischaemia and viability in a rat model [5]. They tested whether 23Na MR imaging can be used to assess viability after low-flow ischaemia. 23Na MR chemical shift imaging was alternated with 23Na MR spectroscopy. Na image intensity increased with increasing severity of ischaemia. 23Na image intensity at end low-flow ischaemia was well correlated with CK release during reperfusion, as well as with infarct size. Therefore, their study indicates that 23Na MR imaging is a promising tool for the assessment of myocardial viability. Ouwerkerk et al. applied 23Na MR imaging to measure cardiac tissue sodium concentrations in the human myocardium [6]. They used a surface coil at 1.5\u00a0T MR to non-invasively quantify regional myocardial sodium concentrations in the left ventricular free wall, septum and adipose tissue. Their 23Na MR imaging results were consistent with prior invasive measurements on biopsy and autopsy specimens.\nIn the past, 1H-MRS of the myocardium was first applied to non-invasively study creatine depletion in non-viable infarcted myocardium [7]. Total creatine was measured in the posterior and anterior left ventricle and septum, and was significantly lower in regions of infarction than in non-infarcted regions of myocardium in patients or in the myocardium of healthy controls. Therefore, they showed for the first time that spatially localised 1H-MRS can be used to measure total creatine non-invasively throughout the human heart. The detection of regional creatine depletion may provide a metabolic means to distinguish healthy from infarcted non-viable myocardium. Szczepaniak et al. used 1H-MRS to measure myocardial lipid content [8]. Studies in rat tissue ex vivo and in healthy humans in vivo provided evidence that 1H-MRS constitutes a reproducible technique for the measurement of myocardial triglyceride. Increased myocardial triglyceride content was accompanied by elevated LV mass and suppressed septal wall thickening as measured by cardiac imaging. More recently, 1H-MRS of the human myocardium was improved by implementing the respiratory navigator technique to monitor diaphragmatic motion, and thereby correct data acquisition prospectively for breathing motion. First (unpublished) results from our institution show improved reproducibility of human cardiac 1H-MRS measurements when using the respiratory navigator technique, as compared with conventional continuous breathing. Respiratory navigator gated 1H-MRS was recently applied in an experimental setting in our institution to evaluate the effects of a very low calorie diet on myocardial triglyceride content. First (unpublished) results show that after a short very low calorie diet, there is an increase in intramyocardial triglyceride content (Fig.\u00a02).\nFig.\u00a02The surface coil was positioned just below the mitral valve level of the heart (a, b). Spectroscopic volume localisation in the interventricular septum on four-chamber (c) and short axis (d) views. Special care was taken to avoid contamination from epicardial fat. (e) Typical water-suppressed 1H-MR spectrum of myocardial tissue located in the interventricular septum. Peak heights are in arbitrary units\nGadolinium-based contrast agents\nGadolinium-based contrast agents can be applied to study regional myocardial perfusion. After a rapid intravenous contrast injection, there is marked signal enhancement first in the RV cavity, then in the LV cavity, and subsequently in the LV myocardium [9]. The peak signal intensity is related to the concentration of the contrast agent in the local tissue and is directly proportional to the coronary blood flow. Perfusion MR at rest and after infusion of pharmacological agents (adenosine and persantine) have been compared with standard methods (angiography or radionuclide scintigraphy) and demonstrated reasonable sensitivity (67\u201383%) and specificity (75\u2013100%) [9].\nCurrently, multiple MR imaging techniques are available to assess myocardial viability. Cardiovascular MR imaging can be used to assess end-diastolic wall thickness and contractile function at rest [10]. Segments with an end-diastolic wall thickness <6\u00a0mm most likely represent transmural scar formation, and contractile function will not improve after myocardial revascularisation. Dobutamine MR imaging can be used to evaluate contractile reserve, in a similar manner to dobutamine echocardiography. Gadolinium contrast-enhanced MR imaging [11] allows for detection of the extent and transmurality of scar tissue (Fig.\u00a03). Recently reported sensitivity and specificity are in the range of 74% and 82% respectively.\nFig.\u00a03Left panel: short-axis MR images at rest and during dobutamine stress. Note the lack of improvement in myocardial wall motion in the anteroseptal region when dobutamine stress is applied. Right panel: delayed gadolinium contrast-enhanced MR images in two-chamber, four-chamber and short-axis views. Note the almost transmural delayed enhancement of the anteroseptal\/apical region, corresponding to the region without contractile response due to dobutamine stress in the left panel. The anteroseptal region is considered as \u2018non-viable\u2019 myocardial tissue\nBased on as yet unpublished scientific developments, it is expected that gadolinium-based delayed enhancement of the vessel wall may become reality. This MR imaging technique may allow fast total body screening for total plaque burden, an important predictor for morbidity and mortality.\nIn general, gadolinium-based contrast agents are not perfectly suited for molecular imaging because of the inherent high threshold of detectability. Therefore, new contrast agents are under development to potentiate the effect of distortion of the magnetic field.\nIron oxide MR contrast agents\nSuperparamagnetic iron oxide (SPIO) particles can be detected at micromolar concentrations of iron, and offer sufficient sensitivity for MR imaging [12]. SPIO-based cellular imaging has become an established technique to label and detect cells of interest. Imaging of macrophage activity was the initial and is still the most significant application, with several products either approved for or in clinical trials [12]. Another exciting application of SPIOs is labelling of myocardial stem cells. In a swine model for myocardial infarction, magnetically labelled stem cells were injected in the infarcted myocardial region (Fig.\u00a04). Using delayed contrast-enhanced MR imaging, the infarcted area can be identified with high accuracy. New technical developments may even allow specific delivery of magnetically labelled therapeutics to the infarcted myocardial region [12]. Combined with measurements of myocardial function, MR imaging seems an excellent modality for planning, delivery and follow-up of myocardial stem cells as therapy for ischaemic heart disease.\nFig.\u00a04Detection of delivery and migration of Feridex-labelled myocardial stem cells in a swine model. Hypointense lesions in spin-echo (SE),gradient-echo (GRE) and delayed-enhanced (Delayed) MR imaging (upper panel) of injection sites (arrows) within 24\u00a0h of intramyocardial injection. Cells were injected in the myocardial infarct (MI). Long-axis MR images (lower panel) show hypointense lesions (arrows) caused by labelled myocardial stem cells acquired within 24\u00a0h and 1\u00a0week. LV left ventricle, RV right ventricle. Courtesy of Bulte andKraitchman [12]\nAnother promising application of SPIO MR imaging is visualisation of vessel wall inflammation. SPIOs are \u2018digested\u2019 by macrophages, involved in inflammatory processes. Imaging of the SPIO-induced magnetic inhomogeneities allows for imaging of inflammation. Such an approach is currently only available in a research setting; it is, however, expected that these contrast agents will become available for clinical application soon.\nFibrin-targeted MR contrast agents\nExciting recent developments allow selective and non-invasive molecular MR imaging of thrombus [13]. The principle of the contrast agent is that it is targeted to fibrin. In an elegant study by Spuentrup et al., a swine model was used to test the innovative fibrin-targeted MR imaging contrast agent, which can be administered intravenously [14]. The imaging protocol consisted of coronary MR angiography to demonstrate the lumen of a coronary artery, combined with molecular thrombus MR imaging. Thereby, anatomical information could be linked to specific information of vessel wall components. In an area of focal coronary artery stenosis, intraluminal thrombus could be detected (Fig.\u00a05).\nFig.\u00a05Coronary thrombus visualisation with a fibrin-targeted molecular MR imaging contrast agent. Left panel: double-oblique white blood coronary MR angiography (multiplanar reconstruction) demonstrating the lumen of the left anterior descending artery with bright signal (arrowheads). Right panel: double-oblique MR images after administration of the fibrin-targeted contrast agent (multiplanar reconstruction, same orientation). Note the increased signal (arrow) in the left anterior descending coronary artery, corresponding to thrombus. Courtesy of Spuentrup et al. [14]\nThe same contrast agent can be applied to detect, for example, right atrial thrombus, a potential source of more distal emboli (Fig.\u00a06). An atrial clot could be visualised easily with this molecular MR imaging technique, by intravenous administration of the fibrin-targeted contrast agent. Furthermore, MR clot imaging can be combined with functional imaging of the heart in the same imaging session. An even more exciting application of this fibrin-targeted contrast agent is detection of pulmonary emboli (Fig.\u00a07).\nFig.\u00a06Molecular MR imaging of atrial clot in a swine model. The left panel shows pre-contrast (upper) and post-contrast (lower) coronal images. Note the presence of high MR signal in the area of the right atrium, indicating an atrial clot. The right panel shows increased MR signal in the left atrium (LA), corresponding to a left atrial clot (arrow). These clots are potential sources for more distal emboli. LV left ventricle. Courtesy of Spuentrup et al. [14]Fig.\u00a07Examples of molecular MR imaging of pulmonary embolus. Two examples are shown, each consisting of three adjacent coronal slices (horizontal). The MR imaging technique is such that signal from surrounding blood pool and lung parenchyma is suppressed. The upper row shows pulmonary embolus (arrow) in the right lower lobe. The lower panel shows bilateral pulmonary emboli (arrows) after intravenous administration of fibrin-targeted MR imaging contrast agent. Courtesy of Spuentrup et al. [14]\nThe above-described applications of molecular MR imaging may be especially suitable for fast screening for cardiovascular disease in an emergency setting. Patients presenting with chest pain in the emergency room can be studied by MR imaging to confirm or rule out ischaemic heart disease or pulmonary embolus. Molecular MR imaging using fibrin-targeted contrast agents allows selective visualisation of acute coronary, cardiac and pulmonary thrombi. Additional functional cardiac imaging can help determine the functional effects of detected thrombi.\nConclusion\nMolecular MR imaging is an exciting and rapidly evolving new area of cardiovascular imaging. MR imaging seems very suitable for molecular imaging, although many technical difficulties have to be overcome. The main current limitation is the low sensitivity of MR imaging to detect small changes in magnet homogeneity. We expect that in the next decade, currently promising MR molecular imaging agents will be introduced into the clinical arena to guide diagnosis and therapy of cardiovascular disease.","keyphrases":["cardiovascular molecular imaging","cardiovascular disease","contrast agents","mr spectroscopy"],"prmu":["P","P","P","P"]}
{"id":"Int_J_Biol_Sci-3-3-1802012","title":"Advances in Swine Transcriptomics\n","text":"The past five years have seen a tremendous rise in porcine transcriptomic data. Available porcine Expressed Sequence Tags (ESTs) have expanded greatly, with over 623,000 ESTs deposited in Genbank. ESTs have been used to expand the pig-human comparative maps, but such data has also been used in many ways to understand pig gene expression. Several methods have been used to identify genes differentially expressed (DE) in specific tissues or cell types under different treatments. These include open screening methods such as suppression subtractive hybridization, differential display, serial analysis of gene expression, and EST sequence frequency, as well as closed methods that measure expression of a defined set of sequences such as hybridization to membrane arrays and microarrays. The use of microarrays to begin large-scale transcriptome analysis has been recently reported, using either specialized or broad-coverage arrays. This review covers published results using the above techniques in the pig, as well as unpublished data provided by the research community, and reports on unpublished Affymetrix data from our group. Published and unpublished bioinformatics efforts are discussed, including recent work by our group to integrate two broad-coverage microarray platforms. We conclude by predicting experiments that will become possible with new anticipated tools and data, including the porcine genome sequence. We emphasize that the need for bioinformatics infrastructure to efficiently store and analyze the expanding amounts of gene expression data is critical, and that this deficit has emerged as a limiting factor for acceleration of genomic understanding in the pig.\n1. Overview\nDramatic advances in our understanding of the porcine transcriptome have occurred over the past decade, and especially in the past few years. The pig transcriptome has been analyzed to address biomedical, agricultural and fundamental biological questions, using more and more sensitive and comprehensive tools. Few reviews of porcine transcriptional profiling have been published. Blomberg and Zuelke 1 recently outlined several techniques that have been used for porcine profiling, although their focus was on serial analysis of gene expression (SAGE). This review will specifically discuss results in pigs using the major technologies of EST sequencing; PCR-centric screening, analysis and assay approaches; and array hybridization methods. Due to space limitations, in this review we will not describe the technical aspects of each technique in detail; there are many reviews and specialized resources for cDNA library and EST production; differential display PCR; SAGE; suppression subtractive hybridization (SSH); real-time quantitative PCR; and of course microarray based techniques. Further, many reports have been made using techniques such as differential display or microarrays to identify genes differentially expressed in biological states of interest. The majority of these reports use a porcine tissue or cell line to ask questions of biomedical interest, and the use of porcine-derived biological material is not directly relevant to the study. Space limitations require us to describe in detail only those efforts with broad interest to the pig genomics community. All papers found that differential display screening or the use of microarrays in assaying expression in porcine tissues report are listed in Supplementary Tables 1 and 2, respectively. Reports on EST sequencing will be discussed, but we will not discuss publications that focus on physical and\/or genetic mapping of such ESTs. Finally, while still in its infancy, we will discuss published and other publicly reported work on the development and use of bioinformatics and databases to analyze porcine gene expression data.\n2. Tools, Techniques and Results for Porcine Transcriptome Analysis\nPorcine Expressed Sequence Tag Projects describe Significant Portions of the Swine Transcriptome\nSoon after Adams and co-workers 2 suggested that the sequencing of random clones from cDNA libraries would be an effective means to obtain human gene sequence data rapidly, researchers have been reporting porcine ESTs 3, 4, 5. Many groups have now submitted EST sequence data; Table 1 shows the projects with major contributions to global EST sequencing efforts (totaling over 564,000 entries). The NCBI UniGene website for pig (http:\/\/www.ncbi.nlm.nih.gov\/UniGene\/UGOrg.cgi?TAXID=9823) shows over 100 libraries have more than 250 EST sequences deposited (over 150 additional libraries with less than 250 sequences\/library have been reported). These sequences come from thirteen categories of tissues, ranging from a low of 3 libraries found for the adipose, brain, and conceptus categories to 31 libraries in the genito-urinary category. In NCBI Build #25 (as of July 30, 2006), the mRNAs (4,410), high-throughput cDNAs (1,153) and ESTs (463,885) have been clustered into 37,861 UniGene sets. On the other hand, a slightly earlier build of the Porcine Gene Index (SsGI Release 12.0; June 20, 2006 (recently moved from TIGR to Dana Farber Cancer Institute; http:\/\/compbio.dfci.harvard.edu\/tgi\/cgi-bin\/tgi\/gimain.pl?gudb=pig) shows 575,730 ESTs and 6,854 expressed transcripts from a total of 257 cDNA libraries. This release identifies 64,746 tentative clusters (TCs) and 88,117 singleton ESTs and ETs. Many of these ESTs have been submitted within the past three years, showing the great strides taken recently; since January 2004 (Release 8.0), the number of TCs in the Pig Gene Index has nearly tripled. Additional information on these databases and others is discussed in the section on bioinformatics.\nIn addition to the many uses for such sequencing data that can be made in structural biology and in mapping genes, this large amount of data has also been used to estimate the level of expression of a gene by calculating the number of instances when the transcript has been randomly sequenced in a library. While care must be taken in such comparisons to use non-normalized libraries and EST data of sufficient size, several authors have discussed such estimates. Davoli and co-workers 6 used EST sequence frequencies to compare to equivalent data for human transcripts to characterize their skeletal muscle library; similar estimates were performed by Smith and others 7 to identify highly expressed genes in early embryo. Several groups 8, 9, 10 have used data from non-normalized libraries to estimate such \u201cvirtual expression patterns\u201d across multiple tissues or developmental stages. Using sequence frequencies, we were able to identify genes that are expressed highly selectively in one tissue, as well as genes changing expression during embryonic development through calculating frequencies in libraries created from different embryonic stages 8. Jiang and colleagues 9 identified sequence clusters that were over-represented across specific stages of ovarian follicle development and between growing follicles and corpora lutea. Whitworth and others 10 recognized genes showing differential frequency in very early embryogenesis, finding many transcripts with different frequency in comparisons between unfertilized oocytes and 4-cell embryos, and between these embryos and the more differentiated blastocysts. Interestingly they found transcripts with different frequency between in vivo and in vitro produced four cell embryos as well as between in vivo and in vitro blastocysts. Differential frequency has also been used by several groups 8, 11, 12 to determine the efficacy of normalization of cDNA libraries, which is performed to remove the highly expressed (and therefore high frequency) transcripts, allowing for increased efficiency in finding new gene transcripts through random EST sequencing. Finally, both UniGene and SsGI (now the Dana Farber Cancer Institute Pig Gene Index) provide global views of gene expression levels based on EST frequency (see later section).\nThrough comparison of frequencies of ESTs across species, it is possible to identify species-specific differences in expression. Zhao et al. 13 identified a highly expressed transcript in porcine placenta (1 % of ~4,500 placenta and early conceptus sequences available) which had no ortholog in the human EST database, although hundreds of thousands of placenta sequences had been deposited. A highly similar sequence was present in the human genome, with a conserved open reading frame. Further cDNA sequencing and RACE-based transcript mapping analysis of this gene (PLET1) in human, mouse and pig placenta was performed and it was determined that the human gene is expressed, but not spliced due to mutations in splicing signals, thus very few transcripts for human PLET1 appear in cDNA libraries. This study is one of the first examples of the use of porcine EST frequency data to identify a splicing-defective gene in the human genome 13.\nSerial Analysis of Gene Expression in Porcine Tissues\nAnother sequencing-based method to estimate transcript abundance is serial analysis of gene expression (SAGE). This is a powerful approach whose sole purpose is developing a statistically robust estimate of the relative amounts of expressed sequences in the genome of interest; no cDNA clones or libraries are created. Double-stranded cDNA is digested with specific restriction enzymes, adapters are ligated to cDNA fragments, and the resulting fragments are concatenated and sequenced extensively to obtain sequences for tens to hundreds of thousands of 10-20 bp sequence \u201ctags\u201d. These tags can be mapped back to known cDNA sequence and the resulting data is analyzed for relative abundance. Blomberg and colleagues 14 have applied SAGE to determine the content and expression pattern of developing porcine conceptus at the critical stage of peri-implantation, where the developing conceptus goes through a remarkable morphological elongation stage just prior to implantation. They identified over 400 putative transcripts with differential expression between ovoid (before elongation) and filamentous concepti, and identified several pathways involved in this process, including steroidogenesis and oxidative stress response. In a follow-up paper, they describe the production of SAGE libraries for tubular conceptus and a comparison of non-amplified and amplified SAGE libraries, which showed that amplification, a useful procedure when tissue is limiting, can be used to generate SAGE data accurately reflecting transcript levels similar to non-amplified libraries 15. They then compared expression patterns between this intermediate conceptus form and the ovoid and filamentous expression patterns, and identified over 600 genes with putative differentially expression between at least two stages. Genes differentially expressed at all stages had GO annotations for involvement in cell cycle, cellular organization, cell-cell interaction and general metabolism. Specific differentially expressed or constitutively expressed genes involved in tissue remodeling, glycolysis, cell cycle and tissue\/cell type differentiation were further studied; many patterns, but not all, were confirmed by QPCR. In most cases of dissimilar results between SAGE and QPCR, the QPCR results was in agreement with one but not both stage-differences (ovoid versus tubular but not tubular to filamentous, etc.), and the authors remark that these differences may be due to unknown splicing variants which may be resolved once further information on the porcine genome sequence becomes available 15.\nScreening for Differentially Expressed Genes using Differential Display and Suppression Subtractive Hybridization Technology\n\u201cOpen-ended\u201d techniques such as differential display (DD) and suppression subtractive hybridization (SSH) have been used to profile the transcriptional response to various treatments or to contrast tissues or other variables. Such screening techniques are complementary to \u201cclosed\u201d profiling technologies such as microarrays as they allow the discovery of novel, heretofore un-sequenced transcripts. The DD technique uses specific primers to amplify specific subsets (reduced representations) of the total mRNA pool of a cell or tissue, allowing resolution of a limited number of PCR products for each specific primer pair on a sequencing gel. These PCR products can be compared by side-by-side electrophoresis of PCR products amplified from RNAs representing different tissues or treatments to be compared\/screened. Research using DD to screen porcine transcripts have been published starting in 1996 and reported from 1-20 differentially expressed products\/genes. The majority of these reports (over 20) focused on biomedical research question, using porcine cells or tissues. Due to space limitations, these papers will not be discussed here, although all papers are listed in Supplementary Table 1. On the other hand, papers reporting the characterization of larger sets of genes, from about 50 to 200 or more genes, began appearing in 2001. These reports describe the use of DD to study response to viral infection; liver-specific expression and hepatic response to dietary changes; and gene expression changes in response to selection for improved reproductive traits. These six papers will be summarized here.\nWang and colleagues 16 identified 48 total ESTs that increased (35) or decreased (13) their expression in alveolar macrophage cells as a result of porcine reproductive and respiratory syndrome virus (PRRSV) infection. Differential expression for seven genes was tested and all confirmed by Northern blot analysis of infected or mock-infected macrophages. Of interest was the finding that the majority of the annotated genes in this group were mapped to metabolic and physiological pathways; only one EST was clearly annotated as an immune response gene (inflammatory mediator gene chemotactic factor II) and this gene was down-regulated by PRRSV 16. In a recent report, also investigating host response to viral infection, Bratanich and Blanchetot 17 used DD to study genes differentially expressed in lymph nodes between healthy piglets and those suffering from porcine multi-systemic wasting syndrome (PMWS). These authors sequenced and confirmed nine DD products that were found only in the affected animals or showed an increase in PMWS animals as compared to controls. Northern analysis of two genes, hyaluronan-mediated motility receptor (RHAMM) and RNA splicing factor, further confirmed the expression response for these two genes 17.\nTwo groups have successfully applied DD to study liver gene expression. Ponsuksili and co-workers 18 used DD to screen for genes selectively expressed in liver, in order to identify candidate genes for metabolic traits. In a comparison of normal liver and nine other tissues, they found 240 candidate EST fragments that were expressed only in liver or predominately in liver with up to 4 tissues showing expression. These fragments represented 200 known (91) or unknown (109) transcripts. Most of the genes represented by multiple clones were known to encode proteins that are secreted by the liver into the bloodstream. Multi-tissue Northern blot analysis confirmed the expression pattern for complement component 3 (C3), while semi-quantitative PCR confirmed C3 and an additional 14 genes 18. A second group 19 exploited the DD technique to find genes whose hepatic expression was altered by feeding pigs on a restricted protein diet formulated using either casein (CAS) or soy protein isolate (SPI). This group identified and confirmed 86 differentially displayed PCR products, of which 44% has similarity to known genes. Nearly all of the known genes were annotated to pathways expected to be involved in the restricted diet, including protein and amino acid metabolism, oxidative stress, regulation of gene expression, fat and energy metabolism and others. Schwerin and collaborators further showed that six genes involved in stress response were differentially expressed between pigs fed the two diets, with five of six genes showing higher liver expression in the SPI-fed pigs over the CAS-fed pigs. The authors proposed that one reason for this stress response in the SPI-fed animals is the amino acid imbalance in this diet, as compared to the CAS-based diet 19.\nFinally, two reports looking at gene expression changes due to selection for improved reproductive traits were published recently. Gladney and colleagues 20 used DD of ovarian follicles to identify genes differentially expressed in control animals as compared to animals in a population selected for improved litter size based on ovulation rate and embryo survival. Tissue was collected at 2 or 4 days post prostaglandin F2alpha injection, which was used to synchronize follicular development. Overall, 84 DD products were identified and sequenced; the majority of which were predicted to differentially expressed although several equally expressed transcripts were also characterized. Four genes were confirmed as DE by Northern blot. Of interest was the finding that calpain I light subunit (CAPN4) expression was lower in the select line as compared to controls. As the similar small subunit calpain gene ACG-2 has been linked to apoptosis, the authors speculate that a decrease in CAPN4 expression in the select line could lead to decreased apoptosis and to a larger number of follicles available for ovulation 20. The same group published a companion paper, using DD to look at changes in anterior pituitary expression in the same selection line and controls 21. Anterior pituitary tissue was collected similarly to the follicles above, and DD analysis was performed on tissue pooled across PGF2alpha treatment days. A total of 162 bands were successfully sequenced, representing 125 distinct sequences, a majority of which (58%) had sequence similarity to annotated genes. Three genes were validated by Northern blot, demonstrating that follicle stimulating hormone beta (FSHB) increased expression in the selection line, while both ferritin heavy chain and G-beta-like protein genes decreased expression relative to controls. The former finding is interesting, as FSHB has a direct role in increasing the development of mature ovarian follicles, and this clearly indicates FSHB may have been a direct target of the selection applied to this population 21.\nAnother useful approach to gene discovery is SSH. This approach uses hybridization of an excess of \u201cdriver\u201d cDNA (from one state\/treatment) to remove sequences in the \u201ctester\u201d cDNA (from the other state\/treatment) that are present in both populations in similar amounts. Highly differentially expressed sequences can be enriched for by preferential recovery of the resulting set of un-hybridized \u201ctester\u201d cDNAs. The tester and driver cDNA populations can be reversed, to allow screening for both up- and down-regulated genes. This technique was used by Narayanan and co-workers to 22 identify genes with altered abundance at different stages of the conversion of hepatocyte cells in culture into spheroids, an aggregate of cells that is functionally similar to liver tissue. This group identified 65 genes and 14 novel sequences and deposited 70 ESTs; demonstrating the down-regulation of cytochrome P450 family members as well as genes involved in heme biosynthesis, and the up-regulation of acute phase proteins and genes encoding proteins involved in Ca-dependent vesicle trafficking. Ross and co-workers 23 have used SSH to screen for genes differentially expressed during porcine conceptus elongation, a process important for efficient implantation in the pig. This group reported 142 sequences that changed expression levels during elongation; they deposited 16 distinct sequences to GenBank. Decreased expression during elongation was observed for several ribosomal RNAs, and an increase was seen for IL1B, thymosin beta 4, mitochondrial proteins, HSP70 and S-adenosyl homocysteine hydrolase (SAHH). The latter two genes, as well as an un-annotated transcript, were verified by QPCR. SAHH is part of the pathway to generate folate, an essential metabolite for embryogenesis, which is known to increase in the uterine lumen at the elongation stage of conceptus development 23. Other studies of reproductive biology have used SSH as well. Bonnet and colleagues 24 isolated transcripts responding to follicle stimulating hormone (FSH) treatment of granulose cells in culture. Sixty-four independent sequences predicted to respond to FSH were further analyzed and 25 transcripts were confirmed by Northern or QPCR to be regulated by FSH. Annotation of these 25 genes shows FSH may alter pathways controlling peroxidase activity and chromatin remodeling 24. To study the mechanism of heterosis, Xu and collaborators 25 exploited SSH to identify genes expressed differently in Longissimus dorsi tissue between Yorkshire dams and their Landrace x Yorkshire offspring. The HUMMLC2B gene was identified and confirmed by semi-quantitative PCR methods to be higher in the dam compared to her cross-bred offspring. The HUMMLC2B gene was expressed equally throughout muscle development (fetal to 6 month old samples were tested) and expression was found primarily in skeletal muscle tissue. As HUMMLC2B has been implicated in Ca+2 dependent signaling pathways, and a down-regulation was seen in the F1 hybrid offspring, these results may indicate Ca signaling influences porcine heterosis traits 25.\nFinally, researchers screening for genes responding to Salmonella enterica serotype Choleraesuis (SC) inoculation have reported the use of SSH to identify 88 sequences that change expression at 24-48 hours post-inoculation in mesenteric lymph nodes 26. Seven SSH-identified and two related genes were selected for further study and all were verified by quantitative real-time PCR. The differential expression of these genes indicate that modulation of cytoskeletal components and heat shock pathways are involved in the host immune response to SC in the porcine mesenteric lymph tissues 26.\nQuantitative PCR Methods as the \u201cGold\u201d Standard in Confirming Differential Expression Results\nThere have been many reports of quantitative real-time PCR (QPCR) being used to measure the abundance of porcine transcripts. Many recent publications focus on a small number of genes in the context of verifying differential display results 27, 28, as well as microarray or other profiling data (see specific results in the following microarray section), as QPCR has been the standard technique used to validate such results. This comparison is not as direct as one might wish, as properly run QPCR measures the abundance of a single transcript or transcript segment. Thus microarray data, which may result form hybridization of multiple transcript isoforms to a probe or probes on an array, may not be confirmed by a QPCR assay even though the microarray data is not necessarily incorrect. In fact, some microarray-based technologies, such as the Affymetrix platform, have multiple oligonucleotides that interrogate multiple locations along a gene transcript. Thus data may be available to identify differential abundance of different segments of transcripts, allowing measurement of differential expression of splice variants. On the other hand, QPCR is clearly more sensitive and often shows an increase in the level of difference (fold change) between the class or treatments being compared than seen in microarray data. It is therefore important to recognize both the strengths and weaknesses of each of these techniques in such confirmation studies.\nQPCR is traditionally not viewed as a high-throughput screening tool, due to lack of a large sets of specific assays available for porcine transcripts as well as the relatively high cost per gene and biological sample assayed. Recently, however, a large number of validated QPCR assays have become publicly available (http:\/\/www.ars.usda.gov\/Services\/docs.htm?docid=6065) through the efforts of a collaborative group headed by Harry Dawson and Joan Lunney at USDA-ARS-Beltsville. This group has developed and tested 474 QPCR assays based on the Taqman fluorescent dual-labeled probe technology (PIN database v. 3.7 as of October 2006; H. Dawson, personal communication). These assays focus on genes in immune and nutrition\/metabolic pathways, but include assays for many signaling molecule of general interest. Dawson, Lunney and co-workers have used such high-throughput QPCR to assay expression of many genes responding to pathogenic parasites 29, PRRSV vaccination 30, and have contributed nearly 150 QPCR assays to recent microarray confirmation studies by our group [31, 32, Wang et al., submitted] described below. Other groups have used a significant number of QPCR assays to validate microarray data, including Passerini et al. 33 (27 genes); Ponsuksili et al. 34 (10 genes); and Okomo-Adhiambo et al. 35 (10 genes); these papers are discussed in the microarray section.\nThe QPCR technique can, of course, be used to measure gene expression independently of microarray confirmation. Examples where 4 or more genes were quantitatively assayed with or without microarray data include measurement of response to LPS 36, to infection 26, 29, 37, 38, or to islet graft rejection 39; measurement of promoter activity in vitro 40, 41; assessing expression of gene family transcript isoforms 42; measuring responses during parthenogenesis 43, 44 or oocyte\/conceptus development 23, 45; brain response to early weaning\/isolation 46, 47; multi-tissue response to carnitine treatment 48 or liver response to dietary treatments 28, 49; differences across specific muscle types 50; and differences between stented and unstented arteries 51.\nMicroarray Hybridization Studies have Dramatically Expanded our Knowledge of the Porcine Transcriptome\nThe use of miniaturized arrays of individual gene sequences to survey or \u201cprofile\u201d the expression levels of hundreds (later; thousands) of transcripts within a cell or tissue was first published by Schena et al. 52, although reports of larger dimension element arrays (dot blots, etc.) had already been published, including early work on porcine muscle EST expression patterns 3. The main technological advances by Schena and colleagues were 1) the use of robotics to place very small amounts of gene sequences very closely together, minimizing reagent use and allowing the use of standard size glass microscope slides as the solid substrate, and 2) the independent labeling, by fluorescent substrates, of the of the RNA samples to be compared, allowing for simultaneous measurement of hybridization of labeled cDNA to each probe on the array. Initially, the criteria for finding DE genes in the comparison at hand was limited to an intuitive but statistically unsupported two-fold difference in fluorescence levels. Later, additional experimental designs and data analyses became statistically rigorous. Such analytical methods have become a significant field unto themselves 53.\nMuch of the data and publications described below are in the very early stages of understanding RNA expression profiles, as the field of transcriptional profiling is relatively new, and especially so for work in the livestock species. Many early papers were limited in scope, with relatively few elements and scarce biological replication, which is critical for adequate statistical rigor. Initial insights as to new aspects of tissue and cellular function have been gleaned from pig microarray data, and connections between datasets are made where possible and appropriate. However, an increased depth of data as well as more comprehensive tools will be required to fully comprehend and integrate the massive amounts of data already collected across many different tissues, systems and perturbations. Different platforms for porcine expression profiling have been used; these are listed in Supplementary Table 2 and briefly described below. Most of this work used two-color analyses, labeling RNA samples using fluorescent Cy3 or Cy5 conjugated nucleotides and performing a dye-swap, in which samples are labeled with each dye and used in separate hybridizations to eliminate dye effects. Cy3 and Cy5 have different excitation\/emission wavelengths, allowing for independent collection of hybridization data from each sample on the same array element. Further, most work described below used a normalization method called LOWESS, (Locally Weighted Scatterplot Smoothing), which corrects for intensity-dependent variation in data. These methods will serve as the default in the discussions below and are not described; however, if some other system and analysis is used, it is mentioned below. Statistical methods to identify DE genes across treatments vary, although ANOVA methods and corrections for multiple testing using false discovery rate calculations appear to becoming the standard. As these methods greatly affect the results, they are noted in the descriptions of experiments. Finally, annotations of DNA elements on the array are an important and growing area of research. Such annotations are most often based on sequence similarity to genes whose function is known or has been inferred, in turn, from other gene with similar sequence in another species. Functional annotations can also be obtained from curation of available biological data as complied for genes and pathways at NCBI (http:\/\/www.ncbi.nlm.nih.gov\/Entrez\/query.fcgi?db=gene) or at Mouse Genome Informatics; http:\/\/www.informatics.jax.org\/). At the Gene Ontology (GO) Consortium site (http:\/\/www. geneontology.org\/), annotation of a gene's function, where in the cell it performs this function, and its involvement in specific biological processes, can be found. Software to assign GO annotation to gene sequences, such as the DAVID software at NIH (http:\/\/david.abcc.ncifcrf.gov\/), are very useful and many are freely available. Such functional annotation can be especially helpful in annotating a set of gene sequences that respond similarly to specific treatments; such sequence groups or \u201cclusters\u201d can be identified using a number of clustering algorithms; see the review by Quackenbush 53 for a discussion of current work in this area. In the database section below, we discuss some of the methods being used to evaluate the biological knowledge available on specific genes and groups of genes that are found to be co-expressed. Specific methods used by investigators are also noted in the descriptions below.\nEvery effort was made to include work published that used microarrays to determine porcine expression profiles. Seven different groups also graciously provided summary details of fourteen different unpublished microarray projects (see Supplementary Table 2). These unpublished projects include studies on reproduction (5); muscle traits (3); genetic effects on expression (2), and host response to infection (2), antibody treatment (1), or stress (1).\n3. Current Expression Profiling Results in Porcine Tissues and Cells using Microarrays\nMuscle Expression Profiling\nMicroarray studies have been used in several species to better understand the changes in gene expression during livestock muscle growth and development 54, and the pig is no exception. One of the first large-scale profiles of porcine skeletal muscle was published by Moody and colleagues 55. They used a nylon membrane spotted with human cDNA fragments and hybridized this membrane with human and porcine skeletal muscle cDNA. They found that approximately 48% of the spots provided no signal; however, they also established that the hybridization signals were reproducible within sample for both human and pig RNA, and that concordance of results between species high. As this was a test of cross-hybridization, no expression contrasts were reported, but the authors concluded that cross-hybridization appeared promising 55.\nAnother group has used a cross-species approach to investigate porcine skeletal muscle gene expression. Lin and Hsu 56 used a human cDNA microarray to identify genes differentially expressed (DE) between Duroc and Taoyuan Longissimus dorsi (LD) tissue. They identified 6,400 DE genes with signal 2.5 fold higher that background for both breeds. They used correlation analysis to show a high level of reproducibility as had been found by Moody et al. Among these expressed genes, 117 were found to differ between breeds (p < 0.05), less that one would find by chance (~ 460 genes). Nevertheless, of the eight genes selected for verification by QPCR, all were confirmed. The majority of DE genes had higher expression in Duroc pigs, including genes for ribosomal proteins, heat shock proteins and myofibrillar proteins, and genes involved in transcription\/translation and metabolism-related genes. The authors indicated that the higher proportion of proteins involved in myofibrillar structure in DE genes in Duroc over Taoyuan could explain Duroc higher muscle growth. They further showed that SLIM1, a known myogenesis control factor, was over-expressed in Duroc, providing further clues as to the increased muscle growth rate in Duroc 56.\nA different pioneering approach for skeletal muscle profiling was reported by Bai and others 50, where a porcine microarray, consisting of un-sequenced cDNA clones from fetal and neonatal LD tissue libraries, was used for expression profiling of LD (as a white muscle type) as compared to psoas muscle (as a red muscle type). Genes identified as having muscle-type specific expression were defined as those having a normalized psoas\/LD ratio of > 2.0 or < 0.7, and these cDNAs were then sequenced and annotated. Genes with a high psoas\/LD ration (70 clones) included 16S and 18S ribosomal RNAs and NADH dehydrogenase subunits 3 and 6, fructose-1,6 bisphosphatase and members of the casein kinase 2 complex or targets of that pathway. Many genes highly expressed in LD (50%) included many of the fast isoforms of muscle fiber proteins and about 25% included genes involved in glycolysis such as GAPDH. In the last 25%, the authors highlighted the tumor suppressor gene, bin1, as it has been linked to myoblast differentiation in culture. Four genes predicted to be DE (GAPDH, bin1, MyHC2b and one novel gene) were tested by QPCR and all were confirmed, although bin1 was higher in LD in only 3 or 4 pigs tested. The authors propose that this method of arraying non-sequenced clones is useful in finding genes DE in species with little genome characterization 50. However, as this lack of information eases, this advantage becomes less important and the inefficiencies on spotting duplicate unknown cDNAs become significant. Members of this group used the same array to investigate the molecular changes in response to dietary restriction (20% less protein, 7% less energy, termed the LPE diet) in porcine LD and psoas muscles 57. In both muscle types, the LPE diet resulted in higher intra-muscular fat and caused twice as many genes to show higher expression as compared to the control diet treatment. The authors sequenced these DE genes and identified genes involved in turnover of protein, fat and carbohydrate, as well as genes involved in growth, mitochondrial function, translation and glycolysis. QPCR analysis verified the microarray data for several genes and indicated several systems were involved: the increase in intramuscular lipid (phytanoyl-CoA- hydroxylase and delta 9 desaturase); energy (creatinine kinase); fiber type specificity (MyHC2a, 2b); and muscle growth (cbl-b, kc2725, P311). The P311 gene, increased by LPE diet in both muscle types, is interesting as the authors further showed that P311 was increased during C2C12 myoblast differentiation 57.\nThe use of sequenced porcine ESTs to create an expression profiling tool was first published by Zhao and others 58. This group initially arrayed known ESTs and tested for detectable hybridization signal using muscle RNA from two fetal ages [day (d) 75 and d105] and two postnatal ages (1 week and 7 week) as targets. The ESTs that provided a clear signal were re-arrayed to generate the test array, which was then used to profile the fetal (d75) and postnatal (1 week) targets. A mixed linear model was employed to identify 28 genes with stage-specific expression (defined as two fold difference in expression between stages with P < 0.01). Of these, 19 genes matched known human genes, and included elongation factor 1 alpha, a number of ribosomal proteins (12), GAPDH, and structural proteins such as vimentin and tubulin. Five of these genes were tested and confirmed by Northern blot analysis of RNA representing seven stages of muscle development, from d45 to 7 week postnatal. The results with intermediate filament (IF) protein family members vimentin and desmin (tested as an additional control) confirmed protein-level data 59 that vimentin expression dramatically decreases during prenatal muscle development while muscle-specific IF protein desmin increases. The authors conclude their membrane and radioactive-labeling approach is a low cost and accurate expression profiling method to find DE genes of biological interest 58.\nTwo papers that appeared recently studied myogenesis by using an array containing cDNAs selected for their roles in myogenesis, energy metabolism, and myofibrillar structure, as well as additional skeletal muscle ESTs. The first paper 60 reported the expression pattern for these genes during seven stages of gestation, d14 to d91, covering the two stages of myogenesis in the pig (d30-60 and d54-90). Taking advantage of known annotations, genes were divided into functional groups for analysis. Genes both stimulating and inhibiting muscle differentiation as well as muscle structural genes had a peak of expression at d35, and declined thereafter. Genes in the glycolysis pathway decreased early then had a peak of expression at d77. The number of genes showing expression from d14 to d91 increased for the myofibrillar group, while the number of expressing myogenesis-affecting genes and differentiation-stimulating genes decreased from d14 to d49, then increased from d49 to d91. The expression pattern of five genes presenting the major annotation groups (myogenesis: EPO-receptor, beta-catenin, and TGF beta2; energy metabolism: GAPDH; and structural: COL3A1) was tested and confirmed by QPCR, although statistical analysis of these data was not described 60. In a second paper, Cagnazzo and collaborators used the same microarray and tissue sampling to compare expression between the Duroc and Pietrain breeds 61. The authors found that energy metabolism genes were consistently higher in Pietrain over Duroc for all ages except d35 in which this ratio was reversed. On the other hand, fatty acid metabolism genes had the opposite profile: higher levels in Duroc from d14 to d49, then higher in Pietrain at later ages. They found myogenesis apparently initiated earlier in Duroc pigs, as the expression of proliferation and differentiation genes was higher at d14 and d35 in this breed; this was reversed at later stages. Muscle structural genes also showed higher expression in early Duroc embryos, and after d49 expression was higher in Pietrain samples. The same five genes as assayed in te Pas et al. 60 were tested by QPCR and all results were consistent with the microarray data, although, as for the te Pas paper, no statistical analysis was discussed. The authors conclude that, as a delay in fiber formation has been associated with higher fiber numbers in other species, their finding that Pietrain myogenesis is delayed relative to Duroc may explain the Pietrain higher adult muscle mass 61.\nTwo reports have been published looking at porcine heart gene expression as a model for human disease. Lahmers and co-workers 62 used a novel array consisting of 50 mer oligonucleotides representing each of the 363 exons of the enormous human titin gene. They investigated rabbit, mouse, rat, and pig myocardial gene and protein expression from adult left ventricular tissue. In preliminary validation using pig soleus adult tissue, they found that 92% of the exons expressed in human soleus were also expressed in the corresponding pig muscle. For pig myocardium, the authors reported ~20 exons were fetal-specific and these were very similar to human fetal-specific exons 62. In the second report, Lai et al. 63 used a human cDNA microarray to determine genes whose expression changes during atrial fibrillation. Nearly 500 genes showed DE, with genes annotated in cell signaling and cell communication over-represented, while the second largest group were genes involved in gene regulation. A single gene, MLC-2V, was tested and confirmed by QPCR. The authors indicate that many of these genes had not previously been associated with atrial fibrillation, and thus further study on these genes is warranted 63.\nReproductive Tissue Expression Profiling\nA number of papers have been published in the past two years on the use of microarray to study reproductive tissues, primarily the ovary (3) uterus (1) and testis (1), but also on the developing embryo\/conceptus (2). As described above in the DD section, Gladney and colleagues 20 have studied the expression profile in ovarian follicles collected from animals selected for improved litter size and in a random-bred control population. In addition to DD, microarray analysis of pooled follicles from these two populations was performed by using two different Incyte UniGEM human cDNA microarrays. Based on two-fold differences only, they identified 33 and 21 DE genes using these microarray platforms, and found little overlap in the two lists of genes. Northern blot analyses of three genes (follistatin (FST); early growth response 1, nuclear family receptor 4A1(NR4A1)) confirmed the microarray results for FST and NR4A1 20. This group has built on this early work, using a porcine cDNA microarray to profile the expression pattern of ovary and follicle tissue from the select and control lines of pigs 64. A mixed-model analysis identified 72 ovary and 59 follicle genes DE between these two lines. In contrast to the data obtained with the human microarrays, 32 genes (about half of the total) were found DE in both tissues, providing a measure of confidence in these results. Northern hybridization using probes for calpain light subunit I and cytochrome p450scc (P450scc) genes were used to confirm the microarray data for both genes. Overall, the lists of DE genes contained both genes known to be expressed during folliculogenesis as well as genes not previously associated with this process. The authors highlighted DE genes involved in steroid biosynthesis (Collagen type I receptor, P450scc, STAR, 3betaHSD, CYP17 and CYP19) and tissue remodeling (PAI1) that could provide mechanistic clues for the difference in ovulation rate seen between these lines, and they indicate further ontogenic studies of these candidates would be helpful in such interpretations 64.\nGene profiling was used successfully to study the process of luteinization by comparing pre-estrous preovulatory ovarian follicles to luteinized follicles on day 2 of estrous prior to ovulation 65. A custom porcine cDNA microarray was created from clones selected from the ovary EST library project described above 9. Microarray data collected on these two types of follicle was produced using a reference design; the reference was an RNA mixture from all follicular stages, and data normalization and the Welsh t-test were used to identify 150 DE genes (P < 0.01) between follicle stages. The authors reported the major classifications for the 107 genes decreased from pre-estrous to luteinized post-estrous follicles were cytoskeletal structural and regulatory proteins, chromatin component and nucleic acid-binding proteins, metabolic enzymes, oxidative response proteins, cAMP receptor pathways proteins, and cell proliferation\/differentiation pathways proteins. For the 43 genes increased in the same comparison, the major annotation groups were cell adhesion; migration, growth inhibition; and angiogenesis. Some functional categories were observed in both lists, including different genes involved in steroidogenesis, proteolysis and metabolism. In validation work, four genes (CYP17A1, 3beta-HSD, LHCGR, and PLANH1) were tested by QPCR and results showed similar patterns to the microarray data, although statistical evidence was not discussed. The authors note that many genes, whose function and expression patterns were known previously to be involved in folliculogenesis, were identified in this analysis and this further validates their results. Many of the identified genes were novel with respect to luteinization, however, and are interesting candidates for future study of this process 65.\nTwo manuscripts have been published on expression profiling in the developing porcine embryo. Lee et al. 66 used a custom cDNA membrane array to initiate microarray studies on the elongating, peri-implantation embryo, a morphological transition critical for successful implantation. Concepti from four stages of this transition (small spherical, large spherical, tubular and filamentous forms) were collected and profiled using the above array. The minimal quantities of embryo tissue available required an amplification step; preliminary hybridizations with un-amplified and amplified material indicated amplification did not introduce bias. Nine genes were declared as DE using three criteria. Two genes met the stringent Bonferonni test for multiple testing, four genes were DE at p <0.001, while the remaining three genes had p < 0.01 and at least a two-fold difference in expression between stages. Four genes covering these three categories (STAR, TGFbeta3, interleukin 1 beta (IL1B), and thymosin beta 10) were selected for QPCR confirmation and all four showed statistically significant differential expression in both QPCR and hybridization data. The IL1B results confirm reported SSH 23 and EST frequency data 7, 8 and the thymosin beta 10 results agreed with data reported by Smith et al. 7. Further, the STAR gene has been shown to increase in expression by SAGE data 14 and by follow-up Northern, QPCR and protein level analysis by the same group 15. These confirmations verify the results of the Lee et al. study 66, which contributed additional genes for understanding of the rapid transition spherical to filamentous conceptus which is nearly unique to the pig.\nWhitworth and others 67 developed a custom cDNA array containing genes collected from cDNA libraries from ovary, embryo, oocyte, oviduct, uterus, conceptus and fetus, to determine genes DE across several stages and modes of embryogenesis. These included in vivo produced germinal vesicle oocytes (pgvo), four cell embryos (p4civv) and blastocysts (pblivv), as well as in vitro produced four cell embryos (p4civp) and blastocysts (pblivp). An ontogenic study from oocyte to blastocysts (all in vivo samples) showed ~ 2,000 to 4,500 DE genes. A comparison of in vivo to in vitro embryos identified 1,409 (p4civv versus p4civp) and 1,696 (pblivv versus pblivp) DE genes. False discovery calculations for the latter two comparisons eliminated nearly all of the DE genes, and a condition tree clustering did not show evidence that in vitro expression patterns could be easily distinguished from the in vivo patterns. In the ontogeny study results, similar clustering showed each stage correctly clustered together and that, as expected, the two earlier stages clustered more closely together. QPCR validation of seven genes from these comparisons was performed and of the 21 pair-wise comparisons available in the microarray data for these seven genes, 20 were confirmed by QPCR. The authors found that genes induced in the transition from germinal vesicle to four cell embryo were similar to those reported for the same transition in the mouse, and included nuclear structural and functional proteins, while genes decreased during this transition included those involved in cell adhesion receptor activity, mitotic cell cycle, transporter activity and M-phase microtubule activity. Genes increased in blastocyst as compared to 4 cell embryos included ribosome, hydrogen ion transport and cation transport activities. Many examples were discussed showing much of this data is similar to that reported in the mouse, although many differences between pig and mouse profiles were observed. The authors correctly note that this analysis is just the beginning of understanding the transcriptional program during early embryogenesis 67.\nThis group has also used this array to investigate gene expression changes during the estrous cycle and pregnancy in the porcine endometrium 68. They collected RNA at seven times during the estrous cycle (0, 3, 6, 10, 12, 14, and 18 days post-estrous), and used a reference design to identify genes DE across the estrous cycle. The reference RNA consisted of a mixture of RNA from the tissues used to make the array, see above. Genes were identified as DE using ANOVA with Benjamini-Hochberg correction to control for multiple testing, with the added criteria that the gene must be at least twice as abundant as seen in the reference. The number of DE genes varied by day of estrous, with a minimum of 118 genes on day 0 and a maximum of 542 genes on day 12. Clustering of these genes using k-means methods identified six main patterns of expression across the estrous cycle. Several of these patterns (i.e., DE on day 0; DE on days 3 and 6; DE on days 10-14) are coincident with specific known functions of the endometrium: a) sperm maturation; b) blastocyst growth and position; c) conceptus development and attachment, respectively. They also used EASE software to connect GO terms to these clusters, where, for example, in the day 0 cluster immune cell markers and cytokine genes predominated; within the day 10-14 cluster, many DE genes were annotated with tyrosine receptor kinase activity function. Using cluster and EASE results as a guide, seven genes were selected for QPCR based validation; all resulting QPCR data was consistent with the microarray results, although statistical significance of the QPCR results was not discussed. The authors conclude that these data on the global expression patterns with the cycling uterus will guide transgenetic and cell transfer approaches to improve reproduction efficiency 68.\nFinally, Stewart and colleagues have reported 69 the use of a human cDNA microarray to assess testicular expression patterns in boars differing in steroidogenesis levels. The RNA from animals (n=4) with known high plasma estrone was directly compared to RNA from low plasma estrone boars in paired hybridizations. Genes with statistically significant differences between the two states were identified using Student's t-test and Benjamini-Hochberg correction for multiple testing. Seven genes were found by this method to be more highly expressed in the high estrone boars (p< 0.05). Five of the genes tested were verified as DE using QPCR, and included CyB5, Cyp19A1, SAT, FTL, and DNASE1L1; the authors note that the fold changes were much higher than those observed in the microarray data 69.\nImmune Response Expression Profiling\nThe immune response is highly complex, with multiple tissues and cell types communicating information that is contextual and changes during early inflammatory stages as well as during inflammatory resolution and the adaptive immune response, and changes can also be long-term through immune memory. Hammamieh et al. 70 have developed useful data on variability among pigs within peripheral blood mononuclear cells (PBMC). Using a human cDNA membrane array, they measured the PBMC expression profiles of ten Yorkshire piglets. They found 19 DE genes, which were annotated as stress response, immune response, and genes involved in transcription. This group then used this same array in a follow-up paper comparing expression profiling of human PMBC exposed in vitro to staphylococcal enterotoxin B (SEB) to profiling data of PBMC from piglets intoxicated with SEB to LD95 in vivo. Using a supervised learning procedure for class prediction to compare these two datasets and by using a separate training dataset of gene expression profiles of human PBMC infected with eight separate pathogens, they were able to identify a set of 16 genes predictive for SEB exposure 71.\nA separate group has also reported 72 the development of tools for assaying gene expression responses in immune cells. This group selected 20 cytokines, 11 chemokines and 12 receptors relevant to immune response, and developed a cDNA array containing these genes. They then validated the resulting array by interrogating RNAs isolated from control or phorbol ester\/ionomycin stimulated PBMC from pigs. Expression patterns obtained through hybridization of radioactively labeled cDNAs to the array followed closely the expected expression patterns published previously for most of these genes, indicating this focused immune array can be used for profiling the porcine immune response 72.\nAfonso and co-workers 73 have used a cDNA microarray constructed from a swine macrophage library to compare expression profiles from macrophage cultures infected with two forms of African swine fever virus (ASFV). One infection used a parental ASFV (Pr4) while the alternate infection used a mutant virus (Pr4delta35) deficient in two genes that control growth of ASFV in macrophages. RNA was collected at 3 and 6 hours post infection (hpi), and t-test analysis of the 3 hpi hybridization data indicated a total of 38 genes were found to be up-regulated 2 fold or more in the Pr4delta35 infections as compared to the Pr4 infections, while 133 genes were down-regulated. The up-regulated genes, responding only to the gene-deleted virus, included many interferon-regulated genes. Control experiments with mock-infected cultures, as well as Northern blots and or QPCR of 14 test genes, showed this data to be reliable. Interferon regulation of response to Pr4delta35 virus was further confirmed by the expression profile observed for an inhibitor of IFN-induced antiviral protein, which was decreased in Pr4delta35 infected cells 73.\nAnother analysis of blood cell response to infection has been published by Moser et al. 74. This group is interested in finding genes that may control susceptibility to bacterial disease. A microarray containing cDNAs from ten immune cell types and tissues was used to interrogate expression in peripheral blood leukocytes isolated from the two most extreme responders to an Actinobacillis pleuropneumoniae (A.p.) infection. The RNA was collected from all 18 pigs in the infection study just before (T=0) and 24 hpi (T=24). The experimental design was a reference design and used either uninfected leukocyte RNA or a 1:2 mixture of muscle and leukocyte RNA. A sophisticated mixed model analysis was used to identify DE genes from T=0 to T=24 for each pig. The authors reported a list of 128 genes was decreased during infection in the resistant animal and increased in the susceptible animal, and a second cluster of 179 genes with the reverse pattern. The origin of the genes in the first cluster was predominately a cDNA library created by subtraction of liver expressed genes from genes expressed in lymphocytes, indicating an immune system origin. The cDNAs found in the second cluster primarily arose from a library created from A.p. infected leukocyte RNA subtracted by uninfected leukocytes. No specific genes were mentioned; however, the apparent immune response indicated genes relevant to this infection were identified 74.\nSeveral papers have been recently published that use microarray analysis to investigate the transcript profile of the intestine and associated lymph tissue. Dvorak and colleagues 75 describe the generation of over 3,000 ESTs from a cDNA library of Peyer's Patch, a mucosal tissue of the intestine, and the development of a custom cDNA microarray from these clones. The microarray was used to assess expression across total library cRNA from un-stimulated Peyer's Patch tissue compared to stimulated Peyer's Patch total cRNA. The latter cRNA was a pool of RNAs from cell cultures stimulated for 3 hours in tissue culture chambers with SC, lipopolysaccaride + cholera toxin, or Phorbol ester + homocyclic AMP + concanavalin A. Additional preliminary hybridizations comparing the normal un-stimulated library, the stimulated library and the pooled and subtracted library were run in duplicate with dye swap. A number of genes were found to be differentially represented among these three libraries, indicating the pooling and subtractions was successful 75. This group then used this microarray to examine expression differences between juvenile Peyer's Patch (JPP) and adult Peyer's Patch tissue 76. The hybridization analyses used a reference design, with the reference being pooled mesenteric lymph nodes (MLN) from young pigs. The Student's t-test with Benjamini-Hochberg correction was used to identify DE genes in JPP compared to reference. Using as minimum fold change value of 1.5 (based on JPP:JPP self hybridization analysis) as an additional criteria, 24 genes were identified as DE, and all but 2 of these were more highly expressed in JPP compared to MLN. Hierarchical clustering of these DR genes across the four individual pig samples indicated significant variation among animals. Seven of these ESTs and thirteen additional non-differentially expressed ESTs were tested by QPCR in JPP, MLN and adult PP. Of 18 ESTs with QPCR data for JPP, there was a positive correlation (correlation coefficient = 0.48) between the JPP\/MLN rations for QPCR and microarray. Several genes (AECC, GW112, SPAI, PSP) had much higher rations in the QPCR data (100 to 1,000 fold) than seen in the microarray data. In QPCR comparison between JPP and adult PP, several genes were highlighted. Five genes, MARCKS, CIDE-B, GW112, PSP, and PROLI, were much more highly expressed in JPP as compared to adult PP, while MHCII-DR was more highly expressed in adult PP. The genes CIDE-B, GW112, and PSP are interesting as these are annotated as growth and apoptosis-regulating genes associated with responses to normal and\/or abnormal intestinal microflora 76.\nAdditional microarray-based analysis of the intestinal response to bacteria, in this case Escherichia coli and S. enterica serotype Typhimurium (ST), has recently been published by Niewold and collaborators 77, 78. In these reports, this group used a model of in vivo infection, called small intestinal segment perfusion (SISP). They created a cDNA array using jejunal intestine ESTs and have used this array to measure expression in jejunal tissue after infection with an enterotoxigenic E. coli (ETEC) strain, or mock-infected pigs 77. Fifteen genes were declared DE when the fold change in expression between uninfected and infected tissue (within a SISP loop in the same animal) was >4 or <-4 and the false discovery rate, as calculated by significance of microarray, was < 0.02. One of these, PAP, was thirty-fold higher in infected tissue; the other genes were not identified. The PAP expression pattern was confirmed by Northern blot analysis 77. In a second report 78 using the same array and SISP technique, the same group looked at the response to ST. Jejunal RNA collected from several locations in the jejunum after mock-infected or ST infected jejunum at 2, 4 or 8 hpi was used in microarray hybridizations; they pooled tissue samples from 4 animals so unfortunately there was no biological replication. Spotfire software was used to find DE genes with fold differences <-1.58 or >1.58 and p < 0.025 relative to mock-infected jejunal RNA. Seven genes were found DE between either the 4 or 8 hpi samples as compared to mock-infected tissue. Three genes (MMP1, PAP, and STAT3) were DE at both time points, while IL8 and TM4SF20 were DE at only the 4 hpi time, while THO4 and an unknown EST were increased at 8 hpi only. No down-regulated genes were found. One gene (PAP) was tested using QPCR and was confirmed although the data was not shown. The most interesting result was that STAT3, known to function in an immunosuppressive pathway involving SOCS3, was up-regulated and thus could indicate resolution of inflammation was occurring by 4 hours. Alternatively, the authors suggest that immunosuppression by ST may also be occurring, and that such immunosuppression could be a reason for the very low numbers of identified DE genes by this model. They also acknowledge that, as they collected whole jejunal tissue, the DE gene signal may be swamped by the large number of other cell types in the tissue isolated 78.\nAnother microarray was recently used to study the transcriptional response to SC in the gut. Zhao and others 31 used a novel broad-coverage oligonucleotide array (further discussed below in the Tissue Expression Pattern section) to investigate changes in lung expression at 24 and 48 hpi relative to uninfected pigs. A loop design was used in array hybridizations to identify DE genes across these time points. Fifty-seven genes showed DE (P< 0.001, maximal FDR 27%) between uninfected and infected lung. Cluster analysis of these 57 genes showed that, of the 33 genes with annotation, 17 (52%) were related to immune response, apoptosis or tumorigenesis, clearly indicating that the array was useful in identifying relevant genes for this infection. Several genes showed dramatic increases in expression level relative to controls; 25 genes showed >4.5 fold greater expression at 48 hours. These included INDO, IRF1, HSPA6 (known immune response genes), and GBP1, GBP2 and GBP3 (a known interferon-inducible gene family). Interestingly, TGM1 and TGM3, members of a transglutaminase gene family with possible roles in apoptosis and\/or antigen processing, increased 31 fold and >1,000 fold, respectively. This gene family has not directly been implicated in the immune response to bacteria in any species, but the known role in apoptosis for TGM genes indicates this pathway is important in the response of the lung to infection. A large number of genes, 61, were chosen for QPCR for two purposes: a) to validate microarray expression patterns (33 genes) and b) to characterize more fully the transcript response to SC (28 genes). The QPCR confirmed 23 of the 33 DE genes tested, and identified six additional DE genes, validating the microarray data from this oligonucleotide array. Overall, the QPCR results showed a strong T helper 1 cell type response in the lung to Salmonella (exemplified by induction of IFNG, IL15, INDO, IRF1, SOCS1, TNF, and WARS). These results also demonstrated a strong apoptotic response (exemplified by TGM3, TNFRSF5, TNFSF6, and CASP1 induction) and an antigen processing response (exemplified by MHC2TA, PSMB8, TAP1, TAP2 induction). The classical complement pathway (C1s, C1r) and type 1 interferon pathway (GBP1, GBP2) were were also confirmed as strongly induced, while decrease of T helper 2 cell type response genes (IL4, TPS1, IL13) by 48 hpi was confirmed 31. These data clearly show the value of the microarray, as well as extensive QPCR analyses, to determine expression profiles during infection that inform us of the conserved and potentially pig-specific biological pathways involved.\nA first generation Affymetrix porcine GeneChip\u00ae with probe sets to assay over 23,000 transcripts, was recently used to study host mesenteric lymph nodes (MLN) transcriptional response to ST [Wang et al., submitted]. Animals were infected with ST and tissues collected after 8 hpi, 24 hpi, 48 hpi, or 21 days post-infection. RNA was used in standard Affymetrix analyses to produce profiling data analyzed by MAS5.0 and a mixed model ANOVA with false discovery rate control to identify genes DE across stages within infection. Results showed that 848 genes changed their MLN expression level across one or more pair-wise time-point comparisons in the ST infection (p<0.01, fold change >2, q <0.24); about 100-150 genes were found DE at each time relative to uninfected pigs. Of interest was the finding that, in contrast to the strong transcriptional response to SC seen in the lung by 48 hpi 31, a limited induction of genes at 24 hpi with ST was observed. In fact, from 24 hours to 48 hours post infection, many genes decreased their expression. To study this further, cluster analysis and analysis of specific pathways were used to reveal common expression patterns for sets of genes, and identify specific features of the host response to ST infection. In Figure 1, the cluster analysis of all genes found DE in the ST infection is shown; one particular cluster (#4) is highlighted. This cluster, the genes within which on average rose in expression by 24 hours and then decreased by 48 hours, contains a large number of cytokine genes and NFkB-dependent genes known to be involved in the inflammatory response. This result indicates that, in parallel to the clinical features of ST infection where inflammation is mild and peaks at 24 hpi, the NFkB pathways appears to be activated between 8 and 24 hpi, and then suppressed thereafter. The expression profile of 22 genes (seven of which are present in cluster #4 in Figure 1) was analyzed by Q-PCR, and 95% showed statistically significant confirmation of the expression pattern observed in the microarray data [Wang et al., submitted].\nToxoplasma gondii is a protozoan parasite that infects a significant portion of the world-wide human population. A secondary host reservoir for this parasite is the pig, and the host interaction between T. gondii and porcine cells was recently investigated using cDNA microarrays 35. The ESTs on the array were selected from immune libraries for likely involvement in immune response, and this selected set was used to assay expression in uninfected kidney epithelial cells (PK13) or after T. gondii infection at eight time points after infection (1 hour to 72 hours). Relative to uninfected cells, a total of 263 genes DE genes identified using Student's t-test were found to be induced, and 48 more were decreased, at one or more times after infections. The majority of the induced genes responded to infection within the first 4 hours, and 12 different functional classes of DE genes responded, including transcription and signaling, metabolism, immune response, cell cycle, and apoptosis. Eleven DE genes were tested and confirmed by QPCR. The authors concluded that this approach identified many classes of genes that will be useful candidates to study cell-mediated responses, especially apoptosis and NFkB-dependent pathways, to this parasitic infection 35.\nUsing Microarrays to Determine Tissue-Selective Gene Expression Patterns and Microarray Applications in Other Research Areas\nMost of the microarrays discussed above were developed to ask specific questions in biology. When tools for a specific tissue were not available, some researchers tested the utility of human arrays to profile porcine expression (55; see additional papers in Supplementary Table 2). While these cross-species tools can be successful, large-scale porcine-specific tools useful in studying a wide variety of biological questions are available. As discussed above in the infection section, a first-generation porcine oligonucleotide set, representing 13,297 cDNAs and ESTs with broad coverage across tissues, has been designed by Qiagen-Operon in collaboration with researchers in the Swine Sub-committee of the USDA-NRSP8 research project. Zhao and collaborators 32 validated the novel 70-base oligonucleotides on the array by hybridized with targets from porcine adult liver, lung, muscle, or small intestine. A loop design were utilized to collect transcriptome data for each tissue and to identify DE genes across tissues. Using available negative controls (average signal of five Arabidopsis gene oligonucleotides) to calculate a true background level, the large-scale transcriptome for each adult tissue (from 8,358 in muscle to 10,556 in lung) was established as those genes with greater than 3 fold background signal (q < 0.01). Using a criteria of P < 0.001 and q < 0.003, tissue-selective gene lists were produced, from a low of 147 genes in small intestine to a high of 405 genes in liver. Clustering results of the expressed genes identified a number of patterns across these four tissues that are useful in annotating the array ESTs. QPCR analysis of 11 selected genes across the four tissues was used to verify tissue expression and oligonucleotide specificity (multiple members of gene families tested) and showed statistically significant confirmation of all but 2 genes. These results demonstrated that this porcine oligonucleotide array is informative and the oligonucleotide specificity is high, thus the Qiagen-Operon\u2013NRSP8 porcine array can be used for porcine functional genomics analysis 32.\nA small number of papers have been published recently on explorations of porcine brain (2), liver (2), and adipose (1) tissues. Nobis and coauthors are interested in the expression pattern of genes in the porcine brain and have submitted ESTs from a brain cDNA library constructed from pooled brain regions 79. In addition, they constructed a brain cDNA microarray, and demonstrated with self-hybridizations of normal pooled brain cDNA (and appropriate statistical corrections) that the microarray can provide quality data when a fold cut-off of 2.0 is used to control false positives 79. Members of this group have also used this microarray to investigate frontal cortex expression profiles in early-weaned (EWC) pigs compared to nonweaned (NWC) pigs as well as weaned\/nonweaned pigs isolated socially (EWI, NWI) in a 2x2 factorial design 46. Using a replicate loop design to collect data for all comparisons, they found 103 DE genes using a modified t-test that accounts for multiple testing (P < 0.05, fold change > 1.25). Of these genes, they found 24 of 42 annotated genes had relevant brain functions. Six DE genes were selected for QPCR validation based on DE in specific contrasts, including social isolation (NWI vs. NWC; 14-3-3, CPE, and PEA-15), or social isolation in early weaned pigs (EWI vs. EWC; DBI and ARP2\/3) and early weaning in the presence of social isolation (EWI vs. NWI; OAZ2). The QPCR results confirmed the first five genes, and indicated that social isolation in either weaned or nonweaned pigs has effects on neuronal gene expression, but that they did not detect gene expression differences due to weaning alone. The authors postulate this latter result is potentially due to the small study size 46.\nA custom oligonucleotide microarray was used to study porcine adipose tissue and stromal\/vascular (SV) gene expression 80. The SV cultures were collected from three stages of growth and development (90-day and 102-day fetal stages, and 5-7 day neonatal stage), and adipose tissue was collected from 105 day fetal and 5-7 day neonatal stages. They identified expression above background for 200 genes in SV cultured cells and 160 genes in the adipose tissue samples. Many of the these genes have been reported to be expressed in adipose cells previously; however the authors highlighted eleven genes not previously so reported, including relaxin, chromogranins A and B, INSL3, FGF12, IGFBP7, GDF9B, BDNF, IL12, and APOR). Three genes were reported as more highly expressed in fetal SV cells as compared to neonatal adipose tissue (IL4, IFNG and IGFBP5) using a t-test of normalized spot intensities 80.\nSeveral papers have reported changes in liver gene expression under a number of treatments. To profile the response to fasting and to treatment with a peroxisome proliferator-activated receptor alpha (PPARG) ligand (Clofibric acid, CA), Cheon and collaborators 49 used a pig skeletal muscle EST array to measure the liver transcriptional profile in normal fed pigs compared to fasted or CA-treated pigs. Liver RNA was pooled within treatment thus no biological replication was available. Genes DE between treatments were identified by as criteria: a) P < 0.1, b) minimum fluorescent intensity of 1,000 units, and c) 2 fold or greater difference between treatments. In contrast to results from rodents, there was little evidence from the microarray data that fatty acid oxidation related genes were highly induced by either CA treatment or fasting. QPCR assays showed statistically significant increased expression for ABCD3, CAT, CYP4A, ACOX1, and EHHADH by CA treatment, and all but the last two genes were also up-regulated by fasting. The QPCR data showed glucokinase was decreased only in the fasting treatment, while glycogen synthase was increased by both treatments. In fatty acid metabolic pathways, both QPCR and microarray data showed that steroyl-CoA desaturase and fatty acid desaturase 1 were decreased by fasting, but these two genes, as well as fatty acid desaturase 2, were increased by CA treatment. The authors conclude that there are major species differences in the liver RNA response to peroxisome proliferator ligands like CA, although they provide additional evidence that the response to fasting, especially for mitochondrial fatty acid oxidation and ketogenic pathways, is more similar across species 49.\nA recently published study 34 combined microarray analysis, SNP detection within expression candidates, and association and physical mapping analyses to find liver genes affecting carcass traits. These authors intended to capture expression differences related to phenotypic traits (and minimize other genetic differences) by using discordant sib pair analyses. They identified sibs within an F2 population from a Duroc x German miniature pig cross that were distinctly different for eye muscle area and backfat thickness. To profile the liver expression in these sib pairs, the microarray contained well-annotated genes, including liver genes from earlier work 18, 19 described above, as well as genes known to be important in hepatic metabolism. The liver mRNA from 4 sib pairs (high performing (HP) and low performing (LP)) was pooled by trait, and hybridized to this array. Overall, nine genes were identified by t-test as DE at least two fold between HP and LP groups; four were higher in HP (TBG, PIGSPI, DBI, and SLC01B3) and five were lower (PLA2G6, CPS1, PTN, NAN1, and BHMT1). Ten genes, including six DE genes, were tested by QPCR on liver RNA from the eight individual pigs from the pooled samples. Although good agreement with the microarray data was seen for most genes, only four (TBG, SLC01B3, PEDF and APOH) were statistically different between HP and LP individuals. Two confirmed genes, TBG (higher in HP) and APO (higher in LP) were screened for SNPs by re-sequencing of cDNAs for all eight HP\/LP animals. Of three SNPs found in the TBG cDNA sequence, one (A>C at nt 778) was found associated (P < 0.0001) with variation in three fat traits, with the C allele associated with higher fat. As the SNP at nt 718 changes the amino acid at this position, the authors also checked TBG protein expression and function. Available serum data on TBG and TBG bound metabolite levels showed an association of TBG concentration (P < 0.12) and function (P < 0.06 to 0.11) with TBG genotype. Thus this group was able to use discordant sib pair microarray expression analysis to identify expression candidates, identify SNPs in one candidate that was associated with quantitative phenotypes for fatness and with relevant biochemical differences in serum. The combination of evidence at the genetic, expression and functional levels makes a strong case that TBG variation directly controls part of the variation in fatness in this population 34.\n4. But What Does It All Mean? Pig Expression Bioinformatics and Databases\nAt this juncture, available swine transcriptomic data, especially for microarray projects, is somewhat fragmented and sparse. Many different platforms are being used and the data is not always being submitted to a common repository. The recent public disclosure of nearly a million additional ESTs from 97 different non-normalized libraries by the Sino-Danish consortium (Gorodkin et al., submitted) will certainly improve the accuracy of EST frequency data as an estimate of expression level. To become more efficient at drawing biological meaning out of such data, more attention needs to be paid to public sharing of data and integration of that data so that an increase in power is possible. In this section, we discuss the available public resources for pig microarray and other transcriptomic data and discuss some of our efforts to integrate these platforms and data sources.\nThe following information does not include several storage\/analysis efforts on genomic data that includes Sus scrofa as one of the species. These include mapping databases such as ArkDB and databases that link traits to phenotypes without expression data, such as OMIA. Links to these and other sites of interest to pig genomics researchers can be found at the U.S. Pig Genome Coordination website (http:\/\/www.animalgenome.org\/pigs\/).\nTwo groups have databases available for both expression and sequence information. The National Center for Biotechnology Information (NCBI; http:\/\/www.ncbi.nlm.nih.gov) and the European Bioinformatics Institute (EBI; http:\/\/www.ebi.ac.uk) have multiple databases, each designed for a specific purpose. At NCBI, UniGene takes the sequence data in the general EST and nr databases and clusters them into single gene units by species (http:\/\/www.ncbi.nlm.nih.gov\/UniGene\/UGOrg.cgi?TAXID=9823). The expression database at NCBI is Gene Expression Omnibus (GEO; http:\/\/www.ncbi.nlm.nih.gov\/geo\/), which uses a specific format for loading and exporting data. As of October 2006, GEO currently includes 18 porcine experiments, and the platform designs for 19 platforms, including the two large commercial platforms: Affymetrix 24k Porcine GeneChip\u00ae and several versions of spotted platforms using the Qiagen-Operon-NRSP8 13k oligonucleotide set. Of the 18 experiments, the majority were performed on microarrays, though three used SAGE. The user interface at NCBI for text or BLAST-based searches is advanced, allowing searches to be refined to search specific sub-databases or with other limits.\nIn contrast, at EBI all the sequences are in a central repository and there is no species-specific clustering database as at NCBI\/UniGene. The expression database at EBI is called ArrayExpress (http:\/\/www.ebi.ac.uk\/arrayexpress\/), and uses the Minimal Information About Microarray Experiments (MIAME) standards created by the Microarray Gene Expression Data Society (MGED). ArrayExpress, as of October 23, 2006, has a single porcine expression study, the main bulk of the data being human and mouse.\nAnother comprehensive EST database is the Dana Farber Cancer Institute Computational Biology and Functional Genomics Laboratory's Pig Gene Index (PGI; http:\/\/compbio.dfci.harvard.edu\/tgi\/). The PGI database is the TIGR Gene Indices information that was brought to Dana Farber by John Quackenbush. The PGI database is similar to UniGene in that it clusters ESTs together, but goes a step further and provides a tentative consensus sequence for each cluster. Also, unlike UniGene which tries to group alternative spliced genes together, PGI attempts to separate them into their own, unique clusters. However, it does provide a link that clusters the tentative alternatively spliced sequences together. The DF database also provides Gene Ontology (GO) annotations, metabolic pathway information, and predicted 70-mer oligonucleotide sequences and SNP information for each of their TCs. Both UniGene and DFGIP data collections can be searched using BLAST, and an expression summary of each of the EST's based on the libraries, and EST count within the libraries, is provided. Both data collections can also be downloaded for local investigator use.\nRecently the Sino-Danish group has begun to release their EST sequence information, including alignments into clusters, which is based on over one million ESTs (http:\/\/pigest.kvl.dk\/index.html). They have submitted their sequences to the Trace Archive at NCBI, but these data have not, as of October 2006, been integrated into dbEST. They have made their clusters available online, and have provided a bulk download website for both the ESTs and the consensus sequences of their clusters. Expression profiles for all the genes are also provided, based on EST frequency within the 97 non-normalized libraries. They predict SNPs based on sequence discrepancies within alignments, as the libraries were created from tissues from multiple breeds. They provide some online tools, including viewing the aligned and clustered sequences as well as searches against the database using BLAST. The website indicates these data have been submitted for publication (Gorodkin et al., submitted).\nThere are non-comprehensive EST databases, which focus on a select number of tissues. Among these is the Michigan State Center for Animal Functional Genomics (CAFG; http:\/\/gowhite.ans.msu.edu\/public_php\/showPage.php). This database contains tissue specific porcine EST libraries: brain, adipose, and skeletal muscle, the use of which was discussed above in the microarray section 12, 46, 79. It also contains a mixed tissue porcine library. To annotate the ESTs, the database uses homology searches to various NCBI databases, including RefSeq and Gene. To search the libraries, keywords based on this information transferred from GenBank can be searched; there is also an option to use BLAST to search the different libraries. Clones and clusters can be viewed to determine which libraries have sequences donating to the clusters, and clones are available for order. In addition to the EST information, there is a private internal section for microarrays.\nAnother focused database is the Pig Expression Data Explorer (PEDE; http:\/\/pede.dna.affrc.go.jp\/). PEDE also uses homologues in RefSeq and UniGene to annotate the EST sequences that primarily are derived from tissues of interest to porcine immunology research (81 and references therein). They identified the full-length cDNAs within the ESTs. Their online interface allows a user to search their EST\/cDNA clusters using keywords, library, their accession numbers, and the corresponding human chromosome. It allows filtering the results based on evidence of SNP in a specific breed, and provides suggested primers for detection of the SNP. Like the Sino-Danish EST database and the PGI database, it provides a visual alignment of the ESTs to each other and to the cluster consensus sequence. A unique feature of this database is a more in-depth analysis of artiodactyl-specific antigens for furthering the development of xenotransplantation.\nSeveral databases are also in production at the moment, and at various stages of development. One of these is under development at the University of Minnesota (http:\/\/gnomix.ansci.umn.edu\/bioinf.htm). Their database focuses on sequence data currently, though they indicate they plan on integrating expression data in the future, and currently have annotation provided for the Qiagen-Operon-NRSP8 13K array. Most recently a new database annotating the new Swine Protein Annotated oligonucleotide Microarray (SPAM) has become available (http:\/\/www.pigoligoarray.org\/). The database describes the set of 18,254 oligonucleotide sequences in the SPAM, and provided the best Ensembl protein matches and Gene Ontology (GO) annotations for each sequence. Another database is being developed at the Advanced Food and Materials in Canada (http:\/\/www.afmnet.ca\/index.php?fa=Research.myProject&project_id=77&page=1). Their aim is to integrate various types of expression data, including microarray, proteomics, and metabolomics, to compare genetically modified foods. Their first goal is to compare the EnviroPig to the Yorkshire.\nA fourth database currently under construction is at Iowa State University (URL pending). Our focus is storage and analysis of data from the Affymetrix platform, although Qiagen-Operon-NRSP8 platform data is also curated. One specific interest is using expression data to help identify tissue-selective genes and across-species expression comparison of such genes to recognize evolutionarily conserved regulatory modules of interest to pig genome scientists. Here we describe some of our efforts in this area; integration and comparison of data from the two broad-coverage platforms that currently exist for the pig; the Qiagen-Operon-NRSP8 13K oligonucleotide array (hereafter abbreviated the Operon array) and the Affymetrix 23K Porcine GeneChip\u00ae (abbreviated the Affymetrix chip), both of which were discussed above.\nTo integrate the expression data for these two platforms, we need first to determine which Operon oligonucleotide probes have a sufficient sequence similarity to the consensus sequences used to create the probesets on the Affymetrix chip so that the two elements will recognize the same transcript(s). The Operon 70-mer probe sequences were used as a BLAST query against the Affymetrix porcine consensus sequences (Figure 2). We set a cutoff criterion of an alignment length \u2265 67 nucleotides with an alignment identity of \u2265 97% for pairings between the 70-mer probes and the consensus sequences. This resulted in a total of 8,317 cross platform mappings (Table 2), which fall on six different match classes: (1) an Operon probe not matching any Affymetrix consensus sequence, (2) an Affymetrix consensus sequences without an Operon probe match, (3) the ideal case when a single Operon probe matches a single Affymetrix consensus sequences, (4) two or more Operon probes map to a single Affymetrix consensus sequence, (5) a single Operon probe maps to multiple Affymetrix consensus sequences, and (6) multiples of each match creating a cluster of probes and probe sets (Figure 2, Table 2). The different classes most likely result from the difference in the known amount of porcine sequence during the development of the probes\/probe sets; the Operon probes being designed in 2002, while the Affymetrix probe sets were designed in 2004. This helps explains the different classes: Class 4 from the merging of old clusters, Class 5 from Affymetrix designing separate probe sets for alternative transcripts (which might not have been known in 2002) or that the Operon probe is no longer unique (for example, now targeting multiple genes in the same family), and Class 6 from the joining of different consensus sequences into single genes, but then having alternative splicing.\nNow that we had the ability to directly compare platform elements, we determined the extent to which the two platforms agree on assaying for the presence or absence of expression for these genes in a common tissue. Two Affymetrix experiments and one Operon experiment were used to calculate the agreement of the presence\/absence calls of the platform pairings created by BLAST. All three experiments were performed on normal liver tissue; the first Affymetrix experiment (A1) collected data from the same RNA on six different chips; the second Affymetrix experiment (A2) data was collected using four separate RNA samples on four different arrays. The Operon (O) experimental data on an additional six RNA samples was available from our previous study 32. MAS5 provides the present\/absent calls for the Affymetrix GeneChip\u00ae, and for the Operon array we used presence\/absence as calculated by Zhao et al. 32. To take a step beyond simple agreement of presence\/absence, we also calculated the correlation of expression level for each gene as estimated by each platform. We ranked each gene by expression level and used the Spearman's Rank correlation to determine the extent of agreement for relative expression of each gene within the common set of genes.\nAcross the two platforms, we saw a significant amount of agreement in declaring a gene expressed (Table 2). Of the different classes, Class 4 had both the highest agreement (89%) and highest correlation (r2 = 0.77 for Operon to either array and r2 = 0.97 between Affymetrix experiments). The other three classes had similar correlations: r2 = 0.62 for the Operon to A1 experiment, r2 = 0.55 to 0.61 for the Operon to A2 experiment, and r2 = 0.94 to 0.97 for the A1 to A2 comparison. However, they differed in their agreement: 71% for Class 5, 82% for Class 3, and 84% for Class 6. When all pairs are considered, we found an r2 = 0.71 to 0.72 for the cross-platform comparisons and r2 =0.97 for the within platform comparison, with an present\/absent agreement of 82%.\nSince Class 5 has the lowest agreement and correlations (equal to Class 3 for cross platform correlations, but with a lower within platform correlation), this result indicates that the Operon probes could cross-hybridize to either alternative splice variants or gene family members with close sequence homology that the Affymetrix platform was designed to assay separately. Further investigation is needed to see if the Operon probe is present, while the Affymetrix probe sets are absent, or if there are multiple Affymetrix probe sets present while the single Operon is absent. The results for Class 4, which have the highest agreement and the best correlation, lends support to the proposal that multiple Operon probes target the same gene product\u2014likely by the clustering of sequences from the time the Operon chip was developed to the time the Affymetrix chip was developed. While these results already show good agreement between platforms, it will be important to update the sequence comparisons on a regular basis, especially with the Sino-Danish data, as well as the genome sequence, coming online. As part of our database we plan to develop the means to regularly develop a consensus sequence for each gene from all available sequences to map the various probes and probe sets to each other.\nBeyond EST and microarray databases, as noted above, the USDA Beltsville group has set up a database for quantitative real-time PCR assays for genes related to nutrition and immunity (http:\/\/www.ba.ars.usda.gov\/nrfl\/nutri-immun-db\/nrfl_query1.html). All assays are based on the Taqman technology, so sequences for both a primer pair as well as the dual-labeled probe are presented. Of the almost 3,000 genes identified, they have validated assays for 474 available, of which 237 are known to be cross-reactive in humans, and another 771 candidate assays are being validated. Many of these assays have been published by this group and their collaborators 29, 31, 32.\nIn addition to creating databases for porcine transcriptomics, groups are using available stored information for addressing biological information and additional annotation. In a purely bioinformatic analyses of porcine expressed sequences, Jiang and collaborators 82 used 33,308 human gene sequences and mapped, by using BLAST against the est_other database at NCBI, nearly 14,000 of them to a porcine EST in either embryonic or reproductive tissue. Of these, 2,167 were found only in the embryo and 4,552 were only in reproductive tissues, while 7,243 were found in both tissues. Therefore they found a total of 9,410 ESTs present in embryos and 11,795 in reproductive tissues 82.\n5. Conclusions\nFunctional genomics data, primarily at the RNA level currently, is accumulating rapidly for the pig species. Excellent, sensitive and broadly useful tools are already available and more will be become available within the next year. Annotation of the draft porcine genome sequence, expected in late 2007 and into 2008, will allow rapid integration of the gene expression data discussed above with gene sequences, potential splice sites, and gene families within the draft sequence. Advances in other areas of investigation in pig genetics and genomics can be anticipated. One such area would be the ability to find common regulatory sites within flanking DNA of co-expressed\/co-regulated genes; leading to the identification of critical regulatory proteins in common with, or distinct from, those found in other species. Such information will reinforce the discovery of pathways through gene list annotations, improve pathway understanding through differentiation of direct targets from indirect targets of transcriptional signals, and would identify targets for manipulation of complete pathways and systems. We can also anticipate the comprehensive integration of linkage mapping and expression profiling of the same population, termed eQTL studies. Such integration of functional and structural genomic data will dramatically improve our understanding of the genetic architecture controlling quantitative traits in pigs. eQTL analyses may lead to the first application of \u201csystems biology\u201d to genetic improvement in the pig through the identification of cis-regulatory variation controlling an economically important phenotype.\nAs we move toward such a \u201csystems biology\u201d approach in animal genomics, access to and integration of these data sources will become critical. Thus a continuing need is for bioinformatics to integrate the structural and functional data we are generating to inform our investigations 83. It is unfortunate that many datasets are not being submitted to public repositories; it is our hope and expectation that journals will both facilitate and require such public submissions, as is more the norm in the biomedical fields. Many groups are working on this bioinformatics effort and therefore the near future in pig genomics is especially exciting. The long-term goal of the application of genome data to improve pig genetics will be reached when we can apply a more robust understanding of pig genetic pathways to identify variation at genes controlling important traits of interest in the pig.\nSupplementary Material\nSupplementary Table 1\nClick here for additional data file.\nSupplementary Table 2\nClick here for additional data file.","keyphrases":["transcriptomics","porcine","microarray","bioinformatics","quantitative pcr"],"prmu":["P","P","P","P","P"]}
{"id":"J_Neurooncol-4-1-2295256","title":"The influence of low-grade glioma on resting state oscillatory brain activity: a magnetoencephalography study\n","text":"Purpose In the present MEG-study, power spectral analysis of oscillatory brain activity was used to compare resting state brain activity in both low-grade glioma (LGG) patients and healthy controls. We hypothesized that LGG patients show local as well as diffuse slowing of resting state brain activity compared to healthy controls and that particularly global slowing correlates with neurocognitive dysfunction. Patient and methods Resting state MEG recordings were obtained from 17 LGG patients and 17 age-, sex-, and education-matched healthy controls. Relative spectral power was calculated in the delta, theta, upper and lower alpha, beta, and gamma frequency band. A battery of standardized neurocognitive tests measuring 6 neurocognitive domains was administered. Results LGG patients showed a slowing of the resting state brain activity when compared to healthy controls. Decrease in relative power was mainly found in the gamma frequency band in the bilateral frontocentral MEG regions, whereas an increase in relative power was found in the theta frequency band in the left parietal region. An increase of the relative power in the theta and lower alpha band correlated with impaired executive functioning, information processing, and working memory. Conclusion LGG patients are characterized by global slowing of their resting state brain activity and this slowing phenomenon correlates with the observed neurocognitive deficits.\nIntroduction\nLow\u2013grade glioma (LGG) patients constitute 25% [1] of the glioma patient population and have a survival of 5\u201310\u00a0years [2]. The optimal treatment for this patient group is still a matter of debate [3\u20135]. The first clinical feature of this disease is epilepsy in two-third of the patients [1, 4]. The majority of LGG patients also suffer from cognitive deficits, which tend to have a global character and cannot only be explained by the tumor localization alone [6, 7].\nHigher cognitive functions depend on the integrated activity of several specialized brain areas. Functional imaging techniques such as electroencephalography (EEG) and magnetoencephalography (MEG) are used to characterize oscillatory activity in patients with neurodegenerative diseases, including Alzheimer\u2019s [AD; 8\u201310] and Parkinson\u2019s disease [PD; 11\u201313]. Oscillations are a feature of neuronal brain activity and the synchronization of the oscillatory activity (reflecting the interactions of neuronal activity) is a likely mechanism for neuronal communication. For an overview of the relation between oscillatory brain activity and neurocognitive function [see 14, 15].\nMagnetoencephalography (MEG) is a relatively novel way to capture the dynamics of the electromagnetic fields within the brain, and combines excellent temporal and spatial resolution. Comparison of MEG in AD patients and healthy controls showed more delta and theta activity in the patient population especially in the temporo-parietal region [16]. Another study compared spectral power in four different groups (severe AD, moderate AD, Lewy Body dementia, and healthy controls) and showed that the healthy control population had the largest rhythmic activity in the alpha band. The moderate AD population and the patients with Lewy Body dementia had the rhythmic predominance in the pre-alpha frequency band (7\u20139\u00a0Hz), whereas the severe AD showed a shift towards the slow-band (3\u20137\u00a0Hz) [17]. In PD patients, MEG has been used by Kotini [18], who showed slowing of the alpha rhythm in the patient population compared to healthy controls, and Bosboom [19], who demonstrated slowing in the theta, beta, and gamma frequency band in non-demented PD patients with a further slowing (and involvement of delta and alpha band) in the demented PD patient population.\nIt is suggested that the increase in slow wave activity corresponds with neurocognitive deficits in AD as well as in PD [13, 16, 19, 20].\nMEG-studies have previously been performed in brain tumor patients. Oshino [21] found an increase in delta and theta band activity especially in the vicinity of the tumor and in the surrounding regions of edema. In the majority of these patients, the increase correlated with the clinical symptoms of these patients, assessed with a routine neurological examination, but neurocognitive function were not formally investigated. Although the focal lesion in brain tumor patients contrasts with the generalized brain degeneration in AD and PD, there is evidence that brain tumors can cause functional disturbances in areas remote from the tumor [22\u201325]. De Jongh [22] found focal clusters of delta activity near the tumor area but also in the contralateral hemisphere. An evaluation of fast MEG waves in brain tumor patients showed dipoles located in the parietal and occipital area, whereas the tumors were located in the parietal, temporal, and frontal area [23]. In this study, these dipoles were found remote from the tumor area and even in the contralateral hemisphere. Bartolomei [24, 25] found differences in resting state functional connectivity in the same brain tumor population within several frequency bands when compared to healthy controls. Interestingly, in agreement with de Jongh\u2019s results, these differences were not limited to the tumor area and were more obvious in those with a tumor in the left hemisphere.\nSince higher cognitive functions are presumed to depend on the integrated activity of several specialized brain areas, it is suggested that neurocognitive deficits may have a stronger correlation with diffuse alterations in resting state brain oscillatory activity than focal abnormalities in the brain tumor population.\nIn the present MEG-study, power spectral analysis of oscillatory brain activity was used to compare resting state brain oscillatory activity in both LGG patients and healthy controls.\nWe hypothesize that (1) LGG patients show, in addition to the local abnormalities at the tumor site, global slowing of the resting state brain activity compared to healthy controls which will vary between patients with a tumor in the left or right hemisphere (2) changes in resting state brain oscillatory activity reflect an intermediate level between the impact of tumor and tumor-related treatment on the one hand (\u2018input\u2019) and the neurocognitive deficits (\u2018output\u2019) on the other hand and that diffuse slowing is correlated with higher neurocognitive dysfunction.\nMaterials and methods\nPatients and controls\nTwenty-three LGG patients were asked to participate in this study. Patients were eligible if: (a) they had a suspected or histologically confirmed LGG; (b) there was no radiological (confirmed by MR or CT scan) and\/or clinical tumor progression in the previous 6\u00a0months; (c) they did not use medication possibly interfering with neurocognitive function, other than anti-epileptic drugs (AEDs).\nPatients were recruited from the VU University Medical Center (VUmc) and the Academic Medical Center (AMC), both tertiary referral centres in Amsterdam for brain tumor patients, after the institutional ethical review boards of both participating hospitals approved the study protocol. Relatives of the patients were asked to participate as healthy controls. Healthy controls were eligible if they: (a) did not suffer from any neurological disease; (b) did not use any medication that might influence cognitive function. For patients who could not provide a healthy control participant, VU University Medical Center staff members were included.\nMagnetoencephalography\nMEG recordings were obtained using a 151-channel whole-head MEG system (CTF systems; Port Coquitlam, British Columbia, Canada) while participants were seated inside a magnetically shielded room (Vacuumschmelze GmbH, Hanau, Germany). Magnetic fields were recorded during a no-task, eyes-closed resting state. Metal artefacts were avoided as much as possible. A third-order software gradient [26] was used with a recording passband of 0.25\u2013125\u00a0Hz and a sample frequency of 312.5\u00a0Hz. At the beginning, middle and end of each recording, the head position relative to the coordinate system of the helmet was recorded by leading small alternating currents through three head position coils attached to the left and right preauricular points and the nasion on the subject\u2019s head. Head position changes up to approximately 1.5\u00a0cm during a recording condition were accepted.\nFor this study, 149 of the 151 channels could be used. MEG recordings were converted to ASCII files. From these ASCII files four artefact free epochs of 13\u00a0s per subject (4096 samples) were carefully selected by visual analysis by one of the authors (IB).\nMagnetic field frequencies ranging from 0.5 to 80\u00a0Hz were recorded. The MEG data were digitally filtered off-line in the following frequency bands: delta (0.5\u20134\u00a0Hz), theta (4\u20138\u00a0Hz), lower alpha (8\u201310\u00a0Hz), upper alpha (10\u201313\u00a0Hz), beta (13\u201330\u00a0Hz), gamma (30\u201350\u00a0Hz).\nThe MEG channels were grouped into 10 distinct regions, respectively left and right frontal region, left and right temporal region, left and right parietal region, left and right occipital region and left and right central region, as shown in Fig.\u00a01. Fast Fourier transformation was separately applied for every patient and control on the four epochs in the above mentioned frequency bands. The results of the four epochs were averaged for each participant and the mean relative power for each MEG region were used for the analysis.\nFig.\u00a01Distribution of MEG regions\nNeurocognitive assessment\nPatients and controls participated in neurocognitive assessment (see Table\u00a01). The total duration of the assessment varied between 1 and 2\u00a0h. Individual patient\u2019s test scores were converted to z-scores, using the means and standard deviations of the age-, sex- and education-matched healthy controls as a reference.\nTable\u00a01Description of neuropsychological test batteryLetter-digit substitution test [27] This test provides a measure of psychomotor performance that is relatively unaffected by intellectual prowess and is suitable for groups with an age range exceeding 60\u00a0years. The number of items written down in 90\u00a0s is registered, as in the decrease in performance when graphomotor speed is involvedVisual verbal learning test [27] This version of the Rey Auditory Verbal Learning Test calls for various aspects of verbal learning and recall. Measures used for analysis are memory performance on trial 1 as indicator of immediate recall, total recall after five trials, delayed recall and recognition after 20\u00a0min as indicators of memory consolidation into long-term memory, and a delta score as a measure of learning capacityStroop color-word test [27] This test is a selective attention task aiming at measuring interference susceptibility and consists of three subtasks with increasing task complexityCategoric word fluency [28]A simple task requiring the generation of words from semantic categories (animals) within a limited timeConcept shifting test [29]This test, which has two conditions of complexity, predominately measures functions associated with executive function, especially visual scanning and conceptual tracking. The motor component of this task is measured by three dummy conditions in which no cognitive capacity except for graphomotor speed is requiredMemory comparison test (MCT) Selective attention, mental concentration, memory and information processing\nTo reduce data, individual scores on these tests were summarized into six cognitive domains, namely information processing speed, psychomotor function, attention, verbal memory, working memory, and executive functioning. Construction of these domains has previously been reported [30], and was based on a Principal Component Analysis using Varimax rotation with Kaiser normalization performed on the z-scores of a large group of healthy controls [31]. The domains found are commonly used in neurocognitive practice and research.\nStatistical analysis\nDifferences between both groups in the distribution of age, sex, and education were analyzed by means of chi-square tests. Mann\u2013Whitney nonparametric U-tests were used to investigate whether patients\u2019 standardised z-scores on neurocognitive tests in the overall measure of cognition differed significantly from healthy control z-scores.\nBecause of the non-normal distribution of the relative power, Mann\u2013Whitney nonparametric U-tests were used to determine possible differences between the patient population and healthy controls.\nTo assess the association between relative power and cognition within the patient group, Spearman\u2019s correlation coefficient (\u03c1) was calculated between the relative power in the different MEG regions and z-scores of the six neurocognitive domains for all separate frequency bands.\nResults\nPatient characteristics\nFrom the initial patient group, six patients were excluded, four patients due to metal artefacts on the MEG measurements, one due to severe epileptic seizures, and one due to tumor progression at the time of registration. The final analyses were performed on a sample of 17 patients and 17 matched healthy control participants.\nDue to the matching procedure, there were no significant differences between patients and healthy controls in age (M\u00a0=\u00a042.7, SD\u00a0=\u00a011.2 in patients, M\u00a0=\u00a042.6, SD\u00a0=\u00a012.7 in healthy controls, p\u00a0=\u00a00.99), and educational level (M\u00a0=\u00a05.2, SD\u00a0=\u00a01.8 in patients, M\u00a0=\u00a05.5, SD\u00a0=\u00a01.8 in healthy controls, p\u00a0=\u00a00.64). The male\u2013female ratio between the two groups did not differ significantly (p\u00a0=\u00a00.37). Fourteen of the 17 LGG patients had a histologically confirmed LGG, clinically and radiologically stable for more than six months before inclusion, whereas the other 3 patients were suspected of having LGG, and stable for more than six months. The patients in our study were diagnosed several years ago (mean 8\u00a0years, range: 1\u201319\u00a0years). One year after MEG registration two of the three patients with suspected LGG were operated on because of increasing epilepsy frequency.\nEleven of those 16 patients underwent debulking, whereas 3 patients underwent a stereotactic biopsy and another two patients had an open biopsy. Of the 16 patients with a histologically confirmed LGG, the pathological diagnosis was grade II astrocytoma in ten patients, oligodendroglioma grade II in four patients and oligoastrocytoma grade II in two patients.\nSeven of the seventeen patients underwent radiotherapy with prior chemotherapy in two patients (1 patient with 5 cycles of PCV and 1 patient with 2 cycles of PCV and 3 cycles of temozolomide). Eleven patients had left hemisphere tumors and 6 patients had right-sided tumors. The specific localisation of the tumor is shown in Table\u00a02.\nTable\u00a02Tumor lateralization and localizationLeft hemisphereRight hemisphereTumor locationNo. of patientsTumor locationNo. of patientsLeft frontal4Right frontal2Left parietal3Right frontoparietal3Left temporal3Right insular region1Left parieto-occipital 1Total11Total6\nIn the patient group, all but one patient used AED mono- or poly-therapy. Six of the 16 patients on AED were free of seizures, while the other 10 patients were still having seizures.\nDifferences between patient and healthy control group\nNeurocognitive functioning\nAs expected, patients performed poorer than healthy controls on the neurocognitive test battery (controls M\u00a0=\u00a00.00). More specifically, patients showed a significantly lower psychomotor function (M\u00a0=\u00a0\u22120.50, SD\u00a0=\u00a00.75 versus SD\u00a0=\u00a00.55, p\u00a0=\u00a00.044), working memory capacity (M\u00a0=\u00a0\u22121.43, SD\u00a0=\u00a01.62 versus SD\u00a0=\u00a00.91, p\u00a0=\u00a00.002), information processing speed (M\u00a0=\u00a0\u22120.85, SD\u00a0=\u00a00.88 versus SD\u00a0=\u00a00.97, p\u00a0=\u00a00.018) and attention (M\u00a0=\u00a0\u22121.92, SD\u00a0=\u00a03.87 versus SD\u00a0=\u00a00.68, p\u00a0=\u00a00.003). Patients\u2019 performance on the other two cognitive domains did not differ significantly from controls (Fig.\u00a02).\nFig.\u00a02Patients\u2019 z-scores on the six neurocognitive domains and on total neurocognitive functioning. Note: *\u00a0p\u00a0<\u00a00.05, **\u00a0p\u00a0<\u00a00.01. A\u00a0=\u00a0attention, EF\u00a0=\u00a0executive functioning, IPS\u00a0=\u00a0information processing speed, PF\u00a0=\u00a0psychomotor function, VM\u00a0=\u00a0verbal memory, WM\u00a0=\u00a0working memory. Performance is relative to that of age-, sex-, and education-matched healthy controls (represented by the 0-line). A higher score (i.e. approaching 0) means better performance\nSpectral analysis\nGlobal spectral analysis. The mean relative power of the LGG patient population showed a significant decrease (Mann\u2013Whitney U, p\u00a0=\u00a00.034) in the gamma band (30\u201350\u00a0Hz) compared with healthy controls.\nSpectral analysis of frequency bands within each MEG region. An increase (Mann\u2013Whitney U; p\u00a0=\u00a00.014) in relative power in the LGG patients compared to the healthy controls was found in the left parietal region within the theta frequency band (4\u20138\u00a0Hz). A decrease in relative power was seen in the left and right central region (Mann\u2013Whitney U; both p\u00a0=\u00a00.024) and left and right frontal region (Mann\u2013Whitney U; both p\u00a0=\u00a00.005) of the LGG patient population compared to the healthy controls within the gamma band (30\u201350\u00a0Hz), as shown in Table\u00a03 and Fig.\u00a03.\nTable\u00a03Significant differences in relative power between patients and controls per frequency band and accompanying statisticsPatientsControlspMSDMSDThetaLeft parietal0.1310.0530.0920.0260.014GammaLeft central0.0640.0270.0950.0410.024Right central0.0660.0330.1020.0470.024Left frontal0.0480.0270.0880.0430.005Right frontal0.0500.0300.0830.0370.005Significant higher relative power is depicted in bold and in italicsFig.\u00a03The significant differences in relative power between the patient group and the healthy controls within the different frequency bands. Green area: significant higher relative power in the patient group compared to the healthy controls. Red area: significant lower relative power in the patient group compared to the healthy controls\nInfluence of tumor lateralization on relative power. Eleven patients had left hemisphere tumors and 6 patients had a tumor in the right hemisphere. Patients with a tumor in the left hemisphere showed an increase in theta activity in the left and right central region (Mann\u2013Whitney U; respectively p\u00a0=\u00a00.001 and p\u00a0=\u00a00.014), left and right parietal region (respectively p\u00a0=\u00a00.004 and p\u00a0=\u00a00.029), and left and right temporal region (respectively p\u00a0=\u00a00.022 and p\u00a0=\u00a00.027) when compared to the healthy control population. Patients with left-sided tumors also showed a decrease in relative power in the gamma band in the left and right central region (respectively p\u00a0=\u00a00.023 and p\u00a0=\u00a00.048) and left and right frontal region (respectively p\u00a0=\u00a00.011 and p\u00a0=\u00a00.019).\nPatients with a tumor in the right hemisphere showed a significant increase in relative power of delta activity in the right parietal region (Mann\u2013Whitney U; p\u00a0=\u00a00.050) compared to the healthy control population. These patients also showed a decrease in relative power in the gamma band in the left and right frontal region (respectively p\u00a0=\u00a00.005 and p\u00a0=\u00a00.025), right temporal region (p\u00a0=\u00a00.042), and left and right occipital region (respectively p\u00a0=\u00a00.025 and p\u00a0=\u00a00.014), as shown in Table\u00a04 and Fig.\u00a04.\nTable\u00a04Significant differences in relative power between patients with a tumor in the left or right hemisphere and controls per frequency band and accompanying statisticsPatientsControlspMSDMSDLeft hemisphereThetaLeft central0.1410.0390.0950.0180.001Right central0.1330.0410.0960.0220.014Left parietal0.1490.0570.0920.0260.004Right parietal0.1410.0650.0890.0290.029Left temporal0.1070.0490.0720.0190.022Right temporal0.1000.0430.0710.0230.027GammaLeft central0.0630.0260.0950.0410.023Right central0.0690.0320.1020.0470.048Left frontal0.0480.0270.0880.0430.011Right frontal0.0530.0310.0830.0370.019Right hemisphereDeltaRight parietal0.4240.1440.3030.1370.050GammaLeft frontal0.0500.0280.0880.0430.005Right frontal0.0450.0310.0830.0370.025Right temporal0.0420.0220.0700.0450.042Left occipital0.0400.0290.0770.0320.025Right occipital0.0480.0360.0990.0370.014Significant higher relative power is depicted in bold and in italicsFig.\u00a04The significant differences in relative power between the patients with a tumor in the left or right hemisphere compared to the healthy controls within the different frequency bands. Green area: significant higher relative power in the patient group compared to the healthy controls. Red area: significant lower relative power in the patient group compared to the healthy controls\nSince these results could be influenced by differences in tumor volume, we evaluated the tumor size of participating patients. We used T1-weigthed MRI and defined tumor size as the product of the two largest perpendicular diameters of tumor hypointensity [32]. We found no significant differences in the tumor size between left- and right-sided tumors.\nAssociations between patient\u2019s neurocognitive functioning and relative power in distinct MEG regions\nIn the patient population, negative associations between neurocognitive functioning and relative power were found in the theta and lower alpha band. An increase in the relative power in the theta band within the left frontal (\u22120.606; p\u00a0=\u00a00.010) and the right frontal region (\u22120.490; p\u00a0=\u00a00.046) was correlated with a poorer executive functioning. In the same frequency band (4\u20138\u00a0Hz), an increase in theta band power was correlated with poorer information processing within the left central and left frontal region (respectively \u22120.515; p\u00a0=\u00a00.034 and \u22120.592; p\u00a0=\u00a00.012). Within the lower alpha band in the right temporal region, an increase in relative power was correlated with a disturbed working memory (\u22120.508; p\u00a0=\u00a00.037).\nDiscussion\nThe primary goal of the present study was to evaluate whether LGG patients show (in addition to the well known MEG slowing around the tumor) diffuse slowing in resting state brain activity. The secondary goal of the study was to investigate whether this slowing is correlated with neurocognitive dysfunction. By means of MEG-registrations we have demonstrated that LGG patients have slowing of the resting state brain activity when compared to healthy controls. The decrease in relative power was mainly found in the gamma frequency band in the bilateral frontocentral MEG regions. Regarding the low frequency bands, an increase in relative power was found in the theta frequency band in the left parietal region. Correlations of neurocognitive functioning with the relative power in the patient population showed clear associations in the lower alpha and theta band, increased slowing correlating with poorer performance.\nWe chose to use the relative power instead of the absolute power, because relative power is less influenced by the distance between the MEG sensor and the underlying neural populations. The distance is variable due to head position in the helmet and the thickness of the skull. Lower variance of power values in subject groups are expected by using the relative power instead of the absolute power. A second reason for us to use this method is the fact that by using this method we are able to compare our results with those of other patient cohorts at our MEG center [19, 20]. This includes comparisons with other brain tumor data sets but also the comparison of our results with neurodegenerative diseases (including AD and Parkinson\u2019s disease (PD)). This will give us an opportunity to learn more about differences in resting state oscillatory brain activity in the different diseases and at different disease stages. A limitation of this method of analysis is that an increase in one frequency band can cause a decrease in another band, although this might not be the observed pattern in absolute measures.\nIn previous studies, MEG has been used for power analysis in patients with neurodegenerative diseases, such as AD and PD. They showed similar results, namely a rhythmic predominance in the lower frequency bands, as was found in our study with LGG patients [16\u201320]. There are only few studies performed in brain tumor patients, however.\nDe Jongh [22] demonstrated that both a higher tumor malignancy and larger tumor volume were associated with higher signal powers in the delta band. In our study, we did not find increased oscillatory brain activity in the delta band. It might be that the conflicting results are due to differences in methodology of both studies. In contrast with De Jongh\u2019s study [22], we analysed the relative power, which means that the power is influenced by the power in the other frequency bands and therefore group differences in a specific frequency band could results from changes in that specific band or in the absolute power of the other frequency bands. Furthermore, we evaluated diffuse delta activity instead of clusters of dipoles in the vicinity of the tumor. Apart from these technical differences, De Jongh\u2019s patient population consisted of a more heterogeneous group of brain tumor patients. They found a higher delta power in the high-grade gliomas compared to those patients with a low-grade glioma and a delta signal power decrease after surgery. Our patient population consisted exclusively of patients with a low-grade glioma, and all but three MEG registrations were done after surgery, which might explain the lack of changes in the delta band in the present study. It also suggests that changes in the gamma and theta band may be more sensitive indicators of brain dysfunction in LGG.\nIn another study of De Jongh [23], the authors found focal clusters of fast MEG waves outside the tumor area and even in the contralateral hemisphere. They concluded that the asymmetry possibly reflects features of normal background activity, since asymmetry has already been demonstrated in healthy subjects for the alpha activity which is usually dominant in the right hemisphere. Since De Jongh used a single dipole analysis instead of our analysis of diffuse power changes and, again, analysed a group of patients with diverse brain tumors, it is difficult to compare the results of both studies.\nOshino and colleagues [21] used synthetic aperture magnetometry (SAM) to analyze oscillatory activity recorded by the MEG. SAM is an alternative method investigating the distribution of reconstructed sources over different cortical regions. In contrast to other source localization methods, beamforming does not rely on averaging and therefore allows analysis of evoked and induced brain activity. In a group of 15 patients with various types of primary or secondary brain tumors, they found an increase in delta and theta band activity especially in the vicinity of the tumor and in the surrounding regions of edema. In the majority of these patients, the increase in the delta band correlated well with the clinical symptoms, assessed with a routine neurological examination. The results were not correlated with neurocognitive functioning because they stated that there is much individual variation in brain activity related to neurocognitive function and they expected it difficult and sometimes impossible for these patients to undergo the neurocognitive battery. In contrast to their results, we only found an increase in theta activity and decrease in the gamma frequency band. As mentioned before we analyzed the relative power and also our patient population with only LGG patients is not comparable to the patients with various brain tumors in the study of Oshino. Although, different methods to reconstruct sources can show significant discrepancies, we also found spectral changes in the contralateral hemisphere.\nBy evaluating separately those patients with a tumor localisation in the left or the right hemisphere, we found more significant differences in patients with a tumor in the left hemisphere compared to healthy controls. In agreement with our previous study [24], in which we evaluated the functional connectivity (functional connectivity is a statistical correlation between time series of brain activity recorded over distinct regions which are assumed to reflect interactions between the brain regions) in patients with diverse primary brain tumors, we also found that patients with a tumor in the left hemisphere showed more differences in this functional connectivity compared to those with right-sided tumors.\nWhy those patients with a tumor in the left hemisphere do show more differences in oscillatory activity is not known. It is possible that the left hemisphere (usual the dominant hemisphere) acts differently to the tumor compared to those located in the right hemisphere. It is also possible that patients with a tumor in the left hemisphere are in a different stage of their disease compared to those with a tumor in the right hemisphere. Since our sample size is rather small, this could be reflected on the observed results. On the other hand is it possible that our results reflect hemispheric asymmetry which has been found in the healthy population previously. Gootjes et\u00a0al. [33] found hemispheric differences in functional connectivity in healthy young adults during resting state.\nThe neurocognitive domains of psychomotor function, working memory, information processing speed and attentional tasks were impaired in LGG patients. These results corroborate previous studies indicating a decline in neurocognitive functioning in LGG patients [6, 34\u201338]. Several research groups have suggested that the increase in slow wave activity corresponds with neurocognitive deficits in AD and PD. Fernandez [16] found right parietal delta activity to be significantly associated with variability in the Cambridge Examination for Mental Disorders in the elderly (CAMCOG) score, whereas the left temporal theta activity predicted the variability of Mini Mental State Examination (MMSE) results. Sinanovic [13] found a positive correlation between diffuse changes in spectral power and MMSE scores in the PD patients with dementia. Bosboom [19] found a negative correlation in the non-demented PD patients between theta power and CAMCOG scores in both occipital and right temporal region but expected these associations, given the large number of relations that were analyzed, likely to be caused by coincidence. In the demented PD group they found no significant correlations. Stoffers [20] showed a negative association between lower alpha power and the performance on perseveration-related tasks in the non-demented PD patients.\nEvidently, this study has its limitations. First of all, the patient group is rather small as mentioned before. Secondly, although our patient population consisted of only LGG patients, they did not all receive the same treatment. In our study we hypothesized changes in oscillatory brain activity to be the intermediate between the impact of the tumor and its treatment on the one hand and the neurocognitive deficits as the output on the other hand. This study showed that irrespective of the different treatment options, changes in oscillatory brain activity can be found in brain tumor patients which is associated with neurocognitive function. To get informed on the influence of the different tumor treatments on brain activity and its relation with neurocognitive function is very interesting. A longitudinal study of these effects (e.g. surgery, radiotherapy and chemotherapy) on brain activity is currently under way. The goal of that study is to explore correlations between changes in neurocognitive function and changes in functional brain dynamics during the disease course.\nOur study is the first to correlate the relative power with neurocognitive functioning in brain tumor patients. The observed correlations were quite strong and showed that an increased activity in the theta and lower alpha band is correlated with impaired executive functioning, information processing and working memory. This is a first step in unraveling the underlying mechanisms of neurocognitive dysfunction in brain tumor patients. MEG power analysis gives us an interesting tool to assess functional alterations in the patient\u2019s brain in the course of disease and to evaluate its relationship with neurocognitive functioning.","keyphrases":["low-grade glioma","meg","cognition","power analysis"],"prmu":["P","P","P","P"]}
{"id":"Matern_Child_Health_J-2-2-1592151","title":"What Obstetrician-Gynecologists Think of Preconception Care\n","text":"Objectives: To describe obstetrician-gynecolog-ists\u2019 opinions of preconception care (PCC) and ascertain patient uptake for this service. Methods: A questionnaire was mailed to 1105 ACOG members in August 2004. Results: There was a 60% response rate. Most physicians think PCC is important (87%) and almost always recommend it to women planning a pregnancy (94%); 54% do so with women who are sexually active. Around a third (34%) thought their patients usually do not plan their pregnancies and 49% said very few pregnant patients came in for PCC. Of those who obtain PCC, they were believed to do so more likely to assure a healthy pregnancy (83%) than because of an elevated risk for birth defects (20%). Of 11 issues presented, cigarette smoking and folic acid supplementation were rated the most important for PCC counseling; exercise and environmental concerns were the least important. Conclusions: Physicians are willing to provide PCC but few patients are accessing such services.\nIntroduction\nWhen poor pregnancy outcomes occur, they frequently have been set in motion long before the first prenatal visit. Fortunately, many of the factors contributing to less than ideal birth outcomes can be managed and brought under control prior to conception. Preconception care (PCC) consists of the identification of those conditions that could affect a future pregnancy or fetus and that may be amenable to intervention [1]. ACOG recommends that all health encounters during a woman's reproductive years, particularly those that are a part of PCC, should include counseling on appropriate medical care and behavior to optimize pregnancy outcomes [1]. Through PCC, physicians can advise patients on numerous behavioral and lifestyle changes, ranging from exercise and weight control to the use of prescribed medications and illicit drugs. They may also address carrier screening for heritable genetic disorders and the options available for helping to avoid an affected birth. For example, it is well known that women with a chronic disease such as diabetes have an increased risk of congenital abnormalities in their offspring, and they are known to have improved birth outcomes when they plan their pregnancies and utilize PCC [2]. Another prime example of the success of PCC efforts is the reduction in the number of pregnancies affected by neural-tube defects following national efforts to increase consumption and supplementation of folic acid by women of childbearing potential [3] through education and food fortification. Despite such improvements, fewer than 50% of women are aware of the benefits of taking folic acid supplements around the time of conception [4], the majority of women with diabetes do not plan their pregnancies [5], many women continue to drink alcohol regularly during pregnancy [6] and almost 50% of pregnancies in the U.S. are unplanned [7].\nPCC has had its successes, but clearly more work needs to be done. The importance of PCC in promoting maternal and fetal health has long been recognized, and national recommendations and guidelines for PCC are being developed [8]. One aspect of successful development of PCC guidelines involves knowledge of obstetrician-gynecologists\u2019 current practices and opinions regarding PCC and what they perceive to be barriers to successful implementation of PCC. The purpose of this study was to assess the practices and opinions of obstetrician-gynecologists regarding PCC, and how frequently they perceive their patients to avail themselves of such services.\nMaterials and methods\nQuestionnaires were mailed in August 2004 to 1105 American College of Obstetricians and Gynecologists (ACOG) Fellows and Junior Fellows in Practice. Of these subjects, 605 were members of the Collaborative Ambulatory Research Network (CARN), and 500 were a computer-generated random sample of ACOG members who had not received a survey from ACOG during the previous two years (Non-CARN). Members of CARN are practicing obstetrician-gynecologists who have volunteered to participate in survey studies on a regular basis. CARN was established to facilitate assessment of clinical practice patterns and aid the development of educational materials. Two reminder mailings were sent to non-respondents and questionnaires returned by December 31, 2004 were included in the survey. This protocol has typically resulted in a total sample size of >450, which is sufficient to detect differences between groups of <0.5 standard deviation with power of 80% and significance at the 0.05 level [9].\nThe survey recorded demographic details of physicians and their patient population, and assessed practices and opinions regarding PCC. A questionnaire concerning screening for aneuploidy was included in the same mailing; both questionnaires shared a demographics section containing 9 questions on one page. The PCC questionnaire contained 11 questions, some multi-part, on two pages. Both questionnaires were brief and resulted in a final document of 3 double-sided pages, a standard length for our full-length questionnaires. Seven of the 11 questions on PCC involved rating the frequency of an activity (e.g., always, usually, occasionally, never) or degree of agreement with a statement, and three questions were of a multiple-choice format. The questionnaires were developed in consultation with medical specialists and were pilot tested on a sample of practicing obstetrician-gynecologists prior to final distribution.\nThe data were analyzed using a personal computer-based software package (SPSS\u00ae 12.0, SPSS Inc., Chicago, IL). Descriptive statistics were computed for the measures used in the analyses, which are reported as mean \u00b1 SEM. Student's t test was used to compare group means of continuous variables. Factor Analysis was conducted on two sets of rating variables (Agreement with 6 statements regarding PCC, and Importance of counseling on 11 issues during PCC and during routine care), and factors with eigenvalues greater than one were treated with a varimax rotation. Differences in ratings on the Importance scales were examined with multivariate analysis of variance. Differences on categorical measures were assessed using \u03c72. Group differences on ordinal measures were assessed using the Mann-Whitney U test or Kruskal Wallis \u03c72. Related-sample differences on ordinal measures used the Wilcoxon signed ranks test. Correlations including an ordinal measure used the Spearman's rho coefficient. All analyses were tested for significance using an alpha of 0.05.\nResults\nDemographics\nA total of 670 questionnaires were returned. Data from 18 respondents were judged invalid (physician retired, returned to sender), resulting in a valid response rate of 60% (652\/1087), 432 from CARN members (72.1% response rate) and 220 from Non-CARN (45.1% response rate). Physicians responded from every state of the United States except Maine, as well as the District of Columbia, Puerto Rico, Canada, and overseas military installations. The respondents\u2019 mean age (47.32\u00b10.39) and the proportion of males to females (males = 55%) closely matched those of the larger population to whom the survey was sent (46.99\u00b10.32, males = 55%) and of ACOG Fellows and Junior Fellows in Practice as a whole (47.64, males = 58%).\nThe remaining analyses are limited to the 88.8% (579) of respondents who indicated that their primary medical specialty was gynecology or general obstetrics and gynecology. This selection was made to best assess the practices of non-subspecialist obstetricians and gynecologists, rather than those in maternal fetal medicine, reproductive endocrinology, or other subspecialties, to whom patients may be referred after pregnancy has already been attempted. Of these physicians, 83.9% (486) practice obstetrics (ObGyn), and 16.1% (93) do not (GynOnly); the term \u2018physicians\u2019 is used when referring to both ObGyns and GynOnlys. See Table 1 for physician demographics. CARN and Non-CARN differed on only one non-demographic item (CARN were more likely than Non-CARN to discuss carrier screening with all patients who present for PCC. See results.); thus data were collapsed across these two groups.\nTable 1Physician demographicsTotal (n=579)Gender (%)\u2003Males53.9\u2003Females46.1CARN67.5Non-CARN32.5Age in years, mean (SEM)*47.12 (0.42)\u2003Males50.88 (0.54)\u2003Females42.76 (0.54)Years in practice, mean (SEM)15.22 (0.41)Deliveries in 2003, mean (SEM)129.65 (3.13)Practice location (%)\u2003Urban, inner city10.7\u2003Urban, non-inner city29.3\u2003Suburban32.1\u2003Mid-sized town19.4\u2003Rural6.9\u2003Other1.6Practice type (%)\u2003Ob\/Gyn partnership\/gp51.4\u2003Solo practice22.5\u2003Multi-specialty10.4\u2003University full-time faculty and practice8.5\u2003Other7.2Patient ethnicity\u2014mean (SEM) % of patients\u2003Non-Hispanic white62.29 (1.10)\u2003African-American16.78 (0.79)\u2003Hispanic13.35 (0.78)\u2003Asian\/Pacific Islander3.83 (0.29)\u2003Native American1.29 (0.23)*P < 0.001: Males older than females; CARN older than non-CARN.\nDefining preconception care\nPhysicians were asked whether they would define PCC as specialized or routine care. The great majority (86.7%) defined it \u201cAs specialized pre-pregnancy care that focuses on issues not typically addressed during a routine exam which are specific to ensuring an optimal pregnancy outcome.\u201d Far fewer (13.3%) defined it as \u201cThe same as routine well-woman care that occurs during the reproductive years, prior to a pregnancy.\u201d Those who defined PCC as routine tended to be in practice longer than those defining it as specialized (17.55\u00b11.16 yrs versus 14.84\u00b10.45 yrs. t(555)=2.22; P < 0.05), independent of gender.\nRecommending PCC\nThe vast majority of physicians (97.3%) indicated that they provide PCC for their patients. Physicians were asked how frequently they recommend PCC to different groups of women of childbearing age (see Table 2). Three quarters (75%) always recommend PCC to patients planning a pregnancy, and 9 in 10 (89.9%) always recommend it to diabetic patients planning a pregnancy. Physicians who defined PCC as routine were more likely than those who defined it as specialized to recommend PCC to women who are sexually active (\u2018always\u2019 or \u2018usually\u2019: routine = 63.4%, specialized = 52.1%. M-W U=13989.5; P<0.05) and to women who are using birth control (\u2018always\u2019 or \u2018usually\u2019: routine = 48.6%, specialized = 32.8%. M-W U=13002; P < 0.01).\nTable 2Percent of physicians indicating how frequently they recommend PCC to different groups of womenHow frequently do you recommend preconceptioncare to the following women of childbearing age?AlwaysUsuallyOccasionallyNeverDiabetic women planning a pregnancy89.97.91.70.5Women who are planning a pregnancy75.019.25.00.8Obese women planning a pregnancy61.527.09.52.0Women indicating they want children in the future38.543.516.02.0Women who are sexually active19.134.938.67.4Women who are using birth control11.524.145.918.5\nOpinions about PCC\nPhysicians were asked their level of agreement with several statements regarding PCC (see Table 3). The majority agreed (4\u20135 on 5 point scale) that PCC is an important issue (87.3%) and that it has a positive effect on pregnancy outcomes (83.5%), though only 20.7% agreed that it is a high priority in their workload. Three quarters (76.8%) thought they have appropriate training to provide PCC. Half agreed that there is not enough time to provide preconception care visits to all women of childbearing age (51.4%), and that time devoted to PCC is not reimbursed (49.8%). Based on factor analysis, the first three items were combined to produce a single mean score for the \u2018positive\u2019 aspects of PCC (Factor I), and the second set of three items for the \u2018negative\u2019 aspects of PCC (Factor II). There were significant positive correlations between agreement on Factor I and frequency of recommending PCC in all groups of women (all P\u2019s < 0.001); there were significant negative correlations between agreement on Factor II and frequency of recommending PCC in all groups of women (all P\u2019s < 0.01). In other words, the more strongly they agreed that PCC was important\/positive\/high priority, the more frequently they recommended PCC. Looking at the individual components of the two factors, lack of reimbursement for PCC was the only item that did not correlate with frequency of recommending PCC in any group of women.\nTable 3Percent of physicians indicating how strongly they agree with several statement regarding PCCStronglyStronglyagreedisagreeMean on 5 pt Six statements regarding opinions about PCC54321scale (SEM)Factor I: positive aspects\u2003Preconception care is an important issue47.439.910.51.30.94.32 (0.034)\u2003Preconception care has a positive effect on pregnancy outcomes44.738.814.31.11.14.25 (0.035)\u2003Preconception care is a high priority in my workload6.813.942.327.99.12.82 (0.043)Factor II: negative aspects\u2003Time devoted to preconception care is not reimbursed24.725.130.814.05.53.50 (0.051)\u2003There is not enough time to provide preconception care visits to all women of childbearing age22.628.819.718.910.03.35 (0.056)\u2003I do not have appropriate training to provide preconception care3.05.214.735.741.41.92 (0.044)\nCounseling in PCC\nAlmost a third (31.9%) of physicians said they discuss screening for aneuploidy with All patients who present for preconception counseling. Of those not selecting All, 80.7% said they did so with patients at risk for aneuploidy. Almost one-in-ten (9.8%) said they did not discuss aneuploidy. The majority (54.4%) of physicians said they discuss carrier screening for heritable genetic disorders (e.g., CF, sickle cell anemia) with All patients who present for preconception counseling, and, of those not selecting All, 84.8% said they did so with patients who have a family history of heritable genetic disorders or other risk factors. Only 4.3% said they did not discuss carrier screening.\nPhysicians were presented with 11 health-related issues (e.g., exercise, nutrition, over the counter drug use) and were asked to rate how important counseling was for each issue during a routine office exam and during preconception care (see Table 4). (Note: Physicians were not asked to rank order the issues in terms of relative importance, nor were they asked to compare the issues in terms of routine or PCC; such calculations were made based on analyzing the mean ratings for each issue.) Physicians rated all issues as more important in PCC than in routine care, although the difference for Exercise was marginal (Wilcoxon signed ranks test. Exercise: P=0.052. All other P\u2019s < 0.001). All issues were rated as very important for PCC, with cigarette smoking and folic acid supplementation rated the most highly important issues in PCC. Cigarette smoking and illegal drug use were rated the most important issues for counseling in routine care. Whereas folic acid supplementation ranked second in importance for PCC, it ranked tenth for routine care.\nTable 4Percent of physicians indicating how important they think counseling is on several issues for patients who (a) come in for a routine office exam, and (b) those who are planning a pregnancy(Rank of meanImportanceVery importantNot importantMean (SEM) within a or b)Neutral [3\u20135]on 7 pt scaleFactor I\u2003Folic acid supplements:\u2003\u2003(a) routine exam(10)36.852.510.74.88 (0.07)\u2003\u2003(b) preconception care(2)96.43.4.26.83 (0.03)Factor II\u2003Cigarette smoking:\u2003\u2003(a) routine exam(1)89.210.60.26.57 (0.03)\u2003\u2003(b) preconception care(1)98.41.60.06.86 (0.02)\u2003Illegal drug use:\u2003\u2003(a) routine exam(2)83.215.21.66.40 (0.05)\u2003\u2003(b) preconception care(3)94.55.10.46.74 (0.03)\u2003Alcohol consumption:\u2003\u2003(a) routine exam(7)49.146.54.45.40 (0.06)\u2003\u2003(b) preconception care(4)88.511.50.06.57 (0.03)Factor III\u2003Obesity:\u2003\u2003(a) routine exam(3)76.123.70.26.11 (0.04)\u2003\u2003(b) preconception care(7)82.717.30.06.30 (0.04)\u2003Exercise:\u2003\u2003(a) routine exam(4)67.031.41.65.87 (0.05)\u2003\u2003(b) preconception care(10)68.331.00.75.94 (0.05)\u2003General Nutrition:\u2003\u2003(a) routine exam(6)51.246.72.15.51 (0.06)\u2003\u2003(b) preconception care(8)82.217.50.46.28 (0.04)Factor IV\u2003Chronic diseases:\u2003\u2003(a) routine exam(5)63.834.91.25.79 (0.05)\u2003\u2003(b) preconception care(6)88.310.80.96.45 (0.04)\u2003Family health history (inherited disorders):\u2003\u2003(a) routine exam(8)51.543.94.65.38 (0.06)\u2003\u2003(b) preconception care(5)90.69.20.26.55 (0.04)\u2003Over the counter and prescription drug use:\u2003\u2003(a) routine exam(9)42.051.46.55.08 (0.06)\u2003\u2003(b) preconception care(9)81.617.70.76.27 (0.04)\u2003Environmental concerns:\u2003\u2003(a) routine exam(11)19.966.313.94.25 (0.06)\u2003\u2003(b) preconception care(11)56.139.14.85.47 (0.06)\nThe 11 issues were grouped into 4 factors, for both routine care and PCC (see item labels, Table 4). How physicians defined PCC had a significant effect on importance ratings overall, controlling for years in practice [F(8, 539)=3.437; P < 0.005]. However, the only individual factor on which ratings were distinctly different was folic acid supplementation during routine care: physicians who defined PCC as routine were more likely than those defining it as specialized to rate it as very important (49.3% vs 34.8%) during routine care [t(556)=2.53; P < 0.02].\nGender, controlling for years in practice, also had an overall effect on ratings [F(8, 541)=4.066; P < 0.001]: females tended to rate most items as more highly important than did males.\nPatient use of PCC\nOf the physicians who provide PCC, a third (32.3%) said that patients \u2018rarely\u2019 present for PCC, almost half (48.5%) said they \u2018sometimes\u2019 do, and 17.3% said patients \u2018frequently\u2019 present for PCC. Over a third (34.7%) agreed that their patients usually do not plan their pregnancies. Almost half (49.1%) of ObGyns said \u2018few\u2019 or \u2018none\u2019 of their pregnant patients came in for PCC prior to the pregnancy, and almost two thirds (63.1%) said \u2018most\u2019 or \u2018all\u2019 of their pregnant patients initially made contact with them once they were already pregnant. See Table 5. The frequency with which patients reportedly present for PCC was positively correlated with physician agreement that their patients plan their pregnancies (P < 0.001), as well as with how frequently physicians recommend PCC to the 6 different patient groups listed in Table 2 (all P\u2019s < 0.001, except \u2018Diabetic,\u2019 P=0.01).\nTable 5Patient use of pre-pregnancy planningPercent of physicians selecting a particular response to questionnaire items:How frequently do you have patients present for PCC?Never or rarelySometimesFrequently32.348.517.3Women that I see usually do not plan their pregnancies Agree (1\u20132)Neutral (3)Disagree (4\u20135)\u2003(on a 5 point scale)34.733.831.6How many of your pregnant patients came in for NoneFewSomeHalf or More\u2003preconception care before they became pregnant? (OB)3.345.838.312.6How many of your pregnant patients initially made AllMostManyHalf or Fewer\u2003contact with you once they were already pregnant? (OB)9.453.718.018.9\nPhysicians were asked to indicate how many of the patients who presented for PCC did so for each of three reasons (7 point scale: None Few Some Half Many Most All). Over four-fifths (82.7%) of physicians indicated that more than half of the patients do so to ensure a healthy pregnancy (mean 5.63\u00b10.062 on 7 pt scale). In contrast, 41.9% said more than half do so because of difficulties conceiving (mean 4.05\u00b1.058), and 20.0% said more than half do so because of an elevated risk of a birth defect or developmental disorder (mean 3.13\u00b10.064).\nDiscussion\nThe purpose of this study was to examine obstetrician-gynecologists\u2019 opinions and beliefs about PCC, and how frequently they perceived their patients to be utilizing PCC. Our findings document that most physicians think it is an important issue, that it has a positive effect on pregnancy outcomes, and that they are appropriately trained to provide it (Table 3). However, respondents reported that few of their patients seek PCC. Explanations for this lack of patients seeking PCC likely include the fact that almost 50% of pregnancies in the U.S. are unplanned [7], the lack of third party reimbursement for PCC visits, and patients\u2019 rather poor understanding of the potential benefits of PCC to the lifetime health of future offspring. We did find an association between the frequency with which physicians recommend PCC and the frequency with which patients present for PCC. It is also possible that some women receiving routine health care prior to a first pregnancy see an internist, family physician, or nurse practitioner for such care, and only seek the care of an obstetrician-gynecologist once they become pregnant. Thus, it is possible that our study of obstetrician-gynecologists does not reflect the full extent to which women may be seeking PCC. However, a recent study of primary care providers (not obstetrician-gynecologist) also found that few patients were receiving PCC [10].\nStumbling blocks to increasing utilization of PCC may include physicians\u2019 beliefs that time devoted to PCC is not reimbursed and that there is not enough time to provide PCC to all women of childbearing age. Regarding this latter point, opinions differ within the medical community as to who the target population for PCC should be. Three general target groups include women at high risk for poor birth outcome, women planning a pregnancy, and all women of childbearing age [11]. In this study, the vast majority of physicians said they frequently recommend PCC to diabetic and obese women planning a pregnancy, as well as to women generally who are planning a pregnancy (see Table 2). Far fewer obstetrician-gynecologists said they frequently recommend PCC to women described simply as sexually active (54%) or to those using birth control (36%). This suggests that our survey sample population views women who have consciously decided to plan a pregnancy as the most appropriate target group for PCC. Almost 9 in 10 (87%) physicians defined PCC as specialized pre-pregnancy care focusing on issues not typically addressed during a routine exam, and the remaining 13% defined it as routine well-woman care occurring during the reproductive years, prior to a pregnancy. Those who defined it as routine were more likely to offer such counseling to women who were sexually active or using birth control than were those defining it as specialized care.\nOne aspect of PCC includes counseling women on topics that encourage a healthy pregnancy, although many such topics are clearly relevant to the general health of any woman. Physicians were asked to rate eleven topics in terms of their importance for counseling during PCC and routine care, such as exercise, nutrition, and over the counter drug use (see Table 4). All were considered very important topics for counseling in PCC, with cigarette smoking and folic acid supplementation rated the most highly important for PCC. For counseling in routine care, folic acid supplementation received the second lowest score of all, with under two-fifths (37%) of physicians rating it as important. This points to the need for continued national attention to increase consumption and supplementation of folic acid.\nOur study has limitations that should be acknowledged. The response rate was 60%, and our findings are based on the responses of 579 non-subspecialist obstetricians and gynecologists. While our study may be subject to non-response bias, we believe that our findings are reliable. The typical response rate in these survey studies is approximately 35\u201360%, and our response rate was at the high end of expected participation. In addition, the responses were derived from geographically diverse locations and from physicians from different practice types reflecting the influence of physician location and practice type. Whereas physicians who were more interested in the topic of the survey may have been more likely to respond, a subset of our subject pool was comprised of CARN members who respond to several questionnaires a year covering a wide variety of topics; it is unlikely that Preconception Care is a topic of greater interest to this group than to the group of randomly selected ACOG members. CARN members differed significantly from non-CARN subjects on only one non-demographic response, and the mean age and male to female ratio of respondents closely matched those of the larger group to whom the survey was sent and of ACOG Fellows and Junior Fellows as a whole, all of which suggests that response bias was minimized.\nRecent studies have recommended offering PCC \u201copportunistically,\u201d incorporating information relevant to preconception care into routine well-woman visits [10, 12]. Most topics relevant to preventing congenital birth defects are also important to the overall health of women, pointing out the compatibility of the two counseling contexts. The one exception to this in our questionnaire was folic acid supplementation, and it would need greater emphasis in routine care to take advantage of opportunistic counseling. We found that those physicians who defined PCC as \u201cthe same as routine well-woman care\u201d differed from those defining it as \u201cspecialized\u201d in several ways, including increased importance of counseling on folic acid supplementation during routine care. A recent study found that topics such as vitamin supplements and alcohol use were rarely discussed during routine care by a high proportion of obstetrician-gynecologists, and by an even higher proportion of family practitioners [13]. They also found that physician recommendations influenced women's decisions to take folic acid.\nObstetrician-gynecologists recognize the importance of preconception care and provide this care for their patients. However, lack of third party reimbursement, lack of time during office visits due to competing demands, and lack of consumer awareness pose barriers to effective implementation of PCC. The fact that almost half of all pregnancies in the U.S. are unplanned poses an even greater challenge. Continued efforts are needed to raise awareness of the importance of PCC by consumers, health care providers, third party carriers, and policy makers.","keyphrases":["obstetrician-gynecologist","preconception care","folic acid","survey","routine care"],"prmu":["P","P","P","P","P"]}
{"id":"Planta-4-1-2270913","title":"Identification of an OsPR10a promoter region responsive to salicylic acid\n","text":"Orysa sativa pathogenesis-related protein 10a (OsPR10a) was induced by pathogens, salicylic acid (SA), jasmonic acid (JA), ethephon, abscisic acid (ABA), and NaCl. We tried to analyze the OsPR10a promoter to investigate the transcriptional regulation of OsPR10a by SA. We demonstrated the inducibility of OsPR10a promoter by SA using transgenic Arabidopsis carrying OsPR10a:GFP as well as by transient expression assays in rice. To further identify the promoter region responsible for its induction by SA, four different deletions of the OsPR10a promoter were made, and their activities were measured by transient assays. The construct containing 687-bp OsPR10a promoter from its start codon exhibited a six-fold increase of induction compared to the control in response to SA. Mutation in the W-box like element 1 (WLE 1) between 687 and 637-bp from TGACA to TGAAA completely abolished induction of the OsPR10a promoter by SA, indicating that the WLE 1 between \u2212687 and \u2212637 of OsPR10a promoter is important in SA-mediated OsPR10a expression. We show for the first time that the W-box like element plays a role in SA mediated PR gene expression.\nIntroduction\nPlants have developed defense mechanisms to recognize pathogens and subsequently activate defense-related genes, such as pathogenesis-related proteins (PR proteins). The major families of PR proteins have been grouped into at least 14 different classes, primarily on the basis of their amino acid sequences (Van Loon and Van Strien 1999). Although the biological and\/or biochemical functions of many PR proteins remain unclear, PR2 (\u03b2-1, 3-glucanase activity) and PR3 (chitinase) proteins have been shown to inhibit fungal growth (Woloshuk et al. 1991; Sela-Buurlarge et al. 1993). These responses are not limited to pathogen attack and can be induced by defense signaling molecules such as SA, JA and ET (Dempsey et al. 1999; Pieterse and van Loon 1999). To study the defense signaling in plants, many groups have isolated promoters of PR proteins in several plant species, such as Arabidopsis, tobacco, pepper, and rice (Malnoy et al. 2003; Hong et al. 2005; Li et al. 2005; Liu et al. 2005a; Lee and Hwang 2006).\nExpression profiles of PR proteins, such as OsPR1, OsPR10 and OsPR1b were reported. Originally, OsPR10a was known to be induced by probenazole and thus, was called a probenazole-inducible gene, PBZ1 (Midoh and Iwata 1996). Later, PBZ1 was renamed as OsPR10a because it shares a similar sequence with (has sequence similarity to) PR-10 proteins. The investigators reported that OsPR10a is only induced by probenazole but not by ethephon, NAA, SA, NaCl or mannitol in rice leaves. In contrast, Rakwal et al. (2001) reported that OsPR10a is induced by JA, SA, and ABA but not by IAA or GA in light. Ryu et al. (2006) found similar results using RT-PCR. Chen et al. (2006) reported that the elicitor derived from Magnaporthe grisea induces OsPR10a. However, there has been only one study on OsPR10a and OsCHNIII promoters, even though many reports are available for the expression profile of PR genes in rice (Rakwal et al. 2001; Hashimoto et al. 2004; Chen et al. 2006; Ryu et al. 2006). The authors reported that OsPR10a and OsCHNIII promoters are induced by an elicitor derived from Magnaporthe grisea by a transient assay in vitro. However, the cis elements were not analyzed.\nMost promoters induced by pathogens or SA contain the W-box, GCC box, RAV1 AAT, or ASF1 motif, etc. (Li et al. 2005; Lee and Hwang 2006; Sohn et al. 2006). Their cis-elements have been identified by series deletion of the promoter and site directed mutagenesis of its plausible site. Maleck et al. (2000) analyzed the transcriptome of Arabidopsis under defense inducing conditions, and they studied induced promoters such as PR1. The W-boxes ((T)TGACC\/T) are enriched in the PR1 regulon promoter. They also described that the W-box like element ((T)TGACA) is also enriched in PR-1 regulon promoters even though there is no evidence that WRKYs bind to this motif (Maleck et al. 2000).\nTranscription factors that can recognize the cognate cis element were identified by methods such as the gel-mobility shift assay, yeast-one hybrid, transient assay in plant. WRKY, ERF, RAV, bZIP, MYB, etc. have been shown to be involved in the defense signaling (Rushton et al. 1996, 2002; Eulgem et al. 1999; Kirsch et al. 2001; Heise et al. 2002). The interaction of a transcription factor to its cognate cis element is a key step in the process of defense signaling. Among transcription factors, WRKY proteins are the most extensively studied in defense signaling (Eulgem et al. 1999; Robatzek and Somssich 2001; Shimono et al. 2007). Asai et al. (2002) reported that AtWRKY22 and AtWRKY29 regulate FLS2-mediated defense signaling. The complex of TGA factor and NPR1 binds to the LS7 in the PR-1 promoter of Arabidopsis (Johnson et al. 2003). Furthermore, the TGA\/NPR1 complex is as well conserved in rice as in Arabidopsis (Fitzgerald et al. 2005). Recently, there are three reports that OsWRKY45, OsWRKY71 and OsWRKY03 regulate the defense signaling in rice, respectively (Liu et al. 2005b, 2006; Shimono et al. 2007), implicating that WRKYs also binds to the W boxes in rice as it does in Arabidopsis.\nIn this study, we analyzed the expression profile of OsPR10a. We isolated its promoter and analyzed its cis-elements. We also identified the WLE1 (TGACA) controlling induction of OsPR10a promoter by SA. This is the first report that the W-box like element actually plays a role in SA-mediated defense signaling.\nMaterial and methods\nPlant materials\nRice seedlings (Oryza sativa cv. Hwachung; seeds from Dr. Wan-He Ye, NIAST, Suwon, South Korea) were grown in a greenhouse at 28\u00b0C for 3\u00a0weeks. Three-week-old rice seedlings were washed, incubated in tap water for 2\u00a0days, and then treated with SA, JA, ethephon, ABA, or NaCl at 1\u00a0mM, 100\u00a0\u03bcM, 100\u00a0\u03bcM, 100\u00a0\u03bcM, and 200\u00a0mM, respectively. Rice leaves were harvested at the times indicated in the figures. For bacterial inoculations, a strain of Xanthomonas oryzae pv oryzae KXO98 (Xoo; obtained from Korean Agricultural Culture Collection, KACC, Suwon, South Korea) incompatible to O. sativa cv. Hwachung was grown in PSA medium (10\u00a0g peptone, 10\u00a0g sucrose, 1\u00a0g sodium\u2013glutamate, and 15\u00a0g agar per L) for 2\u00a0days and then resuspended in 1 mM MgCl2 to a final OD600 of 0.5. Xoo was sprayed on 3-week-old rice seedlings. After inoculation, plants were incubated in a humidity chamber for 24\u00a0h. Samples were taken at the times indicated in the figures and were immediately frozen in liquid nitrogen and stored at \u221280\u00b0C until further analysis.\nIsolation of OsPR10a promoter \nBased on an annotation of the rice genome, a \u22121000\u00a0bp fragment of the OsPR10a promoter was obtained by PCR from rice genomic DNA using an OsPR10a gene-specific primer sets. These primers were designed from the Genbank sequence AL845342. PCR was performed for 30 cycles under the following condition: 94\u00b0C for 30\u00a0s, 53\u00b0C for 1\u00a0min, and 72\u00b0C for 1\u00a0min, followed by a final extension at 72\u00b0C for 7\u00a0min. The primer sets are as follows:\n5\u2032-AAAAAGCAGGCTTGTTTTGAATGCTGGAATGATAA-3\u2032, and 5\u2032-AGAAAGCTGGGTCACTGAAGATATAATCTA-3\u2032.\nThe underlined sequences match the attB1 and attB2 sites for the Gateway cloning system (Invitrogen, Carlsbad, CA, USA). A 1000\u00a0bp amplified PCR product was cloned into pDONR221 to make an entry clone by BP clonase (Invitrogen); successful insertion was confirmed by sequencing.\nPromoter-LUC constructs\nThe reporter constructs used in the transient expression assays in this study were prepared according to the following procedure. For a 1.0\u00a0kb OsPR10a promoter, 1000-bp upstream from the start codon of OsPR10a was cloned by BP reaction into pDONR221 to make the \u22121000\u00a0bp PR10a promoter entry clone described in the previous section. \u22121000-OsPR10a:LUC was created by LR reaction with the \u22121000-PR10a entry clone and promoter destination vector (attB1-ccdB-Cmr-attB2-LUC, unpublished results; Invitrogen). On the basis of W-boxes involved in the activation of defense genes in plants to construct the deleted OsPR10a:LUC construct, we amplified the OsPR10a promoter region using these sense primers: \u2212818: 5\u2032-AAAAAGCAGGCTCGTGACATCAGATTGAGTAT-3\u2032\u2212687: 5\u2032-AAAAAGCAGGCTCGATAAAGGGTATTTGTTTA-3\u2032\u2212637: 5\u2032-AAAAAGCAGGCTACCTATCATCTAAAAGCATT-3\u2032.\nWe also used the antisense primer 5\u2032-AGAAAGCTGGGTCACTGAAGATATAATCTA-3\u2032. PCR was performed for 30 cycles under the following condition: 94\u00b0C for 30\u00a0s, 53\u201355\u00b0C for 1\u00a0min, and 72\u00b0C for 1\u00a0min, followed by a final extension at 72\u00b0C for 7\u00a0min.\nThe sequences underlined match attB1 and attB2 sites in the Gateway cloning system. These \u2212818, \u2212687, and \u2212637\u00a0bp amplified PCR products were cloned into pDONR221 to make entry clones by BP clonase and confirmed by sequencing. 818-, 687-, and 637-OsPR10a:LUC were created by LR reaction with the 818-, 687-, and 637-OsPR10a entry clones and the promoter destination vector (attB1-ccdB-Cmr-attB2-LUC, unpublished results).\nSite directed mutagenesis\nThe mutagenized reporter constructs used in the transient expression assays in this study were prepared according to the manufacturer\u2019s instruction (Stratagene, La Jolla, CA, USA). For the mutagenized 687 bp-OsPR10a promoter, \u22121000-OsPR10a entry clone (20\u00a0\u03bcg) was added to 1\u00a0\u03bcL of 10\u00d7 reaction buffer, 1\u00a0\u03bcL (1\u00a0\u03c1mole) phosphorylated specific mutagenic primer sets, 0.5\u00a0\u03bcL 10\u00a0mM dNTPs, 0.6\u00a0\u03bcL Quick solution; and the volume was adjusted to 10\u00a0\u03bcL with sterile deionized water. The solution was added to 0.3\u00a0\u03bcL of Pfu Turbo DNA polymerase (2.5\u00a0units\/\u03bcL). The reaction matrix mix was used for subsequent PCR. Specific PCRs were performed for 18 cycles under the following conditions: 95\u00b0C for 50\u00a0s, 60\u00b0C for 50\u00a0s, and 68\u00b0C for 1\u00a0min, followed by a final extension at 68\u00b0C for 7\u00a0min. After generation of the mutgenic double stranded plasmid containing staggered nicks, the product was treated with Dpn I and incubated at 42\u00b0C for 60\u00a0min. The nicked plasmid incorporating the desired mutations was purified with phenol and chloroform extraction and ligated with T4 DNA Ligase at 16\u00b0C for overnight. Five microliters of the mutated plasmid was transformed into E.coli (DH5\u03b1) cells. After transformation, the plasmid DNA was isolated from the mutagenic transformant and confirmed by sequencing. For PCR of mutant strand synthesis reaction, the following mutagenic primer pairs were used: 5\u2032-TGAAATGTAGTCGTACCTATCA-3\u2032 and 5\u2032-TGCTCTGAGATGGGTCTAAACA-3\u2032.\nParticle bombardment and transient expression assays\nLeaf bombardments were performed in a Biolistic PDS-1000\/He particle delivery system using 1100-p.s.i. rupture disks (BioRad, Hercules, CA, USA). Plasmid DNAs for particle bombardment were prepared as described by the manufacturer\u2019s instructions. For reporters, \u22121000-bp OsPR10a:LUC and 818-, 687-, 637-OsPR10a:LUC and m687-OsPR10a:LUC were used; 35S:RLUC was used as an internal control to normalize LUC activities between samples after bombardments. About 2\u00a0cm lengths of one-week old rice seedlings grown in the dark were cut and incubated on a plate in 1\/2 MS medium overnight (Murashige and Skoog 1962). Tungsten particles coated with 1000-, 818-, 687-, 637-OsPR10a:LUC, or m687-OsPR10a:LUC, and the internal control were delivered into leaf segments by the particle delivery system (BioRad). Leaf segments were incubated at 28\u00b0C for 24\u00a0h with buffer (1\/2\u00a0MS medium) or with 1\u00a0mM SA and then harvested. Leaf segments were ground in liquid nitrogen and dissociated in 1\u00d7 passive buffer. The luciferase activities from protein extracts were measured by a dual luciferase system (Promega, Madison, WI, USA) with a luminometer (Aureon Biosystems, Vienna, Austria).\nRT-PCR analysis\nLeaf samples were ground to powder in liquid nitrogen, and total RNA was extracted using the Trizol reagent according to the manufacturer\u2019s instructions (Invitrogen). For reverse transcription, total RNA (1\u00a0\u03bcg) was added to 1\u00a0\u03bcL of oligo (dT)16 and 1\u00a0\u03bcL of gene specific primer sets (0.5\u00a0\u03c1mole); and the volume was adjusted to 15\u00a0\u03bcL with sterile deionized water. The solution was incubated at 70\u00b0C for 5\u00a0min, then immediately transferred to ice before the addition of 35\u00a0\u03bcL of reverse transcriptase master mix containing 10\u00a0\u03bcL 5\u00d7 buffer, 3\u00a0\u03bcL 0.1\u00a0M DTT, 5\u00a0\u03bcL 10\u00a0mM dNTPs, 1\u00a0\u03bcL (200\u00a0units\/\u03bcL) M-MLV RTase (Promega) and 0.2\u00a0\u03bcL (40\u00a0units\/\u03bcL) RNasin (Promega). The reaction was incubated at 42\u00b0C for 90\u00a0min before heat inactivation at 65\u00b0C for 10\u00a0min. Two microliters of each reverse transcriptase reaction was used for subsequent PCR. Gene specific PCRs were performed for 35 cycles under the following conditions: 94\u00b0C for 30\u00a0s, 53\u00b0C for 1\u00a0min, and 72\u00b0C for 1\u00a0min, followed by a final extension at 72\u00b0C for 7\u00a0min. Samples were visualized on 1.2% agarose gels. For RT-PCR analysis of OsPR10a genes in rice, the following primer pairs were used: OsPR10a (D38170)5\u2032-GCTACAGGCATCAGTGGTCA-3\u2032 and 5\u2032-GACTCAAACGCCACGAGAAT-3\u2032,OsActin (XM469569) 5\u2032-TCCATCTTGGCATCTCTCAG-3\u2032and 5\u2032-GTACCCGCATCAGGCATCTG-3\u2032.\nGeneration of transgenic Arabidopsis, induction with SA, and fluorescence microscopy\nOsPR10a:GFP was constructed by LR reaction with pBGWFS7 (Gateway\u2122; Department of Plant Systems Biology, VIB-Ghent University, Belgium) and the OsPR10a promoter entry clone described in the previous section, and then transformed into Agrobacterium tumefaciens GV3101 for Arabidopsis. Arabidopsis (Columbia ecotype) was transformed with A. tumefaciens GV3101 carrying OsPR10a:GFP::GUS and 35S:GFP(35S:pBGWFS7::GUS) as an internal control. A bacterial suspension of A. tumefaciens GV3101 carrying OsPR10a:GFP::GUS and 35S:GFP::GUS was sprayed on the unopened flowers of Arabidopsis. T1 plants were screened by 0.3% Barstar spray (Misung, Daejeon, South Korea). Samples were taken from independent T1 plants for RT-PCR analysis. T2 plants were also screened by 0.3% Barstar spray. Three individual T2 plants in each line were used for induction with 1\u00a0mM SA at 28\u00b0C for 72\u00a0h. After induction, transgenic plants carrying OsPR10a:GFP and 35S:GFP were examined by fluorescence microscopy using an Olympus SZX-RFL3 (Olympus Optical Co., LTD, Tokyo, Japan). Excitation and emission filters SZX-FGFP and SZX-FGFPA were used for GFP and GFPA (Ex 460\u2013490\/Em510- for GFP and Ex460\u2013490\/Em510\u2013550 for GFPA). Images were captured with a JP\/FV300 camera (Olympus).\nResults\nExpression patterns of OsPR10a in response to different stimuli\nSeveral research groups have reported some discrepancy for the expression patterns of OsPR10a to some stimuli. Here we looked at the expression of OsPR10a by RT-PCR, which is a more sensitive method than reported previously. First, we tested whether OsPR10a is induced by a pathogen, as reported previously (Midoh and Iwata 1996; Ryu et al. 2006; Fig.\u00a01a). Induction of OsPR10a started at 6\u00a0h and reached a maximum at 48\u00a0h after Xoo infection, as reported previously (Ryu et al. 2006). We also determined whether OsPR10a was induced by biotic elicitors such as SA, JA, and ethephon. For the fist time, we show that OsPR10a is induced by ethephon (Fig.\u00a01b). Witzh regard to abiotic stress treatments, OsPR10a was induced by NaCl and ABA (Fig.\u00a01c). Taken together, we conclude that OsPR10a is induced by the pathogen Xoo, SA, JA, ethephon, NaCl, and ABA.\nFig.\u00a01Expression pattern of OsPR10a in rice leaves treated with Xoo and five compounds. a Three-week-old rice seedlings were infected with Xoo and were harvested at 0, 6, 12, 24, and 48\u00a0h. b, c Three-week-old rice seedlings were treated with SA, JA, ethephon, ABA, or NaCl and were harvested at 0, 6, 12, 24, and 48\u00a0h. Total RNA was isolated from each sample, and RT-PCR was performed using OsPR10a specific primer pair. Transcript levels of OsActin show that equal amounts of RNA were used in the RT-PCR samples\nThe OsPR10a promoter is induced by SA treatment as shown in transient-assay system\nWe analyzed the expression of the OsPR10a gene to pathogens and various phytohormones. We focused on the SA-mediated response of the OsPR10a gene. To investigate how the OsPR10a gene was transcriptionally regulated by SA, we isolated the OsPR10a promoter in a 1.0\u00a0kb genomic DNA fragment upstream from the start codon of the OsPR10a gene by PCR. To analyze whether the 1.0\u00a0kb OsPR10a promoter was activated by SA as expected by its expression pattern, we carried out a transient assay using particle bombardment. The 1.0\u00a0kb fragment of OsPR10a promoter was used to make a reporter construct (OsPR10a:LUC). Its schematic diagram is shown in Fig.\u00a02a. OsPR10a:LUC was introduced into rice leaves by particle bombardment; leaf segments were then treated with either buffer or SA. Protein extracts were prepared from samples after 24\u00a0h post-treatment, and their relative luciferase activities were measured. OsPR10a promoter activities were expressed as relative luciferase activities. Figure\u00a02b shows a representative graph out of more than three independent experiments. The absolute values from each experiment were different, but the relative ratios from each sample were similar. Luciferase activity in the SA-treated sample was about two-fold higher than in non-treated (control) samples (Fig.\u00a02b). This result indicates that the OsPR10a promoter is activated by SA, based on its expression profile.\nFig.\u00a02OsPR10a promoter activity in response to SA: a schematic representation of the OsPR10a promoter in the reporter construct. b A transient assay showing the OsPR10a promoter in response to SA. OsPR10a:LUC was bombarded into rice leaves, which were then incubated in MS medium or MS medium containing 1\u00a0mM SA at 28\u00b0C for 24\u00a0h. Protein extracts were made by dissociation in passive lysis buffer as described in \u201cMaterials and methods\u201d. Relative luciferase activity is the ratio of the value obtained with the SA-treated OsPR10a:LUC divided by the value obtained with the buffer-treated OsPR10a:LUC. Bars indicate the standard error of three replicates\nThe OsPR10a promoter is induced by SA treatment in stably transformed Arabidopsis \nWe further investigated whether SA, as seen in the transient assay, activates the OsPR10a promoter using a transgenic approach. A 1.0\u00a0kb fragment of the OsPR10a promoter was cloned into a promoter-less GFP::GUS expression vector to make a OsPR10a:GFP::GUS construct, and was then introduced into Arabidopsis by Agrobacterium-mediated transformation (Fig.\u00a03a). Transgenic Arabidopsis plants (T1) were screened by spraying with 0.3% Barstar and then with self-crossing. Induction of the OsPR10a promoter by SA was analyzed by GFP fluorescence in T2 transgenic Arabidopsis seedlings treated with either SA or buffer (Fig.\u00a03b). In these GFP filter images (>510\u00a0nm), transgenic Arabidopsis carrying OsPR10a:GFP::GUS exhibited an orange fluorescence in the SA-treated sample because the green fluorescence from GFP was mixed with the red fluorescence from plants themselves (Fig.\u00a03b, middle panel). The green fluorescence from GFP is shown more clearly using a GFPA filter (510\u2013550\u00a0nm; bottom panel of Fig.\u00a03b). As shown in Fig.\u00a03b, the OsPR10a promoter was clearly activated by SA in transgenic Arabidopsis.\nFig.\u00a03Fluorescence images of Arabidopsis transgenic plants carrying OsPR10a:GFP: a schematic diagram of OsPR10a:GFP::GUS fusion construct. b Induction of OsPR10a promoter by SA. OsPR10a:GFP::GUS was introduced into Arabidopsis by Agrobacterium-mediated transformation. Transgenic Arabidopsis seedlings carrying the OsPR10a:GFP::GUS was examined using fluorescence microscopy after SA treatment at 72\u00a0h. Non-transgenic Arabidopsis seedling was used as a control (left panel at mock and SA treatments). Shown are the bright-field images (upper panel Bright), the green fluorescent images using GFP filter (middle panel GFP) and the GFPA filter (bottom panel GFPA). Images are representatives from two independent experiments. The experiments were repeated at least twice\nAnalysis of cis-elements of OsPR10a promoter\nIn order to find a cis-acting element of the promoter in response to SA, an analysis was done using the PLACE program (a database for PLAnt Cis-acting Elements located at http:\/\/www.dna.affrc.go.jp\/cDNA\/place) (Fig.\u00a04). Among many putative cis elements, we only indicated cis-elements in boxes known to be related to defense inducers, such as the pathogens, SA, JA, and ethephon, of the OsPR10a gene shown in Fig.\u00a01 (Shinshi et al. 1995; Eulgem et al. 1999; Kagaya et al. 1999). The OsPR10a promoter analyzed by the PLACE program contains four W-boxes, whose detail sequences are different; there are one canonical W-box ((T)TGACC\/T) and three W-box like elements (WLE 1) containing TGAC core (TGACA). There would be more W-box like elements in defense gene regulon promoters. Therefore, we decided to name TGACA as the W-box like element 1 (WLE1). In addition, there are three RAV1AAT elements, and one ASF1 motif element (Fig.\u00a04). The W-box, RAV1AAT, and ASF1 motif are known to be cis-elements of the WRKY, RAV1, and bZIP proteins, respectively (Abe et al. 1997; Chen and Chen 2002; Yamamoto et al. 2004). The WRKY, RAV1, or bZIP proteins might be involved in the response of the OsPR10a promoter to SA. In addition to them, there are many cis-elements involved in ABA responsiveness, even though they are not indicated in Fig.\u00a04. These elements might be involved in the induction of OsPR10a by ABA as shown in Fig.\u00a01c. Interestingly, there is no cis-element, such as the JA responsive element (JERE) (AGACCGCC) or the ethylene response element (ERE) (AGCCGCC), which is the binding site for ethylene response element binding proteins (EREBP), despite the fact that OsPR10a was induced by JA and ethephon.\nFig.\u00a04Putative cis-acting elements in 1.0\u00a0kb OsPR10a promoter. The putative cis-elements are indicated in boxes and its name is given above each element. Arrows indicate the direction of the cis-element. W-box WRKY transcription factor binding site; RAV1AAT RAV transcription factor binding site; ASF1 motif bZIP factor binding site; WLE1 putative WRKY transcription factor binding site \nDeletion analysis of the OsPR10a promoter to identify the regions responsible for the induction by SA\nTo identify the region of the OsPR10a promoter involved in the response to SA, we made serial deletions of the OsPR10a promoter by PCR (Fig.\u00a05a). Deletions, beginning with the locations \u2212818, \u2212687, and \u2212637, were fused to the LUC coding sequences and 3\u2032 nopaline synthase gene terminator (Fig.\u00a05a). These four constructs were tested for SA inducibility of the OsPR10a promoter by introducing them into rice leaves using particle bombardment and then treating them with either buffer or SA for 24\u00a0h. Protein extracts were made from the bombarded leaves and their luciferase activities were measured (Fig.\u00a05b). In the case of the 1.0\u00a0kb OsPR10a:LUC construct, luciferase activity was increased up to two fold over the control with SA treatment but not in the 818:LUC construct, indicating that there is a weak positive cis-element in region I between \u22121000 and \u2212818\u00a0bp of OsPR10a promoter (Fig.\u00a05a). One ASF1 motif was found in region I. The exact positive element in this region has not yet been identified. Luciferase activity in the 687:LUC construct was increased up to sixfold with SA treatment, indicating that there is a negative element in region II between \u2212818 and \u2212687\u00a0bp of the OsPR10a promoter. There is only one WLE1 with the TGAC core (TGACA) and one RAV1AAT element in region II that is known to be bound by transcription factors associated with the defense signaling (Fig.\u00a04). Besides this, there are many putative cis-elements in region II (data not shown). Therefore, the exact negative element has not yet been determined. In the 637:LUC construct, there was only about a two-fold increase in luciferase activity with SA treatment, indicating that there is at least one positive element between \u2212687 and \u2212637 bp (region III) and another one between \u2212637 and 1\u00a0bp (region IV) of the OsPR10a promoter. There is only one WLE1 containing the TGAC core (TGACA) in region III, suggesting that this element may play an important role in the strong inducibility of the 687:LUC construct by SA (Table\u00a01). The W-box, RAV1AAT, and WLE1 are found in region IV of the OsPR10a promoter, and at least one of them can act as a weak positive element.\nFig.\u00a05Deletion analysis of OsPR10a promoter: a schematic diagrams of serial deletion constructs of OsPR10a promoter. The numbers to the left of each construct indicate the distance from the start codon ATG. The predicted cis-elements ( W-box, RAV1AAT, and ASF1motif) are indicated by their respective abbreviations. The start codon, ATG, is written in bold. b Luciferase activity in deletion constructs of the OsPR10a promoter. Each deletion construct OsPR10a:LUC was bombarded into rice leaves, which were incubated in MS liquid medium or MS medium containing 1\u00a0mM SA at 28\u00b0C for 24\u00a0h. Protein extracts were made by dissociation in passive lysis buffer as described in \u201cMaterials and methods\u201d. Bars indicate the standard error of three replicates. The values are the ratio of the value obtained from each deletion constructs of OsPR10a promoter treated with SA or buffer divided by the value obtained from 1.0\u00a0kb OsPR10a promoter construct treated with bufferTable\u00a01The list of putative cis-acting elements of the OsPR10a promoter in region IIIRegionaPositionbcis-Elements (#)\/putative factor cIII\u2212687 to \u2212637GT1CONSENSUS(1)\/GT-1, WLE1 (1)\/WRKYaOsPR10a promoter was divided into four regions depending on the presence of W-box. The region III between \u2212687 and \u2212637\u00a0bp of OsPR10a promoterbindicates the distance of upstream from the start codon of OsPR10acPutative cis-acting elements in region III of OsPR10a promoter were analyzed using PLACE (a database for PLAnt Cis-acting Elements located at the web site (http:\/\/www.dna.affrc.go.jp\/cDNA\/place)\nMutation of a W-box like element in OsPR10a promoter abolished its SA inducibility\nSA inducibility of the 687:LUC construct is the highest among deletion constructs, and only one WLE1 with the TGAC core is present in region III. To further verify this, the WLE1 in region III was mutagenized from TGAC to TGAA (Fig.\u00a06a). Eulgem et al. (1999) reported that the WRKY protein couldn\u2019t bind to a TGAA sequence; therefore, this mutation prevents the association of WRKY to the WLE1 of the OsPR10a promoter. Interestingly, SA inducibility of 687\u00a0bp-OsPR10a promoter was completely abolished in the mutagenized 687\u00a0bp-OsPR10a promoter, indicating that this WLE1 is involved in the SA inducibility of OsPR10a.\nFig.\u00a06The effect of the mutation in the WLE1 of OsPR10a promoter region III. a Sequences of the WLE1 (the \u2212659 to \u2212644\u00a0bp) in the OsPR10a promoter and the mWLE1 with the TGAAA instead of TGACA. The WLE1 sequence is underlined and bolded. The asterisk represents the mutated base in the WLE1. b Luciferase activity in 687:LUC and m687:LUC in rice leaves. Bar indicates the standard error of the three replicates\nDiscussion\nPlant defense mechanisms to pathogen attack have been extensively studied in Arabidopsis; however, it is not well studied in rice. To study the defense mechanisms in rice, we tried to understand the transcriptional regulation of OsPR10a because OsPR10a has been used as a marker of induction for the defense response in rice (Ryu et al. 2006; Chen et al. 2006). OsPR10a was originally cloned by Midoh and Iwata (1996). They reported that OsPR10a was induced by Magnaporthe grisea and probenazole but not by ethephon, NAA, SA, NaCl, mannitol, and wound. More recently, Rakwal et al. (2001) reported that OsPR10a was induced by various phytohormones, such as SA, JA, and ABA, but not by IAA and GA. There were discrepancies between these two reports. In our study, we have shown that OsPR10a was induced by Xanthomonas oryzae pv. oryzae, phytohormones, such as JA, SA, ethephon, ABA and NaCl, but not by IAA and GA. In the case of JA, our result is consistent with the previous report (Rakwal et al. 2001). However, in the case of SA, our result is consistent with the findings by Rakwal et al. (2001), but not with that one by Midoh and Iwata (1996). There might be some differences in the method of SA treatment. We treated rice seedlings with SA by the soil drenching method because our previous result, based on the expression of OsPR1 gene, indicated that spraying rice leaves with SA does not reliably induce the defense response. In the case of ethephon, our data are also not consistent with the results from Midoh and Iwata (1996). Their data on the expression of OsPR10a were generated by Northern blots, whereas our results were generated by a more sensitive method, RT-PCR. Our result is the first report on the response of OsPR10a to ethephon. Our data suggest that OsPR10a is induced by three different defense signaling transducers (SA, JA, and ethephon). For abiotic stress treatments, OsPR10a was induced by NaCl and ABA. In the case of NaCl, our result also differs from the data shown by Midoh and Iwata (1996). We think that there is a sensitivity difference due to the detection methods of OsPR10a mRNA between RT-PCR and Northern hybridization as in the case of ethephon. In the case of ABA, our result is consistent with a report by Rakwal et al. (2001). Taken together, we conclude that OsPR10a is induced by the pathogens, SA, JA, ethephon, NaCl, and ABA.\nIn this study, we focused on SA mediated induction of OsPR10a because SA mediated defense signaling is the most well studied in Arabidopsis. The OsPR10a promoter was isolated to study the transcriptional regulation of OsPR10a gene. Gene activity was induced by SA in a transient assay system as expected by its expression profile. Chen et al. (2006) reported that it was induced by an elicitor derived from Magnaporthe grisea as shown in a transient assay system. SA might be involved in elicitor-mediated defense signaling, yet there was no report on the activity of OsPR10a promoter in plants. Our data now have shown that the OsPR10a promoter was also activated by SA in stably transformed plants, as we have seen in a transient-assay system.\nThe cis-acting elements of the OsPR10a promoter were analyzed to find the elements responsible for its induction by SA. It resulted in many putative cis-acting elements. The transcription factors which play an important role in defense signaling are WRKY, ERF, bZIP, MYB, RAV1, etc. (Ruston and Somssich 1998; Singh et al. 2002; Sohn et al. 2006). Therefore, we searched binding sites in the OsPR10a promoter for WRKY, ERF, bZIP, MYB, and RAV1. We found W-box, RAV1AAT element, and ASF1 motif element. The W-box, RAV1AAT, and ASF1 motif are known to be the binding sites of the WRKY, RAV1, and bZIP proteins, respectively (Abe et al. 1997; Chen and Chen 2002; Yamamoto et al. 2004). This suggests that the WRKY, RAV1, or bZIP proteins might be responsible for the induction of OsPR10a promoter by SA. We also found several ABRE sequences that are known to be responsible for ABA responsiveness of the gene (Shinozaki and Yamaguchi-Shinozaki 1996). This element might be involved in the induction of OsPR10a by ABA. There is no cis-element, such as JERE or ERE even-though OsPR10a was induced by JA and ethephon. Induction of OsPR10a by JA and ethephon appears to occur indirectly through some other transcription factors bound to the OsPR10a promoter.\nBased on cis-elements found in OsPR10a promoter, three different deletion constructs (818:LUC, 687:LUC, and 637:LUC) were made. Induction of the OsPR10a promoter by SA was completely abolished using the 818:LUC construct, indicating that at least one weak positive element exists in region I. In the 687:LUC construct, there was approximately a sixfold increase compared to the 1.0\u00a0kb OsPR10a promoter construct. This suggests that at least one negative element exists in region II. In the 637:LUC construct, its activity was dramatically reduced compared to the 687:LUC construct, suggesting that there is a positive element in region III. Induction of the promoter by SA was also maintained in the 637:LUC construct, suggesting a positive element is present in region IV. In region III, there were a number of available cis elements in the OsPR10a promoter (Table\u00a01). However, only one WLE1 with the TGAC core was present in region III. Its nucleotide sequence is different from the canonical W-boxes ((T)TGACC\/T) (Maleck et al. 2000). However, they also described that the WLE1 (TGACA) is enriched in PR-1 regulon promoters. To verify involvement of the WLE1 in response to SA, its sequences were mutagenized (Eulgem et al. 1999). The mutation of the WLE1 from TGAC to TGAA in region III completely abolished the induction of the 687:LUC construct by SA. This suggests that the WLE1 is important in the expression of the OsPR10a gene in response to SA. This is the first finding that the WLE1 (TGACA) is important in SA mediated PR gene expression. Interaction of transcription factors and cis-acting elements constitute a key step in the defense signaling. The OsTGA factor interacts with OsNPR1 as reported in Arabidopsis (Chern et al. 2001; Yu et al. 2001). These authors suggest that NPR1-mediated defense signaling in Arabidopsis is conserved in rice. However, they did not report the identity of the target gene of this complex. Liu et al. (2005b) reported that OsWRKY12 induces the expression of OsNPR1 and OsPR1b; however, they did not show evidence that OsWRKY12 directly regulates the expression of OsNPR1 and OsPR1b since their experiments utilized transgenic plants over-expressing OsWRKY12. Here, we suggest that WRKY may play a major role in SA-mediated OsPR10a expression in rice. However, we cannot exclude involvement of other transcription factors in SA-mediated expression of OsPR10a. In the near-future, we will carry out electrophoretic mobility assays of the WRKY proteins to the WLE1 described in this study. We will further address what kinds of WRKY proteins regulate the OsPR10a promoter and identify the different partners required for SA-mediated OsPR10a expression.","keyphrases":["ospr10a promoter","salicylic acid","cis-acting element","salicylic acid induction"],"prmu":["P","P","P","R"]}
{"id":"Plant_Mol_Biol-3-1-2140092","title":"PsRBR1 encodes a pea retinoblastoma-related protein that is phosphorylated in axillary buds during dormancy-to-growth transition\n","text":"In intact plants, cells in axillary buds are arrested at the G1 phase of the cell cycle during dormancy. In mammalian cells, the cell cycle is suppressed at the G1 phase by the activities of retinoblastoma tumor suppressor gene (RB) family proteins, depending on their phosphorylation state. Here, we report the isolation of a pea cDNA clone encoding an RB-related protein (PsRBR1, Accession No. AB012024) with a high degree of amino acid conservation in comparison with RB family proteins. PsRBR1 protein was detected as two polypeptides using an anti-PsRBR1 antibody in dormant axillary buds, whereas it was detected as three polypeptides, which were the same two polypeptides and another larger polypeptide 2 h after terminal decapitation. Both in vitro-synthesized PsPRB1 protein and lambda protein phosphatase-treated PsRBR1 protein corresponded to the smallest polypeptide detected by anti-PsRBR1 antibody, suggesting that the three polypeptides correspond to non-phosphorylated form of PsRBR1 protein, and lower- and higher-molecular mass forms of phosphorylated PsRBR1 protein. Furthermore, in vivo labeling with [32P]-inorganic phosphate indicated that PsRBR1 protein was more phosphorylated before mRNA accumulation of cell cycle regulatory genes such as PCNA. Together these findings suggest that dormancy-to-growth transition in pea axillary buds is regulated by molecular mechanisms of cell cycle control similar to those in mammals, and that the PsRBR1 protein has an important role in suppressing the cell cycle during dormancy in axillary buds.\nIntroduction\nIn intact plants, the terminal bud predominantly grows, while axillary bud growth is suppressed. This phenomenon is called apical dominance. After terminal bud decapitation, however, the axillary buds grow rapidly and develop in the same manner as the terminal bud. Apical dominance was one of the first developmental phenomena shown to be regulated by plant hormones (Thimann and Skoog 1934). The outgrowth of axillary buds is inhibited by auxin flow from the terminal bud (Booker et\u00a0al. 2003), and is promoted by cytokinin biosynthesized in the stem after decapitation (Tanaka et\u00a0al. 2006). Recently, a variety of experimental approaches were used to examine the molecular mechanisms that control shoot branching (Bennett and Leyser 2006; Dun et\u00a0al. 2006; Shimizu-Sato and Mori 2001). Some mutants were isolated and characterized in Arabidopsis (max1, 2, 3, and 4), pea (rms1, 2, 3, 4, and 5), and petunia (dad1 and 2). Findings from these molecular genetic approaches combined with conventional physiologic studies, such as grafting experiments, suggested that novel signals regulate axillary bud dormancy. The molecular mechanisms of cell cycle control in axillary buds, however, are still not well understood.\nMost cells in pea axillary buds are arrested at the G1 phase in the cell cycle, and axillary buds have the capacity to undergo multiple cycles of dormancy and growth (Devitt and Stafstrom 1995; Shimizu and Mori 1998). Arrest and progression of the cell cycle in axillary buds are controlled in response to environmental signals and by developmental programs. Thus, we hypothesized that cells in dormant axillary buds are regulated through suppression mechanisms of cell cycle control suppression mechanisms similar to those in mammals.\nIn mammals, arrest and progression of the cell cycle are also controlled during the G1 phase in response to environmental signals and by developmental programs (Sherr 2004). This process is governed by both positive- and negative-regulatory factors. One of the most important key regulators is the product of the retinoblastoma tumor suppressor gene (RB). The human RB gene encodes a 110-kDa nuclear phosphoprotein (pRB). pRB activities are regulated by phosphorylation in a cell cycle-dependent manner. Phosphorylation states of pRB regulate the interactions of multiple cellular proteins. In the G1 phase, hypo-phosphorylated pRB (hypo-ppRB) binds to a transcriptional factor, E2F family protein, and suppresses its transcriptional activity. E2F family proteins regulate the transcription of several genes (e.g., dihydrofolate reductase, thymidine kinase, DNA polymerase \u03b1, and proliferating cell nuclear antigen [PCNA]) whose products are required for either G1\/S transition or DNA replication. Thus, by negatively regulating E2F family proteins, pRB negatively controls cell cycle progression. pRB is highly phosphorylated by several types of cyclin and cyclin-dependent kinase (CDK) complexes in response to environmental signals or developmental programs to progress the cell cycle. E2F family proteins are released from hyper-ppRB and promote the transcription of genes for G1\/S transition or DNA replication. Thus, pRB regulates the arrest and progression of the cell cycle based on its phosphorylation state.\nIn mammals, there are pRB-related proteins, p130 and p107 (Gra\u00f1a et\u00a0al. 1998). RB family proteins regulate the progression of the cell cycle as well as the entry\/exit transition of the cell cycle. RB family proteins have the same pocket protein feature; that is, they interact with a multitude of cellular proteins in a phosphorylation-dependent manner. RB family proteins are, however, modulated by differential regulation of phosphorylation states and protein levels in quiescent cells re-entering the cell cycle. In quiescent cells and in cells at the G0\/G1 transition, pRB is in a hypo-phosphorylated form, and p130 is detected in non- and low-phosphorylated forms, whereas p107 is not detectable (Lin and DeCaprio 2003). In mid-G1\/S transition, RB family proteins are phosphorylated by cyclin\/CDK complexes. Hypo-ppRB and non- and low-phosphorylated p130 change to hyper-ppRB and high-phosphorylated p130, respectively. p107 is synthesized de novo, followed by phosphorylation to a high-phosphorylated form. Protein levels of pRB remain relatively constant throughout the cell cycle, compared to either p130 or p107. Although there is abundant p130 in the G0\/G1 phase, it is dramatically down-regulated in the mid-G1\/S phase. p130 down-regulation appears to be mediated by a cell cycle stage-dependent proteasome degradation pathway (Tedesco et\u00a0al. 2002). These observations suggest that RB family proteins have overlapping functions, as well as different functions in the cell cycle regulation of quiescent cells.\nIn plants, knowledge of cell cycle control has increased in recent years (Gegas and Doonan 2006). Some basic mechanisms that regulate the cell cycle appear to have been conserved throughout eukaryotic evolution (Dewitte and Murray 2003). To date, plant cDNAs encoding RB-related proteins have been reported in Zea mays (Ach et\u00a0al. 1997; Grafi et\u00a0al. 1996; Xie et\u00a0al. 1996), Nicotiana tabacum (Nakagami et\u00a0al. 1999), Chenopodium rubrum (Fountain et\u00a0al. 1999), Arabidopsis thaliana (Kong et\u00a0al. 2000), Populus tremula\u00a0\u00d7\u00a0Populus tremuloides (AF133675), Cocos nucifera (AY117036), and Oryza sativa (AP004592). The amino acid conservation between animal and plant RB-related proteins suggests that the proteins have similar biochemical properties. Like animal RB family proteins, plant RB-related proteins also interact with various cellular proteins, such as E2F family proteins, D-type cyclin, mammalian viral oncoproteins (SV40 large-T antigen, adenovirus E1a, and HPV E7), and the plant virus proteins (wheat dwarf virus RepA, and the tomato golden mosaic virus replication factor AL1) (Ach et\u00a0al. 1997; Grafi et\u00a0al. 1996; Huntley et\u00a0al. 1998; Nakagami et\u00a0al. 1999; Ramirez-Parra et\u00a0al. 1999; Sekine et\u00a0al. 1999; Xie et\u00a0al. 1996).\nBecause the functions of the mammalian RB family proteins depend on their phosphorylation state, the phosphorylation states of plant RB-related proteins were analyzed. ZmRBR1 protein undergoes changes in phosphorylation states concomitant with endoreduplication in maize (Grafi et\u00a0al. 1996). The human G1\/S protein kinases cyclinD\/CDK4, cyclinE\/CDK2, and cyclinA\/CDK2 can phosphorylate ZmRBR1 protein in\u00a0vitro (Huntley et\u00a0al. 1998), and NtRBR1 protein is phosphorylated by tobacco cyclinD\/CDC2 in\u00a0vitro (Nakagami et\u00a0al. 1999). The ZmRBR1 kinase activity correlates with the proliferation state in maize leaf (Boniotti and Gutierrez 2001), and NtRBR1 kinase activity is detected only during the mid-G1\/S phase in tobacco BY-2 cells (Nakagami et\u00a0al. 2002). These observations also suggest that plant RB-related proteins have biochemical properties similar to those of mammalian RB family proteins. Plant RB-related proteins seem to control not only cell cycle arrest\/progression, but also development and cellular differentiation in endosperm, leaf, and root (Ebel et\u00a0al. 2004; Desvoyes et\u00a0al. 2006; Wildwater et\u00a0al. 2005). Only limited information is available, however, on the development-dependent phosphorylation states of plant RB-related protein. In this paper, we describe the isolation and functional characterization of a pea (Pisum sativum L. cv. Alaska) cDNA encoding an RB-related protein (PsRBR1), which has biochemical properties similar to those of mammalian RB family proteins. PsRBR1 protein undergoes changes in its phosphorylation state concomitant with dormancy-to-growth transition in pea axillary buds.\nMaterials and methods\nPlant growth and tissue collection\nSeeds of Pisum sativum L. cv. Alaska were soaked in running tap water for 24\u00a0h and sown in trays of rockwool. Plants were grown at 25\u00b0C in the dark for 4\u00a0days, and then in a 16\u00a0h light\/8\u00a0h dark photoperiod for 3\u00a0days. Tissues studied were axillary buds at the second node in 7-day-old seedlings. Plants were decapitated 1\u00a0cm above the second node to stimulate outgrowth of axillary buds.\nPCR and cloning of PsRBR1 cDNA\nDegenerate oligonucleotide primers were designed for conserved regions of published amino acid sequences of the RB family proteins (sense; 5\u2032-TT(T\/C)TT(T\/C)AA(T\/C)C GNCA(T\/C)AT(T\/C\/A)GA(T\/C)CA-3\u2032 and antisense; 5\u2032-AC(T\/C)TC(G\/A)TT(G\/A)TA(G\/A)AANGT(T\/G\/A)AT(T\/G\/A)AT-3\u2032), where the N in the parentheses indicates all four deoxyribonucleotides. Polymerase chain reaction (PCR) amplification was performed with cDNA from total RNAs of shoot apices. The amplified fragments (237\u00a0bp) were cloned into a BSII TSK-plasmid vector (Ichihara and Kurosawa 1993) and sequenced. A pea cDNA library was constructed using poly (A)+ RNA prepared from dormant axillary buds with a HybriZAP-2.1 A Two-Hybrid cDNA Gigapack Cloning Kit (Stratagene, La Jolla, CA). Approximately 1\u00a0\u00d7\u00a0106 phage recombinants derived from the pea cDNA library were screened with the selected PCR products labeled with [32P]-dCTP. Hybridization and subsequent washing were performed as described by Shimizu and Mori (1998).\nDNA sequencing\nThe cDNAs were sequenced by the dideoxy chain termination method using an automatic DNA sequencer (LIC-4000, LI-COR Inc., Lincoln, NE) according to the manufacturer\u2019s protocol. Both strands were entirely sequenced.\nYeast two-hybrid system\nThe vectors and strains were provided in the MATCHMAKER Two-Hybrid System 2 (Clontech, Palo Alto, CA). DNA manipulations and yeast methods were performed according to the manufacturer\u2019s instructions. PsRBR1 cDNA was introduced into the pAS2-1 vector, and another cDNA was introduced into the pACT2 vector. Saccharomyces cerevisiae Y187 was transformed with combinations of these plasmids. For quantitative liquid assay, \u03b2-galactosidase activity was assayed with O-nitrophenylgalactoside as a substrate.\nAntibody preparation and immunoblot analysis\nFor an anti-PsRBR1 antibody, full-length PsRBR1 protein was expressed in Escherichia coli BL21 pLysS as a 6xHis-tagged protein in pET32a (Novagen, Madison, WI). The antigen was purified with TALON Metal Affinity Resin (Clontech) according to the manufacturer\u2019s protocol. The antigen was injected into a mouse using conventional procedures.\nProteins from axillary buds were extracted by grinding with aluminum oxide in extraction buffer (20\u00a0mM Tris\u2013HCl [pH 8.0], 100\u00a0mM NaCl, 1\u00a0mM EDTA, 0.5% NP-40, 50\u00a0\u03bcM NaF, and 1\u00a0\u03bcM Na3VO4, and the proteinase inhibitor cocktail Complete [Roche, Penzberg, Germany]). Insoluble materials were removed by microcentrifugation. Protein concentrations were determined with the DC Protein Assay (Bio-Rad, Hercules, CA). Proteins were separated using sodium dodecyl sulfate\u2014polyacrylamide gel electrophoresis (SDS-PAGE, 10% acrylamide gels [acrylamide:bis-acrylamide\u00a0=\u00a037.5:1]), or 10% low bis-acrylamide gels [acrylamide:bis-acrylamide\u00a0=\u00a0142:1]), and blotted onto a nitrocellulose membrane (BA-S 85, Schleicher & Schuell, Dassel, Germany). The membrane was blocked with 5% skim milk and 0.05% Tween 20 in Tris-buffered saline (50\u00a0mM Tris\u2013HCl [pH 8.0], and 150\u00a0mM NaCl), and incubated with the anti-PsRBR1 antibody at a dilution of 1:1000. Horseradish peroxidase-conjugated goat anti-mouse IgG (Pierce, Rockford, IL) was used as the secondary antibody at a dilution of 1:2000. The signals were detected with the SuperSignal ULTRA (Pierce).\nIn\u00a0vitro-coupled transcription and translation\nA cDNA fragment corresponding to PsRBR1 cDNA, encoding the full-length protein, was cloned into the pBluescript SK+ vector (Stratagene). The template was digested with a restriction enzyme for linearization. In\u00a0vitro-coupled transcription-translation was performed with the digested template (1\u00a0\u03bcg) in a TnT\u00ae T3 Coupled Wheat Germ Extract System (Promega, Madison, WI) in the presence of [35S]-methionine according to the manufacturer\u2019s protocol. As a control, the in\u00a0vitro-coupled transcription-translation reaction was performed without the DNA template. Samples were separated by SDS-PAGE. The gel was dried and exposed to a Fuji Imaging Plate (Fuji Photo Film, Tokyo, Japan) overnight. The radiographic image was visualized with a BAS2000 Image Analyzer (Fuji Photo Film).\nDephosphorylation\nProtein extracts were incubated in reaction mixture (50\u00a0mM Hepes [pH 7.5], 2\u00a0mM MnCl2, 0.1\u00a0mM EDTA, 5\u00a0mM dithiothreitol, 0.01% Brij35, and the proteinase inhibitor cocktail Complete) with 1 unit of lambda protein phosphatase (\u03bb-PPase, New England Biolabs, Beverly, MA) for 30\u00a0min at 30\u00b0C. Samples were immunoblotted by the anti-PsRBR1 antibody as described above.\nRNA extraction and blot analysis\nTotal RNA was isolated from the tissues by SDS-phenol extraction, followed by LiCl precipitation. Formaldehyde agarose gel electrophoresis of total RNA was performed using standard procedures. The RNAs were blotted onto a Hybond N+ membrane (Amersham Biosciences), and hybridized with PsRBR1, PCNA, and Histone H4 cDNA labeled with [32P]-dCTP, respectively. Hybridization was performed in 0.25\u00a0M NaH2PO4 buffer (pH 7.2), 0.25\u00a0M NaCl, 7% SDS, 10% dextran sulfate (Amersham Biosciences), and 1% polyvinylpyrrolidone K30 at 65\u00b0C for at least 16\u00a0h. The membrane was washed in 2xSSPE (1xSSPE is 0.15\u00a0M NaCl, 10\u00a0mM NaH2PO4, and 1\u00a0mM EDTA [pH 7.4]) and 0.1% SDS at 50\u00b0C twice for 15\u00a0min each, and visualized by autoradiography at \u221280\u00b0C using Kodak XAR-5 film and an intensifying screen (Kodak, Rochester, NY).\nPhospholabeling and immunoprecipitation\n[32P]-inorganic phosphate (Amersham Biosciences, Piscataway, NJ) was applied to axillary buds (10\u00a0\u03bcCi per bud) 1\u00a0h before collection of the buds. Protein extraction was performed as described above. Protein extracts were incubated with both preimmune serum and Protein A Sepharose 4 Fast Flow (Amersham Biosciences) for 1.5\u00a0h at 4\u00b0C, and then non-specific associated materials were removed by microcentrifugation. The anti-PsRBR1 antibody was added to the clarified extracts and incubated for 1\u00a0h on ice. Protein A Sepharose 4 Fast Flow was added, and incubated for 30\u00a0min at 4\u00b0C. The complex of immunoprecipitates was washed three times with extraction buffer with 1\u00a0mg\/ml bovine serum albumin, resuspended in SDS sample buffer (20\u00a0mM Tris-HCl [pH 6.8], 40% glycerol, 2% SDS, 2% \u03b2-mercaptoethanol, and bromophenol blue), and separated by SDS-PAGE. Dried gels were placed on a Fuji Imaging Plate, and the radiographic images were analyzed using the BAS2000 Image Analyzer.\nResults\nIsolation of a pea cDNA encoding RB-related protein\nTo analyze the molecular mechanisms of cell cycle control in axillary buds, we focused on the function of plant RB-related proteins during dormancy-to-growth transition. Based on the amino acid sequences of reported RB-related proteins, we designed degenerate primers and performed reverse transcription PCR with total RNA prepared from pea shoot apices. Using the amplified DNA fragments as a probe, we screened the cDNA library prepared from pea dormant axillary buds. We obtained 14 positive clones by screening, and partially sequenced their 5\u2032 ends. The clone that had the longest 5\u2032 sequence (3540\u00a0bp) was used for further analysis because the DNA sequences of the other clones corresponded to those of the longest clone. This cDNA contained a single open reading frame capable of encoding a 1026-amino acid residue protein, PsRBR1. The molecular mass of PsRBR1 protein was estimated to be 114-kDa. The PsRBR1 cDNA contained a stop codon (TAG) four amino acids upstream of the putative initiation methionine codon within the reading frame, indicating that the PsRBR1 cDNA contained the entire coding region.\nThe alignment of the A and B conserved domains and the N-terminal leucine-rich domain of PsRBR1 protein with maize ZmRBR1 protein and human RB family proteins is shown in Fig.\u00a01(A). The A and B conserved domains of the PsRBR1 protein share 43% identity and 71% similarity, respectively with the A and B conserved domains of the ZmRBR1 protein. The conserved cysteine residue corresponding to Cys 706 of human pRB was present as Cys 798 in PsRBR1 protein (shown by an asterisk in the B conserved domain). This conserved cysteine residue is essential for the function of mammalian and plant RB proteins (Ach et\u00a0al. 1997; Grafi et\u00a0al. 1996; Kaye et\u00a0al. 1990).\nFig.\u00a01Comparison of deduced amino acid sequences of pea PsRBR1 with selected homologous sequences. (A) Sequence alignments of the A and B conserved domains, and the N-terminal leucine-rich domain of PsRBR1 protein and ZmRBR1 protein with human RB family proteins. Black boxes indicate conserved amino acid residues, and dashes indicate gaps in the sequences. The asterisk in the B conserved domain denotes a conserved cysteine residue (798 of PsRBR1 protein), essential for RB function in mammalian cells. (B) Phylogenic analysis of the RB-related proteins under neighbor-joining analysis, and schematic of the salient features of RB-related proteins. The phylogeny is based on the amino acid sequences from the A conserved domain of RB-related proteins. Branch lengths are indicated above each branch. Bootstrap values from the results of 1000 replicates are shown below a node if the node is present in more than 50% of the bootstrap replicate analyses. The three conserved regions (N, N-terminal leucine-rich domain; A, A conserved domain; B, B conserved domain) between plants and mammals are indicated in boxes. Bars represent potential CDK phosphorylation sites. Numbers refer to protein length in amino acids, and numbers in parentheses show potential CDK phosphorylation sites. The accession numbers are AtRBR1 (AC069472), CnRBR1 (AY117036), CrRBR1 (AJ011681), NtRBR1 (AB015221), OsRBR1 (AP004592), PsRBR1 (AB012024), PtRBR1 (AF133675), ZmRBR1 (AF250050), p107 (L14812), p130 (X76061), and pRB (AF551763)\nThe phylogenic analysis of plant RB-related proteins reported so far and human RB family proteins is shown in Fig.\u00a01(B). The scheme on the right in Fig.\u00a01(B) shows a diagram of the characteristic features of RB-related proteins. PsRBR1 protein was most similar to Populus tremula\u00a0\u00d7\u00a0Populus tremuloides PtRBR1 protein. PsRBR1 protein has 16 potential phosphorylation sites (Ser-Pro or Thr-Pro) for CDKs, which are highly clustered in regions flanking the A and B conserved domains. These conserved primary structures strongly suggest that RB-related proteins, including PsRBR1 protein, have similar biochemical properties in plant and animals.\nGenomic Southern blot analyses were performed to estimate the copy number and closely related genes of PsRBR1 in pea. Full-length PsRBR1 cDNA was used as a probe. Hybridization at both high- and low-stringencies yielded only signals derived from PsRBR1 (data not shown). These results suggested that PsRBR1 gene exists as a single-copy gene in pea.\nInteractions of PsRBR1 protein with both plant D-type cyclins and a mammalian oncoprotein\nMammalian and plant RB proteins bind to a large number of cellular and viral proteins, such as D-type cyclin and SV40 large-T antigen. To confirm that PsRBR1 protein acts in the same manner as the mammalian and various plant RB proteins, we tested the ability of PsRBR1 protein to interact with these proteins using a yeast two-hybrid system. The genes for the tested proteins were fused to the GAL4 DNA-binding domain or the GAL4 activation domain. The data shown in Table\u00a01 are the averages of three independent transformants. We verified that PsRBR1 protein alone did not activate the reporter gene. PsRBR1 protein interacted with both pea and Arabidopsis D-type cyclin (Pissa;CycD3;1 and Arath;CycD3;1). Both D-type cyclins contain the canonical RB-binding motif, LXCXE, in their N-terminus (Shimizu and Mori 1998; Soni et\u00a0al. 1995). PsRBR1 protein interacted with SV40 large-T antigen, which is a mammalian DNA tumor virus oncoprotein. The similarities in primary structures and the coincidence of interacting partners of RB-related proteins suggest that animal and plant RB-related proteins, including PsRBR1 protein, have similar functions in cell cycle regulation.\nTable\u00a01PsRBR1 protein binding in the two-hybrid assayPlasmida\u03b2-galactosidase activitybPsRBR10.11\u00a0\u00b1\u00a00.01PsRBR1\u00a0+\u00a0Pissa;CycD3;113.9\u00a0\u00b1\u00a00.2PsRBR1\u00a0+\u00a0Arath;CycD3;110.6\u00a0\u00b1\u00a00.1PsRBR1\u00a0+\u00a0SV 40 large-T antigen31.5\u00a0\u00b1\u00a00.1aSaccharomyces cerevisiae Y187 was transformed with the indicated plasmidsb1 unit of \u03b2-galactosidase is defined as the amount that hydrolyzes 1 \u03bcmol of O-nitrophenylgalactoside to O-nitrophenyl and d-galactose per min. LacZ assays were done in triplicate and the data represent mean\u00a0\u00b1\u00a0standard deviation\nImmunochemical detection of PsRBR1 protein in pea axillary buds\nTo investigate protein levels and phosphorylation states of PsRBR1 protein in axillary buds during dormancy-to-growth transition, we prepared an anti-PsRBR1 antibody. The specificity of the anti-PsRBR1 antibody against PsRBR1 protein was examined (Fig.\u00a02A). Protein extracted from growing axillary buds was detected by immunoblotting with the anti-PsRBR1 antibody. The anti-PsRBR1 antibody cross-reacted with three polypeptides (Fig.\u00a02A, lane 1, filled circle, empty circle, and arrowhead), with molecular masses of approximately 150, 140, and 114-kDa. The pre-immune serum did not cross-react with any polypeptides in the protein extracts (Fig.\u00a02A, lane 2). The anti-PsRBR1 antibody was incubated with an excess of purified recombinant PsRBR1 protein as a competitor, and then used to detect PsRBR1 protein in pea extract (Fig.\u00a02A, lane 3). In this case, no polypeptides were detected with the anti-PsRBR1 antibody. These results indicate that the anti-PsRBR1 antibody specifically cross-reacts with PsRBR1 protein in axillary bud extracts.\nFig.\u00a02Immunochemical detection of PsRBR1 protein in pea axillary buds. (A) The terminal bud was cut 1\u00a0cm above the second node. After 24\u00a0h, the growing axillary buds were collected. Proteins in the protein extracts (30 \u03bcg) prepared from axillary buds were detected by immunoblotting with the anti-PsRBR1 antibody (lane 1), pre-immune serum (lane 2), and anti-PsRBR1 antibody preincubated with excess recombinant PsRBR1 protein (lane 3). Molecular mass markers are indicated at the left of the lane. The anti-PsRBR1 antibody cross-reacted with three polypeptides, indicated by the filled circle, empty circle, and arrowhead. (B) Comparison of molecular mass of authentic PsRBR1 protein with that of PsRBR1 synthesized by in\u00a0vitro-coupled transcription-translation reaction. The plasmid DNA containing the T3 promoter and the PsRBR1 coding sequence was transcribed, and the resultant RNA was translated. The translation products labeled with [35S]-methionine (lane 2) were immunoprecipitated by the anti-PsRBR1 antibody (lane 3). Proteins in the protein extracts prepared from growing axillary buds were detected by immunoblotting with the anti-PsRBR1 antibody (lane 4). The in\u00a0vitro-coupled transcription-translation reaction was performed without the plasmid DNA (lane 1). Molecular mass markers are indicated at the left of the lane. The product synthesized in\u00a0vitro was a 114-kDa polypeptide, indicated by the arrowhead (lane 2), which is consistent with the calculated values based on the deduced amino acids sequences of PsRBR1 cDNA. (C) Dephosphorylation of authentic PsRBR1 protein. Protein extracts prepared from growing axillary buds were dephosphorylated with \u03bb-PPase treatment, separated by SDS-PAGE in either a standard acrylamide gel (left) or low bis-acrylamide gel (right), and proteins were detected by immunoblotting with the anti-PsRBR1 antibody. Numbers in parentheses indicate the ratio of acrylamide to bis-acrylamide. Lane 1, untreated samples; lane 2, samples incubated without \u03bb-PPase; lane 3, samples incubated with \u03bb-PPase\nComparison of molecular mass of authentic PsRBR1 protein in pea with that of in\u00a0vitro-synthesized PsRBR1 protein\nThree polypeptides were detected in protein extracts from pea axillary buds by immunoblotting with the anti-PsRBR1 antibody. The molecular mass of PsRBR1 protein was estimated to be 114-kDa based on the deduced amino acid sequences of PsRBR1 cDNA. To clarify these differences in the molecular mass of PsRBR1 protein, we compared the molecular mass of authentic PsRBR1 protein in pea with that of in\u00a0vitro-synthesized PsRBR1 protein (Fig.\u00a02B). PsRBR1 protein was synthesized using an in\u00a0vitro-coupled transcription-translation system with [35S]-methionine. In\u00a0vitro-synthesized PsRBR1 protein produced a major 114-kDa polypeptide (Fig.\u00a02B, lane 2, arrowhead). The molecular mass of the PsRBR1 protein was consistent with that predicted from the deduced amino acid sequences of PsRBR1 cDNA. The 114-kDa polypeptide was not detected in protein products prepared from the in\u00a0vitro-coupled transcription-translation system without the PsRBR1 DNA template, which was used as a negative control (Fig.\u00a02B, lane 1). To confirm whether the 114-kDa polypeptide was PsRBR1 protein, in\u00a0vitro-coupled transcription-translation products were immunoprecipitated with the anti-PsRBR1 antibody (Fig.\u00a02B, lane 3). The 114-kDa polypeptide was immunoprecipitated with the anti-PsRBR1 antibody, indicating that in\u00a0vitro-synthesized PsRBR1 protein is 114-kDa and corresponds to the smallest polypeptide detected by anti-PsRBR1 antibody in growing axillary buds (Fig.\u00a02B, lane 4). These results suggested that the smallest polypeptide (114-kDa) detected by the anti-PsRBR1 antibody was non-modified PsRBR1 protein in axillary buds, and the two larger polypeptides of PsRBR1 proteins were modified by post-translational regulation in axillary buds.\nDephosphorylation of authentic PsRBR1 protein\nThe activity of mammalian RB family proteins is regulated by their phosphorylation states. Plant RB-related proteins are also phosphorylated by cyclin\/CDK complexes (Huntley et\u00a0al. 1998; Nakagami et\u00a0al. 1999). PsRBR1 protein has some putative CDK phosphorylation sites (Fig.\u00a01B). Thus, we expected that the modification of authentic PsRBR1 protein in the axillary buds caused its phosphorylation. To examine this, we performed in\u00a0vitro dephosphorylation reactions against PsRBR1 protein using (\u03bb-PPase. Proteins were separated by two types of SDS-PAGE gels, because some phosphorylated proteins were more clearly separated by low bis-acrylamide gel (Fig.\u00a02C, right) than by standard acrylamide gel (Fig.\u00a02C, left) (Iwasaki et\u00a0al. 2002). Immunoblotting with anti-PsRBR1 antibody indicated that there were three polypeptides corresponding to PsRBR1 protein in growing axillary buds (Fig.\u00a02C, lane 1), but after \u03bb-PPase treatment of the protein extract, PsRBR1 protein was detected as the smallest of the polypeptides (Fig.\u00a02C, lane 3). On the other hand, the molecular mass of PsRBR1 protein did not change after treatment without \u03bb-PPase added to the protein extract as a negative control (Fig.\u00a02C, lane 2). In addition, the low bis-acrylamide gel separated the protein more clearly than did the standard acrylamide gel. These results suggested that PsRBR1 protein was controlled by post-translational modification, and exists in non-phosphorylated form of PsRBR1 protein, lower-molecular mass form of phosphorylated PsRBR1 protein (Lp-form), and higher-molecular mass form of phosphorylated PsRBR1 protein (Hp-form) in growing axillary buds.\nProtein levels and phosphorylation states of PsRBR1 protein in axillary buds during dormancy-to-growth transition\nBecause suppression of the cell cycle is regulated by the phosphorylation states of mammalian RB family proteins, we evaluated the phosphorylation states of PsRBR1 during dormancy-to-growth transition in pea axillary buds by immunoblotting using the anti-PsRBR1 antibody (Fig.\u00a03A). During dormancy, PsRBR1 protein was detected as two polypeptides, which are non-phosphorylated form of PsRBR1 protein and the Lp-form (Fig.\u00a03A, 0\u00a0h after decapitation). In addition to these non-phosphorylated form of PsRBR1 protein and the Lp-form, the Hp-form was detected 2\u00a0h after decapitation (Fig.\u00a03A, indicated by the arrow). These results suggested that PsRBR1 protein was shifted from non-phosphorylated form of PsRBR1 protein and the Lp-form to the Hp-form; that is, the PsRBR1 protein was more phosphorylated after decapitation. The protein levels of these three polypeptides were increased 12\u00a0h after decapitation. Non-phosphorylated PsRBR1 protein and the Lp-form existed during late stage (12 to 24\u00a0h) after decapitation. PsRBR1 mRNA level was increased 12\u00a0h after decapitation (see Fig.\u00a03B). It seems that non-phosphorylated PsRBR1 protein and the Lp-form were synthesized and phosphorylated de novo after decapitation. Moreover, because the axillary bud cell cycle is not completely synchronized, the non-phosphorylated PsRBR1 protein and the Lp-form were included in the protein extracts prepared from growing axillary buds.\nFig.\u00a03Protein levels and phosphorylation states of PsRBR1 protein in axillary buds during the dormancy-to-growth transition. (A) Proteins were extracted from axillary buds after terminal bud decapitation. Numbers below each lane indicate the hours after decapitation. Proteins in the protein extracts (30\u00a0\u03bcg) were detected by immunoblotting with the anti-PsRBR1 antibody. Molecular mass markers are indicated at the left of the lane. The bottom panel shows the Coomassie Brilliant Blue (CBB) staining around 100\u00a0kDa as a loading control. The filled circle, empty circle, and arrowhead indicate higher-molecular mass form of phosphorylated PsRBR1 protein (Hp-form), lower-molecular mass form of phosphorylated PsRBR1 protein (Lp-form) and non-phosphorylated PsRBR1 protein, respectively. The arrow shows more phosphorylated PsRBR1 protein (Hp-form) appearing 2\u00a0h after decapitation. (B) Northern blot analyses of total RNA isolated from pea axillary buds before and after decapitation. Plants were decapitated 1\u00a0cm above the second node. After decapitation, axillary buds were collected at several time points, as indicated below each lane. Total RNA (10\u00a0\u03bcg) was separated by denaturing formaldehyde gel electrophoresis, and transferred to a Hybond N+ membrane. The membranes were hybridized with the cDNA of PsRBR1, PCNA, and histone H4. The bottom panel shows the ethidium bromide-stained RNA gel as a loading control\nAccumulation patterns of PsRBR1 transcripts in axillary buds during dormancy-to-growth transition\nTo investigate the relationship between different phosphorylation states of PsRBR1 protein with the transcriptional control of cell cycle regulators, we examined the accumulation patterns of PsRBR1, PCNA, and Histone H4 transcripts during dormancy-to-growth transition in axillary buds. The results of Northern blot analyses using PsRBR1, PCNA, and histone H4 cDNAs (Shimizu and Mori 1998) are shown in Fig.\u00a03B. PsRBR1 transcripts were detected at 3.6\u00a0kb. The mRNA levels of the three genes were very low during dormancy in axillary buds. When the axillary buds were stimulated to grow by terminal bud decapitation, mRNA levels of these genes increased markedly. PsRBR1 and histone H4 mRNA increased 12\u00a0h after decapitation. Histone H4 was specifically expressed during the S phase. The accumulation patterns of PsRBR1 mRNA are similar to that of histone H4 mRNA, suggesting that PsRBR1 is predominantly expressed at the S phase in pea axillary buds at least during the first 24\u00a0h after terminal bud decapitation. Because PsRBR1 protein was increased 12\u00a0h after decapitation (see Fig.\u00a03A), the expression patterns of PsRBR1 are coincident with those of PsRBR1 protein. The mRNA expression of PCNA seems to be regulated by RB-related proteins. PCNA mRNA increased significantly 4\u00a0h after decapitation, and PCNA mRNA accumulated after the appearance of the Hp-form.\nDetection of phosphorylated PsRBR1 protein in axillary buds after decapitation\nWe confirmed that PsRBR1 protein was phosphorylated in the axillary buds just after decapitation with in\u00a0vivo labeling followed by immunoprecipitation using the anti-PsRBR1 antibody (Fig.\u00a04). [32P]-inorganic phosphate was incorporated into growing axillary buds. Many proteins were phosphorylated in growing axillary buds after decapitation (Fig.\u00a04A, lane 1). Immunoprecipitation with the anti-PsRBR1 antibody revealed a [32P]-labeled 150-kDa phosphoprotein (Fig.\u00a04A, lane 3, arrow). The size of the [32P]-labeled PsRBR1 protein was consistent with the Hp-form detected by immunoblotting analysis. The signal was not detected by immunoprecipitation with preimmune serum used as a negative control (Fig.\u00a04A, lane 2).\nFig.\u00a04Detection of phosphorylation of PsRBR1 protein in axillary buds during dormancy-to-growth transition. (A) Eight hours after decapitation of the terminal bud, [32P]-inorganic phosphate was applied to axillary buds to be incorporated in\u00a0vivo. One hour after the application of [32P]-inorganic phosphate, proteins were extracted from the axillary buds. The protein extracts were immunoprecipitated with the anti-PsRBR1 antibody (lane 3) or preimmune serum (lane 2), and separated by SDS-PAGE. Total proteins without immunoprecipitation were loaded in lane 1. The dried gel was exposed to a Fuji Imaging Plate. Molecular mass markers are indicated on the left of the lane. (B) [32P]-inorganic phosphate was applied to axillary buds 1\u00a0h before collection of the buds. The axillary buds were collected at 0, 2, and 4\u00a0h after terminal bud decapitation (lanes 1 to 3, respectively), and proteins were extracted from the axillary buds. Other details are as described in (A)\nTo confirm whether PsRBR1 protein is more phosphorylated in axillary buds just after decapitation, we examined dormant and outgrowing axillary buds after decapitation. No signal was detected before decapitation (Fig.\u00a04B, lane 1), suggesting that PsRBR1 protein was not highly phosphorylated de novo before decapitation. Two hours after decapitation, the 150-kDa polypeptide was detected (Fig.\u00a04B, lane 2) and the signal was increased 4\u00a0h after decapitation (Fig.\u00a04B, lane 3, arrow). These results suggested that the level of phosphorylation of PsRBR1 protein increased immediately after terminal bud decapitation, producing the Hp-form.\nDiscussion\nIn intact plants, the pea shoot apex inhibits growth; i.e., cell elongation and cell division of the dormant axillary buds. The cell cycle of dormant axillary buds is arrested at the G1 phase. Therefore, from the point of view of cell cycle control, the transition from dormancy to outgrowth in axillary buds seems to be from the G1 phase to the S phase. In animal cells, RB family proteins function as negative controllers in G1 arrest, and the activities of RB family proteins are controlled by their phosphorylation states. Here, we investigated phosphorylation states of the pea RB-related protein, PsRBR1, during the dormancy-to-growth transition in axillary buds. The results of the present study provide a basis for further studies of the molecular mechanisms underlying apical dominance and cell cycle control.\nMammalian and plant RB proteins have several highly conserved regions, which are the N-terminal leucine rich, A and B conserved domains, multiple potential CDK phosphorylation sites, and a conserved cysteine residue. PsRBR1 protein, like other plant RB-related proteins, has the ability to interact with both plant D-type cyclins and a mammalian oncoprotein. Plant RB-related proteins have the same properties and activities as animal RB family proteins. For example, ZmRBR1 protein binds to human and Drosophila E2F, and inhibits transcriptional activation of human E2F (Huntley et\u00a0al. 1998; Ramirez-Parra et\u00a0al. 1999; Sekine et\u00a0al. 1999). Geminivirus DNA replication is reduced in plant cells transfected with plasmids encoding human p130 (Xie et\u00a0al. 1996). Human cyclinD\/CDKA phosphorylates NtRBR1 protein in\u00a0vitro (Nakagami et\u00a0al. 1999). Thus, plant RB-related proteins can function not only as plant factors, but also as animal factors. These results strongly suggest that the functions of plant RB-related proteins, including PsRBR1 protein, are similar to those of the mammalian RB family proteins in G1\/S transition, and that plant and animal cells might use similar regulatory proteins and pathways for cell cycle control during the G1 phase. Therefore, the maintenance of this high degree of similarity throughout evolution (in plants, insects, and vertebrates), further emphasizes the critical roles of RB-related proteins.\nThree human RB family proteins (pRB, p130, and p107) have individual functions in cell cycle regulation and development. Two RB-related genes were isolated from Drosophila melanogaster. To date, plant RB-related proteins have been reported in Zea mays, Nicotiana tabacum, Chenopodium rubrum, Arabidopsis thaliana, Populus tremula x Populus tremuloides, Cocos nunucifera, and Oryza sativa (Ach et\u00a0al. 1997; Fountain et\u00a0al. 1999; Grafi et\u00a0al. 1996; Kong et\u00a0al. 2000; Nakagami et\u00a0al. 1999; Xie et\u00a0al. 1996). Of these, Arabidopsis thaliana and Oryza sativa might contain only one copy of the RB-related gene in the whole genome based on the similarity of amino acid sequences of RB-related proteins. Here, our genomic Southern blot analysis suggested the presence of only one copy of the PsRBR1 gene in pea. The dicot RB-related gene seems to contain only one copy in the genome. By contrast, maize contains at least three RB-related genes. ZmRBR3 protein also functions to promote endosperm development (Sabelli et\u00a0al. 2005), suggesting that maize RB family proteins have special roles in endosperm development.\nWhen the protein extracts in growing axillary buds were treated with \u03bb-PPase, the Lp- and Hp-forms should have been dephopshorylated and stoichiometrically shifted to non-phosphorylated forms. As shown in Fig.\u00a03(C), however, it appears that there was no increase in the non-phosphorylated form (lane 3) compared to the other lanes. The reason for this might be that a 40-kDa polypeptide was also detected by immunoblotting with the anti-PsRBR1 antibody (data not shown). The 40-kDa polypeptide was probably derived from in\u00a0vitro-dephosphorylated PsRBR1 protein by degradation during \u03bb-PPase treatment. In\u00a0vitro-dephosphorylated PsRBR1 protein might be more unstable than in\u00a0vivo-dephosphorylated PsRBR1 protein, despite the presence of proteinase inhibitors, though the reason for this is not clear.\nThe results of the immunoblotting experiments indicated that the migration patterns of mammalian RB family proteins differ depending on their phosphorylation states. According to the reports (Farkas et\u00a0al. 2002), the faster migrating proteins were the non- and hypo-phosphorylated forms in the G0\/G1 phases. The slower migrating protein was the hyper-phosphorylated form during the G1\/S transition. In plant, immunoblotting with the anti-ZmRBR1 antibody revealed the ZmRBR1 protein to be a diffuse protein band of approximately 110-kDa during endoreduplication in maize endosperm, and there are some polypeptides of approximately 110-kDa in maize leaf (Grafi et\u00a0al. 1996; Huntley et\u00a0al. 1998). The diffused protein bands of ZmRBR1 were confirmed by the use of \u03bb-phosphatase to result from phosphorylation. (Grafi et\u00a0al. 1996). In the present paper, using \u03bb-PPase treatment and in\u00a0vivo labeling followed by immunoprecipitation with the anti-PsRBR1 antibody, we showed that PsRBR1 protein exists in three phosphorylated forms in axillary buds. Although we could not determine the number of phosphorylation sites in the Lp- and Hp-forms, the Lp- and Hp-forms might correspond to the hypo- and hyper-phosphorylated forms of PsRBR1 protein, respectively.\nPlant E2F isolated from tobacco and wheat has transcriptional activity and interacts with RB-related protein (Ramirez-Parra et\u00a0al. 1999; Sekine et\u00a0al. 1999). There are E2F-binding sites in the promoter region of PCNA, whose transcripts are induced in the late-G1\/S phase (Kodama et\u00a0al. 1991). These observations suggest that plant E2F, like that in animal, induces gene expression of PCNA. Further phosphorylation of PsRBR1 protein was induced 2\u00a0h after terminal bud decapitation, and PCNA transcripts were induced 4\u00a0h after terminal bud decapitation in pea axillary buds. Although it is unclear whether the transcriptional activation of plant PCNA is regulated by E2F, E2F released from RB-related protein might promote PCNA mRNA expression, as in mammals. This possibility is supported by the observation that ZmRBR1-associated kinase activity peaks at the G1\/S transition in synchronized tobacco BY-2 cells (Boniotti and Gutierrez 2001). It is likely that increased phosphorylation states of PsRBR1 protein have an important role in the regulation of E2F-mediated gene expression involved in the G1\/S transition and DNA replication during dormancy-to-growth transition in pea axillary buds.\nIn some plants, the gene expression of cyclins and CDK activities are regulated by plant hormones, e.g., cytokinin, auxin, and gibberellin (Miao et\u00a0al. 1993; Sauter 1997; Soni et\u00a0al. 1995). In Arabidopsis suspension-cultured cells, D-type cyclin mRNA is rapidly induced by the addition of cytokinin or glucose, and repressed by the addition of auxin (Soni et\u00a0al. 1995). Cytokinin is biosynthesized at the stem after decapitation and moves into the axillary buds for outgrowth (Tanaka et\u00a0al. 2006). It is possible that the increasing cytokinin levels caused by decapitation induces the expression of D-type cyclin. D-type cyclin forms a complex with CDKs and immediately phosphorylates RB-related protein. The Hp-form induces gene expression for the G1\/S transition in axillary bud cells. It is likely that RB-related protein controls the cell cycle in axillary buds during the dormancy-to-growth transition. Further analyses will provide more insight into the regulation of dormancy-to-growth transition in axillary buds.","keyphrases":["pea","retinoblastoma-related protein","phosphorylation","axillary buds","dormancy","cell cycle"],"prmu":["P","P","P","P","P","P"]}
{"id":"J_Med_Internet_Res-4-2-1761932","title":"Using the Internet for Surveys and Health Research\n","text":"This paper concerns the use of the Internet in the research process, from identifying research issues through qualitative research, through using the Web for surveys and clinical trials, to pre-publishing and publishing research results. Material published on the Internet may be a valuable resource for researchers desiring to understand people and the social and cultural contexts within which they live outside of experimental settings, with due emphasis on the interpretations, experiences, and views of `real world' people. Reviews of information posted by consumers on the Internet may help to identify health beliefs, common topics, motives, information, and emotional needs of patients, and point to areas where research is needed. The Internet can further be used for survey research. Internet-based surveys may be conducted by means of interactive interviews or by questionnaires designed for self-completion. Electronic one-to-one interviews can be conducted via e-mail or using chat rooms. Questionnaires can be administered by e-mail (e.g. using mailing lists), by posting to newsgroups, and on the Web using fill-in forms. In \"open\" web-based surveys, selection bias occurs due to the non-representative nature of the Internet population, and (more importantly) through self-selection of participants, i.e. the non-representative nature of respondents, also called the `volunteer effect'. A synopsis of important techniques and tips for implementing Web-based surveys is given. Ethical issues involved in any type of online research are discussed. Internet addresses for finding methods and protocols are provided. The Web is also being used to assist in the identification and conduction of clinical trials. For example, the web can be used by researchers doing a systematic review who are looking for unpublished trials. Finally, the web is used for two distinct types of electronic publication. Type 1 publication is unrefereed publication of protocols or work in progress (a `post-publication' peer review process may take place), whereas Type 2 publication is peer-reviewed and will ordinarily take place in online journals.\nIdentifying issues for qualitative research\nAs the most comprehensive archive of written material representing our world and people's opinions, concerns, and desires (in industrialized countries), the Internet can be used to identify `issues' for qualitative (descriptive) research and to generate hypotheses. Material published on the Internet may be a valuable resource for researchers desiring to understand people and the social and cultural contexts within which they live--outside of experimental settings--with due emphasis on the interpretations, experiences, and views of `real world' people. Reviews of information posted by consumers on the Internet may help to identify health beliefs, common topics, motives, information, and emotional needs of patients, and point to areas where research is needed. Comparing recommendations found on the Web against evidence-based guidelines is one way to identify areas where there is a gap between opinion and evidence, or where there is a need for clinical innovation.\nThe accessibility of information for analysis and the anonymity of the Internet allow researchers to analyse text and narratives on Web sites, to use newsgroups as global focus groups, and to conduct interviews and surveys via e-mail, chat rooms, Web sites, or newsgroups.Topics suited to qualitative research include:\nAnalysis of interactive communications (e.g. e-mail).\nStudy of online communities (virtual self-help groups, newsgroups, mailing lists).\nInvestigation of communication processes between patients and professionals.\nStudy of consumer preferences, patient concerns, and information needs.\nExploration of the `epidemiology of health information' on the Web [1-2].\nThe Internet population is unrepresentative of the general population, restricting the use of the Internet for quantitative studies (i.e. studies focusing on measurement). Qualitative studies, however, do not require representative samples:`In qualitative research we are not interested in an average view of a patient population, but want to gain an in-depth understanding of the experience of particular individuals or groups; we should therefore deliberately seek out individuals or groups who fit the bill' [3]. Three different research methodologies for qualitative research on the Internet may be distinguished:\nPassive analysis: For example, studying information on Web sites or interactions in newsgroups, mailing lists, and chat rooms--without researchers actively involving themselves.\nActive analysis: Also called participant observation; the researcher participates in the communication process, often without disclosing their identity as researcher. For example, they may ask questions in a patient discussion group implying that she or he is a fellow patient. Such studies often involve elements of deception, unless the researcher is a sufferer him- or herself.\nInterviews and surveys: See below.\nExamples of these three types of qualitative research on the Internet are available elsewhere [1].\nUsing the Internet for surveys\nUsing the Internet for surveys requires an awareness of methodologies, selection bias, and technical issues.\nMethodological issues\nInternet-based surveys may be conducted by means of interactive interviews or by questionnaires designed for self-completion. Electronic one-to-one interviews can be conducted via e-mail or using chat rooms. Questionnaires can be administered by e-mail (e.g. using mailing lists), by posting to newsgroups, and on the Web using fill-in forms.\nWhen e-mail is used to administer questionnaires, messages are usually sent to a selected group with a known number of participants, thus allowing calculation of the response rate. Surveys posted to newsgroups may request that the completed questionnaire is posted back to the researcher, but it is impossible to know who and how many people read the questionnaire. If Web-based forms are used, questionnaires can be placed in a password-protected area of a Web site (i.e. participation by invitation or registration only), or alternatively they may be open to the public (i.e. any site visitor can complete the survey). The latter option makes calculation of a response rate more difficult but not impossible: the number of people who access (without necessarily completing) the questionnaire is counted and used as the denominator. Web-based surveys have the advantage that the respondent can remain anonymous (as opposed to e-mail surveys, where the e-mail address of the responder is revealed). Furthermore, they are very convenient for the researcher, as responses can be directly stored in a database where they are immediately accessible for analysis.\nElectronic interviews and surveys (`e-surveys') are emerging scientific research methodologies, pioneered by communication scientists, sociologists, and psychologists, although their use for health-related research is still in its infancy [4-10]. Examples of health-related research include:\nA Web-based survey on the effects of ulcerative colitis on quality of life [11].\nCollection of clinical data from atopy patients [12].\nA Web-based survey looking at complementary and alternative medicine use by patients with inflammatory bowel disease and Internet access [13].\nA survey of dentists regarding the usefulness of the Internet in supporting patient care [14,15].\nGuidelines for Web-based surveys\nScenarios that may be suitable for a Web-based survey\nRespondent features:\nRespondents are already avid Internet users; e-mail addresses known for reminder messages.\nRespondents are enthusiastic form fillers; will not require monetary incentives.\nNeed for respondents covering a wide geographical area (e.g. rare clinical special ties, diseases).\nRespondents are known to match non-respondents and even non-Internet users on key variables.\nSurvey features:\nNeed for complex branching, interactive questionnaire or multimedia as part of the survey instrument.\nSurvey content will evolve fast (e.g. Delphi method surveys use repeating rounds of revised questionnaires delivered over a short period, incorporating aggregate results from previous rounds until convergence is achieved).\nIntent is to document bizarre, rare phenomena whose simple occurrence is of interest.\nNo need for representative results: collecting ideas vs. hypothesis testing.\nInvestigator features:\nLimited budget for mailing and data processing, but good in-house Web skills.\nPrecautions can be taken against multiple responses by same individual, password sharing.\nWeb survey forms have been piloted with representative participants and demonstrate acceptable validity and reliability with most platform, browser, and Internet access provider combinations.\nData is required fast in a readily analysed form.\nScenarios that are unsuitable for a Web-based survey\nRespondent features:\nTarget group is under-represented on Internet; e.g. the underprivileged, elderly people.\nTarget group is concerned, however unreasonably, about privacy aspects.\nTarget group requires substantial incentives to complete the survey.\nNeed for a representative sample.\nSurvey features:\nNeed for very accurate timing data on participants (inaccuracies in the range of seconds are added due to network transmission times, unless JavaScript or Java applets are used; see Glossary) or observational data on participants.\nAn existing paper instrument has been carefully validated on target group.\nNeed to capture qualitative data or observations about participants.\nWish to reach the same group of participants in the same way months or years later.\nInvestigator features:\nLimited in-house Web or Java expertise but existing desktop publishing and mailing facility.\nE-surveys may be part of a qualitative research process, but results can be analysed quantitatively as long as researchers are aware of potential bias (see below). In addition to gathering data, the Internet may also be used in the course of developing questionnaires, as it allows rapid prototyping and pilot testing of instruments, e.g. to evaluate the effect of framing the questions differently [16].\nSeveral studies have checked the validity of Web-based surveys by comparing the results of studies conducted on the Web with identical studies in the real world. These seem to suggest that the validity and reliability of data obtained online are comparable to those obtained by classical methods [4,5,17-19]. However, issues of generalizability (mainly due to selection bias, discussed in detail below) remain important considerations, and the researcher should select his or her research question and interpret the results with care.The benefits and problems of Web-based surveys have been summarized by Wyatt, who suggests guidelines for when they may be appropriate (see Box 1) [20].\nSelection bias\nIn `open' surveys conducted via the Internet where Web users, newsgroup readers, or mailing list subscribers are invited to participate by completing a questionnaire, selection bias is a major factor limiting the generalizability (external validity) of results. Selection bias occurs due to:\nThe non-representative nature of the Internet population.\nThe self-selection of participants, i.e. the non-representative nature of respondents, also called the `volunteer effect' [21].\nThe non-representative nature of Internet demographics was briefly considered earlier. Considering whether the topic chosen for study is suitable for the Internet population is the first and probably the most important step in minimizing bias, thus maximizing response rates and increasing the external validity of the results [20]. For example, targeting elderly homeless alcoholics is unsuitable for an Internet survey and the results are likely to be heavily skewed by hoax responses.\nSelf-selection bias originates from the fact that people are more likely to respond to questionnaires if they see items which interest them, e.g. because they are affected by the items asked about, or because they are attracted by the incentives offered for participating. As people who respond almost certainly have different characteristics than those who do not, the results are likely to be biased.This kind of selection bias is more serious than the bias arising from the non-representative nature of the population, because the researcher deals with a myriad of unknown factors and has little opportunity to interpret his or her results accordingly. Such bias may be exacerbated via loaded incentives (e.g. typical `male' incentives such as computer equipment). Evidence suggests women are generally more interested in health topics and exhibit more active information-seeking behaviour [22], so are more likely to volunteer participation in health questionnaires. For Web surveys, the potential for self-selection bias can be estimated by measuring the response rate, expressed as the number of people completing the questionnaire divided by those who viewed it (cf. the participation rate, expressed as the number of site visitors viewing the questionnaire divided by the total number of site visitors).\nTechnical issues\nAlthough a detailed analysis is beyond the scope of this chapter, a synopsis of important techniques and tips for implementing Web-based surveys provides some insight into the difficulties faced by survey designers (see Box 2).\nTechnical issues in implementing Web-based surveys\nUse of `cookies'\nCookies can assign a unique identifier to every questionnaire viewer, useful for determining response and participation rates, and for filtering out multiple responses by the same person. As cookies may be regarded with suspicion, we recommend that researchers openly state that cookies will be sent (and the reasons for this); set the cookie to expire on the day that data collection ceases; and publish a privacy policy.\nMeasuring response time\nThe time needed to complete a questionnaire can be readily calculated by subtracting the time a form was called up by the browser from the time it was submitted using an automatic time-stamp. The response time may be used to exclude respondents who fill in the questionnaire too quickly: this may identify hoax responses, where respondents don't read the questions.\nAvoiding missing data\nForms can be configured to automatically reject incomplete questionnaires and point out missing or contradictory items. Checks can be made on the client (p. 9) prior to submission, or following submission to the server (where incomplete responses can also be analysed, e.g. during a questionnaire pilot).\nMaximizing response rate\nThe number of contacts, personalized contacts, and contact with participants before the actual survey are the factors most associated with higher response rates in Web surveys [23]. Incentives increase the risk of selection bias (see text), but less so if cash is offered. Perhaps the best incentive (and the easiest to deliver via the Internet) is the promise of survey results or personalized answers (e.g. a score). The option to complete questionnaires anonymously avoids wariness associated with requests for personal information (e.g. an e-mail address), but increases the risk of hoax responses. Researchers should be open about who is behind the study, what the aim is, and provide opportunities for feedback. Although postal surveys are superior to e-mail surveys with regard to response rate, online surveys are much cheaper [24,25]. Schleyer [15] estimated that the cost of their Web-based survey was 38 percent less than that of an equivalent mail survey and presented a general formula for calculating break-even points between electronic and hard-copy surveys. Jones gave figures of 92 p per reply for postal surveys, 35 p for e-mail, and 41 p for the Web [24].\nRandomizing items\nScripting languages may be used to build dynamic questionnaires (as opposed to static forms) that look different for certain user groups or which randomize certain aspects of the questionnaire (e.g. the order of the items). This can be useful to exclude possible systematic influences of the order of the items upon responses.\nEthical issues\nThe ethical issues involved in any type of online research should not be forgotten [1,26-31]. These include informed consent as a basic ethical tenet of scientific research on human populations [32], protection of privacy, and avoiding psychological harm.\nIn qualitative research on the Web, informed consent is required when:\nData are collected from research participants through any form of communication, interaction, or intervention.\nBehaviour of research participants occurs in a private context where an individual can reasonably expect that no observation or reporting is taking place, except when researchers do research `in public places or use publicly available information about individuals (e.g. naturalistic observations in public places, analysis of public records, or archival research)' [33].\nThe question therefore arises of whether researchers analysing newsgroup postings enter a `public place', or whether the space they invade is perceived as private. In the context of research, the expectation of the individual (whether he\/she can reasonably expect that no observation is taking place) is crucial. Different Internet services have different levels of perceived privacy (in decreasing order of privacy: private e-mail; chat rooms; mailing lists; newsgroups;Web sites).The perceived level of privacy is a function of the number of participants, but also depends on other factors such as group norms established by the community to be studied. For example, in a controversial paper, Finn studied a virtual self-support group where the moderator was actively discouraging interested professionals who were not sexual abuse survivors from joining the group [34]. In those cases, obtaining informed consent (or seeking an ethical waiver, if the research could not practicably be carried out were informed consent to be required) is mandatory.\nIn practice, obtaining informed consent, especially for passive research methods, is difficult, as researchers usually cannot post an announcement to a mailing list or newsgroup saying that it will be monitored and analysed for the next few months, as this may greatly influence or even spoil the results, and because the mere posting of such a request may disrupt the community, and therefore be considered unethical. Researchers should therefore first obtain consent from a group moderator in order to explore whether even a request for permission is felt to be disruptive to the group process. If the moderator or person responsible for the list has no objections, one may then post a message to a newsgroup or mailing list explaining the purpose of the research, explaining that one will observe the community, assuring all participants of anonymity, and giving them the opportunity to withdraw from the newsgroup or mailing list or to exclude themselves from the study by writing to the researcher. The fundamental problem is that this may influence the communication process and may even destroy the community. Besides, participants who later join the group need to get the same information. An alternative would be to analyse the communication retrospectively and to write individual e-mails to all participants whose comments were to be analysed or quoted, asking for permission to use them; this technique has been used by Sharf [35].\nIn any case, researchers should make themselves familiar with the virtual community they are approaching; i.e. read the messages in a newsgroup for some time (`lurking'). Under no circumstances should researchers blindly spam (p. 31) or cross-post requests for research participation to various newsgroups.\nInformed consent may also play a role when researchers report aggregate (collated and hence anonymous) data on usage patterns, such as a log-file analysis (reporting data on what Web sites have been accessed by a population). Crucial here is an appropriate privacy statement stating that these data may be analysed and reported in aggregate [28]. Note that aggregate data are exempt from the registration requirements of the UK's Data Protection Act of 1998.\nIn conducting surveys researchers may obtain informed consent by declaring the purpose of the study; disclosing which institutions are behind the study; explaining how privacy will be assured; and detailing with whom data will be shared and how it will be reported, before participants complete the questionnaire.\nWhen reporting results, it is obvious that the total anonymity of research participants needs to be maintained. Researchers have to keep in mind that, by the very process of quoting the exact words of a newsgroup or mailing list participant, the confidentiality of the participant may already be broken as Internet search engines may be able to retrieve the original message, including the e-mail address of the sender. It is essential, therefore, to ask participants whether they agree to be quoted whenever there may be a retrievable archive, pointing out the risk that they may be identifiable. Problems can also potentially arise from just citing the name of the community (e.g. of a newsgroup), which may damage the community being studied.\nFinding methods, protocols, and instruments\nFor laboratory `bench work', researchers often need a protocol for a specific assay method. In addition to the possibility of searching literature databases, there are also specialized services on the Web that can assist in this research, such as MethodsFinder and the `Technical tips online' database at BioMedNet:\nMethodsFinder (BIOSIS): \nhttp:\/\/www.methodsfinder.org\/\nBioMedNet: \nhttp:\/\/www.bmn.com\/\nSometimes asking a specific question on the right newsgroup or mailing list is also very effective. Clinical researchers may be more interested in instruments to measure patient outcomes.An excellent guide to selecting quality-of-life instruments is the Quality of Life Instruments Database at the Mapi Research Institute: http:\/\/www.qolid.org\/\nOnline statistical analysis tools are available at the Simple Interactive Statistical Analysis (SISA) Web site, while background information is available within the online book Statistics at square one:\nSISA (Daan Uitenbroek): \nhttp:\/\/home.clara.net\/sisa\/\nStatistics at square one (British Medical Journal Publishing Group): \nhttp:\/\/www.bmj.com\/collections\/statsbk\/\nProtocols of clinical trials, which may be useful for researchers developing their own protocols, can be found in some of the clinical trial databases available on the Web, as described below.\nClinical trials and the Web\nThe Web is being used to assist in the identification and conduction of clinical trials.\nIdentifying trials\nTo prevent unintended duplication of clinical research, detect underreporting of research, and ease the work of systemic reviewing, it has been suggested that we should prospectively register clinical trials [36-39]. It is, however, unlikely that there will ever be one complete centralized multinational database. Instead, multiple resources set up by numerous different organizations will exist [40]. Internet technology will play a central role in linking these databases and making this information available to researchers and patients. Some scenarios in which a search of trial databases may be useful:\nA researcher wants to conduct a randomized controlled trial and wants to know whether anyone else is already running one on the same topic.\nA physician has a patient who is asking about available trials.\nA patient is looking for ongoing trials.\nA researcher is looking for possible participants for his trial.\nA researcher doing a systematic review is looking for unpublished trials.\nInformation about ongoing and completed clinical trials is increasingly being published on the Internet, and searches on the Web may be a useful means of complementing traditional bibliographic searches if authors of systematic reviews wish to find ongoing or unpublished trials [41].\nResearchers use their personal or department home pages to announce their interest in a certain research area or to recruit patients [42]. Journals like The Lancet have begun to publish research protocols on their Web site [43], and more and more researchers will also publish `pre-prints' (p. 239) of their findings on the Web [44].\nConsumers and patient organizations also have an interest in disseminating information about ongoing trials; e.g. the National Alliance of Breast Cancer Organizations: http:\/\/www.nabco.org\/\nGovernment and funding agencies react to this need by establishing trial databases for consumers; e.g. the US National Institutes of Health searchable database [45]: http:\/\/ClinicalTrials.gov\nCommercial enterprises also help researchers to recruit patients, or help patients to find clinical trials. For example:\nCenterWatch Clinical Trials Listing Service (CenterWatch, Inc.): \nhttp:\/\/www.centerwatch.com\/\nClinicalTrialFinder.com (Clinical Data Technologies Ltd): \nhttp:\/\/www.clinicaltrialfinder.com\/\nCurrent Controlled Trials (BioMed Central): \nhttp:\/\/www.controlled-trials.com\nPharmaceutical companies and industry associations have likewise begun to recognize that openness and access to information on clinical trials and new drug developments can improve patient care and are part of social responsibility [46]. For example:\nClinical Trials Register (GlaxoSmithKline): \nhttp:\/\/ctr.glaxowellcome.co.uk\/\nSearch for Cures (Pharmaceutical Research and Manufacturers of America): \nhttp:\/\/www.phrma.org\/searchcures\/\nFinally, information or databases on ongoing clinical trials can often also be found on disease-specific sites. For example:\nCanadian HIV Trials Network: \nhttp:\/\/www.hivnet.ubc.ca\/ctn.html\nCancerNet (National Cancer Institute): \nhttp:\/\/cancernet.nci.nih.gov\/\nConducting trials on the Web\nThe Web is increasingly being used in the course of conducting large-scale multi-centre clinical trials (e.g. for remote randomization and data entry), and in the distribution of information on trial progress or protocols [47,48]. Trial centres may enter patient data using Java applets (see Glossary) that encrypt data and send it to the data centre via the Internet [49-52], where the data are stored and randomized, returning for example a study number and randomization information.\nPre-publishing and publishing research\nTraditional publication is a well-defined event, whereas `publication' in the electronic age is much more of a continuum [53], reflecting and occurring during the entire research process from hypothesis formulation to data gathering, interpretation, and the presentation and discussion of the final results. In order to distinguish online collaborative `work in progress' from `final' peer-reviewed publication we may term the former `Type 1' and the latter `Type 2' electronic publication [54]. Here, peer review is not the distinguishing characteristic: in Type 1 publication a `post-publication' peer review process takes place. Type 2 publication will ordinarily take place in online journals. The following scenarios illustrate how researchers might use Type 1 electronic publication on the Internet:\nSending and discussing preliminary results on mailing lists.\nPublishing drafts of scientific papers on pre-print\/e-print sites (p. 239) in order to solicit comments and to improve the manuscript.\nPublishing data and information in databases; e.g. nucleotide sequences in the EMBL\/Genbank databases.\nPublishing clinical trial protocols and raw data in a `trial bank' [55].\nCurrent awareness services\nElectronic editions of paper journals and `stand alone' e-journals typically offer subscriptions to `TOC alerts', where users receive a table of contents by e-mail as soon as a new issue appears.The more sophisticated systems allow users to specify their interests using a controlled vocabulary, enabling the system to screen each newly published article for certain keywords or citations. Examples of current awareness services include:\nCustomised @lerts (British Medical Journal): \nhttp:\/\/bmj.com\/cgi\/customalert\/\nJournAlert (Doctors.net.uk): \nhttp:\/\/www.doctors.net.uk\/\nJournal Watch (Massachusetts Medical Society): \nhttp:\/\/www.jwatch.org\/","keyphrases":["internet","qualitative research","clinical trials","survey research","selection bias","data collection","informed consent","confidentiality","ethics, research","evaluation studies","patient selection","research design","research subjects"],"prmu":["P","P","P","P","P","P","P","P","R","R","R","R","M"]}
{"id":"Purinergic_Signal-3-4-2072918","title":"Monitoring the expression of purinoceptors and nucleotide-metabolizing ecto-enzymes with antibodies directed against proteins in native conformation\n","text":"Following their release from cells, ATP and NAD, the universal currencies of energy metabolism, function as extracellular signalling molecules. Mammalian cells express numerous purinoceptors, i.e., the nucleotide-gated P2X ion channels and the G-protein-coupled P2Y receptors. Signalling through purinoceptors is controlled by nucleotide-metabolizing ecto-enzymes, which regulate the availability of extracellular nucleotides. These enzymes include ecto-nucleoside triphosphate diphosphohydrolases (ENTPD, CD39 family) and ecto-nucleotide pyrophosphatase\/phosphodiesterases (ENPP, CD203 family). Investigation of these receptors and enzymes has been hampered by the lack of available antibodies, especially ones that recognize these proteins in their native conformation. This study reports the use of genetic immunization to generate such antibodies against P2X1, P2X4, P2X7, ENTPD1, ENPTD2, ENPTD5, ENPTD6, ENPP2, ENPP3, ENPP4, ENPP5, and ENPP6. Genetic immunization ensures expression of the native protein by the cells of the immunized animal and yields antibodies directed against proteins in native conformation (ADAPINCs). Such antibodies are especially useful for immunofluorescence and immunoprecipitation analyses, whereas antibodies against synthetic peptides usually function well only in Western-blot analyses. Here we illustrate the utility of the new antibodies to monitor the cell surface expression of and to purify some key players of purinergic signalling.\nIntroduction\nFollowing their release from cells, the universal currencies of energy metabolism, ATP and NAD, function as extracellular signalling molecules [1\u20135]. Molecular cloning has identified 7 nucleotide-gated ion channels (P2X purinoceptors) and >8 nucleotide-activated G-protein-coupled receptors (P2Y purinoceptors) [6\u201310]. Signaling through purinoceptors is effectively controlled by nucleotide-metabolizing ecto-enzymes, which regulate the availability of extracellular nucleotides [11]. Prominent roles are played by members of two families of ecto-enzymes: the ecto-nucleoside triphosphate diphosphohydrolases (ENTPD, CD39 family) [12] and the ecto-nucleotide pyrophosphatase\/phosphodiesterases (ENPP, CD203 family) [13].\nThe mammalian P2X family encompasses seven members. All P2X purinoceptors contain cytosolic N- and C-terminal tails, two membrane spanning domains, and an extracellular domain of approximately 280 amino acids containing the ligand-binding site and 10 conserved cysteine residues that likely form five intrachain disulfide bonds [14, 15]. The extracellular domains of the respective mouse and human P2X orthologues show high (ca. 80\u201390%) sequence identity; the paralogues within a given species show lower (40\u201350%) identity.\nThe mammalian ENTPD family encompasses eight members [16]. ENTPD1\u20134, 7 and 8 have a structure similar to that of P2X purinoceptors, i.e., cytosolic N- and C-terminal tails, two transmembrane spanning domains, and an extracellular domain of approximately 440 amino acids, encompassing the ligand-binding site and four conserved cysteines likely engaged in intrachain disulfide bridges [12]. ENTPD1\u20133 and 8 are expressed as cell surface enzymes, ENTPD4 and ENTPD7 as luminal enzymes of intracellular organelles. ENTPDases share structural similarities with the actin\/HSP70\/sugar kinase superfamily [12]. ENTPD5 and ENTPD6 lack the second transmembrane region. Moreover, in ENTPD5, the region corresponding to the first transmembrane domain of the other family members is N-terminal and functions as a signal peptide, resulting in the secretion of ENTPD5 into the lumen of the endoplasmic reticulum. ENTPD6 is a type II transmembrane protein. ENTPD orthologues show high (ca. 80\u201390%) sequence identity; the paralogues within a given species show lower (30\u201350%) identity.\nThe ENPP family encompasses seven members [13]. ENPP1\u20133 are type II transmembrane proteins with a structure analogous to that of ENTPD6, i.e., an N-terminal cytosolic tail, a single transmembrane domain and an extracellular catalytic domain of approximately 400 amino acids. ENPP4\u20137, in contrast, are type I membrane proteins. A hydrophobic N-terminus acts as a signal peptide, the extracellular catalytic domain is followed by a single transmembrane domain and a short C-terminal cytosolic tail. ENPPs 4\u20137 consist of catalytic domains only, the catalytic domains of ENPPs 1\u20133 are flanked by genetically fused upstream somatomedin B and downstream nuclease-like domains [13]. ENPPs share structural similarities with the alkaline-phosphatase superfamily [17, 18]. The catalytic domain of ENPP orthologues shows high (ca. 80\u201390%) sequence identity; the paralogues within a given species show lower (40\u201350%) identity.\nDespite their relevance for purinergic signalling, investigation of these receptors and enzymes has been hampered by the lack of available antibodies, especially ones that recognize the enzymes in their native conformation. Antibodies raised by immunization with synthetic peptides derived from the known amino acid sequence of a protein of interest generally work well in Western-blot analyses but often fail to recognize the native protein on the cell surface (Fig.\u00a01). Such antibodies can be used successfully for monitoring the overall expression level of the protein of interest in a population of cells but cannot be used to assess cell surface expression on individual cells. Genetic immunization, in contrast, ensures that the protein of interest is expressed in native conformation by the cells of the immunized animal and yields antibodies directed against proteins in native conformation (ADAPINCs) [19, 20]. Such antibodies function well in immunofluorescence, FACS, ELISA, and immunoprecipitation analyses, i.e., assays in which antipeptide antibodies often fail. We have previously employed this technique to generate highly specific polyclonal and monoclonal antibodies against ecto-ADP-ribsoyltransferases and against murine P2X7 [20\u201323]. Here we report the generation and characterization of similar antibodies against other key players of purinergic signalling and illustrate the utility of these antibodies for monitoring the cell surface expression of their cognate antigens.\nFig.\u00a01DNA immunization vs. peptide immunization\nMaterials and methods\nCloning of expression vectors and cell transfections\nClones encoding full-length open-reading frames representing members of the P2X, ENTPD, and ENPP families were purchased from the mammalian genome collection (www.rzpd.de). Clones that were not already in an expression vector (pCMV-SPORT6) were subcloned into the pCDNA6 expression vector. Further expression constructs were kindly provided by James Wiley, Sydney (human P2X7), Richard J. Evans, Leicester (human P2X1), Fran\u00e7ois Rassendren, Montpellier (mouse P2X4), Ruth Murrell-Lagnado, Cambridge (rat P2X4), and Astrid Kehlen, Halle (hENPP2). An expression construct for nuclear green fluorescent protein was generated by cloning the DNA-binding domain of the LKLF transcription factor [24] as a C-terminal fusion protein into the pEGFP-N1 vector (Clontech). Expression constructs were transfected into CHO and HEK cells (5\u00a0\u03bcg for purinoceptors and ecto-enzymes and\/or 0.5\u00a0\u03bcg eGFP-LKLF per T25 flask containing 2\u2009\u00d7\u2009106 cells) with the JetPei transfection reagent (Q-Biogen). For stable transfections, plasmids were linearized with Ssp1 or Sca1 before transfection, and stable transfectants were selected by cultivating cells in the presence of 12\u00a0\u03bcg\/ml blasticidin (Sigma).\nGenetic immunization and purification of antibodies\nGenetic immunizations were performed as described [20, 22]. In brief, rabbits or rats were immunized four times by ballistic DNA immunization with plasmid-conjugated gold particles. Serum samples were obtained 10\u00a0days post-immunization. For monoclonal antibody production, animals received a final boost with transiently transfected HEK cells (2\u2009\u00d7\u2009106 cells in 200\u00a0\u03bcl PBS i.v.) 3\u00a0days prior to sacrifice. Splenocytes were fused to Sp2\/0 myeloma cells and screened for production of specific antibodies by immunofluorescence analysis of CHO cells that had been co-transfected 20\u201342\u00a0h earlier with constructs encoding eGFP-LKLF and the protein of interest. Antibodies were purified by affinity chromatography on Protein G Sepharose (Pharmacia), and conjugated to Alexa488 according to the manufacturer\u2019s (Molecular Probes) instructions. Antibodies directed against the C-terminal peptides of P2X purinoceptors were purchased from Caltag (rat P2X1, Cal), ABCAM (rat P2X4, Abc), and Alomone (mouse P2X7, Al). The L4 anti-human P2X7 mAb was kindly provided by Dr. Ian Chessel at Glaxo Wellcome (Middlesex, UK).\nImmunofluorescence analyses\nCHO cells were co-transfected with expression constructs for purinoceptors or ecto-enzymes and nuclear GFP (5 and 0.5\u00a0\u03bcg\/106 cells, respectively). Cells were transferred onto 96-well plates (4\u2009\u00d7\u2009105 cells\/well) 6\u00a0h post-transfection. Then, 20\u00a0h after transfection, cells were fixed for 10\u00a0min in 2% paraformaldehyde (PFA) and then stained with serial dilutions of immune serum for 30\u00a0min at room temperature. To detect ENTPD5, which is not bound to the cell surface, cells were fixed in 4% PFA and subsequently permeabilized with 0.5% IGEPAL (Sigma) before staining. Bound antibody was detected with PE-conjugated antirat or antirabbit IgG (1:100) (Dianova). Cell nuclei were counterstained with Hoechst 33342. Cells were analyzed with a Zeiss Axiovert 25 microscope equipped with the Canon PowerShot G2 digital camera and appropriate filters for visualizing Hoechst stain (Zeiss filter set 01), green fluorescent protein (Zeiss filter set 10) and PE fluorochrome (Zeiss filter set 15). Photographs were taken at 40\u00d7 magnification with fixed camera settings (digital zoom: 4.3\u00d7, aperture: 5.0, exposure time: 2\u00a0s for GFP and PE, 1\/8\u00a0s for Hoechst 33342). GFP and PE images were merged using Adobe Photoshop software.\nFACS analyses\nFor FACS analyses, cells were harvested by brief trypsinization (5\u00a0min at room temperature) and washed in serum-containing medium. Cells (5\u2009\u00d7\u2009105\/100\u00a0\u03bcl) were stained either directly with Alexa-488-conjugated antibodies (1\u00a0\u03bcg) or with serially diluted immune serum followed by PE-conjugated antirat or antirabbit IgG (1:100, Dianova). Gating was performed on living cells on the basis of propidium iodide exclusion.\nImmunoprecipitation analyses\nAntibodies were conjugated to Protein G Sepharose beads (Pharmacia) (1\u00a0\u03bcg antibody\/20\u00a0\u03bcL beads in 200\u00a0\u03bcL PBS containing 1% Triton X-100) by incubation on a roller for 30\u00a0min at room temperature. Beads were washed twice in PBS\/1% Triton X-100 before use for immunoprecipitation. Cells (107\/ml) were lysed in PBS, 1% Triton-X100, 1\u00a0mM AEBSF (Sigma) for 20\u00a0min at 4\u00b0C. Insoluble material was pelleted by centrifugation, and the cleared lysates (2\u2009\u00d7\u2009106 cells in 200\u00a0\u03bcL) were subjected to immunoprecipitation using 20\u00a0\u03bcL antibody-conjugated beads by incubation on a roller for 30\u00a0min at room temperature. Beads were washed four times in PBS\/1% Triton X-100. Proteins were eluted from the beads by incubation in SDS-PAGE sample buffer for 15\u00a0min at 70\u00b0C. Eluted proteins were size-fractionated on precast SDS-PAGE gels (Invitrogen) and blotted onto PVDF membranes. Purinoceptors and ecto-enzymes were detected with serially diluted serum followed by peroxidase (PO)-conjugated antirabbit IgG (1:5,000) using the ECL system (Amersham).\nImmunoblot analyses\nCells (107\/ml) were pelleted by centrifugation and lysed by resuspension in SDS-PAGE sample buffer (Invitrogen). DNA was sheared by brief ultrasonication on ice (two 15-s pulses with an MSE ultrasonicator at maximum power setting). Insoluble material was pelleted by centrifugation, and soluble proteins (5\u2009\u00d7\u2009105 cell equivalents\/lane) were size-fractionated on precast SDS-PAGE gels (Invitrogen) under reducing conditions and blotted onto PVDF membranes. Purinoceptors and ecto-enzymes were detected with serially diluted serum followed by PO-conjugated antirabbit IgG (1:5,000) using the ECL system (Amersham).\nResults and discussion\nUse of transiently transfected CHO cells to assess the specificity and titres of antisera against purinoceptors and ecto-enzymes\nAntibodies raised by genetic immunization of rabbits and rats with cDNA expression constructs for purinoceptors, ENTPDs, and ENPPs were assayed for reactivity and specificity by indirect immunofluorescence staining of CHO cells, 24\u00a0h after cotransfection with a construct encoding nuclear GFP and the cDNA expression construct used for immunization (Fig.\u00a02 and Table\u00a01). By this time, transfected cells were clearly distinguishable from untransfected cells by virtue of their green fluorescent nuclei (Fig.\u00a02). Specific antibodies were detected on the basis of bright cell surface staining of transfected cells and lack of reactivity with untransfected cells. Antipeptide antisera against the same antigens, in contrast, did not show any detectable reactivity in this assay (Table\u00a01).\nFig.\u00a02Immunofluorescence analyses of CHO cells following transient cotransfection with expression constructs for nuclear green fluorescent protein (GFP) and purinoceptors (a), CD39-like ENTPDs (b), and CD203-like ENPPs (c). CHO cells were cotransfected with nuclear GFP and expression constructs for the indicated purinoceptors or ecto-enzymes. Twenty hours after transfection, cells were stained with serial dilutions of the indicated antisera followed by PE-conjugated antirabbit IgG. To detect non-membrane-bound ENTPD5, intracellular staining was performed by fixing and permeabilizing cells. Cell nuclei were counterstained with Hoechst 33342. Cells were analyzed with a Zeiss Axiovert 25 microscope equipped with filters for visualizing Hoechst dye, GFP, and PE fluorochrome. Panels show merged images for Hoechst dye and GFP (b, c, left panels) or for GFP and PE (a\u2013c, right panels)Table\u00a01Titres, specificities, and applications of antibodies against members of the families of P2X purinoceptors, CD39-like ENTPDs, and CD203-like ENPPsAntibodyHostTitreSpecificityFACS\/IFTWestern blotP2X family9316rb1:800hP2X1+++(+)RG2rt1:800hP2X1+++++CR29rb1:1600hP2X4+++++CR30rb1:1600mP2X1+++++116rb1:800mP2X4RG22rt1:1600mP2X4++++++K1Grb1:1600mP2X7+++\u2212RH23rt1:1600mP2X7++\u2212Hano43rtmAbmP2X7++\u22123428rb1:1600rP2X7++\u2212Caltrb1:2000rP2X1\u2212+++Abcrb1:2000rP2X4\u2212+++Alrb1:5000mP2X7\u2212+++ENTPD family5441rb1:1600hENTPD2+++\u2212RG10rt1:1000h\/mENTPD2+++ndRG10-B28rtmAbhENTPD2+++nd5446rb1: 400hENTPD5+++\u2212CR44rb1:6,400mENTPD1++++++RG9rt1:1600mENTPD1+++ndRG9-A59rtmAbmENTPD1+++\u2212CR45rb1:1600mENTPD2++++CR63rb1:1600mETNTPD2+++ndCR46rb1:400mENTPD5+++\u2212CR47rb1:200mENTPD6+++\u2212ENPP familyK2Grb1:200hENPP2+++nd7619rb1:800hENPP2+++ndCR65rb1:800mENPP4+++ndCR66rb1:400mENPP5+++ndCR67rb1:800mENPP6+++nd7620rb1:3200rENPP3+++nd\nMonitoring cell surface expression of purinoceptors and ecto-enzymes by flow cytometry\nThe utility of the ADAPINC antisera for monitoring cell surface expression was assessed further by flow cytometry (FACS analyses) of transiently transfected HEK cells (Fig.\u00a03). The results confirm that the antisera react specifically with appropriately transfected cells and that they do not react with untransfected HEK cells. Importantly, the antisera recognize the respective purinoceptor\/ecto-enzyme on the cell surface of living cells. In contrast, antipeptide antisera against the same antigens did not show any detectable reactivity with the transfected cells in these assays. Further, the use of the ADAPINC antisera for FACS analyses permits an assessment of the relative expression level of the cognate protein on the cell surface of individual cells as well as an assessment of the number of transfected vs. untransfected cells in the cell population. These parameters cannot be quantified appropriately by Western-blot analyses, since for these analyses, lysates are prepared from cell populations. We further used the FACS-based assay system to test whether the antisera crossreact with other members (paralogues) of the receptor\/enzyme family and\/or the same receptor\/enzyme (orthologue) in other species (human, rat, or mouse). The results reveal that most antisera are specific for the receptor\/enzyme used for immunization and do not cross-react with other members of the same protein family. Some antisera, however, did react with the orthologous protein, in particular at low dilutions, e.g., antiserum CR30 raised against mouse P2X1 shows some reactivity with the human P2X1 orthologue (Fig.\u00a03a).\nFig.\u00a03FACS analyses of purinoceptor (a) and ecto-enzyme (b) expression by transfected HEK cells. HEK cells were transfected with expression constructs for the indicated purinoceptors and ecto-enzymes. Then, 24\u00a0h post-transfection, cells were harvested by brief trypsinization and single cell suspensions were stained with serial dilutions of the indicated antisera followed by PE-conjugated antirabbit IgG before FACS analyses. Control stainings were performed with untransfected cells (shaded histograms in a, dotted lines in b) and with cells transfected with the species orthologue (red lines in a)\nSome of the antisera recognize the denatured cognate antigen in Western-blot analyses\nWe next tested the utility of the ADAPINC antisera for monitoring overall protein expression levels in immunoblot analyses (Fig.\u00a04). To this end, we size-fractionated the proteins from lysates of untransfected and of transiently transfected HEK cells by SDS-PAGE followed by immunoblot analyses. The results show that only some of the ADAPINC antisera detected bands of the expected size. For comparison, we again performed parallel analyses with antisera raised against the same proteins by peptide immunization. The results show that the antipeptide sera generally detected bands of the expected size. The C-terminal peptide is commonly used for peptide immunization. In the case of P2X7, this sequence is highly conserved between rat, mouse and human orthologues, and the corresponding antipeptide serum recognized the respective orthologues (not shown). In contrast, in the case of P2X4, the mouse and human sequences differ in two and five positions, respectively, from the rat peptide used for immunization, and this antipeptide serum shows only weak cross-reactivity with these orthologues (Fig.\u00a04a, Abc \u03b1-mP2X4).\nFig.\u00a04Immunoblot analyses of purinoceptor (a) and ecto-enzyme (b) expression by transfected HEK cells. Untransfected (u) or HEK cells transfected with mouse (m) or human (h) purinoceptors or ecto-enzymes were solubilized 24\u00a0h post-transfection with SDS-PAGE sample buffer. Proteins in cell lysates were size-fractionated by SDS-PAGE and subjected to immunoblot analyses using the indicated antisera. Bound antibodies were detected with peroxidase-conjugated secondary antibody and the ECL system\nAffinity purification of purinoceptors and ecto-enzymes\nFinally, we tested the utility of the ADAPINC antisera for immunoprecipitation of the cognate proteins from cell lysates (Fig.\u00a05). To this end, we prepared lysates of untransfected and of transiently transfected HEK using the nondenaturing nonionic detergent Triton-X-100. Lysates were incubated with ADAPINC antibodies immobilized on protein-G sepharaose beads, and proteins bound to washed beads were subsequently analyzed by SDS-PAGE followed by immunoblot analyses with antipeptide antibodies. The results show that all of the ADAPINC antisera efficiently precipitated proteins of the expected size from cell lysates. This underscores the utility of these antibodies for purifying native receptors and ecto-enzymes from lysed cells.\nFig.\u00a05Immunoprecipitation of purinoceptors by antibodies raised via genetic immunization. Untransfected (u) and transfected (t) HEK cells were solubilized 24\u00a0h post-transfection with PBS\/1% Triton X-100. Purinoceptors were immunoprecipitated from cell lysates by incubation with the indicated antisera bound to Protein G Sepharose. Precipitates were subjected to immunoblot analysis as in Fig.\u00a04, using the indicated antipeptide antisera for detection\nConclusion\nUsing genetic immunization, we have raised highly specific polyclonal and monoclonal antibodies against key players of purinergic signalling, i.e., P2X1, P2X4, and P2X7 purinoceptors and ENTPD1, ENPTD2, ENPTD5, ENPTD6, ENPP2, ENPP3, ENPP4, ENPP5, and ENPP6 enzymes. Our findings underscore the utility of genetic immunization for generating highly specific polyclonal and monoclonal antibodies directed against proteins in native conformation (ADAPINCs) [19, 20]. These antibodies are valuable tools for assessing the expression levels of the native protein by immunofluorescence analyses and flow cytometry, i.e., assays in which antipeptide antibodies often fail. The antibodies described here provide useful tools for further characterization of the structure and function of these purinoceptors and ecto-enzymes.","keyphrases":["purinoceptor","genetic immunization","monoclonal antibodies","ecto-nucleotidases"],"prmu":["P","P","P","U"]}
{"id":"Int_J_Colorectal_Dis-3-1-2077921","title":"The artificial bowel sphincter for faecal incontinence: a single centre study\n","text":"Background and aims Faecal incontinence (FI) is a socially devastating problem. The treatment algorithm depends on the aetiology of the problem. Large anal sphincter defects can be treated by sphincter replacement procedures: the dynamic graciloplasty and the artificial bowel sphincter (ABS).\nIntroduction\nFaecal incontinence (FI) is a complex problem. The resulting social isolation is a major concern, which results in a reduced quality of life [1]. The real prevalence is unknown, but studies show a higher prevalence than expected [2\u20135]. Most patients are females with one or more vaginal deliveries in the past. Direct trauma to the anal sphincter complex can give immediate problems or problems later in life [6, 7].\nThe initial therapy should be conservative, e.g. diet modifications, medication, biofeedback physiotherapy or retrograde irrigation. Surgical intervention is indicated when conservative treatment fails. An anal repair is usually the first choice of treatment for a minor sphincter defect. Satisfactory results are achieved in a tension-free repair in 47\u2013100% of the cases [8]. Long-term results are less satisfying [9]. Sacral nerve modulation (SNM) has proven to be effective for treating faecal incontinence in patients with an intact sphincter complex [10]. Sphincter replacing therapy is indicated in patients with large sphincter defects or completely disrupted sphincters and in case of SNM failure. The sphincter replacement procedures are grossly divided in the dynamic graciloplasty (DGP) [11, 12] or the artificial bowel sphincter (ABS). The first artificial bowel sphincter for faecal incontinence was a urinary prosthesis (AMS 800, AMS) placed by Christiansen in 1987 [13]. Modifications had to be made to suit the anal sphincter for use in patients with faecal incontinence.\nUntil 1997, patients with faecal incontinence due to large anal sphincter defects were treated with DGP in our institution [16]. Since then, the ABS was introduced in our institution for the same indication. Because the operating technique is similar, there was no learning curve to be dealt with. Is this study, the results of the ABS implantations for the treatment of feacal incontinence in a large volume centre are presented.\nMaterials and methods\nThis study is a non-randomised, non-controlled, prospective single-centre study. Thirty-four patients with persisting or recurrent end-stage FI were included between 1997 and 2006.The majority of patients had large (>33% of circumference) anal sphincter defects. A sufficient length of the perineum was a prerequisite for ABS implantation. Previous sphincter replacement surgery was no exclusion criterion for implantation of an ABS. All patients underwent a full preoperative examination consisting of a defaecography, endo-anal ultrasound (SDD 2000, Multiview, Aloka, Japan, 7,5\u00a0Mhz endo-anal transducer), pudendal nerve terminal motor latency measurement (St Mark\u2019s pudendal electrode) and anal manometry using a Konigsberg catheter (Konigsberg Instrument, Pasadena CA, USA) connected to a polygraph (Synectics Medical, Stockholm, Sweden). An Acticon artificial bowel sphincter (ABS, American Medical Systems, Minneapolis, MN, USA) was used in all patients. The Williams incontinence score was used to classify the symptoms. Anal manometry was routinely performed during the follow-up and used to objectivity ABS function. The follow-up appointments were scheduled at 1, 3, 6, 12\u00a0months and annually. Infection necessitating explantation was a primary endpoint. A re-intervention was a secondary endpoint.\nThe system implantation has been described extensively elsewhere [14, 15], but will be summarised here. The ABS implant consists of three parts: an inflatable balloon, a cuff and a pump. Under strict systemic and local antibiotic prophylaxes, the cuff is placed around the anus using two lateral incisions. The pump is placed in the labia majora or scrotum, and the pressure-regulating balloon is placed in cavum Retzii. Care is taken not to perforate the rectum. If a perforation occurs, the procedure is stopped. After proper wound healing, the patient is eligible for another implantation procedure.\nData are expressed as the mean with the range between parenthesis. Data were analysed using the commercially available GraphPad Prism 4.00 software (GraphPad Software, San Diego, USA). The Wilcoxon signed rank test was used for non-parametric paired values. Statistical significance was set at p\u2009<\u20090.05.\nResults\nThe patient population existed of 25 women and nine men. The aetiology of the faecal incontinence is shown in Table\u00a01. Three patients were previously treated with a DGP. The average age was 55.3 (23.8\u201375.6) years. The mean period of faecal incontinence before the placement of the ABS was 11.0 (1.0\u201348.0) years. One patient had a rectum perforation during the initial surgery, and placement of the ABS was abandoned. She awaits a second implant attempt. Thirty-three patients were implanted. The mean follow-up was 17.4 (0.8\u2013106.3) months. The mean procedure time was 68.1\u00a0min (38.0\u2013105.0). In 24 patients, the length of the cuff was 11\u00a0cm, in three patients 10\u00a0cm, in two patients 13\u00a0cm, in two patients 12\u00a0cm, in one patient 14 and in one patient 9. The width of the cuff was in all, but one patient, 2.9\u00a0cm. There was one patient with a cuff off 2.0\u00a0cm. All patients received a pressure-regulating balloon of 91\u2013100\u00a0cm H2O. The mean postoperative hospital stay was 3.5 (2.0\u201312.0) days.\nTable\u00a01Aetiology and previous surgical treatmentNumberSexEtiologyPrevious treatment1FHysterectomy, cervix carcinoma, radiotherapy2MAnal atresiaDGP3FTwo breech deliveries: ruptureAnal repair, SNM4FEpisiotomy, hysterectomyPNE5MPelvic trauma: urethra\/rectum ruptureRepair and colostomy6MTrauma, partial spinal cord lesionPNE7FDelivery trauma: total rupture, hysterectomyAnal repair, SNM8FDelivery trauma: rupture9MAnal atresia10MClassical hemorroidectomy11FEpisiotomy, hysterectomyPNE12FDelivery trauma: ruptureTwo anal repairs, PNE13FDelivery trauma: total ruptureTwo anal repairs, 14FDelivery trauma: ruptureAnal repair15FDelivery trauma: rupture, cauda syndromeAnal repair16FDelivery trauma: rupture, hysterectomyAnal repair17FDelivery trauma: rupture, hysterectomyAnal repair18MAnal atresiaDGP19FDelivery trauma: ruptureAnal repair20FDelivery trauma: rupturePre-\/post-anal repair, PNE21FDelivery trauma: rupture, hysterectomyAnal repair, PNE22FDelivery trauma: rupturePost-anal repair, SNM22MLow anterior resection T2NOM023FDelivery trauma: rupture24MPelvic crush trauma: urethra\/rectum ruptureRepair and colostomy25FDelivery trauma: rupture26FDelivery trauma: total rupture12 anal repairs27FDelivery trauma: ruptureAnal repair, colostomy28FDelivery trauma: ruptureAnal repair29MPelvic trauma30FDelivery trauma, uterus extirpatie DGP31FDelivery traumaPNE32FClassical hemorroidectomySECCA33FDelivery trauma, total ruptureAnal repair, Thiersch wire34FDelivery trauma, hysterectomyPNEF Female, M male, DGP dynamic graciloplasty, SNM sacral neuromodulation, PNE percutaneous nerve evaluation\nThe mean preoperative Williams score of 4.8 (4\u20135) decreased significantly after ABS placement to 2.1 (1\u20135; Fig.\u00a01). The mean preoperative anal resting pressure was 58.1 (17.0\u2013128.0) mmHg. This was not significantly altered after implantation (60.3 (21.0\u201393.0\u00a0mmHg; p\u2009=\u20090.89). The mean preoperative squeeze pressure was 80.1 (25.0\u2013149.0) mmHg, which increased to 120.5 (65.0\u2013154.0) mmHg after implantation (p\u2009=\u20090.003; Fig.\u00a02).\nFig.\u00a01Mean pre- and postoperative Williams score (1 = continent, 2 = incontinent to flatus, 3 = incontinent to liquid stool, 4 = occasional incontinence to normal stool <1, 5 = fully incontinent)Fig.\u00a02Baseline resting pressure versus deflated ABS pressure and baseline squeeze pressure vs inflated ABS pressure pre- and postoperatively (at last follow-up)\nThirteen patients (39%) complained about a rectal evacuation problem. In 12 patients, this could be managed conservatively. One patient had a revision of the system with placement of a wider anal cuff. Seven patients (21.2%) had an infection of the system, which led to seven explantations. One of these patients has been implanted successfully with a new ABS (Fig.\u00a03).\nFig.\u00a03Flow chart of implanted patients\nIn one patient, the ABS was successfully converted to a dynamic graciloplasty. In two patients, a colostomy was performed. The other three patients had no other interventions.\nOne patient was explanted due to persisting peri-anal pain without an infection. She received a colostomy. Twenty-six reinterventions (including explantations) had to be performed. This means 0.79 re-intervention per implanted patient.\nDiscussion\nThere is a large experience in our institution with the DGP [16]. However, since 1997, the ABS is also performed in our institution for the same area of indications as the DGP. When a patient qualifies for a sphincter replacement procedure, he or she can decide whether an ABS or DGP will be performed. Nonetheless, sufficient perineal length is a prerequisite for ABS implantation in a female patient. We believe that the risk for late erosion of the ABS is higher in the case of severe, cloaca-like malformations of the perineum. In these cases, a DGP is the preferred procedure. All patients in this study had an adequate perineal length.\nIn the beginning, the initial infection rate of the DGP was a problem, but improved as a result of technical modifications and the introduction of systemic and local antibiotic prophylaxis. The same prophylaxis protocol was used for the implantation of ABS. However, despite meticulous application of the antimicrobial protocol, the infection rate of the ABS implantations in our patient population remains high and is comparable with other series [14, 15]. We believe that this infection rate is likely to remain a serious problem in every attempt to place a corpus alienum around the anus through peri-anal incisions.\nTo overcome this problem of infection, Finlay et al. [17] have developed a new prosthetic sphincter, which is placed above the pelvic floor musculature by means of a laparotomy. It was hypothesised that this sphincter will function as a new puborectal sling in this position. Till now, 12 patients are implanted. Infectious complications, however, occurred in three patients (25%), with subsequent removal of the system. Technical problems occurred in five of the nine remaining patients during follow-up. Technical failure is also one of the main problems of the ABS. Twelve of our patients had some sort of technical failure. This is also known from other studies concerning the ABS [18].\nOnly limited data on long-term follow-up of a sufficient number of ABS sphincters are available. There is one multicentre study with disappointing long-term data where the initial data were promising [19]. The anal manometry data of this patient population suggest poor action of the ABS. The authors conclude that the ABS acts as a passive barrier causing a rectal outlet obstruction. Our manometry data contradict with this conclusion. We strongly believe that the ABS acts as an active sphincter. In our experience, the patients need to deflate the anal cuff to defecate. Nevertheless, constipation can be a problem. Thirteen of our patients complained about constipation. This could be solved in the majority of patients by conservative means. One patient needed a wider anal cuff to treat an outlet obstruction.\nThe indications for sphincter replacement surgery are decreasing in our institution since the introduction of SNM. The relative numbers of DGPs and ABSs decreased, while the number of SNM has increased. This implicates that ABS and DGP are reserved for the more severe complicated cases of faecal incontinence. A higher complication rate is therefore expected. However, the placement of an ABS remains an alternative to a colostomy in the well-informed and motivated patient even if a DGP has failed.\nConclusion\nThe artificial bowel sphincter is an effective treatment option for severe faecal incontinence. Even in experienced hands, the risk of infection, explantation and system malfunctioning remain high. In well-informed and motivated patients, it is worthwhile to proceed to implantation, as the alternative is a colostomy. Our data suggest that the ABS acts as an active sphincter and not as a passive barrier.","keyphrases":["artificial bowel sphincter","faecal incontinence","single centre"],"prmu":["P","P","P"]}
{"id":"Eur_J_Health_Econ-_-_-1388081","title":"The \u201cHealth Benefit Basket\u201d in France\n","text":"The French \u201cHealth Benefit Basket\u201d is defined principally by positive lists of reimbursed goods and services; however, global budget-financed hospital-delivered services are more implicitly defined. The range of reimbursable curative care services is defined by two coexisting positive lists\/fee schedules: the Classification Commune des Actes M\u00e9dicaux (CCAM) and the Nomenclature G\u00e9n\u00e9rale des Actes Professionnels (NGAP). The National Union of Health Insurance Funds has been updating these positive lists since August 2004, with the main criterion for inclusion being the proposed procedure\u2019s effectiveness. This is assessed by the newly created High Health authority (replacing the former ANAES). In addition, complementary health insurers are consulted in the inclusion process due to their important role in French healthcare financing.\nThe French health insurance system, a mix of explicit and implicit regulations, offers wide-ranging reimbursement in the fields of preventive, curative, rehabilitative, and palliative care. The present analysis of the health benefit basket in France is being carried out during a period of reforms. This contribution describes the structure of the statutory health insurance system, followed by a description of the entitlements and benefits and of the actors involved in the decision-making process with its criteria for services of curative care, HC1. Finally, we analyze the main changes which may affect the health basket in France.\nThe structure of the French statutory health insurance system\nThe financial management of health care in France is undertaken mainly by the statutory health insurance system as a branch of the wider social security system. The health insurance system\u2019s current structure is based on its founding text, the Ordinance of 4 October 1945 and subsequent legislative measures. This system has covered the entire population of France since 1 January 2000 when the Couverture Maladie Universelle (CMU, Universal Health Coverage Act) extended basic health insurance cover to all legal residents of France. The French statutory health insurance system is a compulsory scheme and covers all households regardless of health status, income, number of persons, etc. It provides a somewhat uniform field of reimbursement, with the \u201cbasket of goods and services\u201d covered by the insurance funds being identical for all the statutory schemes with the reimbursement rate being the same for the three main insurance schemes since 2000. The exceptions are the Alsace-Moselle region\u2019s local scheme and certain public sector schemes [1]. Membership in one of the health insurance funds of the statutory health insurance depends on the profession of each person. In the context of the CMU, however, participation depends on legal residence in France and on the level of income. Any dependants of the insured person are also covered by his\/her health insurance.\nThe three main health insurance schemes are as follows: (a) The General Scheme (R\u00e9gime g\u00e9n\u00e9ral) covers employees in commerce and industry and their families (about 84% of the population) as well as persons receiving CMU, estimated in late 2003 at about 1,500,000 person (2.4% of the population) [2]. (b) The Agricultural Scheme (Mutualit\u00e9 sociale agricole) covers farmers and agricultural employees and their families (about 7.2% of the population). (c) The Scheme for the Non-agricultural Self-Employed (CANAM) covers craftsmen and self-employed persons, including self-employed professionals such as lawyers (about 5% of the population). Another ten work-related schemes cover specific sections of the population.\nStatutory health insurance funds three-quarters of the health expenditure and therefore leaves considerable scope for complementary sources of funding. An estimated 85% of the population have complementary health insurance. Taking into account recipients of Couverture Maladie Universelle Compl\u00e9mentaire (Complementary Universal Health Coverage 6% of the population), about 91% of the French population are covered by the complementary health insurance scheme, which covers the same health basket as the statutory schemes and not other goods and services [3]. The most important benefit catalogues for France and their underlying criteria are displayed in Tables\u00a01 and 2 while the following sections are confined to curative health care services.Table\u00a01 Catalogues and implicit regulationGBRCCAMNGAPLSAC ATUMed. dev.NABM Sp. reg.HC.1 Curative care services\u00a0\u00a01. Inpatient curative care++\u2013\u2013\u2013\u2013\u2013\u00a0\u00a02. Day-patient curative care++\u2013\u2013\u2013\u2013\u2013\u00a0\u00a03. Outpatient curative care+\u2013\u2013\u2013\u2013\u2013\u00a0\u00a0\u00a0\u00a03.1. Basic medical and diagnostic services++\u2013\u2013\u2013\u2013\u2013\u00a0\u00a0\u00a0\u00a03.2. Outpatient dental care++\u2013\u2013\u2013\u2013\u2013\u00a0\u00a0\u00a0\u00a03.3. All other specialized health care++\u2013\u2013\u2013\u2013\u2013\u00a0\u00a0\u00a0\u00a03.4. All other outpatient curative care (paramedical)\u2013\u2013+\u2013\u2013\u2013\u2013\u00a0\u00a0\u00a0\u00a03.5. Services of curative home care+\u2013+\u2013\u2013\u2013\u2013HC.2 Services of rehabilitative care\u00a0\u00a01. Inpatient rehabilitative care+\u2013\u2013\u2013\u2013\u2013+\u00a0\u00a02. Day cases of rehabilitative care+\u2013\u2013\u2013\u2013\u2013+\u00a0\u00a03. Outpatient rehabilitative care+\u2013+\u2013\u2013\u2013\u2013\u00a0\u00a04. Services of rehabilitative home care+\u2013+\u2013\u2013\u2013+HC.3 Services of long-term nursing care\u00a0\u00a01. Inpatient long-term nursing care+\u2013\u2013\u2013\u2013\u2013+\u00a0\u00a02. Day cases of long-term nursing care+\u2013\u2013\u2013\u2013\u2013+\u00a0\u00a03. Long-term nursing care: home care+\u2013+\u2013\u2013\u2013\u2013HC.4 Ancillary health care services\u00a0\u00a01. Medical analysis laboratory+\u2013\u2013\u2013\u2013+\u2013\u00a0\u00a02. Diagnostic imaging++\u2013\u2013\u2013\u2013\u2013\u00a0\u00a03. Patient transport and emergency rescue +\u2013\u2013\u2013\u2013\u2013+HC.5 Medical goods dispensed to outpatients\u00a0\u00a01. Prescribed medicines+\u2013\u2013+\u2013\u2013\u2013\u00a0\u00a02. Other medical nondurables+\u2013\u2013\u2013+\u2013\u2013\u00a0\u00a03. Therapeutic devices and other medical durables+\u2013\u2013\u2013+\u2013\u2013HC.6 Prevention and public health services\u2013\u2013\u2013\u2013\u2013\u2013+GBR General Benefit Regulation, CCAM list of physicians\u2019 and dentists\u2019 technical procedures, NGAP list of physicians\u2019 consultations and other health professionals\u2019 activity, LSAC ATU positive lists of drugs, Med. dev. positive lists of medical devices, NABM positive lists NABM biology procedure, Sp. reg. specific regulationTable\u00a02 Benefit-defining laws\/decrees, catalogues and implicit regulationGBRCCAM, NGAPLSAC ATUMed. dev.NABM Sp. reg.Legal status: law, decreeLawUNCAM decisionMinisterial orderMinisterial orderUNCAM decisionAdministrative documentDecision makerParliament UNCAM, on the advice of HAS and UNOCMinisters of health and social security, on the advice of the Transparency CommissionMinisters of health and social security on the advice of the ad hoc CommissionUNCAM, on the advice of HAS and UNOCOriginal purpose: entitlements, reimbursement, target settingReimbursementPositive listPositive listPositive listPositive listPositive listTariffsFee schedulePrices or reference pricesReference pricesFee schedulePositive-negative definition of benefitsPositivePositivePositivePositivePositivePositiveDegree of explicitnessa3 (except inpatient care: 1)33332 or 3If itemized: goods\/procedures only; linked to indicationsMainly goods and procedures; linked to indicationsProcedures, sometimes linked to indicationsPharmaceutical products, linked to indicationsGoods linked to indicationsProcedures linked to indicationsMainly indicationsUpdating\u2013RegularlyRegularlyRegularlyRegularly\u2013GBR General Benefit Regulation, CCAM list of physicians\u2019 and dentists\u2019 technical procedures, NGAP list of physicians\u2019 consultations and other health professionals\u2019 activity, LSAC ATU positive lists of drugs, Med. dev. positive lists of medical devices, NABM positive lists NABM biology procedure, Sp. reg. specific regulationa 1, \u201call necessary\u201d; 2, \u201careas of care\u201d; 3 \u201citems\u201d\nOrganizational structure and actors involved in the definition of the benefit basket for curative services\nIn France the general conditions of the reimbursement system are established by law. Health benefit catalogues are drawn up at national level with the whole range of goods and services reimbursed by the statutory scheme being specified in Article L.321-1 of the Social Security Code (SSC) [4]. The reimbursement of goods and services depends on their inclusion in positive lists, according to Articles L.162-1-7, L.162-17, and L. 165-1 of the SSC. Until 2004 positive lists were officially enforced by ministerial orders detailing the inclusion of new goods and services. Ministers made their decisions upon the advice of ad hoc scientific commissions and agencies, for example, the National Agency for Accreditation and Evaluation in Health Care (ANAES). The inclusion of all the listed procedures depends on ANAES advice which considers the effectiveness and\/or safety of these procedures and the conditions under which they need to be performed.\nReimbursement is legal only when goods or services are provided in an appropriate medical context. Reimbursement for all goods and all paramedical procedures depends on the provision of a prescription, which serves as confirmation of the necessity of such goods and services. For some types of treatment, such as physiotherapy and spa treatment, the prescription from a physician does not provide the needed status for reimbursement. Coverage by statutory health insurance is subject to the prior authorization (entente pr\u00e9alable) of the physicians advising the health insurance funds, after examination of the patient\u2019s case history and a possible interviewing of the patient.\nAccording to Article L.322-2 of the SSC, the insured person\u2019s copayment is fixed by a decision of Union Nationale des Caisses d\u2019Assurance Maladie (UNCAM, National Union of Health Insurance Funds) within rate limits defined by a Council of State decree. The copayment can be either a percentage of the charges or a lump sum. The patient\u2019s contribution to the total cost of treatment varies according to the type of treatment, being higher for outpatient care and drugs compared to hospital treatment. The copayment must be paid by the insured person or, where applicable, by their complementary health insurance fund.\nCouncil of State decrees also define copayment exemption conditions (Article L.322-3 of the SSC). The most important exemptions are linked to the health status, especially in the case of one person suffering from one of 30 specified long-term illnesses, for example, diabetes, AIDS, cancer, and psychiatric illness, and if the patient is suffering from one or several incapacitating diseases. Other health status-based exemptions concern, for example, disabled persons under the age of 20\u00a0years and pregnant women during the final 4\u00a0months of pregnancy.\nThe health benefit basket is explicitly defined by positive lists of goods for both the public and the private sectors. Positive lists only apply to services delivered by private sector professionals in their own practices or in private for-profit hospitals. Conversely, services dispensed in public hospitals or private not-for-profit hospitals were mainly the subject of implicit definition since they were paid for by a global budget, which means in practice that basically every dispensed service was reimbursed. The situation is currently undergoing an important reform.\nHC1 services qualifying for reimbursement by the health insurance system include: (a) inpatient care and treatment in public or private healthcare institutions; (b) outpatient care provided by general practitioners, specialists, dentists, and midwives; and (c) diagnostic services and care prescribed by physicians and performed by laboratories and paramedical professionals, such as nurses, physiotherapists and speech therapists.\nIn July 2005, two health benefit catalogues for curative services exist: (a) The Classification Commune des Actes M\u00e9dicaux (CCAM), which revises the previous medical services catalogue, has been adopted in 2005 with its application still only partial [5]. (b) The general fee schedule (Nomenclature G\u00e9n\u00e9rale des Actes Professionnels, NGAP), which is the medical procedures positive list for health professionals in private practice, in their own surgeries and consulting rooms and in private-for-profit hospitals, remains in force until the CCAM is fully implemented.\nInpatient curative service\nThe Social Security Act of 18 December 2003 (Loi de financement de la s\u00e9curit\u00e9 sociale, LFSS) changed the inpatient acute care funding rules, but implementation is still in progress. The situation will continue to change during the next few years for at least two reasons. Firstly, the positive list for physicians\u2019 procedures, the CCAM, applies to both private and public hospitals (Article 162-1-7 of the SSC), which had not been the case before. Secondly, the implementation of the per-case payment reform that will lead to the result of both sectors being brought into line. These situations before and after the reforms of 2004-2005 are shown in Table\u00a03.Table\u00a03 Definition of benefit catalogues for inpatient care Before 2004\u20132005After 2004\u20132005Funding rules depend upon hospital status: PH and PNFPH, vs. PFPHNew funding rules making the basket more explicitRange of reimbursed medical procedures defined at least for PFPH but not for other services, e.g., nursing careSame list of medical procedures for both private and public hospitals (Article 162-1-7 of the SSC),No positive list for PH, an implicit coverage for all services in PH and PNFPH mainly financed by the global budgetSame prospective per-case payment system for PH and PFPH for all medical, surgery and obstetrics services, based on DRG-type classification of GHS, but tariffs still differ from PH to PFPH.Basket of reimbursable drugs defined for PH and PFPHBasket of reimbursable drugs defined for PH and PFPHPH public hospital,\nPNFPH private non-for-profit hospital, PFPH private-for-profit hospital, DRG diagnosis-related group, GHS homogeneous stay group\nAs seen in Table\u00a03, there has been no positive list to define reimbursable services in public and in private not-for-profit hospitals, in which inpatient services were regulated by implicit coverage. However, the implementation of the per-case payment system will simplify the situation as all medical, surgery and obstetrics services in all hospitals will be included in it. The reform will also change the remuneration schemes of inpatient and outpatient care. Services provided in inpatient or outpatient acute care will be financed through a payment-per-case system. This is based on a diagnosis-related group type of classification of 700 Groupes Homog\u00e8nes de Malades (GHM), considering comorbidities. A nationally fixed tariff (Groupe Homog\u00e8ne de S\u00e9jours, GHS, Homogeneous Group of Stays) is applied to each GHM [6]. Outpatient procedures will be paid on a fee-for-service basis, and organ retrieval and emergency services by annual lump sum payments. This payment system also includes earmarked funding (Missions d\u2019int\u00e9r\u00eat g\u00e9n\u00e9ral et Aide \u00e0 la contractualisation, MIGAC) to finance other activities, such as research and education, but also certain healthcare activities promoted by contracts between hospitals and regional health agencies. The new GHS payment system has been in operation since 1 March 2005 for private-for-profit hospitals. Procedures carried out by physicians are always paid separately and directly to the physicians concerned on a fee-for-service basis, while in public hospitals, tariffs include specialists\u2019 salaries. Since July 2005, the CCAM is used as the general fee schedule and can be considered as the positive list for private for hospitals. Health insurance funds will not finance any stay if one of these procedures does not apply. CCAM is currently available only for technical procedures such as surgery, medical imaging, and radiotherapy performed by physicians. Paramedical services, including, physiotherapy, which are linked to inpatient medical procedures, are reimbursed only if they are an essential part of the medical treatment. Regarding the definition of the health basket, the rules for day-case patient care are the same as those applicable to inpatient care. GHS tariffs refer to day hospital treatment.\nOutpatient curative care\nPatients who need health care have until now been free to choose which physician to consult and have also been allowed to refer themselves to specialists. Only few general practitioners have begun playing the role of gatekeepers, although a few of them have been making some attempts in this regard since 1996. Recent health insurance reforms are about to change this situation. Since 1 July 2005 all those benefiting from health insurance coverage in France are obliged to choose their main physician (m\u00e9decin traitant). As a result there will be higher user charges if a person chooses to consult a specialist directly without being referred by her\/his \u201cgate-keeping\u201d m\u00e9decin traitant general practitioner [7]. The basket of reimbursable outpatient curative services is defined by a positive list which is also used as a fee-schedule for health professionals in private practice. Until March 2005 the NGAP was the only list and applied to all services. This will be replaced by the new CCAM classification. In a rather lengthy process CCAM is currently being developed. At this stage CCAM specifies only technical procedures such as diagnosis, surgery, and radiology performed by specialists and dentists. CCAM is also being used in public hospitals to charge for outpatient care and to specify the reimbursement rate. This classification is not yet used for specialist consultations or procedures carried out by other healthcare professionals who are still in use of the NGAP. The CCAM is fully comprehensive in content as it contains details of all medical procedures, even those that are not reimbursable. Each procedure corresponds to only one label and one code, eliminating any ambiguity and making it easy to use. The classification is structured according to the anatomical classification and specialities. There are 17 chapters which are based on the organ system to which the procedures refer. The CCAM is based on the rule of procedural totality, meaning that each label implicitly contains all the operations necessary for the performance of the medical procedure. It was drawn up as a resource-based relative value scale (RBRVS) by the main health insurance fund (CNAMTS) with the collaboration of health professionals, and the ANAES being involved in the selection of effective procedures.\nSince the reform of 13 August 2004 the UNCAM, which includes representatives of the three main sickness funds, is in charge of preparing and updating the positive lists. UNCAM will be assisted in its decision making by the advice of the two newly created bodies, the High Health Authority (Haute Autorit\u00e9 de Sant\u00e9), which replaced the ANAES and the National Union of Complementary Health Insurance Organizations (Union Nationale des Organismes d\u2019Assurance Maladie Compl\u00e9mentaire, UNOC). The same criteria used for the NGAP will be used for the taxonomy of medical procedures leaving room for improvements in medical services and achieving cost-containment at the same time [8]. Nevertheless, the ministries of health and social security retain the right to reject UNCAM\u2019s decisions and to include or exclude goods and services in or from the list, especially if public health issues are concerned. A commission comprising healthcare professionals\u2019 unions and representatives of UNCAM has been created to determine the general rules for drawing up the RBRVS and then for validating the scale proposed by the health insurance fund services. In addition, UNCAM is responsible for negotiating the tariffs of medical procedures with healthcare professionals\u2019 unions and for determining the levels of copayment and coinsurance.\nOther outpatient services such as auxiliary services provided by nurses, physiotherapists, orthoptists, speech therapists and chiropodists must be provided on medical prescription in order to be reimbursed. In the area of complementary and alternative medicine (CAM) special recognition is given to acupuncture and homeopathy. These two therapies are thus recognized and may be legally practiced, but only by physicians or licensed physiotherapists on medical prescriptions for acupuncture. A few osteopathy and chiropractic procedures have recently been included in the CCAM in addition to certain vertebral manipulations which were already listed in the NGAP. The range of services covered by statutory health insurance does not include cosmetic surgery and some other treatments and services of uncertain effectiveness such as spa treatments. The issue of the allocation of scarce resources means that choices must be made which may result in the nonreimbursement of certain procedures, for example, bone density measurement when performed in the private sector as a preventive measure, and the imposition of limits on the frequency for which they can be reimbursed, such as mammography for cancer screening purposes.\nHome care services\nThe entitlements laid down in the SSC include services provided by independent physicians (home visits) and by other health professionals such as nurses and hospital-at-home services. Regarding palliative care at home, according to Article R 162-1-10 of the SSC, a multidisciplinary team of health professionals can be organized at the special request of a person whose state of health demands it, to provide palliative home care. The remuneration can be paid on a fee-for-service basis or as a lump sum.\nDiscussion\nThis study faces a number of limitations which may affect the analysis of the French health benefit basket. Firstly, the study was carried out in a period of changes related to the redefinition of the medical procedures for both inpatient and outpatient care. This will make some statements in the previous catalogue become obsolete. Furthermore, the introduction of the new CCAM catalogue has been postponed several times due to conflicts of interest between health professionals, especially those between the physicians\u2019 unions and the health insurance funds. The catalogue deals with the two issues of the remuneration of healthcare professionals and the state regulation. Secondly, the recent health insurance reform (Health Insurance Act, 13 August 2004) affects all actors involved in the definition of the benefit catalogue and thus the decision criteria. The High Health Authority, for instance, is expected to have considerable powers. At the same time, UNCAM is also playing an important role in the drawing up of the positive lists of procedures. However, the ministers of health and social security still retain their right to reject the UNCAM\u2019s decisions. It could be asked whether the change regarding the delegation of the task of drawing up benefit catalogues to a so-called \u201cself-governing body\u201d is fully effective, as this is the case in Germany. It is too early to assess the consequences that these new regulations will bring about, but it can be anticipated that it might well be a case of plus \u00e7a change, plus c\u2019est la m\u00eame chose, as both the French government and the physicians have always played a major role in the healthcare decision-making process. Thirdly, the French system offers a mix of explicit regulation for ambulatory care, pharmaceuticals and medical devices, dental care, etc., with benefit catalogues and positive lists and of implicit regulation for a large part of inpatient care [9]. Altogether the package of health care services covered is comprehensive and wide-ranging although that the additional billing is rather high for certain types of goods and services. As a consequence of the implicitness of the coverage with particular respect to inpatient care, we have noted some differences between principles and practice, indicating that not all services are actually covered to the same extent. The interviews carried out at the Ministry of Health confirmed this finding.\nFinally, the drawing-up of the positive lists still remains hotly contested among those at the health and social security ministries who were interviewed for this study. Current French health policy combines the harmonization of regulation with the reduction in health inequalities on the basis of improved knowledge of public health needs. However, this policy is being implemented in a context of increasing health expenditure and especially of increasing user charges. It is a contradictory situation in which things that have been received by the one hand have again been taken away by the other one.","keyphrases":["france","health benefit plans","health services","health priorities","national health programs"],"prmu":["P","M","R","M","M"]}
{"id":"Exp_Brain_Res-4-1-2315690","title":"Eye\u2013hand coordination during manual object transport with the affected and less affected hand in adolescents with hemiparetic cerebral palsy\n","text":"In the present study we investigated eye\u2013hand coordination in adolescents with hemiparetic cerebral palsy (CP) and neurologically healthy controls. Using an object prehension and transport task, we addressed two hypotheses, motivated by the question whether early brain damage and the ensuing limitations of motor activity lead to general and\/or effector-specific effects in visuomotor control of manual actions. We hypothesized that individuals with hemiparetic CP would more closely visually monitor actions with their affected hand, compared to both their less affected hand and to control participants without a sensorimotor impairment. A second, more speculative hypothesis was that, in relation to previously established deficits in prospective action control in individuals with hemiparetic CP, gaze patterns might be less anticipatory in general, also during actions performed with the less affected hand. Analysis of the gaze and hand movement data revealed the increased visual monitoring of participants with CP when using their affected hand at the beginning as well as during object transport. In contrast, no general deficit in anticipatory gaze control in the participants with hemiparetic CP could be observed. Collectively, these findings are the first to directly show that individuals with hemiparetic CP adapt eye\u2013hand coordination to the specific constraints of the moving limb, presumably to compensate for sensorimotor deficits.\nIntroduction\nCerebral palsy (CP) is an umbrella-term describing a group of disorders of movement and posture leading to activity limitations, and that are caused by damage to the fetal or infant brain (Bax et al. 2005). In this study, we focused on the most common subtype of cerebral palsy, spastic hemiparesis, which typically occurs after unilateral lesions to the cerebral cortex or corticospinal pathways (Kwong et al. 2004). This condition is characterized by impaired control of muscle tone and spasticity in the upper and lower limbs of the contralesional, \u201caffected\u201d, side of the body (Albright 1996), generally progressing from proximal to distal (Freund 1987; Steenbergen et al. 2000b), and often accompanied by sensory deficits of proprioception and tactile perception (Cooper et al. 1995). These symptoms induce limitations on manual actions performed with the affected hand (AH), in particular related to fine motor skills such as fingertip force control (Eliasson et al. 1991). These limitations are further amplified by more central deficits in the integration of different modalities, such as matching proprioceptive and visual spatial information (Wann 1991), and integrating sensory information with motor output (Gordon et al. 2006). While the less affected hand (LAH) is usually spared from severe sensorimotor impairments (but see, Steenbergen and Meulenbroek 2006 for subtle deficits at the less affected body side), there is converging evidence for more high-level deficits in prospective control, or motor planning, in particular, in individuals with right hemiparesis (Mutsaarts et al. 2005, 2006; Steenbergen et al. 2000a, 2004; te Velde et al. 2005).\nThese studies on manual control have advanced our knowledge about the different levels at which the motor system affects CP as well as the behavioural reorganisation during the performance of actions (e.g., van Roon et al. 2005b). Yet, up to date, eye\u2013hand coordination has not been investigated, despite preliminary evidence suggesting that it may contribute to deficits in action performance in individuals with CP (van der Meer et al. 1995; van der Weel et al. 1996). Even indirect evidence regarding the role of visual feedback for manual action performance in CP is scarce. Wann (1991) investigated visuo-proprioceptive integration in adolescents with CP. Comparing arm postures in a bimanual position-matching task under different conditions of sensory feedback, he found that, in contrast to adult controls, participants with CP employed very similar end postures (joint angle configurations) with the left and right arm in conditions in which vision of one or both arms was prevented. This was interpreted as evidence that they relied more strongly on purely proprioceptive information rather than on a shared visuospatial-proprioceptive representation and that the encoding of visual and proprioceptive information into a common egocentric frame may be problematic in these individuals. This would suggest that individuals with CP more strongly depend on continuous visual feedback for guiding manual actions to visual targets, compared to neurologically healthy adults.\nMoreover, as the severity of the motor impairment, and concomitant proprioceptive impairment, increases from proximal to distal (e.g., Steenbergen et al. 2000b), it may be expected that actions with the affected-hand involving more fine motor control, such as grasping, need closer visual monitoring to provide additional feedback. In line with this, Steenbergen and colleagues (1996), studying bimanual actions in participants with hemiparetic CP, made the anecdotal observation that (overt) visual attention seemed to be drawn to the affected side of the body. However, in contrast to the reasoning above, a recent study that scrutinized the effect of removing visual information of the moving limb on end-point accuracy in a straight-line drawing task (van Roon et al. 2005a) found no specific adverse effect of this manipulation in participants with tetraparetic cerebral palsy when using their relatively less-impaired hand compared to control participants. Thus, up to date, there is no consensus on the role of visual guidance for the control of manual actions in CP.\nBesides the sensorimotor impairments of the AH, more high-level aspects of action control, such as motor planning and prospective control, have been shown to be compromised as well in hemiparetic CP. In research on motor planning in individuals with CP, the LAH is used, as this hand has no (or small) movement restrictions, as is often the case with the AH. Hence, the higher-order process of motor planning can be studied without the confounding effects of possible movement restrictions. Atypical performance with the LAH was found for instance in tasks involving planning of the hand\u2019s posture at the end of the action (Steenbergen and Gordon 2006; Steenbergen et al. 2000a), in particular, in individuals with right hemiparesis (Steenbergen et al. 2004). In these tasks, individuals have to pick up objects and place them in another orientation on a designated target. Ample evidence in controls suggest that individuals pick-up objects with a grip that allows them to end the task with a comfortable posture, even when this means that they have to sacrifice comfort of the start grip (see Steenbergen and Gordon 2006). This \u201cend state comfort\u201d effect was not present in individuals with CP, indicating a higher-level impairment of movement planning. Prospective control in individuals with hemiparetic CP was recently investigated in a collision avoidance task (te Velde et al. 2005). This study found later, hand-movement initiation in individuals with right as compared to left hemiparesis, again indicating reduced anticipation of action requirements. The extent to which deficits in motor planning or prospective control may be related to deviations in visual monitoring, with its typically anticipatory role for action control (Johansson et al. 2001) has not been investigated yet in individuals with CP.\nIn the present study, we examined eye\u2013hand coordination in an unconstrained object prehension task in individuals with hemiparetic CP and neurologically healthy controls. To manipulate task difficulty, an obstacle was present in half of the trials, inducing a more complex transport trajectory. Participants performed the task with each hand separately. Based on the registration of eye and hand movements, we calculated several measures of temporal and spatial aspects of eye\u2013hand coordination. Besides giving a first descriptive account of gaze patterns during object manipulation in a population with congenital hemiparesis, we addressed two hypotheses. Our first and main research question concerned the way in which individuals with hemiparetic CP adapt eye\u2013hand coordination to the sensorimotor impairments of their affected hand. We hypothesized that individuals with CP would use increased visual monitoring when they perform the task with their AH as compared to performance with the LAH and to performance of control participants. Second, we examined eye\u2013hand coordination when the task was performed with the LAH in participants with CP. Based on the previously established deficits in action planning, we hypothesized that gaze patterns are less anticipatory in general, in individuals with CP. Given the limited knowledge on visumotor control in CP and on the role of eye-movements for action planning, this second research question was much more exploratory than the first.\nMethods\nParticipants\nIn total, 16 individuals took part in the study. Six participants with hemiparetic CP constituted the experimental group and ten participants with no known history of neurological disorders served as controls. Control participants were all right handed (Dutch version of the Edinburgh handedness inventory, Oldfield 1971) University students (9 females, 20\u201325\u00a0years). The six participants of the experimental group (5 females, 14\u201319\u00a0years) were students at the Werkenrode Institute (Groesbeek, The Netherlands), where they followed an adapted educational programme. They had been asked to take part in the study based on their condition (viz. congenital spastic hemiparesis). Additional requirements were no known oculomotor disturbance (such as nystagmus), and having the motor, cognitive and attentional capacities to perform the experimental task as assessed by initial screening. All were able to walk and sit independently, and able to read and write with their LAH.\nConsent was obtained prior to the experiment, both in verbal form from the participants during the initial screening session, as well as in written form from their parents or caregivers. In a separate session taking place after the experiment, the participants with CP performed two standardised dexterity tests, the Box and Block test for gross dexterity (Mathiowetz et al. 1985) and the Purdue pegboard test for fine dexterity (Tiffin 1968). One of the participants was not available for the manual dexterity testing afterwards. Table\u00a01 shows the results of these tests as well as other participant information. For the Purdue\u2013Pegboard test, the norm score for individuals at the age of 16\u201317\u00a0years is 49.5 for the preferred hand. Standardised norm scores for the Box and Block test are not available for individuals younger than 20\u00a0years. For the youngest age group available, individuals between 20 and 24, the norm score is 88.2 for the preferred hand. The performance of the participants with CP was consistently below these norms, even when using the LAH.\nTable\u00a01Participant information for the participants with cerebral palsyParticipantAgeParetic sideB and Ba, cPPb, cCP115Right15\/380\/33CP217Left24\/520\/42CP317Left16\/326\/27CP415Right15\/430\/33CP514Left18\/423\/33CP619RightNAdNAdaBox and Blocks testbPurdue\u2013Pegboard testcAffected hand\/less affected handdNot available for testing\nAll participants received the same reimbursement (\u20ac6 per hour) for taking part. The study was approved by the local ethics committee and performed in accordance with the standards laid down in the 1964 Declaration of Helsinki.\nSetup and procedure\nThe experimental setup is depicted in Fig.\u00a01. Participants were comfortably seated on a chair approximately centered at the midline of the table, which constituted the working region for the experiment. Two objects (blue and green), two target regions, and an optional obstacle were placed on the table, as well as a fixation target (see Fig.\u00a01 and caption for details). Two loudspeakers, placed below the table, were used to verbally instruct the participant through a pre-recorded voice. The experimenter sat facing the participant at a separate table. From this position he was able to supervise the experiment by instructing the participant, starting and terminating the trials, and monitoring participants\u2019 performance (directly and via a computer monitor that showed the eye movement recording system\u2019s output in real time).\nFig.\u00a01Experimental setup (top view). The participant is seated at the table, with the right hand resting at the starting position. Positions of objects (blue and green cylinder, 6\u00a0cm in height, 3\u00a0cm in diameter, with an enlarged stabilizing basis of 6\u00a0cm in diameter), target regions (blue and green disc, 10\u00a0cm in diameter, contralateral to the corresponding object), optional obstacle (cylinder, 15\u00a0cm in height, 6\u00a0cm in diameter), and the pre-trial fixation target (red ball 2\u00a0cm in diameter mounted on a frame at about eye height) are shown. The experimenter was seated at a separate desk facing the participant. The dashed line represents the object transport movement from object to target region in a \u201cblue\u201d trial. The x- and z-axis of the Optotrak system were approximately aligned to the axes indicated here, the y-axis was orthogonal to both (vertical axis)\nFor each participant, the experiment consisted of eight conditions in a 2\u00a0\u00d7\u00a02\u00a0\u00d7\u00a02 factorial design, with the factors hand (AH\/LAH for the participants with CP, NPH\/PH for controls), obstacle (present, absent) and side (blue, green). In each of these conditions, five trials were performed, yielding a total of 40 trials per subject. The factors hand and obstacle were blocked while side was randomized within blocks. The order of the blocks was partially counterbalanced between participants. Prior to each trial, the participant was instructed to rest the \u201cinactive\u201d hand in his\/her lap, such that it did not interfere with the task.\nEach trial started with the task-hand resting at the starting position, and with gaze directed to the fixation target. After a variable delay (500\u20131,500\u00a0ms), the pre-recorded instruction \u201cgreen\u201d or \u201cblue\u201d (in Dutch) was played, upon which, the participant was to grasp the corresponding object and transport it to and place it on the contralateral target region of the same color. When an obstacle was present, the object had to pass in front of it, that is, between obstacle and participant (see Fig.\u00a01, dashed arrow-line). After placing the object on the target region, the participant moved his\/her hand back to the starting position and the experimenter put the cylinder back to its original location. When a trial failed, for instance because the cylinder slipped out of the participant\u2019s hand, it was immediately repeated. This never occurred more than twice in a single participant.\nData acquisition\nDuring the experiment, hand and head movements were recorded using an Optotrak 3020 system (sampling rate set at 125\u00a0Hz). Four Optotrak markers were located on the Eyelink helmet (see below) to monitor position and orientation of the head. Hand movements were recorded by means of a marker placed on the back, each of the participant\u2019s hands near the head of the second metacarpal. For two participants with CP, this position did not guarantee sufficient visibility of the marker on the AH due to individual grasping patterns (excessive arm pronation leading to a thumb-down grasping posture). In these instances, the marker was placed more laterally on the hand. As we were mainly interested in the temporal aspects of the movements kinematics, this adjustment did not affect the measurement in a relevant way. The coordinate frame of the Optotrak system was oriented such that x, y and z axes roughly corresponded to the horizontal, vertical and posterior\u2013anterior dimension with respect to the participant\u2019s position (see Fig.\u00a01).\nGaze direction was assessed using a head mounted Eyelink II system (sampling frequency 500\u00a0Hz), which was calibrated twice during the experiment, before each of the main blocks that corresponded to the obstacle present\/absent conditions. To calibrate the system, the subject was asked to look at 10\u201315 points covering the frontal working plane. These points were manually indicated by the experimenter by using a stick on the end of which both a fixation target (small red ball with a diameter of 13\u00a0mm) and an Optotrak marker were attached. This \u201cinteractive\u201d calibration method was chosen because pilot recordings revealed that two participants with CP failed to follow the instructions of the standard calibration procedure (employing a calibration board with LEDs). In addition, the \u201cinteractive\u201d method allowed for constant monitoring of the Eyelink system\u2019s output by the experimenter (see \u201cSetup and procedure\u201d).\nThe position of the fixation target and the position of the eye relative to the Eyelink helmet were used to compute gaze direction. Gaze direction was subsequently transformed to a 2D signal by projecting it to the xy-plane. Linear regression between this direction and the raw Eyelink data was used to calibrate the system. Although calibration error (deviation between actual and reconstructed gaze direction for the calibration targets) was relatively large\u2014on average, 5\u00b0 of visual angle, increasing towards the periphery\u2014it was well within the precision requirements needed to answer our research questions. Particular care was taken in the analysis of eye movements (see \u201cEye movements\u201d) to minimize the effect of this inaccuracy.\nPreprocessing\nThe raw Optotrak data were partially interpolated with cubic spline interpolation (up to ten successive samples, corresponding to 80\u00a0ms) and low-pass filtered (third order Butterworth filter with cut-off frequency of 10\u00a0Hz). Hand velocity was computed by 3-point numerical differentiation.\nThe raw Eyelink signal was transformed to relative gaze direction and integrated with the helmet position and orientation to yield absolute gaze direction. Approximate fixations were computed as the intersection of gaze direction with the plane parallel to the xy-plane (see Fig.\u00a01) and containing the centers of objects and target regions. Only data from one eye (selected by visual inspection for each individual participant) were used for further analysis. Gaze data were partially interpolated (up to four successive samples, corresponding to 8\u00a0ms, using cubic spline interpolation) and low-pass filtered (third order Butterworth with cut-off frequency of 20\u00a0Hz). Subsequently, gaze velocity, acceleration, and jerk (second time differential of velocity) were computed by repeated 3-point numeric differentiation.\nData reduction\nHand movements\nHand movements were identified using an absolute velocity threshold (0.20\u00a0m\/s) and a direction criterion (start of a new movement indicated by a reversal of horizontal direction). These criteria provided the algorithms for the semi-automatic custom-written selection routines for the segmentation of the trials. Based on this segmentation, the grasp time (interval between the hand reaching and leaving the object region) and hand movement duration (interval between the hand leaving the object and reaching the target region) were determined. Analysis of eye\u2013hand coordination was confined to the object transport phase that is from onset of the hand movement away from the object region till the end, reaching the target region. The duration of this movement was determined.\nEye movements\nSaccades were detected using a jerk (second time differential of velocity) criterion, which has been shown to be more reliable than methods based purely on gaze velocity or acceleration, especially for head-free eye movement recordings. A saccade was scored whenever jerk exceeded 200.000\u00b0\/s3, with an additional velocity requirement (50\u00b0\/s) (see Wyatt 1998, for similar analaysis). Saccade termination was determined as the first moment after saccade onset at which velocity dropped below 50\u00b0\/s. The total number of saccades registered in the experiment was 4,613. Saccade initiation\/termination time was determined when none of the first or last eight samples (16\u00a0ms) were missing. Saccade peak velocity was determined, when in addition no more than four samples were missing in a row throughout the entire saccade. According to these criteria, saccade initiation and termination time could not be determined in 5.5% of all saccades, and peak velocity could not be determined in 10.5% of all saccades.\nTo test for potential group differences in basic eye movement kinematics, saccade main sequences were plotted, based on the saccades for which the peak velocity could be determined. Main sequences showed, for both groups, the typical linear relationship between amplitude and duration (e.g., Carpenter 1988), and the log-linear relationship between amplitude and peak velocity for all participants of both groups. No significant group differences were found in gradient or intercept of linear regressions of these relationships (all p\u00a0>\u00a00.05, Welch\u2019s unpaired t-test).\nSubsequently, for each trial the \u201cobject-leaving\u201d and the \u201ctarget-reaching\u201d saccade were determined (in many trials these saccades coincided since there was a single gaze shift from object to target). Automatic detection of these saccades was complicated by the fact that precision of gaze data was not sufficient for a procedure based on landmark regions around object and target. Therefore, the following semi-automatic two-step procedure was adopted. First, fixations to object and target were determined automatically, assuming that these occurred before (up to \u2212600\u00a0ms) the beginning and at the end of object transport. Fixation periods were defined as intervals of at least 200\u00a0ms, during which, the standard deviation of absolute gaze fixation was smaller than 5\u00a0mm. Note that this automatic procedure did not use absolute landmark regions but relative position information\u2014this was possible because the object and target were placed at the lateral extremes of the working region of the experiment. For the obstacle, an analogous procedure was not possible.\nChoices of this first step were inspected trial by trial and manually corrected if necessary. Obstacle fixations were not taken into account since it was not always possible to reliably distinguish them from other, frequently occurring intermediate fixations between object and target. Second, an automatic routine was used to detect the object-leaving saccade, defined as the first saccade, at the end of which, gaze had moved more than 10\u00b0 horizontally relative to the object fixation. Based on an analogous 10\u00b0 criterion, the target-reaching saccade was determined at the end of object transport. Thus, small (<10\u00b0) corrective saccades at the end of object transport were ignored in the definition of the target-reaching saccade.\nEye\u2013hand coordination\nVisual monitoring during object transport was quantified by the number of intermediate fixations, that is, the number of gaze shifts minus one occurring between the object-leaving and the target-reaching saccade. Note that this number does not include potential small corrective saccades at the end of object transport, since the target-reaching saccade was defined as the last saccade of horizontal amplitude >10\u00b0 reaching the target region.\nTo examine temporal coordination between eye and hand at the beginning and end of object transport, two variables were computed: the movement onset asynchrony (MOA) and the movement termination asynchrony (MTA). The MOA was defined as the interval between the start of the object transport movement and the onset of the object-leaving saccade, with larger (positive) values corresponding to later gaze departure. Similarly, the MTA was determined by the delay between the termination of the object transport movement and the end of the target-reaching saccade, with smaller (negative) values corresponding to earlier target fixation. To eliminate a potential confounds with hand kinematics (in particular in the comparison of AH and LAH in participants with CP), both MOA and MTA were normalized with respect to hand movement duration (expressed as percentage of the latter).\nWith respect to our experimental hypotheses, we predicted that closer visual monitoring would be reflected in greater gaze-hand proximity (smaller average angular distance), larger number of intermediate gaze fixations, and delayed gaze departure from the object region (that is, a longer MOA). Regarding prospective control, we assumed that less anticipatory gaze patterns would be reflected in delayed departure from the object region (longer MOA) and delayed arrival on target (shorter\/less negative MTA).\nStatistical analysis\nData from one control participant were excluded due to insufficient quality of the Eyelink data, leaving data from six experimental and nine control subjects for statistical analysis. For each of the five variables of hand kinematics and eye\u2013hand coordination introduced in \u201cData reduction\u201d, the means averaged across all replications in each condition were submitted to a three-way repeated measures ANOVA with one between-subject factor (group: CP\/control) and two within-subject factors (hand: AH\/LAH, NPH\/PH; obstacle: present\/absent). Subsequent analyses of simple effects were performed using paired t-tests for within-subject factors, and Welch\u2019s unpaired t-test (no equal variance assumed) for the between-subject factor. To correct for multiple (five) ANOVAs, the critical significance level was set to 0.01.\nResults\nOf the total 15\u00a0\u00d7\u00a040\u00a0=\u00a0600 trials, seven trials were entirely rejected due to insufficient data quality (no more than two trials in any individual participant).\nSample trials\nFigures\u00a02 and 3 show representative trials from participants with CP and control participants. Horizontal components of head and hand position and gaze fixation (see \u201cData reduction\u201d) are plotted on the vertical axis, as a function of time. The intervals and durations of the (non-normalized) MOA and MTA are indicated by vertical arrows and numerically in the plots. To allow comparisons between the plots, all data shown are from obstacle present trials. Visual inspection indicates qualitatively similar patterns (saccadic gaze shifts to future hand targets) in all conditions, and for both participant groups. Complete visual tracking of the trajectory of the hand was found only in a few trials (detected by visual inspection) in two participants with CP when using their AH. For these trials, MOA and\/or MTA could not be determined.\nFig.\u00a02Sample trials of two control participants, using their PH (a, c) and NPH (b, c) in trials with the obstacle present. Interpolated data points are represented by dashed lines. Vertical arrows at the top and bottom of the plots specify the intervals defining the MOA and MTA (beginning\/end of hand and eye movement), with numerical values of these measures addedFig.\u00a03Sample trials of two participants with CP, using their LAH (a, c) and AH (b, d) in trials with the obstacle present. Note the delayed gaze departure (longer MOA) and increased number of intermediate fixations for the AH compared to the LAH and to both hands of control participants (Fig.\u00a02). Interpolated data points are represented by dashed lines. Vertical arrows at the top and bottom of the plots specify the intervals defining the MOA and MTA (beginning\/end of hand and eye movement), with numerical values of these measures added\nThe representative trials shown in Fig.\u00a02 (control participants using their PH and NPH) and Fig.\u00a03 (participants with CP using their LAH and AH) illustrate some general trends when participants with CP use their AH. These are a prolonged MOA, a larger number of intermediate fixations and a greater gaze-hand proximity compared to the other combinations of group and task hand. These observations are in agreement with the hypothesis of increased visual monitoring (see Eye\u2013hand coordination\u201d), and this is corroborated by the statistical analysis presented below. In contrast, no clear pattern with respect to the MTA is obvious from these data plots, nor from the statistical analysis.\nDependent variables\nValues for the different dependent variables are summarized in Table\u00a02. Due to missing samples, some of the measures reported below could not be determined in a number of trials. This number did not exceed 40 out of 600 trials (never more than five out of ten in a single condition and participant) for any of the variables.\nTable\u00a02Mean and standard deviation (between participants) of dependent variablesDependent variableParticipants with CPControl participantsLAHaAHbPHcNPHdNo obstacleObstacleNo obstacleObstacleNo obstacleObstacleNo obstacleObstacleHand movement duration (s) \u00a0Mean0.811.051.051.600.610.83 0.650.87\u00a0SEM0.030.050.030.150.010.020.020.03Object grasp time\u00a0Mean0.220.251.071.160.0630.0710.0860.094\u00a0SEM0.030.040.140.160.0090.010.0170.015Number of intermediate fixations\u00a0Mean0.240.820.76 1.300.06 0.73 0.07 0.61\u00a0SEM0.090.340.241.210.040.220.060.26Movement onset asynchrony (% hand movement duration)\u00a0Mean\u22126.881.5313.4 14.62\u22124.45 0.02\u22123.143.84 \u00a0SEM5.814.964.372.124.844.214.844.37Movement termination asynchrony (% hand movement duration)\u00a0Mean\u221276.8 \u221261.2 \u221249.5 \u221251.4 \u221275.7 \u221265.6 \u221271.5 \u221262.2 \u00a0SEM5.396.872.843.644.393.784.643.56aLess affected handbAffected handcPreferred handdNon-preferred hand\nGrasp time showed main effects of group, hand as well as a group\u2013hand interaction (ANOVA, all p-values\u00a0<\u00a00.0001). Both, with the AH and the LAH participants with CP showed a longer grasp time than controls using either hand. The simple effect of hand was only present in participants with CP (longer grasp time with the AH compared to the LAH).\nHand movement durations for object transport showed main effects of group, hand, and obstacle, as well as a group\u2013hand interaction (ANOVA, all p-values\u00a0<\u00a00.0001). Simple effect analysis revealed that the effect of hand was present in both groups, but more pronounced in participants with CP. Moreover, the movement duration was longer in participants with CP and in the presence of an obstacle (Fig.\u00a04).\nFig.\u00a04Mean\u00a0\u00b1\u00a0SEM of the movement onset asynchrony (MOA) as a function of participant group, task hand and obstacle presence. MOA was normalized with respect to hand movement duration\nRegarding the number of intermediate fixations during object transport, a main effect of obstacle [F(1,13)\u00a0=\u00a015.06, p\u00a0<\u00a00.002] was found, as well as a group\u2013hand interaction [F(1,13)\u00a0=\u00a012.36, p\u00a0<\u00a00.005]. Analysis of simple effects showed that the number of intermediate fixations was larger in the obstacle present condition than in the obstacle absent condition. With respect to the group\u2013hand interaction, significant differences were found between participants with CP using their AH and control participants using either hand (higher number of intermediate fixations for CP using AH), but not between the AH versus LAH of participants with CP.\nTemporal eye\u2013hand coordination at the beginning of object transport, as quantified by MOA (see Fig. 4), showed main effects of hand [F(1,13)\u00a0=\u00a017.25, p\u00a0<\u00a00.002] and a group\u2013hand interaction [F(1,13)\u00a0=\u00a012.55, p\u00a0<\u00a00.005]. The simple effect of hand was present in participants with CP, with longer MOA in the AH condition, but not in control participants. Moreover, there was a significant difference (prolonged MOA) between participants with CP using their AH and control participants using either hand.\nWith respect to MTA, a main effect of hand [F(1,13)\u00a0=\u00a025.68, p\u00a0<\u00a00.0005], a group\u2013hand interaction [F(1,13)\u00a0=\u00a014.189, p\u00a0<\u00a00.005] were found, as well as a marginally significant group\u2013hand\u2013obstacle interaction [F(1,13)\u00a0=\u00a08.19, p\u00a0=\u00a00.013]. The simple effect of hand was present only in participants with CP, with a shorter (less negative) MTA when using the AH. Also the difference between participants with CP using the AH and control participants using either hand was significant (shorter MTA in participants with CP using the AH).\nDiscussion\nThe main question we pursued in the present study was whether and in which way individuals with hemiparetic CP adapt eye\u2013hand coordination to their sensorimotor impairments, in particular, when actions are performed with the affected hand (AH). We hypothesized that participants with CP would more closely monitor actions performed with their AH, compared to actions performed with their less affected hand (LAH) and compared to neurologically healthy control participants using either hand. Qualitatively, eye\u2013hand coordination patterns were similar among control participants and participants with CP, regardless of the hand used to perform the task. That is, anticipatory saccadic gaze shifts were used to fixate future \u201caction sites\u201d\u2014such as object, target, or intermediate fixations\u2014in advance. Notwithstanding these qualitative resemblances, a more fine-grained analysis of temporal and spatial aspects of eye\u2013hand coordination did confirm our hypothesis of increased visual monitoring when moving with the AH. Depending on the measure employed, this effect was present when comparing performance with the AH to control participants using either hand (increased number of intermediate fixations), or in addition for comparing performance with the AH to performance with the LAH in participants with CP (delayed gaze departure from object and hand, i.e., a longer MOA).\nIt is important to note that our main result regarding visual monitoring is an interaction effect of group and task hand (effect of task hand was present only in the experimental group). Therefore, it cannot be explained by potential group differences in oculomotor control or gaze data quality. Moreover, despite previous findings that did not find a strong relationship between gaze-exit time relative to hand action and task speed (Flanagan and Johansson 2003), we decided to normalize our measures of temporal eye\u2013hand coordination in order to exclude any potential confounding effects of hand kinematics. This normalization may be considered rather conservative. Without normalizing, the effects of task and participant group on MOA may have even been stronger.\nA second, and more speculative hypothesis of the present study was that in general, gaze patterns might be less anticipatory in participants with CP, contributing to deficits in prospective control and action planning that have been reported in this population (Steenbergen et al. 2000a, 2004; te Velde et al. 2005), in particular, individual with right hemiparesis. The present study found no evidence in support of this hypothesis. Although participants with CP did show a prolonged MOA, that is, a delayed gaze departure that may indicate less anticipation, this effect was restricted to the AH. No significant differences in the MOA were found between actions of participants with CP using their LAH and control participants using their PH. Similarly, for our measure of temporal eye\u2013hand coordination at the end of object transport (the movement termination asynchrony, MTA), a delayed gaze arrival on target was only present in participants with CP using their AH, not as a general group effect. In addition, preliminary analyses had shown no difference between individuals with left and right hemiparesis with respect to any of our dependent variables. Thus our results suggest that deficits in prospective action control found in CP are not directly related to atypical eye\u2013hand coordination. However, this statement is made with caution as we only tested a small number of participants (n\u00a0=\u00a06 in total, n\u00a0=\u00a03 for each left and right hemiparesis).\nAn unexpected finding of the present study was that gaze frequently departed from the object region only after the hand movement had started, in particular, in the presence of the obstacle. This was also the case in control participants and is in contrast to the results of Johansson et al. (2001), who reported that in the majority of the trials gaze departure from the grasp site occurred even before the fingertips had reached the object. These contrasting findings may in part be explained by differences in the experimental set-up. In the present study the distance between initial object position and the target was much larger compared to the one used by Johansson et al. (2001), leading to longer hand movement durations. This potential confounding effect of hand movement duration needs to be addressed in future research, for instance, by systematically manipulating hand movement distance in order to disentangle components of online and prospective control in the MOA. Also, task complexity might play a role. In a block-stacking task, Flanagan and Johansson (2003) found gaze to depart on average just before hand movement onset, which is more consistent with our present findings.\nIn sum, we found evidence in support the hypothesis of increased visual monitoring of manual actions of individuals with hemiparetic CP performing actions with their AH, both at the beginning and during object transport. This may reflect a strategy of visual compensation for sensorimotor deficits (see also, Mulder et al. 2001). However, since the functional significance of the observed gaze patterns was not addressed in the present study, the question whether and by which neurophysiological mechanisms vision enhances manual performance requires further research. Generalizability of our results is certainly limited by the fact that our experimental group was relatively small (n\u00a0=\u00a06) and highly selective, considering the high variability in the CP population regarding intellectual and motor abilities. In particular, the null-findings on anticipatory control and the comparison of individuals with left and right hemiparesis should be considered preliminary and require further investigation. On the other hand, the fact that we did find significant effects with respect to online visual control (number of intermediate fixations and MOA) despite the small number of participants suggests that the observed effects are robust.","keyphrases":["prehension","visuomotor control","sensorimotor impairment","adaptation","affected side","congenital brain damage"],"prmu":["P","P","P","P","P","R"]}
{"id":"Purinergic_Signal-2-2-2096640","title":"E-NTPDases in human airways: Regulation and relevance for chronic lung diseases\n","text":"Chronic obstructive lung diseases are characterized by the inability to prevent bacterial infection and a gradual loss of lung function caused by recurrent inflammatory responses. In the past decade, numerous studies have demonstrated the importance of nucleotide-mediated bacterial clearance. Their interaction with P2 receptors on airway epithelia provides a rapid \u2018on-and-off\u2019 signal stimulating mucus secretion, cilia beating activity and surface hydration. On the other hand, abnormally high ATP levels resulting from damaged epithelia and bacterial lysis may cause lung edema and exacerbate inflammatory responses. Airway ATP concentrations are regulated by ecto nucleoside triphosphate diphosphohydrolases (E-NTPDases) which are expressed on the mucosal surface and catalyze the sequential dephosphorylation of nucleoside triphosphates to nucleoside monophosphates (ATP \u2192 ADP \u2192 AMP). The common bacterial product, Pseudomonas aeruginosa lipopolysaccharide (LPS), induces an acute reduction in azide-sensitive E-NTPDase activities, followed by a sustained increase in activity as well as NTPDase 1 and NTPDase 3 expression. Accordingly, chronic lung diseases, including cystic fibrosis (CF) and primary ciliary dyskinesia, are characterized by higher rates of nucleotide elimination, azide-sensitive E-NTPDase activities and expression. This review integrates the biphasic regulation of airway E-NTPDases with the function of purine signaling in lung diseases. During acute insults, a transient reduction in E-NTPDase activities may be beneficial to stimulate ATP-mediated bacterial clearance. In chronic lung diseases, elevating E-NTPDase activities may represent an attempt to prevent P2 receptor desensitization and nucleotide-mediated lung damage.\nIntroduction\nAirway epithelia constitute an essential protective barrier against lung infection, coordinating luminal and interstitial responses to inhaled pathogens through signals (growth factors, cytokines and nucleotides) provided by epithelial, inflammatory and immune cells [1]. It is now widely accepted that extracellular nucleotides provide an elaborated cell communication system in mammalian tissues [2, 3] including the airways [4, 5]. Each signaling event constitutes a brief \u2018on-and-off\u2019 switch mechanism allowing the target cells to perceive the subsequent signal. The major source of extracellular nucleotides in normal airways is the epithelium, releasing ATP under resting conditions and in response to mechanical or osmotic stress [6]. While basal ATP concentrations maintained under resting conditions are insufficient to activate surface receptors, the levels reached near the site of stimulated release initiate a variety of P2 receptor-mediated responses [7]. Two P2 receptor families have been identified: Fast-acting (ionotropic) P2X ligand-gated cation channels and slow-acting (metabotropic) G protein-coupled P2Y receptors [8]. Although ATP activates members of both families, P2X receptors generally respond to higher concentrations (EC50 = 1\u201310 \u00b5M) than P2Y receptors (EC50 = 0.1\u20131.0 \u00b5M). Each signal is promptly terminated by surface conversion of ATP to adenosine [9, 10] and dispersal into the interstitial fluid. Adenosine also initiates cellular responses through P1 (A1, A2A, A2B or A3) surface receptors [11]. In the airways, nucleotide- and adenosine-mediated communications are involved in wound healing [4], bacterial clearance [12] and inflammatory responses [1, 3, 13, 14].\nCell surface nucleotide concentrations are regulated by three families of ectonucleotidases in mammalian tissues: Ecto-nucleotide pyrophosphatase\/phosphodiesterases (E-NPPs: ATP \u2192 AMP), alkaline phosphatases (APs: ATP \u2192 AMP \u2192 AMP \u2192 adenosine) and ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases: ATP \u2192 AMP \u2192 AMP) [15]. This review describes the importance of regulating extracellular nucleotide concentrations for airway homeostasis and the impact of chronic lung diseases on E-NTPDases. Few studies have addressed the activity or expression of airway E-NTPDases. In this review, the available information is complemented by original data.\nNucleotide-mediated bacterial clearance\nThe importance of purine signaling in the airways is well illustrated in nucleotide-mediated mucociliary clearance (MCC), which constitutes the first line of defense against bacterial infection [16]. Mucosal epithelial surfaces are protected by an airway surface liquid (ASL) layer containing a thin mucin sheath kept above the cilia by a periciliary liquid (PCL) layer (Fig. 1). Inhaled bacteria adhere to the mucin and are continuously displaced upward by coordinated cilia beating activity [17, 18]. Comparative analysis of the bioelectric properties of airway epithelia from normal donors and CF patients revealed that PCL height is regulated by a delicate balance between the activities of the epithelial sodium channel (ENAC) and the cystic fibrosis transmembrane regulator (CFTR) [19]. Cystic fibrosis is characterized by mutations in the gene encoding CFTR [16]. The lack of ion secretion through CFTR is associated with Na+ hyperabsorption by ENAC and water influx, resulting in the depletion of the PCL layer essential for cilia beating activity. The cilia become collapsed under a thick layer of mucus filled with bacteria and leukocytes, leading to severe inflammatory responses and progressive loss of lung function.\nFig. 1Impact of chronic obstructive lung diseases on mucociliary clearance. The polarized epithelium is composed of columnar (ciliated or mucin-secreting) cells facing the lumen and basal cells facing the serosal compartment. Bacterial clearance in healthy lungs is maintained by coordinated cilia beating activity within the PCL layer, moving upward the overlying mucus and pathogens. In chronic obstructive lung diseases, cilia are collapsed under a thick layer of mucus containing bacteria and leukocytes.\nExtracellular nucleotides and nucleosides regulate CFTR and ENAC [20]. The basal activity of CFTR is maintained by adenosine (A2B) receptors, which induce G protein-coupled adenylate cyclase, followed by cAMP-dependent activation of type II protein kinase A [5]. In contrast, ATP stimulates Cl\u2212 secretion through Ca2+-activated Cl\u2212 channels. This signaling pathway involves P2Y2 receptors, leading to G protein-coupled phospholipase C activation and cytosolic Ca2+ mobilization. Water efflux is also stimulated by P2Y2 receptor-mediated inhibition of Na+ absorption by ENAC using Ca2+-dependent signaling pathways [21]. Members of the P2X receptor family also support luminal Cl\u2212\/water efflux in human airway epithelia, as demonstrated in primary cultures and cell lines (Beas2B, 16HBE14o\u2212, Calu-3, (\u03a3CFTE-29o\u2212) by Ussing chamber and patch-clamp experiments [22]. The receptors identified in airway epithelia by RT-PCR were P2X2, P2X4 and P2X5. These non-selective cation channels allow Ca2+ influx, which then stimulates Ca2+-activated Cl\u2212 channels. Interestingly, P2X7 receptors are only detected in CF airway epithelia, primarily in nasal polyps [22]. Because they are known to induce apoptosis in response to high ATP concentrations [23], P2X7 receptors may contribute to disease-associated epithelial damage.\nThe central role of ASL nucleotides for MCC was further substantiated by the observation that P2Y2 receptor activation constitutes the most potent stimulus for cilia beating activity [24\u201326] and mucin secretion [27, 28]. In cultured CF airway epithelia, P2Y2 receptor activation restores normal PCL height and rotational mucus transport [29]. Based on these findings, aerosolized nucleotides were proposed for the treatment of chronic obstructive lung diseases [16]. However, clinical studies showed that patients with asthma or chronic obstructive pulmonary disease exposed to aerosolized metabolites of ATP (AMP and adenosine) experience coughing and bronchoconstriction [30, 31]. Furthermore, aerosolized ATP (not adenosine) induced similar reactions from healthy subjects [32, 33]. In developing nucleotide-mediated MCC therapies, the equally potent P2Y2 receptor agonist, UTP, was evaluated [6]. Airway clearance in sheep, measured with nebulized technetium-labeled albumin, was transiently increased by UTP [34]. Aerosolized UTP also improved MCC in patients with mild chronic bronchitis [35], CF [36, 37] and primary ciliary dyskinesia [38].\nA major obstacle to the treatment of chronic lung diseases with aerosolized UTP is the rapid disappearance of therapeutic concentrations (0.1 mM) from the ASL layer (<5 min) [10, 29]. Incidentally, the metabolically stable UTP analogs INS365 (diquafosol) and INS37217 (denufosol) developed by Inspire Pharmaceuticals Inc. [39] were reported to improve cough induced-sputum clearance in CF patients [40, 41]. However, long-term treatments with these drugs may require dose optimization to minimize P2Y2 receptor desensitization [42].\nP2 receptors and airway inflammation\nIn vivo studies conducted on the injurious effects of mechanical ventilation demonstrated that endogenous ATP may reach ASL concentrations sufficiently high to trigger inflammatory responses and lung damage in the absence of infection [43]. The bronchoalveolar lavage fluid of rats subjected to positive-pressure mechanical ventilation contains significantly higher protein, ATP and cytokine (IL-6, TNF\u03b1) levels than control animals. Instillation of an equivalent ATP concentration increased lung fluid volume, supporting the existence of P2 and\/or P1 receptor-mediated lung edema [43]. Analysis of bronchoalveolar lavage fluid nucleotide content by etheno-derivatization revealed an increase in AMP + adenosine\/ADP + ATP ratio [44], suggesting that mechanical ventilation up-regulates airway ectonucleotidases. Real-time PCR on total lung tissue indicated that mechanical ventilation increases A2B, but decreases P2X7, receptor expression. Rats subjected to both mechanical ventilation and positive end-expiratory pressure exhibited normal bronchoalveolar lavage composition and receptor expression. These findings suggest that patients subjected to positive end-expiratory pressure during large-volume ventilation may avoid ATP-mediated lung injuries. On the other hand, the fact that adenosine levels and A2B receptor expression were raised, whereas ATP levels and P2X7 receptor expression were decreased, by mechanical ventilation supports a dynamic role for ectonucleotidases in the regulation of purine signaling in the airways.\nThe role of P2 receptors in innate defense against bacterial infection was investigated using mice deficient in P2Y1, P2Y2 or both receptors [45]. All these mice exhibited lower survival and lower cytokine levels in lung homogenates than wild-type animals measured 24 h after intranasal instillation of P. aeruginosa. These results suggest a protective role for ATP-induced inflammation [45]. In human airways, P2Y2 receptor activation amplified inflammatory responses to bacterial infection. Primary cultures of bronchial epithelial cells exposed to UTP and sputum from CF patients exhibited an eight-fold increase in IL-8 secretion, compared to two-fold with UTP alone [46]. Furthermore, P2X7 receptors are known to support several processes relevant to inflammation, including LPS neutralization [47], pathogen killing, nitric oxide production [1] and cytokine release from mast cells, leukocytes, dendritic cells and neurons [3, 14]. In human blood, LPS-induced IL-1\u03b2 release is increased 5-fold by P2X7 receptor activation [48]. Altogether, these studies entail that P2 receptor activation may not only induce innate immunity but also amplify pathogen-induced inflammatory responses in chronic infectious lung diseases.\nToo much of a good thing..\nExcessive inflammatory response to recurrent bacterial infection is a major component in the pathogenesis of chronic lung diseases, irreversibly damaging the airways and leading to bronchiectasis and respiratory failure [49\u201351]. With respect to purine signaling, nucleotide-mediated cell communications may be overwhelmed by massive ATP release from cell lysis of bacteria and damaged epithelia, as well as stimulated leukocytes and epithelia [6]. The sensitivity of certain P2 receptors (P2Y2 [42], P2X1 and P2X3 [52]) to agonist-induced desensitization may reduce the efficiency of nucleotide-mediated MCC [53]. On the other hand, chronically elevated ATP may recruit signaling pathways normally dormant in healthy lungs. For instance, P2X7 receptor activation only induces cytokine release and cell death in response to high ATP concentrations encountered in damaged tissues [23]. On astrocytes, LPS-induced TNF\u03b1 secretion is increased by P2Y receptor activation at low micromolar ATP, but reduced by P2X7 receptor activation at high ATP concentrations [54]. The authors propose a beneficial effect for the potentiation of TNF\u03b1 secretion by P2Y receptors during mild or acute inflammation and a protective role for P2X7 receptors in damaged tissues or chronic infectious diseases. These studies suggest that P2X7 receptor-mediated responses may be tissue-specific and\/or influenced by bacterial infections. In the respiratory system, P2Y2 receptors are expressed throughout airway epithelia [39] while P2X7 receptors have only been reported in nasal polyps of CF patients [22]. The impact of P2X and P2Y receptor activation on inflammatory responses mediated by airway epithelia under normal and pathological conditions remains to be investigated.\nE-NTPDases regulate airway ATP\nSeveral studies demonstrate the presence of ectonucleotidase activities on human airway epithelia [9, 10, 55\u201358]. Time-course experiments conducted on polarized primary cultures of human bronchial epithelial cells showed that mucosal ATP is dephosphorylated into ADP, AMP and adenosine (Fig. 2a). Experiments repeated with nasal, bronchial and bronchiolar epithelial cultures indicated that ATP metabolism accelerates toward the alveolar region (Fig. 2b). Such gradient distribution was also reported for non-specific alkaline phosphatase (NS AP) in human airways [9], an ectonucleotidase capable of sequentially dephosphorylating ATP to adenosine [59]. However, complete inhibition of NS AP with 10 mM levamisole [9] only reduced by 25% total ectoATPase activities [10]. Other ATP-regulating ectonucleotidases were identified in mammalian tissues as members of the ENTPDase family [15, 60]. They catalyze the hydrolysis of \u03b3- and\/or \u03b2-phosphate residues, resulting in the formation of nucleoside monophosphates. Their activities are Ca2+ and Mg2+-dependent and insensitive to inhibitors of P-, F- and V-type ATPases or APs [61]. Four E-NTPDases are localized to cell surfaces: NTPDase 1, 2, 3 and 8. Functional studies showed that NTPDase 1 [62] and NTPDase 3 [63] are inhibited by azide, whereas NTPDase 2 [64] and NTPDase 8 [65] are insensitive. The mRNA expression of NTPDase 1, 2 and 3 in human airways was reported in total lung RNA [66] and cultured bronchial epithelial cells [67]. We now provide evidence that NTPDase 1 and NTPDase 3 are both expressed throughout human airways by real-time PCR on freshly excised epithelial cells (Fig. 3). Their expression distribution followed surface ectoATPase activities (Fig. 2b), exhibiting higher mRNA levels in smaller airways (Fig. 3). The fact that 20 mM azide reduced total ectoATPase activities by 45% on human bronchial epithelial cells [67] suggests that the high affinity (Km < 15 \u00b5M) [68] NTPDases 1 and\/or 3 play major roles in the regulation of physiological ASL nucleotide concentrations.\nFig. 2Ectonucleotidases regulate nucleotide concentrations on airway epithelial surfaces. a Polarized primary cultures of human bronchial epithelial cells were assayed with 100 \u00b5M ATP added to the mucosal surface, as we previously described [10]. Buffer sample analysis by high-pressure liquid chromatography (10) shows that exogenous ATP (\u2022) is dephosphorylated into ADP (\u25aa), AMP (\u2246) and adenosine (\u25cb). b Similar experiments repeated on primary cultures of human nasal, bronchial and bronchiolar epithelial cells show that the elimination rate of ATP increases toward alveoli (N = 3, *, P < 0.05; Mann-Whitney test).Fig. 3Expression level of NTPDase 1 and NTPDase 3 along human airways. Total RNA from excised epithelia was analyzed by real-time PCR using SYBR green assays and normalized to the expression level of the house-keeping gene, 18S, as we previously described [89]. The mRNA level of E-NTPDase 1 (\u25a1) and NTPDase 3 (\u25aa) increases toward alveoli (N = 4, *, P < 0.05; Mann-Whitney test).\nThe physiological importance of azide-sensitive E-NTPDases for nucleotide-mediated MCC was recently established using an in vitro model of rhythmic breathing. Airway epithelia are continuously subjected to mechanical stress generated by breathing, coughing or chest movement. Since mechanical stress induces epithelial ATP release [6], static culture conditions may underestimate the lungs capacity to regulate MCC in vivo. Rhythmic pressure changes mimicking normal tidal breathing were reproduced in vitro by a system applying cyclic compressive stress (CCS) to the mucosal surface of primary bronchial epithelial cultures [69]. Whereas CF cultures under static conditions exhibit a depleted PCL layer and mucostasis, CCS mimicking normal tidal breathing (20 cmH2O; 15 cycles\/min) restored normal PCL height and mucus transport through ATP release and P2Y2 receptor activation. These results also suggest that purine signaling may provide an explanation for the beneficial effects of oscillatory therapeutic devices clinically used to stimulate sputum clearance [70].\nCyclic compressive stress also enhances MCC through a reduction in ASL nucleotide metabolism. We recently demonstrated that CCS decreases the rate of ATP hydrolysis on the mucosal surface of normal and CF bronchial epithelial cultures [67]. More importantly, CCS restored normal ectoATPase activities on CF epithelial surfaces. The inhibitory effect of CCS on ATP metabolism was abrogated by 20 mM azide, indicating that E-NTPDase 1 and\/or E-NTPDase 3 are key components in the regulation P2 receptormediated MCC. Perhaps the ability of CF airway epithelia to provide adequate nucleotide-mediated MCC under conditions mimicking normal breathing may contribute to the relatively healthy state of young patients before the establishment of chronic infection [71].\nChronic lung diseases shorten the signals\nRecent studies demonstrate that chronic lung diseases accelerate all metabolic steps supporting the mucosal conversion of ATP to adenosine [10, 72]. We identified the ectonucleotidases involved using primary bronchial epithelial cultures from healthy donors and patients with primary ciliary dyskinesia, CF or \u03b11-antitrypsin deficiency. All airway diseases were characterized by a two- to four-fold increase in the elimination rate of ATP on the mucosal surface, without significant effect on the serosal surface (Fig. 4). Selective inhibitors of NS AP (10 mM levamisole), NTPDase 1 and NTPDase 3 (20 mM azide) partially restored normal activity levels. However, the lower substrate affinities of airway NS AP (36 \u00b5M and 717 \u00b5M) [9] suggest that NTPDase 1 and\/or NTPDase 3 are responsible for the accelerated clearance of P2 receptor agonists in diseased airways.\nFig. 4Chronic lung diseases enhance azide-sensitive E-NTPDases. Primary bronchial epithelial cultures from healthy donors (N) and patients diagnosed with primary ciliary dyskinesia (PCD), CF or \u03b11-antitrypsin deficiency (\u03b1 AT) were assayed with bilateral 1 mM ATP in the absence (\u25aa) or presence (\u25a1) of 20 mM azide, as we previously described [10]. Analysis of buffer samples by high-pressure liquid chromatography showed that azide-sensitive E-NTPDases are concentrated on the mucosal surface under normal and pathological conditions (N = 4, *, P < 0.05; Mann-Whitney test).\nPseudomonas aeruginosa is among the most frequently isolated pathogens from the airways of patients with gram-negative infections [51]. In patients with damaged airways from mechanical ventilation, trauma or viral infection, airway colonization by P. aeruginosa is often followed by pneumonia, sepsis and death. Much of the airway inflammation induced by these organisms is caused by released bacterial products adhering to the epithelial surface, such as LPS. The contribution of bacterial infection to the deregulation of E-NTPDase activities in lung diseases was examined using P. aeruginosa LPS. Primary cultures of human bronchial epithelial cells exposed 24 h on the mucosal surface to an optimum LPS concentration (100 ng\/ml) [73] exhibited a biphasic response over time (Fig. 5A). Azide-sensitive E-NTPDases were transiently reduced over 8 h, without affecting mRNA expression. In contrast, prolonged exposures (24 h) raised the activities two-fold above control levels and increased NTPDase 1 and NTPDase 3 expression by 4 and 10-fold (Fig. 5B). These opposite effects suggest that two regulatory mechanisms may be involved in the acute and chronic effects of LPS on airway E-NTPDases.\nFig. 5Impact of P. aeruginisa LPS on NTPDase 1 and NTPDase 3. A The mucosal surface of primary bronchial epithelial cultures were exposed 0, 1, 8 or 24 h to 100 ng\/ml LPS, then assayed for surface activity by high-pressure liquid chromatography, as we previously described [10]. The activities of azidesensitive mucosal E-NTPDases measured with 0.03 mM ATP were transiently reduced by LPS. B Quantification of their mRNA expression by real-time PCR using the house-keeping gene 18S, as we previously described (89). LPS induced a delayed increase in the expression level of both E-NTPDases (N = 5, *, P < 0.05; Mann-Whitney test).\nOxidative stress is one of the major causes of epithelial injury in chronic lung diseases [74]. Reactive oxygen and nitrogen species released from airway epithelia or activated leukocytes damage cell membranes by peroxidation of lipids, amino acids and carbohydrates. P aeruginosa LPS was reported to induce their release from airway epithelia [75]. Incidentally, intrahepatic cholestasis induced by intraperitoneal injection of LPS reduced total liver NTPDase 1 activity within 2 h [76]. In a rat model of glomerulopathy, short-term exposure (1 h) to LPS inactivated endothelial NTPDase 1, which was prevented by pre-treatment with the anti-oxidant superoxide dismutase [77]. Oxidative stress induced by membrane depolarization [78] or ischemia\/ reperfusion [79] also inhibited endothelial NTPDase 1. Furthermore, short exposures (4 h) to the pro-inflammatory cytokine, TNF\u03b1, inhibited endothelial NTPDase 1, which was mimicked by hydrogen peroxide but prevented by superoxide dismutase [80]. Altogether, these studies suggest that oxidative stress may be responsible for the early and transient reduction in azide-sensitive E-NTPDase activities detected in LPS-treated airway epithelia. Since the mRNA expression of E-NTPDases is unaffected by acute oxidative stress [80] or LPS (Fig. 5B), and the inactivation of NTPDase 1 by ischemia\/reperfusion was prevented by inhibitors of lipid peroxidation [79], the early loss of surface activity may involve local peroxidation of membrane lipids.\nThe long-term effects of LPS on the azide-sensitive E-NTPDases have not been documented in mammalian tissues. On the other hand, a 24 h exposure to endotoxin stimulated NTPDase 2 on astrocytes [81], as well as NS AP on endothelial, mesengial [82, 83] and bronchial epithelial [73] cells. Additionally, the mRNA expression of NTPDase 1 in rat forebrain was up-regulated seven days after transient ischemia [84]. P. aeruginosa LPS [85, 86] is well-known to influence gene expression through the activation of the transcription factors: Nuclear factor-kappa B and activator protein-1 [87]. Recent studies have shown that CFTR acts as a pattern recognition molecule for P. aeruginosa LPS [88]. Endocytosis of the complex triggers translocation of these transcription factors to the nucleus, which initiates innate immunity through the expression of numerous mediators recruiting and activating inflammatory cells. These signaling pathways could be involved in the up-regulation of NTPDase 1 and NTPDase 3 expression resulting from long-term exposures to P. aeruginosa LPS in human airways.\nAdaptation of purine signaling to airway diseases\nIn past decade, the therapeutic potential of aerosolized nucleotides for the treatment of chronic obstructive lung diseases has motivated numerous studies on the importance of purine signaling in the airways. This elaborated cell communication system is involved in major defense mechanisms including bacterial clearance and inflammatory responses to bacterial infection. Each signaling event constitutes a brief \u2018on-and-off\u2019 switch mechanism allowing the target cells to perceive the subsequent signal. Environmental changes are communicated to target cells in the form of released ATP, which binds surface receptors and triggers an appropriate response. The efficiency of this communication system depends on the cells capacity to rapidly eliminate these local bursts in extracellular ATP concentrations to prevent receptor desensitization. This review establishes the pivotal role of E-NTPDases in the regulation of airway defenses mediated by purine signaling, namely bacterial clearance (Fig. 6). Cyclic compressive stress, mimicking normal breathing or clinical oscillatory devices, stimulates P2 receptormediated MCC through ATP release and reduced azide-sensitive E-NTPDase activities. We also presented original data indicating that NTPDase 1 and 3 activities and expressions are up-regulated in chronic lung diseases by inflammatory mediators, including endotoxin. Interestingly, P. aeruginosa LPS generated a biphasic modulation of airway E-NTPDases which may reconcile previous conflicting reports based on exposure duration. Acute LPS exposures reduced ENTPDase activities, as observed during CCS, most likely to clear a pathogen through P2 receptor-mediated MCC. The signaling pathways activated by P. aeruginosa LPS and the lack of concomitant reduction in ENTPDase mRNA levels support a local inhibitory mechanism that may involve oxidative stress-mediated membrane damage. On the other hand, prolonged endotoxin exposures mimicked the impact of chronic lung diseases on NTPDases 1 and 3, characterized by an up-regulation of both activities and mRNA expression. In chronically infected lungs, an increase in E-NTPDase activities may represent an attempt to prevent lung damage, excessive inflammation and P2 receptor desensitization by chronically elevated ATP concentrations.\nFig. 6Purine signaling and nucleotide salvage pathways on human airway epithelia. In normal lungs, basal adenosine levels maintain adequate PCL height for mucus transport through A2B receptor-mediated CFTR activity. Mechanical stimulation of the epithelial surface by an irritant, a pathogen, mechanical ventilation or cyclic compressive stress induces ATP release and elevates ASL concentrations above activation threshold level for P2Y2 receptor activation. This autocrine signal transiently enhances basal MCC through cilia beating, mucin secretion and ion\/water efflux into the lumen. The signal is rapidly terminated by NTPDase 1 and\/or NTPDase 3 dephosphorylating excess ATP to AMP. Ecto 5\u2032-nucleotidase (ecto 5\u2032-NT; CD73) produces ASL adenosine from the resulting AMP, and excess adenosine is transported back to the cytosol. Chronic lung diseases burdened by considerable tissue damage recruits additional receptors (P2X4, 5, 7) responding to higher ATP cocnentrations.\nThe information provided in this review also exposes the complexity of the signaling interactions taking place between different classes of mediators, including bacterial products, cytokines and extracellular nucleotides, as combinations of mediators may induce different responses than when tested individually. Furthermore, communication networks in the airways may evolve with time during the establishment of a chronic infection, as new target cells (bacteria, leukocytes, lymphocytes) agglomerate on either side of the epithelial barrier. It is proposed that nucleotide-mediated airway functions defined in normal tissues or aseptic culture conditions may require reassessment under conditions mimicking chronic lung diseases before their therapeutic potential may be clearly established.","keyphrases":["bacterial clearance","diphosphohydrolase","oxidative stress","endotoxin","apyrase","cd39","chrosobstructive lung diseases","purinergic signaling"],"prmu":["P","P","P","P","U","U","M","M"]}
{"id":"Hum_Reprod-1-1-2387222","title":"Myeloid ecotropic viral integration site 1 (MEIS) 1 involvement in embryonic implantation\n","text":"BACKGROUND The HOXA10 homeobox gene controls embryonic uterine development and adult endometrial receptivity. The three-amino-acid loop extension (TALE) family homeobox genes like myeloid ecotropic viral integration site 1 (MEIS) provide enhanced target gene activation and specificity in HOX-regulated cellular processes by acting as HOX cofactors.\nIntroduction\nThe molecular mechanisms responsible for endometrial development and receptivity to embryonic implantation are poorly understood (Cross et al., 1994; Wang and Dey, 2006). In the last few years, therefore, a large number of high-throughput expression profiling studies have been performed on both human and animal model tissues to identify the molecular signaling involved in the endometrial development and embryo-endometrium interactions leading to successful embryonic implantation. These include DNA-microarray studies (reviewed in Horcajadas et al., 2007; see also Chen et al., 2006; Pan et al., 2006), as well as SAGE (serial analysis of gene expression) studies (Blomberg et al., 2005; Ma et al., 2006). In addition, two databases have been set up that contain expression data on genes thought to be involved in endometrium development and function: www.endometrialdatabase.com and endometrium.bcm.tmc.edu.\nHOX-class homeobox genes are leading candidates for the regulation of endometrium differentiation in preparation for embryonic implantation (Taylor, 2000a; Eun Kwon and Taylor, 2004). HOX genes assign the correct identity to undifferentiated body segments along several developmental axes (Krumlauf, 1994; Carroll, 1995; Moens and Selleri, 2006; Wellik, 2007; Zakany and Duboule, 2007). They are, however, not only typically expressed during embryogenesis but also persistently active in both the mouse and human female reproductive tract (Taylor et al., 1997; Taylor, 2000b). The continued expression of these HOX genes allows the reproductive tract to maintain developmental plasticity and to differentiate appropriately during each menstrual cycle and throughout pregnancy.\nThe development of the para-mesonephric duct in the embryo is dictated in part by the abdominal-B group of HOX genes (Taylor et al., 1997, 1998; Taylor 2000b; Eun Kwon and Taylor, 2004). Specifically, HOXA10 is responsible for proper uterine development in both mouse and man (Taylor et al., 1997; Block et al., 2000). Female Hoxa10 null-mutant mice demonstrate uterine factor infertility (Satokata et al., 1995). These mice ovulate normally and produce normal embryos, but their uteri will not support the implantation of their own or transplanted wild-type embryos. In humans, HOXA10 expression is up-regulated in the mid-luteal phase at the time of implantation, and its expression is driven by estrogen and progesterone (Taylor et al., 1998). HOXA10 is a well-characterized marker of endometrial receptivity, which exerts pleiotropic effects on multiple aspects of adult endometrial development such as stromal decidualization, leukocyte infiltration and pinopode development (Bagot et al., 2001; Daftary and Taylor, 2004; Qian et al., 2005).\nPinopodes (also called uterodomes) are the best-characterized structural markers of endometrial receptivity in rodents and humans (Nikas, 2000; Nardo et al., 2002; Nikas and Makrigiannakis, 2003). Defective endometrial HOXA10 expression has been described in several conditions that result in diminished human embryo implantation such as endometriosis, polycystic ovarian syndrome, and hydrosalpinx fluid (Taylor et al., 1999; Daftary and Taylor 2002; Cermik et al., 2003). One of the known downstream target genes of Hoxa10 in endometrial cells is Itgb3 (Daftary et al., 2002).\nHOX genes perform their function by acting as transcription factors. They bind to the regulatory regions of downstream target genes through their homeobox domain, and thereby activate or repress transcription. HOX proteins bind only weakly to DNA by themselves and\/or exhibit a high degree of redundancy in binding site specificity (Pellerin et al., 1994). It has become increasingly evident that HOX proteins usually act as a part of hetero-dimeric (van Dijk et al., 1995; Kurant et al., 1998) or hetero-trimeric complexes (Jacobs et al., 1999; Shen et al., 1999; Schnabel et al., 2000). The TALE (three-amino-acid loop extension) family of homeodomain proteins represents the most important group of HOX cofactors. The unique TALE motif within their homeodomain allows TALE proteins to interact with other homeobox-containing proteins (B\u00fcrglin, 1997; Piper et al., 1999). Unlike most other homeobox gene families, TALE genes share sequence homology outside of the homeobox domain. This extensive homology appears linked to their function. Single TALE proteins have only low DNA-binding activity and specificity, but once complexed with a TALE protein from another subfamily and a HOX-class homeobox protein via these homologous domains, they show powerful and specific downstream target promoter regulation (reviewed in Geerts et al., 2003, 2005). Abd-B-like HOX transcription factors, like HOXA10, can form dimers or trimers with TALE proteins that thereby regulate the multiple HOX downstream target genes necessary for the implantation process.\nThe myeloid ecotropic viral integration site 1 (MEIS) genes belong to the TALE homeobox family. MEIS proteins are not just involved in TALE complex DNA binding and complex stabilization on the target DNA, but can also recruit other TALE partner proteins into the nucleus (Rieckhof et al., 1997). Meis1 was first identified as a major integration site for leukemogenic virus in a murine leukemia model (Moskow et al., 1995). Also in human leukemia, high expression of MEIS1 was found in bone marrow cells of acute myelogenous leukemia (AML) patients, where it was always co-expressed with HOXA9 (Smith et al., 1997; Lawrence et al., 1999). HOXA9 expression was found to cause transformation of normal hematopoietic cells to AML cells only when expressed together with MEIS1 (Kroon et al., 1998). Specifically, MEIS1 expression is vital for keeping HOXA9-immortalized myeloid cells in an undifferentiated, proliferating state, refractory to granulocyte-colony stimulating factor-induced terminal differentiation (Calvo et al., 2001).\nThough co-expression and interaction of MEIS1 with Abd-B-like HOX transcription factors was convincingly demonstrated in leukemia, the data on MEIS1 gene expression and function in the endometrium were still scarce. In the current study, we performed data-mining on gene expression sets in the public domain to investigate the expression of the MEIS genes (MEIS1\u20133) in the human female reproductive tract. We next characterized the menstrual cycle-specific pattern of MEIS1 in human endometrium. To block or over-express maternal Meis1 expression with Meis1 small interfering RNA (siRNA) or over-expression constructs in a mouse model, we used liposome-mediated gene transfection in the murine uterus, to investigate whether maternal Meis1 expression in the endometrium contributes to implantation. We conclude from these experiments that manipulation of adult Meis1 expression significantly affected fertility in the mouse.\nMaterials and Methods\nMeis gene expression in normal tissue\nAffymetrix data for a set of 87 different normal human tissue types (the \u2018human body index\u2019 set) representing a total of 504 tissue samples were retrieved from public gene expression omnibus (GEO) data sets on the National Center for Biotechnology Information (NCBI) website (Barrett et al., 2005, 2007). CEL data from the Affymetrix GeneChip Human Genome U133 Plus 2.0 array data sets were downloaded, and intensity values and the accompanying P-values assigned with GeneChip\u00ae Operating Software (GCOS) using the MASS5.0 algorithm (Affymetrix, Santa Barbara, CA, USA). Annotations and clinical data for the tissue samples analyzed were available from http:\/\/www.ncbi.nlm.nih.gov\/geo\/query\/ through its GEO ID: GSE7303.\nHuman tissue collection\nEndometrial tissue was collected during routine endometrial biopsy from women with a normal menstrual cycle who were eligible for IVF and embryo transfer because of (partial) tubal blockage. Informed consent was obtained from all patients. The tissue collection met with the conditions described in the \u2018Declaration of Helsinki for Medical Research involving Human Subjects\u2019 as described in (http:\/\/www.wma.net\/e\/plicy\/pdf\/17c\/pdf). Also, approval was obtained from the Tongji Hospital research and ethics committee. One half of the tissue sample was immediately frozen in liquid nitrogen and stored at \u221272\u00b0C. The other half was fixed in formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin. Menstrual cycle dating was determined by menstrual history and confirmed by histological examination using the Noyes criteria (Noyes et al., 1955). The early and late proliferative phase, and the early, mid and late secretory phase were defined as Days 5\u20139, 10\u201314, 15\u201319, 20\u201324 and 25\u201328 of the menstrual cycle, respectively.\nRNA isolation and RT\u2013PCR\nTotal RNA was extracted using the total RNA isolation reagent (TRI reagent, Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer\u2019s instructions. Four microgram total RNA from each sample was denatured at 70\u00b0C for 5 min and chilled rapidly on ice. The RNA was then reverse transcribed in a 20 \u00b5l reaction mixture containing 4 \u00b5l 5 \u00d7 RT buffer, 2 \u00b5l 10 mM dNTP, 1 \u00b5l 0.5 \u00b5g\/\u00b5l Oligo(dT15), 0.5 \u00b5l 50 U\/\u00b5l ribonuclease inhibitor, 1 \u00b5l 200 U\/\u00b5l Moloney murine leukemia virus transcriptase (Promega, Madison, WI, USA) with the following reaction condition: 42\u00b0C for 60 min; 95\u00b0C for 5 min in a Biometra T Gradient Thermocyler (Biometra, G\u00f6ttingen, Germany). After first strand complementary DNA (cDNA) synthesis, 2 \u00b5l of cDNA was amplified in 50 \u00b5l of PCR mixture containing 5 \u00b5l 10 \u00d7 PCR buffer, 4 \u00b5l 25 mM MgCl2, 1 \u00b5l 10 mM dNTP, 1.5 \u00b5l (1 U\/\u00b5l) Taq polymerase (Fermentas, Foster City, CA, USA) and 1 \u00b5l (10 pmol\/\u00b5l) of each primer pair. The primers used were (forward, reverse): for human MEIS1 (5\u2032-TCCATAGCTCTTCACTTC-3\u2032, 5\u2032-GCTTGATGTGACAATTAG-3\u2032), for mouse Meis1 (5\u2032-ACGGCATCCACTCGTTCA-3\u2032, 5\u2032-TGGCTGTCCATCAGGGTT-3\u2032), for mouse integrin \u03b23 (Itgb3) (5\u2032-GCCTTCGTGGACAAGCCTGTA-3\u2032, 5\u2032-GGACAATGCCTGCCAGTCTTC-3\u2032), for human Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5\u2032-GGTCGGAGTCAACGGATTTGGTCG-3\u2032, 5\u2032-CTTCCGACGCCTGCTTCACCAC-3\u2032) and for mouse \u03b2-actin (5\u2032- TTCCAGCCTTCCTTCTTGGG-3\u2032, 5\u2032-TTGCGCTCAGGAGGAGCAAT-3\u2032). The PCR reactions were performed as follows: 94\u00b0C for 5 min, followed by a number of cycles of 94\u00b0C for 40 s, annealing for 50 s at different annealing temperatures, and 72\u00b0C for 1 min, followed by incubation at 72\u00b0C for 10 min. Annealing temperatures for human MEIS1, mouse Meis1, mouse Itgb3, mouse \u03b2-actin and human GAPDH were 55\u00b0C, 50\u00b0C, 55\u00b0C, 50\u00b0C, and 55\u00b0C, respectively, and the numbers of cycles were 25, 30, 28, 30 and 25, respectively. The PCR products were separated on 2% agarose gels and visualized by ethidium bromide (EtBr) staining under UV light. The resulting PCR products were 411 bp for human MEIS1 [representing nucleotides (nt) 2261\u20132671 of RefSeq NM_002398), 405 bp for mouse Meis1 (nt 1106\u20131510 of NM_010789), 337 bp for mouse Itgb3 (nt 571\u2013907 of NM_016780), 786 bp for human GAPDH (nt 117\u2013902 of NM_002046) and 224 bp for mouse \u03b2-actin (nt 850\u20131073 of NM_007393). Mouse Meis1 primers used in the experiments described yielded a 405 bp PCR product containing endogenous mouse Meis1 as well as human MEIS1 (nt 1115\u20131519 of NM_002398), resulting from the pcDNA4-encoded transgene. Human MEIS1 or mouse Meis1 and Itgb3 mRNA levels were calculated as a ratio of the densitometric values for MEIS1, Meis1, or Itgb3 to their corresponding GAPDH or \u03b2-actin values. Care was taken to ensure a logarithmic signal for the PCR products measured. RT\u2013PCR reactions performed for human MEIS1\/GAPDH showed differential MEIS1 and equal GAPDH expression for all samples when RT\u2013PCR was performed for 28, 25 or 22 cycles. For all three cycle numbers used, MEIS1 and GAPDH signal intensities were different between cycle numbers. Similar results were obtained for mouse Meis1\/\u03b2-actin (35, 30 or 25 cycles) and mouse itgb3\/\u03b2-actin (30, 28 or 25 cycles) (results not shown).\nIn situ hybridization\nDiethyl pyrocarbonate-treated Milli-Q water was used in each step. Sections (4 \u00b5m thick) were immersed in 30% H2O2 solution for 30 min to eliminate endogenous peroxidase activity. After rinsing in 100 mM phosphate-buffered saline (PBS), sections were treated with pepsin [0.02% (w\/v) in 3% citric acid] at 37\u00b0C for 10 min. Thereafter, sections were fixed in freshly prepared 4% (w\/v) paraformaldehyde in PBS for 20 min at 4\u00b0C, rinsed in PBS and immersed in 0.25% acetic anhydride, 100 mM triethanolamine and 0.9% (w\/v) NaCl consecutively, each for 10 min at room temperature. The probe for human MEIS1 was a 1:1 molar mixture of single stranded DNA oligonucleotides complementary to the 5\u2032-CACTCGCATCAGTACCCGCACACAGCTCATACCAA-3\u2032 (nt 593\u2013627 of NM_002398) and 5\u2032-ACACCTTATAATCCTGATGGACAGCCCATGGGAGG-3\u2032 (nt 1493\u20131527 of NM_002398) MEIS1 sequences, labeled with digoxigenin. Sections were hybridized overnight at 40\u00b0C in hybridization buffer containing digoxigenin-labeled oligonucleotide probe at a final concentration of 400 mg\/l. After hybridization, sections were washed in fresh 2 \u00d7 SSC (saline sodium citrate) at 37\u00b0C for 10 min, 0.5 \u00d7 SSC at 37\u00b0C for 15 min, and 0.2 \u00d7 SSC at 37\u00b0C for 15 min, then incubated in 5% (w\/v) bovine serum albumin for 30 min at room temperature, and in biotin-anti-digoxigenin at 37\u00b0C for 1 h, and finally washed and incubated in streptavidin\u2013biotin complex (SABC) for 20 min at 37\u00b0C. Probe labeling, and digoxigenin, biotin-anti-digoxigenin and SABC incubations were performed using the MK2502 kit (Boster Bio-technology, Wuhan, Hubei, China). The brown precipitate generated at the site of digoxigenin-probe hybridization by reaction of the anti-digoxigenin antibody bound peroxidase with diaminobenzidine tetrahydrochloride (DAB) (AR1022, Boster Bio-technology) was scored as presence of MEIS1 mRNA. Hematoxylin was used for counterstaining. A total of 33 samples were examined. The staining of 100 consecutive cells in five non-adjacent microscopic fields was evaluated to quantify the expression of MEIS1.\nWestern blot analysis\nTissues from each mouse uterus were washed twice with PBS and lysed on ice in lysis buffer [50 mM Tris\u2013HCl (pH 8.0), 150 mM NaCl, 0.1% (w\/v) sodium dodecyl sulfate (SDS), 0.5% (w\/v) sodium deoxycholate, 1% (v\/v) Nonidet-P40, 0.02% (w\/v) sodium azide and freshly added protease inhibitors (phenylmethylsulphonyl fluoride to 10 \u00b5g\/\u03bcl and aprotinin to 1 \u00b5g\/ml)]. Solid cellular debris was removed by centrifugation at 1200g for 5 min. Protein concentration was measured by the Coomassie Brilliant Blue G-250 assay. Protein samples (45 \u00b5g each) were subjected to 10% SDS\u2013polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membrane using a Bio-Rad electroblot apparatus (Bio-Rad, Beijing, China). Non-specific binding sites were blocked in 5% (w\/v) non-fat dry milk in PBS containing 0.05% (v\/v) Tween-20. The primary goat anti-MEIS1 polyclonal antibody (sc-10599, Santa Cruz, CA, USA) was used at 1:100 and incubated at 4\u00b0C overnight. This antibody was raised against a C-terminal epitope of human MEIS1, and recognizes both human MEIS1 and murine Meis1, since their protein sequences are identical in that region (also see below, section MEIS1 constructs). As secondary antibody rabbit peroxidase-conjugated anti-goat immunoglobulin (Ig)G (ZDR-5308, Zhongshan Biotechnology, Beijing, China) was used at 1:200, and incubated at 37\u00b0C for 1.5 h. Rabbit anti-actin antibody (sc-101616-R, Zhongshan Biotechnology, as a loading control) was used at 1:500 and incubated at 4\u00b0C overnight, and the secondary antibody goat anti-rabbit IgG (ZDR-5306, Zhongshan Biotechnology) was used at 1:500, and incubated at 37\u00b0C for 1.5 h. Protein bands were visualized by enhanced chemiluminescence (32109, Pierce Biotechnology, Rockford, IL, USA).\nMEIS1 constructs\nThe complete human MEIS1 coding sequence was isolated by PCR using neuroblastoma tumor cDNA as a template with forward primer 5\u2032-ccggaattccggATGGCGCAAAGGTACGAC-3\u2032 and reverse primer 5\u2032-ccgcgtcgacggCTACTGAGCATGAATGTCCAT-3\u2032. The forward primer consisted of a non-coding flag containing an EcoRI recognition site (underlined) and the ATG initiation codon (in bold capitals) followed by codons 2\u20136 (in capitals), representing nt 458\u2013475 of the human MEIS1 NCBI RefSeq NM_002398 coding sequence. The reverse primer consisted of a non-coding flag containing a SalI recognition site (underlined) and the TAG stopcodon (in bold capitals) preceded by the final codons 385\u2013390 (in capitals), representing nt 1630\u20131610 of the NM_002398 coding sequence. This sequence encoded MEIS1B, the most widely expressed MEIS1 splice variant (see also Geerts et al., 2005 and the MEIS1 locus in TranscriptView at http:\/\/bioinfo.amc.uva.nl\/human-genetics\/transcriptviewb\/). The derived human MEIS1B protein (NCBI sequence NP_002389, 390 residues) was identical to mouse Meis1B protein (NCBI sequence Q60 954), except for substitution of the Ile residue at position 201 in the human sequence by an analogous Val residue at position 201 in the mouse sequence. The 1196 bp MEIS1 PCR product was restricted with EcoRI and SalI, purified by agarose gel electrophoresis using Qiaex II gel extraction (20021, Qiagen, Venlo, The Netherlands) and ligated into EcoRI\u2013SalI restricted pCI-neo vector (E1841, Promega Benelux, Leiden, The Netherlands) backbone. The MEIS1 sequence was excised from this construct using EcoRI and the 3\u2032 polylinker NotI site and subcloned into the pcDNA4\/TO\/myc-HisA mammalian expression vector (V1030-20, Invitrogen, Breda, The Netherlands) restricted with EcoRI and NotI, resulting in pcDNA4\/MEIS1. The construct was verified by sequencing, and expression of MEIS1 protein by the construct was demonstrated by western blot analysis of transiently transfected HEK293T cells and stably transfected human neuroblastoma cells (Revet et al., manuscript in preparation, see also Fig.\u00a02 in Geerts et al., 2005).\nThe Meis1 siRNA expression constructs were prepared as follows: oligos T14F 5\u2032-gatcccTCCAGAACTGGATAACTTGttcaagagaCAAGTTATCCAGTTCTGGAtttttggaaa-3\u2032 and T14R 5\u2032-agcttttccaaaaaTCCAGAACTGGATAACTTGtctcttgaaCAAGTTATCCAGTTCTGGAgg-3\u2032, targeting the TCAAGAACTGGATAACTTG sequence in MEIS1 mRNA (nt 871\u2013889 in the human MEIS1 RefSeq NM_002398, and 862\u2013880 in the murine Meis1 RefSeq NM_010789) or 16F 5\u2032-gatcccCTTGATGATTCAAGCCATAttcaagagaTATGGCTTGAATCATCAAGtttttggaaa-3\u2032 and 16R 5\u2032-agcttttccaaaaaCTTGATGATTCAAGCCATAtctcttgaaTATGGCTTGAATCATCAAGgg-3\u2032, targeting the CTTGATGATTCAAGCCATA sequence in MEIS1 mRNA (nt 886\u2013904 in NM_002398, and 877\u2013895 in NM_010789) were annealed and ligated into pTER restricted with BglII and HindIII as described (van de Wetering et al., 2003), to create pTER\/Meis1-T14 and pTER\/Meis1-T16, respectively. A non-targeting pTER construct contained a similar sequence that had no homology to the human or murine genome, and was used as a negative control. The constructs were verified by sequencing and siRNA-mediated MEIS1 protein and mRNA knockdown by both constructs was demonstrated (Revet et al., manuscript in preparation, see also Fig.\u00a02 in Geerts et al., 2005). The T14 and T16 constructs have complete homology to human MEIS1 as well as to murine Meis1, and target all annotated MEIS1 splice variants (i.e. isoforms A-D, see also Geerts et al., 2005).\nDNA\/liposome preparation\nPlasmid DNA (1\u20132 \u00b5l) was mixed with 50 \u00b5l Opti-MEM reduced serum medium (31985, Invitrogen, Carlsbad, CA, USA), added to 4 \u00b5l liposome [a 3:1 (w\/w) formulation of 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N-N-dimethyl-1- propanaminium trifluoroacetate (DOPSA) and dioleoylphosphatidyl ethanolamine (DOPE) (11668-027, Invitrogen)] in 50 \u00b5l OptiMEM and incubated for 20 min at room temperature. Final concentrations were 16 \u00b5g\/ml DNA and 40 \u00b5g\/ml liposome.\nIn vivo gene transfection\nNulliparous female and male Kunming mice of reproductive age were supplied by Center of Experimental Animals, Tongji Medical College (Wuhan, China). Female mice were mated and examined every 8 h until detection of a vaginal plug, which was designated Day 1 of the pregnancy. The mice were anesthetized 30\u201360 h following plug detection with 1% butaylone given by i.p. injection. A laparotomy was performed to expose the uterus and 25 \u00b5l of the DNA\/liposome complex was injected into the base of each uterine horn using a 27-gage needle, as described (Bagot et al., 2000; Hsieh et al., 2002). The incision was closed in two layers (peritoneal and cutaneous) with 4\u20130 vicryl suture. The mice were euthanized by cervical vertebrae dislocation under anesthesia before excision of the uteri. For the experiments studying the effect of MEIS1 expression manipulation on MEIS1 expression, litter size and pinopode formation, the group sizes were five mice each transfected with pcDNA4\/MEIS1, empty pcDNA4, pTER\/Meis1 siRNA construct or non-targeting pTER, respectively (20 mice in total). For the experiments studying the effect of MEIS1 expression manipulation on Itgb3 expression, 25 mice each were transfected with pcDNA4\/MEIS1, empty pcDNA4, pTER\/Meis1 siRNA construct or non-targeting pTER, respectively (100 mice in total). The numbers of mice available for Meis1 expression analysis in Fig. 5 were 19, 17, 19 and 19, respectively (the remainder were not pregnant in spite of the detection of the vaginal plug or died of post-surgical complications). The presented work conformed to the \u2018Guiding principles in the Care and Use of Animals\u2019 as described in DHEW publication No. (NIH) 85-23, Revised 1985, Office of Science and Health Reports, DRR\/NIH, Bethesda, MD 20982, USA, was approved by the ethical committee of Tongji Medical College and was also covered by Chinese animal husbandry legislation.\nScanning electron microscropy\nUteri were dissected from mice 90 h after vaginal plug detection. The specimens were dehydrated in an acetone series, dried in a critical point drier using carbon dioxide, mounted on the specimen holder, coated with gold and examined under a Stereoscan 520 Scanning Electron Microscope (Hitachi, Tokyo, Japan). From each biopsy, six to eight tissues fragments, measuring \u223c2 mm in thickness and 10 mm in length, were evaluated. A minimum of an aggregated 4\u20135 mm2 of well-preserved epithelial surface was analyzed, to increase the likelihood that the observations were representative by negating the influence of local differences in endometrial morphology. The evaluations were performed with coded tissue and read blindly by an experienced investigator according to Nikas and Makrigiannakis (2003).\nImmunohistochemistry\nThe expression of mouse Itgb3 protein in the uterus was evaluated by immunohistochemistry using a goat polyclonal antibody to Itgb3 (sc-6626, Santa Cruz). The full-thickness biopsies were obtained at the time of histological evaluation from each uterine horn. The specimens were embedded in paraffin and serial 5 \u00b5m sections obtained. The sections were deparaffinized in xylene and ethanol. Endogenous peroxidase was blocked with 3% H2O2 at room temperature for 10 min. After blocking for 45 min with 1.5% normal horse serum in PBS, the sections were incubated overnight at 4\u00b0C with primary Itgb3 antibody (1:200) in PBS. The sections were then incubated with biotinylated rabbit anti-goat IgG (ZDR-5308, Zhongshan Biotechnology) as a secondary antibody at 1:50 and incubated at 37\u00b0C for 30 min, subsequently with avidin and biotinylated peroxidase at room temperature for 45 min, and finally with DAB (400 mg\/ml) at room temperature for 5 min. Hematoxylin was used for counterstaining. The staining of 100 consecutive cells in five non-adjacent microscopic fields was evaluated. Immunoreactivity of Itgb3 was scored as follows: 1+, <25% of cells stained; 2+, >25% but <50% of cells stained; 3+, >50% but <75% of cells stained; and 4+, >75% of cells stained. Overall intensity was scored as weak (1+), moderate (2+) or strong (3+). The sum of cell stained scores and intensity scores was calculated to quantify the expression of Itgb3.\nStatistical analysis\nAll numerical results are expressed as the mean value \u00b1 SD, except for the Affymetrix expression values of MEIS1\u20133 mRNAs in normal human tissue, which are represented as mean value \u00b1 SEM. The data of MEIS1 mRNA in endometrium during the menstrual cycle were analyzed by analysis of variance. Comparisons between the expression of Meis1 in each group of murine tissue, Itgb3 in each group of murine tissue and litter size of each group of mice were performed using two-tailed unpaired t-tests. The differences of pinopode development between each group of murine tissue were analyzed using a chi-square test. P < 0.05 was considered significant in all tests. The Statistical Package for the Social Sciences software package for Windows (Version 13.0) was used for all statistical analyses.\nResults\nExpression of MEIS1, MEIS2 and MEIS3 genes in the normal human uterus\nAffymetrix data for a set of 87 different normal human tissue types (the \u2018human body index\u2019 set) representing a total of 504 tissue samples were retrieved from the NCBI website. As shown in Fig. 1, MEIS1 was highly expressed in both uterine tissues present in the data set: myometrium and endometrium. In fact, the MEIS1 expression in these two tissues was the highest observed in all 87 tissue types investigated. In contrast, MEIS2 expression was high in myometrium, although several other tissues show even higher expression, but only moderate in endometrium. Finally, the MEIS3 gene showed overall much lower expression (5\u20137-fold) than the MEIS1 and MEIS2 genes. The high expression of MEIS1 compared with MEIS2 and MEIS3 in endometrium prompted us to investigate the expression and function of the MEIS1 gene in the reproductive cycle.\nFigure 1:\nExpression of MEIS1-3 mRNAs in normal human tissue.\nVisual representation of MEIS1, MEIS2 and MEIS3 expression in the \u2018human body index\u2019 Affymetrix data set in the public domain: 504 samples representing 87 different normal human tissues. For reasons of presentation, only tissues consisting of five or more different samples were shown: 40 tissues representing 329 samples. MEIS1 expression in endometrium and myometrium was higher than in any of the non-shown sets. Average MEIS expression in all 87 tissues was: MEIS1 338.3, MEIS2 516.6 and MEIS3 77.1. For comparison: average expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and \u03b2-actin in this data set was 9240 and 11 890, respectively. Average MEIS gene expression in endometrium (18 samples) was: MEIS1 1241 \u00b1 83, MEIS2 358 \u00b1 99 and MEIS3 149 \u00b1 16. Average MEIS expression in myometrium (27 samples) was: MEIS1 1330 \u00b1 90, MEIS2 1273 \u00b1 84 and MEIS3 254 \u00b1 26. The average expression over the different samples per tissue is shown. The error bars represent the SEM\nExpression of MEIS1 mRNA during the human reproductive cycle\nMEIS1 expression has been previously demonstrated in epithelial cells of the developing female reproductive tract (Dintilhac et al., 2005). To investigate the expression of MEIS1 in cyclic development of the endometrium, the menstrual cycle stage-specific expression was determined by RT\u2013PCR. Representative EtBr stained agarose gel pictures of RT\u2013PCR are shown in Fig. 2. MEIS1 mRNA expression was evident throughout the menstrual cycle.\nFigure 2:\nMEIS1 mRNA expression in human endometrium during the menstrual cycle.\nRT\u2013PCR analysis of MEIS1 expression. A representative agarose gel stained with ethidium bromide (EtBr) picture is shown. MEIS1 was expressed in all stages of the human menstrual cycle investigated; the first half of the proliferative phase (P1), the second half of the proliferative phase (P2) and the first third of the secretory phase (S1), the mid-secretory phase (S2) and the late secretory phase (S3). The sample numbers for the P1, P2, S1, S2 and S3 groups were 5, 7, 7, 8 and 6, respectively. M, size markers. No PCR product was observed in control samples that did not contain RT enzyme during RT synthesis or template DNA during PCR (not shown). RT\u2013PCR analysis was repeated at least three times per sample. MEIS1 expression was analyzed by densitometry and normalized to GAPDH in 33 endometrial samples. The densitometric analysis was repeated three times, with similar results. The data were analyzed using analysis of variance. The average MEIS1\/GAPDH ratio in each group was: P1 0.20 \u00b1 0.02, P2 0.26 \u00b1 0.03, S1 0.41 \u00b1 0.07, S2 0.70 \u00b1 0.10 and S3 0.44 \u00b1 0.09. There was no statistical difference within the proliferative phase (P = 0.47), but the difference between the proliferative phase and the secretory phase was significant (P = 0.00001)\nTo assess potential regulation of MEIS1 expression during the reproductive cycle, densitometric analysis was performed on all samples, and the average abundance of MEIS1 during each stage of the menstrual cycle normalized to GAPDH, calculated. No difference was apparent between the levels of expression in the segments of the proliferative phase. In contrast, expression at the mid secretory phase was elevated relative to other stages (see legend to Fig. 2). These results were a first indication that MEIS1 expression varied during the menstrual cycle, markedly increasing at the mid secretory phase (S2), which corresponded to the time of implantation.\nMEIS1 mRNA is expressed in the human endometrial stroma and glands\nTo investigate the site of MEIS1 expression in the human endometrium, and as a support of the MEIS1 expression detected with RT\u2013PCR described above, in situ hybridization was performed. Fig. 3a and b shows high-power views of MEIS1 expression in proliferative and secretory endometrium. MEIS1 was expressed in both glandular and stromal cells. This supports the results of RT\u2013PCR in Fig. 2, and further suggests that MEIS1 may play a role in embryonic implantation.\nFigure 3:\nMEIS1 mRNA expression in human endometrial tissue.\nIn situ hybridization analysis of MEIS1 expression in human endometrial samples. Views of two representative samples demonstrate MEIS1 expression in both the glandular and stromal cells of (a) proliferative phase and (b) secretory phase endometrium. MEIS1 mRNA is stained brown. No staining was observed when the MEIS1 probe was omitted (not shown). Scale bar = 100 \u00b5m. Each sample was analyzed at least in triplicate\nDown-regulation of Meis1 expression affects murine embryo implantation rate\nTo assess whether an up-regulation of Meis1 expression during embryo implantation was essential to this process, the expression of maternal Meis1 was down-regulated in a mouse model by transfection in vivo, as described by (Bagot et al., 2000; Hsieh et al., 2002). A first indication of decreased Meis1 expression in the uterus was obtained by RT\u2013PCR, and this was confirmed by western blot on Day 3 after transfection. Representative results for mRNA are shown in Fig. 4a and b. Densitometric analysis was performed on all samples, and the average abundance of Meis1 mRNA in each group normalized to \u03b2-actin mRNA was calculated. Levels of Meis1 mRNA and protein (Fig. 4b and c) were significantly decreased in endometrium transfected with pTER\/Meis1 siRNA compared with the control.\nFigure 4:\nMeis1 expression manipulation in murine uterine tissue.\n(a) RT\u2013PCR analysis of Meis1 expression in 20 murine endometria transfected with pcDNA4\/MEIS1, Meis1 small interfering RNA (siRNA) constructs or respective controls (five animals in each group). Shown is a representative EtBr picture. (b) Meis1 mRNA expression was analyzed by densitometry and normalized to \u03b2-actin as described in Fig. 2, except that data were analyzed using a two-tailed unpaired t-test. No PCR product was observed in control samples that did not contain RT enzyme during RT synthesis or template DNA during PCR (not shown). RT\u2013PCR and quantification was repeated at least three times, with similar results. Average Meis1\/\u03b2-actin ratios in endometrium transfected with pcDNA4\/MEIS1 (U) and control pcDNA4 DNA (Uc) were 0.90 \u00b1 0.09 and 0.48 \u00b1 0.07, respectively. There was a significant increase in Meis1 mRNA in the up-regulation group (U) compared with the control (Uc) (P = 0.0001). Average Meis1\/\u03b2-actin level in endometrium transfected with Meis1 siRNA (D) and control pTER constructs (Dc) were 0.27 \u00b1 0.05 and 0.40 \u00b1 0.03, respectively. Levels of Meis1 mRNA were significantly decreased in Meis1 siRNA-transfected samples (D) compared with the control samples (Dc) (P = 0.002). (c) Western analysis performed on 20 murine endometria transfected with pcDNA4\/MEIS1, Meis1 siRNA or respective controls (five animals in each group). A representative western blot image is shown. (d) Meis1 protein expression was analyzed by densitometry and normalized to \u03b2-actin values as described in Fig. 4b. No Meis1 band was detected on western blot in the absence of primary Meis1 antiserum (not shown). Western blot analysis and quantification was repeated at least three times, with similar results. The average Meis1\/\u03b2-actin ratio in each group was: U 0.94 \u00b1 0.05, Uc 0.57 \u00b1 0.06, D 0.37 \u00b1 0.05 and Dc 0.57 \u00b1 0.08. There was a significant increase in Meis1 protein in endometria transfected with pcDNA4\/MEIS1 (U) compared with pcDNA4-transfected controls (Uc) (P = 0.00001). Endometria transfected with pTER\/Meis1 siRNA constructs (D) demonstrated a significant decrease in Meis1 protein compared with pTER controls (Dc) (P = 0.01)\nAt Day 9 of pregnancy, mice were killed and the uteri removed and examined for normal histology and for the number of implantation sites per uterus as a measure of litter size. The endometrium appeared histologically normal, and demonstrated adequate decidualization. Also the total number of pregnant mice did not vary significantly between Meis1 siRNA-treated mice and the control. Pups of mice transfected with Meis1 siRNA were born following a normal length gestation of 18\u201322 days, were of normal size and demonstrated no morphological abnormalities. However, the average number of implantation sites per uterus was 12.2 \u00b1 1.5 in the mice transfected with the control pTER plasmid and 7.6 \u00b1 2.7 in the mice transfected with a pTER\/Meis1 siRNA construct (Fig. 5a). Decreased expression of Meis1 therefore resulted in a significant decrease in litter size (see legend to Fig. 5a). We concluded that the naturally occurring up-regulation of Meis1 in the reproductive cycle contributes to normal embryo implantation.\nFigure 5:\nEffect of Meis1 expression on mouse litter size.\nLitter size, as determined by counting the number of implantation sites per uterus examined, at Day 9 of pregnancy. Mice were transfected 1 day after detection of a vaginal plug with pTER\/Meis1, control pTER, pcDNA4\/MEIS1 or empty pcDNA4. (a) Litter size was reduced by 40% in the group of mice transfected with pTER\/Meis1 siRNA compared with pTER controls. This difference was statistically significant according to a two-tailed unpaired t-test (P = 0.00001). (b) Litter size at Day 9 of gestation was not affected by transfection with pcDNA4\/MEIS1 compared with pcDNA4-transfected controls (P = 0.08). The experiment was repeated three times, with similar results\nTo evaluate the effect of up-regulation of Meis1 expression in the adult murine uterus, mice were transfected with the pcDNA4\/MEIS1 expression construct, or with empty pcDNA4 plasmid (as a negative control). A first indication of increased Meis1 expression in the uterus was obtained by RT\u2013PCR and this was confirmed by western blot. Levels of Meis1 mRNA and protein were significantly increased in endometrium transfected with the pcDNA4\/MEIS1 expression construct compared with the control (Fig. 4c and d). As in the experiment described above, uterine histology was not affected by the transfection, and the number of mice pregnant and the weight and morphology of the pups did not differ between the groups. The average number of implantation sites was 13.4 \u00b1 1.1 in the pcDNA4\/MEIS1 treated mice and 12.6 \u00b1 1.1 in the control mice (Fig. 5b). In contrast to Meis1 down-regulation, up-regulation of Meis1 expression by pcDNA4\/MEIS1 treatment did not significantly alter litter size.\nEffect of Meis1 on murine pinopode formation\nFor further clues as to a possible function for Meis1 in murine implantation, uterine epithelial morphology was investigated. As described above, no uterine histological changes could be detected at the light microscopy level in mice transfected with Meis1 DNA constructs. To evaluate the effect of Meis1 on epithelial morphology, the uteri of these mice were examined with scanning electron microscopy for evidence of altered formation of pinopodes.\nMice which were transfected in vivo as described above were killed 4.5 days after vaginal plug detection, the uteri removed and prepared for electron microscopy analysis. ***Fig. 6a and b shows a typical section of endometrium from a pcDNA4\/MEIS1-treated mouse, and an empty pcDNA4-treated control mouse; there was a significant increase in the development of pinopodes in the mouse transfected with MEIS1 over-expression plasmid. Fig. 6c and d shows a typical section of endometrium from pTER\/Meis1 and control pTER-treated mouse uteri. None of the sections from Meis1 siRNA-treated mice contained normal pinopodes. The secretory cells were covered with dense microvilli, instead of with pinopodes, and were slightly bulging. In contrast, all sections from control animals contained a normal number of pinopodes. Apparently, decreasing Meis1 expression had a negative effect on efficient pinopode maturation. Absence of a normal amount of functioning pinopods in mice transfected with Meis1 siRNA could be a mechanism contributing to a less efficient embryonic implantation rate and diminished litter size in these animals.\nFigure 6:\nEffect of Meis1 expression on murine pinopode formation.\nPinopode development in transfected mouse uteri, examined with scanning electron microscopy. (a) Transfection with pcDNA4\/MEIS1 demonstrated fully developed pinopodes on most (\u226560%) secretory cells. (b) Transfection with empty pcDNA4 plasmid as a negative control showed a wide distribution of developing pinopodes, covering \u223c50% of the endometrial luminal surface. However, there was a much lower occurrence (20%) of fully developed pinopodes in this group. (c) Transfection with Meis1 siRNA plasmid resulted in the majority of the endometrial surface expressing few, to no, pinopodes. Instead, the epithelial cells were covered with short and thick microvilli. (d) Transfection with non-targeting pTER plasmid as a control resulted in developing pinopodes on about half of the secretory cells. Scale bar = 10 \u00b5m. The experiment was repeated three times. The data were analyzed using a chi-square test\nEffect of Meis1 on Itgb3 expression in the murine uterus\nMaternal Hoxa10 has earlier been shown to be involved in pinopode formation in the mouse uterus (Bagot et al., 2001), in a manner resembling the effect of Meis1 on pinopode formation observed in this study. To investigate a possible connection between Meis1 and Hoxa10 function in murine uterine development and embryonic implantation, we evaluated the effect of Meis1 expression manipulation on Itgb3 expression. To obtain a first indication of differential Itgb3 mRNA expression, peri-implantation uteri from mice treated with pcDNA4\/MEIS1 or empty pcDNA4 (as a negative control), or with pTER\/Meis1 or non-targeting pTER (as a negative control) were analyzed by RT\u2013PCR. Fig. 7a shows a representative EtBr gel picture of the RT\u2013PCR. Densitometric analysis was performed on all samples, and the average abundance of Itgb3 in each group normalized to \u03b2-actin was calculated. Levels of Itgb3 mRNA appeared significantly decreased in endometrium transfected with pTER\/Meis1 siRNA compared with the control. In contrast, levels of Itgb3 mRNA increased in endometrium transfected with the pcDNA4\/MEIS1 expression construct compared with the control (see legend to Fig. 7).\nFigure 7:\nEffect of Meis1 expression manipulation on murine uterine integrin \u03b23 (Itgb3) expression.\n(a) RT\u2013PCR analysis of Itgb3 expression in murine endometria transfected with pcDNA4\/MEIS1 (U), empty pcDNA4 (Uc), pTER\/Meis1 siRNA (D) or control pTER constructs (Dc). A representative agarose EtBr picture is shown. No PCR product was observed in control samples that did not contain RT enzyme during RT synthesis or template DNA during PCR (not shown). RT\u2013PCR was repeated three times, with similar results. Itgb3 expression was analyzed by densitometry and normalized to \u03b2-actin as described in Fig. 4. Average Itgb3\/\u03b2-actin ratio in each group was: U 0.83 \u00b1 0.17, Uc 0.50 \u00b1 0.07, D 0.28 \u00b1 0.06 and Dc 0.44 \u00b1 0.04. There was a significant increase in Itgb3 mRNA in endometrium transfected with pcDNA4\/MEIS1 compared with endometrium transfected with empty pcDNA4 DNA (P = 0.02). Levels of Itgb3 mRNA in endometrium transfected with pTER\/Meis1 siRNA were significantly decreased when compared with endometrium transfected with control pTER (P = 0.006). (b) Immunohistochemistry of Itgb3 protein expression in the endometrium of mice transfected with either pcDNA4\/MEIS1 or pTER\/Meis1 siRNA plasmid. Shown are representative images of each group: uterine sections from mice transfected with pcDNA4\/MEIS1 (U), with empty pcDNA4 vector (Uc), with pTER\/Meis1 siRNA (D) or with non-targeting pTER vector (Dc). No staining was observed in a negative control without Itgb3 antibody as the primary antiserum (not shown). Scale bar = 100 \u00b5m. Statistical analysis of Itgb3 immunoreactivity was performed as described in the \u2018Materials and Methods\u2019 section. The data were analyzed using a two-tailed unpaired t-test. The immunoreactivity analysis was repeated three times, with similar results. The average Itgb3 immunoreactivity scores in each group were: U 6.4 \u00b1 0.5, Uc 4.0 \u00b1 0.8, D 2.4 \u00b1 0.9 and Dc 4.8 \u00b1 0.7. Uterine sections from pcDNA4\/MEIS1-transfected mice showed significantly increased glandular and stromal Itgb3 expression compared with control mice treated with empty pcDNA4 vector (P = 0.0015). Uterine sections from pTER\/Meis1 siRNA-transfected mice showed significantly reduced glandular and stromal Itgb3 expression compared with control mice treated with non-targeting pTER vector (P = 0.03)\nTo support the results of the RT\u2013PCR described above, the effect of Meis1 expression on Itgb3 protein levels was determined by immunohistochemistry on transfected uterine tissue of each group. Representative pictures are shown in Fig. 7b. Itgb3 expression levels appeared higher in mice transfected with pcDNA4\/MEIS1 expression construct compared with the pcDNA4 control. In contrast, Itgb3 expression was lower in mice transfected with Meis1 siRNA expression constructs than in those transfected with the non-targeting siRNA construct. Statistic analysis of the Itgb3 immunoreactivity was performed on the samples, and the average abundance of Itgb3 in each group was calculated. Levels of Itgb3 expression were significantly decreased in endometrium transfected with pTER\/Meis1 siRNA compared with the control. In contrast, Itgb3 protein levels were increased in endometrium transfected with the pcDNA4\/MEIS1 expression construct compared with the control (see legend to Fig. 7). This was consistent with the results of the RT\u2013PCR.\nTogether, these results showed that, in a murine uterine receptivity model, the expression of the known Hoxa10 target Itgb3 could be regulated by the manipulation of Meis1 expression, suggesting Meis1 action through co-operation, or possibly interaction, with Hoxa10.\nDiscussion\nHOXA10 is essential for normal embryonic uterine development. In the developing mouse embryo, altered Hoxa10 expression as a result of targeted mutation (Satokata et al., 1995) or exposure to diethylstilbestrol leads to abnormal uterine development (Block et al., 2000). Hoxa10 expression persists in the adult uterus and is important for implantation of the embryo. Hoxa10 null-mutant mice or mice in which Hoxa10 has been blocked using antisense oligonucleotides demonstrate decreased litter size due to failure of implantation (Benson et al., 1996; Bagot et al., 2000).\nHOX genes function as transcription factors and either activate or repress target genes. The pleiotropic effects of HOXA10 on endometrial differentiation and function are likely modulated through the regulation of multiple downstream targets that are necessary for the implantation process (Daftary et al., 2002; Troy et al., 2003). HOXA10 has been demonstrated to be involved in the regulation of pinopode development and expression of other genes involved in endometrial development such as ITGB3 and EMX2 (Bagot et al., 2001; Daftary et al., 2002; Troy et al., 2003).\nHOX proteins require co-operation with other proteins to bind to their target DNA. MEIS1 in particular aids 5\u2032 HOX proteins, like HOXA10, to gain this specificity. MEIS1 physically interacts with 5\u2032 HOX proteins HOXA9\u201311 by forming heterodimeric binding complexes on a DNA target containing a MEIS1 site (TGACAG) and an Abd-B-like Hox site (TTTTACGAC) (Shen et al., 1997). Studies demonstrated that MEIS1 gene and HOXA9-13 genes are co-expressed throughout M\u00fcllerian duct differentiation (Dintilhac et al., 2005), which suggests that MEIS1 might play a role in embryonic female genital tract development. We have performed data-mining on Affymetrix gene expression database in the public domain showing that MEIS1 was highly expressed in human endometrium as well as in myometrium (Fig. 1), implying it may play a role in reproductive function in the adult human uterus.\nWe therefore investigated the expression pattern of the MEIS1 gene at different stages of the human menstrual cycle, demonstrating that MEIS1 appeared to be expressed in the endometrium at different levels, depending on the menstrual cycle stage. MEIS1 mRNA levels appeared significantly increased in the mid secretory phase, closely resembling the expression pattern of HOXA10. This observation pointed to the possibility that MEIS1 is an active cofactor of HOXA10 in the human endometrium.\nOn the basis of the results described above, we postulated that Meis1 and Hoxa10 might have closely related roles in vertebrate reproduction and in the regulation of endometrial cell proliferation or differentiation. Meis1 null-mutant mice die between embryonic days 11.5 and 14.5, because of internal hemorrhage, liver hypoplasia and anemia (Azcoitia et al., 2005). The embryo lethal phenotype has precluded investigation of the function of Meis1 in the adult reproductive tract of Meis1 mutant mice. Therefore, to investigate the role of Meis1 in adult reproductive function in a mouse model, we altered the expression of the Meis1 gene in the adult murine endometrium. When mouse endometrium was transfected with Meis1 siRNA at the time of implantation, the number of embryos that could implant in the uterus was reduced by 40%. This significant reduction in implantation sites demonstrated that, in normal embryonic development, optimal implantation benefits from maternal Meis1 expression.\nWe also investigate the effect of Meis1 on pinopodes. Pinopode formation is directly regulated by HOXA10 (Bagot et al., 2001). In this study, we examined whether the expression of pinopodes could be also affected by altering Meis1 expression levels. We demonstrated that blocking Meis1 expression reduced pinopode numbers and reduced their ability to differentiate into fully mature forms. In contrast, supernumerary pinopodes could be induced by over-expression of Meis1.\nTo elucidate potential mechanisms by which Meis1 might control endometrial function and receptivity to the implanting blastocyst, we tested the effect of altering Meis1 expression on Itgb3 expression. The ITGB3 gene has been identified as the first known target of HOXA10 gene regulation in the endometrium (Daftary et al., 2002). As a bridging molecule between the endometrium and trophoblast (Kiefer et al., 1989; Van Dijk et al., 1993; Sueoka et al., 1997), ITGB3 plays an important role in embryonic implantation. We found that a blockade of Meis1 expression with Meis1 siRNA was associated with a decrease in Itgb3 mRNA. Itgb3 mRNA levels significantly increased after up-regulation of Meis1 expression with MEIS1 cDNA. It appeared likely that Itgb3 was not regulated by Hoxa10 monomer, but by a heterodimeric or heterotrimeric transcription complex containing Meis1. The interaction of Abd-B-like HOX transcription factors and MEIS1 at the HOX site in the target gene promoter has been demonstrated in myeloid differentiation. The co-activation of HOXA9 and MEIS1 is a common event in AML (Lawrence et al., 1999; Cam\u00f3s et al., 2006), but co-regulation of MEIS1 and HOXA9, and also of HOXA10, might play a role in pathogenesis of acute lymphocytic and acute myeloid leukemias associated with ALL-1 fusions (Rozovskaia et al., 2001; Ferrando et al., 2003; Dik et al., 2005). In addition, Pineault et al. (2004) demonstrated that MEIS1 enhances the effect of a HOXA10 rearrangement releasing HOX expression necessary for leukemia induction. Finally, binding of MEIS1 to HOXA10 could be demonstrated in vitro (Williams et al., 2005).\nCDKN1A (also known as P21 or CIP1) is a transcriptional target of HOXA10 in differentiating myelomonocytic cells. CDKN1A reporter expression is enhanced after transfection with PBX1 and HOXA10, especially when MEIS1 was co-transfected (Bromleigh and Freedman, 2000). It is likely that a similar regulation occurs with HOXA10 and MEIS1 cofactor in the endometrium. Recently, Taylor et al. have confirmed that this potential protein\u2013protein interaction does occur in endometrial cells. Ablation of the PBX cofactor half-site results in a loss of HOXA10\u2013PBX2 binding to the EMX2 probe, suggesting the importance of the PBX2\/HOXA10 protein\u2013protein interaction in endometrium for high affinity HOXA10 binding to its target genes (Sarno et al., 2005).\nIn conclusion, this study shows for the first time that MEIS1 expression contributes to endometrial receptivity in mouse. MEIS1 can function in cancer as an inhibitor of differentiation, and have a positive influence on cellular proliferation (see \u2018Introduction\u2019). In addition, the MEIS genes are potent enhancers of early cell proliferation during development (reviewed in Geerts et al., 2003, 2005). MEIS1 could have a similar proliferative function during endometrial development. The expression pattern of MEIS1, its contribution to pinopode formation in mouse and its effect on Itgb3 expression suggest that the effects of MEIS1 on endometrial function occur through affecting the affinity and specificity of HOXA10\u2013DNA interactions, leading to the regulation of downstream targets of HOXA10. The identification of MEIS1 as a potential HOXA10 co-operating gene could lead to elucidation of a partial HOXA10 network in endometrial development, but could also establish MEIS1 in a signaling network in its own right.\nAuthor\u2019s Role\nB.X.: conception and design of experiments, acquisition of data, analysis and interpretation of data; writing and revising of manuscript; final approval of manuscript to be published.\nD.G.: design of experiments, acquisition, analysis, and interpretation of data; writing and revising of the manuscript; final approval of manuscript to be published.\nK.Q.: conception and design of experiments; revision of manuscript; final approval of manuscript to be published.\nH.Z.: conception and design of experiments; final approval of manuscript to be published.\nG.Z.: interpretation of data; revision of manuscript; final approval of manuscript to be published.\nFunding\nDutch Cancer Society (\u2018KWF Kankerbestrijding\u2019) (UVA2003\u20132849, UVA2005\u20133665 to D.G.); The \u2018973\u2019 Program of China (No. 2007CB948104).","keyphrases":["embryonic implantation","hoxa10","endometrium","meis1","integrin \u03b23"],"prmu":["P","P","P","P","P"]}
{"id":"Glycobiology-1-1-2430009","title":"Glycation does not modify bovine serum albumin (BSA)-induced reduction of rat aortic relaxation: The response to glycated and nonglycated BSA is lost in metabolic syndrome\n","text":"The effects of nonglycated bovine serum albumin (BSA) and advanced glycosylation end products of BSA (AGE-BSA) on vascular responses of control and metabolic syndrome (MS) rats characterized by hypertriglyceridemia, hypertension, hyperinsulinemia, and insulin resistance were studied. Albumin and in vitro prepared AGE-BSA have vascular effects; however, recent studies indicate that some effects of in vitro prepared AGEs are due to the conditions in which they were generated. We produced AGEs by incubating glucose with BSA for 60 days under sterile conditions in darkness and at 37\u00b0C. To develop MS rats, male Wistar animals were given 30% sucrose in drinking water since weanling. Six month old animals were used. Blood pressure, insulin, triglycerides, and serum albumin were increased in MS rats. Contraction of aortic rings elicited with norepinephrine was stronger. There were no effects of nonglycated BSA or AGE-BSA on contractions in control or MS rats; however, both groups responded to L-NAME, an inhibitor of nitric oxide synthesis. Arterial relaxation induced using acetylcholine was smaller in MS rats. Nonglycated BSA and AGE-BSA significantly diminished relaxation in a 35% in the control group but the decrease was similar when using nonglycated BSA and AGE-BSA. This decrease was not present in the MS rats and was not due to increased RAGEs or altered biochemical characteristics of BSA. In conclusion, both BSA and AGE-BSA inhibit vascular relaxation in control artic rings. In MS rats the effect is lost possibly due to alterations in endothelial cells that are a consequence of the illness.\nIntroduction\nReducing sugars including glucose, fructose, trioses, and glyceraldehyde alter proteins by nonenzymatic glycosylation known as glycation or Maillard reaction. This reaction occurs in several steps, the initial ones being relatively fast and reversible, while the latter ones are slower and irreversible originating products known as advanced glycosylation end products (AGEs). AGEs were originally characterized by their yellow-brown fluorescent color and by their ability to cross-link with and between amino groups (Reynolds 1963). However, the term is now used for a broad range of advanced products of the Maillard reaction, including N-carboxymethyllysine (CML) and pyrroline (Reddy et\u00a0al. 1995; Baynes and Thorpe 1999).\nAGEs exert their actions by two different mechanisms: (1) modifying structural intra- and extracellular proteins and (2) binding to their receptors (RAGEs) that belong to the immunoglobulin family and are located in the plasma membranes of monocytes, macrophages, endothelial cells, and vascular smooth muscle cells (Brownlee 1995; Basta et\u00a0al. 2002). When AGEs bind to their receptors they initiate second messenger cascades. They also generate reactive oxygen species which modulate cellular function and can induce inflammatory processes (Krieglstein and Granger 2001; Basta et\u00a0al. 2002; Ramasamy et\u00a0al. 2005).\nAlbumin is one of the main proteins undergoing glycation reactions due to its abundance in serum and to the fact that it can be glycated at multiple sites (Wautier and Guillausseau 1998). Both bovine serum albumin (BSA) and AGE-BSA have been reported to have vascular effects. BSA reacts with nitric oxide (NO) therefore modulating its biological actions and increasing endothelial permeability (Predescu et\u00a0al. 2002). Some forms of AGEs also diminish vascular relaxation by decreasing the NO production of endothelial cells (Hogan et\u00a0al. 1992; Xu et\u00a0al. 2003). Nevertheless, there have been recent reports that other forms of in vitro prepared AGEs have different effects from the naturally produced AGEs (Valencia et\u00a0al. 2004).\nAGEs have been implicated in aging processes and in the development of chronic complications of diabetes such as renal and vascular damage and could also participate in complications of metabolic syndrome (Brownlee 1995; Wautier and Guillausseau 1998; Valencia et\u00a0al. 2004). There is a significant association between a high incidence of cardiovascular events and endothelial damage in animal models and diabetic patients and one of the mechanisms underlying this association is the presence of AGEs in plasma (Tan et\u00a0al. 2002).\nA variant of fructose-induced hypertension by giving sucrose to rats has been developed in our institution. The fructose-fed rat becomes hypertensive, hypertriglyceridemic, hyperinsulinemic and has insulin resistance; it exhibits what is known as metabolic syndrome (MS) or \u201cSyndrome X\u201d (Hwang et\u00a0al. 1987). Some of the characteristics of the model developed in our institution have already been described (Ba\u00f1os et\u00a0al. 1997) and they coincide with those of fructose fed animals, i.e., our rats also develop hypertension, hypertriglyceridemia, hyperinsulinemia, and insulin resistance. They also show increased oxidative stress (Ba\u00f1os et\u00a0al. 2005). Some of the characteristics of these animals are accentuated by age and have been associated with renal failure and cardiovascular diseases which are related to alterations in the structure and function of endothelial cells (Rubio et\u00a0al. 2006). AGEs could play a role in the vascular damage found in MS rats. Therefore, the aim of the present paper was to determine the effect of nonglycated BSA and AGE-BSA on aortic rings from control and MS rats.\nResults\nChanges in body weight, arterial pressure, triglycerides, glucose, insulin, and serum albumin\nTable I shows the values of body weight, arterial pressure, glucose, insulin, homeostasis model assessment (HOMA), triglycerides, and serum albumin for control and MS rats. Experimental animals developed metabolic syndrome characterized by arterial hypertension, hypertriglyceridemia, hyperinsulinemia, and insulin resistance. There was a statistically significant increase in the serum albumin values in MS rats. Although there was no significant change in body weight, the animals with MS clearly developed central adiposity.\nTable\u00a0I\nClinical characteristics and biochemical parameters from control and MS rats\nControl\nSM\nBody weight (g)\n558.3 \u00b1 14.9\n547.5 \u00b1 38.1\nArterial pressure (mm of Hg)\n101.9 \u00b1 1.4\n138.9 \u00b1 0.8*\nTriglycerides (mg\/dL)\n55.6 \u00b1 4.7\n109.1 \u00b1 12.8*\nGlucose (mmol\/L)\n5.9 \u00b1 0.3\n4.8 \u00b1 0.7\nInsulin (\u03bcU\/mL)\n6.5 \u00b1 0.9\n24.2 \u00b1 5.7*\nHOMA\n1.02 \u00b1 0.5\n5.02 \u00b1 2.1*\nAlbumin (g\/dL)\n3.3 \u00b1 0.1\n3.6 \u00b1 0.1*\nValues are mean \u00b1 SEM, n = 8; *P < 0.01.\nAGE-BSA preparation\nIt was possible to produce AGE-BSA under the previously described condition. After incubation with glucose, the albumin solution took a yellow-brown color and showed the characteristic fluorescence spectrum of AGEs. Albumin incubated without glucose did not significantly change its appearance and did not have the characteristic fluorescence spectrum of AGEs (Figure\u00a01).\nFig. 1\nFluorescence spectrum of AGE-BSA and nonglycated BSA. Both solutions were tested at 2 mg\/mL of protein. The peak of fluorescence is not present in the nonglycated BSA. The interrupted line represents the saturation level of the fluorescence detection by the equipment employed.\nThe purification of AGE-BSA on Affi-Gel-Blue is shown in Figure 2; three peaks were observed which corresponded to the highly glycated BSA (first one), the less glycated BSA moderately, and stronger bound to the chromatographic matrix (second and third peaks, respectively). The first peak was employed for subsequent analysis and tested for vasoreactivity.\nFig. 2\nPurification of AGE-BSA on Affi-Gel-Blue. Three peaks were observed: the first one corresponds to highly glycated BSA which did not bind to the matrix, the second one corresponds to the less glycated BSA which bound moderately, and the third peak corresponds to the stronger bound BSA. The first peak was the only one to be further analyzed. The arrows indicate the buffer used at each stage.\nThe AGE-BSA produced had a protein concentration of 53.8 mg\/mL and a specific activity, before its purification in Affi-Gel Blue, of 7.9 AU\/mg of protein and of 10.5 AU\/mg of protein, after purification. Fluorescence was not significantly elevated when protein concentration of nonglycated BSA was increased (Figure 3). The value of specific activity was similar to that obtained by other authors using similar procedures (Verbeke et\u00a0al. 1997).\nFig. 3\nEvaluation of the specific activity (SA) of AGE-BSA. The increase of specific activity obtained by the purification process on Affi-Gel-Blue is observed. Bold circles represent the nonglycated BSA. Bold squares represent the unpurified AGE-BSA and bold triangles represent the purified AGE-BSA. Specific activity increased in approximately 33% by the purification process.\nChanges in mass and isoelectric point of BSA and glycated BSA\nTable II shows the results of the analysis of the main glycation sites of the BSA structure. The analysis was done using the sequence of bovine serum albumin reported in the GenBank with number CAA76847 (gi:3336842) and the PyMOL v0.99 program (DeLano Scientific LLC, CA). The disposition of the lysine and arginine residues in the three-dimensional structure was evaluated. The analysis shows that a maximum of 52 residues from the 79 present in the molecule can be glycated. The rest of the residues are in the inside of the structure of BSA.\nTable\u00a0II\nDistribution of the amino acids lysine and arginine in the three-dimensional structure of BSA\nAmino acid position in the three-dimensional structure\nAmino acid\nTotally exposed\nPartially exposed\nNonexposed\nLysine\n4,12,20,41,51,64,76, 116,127,132,136,180,187,211,224, 261,273,275,285,312,316,322,350,362,375,377,388,396, 465,474,499,504,520,523,524,535,537,556, 563,573.\n159,413,431,533.\n93,106,114,131, 204,221,232,239, 242,279,294,439, 544.\nArginine\n194,196, 198.\n143,185,409,412, 444.\n10,81,98,144,208, 217,256,335,336, 347,427,458,483, 484.\nTotal\n43\n9\n27\nThe amino acids localized in depression areas are considered as partially exposed.\nFigure 4 shows a molecular mass of 66,655.58 Da for nonglycated BSA (Figure 4A). After glycation, the molecular mass increased to 74,461.15 Da (Figure 4B). The net increase of 7,805.57 Da observed corresponds to the addition of 48 glucose molecules, considering that the molecular mass of glucose is 180.2 Da and that during each reaction of Amadori product, a water molecule is liberated (18 Da). The amount of glucose bound to BSA in our preparation was 48, very close to the 52 theoretically possible; therefore, we consider that our AGE-BSA is a highly glycated product.\nFig. 4\nAnalysis of BSA and AGE-BSA by mass spectrometry. The mass increase of protein after glycation is observed. The mass of nonglycated BSA was 66,655.58 Da (A) and that of AGE-BSA was 74,461.15 Da (B). The net increase of mass was 7,805.57 Da, corresponding to the addition of 48 molecules of glucose to each BSA molecule. Inset: change of isoelectric point of BSA after glycation. Nonglycated BSA had a pI of 4.2 while AGE-BSA had a pI of 6.3. Line 2 is the broad range pI standard kit (pH 3\u201310).\nNonglycated BSA had an isoelectric point of 4.2. The glycation process changed the isoelectric point to 6.3 (Figure 4, inset).\nVascular responses\nNorepinephrine-induced vasoconstriction was higher in MS rats than in control rats (Table III); nevertheless, vasoconstriction was not significantly modified in aortic rings from control or MS rats in the presence of nonglycated BSA or AGE-BSA. L-NAME significantly increased vascular contraction in both groups (154.41 \u00b1 10.35% versus 186.18 \u00b1 7.58%, respectively) since inhibition of NOS induces an imbalance in vasoconstriction and vasodilation which is greater in MS than in control rats (Figure 5).\nFig. 5\nEffect of AGE-BSA on vascular contraction in aortic rings from control (solid bars) and MS rats (open bars). The contractions were induced by NE 1 \u03bcM and basal tension was normalized to 100% in control and MS rats. Tension values in grams are shown in Table III. Results are the means \u00b1 SEM of six independent experiments. *P < 0.05 between MS and control; **P < 0.001 between the Tyrode solution and L-NAME.\nTable\u00a0III\nValues of contraction (g) and relaxation (%) of aortic rings in control and experimental rats\nControl\nMS\nVascular contraction\n1.5 \u00b1 0.2\n2.2 \u00b1 0.2*\nVascular relaxation\n84.4 \u00b1 2.6\n64.9 \u00b1 3.3*\nThe results are expressed as relaxation percentage of the initial precontraction level with NE (1 \u03bcM). Final relaxation with Ach (1 \u00d7 10\u22126 M) is reported. Values are mean \u00b1 SEM, n = 8; *P < 0.001.\nBasal relaxation was diminished in MS rats when compared to control rats (Table III). Figure 6 shows the effect of nonglycated BSA and AGE-BSA upon vascular relaxation in aortic rings from control and MS rats. In control rings nonglycated BSA and AGE-BSA at 40 AU\/mL (3.8 mg\/mL) significantly inhibited the endothelium-dependent vasorelaxation in a 35%. This effect was not present when a lower dose was used (10 AU\/mL; 0.95 mg\/mL). Fresh albumin, fatty acid free, diminished vasorelaxation in aortic rings from control rats in the same proportion as pre-incubated control BSA and AGE-BSA at a concentration of 3.8 mg\/mL (data not shown).\nFig. 6\nEffect of nonglycated BSA and AGE-BSA at 40 AU\/mL (3.8 mg\/mL) on endothelium-dependent vasorelaxation in aortic rings from control (A) and MS rats (B). Results are expressed as relaxation percentage from the initial precontraction level with NE 1 \u03bcM. *P < 0.05 between the Tyrode solution and nonglycated BSA and AGE-BSA; #P < 0.05 between the Tyrode solution in MS and control rats.\nIn MS aortic rings vasorelaxation was not significantly affected by BSA or AGE-BSA at any of the doses tested.\nImmunofluorescence of RAGEs\nResults of expression of RAGE are shown in Figure 7. There is an increased RAGE immunoreactivity in aortas from MS rats when compared to control rats. In aortas from C rats fluorescence is observed mainly in the endothelium while in MS aortas both the endothelium and muscular fibers fluoresce. Damage due to the development of the metabolic syndrome can also be appreciated by the presence of buckles and a loss of continuity in elastic fibers.\nFig. 7\nImmunohistochemistry for RAGE in aortas of control (Panel A) and MS (Panel B) rats. Negative control (Panel C). Magnification 40\u00d7. Asterisk indicates internal elastic lamina. There is an increased RAGE immunoreactivity in MS rats.\nDiscussion\nThe aim of the present paper was to determine the effect of nonglycated BSA and AGE-BSA on vascular aortas from control and MS rats in which the condition is induced by a high ingestion of sucrose and where the structure and function of endothelial cells are altered. Both albumin and AGEs have been reported to have vascular effects (Vlassara et\u00a0al. 1992). However, the vascular effects of AGEs might not be produced by all types of AGEs, since AGEs may have very different structures depending on the reactions generated during the in vitro glycation process of the protein by reducing sugars (Katchalsky and Sharon 1953). Some of the AGEs generated are capable of cross-linking to proteins and others have the capacity to interact with specific receptors (RAGEs) present in some cellular types (Valencia et\u00a0al. 2004).\nThere have also been recent reports that some of the effects of in vitro prepared AGEs are due to metal ions which are contaminants commonly found in AGE preparations. Various metal ions have been shown to drive inflammatory responses via generation of oxidative stress (Valencia et\u00a0al. 2004). The use of a chelator such as EDTA to facilitate the reaction could induce some of the effects reported to be produced by AGEs (Al-Abed et\u00a0al. 1999).\nOur results show that body weight was not significantly modified in MS rats even if the diet of the sucrose-fed rats was hypercaloric (Ba\u00f1os et\u00a0al. 1997) (Table I). However, sucrose-fed animals showed increased central adiposity. Insulin was increased in MS rats and this increase accompanies an increase in blood pressure. Triglycerides were also elevated. Therefore the MS rats show many of the characteristics of metabolic syndrome. Serum albumin concentration was significantly higher in MS rats when compared to control rats. However, Rumble et\u00a0al. (1997) reported that diabetic and hypertensive rats had lower serum albumin levels than the normotensive Sprague Dawley rats.\nVascular contraction to norepinephrine increased MS rats in this study. This has been previously described for this model (Ba\u00f1os et\u00a0al. 1997) and additionally we have previously found that contraction to KCl in MS rats is also increased and that this is due to an elevated response to endothelin (Nava et\u00a0al. 1999; Rubio et\u00a0al. 2006). The increase in the circulating levels of albumin in the MS group may contribute to the increased vascular tone observed in this group. Indeed, an increase in circulating albumin levels has been observed in some forms of hypertensive pathology, due to the hemoconcentration induced by edema, which positively correlates with the hypertension levels (Allen and Patterson 1995). AGEs could also participate in the increase in vascular contraction in MS rats since they increase the production of endothelin-1 by endothelial cells (Tan et\u00a0al. 2002). Our results show that vasoconstriction was not modified by nonglycated BSA or by AGE-BSA (Figure 5). This is in accordance with the results of Xu et\u00a0al. (2003).\nVasorelaxation in MS rats was significantly diminished in comparison to control rats and could be attributed to an imbalance of constricting and relaxing factors as has been previously reported (Ba\u00f1os et\u00a0al. 1997). Furthermore, as our model of MS rats is caused by the chronic ingestion of sucrose, vascular damage could be a probable consequence of AGEs increased production. AGEs deposit inside smooth muscle and endothelial cells as well as in extracellular matrix proteins increasing rigidity. Hogan et\u00a0al. (1992) have reported that AGEs react with NO produced by endothelial cells. They also reported that AGEs reduce the antiproliferative effect of NO on aortic smooth muscle vascular and renal mesangial cells.\nInsulin, which is also increased in our model, stimulates collagen synthesis in vascular smooth muscle cells (Ruiz Torres et\u00a0al. 1998) and could contribute to diminished vasorelaxation in MS rats. Arterial endothelium-dependent relaxation is also diminished hypertensive rats (Ibarra et\u00a0al. 1995; K\u00fcng and L\u00fcscher 1995; Challah et\u00a0al. 1997; Freitas et\u00a0al. 2003; Shipley and Muller-Delp 2005). These authors have proposed that the reduced response to Ach could be a consequence of an impairment of either the generation (synthesis or release) of relaxant factors or the cellular response to them.\nIn our study, both nonglycated BSA and AGE-BSA decreased vasorelaxation in the same proportion in control rats. In arterial rings incubated in the presence of glycated and nonglycated BSA the response to acetylcholine was 35% reduced in comparison to control rings incubated in the absence of BSA (Figure\u00a06A). The reduction in vascular relaxation in the presence of nonglycated BSA is in accordance with the report by Predescu et\u00a0al. (2002). These authors suggested that BSA reacts with NO therefore modulating its biological actions and increasing endothelial permeability. Although in our experiments we also found a decrease by AGE-BSA, it seems to be due to BSA since there was not a significant difference in the reduction produced by AGE-BSA and by nonglycated BSA.\nPreviously, Xu et\u00a0al. (2003) studied the vascular effects of AGEs produced in vitro testing vasoreactivity in rings of thoracic aorta from New Zealand white rabbits and in vitro production of NO in endothelial cells from human umbilical veins. These authors reported that there was not a significant effect of AGEs upon vasoconstriction induced by NE and our results are in accordance with this result. However, they found that AGEs diminished the NO production of isolated endothelial cells and that vasodilation was reduced in thoracic aortic rings while we found that AGEs did not modify vasodilation. Our data suggest that it is BSA that exerts the diminished vasorelaxing effect and not AGEs. In their study the authors produced AGEs incubating BSA with glucose 6-phosphate instead of glucose which is a faster reaction. Glucose 6-phosphate is a reducing sugar of great intracellular importance and can glycate easier than glucose since it is present in a higher proportion in the open-chain form (Hogan et\u00a0al. 1992). Their reaction was facilitated by the use of EDTA which could induce the effect reported by them (Al-Abed et\u00a0al. 1999). AGEs derived from a long period of incubation of BSA with glucose, as was done in this paper, conduces mainly to fluorescent cross-linking AGE production. Using specific antibodies, these authors found high levels of carboximetillysine (CML) in their preparation and identified CML as the AGE responsible of the NO synthesis inhibition. Therefore, the kind of AGEs used by them could explain the contradiction between their results and what we observed in the present paper. Furthermore, the difference of the species used for testing vasodilation in both papers (New Zealand rabbits and Wistar rats) might account for the contradicting results found.\nThe same authors (Xu et\u00a0al. 2003) did not find the expected previously reported decrease in vascular relaxation when they added nonglycated BSA. The absence of a vascular effect of nonglycated BSA in the above-mentioned study is probably due to the very low doses assayed, in the order of \u03bcg\/mL. It is known that albumin has a physiologic effect at higher concentration. Indeed, our own results show that the inhibitory effect of BSA or AGE-BSA was only observed when the protein concentration assayed was about of 3.81 mg\/mL, but not when it was only of 0.95 mg\/mL.\nThe response to nonglycated BSA and AGEs disappeared in the MS rats (Figure 6B). Alterations in the structure and function of the vascular structures caused by the metabolic syndrome could be the underlying cause of the lack of response in these animals. This physiological adaptation could be considered as a protector effect to counteract the increased basal vasoconstriction observed in metabolic syndrome rats, and must be better studied. We evaluated the presence of AGEs\u2019 receptors (RAGEs) from aortas in MS rats to better explore the mechanism by which AGEs participate in this pathology. Many of the deleterious effects of AGEs are mediated by binding to their receptors which are present in various cellular types. In endothelial cells, the interaction AGE\u2013RAGE modulates the expression of citocynes and adhesion molecules and increases the production of oxygen reactive species (ROS) generating oxidative stress (Ramasamy et\u00a0al. 2005). Koyama et\u00a0al. (2005) demonstrated that an alternative form of RAGE, the endogenous secretory RAGE (esRAGE), is associated with the metabolic syndrome or atherosclerosis in the absence of diabetes. Inflammation might be the factor linking the RAGE system with MS. Although a higher number of RAGEs were observed in the aortas from MS rats, AGE-BSA did not have any effect on arterial relaxation in this group. Moreover, the absence of differences in arterial relaxation induced by AGE-BSA or BSA in the control group suggests that the possible effect of AGE-BSA on vascular function in not mediated by RAGEs.\nThe exposition of the vascular tissue to AGE-BSA during the development of MS could also cause the loss of the relaxation capacity of aortas. The changes in the charge and composition of the BSA molecule produced by glycation increase its aggregation capacity. When the isoelectric point is closer to physiological pH, hydrophobic interactions are favored. These changes could increase the cross-linking activity of AGEs to proteins and the capacity to deposit in the extracellular matrix of the tissues. Correspondingly, we observed an increased aggregation capacity of AGE-BSA during alcohol precipitation or desalting. However, the loss of the relaxing capacity due to biochemical changes of AGE-BSA is not favored by our results since there were no differences in the relaxation in the presence of BSA or AGE-BSA.\nWe speculate that the cause could be the altered albumin internalization to endothelial cells and therefore a modified neutralization of NO. This should be further evaluated. The molecular structure and charge of albumin facilitate the cotransport of a number of hydrophobic molecules, enzymes, and hormones across the endothelium and the maintenance of vascular integrity and transvascular oncotic pressure gradient (Metha and Malik 2006). Endothelial permeability to plasma proteins and liquid is increased in inflammation, a condition manifested by protein-rich edema (Metha and Malik 2006). Inflammation is present in metabolic syndrome and therefore alterations in albumin transport across endothelial cells are likely to be present. Bevers et\u00a0al. (2006) reported that in bEnd.3 endothelial cells, which only produce eNOS and have higher levels of NO than other endothelial cells, incubation with low albumin levels increased eNOs activity and the production of NO.\nIn conclusion both BSA and AGE-BSA inhibit vascular relaxation mediated by NO in control artic rings in the same proportion. This inhibitory effect is likely to be due to the structure of BSA and not to the fluorescent AGEs synthesized by the employed procedure. However, in MS rats this inhibitory vascular relaxation effect is lost, possibly due to alterations in endothelial cells that are a consequence of the illness.\nMaterials and methods\nAnimals and arterial pressure determination\nExperiments in animals were approved by the Laboratory Animal Care Committee of our institution and were conducted in compliance with our institution's ethical guidelines for animal research.\nWeanling male Wistar rats aged 25 days and weighing 50 \u00b1 4 g were separated into two groups\u2014group 1: control rats (C) were given tap water for drinking and group 2: MS rats were given 30% sucrose in drinking water during a 24-week period. All animals were fed Purina 5001 rat chow (Purina Mills Inc., Richmond, IN) ad libitum, which provides 14.63 kJ\/g, with 23% protein, 12% fat, and 65% carbohydrate. Animals were kept at controlled temperature and a 12:12-h light-dark cycle.\nSystolic arterial blood pressure was measured in conscious animals using the tail cuff method; the cuff was connected to a pneumatic pulse transducer (Narco Bio-Systems Inc., Healthdyne Co., Austin, TX) and a programmed electrosphyngomanometer. The mean of five independent determinations was calculated.\nAGE-BSA preparation\nAGE-BSA were prepared, purified, and characterized in the reproductive biology laboratory of Instituto Nacional de Ciencias M\u00e9dicas y Nutrici\u00f3n \u201cSalvador Zubir\u00e1n\u201d following the technique previously described (Makita et\u00a0al. 1991, 1992).\nAGE-BSA was prepared by incubating 5% of bovine serum albumin (BSA fraction V; Sigma Chemical, St. Louis, MO) in 200 mM of sodium phosphate buffer (pH 7.4) with 500 mM glucose for 60 days under sterile conditions, in darkness and at 37\u00b0C. After incubation, the product was reduced with 100 mM sodium borohydride (NaBH4, Sigma Chemical) for 30 min at room temperature to stabilize the Schiff bases formed (Eble et\u00a0al. 1983; Fluckiger and Gallop 1984; Verbeke et\u00a0al. 1997). Then, the unbound glucose and unreacted NaBH4 were removed by extensive dialysis against a 500 mM sodium phosphate buffer (pH 7.4). Less glycosylated BSA was removed from AGE-BSA by affinity chromatography on Affi-Gel Blue (Bio-Rad Laboratories, Richmond, CA) (Travis et\u00a0al. 1976; Verbeke et\u00a0al. 1997). The Affi-Gel matrix has a blue dye named Cibacron Blue F3GA, with natural affinity for native albumin. A column with 5 mL of matrix with a binding capacity of 170 mg of BSA was employed. 2 mL of AGE-BSA solution with a concentration of 53.8 mg\/mL was applied in each purification process, using a flow rate of 1 mL\/min. The chromatogram was performed evaluating a dilution 1\/10 of each tube.\nProtein concentration of AGE-BSA was evaluated by the Lowry method using BSA as standard (Lowry et\u00a0al. 1951). Finally, AGE-specific fluorescence determination was performed by measuring emission at 440 nm upon excitation at 370 nm using an Aminco fluorescence spectrometer (Aminco-Bowman Series 2 SLM Instruments. Inc., Rochester, NY) (Monnier and Cerami 1981; Makita et\u00a0al. 1992). Fluorescence value of AGE-BSA was measured at a protein concentration of 1 mg\/mL and expressed in arbitrary units (AU). The specific activity (SA) was expressed as arbitrary units of fluorescence per mg of protein (AU\/mg).\nThe control BSA sample (nonglycated BSA) was incubated under identical conditions but without glucose. AGE-BSA and its control (nonglycated BSA) were maintained at \u221270\u00b0C until used.\nMass increase analysis of glycated BSA\nThe mass change of BSA after glycation was analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF) (Voyager DE-PRO from Applied Biosystems, CA), equipped with a nitrogen laser (337 nm), operating in a positive high-energy linear mode. This analysis was made in the Analysis and Molecular Diagnose Unit of the Insituto Nacional de Salud P\u00fablica de M\u00e9xico.\nAliquots of 500 \u03bcL containing 10 mg\/mL of BSA or AGE-BSA were diluted to 1.5 mL with deionized water and centrifuged in Amicon Ultracel-10 K (Millipore Corporation, MA) at 2,000 \u00d7 g for 30 min to a final volume of 500 \u03bcL. Washing was repeated three times. A 10 \u03bcL aliquot was taken for MALDI-TOF analysis. The matrix solution employed for BSA analysis contained 10 mg\/mL 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid) in 30% vol\/vol acetonitrile in 0.1% vol\/vol aqueous trifluoroacetic acid. For AGE-BSA the matrix solution contained 2,5-dihydroxybenzoic acid (gentisic acid, which is employed for analysis of glycoproteins) to 10 mg\/mL in 50% vol\/vol acetonitrile. A sample protein solution (1 \u03bcL) of a 1:1 vol\/vol mixture of 1 mg\/mL of total protein and the matrix solution were applied on a sample spot in a steel plate slide and dried in a warm air stream. Bovine's insulin (5,734.59 Da), E. coli's Thioredoxin (11,674.48 Da), and horse's Apomyoglobyn (16,952.56 Da) were used as standard for calibration.\nIsoelectric point of glycated BSA\nThe change in the isoelectric point of BSA after glycation was analyzed by isoelectric focusing in polyacrylamide gels using a FastSystem (Pharmacia, Uppsala, Sweden). The bands were dyed with silver nitrate, and the isoelectric point was evaluated using the analytical unit of FastSystem employing a broad range standard kit (pH 3\u201310) (Amersham Biosciences, Buckinghamshire, United Kingdom) as a protein calibrator.\nBlood sample collection and determination of glucose, insulin, triglycerides, and serum albumin\nAfter overnight fasting (12 h), the animals were killed by decapitation and blood was collected. It was spun and the serum was separated by centrifugation at 600 \u00d7 g during 15 min at room temperature and stored at \u221270\u00b0C until needed. Serum insulin was determined using a commercial radioimmunoassay (RIA) specific for rat (Linco Research, Inc., Missouri); its sensitivity was 0.1 ng\/mL and intra- and interassay coefficients of variation were 5 and 10%, respectively. Glucose concentration was assayed using an enzymatic SERA-PAKR Plus from Bayer Corporation (S\u00e9es, France). Homeostasis model assessment (HOMA) was used as an index to measure the degree of insulin resistance and was calculated by the formula [insulin (\u03bcU\/mL) \u00d7 glucose (mmol\/L)\/22.5] (Matthews et\u00a0al. 1985; Pickavance et\u00a0al. 1999; Nandhini et\u00a0al. 2005). Triglycerides (TGs) were determined by commercially available procedures (Randox, Laboratories LTD, Antrim, United Kingdom). Serum albumin was evaluated in both groups of animals using a kit commercialized by Hycel of Mexico which uses bromocresol green (IL-test Instrumentation Laboratory Company, Lexington, MA). Samples were read using an Ilab-600 automatic system.\nSample preparation and tension recording\nThe animals were killed by decapitation, and aortas were immediately dissected and placed in an oxygenated normal Tyrode solution (mM: 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 5 Hepes, and 5.5 glucose) pH 7.4. Arteries were carefully cleaned from connective and adipose tissue, taking care not to damage the endothelium. Tension measurements were made as previously described (Nava et\u00a0al. 1999). Briefly, segments of about 2 to 3 mm long were cut and two 250-\u03bcm-diameter S-shaped silver wires (Medwire Corp, Mount Vernon, NY) were inserted into the lumen to measure tension developed transversely by rings of the vessel. One of the silver wires was fixed to the bottom of an in vitro chamber, and the other was attached to a tension transducer that was connected to a Grass polygraph model 79D. The chamber was filled with a Tyrode solution, and termorregulated and bubbled with carbogen (95% oxygen, 5% carbon dioxide). A basal passive tension of 2 g was applied after determination in preliminary tests that this was the optimal resting tension under our experimental conditions. Arteries were allowed to rest for 1 h and the solution was changed every 20 min. Contraction was induced twice by the addition of norepinephrine (NE) (1 \u03bcM). Arteries were washed by adding a fresh Tyrode solution to the chamber allowing the rings to return to their basal tension (2 g). The mean contraction value was considered as 100% of response. The vasodilator activity was studied by cumulative concentration\u2013response curves to acetylcholine (Ach) (10\u22129\u221210\u22124 M) on precontracted aortic rings in the following experimental groups: (A) incubated only with the Tyrode solution, (B) incubated during 40 min with AGE-BSA at 10 AU\/mL, (C) incubated with AGE-BSA at 40 AU\/mL, (D) incubated with nonglycated BSA at a protein concentration equivalent to 10 UA\/mL of AGE-BSA (0.95 mg\/mL), (E) incubated with nonglycated BSA at a protein concentration equivalent to 40 AU\/mL of AGE-BSA (3.81 mg\/mL), and (F) incubated with L-NAME at 300 \u03bcM, in both control and MS rats. The percentage of the response in each experimental group was calculated in relation to the tension developed by the same ring during its basal contraction.\nImmunohistochemistry of RAGEs\nAortic rings of both MS and C rats were quickly frozen in Tissue-Tek (Sakura Finetek USA, Inc., Torrance, CA). Sections were fixed with acetone and were blocked with PBS\/Azide 0.02%\/BSA 1% for 30 min.\nSubsequently, sections were incubated during 2 h at room temperature with a rabbit polyclonal antibody against RAGE (1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Primary antibodies were detected by using goat anti-rabbit FITC (Jackson ImmunoResearch Laboratories Inc. West Grove, PA), at room temperature for 60 min. Negative controls were prepared by substituting the primary antibody with an irrelevant antibody. Staining was analyzed using fluorescence microscopy.\nStatistical analysis\nResults are expressed as mean \u00b1 standard errors of the mean (SEM) from 6 to 10 different artery preparations. The percentage of contraction in each experiment was calculated, and the mean was then determined. When applicable (comparisons between two values; MS and controls), statistical analysis was done by Student's t-test. Comparisons between groups were done by analysis of variance (ANOVA) or Anova on ranks followed by Student\u2013Newman\u2013Keuls or Dunn's tests, depending on whether the data were normally distributed or not, using the SigmaStat 2.0 program (Jandel Scientific, San Rafael, CA). Differences were considered statistically significant when P < 0.05.","keyphrases":["bsa","metabolic syndrome","age-bsa","vascular relaxation"],"prmu":["P","P","P","P"]}
{"id":"Biochim_Biophys_Acta-2-1-2428106","title":"Regulation of intestinal hPepT1 (SLC15A1) activity by phosphodiesterase inhibitors is via inhibition of NHE3 (SLC9A3)\n","text":"The H+-coupled transporter hPepT1 (SLC15A1) mediates the transport of di\/tripeptides and many orally-active drugs across the brush-border membrane of the small intestinal epithelium. Incubation of Caco-2 cell monolayers (15 min) with the dietary phosphodiesterase inhibitors caffeine and theophylline inhibited Gly\u2013Sar uptake across the apical membrane. Pentoxifylline, a phosphodiesterase inhibitor given orally to treat intermittent claudication, also decreased Gly\u2013Sar uptake through a reduction in capacity (Vmax) without any effect on affinity (Km). The reduction in dipeptide transport was dependent upon both extracellular Na+ and apical pH but was not observed in the presence of the selective Na+\/H+ exchanger NHE3 (SLC9A3) inhibitor S1611. Measurement of intracellular pH confirmed that caffeine was not directly inhibiting hPepT1 but rather having an indirect effect through inhibition of NHE3 activity. NHE3 maintains the H+-electrochemical gradient which, in turn, acts as the driving force for H+-coupled solute transport. Uptake of \u03b2-alanine, a substrate for the H+-coupled amino acid transporter hPAT1 (SLC36A1), was also inhibited by caffeine. The regulation of NHE3 by non-nutrient components of diet or orally-delivered drugs may alter the function of any solute carrier dependent upon the H+-electrochemical gradient and may, therefore, be a site for both nutrient\u2013drug and drug\u2013drug interactions in the small intestine.\n1\nIntroduction\nThe human intestinal H+-coupled di\/tripeptide transporter (hPepT1 or SLC15A1) is responsible for the transport of a significant proportion of dietary protein across the brush-border membrane of the small intestinal epithelium. hPepT1 is also of pharmacological interest as it can transport orally-active drugs such as \u03b2-lactam antibiotics and angiotensin converting enzyme inhibitors [1\u20134]. More recently a number of amino acid modified pro-drugs such as val-acyclovir, l-DOPA-l-Phe and the anti-hypotensive midodrine have been designed specifically to target hPepT1 and thus to enhance oral bioavailability [3\u20135].\nWhen expressed heterologously, hPepT1 functions as a Na+-independent, pH-dependent, H+-coupled transporter [6,7]. However, until the mid-1980s, it was considered widely that di\/tripeptide transport was via a Na+-coupled transport mechanism. Indeed, dipeptides can stimulate Na+ absorption in the human small intestine and Na+ uptake in human intestinal Caco-2 cell monolayers [8\u201310]. Ganapathy and Leibach proposed a model to account for the interaction of H+-coupled di\/tripeptide transport and Na+ absorption [11] whereby the intracellular acidification caused by di\/tripeptide associated H+ influx activated an apical Na+\/H+ exchanger and thus Na+ absorption. This model also explains the partial Na+-dependence of di\/tripeptide uptake observed in mammalian small intestine and Caco-2 cell monolayers [11,12]. Inhibition of Na+\/H+ exchange by removal of extracellular Na+, prevents the enterocyte from maintaining intracellular pH (pHi) during H+\/solute influx and, therefore, abolishes the driving force (the transmembrane H+ electrochemical gradient) for further transport. The apical Na+\/H+ exchanger specifically activated by di\/tripeptide uptake is NHE3 (SLC9A3) which plays a significant role in Na+ absorption in the small intestine [7,10,13].\nAs hPepT1 is involved in both protein assimilation and drug delivery it is important to elucidate how altering hPepT1 function may affect nutritional status and oral bioavailability. Progress has been made in understanding how intestinal di\/tripeptide transport is regulated by hormonal and neural signals as well as by disease states and surgical intervention (for review see [14]). However, little is known about how co-administered drugs or dietary factors affect hPepT1 function or the function of other brush-border membrane transport proteins. Alteration of cAMP levels within enterocytes by hormonal, neural and paracrine signals is a key step in the regulation of many brush-border membrane nutrient and electrolyte transporters. NHE3 is inhibited acutely by increasing [cAMP]i\n[15]. Therefore, the aim of this study was to identify how dietary and orally-delivered compounds, which act as phosphodiesterase inhibitors (and thus increase [cAMP]i by preventing breakdown), affect hPepT1 function. The dietary phosphodiesterase inhibitors caffeine and theophylline and the orally-delivered drug pentoxifylline all inhibit dipeptide uptake across the brush-border membrane of human intestinal epithelial Caco-2 cell monolayers. This inhibition is not a direct effect on hPepT1 but rather is indirect through inhibition of NHE3.\n2\nMaterials and methods\n2.1\nMaterials\nGlycyl[1-14C]sarcosine (specific activity 57\u00a0mCi mmol\u2212\u00a01)] was obtained from Cambridge Research Biochemicals (Billingham, UK). [3-3H]\u03b2-Alanine (specific activity 50 Ci mmol\u2212\u00a01)] was obtained from American Radiolabeled Chemicals (St. Louis, MO, USA). PACAP (pituitary adenylate cyclase-activating polypeptide) was from Bachem (St. Helens, UK). Biotrak cAMP enzyme immunoassay was from GE Healthcare (Chalfont St. Giles, UK). 2\u2032,7\u2032-Bis(2-carboxyethyl-5(6)-carboxyfluorescein) acetoxymethyl ester (BCECF-AM) was from Invitrogen (Paisley, UK). Transwell polycarbonate filters were from Corning (Schiphol-Rijk, The Netherlands). S1611 was obtained from H.J. Lang (Aventis Pharma Deutschland GmbH, Frankfurt\/Main, Germany). Forskolin, phosphodiesterase inhibitors, cell culture media and supplements were from Sigma-Aldrich (Poole, UK). All other chemicals were from Sigma-Aldrich or VWR (Lutterworth, UK) and were of the highest quality available.\n2.2\nCell culture\nCaco-2 cells (passages 100\u2013119) were cultured as confluent monolayers on permeable polycarbonate filters, as described previously [16]. Monolayers were used at 14\u201322\u00a0days post-seeding and fed approximately 24\u00a0h prior to use.\n2.3\nMeasurement of dipeptide uptake\n[14C]Gly\u2013Sar (0.5\u00a0\u03bcCi ml\u2212\u00a01, 0.1 \u2013 10\u00a0mM) or [3H]\u03b2-alanine (0.5\u00a0\u03bcCi ml\u2212\u00a01, 0.1\u00a0mM) uptake across the apical membrane of Caco-2 cell monolayers was measured, essentially as described previously [16]. Briefly, Caco-2 cells were washed (in 4\u00a0\u00d7\u00a0500\u00a0ml) and bathed in modified Krebs' solution (composition (mM): NaCl, 137; KCl, 5.4; MgSO4, 0.99; KH2PO4, 0.34; NaH2PO4, 0.3; CaCl2, 2.8; Glucose, 10) or Na+-free solution (identical except choline chloride replaced NaCl and NaH2PO4 was omitted). Uptake was measured at apical pH 5.5 or pH 6.5 (10\u00a0mM MES, adjusted to the correct pH by Tris) with the basolateral solution being pH 7.4 (10\u00a0mM HEPES, adjusted to the correct pH by Tris) in all experiments. Uptake was measured over 15\u00a0min (37\u00a0\u00b0C) at the end of which the monolayers were rinsed thoroughly (in 3\u00a0\u00d7\u00a0500\u00a0ml, ice-cold, pH 7.4 Krebs' solution). Various compounds were added to the buffers for the duration of uptake (see figure legends for details). Cell monolayer-associated radioactivity was determined by scintillation counting.\n2.4\nMeasurement of pHi\nIntracellular pH (pHi) was measured in Caco-2 cell monolayers by microspectrofluorimetry using the pH-sensitive dye BCECF, essentially as described previously [10,17]. Cell monolayers were acidified by superfusion with apical Gly\u2013Sar (20\u00a0mM, 5\u00a0min) in modified Krebs' solution (Na+-free, apical pH 5.5). The basolateral solution was Na+-free, pH 7.4 throughout all experiments. pHi recovery was then measured in Na+-containing pH 7.4 apical solution until pHi returned to baseline. The monolayers were incubated with caffeine (5\u00a0mM, apical and basolateral) for 10\u00a0min. The cells were then exposed to Gly\u2013Sar once again but in the continued presence of caffeine (thus total exposure time to the compound before pHi recovery was 15\u00a0min). pHi recovery was then measured in the continued presence of caffeine. The initial rate of pHi recovery is expressed as H+ efflux calculated from the gradient of the first 30\u00a0s of recovery [18].\n2.5\nMeasurement of intracellular cAMP levels\nCaco-2 cell monolayers were washed in 4\u00a0\u00d7\u00a0500\u00a0ml Krebs' solution (pH 7.4). Monolayers were incubated for 15\u00a0min in the presence or absence of caffeine, pentoxifylline, theophylline (all 5\u00a0mM), or forskolin (10\u00a0\u03bcM) at apical pH 6.5 and basolateral pH 7.4. Monolayers were then washed in 3\u00a0\u00d7\u00a0500\u00a0ml, ice-cold Krebs' (pH 7.4) and lysed in 500\u00a0\u03bcl lysis buffer (from the Biotrak enzyme immunoassay kit) for 10\u00a0min. [cAMP]i was measured following the kit protocol.\n2.6\nStatistical analysis\nData are expressed as mean\u00a0\u00b1\u00a0SEM unless stated otherwise. Statistical comparisons of mean values were made using paired two-tailed Student's t-test or one-way analysis of variance (ANOVA) (using the Tukey\u2013Kramer or Bonferroni's multiple comparisons post-test) as appropriate. Significance was assumed if p\u00a0<\u00a00.05. Curves were fitted using FigP software.\n3\nResults\n3.1\nInhibition of dipeptide uptake by caffeine is via inhibition of NHE3\nUptake of the dipeptide Gly\u2013Sar (glycylsarcosine) across the apical membrane of Caco-2 cell monolayers was measured over 15\u00a0min at an apical pH (pH 6.5) representative of that found at the surface of the proximal small intestine. Incubation of Caco-2 cell monolayers with the dietary phosphodiesterase inhibitor caffeine (5\u00a0mM) at both the apical and basolateral surfaces (for the duration of the 15\u00a0min uptake period) reduced Gly\u2013Sar uptake by around 50% (from 683.6\u00a0\u00b1\u00a018.9 to 351.1\u00a0\u00b1\u00a010.7\u00a0pmol cm\u2212\u00a02 [15\u00a0min]\u2212\u00a01, n\u00a0=\u00a017\u201318, p\u00a0<\u00a00.001, Fig. 1A). Caffeine was equally effective at inhibiting Gly\u2013Sar uptake when added solely to either the apical or basolateral chamber (data not shown). Removal of extracellular Na+ reduces Gly\u2013Sar uptake (as described previously [12]) (Fig. 1A). However, in the absence of extracellular Na+, caffeine had no effect on Gly\u2013Sar uptake (uptake being 244.0\u00a0\u00b1\u00a09.1 and 242.0\u00a0\u00b1\u00a09.5\u00a0pmol cm\u2212\u00a02 [15\u00a0min]\u2212\u00a01 in the absence and presence of caffeine respectively, n\u00a0=\u00a017\u201318, p\u00a0>\u00a00.05).\nPreviously we have shown that the Na+-dependence of Gly\u2013Sar uptake can be attributed to a lack of NHE3 activity in the absence of extracellular Na+\n[10,12,18]. To test if the lack of effect of caffeine in the absence of extracellular Na+ was due to a requirement for NHE3 activity, dipeptide uptake was measured under other conditions where NHE3 would be inactive (Fig. 1B). At apical pH 6.5, the NHE3-selective inhibitor S1611 (3\u00a0\u03bcM) significantly reduced Gly\u2013Sar uptake in a manner similar to that caused by the removal of extracellular Na+ (p\u00a0<\u00a00.001). This concentration of S1611 is sufficient to inhibit NHE3 completely but would not inhibit NHE2 or NHE1 [19]. Previous work has shown that in the absence of Na+, S1611 has no effect on Gly\u2013Sar uptake into Caco-2 cells [12]. In addition, when hPepT1 is expressed in isolation in Xenopus laevis oocytes, dipeptide uptake is not inhibited by S1611 [7]. This observation confirms that the inhibition of dipeptide uptake by S1611 into Caco-2 cells is an indirect effect via inhibition of NHE3. In the presence of S1611, Gly\u2013Sar uptake was no longer inhibited by caffeine (5\u00a0mM). The level of inhibition caused by caffeine alone, by S1611 alone and by both compounds together were not significantly different (p\u00a0>\u00a00.05) suggesting that caffeine and S1611 inhibit hPepT1 activity through a common mechanism (i.e. NHE3 inhibition). Gly\u2013Sar uptake increases with decreasing apical pH, consistent with hPepT1 being a H+\/dipeptide symporter. However, NHE3 activity decreases with acidification of the apical solution and is inactive by pH 5.5 due to the unfavourable transmembrane pH gradient [12]. Previous studies have shown that Gly\u2013Sar uptake at apical pH 5.5 is not inhibited by S1611 or by the removal of extracellular Na+\n[7,12]. Similarly, Fig. 1B shows that caffeine (5\u00a0mM) has no effect on Gly\u2013Sar uptake at apical pH 5.5 (p\u00a0>\u00a00.05) suggesting again that caffeine is inhibiting Gly\u2013Sar uptake through inhibition of NHE3.\n3.2\nThe inhibition of hPepT1 by caffeine and related compounds is consistent with inhibition of phosphodiesterase activity\nPhosphodiesterase inhibitors structurally-related to caffeine and either found in diet (theophylline), or used as orally-delivered therapeutics (pentoxifylline) or used as laboratory tools (IBMX, 3-isobutyl-1-methylxanthine) were tested for inhibition of hPepT1 (Fig. 2). Theophylline (5\u00a0mM, Fig. 2A), pentoxifylline (5\u00a0mM, Fig. 2B) and IBMX (1\u00a0mM, Fig. 2C) reduced dipeptide uptake in the presence of extracellular Na+ (p\u00a0<\u00a00.001) but not in Na+-free conditions (p\u00a0>\u00a00.05). Previously, we have shown that the enteric neuropeptide PACAP (pituitary adenylate cyclase-activating polypeptide) and the related neuropeptide VIP (vasoactive intestinal peptide) reduce dipeptide uptake across the apical membrane of Caco-2 cell monolayers by inhibition of NHE3 [18]. PACAP activates the VPAC1 receptor which is coupled to adenylate cyclase and, therefore, increases [cAMP]i. Fig. 2C shows that when Caco-2 cell monolayers were incubated with PACAP (5\u00a0nM) in the basolateral solution for the duration of uptake, Gly\u2013Sar uptake was reduced. Incubation of the cells with both PACAP and IBMX resulted in a reduction in uptake which was not significantly different from the uptake in the presence of either PACAP or IBMX alone suggesting IBMX, like PACAP, is inhibiting the NHE3-dependent component of Gly\u2013Sar uptake.\nPhosphodiesterase inhibitors increase the levels of cAMP within cells by preventing cAMP breakdown. However, caffeine can also elicit cell signalling effects such as antagonism of adenosine receptors and activation of calcium release from intracellular stores [20\u201322]. To confirm that the compounds used in this study inhibit hPepT1 function through increasing cAMP levels, [cAMP]i in Caco-2 cell monolayers was measured. Monolayers were incubated with caffeine (5\u00a0mM), theophylline (5\u00a0mM), pentoxifylline (5\u00a0mM) or the adenylate cyclase activator forskolin (10\u00a0\u03bcM) which can also inhibit dipeptide uptake [12]. Cells were lysed and [cAMP]i measured by enzyme immunoassay. Caffeine, theophylline and pentoxifylline caused [cAMP]i to more than double from control levels of 950\u00a0\u00b1\u00a0230\u00a0fmol cm\u2212\u00a02 (mean\u00a0\u00b1\u00a0SD, n\u00a0=\u00a03) to 2200\u00a0\u00b1\u00a0830, 2780\u00a0\u00b1300 and 2330\u00a0\u00b1\u00a0150\u00a0fmol cm\u2212\u00a02 (mean\u00a0\u00b1\u00a0SD, n\u00a0=\u00a03), respectively. However, the change in cAMP was modest compared to [cAMP]i detected in the presence of forskolin (92500\u00a0\u00b112990\u00a0fmol cm\u2212\u00a02 [mean\u00a0\u00b1\u00a0SD, n\u00a0=\u00a03]).\nInhibition of the Na+-dependent (NHE3-dependent) component of Gly\u2013Sar uptake by phosphodiesterase inhibitors was concentration-dependent (Fig. 3). The IC50 values were 1.0\u00a0\u00b10.2\u00a0mM for caffeine, 0.6\u00a0\u00b1\u00a00.1\u00a0mM for theophylline and 0.5\u00a0\u00b10.1\u00a0mM for pentoxifylline. The sensitivity of hPepT1 to these compounds is consistent with the effect being through an inhibition of phosphodiesterase activity [20,21,23\u201325]. At a concentration of 5\u00a0mM all three phosphodiesterase inhibitors almost completely inhibited Na+-dependent Gly\u2013Sar uptake.\n3.3\nPentoxifylline decreases the maximal capacity for dipeptide uptake\nGly\u2013Sar uptake across the apical membrane was measured at apical pH 6.5 at various concentrations of Gly\u2013Sar (Fig. 4). In the presence of pentoxifylline (5\u00a0mM) Gly\u2013Sar uptake was reduced compared to control at all Gly\u2013Sar concentrations. Analysis of the Michaelis\u2013Menten kinetics confirmed that the maximal capacity (Vmax) for Gly\u2013Sar uptake was significantly reduced from 8246\u00a0\u00b1\u00a0913 to 5369\u00a0\u00b1\u00a0117\u00a0pmol cm\u2212\u00a02 [15\u00a0min]\u2212\u00a01 (p\u00a0<\u00a00.05, n\u00a0=\u00a06) by pentoxifylline but the affinity (Km) was not affected (1.0\u00a0\u00b1\u00a00.2\u00a0mM and 1.6\u00a0\u00b1\u00a00.6\u00a0mM in the absence and presence of pentoxifylline, respectively, p\u00a0>\u00a00.05). Reduction in the capacity for dipeptide uptake is consistent with a reduction in the driving force (the H+-electrochemical gradient) for uptake through inhibition of NHE3. Previously, we have shown that S1611 and forskolin similarly decrease the Vmax for dipeptide uptake across the apical membrane of Caco-2 cell monolayers [12].\n3.4\nCaffeine inhibits pHi recovery (NHE3 function) after dipeptide-induced acidification\nPrevious work demonstrated that NHE3 (but not NHE1 or NHE2) is selectively activated by the intracellular acidification associated with H+\/dipeptide symport in Caco-2 cell monolayers [10,12]. S1611, the removal of extracellular Na+, or factors that increase [cAMP]i, such as forskolin and VIP, all inhibit the ability of the cell to regulate pHi after H+\/solute-induced intracellular acidification [10,12,18,26]. pHi was measured using the pH-sensitive dye BCECF (Fig. 5). Caco-2 cell monolayers loaded with BCECF were superfused across the apical membrane with Na+-containing, pH 7.4 modified Krebs' solution. The basolateral solution was maintained as Na+-free, pH 7.4 solution throughout the experiment. Cells were exposed to Gly\u2013Sar (20\u00a0mM) under conditions where hPepT1 activity would be maximal but where there would be no concurrent NHE3 activity (pH 5.5, Na+-free). After 5\u00a0min, Gly\u2013Sar had induced a large intracellular acidification. When Gly\u2013Sar was removed and the apical solution returned to a Na+-containing, pH 7.4 solution, pHi recovered rapidly back to baseline levels. The same monolayers were then incubated with caffeine (5\u00a0mM) in both the apical and basolateral solutions for 10\u00a0min. Gly\u2013Sar was then reintroduced for 5\u00a0min in the continued presence of caffeine. The degree and rate of acidification caused by Gly\u2013Sar was not altered by caffeine suggesting that hPepT1 is not directly regulated by phosphodiesterase inhibitors. However, the ability of the cell to recover pHi after Gly\u2013Sar-induced acidification was attenuated after the 15\u00a0min exposure to caffeine (Fig. 5A). The degree of attenuation was quantified by calculating the H+ efflux rate over the first 30\u00a0s of pHi recovery (Fig. 5B). In the presence of caffeine H+ efflux was reduced from 57.6\u00a0\u00b1\u00a05.8 to 20.5\u00a0\u00b1\u00a03.9\u00a0\u03bcM s\u2212\u00a01 (n\u00a0=\u00a04, p\u00a0<\u00a00.01).\nTo confirm that phosphodiesterase inhibitors do not have any direct inhibitory effect on dipeptide transport, hPepT1 was expressed in Xenopus laevis oocytes, as described previously [7]. Incubation of hPepT1-expressing oocytes with phosphodiesterase inhibitors such as caffeine (5\u00a0mM) had no effect on dipeptide uptake (data not shown).\n3.5\nInhibition of NHE3 by caffeine also inhibits the H+\/amino acid transporter hPAT1 (SLC36A1)\nThe indirect nature of the inhibition of hPepT1 by phosphodiesterase inhibitors suggests that any apical solute transporter dependent upon the transmembrane H+-electrochemical gradient will similarly be regulated by these compounds. The H+-coupled amino acid transporter hPAT1 (SLC36A1) has been isolated from Caco-2 cell monolayers [27]. As well as mediating the uptake of a wide variety of amino acids, hPAT1 can also transport orally-active drugs such as the anti-epileptic vigabatrin [28]. Previously we have identified that amino acid uptake into hPAT1-expressing oocytes is Na+-independent but hPAT1-mediated amino acid uptake into Caco-2 cells is partially Na+-dependent [26,29,30]. Intracellular acidification caused by the hPAT1 substrate \u03b2-alanine selectively activated Na+\/H+ exchange by NHE3 [26]. Like H+-coupled dipeptide uptake, H+-coupled amino acid uptake into Caco-2 cells is inhibited by forskolin, S1611 and VIP in a Na+ and pH-dependent manner via inhibition of NHE3 [26,29,30]. Uptake of the hPAT1 substrate \u03b2-alanine [16] was measured across the apical membrane of Caco-2 cell monolayers at apical pH 6.5 for 15\u00a0min (Fig. 6). Caffeine (5\u00a0mM) reduced \u03b2-alanine uptake in the presence (p\u00a0<\u00a00.001) but not the absence of extracellular Na+ (p\u00a0>\u00a00.05) suggesting that H+-coupled amino acid uptake via hPAT1 is also modulated indirectly through regulation of NHE3.\n4\nDiscussion\nThe di\/tripeptide transporter hPepT1 acts as a high-capacity route for solutes across the first barrier to oral-bioavailability, the brush-border membrane of the small intestine. Many, orally-active peptidomimetics and amino acid-conjugated pro-drugs have been identified as hPepT1 substrates [3,4]. There is an increasing number of examples of physiological regulation (hormonal, neural, paracrine) of hPepT1 and of regulation of hPepT1 in certain disease states and after surgery (reviewed by [14]). Another, less studied, factor which may affect the degree to which drugs are absorbed across the small intestinal epithelium is interaction with co-administered drugs or components of diet. Exposure of Caco-2 cell monolayers to the hPepT1 substrate Gly\u2013Gln for 4\u00a0days resulted in a subsequent increase in capacity for dipeptide uptake and in hPepT1 expression [31]. Another study found that an array of flavonoids, which are found ubiquitously in foods of plant origin, either inhibit, have no effect or increase the hPepT1-mediated uptake of the antibiotic cefixime into Caco-2 cell monolayers [32]. In this study we identify that incubation of human intestinal epithelial cells with either dietary or orally-active therapeutic phosphodiesterase inhibitors reduces Gly\u2013Sar uptake through a reduction in hPepT1 capacity.\nThe data presented here show that the inhibition of Gly\u2013Sar uptake by phosphodiesterase inhibitors is both Na+- and pH-dependent (Figs. 1 and 2) suggesting that inhibition is not a direct effect on hPepT1 but rather through NHE3. When NHE3 is inhibited (e.g. by the removal of extracellular Na+ or by addition of S1611) the cells are no longer able to maintain pHi during solute-induced acidification and, therefore, the driving force (the transmembrane H+ electrochemical gradient) for further dipeptide uptake is reduced. Previously, we have shown that hPepT1 can be inhibited by other factors which are known to increase cAMP in intestinal epithelial cells such as the enteric neuropeptides VIP and PACAP [18]. Although caffeine, theophylline and pentoxifylline can elicit effects through pathways other than increasing cAMP, a number of factors suggest that they are acting here as phosphodiesterase inhibitors. Firstly, incubating Caco-2 cell monolayers with all three compounds produced an increase in [cAMP]i. The increase is relatively small compared to that produced by forskolin. However, this could be due to the cAMP signal being compartmentalised (as demonstrated in cardiomyocytes [33]) and thus the local change may be much greater than the measured, global change. Secondly, phosphodiesterases can also regulate the levels of cGMP but none of the compounds tested here produced a significant change in [cGMP]i (data not shown). Lastly, the concentrations of caffeine, theophylline and pentoxifylline required to produce inhibition of hPepT1 (IC50\u00a0=\u00a01.0, 0.6 and 0.5\u00a0mM, respectively, Fig. 3) are much closer to those for inhibition of phosphodiesterases than for other effects such as antagonism of adenosine receptors (IC50\u2248\u00a02\u201380\u00a0\u03bcM) and release of intracellular calcium (EC50\u2248\u00a05\u201320\u00a0mM) [20\u201322,34\u201336]. Theophylline (100\u00a0\u03bcM) has been shown to abolish increased glucose absorption resulting from activation of A2 receptors by luminal adenosine (5\u00a0mM) in mouse small intestine in vivo [37]. However, in addition to the significantly higher affinity for theophylline, A2 receptors are positively coupled to adenylate cyclase so antagonism of the receptor should cause a decrease in [cAMP]i and not the increase noted here [22,37].\nCaffeine (and to a lesser extent theophylline) is found in high levels in many beverages either naturally or by addition. For example, the average cup of coffee contains 85\u00a0mg of caffeine [38]. Pentoxifylline is given orally as 400\u00a0mg tablets to treat intermittent claudication as part of peripheral arterial disease [39]. Although it is difficult to predict the actual concentration at the surface of the small intestinal epithelium it is conceivable that caffeine and pentoxifylline may reach concentrations high enough to elicit an effect on hPepT1 similar to that measured here using Caco-2 cell monolayers. There is evidence that caffeine given orally in doses equivalent to diet can elicit changes in small intestinal function. A study using human volunteers found that ingestion or intraluminal jejunal perfusion of caffeine (75\u2013300\u00a0mg) caused a rapid reversal of net fluid absorption to net secretion [40]. Although the cell signalling pathway by which caffeine stimulated fluid secretion was not investigated, this study does indicate that the concentrations of caffeine used here in vitro may be sufficient to cause changes in the small intestinal epithelium in vivo.\nSince the initial reports of phosphodiesterase activity it has become evident that there are many types of phosphodiesterase. Currently there are 11 different PDE families each of which can contain several isoforms and splice variants [24,41]. Many of these isoforms are tissue or cell-specific and recent studies have shown that they can be expressed in distinct sub-cellular locations where they act to limit site, amplitude and duration of cyclic nucleotide signals [24,33]. Little is known about which PDE isoforms are found in the small intestinal epithelium. Studies reporting tissue distribution of PDE mRNA expression often include a small intestinal sample but generally it is not clear whether the PDE is expressed specifically in enterocytes. A single study of PDE3 isoforms identified that PDE3B but not PDE3A mRNA was highly abundant in the epithelium and underlying smooth muscle layers of developing small intestine of rat [42].\nCaffeine, theophylline, IBMX and pentoxifylline generally inhibit all PDE isoforms with roughly equal efficacy [25,43,44]. However, since many PDE isoforms and variants are located in distinctive cell and sub-cellular locations they are now considered to be good targets for specific therapeutic agents. Often the resulting drugs are given orally, for example, the PDE5 inhibitor sildenafil used to treat erectile dysfunction [45]. PDE5 is expressed at the mRNA level in the small intestine [46,47]. Further work is required to identify which PDE isoforms are expressed in small intestinal epithelial cells both along the proximal\u2013distal and crypt\u2013villus axes and which isoforms play a functional role in regulating solute transport at the brush-border membrane.\nIn conclusion, we have shown that non-nutrient components of diet and orally-delivered therapeutic agents both regulate activity of the nutrient and drug transporter hPepT1. This regulation is an indirect effect through inhibition of apical NHE3 function, which acts as a pH homeostatic mechanism during H+-coupled dipeptide transport. Functional cooperativity between hPepT1 and NHE3 results not only in the absorption of di\/tripeptides but also stimulation of Na+ absorption [8\u201310]. The H+-coupled amino acid transporter hPAT1 (SLC36A1) is regulated by caffeine in a similar, indirect manner to hPepT1 (Fig. 6). Thus, through modulation of a single homeostatic mechanism (NHE3), a luminal compound may influence several different absorptive mechanisms. The results presented here suggest that any regulation of NHE3 activity by luminal contents, co-administered drugs or by neurohormonal signals may have significant impact on the general absorptive state of the cell. Any other nutrient transporter dependent on the maintenance of the H+ electrochemical gradient for function may be regulated in a similar manner.","keyphrases":["nhe3","dipeptide transport","na+\/h+ exchange","drug transport","pept1","intestinal absorption"],"prmu":["P","P","P","P","U","R"]}
{"id":"J_Affect_Disord-1-5-1894757","title":"Outcome of prenatal depression and risk factors associated with persistence in the first postnatal year: Prospective study from Rawalpindi, Pakistan\n","text":"Background Rates of prenatal and postnatal depression in developing countries are high. Prolonged depression during the postnatal period is associated with impaired infant growth and development. Little is known about the factors predicting the persistence of prenatal depression beyond the first few postnatal months.\n1\nIntroduction\nThe rate of postnatal depression in developing countries ranges from 16% to 35% (Ghubash and Abou-Saleh, 1997; Patel et al., 2002; Aydin et al., 2005; Cooper et al., 1999) and is a major contributor to the \u2018burden of disease\u2019 in these countries. Depression around childbirth is associated with low birth weight and impaired weight gain in the first year of the infant's life (Rahman et al., 2004). The outcome is worse in infants whose mothers remained persistently depressed from the third trimester throughout the first postnatal year. It is, therefore, important to predict which mothers, depressed prenatally, are likely to remain depressed throughout the next year.\nRelatively fewer studies have examined the long-term outcome of postnatal depression. A recent review of studies from developed countries concluded that in about 30% women with postnatal depression, symptoms persist for up to a year after giving birth (Goodman, 2004). There are no studies exploring the long-term outcome of postnatal depression in developing countries. Such longitudinal studies identify the course of depressive disorder and the factors that promote persistence thus helping to formulate effective preventive and treatment strategies (Weich and Araya, 2004). They can also help in targeting limited health resources in developing countries towards those at the greatest risk of poor outcome.\nIn an earlier paper (Rahman et al., 2003a,b), we assessed 632 women in rural Pakistan and found that 160 (25%) of the sample had ICD-10 Depressive disorder. In this paper, we present the results of the one-year follow-up of those mothers who were depressed in the third trimester of pregnancy.\n2\nMethod\n2.1\nStudy area, subjects and sampling\nThe study was carried out in a rural sub-district of Rawalpindi, Pakistan. This is a mainly agrarian low-income rural area about 60\u00a0km south-east from the city of Rawalpindi. All married women aged 17 to 40 in their third trimester of pregnancy (n\u00a0=\u00a0701) were identified from 10 Union Councils (each consisting of 5\u201310 villages; total population 150,000) over a period of 4\u00a0months (Fig. 1). Subjects were identified by obtaining official lists from 120 government-employed Lady Health Workers (LHWs) working in the area, who routinely collect data on new pregnancies. Six-hundred and seventy out of 701 (95%) agreed to take part. Written informed consent was obtained from all subjects after the procedure had been fully explained. Fourteen (2%) were excluded because of a physical illness or complication of pregnancy, 21 (3%) had anxiety disorder and 3 (0.5%) had learning disability and were excluded. Out of the remaining, 160 were diagnosed with ICD-10 Depressive Episode, giving a prevalence rate of depressive disorder in the prenatal period (T1) of 25%. Out of these 160, four had infants born prematurely and were excluded from this study. Two mothers discontinued due to severity of depression. Ten mothers had stillbirths or neonatal deaths, and 1 newborn had a congenital abnormality and were excluded. Fourteen subjects dropped out of the study. Thus, one hundred and twenty-9 mothers were assessed at 3\u00a0months (T2), 6\u00a0months (T3) and 12\u00a0months (T4) postnatal.\n2.2\nData collection\nMental state assessments were carried out at all time points by two trained and experienced clinicians using the Schedules for Clinical Assessment in Neuropsychiatry (SCAN), developed by the World Health Organization as an internationally validated semi-structured interview generating ICD-10 diagnoses of Depressive Disorder (World Health Organization, 1994a). All interviews were carried out after translation, back-translation and cultural adaptation of the interview schedule using an established procedure (Rahman et al., 2003a,b). Interrater reliability was established prior to the study when both interviewers independently assessed 20 women (10 had clinical depression) and agreed on the diagnosis of 19 (\u03ba\u00a0=\u00a00.90).\nPsychological symptoms were assessed at T1 by the same interviewers using the Self-Reporting Questionnaire (SRQ-20) (World Health Organization, 1994b). This consists of twenty items designed to identify psychological symptoms associated with anxiety and depression. Each item has a yes\/no answer. The time span refers to the individual's feelings over the past 30\u00a0days. Each item is scored 0 or 1. The maximum score is therefore 20.\nDisability in mothers was assessed at T1 using the Brief Disability Questionnaire (BDQ) (VonKorff et al., 1996). This is an 8-item questionnaire that rates current problems in carrying out daily activities on a scale of 0 (not at all) to 2 (definitely), with a maximum score of 16. This instrument has been validated in a 15-center cross-national, multilingual study (VonKorff et al., 1996).\nSocio-demographic variables (age, education, employment, family structure and composition) were assessed at T1 by the same interviewers using a specially designed Personal Information Questionnaire (PIQ). Education was categorised into no education versus at least four years primary education. Four years of schooling was chosen as a cut-off because many Pakistani female children attend primary school for four years, after which many children from low-income families stop attending. Family structure was categorised into nuclear family (parents and children only) or extended family (three generations, or one or both parents with married sons, their wives and children).\nSocioeconomic status was assessed at T1 by inquiring if the household was in debt and by asking Lady Health Workers, who lived in the same locality and had intimate knowledge of the families being studied, to rate the household on a 5-point Likert scale ranging from 1 (richest) to 5 (poorest). A single dichotomous variable of \u2018poverty\u2019 was created by combining these 2 measures, i.e., subjects who were both in debt and rated below 3 on the socioeconomic 5-point Likert scale were classified as being poor.\nMaternal financial empowerment within the household was measured at T1 by asking the mothers if they were given a lump-sum amount of money for day-to-day household expenses, and whether they could take independent decisions about its use. Mothers who answered \u2018yes\u2019 to both questions were classified as financially empowered within the household. Social support was assessed by inquiring if the woman received any support during pregnancy from relatives or friends.\nA brief list of life events and difficulties was administered at T1. These were derived from the Life Events and Difficulties Schedule (LEDS) (Brown and Harris, 1989), a semi-structured instrument that measures events and difficulties experienced during the previous year. LEDS has been translated and culturally adapted for use in the study area (Husain et al., 2000). Based on the data from this study, 9 types of events or difficulties that accounted for the majority of the severe events and difficulties reported in that population were used in a modified semi-structured interview. We recorded only those events and difficulties which rated as severe according to the Brown and Harris Rating Scale. In order to do this we discussed the context in which they occurred with the local lady health worker (who lived in the same community and had intimate knowledge of the families being studied).\n2.3\nStatistical analysis\nAll analyses were carried out with STATA, version 7 (StataCorp., 2001) Subjects who were depressed at all 4 time points (persistent cases) were compared with the rest (non-persistent cases). T-test was used to compare psychological symptoms, disability and life event scores. Univariate analyses (relative risk, Fisher's two sided exact p) was performed between potential risk factors and chronic depression. Associations were considered significant at the 5% level. The simultaneous effects of the measured risk and protective factors on persistent depression were analyzed using logistic regression analysis, including all the variables studied in the model (listed in Table 1).\nThe study was approved by the ethics committees of Rawalpindi Medical College, Pakistan and University of Manchester, UK.\n3\nResults\n3.1\nSample characteristics\n129 depressed women completed the one-year follow-up. Their average age was 27.5\u00a0years (SD\u00a0=\u00a05.3). All were married, the average age of marriage being 20\u00a0years (SD\u00a0=\u00a02.8). Forty-four percent were uneducated; only 4% were employed outside the home. Eighty-eight percent of the fathers were employed and about 22% of them were absent from home for 6\u00a0months or more due to employment in the cities. The average reported monthly family income was 2500 rupees (US$42). Sixty-seven percent of the families were rated below 3 on the 5-point socioeconomic scale by the LHWs and 53% of the households were in debt. Sixty-two (48%) were in debt and rated below 3 by the LHWs and were classified as \u2018poor\u2019 in this study.\nNine percent were primigravid, 16% already had one child, 21% two children, 17% three children and the remaining 37% had three or more. Thirty-seven percent lived in nuclear families (parents and children only) while the remaining lived in extended families (three generations, or one or both parents with married sons, their wives and children). Fifty-seven percent delivered at home with a traditional birth attendant, 98% without any reported complication. The gender of newborns was equally distributed.\nOf the 129 women, 121 (94%) were depressed at 3\u00a0months, 98 (76%) at 6\u00a0months and 80 (62%) at 12\u00a0months. Eighty out of 129 (62%) mothers depressed during the third trimester of pregnancy were still depressed at 12\u00a0months postnatally but 7 of these had not been depressed at 6\u00a0months; thus 73 (57%) were depressed at all time points.\nA comparison of the 73 women who had persistent depressive disorder with the remainder on scores of the Self-Reporting Questionnaire, Brief Disability Questionnaire and Life Events Checklist is shown in Table 1. Persistent depression was significantly associated with SRQ score and BDQ score, and weakly associated with Life Events score.\nUnadjusted relative risks with other factors are shown in Tables 2 and 3. Persistent depressive disorder was associated with having an uneducated husband, family size (5 or more children), poverty and lack of a confiding relationship.\nIndependent predictors of persistent depression selected by multiple logistic regression were: high score on the SRQ in the third trimester of pregnancy (odds ratio (OR) 1.3, 95% CI 1.1 to 1.6, p\u00a0<\u00a00.01) and poverty (OR 3.1, 95% CI 1.2\u20138.4, p\u00a0>\u00a00.05) (Table 4).\n4\nDiscussion\nTo our knowledge, this is the first study from the developing world exploring the course of prenatal depression and factors predicting its persistence during the first year of the newborn's life. The study was community based and used standardised and valid instruments to diagnose depression. Depression around childbirth is a serious public health problem in south Asia, affecting about one in four women (Patel et al., 2002; Rahman et al., 2003a,b). Recent studies also provide strong evidence that maternal depression is associated with poor growth in infants living in poor communities in developing countries (Rahman et al., 2004; Patel et al., 2004), and the outcome is worse in infants of mothers with persistent depression. In developed countries, maternal depression is associated with long-term cognitive, emotional and behavioural problems in children, and the impact is worse where the depressive episode is severe or prolonged (Grace et al., 2003; O'Connor et al., 2002). Therefore, prolonged maternal depression has serious consequences not only for the mother but also for infant growth and development.\nThe main findings of this study are that over half of mothers depressed in the third trimester of pregnancy continued to be depressed one year after giving birth. In similar studies from developed countries, depressive levels have been found to decrease steadily over time. In an early prospective study from USA, O'Hara et al. (1984) followed up 99 women from second trimester of pregnancy to 6\u00a0months after giving birth, and found that although almost one half of the subjects had depressive scores that would place them in the mildly depressed range during the second trimester, less than 12% of the subjects were in the mildly depressed range at the 9-week and 6-month follow-ups. In a more recent American study, Campbell and Cohn (1997) followed up 70 women meeting the criteria for clinical depression at 2\u00a0months postnatal and found that at 4\u00a0months postnatal, 48% continued to be depressed; at 6\u00a0months 30% and at 12\u00a0months, 24% continued to meet the criteria for depression.\nBeeghly et al. (2002) followed up 106 women with high depression scores at 2\u00a0months postnatal and found that 35% and 31% continued to have high scores at 6 and 12\u00a0months respectively. Rubertsson et al. (2005) followed up a national cohort of Swedish women and found that out of 333 women with high depression scores in early pregnancy, 79 (24%) continued to have high scores at one year postpartum. A follow-up study of an Australian cohort from early pregnancy till five years after birth suggested that in the majority of women who experienced depressed mood after birth, the symptoms were not severe and did not continue beyond a few weeks (Najman et al., 2000). In our study, prevalence of antenatal depression is high and comparatively fewer women recover in the first year after childbirth.\nThe low rate of recovery could be due to the adverse circumstances experienced by many women in developing countries. Persistent depression was associated with several factors that preceded the birth: poverty, already having 5 or more children, an uneducated husband, and lack of a friend or confidant. Similar factors have been reported in studies from poor communities in developed countries. Horowitz and Goodman (2004) found that women still depressed two years after giving birth were more likely to be poor and have less social support. Bernazzani et al. (1997) found that lower occupational status, lower income, prenatal depression and stressful life events in 12\u00a0months prepregnancy were associated with depression persisting at 6\u00a0months postnatal. Yonkers et al. (2001) found that persistent postnatal depressive symptoms were linked with the presence of other young children at home. In the current study, multivariate analyses suggested poverty to be the major predictor of persistence of depression after severity of depressive symptoms in the prenatal period was adjusted for.\nThe gender of the newborn did not predict persistence. In south Asia, giving birth to a female infant was found to be associated with postnatal depression (Patel et al., 2002), especially in mothers who already had more than two girl-children (Rahman et al., 2003a,b). The preference for male children is deeply rooted in South Asia. Women are often blamed for the birth of girls. However, this study suggests that the effects of having a female child on the mother's mood may only be transient. Similarly, lack of social support during pregnancy (in terms of assistance provided in daily activities) was not associated with persistence while lack of a confidant or friend was. It may be that there are qualitative differences in the type of social support that predicts a worse outcome of depression, and studies using better measures of social support may be required.\nWhile only physically healthy mothers were included in the study, we did not include any physical measures to screen mothers for common physical problems such as anaemia or iodine-deficiency. However, chronic malnutrition in the mothers indicated by a low body-mass index was not significantly associated with persistent depression. Other limitations include a relatively small sample size and the fact that all the women came from one sub-district of Rawalpindi.\nCross-sectional epidemiological studies in Pakistan suggest that the prevalence rate of depressive disorder in women is high even in the non-postnatal period (Husain et al., 2000; Mirza and Jenkins, 2004). Other longitudinal studies would be required to try and understand the origins of depression in these women, which may well pre-date the first pregnancy, or even have its onset in adolescence. However, the postnatal period assumes importance because of the impact of maternal depression on the infant's development. Thus, early identification of mothers whose depression is likely to persist and providing extra support could improve outcomes in both mothers and their infants.\nThe strongest predictor of persistent depression in this study was a high score on the SRQ-20 in the third trimester pregnancy. Recent meta-analyses suggest that psychological symptoms (mainly depression and anxiety) during pregnancy, along with past history of psychiatric illness are primary risk factors for postnatal depression (O'Hara and Swain, 1996; Beck, 2001). The few studies that have explored the long-term outcome of postnatal depression suggest that depressive symptoms during pregnancy (Bernazzani et al., 1997; Verkerk et al., 2003) not only predict early postnatal depression but are also associated with persistence of depression. The utility of the SRQ-20 in predicting persistent depression suggests that this questionnaire, which has been specifically developed for use in primary care in developing countries, could also serve as a useful screening instrument during the antenatal period.","keyphrases":["pakistan","postnatal depression","developing countries","depression women","mental health"],"prmu":["P","P","P","P","R"]}
{"id":"Int_Arch_Occup_Environ_Health-4-1-2175018","title":"Quality of life and illness perception in working and sick-listed chronic RSI patients\n","text":"Objective To study differences between working and sick-listed chronic repetitive strain injury (RSI) patients in the Netherlands with respect to indices of quality of life and illness perception.\nIntroduction\nThe growing global concerns in the 1990s about the effects of work-related upper extremity musculoskeletal disorders (i.e. Repetitive Strain Injuries, or RSI) on the health and well-being of workers, and about the economic and social impact of these conditions, has led to a variety of research on the international level (e.g. Sluiter et al. 2001; Lee et al. 2005; Bongers et al. 2006; Waters et al. 2007), and in the Netherlands governmental actions, new rules, regulations, and professional guidelines were developed (Sluiter et al. 2001; Douwes et al. 2001; NVAB 2003; Blatter et al. 2004). In addition, the epidemiological studies begun in the 1990s that tried to unravel the multifactorial predisposing and precipitating factors of RSI complaints managed to find several significant factors, albeit the perceived relationships were neither very pronounced nor very specific (e.g. Bongers et al. 2006).\nIn contrast to the recently expressed concerns about multidisciplinary curative actions in the US (Feuerstein and Harrington 2006), many preventive ergonomic actions have been introduced in high risk sectors in the Netherlands during the \u201cCovenant\u201d periods. To prevent long-term work disability, occupational physicians have learned to start taking action as early as three weeks after onset of sickness absence for RSI-like complaints. Long-term sickness absence because of RSI-complaints has decreased in the past few years (Bongers et al. 2006). Moreover, when workers\u2019 sickness absence is of very long duration, some employers invest in their workers\u2019 health by paying for intensive multidisciplinary treatments aimed at a return to work (Meijer et al. 2006).\nDespite all these changes, the number of members in, for example, the Dutch RSI Patients\u2019 Association doubled during the last seven years, and prevalences of over 25% for upper extremity complaints are still found; rates are comparable to other European countries (Bongers et al. 2006). All medical professionals deal with patient groups that have medical disorders that may be cared for but not cured with additional costs (Meerding et al. 1998), and also therefore, increasing interest in the patient\u2019s perspectives has grown in the last decade. Perspectives about what are important points of interest to deal with in treatment and knowledge about their quality of life both reveal interesting information for specific focus in treatment (Gafni et al. 1998). Illness perception may be distorted more in work-related disorders compared to other diseases when the ability to return and to keep on working in the job that may have caused the health complaints is questioned. Up to now, only little or even no information was available on chronic RSI patients\u2019 quality of life and illness representation (Picavet and Hoeymans, 2004; Broadbent et al. 2006). Therefore, in 2005 a study that aimed to describe the working vs. sick-listed RSI patients\u2019 perspectives in the Netherlands with respect to their complaints, quality of life, and illness perception was begun in a large group of chronic RSI patients.\nMethods\nPopulation\nPatients with RSI were identified by approaching all 3,250 members of the Dutch RSI patients\u2019 association. The Dutch RSI patient association was started 11\u00a0years ago. In the Netherlands, patients with the same disease are allowed and administratively helped by Governmental Services to join and start an association. Their goal is to gather and spread relevant information to all patients with the same disease in The Netherlands. In addition, their input in sickness-related policy matters is increasingly asked for. Members that were registered in March 2005 were eligible for inclusion. This group defined the study population in this cross-sectional study.\nData collection\nIn April 2005 questionnaires were sent by the secretariat of the RSI patients\u2019 association to the home addresses of all their current members. An accompanying letter was included from the patients\u2019 association. Patients were requested to complete a set of self-report measures. Return envelopes that were addressed directly to the research institute were included as well. One week later, a reminder letter was sent to all members. Data collection was stopped four weeks after sending the reminder. A total of 1,185 (36%) questionnaires were returned by mail.\nOutcome measurements\nQuality of life was assessed in three ways: Through seven subscales of the Dutch version of the 36-item Short Form Health Survey (Ware and Sherbourne 1992; Aronson et al. 1998): physical role functioning, emotional role functioning, social functioning, pain, mental health, vitality, and physical functioning. Scores ranged between 0 and 100 (higher scores indicated better functioning). Visual analogue scales (VAS) (Streiner and Norman 2003) were used to rate the level of present general quality of life with respect to health (0\u00a0=\u00a0the worst imaginable; 10\u00a0=\u00a0the best imaginable) and to rate the level, retrospectively, of the estimated general quality of life with respect to health before the RSI complaints started (0\u00a0=\u00a0the worst imaginable; 10\u00a0=\u00a0the best imaginable). The patients were also asked to report on their current work-ability by means of an 11-point scale (ranging from 0 for the least ability imaginable to the participant\u2019s own maximum level of 10, the highest level imaginable) (e.g. Croon et al. 2005).\nThe brief illness perception questionnaire (IPQ-B) (Broadbent et al. 2006) assessed cognitive illness perception through items on illness \u201cconsequences\u201d(how much does your illness affect your life?), \u201cidentity\u201d (how much do you experience symptoms from your illness?), \u201ctimeline\u201d (how long do you think your illness will continue?), \u201cpersonal control\u201d (how much control do you feel you have over your illness?), \u201ctreatment control\u201d (how much do you think your treatment can help your illness?). It assessed emotional illness perception with items on illness \u201cconcern\u201d (how concerned are you about your illness?) and \u201cemotions\u201d (how much does your illness affect you emotionally? [e.g. does it make you angry, scared, upset, or depressed?]), and it assessed illness understanding with an item on illness \u201ccomprehensibility\u201d (how well do you feel you understand your illness?). Scores ranged from 0 to 10, and averages on the group level were calculated (Broadbent et al. 2006). The last question of the IPQ-B is open ended, centring on the most important factors believed to cause the illness.\nThe demographic, complaint-related, and work activities characteristics were used to describe the two groups. Demographics included gender, age, education, and current work activities. Complaint-related variables were pain and stiffness\/tingling intensity (assessed with VAS scales ranging from 0\u00a0=\u00a0no complaints at all to 10\u00a0=\u00a0the worst complaints imaginable) (Streiner and Norman 2003), pain duration (in years), extent of pain complaints (number of upper body regions affected) (Sluiter et al. 2001), ranging between 0 and 16. Work activity characteristics were assessed by four psychosocial work characteristics subscales: work pressure, social support from colleagues or supervisor, and job control (from the \u201cDutch Questionnaire on the Experience and Assessment of Work\u201d, VBBA) (Van Veldhoven and Meijman 1994; Sluiter et al. 2003). VBBA subscale scores ranged between 0 and 100, with higher scores being more unfavourable.\nIn order to compare the two groups of RSI patients, we classified the \u201cworking\u201d group as those patients that had been present at their work for a minimum of 8\u00a0h during the previous week, and the \u201csick-listed\u201d group were RSI patients with a sickness absence certificate following the Dutch social system (i.e. longer than 1\u00a0year sick-listed, or those that stated that they did not work according to their contract hours during the previous week before they filled in the questionnaire).\nStatistical analysis\nScale scores were calculated according to the original descriptions of the scales used. VAS scores were calculated in mm, ranging from 0 to 100. Means and standard deviations were calculated for every outcome. We divided the respondents in two groups, one group of patients that indicated they worked in a paid job, and one group of patients that indicated they were sick-listed. After testing assumptions, either multivariate ANOVA controlling for age, gender, and education level or nonparametric tests (Mann\u2013Whitney) were performed to test differences between the two groups of RSI patients; P-values\u00a0<\u00a00.01 were considered significant. Average differences over 1 on a 10-point scale and over 10 on a 100-point scale were considered clinically important in terms of effect size (Streiner and Norman 2003).\nResults\nA total of 1,185 respondents responded, and data from 1,121 questionnaires could be used in the analyses for this study. Table\u00a01 provides a description of demographic and both complaint-related and work-related variables for the groups under study.\nTable\u00a01Total study population and working vs. sick-listed groups: gender, education, age, pain intensity, pain duration, work status, and work-abilityTotal group (n\u00a0=\u00a01,121)Working group (n\u00a0=\u00a0745)Sick-listed group (n\u00a0=\u00a0376)Difference between groups: P-valueGender (% females)676278a\u00a0<\u00a00.001Education (% high )6771 60a\u00a0<\u00a00.001Age in years (mean (SD))40.8 (8.7)40.0 (8.3)42.3 (9.4)a\u00a0<\u00a00.001Pain intensity (0\u2013100) (mean (SD))41.3 (25.4)36.2 (24.7)51.6 (23.9)a\u00a0<\u00a00.001Stiffness\/tingling intensity (0\u2013100) (mean (SD))37.8 (26.8)33.7 (25.8)45.7 (27.0)a\u00a0<\u00a00.001Pain duration in years (mean (SD))5.8 (3.2)5.6 (3.1)6.2 (3.3)0.01Pain extent (mean (SD) number of body regions)6.4 (3.9)5.7 (3.6)7.7 (4.1)a\u00a0<\u00a00.001Work pressure (0\u2013100) (mean (sd))b48.4 (21.1)48.7 (20.8)45.9 (23.6)0.885Social support from colleagues (0\u2013100) (mean (SD))b27.8 (20.1)27.4 (20.0)31.5 (20.4)0.157Social support from direct supervisor (0\u2013100) (mean (SD))b35.1 (26.2)34.4 (26.1)42.0 (26.1)0.138aKolmogorov\u2013Smirnov test P-values\u00a0<\u00a00.01bFilled in by n\u00a0=\u00a084 in the sick-listed group\nRSI patients in the sick-listed group were two years older on average, and this group contained a relatively higher proportion of women and lower proportion of highly educated persons. In the sick-listed group, 76% was sick-listed longer than one year.\nIn both groups, complaints were present for several years and extended over several upper body regions. In both groups, over 85% received a diagnostic label, provided by a medical professional, that belong to the umbrella term RSI. With respect to complaint-related variables, the patients in the sick-listed group reported significantly more pain in more regions, and they reported more stiffness\/tingling complaints. The sick-listed group reported significantly lower ability to work, and no differences between groups were found in the psychosocial work characteristics experienced. With respect to co-morbidity, no other diseases were reported by 46% in the sick-listed group compared to 64% in the working group; depression and burnout were reported by 12 and 11% in the sick-listed group versus 6 and 5% in the working group. In the working patient group, 35% filled some kind of administrative function, 11% worked in IT, 10% worked in a physical job, and 10% in a management function. In both groups, almost 80% reported long- or short-term overexertion during work as the only main cause of the onset of their illness.\nExperienced quality of life on the studied SF-36 subscales is shown in Tables\u00a02 and 3. In Table\u00a02, a matrix of zero-order correlation coefficients is shown for the complete population between work-ability, pain intensity, pain duration, the SF-36 subscales, and the eight B-IPQ dimensions. The highest correlation coefficients were found between the two SF-36 subscales mental health and vitality (r\u00a0=\u00a00.75), and the SF-36 subscale pain and VAS scale pain intensity (r\u00a0=\u00a00.72).\nTable\u00a02Matrix of zero-order correlation coefficients between 1: work-ability, 2: pain intensity, 3: pain duration, 4\u201310: SF-36 subscales, and 11\u201318: the B-IPQ outcomes (n varies between 1,027 and 1,114)1234567891011121314151617181 work-ability\u20132 pain intensiy\u22120.48\u20133 pain duration\u22120.080.12\u20134 sf physical role0.54\u22120.45\u22120.02\u20135 sf mental health0.34\u22120.250.030.32\u20136 sf emotional role0.26\u22120.180.050.31\u20137 sf social functioning\u22120.010.05\u22120.01\u22120.07\u22120.010.09\u20138 sf pain0.61\u22120.72\u22120.050.590.310.24\u22120.03\u20139 sf vitality0.40\u22120.300.010.400.750.47\u22120.080.39\u201310 sf physical functioning0.60\u22120.52\u22120.180.480.270.22\u22120.020.610.38\u201311 consequences\u22120.610.510.08\u22120.58\u22120.35\u22120.27\u22120.001\u22120.62\u22120.41\u22120.49\u201312 timeline\u22120.130.180.32\u22120.10\u22120.030.010.01\u22120.15\u22120.07\u22120.160.19\u201313 personal control0.44\u22120.36\u22120.0040.350.330.18\u22120.0010.420.350.34\u22120.36\u22120.06\u201314 treatment control0.29\u22120.19\u22120.090.160.100.050.040.210.150.15\u22120.16\u22120.160.33\u201315 identity\u22120.510.640.07\u22120.54\u22120.29\u22120.230.04\u22120.67\u22120.35\u22120.490.680.23\u22120.33\u22120.10\u201316 concern\u22120.380.46\u22120.03\u22120.46\u22120.35\u22120.270.01\u22120.49\u22120.37\u22120.300.570.16\u22120.36\u22120.100.58\u201317 comprehensibility0.20\u22120.160.040.220.200.14\u22120.030.190.210.12\u22120.210.000.310.22\u22120.16\u22120.29\u201318 emotional response\u22120.380.35\u22120.07\u22120.46\u22120.51\u22120.390.01\u22120.41\u22120.47\u22120.250.590.08\u22120.35\u22120.080.480.64\u22120.27\u2013Table\u00a03Mean (SD) scores by groups on SF-36 subscales physical role functioning, mental health, emotional role functioning, social functioning, pain, vitality, physical functioning, and work-abilityTotal group (n\u00a0=\u00a01,121)Working group (n\u00a0=\u00a0745)Sick-listed group (n\u00a0=\u00a0376)Difference between groups: P-valueSF-36 scales (0\u2013100) (mean (SD))\u00a0Physical role functioning34.7 (38.2)43.4 (39.2)17.0 (29.0)a\u00a0<\u00a00.001\u00a0Mental health68.5 (16.7)70.6 (15.6)64.3 (18.0)a\u00a0<\u00a00.001\u00a0Emotional role functioning73.9 (38.3)78.7 (34.8)64.1 (43.1)a\u00a0<\u00a00.001\u00a0Social functioning54.5 (9.4)54.6 (8.9)54.2 (10.4)0.208\u00a0Pain54.7 (21.6)60.7 (19.2)42.7 (22.0)a\u00a0<\u00a00.001\u00a0Vitality53.6 (17.9)55.7 (17.1)49.3 (18.6)a\u00a0<\u00a00.001\u00a0Physical functioning76.5 (18.0)81.7 (14.6)65.9 (19.6)a\u00a0<\u00a00.001\u00a0Work-ability (0\u201310) (mean (SD))5.4 (2.7)6.6 (1.8)3.1 (2.6)a\u00a0<\u00a00.001aKolmogornov\u2013Smirnov test P-values\u00a0<\u00a00.01\nAs shown in Table\u00a03, the average scores of all scales but social functioning differed significantly between the two groups to the detriment of the sick-listed RSI patients on the other SF-36 subscales. Clinically relevant differences were found in work-ability and in the SF-36 scales for physical role functioning, emotional role functioning, pain, and physical functioning.\nGeneral quality of life with respect to present health was rated as 52.7 on average (SD: 24.4) in the total group of RSI patients, and the average in the working group and sick-listed groups were 57.9 and 42.2, respectively. The group difference was significant (P-value Kolmogornov\u2013Smirnov test\u00a0<\u00a00.001). In contrast, the estimated general quality of life with respect to health before the onset of RSI complaints was rated as 83.1 on average (SD: 15.4) in the total group of RSI patients. There was only a non-significant difference in the average scores between groups (83.1 in the working group and 83 in the sick-listed group (P-value Kolmogornov\u2013Smirnov test\u00a0=\u00a00.796)). Combining these results, the general quality of life with respect to health was subjectively decreased by 37% in the total group over time. However, the difference in decrease was significant (Kolmogornov\u2013Smirnov test, P-value\u00a0<\u00a00.001): only 31% in the working group but 49% in the sick-listed group of patients.\nScores on the eight illness perception dimensions are shown in Table\u00a04.\nTable\u00a04Mean (SD) illness perception scores (B-IPQ) by groupsIllness perception dimensionsTotal group (n\u00a0=\u00a01,121)Working group (n\u00a0=\u00a0745)Sick-listed group (n\u00a0=\u00a0376)Difference between groups: P-valueConsequences6.3 (2.6)5.6 (2.5)7.6 (2.1)a\u00a0<\u00a00.001Timeline8.3 (2.0)8.2 (2.1)8.5 (1.7)0.557Personal control 6.3 (2.0)6.7 (1.8)5.6 (2.1)a\u00a0<\u00a00.001Treatment control 5.2 (2.6)5.7 (2.5)4.4 (2.6)a\u00a0<\u00a00.001Identity6.2 (2.4)5.8 (2.4)7.1 (2.1)a\u00a0<\u00a00.001Concern5.5 (2.6)5.2 (2.6)6.1 (2.6)a\u00a0<\u00a00.001Comprehensibility6.9 (2.1)7.1 (2.0)6.6 (2.3)0.014Emotional response5.4 (2.6)5.1 (2.6)6.0 (2.5)a\u00a0<\u00a00.001aKolmogornov\u2013Smirnov test P-values\u00a0<\u00a00.01\nWith the exception of the dimension timeline and comprehensibility, there was a significant difference in the six other illness perception dimensions between the two groups. Clinically relevant differences between the two groups to the detriment of the sick-listed RSI patients were found for the illness perception dimensions of \u201cconsequences\u201d, \u201cpersonal-\u201d and \u201ctreatment-control\u201d, and \u201cidentity\u201d.\nDiscussion\nThe aim of the present study was to describe working vs. sick-listed RSI patients\u2019 perspectives in the Netherlands in 2005 with respect to their complaints, quality of life, and illness perception. The sick-listed RSI patients reported more severe and extensive complaints in the upper extremity, but the long duration (6\u00a0years) of their complaints was comparable to the working RSI patients. The sick-listed patients, however, experienced a considerably more decreased quality of life because of their complaints, as well as more distorted illness perceptions.\nThe quality of life in sick-listed patients was lower than that of the working patients when the SF-36 subscales scores were taken into account. Interestingly, both groups showed comparable but low social functioning scores (averaging around 55). This was also actually quite low compared to a group of patients with rheumatoid arthritis, who averaged over 70 and compared with a small sample of Dutch workers with RSI who averaged 79 (Sprangers et al. 2000; Picavet and Hoeymans 2004). The experienced quality of life decrease over time was found to be substantial in both groups, which might be explained by the severity, duration, and extent of the reported complaints. It is possible to argue that the anchor question that was used to calculate the quality of life decrease was posed post-hoc. But that was one of the proposed solutions among quality of life researchers in the debate about changed perspectives and response shift: by asking for the two rates at a given time in the form of an existing test, there cannot be any recalibration, re-evaluation of re-prioritization, and re-conceptualization (Osborne et al. 2006; Visser et al. 2005).\nThis may have been the first time that illness representation was assessed in a large group of RSI patients using the recently described brief illness perception questionnaire (IPQ-B) (Broadbent et al. 2006). Illness representation covers the dimensions of illness identity, consequences, cause, timeline, and cure or control. These perceptions about the illness influence patient behaviours, and changing perceptions may improve recovery (e.g. Petrie et al. 2002). If an average difference of over 1 on a 0\u201310 point scale is considered clinically relevant, illness consequences, personal control, treatment control, and identity (i.e. the number of symptoms because of the illness) clearly differed between sick-listed and working RSI patients. We believe that it would be useful to focus future informational treatment strategies in the sick-listed patient group on changing their illness perception.\nWe successfully compared two fairly large groups of chronic RSI patients with respect to their current complaints, quality of life, and illness perception. However, some methodological considerations are in place: (1) the used case-definition to identify sick-listed RSI patients may have influenced the outcomes although it is not clear in what direction this may have caused a bias; (2) because not all variables complied with all assumptions for multivariate testing, that would have allowed for controlling for different possible confounders, non-parametric test outcomes have been presented. Post-hoc multivariate testing did not show any differences in the outcomes as were presented. (3) It is acknowledged that a relatively modest proportion of the total study population responded. Considering the variation in responses and the main purpose of this study, the response should not be considered as a bias. Our population, however, is a selected population in several ways and this has been described in the methods section: participants were members of the RSI patients association, they had long-term complaints, it was not possible to take a random sample in the theoretically existing Dutch group of chronic RSI patients who work or are sick-listed, and the number of responders of the total sample was not as much as would be desirable. However, the diagnostic label that was communicated with over 85% of these chronic patients by different physicians was one of the syndromes that fall under the umbrella diagnosing label of \u201cRSI\u201d (Sluiter et al. 2001). In addition, comparisons between two groups of this specific group of patients were reported upon for this study. Summarizing, besides its possible shortcomings, this study is thought to be unique with respect to studying a large group of chronic RSI patients as most of the published literature on work-related upper extremity musculoskeletal disorders deal with acute and sub-acute populations.\nIt would be interesting to study how the relation between the variables under study developed over time: if the severity of complaints, quality of life level, and illness perception dimensions are relatively stable over time in this subgroup of chronic patients, they could be used as prognostic factors to decide on interventions with respect to future ability to work. It may also be that if activities are increased because of work resumption, they will act as \u201cintervention\u201d in the place-then-train ratio and influence the measures over time. We do know that the differences in the severity of RSI complaints and quality of life indices, to the detriment of the sick-listed group of RSI patients, could be used as an indication for referral to multidisciplinary treatment programmes, because all of these parameters have recently been shown to be influenced positively, even in patients with long-term complaints (Meijer et al. 2006).","keyphrases":["quality of life","illness perception","repetitive strain injuries","work-related upper extremity musculoskeletal disorders"],"prmu":["P","P","P","P"]}
{"id":"Pediatr_Nephrol-3-1-1766479","title":"The management of anemia in pediatric peritoneal dialysis patients\n","text":"Anemia is common in chronic renal failure. Guidelines for the diagnosis and treatment of anemia in adult patients are available. With respect to the diagnosis and treatment in children on peritoneal dialysis, the European Pediatric Peritoneal Dialysis Working Group (EPPWG) has produced guidelines. After a thorough diagnostic work-up, treatment should aim for a target hemoglobin concentration of at least 11 g\/l. This can be accomplished by the administration of erythropoietin and iron preparations. Although there is sufficient evidence to advocate the intraperitoneal administration of erythropoietin, most pediatric nephrologists still apply erythropoietin by the subcutaneous route. Iron should preferably be prescribed as an oral preparation. Sufficient attention has to be paid to the nutritional intake in these children. There is no place for carnitine supplementation in the treatment of anemia in pediatric peritoneal dialysis patients.\nThe treatment of anemia in chronic kidney disease has been summarized in the NKF\/DOQI guidelines [1], which have recently been updated [2]. European best practice guidelines have also been recently published [3]. These published guidelines pay no, or very limited, attention to the special situation in children.\nThe European Pediatric Peritoneal Dialysis Working Group (EPPWG) was established in 1999 by pediatric nephrologists with a major interest in peritoneal dialysis and has, among other things, published guidelines on commencing elective chronic peritoneal dialysis [4]. One of the functions of the group is to establish expert guidance in important clinical areas associated with peritoneal dialysis in conjunction with other members of the multidisciplinary team. These guidelines were initiated and discussed at meetings of the group and developed by e-mail discussion to develop a consensus of opinion based upon cumulative clinical experience and reported studies. The present guidelines apply to the management of anemia in pediatric peritoneal dialysis patients.\nDefinition of anemia\nMore than in adult patients, hemoglobin and hematocrit values are age dependent in children [5], (Tables\u00a01, 2, 3, 4). Also, concentrations of ferritin, transferrin, and iron are dependent on age. Early iron deficiency may be diagnosed by an increase in hypochromic red blood cells [6]. The technique for measuring this parameter is not available everywhere. Since children with renal failure generally do not have underlying co-morbidity contributing to anemia, it is obvious that in children on peritoneal dialysis normal hematocrit levels should be aimed for (opinion). The work-up of the anemic child is not different from the work-up that is advocated for adult patients by the DOQI guidelines, although the importance of a stool test for occult blood may be less useful in this age group [2, 7] (opinion):Iron deficiencyClinical historyAssessment of nutritional statusHemoglobin and hematocritRed blood cell indicesReticulocyte countIron parametersSerum ironTable\u00a01. Reference ranges of anemia parameters in children [5]: hemoglobin and hematocrit (M male; F female)Hemoglobin (g\/dl)Hematocrit (%)1\u20133\u00a0days14.5\u201322.545\u2013702\u00a0months9.0\u201314.028\u2013426\u201312\u00a0years11.5\u201315.535\u20134512\u201318\u00a0years, M13.0\u201316.037\u201349\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0F12.0\u201316.036\u201346Table\u00a02. Reference ranges of anemia parameters in children [5]: ferritinFerritin (ng\/ml)Newborn25\u20132001\u00a0month200\u20136002\u20135\u00a0months50\u20132006\u00a0months\u201315\u00a0years7\u2013140Table\u00a03. Reference ranges of anemia parameters in children [5]: iron (M male; F female)Iron (\u03bcg\/dl)0\u20132 months100\u20132502\u201312 months40\u20131001\u201312\u00a0years50\u2013120Thereafter, M50\u2013160\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0F40\u2013150Table\u00a04. Reference ranges of anemia parameters in children [5]: transferrinTransferrin (mg\/dl)Newborn130\u2013275Adult200\u2013400\nAfter this work-up has been completed, iron and\/or erythropoietin therapy should be initiated to obtain a target hemoglobin concentration of at least 11\u00a0g\/dl (hematocrit 33%), although there is a tendency to increase this target [8] (opinion).\nA target hemoglobin concentration of at least 11\u00a0g\/l should be aimed for in children on peritoneal dialysis (opinion).\nErythropoietin\nMost experience has been obtained with subcutaneous administration of recombinant human erythropoietin [9, 10]. Erythropoietin is available as erythropoietin alpha and beta. Erythropoietin alpha and beta differ noticeably in their formulation excipients. No clear prescription schedules for starting erythropoietin are available for children, and schedules provided in the literature vary greatly. It seems reasonable to start with a subcutaneous dosage of 50\u2013100\u00a0U\/kg body weight 2 or 3 times per week. In some selected patients (particularly if started in the predialysis period) a lower frequency or dosage can be attempted. In studies in adult patients once weekly subcutaneous administration of erythropoietin was effective for the treatment of renal anemia [11, 12, 13]. It is well known that younger children need relatively more erythropoietin than older ones [14] (evidence). Maintenance recommendations vary from 300\u00a0U\/kg\/week for a child with a weight of <20\u00a0kg to 120\u00a0U\/kg\/week for a child with a weight of >30\u00a0kg [10] (opinion). Each child will need the dosage titrated to achieve the target hemoglobin concentrations. For the titration of erythropoietin the DOQI guidelines can be used [2]. If the increase in hematocrit after initiation of erythropoietin therapy is less than 2% over a 2- to 4-week period, the dose should be increased by 50%. If the absolute rate of increase in hematocrit after initiation of erythropoietin therapy or after a dose increase exceeds 8% per month, the weekly dose of erythropoietin should be decreased by 25%.\nSide effects of erythropoietin therapy are rare; increased clotting tendency, hypertension, and seizures are to be considered the consequence of the therapeutic effect rather than an adverse effect of the preparation. Nevertheless, blood pressure should be carefully monitored during therapy.\nRecently, attention was drawn to a severe side effect of erythropoietin therapy, pure red blood cell aplasia due to the occurrence of neutralizing antierythropoietin antibodies [15, 16]. Subsequent investigations showed that this was in most cases associated with subcutaneously administered erythropoietin alpha from one brand, although some cases have also been described using the other brand available on the market. The application of erythropoietin beta is associated with a much lower incidence of this severe side effect. In many countries the subcutaneous administration of erythropoietin alpha is discouraged at present. The administration of darbepoetin alpha is not associated with pure red blood cell aplasia, but it should be remembered that experience with this drug is still limited.\nSubcutaneous administration of drugs is psychologically distressing, especially for children (opinion). The use of ultrafine needles and special injection pens may help to alleviate the upset. In adult peritoneal dialysis patients a noncompliance with erythropoietin administration was reported in between 35% and 55% of patients [17, 18]. For children no data on erythropoietin are available, but from clinical practice it is known that subcutaneous administration is frightening and a source of conflict between child and caregivers. Limited data are available on the compliance with another drug which has to be administered subcutaneously, recombinant human growth hormone. Noncompliance was reported to be between 50% and 91% [19, 20, 21]. In one study it was reported that noncompliance increased significantly from 41% at 1\u00a0year to 91% at 2\u00a0years [21]. Intravenous administration is a more expensive alternative, but will rarely be applied in pediatric peritoneal dialysis patients [22].\nDespite some initial discouraging but still frequently cited reports in the literature, erythropoietin can be administered very well by the intraperitoneal route [23, 24]. If erythropoietin is administered in a small volume of dialysis fluid (50-ml bags are commercially available), bioavailability is similar to that after subcutaneous administration of the same amount of hormone [25] (evidence). Mean dosage needed for maintaining the target hematocrit decreased from 279 to 194\u00a0U\/kg\/week if the drug was administered in a 50-ml dialysis bag during the daytime [26, 27]. In a more recent study in 20 patients on nightly intermittent peritoneal dialysis mean dosage was 179\u00a0U\/kg\/week [28]. Dialysis adequacy is a major factor of concern in such a regimen: KT\/V urea was \u22652.2 in the group studied. However, in some patients the application of intraperitoneal erythropoietin may be limited by the inability to obtain adequate dialysis (opinion). In patients not achieving adequate dialysis, the daytime period should be used for additional dialysis exchanges. In this category, that period will not be available for intraperitoneal erythropoietin therapy. Peritonitis frequency does not need to increase using intraperitoneal erythropoietin: the two pediatric studies reporting peritonitis rates mention one episode every 14.7 and 11.2 treatment months, respectively [19, 21]. One early study in children was broken off because of a high peritonitis rate [29]; possibly this was due to insufficient training of the caregivers.\nRecently, an erythropoietin analogue was developed (darbepoetin alpha or NESP = novel erythropoiesis stimulating protein), which is a hyperglycosylated erythropoiesis-stimulating protein with a presumed threefold longer half-life than erythropoietin in man [30, 31, 32, 33, 34] (evidence). In children a two- to fourfold longer half-life was reported [35]. The pharmacokinetics when administered intravenously and subcutaneously appear to be the same in adult and pediatric patients. A randomized comparative study of darbepoetin and erythropoietin in pediatric patients with chronic or end-stage renal disease is just starting. There are no data available with respect to the intraperitoneal administration of darbepoetin.\nThe development of an orally active agonist of the erythropoietin receptor will be an interesting future feature [36].\nErythropoietin resistance may be due to a number of causes:InfectionHyperparathyroidismMalnutritionHemolytic disordersFolate or vitamin B12 deficiencyUnderdialysisVitamin C deficiencyACE inhibitorsAnti-erythropoietin antibodies\nSince there is an excellent review of these in the adult guidelines [2, 3, 37], and they are not essentially different in children, they are not discussed in detail here. In children responding poorly to erythropoietin therapy, special emphasis should be put on the possible contribution of inflammation or hyperparathyroidism [38]. The possible occurrence of pure red cell aplasia due to the development of neutralizing antierythropoietin antibodies has been discussed before.\nErythropoietin should be administered by the subcutaneous or intraperitoneal route in children on peritoneal dialysis (evidence). Although there is sufficient evidence to advocate the intraperitoneal administration of erythropoietin, most pediatric nephrologists still apply erythropoietin by the subcutaneous route in their peritoneal dialysis patients.\nIron\nIron supplementation is indicated in virtually all pediatric patients with renal anemia who are treated with erythropoietin. According to the DOQI guidelines, transferrin saturation should be maintained above 20% and serum ferritin concentration above 100\u00a0ng\/ml [2] (evidence). There is no reason to anticipate that these guidelines should be different for children and for adults (opinion). It may be difficult to maintain sufficient iron stores in children on peritoneal dialysis, using oral iron preparations only. Iron supplements should be given to prevent iron deficiency and to maintain adequate iron stores. A dosage of 2\u20133\u00a0mg\/kg body weight per day is recommended and administered in two to three divided doses either 1\u00a0or 2\u00a0h after food.\nIron supplements should not be added to the infant formula or nutrition supplements [39] (evidence). If possible, they should be prescribed with vitamin C to enhance absorption (opinion). However, it is important not to oversupplement with vitamin C for risk of increased oxalate formation. Iron supplements should ideally not be taken with cereals and legumes, tannins (tea, cocoa, chocolate) and dairy products as these interfere with absorption. Micronutrient supplements should also be prescribed following individualized dietary assessment and should account for the potential peritoneal dialysis losses of folic acid and vitamins C and B6. Compliance with oral iron preparations for micronutrient supplements may be difficult and must be reinforced by both medical and dietetic staff [39, 40].\nParenteral iron preparations, commonly used in patients on hemodialysis, are more difficult to apply in children on peritoneal dialysis. After the recent approval of iron gluconate, iron sucrose, and iron saccharate, the application of iron dextran should also be abandoned in the United States [41, 42, 43, 44] (evidence). Recommended dosage is 2\u00a0mg iron per kg per week for intravenous iron sucrose treatment [7]. Before starting intravenous therapy the administration of a test dosis is recommended. It is clear, however, that the intravenous administration of iron preparations is cumbersome, and oral administration is preferred [39] (opinion). In rare cases, for example with noncompliance with oral medication, intermittent intravenous administration will be indicated.\nLimited but positive experience has been obtained with the intraperitoneal administration of iron dextran, both in rats and men [45, 46, 47]. Reports on the intraperitoneal administration of iron gluconate or iron sucrose are lacking.\nIron should preferably be prescribed as an oral preparation (evidence). In rare cases intermittent intravenous administration will be indicated.\nCarnitine\nSeveral studies in adult patients suggest that intravenous supplementation with l-carnitine reduces requirements for erythropoietin by 38\u201350% [48, 49, 50] (opinion). Published data on the effect of l-carnitine supplementation on the treatment of anemia in children are very scarce. One study showed an increase in the hematocrit by 34% in two children on hemodialysis with l-carnitine supplementation without modification of erythropoietin dosage [51]. Another study in 16 children on dialysis, of whom five were on peritoneal dialysis, showed no beneficial effect of oral supplementation with l-carnitine on erythropoietin requirement [52].\nThere is no precise place for carnitine supplementation in the treatment of anemia in pediatric peritoneal dialysis patients (opinion).","keyphrases":["peritoneal dialysis","children","erythropoietin","iron","carnitine"],"prmu":["P","P","P","P","P"]}
{"id":"Diabetologia-3-1-2039833","title":"Fatty acid-induced mitochondrial uncoupling in adipocytes as a key protective factor against insulin resistance and beta cell dysfunction: a new concept in the pathogenesis of obesity-associated type 2 diabetes mellitus\n","text":"Type 2 diabetes is associated with excessive food intake and a sedentary lifestyle. Local inflammation of white adipose tissue induces cytokine-mediated insulin resistance of adipocytes. This results in enhanced lipolysis within these cells. The fatty acids that are released into the cytosol can be removed by mitochondrial \u03b2-oxidation. The flux through this pathway is normally limited by the rate of ADP supply, which in turn is determined by the metabolic activity of the adipocyte. It is expected that the latter does not adapt to an increased rate of lipolysis. We propose that elevated fatty acid concentrations in the cytosol of adipocytes induce mitochondrial uncoupling and thereby allow mitochondria to remove much larger amounts of fatty acids. By this, release of fatty acids out of adipocytes into the circulation is prevented. When the rate of fatty acid release into the cytosol exceeds the \u03b2-oxidation capacity, cytosolic fatty acid concentrations increase and induce mitochondrial toxicity. This results in a decrease in \u03b2-oxidation capacity and the entry of fatty acids into the circulation. Unless these released fatty acids are removed by mitochondrial oxidation in active muscles, these fatty acids result in ectopic triacylglycerol deposits, induction of insulin resistance, beta cell damage and diabetes. Thiazolidinediones improve mitochondrial function within adipocytes and may in this way alleviate the burden imposed by the excessive fat accumulation associated with the metabolic syndrome. Thus, the number and activity of mitochondria within adipocytes contribute to the threshold at which fatty acids are released into the circulation, leading to insulin resistance and type 2 diabetes.\nIntroduction\nType 2 diabetes mellitus is generally associated with an enhanced energy intake and too little physical exercise. Also genetic factors determine the susceptibility to develop this disease. Whole-body insulin resistance and a state of low grade inflammation are early marker of the disease process [1]. Subsequently, hyperglycaemia develops due to an accelerated decline in beta cell function [2\u20134]. Type 2 diabetes mellitus is often accompanied by other co-morbidities such as hypertension and dyslipidaemia. Together, they constitute the metabolic syndrome [2].\nHere we present the concept that removal of fatty acids within white adipocytes by fatty acid-induced uncoupled mitochondrial \u03b2-oxidation protects the organism against fatty acid leakage out of adipocytes, thereby preventing fatty acid-induced insulin resistance in liver and muscle and lipotoxicity in pancreatic beta cells. A consequence of this concept is that mitochondrial dysfunction in adipocytes, either inherited or acquired, makes the organism more prone to develop insulin resistance and type 2 diabetes.\nMitochondria and fatty acids\nOne of the main functions of mitochondria, in addition to producing ATP, is to remove fatty acids by \u03b2-oxidation. In this way, mitochondria are able to remove NEFA and to protect the organism against fatty acid-induced insulin resistance and pancreatic beta cell lipotoxicity. \u03b2-Oxidation can take place in all cell types relevant for glucose homeostasis, including muscle, liver and adipocytes. During this process, fatty acids are oxidised in the mitochondrial matrix by NAD+ and flavin adenine dinucleotide (FAD), yielding acetyl coenzyme A, which is further degraded to CO2 by the citric acid cycle. The resulting NADH and FADH2 need to be recycled into NAD+ and FAD by the respiratory chain so that additional fatty acid molecules can be oxidised. This requires a supply of ADP, which is converted into ATP by respiratory chain activity. The rate of conversion of ATP back into ADP, which is determined by the metabolic activity of the cell, determines the rate at which fatty acids can be removed by mitochondrial activity. If the capacity of the cell to oxidise fatty acids is to be enhanced, ATP needs to be reconverted into ADP at an increased rate. This occurs during exercise in contracting muscle. The resulting enhanced rate of ADP generation provides a major additional sink for the oxidation of fatty acids within the body. Only when mitochondria are in the uncoupled state can regeneration of NAD+ and FAD occur without the conversion of ATP into ADP. The released energy is then converted into heat. Uncoupling is a physiological adaptation process, normally regulated by specific proteins [5]. It is remarkable that, like uncoupling proteins, fatty acids are capable of inducing mitochondrial uncoupling, especially when their concentrations exceed the binding capacity of fatty acid binding proteins [6\u20138]. This implies that when unbound fatty acids are present in the cytosol of cells, mitochondria become less efficient at ATP production and generate more heat during the oxidation of NADH and FADH2 derived from food.\nMitochondrial uncoupling in adipocytes by fatty acids may protect the organism against fatty acid-induced insulin resistance and lipotoxicity\nWhite adipocytes contain large amounts of mitochondria in their tiny cytosolic compartment [9, 10]. There is no obvious reason why these cells need such a large capacity to produce ATP. We propose that, when uncoupled by fatty acids, these mitochondria prevent the release of fatty acids out of the adipocytes when the antilipolytic action of insulin is attenuated.\nEvidence is accumulating that inflammation of adipose tissue is an early step in the pathogenesis of type 2 diabetes. The combination of excessive food intake with a sedentary lifestyle results in the formation of large adipocytes overloaded with triacylglycerols. Through as yet unknown mechanisms the adipose tissue becomes inflamed and infiltrated by leucocytes. This leads to the release of inflammatory cytokines such as TNF-\u03b1 [11\u201314], a potent inducer of insulin resistance in adipocytes which also induces lipolysis. As a result, fatty acids are released out of the large triacylglycerol pool [15]. We propose that, initially, a part of these fatty acids are removed inside adipocytes by partially uncoupled mitochondrial \u03b2-oxidation. In doing so, these mitochondria create a threshold for fatty acid release into the circulation, which would otherwise trigger the development of whole-body insulin resistance and pancreatic beta cell lipotoxicity, the latter expected to occur when the body cannot utilise these released fatty acids as fuel, for example, in response to high food intake and little physical exercise. Our proposed mechanism is outlined in Fig.\u00a01. When low amounts of fatty acids are released out of the triacylglycerol pool in insulin-resistant adipocytes, these fatty acids can be removed by coupled \u03b2-oxidation. The rate of fatty acid removal by this pathway is determined by the rate of ADP regeneration within adipocytes, which is likely to be a more or less constant factor, merely determined by cellular metabolism and not affected by variations in the lipolytic rate. We propose that, when the rate of fatty acid release exceeds the rate of fatty acid removal by coupled \u03b2-oxidation, the increase in cytosolic concentrations of fatty acids not bound to binding proteins (unbound) induces a partial uncoupling of the mitochondria. Uncoupling by fatty acids has been shown in multiple in vitro experiments [5\u20138, 16]. Evidence of uncoupling in vivo is provided by a study in which exposure of heart muscle to fatty acids resulted in a decrease in ATP production and a concomitant increase in oxygen consumption [17]. When uncoupled, the mitochondria are able to remove much larger quantities of fatty acids through generation of heat. The degree of uncoupling increases in with further elevations in fatty acid concentration, thus generating more heat. This represents a dynamic adaptation of the efficiency of the mitochondrion, which is determined by the intracellular concentration of fatty acids. If, however, the rate of fatty acid release exceeds the maximum clearance rate, fatty acids may reach concentrations that are toxic to the mitochondrion [18]. At this point \u03b2-oxidation capacity collapses and fatty acids are released out of adipocytes and redistributed over other tissues in the body. These ectopic triacylglycerol deposits are associated with the development of insulin resistance in muscle and liver and lipotoxicity in pancreatic beta cells [19]. At this stage of the disease process physical exercise will, by virtue of the generation of large amounts of ADP in muscle tissue, protect against lipotoxicity and the development of insulin resistance. This is because the muscle switches to \u03b2-oxidation to regenerate ATP when fatty acids reach a concentration sufficient to make the muscle resistant to insulin-stimulated glucose uptake [20, 21].\nFig.\u00a01Consequences of fatty acid release from the adipocyte triacylglycerol pool. In insulin-sensitive adipocytes, fatty acid concentrations are kept low by insulin-induced antilipolytic action, re-esterification of fatty acids and mitochondrial \u03b2-oxidation. TNF-\u03b1 induces insulin resistance and lipolysis. At low cytosolic concentrations of unbound fatty acids, the flux through mitochondrial \u03b2-oxidation is limited by the rate of ADP generation by cellular metabolism. At intermediate concentrations of unbound fatty acids, uncoupling of mitochondria is induced leading to continuous oxidation of fatty acids, independent of ADP supply. This process generates heat and keeps cytosolic fatty acid concentrations low. When the rate of fatty acid release from the triacylglycerol pool exceeds the rate of fatty acid removal, high cytosolic concentrations of unbound fatty acids develop, which induce mitochondrial damage [18]. This results in a decline in the capacity to remove fatty acids and the release of large amounts of fatty acids into the circulation. Unless these are removed by muscle activity they form ectopic triacylglycerol deposits and induce whole-body insulin resistance and beta cell damage. Dotted arrows indicate consequences; continuous arrows, fluxes\nDiscussion\nIn our model for the development of obesity-induced whole-body insulin resistance and beta cell damage leading to type 2 diabetes, we propose a key role for fatty acid removal by adipose tissue mitochondria. The disease process, as outlined in Fig.\u00a02, is initiated by the development of cytokine-induced adipose tissue insulin resistance [11\u201315] and results in the release of fatty acids into the cytosol of the adipocytes. Further increases in cytosolic fatty acids induce mitochondrial dysfunction [18], decreasing the rate of \u03b2-oxidation, augmenting the fatty acid concentration, leading to their release into the circulation. These fatty acids are responsible for the development of insulin resistance in liver and muscle and lipotoxicity in pancreatic beta cells. Fatty acids do not appear to induce insulin resistance in adipocytes [22], which otherwise would create a positive feedback loop whereby the release of fatty acids induces additional insulin resistance and further promotes the release of fatty acids.\nFig.\u00a02Proposed sequence of events leading to the development of hyperglycaemia during the metabolic syndrome. When adipocytes become overloaded with triacylglycerol, low-grade inflammation develops and inflammatory cytokines such as TNF-\u03b1 induce insulin resistance in the adipocytes. This results in an elevated state of lipolysis. When fatty acids are inadequately removed within adipocytes because of mitochondrial dysfunction (for example, induced by fatty acids or HAART therapy, fatty acids appear in the circulation, where they induce insulin resistance of muscle and liver and malfunction of pancreatic beta cells. The elevated circulating fatty acid concentrations may also uncouple mitochondria in artery wall smooth muscle cells, thereby elevating the risk of hypertension [46]. Thiazolidinediones (TZDs) ameliorate the disease process in two ways: (1) by creating more mitochondria in adipose cells [23, 36, 37], thereby enhancing the capacity for oxidation of fatty acids; and (2) by enhanced re-esterification of fatty acids [39, 40]\nIs there any evidence to support our model? Several studies have shown that in adipose tissue from obese animal models and humans, mitochondrial (mt) DNA copy number and expression of mitochondrial genes is decreased [23, 24].\nIn the diabetic state, mitochondrial \u03b2-oxidation of fatty acids was found to be attenuated. In addition, mitochondria showed an abnormal morphology [24]. These data support the concept of mitochondrial dysfunction in adipose tissue in states of obesity and type 2 diabetes.\nFurthermore, intake of high-fat food by healthy persons rapidly increases the basal metabolic rate. This increase is blunted in obese individuals, whereas obese individuals exhibit an increased thermogenesis. These observations are suggestive for a rapid effect of ingested fatty acids on thermogenesis by mitochondrial uncoupling. The data also suggest that, in obese individuals, mitochondria are already uncoupled or damaged and less responsive to further fatty acid-induced uncoupling [25].\nIn vitro studies have shown that fatty acid-induced uncoupling of the mitochondrial respiratory chain depends on the chemical nature of these fatty acids. Unsaturated fatty acids, such as oleic acid, are better uncouplers than saturated fatty acids [8]. This could imply that the inclusion of saturated fatty acids in the diet makes mitochondria less prone to uncoupling, resulting in the earlier release of fatty acids, i.e. at a lower fatty acid level.\nA further implication of our model is that a decline in mitochondrial function in adipocytes reduces the ability of these cells to store triacylglycerol. Inadvertently released fatty acids out of the triacylglycerol pool enter the circulation and are redistributed in other tissues. This occurs, for example, in individuals starting on highly active antiretroviral therapy (HAART) [26\u201329]. The nucleoside analogues included in HAART inhibit mtDNA polymerase and induce a \u223c30\u201350% reduction in mtDNA content in adipocytes and other tissues. HAART is associated with the redistribution of peripheral fat to the central and other compartments and with an elevated risk of developing the metabolic syndrome and type 2 diabetes. We see this clinical phenotype as a result of release of fatty acids by peripheral adipocytes, which in turn is due to a decrease in mitochondrial capacity to remove fatty acids. The fatty acids, after entering the circulation, induce insulin resistance and lipotoxicity to pancreatic beta cells and become stored as ectopic triacylglycerol deposits.\nAnother example of a mitochondrial dysfunction is represented by patients carrying a 3243A>G mutation in mtDNA. This mutation results in an attenuated mitochondrial function. Most of these patients develop the maternally inherited diabetes and deafness (MIDD) syndrome. Remarkably, these patients usually have a BMI of <25\u00a0kg\/m2. Furthermore, they exhibit ectopic triacylglycerol deposits in multiple tissues [30\u201333]. This clinical picture suggests the attenuated storage of triacylglycerol in adipose tissue. Around mid-life, these patients show a decrease in insulin secretion, which may result from lipotoxicity to pancreatic beta cells.\nCongenital lipodystrophy is another clinical example showing that an inadequate storage of triacylglycerol in adipocytes contributes to ectopic triacylglycerol deposits and the development of severe whole-body insulin resistance and diabetes [34].\nIn contrast, improved mitochondrial function specifically in adipocytes is seen in individuals taking thiazolidinediones. These drugs are widely used to ameliorate whole-body insulin resistance in patients suffering from type 2 diabetes mellitus and the metabolic syndrome. These drugs bind to peroxisome proliferator-activated receptor-\u03b3 receptors which are highly expressed in adipocytes and in cells from the immune system, but low in liver and muscle [35]. These drugs increase mitochondrial copy number and mitochondrial gene expression specifically in adipose tissue [23, 36, 37]. Clinically, these drugs improve storage of triacylglycerol in peripheral adipocytes and ameliorate fatty acid-related insulin resistance. Patients taking these drugs tend to store more triacylglycerol in their adipocytes, leading to weight gain [38]. Together, these clinical data suggest that correct mitochondrial function is needed for adequate storage of fatty acids as triacylglycerol in adipocytes. By this means the organism is protected against the fatty acid-induced development of insulin resistance and lipotoxicity to the pancreas.\nAs thiazolidinediones also induce the expression of genes involved in the neogenesis of glycerol, these drugs also enhance the re-esterification of fatty acids [36, 39, 40]. This also contributes to the removal of fatty acids from the circulation and protects adipocyte mitochondria against fatty acid-induced damage. Because part of the glycerol in adipocytes is synthesised through pathways that are dependent upon mitochondrial function [36], this suggests that changes in mitochondrial function also may affect the rate of glycerol production and thereby the rate of fatty acid esterification.\nTo be able to perform, uncoupled \u03b2-oxidation mitochondria require the presence of sufficient amounts of oxygen. It has been suggested that a state of hypoxia occurs in expanding adipose tissue during the development of obesity and that this state may contribute to the development of the metabolic syndrome [41, 42]. Our model predicts that hypoxia in adipocytes would lead to a reduced rate of fatty acid removal inside these cells and to more fatty acid release into the circulation.\nThe sensitivity of an individual to the development of type 2 diabetes as a result of lifestyle is determined by genetic factors. Multiple genetic variants that modulate the risk of an individual for the development of diabetes have been identified, including genetic variants related to mitochondrial function. A high penetrance mutation in mtDNA has been found to be associated with the MIDD syndrome. This mutation predominantly affects the activity of complex I of the respiratory chain [43]. In the same biochemical pathway a mutation in the LARS2 gene, which encodes mitochondrial leucyl-tRNA synthetase 2, also modulates the risk for diabetes [44]. Furthermore, genetically determined changes in expression levels of components of the mitochondrial respiratory chain have been found to be associated with an increased risk of developing type 2 diabetes mellitus [45]. These genetic factors are likely to result in a decreased capacity of the mitochondria to remove fatty acids through uncoupled \u03b2-oxidation. Thereby, a genetic predisposition can lower the threshold for fatty acid release by adipocytes into the circulation and as a result enhance the risk for type 2 diabetes.\nOur model of the pathogenesis of type 2 diabetes mellitus requires experimental verification of several points, and it certainly does not exclude the involvement of additional factors such as the coregulation of whole-body insulin action and insulin secretion through adipokines and the involvement of uncoupling proteins in setting the threshold for fatty acid-induced uncoupling of mitochondria. However, we see the way fatty acids interact with mitochondria in the cytosol of adipocytes as a major initiating event in the disease process leading to the metabolic syndrome and type 2 diabetes mellitus.","keyphrases":["fatty acids","adipocytes","type 2 diabetes mellitus","mitochondria","thiazolidinedione","haart"],"prmu":["P","P","P","P","P","P"]}
{"id":"Sex_Abuse-2-2-1764595","title":"Assessing Sexual Arousal with Adolescent Males Who Have Offended Sexually: Self-Report and Unobtrusively Measured Viewing Time\n","text":"Sexual arousal was assessed using three approaches: the Affinity (Version. 1.0) computerized assessment of unobtrusively measured viewing time (VT), Affinity self-report ratings of sexual attractiveness, and a self-report sexual arousal graphing procedure. Data were collected from 78 males, aged 12\u201318 (M=15.09; SD=1.62), who acknowledged their sexual assaults. The pattern of responses to all three assessment techniques was remarkably similar, with maximal sexual interest demonstrated and reported for adolescent and adult females. Both self-report procedures could significantly distinguish those adolescents who assaulted a child from those who assaulted peers or adults. The self-report procedures could also significantly discriminate those adolescents with male child victims. The Affinity VT approach significantly differentiated those adolescents who assaulted male children from those who assaulted other individuals. No assessment technique could accurately identify those adolescents with exclusively female child victims. Overall, the results suggest that structured, self-report data regarding sexual interests can be useful in the assessment of adolescents who have offended sexually.\nIntroduction\nMuch of the treatment that was provided in the 1980's and 1990's to adolescents who offended sexually was predicated on the notion that deviant sexual interests played a critical role in the sexual assaults. For example, adolescents were often asked to track their sexual interests in violence and\/or prepubescent children for lengthy time periods by completing deviant fantasy logs and charts and to participate in punishment-based procedures, such as covert sensitization, to reduce the strength and frequency of their presumed deviant sexual interests. In the last several years, however, this heightened focus on the role of deviant sexual arousal has shifted considerably; in part because it is now recognized that most adolescents who commit a sexual offense do not display primarily deviant sexual interests. For example, in their analysis of archival penile plethysmograph (PPG) data, Seto, Lalumi\u00e8re, and Blanchard (2000) reported that 25% (or 10 out of 40) of a small sample of adolescents who had offended sexually demonstrated maximal sexual interest in prepubescent children. With an overlapping yet augmented sample, Seto, Murphy, Page, and Ennis (2003) found that 30% (76 out of 253) of adolescents who had offended sexually responded equally or more to child stimuli during PPG assessments. In a study of 136 adolescents who had committed a sexual offense, Worling (2004) found that 36% of the adolescents were rated by clinicians as having sexual interests in prepubescent children and\/or sexual violence. Although deviant sexual interests likely play a role in the etiology and\/or maintenance of adolescent sexual offending for some adolescents, there are likely other factors to consider such as intimacy deficits, antisociality, attitudes supportive of sexual offending, and opportunity, for example.\nMeasurement of Sexual Arousal\nGiven the assumption that individuals will be reluctant to reveal their sexual thoughts and feelings during an interview, some believe that it is necessary to utilize a physiological measure of sexual interest. The most popular technique for measuring sexual arousal with adult males is the PPG. Although it has been argued by some that the PPG can provide valuable information for adult male clients who have offended sexually (Lalumi\u00e8re & Harris, 1998; Seto, 2001), there are some concerns regarding the reliability and validity of PPG data collected from adult males (Konopasky & Konopasky, 2000; Marshall & Fernandez, 2000). There are also several ethical and empirical concerns regarding the use of the PPG with adolescents (Becker & Harris, 2004; Hunter & Lexier, 1998; Worling, 1998). For example, adolescence is a period of emerging sexual development and teenagers are continuously developing and refining their sexual scripts, identities, and preferences. The potential for iatrogenic harm from exposing adolescents to visual and\/or auditory depictions of deviant sexual activities is considerable, yet this has never been examined. Second, there is little compelling evidence regarding the reliability or validity of a PPG procedure with adolescents. Indeed, in studies with adolescents, PPG data are significantly influenced by such variables as the adolescent's age (Kaemingk, Koselka, Becker, & Kaplan, 1995) and history of physical and sexual abuse (Becker, Hunter, Stein, & Kaplan, 1989; Becker, Kaplan, & Tenke, 1992). Furthermore, although PPG data are predictive of subsequent sexual offending for adults (Hanson & Bussi\u00e8re, 1998; Hanson & Morton-Bourgon, 2004), the available research with adolescents indicates that there is no significant relationship between phallometrically-measured sexual deviance and sexual assault recidivism (Gretton, McBride, Hare, O\u2019Shaughnessy, & Kumka, 2001; Gretton et\u00a0al., 2005). Becker et\u00a0al. (1992) also found that a majority of adolescents who denied their sexual offenses provided invalid deviant-arousal data. In a chart-review study by Seto et\u00a0al. (2000), it was found that PPG data were only moderately discriminative for adolescent males who sexually assaulted male victims and that adolescents who offended against female children could not be differentiated from a nonoffending population. Hunter, Becker, and Goodwin (1994) similarly found that only those adolescents with male victims demonstrated significant deviant arousal using the PPG. In their review of the research, Becker and Harris (2004) concluded that the PPG may be useful only for the following adolescent clients: (i) older teenage males, (ii) individuals who acknowledge their sexual assaults, and (iii) those who offend sexually against males.\nGiven the scientific and ethical concerns regarding phallometric assessment, a number of researchers and clinicians have begun to use an alternative physiological measure: unobtrusively measured viewing time (VT). With this procedure, clients are asked to rate the sexual attractiveness of photographs of a variety of models while the response time to provide the ratings is unobtrusively recorded. The assumption underlying this technique is that people will look longer at stimuli that they find sexually attractive relative to stimuli that they find sexually unattractive. In several VT assessment systems that are commercially available, the models in the photographs are clothed and are not displayed in sexual poses. For many clinicians, therefore, the VT assessment addresses some of the ethical concerns raised by the PPG.\nIn studies with nonoffending adults, it has been demonstrated that VT is significantly correlated with self-reported ratings of sexual arousal (e.g., Harris, Rice, Quinsey, & Chaplin, 1996; Lang, Searles, Lauerman, & Adesso, 1980; Quinsey, Ketsetzis, Earls, & Karamanoukian, 1996) and with sexual arousal as measured by the PPG (Harris et\u00a0al., 1996; Quinsey et\u00a0al., 1996). There have also been several investigations of the efficacy of VT with adults who have offended sexually. For example, Harris et\u00a0al. (1996) found that men who sexually assaulted children viewed slides of children longer than they viewed slides of adults. In several studies conducted with the Abel Assessment for Sexual InterestTM (AASI), a VT assessment methodology using photographs of clothed models, Abel and his colleagues have reported encouraging data with respect to the internal consistency and discriminant validity of the AASI with adult males who acknowledged sexual assaults against children (Abel, Jordan, Hand, Holland, & Phipps, 2001; Abel, Lawry, Karlstrom, Osborn, & Gillespie, 1994; Abel, Huffman, Warberg, & Holland, 1998). In one of the few studies of the utility of the AASI conducted outside of the developer's laboratory, Letourneau (2002) found that PPG and AASI responses from 57 adult males were significantly correlated for most stimulus categories, and that both assessment approaches significantly differentiated those participants with male child victims from those with victims from other groups. Furthermore, the AASI VT procedure could identify those men with adolescent female victims. More recently, Gray and Plaud (2005) compared the AASI and the PPG using data collected from 39 men who had offended sexually against a child less than 11 years of age. They found that both procedures significantly identified sexual interest in children.\nThere have been few published studies of the utility of VT with adolescents. In one paper, Smith and Fisher (1999) used Abel's VT system (AASI) with 81 adolescent males. They concluded that this particular approach yielded little convincing evidence with respect to reliability or validity. However, it is important to note that Abel (2000) later disputed many of the results and interpretations presented by the investigators. More recently, Abel et\u00a0al. (2004) evaluated the AASI with data collected from 1,704 males aged 11 to 17. The authors reported that VT for images of children was moderately correlated to the number of child victims (r=.18) and the number of acts of child sexual offending (r=.23). It was also noted that VT for child stimuli could moderately differentiate those adolescents who offended sexually against children from those who offended against peers or adults (AUC=.64).\nDespite the prevailing assumption that self-reported sexual interests from individuals who commit sexual offenses will be necessarily biased and prejudicial, there is growing evidence of the utility of self-reported data. For example, Laws, Hanson, Osborn, and Greenbaum (2000) found that self-reported sexual interests showed more classification accuracy than PPG data with respect to victim gender in a sample of men who offended against children. Laws et\u00a0al. also found that the self-report methodology used (card sort) had excellent levels of internal consistency. In a similar study, Day, Miner, Sturgeon, and Murphy (1989) found that self-report data from a structured questionnaire regarding sexual thoughts, feelings, and behaviors could accurately classify men according to the gender of their child victims.\nTurning to research with adolescents, Seto et\u00a0al. (2000) found that most participants who acknowledged a sexual interest in children were classified as \u201cpedophilic\u201d using the PPG. Furthermore, Seto et\u00a0al. found that those participants who offended sexually against children but who denied sexual interest in children scored significantly lower on the pedophilic index. Daleiden, Kaufman, Hilliker, and O\u2019Neil (1998) found that adolescents who offended sexually disclosed significantly more deviant sexual behaviours relative to both nonsexual offenders and a nonoffending group. Results such as these suggest that individuals may be quite open regarding deviant sexual thoughts, interests, and\/or behaviors.\nMethod\nParticipants\nAfter obtaining informed consent (and parental consent when required), data were collected from 78 males aged 12\u201318 (M=15.09; SD=1.62). Participants were assessed at a residential treatment facility in Minnesota (n=44) or at one of three community-based treatment centers in the Greater Toronto (Ontario, Canada) area (n=34). Females were not specifically excluded; rather the facility in Minnesota provides services only to males, and the 34 consecutive Canadian referrals who chose to participate were male.\nAll participants for this study acknowledged a contact sexual offense. During the course of this investigation, none of the adolescents who completely denied their sexual offenses and who were approached to participate (n=5) volunteered for this study. Sixty-seven percent (52\/78) of the adolescents committed a sexual offense against at least one child (defined as under the age of 12 and 4 or more years younger than that adolescent at the time of the offense); the remainder (33% or 26\/78) of the adolescents committed sexual assaults against peers or adults exclusively (5 of these 26 offended against male peers). With respect to the participants' ethnic origin, 81% (63\/78) were Caucasian, 12% were African American\/Canadian, 5% (4\/78) were native American\/Canadian, and 2% (2\/78) were Hispanic. Information regarding intellectual functioning was available for 52 of the 78 adolescents: 19% (10\/52) of these adolescents had IQ's less than 80, 6% (3\/52) of the adolescents had IQ's above 120, and 75% (39\/52) had an IQ's within the average range (i.e., IQ between 80 and 120). Assessors reported that 50% (39\/78) of the participants disclosed a childhood sexual victimization history and 42% (33\/78) of the adolescents disclosed physical abuse within their families.\nSelf-Report Sexual Arousal Graphs\nTo collect self-reported sexual arousal data, adolescents were asked to complete 2 graphs (1 graph for each gender) on which they rated their sexual arousal for 8 age-based categories: 0\u20133 years, 4\u20136 years, 7\u20139 years, 10\u201312 years, 13\u201315 years, 16\u201318 years, 18\u201324 years, and over 24 years (see Appendix A for self-report arousal graphs and instructions). Adolescents were first asked to rate their level of sexual arousal to the various age groups if there was no physical force or violence involved in the sexual interaction; they were then asked to use a different color of pen and indicate how their sexual arousal would change for each age group when the thought of forced sexual contact was introduced. All ratings were made on a scale from 0 (low) to 10 (high). For all participants, the graph to address sexual arousal to males was completed first, followed by the graph to capture sexual arousal to females. The graphing procedure was completed with the adolescents by a psychologist or social worker during the completion of a comprehensive assessment. There were 7 different clinicians (1 in Minnesota and 6 in Ontario) at the various agencies who assisted the adolescents to complete the graphs.\nAffinity Assessment of Sexual Interest\nThe Affinity procedure is explained in detail elsewhere (Glasgow, Osborne, & Croxen, 2003), and assessors followed the assessment protocol outlined in the Affinity 1.0 manual (Glasgow, 2001). Briefly, the assessment involves the computerized presentation of photographs of 28 males and 28 females in 4 age categories: toddlers, preadolescents, adolescents, and adults. All of the photographs are of clothed individuals, and none of the models are depicted in sexual poses. The 56 images are presented in a fixed, random order, and participants are asked to rate the sexual attractiveness of each image using a mouse pointer. The on-screen self-report rating scale accompanying each photograph includes the anchors \u201cVery unattractive,\u201d \u201cNeutral,\u201d and \u201cVery attractive,\u201d and there are 19 different, unnumbered gradients on the scale. The Affinity computer program registers a self-report rating score for each photograph ranging from 0 to 18, and it records the time taken (in seconds; accurate to \u00b10.02\u00a0s) to provide each attractiveness rating. The later measure is referred to as on-task latency (OTL). Immediately prior to the debriefing procedure where the nature of the Affinity program was explained, participants were asked to rate\u2014on a scale from 1 (Not at all upsetting) to 10 (Very upsetting)\u2014how upsetting they found the experience of rating the Affinity photographs. They were also asked to indicate how enjoyable the experience was using a scale from 1 (Not at all enjoyable) to 10 (Very enjoyable), and participants\u2019 comments regarding the assessment process were recorded by the assessor.\nAverage Sexual Arousal Scores\nAs a result of the fact that the Affinity program uses four age groups (toddler, preadolescent, adolescent, and adult) for each gender, the eight age groups examined using the self-report graphing procedure were collapsed to approximate the same four Affinity groups to facilitate raw-score comparisons. Specifically, self-report sexual arousal graph data were collapsed as follows: ages 0\u20133 and 4\u20136 (toddler); ages 7\u20139 and 10\u201312 (preadolescent); 13\u201315 and 16\u201318 (adolescent); 18\u201324 and 24 and over (adult).\nTo facilitate visual comparisons between the three assessment techniques, raw scores were converted to z scores. The average standardized raw scores for the three assessment approaches are displayed in Fig. 1. The raw data distributions are remarkably similar across approaches, despite the differences in measurement scales and assessment methodology. Specifically, average self-reported sexual arousal ratings and VT scores for female adults and adolescents are significantly higher than the arousal data for all remaining stimulus categories. Furthermore, there was significant overlap in the arousal data provided for males of any age and for female toddlers and preadolescents.Fig. 1Average standardized raw scores for Affinity On-Task Latency (OTL; n=78), Affinity Self-Report (n=78), and Self-Report Sexual Arousal Graph (n=72) procedures across stimulus groups. Bars represent 95% confidence intervals. MTOD: Male Toddler; MPRE: Male Preadolescent; MADO: Male Adolescent; MADU: Male Adult; FTOD: Female Toddler; FPRE: Female Preadolescent; FADO: Female Adolescent; FADU: Female Adult\nInternal Consistency\nInternal consistency estimates (Cronbach's \u03b1) for the 8 Affinity stimulus categories are displayed in Table 1. With the exception of the \u03b1 for OTL measure for the 7 photographs of female adolescents, all internal consistency estimates were above .70 for the OTL values across the age\/gender categories. Note, however, that higher internal consistency estimates were found for all Affinity stimulus categories based on self-report ratings.Table 1Internal consistency estimates for Affinity on-task latency and Affinity self-report ratingsAffinity on-task latency (measuredAffinity self-report ratings (scaleStimulus categoryin seconds) Cronbach's \u03b1from 0\u201318) Cronbach's \u03b1Female toddlers.82.96Female preadolescents.79.95Female adolescents.62.87Female adults.72.94Male toddlers.73.97Male preadolescents.82.97Male adolescents.77.94Male adults.77.94\nInternal consistency estimates (Cronbach's \u03b1) for the self-report sexual arousal graphs were .83 for females (collapsed across the four age groups) and .89 for males (collapsed across the four age groups). For children 12 and under (for both males and females), the internal consistency estimate was .92; for the 4 age groups of both males and females over the age of 12, internal consistency was .77.\nValidity\nThe assumption underlying VT is that, when asked to rate the sexual attractiveness of a particular model, individuals look longer at photographs that they find sexually arousing relative to the time that they spend looking at photographs that they find not sexually arousing. The correlations pertaining to this issue are displayed in Table 2, and it can be seen that for 7 of the 8 stimulus categories, the time spent viewing the slides (Affinity OTL) was significantly correlated with self-report ratings for the same slides (Affinity Self-Report). The average correlation between Affinity OTL and Affinity self-report ratings across all slide categories was r=.31, p < .01. There was, however, a significant, negative correlation between OTL and Affinity Self-Report for the slides of the adult females. In other words, there was a marked tendency for those adolescents who rated the photographs of adult females as sexually attractive to provide their ratings more quickly than those adolescents who found the photographs less sexually attractive. This, of course, is counter to the assumption that forms the premise of the VT approach.Table 2Correlations between Affinity on-task latency, Affinity self-report, and self-report sexual arousal graph proceduresSelf-report sexualStimulus groupAffinity self-reportArousal graphsMale Toddler\u2003Affinity OTL.38**.35**\u2003Affinity self-report.64**Male preadolescent\u2003Affinity OTL.67**.55**\u2003Affinity self-report.79**Male Adolescent\u2003Affinity OTL.61**.46**\u2003Affinity self-report.63**Male adult\u2003Affinity OTL.52*.21*\u2003Affinity self-report.50**Female toddler\u2003Affinity OTL.49**.44**\u2003Affinity self-report.80**Female preadolescent\u2003Affinity OTL.48**.38**\u2003Affinity self-report.73**Female Adolescent\u2003Affinity OTL.24*.16\u2003Affinity self-report.34**Female Adult\u2003Affinity OTL\u2212.26*\u2212.17\u2003Affinity self-report.48**Note. OTL: On-task latency. n=78 for all correlations between Affinity self-report and Affinity OTL. n=72 for all correlations with the Self-report sexual arousal graphs.*p < .05; **p < .01.\nAlthough the Affinity self-report is based on photographs of various models, and the self-report sexual arousal graphs simply require a rating in response to a numeric age group, the correlations between these two measures of sexual arousal were significant for all 8 stimulus categories. The correlations between OTL and the self-report graphs were also significant for 6 of the 8 stimulus categories.\nThe validity of the two Affinity procedures (OTL and self-report) and the self-report graphing procedures was examined by comparing subgroups of adolescents formed on the basis of the age and gender of their victims. First, an Affinity OTL deviance index was calculated for each participant by dividing the highest mean OTL (in seconds) for male or female toddlers or preschoolers by the highest mean for male or female adolescents or adults.1 An Affinity Self-Report deviance index was calculated using the same mathematical procedure: the highest mean Affinity self-report rating for male or female toddlers or preschoolers was divided by the highest mean self-report rating for male or female adolescents or adults. Finally, a deviance index was calculated in the same fashion for the self-report graphing procedure by dividing the highest rating for a male or female child aged 0 to 12 by the highest rating for a male or female aged 13 and over.\nTo examine the discriminative validity of the three assessment procedures, the area under the Receiver Operating Characteristic (ROC) curve (AUC) was calculated for several different between-group comparisons (see Table 3). Both of the self-report procedures could significantly identify those adolescents with single child victims, multiple child victims, or male child victims (whether defined as ever a male child or exclusively male child victims), and there were no significant differences between the two self-report methodologies, all Z\u0394\u2019s \u2264 .03, all p\u2019s \u2264 .05. The Affinity OTL deviance index was only able to discriminate those adolescents with a male child victim from those adolescents with victims from all other age\/gender groups, and neither self-report measure was superior to the Affinity OTL for this discrimination, all Z\u0394\u2019s \u2264 .03, all p\u2019s > .05. Interestingly, none of the deviance indices examined correctly identified those adolescents with female child victims: whether this was defined as if ever a female child victim or exclusively female child victims. It should also be pointed out that of the 30 adolescents who had 2 or more child victims, 23\/30 (or 77%) had at least one male child victim. Only 7\/30, or 23%, had 2 or more child victims that were exclusively female.Table 3Area under the ROC curve (AUC) classification data for three measures of deviant sexual interestSexual assault victim(s)Affinity on-task latency deviance index AUC (n=78)95% CIAffinity self-report deviance index AUC (n=78)95% CISelf-report sexual arousal graph deviance index AUC (n=72)95% CIEver a child victim.61.47\u2013.75.67*.54\u2013.79.66*.53\u2013.792 or more child victims.60.47\u2013.73.73**.61\u2013.86.70**.59\u2013.82Ever a male child victim.69**.57\u2013.81.72**.60\u2013.84.72**.60\u2013.84Only male child victim(s).73**.60\u2013.86.74**.60\u2013.89.76**.64\u2013.88Ever a female child victim.42.30\u2013.55.48.35\u2013.61.45.33\u2013.58Only female child victim(s).43.29\u2013.56.42.28\u2013.56.41.27\u2013.56Note. Affinity On-Task Latency Deviance Index = (highest mean on-task latency for male or female toddlers or preadolescents)\/(highest mean on-task latency for male or female adolescents or adults). Affinity Self-Report Deviance Index = (highest mean self-reported ratings for male or female toddlers or preadolescent)\/(highest mean self-reported ratings for male or female adolescents or adults). Self-Report Sexual Arousal Graph Deviance Index = (highest rating for male and female children aged 0\u20133, 4\u20136, 7\u20139, and 10\u201312)\/(highest rating for males and females aged 13\u201315, 16\u201318, 18\u201324, and 24 and over).*p < .05; **p < .01.Table 4Mean (and SD) deviance indexes for between-group comparisonsComparisonAffinity on-task latency deviance index (n=78)FAffinity self-report deviance index (n=78)FSelf-report sexual arousal graph deviance index (n=72)FEver a child victim\u2003Yes.72 (.20)1.6.17 (.27)2.2.29 (.29)1.79\u2003No.65 (.24).08 (.24).19 (.32)2 or more child victims\u2003Yes.74 (.20)1.6.24 (.31)7.4 **.34 (.25)4.1*\u2003No.67 (.23).08 (.20).19 (.31)Ever a male child victim\u2003Yes.77 (.20)7.1 **.24 (.31)7.4 **.37 (.28)7.1 **\u2003No.65 (.21).08 (.20).18 (.29)Only male child victim(s)\u2003Yes.84 (.20)10.9 **.32 (.37)11.7 **.39 (.29)4.2*\u2003No.66 (.21).09 (.19).20 (.29)Ever a female child victim\u2003Yes.66 (.18)1.9.10 (.16)1.6.24 (.27)0.2\u2003No.73 (.24).18 (.31).27 (.32)Only female child victim(s)\u2003Yes.64 (.19)0.1.08 (.13)0.2.17 (.26)0.2\u2003No.72 (.22).17 (.29).28 (.31)Participant a victim of sexual abuse\u2003Yes.68 (.22)0.4.20 (.03)0.9.24 (.29)0.7\u2003No.72 (.21).31 (.05).27 (.30)Participant a victim of physical abuse\u2003Yes.67 (.23)0.7.13 (.22).07.20 (.25)1.7\u2003No.71 (.21).15 (.28).29 (.32)Location of participant\u2003Minnesota.70 (.22)0.0.23 (.03)0.1.26 (.31)0.2\u2003Greater Toronto area.70 (.22).33 (.07).23 (.28)Note. Affinity On-Task Latency Deviance Index = (highest mean on-task latency for male or female toddlers or preadolescents) \/ (highest mean on-task latency for male or female adolescents or adults). Affinity Self-Report Deviance Index = (highest mean self-reported ratings for male or female toddlers or preadolescent)\/(highest mean self-reported ratings for male or female adolescents or adults). Self-Report Sexual Arousal Graph Deviance Index = (highest rating for male and female children aged 0\u20133, 4\u20136, 7\u20139, and 10\u201312)\/(highest rating for males and females aged 13\u201315, 16\u201318, 18\u201324, and 24 and over).*p < .05; **p < .01.\nGiven that there are few published data using the Affinity assessment system, one-way ANOVAs were also computed for the 3 deviance measures for each of the comparisons described above. The means and standard deviations for these comparisons are presented in Table 4 to facilitate the calculation of effect sizes and comparisons with future investigations. The pattern of group differences is similar to that found with the ROC analyses. Also note in Table 4 that there were no significant differences on any of the deviance measures as a function of sexual or physical victimization history or location of data collection. It should also be pointed out that there was no significant correlation between participant age and the deviance index calculated for Affinity OTL (r=.02), Affinity Self-Report (r=.09), or the self-report sexual arousal graphs (r=\u22121.1), all p\u2019s > .05. Correlations between the three deviance measures and the number of known child victims are displayed in Table 5.Table 5Correlations between deviance indices and number of known child victimsVariableAffinity on-task latency deviance index (n=78)Affinity self-report deviance index (n=78)Self-report sexual arousal graphs deviance index (n=72)Number of child victims.07.23*.14Number of male child victims.33**.50**.34**Number of female child victims\u2212.13\u2212.04\u2212.05Note. Affinity On-Task Latency Deviance Index = (highest mean on-task latency for male or female toddlers or preadolescents)\/(highest mean on-task latency for male or female adolescents or adults). Affinity Self-Report Deviance Index = (highest mean self-reported ratings for male or female toddlers or preadolescent)\/(highest mean self-reported ratings for male or female adolescents or adults). Self-Report Sexual Arousal Graph Deviance Index = (highest rating for male and female children aged 0\u20133, 4\u20136, 7\u20139, and 10\u201312)\/(highest rating for males and females aged 13\u201315, 16\u201318, 18\u201324, and 24 and over).*p < .05; **p < .01.\nCombinatory deviance scores were also computed to determine whether the results from multiple assessment methods could enhance discriminatory power. Specifically, the following combined deviance scores were examined: Affinity OTL + Affinity self-report; Affinity OTL + self-report arousal graph; Affinity self-report + self-report arousal graph; and Affinity OTL + Affinity self-report + self-report arousal graph. For the six between-groups comparisons outlined in Table 3, there was no significant increase in predictive efficiency for any of the combinatory deviance scores, all Z\u0394\u2019s \u2264 .06, all p\u2019s > .05. The highest AUC value obtained for any comparison was 0.79 (95% CI; .66\u2013.91), and this represented the ability to detect multiple child victims using the combination of Affinity self-report plus the self-report sexual arousal graphing procedure.\nGiven the relative novelty of the self-report sexual arousal graphs, a final examination of the validity of this technique involved an analysis of the frequency with which adolescents ever rated their sexual arousal over the \u201c0\u201d line for male or female children aged 0 to 12 years. The data related to this analysis are presented in Table 6, and it can be seen that if an adolescent ever provided a rating over \u201c0\u201d for children aged 0 through 12 years, this was significantly related to the choice of a single child victim, multiple child victims, and male child victims. As with all previous analyses, however, this particular procedure could not discriminate those adolescents with female child victims.Table 6Frequency of self-report sexual arousal graph ratings over \u201c0\u201d for children aged 0 to 12Ever more than \u201c0\u201d sexual arousalNoYesTotalEver a child victim\u2003No16925\u2003Yes153247\u2003Total314172\u03c72 = 6.9*Ever 2 or more child victims\u2003No261945\u2003Yes52227\u2003Total314172\u03c72 = 10.6**Ever a male child victim\u2003No261844\u2003Yes52328\u2003Total314172\u03c72 = 11.87**Only male child victim(s)\u2003No282755\u2003Yes31417\u2003Total314172\u03c72 = 5.86*Ever a female child victim\u2003No192342\u2003Yes121830\u2003Total314172\u03c72 = 0.20Only female child victim(s)\u2003No213253\u2003Yes10919\u2003Total314172\u03c72 = 0.97Note. Ratings were provided on a scale from 0 (low) to 10 (high).*p < .05; **p < .01.\nFinally, participants were asked to rate how upset they were after completing the Affinity rating task. On the scale from 1 (Not at all upsetting) to 10 (Very upsetting), the mean rating was 2.38 (SD=1.88). Only 6 adolescents provided a rating of 5 or higher, and 66% of the participants provided a rating of 1 or 2. Anecdotally, those adolescents who rated the Affinity procedure as somewhat upsetting most often commented that there were too many photographs of young children or that there were too many photographs of males. Participants were also asked to rate how enjoyable they found the Affinity computerized assessment using a scale from 1 (Not at all enjoyable) to 10 (Very enjoyable). The mean rating for this variable was 3.70 (SD=2.10). Those adolescents who found the procedure enjoyable most often commented that it was \u201cinteresting\u201d; those who rated the Affinity assessment as Not at all enjoyable most often mentioned that it was \u201cboring.\u201d\nDiscussion\nAdolescent sexual interest was assessed using three different assessment procedures: the Affinity (version 1.0) VT procedure, the Affinity self-report procedure, and a self-report sexual arousal graphing procedure. Overall, the internal consistency estimates for all three measures were acceptable for most age and gender groups, and all three assessment approaches significantly differentiated those adolescents with male child victims from those adolescents who never offended sexually against a male child. On the other hand, none of the assessment techniques could successfully differentiate adolescents with female child victims from those who offended sexually against other groups.\nThe finding that those adolescent males with male child victims demonstrated the highest interest in prepubescent children\u2014either through VT or self-report\u2014is consistent with results from several studies in which the PPG was used (Becker et\u00a0al., 1989; Hunter et\u00a0al., 1994; Murphy, DiLillo, Haynes, & Steere, 2001; Seto et\u00a0al., 2000). As has been noted by many of these researchers, it is quite likely, therefore, that many adolescents who commit a sexual offense against a male child are motivated, at least in part, by a sexual interest in prepubescent children. Of course, not all adolescents who target male children demonstrate deviant sexual arousal, and other factors, such as opportunity or antisocial attitudes, for example, need to be considered in the etiology and\/or maintenance of their sexual offenses.\nSimilarly, the fact that many participants who offended sexually against female children did not demonstrate or report significant sexual interest in prepubescent children is also consistent with PPG data collected from adolescents (e.g., Seto et\u00a0al., 2000) and with both PPG and VT data collected from adults (e.g., Letourneau, 2002). As has been noted by others, this may be a result of the fact that the various assessment procedures are not yet able to discern such a sexual interest. Alternatively, given the fact that this finding has been observed with both adults and adolescents\u2014using PPG, VT, and self-report\u2014it is possible that there are many males who commit sexual offenses against prepubescent girls for reasons other than deviant sexual interest. As noted with respect to some of those who offend against boys, it could be that factors such as opportunity or abuse-supportive attitudes, for example, are critical in the formation and\/or continuation of offending behaviors.\nThe AUC reported by Abel et\u00a0al. (2004) for the AASI to differentiate those adolescents who ever assaulted a child from those who sexually assaulted a peer or adult was .64. The AUC for the Affinity VT in the present study (AUC=.61) was remarkably similar for the same comparison. As noted clearly by Abel et\u00a0al. (2004), this suggests that VT alone should not be used to classify adolescents who have offended sexually with respect to victim age. Of course, despite the fact that both of the self-report procedures significantly differentiated adolescents with versus without child victims, no one assessment methodology produced an AUC over .74 regardless of the discrimination examined, and no combinatory deviance score produced an AUC over .79. This indicates that there is certainly much room for improvement, and that neither self-report procedure should be used on its own, or in combination, to determine whether or not an adolescent has committed a sexual offense against a person from a specific age\/gender category. Of course, it is also possible that researchers will never find that they can get better discrimination between subgroups on the basis of victim gender and\/or age because of the fact that sexual interests and attitudes are still being formed during adolescence. Although victim gender and\/or age may be proxy markers of sexual interest for some adults who offend sexually, particularly those who offend against boys, it is likely that the choice of victim is less often related to fixed sexual interests for adolescents.\nDespite the encouraging results reported regarding both self-report procedures, it should be stressed that there were no adolescents who completely denied their sexual crimes included in this study. As such, these results should not be generalized to that subgroup. It would be interesting to investigate the utility of both VT and self-report with those who deny their sexual offenses as Becker et\u00a0al. (1992) found that the PPG was not useful with this group as most of these adolescents were classified as \u201cnonresponders.\u201d There were also no female participants included in this study. Given the relative ease with which both Affinity and the self-report graphing procedures can be carried out with both male and female adolescents, it would be beneficial to learn about the psychometric properties of these assessment techniques with female participants. It would also have been ideal to have included a group of adolescents without a history of sexual offending. This would have permitted analyses regarding normative responses for both VT and self-report, and there may have been greater discrimination accuracy for specific subgroups if nonoffending youth were included. Finally, it should also be pointed out that there were only 78 participants in this investigation; therefore, the probability of a Type 1 error should be considered given the number of statistical tests herein.\nAlthough VT was significantly correlated with ratings of sexual interest for the same photographs within most of the stimulus categories, there was a significant negative correlation between VT and sexual attractiveness ratings for photographs of adult females. It is difficult to comment on the uniqueness of this finding, however, as few researchers have published correlations between self-reported ratings and VT scores for separate stimulus categories. Rather, it is more common for researchers to report an average correlation across the various age\/gender categories (r=.31 in the present study). One can quickly see in Fig. 1 that, on average, adolescents viewed photographs of adolescent and adult females for approximately the same amount of time, but that the sexual attractiveness ratings provided for the photographs of the adult females were significantly higher than those ratings given to the photographs of adolescent females. Given that the relationship between VT and self-reported attractiveness ratings were in the expected direction for most stimulus categories, it may be that this result is spurious and sample specific. On the other hand, there may be something about the seven photographs of adult women used in the Affinity program that contributed to this unexpected result. Alternatively, it is possible that some participants provided exaggerated ratings to the slides depicting adult females in an effort to appear more socially desirable. Finally, it is possible that there is an inverted-U-shaped distribution of VT responses such that respondents provide the fastest ratings to categories that they find both highly attractive and highly unattractive. Given that the majority of adolescents in this study found only 2 of the 8 categories of photographs as highly sexually attractive (adolescent and adult females), the fast VT responses to the 6 remaining categories would account for an overall positive correlation between low VT and low attractiveness ratings. In other words, participants provided low average attractiveness ratings after low average viewing times for 75% (42\/56) of the slides. It would be ideal for researchers to examine the possibility of such an inverted-U-shaped distribution in future research with VT technology; particularly given the assumption underlying the technique that there is a linear relationship between viewing times and attractiveness ratings for all age\/gender categories.\nMost of the adolescents found that participating in the Affinity VT assessment was not very upsetting, and many even found the process to be at least somewhat enjoyable. Given the potential intrusiveness of the PPG procedure, it is interesting that researchers have never collected information from adolescents regarding their experience of providing PPG data. Given the current scientific limitations of the PPG with adolescents, and the ethical concerns involved in conducting such an assessment, it is encouraging that at least one VT approach (Affinity 1.0) can produce some useful information regarding sexual interests without significant negative impact. Furthermore, it is important to point out that age at time of assessment, childhood sexual victimization history, and physical abuse history were not significantly related to deviance indices calculated for Affinity VT, Affinity Self-Report, or the self-report graphing procedure. Recall that some researchers have found that PPG data are correlated with these factors.\nThe results of the present study also lend support to the growing evidence that structured, self-report methodologies can provide the assessor with valuable information regarding an adolescent's sexual interests. For example, adolescents with a history of sexual offenses who rated their sexual arousal to children 12 years of age and under over the \u201c0\u201d mark on the self-report arousal graphs were significantly more likely to have offended against a single child victim, multiple child victims, or male child victims. Of course, regardless of the particular assessment methodology chosen, it is always prudent to supplement any sexual-interest data with information from official documentation and with reports from parents and other adults who may be familiar with the adolescent. It is also essential to be mindful of the fact that sexual interests are not necessarily crystallized for many adolescents and, furthermore, that victim choice may not necessarily be a proxy for sexual preferences. Even when deviant sexual interests are identified, however, there is very little research regarding how best to help adolescents in this regard. Although covert sensitization is by far the most popular behavioral treatment for deviant-arousal reduction in programs in the United States for adolescents who have offended sexually (McGrath, Cumming, & Burchard, 2003), there are no published data regarding the efficacy of this approach for adolescents. In addition to the continued refinement of measurement strategies, therefore, it is critical that we examine the impact of treatment and management techniques for those adolescents who evidence deviant sexual interests.","keyphrases":["sexual arousal","adolescents","self-report","viewing time","sexual interests","sexual offending"],"prmu":["P","P","P","P","P","P"]}
{"id":"Eur_Child_Adolesc_Psychiatry-3-1-1914287","title":"Problem behavior in a community sample of 14- and 19-month-old children\n","text":"Few studies have examined the presence, structure, and stability of behavior problems in a community sample of 14- and 19-month-old infants. A questionnaire with items on emotional, attentional, and impulsive behavior and social communication was completed by the parents of 6,491 infants aged 14 months and 1,803 infants aged 19 months. Particularly externalizing behavior problems were reported to present sometimes or often for 50% of more of the sample and could be considered as common. In contrast, social communication problems were reported to be present in less than 10% of the sample. Overall, boys showed more problem behaviors than girls. Principal component analysis at the 14 months data revealed seven factors, which could all be replicated in the 19 months data. Pearson correlations between scores at 14 months and 19 months were highest for oppositional and attention factors (0.68 and 0.63) and lowest for the inhibiton factor (0.38). More than 50% of those scoring in the top 10% for total problem score at 14 months were in the top 10% at 19 months. These results will facilitate the recognition of psychopathology at very early age and the study of its development over time.\nIntroduction\nPsychopathological research has only recently started to focus on the expression of problematic development at a very young age, i.e., younger than 18\u00a0months, and few data are available on the expression of problem behavior in somewhat older children between 1- and 3-year-old [5, 9, 23]. Indeed, the lack of normative data for normal and deviant development in children younger than 18\u00a0months may lead to inadequate early recognition of serious behavior problems [8, 33, 34].\nThe differentiation of normal behavior from problem behavior is particularly challenging in young children [19]. Many symptoms of psychiatric disorders in childhood and adolescence are considered to be relatively normal in preschool-age children. For example, during the preschool years, aggressive, hyperactive, and non-compliance behaviors and separation problems are relatively common and can be viewed as relatively normal within a developmental perspective [6]. Still, these behaviors can be quite stressing for parents and are generally referred to as problem behaviors. Children younger than 18\u00a0months are learning to control themselves, to become attached to people and objects, and to explore, and are only beginning to form stable behavior patterns. Earlier studies reported that problem behavior affected between 7.3% and 24% of 2- and 3-year-old children [5, 12, 14, 18, 20, 22, 24, 26, 27, 31, 32], but there are few data for children younger than this age. One developmental screening study reported that about 10% of infants had emotional and behavioral problems in the first year of life [15], and another study reported that about 6% of the 1-year-old children had high scores on the Difficult Child domain of the Parenting Stress Index [5].\nAn important reason to study behavior at a young age is that it may predict problem behavior at a later age. For example, indices of temperament at 3\u00a0years were found to predict the presence and the type of psychiatric disorder in young adulthood [10], and behavior problems at 2\u00a0years, such as aggression, non-compliance, and difficult temperament, were valuable predictors of lasting problems at a later stage of development [17]. Approximately 50% of the 2- and 3-year-old children with externalizing psychiatric disorders continued to have these problems later in life [6, 21]. Such problems appear to be stable from an age of 18\u00a0months onward [23], but to our knowledge stability has not been evaluated in younger children.\nMore knowledge on the expression and stability of problem behaviors before the age of 18\u00a0months would be advantageous, especially for doctors and nurses working in well baby clinics. In the Netherlands, infants visit well baby clinics according to a fixed scheme. At 14\u00a0months there is a relatively extensive check up. Since this is a moment for vaccination the attendance rate for this check up is high. Therefore, more knowledge on problem behaviors at especially 14-month-old was desirable. The current study was designed to investigate behaviors at this young age and had the following aims.\nThe first aim was to examine the occurrence of problem behaviors or lack of competences at younger age that are considered to be psychopathological in later childhood. A second aim was to examine whether the constructs of problem behaviors and competences usually found in older children are already present at 14 and 19\u00a0months of age. A third aim was to examine the short-term stability of parent-reported problem behavior and lack of competences between age 14 and 19\u00a0months. To achieve these aims, we used a questionnaire with 55 items on mainly externalizing and social communication behaviors.\nMethod\nSampling\nThe sample was a total population birth cohort of children born between August 2000 and August 2001 (N\u00a0=\u00a012,297) in the province of Utrecht in the Netherlands. The province has a population of about 1.117.997 and includes rural and highly urbanized areas. Parents of 14-month-old children received a request from the provincial inoculation center to complete our questionnaire concerning infant problem behavior. This request was accompanied by an information brochure that explained the purpose of this study (at time t1). Parents of 6,491 infants (i.e., 53% of the 12,297 infants eligible) returned the questionnaire and consented to participate. About equal numbers of boys and girls participated. Unfortunately, we were not allowed access to information on non-responders, and so we investigated potential selection bias by comparing the data of responders to demographic data for the province of Utrecht [25]. Responders lived somewhat less often in urban areas (responders 46.6%, province of Utrecht 48.1%) and had a higher educational level, which was defined as parents with at least a high school or college degree (responders sample: mothers 43.4%; fathers 44.2%; province of Utrecht: mothers 38.9%; and fathers 36.0%). Chi-square test for differences confirmed significant differences with respect to urbanization grade (\u03c72\u00a0=\u00a086.437, df 4, P\u00a0<\u00a00.000) and educational level of the mother (\u03c72\u00a0=\u00a0328.56, df 2, P\u00a0<\u00a00.000) and of the father (\u03c72\u00a0=\u00a0313.18, df 2, P\u00a0<\u00a00.000). Further, our sample contained significantly fewer children (responder sample: 5.3%, province of Utrecht: 17.94%) of non-Dutch descent (\u03c72\u00a0=\u00a0695.785, df 5, P\u00a0<\u00a00.000).\nTo parents of children born in June, July, and August 2001 (N\u00a0=\u00a04,051) who had also received the questionnaire at 14\u00a0months and had returned the questionnaire the first time, the questionnaire was sent again (at time t2) when the children were 19\u00a0months of age. This time 1,803 subjects (44%, 919 boys, 889 girls) participated. There were no significant differences in demographic characteristics between the 19-month-old group and the 14-month-old group. For further analyses of the 14- and 19-month-old data, we used the 14-month-old data (N\u00a0=\u00a06,491), and the 19-month-old sample (N\u00a0=\u00a01,803), respectively. When both samples were compared we used the combined sample (N\u00a0=\u00a01,803).\nDescription of participants\nApproximately 88% of the parents were Dutch, 1.3% were Turkish, 1.8% were Moroccan, 1.3% were from the Caribbean (Surinam or the Dutch Antilles), 0.4% were from other Western countries, and 0.5% were of non-Western origin. Questionnaires were completed by mothers (80.5%), fathers (10.7%), and by both (0.6%). Mean child age was 14.80 months (SD\u00a0=\u00a01.84). The mean number of children in the family was 1.80 (SD\u00a0=\u00a01.03). Mean birth weight was 3,537.5\u00a0g (SD\u00a0=\u00a0823.9). Of all infants, 1% had a physical or mental disability, 4.1% had a physical disease, and 6.3% used medication. Parental education level varied: 43.8% had a high level of education, 38.1% had a medium level of education, and 15.5% had a low level of education.\nMeasures\nBehavior problem questionnaire\nWe used a combination of 55 items on a 3-point Likert scale (0 \u2018\u2018never,\u2019\u2019 1 \u2018\u2018sometimes,\u2019\u2019 2 \u2018\u2018often\u2019\u2019) focusing primarily on externalizing behavior, social communication problems, and internalizing items. In selecting items, we first composed a large pool of potentially interesting items concerning children younger than 18\u00a0months by reviewing instruments for older children, namely The Infant-Toddler Social and Emotional Assessment; The Child Behavior Checklist for 1.5\u20135; and the Vineland Social-Emotional Early Childhood Scales [1, 7, 29, 30] and based on our clinical experience with very young children. Then a multidisciplinary panel of experts with clinical and research experience with infants and toddlers made a final selection of items based on two criteria, namely, the item had to be specific for externalizing, social communication or internalizing problem behaviors and to be usable for infants younger than 18\u00a0months. The final selection included items that covered attentional problems, hyperactivity, oppositional behavior, aggressive behavior, verbal and non-verbal aspects of social communication (such as initiating and responding) and internalizing behaviors such as problems with eating or sleeping.\nData analyses\nItems were recoded in such way that a score of two represented the complete presence of an unwanted behavior or the complete absence of a wanted behavior. Common and uncommon problems were identified using the approach of Koot and Verhulst [18]. Common problems were defined as all items that scored 1 (present sometimes) or 2 (present often) for 50% or more of the sample. Uncommon problems were defined as all items that scored 1 or 2 for less than 10% of the sample.\nThe structure of problem behavior was examined by entering all items in a principal component analysis (PCA) with varimax rotation. The number of factors was based on the scree test [13]. Items with a minimum factor loading of \u22650.30 were included [2, 4]. To validate the findings, PCA was performed on two split half samples Pearson correlations were calculated to examine the interrelationship between the factor sumscores. Finally, in order to examine possible two higher-order factors, PCA was performed requiring all items to load on two factors and Pearson correlations were calculated to examine their interrelationship.\nStability was examined parametrically by calculating Pearson correlations between scores at 14 and 19\u00a0months. Further, stability was examined non-parametrically by examining how many children with high scores (above the 75th or 90th percentile) on Total Problem scores and single factor scores at 14\u00a0months still had high scores at 19\u00a0months. ANOVA was performed to assess the effect of sex on the scores of each problem item and for the factor sum scores. Cohen\u2019s d value is reported as an index of effect size (small, d\u00a0=\u00a00.2; medium, d\u00a0=\u00a00.5; or large, d\u00a0>\u00a00.8) [11]. All analyses were two-tailed and a 0.05 level of significance was used. These analyses were performed using the Statistical Package for Social Sciences (SPSS for Windows, version 11.5).\nResults\nFrequencies\nCommon and uncommon problem behaviors and their frequency of occurrence are shown in Table\u00a01. Almost all common problem behaviors were externalizing items, whereas uncommon problems were social communication and internalizing items. Seven of 13 common behavior problems and two of eight uncommon behavior problems were more common among boys than among girls.\nTable\u00a01Common and uncommon problem behavior at 14\u00a0months% With score 1 (sometimes)% With score 2 (often)% With score 1 or 2Common problem behavior\u00a0\u00a0Quickly shifts from one thing to othera61.926.087.9\u00a0\u00a0Angry moodsa63.59.673.1\u00a0\u00a0Demands must be met56.814.170.9\u00a0\u00a0Can\u2019t stand waiting, wants everything now51.116.267.3\u00a0\u00a0Uses toys for fantasy playa35.229.865.0\u00a0\u00a0Wants a lot of attention50.314.464.7\u00a0\u00a0Accident prone56.46.462.8\u00a0\u00a0Stays close to parent54.04.558.5\u00a0\u00a0Easily upset46.37.553.8\u00a0\u00a0Doesn\u2019t sit still during storya39.414.053.4\u00a0\u00a0Won\u2019t share toys or other thingsa46.15.952.0\u00a0\u00a0Doesn\u2019t keep trying46.75.151.8\u00a0\u00a0Can\u2019t sit still, restless, hyperactivea40.011.051.0Uncommon problem behavior\u00a0\u00a0Fails to follow with eyes8.01.49.4\u00a0\u00a0Doesn\u2019t show interest in new objects7.10.67.7\u00a0\u00a0Doesn\u2019t make happy noises6.70.87.5\u00a0\u00a0Doesn\u2019t make noises spontaneously5.81.16.9\u00a0\u00a0Has less fun than others4.51.56.0\u00a0\u00a0Doesn\u2019t react at calling namea5.30.65.9\u00a0\u00a0Seems unhappy without good reason4.90.75.6\u00a0\u00a0Doesn\u2019t imitate simple gesturesa4.30.95.2Note: Common is defined as >50% with score 1 or 2. Uncommon is defined as <10% with score 1 or 2. Please note further that items covering lack of competences were recoded in such way that a score of 2 represented the complete presence of an unwanted behavior or the complete absence of a wanted behavior. This explains why all items have the same-sign factor loadingsa\u00a0Significantly more boys (P\u00a0<\u00a00.001)\nFactor structure of problem behavior\nThe principal component analysis with varimax rotation with eigenvalues >1.5 for two equally sized split half samples resulted in two very similar solutions. The scree test suggested a seven-factor PCA solution (eigenvalue >1.5) with a percentage explained variance of 34.6% (see Appendix to this paper for details). The PCA for the 19-month-old children showed the same factors as the analysis at 14\u00a0months, with 37.7% of explained variance. Five of the 55 items had higher loadings at 19\u00a0months on other factors than at 14\u00a0months.\nPearson correlations between the factor scores (Table\u00a02a) suggested a higher-order division into two problem domains, one reflecting total problem behaviors and one reflecting competences. Oppositional problem behavior at 14\u00a0months of age was mostly related to the factors Attention, Inhibition, and Dysregulation, with r values varying between 0.37 and 0.52 and the factor Language Development was mostly related to the factors Explorative Behavior and Communicative Intent (r values of 0.41 and 0.42, respectively). Indeed, principal component analysis confirmed the underlying structure of the higher-order factors when all items were forced to load on two factors and were named Total Problem Behavior (eigenvalue 6.9, explained variance 11.5%) and Competences (eigenvalue 4.5, explained variance 7.5%). Pearson correlation of these higher order constructs revealed a low correlation (0.12).\nTable\u00a02Pearson correlation matrix of factor sumscores of (a) 14-month-old and (b) 14- and 19-month-oldQuestionnaire domainsOppositional behaviorLanguage developmentAttentionExplorative behaviorCommunicative intentDysregulationInhibition14\u00a0monthsaOppositional behavior1Language development\u22120.0011Attention0.522**0.106**1Explorative behavior0.0050.412**0.167**1Communicative intent0.102**0.422**0.142**0.351**1Dysregulation0.436**0.0220.3150.060**0.124**1Inhibition0.379**0.098**0.235**0.076**0.308**0.308**114\u00a0months19\u00a0monthsbOppositional behavior0.678Language development0.0290.476**Attention0.498**0.112**0.625**Explorative behavior0.0890.264**0.188**0.452**Communicative intent0.057**0.255**0.131**0.206**0.466**Dysregulation0.353**0.069**0.300**0.080**0.127**0.445**Inhibition0.306**0.100**0.247**0.086**0.120**0.226**0.380Note. *Correlation is significant at the 0.05 level (2-tailed)**Correlation is significant at the 0.01 level (2-tailed)\nA significant sex difference (boys had higher scores than girls) was found on several factors, namely Oppositional Behavior (F\u00a0=\u00a015.20, df 1, P\u00a0<\u00a00.000), Language Development (F\u00a0=\u00a0108.88, df 1, P\u00a0<\u00a00.000), Attention (F\u00a0=\u00a022.56, df 1, P\u00a0<\u00a00.000), Explorative Behavior (F\u00a0=\u00a018.05, df 1, P\u00a0<\u00a00.000), and Communicative Intent (F\u00a0=\u00a015.76, df 1, P\u00a0<\u00a00.000). The effect sizes were small (Oppositional Behavior, d\u00a0=\u00a00.09; Language Development, d\u00a0=\u00a00.26; Attention, d\u00a0=\u00a00.11; Communicative Intent, d\u00a0=\u00a00.10). No significant sex differences were found for Dysregulation and Inhibition. At 19\u00a0months, the same sex differences were found, except for Communicative Intent.\nStability over 5\u00a0months\nPearson correlations between the factor sum scores at 14 and 19\u00a0months varied between 0.38 and 0.68, with the highest correlation being found for the Oppositional (0.68) and the Attention factors (0.63) and the lowest for the Inhibition factor (0.38). This indicates moderate stability of the constructs of problem behavior over the 5-month period (See Table\u00a02b).\nThe most stable problems were problems of Communicative Intent, Oppositional Behavior, and Attention (see Table\u00a03). At least half of the group with scores at or above the 75th percentile range at 14-month-old had high scores on problem behaviors at 19\u00a0months. With regard to the Total Problem sum scores, 75.5% of the group with scores at and above the 90th percentile of the Total Problem score at 14\u00a0months still had problem behaviors at 19\u00a0months within the75th percentile range. Also, 53.1% of the children at or above the 90th percentile still had high scores within the highest range 5\u00a0months later. On the other hand, about 88.1% of the children with scores below the 75th percentile at 14\u00a0months remained below the 75th percentile at 19\u00a0months. However, 3.4% of the infants with the least problems on the Total Problem score at 14\u00a0months scored at or above the 90th percentile at 19\u00a0months (see Fig.\u00a01).\nTable\u00a03Mean factor scores at t1 (14\u00a0months) and t2 (19\u00a0months) and percentage of children with scores above 90th and 75th percentile on both 14\u00a0months t1 and 19\u00a0months t2 (N\u00a0=\u00a01,803)Behavioral dimensionMean factor scores at t1 and t2% Of children scoring >90th percentile at both t1 and t2% Of children scoring >75th percentile at both t1 and t2Oppositional behavior0.370.4244.860.3Language development0.470.4431.355.8Explorative behavior0.420.4434.250.3Attention0.420.4245.056.7Inhibition0.640.6529.043.7Communicative intent0.500.9246.851.3Dysregulation0.730.3527.551.1Note. The columns represent the percentage, of those children scoring more than 90th\/75th percentile at t1, who were also above 90th\/75th percentile at t2Fig.\u00a01Transitions (expressed as percentages of children) between 14\u00a0months and 19\u00a0months for the 90th and 75th percentile cutoff of the total problem behavior score\nDiscussion\nRecently, more interest has been paid to the manifestation of early problem behavior in infants and toddlers. As expected, we found externalizing behaviors to be among the most common problem behaviors in 14-month-old infants. Indeed, more than 70% of the parents reported that their children often or very often showed behavioral items such as \u201cquickly shifts activities,\u201d \u201cangry moods,\u201d and \u201cdemands must be met immediately.\u201d These rates are fairly comparable with the prevalence rates reported earlier for somewhat older children [18]. Although these and other common oppositional behaviors are symptoms of DSM-IV axis-I disorders such as Attention Deficit Hyperactivity Disorder, Oppositional Defiant Disorder, and Conduct Disorder at older ages, these high scores for 14-month-old infants are not necessarily psychopathological [6, 18]. Instead, the high occurrence of parent-reported problem behaviors at this early age can best be interpreted as \u201crelatively normal\u201d [18, 23]. While the symptoms and frequency of externalizing problem behaviors change with age, it is of interest to categorize these changes by their severity and to study their developmental trajectories in order to classify expected abnormal outcomes [3]. Further work should therefore focus on longitudinal studies to decide where the cut-off point is and then start to index a risk for psychopathology.\nLack of competences in social communication and internalizing problem behaviors were relatively rare, occurring in about 5\u20139% of the community sample. These results confirm that internalizing symptoms with possible underlying constructs of withdrawal and depression are less prevalent at this early age [4]. This is not to say that these behaviors are unimportant or should be neglected. For example, other social communication items such as \u201cfailure to follow with the eyes,\u201d \u201cdoes not react to calling name,\u201d and \u201cdoes not imitate simple gestures\u201d may indicate the presence of more serious psychopathology, such as autistic spectrum disorder. It would be interesting to determine whether the risk of later psychopathology is significantly higher for a combination of problem behaviors and for which combination this accounts. Also, the frequency or severity of these social communication problems is associated with a serious risk of problems later in development.\nPrincipal component analysis of the data for the 14-month-old children indicated that seven factors could identify important behavioral dimensions. The observation that PCA revealed the same factor solutions for the original as well as for both split half samples is strong evidence for the presence of these distinct behavioral dimensions at 14\u00a0months. While the CBCL 1.5\u20135 [1] can detect externalizing problem behavior in 18-month-old children, our study indicates that these behaviors are present at an even younger age, 14\u00a0months. The two behavioral dimensions with the highest explained variance were dimensions describing problems in oppositional behaviors and language development. The dimension Language Development was correlated with the behavioral dimension Communicative Intent, which covers items of more nonverbal-related behaviors, and with Explorative Behavior. Ever since the work of Piaget, explorative behavior has been considered crucial for cognitive development. Explorative behavior is also an important construct in the field of attachment theory, where the infant is thought to use the attachment figure as a secure base for exploring its environment. Interestingly, the finding of a separate attentional factor refers to the emergence of attentional systems of higher-level control at 12\u201336\u00a0months, which contribute to the development of more goal-oriented behaviors and the ability to inhibit actions [28]. The behavioral dimension termed Inhibition consisted of items consistent with the construct of passive inhibition, namely items focusing on inhibition to novelty, separation distress, and affect-relating internalizing behaviors [16]. Eating and sleeping problems as well as more anxiety-related problem behavior formed a Dysregulation dimension, as found in other studies [4]. Our findings suggest that even at this young age the structures that underlie behavior problems are becoming clearer.\nThese behavioral dimensions are in line with dimensions described in other questionnaires. For example, our Oppositional Behavior dimension includes many items found in the Aggressive behavior factor of the CBCL 1.5\u20135 [1]. Items of the Communication scale of the Vineland are found in our dimension Language Development and Communicative Intent in our analysis. The latter dimension also includes some items of the Socialization scale of the Vineland [29, 30]. Next, our dimension measuring Attention consists of items from Attention dimensions of other scales (the Attention Problems of the CBCL 1.5\u20135 and the Attention scale of the ITSEA) [7]. Also, our dimension Explorative behavior is partly in line with ITSEA\u2019s Mastery motivation and partly with the Socialization scale of the Vineland. Furthermore, our dimension Dysregulation consists mainly of items of the Internalizing dimension of the CBCL 1.5\u20135. Finally, most of the items seen in our dimension Inhibition are found in the Other Problem dimension of the CBCL 1.5\u20135 and some are from the Internalizing and Competence domain of the ITSEA.\nInspection of the factorsumscores correlation matrix suggested two higher-order dimensions, one consisting of problems behaviors (Oppositional behavior, Attention, Dysregulation, and Inhibition) and a second one covering competences (Language Development, Explorative Behavior, and Communicative Intent). This higher order division was confirmed in a factor analysis forcing a two-factor solution. The two higher-order factors Total Problem Behavior and Competences correlated only very weakly with each other (0.12), suggesting a fairly amount of independence. Apparently a relative high level of problem behaviors is not necessarily accompanied by a relative low level of competences and vice versa. This finding is in accordance with studies using the ITSEA in older children. In these studies too, competences emerged as a separate factor from factors on problem behaviors [4, 5, 9]. For the clinical practice our result indicates that even at a very young age, a thorough evaluation of a child\u2019s functioning should include both an investigation of problem behaviors and an evaluation of competences.\nWith regard to sex differences, boys were reported to have significantly more problem behaviors than girls on all factors, except for dysregulation and inhibition, consistent with the finding that boys show more externalizing behaviors [18]. We found no sex differences in dysregulation and inhibition, whereas other studies report girls to be more fearful and to have more sleeping problems than boys [14, 27]. Boys were reported to have more problems in social communication behaviors, which suggests that boys acquire these competencies later than girls. This could play a role in the emergence of problem behavior among boys, whereas the early acquisition of competencies could be a protective factor for girls [4]. Contradictory reports on the presence of sex differences in problem behaviors may also be due to cultural or social factors, such as social role expectancy issues, and these may well complicate the interpretation of results.\nProblems of communicative intent, oppositional problems, and attentional problems had the highest correlations and remained moderately stable over the 5-month period between 14 and 19\u00a0months of age. The least stable were inhibition and dysregulation problems, perhaps reflecting a more temporary nature of these behaviors, as reported earlier by Briggs-Gowan and Carter [4]. At least 53% of the infants with scores within the 90th percentile of each factor sum score at 14\u00a0months still exhibited these problem behaviors at 19\u00a0months of age. These results are in accordance with other studies that have also shown substantial stability in problem behavior [21, 23]. These studies investigated children older than 19\u00a0months of age, whereas our results suggest that these problem behaviors are already moderately stable at an even younger age, emphasizing the need for early assessment. We can only speculate on the determinants of this stability, such as the child\u2019s temperament, family environment, or parental variables. It would be interesting to investigate these determinants in relation to the frequency, severity, and pervasiveness of behavior problems over a longer period of time, for example, at 14, 19, and 36\u00a0months of age, in order to gather information on how problem behavior evolves.\nLimitations\nOne should note that we did not aim to present a new instrument to measure problem behaviors at this young age. Instead, we wanted to report on common and uncommon problem behaviors in this young age group. Although the study population was large and community based, it was not fully representative of the general population. Our sample contained more parents with a higher socioeconomic status than expected compared to the general population. Thus scores for problem behaviors might have been higher in the non-responder group. A second limitation is that we only used parent reports. Information from other sources can be of additional value to evaluate the level of problem behavior [8, 18]. Future work should include measures of impairment of functioning and should focus on more in-depth assessments of psychopathology and level of cognitive development. Finally, it may be informative to compare a population-based sample with a clinically referred group.\nClinical implications\nThis study underlines the growing need to identify problem behavior in early development. A better understanding of what is normal and abnormal in early infant-toddler development is of great value for the recognition of early psychopathology. Normative data for the prevalence and stability of behavior problems of 1- and 2-year-old children in a normal population may make it possible to provide early effective preventive intervention programs, in addition to background information for clinicians and researchers in order to develop such programs.\nIn summary, externalizing behaviors were the most common, and social communication the least common, problems reported in a population sample of 14-month-old children. Boys were reported to show significantly more problems of externalizing behavior and competences. These behaviors were moderately stable over a 5-month period, between the ages of 14 and 19\u00a0months.","keyphrases":["problem behavior","infant development"],"prmu":["P","R"]}
{"id":"J_Med_Internet_Res-9-2-1913940","title":"Language Preferences on Websites and in Google Searches for Human Health and Food Information\n","text":"Background While it is known that the majority of pages on the World Wide Web are in English, little is known about the preferred language of users searching for health information online.\nIntroduction\nThe World Wide Web has more than 15 billion pages [1] and has become an important source of health-related information [2-5]. In the United States, for example, it has been reported that 82% of female users and 77% of male users used the Internet to obtain medical information on a routine basis [6]. Google searches have even been shown to assist physicians in the correct diagnosis of medical ailments [7]. However, two thirds of the pages on the Web are published in English [8,9], even though the world has over 5 billion non-English speakers [10], including approximately 700 million non-English-speaking Internet users [8]. In fact, the vast majority of the world\u2019s 6900 living linguistic groups [10] have little Web content available in their language [8,9]. Adding to this problem, search engines such as Google do not translate search terms into other languages [9]\u2014perhaps a surprise to many users. To overcome linguistic barriers, the World Health Organization (WHO) and the Food and Agricultural Organization (FAO) now publish their websites in six and four major languages, respectively. However, other globally authoritative organizations, such as the Centers for Disease Control (CDC) in the United States, primarily publish online information in only one or two languages of domestic importance.\nIn spite of the significant challenges created by linguistic differences in effectively communicating health information to the world\u2019s peoples online, we could find little quantitative data on this issue. Do the world\u2019s online users, presumably wealthier and more educated than the general population, primarily search online for health information in their local language, or do they employ Web-prevalent languages such as English? Are current online translation efforts by the world\u2019s health and food agencies beneficial, and should these agencies be spending more resources on these efforts? In a world of human migration, which language(s) should domestic governments and international agencies use in order to communicate online health information to target populations? To transmit information to front-line health professionals in developing nations, a group that can include international aid workers from wealthy nations, which language(s) should be employed to target a particular nation? Do indigenous peoples search for online health-related information using search terms belonging to their own language or the colonial language? Real-time, accurate communication of health information might be especially critical during a pandemic infectious disease outbreak or famine. To begin to answer these questions, we have used a case-study approach that examines linguistic preferences in Internet search engine queries.\nSpecifically, we measured search patterns on Google for four health- and food-related terms in seven languages in 227 nations. The four search terms we chose for our study were \u201cavian flu,\u201d \u201ctuberculosis,\u201d \u201cschizophrenia,\u201d and \u201cmaize\u201d (corn). We chose \u201cavian flu\u201d because it is an ideal model for searching for online information concerning an emerging infectious disease pandemic; as of August 2006, avian influenza (virus subtype H5N1) had killed 141 people in 10 countries [11,12] in addition to prompting the slaughter of millions of animals [13]. We chose \u201ctuberculosis\u201d because it is a good model for searching an established global infectious disease, as it is a major cause of death for HIV-positive patients [14] and currently afflicts about 15 million people in 207 nations [15]. We chose \u201cschizophrenia\u201d because psychiatric and neurological diseases affect more than 450 million people globally [16,17] and because online mental health information has the potential to help nations that have few mental health specialists [17]. Schizophrenia afflicts over 24 million people worldwide [18]. Finally, because there are currently around 850 million chronically undernourished people in the world [19], under frequent threat of famine, and because malnutrition is a major underlying contributor to infectious disease susceptibility [20], we chose \u201cmaize\u201d (corn) because it is an important search term for global food security agencies. Maize supplies one third of all human calories in Latin America and Sub-Saharan Africa [19] and, combined with its genetic relatives rice and wheat, supplies approximately 50% of all human calories globally, either directly or via animal feed [19].\nIn this paper, we quantitatively demonstrate the need for health and food website translation that not only targets the world\u2019s major languages, but also linguistic minorities within nations, including immigrants, foreign aid workers, and indigenous groups.\nMethods\nMeasuring the Extent to Which Health and Food Agencies Translate Websites\nWhen an online publisher translates its Web site across languages, the same source domain name can be found in the different result lists of a search engine when entering different translated search terms. In an URL, the domain name is the identifier before the first slash (eg, for the WHO, the domain name is www.who.int). Therefore, to estimate the percentage of institutions, news agencies, and other sources that translate their health and food websites, we measured the frequency that domain names overlap (institutional sources overlap) in different results lists when Google is queried using different language translations of the same search term (eg, English \u201ctuberculosis\u201d versus Bahasa Indonesia \u201ctuberkulosa\u201d). The language translations used in this study are shown in Multimedia Appendix 1. We first extracted URLs from Google.com listed as \u201cthe most relevant\u201d (about 500-1000 URLs) based on the Google page rank algorithm [21] on July 10, 2006. We then excluded nonunique domain names from within each query group; the remaining unique subset was then compared to the URLs retrieved using the comparison language.\nMeasurements of Online Search Rates and Language Choice\nFor each of the four terms and their translations, we measured the rate at which users from 227 different countries searched Google (Multimedia Appendix 2). The country of origin for each search was identified using the geographic locations of Internet Protocol (IP) addresses. The search rates were based on proprietary Google Inc. (Mountain View, CA) data using an algorithm that measured 20% of all first-page Web search queries from January 2004 to April 2006.\nThe search rate, M, for a term, T, originating from a specific country was then estimated from this large sample: the algorithm calculated the ratio of searches using a specific term (T) divided by all searches (A) from that nation during the time period, such that M = T\/A. Multimedia Appendix 2 contains the standard error (SE) for each search rate. To calculate the standard error, a normal distribution for the statistic was assumed. To convert standard error to the 95% confidence interval of the estimate, the following formula was used: 95% CI = M \u00b1 (1.96 \u00d7 SE). Only the search rates for selected languages are shown for each nation, not the total search rate across languages or the total number of searches, which are proprietary.\nFor ease of database searching and validation, we chose languages that share a Latin-based script similar to English. For detailed analysis, the languages chosen were English, Bahasa Indonesia, Spanish, Portuguese, German, and French, as they represent the primary or secondary language of about 100-500 million people worldwide and\/or are significant post-colonial languages [10]. Because Turkey has reported cases of avian influenza [11,12], Turkish was added as a language of interest. A search term may be common to multiple languages, not all of which are noted here. Multimedia Appendix 1 contains a complete list of the language translations used to retrieve search rate data from the Google database.\nHealth and Food Security Indicators\nFor the number of human avian influenza cases (subtype H5N1), we employed the WHO Epidemic and Pandemic Alert and Response Database [12], updated on August 9, 2006. For the number of poultry outbreaks of type H5 since 2003, we used the August 16, 2006 Avian Flu Update from the Organisation Mondiale de la Sant\u00e9 Animale (OiE) [World Organisation for Animal Health] [13]. For tuberculosis (TB), we used Millennium Development Goal (MDG) Indicator 23, the estimated TB prevalence in 2004 from the Global Tuberculosis Database of the WHO Global Health Atlas [15]. The number of psychiatrists per 100000 people in 2005 was obtained from Project Atlas: Resources for Mental Health and Neurological Disorders, from the WHO Global Health Atlas Database [17]. The most recent maize consumption data (kcal\/person\/day in 2004) was from the Supply Utilization Accounts (SUA) Database of the Food and Agricultural Organization Statistical Division (FAOSTAT) [19].\nResults\nMeasuring the Extent to Which Institutions Currently Translate Online Information\nWe measured the extent to which the world\u2019s institutions, including health and food agencies, news organizations, and other sources, currently publish Web content in multiple languages. To quantify this, we measured the institutional source (host-domain) overlap between search results (first 500-1000 URLs) retrieved using English-language queries versus select comparison languages (Table 1). For example, as shown in Table 1, \u201cavian flu\u201d resulted in 906 hits, which included 539 unique hosts. The same search in French resulted in 801 hits, which included 375 hosts. Only 7.1% of the 539 hosts found in the English search were also found in the French search, while 10.1% of the 375 hosts found with the French search were also found in the English search. This means that approximately 7.1%-10.1% of all hosts have an English\/French translation.\nFor \u201cavian flu,\u201d which has afflicted 56 people in Indonesia [12], only 6.8% and 2.4% of institutional domain names overlapped between Bahasa Indonesia searches and English-language searches, respectively (Table 1). For \u201ctuberculosis,\u201d we discovered only about 9% overlap in the institutional domain names retrieved for English and French\/Portuguese translations: French and Portuguese are widely spoken by TB-afflicted nations in Africa and Brazil [15]. The host domain overlap was typically less than 10% between European languages; we were surprised by this low rate given the presence of common governmental institutions in Europe (eg, European Union). We extrapolate that 80%-90% of health- and food-related institutions do not translate their websites into multiple languages, even when the information concerns pandemic disease. This does not exclude the possibility that other agencies, such as domestic health agencies, might be translating this information.\nWe then specifically screened for Web pages belonging to the WHO and the CDC, both authoritative agencies for infectious disease information. Because the first page of search results is the most viewed [22], the rank order in which search results appear is critical. When we searched for \u201cavian flu\u201d in English on Google.com, the CDC website was the first hit, followed second by the WHO website. In the French translation, however, no CDC-affiliated Web pages appeared in the first 100 hits. When we searched Google Indonesia using the Bahasa Indonesia translation of \u201cavian flu,\u201d a WHO-affiliated Web page did not appear until page three of the search results (rank 21), and the first CDC-affiliated page appeared on page six (rank 63). Most significant, by searching for \u201cavian flu\u201d in Turkish using Google Turkey, we were unable to retrieve the websites of either the WHO or CDC in the first 500 search results.\nAs a cautionary note, we found that a single accent or special character in the search term sometimes changed the rank order of health search results significantly, consistent with more detailed analysis conducted for nonmedical terms [9,23]: for example, for avian flu, we found that \u201cgripe avi\u00e1ria\u201d (accent, Portuguese) yielded 49\/100 top search results of Brazilian origin (ie, \u201cbr\u201d in domain name), whereas \u201cgripe aviaria\u201d (no accent, Portuguese) yielded 40\/100 top search results from Spanish-speaking nations and only 4\/100 search results from Brazil. This is significant, because we found that Brazil, Portugal, and other Portuguese-speaking nations (Angola, Mozambique) searched Google for this term with and without the accent at nearly equal rates (Multimedia Appendix 2).\nTable 1\nOverlap in the institutional domain names retrieved by Google to measure the extent to which institutions translate websites across languages\nEnglish\nSearch\nTerm\nComparison\nLanguage\nEnglish Language Search\nComparative Language Search\nURLs\nUnique\nHosts\nHost Overlap with\nComparison Languages\nURLs\nUnique\nHosts\nHost Overlap with\nEnglish\nAvian flu\nFrench\n906\n539\n7.1%\n801\n375\n10.1%\nAvian flu\nIndonesian\n906\n539\n2.4%\n472\n190\n6.8%\nTuberculosis\nFrenchPortugueseDutch\n834\n473\n8.5%\n790\n426\n9.4%\nTuberculosis\nGermanDanishAfrikaans\n834\n473\n0.0%\n809\n443\n0.0%\nTuberculosis\nBahasa Indonesia\n834\n473\n0.8%\n462\n273\n1.5%\nSchizophrenia\nSpanishPortuguese\n813\n444\n10.8%\n764\n518\n9.3%\nSchizophrenia\nBahasa IndonesiaMalay (Bahasa Malayu)\n813\n444\n17.1%\n748\n463\n16.4%\nMaize\nSpanish\n828\n421\n7.8%\n830\n543\n6.1%\nLanguage-Specific Searching of Infectious Disease Information\nThough we did not retrieve many WHO- and CDC-affiliated Web pages when we searched across different languages, one possibility is that Internet users, many of whom are well-educated, are supplementing their Google searches for online health information by searching in English. If true, then there would be less of a need for the WHO and other global agencies to translate online information into diverse languages.\nIn our case study, we found the actual results to be variable (Table 2; Multimedia Appendix 2): for \u201cavian flu,\u201d we often found that only 1% of searches in non-English-speaking nations were in English, whereas for \u201ctuberculosis\u201d or \u201cschizophrenia,\u201d about 4%-40% of searches in non-English countries employed English. Brazil, which had an estimated 141000 cases of TB in 2004 [15], had an 18-fold higher query rate for \u201ctuberculose\u201d (Portuguese) than \u201ctuberculosis\u201d (English). However, the comparison is more important for languages that have much less Web content, such as Bahasa Indonesia and Turkish. Indonesia, which had the highest number of reported cases of human H5N1 viral infections in 2006 [12], had a 15-fold higher query rate for \u201cavian flu\u201d in Bahasa Indonesia (\u201cflu burung\u201d) than in English. We found that Turks used Turkish to search Google for health terms at 3- to over 1000-fold higher rates than English, French, German, or Spanish (Table 2; Multimedia Appendix 2). We did, however, find sites in Turkish that had translated information from the WHO. Whether or not the WHO, CDC, and FAO wish to leave it up to others to translate their information accurately and rapidly must be decided based on their confidence level of the eHealth capabilities of each target nation. Given the Google language-based search patterns, we conclude that during times of infectious disease outbreaks, though English may be useful, global agency\u2013affiliated Web pages translated into local languages would likely be highly accessed and would have the benefits of being viewed as authoritative and accurate and of being transmitted in real time.\nLanguage of Online Mental Health Information Searches\nIn terms of mental health, many developing nations have 10- to 100-fold fewer psychiatrists per capita than many developed nations [17] (Table 2). For this reason, global accessibility of online mental health information has the potential to be very beneficial to physicians and families of patients in developing nations [24]. Given ethnic taboos [24], we first asked whether or not Internet users from developing nations are searching online for mental health information\u2014potentially useful information for global mental health experts. Indeed, we found that the search rate for \u201cschizophrenia\u201d was similar between developed and developing nations in the local language, demonstrating an active need for online mental health information in poor countries. We have made the full mental health dataset available (Multimedia Appendix 2). Given that Google estimates 3-fold more search results concerning schizophrenia in English versus the next 10 languages combined (data not shown), we asked whether middle- and low-income nations searched for this topic in English. We found that people from Brazil, a nation of about 180 million people, searched for \u201cschizophrenia\u201d in Portuguese at a 28-fold higher rate than in English (Table 2). \nTable 2\nGoogle search rates for selected health terms in local languages relative to English*\nSearch Term\nCountry\nPublic Health Comparison Metric\nLocal Language\nEnglish Searches (% of Local Language Searches)\nAvian flu\nHuman Cases\u2020 (Poultry Outbreaks)\u2021\nUnited States\n0 (0)\nEnglish\n100.0\nFrance\n0 (1)\nFrench\n1.6\nGermany\n0 (1)\nGerman\n0.8\nTurkey\n12 (176)\nTurkish\n0.9\nDemocratic Republic of Congo\n0 (0)\nFrench\n1.1\nCote d\u2019Ivoire\n0 (3)\nFrench\n1.4\nBurkina Faso\n0 (4)\nFrench\n100.0\nMozambique\n0 (0)\nPortuguese\n143.8\nMexico\n0 (0)\nSpanish\n3.3\nBrazil\n0 (0)\nPortuguese\n1.0\nIndonesia\n56 (211)\nBahasa Indonesia\n6.5\nTuberculosis\nTB Cases\n\u00a7\nUnited States\n10510\nEnglish\n100.0\nFrance\n5901\nFrench\n9.8\nGermany\n5243\nGerman\n14.3\nTurkey\n32371\nTurkish\n29.1\nDemocratic Republic of Congo\n307554\nFrench\n23.6\nCote d\u2019Ivoire \n116349\nFrench\n8.6\nBurkina Faso\n46815\nFrench\n15.9\nMozambique\n123360\nPortuguese\n28.4\nMexico\n45710\nSpanish\nsame term\nBrazil\n141115\nPortuguese\n5.6\nIndonesia\n605759\nBahasa Indonesia\n1779.3\nSchizophrenia\nPsychiatrists per 100000|| People\nUnited States\n13.70\nEnglish\n100.0\nFrance\n22.00\nFrench\n6.6\nTurkey\n1.00\nTurkish\n10.5\nDemocratic Republic of Congo\n0.04\nFrench\n43.0\nCote d\u2019Ivoire \n0.20\nFrench\n7.1\nBurkina Faso\n0.05\nFrench\n9.5\nMozambique\n0.04\nPortuguese\n24.4\nMexico\n2.70\nSpanish\n4.1\nBrazil\n4.80\nPortuguese\n3.6\nIndonesia\n0.21\nBahasa Indonesia\n115.4\n*Based on sampling 20% of all searches on Google.com from January 2004 to April 2006.\n\u2020Subtype H5N1, from the WHO Epidemic and Pandemic Alert and Response Database [12], updated August 9, 2006.\n\u2021Type H5 outbreaks since 2003, from the OiE [13], updated August 16, 2006.\n\u00a7Estimated TB prevalence in 2004 from the WHO Global Tuberculosis Database [15].\n||2005 data from Project Atlas: Resources for Mental Health and Neurological Disorders, from the WHO Global Health Atlas Database [17].\nIn contrast, people from Indonesia, a nation of 220 million people, searched for \u201cschizophrenia\u201d at similar rates in Bahasa Indonesia as in English. As with infectious disease searching, our data would suggest that users from most nations tend to search Google for \u201cschizophrenia\u201d in their official language at up to 10-fold higher rates than other languages (Multimedia Appendix 2). Many of the world\u2019s people might therefore benefit if the world\u2019s most authoritative mental health agencies (eg, US National Institute of Mental Health) translated information into other languages, even though this is not part of their domestic mandate.\nThe Online Search Rates of Immigrant Minorities\nIn some developed nations, there is concern that immigrant groups might spread infectious diseases. In Europe, the TB prevalence is 43\/100000 people in Turkey but lower in wealthier nations such as Germany (6\/100000); in Asia, the TB prevalence is 273\/100000 people in Indonesia, compared to 48\/100000 in Singapore [15] (Table 2). Unlike many of their neighbors, both Turkey and Indonesia have reported human cases of avian influenza [12]. Governments may be interested to know whether their immigrant communities consult infectious disease\u2013related websites originating from their adopted country or their native country. As a case study, we examined the search rates of Turks and Indonesians after they migrated to other nations in Europe or Asia, respectively, as determined by searches in Turkish and Bahasa Indonesia originating outside Turkey and Indonesia. The Turkish and Indonesian languages are distinct relative to many of their surrounding nations, permitting us to discern the search behavior of these populations after they have emigrated, provided they search in their native language. As shown in Table 3, we detected searches in Turkish for \u201cavian flu\u201d and \u201ctuberculosis\u201d throughout Europe, including high rates in Belgium and Austria, respectively. In Asia, we could detect searches in Bahasa Indonesia for \u201ctuberculosis\u201d and very high rates for \u201cavian flu\u201d throughout Asia and the Pacific region, including Hong Kong, Singapore, and Australia. We cannot exclude that these (presumed) Turkish and Indonesian immigrants also searched online in their adopted language(s), nor could we measure the fraction of their searches in their native language versus adopted language(s). However, based on the fraction of a country\u2019s population that belongs to a particular immigrant group versus the fraction of searches conducted in the immigrant language compared to the adopted language, then if every person in a country searched the Internet for the same term at the same rate, but in different languages, we could then extrapolate that Turkish immigrants in Belgium searched for \u201cavian flu,\u201d \u201cschizophrenia,\u201d and \u201ctuberculosis\u201d 100%, 40%, and 15% of the time, respectively, in Turkish rather than French (Table 3). Using the same simplistic assumptions, in Austria, 39% of searches for \u201ctuberculosis\u201d by Turkish immigrants were in Turkish rather than German. In reality, immigrant groups are likely searching for a term such as \u201cavian flu\u201d at a higher rate than the general population when the corresponding disease affects their homeland and is in the news. However, we also found high search rates for \u201ctuberculosis\u201d and even \u201cschizophrenia\u201d in Turkish in these nations, which are less featured in the news. We conclude that it is important for health officials to be aware that if they wish to disseminate health information to susceptible immigrant groups, they should not rely on websites published in the majority language(s) of the nation. High priority domestic health-related websites should be multilingual, particularly those that concern infectious disease.\nOur analysis also revealed surprises: for example, in Bangladesh, one of the world\u2019s poorest and most populated nations, with 136 million residents, we found that the search rate for \u201cavian flu\u201d in Bahasa Indonesia (\u201cflu burung\u201d) was equivalent to the search rate in English (Table 3); the Bahasa Indonesia translation of \u201cschizophrenia\u201d was also high in Bangladesh. The high Bahasa Indonesia search rate may reflect the fact that many Bangladeshi citizens work in Singapore and Malaysia, nations that speak a similar language, Malay (Bahasa Malayu) [25]; it would appear that when they return to Bangladesh, Bangladeshis continue to use the terminology they learned while away, but the reason is unclear. We suggest that international health organizations aiding Bangladesh should publish or meta tag health information in Malay (Bahasa Malayu) to reach health practitioners in that nation. This result also highlights the practical value of analyzing linguistic preferences during online searching.\nFinally, we were surprised to find that online searches for \u201cavian flu,\u201d \u201cschizophrenia,\u201d and \u201cmaize\/corn\u201d in Spanish in the United States occurred at less than 1% of the English search rate (Table 3). The US online Hispanic population (legal and illegal) is estimated to be 12% of the total US online population, of which nearly half use Spanish for some (28%) or all (21%) of their Internet usage [8,26]. Therefore, our data suggest that Latin American immigrant groups in the United States search for health information to a lesser degree in Spanish than might be predicted, although these data could also be a result of the digital divide between the groups.\nTable 3\nOnline search rates of immigrant minorities\nSearch Term\nCountry\nImmigrant Language\nComparative Major Language* of Adopted Country\nImmigrant Language Searches per 10000 Major Language Searches\nRelevant Immigrants per 10000 Total Population\u2020\nTurkish immigrants in Europe\nAvian flu\nBelgium\nTurkish\nFrench\n56.1\n39.7\u2021\nSwitzerland\nTurkish\nGerman\n9.2\nN\/A\nUnited Kingdom\nTurkish\nEnglish\n31.2\nN\/A\nTuberculosis\nAustria\nTurkish\nGerman\n60.2\n155.4\u00a7\nBelgium\nTurkish\nFrench\n5.8\n39.7\u2021\nGermany\nTurkish\nGerman\n11.8\n212.2||\nSwitzerland\nTurkish\nGerman\n10.7\nN\/A\nUnited Kingdom\nTurkish\nEnglish\n2.8\nN\/A\nSchizophrenia\nBelgium\nTurkish\nFrench\n15.9\n39.7\u2021\nSwitzerland\nTurkish\nFrench\n62.2\nN\/A\nUnited Kingdom\nTurkish\nEnglish\n2.9\nN\/A\nIndonesian\/Malaysian immigrants in Asia\/Pacific\nAvian flu\nAustralia\nBahasa Indonesia\nEnglish\n234.7\n625.2\u00b6\nBangladesh\nBahasa Indonesia\nEnglish\n10474.0\nN\/A\nHong Kong\nBahasa Indonesia\nEnglish\n3424.4\nN\/A\nIndia\nBahasa Indonesia\nEnglish\n154.4\nN\/A\nSingapore\nBahasa Indonesia\nEnglish\n2268.0\nN\/A\nTuberculosis\nHong Kong\nBahasa Indonesia\nEnglish\n23.3\nN\/A\nSingapore\nBahasa Indonesia\nEnglish\n17.0\nN\/A\nSchizophrenia\nAustralia\nBahasa Indonesia\nEnglish\n6.0\n625.2\u00b6\nBangladesh\nBahasa Indonesia\nEnglish\n687.4\nN\/A\nHong Kong\nBahasa Indonesia\nEnglish\n295.1\nN\/A\nIndia\nBahasa Indonesia\nEnglish\n16.0\nN\/A\nSpanish immigrants in English-Speaking North America#\nAvian flu\nUnites States\nSpanish\nEnglish\n102.8\n> 503.0**\nSchizophrenia\nUnites States\nSpanishPortuguese\nEnglish\n91.8\n> 503.0**\nCorn (maize)\u2021\u2021\nUnites States\nSpanishIndigenous (\u201cchoclo\u201d\/\u201delote\u201d)\nEnglish\n37.9\n> 503.0**\n*a major language for which Google search rates were accessible, not necessarily the largest linguistic group of the nation.\n\u2020Total population data are from United Nations Population Division, 2005 data. URL: http:\/\/www.un.org\/esa\/population\/unpop.htm.\n\u2021Data are from Institut National De Statistique, 2003 data. Population et M\u00e9nages: Mouvement de la population et migrations, \"Immigrations ext\u00e9rieures par nationalit\u00e9 et groupe d'\u00e2ges \u2013 Belgique.\" URL: http:\/\/www.statbel.fgov.be.\n\u00a7Data are from Statistics Austria, Volksz\u00e4hlung. Hauptergebnisse I \u2013 \u00d6sterreich, 2001 census data. URL: statistik.at\/neuerscheinungen\/vzaustria.shtml.\n||Data are from Statistisches Bundesamt (Federal Statistical Office, Germany), 2006 data. URL: http:\/\/www.destatis.de\/themen\/e\/thm_bevoelk.htm.\n\u00b6Data are from Australia Bureau of Statistics, Cultural and Language Diversity, 2001 Census data. URL: http:\/\/www.abs.gov.au\/.\n#\u201cTuberculosis\u201d is not included as it is the same term in both English and Spanish.\n**Data are from Department of Homeland Security Yearbook of Immigration Statistics. Legal immigrants, 2005 data. URL: uscis.gov\/graphics\/shared\/aboutus\/statistics\/ybpage.htm.\n\u2021\u2021The search term used was \u201ccorn.\u201d\nTable 4\nSearch rates in European languages in Sub-Saharan Africa\nCountry\nColonial\nLanguage*\nMinority Language\nMinority Language Searches per 10000 Colonial Language Searches\nSearch Term: Avian Flu\nAngola\nPortuguese\nEnglish\n6629\nCameroon\nFrench\nGerman\n323\nGhana\nEnglish\nFrench\n2024\nGhana\nEnglish\nGerman\n1744\nGhana\nEnglish\nDutch\n498\nKenya\nEnglish\nFrench\n2941\nMozambique\nPortuguese\nEnglish\n14286\nMozambique\nPortuguese\nFrench\n4444\nNigeria\nEnglish\nFrench\n4235\nNigeria\nEnglish\nGerman\n897\nRwanda\nFrench\nEnglish\n1481\nSenegal\nFrench\nEnglish\n732\nSearch Term: Tuberculosis\nAngola\nPortuguese French\nEnglishSpanish\n1504\nCameroon\nFrench Portuguese\nEnglish\n2405\nDemocratic Republic of Congo\nFrench Portuguese\nEnglishSpanish\n2364\nGhana\nEnglish\nFrenchPortuguese\n836\nKenya\nEnglish\nFrenchPortuguese\n1128\nMozambique\nPortuguese French\nEnglishSpanish\n2842\nNigeria\nEnglish\nFrenchPortuguese\n506\nRwanda\nFrench Portuguese\nEnglishSpanish\n2808\nSouth Africa\nEnglish\nAfrikaans\n1547\nSearch Term: Schizophrenia\nAngola\nPortuguese Spanish\nEnglish\n1547\nCameroon\nFrench\nEnglish\n1922\nDemocratic Republic of Congo\nFrench\nEnglish\n4304\nGhana\nEnglish\nFrench\n488\nMozambique\nPortuguese Spanish\nEnglish\n2441\nRwanda\nFrench\nEnglish\n18453\nSenegal\nFrench\nEnglish\n3287\n*When two languages are noted, the term is the same in both languages.\nSearch Rates in European Languages in Sub-Saharan Africa\nSub-Saharan Africa suffers from high rates of infectious disease, including TB and HIV [18], and high rates of malnutrition [19]. As demonstrated in Table 4, our search results suggest that the dissemination of online health or food security information to this region by international agencies should not be limited to the colonial language(s) of the target nations. For example, in English-speaking Ghana, 20% of searches for avian flu were in French (\u201cgrippe aviaire\u201d), 17% in German (\u201cvogelgrippe\u201d), and 5% in Dutch (\u201cvogelgriep\u201d) relative to English (Table 4). In Mozambique, a former Portuguese colony, we found 1.4 times more searches for \u201cavian flu\u201d in English relative to Portuguese, and a high rate in French. In the Democratic Republic of Congo (DRC), a former French colony with more than 300000 TB infections in 2004 [15], 24% of the searches for tuberculosis were in English relative to French. Because Internet use is only 1.9% is Ghana, 0.7% in Mozambique, and 0.2% in DRC [8], it is plausible that these high rates of non-colonial language searches using Google may reflect searches by health professionals trained in other nations, including workers from international agencies who would be expected to have better Internet access than the general population.\nThe Effect of Region-Specific Cultural and Indigenous Terminology\nFinally, we measured the effects of cultural bias within the same linguistic group, using a term important for human nutrition and food security, \u201cmaize.\u201d Maize is eaten directly, but it is also a major source of animal feed worldwide. Maize is known as \u201ccorn\u201d in the United States and the United Kingdom, but as \u201cmaize\u201d in many other English-speaking nations. We found that the US searches for \u201ccorn\u201d were at a 28-fold higher rate than for \u201cmaize,\u201d while other English-speaking nations such as Nigeria and Zimbabwe queried \u201cmaize\u201d at a 1.5- to 4-fold higher rate than \u201ccorn,\u201d respectively (Table 5); in the latter nations, \u201ccorn\u201d may also refer to any large cereal grain (eg, wheat). This search behavior has consequences, as we found only three domain names that overlapped between searches for \u201cmaize\u201d versus \u201ccorn\u201d out of the first 50 unique Google search results.\nTherefore, when African nations attempt to retrieve information about growing maize in English, they may unknowingly be excluding authoritative information from the United States and other Western English-speaking nations, such as practical information from the US National Corn Growers\u2019 Association, whose website does not appear in the first 100 Google hits for \u201cmaize,\u201d but ranks sixth when \u201ccorn\u201d is searched. Similarly, when international organizations such as the FAO wish to transmit knowledge, for example, using Spanish or Portuguese in Latin America, indigenous terminology usage may make some of this information inaccessible. Maize originated from Southern Mexico and fed indigenous Latin American civilizations [27]. In Mexico and Guatemala, we found that an indigenous synonym for maize, \u201celote,\u201d was searched at a similar rate to the Spanish term, \u201cma\u00edz\u201d (Table 5). In Peru, however, we found that a different indigenous term, \u201cchoclo,\u201d was searched at a 2-fold higher rate than \u201cma\u00edz.\u201d We conclude that cultural and indigenous linguistic divisions may be preventing large numbers of food security and nutrition websites from reaching those people aiding 800 malnourished people or 1.2 billion agricultural workers that live in developing countries [19]. Cultural and indigenous bias may be particularly prevalent for terms related to crops, diseases, or pathogens that have pre-colonial origins.\nTable 5\nThe effect of region-specific cultural and indigenous terminology\nCountry\nCalories from Maize (kcal\/person\/day)*\nSearch Term Comparison\nSearch Rate Ratio\nCanada\nN\/A\u2020\ncorn:maize\n36:1\nUnited States\n512\ncorn:maize\n28:1\nUnited Kingdom\n115\ncorn:maize\n7:1\nIndia\n38\ncorn:maize\n2:1\nNigeria\n179\ncorn:maize\n1:2\nKenya\n775\ncorn:maize\n1:3\nTanzania\n646\ncorn:maize\n1:4\nZimbabwe\n720\ncorn:maize\n1:4\nSpain\nN\/A\u2020\nma\u00edz:elote:choclo\n10:1:4\nVenezuela\n467\nma\u00edz:elote:choclo\n16:1:3\nColombia\n312\nma\u00edz:elote:choclo\n20:1:5\nMexico\n1081\nma\u00edz:elote:choclo\n14:16:1\nGuatemala\n869\nma\u00edz:elote:choclo\n12:10:1\nPeru\n145\nma\u00edz:elote:choclo\n11:1:25\nArgentina\n132\nma\u00edz:elote:choclo\n41:1:51\n*Data from FAOSTAT [19].\n\u2020N\/A: Data not available.\nDiscussion\nPrincipal Results\nIn a world where infectious disease pandemics and threats of famine are always present, and in spite of the fact that the World Wide Web offers great hope for rapid and accurate sharing of information between peoples, we have demonstrated that one linguistic group does not or cannot access the health and food security websites of a different linguistic group. Our data suggest at least three reasons for this.\nThe first reason is that the websites of most institutions are not published in more than one or two languages. When we sampled Web pages found by the English-language queries \u201cavian flu,\u201d \u201ctuberculosis,\u201d \u201cschizophrenia,\u201d and \u201cmaize\/corn\u201d and their counterpart queries in other languages, the Google search results typically overlapped by only less than 10% in terms of the domain names retrieved, indicating that 90% of the relevant Web pages had not been translated into at least two languages (see Table 1). For example, when Turkish or Bahasa Indonesia was used as the search language, Web pages from very authoritative sources, such as the CDC or WHO, were not retrieved by Google. We also found that a single linguistic accent or special character in the search query could significantly alter the number and content of health-related search results retrieved by Google. Therefore, one reason for the linguistic digital divide is that the majority of health and food Web pages are not translated into multiple languages and\/or that their cross-language retrieval by search engines is poor.\nThe first problem would not be important if the world\u2019s online community, better educated than the general public, searched in English, since the majority of the world\u2019s Web pages are published in English [8,9]. However, we found that there was a 2- to 100-fold higher Google search rate for health and food terms in the native language of a country compared to English (see Table 2).\nFinally, within a nation, it might be assumed that language would not be a problem if a domestic agency only published their health websites in the majority language of their own people. However, we found that in Asia and Western Europe, a subset of immigrants from Indonesia and Turkey, respectively, searched Google for health and food information in their native language, not the language(s) of their adopted countries (see Table 3). In Sub-Saharan Africa, we detected unexpectedly high search rates for health information in non-colonial European languages (see Table 4), perhaps reflecting the presence of international aid workers. Finally, in Latin America, we found that indigenous words were used to search Google for information about food, rather than the colonial language of Spanish (see Table 5). Therefore, domestic agencies, in addition to global agencies, face a linguistic challenge when publishing information online: their target audiences still require information to be published in different languages, even though Internet users are presumably more educated and thus more multilingual than the general population.\nRecommendations\nGiven our observation that the world\u2019s peoples appear to be searching for health and food terms in their local language or mother tongue, not in English, previous online language translation efforts by the WHO and FAO have no doubt been worthwhile. This is also revealed by the high page rank (first page results) of WHO-affiliated search results when Google is searched for infectious disease information in one of the WHO\u2019s six online languages. We recommend that these efforts be continued and further expanded to include more languages; this recommendation applies to global agencies, but also to domestic agencies, in order to meet immigrant or indigenous needs and\/or to make information accessible to other nations. To achieve such translation goals, better health-specific translation software must be developed and more translators are needed who specialize in human health and food security terminology. For example, improved cross-language search retrieval [23] of health information by online search engines would be beneficial. These investments should then be used to initially target health and food security information to the world\u2019s most important linguistic groups, which include speakers of Chinese (1080 million people), Hindi (about 500 million), English (350-500 million), Spanish (390 million), Arabic (255 million), Portuguese (190 million), Bengali (215 million), Russian (255 million), Bahasa Indonesia (200 million), Japanese (127 million), Punjabi (104 million), German (123 million), and French (119 million) [10]. In a world that is primarily non-English speaking, such attempts will help to reduce the linguistic digital divide in health and food information on the World Wide Web.\nFurthermore, as we did in this study (see Table 2 to Table 5), we recommend that when global or domestic health and food security organizations wish to use the Internet to disseminate information to other nations [28] or to their own immigrant or indigenous communities, they should first consult search engine query rates for different translations of possible search terms in order to determine which online languages are most needed. Multimedia Appendix 2 contains extensive linguistic online search pattern data to help health and food agencies better select languages for targeted website publishing. In order to measure search rates for other subjects of interest, we note that free online tools exist, such as Google Trends. In some situations, such as targeting indigenous groups, who often speak the majority language of a nation, all that may be needed is to imbed translated keywords into a majority-language website (eg, Spanish) so that search engines such as Google can cross-retrieve relevant information.\nFuture Studies\nThough this study examined the extent to which agencies such as the WHO are publishing information in multiple languages, we did not systematically address the quality of health and food information available in different languages. Quality analysis would especially be important for minor languages that have little content available on the Web: for example, we estimate that there are more than 4000-fold fewer search results in Bahasa Indonesia than in English for \u201ctuberculosis,\u201d more than 200-fold fewer search results for \u201cavian flu\u201d in Arabic or Japanese than in English, and about 500-fold fewer search results for \u201cschizophrenia\u201d in Arabic than in English. Though these precise numbers are considered to be unreliable [29], they do illustrate the point that most of the world\u2019s linguistic groups, even major ones, have much less available online health information relative to English. As to the quality of the websites that are available, this will require systematic analysis, which poses significant methodological problems [30]. One could, however, perform a subjective survey-based evaluation by multilingual physicians, as has been performed to evaluate disease-specific websites published in English [31]. Such a survey should include quantifying to what extent online information from the WHO, CDC, and FAO is being translated into the world\u2019s minor languages in order to help these agencies determine where they need to target their online translation efforts.","keyphrases":["language","google","health","internet","linguistic","indigenous","avian flu","tuberculosis","schizophrenia","maize","nutrition","food security","immigrant"],"prmu":["P","P","P","P","P","P","P","P","P","P","P","P","P"]}
{"id":"J_Neurol-2-2-1705504","title":"Genetics of human hydrocephalus\n","text":"Human hydrocephalus is a common medical condition that is characterized by abnormalities in the flow or resorption of cerebrospinal fluid (CSF), resulting in ventricular dilatation. Human hydrocephalus can be classified into two clinical forms, congenital and acquired. Hydrocephalus is one of the complex and multifactorial neurological disorders.\nIntroduction\nHuman hydrocephalus is a significant medical condition with an estimated incidence of 1 in 1500 births [1]. Hydrocephalus is characterized by abnormalities in the flow or resorption of cerebrospinal fluid (CSF), resulting in ventricular dilatation. However, hydrocephalus is far more complicated than a simple disorder of CSF circulation [2]. Although commonly considered a single disorder, human hydrocephalus is a collection of a heterogeneous complex and multifactorial disorders [3]. Genetic factors are involved in the pathogenesis of hydrocephalus [4\u20136]. For the purposes of this review, we categorize hydrocephalus as congenital, which is present at birth and often associated with developmental defects; and acquired, which occurs after development of the brain and ventricles [7\u201312].\nThe development and progression of congenital hydrocephalus is a dynamic process that is not yet well understood. It is thought that it may develop at an important and specific embryonic time period of neural stem cell proliferation and differentiation in the brain [13, 14]. Congenital hydrocephalus may occur alone (non-syndromic) or as part of a syndrome with other anomalies (syndromic) [15, 16]. In syndromic forms, it is hard to define the defective gene because of the association with other anomalies. We will mainly focus on isolated forms of hydrocephalus. In genetic terms, the isolated (non-syndromic) form of hydrocephalus is a primary and major phenotype caused by a specific faulty gene.\nIt is estimated that about 40% of hydrocephalus cases have a possible genetic etiology [10]. In humans, X-linked hydrocephalus (HSAS1, OMIM) comprises approximately 5\u201315% of the congenital cases with a genetic cause [10, 17\u201320]. Although there is strong evidence for genetic causes, only one hydrocephalus gene (X-linked) has been identified in humans.\nBesides genetic factors, many other factors influence the development of congenital hydrocephalus, such as congenital malformations, intracerebral hemorrhage, maternal alcohol use [21, 22], infection [6, 23\u201325], and X-ray radiation during pregnancy [26, 27].\nGenetics in hydrocephalus\nCongenital hydrocephalus is the more common of the two forms of hydrocephalus, and is probably the consequence of abnormal brain development and perturbed cellular function, which emphasizes the important roles that congenital hydrocephalus genes play during brain development. In general, the recurrence risk for congenital hydrocephalus excluding X-linked hydrocephalus is low. Empiric risk rates range from\u00a0<1% to 4% [28\u201330], indicating the rarity of autosomal recessive congenital hydrocephalus [10, 20, 31, 32]. However, multiple human kindreds with congenital hydrocephalus have been reported [10, 15, 20, 32\u201343]. The loci or genes for human autosomal recessive congenital hydrocephalus have not yet been identified, but there is at least one locus for this trait. Furthermore, like animal models, since there is heterogeneity among clinical phenotypes, there may be more genetic loci in human autosomal recessive congenital hydrocephalus.\nOne kindred was reported in which congenital hydrocephalus was transmitted in an autosomal dominant fashion. This condition was associated with aqueductal stenosis but was not associated with mental retardation or pyramidal tract dysfunction. The lack of mental retardation and pyramidal tract dysfunction was in contrast to X-linked or recessive congenital hydrocephalus with stenosis of the aqueduct of Sylvius (HSAS), in which these abnormalities are commonly seen [44]. Another study identified a kindred with a microdeletion of 8q12.2-q21.2 which subsequently developed hydrocephalus. This trait was also transmitted in an autosomal dominant fashion [45]. Molecular genetic studies have revealed that the responsible gene for X-linked human congenital hydrocephalus is at Xq28 encoding for L1CAM (L1 protein) [46]. The mutations are distributed over the functional protein domains. The exact mechanisms by which these mutations cause a loss of L1 protein function are still under investigation.\nAnother form of this disorder, acquired or adult-onset hydrocephalus is mostly sporadic and characterized by ventricular enlargement in the absence of significant elevations of intracranial pressure; therefore this form is termed normal pressure hydrocephalus (NPH). Definite changes in CSF flow, resorption, and associated dynamics have been found in NPH patients, and these changes may represent a pathogenic mechanism or a secondary phenomenon [47]. Adult-onset hydrocephalus may develop either as a result of decompensation of a \u201ccompensated\u201d congenital hydrocephalus, or it may arise de novo in adult life secondary to an acquired disturbance of normal CSF dynamics. The latter may be due to late-onset aqueductal stenosis or disruption of normal CSF absorptive pathways [11, 48]. Acquired (adult-onset, or NPH) form of inherited hydrocephalus is very rare. Recently, an X-linked adult-onset NPH [49] and a form of familial NPH that is transmitted in autosomal dominant fashion [50] have been reported, but detailed genetic linkage studies have not been carried out yet. The genetic etiology of this form is therefore totally unknown.\nHydrocephalus has been observed in many mammals [51\u201359]. Animal hydrocephalus models have many histopathological similarities to humans and can be used to understand the genetics and pathogenesis of brain damage [59\u201364]. It has been well documented in the animal models that in the majority of cases, congenital hydrocephalus is a genetic disease. Furthermore, many congenital hydrocephalus loci have been mapped and identified in the animal models.\nHydrocephalic Texas strain (HTX) rat model of inherited congenital hydrocephalus is characterized by onset in late gestation, a complex mode of inheritance, and ventricular dilatation associated with abnormalities in the cerebral aqueduct and subcommissural organ (SCO), a structure that is important for the patency of the aqueduct of Sylvius and normal CSF flow in the brain. Quantitative trait locus (QTL) genetic mapping has been performed from the progeny of a backcross of HTX rat with the non-hydrocephalic Fischer F344 strain. The disease has been linked with loci on chromosome (Chr) 9 (peak markers D9Rat2), 10 (between markers D10Rat136 and D10Rat135), 11 (peak markers D11Arb2 and D11Rat46) and 17 (peak markers D17mit4 and D17Rat154) respectively. The severity of hydrocephalus in HTX rat seems to be influenced by different genetic loci [65\u201368]. Another study suggested that the HTX strain is homozygous carrier of an autosomal recessive hydrocephalus gene with incomplete penetrance [69]. The genetics of another hydrocephalus inbred strain, Wistar-Lewis rats (LEW\/Jms) which demonstrate inherited congenital hydrocephalus, is less clear with possible traits as an autosomal recessive [70] or semidominant or multigenic (possible QTL) with a possible locus on sex chromosomes [71], but none of the loci has been localized.\nIn mouse models, three QTL loci associated with congenital hydrocephalus have been identified and labeled as Vent8a, Vent4b, and Vent7c. As a major QTL controlling variance in ventricular size, Vent8a is located on Chr 8 (near the markers D8Mit94 and D8Mit189). The Other two loci, Vent4b and Vent7c, show strong epistatic interactions affecting ventricular size in the developing embryo. Vent4b is located on Chr 4 (near D4Mit237 and D4Mit214), and Vent7c is located on Chr 7 (between D7Mit178 and D7Mit191) [72].\nThe autosomal recessive congenital hydrocephalus-1 (hy1) mouse has been characterized phenotypically by a dome-shaped head that is sometimes seen at birth or develops during the first 2\u00a0weeks. Internally, dilatation of the entire ventricular system is observed [73, 74]. A more severe phenotypic form, hydrocephalus-2 (hy2) mouse [75, 76], and an obstructive hydrocephalus (oh) mouse with communicating hydrocephalus and secondary aqueductal stenosis have also been described [77, 78]. Unfortunately subsequent efforts to identify genetic loci have not been done on these non-inbred mouse strains.\nIn mouse targeted insertional mutagenesis, the accidental insertion of a transgene into a crucial genomic locus could yield important information, which has happened twice in hydrocephalus genetic studies. The transgenic mouse line OVE459 demonstrates autosomal recessive congenital hydrocephalus. This is caused by a Bdnf transgene-induced insertional mutation on a single locus on mouse Chromosome 8 (near marker D8Mit152). The OVE459 insertion locus is overlapped with that of autosomal recessive hydrocephalus-3 (hy3) mouse that phenotypically shows lethal communicating hydrocephalus with perinatal onset [79, 80]. The transgene insertion resulted as a rearrangement of Hydin exons in OVE459 mice. Subsequently, a single CG base-pair deletion in exon 15 of Hydin was also discovered in hy3 mice carrying the spontaneous hy3 mutant allele [81, 82].\nIn another targeted insertional mutagenesis resulting in congenital hydrocephalus, the CYP2J2 transgene interferes with the expression of a brain-specific isoform of the regulatory factor X4 (RFX4), which belongs to the winged helix transcription factor family. This brain specific isoform is called variant transcript 3 or RFX4_v3 and is crucial for normal brain development as well as for the genesis of the SCO. Loss of a single allele prevents formation of the SCO and leads to an autosomal dominant congenital hydrocephalus. This obstructive hydrocephalus appeared to be secondary to failure of development of the SCO [83].\nThe autosomal recessive congenital hydrocephalus (ch) mouse was reported decades ago [79]. Recently this mouse has been shown to have a mutation on another winged helix\/forkhead transcription factor gene, Foxc1 (Mf1) on mouse Chromosome 13 [84, 85]. There is a recent report of 6 children with hydrocephalus from 3 different families with subtelomeric deletions from chromosome 6p. Three forkhead genes within this region (FOXF1 and FOXQ1) or proximal to it (FOXC1) were evaluated as potential candidate disease genes but no disease causing mutations were identified [86].\nThe autosomal recessive hydrocephalus with hop gait (hyh) mouse exhibits dramatic dilation of the ventricles at birth and invariably develops hopping gait. The hyh mouse shows a markedly small cerebral cortex at birth and dies postnatally from progressive enlargement of the ventricular system. The small cortex in hyh mouse reflects altered development of the neuronal cells. In this mouse, it is postulated that neural progenitor cells withdraw prematurely from the cell cycle, producing more early-born, deep-layer cerebral cortical neurons but depleting the cortical progenitor pool, and creating a small cortex. Genetic linkage analysis localized the hyh locus between markers D7Mit75 and D7Mit56 on mouse Chr 7. Later, the hyh gene was identified as \u03b1-SNAP (soluble NSF attachment protein \u03b1) [87]. Homozygous mutant mice harbor a missense mutation M105I in a conserved residue in one of the \u03b1-helical domains. The hyh mutant was not a null allele and is expressed; however, the mutant protein is 40% less abundant in hyh mice.\nThe autosomal recessive hemorrhagic hydrocephalus (hhy) homozygous mutant mouse has dilated lateral ventricles and a patent aqueduct, with no histological abnormalities either in the subarachnoid space or in the choroid plexus. Multiple hemorrhages in the meninges and throughout the brain parenchyma can be observed in the advanced stages of hydrocephalus. The hhy locus has been localized on mouse Chr 12 [88].\nRecently, several new congenital hydrocephalus models have emerged in zebra fish mutagenesis screening. These models have been shown to have the defects in embryogenesis and early development leading to enlarged brain ventricles. However, genetic loci for these models have not been identified yet [89, 90].\nGenetic studies in animal models have started to open the way for understanding the underlying pathology of hydrocephalus. In contrast to research with animal models, human hydrocephalus genetic research has lagged far behind. To date, at least 43 mutants of hydrocephalus have been described, and 10 congenital hydrocephalus genes have been identified. Among them, only one hydrocephalus gene has been identified in humans (see Table\u00a01).\nTable\u00a01Summary of current known loci (or mutants) of hydrocephalus in vertebratesSpeciesStrainClinical formTrait*LocusChromosomeHuman syntenic regionHuman GeneReferencesHumanCARUnknownunknown10,15,27,32\u201343HumanCADUnknown8q12.2\u201321.2 or unknown 44,45HumanAOADNPHunknown50HumanCX-linkedL1camXXL1CAM46HumanAOX-linkedUnknownXX49RatHTXCQTLD9Rat2 9q38 5q21.1, 18p11.22\u201331 65\u201368RatHTXCQTLD10Rat136, D10Rat13510q32.1\u201310q32.317q21.3\u2013q25.365\u201368RatHTXCQTLD11Arb2, D11Rat4611q233q27\u201328, 22q11.21,10p12.2 65\u201368RatHTXCQTLD17Mit4, D17Rat15417q12.1 1q43, 10p11.21\u2013p13 65\u201368RatLEW\/JmsCAR, (QTL)unknownunknown70,71MouseC57BL\/6JCQTLVent8a88p11\u201323, 13q11\u20133472MouseC57BL\/6JCQTLVent4b46p, 972MouseC57BL\/6JCQTLVent7c719q10\u20131372Mousehy1CARunknownunknown73,74Mousehy2CARunknownunknown75,76Mousehy3CARHydin816q22.2HYDIN79\u201382MouseC57BL\/10JCARhyh719q13.3a-SNAP87, 100MouseC57BL6\/JCADRfx41012q24RFX483MouseBALB\/cHeACARhhy1214q3288MousechCARMf1136p25FREAC-384MouseSTOCK tbCARohunknown77,78MouseC57BL\/6*CBA\/JCARMdnah5155p15.2DNAH5106MouseC57BL\/CBACADOtx21414q21-q22OTX2122Mouse129P2\/OlaHsdCARMsx154p16.3-p16.1MSX1107,108MouseC57BL\/6CARSocs71117q12SOCS7118MouseC57BL\/6JCARNmhc-b1117q13MYH10121Zebrafishm404\/m491CARapounknown89,90Zebrafishm409\/m432CARcudakunknown89,90Zebrafishm691CAReagleunknown89,90Zebrafishm591CARendeavorunknown89,90Zebrafishm584CARenterpriseunknown89,90Zebrafishm492\/m510CARgalileounknown89,90Zebrafishm445\/m585\/m700CARgumowyunknown89,90Zebrafishm727CARhubbleunknown89,90Zebrafishm221\/m470\/m680CARinterrailunknown89,90Zebrafishm733CARkeplerunknown89,90Zebrafishm728CARneilunknown89,90Zebrafishm481CARpan twardowskiunknown89,90Zebrafishm172\/m476CARuchu hikoushiunknown89,90Zebrafishm766CARvoyagerunknown89,90Zebrafishm331CARvikingunknown89,90Zebrafishm479\/m627CARyuraunknown89,90Zebrafishm111\/m307\/m512\/m97CARzezemunknown89,90* Genetic trait, AR: autosomal recessive, AD:autosomal dominant, QTL: Quantitative trait locus, ** clinicalform, C: congenital, AO: Adut-Onset\nDevelopmental, physiological and anatomical pathology of hydrocephalus\nThe neuropathology of hydrocephalus has been adequately elucidated. Cerebral ventricle dilatation secondary to disturbed CSF flow has been observed as an inheritable trait in a variety of laboratory animals (as well as in humans). In most cases, defective development of the cerebral aqueduct or  the subarachnoid space has been observed [61]. Affected individuals may have severe developmental delay and radiographic findings of hydrocephalus [91].\nThe morphological and developmental changes in the ventricular system have been well studied in three major rat models of congenital hydrocephalus: 6-aminonicotinamide (6-AN)-induced, LEW\/Jms and HTX mutant rats. Comparative morphological studies revealed that 6-AN-induced hydrocephalus was comparable to the Dandy-Walker syndrome. The LEW\/Jms and HTX mutant models were identical with regard to the form of presentation and progression of hydrocephalus in the postnatal period; but the pathogenesis of these two conditions in the fetal period was different. The LEW\/Jms rats showed primary congenital aqueductal stenosis in early prenatal life and the hydrocephalic state appeared before pulmonary maturation was completed. However, although the model has been considered to be of congenital communicating hydrocephalus [64], the HTX fetuses demonstrated secondary closure of the aqueduct in the perinatal period. This secondary closure of the aqueduct in HTX rats is believed to be due to retrograde degeneration of the thalamus caused by apoptotic cell death [92, 93] and failure in cell proliferation [94, 95]. The HTX rat also shows a reduction in the secretory cells of the SCO. Regarding the role of the SCO in hydrocephalus pathogenesis, serial brain sections through aqueduct regions containing the SCO from HTX rats, in comparison with normal Fischer F344 strain, have been studied and found that reduced SCO glycoprotein immunoreactivity precedes both aqueduct closure and expansion of the lateral ventricles in the HTX rate (as it\u2019s redundant) [96, 97].\nAlthough some studies have addressed the activation of macrophages and microglia (the resident mononuclear phagocytes of the brain) within the brain in animal hydrocephalus models, little is known of their state of activation or regional distribution in human congenital hydrocephalus. In one experiment, brain tissue samples of 10 human fetal cases with hydrocephalus and 10 non-hydrocephalic controls were stained immunohistochemically with antibodies directed against MHC class II and CD68 antigens, and lectin histochemistry was done with tomato lectin. Hydrocephalus cases showed focal collections of CD68 and tomato lectin-positive macrophages along the ependymal lining of the lateral ventricles, particularly within the occipital horn. By comparison, brain tissue samples from controls showed few or no ependymal or supraependymal macrophages and the few macrophages that were present were not as intensely immunoreactive as in the hydrocephalus cases. The macrophage response detected at the ependymal lining of the ventricles and within the periventricular area in hydrocephalus may be related both to the severity of hydrocephalus and the age of the fetus [98]. Microglia that are normally interspersed throughout the intermediate zone and circumscribing the basal ganglia were within normal confines in all cases examined. Unexpectedly, hydrocephalic cases also showed focal regions of hypovascularization or alterations in the structure and orientation of capillaries within periventricular areas, compared with controls [98].\nIn summary, the pathological studies of hydrocephalus clearly indicate that impaired and abnormal brain development in the early development stage caused by altered neural cell fate and perturbed regulation of cellular proliferation and apoptosis. The abnormal brain development subsequently leads to the accumulation of the CSF in cerebral cavities. All these cellular and developmental events eventually lead to the congenital hydrocephalus accompanied by possible secondary inflammatory reaction and neurovascular pathogenesis.\nThe molecular and cellular etiology of hydrocephalus\nOne of the possible mechanisms leading to the pathogenesis of hydrocephalus is the disruption of neural cell membrane proteins that play an important function during brain development. The L1 protein coded by human X-linked hydrocephalus gene is a member of the immunoglobulin superfamily of neural cell adhesion molecules that is expressed in neurons and Schwann cells, and seems to be essential for the brain development and function. Hirschsprung\u2019s disease (HSCR) is characterized by the absence of ganglion cells and the presence of hypertrophic nerve trunks in the distal bowel. There have been several reports of patients with X-linked hydrocephalus and HSCR with a mutation in the L1CAM gene. Therefore, decreased L1CAM may also be a modifying factor in the development of HSCR [99].\nThe gene carrying the mutation for autosomal recessive hydrocephalus in the hyh mouse codes \u03b1-SNAP protein. \u03b1-SNAP is essential for apical protein localization and cell fate determination in neuroepithelial cells [100]. \u03b1-SNAP plays a key role in a wide variety of membrane fusion events in eukaryotic cells. Membrane fusion is required for two main cellular functions: 1) the transport of molecules to distinct inter- and intracellular compartments and thereby maintenance of the functional and structural organization of eukaryotic cells; 2) the intercellular communication such as the regulated exocytosis (secretion) of neurotransmitters by neuronal cell, which occurs temporally and spatially as the precise sequential regulation events at the plasma membrane during the early brain development [87]. In the hyh mouse model, altered neural cell fate is also accompanied by abnormal localization of many apical proteins implicated in regulation of neural cell fate, including E-cadherin, beta-catenin, atypical protein kinase C (aPKC), inactivation-no-afterpotential D-like (INADL), SNAP receptor (SNARE), and vesicle-associated membrane protein-7 (VAMP-7) [100]. Furthermore, disturbed astrocyte metabolism in the early brain development in the kaolin-induced rat model of hydrocephalus has also been reported [101].\nHydrocephalus may also be caused by a malfunction of the ependymal cells [102\u2013105]. Within the neonatal brain of the mouse, the hy3 protein (hydin) is confined to the ciliated ependymal cell layer lining the lateral, third and fourth ventricles. Hydin is not closely related to any previously known protein, with the exception of a 314 amino acid domain with homology to caldesmon, an actin-binding protein, suggesting that hydin interacts with the cytoskeleton [81, 82]. The protein of axonemal heavy chain 5 gene (Mdnah5), dynein is also specifically expressed in ependymal cells, and is essential for ultra structural and functional integrity of ependymal cilia. In Mdnah5-mutant mice, lack of ependymal flow causes closure of the aqueduct and subsequent formation of triventricular hydrocephalus during early postnatal brain development. The higher incidence of aqueductal stenosis and hydrocephalus formation in patients with ciliary defects proves the relevance of this novel mechanism in humans [106].\nHydrocephalus may be caused by malfunction of mesenchymal cells. In mice, Msx1 is a regulatory gene involved in epithelio-mesenchymal interactions in limb formation and organogenesis. In the embryonic brain, the Msx1 gene is expressed along the dorsal midline. The most important features observed in homozygous Msx1 mutants were the absence or malformation of the posterior commissure (PC) and of the SCO, the collapse of the cerebral aqueduct, and the development of hydrocephalus. The heterozygous mutants developed an abnormal PC and smaller SCO, as revealed by specific antibodies against SCO secretory glycoproteins. About one third of the heterozygous mutants also developed hydrocephalus; therefore the phenotype may be determined by the Msx1 gene dosage during a specific developmental period [107, 108]. In the autosomal recessive congenital hydrocephalus (ch) mouse model, a truncated protein lacking the DNA-binding domain of the forkhead\/winged helix gene, Mf1, was generated. Mesenchymal cells from Mf1lacZ embryos differentiate poorly into cartilage in micromass culture and do not respond to added bone morphogenetic protein 2 (BMP2) and transforming growth factor-beta 1 (TGFB1). The differentiation of arachnoid cells in meninges of the mutant mice is also abnormal. The levels of developmental growth factors such as TGFB1 and BMP2 are dramatically increased in the ch mouse, and it is possible that phenotypic hydrocephalus of Mf1 mouse is due to the secondary effect of these elevated growth factors. Corresponding to studies in the ch mouse, human patients with deletions in the region containing human Mf1 homolog FREAC3 were found to develop multiple developmental disorders, including hydrocephalus [84]. Another winged helix transcription factor causing congenital hydrocephalus when mutated, RFX4_v3 transcript, is dynamically expressed in the developing brain from the neural plate stages. The RFX proteins belong to the winged-helix subfamily of helix-turn-helix transcription factors, and bind to \u2018X-boxes\u2019 in target DNA sequences and regulate expression of the downstream target genes [83]. Disruption of both RFX4_v3 alleles by insertional mutagenesis severely alters early brain morphogenesis, reduces Msx2 expression, and causes a deficiency in WNT signaling [83]. This may suggest that RFX4_v3 is probably upstream of Mf1 in the signaling pathway during early brain development.\nHydrocephalus may be caused by perturbation of growth factor signaling [109, 110]. Developmental abnormalities in congenital hydrocephalus provide the clues for the perturbation of major signaling pathways in the development [111]. TGFB is an important cytokine and growth-signaling molecule in the brain. In mouse models, severe hydrocephalus has been observed in transgenic mouse overexpression of TGFB1 in astrocytes [112, 113]. In the HTX rat, increased level of TGFB3 may contribute to the development of hydrocephalus [114]. In mouse models, fibroblast growth factor-2 (FGF-2) seems to play a predominant role in the proliferation of neuronal precursors and in neuronal differentiation in the developing cerebral cortex even at relatively late stages of brain neurogenesis. Administration of FGF-2 to embryonic brain induces hydrocephalic brain morphology and aberrant differentiation of neurons in the postnatal cerebral cortex [110]. IGF binding protein-1 (IGFBP-1) modulates the cellular action of insulin-like growth factors (IGFs), some of which are expressed in the fetal brain. Hydrocephalus has been observed in mouse models that overexpress liver-specific IGFBP-1 during fetal life. The hepatic over-expression of IGFBP-1 may have endocrine effects on brain development and induction of congenital hydrocephalus [115]. Other studies have shown that up-regulation of certain growth factors in the brain could lead to altered brain fluid dynamics [116, 117]. SOCS7 is a member of the suppressor of cytokine signaling (SOCS) protein family. SOCS proteins have a similar structure: an N-terminal domain of variable length, a central Src homology-2 domain, and a C-terminal SOCS box. Biochemical and genetic studies have revealed that SOCS family members play an important role in the termination of cytokine and growth factor signaling. Homozygous Socs7 mutant mice were born in expected numbers, were fertile, and did not exhibit defects in hematopoiesis or circulating glucose or insulin concentrations. Strikingly however, these homozygous Socs7 mice were 7\u201310% smaller than their wild-type littermates, and within 15\u00a0weeks of age approximately 50% of the homozygous Socs7 mice died as a result of hydrocephalus. In situ hybridization studies in normal mice have revealed that Socs7 is prominently expressed in the brain, suggesting that SOCS7 plays an important functional role in early brain development [118]. We can therefore hypothesize that loss of SOCS7 function will lead to increased expression of cytokines resulting in developmental abnormalities and congenital hydrocephalus due to its inhibitory role in cytokine signaling.\nHydrocephalus may also be caused by the disruption of extracellular matrix (ECM). In the TGFB1 over-expression mouse model, the changing expressions of a remodeling protein - matrix metalloproteinase-9 (MMP-9) and its specific inhibitor- tissue inhibitor of metalloproteinases-1 (TIMP-1) were also found to be important factors in the spontaneous development of hydrocephalus by altering the ECM environment [119]. Furthermore, increased expression of cytokines such as TGFB1 might also reciprocally play an important role by disrupting the vascular ECM remodeling, promoting hemorrhages, and altering the re-absorption of CSF [120]. In another mouse model, ablation of the nonmuscle myosin heavy chain II-B (NMHC-B) results in severe hydrocephalus with enlargement of the lateral and third ventricles. These defects may be caused by abnormalities in the cell adhesive properties of neuroepithelial cells and suggest that NMHC-B is essential for both early and late developmental processes in the mammalian brain [121].\nHydrocephalus may also be caused by the disruption of major early brain developmental patterning molecules. Autosomal dominant hydrocephalus in Otx2 mutation mice is characterized by eminent dilatation of lateral ventricles and a ballooned cerebrum. Histopathology shows edematous change of the periventricular white matter, suggesting that Otx2 functions as a brain developmental organizer, and a disruption of this gene is a likely cause of hydrocephalus [122].\nIn conclusion, many genetic loci of hydrocephalus have been defined in animal models, which is building a foundation for better understanding of molecular etiology of hydrocephalus; however, genetic research of hydrocephalus in humans is limited. The histopathological similarities of animal models can be used to understand the genetics and pathogenesis of human hydrocephalus. For example, the histopathological and morphological appearance of hydrocephalic HTX rats is demonstrated in Fig.\u00a01, in which hydrophilic HTX rat exhibits large cerebral ventricles, a progressively-thinned cortical mantle, and stretched internal capsule fibers[123]. Review of the molecular etiologies shows a very diverse set of pathogenetic mechanisms. Perturbation of almost any molecule that plays a crucial role in early brain development, and sequentially regulation of dynamics of cerebrospinal fluid, could lead to the pathogenesis of congenital hydrocephalus. The 10 known hydrocephalus genes mostly code for important cytokines, growth factors, or related molecules in the cellular signal pathways during early brain development.\nFig.\u00a01Comparison of rat brain morphology by MRI and histology at 4, 11 and 21\u00a0days of age in non-hydrocephalic and hydrocephalic HTX rats. T2-weighted MRI scans of coronal sections from a non-hydrocephalic HTX rat at the level of the thalamus at 4, 11 and 21\u00a0days (A, D, G) shows small ventricular and subarachnoid spaces compared to an age-matched HTX littermate (B, E, H) that exhibits large cerebral ventricles, a progressively-thinned cortical mantle, and stretched internal capsule fibers (*). Congenital closure of the cerebral aqueduct becomes life-threatening by 21\u00a0days of age. Histological sections (C, F, I) at the level of the midbrain at the same ages demonstrate extreme thinning of the cortical mantle. MRI images are modified from Jones et al (2000) [123] with permission by Maney Publishing; histological samples are from the doctoral thesis of Janet M. Miller, PhD\nFuture prospects\nIt is essential to recognize that molecular genetics is the only current scientific approach that can be used to study hydrocephalus in which the usual concern about whether an observed phenomenon is a consequence or a cause is completely addressed.\nDespite our knowledge of the genetics of hydrocephalus in animal models, we have very limited knowledge about the genetic and molecular mechanisms that cause human hydrocephalus. Without this knowledge, it is impossible to say whether the pathogenesis of human hydrocephalus is comparable to that seen in animal models, and impractical to extrapolate data gained from animal models to humans. In order to better understand human hydrocephalus and to develop more appropriate translational research, it will be necessary to conduct large-scale genetic studies of human hydrocephalus. If, and when, more heritable forms of human hydrocephalus are identified, and underlying genes and their functions are characterized, then this knowledge could be used to improve patient care in a variety of different ways such as prenatal diagnosis and new potential therapeutic approaches. Possible new mechanisms other than altered CSF circulation and resorption, if uncovered via the genetic research, may also help explain why patients with hydrocephalus may experience symptomatic progression despite functioning shunts.\nEfforts to identify genetic variants associated with susceptibility to genetic diseases rely on three major approaches: pedigree and sib-pair linkage analysis and population association studies. The differences among these study designs reflect their derivation from biological versus epidemiological traits. Like most common diseases, it would be very difficult to identify and recruit large pedigrees in hydrocephalus that show hereditary transmission of the condition. Therefore, the last two approaches, sib-pair linkage analysis and population association studies, are the best options for the genetic study on this disease. For any study, but particularly in the case of genetic mapping for a common disease, a large sample size is crucial in achieving statistical significance. Recent advances in the genomics and statistical methodology in genetic mapping will certainly help in making well-powered studies more feasible, by reducing the number of genetic markers or workload required for these studies. For example, since many genes and loci response for hydrocephalus in animal models have been mapped, candidate genes approach will certainly be the very first choice to test the collected human hydrocephalus population for linkage and association analysis.\nIn collaboration with the Hydrocephalus Association (HA), our group has initiated a genetic study of human hydrocephalus. As part of a prospective study that has been approved by the Johns Hopkins Institutional Review Board, we are collecting blood samples from both congenital and acquired NPH hydrocephalus patients. The objectives of this study are to identify the genetic loci responsible for the development of hydrocephalus, to examine the relationship between genotype and phenotype and to define the functions of these genes during early development. This is the first large-scale research study of its kind and information gained from this study will undoubtedly provide invaluable information concerning the developmental mechanisms of this disease in humans. Such knowledge will hopefully lead to the more reasonable treatment schemes, the better diagnostic tools, and the more effective therapeutic modalities.","keyphrases":["genetic of","hydrocephalus","congenital","acquired","multifactorial disorder"],"prmu":["P","P","P","P","P"]}
{"id":"Pediatr_Nephrol-4-1-2259254","title":"Muscle wasting in chronic kidney disease: the role of the ubiquitin proteasome system and its clinical impact\n","text":"Muscle wasting in chronic kidney disease (CKD) and other catabolic diseases (e.g. sepsis, diabetes, cancer) can occur despite adequate nutritional intake. It is now known that complications of these various disorders, including acidosis, insulin resistance, inflammation, and increased glucocorticoid and angiotensin II production, all activate the ubiquitin\u2013proteasome system (UPS) to degrade muscle proteins. The initial step in this process is activation of caspase-3 to cleave the myofibril into its components (actin, myosin, troponin, and tropomyosin). Caspase-3 is required because the UPS minimally degrades the myofibril but rapidly degrades its component proteins. Caspase-3 activity is easily detected because it leaves a characteristic 14kD actin fragment in muscle samples. Preliminary evidence from several experimental models of catabolic diseases, as well as from studies in patients, indicates that this fragment could be a useful biomarker because it correlates well with the degree of muscle degradation in dialysis patients and in other catabolic conditions.\nMaintenance of protein stores in chronic kidney disease\nIn uremic patients, protein stores are frequently depressed when assessed by a low serum prealbumin and weight loss, which includes loss of muscle mass [1, 2]. In pediatric patients with chronic kidney disease (CKD), linear growth is impaired and muscle mass is reduced. Although these findings have been attributed to \u201cmalnutrition\u201d, many uremic patients with muscle wasting have not developed the problems because of inadequate diet; instead, they have complications that induce a complex series of physiological and biochemical adaptations, resulting in protein catabolism [3, 4]. In children and adults with CKD, these complications include metabolic acidosis, insulin resistance, increased glucocorticoid production, high levels of angiotensin II (Ang II), and inflammation [5\u20138]. Many observational studies and mechanistic investigations have attempted to explain this loss of protein stores, and especially the loss of muscle mass. There are at least three conclusions from these studies. First, rates of protein synthesis in muscle are generally unchanged, whereas rates of protein degradation tend to increase [5, 9]. Second, the daily rates of protein turnover in cells are so high (3.5\u20134.5\u00a0g protein\/kg per day) that even a small increase in protein degradation (and\/or a decrease in protein synthesis) will cause marked protein depletion over time [10]. Third, the increase in muscle protein degradation in uremia and most other catabolic disease states is mostly due to programmed activation of the ubiquitin\u2013proteasome system (UPS) [5, 11]. Therefore, to understand muscle wasting, one must understand the UPS.\nThe ATP-dependent, ubiquitin\u2013proteasome system (UPS)\nOver the past two decades, progress in understanding the action and regulation of the UPS has been at the center of attempts to understand the control of protein turnover. The UPS includes concerted actions of enzymes that link ubiquitin (Ub), a member of the heat-shock protein family, to protein substrates that are destined for degradation (Fig.\u00a01). When a chain of at least four to five ubiquitins are linked to a protein, it is marked for degradation in a second step mediated by the proteasome [12, 13]. Specifically, the tagged protein will be recognized by the 26S proteasome, a very large multicatalytic protease complex that not only recognizes Ub-conjugated proteins but also removes Ub, unwinds the protein, and injects it into the central core of the 26S proteasome. Once inside this central, tube-like structure, the protein substrate is degraded into small peptides [14].\nFig.\u00a01The ubiquitin\u2013proteasome pathway of protein degradation. Ubiquitin (Ub) is conjugated to proteins destined for degradation by an ATP-dependent process that involves three enzymes (E1\u2013E3). A chain of five Ub molecules attached to the protein substrate is recognized by the 26S proteasome, which removes Ub and digests the protein into peptides. The peptides are degraded to amino acids by peptidases in the cytoplasm or used in antigen presentation. (Reproduced with permission from [15])\nThree enzymatic components are required to link Ub to proteins that are destined for degradation. There appears to be only one E1 (Ub-activating) enzyme and around 40\u00a0E2 (Ub-carrier or conjugating) proteins. The key enzyme, however, is the E3 enzyme, which accounts for the exquisite specificity of proteins to be degraded. There are at least a thousand E3 enzymes (Ub ligase), and each can recognize a specific protein substrate and catalyze the transfer of an activated Ub from the E2 carrier protein to the substrate protein [10, 15].\nSince the initial reports that the UPS recognizes specific proteins and tags them for destruction, knowledge about proteolytic processes in the proteasome has exploded. Thousands of proteins have been recognized as being degraded by the UPS, and novel cellular functions are now known to be regulated by Ub conjugation. In terms of protein breakdown, the major functions of the pathway are:\nRapid removal of proteins Protein degradation is irreversible, and hence, destruction of a protein generally leads to a complete termination of cellular process mediated by the protein. Consequently, protein degradation is critical for the regulation of metabolism and cell turnover. The rapid degradation of specific proteins also permits cells (as well as the organism) to rapidly adapt to a change in physiological conditions (e.g. requiring a switch to glucose as an energy source involves converting protein stores into amino acids that can be used in gluconeogenesis).\nRegulation of gene transcription Ub conjugation affects gene transcription because many transcription factors become conjugated to Ub, and transcription activators are degraded by the proteasome [16]. This process regulates transcriptional activity by removing \u201cspent\u201d activators and resetting a promoter for additional rounds of transcription [17]. Second, the ability of transcription factors to function varies with their location within the cell. For example, nuclear factor (NF)-\u03baB, a proinflammatory transcriptional factor, is kept outside the nucleus because movement into the nucleus is blocked by its association with an inhibitory protein, I\u03baB. Destruction of I\u03baB, initiated by the IKK kinase and carried out by the UPS, frees NF-\u03baB, which then translocates to the nucleus to stimulate gene transcription [18]. A clinical application of this function of the UPS has developed in oncology. Bortezomib (Velcade, PS-341), a proteasome inhibitor, has proven to be beneficial in patients with multiple myeloma and is currently in clinical trials for the treatment of other cancers [19, 20]. The proposed mechanism of action involves the ability of bortezomib to prevent the UPS-induced destruction of I\u03baB, thereby blocking the activation of NF-\u03baB (an antiapoptosis transcription factor), leading to an increase in apoptosis [21]. Inhibition of the proteasome, therefore, will induce apoptosis of the neoplastic cells [22]. In addition, myeloma cells are also particularly dependent upon NF-\u03baB to produce essential growth factors [especially inerleukin 6 (Il-6)]; when NF-\u03baB is inactive, the growth of myeloma cells is depressed.\nQuality-control mechanism The UPS selectively eliminates abnormally folded or damaged proteins that have arisen because of missense or nonsense mutations, biosynthetic errors, proteins damaged by oxygen radicals, or by denaturation. For example, in cystic fibrosis, the mutant form of the transmembrane conductance regulator protein (CFTR) is selectively degraded before it reaches the cell surface [23]. The UPS catalyzes destruction of this mutant CFTR because its tertiary structure is abnormal. Another example is the degradation of misfolded proteins within the endoplasmic reticulum. Endoplasmic-reticulum-associated degradation (ERAD) of proteins removes misfolded proteins by targeting them for destruction by proteasomes in the cytoplasm [24].\nInfluencing the function of the immune system The UPS is responsible for creating antigens from the degradation of foreign proteins (e.g. viral particles). The antigens are presented on the major histocompatibility complex as class I molecules. In this way, the 26S proteasome exerts dual roles of removing foreign proteins and creating a stimulus of the immune system [15, 25].\nAs a source of amino acids When carbohydrate calories are rapidly needed or when cells must respond to catabolic diseases\/conditions, there is breakdown of cell proteins, especially skeletal muscle proteins. The UPS degrades muscle proteins to provide amino acids that can be converted to glucose (i.e. gluconeogenesis). An undesired consequence of this activity could be an inappropriate loss of muscle protein.\nFunctions of Ub not associated with proteolysis Ub can also be conjugated to proteins as a monomer (rather than as the typical Ub chain). When this occurs on cell-surface proteins, the protein is internalized into the endocytic pathway to be degraded in lysosomes [26, 27].\nUremia-activated mechanisms that accelerate loss of muscle protein\nResults from rodent models of CKD have established that accelerated muscle protein catabolism involves many of the same cellular mechanisms that cause muscle wasting in other catabolic conditions, such as cancer, starvation, insulin deficiency\/resistance, or sepsis [10, 28]. The principal mechanism causing muscle atrophy in CKD involves activation of the UPS. Evidence for this includes the presence of higher levels of mRNAs encoding certain components of the UPS, as well as a similar pattern of increases and decreases in the expression of about 100 atrophy-related genes (also called atrogenes) [5, 28]. Changes in atrogenes include decreased expression of various growth-related genes and increased expression of components of the UPS. Patients with different clinical conditions associated with muscle atrophy exhibit similar increases in mRNAs encoding components of the UPS (e.g. an increase in mRNAs encoding Ub and proteasome subunits) [11, 29\u201331]. In these cases, changes in gene expression are most likely due to transcriptional regulation because we have shown that uremia or abnormal insulin responses increase the transcription of Ub and subunits of the proteasome [5, 6, 32]. Additional evidence linking the UPS to protein degradation in catabolism is the finding that the increase in protein degradation in the muscle of rats with CKD (and other muscle-wasting conditions) can be blocked by inhibitors of the proteasome [5, 6, 33]. Considered together, these results indicate that muscle wasting is a specific and carefully orchestrated program.\nOther questions are why are proteins degraded, and how is the complex program triggered in widely varied pathological conditions (e.g. acidosis in renal failure, low insulin levels in fasting and diabetes, inactivity, or glucocorticoids and cytokines in sepsis and other inflammatory conditions)? In fasting and in other disease states, acceleration of muscle-protein breakdown mobilizes amino acids, which are used for protein synthesis in tissues and for conversion to glucose in the liver [10]. However, if excessive protein degradation persists, the protein loss will have deleterious effects. In CKD, the breakdown of tissue protein produces nitrogenous waste products, which must be excreted to prevent the accumulation of uremic toxins [3, 10]. Finally, in muscle wasting conditions, contractile proteins are lost differentially, whereas in conditions causing atrophy (e.g. aging), all components of muscle cells seem to be affected.\nWhat accounts for muscle-specific response? The answer to this question lies in the involvement of the UPS. The UPS degrades a specific protein depending on which E3 ubiquitin ligase is activated. For example, two Ub ligases, atrogin-1 (also known as MAFbx) and MuRF-1, are found specifically in muscle, and their expression increases dramatically (8- to 20-fold) in catabolic states, causing loss of muscle protein [28, 34, 35]. In mice lacking the genes for either atrogin-1 or MuRF-1, muscles grow normally, but in response to muscle denervation, the ensuing atrophy is 30\u201350% slower [34]. In addition, the muscle\u2019s content of atrogin-1\u00a0mRNA can be considered a biomarker for the rate of proteolysis in muscles responding to a catabolic condition [36\u201338].\nIn uremia, the initial cleavage of myofibrillar proteins is mediated by caspase-3\nMyofibrillar proteins (including actomyosin) comprise about two thirds of the protein in muscle, the major store of amino acids for new protein synthesis and for gluconeogenesis. The UPS readily degrades the main proteins in myofibrils (i.e. actin, myosin, troponin, or tropomyosin), but it does not readily break up the myofibril into its main component proteins [39]. This means that another proteolytic system must initially digest myofibrils to create substrates that can be degraded by the UPS [40].\nMany catabolic conditions are associated with inflammation and\/or cell injury, and these conditions activate a cysteine protease called caspase-3. We tested caspase-3 in an in vitro system using purified actomyosin and found that caspase-3 cleaves actomyosin and leaves a characteristic 14kD actin fragment [40]. When we activated caspase-3 in cultured muscle cells, we found that UPS rapidly degraded myofibrillar component proteins, and again, the 14kD C-terminal fragment of actin was left in the muscle cells [40]. The protein-cleaving action of caspase-3 is important because blocking caspase-3 will reduce overall protein degradation in muscle [40]. In addition, we have found the same cleavage processes are present in muscles of rodent models of uremia, type-1 or type-2 diabetes, or in Ang-II-induced hypertension [7, 35, 40, 41]. Moreover, we found that the 14kD actin fragment also accumulates in muscles of patients with loss of muscle mass due to painful osteodystrophy, uremia, or burn injury [42]. In the latter study, we found that the rate of protein degradation in muscle (measured from the turnover of labeled amino acids) directly correlated (r\u2009=\u20090.78) with the level of the 14kD actin fragment in the same muscle. In addition, there was a lower level of the 14kD actin fragment in muscle of hemodialysis patients who participated in 18\u00a0weeks of an endurance exercise training program. In summary, the level of the 14kD actin fragment is directly associated with measured protein degradation in muscle, and the accumulation of the fragment responds to a beneficial therapeutic intervention. If these properties hold up in other trials, the level of the 14kD actin fragment could be used as a \u201cbiomarker\u201d of increased muscle protein degradation [42].\nSignals triggering muscle atrophy in kidney disease and other catabolic states\nComplications of CKD, as well as the complex syndrome of uremia, can trigger muscle protein breakdown. The triggering complications include metabolic acidosis, decreased insulin action (including insulin resistance), increased glucocorticoid production, high levels of Ang II, and inflammation [5\u20138]. Metabolic acidosis is known to cause accelerated protein degradation in infants, children, adults, the elderly, and patients with CKD (Table\u00a01). The mechanism by which metabolic acidosis causes muscle wasting involves activation of the UPS and caspase-3 [40, 43]. In addition, acidosis changes hormone actions, such as insulin resistance and increased glucocorticoid production, which are involved in activating protein degradation [32, 41, 43\u201345] (Table\u00a02). It is important to emphasize that the correction of metabolic acidosis has been shown to decrease protein breakdown in various clinical trials, indicating why maintaining normal serum bicarbonate levels should be part of standard clinical care (Table\u00a01).\nTable\u00a01Evidence that metabolic acidosis induces catabolism of protein and amino acids in normal infants, children, and adults, as well as in patients with chronic kidney disease (CKD)Subjects investigatedOutcome measurementsTrial outcomeInfants [68]Low-birth-weight, acidotic infants were given NaHCO3 or NaClNaHCO3 supplement improved growthChildren with CKD [69]Measured rates of protein degradation in children with CKDProtein loss was \u223c 2-fold higher when HCO3 was < 16 mM compared with > 22.6 mMNormal adults [70]Acidosis induced and then measured amino acid and protein metabolismAcidosis increased amino acid and protein degradationNormal adults [71]Induced acidosis and then measured nitrogen balance and albumin synthesisAcidosis induced negative nitrogen balance and suppressed albumin synthesisChronic renal failure [72]Nitrogen balance before and after treatment of acidosisNaHCO3 improved nitrogen balanceChronic renal failure [73]Essential amino acid and protein degradation before and after treatment of acidosisNaHCO3 suppressed amino acid and protein degradationChronic renal failure [74]Muscle protein degradation and degree of acidosisProteolysis was proportional to acidosis and blood cortisolChronic renal failure [75]Nitrogen balance before and after treatment of acidosisNaHCO3 reduced urea production and improved nitrogen balanceHemodialysis [76]Protein degradation before and after treatment of acidosisNaHCO3 decreased protein degradationHemodialysis [77]Serum albumin before and after treatment of acidosisNaHCO3 increased serum albuminCAPD [78]Protein degradation before and after treatment of acidosisNaHCO3 decreased protein degradationCAPD [79]Weight and muscle gain before and after treatment of acidosisRaising dialysis buffer increased weight and muscle massCAPD continuous ambulatory peritoneal dialysisTable\u00a02Metabolic acidosis in otherwise normal humans changed hormonal levels or responses to hormonesHormoneAcidosis-induced responseGrowth hormone (GH) [80\u201384]Suppressed GH secretionLower IGF-1 responseInsulin [44, 85, 86]Suppressed insulin-stimulated glucose metabolismInsulin-like growth factor (IGF)-1 [81, 84, 87]Decreased IGF-1 in plasma, and kidney and liver (but not in muscle)Thyroid hormone [82, 88]Decreased plasma T3 and T4 levels plus a higher plasma thyroid-stimulating hormoneGlucocorticoids [89]Increased glucocorticoid productionParathyroid hormone (PTH) [90, 91]Decreased sensitivity of PTH secretion to changes in plasma calciumVitamin D [91]Suppressed activation to 1,25 (OH)2 cholecalciferol\nThe finding that various diseases with muscle wasting are caused by activation of the UPS, plus the fact that coordinated changes in the expression of genes in muscle occur in different catabolic states, suggest that catabolic states activate a common cellular signaling pathway [28]. One signaling pathway is a decrease in phosphatidylinositol 3-kinase (PI3K) activity (Fig.\u00a02). The involvement of this signaling pathway follows from the finding that several catabolic illnesses, including sepsis, acidosis, uremia, and diabetes, are characterized by insulin resistance [41, 44, 46, 47]. In normal muscle, binding of insulin or insulin-like growth factor (IGF)-1 to their receptors increases the activities of PI3K and its downstream target, Akt. In insulin-resistant conditions or with deficiency of IGF-1, the activity of this signaling pathway is depressed [35, 48, 49]. When PI3K activity falls, there is decreased production of phosphatidylinositol-3,4,5 phosphate (PIP3), leading to decreased phosphorylation and activity of the downstream serine\/threonine kinase, Akt. This is a key step, because activated Akt is a major stimulator of growth-related processes via phosphorylation of the downstream kinases, GSK1 and mTOR\/S6kinase, stimulating protein synthesis. On the other hand, reduced PI3K-Akt signaling (as occurs in insulin resistance) enhances protein breakdown in muscle [41]. The rise in muscle protein losses is associated with two catalytic processes: first, caspase-3 is activated to break down the complex structure of muscle; second, there is increased expression of the E3 ubiquitin ligases, atrogin-1 and MuRF-1, to degrade the proteins made available by caspase-3 [35\u201337] (Fig.\u00a02). The result is muscle wasting.\nFig.\u00a02The balance between muscle hypertrophy and atrophy depends on whether protein synthesis is more active than degradation or vice versa. In protein synthesis, insulin-like growth factor (IGF)-1 and insulin signaling leads to increased phosphatidylinositol 3-kinase (PI3K), which promotes Akt phosphorylation (Akt-P). Akt-P promotes phosphorylation of GSK1 and mTOR\/S6 kinases, leading to increased protein synthesis. Akt-P also phosphorylates the forkhead (FoxO) transcription factor, preventing it from entering the nucleus to promote expression of atrogin-1, MuRF-1, and other atrogenes, thereby blocking protein degradation. In protein degradation, the opposite pathway happens, but additionally, decreased Akt-P leads to increased caspase-3 activity, further promoting degradation. In inflammation, it is thought that tumor necrosis factor (TNF)-\u03b1 and other inflammatory cytokines phosphorylate the inhibitor of nuclear factor (NF)-\u03baB (I\u03baB), to free NF-B to enter the nucleus and promote MuRF-1 expression, and ultimately, muscle protein degradation. (Reproduced with permission from [15])\nHow are the two mechanisms activated? In studies of muscles from insulin-deficient rats or db\/db mice (a model of insulin resistance), we found that accelerated muscle protein degradation increases the level of the proapoptotic factor, Bax [35, 41]. This is relevant because activated Bax causes the release of cytochrome C from mitochondria. Cytochrome C release, in turn, activates caspase-3 to trigger actomyosin\/myofibril cleavage, leaving behind the 14kD actin fragment marker [35]. The activation of atrogin-1, and hence the UPS, involves another mechanism: changes in activity of the fork-head transcription factors (FoxO 1, 3 and 4). When these transcription factors are phosphorylated by Akt, they cannot enter the nucleus to stimulate transcription of atrogin-1. However, when PI3K\/Akt activities are low, the FoxOs are not phosphorylated, so they can enter the nucleus to increase the transcription of atrogin-1, resulting in an increase in muscle protein degradation [35\u201337].\nThe influence of Akt on the other E3 ubiquitin ligase involved in muscle proteolysis, MuRF-1, is not as clearly established, and it may be linked to inflammation, as activation of NF-\u03baB will cause overproduction of MuRF1 and muscle atrophy [37].\nBesides acidosis and depressed insulin\/IGF-1 action, another complication of CKD that participates in muscle wasting is increased glucocorticoid production. The complexity of these interactions is great because increased glucocorticoids can cause insulin insensitivity [32, 41, 50], and both insulin deficiency and insulin resistance increase glucocorticoid production. Glucocorticoids exert a permissive effect on protein degradation in muscle caused by several catabolic conditions. For example, activation of muscle protein breakdown does not occur in adrenalectomized animals with metabolic acidosis or with acute diabetes unless the animals are also given a physiological dose of glucocorticoids [32, 51\u201353]. Similarly, the increase in muscle proteolysis induced by Ang II or sepsis is largely eliminated by inhibiting the glucocorticoid receptor [7, 54]. This response to glucocorticoids is \u201cpermissive\u201d because the same physiological level of glucocorticoids does not stimulate muscle protein degradation unless the animals are either acidotic or insulin deficient. These complex interactions actually make \u201cphysiological sense\u201d because glucocorticoids evolved to integrate stress responses in different tissues. When glucose is needed, glucocorticoids mobilize amino acids from muscle protein. At the same time, glucocorticoids induce gluconeogenic enzymes in liver to catalyze the conversion of the amino acids to glucose and urea.\nIn children, accelerated loss of protein stores associated with glucocorticoid therapy frequently results in impaired linear growth. One glucocorticoid-dependent mechanism causing these defects is impaired response to insulin (insulin insensitivity). An allied mechanism is impaired response to IGF-1 arising from decreased or impaired action of growth hormone. Indeed, administration of growth hormone has been shown to improve linear growth of children being treated chronically with glucocorticoids [55]. Moreover, growth hormone has been shown to improve growth of children with chronic catabolic diseases such as CKD; whereas growth hormone improves growth, it does not improve growth in children to (or close to) a normal height for age [56\u201358]. The impaired response is likely due to the multiple complications of CKD, resulting in retarded growth. Interestingly, there is a small uncontrolled study of five children who were being treated with chronic intermittent hemodialysis and growth hormone. The children were then placed on an intensified daily hemodialysis regimen (3\u00a0h\/day, five to six times a week) for a median time of 1.5\u00a0years [59]. The new regimen led to significant catchup growth into the range of a normal height for age. Intensified dialysis plus growth hormone could correct acidosis, improve insulin and IGF-1 signaling and responses to other hormones, and remove unidentified uremic toxins. These responses emphasize the complexity of sorting out mechanisms for muscle wasting in CKD.\nAnother catabolic factor associated with CKD is Ang II. Infusion of Ang II into rodents causes both anorexia and muscle protein loss by mechanisms that depend on glucocorticoids [7]. Also, there is the knotty problem of understanding the influence of inflammation. In this case, the mechanism(s) causing muscle wasting in inflammation is not clear. Suggested responses include the ability of certain inflammatory mediators to cause insulin resistance, as well as a more direct influence of inflammatory mediators on muscle protein metabolism (Fig.\u00a02). The problem is difficult because the link between an increase in inflammatory markers in uremic patients (e.g. C-reactive protein) to loss of muscle mass has not been established [60, 61].\nFinally, there is an intriguing protein\u2014myostatin\u2014a member of the transforming growth factor (TGF)-\u03b2 family of cytokines. It is produced in skeletal and cardiac muscle and regulates muscle growth by limiting it [62]. Overexpression of myostatin in muscle leads to loss of protein mass via inhibition of Akt phosphorylation with an increase in active FoxO1; this increases the expression of atrophy-related genes [63, 64]. Underexpression of myostatin results in skeletal muscle hypertrophy [65]. An increase in myostatin expression is found in several cachexia-associated disease states. However, there is limited information about the influence of kidney disease on myostatin expression and function beyond changes in myostatin mRNA [66, 67].\nConclusion\nIn this brief review of mechanisms causing muscle protein losses, we discussed how a complex series of biochemical reactions are coordinated to create a genetic program that degrades muscle proteins. We also identified an initial step in muscle proteolysis that leaves behind a biomarker in muscle, the 14kD actin fragment, resulting from caspase-3. We emphasized how the UPS causes muscle wasting in uremia, as well as the role UPS plays in the regulation of cellular functions, ranging from the control of the cell cycle to activities that promote cancer. Indeed, inhibitors of proteasome activity have emerged as novel chemotherapeutic agents. Involvement of the UPS in such a wide range of functions explains why the 2004 Nobel Prize in Chemistry was awarded to Avram Hershko, Aaron Ciechanover, and Irwin Rose for their discovery of Ub and its role in orchestrating cellular protein turnover (http:\/\/nobelprize.org\/chemistry\/laureates\/2004\/).","keyphrases":["muscle wasting","chronic kidney disease (ckd)","caspase-3","14kd actin fragment","protein degradation","uremia","muscle atrophy","ubiquitin-proteasome system (ups)"],"prmu":["P","P","P","P","P","P","P","M"]}
{"id":"J_Fluoresc-3-1-2064943","title":"Single-Pair FRET Microscopy Reveals Mononucleosome Dynamics\n","text":"We applied spFRET microscopy for direct observation of intranucleosomal DNA dynamics. Mononucleosomes, reconstituted with DNA containing a FRET pair at the dyad axis and exit of the nucleosome core particle, were immobilized through a 30 bp DNA tether on a polyethyleneglycol functionalized slide and visualized using Total Internal Reflection Fluorescence microscopy. FRET efficiency time-traces revealed two types of dynamics: acceptor blinking and intramolecular rearrangements. Both Cy5 and ATTO647N acceptor dyes showed severe blinking in a deoxygenated buffer in the presence of 2% \u03b2ME. Replacing the triplet quencher \u03b2ME with 1 mM Trolox eliminated most blinking effects. After suppression of blinking three subpopulations were observed: 90% appeared as dissociated complexes; the remaining 10% featured an average FRET efficiency in agreement with intact nucleosomes. In 97% of these intact nucleosomes no significant changes in FRET efficiency were observed in the experimentally accessible time window ranging from 10 ms to 10\u2019s of seconds. However, 3% of the intact nucleosomes showed intervals with reduced FRET efficiency, clearly distinct from blinking, with a lifetime of 120 ms. These fluctuations can unambiguously be attributed to DNA breathing. Our findings illustrate not only the merits but also typical caveats encountered in single-molecule FRET studies on complex biological systems.\nIntroduction\nFluorescence (or F\u00f6rster) Resonance Energy Transfer (FRET) is a process in which the energy of an excited donor fluorophore is transferred non-radiatively to an acceptor molecule [1]. The efficiency of energy transfer E is given by:\nwhere R is the distance between donor and acceptor and R0 is the F\u00f6rster radius, at which 50% energy transfer occurs (typically 5\u00a0nm for Cy3\u2013Cy5, a commonly used FRET pair). FRET is a powerful tool to study the structure and function of biological molecules, such as DNA. When extended to the single-molecule level, single pair FRET (spFRET) can potentially be applied to determine the conformational distribution of an ensemble of molecules and the dynamics of individual molecules [2\u20134]. We exploited spFRET to study the structure and dynamics of single nucleosomes, the fundamental units of compaction and organization of eukaryotic DNA.\nThe nucleosome core particle consists of \u223c 50\u00a0nm DNA wrapped nearly twice around a histone-octamer protein-core [5]. Nucleosomal DNA has to unwrap from the nucleosome core to sterically allow processes such as transcription, replication and repair. Accessibility to nucleosomal DNA is facilitated by ATP-dependent remodeling enzymes in vivo [6]. However, it is known that spontaneous conformational changes of the nucleosome expose occluded sites in the DNA as well [7]. DNA breathing, the transient unwrapping and rewrapping of a stretch of DNA from the nucleosome core, has recently been studied in detail with a variety of fluorescence techniques. The equilibrium constant of this process was determined with bulk FRET measurements [8]. Unwrapping lifetimes of 10\u201350\u00a0ms were obtained with stopped-flow FRET measurements and Fluorescence Cross Correlation Spectroscopy (FCCS) [9]. Interestingly, based on their single pair FRET work, Tomschik et al. concluded that unwrapping of nucleosomal DNA occurs to a much larger extent than was previously anticipated [10]: they suggested that 30\u201360% of the nucleosomal DNA was unwrapped with a lifetime on the order of \u223c150\u00a0ms before rewrapping.\nAlthough the conceptual beauty of FRET studies is undisputed, there are a number of important caveats in single-molecule FRET studies of biomolecules, such as fluorophore blinking, photobleaching and sample immobilization. Here, we addressed these issues. spFRET microscopy on mononucleosomes revealed two dominant types of dynamics: acceptor blinking and intramolecular rearrangements that we attribute to DNA breathing, which only became apparent after suppression of blinking. Upon immobilization, we observed three different populations: 90% of the nucleosomes dissociated or represented donor-only species, and 10% remained intact. Of these fully wrapped nucleosomes, 97% showed stable FRET on timescales between 0.01\u201310\u00a0s, while 3% showed dynamics with a dwell time of 120\u00a0ms that we attribute to conformational changes in the nucleosome.\nMaterial and methods\nDNA preparation\nA 177 base pair (bp) DNA was constructed by PCR using the 601 nucleosome positioning element [11] as template. PCR primers were as follows. Forward primer: Biotin-TTTGAATTCC CAGGGAATTG GGCGGCCGCC CTGGAGAATC CCGGTGCCGA GGCCGC (acceptor labeled nucleotide is underlined). Reverse primer: ACAGGATGTA TATATCTGAC ACGTGCCTGG AGACTAGGGA GTAATCCCCT TGGCGGTTAA AACGCGGGGG ACAGCGCGTA CG (donor labeled nucleotide is underlined). We used either Cy3\u2013Cy5 or ATTO550-ATTO647N as donor-acceptor FRET pair. PCR products were purified with a GFX PCR DNA & Gel Band Purification Kit (GE Healthcare). The position of the labels was chosen such that after reconstitution the acceptor was located at the nucleosome exit, and the donor near the dyad axis, as illustrated in Fig.\u00a01. Donor and acceptor were predicted to be \u223c4\u00a0nm apart, as deduced from the nucleosome crystal structure [12], resulting in a FRET efficiency E of approximately 0.8 for the Cy3\u2013Cy5 pair (R0 \u223c5\u00a0nm), and of approximately 0.9 for the ATTO550-ATTO647N pair (R0 \u223c6\u00a0nm).\nFig.\u00a01FRET system for the study of mononucleosome dynamics. a The 177\u00a0bp DNA construct, indicating the position of the labels 80\u00a0bp apart in a fragment containing the 601 nucleosome positioning sequence. A biotin label allowed for immobilization of the construct. b, c Illustrations of the mononucleosome structure, indicating the position of donor and acceptor upon reconstitution. The distance between the labels was \u223c4\u00a0nm, at which efficient FRET takes place. Unwrapping of the DNA from the nucleosome core will be accompanied by a decrease in FRET due to increasing separation between donor and acceptor\nNucleosome reconstitution\nRecombinant histone octamers were mixed with the DNA construct at a 1:1 ratio, in TE (1\u00a0mM EDTA, 10\u00a0mM TRIS pH\u00a08.0) and 2\u00a0M NaCl. Mononucleosomes were reconstituted by salt dialysis against 0.85, 0.65, 0.5 and finally against 0.1\u00a0M NaCl, all buffered with TE.\nBulk fluorescence measurements\nBulk fluorescence experiments were carried out on a Luminescence Spectrometer (LS55, Perkins Elmer). All experiments were performed at room temperature (22\u00b0C). The nucleosome concentration was 10\u201350\u00a0nM. The donor dye was excited at 515\u00a0nm and the emission was recorded from 535 to 700\u00a0nm. The acceptor dye was excited at 615\u00a0nm and the emission was recorded from 635 to 700\u00a0nm, to obtain acceptor-only emission spectra. The FRET efficiency was determined from the enhanced fluorescence of the acceptor using the (ratio)A method [13]:\nwhere  and  are the acceptor and donor extinction coefficient respectively at wavelength \u03bb,  is the fluorescence intensity of the acceptor when excited at wavelength \u03bb, and d+ is the fractional labeling coefficient of the donor. The fluorescence intensity of the acceptor was determined at its maximum value. d+ was determined from DNA and fluorophore absorption peaks in an absorption spectrum of the labeled DNA, measured from 230 to 700\u00a0nm with a spectrophotometer (Pharmaspec UV-1700, Shimadzu).\nSingle-molecule FRET measurements\nCleaned glass slides were amino functionalized with 10\u00a0\u03bcg\/ml poly-d-lysine, and subsequently incubated for 4\u00a0h with an amine reactive polyethylene glycol (PEG) mixture: 20% mPEG-succinimidyl propionate 5,000 molecular weight (Nektar Therapeutics) and 0.2% biotin-PEG-n-hydroxysuccinimide 3,400 molecular weight (Nektar Therapeutics) in 0.1\u00a0M sodium carbonate buffer (pH\u00a08.2). A flow cell was assembled by sealing a poly-dimethylsiloxane channel with a PEG functionalized slide. A 0.1\u00a0mg\/ml streptavidin (Roche) solution was incubated for 5\u00a0min, and subsequently washed away. A sample, which typically consists of 10\u201350\u00a0pM of labeled mononucleosomes in 50\u2013200\u00a0mM NaCl, 10\u00a0mM TRIS.HCl pH\u00a08.0, 0.03% NP-40, and 10\u201350\u00a0nM unlabeled mononucleosomes, was injected in the channel and immobilized. An enzymatic oxygen scavenger system (1% glucose, 2% \u03b2-mercaptoethanol (\u03b2ME) or 1 to 2\u00a0mM Trolox (Sigma), 0.2\u00a0mg\/ml glucose oxidase, and 0.04\u00a0mg\/ml catalase), was added to the buffer to extend the lifetime of the fluorophores before photobleaching. The buffer was degassed prior to use to further reduce the oxygen concentration. The flow cell was mounted on a microscope equipped with a 100X oil-immersion TIRF microscope objective (NA\u2009=\u20091.45, NIKON) and temperature-stabilized at 22\u00b0C using a water circulating bath connected to all parts of the setup in contact with the sample. The 514\u00a0nm line of an Ar+ laser (Coherent) was used to illuminate an area of \u223c 600\u00a0\u03bcm2 with a power of 0.9\u00a0mW. In the case of alternating excitation, a 636\u00a0nm diode laser (Power Technology) was used to illuminate an area of \u223c900\u00a0\u03bcm2 with a power of 0.3\u00a0mW. Both beams were circularly polarized and were displaced parallel to the optical axis of the objective, so that an evanescent excitation field was generated by total internal reflection of the light at the glass-water interface. The excitation intensity at the interface in the evanescent field is \u223c4 times higher than the incident beam intensity at the critical angle [14]. We therefore estimated that the resulting excitation intensities at the interface were \u223c0.6\u00a0kW\/cm2 for the 514\u00a0nm excitation and \u223c0.13\u00a0kW\/cm2 for the 636\u00a0nm excitation respectively.\nThe fluorescence was collected by the objective and filtered through a custom-made dual color band pass filter (Chroma), that rejects scattered laser light, and a long pass filter (OG530, Schott). The fluorescence was further split into a donor and an acceptor channel by a custom-made dichroic wedge mirror (0.5\u00b0 angle, center wavelength of 630\u00a0nm, Chroma) placed in the infinity path of the microscope [15]. A +150\u00a0mm achromatic lens (Thorlabs) projected the separate images on a multiplication gain CCD camera (Cascade 512B, Roper Scientific) operating at a frame rate of 20 to 100\u00a0Hz.\nData analysis\nThe simultaneously acquired donor and acceptor images (typically 80 by 80 pixels) were aligned with respect to one another through their cross correlation. The first 50 donor and acceptor frames were overlaid, and their intensities averaged. Low frequency background signal was filtered out with a high-pass FFT filter. The location of the fluorophores was then determined by applying a threshold of 2 times the background noise level. A time-trace of donor and acceptor intensities was then calculated by integrating the pixel intensities 1.5 pixel around the fluorophore center for each frame and each image. In the case of alternating excitation, the acceptor intensity upon direct excitation was retrieved by deinterleaving the acceptor time-trace. The FRET efficiency was calculated from [2]:\nwhere IA and ID are acceptor and donor intensity respectively, and  is a parameter to correct for photophysical properties of the dyes. \u03a6A and \u03a6D are acceptor and donor quantum yield, and \u03b7A and \u03b7D are acceptor and donor detector efficiency respectively. As a first approximation \u03b3 was set to unity.\nA more accurate estimate for \u03b3 was obtained from experimental intensity time traces where donor bleaching took place after acceptor bleaching. In these cases the FRET efficiency could also be calculated from donor quenching:\nwhere ID0 is the donor intensity after bleaching of the acceptor. Combining Eqs.\u00a03 and 4 results in:\nExperimental results\nBulk fluorescence spectra reveal proper reconstitution of mononucleosomes\nThe results of bulk fluorescence and absorption experiments on reconstituted mononucleosomes are shown in Fig.\u00a02. The reconstituted sample showed efficient FRET, indicated by a distinct peak of fluorescence at the acceptor maximum emission wavelength (670\u00a0nm for ATTO647N, see Fig.\u00a02a). This peak was not present in the labeled DNA-only sample, confirming that the donor and the acceptor were in close proximity due to mononucleosome reconstitution. As a control, we diluted the mononucleosome sample in 2\u00a0M NaCl, as this high ionic strength disrupts nucleosome structure [16]. As predicted, over 90% of the energy transfer signal was lost.\nFig.\u00a02Bulk fluorescence emission and absorption spectra revealed proper reconstitution of mononucleosomes. a Bulk fluorescence emission spectra. A distinct peak of fluorescence at the acceptor emission wavelength was seen after reconstitution, which was not present for the labeled DNA only. The peak disappeared upon dilution of the reconstituted material in 2M NaCl, an ionic strength at which nucleosome structure is disrupted. b Bulk absorption spectrum of the fluorescently labeled DNA construct. The stoichiometry of the labels was obtained by comparison with the absorption spectra of ATTO550 and ATTO647N (as provided by the manufacturer), which are plotted with dotted lines\nThe observed average FRET efficiency in the reconstituted mononucleosomes was 0.75\u2009\u00b1\u20090.1, which was in good agreement with FRET values predicted by the position of the FRET pair in the nucleosome. From the bulk FRET experiments, and the predicted maximum FRET efficiency of \u223c0.9 for a mononucleosome with the ATTO550-ATTO647N FRET-pair, we estimated the reconstitution yield to be at least 85%. Some residual donor emission can be accounted for by incomplete acceptor labeling. With absorption measurements on the DNA construct (Fig.\u00a02b) we determined that the acceptor:donor:DNA stoichiometry was \u223c0.7:0.9:1. Together, these bulk data show that the labeled DNA construct and the histone proteins properly formed mononucleosomes upon reconstitution (see Fig.\u00a01).\nspFRET microscopy reveals individual nucleosomes together with a large population of dissociated nucleosomes\nTo investigate mononucleosome subpopulations and dynamics, spFRET measurements were performed in a wide field microscope. Figure\u00a03 shows an example of typical single-molecule fluorescence images of immobilized mononucleosomes. In Fig.\u00a03a, an acceptor channel image was superimposed on a donor channel image. 10% of the immobilized fluorophores showed efficient FRET, as indicated by colocalized fluorescent spots in the acceptor channel upon donor excitation, and thus represent fully reconstituted mononucleosomes.\nFig.\u00a03Single molecule fluorescence image of immobilized mononucleosomes. a False color representation of averaged donor and acceptor channel images, excited at 514\u00a0nm. The arrows point at molecules that featured efficient FRET from donor to acceptor. The majority of the molecules however did not show FRET and appears in red. b The same field of view excited at 636\u00a0nm, allowing for unambiguous identification of acceptor fluorophores\nIn contrast, 90% of the fluorophores did not show FRET at all. This conflicts with the bulk experiments, where after correction for incomplete labeling an average FRET efficiency of 0.75 was found. As mentioned before, there was a fraction of donor only labeled species (\u223c30%), but this alone could not explain the observed discrepancy between bulk and single-molecule measurement. When we directly excited the acceptor fluorophores (see Fig.\u00a03b), we found that most of the donors were colocalized with an acceptor. Therefore we conclude that FRET signal was lost during the single-molecule measurement, due to disassembly of a large fraction of the nucleosomes.\nIt is known that nucleosomes become unstable and dissociate when they are diluted to low concentrations [17, 18]. For our wide field spFRET measurements we diluted to pM fluorophore concentrations to resolve individual fluorophores. We ensured that the nucleosome concentration was always above 10\u201350\u00a0nM. By adding an excess of unlabeled mononucleosomes and 0.03% non-ionic detergent (NP-40) to our buffer. Thastrom et al. [19] reported that under these conditions nucleosomes do not dissociate in bulk solutions. We found that even 50\u00a0nM of unlabeled nucleosomes, far above the dilution-driven dissociation threshold, did not retain proper nucleosome folding, excluding dilution effects to be the cause.\nIt is known that H2A\u2013H2B histone dimers can spontaneously be exchanged from the protein core [20], which in our case would result in a transient loss of FRET. However, we found the same amount of disassembled nucleosomes upon immobilization when the octamer protein core was crosslinked by dialysis against 0.05% glutaraldehyde in 1\u00a0mM EDTA. We confirmed that the crosslinking itself did not dissociate nucleosomes with bulk fluorescence experiments. This suggests that not the histone protein core dissociates, but rather that the wrapped DNA loosens or significantly rearranges itself around the protein core. We confirmed that mononucleosomes in free solution (in the same buffer used for single-molecule experiments) remain stable for hours at room temperature using bulk fluorescence measurements. Therefore we consider the dissociation of the nucleosomes described here to be associated with their immobilization to the functionalized cover glass. As an alternative immobilization strategy we performed experiments with biotinylated BSA-functionalized cover glasses instead of PEGs. Biotinylated BSA is often used for single-molecule studies involving nucleic acids, whereas PEGs are often used for studies involving DNA-protein complexes [21]. Biotinylated BSA yielded even less intact nucleosomes. The exact nature of the interactions of the nucleosomes with the modified cover slides remains unclear, but the destabilizing effect of the surface forms a hurdle for obtaining large datasets of spFRET measurements.\nOn the 10% immobilized mononucleosomes showing FRET, irreversible loss of FRET was only found after photobleaching, implying that their nucleosomal structure remained intact after immobilization.\nSingle-molecule fluorescence footprint of individual nucleosomes\nExample intensity time-traces of intact single nucleosomes are shown in Fig.\u00a04a and b. Donor and acceptor intensity were clearly anti-correlated, indicative of their FRET interaction. The intensity of a single donor (Cy3) was 1.4\u2009\u00b1\u20090.3\u2009\u00d7\u2009103 counts\/10\u00a0ms at a signal to noise ratio (SNR) of 5. When the donor was quenched by FRET, both the intensity and SNR decreased. The intensity of an acceptor (Cy5) excited via FRET was 0.9\u2009\u00b1\u20090.2\u2009\u00d7\u2009103 counts\/10\u00a0ms at a SNR of 3. After \u223c4\u00a0s of continuous illumination at \u223c0.6\u00a0kW\/cm2 either donor or acceptor photobleached, limiting the observational window to a few seconds. The total number of emitted photons until bleaching from a FRET pair was \u223c105 (calculated with gain G\u2009=\u200933 counts\/photon, detection efficiency \u03b7D and \u03b7A \u223c15%). The observed average FRET efficiency of the high FRET level was \u223c0.5\u2009\u00b1\u20090.13, slightly lower than the values measured in the bulk. From the traces where donor bleaching takes place after acceptor bleaching, we estimated the correction factor \u00e3 for photophysical parameters of Cy3\/Cy5 to be \u223c0.7\u2009\u00b1\u20090.3 (Eq.\u00a04). The corrected FRET efficiency was then \u223c0.6\u2009\u00b1\u20090.3, in good agreement with values obtained from bulk measurements.\nFig.\u00a04Single molecule FRET traces from individual mononucleosomes. The top panels in a and b show the intensity time traces of donor and acceptor for green excitation; the middle panel shows the intensity time traces of donor and acceptor for red excitation, which were acquired in alternation with the green excitations. The bottom panels show the calculated FRET efficiency. The fluctuations between high and low FRET states featured perfect correlation with the corresponding acceptor intensity traces excited at 636\u00a0nm. c Histogram of the FRET efficiencies of multiple single molecule traces. d Histogram of the FRET efficiencies of a single trace. The spread in FRET efficiency was larger between different traces than within a single trace\nWe found that the standard deviation in FRET efficiency of the high FRET state of the entire population (0.13) was larger than the standard deviation within individual traces (0.06), as shown in the histograms in Fig.\u00a04c and d. This observation can be accounted for either by different nucleosome populations with slight variations in FRET efficiency, or by local differences in rotational freedom of the dyes due to immobilization.\nAcceptor blinking is the dominant source of spFRET dynamics\nThe FRET traces shown in Fig.\u00a04a and b are highly dynamic and fluctuate between a high FRET state (E \u223c0.6, lifetime 2.5\u00a0s) and a low FRET state (E \u223c0.1, lifetime 0.13\u00a0s). Interestingly, the characteristics of these fluctuations, i.e. both on and off time, and the low FRET level, are remarkably similar to those observed by Tomschik et al. [10] who performed analogous experiments. This similarity however, is remarkable in view of the completely different FRET-label location. Tomschik et al. labeled the nucleosome opposite to the dyad axis and probed the DNA at the most internal position of the nucleosome, whereas our substrate has labels at the most exterior position. Widom et al. have previously shown that the enzymatic accessibility of the DNA inside a nucleosome strongly reduces as the DNA is more internal in the nucleosome [7], suggesting a higher frequency of unwrapping events in our experiments.\nBecause of the nearly complete absence of acceptor emission, we investigated the nature of these fluctuations in order to exclude reversible transitions of the acceptor to an inactive state (acceptor blinking, resulting in a F\u00f6rster radius of effectively zero [22]) as the origin of these events. By alternating donor excitation with direct acceptor excitation we could directly monitor the acceptor condition as shown in Fig.\u00a03b. After deinterleaving the data into two time-traces, one for green excitation and one for red excitation, it became obvious that the fluorescence intensity of the acceptor upon direct excitation correlated perfectly with the enhanced emission of the acceptor due to FRET. Thus, the low FRET state must be attributed to blinking, due to a dark-state level of the acceptor. Further evidence that these fluctuations were caused by acceptor blinking was provided by experiments with alternative acceptor dye (ATTO647N, emission spectrum similar to Cy5). Alternating excitation of the acceptor dye revealed a strong positive correlation between sensitized emission of the acceptor and direct excitation of the acceptor. In this case the low FRET state was also present, but with a much shorter lifetime of 0.046\u00a0s. In conclusion, our data confirm that the fluctuations between a high and a low FRET state reflect photophysical processes in the acceptor dye rather than nucleosome conformational changes.\nWe further analyzed the single-molecule FRET traces for dynamics other than blinking. Therefore we filtered out blinking events by the application of a threshold on low FRET efficiencies (\u2264 \u223c0.1\u20130.2, dependent on the noise in the measurement). Although careful inspection did occasionally reveal anticorrelated features of donor and acceptor channel, these features had a lifetime below the time resolution of our measurements. To confirm that we did not overlook any dynamics, we analyzed the fluorophore intensity noise in the high FRET state, which in the absence of dynamics should be limited by shot noise. The theoretical noise \u03c3tot in the measurement was estimated by [23]:\nwhere G is the multiplication gain factor, F is the excess noise factor due to the multiplication gain register, S is the number of photons that reach the camera, \u03a6 is the camera quantum yield, D is the dark count, and \u03c3R is the readout noise. The first contribution represents photon shot noise after multiplication, the second contribution represents the camera dark noise after multiplication, and the third the ADC converter electronic noise. Readout noise and dark noise were calculated from the standard deviation of an area of the chip that was not illuminated by fluorescence to be 130 counts\/10\u00a0ms. The actual noise \u03c3 in the single-molecule fluorescence traces was estimated by the standard deviation of the measured fluorophore intensity. The measured and calculated noise were tested for equality with an F test:  where \u03b1 is the significance level at which the test was performed (0.05), and \u03bd1, \u03bd2 are the degrees of freedom used to calculate \u03c3 and \u03c3tot respectively. We found that the total measured noise was significantly (typically 1.5 times) higher than that predicted by photon statistics and camera noise only. This implied that the traces contained dynamic events that cannot be fully resolved, originating from either photophysical processes (short blinking events, or intersystem crossing), or fast nucleosome dynamics. Hence, to accurately capture these events, blinking had to be further suppressed, and the sampling frequency had to be increased.\nSuppression of blinking\nIn order to suppress blinking, we first tested a different acceptor dye (ATTO647N), which was reported to have superior photochemical stability compared to Cy5 [24]. As mentioned before, this acceptor dye showed blinking as well, as seen in the example traces and histograms of Fig.\u00a05a and b. Although a small fraction of molecules did not show any dynamics in FRET, the majority significantly blinked. In the case of Cy5 93% of all acceptors excited via FRET showed significant blinking, with a lifetime of \u223c0.13\u2009\u00b1\u20090.05\u00a0s, and lifetime of the high state of \u223c0.8\u2009\u00b1\u20090.1\u00a0s. In the case of ATTO647N, 94% of all acceptors excited via FRET showed blinking, with a lifetime of \u223c0.046\u2009\u00b1\u20090.02\u00a0s, and lifetime of the high state of \u223c1.2\u2009\u00b1\u20090.2\u00a0s. In conclusion, the use of a different dye did not suppress blinking to the required level, but just yielded different blinking statistics.\nFig.\u00a05Fluorophore blinking in spFRET traces obtained from mononucleosomes. In the presence of \u03b2ME, nucleosomes labeled with Cy5 a or ATTO647N b both show severe blinking in 95% of the traces. Example traces (top) and blinking lifetime histograms (bottom) are shown. c,d In the presence of an alternative triplet quencher, Trolox, blinking of both dyes is significantly suppressed. The example traces (top) show the absence of blinking in 90% of the traces, while the blinking lifetime histograms (bottom) show a small but finite amount of fast blinking still present in \u223c10% of the traces\nRecently a different approach to reduce blinking was described by Rasnik et al. [25]. They replaced the triplet quencher \u03b2ME in the oxygen scavenger system by a water-soluble analog of vitamin E, Trolox. Using this approach, Cy5 blinking in single-molecule FRET measurements on DNA constructs was eliminated. We tested the effect of Trolox in the imaging buffer on blinking of FRET pair labeled mononucleosomes. Results are shown in example traces and histograms in Fig.\u00a05c and d. Both for Cy5 and ATTO647N blinking was dramatically suppressed in the presence of 1 to 2\u00a0mM Trolox: over 90% of the traces showed no observable blinking. Noise analyses of the intensity fluctuations in most of these traces were fully accounted for by camera noise and photon statistics (shot noise) only. Thus, within our time resolution (10\u00a0ms), no effect of short time scale blinking, or inter system crossing, was detected.\nSurprisingly, less than 10% of the observed FRET pairs still showed some extent of blinking indicated by fast excursions into a FRET state below 0.2, with a typical off time of 14\u2009\u00b1\u20091 and 13\u2009\u00b1\u20091\u00a0ms respectively, as shown in the histograms in Fig.\u00a05c and d. Because the lifetime of these blinking events was on the order of the smallest sampling time used, blinking events were not identified by alternating excitation of the acceptor dye, but only by FRET efficiencies below the noise threshold. Direct excitation of the acceptor did only reveal some occasional blinking in the acceptor traces, with the same lifetime of 13\u201314\u00a0ms. To confirm that a small but finite amount of fast blinking still occurred in the presence of Trolox, we performed spFRET measurements on a FRET pair that was separated by 11 basepair duplex DNA. This construct does not exhibit structural changes that affect the FRET intensity. In this case we also observed a small, but finite amount of blinking in a number of traces (data not shown), with a lifetime similar to that measured on mononucleosomes.\nA fraction of the immobilized nucleosomes shows dynamics clearly distinct from blinking\nThe suppression of blinking finally allowed us to unambiguously identify non-blinking dynamic events in the FRET traces. From a sample of 236 mononucleosomes that showed FRET, we found that over 95% of the traces essentially show stable FRET efficiency, as illustrated in Fig.\u00a06a and b; all anti-correlated features in the FRET efficiency were short-lived and fall within the noise of the measurement. Thus, the upper limit for dynamic events that could have been missed in this population was 10\u00a0ms (the sampling time used). Interestingly, 3% of the traces showed dynamic events clearly distinct from blinking (examples shown in Fig.\u00a06c and d), as judged by the following criteria: (1) the acceptor signal of a low FRET event was significantly higher than zero. (2) No correlated change in acceptor intensity was detected using alternating excitation. (3) Events persisted for at least two data-points. We found 14 events with an average FRET change \u0394E of \u22120.23 and an average dwell time of 120\u2009\u00b1\u20095\u00a0ms, as summarized in the histogram and cumulative distribution function in Fig.\u00a06e and f respectively. The lifetime was determined by fitting a cumulative exponential distribution to the data, independent of binning and therefore a more accurate way of determining the lifetime when using small datasets (Fig.\u00a06f) than fitting a distribution to binned data. The lifetime of the high FRET state could not be determined accurately, due to the short time window that was available due to photobleaching. Since we explicitly checked the vitality of both fluorophores, we ruled out photodynamics and we could unambiguously attribute the observed features to DNA breathing dynamics.\nFig.\u00a06A fraction of the immobilized nucleosomes showed dynamics clearly distinct from blinking. a,b After suppression of blinking with Trolox, over 95% of the FRET traces do not show FRET dynamics. The theoretical photon and instrument noise is approximately indicated by the grey bars. c,d \u223c3% of the intensity traces (top panels) showed FRET fluctuations (bottom panels) clearly distinct from blinking: the acceptor intensity was significantly higher than zero, and events persisted multiple data points (see insets). These fluctuations clearly exceeded the noise. e Histogram and cumulative distribution plot f of the lifetime of the dynamic events. An exponential fit to the data gave an average lifetime of 120\u00a0ms\nDiscussion and conclusion\nTime-traces of spFRET microscopy on single reconstituted mononucleosomes revealed two types of dynamics: acceptor blinking and intramolecular rearrangements. Intramolecular rearrangements became only apparent after suppression of blinking. Both Cy5 and ATTO647N showed severe blinking in a deoxygenated buffer in the presence of 2% \u03b2ME. Replacing the triplet quencher \u03b2ME with Trolox effectively eliminated most blinking effects. The lifetime of DNA unwrapping that we obtained after rigorous elimination of blinking events (\u223c120\u00a0ms) was comparable to the 150\u2013180\u00a0ms obtained by Tomschik et al. [10], despite the very different location of the labels in the nucleosome, and probably less important, the different DNA sequence and origin of the histones. However, we observed a very similar lifetime (\u223c130\u00a0ms) for Cy5 blinking under comparable buffer conditions (2% \u03b2ME). The FRET efficiency of the open states in our experiments was significantly above the detection threshold, so we can explicitly exclude photophysics as the origin of the observed changes in FRET efficiency.\nOur single-molecule measurements revealed at least three subpopulations in the reconstituted and immobilized nucleosome sample: 90% of the fluorophores represented dissociated nucleosomes or donor only species, 10% represented intact nucleosomes. Of these, 97% remained stable on time-scales ranging from 10\u00a0ms to 10\u00a0s of seconds, while 3% showed intervals with reduced FRET efficiency and a lifetime of 120\u00a0ms clearly distinct from blinking.\nWhy most nucleosomes dissociate upon immobilization to the cover slip remains unknown. Immobilization of the molecules is necessary for extension of the available observation time. The time limit is given by photobleaching, one of the key advantages of this method with respect to, for example, Fluorescence Correlation Spectroscopy. However, the close proximity to the surface provides ample opportunity for interactions with it. Surface induced nucleosome dissociation has been reported before in Atomic Force Microscopy (AFM) studies. Using AFM in liquid, Nikova et al. observed an unwrapping of \u223c25\u00a0nm of DNA from nucleosomes absorbed to a mica surface [26]. This unwrapping was attributed to a depletion of H2A\u2013H2B histone dimers induced by the high surface charge of the mica, resulting in unwrapping of DNA. Although PEGs are neutral polymers that are commonly used to reduce non-specific surface binding of proteins, they may affect nucleosomes in different ways: PEG molecules have been reported to interact strongly with unfolded proteins [21], and could therefore possibly interact with histone tails. Furthermore, histone proteins are known to be adhesive to glass or plastic [27].\nThe large fraction of dissociated nucleosomes we report here was not observed by Tomschik et al. [10]. Because of the internal position of the labels they used, at least 50\u00a0bp of DNA had to be detached from the histone core before FRET was completely lost. We labeled the DNA at the very end of the histone bound part, and accordingly a detachment of 10\u201320\u00a0bp of DNA would already result in complete loss of FRET. Furthermore, the exterior part of the DNA is largely constrained by the mobile H2A\u2013H2B dimer, whereas the labeled part of the DNA in the nucleosomes used by Tomschik et al. is mostly constrained by the more stable H3\u2013H4 tetramer.\nA labeling strategy by Li et al. [8, 9], who end-labeled a 601 nucleosome positioning element together with either histone H3 or H2A, provides a more comparable construct. Based on stopped-flow FRET and FCCS experiments they deduced an unwrapping rate of 4\u00a0s\u22121 an unwrapping lifetime of 10\u201350\u00a0ms. The 3% of our traces that showed dynamics typically featured multiple unwrapping events before photobleaching. Though photobleaching obstructs quantification of the unwrapping rate, it is of the same order of magnitude as observed by Li et al. The lifetime of the unwrapped state we observed is five to ten times larger. This discrepancy may in part be explained by differences in experimental conditions and nucleosome constructs; we can however not exclude the possibility that we overlook short-lived unwrapped states, biasing our data to a longer lifetime.\nThe absence of observations of DNA unwrapping in the majority of the intact nucleosomes reported in this study is in strong contrast with the extent of DNA breathing dynamics found by Li et al. [8, 9]. Two possible explanations could account for this difference: (1) The most frequently occurring DNA unwrapping occurs at a rate that exceeds the time resolution of our experiment. The rare dynamics (3%) that we observe would reflect the release of multiple histone-DNA contacts, a process that would occur less often and on longer time scales than unwrapping of only the first DNA-octamer. However, unwrapping of 10\u201320\u00a0bp of DNA would induce a more dramatic reduction in FRET efficiency than the reduction we observed, which is consistent with unwrapping of 10\u00a0bp or less. (2) The immobilized nucleosomes did not undergo breathing dynamics. It should be kept in mind that because of the disruption of 90% of the nucleosome upon immobilization, we only probed a subset of nucleosomes that do not dissociate upon immobilization. These nucleosomes could either be resistant to unwrapping of the DNA, or immobilized in such a way that DNA dynamics are inhibited due to interactions with the surface, while still retaining proper folding. In either case, immobilization is expected to have major impact on nucleosome dynamics, emphasizing the need for a more inert immobilization than point attachment to a PEG coated surface.\nOur findings demonstrate that experimental conditions can have a profound impact on the data obtained when probing nucleosome structure and conformational dynamics. Immobilization effects and blinking dynamics have to be accounted for, and where possible suppressed in order to extract biologically relevant data from spFRET experiments. We have shown that DNA breathing kinetics obtained from carefully optimized spFRET experiments approaches values obtained from bulk experiments, opening the way to more complex single-molecule studies of chromatin dynamics.","keyphrases":["fret","nucleosome","blinking","single-molecule"],"prmu":["P","P","P","P"]}
{"id":"Cardiovasc_Intervent_Radiol-3-1-2039794","title":"Sexuality and Body Image After Uterine Artery Embolization and Hysterectomy in the Treatment of Uterine Fibroids: A Randomized Comparison\n","text":"In this paper the effect of uterine artery embolization (UAE) on sexual functioning and body image is investigated in a randomized comparison to hysterectomy for symptomatic uterine fibroids. The EMbolization versus hysterectoMY (EMMY) trial is a randomized controlled study, conducted at 28 Dutch hospitals. Patients were allocated hysterectomy (n = 89) or UAE (n = 88). Two validated questionnaires (the Sexual Activity Questionnaire [SAQ] and the Body Image Scale [BIS]) were completed by all patients at baseline, 6 weeks, and 6, 12, 18, and 24 months after treatment. Repeated measurements on SAQ scores revealed no differences between the groups. There was a trend toward improved sexual function in both groups at 2 years, although this failed to reach statistical significance except for the dimensions discomfort and habit in the UAE arm. Overall quality of sexual life deteriorated in a minority of cases at all time points, with no significant differences between the groups (at 24 months: UAE, 29.3%, versus hysterectomy, 23.5%; p = 0.32). At 24 months the BIS score had improved in both groups compared to baseline, but the change was only significant in the UAE group (p = 0.009). In conclusion, at 24 months no differences in sexuality and body image were observed between the UAE and the hysterectomy group. On average, both after UAE and hysterectomy sexual functioning and body image scores improved, but significantly so only after UAE.\nIntroduction\nWhen hysterectomy looms as a definite treatment for fibroid related menorrhagia, women and their sexual partners often express their concern about negative effects of the operation on their sexual wellbeing [1\u20134]. Furthermore, the uterus is considered important for a woman\u2019s self-image and sexual image and some women fear that they will be \u201cless of a woman\u201d to their sexual partners [2, 4, 5]. Therefore a hysterectomy may also interfere with the woman\u2019s body image. These concerns may cause some women to choose alternative treatment options [5]. During the last decade, uterine artery embolization (UAE) has emerged as an alternative treatment for hysterectomy [6]. UAE has been investigated in several case series and three randomized controlled trials [7\u201313], but the advantage of UAE in terms of sexual wellbeing and body image in comparison to hysterectomy remains unknown, since randomized controlled trials on this subject are not available as yet. Randomized controlled data are preferential to nonrandomized data, since nonrandomized data tend to overestimate treatment effect and are prone to selection bias [14]. We initiated a randomized trial comparing UAE and hysterectomy and have published several short-term results previosly [15\u201317]. In the present paper, we compare changes in sexuality and body image in patients with symptomatic uterine fibroids who were randomly assigned to either UAE or hysterectomy.\nMaterials and Methods\nStudy Design\nThe details of our study design have been described elsewhere and are discussed here briefly [15]. The EMMY trial (EMbolization versus hysterectoMY) is a prospective randomized multicenter clinical trial with 28 participating hospitals in the Netherlands. Eligible patients met the following inclusion criteria: they were clinically diagnosed with uterine fibroids (confirmed by ultrasonography), had menorrhagia as the major complaint, were premenopausal, and had no wish to conceive. All patients were candidates for undergoing a hysterectomy: other treatment options either had failed, were undesired, or had provided unsatisfactory results.\nEligible patients were informed verbally and in writing about possible risks and benefits of both procedures, and were invited to participate in the trial. After written informed consent had been obtained, patients were randomly assigned (1:1) to UAE or hysterectomy by computerized randomization, stratified for hospital. The study was approved by the Dutch Central Committee Involving Human Subjects (www.ccmo.nl) and the local ethics committees of all participating hospitals.\nPre-assessment\nAll patients were assessed by the attending gynaecologist. Current symptoms and a complete medical and gynaecological history were recorded. All patients underwent a pelvic ultrasound to determine the number of fibroids and the size of the largest fibroid. Sociodemographic characteristics were assessed by means of a questionnaire.\nProcedures\nUterine artery embolization\nUAE was performed by an interventional radiologist. A catheter was introduced into the femoral artery and advanced over the aortic bifurcation to the contralateral internal iliac artery, and digital subtraction angiography was performed to identify the origin of the uterine artery. When catheters were placed correctly, the UAE was carried out. Polyvinyl alcohol (PVA) particles of 355\u2013500\u00a0\u03bcm were used in all procedures. UAE patients were advised to refrain from sexual intercourse for at least 2 weeks and thereafter, depending on their complaints.\nHysterectomy\nThe type of hysterectomy and the route of access were determined by the gynecologist. The following procedures were allowed: abdominal hysterectomy, vaginal hysterectomy, laparoscopically assisted vaginal hysterectomy, and laparoscopic hysterectomy. Both supravaginal and total hysterectomies were allowed. After discharge hysterectomy patients were advised not to have sex until the first outpatient visit at 6 weeks.\nQuestionnaires\nSexual functioning and body image were assessed by means of patient questionnaires. At baseline, questionnaires were completed by all patients before randomization. Follow-up questionnaires were completed at 6 weeks and 6, 12, 18, and 24 months after treatment. The questionnaire consisted of the Sexual Activity Questionnaire (SAQ), the Body Image Scale (BIS), and the Mental Component Summary (MCS; as part of the Medical Outcome Study Short Form [MOS SF-36]).\nThe SAQ, originally designed to investigate sexual functioning in women treated with tamoxifen because of a positive family history of breast cancer [18], is also useful in women with nonmalignant gynecological disorders [19]. In Table\u00a01 the SAQ is displayed. The questionnaire consists of three dimensions, and each dimension yields its own score: pleasure from sexual intercourse (desire, enjoyment, and satisfaction; score range, 0\u201318), discomfort during intercourse (score range, 0\u20136), and habit (frequency; score range, 0\u20133). Pleasure and habit are good with higher scores, while discomfort is worse with higher scores. The SAQ was only filled out by patients who were sexually active during the month before receiving the questionnaire.\nTable\u00a01The Sexual Activity Questionnaire (SAQ) and the Body Image Scale (BIS)SAQ\u00a0Dimension pleasure\u00a0\u00a01. Was having sex an important part of your life in the last month?\u00a0\u00a02. Did you enjoy sexual activity in the last month?\u00a0\u00a03. Did you desire to have sex with your partner in the last month?\u00a0\u00a04. In general were you satisfied after sexual activity in the last month?\u00a0\u00a05. How often did you engage in sexual activity in the last month?\u00a0\u00a06. Were you satisfied with the frequency of sex in the last month?\u00a0Dimension discomfort\u00a0\u00a01. Did you notice dryness of your vagina in the last month?\u00a0\u00a02. Did you feel pain or discomfort in the last month?\u00a0Dimension habit\u00a0\u00a01. How did the frequency of sexual behavior in the last month compare with what is usual for you?BIS\u00a0In the last month\u00a0\u00a01. Have you been feeling self-conscious about your appearance?\u00a0\u00a02. Have you felt less physically attractive as a result of your menstrual bleeding problem?\u00a0\u00a03. Have you been dissatisfied with your appearance when dressed?\u00a0\u00a04. Have you been feeling less feminine as a result of your menstrual bleeding problem?\u00a0\u00a05. Did you find it difficult to look at yourself naked?\u00a0\u00a06. Have you been feeling less sexually attractive as a result of your menstrual bleeding problem?\u00a0\u00a07. Did you avoid people because of the way you felt about your appearance?\u00a0\u00a08. Have you been feeling the treatment has left your body less whole?\u00a0\u00a09. Have you felt dissatisfied with your body?\u00a0\u00a010. Have you been dissatisfied with the appearance of your scar? Questions on the BIS shown in italics have been omitted\nFurthermore, participating patients were asked to judge their current sexual life in two questions: \u201cHow would you describe the quality of your current sexual life?\u201d and \u201cHow well can you live with your current sexual life?\u201d One of the following responses could be ticked: \u201cvery good,\u201d \u201cgood,\u201d \u201cmoderately good,\u201d \u201cneither good nor bad,\u201d \u201cmoderately bad,\u201d \u201cbad,\u201d and \u201cvery bad.\u201d\nThe BIS was designed in order to asses changes in body image among cancer patients [20]. The questionnaire was also found reliable and valid in women with benign gynecological conditions [21]. Table\u00a01 displays the questions of which the BIS consists. Unfortunately, questions 2, 4, and 6 were not represented correctly in the questionnaire due to a translation error. Therefore we decided to omit these questions in order to attain a comparable outcome between groups. The BIS score ranges from 0 to 30. A higher BIS score indicates a worse body image.\nIn order to assess mental health before treatment as a predictor of worse sexual function after treatment, the MCS score of the 36-item MOS SF-36 was used [22]. This was done because bad preoperative mental health is associated with bad sexual life outcome [23].\nSample Size and Endpoints of the Present Study\nThe sample size was based on the primary endpoint of the clinical study, published elsewhere [24]: the elimination of menorrhagia in such way that hysterectomy could be avoided in the UAE group in at least 75% of patients within 2 years after the primary interventions.\nThe objective of the present study was to compare the following endpoints between both interventions: the number of sexually active patients, the scores yielded by the SAQ and the BIS, the subjective quality of sexual life. For this analysis, no separate power calculation was made.\nStatistical Analysis\nData entry was performed using SPSS data entry for Windows 3.0. A random sample of 10% of the questionnaires was visually double checked by an independent second investigator, revealing a false entry level of 0.3%. All false data entries were corrected.\nAll analyses were performed using SPSS (release 11.5.1) statistical software. Missing items in the questionnaire were treated as follows. For the SAQ no advice is available on scoring missing items. If one item was missing, we regarded the dimension score (which the item was part of) as being missing completely, according to earlier research [25]. For the BIS no missing values were allowed since three questions were omitted already. In these cases, the entire score was omitted. For the three omitted questions, the individual mean score of the remaining seven questions was imputed.\nFirst, we determined how many patients in both treatment groups were sexually active before and after treatment. An overall percentage per treatment group was calculated, after which sexual activity in subgroups was assessed: those being sexually active and those not being sexually active before treatment. Second, the mean of the dimension scores were plotted for the hysterectomy and UAE group. Third, the mean differences between all follow-up SAQ dimension scores and baseline were compared between treatment groups. This was only possible for patients who were sexually active both before and after treatment. Within group changes were analyzed as well. Similar analyses were performed for the BIS. Differences in pleasure, discomfort, habit (SAQ), and body image (BIS) between groups over time were tested with repeated measurement analysis, excluding the 6-week measurement for the SAQ, since a high proportion of patients was expected not to be sexually active at that time and therefore not to yield a score. Differences in continuous variables were tested with Student\u2019s t test. Differences in data with skewed distributions were tested with the nonparametric Mann-Whitney U test.\nThe two added questions on the quality of current sexual life were compared between the groups using the chi-square test (or Fishers\u2019 exact test when appropriate). Furthermore, the quality of sex life and the additional questions at the various follow-up moments were compared to baseline, yielding two different options: \u201cworse,\u201d \u201cthe same,\u201d or \u201cbetter\u201d compared to baseline. Differences between the groups were compared with the chi-square test (or Fishers\u2019 exact test when appropriate).\nLogistic regression analysis was performed to investigate variables associated with a worse sexual life quality at 24 months after treatment compared to baseline versus unchanged or improved quality of sexual life. First, univariate analysis was performed with the following baseline characteristics: intended treatment, age, BMI, parity, ethnicity, having a partner, employment status, smoking status, number of fibroids, uterine volume measured by ultrasound, dominant fibroid volume measured by ultrasound, existence of any comorbid disease, and mental health status (MCS-MOS-SF36) at baseline. Second, covariates with p values <0.1 in the univariate analysis were selected for multivariate analysis.\nAll analyses were two-tailed and carried out based on the intention-to-treat principle. A p value <0.05 (two-tailed) was considered statistically significant.\nResults\nPatients were enrolled between March 2002 and February 2004. Of 349 eligible patients, 177 were randomized: 89 were allocated hysterectomy and 88 were allocated UAE. In the hysterectomy group 14 patients refused the allocated treatment, compared to 7 patients in the UAE group, and these patients withdrew from participation. In 4 UAE patients embolization failed bilaterally. These patients subsequently had a hysterectomy but were analyzed in the UAE group according to the intention-to-treat principle (Fig.\u00a01) [15]. The baseline characteristics of all randomized patients are reported in Table\u00a02. No differences between the groups are apparent, as expected considering the randomized design.\nFig.\u00a01FlowchartTable\u00a02Baseline characteristicsUAE (N = 88)Hysterectomy (N = 89)Age (yr), mean (SD)44.6 (4.8)45.4 (4.2)Body mass index (weight [kg]\/length [m]2), mean (SD)26.7 (5.6)25.4 (4.0)Parity (n)\u00a0\u00a0030 (34.1%)20 (22.5%)\u00a0\u00a0\u2265158 (65.9%)69 (77.5%)Ethnicity (n)\u00a0\u00a0Black24 (27.3%)20 (22.5%)\u00a0\u00a0White54 (61.4%)57 (64.0%)\u00a0\u00a0Other10 (11.4%)12 (13.5%)Marital statusa (n)\u00a0\u00a0Single16 (18.2%)13 (14.8%)\u00a0\u00a0Married55 (62.5%)54 (61.4%)\u00a0\u00a0Living apart together5 (5.7%)4 (4.5%)\u00a0\u00a0Divorced12 (13.6%)15 (17.0%)\u00a0\u00a0Widow0 (0%)2 (2.3%)Partner relationshipa (n)\u00a0\u00a0No partner13 (15.3%)19 (22.4%)\u00a0\u00a0Partner72 (84.7%)66 (77.6%)Employment statusa (n)\u00a0\u00a0Employed68 (77.3%)69 (78.4%)\u00a0\u00a0Unemployed20 (22.7%)19 (21.6%)Smoking status (n)\u00a0\u00a0Current smoker21 (23.9%)23 (25.8%)\u00a0\u00a0Former smoker11 (12.5%)14 (15.7%)\u00a0\u00a0Nonsmoker56 (63.6%)52 (58.4%)Comorbid diseaseb\u00a0\u00a0Any comorbid disease24 (27.3%)22 (24.7%)Number of fibroids, median (range)2 (1\u201320)2 (1\u20139)Uterine volume (cm3), median (range)321 (31\u20133005)313 (58\u20133617)Fibroid volume (dominant fibroid; cm3), median (range)59 (1\u2013673)87 (4\u20131641)Mental Component Summary (SF-36), mean (SD)40.9 (10.7)41.5 (11.0)aSome values are missingbAny of the following: hypertension, diabetes, astma, clotting disease, system disease, or other\nThe proportion of questionnaires available for analysis ranged from 96.0% (baseline) to 98.7% (6 weeks, 24 months). Before treatment 54 of 81 (67%) and 46 of 75 (61%) of the participating patients were sexually active in the UAE and hysterectomy groups, respectively (Fig.\u00a02A). Six weeks after treatment, sexual activity had decreased in both groups, with significantly more patients showing sexual activity in the UAE group compared to hysterectomy (53% versus 29%; p = 0.004). Hereafter sexual activity was restored in both groups, with no significant differences between the groups at 24 months (p = 0.07).\nFig.\u00a02Proportion of sexually active women, by treatment strategy, over time. (A) The weighted average of women who were not sexually active at baseline (B) and those who were sexually active at baseline (C)\nPatients who were not sexually active before treatment gradually resumed sexual activity after both UAE and hysterectomy (Fig.\u00a02B). After 24 months 31% (hysterectomy) and 52% (UAE) were sexually active (p = 0.118). No significant differences were observed between the groups.\nAmong those patients who were sexually active before treatment, there was a major drop in activity at 6 weeks after treatment, which was partially restored. After 2 years, 86% (UAE) and 90% (hysterectomy) of these patients had resumed their sexual activities (Fig.\u00a02C). Only at 6 weeks had significantly more UAE patients resumed sexual activity (p = 0.01).\nFigure\u00a03 shows the course of the SAQ dimensions pleasure, discomfort, and habit and the BIS scores over time. There were no differences between the groups at baseline (pleasure, p = 0.76; discomfort, p = 0.44; habit, p = 0.77; body image,: p = 0.60). Repeated measurements revealed no differences between the groups for the SAQ dimensions pleasure (p = 0.343), discomfort (p = 0.246), and habit (p = 0.453) or for body image scores (p = 0.359). Table\u00a03 shows the changes in dimension scores per treatment group for patients who were sexually active at baseline. Positive numbers for pleasure and habit and negative numbers for discomfort and body image are indicative for improvement compared to baseline. No significant differences were found between the two treatment groups at all time points, except for body image at 6 months after treatment: body image had improved significantly more in UAE patients than in hysterectomy patients (UAE, \u20131.34, versus hysterectomy, no change; difference, \u20131.34; 95% CI, \u20132.50 to \u20130.18; p = 0.02). Within the groups, however, a significant improvement of various dimension scores was noted (indicated in boldface): at 24 months UAE patients reported significantly less discomfort, a higher frequency of intercourse, and a better body image compared to baseline (p = 0.022, p = 0.022, and p = 0.009 respectively). At 24 months hysterectomy patients improved on all dimensions compared to baseline, but differences were not statistically significant.\nFig.\u00a03Pleasure, discomfort, habit, and body image, by treatment strategy, over timeTable\u00a03Mean differences in pleasure, discomfort, habit, and body image compared to baseline, by treatment strategy, over timeUAEHysterectomyp valueSAQ: pleasure (0\u201318)a\u00a0\u00a06 wk0.29\u22120.500.47\u00a0\u00a06 mo1.631.230.61\u00a0\u00a012 mo0.951.490.50\u00a0\u00a018 mo1.860.680.13\u00a0\u00a024 mo0.891.180.74SAQ: discomfort (0\u20136)b\u00a0\u00a06 wk\u22120.25\u22120.210.96\u00a0\u00a06 mo\u22120.58\u22120.320.41\u00a0\u00a012 mo\u22120.47\u22120.470.98\u00a0\u00a018 mo\u22120.51\u22120.290.51\u00a0\u00a024 mo\u22120.43\u22120.490.88SAQ: habit (0\u20133)a\u00a0\u00a06 wk\u22120.030.000.92\u00a0\u00a06 mo0.480.280.30\u00a0\u00a012 mo0.180.420.24\u00a0\u00a018 mo0.270.190.70\u00a0\u00a024 mo0.280.220.74BIS (0\u201330)b\u00a0\u00a06 wk\u22121.27\u22120.280.10\u00a0\u00a06 mo\u22121.340.000.02\u00a0\u00a012 mo\u22120.240.080.64\u00a0\u00a018 mo\u22121.24\u20130.280.15\u00a0\u00a024 mo\u22121.06\u20130.500.36Note. SAQ, Sexual Activity Questionnaire; BIS, Body Image Scale. Boldface numbers indicate a significant difference from baseline within group (p < 0.05)aA higher score represents more favorable sexual functioning (pleasure, habit)bA lower score represents more favorable sexual functioning (discomfort) or body image\nTable\u00a04 reports the patients\u2019 experienced quality of their sexual life at 6 weeks and 12 and 24 months. No differences between groups were observed. Table\u00a05 presents the proportion of patients who judged the quality of sexual life as better, comparable to, or worse compared to baseline based on the question: \u201cHow is the quality of your current sexual life?\u201d A minority of patients reported worsened sexual life quality compared to baseline at all time points. At 24 months the proportion of patients reporting a worse sexual life compared to baseline was slightly higher in the UAE than in the hysterectomy group, but the difference was not statistically significant (29.3% versus 23.5%; p = 0.43).\nTable\u00a04Satisfaction with sexual life and ability to cope with sexual life, by treatment strategy, over timeBaseline6 weeks12 months24 monthsUAE (N = 81)Hyst. (N = 75)p valueUAE (N = 81)Hyst. (N = 75)p valueUAE (N = 81)Hyst. (N = 75)p valueUAE (N = 81)Hyst. (N = 75)p valueHow is the quality of your current sex life?\u00a0\u00a0Very good1580.84710.487110.358110.54\u00a0\u00a0Good2227252231182921\u00a0\u00a0Somewhat good15131213911812\u00a0\u00a0Neither good nor bad1212231915211615\u00a0\u00a0Somewhat bad66445388\u00a0\u00a0Bad43583444\u00a0\u00a0Very bad33214251How well can you live with your current sex life?\u00a0\u00a0Very good930.771160.3318170.9320170.78\u00a0\u00a0Good2021373035303431\u00a0\u00a0Somewhat good1012151611131011\u00a0\u00a0Neither good nor bad201710564106\u00a0\u00a0Somewhat bad89251325\u00a0\u00a0Bad44252222\u00a0\u00a0Very bad65011100Table\u00a05Changes in sexual wellbeing compared to baseline, by treatment strategy, over timeUAE (n = 81)Hysterectomy (n = 75)p value6 months\u00a0\u00a0Worse16130.68\u00a0\u00a0The same2527\u00a0\u00a0Improved322512 months\u00a0\u00a0Worse17140.81\u00a0\u00a0The same2926\u00a0\u00a0Improved252724 months\u00a0\u00a0Worse22160.32\u00a0\u00a0The same2720\u00a0\u00a0Improved2632\nUnivariate logistic regression analysis revealed BMI, number of fibroids, and presence of comorbid disease to be associated with a worsened quality of sexual life at 24 months compared to baseline (p < 0.1). Allocated treatment and mental health at baseline were not associated with worse outcome.\nBMI was not significant in the multivariate analysis (p = 0.163). The other two parameters remained significantly associated with worse outcome: a higher number of fibroids (OR, 0.69; 95% CI, 0.51\u20130.94; p = 0.018) predicted a decreased risk of a worsening sexual life at 24 months compared to baseline, while the presence of comorbid disease (OR, 3.20; 95% CI, 1.38\u20137.41; p = 0.007) was associated with an increased risk of a worse sexual life quality at 24 months after treatment compared to baseline.\nDiscussion\nIn our trial no differences, on average, in sexual function at 24 months were observed between UAE and hysterectomy. After both treatments the dimensions \u201cpleasure\u201d and \u201chabit\u201d improved, while the dimension \u201cdiscomfort\u201d decreased at 24 months compared to baseline, although only the UAE group showed significant improvement in discomfort and frequency of intercourse.\nSexuality and body image after UAE have never been evaluated in a randomized controlled trial before. However, some nonrandomized series have addressed this subject. The improvement of sexual functioning after UAE was in accordance with the limited number of earlier studies on this subject [26\u201328], which reported a significant improvement in sexual functioning as well. Sexual functioning after hysterectomy has received much attention in the literature. Improvement in sexual functioning after hysterectomy might indirectly be associated with the consequences of surgery, such as less worry about unwanted pregnancy, absence of vaginal bleeding, and more time for sexual activities by the cessation of monthly periods [29]. Most reports on sexual functioning after hysterectomy found significant benefits in various aspects of sexual functioning [25, 30\u201335]. In contrast, the improvement in sexual functioning within our hysterectomy group at 24 months of follow-up was not statistically significant in our trial. This might be explained by insufficient power, which is partly due to the relatively small sample size for the SAQ scores in our population: change scores were only available from women who were sexually active at baseline and at follow-up. Furthermore, the studies on sexuality after hysterectomy used varying follow-up intervals, ranging from 6 months to 2 years. In our series, hysterectomy patients experienced significantly improved pleasure and habit scores at 12 months of follow-up. These results might indicate that improvement in sexual functioning after hysterectomy could be temporary. On the other hand, a review article recently suggested that there is no scientific proof for either improvement or deterioration of sexual functioning after hysterectomy, unless hysterectomy was performed based on a sound clinical indication [29]. In our patient group, however, there was no doubt about the indication: complaints were serious and lengthy enough to warrant a hysterectomy.\nAlthough, on average, SAQ scores for the group as a whole improved, there was a small group of women who reported a deterioration in self-reported quality of sexual life, consistent with other reports [30\u201332, 34, 36]. For obvious reasons, the prospects of deterioration of sexual life might be of greater concern to patients than the improvement after treatment. Regression analysis of worsened sexual life revealed that women with a low number of fibroids (possibly representing a group of women with less severe complaints) and women with other concomitant chronic diseases might not benefit from fibroid treatment in terms of sexual life improvement.\nThe improved body image after UAE in our series is in accordance with another study that reported higher self-consciousness after UAE [26]. After hysterectomy a period of temporary worsened body image following the operation has been described previously [25, 36, 37]. This was not confirmed by our results, but in accordance with these studies, a normal or even improved body image was found 1 year after hysterectomy.\nThere are several limitations of our study that need to be addressed. First, the values of the SAQ dimensions were based on all sexual active women at the various points in time. Reported values may reflect an ever-changing group of sexually active women. At any stage, some women may cease while others may initiate sexual activity. Of all non-sexually active women before treatment, 50% became active after treatment. When comparing pre- and posttreatment scores, these women cannot be included, since their baseline scores were missing.\nSecond, the significant difference between resumption of sexual activity between the groups at 6 weeks is probably explained by differences in counseling between both treatments as mentioned earlier: hysterectomy patients in our trial were told not to have sex until the first outpatient visit at 6 weeks, while UAE patients were advised to refrain from intercourse for at least 2 weeks and thereafter, depending on their complaints.\nThird, we allowed all kinds of hysterectomies, thereby possibly biasing the results in the hysterectomy group. However, various reports found no long-term differences in sexual functioning after various surgical routes of hysterectomy [25, 32, 35].\nFinally, as described above, three questions on the BIS were omitted because of wrongly posed questions, thereby creating asymmetry in the treatment arms. Since the remaining questions in both groups were identical, the comparison between the groups is still valid but reduces the comparability with other series.\nIn conclusion, at 24 months no differences in sexuality and body image were observed between the UAE and the hysterectomy groups. After both UAE and hysterectomy, on average, sexual functioning and body image scores improved, but significantly so only after UAE.","keyphrases":["sexuality","body image","uterine artery embolization","hysterectomy","fibroid","leiomyoma"],"prmu":["P","P","P","P","P","U"]}
{"id":"Pediatr_Nephrol-3-1-1805047","title":"Normative data on the Bonn Risk Index for calcium oxalate crystallization in healthy children\n","text":"Bonn Risk Index (BRI) is being used for the assessment of urinary calcium oxalate (CaOx) crystallization. There are no published data regarding BRI during growth. The objective of this study was to establish age- and sex-dependent BRI values in healthy children and adolescents. A total of 1,050 Caucasian subjects aged 3\u201318 years (525 males, 525 females) without a history of kidney stone disease were enrolled in the cross-sectional study. The study group was divided into 15 ranges according to age, each comprising 70 subjects. Urinary ionized calcium [Ca2+] was measured using a selective electrode while the onset of spontaneous crystallization was determined using a photometer and titrating with 40 mmol\/L ammonium oxalate (Ox2\u2212). The calculation of BRI value was based on the ratio of [Ca2+] to the required amount of ammonium oxalate added to 200 ml of urine to induce crystallization. The median BRI was 0.26 1\/L and the values of the 5th and 95th percentiles were 0.06 1\/L and 1.93 1\/L, respectively. BRI correlated positively with body-area-related BRI (1\/L \u00d7 1.73 m2) (R = 0.18; P < 0.05), whereas a negative correlation was found between BRI and body weight (1\/L \u00d7 kg) (R = \u22120.85; P < 0.05). Neither sex nor age differences were detected in BRI across studied children and adolescents. The values of Bonn Risk Index were constant during growth and there was a limited influence of age and sex on BRI in children over 3 years of age. The BRI may be valuable in the evaluation of pediatric patients at risk for kidney stones, particularly if the BRI from stone formers is demonstrated to be higher than in normal children.\nIntroduction\nUrolithiasis is a frequently reported condition in children and is diagnosed even in neonates and infants [1, 2]. The disease may be the first sign of congenital and acquired metabolic disturbances, or the consequence of anatomic or genetic abnormalities [2]. The majority of kidney stones are composed of calcium oxalate and calcium phosphate [1]. Insight into this pathology is increasing and is focused on the pathogenesis of deposit formation in the urinary tract [3\u20135]. Investigation is being conducted into more effective methods which would enable the detection of risk factors for urolithiasis. One of the risk factors is an increase in the crystallization of calcium oxalate (CaOx) in urine. Kavanagh and Laube recently published a review of methods used to assess the crystallization of CaOx in urine [6]. During the last few years, activity products (AP) of crystallization have usually been assessed by means of the APCaOx index or as the relative supersaturation (RS) of urinary calcium oxalate (RSCaOx) using the computer program EQUIL [7, 8].\nLaube et al. showed that the ratio of calcium ions [Ca2+] to the amount of ammonium oxalate added to 200\u00a0ml of urine to induce crystallization [designated here as (Ox2\u2212)] may be, at the moment of spontaneous crystallization of CaOx, an indicator of the risk of CaOx crystal formation [9]. The authors made this determination with a direct urine collection, without an initial processing. This ratio is known as the Bonn Risk Index (BRI): BRI=[Ca2+]\/(Ox2\u2212) 1\/L [9, 10]. Due to its characteristics, this index is an accurate indicator of the individual state of balance between the quantities of the most important promoters and inhibitors of the crystallization process within urine [9, 10]. Measuring BRI is simple, cost-effective, and the results are repeatable. In patients with calculi formation, CaOx assessed using BRI is significantly higher when compared to healthy subjects [9]. A lack of published studies concerning BRI in pediatric patients led us to conduct the study in children and adolescents. The purpose of this study was to define the BRI value in healthy subjects aged 3\u201318\u00a0years, in relation to age and sex.\nMaterials and methods\nThe study was performed on a group of 1,050 healthy Caucasian children and adolescents (group I) aged 3\u201318\u00a0years (mean\u2009\u00b1\u2009SD: 10.51\u2009\u00b1\u20094.33), comprised of 525 boys and 525 girls. The study population was divided into 15 age groups, consisting of 70 children in each 1-year group (35 boys and 35 girls). The children reported no history of dietary restrictions. All participants met the criteria of the standard dietary energy and nutrient intakes recommended in Poland [11]. These children were free of infection at the time of the examination (serum C-reactive protein CRP <0.4\u00a0mg\/dL, blood leukocyte count <10\u2009\u00d7\u2009109\/L). Prior to inclusion, all participants were screened regarding serum levels of protein, albumin, calcium, phosphate, potassium, uric acid, creatinine and alkaline phosphatase as well as urine concentrations of citrate, oxalate, potassium, calcium and phosphate. Urinary dipstick testing (Bayer Diagnostics, Bridgend, UK) detecting nine parameters, including leukocytes, protein and blood, did not reveal any abnormalities. Children with a family history of kidney stones were excluded from the study. Subjects with diseases known to affect oxalate, calcium and phosphate metabolism and children treated with antibiotics were excluded. All children were screened using renal ultrasound examination to exclude urolithiasis (Toshiba SSH-140A apparatus; probe Convex 3.75\u00a0MHz). Participants and their legal guardians gave informed consent, and the study was approved by the Ethical Committee of the Medical University of Bialystok.\nBonn Risk Index\nThe Bonn Risk Index was assessed using the method of Laube [12]. Each child had a 24-h urine collection into sterile containers, without additional preserving substances, which was stored at 4\u00b0C. The testing was always performed twice using the same urine collection from each subject. Two consecutive urine samples (each 200\u00a0mL) were incubated immediately after collection, at a temperature of 37\u00b0C and the calcium ion concentration was measured using calcium ion-selective electrodes of type Rapilab 855 (Bayer, Germany) and titrated with ammonium oxalate solution (40\u00a0mmol\/L) at a rate of 0.75\u00a0mL\/min. The onset of spontaneous crystallization was detected using an Eppendorff photometer (filter 585\u00a0nm) with a decrease in light transmission to 98% of the initial value. Each analysis was repeated twice. The BRI is presented as [Ca2+] mmol\/L \/(Ox2\u2212) mmol\u00a0=\u00a01\/L. Calibration and quality assurance procedures, based on the calibration curves, were conducted every day.\nStatistical analysis was performed using the program Statistica 6.0\u00a0PL. Mann-Whitney test was used for the analysis of two non-parametric independent variables, with P\u2009<\u20090.05 considered statistically significant. Assessment of the rank of two independent variables was conducted using Spearman correlation, with P\u2009<\u20090.05 considered statistically significant. For the purpose of plotting the curve of spontaneous crystallization (an association between the number of calcium ions and the amount of added ammonium oxalate leading to the spontaneous crystallization), we used the computer program with the range of values as a scatterplot with the option of adding curves.\nResults\nThe characteristics of the study group are presented in Fig.\u00a01. The anthropometric traits of participants, based on weight and height measurements and body mass index (BMI), were within the normal range in each subgroup, relative to the Polish references described by other authors [13].\nFig.\u00a01Body mass index (BMI) in studied girls and boys aged 3\u201318\u00a0years compared to age and sex-matched reference range\nFigure\u00a02 shows the detailed results for the whole study group for BRI, defined as the ratio of [Ca2+] concentration in urine to the amount of added ammonium oxalate (Ox2\u2212) necessary for the spontaneous crystallization of CaOx. BRI in healthy children ranged between 0.06 and 1.93 1\/L. The values between the 5th and 95th percentiles are found between the two borderline arrows. This diagram also presents the minimum and maximum concentrations of [Ca2+] and the required amount of added oxalate (Ox2\u2212). The concentration of [Ca2+] in urine ranged from 0.25\u00a0mmol\/L (5th percentile) to 0.89\u00a0mmol\/L (95th percentile) with a median of 0.42\u00a0mmol\/L, and an amount of added (Ox2\u2212) ranging from 0.46\u00a0mmol (5th percentile) to 3.53\u00a0mmol (95th percentile) with a median of 1.65\u00a0mmol.\nFig.\u00a02The spontaneous crystallization of CaOx in urine of 1,050 children aged 3.00\u201317.99\u00a0years. The x-axis shows amount of ammonium oxalate (Ox2\u2212) in mmol necessary for the onset of spontaneous crystallization. The y-axis shows the concentration of calcium ions [Ca2+] before adding Ox2\u2212. The extremes of the hyperbola represent minimum and maximum values. The minimum hyperbola is defined by the equation [Ca2+]=0.27\/(Ox2\u2212), and the maximum is [Ca2+]=1.67\/(Ox2\u2212). The BRI values for the 5th to the 95th percentile are found between the hyperbolas [Ca2+]=0.39\/(Ox2\u2212) and [Ca2+]=0.94\/(Ox2\u2212), median [Ca2+]=0.65\/(Ox2\u2212)\nFigure\u00a03 presents the BRI values in children representing various age groups. We considering the subjects in two main subgroups, younger children aged 3\u20139\u00a0years, and older children and adolescents aged 10\u201318\u00a0years. In children 3\u20139\u00a0years of age, the lowest values of BRI were found in the youngest children, i.e. those 3\u00a0years old (median 0.18 1\/L), whilst the highest values were found in 9-year-old children (median 0.34 1\/L). However, statistical analyses did not show a difference between the values across the various age groups. The lowest median BRI value was 0.03 1\/L in 9-year old children, whilst the highest median value was 2.48 1\/L in 4-year-old children. The BRI values for younger children corresponding to the 5th percentile ranged from 0.03\u20130.08 1\/L, and the 95th percentile from 1.38\u20132.27 1\/L.\nFig.\u00a03Bonn Risk Index (BRI) in the whole studied group (left box) and in separate 1-year age groups for children aged 3\u201318\u00a0years\nIn older children and adolescents aged 10\u201318\u00a0years, the lowest BRI values were found in the participants who were 12\u00a0years old (median 0.17 1\/L), whilst the highest were in 17-year-old adolescents (median 0.31 1\/L) (Fig.\u00a03). However, statistical analysis did not show significant differences between the age subgroups. The minimum value of BRI was 0.02 1\/L, and was found in 11-year-old children, whereas the maximum value was 3.1 1\/L in children aged 10 and 17\u00a0years. The BRI values for older children corresponding to the 5th percentile ranged between 0.05 and 0.13 1\/L, whilst the 95th percentile was from 1.69\u20132.66 1\/L.\nThe crystallization values of CaOx, based on BRI in healthy children aged 3\u201318, did not exceed 2.66 1\/L. No significant differences were found in the BRI values between boys and girls in either age group.\nTable\u00a01 presents the BRI and BRI related to the 1.73\u00a0m2 body surface area and body mass (kg). The median BRI in relation to body surface area (1\/L\u2009\u00d7\u20091.73\u00a0m2) was 0.39 1\/L\u2009\u00d7\u20091.73\u00a0m2, with 5th and 95th percentile medians of 0.09 and 3.01 1\/L\u2009\u00d7\u20091.73\u00a0m2, respectively. However, the median BRI in relation to 1\u00a0kg of weight was 0.008 1\/L\u2009\u00d7\u2009kg with corresponding 5th and 95th percentiles of 0.0015 and 0.06 1\/L\u2009\u00d7\u2009kg, respectively. A weak positive correlation was found between BRI and BRI\/1.73\u00a0m2 (R\u2009=\u20090.18, P\u2009<\u20090.05), and a negative correlation was found between BRI\/1.73\u00a0m2 and BRI\/kg (R\u2009=\u2009\u22120.86, P\u2009<\u20090.05). No differences were found between the values of BRI\/1.73\u00a0m2 and BRI\/kg in the various age groups or in relation to sex. \nTable\u00a01Bonn Risk Index values in children aged 3\u201318\u00a0years in relation to body surface area and body weight\u00a0MedianMinimumMaximum5th percentile95th percentileBRI (1\/L)0.260.023.100.061.93BRI\/1.73\u00a0m2(1\/L\u2009\u00d7\u20091.73\u00a0m2)0.390.036.650.093.01BRI\/kg (1\/L\u2009\u00d7\u2009kg)0.0080.00060.150.00150.06\nDiscussion\nUrinary stones are considered a major health problem in society, both in adults and in children, due to their recurrent nature and the cost of treatment [14]. The pathogenic pathways leading to stone formation in kidneys have not been fully explained. During processes of calculus formation, a number of phenomena have been reported such as excess of crystallizing substances, nucleation, crystallization, aggregation and stone formation [15, 16]. However, it has not been explained why calculi do not form in all subjects, despite a large amount of urinary crystallization products. Over the past few years, there have been many attempts to define the risk factors leading to urinary stone formation on the basis of the ability to form oxalate crystals, a main component of calculi [7, 8, 17]. Tiselius et al. described the practical importance of assessing the activity of calcium oxalate ions in urine using the APCaOx index [7]. \nStone formation has also been evaluated in terms of excess urinary calcium oxalate, using the computer program EQUIL [18, 19]. Laube et al. showed a strong correlation between the concentration of free calcium ions [Ca2+] in urine and the quantity of ammonium oxalate (Ox2\u2212) added to invoke spontaneous urinary crystallization of CaOx [9]. The authors suggested that the BRI index in healthy subjects was significantly lower than in those forming urinary calculi. The study, based on a group of 72 healthy subjects, provided mean BRI values of 1.05\u2009\u00b1\u20091.038 1\/L with a range of 0.06\u20134.89 1\/L. In other studies involving 85 healthy adult subjects, the mean BRI values were similar, 0.89\u2009\u00b1\u20090.91 1\/L. Higher BRI values have been described by Lewandowski et al. whose study group of 15 Caucasian adults demonstrated a mean BRI of 4.90 1\/L, and 15 African adults had a mean BRI of 2.04 1\/L [10]. The literature review did not reveal any larger or more comprehensive studies regarding BRI in children.\nOur findings in the large group of healthy children showed that the normal BRI value ranged from 0.06 1\/L (5th percentile) to 1.93 1\/L (95th percentile). The maximum values obtained in children were lower than those described by other authors in adult populations [6, 9, 20]. This may be due to the fact that the mechanism which inhibits crystal formation in children\u2019s urine is more effective when compared to adults. Teller et al. observed that urinary glycosaminoglycans in children reduced the aggregation of crystals more effectively than in adults [21]. A similar age-related effect regarding stone formation has been observed by Bergsland et al., who showed that the urine of subjects aged less than 20\u00a0years produced a strong inhibitory effect on crystal formation [22]. Ricchiuti et al. investigated the process in boys aged less than 10\u00a0years and found increased activity of plasma protease inhibitor (inter-alpha inhibitor), blocking urinary crystallization during various stages [23].\nOur results did not show a significant difference in BRI values between boys and girls. A minor difference was found in relation to age, but this was not significant. The BRI\/1.73\u00a0m2 and BRI\/kg behaved similarly. High correlations between BRI, BRI and body weight, and BRI and body surface area of studied subjects enable practical, more objective use of BRI and allow for comparisons between individuals. For this reason, these results are presented as median values with ranges for the 5th to 95th percentiles for the whole study group.\nThe study has several limitations. So far, the use of our normative data appears to be limited to the Polish population. It is difficult to relate the results to other geographical regions or to different ethnic groups as there are no published data regarding BRI in children. Further comparative and prospective investigations are needed because a cross-sectional study is not able to reveal whether some children with a normal BRI value will be at risk for stone disease in the future. Moreover, some studied children may have had urolithiasis despite normal results of renal ultrasound. However, this study provided consistent normative data on BRI, due to both the large age representation and the stringent selection criteria.\nIn summary, the Bonn Risk Index, expressed as the ratio of ionized calcium to the amount of ammonium oxalate necessary to initiate spontaneous CaOx urinary crystal formation, is lower in healthy children and adolescents aged 3\u201318\u00a0years than in studies of adults by other authors. The BRI during growth appears to be independent of age and sex. Thus, our results may contribute to the effective screening of kidney stone disease in pediatric subjects. We conclude that the BRI may be valuable in the evaluation of pediatric patients at risk for kidney stones, particularly if the BRI from stone formers is demonstrated to be higher than in normal children.","keyphrases":["oxalate crystallization","children","bonn risk index (bri)","normative values"],"prmu":["P","P","P","R"]}
{"id":"Crit_Care-6-3-137445","title":"Clinical review: Complications and risk factors of peripheral arterial catheters used for haemodynamic monitoring in anaesthesia and intensive care medicine\n","text":"In order to evaluate the complications and risk factors associated with peripheral arterial catheters used for haemodynamic monitoring, we reviewed the literature published from 1978 to 2001. We closely examined the three most commonly used arterial cannulation sites. The reviewed papers included a total of 19,617 radial, 3899 femoral and 1989 axillary artery catheterizations. Factors that contribute to higher complication rates were investigated. Major complications occurred in fewer than 1% of the cases, and rates were similar for the radial, femoral and axillary arteries. We conclude that arterial cannulation is a safe procedure.\nIntroduction\nIndwelling arterial catheters are used routinely for continuous haemodynamic monitoring in the operating room during major surgery and in critically ill patients. Arterial cannulation provides easy and convenient access, allowing multiple blood samplings and blood gas analysis. Arterial cannulation has generally been found to be a safe procedure, with few serious complications [1,2,3,4,5,6]. Approximately 8 million and 2.5 million arterial catheters are placed yearly in the USA and Europe, respectively [7]. Despite the frequency with which arterial catheters are employed, there is little information on the impact of cannulation site on risk for complications [8]. We reviewed reports concerning arterial cannulation sites such as the radial, femoral, axillary, brachial, ulnar, dorsal pedis, tibial posterior and temporal arteries with regard to thrombotic, infectious and vascular complications.\nMaterial and methods\nThe PubMed, Medline Express and Winspirs databases (publication years 1978-2001) were searched to identify pertinent articles. The keywords 'artery catheter', 'radial artery catheter', 'femoral artery catheter', 'axillary artery catheter', 'catheter complication', 'hemodynamic monitoring' and 'peripheral methods' were used. We reviewed the papers and identified further articles from the references of the papers found in the initial search.\nWe reviewed all studies concerning the use of radial, femoral, axillary, brachial, ulnar, dorsal pedis, tibial posterior and temporal artery catheters for haemodynamic monitoring that dealt with complications and risk factors in adults. We excluded studies conducted in the paediatric patient population.\nWe opted not to perform statistical analyses because the data selection of the published reports do not follow the same criteria, and most of them give no specific information regarding patient population, catheter material, cannulation technique and times. Therefore, comparison between studies and statistical analyses are unlikely to be valid.\nResults\nWe identified 78 studies that met our criteria.\nRadial artery\nThe radial artery is the most common site for arterial cannulation, and the studies that reported complication rates are listed in Table 1 [9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]. The most common complication was temporary occlusion of the artery, the incidence of which ranged from 1.5% [17] to 35% [10] (mean 19.7%), the variability being due to the different methods employed and the degree of effort invested in detecting this complication.\nTable 1\nComplications following radial artery cannulation\nReference\nCases\nPermanent ischaemic damage (n)\nTemporary occlusion (n)\nSepsis (n)\nLocal infection (n)\nPseudoaneurysm (n)\nHaematoma (n)\nBleeding (n)\n[9]*\n80\n1\n9\n0\n0\n-\n-\n-\n[9]*\n34\n1\n10\n0\n2\n-\n-\n-\n[10]\n100\n0\n35\n-\n-\n-\n-\n-\n[11]\n148\n0\n27\n-\n-\n-\n-\n-\n[12]\n29\n0\n5\n-\n-\n-\n-\n-\n[13]\n62\n0\n16\n0\n0\n-\n-\n-\n[14]\n333\n0\n100\n0\n16\n-\n29\n-\n[15]\n40\n0\n2\n-\n-\n-\n-\n-\n[16]\n100\n2\n14\n0\n1\n-\n31\n-\n[17]\n197\n0\n3\n3\n4\n2\n-\n1\n[18]\n178\n0\n7\n1\n3\n-\n-\n1\n[5]\n1699\n0\n360\n0\n1\n-\n206\n-\n[19]\n26\n0\n6\n0\n1\n1\n1\n-\n[20]\n100\n0\n14\n-\n-\n-\n-\n-\n[21]\n100\n0\n33\n0\n1\n-\n13\n-\n[22]\n118\n0\n18\n0\n0\n-\n21\n-\n[23]\n200\n0\n15\n-\n-\n-\n61\n-\n[24]\n25\n0\n3\n0\n0\n-\n-\n-\n[25]\n200\n0\n60\n0\n0\n-\n32\n-\n[26]\n193\n0\n50\n0\n4\n-\n-\n-\n[27]\n88\n0\n15\n-\n-\n-\n-\n-\n[28]\n2900\n-\n-\n4\n12\n5\n-\n-\n[29]\n38\n0\n3\n-\n-\n-\n5\n-\n[30]*\n45\n0\n4\n-\n-\n-\n10\n-\n[30]*\n44\n0\n11\n-\n-\n-\n9\n-\n[31]\n12,500\n-\n-\n-\n-\n6\n-\n-\n[32]\n40\n0\n11\n-\n-\n-\n-\n-\nMean incidence (%)\n0.09 (4\/4217)\n19.7 (831\/4217)\n0.13 (8\/6245)\n0.72 (45\/6245)\n0.09 (14\/15,623)\n14.40 (418\/2903)\n0.53 (2\/375)\nThe total number of cannulations was 19,617. *One study examining two different populations. -, Not investigated.\nGenerally, temporary occlusion of the artery has no serious sequelae. Permanent occlusion of the radial artery appears to be rare because it was reported in only four patients (mean incidence 0.09%).\nAnother serious complication is pseudoaneurysm, which was reported in 14 patients (mean incidence 0.09%). Pseudoaneurysm poses a risk for infection, sepsis, rupture [33,34,35] and formation of an extracorporeal pseudoaneurysm [36]. Radial catheterization was associated with sepsis in eight patients (mean incidence 0.13%), whereas local infection at the cannulation site was identified in 45 patients (mean incidence 0.72%).\nMinor complications such as haematoma formation or bleeding at the puncture site are also shown in Table 1. Other complications include abscess, cellulitis, paralysis of the median nerve [37,38,39], suppurative thrombarteritis [40], air embolism [41], compartment syndrome and carpal tunnel syndrome [42,43,44]. Other rarely reported complications include catheter failure as result of manufacturing defect or incorrect use with resultant catheter replacement [45,46,47,48].\nFemoral artery\nWe identified 11 studies that used the femoral artery for haemodynamic monitoring, which are listed in Table 2 [17,18,19,49,50,51,52,53,54,55,56]. Temporary occlusion was reported in 10 patients (mean incidence 1.45%), and serious ischaemic complications requiring extremity amputation was reported in three patients (mean incidence 0.18%) [56].\nTable 2\nComplications following femoral artery cannulation\nReference\nCases\nPermanent ischaemic damage (n)\nTemporary occlusion (n)\nSepsis (n)\nLocal infection (n)\nPseudoaneurysm (n)\nHaematoma (n)\nBleeding (n)\n[49]\n46\n-\n-\n0\n-\n-\n-\n-\n[50]\n50\n0\n1\n0\n-\n-\n5\n-\n[51]\n85\n0\n0\n0\n-\n-\n10\n-\n[17]\n113\n0\n0\n0\n0\n-\n-\n4\n[52]\n89\n-\n-\n0\n0\n-\n-\n1\n[53]\n2100\n-\n-\n6\n-\n6\n-\n-\n[18]\n114\n0\n4\n2\n0\n-\n-\n0\n[54]\n42\n0\n1\n0\n1\n-\n3\n-\n[55]\n220\n0\n1\n4\n4\n-\n8\n-\n[19]\n64\n0\n3\n1\n0\n-\n2\n-\n[56]\n976\n3\n-\n-\n-\n-\n-\n-\nMean incidence (%)\n0.18 (3\/1664)\n1.45 (10\/688)\n0.44 (13\/2923)\n0.78 (5\/642)\n0.3 (6\/2100)\n6.1 (28\/461)\n1.58 (5\/316)\nThe total number of cannulations was 3899. -, Not investigated.\nPseudoaneurysm formation occurred in six patients (mean incidence 0.3%), sepsis was observed in 13 patients (mean incidence 0.44%) and local infection was reported in five patients (mean incidence 0.78%). Bleeding (generally minor) was observed in five patients (mean incidence 1.58%), and haematoma formation was reported in 28 (mean incidence 6.1%). One patient developed an infected haematoma and needed blood transfusion [17] and another patient eventually died from massive retroperitoneal bleeding [57].\nAxillary artery\nThe axillary artery was cannulated in a total of 1989 reported cases. The complication rates at this site are summarized in Table 3 [54,55,58,59,60,61]. Serious complications included permanent ischaemic damage in two patients (mean incidence 0.20%), pseudoaneurysm formation in one patient (mean incidence 0.1%) and sepsis in five patients (mean incidence 0.51%). Paresthesia of the hand due to pressure on the brachial nerve plexus was also described [54,59].\nTable 3\nComplications following axillary artery cannulation\nReference\nCases\nPermanent ischaemic damage (n)\nTemporary occlusion (n)\nSepsis (n)\nLocal infection (n)\nPseudoaneurysm (n)\nHaematoma (n)\nBleeding (n)\n[58]*\n1000\n-\n-\n-\n-\n1\n-\n-\n[58]*\n245\n0\n1\n0\n-\n-\n-\n6\n[59]\n120\n0\n1\n0\n3\n-\n4\n-\n[60]\n435\n2\n9\n3\n7\n-\n4\n3\n[54]\n28\n0\n-\n0\n1\n-\n3\n-\n[61]\n31\n0\n-\n0\n-\n-\n1\n1\n[55]\n130\n0\n0\n2\n5\n-\n5\n-\nMean incidence (%)\n0.20 (2\/989)\n1.18 (11\/930)\n0.51 (5\/989)\n2.24 (16\/713)\n0.1 (1\/1000)\n2.28 (17\/744)\n1.41 (10\/711)\nThe total number of cannulations was 1989. * One study examining two different populations. -, Not investigated.\nOther sites for arterial cannulation\nOther less frequently reported cannulation sites are the brachial, dorsal pedis, ulnar, tibial posterior and temporal arteries.\nOnly one serious complication was found in a study of 1000 patients [62] in which the brachial artery was used for invasive monitoring in ambulatory patients. This complication was an infected haematoma arising from a pseudoaneurysm. Another study that employed the brachial artery for arterial blood sampling in 6185 patients [63] also showed a small number of complications (incidence 0.2%), mainly paresthesias.\nThe dorsal pedis artery has also been cannulated without any major complications [24,54]. However, because of its distance from the central circulation and associated difficulties in hypotensive patients it is generally avoided [64]. The ulnar artery has been used without serious complications, and in a direct comparison [29] this site exhibited similar complication rates to those encountered with the radial artery.\nThe tibial posterior artery has been used in paediatric patients, without major complications [65,66]. However, there also exists a case report of limb amputation secondary to a tibial artery catheterization [67]. The temporal artery is an end artery of the external carotid artery, and is usually avoided for fear of serious complications. There is a report of three cases of cerebral embolization in paediatric patients [68].\nFurther studies\nOther studies that examined arterial catheter complications but that did not specifically attribute their occurrence to one specific site are listed in Table 4 [49,69,70,71]. One study [69] used mainly the radial (n = 210), ulnar (n = 158) and dorsalis pedis arteries (n = 82), whereas another [49] used mainly the femoral (n = 46), brachial (n = 9), axillary (n = 3) and radial arteries (n = 1). One serious complication was found at the femoral site, with compromised circulation to the leg [70]; eventually, thrombectomy was required. Another reported serious complication is air embolism caused by improper use of the flushing device [70].\nTable 4\nStudies examining arterial catheter complications without specifically attributing them to any specific site\nReference\nCases\nAir embolism (%)\nPermanent occlusion (%)\nTemporary occlusion (%)\nHaematoma (%)\nBleeding (%)\nSepsis (%)\nAbscess (%)\n[69]\n450\n0\n0\n18.4\n-\n8.7\n0\n-\n[49]\n59\n0\n1.7\n6.8\n5.1\n1.7\n0\n-\n[70]\n506\n0.2\n0\n-\n-\n0\n0\n-\n[71]*\n1556\n0\n0\n3.4\n0.2\n1.5\n0.06\n0.3\n[71]\ufffd\n565\n0\n0\n4.6\n0.3\n2.3\n0\n0.5\n*Medical intensive care unit (radial artery 52%, femoral artery 45%). \ufffdSurgical intensive care unit (radial artery 78%, femoral artery 11%). -, Not investigated.\nA recent study [71] examined complication rates, comparing medical with surgical intensive care units. It also directly compared radial and femoral artery catheters. There were no significant differences in terms of sepsis and local infection rates between the femoral and radial arteries, and no significant difference between the medical and surgical intensive care units.\nAnother nine studies specifically looked for septic complications associated with arterial catheter placement, and included a total number of 1681 patients (Table 5) [52,54,72,73,74,75,76,77,78]. No septic complications were identified in seven of those studies [52,72,73,74,75,76,77], and nine cases of sepsis were reported in the remaining two studies [54,78]. All of the cases of sepsis were identified after the line had been in place for longer than 4 days. The studies in which sepsis was reported predominately used the radial artery.\nTable 5\nIncidence of sepsis following arterial cannulation\nReference\nCases\nSepsis (%)\n[54]\n130\n4\n[72]\n172\n0\n[52]\n186\n0\n[73]\n19\n0\n[74]\n155\n0\n[75]\n643\n0\n[76]\n230\n0\n[77]\n75\n0\n[78]\n71\n5.5\nRisk factors\nA further objective of the present review was to consider risk factors that might have impacted on complication rates.\nThrombotic complications\nThrombus formation and occlusion of the artery appear to be caused by changes in the integrity of the vessel wall induced by the presence of the catheter [79]. Recannulation of the occluded artery generally occurs, but this may take up to 75 days [80]. The incidence of thrombus appears to be related to the degree to which the catheter fills the arterial lumen [3]. It has been shown that the incidence of radial obstruction increases linearly with the ratio of outer diameter to vessel lumen diameter [81]. This might explain the higher incidence of radial occlusion in female patients, who generally have vessels of smaller diameter [5,10,14,22,25]. This has led to a preference for 20-gauge catheters for radial artery cannulation [11,14]. Teflon catheters also appear to be associated with a lower incidence of occlusion [11], but in another study [5] this could not be confirmed. Patients with a low cardiac output have a higher incidence of radial artery occlusion [5,14], but no difference was found among patients with episodes of hypotension [21]. Multiple punctures for catheter insertion was shown to be a risk factor for complications in one study [14], but this could not be confirmed in another [5]. A higher incidence of occlusion is found in the presence of a haematoma [5,14,21]. Patients who are treated with aspirin or low-dose heparin have fewer vessel occlusions [13,14]. The age of adult patients does not appear to be a risk factor [14]. An important factor with regard to arterial occlusion is the duration of cannulation. A higher incidence of occlusion was reported when the cannula was left in place longer than 48 or 72 hours [5,9,14,16,25].\nInfectious complications\nImportant factors in decreasing the incidence of catheter-related infections are aseptic insertion technique and adequate disinfection of the insertion site [82]. The risk for infectious complications and sepsis increases with the duration of cannulation, especially if the catheter is left in place for longer than 96 hours [78,83]. The catheter material also appears to be of importance, because Teflon has a greater resistance to Escherichia coli and Pseudomonas aeruginosa [84] and a lower affinity for stapylococci [85]. Prophylactic treatment with antibiotics does not appear to reduce the risk for catheter-related infections [78,83], but antibiotic bonding of the catheter may confer a degree of protection [86,87]. Arterial catheter placement via the cut-down technique is associated with a higher risk for infection [83]. Furthermore, sepsis occurs more frequently in the presence of local site inflammation [83]. Infection may also occur through contamination by caregivers of the monitoring system, the flushing device and the infusion system [88,89,90].\nDiscussion\nThe use of peripheral arterial catheters for haemodynamic monitoring is widespread. The most frequently used site is the radial artery because of its well documented low complication rates and easy access [9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]. In the present review we report on 19,617 radial artery cannulations, and the main complication was temporary occlusion of the artery (incidence 19.70%). This complication rate is close to the finding of Wilkins [91], who reviewed reports of catheterization of the radial artery with regard to ischaemic complications published between 1969 and1983. He reported a temporary occlusion rate of 23%. In an extensive study of 1699 patients, Slogoff and coworkers [5] reported a 21.2% rate of temporary occlusion. Although temporary occlusion is reported quite frequently, serious ischaemic damage was reported in only two studies [9,16], with a mean complication rate of 0.09%. Nevertheless, there have been reports of serious ischaemic damage after radial artery cannulation that led to necrosis, and amputation of fingers or the whole hand [92,93,94,95,96,97,98,99].\nOther major complications such as pseudoaneurysm and sepsis were reported to occur in a mean of 0.09% and 0.13% of cases, respectively.\nThe second most cannulated artery for haemodynamic monitoring is the femoral artery, and we reviewed 3899 cases. Temporary occlusion was reported in only 1.18% of the reviewed cases. The incidence of this complication in the femoral artery is much lower than that in the radial artery. We attributed this to the larger vessel diameter of the femoral artery. Serious ischaemic complications were reported in only one study [56], and the mean complication rate was 0.18%. The incidence rates for other major complications such as pseudoaneurysm and sepsis (0.3% and 0.44%, respectively) were similar to our findings in the radial artery. Some authors caution against use of the femoral artery for cannulation for fear of higher sepsis rates because of the close proximity to the perianal region [52]. We are unable to corroborate this on the basis of the reviewed literature, and recent studies that directly compared the radial and femoral arteries with regard to septic complications identified similar rates of sepsis [53,71]. In particular, in an extensive study of 4932 patients, Frezza and Mezghebe [71] were unable to identify a difference in complication rates between the radial and femoral arteries. In that study the femoral artery was actually the preferred site in the medical intensive care unit. Other authors prefer the femoral artery over the radial artery because the femoral artery is usually palpable even in hypotensive patients and may be the only accessible route for haemodynamic monitoring [64,100,101,102,103]. The blood pressure curve that is obtained from this larger artery is generally more accurate and gives a closer estimation of the aortic blood pressure [64,100,101,102,103].\nThe third most cannulated artery in the present review of the literature was the axillary artery, with almost 2000 reported cases. Some authors prefer not to cannulate the axillary artery because of its close location to the carotid artery and because of fear of embolism to the brain [101]. On the basis of the data summarized here we cannot confirm this, and no case of embolism to the brain was reported. Sometimes the axillary artery is avoided because of the more difficult approach required, although (particularly in anaesthesia) it is a well known route because it requires the same approach as that for axillary nerve block [3]. The major complications encountered with the axillary artery were similar to those for the radial and femoral arteries, and we conclude that it is a safe route for arterial cannulation.\nOther reviewed arteries employed for catheterization, such as the brachial, dorsal pedis, ulnar, tibial and temporal arteries, have been used without serious complications, but published reports of their use are limited.\nConclusion\nIncidence rates for major complications such as permanent ischaemic damage, sepsis and pseudoaneurysm formation are low and similar for the radial, femoral and axillary arteries. They occur in fewer than 1% of cases.\nOn the basis of the present systematic review we can conclude that serious complications of the radial, femoral and axillary artery are rare, and that arterial cannulation is a relatively safe procedure. Nevertheless, one must be aware of the possible serious complications that might arise. Therefore, it is important to use an arterial catheter only when it is indicated and to select carefully the best cannulation site individually for each patient, because the different cannulation sites have their specific advantages and attendant risks.\nCompeting interests\nUJP is President of Pulsion Medical Systems AG, and AP is on the medical advisory board of Pulsion Medical Systems AG, whose PiCCO product uses a femoral arterial line.","keyphrases":["complication","peripheral","catheter","haemodynamic monitoring","axillary artery","radial artery","femoral artery"],"prmu":["P","P","P","P","P","P","P"]}
{"id":"J_Gastrointest_Surg-3-1-1852380","title":"Management of Massive Arterial Hemorrhage After Pancreatobiliary Surgery: Does Embolotherapy Contribute to Successful Outcome?\n","text":"Massive arterial hemorrhage is, although unusual, a life-threatening complication of major pancreatobiliary surgery. Records of 351 patients who underwent major surgery for malignant pancreatobiliary disease were reviewed in this series. Thirteen patients (3.7%) experienced massive hemorrhage after surgery. Complete hemostasis by transcatheter arterial embolization (TAE) or re-laparotomy was achieved in five patients and one patient, respectively. However, 7 of 13 cases ended in fatality, which is a 54% mortality rate. Among six survivors, one underwent selective TAE for a pseudoaneurysm of the right hepatic artery (RHA). Three patients underwent TAE proximal to the proper hepatic artery (PHA): hepatic inflow was maintained by successful TAE of the gastroduodenal artery in two and via a well-developed subphrenic artery in one. One patient had TAE of the celiac axis for a pseudoaneurysm of the splenic artery (SPA), and hepatic inflow was maintained by the arcades around the pancreatic head. One patient who experienced a pseudoaneurysm of the RHA after left hemihepatectomy successfully underwent re-laparotomy, ligation of RHA, and creation of an ileocolic arterioportal shunt. In contrast, four of seven patients with fatal outcomes experienced hepatic infarction following TAE proximal to the PHA or injury of the common hepatic artery during angiography. One patient who underwent a major hepatectomy for hilar bile duct cancer had a recurrent hemorrhage after TAE of the gastroduodenal artery and experienced hepatic failure. In the two patients with a pseudoaneurysm of the SPA or the superior mesenteric artery, an emergency re-laparotomy was required to obtain hemostasis because of worsening clinical status. Selective TAE distal to PHA or in the SPA is usually successful. TAE proximal to PHA must be restricted to cases where collateral hepatic blood flow exists. Otherwise or for a pseudoaneurysm of the superior mesenteric artery, endovascular stenting, temporary creation of an ileocolic arterioportal shunt, or vascular reconstruction by re-laparotomy is an alternative.\nRecent advances in surgical technique and postoperative management of major pancreatobiliary surgery have reduced the morbidity and mortality. However, the problem of life-threatening postoperative arterial hemorrhage caused by pseudoaneurysm rupture, although uncommon, has received little attention.1\u20136 Immediate arteriography to identify the site of bleeding and subsequent treatment by radiological intervention have been the first approach for the last decade.7\u20139 The efficacy of transcatheter arterial embolization (TAE) is well established.8\u201318 Nevertheless, occlusion by TAE sometimes causes distal end-organ damage, even though bleeding has been controlled.9\u201311 Indeed, complete interruption of hepatic inflow may lead to fatal hepatic necrosis, but risk factors for whole liver infarction have yet to be identified. Knowledge of which sites are associated with a high risk of hepatic necrosis would enable surgeons to choose an alternative to TAE, such as vascular reconstruction. Recent reports have documented an advantage of endovascular stenting over TAE.19\u201322 We reviewed the records of patients who experienced massive arterial hemorrhage after pancreatobiliary surgery to help generate guidelines for the management of postoperative pseudoaneurysm rupture.\nPatients and Methods\nFrom January 1993 to December 2005, 351 patients underwent major surgery for malignant pancreatobiliary disease in the Department of Gastroenterological Surgery, Yokohama City University. The cases were pancreatic cancer (n\u2009=\u2009139), distal bile duct cancer (n\u2009=\u200958), ampullary cancer (n\u2009=\u200946), hilar bile duct cancer (n\u2009=\u200947), advanced gallbladder cancer (n\u2009=\u200942), and intrahepatic cholangiocarcinoma (n\u2009=\u200919). Procedures included pylorus-preserving pancreatoduodenectomy (PPPD) in 113, conventional pancreatoduodenectomy (PD) in 84, distal pancreatectomy (DP) in 35, segmental resection of the pancreas in 4, total pancreatectomy in 6, bile duct resection with partial hepatectomy in 98, and PD combined with partial hepatectomy (HPD) in 11. Patients undergoing isolated bile duct resection or partial hepatectomy without bile duct resection were excluded. Except for the patients who underwent DP, all patients received biliary tract reconstruction and skeletonization of the hepatic arteries to complete lymphadenectomy within the hepatoduodenal ligament. Pancreatojejunostomy was created as an end-to-side, duct-to-mucosa anastomosis with a stenting tube, or via the pancreatic duct insertion technique with total tube drainage when the pancreatic duct was smaller than 3\u00a0mm. Hepaticojejunostomy was created end-to-side in a single layer.\nMassive postoperative arterial hemorrhage was defined as bleeding requiring a transfusion of 2 or more units of packed red blood cells, an invasive intervention such as laparotomy or TAE and monitoring in the surgical intensive care unit within 24\u00a0h of the onset of hemorrhage. The medical records of these patients were analyzed retrospectively.\nResults\nOf the 351 patients who underwent a major procedure, 13 (3.7%) presented with massive postoperative arterial hemorrhage (10 men and 3 women; average age, 66\u2009\u00b1\u200910\u00a0years). The demographic and clinical characteristics are summarized in Table\u00a01.\nTable\u00a01Baseline Characteristics of PatientsCaseDiseaseOrigin of bleedSurgeryInterval (days)aSite of TAERe-laparotomyCause of bleedOutcome1Ampullary cancerRHAPPPD10RHANoPancreatic leakAlive2Gallbladder cancer RHA and RHAHPD8 and 12RHA and PHANoMinor injuryHepatic failure died3Hilar bile duct cancerRHAExtended left hepatectomy9NoneNoUnsuccessful reconstructionCancer recurrence, died4Gallbladder cancerGDAHPD10GDANoPancreatic leakAlive5Distal bile duct cancerGDAPD11GDAYesMinor injuryAlive6Distal bile duct cancerCHAPPPD24CHANoPancreatic leakAlive7Distal bile duct cancerPHAPD7NoneNoPancreatic leakSepsis, died8Hilar bile duct cancerGDARight hepatectomy7CHAYesMinor injuryHepatic failure died9Gallbladder cancerMHA and GDARight hepatectomy13 and 27MHA and CHANoPancreatic leakHepatic failure died10Gallbladder cancerGDARight hepatectomy7GDAYesPancreatic leakHepatic failure died11Pancreatic cancerSPADP17CAYesPancreatic leakAlive12Intrahepatic cholangiocarcinomaSPAHPD9NoneYesMinor injuryHepatic failure died13Pancreatic cancerSMAPPPD34NoneYesPancreatic leakMOF, diedTAE Transcatheter arterial embolization, RHA right hepatic artery, PHA proper hepatic artery, GDA gastroduodenal artery, CHA common hepatic artery, MHA middle hepatic artery, SPA splenic artery, CA celiac axis, SMA superior mesenteric artery, PPPD pylorus preserving pancreatoduodenectomy, HPD pancreatoduodenectomy combined with partial hepatectomy, DP distal pancreatectomy, PD pancreatoduodenectomy, MOF multiple organ failureaDays from surgery to hemorrhage\nAfter restoration of hemodynamic stability by volume loading, 10 of 13 patients underwent emergency pan-abdominal angiography visualizing the celiac axis (CA) and superior mesenteric artery (SMA) by standard Seldinger technique. The other three patients required emergency laparotomy without angiography because their clinical status was deteriorating.\nOf the 10 patients who underwent angiography, an arterial pseudoaneurysm was detected in 7: right hepatic artery (RHA) in two, gastroduodenal artery (GDA) in three, common hepatic artery (CHA) in one, and splenic artery (SPA) in one. Three patients had extravasation in the area of the middle hepatic artery (MHA), proper hepatic artery (PHA), or GDA without a clear source of bleeding (see Table\u00a01).\nTAE was attempted in nine patients using various coil occlusion devices. Table\u00a01 details the origin of bleeding and the sites of TAE. Complete hemostasis was achieved by TAE in five patients, but hemostasis was only temporary in four: two required a second TAE and two required laparotomy to control rebleeding.\nOf the three patients with a pseudoaneurysm distal to the PHA, one received selective TAE of the RHA with a successful outcome (patient 1). A second patient had recurrent hemorrhage after TAE of the RHA and second TAE of the PHA was required. Unfortunately, the cluster of coils compressed the portal vein and portal flow was disrupted after the second TAE. This patient died of hepatic failure secondary to hepatic hypoperfusion (patient 2). The third patient had undergone extended left hepatectomy with resection of the RHA and had bleeding from the site of the RHA reconstruction (patient 3). If TAE had been performed at the RHA, inflow to the small remnant liver would be completely interrupted, and fatal hepatic failure would have been the most likely outcome. Instead, we re-operated, ligated the RHA, and created an ileocolic arterioportal shunt to supply the hepatic remnant. The patient survived without hepatic failure. However, portal hypertension developed because this shunt remained patent 6\u00a0months after reoperation, and the patient died of spinal metastasis 11\u00a0months after the initial operation.\nIn seven patients, the pseudoaneurysm was proximal to the PHA and involved the GDA or CHA. Two patients successfully underwent selective TAE of the GDA for a pseudoaneurysm that originated from the ligated GDA. One patient had a replaced RHA from the CA, which contributed to a favorable outcome (patient 4, Fig.\u00a01). One patient underwent TAE of the CHA uneventfully because the hepatic inflow was narrowly maintained by the left subphrenic artery (patient 6, Fig.\u00a02). In contrast, the patient who had extravasation of the PHA without a discrete source had intimal injury and occlusion of the CHA during angiography. The patient experienced a liver abscess that was difficult to treat and died of sepsis and cancer recurrence during a long hospital stay (patient 7). Of the three patients who underwent right hepatectomy, two had complete disruption of the inflow to the hepatic remnant by TAE of the CHA, leading to fatal hepatic failure (patients 8 and 9), and one underwent unsuccessful TAE of the GDA and required a laparotomy. Vascular ligation at the pancreatic head was successful in achieving hemostasis, but hepatic failure secondary to hemorrhagic shock proved fatal 59\u00a0days after the initial surgery (patient 10).\nFigure\u00a01Case 3. A 74-year-old woman with advanced gallbladder cancer presented with massive hemorrhage 10\u00a0days after pancreatoduodenectomy combined with partial hepatectomy of the segments (Couinaud segments) IVb and V. Angiogram of the common hepatic artery [the right hepatic artery (RHA) replaced from the celiac axis (CA)] showed a pseudoaneurysm (arrow) originating from the stump of the gastroduodenal artery (GDA). (A) Complete hemostasis was obtained using transcatheter arterial embolization of this stump. The patient survived without hepatic failure because the hepatic inflow was maintained by the replaced RHA (B). LHA Left hepatic artery, SPA splenic artery.Figure\u00a02Case 4. A 69-year-old man who had undergone a pylorus preserving pancreatoduodenectomy for distal bile duct cancer had massive hemorrhage 24\u00a0days after surgery. Angiogram of the celiac axis (CA) showed a pseudoaneurysm (arrow) originating from the common hepatic artery (CHA) (A) Complete hemostasis was obtained using transcatheter arterial embolization proximally and distally to the origin of the pseudoaneurysm, but the proper hepatic artery was occluded. The hepatic arterial inflow was narrowly maintained via the left subphrenic artery (B). The patient had an uneventful course. RHA Right hepatic artery, LHA left hepatic artery, SPA splenic artery.\nOf the two patients with a pseudoaneurysm originating from the SPA, one underwent TAE of the CA with a favorable outcome because the pancreatoduodenal arcades around the pancreatic head maintained hepatic inflow after DP (patient 11, Fig.\u00a03). The other patient underwent an emergency laparotomy. Complete hemostasis was achieved by ligation, but hemorrhagic shock resulted in subsequent hepatic failure (patient 12).\nFigure\u00a03Case 8. A 77-year-old man who had undergone a distal pancreatectomy for pancreatic tail cancer had massive hemorrhage 17\u00a0days after surgery. Angiogram of the celiac axis (CA) showed a pseudoaneurysm (arrow) originating from the splenic artery (A). Hemostasis was obtained using transcatheter arterial embolization (TAE) of the CA. The common hepatic artery (CHA) was occluded by TAE but the hepatic inflow was maintained via the arcades of the pancreatic head from the superior mesenteric artery (B). The patient had a favorable course. RHA Right hepatic artery, LHA left hepatic artery, GDA gastroduodenal artery.\nOne patient with a pseudoaneurysm of the SMA underwent surgical resection with vascular reconstruction. However, the patient died of recurrent pancreatic cancer and multiple organ failure during the hospital stay (patient 13).\nSeven of 13 patients died in the hospital; thus, the mortality rate for massive arterial hemorrhage was 54%, and the overall mortality rate was 2.0%.\nCase reports of some patients are illustrated in Figs.\u00a01, 2, and 3.\nDiscussion\nRupture of a pseudoaneurysm, although uncommon, can cause life-threatening hemorrhage even comparatively late after pancreatobiliary surgery.1\u20136 This morbidity rate for pancreatectomy was reported to range from 2.0% to 4.6%.4\u20136,11,12 A pseudoaneurysm is a pulsatile hematoma surrounded by fibrous tissue that communicates with the artery via a disruption of the arterial wall. It can rupture into the peritoneal cavity, the gastrointestinal tract, or biliopancreatic ducts through a point of weakness, most commonly the anastomotic site. The Japanese Multi-institutional Study of 1,066 patients who underwent PPPD reported that the incidence of intra-abdominal hemorrhage was 3.5% and that of upper gastrointestinal hemorrhage was 3.2%.23\nThe etiology of pseudoaneurysm formation has yet to be clearly delineated. It is believed to be most commonly due to pancreatic fistula or anastomotic dehiscence.1\u20134,10\u201312 However, pseudoaneurysm can develop far from the pancreatic cut surface, and there is no evidence of a pancreatic leak in some cases. It has been suggested that skeletonization of the visceral arteries may result in iatrogenic vascular injury (e.g., secondary to diathermy).10,14 In three patients in our series, the etiology was thought to be a minor vascular injury that occurred during dissection (see Table\u00a01). Previous reports found that the patients with massive arterial bleeding frequently had septic complications.3,4,17 Therefore, both arterial injury and infection can contribute to massive arterial hemorrhage. Whether or not preoperative obstructive jaundice is an etiologic fact remains controversial.3,24\nSome clinical studies found that a preliminary warning bleeding (sentinel bleeding) precedes major hemorrhage.1,2,9\u201313 This sentinel bleeding probably indicates local infection and an anastomotic leak.2 So, recognition of a sentinel bleeding and prompt intervention can be life-saving.\nAngiography is necessary to identify the site of bleeding, and TAE is the treatment of choice to control massive bleeding and achieve hemodynamic stabilization. When performing TAE for a pseudoaneurysm, microcoils must be placed both proximally and distally to the origin, not within the pseudoaneurysm itself.11,25\nWhen the origin of the pseudoaneurysm is distal to the PHA, left hepatic artery, RHA, or MHA, TAE should be highly selective to preserve the other branch to the liver. Selective TAE without devascularization of the other lobe provides optimal protection of the liver parenchyma (patient 1).15 Nevertheless, superselective TAE frequently is not possible. Inadvertent occlusion of PHA, even when bleeding is controlled, may cause fatal hepatic infarction (patient 2).10 Therefore, the success of superselective TAE distal to the PHA depends on the expertise of the radiologist.\nSuperselective TAE is difficult or even impossible for a pseudoaneurysm proximal to the PHA.24 When the GDA is ligated close to its divergence from the CHA, you must embolize the CHA to obtain hemostasis. The proximal and distal control of a bleeding pseudoaneurysm of the PHA or CHA usually results in complete occlusion of hepatic arterial flow. Fortunately, we successfully performed TAE of the GDA in patient 4 because the patient had the replaced RHA from CA, and it was unaffected by TAE. Even if the CHA is occluded, the prognosis is good when hepatic inflow can be maintained by a replaced hepatic artery,6,13,14 but success is not uniform. In another case, hepatic inflow was narrowly maintained by the left subphrenic artery after TAE of the CHA (patient 6). A similar case has been described previously.16 The liver can tolerate considerable arterial embolization because of its multiple collateral pathways, mainly via the subphrenic arteries. Unless the surgical procedure includes mobilization of both lobes of the liver, as long as the subphrenic arteries are well developed, TAE proximal to the PHA should lead to a successful outcome. However, TAE proximal to the PHA usually risks occlusion of the CHA with the attendant risk of necrosis and liver failure.9,17 The authors believe that the presence of a few collateral pathways might make liver abscesses difficult to treat and may be associated with high morbidity and mortality rates (patient 7).10 Complete interruption of arterial inflow to the remaining liver after major hepatectomy usually causes imminent hepatic failure (patients 8 and 9). Emergency laparotomy and vascular reconstruction are the most certain treatments for this type of bleeding, but preoperative angiography is highly recommended to identify the bleeding site, although surgery should not be excessively delayed. Endovascular stenting is another option in reestablishing the continuity of the bleeding artery, such as GDA, PHA, or CHA.19\u201322 A covered stent makes it possible to arrest the bleeding, while preserving patency. Potential disadvantages include longer duration to obtain hemostasis than TAE, technical difficulties in negotiating tortuous arteries, and the risk of arterial rupture due to low flexibility and fragile vascular walls. In the case of a stented hepatic artery, the progressive occlusion of the stent due to intimal hyperplasia would not influence outcome because this process is gradual and allows for the formation of collateral pathways.21 Therefore, a stent-graft delivered into a bleeding artery proximal to PHA may be the treatment of choice. When this procedure is technically difficult, vascular reconstruction should be performed after temporary occlusion by interventional radiology. When vascular reconstruction is difficult and ligation is required to obtain hemostasis, creation of an ileocolic arterioportal shunt is another option,26 which we performed successfully in patient 3. Thus, the indications for TAE proximal to the PHA are limited to cases where the replaced hepatic artery exists or the subphrenic arteries are well developed.\nPseudoaneurysm originating from the SPA is well described as a complication of pancreatitis,27,28 but this is an unusual source of bleeding after pancreatobiliary surgery. Selective TAE is indicated because ischemia of the spleen is rare. However, when the origin of the pseudoaneurysm is close to the CA, selective TAE proximal to the SPA is as difficult as the case of bleeding from the GDA. Therefore, when the SPA is divided in surgery, it may be better to leave the proximal part of the ligation site in some degree. TAE of the CA is contraindicated as it will completely interrupt hepatic arterial flow in most cases (patient 11).\nWhen the origin of a pseudoaneurysm is the SMA, it may be extremely difficult or even impossible to preserve mesenteric arterial flow. Hence, resection of the pseudoaneurysm with vascular reconstruction is indicated (patient 11). Endovascular stenting may be an alternative deserving further study.\nHemorrhagic shock is a potentially fatal complication that may result in hepatic failure (patient 10). Rebleeding after TAE is a poor prognostic factor. As rebleeding is often due to a pancreatic leak or intraperitoneal septic condition, pancreatic drainage and loculated fluid collections are mandatory.6 The management of anastomotic dehiscence after pancreatectomy, whether a completion pancreatectomy is necessary or not, is controversial.1,6,29\u201333 When management of the dehiscence is difficult or when severe organ failure is present, completion pancreatectomy is probably necessary.1,6,29\u201331\nAnalysis of our experience and literature review suggests that management of a pseudoaneurysm must be individualized according to the site of bleeding (Fig.\u00a04). We have developed a protocol for managing massive postoperative arterial hemorrhage and are evaluating it postoperatively in a prospective fashion. We hope to report the value of this approach in the future.\nFigure\u00a04Scheme for an approach to the management of a pseudoaneurysm according to the site of bleeding. PHA Proper hepatic artery, RHA right hepatic artery, MHA middle hepatic artery, LHA left hepatic artery, SPA splenic artery, GDA gastroduodenal artery, CHA common hepatic artery, CA celiac axis, SMA superior mesenteric artery, TAE transcatheter arterial embolization.","keyphrases":["hemorrhage","transcatheter arterial embolization","pseudoaneurysm","pancreato-biliary surgery"],"prmu":["P","P","P","M"]}
{"id":"Eur_Spine_J-2-2-1602189","title":"Giant cervicothoracic extradural arachnoid cyst: case report\n","text":"The pathogenesis, etiology, and treatment of the spinal arachnoid cyst have not been well established because of its rarity. A 57-year-old male was presented with spastic quadriparesis predominantly on the left side. His radiological examination showed widening of the cervical spinal canal and left neural foramina due to a cerebrospinal fluid - filled extradural cyst that extended from C2 to T2 level. The cyst was located left anterolaterally, compressing the spinal cord. Through a C4\u2013T2 laminotomy, the cyst was excised totally and the dural defect was repaired. Several features of the reported case, such as cyst size, location, and clinical features make it extremely unusual. The case is discussed in light of the relevant literature.\nIntroduction\nVarious types of cystic lesions are confronted in the spinal canal and are classified in respect of their relationship to the adjacent structures and nature of the cyst content. Amongst them, cerebrospinal fluid (CSF)-filled meningeal cysts constitute for the majority of the cases [11, 18] and are called as \u201carachnoid cysts,\u201d \u201cdiverticula,\u201d [5, 9] or \u201cpouches\u201d [9]. They may be located in the intradural, extradural, or perineural spaces. Depending on the location, size, and mechanism of origin, the clinical course varies between asymptomatic, incidentally-diagnosed cases [17] to severe myelopathy [11]. When they communicate freely with subarachnoid space, fluctuating symptoms related to changes in CSF pressure may occur [8, 9]. Although arachnoid cysts can be observed anywhere along the length of the spinal canal, middle, and lower thoracic regions constitute for the most frequently involved areas. When progressive neurological findings exist, surgical treatment is warranted. Surgical techniques include excision, fenestration, or placement of a cysto - subarachnoid shunt [6, 11], as well as some less invasive techniques that have been proposed recently [16].\nWe present a case with a giant cervicothoracic extradural arachnoid cyst and its surgical management. Several features of the cyst, such as its size, location, and clinical features make the case extremely unusual.\nCase Report\nA 57-year-old male was referred with symptoms of difficulty in walking, weakness, numbness, and thinning of arms and legs, and impotency, which had all begun two years before. The patient had suffered a serious traffic accident when he was 10\u00a0years old, and has had a contracture of his right hand since then. Neurological examination revealed spastic quadriparesis predominantly on the left side (left upper extremity proximally had 2\/5 muscle strength, distally had 1\/5, and left lower extremity globally had 4\/5), left-sided atrophy, hypoesthesia and hypoalgesia in C5 - C8 dermatomes, hypoactivity of deep tendon reflexes at the upper extremity, hyperactivity in patellar reflex, and Babinski sign. Muscle tonus of bilateral lower extremities was increased. The anal sphincter tonus was weak, and the patient reported an intermittent sphincter control defect.\nCervical radiographs showed widening of the left neural foramina and the spinal canal. Magnetic resonance imaging (MRI) revealed an extradural cystic mass extending from C2 to T2 vertebral bodies, located left anterolaterally, compressing the spinal cord (Figs.\u00a01a, b, c and 2). It had the same CSF intensity on T1 and T2-weighted images and showed no contrast enhancement. The lesion had caused a concavity in the related vertebral bodies and widening of the neural foramina on the left side. Electrophysiological tests reported chronic severe neurogenic involvement and a delay in the somato - sensorial evoked potential responses in C6 - T1 innervated muscles.\nFig.\u00a01T2-weighted median (a), slightly left paramedian (b), and left paramedian (c) sagittal magnetic resonance images of the cervical region shows a cystic mass extending from C2 to T2 vertebral bodies, located left anterolaterally, enlarging left neural foramina (arrows). (Arrowheads spinal cord)Fig.\u00a02Axial magnetic resonance image taken from T1 level shows the CSF-filled extradural cyst, located left anterolaterally, compressing the spinal cord (SC spinal cord)\nA C4 \u2013 T2 laminotomy was performed. An extradural cystic lesion, full of CSF and having a thin and transparent membrane, was observed compressing the dural sac. The lesion had caused widening of C5 \u2013 C7 foramina and there was an active flow of CSF to the cyst through the defects located at the exiting points of these three roots. The cyst wall was excised and the dural defects were repaired using muscle pieces and sutures. Despite some degree of the spinal cord and dural sac expansion, the epidural and subarachnoid spaces left enlarged, since the canal was widened. The epidural space was left unfilled for the fear of compression. The samples taken from the cyst wall were reported as fibrohyalinized meningeal tissues.\nPostoperative course was uneventful. Although the spasticity gradually improved during two years of follow-up, muscle strength and other neurological findings did not change.\nDiscussion\nSpinal arachnoid cyst is a rare entity. There are various classifications based on etiology, histopathology, or localization of the cysts. Many authors classify spinal meningeal cysts as both intradural and extradural.\nExtradural cysts include arachnoid cysts, synovial cysts, ganglias, cysts of ligamentum flavum, and cysts originating from the intervertebral discs. Intradural cysts include arachnoid cysts, enterogen (endodermal, neuroenteric) cysts, and ependymal cysts [3, 6, 7, 10, 12 \u2013 15, 18]. The pathogenesis of spinal arachnoid cyst is not clear and probably not homogenous. Congenital [13], traumatic [1], or inflammatory [15] mechanisms were proposed. It was documented that congenital asymptomatic cysts could enlarge due to trauma and become symptomatic [3]. The size of arachnoid cysts may vary in relation to underlying mechanisms. For example, the arachnoid cysts related to arachnoiditis are usually localized and small [6, 18]. Pulsatile CSF dynamics [9], osmotic gradient between the subarachnoid space and cyst [7], and the valve - like mechanism between the cyst and subarachnoid space [10] may play an important role in the enlargement of spinal extradural arachnoid cysts. Doita et al. [4] demonstrated that increased intra abdominal and intrathoracic pressure may lead to size changes of the spinal arachnoid cyst.\nA review demonstrated that arachnoid cysts involving the cervical region are extremely rare, that they never exceed three vertebral bodies in length, and that none extended into the thoracic region [6], except the case reported by Safriel et al. [14]. That case was an intradural arachnoid cyst situated anteriorly to the spinal canal extending between C5 and T3 levels. The presented cyst was extradural, located at the cervicothoracic region, anterolateral to the spinal cord, and involved eight vertebral bodies extending from C2 to T2 level. To the authors\u2019 knowledge, there is no extradural arachnoid cyst reported in the literature matching that size. The patient had no disposing factor other than trauma. He had suffered a serious traffic accident when he was a child and consequently had a contracture in his left upper extremity. Although there was an excessively long duration (45\u00a0years) between trauma and appearance of new symptoms attributable to the cyst, the concordance of the former neurological deficit and location of the cyst convinced us that the etiology of the cyst might have been the childhood trauma. During the surgery, we did not observe any laminar signs that indicated a sustained compression fracture on the posterior elements. Thus, stretching and partial avulsion of the roots may have caused arachnoid and dural tearing during the childhood trauma, causing subsequent collection of the CSF at the epidural space, communicating freely both subarachnoid space and dural sleeves of the roots. Indeed, the occurrence of multiple dural defects, through which the CSF exits, is very rare. This point makes an extra implication on the case and further supports the avulsion theory as the cause of multiple dural tearings, since the neurological deficit due to childhood trauma is concordant with multiple root involvement. Long-standing progressive enlargement of the cyst explains radiological signs such as eroded vertebral bodies and enlarged intervertebral foramina.\nConclusion\nThe pathogenesis, etiology, and treatment of the spinal arachnoid cyst have not been well established because of its rarity. Symptomatic cases require surgical intervention. For long segment involvements, hemilaminectomy or laminotomy, as was performed in the current case is preferable to laminectomy in order to prevent postoperative deformity. In surgery, dural defect should be closed after excision. Neurological recovery depends on the size of the cyst and degree and duration of the spinal cord compression. A long-standing spastic myelopathy is unlikely to have a significant improvement [2], as we observed in the current case. Except spasticity, the patient had no improvement on his neurological findings. Thus, in delayed cases, surgery may be taken and should be offered as a prophylactic intervention to prevent further impairment, rather than a curative one.\nIn conclusion, the case presented here is a very unusual occurrence of this rare entity. Although probably traumatic, it had extremely delayed clinical presentation, its location was unusual, and as an extradural cervicothoracic arachnoid cyst, it was the largest amongst those reported.","keyphrases":["arachnoid cysts","surgical treatment","magnetic resonance imaging","spinal cord compression","spine"],"prmu":["P","P","P","P","U"]}
{"id":"Virchows_Arch-3-1-2082069","title":"Pseudoneoplastic lesions of the testis and paratesticular structures\n","text":"Pseudotumors or tumor-like proliferations (non-neoplastic masses) and benign mimickers (non-neoplastic cellular proliferations) are rare in the testis and paratesticular structures. Clinically, these lesions (cysts, ectopic tissues, and vascular, inflammatory, or hyperplastic lesions) are of great interest for the reason that, because of the topography, they may be relevant as differential diagnoses. The purpose of this paper is to present an overview of the pseudoneoplasic entities arising in the testis and paratesticular structures; emphasis is placed on how the practicing pathologist may distinguish benign mimickers and pseudotumors from true neoplasia. These lesions can be classified as macroscopic or microscopic mimickers of neoplasia.\nIntroduction\nThere are many lesions that can simulate a neoplasm in the testis or paratesticular structures. Their incidence among tumors arising within the scrotal sac varies according to different series from 6 to 30% [16, 32]. These pseudoneoplastic lesions can be divided into those that only macroscopically imitate neoplasia (Table\u00a01) and those that microscopically imitate neoplasia, regardless of whether they form a macroscopic mass (Table\u00a02). The latter group causes more problems to the practicing pathologist in terms of the correct classification of a giving lesion. Hereby, we summarize main tumor-like lesions and benign mimickers that may be seen in the testis and paratesticular coverages, with emphasis on morphologic criteria for the differential diagnosis from true neoplasia.\nTable\u00a01Macroscopic mimickers (pseudotumors) of testicular and paratesticular neoplasiaVascular lesions\u00a0\u00a0Intratesticular hemorrhage\u00a0\u00a0Segmental testicular infarction\u00a0\u00a0Organizad testicular hematocele\u00a0\u00a0\u00a0\u00a0Cholesterol granuloma of the tunica vaginalisInflammatory lesions\u00a0\u00a0Nonspecific infectious inflammatory lesions\u00a0\u00a0Specific infectious inflammatory lesions\u00a0\u00a0Non-infectious inflammatory lesions\u00a0\u00a0\u00a0\u00a0Idiopathic inflammatory lesions\u00a0\u00a0\u00a0\u00a0\u00a0Idiopathic granulomatous orchitis\u00a0\u00a0\u00a0\u00a0\u00a0Testicular malakoplakia\u00a0\u00a0\u00a0\u00a0\u00a0Testicular sarcoidosis\u00a0\u00a0Meconium periorchitis\u00a0\u00a0Sperm granulomaCysts\u00a0\u00a0Testicular cysts\u00a0\u00a0\u00a0\u00a0Albuginea cysts\u00a0\u00a0\u00a0\u00a0Parenchymal cysts (Epidermoid cysts)\u00a0\u00a0\u00a0\u00a0Rete-testis cysts- Cystic displasia of the rete testis\u00a0\u00a0Epididymal cysts and Spermatoceles\u00a0\u00a0Spermatic cord cystsEctopic tissues\u00a0\u00a0Adrenal cortical rests\u00a0\u00a0Spleno-gonadal fusionTesticular appendagesMiscellaneous other lesionsFibrous pseudotumors; (Fibromatous periorchitis-Nodular periorchitis)\u00a0\u00a0Amyloidosis\u00a0\u00a0Polyorchididm\u00a0\u00a0Sclerosing lipogranulomaTable\u00a02Microscopic mimickers of testicular and paratesticular neoplasiaTesticular\u00a0\u00a0Inflammatory-reactive lesions\u00a0\u00a0\u00a0Lymphocitic orchitis (Testicular pseudolymphoma)\u00a0\u00a0\u00a0Rosai-Dorfman disease\u00a0\u00a0Sertoli cell hyperplasia\u00a0\u00a0\u00a0\u00a0Pick adenoma\u00a0\u00a0\u00a0\u00a0Hamartomatous proliferation testicular feminization syndrome\u00a0\u00a0Interstitial cell hyperplasia\u00a0\u00a0\u00a0\u00a0Leydig cell hyperplasia\u00a0\u00a0\u00a0\u00a0Testicular \u201ctumor\u201d of the adrenogenital syndrome\u00a0\u00a0Hyperplasia of the rete testisEpididymis\u00a0\u00a0Adenomatoid hyperplasiaTunicas albuginea-vaginalis\u00a0\u00a0Mesothelial hyperplasiaSpermatic cord\u00a0\u00a0Vasitis nodosa\u00a0\u00a0Inflammatory pseudotumor (Funiculitis proliferans)Miscellaneous other lesions\nMacroscopic mimickers (pseudotumors) of testicular and paratesticular neoplasia\nThis group of lesions refers to those that macroscopically imitate a neoplasia. Frequently, these lesions affect both compartments and are therefore difficult to establish if one or both are affected. For this reason, we classify the tumor-like lesions according to their etiology.\nVascular lesions\nThe majority of the vascular disorders of the male gonad are not confused with a tumor because, in addition to causing acute symptoms, they usually affect the entire gonad. However, in the rare situation in which the vascular lesion is segmental, it can simulate neoplasia, as follows:\nIntratesticular hemorrhage, which occurs spontaneously [24] or in the context of anticoagulation treatment [15], may simulate a tumor at ultrasound examination.Segmental testicular infarction, a lesion clinically characterized by slight local pain unrelated to any acute episode [20], may be related to isolated or systemic vasculitis [43, 103] with morphology of polyarteritis nodosa [21], giant cell vasculitis [94], or Wegener granulomatosis [45]. Up to year 2000, 81 cases of systemic vasculitis with testicular tumor-like lesion have been recorded [45]. Other cases are seen in the context of a hematological disease (sickle cell anemia) [53] or associated with nonspecific perivascular fibrosis. Any of the phases of an infarction can be observed from acute (with hemorrhage) to healing stage (Fig.\u00a01). Currently, the clinical diagnosis can be suspected with Doppler sonography [81], avoiding orchiectomy.Organized testicular hematocele and other hemorrhages in the tunica vaginalis are rarely confused with a neoplasia [89], but this can happen occasionally in long-standing cases because of fibrous thickening with cholesterol granuloma formation in the tunica vaginalis (Fig.\u00a02) [61] Exceptionally, a true testicular neoplasia presents clinically with a hematocele [86].Fig.\u00a01Segmental testicular infarction. a Acute hemorrhagic with reinforcement of the peripheral area (arrows). b Healing fibrosisFig.\u00a02Cholesterol granuloma in the tunica vaginalis (H&E)\nInflammatory lesions\nSimilar to what was stated for vascular tumor-like lesions, inflammation that simulates a neoplasia usually has atypical clinical features; these entities can be grouped as follows:\nNonspecific infectious inflammatory lesions with a tumor-like presentation are frequently chronic processes causing progressive fibrosis, which may clinically [38] or sonographicaly [23] simulate neoplasia. Rarely a testicular, epididymal, or vas deferens abscess can look like a neoplasia [84]. Very occasionally, testicular neoplasia can clinically imitate an acute inflammatory process [44].Specific infectious inflammatory lesions. The entities of this group that most often have been confused with neoplasias are granulomatous inflammation in tuberculosis [87], brucellosis [49], syphillis [5], fungal infections [42], and parasite diseases [9]. These lesions are usually not problematic for the pathologist.Noninfectious inflammatory lesions. This group of tumor-like lesions include different entities among which can be highlighted:Idiopathic granulomatous orchitis, probably of autoimmune aetiology, of which around 230 cases have been published [4], is characterized by tubular granulomas (tubular orchitis; Fig.\u00a03) or interstitial granulomas (interstitial orchitis). The presence of intratubular giant cells differentiate this entity from infectious granulomatous orchitis [70]. Diffuse testicular hypoechoic involvement with only peripheral low-resistance flow on color Doppler sonography is a typical but not pathognomonic pattern [72].Malakoplakia. This lesion is secondary to a mononuclear decrease in cyclin\u2013guanine monophosphate that impairs the killing of bacteria. Fusion of the phagolysosomes with bacterial rests produces the characteristic Michaelis\u2013Gutman bodies in the cytoplasm of the macrophages (von Hansemann cells) [1] (Fig.\u00a04). Giant cells are occasional or absent. Testicular involvement represents only 12% of genital malakoplakia with around 388 cases in the literature [19, 56]; exceptionally, it may affect the epididymis only [31]. It has been related to idiopathic granulomatous orchitis [56, 57] and chronic xantogranulomatous inflammation of testis [85, 109, 110]; this last also reported in the spermatic cord [64, 100].Sarcoidosis. Testicular involvement in a systemic sarcoidosis [39] is exceedingly rare, and its presentation as the primary form is even rarer; in these cases, the epididymis is affected more with the testicle being involved by contiguity [83].Meconium periorchitis. This is an infrequent lesion (around 30 cases being reported) that typically presents in the first months of life; most times there is a clinical history with obstetric problem that has caused the passing of meconium toward the testicular surrounding structures [29]. The macroscopic appearance is a myxoid material with calcified pearls resulting from the calcification of the remains of squamous cells or lanugo hairs [107] (Fig.\u00a05). Suspicion of neoplasia, although very uncommon in this period of life, may be caused by a scrotal mass or sonographically detectable calcifications [6]. Clinically, the peritesticular and spermatic cord enlargement can simulate a paratesticular rhabdomyosarcoma.Sperm granuloma. As its name indicates, this granulomatous lesion with few giant cells is the consequence of extravasation of spermatozoa generally postvasectomy (40% of vasectomized men and 2.5% of general population) [108]. When it produces a tumor-like lesion, it is usually located in the deferent duct or the epididymis [22] with firm nodules of 0.7 to 4\u00a0cm with occasional cysts formation.Fig.\u00a03Idiopathic granulomatous orchitis characterized by tubular granulomas (tubular orchitis)Fig.\u00a04Malakoplakia. a Macroscopic appearance with a homogeneous aspect. b The characteristic Michaelis\u2013Gutman bodies in the cytoplasm of the macrophages (von Hansemann cells) (PAS)Fig.\u00a05Meconium periorchitis. a calcified pearls resulting from the calcification of the remains of squamous cells or lanugo hairs. b Keratin rest in one of the calcifications (H&E)\nCysts\nThe majority of the cysts with a tumor-like appearance are paratesticular structures, but the testicle may occasionally have some cystic lesions that can be confused with a neoplasia.\nTesticular cysts occur in approximately 8\u201310% of patients with a lump in the testis, including those of the tunica albuginea or the parenchyma [33].Tunica albuginea cysts do not usually cause any diagnostic problem, unless if they are complex cysts [74].Parenchyma testicular cysts can be more difficult to distinguish from a neoplasia and if there is the slightest suspicion of an intracystic content, one must suspect a malignant neoplasia [107]. Special consideration deserves the testicular Epidermoid cyst, which must only be lined with squamous epithelium (Fig.\u00a06). It is recommended that the specimen is examined \u201cin toto\u201d to avoid underdiagnosis of any area of teratoma (especially among postpuberal patients) or intratubular germ cell neoplasia, as sonographically it is not possible to distinguish between these lesions [54, 106]. Epidermoid cysts represent 1% of the masses of the testes. Recent genetic studies have shown that there is no chromosome 12p abnormality [14], thus supporting its distinction from teratoma.Tubular ectasia of the rete testis secondary to obstruction and generally located in the mediastinum area of the testes (Fig.\u00a07) [69], is usually bilateral and very different from cystic dysplasia of the rete testis, a congenital lesion with complete testicular parenchyma substitution [68].Epididymal cysts and spermatoceles are relatively frequent and the majority are in relation to the inflammatory processes. The differential diagnosis with other entities are related to its size, and similar to the other cystic formations a true neoplasia must be considered in case of observing any content in its interior [108].Spermatic cord cysts. The majority of these cysts do not cause any diagnostic doubts, only the occasional Epidermoid cysts [46, 105] can simulate a neoplasia. These cysts can be unilocular or multilocular depending on their origin [65], and the multilocular must be distinguished from the exceptional cystadenomas of probably M\u00fcllerian origin [55].Fig.\u00a06Epidermoid cyst, which must only be lined with squamous cell epithelium (H&E)Fig.\u00a07a Tubular ectasia of the rete testis located in the mediastinum area of the testes (arrows). b Microscopic aspect (H&E)\nEctopic tissues\nEctopic adrenocortical tissue is relatively frequent in the tunica albuginea, rete testis, epididymis, and spermatic cord and occasionally reaches the size to be symptomatic. Its incidence ranges from 2.5 to 15% [17, 102]. It is made up of adrenal cortical nodes surrounded by a connective tissue band and of about 5\u00a0mm in diameter on average (Fig.\u00a08); for which reason, they are not clinically palpable. Only in cases of congenital adrenal hyperplasia or Cushing syndrome, ectopic adrenocortical tissue can be prominent and appear as a tumor-like lesion [90].\nSplenic\u2013gonadal fusion, as its name indicates, is the fusion of spleen and gonad. It is more frequent on the left side, with about 148 published cases [41]. Morphologically, the ectopic splenic tissue can be in close relation to the head of the epididymis or the upper pole of the testis (Fig.\u00a09) or being separated from it; likewise, there may or may not be a structural continuity between the normal spleen and the ectopic tissue [30]. The same alteration has been described in women, but it is much less frequent that in men, probably because it is much easier to find it clinically in men. In about 30% of the cases, it is associated with complex malformations such as micrognathia, peromelia, or phocomelia (absence of upper portion of a limb) [30, 60, 95]. In three of the reported cases, the fusion was associated with a germ cell tumor of the testis [41].\nAn exceptional hepato-gonadal fusion is reported [26].\nFig.\u00a08\nEctopic adrenocortical tissue. Adrenal cortical nodes surrounded by a connective tissue band in continuity with epididymis. (H&E)\nFig.\u00a09\nSplenic\u2013gonadal fusion. The ectopic splenic tissue in close relation with the upper pole of the testis (H&E)\nTesticular appendages\nThere are five testicular appendages, but for the surgical pathologist, only three can be of interest (appendix testis or hydatid of Morgagni, appendix epididymis, paradidymis or organ of Giraldes; Fig.\u00a010). These structures are not usually the origin of a tumor-like lesion, but in rare cases of large-sized cysts, it may presents as a paratesticular mass [93]. A case of ectopic epididymal tissue in an appendix testis [101] has recently been reported.\nFig.\u00a010Normal testicular appendages\nMiscellaneous other lesions\nFibrous pseudotumors. This name refers to a fibrosis phenomenon with paucicellular hyalinized collagen (Fig.\u00a011) presenting as nodular (single or multiple) or diffuse lesion of the testicular tunics [96, 71]. Sometimes, a node can be free (scrotal mouse) [108]. This broadly considered spectrum of lesions has received diverse names: chronic periorchitis, fibromatous periorchitis, nodular periorchitis, fibrous proliferation of the tunica, nonspecific paratesticular fibrosis, granulomatous periorchitis, nodular fibrous pseudotumor, fibrous pseudotumor, inflammatory pseudotumor, fibroma, reactive periorchitis [108], indicating its controversial pathogenesis. Some cases have been reported preceded by trauma or infection, and on occasion, an inflammatory component can be observed and granulation tissue suggesting the possibility that there might be the healing of an inflammatory pseudotumor, (which will be described later on) [10]. Although radiologically, it is not difficult to recognize, upon occasion, an intraoperative frozen section becomes necessary.\nAmyloidosis. It is usually bilateral and present in a patient with a prior history of amyloidosis [34]; more rarely, it is a primary form that by being a cryptorquidic patient simulates a testicular tumor [13].\nPolyorchidism or supernumerary testes is a rare condition, which is easy to recognize. However, the sonography can occasionally be different from that of the normal testis, and the condition may then be tumor suspicious [79].\nFig.\u00a011\nFibrous pseudotumor. a Well outlined fibrous-like nodule. b Paucicellular hyalinized collagen with calcification\nMicroscopic mimickers of testicular and paratesticular neoplasia\nLesions or cellular changes that microscopically imitate a neoplasia are included under this category, whether or not they make a clinical mass. These changes are closely related to the structure of the organ in which they arises, as follows.\nTesticular\nInflammatory-reactive lesions Some lesion, already described above such as xantogranulomatous orchitis, idiopathic granulomatous orchitis and malakoplakia could be considered under this category; their microscopic confusion with a neoplasia (seminoma for example) is not currently a usual event; therefore, we preferred to include them in the macroscopic mimickers (pseudotumors). The situation is different with lymphocitic orchitis or testicular pseudolymphoma [2, 3], which is characterized by a lymphocytic and plasmocellular reaction that may be confused with a lymphoma, but immunohistochemistry will show that the cellular infiltrate is polyclonal (Fig.\u00a012). Among these idiopathic lesions, we can include Rosai\u2013Dorfman disease; histological examination of the testicular mass reveals an inflammatory lesion comprising lymphocytes, plasma cells, and sheets of pale staining histiocytes, some containing lymphocytes within their ample cytoplasm, suggestive of emperipolesis. The histiocytes stained positive for CD68, S100 by immunohistochemistry and negative for CD1a, while ultrastructural examination confirmed emperipolesis [25].\nFig.\u00a012Lymphocitic orchitis. Lymphocitic cellular infiltration of polyclonal type. a CD45RA (lymphocytes B). b UCHL-1 (lymphocytes T)\nSertoli cell hyperplasia In a series of situations, nonencapsulated nodules of Sertoli cells can be found, especially known in cryptorchid testes as Pick\u2019s adenomas (Fig.\u00a013a) [77]. Because of their appearance, these should be distinguished from the actual Sertoli cell tumors that generally are larger and sometimes there are areas that mimic Call\u2013Exner bodies (Fig.\u00a013b) A differential diagnosis with a yolk-sac tumor is not usually in the scope, but the immunohistochemistry study with AFP, calretinin, \u03b1-inhibin, and CD 99 can help [40].\nFig.\u00a013Sertoli cell hyperplasia. a Nonencapsulated nodules of Sertoli cells (Pick\u2019s adenoma). b Sometimes there are areas that mimic Call\u2013Exner bodiesA special consideration merit the androgen insensitivity syndrome or testicular feminization (male pseudohermaphrodism, caused by a failure of androgen receptor binding) that in 63% of cases can have tubular hamartomas (tubules lined by immature Sertoli cells) [82] that must be differentiated from the Sertoli cell adenomas and sex cord tumor with annular tubules [76].\nInterstitial cell hyperplasia In testicles with marked tubular atrophy, such as in the Klinefelter\u2019s syndrome, it is possible to see Leydig cell nodules that must be distinguished from Leydig cell tumors. An interstitial growth without expansive pattern favors hyperplasia (Fig.\u00a014) [62].\nFig.\u00a014Leydig cell hyperplasiaNodules of eosinophilic cells appearing to be Leydig cells are found in the patients with adrenogenital syndrome [18, 48] and Nelson\u2019s syndrome (adrenocorticotropic hormone-secreting pituitary adenoma after bilateral adrenalectomy), some of this last syndrome with excessive testosterone production [91]; proof that these interstitials cells are not only morphologically similar to Leydig cells but also have the functional property of these cells. These nodules are usually bilateral and of a large size with cellular pleomorphism and pigmentation. The clinical history and a complete endocrinological profile avoid an unnecessary orchiectomy [78] because only one case of aggressive behavior is published [18].\nHyperplasia of the rete testis The normal rete testis epithelium is flat, but in some hyperestrogenic situations (treatment or hepatic dysfunction), the epithelium may become columnar and rarely a micropapillary growth of bland cells can be observed. The diagnosis of rete testis hyperplasia is subjective, and adenomatous lesions are rarely seen [35, 63]. In some cases, there are intracytoplasmic hyaline eosinophilic globules resembling a yolk sac tumor, but the negative stains of \u03b1-fetoprotein or placental alkaline phosphatase help to rule this differential diagnosis [99].Pseudohyperplasia of the rete testis and epithelial reaction in case of germ cell invasion and cryptorchidism must be differentiated from real hyperplasia of the rete testis [62].\nEpididymis\nBenign microscopic mimickers of cancer in the epididymis are very rare. Cysts may occur but do not resemble tumors microscopically. However, some cases of adenomatoid hyperplasia of the rete testis can involve the epididymis, and occasionally, they may become macroscopically apparent [93].\nTunica albuginea and vaginalis\nNon-neoplastic mesothelial lesions involving the paratesticular region include mesothelial cysts and reactive mesothelial hyperplasia [73].\nMesothelial hyperplasia is the most important benign mimicker of the testicular tunics. It is present as a reactive lesion in hydrocele or hernia but may also be found microscopically in older men [80]. The mesothelial proliferation has an epithelial appearance, and rarely, a spindle cell proliferation can be present. In the differential diagnosis with mesothelioma, the bland nucleus, no true invasion, and associated inflammatory elements can be useful (Fig.\u00a015) [11, 98]. Recently, a case was published with \u201catypical\u201d mesothelial hyperplasia on one side and \u201cwell differentiated\u201d mesothelioma on the contalateral [97]. This case is an example of the subjective interpretation of some mesothelial proliferative lesions because some malignant mesotheliomas lack cellular atypia. In these cases, an extensive confluence or prominent infiltration favors a malignant diagnosis. Unfortunately, the immunohistochemical expression of benign and malignant mesothelial proliferations are similar (low- and high-molecular-weight cytokeratins and vimentin) [11, 28], and only the metastatic neoplasias can be differentiated by the CEA, Ber-EP4, and B72.3 expression [27]. Only deoxyribonucleic acid ploidy can distinguish some borderline lesions [28].\nFig.\u00a015Mesothelial hyperplasia. The bland nucleus, no true invasion and associated inflammatory elements, can be useful to distinguish from malignant mesothelioma\nSpermatic cord\nThe vas deferens and the soft tissues of spermatic cord can have benign mimickers.\nVasitis nodosa is a ductular proliferation, generally after vasectomy [36], although it can be seen following other trauma on that area, [75]. It has a microglandular morphology (Fig.\u00a016) with mild nuclear atipia and perineural growth [7] or benign vascular invasion [8] that may be mistaken for malignancy [104]. The frequent hyperplasia of nerve fibers in the adventitia can explain the painful symptoms in some patients [36]. The coincidence with microscopic sperm granulomas and inflammatory reaction can help in the correct diagnosis. An analogous epididymal lesion also occur [88].Proliferative funiculitis is the inflammation of the spermatic cord usually the result of an extension of vasitis. The soft tissues of the spermatic cord are also the most common site of an inflammatory pseudotumor in the male genital tract [37, 50, 58]. The lesion is ill defined, myxoid with white-gray color, and a moderate cellular proliferation with loose collagen fibers and irregular infiltration of inflammatory cells. An exceptional case has been reported that was largely infiltrated by mast cells [92]. In some cases, a prominent spindle cell proliferation mimics a sarcoma (pseudosarcomatous myofibroblastic proliferation), but low mitotic index, a capillary pattern, inflammatory cells, and absence of atypical mitoses speak against a diagnosis of sarcoma. Unfortunately, immunohistochemistry is only partly helpful because the cells express actin and vimentin, less strongly desmin and exceptionally cytokeratin. Two cases of this lesion in epididymis [12, 51] and one in the rete testis [47] have been reported.Fig.\u00a016Vasitis nodosa. Ductular proliferation with a microglandular morphology\nEmbryonic remnants\nAlthough ectopic tissue usually is more problematic for constituting a mass, some of them can cause microscopic diagnostic doubts for which reason they can be included in this group of benign mimics. The presence of seminiferous tubules within the tunica albuginea [67], Leydig cells in rete testis, albuginea, spermatic cord, or within sclerotic tubules [59, 66], prostate gland in the epididymis [52], and special circumstances with muscular rete testis hypertrophy [27] can mimic a neoplasia.\nThe lesions described constitute a large heterogeneous group, without etiological or pathogenic relations among them or with true neoplasias. However, in spite of that, the patient with one or more of these tumor-like and\/or benign mimickers can have a concomitant or ulterior true neoplasm.","keyphrases":["testis","paratesticular structures","pseudotumor","tumor-like","benign mimics"],"prmu":["P","P","P","P","P"]}
{"id":"Graefes_Arch_Clin_Exp_Ophthalmol-4-1-2292474","title":"The effect of acute hyperglycemia on retinal thickness and ocular refraction in healthy subjects\n","text":"Purpose To quantify the retinal thickness and the refractive error of the healthy human eye during hyperglycemia by means of optical coherence tomography (OCT) and Hartmann\u2013Shack aberrometry.\nPatients with diabetes mellitus (DM) often experience subjective symptoms of blurred vision associated to hyperglycemia. The nature and origin of this phenomenon are still unclear. Blurred vision during hyperglycemia could be a result of transient refractive alterations due to changes in the lens [5, 12, 15, 25, 27, 36, 37], but it could also be caused by changes in the retina. Macular edema, or retinal thickening due to abnormal fluid accumulation within the macula, is a common cause of visual loss [1, 14, 22]. The degree of retinal thickening has been found to be significantly correlated with visual acuity [24]. Furthermore, a change in retinal thickness, resulting in a change in axial eye length, could also induce a change in ocular refractive error. For instance, it can be calculated that with a 50\u00a0\u03bcm increase in retinal thickness, the ocular refractive error becomes 0.15\u00a0D more hyperopic [30].\nSeveral studies have demonstrated that retinal thickness is affected by DM [2, 7, 8, 18, 21, 23, 26, 32, 33, 35, 38]. In general, an increase in retinal thickness has been reported in patients with long-term DM and advanced stages of retinopathy [7, 8, 18, 32, 33, 35]. However, in diabetic patients with and without minimal diabetic retinopathy, a decrease in retinal thickness has been observed [2, 23]. In healthy subjects, it has been shown that the average retinal thickness did not change during normo-insulinaemic hyperglycemia [13].\nIt is unclear whether the thickness of the different retinal areas, such as the foveal area, the pericentral foveal area, and the peripheral foveal area, changes during acute hyperglycemia and suppression of insulin. A change in retinal thickness and\/or ocular refractive error could explain the subjective symptoms of blurred vision in patients with DM and hyperglycemia. Therefore, in the present study the effect of hyperglycemia on retinal thickness and ocular refractive error was investigated in healthy subjects during suppression of endogenous insulin. Retinal thickness was measured by means of optical coherence tomography (OCT), which is a non-invasive technique that provides cross-sectional retinal images, and produces an objective measurement of the retinal thickness, independent of the refractive status of the eye [10, 11, 29]. Furthermore, aberrometry was used to measure the ocular refractive error. This technique makes it possible to detect small changes in ocular refraction [19].\nSubjects and methods\nFive healthy subjects (two males and three females) participated in the study. The mean age of the subjects was 24.8\u00a0years (range 21.2\u201332.6), and their mean body mass index (BMI) was 24.2\u00a0kg\/m2 (range 21.4\u201329.7). The subjects were screened during a first visit, which included medical history-taking, a physical examination (measurement of visual acuity, weight, height and blood pressure) and collecting a fasting blood sample. Exclusion criteria were a history of DM (or a fasting plasma glucose >5.5\u00a0mmol\/l), a BMI of >30\u00a0kg\/m2, elevated blood pressure (>140 \/ 85\u00a0mmHg), a visual acuity of <0.5 (Snellen) or a history of ocular pathology. The investigators of the ocular parameters (NW and MD) were not informed about the blood glucose levels. Furthermore, the investigators who induced hyperglycemia (EE and SS) were not informed about the results of the ocular measurements. The study protocol was approved by the Medical Ethics Committee of the VU University Medical Center in Amsterdam, and written informed consent was obtained from all subjects after the purpose and nature of the study had been explained to them.\nProcedure to induce hyperglycemia\nAfter a 10-hour overnight fast, the subjects were given a subcutaneous injection of a low dose (100\u00a0\u03bcg) of synthetic somatostatin (Sandostatin, Novartis, Basel, Switzerland) in order to suppress endogenous insulin secretion. Each subject underwent an oral glucose tolerance test (OGTT) (75\u00a0g glucose) 30\u00a0minutes after the somatostatin injection, and blood glucose levels were measured with a blood glucose analyzer (HemoCue Diagnostics BV, Oisterwijk, the Netherlands). Endogenous insulin levels were measured by means of immunometric assays (Luminescence, Bayer Diagnostics, Mijdrecht, the Netherlands) in the Endocrinology Laboratory at the Department of Clinical Chemistry of the VU University Medical Center. The subjects remained in fasting state during the entire procedure.\nOcular measurements\nRetinal thickness was measured with the Stratus OCT (Model 3000, Carl Zeiss Meditec, Dublin, CA, USA), which combines a low-coherence scanning interferometer (wavelength 820\u00a0nm) with a video camera to visualize the fundus of the eye. The fast macular thickness OCT scan protocol was used to obtain six cross-sectional macular scans, 6\u00a0mm in length, which were positioned at equally spaced angular orientations (30\u00b0) centred on the fovea. The cross-sectional images were analyzed with OCT3 mapping software that uses an edge-detection technique to locate the vitreoretinal interface and the anterior surface of the retinal pigment epithelium. Retinal thickness was defined as the distance between these two surfaces. Two OCT scans were made of each subject before, and every 30\u00a0minutes during the period of hyperglycemia. In order to quantify the retinal thickness, the foveal map constructed by the software was divided into nine Early Treatment Diabetic Retinopathy Study (ETDRS) areas [6]: the central fovea (central circle with a diameter of 1\u00a0mm), and the pericentral area (donut-shaped ring with an inner diameter of 1\u00a0mm and an outer diameter of 3\u00a0mm) and peripheral area (donut-shaped ring with an inner diameter of 3\u00a0mm and an outer diameter of 6\u00a0mm), both of which were divided into four quadrants. Retinal thickness was calculated for all separate areas, and for the average pericentral and peripheral regions.\nOcular refractive error was determined with an IRX3 aberrometer (Imagine Eye Optics, Paris, France), which performs wavefront analysis of the eye according to the Hartmann-Shack principle [19]. After pupil dilation and paralysis of accommodation with 1.0% cyclopentolate and 5.0% phenylephrine eye-drops, a series of three aberrometry measurements was made before, and every 30\u00a0minutes during the hyperglycemic condition. From these measurements, the equivalent refractive error was calculated as: equivalent refractive error (ERE) = sphere + (cylinder \/ 2).\nThe measurements at baseline and during maximal hyperglycemia were analyzed, and any change was considered to be meaningful if the difference between the measurements was greater than the threshold of 50\u00a0\u03bcm for retinal thickness and 0.2 diopters (D) for ERE. The threshold of 50\u00a0\u03bcm exceeded the 95% confidence interval for the detection of a change in retinal thickness, which has been reported to be approximately 40\u00a0\u03bcm [4, 20, 28]. A refractive change of more than 0.2\u00a0D also surpasses the precision (defined as 95% confidence interval) of the aberrometer for measuring sphere, cylinder, and consequently ERE [3, 31]. In each subject, the significance of a change was obtained from the precision of the measurement instruments and the difference in the ocular parameters at baseline and during hyperglycemia. In the whole group, the significance of a change could be determined by means of Wilcoxon matched pairs signed rank sum tests. P-values \u22640.05 were considered to be statistically significant.\nResults\nThe changes in blood glucose after the administration of somatostatin and glucose are shown in Fig.\u00a01. Mean blood glucose levels rose from 4.0\u00a0mmol\/l (range 3.4\u20134.5\u00a0mmol\/l) to 18.4\u00a0mmol\/l (range 16.1\u201322.0\u00a0mmol\/l) after the OGTT. Endogenous insulin was suppressed by the subcutaneous injection of somatostatin during the glucose load to a mean value of 2.1\u00a0pmol\/l (range 0.4\u20134.5\u00a0pmol\/l), and remained below basal secretion level (<110.0\u00a0pmol\/l) for 147\u00a0minutes (range 75\u2013270\u00a0minutes). Subject 01 had a delayed elevation of blood glucose level, compared to the other subjects. This person received a second 75\u00a0g oral glucose load after 30\u00a0minutes in order to induce a rise in the blood glucose level. In all subjects, the blood glucose and endogenous insulin levels normalized within 6\u00a0hours after the OGTT.\nFig.\u00a01Graph of normalized blood glucose levels in the five subjects after the administration of somatostatin and glucose. Data are normalized by subtracting the value at baseline from the measured value in each subject. The oral glucose load (75\u00a0g) was administered at T 0. Subject 01 received an extra 75\u00a0g oral glucose load at T 30\nFigure\u00a02 shows the normalized ERE of the five subjects during hyperglycemia. Mean ERE at baseline was 0.6\u00a0D (SD 0.6) and 0.7\u00a0D (SD 0.6) during maximal hyperglycemia; no significant change was found in the group as a whole. A small, but significant hyperopic shift of 0.4\u00a0D (SD 0.2) in ERE was measured in subject 01 during maximal hyperglycemia, compared to the start of the procedure (p\u2009<\u20090.001). No significant change in ERE was found in any of the other subjects. Normalized retinal thickness parameters are shown in Fig.\u00a03a (central foveal area), Fig.\u00a03b (average pericentral foveal area) and Fig.\u00a03c (average peripheral foveal area). Average central foveal thickness, average pericentral foveal thickness, and average peripheral foveal thickness at baseline were 202\u00a0\u03bcm (SD 8), 277\u00a0\u03bcm (SD 5), and 243\u00a0\u03bcm (SD 8). During maximal hyperglycemia, average central foveal thickness, average pericentral foveal thickness, and average peripheral foveal thickness were 203\u00a0\u03bcm (SD 7), 275\u00a0\u03bcm (SD 3), and 242\u00a0\u03bcm (SD 9). No significant differences were found in the group as a whole. Furthermore, none of the subjects had any significant changes in the thickness of the central fovea, the pericentral fovea, or the peripheral fovea during maximal hyperglycemia, compared to baseline. The nine separate ETDRS areas were not affected by hyperglycemia. At baseline and during hyperglycemia, any change in retinal thickness that occurred in the various areas was less than 15\u00a0\u03bcm, which was within the previously defined threshold of 50\u00a0\u03bcm.\nFig.\u00a02Graph of the normalized equivalent refractive error (ERE) in diopters (D) of the five subjects. Data are presented as mean\u00b1SD; three measurements were made of each subject every 30\u00a0minutes during the procedure. Data are normalized by subtracting the value at baseline from the measured value in each subject. The oral glucose load was administered at T 0. * Significant difference between ERE at T 0 and T 210 (maximal hyperglycemia), p\u2009<\u20090.001Fig.\u00a03Graphs and maps of normalized retinal thickness parameters in the five subjects during hyperglycemia: (a) central fovea, (b) pericentral fovea, (c) peripheral fovea. Data are normalized by subtracting the value at baseline from the measured value in each subject. Each measured area has been indicated by a dark grey area on the retinal maps. No significant changes in retinal parameters were found in any of the subjects. The oral glucose load was administered at T 0\nDiscussion\nBlurred vision is a symptom that occurs frequently in patients with DM and hyperglycemia. The underlying mechanism is still unclear, and therefore the present study was carried out in an attempt to identify a possible cause of this symptom. The effect of reproducible hyperglycemia on retinal thickness and refractive error was studied in healthy young subjects who did not suffer from the systemic effects of DM.\nNo changes in the thickness of the central, pericentral or peripheral foveal areas were found in any of the subjects during hyperglycemia. In addition, no significant change was measured in any of the nine different ETDRS areas of the macula. In their study, Jeppesen et al. [13] also found no significant difference in retinal thickness in healthy subjects during normo-insulinaemic hyperglycemia. Before and 180\u00a0minutes after the start of a hyperglycemic clamp they measured the average thickness of the retina, and found that retinal thickness was not affected by hyperglycemia. Although in the present study retinal thickness was measured under different circumstances than in the study of Jeppesen et al. (hypo-insulinaemic hyperglycemia instead of normo-insulinaemic hyperglycemia), the results confirm those of Jeppesen et al.\nRetinal thickness has been reported to change in patients with long-term DM and retinopathy. A morphological change in the retina may even occur in the early stages of diabetic retinopathy [2, 7, 8, 18, 21, 23, 26, 32, 33, 35, 38]. These changes in retinal thickness are usually due to abnormal fluid accumulation resulting from a breakdown of the blood-retinal barrier [34]. Goebel et al. [8] measured retinal thickness by means of OCT in 136 patients with different stages of diabetic retinopathy and with a mean DM duration of 16\u00a0years. Mean foveal thickness was 307\u2009\u00b1\u2009136\u00a0\u03bcm in the diabetic subjects, compared to 153\u2009\u00b1\u200915\u00a0\u03bcm in healthy subjects. It seems that only long-term hyperglycemia and\/or long-term fluctuations in blood glucose levels have any significant influence on the blood\u2013retina barrier and retinal thickness. From the findings of the present study, it appears that the blood\u2013retina barrier does not seem to be affected by a single episode of acute hyperglycemia. Nevertheless, the fact that no change in retinal thickness could be determined does not exclude the possibility that there could be early dysfunction of the blood retina barrier. Other means of examination could evidence such a dysfunction of the blood retina barrier following acute hyperglycemia.\nA factor that could have biased the results of this study is the administration of a synthetic somatostatin analogue to the subjects. Somatostatin is a peptide hormone that inhibits several hormones, including IGF-1 and insulin. IGF-1 is a growth factor that is produced by the hypoxic retina to mediate angiogenesis, resulting in neovascularisation. Somatostatin analogues not only inhibit neovascularisation in patients with advanced diabetic retinopathy, but also stabilize the blood\u2013retinal barrier in patients with diabetic macular edema [16, 17]. It could have been possible that in the present study an increase in retinal thickness during hyperglycemia was prevented by somatostatin. Nevertheless, the efficacy of synthetic somatostatin in the treatment of advanced diabetic retinopathy was investigated by Grant et al. [9]. With maximally tolerated doses of somatostatin (ranging from 200 to 5000\u00a0\u03bcg\/day), after a period of 15\u00a0months one out of 22 eyes required panretinal photocoagulation, compared to nine of 24 eyes that were not treated with somatostatin. From the results of the Grant et al. study, it seems that only frequent, large doses of somatostatin over a long period of time have any significant effect on the progression of diabetic retinopathy. Although the effect of somatostatin on the healthy retina has not been investigated yet, it seems to be unlikely that the results of the present study were biased by the administration of one single, low dose (100\u00a0\u03bcg) of somatostatin.\nIn conclusion, the results of this study indicate that in healthy subjects, hyperglycemia does not cause any change in retinal thickness. Furthermore, ocular refraction in general was not affected by hyperglycemia. However, there were interindividual variations, as illustrated by subject 01, who had a hyperopic shift of refraction during hyperglycemia. Therefore, it seems that a refractive change during hyperglycemia cannot be explained by a change in retinal thickness. It could well be that other refractive components, such as the lens, are involved in causing blurred vision and refractive alterations during hyperglycemia.","keyphrases":["hyperglycemia","refractive errors","optical coherence tomography","diabetes mellitus"],"prmu":["P","P","P","P"]}
{"id":"Eur_J_Pediatr-4-1-2413085","title":"Rab proteins and Rab-associated proteins: major actors in the mechanism of protein-trafficking disorders\n","text":"Ras-associated binding (Rab) proteins and Rab-associated proteins are key regulators of vesicle transport, which is essential for the delivery of proteins to specific intracellular locations. More than 60 human Rab proteins have been identified, and their function has been shown to depend on their interaction with different Rab-associated proteins regulating Rab activation, post-translational modification and intracellular localization. The number of known inherited disorders of vesicle trafficking due to Rab cycle defects has increased substantially during the past decade. This review describes the important role played by Rab proteins in a number of rare monogenic diseases as well as common multifactorial human ones. Although the clinical phenotype in these monogenic inherited diseases is highly variable and dependent on the type of tissue in which the defective Rab or its associated protein is expressed, frequent features are hypopigmentation (Griscelli syndrome), eye defects (Choroideremia, Warburg Micro syndrome and Martsolf syndrome), disturbed immune function (Griscelli syndrome and Charcot\u2013Marie\u2013Tooth disease) and neurological dysfunction (X-linked non-specific mental retardation, Charcot\u2013Marie\u2013Tooth disease, Warburg Micro syndrome and Martsolf syndrome). There is also evidence that alterations in Rab function play an important role in the progression of multifactorial human diseases, such as infectious diseases and type 2 diabetes. Rab proteins must not only be bound to GTP, but they need also to be \u2018prenylated\u2019\u2014i.e. bound to the cell membranes by isoprenes, which are intermediaries in the synthesis of cholesterol (e.g. geranyl geranyl or farnesyl compounds). This means that isoprenylation can be influenced by drugs such as statins, which inhibit isoprenylation, or biphosphonates, which inhibit that farnesyl pyrophosphate synthase necessary for Rab GTPase activity. Conclusion: Although protein-trafficking disorders are clinically heterogeneous and represented in almost every subspeciality of pediatrics, the identification of common pathogenic mechanisms may provide a better diagnosis and management of patients with still unknown Rab cycle defects and stimulate the development of therapeutic agents.\nIntroduction\nGermline mutations in the genes involved in the Ras\u2013mitogen-activated protein kinase (MAPK) pathway explain a number of neuro\u2013cardio\u2013facio\u2013cutaneous syndromes, as reported in a paper recently published in this journal [10].\nRas-associated binding (Rab) proteins are small GTPases of the Ras superfamily that continuously cycle between the cytosol and different membranes. In 2002, Pfeffer described 20 different Rab GTPases that have been identified as prenylated proteins localized in distinct membrane-bound compartments [25]. Prenylated proteins are proteins bound to isoprenes, which are intermediaries in the cholesterol synthesis. At the time of the writing this article, more than 60 human Rabs had been identified [26]. This high number of different Rab proteins highlights their importance in the regulation of vesicle trafficking processes, including vesicle formation, motility, tethering and fusion to the acceptor membrane and signaling to other organelles. The ability of Rabs to perform these different tasks in a co-ordinated and regulated manner originates in their highly dynamic conformation and mobility and from their interaction with different Rab-associated proteins, as illustrated in the Rab GTPase cycle (Fig.\u00a01). Rabs can cycle between the active GTP-bound and the non-active GDP-bound forms, assisted by different Rab-associated proteins, such as the GEFs (guanine nucleotide-exchange factors) and GAPs (GTPase-activating proteins), respectively [24, 26]. This conformational cycle is the main driving force for the on\/off \u2018switch\u2019 mechanism of Rabs and their ability to both regulate binding to downstream effectors and perform the desired function. Rabs are initially synthesized as soluble proteins in the cytosol where they are first recognized by a soluble chaperone-like protein named REP (Rab escort protein) [3]. The REP brings the Rab to the RabGGT (Rab geranylgeranyl transferase) for the addition of\u2014generally\u2014two geranylgeranyl groups. These prenyl groups are added, by covalent thioether bonds, to cysteine residue(s) located at the C-terminus of the Rab protein [7]. This post-translational modification of Rabs is needed to allow for the attachment of the Rab proteins into the lipid bilayer of the organelle; consequently, Rabs are considered to be peripheral membrane proteins (Fig.\u00a01). After performing their function, Rab proteins are extracted from the membranes by RabGDI (GDP dissociation inhibitor), and they remain in the cytosol until they are needed again [7, 24, 37]. Statins are known to inhibit protein isoprenylation. As such, they are able to reduce the production of amyloid-beta and inhibit preamyloid catabolism in the lysosomes, which suggests their possible application in the treatment of Alzheimer [20]. However, the therapeutic role of statins in Rab-related disorders has not yet been established. The potent nitrogen-containing biphosphonates, such as pamidronate, alendronate, ibandronate and zoledronate, inhibit a key enzyme, farnesyl pyrophosphate synthase, which is necessary for Rab activity, hence their inhibitory action on osteoclast activity [29].\nFig.\u00a01The Ras-associated binding (Rab) protein\u2013GTPase cycles. Rab proteins are intrinsically soluble and require a post-translational modification for membrane association. They first associate with a Rab escort protein (REP) and form a stable complex that is the substrate for the subsequent dual prenylation of C-terminal cysteine motifs via Rab geranylgeranyl transferase (RabGGT). RabGGT consists of two different functional subunits (RabGGT\u03b1 and RabGGT\u00df). After lipid tranfer, REP delivers the prenylated Rab to the donor membrane (the REP cycle is shown with blue arrows). In the absence of REP or RabGGT, Rab proteins remain in the cytosol in an inactive state. The transfer of Rab proteins between acceptor and donor membranes is facilitated by the GDP dissociation inhibitor (GDI) (the GDI cycle is shown with red arrows). Both REP and GDI bind the GDP-bound inactive form of Rab. After REP or GDI dissociate from Rabs at the donor membrane, Rabs can cycle between the inactive (GDP-bound) and active (GTP-bound) states. Rab proteins are activated by guanine nucleotide exchange factors (RabGEFs) and deactivated by GTPase activating proteins (RabGAPs), which accelerate the slow intrinsic rates of nucleotide exchange and GTP hydrolysis. In the active state, Rabs interact with structurally and functionally diverse effectors, including cargo sorting complexes on donor membranes, motor proteins involved in vesicular transport and tethering complexes that regulate vesicle fusion with acceptor membranes\nEvolutionarily conserved Rabs are usually expressed in all cell and tissue types and regulate more fundamental vesicle transport pathways whereas the less conserved Rab family members function in more specific pathways, which are often organ-dependent. Defects in intracellular vesicle trafficking underlie a large variety of human diseases, including pathologies associated with defects in Rabs or Rab-associated proteins [14, 32]. In this review, we deal with the role of different Rab and rab-associated proteins in inherited (Table\u00a01) as well as certain multifactorial human diseases in different pediatric disciplines. In addition, the review will also focus briefly on recent advances in understanding human disease through the study of Rab proteins from in vitro and mouse knockout studies.\nTable\u00a01Human monogenic diseases caused by a Ras-associated binding (Rab) protein or Rab-associated protein defectDiseaseOMIMGeneRabRab-associatedDescriptionGriscelli syndrome type I214450MYO5AxAutosomal recessive, albinism, neurological impairmentGriscelli syndrome type II607624RAB27AxAutosomal recessive, albinism, hemophagocytic syndrome, sometimes secondary neurological impairmentGriscelli syndrome type III609227MLPHxAutosomal recessive, albinismChoroideremia303100REP1xX-linked, progressive loss of vision beginning at an early age, and the choroid and retina undergo complete atrophyNon-specific mental retardation300104GDI1xX-linked, affected males show moderate to severe mental retardation, carrier females may also be mildly affectedCharcot-Marie-Tooth disease type IIb608882RAB7xAutosomal dominant, peripheral sensory neuropathy with late onset muscle weakness, foot ulcers and infectionsWarburg Micro syndrome600118RAB3GAP1xAutosomal recessive, microcephaly, microcornea, congenital cataract, mental retardation, optic atrophy, and hypogenitalismMartsolf syndrome212720RAB3GAP2xAutosomal recessive, congenital cataracts, hypogonadism, and mild mental retardation\nRab proteins in skin and hear pigmentation\nGriscelli syndrome (GS) is an autosomal recessive disorder that causes partial albinism [15]. There are three variants of this disease: one is a purely hypopigmentation disorder (GS type III), and two, in addition to the pigmentation defect, are also associated with immunological defects (GS type II) or with primary neurological dysfunctions (GS type I). Griscelli syndrome type II with immunological defects is caused by missense mutations in the gene encoding Rab27a [18]. This Rab regulates the movement of melanosomes to the cell periphery of melanocytes and also regulates the secretion of lytic granules in cytotoxic T lymphocytes (CTL) [35]. The lack of Rab27a thus causes pigment anomalies and dysfunctional T lymphocytes, which is in agreement with the defects observed in the patients that present with albinism and hemophagocytic syndrome. The GS type I with neurological symptoms is caused by mutations in the MYO5A gene encoding the motor protein myosin Va [22], a putative Rab27a effector that drives the peripheral distribution of melanosomes along actin filaments [15]. As myosin Va does not participate in the exocytosis of lytic granules of CTL, the inactivation of this protein does not lead to immunological symptoms. Finally, GS type III in patients with typically pigment abnormalities without additional features is caused by a mutation in the MLPH gene encoding melanophilin. A direct interaction exists between the SHD domain of melapholin and Rab27a and between the C-terminal domain of melanophilin and myosin Va [36]. Therefore, although the Rab27a\/Mlph\/MyoVa complex is crucial for the trafficking of melanosomes, neurodevelopmental abnormalities are specific for the Myo5a deficiency, and Rab27a uses a different effector for its function in the CTL.\nRab proteins in immunology\nIt has already been mentioned that GS type II patients with Rab27a mutations develop the hemophagocytic syndrome, which is characterized by episodes of life-threatening uncontrolled T lymphocyte and macrophage activation [17]. During these episodes, activated T cells and macrophages infiltrate various organs (including the brain), leading to tissue damage, organ failure and death in the absence of immunosuppressive therapy. In practice, only bone-marrow or cord blood stem cell transplantation can be curative for this condition [33].\nThe important function of Rab proteins in intracellular traffic, endocytosis and exocytosis explains their role in counteracting bacterial and viral infections. Phagocytosis by macrophages leads to the destruction of bacteria. Endocytosis leads to the formation of phagosomes, but phagosomes fuse with lysosomes (phagolysosomes), resulting in the release of toxic products that kill the bacterium as well as the release of the degradation products by exocytosis [19]. However, some bacteria survive this process and avoid bactericide by escaping from the phagosomes into the cytoplasm [19]. Listeria monocytogenes is a paramount example. It has been shown that the stimulation of macrophages with interferon (IFN)-\u03b3, a cytokine that is secreted by natural killer and T helper cells, results in the expression of at least 200 proteins, including Rab5a [11]. In L. monocytogenes-infected macrophages, overexpression of Rab5a stimulates the intracellular degradation of the pathogen [6]. It has been suggested that Rab5a participates in the active recruitment of Rac2 to phagolysosomes, which is important for the destruction of the bacterium [27]. Other microbes develop antiphagocytosis (e.g. Yersinia enterocolitica) or inhibit the formation of phagosomes (e.g. Salmonella typhimurium). In Salmonella infections, there is evidence that Rab5a is stabilized on the phagosome by the Salmonella type III secretory protein SopE [23]. SopE has GEF activity for Rab5a, stimulating activated GTP-bound Rab5a and preventing its membrane extraction by RabGDI. A general conclusion that can be drawn is that parasites recruit endocytic Rabs to prevent or delay the formation of the degradative phagolysosome in the host organism.\nViruses enter host cells by receptor-mediated endocytosis, but they usually escape from endosomal vesicles to enter either the cytosol or the nucleus where they replicate. The binding of hepatitis C virus (HCV) non-structural protein (NS) with Rab1 GTPase-activating protein suggests that viruses are able to subvert host cell machinery in this way because this binding is necessary for HCV replication.\nRab1 depletion decreases HCV-RNA levels, which could lead to therapeutic applications [34].\nRab proteins in vision\nChoroideremia is an X-linked disease that involves the degeneration of the retinal pigment epithelium and the adjacent choroid and retinal photoreceptor cell layers, leading to blindness. The mutated gene in the choroideremia is one of the two REP isoforms, REP1 [3, 30]. Although the other isoform, REP-2, seems to be sufficient for the geranylgeranylation of all Rab GTPases in all tissues, the retinal pigment epithelium seems to be the exception. A role for REP-2 in human disease has not yet been discovered. REP-1 is essential for the efficient geranylgeranylation of Rab27a in the retinal pigment epithelium. Thus, a lack of REP1 leads to a lack of functional Rab27a specifically in the retinal pigment epithelium [30, 31]. The degeneration of this epithelium and its adjacent layers may be due to deficient melanosome transport and, consequently, a lack of protection against harmful light exposure.\nRab proteins in neurology\nA subgroup of patients with X-linked non-specific mental retardation have mutations in the GDI1 gene for one of the GDI isoforms, GDI-\u03b1 [9]. This isoform is particularly abundant in the brain, and dysfunctional membrane recycling of one or more Rab GTPases in brain synapses, leading to aberrant neurotransmission, is likely to underlie the symptoms in this disease.\nHereditary sensory and autonomic neuropathies (HSAN), known for many years as familial dysautonomia in Jewish patients, are characterized by the loss of sensation for pain and temperature, alacrima, excessive sweating and the absence of fungiform tongue papillae. The onset is congenital, and the transmission is autosomal recessive. The clinical symptoms of HSAN have been classified into five types [21]. HSAN type II encompasses patients with Charcot\u2013Marie\u2013Tooth disease type II. This is an axonal neuropathy with a median motor conduction velocity of >38\u00a0m\/s. This peripheral neuropathy starts in the second or third decade of life and presents as mixed motor and sensory involvement associated with ulcers. Four mutations in the Rab7 gene have been identified in four families with Charcot\u2013Marie\u2013Tooth disease type 2B (CMT2B), with symptoms leading to ulcerations and amputations [38, 39]. However, a hallmark feature of patients with HSAN type 1 is lancinating pain, which is not commonly observed in CMT2B. Moreover, HSAN type 1 is associated with a mutation in serine palmitoyl transferase (SPT), a rate-limiting step in the sphingo-lipid synthesis. This overlapping of the two syndromes is possibly explained by Rab7 being involved in both the endocytosis and transport of sphingolipids [39].\nThe importance of Rab proteins in brain development and function is even more emphasized by the recent discoveries of the involvement of Rab3-associated proteins in human disease [4, 5]. Rab3A is the most abundantly expressed protein in the brain, where it is present in all synapses and involved in calcium-dependent neurotransmitter release [13]. The activity of Rab3 proteins is tightly regulated by Rab3GAP, which specifically converts active Rab3-GTP to the inactive-GDP form. Germline-inactivating mutations in the catalytic subunit of Rab3GAP (Rab3GAP1) cause Warburg Micro syndrome, an autosomal recessive disorder characterized by ocular defects (microphthalmos, microcornea, congenital cataracts and optic atrophy), neurodevelopmental defects (microcephaly, cortical gyral abnormalities, such as pachygyria and polymicrogyria, hypoplasia of the corpus callosum, severe mental retardation, and spastic cerebral palsy) and hypothalamic hypogenitalism [4]. Mutations in the non-catalytic subunit of Rab3GAP (Rab3GAP2) have been described in patients with Martsolf syndrome, an autosomal recessive disorder with a similar but milder phenotype than the Warburg Micro syndrome, including congenital cataracts, hypogonadism and mild mental retardation [5]. The precise mechanisms whereby the Rab3GAP1 and Rab3GAP2 mutations cause the human phenotype is still unclear.\nRab proteins in endocrinology\nAbnormal trafficking of the insulin-sensitive glucose transporter GLUT4 has been described in patients with diabetes type 2. Glucose transporter 4 accumulates in the dense membrane compartments, suggesting that defects in membrane trafficking may be involved in insulin resistance [12]. Rab4 has been found to be implicated in GLUT4 biogenesis, sorting and exocytosolic movement but more studies are needed to define the exact role of this and other Rabs and Rab-associated proteins in GLUT4 trafficking [16]. Rab4 mutations in humans or mice do not yet exist.\nRab27a is necessary for insulin secretion [2]. Diabetes mellitus is characterized by both low insulin and high glucagon levels. These conditions have been shown recently to be due to the expression of an inducible cAMP early repressor (ICER) [1], resulting in a repression of the transcription of the insulin gene, and associated with a diminished level of granuphilin, a Rab27a-dependent protein. However, patients with GS type II-homozygous deficiency in Rab27a do not have diabetes mellitus.\nThe control of thyroid hormone production depends on endocytic catalysts and the tandem regulators Rab5a and Rab7, which have been found in excess in thyroid adenomas [8].\nRab proteins in nephrology\nNephrogenic diabetes insipidus (NDI) is characterized by the inability to concentrate the urine in response to the antidiuretic hormone vasopressin (AVP). Under normal conditions, binding of AVP to the vasopressin receptor (V2R) leads to the insertion of aquaporin 2 (AQP2) water channels in the apical membrane of the renal collecting duct [28] (Fig. 2). The defect can be inherited and is either X-linked due to mutations in the genes encoding the V2R or autosomal recessive or dominant in mutations in the AQP2 gene. The trafficking of AQP2 in the cell and its internalization are cAMP and especially Rab11-dependent [40]. However, Rab11 mutations in humans or mice are not yet described.\nFig.\u00a02Regulation of aquaporin-2 (AQP2)-mediated water transport by vasopressin (AVP). Shown is a nephron with a magnified principal cell. In this cell, the vasopressin V2 receptor (V2R), stimulatory GTP binding protein (Gs), adenylate cyclase (AC), ATP, cAMP, and phosphorylated proteins (O-P) are indicated. This figure has been used with permission of the American Journal of Physiology and Renal Physiology\nConclusion\nThe Rab GTPases are a large family of proteins with a variety of regulatory functions in membrane trafficking. The central role of these proteins has become clear during the past decade, as part of the progress that has been made in understanding in detail the mechanistic principles of transport vesicle formation, movement, and fusion. Sequencing of the human genome has enabled researchers to realize the diversity of the Rab gene family, although the functions of the majority of the gene products are still unknown. The availability of complete genomic sequences as well as the important advances in molecular and cell biological methods that have already been made hold the promise of significant progress being booked in our understanding of Rab function in the near future. The identification of further genes involved not only in monogenic but also in common multifactorial human vesicle-trafficking disorders will result in a better understanding of this complex transport pathway but, more importantly, will also lead to opportunities to develop novel treatments.","keyphrases":["rab proteins","vesicle transport"],"prmu":["P","P"]}
{"id":"Mol_Genet_Genomics-2-2-1705487","title":"The fission yeast Rpb4 subunit of RNA polymerase II plays a specialized role in cell separation\n","text":"RNA polymerase II is a complex of 12 subunits, Rpb1 to Rpb12, whose specific roles are only partly understood. Rpb4 is essential in mammals and fission yeast, but not in budding yeast. To learn more about the roles of Rpb4, we expressed the rpb4 gene under the control of regulatable promoters of different strength in fission yeast. We demonstrate that below a critical level of transcription, Rpb4 affects cellular growth proportional to its expression levels: cells expressing lower levels of rpb4 grew slower compared to cells expressing higher levels. Lowered rpb4 expression did not affect cell survival under several stress conditions, but it caused specific defects in cell separation similar to sep mutants. Microarray analysis revealed that lowered rpb4 expression causes a global reduction in gene expression, but the transcript levels of a distinct subset of genes were particularly responsive to changes in rpb4 expression. These genes show some overlap with those regulated by the Sep1-Ace2 transcriptional cascade required for cell separation. Most notably, the gene expression signature of cells with lowered rpb4 expression was highly similar to those of mcs6, pmh1, sep10 and sep15 mutants. Mcs6 and Pmh1 encode orthologs of metazoan TFIIH-associated cyclin-dependent kinase (CDK)-activating kinase (Cdk7-cyclin H-Mat1), while Sep10 and Sep15 encode mediator components. Our results suggest that Rpb4, along with some other general transcription factors, plays a specialized role in a transcriptional pathway that controls the cell cycle-regulated transcription of a specific subset of genes involved in cell division.\nIntroduction\nRNA polymerase II (pol II) transcribes the coding genes, and its activity is the main target for transcriptional control in all eukaryotes. The pol II enzyme consists of 12 subunits, which are highly conserved during evolution. The specific functions of the smaller subunits such as Rpb4 are relatively poorly understood, and they can show differences between organisms. For example, Rpb4 is essential in mammals and fission yeast (Schizosaccharomyces pombe), but not in budding yeast (Saccharomyces cerevisiae). Here we use fission yeast as a model to learn more about particular roles of Rpb4 in genome-wide transcription.\nMuch of our current understanding of the mechanism of transcription and the transcription machinery comes from studies using budding yeast. The distantly related fission yeast provides a valuable complementary model system to dissect the functions of different components of the transcriptional apparatus, because its transcription mechanism is more similar to that of mammals in some respects. For example, the initiation of transcription in both mammalian cells and S. pombe occur \u223c30\u00a0bp downstream of the TATA box, whereas in S. cerevisiae this distance can vary between 40 and 120\u00a0bp (Li et al. 1994). Moreover, transcriptional initiation from mammalian promoters introduced into S. pombe occurs at the same site as in mammalian cells (Toyama and Okayama 1990).\nAs in other eukaryotes, S. pombe pol II is a multi-subunit enzyme containing the 12 subunits Rpb1 to Rpb12. The four core subunits, Rpb1, Rpb2, Rpb3 and Rpb11, are homologous to the \u03b2\u2032, \u03b2, \u03b1\u2032 and \u03b1 subunits, respectively, of the eubacterial RNA polymerase. Five subunits, Rpb5, 6, 8, 10 and 12, are shared by the three eukaryotic RNA polymerases I, II and III, whereas Rpb4, Rpb7 and Rpb9 are subunits specific to pol II (Mitsuzawa and Ishihama 2004). The functions of most of these smaller subunits are not well characterized.\nRpb4 and Rpb7 form a conserved heterodimer complex in archaebacteria, yeast, plants and humans (Choder 2004). Crystallographic studies of pol II and biochemical data provide evidence for a role of the Rpb4\/7 heterodimer in transcription initiation and RNA binding in S. cerevisiae (Armache et al. 2005; Bushnell and Kornberg 2003; Edwards et al. 1991; Orlicky et al. 2001). S. pombe Rpb7 interacts with an RNA-binding protein, implying a role in the coupling of RNA processing to transcription, and it associates with glyceraldehyde-3-phosphate dehydrogenase and actin (Mitsuzawa et al. 2003, 2005).\nThe in vivo functions of Rpb4 have been characterized in S. cerevisiae, and several lines of evidence suggest that it plays an important role in the response and survival to stress (reviewed by Choder 2004). S. cerevisiae Rpb4 is also important for activated transcription from a subset of promoters and in carbon and energy metabolism at moderate temperatures (Pillai et al. 2001, 2003). Recent data show that it is required for the decay of a class of mRNAs whose products are involved in protein synthesis, and it interacts with subunits of the mRNA decay complex Pat1\/Lsm1-7 that enhances decapping (Lotan et al. 2005).\nWe are only beginning to understand the role of the Rpb4 subunit in S. pombe: it binds to the TFIIF-interacting carboxyl-terminal domain (CTD) phosphatase Fcp1, and it has been proposed to play a role in the assembly of the Fcp1\u2013pol II complex, thereby promoting CTD phosphorylation for the reutilization of pol II in a new cycle of transcription (Kimura et al. 2002). S. pombe Rpb4 exhibits features that resemble its budding yeast counterpart, while others place it closer to its orthologs in multicellular eukaryotes. It is essential for viability, whereas in S. cerevisiae it is not required for cell growth under optimal conditions (Sakurai et al. 1999; Choder 2004). S. pombe Rpb4 contains 135 amino acids, placing it closer in size to its counterparts in humans (142 amino acids) and plants (138 amino acids) than to S. cerevisiae (221 amino acids). Stoichiometric amounts of Rpb4 associate with pol II in S. pombe and higher eukaryotes, while the fraction of pol II molecules containing Rpb4 in optimally growing S. cerevisiae cells is only about 20%. Finally, Rpb4 is more tightly associated with S. pombe pol II than with S. cerevisiae pol II (Sakurai et al. 1999).\nHere, we provide evidence that Rpb4 influences the growth and gene expression of S. pombe cells in a dose-dependent fashion under optimal conditions. We show that lower levels of rpb4 expression do not affect the survival of cells under several stress conditions. Our results imply a particularly important role for Rpb4 in the expression and regulation of a subset of genes involved in cell separation. The defects in cell growth and separation as well as the gene expression signatures associated with low levels of rpb4 expression are similar to the phenotypes and expression signatures of a range of transcriptional regulatory mutants with defects in cell separation.\nMaterials and methods\nStrains and molecular genetic methods\nThe S. pombe strains used in this study are listed in Table\u00a01. JB22 was the parental haploid strain used to generate the strains expressing rpb4 under the P3nmt1, P41nmt1 and P81nmt1 promoters (Basi et al. 1993). The strains were constructed by PCR using the pFA6a-kanMX6-P3\/P41\/P81nmt1 constructs as described (B\u00e4hler et al. 1998), and the kanamycin-resistant transformants were selected on KsNoT media (Linder et al. 2002). Recombinant DNA methods were performed as described (Sambrook et al. 1989) and standard methodology and media for the manipulation of S. pombe were used (Moreno et al. 1991).\nTable\u00a01Strains used in this studyStrainRelevant genotypeSourceJB22972 h\u2212Leupold (1970)JB394 kanMX6-p3nmt1-rpb4 h\u2212This studyJB395KanMX6-p41nmt1-rpb4 h\u2212This studyJB396kanMX6-p81nmt1-rpb4 h\u2212This study\nGrowth experiments\nPre-cultures for the growth curves were made by inoculating the respective strains in Edinburgh Minimal Medium (EMM) without thiamine or with 15\u00a0\u03bcM thiamine and growing the cultures for \u223c17\u00a0h at 32\u00b0C with shaking. Subsequently, the pre-culture was diluted into 50\u00a0ml of fresh EMM medium at an OD600\u00a0=\u00a00.05\u20130.1 and allowed to grow at 32\u00b0C with shaking. Samples were removed at various timepoints thereafter for measuring ODs at 600\u00a0nm, microscopic examination, and microarray hybridization experiments. The low OD measurements in cells expressing rpb4 under the P41nmt1 and P81nmt1 promoters is not caused by the phenotype, because mutants with similar phenotypes such as sep1 show strong increases in OD during growth (unpublished data).\nQuantitative RT-PCR experiments\nTo measure rpb4 expression, the strains expressing rpb4 under the three promoters were grown for 24\u00a0h in EMM medium at 32\u00b0C (promoter on) or cultivated for a further 21\u00a0h in the presence of 15\u00a0\u03bcM thiamine (promoter off). RNA was isolated by phenol extraction, purified using the RNeasy kit (Qiagen), and treated with RNase-free DNase (Roche). The reverse transcription reactions were performed using Superscript III (Invitrogen). Real-time PCR was performed using the Brilliant\u00ae SYBR\u00ae Green QPCR Core Reagent Kit (Stratagene) on an Mx3000P QPCR system (Stratagene) with primers specific for rpb4 or fba1 (control) (sequences available on request). Arbitrary expression units were calculated using a standard curve made from serial dilutions of fission yeast genomic DNA.\nStress response assays\nSerial dilutions (1\/10) of rpb4 strains generated in our study were spotted on EMM plates with or without 15\u00a0\u03bcM thiamine. The plates were supplemented with 0.5\u00a0mM hydrogen peroxide or different concentrations of sorbitol (1, 2, 3 and 4\u00a0M) to test the response to oxidative and osmotic stress, respectively. These plates were incubated at 32\u00b0C for 2\u00a0days and photographed. The effect of temperature stress was tested by incubating the strains at 36\u00b0C for 2\u00a0days.\nMicroscopy\nUnfixed cells were observed at 2-h intervals for 25\u00a0h by light microscopy using a Zeiss Axioscope fluorescence microscope set up for differential interference contrast (DIC) with a 40\u00d7 objective and Axiovision digital imaging system. To stain the nuclei, cells were spread onto microscopic glass slides. Subsequently, the cells were fixed by heating at 70\u00b0C for 1\u00a0min and DAPI (4\u2032,6\u2032 diamidino-2-phenylindole) was added at a final concentration of 1\u00a0\u03bcg\/ml. To view the division septa, calcofluor was added to cultures at a final concentration of 5\u00a0mg\/ml and the cultures were incubated in the dark at room temperature for 5\u00a0min. The cells were washed twice with phosphate buffer saline, and 5\u00a0\u03bcl of cell suspension was spotted onto microscopic glass slides. DAPI- and calcofluor-treated cells were visualized under a Zeiss Axioscope microscope as above.\nMicroarray experiments\nTo compare the gene expression profiles as a function of rpb4 expression, cells were grown in the presence of thiamine to OD600 of 0.2, and 50\u00a0ml of culture was centrifuged. The remaining culture was washed four to five times with EMM and subsequently grown in EMM medium without thiamine for 17\u00a0h and centrifuged. To identify transcripts affected by lowered rpb4 expression, the desired strains were grown in the presence of thiamine for 21 and 23\u00a0h and harvested. A wild-type parental strain grown in the presence of thiamine served as a reference. Although thiamine leads only to minimal expression changes (Jenkins et al. 2005), this experimental design corrects for effects caused by the presence of thiamine. Pellets were frozen in liquid nitrogen and kept at \u221270\u00b0C until further processing. RNA was isolated by phenol extraction and purified by the RNeasy kit (Qiagen). cDNA probes were prepared with Superscript (Invitrogen) and labeled with Cy3 or Cy5. Detailed methods were as described (Lyne et al. 2003; http:\/\/www.sanger.ac.uk\/PostGenomics\/S_pombe). Microarrays were scanned with a Genepix 4000B scanner and analyzed with Genepix software (Axon Instruments). Data filtering, normalization and quality control were performed as described (Lyne et al. 2003). The data are based on two to three independent biological repeats with dye swaps. To quantify global effects on gene expression, we repeated microarray analysis spiked with external control RNAs. The expression data were normalized using Bacillus subtilis spikes added in equal quantities to the RNA samples before labeling (Lee et al. 2005). Each microarray contains 720 control elements for the spikes, which are spread across the complete grid of the array in a spatially even manner. Clustering and visualization were done with GeneSpring. Gene annotations were taken from the S. pombe GeneDB database (www.genedb.org\/genedb\/pombe\/index.jsp). Overlaps between different gene lists were calculated using the hypergeometric distribution. All normalized data sets are available from our website: http:\/\/www.sanger.ac.uk\/PostGenomics\/S_pombe\nResults\nDosage-dependent effect of Rpb4 on cell growth\nRpb4 is an essential subunit of S. pombe pol II. To elucidate its in vivo function(s), we constructed haploid strains expressing rpb4 from its normal chromosomal location using the thiamine-regulated nmt1 promoter. Three different versions of this promoter were used: the wild-type P3nmt1 promoter has the highest level of promoter activity and maintains substantial basal levels of expression even under repressed conditions; the two derivatives, P41nmt1 (medium strength) and P81nmt1 (weakest strength) have reduced levels of activity due to mutations in the TATA box (Basi et al. 1993).\nWe performed quantitative PCR of cells expressing rpb4 under the three promoters, both in the absence (nmt1 promoter \u2018on\u2019) or presence (nmt1 promoter \u2018off\u2019) of thiamine to verify changing levels of rpb4 mRNA (Fig.\u00a01). Expression levels of rpb4 showed a range of more than 1,000-fold in the different conditions, and the relative expression levels were as expected for the three promoters of different strength. The rpb4 levels were especially low when expressed from the P41nmt1 and P81nmt1 promoters in the presence of thiamine. The fba1 gene is highly expressed and was used as a control gene. Although it shows much less variation in expression levels than rpb4, there are some changes in accordance with rpb4 levels (Fig.\u00a01). This probably reflects effects on global transcription as a function of rpb4 expression levels (see below).\nFig.\u00a01Expression levels of rpb4 mRNA when transcribed from different promoter constructs. Cells expressing rpb4 from the P3nmt1, P41nmt1 or P81nmt1 promoter were grown for 24\u00a0h in EMM medium at 32\u00b0C (promoter on; gray bars) or cultivated for a further 21\u00a0h in the presence of thiamine (promoter off; white bars). rpb4 (left) and fba1 (right; control) mRNA levels from the same cells were measured by quantitative RT-PCR. The PCR was performed on reverse transcription reactions, which were carried out in the presence (RT+) or absence (RT\u2212) of reverse transcriptase\nTo analyze effects of rpb4 expression levels, the three strains were grown at 32\u00b0C either in the absence or presence of thiamine, and cell growth was monitored. In the absence of thiamine, all strains grew similar to each other and to the wild-type strain (Fig.\u00a02a). This shows that rpb4 expressed from the weak P81nmt1 promoter under induced conditions was sufficient to sustain normal cell growth. When these strains were grown in the presence of thiamine, however, there was a direct correlation between residual promoter activity and growth rate: cells expressing rpb4 from the P3nmt1 promoter showed the same growth rate as wild-type cells, while cells expressing rpb4 from the P41nmt1 promoter and, even more so, from the P81nmt1 promoter showed reduced growth rates (Fig.\u00a02b). Thus, in the presence of thiamine, the lowered Rpb4 levels become limiting for cell growth, and residual growth directly reflects basal expression levels from the different promoter constructs.\nFig.\u00a02Effect of Rpb4 on cell growth. Wild-type cells (filled circle) and cells expressing rpb4 from either the P3nmt1 (filled square), P41nmt1 (filled triangle) or P81nmt1 (crosses) promoter were grown in EMM medium at 32\u00b0C. OD600 was measured at the indicated times. a In the absence of thiamine, all strains grew at similar rates. b In the presence of thiamine, the growth rates were dependent on the residual levels of rpb4 being expressed from the different nmt1 promoters\nEffect of Rpb4 on cell growth under stress conditions\nRpb4 is required for stress response in S. cerevisiae (Choder 2004). We therefore, determined if Rpb4 played a similar role under stress conditions in S. pombe. Cells expressing varying levels of rpb4 were subjected to temperature stress (36\u00b0C), osmotic stress (1\u00a0M sorbitol) or oxidative stress (0.5\u00a0mM hydrogen peroxide). We found that neither in the presence nor absence of thiamine, the cells exhibited any increased stress sensitivity compared to control cells when grown under the different stress conditions (data not shown). Similar results were obtained when cells were grown in up to 2\u00a0M sorbitol. No significant growth of either the wild-type or Rpb4 expressing cells was observed in the presence or absence of thiamine at 3 and 4\u00a0M concentrations of sorbitol. These observations indicate that low levels of Rpb4 do not affect the survival of cells under any of the tested stress conditions.\nRepression of rpb4 transcription causes defects in cell separation\nWe next examined whether different levels of rpb4 expression would influence specific cellular processes. Cells expressing rpb4 from any of the three nmt1 promoters in the absence of thiamine did not lead to any obvious phenotypes even after 25\u00a0h of growth in medium without thiamine (Fig.\u00a03a).\nFig.\u00a03Rpb4 affects cell separation and morphology. Cells expressing rpb4 from either the P3nmt1 or P81nmt1 promoter were analyzed by microscopy. a Cells grown in EMM in the absence of thiamine (promoter \u2018on\u2019) at 32\u00b0C appear like normal, wild-type cells. b When grown in the presence of thiamine (promoter \u2018off\u2019), cells expressing rpb4 from the P3nmt1 promoter appear normal, while those expressing rpb4 under the P81nmt1 promoter display defects in cell morphology and septation. Multiple phenotypes including elongated cells with single or multiple septa and growth by branching are evident. c Cells expressing rpb4 under the P81nmt1 promoter were analyzed by fluorescence microscopy in the presence of thiamine. DAPI staining revealed single nuclei in each cell compartment, while calcofluor staining showed septa (some of them aberrant) separating the cell compartments. d Control cells and mutants showing similar phenotypes as cells expressing rpb4 under the P81nmt1 promoter cells. From left to right: wild-type cells, sep1 deletion mutants, ace2 deletion mutants and sep1 ace2 double deletion mutants\nIn thiamine-containing medium, however, cells expressing rpb4 from the weak P81nmt1 promoter became more elongated than the wild-type cells after 19\u00a0h of growth (Fig.\u00a03b). Moreover, after continued incubation in the same medium for another 2\u00a0h (21\u00a0h with thiamine), cells also showed defects in cell separation, which became more evident after an additional 4\u00a0h of incubation (25\u00a0h with thiamine) (Fig.\u00a03b). Most cells were elongated with either a single or multiple septa, and some cells also displayed bent morphologies and\/or growth by branching. Cells expressing rpb4 from the medium strength P41nmt1 promoter displayed no obvious cell separation defects until 23\u00a0h of growth in the presence of thiamine. Similar defects as described above, however, became apparent after 23\u00a0h of growth in thiamine (data not shown). In contrast, cells expressing rpb4 from the strong P3nmt1 promoter did not exhibit any aberrations in morphology or division after growth in thiamine-containing medium for 25\u00a0h (Fig.\u00a03b) and continued to look like wild-type cells even at 40\u00a0h of growth in thiamine-containing medium (data not shown). Thus, the basal level of expression from the strongest promoter is sufficient to prevent the cell separation defects seen with the weaker promoters.\nWe investigated these phenotypes in more detail for cells expressing rpb4 under the P81nmt1 promoter by staining the division septa and cell nuclei (Fig.\u00a03c). Several nuclei were present separated from each other by either a single septum or multiple, aberrant septa. Some cells also contained large deposits of septal material at random positions (Fig.\u00a03c). We conclude that lowered rpb4 expression levels lead to filamentous growth with multiple cells remaining attached to each other. Similar phenotypes to those of cells expressing rpb4 under the P81nmt1 promoter have observed in several different mutants (see Discussion); for example, deletions in two transcription factor genes, sep1 and ace2, also lead to defects in cell separation (Fig.\u00a03d; B\u00e4hler 2005). These findings raise the possibility that Rpb4 plays a specific role in cell separation after cytokinesis.\nLow levels of rpb4 transcripts most strongly affect expression of genes involved in septation\nWe used S. pombe whole-genome microarrays to investigate the effects on gene expression caused by low levels of rpb4. Wild-type cells and cells expressing rpb4 under the different regulatable promoters were grown in the presence and absence of thiamine followed by RNA extraction. Cells expressing rpb4 under the P3nmt1 promoter did not show any significant differences in gene expression profiles when compared with wild-type cells, either in the presence or absence of thiamine (data not shown), consistent with our earlier observations that these cells show normal growth and phenotypes. If rpb4 was expressed under the weakest promoter in the presence of thiamine, however, gene expression changed substantially. As down-regulation of rpb4 is expected to have a global effect on transcription, we used external spikes for normalization (Materials and methods). This revealed that mean transcript levels were 26\u201336% lower compared to wild-type cells grown under the same conditions (Fig.\u00a04a). The expression levels of rpb4 were two- to threefold lower under these conditions compared to the levels under its own promoter. Thus, lowered rpb4 expression leads to reduced overall transcript levels.\nFig.\u00a04Effects on global and specific gene expression as a function of rpb4 levels. a Microarray experiments of cells expressing rpb4 under the weakest promoter. The histograms show the distribution of gene expression ratios of P81nmt1-rpb4 cells grown in the presence of thiamine for 21 and 23\u00a0h relative to wild-type cells grown in the presence of thiamine. Genes in both histograms are colored by their relative expression levels at 21\u00a0h as indicated at upper right. External spikes have been used for normalization, revealing a global down-regulation of transcript levels by 26% (21\u00a0h) to 36% (23\u00a0h) on average when rbp4 expression is lowered. The averages of two independent repeats for each timepoint are shown. b Overlap between the genes downregulated when rpb4 is expressed under the weakest promoter in the presence of thiamine and genes downregulated in both sep10 and sep15 mutants (Lee et al. 2005). The number in brackets represents the overlap expected by chance, given the sizes of the gene sets considered and the total number of 5,180 genes. The overlap is highly significant as calculated using the hypergeometric distribution (P\u00a0\u223c\u00a01\u00a0\u00d7\u00a010\u221230). c Hierarchical cluster analysis of 249 genes that are either 1.5-fold upregulated or threefold downregulated when rbp4 is expressed under the weakest promoter in presence of thiamine. Horizontal rows represent the profiles of hierarchically clustered genes. Columns represent experiments of different mutant strains and different time-points for the same strain. The transcript levels for each strain (indicated at bottom) relative to levels in wild-type cells are color-coded as indicated at right, and missing data are shown in gray. The microarray data from mcs6, pmh1, sep10 and sep15 mutants are from Lee et al. (2005), and the data from sep1 and ace2 mutants are from Rustici et al. (2004)\nOn top of this genome-wide down-regulation of transcription, 96 transcripts were at least 1.5-fold upregulated in three of the four samples tested, while 150 genes were at least threefold downregulated in three of the four samples tested upon lowering rpb4 expression (Supplementary Tables\u00a0S1 and S2). These genes were categorized according to their functions (Supplementary Table\u00a0S3). The majority of the genes whose expression levels were affected by rpb4, encoded proteins involved in metabolism and transport. Many of the upregulated transcripts in cells with lowered rpb4 expression levels overlapped with genes upregulated during different environmental stresses (Chen et al. 2003) and nitrogen starvation (Mata et al. 2002) (P\u00a0\u223c\u00a01\u00a0\u00d7\u00a010\u221213 to 1\u00a0\u00d7\u00a010\u221236). This probably reflects an indirect effect caused by the defects in these cells. A substantial number of the downregulated genes in the category of cell cycle and cell division played specific roles in cytokinesis and cell separation. For example, the two genes eng1 and agn1 encode \u03b2-glucanase and \u03b1-glucanase, respectively; Eng1 degrades the primary division septum between the new ends of daughter cells and Agn1 hydrolyzes the old cell wall surrounding the septum leading to full separation of daughter cells (Mart\u00edn-Cuadrado et al. 2003; Dekker et al. 2004). The genes adg1 and adg3 have also been shown to be involved in cell separation (Alonso-Nunez et al. 2005), and rng3 encodes a protein involved in formation of contractile ring during cytokinesis (Wong et al. 2000).\nBoth the lists of genes being upregulated and downregulated after rpb4 down-regulation showed highly significant overlaps with lists of genes upregulated or downregulated, respectively, in sep10, sep15, mcs6 and pmh1 mutants (P\u00a0\u223c\u00a01\u00a0\u00d7\u00a010\u221216 to 1\u00a0\u00d7\u00a010\u221247) (Lee et al. 2005), whose genes encode different proteins involved in transcriptional regulation (see Discussion). Among the downregulated genes, the most pronounced overlap was observed with sep10 and sep15 mutants (Fig.\u00a04b). The downregulated genes also showed some overlap with genes downregulated in sep1 and ace2 mutants (P\u00a0\u223c\u00a01\u00a0\u00d7\u00a010\u221214) (Rustici et al. 2004), which encode cell-cycle transcription factors. Figure\u00a04c shows a cluster analysis of the genes most strongly regulated by rpb4 repression and their expression levels in the various transcriptional mutant backgrounds. The sep10, sep15, mcs6 and pmh1 mutants show highly similar expression signatures to cells with compromised rpb4 expression, while the overlap with sep1 and ace2 mutants is less evident. Intriguingly, if rpb4 was overexpressed using the medium strength promoter, the expression levels of these genes were reversed, with downregulated genes becoming upregulated and upregulated genes becoming downregulated (Fig.\u00a04c). Thus, transcript levels of this gene set are particularly responsive to the transcript levels of rpb4.\nDiscussion\nWe have analyzed functions of the Rpb4 subunit of pol II in S. pombe. Despite the overall conservation in structure and function of pol II across species, the Rpb4 subunit presents a special case. S. pombe Rpb4 is similar to its orthologs in higher eukaryotes with respect to its structure and essential nature (Sakurai et al. 1999), but in contrast to its human ortholog, it can form stable hybrid dimers with S. cerevisiae Rpb7 (Sakurai et al. 1999) and can rescue the temperature-sensitive phenotype associated with the lack of S. cerevisiae Rpb4 (Shpakovski et al. 2000). Thus, S. pombe Rpb4 may have intermediate characteristics useful to bridge the knowledge from S. cerevisiae and multicellular eukaryotes.\nWe used regulatable gene expression constructs to study the in vivo roles of Rpb4. Under optimal conditions, rpb4 influences the growth of cells in a dose-dependent manner when expression drops below a certain level using the regulatable promoters (Figs.\u00a01, 2). In S. cerevisiae, deletion of the RPB4 gene leads to slow cell growth under optimal conditions (Woychik and Young 1989; Choder 2004), while in S. pombe rpb4 is essential (Sakurai et al. 1999). We found that low levels of rpb4 expressed from the weakest promoter were sufficient for the cells to survive a range of different stress conditions including temperature stress. Accordingly, core stress genes were actually upregulated in rpb4 mutants, and S. pombe Rpb4 is therefore unlikely to play any specialized role during environmental stress. S. cerevisiae Rpb4 is dispensable for oxidative and osmotic stress (Maillet et al. 1999), but unlike in S. pombe, it is required for survival of temperature stress (Woychik and Young 1989).\nFission yeast cells grown under low expression of rpb4 were elongated and showed defects in cell separation as indicated by the accumulation of division septa, some of them highly aberrant (Fig.\u00a03). Strains with cell separation defects similar to the ones described here include mutants in components of the exocyst complex (sec6, sec8, sec10 and exo70), an anillin homolog (mid2), septins (spn3, spn4), an endo-\u03b1-1,3-glucanase (agn1), an endo-\u03b2-1,3-glucanase (eng1), calcineurin (ppb1), a MAP kinase and phosphatase (pmk1, pmp1), two transcription factors (sep1, ace2), and subunits of the mediator complex (sep10, sep11 and sep15) (Sipiczki et al.1993; Yoshida et al. 1994; Longtine et al. 1996; Toda et al.1996; Sugiura et al. 1998; Grallert et al. 1999; Wang et al. 2002; Mart\u00edn-Cuadrado et al. 2003; Sp\u00e5hr et al. 2003; Tasto et al. 2003; Dekker et al. 2004). In S. cerevisiae, haploid rpb4 deletion mutants exhibit an axial budding pattern with normal yeast cell morphology, but diploid rpb4 mutants are more elongated than wild-type cells during nitrogen starvation and grow in a unipolar pattern, resembling pseudohyphae formation (Pillai et al.2003).\nLowered expression of rpb4 causes relatively modest reductions in global transcription, which probably leads to the reduced growth rates under these conditions (Fig.\u00a04a). In addition, low rpb4 transcription results in substantially lowered expression of a distinct subset of genes. Among the strongly affected genes were eng1, agn1, adg1 and adg3 with known functions in cell separation, which are targets of a transcriptional cascade regulated by the Sep1 and Ace2 transcription factors (Rustici et al. 2004; Alonso-Nunez et al. 2005). Accordingly, the genes affected by lowered rpb4 expression significantly overlapped with genes affected in sep1 and ace2 mutants. Much stronger similarities, however, were evident with the gene expression signatures of several strains mutated in the sep10, sep15, mcs6 and pmh1 genes, which encode various components of the transcriptional machinery. Sep10 and Sep15 are essential components of the mediator complex of pol II, while Mcs6 and Pmh1 are components of a complex homologous to metazoan Cdk7-cyclinH-Mat1, a cyclin-dependent kinase-activating kinase (CAK) and a pol II CTD kinase associated with transcription factor IIH (Sp\u00e5hr et al. 2001, 2003; Lee et al. 2005). The overlap in expression signatures between these four mutants and cells with lowered rpb4 expression was evident for both upregulated and downregulated genes. These differentially regulated genes showed reverse regulation (upregulated genes became downregulated and vice versa) when rpb4 was overexpressed from the medium strength promoter (Fig.\u00a04c). Thus, the expression of this gene set is particularly responsive to rpb4 expression levels and also depends on a range of other factors involved in general transcription. This could either reflect a specific transcriptional pathway involving Rpb4 and the other factors (either directly or indirectly via the control of a regulator), or the affected genes could be generally sensitive to transcriptional interference. In any case, these data show that the transcriptional pathway required for cell separation in fission yeast (B\u00e4hler 2005) is strongly affected when interfering with rpb4 expression levels.\nWe speculate that the Rpb4\/Rpb7 complex is involved in linking the transcriptional output to the metabolic status of the cell. The rbp4 and rpb7 transcripts are down-regulated when cells enter stationary phase (unpublished observations), Rpb7 interacts with the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (Mitsuzawa et al. 2005), and global transcription is sensitive to rpb4 expression levels. It is possible that the transcriptional pathway required for cell separation is particularly sensitive for compromised transcription to adjust the growth mode to availability of nutrients: in abundant nutrients, global transcription is efficient and growth is best as single cells, while in limiting nutrients, global transcription and cell separation are compromised, and cells grow as multicellular pseudohyphae, which may allow a more efficient grazing for new nutrients as growth is directed. In the first description of S. pombe, multi-cellular pseudohyphae similar to those seen in rpb4 mutants have been observed (Lindner 1893); the natural capacity to switch to multi-cellular growth might have been lost in the yeast strains cultured in the laboratory.\nTaken together our data suggest that S. pombe Rpb4, besides its general role in transcription, performs a more specialized function in regulating a specific gene expression program required for cell separation at the end of the cell cycle. It is possible that this role is carried out by interacting with other general factors of the transcriptional machinery such as the mediator and CTD kinase as suggested by similarities in phenotypes and expression signatures in our study. Accordingly, a comparison of the X-ray crystallographic structure of the complete pol II, including the Rpb4\/7 heterodimer, with an electron microscopic structure of the pol II-mediator complex, has indicated that Rpb4\/7 may interact with the mediator (Bushnell and Kornberg 2003). Alternatively, the subset of genes affected by changes in rpb4 expression (and by manipulating other general transcription factors) could reflect genes that are especially sensitive to transcriptional stress. This interpretation does not imply any specific gene expression program, although it could be used by the cell to adjust transcription to available nutrients. Given the conservation of Rpb4, it will be interesting to see whether the human ortholog plays a similar specialized role to transcribe particular subsets of genes.\nElectronic supplementary material\nSupplementary material","keyphrases":["transcription","growth","cell division","s. pombe"],"prmu":["P","P","P","P"]}
{"id":"Health_Care_Anal-3-1-2045690","title":"Information Rx: Prescribing Good Consumerism and Responsible Citizenship\n","text":"Recent medical informatics and sociological literature has painted the image of a new type of patient\u2014one that is reflexive and informed, with highly specified information needs and perceptions, as well as highly developed skills and tactics for acquiring information. Patients have been re-named \u201creflexive consumers.\u201d At the same time, literature about the questionable reliability of web-based information has suggested the need to create both user tools that have pre-selected information and special guidelines for individuals to use to check the individual characteristics of the information they encounter. In this article, we examine suggestions that individuals must be assisted in developing skills for \u201creflexive consumerism\u201d and what these particular skills should be. Using two types of data (discursive data from websites and promotional items, and supplementary data from interviews and ethnographic observations carried out with those working to sustain these initiatives), we examine how users are directly addressed and discussed. We argue that these initiatives prescribe skills and practices that extend beyond finding and assessing information on the internet and demonstrate that they include ideals of consumerism and citizenship.\nIntroduction\nIn discussions about the increasing use of internet technologies in health care a new kind of patient has slowly been constructed\u2014a patient that is reflexive and informed, with highly specified information needs and perceptions, as well as highly developed skills and tactics for acquiring information. This specific depiction of the patient transforms the patient into an active participant in his or her care, \u201cempowered\u201d through training, skill development and the use of technologies such as the world wide web, all of which can be provided or enabled by government organizations or other political actors. Through increased access to medical information coming from outside of the health care arena the boundary between lay and expert is breaking down, enabling patients to become experts, be more assertive in managing their own care and change the nature of their existing relationships with health care professionals [6, 27, 28].\nAs Henwood et\u00a0al. [31] point out, however, just because the potential for empowerment exists, it does not necessarily occur. Patients conform only with difficulty to the images associated with the reflexive consumer, and the concept of the \u201cinformed patient\u201d is empirically difficult to sustain [41]. Becoming informed requires skills related both to information and to the various media that can be used to access that information. Patients reflect low-level skills with respect to searching for information, rely on intermediaries, and experience concrete barriers during the medical encounter, all of which act as actual constraints on the emergence of informed patients [31, 32].\nIn literature about the questionable reliability of web-based information, the suggestion that lay information seekers are constrained in conforming to this image is too mild. This body of literature asserts the stronger position that they are actually prevented from being empowered because of the large amount of information and the questionable quality of much of that information. Central in this discussion is the concern that even if patients manage to find web-based medical information, they lack the skills necessary to evaluate the quality of that information, and therefore are especially vulnerable to harmful information in the form of errors, fraud and \u201cquackery\u201d [16, 17, 34, 35, 50, 51]. This concern is answered by the argument that a pressing need exists for educating patients in how to judge a website\u2019s reliability [10, 18, 19, 23, 33, 48, 50, 54].\nTaken together, these discourses construct patients-as-web-users that are simultaneously skilled and capable, but also incompetent and unskilled. The informed patient must be educated in how to search for and assess information in order to conform to the definition of a reflexive, empowered consumer. Patients are not empowered merely through access to the internet or web-based information [1, 49], but, this literature suggests, must be constructed in the process of being led to pre-selected information by health professionals and information specialists. In suggesting the need to create user tools that have pre-selected information, as well as guidelines for patients to use to check the individual characteristics (author, sources, date, underlying financial sponsorship, etc.) of the information they encounter, authors emphasize the boundary between lay and expert assessments of information, privileging the latter over the former. Existing literature prescribes that patients utilize government-provided medical\/health portals, click on hyperlinked icons (seals of approval or \u201ctrust marks\u201d) provided by non-profit or non-governmental organizations, follow checklists created by professionals or health educators, and\/or download special toolbars, all of which will assist them in finding and evaluating information on the web.\nBecause web pages provide powerful examples of pre-defining (or pre-confining) how information technologies should work, how they should be perceived and how different actors should utilize them, it is important to study the discourses that they carry [43]. However, little attention has been given to the underlying prescriptions about skills and use, as well as additional political agendas and messages about individual behavior, that individual web-based reliability initiatives, such as portals, seals and special toolbars, convey. In this article, we look not at how patients assess information (or what skills they do\/not reflect), but rather, at what health educators, medical professionals and review organizations suggest the skills of a reflexive consumer should be.1 We view the various user tools, such as guidelines, checklists portals, and clickable seals, as artifacts with politics [56]\u2014particularly, the politics of building consumer-citizens and shaping their skills, perceptions and behaviors. We begin by looking at more general literature about the construction of users and designers, followed by a review of literature that transforms potentially informed patients into reflexive consumers. Using two types of data: \u201cfront page\u201d data (information, images, quotes gleaned directly from websites and their accompanying promotional items) and supplementary \u201cbehind the scenes\u201d data from interviews and observations carried out with those working to sustain given initiatives, we examine how users are directly addressed and discussed. How are users enrolled by sites and promotional items? What ideals are represented in these user tools and in what kind of practices are they embedded? We argue that, although these examples claim to target \u201call citizens,\u201d the prescriptions for action that these user tools carry suggest that the user envisioned by the developers of these tools is not everybody, but rather a specific, ideal type of user: the good consumer\/ responsible citizen.2 In order to develop skills for finding and assessing information on the internet, patients must engage in certain practices\u2014practices that also enable developing the skills necessary to share the responsibility for information reliability and to be an empowered health consumer and responsible citizen.\nThe Importance of the State in Configuring Users of Technologies\nStudies of science, technology and society (STS) have a long tradition of reflecting on how users are or are not included in the design and implementation of different technologies.3 Woolgar [58] argues that designer preconceptions about use can shape what counts as legitimate behavior and that users are \u201cconfigured,\u201d i.e.: their identity and skills are defined and constraints upon their (possible, future) actions are set, before the technologies ever reach the anticipated user group. Because of this configuring, technologies can be read as carrying specific scripts for use [5]. Rose and Blume [52], however, have criticized much of the subsequent work on configuring users because it attributes configuration largely to market-driven design and pays too little attention to the possibility of states as providers or enablers of technology use, and therefore neglects to consider the potential role states have in user configuration.\nAccording to Rose and Blume, focusing on technologies that are developed or facilitated by the state and its institutions may highlight significant tensions between individuals as users and the state of which they are members and citizens. Policy documents can develop meanings for information technologies by providing, for example, the language of discourse about those technologies [38]. In their behavior and discourse, collective providers, much like market-like providers, presume that individuals will be active consumers of technologies, meaning that the state may enact policies that, while not overtly or explicitly configuring the user, do create or maintain an environment that helps to shape how users are configured. In using certain technologies (or by using them in the specified ways), individuals fit with their configurations and follow the technologies\u2019 scripts and actualize their potential as \u201cgood\u201d citizens [52].\nSingleton [53] makes a similar point with her suggestion that the New Public Health in the UK seeks to train citizens in more ways than one. She asserts that training programs for healthy citizens are not just about physical health but also about practical skills and about distinguishing between active and inactive citizens. Klecun-Dabrowska and Cornford [38] have also looked at the recent discourses on health in the UK, specifically focusing on the role of telemedicine within wider national and international debates on health. They, too, saw that documents convey a vision of a refocused health care system to serve a population that has enough information resources to enable individuals to manage their own care and participating more actively in the health care process. Information, they argue, is transformed into something that is easily captured, understood and transformed to achieve wider goals\u2014goals related to social responsibility and community values.\nAt the same time, states want to forward their political agendas and invent new modes of indirect steering for empowering their members such that they freely, willingly and self-reflexively choose the paths toward the desired outcomes of the state. The possibility of the state to directly influence its citizens is made relative, as the state competes with other types of influences. This has led to the development of new types of steering, where governments seek to rule the social indirectly through designing, facilitating and moderating processes of self- and co-governance [8]. More communicative and cooperative modes of re-centering allow for bottom up articulation, without relinquishing everything to citizen preferences. Again, these programs are not just about the empowerment of individuals or about the distribution of expertise, but are also about distinguishing between active and inactive citizens. Although states demonstrate a readiness to engage in active teamwork and argue the need for collaborative solutions, Bang argues that they nonetheless reveal how key relationships are re-imagined and redrawn.\nTwo things are worthy of note: first, the terms \u201cre-imagining\u201d and \u201credrawing\u201d suggest that something new can emerge, which is a distinct possibility. However, with respect to the internet, at least, studies have shown that the boundaries that are constructed around information, for example, tend to reinforce existing social ideas [49] and geographic borders [26]. The lines that are redrawn serve only to reinforce what is already there. Second, the exclusive focus on states tends to neglect the role of other political actors. Especially in activities regarding reliable medical information, states are not alone, but are joined by non-governmental organizations (NGOs) and inter-governmental organizations (IGOs), as well as non-profit organizations voicing their own political advocacy agendas.\nWe extend the analysis in this paper to look at both programs from federal agencies and initiatives created by other types of political institutions. These programs and initiatives provide tools for accessing and assessing information on the web, in some cases enabling information provision. (See Textbox\u00a01 for an overview of the different initiatives that are used as examples).4 For example, the US Department of Health and Human Services provides a portal with contact information for organizations (http:\/\/www.healthfinder.gov) and the US National Library of Medicine (NLM) provides a portal with health content from the National Institutes of Health (http:\/\/www.medlineplus.gov). Outside of the US, the Health on the Net Foundation (HON) in Geneva seeks to raise awareness about ethical issues related to providing medical information, while the World Health Organization (WHO) proposes the creation of a \u201c.health\u201d domain afforded only to sites meeting specific criteria.5, 6Textbox\u00a01Overview of home pages for cited examplesHealthfinder is the official Federal Gateway to health information, provided by the US Department of Health and Human Services http:\/\/www.healthfinder.govMedlineplus is a portal provided by the US National Library of Medicine http:\/\/www.medlineplus.govQuackwatch seeks to combat health fraud and is run by Dr. Stephen Barrett http:\/\/www.quackwatch.orgHealth on the Net is a Geneva-based non-governmental organization that provides diverse user tools. The focus of this study is the 8 principle HON Code of Conduct and accompanying hyperlinked seal http:\/\/www.hon.chMedCERTAIN was funded by the EU from 2000-2002under the \u201cAction Plan on promoting safer use of the Internet by combating illegal and harmful content on global networks\u201d. The collaborative MedCIRCLE is a follow-up that provides users with a downloadable toolbar http:\/\/www.medcertain.org; http:\/\/www.medcircle.orgTNO\u2019s QMIC is a three-tiered trust mark for medical websites in the Netherlands. The third tier signifies content review of information http:\/\/www.qmic.nl\/qmic\/home.doThe World Health Organization recently released a list of approved sites for vaccine safety http:\/\/www.who.int\/immunization_safety\/safety_quality\/approved_vaccine_safety_websites\/en\/\nThese self-ascribed roles lead to numerous questions, such as why UN NGO status (in the case of HON) or a \u201cneutral position\u201d (claimed by the WHO) is crucial to building claims about the reliability of medical information. Why is a \u2018.gov\u2019 domain \u201cmore reliable\u201d than a \u2018.com\u2019 and where does the \u2018.org\u2019 domain rank in relation to other existing and proposed domains? What potential tensions arise between states and citizens (or even NGOs and citizens) in these settings? Although these questions would be difficult to answer, they do give us cause to think about the political messages that are sent to lay information seekers when they are instructed on how to search for, evaluate and use web-based medical information.\nInformed Patients...Reflexive Consumers...Ideal Citizens?\nThis entire line of discourse about empowering patients and the role of web-based information has led to the semantic challenge of properly naming those non-medically trained individuals searching for information online. For example, the term patient does not encapsulate those persons who search for information regarding the health situation of a family member or friend. Miller and Reents\u2019 [44] alternative, \u201cinformation retrievers\u201d, makes the user too passive, while the suggested informatics alternative \u201cmedical end-users,\u201d [22] makes the route to the information too technology specific, without reflecting the social aspects of information use and broader information \u201clandscapes\u201d [13, 29, 32] that persons can access. Furthermore, the term medical does not reflect broader issues related to health and fails to allow for use of information by those that Kivits [36] calls \u201chealthy internet users,\u201d those not necessarily afflicted by illness, but nonetheless interested in health information.\nThe concepts \u201ccitizen\u201d and \u201cconsumer\u201d are terms for users that are used most often by those creating different web-based reliability initiatives, working under the suggestion that these terms are neutral and avoid the semantic problems mentioned above [see, for example, 7, 9, 45]. However, these words also carry connotations; Anderson et\u00a0al. even go so far as to state, \u201cWhen speaking about consumers, from this model it should become clear that we mainly refer to educated patients with chronic diseases from developed countries\u201d (2003: 72). Using these two terms more generally implies certain rights, such as Gustafson and Wyatt\u2019s [25] assertion that consumers deserve both high quality content and a certain degree of confidence in the information they use, but such use also connotes certain responsibilities. Each of the initiatives to which the usage of these terms is attached defines particular types of participation that extend beyond gathering information to include activities in the health care process and social community at large. The consequence, of course, is that teaching patients how to search for and assess information on the web becomes more than just a process of constructing empowered patients\u2014it extends further to inscribe notions of good consumerism and responsible citizenship.\nWhen we couple the discussion about empowerment on the practical skills that users are expected to develop, we see that it takes place within an overt and dominant biomedical discourse [24], which is framed not only by medical professionals, but also by political actors. For example, in relation to assessing web-based health information, Edgar et\u00a0al. [15] describe three essential skills: the ability to conduct a search and find the \u201cright\u201d sites; the ability to judge the quality of information found on a given site and the ability to synthesize that information into a useful context for personal\/individual health. Eng and Gustafson [16] argue, however, that the skill is more than just assessing and using information, but is actually about deciding which of the existing tools works best for the individual. Only in finding the right tool can s\/he implement a personal evaluative framework and learn how to be an educated consumer.\nHow the Initiatives Enroll Citizens and Consumers\nEach of the initiatives discussed in this paper attempts to enroll internet users in specific activities of finding and assessing health information. The most prominent strategy of enrollment that we see is the suggestion of abounding risks to personal health and the simple ways to combat them. Involvement\u2014learning how to check information actively and always\u2014becomes a matter of personal responsibility and an identifying component of reflexive information consumption. Failure to learn about user tools and use them as prescribed is to be inactive, and therefore, deviant or lacking [52, 53].\nHighlighting Risks and Providing Simple Instructions for Combating Them\nOne evident strategy for interesting patients in the need to check the reliability of the information is the construction of web-based information as information that puts individuals at risk. Because of the nature of the web, information can come from anyone, anywhere\u2014it originates outside of a given country, in another medical tradition or health care context and as such it is foreign and worthy of suspicion. If information isn\u2019t pre-approved, individuals could be making their decisions based on wrong information, fraud, or quackery. An individual\u2019s health is potentially endangered because this information is always potentially just one mouse-click away and is difficult to distinguish from \u201ctruly reliable\u201d information. In the language of this discourse, boundaries are constructed around information\u2014these boundaries distinguish, for example, between geographic locations, but also reinforce more traditional ideas by distinguishing between lay persons and experts.\nThe proposed need to implement user education programs for checking the reliability of the information they encounter suggests that anyone online should be aware of this potential for harm and, therefore, actively involved in policing that information [21, 35]. HON, for example, currently includes information on its website about being a \u201cvigilant user\u201d. Instructions about clicking and verifying proper use of its HONcode icon is prefaced by the following:Unfortunately, we cannot banish incompetence or fraud from the medical Internet. If you come across a healthcare Web site that you believe is either possibly or blatantly fraudulent and does NOT display the HONcode, please alert Quackwatch. Of course, if such a site DOES display the HONcode, alert us immediately. HON cannot prevent dishonest operators from simply cutting and pasting the HONcode seal onto their Web sites in a bid to enhance their credibility. We do conduct our own random checks on subscribers to ensure they remain compliant with the HONcode. But we also rely heavily on vigilant Web surfers to alert us to dubious sites\u2014and they do. ...There are three quick ways users can check whether a chosen site featuring our seal is a bona fide HONcode subscriber.7\nHON points out to users that it needs its users to help police information on the internet. It invites them to join in a partnership with two types of authorities, the first being itself, an overarching political organization and the second being participants from the community of medical professionals. Active policing on the part of the user is made extremely simple and practically effortless\u2014checking information is also as easy as a single mouse click (merely clicking on a small icon) or just running through a short checklist.8 Taking a few seconds can prevent all users from encountering bad information, whereas not checking could subject the user to fraudulent or otherwise harmful information. With its clickable trust mark9 HON provides users with an easy tool to double check the background of the web information provider. This type of tool makes the action of checking information is made simple and non-time consuming\u2014as easy as a mouse click. The MedCERTAIN project, which proposed a mechanism similar to HON\u2019s clickable seal, except that it proposed to include content review in addition to review of ethical principles, produced a prototype with the instruction, \u2018Remember to verify by simply clicking on it.\u201910 Similarly, the Dutch QMIC trust mark states, \u2018In the blink of an eye, you can see that information is reliable and correct.\u201911\nThe user that all of these organizations presuppose is someone who, like the project reviewers, is (or should be) familiar with the ethical issues underlying the provision of any, but especially medical, information on the web. We begin to see how political actors invent new modes of indirect steering for empowering users such that they choose specific paths. They attribute to these users skills and practices more in keeping with professional levels of information assessment and action. But they also make these practices \u201cuser friendly\u201d by emphasizing the ease with which information can be checked, with little additional thought or demands on time, and by encouraging participation in partnerships with them.\nDirecting Paths Toward Information\nThe various initiatives also emphasize their links to the medical community and partnerships with one another. This is evident in at least three forms: logos from other departments and organizations placed on websites, joint projects with physician\u2019s organizations and overt statements about the best course of action that are located on websites. At the time of writing, HON\u2019s website includes the logos of the University Hospital, the EU, the city of Geneva and Sun Microsystems. The tag lines for each individual logo identify how these different groups provide social and technical support for HON and its web activities. These logos show that HON is part of a larger national and international community that transcends medical practice, politics and businesses\u2014and suggest that evaluating medical information on the web can only be done in this context.\nSome studies of patient searching behaviors have indicated that patients refer to their physician for information about where to search for information on the web. [See, for example, Cotton and Gupta 12] Recognizing the potential of taking advantages of this preference for physicians as trusted sources of website recommendations, but also recognizing that physicians probably do not have time to review scales of sites, some initiatives have developed (or furthered existing) relationships with professionals. The US National Library of Medicine (NLM) has worked with professional communities to develop prescription pads for web-based information (see Fig.\u00a01).\nFig.\u00a01Example of one version of the prescription pad that physicians can give to patients during the consultation to refer them to web-based information about a specific health topic\nThis project enabled NLM to inform physicians about the topics available on medlineplus. Additionally, it supports the physician and patient in post-consultation searches for health information. With this information prescription, the physician gives the patient a place to start and the term to use when searching on the web for information specific to his or her health situation\u2014and that starting place is a government portal:In this trial, the doctors each had their own pre-printed prescription pad. The prescription pad that we used in the first part of the pilot had room for 12 URLs. There were about 40 to choose from and they chose the ones that they saw most often in their practices. What we found is that the doctors don\u2019t want to bother with all that. They want to have their own prescription pad, they really like that. But they just want to be able to write something on it and then send people to Medlineplus.12\nFinally, websites send patients back and forth between the various initiatives and tools, implicitly and explicitly endorsing one another. HON, for example, recommends that English-speaking site visitors follow a specific and narrow path to information:To find good (English-language) healthcare information, you can bypass the all-purpose commercial search engines and go straight to healthcare portals like Health on the Net (http:\/\/www.hon.ch) or Healthfinder (http:\/\/www.healthfinder.org\/). These portals have already eliminated the irrelevant for you. A useful rule of thumb is first check out the governmental, not-for-profit and hospital Web sites, or those carrying an immediately recognizable and trusted name.13\nIt is interesting in this case that HON excludes more commercial search engines as a valid route to information. The message is that web tools that are general in scope, rather than specifically directed toward health information, or that are funded by commercial interests (or both) are incorrect choices because they contain too much extra \u201cstuff\u201d14 and will not enable the user to find what s\/he is looking for. Reliability is redefined as an issue of what is \u201crelevant\u201d and proper behavior involves not wasting time sifting through information and other materials that are, according to HON de facto irrelevant. Furthermore, in emphasizing that HON and the US gateway are the best types of sites, HON privileges state (implicitly working with medical) actors over other types of actors providing medical information on the web. The suggestion, then, is that engaging in good searching practices and finding\/using the \u201cright\u201d information means prioritizing government and medical channels.\nThrough these statements, the organizations affirm each other, arguing not only the importance of a joint approach to reliability, but also the importance of collaboration between different countries and regions. The risks associated with misinformation are countered with initiatives that seek to rein in information, centralizing it at certain web addresses and judging it according to politically and professionally defined criteria. One example would be the criterion for levels of readability [16] applied to sites such as Medlineplus. Another would be the use of selection criteria for information that derive from similar roots [9] or the transfer of existing publishing standards to evaluate how information is produced [1]. Website providers enable access to information that is consolidated and repackaged in a uniform format for readability and then placed on the respective site under a uniform design and structure for ease of navigation. Once again finding and using good information is made \u201ceasy\u201d in the design of technology-specific tools.\nWhat is interesting are the personalized messages and rhetoric of choice in which these standardized formats are embedded. Healthfinder is \u201cyour guide to reliable information,\u201d while Medlineplus provides \u201cTrusted health information for you.\u201d Access to information gives users new choices, but these initiatives convey that responsible consumerism and reflexive use of information mean choosing narrow routes to prepackaged information. The internet offers numerous avenues to information, but \u201cgood\u201d participation means utilizing choice to choose the routes provided by one\u2019s own government and combining these with overarching non-governmental organizations or other non-profit organizations.\nIndividualizing Choice Along the Way\nBoth HON and the MedCIRCLE initiative have taken this one step further\u2014emphasizing the need to combine top-down control of information with bottom-up user choices. They have both developed special toolbars that can be downloaded and coupled on the user\u2019s internet browser (see Fig.\u00a02 for the MedCIRCLE prototype). Such a toolbar allows the user to set his\/her preferences for which components of information are most important and then gives a confidence rating for how well an individual website answers to these preferences. The toolbar gives a message that user preferences about the reliability of information are important and that users should actively set their own criteria for assessment. However, this is acceptable only once users are already searching within the confined space of pre-approved sites.\nFig.\u00a02Patients can download this toolbar from http:\/\/www.medcircle.org and set their own preferences for characteristics that they find important in the provision of health information\nThe confidence rating and other information provided by the tool are only available for sites already in the HON or MedCIRCLE databases. While these types of tools suggest the importance of individual choice, they nonetheless hold individuals responsible for following the \u2018right\u2019 paths to information. In this sense, the discussion is not merely an issue of creating reflexive consumers, but also of judging non-reflexivity as irresponsible and deviant.\nFollowing the Path Back to the State\nThe responsibility for finding good information and avoiding the risks associated with bad information on the internet becomes collaborative and distributed. Within this context, individuals are held responsible for the choices they make, how they engage with available information and how they engage with other actors in the collaborative process. This process involves new actors, such as HON and MedCERTAIN\/MedCIRCLE, but also longer standing actors, such as the WHO, state governments and medical associations. Reliability is created through distributing skills, information, and practices, and is intertwined with the creation of new networks that bind together the technical and social. We see this in especially in uses of internet-based technologies to further public health education.\nHealthfinder, for example, sends monthly newsletters to inform its public about changes and updates to the site and about different services it provides. Each newsletter also contains a list of \u201chealth observances\u201d for that month, each of which includes hyperlinks to special sites with more information. These observances are, where possible, coupled on the (national) holidays being celebrated in that month. For example, February is \u2018the month for all kinds of hearts\u2019 and the newsletter\u2019s healthy observances are all related to cardiac issues, while July\u2019s newsletter focuses on food safety tips for warm summer days and firework safety tips specifically related to the July 4th holiday. Although other months are more general, all months prescribe topics of suggested interest for the general population:All of us, in all stages of life, can find a health observance of interest this month. April\u2019s observances range from National Donate Life Month, a request for us to consider giving another person life, to WalkAmerica, a campaign to promote the benefits of carrying babies to term, sponsored by March of Dimes, to many observances in between.15\nThe observances further point out special days of awareness and political activism, and also broadly publicize public health education opportunities:The National Headache Foundation is sponsoring National Headache Awareness Week June 5\u201311 to recognize headache pain as a real and legitimate condition and encourage those who suffer with headaches to see a physician for proper diagnosis and treatment. This year\u2019s campaign, \u201cStop Migraines Before They Stop You,\u201d will feature public education activities nationwide.\u201d16\nThe links that are selected for inclusion in the newsletters link largely to other parts of the Department of Health and Human Services and to other health-related government institutions (.gov domain endings), such as the National Institutes of Health (of which NLM is also a part) or the Centers for Disease Control and Prevention.\nAs these examples show, different actors have developed practices that place various elements in new and unique relationships with one another. Issues related to health become intertwined with, for example, federal holidays. The health practices of the individual are now inextricably linked with diverse forms of community participation. In this sense, health education and enrollment is no longer only or primarily about verifying online health information, but rather about diverse activities that extend beyond the online realm.\nDirectly Linking Health Practices and Citizenship\nThese initiatives are tools to help people find and\/or check information and about helping lay persons become empowered, not merely through distribution of information, but also through coaching or education in how to participate more actively in the search for and use of information. Users are invited to join specific, defined communities that will, in one way or the other, protect them from misinformation and inform them about better ways to act. HON even emphasizes the strength and necessity of its partnership with lay users in able do what it sets out to do\u2014HON cannot help lay users if the lay users do not help HON.\nAdditionally, there is a normative message about responsible citizenship. HON, for example, responds to individuals who follow their instructions and report (suspected) misuse of the HONcode icon by thanking them for their \u201csense of civic duty\u201d and healthfinder\u2019s newsletters make the initiative much more than just a gateway, or portal, to links of organizations with information. They also serve a promotional function to keep visitors returning to the healthfinder website and getting them to link through to other government sites. Further still, they show that being an active consumer and responsible citizen involves more than merely checking the information one encounters on sites or reporting misinformation and\/or misuse of seals through certain channels. The responsibility extends into all areas of daily life (from good nutrition to awareness about possible diseases) and extends beyond individuals to include not only family or friends, but also the community-based or social responsibility that accompanies participation in educative programs, activism, or fund-raising events.\nWhat begins as a prescription for how to search for and assess information on the web actually extends to the entire lifestyle in which these searching activities are contextualized (As Dessauer [14] notes, much of what we think of with cyberspace loses meaning and referents if it excludes the external context). Interestingly, despite the varied forms of the messages emanating from the different initiatives, each of the prescribed behaviors, such as entering the web at a location of geographical significance (within government borders), participating in a chain that actively polices adherence to an ethical code and reports misuse, or even downloading and using a browser with a special toolbar, emerges as simultaneously optional and obligatory [53]. Herein lie strains of the tensions that are suggested by both Rose and Blume and Singleton: initiatives are created and promoted under the guise of increasing choice and democratization, but the actual prescriptions that they carry entail more powerful suggestions about which choices (both online and off) are the \u201cright\u201d or \u201cbetter\u201d choices\u2014with alternatives to those choices being (explicitly or implicitly) discouraged.\nDiscussion\nIn this paper, we discuss how political actors have enabled access to web-based information largely in terms of specific user tools; however, through these examples, we see that, as Markham [42, 43] argues, access to the internet is access not just to a tool, but to a place (emergent in for example, how the interface is designed or the level of engagement in a given activity) and a way of being (dependent on the degree to which the individual integrates the technology into his or her understanding of social construction). As providers or enablers of technology use, political actors at various levels (primarily states, but also NGOs and IGOs that provide different types of sites and tools) provide points of entry and create avenues not just to online information, but to online and offline social communities with specific rules for behaviour. This has implications for how technology is configured within the relationship between individuals and the state and other political actors. Especially important is that it highlights how the emphasis on claims of \u201cneutrality\u201d within the reliability debate is misplaced. Organizations such as HON or WHO, who claim their neutrality based on their international scope and\/or their position outside of national governments, nonetheless carry specific normative ideas of acceptable politically or socially related user behavior. Likewise, individual state governments that claim to represent the needs of their own \u201cgeneral public\u201d impose values on the information that they recommend to that public \u2013 for example, in privileging a .gov above all else, followed by a \u201c.org\u201d or \u201c.edu\u201d and reducing as much as possible endorsement of a \u201c.com.\u201d\nExisting literature arguing the need for collective solutions to reliability problems, together with the abundant presence of initiatives offering different options for interactions between lay end users and those providing assessment tools, indicate a readiness among political actors and some health professionals to engage in active teamwork with the public. There is even the possibility that this language of collaborative efforts suggests that the public is\/can be seen as just another interest group balanced with physicians [30]. However, in the examples provided above, we see that partnership does not necessarily imply equal footing for each of the three types of actors. Organizations use the catch-word reliability as a rhetorical device to capture attention and enroll users\u2014redirecting their information-seeking behaviors, but also emphasizing deeper-rooted values that situate individual health-related practices within a greater context of idealized citizenship. As such, in addition to prescriptions for use of specific tools, we find underlying prescriptions for practices that reflect good citizenship.\nWhat, then, comprises the construction of a \u2018good\u2019 citizen? First and foremost a good citizen is one who is interested in a healthy lifestyle and reflects this through choosing to participate in activities such as searching for information about his\/her health and the health of loved ones. Secondly, in searching for this information, the citizen reflects the possession of basic practical skills by choosing to align his\/her practices with political actors, thereby choosing for sites that have been created or in some way reviewed by these actors. Thirdly, the citizen actively participates in the online community in which these sites are embedded. Active participation includes helping to \u201cpolice\u201d the information online by checking links and actively reporting misuse. Additionally, active participation includes extending knowledge accrued online to participate in offline community activities (activism, education, and fund-raising).\nThis participation also forms a reciprocal relationship between the participant and the given organization\u2014in return for making oneself available to the community, the burden to search for, find, and evaluate information is made easier. Searching can be done using tools within which information has been pre-selected and\/or information can even be sent directly to the individual\u2019s personal e-mail. Both of these technical options for acquiring information also carry an implicit social promise of being taken care of\u2014protection from misinformation or mis-action due to lack of information. The readiness to participate in this partnership\u2014the commitment of the organization to helping protect its user community is made explicit.\nThrough the creation of initiatives that respond to debates about the reality of information online, organizations prescribe both online and offline behaviors, such that these are inextricably intertwined. This leads to the emergence of new collectives, counteracting the idea that internet enables people to separate activities with ease, compartmentalizing on and offline activities and leading to division.17 These collectives further counteract notions of individualization and separation from the community, as well as arguments that certain actors can be rendered irrelevant. Existing relationships become re-conceived as new alliances between medical and political actors, where internet technologies can be used to strengthen existing public education activities. Additionally, new (types of) communities emerge: families and friends or geographically concentrated communities are supplemented by communities that share aspects of visiting the same web sites and participating in the same self-policing chains\u2014online and offline, individuals participate in communities by watching out for one another and sharing in the same activities.\nAlthough these initiatives claim to target \u201call users, everywhere,\u201d they actually idealize use such that discussions about increased choice (and how this leads to patient empowerment) also carry the normative implications that \u201ctrue\u201d empowerment is only possible when the \u201cright\u201d choices are made. Within these discussions, individuals have the responsibility to develop skills that make them informed consumers, and the process of acquiring these skills also enables them to contribute their part to the greater civic communities in which they engage. The additional implications connoted by the terms \u201ccitizen\u201d and \u201cconsumer\u201d raise the question of whether these really are more suitable as replacements for the word \u201cpatient\u201d than other suggested alternatives.\nConclusion\nWhereas discussions of technologies and their users have largely neglected to consider the role of states (and other political actors), discussions of the relations of states and individuals have not always considered the important reciprocal effects of and on changing technologies. Discussions of skill manage to link individuals to individual technologies, but they have not successfully coupled this back on the role of states. This paper has used the discussion about the reliability of web-based information to attempt to bridge these gaps and demonstrate the integral relationship between technologies and users, states and individuals, and individuals and the development of skills. It shows that political actors, including both governmental and non-governmental organizations, make presumptions about citizen willingness to use certain technologies and, as such, utilize soft power to encourage these citizens-as-users-of-technologies to forward certain agendas.\nUnder the guise of democratizing ideals and suggestions of empowerment, they use policy, programs and web-based tools to encourage citizen participation. Underlying these different modes of participation, these actors are able to re-create boundaries both online and off, not only by demarcating geographical boundaries on the web, but also by supporting conventional understandings of social norms, such as the role of the medical expert as a primary leader. In so doing, they give patients the \u201cchoices\u201d about how to behave, with strong normative undertones about rights and responsibilities associated with being informed and reflexive consumers. As others have pointed out, where medical or health-related information is discussed, an issue of power is usually at stake [37]. The power in question is generally interpreted as that of medical professionals, but this is not necessarily the only interpretation, as political actors clearly use this for their own means and ends, as well.","keyphrases":["reflexive consumerism","internet","reliability initiatives"],"prmu":["P","P","P"]}
{"id":"J_Headache_Pain-4-1-2386847","title":"Involvement of calcitonin gene-related peptide in migraine: regional cerebral blood flow and blood flow velocity in migraine patients\n","text":"Calcitonin gene-related peptide (CGRP)-containing nerves are closely associated with cranial blood vessels. CGRP is the most potent vasodilator known in isolated cerebral blood vessels. CGRP can induce migraine attacks, and two selective CGRP receptor antagonists are effective in the treatment of migraine attacks. It is therefore important to investigate its mechanism of action in patients with migraine. We here investigate the effects of intravenous human alpha-CGRP (h\u03b1CGRP) on intracranial hemodynamics. In a double-blind, cross-over study, the effect of intravenous infusion of h\u03b1CGRP (2 \u03bcg\/min) or placebo for 20 min was studied in 12 patients with migraine without aura outside attacks. Xenon-133 inhalation SPECT-determined regional cerebral blood flow (rCBF) and transcranial Doppler (TCD)-determined blood velocity (Vmean) in the middle cerebral artery (MCA), as well as the heart rate and blood pressure, were the outcome parameters. No change of rCBF was observed at the end of infusion [1.2% \u00b1 1.7 with h\u03b1CGRP, vs. \u22121.6% \u00b1 3.1 with placebo (mean \u00b1 SD)] (P = 0.43). Vmean in MCA decreased to 13.5% \u00b1 3.6 with h\u03b1CGRP versus 0.6% \u00b1 1.8 with placebo (P < 0.005). Since rCBF was unchanged, this indicates a dilation of the MCA. h\u03b1CGRP induced a decrease in MAP (12%) (P < 0.005) and an increase in heart rate (58%) (P < 0.0001). CGRP dilates cerebral arteries, but the effect is so small that it is unlikely to be the only mechanism of CGRP-induced migraine.\nIntroduction\nBased on animal research showing calcitonin gene-related peptide (CGRP) in perivascular nerves [1] and a strong vasodilator effect of CGRP on cerebral blood vessels [2], a role for CGRP in the pathogenesis of migraine pain was first suggested by reports of increased CGRP in external jugular venous blood during migraine attacks [3\u20135]. In one recent study, there was, however, no increase of CGRP [6]. More solid evidence was presented in a study which demonstrated that, CGRP infused intravenously in patients with migraine was able to induce a vascular type headache in great majority of patients and in some patients this headache fulfilled all diagnostic criteria for migraine without aura [7]. The final proof of the involvement of CGRP in migraine mechanisms was provided by two phase II clinical trials [8, 9], demonstrating significant efficacy of the specific CGRP antagonists, BIBN4096BS [10] and MK0974 [9]. Thus, CGRP is not only able to induce attacks, but it seems to be continuously important throughout the entire migraine attack. CGRP is one of the most potent dilators of isolated cerebral arteries known today [2, 11], but possible species differences make it difficult to predict the effect of CGRP in the human cerebral circulation. Although the effect of CGRP on the cerebral circulation has been studied in normal subjects [12], it is important to study it also in patients with migraine in whom migraine-like headache and migraine attacks are induced by CGRP [7]. The aim of the present study was therefore to investigate the cerebral hemodynamic effects of CGRP in patients with migraine outside of attack.\nPatients and methods\nPatients\nTwelve patients with migraine (11 females, 1 male; mean age 39.5\u00a0years, range 31\u201347\u00a0years; mean weight 69.7\u00a0kg, range 51\u201389\u00a0kg) were included. All suffered from migraine without aura according to criteria of the International Headache Society [13]. The subjects were not allowed to take medication, coffee, tea, alcohol or tobacco for 12\u00a0h before the study, and they were not allowed to take a triptan 24\u00a0h or ergotamine 48\u00a0h before the study.\nExclusion criteria were as follows: use of any kind of daily medication including prophylactic headache therapy but excluding oral contraceptives; pregnancy or breastfeeding; excessive use of analgesics or alcohol; serious somatic or psychiatric disorders; ischaemic heart disease; a supine systemic blood pressure more than 160\/90 or less than 110\/75\u00a0mmHg at entry of study.\nPatients were informed that they were free to withdraw at any time and all gave written informed consent. The study was approved by the local ethics committees of Copenhagen and Copenhagen County (KA 96054) and complied with the Declaration of Helsinki.\nDesign and procedure\nThe patients with migraine were studied outside of attacks. The study used a double-blind, placebo-controlled crossover design. Subjects were randomized to receive 2\u00a0\u03bcg\/min human \u03b1CGRP (h\u03b1CGRP; Clinalfa, Switzerland) or placebo (0.9% NaCl) infused intravenously for 20\u00a0min on 2\u00a0days separated by at least 1\u00a0week. The dose was chosen as the highest tolerated dose (because of blood pressure reduction) based on reports in the literature [14, 15]. Patients were randomly assigned by computer (Med. Stat\u00ae, version 2.12). Randomization and preparation of study drugs was done by medical staff, who were not involved in the study. Five patients started with placebo and seven with h\u03b1CGRP. This quota was chosen by the computer and not known before the study. The effectiveness of blinding was not estimated.\nWhen subjects arrived at the laboratory, a cannula (Viggo Venflon\u00ae, 1.4\u00a0mm) was placed in the right cubital vein for h\u03b1CGRP\/placebo infusion. Baseline values of mean maximal blood velocity (Vmean) in the middle cerebral artery (MCA), regional cerebral blood flow (rCBF), blood pressure, heart rate and pCO2 were recorded after 30\u00a0min of rest in the supine position in a quiet room. Then, h\u03b1CGRP or placebo was infused intravenously for 20\u00a0min by a volumetric pump (Braun Perfuser). Vmean, blood pressure, heart rate and end tidal pCO2 were recorded every 10\u00a0min during and after infusion until 80\u00a0min after start of the h\u03b1CGRP infusion. rCBF was measured again during the last 5\u00a0min of infusion and at 75\u201380\u00a0min after start of h\u03b1CGRP\/placebo infusion. At every recording, it was noticed if volunteers were flushing and information about headache presence, intensity (measured on a 0\u201310 scale) and characteristics were obtained before, during and after the infusions. The results concerning headache response have been published elsewhere [7].\nMethods\nrCBF was measured with a highly sensitive, brain-dedicated, fast-rotating, single photon emission computerized tomograph (Tomomatic 232). Each study lasted 4.5\u00a0min. A mixture of atmospheric air and 133Xenon was rebreathed during the first 1.5\u00a0min through a closed system from a 4-L reservoir (740\u00a0Mbq\/L). During the last 3\u00a0min, the 133Xenon mixture was expired against atmospheric air. rCBF was recorded simultaneously in two slices positioned 50 and 90\u00a0mm above and parallel to the orbito-meatal plane (OM). Each slice was 16\u00a0mm thick and the distance between the centers of slices was 40\u00a0mm. The full width half maximum resolution of the instrument is about 16\u00a0mm in the horizontal plane. rCBF was calculated according to Celsis et al. [16].\nA fixed matrix of regions of interest was superimposed on the rCBF picture. The shape and size were fitted to the outlines on the brain excluding extracranial flow. Regional mean values were calculated within the predefined regions of interest. The matrix was divided into regions of interest representing the hemispheric rCBF regions and the vascular territories of supply by the anterior-, middle- and posterior cerebral arteries. The maximum whole body radiation was approximately 0.6\u00a0mSV per rCBF measurement [17].\nTime averaged mean of the maximal blood velocity (Vmean) in MCA at the usual headache side was measured with transcranial Doppler (TCD) ultrasonography (2\u00a0MHz, Multidop X Doppler: DWL, Sipplingen, Germany). A mean of four consecutive values of Vmean was taken (each representing the mean value of typically four to five heart beats automatically calculated by the computer). Positions of the measurements were reproduced from day to day by recording the position in relation to the angle and distance relative to the orbito-meatal line. Blood pressure and heart rate were measured with an automatic inflatable arm cuff (Omega 1400, Invivo Research Laboratory, New York, USA). Simultaneously, with each rCBF and TCD measurement, the end expiratory pCO2 was recorded by means of a capnograph (Datex OY, CD 101).\nStatistics\nHemodynamic responses are given as mean and standard deviation (\u00b1SD). Differences in blood velocity, rCBF, blood pressure, heart rate and pCO2 over time within the group were analyzed with analysis of variance (MANOVA, Statgraphics\u00ae 7.0), and changes were then located with a multiple range test (Confidence intervals, Statgraphics\u00ae 7.0).\nDifferences in the delta values (baseline\u2014the last measurement during infusion and baseline\u2014the last measurement during the study) in Vmean, rCBF, blood pressure, heart rate and pCO2 between the effect of h\u03b1CGRP and the effect of placebo were tested with a paired t test (Statgraphics\u00ae 7.0). In all tests, P\u00a0<\u00a00.05 was considered statistically significant.\nResults\nDuring CGRP infusion, two volunteers experienced a substantial decrease in blood pressure (70\/50 and 65\/25, respectively) causing the infusion to be terminated ahead of time. Signs and symptoms were pallor, cold sweat, stomach ache, nausea and palpitation. The situation was restored after placing the volunteers in head-down tilt for 7 and 20\u00a0min, respectively. No rCBF and TCD measurements were taken during this time period, and therefore, results from these two volunteers were excluded from the calculations below.\nChanges in end tidal pCO2\nEnd-tidal pCO2 decreased significantly over time when volunteers were treated with h\u03b1CGRP (P\u00a0<\u00a00.0001) but not when treated with placebo (P\u00a0<\u00a00.26, MANOVA). The pCO2 decrease at the last measurement during infusion was \u22124.0%\u00a0\u00b1\u00a00.8% when treated with h\u03b1GCRP versus 0.7%\u00a0\u00b1\u00a01.0% when treated with placebo (P\u00a0<\u00a00.05, paired t test). The peak decrease in end-tidal pCO2 induced by h\u03b1CGRP (\u22126.8%\u00a0\u00b1\u00a02.6%) occurred at 25\u00a0min, 5\u00a0min after end of infusion.\nTranscranial Doppler measurements\nThe TCD examination was performed on the side of usual migraine. The following data for Vmean in MCA are corrected for changes in pCO2 according to Markwalder et al. [18]: \nVmean changed significantly over time when patients were treated with h\u03b1CGRP (P\u00a0<\u00a00.0001, MANOVA), but not when treated with placebo (P\u00a0=\u00a00.26, MANOVA). At the last measurement during infusion, 15\u00a0min after its beginning, Vmean compared to baseline was \u221213.5%\u00a0\u00b1\u00a011.4 when treated with hCGRP, versus 0.6%\u00a0\u00b1\u00a05.6 when treated with placebo (P\u00a0<\u00a00.005, paired t test). At the end of the in-hospital period (75\u00a0min after start of the infusion), there was no difference between the two groups (P\u00a0=\u00a00.97, paired t test) (Fig.\u00a01, Table\u00a01). There was a positive correlation between \u0394Vmean and \u0394MABP (r\u00a0=\u00a00.703, P\u00a0<\u00a00.05).\nFig.\u00a01The effect of hCGRP (2\u00a0\u03bcg\/min) for 20\u00a0min and the effect of placebo on the mean velocity (Vmean) in middle cerebral artery (squares) and the regional cerebral blood flow (rCBF) (triangles) in the territory of the middle cerebral artery (MCA) used for transcranial Doppler examination. X-axis: time from start of infusion (min), y-axis: changes in percent of baseline. Vmean (squares) decreased \u221213.5%\u00a0\u00b1\u00a03.6 compared to baseline with hCGRP treatment versus 0.6%\u00a0\u00b1\u00a01.8 with placebo treatment (P\u00a0<\u00a00.0001). After 75\u00a0min, there was no difference between the two groups (P\u00a0=\u00a00.97). No change of rCBF (triangles) was observed at the end of infusion (P\u00a0=\u00a00.43) or at the end of study period (P\u00a0=\u00a00.12)Table\u00a01The maximum mean blood velocity (Vmean) in the middle cerebral artery (MCA)Time (min)Vmean in MCA (cm\/s)No pCO2 correctionCorrected for pCO2 changesCGRPPlaceboCGRPPlaceboCGRP%Placebo%081.6 (10.7)79.9 (12.3)81.6 (10.7)79.9 (12.3)0015\u20132066.9 (13.5)*79.5 (12.4)70.7 (14.2)*80.3 (12.7)\u221213.5 (11.4)*0.6 (5.6)75\u20138076.6 (9.9)77.4 (12.7)80.6 (11.3)79.2 (13.6)\u22120.7 (11.6)\u22120.9 (6.4)Values within parentheses represent the standard deviation (SD)Data is shown with and without pCO2 correction according to Markwalder et al. [18]The pCO2-corrected data is shown as absolute data and in percent change from baseline (P\u00a0<\u00a00.001)*\u00a0Significant changes compared with baseline\nRegional cerebral blood flow\nAll data have been corrected for changes in pCO2: rCBF(korr)\u00a0=\u00a0rCBF(meas)\u00a0\u00d7\u00a0exp(0.04(pC02(Basal)\u00a0\u2212\u00a0pCO2(n)). For comparison of Vmean and rCBF, we calculated rCBF in the territory of MCA, the artery used for TCD examination. Area-weighted data from the OM\u00a0+\u00a050\u00a0mm and OM\u00a0+\u00a090\u00a0mm were used for the results given below.\nThere was no change over time neither when patients were treated with h\u03b1CGRP (P\u00a0=\u00a00.26) nor when they were treated with placebo (P\u00a0=\u00a00.88, MANOVA). During infusion, rCBF compared to baseline was +1.2%\u00a0\u00b1\u00a05.5 when treated with h\u03b1CGRP versus \u22121.6%\u00a0\u00b1\u00a09.8 when treated with placebo. The difference between CGRP and placebo was not significant (P\u00a0=\u00a00.43, paired t test). Furthermore, no significant difference between CGRP and placebo was found at the end of the study period (P\u00a0=\u00a00.12, paired t test) (Fig.\u00a01, Table\u00a02).\nTable\u00a02Regional cerebral blood flow rCBF in the MCA territory recorded 50 and 90\u00a0mm above the orbitomeatal plane (OM)Cerebral blood flow (ml blood\/100\u00a0g\/min) Time (min)MCA (OM 50\u00a0+\u00a0OM 90) in the territory used for TCD examinationNo pCO2 correctionCorrected for pCO2 changesAbsolute dataAbsolute dataData in % of baselineCGRPPlaceboCGRPPlaceboCGRPPlacebo070.8 (13.7)67.7 (9.3)70.8 (13.7)67.7 (9.3)0015\u20132066.8 (9.4)65.3 (11.6)71.1 (10.5)66.7 (11.4)1.2 (1.7)\u22121.6 (3.1) 75\u20138069.3 (7.9)65.0 (9.8)72.8 (8.7)66.7 (10.4)4.2 (3.0)\u22121.6 (2.8)Values within parentheses represent the standard deviation (SD)Data is shown with and without pCO2 correction according to Markwalder et al. [18]The pCO2-corrected data is shown as absolute data and in percent change from baseline (P\u00a0<\u00a00.001)\nThe absolute rCBF data and the rCBF data with pCO2 correction concerning the MCA area relevant for TCD examination are given in Table\u00a02. There was also no significant difference in pCO2-uncorrected rCBF between CGRP and placebo during (P\u00a0=\u00a00.78, paired t test) or after the infusion (P\u00a0=\u00a00.357, paired t test). There was a negative correlation between \u0394rCBF(MCA) and \u0394MABP (r\u00a0=\u00a0\u22120.654, P\u00a0<\u00a00.05). rCBF during h\u03b1CGRP infusion was not significantly different in any territory or at any time point.\nBlood pressure\nThe mean arterial blood pressure (MABP) decreased significantly over time when patients were treated with h\u03b1CGRP (P\u00a0<\u00a00.0001), but not when they were treated with placebo (P\u00a0=\u00a00.48, MANOVA). The maximal decrease occurred at the last measurement during infusion (20\u00a0min after start of infusion) and amounted to \u221212.3%\u00a0\u00b1\u00a08.1% with h\u03b1CGRP treatment versus +2.4%\u00a0\u00b1\u00a05.9% with placebo treatment (P\u00a0<\u00a00.005, paired t test). MABP had normalized at the end of the study period (80\u00a0min from start of infusion) (P\u00a0=\u00a00.49) (Table\u00a03). Both systolic and diastolic blood pressures decreased during infusion of CGRP compared to that during infusion of placebo (P\u00a0<\u00a00.05).\nTable\u00a03The mean arterial blood pressure (MABP), systolic and diastolic blood pressure (mmHg) and heart rate (bpm) after CGRP and placebo treatmentBlood pressure (mmHg)Heart rate (bpm)Time (min)MABPSystolic BPDiastolic BPCGRPPlaceboCGRPPlaceboCGRPPlaceboCGRPPlacebo087.8 (9.6)91.6 (9.5)117.0 (11.1)119.0 (12.6)73.2 (9.5)77.9 (9.4)63.0 (6.0)64.4 (7.2)1578.0 (7.5)*90.0 (9.2)110.7 (9.6)117.2 (12.5)61.7 (7.8)76.4 (8.7)92.9 (10.2)*63.8 (6.8)2076.9 (10.1)*93.5 (8.3)112.2 (13.3)123.9 (11.6)59.3 (9.1)78.3 (7.6)98.9 (11.2)*65.5 (7.1)7586.5 (9.2)91.4 (8.7)117.6 (12.0)121.9 (13.4)71.0 (8.9)76.2 (7.2)76.1 (7.0)*62.9 (6.0)8085.3 (9.2)91.3 (9.9)116.4 (13.2)123.3 (11.3)69.7 (8.4)75.3 (9.7)75.3 (7.7)*64.4 (5.3)Values within parentheses represent \u00b1standard deviation (\u00b1SD)*\u00a0P\u00a0<\u00a00.05\nHeart rate\nWhen patients were treated with h\u03b1CGRP, heart rate increased significantly over time (P\u00a0<\u00a00.0001, MANOVA), but not when they were treated with placebo (P\u00a0=\u00a00.44, MANOVA). The peak increase in heart rate after h\u03b1CGRP treatment occurred 20\u00a0min after start of infusion reaching 58.1%\u00a0\u00b1\u00a022.7% versus 1.9%\u00a0\u00b1\u00a05.58% with placebo treatment (P\u00a0<\u00a00.0001, paired t test). Heart rate after h\u03b1CGRP treatment was still different from that after placebo treatment at the end of the study period (P\u00a0<\u00a00.005, paired t test) (Table\u00a03).\nOther signs\nFlushing after h\u03b1CGRP treatment was pronounced and appeared only in the face, neck and upper chest. It appeared from 10\u00a0min (time of first observation) after start of the infusion of h\u03b1CGRP in all patients until median 70\u00a0min after start of infusion (range 20\u201380\u00a0min). There was no flushing when the patients were treated with placebo. The median headache during infusion of h\u03b1CGRP was 1 versus 0 in the placebo-treated group (P\u00a0<\u00a00.01). During the following 11\u00a0h, all patients experienced headache after h\u03b1CGRP treatment versus the one after placebo treatment (P\u00a0<\u00a00.001). The median headache score was 4 after h\u03b1CGRP treatment and 0 after placebo treatment. For details, see [7].\nDiscussion\nThe main finding of the present study was that CGRP-infusion dilated the MCA in patients with migraine, while cerebral blood flow remained unchanged. The dose of 2\u00a0\u03bcg\/min of CGRP was chosen from the literature in which the same dose of 545\u00a0pmol\/min was given in healthy volunteers without any reported adverse events [14]. In another study, up to 25\u00a0\u03bcg CGRP was given as an intravenous bolus injection and all subjects had facial flushing [15]. The dose of 2\u00a0\u03bcg\/min for 20\u00a0min is probably the maximally tolerated dose of CGRP in patients with migraine, since it resulted in a substantial decrease of MABP and a marked increase in heart rate. Two subjects were near fainting and had to be withdrawn. In healthy volunteers, a dose of 1.5\u00a0\u03bcg\/min was used without adverse events apart from flushing and without any effect on mean blood pressure [12]. It remains uncertain whether the stronger circulatory response in the present study was exclusively due to the higher dose or whether patients with migraine are more sensitive to CGRP. After 20\u00a0min infusion at 1.5\u00a0\u03bcg\/min, the mean plasma level was 340\u00a0pmol\/L in healthy volunteers [12]. It can be calculated from these results that the plasma level would be 450\u00a0pmol\/L in our patients with migraine. The EC50 for CGRP for the dilatory effect on human pial arteries is 500\u00a0pmol\/L [19]. The dose of CGRP used in the present study thus resulted in a plasma level very near to the EC50 for cerebral arteries in vitro.\nThe blinding of the present study can be criticized. Signs (increased heart rate) and symptoms (facial flushing and feeling of warmth) during CGRP infusion made it difficult to keep the study completely blinded. However, the present design is the best one available, because no technique is available to disguise such symptoms.\nAn interesting finding of the present study is the significant reduction of blood velocity (13.5% decrease) in the MCA, but rCBF remained unchanged during intravenous h\u03b1CGRP-infusion. A reduced velocity in an artery, with unchanged regional blood flow in its territory of supply, reflects dilation [20]. The relative diameter change can be roughly estimated from the relation: flow\u00a0=\u00a0mean velocity multiplied by the cross-sectional area of the artery (2\u03c0r2). When the flow is constant, velocity is a function of the reciprocal value of r2 and Va\/Vb\u00a0=\u00a0rb2\/ra2 [20]. We can thus estimate from the change in mean velocity that the h\u03b1CGRP infusion caused a 7.5% increase in MCA diameter corresponding to 17% increase of its cross sectional area.\n\u03b1CGRP infused in a considerably lower dose (0.6\u00a0\u03bcg\/min) did not change MCA velocity or rCBF in healthy volunteers [21], while the dose of 1.5\u00a0\u03bcg\/min in volunteers dilated the MCA (9% increase in diameter) to the same extent as in the present study [12]. The dilatation of MCA in the present study could, theoretically, be due to cerebrovascular autoregulation secondary to decreased blood pressure. However, conduction arteries like MCA do normally only autoregulate due to blood pressure changes to a much lesser extent than arterioles (for review, see [22]). A positive correlation (r\u00a0=\u00a00.7, P\u00a0<\u00a00.05) between changes in MCA and changes in MABP seems to support this possibility. However, this correlation could also reflect that MCA and systemic circulation vary in parallel. In healthy volunteers, using a slightly lower CGRP dose of 1.5\u00a0\u03bcg\/min, blood pressure was unchanged but MCA dilated to the same extent as in the present study [12]. This strongly indicates that MCA dilation is a direct effect of CGRP.\nIn rat MCA, using the in vitro-pressurized arteriographic model, luminal CGRP was without any dilating effect, whereas abluminal CGRP dilated the artery [23]. This indicated that CGRP was unable to cross the blood\u2013brain barrier in rat MCA. In contrast, both in patients with migraine (Fig.\u00a01) and in healthy volunteers [12], a dilation of MCA was observed. Theoretically, CGRP could act on an endothelial receptor, but this is unlikely because the CGRP antagonist BIBN4096BS could not block the effect on MCA in man [12]. Furthermore, in man, the calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein (RAMP) are located mainly in the muscular layer of the human MCA [24]. In the endothelium, there was only a minor amount of CLR and largely absent RAMP1 [24]. There is thus no CGRP receptor (CLR plus RAMP1) on the endothelium. The present results with a dilation of MCA could indicate that the blood\u2013brain barrier to CGRP in the MCA is less tight in man than in rats, possibly related to the large difference in size.\nIn healthy volunteers, dilation of MCA was accompanied by a modest (14%) increase in rCBF [12], whereas in the present study, in patients with migraine, a 5% increase in rCBF was not statistically significant (Fig.\u00a01). The discrepancy may be due to random variation. Altered cerebrovascular reactivity in patients with migraine compared to controls is another possibility. The decrease of mean MABP from 88 to 77\u00a0mmHg observed in the present study could also be a factor if autoregulation was disturbed by CGRP.\nIn patients with subarachnoid hemorrhage, a CGRP-infusion concomitant with intravenous fluid to correct any drop in blood pressure resulted in a dilation of the MCA on the vasospasm side [25]. Blood pressure was unchanged while heart rate and cardiac output were increased and total peripheral resistance was decreased [25]. In two studies in man [14, 15], an increase in noradrenaline in plasma was observed after CGRP. We found in the present study a decrease in mean blood pressure and an increase in heart rate as would be expected after administration of a potent vasodilator such as CGRP.\nIn conclusion, the potent endogenous migraine-inducing molecule CGRP resulted in a dilation of MCA and unchanged rCBF. CGRP can thus most likely cross the blood\u2013brain barrier to some extent in the large human cerebral arteries. The vasodilator effect on these arteries is in our opinion, however, so small that it is unlikely to be the only mechanism of CGRP-induced migraine observed in our patients with migraine [7].","keyphrases":["migraine","regional cerebral blood flow","transcranial doppler","cerebral arteries","human calcitonin gene-related peptide"],"prmu":["P","P","P","P","R"]}
{"id":"BMC_Med_Educ-4-_-328087","title":"Learning from the problems of problem-based learning\n","text":"Background The last decade has witnessed a rapid expansion of biomedical knowledge. Despite this, fashions in medical education over the same period have shifted away from factual (didactic) teaching and towards contextual, or problem-based, learning (PBL). This paradigm shift has been justified by studies showing that PBL improves reasoning and communication while being associated with few if any detectable knowledge deficits.\nBackground\nMany doctors have commented that their medical education began in earnest on the first day that they entered the hospital wards as a hands-on practitioner. Claims of this kind support the view that the apprenticeship model of professional learning \u2013 which has been the backbone of training in the healing arts for thousands of years [1] \u2013 remains as central to medical career development today as ever [2]. A perennial complaint of the medical apprentice-in-training is that there are too few structured teaching activities within the busy world of postgraduate work [3], a concern which many institutions have addressed by developing formalised continuing education initiatives reminiscent of medical school courses [4-6]. Predictably, different complaints prevail at the pre-licensure phase of the training spectrum, where students often feel more motivated to acquire the (implicit) competence of the practising doctor [7,8] than to absorb large volumes of (explicit) scientific and\/or humanistic theory [9,10].\nSuch feelings underlie an unresolved debate over the optimal balance between factual ('teaching', or content-based) and practical ('training', or performance-based) components of professional development [11] and, as such, could explain a recent drift away from the didactic emphasis of older biomedical educative approaches [12] and towards quasi-experiential, or problem-based, learning [13,14] (PBL; Figure 1A). This hypothesis cannot fully account for the PBL-led transformation of medical teaching in the 1990s, however, overlooking as it does a powerful contrary trend: the explosive proliferation of biomedical knowledge [15,16] as epitomized by the completion of the Human Genome Project [17]. Although at first sight contradictory, this reciprocal relationship between knowledge growth and didactic teaching invites a unifying explanation: namely, that the switch of educational philosophy to non-didactic methods represents a strategy for teachers and students to cope with the expansile information environment [18,19].\nFigure 1\nComparison of growth rates of PBL and biomedical knowledge. (A), Relative growth of interest in PBL versus lecture-based teaching based on PubMed keyword frequencies between 1975 and 2000. The Title fields of the journal database were searched each year for the strings \"problem-based learning\" (open squares) or \"lecture\" (open diamonds); the retrieved items were then scrutinized to determine those dealing with the subject of teaching style (e.g., eponymous \"lectures\" were excluded). (B), Growth rates of journal articles dealing with subject matter relating to science, medicine and education. The strings \"gene\" (solid diamonds), \"clinical\" (open squares) and \"medical education\" (solid triangles) are shown here, illustrative of the frequencies of many other keywords searched.\nThere are many things which a fresh medical student, unburdened by factual knowledge, can begin to learn: basic surgical methods, resuscitation interventions, generic reasoning skills, and counselling techniques, to name a few. The pivotal question, then, is not whether such context-dependent (but sequence-independent) learning will prove effective [20]; rather, it is why this reorientation of teaching philosophy has occurred at all, and at this time. Or to put the issue another way: what is the hard evidence indicating that the original educational system was broken and that the new system is likely to fix it?\nA secondary issue, which has been a prime concern of PBL critics [21], is whether there may prove to be long-term hidden costs payable for the clear short-term benefits afforded by the PBL teaching philosophy. Since there are major differences in the way that PBL is implemented between schools, evidence to confirm or refute such hypotheses may be impossible to assemble. By the same token, it is an oversimplification to view all PBL as having low fact-based content, just as it is to equate all older teaching methods with rote learning. Nonetheless, since PBL veers more to the active\/contextual, and didactic teaching to the passive\/factual, it is plausible that one bias occurs at the expense of the other.\nDiscussion\nWhat is knowledge \u2013 anyone know?\nThe traditional educational sequence involves theory preceding application, an accelerated model of which has long been satirized in clinical circles as \"watch one, do one, teach one\". As noted above, however, some applications may be learned in the absence of theoretical knowledge, just as some subsets of theoretical knowledge may be unassociated with any obvious application. Is it possible, then, to define a minimum essential \"core knowledge\" spectrum for the student of biomedicine? If so, should such knowledge expand in parallel with other biomedical information, or should any such expansion be restricted by its relevance to changes in clinical performance (the 'barefoot doctor' model)? In the latter case, how long can healthcare competence and credibility be maintained in the face of rising constraints on scientific core knowledge [22]?\nIf core knowledge is indeed expanding at a rate similar to that of non-core knowledge, then the strategy of solving the broad problem of knowledge expansion by defining a narrower core can only be a temporizing measure. On the other hand, if the quantum of core knowledge is deemed non-expansile \u2013 arbitrarily defined, for example, to represent the amount of knowledge capable of being instilled in an average student by x teaching hours per week spread over y years \u2013 then any expansion of non-core knowledge will cause the core to shrink as a proportion of total knowledge. In 1984, for example, a list of two hundred drugs was hailed as a solution to information overload in the field of pharmacology [23]; but by 2000 the overload problem in this discipline was perceived to have deteriorated despite both the embracement of PBL and relentless efforts to re-define a core curriculum [24].\nA key difficulty in addressing this problem is that expansion of biomedical information is asymmetric \u2013 different areas of knowledge grow at different rates which in turn vary (and are ascribed differing priorities) during different periods (Fig. 1B). In practice, most curricula cope with differential knowledge growth by adding new core modules to cover areas of rapid growth [25]; the problem with this approach is that the notion of \"core\" becomes fluid, invalidating the concept. Moreover, it is difficult to discard ageing core knowledge at the same rate as adding new information, since the credibility of newer information tends by its nature to be weaker than that of older content. Rigid conservation of the core leaves trainees selectively deficient in new knowledge areas, on the other hand, making them less competitive in the marketplace. Discrepancies emerging between planned (taught) and actual (learned) medical curricula [26] further weaken the practicability of paradigms based on core knowledge.\nThe concept of core knowledge as a stand-alone solution to the problem of information inflation thus appears flawed [27]. Although at any one time certain knowledge subsets may be deemed dispensable for learning purposes, a continuous expansion of knowledge must imply a comparable expansion of knowledge essential for maintenance of professional competence [28]. Moreover, practicality should not be the sole criterion by which core knowledge is judged; a medical training system cannot succeed by simply cloning service-based doctors, but must also produce academics, researchers, visionaries and leaders able to develop the service infrastructure [29]. It is against the background of these diverse challenges that the recent growth of PBL should be appraised.\nThe lure of the non-expert\nA traditional authoritative doctor who dares challenge the information explosion faces the same risk as the immovable object confronting the irresistible force. The outcome has been to shatter the image of the omniscient doctor, as well as to dent the plausibility of experts in all fields. This slide from grace of the specialist \u2013 formerly a dominant figure in the medical educational pantheon, and a revered colleague in the battle against information overload [30] \u2013 has paralleled the decline of the lecture as a teaching medium. These linked trends suggest that the internet-empowered medical customer of the 21st century (whether patient or student) now questions information promulgated by mere individuals, thus threatening clinical and teaching paradigms formerly assumed unassailable.\nThis problem has created a niche for PBL, rejecting as it has the old curriculum's reliance upon experts and specialists [31]. The reported disadvantages of non-expert biomedical teaching with respect to knowledge transmission [32] and disease understanding [33] have been parried by numerous studies showing no detectable information deficits in PBL-trained (compared to lecture-taught) students [34-38]. Such negative data may be of limited reassurance, however, given the insensitivity of the endpoints used to measure what is in practice a rather limited curricular divergence [39]. It is likewise arguable that endpoints such as knowledge acquisition and clinical skills are surrogates, and that the most critical deliverable of medical training \u2013 namely, the quality of patient outcomes \u2013 has not been measured in any controlled trial of PBL [40]. These points lend credence to criticisms that the present-day popularity of PBL has so far been driven more by individual enthusiasm and conjecture than by objective scientific evidence [41].\nWho, then, stands to gain from PBL? Medical teachers are perhaps the most immediate beneficiaries [42]. Reduced self-perceptions of fallibility may be one attraction for teachers, as new PBL supervisors find that their educational contributions are no longer falsifiable by their pupils. In addition, responsibility for providing a sufficient knowledge base can be passed from teacher to student under the PBL auspices of 'self-directed learning' [43]. Yet another benefit relates to the lack of formal preparation required to initiate a PBL session [44] \u2013 an advantage which suggests a gain in efficiency. But does this bear scrutiny [45]?\nThings have changed\nEfficiency can be calculated by dividing (productivity) by (time and effort). What do we mean in this context by productivity? A half-century ago, the only responsibility of a medical school was to produce clinicians to serve the local community; today, however, teaching activities incorporate postgraduate specialist education, continuing medical education, professional and career development, public and patient awareness programmes, education-related research, conference and workshop organization, national and international collaborative initiatives, professional accreditation and audit activities, development of electronic teaching resources, and so on. Hence, a modern faculty's teaching productivity is not able to be gauged exclusively (or even predominantly) by the number and quality of its outgoing medical graduates, but rather must be judged by the sum total of its useful educational output.\nThis raises short- and long-term issues as to the most efficient ways to utilize faculty time and effort: traditional teaching service activities (e.g., tutorials, mentoring) must compete with more ambitious developmental activities (e.g., production of journal articles, books, software or web resources). Small-group tutorials are a time-honoured teaching modality, but the opportunity cost is high; while there must surely remain a place for personalized teaching, it seems doubtful whether the modern academic system can tolerate the luxury of an accelerating trend in this direction [46]. In contrast, the traditional apprenticeship training approach seems cost-effective, relying as it does upon the learner assisting a professional in the execution of his\/her paid duties.\nIn this context it is worth noting that the development of PBL \u2013 growth of which during the 1990s coincided with similar trends favouring noncognitive-based medical school admissions [47-49] and humanities-rich preclinical experience [50-52] \u2013 was spawned a quarter of a century ago in a regional medical school in Canada [53]. One need scarcely point out that the 1975 academic environment responsible for this educational breakthrough bears little resemblance to the market-driven imperatives that preoccupy most medical faculty members today, both in Canada [54-56] and elsewhere [57-62]. A changing environment not only justifies, but mandates, adaptation; if the 1990s trends do indeed represent a retreat from an information-dominated world, then the substitution of a PBL-dominated philosophy could be fraught with significant longterm perils.\nFrom words to actions\nSolutions lie in compromise. Such change is painful because it involves the abandonment of ideals formerly attainable; the vision of a one-size-fits-all medical school becomes no longer practical, and ever more difficult decisions will be needed as to what style(s) of graduate is most urgent for a faculty to produce. This process of curricular differentiation has started, but the pace is set to quicken as medical markets emerge and diverge, and as competition for faculty survival sharpens. To what extent, though, should these divisive educational decisions be made by markets, faculties, students, patients or governments?\nContrary to popular thought, there will remain a strong need \u2013 and possibly an enlarging one \u2013 for a subset of highly-trained medical graduates from a knowledge-intensive learning environment who are capable of assimilating the complexities of science, informatics, humanities and logistics that comprise modern medicine. Since the proportion of individuals and faculties suitable for this leadership mission looks set to decline, however, a larger number will need to accept the equally daunting compromise of skills prioritization.\nTeachers cannot teach without students, but students can learn without teachers. This belated insight has transformed the role of teachers into that of learning facilitators, akin to a culture of \"thinking apprenticeship\". Paradoxically, in an age when even complex skills such as landing aircraft are learned using robotic simulators, the trend in medical education has switched back to labor-intensive small-group teaching under the guise of PBL. This at first seems all the more curious given the unprecedented availability of alternative technologies for teaching clinical reasoning, the increasing importance of an adequate knowledge base in an ever more sophisticated professional environment, the growing pressures on faculties to use limited fiscal resources in the most cost-effective manner, and the novel opportunities for commercializing educational activities and products via the development of software and web-based resources.\nThe rise in PBL popularity over the 1990s thus suggests a retreat from the fallout of the biomedical information explosion. Although this response seems rational enough as a short-term adaptive measure, it should not be regarded as a solution to the problem of knowledge expansion. Just as PBL was originally pioneered as a reaction against complacency in traditional pedagogy, so must today's medical schools reject expediency and confront the unresolved information-management challenges of 21st-century medical education. The formulation of more efficient techniques for imparting factual knowledge, a greater emphasis on directing limited resources to the production of reusable teaching tools [63], and a willingness to experiment with differentiated medical curricula that prioritise graduate skill subsets, can all play a role in driving educational reform as a positive and ongoing adaptive process.\nSummary\nThe knowledge explosion of the last two decades has been accompanied by a decreasing reliance on didactic teaching. This educational paradigm shift has been led by widespread embracement of PBL, the original rationale of which was to improve students' ability to reason and communicate. In recent years, however, PBL has grown more rapidly in apparent response to information overload in medical school curricula, and may thus be viewed as a symptom of the problem of biomedical knowledge expansion.\nThe challenge of defining the right balance between what is taught, what is learned, and what remains unlearned will not disappear. Although few knowledge deficits have been detected in today's PBL-educated students, a decreasing concern with the adequacy of the professional knowledge base could yet erode the future credibility of the medical profession. By continuing to rely on popular PBL escape clauses such as 'self-directed learning' and 'information management', today's medical educators risk losing sight of this longterm threat.\nThe era of active learning began thousands of years ago with the first apprentice. We now live in a new era with new challenges, one of which is exponential information expansion. PBL provides one way for faculty and students to cope with this challenge, but sidesteps deeper issues relating to the widening core of essential professional knowledge. Innovative curricular experiments using educational strategies complementary to PBL would therefore appear timely.\nAbbreviations\nPBL, problem-based learning\nCompeting interests\nNone declared.\nPre-publication history\nThe pre-publication history for this paper can be accessed here:","keyphrases":["knowledge management, medical education"],"prmu":["R"]}
{"id":"Qual_Life_Res-3-1-2039864","title":"The effect of walking and vitamin B supplementation on quality of life in community-dwelling adults with mild cognitive impairment: a randomized, controlled trial\n","text":"Objectives To examine the effect of walking and vitamin B supplementation on quality-of-life (QoL) in community-dwelling adults with mild cognitive impairment.\nIntroduction\nEspecially in older people, both mental and physical function decrease due to multiple age related changes, which in turn may affect quality of life (QoL). The most obvious decrease in mental function is cognitive decline, which is a common aspect of aging. However, in some cases decline is more serious than expected for a certain age. This is specified as Mild Cognitive Impairment (MCI). MCI is considered to be a potential transitional stage between normal cognitive function and Alzheimer\u2019s disease, characterized by (1) subjective memory complaint (2) objective memory impairment (3) normal mental status (4) intact activities of daily living (ADL) (5) absence of dementia [1]. Independent of the latter four criteria, subjective memory complaints are related to lower QoL [2]. Moreover, MCI is associated with poor physical health and high risk of ADL dependence [3, 4]. Since both cognitive and physical decline belong to the most important determinants of QoL in community dwelling elderly subjects [5], subjects with MCI are likely to be susceptible to a decrease in QoL.\nThe number of adults with MCI is increasing considerably due to the aging population. For multiple reasons, it is important to prevent a decrease in QoL. Apart from the personal benefits, a high rated QoL also reduces medical consumption and helps to maintain independency as long as possible [6]. This in turn may relieve significant others, caregivers and medical society in general. For this reason, attention should be paid to possible interventions contributing towards a higher level of overall QoL and it\u2019s mental and physical components. In this respect, physical exercise and vitamin supplementation are interesting interventions worth investigating. Regular participation in moderate intensity aerobic training is reported to be beneficial in improving QoL and wellbeing, which is an important aspect of QoL [7, 8]. Since walking is the most prevalent physical activity among older adults [9], improving QoL by increasing the time spent on moderate intensity walking seems promising. Indeed, a community based walking program significantly improved both the physical and mental components of health-related QoL in older adults (n\u00a0=\u00a0582) [10]. Inconclusive evidence has been reported on the influence of vitamin B supplementation on QoL. Different aspects of QoL were not responsive to short term supplementation (range 4\u201312\u00a0weeks) with different doses and combinations of B vitamins in men and women [11\u201313].\nNot much is known about QoL in community dwelling elderly with MCI. Moreover, no trials on the effect of exercise and vitamin B supplementation on QoL have been carried out yet in adults with MCI. The FACT-study (Folate physical Activity Cognition Trial) was developed to examine the effect of these interventions on cognition [14]. Aspects of QoL were measured as a secondary outcome. In the present paper, the effectiveness of 1\u00a0year moderate intensity walking (two sessions of 60\u00a0min per week) and daily vitamin supplementation (5\u00a0mg folate, 50\u00a0mg vitamin B6 and 0.4\u00a0mg B12) on both overall QoL and it\u2019s health-related components is examined in community dwelling older adults with MCI. We hypothesize that 1\u00a0year moderate intensity walking benefits QoL. Concerning the effect of vitamin supplementation, this paper should be considered as explorative.\nMethods\nStudy design \nThe study was designed as a randomized, placebo controlled intervention trial, based on a two-by-two factorial design. The study-protocol has been described in detail elsewhere [14] and was approved by the VU University Medical Center medical ethics committee. Written informed consent was obtained from all participants.\nParticipants \nIn a medium-sized Dutch town community dwelling subjects aged 70\u201380\u00a0years with MCI were identified using a population based two-step-screening [15]. The operational criteria for MCI according to the criteria of Petersen et\u00a0al. [1] and additional inclusion criteria for the RCT are described in Table\u00a01. Subjects were not paid to participate in the study.\nTable\u00a01Inclusion and exclusion criteria for participation in the trial Operationalization of Petersen criteria for MCI (1\u20135) and additional inclusion criteria for the RCT (6\u201312)1. Memory complaints (answer yes to question \u2018do you have memory complaints\u2019, or at least twice sometimes at cognition scale of Strawbridge [16]2. Objective memory impairment; 10 WLT delayed recall \u22645\u00a0+\u00a0percentage savings\u00a0\u2264\u00a0100 [17]3. Normal general cognitive functioning; TICS\u00a0\u2265\u00a019\u00a0+\u00a0MMSE\u00a0\u2265\u00a024 [18, 19]4. Intact daily functioning: no report of disability in activities of daily living on GARS-scale, except on the item \u2018taking care of feet and toe nails\u2019 [20]5. Absence of dementia; TICS\u00a0\u2265\u00a019\u00a0+\u00a0MMSE\u00a0\u2265\u00a0246. Being able to perform moderate intensity physical activity, without making use of walking devices, e.g., a rollator or a walking frame 7. Not using vitamin supplements\/vitamin injections\/drinks with dose of vitamin B6, B11 or B12 comparable to vitamin supplement given in intervention8. Not suffering from epilepsy, multiple sclerosis, Parkinson\u2019s disease, kidney disorder requiring haemodialysis, psychiatric impairment9. Not suffering from depression as measured by the GDS (cut off \u22645) [21]10. Not using medication for rheumatoid arthritis or psoriasis interfering with vitamin supplement11. No alcohol abuse (men\u00a0<\u00a021 consumptions a week, women\u00a0<\u00a015 consumptions a week)12. Not currently living in a nursing home or on a waiting list for a nursing homeMCI\u00a0=\u00a0Mild Cognitive Impairment, RCT\u00a0=\u00a0Randomized Controlled Trial, 10 WLT\u00a0=\u00a010 Word Learning Test, TICS\u00a0=\u00a0Telephone Interview for Cognitive Status, MMSE\u00a0=\u00a0Mini Mental State Examination, GARS\u00a0=\u00a0Groningen Activity Restriction Scale, GDS\u00a0=\u00a0Geriatric Depression Scale \nRandomization \nAfter the baseline interview, subjects were randomly assigned to the interventions using the statistical computer program SPSS. Intervention groups were: (1) walking program or placebo activity program; and (2) vitamin B supplementation or placebo supplementation. Randomization was stratified for physical activity level at baseline in minutes per day as measured by the LASA physical activity questionnaire [22]. For the flow of participants see Fig.\u00a01.\nFig.\u00a01Flow chart. TI\u00a0=\u00a0Telephone Interview, WP\u00a0=\u00a0Walking Program, PAP\u00a0=\u00a0Placebo Activity Program, FA\/B12\/B6\u00a0=\u00a0Folic Acid, Vitamin B12, Vitamin B6 supplementation, SO\u00a0=\u00a0significant other, T6\u00a0=\u00a0follow-up after 6\u00a0months, T12= follow-up after 12\u00a0months, *reason for exclusion: only baseline data available\nExercise intervention\nSubjects assigned to the walking program (WP) participated twice a week 60\u00a0min in group-based moderate intensity walking during 1\u00a0year. Each session consisted of a warming up, moderate intensity walking exercises and a cooling down. The WP was based on \u2018Sportive Walking,\u2019 an existing aerobic walking program [23] aimed at improving cardiovascular endurance. Therefore, duration and intensity of the walking exercises increased gradually during the program. Sessions took place outdoor in municipal parks. Subjects not assigned to the WP participated in a placebo activity program (PAP) with the same frequency, session duration and program duration. However, the PAP consisted of low intensity exercise, such as light range of motion movements and stretching. Sessions were divided into five themes: relaxation, activities of daily living, balance, flexibility, posture and a combination of all. For each theme three sessions were developed and the entire series of 18 sessions was repeated during the intervention period. The PAP was carried out in community centers. Both programs were supervised by qualified and trained instructors. Attendance to both programs was assessed by the percentage of attended sessions.\nVitamin supplementation (FA\/B12\/B6)\nSubjects in the vitamin supplementation group took one pill containing 5\u00a0mg vitamin B11 (Folic Acid), 0.4\u00a0mg vitamin B12 (Cyanocobalamin) and 50\u00a0mg vitamin B6 (Pyridoxine-hydrochloride) daily during 1\u00a0year. This vitamin supplement is available on prescription in The Netherlands. Subjects randomized to the control group took an identically looking placebo pill. The pills were packed in blister packs for 1\u00a0week, which were labeled for each day of the week. Compliance with the vitamin supplementation was verified by pill counts in returned blister packs during the intervention.\nOutcome measures\nBaseline data on sociodemographic and background variables were collected using a postal screening questionnaire. The measurement of other baseline variables, as reported in Table\u00a02, has been described elsewhere [14]. In the present manuscript a distinction was made between \u2018overall quality of life,\u2019 referring to a subjects overall enjoyment of life and \u2018health-related quality of life,\u2019 referring to health-related factors affecting quality of life. The term QoL was used as an umbrella term for both overall and health-related QoL. The population-specific Dementia Quality of Life questionnaire (D-QoL) [24] was used to assess overall QoL and the generic Short Form 12 (SF12) [25] to assess health-related QoL. The D-QoL is a 29 item measure especially developed for elderly with cognitive decline and dementia. The participant is asked about how much they enjoyed activities that were reported to be important for elderly such as \u2018watching animals.\u2019 Moreover, the frequency of certain positive and negative feelings such as \u2018lovable\u2019 or \u2018worried\u2019 were asked for. Finally, they were asked to rate their overall quality of life. The participant was instructed to choose the best fitting answer from five item response scales. The answers were divided into five domains of QoL measuring sense of aesthetics, feelings of belonging, negative affect, positive affect\/humor and self esteem. A mean score ranging from one to five was calculated for these subscales and for the total D-QoL. A higher score indicated better quality of life. Median internal consistency reliability of the D-QoL was 0.80 and median test\u2013retest reliability was 0.72 in a sample of 95 older adults with different stages of cognitive decline [24]. The SF12 consists of 12 items measuring eight concepts of both mental and physical health, i.e., physical functioning, role-physical, bodily pain, general health, energy, social functioning, role emotional and mental health. These concepts are subdivided into two summary scores using a norm-based criterion: i.e., mental and physical component summary scales (SF12-MCS and SF12-PCS). The mean score is 50 with a standard deviation of 10. For example, a score of 60 corresponds to a-QoL rating of one standard deviation above the average ratings in the general population. Test retest reliability for the SF-12 MCS was 0.76 in a sample of 187 adults in the United Kingdom and 0.77 in a sample of 232 adults in the United States. Reliability coefficients of the SF-12 PCS in these populations were 0.86 and 0.89, respectively [25]. In the present study, measurement took place during a personal interview at baseline and after 6 and 12\u00a0months. Both the D-QoL and the SF-12 were administered by a trained interviewer who was unaware of the participants\u2019 group allocation.\nTable\u00a02Baseline characteristics of participants (n\u00a0=\u00a0152)Exercise interventionVitamin interventionWP (n\u00a0=\u00a077)PAP (n\u00a0=\u00a075)FA\/B12\/B6 (n\u00a0=\u00a078)Placebo-pill (n\u00a0=\u00a074)Age (Mean (SD))75 (2.9)75 (2.8)75 (2.8)75 (2.9)Gender (% male)48*645655MMSE (Median (10th\u201390th \u2030))29 (26\u201330)29 (27\u201330)29 (25\u201330)29 (27\u201330)Education (% low\/middle\/high) 61\/22\/1752\/29\/1957\/26\/1755\/26\/19Marital status (% living together)75686973Physical activitya (min\/day) (Median (10th\u201390th \u2030))44 (10\u2013155)39 (11\u2013120)45 (13\u2013155)38 (9\u2013111)Vitamin status (% deficient FA\/B12\/B6)b46\/8\/048\/8\/049\/9\/045\/7\/0Homocysteine statusc (% hyperhomocysteinemia)27232723Blood pressure (% hypertension)d27*142516BMI (kg\/m2) (Median (10th\u201390th p\u2030))26.7 (23.1\u201331.5)26.6 (23.5\u201332.7)26.5 (23.3\u201332.8)26.7 (23.5\u201331.2)Smoking (% smokers)13151711Number of self-reported diseases (% 0,1,2)e52\/42\/669\/27\/466\/28\/655\/41\/4WP\u00a0=\u00a0Walking Program, PAP\u00a0=\u00a0Placebo Activity Program, FA\/B12\/B6\u00a0=\u00a0Folic Acid, Vitamin B12, Vitamin B6 supplementation, MMSE\u00a0=\u00a0Mini Mental State Examination, Education: low\u00a0=\u00a0no education, primary education, lower vocational training; intermediate\u00a0=\u00a0intermediate level secondary education, intermediate vocational training; high\u00a0=\u00a0higher level secondary education, higher vocational training, university training. BMI\u00a0=\u00a0Body Mass Indexa \u22653.0 metabolic equivalents b Cut off points: FA red blood cell <337\u00a0nmol\/l or FA plasma\u00a0<\u00a06,3\u00a0nmol\/l; B12\u00a0\u2264\u00a0150 pmol\/l; B6\u00a0<\u00a020\u00a0nmol\/lc Homocysteine\u00a0>\u00a014\u00a0mmol\/ld Hypertension\u00a0=\u00a0diastole\u00a0\u2265\u00a090 and systole \u2265160e Cardiovascular disease, chronic obstructive pulmonary disease, diabetes, epilepsy, multiple sclerosis, Parkinson\u2019s disease, psychiatric disease, renal failure requiring dialysis and\/or rheumatoid arthritis * Significantly different from PAP (P\u00a0<\u00a00.05)\nStatistical analyses\nDifferences between groups on baseline characteristics were tested using independent t-tests (normally distributed variables), Mann\u2013Whitney U tests (not normally distributed variables) and \u03c72 (categorical variables). Within group differences were tested using dependent t-tests.\nSubsequently, data were analyzed according to a modified intention-to-treat principle, based on data from all randomized participants who provided data at baseline and at least one follow-up measurement. To evaluate the effects of the walking program and the vitamin supplementation on QoL, longitudinal regression analysis was used. The two follow-up measurements were defined as dependent variable and multi level analysis with two levels was used, (1) time of follow-up measurement (values corresponding with performance after six and 12\u00a0months intervention); (2) individual. According to the study protocol [14], the effect of both interventions was examined independently from each other. Data were analyzed using a crude and an adjusted model. Independent variables were exercise intervention and vitamin intervention. By analyzing both interventions in the same model, results were adjusted for the possible influence of the other intervention. Moreover, all analyses were adjusted for baseline performance on the outcome measure by adding this as a covariate. In the adjusted model, education, baseline activity level, baseline vitamin status, attendance to the exercise program and compliance with the supplementation were added as covariates. Interaction between gender and the WP or FA\/B6\/B12-supplementation was checked in the adjusted model. In the case of significant interaction, results were reported for men and women separately. In the case of no interaction, gender was added to the adjusted model as an additional covariate. Also, in the \u2018adjusted model\u2019 an interaction effect of the exercise program with attendance to the exercise program was checked. Finally, data were analyzed according to the per protocol principle, including all participants who attended at least 75% of the sessions. This cut-off point is in concordance with previous exercise intervention studies in older adults [26, 27].\nData were analyzed using SPSS for Windows (release 12.0.1). A significance level of 5% was used for between group comparisons and of 10% for interaction terms. For all analyses, regression coefficients and 95% confidence intervals for the adjusted models were reported, with the regression coefficients directly indicating the difference in QoL ratings between the WP and the PAP or the FA\/B12\/B6-supplementation versus placebo supplementation. In the case of significant interaction, regression coefficients and the 95% confidence intervals of the interaction terms were reported.\nResults\nPatient characteristics\nHundred-seventy-nine participants were randomized to the interventions. Twenty-seven of them were excluded from the analyses, because they only provided baseline data. These subjects were more often married (71% vs. 52%, P\u00a0=\u00a00.05) and less often current smokers (0% vs. 14%, P\u00a0=\u00a00.04) than the remaining 152 participants who provided QoL data at baseline and at at least one follow-up measurement. The latter 152 participants were included in the analyses (see Fig.\u00a01). Their mean age (SD) was 75 (2.9) years. Fifty-six percent was male. Additional baseline variables are described per factor in Table\u00a02. Compared to the PAP, the WP included fewer men (48% in WP vs. 64% in PAP) and more subjects with hypertension (27% in WP vs. 14% in PAP). Ratings for both overall and health-related QoL at baseline and after 6 and 12\u00a0months intervention are presented in Table\u00a03. No baseline differences were observed on these measures, except for a higher rating of D-QoL self-esteem in subjects in the FA\/B12\/B6-group compared to subjects in the placebo-supplementation group.\nTable\u00a03Means (standard deviations) of QoL ratings at baseline and after 6 and 12\u00a0months in older adults with MCIaWP PAPFA\/B12\/B6 Placebo T0 (n\u00a0=\u00a077)T6 (n\u00a0=\u00a077)T12 (n\u00a0=\u00a071)T0 (n\u00a0=\u00a075)T6 (n\u00a0=\u00a075)T12 (n\u00a0=\u00a067)T0 (n\u00a0=\u00a078)T6 (n\u00a0=\u00a078)T12 (n\u00a0=\u00a071)T0 (n\u00a0=\u00a074)T6 (n\u00a0=\u00a074)T12 (n\u00a0=\u00a067)D-QoL sumscore3.5 (0.26)3.5 (0.29)3.5 (0.27)3.5 (0.32)3.5 (0.34)3.5 (0.34)3.5 (0.32)3.5 (0.32)3.5 (0.33)3.4 (0.24)3.5 (0.31)3.5 (0.27)D-QoL aesthetics3.5 (0.63)3.5 (0.64)3.6 (0.60)3.5 (0.70)3.5 (0.71)3.5 (0.65)3.5 (0.64)3.5 (0.68)3.6 (0.61)3.4 (0.68)3.5 (0.67)3.6 (0.64)D-QoL belonging3.7 (0.50)3.7 (0.49)3.7 (0.44)3.8 (0.45)3.7 (0.47)3.7 (0.46)3.8 (0.50)3.6 (0.50)3.6 (0.48)3.7 (0.44)3.8 (0.45)3.8 (0.40)D-QoL negative affect2.7 (0.45)2.7 (0.46)2.8 (0.50)2.7 (0.55)2.8 (0.54)2.8 (0.52)2.7 (0.54)2.8 (0.47)2.8 (0.53)2.7 (0.47)2.7 (0.53)2.8 (0.49)D-QoL positive affect3.8 (0.39)3.7 (0.46)3.8 (0.40)3.8 (0.40) 3.7 (0.44)3.8 (0.43)3.8 (0.41)3.7 (0.47)3.8 (0.44)3.8 (0.39)3.8 (0.43)3.8 (0.39)D-QoL self esteem3.6 (0.45)3.8 (0.41)3.8 (0.40)3.7 (0.48)3.7 (0.49)3.8 (0.48)3.8 (0.48)*3.8 (0.48)3.9 (0.48)3.6 (0.43)3.7 (0.43)3.7 (0.38)SF12-MCS54.6 (6.85)55.6 (6.40)55.3 (4.39)54.7 (8.07)55.0 (7.34)55.3 (6.24)55.5 (7.49)55.9 (6.91)55.8 (4.90)53.8 (7.36)54.6 (6.86)54.8 (5.76)SF12-PCS48.2 (7.15)48.1 (7.57)50.5 (6.13) 48.7 (7.86)48.8 (8.47)49.8 (7.04)47.9 (8.20)47.4 (8.79)49.8 (6.68)49.1 (6.67)49.6 (7.00)50.6 (6.49)MCI\u00a0=\u00a0Mild Cognitive Impairment, WP\u00a0=\u00a0Walking Program, PAP\u00a0=\u00a0Placebo Activity Program, FA\/B12\/B6= Folic Acid, Vitamin B12, Vitamin B6 supplementation, D-QoL\u00a0=\u00a0Dementia Quality of Life, SF12-MCS\u00a0=\u00a0Short Form 12 Mental Component Summary, SF12-PCS\u00a0=\u00a0Short Form 12 Physical Component Summarya Higher rating indicates better QoL* P\u00a0<\u00a00.05 (difference between FA\/B12\/B6 and placebo)\nAttendance to the WP and the PAP\nOverall median attendance to the exercise programs (10th\u221290th percentile) was 63 (0\u201389) percent and did not differ between the WP and the PAP. Especially in the first weeks, a considerable number of subjects discontinued participation, mostly because they did not want to participate in the exercise programs after all. Most frequent reasons for discontinuation of the program after the first weeks were health-related problems. No adverse events of the WP or PAP itself were reported. Adherent subjects attending at least 75% of the sessions (n\u00a0=\u00a051) were more often living together (82% vs. 65%, P\u00a0=\u00a00.03) and less physically active than non-adherers (n\u00a0=\u00a0101), (median [10th\u201390th percentile] was 36 [13\u201382] vs. 44 [10\u2013169] min\/day, P\u00a0=\u00a00.02). At baseline, adherers also had lower ratings of D-QoL-belonging (3.6 [0.41] vs. 3.8 [0.49], P\u00a0=\u00a00.02) and higher SF12-MCS values (56.5 [5.6] vs. 53.7 [8.1], P\u00a0=\u00a00.02). Other baseline and QoL characteristics did not differ significantly.\nCompliance with the (FA\/B12\/B6)supplementation\nFour participants did not return the blister packs. On the basis of pill counts in returned blister packs, median compliance (10th\u201390th percentile) with the FA\/B12\/B6-supplementation was 100 (97\u2013100) percent and compliance with placebo-supplementation was 100 (35\u2013100) percent. Even though median compliance in both groups was 100%, compliance in the placebo-group was significantly lower (P\u00a0<\u00a00.05). Eight subjects, one in the FA\/B12\/B6-group and seven in the placebo-group, did not take (vitamin)supplementation. Seven of them decided immediately after randomization not to participate in the interventions. The other wanted to participate in the exercise intervention only. Two participants discontinued taking vitamin pills during the trial after reporting sleep problems and increased forgetfulness; one participant discontinued taking the placebo pills after reporting not feeling well.\nModified intention to treat analyses\nResults of the walking program and FA\/B6\/B12 supplementation are presented in Table\u00a04. With respect to overall QoL, no positive significant main effect of the WP or FA\/B6\/B12 supplementation was found. A significantly detrimental effect of FA\/B6\/B12 supplementation was observed on D-QoL-belonging, (beta (95%CI)\u00a0=\u00a0\u22120.18 (\u22120.29; \u22120.07), P\u00a0<\u00a00.01). A positive interaction between the WP and attendance to the WP was observed on D-QoL-belonging and D-QoL-positive affect. With each percent increase in attendance, D-QoL-belonging increased with 0.003 points (P\u00a0=\u00a00.04) and D-QoL-positive affect with 0.002 points (P\u00a0=\u00a00.06) in the WP compared to the PAP. With respect to health-related QoL, an interaction between the WP and gender was observed on the SF12-MCS (P\u00a0=\u00a00.06) and therefore analysis for the SF12-MCS was stratified for gender. No main effects of the WP or FA\/B12\/B6-pills were observed. However, in men in the WP, SF12-MCS increased with 0.03 points with each percent increase in attendance (P\u00a0=\u00a00.08).\nTable\u00a04Results of longitudinal multi level analyses on the effect of the WP and FA\/B6\/B12 supplementation on change in QoL (adjusted model) WP versus PAP Beta (95%CI)P-valueFA\/B12\/B6 versus placebo Beta (95%CI)P-valueD-QoL sumscore0.04 (\u22120.03;0.10)0.25\u22120.06 (\u22120.12;0.004) 0.07D-QoL aesthetics0.06 (\u22120.07;0.20)0.37\u22120.07 (\u22120.20;0.07)0.33D-QoL belonging0.00 (\u22120.11;0.11)0.96\u22120.18 (\u22120.29; \u22120.07)0.00D-QoL negative affect\u22120.02 (\u22120.12;0.08)0.650.04 (\u22120.05;0.14)0.37D-QoL positive affect0.04 (\u22120.04;0.13)0.34\u22120.04 (\u22120.12;0.04)0.33D-QoL self esteem0.08 (\u22120.02;0.18)0.110.00 (\u22120.10;0.11)0.94SF12-PCS0.66 (\u22121.23;2.54)0.49\u22120.73 (\u22122.65;1.19)0.45SF12-MCS*Men\u22120.82 (\u22122.24;0.60)0.250.25 (\u22121.31;1.81)0.76Women1.66 (\u22121.50;4.81)0.301.32 (\u22121.93;4.56)0.42WP\u00a0=\u00a0Walking Program, PAP\u00a0=\u00a0Placebo Activity Program, FA\/B12\/B6= Folic Acid, Vitamin B12, Vitamin B6 supplementation, D-QoL\u00a0=\u00a0Dementia Quality of Life, SF12-MCS\u00a0=\u00a0Short Form 12 Mental Component Summary, SF12-PCS\u00a0=\u00a0Short Form 12 Physical Component Summary* Interaction WP and gender\nPer protocol analyses\nSubgroup analyses were performed in subjects attending 75% or more of the WP or PAP sessions (n\u00a0=\u00a051, 33 men and 18 women). No between group differences were observed for FA\/B12\/B6-pills versus placebo-pills. A significant positive effect of the WP compared to the PAP was observed on D-QoL-positive affect, beta (95%CI)\u00a0=\u00a00.23 (0.06; 0.39), P\u00a0<\u00a00.01 and a borderline significant positive effect on D-QoL-self esteem, beta (95%CI)\u00a0=\u00a00.17 (0.001; 0.34), P\u00a0=\u00a00.05.\nDiscussion\nNo positive main effect of walking or daily FA\/B6\/B12 supplementation was observed on QoL in community-dwelling adults with MCI. However, ratings of overall QoL (i.e., feelings of belonging, positive affect) and the mental component of health-related QoL improved slightly with increasing attendance to the walking program. In a subgroup that attended at least 75% of the sessions, a beneficial effect of the walking program was observed on positive affect and self esteem.\nTo our knowledge, this is the first intervention study on QoL in community-dwelling adults with MCI. While memory complaints are reported to be negatively associated with QoL in healthy older adults with subjective memory complaints [2], QoL ratings in our study population were already quite high at baseline. Baseline ratings on the DQOL sumscore and subscales fell ample above the midpoint of the scale, except for negative affect. Baseline scores on the SF-12MCS fell around a half standard deviation above the average in the general population and SF-12PCS fell about the average ratings. QoL-ratings have been reported to decrease as the severity of cognitive decline increases [28]. The possibility exists that MCI as operationalized in the present study may not have been serious enough to negatively influence overall and health-related QoL. In spite of the high baseline values, the QoL scales still allowed for further improvements, i.e., there was no ceiling effect. However, it has been discussed before that QoL may represent a stable concept, which is difficult to change or that existing measures may not be responsive to subtle changes [29].\nThe relationship between physical activity and QoL has been studied extensively. However, it is difficult to draw a clear conclusion, since various definitions and operationalizations of QoL circulate. Moreover, comparisons between studies are being complicated by the wide variety of study populations and features of exercise intentions such as intensity, exercise mode, frequency and session and total duration [30]. However, Rejeski et\u00a0al. [31] concluded in a review including 28 studies, of which 11 RCT\u2019s, that physical activity positively influenced aspects of health-related QoL. In a recent meta-analysis of Netz et\u00a0al. [7] including 36 studies, a small positive effect of exercise was observed on wellbeing in healthy older adults. In that meta-analysis four components of wellbeing were considered, including aspects that were also measured in the FACT-study, such as positive and negative affect, perception of physical fitness and physical symptoms.\nIn the present study no main effects of the WP were observed in the modified intention to treat analyses. First, a possible explanation for the lack of effect may be that only participants with good QoL were able to attend enough sessions. In contrast to an earlier study, no baseline differences in number of chronic diseases, physical health-related QoL and endurance were observed between adherers (attending \u226575% of the sessions) and non-adherers (attending <75% of the sessions) [32]. However, adherers rated their mental health-related QoL at baseline significantly better than non-adherers. The difference was three points, which approximately equaled a difference of 5%. The possibility exists that subjects with lower mental health-related QoL were inclined to attend less sessions. Nevertheless, it is not likely that this biased our results, because non-adherers and drop-outs from the exercise programs were included in the modified intention to treat analyses. In future studies in subjects with cognitive decline, session attendance may be improved by informing subjects extensively about the study aims and the consequences of participation. Moreover, if possible with respect to logistic and financial issues, we advice to schedule time and staff for the close personal follow-up of temporary drop-outs.\nSecond, it has been reported that the association between physical activity and QoL is lower among older adults who function at or above the norm [31]. By applying inclusion criteria for the present trial (e.g., community dwelling, no ADL disabilities, being able to perform moderate intensity physical activity), we presumably selected physically healthy and active subjects. This is supported by the high baseline activity levels. Two-thirds of the participants reported to be physically active at moderate intensity for 30\u00a0min or more per day. Subjects meeting this guideline are reported to have better health-related QoL than physically inactive adults [8]. Additionally, Netz et\u00a0al. found that larger effects of exercise on wellbeing were observed in sedentary adults [7]. However, in the present study, no interaction between the walking program and baseline physical activity level was observed (results not presented), indicating that inactive participants did not benefit more from the WP than active participants. Therefore, it is not likely that baseline physical activity level was a main cause of the lack of main effects.\nFinally, inconclusive evidence is available about the intensity and exercise mode of physical activity required to benefit QoL. Netz et\u00a0al. [7] concluded that aerobic training of moderate intensity was most beneficial for wellbeing. In a cross-sectional study, it was also observed that moderate intensity physical activity was positively related to health-related QoL [8]. In contrast, in a review by Spirduso and Cronin [33] no evidence of a relationship between exercise intensity and the rate of improvement in QoL was found. If the former would be true, the possibility exists that the contrast between both programs in the present study would not have been large enough to induce differences in QoL. If the latter would be true, participants would have benefited from participation in both exercise programs regardless of intensity. Both programs may either have added to better self-efficacy, or may prevented a decline in self-efficacy. The walking program by training cardiovascular endurance; the placebo activity program by training e.g., balance and ADL. Self-efficacy refers to somebody\u2019s belief that one has the capabilities to successfully manage situational demands and is mentioned to be a mediating mechanism for the effect of physical activity on QoL [7, 30, 34, 35]. Thus, the presence of the low intensity placebo activity program in our study may have contributed towards the lack of between group differences.\nNevertheless, several outcomes improved with increasing attendance to the walking program. In the per protocol analyses a beneficial effect was observed on positive affect. Self esteem also tended to improve. However, observed differences were small and approximated 5% differences from baseline QoL ratings. As a rule of thumb, a minimal change of 5% has been mentioned to signify clinical relevance. To obtain a change of 5% by increasing attendance, the required increase in attendance would be 62% for D-QoL-belonging and 94% for D-QoL-positive affect and the SF12-MCS. Therefore, it can be questioned whether the observed effects are clinically relevant.\nNo effect of the FA\/B12\/B6 supplementation was observed except for a negative effect on feelings of belonging. However, no theoretical rationale exists for this effect. Our findings are in line with previous RCT\u2019s on the effect of vitamin B supplementation on aspects of QoL. Deijen et\u00a0al. [11] observed no effect of supplementation with 20\u00a0mg vitamin B6 for 3\u00a0months on mood in healthy men (n\u00a0=\u00a076). Also no effect of supplementation with 750\u00a0\u03bcg folate, 15\u00a0\u03bcg vitamin B12 or 75\u00a0mg vitamin B6 daily for 35\u00a0days was observed on mood in women aged 65 or over (n\u00a0=\u00a075) [11]. Finally, no effect on health-related QoL was observed of a weekly injection with 1\u00a0mg vitamin B12 for 4\u00a0weeks in adults with vitamin B12 deficiency (n\u00a0=\u00a0140) [13]. These findings may find its origin in the used operationalizations and measures of QoL that include very few items that directly relate to nutrition. Amarantos et\u00a0al. [36] underline the need to develop QoL measures including items that relate nutrition to QoL.\nTo conclude, the walking program and vitamin B supplementation were not effective in improving QoL in community-dwelling older adults with MCI within 1\u00a0year. However, increasing attendance to moderate intensity physical activity may benefit certain aspects of QoL.","keyphrases":["quality of life","aged","exercise","randomized controlled trial","dietary supplements"],"prmu":["P","P","P","P","M"]}
{"id":"Neurosci_Lett-2-1-2330064","title":"Contribution of P2Y1 receptors to ADP signalling in mouse spinal cord cultures\n","text":"Mixed neuronal and glial cell spinal cord cultures from neonates express ADP sensitive P2Y1,12&13 receptors. ADP (10 \u03bcM) evoked increases in intracellular calcium that were essentially abolished by the P2Y1 receptor antagonist MRS2179 (10 \u03bcM), responses were also absent in preparations from P2Y1 receptor deficient mice however UTP (100 \u03bcM) evoked calcium rises were unaffected. ADP also evoked a robust increase in extracellular signal-regulated protein kinase (ERK) phosphorylation that was of similar magnitude in the cultures from wild type and P2Y1 receptor deficient mice. These results suggest that ADP acts through P2Y1 receptors to mediate an increase in intracellular calcium but not to stimulate ERK phosphorylation in the spinal cord.\nNucleotides released from neurons, glia and damaged cells play a signalling role in the nervous system. P2Y receptors are a family of G-protein coupled nucleotide receptors, and several of the eight subtypes (P2Y1,2,4,6,11,12,13,14) are expressed in the nervous system [11]. P2Y1 receptors have been shown to be involved in calcium signalling in a range of neurons and glial cells (e.g. [3,4,9,12]). In addition signalling through recombinant P2Y1 receptors stimulates mitogen-activated (MAP) kinases [10] and in native cells P2Y1 receptors have been implicated to play a role in extracellular signal related kinases (ERK) activation and stretch induced injury in astrocytes [8]. In this study the aim was to determine whether ADP evoked calcium and MAP kinase responses in cultured spinal cord neurons and glial cells and use knockout mice to determine the contribution of P2Y1 receptors to ADP evoked signalling.\nWild type or P2Y1 receptor deficient C57 neonatal mice [7] (3\u20137 days) of either sex were decapitated and the spinal column was removed and washed twice in a calcium\/magnesium-free Hanks balanced salt solution (HBSS) containing HEPES (0.3\u00a0M), sodium pyruvate and penicillin\/streptomycin (50\u00a0\u03bcg\/ml each). The spinal cord was then cut into small pieces and placed in ice cold HBSS. The tissue pieces were then placed in a HBSS-trypsin solution (2.5%) and incubated at 37\u00a0\u00b0C for 30\u00a0min. The tissue was then transferred to a HBSS-trypsin inhibitor solution (1\u00a0mg\/ml) for 5\u00a0min before being replaced with 1\u00a0ml Neurobasal media containing glutamine and B27 supplement. Mechanical trituration was used to dissociate the spinal cord cells. The resulting cell suspension was seeded onto poly-d-lysine-coated 16\u00a0mm coverslips and maintained at 37\u00a0\u00b0C in a humidified incubator with a 95% O2\/5% CO2 atmosphere. After 2\u00a0h an additional 2\u00a0ml of Neurobasal media was added and cells left to grow for 2\u20135 days.\nSpinal cord cultures were ester loaded with a calcium sensitive dye, Fluo-3 acetoxymethyl ester (Fluo-3-AM, final concentration 1\u00a0\u03bcM) for 30\u00a0min at 37\u00a0\u00b0C in a humidified incubator. The coverslips were then washed in extracellular solution (in mM; 150 NaCl, 10 HEPES, 10 Glucose, 2.5 KCl, 2.5 CaCl2, 1 MgCl2, pH 7.3) to remove any extracellular dye and maintained at room temperature. Coverslips were mounted in a perfusion chamber on the stage of a Fluoview FV300 confocal microscope and were continuously perfused with extracellular solution. The argon laser excitation of the dye was at 488\u00a0nm and the emissions were captured at wavelengths greater than 510\u00a0nm by Olympus Fluoview v4.2 software at a frequency of 0.5\u00a0Hz for a continuous time period of 280\u00a0s. Agonist application was via a U-tube [2]. ADP stocks were treated with hexokinase to remove any ATP contamination. Agonist applications were at intervals of \u223c10\u00a0min. Antagonists were perfused for 8\u00a0min before test solution application so as to allow complete equilibration. Cells displaying \u2018puff\u2019 artefacts to just extracellular solution were discounted from analysis [1]. Baseline measurements of fluorescence were calculated as a mean of the 10\u00a0s of recording prior to agonist application. Peak changes in fluorescence for neuronal and glial cells were expressed as a ratio of the baseline (increases in fluorescence are expressed as a self-ratio, baseline value is 1).\nSpinal cord cells were cultured for 2\u20135 days as described previously and agonists were applied to individual wells and incubated for 10\u00a0min. The cells were subsequently lysed in a solution (in mM; 10 \u03b2-glycerophosphate, 1 EDTA, 1 EGTA, 50 Tris\u2013HCl, 1 benzamidine, 1 sodium orthovanadate, 0.2 PMSF, 50 sodium fluoride, 1\u00a0mg\/ml pepstatin A, 1\u00a0mg\/ml leupeptin, 0.1% \u03b2-mercaptoethanol, 1% Triton X-100). The samples were then mixed with sodium dodecyl sulphate sample buffer (containining \u03b2-mercaptoethanol) at a ratio of 1:1 and denatured for 5\u00a0min at 95\u00a0\u00b0C prior to loading on a 10% sodium dodecyl sulphate polyacrylamide gel. The protein was then transferred onto a nitrocellulose membrane and subsequently blocked with blocking medium (5% non-fat milk powder and Tris-Tween 20, buffered salts-TTBS) overnight at 4\u00a0\u00b0C. Nitrocellulose membranes were then incubated in 5% blocking medium containing either anti-phospho-ERK antibody (Cell Signaling Technology, MA, USA) at a dilution of 1:1000 or anti-non-phosphorylated ERK antibody (Cell Signaling Technology, MA, USA) at a dilution 1:1000. Immunoreactive bands were visualised by incubation of nitrocellulose membranes with anti rabbit peroxidase conjugate at 1:1500 for 2\u00a0h at room temperature and then detected with chemiluminiscence (ECL Plus, Amersham, UK) and recorded onto hyperfilm (Amersham, UK). Western blots were analysed using Image J, NIH, USA\u2014http:\/\/rsb.info.nih.gov. Phospho ERK intensity was corrected for loading using non-phosphorylated ERK blotting and expressed as fold over basal.\nTotal RNA was isolated from either intact spinal cord or spinal cord using the RNeasy Mini Kit (QIAGEN, Valencia, CA). The RNA was treated with DNase I (Sigma) to remove any DNA contamination. Half of the purified RNA was reverse-transcribed to cDNA using SuperScript II-reverse transcriptase (Invitrogen, Carlsbad, CA), and the remainder of the RNA was used as a negative control to ensure that there was no genomic contamination. The primers have been described previously [1]. PCR reactions were carried out with 2\u00a0\u03bcl of cDNA and a final concentration of 500\u00a0nM of each primer. Amplification took place in a Techne Genius PCR machine (Techne, Cambridge, UK) using the following protocol: denaturation at 94\u00a0\u00b0C for 5\u00a0min; repeated cycles of denaturation at 94\u00a0\u00b0C, followed by annealing at 57\u00a0\u00b0C and extension at 72\u00a0\u00b0C, each for 30\u00a0s; final extension at 72\u00a0\u00b0C for 10\u00a0min. Thirty-five cycles were used. The amplification products were examined by electrophoresis on a 1% agarose gel and visualized using ethidium bromide under UV light.\nStatistical tests were preformed using an unpaired t-test with a p-value of <0.05 considered as significant. Data are expressed as mean\u00a0\u00b1\u00a0standard error of the mean.\nSpinal cord cells cultured from neonates for 3\u20137 days contained both neuronal and glial cells that could be distinguished by their morphology and staining with glial fibrillary acidic protein; approx 43% of cells were glia and 57% neuronal (n\u00a0=\u00a0576 cells from 4 coverslips). We have previously used calcium imaging to look at P2Y receptor signalling in mouse superior cervical ganglia [1] and similar protocols were used in this study. ADP (10\u00a0\u03bcM) evoked robust increases in intracellular calcium for both neurons (64% cells responding with peak self-ratio amplitude of 2.20\u00a0\u00b1\u00a00.14, n\u00a0=\u00a0120 cells from 12 coverslips) and glial cells (57% of cells responded with a peak self-ratio amplitude of 2.17\u00a0\u00b1\u00a00.2, n\u00a0=\u00a0120 cells from 12 coverslips). Responses were not sustained and generally decayed during the time-course of application (self-ratio at end of 30\u00a0s application 1.34\u00a0\u00b1\u00a00.08 and 1.27\u00a0\u00b1\u00a00.04 for neurons and glia, respectively, n\u00a0=\u00a020 for each).\nThe selective P2Y1 receptor antagonist MRS2179 reduced ADP evoked calcium rises in a concentration-dependent manner and these effects were reversed on antagonist washout (Fig. 1). ADP evoked calcium rises were essentially abolished in spinal cord cultures from P2Y1 receptor deficient mice with small residual responses in 11% of neurons (self-ratio 1.14\u00a0\u00b1\u00a00.05, n\u00a0=\u00a0100 cells from 10 coverslips) and 17% of glial cells (self-ratio 1.21\u00a0\u00b1\u00a00.05, n\u00a0=\u00a0115 cells from 11 coverslips)(Fig. 2). Responses to the P2Y2\/4 receptor agonist UTP (100\u00a0\u03bcM) in cultures from P2Y1 receptor deficient mice were the same as for WT for neurons (45 and 48% responding with amplitudes of 1.96\u00a0\u00b1\u00a00.09 and 1.79\u00a0\u00b1\u00a00.08, n\u00a0=\u00a060, 114 from 6 and 12 coverslips, respectively for WT and P2Y1 receptor deficient mice) and glial cells (58 and 62% responding with amplitudes of 1.88\u00a0\u00b1\u00a00.13 and 1.67\u00a0\u00b1\u00a00.04, n\u00a0=\u00a060, 119 from 6 to 12 coverslips, respectively for WT and P2Y1 receptor deficient mice) indicating that G\u03b1q coupled receptor calcium signalling was not affected in the P2Y1 receptor deficient mouse (Fig. 2). Taken together these results define a role of the P2Y1 receptor in calcium signalling in spinal cord neurons and glial cells. These studies support the role of P2Y1 receptors in calcium signalling in the nervous system [3,9,12] and corroborate previous studies using sympathetic ganglion cultures from the P2Y1 receptor deficient mouse [1].\nP2Y1 receptor stimulation has also been reported to mediate ERK phosphorylation in recombinant [10] and native systems [8] and this may contribute to changes in the spinal cord following injury. In spinal cord cultures ADP evoked a time-dependent phosphorylation of p44\/42 ERK that was detected at 5\u00a0min, peaked at 10\u00a0min, and declined back to basal levels by 30\u00a0min (Fig. 3A). When just media was added to the cultures there was no change in ERK phosphorylation (Fig. 3B) indicating that ERK stimulation does not result from a mechanical artefact. Adenosine (10\u00a0\u03bcM) also had no effect demonstrating that adenosine receptors are not involved. We therefore compared ADP evoked ERK phosphorylation in spinal cord cultures from WT and P2Y1 receptor deficient mice. Surprisingly ADP (10\u00a0\u03bcM) evoked similar levels of ERK phosphorylation (\u223c3-fold increase over basal) in WT and P2Y1 receptor deficient cultures (Fig. 3) demonstrating that a receptor other than the P2Y1 receptor mediates this ADP response.\nOf the currently eight identified P2Y receptor genes ADP is an agonist at G\u03b1q coupled P2Y1, and G\u03b1i coupled P2Y12 and P2Y13 receptors [11]. ADP is also an agonist at the human P2Y11 receptor; however a corresponding mouse homologue of the P2Y11 receptor has not been detected suggesting that mice do not express P2Y11-like receptors [11]. Transcripts for P2Y1,12&13receptors were detected in 3\u20137 days old spinal cord cultures (data not shown). Amplification of RNA for ADP sensitive P2Y12&13 receptors raised the possibility that these may contribute to the ERK phosphorylation. AR-C66931MX [6] is an antagonist of P2Y12 and P2Y13 receptors with a reported pA2 of 8.7 at P2Y12 receptors and 0.01\u00a0\u03bcM reduced ADP responses by \u223c80% at recombinant P2Y13 receptors (see review [11]). AR-C66931MX (10\u00a0\u03bcM a supra-maximal concentration at P2Y12 receptors) had no effect on ADP evoked ERK phosphorylation indicating that P2Y12 receptors did not mediate the ERK phosphorylation. AR-C66931MX may have complex actions at P2Y13 receptors, possibly dependent on cell type, and has been described as an activator of P2Y13 receptor dependent high density lipoprotein endocytosis in hepatocytes [5] questioning the use of this compound as a selective antagonist at the P2Y13 receptor. Thus it remains to be determined whether the P2Y13 receptor, or a novel ADP sensitive receptor, mediates the ADP evoked ERK phosphorylation in the spinal cord.","keyphrases":["p2y1","adp","spinal cord","neurons","glia","calcium","phosphorylation","p2y12","p2y13"],"prmu":["P","P","P","P","P","P","P","P","P"]}
{"id":"Ann_Biomed_Eng-2-2-1705493","title":"Dependence of Intramyocardial Pressure and Coronary Flow on Ventricular Loading and Contractility: A Model Study\n","text":"The phasic coronary arterial inflow during the normal cardiac cycle has been explained with simple (waterfall, intramyocardial pump) models, emphasizing the role of ventricular pressure. To explain changes in isovolumic and low afterload beats, these models were extended with the effect of three-dimensional wall stress, nonlinear characteristics of the coronary bed, and extravascular fluid exchange. With the associated increase in the number of model parameters, a detailed parameter sensitivity analysis has become difficult. Therefore we investigated the primary relations between ventricular pressure and volume, wall stress, intramyocardial pressure and coronary blood flow, with a mathematical model with a limited number of parameters. The model replicates several experimental observations: the phasic character of coronary inflow is virtually independent of maximum ventricular pressure, the amplitude of the coronary flow signal varies about proportionally with cardiac contractility, and intramyocardial pressure in the ventricular wall may exceed ventricular pressure. A parameter sensitivity analysis shows that the normalized amplitude of coronary inflow is mainly determined by contractility, reflected in ventricular pressure and, at low ventricular volumes, radial wall stress. Normalized flow amplitude is less sensitive to myocardial coronary compliance and resistance, and to the relation between active fiber stress, time, and sarcomere shortening velocity.\nIntroduction\nCoronary arterial inflow varies in time during the cardiac cycle. Systolic inflow is smaller than diastolic inflow, demonstrating that the pulsatility of coronary flow is not caused by variation of the arterial venous pressure difference. Instead, the phasic pattern of coronary inflow has been attributed to changes in cross-sectional area of the myocardial vessels. For example, a transient decrease in cross-sectional area affects coronary flow in two ways. First, coronary inflow decreases and outflow increases because blood is squeezed out of the coronary bed. Second, both coronary inflow and outflow decrease because of the increase of coronary resistance.\nVessel cross-sectional area depends on the local coronary pressure, the vessel wall mechanical properties and the embedment of the vessel in the myocardial tissue. The relation between vessel cross-sectional area and transmural pressure is nonlinear,11 and changes due to autoregulation. The embedment in the myocardial tissue can be represented by myocardial wall stress, which consists of two contributions: the stress in the collagen matrix, through which the vessel is tied to the surrounding tissue, and the intramyocardial pressure in the interstitial fluid. In the end, the pattern of coronary flow is mainly determined by contraction of the myofibers in the cardiac wall, since myofiber contraction determines the level of both myocardial wall stress and coronary pressure.\nMany experiments have been performed to elucidate the interplay between the factors governing coronary flow. It has been observed that the pulsatile component of the coronary inflow signal (1) is about proportionally related to left ventricle (LV) contractility, flow amplitude being about zero when contractility is about zero,16 (2) is virtually independent of systolic LV pressure in isovolumic beats, executed at various LV volumes, for pressures up to about 13\u00a0kPa,15,17 and (3) is about the same in isobaric beats at low LV pressure as in isovolumic beats at LV pressures up to about 13\u00a0kPa.15 It was observed also that minimum systolic flow (4) is virtually independent of LV pressure for pressures below 13\u00a0kPa, but (5) decreases with increasing LV pressure for pressures above about 13\u00a0kPa.20 In addition, it has been found that (6) systolic intramyocardial pressure may exceed left ventricular pressure in subendocardial layers in low afterload beats,18 and (7) that an increase of coronary perfusion pressure leads to an increase of intramyocardial pressure.18\nMathematical models have been proposed as a tool to interpret the experimental data. In early models, the interaction between the coronary vessel and the myocardium was modeled through the intramyocardial pressure only. In the waterfall model9 and the intramyocardial pump model,1,2,6,22 this pressure was assumed to be determined completely by left ventricular pressure, with intramyocardial pressure decreasing linearly from left ventricular pressure at the endocardial surface to zero at the epicardial surface. These models can explain the pulsatility of coronary inflow under normal physiological conditions, and the experimental observations (1) and (5), listed above.\nIn later models, the interaction between vessel and tissue was still modeled through intramyocardial pressure only, but intramyocardial pressure was assumed to depend both on left ventricular pressure and on transverse tissue stress, i.e. stress perpendicular to the muscle fiber direction. In a finite element model of the beating heart, incorporating transverse tissue stress, observation (6) was replicated.12 Transverse stress is also included in models by Beyar et\u00a0al., together with nonlinear characteristics of the properties of the coronary bed, and exchange of fluid between the coronary vessels and the myocardial interstitium.5,29,30 In these models experimental observations (1) through (6) are replicated.\nVis et\u00a0al.26,27 extended the model for interaction between the vessel and the cardiac wall, by assuming that the coronary vessel is subject to an extravascular pressure that depends both on intramyocardial pressure and local tissue stress. So the effective compliance of the coronary vessel depends both on the compliance of the vessel wall and the stiffness of the myocardium, thus implementing an idea introduced before as the time-varying elastance concept.15,17 In a finite element model of one vessel in the myocardial wall Vis et\u00a0al. investigated the influence of contractility, pressure, and circumferential wall stretch on vessel area, for static diastole and systole. With the model, observations (6) and (7) were reproduced.\nWith the increasing complexity of the models, more experimental observations have been replicated. Simultaneously, the number of model parameters has increased, making it difficult to perform a detailed parameter sensitivity analysis and identify the critical model parameters. Therefore, our aim was to study the primary relations between left ventricular pressure and volume, wall stress in fiber and transverse direction, intramyocardial pressure and the coronary blood flow, with a mathematical model with a limited number of parameters, and to assess the sensitivity of the model results to the model parameter settings.\nMaterial and methods\nThe complete model consists of four parts, describing ventricular wall mechanics, myocardial constitutive properties, intramyocardial pressure, and the coronary and systemic circulation.Figure\u00a01.Passive material behavior. Top: passive stress as a function of stretch ratio \u03bb for uniaxial stretch along fiber direction (\u03c3m,f) and radial direction (\u03c3m,r). Bottom: passive left ventricular pressure\u2013volume relation according to model (solid line) and experimental data;19 experimental data given for average minimum and maximum volume (dashed line), and minimum and maximum volume\u00a0\u00b1\u00a01\u00a0SD (dash-dot lines).\nVentricular Wall Mechanics\nThe model of ventricular wall mechanics describes how left ventricular pressure and volume are related to local tissue properties, i.e. fiber stress and strain, and radial wall stress and strain. The model is based on a previously published model,3 which is extended to describe the influence of radial wall stress. While the original model is derived for arbitrary ventricular geometries with rotational symmetry, here we will derive the equations for the special case of a thick walled sphere. We consider the sphere to consist of a set of nested thin spherical shells. In each shell, stresses satisfy the condition of force equilibrium in radial direction:\nwhere \u03c3r, \u03c3c, and \u03c3l denote the radial, circumferential and longitudinal component of the tissue stress tensor, respectively, and r indicates the radial position in the wall. We neglected shear stress components in view of the low shear stiffness of the tissue. The myocardial tissue was assumed to be incompressible, and to consist of myofibers, embedded in a collagen matrix. The Cauchy stress tensor\nin tissue is written as:\nwhere pim denotes the intramyocardial pressure,\n, the stress in the collagen matrix, and \u03c3a the stress generated in the myofibers along the myofiber direction\nWe assume that the fibers are located in spherical shells at an angle \u03b1 with the circumferential direction, and adopt the assumption by Arts,3 that the stress in the collagen matrix in the plane of a shell is completely determined by the passive stress \u03c3m,f in the fibers. Then it holds:\nwhere \u03c3m,r represents the radial wall stress, generated in the collagen matrix, and the total fiber stress \u03c3f is introduced as the sum of passive and active fiber stress. Substitution in (1) yields:\nThis equation describes how, in each shell, fiber stress \u03c3f and radial wall stress in the collagen matrix \u03c3m,r contribute to the variation of total radial wall stress \u03c3r in radial direction. Together, all shells increase radial wall stress from zero stress at the outer surface (r\u00a0=\u00a0ro) to minus left ventricular pressure at the inner surface (r\u00a0=\u00a0ri):\nThe relation between wall stress and left ventricular pressure is found by integrating Eq. (4) from the endocardial to the epicardial surface. To evaluate the integral, we first adopt the assumption by Arts3 that \u03c3f is constant across the wall. Secondly, we assume that a representative position\ncan be found, such that:\nEvaluation of the integral and substitution of the boundary conditions (5) yields:\nwhere ri and ro are rewritten in terms of the cavity volume Vlv and wall volume Vw, and\nis introduced as a short notation of\nApart from the term\nthis relation is identical to that derived in Arts et al.3 To complete the model relating wall mechanics to cavity mechanics, Eq.\u00a0(7) is complemented by a relation between ventricular volume and tissue strain. We choose the passive ventricle at zero transmural pressure as a reference state. In this state, we assume a sarcomere length ls0 and a cavity volume Vlv0. It has been shown,3 that the fiber stretch ratio \u03bbf can be approximated by:\nThis ratio corresponds to the circumferential stretch ratio at the outer surface of a shell that contains the left ventricular cavity volume and one third of the wall volume. At the same location, the radial stretch ratio \u03bbr equals:\nwhere we assumed incompressibility of the myocardial tissue. Equations (7)\u2013(9) describe how global ventricular properties Vlv and plv are related to local tissue properties \u03bbf, \u03bbr, \u03c3f and\nMyocardial Constitutive Properties\nThe model of ventricular mechanics is completed with constitutive laws for fiber stress and radial stress. The active fiber stress \u03c3a was modeled to depend on contractility c, sarcomere length ls, time elapsed since activation ta and sarcomere shortening velocity vs as:\nwith contractility c (0 \u2264 c \u2264 1), scaling constant \u03c3ar, and:\nHere, ls,a0 denotes the sarcomere length below which active stress becomes zero and ls,ar represents the sarcomere length to which the reference stress \u03c3ar is referred to. Times ta and tmax denote the time elapsed since activation, and the duration of the twitch, respectively. Velocity v0 represents the unloaded sarcomere shortening velocity, while cv governs the shape of the stress\u2013velocity relation. Parameter values in the active stress law (Table\u00a01) are chosen in agreement with experimental data.8,13Table\u00a01.Reference settings for parameters in the model of the left ventricle.ParameterValueUnitParameterValueUnitVw20010\u22126\u00a0m3\u03c3ar55103\u00a0PaVlv,06010\u22126\u00a0m3c1\u2013ls,01.910\u22126\u00a0mls,a01.510\u22126\u00a0m\u03c3f00.9103\u00a0Pals,ar2.010\u22126\u00a0m\u03c3c00.2103\u00a0Patmax40010\u22123\u00a0scf12\u2013v01010\u22126\u00a0m\u00a0s\u22121cr9\u2013cv0\u2013\nThe passive stress along the fiber direction is modeled as:\nwhere it was assumed that no stress can be transmitted in compression. Passive transverse stress is modeled similarly. Since the radial direction at ventricular level coincides with the transverse direction at the tissue level, we write the transverse stress model in terms of radial stress and stretch ratio:\nSettings of parameter values \u03c3f0, cf, \u03c3r0 and cr are based on experimental data on the pressure volume relation of the passive left ventricle.19 In the latter study, the pressure volume relation was described by a logarithmic relation, involving chamber stiffness at positive and negative pressures, and maximum and minimum attainable volume. The bottom panel of Fig.\u00a01 shows the experimental data, and our model prediction for parameter settings in Table\u00a01. Uniaxial stress\u2013strain responses are shown in the top panel of Fig.\u00a01.Figure\u00a02.Schematic representation of the left ventricle (LV) in the systemic circulation and coronary circulation; Rart, Rper and Rven represent the systemic arterial, peripheral and venous resistance, respectively; MV and AV represent the mitral and aortic valve, respectively; Cart and Cven the systemic arterial and venous compliance; Lven and Lart the inertia of the blood in the venous and arterial system; Rart,c, Rmyo,1, Rmyo,2 and Rven,c represent the coronary arterial, the two intramyocardial and venous resistances; Cart,c, Cmyo,c and Cven,c the coronary arterial, intramyocardial and venous compliance; part,c, pmyo,c and pven,c the pressure in the coronary arteries, myocardium and veins, respectively; pao and pla the aortic and left atrial pressure, respectively; qao, qm represent the AV flow and MV flow; qart,c and qven,c the coronary arterial inflow and venous outflow;\nrepresents the intramyocardial pressure at the representative radial position\nWhen simulating isovolumic and isobaric contractions, the pressure drop across the coronary circulation is switched from pao\u2212\u00a0pla to a constant perfusion pressure pper.Figure\u00a03.Hemodynamics in the beating heart. Left: simulation of the physiological state with aortic pressure driving the coronary circulation. Right: simulation of isolated heart experiment with a constant perfusion pressure and maximum vasodilation. From top to bottom: ventricular pressure plv, intramyocardial pressure\n(bold) and radial wall stress\naortic valve (AV) flow qao (bold) and mitral valve (MV) flow qm; left ventricular cavity volume Vlv; coronary perfusion pressure pper, myocardial coronary pressure pmyo,c (bold) and left atrial pressure pla; coronary inflow\n(bold) and outflow\nper 100\u00a0g wall volume; volume of myocardial coronary bed Vmyo,c.Figure\u00a04.Hemodynamics at maximal vasodilatation and constant perfusion pressure, expressed in time courses of ventricular pressure plv, ventricular volume Vlv, radial wall stress\nintramyocardial pressure\nand coronary arterial inflow\nLeft: isovolumic beats for ventricular volumes ranging from 120 to 20\u00a0ml. Middle: isovolumic beats at 60\u00a0ml for contractility parameter c ranging from 1.0 to 0.1. Left: isobaric beats for systolic pressures ranging from 16 to 2\u00a0kPa.\nIntramyocardial Pressure\nTo derive the expression for the intramyocardial pressure, again we consider the ventricular wall to be composed of a number of nested shells. The pressure in between two shells is assumed to be a fraction \u03b2 of the left ventricular pressure. In analogy to Eq.\u00a0(5), this pressure is in equilibrium with radial tissue stress \u03c3r:\nIn the model, we assume a linear variation of \u03b2 with the transmural position in the wall, with \u03b2\u00a0=\u00a01 at the endocardial surface, and \u03b2\u00a0=\u00a00 at the epicardial surface. Again stress at the shell containing the LV cavity volume and one third of LV wall volume is considered representative. For this location at a radius\nin the wall we find:\nwhere we introduced the notation\nand the radial positions ri, ro and\nare defined as:\nEquation (17) describes intramyocardial pressure as a function of LV pressure and volume: the volume dependency enters the equation through the radial stress \u03c3m,r [Eq.\u00a0(15)], which depends the radial stretch ratio \u03bbr [Eq.\u00a0(9)], which in turn depends on LV volume [Eq. (8)].\nSystemic and Coronary Circulation\nThe model of left ventricular wall mechanics is incorporated in lumped parameter models for the coronary and systemic circulation (Fig.\u00a02). The aortic (AV) and mitral valve (MV) are modeled as an ideal diode. Vessels are modeled with constant resistances R, inertances L and capacitances C. The pressure drop \u0394p across each of these components is given by\nwith V the volume in the capacitance and q the flow through a resistance or an inertance. The pressure\u2013volume relation of the capacitance represents a linearization around the physiologic working point, V0 representing the volume at zero pressure. Values of parameters in the circulation model were based on literature (Table\u00a02).Table\u00a02.Reference settings for parameters in the circulation model; coronary resistance values in parentheses represent maximum vasodilation.Systemic circulationCoronary circulationParameterValueUnitParameterValueUnitRart5106\u00a0Pa\u00a0s\u00a0m\u22123Rart,c700 (200)106\u00a0Pa\u00a0s\u00a0m\u22123Rper120106\u00a0Pa\u00a0s\u00a0m\u22123Rmyo,1900 (100)106\u00a0Pa\u00a0s\u00a0m\u22123Rven5106\u00a0Pa\u00a0s\u00a0m\u22123Rmyo,2900 (100)106\u00a0Pa\u00a0s\u00a0m\u22123Cart2010\u22129\u00a0m3\u00a0Pa\u22121Rven,c200106\u00a0Pa\u00a0s\u00a0m\u22123Cven80010\u22129\u00a0m3\u00a0Pa\u22121Cart,c0.0310\u22129\u00a0m3\u00a0Pa\u22121Vart,050010\u22126\u00a0m3Cmyo,c1.410\u22129\u00a0m3\u00a0Pa\u22121Vven,0300010\u22126\u00a0m3Cven,c0.710\u22129\u00a0m3\u00a0Pa\u22121Lart60103\u00a0Pa\u00a0s\u00a0m\u22123Vart,c0610\u22126\u00a0m3Lven60103\u00a0Pa\u00a0s\u00a0m\u22123Vmyo,c0710\u22126\u00a0m3Vblood500010\u22126\u00a0m3Vven,c01010\u22126\u00a0m3\nThe connection between the model of LV mechanics and the coronary circulation model is made through the intramyocardial pressure, that acts on the myocardial capacitance Cmyo,c. The values of the coronary capacitances were based on measurements by Spaan et\u00a0al.:23 0.0022, 0.091 and 0.045\u00a0ml\u00a0mm\u00a0Hg\u22121\u00a0100\u00a0g\u22121\u00a0LV in large coronary arteries, myocardial coronary bed, and coronary veins, respectively. Zero pressure volumes were chosen such that under normal physiological conditions, time-averaged coronary volume was about 15\u00a0ml\u00a0100\u00a0g\u22121 of LV tissue, distributed over arterial, myocardial and venous vessels in a ratio of 1:2:2.23\nIn the coronary circulation, resistance values during normoxia and hyperemia were derived from Chilian et\u00a0al.7 In that study, total coronary resistance under normal and vasodilated conditions was measured to be 66 and 14\u00a0mm\u00a0Hg\u00a0min\u00a0g\u00a0ml\u22121, respectively. Distribution of resistance over the arterial, myocardial and venous compartment was measured to be 25, 68 and 7% under normal conditions, and 42, 27 and 31% under maximal vasodilation.\nSystemic parameters are chosen to yield representative function curves for a 70\u00a0kg adult, at a heart rate of 75\u00a0bpm. LV wall volume was set to 200\u00a0ml, and cavity volume at zero pressure was set to 30% of this volume. The arterial load was modeled by a three-element windkessel model, consisting of a characteristic aortic impedance Rart, an arterial compliance Cart, and a peripheral resistance Rper. The peripheral resistance was chosen to yield realistic time-averaged values of aortic pressure and aortic flow. Next, arterial capacitance was chosen to yield realistic values of minimum and maximum aortic pressure. Total blood volume was set to 5000\u00a0ml. The blood volume at which mean systemic pressure is zero was assumed to be equal 70% of total blood volume, about 85% of which is contained in the venous system. Venous capacitance was chosen such that the additional 30% of blood volume leads a mean systemic pressure of about 2\u00a0kPa.\nSimulations Performed\nWith the model, a number of simulations were performed. First we considered the normal physiological situation, with normal vessel tone and coronary flow driven by the difference between aortic and left atrial pressure. Next we investigated the changes induced in the isolated heart setup used by Krams et\u00a0al.,16,17 by the combination of maximum vasodilation, constant perfusion pressure and zero coronary outflow pressure. Then, under these conditions we simulated isovolumic and isobaric beats, for various settings of LV volume, pressure and contractility. Following Krams et\u00a0al.16,17 and Pagliaro et\u00a0al.,20 results were analyzed in terms of minimal systolic coronary flow\nand the normalized coronary flow amplitude (NFA):\nFinally, we investigated sensitivity of NFA to changes in myocardial radial stiffness, active material properties, coronary myocardial resistance, and coronary capacitance.Figure\u00a05.Analysis of coronary inflow data from simulations in Fig.\u00a04, expressed in normalized arterial coronary flow amplitude (NFA), defined in Eq.\u00a0(22), and minimal coronary arterial inflow\nLeft: NFA (top) and\n(bottom) in isovolumic beats as a function of maximum left ventricular pressure for contractility parameter c varying from 0.1 to 1.0; in top panel experimental data15 are given by the dashed line (reference), dash-dotted line (high contractility) and dotted line (low contractility). Middle: NFA and\nin isovolumic beats as a function of contractility parameter c for LV volumes of 40, 60, and 100\u00a0ml. Right: NFA and\nin isobaric beats as a function of maximum ventricular pressure for contractility parameter c ranging from 0.6 to 1.0.\nResults\nThe Normal Beating Heart\nTo illustrate the behavior of the model, first the normal physiological situation was simulated, with normal vessel tone and coronary flow driven by the difference between aortic and left atrial pressure. Ventricular and intramyocardial pressure rise to 16.1 and 9.1\u00a0kPa, respectively (Fig.\u00a03, left panel). Intramyocardial pressure is almost completely determined by ventricular pressure, since radial wall stress is virtually zero. Maximum aortic and mitral flow are 646 and 307\u00a0ml\/s, respectively. Stroke volume is 75.6\u00a0ml at an ejection fraction of 64%. Perfusion pressure equals aortic pressure and varies between 11.4 and 16.3\u00a0kPa. Myocardial coronary pressure varies between 3.0 and 12.3\u00a0kPa. Left atrial pressure remains about constant at 1.45\u00a0kPa. Mean coronary arterial inflow and venous outflow are 2.2\u00a0ml\u00a0s\u22121\u00a0100\u00a0g\u22121\u00a0LV wall volume. Coronary arterial inflow displays a two peaked pattern, the peaks occurring in early filling and in ejection. Diastolic inflow is larger than systolic inflow. Coronary arterial outflow shows only one peak per cycle, occurring during ejection. Myocardial coronary volume varies between 10.7 and 12.5\u00a0ml.\nConstant Perfusion Pressure and Maximal Vasodilation\nThis simulation represents the experimental situation in the isolated heart with a constant perfusion pressure of 10\u00a0kPa and maximal vasodilation. Myocardial coronary pressure varies between 1.5 and 10.0\u00a0kPa (Fig.\u00a03, right panel). Arterial inflow varies between \u22120.1 and 14.2\u00a0ml\u00a0s\u22121\u00a0100\u00a0g\u22121\u00a0LV wall volume, with a mean of 8.3\u00a0ml\u00a0s\u22121\u00a0100\u00a0g\u22121 LV wall volume. In the arterial inflow curve, the positive flow peak in early systole has disappeared. Myocardial coronary volume varies between 5.8 and 12.4\u00a0ml.\nThe Isovolumic Beating Heart\nThe isovolumic experiments by Krams et\u00a0al.15\u201317 were simulated at a constant perfusion pressure of 10\u00a0kPa and maximal vasodilation. Maximum LV pressure decreases with decreasing LV volume (Fig.\u00a04, left panel). As LV volume decreases below 60\u00a0ml, the volume at which pressure in the passive LV is zero, diastolic LV pressure becomes negative. At these volumes, radial wall stress becomes positive, while being constant during a beat. With decreasing volume, maximum intramyocardial pressure and coronary arterial inflow decrease, whereas minimum coronary arterial inflow increases.\nThe normalized coronary flow amplitude (NFA) [Eq. (22)] decreases with decreasing LV pressure (Fig.\u00a05, top left panel), but pulsatility persists at zero ventricular pressure. NFA decreases with decreasing contractility as well. Minimum coronary arterial inflow increases with decreasing LV pressure (Fig.\u00a05, bottom left panel). At low pressures, the slope of the pressure\u2013flow relation decreases.\nFor a constant LV volume of 60\u00a0ml, developed LV pressure decreases with decreasing contractility (Fig.\u00a04, middle panel). At this volume of 60\u00a0ml, radial wall stress is zero. Maximum intramyocardial pressure decreases with maximum LV pressure. Maximum coronary arterial inflow decreases slightly, whereas minimum coronary arterial inflow increases strongly. With decreasing contractility, normalized coronary flow amplitude (NFA) decreases about linearly, while minimal arterial inflow increases linearly (Fig.\u00a05, middle panel). For other volumes, a similar behavior is found.\nThe Isobaric Beating Heart\nSimulations of hemodynamics in the isobaric beating heart were performed at a filling pressure of 1\u00a0kPa, a constant perfusion pressure of 10\u00a0kPa, maximal vasodilation and contractility parameter c\u00a0=\u00a01. With decreasing LV pressure during ejection, minimal LV volume decreases and radial wall stress increases (Fig.\u00a04, right panel). Reduction of maximum LV pressure from 16 to 12\u00a0kPa is accompanied by a reduction of maximum intramyocardial pressure to 7.6\u00a0kPa. Maximum arterial inflow decreases, whereas minimum arterial inflow increases. Further reduction of LV pressure below 12\u00a0kPa hardly affects maximum intramyocardial pressure, due to the increasing contribution of radial wall stress. Maximum and minimum arterial inflow remain about constant. These changes are reflected in the right panel of Fig.\u00a05. With decreasing left ventricular pressure, initially NFA decreases and minimum arterial inflow increases. Below a threshold pressure of about 10\u00a0kPa, these quantities become about constant. A similar behavior is found for lower contractilities.\nSensitivity Analysis\nResults of the sensitivity analysis are shown in Fig.\u00a06. Sensitivity to settings of parameters in the model for radial wall stress was investigated, since in our model radial wall stress is an important determinant of intramyocardial pressure. If the radial stress parameter cr0 [Eq. (15)] is decreased or increased by a factor 2, the pulsatile character of the coronary inflow at lower pressures persists, both for isovolumic and isobaric beats (Fig.\u00a06, top panel). In isovolumic beats, maximum LV pressure increases with decreasing cr0. At the lowest LV volume simulated, 15\u00a0ml, maximum left ventricular pressure does not drop below 14\u00a0kPa, when cr0 is set to zero. In isobaric beats, at high pressures NFA is not affected by changes in cr0. Below an ejection pressure of 10\u00a0kPa, NFA increases with increasing radial stiffness. When cr0 is set to zero NFA decreases proportionally with left ventricular pressure, until pulsatility is lost at zero left ventricular pressure.\nSensitivity to settings of parameters in active stress model was investigated, since myofiber contraction is the origin of both left ventricular pressure and radial wall stress [Eq. (7)], and thus of intramyocardial pressure and of coronary flow impediment. In both isovolumic and isobaric beats, NFA increases slightly if the twitch duration tmax [Eq. (12)] is reduced from 400 to 300\u00a0ms (Fig.\u00a06, 2nd panel from top). Changing the linear relation between fiber stress and sarcomere shortening velocity into a hyperbolic one (by setting cv\u00a0=\u00a01 in Eq. (13) does not affect NFA in isovolumic beats, where shortening velocity is zero. NFA in isobaric beats is hardly affected. Increasing the sarcomere length ls,a0, below which active stress is zero [Eq. (11)], from 1.5 to 1.6\u00a0\u03bcm yields a reduction in NFA in isovolumic and isobaric beats with a ventricular pressure below about 10\u00a0kPa (Fig.\u00a06).Figure\u00a06.Sensitivity of relation between maximum left ventricular pressure and normalized flow amplitude (NFA) to settings of model parameters, for isovolumic beats (left) and isobaric beats (right). Results for reference parameter settings are indicated with \u2018ref\u2019. Top: radial stiffness parameter cr0 set to twice (\u2018high\u2019) or half (\u2018low\u2019) the reference value, or set to zero (\u2018zero\u2019). Next: variation of active stress model: twitch duration reduced from 400 to 300\u00a0ms (\u2018tim\u2019), stress\u2013velocity relation changed from linear into hyperbolic (\u2018vel\u2019), and stress-free sarcomere length increased from 1.5 to 1.6\u00a0\u03bcm (\u2018len\u2019). Next: all coronary resistances Rart,c, Rmyo,1, Rmyo,2 and Rven,c set to twice (\u2018high\u2019) or half (\u2018low\u2019) their reference values. Bottom: coronary myocardial capacitance Cmyo,c set to twice (\u2018high\u2019) or half (\u2018low\u2019) the reference value.\nSensitivity to coronary resistance was studied to mimic change in resistance due to variation in perfusion pressure. A two-fold simultaneous increase or decrease of all resistances in the coronary circulation model (Rart,c, Rmyo,1, Rmyo,2 and Rven,c) does affect NFA more in isovolumic beats than in isobaric beats (Fig.\u00a06, 3rd panel from top). Still, changes in NFA are limited, and pulsatility at low pressures is maintained.\nFinally, sensitivity to myocardial compliance Cmyo,c was studied, since pulsatility of coronary inflow is closely related to changes in coronary volume. When myocardial compliance Cmyo,c is set to twice or half its reference value, NFA-values change quantitatively, but the phasic nature of coronary flow at low left ventricular pressure remains, both in isovolumic and isobaric beats (Fig.\u00a06, bottom panel). Again, the effect is larger in isovolumic beats than in isobaric beats.\nDiscussion\nModel Setup\nThe aim of this study was to design a model with a limited number of parameters for investigation of the primary relations between left ventricular pressure and volume, wall stress in fiber and transverse direction, intramyocardial pressure and the coronary blood flow. Central to the model are Eq. (7), which shows how fiber stress, that ultimately drives the cardiac cycle, is converted into both LV pressure and radial wall stress, and Eq. (17), that describes how LV pressure and radial wall stress contribute to intramyocardial pressure. Finally, it is the variation of intramyocardial pressure that causes a variation in arterial coronary inflow through a change in coronary volume, represented by intramyocardial compliance. Since the use of a simple model is not without danger, we will address the impact of the main model simplifications.\nIn the model of ventricular wall mechanics spatial homogeneity of fiber stress and strain was assumed. The plausibility of this assumption has been illustrated in finite element models of ventricular mechanics.21,24 The assumption that in plane stress is dominated by fiber stress [Eq. (3)] is realistic during systole, when myofibers are active and the main flow impediment occurs. Obviously, radial wall stress is spatially inhomogeneous, since the ventricle is thick walled. Yet we introduced a representative stress\ncomputed at a representative radial position enclosing one third of the wall volume, in evaluating the integral of \u03c3m,r over the wall thickness [Eq. (6)]. In principle, since the transmural variation of radial strain is known once LV cavity volume is known, using the constitutive Eq. (15), the integral in Eq. (6) can be determined exactly. Due to the strong increase of radial wall strain from the outside to the inside surface and the nonlinearity of the constitutive law, its value would be dominated by the inner layers. However, in those layers the assumption of a solid wall with a smooth endocardial surface is in error: the endocardial surface shows many invaginations, relieving any radial stress that would build up in the subendocardial tissue. The choice to use representative positions, strains and stresses is compatible with the aim of the study, to investigate determinants of the coronary flow signal. Obviously the model cannot describe spatial differences in coronary perfusion,4 which are clinically relevant in relation to vulnerability to ischemia.10 In view of the use of a representative intramyocardial pressure, and the use of average values for the coronary compliances and resistances, the flow in the model should be regarded as a mean flow over the myocardial wall.\nIn the constitutive model for the cardiac tissue, the description of the active stress component is chosen as simple as possible, while maintaining the basic dependence of active stress on time, sarcomere length, and sarcomere shortening velocity. Within the limitations of the model, parameter settings are chosen to mimic experimental data.8,13 The model could be extended to incorporate the sigmoidal relation between stress and sarcomere length, or the increase of twitch duration with increasing sarcomere length. However, we prefer the current version with a limited number of parameters, in view of the sensitivity analysis. The model for passive tissue stress is simplified, since we do not model a complete three-dimensional state of stress. Yet, the behavior shown in Fig.\u00a01 agrees well with experimental data.28\nWe approximate extravascular pressure by intramyocardial pressure, thus neglecting the contribution of local tissue stress.26 In fact this assumption is made in many other models, including the waterfall model,9 the intramyocardial pump model,1,2,6,22 and the models by Huyghe et\u00a0al.12 and Beyar et\u00a0al.5,22,30 In addition, in our model intramyocardial pressure is determined completely by the model of LV wall mechanics. Thus, we can not replicate the observation that intramyocardial pressure depends on perfusion pressure.18 We feel this simplification is allowed in our simulations of isovolumic and isobaric beats, in which we assumed a constant perfusion pressure.\nIn the coronary circulation model resistances are constant. This is an approximation to the real situation, in which vessel diameter and therefore vessel resistance depend on vessel transmural pressure. The nature of the approximation is two-fold. First, we neglect the chronic change in resistance due to a chronic change in perfusion pressure. This is not critical for the value of NFA, as is apparent from our sensitivity analysis where we applied a two-fold change of coronary resistance (Fig.\u00a06). Second, we neglect cyclic changes in resistance during the cardiac cycle, which seems incompatible with the ability in the model to temporarily store blood in the coronary capacitance. Yet, the phasic nature of coronary flow is reproduced in the model. We explain this by noting that a change in vessel diameter during the cardiac cycle is associated with change in coronary volume, that affects coronary inflow irrespective of the location in the coronary tree where the volume change takes place. However, the associated change in resistance is important in the smallest vessels only, due to the nonlinear relation between vessel diameter and resistance. Obviously, the effect of changes in coronary resistance during the cycle may show up in aspects of the coronary flow signal, that are not captured by the normalized flow amplitude NFA.\nThe simplifications in the model may be seen as limitations, if one has the goal to explain all experimental observations available. In this study, we consider it a strength of the model, in view of our aim to investigate primary interactions and dependence on parameter settings.\nResults\nIn contrast to the situation in all other organs, coronary inflow of the heart muscle occurs mainly in diastole and is significantly impeded in systole. Experimentally, the amplitude of the coronary inflow signal was found to be only weakly coupled to systolic LV pressure in isovolumic beats at various LV volumes.15 This behavior is illustrated by the three fits to experimental data sets, shown in top left panel of Fig.\u00a05. As in the experiment, in our model pulsatility of coronary inflow is maintained at low left ventricular pressure, although dependence of NFA on pressure is slightly higher than in the experiment.\nThe mechanism by which pulsatility of coronary inflow is maintained in isovolumic beats is explained as follows (Fig.\u00a04, left panel). Total LV pressure is the sum of diastolic pressure in the passive ventricle, and the extra pressure, generated by muscle contraction. Diastolic pressure decreases with decreasing volume and becomes negative as volume decreases below the resting volume of 60\u00a0ml. We note that in the experiments by Krams et\u00a0al. negative diastolic pressure was induced as well, by applying suction to the balloon, inserted in the LV cavity.16 Since LV volume is constant during a beat, radial wall stress and its contribution to LV pressure are constant as well. The extra pressure, related to muscle contraction, reduces with decreasing volume, but at the lowest volume of 20\u00a0ml it still is about 15\u00a0kPa, and maximum total LV pressure is still about 11\u00a0kPa. The fact that the variation in LV pressure, which determines pulsatility of coronary flow, decreases more slowly with decreasing volume than maximum LV pressure, is reflected in the value of NFA shown in Fig.\u00a05.\nThe relation between maximum LV pressure and minimum coronary inflow, shown in the bottom left panel of Fig.\u00a05, qualitatively corresponds with experimental data by Kouwenhoven et\u00a0al.14 and Pagliaro et\u00a0al.20 In the latter study, minimum coronary inflow was found to decrease with increasing maximum LV pressure for pressures above 13\u00a0kPa, but was virtually independent of systolic LV pressure below 13\u00a0kPa. It was suggested that at low pressures \u2018the shielding effect of the contracting ventricle prevents ventricular pressure from being transmitted in the myocardial wall\u2019.20 In our model, this shielding effect is identified as radial wall stress.\nExperimentally, NFA in isobaric beats at low LV pressure was found to be about equal to NFA in isovolumic beats.15 This finding is replicated in our model (Fig.\u00a05, right panel). In contrast to the isovolumic case, the relation between maximum pressure and NFA is non monotonous, with distinct behavior at high and low pressures. At high pressures, LV volume remains mostly above the passive resting volume of 60\u00a0ml, and intramyocardial pressure is dominated by LV pressure. At low pressures, the LV ejects to far below the passive resting volume of 60\u00a0ml, and the decreasing contribution of LV pressure to intramyocardial pressure is compensated by the increasing contribution of radial wall stress.\nIt is to be noted that in the isobaric beats both contributions to intramyocardial pressure, radial wall stress and left ventricular pressure, vary in time, while in the isovolumic beats radial wall stress is constant and cyclic flow impediment is due to the varying contribution of left ventricular pressure only (Fig.\u00a04). This explains the two peaks in the intramyocardial pressure-signal in Fig.\u00a04: the first one is related to the rise in left ventricular pressure, while the second one arises from the rise in radial wall stress, related to the decrease in left ventricular volume.\nAnother experimental observation is that NFA is about proportionally related to LV contractility, expressed by maximum elastance, NFA being about zero when contractility was about zero.17 This observation is replicated by our model, in which NFA changes about proportionally with the contractility parameter c, irrespective of the volume setting (Fig.\u00a05). For low contractilities, minimum coronary inflow approaches the theoretical value of 8.3\u00a0ml\u00a0s\u22121\u00a0100\u00a0g\u22121 of tissue, obtained from the coronary perfusion pressure of 10\u00a0kPa, the total coronary resistance of 6\u00a0\u00d7\u00a0108\u00a0Pa\u00a0s\u00a0m\u22123, and a wall volume of 200\u00a0g.\nFinally, it has been observed that intramyocardial pressure may exceed left ventricular pressure in low afterload beats.18 This is also the case in our model (Fig.\u00a04, right panel), and explained again from the contribution of radial wall stress at the low ventricular volumes associated with the low LV ejection pressures.\nSensitivity Analysis\nSensitivity of NFA to the radial stiffness parameter cr0 in isovolumic beats is low (Fig.\u00a06, top left). This can be best understood from Eq. (17), which shows that only the constant level of radial wall stress is affected by changing cr0, while the variation of left ventricular pressure, and thus of intramyocardial pressure and arterial inflow, is unaffected. The increase in left ventricular pressure with decreasing stiffness is explained from Eq. (7), which shows that fiber stress is converted into both LV pressure and radial wall stress. With decreasing radial stiffness, an increasing part of fiber stress is converted into LV pressure. In the extreme case of zero radial stiffness, minimum left ventricular pressure is about 14\u00a0kPa, which is higher than the pressure measured in the experiment (Fig.\u00a06).15\nVariation of NFA with radial stiffness is more prominent in isobaric beats at low LV pressure (Fig.\u00a06, top right). An increase of radial stiffness causes a more rapid increase of radial wall stress with decreasing volume below the equilibrium volume. Thus the pressure at which the contribution of radial wall stress to intramyocardial pressure takes over from the contribution of ventricular pressure is increased (17), and pulsatility of coronary inflow is maintained. At zero radial stiffness, intramyocardial pressure is determined fully by left ventricular pressure, explaining loss of pulsatility of coronary flow with decreasing ejection pressure, in disagreement with experimental findings.15\nSensitivity of NFA to settings of the active stress model (Fig.\u00a06) is low, as far as the velocity and time dependence is concerned. NFA was more affected by increasing the stress-free sarcomere length from 1.5 to 1.6\u00a0\u03bcm. In fact this variation has the same effect as decreasing contractility at low sarcomere lengths. Thus, in this case the decrease of NFA is explained from the decrease in variation of left ventricular pressure, which is reflected in a decreased variation of intramyocardial pressure.\nThe increase of NFA with increasing myocardial compliance (Fig.\u00a06), both in isovolumic and isobaric beats, is explained from the fact that, at the same variation of intramyocardial pressure, the volume change of the myocardial bed increases. Hence the pulsatility of arterial inflow is increased as well. A decrease of myocardial compliance has the opposite effect.\nThe first order effect of a variation of coronary resistance is a change in both the average and pulsatile component of coronary flow, as was illustrated already in Fig.\u00a03. In this approximation, the relative changes in average and pulsatile flow component are equal, causing NFA to remain unaffected. However, since coronary compliance is kept constant, the relative contribution of flow related to volume change of the coronary bed becomes less when decreasing coronary resistance, causing the decrease of NFA, observed in Fig.\u00a06. Increasing coronary resistance has the opposite effect.\nRelation with Other Models\nIn comparison to the waterfall model9 and the intramyocardial pump model,1,2,6,22 our model is more advanced in its description of intramyocardial pressure, adding the effect of radial wall stress. Radial wall stress becomes important only below the zero-pressure volume of the passive ventricle. Thus, the early models still are applicable in normal physiological conditions, in which ventricular volume is higher than this zero-pressure volume during the major part of the cardiac cycle.\nIn comparison to the models by Beyar et\u00a0al.5,29,30 our model is simpler, emphasizing primary interactions, at the cost of information on transmural variation of coronary flow and long-term exchange of fluid between the coronary vessels and the interstitium. However, the reduced complexity yields increased insight into primary determinants of coronary flow impediment.\nMain limitation of our model with respect to the model presented by Vis et\u00a0al.,26,27 lies in neglecting the direct contribution of tissue stress to the extravascular pressure and effective compliance of the coronary vessel. The approach of Vis et\u00a0al. requires a detailed analysis of the equilibrium between the pressure in a cavity in the LV wall, and the stress in the surrounding tissue. However, such an analysis would compromise the simplicity, that we aim for in this study.\nConclusion\nIn conclusion, a mathematical model of the interaction between coronary flow and cardiac mechanics is presented, with a limited number of model parameters. The model replicates the experimental observations, that the phasic character of coronary inflow is virtually independent of maximum left ventricular pressure, that the amplitude of the coronary flow signal depends linearly on cardiac contractility, and that intramyocardial pressure in the left ventricular wall may exceed left ventricular pressure. The normalized amplitude of coronary inflow is mainly determined by contractility, reflected in dependence of active fiber stress on sarcomere length, and maintained at low ventricular volumes by radial wall stress. The sensitivity of the NFA to myocardial coronary compliance and resistance, and to the relation between active fiber stress, time, and sarcomere shortening velocity is low.","keyphrases":["intramyocardial pump","radial wall stress","ventricular mechanics","lumped parameter model","empty beating heart"],"prmu":["P","P","P","P","M"]}
{"id":"Ann_Biomed_Eng-2-2-1705509","title":"Remote Non-invasive Stereoscopic Imaging of Blood Vessels: First In-vivo Results of a New Multispectral Contrast Enhancement Technology\n","text":"We describe a contactless optical technique selectively enhancing superficial blood vessels below variously pigmented intact human skin by combining images in different spectral bands. Two CMOS-cameras, with apochromatic lenses and dual-band LED-arrays, simultaneously streamed Left (L) and Right (R) image data to a dual-processor PC. Both cameras captured color images within the visible range (VIS, 400\u2013780 nm) and grey-scale images within the near infrared range (NIR, 910\u2013920 nm) by sequentially switching between LED-array emission bands. Image-size-settings of 1280 \u00d7 1024 for VIS & 640 \u00d7 512 for NIR produced 12 cycles\/s (1 cycle = 1 VIS L&R-pair + 1 NIR L&R-pair). Decreasing image-size-settings (640 \u00d7 512 for VIS and 320 \u00d7 256 for NIR) increased camera-speed to 25 cycles\/s. Contrasts from below the tissue surface were algorithmically distinguished from surface shadows, reflections, etc. Thus blood vessels were selectively enhanced and back-projected into the stereoscopic VIS-color-image using either a 3D-display or conventional shutter glasses.\nIntroduction\nVisually guided procedures provide instant feedback and meantime afford insights that would otherwise be difficult or even impossible to obtain.26 This is a primary reason for mankind\u2019s continuous desire to extend vision beyond the boundaries of the human eye, which has resulted in many successful diagnostic imaging modalities like X-ray imaging, endoscopy, thermography, ultrasound scans and MRI that all found their way into the clinic.\nWhile investigating the principal feasability of a camera for imaging blood oxygenation levels, we noticed that our multispectral images also contained information about subcutaneous vasculature, with improved contrast in the near infrared.24 Near infrared imaging of superficial blood vessels in itself is not new; soon after infrared photography2,9,25 early electronic cameras were used for experiments.13,15 Since then, numerous near infrared imaging methods have been developed using transillumination mode and\/or reflection mode.5,6,10,14, 22 Our multispectral camera, however, provides the opportunity to obtain normal color images within the visible range (VIS) which are pixel-to-pixel matched with images obtained in the invisible near infrared range (NIR). By combining the image information content of both spectral bands with a processing algorithm, we developed a new technique that allows selective enhancement of superficial blood vessels with selectable pigment suppression within a normal color image.23\nThe underlying principle does not necessarily require stereoscopic image acquisition to derive increased vessel contrasts from the tissue. When, however, developing a new medical imaging technique, deriving image information from patient tissue is not the only issue. Especially for visually guided procedures it is also crucial to put effort in an ergonomic human interface and avoid conflicts between the proprioceptional and visual perceptions of the user. The added value of stereoscopic image recordings already was recognized and successfully used to document medical cases more than a century ago.4 We likewise reasoned that offering enhanced blood vessel contrast at the cost of depth perception would restrict the usefulness of our technique and therefore decided to realize a stereoscopic version of our vessel contrast enhancement device.\nVisually guided procedures typically require a combination of well trained eyes and specific fine-motoric skills, since the coordination between eyes and hands is task dependent.16 For eye-hand coordination, brain processes for perception and action interact so closely that they cannot be separated and the influence of visual illusions (like human depth perception) to motoric tasks becomes stronger when input takes place via lower levels in the brain.3 The match between a human interface device and the visual and motoric brain processes strongly defines whether a technology can be applied intuitively or not. A new technology can be classified as intuitive if it speeds up the learning process for novices in a certain skill, without impairing the performance of persons already skilled in the existing art.20 Thus a useful increase in vascular contrast should neither imply a task-impairing decrease in depth perception (like in monoscopic techniques) nor distort spatial clues for human vision (like shadows) nor introduce false depth information (like projection parallax).\nAs a first reconnaissance of practical feasibility, we applied our new technique to several visual clinical procedures for which we expected that improved visualization of blood vessels would be of interest, being:blood withdrawal;vein inspection in dark skin;detection of veins through iodide;inspection of varicose veins and nevi pigmentosum.\nMethods\nInstrumental Setup\nThe instrumental hardware setup is schematically drawn in Fig.\u00a01. Two synchronized identical single-chip CMOS-cameras (Vector Technologies Belgium, custom built) were equipped with apochromatic lenses and two identical custom-built dual-band LED-arrays (O2-View, the Netherlands). These LED-arrays were current controlled and each had two individually programmable channels for the emission of in total \u00b11.2 Cd visible white light with adjustable color temperature (consisting of 40 broadband white LEDs with a yellow accent and 20 broadband white LEDs with a blue accent) as well as one programmable channel for the emission of near infrared radiation (20 LEDs, 920\u00a0nm, max. 32\u00a0mW per LED). The light sources were constructed so that the geometrical beam profiles of VIS and NIR matched very closely (and thus also any resulting shadows and\/or reflections). Left (L) and Right (R) image data was simultaneously acquired and streamed to a dual processor PC equipped with a stereoscopic monitor (Sharp LL-151\u20133D). It was also possible to connect a conventional cathode ray tube (CRT) monitor equipped with shutter glasses, for comparison of stereoscopic representation.\nFigure\u00a01.Experimental setup. Two CMOS-cameras, with apochromatic lenses and dual-band LED-arrays, simultaneously stream Left (L) and Right (R) image data to a dual processor PC. Both cameras captured color images within the visible range (VIS, 400\u2013780\u00a0nm) and grey-scale images within the near infrared range (NIR, 910\u2013920\u00a0nm) by sequentially switching between LED-array emission bands.\nA schematical representation of image aquisition and data structure is drawn in Fig.\u00a02. The CMOS-camera detector chips were equipped with a Bayer filter mosaic to obtain an RGB color image within the VIS. The camera pixels, however, were also sensitive to near infrared radiation because all three filter channels of the Bayer mosaic (red, green and blue) were designed as highly transparant within the NIR. Thus four NIR-pixels were acquired for each VIS RGB color pixel group. By sequentially switching between emission bands of the LED-arrays, images within the Visual range (VIS, 400\u2013780\u00a0nm) and Near Infrared range (NIR, 910\u2013920\u00a0nm) were acquired in an alternating fashion. VIS and NIR image-size-settings could be varied independantly. For 8-bit encoding depth, at image-size-settings of 1280\u00a0\u00d7\u00a01024 for VIS and 640\u00a0\u00d7\u00a0512 for NIR, 12\u00a0cycles\/s were obtained (1\u00a0cycle\u00a0=\u00a01 VIS L&R-pair\u00a0+\u00a01 NIR L&R-pair). At the cost of decreasing image-size-settings downto 640\u00a0\u00d7\u00a0512 for VIS and 320\u00a0\u00d7\u00a0256 for NIR, camera speed could be increased up to 25\u00a0cycles\/s. Encoding depths of 10-bit and 12-bit were also available, but only used for stills (due to the lower obtainable framerate).\nFigure\u00a02.Schematic diagram of image aquisition. The sequentially acquired alternating VIS and NIR raw image frames form 3D-matrices for the Left and Right channel. The NIR image size is smaller than the VIS image size to increase framerate while maintaining an overview of the imaged area. Enlarged details illustrate the NIR transparency of the Bayer pattern RGB-filters which are applied to obtain a VIS color image. The time domain axis is expressed in image cycles. Along this image cycle axis, the control signals for NIR and VIS LEDs (synchronized with respectively NIR and VIS camera exposures) are visualized.\nData Aquisition\nMultispectral stereoscopic movies were recorded in several typical clinical settings for which the technique was considered as possibly useful. LED currents and diaphragm settings were chosen so that for each movie saturated pixels were avoided. A preview mode allowed aiming, adjustment of converging angle \u03b1 and focusing of the cameras. After software triggering the stereo-camera streamed a sequence of 8-bit digitally encoded image cycles to PC-memory (using auto-incremental numbering). All images were automatically saved on a fast SATA harddisk-array. Camera and light source settings were automatically stored in a text file and located in the same directory.\nAll patients and volunteers gave their informed consent for filming as well as for publishing the resulting image material. No diagnosis or therapy was based upon any of our results.\nData Processing\nGeneral Aspects\nProcessing was performed with custom developed software (programming language C++) using the stored text file with camera and light source settings as additional input values. Figure\u00a03 schematically represents this process.\nFigure\u00a03.Schematic diagram of image processing. The images captured within the visible range (VIS) and the images captured within the near infrared range (NIR) are combined, which reveals blood vessel patterns below the skin. The left and middle column focus on edge-enhancement and suppression of superficial artifacts, the right column serves to fill-in the blood vessel lumen. For raw VIS & NIR images as well as processed results see figures 6, 7, 8 and 9.\nThe processing method allowed discrimination between image information obtained from the tissue surface versus image information obtained from within the tissue. In order to achieve this, a distinction was made between shadows, reflections and absorption contrasts for both VIS and NIR.\nSuppressing Shadows on the Surface\nSince NIR and VIS beams were matched closely, shadows produced by irregular shapes at the tissue surface (e.g. skin structure, skin folds, nevi, hair, etc.) or by objects between the light sources and the tissue (fingers, needles, surgical tools, etc.) also matched well in both wavelength ranges. Our algorithm excluded such matching shadows from enhancement and left all useful aspects of shadows unaffected (e.g. depth clues).\nUseful Effects of Shadows and Lighting Geometry\nDue to the fact that the VIS and IR shadows matched very closely, the algorithm was able to selectively enhance contrast from below the surface while leaving shadows on the surface unaffected (thus not distorting these important depth clues).\nDue to the shallow angle of the lightbeams, the skin texture was pronounced. Due to the lighting from two sides, objects that were brought towards the tissue surface (e.g. needles, scalpels, probes, etc.) when lighted from both sides, could produce two separate (not too heavy) shadows. These shadows met and typically formed a \u201cV\u201d pattern when an object touched the surface in the middle of the field of view, thus providing extra information for depth perception.\nEnhancing Absorption Contrast of Blood Vessel Walls\nEdge detection by a Prewitt image filter was performed on each VIS and NIR image. Pixel positions containing edge information above a certain adjustable threshold in both spectral regions were classified as surface artefacts and excluded from enhancement. The boundary regions of absorption contrasts caused by structures below the tissue surface, produced edges that were mainly present in the NIR image. Enhancement was selectively performed only for pixel positions where the NIR image contained more edge information than the corresponding VIS image. These \u201cvalid\u201d pixels identified the vessel boundaries. The positions of these valid pixels were stored in a 1st NIR mask.\nDiscarding Reflections on the Surface\nShiny areas that produced reflections and\/or saturated pixels also matched in both wavelength ranges. This characteristic allowed to calculate a 2nd NIR mask in which superficial reflections were also excluded from enhancement. Neighboring pixels of identified saturated pixels were excluded from enhancement. The radius of this exclusion region was programmable, but due to the favorable anti-blooming behavior of the CMOS-camera chips, suppressing direct neighbor pixels appeared sufficient.\nEnhancing Absorption Contrast of Blood Vessel Lumen\nThe \u201ccontent\u201d of blood vessels was separately enhanced by raising pixel values of the normalized NIR image to the power of N (with N user adjustable between 0.5 and 2.5) while discriminating NIR pixels below a freely adjustable noise threshold and excluding information from identified shadows. Multiplication with the 2nd NIR mask then produced a final enhancement mask for subsequential backprojection into the VIS image by pixel-to-pixel multiplication.\nSuppressing Contrasts Originating from Melanin Pigment\nSuperficial contrasts within the VIS, originating from melanin pigment concentrations, could either be filtered out or left unchanged. Figure\u00a04 illustrates the normalized distribution of the intensities for the aquired separate spectral bands (R, G, B and NIR) in relation to the intensity (IVIS) of the composed visible RGB-image. Intensities were calculated using the Intel ippiRGBtoGray function.11 Four clouds of data points can be discerned, being IR\/IVIS (red channel), IG\/IVIS (green channel), IB\/IVIS (blue channel) and INIR\/IVIS (NIR channel). By calculating the ratio of (IR\/IVIS )\/(INIR\/IVIS ) and comparing the result for each pixel with an adjustable threshold, it can be decided whether or not to apply backprojection to a pixel. This concerns the pixels located within the overlapping region of IR\/IVIS and INIR\/IVIS within Fig.\u00a04. The resulting difference is clear when comparing between Fig.\u00a09c and d with regard to the visualization of nevi pigmentosum and hair.\nFigure\u00a04.Relative intensity distribution of the different spectral bands. Dimensionless normalized distribution of the intensities for the 4 aquired red (IR), Green (IG), Blue (IB) and near infrared (INIR) individual spectral bands expressed in ratio to the normalized intensity (IVIS) of the composed RGB-image. The four data clouds (R, G and B labeled by their natural colors and NIR labeled as pink) show a generally marked separation, but especially for a number of pixels within the red and infrared the data clouds partly overlap. This indicates regions where the VIS contrast potentially is superior to the NIR contrast. By comparing an adjustable threshold with the calculated ratio of (IR\/IVIS)\/(INIR\/IVIS) it can be decided which spectral band provides superior contrast for the pertaining pixel and thus whether or not it is used for enhanced backprojection.\nData Presentation\nThe images could either be displayed on an auto-stereoscopic liquid crystal display (LCD) monitor or on a conventional CRT monitor equipped with shutter glasses.\nThe auto-stereoscopic LCD-monitor (Sharp LL-151\u20133D) was equipped with software controllable switching between stereoscopic (or 3D) and normal (monoscopic or 2D) mode. Monitor resolution was 1280\u00a0\u00d7\u00a0768 in monoscopic mode (XGA). In stereoscopic mode the available pixels were split-up in two separate images (L&R) by activation of a vertical LCD parallax barrier. The principle behind this technology is illustrated in Fig.\u00a05.\nFigure\u00a05.Principle of applied autostereoscopic LCD-monitor (reprinted with permission from Sharp). In 2D mode, only one camera-channel is displayed (either from the L or R camera) and the parallax barrier is not actuated. Both eyes of an observer therefore receive the same image and a conventional flat image with full resolution is seen. In 3D mode, both camera channels are displayed (L&R) and the parallax barrier is actuated. The left eye and right eye of an observer now receive different images, and a stereoscopic (in-depth image) with halve resolution is seen.\nThe CRT-monitor (iiyama vision master 21) was used at a resolution setting of 1280\u00a0\u00d7\u00a01024 in combination with wireless shutter glasses (e-Dimensional) and thus provided stereoscopic information without sacrificing resolution.\nThe enhancement algorithm occurs on a pixel-to-pixel basis and does not affect resolution.\nOur device offered several imaging modes for data presentation:Monocopic raw VIS preview (normal full color vision)Monoscopic raw NIR preview (greyscale)Off-line stereoscopic looped VIS view with and without enhanced blood vessel back-projection (with freely adjustable enhancement settings).Off-line stereoscopic looped raw NIR view or enhanced NIR view (with freely adjustable enhancement settings)Stereoscopic stills in all modesMonoscopic stills in all modes (freely switchable between Left and Right)\nAll modes offered the possibility for pause and scrolling forward or reverse frame-by-frame. By means of virtual slider controls and virtual pushbuttons the user interface allowed freely adjustable settings for shadow suppression, pigment suppression, noise threshold and vessel lumen fill-in contrast.\nResults\nResults for Blood Withdrawal\nDuring routine blood withdrawal the inserted needle tip remained only slightly visible on the VIS image due to skin surface deformation (see Fig.\u00a06a). On the raw NIR image, however, the inserted needle remained visible within the tissue for a few mm with some metallic reflection. Subcutaneous bleeding during needle removal could be detected in the raw NIR image while the needle tip was still in the tissue (see Fig.\u00a06b). This was clearly highlighted by backprojection in the VIS image (see Fig.\u00a06c). The processing settings used to obtain this backprojection mainly laid the accent upon enhancing the absorption contrast of blood vessel lumen, whereas edge enhancement was set to minimum. Reflections on the thumb nail did not lead to image distortion and the blood volume in the nail bed showed more contrast.\nFigure\u00a06.Routine blood withdrawal. Image pairs showing unprocessed images for VIS (a) and NIR (b) as well as the result after application of the new image processing method (c). Note the forked shadow (which is not effected by the enhancement algorithm), the clearly visualized subcutaneous bleeding and the improved visibility of the needle tip.\nResults for Dark Skin\nHardly any vascular contrast is present within the VIS image (see Fig.\u00a07a). The NIR image, however, is not affected by skin pigmentation and reveals a subcutaneous vascular pattern (see Fig.\u00a07b). The combined information results in an enhanced image (see Fig.\u00a07c).\nFigure\u00a07.Influence of skin pigmentation. A dark skin color (a) has no significance for the applied NIR wavelength of 920\u00a0nm. Blood vessels provide good contrasts (b) and the resulting enhanced image (c) offers an improved visualization of the vasculature.\nResults for Vein Detection Through Iodide\nIodizing skin portions before surgery is a common clinical procedure which darkens the skin and lowers the visibility of blood vessels. Our method offers imaging right through iodide. To demonstrate this, we filmed a glass Petri dish, placed horizontally upon a volunteers arm while filling it up to a 3\u00a0mm thick layer of iodide solution. From the VIS image no vessels could be detected through the iodide solution (see Fig.\u00a08b) and hardly any through the empty Petri dish (see Fig.\u00a08a). The raw NIR image, however, clearly showed superficial blood vessels even through the Petri dish (see Fig.\u00a08c).\nFigure\u00a08.Vein detection through iodide. Within the visible range, the superficial vasculature is only vaguely discernable (a). After filling the Petri-dish with a 3\u00a0mm thick layer of iodide solution, this fully blocks out the tissue view within the visual range (b), whereas a clear view of the vasculature remains possible at the applied NIR wavelength of 920\u00a0nm (fig 8c).\nResults for Varicose Vein and Nevi Pigmentosum Inspection\nThe device was used to film varicosis patients during dermatological outpatient clinic. The visualization of varicose veins could be drastically improved. Even in cases where the blood vessels were covered by subcutaneous fat, they could be detected quite well (see Fig.\u00a09). By either de-activating or activating the first NIR mask in the processing algorithm (see Fig.\u00a03) it was also possible to choose whether pigment contrasts in the visual range were left intact or filtered out. The difference is clear when comparing between Fig.\u00a09c and d with regard to the visualization of nevi pigmentosum and hair. By clicking a software screen button, this suppression of pigment contrast could be freely switched on and off during viewing.\nFigure\u00a09.The VIS image does not contain much information about the underlying vascular pattern (a). The NIR image, however, clearly shows what\u2019s hiding beneath the surface (b). Note that, when building the enhanced image, the nevus which is present in the VIS image can freely either be suppressed as a surface contrast (c) or be kept visible (d).\nDiscussion\nFrom the figures presented in this article, it is clear that all experiments resulted in improved visualization of superficial blood vessels. Based upon the absorption coefficients of Hb versus HbO2, arteries provide superior contrasts at 920\u00a0nm. The presented images, however, show enhanced venous contrasts, because the larger arteries are buried deeper underneath the skin. Stereoscopic movies provide a more lifely impression of the underlying, but presentation of stereoscopic movies is not possible in a printed journal. We therefore offer the possibility to download viewer software and movies via the internet http:\/\/www.erasmusmc.nl\/ThoraxcenterBME\/html\/research\/additional\/bloodvesselcamera.htm\nFor both the autostereoscopic LCD and the CRT display plus shutter glasses, we found that the perception of depth was less for stills than for movies. This qualitative observation matches research findings on stereoscopic display techniques in X-ray technology8 and is also consistent with the differences between static and dynamic stereoacuity described by Mathias and Rudolf Sachsenweger.17 Stereoscopic movies also showed an increase in apparent image sharpness compared to stereoscopic stills, which may be explained by Shipley\u2019s description of stereoscopic contour integration over blur.18 Compared to the autostereoscopic LCD-display, the increase in perceived sharpness was much stronger when using a CRT with shutter glasses. For monoscopic stills, however, the LCD-display (switched to monoscopic mode) was superior to the CRT. These observations are in agreement with the technology overview by Szold19 and can be explained by the sacrifice of resolution when using the LCD display in autostereoscopic mode, which price has not to be paid when using the CRT with shutter glasses.\nThe beneficial effect of separately adjustable enhancement paths for blood vessel contours and blood vessel lumen, which we experienced during the development of our processing algorithm, is consistent with the findings of Yin et\u00a0al. that surface-features and edge processes make different contributions in determining an object\u2019s unity and shape.27 The perceived usefulness of stereoscopic information, however, also supports the concept formulated by Tse that \u2018\u2018mergeable\u2019\u2019 volumes, rather than relatable contours, are the critical elements in completion.21\nWith our technique we have aimed to avoid parallax errors and loss of vessel contrast by shadows which are inherent to other blood vessel contrast enhancement techniques projecting the vascular pattern via a projector onto the skin.28 We exclude parallax errors because the user truly looks beneath the tissue surface trough two camera\u2019s. Absence of vessel contrast in shadows is avoided by lighting from two sides and by discrimination of superficial artifacts versus contrasts originating from below the surface.\nThis ability to selectively enhance contrasts from beneath the surface, while preserving the natural depth clues of shadows on the surface, supports both static and dynamic transmittance anchoring of the visual system which is crucial for depth perception.1 Preservation of natural shadows is furthermore important for the correct interpretation of depth clues by occlusion (e.g. from a hand or an instrument positioned between observer and tissue) thus precluding errors in depth perception.12\nSince only carefully balanced white light is projected on the skin, there is no impairment of color perception. It is up to the user to freely switch between normal full color vision, color vision plus superimposed blood vessel backprojection and stereoscopic near infrared greyscale vision (with or without enhancement features).\nThe embodiment described in this article still has some drawbacks:Due to the fact that the pertaining configuration aquires VIS and NIR images sequentially, motion artifacts can lead to a backprojection shift.The deeper the vessels lie under the surface, the less we can visualize them. In the pertaining configuration, the depth range is limited to about 1\u00a0mm. Based upon the absorption coefficients of Hb and HbO2, arteries provide superior contrasts at 920\u00a0nm. The presented images, however, show enhanced venous contrasts, because the larger arteries are buried deeper underneath the skin.\nThe above mentioned drawbacks require further development. Fortunately, many technological improvents still can be added. Allthough the device worked quite well under normal ambient lighting conditions (100\u2013200 Lux), the use of a switchable filter might further improve spectral separation. Furthermore, known techniques like application of crossed polarizers, in combination with more powerful light sources, can offer a considerably larger penetration depth.7\nConclusions\nCompared to inspection with the naked eye under normal lighting conditions, the tested stereoscopic blood vessel contrast enhancer offered improved visualization in all investigated settings, providing the best stereoscopic image quality when using the CRT monitor with shutter glasses and the best monoscopic image quality when using the LCD set to monoscopic mode.\nOur technique supports perception of depth, 3-dimensional motion and discrimination between tissue surface and underlying structures. It also has potential as an educational tool by offering the possibility to look and record trough the eyes of an experienced specialist. Further improvements on penetration depth, frame-rate and focus depth-of-field form targets for momentary ongoing further research.","keyphrases":["3d-display","multispectral stereoscopy","contactless enhanced viewing of superficial vasculature","intuitive technology","reconnaissance of feasibility for various clinical applications"],"prmu":["P","M","R","R","M"]}
{"id":"Exp_Brain_Res-3-1-1914290","title":"Regularity of center-of-pressure trajectories depends on the amount of attention invested in postural control\n","text":"The influence of attention on the dynamical structure of postural sway was examined in 30 healthy young adults by manipulating the focus of attention. In line with the proposed direct relation between the amount of attention invested in postural control and regularity of center-of-pressure (COP) time series, we hypothesized that: (1) increasing cognitive involvement in postural control (i.e., creating an internal focus by increasing task difficulty through visual deprivation) increases COP regularity, and (2) withdrawing attention from postural control (i.e., creating an external focus by performing a cognitive dual task) decreases COP regularity. We quantified COP dynamics in terms of sample entropy (regularity), standard deviation (variability), sway-path length of the normalized posturogram (curviness), largest Lyapunov exponent (local stability), correlation dimension (dimensionality) and scaling exponent (scaling behavior). Consistent with hypothesis 1, standing with eyes closed significantly increased COP regularity. Furthermore, variability increased and local stability decreased, implying ineffective postural control. Conversely, and in line with hypothesis 2, performing a cognitive dual task while standing with eyes closed led to greater irregularity and smaller variability, suggesting an increase in the \u201cefficiency, or \u201cautomaticity\u201d of postural control\u201d. In conclusion, these findings not only indicate that regularity of COP trajectories is positively related to the amount of attention invested in postural control, but also substantiate that in certain situations an increased internal focus may in fact be detrimental to postural control.\nIntroduction\nBy now, it is well established that maintaining and controlling an upright posture requires a certain amount of attention (for a review see Woollacott and Shumway-Cook 2002). The relation between attentional resources and the processing of information from somatosensory, visual, and vestibular systems is readily apparent in cases of reduced or conflicting sensory information (e.g., Redfern et al. 2001, 2004; Shumway-Cook and Woollacott 2000; Teasdale and Simoneau 2001). On the one hand, the degree of attention, or cognitive involvement, required for controlling posture increases with task difficulty. This has been (indirectly) illustrated by, for example, Lajoie et al. (1993) and Vuillerme and Nougier (2004), who both found that the reaction time of a verbal response to an auditory stimulus increased with the difficulty of the postural task. On the other hand, the amount of attention required to perform a secondary suprapostural task is known to influence posture (e.g., Balasubramaniam et al. 2000; Huxhold et al. 2006; Pellecchia 2003; Riley et al. 2003). The dependency of posture on attention is even more prominent in the presence of pathology or aging, when both peripheral and central changes occur that decrease the (physical) capability needed to maintain balance during standing or walking (e.g., Brown et al. 1999; Lindenberger et al. 2000; Marchese et al. 2003; Rankin et al. 2000; Redfern et al. 2004). In balance-impaired elderly individuals, for example, the performance of a secondary cognitive task (i.e., dual-task paradigm) may promote postural instability and even falls (Barra et al. 2006; Brauer et al. 2001). Hence, the amount of attention required for maintaining and controlling upright posture is indicative of the degree of \u201cautomaticity\u201d of postural control and, for that reason, has been advocated and used as an important tool in clinically oriented studies (cf., Geurts et al. 1991; Melzer et al. 2001).\nCenter-of-pressure (COP) fluctuations measured while standing on a force platform provide a complex output signal of the postural control system in which various pertinent cognitive, perceptual, and motor processes are reflected. Recently, COP measures pertaining to the dynamical structure of COP fluctuations have helped to understand the inherent complexity of the postural control system and its constituent processes (e.g., Baratto et al. 2002; Collins and De Luca 1993; Newell et al. 1993; Pascolo et al. 2005; Peterka 2000; Yamada 1995a; cf., Riley and Turvey 2002 for a review). Inspired by this development in modern posturography, Roerdink et al. (2006)\u2014in a study on the functional recovery of posture in stroke patients\u2014proposed a direct relation between the amount of attention invested in postural control and the regularity of COP fluctuations. COP trajectories were more regular (as indexed by reduced sample entropy) in stroke patients than in healthy elderly and became less regular when performing a secondary cognitive task while standing. These results were interpreted to imply that postural sway regularity is positively correlated with the degree of cognitive involvement in postural control. Interestingly, the regularity of the COP fluctuations decreased with rehabilitation, whereas postural stability (as indexed by the largest Lyapunov exponent) increased, suggesting that the required degree of cognitive involvement in postural control decreased during the course of rehabilitation. Hence, these progressively more irregular COP fluctuations (as indexed by an increase in sample entropy) may be interpreted as an increase in the efficiency or \u201cautomaticity\u201d of postural control. This interpretation is in line with physiological studies showing that a decrease in \u201ccomplexity\u201d or \u201cirregularity\u201d of a physiological time-series is indicative of a decrease in healthiness or effectiveness of the physiological control system (cf., Goldberger et al. 2002), a phenomenon known as \u201cdynamical diseases\u201d (cf. Belair et al. 1995). In other words, increased COP regularity may be explained as an indication of an increasingly ineffective postural control strategy.\nThe aim of the present study was to examine the role of attention in the regulation of posture. To this end, the amount of attention invested in postural control was manipulated experimentally in a large group of young healthy adults. In particular, we increased the difficulty of the postural task through visual deprivation under the assumption that an increase in task difficulty is associated with an increase in cognitive involvement in postural control, creating a so-called internal attentional focus (cf., Andersson et al. 1998; Teasdale et al. 1993; Teasdale and Simoneau 2001). In contrast, we used a concurrent attention demanding cognitive task in order to withdraw attention from postural control, creating an external focus of attention (cf., Huxhold et al. 2006). These manipulations of attention allowed us to examine the proposed direct relation between COP regularity and the amount of attention invested in postural control (Roerdink et al. 2006). In line with this relation, we hypothesized that: (1) increasing postural task difficulty (i.e., by standing with eyes closed, creating an internal focus) increases the cognitive involvement in postural control and hence the regularity of COP fluctuations, and (2) reduced attention to postural control (i.e., by performing a cognitive dual task, creating an external focus) decreases the regularity of COP fluctuations. In addition, based on the suggestion that posture is mainly controlled in the direction of the largest postural sway (Roerdink et al. 2006) and the common finding that in healthy young adults postural sway is largest in the sagittal plane relative to the frontal plane (e.g., Gatev et al. 1999; Winter et al. 1998), we expected COP regularity to be largest in the sagittal plane.\nRegularity of COP trajectories was quantified by the sample entropy (Richman and Moorman 2000; Roerdink et al. 2006). In order to examine the structure of COP fluctuations in more detail, we further used a combination of more traditional (i.e., based on summary statistics) and dynamical measures that are all defined operationally in terms of readily interpretable features of motor control (see also Table\u00a01): standard deviation of COP time-series (indexing variability or the amount of postural sway), sway-path length of the normalized posturogram (indexing the amount of twisting and turning of the COP trajectory), largest Lyapunov exponent (Rosenstein et al. 1993; indexing local stability), correlation dimension (Grassberger and Procaccia 1983; indexing the number of active, dynamical degrees of freedom involved in postural control and hence its dimensionality, e.g., Kay 1988) and the scaling exponent (e.g., Peng et al. 1995; indexing long-range correlations in COP time-series). Based on previous research, we expected visual deprivation to decrease local stability and to increase variability of COP time-series (cf., Roerdink et al. 2006; Woollacott and Shumway-Cook 2002; Yardley et al. 1999a). Moreover, we anticipated the attention manipulations to induce adjustments in the dynamical structure of postural control leading to changes in scaling exponent, sway-path length and dimensionality of COP fluctuations.\nTable\u00a01Abbreviations and meaning of interest of the calculated COP measuresVariableMeaning of interestSample entropy, SEnNegatively related with the regularity of COP trajectoryStandard deviation, \u03c3 (mm) Positively related with the variability of COP trajectorySway-path length, SPn (s\u22121)Positively related with the curviness of COP trajectoryLargest Lyapunov exponent, \u03bbmaxNegatively related with the local stability of COP trajectoryDimensionality, D2Positively related with the number of active control variablesScaling exponent, \u03b1Long-range correlations: \u03b1 = 0.5 for uncorrelated data (i.e., white noise),\u03b1 = 1.5 for \u201cBrown noise\u201d, the integration of white noise\nMethods\nParticipants and procedures\nA total of 30 healthy young adults (10 males, 20 females; mean age\u00a0=\u00a024\u00a0years, range\u00a0=\u00a019\u201330\u00a0years), without known motor impairments or movement-related disorders, volunteered to participate in the experiment. Participants stood barefoot on a 1\u00a0\u00d7\u00a01\u00a0m custom-made strain gauge force plate1 with their arms hanging relaxed alongside their body. The medial sides of the heels were separated by about 8\u00a0cm and each foot was placed with the toes outward at a 10\u00b0 angle from the sagittal midline (i.e., standard Romberg position). In order to examine the role of attention in the regulation of posture we carried out two manipulations. On the one hand, we increased the postural task difficulty by inviting participants to stand with eyes closed, while on the other hand, we withdrew attention from postural control by inviting them to perform a cognitive dual task. In particular, the participants were invited to stand upright with (1) eyes open (EO-ST), (2) eyes closed (EC-ST), (3) eyes open while performing a dual task (EO-DT) and (4) eyes closed while performing a dual task (EC-DT). The dual task consisted of uttering backwards names read out aloud by the investigator (e.g., \u201cSimon\u201d had to be repeated as \u201cnomis\u201d). The sole aim of this cognitive dual task was to withdraw attention from the postural task. Therefore, the participants were instructed to perform the task to the best of their ability. No feedback on the accuracy with which they performed this task was provided. Each participant performed the four task conditions in random order and once in reverse order, resulting in a total of eight recordings. COP trajectories were collected for 35 s at a sampling rate of 100\u00a0Hz.\nAfter the local ethics committee had approved the study, all participants gave their informed consent prior to their participation.\nData analysis\nIn order to examine whether posture is actively controlled in the direction of largest postural sway (Roerdink et al. 2006), we analyzed both the registered x (mediolateral ML) and y (anterioposterior AP) COP time-series. After omitting the first 5 s of each recording, leaving 30 s of data for further analyses (i.e., 3,000 samples), the time-series were bi-directionally filtered (second-order low-pass Butterworth filter, cut-off frequency of 12.5\u00a0Hz) to eliminate low amplitude measurement noise.2 First, we calculated the conventional standard deviation \u03c3 of x and y COP trajectories to quantify the variability, or amount, of postural sway. Subsequently, we normalized the x and y trajectories to unit variance (i.e., by dividing the time-series in question by their respective standard deviation) and calculated the sway path defined as the length of the COP trajectory traveled per second. The applied normalization procedure enabled us to determine the sway path in the normalized posturogram, abbreviated as SPn, providing a scale-independent measure of the amount of twisting and turning of the COP trajectory.\nApart from these summary statistics of postural sway (i.e., SPn and \u03c3), which by definition ignore the temporal structure of the COP time-series, we assessed COP dynamics by means of sample entropy, largest Lyapunov exponent, correlation dimension and scaling exponent, which will be briefly explained in the following (for a more extensive description we refer to Roerdink et al. (2006) and references therein). Table\u00a01 presents the six different measures and their interpretation.\nSample entropy, SEn\nHealthy physiological systems are often characterized by an irregular and complex type of variability, whereas disease or aging is often associated with greater regularity and less complexity (cf., Goldberger 1996; Goldberger et al. 2002; Pincus et al. 1991). A method to quantify the regularity of time-series is the sample entropy analysis (Lake et al. 2002; Richman and Moorman 2000). Sample entropy indexes the regularity of a given time-series, and is used to analyze complex stochastic systems that (by definition) include both deterministic and random processes (Pincus 1991). Specifically, sample entropy calculates the probability that a sequence of data points, having repeated itself within a tolerance r for a window length M, will also repeat itself for M\u00a0+\u00a01 points, without allowing self-matches (see Lake et al. 2002; Richman and Moorman 2000).3 Smaller sample entropy values are associated with greater regularity. In the present study, a decrease in sample entropy (i.e., more regular sway fluctuations) was interpreted as a decrease in the effectiveness of postural control.\nLargest Lyapunov exponent, \u03bbmax\nThe largest Lyapunov exponent4 provides a measure of the local stability of a dynamical system (e.g., Abarbanel et al. 1996), i.e., the system\u2019s sensitivity to initial conditions or its resistance to small internal perturbations, such as the natural fluctuations that occur while maintaining an upright stance. It quantifies the exponential divergence or convergence of initially nearby trajectories in state space as time progresses (e.g., Rosenstein et al. 1993). If nearby points diverge, they produce instability. The exponent \u03bbmax indexes this instability: positive values of \u03bbmax indicate either the presence of deterministic chaos (i.e., a form of variability that is brought about by an underlying lawful nonlinear dynamical structure) or complete randomness (i.e., noise), implying that nearby points diverge rapidly, reflecting local instability and lack of predictability. In order to distinguish a deterministic component from genuine randomness, it is necessary to validate results against surrogate data (cf., Theiler et al. 1992).\nDimensionality, D2\nThe dimensionality of all COP time-series was calculated using the correlation dimension algorithm of Grassberger and Procaccia (1983).5 The correlation dimension provides an index of the number of independent degrees of freedom (equations of motion) that are required to reproduce the time evolutionary properties of the COP time-series. Note that this analysis of the dynamical degrees of freedom is different from the analysis of the (mechanical) degrees of freedom of the joints as commonly applied in the study of motor control, and that no straightforward or uniform relation exists between the number of component degrees of freedom in motion and the dimension of the organizational dynamic in controlling those components (Newell and Vaillancourt 2001).\nScaling exponent, \u03b1\nIn order to determine whether the measured COP time-series were characterized by the presence of long-range correlations, we applied a fractal analysis method for biological time-series called detrended fluctuation (DFA) analysis (Peng et al. 1995).6 The scaling exponent \u03b1 as determined by this method indicates the presence or absence of (long-range) correlations in the COP trajectories, as defined by Peng et al. (1995). For uncorrelated data, (e.g. white noise) \u03b1 = 0.5. An \u03b1 greater than 0.5 and less than or equal to 1.0 indicates persistent long-range power-law correlations. In contrast, 0 < \u03b1 < 0.5 indicates a different type of power-law correlation such that large and small values of the time series are likely to alternate. For \u03b1 > 1 correlations exist but cease to be of a power-law form; \u03b1 = 1.5 indicates Brown noise, i.e., integrated white noise. The scaling exponent \u03b1 can also be viewed as an indicator that describes the roughness of the time series: the larger the value of \u03b1, the \u201csmoother\u201d the time series (Peng et al. 1995).\nSurrogate analysis\nTo test for spurious effects and to distinguish between deterministic features and randomness, the scaling exponents, dimension estimates, Lyapunov exponents and entropy values were also computed for surrogate data (Theiler et al. 1992). In particular, we generated both time- and phase-randomized surrogate data of the filtered COP time-series (Fig.\u00a01). In time-randomized surrogate data, the distribution of the original data is being preserved (i.e., mean, variance, etc. are unaltered), whereas the temporal correlations in the COP time-series are destroyed. The absence of temporal correlations will result in a scaling exponent \u03b1 close to 0.5 and very large values for dimension and sample entropy. Phase-randomized surrogate data are obtained by randomizing the data\u2019s Fourier phases. In contrast to time-randomization, this procedure does not alter the spectral power distribution and preserves the data\u2019s auto-correlation function. Consequently, scaling exponents of phase-randomized and original data should match, whereas estimates of correlation dimension and sample entropy should be largely increased in the surrogate data.Fig.\u00a01An example of the surrogate analysis, as applied to all time-series. Surrogate data of a measured COP trajectory (upper panel) were constructed by randomizing the Fourier phase (middle panel) and the temporal order (lower panel)\nStatistical analysis\nFor all dependent variables, the first and second trials were averaged. To test for differences between AP and ML COP measures and to examine the effect of the different experimental tasks, we used a repeated measures analysis of variance (ANOVA) design with within-subject factors vision (2 levels: standing with eyes open EO, and standing with eyes closed EC), dual task (2 levels: standing without cognitive dual task performance, i.e., single task, ST, and standing with cognitive dual task performance, DT), and plane (2 levels: sagittal and frontal plane).7 Subsequently, we tested for differences between surrogate and original data using a design involving a within-subject factor surrogate (3 levels: original data and time- and phase-randomized surrogate data). To assess the strength of the (main and interaction) effects, we determined the eta squared (\u03b72), a commonly used measure of effect size in AVOVAs, reflecting the proportion of variance in the dependent variable that is attributable to each effect. Subsequently, \u03b72 was converted into Cohen\u2019s f according to:  An effect size (f) of > 0.4 was considered to reflect a strong effect (Cohen 1988). The analyses were performed using SPSS (SPSS, Inc., Chicago, IL, USA).\nResults\nThe result section is organized as follows. We first report possible differences in the dependent variables between the eyes open and eyes closed conditions (i.e., main effect of vision, hypothesis 1). Second, we describe the effect of experimentally withdrawing attention from postural control by comparing single task and dual task conditions (i.e., main effect of dual task, hypothesis 2). Third, we report whether significant vision\u00a0\u00d7\u00a0dual task interaction effects were present. Finally, we present the effects of plane, which may reveal possible directional differences in control. In this context, we also report the effects of randomization on the dependent variables to ensure that the observed changes in the dynamics of COP trajectories were genuine effects.\nTable\u00a02 presents the results of the vision\u00a0\u00d7\u00a0dual task ANOVA for the six dependent posturographic measures (i.e., interindividual means corresponding to the main effects of vision and dual task, collapsed over x and y time-series, as well as F, P and f values for main and interaction effects). Significant vision\u00a0\u00d7\u00a0dual task interaction effects are presented in Fig.\u00a02.\nTable\u00a02Main and interaction effects of vision and dual task (i.e., collapsed over x and y time-series) of sample entropy (SEn), standard deviation (\u03c3), sway-path length of the normalized (by the standard deviation) posturogram (SPn), largest Lyapunov exponent (\u03bbmax) and scaling exponent (\u03b1) of COP time-series for 30 healthy individualsConditionMeanVision (EO vs. EC)ConditionMeanDual task (ST vs. DT)Vision\u00a0\u00d7\u00a0dual taskaF(1, 29)P\u0192F(1, 29)P\u0192F(1, 29)P\u0192SEnEO0.723.83=0.060*0.36ST0.701.45ns0.256.72<0.050.48EC0.70DT0.72\u03c3EO3.5211.82<0.0050.64ST3.892.45ns0.293.18=0.0850.33EC4.01DT3.64SPnEO4.275.28<0.050.43ST4.1313.57<0.0050.686.98<0.050.49EC4.52DT4.66\u03bbmaxEO1.5636.23<0.0011.12ST1.710.10ns0.064.26<0.050.38EC1.88DT1.73D2EO2.2323.58<0.0010.90ST2.2045.70<0.0011.266.15<0.050.46EC2.48DT2.51\u03b1EO1.3913.70<0.0010.69ST1.3924.57<0.0010.921.80ns0.25EC1.34DT1.35*\u00a0Significant vision\u00a0\u00d7\u00a0plane interaction (F(1, 29)\u00a0=\u00a05.48, P\u00a0<\u00a00.05, \u0192\u00a0=\u00a00.44), which was caused by the fact that, in contrast to that in the frontal plane, the effect of vision was significant in the sagittal plane (F(1, 29)\u00a0=\u00a06.47, P\u00a0<\u00a00.05, \u0192\u00a0=\u00a00.47)a \u00a0See Fig.\u00a02 for mean values of the conditions EO-ST, EC-ST, EO-DT and EC-DTFig.\u00a02Interindividual averages, collapsed over x and y time-series, of sample entropy (SEn), standard deviation (\u03c3), sway-path length of the normalized (by the standard deviation) posturogram (SPn), local stability (\u03bbmax) and dimensionality (D2) for the four experimental conditions: standing with eyes open (EO-ST), eyes closed (EC-ST), eyes open while performing a cognitive dual task (EO-DT) and eyes closed while performing a cognitive dual task (EC-DT). The asterisks indicate significant (P\u00a0<\u00a00.05) differences between conditions\nIncreased postural task difficulty (EO vs. EC)\nAs is apparent from the significant main effects of vision in Table\u00a02, standing with eyes closed resulted in more regular sway fluctuations, as indexed by a decrease in SEn. In addition, sway variability (\u03c3), dimensionality (D2) and sway-path length (SPn) were increased, whereas \u03b1 and local stability (as indexed by an increase in \u03bbmax) decreased.\nDecreased attention to posture (ST vs. DT)\nCollapsed over x and y time-series and visual conditions, no significant main effects involving dual task were found for \u03c3, SEn or local stability. In contrast, a main effect of dual task was found for both SPn and D2 in that performing a cognitive dual task brought about an increase in both variables as compared to the single task condition (Table\u00a02). Moreover, as shown in Table\u00a02, performing a dual task resulted in a significant decrease of \u03b1 indicating that dual task performance brought about changes in the time-varying structure of sway fluctuations.\nVision\u00a0\u00d7\u00a0dual task interaction effects\nSignificant dual task\u00a0\u00d7\u00a0vision interaction effects (Table\u00a02) revealed that the effects of visual deprivation on SEn, \u03c3 and SPn were different for single and dual task conditions (see Fig.\u00a02). In particular, the observed effect of visual deprivation on \u03c3 and SEn was significant only for the single task condition, whereas the observed increase in SPn only existed for dual task performance (see Fig.\u00a02). On the other hand, as can be appreciated from both Table\u00a02 and Fig.\u00a02, the effects of introducing a cognitive dual task on SEn, \u03c3 and SPn depended on eye closure. Although no significant main effect of dual task was found for SEn, there was a significant dual task\u00a0\u00d7\u00a0vision interaction. This effect occurred because SEn increased significantly from 0.68 to 0.72 through the introduction of a dual task when standing with eyes closed, whereas such an increase was absent when standing with eyes open (0.72 for both single and dual task conditions). Similarly, when standing with eyes open, dual task performance had no significant effect on \u03c3, whereas dual task performance resulted in a decrease in \u03c3 when standing with eyes closed (see Fig.\u00a02).\nThe observed significant dual task\u00a0\u00d7\u00a0vision interaction effects for \u03bbmax were brought about by the fact that the increase (i.e., decrease in local stability) when standing with eyes closed was stronger for the single task condition than for the dual task condition. A similar asymmetric effect was found for D2, in that the increase when standing with eyes closed was stronger for the dual task condition than for the single task condition. Moreover, as can be observed in Fig.\u00a02, the effect of performing a dual task (i.e., increase in D2) was larger when standing with eyes closed than with eyes open.\nEffects of plane and randomization\nCollapsed over conditions, significant differences between sagittal and frontal plane were found for all variables, except \u03b1. In particular, sway variability, \u03c3, and local stability, \u03bbmax, were significantly larger in the sagittal than in the frontal plane (F(1, 29)\u00a0=\u00a020.94, P\u00a0<\u00a00.001, f\u00a0=\u00a00.82 and F(1, 29)\u00a0=\u00a015.19, P\u00a0<\u00a00.005, f\u00a0=\u00a00.72, respectively). In contrast, sample entropy, SEn, and dimensionality, D2, were significantly lower in the sagittal than in the frontal plane (F(1, 29)\u00a0=\u00a0145.38, P\u00a0<\u00a00.001, f\u00a0=\u00a02.31 and F(1, 29)\u00a0=\u00a016.01, P\u00a0<\u00a00.001, f\u00a0=\u00a00.74, respectively). A significant vision\u00a0\u00d7\u00a0plane interaction was found for SEn (F(1, 29)\u00a0=\u00a05.48, P\u00a0<\u00a00.05, f\u00a0=\u00a00.44), which was caused by the fact that SEn was smaller when standing with eyes closed (EC-ST, see Fig.\u00a02) for COP fluctuations in the sagittal plane, whereas this was not the case for COP fluctuations in the frontal plane.\nFigure\u00a03 shows the results of the interindividual means of the surrogate analyses. Sample entropy measures of both phase- and time-randomized surrogate data were significantly higher compared to the original COP time-series (F(2, 58)\u00a0=\u00a010985.1, P\u00a0<\u00a00.001, f\u00a0=\u00a022.34). Whereas scaling exponents of phase-randomized surrogate data and original COP time-series did not differ, correlations were completely absent when the data were time-randomized as evidenced by \u03b1 values around 0.5 (F(2, 58)\u00a0=\u00a05436.7, P\u00a0<\u00a00.001, f\u00a0=\u00a014.12). As a result of very high-dimensional noise in the time-randomized data, no embedding dimension could be estimated and hence no dimensionality estimates and Lyapunov exponents could be determined for the time-randomized surrogate data.8 Randomizing the phases of original COP data significantly increased the dimensionality (F(1, 29)\u00a0=\u00a050.07, P\u00a0<\u00a00.001, f\u00a0=\u00a01.31). In addition, the Lyapunov exponent of the phase-randomized surrogate data was significantly higher than that of the original time-series (F(1, 29)\u00a0=\u00a053.44, P\u00a0<\u00a00.001, f\u00a0=\u00a01.36).\nFig.\u00a03Grand means, collapsed over all conditions, planes and participants, of sample entropy, scaling exponent, dimensionality and local stability for the original (OR) COP time-series and their phase-randomized (PHASE) and time-randomized (TIME) surrogate counterparts. The error bars represent the interindividual standard deviations. The asterisks represent significant (P\u00a0<\u00a00.05) differences between the surrogate data and the original time-series\nDiscussion\nThe present experiment was conducted to investigate the role of attention in the regulation of posture. Specifically, we examined whether an increase in postural sway regularity (i.e., as indexed by a decrease in SEn) is representative of an increase in cognitive investment in postural control. We hypothesized that COP trajectories become more regular (i.e., SEn decreases) when task difficulty is increased (EC vs. EO) and, conversely, become less regular (i.e., SEn increases) when an attention-demanding cognitive dual task is introduced (DT vs. ST). We further expected that these changes in regularity of COP fluctuations would be accompanied by changes in variability, local stability, sway-path length, dimensionality and scaling exponent reflecting functional modifications of postural control. For the proper interpretation of the present findings, however, it was necessary to ascertain that the observed structure (and changes herein) of the COP fluctuations did not result from noise, but was indeed brought about by deterministic processes. Therefore, we will first discuss the results of the surrogate analyses before discussing the respective effects of vision, dual task and plane on the dynamical structure of postural sway.\nSurrogate analyses\nAlthough nonlinear estimates of dynamical structure are not readily interpretable in an absolute sense, they can be meaningfully interpreted by comparing them across conditions, (see e.g., Newell et al. 1993), as well as with surrogate data. For example, entirely random data are characterized by large (theoretically infinite) dimensionality and large \u03bbmax values, whereas chaotic\/deterministic data have smaller dimensionality and smaller \u03bbmax values. In the present analysis, the surrogate data had greater dimensionality and larger \u03bbmax values than the original data, implying that the latter had considerable deterministic structure (cf., Theiler et al. 1992). Moreover, both phase- and time-randomized surrogate data showed increased sample entropy values. Hence, the original COP fluctuations clearly had a deterministic component, which was evidenced further by the fact that the scaling exponents became 0.5 after time-randomization (resulting from a loss of temporal correlations in the shuffled time-series), but remained unaffected by phase-randomization (i.e., preserving temporal correlations). These findings are consistent with those of previous studies suggesting that COP fluctuations are (largely) of deterministic origin (e.g., Doyle et al. 2004; Riley et al. 1999; Yamada 1995b), and testify to the relevance and need of including dynamical measures in posturography.\nIncreasing postural task difficulty (EO vs. EC)\nIn the present study, standing with eyes closed brought about an increase of sway variability, which was accompanied by an increase in dimensionality and \u03bbmax, implying that local stability decreased. Possibly, the observed increase in dimensionality may serve as a mechanism to enrich information so as to facilitate the control of standing and to cope with the reduced (local) stability (cf., Riley and Clark 2003; van Emmerik and van Wegen 2002). These observations are in line with the common notion that visual deprivation increases the task difficulty of postural control, and, consequently, requires cognitive monitoring of postural control. We expected that this increase in cognitive investment would be accompanied by a decrease in SEn (i.e., an increase in regularity), which was indeed the case (see Fig.\u00a02, compare EO-ST with EC-ST), thus confirming hypothesis 1. These findings are consistent with the results of other studies showing that COP variability tends to increase as experimental task conditions become increasingly difficult, whereas the temporal structure of postural sway tends to become increasingly regular (Riley and Clark 2003; Roerdink et al. 2006; Thurner et al. 2002).\nIn the present study, visual deprivation resulted in qualitatively similar changes in the structure of COP trajectories as reported in the study of Roerdink et al. (2006) for stroke patients (i.e., increased \u03c3, \u03bbmax, D2, and decreased SEn and \u03b1 with respect to healthy elderly adults). In healthy young adults, the qualitatively similar changes in COP dynamics with visual deprivation as compared to standing with eyes open may, likewise, be interpreted to indicate that postural control is performed less automatically and effectively. Whereas in stroke patients such modifications in postural control may be due to a defect or slowing down of the central processing of sensory information (cf., Teasdale et al. 1991; Woollacott et al. 1986), in young healthy adults a more regular sway, resulting from standing with eyes closed, implies increased \u201cactive\u201d monitoring of postural control with increasing task difficulty (cf., Andersson et al. 1998; Nashner and McCollum 1985; Redfern et al. 2001; Teasdale et al. 1993; Teasdale and Simoneau 2001). If the proposed relation between cognitive investment in postural control and postural sway regularity does indeed exist, then the performance of a cognitive dual task should result in less regular COP trajectories. That this was indeed the case will be discussed in the next two subsections.\nWithdrawing attention from posture\nThe performance of a concurrent dual task led to changes in scaling exponents and an increase in dimensionality, reflecting cognition-invoked adjustments of postural control. These changes under dual task performance may have served to enrich the information captured in sway fluctuations without increasing the amount of sway (i.e., variability remained unaltered). This interpretation is amplified by the observation that the sway-path length of the normalized posturogram increased, indicating more twisting and turning in the COP trajectories. Interestingly, despite the fact that attention was withdrawn experimentally from postural control, local stability remained unaltered. In contrast to what we expected, no main effect of dual task was found for SEn. However, this finding does not necessarily militate against the proposed relation between the regularity of COP fluctuations and the amount of attention directed to postural control, as will be argued in the following subsection.\nVision\u00a0\u00d7\u00a0dual task interaction\nEspecially noteworthy in this context and in view of our expectations is that while standing with eyes closed postural sway regularity decreased (i.e., sample entropy increased) when performing a cognitive dual task (compare EC-DT and EC-ST in Table\u00a02 and Fig.\u00a02). This finding is consistent with hypothesis 2 and supports the proposed positive correlation between COP regularity and the degree of attention involved in postural control. Apparently, the fact that regularity remained unaltered (i.e., 0.72) when performing a dual task while standing with eyes open (EO-DT) implies that, for young healthy adults, standing with eyes open is not very attention demanding. Conversely, during the more challenging task of standing with eyes closed (EC-ST) COP fluctuations became more regular (i.e., 0.68; Fig.\u00a02). However, the finding that sample entropy again increased to its \u201cnormal\u201d (EO-ST) level when withdrawing the focus of attention from the postural task (EC-DT) indicates that the increased cognitive monitoring of posture during EC-ST had a detrimental effect. Visual deprivation increased the awareness of the postural task (i.e., creating an internal focus), resulting in efforts to actively (consciously) control posture and, as such, preventing the postural control system to work in a relatively automatic and efficient manner (viz., Andersson et al. 2002; Hunter and Hoffman 2001; McNevin and Wulf 2002; Milton et al. 2004).\nSimilarly, when standing with eyes open the attention-demanding dual task had no significant effect on \u03c3, whereas when standing with eyes closed the dual task did result in a decrease in \u03c3 (see Fig.\u00a02), corroborating the findings of e.g., Andersson et al. (2002), McNevin and Wulf (2002) and Morioka et al. (2005). In contrast, many authors have found an increase in postural sway variability when performing a cognitive dual task (see Shumway-Cook and Woollacott 2000). It has been suggested that this effect of dual task on the amount of sway may, in part, result from articulation (Dault et al. 2003; Yardley et al. 1999b). In this context, it is important to note that the present finding that variability actually decreased with the introduction of a cognitive dual task indicates that articulation played no significant role in the present study.\nSagittal versus frontal plane\nThe increased variability of the COP trajectories in the sagittal plane (as compared to the frontal plane) was accompanied by reduced local stability and greater regularity (i.e., a decrease in sample entropy). These observations are consistent with the findings of Roerdink et al. (2006), which showed that in healthy elderly adults local stability was reduced in the sagittal plane, whereas regularity and variability were elevated in this plane. Based on these results, they suggested that posture is mainly controlled in the direction of largest postural sway (i.e., sagittal plane), which required a certain amount of attention as reflected by the regularity findings. The present observation that during standing with eyes closed (EC-ST) regularity increased significantly in the sagittal plane, whereas no effect was found for the frontal plane, is in line with this suggestion: young healthy adults mainly control posture in the sagittal plane, which becomes particularly attention demanding when task difficulty is increased (see also Pellecchia 2003; Riley et al. 2003).\nConclusion\nThe present study showed that the amount of attention invested in postural control is positively correlated with sway regularity. Specifically, the present study showed that increasing postural task difficulty by means of visual deprivation (EC-ST) not only resulted in an increase of COP variability and a decrease in local stability, but also in more regular COP trajectories. These findings could be taken to imply that the participants actively monitored their posture in order to cope with the increased postural task difficulty. However, when the amount of attention invested in postural control was experimentally reduced by introducing a cognitive dual task (EC-DT), both regularity and variability of sway fluctuations returned to values observed when standing with eyes open (EO-ST). This finding suggests that during standing with eyes closed (EC-ST) the increase in monitoring posture was due to an increase in awareness of the postural task (i.e., internal attentional focus), preventing postural control from working in an automatic and efficient manner (e.g., Hunter and Hoffman 2001). All in all, it is fair to conclude that the methodological and analytical approach adopted in the present study allows for disentangling whether or not there is an increase in cognitive involvement and to what extent an increase in cognitive involvement has a detrimental or beneficial effect.","keyphrases":["regularity","center-of-pressure","attention","postural control"],"prmu":["P","P","P","P"]}
{"id":"Plant_Mol_Biol-3-1-1805039","title":"Global genome expression analysis of rice in response to drought and high-salinity stresses in shoot, flag leaf, and panicle\n","text":"To elucidate genome-level responses to drought and high-salinity stress in rice, a 70mer oligomer microarray covering 36,926 unique genes or gene models was used to profile genome expression changes in rice shoot, flag leaf and panicle under drought or high-salinity conditions. While patterns of gene expression in response to drought or high-salinity stress within a particular organ type showed significant overlap, comparison of expression profiles among different organs showed largely organ-specific patterns of regulation. Moreover, both stresses appear to alter the expression patterns of a significant number of genes involved in transcription and cell signaling in a largely organ-specific manner. The promoter regions of genes induced by both stresses or induced by one stress in more than one organ types possess relative enrichment of two cis-elements (ABRE core and DRE core) known to be associated with water stress. An initial computational analysis indicated that novel promoter motifs are present in the promoters of genes involved in rehydration after drought. This analysis suggested that rice might possess a mechanism that actively detects rehydration and facilitates rapid recovery. Overall, our data supports a notion that organ-specific gene regulation in response to the two abiotic stresses may primarily be mediated by organ-specific transcription responses.\nIntroduction\nDrought and highly saline soils are among the most serious challenges to crop production in the world today. This is particularly the case in developing countries, where these abiotic stresses severely limit crop growth and productivity. Both traditional breeding and genetic engineering of crop plants have been utilized to improve drought and high-salinity tolerance or resistance with the goal of increasing agricultural productivity in affected regions. Understanding plant responses to abiotic stresses at the genomic level provides an essential foundation for future breeding and genetic engineering efforts.\nRecent research on drought and high-salinity responses in Arabidopsis implied that a large proportion of the genome is involved in drought (Shinozaki et al., 2000, 2003) or high-salinity stress responses (Xiong et al., 2002; Zhu, 2001, 2002). In several cases, it has been shown that alteration of individual gene expression level can significantly impact responses to drought (Garg et al., 2002; Haake et al., 2002) or high-salinity stresses in plants (Kasuga et al., 1999; Shi et al., 2003; Xu et al., 1996; Zhang et al., 2004). Genome-wide identification of genes regulated by drought or high-salinity conditions has manifold significance. First, it provides a more comprehensive understanding of the transcriptional responses to those stresses. Second, it provides a starting point for further elucidating the role of individual genes in stress responses, which will be of great value in crop engineering. Third, it aids in the identification of stress responsive promoters and responsible cis-elements within them that are important both for basic study and crop engineering applications.\nDNA microarrays provide a high throughput means of analyzing genome expression, which has been used to study patterns of gene expression in response to drought or high-salinity stresses in several plant species (Seki et al., 2003, 2004). Initially, a microarray containing \u223c1,300 full-length cDNA clones from Arabidopsis was used to study gene expression under drought and cold stresses. This study resulted in the identification of 44 and 19 cDNA clones as drought and cold-inducible genes, respectively (Seki et al., 2001). Other studies employed an improved microarray containing around 7,000 Arabidopsis full-length cDNA clones to profile gene expression in response to abscisic acid (ABA) treatment (Seki et al., 2002a) as well as cold, drought, and high-salinity stresses (Seki et al., 2002b). Another study employed an Affymetrix GeneChip covering approximately 8,100 genes from Arabidopsis to monitor changes in gene expression under salt, osmotic, and cold stresses. This study revealed that resulting expression changes varied significantly between root and leaf, with only minor overlap (Kreps et al., 2002). Similar studies have also been performed in barley to assess the drought and high-salinity gene expression responses using a microarray containing 1,463 DNA elements (Ozturk et al., 2002).\nRice (Oryza sativa) is a model plant for cereal crops and has perhaps the richest set of resources available for plant genomic studies (Feng et al., 2002; Goff et al., 2002; RCSC, 2003; Sasaki et al., 2002; Yu et al., 2002). In rice, the high-salinity stress response has been analyzed with a microarray containing 1,728 cDNA clones from a root cDNA library of salt-tolerant rice (var Pokkali) (Kawasaki et al., 2001). Another study with a microarray containing 8,987 DNA elements detected 509 possible abscisic acid (ABA) or gibberellin-responsive genes (Yazaki et al., 2003). Later, the same group used a 22,000 rice cDNA based oligonucleotide array to identify ABA and GA responsive genes, made comparison of ABA-responsive genes and their putative responsible promoter elements between rice and Arabidopsis (Yazaki et al., 2004). This analysis gave an initial global view of the ABA- and GA-responsive genes in rice. A total of 73 genes induced by cold, drought, high-salinity and ABA treatment were further identified using a microarray containing 1,700 full-length cDNA clones (Rabbani et al., 2003). Recently, an Affymetrix rice genome array containing 55,515 probe sets was used to profile the transcriptomes of rice strains with salt-tolerant and salt-sensitive genotypes, revealing genome-wide differential transcription under salinity treatments during vegetative growth (Walia et al., 2005). Taken together, previous studies in rice have identified various genes regulated by different stress conditions. However, a systematic comparison of whole-genome expression responses to drought and high-salinity stresses in various organs has not yet been performed.\nThe availability of the complete genome sequences of two rice sub-species (Yu et al., 2005) makes the construction of a whole-genome microarray possible. A whole-genome 70mer oligomer microarray for rice was developed and successfully employed to obtain genome expression profiles (Ma et al., 2005b; Jiao et al., 2005). Here, we use this whole-genome microarray to monitor expression changes for a total of 36,926 genes in response to drought and high-salinity stresses in shoots at the four-tiller stage and in flag leaves and panicles at 1\u00a0week prior to heading. This analysis revealed the extent of reprograming and alteration of cellular pathways in response to drought and high salinity stresses. The possible underlying mechanism for the observed reprograming of the genome expression was examined.\nMaterials and methods\nPlant material and stress treatments\nPlant material used in this study was O. Sativa L. ssp. indica (cv. Minghui 63). Shoot samples were selected at the four-tiller stage (vegetative stage) and flag leaf and panicle samples were selected at one-week-before-heading (reproductive stage). The plants used for shoot samples were grown in hydropolic half-strength Hoagland solution up to the four-tiller stage, and then plants allowed to reach the reproductive stages were grown in soil.\nFor drought treatment samples, plants were taken out from the Hoagland solution directly and their roots were put on filter paper for drought treatment. Under this condition, the phenotype of shoot was observed. Materials at the following stages were collected: Stage 1 (D1), when leaves were slightly rolled and leaf relative water content (RWC) was around 90\u201395%; Stage 2 (D2), when leaves were half-rolled and leaf RWC was around 80\u201385%; Stage 3 (D3), when leaves were completely rolled and leaf RWC was around 70\u201375%. For most of the plants, usually it took half to 1\u00a0h to reach stage 1, and then after another 2\u00a0h or 4\u00a0h, they could reach stage 2 or stage 3 respectively. Rehydration samples were prepared by first subjecting the rice plants to drought treatment until leaves were completely rolled (D3), then supplying enough water and samples were collected after 48\u00a0h. For each treatment stage, three independent replicates were collected. For sample collection under high-salinity treatment (200\u00a0mM NaCl), rice plants were collected when plants reached stages 1, 2, or 3 as outlined above for drought treatment. Rice plants were taken out from Hoagland solution and were directly put into Hoagland solution with 200\u00a0mM NaCl. For most of plants, usually it took 5\u00a0h to reach stage 1, and then after another 12 and 24\u00a0h, they could reach stage 2 and stage 3 respectively. For each stage, three independent replicates were collected. Samples from normal grown plants corresponding to each drought and high-salinity stages and rehydration stage were also collected at the same time as respective controls. Upon collection, samples were frozen immediately in liquid nitrogen and stored in the \u201380\u00b0C freezer. The estimation of RWC in rice plants was prior reported method (Bare and Weatherley, 1962).\nRNA isolation, probe labeling, and hybridization\nRNA preparation, fluorescent labeling of probes, slide hybridization, washing and scanning were performed as described previously (Ma et al., 2005b). Total RNA was prepared from frozen samples using the RNAwiz reagent (Ambion). Each RNA preparation was used to generate a labeled cDNA probe for hybridization. There were at least three high-quality replicate data sets for each experiment, with each data set obtained from an independent biological sample.\nThe cDNA synthesis, probe labeling, and microarray hybridization, microarray slide washing, and array scanning were performed following previously described protocols (Jiao et al., 2005). Hybridized microarray slides were scanned with an Axon GenePix 4000B scanner, and independent TIFF images for both Cy3 and Cy5 channels were used for subsequent analysis.\nMicroarray and initial data analysis\nThe microarray construction was described previously (Ma et al., 2005b, Jiao et al., 2005). Microarray data were preprocessed with Limma R Package (Smyth, 2005). We first performed Loess normalization to balance the dye-bias, and to remove the print-tip effect within each array. Quantile normalization was performed to remove the experimental variances across replicate arrays. Based on the distribution of negative control oligos within each slide, we determined an experimental threshold for minimum non-specific hybridization intensity, and the gene oligos above 90-percentile of negative oligos were set as to be detected in each experiment.\nTo identify the genes that respond to drought and salinity stress, we used the Limma R implementation to perform hypothesis tests by fitting a linear model to the expression data. During pair-wise comparison between each treated sample versus control, a moderated t-statistics were computed, by using an empirical Bayes method to shrink the gene-wise sample variances towards a common value (Smyth, 2004), and the differential level was represented by a log (2) of intensity ratio. The P value adjustment used in false discovery rate control for multiple testing is Benjamini and Hochberg method (Benjamini and Hochberg, 2000; Reiner et al., 2003). The genes with significantly differential expression were selected by the adjusted P values less than 0.05.\nRT-PCR analysis of genes\nThe expression profiles were further quantified by RT-PCR and compared to results obtained by chip hybridization. The first strand of cDNA was generated from 1\u00a0\u03bcg of total RNA isolated independently from each sample in a 100\u00a0\u03bcl volume and 1\u00a0\u03bcl was used as template in each PCR reaction (25 cycles of 1\u00a0min at 94\u00b0C, 1\u00a0min at 58\u00b0C, 1\u00a0min at 72\u00b0C). A total of eight drought-induced genes were selected for RT-PCR analysis (the primers of these genes are listed in Table S18). The Actin1 gene of rice was used as a control for RT-PCR experiments (forward primer, 5\u2032-cgcagtccaagaggggtatc-3\u2032; reverse primer, 5\u2032-tcctggtcatagtccagggc-3\u2032).\nFunctional classification\nGO terms used in rice gene functional annotations were downloaded from the BGI-RIS database at http:\/\/rise.genomics.org.cn (Zhao et al., 2004). For biochemical pathway analysis, we classified genes by associated biochemical pathway(s) using the AraCyc database (http:\/\/www.arabidopsis.org\/tools\/aracyc) for Arabidopsis, which is based on MetaCyc pathway collections (Mueller et al., 2003). A rice gene was considered to be associated with a biochemical pathway if it had an Arabidopsis homolog in that pathway. To compare the expression profiles of conserved genes between rice and Arabidopsis, we selected reciprocal best-matched gene pairs between rice and Arabidopsis, which are based on a TBLASTN search of both rice and Arabidopsis gene sequences. This method used to identify highly homologous genes between rice and Arabidopsis has been described in previous reports (Jiao et al., 2005; Ma et al., 2005b). For the diagrams in Fig.\u00a0S3, the expression ratios of the selected rice genes at D3 and S3 stage only were used.\nAnalysis of cis-acting regulatory elements\nDifferentially expressed genes supported by available full-length cDNA\u00a0sequences were used to further elucidate regulatory cis-elements in the putative promoter regions. After mapping the FL-cDNAs on indica rice 12 pseudo-molecules, 2\u00a0kb DNA sequences upstream from the 5' end of the available cDNA sequences were extracted and searched against a plant cis-regulatory database (Higo et al., 1999) (http:\/\/www.dna.affrc.go.jp\/htdocs\/PLACE\/signalscan.html). The abundance of known ABRE core and DRE core elements in each query set were counted.\nIdentification of novel cis-regulatory elements was performed using the Improbizer tool (cis-Site Seeker) by Jim Kent (http:\/\/www.cse.ucsc.edu\/\u223ckent\/improbizer\/), which is based on ab-initio prediction of consensus binding sites among each co-regulated gene set. The upstream 2\u00a0kb regions of co-regulated FL-cDNA genes were used for common motif searching. To evaluate the significance for each putative novel motif, we generated an equal number of 2\u00a0kb contrast sequences in each query set by randomly arranging the four nucleotides. We performed the standard Student\u2019s t-test on motif score tables, comparing query set versus contrast set. The known ABRE motif predicted by Improbizer has a pretty high confidence level (above 0.999), and the other two novel motifs were calculated to above a 0.95 confidence level. Motif consensus sequences were displayed by WEBLOGO program at http:\/\/weblogo.berkeley.edu\/ by Crooks et al. (2004).\nGel shift assays\nNuclear protein extracts and gel mobility shifts were performed as described previously (Gupta et al., 1998). Oligomers were synthesized using the following sequences: OsJRFA070715: GCAGATACTGCAGCCAACCTCTCT, OsJRFA070715M: GCAGATACTGAAGCCAACCTCTCT. The complement sequence for each oligo was also synthesized. After denaturing and annealing, double-stranded probes were used for labeling.\nClustering and chromosomal location analysis of genes involved in stress responses\nUnsupervised classification of stress-response genes was based on log-transformed ratios of all up- or down-regulated genes detected in at least one stage of drought treatment. We performed hierarchical clustering analysis to display the expression pattern and tree diagram at different stress stages by employing Cluster and TreeView (Eisen et al., 1998). Classification of rehydration-induced genes was based on the log-transformed ratios of 48-h rehydration and three stages of drought treatment in the three organs, and 807 genes in flag leaf, 281 genes in shoot, and 224 genes in panicle were grouped with the above method.\nTo analyze the genome-wide distribution of rehydration-induced genes in the three organs and drought and high-salinity induced genes in shoot, as well as identify potential clusters of co-regulated genes, we mapped these genes to the indica 12 chromosomes using the BLAT program. The expression profiles in Fig.\u00a09 were drawn using a customized Perl script plus a scalable vector graphics (SVG) bundle.\nResults\nIdentification of drought and high-salinity stress responsive genes using a rice whole genome oligomer microarray\nA previously reported rice genome microarray containing 70mer oligomer probes for 36,926 unique genes or gene models (Ma et al., 2005b; Jiao et al., 2005) was used for genome-wide profiling of gene expression responses to drought and high-salinity stresses. Total RNAs were isolated from four-tiller stage shoots and from flag leaves and panicles (as described in Experimental procedures) at three different time points (or sampling stages) after initiation of drought or high-salinity stress treatments (Fig.\u00a0S1). The sampling stages for both drought and high-salinity treatments were based on morphological phenotypes (leaf rolling state) and physiological states (relative water content, RWC, see Bare and Weatherley, 1962). Stage one is defined as exhibiting slightly rolled leaves with 90\u201395% RWC. Stage two is defined as exhibiting halfway rolled leaves with 80\u201385% RWC. Stage three is defined as exhibiting completely rolled leaves and with 70\u201375% RWC. For drought treatment, the three stages were designated as D1, D2, and D3, while the three stages for the high-salinity treatment were designated as S1, S2, and S3 (see Fig.\u00a0S1). The cDNA probes produced from the stress-treated and corresponding untreated control sample pairs were hybridized to microarray slides. For each sample point, three replicates were performed with dye-swap to correct for uneven dye effects.\nThe microarray data processing includes LOESS normalization within slide and quantile normalization between slides to remove the systematic errors (see Experimental procedures for details). To identify the differentially expressed genes, Limma R implementation was used to perform pair-wise comparison, and a moderated t-statistics was computed for each gene of treated sample versus control sample. Genes with FDR-adjusted P value less than 0.05 were considered to indicate significant differences (see Experimental procedures for details). Numbers of genes up- or down-regulated by drought or by high-salinity stress at each stage in each organ were shown in Fig.\u00a01. In total, 582, 1,257 and 614 drought up-regulated genes and 795, 646 and 1,305 drought down-regulated genes were identified in flag leaf, shoot and panicle, respectively; and 1,676, 817 and 1,310 high-salinity up-regulated genes and 1,270, 1,323 and 2,284 high-salinity down-regulated genes were identified in flag leaf, shoot and panicle, respectively (Fig.\u00a02A, B). The complete lists of these genes are available in the supplemental data (Tables\u00a0S2\u2013S13).\nFig.\u00a01The number of differentially expressed genes at each stage of drought and high-salinity treatments in three organs. Differentially expressed genes (both induced or repressed) are defined as those with P\u00a0<\u00a00.05. D1, D2 and D3: three stages of drought stress, S1, S2, and S3: three stages of high-salinity stress. y-axis shows the gene number. (A) Number of genes induced or repressed in flag leaf. (B) Number of genes induced or repressed in panicle. (C) Number of genes induced or repressed in shoot.Fig.\u00a02Comparison of drought and high-salinity genome expression responses in the three rice organs. (A) Total number of drought and high-salinity stress inducible genes and the number of genes induced in response to both stresses in each of the three rice organs. Genes induced at least at one stage under drought or high-salinity treatments are included in the analysis. y-axis shows the gene number. (B) The total number of drought and high-salinity stress repressed genes and the number of genes repressed in response to both stresses in each of the three rice organs. Genes repressed at least at one stage under drought or high-salinity treatments are included in the analysis. y-axis shows the gene number. (C) Clustering analysis of all drought and high salinity responsive genes in each of the three rice organs. The differentially expressed genes (both induced or repressed) with P\u00a0<\u00a00.05 at least at one stage of drought treatment are included. The median ratio (treated\/untreated sample) is log2 transformed and subject to complete linkage hierarchical clustering. D1, D2, and D3: three stages of drought treatment; D3R: 48-h water recovery after drought; S1, S2, and S3: three stages of high-salinity treatment. In total, 1,377, 1,903, and 1,919 genes from flag leaf, shoot, and panicle, respectively, were included in this analysis. (D) Relatedness of the genome expression patterns across selected stress-treated rice organs. A complete-linkage hierarchical clustering analysis of overall relatedness for expression ratios from selected organs at the stage 3 of both abiotic stress treatments. The abbreviations for different sample types are the same as in panel C\nMost of the previously known genes responsive to both drought and high-salinity stress have been recovered from our microarray analysis. Those include the LEA protein (OsJRFA063984), aquaporin (OsIRUA001311), OsNAC1 (OsJRFA108080), dehydrin rab 16b (OsIFCC035025) and DREB1 (OsJRFA067313). Many other responsive genes have been identified for the first time in this study (Table\u00a0S1C). Among those newly revealed genes, some are expected to be involved in general cell function or known to be involved in other stress responses, while others have putative or unknown function. The annotation for some of those genes suggested that they include transcription factors from multiple families, heat shock proteins, various stress (drought, high-salinity, disease, cold, and ABA) responsive genes, protein kinases, transporters, photosynthesis enzymes, and other metabolic pathways (Table\u00a0S1C). The diversity of affected processes suggests a high level of complexity in regulation.\nThere is a significant overlap between genome expression profiles in response to drought and high-salinity stress\nTo compare the gene expression profiles of the drought and high-salinity stress responses, genes commonly regulated by both stresses at each organ type were examined. A total of 322, 415, and 174 genes were up-regulated and 215, 173, and 372 genes were down-regulated by both stresses in flag leaves, shoots and panicles, respectively (Fig.\u00a02A, B). The common genes represent 55, 33 and 28% (induced) and 27, 27 and 29% (repressed) of all drought-responsive genes in flag leaf, shoot and panicle, respectively. These results indicate that about one-third, even half in flag leaf, of the drought responsive genes in each organ are also regulated by high salinity stress.\nTo further compare gene expression under the two abiotic stresses, all drought regulated genes were selected for cluster analysis (see Experimental procedures). As shown in Fig.\u00a02C, up to half of the genes up- or down-regulated by drought stress also exhibit a similar expression pattern under high-salinity stress. The remainder of the drought responsive genes exhibits minimal expression changes or distinct expression patterns in response to high-salinity stress.\nAmong genes specifically induced by drought stress, some have known functions while others were previously predicted to have functions related to drought stress or the ABA response. For example, our experiments showed that genes with putative functions including NAM, HLH, G-box binding, Zn-finger, AP2 transcription factors, and protein kinases (including MAPK family genes) were all affected by drought stress. Quite a few known drought-responsive genes such as DREB1A, LEA protein, WSI76 protein, MAP65 (microtubule associated protein), and ubiquitin were also included. These genes were specifically induced by drought and may function exclusively in the response to drought stress in rice. To gain an overall view of the effects of drought and high-salinity stress on various functional gene groups, gene ontology (GO) categories for drought and high-salinity stress induced genes in three organs were examined (Fig.\u00a0S2). For the majority of these functional categories, the relative numbers of genes responsive to the two stresses were similar.\nWe also examined the effects of drought and high-salinity on rice genes coding enzymes in known metabolic pathways using shoot as a model (Fig.\u00a0S3). Some pathways are similarly regulated by the two stresses, including the Calvin cycle, TCA cycle variation I, brassinosteroid biosynthesis II, gibberellin biosynthesis, and IAA biosynthesis I, and the de novo biosynthesis of pyrimidine ribnucleotides\/pyrimidine deoxyribonucleotides\/purine nucleotides (Fig.\u00a0S3 and data not shown). While other pathways respond differentially to drought or high-salinity stresses, the representative pathways include sterol biosynthesis, sugars and polysaccharides, ABA biosynthesis, lignin biosynthesis, octane oxidation, cutin biosynthesis, and starch degradation (Fig.\u00a0S3). For example, some steps in these pathways were repressed or invariable under high-salinity stress, but were induced by drought stress. It is noticeable that one step in the ABA-biosynthesis pathway (corresponding to Arabidopsis NCED3) showed slightly inhibited in gene expression under high-salinity stress (only at stage 3), but was induced under drought treatment (Fig.\u00a0S3c). Our informatic analysis suggested that OsJRFA107649 is the closest Arabidopsis NCED3 homolog, with 66% identity (E\u00a0=\u00a01.8E\u2013195). From our analysis, OsJRFA107649 expression ratios (log2 transformed) were 0.278 (D1), 3.461 (D2) and 2.53(D3) under drought treatment and 3.626 (S1), 2.283 (S2) and \u20131.958 (S3) under high salinity treatment. The expression ratios of rice gene at D3 and S3 stage were used for the diagram in Fig.\u00a0S3. It is interesting to note that under high salinity this gene was initially induced and then inhibited at stage 3 (S3 ratio: \u20131.958), while under drought treatment this gene was induced starting at stage 2 and maintained after stage 3 (D3 ratio: 2.53). This example illustrated a distinct response kinetics for different responsive genes in response to drought and high-salinity stresses.\nBy comparing transcriptomes under drought and high-salinity stresses in three distinct organs, we found that drought responsive genome expression in flag leaf is closer to that for high salinity stress than to the drought expression response observed in any other organ (Fig.\u00a02C, D). In general, there seems to be a closer relationship between the transcriptome-level responses to the two abiotic stresses in the same organ than between or among transcriptomes in distinct organs in response to the same stress. However, the extent of overlap in the responses to drought or high-salinity stresses varies for the different rice organs. For example, it is more divergent in the responses to the two abiotic stresses in panicle, while flag leaves show the greatest overlap.\nDistinct groups of genes are induced during rehydration after drought stress\nIn its natural environment, a plant\u2019s ability to respond to rehydration after drought stress is important for its survival, although little is known about the gene expression changes that occur during rehydration. Our experiments showed that after a period of drought (third stage), all the rice leaves rolled and turned yellow, and the RWC dropped to 70\u201375%. When we applied water to those severely stressed plants, rice leaves started to unroll after 5\u00a0h, and exhibited normal flatness within 24-48\u00a0hours of rehydration. We collected tissue samples 48\u00a0h after rehydration and extracted total RNA for gene expression analysis (Fig.\u00a02C).\nComparing to samples from normal grown rice plants, in flag leaf, shoot and panicle, 807, 281 and 224 genes were up-regulated after 48\u00a0h of rehydration following drought stress, respectively (Fig.\u00a03, Table S14\u2013S16). We further compared gene expression profiles during drought treatment with those after rehydration (Fig.\u00a03A\u2013C). The expression patterns of those genes seem to belong to three groups, with flag leaf exhibiting the greatest number of gene expression changes following rehydration. Group I, which included 71, 98 and 68 genes from flag leaf, shoot and panicle, respectively, was induced during both drought treatment and rehydration. Group II, which included 71, 8 and 21 genes in flag leaf, shoot and panicle, respectively, was repressed during drought stress but induced by rehydration (for shoot and panicle, see Table\u00a01). Group III, which included all remaining 665 genes in flag leaf, 175 genes in shoot, and 135 genes in panicle, showed no significant expression changes under drought stress but were up-regulated during rehydration. We suspected that group I genes must be induced by drought stress, but have yet to return to normal levels after 48-h rehydration. Those genes in groups II and III were specifically induced by rehydration.\nFig.\u00a03Cluster analysis of genes exhibiting elevated expression after 48-h rehydration following drought. (A) (B) and (C) Cluster analysis of the genes exhibiting significant elevation of expression (with P\u00a0<\u00a00.05 as threshold) after 48\u00a0h of rehydration treatment for flag leaf, shoot, and panicle. The median ratio is log2 transformed and subject to complete linkage hierarchical clustering. A total of 807 genes in flag leaf (A), 281 genes in shoot (B), and 224 genes in panicle (C) are included in this analysis. D1, D2, and D3 represent three stages of drought treatment, and D3R represents 48-h rehydration of the D3 stage. (D) The histogram shows the number of genes in each of the three rice organs induced under drought treatment at least at one drought stage, after 48-h rehydration following the D3 stage, and the number of genes with overlapping expression in various pairs of organs. y-axis shows the gene numberTable\u00a01Genes induced by 48-h rehydration and inhibited by drought stress in shoot and panicleGene namePutative functionsD1D2D3D3RShootOsIFCC006273Unknown0.596\u22121.611\u22123.4971.920OsIFCC021002Glycosyl hydrolases family 170.440\u22121.105\u22121.7752.470OsJRFA107373Unknown\u22121.521\u22120.487\u22124.3482.470OsJRFA101949AP2 domain0.216\u22120.166\u22122.4562.026OsJRFA106016Kelch motif\u22120.315\u22120.582\u22122.6041.865OsJRFA070715Peroxidase\u22120.189\u22120.499\u22122.4562.215OsJRFA065471Folate\/biopterin transporter0.331\u22120.533\u22122.5953.095OsIFCC041459Chitinase\u22120.460\u22120.136\u22122.1251.972PanicleOsIFCC023423Photosystem I reaction center subunit IV\u22120.067\u22120.293\u22122.6102.206OsIFCC032344Photosystem I reaction center subunit n\u22120.141\u22120.626\u22121.7002.390OsIFCC001915Photosystem II polypeptide\u22120.119\u22120.934\u22121.7741.787OsJRFA061606Pathogenesis\u2013related protein PR-10a\u22121.811\u22120.007\u22121.8414.117OsJRFA107373Unknown\u22123.6930.395\u22121.9785.605OsJRFA109398Chlorophyll a\/b binding protein\u22121.867\u22121.348\u22121.8991.905OsIFCC025509Photosystem II 10\u00a0kDa polypeptide PsbR\u22121.077\u22120.966\u22121.7351.831OsIFCC038501Chloroplast precursor\u22121.087\u22120.966\u22121.8051.837OsIFCC022062Photosystem I reaction center subunit VI\u22120.540\u22120.206\u22121.9961.811OsIFCC024081Chlorophyll a\/b-binding protein\u22120.595\u22120.685\u22121.8142.298OsIFCC014461Unknown\u22120.794\u22120.660\u22121.7221.790OsIFCC017469Unknown\u22120.879\u22121.619\u22121.7642.417OsIFCC012276Catalase\u22121.567\u22120.594\u22121.8932.592OsJRFA060135Unknown\u22120.922\u22120.491\u22122.5401.793OsIFCC033400Pathogenesis-related protein PR-10a\u22120.115\u22120.032\u22122.5502.008OsJRFA059435Magnesium-protoporphyrin IX monomethyl esteraerobic oxidative cyclase\u22120.344\u22120.348\u22121.8372.851OsIFCC022709Protease inhibitor\/seed storage\/LTP family\u22120.091\u22120.300\u22121.6841.842OsJRFA061968Geranylreductase\u22120.334\u22120.244\u22121.8042.001OsJRFA071762Aluminium-induced protein0.217\u22120.594\u22121.8491.752OsIFCC029079Blight-associated protein p12 precursor\u22121.1100.379\u22121.8482.980OsJRFA106991Unknown\u22120.899\u22120.142\u22121.8741.715Log2 transformed ratios of all genes at three stages of drought stress and rehydration stage were listed\nThe down-regulation of group II genes in response to drought led us to speculate that they may play an important role in conferring drought stress tolerance, whereas the up-regulation of group II and group III genes during rehydration could be important for recovery. In both group II and III genes, we found that two classes of genes were over-represented among the rehydration-inducible genes in shoot and panicle: transporter genes and photosynthesis-related genes. These transporter genes included: OsJRFA065471 (folate\/biopterin transporter), OsJRFA066919 (putative potassium transporter), OsJRFA067899 and OsIFCC019970 (ABC transporter, putative), OsJRFA068003 and OsJRFA106202 (Transmembrane amino acid transporter protein), OsJRFA068765 (H-ATPase), OsIFCC040482 (phosphate:H+ symporter), OsJRFA072183 (Sodium:sulfate symporter transmembrane region), OsJRFA102086 (putative lipid transfer protein) and OsJRFA103807 (aquaporin). The photosynthesis related genes cover most of the gene components of the two photosystems, such as genes for putative chlorophyll a\/b-binding protein, Photosystem I or II reaction center subunits, and plastocyanin. These photosystem genes and transporter genes represent a large portion of all genes induced by rehydration, and their physiological roles in plants fit well with a potential contribution toward plant recovery from drought stress.\nLimited overlap of stress responsive genes among rice organs\nWe also examined the degree of overlap in expression of stress responsive genes in two or more rice organs under drought or high-salinity stress. Venn diagrams revealed that only a small portion of genes was shared between each pair of organs (Fig.\u00a04). The greatest overlap occurred between shoot and flag leaf, which shared more inducible genes than shoot and panicle or flag leaf and panicle under both drought and high-salinity conditions. Under drought stress, one-third of the genes up-regulated in flag leaf (197\/582) were also induced in shoot (Fig.\u00a04A), while under high salinity stress, over half of the induced genes (494\/817) in shoot (mostly young leaves) were also up-regulated in flag leaf (Fig.\u00a04B). Only 21 and 23% of genes up-regulated in panicle were also induced in flag leaf under drought and high salinity stresses respectively. The percentage of genes shared between shoot and panicle is similar to that between flag leaf and panicle (Fig.\u00a04A, B).\nFig.\u00a04Comparison of gene expression patterns among the three rice organs in response to drought and high-salinity stresses. (A) and (B): Venn diagram of drought (A) and high-salinity (B) induced genes among the three rice organs. (C) and (D): Venn diagram of drought (C) and high-salinity (D) repressed genes among the three rice organs\nInterestingly, only a small fraction of stress responsive genes were expressed in all three organs examined. For example, 42 and 151 genes were induced in all three organs under drought and high-salinity stress respectively. In general, most of those genes exhibited high levels of expression as well as strong inducibility. Among them, 27 genes were induced by both drought and high-salinity stress in all three organs. This group of 27 genes includes a protein kinase (OsJRFA058518), chlorophyll a\/b binding protein (OsJRFA062972), ABA-responsive protein (OsJRFA063578, OsIFCC018156), LEA protein (OsJRFA063984), dehydrin (OsIFCC035028), CHY zinc finger protein (OsIFCC003263), and homeobox protein (OsIFCC018343) (Table\u00a02).Table\u00a02Genes up- or down-regulated by both drought and high-salinity stresses in all three organsGene namePutative functionsFlag leafShootPanicleD(h)S(h)D(h)S(h)D(h)S(h)Up-regulatedOsJRFA058518Protein kinase domain2.5911.9393.9393.4433.1343.553OsJRFA058851Unknown5.4303.9005.8944.6002.0423.878OsJRFA062356Unknown4.9592.4774.6994.6603.3145.727OsJRFA062972Chlorophyll a\/b binding protein4.7582.5421.8952.9312.2912.204OsJRFA063156Unknown1.8371.7434.4053.9212.6062.866OsJRFA063334Unknown3.2082.1404.5213.1881.8092.167OsJRFA063578ABA-responsive protein2.7433.3524.1693.7331.8592.748OsJRFA063889Unknown3.3403.2632.1863.3442.0613.712OsJRFA063984LEA protein5.5134.7303.7634.6293.9716.773OsJRFA068381Unknown2.6481.8303.6302.8331.8201.786OsJRFA070577Unknown3.0362.8773.0652.8991.8832.372OsJRFA070872Unknown8.4857.7333.4466.3463.9634.107OsJRFA071812Unknown2.7482.2683.0152.2441.7672.559OsJRFA072568Unknown3.0013.1212.4113.3261.7142.005OsJRFA106307Unknown4.2843.4934.3992.5112.3162.960OsJRFA106562Unknown3.7974.0092.5443.4672.0103.978OsJRFA107065Unknown2.7701.7514.8593.6482.9842.292OsJRFA108083Unknown2.2742.5974.8534.2413.0774.631OsIFCC031279Alpha-galactosidase3.4073.0873.8193.5382.3892.206OsIFCC036408Unknown3.1373.9873.0383.1114.7424.192OsIFCC035028Dehydrin7.9747.6895.1486.7678.0456.382OsIFCC018156Abscisic acid-induced protein5.1677.2243.2434.9112.3292.327OsIFCC003263CHY zinc finger1.7092.1471.9942.2823.7911.956OsIFCC018343Homeobox domain4.2903.9553.4323.9592.6833.017Down-regulatedOsIFCC033098Similar to Arabidopsis F16L1.3 protein\u22122.676\u22124.719\u22122.542\u22122.346\u22121.701\u22122.519OsIFCC015113Phosphoribulokinase \/Uridine kinase family\u22122.456\u22122.689\u22121.863\u22121.717\u22121.853\u22121.899The highest ratio of each gene at three stages under drought (D(h)) or high-salinity (S(h)) were log2 transformed and listed\nAnalysis of stress down-regulated genes (Fig.\u00a04C, D) revealed a similar small overlap among the three organs. Only 68 and 129 out of 795 total genes down-regulated in flag leaf under drought stress were also inhibited in shoot and panicle, respectively. For high-salinity stress, 135 and 214 out of 1,270 down-regulated genes in flag leaf were also repressed in shoot and panicle respectively. Only 16 and 38 genes were down-regulated in all three organs under drought and high salinity stress respectively, while only two genes were inhibited in all three organs under both drought and high salinity stress (Table\u00a02).\nThe shoot samples we used consisted largely of young leaves and were thus physiologically closer to flag leaf than panicle. Indeed, shoot and flag leaf generally exhibited similar patterns of gene up-regulation in response to drought and high-salinity stress. However, this does not appear to be the case for repressed genes, where overlap between shoot and flag leaf gene expression is minimal.\nTo validate the microarray data, RT-PCR analysis was used to verify the transcription response of representative genes from microarray results. For this purpose, we picked a group of eight representative genes and designed primers for RT-PCR analysis (Table\u00a0S18). The cDNA templates were synthesized from total RNA samples prepared from shoot, flag leaf and panicle under drought and unstressed controls. The semi-quantitative RT-PCR results for the eight representative genes were shown in Fig.\u00a05. The expression patterns of all eight genes reflected changes observed by microarray analysis fairly and accurately for all three organs examined, except in three cases where RT-PCR failed to confirm the microarray results (Fig.\u00a05), indicating that our microarray data is reliable.\nFig.\u00a05RT-PCR analysis of the representative drought-induced genes among the three rice organs. Total RNA samples were prepared from the shoot (S1, S2, and S3), flag leaf (F1, F2, and F3), and panicle (P1, P2, and P3) and taken from plants at three stages of drought treatment and the untreated control plants (S0, F0, and P0). The corresponding log2 transformed median ratio of microarray data is shown at the bottom of each mRNA blot line. N\/A: no expression. The * corresponds to cases where the microarray data was not confirmed by the mRNA blot results\nEnrichment of ABRE and DRE cis-regulatory elements in abiotic stress-induced gene promoters\nPrevious reports have suggested that several well-characterized drought-, high-salinity and cold inducible gene promoters contain two common cis-regulatory elements, the ABA-responsive element (ABRE) and the dehydration-responsive element (DRE) (Seki et al., 2001, 2002a, b; Yazaki et al., 2003; Rabbani et al., 2003; Dubouzet et al., 2003; Nakabayashi et al., 2005; Maruyama et al., 2004; Yamaguchi-Shinozaki and Shinozaki, 2005). ABRE and DRE confer ABA-dependent and ABA-independent gene expression in response to water stress. It has been reported that a single copy of ABRE in the promoter region only induces relatively minor elevation of expression level, while multiple copies of ABRE strongly activate expression (Shen and Ho, 1995; Nakabayashi et al., 2005).\nTo elucidate the relationship between copy numbers of DRE and ABRE cis-regulatory elements in gene promoter regions and abiotic stress-induced gene transcription, we divided the genes into groups based on their expression patterns. Genes induced specifically by drought or high-salinity stress in each of the three organs defined six groups, while genes induced by both drought and high-salinity stress in each organ defined another three groups. Common inducible genes shared by at least two organs under drought or high-salinity stress defined another nine groups. We only selected those genes among all those groups with full-length cDNA sequences available for further promoter analysis. In brief, the 2\u00a0kb upstream regions (\u20131 to \u20132,000\u00a0bp) preceding the ATG start codon of genes were selected for promoter motif analysis (see Experimental procedures). A similar number of rice genes with full length cDNA available that lack stress responsive expression were used as control. Copy number of ABRE and DRE core motifs on each promoter region were counted. The percentages of genes in each group with 1 to 6 copies of ABRE core or DRE core elements were then calculated.\nCompared to control genes, copy numbers of the ABRE and DRE core motifs in the promoter regions of genes with organ-specific expression in response to drought or high-salinity stress were not markedly different (Fig.\u00a06A, D). The promoter regions of genes responsive to both drought and high-salinity stress in each organ were enriched for ABRE and DRE core motifs compared to the promoters of control genes (Fig.\u00a06B, E). For example, only 25% of control genes have over four copies of ABRE core motif in their promoter regions, but 48, 44 and 48% of genes induced by both drought and high-salinity stresses in flag leaf, panicle and shoot respectively have over four copies of the ABRE core motif in their promoter regions (Fig.\u00a06B).\nFig.\u00a06Distribution of the ABRE and DRE core motif sequences among different groups of drought and high-salinity stress responsive genes. In all panels, the x-axis shows the copy number of ABRE or DRE core motifs and the y-axis shows the relative number of genes containing different copy numbers of ABRE or DRE core motifs.(A) ABRE core motif distribution among promoters of the genes induced only by drought or only by high-salinity stress in each single rice organ. (B) ABRE core motif distribution among promoters of genes induced by both drought and high-salinity stress in each rice organ. (C) ABRE core motif distribution among promoters of genes induced in two or three organs. (D) DRE core motif distribution among promoters of genes induced only by drought or only by high-salinity stress in each rice organ. (E) DRE core motif distribution among promoters of genes induced by both drought and high-salinity stress in each rice organ. (F) DRE core motif distribution among promoters of genes induced in two or three organs or by both stresses in more than one organ. The following 18 gene groups (with number of genes with full-length cDNA gene number) were analyzed. (1) Genes induced by drought stress only in flag leaf (F-D123, 82). (2) Genes induced by high-salinity stress only in flag leaf (F-S123, 433). (3) Genes induced by drought stress only in shoot (S-D123, 272). (4) Genes induced by high-salinity stress only in shoot (S-S123, 77). (5) Genes induced by drought stress only in panicle (P-D123, 139). (6) Genes induced by high-salinity stress only in panicle (P-S123, 420). (7) Genes induced by both stresses in flag leaf (F-D123S123, 190). (8) Genes induced by both stresses in shoot (S-D123S123, 252). (9) Genes induced by both stresses in panicle (P-D123S123, 77). (10) Genes induced by drought stress in both flag leaf and shoot (F-S-D123, 123). (11) Genes induced by drought stress in all three organs (F-S-P-D123, 59). (12) Genes induced by high-salinity stress in all three organs (F-S-P-S123, 97). (13) Genes induced by high-salinity stress in both flag leaf and shoot (F-S-S123, 309). (14) Genes induced by drought stress in both flag leaf and panicle (F-P-D123, 59). (15) Genes induced by high-salinity stress in both flag leaf and panicle (F-P-S123, 184). (16) Genes induced by drought stress in both shoot and panicle (S-P-D123, 51). (17) Genes induced by high-salinity stress in both shoot and panicle (S-P-S123, 124). (18) Genes induced by both stresses in all three organs (F-S-P-D123S123, 20)\nWhen genes expressed in at least two organs under drought or high-salinity stress were compared to control genes, the ABRE and DRE core motif copy numbers also showed significant differences (Fig.\u00a06C, F). For example, 50% of these genes contain over four copies of the ABRE core motif in their promoter regions, compared with 25% for the control genes (Fig.\u00a06C). A similar pattern was found for the DRE core element (Fig.\u00a06F).\nGenes induced in an organ-specific manner by drought or high-salinity stresses did not contain a significantly different number of copies of ABRE or DRE core motifs compared to control genes (Fig.\u00a06A, D). Thus it is plausible that the stress-induced expression in this fraction of genes may rely less on transcriptional activation mediated by ABRE and DRE motifs. Unidentified organ-specific cis-regulatory elements may exist in the promoter regions of these genes and play a more important role in transcription activation under drought or high-salinity stress.\nIdentification of two novel cis-regulatory elements that respond to rehydration after drought stress\nAs shown in Table\u00a01, there are 8 and 21 genes with full-length cDNA sequences repressed by drought stress but induced by rehydration in shoot and panicle, respectively. An analysis of the upstream region of these eight shoot genes identified a novel motif (motif-SP, GGCAGCCG) located near to the translation start codon (\u201373 to \u2013250) in five of them (Fig.\u00a07A). To investigate the potential functional role of this novel motif, we performed a gel shift mobility assay which involved incubating a 24\u00a0bp probe containing motif-SP from one of those five genes (OsJRFA070715) and a control containing a single base mutation at an invariable position with nuclear extracts (see Experimental procedures). Only nuclear extracts from drought-treated shoot (at the third stage), but not from unstressed shoot or shoot under rehydration, showed a sequence specific binding activity with the probe (Fig.\u00a07C). A single base mutation at an invariable position in the motif-SP core of the probe completely abolished protein binding activity, suggesting high specificity of interaction with the core motif included in this 24\u00a0bp sequence. This specific binding activity was only observed in drought-treated plants, suggesting that it is involved in transcriptional repression under drought conditions.\nFig.\u00a07A novel promoter motif associated with genes repressed by drought but induced after rehydration in rice shoot. (A) The core sequence of the motif and the position of the motif in each promoter. (B) The expression pattern of a representative gene containing motif-SP. The histogram shows the log2 transformed ratio at three drought stress stages (D1, D2, D3) and 48-hour rehydration (D3R). (C) Gel shift assay of motif-SP. Nuclear proteins were extracted from shoots of untreated plants (C), plants at stage 3 of drought treatment (D3), and plants after 48-h rehydration following stage 3 (D3R). Core sequences of the probe OsJRFA070715: TGCAGCCA, and core sequence of the probe with a single base point mutation OsJRFA070715M: TGAAGCCA\nWe also searched promoter regions of 21 genes inhibited by drought stress but induced by rehydration in panicle. Three conserved motifs were found through this blind search in 11 of the 21 genes: ABRE motif, the above-mentioned motif-SP, and another novel element, motif-P (Fig.\u00a08). The presence of the ABRE motif suggests that a gene may respond to drought stress by employing an ABA-dependent pathway, while motif-SP may play an important role in drought-stress repression and subsequent activation during rehydration. We failed to detect specific binding activity using similar nuclear extracts (data not shown) and thus the role of motif-P remains to be defined.Fig.\u00a08Three motifs associated with genes repressed during drought and induced by rehydration in panicle. The core sequence logos and the position of the motifs in each of the 11 promoters are listed\nTranscription factor genes under drought and high salinity stress are expressed in a largely organ specific manner\nWe further examined transcription factor genes whose expression are regulated by drought and high-salinity stresses. Among all genes induced by drought or high-salinity treatment in the three organs, a total of 186 genes (Table\u00a0S17) were predicted to be transcription factors with a DNA-binding domain. These transcription factors belong to various families, including AP2, bHLH, MYB, HB, NAC, zinc finger, MADS, bZIP, WRKY and HSF families. These transcription factors could also be divided into several groups depending on their expression patterns. Among the 186 transcription factor genes, 12 genes were induced in all three organs in at least one stage following either drought or high-salinity stress, whereas the remaining 174 genes were mainly up regulated in one or two organs. Transcription factors induced only in one individual organ were listed in Table\u00a03. It is interesting to note that over half of the transcription factors were expressed in at least two organs, while other transcription factors were activated in an organ-specific manner. This suggests that the expression of different transcription factors may play a key role in common or organ-specific gene expression in response to drought or high-salinity conditions.\nTable\u00a03Transcription factor genes induced in each organ by drought or high-salinity stressGene name Putative functionPanicle: induced by high-salinity onlyS(h)D(h)OsIFCC018668bHLH transcription factor1.673\u22120.850OsIFCC029156Helix-loop-helix DNA-binding domain1.720\u22120.599OsJRFA110611No apical meristem (NAM) protein2.234\u22121.500OsJRFA105079CCAAT-box binding factor HAP5 homolog3.521\u22120.850OsJRFA070817RING zinc finger protein1.708\u22120.685OsIFCC039583Zinc finger, C3HC4 type (RING finger)2.2780.530OsJRFA108605Helix-loop-helix DNA-binding domain4.7130.240OsJRFA108208AP2 domain2.429N\/AOsJRFA101136C3HC4-type zinc finger5.559\u22120.753OsIFCC008718No apical meristem (NAM) protein2.542N\/AOsJRFA066984Dof domain, zinc finger1.8460.168OsJRFA110661Zinc finger C-x8-C-x5-C-x3-H type1.732N\/AOsJRFA106969Myb-like DNA-binding domain2.010N\/AOsIFCC016263Zinc finger, C2H2 type2.636N\/APanicle: induced by drought onlyS(h)D(h)OsIFCC042866AP2 domainN\/A3.050Panicle: induced by both drought and high-salinityS(h)D(h)OsJRFA107283NAM-like protein7.7732.080Shoot: induced by drought onlyS(h)D(h)OsJRFA105599DRE-binding protein 1AN\/A1.784OsIFCC031932WRKY DNA -binding domain\u22122.7462.186OsIFCC031182Myb factorN\/A2.565OsIFCC042758Helix-loop-helix DNA-binding domain\u22120.0242.367OsJRFA100208Helix-loop-helix DNA-binding domain\u22120.8363.132OsJRFA107524Dof domain, zinc fingerN\/A2.294OsJRFA106333Helix-loop-helix DNA-binding domain0.4301.762OsJRFA106282WRKY DNA -binding domainN\/A2.026OsIFCC043271Helix-loop-helix DNA-binding domain0.2452.259OsIFCC000715Myb-like DNA-binding domain0.9492.617OsJRFA110587Similar to DNA-binding protein WRKY30.9054.075OsJRFA107146AP2 domain0.6252.746OsIFCC038336Zinc finger transcription factor ZF10.5292.407Shoot: induced by both drought and high-salinityS(h)D(h)OsJRFA072192Zinc-finger protein2.0742.059OsIFCC000984WRKY DNA -binding domain1.7442.153Shoot: induced by high-salinity onlyS(h)D(h)OsIFCC017057B3 DNA binding domain2.026N\/AOsJRFA067496TRAF-type zinc finger2.1990.801Flag leaf: induced by drought onlyS(h)D(h)OsIFCC029554Zinc finger, C2H2 type\u22120.5532.032Flag leaf: induced by high-salinity onlyS(h)D(h)OsIFCC001054Zinc finger C-x8-C-x5-C-x3-H type1.7050.582The highest ratio of each gene among three stages of drought (D(h))or high-salinity (S(h)) were log2 transformed and listed\nMinimal co-regulation of neighboring genes in drought or high-salinity stress responses\nCo-regulation of adjacent genes has been observed in several organisms (Hurst et al., 2004), including human (Caron et al., 2001; Lercher et al., 2002), Drosophila (Spellman and Rubin, 2002), Arabidopsis (Birnbaum et al., 2003, Ma et al., 2005a), and yeast (Cohen et al., 2000). All these studies identified co-regulation of neighboring gene clusters on chromosomes (chromatin domains). In rice, analysis of organ-specific gene expression revealed that about 10% of the genome belongs to chromatin domains that exhibit this co-expression pattern (Ma et al., 2005b). However, similar analysis of light-regulated genes failed to reveal significant co-regulation (Jiao et al., 2005).\nTo investigate possible co-regulation of neighboring genes in the drought and high-salinity responses, we mapped rehydration induced genes in three organs and drought and high-salinity induced genes in shoot to all 12 indica chromosomes (Figs.\u00a09, S4). This analysis indicated that significant gene clustering among rehydration, drought, and high-salinity induced genes did not occur at the chromosomal level, similar to the pattern characteristic of light-regulated genes (Jiao et al., 2005). Among the 12 rice chromosomes, only three regions showed significant enrichment of 10 or more co-regulated genes within any given 2\u00a0Mb domain. For example, the 33\u201335\u00a0Mb region on chromosome 2, the 11\u201313\u00a0Mb region in chromosome 3, and the 31\u201333\u00a0Mb region in chromosome 6 (Fig.\u00a09B) contain significant enrichment of rehydration regulated genes in all three organs, although even in those cases genes are not directly adjacent.\nFig.\u00a09Distribution of drought, high salinity and rehydration regulated genes in a representative rice chromosome. (A) Map of chromosome 3 genes differentially expressed at the first stage of drought, first stage of high-salinity stress, and 48\u00a0h after rehydration in shoot. (B) Map of chromosome 3 genes differentially expressed at 48\u00a0h after rehydration in the three rice organs\nDiscussion\nThis study provides new insight into the rice response to drought and high salinity stresses at the whole genome level. Using a whole genome microarray, we monitored the expression of 36,926 unique or known rice genes or gene models in three different organs under drought and high salinity stress. Our work thus offers the first comprehensive picture of genome expression modulation in response to drought and high salinity stress in three distinct rice organs.\nGenome expression reprograming showed significant overlap between drought and high salinity responsive genes \nUsing the criteria outlined above, a total of 2,957 and 2,090 rice genes showed significant up- or down-regulation in response to high salinity stress and drought stress in at least one of the three organs. Our analysis suggested that 927 out of 2,090 (44%) genes induced by drought were also up regulated by high-salinity stress in the same organs. This number is consistent with previous studies reported for Arabidopsis (Seki et al., 2002a, b; Shinozaki et al., 2003). In rice, an even higher percentage of drought induced genes were also up-regulated under high-salinity stress, as reported previously on a smaller scale (Rabbani et al., 2003), which covered only cDNAs from plants under drought, cold and high-salinity treatment. Thus our analysis should be more representative of the overall rice genome response to drought and high salinity stresses without such bias. However, drought and salt stress conditions in the field may be more subtle and less regular as in our experimental conditions, thus our results are only meant to illustrate some particular situations rice genome expression responses to stresses.\nIt is interesting to note an overlap of up to about half of the genes induced or inhibited by drought and high-salinity stress in rice. This observation is consistent with current understanding that these two stresses affect plants in overlapping but not identical ways. At a physiological level, both stresses cause water depletion in the above-ground portion of the plants and induce similar morphological responses (Fig.\u00a0S1). At the molecular level, half of the transcriptional factor genes identified in our results were shared by both stresses in each organ, which is consistent with the observation that about half of the genes that respond to the two stresses were shared at the whole genome level.\nReprograming of genome expression in response to drought and high-salinity stresses is largely organ specific\nIn this study, we examined whole genome expression profiles under drought and high-salinity conditions in three organs: four-tiller stage shoot, filling stage flag leaf and panicle. Rice plants at these two growth stages, particularly the late one, are sensitive to drought and high-salinity stress. It is well known that drought or high-salinity stress at the heading and early panicle stages can severely compromise rice growth and development and reduce crop yield even with late rehydration. It is evident that the rice genome is subject to significant reprograming with regard to which portion of genome is expressed under drought or high salinity stress.\nOur results showed that only a limited number of drought and high salinity responsive genes were shared between any two organs. We found that only 13.5 and 20.5% of drought-induced genes in panicle were shared with those induced in shoot and flag leaf respectively, and 33.8% of drought-induced genes in shoot were also activated in flag leaf (Fig.\u00a04). The percentages of genes shared between any two organs under high-salinity conditions were similar to those under drought stress, suggesting that responses to drought or high-salinity stress in different organs were independently regulated. This observation is similar to the observed small overlap of cold responsive gene expression between root and leaf in rice (Kreps et al., 2002).\nGenome expression reprograming under either drought or high salinity stress entails a large number of genes involved in many aspects of cellular function. A GO analysis of stress responsive genes (Fig.\u00a0S2) indicates that, in most categories, three organs activated similar scale (number) of genes in response to drought or high salinity stress, while only small percentage of responsive genes were overlapped between or among organs (Fig.\u00a04). This suggests that responsive genes under the same category are largely distinct individuals under distinct organ types. It is thus possible that homologous or functionally similar gene family members are responsive to the same stresses in each organ. This is consistent with our observation of organ-specific transcription factor gene expression in response to both drought and high salinity stresses (Table\u00a03). The fact that genes specifically induced in each organ do not exhibit enrichment of DRE and ABRE motifs (Fig.\u00a06) suggests that organ-specific transcriptional factor gene expression may be responsible for activating organ-specific downstream genes in a secondary transcriptional response to stress. For these genes, it is likely that certain organ-specific promoter elements mediate this response pathway. According to the GO analysis, under high salinity stress, about 10% storage proteins were up-regulated in panicle, while in flag leaf and shoot only less than 1% storage protein genes were induced (Fig.\u00a0S2). This kind of difference among organs may play a role in organ specific response to two stresses; further function analysis of these differentially regulated genes among organs are needed.\nRice may possess specific mechanisms to facilitate plant recovery during rehydration after drought\nAccording to our microarray analysis, rice genes induced by 48-h rehydration were divided into three groups according to their expression patterns (Fig.\u00a03), somewhat similar to a previous report for Arabidopsis (Oono et al., 2003). This observation suggested that monocotyledonous and dicotyledonous plants share similar gene expression responses to rehydration after drought. Analysis of the genome-wide distribution of rehydration-regulated genes in the three organs showed no significant co-regulation of neighboring genes at the chromosomal level. This observation is similar to the case of light-regulated genes (Jiao et al., 2005) but different from general gene transcription from organ samples (Ma et al., 2005b). This distinction in the modulation of genome expression may reflect different mechanisms employed in response to different developmental or environmental signals.\nUp to now only one possible cis-element has been reported to be involved in the rehydration process after dehydration in Arabidopsis (Satoh et al., 2002; Oono et al., 2003). In rice, at the whole genome level we identified 807, 281 and 224 genes induced by rehydration in flag leaf, shoot and panicle separately (Fig.\u00a03). These genes provided us an important starting point to study rehydration mechanisms and to search for novel cis-regulatory promoter elements associated with dehydration or rehydration. Two novel cis-regulatory elements (motif-SP and motif-P) were identified (Fig.\u00a08). Motif-SP was also found in shoot-specific genes with similar expression patterns in response to drought and rehydration. Gel shift assays provided evidence that motif-SP may function as a cis-element to mediate the drought-induced repression and late de-repression (activation) during rehydration in rice. Further functional analysis of the promoter elements may substantiate the role of those novel promoter motifs.\nPrevious studies reported that drought stress suppressed plant photosynthesis systems and significantly modulated the activity of some membrane transporters (Rizhsky et al., 2002; Ramachandra et al., 2004; Johansson et al., 2004; Becker et al., 2003). Here microarray analysis revealed that genes involved in photosynthesis and genes encoding transporters were repressed or maintained at low levels of expression under drought but were then strongly activated after rehydration. The repression of metabolic genes during drought stress allows the plant to conserve energy and subsist on less water, conferring better drought tolerance. When supplied plenty of water upon rehydration, activation of these genes could aid in the recovery of full photosynthesis activity and transmembrane solute\/water exchange, thus helping plant resume its normal growth and development quickly.\nElectronic supplementary material\nBelow are the electronic supplementary materials.\nFig. S1 (JPG 3,830 kb)\nFig. S2 (JPG 4,490 kb)\nFig. S3 (JPG 2,773 kb)\nFig. S4 (JPG 15,611 kb)\nESM 5 (XLS 7,081 kb)\nESM 6 (XLS 37 kb)\nESM 7 (XLS 48 kb)\nESM 8 (XLS 71 kb)\nESM 9 (XLS 40 kb)\nESM 10 (XLS 39 kb)\nESM 11 (XLS 73 kb)\nESM 12 (XLS 92 kb)\nESM 13 (XLS 72 kb)\nESM 14 (XLS 49 kb)\nESM 15 (XLS 74 kb)\nESM 16 (XLS 74 kb)\nESM 17 (XLS 122 kb)\nESM 18 (XLS 48 kb)\nESM 19 (XLS 22 kb)\nESM 20 (XLS 19 kb)\nESM 21 (XLS 23 kb)\nESM 22 (XLS 10 kb)","keyphrases":["rice","drought","salinity","promoter","cis-element","rehydration"],"prmu":["P","P","P","P","P","P"]}
{"id":"J_Chem_Ecol-4-1-2373416","title":"The Chemistry of the Postpharyngeal Gland of Female European Beewolves\n","text":"Females of the European beewolf, Philanthus triangulum, possess a large glove-shaped gland in the head, the postpharyngeal gland (PPG). They apply the content of the PPG to their prey, paralyzed honeybees, where it delays fungal infestation. Here, we describe the chemical composition of the gland by using combined GC-MS, GC-FTIR, and derivatization. The PPG of beewolves contains mainly long-chain unsaturated hydrocarbons (C23\u2013C33), lower amounts of saturated hydrocarbons (C14\u2013C33), and minor amounts of methyl-branched hydrocarbons (C17\u2013C31). Additionally, the hexane-soluble gland content is comprised of small amounts of an unsaturated C25 alcohol, an unknown sesquiterpene, an octadecenylmethylester, and several long-chain saturated (C25, C27) and unsaturated (C23\u2013C27) ketones, some of which have not yet been reported as natural products. Surprisingly, we found a dimorphism with regard to the major component of the PPG with some females having (Z)-9-pentacosene, whereas others have (Z)-9-heptacosene as their predominant component. The biological relevance of the compounds for the prevention of fungal growth on the prey and the significance of the chemical dimorphism are discussed.\nIntroduction\nHymenoptera possess a huge variety of exocrine glands (e.g., H\u00f6lldobler and Wilson 1990). The chemistry and function of different types of these have been studied for a number of social species, whereas comparatively little is known from solitary bees and wasps. Recently, a postpharyngeal gland (PPG) has been described from a species of digger wasp (Strohm et al. 2007), the European beewolf, Philanthus triangulum Fabricius 1775 (Hymenoptera: Crabronidae, formerly Sphecidae, Melo 1999). The occurrence of this gland is surprising since the PPG was assumed to be restricted to ants (H\u00f6lldobler and Wilson 1990; Schoeters and Billen 1997; Lenoir et al. 1999) where it functions in generating the colony odor (e.g., Hefetz et al. 1992, 1996; Soroker et al. 1994, 1995, 1998; Vienne et al. 1995; Dahbi et al. 1998; Lenoir et al. 1999, 2001; Oldham et al. 1999; Soroker and Hefetz 2000; for a review of other proposed functions, see Eelen et al. 2006).\nIn beewolves, the PPG has a unique function in protecting the larval provisions from microbial attack (Strohm and Linsenmair 2001; Herzner and Strohm 2007; Herzner et al. 2007a). Female European beewolves hunt and paralyze honeybees, bring them to their nest burrow, and provision one to six bees in a brood cell as larval food for one progeny. Due to the humid and warm conditions in the brood cell, the highly nutritive provisions are prone to detrimental microbial attack (Strohm and Linsenmair 2001). Early fungus infestation inevitably destroys the food resources, and larvae are killed by fungal toxins or starve to death. Observations in special cages (Strohm and Linsenmair 1994\u20131995) revealed that beewolf females intensively lick the bodies of the paralyzed bees and apply the secretion from the PPG to the bees\u2019 surface (Strohm and Linsenmair 2001; Herzner et al. 2007a). This treatment has the effect of delaying fungus growth for 2 to 3\u00a0days, which is a highly relevant effect given the short larval feeding period of only 8 to 11\u00a0days. The primary mechanism of this delay is not a direct chemical effect of the secretion on fungi, but the prevention of water condensation on the bees that in turn impairs the germination and growth of fungal spores (Herzner and Strohm 2007). Male European beewolves also have a PPG that is even larger than in females (Herzner et al. 2007b). However, it serves as a reservoir for the scent marking pheromone that males apply to their territories to attract females (Kroiss et al. 2006).\nDespite the advanced understanding of the function of the secretion of the PPG of female European beewolves, there has been no detailed investigation of its chemistry. Therefore, we analyzed the chemical composition by using combined gas chromatography-mass spectrometry (GC-MS), gas chromatography-Fourier transform infrared spectroscopy (GC-FTIR), fractionation, and derivatizations.\nMaterials and Methods\nSampling Beewolf females were taken either from a field population close to the Biocenter of the University of W\u00fcrzburg, Germany, or from a laboratory population kept at the University of W\u00fcrzburg (daughters of field caught females). They were all mated with actively provisioned nests with honeybees and were between 1 and 4\u00a0weeks old. To identify the chemicals of the PPG, females that were freshly killed by CO2 were decapitated, and their PPGs were removed from the heads by grasping the hypopharynx with tweezers and gently pulling the attached gland out through the mouth (Strohm et al. 2007). The glands were immersed immediately in 0.25\u00a0ml n-hexane (Fluka Chemie GmbH, Buchs, Switzerland) that had been distilled prior to use. For the identification of the components in the PPG, the glands of four females were pooled. This enabled us to identify minor components that were not reliably detectable in the extracts of individual PPGs. In order to have an easier method to obtain the content of the PPG, we investigated whether extracts of whole heads differed in composition from dissected glands. There was a large amount of hydrocarbons in the PPG, and extracts of entire heads were identical to extracts of the dissected glands. There were no additional peaks detectable and the proportions were the same (see Herzner et al. 2007b for an analogous procedure for male PPGs). Thus, as an easier alternative to the dissection of the PPG, entire heads were extracted. To obtain data on PPG content variability, we analyzed individual extracts made from the heads of 37 females. Heads of freshly killed females were cut off and extracted in distilled hexane for 4\u00a0h. An internal standard (octadecane, Sigma, St Louis, MO, USA) was added to assess the absolute amount of compounds in the PPG (although octadecane could be detected in pooled samples, it was not detectable in individual samples and was therefore employed as an internal standard).\nIdentification Identification of the chemicals was accomplished by GC-MS. Head extracts were fractionated by solid phase extraction (SPE, Chromabond, unmodified silica, 3\u00a0ml, 500\u00a0mg, Macherey-Nagel, D\u00fcren, Germany) with hexane as the first and dichloromethane as the second eluent to separate nonpolar and polar fractions. Alkanes were characterized by comparison of mass spectra and retention indices with those of purchased standard alkanes (Aldrich, Deisenhofen, Germany). Corresponding alkenes were tentatively identified by their typical mass spectra, their retention indices, and (depending on availability) with commercially available (Aldrich) and synthesized standards. Dimethyl disulfide (DMDS) derivatization was carried out to determine the position of double bonds according to the method of Dunkelblum et al. (1985). The configurations of the double bonds were determined by using GC-FTIR (Attygalle 1994). Methyl-branched alkanes were identified by using MS databases and diagnostic ions, and by determining retention indices (Carlson et al. 1998). Details on the identification of polar compounds are given in the results section.For the identification of the hydrocarbons, we used a Hewlett Packard HP 6890 Series GC System coupled to a Hewlett Packard HP 5973 Mass Selective Detector (Agilent Technologies, B\u00f6blingen, Germany). The GC was equipped with a DB-1 fused silica capillary column (30\u00a0m\u2009\u00d7\u20090.25\u00a0mm i.d., 0.25\u00a0\u03bcm film thickness; J & W, Folsom, CA, USA). Temperature was programmed from 100\u00b0C to 300\u00b0C with a 6\u00b0C\/min heating rate, held for 20\u00a0min at 300\u00b0C. Helium was used as carrier gas with a constant flow of 1\u00a0ml\/min. Injection was carried out at 300\u00b0C in the splitless mode for 2\u00a0min. The electron impact mass spectra (EI-MS) were recorded with an ionization voltage of 70\u00a0eV and a source temperature of 230\u00b0C. The software ChemStation (Agilent Technologies) for windows was used for data acquisition.GC-FTIR spectra were obtained by using an HP 5890 GC (Agilent Technologies) coupled to an FTS 575C Tracersystem (BioRad, Hercules, CA, USA). The GC was equipped with the same column as described above. Temperature was programmed from 80 to 270\u00b0C with a 4\u00b0C\/min heating rate. Helium was used as carrier gas with a constant flow of 1\u20132\u00a0ml\/min. Injections were carried out by using a split\/splitless injector at 250\u00b0C in the splitless mode for 60\u00a0s. Injection volume was 0.1\u00a0\u03bcl. IR spectra were recorded by scanning 256 times in a frequency range from 4,000 to 700\u00a0cm-1 with a resolution of 1\u00a0cm-1. Data system was a Dell Optiplex GX110-PC with BioRad WinIR Pro (Version 2.7) Tracer Software and Sadtler IRSearchMaster.\nData Handling and Statistical Analysis Because the relative amounts of compounds constitute compositional data, they were transformed to logcontrasts according to Aitchison (1986): Zij\u2009=\u2009log10(Yij\/g(Yj)) where Zij is the standardized peak area i for individual j, Yij is the peak area i for individual j, and g(Yj) is the geometric mean of all peaks for individual j, prior to statistical analysis. Several peaks had to be combined for the quantitative analysis of individuals because they were not always clearly separated in the chromatograms. To test for differences in the proportions of components between groups, we subjected the transformed data to exact tests for two independent groups (SPSS 13.0, SPSS Inc. 2004).\nResults\nIdentification and Analysis of Hydrocarbons in the PPG The chromatograms of the crude hexane extracts of the PPG showed a total of 53 peaks that represented 62 different compounds (Fig.\u00a01, Table 1). The mean amount of all compounds was 337\u2009\u00b1\u2009292\u00a0\u03bcg (minimum, 36.7\u00a0\u03bcg; maximum, 1,410\u00a0\u03bcg, N\u2009=\u200937). All alkanes as well as (Z)-9-tricosene, (Z)-9-pentacosene, and (Z)-9-heptacosene were identified by comparison of mass spectra and retention indices with those of available standards. The location and configuration of double bonds of other alkenes were determined with DMDS derivatives and GC-FTIR data (band at 721\u00a0cm-1: cis configuration of RCH\u2009=\u2009CHR\u2032; Attygalle 1994). Some components could, however, not be completely characterized (location of methyl groups or double bond, configuration of double bond) due to the small amounts in the extracts. One component could not be identified at all. There were more unsaturated (81.5%) than saturated compounds in the nonpolar fraction of the PPG extract from beewolf females.\nFig.\u00a01Total ion chromatograms of the hexane extract of the PPG from individual female European beewolves, Philanthus triangulum, with a (Z)-9-pentacosene (C25-type) and b (Z)-9-heptacosene (C27-type) as the predominant hydrocarbon peak. Numbers correspond to the numbers in the peak list (Table\u00a01). Some compounds listed in Table\u00a01 were present in quantities too low to be visible in these chromatogramsTable\u00a01List of compounds in the postpharyngeal gland of females of the European beewolf, Philanthus triangulumPeak no.RICompound nameDiagnostic MS ions11400Tetradecanea19821500Pentadecanea21231600Hexadecanea22641700Heptadecanea24051710X-Methylheptadecane\u201361800Octadecanea25471814X-Methyloctadecane\u201381900Nonadecanea26891906Sesquiterpene41, 69, 81, 93, 107, 120, 133, 222102000Eicosanea282112080(Z)-9-Octadecenylmethylestera222, 264, 296122100Heneicosanea296132200Docosanea310142267\u0394-X-Tricosene322152273(Z)-9-Tricosenea322162286\u0394-7-Tricosene322172300Tricosanea32418233811-; 9-Methyltricosane168\/169, 196\/197; 140\/141, 224\/2251923437-Methyltricosane112\/113, 252\/2532023525-Methyltricosane84\/85, 280\/281212371\u0394-X-Tetracosene336222400Tetracosanea338232436\u0394-14-Tricosen-6-one99, 115, 127, 181, 265, 336242464\u0394,\u0394-X,Y-Pentacosadiene348252471(Z)-9-Pentacosenea350262492\u0394-Pentacosene350272500Pentacosanea35228253713-; 11-; 9-Methylpentacosane196\/197; 168\/169, 224\/225; 140\/141, 252\/253292538\u0394-15-Tetracosen-7-one94, 113, 141, 181, 195, 265, 3503025437- Methylpentacosane112\/113, 280\/281312554\u0394-16-Pentacosen-8-ol67, 348322572(Z)-9-Hexacosene364332600Hexacosanea366342639\u0394-16-Pentacosen-8-one127, 155, 195, 209, 265, 364352668Pentacosan-8-one127, 143, 155, 267, 283, 366362672(Z)-9-Heptacosenea378372700Heptacosanea380382714NI\u201339273513-; 11-; 9-Methylheptacosane196\/197, 224\/225; 168\/169, 252\/253; 140\/141, 280\/281402740\u0394-17-Heptacosen-9-one141, 169, 209, 223, 265, 378412800Octacosanea394422840\u0394-18-Heptacosen-10-one155, 171, 183, 223, 237, 265, 392432868Heptacosan-10-one127, 143, 155, 171, 267, 283, 295, 394442872(Z)-9-Nonacosene406452900Nonacosanea40846293315-; 13-; 11-Methylnonacosane224\/225; 196\/197, 252\/253; 168\/169, 280\/281473000Triacontanea422483076(Z)-9-Hentriacontene434493100Hentriacontanea43650313415-; 13-Methylhentriacontane224\/225, 252\/253; 196\/197, 280\/281513200Dotriacontanea450523275(Z)-9-Tritriacontene462533300Tritriacontanea464RI retention index; X, Y position of the methyl-group or double bond not known; \u0394 configuration of the double-bond not known; NI not identifiedaIdentification with available standards.\nIdentification of the Constituents in the Polar Fraction The polar fraction contained a group of uncommon constituents. Hydrogenation of double bonds with H2 and palladium on carbon (Attygalle 1998) as well as DMDS adducts were used to obtain sufficient information by mass spectrometry to identify these compounds. 18-Heptacosen-10-one was identified by comparison with an already published mass spectrum (Yasui et al. 2003). The mass spectrum of this compound showed a molecular ion of m\/z 392 and two diagnostic fragment ions at m\/z 155 for [C9H19\u2013CO]+ and at m\/z 265 for [C17H33\u2013CO]+ indicating the carbonyl position at C10. After hydrogenation of this unsaturated ketone, the resulting 10-heptacosanone as well as the 10-heptacosanone of the untreated extract showed identical mass spectra (m\/z: 155 [C9H19-CO]+ , 267 [C17H35-CO]+, 394 M+) and identical retention indices (2,868) with that already published by Yasui et al. (2003). 14-Tricosen-6-one was characterized by its diagnostic masses (m\/z: 99 [C6H11-CO]+, 265 [C17H33-CO]+, 336 M+). Also, hydrogenated 14-tricosen-6-one was tentatively identified as 6-tricosanone by comparison with a commercially available MS library (NIST 2.0, Stein et al. 2001). The double bond positions in these and additional unsaturated ketones were determined from mass spectra of DMDS adducts. Additionally, three unsaturated and one saturated ketone were tentatively identified as 15-tetracosen-7-one, 16-pentacosen-8-one, 17-hexacosen-9-one, and 8-pentacosanone. The corresponding alcohol 16-pentacosen-8-ol was tentatively identified by its diagnostic masses after hydrogenation of the polar fraction with lithium aluminum hydride (Attygalle 1998; Table 1). (Z)-9-Octadecenylmethylester was characterized by using a commercially available standard (Aldrich). Finally, a compound with a typical sesquiterpene mass spectrum was found in small amounts in the polar fraction of the extract. We could not determine the type of functional group in this sesquiterpene.\nChemical Dimorphism One alkene dominated the composition of the PPG, and surprisingly, this major compound differed among individuals (Fig.\u00a02, Table 2). Among the 37 females whose PPG content was analyzed, 29 had (Z)-9-pentacosene and eight had (Z)-9-heptacosene as their major peak (from here on referred to as the C25- and C27-type, respectively). The frequency of the two types differed significantly from equality (\u03c72\u2009=\u200915.4, df\u2009=\u20091, P\u2009<\u20090.001). By using exact tests for independent samples to assess whether the proportions of other components were also affected by the major peak, we revealed that 14 of the 21 peaks differed significantly between the C25- and the C27-type (Table 2). Generally, the C27-type had larger proportions of components with longer chain lengths.\nFig.\u00a02Frequency distribution (population of N\u2009=\u200937) of individual female European beewolves, Philanthus triangulum, based on the proportion of (Z)-9-heptacosene in their PPG secretion (values transformed to logcontrasts (Aitchison 1986): Zij\u2009=\u2009log10(Yij\/g(Yj)) where Zij is the standardized peak area i for individual j, Yij is the peak area i for individual j, and g(Yj) is the geometric mean of all peaks for individual j). There is a clear bimodal distribution, i.e., some individuals have a large proportion of (Z)-9-heptacosene, whereas some have a small proportion; there are no intermediate typesTable\u00a02Mean proportions of selected hydrocarbons and ketones from the postpharyngeal glands of individual female European beewolves, P. triangulum, that had either (Z)-9-pentacosene (C25-type) or (Z)-9-heptacosene (C27-type) as the major componentaCompound nameC25-typeC27-typeDifferencesbP values(Peak no. in Table 1)MeanSDMeanSDPent-HeptHeneicosane (12)0.200.140.180.130.020.086Docosane (13)0.160.090.130.080.040.001Tricosenes (14, 15, 16)0.360.270.080.070.28<0.001Tricosane (17)14.063.7412.933.021.130.003Methyltricosanes (18, 19, 20)0.100.100.050.040.050.003Tetracosene (21)1.190.650.140.081.06<0.001Tetracosane (22)0.170.100.140.070.020.003Pentacosenes (25, 26)77.487.968.295.5069.19<0.001Pentacosane (27)2.050.922.821.20\u22120.780.335Methylpentacosanes (28)0.060.030.080.03\u22120.020.704Hexacosene (32)0.130.051.620.72\u22121.50<0.001Hexacosane (33)0.050.030.050.020.000.06716-Pentacosen-8-one (34)1.130.730.250.150.88<0.001Heptacosene (36)1.000.3969.808.86\u221268.80<0.001Heptacosane (37)0.380.220.310.120.070.032Octacosane (41)0.030.030.030.020.000.18218-Heptacosen-10-one (42)1.025.181.730.75\u22120.70<0.001Nonacosene (44)0.040.060.950.43\u22120.90<0.001Nonacosane (45)0.300.300.320.11\u22120.020.550Hentriacontene (48)0.010.040.040.02\u22120.03<0.001Hentriacontane (49)0.070.080.060.020.010.062aTable entries include mean and one standard deviation (SD) of the proportion (%) of the components for females of the C25 (N\u2009=\u200929) and of the C27-type (N\u2009=\u20098), as well as the difference (C25\u2013C27) and the significance level for the difference according to an exact test (P, significant differences in bold).bDifferences were calculated from the original data, rounding of values produced some rounding error.\nDiscussion\nThe PPG secretion of beewolf females contains predominantly unbranched unsaturated long chain hydrocarbons (C23\u2013C33, mainly either C25 or C27), smaller amounts of saturated hydrocarbons (C14\u2013C33), and small or trace amounts of methylalkanes (C17\u2013C31), unsaturated ketones (C23\u2013C27), saturated ketones (C25, C27), a sesquiterpene, an unsaturated C18 methylester, and an unsaturated C25 alcohol.\nThe secretion of the PPG of beewolf females is involved in the preservation of their honeybee prey that serves as larval food (Herzner and Strohm 2007). Females apply large amounts of the PPG secretion to the prey prior to oviposition (Strohm and Linsenmair 2001; Herzner et al. 2007a). The prevalence of unsaturated hydrocarbons in the PPG of beewolf females is probably related to the preservation function. The preservation seems to be mainly accomplished by a physical mechanism (Herzner and Strohm 2007): the secretion prevents the condensation of water on the bees and in this way renders the microclimatic conditions unsuitable for the growth of fungi. Possibly, the PPG secretion covers structures on the paralyzed bee that would otherwise function as effective nuclei for the condensation of water. Scanning electron microscopy revealed that the PPG secretion forms a contiguous layer over the whole surface of the prey (Herzner and Strohm 2007). A hydrocarbon mixture of predominately alkenes might be an ideal means to build up such layers, because at the temperatures that prevail in beewolf brood cells, the unsaturated hydrocarbons might be in a more or less liquid state and can be spread easily over the bee surface.\nThe composition of hydrocarbons on the cuticle of paralyzed honeybees is dramatically modified due to the treatment by beewolf females (Herzner et al. 2007a, b). Untreated honeybees and most other insects bear predominantly saturated straight or branched hydrocarbons (e.g., Howard and Blomquist 1982, 2005; Schmitt et al. 2007). Less frequently, alkenes constitute large proportions of cuticular hydrocarbons (e.g., on a termite, Howard et al. 1978; on an aphid parasitoid, Liepert and Dettner 1996; on workers of European hornets, Ruther et al. 2002; on stingless bee foragers, Abdalla et al. 2003; on diapausing butterfly pupae, Kaneko and Katagiri 2004; or on nesting females of the burrowing bee, Simmons et al. 2003). The reasons for the prevalence of saturated hydrocarbons in some species and unsaturated hydrocarbons in other species are unknown. Possibly, the physicochemical properties of the surface can be adjusted to specific requirements by a particular mixture of saturated and unsaturated compounds (e.g., Gibbs and Pomonis 1995; Gibbs 1998).\nIn addition to the ubiquitous alkanes and alkenes that we found in the PPG, we also identified long chain unsaturated ketones, some of which had not yet been described as natural products. Only (Z)-18-heptacosen-10-one and 10-heptacosanone had previously been reported as components of a contact sex pheromone from females of the white-spotted longicorn beetle, Anoplophora malasiaca (Yasui et al. 2003). The function of these compounds in the secretion of beewolf females is unclear. Besides a not-completely identified sesquiterpene, (Z)-9-octadecenylmethylester, and 16-pentacosen-8-ol, these ketones are the only identified components in the PPG of beewolf females that have a functional group. They might be likely candidates to exhibit an antifungal effect. However, in bioassays, no direct antifungal effect of the PPG content could be detected (Herzner et al. 2007a). Male European beewolves have some of the same ketones and a slightly shorter unsaturated ketone in their marking secretion (Schmitt et al. 2003; Kroiss et al. 2006). Their exact function in males is also unknown (see below).\nThe estimated amount of hydrocarbons in the secretion of the PPG differed considerably among females. This might be due to differences in physiological status, size, and age. Interestingly, the mean (approximately 330\u00a0\u03bcg) and maximum (approximately 1,400\u00a0\u03bcg) amounts match the quantities found on honeybees that were embalmed with the PPG secretion by beewolf females. Bees are each embalmed with approximately 110\u00a0\u03bcg (Herzner et al. 2007a) and females provision on average three bees per day and a maximum of ten bees per day (Strohm and Linsenmair 1997). Thus, an average female has available the necessary amount of PPG secretion for the embalming of the average number of bees caught on 1\u00a0day. Likewise, the maximum amount found in some individuals would be sufficient to embalm the maximum number of bees that the most successful females hunt per day. Moreover, according to a three-dimensional reconstruction based on histological sections, the maximum volume of the gland was estimated to be 3\u20134\u00a0\u03bcl (Strohm et al. 2007). The comparatively large size of the gland is explained by the need to provide enough secretion for the treatment of several prey items per day. The supply with such large amounts of unsaturated hydrocarbons might be costly and might represent a considerable part of the cost of parental care in this species (Strohm and Linsenmair 1999, 2000; Strohm and Marliani 2002).\nRemarkably, beewolf females show a striking dimorphism with regard to the chemical composition of the PPG secretion. The major component is either (Z)-9-pentacosene or (Z)-9-heptacosene, and there are no intermediate individuals. Both compounds are widespread among aculeate Hymenoptera and other insects (e.g., Ruther et al. 2002; Simmons et al. 2003). Most other components of the PPG secretion also differ between the two morphs, in that C27-types tend to have larger proportions of long-chain compounds. Possibly, the whole metabolism of hydrocarbons is adjusted to longer chain lengths in the C27-types. Why females have either (Z)-9-pentacosene or (Z)-9-heptacosene as the major compound is unclear. Preliminary analyses (E. Strohm, G. Herzner, M. Kaltenpoth, unpublished data) suggest that there is no effect of age or physiological status on the expression of the major component. One proximate explanation could be that conditions during development differ between the females and cause differential gene activation [for example, the synthesis of (Z)-9-heptacosene might be induced by high temperatures because of the presumably higher melting point, e.g., Gibbs et al. 1998; Rouault et al. 2000]. However, our study specimens were bred under identical temperature conditions in the same climate chamber with the same diet (honeybees from the same population) and, nevertheless, showed this dimorphism. Furthermore, an analysis of beewolf females from different populations ranging from northern Germany to the southern valleys of the Alps not only showed both types of females, but also revealed that they occurred in similar proportions (Strohm et al. 2008). Together with the lack of intermediate individuals, this might suggest that the dimorphism has a genetic basis. Such a dimorphism would have to be balanced because otherwise one morph would disappear at least from some populations either because it has a selective disadvantage or because of genetic drift. One possible explanation for a balanced dimorphism is a spatial difference in the suitability of the two alleles. Such spatial heterogeneity might either be generated by differences in abiotic or biotic conditions. There are specialized cuckoo wasps that enter the nests and oviposit on the bees (Strohm et al. 2001). These wasps seem to employ chemical mimicry in order not to leave traitorous signs in the nest. Most notably, the chemical mimicry only refers to the C27-type of females (Strohm et al. 2008). Thus, the C27-type might be disadvantaged with regard to the rate of parasitism, but this could be balanced by other advantages. Again, this hypothesis is weakened by the fact that the proportions of both types of individuals are similar over a wide geographical range despite probable differences in the abundance of the cuckoo wasps.\nCompared to the content of the PPG of males of the European beewolf (Schmitt et al. 2003; Kroiss et al. 2006), females have fewer components with functional groups and lower proportions thereof, whereas the composition of the alkanes and alkenes is similar. The function of the secretion of the male PPG is also quite different. Males use this secretion to scent mark their territories and to attract receptive females (Simon-Thomas and Poorter 1972; Evans and O\u2019Neill 1988; Strohm 1995; Strohm and Lechner 2000; Schmitt et al. 2003). The marking secretion is stored in and delivered from the PPG (Kroiss et al. 2006). Therefore, the compounds with functional groups may play a role in the attraction of females (Herzner et al. 2005; Kroiss et al. 2006) and may also convey some additional information on male quality and suitability as a mate (Herzner et al. 2006; Kaltenpoth and Strohm 2006).\nIn summary, females of the European beewolf, P. triangulum, have large amounts of mainly unsaturated hydrocarbons in their PPG. The composition of the secretion is probably shaped by its function as an antifungal coating of the prey, paralyzed honeybees. Thus, the function of the PPG of beewolf females differs entirely from this gland in ants. However, the general chemistry is consistent with that found in ants. This supports earlier arguments based on morphology, ultrastructure, and behavioral context (Strohm et al. 2007) that the PPGs of these two taxa are homologous. Comparison of physiological aspects of the PPGs of ants and beewolves, as well as the investigation of other aculeate Hymenoptera is necessary to obtain further insights into the evolution and function of the PPG in this group of insects.","keyphrases":["postpharyngeal gland","philanthus triangulum","ppg","gc-ftir","hymenoptera","crabronidae","sphecidae","antifungal"],"prmu":["P","P","P","P","P","P","P","P"]}
{"id":"Dev_Genes_Evol-4-1-2292473","title":"Analysis of the Tribolium homeotic complex: insights into mechanisms constraining insect Hox clusters\n","text":"The remarkable conservation of Hox clusters is an accepted but little understood principle of biology. Some organizational constraints have been identified for vertebrate Hox clusters, but most of these are thought to be recent innovations that may not apply to other organisms. Ironically, many model organisms have disrupted Hox clusters and may not be well-suited for studies of structural constraints. In contrast, the red flour beetle, Tribolium castaneum, which has a long history in Hox gene research, is thought to have a more ancestral-type Hox cluster organization. Here, we demonstrate that the Tribolium homeotic complex (HOMC) is indeed intact, with the individual Hox genes in the expected colinear arrangement and transcribed from the same strand. There is no evidence that the cluster has been invaded by non-Hox protein-coding genes, although expressed sequence tag and genome tiling data suggest that noncoding transcripts are prevalent. Finally, our analysis of several mutations affecting the Tribolium HOMC suggests that intermingling of enhancer elements with neighboring transcription units may constrain the structure of at least one region of the Tribolium cluster. This work lays a foundation for future studies of the Tribolium HOMC that may provide insights into the reasons for Hox cluster conservation.\nIntroduction\nHox clusters arose near the origins of the animal kingdom (Larroux et al. 2007; Ryan et al. 2007). The last common ancestor of the protostomes and deuterostomes is thought to have had a cluster of at least seven genes characterized by a common transcriptional orientation and by colinearity in the order of the genes and their expression domains along the anterior\u2013posterior axis (reviewed in Garcia-Fernandez 2005).\nIn various metazoan lineages, Hox clusters have gained or lost genes by duplication and deletion but often have maintained their chromosomal order, transcriptional orientation, and both spatial and temporal colinearity of expression patterns (reviewed in Ferrier and Minguillon 2003), suggesting that Hox cluster organization has been subject to strong constraints during evolution. Classical model systems, such as Drosophila and Caenorhabditis elegans, have provided many important insights into the developmental functions of Hox genes but do not provide particularly good examples of Hox cluster conservation. The Hox cluster of Drosophila melanogaster is split into two parts (the Antennapedia (ANTC) and bithorax (BXC) complexes), shows changes in transcriptional orientation of some genes, and includes interspersed genes of independent origin as well as Hox-derived genes that have evolved novel developmental roles (reviewed in Ferrier and Minguillon 2003). These alterations suggest that the constraints keeping the Hox cluster intact may have been lost in the lineage leading to Drosophila. Additional Hox cluster rearrangements (breaks, microinversions, and gene transpositions) have been found in other Drosophila species (Negre et al. 2003; Negre and Ruiz 2007; Von Allmen et al. 1996) as well as in the silk moth Bombyx mori (Yasukochi et al. 2004). The Hox genes of C. elegans (reviewed in Aboobaker and Blaxter 2003) and the tunicate Oikopleura dioica (Seo et al. 2004) have undergone even more extreme loss and rearrangement such that none of their remaining Hox genes are clustered. In most cases, the Hox genes of these organisms still show spatial but not temporal colinearity. Rapid development seems to be the common denominator among most of these organisms, perhaps making temporal colinearity of Hox genes unnecessary, or even undesirable (Ferrier and Holland 2002; Ferrier and Minguillon 2003; Negre et al. 2005).\nWhile studies of disrupted Hox clusters have provided some insights into Hox cluster maintenance, a more complete understanding will require analysis of organisms where they are still intact. Studies of vertebrate Hox clusters have uncovered several potential mechanisms that may promote temporal colinearity and therefore constrain the organization of these clusters (reviewed in Kmita and Duboule 2003). These include progressive changes in chromatin state along the length of the cluster, varying affinity of regulatory elements to a gradient of signal, and the presence of global enhancer elements outside the cluster that regulate multiple genes within the cluster. However, it is not clear whether these mechanisms apply to other organisms.\nDuboule (2007) has suggested that the modern vertebrate Hox clusters are actually more organized than the ancestral cluster. Some of the mechanisms constraining the organization of vertebrate Hox clusters likely evolved concomitant with the co-option of Hox genes for functions such as limb development (Duboule 2007; Kmita and Duboule 2003) and, therefore, may not be applicable to other lineages. Based on this model, we might expect to gain a better understanding of the ancestral constraints on Hox clusters by studying a less organized but still intact cluster. Such clusters have been described in organisms as diverse as the cephalochordate amphioxus (Garcia-Fernandez and Holland 1994; Minguillon et al. 2005), sea urchins (Cameron et al. 2006), and the insects Apis (Honey Bee Genome Sequencing Consortium 2006; Dearden et al. 2006) and Anopheles (Holt et al. 2002; Negre and Ruiz 2007). Evidence also suggests that the red flour beetle, Tribolium castaneum, has an intact Hox cluster. Conventional cloning and sequencing of the portion of the cluster corresponding to the Drosophila Antennapedia complex has shown that this region of the homeotic complex (HOMC) is intact in Tribolium (Brown et al. 2002). Genetic mapping also suggests that the integrity of the Tribolium Hox cluster has been maintained (Beeman 1987). Moreover, the genetic methodologies possible with Tribolium, as well as the application of RNAi, have provided a comprehensive description of the full repertoire of Hox genes and their functions (e.g., Beeman et al. 1993; Beeman et al. 1989; Brown et al. 2000; Shippy et al. 2000; Shippy et al. 2006; Stuart et al. 1991; Stuart et al. 1993; Tomoyasu et al. 2005). The Tribolium genome has recently been sequenced, giving us the opportunity to explore the structure of its Hox cluster in detail. Here, we present an analysis of several Hox mutations along with the transcriptional profile of the cluster during embryonic development. We discuss these results with respect to potential mechanisms of Hox cluster organization and constraint.\nMaterials and methods\nSequence and transposable element analysis\nSequence analysis was performed using Vector NTI Advance 10 (Invitrogen). Basic Local Alignment Search Tools (BLASTs) against Tribolium genome sequence (Tcas_2.0) were performed at http:\/\/www.hgsc.bcm.tmc.edu\/blast\/blast.cgi?organism= Tcastaneum or http:\/\/www.ncbi.nlm.nih.gov\/genome\/seq\/BlastGen\/BlastGen.cgi?taxid= 7070, and subsequent analysis was performed using Genboree (http:\/\/www.genboree.org\/java-bin\/login.jsp) or NCBI Map Viewer (http:\/\/www.ncbi.nlm.nih.gov\/mapview\/). The entire HOMC sequence was used as a BLASTn query against a collection of expressed sequence tags (ESTs) provided by Dr. Yoonseong Park (Department of Entomology, Kansas State University, Manhattan, KS, USA). Transposable elements were identified and classified using Censor to search the arthropod subset of Repbase (Kohany et al. 2006).\nArray design and probe synthesis\nSequence for the Tribolium HOMC was taken from the Tcas_2.0 Baylor HSGC assembly. The tiled region consists of \u223c810,000 bases from LG2 (2,290,000 to 301,000) stretching between the two non-Hox genes flanking the complex. NimbelGen designed \u223c50\u00a0mer oligos covering this region at two densities: (1) one feature per 91\u00a0bp and (2) one feature per 70\u00a0bp. An additional region spanning 5\u00a0kb on either side of the putative homolog of dme-miR-iab-4 was tiled at a higher density of one feature every 10\u00a0bp. Visualization and scaling of tiling data was performed using Integrated Genome Browser (Affymetrix, http:\/\/www.affymetrix.com\/support\/developer\/tools\/download_igb.affx).\nTribolium 0\u201372-h-old embryos, grown at 30\u00b0C in standard media, were collected by sieving, dechorionated for 2\u00a0min in 100% bleach, and homogenized in 200\u00a0\u03bcl of Trizol using a teflon pestle. Total RNA was then extracted using the standard Trizol protocol (Invitrogen). dsDNA was prepared from \u223c10\u00a0\u03bcg of total RNA with random hexamers according to Kapranov et al. (2002), with the following modifications. Primers were annealed using a 20-min ramp to 15\u00b0C, and the first strand reaction was not subdivided for second strand synthesis. The resulting cDNA was used as template by NimbelGen for labeling and hybridization (Squazzo et al. 2006).\nFluorescent in situ hybridization\nProbe labeling, embryo fixation, and RNA FISH were performed according to Kosman et al. (2004) with the following modifications. Tribolium embryos were dechorionated in 100% bleach for 2\u00a0min and agitated for 45\u00a0min to 1\u00a0h. Embryos were devitellinized by alternating 1\u00a0min vortexing with 1\u00a0min shaking for 5\u00a0min after the addition of cold methanol, followed by passage through an 18-gauge syringe three to five times. Primers used for making the TcNC-1 and iab-4 probes are as follows: TcNC5\u2032:AGATAAGATATAATGAGGTGTAGAGTTG, TcNC3\u2032: TGATTAACATGGACGGCTTCATTAG, iab-45\u2032: CATCCTATGCACATGCGTTC, iab-43\u2032: CGTTTTAATGGGTGCATCGT. Dig-labeled RNA probes were detected using sheep\u03b1DIG (Roche) primary and donkey\u03b1sheep Alexa Fluor 555 (Molecular Probes) secondary antibodies.\nGenetics\nBeetles were cultured at 30\u00b0C on whole wheat flour supplemented with 5% brewer\u2019s yeast as described by Beeman et al. (1989). Strains used were: Ga-1 and Ga-2 (wild type); mxpDch-3\/Ey; Cx6\/AEs; ptlKT76\/+; Cx61\/AEs; ptlD60\/Ey and Dfd1\/AEs. Eyeless (Ey; Beeman et al. 1996) and AbdominalExtra sclerite (AEs; Beeman et al. 1989) are dominantly marked balancer chromosomes that suppress crossing over within the HOMC.\nCuticle preparations were performed as described by Shippy et al. (2000). For documentation, cuticles were placed in 9:1 lactic acid\/ethanol on a depression slide and covered with a coverslip. Images were captured at several focal planes using a Nikon DXM1200F digital camera and combined into a single image using Auto-Montage software (Syncrosopy).\nRNAi\nParental RNAi for ptl\/Tc-Antp was performed by injection of dsRNA into the abdomens of female pupae. Eggs were collected from injected females at 3-day intervals, aged to hatching, and subjected to cuticle preparation.\nAnalysis of ptlD60 breakpoints\nEggs were collected after overnight incubation at 30\u00b0C and allowed to develop for 3\u00a0days. Genomic DNA was isolated from individual ptlD60 homozygous larvae as described by Gloor et al. (1993).\nPolymerase chain reaction (PCR) surveys of the HOMC were used to identify likely breakpoint positions, and fragments spanning these putative breakpoints were amplified using universal PCR (Beeman and Stauth 1997; Sarkar et al. 1993). PCR products were cloned and sequenced at the Kansas State University DNA Sequencing Center. The resulting sequences were compared to the Tribolium genome sequence to characterize the breakpoints. GenBank accession numbers for these sequences are as follows: ptlD60 A (EF591668) and ptlD60 B (EF591669).\nAnalysis of mxpDch-3 breakpoints\nTribolium genomic DNA was isolated from mxpDch-3\/Ey, AEs\/Ey, and Ga-1 pupae as described by Brown et al. (1990), with the exception that DNA was not purified on a CsCl gradient. Digested DNAs were separated on a 0.7% agarose gel by field inversion gel electrophoresis and transferred to GeneScreen nylon membrane (NEN Life Sciences). To look for restriction fragment length polymorphisms associated with mxpDch-3, the blot was probed with pBmxp2.1, a 5.2\u00a0kb HindIII fragment containing the 5\u2032 end of the mxp\/Tc-pb coding region.\nInverse PCR (Ochman et al. 1988) was used to clone breakpoints associated with mxpDch-3. mxpDch-3\/Ey genomic DNA was digested with a restriction enzyme (EcoRI, HindIII, and RsaI for breakpoint fragments A, B, and C, respectively). After circularization of the fragments, two rounds of PCR were performed with primers designed from known sequence. Resulting fragments were cloned using the TOPO-TA Cloning Kit (Invitrogen), sequenced, and submitted to GenBank under the following accession numbers: Dch3 A (EF591670), Dch3 B (EF591671), Dch3 C (EF591672). Sequences were compared to the Tribolium genome sequence to determine the location of breakpoints.\nAnalysis of ptlKT76 transposon insertion site\nThe ptlKT76 piggyBac-insertion site was amplified by vectorette PCR as described by Lorenzen et al. (2007). The resulting product was sequenced by Elim Biopharmaceuticals, Inc. (Hayward, CA, USA) and the sequence was submitted to GenBank as accession number EU056827.\nResults\nThe Tribolium Hox cluster has retained an ancestral organization\nSeveral bacterial artificial chromosome (BAC) clones encompassing the ANTC-like region of the Tribolium Hox cluster were previously sequenced and annotated (Brown et al. 2002). Using the newly assembled Tribolium genome sequence, we have performed a similar analysis of the BXC-like portion of the cluster and find that this region contains the Tribolium orthologs of Ultrabithorax (Ubx; Bennett et al. 1999), abdominal-A (abd-A; Shippy et al. 1998) and Abdominal-B (Abd-B). As in Drosophila, the transcription units in this part of the complex are larger than those of the ANTC-like portion due to the presence of longer introns.\nAs expected from previous molecular and genetic studies (Beeman 1987; Brown et al. 2002), all of the Tribolium Hox genes map to a single cluster on LG2. This cluster spans approximately 756\u00a0Kb within a single scaffold of the assembled genome sequence. A few small sequencing gaps are present in the assembly, but more than half can be filled by other available sequences (i.e., the three BAC clones previously sequenced for the ANTC-like portion of the cluster and four BACs from the BXC-like region sequenced for verification of the shotgun genome assembly; Tribolium Genome Consortium 2008). The total length of the filled gaps is approximately 2,635\u00a0bp (mismatches in the sequence flanking the gaps lead to some ambiguity), which is only slightly longer than the estimated total length of these gaps (1,938\u00a0bp). Thus, estimation of sequencing gaps in the HOMC region appears to be quite accurate.\nTwo of these gaps are immediately adjacent to transposable element insertion sites and may result from difficulties in assembling repetitive DNA. These two sites account for about 1,300\u00a0bp of the total gaps in the HOMC. In two other cases, gaps in the genome assembly are associated with tandem duplications that are not present in the BAC assemblies: an approximately 160-bp duplication between Tc-Deformed (Tc-Dfd) and Tc-zen1 and an approximately 8.5-kb duplication between prothoraxless\/Tc-Antennapedia (ptl\/Tc-Antp) and Tc-fushi tarazu (Tc-ftz). We designed primers to amplify across the regions in question using Ga-2 genomic DNA (the same inbred strain that was used for the Tribolium genome sequence). In both cases, the size of the resulting fragment is consistent with that predicted from the BAC sequence (data not shown), suggesting that these gaps and duplications are artifacts of the genome assembly process. It is important to note that these artifacts affect only a small fraction of the HOMC sequence, but they underscore the increased quality of finished versus draft sequences.\nThe single Tribolium Hox cluster contains orthologs of all eight Drosophila Hox genes, as well as orthologs of the Hox-derived genes, fushi tarazu and zerkn\u00fcllt (zen; Tribolium Genome Consortium 2008 and Fig.\u00a01). (In the case of zen, Tribolium has two paralogs apparently resulting from a recent duplication in the beetle lineage, independent of the zen duplication that occurred in the Drosophila lineage (Brown et al. 2002)). These genes are arranged in the same order on the chromosome as their counterparts in other insects. As in Apis (Dearden et al. 2006; Negre and Ruiz 2007), but in contrast to Drosophila and Anopheles (Negre and Ruiz 2007), the Hox and Hox-derived genes in the Tribolium cluster are all oriented in the same direction (Fig.\u00a01). In addition, the two miRNA genes (miR-10 and miR-iab-4) that have been described in other insect Hox clusters are found at conserved positions in the Tribolium HOMC (Tanzer et al. 2005 and Fig.\u00a01).\nFig.\u00a01Embryonic transcription across the complete Tribolium Hox complex. The tiling array consists of \u223c50,000 50\u00a0bp probes that estimate degree of transcription. Relative intensities for each probe are represented as peaks correlated with a consensus annotation of the Tribolium Hox complex (below). Peak height, shown as Percentile Probe Intensity (PPI), corresponds to the level of transcription for a particular probe. The nucleotide position for each segment is displayed in the upper left and right corners of the panel (numbers correspond with linkage group 2, release Tcas_2.0). New ESTs (cyan) are displayed in the annotation track along with transposable elements (gray). For annotated genes and ESTs, the arrow indicates the direction of transcription. Red arrows indicate the location of two RNA-FISH probes\nThe ANTC and BXC clusters of Drosophila melanogaster contain a number of non-Hox, protein-coding genes. In contrast, there is no evidence for non-Hox, protein-coding genes in the Tribolium HOMC (The Tribolium Genome Consortium 2008). Here, we corroborate those findings by using several methods to address whether unrelated genes might be interspersed among the Tribolium Hox genes. First, we searched the Tribolium genome for orthologs of genes that are located within the D. melanogaster clusters and determined that none of these genes are located within the Tribolium HOMC. Second, we analyzed predicted proteins within the region to determine whether any of them have recognizable orthologs in other species. Other than the Hox and Hox-derived genes, we found no evolutionarily conserved proteins among either the GLEAN predictions or the GNOMON ab initio predictions that map to the Hox cluster. Third, we searched a collection of Tribolium ESTs for expressed sequences within the HOMC. By this approach, we identified three non-Hox EST clusters that appear to represent noncoding transcripts as well as evidence for a mariner transposase gene (see below), but no other protein-coding genes were found. Finally, we analyzed the embryonically transcribed sequences identified by a tiling array to determine if any were likely to encode proteins. Again, we found no evidence of non-Hox-related protein-coding genes other than those within transposable elements. Although there are caveats to these analyses (e.g., gene prediction methods are imperfect, the tiling array represents only the embryonic transcriptome and EST coverage is incomplete), our results strongly suggest that the protein-coding genes in the Tribolium Hox complex (excluding genes within transposable elements) are all either Hox or Hox-derived genes.\nComparison of transposable element density in the Hox complexes of various animals has led to the suggestion that higher abundance of transposable elements in Hox clusters is correlated with loss of structural integrity. Mammalian Hox complexes have a reduced number of transposons compared to other regions of the genome (Ferrier and Minguillon 2003). Moreover, when transposons are present, they seem to be preferentially inserted into nontranscribed regions of the clusters (Mainguy et al. 2007). In contrast, transposons occur fairly frequently in the split Drosophila clusters (Fried et al. 2004). Though the prediction of three transposable elements in the Tribolium Hox complex (Fig.\u00a01) may be an underestimate, the same method predicts fivefold more in the Drosophila Hox clusters. Additionally, only three transposable elements (all mariners) have been found in the larger but intact Apis Hox complex (Dearden et al. 2006). These numbers are consistent with the apparent inverse correlation between transposon number and the level of Hox cluster organization.\nTaken together, these observations suggest that with respect to gene content, order, and orientation, the Tribolium Hox cluster closely resembles the putative ancestral Hox cluster (Garcia-Fernandez 2005). Thus, the constraints preserving the integrity of the Hox cluster may still be in force in Tribolium.\nTo determine whether these constraints extend outside the Tribolium Hox cluster, we examined synteny beyond the cluster itself. As previously described, Tc-chaoptic partially overlaps the 3\u2032 UTR of Tc-labial (Tc-lab) on the opposite strand (Nie et al. 2001). Working outward, the first gene on the same strand as the Hox genes is Tc_00927, a dolichyl glycosyltransferase orthologous to D. melanogaster CG4542. Beyond the Tc-Abd-B locus is a cluster of putative serine carboxypeptidase genes (Tc_00887, Tc_00664, Tc_00665, and Tc_00666) and the ortholog of D. melanogaster CG3909 (Tc_00886). We identified the orthologs of these genes in D. melanogaster, Anopheles gambiae, and Apis mellifera and determined their map positions. None of these genes map near the Hox cluster in any of the other insects. Likewise, orthologs of the genes adjacent to lab and Abd-B in D. melanogaster do not map near the Hox clusters of the other three insects. These results suggest that the constraints preserving the Hox cluster act only on the Hox genes themselves and not the surrounding region.\nThe Tribolium Hox cluster produces numerous noncoding transcripts\nWe developed a tiling microarray covering the Tribolium Hox complex to identify the transcription units active during a broad window of Tribolium embryonic development. Tiling array signal intensity profiles were compared with previously described Hox cDNA structures. Though the tiling density is not fine-scaled enough to effectively resolve intron\u2013exon boundaries, there is a near-perfect correlation between tiling array-predicted transcription and the position of exons in the well-characterized Hox genes. The only caveat is that the 5\u2032 exons of maxillopedia\/Tc-proboscipedia (mxp\/Tc-pb) and Tc-Abd-B exhibit weaker signals than the other exons of these genes. The most likely explanation is that the 5\u2032 exon is present only in a small subset of the transcripts derived from the gene (i.e., a minor spliceoform).\nDuring the first 3\u00a0days of development, numerous regions of the Hox complex, including intergenic and intronic noncoding regions, are actively transcribed (Fig.\u00a01). Interestingly, neither of the two most likely noncoding candidates, the previously described miRNAs, is robustly identified on the tiling array. Transcription at the tca-miR-10 locus is not detected, and transcription at the tca-miR-iab-4 locus is weak. It may be that tca-miR-10 is not expressed during the stages examined, whereas in situ hybridization assays show that tca-miR-iab-4 is strongly expressed during part of the developmental window examined (Fig.\u00a02b).\nFig.\u00a02Expression pattern of two HOMC noncoding transcripts in Tribolium embryos. Probe positions are shown in Fig. 1. a Expression pattern from a 1-kb probe located \u223c86\u00a0kb 5\u2032 of the start of ptl\/Tc-Antp. b The expression pattern of the Tribolium homolog of the iab-4 miRNA (tca-miR-iab-4)\nThe most intense hybridization signals are detected in the central 250\u00a0kb of the Hox complex, encompassing the ptl\/Tc-Antp and Ultrathorax\/Tc-Ultrabithorax (Utx\/Tc-Ubx) genes. Strikingly, transcription in this region is almost equally intense for coding and noncoding loci, and for both the introns and exons of the protein-coding genes. There are hundreds of discrete regions (500\u00a0bp or longer) where signal intensity is many times greater than for verified Hox gene exons. It is not possible to determine from the single time point we have analyzed if any of these discrete regions are part of larger transcripts. To verify that the observed signals in the tiling array represent authentic transcription, RNA fluorescent in situ hybridization (FISH) was performed with a representative 1-kb region between ptl\/Tc-Antp and Utx\/Tc-Ubx (TcNC-1 in Fig.\u00a01). This region is expressed in a Hox-like pattern with distinct anterior and posterior borders in the posterior region of the elongating germ band (Fig.\u00a02a). Interestingly, signal is detected primarily in two spots per nucleus, presumably at the sites of nascent transcription. This suggests either rapid degradation or processing of a primary transcript as would be seen for a pri-miRNA or an intron.\nIn our search for additional genes within the Tribolium Hox cluster, we identified three ESTs that appear to represent noncoding transcripts. One seems to be a chimeric artifact, arising from the fusion of a tca-miR-10 precursor and part of a 28s rRNA gene. The second, represented by two independent cDNAs, maps between ptl\/Tc-Antp and Utx\/Tc-Ubx while the third is located within the first intron of Utx\/Tc-Ubx. The last two are transcribed from the strand opposite the Hox genes and are correlated with regions of strong signal in the tiling array analysis.\nThe mxpDch-3 mutation affects regulation of both mxp\/Tc-pb and Cx\/Tc-Scr\nBecause complex regulatory regions may act as a constraining force keeping Hox clusters intact, we analyzed the mxpDch-3 mutation, which was shown to have unusual effects on the expression of mxp\/Tc-pb (Shippy et al. 2000); mxpDch-3 homozygotes lack most, if not all, normal mxp\/Tc-pb expression, but both heterozygotes and homozygotes display strong ectopic mxp\/Tc-pb expression in a pattern reminiscent of Cephalothorax\/Tc-Sex combs reduced (Cx\/Tc-Scr) expression (albeit with an apparent posterior shift of some domains). This ectopic expression is sufficient to rescue some aspects of mxp\/Tc-pb function so mxpDch-3 is not an mxp\/Tc-pb null (Shippy et al. 2000). Interestingly, we find that mxpDch-3 fails to complement a null allele of Cx\/Tc-Scr (and, in fact, appears to be null for Cx\/Tc-Scr) but complements null alleles of Tc-Dfd and ptl\/Tc-Antp (data not shown).\nTo better understand this complex mutation, we characterized the breakpoints associated with mxpDch-3. mxpDch-3 is associated with a chromosomal rearrangement involving at least four breakpoints. Although we have not ruled out the presence of additional breakpoints, the simplest interpretation of our data is that a fragment of the HOMC (including Tc-zen1, Tc-zen2, Tc-Dfd, Cx\/Tc-Scr, and Tc-ftz) has been removed from the HOMC and inserted between a fragment of LG9 and a non-HOMC fragment of LG2 (Fig.\u00a03a). This scenario is consistent with previously reported pseudo-linkage of LG2 and LG9 associated with mxpDch-3 (Beeman et al. 1996) and provides an explanation for the mid-embryonic lethality of the mxpDch-3 homozygotes (Shippy et al. 2000). That is, non-HOMC breakpoints interrupt both the TFIIA-L ortholog, which is a component of the transcriptional machinery (Yokomori et al. 1993), and a homolog of groucho, which is an important transcriptional corepressor in Drosophila (Jimenez et al. 1997; Paroush et al. 1994). We conclude that the breakpoint between Tc-ftz and ptl\/Tc-Antp is likely to account for the loss of Cx\/Tc-Scr function associated with mxpDch-3, probably by separating the Cx\/Tc-Scr transcription unit from some or all of its regulatory units. The rearrangement probably juxtaposes these regulatory elements with the mxp\/Tc-pb transcription unit, providing a likely explanation for the Cx\/Tc-Scr-like expression of mxp\/Tc-pb in mxpDch-3 embryos.\nFig.\u00a03Rearrangements of the HOMC. In these schematic diagrams, the positions of cloned breakpoint fragments are underlined. Wild-type chromosomal position (not to scale) on LG2 (purple) is indicated by a gradient of color to illustrate the effects of inversions. a In the mxpDch-3 rearrangement, an approximately 150-kb fragment of the HOMC has been transposed between fragments of LG9 and LG2. b The ptlD60 mutation is a large inversion that splits the Hox cluster into two parts. Small fragments at each end of the inversion appear to have been deleted, including part of the ptl\/Tc-Antp locus\nCx\/Tc-Scr regulatory elements map near or within ptl\/Tc-Antp\nBeeman et al. (1993) observed that mutations in Cx\/Tc-Scr and ptl\/Tc-Antp often partially fail to complement one another. Because this is precisely the type of genetic interaction that would be predicted if the separation of Hox genes has deleterious effects, we decided to analyze ptlD60, an allele of ptl\/Tc-Antp that shows such effects. The ptlD60 mutation, which results in transformation of the larval legs toward antennae as well as reductions of some labial and thoracic tissue, has been proposed to be a null allele of ptl\/Tc-Antp (Beeman et al. 1993). However, adults transheterozygous for ptlD60\/ptlD2 have a very different phenotype from that produced by larval ptl\/Tc-Antp RNAi (Tomoyasu et al. 2005) and, instead, resemble adults in which both ptl\/Tc-Antp and Cx\/Tc-Scr have been knocked down (Tomoyasu, personal communication). This result raises the possibility that the ptlD60 mutation affects the function of both genes. To address this issue, we performed parental RNAi with ptl\/Tc-Antp and found that the resulting larvae (Fig.\u00a04b) show a phenotype almost identical to that of ptlD60 homozygotes (Fig.\u00a04c), suggesting that the ptlD60 mutation primarily affects the function of ptl\/Tc-Antp during embryonic development. However, there is a subtle difference between the ptlD60 and ptl\/Tc-Antp RNAi phenotypes in the positioning of the T1 appendages, which are located near the ventral midline in the RNAi larvae but more laterally in the mutants. This difference is likely attributable to partial loss of Cx\/Tc-Scr function in ptlD60 because Cx\/Scr is responsible for the midline position of the labial appendages in Tribolium (Shippy et al. 2006) and other insects (Hughes and Kaufman 2000; Pattatucci et al. 1991; Rogers et al. 1997). Thus, ptlD60 appears to be not only a null allele of ptl\/Tc-Antp but also a hypomorphic allele of Cx\/Tc-Scr.\nFig.\u00a04Cuticle and enhancer trap phenotypes of ptl\/Tc-Antp mutations. The antennae (ant) and the labial (lab) and thoracic (T1\u2013T3) segments are denoted where relevant. a\u2013e Cuticle preps displaying the phenotypes of wild-type (Ga-1; a), ptl\/Tc-Antp RNAi (b), ptlD60\/ptlD60 (c), ptlKT76\/ptlKT76 (d), and ptlKT76\/ptlD60 (e) first instar larvae. Enhancer trap-driven EGFP expression in a ptlKT76 embryo (f) appears in a very similar pattern to Cx\/Tc-Scr expression (purple) in a wild-type embryo (g). A ptlKT76 larva (h) and pupa (i) also display EGFP enhancer trap expression in parts of the labial and first thoracic segments\nTo understand why the ptlD60 mutation affects both ptl\/Tc-Antp and Cx\/Tc-Scr, we characterized its mutant lesion(s). We found that ptlD60 is associated with an inversion of about 6.6\u00a0Mb, with breakpoints in ptl\/Tc-Antp and a distant region of LG2 (Fig.\u00a03b). In addition, there are small deletions at each end of the inversion (approximately 3.1\u00a0kb of the ptl\/Tc-Antp transcription unit including all of exon 2 and approximately 1.5\u00a0kb at the other end). Consistent with this conclusion, we find that ptlD60 can act as a crossover suppressor for LG2, reducing recombination between Reindeer (a mutation near one end of LG2) and the HOMC from its normal value of 35\u201340\u00a0cM (Beeman et al. 1996) to approximately 20\u00a0cM. These results suggest that breakpoints within the ptl\/Tc-Antp gene affect the function of both ptl\/Tc-Antp and Cx\/Tc-Scr, probably by disrupting the function of Cx\/Tc-Scr regulatory elements (see \u201cDiscussion\u201d).\nAdditional evidence for the presence of Cx\/Tc-Scr regulatory elements in the vicinity of ptl\/Tc-Antp comes from a piggyBac-insertion line recovered during an insertional mutagenesis project. The KT076 line carries a homozygous lethal insertion in the last intron of ptl (Fig.\u00a05). Crosses between KT076 heterozygotes produce a class of embryos (putative homozygotes) with the T1 and T2 legs partially transformed toward antennae (Fig.\u00a04d), a phenotype consistent with partial loss of Ptl\/Tc-Antp function. However, individuals carrying the insertion display an embryonic enhancer trap expression pattern (Fig.\u00a04f) very similar to the expression pattern of Cx\/Tc-Scr (Curtis et al. 2001; Fig.\u00a04g), despite the fact that the insertion site is about 87\u00a0kb upstream of Cx\/Tc-Scr. KT076 larvae and pupae also show weak enhancer trap patterns consistent with predicted Cx\/Tc-Scr domains (Fig.\u00a04h\u2013i). As expected from the phenotype of homozygotes, KT076 fails to complement ptlD60 as assayed by adult viability, and crosses of KT076 to ptlD60 heterozygotes produce embryos with a phenotype similar to, but slightly weaker than, that of ptlD60 homozygotes (Fig.\u00a04e). In contrast, KT076 fully complements both the embryonic phenotype and the adult viability of Cx61, a null allele of Cx\/Tc-Scr (Shippy et al. 2006), indicating that Cx\/Tc-Scr function is not compromised by the insertion (data not shown). These results suggest that KT076 is a hypomorphic allele of ptl\/Tc-Antp, and it will, hereafter, be referred to as ptlKT76. Together with the data from the mxpDch-3 and ptlD60 mutations, the Cx\/Tc-Scr-like enhancer trap phenotype of ptlKT76 provides strong evidence that Cx\/Tc-Scr regulatory elements are located near, and probably within, ptl\/Tc-Antp. Although additional experiments will be necessary to pinpoint the location of regulatory elements and verify this conclusion, it is intriguing to think that overlap of regulatory elements of one Hox gene with the transcription unit of another Hox gene might be an important mechanism of Hox cluster constraint.\nFig.\u00a05Overlap of Cx\/Tc-Scr regulatory elements with the ptl\/Tc-Antp locus. The gene structure of ptl\/Tc-Antp (coding sequence is shaded gray) and the positions of mutant lesions are shown in the diagram. The inferred positions of Cx\/Tc-Scr regulatory elements in the ptl\/Tc-Antp region are indicated below the diagram\nDiscussion\nThe Hox clusters of several insects have now been completely sequenced. While breaks in the cluster seem to have occurred several times in the Drosophila lineage, the clusters of Apis, Anopheles, and Tribolium are intact. This suggests that many insect clusters are still subject to constraints to maintain their organization. Of the insects with intact clusters, Tribolium is, by far, the most genetically tractable and has a strong history of Hox gene studies, thus offering the best system for understanding the constraints acting on an intact insect Hox cluster. Below, we discuss insights provided by our analysis of the Tribolium HOMC into mechanisms that might be responsible for Hox cluster integrity.\nTemporal colinearity\nAmong organisms for which both Hox cluster sequence and expression data are available, the presence of an intact cluster appears to be correlated with temporal colinearity of Hox gene expression, while disrupted clusters are associated with lack of temporal colinearity (Monteiro and Ferrier 2006). This observation has pushed temporal colinearity to the forefront of discussions about Hox cluster maintenance. However, several questions remain to be answered. Does temporal colinearity really require an intact Hox cluster? Is temporal colinearity required for proper Hox gene function in organisms with intact clusters? If temporal colinearity is a key constraint on Tribolium Hox cluster integrity, we might expect rearrangements to affect the function of most or all Hox genes. However, our analysis of Tribolium Hox cluster mutations provides no evidence for such global effects. The ptlD60 inversion splits the complex into two parts but all of the Hox genes except ptl\/Tc-Antp and Cx\/Tc-Scr appear to function normally. In addition, the mxpDch-3 rearrangement results in the translocation of several HOMC genes (Tc-zen, Tc-Dfd, Cx\/Tc-Scr, and Tc-ftz) to a new chromosomal location. At least one of these genes, Tc-Dfd, is functional because mxpDch-3 fully complements a Tc-Dfd null allele. Likewise, the genes remaining in the HOMC (with the exception of mxp\/Tc-pb) apparently function normally. These limited effects of Hox cluster rearrangements are similar to what has been reported for Drosophila (e.g., Abbott and Kaufman 1986; Pultz et al 1988). Although additional experiments will be required to determine whether Tribolium Hox genes exhibit temporal colinearity, our results suggest that constraints on the Tribolium HOMC are more likely to act locally.\nRegulatory elements\nThe reported breaks and transposition sites in the Hox clusters of Drosophila species are all located in intergenic regions near the 3\u2032 end of a gene (Negre et al. 2005; Negre and Ruiz 2007) and, thus, are presumably less likely to separate a gene from its regulatory elements, which are predominantly located 5\u2032 of each Drosophila Hox gene. Interestingly, the one exception to this rule is the region between abd-A and Abd-B, which contains regulatory regions for both genes and is not split in any of the Drosophila species examined so far (Negre and Ruiz 2007). These observations led to the conclusion that Drosophila Hox clusters have a modular organization (with each gene and its regulatory elements representing a separate module) and that Drosophila Hox genes are still partially clustered simply because the regulatory regions are so large that there are relatively few positions where breaks can occur without disturbing a module. That is, most of the remaining linkage in Drosophila Hox clusters (with the possible exception of abd-A and Abd-B) is due to \u201cphylogenetic inertia,\u201d and, given enough time, the clusters will completely disperse (Negre and Ruiz 2007).\nThe question naturally arises whether phylogenetic inertia could also be the reason for the intact Hox clusters of insects like Tribolium. Lewis et al. (2003) suggested that unusual features of recombination (Ranz et al. 2001) may make drosophilids more tolerant of Hox cluster rearrangements than are most insects. If this is the case, intact clusters may just be the consequence of slower rates of chromosomal rearrangement (Negre and Ruiz 2007). Alternatively, constraints on Hox cluster maintenance may still be functional in Tribolium.\nOur analysis of mutant breakpoints in the ptl\/Tc-Antp region indicates that, in at least one case, Tribolium Hox genes are not modular. That is, at least some of the regulatory elements controlling Cx\/Tc-Scr expression are apparently located within the pt\/Tc-Antp gene (Fig.\u00a05). Most embryonic enhancers of Cx\/Tc-Scr are predicted to lie between the mxpDch-3 and ptlD60 breakpoints because the mxpDch-3 allele appears to lack all Cx\/Tc-Scr function, while ptlD60 has almost normal embryonic Cx\/Tc-Scr function. This conclusion is further supported by the expression of mxp\/Tc-pb in a Cx\/Tc-Scr-like pattern in mxpDch-3 mutants, presumably due to juxtaposition of the mxp\/Tc-pb transcription unit with sequence between Tc-ftz and ptl\/Tc-Antp. Adult Cx\/Tc-Scr regulatory elements are likely located within, or 5\u2032 of, ptl\/Tc-Antp because the ptlD60 rearrangement seems to have a stronger effect on the adult functions of Cx\/Tc-Scr. The presence of Cx\/Tc-Scr regulatory elements near, or within, ptl\/Tc-Antp is supported by the observation that a piggyBac insertion within the ptl\/Tc-Antp transcription unit (ptlK76) shows an enhancer trap phenotype that appears to be driven by Cx\/Tc-Scr regulatory elements.\nIn Drosophila, elements which drive Scr-like expression patterns have been found in the region between ftz and Antp (Gorman and Kaufman 1995). However, these elements are apparently redundant because breakpoints in this region only slightly reduce Scr expression levels within its normal domain. Moreover, a deficiency which removes most, if not all, of the Antp transcription unit has no effect on embryonic Scr expression. Thus, it is possible that the modularity of the Drosophila Hox cluster is a recent innovation resulting from changes in the position of cis-regulatory elements. This newly acquired modularity might have allowed breaks in the Hox cluster in the Drosophila lineage. In contrast, overlap of regulatory elements with neighboring genes might still act as a constraint on the integrity of the Hox cluster in Tribolium.\nNoncoding transcripts\nAlthough noncoding transcripts within Hox clusters have been recognized for many years (Cumberledge et al. 1990; Lipshitz et al. 1987; Sanchez-Herrero and Akam 1989), there has recently been renewed interest in these enigmatic RNAs. At least two of these ncRNAs, miR-196 and miR-iab-4, can have homeotic function and attenuate the actions of protein-coding Hox genes (Hornstein et al. 2005; Ronshaugen et al. 2005).\nIn addition to the miRNAs, numerous long noncoding RNAs have been identified within the Hox clusters of both flies and mammals (Bae et al. 2002; Rinn et al. 2007). ncRNAs are implicated in a vast array of processes, including regulation of transcription, translation, epigenetic control of chromatin, mono-allelic expression, dosage compensation, and silencing (reviewed in Mattick and Makunin 2006). In the Drosophila BXC, these transcripts have been implicated in the control of Hox gene expression, although there is some controversy as to whether they promote (Sanchez-Elsner et al. 2006) or repress (Petruk et al. 2006) Hox gene transcription. Mainguy et al. (2007) found evidence for extensive noncoding transcription in the mammalian Hox clusters and proposed that polycistronic and antisense transcription might play a role in keeping Hox genes clustered. Our transcriptional profiling data demonstrates that Tribolium also shows considerable noncoding transcription in the Hox complex. While the tiling array has provided a revealing snapshot of transcription levels during embryonic development, much additional work will be necessary to characterize the actual transcripts. For example, the high levels of transcription in the ptl\/Tc-Antp and Utx\/Tc-Ubx regions could be the result of several individual transcripts or one long transcript. Interestingly, dicistronic transcripts spanning the Antp and Ubx orthologs have been reported in crustaceans (Shiga et al. 2006) and centipedes (Brena et al. 2006). If such transcripts have an important function, they might constrain linkage in at least some parts of the Hox cluster.\nThe data available thus far indicate that noncoding transcripts are prevalent in both intact and broken Hox clusters. However, it is not clear whether these transcripts perform the same functions in all organisms. Perhaps, noncoding RNAs play a different or more critical role in organisms with intact clusters. Future studies in this area are likely to provide important insights into Hox gene function and possibly into Hox cluster conservation.\nConclusions\nThe results presented here provide a foundation for further studies of the constraints acting on Hox clusters. While it is important to keep in mind that multiple factors may have contributed to the maintenance of Hox clusters during evolution (Kmita and Duboule, 2003), the intact structure of the Tribolium Hox cluster and the suite of tools now available for this insect makes it an ideal candidate for such research.","keyphrases":["tribolium","homeotic","insect","hox cluster","tiling"],"prmu":["P","P","P","P","P"]}
{"id":"Knee_Surg_Sports_Traumatol_Arthrosc-3-1-2082657","title":"Description of the attachment geometry of the anteromedial and posterolateral bundles of the ACL from arthroscopic perspective for anatomical tunnel placement\n","text":"The anterior cruciate ligament (ACL) consists of an anteromedial bundle (AMB) and a posterolateral bundle (PLB). A reconstruction restoring the functional two-bundled nature should be able to approximate normal ACL function better than the most commonly used single-bundle reconstructions. Accurate tunnel positioning is important, but difficult. The purpose of this study was to provide a geometric description of the centre of the attachments relative to arthroscopically visible landmarks. The AMB and PLB attachment sites in 35 dissected cadaver knees were measured with a 3D system, as were anatomical landmarks of femur and tibia. At the femur, the mean ACL centre is positioned 7.9 \u00b1 1.4 mm (mean \u00b1 1 SD) shallow, along the notch roof, from the most lateral over-the-top position at the posterior edge of the intercondylar notch and from that point 4.0 \u00b1 1.3 mm from the notch roof, low on the surface of the lateral condyle wall. The mean AMB centre is at 7.2 \u00b1 1.8 and 1.4 \u00b1 1.7 mm, and the mean PLB centre at 8.8 \u00b1 1.6 and 6.7 \u00b1 2.0 mm. At the tibia, the mean ACL centre is positioned 5.1 \u00b1 1.7 mm lateral of the medial tibial spine and from that point 9.8 \u00b1 2.1 mm anterior. The mean AMB centre is at 3.0 \u00b1 1.6 and 9.4 \u00b1 2.2 mm, and the mean PLB centre at 7.2 \u00b1 1.8 and 10.1 \u00b1 2.1 mm. The ACL attachment geometry is well defined relative to arthroscopically visible landmarks with respect to the AMB and PLB. With simple guidelines for the surgeon, the attachments centres can be found during arthroscopic single-bundle or double-bundle reconstructions.\nIntroduction\nThe anterior cruciate ligament (ACL) consists of two functional bundles [2, 4, 16]. The anteromedial bundle (AMB) originates anteroproximal in the intercondylar notch, close to the over-the-top position at the posterior edge of the notch, from the deep high part of the femoral attachment area and inserts anteromedial on the anterior intercondylar area of the tibia. The posterolateral bundle (PLB) originates more posteriorly and distally in the notch, from the shallow low part of the femoral attachment area and inserts posterolateral on the anterior intercondylar area of the tibia. The ACL reconstruction aims at restoring normal knee function. Most ACL replacements are performed with the isometric single-bundle technique. Isometric positioning of a single-bundle graft results in replacement of the AMB only. Although tensioned over the complete range of motion, the fibres are mostly tight in flexion. The AMB is the major constraint for anterior tibial displacement of the flexed knee [30], but cannot restore normal knee laxity and kinematics near extension [4, 28, 41]. In an effort to improve knee mechanics, double-bundle anatomic ACL reconstructions are now developed with reconstruction of both AMB and PLB [9, 10, 13, 19, 28, 35, 40, 46, 49, 50, 52, 53]. As presented in previous studies, a reconstructed PLB is able to restore stability in knee angles where an isometrically placed graft fails [28, 41, 50]. Additional restraint against anterior displacement in 15\u00b0 of flexion [48] as well as prevention of the pivot shift is demonstrated [28, 50]. Therefore, a reconstruction with two bundles should be able to approximate normal ACL function over the complete range of motion [39, 41, 51].\nTunnel positioning is an important factor for clinical success of ACL reconstructions. Incorrect tibial [23] and femoral [29, 55] tunnel placements result in abnormal knee mechanics. Anatomical placement restores normal knee function better than isometric placement [36, 55]. However, accurate tunnel placement seems difficult. Misplacement between 25 and 65% of the tibial and femoral tunnels is reported [8, 27, 47]. Double-bundle ACL reconstructions require an anatomical placement of the bone tunnels. It is difficult to identify the ACL remnants in chronic ACL-injured knees. Therefore, detailed information about the approximate native position is essential to determine proper anatomic tunnel placement for the two bundles during arthroscopy [17]. The anatomical position has been the subject of many studies [3, 5, 12, 14\u201316, 20, 26, 33, 34, 37, 38, 44]. Only a few recent studies have described the anatomic positions of the AM and PL bundles [11, 32, 45, 52]. Due to the two-dimensional and limited view on the arthroscopic monitor, the landmarks and descriptions used in the above-mentioned studies seem not sufficient for correct positioning of the two separate bundles in all planes during arthroscopic surgery.\nThis study is aimed at acquiring quantitative geometric data of the ACL attachments on tibia and femur, such that these data can be used in an arthroscopically guided procedure for reconstruction of the ACL. We hypothesized that reliable guidelines to find the centres of the AMB, PLB and ACL relative to arthroscopically visible landmarks can be established. For the purpose of an anatomically accurate reconstruction of the ACL, the variations should be equal to or less than reported in other studies. As regard to the dimensions of drilled tunnel holes, normally 10 up to 12\u00a0mm, 95% (mean\u00a0\u00b1\u00a02SD) of the attachment centres should be within this range. Therefore the a priori set assumption is that the maximally acceptable SD is 2.5 up to 3\u00a0mm.\nMethods\nDissection\nThirty-five intact human cadaveric knee joints of elderly donors preserved in formalin, without signs of gross bony deformity, previous fracture or degenerative disease and with intact knee ligaments were dissected. Because of local post-dissection handling procedures, no exact data on gender and age of the donors were available, but they were older than 60\u00a0years. The muscles and anterior capsule were removed. The ligaments were left intact in order to preserve controlled motion of the knee. The femur was fixated in a clamp, the tibia was moved, resulting in flexion and extension of the knee joint. During this repeated passive movement, the two functional components of the ACL were identified, based on a visually detectable difference in their tensioning patterns as described by Girgis et al. [16]. In 90\u00b0 of flexion, an anterior load was manually applied. This caused tension in the fibres of the medial tibial attachment site, the AMB. The fibres of the PLB remained slack. This procedure enabled a separation of the two bundles at the tibial attachment from ventral. The femur was turned in the clamp to enable a posterior approach and the posterior cruciate ligament (PCL) was removed. In this position the initial division of the ACL at the tibial attachment was visible and used to extend the division towards the femoral origin. The tibia was moved towards extension. The PLB-fibres, inserting at the lateral femoral ACL attachment site, tightened, enabling to complete the separation as far as the femoral attachment. The outline contours of both AMB and PLB attachment areas were marked, with a waterproof felt pen.\nAnatomical position\nTo quantify the position of the centres of the attachment sites relative to arthroscopically visible landmarks, three-dimensional (3D) measurements were made with a 3Space Fastrak electromagnetic tracking system (Polhemus Navigation Sciences, Colchester, VT, USA). The x, y and z co-ordinates of each measured point were recorded with an accuracy of 0.35\u00a0mm [42]. On the femur and tibia, the attachment sites of the AMB, PLB and the entire ACL were digitized by means of a collection of points placed at equal distances on the marked outlines. The 3D position of the centres of the ACL, AMB and PLB attachments were calculated by the geometric mean of all points on the outlines. The 3D distances between the centres of the two bundles were calculated. On both femur and tibia, an arthroscopically visible landmark was digitized that served as the origin of a local coordinate system. The absolute two-dimensional (2D) positions of the centres relative to these landmarks were calculated. The position in the third dimension was determined by the surface geometry of the femoral condyle and the intercondylar tibial area, respectively.\nAbsolute positions can depend on different knee sizes and the dimensions of the femoral notch and the intercondylar tibial area. To correct for this, the relative centre positions were also calculated. Additional reference points near the attachment area on femur and tibia were digitized. Between these points two reference lines were defined to create a 2D coordinate system. The distances defined the dimensions of the femoral notch and intercondylar tibial area. Absolute centre positions were transformed in positions relative to the reference lines (%) within femur and tibia. To detect whether the absolute position of the attachment centres was actually influenced by knee size, statistical analyses were performed. The relation between absolute position and knee size can be demonstrated with a correlation coefficient. The Pearson\u2019s correlations between the distances of the absolute positions and the length of the reference lines, representing knee size, were calculated. Correlations with a Pearson\u2019s correlation coefficient r\u00a0>\u00a00.6 and a significance level P\u00a0<\u00a00.05, for a 95% alpha level were considered relevant and confirmed the relation between knee size and absolute centre position.\nFemur\nIn the anatomical nomenclature the femoral ACL attachment uses anterior\/posterior and proximal\/distal positions, relative to the extended knee [17] (Fig.\u00a01a). Since this study aimed at describing the positions of the AMB and PLB of the femoral attachment from an arthroscopic perspective, the arthroscopic nomenclature was used as recommended by the \u2018\u2018ESSKA Scientific Workshops\u2019\u2019 in 1998 [4]. The definitions, shallow\/deep and high\/low, refer to the position along the wall and from the roof of the intercondylar notch in a 90\u00b0 flexed knee (Fig.\u00a01b).\nFig.\u00a01The orientation in the femoral notch, used in this study, is based on the recommendations of the ESKKA 1998 [4]. The notch depth (ND) is directed from shallow to deep in anatomic distal\u2013proximal direction. The notch height (NH) is directed from low to high in anatomic dorsal\u2013ventral direction. The notch wall is located at the medial side of the lateral condyle. The notch roof is the connection between the two condyles\nThe digitized points on the femur are represented in Fig.\u00a02. The main femoral landmark was derived from the over-the-top position, located at the posterior edge of the intercondylar notch. The most proximal high deep point (D) on the lateral condyle was found at the 10.30 o\u2019clock position in the arc of the femoral notch, in a right knee (at 1.30 in a left knee) [17] (Fig.\u00a02). The circumferences of the ACL attachments were digitized, as was the additional reference point, the most distal high shallow point (S). This point is positioned on the distal cartilage edge of the lateral condyle in the anterior notch outlet (Fig.\u00a02a). The femoral coordinate system was defined with two reference lines (Fig.\u00a03). The first-defined femoral reference line (from D to S) divided the notch roof from the notch wall and defined the length of the lateral notch depth (ND). The lowest, dorsal point (L) on the posterior joint cartilage edge was found using a line parallel to the line DS. The second reference line (from L to H) was defined from point L perpendicular to crossing point H, high in the notch at the line DS. It indicated the magnitude of the notch height (NH). After creating this coordinate system, the calculated attachment centres (C) of the ACL, AMB and PLB were projected on the line DS at point P. The absolute centre position (mm) was composed of the distance between the main femoral landmark, the high deep point D and the point P and the distance between the point P and the centre C. The relative (%) position was calculated by dividing the distance DP by DS (notch depth) and distance CP by LH (notch height). Finally the individual results were displayed in a diagram to define the distribution of the centres relative to the means and the advised tunnel position.\nFig.\u00a02a Distal view at a left femur. The high shallow and high deep points of the cartilage border were determined by placing the stylus at the point of an imaginary rectangular corner, indicating the separation between wall and roof, this corresponds with the 1.30 o\u2019clock position in a left knee (10.30 o\u2019clock in a right knee). b View at the medial side of a left lateral femoral condyle. The points that were digitized: AMB (white rounds) and PLB (black rounds) attachments; the cartilage border (grey asterisks), with the most distal, high shallow point (black asterisk) and most proximal, high deep point (white asterisk) indicating the separation between the notch wall on the lateral condyle and the notch roofFig.\u00a03A schematic arthroscopic femoral view through the anteromedial portal. The femoral coordinate system is indicated. The first reference line, from the high deep point (D) to the high shallow point (S) on the cartilage edge separated the notch wall on the lateral condyle from the notch roof and defined the notch depth (ND). The second reference line, a perpendicular line, from the lowest point on the posterior cartilage edge (L) to the crossing point on the line DS (H) and defined the notch height (NH). The calculated attachment centre of the ACL (C) was projected on line DS (P). The absolute distances DP and CP were calculated, as were the distances relative to the reference lines (DP\/DS and CP\/LH). This was also done for the AMB and PLB, for the sake of clearness, only the centre of the entire ACL is depicted\nTibia\nThe digitized points on the tibia are represented in Fig.\u00a04. The medial tibial spine (M) was determined as the main tibial landmark. The circumferences of the ACL attachments were digitized, as were the additional reference points. Those were, besides the lateral tibial spine (L), the most anterior points on the margin of the articular surfaces of the medial tibial condyle (MA) and lateral tibial condyle (LA). The tibial coordinate system was defined as follows (Fig.\u00a05). The first tibial reference line (from M to L) was defined between the medial and the lateral tibial spine representing the interspinal distance (ID). A line connecting the anterior points MA and LA indicated the anterior margin of the articular surface of the medial and lateral condyles of the tibia. The second reference line (from M to Q) connected the medial spine (M) perpendicular with the anterior margin at crossing point Q. The length of the second reference line represents the length of the anterior intercondylar area (AL). After creating this coordinate system, the calculated attachment centres of the ACL, AMB and PLB were projected on the line ML at point P. The absolute centre position (mm) was composed of the distance between the major tibial landmark, the medial tibial spine M and the point P, and the distance between the point P and the centre C. The relative position (%) was calculated by dividing the distance MP by ML (interspinal distance) and distance CP by MQ (anterior length). Finally the individual results were displayed in a diagram to define the distribution of the centres relative to the means and the advised tunnel position.\nFig.\u00a04Proximal view at the articular surfaces of the tibial condyles and the anterior intercondylar area. The points that were digitized: AMB (white rounds) and PLB (black rounds) attachments; the lateral tibial spine (light grey oval) and medial tibial spine (dark grey oval) and the most anterior points on the margin of the articular surface of the medial and lateral tibial condyles (white asterisks)Fig.\u00a05A schematic arthroscopic tibial view. The tibial coordinate system is indicated. The first reference line, from the medial spine (M) to the lateral spine (L), defined the interspinal distance (ID). The second reference line, from the medial spine (M) perpendicular to the crossing point (Q) at the anterior line between the most anterior points on the margin of the articular surface of the medial and lateral tibial condyles defined the anterior intercondylar length (AL). The calculated attachment centre (C) was projected on line ML (P). The absolute distances MP and CP were calculated, as were the distances relative to the reference lines (MP\/ML and CP\/MQ). This was also done for the AMB and PLB, for the sake of clearness, only the centre of the entire ACL is depicted\nSurface\nTo determine the surface dimensions of the bony attachment areas, a line was fitted through the digitized points on the outlines of both the AMB and PLB attachments on femur and on the tibia. The enclosed surface of all areas was calculated (in mm2), with the aid of a Delaunay triangulation based on the Qhull algorithm as provided by Matlab\u00ae (version 7 The MathWorks, Inc, more details provided on http:\/\/www.mathworks.com). The surface of the entire ACL attachment was calculated as the sum of AMB and PLB. Also the percentage of AMB and PLB surfaces was calculated relative to the ACL attachment surface. Statistical analyses to detect differences in size of the attachment surfaces were performed. Femoral AMB and femoral PLB, tibial AMB and tibial PLB and finally femoral ACL and tibial ACL were compared. A 2-tailed Student\u2019s t test for paired data was used. Statistical significance was defined as P\u00a0<\u00a00.05, for a 95% alpha level.\nResults\nFemur\nThe oval-shaped attachment of the anterior cruciate ligament was situated on the medial surface of the lateral femoral condyle. It was positioned deep in the notch, covering most of the proximal half of the wall. The fibres of the deep low border attached to the edge of the joint cartilage, following the contour of this edge posteriorly on the condyle. In only 4 of the 35 specimens, the attachment site was completely limited to the medial wall of the lateral condyle and had no footprint in the notch roof. In 31 femurs, a small part of the deep high AMB attachment extended into the intercondylar notch roof.\nThe notch depth (DS\u00a0=\u00a031.8\u00a0\u00b1\u00a02.6\u00a0mm) was the largest distance of the femoral dimensions. On average it was 2\u00bc times the length of the notch height (LH\u00a0=\u00a014.3\u00a0\u00b1\u00a01.5\u00a0mm). In shallow\u2013deep direction measured along the notch, relative to the notch depth, the centres of the two bundles were more close to each other, than in high\u2013low direction measured from the roof, relative to the notch height. The mean position of all AMB centres along the roofline of the notch was 7.2\u00a0mm shallow from the high deep corner at \u00bc of the ND-line (Table\u00a01; Fig.\u00a06). In 9 of the 35 femurs the centre of the AMB was not situated on the condyle wall, but above the transition line on the notch roof. Therefore, the average centre was high in the notch at 1\/10 of the NH-line, 1.4\u00a0mm from the roof. The centre of the PLB was positioned slightly more shallow at less than 2\u00a0mm from the AMB (Table\u00a01; Fig.\u00a06). However, it was situated clearly lower, about 5\u00a0mm, on the femoral condyle wall, approximately halfway the notch height. The centre position of the entire ACL was in the middle between the AMB and the PLB at \u00bc of both reference lines. Approximately 96% of the mean centres were inside a 12\u00a0mm drill hole, if positioned at the mean centre of the ACL attachment. The Pearson\u2019s correlation coefficients between the absolute positions and the reference lines were smaller than 0.3 and not significant (P\u00a0>\u00a00.05).\nTable\u00a01The positions of the ligament centres in the femoral notch Absolute distances (mm) mean (SD)Relative distances (%) mean (SD)DPCPDP\/DSCP\/LHAMB7.2 (1.8)1.4 (1.7)23 (6)10 (12)PLB8.8 (1.6)6.7 (2.0)28 (6)47 (13)ACL7.9 (1.4)4.0 (1.3)25 (5)28 (9)DP The absolute distance from the high deep point D until point P, the projection of the mean ligament centre at the DS-line, in the notch depth direction; CP the absolute distance from the mean ligament centre until point P at the LH-line, in notch height direction; DP\/DS The relative position of the centre to the notch depth; CP\/LH The relative position of the centre to the notch heightFig.\u00a06a A two-dimensional graph of the medial side of a right lateral femoral condyle with the individual centres, and the mean centres with the 95% Confidence Interval of the AMB (red squares), PLB (blue diamonds) and ACL (green dots). The position of a 12\u00a0mm drill hole at the ACL centre is also displayed. b A schematic arthroscopic view through the anteromedial portal with the mean centres and the 95% Confidence Interval areas of the AMB (red square with line), PLB (blue diamond with line) and ACL (green dot with line)\nThe mean centres of the AMB and PLB were situated 6.2\u00a0\u00b1\u00a01.2\u00a0mm from each other. The AMB attachment area, 45% of the total ACL, was significantly (P\u00a0=\u00a00.005) smaller than the PLB attachment area (Table\u00a02).\nTable\u00a02The surface dimensions of the ACL and the two bundles at femur and tibiaAbsolute surface area (mm) mean (SD)Relative surface to total ACL (%) mean (SD)ACLAMBPLBAMBPLBFemur184 (52)81 (27)103 (39)45 (11)55 (11)Tibia229 (53)136 (37)93 (33)59 (9)41 (9)\nTibia\nThe tibial attachment area of the ACL was situated between the medial and lateral tibial condyle covering the medial part of the anterior intercondylar area. It was stretched out from the region between the tibial spines to anterior with various extensions, more or less shaped as a footprint. The fibres of the AMB inserted medially along the cartilage edge of the articular surface of the medial tibial condyle. The PLB covered the lateral side of the attachment area and was bounded by the attachment of the anterior horn of the lateral meniscus.\nThe interspinal distance (ML\u00a0=\u00a012.9\u00a0\u00b1\u00a01.6\u00a0mm) between the medial and the lateral spine was on the average half times the length of the anterior intercondylar area (MQ\u00a0=\u00a024.8\u00a0\u00b1\u00a02.7\u00a0mm). The average centre of the AMB was situated closest to the medial spine, lateral at \u00bc of the ID. The PLB was 4\u00a0mm more lateral, approximately halfway between the medial and lateral spine (Table\u00a03; Fig.\u00a07). The ACL was in between at 2\/5 of the ID. In anterior direction the AMB, ACL and PLB were close to each other, just over 1\/3 of the anterior length. Approximately 94% of the mean centres were inside a 10\u00a0mm drill hole, if positioned at the mean centre of the ACL attachment (Table\u00a03; Fig.\u00a07). The Pearson\u2019s correlations coefficients between the absolute positions and the interspinal distance (reference line ML) were smaller than 0.3 and not significant (P\u00a0>\u00a00.05). The Pearson\u2019s correlations coefficients between the absolute positions and the anterior length (reference line MQ) were significant (P\u00a0<\u00a00.05), however smaller than 0.6.\nTable\u00a03The positions of the ligament centres at the anterior intercondylar tibial areaAbsolute distances (mm) mean (SD)Relative distances (%) mean (SD)MPCPMP\/MLCP\/MQAMB3.0 (1.6)9.4 (2.2)23 (12)37 (9)PLB7.2 (1.8)10.1 (2.1)55 (13)38 (9)ACL5.1 (1.7)9.8 (2.1)39 (12)38 (8)MP The absolute distance from the medial spine (M) until the point P, the projection of the mean ligament centre at the line ML in lateral direction; CP the absolute distance from the mean ligament centre until point P on the line ML, in anterior direction; MP\/ML The relative position of the centre to the interspinal distance; CP\/MQ The relative position of the centre to the anterior lengthFig.\u00a07a A two-dimensional graph of a right anterior intercondylar tibial area with the individual centres, and the mean centres with the 95% Confidence Interval of the AMB (red squares), PLB (blue diamonds) and ACL (green dots). The position of a 10\u00a0mm drill hole at the ACL centre is also displayed. b A schematic arthroscopic view with the mean centres and the 95% Confidence Interval areas of the AMB (red square with line), PLB (blue diamond with line) and ACL (green dot with line)\nThe mean centres of the AMB and PLB were situated 4.5\u00a0\u00b1\u00a00.1\u00a0mm from each other. The tibial AMB attachment area of 59% was significantly larger than the PLB attachment area (P\u00a0<\u00a00.001). The tibial attachment area of the ACL and of the AMB was significantly larger than on the femur (P\u00a0<\u00a00.001) (Table\u00a02).\nDiscussion\nIncorrect tunnel placement, tibial [22, 25] as well as femoral [43], is seen as one of the most important causes of clinical failure in single-bundle ACL reconstructions [1]. In double-bundle ACL reconstruction, exact anatomic tunnel placement seems to be even more essential. Two bundles must be accurately placed relative to the surrounding structures and relative to each other. Although exact tunnel positioning is important, it seems difficult, even for experienced surgeons [8, 27, 47]. A clear description of the anatomic centres with guidelines to determine the correct tunnel position during arthroscopic procedures can improve the accuracy.\nThe femoral positions of the two distinct bundles found in this study, the AMB deep high and the PLB shallow low in the notch, are broadly in line with literature [11, 20, 32, 45, 52]. Compared to others, the landmarks that are used in the present study are more easy to locate during arthroscopy. Harner et al. [20] quantified the cross-sectional shape and area of the femoral and tibial attachments of both components in 10 knees without describing the positions relative to landmarks. Yasuda et al. [52] limit their study to the femoral attachment, using five specimens. They describe the centre of the PLB 5 to 8\u00a0mm anterior to the edge of the joint cartilage, on the vertical line through the contact point between the femoral condyle and the tibial plateau in a 90\u00b0 flexed knee. Because this point depends on the position of the knee, it can be difficult to locate accurately during an arthroscopic procedure. Colombet et al. [11] examined seven specimens, collecting especially data of the attachment dimensions. The femoral results presented by the studies of Mochizuki et al. [32] and Takahashi et al. [45] are more suitable for practical use during arthroscopy; however, no exactly defined landmarks were used. The tibial results of Takahashi et al. [45] cannot easily be transferred to the arthroscopic situation. Finally, above-mentioned studies did not present the centres of the ACL attachment.\nTunnel positioning in the femoral notch is often determined by the \u2018clock\u2019 method [13, 18, 35, 36, 52, 54]. However, this only determines the high\u2013low position from the roof, in the transversal plane along the arc of the femoral notch [17, 54]. The accuracy in the sagittal plane, deep\u2013shallow along the roof of the notch, seems to affect the functional outcome more [17, 21, 29, 55, 56]. This position is often determined with a femoral guide placed behind the posterior edge of the intercondylar notch, at the over-the-top position, not to be confused with the Resident\u2019s ridge [24]. This seems sufficiently accurate for determining the AMB centre, when a guide with a 7\u00a0mm offset is placed in the 10.30 or 1.30 o\u2019clock position. The 7\u00a0mm offset of the guide places the tunnel in the sagittal plane close to the AMB centre position, found in this study, i.e. 7.2\u00a0mm shallow along the roof. Although we did not translate the measured distance in a clock position, the 10.30 clock position seems close to the measured position in the transversal plane, 1.4\u00a0mm low from the roof on the condyle wall. This corresponds with the results of Mochizuki et al. [32] and Yasuda et al. [52]. However, the clock method is sensitive to subjective interpretation of positioning the face of the clock [7]. It is not sufficient to find the correct position for the PLB, especially in the sagittal plane. Therefore Colombet et al. [11] defined an extra guideline to position the PLB tunnel: 8\u00a0mm lower and \u2018shallower\u2019 relative to the AMB centre, found with the clock method. The present study is more precise: 1.3\u00a0mm more shallow along the notch and 5.3\u00a0mm lower from the roof on the condyle wall, relative to the above-described position of the AMB centre. These positions can best be approached through an anteromedial arthroscopic portal [6].\nVarious methods can be used to determine the correct drill hole position at the tibia. Some authors prefer placement of the tibial tunnel based on avoiding graft impingement against the roof of the femoral notch with knee extension [22, 34, 44]. Others prefer guides that use the posterior cruciate ligament (PCL) attachment as reference point [31]. Based on these methods, positioning in anteroposterior direction, the sagittal plane, is defined. However, placement of the tunnel in mediolateral direction, the transversal plane, which is also important [26], is not determined. Some studies describe the mediolateral position, relative to the width of the tibial plateau [23, 45]. However, this guideline cannot be used during an arthroscopic procedure. The results of this study can be used for arthroscopic positioning in both directions.\nThe division of the AMB and PLB on the tibia in anteroposterior direction is similar to Harner et al. [20], resulting in a medial AMB and lateral PLB. This deviates only marginally from other definitions, where the division is in an anteromedial and a posterolateral part [3, 16, 38]. The position of the centres in anteroposterior direction correspond with the results of Takahashi et al. [45], who used similar reference points to define the anterior margin of the articular surface of the tibial condyles. The centres of the two bundles were more close to each other on the tibia, than on the femur.\nThe size of the femoral ACL attachment 184\u00a0\u00b1\u00a052\u00a0mm2 was similar to the result of Odensten and Gillquist (200\u00a0mm2) [38]. Other studies found smaller attachment areas, 132\u00a0mm2 [45] and 113\u00a0mm2 [20]. This difference may be caused by a different measuring method, using the largest projection in a 2D image plane. Our study measured the actual three-dimensional attachment area. The partition of the femoral attachment area was nearly equal with 45%, 55%, respectively, for the AMB and PLB. This was comparable to Harner et al. [20] (AMB\u00a0=\u00a052%, PLB\u00a0=\u00a048%) and Takahashi et al. [45] (AMB\u00a0=\u00a050%, PLB\u00a0=\u00a050%). The average tibial attachment area found in this study was larger than the femoral attachment. This is in line with the results of Girgis et al. [16] and Fuss et al. [15]. On the other hand, Takahashi et al. [45] found that the femoral attachment was larger than the tibial attachment (132 vs 119\u00a0mm2). Harner et al. [20] and Odensten and Gillquist [38] found similar sizes for the tibial and femoral attachment areas. In our study the AMB occupied 59% of the tibial attachment, nearly similar to the 57% of Takahashi et al. [45], slightly more than the 52% Harner et al. [20] found.\nThere was a large variation in knee sizes and dimensions of the reference lines. However, we did not find a correlation between the absolute distance of the attachment centres and the size of the reference lines, i.e. knee size. The absolute positions of the attachment centres were more or less similar for large and small knees. Therefore both absolute as well as relative data can be used as guidelines to find the anatomical positions. At the arthroscopic view, the absolute positions are more useful than the relative positions.\nAs regards to the dimensions of drilled tunnel holes (10\u201312\u00a0mm), the standard deviations of the attachment centres within 3\u00a0mm are acceptable. The femoral positions had less variations than the tibial positions. The positions for the entire ACL attachment had less variations than the attachment positions for the separate bundles. The results of this study seem to produce accurate guidelines to find the anatomic tunnel position during arthroscopic reconstruction. However, some limitations must be mentioned. The subdivision of the ACL in AMB and PLB is not based on anatomically distinct fibre bundles surrounded with fibrous issue [5, 15, 33, 38]. Fibres of both bundles are twisted around each other [33]. Division of the ACL in separate bundles is not easy [3]. Therefore utmost care was taken to divide the two functional bundles according to a previously described method [11, 20]. The method, based on observed tension variation during passive flexion\u2013extension movement was reported to be consistent [32]. This is expressed in the standard deviations (SD) of the mean attachment centres. The variation of the population is expressed by the SD of the ACL centre. The SD of the mean PLB and mean AMB centres show this variation combined with the division error. For the tibia the SDs of the PLB and AMB attachment centres are equal to that of the ACL. For the femur the SDs of the two bundle centres are slightly larger. However, these results do not point to large errors in the identification of the two bundles. The variations of attachment centre positions are actually rather small and similar to other studies [11, 45].\nThe age of the cadaveric knee specimens was more than 60\u00a0years. However, only knees without severe arthritic signs were evaluated. It seems likely that the data can be transferred to the, mostly younger, population receiving an ACL reconstruction [3, 11, 34, 44]. Perfusion fixation preserves the outer contours of shapes i.e. ligament bundles, so neither the identification nor the measurements of the various bundles can be assumed to have been hampered by the fixation.\nConclusions\nThis study was performed because no earlier study described the ACL, AMB and PLB attachment centres relative to arthroscopically visible landmarks. This resulted in quantitative data of the positions of the attachment centres of the ACL and its two bundles, relative to bony landmarks on femur and tibia, visible through an arthroscope. Using these results, the surgeon will be able to determine the anatomical position of the ACL and the two functional bundles during arthroscopy without additional images or fluoroscopic support. The results can be applied for anatomic single-bundle or anatomic double-bundle techniques.","keyphrases":["posterolateral bundle","tunnel placement","anterior cruciate ligament","anteromedial bundle","anatomic acl reconstruction","double-bundle acl reconstruction","arthroscopic view","acl anatomy"],"prmu":["P","P","P","P","P","P","P","M"]}
{"id":"Int_Urogynecol_J_Pelvic_Floor_Dysfunct-4-1-2259253","title":"The diagnostic strength of the 24-h pad test for self-reported symptoms of urinary incontinence in pregnancy and after childbirth\n","text":"The clinical impact of incontinence in pregnancy and after childbirth is growing because some studies report the efficacy of physiotherapy in pregnancy and because obstetric choices are supposed to have significant impact on post-reproductive urinary function (Goldberg et al. in Am J Obstet Gynecol 188:1447\u20131450, 2003). Thus, the need for objective measurement of urinary incontinence in pregnancy is growing. Data on pad testing in pregnancy are lacking. We assessed the clinical relevance of the 24-h pad test during pregnancy and after childbirth, compared with data on self-reported symptoms of urinary incontinence and visual analogue score. According to the receiver operating characteristic curve, the diagnostic value of pad testing for measuring (severity of) self-reported incontinence during pregnancy is not of clinical relevance. However, for the purposes of research, pad tests, combined with subjective\/qualitative considerations, play a critical role in allowing comparisons across studies, quantifying the amount of urine loss and establishing a measure of severity.\nIntroduction\nIncontinence is reported frequently in pregnancy and after childbirth [2\u20134]. It has been suggested that urinary incontinence in pregnancy is a predictor of the chance to develop post-partum urinary incontinence [5]. In that respect, prevention such as physiotherapy during pregnancy is advised in women with positive symptoms of urinary incontinence in pregnancy [6\u20139]. Based on questionnaires on symptoms of post-partum urinary incontinence, Goldberg et al. found a strong protective effect of cesarean delivery against the development of post-partum urinary incontinence and highlighted the impact of obstetric choices on post-reproductive urinary function [1]. When the possible consequences of the fact that a pregnant women reports urinary incontinence grow, such as an advise for preventive physiotherapy or an advice regarding the mode of delivery, there is a greater need for objectivity in diagnosing the problem. Pad testing yields an objective measurement of fluid loss over a certain period. In non-pregnant women, the diagnostic value of pad testing for self-reporting of symptoms of urinary incontinence has been questioned [10, 11]. These data are lacking for pregnant women. Most common used types of pad tests are the 1-h and the 24-h test [12\u201315]. The 24-h pad test is almost certainly more representative to the patients\u2019 day-to-day experiences and is more likely to correlate with self-reported symptoms [16]. The 24-h pad testing has been studied and described exclusively in non-pregnant women. In this study, we focussed on the diagnostic strength of pad testing to measure (the severity of) urinary incontinence in pregnancy and after childbirth. The aims of this study were (1) to describe pad weight gain as measured by the 24-h pad test in a cohort of pregnant women and (2) to assess the clinical usefulness of the 24-h pad test in pregnancy and after childbirth in terms of the relationship between objective urine loss and the self-reported symptoms of urinary incontinence.\nMaterials and methods\nOne hundred and seventeen women who attended the outpatient clinics of the University Hospital Groningen and the Martini Hospital Groningen enrolled for the study, with a mean age of 30\u00a0years (range 17\u201341). All women were of Caucasian origin, except three who were of Mediterranean origin. All women were nulliparous and had no history of incontinence, pelvic operations or neurological disease. Written informed consent was obtained from all participating women. The study was approved by the medical ethical committees of both hospitals. For this study, the women were investigated at 28\u201332 and at 36\u201338\u00a0weeks of pregnancy and 6\u00a0weeks and 6\u00a0months post-partum.\nAt each visit, all women completed a questionnaire and a visual analogue score (VAS) on symptoms of urinary incontinence. Complaints of more than five on the VAS scale 0\u201310 were defined as severe complaints. Women were asked to classify their incontinence as mainly: (1) stress urinary incontinence, involuntary leakage on effort or exertion or on sneezing or coughing, and (2) urge urinary incontinence, involuntary leakage accompanied by or immediately preceded by a strong desire to void [17]. Three pads were packed, each in a plastic bag, and weighed by the investigators before and after use. The women received a written instruction and were free to wear one, two or three pads. It was emphasised that the bag should be closed carefully every time a pad was changed to prevent evaporation. If the bags were open or less than three pads were returned, the test was excluded from evaluation. Pads were given to all women, to be worn for 24\u00a0h preceding their appointment. The outcome of the 24-h pad test was recorded as the weight gain as measured by a verified spring balance. Weighing was done by the first or second author, within 3\u00a0days after the pad test was carried out. According to the literature, pads were assigned as wet if the total weight gain per 24\u00a0h was \u22659\u00a0g [11].\nStatistical analysis\nPad test results have a non-parametric distribution. For continuous variables, non-parametric tests are used. Numeric data are analysed by cross tabulation, chi-squared test and risk analysis. Pearson correlation test was used to identify significant relationships between variables. Data are presented as median or numbers.\nResults\nPregnancy, pad test At 28\u00a0weeks of pregnancy, 115 of 117 patients (98%) returned their pads according to the protocol. The median weight gain was 5\u00a0g (range 0\u201336). At 38\u00a0weeks, data were available from 98 women (84%). Two patients had withdrawn from the study because of inconvenience, while 17 pads were not, or were not according to the protocol returned. At this stage of pregnancy, the median weight gain was also 5\u00a0g (range 0\u201322). Distributions of pad test weight gain are given in Fig.\u00a01.\nFig.\u00a01Distribution of 24-h pad test results at 28 and 38\u00a0weeks of pregnancyPad test results at 28 and 38\u00a0weeks of pregnancy were related (r\u2009=\u20090.452, p\u2009<\u20090.0001). Twenty-seven out of 115 (23%) and 17out of 98 pads (17%) were wet at 28 and 38\u00a0weeks of pregnancy, respectively, and again, results at 28 and 38\u00a0weeks were related (r\u2009=\u20090.317, p\u2009<\u20090.001).\nPregnancy, questionnaire At 28\u00a0weeks of pregnancy, 35 of 117 women (30%) reported incontinence (stress, 28 of 35, and urge, 7 of 35), and at 38\u00a0weeks, 40 of 115 (35%) did (stress, 33 of 40 [82%], and urge, 7 of 40 [8%]). Reported incontinence at 28 and 38\u00a0weeks of pregnancy was related (r\u2009=\u20090.55, p\u2009<\u20090.001). Severe complaints of incontinence were reported in 17 of 117 (15%) and 22 of 115 cases (19%), which were also related (r\u2009=\u20090.482, p\u2009<\u20090.001).\nPregnancy, pad test and questionnaire Women with self-reported incontinence at 28 and 38\u00a0weeks of pregnancy had a median pad weight gain of 6.0 (range 0\u201336) and 6.0\u00a0g (range 0\u201322), respectively, while women without self-reported incontinence had a pad test result of 4.0 (range 0\u201322) and 4.0\u00a0g (range 0\u201322), respectively, showing no differences between these groups of women. To evaluate the diagnostic value of the pad test for measuring self-reported incontinence, the sensitivity and specificity for several cutoff levels for the pad test was calculated, graphically known as the receiver operating characteristic (ROC) curve. The ROC curve for pad tests and self-reported incontinence at 28\u00a0weeks of pregnancy did not differ from the reference line (area 0.575 vs 0.5, p\u2009=\u20090.207), showing a non-diagnostic test (Fig.\u00a02). At 38\u00a0weeks of pregnancy, the ROC curve showed a significant difference from the reference line (area 0.663 vs 0.5, p\u2009=\u20090.008), the optimum cutoff point at 5.5\u00a0g with a sensitivity of 0.629 and a specificity of 0.619 (Fig.\u00a02). When stratified for stress incontinence or for severe symptoms of urinary incontinence, the ROC curve did not improve.\nFig.\u00a02ROC curve, the diagnostic value of the pad test at different cutoff levels (g\/24\u00a0h) for measuring self reported incontinence at 28 (a) and 38 (b) weeks\nPuerperium, pad test At 6\u00a0weeks post-partum, 80 of 117 patients (68%) returned their pads according to the protocol. The median weight gain was 3\u00a0g (range 0\u201340). At 6\u00a0months post-partum, pad data were available from 76 patients (65%). Six patients had withdrawn from the study because of inconvenience, three patients had their delivery preterm, 32 pads were not, or were not according to the protocol returned. These women did not want to participate in the pad study anymore or did not return their pads according to the protocol. The women that withdrew from the pad test study were asked to fill up the questionnaires on symptoms of urinary incontinence to check for bias. The women did not differ for self-reported symptoms on urinary incontinence from the women that continued the study on pads. In the study group, the median weight gain was again 3\u00a0g (range 0\u201340). Distributions of the pad test weight gain are given in Fig.\u00a03. Pad test results at 6\u00a0weeks and 6\u00a0months post-partum were related (r\u2009=\u20090.792, p\u2009<\u20090.0001). Seven out of 80 (9%) and 3 out of 76 pads (4%) were wet at 6\u00a0weeks and 6\u00a0months post-partum, respectively, and again, the results at 28 and 38\u00a0weeks were related (r\u2009=\u20090.320, p\u2009=\u20090.017).\nFig.\u00a03Distribution of 24-h pad test results at 6\u00a0weeks and 6\u00a0months post-partum\nPuerperium, questionnaire At 6\u00a0weeks post-partum, 21 of 115 women (18%) reported incontinence (stress, 16 of 21, and urge, 5 of 21), while at 6\u00a0months post-partum, 16 of 109 (15%) did (stress, 12 of 16, and urge, 4 of 16). Reported incontinence at 6\u00a0weeks and 6\u00a0months post-partum is related (r\u2009=\u20090.61, p\u2009<\u20090.001). Severe complaints of incontinence was reported in 15 of 115 (13%) and 11 of 109 cases (10%), which were also related (r\u2009=\u20090.691, p\u2009<\u20090.001).\nPuerperium, pad test and questionnaire Women with self-reported incontinence at 6\u00a0weeks and 6\u00a0months post-partum had a median pad weight gain of 5.5 (range 0\u201340) and 6.0\u00a0g (range 0\u201340), respectively; women without self-reported incontinence had a pad test result of 3.0 (range 0\u201316) and 3.0\u00a0g (range 0\u20139), showing a differences between these groups of women (p\u2009=\u20090.01 and p\u2009=\u20090.045, respectively). The ROC curve for pad tests and self-reported incontinence at 6\u00a0weeks post differed from the reference line (area 0.767 vs 0.5, p\u2009=\u20090.001), the optimum cutoff point at 4.5\u00a0g and the sensitivity of 0.722 and specificity of 0.742 (Fig.\u00a04). At 6\u00a0months post-partum, the ROC curve shows also a significant difference from the reference line (area 0.666 vs 0.5, p\u2009=\u20090.047), the optimum cutoff point at 5.5\u00a0g and the sensitivity of 0.629 and specificity of 0.619 (Fig.\u00a04). Again, stratifying for stress incontinence or for severe symptoms of urinary incontinence, the ROC curve did not improve.\nFig.\u00a04ROC curve, the diagnostic value of the pad test at different cutoff levels (g\/24\u00a0h) for measuring self-reported incontinence at 6\u00a0weeks (a) and 6\u00a0months post-partum (b)\nDiscussion\nIn this study, we focussed on the use of pad testing to investigate its prognostic value for objectively measuring (the severity of) self-reported urinary incontinence during pregnancy and after childbirth. The clinical impact of incontinence in pregnancy and after childbirth is growing because some studies report the efficacy of physiotherapy in pregnancy and because obstetric choices are supposed to have a significant impact on post-reproductive urinary function [1]. This growing impact requires objective measurement. In a meta-analysis, the symptom of stress incontinence was 91% sensitive but only 51% specific for detecting genuine stress urinary incontinence as defined by the International Continence Society, based on history and urodynamic testing [18]. Because of pregnancy and because we are interested in an instrument for screening, it is obvious that urodynamic testing cannot be the instrument of choice. Pad testing is an objective, simple and non-invasive instrument capable of measuring fluid loss in a certain period. First of all, we need to define normal values, data that describe the results of pad testing in a cohort of pregnant women without a history of incontinence before pregnancy. Secondly, we need comparison with the criterion standard in pregnancy and the patients\u2019 history.\nThe median weight gain in the 24-h pad test in pregnancy as reported in our study is in accordance with results reported in a group of non-pregnant, not incontinent, premenopausal women, 2.6\u20137.0\u00a0g, with an upper confidence limit of 5.5\u20138\u00a0g [16, 19]. In post-menopausal continent women, a much lower weight gain of 0.3\u00a0g is reported [20]. When compared to the pre-menopausal women, the state of pregnancy does not lead to a higher weight gain in the 24-h pad test nor does the puerperal state.\nIt is remarkable that the group of controls as referred to had similar pad test results but did not report incontinence, whereas in our pregnant group, 30 (28\u00a0weeks of pregnancy) and 35% (38\u00a0weeks of pregnancy) of the women did. With the same weight gain in pregnancy, women report more incontinence than in the sample of non-pregnant women. It seems therefore that not the amount of weight gain in the pad test but the pregnancy state itself is more discriminating for the chance that a woman qualifies herself as incontinent.\nIn our study, during pregnancy, pad test results had only limited diagnostic value for self-reporting of incontinence. In their review article, Ryhammer et al. [21] stated that \u201cincontinence is a complex condition in which differences in the individual patients\u2019 personal characteristics influence the perception of leakage and the identification of the problem. Pregnancy seems to modulate this perception in such a way that it cannot be measured by pad test. As pad testing did not show to have high sensitivity and specificity for self-reported urinary incontinence in pregnancy and after childbirth, there remains confusion about the accurate diagnosis. This becomes important in deciding on management options such as offering preventive physiotherapy in selected cases or strategies that influence the mode of delivery.\nAfter childbirth, the median weight gain is also in accordance with results in non-pregnant continent women. In our study group, 15 to 18% of the women report positive for symptoms of incontinence. Just like in pregnancy, the pad test result has a significant value for testing self-reporting incontinence but again low figures for sensitivity and specificity. Like us, Morkved and Bo [22] reported a discrepancy between self-reported symptoms and stress urinary incontinence assessed by their (short) pad test, 8\u00a0weeks after delivery. To assign a women to an intervention, one needs a higher specificity, which according to the curves as shown, will rapidly lead to lower sensitivity. Depending on the chosen intervention, this may or may not be accepted.\nThe calculation of the diagnostic strength of our 24-h pad test was made with the self-reporting of symptoms of urinary incontinence as the gold standard. At 28\u00a0weeks, the pad test failed to capture eight subjects who stated they were wet; at 38\u00a0weeks, this was higher. Such results are possibly related to the high threshold for definition of incontinence in the women with some leaking less than 9\u00a0g describing some leakage. The rationale for using a high cutoff is established in both men and women, but subjects themselves may perceive this a severe incontinence. It is possible that pregnant and post-delivery women perceive leakage differently than their non-pregnant counterparts.\nWhen adding severity of symptoms to the gold standard, as reported by VAS, the diagnostic strength of the pad test did not improve.\nIn general practice, questioning about incontinence will provide the clinician with adequate information on the presence, absence or severity of incontinence from a patient perspective, and cumbersome pad tests are unnecessary. In a review article on questionnaires for women with pelvic floor disorders, Barber [23] concludes that measuring symptom severity and quality of life changes in women with pelvic floor disorders is an important part of the evaluation and treatment of women and may be the only practical way to clinically assess symptoms. However, for the purposes of research, pad tests, combined with subjective\/qualitative considerations, play a critical role in allowing comparisons across studies, quantifying the amount of urine loss and establishing a measure of severity. Indeed, the International Continence Society standards for research strongly recommend the pad test as one measure in all incontinence research. The fact that the pad test results and patient-reported incontinence were not strongly correlated illustrates the importance of both quantitative and qualitative measures when considered an intervention trial with pelvic floor muscle exercises, for example.\nFrom our study, we conclude that pad testing measures fluid loss over a certain period but does not quantify self-reported symptoms of urinary incontinence. Both measurements are of interest but cannot replace each other. Stressing of the pelvic floor by pregnancy and childbirth modulates the sensation of urinary leakage in such a way that women in this state do report symptoms of urinary incontinence more frequently than nulliparous pre-menopausal women do.","keyphrases":["diagnostic strength","pad test","urinary incontinence","pregnancy","childbirth"],"prmu":["P","P","P","P","P"]}
{"id":"Bioinformation-2-5-2241933","title":"Functional gene clustering via gene annotation sentences, MeSH and GO keywords from biomedical literature\n","text":"Gene function annotation remains a key challenge in modern biology. This is especially true for high-throughput techniques such as gene expression experiments. Vital information about genes is available electronically from biomedical literature in the form of full texts and abstracts. In addition, various publicly available databases (such as GenBank, Gene Ontology and Entrez) provide access to gene-related information at different levels of biological organization, granularity and data format. This information is being used to assess and interpret the results from high-throughput experiments. To improve keyword extraction for annotational clustering and other types of analyses, we have developed a novel text mining approach, which is based on keywords identified at the level of gene annotation sentences (in particular sentences characterizing biological function) instead of entire abstracts. Further, to improve the expressiveness and usefulness of gene annotation terms, we investigated the combination of sentence-level keywords with terms from the Medical Subject Headings (MeSH) and Gene Ontology (GO) resources. We find that sentence-level keywords combined with MeSH terms outperforms the typical \u2018baseline\u2019 set-up (term frequencies at the level of abstracts) by a significant margin, whereas the addition of GO terms improves matters only marginally. We validated our approach on the basis of a manually annotated corpus of 200 abstracts generated on the basis of 2 cancer categories and 10 genes per category. We applied the method in the context of three sets of differentially expressed genes obtained from pediatric brain tumor samples. This analysis suggests novel interpretations of discovered gene expression patterns.\nBackground\nIn recent years, increasing amounts of biological data have become available through techniques such as DNA microarrays and other high-throughput gene and \nprotein assays. [1,2] As large numbers of genes can be included in such studies, the task of assigning meaningful biological function to gene patterns or gene clusters \nis a considerable challenge. Typical analyses using supervised (classification) or unsupervised (clustering) methods require the user to incorporate the necessary background \nknowledge. [3] This ability to incorporate background knowledge is fundamental to effective and efficient scientific discovery. A substantial amount of biomedical knowledge is \ncaptured in free-text form in abstracts and full-text articles and also in specialized biological information systems such as Gene Ontology (GO) [4], Medical Subject Headings \n(MeSH) [5], Database of Interacting Proteins (DIP) [6] etc. Until only a few years ago, human reasoning was the primary method for the extracting, synthesizing and interpreting \nthe information contained in the biomedical literature and supporting biological information systems. \nHowever, in recent years the number of online documents (and other biological information repositories) has grown tremendously. This is both an opportunity and a challenge. \nOn one hand, such resources facilitate automated processing of the knowledge and information contained in these documents. On the other hand, such processing poses considerable \nalgorithmic and computational challenges [7]. For example, the biomedical abstract database MEDLINE [8] currently contains about 15 million citations and about 40 000 citations \nare added monthly. \nText mining is the application of techniques from machine learning, natural language processing (NLP), information extraction and statistical\/mathematical approaches to \nautomated extraction of useful knowledge from text [9]. Text mining of biomedical literature has been applied successfully to various biological problems. Many studies focus \non protein-protein [10\u201313] and gene-protein interactions. [14] Other specific relationships between biological entities such as sub-cellular localization of proteins [15,16], \nmolecular binding relationships [17] and interaction between genes and drugs [18] are also explored. Text analysis of biomedical literature has also been applied successfully \nto incorporate functional information of gene expression data [19\u201323]. For example, MedMOLE [19] identifies the functions among a group of genes by simple text clustering of \nentire MEDLINE documents associated with the genes. Blaschke et al. [20] extracted information about the common biological characteristics of gene clusters from MEDLINE using \na statistical term weighting approach. This method returns an ordered set of keywords with a high probability of occurrence in abstracts. Liu et al. [24] extended this approach \nby clustering such keywords to find gene-to-gene relationships. Clustering genes by functional keyword association can provide direct information about the nature of genes and \ntheir functional association [25]. However, the quality of the keyword lists extracted from the biomedical literature for each gene significantly affects the clustering results. \nCommonly, these approaches represent genes by extracting keywords from entire abstracts [25]. These keywords may undergo transformations such as weighting or dimension reduction \nwith the goal of improving clustering quality and efficiency. However, gene clustering using entire abstracts has the following main drawbacks. (a) Abstracts normally contain a \nlarge number of irrelevant sentences. These sentences may influence the clustering process and are likely to obscure information useful for gene annotation. (b) The number of \nunique terms in abstracts is typically very large. This requires the ability to deal with sparse data spaces or methods for dimensionality reduction. (c) Dimension reduction \nmethods such as principal component analysis or signal-to-noise methods increase the computational complexity, may lead to the loss of important keywords and do not guarantee \nthat the reduced dimensionality will yield better clustering\/annotation information. Also, the composite features may be hard to interpret. \nTo avoid the above drawbacks and improve the clustering process, we decided to use gene annotation sentences from abstracts instead of using full abstracts to extract the \nkeywords. Current NLP techniques allow such sentence extraction from documents. Using clustering on the basis of sentence-extraction techniques has the advantage of avoiding \ncomplex dimensionality reduction and term weighting techniques. Further, this approach is likely to yield more specific terms which are easier to interpret. We first extracted \nthe potential sentences describing gene annotation information from abstracts using a NLP method utilizing gene\/protein name dictionaries and pattern-matching-based rules. In \naddition to this sentence-keywords approach, we carried out two further experiments involving MeSH terms and GO terms as supplementary keywords. Hence, in our method, each gene \nis represented by set of keywords extracted from sentences, MeSH terms and GO terms. To demonstrate the usefulness of the proposed text mining methods, we performed hierarchical \nclustering of a gene \u00d7 keyword matrix to find functionally discrete sub-groups of genes. The overall experimental design and its components are illustrated in the Figure 1.\nWe validated the performance of keywords extracted by our method using a manually annotated corpus of 200 abstracts. We also evaluated the usefulness of our method by sorting \ndifferentially expressed genes from a microarray experiment into functional sub-groups. The objective of our gene clustering process using functional keywords is to identify and \nsummarize potential functional gene groups and to complement the conventional gene expression data clustering tasks.\nMethodology\nGene\/Protein name and synonym dictionary creation\nOne of the major obstacles in biomedical literature processing is the variety of names each gene or protein is known by. To address this problem in the present study, we \ndeveloped a gene\/protein name dictionary. Essentially, each entry of this dictionary consists of a preferred (or canonical) name for a gene\/protein and a list of synonyms used for \nthis gene\/protein. The dictionary was created on the basis of the Entrez Gene [26] (previously LocusLink) database, one the most stable and complete sources of information on genes. \nSince our study is part of a wider investigation in the context of human brain tumor research, we focused specifically on human genes\/proteins. We developed PERL scripts to extract \nand select from the Entrez Gene entries the official symbols as preferred name of the gene and other aliases as known synonyms. In addition, we augmented the dictionary with relevant \nsynonyms from other publicly available databases including GeneCards [27], SwissProt [28], GoldenPath [29] and HUGO [30]. The final dictionary contains 26 731 unique human gene\/protein \nnames and 274 845 synonym names.\nKeywords extraction from biomedical literature\nIn our study each gene is represented by a list of keywords extracted from MEDLINE abstract sentences, MeSH terms and GO terms. The procedure for extracting keywords from each data \nsource is discussed below.\nMEDLINE abstracts keywords extraction\nTo extract the keywords associated with each abstract, we decided to use gene annotation sentences from the abstracts instead of constructing a large keyword vector based on the entire \nabstract. The assumption is that the information given on sentence-level is much more specific and therefore useful to characterize the function of the genes. Only sentences that contain \none or more genes reference from our gene lists will be considered as gene annotation sentences, all other sentences are discarded from the analysis. We applied the following three steps \nto extract sentence-level keywords (1) gene-name normalization, (2) sentence filtering, and (3) keyword extraction.\nGene-name normalization\nThis process replaces all the gene names in the abstract with its unique canonical identifier (Entrez gene ID) using the gene-synonym dictionary specially constructed for this study.\nSentence filtering\nThis process extracts all the gene annotation sentences from abstracts that contain one or more gene names from our gene lists using regular-expression pattern matching rules. We used different \nregular expressions (which rely on matching of pre-defined patterns or rules such as arrangement of gene\/protein names with articles, prepositions and other keywords) to filter sentences containing \none to three genes. We defined our regular expressions as nouns describing agents, passive verbs, active verbs and nouns describing actions. Table 1 (in supplementary material) depicts an example \nfor each type of expression.  For example, the regular expression \n($gene @{0,6} $action (of|with) @{0,2} $gene)\nextracts sentences that match the structure shown below the expression. The notational construct \u2018A \u2192 B \u2192 ...\u2019 is interpreted as \u2018A followed by B followed by ...\u2019.\ngene name \u2192 0-6 words \u2192 action verb \u2192 \u2018of\u2019 or \u2018with\u2019 \u2192 0-2 words \u2192 gene name\nSentence keyword extraction\nSentences containing one or more gene names were parsed using the Brill part-of-speech tagger. [31] This program labels each word in a sentence with its part-of-speech information such as \nword category like noun, verb, adjective, preposition, etc. This information plays a critical role in identifying corresponding noun and verb phrases. Then, with a simple PERL program, noun \nphrases containing gene names were filtered out and the remaining noun phrases and verb phrases were extracted as keywords. Initial tests showed that certain keywords were common for most of \nthe genes in the list (e.g., activates, associates, stimulates etc.). We manually removed these common keyword words from the list. The following example illustrates this process:\nSentence\nBRCA1 physically associates with p53 and stimulates its transcriptional activity.\nBrill-POS-tagged sentence\nBRCA1\/NNP physically\/RB associates\/VBZ with\/IN p53\/NN and\/CC stimulates\/VBZ its\/PRP$ transcriptional\/JJ activity\/NN. \/.\nSentence keywords\nassociates, stimulates, transcription activity\nSentence keywords after manual curation\ntranscription activity\nMeSH keywords extraction\nTo extract MeSH keywords, we searched for the gene names in our gene lists in the title and abstract of MEDLINE citations related to each gene and extracted the associated MeSH terms for \neach gene. The extracted gene-MeSH term list was represented by scores indicating the frequency of gene-MeSH term co-occurrence. Initial tests showed that certain MeSH keywords in the list \nwere common biological terms and less useful from the point of view of gene annotation (e.g., human, DNA, animal, Support U.S Govt etc.). A collection of MeSH stop words was created manually \nand these terms were removed from the gene-MeSH term lists. Finally, from the thus filtered gene-MeSH lists, the 20 highest-frequency MeSH terms associated with each gene were taken as MeSH \nkeywords associated with each gene. For example the MeSH keywords associated with a gene \u201cFOS\u201d in our gene list are oncogene, felypressin, transcription-factor, thermoreceptors, DNA-binding, \nantibiosis, inflammatory-response, zinc-fingers, gene-regulation, and neuronal-plasticity.\nGO keyword extraction\nWe used the GO keywords information incorporated in Gene Ontology [Error! Bookmark not defined.] to extract GO keywords associated with each gene. Out of the three GO annotation categories \nwe included only molecular function and biological process as we believe that cellular component (e.g. nucleus, cell membrane etc.) is less important for characterizing genes in the context \nof this study. Further, due to the hierarchical nature of GO and multiple inheritance in the GO structure, we consider with every ancestor up to level 2 in the GO tree in assigning GO keywords. \nThis enables us to use more generalized GO terms. For example the GO keywords associated with the gene \u201cFOS\u201d in our gene list are protein-dimerization, DNA binding, RNA polymerase, transcription \nfactor, DNA methylation, inflammatory-response, and nucleus.\nKeyword representation and calculation of numeric vectors\nAfter the keyword extraction phase, each gene was described by a list of keywords extracted from MEDLINE abstract sentences, MeSH terms and GO terms. These keyword vectors then served as a basis \nfor clustering (i.e., unsupervised class discovery). To do this, each term vector needed to be represented by a numeric vector representing the relative importance of keywords for each gene. This \nprocess is concerned with computing the numeric weight, wij, for each gene-term pair (gi, tj) (i = 1, 2,\u2026n and j = 1, 2, \u2026 k) to represent the gene's characteristics in terms of the associated keywords. \nCommon techniques for such numeric encoding includes (1) Binary, the presence or absence of a keyword relative to a gene, (2) Term frequency, he frequency of occurrence of a keyword with a gene (3) \nTerm frequency \u00d7 inverse document frequency (TF*IDF), the relative frequency of occurrence of a keyword with a gene compared to other genes.\nAs we derived the keywords from gene annotation sentences but not from full abstracts, we found the number of keywords associated with each gene is small. We noticed also that absolute frequency of \nmost keywords tended be one. Therefore, we adopted the binary encoding scheme as illustrated in Table 2 in supplementary material, in which each gene is represented by a vector of \u2018normalized\u2019 absolute \nkeywords frequencies. The \u2018normalized\u2019 absolute frequency of each vector element (keyword) is either zero or one. \nGene clustering\nClustering is a data mining technique that groups or clusters data components (typically represented as numeric vectors) according to their similarity or dissimilarity. The goal is to maximize \nintra-cluster and minimize inter-cluster similarity among the components. [32,33] Clustering is typically used to identify sample groups in data. Unlike supervised learning methods that require explicit \nclass label information, clustering is unsupervised and no information about target groups (classes) is used. Two basic approaches to clustering can be distinguished, hierarchical clustering (e.g., agglomerative \nand divisive) and non-hierarchical clustering (e.g., k-means\/c-means clustering). Agglomerative hierarchical clustering starts with each object representing a cluster and then merges the clusters in sequence. \nDivisive hierarchical clustering starts with all samples in one cluster and successively split clusters. In hierarchical clustering the distance (similarity) between clusters is measured using different \ntechniques such as single linkage, average linkage or complete linkage [32] and basic distance and similarity metrics (e.g., Euclidean, Minkowski, Hamming distance). K-means clustering requires a priori \nspecification of the desired number of clusters, k. This method clusters data into groups by iteratively optimizing the positions of cluster centers (means) so that the sum of within-cluster similarities \n(the similarity between data points and their cluster centers) is maximized.\nEssentially, the sentence-level binary coding scheme adopted in this study consists of numeric row vectors representing genes (via the associated biological function\/process terms), and numeric column \nvectors representing annotation terms (via the associated genes). These two sets of vectors can be independently clustered using available clustering algorithms and tools. This approach can produce useful \nand specific information about the biological characteristics of sets of genes. In this study, we have used average linkage hierarchical clustering algorithm. [33] Using this algorithm has two advantages \nfor this study. First, clustograms, a visualization of the substructures contained in a gene collection are produced, and second, individual clusters of genes are identified by clustogram splits at different \nlevels. Clustering was performed using Cluto [34] and Cluster\/Treeview [35] facilitates visualization of the clustograms.\nResults and discussion\nEvaluation\nTo obtain a quantitative measure on the performance of the various keyword encoding schemes, we developed a text corpus of 200 manually annotated abstracts based on two cancer categories brain tumor and breast \ncancer of our interest (see Table 4 under supplementary material). We used the following procedure to establish the corpus: (1)Determine randomly two cancer categories (brain tumor and breast cancer ), (2) For \neach cancer category, select randomly 10 genes from Entrez such that species = human and number of associated abstracts \u2265 50, (3)For each gene identified in this way, select randomly 10 abstracts, resulting in \na total of 200 abstracts; 10 abstracts for each of the 10 genes associated with each of the two cancer categories, (4) For each of the 200 abstracts, identify manually the keywords characterizing biological \nfunction and processes from abstracts, MeSH terms and GO terms.\nWith this text corpus we were able to construct a matrix containing all 20 genes and their associated keywords and keyword frequencies from abstracts, MeSH terms and Go terms. The manually annotated corpus \nof 200 abstracts and the matrix of 20 annotated genes served as gold standard for our evaluation experiments. We carried our four evaluation experiments: (1) Abstract keywords (baseline). Extracts gene annotation \nterms based on term frequencies * inverse document frequencies (TF*IDF) within the entire abstract without regard to sentence structure, (2) Sentence keywords. Extracts gene annotation terms based sentence-level \nkeywords, (3) Sentence + MeSH keywords. As in (2) above plus MeSH terms (see Section MeSH keywords extraction), (4) Sentence + MeSH + GO keywords. As in (2) above plus MeSH terms (see Section MeSH keywords \nextraction) and GO terms (see Section GO keyword extraction).\nEssentially, in each evaluation experiment the input is the text corpus of 200 abstracts and the output is a list of genes with its predicted annotation terms. Informally, the closer the predicted annotation \nterms match the manually established annotation terms, the better is the method. Performance is measured via commonly used criteria such a recall (analogous to sensitivity), precision (analogous to positive \npredictive value) and the F-measure (a score that combines recall and precision). The results we obtained are shown in Table 5 (below in supplementary material).\nWe notice that the baseline method comprising TF*IDF keywords fares worst among all four approaches. We interpret this as evidence for the validity of the methods involving sentence-level processing as this \ninformation is likely to carry most specific characterizing terms. The \u2018brute-force\u2019 abstract-level processing will have difficulty in extracting these terms correctly and consistently. We further notice that \nthe substantial improvements of precision and recall when we include MeSH terms and GO terms. This may be because these two categories are more specific and MeSH and GO annotations were done using full-papers \nand these biological functions and process are not described in all abstracts.\nClustering of genes resulting from microarray experiment\nTo demonstrate the usefulness of the presented keyword-extraction techniques to microarray data analysis, this method was applied to annotate and cluster gene lists that were found differentially expressed in \na microarray experiment investigating the impact of two mitogenic proteins, Epidermal growth factor (EGF) and Sphingosine 1-phosphate (S1P), on glioblastoma cell lines [36]. The microarray data set reveals three \nsets of differentially expressed genes (p<0.05), namely, genes differentially expressed with response to EGF, G(EGF), genes differentially expressed with respect to S1P, G(S1P) and genes differently expressed in \nresponse to both, G(COM).\nGenes were considered differentially expressed if their p-value is smaller than 0.05. We found that, when compared to the resting state, 19 genes were significantly differentially expressed as a response to EGF, \n35 genes as a response to S1P and 30 genes as a response to COM, i.e., combined stimuli of S1P and EGF. The three gene lists are referred to as G(EGF), G(S1P) and G(COM), respectively (see Table 6 in supplementary material).\nUsing these the three gene lists obtained from the microarray experiment (Table 6 shown in supplementary material) as query in MEDLINE returned the three corresponding sets of abstracts A(EGF), A(S1P) and \nA(COM), respectively. The abstracts were processed with the keyword extraction method involving sentence-level, MeSH and GO terms and the resulting representations were clustered using average linkage hierarchical \nclustering algorithm. Our gene clustering strategy and clustering algorithms are explained in the Methodology section. The resulting clustograms are presented in Figure 2, Figure 3, and Figure 4, respectively. \nThe clustograms depict associations between genes and biological function\/process terms derived from the abstracts obtained with the various gene lists. For the investigating scientist, the clustograms fulfill the \nfollowing main functions: (1) Squares highlighted in a horizontal line link a gene to one or more biological functions or processes. This is useful to see which genes are associated with which functions\/processes and \nwhich genes have few or many associations. The interpretation of many and few is very much dependent on the associated biological function\/process categories, the particular scientific question under investigation, \nand also on how extensively a particular gene has been researched and reported in the literature. (2) Users may visually delineate clusters, i.e., rectangular areas with many highlighted squares in them and few \nhighlighted squares around them. Any cluster, small or large, is potentially very useful to have discovered. Each cluster identified in this way relates a set of genes to a group of biological functions and processes. \nIn a sense, each gene in the clustered is characterized by the same set of biological function and process concepts, a kind of \u2018guilt by association\u2019. This information is extremely useful as it provides clues as to \nthe roles genes may play collectively in pathways and functions, processes, and possible phenotypes, that are associated with these pathways. \nSummary of analysis of EGF cluster, G(EGF)\nThe clustograms in Figure 2 show the results obtained from extracting the sentence-level function\/process keywords (plus MeSH and GO terms) from 28,913 abstracts (for the 19 genes detected in response to EGF \nstimulus) and the subsequent clustering. In Figure 2a several individual genes with very many (e.g., CALD1, CLU, FOS) and very few (e.g., HRY, DUSP6) associations stand out. Another interesting feature is the \nlarge cluster at the lower left corner of Figure 2a (reproduced in more detail in Figure 2b) containing the genes DUSP, ID1, KLF2, CALD1, ABCA, CLU, FOS, JUN and SLC5A3. Many genes in this cluster are associated \nwith the same set of keywords (transcription factor, cell death and secretion).\nSummary of analysis of S1P cluster, G(S1P)\nThe clustograms in Figure 3 show the results obtained from extracting the sentence-level function\/process keywords (plus MeSH and GO terms) from 19,705 abstracts (for the 30 genes detected in response to S1P \nstimulus) and the subsequent clustering. In Figure 3a several individual genes with very many (e.g., CCL3, IL6, IL8, F3) and very few (e.g., HERB2, DOC1) associations stand out. Another interesting feature is the \nlarge cluster at the upper left corner of Figure 3a (reproduced in more detail in Figure 3b) containing the genes TNAIP, KLF5, BCL6, NAB1, BTG1, NFKBIA, NR4A1, SOCS5, CITED2, NRG1, JAG1, PLAU, CCL2, IL8, IL6, GLIPR1, \nF3, MAP2K3, and EHD1. Many genes in this cluster are associated with the same set of keywords (atherogenesis, mitogenesis, assemble, inflammation, focal-contact, \u2026, and protein-binding).\nSummary of analysis of the common gene cluster, G(COM)\nThe clustograms in Figure 4 show the results obtained from extracting the sentence-level function\/process keywords (plus MeSH and GO terms) from 39,890 abstracts (for the 30 genes detected in response to EFG and \nS1P stimuli) and the subsequent clustering. In Figure 4a several individual genes with very many (e.g., MYC, MAFF, ATF3) and very few (e.g., DIPA, UGCG, SNARK) associations stand out. Another interesting feature is \nthe large cluster at the upper left corner of Figure 4a (reproduced in more detail in Figure 4b) containing the genes SPRY2, GEM, ZYX, NEDD9, MYC, LIF, SERPINE1, DTR, MUCL1, C8FW, MAFF, ATF3, RTP801, EGR1, JUNB, \nFOSL1, CEPED, TIEG, EGR2, EGR3, and ZFP36. Many genes in this cluster are associated with the same set of keywords (DNA binding, zinc fingers, repressor proteins, \u2026, and mitosis). \nAn important aim in microarray data mining is to bind transcriptionally modulated genes to functional pathways or to understand how transcriptional modulation can be associated with specific biological events \nsuch as genetic disease phenotype, molecular mechanism of drug action, cell differentiation etc. However, the amount of functional annotation available with each transcriptionaly modulated genes is still a limiting \nfactor because not all genes are well annotated. Our functional clustering\/grouping will enable to select literally informative genes (Figure 2b, Figure 3b, and Figure 4b) for further investigations in the above \ndata mining and knowledge discovery pipeline. Our evaluation suggests that this approach will provide more specific and useful information than typical approaches using abstract-level information. This is particularly \nthe case when the sentence-level terms are augmented by MeSH and GO keywords.\nConclusion\nThe sequencing of whole genomes and the introduction high throughput analysis (e.g., oligonucleotide and cDNA chips, MALDI\/SELDI-TOF MS) provides biomedical research with a global perspective, which necessitates \nthe development of novel mining tools to explore and interpret data in timely manner. This paper presents a novel approach to combine sentence-level keywords with GO and MeSH terms. In our evaluation experiment, this \napproach has shown promising results. The present evaluation suggests that this approach will provide more specific information than typical approaches using abstract-level information. This is particularly the case \nwhen the sentence-level terms are complemented by MeSH and GO terms. Further, clustering of genes into different functional groups based on literature keywords has the potential to help biologists identify and characterize \nliterally informative genes of interest for further investigations.\nFuture work\nFuture enhancements of the system will include additional data resources (OMIM. DIP, KEGG) and the generation of association rules to identify correlations among genes in the same cluster. Association rules between \nthe genes in the same cluster seem particularly interesting because it allows one to find the presence of regularities between gene groups. Finally, abstracts were used in this study as they are readily and easily available \nbut they are limited in content. As full-text contains large number of irrelevent sentences compared to abstracts this approach may be useful for full-text analysis too, as it performs filtering of irrelevant sentences \nbefore clustering. The plan to perform the current study with full-text articles and compare the results with that of abstracts is on the way.\nSupplementary material\nData 1","keyphrases":["text mining","microarray data analysis","functional clustering"],"prmu":["P","P","P"]}
{"id":"Qual_Life_Res-3-1-1915653","title":"Small-cell lung cancer patients are just \u2018a little bit\u2019 tired: response shift and self-presentation in the measurement of fatigue\n","text":"Background Response shift has gained increasing attention in the measurement of health-related quality of life (QoL) as it may explain counter-intuitive findings as a result of adaptation to deteriorating health.\nIntroduction \nQuality of life (QoL) is considered an important treatment outcome when the treatment intent is not curative but palliative. However, the expected deterioration in QoL often does not occur, even in cases of serious illness. For example, Groen et\u00a0al. studied patients with inoperable non-small cell lung cancer treated by radiation with and without chemotherapy [7]. QoL was measured with The European Organization for Research and Treatment of Cancer Core Quality of Life Questionnaire (EORTC QLQ-C30) [1], which has been designed specifically for use in clinical trials focusing on cancer patients. However, contrary to expectation, they did not find significant deterioration in the scale scores over the treatment period of 6\u00a0weeks. Although cancer patients are willing to undergo risky and toxic treatments [11], it seemed implausible that the side-effects of treatment had not affected their QoL. Other studies also reported counter-intuitive results. For example, patients with a life-threatening disease or disability were found to report stable QoL, and patients with a severe chronic illness reported QoL levels that were not inferior to that of patients with a less severe illness or to healthy patients [2, 3, 5]. Such counter-intuitive findings, labelled by Breetvelt and Van Dam as \u2018underreporting of problems\u2019 suggest that patients report less distress and dissatisfaction than they actually feel [5].\nIn recent years, response shift theory has gained increasing attention in explaining paradoxical and counter-intuitive findings. Response shift refers to a change in internal standards, values and conceptualization of QoL and is recognized as an important mediator in adaptation to changing health [17]. However, despite the explanatory power of response shift theory, our current understanding of phenomena that can complicate the interpretation of QoL scores is still limited. Therefore, we investigated QoL measurement in small-cell lung cancer (SCLC) patients during 1st line chemotherapy. We were quite surprised when we noticed discrepancies between levels of fatigue measured with the questionnaire and answers spontaneously reported during the interview. We therefore investigated these \u2018conflicting\u2019 findings in the measurement of QoL in more depth.\nThis paper reports the results of an exploratory longitudinal multiple-case study, in which we focused on how patients responded to the EORTC QLQ-C30 question \u2018were you tired\u2019 at different points in their treatment trajectory. We aimed to describe the patients\u2019 explanations when answering the question, and to search for explanations of counter-intuitive findings.\nMethods\nProcedures and study sample\nBetween March 2001 and September 2003, we recruited newly-diagnosed patients with SCLC who were evaluated for 1st line chemotherapy. The patients were attending one of five outpatient clinics for chest diseases in the Netherlands. To maximize the likelihood that we would interview patients from the beginning of their treatment, we were informed about new patients immediately after diagnosis. No restrictions were made with regard to age or treatment (chemotherapy or a combination of chemotherapy and radiotherapy). Participating patients gave written consent and were interviewed at equivalent points in the treatment trajectory. The first interview (T1) was carried out at the start of chemotherapy. In the original plan the second interview was planned after completion of the course of chemotherapy. However, after inclusion and first interviews of 3 patients we made a decision to interview the patients during treatment as well. Therefore the second interview (T2) was conducted 4\u00a0weeks after T1 and the third (T3) 7\u201310\u00a0days after completion of the treatment with chemotherapy and the fourth (T4) 6\u00a0weeks later. Approval for this study was obtained from the Medical Ethics Committees of the research site and the participating hospitals.\nDuring the course of the study, 41 eligible patients were invited to the study. Four patients were unwilling to participate, 3 died before informed consent could be obtained, and 3 were not interviewed because of imminent death. Of the 31 respondents who were interviewed, 8 were excluded from further analysis because their data were incomplete, i.e. they were only interviewed once (six died within a month after T1 and two were too sick at T2 and died before the end of the planned chemotherapy). Consequently, the final study sample consisted of 23 SCLC patients, of whom 12 had limited (3 male and 9 female, mean age 55, range 42\u201369) and 11 had extended disease (8 male and 3 female, mean age 64, range 39\u201372). All patients received standard chemotherapy, except for 7 patients whose chemotherapy was combined with local radiation of the tumour. The majority of the patients were married (19, 83%), and had children (17, 74%).\nOf the 23 patients in our study sample, 15 were interviewed four times, 7 were interviewed three times, and one patient was only interviewed twice resulting in a total of 83 interviews. The interviews were conducted by MW in the homes of the patients. In three cases the 1st interview was held in the hospital. Interviews averaged 80\u2013110\u00a0min.\nMaterials and qualitative method\nIn this exploratory, longitudinal multiple-case study, QoL was assessed with EORTC QLQ-C30 (version 3.0) [1] and the lung cancer module QLQ-CL13 [4]. The EORTC QLQ-C30 is the most widely used cancer-specific QoL instrument in European clinical trials. The questionnaire is composed of several scales, which measure among others physical function, mental health, general health and global QoL. Furthermore, it measures different symptoms such as pain, dyspnoea, nausea and fatigue. The Fatigue Scale consists of 3 items: \u2018did you need to rest\u2019, \u2018have you felt weak\u2019 and \u2018were you tired\u2019 (respectively questions 10, 12 and 18), for which there are 4 response categories: \u2018not at all\u2019, \u2018a little\u2019, \u2018quite a bit\u2019 and \u2018very much\u2019 (respectively scores 1, 2, 3 and 4).\nThe EORTC QLQ-C30 and CL13 were completed in combination with the Three-Step Test-Interview (TSTI) to investigate how respondents interpreted the items and how they responded to them. The TSTI consists of the following steps [8]: (1) concurrent think aloud, aimed at collecting observational data on how a respondent completes the questionnaire, expressing his thoughts aloud; (2) focused interview, aimed at clarifying respondents\u2019 previous expressions while completing the questionnaire; (3) semi-structured interview, aimed at eliciting respondents\u2019 experiences and opinions with regard to the questionnaire.\nInterview protocol\nEach interview was conducted in an identical format. At T1, QoL was measured with the EORTC QLQ-C30, followed by the lung cancer module QLQ-CL13. The questionnaire was conducted in a concurrent think aloud manner and after completion, respondents were asked to clarify previous hesitations, expressions when rating certain items and experiences (i.e. second and third step of the TSTI). Individual QoL was then measured with the Schedule for the Evaluation of Individual Quality of Life\u2014Direct Weighting (SEIQoL-DW) [13, 14]. Finally, we encouraged patients to talk freely about the impact of diagnosis and treatment. At follow-up (i.e. T2, T3 and T4), after the SEIQoL-DW assessment, the EORTC questionnaires were administered a second time as a so-called \u2018then-test\u2019 [15, 16, 18] (i.e. the patients filled out the questionnaire in reference to how they perceived themselves as they were in the previous interview). In these 2nd and following interviews, EORTC assessments were conducted in a concurrent think aloud manner and with the second step of TSTI integrated in the assessment. In fact, we encouraged patients to think aloud and we probed for clarification after each item in the case of extra information was considered useful to understand patients\u2019 answer. We used a flexible approach in order not to interrupt the natural flow of both the assessment as well as the patient-interviewer communication.\nThe interviews were audio-taped and transcribed verbatim. In this article we focus on the EORTC QLQ-C30 question \u2018were you tired\u2019.\nAnalysis\nThree types of data were collected and used for analysis [12]: (1) completed EORTC questionnaires (T1\u2013T4), (2) observed respondent behaviour recorded in field notes, and (3) transcriptions of the interviews, including \u2018think aloud\u2019. The analysis was aimed at identifying discrepancies, response strategies and explanations of response behaviour. We used the qualitative computer package Kwalitan 5.0 (http:\/\/www.kwalitan.net) to extract relevant parts of the transcriptions (1) \u2018think aloud\u2019 of the question \u2018were you tired\u2019 and of other items that were useful in understanding the response behaviour to the question on fatigue (i.e. two other items of the fatigue scale, the general health and the global QoL question of the QLQ-C30), and (2) \u2018comments\u2019 related to fatigue symptoms, impact of treatment on perceived QoL, and attitudes towards life. In order to deal with the still remaining large amount of extracted data, two of the authors (MW, AT) condensed extracted transcripts of the \u2018comments\u2019 into core texts. For each patient, the data (i.e. think aloud combined with scores and core texts of comments) were organized per interview in one mind map (see example in Fig.\u00a01) by means of the computer package Mindjet Mindmanager Pro 6 (http:\/\/www.mindjet.com).\nFig.\u00a01Analysis by means of a mind map. Branch EORTC: scores of GH\/QOL and the fatigue scale are organized per interview and complemented by the think aloud data. Branch Comments: core texts of relevant parts of transcripts are organized per code and per interview. Note: (+) not all branches of the different interview moments are shown\nFurthermore, a different mind map was made to organize think aloud data related to the question \u2018were you tired\u2019 for all patients per response category per assessment, including then-test. For the analysis, three authors (MW, AT, TH) each independently read the mind maps of each patient. They studied patients\u2019 scores, their think aloud responses, and examined whether response shift type explanations would be provided: recalibration (i.e., using different standards of comparison to assess fatigue over time), reprioritization (i.e., changes in the importance attached to fatigue over time) and reconceptualization (i.e., changes in the meaning of fatigue over time). Two researchers (MW, AT) searched for additional explanations in the core texts to account for the response behaviour and the discrepancies. The research team (MW, AT, TH, MS) discussed critically the different response strategies used by the patients and the robustness of the interpretations of response shift.\nResults\nPatients with or without discrepancies\nOf the 23 patients, 15 (5 male and 10 female, age 46\u201372) showed discrepancies at least at one measurement point, i.e. differences between their answer to the EORTC question \u2018were you tired\u2019 and their level of fatigue spontaneously reported during the interview. In their answers to the EORTC questionnaire they all presented themselves positively and said that they were not tired. Mary, for example, was 60\u00a0years old at the time of the first interview. During all her interviews she reported that she was tired, but she consistently answered \u2018not at all\u2019 to the EORTC question (Box 1). Only once she scored \u2018a little bit\u2019. This was at T2 when filling in her questionnaire as a then-test for T1 and she commented \u201ca little, more than at the moment\u201d. \nBox 1Example of a patient with discrepancies in reported level of fatiguePatient MaryMary was 60\u00a0years old and married. She had two sons and two grandchildren. Her answer to the EORTC question was consistently \u2018not at all\u2019, except for the then-test concerning the interview T1. This suggests that she did not suffer from fatigue in the week prior to the interviews and that fatigue due to chemotherapy did not have any impact at all on her energy level. But, during the interview she spontaneously provided information that indicated that chemotherapy had an impact on her life and that she regularly suffered from fatigue.T1 EORTC score \u2018not at all\u2019Think aloud: Were you tired... no not at all, no, no difference compared to the pastInterview: I\u2019m getting tired at the least little thingT2 EORTC score \u2018not at all\u2019Think aloud T2: Not tired, last week, not at allThink aloud then-test T1: a little, more than at the moment.Interview: According to the doctors, the X-rays were very good. I\u2019m very optimistic, sometimes I\u2019m tired but that\u2019s my own fault. I don\u2019t have as much energy as I did before I became ill.T3 EORTC score \u2018not at all\u2019Think aloud: Last week I wasn\u2019t tired, it\u2019s the second week after my chemo, not at all tiredThink aloud then-test for T2:not at allInterview: Yes, it was my last cycle of the chemo, I was afraid that I wasn\u2019t going to be able to carry on through the treatment. But I managed, okay; I\u2019m tired but apart from that... nothing at all.T4 EORTC score \u2018not at all\u2019Think aloud: No I wasn\u2019t tired last week, not at allThink aloud then-test for T3:not at allInterview: Now and then, I\u2019m tired. It\u2019s different to before my chemotherapy. Sometimes I\u2019m so tired, so tired, more than in the past. It comes suddenly...in the middle of the day.\nFor many of our respondents, every new cycle of chemotherapy had a more severe impact on their energy level. Therefore, the highest level of fatigue was to be expected after the 5th and last cycle of chemotherapy, at T3. However, the 15 patients with discrepancies all answered the question \u2018were you tired\u2019 with \u2018not at all\u2019 or \u2018a little\u2019. For example, during her interviews at T2 and T3, Ann reported the growing impact of every cycle of chemotherapy, but her answer to the EORTC question at both interviews was \u2018a little\u2019 (Box 2). \nBox 2Example of a patient using different response strategies, comparison with more sick patients, response shift and self-presentationPatient AnnAnn was 47\u00a0years old and living with a partner. She didn\u2019t have any children.Her scores suggest that the chemotherapy had a slight impact on her energy level during her treatment and a greater impact 6\u00a0weeks after completion of the treatment. But, the interview and the think aloud provided information that indicated that the chemotherapy had a growing impact on her life, and the score \u2018quite a bit\u2019 at T4 was the result of bad news (i.e. a recurrence of the tumour). Furthermore, her data show examples of different response strategies, comparison with more sick patients and self-presentation.T1 EORTC score \u2018not at all\u2019Think aloud: Were you tired.. in principle I wasn\u2019t tired. I was mentally tired, it costs me a lot of energy to talk with my relatives. I think you mean physically tired. You\u2019re tired in the sense that you can hardly put one foot in front of the other. That\u2019s being tired. No I\u2019m not tired.Interview: I want to be realistic, think positively. I try not to worry; there is nothing I can do. I don\u2019t know when I will die. I get angry when people are surprised...if I say I\u2019m doing fine...it\u2019s my decision how I\u2019m feeling.T2 EORTC score \u2018a little bit\u2019Think aloud: I feel it a little bit, compared to other people who are very sick. So, if I have pain or when I\u2019m tired I say to myself don\u2019t complain, so everything I feel, I only feel a little. I\u2019m doing fine.Think aloud then-test for T1:a littleInterview: Yesterday, I worked for three hours and I was exhausted. I went to bed in the middle of the day in order to be able to show my friend that I\u2019m doing fine. She has trouble in coping...me... having cancer.T3 EORTC score \u2018a little bit\u2019Think aloud:A little, yes because I have the feeling that I was able to get over itThink aloud then-test for T2: I think that I\u2019ve said a little last time, it felt a little, but I shift my limit. Interview: It\u2019s a kind of tiredness, I don\u2019t know. I\u2019ve never been like that. To allow yourself to be tired. I think that the story they all tell, that the last cycle of chemo has the most impact, I think that\u2019s very very true.T4 EORTC score \u2018quite a bit\u2019Think aloud: Yes, I was quite a bit tired, but only mentally tired. I have to adjust to the idea of a new course of treatment and radiation. My health is excellent. I\u2019m able to do everything I like, better than 6\u00a0weeks ago. But, with all the medicine I\u2019m taking to suppress the epileptic fits... I\u2019m scared, just like after the start of chemo.Think aloud then-test for T3:a little but I am not really sure, the pain which I had in my ankels made me tired.Interview: The radiation will make me tired, but it has not started yet, so I am not tired yet.\nPatients with discrepancies were identified in both stages of disease and with both treatment regimens, i.e. 12 patients (LD n\u00a0=\u00a05, ED n\u00a0=\u00a07) treated with chemotherapy and 3 LD patients treated with chemotherapy and radiotherapy. During the course of the treatment (T1\u2013T4, 55 interviews) they answered the question \u201cwere you tired\u201d 20\u00a0times with \u2018not at all, 29\u00a0times with \u2018a little\u2019, 4\u00a0times with \u2018quite a bit\u2019 and twice with \u2018very much\u2019 (see individual scores per interview in Table\u00a01)\nTable\u00a01Individual (then-test) scores of patients answering the EORTC QLQ-C30 question \u2018were you tired\u2019. Response categories 1, 2, 3 and 4 are representing respectively the category \u2018not at all\u2019, \u2018a little\u2019, \u2018quite a bit\u2019 and \u2018very much\u2019. Small-cell lung cancer patients (n\u00a0=\u00a023), limited (LD) and extended (ED) disease receiving 1st line chemotherapy were interviewed at equivalent points in treatment: at start of chemotherapy (T1), 4\u00a0weeks later (T2), at end of chemotherapy (T3), and 6\u00a0weeks later (T4). T1t, T2t and T3t are representing then-test scores obtained at respectively T2, T3 and T4, when patients are asked to provide a renewed evaluation of their fatigue at the previous assessment. Two groups were identified: patients with (n\u00a0=\u00a015) and without (n\u00a0=\u00a08) discrepancies between their questionnaire answer and fatigue spontaneously reported in the interviewPatients\u2019 characteristicsNr.M\/FAgeLD\/EDT1T1tT2T2tT3T3tT4DiscrepanciesP 02Male57ED33\u2013\u20131\u2013\u2013P 04Female50LD1223232P 08Female69ED2212122P 09Male66ED3222222P 10Male46LD2222222P 12Female47LD1222223P 15Female69LD1223232P 17Female64ED22222\u2013\u2013P 18Male72ED112\u20131\u20133P 21Male69ED2223222P 22Male55LD411\u20132\u2013\u2013P 24Female56LD1112121P 26Female59LD42122\u2013\u2013P 32Female60LD1211111P 34Female51LD1121211No discrepanciesP 01Female42LD22\u2013\u2013443P 03Female64ED31\u2013\u2013334P 13Male72ED332332P 14Male39LD43223\u2013\u2013P 16Male68LD1133332P 20Female44LD1\u20134\u20134\u20134P 27Male69LD322\u2013\u201342P 29Male63ED2332332\nNo discrepancies were identified in 8 of the 23 patients (LD n\u00a0=\u00a04, ED n\u00a0=\u00a04, age 39\u201372). They answered the question \u2018were you tired\u2019 at the end of chemotherapy (T3) with \u2018quite a bit\u2019 or \u2018very much\u2019. During the course of treatment (T1\u2013T4, 28 interviews) these patients answered the question twice with \u2018not at all\u2019, 9\u00a0times with \u2018a little\u2019, 11\u00a0times with \u2018quite a bit\u2019 and 6\u00a0times with \u2018very much\u2019 (Table\u00a01).\nThen-test scores were dissimilar with scores of the previous assessment in 25 out of 52 cases, with higher then-test scores in 16 cases. Transcripts showed that patients had difficulty remembering either the previous measurement point and\/or their fatigue at that time.\nResponse strategies for the four response options\nThe think aloud texts for the response categories \u2018quite a bit\u2019 and \u2018very much\u2019 were minimal in the entire study population. Only a few patients reacted briefly during the think aloud, e.g. \u201cquite a bit, too tired to keep my eyes open\u201d, \u201cyes quite a bit, very tired\u201d and \u201cnext week it will be better...very much\u201d. The same pattern was found in all patients for all four response options when filling in the questionnaire as a then-test, e.g. \u201ca little bit, I think\u201d, \u201ctired then, no\u201d and \u201cvery much\u201d. However, patients in the group with discrepancies had much more to say in the conventional QoL measurement when choosing the options \u2018not at all\u2019 and \u2018a little\u2019. During \u2018think aloud\u2019, they seemed to justify the chosen response category. They used various strategies to moderate the impact of fatigue on their life. We summarized their strategies in four categories: (1) I am not tired all the time (e.g. \u201conly in the afternoon\u201d); (2) I am not really tired, it\u2019s something else (e.g. \u201c it\u2019s the flu\u201d); (3) I have no problems with it (e.g. \u201cI can still cope with it; I don\u2019t want to exaggerate\u201d); (4) I am a little bit tired but it is due to something else (e.g. \u201cI didn\u2019t have a proper meal\u201d).\nMany of our respondents said that they had expected to become very tired as a result of the treatment, but that they were not as sick as they had expected. They were very happy that they were able to cope with the treatment, and had adjusted to the situation. A male patient, for example, had a score of \u2018not at all\u2019 at T2, and explained: \u201cI\u2019m currently doing nothing, so I\u2019m not tired\u201d. The respondents indicated that they wanted to be honest when filling in the questionnaire and did not want to lie or to exaggerate their fatigue and, because many patients were not tired all the time they considered a score of \u2018not at all\u2019 or \u2018a little\u2019 to be a suitable score (Box 3). \nBox 3Examples of different response strategies used by patients with discrepancies (n\u00a0=\u00a015)Think aloud about the question \u2018were you tired\u2019I\u2019m not tired all the timeI\u2019m only tired in the afternoonNo not at all, I\u2019m not tired at the moment, it comes suddenlyI have to be honest, sometimes I\u2019m tired, I can\u2019t say not at all, otherwise I would be lyingI\u2019m not really tired, it\u2019s something elseNo not tired, it\u2019s the flue, that\u2019s why I\u2019m tiredIt\u2019s not being tired you know, it\u2019s more like being restlessI\u2019m not physically tired, I\u2019m mentally tiredActually, I can\u2019t be tired because the Hb level in my blood is okayI\u2019ve no problems with itI\u2019m currently doing nothing, so I\u2019ve no problems, I\u2019m not tiredOf course, you can make yourself tired, but I\u2019 don\u2019tI can still cope with it; I don\u2019t want to exaggerateI\u2019m a little bit tired but it\u2019s due to something elseA little, but it was my own fault, I did too muchI didn\u2019t have a proper meal, that\u2019s why I was tiredI didn\u2019t have my lady working for me in the house, she went on holidayIt\u2019s because I\u2019ve got problems with my voice caused by the radiation\nOptimism\nMost patients told the interviewer regularly that they had adapted to the situation and had changed their attitude towards a more optimistic perspective. They were not hopeful immediately after diagnosis, but optimism about recovery increased when the tumour was shrinking. Of the 23 patients 17 reported spontaneously that they were optimistic: e.g. \u201cI\u2019ve got good news, I\u2019m as optimistic as can be\u201d, \u201cI\u2019m full of hope because I was diagnosed in an early stage, so I\u2019m good in time\u201d. Furthermore, they said that they felt better off than expected, compared to patients who were worse off: e.g. \u201cI\u2019m lucky not to be very sick, compared to the patients I saw at the hospital\u201d. Although they experienced the impact of every new cycle of treatment as more severe, they said to be able to cope with the treatment and to accept the side-effects: e.g. \u201cit\u2019s part of the package, I\u2019m willing to put up with, knowing the chemo is doing the job properly\u201d. Some patients were actually feeling better after each cycle and happy that they were still alive: e.g. \u201cIt was much worse than I wanted to admit last time, I\u2019m feeling much better\u201d, \u201cI\u2019ve already a couple of months extra\u201d.\nIn contrast, 5 of the 23 patients expressed pessimistic feelings: e.g. \u201cI\u2019m a broken man, hard work all my life and now...I don\u2019t think I\u2019ve much time left\u201d, \u201cI\u2019m a bit depressed, when does it stop, if it doesn\u2019t stop it would be better if my life was over\u201d, \u201cNo plans for the future, you never know when the tumour will come back\u201d. These patients all reported high levels of fatigue during the course of the treatment. One patient did not provide specific comments about optimism or pessimism.\nResponse shift and self presentation \nIn both groups\u2014with and without discrepancies\u2014we found patients who had reported to have changed their reference point after T1 (i.e. recalibration in contrast to T1). They compared their fatigue at the second and following interviews with that of other patients e.g. \u201cI was tired, yes, but compared to the patients I\u2019ve seen in the hospital, I\u2019m just a little bit tired\u201d or, with the period in which they were more tired (e.g. \u201cCompared to the first week after chemo, it\u2019s the second now ... I\u2019m not tired\u201d). In one case, a patient spontaneously re-evaluated her previous measurement: \u201cI told you that I was really tired then, but compared to how I\u2019m feeling now, it was then just a piece of a cake\u201d. Another one spoke about a shift of limits which also suggests recalibration: \u201cI already told you that I would change my standards\u201d.\nWe did not find indications of reconceptualization and reprioritization of fatigue. The only exception was Ann who made a distinction between being physically and mentally tired (see Box 2). At T1, she said that she was mentally tired but not physically and her answer was \u2018not at all\u2019: \u201cTired means that you can hardly put one foot in front of the other\u201d. At T4, she said that she had recovered from chemotherapy and was physically able to do anything she wanted but, unfortunately, suffered from sudden epileptic attacks caused by metastases. She was feeling anxious in the same way as at the start of her chemotherapy, and had to consider further treatment options. Just like in the interview at T1, she said that she was mentally tired but not physically. However, this time her score was \u2018quite a bit\u2019 instead of \u2018not at all\u2019: \u201cPurely, because I was mentally tired last week. I have to adjust to the idea of a new course of treatment. Actually, I have to admit that I really am the cancer patient I never wanted to be\u201d. This response pattern might be interpreted as reprioritization (i.e., changes in the importance attached to mental fatigue over time). \nBox 4Examples of coping strategies used by patients with discrepancies (n\u00a0=\u00a015)Spontaneously reported coping behaviourProtective behaviourI\u2019m trying to avoid or minimize pessimistic thoughtsI don\u2019t think about it, otherwise I can\u2019t cope with itWe don\u2019t talk about it, just follow my every day routineI\u2019m building a wall around myselfAssertive behaviour\/power displayI\u2019ll show others that I\u2019m managing all rightYou have to be positiveYou have to believe in yourself, otherwise you can\u2019t manage it anymoreFighting the stigmaI\u2019m not the cancer patient my neighbour thinks I amThey think I\u2019m lying on my bed all dayPeople look at me, and give advice that I don\u2019t wantI have to admit that I really am a cancer patient... I didn\u2019t want to be (see patient Ann, Box 2).\nBecause the above mentioned response shift type explanations could not adequately explain our conflicting findings in the discrepancy group we questioned: \u201cWhy are patients presenting themselves in the questionnaire more positively than in the informal interview\u201d. In our search for an other explanation, we found that 13 of the 15 patients with discrepancies had spontaneously reported how they dealt with having cancer and the perspective of a short life-expectancy. We summarized their comments in three categories (see examples of coping strategies in Box 4): (1) Protective behaviour (e.g. protecting themselves from harmful thoughts); (2) Assertive behaviour\/power display (e.g. projecting the image of being positive and managing all right); (3) Fighting the stigma (e.g. fighting against being stigmatized).\nTaking these strategies into account, we concluded that a possible mechanism underlying the discrepancies in this group was \u2018self-presentation\u2019. As the questionnaires are explicitly related to cancer and since these patients want to distance themselves from being reduced to only a cancer patient, they want to present themselves as a person who just happened to have cancer. Therefore, they applied various strategies to respond to the question on fatigue in order to produce a score that was as favourable as possible and presented themselves as positive and managing their fatigue.\nDiscussion\nTwo third of the patients showed discrepancies in their reported level of fatigue. They reported a gradual decrease in energy at the end of chemotherapy, but they were \u2018not at all\u2019 or just \u2018a little bit\u2019 tired according to their answer to the EORTC questionnaire, with underreporting as a result. They presented a positive image of themselves and used various strategies to explain their choice of response category. A predominant finding was that patients adopted a more optimistic perspective on the treatment. Interestingly, this was not exclusively found in the discrepancy group. The same was true for recalibration and for the only indication of reprioritization. These response shift type explanations did not sufficiently account for the conflicting findings in our discrepancy group.\nSelf-presentation was found to be an additional (coping) mechanism underlying the discrepancies. Our results suggest that patients are not only concerned about the impression they make on others. They try to protect themselves from negative thoughts and they also feel the need to be positive and to distance themselves from the stereotypical cancer patient. With this strategy they are more capable of coping with a situation that they cannot change.\nThe suggestion that self-presentation is an underlying mechanism is supported, for example in the case of Ann. After a recurrence of the tumour she adopted the realistic perspective by admitting that she really was \u2018the cancer patient\u2019, which she did not want to be before. It seems that she had given up her attitude of showing others that everything was all right, and for the first time she did not present her self as more positive than she actually was as she did before.\nSelf-presentation (also called impression management [6, 9]) is a phenomenon described by Leary et al. in relation to health behaviour [10]. They discussed its implications for research in health psychology. Our study shows that, in addition to response shift, self-presentation may explain unexpected results, at least in SCLC patients. The question \u2018were you tired\u2019 in the EORTC-QLQ-C30 does not unequivocally measure the impact of chemotherapy on the energy level of patients; in fact, with their responses, patients seem to show how that they are managing the situation. From our results we cannot conclude that in the group without discrepancies self-presentation is not present at all, or that whenever self-presentation occurs discrepancies will also be present. However, our study does show that self-presentation affects QoL measurement. These findings must be taken into account when investigating and interpreting QoL data, also in other study populations. Especially after diagnosis and in the initial phase of treatment, self-presentation might be an important coping strategy. In fact, during each phase in which a new equilibrium and a new identity has to be found (e.g. after a recurrence of the tumour, or metastases) self-presentation might affect QoL measurement.","keyphrases":["small-cell lung cancer","response shift","self-presentation","fatigue","quality of life"],"prmu":["P","P","P","P","P"]}
{"id":"Virchows_Arch-3-1-1888716","title":"Expression microarray analysis of papillary thyroid carcinoma and benign thyroid tissue: emphasis on the follicular variant and potential markers of malignancy\n","text":"The most common sub-variant of papillary thyroid carcinoma (PTC) is the so-called follicular variant (FVPTC), which is a particularly problematic lesion and can be challenging from a diagnostic viewpoint even in resected lesions. Although fine needle aspiration cytology is very useful in the diagnosis of PTC, its accuracy and utility would be greatly facilitated by the development of specific markers for PTC and its common variants. We used the recently developed Applied Biosystems 1700 microarray system to interrogate a series of 11 benign thyroid lesions and conditions and 14 samples of PTC (six with classic morphology and eight with follicular variant morphology). TaqMan\u00ae reverse transcriptase-polymerase chain reaction was used to validate the expression portfolios of 50 selected transcripts. Our data corroborates potential biomarkers previously identified in the literature, such as LGALS3, S100A11, LYN, BAX, and cluster of differentiation 44 (CD44). However, we have also identified numerous transcripts never previously implicated in thyroid carcinogenesis, and many of which are not represented on other microarray platforms. Diminished expression of metallothioneins featured strongly among these and suggests a possible role for this family as tumour suppressors in PTC. Fifteen transcripts were significantly associated with FVPTC morphology. Surprisingly, these genes were associated with an extremely narrow repertoire of functions, including the major histocompatibility complex and cathepsin families.\nIntroduction\nPapillary thyroid carcinoma (PTC) is the most common endocrine malignancy and encompasses a variety of morphological\/architectural variants, all of which are characterized by a distinctive nuclear appearance. In recent years, PTC has become an important paradigm of solid tumour molecular pathogenesis principally arising from intensive investigation prompted by the Chernobyl accident.\nThe discovery of ret rearrangements [12, 13, 35] and their association with radiation [42] was followed by the demonstration of the BRAF V600E mutation [21, 30] found more commonly associated with sporadic PTC in non-radiation exposed populations [22, 32]. In the past, our group and others have noted an association between classic morphology and the BRAF V600E mutation and between variant morphology and ret rearrangements particularly ret\/PTC-3 [11, 39]. Similarly ret\/PTC-3 appears to strongly correlate with the solid\/follicular variant seen commonly in children exposed to the Chernobyl fallout [42]. Nevertheless, the utility of these genetic lesions to diagnostic pathology and clinical practice has remained negligible.\nRecently, gene expression microarray technology has been used to attempt to identify clinically relevant biomarkers of malignancy related to the thyroid [2, 6, 9, 10, 20, 28]. The discovery of such a biomarker or panel of biomarkers allied to the gold standard triage method of fine needle aspiration cytology (FNAC) would represent a significant advancement in the treatment of the solitary thyroid nodule.\nAn intriguing but commonly occurring variant of PTC is known as follicular variant (FVPTC). This lesion, which by definition retains the classic nuclear features of PTC, shows no evidence of the architectural papillae. FVPTC may be a controversial lesion due to interobserver variation in its pathological diagnosis [25]. Further, the occurrence of follicular patterned lesions with poorly or incompletely developed nuclear features may occur, which are easily dismissed as benign thyroid nodules. This has led to the controversial designation \u201cwell-differentiated tumour of uncertain malignant potential\u201d for tumours of this type [45].\nIt is clear that this is a complex and contentious area, and that further work needs to be done to ascertain the underlying molecular biology of this particular variant. Recently, inroads into elucidation of molecular pathways underpinning PTC have been carried out using microarray studies. The overriding objective of these investigations was to identify clinically useful biomarkers. However, the majority of these studies have analysed PTC as though it were a homogenous singular entity without deference in a detailed manner to sub-variants and, in particular, the most common variant (FVPTC). The identification of specific biomarkers of FVPTC and a deeper understanding of its origins are clearly warranted.\nThe aim of this expression microarray study using a novel microarray platform was twofold: to identify markers that distinguish PTC from benign thyroid tissue and lesions and, secondly, to identify potential markers and further explore the molecular pathology of FVPTC.\nMaterials and methods\nPatients and tissue samples\nTissue from 25 thyroid resections was collected prospectively from patients undergoing partial or total thyroidectomy for a variety of reasons at St. James\u2019s Hospital, Dublin. The study had approval of the local ethics committee and informed consent was obtained from each patient by the clinical team before surgery. Small samples (<1\u00a0cm) were divided and immediately snap-frozen in liquid nitrogen for storage at \u221280\u00b0C until use. Histopathological examination of formalin-fixed paraffin-embedded sections was performed by two pathologists (SF and MT) for diagnostic categorisation. Classification of neoplastic tissue was made according to a recognised system [24]. The cohort comprised 11 benign lesions or conditions including follicular adenoma, nodular goitre, normal thyroid tissue, and Graves\u2019s thyroiditis. The remaining 14 samples were diagnosed as PTC and comprised six classical morphology PTC and eight FVPTC (see Table\u00a01). Immediately before RNA extraction, frozen sections were cut and stained to confirm the presence of representative lesional tissue with morphology corresponding to that noted in the diagnostic formalin-fixed paraffin-embedded sections. \nTable\u00a01Sample cohortIdentifierDiagnosisN1Normal thyroid tissueN2Normal thyroid tissueN3Lymphocytic thyroiditisN4Nodular hyperplasiaN5Follicular adenomaN6Nodular hyperplasia with focal lymphocytic thyroiditisN7Nodular hyperplasiaN8Follicular adenomaN9Nodular hyperplasiaN10Follicular adenomaN11Grave\u2019s thyroiditisT1Solid\/FVPTCT2FVPTCT3PTC classic morphologyT4FVPTC-oxyphilT5FVPTCT6FVPTCT7PTC classic morphologyT8FVPTCT9PTC classic morphologyT10FVPTCT11PTC classic morphologyT12FVPTCT13PTC classic morphologyT14PTC classic morphologyList of the 11 benign and 14 malignant lesions that were used in this study.FV follicular variant; PTC papillary thyroid carcinoma\nRNA isolation and characterization\nSamples were ground in liquid nitrogen and homogenised in RLT buffer (Qiagen, UK). RNA was then extracted using the RNeasy Mini Kit with optional on-column RNase-free DNase digestion (Qiagen) according to the manufacturer\u2019s instructions. RNA quantity was determined using UV spectrophotometry. RNA quality was assessed using the RNA 6000 Nano LabChip\u00ae Kit in conjunction with the Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany).\nMicroarray analysis\nApplied Biosystems Human Genome Survey Arrays were used to analyse the transcriptional profiles of thyroid RNA samples in this study. Digoxigenin-UTP-labelled cRNA was generated and linearly amplified from 5\u00a0\u03bcg of total RNA using Applied Biosystems Chemiluminescent RT-IVT Labelling Kit v 2.0 using manufacturer\u2019s protocol. 10\u00a0\u03bcg of labelled cRNA were hybridized to each pre-hybed microarray in a 1.5-ml volume at 55\u00b0C for 16\u00a0h. Array hybridization and chemiluminescence detection were performed using Applied Biosystems Chemiluminescence Detection Kit following manufacturer\u2019s protocol. Images were collected for each microarray using the 1700 analyser. Images were auto-gridded and the chemiluminescent signals were quantified, corrected for background and spot, and spatially normalized.\nTaqMan\u00ae PCR validation\nSufficient RNA remained from 20 of the initial 25 samples for TaqMan\u00ae polymerase chain reaction (PCR) validation in a series of 50 targets. RNA was reverse transcribed using a High Capacity cDNA Archive Kit (Applied Biosystems, CA, USA). Primers and probes for TaqMan\u00ae PCR were obtained by using Applied Biosystems\u2019 pre-designed TaqMan\u00ae Gene Expression Assays. PCR was carried out using an ABI PRISM 7900 Sequence Detection System (Applied Biosystems). Analysis of relative gene expression data was performed using the \u0394\u0394CT method [23] with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous control\/reference assay.\nStatistical analysis\nMicroarrays were analysed using Spotfire DecisionSite\u2122 for Functional Genomics (Spotfire AB, Goteborg, Sweden) and R version 1.9.1, a free language and environment for statistical computing and graphics (R Development Core Team, 2004). Arrays were initially normalized, and genes were deemed undetectable and, therefore, excluded from final gene lists if they had a signal-to-noise ratio of less than three (S\/N\u2009<\u20093) in greater than 18 of the 25 arrays.\nAn ANOVA test was used to generate p values for statistical differences between groups. Their p values were then adjusted for multiple comparisons using the technique described by Benjamini and Yekutieli [3]. Genes were deemed statistically different between groups if they had an adjusted p\u2009<\u20090.05 and an average fold-change difference of greater than 2. Hierarchical clustering was performed based on the statistically different genes to determine whether samples grouped appropriately. Gene ontology analysis was performed using an online database known as the Panther classification system (http:\/\/www.pantherdb.org). Correlations between microarray and TaqMan\u00ae expression data were measured using the Pearson coefficient.\nResults\nUnsupervised clustering of all 25 samples demonstrated clustering into two major groups (data not shown) comprising the N group and the T group (see Table\u00a01). There was no tendency for FVPTC to cluster independently with classic morphology PTC, confirming the close relationship of these variants of PTC. To identify potential markers of malignancy, ANOVA with false discovery rate correction was used to compare the benign and malignant thyroid cohorts. A p value cut-off of <0.05 and fold-change difference of \u22652 yielded 236 statistically significant probes. Of these, 172 corresponded to fully annotated probes and are listed in Table\u00a02. \nTable\u00a02Genes differentially expressed in malignant vs benign thyroid tissueGene NameGene SymbolAdjusted p value1700 probe IDGenes up-regulated in malignant vs benign\u00a0Active BCR-related geneABR0.014482154399\u00a0Adaptor-related protein complex 2, alpha 1 subunitAP2A10.0312115368\u00a0Apoptosis, caspase activation inhibitorAVEN0.030998203738\u00a0BCL2-associated X proteinBAX0.009782146510\u00a0BH3 interacting domain death agonistBID0.021424131216\u00a0Brain abundant, membrane attached signal protein 1BASP10.024214198318\u00a0Brain acyl-CoA hydrolaseBACH0.014566133876\u00a0Bromodomain adjacent to zinc finger domain, 1ABAZ1A0.03538209809\u00a0Calcium\/calmodulin-dependent protein kinase ICAMK10.041887157712\u00a0Cathepsin SCTSS0.046544105790\u00a0CD44 antigen (homing function and Indian blood group system)CD440.044181133604\u00a0Chemokine (C-X-C motif) ligand 16CXCL160.043234199059\u00a0Chromosome 1 open reading frame 38C1orf380.049072202924\u00a0CLIP-170-related proteinCLIPR-590.023629102205\u00a0Docking protein 1, 62\u00a0kDa (downstream of tyrosine kinase 1)DOK10.041898204989\u00a0Epidermodysplasia verruciformis 1EVER10.003348175569\u00a0FXYD domain containing ion transport regulator 5FXYD50.01444154607\u00a0FXYD domain containing ion transport regulator 5FXYD50.023629112771\u00a0Galactose-4-epimerase, UDPGALE0.047363141143\u00a0Genethonin 1GENX-34140.016836124360\u00a0Hypothetical gene BC008967BC0089670.015683108526\u00a0Hypothetical protein FLJ10849FLJ108490.013822224983\u00a0Hypothetical protein FLJ22531FLJ225310.024391145918\u00a0Hypothetical protein MGC4607MGC46070.006507211836\u00a0Intercellular adhesion molecule 1 (CD54), human rhinovirus receptorICAM10.028746109070\u00a0Jun dimerization protein p21SNFTSNFT0.043301144215\u00a0Lectin, galactoside-binding, soluble, 3 (galectin 3)LGALS30.034491179836\u00a0Major vault proteinMVP0.0312212354\u00a0Matrix metalloproteinase 14 (membrane-inserted)MMP140.038682152076\u00a0Milk fat globule-EGF factor 8 proteinMFGE80.02392144588\u00a0Mst3 and SOK1-related kinaseMST40.042028112198\u00a0neuronal cell adhesion moleculeNRCAM0.011178106462\u00a0Phospholipase D3PLD30.034491143388\u00a0Promyelocytic leukemiaPML0.016018217558\u00a0Protein inhibitor of activated STAT protein PIASyPIASY0.00536153434\u00a0Protein tyrosine phosphatase, receptor type, EPTPRE0.048653221568\u00a0Rho GDP dissociation inhibitor (GDI) betaARHGDIB0.043853143589\u00a0S100 calcium binding protein A11 (calgizzarin)S100A110.019933145550\u00a0Similar to rat tricarboxylate carrier-like proteinBA108L7.20.025387179870\u00a0SP110 nuclear body proteinSP1100.0312113484\u00a0Stimulated by retinoic acid gene 6FLJ125410.043234193986\u00a0Syndecan 3 (N-syndecan)SDC30.048804143980\u00a0Tax interaction protein 1TIP-10.006673119665\u00a0TBC1 domain family, member 2TBC1D20.029907205982\u00a0Tenascin C (hexabrachion)TNC0.032355143831\u00a0Thymosin, beta 4, Y chromosomeTMSB4Y0.040937193911\u00a0Tissue inhibitor of metalloproteinase 1 (erythroid potentiating activity, collagenase  inhibitor)TIMP10.032306134692\u00a0Topoisomerase (DNA) II alpha 170\u00a0kDaTOP2A0.040937135302\u00a0Transforming growth factor, beta 1TGFB10.016836170749\u00a0Transgelin 2TAGLN20.031971172296\u00a0Tyrosine 3-monooxygenase\/tryptophan 5-monooxygenase activation protein, eta  polypeptideYWHAH0.039009188379\u00a0v-yes-1 Yamaguchi sarcoma viral related oncogene homologLYN0.016175194134Genes down-regulated in malignant vs benign\u00a0Aldehyde oxidase 1AOX10.003227106573\u00a0Ankyrin 2, neuronalANK20.035357155780\u00a0Aspartate beta-hydroxylaseASPH0.002064221656\u00a0Aspartate beta-hydroxylaseASPH0.019666114180\u00a0ATPase, Cu++ transporting, beta polypeptideATP7B0.044511198852\u00a0Brain-specific protein p25 alphap250.023629120622\u00a0Casein kinaseLOC1494200.022382149347\u00a0Cellular retinoic acid binding protein 1CRABP10.008315100295\u00a0Centromere protein JCENPJ0.0312164563\u00a0Ceroid-lipofuscinosis, neuronal 5CLN50.011021205999\u00a0Chloride channel KbCLCNKB0.040418176266\u00a0Chondroitin beta1,4 N-acetylgalactosaminyltransferaseChGn0.013148101140\u00a0Chromosome 11 open reading frame 8C11orf80.001977174025\u00a0Chromosome 11 open reading frame 8C11orf80.004148108279\u00a0Chromosome 21 open reading frame 4C21orf40.0042156895\u00a0Clusterin-like 1 (retinal)CLUL10.019631186062\u00a0Component of oligomeric golgi complex 3COG30.003664129212\u00a0Coxsackie virus and adenovirus receptorCXADR0.004648108284\u00a0Crystallin, alpha BCRYAB0.030418190274\u00a0Cytosolic sialic acid 9-O-acetylesterase homologCSE-C0.040993213856\u00a0Dicarbonyl\/l-xylulose reductaseDCXR0.001977103350\u00a0DnaJ (Hsp40) homolog, subfamily B, member 4DNAJB40.043853103618\u00a0ERO1-like beta (S. cerevisiae)ERO1LB0.013962207998\u00a0Extracellular link domain containing 1XLKD10.039738195865\u00a0Family with sequence similarity 13, member A1FAM13A10.019631116936\u00a0Fatty acid binding protein 4, adipocyteFABP40.014832150137\u00a0Fc fragment of IgG binding proteinFCGBP0.001965118361\u00a0Fibroblast growth factor receptor 2FGFR20.0042110548\u00a0FLJ35740 proteinFLJ357400.020224101102\u00a0Friedreich ataxia region gene X123X1230.032602133505\u00a0Glutamate-ammonia ligase (glutamine synthase)GLUL0.014649175147\u00a0Glycine amidinotransferase (l-arginine\/glycine amidinotransferase)GATM0.013962111904\u00a0Glycoprotein M6AGPM6A0.011739215326\u00a0Growth hormone receptorGHR0.017721190306\u00a0HLA complex group 4HCG40.025807191199\u00a0Hypothetical protein BC009561LOC1197100.003227211319\u00a0Hypothetical protein BC019238LOC1203790.013438201200\u00a0Hypothetical protein FLJ13204FLJ132040.003227145066\u00a0Hypothetical protein FLJ13842FLJ138420.016448208504\u00a0Hypothetical protein FLJ14054FLJ140540.049072202017\u00a0Hypothetical protein FLJ20154FLJ201540.014428143310\u00a0Hypothetical protein FLJ20513FLJ205130.019493154130\u00a0Hypothetical protein FLJ32110FLJ321100.015507229492\u00a0Hypothetical protein FLJ32343FLJ323430.012208116902\u00a0Hypothetical protein FLJ33516FLJ335160.03965224600\u00a0Hypothetical protein FLJ37549FLJ375490.001956218577\u00a0Hypothetical protein FLJ39378FLJ393780.005853163223\u00a0Hypothetical protein FLJ40021FLJ400210.023629174198\u00a0Hypothetical protein LOC134285LOC1342850.018694163671\u00a0Hypothetical protein MGC10946MGC109460.022382195982\u00a0Hypothetical protein MGC14425MGC144250.015445161569\u00a0Hypothetical protein MGC17299MGC172990.026062168452\u00a0Hypothetical protein MGC17943MGC179430.0042147296\u00a0Hypothetical protein MGC23980MGC239800.018694224619\u00a0Hypothetical protein MGC24047MGC240470.001956138122\u00a0Hypothetical protein MGC33607MGC336070.033547100645\u00a0Ionized calcium binding adapter molecule 2IBA20.0042179489\u00a0KIAA0390 gene productKIAA03900.014832119936\u00a0KIAA0703 gene productKIAA07030.032602146652\u00a0Lectin, mannose-binding, 1LMAN10.031092179632\u00a0Leiomodin 1 (smooth muscle)LMOD10.022352120404\u00a0Likely ortholog of rat SNF1\/AMP-activated protein kinaseSNARK0.044605157942\u00a0LIM domain kinase 2LIMK20.002409151439\u00a0Low density lipoprotein-related protein 1B (deleted in tumors)LRP1B0.00536209464\u00a0Low density lipoprotein-related protein 2LRP20.040937114919\u00a0Matrilin 2MATN20.0042167316\u00a0Metallothionein 1A (functional)MT1A0.013822204773\u00a0Metallothionein 1A (functional)|metallothionein 1E (functional)|metallothionein 1K| metallothionein 2AMT1A|MT2A|MT1E|MT1K0.027037146368\u00a0Metallothionein 1A (functional)|metallothionein 1E (functional)|metallothionein 2A| metallothionein 1KMT1A|MT2A|MT1K|MT1E0.043841182305\u00a0Metallothionein 1A (functional)|metallothionein 2A|metallothionein 1K| metallothionein 1E (functional)MT1A|MT1K|MT1E|MT2A0.011739223856\u00a0Metallothionein 1B (functional)MT1B0.019631174119\u00a0Metallothionein 1F (functional)MT1F0.024726144569\u00a0Metallothionein 1GMT1G0.0192164525\u00a0Metallothionein 1GMT1G0.03965171539\u00a0Metallothionein 1JMT1J0.008315227956\u00a0Metallothionein 1XMT1X0.008335119685\u00a0Metallothionein 1XMT1X0.010748173072\u00a0Metallothionein IVMT40.007447223241\u00a0Methionine adenosyltransferase II, alphaMAT2A0.014428158350\u00a0Mitogen-activated protein kinase 4MAPK40.042306131252\u00a0Myc-induced nuclear antigen, 53\u00a0kDaMINA530.011959130284\u00a0NIMA (never in mitosis gene a)- related kinase 11NEK110.001965194628\u00a0OtospiralinLOC1506770.018694182360\u00a0PDZ\/coiled-coil domain binding partner for the rho-family GTPase TC10PIST0.013822103651\u00a0Phospholipase A2 receptor 1, 180\u00a0kDaPLA2R10.004029134379\u00a0Phospholipase C-like 1PLCL10.022657206894\u00a0Phosphotidylinositol transfer protein, betaPITPNB0.014428122698\u00a0Polycystic kidney and hepatic disease 1 (autosomal recessive)-like 1PKHD1L10.0042199896\u00a0Polymerase (DNA directed) iotaPOLI0.003227167492\u00a0Potassium channel, subfamily K, member 9KCNK90.000849108648\u00a0Potassium channel-interacting protein 4KCNIP40.011447147058\u00a0Potassium inwardly-rectifying channel, subfamily J, member 13KCNJ130.008972124187\u00a0pp21 HomologLOC511860.004326127636\u00a0Pre-B cell leukemia transcription factor 4PBX40.030311199118\u00a0Protein kinase, cAMP-dependent, catalytic, betaPRKACB0.023863198878\u00a0Protein phosphatase 4, regulatory subunit 2|hypothetical protein LOC151987PPP4R2|LOC1519870.011338200919\u00a0RAB23, member RAS oncogene familyRAB230.000659122394\u00a0Ras association (RalGDS\/AF-6) domain family 6RASSF60.048804119072\u00a0Sarcoglycan, delta (35\u00a0kDa dystrophin-associated glycoprotein)SGCD0.011178120415\u00a0Serum deprivation response (phosphatidylserine binding protein)SDPR0.011021156433\u00a0SH3 and multiple ankyrin repeat domains 2SHANK20.043996193906\u00a0Solute carrier family 26, member 7SLC26A70.0042225067\u00a0Solute carrier family 26, member 7SLC26A70.005853213530\u00a0Solute carrier family 5 (iodide transporter), member 8SLC5A80.031284231731\u00a0SPARC related modular calcium binding 2SMOC20.021505135930\u00a0Syndecan 2 (heparan sulfate proteoglycan 1, cell surface-associated, fibroglycan)SDC20.001258209676\u00a0Syntaxin 12STX120.01117199949\u00a0T-box 22TBX220.024297177517\u00a0Thioredoxin-like, 32\u00a0kDaTXNL0.001102192552\u00a0Thyroid stimulating hormone receptorTSHR0.02176108606\u00a0Tissue inhibitor of metalloproteinase 4TIMP40.023629184795\u00a0Trefoil factor 3 (intestinal)TFF30.004648114445\u00a0Trefoil factor 3 (intestinal)TFF30.014428100949\u00a0UDP-N-acetyl-alpha-d-galactosamine\/polypeptide N-acetylgalactosaminyltransferase 9 (GalNAc-T9)GALNT90.031284161042\u00a0WEE1 homolog (S. pombe)WEE10.024101123533\u00a0WW domain containing oxidoreductaseWWOX0.012208224298\u00a0WW domain containing oxidoreductaseWWOX0.024101135080\u00a0Zinc finger protein 258ZNF2580.013962225961\u00a0Zinc finger protein 36, C3H type-like 2ZFP36L20.018837210469Two-tail ANOVA with p value correction yielded 173 probes (52 up-, 121 down-regulated in PTC) significantly different (p\u2009<\u20090.05) between the malignant and benign thyroid tissues.\nSupervised hierarchical clustering was performed on the 25 arrays based on the 236 statistically significant probes to determine whether the samples would segregate appropriately. The resulting heat map can be observed in Fig.\u00a01. Benign lesions and tumours clustered together with the exception of one tumour sample that clustered with the benign group (T5).\nFig.\u00a01Hierarchical clustering of samples. This heat map shows the clustering of the 25 samples based on the 236 probes found to be differentially regulated in benign vs malignant thyroid tissue. Clustering was performed using the unweighted pair group method with arithmetic mean, with Euclidian distance as the similarity measure. Average value was used as the ordering function\nA binomial statistics tool was used to compare classifications of multiple clusters of lists to a reference list (i.e. the complete human genome) to statistically determine over- or under-representation of Panther classification categories. Biological processes over-represented in the up-regulated PTC cohort included tumour suppressor, oncogenesis, DNA replication, cell cycle, and cell adhesion (p\u2009<\u20090.0001). Genes involved in homeostasis and other homeostasis activities were highly over-represented in the down-regulated cohort (p\u2009<\u20090.000001).\nANOVA tests were used to determine which genes were differentially regulated in the FVPTC cohort only. Fifteen genes were identified, including cluster of differentiation 14 (CD14), CD74, CTSC, CTSH, CTSS, DPP6, ETHE1, human leucocyte antigen A (HLA-A), HLA-DMA, HLA-DPB1, HLA-DQB1, HLA-DRA, osteoclast stimulating factor 1 (OSTF1), TDO2, and a previously uncharacterized gene (noname).\nMicroarray results were validated using a reverse transcription reaction followed by TaqMan\u00ae PCR for 50 gene targets. The \u0394\u0394CT method [23] was used to analyse relative gene expression data. GAPDH was used as an endogenous control, and T12 was chosen as an arbitrary calibrator sample. Gene expression profiles for TaqMan\u00ae PCR were plotted in conjunction with those for microarray results in Fig.\u00a02. Pearson co-efficient was used to directly compare data from microarray analysis and TaqMan\u00ae RT-PCR. Table\u00a03 depicts genes differentially expressed in both benign vs malignant and FVPTC vs classic morphology PTC.\nFig.\u00a02TaqMan\u00ae PCR validation of microarray experiments. Profile charts of gene expression levels comparing results obtained by microarray analysis (n\u2009=\u200925) to TaqMan\u00ae PCR analysis (n\u2009=\u200920) for six genes. Plots for those genes with multiple probes are also displayed where appropriateTable\u00a03Correlation between TaqMan\u00ae and microarray dataGenePearson\u2019s r coefficienttwo-tailed pGenes differentially expressed in FVPTC vs classic PTCCD140.83<0.0001CD74a0.87<0.00010.740.0002CTSC0.530.0170CTSH0.710.0005CTSS0.620.0037DPP60.760.0001ETHE10.300.2008HLA-A0.77<0.0001HLA-DMA0.750.0001HLA-DPB10.96<0.0001HLA-DQB1a0.700.00060.630.0031HLA-DRA0.82<0.0001NONAME\u22120.020.9323OSTF10.150.5171TDO20.680.0009Genes differentially expressed in benign vs malignantBAX0.200.3997CAMK10.170.4771CD440.570.0094CTSS0.620.0037CXADR0.360.1152FGFR20.84<0.0001GALE0.540.0138ICAM10.620.0038LYN0.450.0483MAPK40.76<0.0001MMP140.360.1175MT1F0.92<0.0001MT1Ka0.680.00110.690.00070.700.0006MT1Xa0.88<0.00010.88<0.0001RAB230.700.0006S100A110.610.0041SDC20.340.1415SDC30.570.0087TFF3a0.97<0.00010.96<0.0001TGFB10.560.0098TIMP10.730.0003TIMP40.80<0.0001TOP2A0.070.7769TSHR0.180.4440Gene expression profiles for TaqMan\u00ae PCR and microarray results were compared using the Pearson coefficient.aGenes have more than one probe ID on microarray\nDiscussion\nThe primary aim of this study was to generate an overview of molecular markers of malignancy in PTC with a view to identifying discriminators between common sub-types (classic PTC and FVPTC), using genome-wide expression microarray technology validated by TaqMan\u00ae RT-PCR. To this end, lesions that were well characterized histologically were selected.\nThe application of microarray analysis designed to identify transcripts strongly associated with each group of interest yielded a gene list of 173 genes that were differentially expressed between cohorts. Significant down-regulation of Coxsackie virus receptor was recorded in the malignant cohort of thyroid lesions. The Coxsackievirus B and adenovirus receptor (CAR) plays a dual role as a homotypic junctional adhesion protein and as a viral receptor. It is biologically plausible that altered expression may impact on the morphology peculiar to PTC given its association with cellular adhesion. CAR has been shown to be differentially expressed in various human adenocarcinomas, and differential expression may represent a new factor in thyroid tumourigenesisigenesis [27].\nRab 23 expression was also down regulated in the malignant cohort. The Rab small G protein family is composed of approximately 40 members. Many of them are ubiquitous and are expressed and participate in transport processes, such as endocytosis and exocytosis [26]. Other gene targets demonstrating significant down-regulation in the malignant group were syndecans 2 and 3. Syndecans are transmembrane proteoglycans expressed on adherent cells. Changes in syndecan expression have been postulated to influence cell adhesion, migration, and the structure of focal contacts and the cytoskeleton [8].\nThe abundance of metallothionein genes in the cohort of genes down-regulated in PTC is interesting partly because of the sheer number of isoforms detected (Table\u00a02). There have been many studies showing increased metallothionein expression in a plethora of cancer types but few showing decreased expression [4, 7, 33]. Although previous microarray experiments showed metallothionein genes to be down-regulated in thyroid tumours to a certain extent [2, 9, 16], none have detected so many as the current study.\nApart from microarray experiments, there has been little reported in the literature regarding metallothioneins in thyroid cancer. An early report by Nartey et al. [31] showed metallothioneins to be expressed actively in certain human thyroid neoplastic tissues but not in normal thyroid tissue, which would seem to contradict the current authors\u2019 findings. In contrast to this, a later immunohistochemical study showed an absence of metallothionein expression in 13 of 20 PTCs [36]. Interestingly, in three of the seven positive PTCs, metallothionein positivity was restricted to areas of follicular differentiation. In one of the only recent studies, Huang et al. [17] followed up their initial microarray experiment by showing that MT1G is down regulated in PTC via hypermethylation. The biological significance of low metallothionein expression in thyroid tumours is therefore still poorly understood; however, it is interesting to speculate that metallothioneins may have roles as tumour suppressors in thyroid carcinoma.\nMany genes identified, such as LGALS3 [9, 16, 20], LYN [46], TFF3 [2, 9, 16], CRABP1 [9, 16], BAX [2], MAPK4 [28], CD44 [16], TIMP1 [20], FGFR2 [9], and S100A11 [20, 40], have been previously reported in both microarray and conventional experiments in thyroid cancer. The data generated in this study corroborates the importance of several biological processes in the progression of thyroid neoplasia. For example, S100A11 expression was up regulated in the PTC cohort compared with benign lesions, paralleling the increased protein expression of this gene target identified at the protein level using immunohistochemistry [29]. S100A11 has also been suggested as a biomarker of malignancy in the context of colorectal carcinoma as long ago as 1995 [41].\nCorrelation of highlighted features with the current state of knowledge of the molecular pathology of thyroid neoplasia goes some way towards providing an external validation of the data obtained from the AB1700 system. However, additional validation using TaqMan\u00ae RT-PCR was performed.\nIn general, microarray and TaqMan\u00ae data correlated well with approx. 80% of comparisons having p\u2009<\u20090.05. Candidate genes were selected contingent on results identified as over-represented biological processes (oncogenesis, cell cycling, DNA replication, and homeostasis) using the Panther binomial statistics tool as opposed to the more traditional method of selecting the most highly disregulated genes. This may account for the poor correlation observed with certain genes. Some genes, such as TFF3 and FGFR2, had excellent correlation between microarray and TaqMan\u00ae results, whereas others, such as BAX and TSHR, showed poor correlation despite previous studies implicating them in thyroid cancer [15, 38]. Where there was discordance in the data, it could be accounted for by differences in the sequences targeted by the TaqMan\u00ae target sequences and the microarray probe. In those cases, the array and pre-designed TaqMan assays interrogated different exons or alternative splice variants. This finding highlights the importance of matching the targets to be validated from microarray data sets.\nAnalysis of differentially expressed transcripts in the FVPTC revealed many transcripts showing similar expression level patterns in both FVPTC and classic morphology PTC. Examples of these transcripts are included in Table\u00a02. In addition, somewhat surprisingly, unsupervised clustering of all samples showed no tendency for FVPTC to cluster independently of classic morphology PTC, emphasizing the very close relationship of these PTC variants (data not shown). However, analysis of differentially expressed genes in FVPTC exclusive of those identified in classic morphology PTC revealed 15 genes exhibiting differential expression in FVPTC compared with benign lesions and conditions outwith of classic morphology PTC. These genes displayed a narrow gamut of function represented by the transcripts involved (Table\u00a03). TaqMan\u00ae RT-PCR was performed for all of these targets to confirm the array findings and correlation with the array data was strong (see Table\u00a03).\nAberrant expression of two major groups of transcripts was noted in FVPTC. Relatively increased expression of class 1 major histocompatibility complex (MHC) genes (HLA-A) and aberrant expression of class 2 MHC genes (HLA-DMA, HLA-DPB-1, HLA-DQB-1, HLA DRA) and associated genes (e.g. CD74 represents the invariant membrane bound moiety of class II HLA molecules and regulates the biology and functions of MHC class II molecules and CD14 is a surface marker of monocytes\/macrophages) were the most significant findings. Additionally, relatively up-regulated expression of members of the cathepsin family (cathepsin C, cathepsin H, cathepsin S, and TDO2) was striking in the FVPTC group. HLA expression is generally associated with immune functions such as T cell interaction and antigen presentation. The presence of prominent HLA transcript expression, especially among class 2 in FVPTC, is intriguing. One potential cause of this was that tumour-infiltrating leucocytes were responsible for this finding. However, haematoxylin\u2013eosin (H&E)-stained slides of each case were reviewed to specifically identify the degree of tumour infiltration by leucocytes. Although a mild lymphocytic infiltrate was noted in some cases, there was no apparent over representation of lymphocytes in the follicular variant compared to benign lesions and classic morphology PTC. This raises the clear possibility that the findings represent aberrant increased expression of class 2 HLA transcripts by the epithelium of FVPTC. This is an unexpected finding, as over-expression of MHC class 2 molecules would be expected to increase tumour immunogenicity. A similar aberrant expression of HLA transcripts has been recently described in ovarian neoplasms [34]. Rangel et al. [34] concluded over-expression of HLA-DRA might represent a novel biomarker for malignancy, and this also seems biologically plausible in the FVPTC setting. A recent paper has also described HLA-DRA expression in ret\/PTC-activated papillary thyroid carcinoma but not in surrounding normal thyroid follicles [19]. Yu et al. [47] showed discordant expression of (CD74Ii) and HLA-DR in Hashimoto thyroiditis, an autoimmune condition associated with increased incidence of PTC and sharing molecular features such as ret\/PTC expression [18, 37]. Hwang et al. [19] have drawn attention to aberrant expression of HLA-DRA in ret\/PTC-activated PTC. Expression of HLA-DRA may in some way explain the propensity for PTC to metastasize to lymph nodes and often apparently reside there without markedly worsening prognosis.\nCathepsins C (dipeptidyl-peptidase I), H, and S showed up-regulation in FVPTC compared to benign lesions. Cathepsins are a family of proteases that play an important role in protein degradation. They are key players in the proliferative, invasive, and metastatic potential of malignant tumour cells. Their expression in the relatively biologically indolent FVPTC is intriguing, and it remains possible that cathepsins have cellular roles outside of those involved in invasion and dissemination of tumour cells as indeed has been suggested by others [43]. For example, cathepsin L has recently been shown to play a role in nuclear transcriptional activation, and cathepsins are now recognized to play a role in MHC class II antigen presentation [44]. OSTF1 has no defined role in carcinogenesis, although outside of its role in ossification, it is also known to have roles in signal transduction and protein binding, which may be relevant to carcinogenesis and, particularly, FVPTC. A recent paper highlights the role of bone mineralization proteins including osteopontin and osteoclast stimulating factors as potential biomarkers of malignant tumours in general [1]. Indeed, in addition to elevated OSTF1 expression, increased expression of osteopontin was seen in PTC (data not shown); however, in our data, osteopontin does not appear to be specifically up-regulated in FVPTC, and this finding has also been noted by other researchers [14]. In any case, osteopontin is known to be a downstream effector of ret\/PTC [5] and mutated BRAF [14], where it acts in association with CD44, another transcript showing increased expression in both classic morphology PTC and FVPTC.\nA particular focus of this study was to compare transcriptome profiles for PTCs with classic morphology and FVPTC given the propensity for FVPTC lesions to prove problematic from a diagnostic perspective. Although the study confirms the close relationship between the two most common variants of PTC, a narrow portfolio of genes and, in particular, gene functions was elucidated in the FVPTC cohort. The targets identified are easily amenable to analysis by more established techniques such as TaqMan\u00ae RT-PCR, with associated potential as additional markers for application in the FNAC setting. Clearly, the potential biomarkers identified in this study will require prospective evaluation in the context of real clinical diagnostic situations in the future to consolidate their merit as adjunctive tests in the diagnostic setting and to validate their altered expression states in the pathobiology of PTC development.","keyphrases":["microarray","papillary thyroid carcinoma","follicular variant","biomarker","taqman\u00ae pcr"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_J_Epidemiol-3-1-2071965","title":"Acute myocardial infarction incidence and hospital mortality: routinely collected national data versus linkage of national registers\n","text":"Background and Objective To compare levels of and trends in incidence and hospital mortality of first acute myocardial infarction (AMI) based on routinely collected hospital morbidity data and on linked registers. Cases taken from routine hospital data are a mix of patients with recurrent and first events, and double counting occurs when cases are admitted for an event several times during 1 year. By linkage of registers, recurrent events and double counts can be excluded.\nIntroduction\nMortality from coronary heart disease, in particular from acute myocardial infarction (AMI), has decreased in many Western countries during the last decades [1]. A decrease in age- and gender-adjusted AMI mortality, assuming a constant quality of diagnosis, is a consequence of either a decrease in incidence, case-fatality or recurrence risk, or a combination of these. Hospital-based registers are often used for surveillance of the morbidity and hospital mortality of AMI [2]. In the Netherlands, the national hospital discharge register has traditionally been used to provide estimates of (trends in) incidence and hospital mortality of AMI [3]. However, in this register, like in many others, a new record is created for each hospital admission. As a consequence, admissions taken from the hospital register will include double counts from patients if they are transferred to a second hospital or if they are admitted for the same event several times during 1\u00a0year. Furthermore, patients taken from the hospital register from 1\u00a0year include a mix of patients with recurrent events (presence of an event in preceding years) and first events (absence of events in preceding years). Tracking individuals over time based on information from the hospital register only is difficult when a unique personal identifier is absent in the hospital register. The effect of both double counting and admixture of first and recurrent events in nationwide registers on (trends) in incidence or hospital mortality has not been well assessed [4, 5]. For the Netherlands, this effect could only be estimated from comparison with results from regional cohort studies, as nationwide estimates of incidence and hospital mortality of first AMI were not available. Furthermore, it has been argued that statistics from routine data could not be used for providing reliable information on (trends in) incidence and hospital mortality. After we recently showed that hospitalized patients in the Netherlands can be followed longitudinally within the national hospital discharge register in a valid way by using information from the Dutch population register [6], we set out to compare the nationwide (trends in) incidence and hospital mortality of first hospitalized AMI based on routinely collected data in the hospital register (double counts and recurrent events included) and based on linkage of the hospital register with the population register (double counts and recurrent events excluded).\nMethods\nData sources\nData on hospital admissions were retrieved from the national hospital discharge register. Since 1986, all general and academic hospitals and most single specialty hospitals participate in the hospital register. There are no private hospitals in the Netherlands that treat patients with AMI. For each hospital admission a new record is created in the hospital register, including the following information: date of birth, gender, numeric part of postal code (since 1991), hospital-specific patient identification code, type of hospital, admission date and principal diagnosis of the admission. The principal diagnosis is determined at discharge and coded using the ninth revision of the International Classification of Diseases (ICD-9-CM) [7]. As the hospital register does not contain a unique personal identifier, we tracked individuals over time within the hospital register by using information from the Dutch population register. This database contains information on all registered persons living in the Netherlands, including date of birth, gender, current address, postal code and nationality. Patients registered in the hospital register were identified in the population register using linkage variables \u2018date of birth\u2019, \u2018gender\u2019 and \u2018numeric part of postal code\u2019. When patients moved, their hospital admissions were recognized by using the new postal code registered in the population register.\nRecently, the validity of the registries and linkage methods was studied. In a random sample of hospital admissions, 99% of the personal, admission and discharge data and 84% of the principal diagnoses (validated through medical record review by medical specialists) were correctly registered [8]. In a random sample of the population register, over 97% of the addresses were shown to be correctly registered [9]. Furthermore, over 97% of the uniquely linked hospital admissions resulting from linkage of the hospital register with the population register were shown to be correctly linked [9].\nThese results are similar to most of the studies that reported on the validity of AMI events in hospital and population based registries [10\u201313].\nAll analyses were performed at Statistics Netherlands in agreement with privacy legislation in the Netherlands [6].\nCohort enrolment from the hospital register \nThe hospital register comprises information based on all admissions in the Netherlands of the entire Dutch population, including double counts, first and recurrent admissions for AMI, and including AMI admissions of non-residents. In the hospital register, 28,733 and 25,864 hospital admissions with a principal diagnosis AMI (ICD-9-CM [7] code 410 and subcategories) were registered in 1995 and 2000.\nCohort enrolment from linked registers \nAfter linkage with the population register using linkage variables \u2018date of birth\u2019, \u2018gender\u2019 and \u2018numerical part of postal code\u2019, 25,142 and 22,470 admissions came from patients with a unique combination of linkage variables in the population register (88% and 87%, respectively). Thus, each remaining admission linked to only one unique individual in the population register (one unique individual in the Netherlands). Admissions linking with more than one person (e.g., administrative twins; two persons with the same date of birth, gender and numeric part of postal code registered in the population register) or with no person at all (e.g., non-residents or administrative errors) in the population register were excluded. Selection of the first admission per person of all subsequent admissions of a person occurring during 1995 and 2000 yielded 23,172 patients in 1995 and 20,414 patients in 2000. Thus, 1,970 double counts had occurred in 1995 (8%) and 2,056 in 2000 (9%). Information on admissions in previous years of the patients in 1995 was obtained by selecting all hospital admissions registered in the hospital register with principal diagnosis AMI in the period 1991\u20131995. These admissions were linked to the cohort of 23,172 patients with linkage variables \u2018date of birth\u2019, \u2018gender\u2019 and one or both of the variables \u2018numerical part of postal code\u2019 and \u2018hospital-specific patient identification code\u2019. Linkage with the population register was not possible, since this register started in October 1994. Subjects who linked in this process were patients with previous hospital admissions for AMI (recurrent AMI patients) and were excluded (1,607 patients (7%)). This resulted in the final cohort of 1995 consisting of 21,565 patients. Information on hospital admissions in previous years of the patients in 2000 was obtained by linking of the hospital register of 1995\u20132000 to the population register with linkage variables \u2018date of birth\u2019, \u2018gender\u2019 and \u2018numerical part of postal code\u2019. All uniquely linked admissions with principal diagnosis AMI were selected and linked to the cohort of 20,414 patients. Patients with previous hospital admissions for AMI (recurrent AMI patients) were excluded (1,356 patients (7%)). This resulted in the final cohort of 2000 consisting of 19,058 patients.\nThus the linked register comprises information for only part of the Dutch population (i.e., those that were unique on date of birth, gender, and postal code), and does not include double counts, and recurrent AMI admissions.\nData analysis\nThe incidence and hospital mortality of AMI (with 95% confidence interval (95% CI)) based on the hospital register (hospital-based) and on linked registers (linkage-based) was computed by year, age and gender. We compared the hospital-based incidence and hospital mortality to the linkage-based incidence and hospital mortality by calculating incidence rate differences or ratios and risk differences or ratios (with 95% CIs) by age and gender. Trends in incidence and hospital mortality were obtained by calculating incidence rate or risk differences and incidence rate or risk ratios (with 95% CIs) by age and gender. Incidence rate differences and ratios were based on the Poisson model, while risk differences and ratios were based on the binomial model [14]. Pooled age-adjusted incidence rate differences or ratios and risk differences or ratios (with 95% CIs) were calculated according to the Mantel Haenszel method [15].\nResults\nIn both 1995 and 2000, the gender and age distribution of the cohort based on the hospital register was comparable to the cohort based on linked registers. In 1995 and 2000, two-thirds comprised men. The mean age in 1995 was 63.7\u00a0years in men and 71.5\u00a0years in women based on the hospital register. This was 64.3 and 71.9\u00a0years, respectively, based on linked registers. In 2000, the mean age based on the hospital register was 63.6\u00a0years in men and 70.9\u00a0years in women. Based on linked registers, this was 64.2 and 71.6\u00a0years, respectively.\nIn men, the (adjusted) hospital-based incidence was 47 per 100,000 person-years or 22% (95% CI 19\u201325%) higher than the (adjusted) linkage-based incidence in 1995 and 43 per 100,000 person-years or 23% (95% CI 20\u201326%) higher in 2000 (Table\u00a01). Age-specific (\u226530\u00a0years) absolute and relative differences ranged from 9\u2013217 per 100,000 person-years or 20\u201328% in 1995 and from 7\u2013220 per 100,000 person-years or 22\u201325% in 2000. The (adjusted) hospital-based incidence was also higher than the (adjusted) linkage-based incidence in women in 1995 (19 per 100,000 person-years or 18%; 95% CI 15\u201322% higher) and 2000 (18 per 100,000 person-years or 20%; 95% CI 16\u201324% higher). Age-specific (\u226530\u00a0years) absolute and relative differences varied from 3\u2013103 per 100,000 person-years or 13\u201320% in 1995 and from 1\u2013116 per 100,000 person-years or 14\u201333% in 2000.\nTable\u00a01Incidence (per 100,000 persons per year) of hospitalized acute myocardial infarction in 1995 and 2000 based on the national hospital discharge register and based on linked registers19952000Hospital registerLinked registersHospital registerLinked registersAgeIncidenceIncidenceID1IR2IncidenceIncidenceID1IR2Men<30211 (0 to 1)1.56 (0.99 to 2.48)210 (0 to 1)1.33 (0.88 to 2.01)30\u20133941329 (4 to 14)1.28 (1.12 to 1.47)40327 (3 to 12)1.23 (1.08 to 1.41)40\u20134920717334 (22 to 45)1.20 (1.12 to 1.27)18014336 (26 to 47)1.25 (1.17 to 1.34)50\u201359509409100 (79 to 121)1.24 (1.19 to 1.30)40733276 (58 to 93)1.23 (1.17 to 1.29)60\u201369887733154 (122 to 186)1.21 (1.16 to 1.26)675551124 (97 to 151)1.23 (1.17 to 1.28)70\u2013791,2261,009217 (169 to 265)1.21 (1.16 to 1.27)1,008824184 (143 to 225)1.22 (1.17 to 1.28)80\u2013891,2541,045209 (126 to 291)1.20 (1.12 to 1.29)1,144925220 (144 to 295)1.24 (1.15 to 1.33)\u226590843660184 (\u221220 to 387)1.28 (0.97 to 1.68)568464104 (\u221259 to 267)1.22 (0.89 to 1.68)TotalCrude25522134 (29 to 39)1.15 (1.13 to 1.18)22219033 (28 to 37)1.17 (1.15 to 1.20)Adjusted347 (42 to 52)1.22 (1.19 to 1.25)43 (38 to 47)1.23 (1.20 to 1.26)Women<30100 (0 to 1)3.59 (1.21 to 10.67)110 (0 to 1)1.31 (0.65 to 2.63)30\u201339981 (\u22121 to 4)1.17 (0.88 to 1.56)1183 (0 to 5)1.33 (1.02 to 1.73)40\u20134938326 (0 to 11)1.17 (1.01 to 1.36)46388 (2 to 13)1.21 (1.06 to 1.38)50\u2013591139519 (9 to 29)1.20 (1.09 to 1.32)958016 (7 to 24)1.20 (1.08 to 1.32)60\u20136930626244 (26 to 62)1.17 (1.10 to 1.25)24319647 (31 to 63)1.24 (1.15 to 1.33)70\u20137958148695 (67 to 123)1.20 (1.13 to 1.26)47239676 (51 to 100)1.19 (1.13 to 1.26)80\u201389750634116 (73 to 160)1.18 (1.11 to 1.26)643540103 (64 to 143)1.19 (1.11 to 1.27)\u22659049143655 (\u221232 to 142)1.13 (0.93 to 1.36)45039456 (\u221221 to 133)1.14 (0.95 to 1.37)TotalCrude11810613 (9 to 16)1.12 (1.09 to 1.16)1049113 (10 to 16)1.14 (1.11 to 1.18)Adjusted319 (16 to 22)1.18 (1.15 to 1.22)18 (15 to 21)1.20 (1.16 to 1.24)1\u00a0Incidence rate difference (with 95% confidence interval) 2Incidence rate ratio (with 95% confidence interval)3\u00a0Pooled age-adjusted incidence rate difference or ratio (with 95% confidence interval) \nThe hospital-based hospital mortality was similar to the linkage-based hospital mortality in men in 1995 (adjusted risk ratio (RR) 1.01; 95% CI 0.95\u20131.07) and 2000 (adjusted RR 1.00; 95% CI 0.94\u20131.07) and in women in 1995 (adjusted RR 0.98; 95% CI 0.92\u20131.05) and 2000 (adjusted RR 0.99; 95% CI 0.93\u20131.06) (Table\u00a02). Also within the age groups, no significant differences between the hospital-based and the linkage-based hospital mortality were revealed.\nTable\u00a02Hospital mortality (%) of acute myocardial infarction in 1995 and in 2000 based on the national hospital discharge register and based on linked registers19952000Hospital registerLinked registersHospital registerLinked registersAgeMortality MortalityRD1RR2MortalityMortalityRD1RR2Men<301.93.6\u22121.6 (\u22129.5 to 6.2)0.54 (0.03 to 8.29)6.910.8\u22123.9 (\u221215.9 to 8.0)0.64 (0.17 to 2.40)30\u2013393.22.40.9 (\u22121.4 to 3.1)1.36 (0.59 to 3.11)2.83.1\u22120.3 (\u22122.6 to 2.0)0.90 (0.42 to 1.95)40\u2013493.33.7\u22120.4 (\u22121.5 to 0.7)0.89 (0.65 to 1.22)3.22.60.7 (\u22120.4 to 1.8)1.26 (0.85 to 1.86)50\u2013594.44.00.4 (\u22120.5 to 1.3)1.10 (0.88 to 1.37)3.73.50.2 (\u22120.6 to 1.1)1.07 (0.84 to 1.37)60\u2013698.48.20.2 (\u22120.9 to 1.3)1.02 (0.89 to 1.16)7.27.10.1 (\u22121.0 to 1.3)1.02 (0.87 to 1.20)70\u20137917.117.00.0 (\u22121.6 to 1.6)1.00 (0.91 to 1.10)15.415.30.1 (\u22121.6 to 1.7)1.00 (0.90 to 1.12)80\u20138928.128.5\u22120.3 (\u22123.6 to 2.9)0.99 (0.88 to 1.11)24.425.5\u22121.1 (\u22124.3 to 2.1)0.96 (0.84 to 1.09)\u22659034.732.62.1 (\u221210.7 to 15.0)1.07 (0.73 to 1.56)47.150.7\u22123.7 (\u221219.5 to \u22122.2)0.93 (0.67 to 1.28)TotalCrude10.710.9\u22120.3 (\u22120.9 to 0.4)0.98 (0.92 to 1.04)9.59.8\u22120.3 (\u22121.0 to 0.4)0.97 (0.91 to 1.04)Adjusted30.1 (\u22120.6 to 0.7)1.01 (0.95 to 1.07)0.0 (\u22120.6 to 0.7)1.00 (0.94 to 1.07)Women<305.925.0\u221219.1 (\u221263.0 to 24.8)0.24 (0.02 to 3.01)5.07.7\u22122.7 (\u221220.0 to 14.7)0.65 (0.04 to 9.50)30\u2013396.96.10.8 (\u22126.1 to 7.7)1.13 (0.38 to 3.33)3.43.30.1 (\u22124.6 to 4.9)1.03 (0.25 to 4.22)40\u2013494.85.1\u22120.3 (\u22123.4 to 2.9)0.95 (0.50 to 1.79)6.26.7\u22120.5 (\u22123.8 to 2.7)0.92 (0.56 to 1.53)50\u2013594.95.3\u22120.4 (\u22122.6 to 1.8)0.92 (0.60 to 1.41)4.95.1\u22120.1 (\u22122.3 to 30.)0.97 (0.63 to 1.49)60\u2013699.68.90.7 (\u22121.2 to 2.5)1.07 (0.88 to 1.31)8.88.10.7 (\u22121.3 to 2.8)1.09 (0.86 to 1.39)70\u20137918.718.30.4 (\u22121.6 to 2.5)1.02 (0.92 to 1.14)16.616.40.2 (\u22121.9 to 2.3)1.01 (0.89 to 1.15)80\u20138930.431.8\u22121.4 (\u22124.3 to 1.5)0.96 (0.87 to 1.05)28.429.3\u22120.9 (\u22124.0 to 2.1)0.97 (0.87 to 1.08)\u22659040.540.6\u22120.1 (\u22129.3 to 9.2)1.00 (0.79 to 1.25)31.534.4\u22122.9 (\u221211.5 to 5.6)0.91 (0.71 to 1.19)TotalCrude17.718.1\u22120.4 (\u22121.6 to 0.8)0.98 (0.92 to 1.05)15.916.5\u22120.5 (\u22121.8 to 0.7)0.97 (0.90 to 1.04)Adjusted3\u22120.1 (\u22121.3 to 1.0)0.99 (0.93 to 1.06)\u22120.2 (\u22121.3 to 1.0)0.99 (0.92 to 1.06)1\u00a0Risk difference (with 95% confidence interval) 2\u00a0Risk ratio (with 95% confidence interval)3\u00a0Pooled age-adjusted risk difference or ratio (with 95% confidence interval) \nFrom 1995 to 2000, the hospital-based decline in incidence was similar to the linkage-based decline (Table\u00a03). In men, the (adjusted) hospital-based incidence declined by 48 per 100,000 person-years or 18% (95% CI 17\u201320%) and the linkage-based incidence declined by 46 per 100,000 person-years or 20% (95% CI 18\u201322%). In women, the (adjusted) hospital-based incidence declined by 18 per 100,000 person-years or 15% (95% CI 13\u201318%) and the (adjusted) linkage-based incidence declined by 18 per 100,000 persons per year or 17% (95% CI 14\u201319%). The age-specific relative changes in hospital-based and linkage-based incidence were largely comparable.\nTable\u00a03Trends in the incidence (per 100,000 persons per year) and hospital mortality (%) of hospitalized acute myocardial infarction from 1995 to 2000 based on the national hospital discharge register and based on linked registersTrends in incidence Trends in hospital mortality Risk difference Relative risk Risk differenceRelative risk AgeHospital registerLinked registersHospital registerLinked registersHospital registerLinked registersHospital registerLinked registersMen<300 (0 to 1)0 (0 to 1)1.16 (0.80 to 1.68)1.36 (0.83 to 2.22)5.0 (\u22122.5 to 12.5)7.2 (\u22124.9 to 19.4)3.59 (0.41 to 31.07)3.03 (0.36 to 25.62)30\u201339\u22121 (\u22126 to 4)1 (\u22124 to 5)0.98 (0.87 to 1.10)1.02 (0.88 to 1.18)\u22120.4 (\u22122.5 to 1.6) 0.7 (\u22121.7 to 3.1)0.87 (0.44 to 1.72)1.30 (0.53 to 3.20)40\u201349\u221228 (\u221239 to \u221217)\u221230 (\u221241 to \u221219)0.87 (0.82 to 0.92)0.83 (0.77 to 0.89)\u22120.1 (\u22121.1 to 0.9)\u22121.2 (\u22122.4 to 0.0)0.97 (0.71 to 1.33)0.69 (0.46 to 1.02)50\u201359\u2212102 (\u2212122 to \u221283)\u221278 (\u221297 to \u221259)0.80 (0.77 to 0.83)0.81 (0.77 to 0.85)\u22120.7 (\u22121.5 to 0.1)\u22120.6 (\u22121.5 to 0.4)0.84 (0.68 to 1.04)0.86 (0.67 to 1.12)60\u201369\u2212211 (\u2212242 to \u2212181)\u2212181 (\u2212210 to \u2212152)0.76 (0.73 to 0.79)0.75 (0.72 to 0.79)\u22121.2 (\u22122.3 to \u22120.2)\u22121.2 (\u22122.4 to 0.0)0.86 (0.75 to 0.98)0.86 (0.73 to 1.00)70\u201379\u2212218 (\u2212265 to \u2212172)\u2212186 (\u2212229 to \u2212143)0.82 (0.79 to 0.86)0.82 (0.78 to 0.86)\u22121.7 (\u22123.2 to \u22120.2)\u22121.7 (\u22123.5 to 0.0)0.90 (0.82 to 0.99)0.90 (0.81 to 1.00)80\u201389\u2212110 (\u2212192 to \u221228)\u2212121 (\u2212196 to \u221245)0.91 (0.85 to 0.98)0.88 (0.82 to 0.95)\u22123.8 (\u22126.8 to \u22120.8)\u22123.0 (\u22126.4 to 0.4)0.87 (0.77 to 0.97)0.89 (0.79 to 1.01)\u226590\u2212276 (\u2212470 to \u221281)\u2212196 (\u2212370 to \u221223)0.67 (0.51 to 0.89)0.70 (0.51 to 0.96)12.3 (\u22121.3 to 26.0)18.1 (2.9 to 33.3)1.35 (0.97 to 1.89)1.56 (1.07 to 2.26)TotalCrude\u221232 (\u221237 to \u221227)\u221231 (\u221236 to \u221227)0.87 (0.86 to 0.89)0.86 (0.84 to 0.88)\u22121.1 (\u22121.8 to \u22120.5)\u22121.1 (\u22121.8 to \u22120.4)0.89 (0.84 to 0.95)0.90 (0.84 to 0.96)Adj1\u221248 (\u221253 to \u221243)\u221246 (\u221251 to \u221241)0.82 (0.80 to 0.83)0.80 (0.78 to 0.82)\u22121.2 (\u22121.8 to \u22120.6)\u22121.2 (\u22121.9 to \u22120.5)0.89 (0.84 to 0.94)0.89 (0.83 to 0.96)Women<300 (0 to 1)0 (0 to 1)1.21 (0.64 to 2.32)3.34 (1.09 to 10.23)\u22120.9 (\u221215.6 to 13.8)\u221217.3 (\u221262.1 to 28)0.85 (0.06 to 12.59)0.31 (0.02 to 3.88)30\u2013392 (\u22121 to 4)0 (\u22122 to 3)1.20 (0.94 to 1.53)1.05 (0.78 to 1.42)\u22123.5 (\u22128.9 to 2.0)\u22122.8 (\u22129.1 to 3.6)0.50 (0.17 to 1.49)0.55 (0.13 to 2.22)40\u2013498 (3 to 13)6 (0 to 11)1.22 (1.07 to 1.38)1.18 (1.01 to 1.37)1.3 (\u22121.5 to 4.2)1.6 (\u22121.9 to 5.1)1.27 (0.75 to 2.17)1.31 (0.71 to 2.40)50\u201359\u221218 (\u221227 to \u22128)\u221215 (\u221224 to \u22126)0.84 (0.77 to 0.92)0.84 (0.76 to 0.94)0.0 (\u22121.9 to 2.0) \u22120.2 (\u22122.6 to 2.1)1.01 (0.68 to 1.04)0.96 (0.61 to 1.51)60\u201369\u221264 (\u221281 to \u221246)\u221266 (\u221283 to \u221250)0.79 (0.74 to 0.84)0.75 (0.69 to 0.80)\u22120.8 (\u22122.6 to 1.1)\u22120.9 (\u22122.9 to 1.2)0.92 (0.75 to 0.98)0.90 (0.71 to 1.15)70\u201379\u2212109 (\u2212136 to \u221283)\u221290 (\u2212116 to \u221264)0.81 (0.77 to 0.85)0.82 (0.77 to 0.86)\u22122.1 (\u22124.0 to \u22120.2)\u22121.9 (\u22124.1 to 0.3)0.89 (0.82 to 0.99)0.90 (0.79 to 1.02)80\u201389\u2212107 (\u2212149 to \u221264)\u221294 (\u2212134 to \u221254)0.86 (0.81 to 0.91)0.85 (0.80 to 0.91)\u22122.0 (\u22124.8 to 0.7)\u22122.5 (\u22125.7 to 0.6)0.93 (0.77 to 0.97)0.92 (0.83 to 1.02)\u226590\u221240 (\u2212125 to 44)\u221241 (\u2212121 to 39)0.92 (0.77 to 1.10)0.90 (0.75 to 1.10)\u22129.0 (\u221217.6 to \u22120.5)\u22126.2 (\u221215.5 to 3.1)0.78 (0.97 to 1.89)0.85 (0.66 to 1.09)TotalCrude\u221215 (\u221218 to \u221212)\u221215 (\u221218 to \u221212)0.87 (0.85 to 0.90)0.86 (0.84 to 0.88)\u22121.8 (\u22122.9 to \u22120.7)\u22121.6 (\u22122.9 to \u22120.3)0.90 (0.84 to 0.95)0.91 (0.85 to 0.98)Adj1\u221218 (\u221222 to \u221215)\u221218 (\u221221 to \u221214)0.85 (0.82 to 0.87)0.83 (0.81 to 0.86)\u22121.6 (\u22122.7 to \u22120.5)\u22121.6 (\u22122.9 to \u22120.4)0.91 (0.85 to 0.97)0.91 (0.85 to 0.98)1\u00a0Pooled age-adjusted risk difference or ratio (with 95% confidence interval)\nThe (adjusted) hospital-based decline in hospital mortality from 1995 to 2000 was similar to the (adjusted) linkage-based decline (Table\u00a03). In men, hospital mortality declined absolutely by 1% and relatively by 11% based on both the hospital register and linked registers. In women, the absolute and relative decline was 2% and 9%, respectively, based on both the hospital register and linked registers. The age-specific relative changes in hospital-based and linkage-based hospital mortality were largely similar.\nDiscussion\nWe combined data from the national hospital discharge register with data from the population register to determine the (trends in) incidence and hospital mortality of first hospitalized AMI (double counts and recurrent AMI cases excluded) and compared the outcomes with the incidence and hospital mortality based on routinely collected data in the hospital register (double counts and recurrent AMI cases included). The incidence based on the hospital register was considerably and significantly higher than the incidence based on linked registers, whereas hospital mortality and trends in incidence and hospital mortality were identical using either approach.\nAlthough we were able to estimate the incidence and hospital mortality of first AMI by linkage of the hospital register with the population register, some aspects of this method should be discussed. First, non-unique persons in the population (register) were excluded from the study population in the linked registry data. If this exclusion produced systematic differences between the linked registry population and the clinically relevant population (i.e., the total Dutch population), it might have affected the incidence estimate in the linked registry to some extent (e.g., an overestimation of incidence resulting from a higher mean age of the study population). A pilot study suggested that non-uniqueness relates to large cities, foreign origin and age [6]. The differences between unique and non-unique persons, however, were small [9] and apply to both 1995 and 2000. Second, information on previous admissions was limited to maximal 5\u00a0years for the patients (as the numeric part of the postal code is registered in the hospital register since 1991). Therefore, it seems likely that some \u201cfirst\u201d AMI patients actually were recurrent AMI patients. However, it has been estimated that most (95%) of recurrent events occur within 5\u00a0years. [4, 16] Third, the outcome measures in the present study were incidence and hospital mortality. Mortality after discharge from hospital was not considered, since this outcome is not registered in the hospital register. Differences in mortality after discharge between patients with a first or a recurrent AMI can only be studied by linkage of national registers (i.e., the hospital register with the population register and the cause of death statistics). A final aspect that needs to be addressed is the generalizability of our findings. The results might differ if a change over time occurs in double-count or readmission fractions. Results might also differ for other diseases than AMI or for specific groups of patients (e.g., non-native patients), hospitals or regions. Such differences will not be apparent from routinely collected data. Since trends in incidence and hospital mortality are often based on national routinely collected data, generalization of our findings would be of great relevance.\nIt has been be argued that routine statistics can not be used for providing information on (trends in) incidence and hospital mortality, because of double counting of cases and admixture of first and recurrent events. In order to prevent erroneous inclusion of prevalent cases (recurrent events) that have had a previous hospitalization for AMI prior to the study period generally an clearance period is employed to overcome overestimation of the incidence [16]. For myocardial infarction, it has been shown that a clearance period of 13\u00a0years should be taken into account to completely overcome inclusion of prevalent cases. In our study we used a 5-year clearance period because of logistical reasons. This however would suggest that in our incidence estimate around 5% of the subjects should be considered as recurrent AMI-patient rather that first ever AMI patient [16]. Although this does affect the absolute incidence estimate, it may not affect trends in incidence and case fatality, assuming that the erroneous inclusion of prevalent cases occurs at both time windows Indeed this has been shown in a Danish study where the incidence based on the number of AMI-patients without an admission for AMI in the previous year overestimated the incidence based on the number of AMI-patients without (an admission for) AMI in the previous 14\u00a0years by 27% in men and 16% in women, but trends reflected trends in true incidence with reasonable accuracy [4]. With respect to double count, in eight states of the USA, it was estimated that double counting of patients resulted in an overestimation of the true incidence of hospitalized AMI and an underestimation of the true hospital mortality. In this study, double counts were defined as readmissions for AMI within 7\u00a0days and thought to result from transfer to a second hospital. Correction for double counting revealed a 10\u201315% lower incidence and a 12% higher hospital mortality [5]. Despite aspects regarding double counts and recurrent events, there have been several consistent reports from different countries using national registries to study trends in case fatality, incidence and survival [17\u201321]. These time trends indicated a decline in incidence of myocardial infarction and in case fatality after AMI. In the present study, we found a significant decline in both incidence and hospital mortality of first AMI between 1995 and 2000. These declines in incidence and hospital mortality appear to mainly reflect declines in first events, as trends were not altered when recurrent cases were excluded from the data, and thus are best explained by advances in primary prevention and acute management of AMI.\nOverall, our results based on deterministic linkages of sources using gender, date of birth and postal code, are in line with earlier reports from other studies, where linkage was performed using unique identification numbers.\nIn conclusion, our study shows that the incidence based on routinely collected data in the national hospital discharge register overestimates the actual incidence of first AMI based on linked national registers by least 22% in men and 18% in women. Yet, the hospital mortality based on the hospital register accurately reflects the actual hospital mortality of first AMI. Furthermore, trends in incidence and hospital mortality based on the hospital register are not changed when double counts and recurrent cases were excluded. Since trends in incidence and hospital mortality of AMI are often based on national routinely collected data, it is reassuring that our results indicate that findings from such studies are indeed valid and not biased because of recurrent events and double counts.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\n(DOC 193 kb)","keyphrases":["acute myocardial infarction","incidence","hospital admissions","registries","medical record linkage","epidemiology"],"prmu":["P","P","P","P","R","U"]}
{"id":"Eur_J_Epidemiol-4-1-2413078","title":"Low migrant mortality in Germany for men aged 65 and older: fact or artifact?\n","text":"Migrant mortality in Europe was found to be lower than mortality of host populations. In Germany, residents with migrant background constitute nearly one tenth of the population aged 65+ with about 40% of them being foreigners. The German Pension Scheme follows vital status of pensioners very accurately. Mortality re-estimation reveals two-fold underestimation of mortality of foreigners due to biased death numerator and population denominator.\nIntroduction\nMigrant mortality has attracted substantial research interest. First, it provides the opportunity to look at the combined influence of earlier life conditions in the country of origin and the more recent life conditions in the receiving country. Second, it allows one to see how cultural and behavioral patterns imported from the country of origin and their further transformation affect migrant health. Third, it enables the researcher to gain insight into selective migration of healthy people (the healthy migrant effect). Indeed, at the very moment of moving, most migrants are in good health because immigration and assimilation to the foreign society are hardly compatible with serious health problems. In addition, migrants often have to pass obligatory medical examinations before they immigrate.\nIn Germany and other European countries, migrant mortality was found to be much lower than the mortality of the host population [1\u20136]. Prior studies have tended to consider the healthy migrant effect as a central explanation of very low immigrant mortality. In addition, the possible role played by behavioral and psychological factors has been emphasized [1, 2, 7].\nAt the same time, there has also been serious concern about the reliability of demographic data on the mortality of migrants. The main problem is related to migrant moves between the home and host country. If departures from the host country and deaths in the country of origin are under-recorded, the moves produce a numerator-denominator bias leading to the under-estimation of migrant mortality. Deaths of migrants in the country of origin unregistered by the population statistics in the receiving countries cause \u201cstatistical immortality.\u201d The results of under-registered return migrations and deaths abroad may be particularly problematic at advanced ages (90+), i.e., at ages when the share of \u201cimmortals\u201d cumulated over time can become significant compared to the relatively small remaining population [4, 8, 9]. Biases in the data can be substantial even when micro-data from population registers or cohort follow-up studies are used [3, 10]. This is so because a segment of migrants (the less-well-integrated and less healthy) may tend to be at higher risk of observation censoring due to departure [3].\nIn Germany in 2005, 15.3\u00a0million people (or 18.6% of the total population) had a migrant background1 and 7.3\u00a0million (8.9%) had foreign citizenship [11]. Among the population aged 65+, 7.6% had a migrant background (mostly first-generation immigrants) and 3.4% of them were foreigners [11, 12]. Many labor migrants (Gastarbeiter) arrived in Germany in the 1950 and 1970s, at a time when the rapidly growing German economy experienced a labor shortage. They came mainly from Southern Europe and took up jobs in the coal, iron, steel, and automobile industries of Germany. Many of them have been granted permanent residence in Germany but not German citizenship. The so-called Aussiedler, i.e., ethnic Germans, mostly coming from the former Soviet Union, were the exception. They were granted the legal right to receive German citizenship upon arrival [13]. On average, foreigners have higher unemployment rates than Germans, their educational level is lower, and they have jobs that require relatively low qualifications [14, 15].\nGerman population statistics are not free of problems, possibly resulting in an overestimation of the foreign population (see also [4]). The last West German and East German censuses took place in the 1980s. Since then, the population has been updated on the basis of vital event registration. Foreigners are also registered by the Central Registry of Aliens (Ausl\u00e4nderzentralregister), but a recent update of this source has not been translated into German population statistics [14]. Departures from Germany to stay abroad for long periods legally should be notified. Nevertheless, allegedly many fail to do so since an eventual return to Germany is facilitated by maintaining a German address.\nIn studies of migrant mortality, data problems are usually not addressed by direct checks since it is difficult to find high-quality data sources alternative to the official population statistics. We are aware of only one study that re-estimated migrant mortality. This was done for Sweden on the basis of income and taxation data [10]. The authors calculated death rates for foreign migrants aged 20\u201364 who had some registered income (either earnings or social benefits) and excluded those who had no registered income. The latter category was considered as the one that included a high proportion of people who (de facto) resided abroad. After the exclusion, the immigrant death rates substantially increased compared to the initial rates calculated from the whole migrant population. Correspondingly, the migrants\u2019 mortality advantage diminished and became statistically insignificant in most immigrant groups.\nOur study is based on data of the scientific use files of the Federal German Statutory Pension Scheme (DRV Bund-Deutsche Rentenversicherung Bund) for the re-estimation of the mortality of the retired German and foreign populations. These data were provided by the DRV Research Data Centre in W\u00fcrzburg [16]. The data cover all public old-age pensions resulting from work careers in Germany and all of the three former branches of the pension insurance: workers, salaried employees, and miners. The data also reflect pension terminations due to the death of pensioners. We consider only men because many women who are now retired have never participated in the labor market. The DRV data cover about 90% of the German male population aged 65+. The remaining people are those whose working careers were entirely related to self-employment or to the German Civil Service.\nThe DRV data allow to identify the narrowly defined migrant population, i.e. pensioners with foreign citizenship. In 1995\u20132004, about 190,000 (or 3.5%) of the 5.4\u00a0million male pensioners aged 65+ had foreign citizenship. The DRV information about pensioners\u2019 nationality is considered to be exact [17]. The DRV data on the insurants\u2019 vital status are also highly reliable. As the function of pension insurers is to provide pension payments, they follow the survival of pensioners very carefully, using several sources: information from the pay-out service of the German Post (Postrentendienst), which receives death notices from the registrar\u2019s office or from the undertaker; information provided by surviving dependents; or information about pensions not delivered. Those receivers of German pensions who live abroad must provide annual confirmation of being alive. Nevertheless, we excluded pensioners with current residence abroad in order to achieve greater data homogeneity.\nThe official population figures, population and deaths by 5-year age groups, split by German and non-German nationality, were obtained from the Federal Statistical Office [12].\nOn the basis of the DRV and official population data, we computed two sets of deaths and population exposures at ages 65\u201369, 70\u201374, \u2026, 90+ for the years 1995, 1998, 2001, and 2004. To estimate life expectancies at age 65, abridged life tables were constructed using Chiang\u2019s method [18, 19].\nTable\u00a01 presents population figures and deaths from official population statistics and DRV pension data. For German men, the death and population ratios of population statistics to DRV statistics are the same. Both official population and official death figures exceed the figures provided by DRV by 9% over the whole period, indicating that DRV data cover 91% of the German population. For foreigners, however, there is a pronounced disagreement between population and death figures. The national figure for the former exceeds the DRV figure by 33%. For the latter, by contrast, the national figure is lower by 33% than that given by the DRV. This gap suggests a major bias in population statistics on foreigners, induced by an over-stated population and under-stated deaths. Table\u00a01 suggests that mortality of foreigners in the official statistics is about two-fold higher than its true value based on the DRV data.Table\u00a01Comparison of male population and death counts (age 65+) between German official population statistics and DRV data for the years 1995\u20132004Nationality and yearPopulationDeathsIn thousandsRatioIn thousandsRatioPopulation statisticsDRV statisticsPopulation statistics\/DRV statisticsPopulation statisticsDRV statisticsPopulation statistics\/DRV statisticsGermans\u00a0\u00a0\u00a0\u00a019954408.04023.11.10277.7255.11.09\u00a0\u00a0\u00a0\u00a019984704.24311.31.09270.7252.41.07\u00a0\u00a0\u00a0\u00a020015208.74760.21.09268.5232.51.15\u00a0\u00a0\u00a0\u00a020045879.45356.61.10277.1264.11.09\u00a0\u00a0\u00a0\u00a01995\u2013200420,200.218,451.31.091,093.91,004.11.09Foreigners\u00a0\u00a0\u00a0\u00a01995109.776.71.432.73.70.74\u00a0\u00a0\u00a0\u00a01998144.7104.21.393.34.60.71\u00a0\u00a0\u00a0\u00a02001190.0144.21.324.05.50.73\u00a0\u00a0\u00a0\u00a02004251.0197.31.274.98.40.58\u00a0\u00a0\u00a0\u00a01995\u20132004695.3522.31.3314.922.10.67Calculated from: official population statistics from the German Federal Statistical Office [14]; pension statistics from scientific use file SUFRTBNRTWF94-04TDemoKibele [5]\nAge-specific death rates (displayed in Fig.\u00a01) accordingly show good agreement for Germans between the official population statistics and the pension statistics except for very old ages, i.e. 90+ at which the official population statistics understate the male mortality [8]. For foreigners, there is a very large disagreement in age-specific death rates. Official population statistics on the death rates for foreigners are extremely low at all ages and do not show the expected increase over age. The corresponding figures of DRV data are similar to those for Germans.Fig.\u00a01Comparison of death rates between German official population statistics and DRV data: pooled data for the years 1995, 1998, 2001, and 2004. Calculated from: official population statistics from the German Federal Statistical Office [14]; pension statistics from scientific use file SUFRTBNRTWF94-04TDemoKibele [5]\nTable\u00a02 shows nearly the same life expectancy figures for Germans at age 65 in both data sources (15.3\u00a0years in the population statistics and 15.6\u00a0years in the DRV statistics for the whole observation period). For foreigners, the population statistics\u2019 figures are unrealistically high (e.g. 30.2\u00a0years) and very different from the figures provided by the DRV data (15.0\u00a0years). Importantly, German life expectancy in the DRV data is slightly higher than that of foreigners. Finally, both in the population statistics and the DRV data, overall life expectancy based on pooled data on Germans and foreigners hardly deviates from the life expectancy of Germans because the impact of the foreign population figure is small.Table\u00a02Life expectancy at age 65 for men in Germany, for the years 1995, 1998, 2001, and 2004, in yearsYearGermansForeignersGermans and foreignersPopulation statisticsDRV statisticsPopulation statisticsDRV statisticsPopulation statisticsDRV statistics199514.714.625.914.314.714.6199815.215.229.214.815.415.2200116.016.434.115.716.216.4200416.616.237.815.016.816.11995\u2013200415.315.630.215.015.415.6Calculated from: official population statistics from the German Federal Statistical Office [14]; pension statistics from scientific use file SUFRTBNRTWF94-04TDemoKibele [5]\nThe life expectancy figures of the order of 30\u00a0years at age 65 calculated from German official population statistics are obviously implausible. They are much higher than the levels of longevity experienced by Japanese women (23.2\u00a0years in 2005), the world\u2019s lowest mortality population. The bias is produced by the inflated population denominator and the under-stated death numerator in German population statistics. By contrast, DRV data (which is based on the rigorous follow-up of people eligible to receive German old-age pensions) show that the mortality of foreigners aged 65+ is actually slightly higher than the mortality of German men. At least among men aged 65+ the seemingly large mortality advantage of foreigners compared to Germans is a statistical artifact.\nOur study makes clear that the inaccuracies in estimation of the population denominator and the death numerator can result in serious underestimation of immigrant mortality. However, a significant mortality advantage of immigrants could be found even in studies where the researchers were well aware of the possible bias [5]. Even in the Swedish study mentioned earlier that revealed a general underestimation of mortality among immigrants, a statistically significant mortality advantage could be observed in some immigrant groups, i.e., women from Southern Europe, men from Latin America, Africa and Asia [10].\nThe underestimated mortality of foreigners should result in the underestimation of mortality in the broader group of German residents who have a migrant background. As mentioned earlier, foreigners constitute about 40% of all people with migrant background at ages 65+. Assuming for simplicity the same level of true mortality among all German residents with migrant background and precisely reported mortality of German nationals with migrant background, it is easy to see that the two-fold underestimation of mortality of foreigners leads to a 25% underestimation of mortality of all residents with migrant background.\nHow can the mortality advantage of the migrant population found elsewhere [1, 2, 4, 5] be reconciled with the complete absence of such an advantage in our analysis? The answer may be found by considering the peculiarities of our data compared to the previous studies. First, our study considers \u201cforeigners\u201d or pensioners with current foreign citizenship. Mostly, these are people from Southern Europe who entered Germany in the 1950\u20131970s and to a much lesser extent more recent immigrants from the former Soviet Union and Eastern Europe. Second, our study includes only people aged 65 and over, while the other studies looked mostly or exclusively at people of working age. Close to the age of their migration, the health advantage of immigrants can be attributed to the selection effect of healthy people, to healthier behaviors and dietary habits, and to positive psychological expectations [1, 2, 7].\nFor many reasons, the initial advantage does not translate into a mortality advantage at ages of 65 and over. Certain factors affecting immigrants in the country of their new residence can lead to a steeper (compared to the host population) health decline with age. In particular, immigrants may gradually adopt less healthy \u201cwestern\u201d lifestyles associated with higher coronary risk. In old age they may also experience higher mortality from conditions related to childhood infections and deprivation, such as stroke and stomach cancer [7, 20, 21].\nMost importantly, immigrants face the health consequences of lower socioeconomic status such as poorer life conditions, restricted life chances, and corresponding psychological stress [22]. According to DRV data, 86% of foreigners but only 45% of Germans belong to the lower 40% of the lifetime income distribution. Our earlier analysis shows that this category of pensioners experience approximately 50% mortality elevation compared to those belonging to the upper lifetime earnings\u2019 quintile [23]. Foreigners are also characterized by a higher share of former manual workers: 80% vs. 58% among Germans. This status is associated with a 35% mortality excess compared to the white-collar occupations [23].\nSpeculation about potential reasons for a steeper health decline with age among the immigrants is supported by empirical evidence that confirms that the initial health advantage of foreigners \u201cwears off\u201d over time [3, 24, 25]. This pattern can lead to mortality convergence and even mortality crossover between the host and migrant populations.\nIn general, our results support the concern about the reliability of very low estimates of migrant mortality at ages 65 and over. It is possible that, despite the initial health advantage, the slightly higher mortality of foreigners in the 65+ population is a result of socioeconomic health inequalities. This is a serious public health concern that deserves further investigation.","keyphrases":["migrants","mortality","germany","pensions","vital statistics registration"],"prmu":["P","P","P","P","R"]}
{"id":"Semin_Immunopathol-4-1-2315689","title":"Mechanisms of leukocyte migration across the blood\u2013retina barrier\n","text":"Immune-mediated inflammation in the retina is regulated by a combination of anatomical, physiological and immuno-regulatory mechanisms, referred to as the blood\u2013retina barrier (BRB). The BRB is thought to be part of the specialised ocular microenvironment that confers protection or \u201cimmune privilege\u201d by deviating or suppressing destructive inflammation. The barrier between the blood circulation and the retina is maintained at two separate anatomical sites. These are the endothelial cells of the inner retinal vasculature and the retinal pigment epithelial cells on Bruch\u2019s membrane between the fenestrated choroidal vessels and the outer retina. The structure and regulation of the tight junctions forming the physical barrier are described. For leukocyte migration across the BRB to occur, changes are needed in both the leukocytes themselves and the cells forming the barrier. We review how the blood\u2013retina barrier is compromised in various inflammatory diseases and discuss the mechanisms controlling leukocyte subset migration into the retina in uveoretinitis in more detail. In particular, we examine the relative roles of selectins and integrins in leukocyte interactions with the vascular endothelium and the pivotal role of chemokines in selective recruitment of leukocyte subsets, triggering adhesion, diapedesis and migration of inflammatory cells into the retinal tissue.\nIntroduction\nImmune-mediated inflammation in the retina is regulated by a combination of anatomical, physiological and immuno-regulatory mechanisms, referred to as the blood\u2013retina barrier (BRB). The BRB is thought to be part of the specialised ocular microenvironment that confers protection or \u201cimmune privilege\u201d by deviating or suppressing destructive inflammation [120, 121]. These mechanisms are designed to prevent normal immune surveillance and delete or inactivate cells migrating across the BRB to mitigate the effects of deleterious immune responses [15, 99]. Nevertheless, retinal inflammation does occur, and in addition to well-defined inflammatory diseases such as uveoretinitis, immune mechanisms affecting the integrity of the BRB and leukocyte infiltration of the retina are implicated in other ocular diseases such as diabetic retinopathy [76] and age-related macular degeneration (ARMD) [72].\nThe barrier between the blood circulation and the retina is maintained at two separate anatomical sites. These are the endothelial cells of the inner retinal vasculature and the retinal pigment epithelial cells (RPE) on Bruch\u2019s membrane between the fenestrated choroidal vessels and the outer retina. For leukocyte migration across the BRB to occur, changes are needed in both the leukocytes themselves and the cells forming the barrier [9, 137]. The retina is an extension of the central nervous system (CNS), and as such, many of the molecular structures making up the inner BRB are similar to those found in the vascular blood\u2013brain barrier (BBB) [1]. However, there are also important differences between the tissues, including vascular heterogeneity in the brain and numbers of microglia in the retina.\nMuch research has been carried out into mechanisms controlling inflammatory cell adhesion and extravasation of leukocytes from the circulation into neural tissues, and a paradigm invoking sequential, separate events involving interacting pairs of selectins and their ligands, chemokines and cell adhesion molecules (CAM) has evolved to be one of the central tenets in immunology (Fig. 1) [12]. Less is understood about the process of extravasation, and there is evidence for leukocyte extravasation in the CNS both through intercellular endothelial tight junctions (paracellular route) and for transcellular migration through the endothelial cell itself [45].\nFig.\u00a01Mechanisms involved in leukocyte trafficking across the blood-retina barrier at the endothelium. Initial interactions between leukocyte and endothelium are usually mediated by selectins that induce tethering and rolling (1). If G-protein-coupled receptors on the leukocyte engage appropriate chemokines on surface of endothelium, then the leukocyte may become activated (2), leading to conformational changes in integrin molecules allowing firm adhesion to the endothelium and leukocyte spreading (3). Diapedesis across the endothelium and into the retina can then take place (4). This is triggered by additional chemokine and cytokine signals and gradients and mediated by matrix metalloproteinases secreted by the leukocyte and alterations in the signalling and regulatory molecules of the TJ that control the interaction between the membranous component and the cytoskeleton of the endothelial cell, leading to breakdown of the BRB (5). How inflammatory responses in the retina are initiated or resolved is still controversial. There is evidence that resident and\/or infiltrating cells may influence the progression (+Ve signal?) or resolution (\u2212Ve signal?) of the inflammatory response\nTo date, many studies have been based on neutrophil migration, or undefined mixed leukocyte populations, but recent research, including our own, has revealed that different leukocyte subsets have distinct requirements for endothelial cell interactions and subsequent migration. These requirements may also vary depending on the type of tissue, whether normal surveillance or inflammatory trafficking is involved and if inflammatory on the type of stimulus. Mononuclear cells including monocytes and T lymphocytes are primarily involved in the pathologies observed in various retinal inflammations and will be the focus for this review. Understanding these processes and whether mechanisms really differ between peripheral tissues and ocular or other CNS sites such as the brain is important as every stage of the process, from initial engagement to migration into the tissue, is a potential target for immunotherapy to control retinal damage [53].\nLeukocyte adhesion cascade\nAdhesion molecules in the leukocyte adhesion cascade\nThe process of the inflammatory response and how blood leukocytes are recruited from the blood to the tissues has been described as the leukocyte adhesion cascade. Rolling of leukocytes on the luminal endothelial cell wall is usually the first step in the adhesion cascade that culminates in leukocyte extravasation, and the mechanisms underlying the various stages involved has been recently reviewed [82]. Selectins, such as L selectin, expressed on microvilli of most leukocytes and P and E selectin, which may be expressed by activated or inflamed endothelium, mediate both tethering and rolling [119]. These molecules interact with ligands such as P-selectin glycoprotein ligand-1 (PSGL-1), expressed on other leukocytes or endothelium only when correctly glycosylated [89]. This initial tethering and fast rolling allows adherence under flow, and shear stress forces support the adhesive and signalling interactions taking place that then mediate slow rolling. Integrins are also involved in rolling, and \u03b14\u03b21-integrin (VLA4) dependent rolling and LFA-1\/ICAM-1 interactions have been shown to be important for firm adhesion and lymphocyte trafficking in the CNS [44, 59]. Leukocyte rolling via low-affinity interactions can then be converted to firm adhesion as chemokine-receptor G-protein dependent activation takes place, resulting in conformational changes in the binding domains of LFA-1 to high-affinity state and in membrane clustering of the integrins giving increased avidity [6, 78]. This leads to firm adhesion and is the prelude to spreading, crawling and migration through the endothelium, either by the paracellular route involving release of endothelial adherens junctional proteins [87, 113, 126] or the transcellular route via small continuous membrane associated structures or vesiculo-vacuolar organelles.\nChemokines in the leukocyte adhesion cascade\nLeukocyte migration is controlled to a large extent by members of a family of chemoattractants, the chemokines [109]. The panel of chemokine receptors that a leukocyte carries enables it to respond to chemokine signals either from normal tissue thus controlling immune surveillance or from an inflammatory site. Forty-three human chemokines have now been officially named, and these do not include isoforms, polymorphisms, splice variants and those chemokines encoded by viruses. There are currently 18 chemokine-specific G-protein-coupled receptors [92]. As these chemokines can be allocated to four groups based on the position of 2 N-terminal conserved cysteines, this has been used as the basis for their systematic nomenclature [7, 143]. The two main groups of chemokines are now designated either CCL, where there is no amino acid separating the cysteines, or CXCL, where there is one amino acid between the cysteine molecules.\nChemokines can bind to more than one receptor, and receptors can bind to more than one chemokine. Receptor affinity varies between the ligands but does not necessarily determine the functional potency of the chemokine [32, 130]. In response to ligand binding, the receptors trigger numerous secondary mediators, which initiate functional responses such as cell migration. The resulting functions may differ depending on the cell type and the prevailing microenvironment [77, 111]. The multiple different combinations of chemokines and receptors, expressed either simultaneously or sequentially [13], plus the cell type and the local microenvironment ensure that the chemokine communication system can deliver a precise and accurate communication message to the required cell based on local circumstances, which may be rapidly changing.\nChemokines add additional specificity to the adhesive interactions initiated by the adhesion molecules during the recruitment of leukocytes from the circulation. Chemokines produced within the tissue are transported by transcytosis to the luminal surface of the endothelium. The transport across the endothelium is active and may involve the chemokine binding protein DARC [110]. Once at the luminal surface, the chemokines are displayed by glycosaminoglycans (GAGs) [124] at the tips of endothelial processes [95, 96]. This prevents the desensitisation of leukocyte chemokine receptors by chemokines in the blood stream and focusses the action of the chemokines on leukocyte adhesion and emigration. Chemokines have a GAG-binding site as well as specific receptor binding site [5], and GAG binding has been shown to be important in vivo [104].\nChemokines presented on the endothelium stimulate firm adhesion by increasing integrin affinity and avidity [6, 20, 26, 74] and then the spreading of cells, their migration to the endothelial cell junction [129] and the initiation of leukocyte transmigration [23, 24]. More than one chemokine is likely to be needed for the complete process of leukocyte extravasation with specific chemokines at distinct steps [142]. It is thought that chemokines do not need to form a gradient across the endothelium to enable adherent cells to move from the luminal surface into the tissue as surface immobilised chemokines and a shear force may be sufficient for transendothelial migration [24].\nNormal retina and immune surveillance\nIn normal physiology, one function of the immune system is to recognise and destroy cells that are abnormal (e.g. transformed tumour cells) or that display foreign or non-self antigens (e.g. virus-infected cells). This entails recirculation of relatively long-lived lymphocytes through the blood stream and lymphatics, moving from one lymph node to another and to peripheral inflammatory sites. In contrast, myeloid monocytes and dendritic cells, generated in the bone marrow, recirculate in the blood for only a few days before migrating into the tissues, forming extensive networks of phagocytic antigen presenting cells (APC). Some of these cells differentiate into long lived, highly specialised cells such as the osteoclasts of bone and microglia of the nervous system. In the tissues, APC may be stimulated by ingesting apoptotic tissue cells or by contact with microbes. They then migrate via the lymphatics to local draining lymph nodes where interaction with T and B lymphocytes can occur, and either tolerance to self-antigens or immunity to altered-self or foreign antigens is generated. Immune surveillance must be regulated in vulnerable tissues such as the brain and retina where tissue repair cannot take place, so structures that anatomically separate the central nervous system have evolved. Thus, the BRB may protect the delicate neural tissue from local, damaging inflammation but at the cost of defective peripheral tolerance to retinal antigens. Consequently, the retina remains vulnerable to autoimmune inflammation, increasing the importance of the BRB in excluding, eliminating or suppressing infiltrating inflammatory cells [15, 99].\nStructure and function of the blood\u2013retina barrier\nThe inner BRB formed at the endothelium is supported by perivascular cells such as smooth muscle cells, pericytes or retinal macrophages [53]. Astrocyte foot process contact is known to be essential for formation of intercellular tight junctions (TJ) and maintenance of structural integrity [1]. Other complex interactions between other constituents of the microenvironment surrounding the endothelial cells, such as the basement membrane, nerve endings, microglial cells and the extracellular fluid, are all required for the proper functioning of the barrier. The outer BRB formed at the RPE cell layer is simpler, the barrier being maintained by tight junctions between polarised epithelial cells on Bruch\u2019s membrane. Barrier function develops during embryogenesis, and once formed, is highly selective excluding molecules above 300\u00a0kDa. It is also effective in excluding most migrating cells, repopulation of the retina with bone-marrow-derived cells such as microglia taking from 6 to 12\u00a0months [4, 132], the rate of haematopoetic-derived cell turnover probably being dependent upon integrity of the RPE cell layer [134].\nIn normal retina, two distinct molecular structures form the physical BRB; these are the TJ and the adherens junctions (Fig. 1). These closely resemble TJ found in neural tissue in the brain [46, 102]. The TJ are highly organised pericellular structures that appear as multilammellar, usually continuous strands containing several integral membrane proteins that seal adjacent endothelial or epithelial cells, creating distinct membrane domains that restrict soluble and ion transport to apical and basal surfaces and block paracellular passage of macromolecular fluids and cells. The two major components are the claudins and the occludins, which are linked via zonula occludin (ZO) proteins to signalling proteins and the actin cytoskeleton. The junctional adhesion molecules (JAMs) are also located in the TJ, JAM A and C being particularly highly expressed in the RPE consistent with a role in establishing and maintaining cell polarity [33, 90]. Adherens junctions are formed mainly by cadherins (mainly VE cadherin), which interact with catenins to bind the cytoskeleton. PECAM\/CD31 expressed by endothelial cells in the junction also binds to the cytoskeleton via catenins and has been implicated in the migration of monocytic myeloid cells [98]. The general structure and regulation of neural tight junctions in retina and brain has been recently reviewed [46, 63].\nLeukocyte infiltration of normal retina\nDespite an apparently intact BRB, lymphocytes have been shown to infiltrate the normal retina. If both the leukocytes and the endothelium are normal, leukocytes do not cross the blood\u2013retina barrier [135, 137], but if lymphocytes are activated, they are able to initiate a transient breakdown in the BRB, enabling sampling of the retinal environment and possibly further recruitment of inflammatory cells [103, 139]. The mechanisms controlling this sampling or immune surveillance of the retina in the absence of retinal inflammation are not clear, and similar observations have been made in the CNS [65, 66]. Using scanning laser ophthalmoscopy, we have shown that circulating, activated T cells induce changes in the retinal vasculature that allow T cell firm adhesion to venule endothelium without rolling (secondary tethering), inferring a role for chemokine or integrin signalling in T cell capture and subsequent diapedesis in the absence of selectin-mediated rolling. Furthermore, recirculation of as few as 1\u2009\u00d7\u2009105 activated cells for 8\u201316\u00a0h is sufficient to allow a permissive endothelium to develop, allowing T cell adhesion and diapedesis. This compares with a minimum of 1\u00a0h for diapedesis when both leukocytes and endothelium are activated [138]. Interestingly, retinal antigens have been identified as having chemotactic properties for T cells via chemokine receptors, providing a mechanism for recruitment of lymphocytes to the retina in the absence of inflammation [68, 101]. Whether this occurs in vivo and contributes to induction of autoimmune inflammation remains to be proven but is consistent with the observation that migration of activated retinal antigen-specific T cells into the retina is not enhanced compared with non-specifically activated T cells [103].\nLeukocyte infiltration of the retina during inflammation\nVarious inflammatory conditions in the eye involve dysfunction of the BRB, and in addition to overt inflammation of the retina in diseases such as uveoretinitis, immune dysfunction leading to vascular permeability and angiogenic changes is linked to pathologies as diverse as retinopathy of prematurity, ARMD and diabetic retinopathy. In premature infants, where the retinal vasculature is underdeveloped, changes in oxygen level in the retina can signal release of vascular endothelial growth factor (VEGF) and development of abnormal vessels [21], and anti-VEGF therapies for ARMD, although possibly harmful to normal circulation, are widely used [88]. VEGF mediates increased vaso-permeability through p38 MAP kinase and ERK1\/ERK2-dependent mechanisms that alter occludin expression. VEGF activity, in turn, is regulated by eNOS-dependent dephosphorylation of the VEGF receptor-2, indicating close regulation of these pathways in normal retina [11, 14]. Decreased localisation of occludin at both endothelial and RPE cell borders has been shown to be linked to increased serine and threonine phosphorylation in occludin, which in turn reduces binding to ZO-1 and connection with the cytoskeleton [46].\nAngiogenesis and inflammation\nVEGF can be secreted by many retinal cell types under stress, including pericytes, astrocytes and RPE, and although not normally considered an inflammatory mediator, VEGF is chemotactic for monocytes, adhesion and migration through endothelium being mediated by increased integrin expression at sites of angiogenesis [64]. VEGF164, in particular, has been shown to induce inflammation and cellular immunity during pathological but not physiological ischaemia-induced sites of retinal angiogenesis [70]. Thus, pathologies involving VEGF are increasingly considered to have an inflammatory component. However, in the absence of adaptive immune responses to antigen in these non-infectious conditions, the inflammatory response is limited to the immediate vicinity by the naturally immunosuppressive ocular microenvironment, avoiding the mononuclear cell influx that characterises infectious or autoimmune inflammation [15, 120].\nDiabetic retinopathy caused by systemic changes in metabolic and cardiovascular control is a well-described complication of diabetes and also linked to ischaemic release of VEGF, leukostasis and BRB breakdown [69, 105]. Hyperglycemia and advanced glycation end products also promote pathology in a number of cell types including endothelium, neurons and glia. Thus, retinopathy may be considered a chronic inflammation involving all major cell types within the retina [52]. What first triggers retinal microvascular changes and breakdown of the BRB in diabetic retinopathy is unclear. In a rat model of diabetes, retinal vascular endothelial cells and RPE showed elevated levels of the matrix metalloproteinases (MMP) MMP-2, MMP-9 and MMP-14 with specific degradation of occludin [56]. Advanced glycation end products also increased leukostasis through NF\u03baB upregulation of integrins as early events [97].\nThere is also a direct relationship between the VEGF and prostaglandin-cyclo-oxygenase (COX-2) pathways. These pathways have vasoactive effects and interact with the nitric oxide synthase pathway (NOS), which in turn also increases vasodilation [57, 128]. Endothelial NOS (eNOS) is constitutively expressed by endothelium and is important for maintaining normal vascular physiology, in part by maintaining smooth muscle tone, but the inducible isoform (iNOS) has also been shown to have a predominant role in leukostasis and BRB breakdown in diabetic iNOS knock-out mice [80]. Expression of ICAM-1 and protein levels of all isoforms of NOS were increased in diabetic mouse retinas whilst occludin and ZO-1 protein decreased. These effects were prevented by the NOS inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME) and absent in the iNOS knock-out diabetic mouse.\nLeukocyte infiltration of the retina in uveoretinitis\nEndogenous posterior uveoretinitis (EPU) or more correctly, posterior intraocular inflammation, can take many clinical forms such as multifocal choroiditis, sympathetic ophthalmia and pars planitis. These clinical forms have features in common, including retinal vasculitis, macular edema, focal chorioretinal infiltrates and inflammatory cells and exudates in the vitreous, which develop as a result of BRB breakdown. EPU appears to be autoimmune- or immune-mediated rather than infectious in nature. However, it is becoming clear that infectious agents may be linked to autoimmunity through molecular mimicry, polyclonal activation and failure of bystander suppression [49].\nExperimental autoimmune uveoretinitis (EAU) is a Th1-type organ-specific autoimmune disease induced by immunisation with retinal antigens such as S antigen and interphotorecepter retinoid-binding protein (IRBP), at distant sites, in susceptible strains of rats or mice. It serves as an animal model of posterior intraocular inflammation, closely mimicking effects seen in the human situation [17, 50]. The BRB is breached in EAU, allowing leukocytes, both CD4+ T lymphocytes and macrophages to move into the retina, resulting in extensive tissue damage [38, 83]. Macrophages are thought to be the main effector cell responsible for tissue damage. EAU can also be induced by adoptive transfer of 5\u2009\u00d7\u2009105 retinal antigen-specific T cells [16, 17, 50, 103, 114, 122], and in particular, Th1 cells [18].\nMechanisms of leukocyte migration across the blood\u2013retina barrier in uveoretinitis\nAdhesion molecules involved in leukocyte\u2013endothelial cell interactions\nAppropriate adhesion molecule expression on both leukocytes and cells of the BRB is clearly important in enabling the migration of leukocytes into the inflamed retina.\nDuring the initial stages of the inflammatory reaction, the endothelium is activated by locally produced cytokines, TNF\u03b1 and IL-1\u03b2 in particular. The cytokines up-regulate adhesion molecules, particularly the selectins [108] and chemokines on the luminal surface of the endothelium. Leukocytes in the circulation then respond to the activated endothelium. In vitro studies have shown clear differences in adhesion molecule expression between human RPE and retinal endothelial cells [85]. ICAM is constitutively expressed at high levels on RPE cells, whereas endothelial cells expressed ICAM-1 only after induction with IFN\u03b3. Binding of CD4+ lymphocytes to RPE was dependent on ICAM-1, although after maximal stimulation, there was also an ICAM-1-independent component. Migration of T lymphocytes across retinal endothelial cells in the rat used both LFA-1-dependent and LFA-1-independent mechanisms [58, 61, 94]. Migration of T lymphocytes across unstimulated RPE was dependent on ICAM-1 and LFA-1, whereas after activation of RPE with IFN\u03b3, migration was dependent on both ICAM-1 and VCAM-1 [37].\nReceptors for various integrins and adhesion molecules are up-regulated on human RPE and vascular endothelial cells in vivo during ocular inflammation such as sympathetic ophthalmia. These include enhanced or de novo expression of ICAM-1, VCAM-1, CD62E and CD44, which in vitro, may be induced by the cytokines IL-1 and TNF-\u03b1. Kuppner et al. [79] compared adhesion molecule expression in acute and fibrotic sympathetic ophthalmia with normal human eyes. Expression of several integrins of the VLA family, which form receptors for extracellular matrix proteins, were found to be expressed by the RPE. However, VLA-4, the ligand for VCAM-1 and fibronectin, VLA-5 (fibronectin) and VLA-6 (laminin), which were increased on endothelium in the inflamed eyes, were not up-regulated on RPE cells. In contrast, ICAM-1 and CD44 expressions were greatly enhanced. Electron microscopy studies have also shown differences in the manner in which leukocytes cross the retinal endothelium and the RPE [40]. Inflammatory cell diapedesis through Bruch\u2019s membrane involved separation of its constitutive layers, migration through the pores in the membrane and between the RPE cells without apparent significant physical disruption of the RPE cell layer. Retinal vascular diapedesis, however, involved changes in post-capillary venules to high endothelial cell morphology, indicating endothelial cell activation [93], with migration appearing to be via the paracellular route [60]. As RPE cells fail to express iNOS during inflammation whilst endothelium does, [19, 67] it has been proposed that the RPE maintains a predominantly immunosuppressive role during inflammation [84].\nMechanisms of leukocyte subset migration into the retina\nTo study the role of adhesion molecules and leukocyte induced breakdown of the BRB, we have developed scanning laser ophthalmoscopy (SLO) methods for use in rodent EAU models. In this technique, syngeneic fluorescently labelled (eGFP CFSE or calcein AM) splenocytes, T helper cell subsets or monocytes are injected into the tail vein of normal or transgenic mice at various disease stages. These transferred cells can then be tracked in vivo in real-time and their interaction with the retinal vascular endothelium quantified in terms of rolling efficiency, rolling velocity and adhesion. These images can then be analysed to investigate the role of various molecules involved in leukocyte recruitment at the BRB [131]. This can be followed by confocal microscopy of retinal wholemounts, which allows both the quantification of cells entering the retina and further immunostaining to reveal changes in phenotype of endothelial cells and trafficking leukocytes.\nThe earliest changes observed are 24\u00a0h prior to detectable leukocyte infiltration. Vascular changes occur, which reduce shear stress in retinal veins and venules from approximately 30 to 20\u00a0dyn\/cm2. Although still considerably higher than in normal fenestrated endothelium, as shear stress falls, rolling and sticking efficiencies of leukocytes increase in the post-capillary venules, correlating with selective upregulation of P and E selectin and ICAM-1 on these vessels, particularly at sites of extravasation [137]. This effect is not observed in arteries or arterioles, although ICAM, VCAM, PECAM, E selectin, P selectin and CD44 are also up-regulated on other retinal endothelium as EAU progresses [135, 140]. As CD44 is widely expressed on most leukocyte subsets, it is required for leukocyte rolling under flow and is involved in early stages of extravasation in non-neuronal tissues [34, 35]. We investigated the role of CD44 in retinal inflammation in our model. Treatment of a mixed leukocyte population with neutralizing antibody against CD44 significantly suppressed the rolling of these cells on inflamed retinal venules and reduced infiltration into the retina. The importance of CD44 in leukocyte recruitment in EAU was also supported by the fact that EAU severity was diminished by administration of the anti-CD44 antibody at the stage of disease in which leukocytes are first seen to infiltrate the retina [140].\nBoth Th1 cells and Th2 subsets of T lymphocyte are implicated in the pathogenesis of uveitis [10, 27]. So to investigate the role of adhesion molecules in the trafficking of different T cell subsets across the BRB in Th1-mediated EAU, naive CD4+ T lymphocytes were isolated from lymph node cells and polarised in vitro into Th1- and Th2-like cells. Surface PSGL-1 and LFA-1 were up-regulated on both populations but expressed at higher levels on Th1-like cells, whereas CD44 expression was up-regulated to a greater extent on Th2 cells. Pretreatment of the polarised T lymphocyte populations with anti-PSGL-1 inhibited rolling and infiltration of Th1-like cells but not Th2, providing direct in vivo evidence for the inability of Th2 to respond to P\/E selectin despite increased expression of PSGL-1. Anti-LFA-1 pretreatment inhibited infiltration of both Th1- and Th2-like cells, but this inhibition was more substantial for the Th-1 cells. Thus, preferential recruitment of Th1 cells in EAU is mediated by PSGL-1:P\/E selectin, whereas non-selective trafficking of activated T cells (both Th1 and Th2) across the BRB is mediated by CD44:CD44r and LFA-1:ICAM-1 [137].\nMonocytes are also necessary for full expression of disease in EAU. We have established a model for studying bone-marrow-derived monocyte trafficking in vivo, and found that, whereas T cells, whether antigen specific or not, roll on inflamed endothelium and rapidly migrate into the retina, monocytes can only traffic into the inflammatory site once they have acquired a specific phenotype [136]. This phenotype was consistent with the CCR2+ phenotype described by others but was also found to be a constitutive time-limited property of the transferred monocytes and independent of an existing inflammation. Monocytes purified from bone marrow failed to roll on inflamed endothelium until they had undergone in vivo recirculation for at least 24\u00a0h, with maximum retinal infiltration not occurring until 48-h post-transfer. Of note was the ability of transferred monocytes to undergo further differentiation during trafficking into the retina. Transferred cells matured into classical tissue macrophages or CD11c+ dendritic cells that persisted within the retina for several days. Several striking contrasts between inflammatory monocyte and Th1 cell trafficking were observed. Monocyte rolling in inflamed venules was faster and CD62-L dependent rather than LFA-1 dependent. While PSGL-1 was found to play a role in regulating diapedesis to the inflammatory site, CD62-L was shown to have a key role in regulating recruitment of monocytes to the lymphoid tissue from the circulation during inflammation (manuscript submitted for publication). These observations reveal that different molecular mechanisms are involved in leukocyte subset adhesion to endothelium and migration into the tissues.\nMechanisms of blood\u2013retina barrier breakdown in EAU\nWhether BRB breakdown is necessary before lymphocytes and monocytes can infiltrate or whether infiltration results in BRB breakdown and free movement of leukocytes between the circulation and the retina is controversial [60, 86]. Blood flow within the CNS and, by extension the retina, is regulated by interactions between neurons, glia and the microvasculature, contact between the microvessel wall and perivascular endfeet of astrocytes, mediated by agrin, being important for maintaining barrier function in the brain [1]. Other cellular components include pericytes and perivascular macrophages, contributing to what has been termed the \u201cneurovascular unit\u201d. Astrocytes can secrete a range of cytokines and chemical mediators that up-regulate TJ and polarised expression of transporters and other enzyme systems that control transendothelial transport of molecules and ions to maintain a metabolic barrier [62]. In BBB disruption, agrin is lost from the abluminal surface of the endothelial cells adjacent to the astrocytic endfeet and contact lost. In the brain, bradykinin activation of astrocytes has been implicated in BBB breakdown in stroke or trauma, but the molecular signals responsible for BRB breakdown in autoimmune inflammation are still not understood [2].\nUsing our in vivo cell trafficking model, we have studied the relationship between changes in vascular permeability, adhesion molecule expression and leukocyte migration into the retina. As described above, circulation of activated lymphocytes induces changes in the endothelium of the retinal venules, allowing transient breakdown of the BRB. Where T cells are non-antigen specific, this occurs in the absence of cell rolling, without any reduction in shear stress and without generalised inflammation of the retina occurring. In contrast, in EAU where retinal autoreactive T cells are present, cell rolling occurs, and extravasation of T cells in the venules (but not arterioles) occurs together with BRB breakdown in these venules as evidenced by dye leakage and an influx of inflammatory cells [135].\nBRB breakdown involves changes in levels of expression and localisation of TJ proteins occludin-1, claudins and ZO-1 [116]. To elucidate mechanisms of BRB and its regulation by inflammatory cells and mediators in vivo, we examined wholemount retinas and choroids from normal and EAU mice. Using confocal microscopy, we showed that in normal retina, TJ proteins were evenly distributed along endothelial cell margins with increased expression in tri-cellular corners, hypothesised to be a \u201cweak spot\u201d in the TJ network. In EAU, disruption of TJ proteins only occurred in the venules where leukocyte infiltration was occurring. Western blot analysis confirmed that claudin 1\/3 and occludin proteins were reduced. ZO-1 protein expression was not reduced but redistributed from the cell membrane and the TJs to the cytoplasm [133]. Examination of venules in retinas in early EAU (with few infiltrating cells) showed that loss of occludin-1 from venule TJ occurred at the point of contact with adherent leukocytes undergoing transendothelial migration. In the absence of other migrating inflammatory cells, TJ appeared to reform. In contrast, in areas of the retina where there was significant inflammatory cell infiltration, confocal microscopy revealed that occludin-1 was lost from venules but not capillaries and arterioles. This correlated with loss of astrocyte foot process contact with the endothelium of the venule [133]. These studies are consistent with a major role for venule endothelium in leukocyte recruitment to the retina and imply that breakdown of the BRB in uveitis is an active event triggered by adherent leukocytes rather than a passive event driven solely by local cytokine microenvironment.\nThe role of chemokines in leukocyte migration across the BRB in EAU\nChemokines are vital for the development of a focussed immune response, and certain chemokines are associated with inflammatory responses and the attraction of inflammatory cells to the site of infection or injury [91, 111]. Chemokines can be produced by both resident cells at the inflammatory site, such as epithelial cells, endothelial cells, fibroblasts and resident macrophages and dendritic cells as well as early infiltrating macrophages and neutrophils and then at later stages, infiltrating T cells. Chemokine production may be via toll-like receptors [36, 71, 115] or via inflammatory cytokines such as IL-1, TNF\u03b1 and IFN\u03b3, in the case of a Th1-type response or IL-4, IL-5 and IL-13 in the case of a Th2-type response [8, 107, 109]. Thus, the inflammatory stimulus will generate a chemokine \u201cfingerprint\u201d, which will be the basis for determining the tailored leukocyte response [22, 111].\nProduction of chemokines is up-regulated in the murine eye in EAU and generally shows, as expected, a Th1-like pattern of expression; for example, CCL2, CCL3 and CCL5 [28]. CCL3 and CCL2 were associated with retinal and choroidal vessels as well as with infiltrating cells, whereas CCL5 was associated predominantly with infiltrating cells [28]. RT-PCR analysis found expression of mRNA transcripts for these and several other chemokines including CXCL9, CXCL10 CCL6, CCL9, CCL19 and CCL22 is also up-regulated, although some of these studies were carried out with whole eyes [31, 51]. Recipients receiving Th1 cells had a similar pattern of expression [51]. Laser capture microdissection showed that both RPE and infiltrating leukocytes expressed chemokine transcripts in distinct but overlapping patterns [51]. In vitro, human cells of the BRB, both RPE and retinal microvascular endothelial cells respond to pro-inflammatory cytokines IL-1\u03b2, TNF\u03b1 and IFN\u03b3 by producing substantial levels of CCL2, CCL5, CXCL8, CXCL10 and CXCL1 [29, 41, 42]. Receptors for these chemokines are also up-regulated in EAU. Expression of mRNA transcripts for CCR1, CCR5, CXCR3, CXCR2, CCR6, CCR8 and CCR2 was detected in whole eyes [51], and cell surface expression of CCR5, CXCR3 and CCR2 on infiltrating cells was detected by immunohistochemistry (pers. comm. IJC).\nWe have investigated the importance of CCL3 in recruitment of leukocytes in vivo at the inflamed BRB using SLO. We showed that CCL3, a ligand for CCR5, was involved in leukocyte recruitment at the BRB and was linked to the inflammatory process and disease pathogenesis in EAU [30]. The effect of short-term anti-CCL3 treatment was examined by injecting anti-CCL3 antibody into mice with EAU 1\u00a0h prior to tracking a labelled population of activated leukocytes (splenocytes from mice with EAU) in real-time using SLO. This treatment inhibited leukocyte slowing and accumulation and subsequent extravasation of leukocytes at the blood\u2013retina barrier. This was effective predominantly in the post-capillary venules, which have been shown to be the main site of passage of leukocytes across the BRB. Long-term anti-CCL3 treatment also prevented decreased leukocyte velocity and reduced disease severity as measured clinically, histologically and in terms of BRB breakdown [30].\nTo define this further, we examined the trafficking of Th1-like cells, polarised in vitro. These cells, which expressed high levels of CCR5, were labelled and adoptively transferred and their trafficking monitored in vivo at an early disease stage in EAU using SLO. Treatment of the cells with antibody against CCR5 prior to transfer resulted in a reduction in their infiltration into the retina. However, rolling velocity, rolling efficiency and adherence of the cells to retinal endothelium was not reduced. CCR5 is clearly important for Th1 cell recruitment at the BRB and acts at the level of transendothelial migration rather than at the earlier stage of rolling on the endothelium [31]. This is consistent with other work on the arrest of monocytes or Th-1-like T cells by CCL5 (RANTES) immobilised to activated endothelium under flow conditions. Arrest was mediated predominantly by CCR1, whereas CCR5 was responsible for spreading. This was irrespective of the degree of expression of the receptors on the different cell types [127].\nInterestingly, studies in which CCR1\/CCR5 was blocked in EAU have emphasised the fact that the action of a chemokine and its receptor in cell recruitment at the BRB may differ dramatically depending on the antigen used to induce uveitis and the stage of the disease and the resulting microenvironment. In Lewis rats in which EAU was induced with S-antigen peptide, treatment with the CCR1\/CCR5 receptor antagonist Met-RANTES was effective in reducing uveitis in the efferent phase, possibly inhibiting migration of activated T cells or monocytes into the retina [39]. When EAU was induced with IRBP peptide, however, Met-RANTES appeared to affect activation of T cells, reducing intraocular inflammation if administered early in the initiation phase of the response but moderately enhancing uveitis if given during the efferent phase [39]. Antibody against RANTES given during the efferent phase in C57BL\/6 mice immunised with IRBP peptide also led to exacerbation of EAU, and this was attributed to a change in the ratio of T cell subsets recruited favouring CD4 over CD8 T cells [117].\nMonocyte trafficking across the BRB has also been examined, and in particular, the role of two chemokine receptors which have been shown to be involved in monocyte recruitment, CCR2 and CX3CR1. This type of experiment is constrained by the number of monocytes, which can be isolated from the blood. However, when in vitro-cultured bone marrow monocytes, non-activated peritoneal monocytes and freshly isolated bone marrow monocyte precursors were compared, only the latter precursors continued to circulate and trafficked efficiently to the inflamed retina in EAU [136]. These were, therefore, labelled and used to investigate trafficking in EAU, and interestingly, it was found that these cells could not initially migrate across the BRB and required 24\u201348\u00a0h in vivo before they started to infiltrate the inflamed retina as described above. This coincided with an increase in expression of CCR2 on these cells. This was only a transient increase, and CCR2 expression and the migratory phenotype were lost 72\u00a0h after adoptive transfer [136]. However, evidence from further studies indicates that although CCR2 may improve the efficiency of monocyte transendothelial migration at the inflamed BRB, it is not essential for monocyte emigration at this site (manuscript in preparation).\nThis would confirm findings from other inflammatory sites in which it has been shown that although CCR2-positive monocytes may be recruited preferentially to an inflammatory site, CCR2-negative monocytes are also able to traffic to these sites [106, 112, 123]. CCR2 also has a role in facilitating the egress of monocytes from the bone marrow, with more monocytes present in the bone marrow in CCR2\u2212\/\u2212 mice and fewer circulating even before infection [112], but its role in monocyte recruitment to an inflammatory site is, in addition to this, and may be mediated by CCL7 (MCP-3) and CCL2 (MCP-1) [125]. In some inflammatory situations, CCR2 is clearly more critical for monocyte recruitment than we show at the BRB; for example, for recruitment into the peritoneum in response to thioglycolate [125]. Thus, reliance on CCR2 for recruitment of monocytes may vary depending on the particular inflammatory stimulus and the microenvironment that is generated.\nCX3CR1 has also been implicated in the trafficking of monocytes, and although it has been proposed to be important for immune surveillance [55], there are also reports that it has a role in recruitment in an inflammatory situation; for example, in atherosclerosis [25, 81], crescentic glomerulonephritis [47] and in cerebral ischaemia [118]. It is possible that this receptor is important for the recruitment of a subpopulation of inflammatory CCR2-negative monocytes. However, recently, it has been shown in an atherosclerosis model that although CX3CR1-positive monocytes can enter atherosclerotic plaques, CX3CR1 was not necessary for this entry. In contrast, CCR2-positive monocytes, which also express substantial levels of CX3CR1 [55], required CX3CR1 in addition to CCR2 for the accumulation of monocytes in these lesions [123]. In EAU, our studies to date indicate that CX3CR1 is not essential for the trafficking of monocytes into the inflamed retina (manuscript in preparation).\nConclusions and implications for design of novel therapies\nCurrent treatment for uveitis is still dependent in the most part on systemic, non-specific immunosuppression. Additional therapies such as cyclosporin A, tacrolimus and rapamycin may act by interfering with the production and action of cytokines, particularly interleukin-2, targeting T cell function. Mycophenolate mofetil, an antimetabolite affecting purine synthesis, is also valuable. Other immunosuppressants such as azathioprine, cyclophosphamide and chlorambucil have severe side effects restricting their use. Blockade of TNF-\u03b1 is also effective at least in the short term and now used clinically in different forms. Despite the usefulness of these therapies, they are all relatively non-specific, and in recent years, the quest for specific, tailored therapies for uveitis has been paramount.\nThe ability of leukocytes to migrate across the BRB depends on many different factors, the importance of which will vary depending on the leukocyte subset, the specific site and the microenvironment created as a result of the inflammatory stimulus and genetic background. Therapies designed to block the passage of cells into the inflamed retina will, therefore, need to be tailored to the situation to increase their potential for success. Understanding in depth the stages in the process of leukocyte migration across the BRB will be fundamental for this. It is clear that there is a degree of redundancy in adhesion molecule use as well as in cytokine, chemokine and integrin signalling, and different inflammatory cell subsets will require different strategies to block their recruitment and effector functions. However, some therapies targeting key intracellular signalling pathways may be beneficial. For instance, lymphocyte trafficking is dependent upon endothelial cell G-protein signalling, and recently, lovastatin has been shown to reduce retinal disease in the mouse model of EAU. Lovastatin inhibited the synthesis of precursors required for prenylation and post-translational activation of endothelial Rho GTPase, an essential step in ICAM-1-mediated leukocyte migration [3, 54]. Although different statins may have different effects in different diseases, they are proven safe drugs and provide attractive therapeutic options.\nSome therapies that target specific aspects of leukocyte migration are already in use and in clinical trials in some inflammatory diseases. Although targeting some adhesion molecules has not always proved successful [100, 141], there are some promising results. These include natalizumab, directed against VLA-4, which is in use for MS and Crohn\u2019s despite a low risk of progressive multifocal leukoencephalopathy, a viral infection of the CNS [73]; and efalizumab, a recombinant humanised antibody, which binds to LFA-1 preventing binding to ICAM-1, is in use for the treatment of psoriasis [75]. The possibility of targetting chemokines and their receptors to prevent recruitment of inflammatory cells has also received much attention from pharmaceutical companies. For example, a number of compounds with anti-CCR2 activity have been developed and patented with use planned in multiple sclerosis and atherosclerosis [48]. An antagonist of CCR5 (maraviroc) is also available, which has been designed for use in HIV [43]. These drugs may have potential use in inflammatory conditions involving the BRB.","keyphrases":["leukocytes","blood\u2013retina barrier","inflammation","tight junctions","selectins","integrins","chemokines","monocytes","lymphocytes","uveitis","retinal pigment epithelium"],"prmu":["P","P","P","P","P","P","P","P","P","P","M"]}
{"id":"Cancer_Immunol_Immunother-3-1-2082063","title":"Association of antigen processing machinery and HLA class I defects with clinicopathological outcome in cervical carcinoma\n","text":"HLA class I loss is a significant mechanism of immune evasion by cervical carcinoma, interfering with the development of immunotherapies and cancer vaccines. We report the systematic investigation of HLA class I and antigen processing machinery component expression and association with clinical outcome. A tissue microarray containing carcinoma lesions from 109 cervical carcinoma patients was stained for HLA class I heavy chains, \u03b22-microglobulin, LMP2, LMP7, LMP10, TAP1, TAP2, ERAP1, tapasin, calreticulin, calnexin and ERp57. A novel staining evaluation method was used to ensure optimal accuracy and reliability of expression data, which were correlated with known clinicopathological parameters. Partial HLA class I loss was significantly associated with decreased 5-years overall survival (61% vs. 83% for normal expression; P < 0.05) and was associated with decreased 5-years disease-free survival (DFS) (65% vs. 82% for normal expression; P = 0.05). All APM components except LMP10, calnexin and calreticulin were down-regulated in a substantial number of cases and, except ERAP1, correlated significantly with HLA class I down-regulation. LMP7, TAP1 and ERAP1 loss was significantly associated with decreased overall and (except LMP7) DFS (P < 0.05 and 0.005, respectively). ERAP1 down-regulation was an independent predictor for worse overall and DFS in multivariate analysis (HR 3.08; P < 0.05 and HR 2.84; P < 0.05, respectively). HLA class I and APM component down-regulation occur frequently in cervical carcinoma, while peptide repertoire alterations due to ERAP1 loss are a major contributing factor to tumour progression and mortality.\nIntroduction\nTumour cells utilize several mechanisms to escape immune-mediated recognition and destruction [1]. Loss of surface-expressed human leukocyte antigen (HLA) class I molecules is particularly important, as this enables tumour cells to evade recognition and lysis by cytotoxic T-lymphocytes [2\u20135]. These molecules consist of a glycoprotein heavy chain (encoded by genes within the HLA regions of chromosome 6p) and a \u03b22-microglobulin (\u03b22-m) light chain (encoded on chromosome 15q) [6, 7].\nDefects in HLA class I expression are caused by multiple mechanisms, including loss of heterozygosity at chromosome 6p; \u03b22-m or HLA class I mutations; and defective expression and\/or function of components of the antigen processing machinery (APM) [8\u201310]. The APM is the combination of cellular processes responsible for the presentation of endogenous peptides by HLA class I molecules. These peptides are generated by specialized immunoproteasomes (composed of LMP2, LMP7 and LMP10 subunits); and transported from the cytoplasm to the endoplasmic reticulum by TAP (transporter associated with antigen presentation) which is composed of TAP1 and TAP2 subunits. Transported peptides undergo length-specific trimming by ERAP1 (endoplasmic reticulum aminopeptidase associated with antigen presentation 1) and are loaded onto empty HLA class I molecules (with involvement of tapasin and the chaperone molecules calnexin, calreticulin and ERp57) [11\u201314].\nAlthough various in vitro studies have demonstrated partial or total APM component loss in human malignancies, the extent to which this occurs in vivo is largely unknown. Two recent reports describe down-regulation of several APM components in head and neck squamous cell carcinoma and astrocytoma, and demonstrate an association of these defects with prognosis [15, 16]. As HLA class I-restricted processing and presentation of endogenous peptides is crucial to the generation of a virus-specific cytotoxic T-lymphocyte-mediated reaction, APM defects are assumed to contribute significantly to virus-mediated carcinogenesis. Several studies have confirmed interference in various APM processes by viral peptides [17]. However, the extent to which defective expression of APM components contributes to HPV-induced cervical carcinogenesis remains to be established. One study has assessed the expression of various APM components, although only at the mRNA level [18]. Other studies of APM component expression in cervical carcinoma were largely limited to TAP1 and TAP2 and encompassed relatively small sample sizes. Moreover, the expression data yielded by these studies are conflicting, with TAP1 loss being reported in 0\u201350% of cases [18\u201320]. Though TAP1 and HLA class I down-regulation has occasionally been shown to be associated with progressive malignant transformation [21\u201324], a comprehensive study of the association of APM defects with prognostic parameters has yet to be performed.\nSince information regarding defective APM component expression in cervical cancer can contribute to the development of T cell-based immunotherapies, we have investigated the expression of all known APM components in a large number of cervical cancer specimens using a tissue microarray (TMA)-based approach [25, 26]. Additionally, we demonstrate for the first time a negative association of APM defects with prognosis and other clinicopathological variables.\nMaterials and methods\nClinical characteristics and tissue samples\nFrom 109 patients with cervical carcinoma who underwent radical hysterectomy with bilateral pelvic lymphadenectomy (by the same surgical team) between 1985 and 1999, formalin-fixed, paraffin-embedded tissue blocks were retrieved from the archives of the Department of Pathology, Leiden University Medical Centre, The Netherlands. Twenty-six adenocarcinomas and 83 squamous carcinomas were obtained. These cases were not consecutive, but chosen on the basis of availability of sufficient material. The use of clinical material was approved by the institutional review board according to the guidelines of the Dutch Federation of Medical Research Associations.\nAll patients were inhabitants of The Netherlands and had not received pre-operative radiotherapy or chemotherapy. Mean age was 48.5\u00a0years, the youngest patient being 24\u00a0years and the oldest 87\u00a0years at the time of surgery. Follow-up of these patients until 2005 provided information concerning recurrence rate and performance state.\nTissue microarray construction\nThe archival slides for all the cases were reviewed; a slide containing representative tumour was selected, and an area of tumour was encircled on the slide. The corresponding tissue blocks were recovered from the archives and the selected area on the slide was circled on the block for tissue microarray construction. Using a manual tissue microarrayer (Beecher Instruments, Silver Spring, MD, USA), the area of interest in the donor block was cored with a 0.6-mm diameter needle and transferred to a recipient paraffin block. The microarray was constructed with a threefold redundancy (three spots for each patient) to increase accuracy. The finalized arrays were then cut into 3\u00a0\u03bcm-thick sections and mounted on glass slides using an adhesive tape-transfer system (Instrumedics Inc., Hackensack, NJ, USA) with ultraviolet cross-linking.\nAntibodies\nThe mouse monoclonal antibody (mAb) HCA2 which recognizes a determinant expressed on \u03b22-m-free HLA-A (excluding HLA-A24), HLA-B7301 and HLA-G heavy chains [27, 28], the mAb HC10 which recognizes a determinant on all \u03b22-m-free HLA-B and HLA-C heavy chains and on \u03b22-m-free HLA-A10, -A28, -A29, -A30, -A31, -A32 and -A33 heavy chains [27, 29], the LMP2-specific mAb SY1 [30], the LMP7-specific mAb HB-2 [30], the LMP10-specific mAb TO-7 [30], the TAP1-specific mAb NOB-1 [31], the TAP2-specific mAb NOB-2 [31], the calnexin-specific mAb TO-5 [32], the calreticulin-specific mAb TO-11 [32], the Erp57-specific mAb TO-2 [32] and the tapasin-specific mAb TO-3 [32] were developed and characterized as described.\nThe rabbit polyclonal anti-\u03b22-m-antibody A-139 was purchased from DAKO, Copenhagen, Denmark. The rabbit polyclonal anti-ERAP1 antibody has been described previously and was kindly provided by Dr. M. Tsujimoto, RIKEN, Wako, Saitama, Japan [33\u201335].\nImmunohistochemical staining\nThe slides were dried for 16\u00a0h at 56\u00b0C before being dewaxed with standard xylene and rehydrated through graded alcohols into water. Standard immunohistochemical staining was performed as described previously [36]. Stromal cells, including infiltrating leukocytes, were used as internal positive controls. Negative control sections of each specimen were processed with omission of the primary antibodies.\nEvaluation of TMA immunostaining\nTwo researchers (A.M. and E.J.) evaluated all patient cores, and scores were averaged to produce a single evaluation with the scorers being blind to the source of the samples. When slides were scored differently, which occurred rarely (<5%), a consensus was reached by simultaneous evaluation. Staining was scored semi-quantitatively by the quality control system proposed by Ruiter et al. [37]. The intensity and percentage of positive cells in the tumour and the stromal cells surrounding the tumour were determined. The intensity of staining was scored as 0, 1, 2, or 3 indicating absent, weak, clear, or strong expression, respectively. The percentage of positive cells was scored as 0 for 0%; 1 for 1\u20135%; 2 for 5\u201325%; 3 for 25\u201350%; 4 for 50\u201375% and 5 for 75\u2013100%. The sum of both scores was used to identify three categories of expression: normal expression (total score 7\u20138), partial loss (3\u20136) and total loss (0\u20132). Immunohistochemical staining demonstrated strong positive expression of all examined markers in stromal tissue and tumour-infiltrating inflammatory cells, thereby providing an internal positive control.\nStatistical analysis\nThe Chi-square test or, where appropriate, Fisher\u2019s exact test was used for evaluation of associations between expression and clinicopathological parameters. All statistical analyses were performed with the SPSS Version 12 software package. Five-year survival rates were calculated and survival rates were compared according to the Kaplan\u2013Meier method using the log rank test, while multivariate analysis of survival was performed according to Cox proportional hazard models. Overall survival (OS) was defined as survival till death due to cervical carcinoma (the two patients that dyed from other causes were excluded), i.e. disease-specific survival, while disease-free survival (DFS) was defined as time to disease recurrence, metastasis, or disease-specific death. All tests were two-sided and the significance level was set to 5%.\nResults\nClinical features\nOf the total group of 109 patients, 52 were diagnosed as FIGO stage IB1, 30 as IB2, 21 as FIGO IIa and 2 as FIGO IIb. For four patients no FIGO staging was available. Fifty-five patients (50%) received post-operative radiotherapy due to narrow tumour-free margins of excision, lymph node metastases, vaso-invasive growth, parametrial infiltration, depth of infiltration exceeding 15\u00a0mm and\/or tumour size exceeding 4\u00a0cm. Twenty-seven patients had lymph node metastases. Fifty-one patients (58%) were HPV16-positive, 21 (24%) were HPV18-positive, while the remaining patients were positive for HPV31, HPV33, HPV45, HPV52 or HPV68. At the end of follow-up, 80 patients were alive, 2 suffered from recurrent disease, 3 had a metastasis, 22 had died of the disease and 2 had died of causes unrelated to the primary disease. The median follow-up time was 45\u00a0months.\nHLA class I and APM component expression\nRepresentative staining patterns for the HLA class I heavy chains and various APM components are shown in Figs.\u00a01 and 2. Expression of the HLA class I heavy chains and \u03b22-m is summarized in Table\u00a01. Of the 109 cases, eight exhibited total \u03b22-m loss; correspondingly, these cases were also negative for HLA heavy chain cell surface expression. As HLA class I expression, and therefore tumour immunogenicity, is not affected by the APM in a \u03b22-m-negative background these eight cases were excluded from further analysis of APM effects. Furthermore, as HLA class I expression is regulated by the APM in a \u03b22-m-independent manner, the overall HLA class I expression score was based on expression of the heavy chains. If either or all the heavy chains were expressed normally, overall HLA class I expression was considered to be normal. Total loss of all heavy chains was counted as total HLA class I loss, and all other cases were counted as partial loss.\nFig.\u00a01Immunohistochemical analysis. Staining patterns of a\u2013c HLA-A, d\u2013f HLA-B\/-C, g\u2013i LMP7 and j\u2013l TAP1 showing a, d, g, j normal expression, b, e, h, k partial loss of expression and c, f, i, l total loss of expressionFig.\u00a02Immunohistochemical staining patterns of ERAP1. a Normal expression. b Partial loss of expressionTable\u00a01Expression of HLA class I heavy chains, \u03b22-microglobulin and overall HLA class IHLA-AHLA-B\/-C\u03b22-microglobulinHLA class ITotal lossPartial lossNormal expressionTotal lossPartial lossNormal expressionTotal lossPartial lossNormal expressionTotal lossPartial lossNormal expressionOverall (n\u00a0=\u00a0109)36 (33)22 (20)51 (47)24 (22)29 (27)56 (51)8 (7)28 (26)73 (67)21 (19)24 (22)63 (59)Tumour type\u00a0AC (n\u00a0=\u00a026)6 (23)5 (19)15 (58)4 (15)8 (31)14 (54)1 (4)7 (27)18 (69)4 (15)5 (19)17 (66)\u00a0SCC (n\u00a0=\u00a083)30 (36)17 (21)36 (43)20 (24)21 (25)42 (51)7 (8)21 (25)55 (67)17 (21)19 (23)46 (56)Data shown as n (%)\nAs shown in Table\u00a02, expression of all APM components except LMP10, calnexin, and calreticulin was lost in some of the cases. This down-regulation was significantly associated with HLA class I down-regulation for all components, except ERAP1. Moreover, defects in overall TAP expression (defined as partial or total loss of either or both of the TAP subunits) were significantly associated with HLA class I down-regulation (P\u00a0<\u00a00.001). Down-regulation of any APM component was significantly associated with HLA class I down-regulation (P\u00a0<\u00a00.001).\nTable\u00a02Overall and histological type-specific expression of APM componentsOverall (n\u00a0=\u00a0101)AC (n\u00a0=\u00a025)SCC (n\u00a0=\u00a076)Total lossPartial lossNormal expressionPTotal lossPartial lossNormal expressionPTotal lossPartial lossNormal expressionPLMP214 (14)30 (30)57 (56)0.0145 (20)11 (44)9 (36)0.7729 (12)19 (25)48 (63)0.001LMP73 (3)16 (16)82 (81)<0.0012 (8)9 (36)14 (56)0.1001 (1)7 (9)68 (90)<0.001LMP1000101 (100)TAP14 (4)19 (19)78 (77)<0.0010 (0)4 (16)21 (84)0.2304 (5)15 (20)57 (75)0.001TAP211 (11)26 (26)64 (63)<0.0014 (16)6 (24)15 (60)0.0057 (9)20 (26)49 (65)<0.001ERAP1014 (15)80 (85)0.3140 (0)2 (9)20 (91)0.582012 (16)60 (84)0.307Tapasin1 (1)24 (24)76 (75)<0.0010 (0)10 (40)15 (60)0.2841 (1)14 (18)61 (81)<0.001Calnexin00101 (100)ERp5712 (12)40 (40)49 (48)0.0171 (4)6 (24)18 (72)0.61511 (15)34 (45)31 (40)0.002Calreticulin00101 (100)Data shown as n (%). P values are shown for association with overall HLA class I down-regulation\nAssociation with histopathological parameters\nDepth of invasion exceeding 15\u00a0mm and vaso-invasive growth are unfavourable prognostic indicators. Of the cases examined, 51 (45%) demonstrated depth of invasion greater than 15\u00a0mm and 38 (33%) demonstrated vaso-invasive growth. Total loss of overall HLA class I expression and of any APM component was found to be significantly associated with depth of invasion exceeding 15\u00a0mm (P\u00a0=\u00a00.034 and 0.045, respectively), while no significant association was found for defective expression of any marker and vaso-invasion.\nNone of the markers was found to be significantly associated with FIGO stage, corresponding to the lack of a significant association with histopathological parameters. No association of any of the markers with specific HPV status of the patients was detected. Interestingly, partial LMP2 and\/or LMP7 loss was significantly associated with an absence of detectable lymph node metastases (P\u00a0=\u00a00.023 and 0.021, respectively).\nAssociation with overall and disease-free survival\nAs shown in Table\u00a03 and Figs.\u00a03 and 4, partial loss of overall HLA class I expression was significantly associated with decreased OS, and exhibited a trend towards a significant association with DFS, whereas total loss was not significantly associated with survival. Moreover, partial HLA-A loss (evaluated by HCA2 reactivity) was significantly associated with decreased OS and DFS, whereas total loss was not significantly associated with survival. Partial, but not total HLA-B\/-C loss (assessed by HC10 reactivity) showed a trend towards significant association with decreased survival.\nTable\u00a03HLA class I and APM component expression and 5-years survival ratesOverall survivalDisease-free survivalNormal expressionPartial lossTotal lossNormal expressionPartial lossTotal lossHLA-A42 (83)12 (55)29 (81)42 (83)13 (60)28 (77)HLA-B\/-C45 (81)19 (64)18 (75)46 (82)19 (67)18 (73)HLA class I52 (83)15 (61)16 (77)52 (82)16 (65)15 (75)LMP724 (74)17 (87)1 (33)62 (76)13 (80)2 (50)TAP161 (78)8 (70)1 (33)63 (81)13 (67)1 (25)ERAP166 (82)5 (38)\u201365 (81)7 (50)\u2013Data shown as n (%)Fig.\u00a03Kaplan\u2013Meier curves for overall survival. Patients with normal expression, partial loss, and total loss of a HLA-A, b HLA-B\/-C, c overall HLA class I, d LMP7, e TAP1 and f ERAP1 expression. P values are shown for comparison with normal expressionFig.\u00a04Kaplan\u2013Meier curves for disease-free survival. Patients with normal expression, partial loss, and total loss of a HLA-A, b HLA-B\/-C, c overall HLA class I, d LMP7, e TAP1 and f ERAP1 expression. P values are shown for comparison with normal expression\nDecreased OS and DFS was significantly associated with total TAP1 loss, but not with normal expression and partial loss. A similar pattern was observed for partial LMP7 loss, though this was not significantly associated with DFS. ERAP1 loss was significantly associated with decreased OS and DFS as compared to normal ERAP1 expression.\nMultivariate analysis\nAs expected, well known prognostic parameters such as depth of infiltration exceeding 15\u00a0mm, presence of vaso-invasive growth, and presence of lymph node metastases were significantly associated with a shorter survival, while FIGO-stages of IB2 or more showed a significant association with shorter DFS (Table\u00a04). HPV type was not significantly associated with survival (data not shown). Subsequently, multivariate analysis was performed on overall HLA class I, TAP1, LMP7 (for OS only) and ERAP1 expression, combined with the aforementioned prognostic factors. ERAP1 down-regulation was demonstrated to be an independent prognostic parameter for shorter OS (hazard ratio (HR) 3.08; 95% CI 1.07\u20138.90; P\u00a0=\u00a00.037) and shorter DFS (HR 2.84; 95% CI 1.01\u20138.03; P\u00a0=\u00a00.049), as was presence of lymph node metastases (HR 7.02; 95% CI 2.55\u201319.32; P\u00a0<\u00a00.001 for OS and HR 6.64; 95% CI 2.53\u201317.44; P\u00a0<\u00a00.001 for DFS). Total TAP1 loss was also an independent predictor for shorter DFS (HR 6.72; 95% CI 1.29\u201335.06; P\u00a0=\u00a00.024).\nTable\u00a04Histopathological parameters and 5-years survival ratesOverall survivalDisease-free survival%P%PDepth of infiltration\u00a0<15\u00a0mm (n\u00a0=\u00a059)8890\u00a0\u226515\u00a0mm (n\u00a0=\u00a051)60<0.00157<0.001Vaso-invasion\u00a0Absent (n\u00a0=\u00a068)8185\u00a0Present (n\u00a0=\u00a038)590.031540.002Lymph nodes\u00a0Negative (n\u00a0=\u00a080)8686\u00a0Positive (n\u00a0=\u00a027)43<0.00139<0.001Histopathological class\u00a0SCC (n\u00a0=\u00a084)7576\u00a0AC (n\u00a0=\u00a026)770.924740.662FIGO stage\u00a0\u2264IB1 (n\u00a0=\u00a052)8291\u00a0\u2265IB2 (n\u00a0=\u00a053)690.060570.002\nDiscussion\nIn the present study we have investigated HLA class I and APM component expression in cervical carcinoma patients and have evaluated the relationship of these factors with survival and other clinicopathological parameters.\nOur results show that HLA class I down-regulation occurs frequently (\u223c40% of cases) in cervical carcinoma, corresponding to previous reports [19, 21]. Synchronous partial or total loss of various APM components was found in a substantial number of cases and was demonstrated to be significantly associated with HLA class I down-regulation. We observed partial HLA class I loss to be significantly associated with decreased OS. Of the APM components, TAP1, LMP7 and ERAP1 down-regulation exhibited significant associations with decreased survival. ERAP1 down-regulation was shown to be an independent, significant predictor of shorter survival.\nThough defective APM component expression is hypothesized to be an important mechanism of HLA class I down-regulation and therefore immune evasion by tumour cells, information regarding APM down-regulation in carcinomas is limited. This is partly due to the scarcity of reliable antibodies for most APM components other than the TAP subunits. Furthermore, most previous studies of APM expression in cervical cancer were limited by small sample sizes. Lastly, the use of various scoring methods for evaluation of immunohistochemical staining has rendered proper interpretation of findings difficult, leading to conflicting data, e.g. regarding the frequency of TAP1 loss [9, 18\u201320].\nIn this study, a panel of recently reported monoclonal antibodies was used to investigate expression of all known APM components in a large set of cervical carcinoma lesions using a TMA-based approach [30\u201333]. Staining was evaluated according to a previously reported quality control system [37], which is more stringent in defining total and partial expression loss than other evaluation methods, which consider \u226425% positive cells as total loss. The system used here defines total loss as complete or near-complete negativity and is therefore more reliable. Moreover, by excluding \u03b22-m-negative cases from the analysis, all outcome parameters can be reliably correlated to expression levels of the various APM components, as any influence of defective APM expression will only be detectable in a \u03b22-m-positive setting.\nAs an APM component, ERAP1 has been shown to be an important determinant of the repertoire of HLA class I-presented peptides [14, 34, 38\u201341]. However, to date no systematic investigation of its potential role in HLA class I down-regulation in tumours has been performed. To our knowledge, this is the first description of ERAP1 down-regulation in a human carcinoma. Additionally, we have identified ERAP1 loss as an independent predictor of shorter survival, in the absence of associated HLA class I down-regulation. A possible explanation for this observation is the role of ERAP1 in shaping the antigen repertoire: ERAP1 down-regulation may lead to the preferential loading and presentation of non-tumour-associated peptides, thereby yielding a less immunogenic phenotype and facilitating tumour growth and progression. This hypothesis is supported by previous reports on the disrupted nature of presented peptide repertoire in ERAP1-deficient mice [41, 42]. Also, the effect of down-regulation of several APM components on the presentation of specific HPV 16 E6-derived epitopes has been firmly established [43\u201345].\nAlthough we found that total TAP1 loss was an independent predictor for shorter DFS, this finding should be interpreted with caution as only four patients exhibited this loss. The decreased significance of ERAP1 as an independent predictor for shorter DFS can be explained by the interdependency of ERAP1-mediated peptide trimming and TAP-mediated peptide transport.\nAn interesting finding in this study is the observed association of LMP2 and LMP7 down-regulation with an absence of lymph node metastasis; this finding is seemingly corroborated by reports that certain immunogenic peptides in melanoma and renal-cell carcinoma are not generated by immunoproteasomes and that presentation of these peptides is reduced upon up-regulation of the immunoproteasomal subunits [46]. Though altered proteasomal cleavage of endogenous proteins can lead to an alternative, less immunogenic antigen repertoire, an adequate mechanism for the association of LMP down-regulation with an absence of lymph node metastases remains to be postulated.\nInterestingly, we have recently observed that genetic variation in various APM components is significantly associated with increased cervical carcinoma risk. Although the underlying mechanisms of APM component loss are largely unknown, the occurrence of single nucleotide polymorphisms in the various APM genes could form a genetic basis predisposing to either mutations, or regulatory down-regulation [47, 48].\nPartial HLA class I loss was observed to be significantly associated with decreased OS; correspondingly, reports in colon cancer have shown partial, but not total HLA class I loss to be associated with worse OS [49]. This finding can be explained by the ability of low levels of HLA class I molecules to provide tumour cells with an escape not only from cytotoxic T lymphocyte-mediated, but also from natural killer (NK) cell-mediated cytotoxicity [2]. This hypothesis is further supported by the association of decreased survival with total TAP1 loss, which has been shown previously to lead to partial HLA class I loss [2, 50\u201352]. In the present study, an association of loss or down-regulation of expression of the various APM molecules with aberrant HLA expression was observed. Moreover, the partial HLA loss observed is most likely an underestimation of the extent of HLA alterations present as immunohistochemical staining on paraffin sections does not reveal loss of specific HLA alleles [8]. However, there are also other aberrations known to lead to HLA class I alterations and it is plausible to hypothesize that all these mechanisms can coexist in the same tumour sample.\nIn conclusion, we have shown here that defective expression of HLA class I and of various APM components occurs frequently in cervical carcinoma. Partial HLA class I loss and total TAP1 loss are associated with decreased survival, while down-regulation of immunoproteasomal subunits is associated with decreased risk of lymphogenic metastasis. Moreover, we report the first description of ERAP1 down-regulation in a human carcinoma, and have found that this is an independent prognostic parameter for decreased survival. A complete understanding of the mechanisms and relevance of HLA class I and APM component down-regulation and immune evasion may contribute to the rational design of tumour vaccines and T-cell-based immunotherapies.","keyphrases":["antigen processing machinery","cervical carcinoma","erap1"],"prmu":["P","P","P"]}
{"id":"Eur_J_Appl_Physiol-3-1-1914232","title":"Reproducibility of onset and recovery oxygen uptake kinetics in moderately impaired patients with chronic heart failure\n","text":"Oxygen (O2) kinetics reflect the ability to adapt to or recover from exercise that is indicative of daily life. In patients with chronic heart failure (CHF), parameters of O2 kinetics have shown to be useful for clinical purposes like grading of functional impairment and assessment of prognosis. This study compared the goodness of fit and reproducibility of previously described methods to assess O2 kinetics in these patients. Nineteen CHF patients, New York Heart Association class II\u2013III, performed two constant-load tests on a cycle ergometer at 50% of the maximum workload. Time constants of O2 onset- and recovery kinetics (\u03c4) were calculated by mono-exponential modeling with four different sampling intervals (5 and 10 s, 5 and 8 breaths). The goodness of fit was expressed as the coefficient of determination (R2). Onset kinetics were also evaluated by the mean response time (MRT). Considering O2 onset kinetics, \u03c4 showed a significant inverse correlation with peak- (R = \u22120.88, using 10 s sampling intervals). The limits of agreement of both \u03c4 and MRT, however, were not clinically acceptable. O2 recovery kinetics yielded better reproducibility and goodness of fit. Using the most optimal sampling interval (5 breaths), a change of at least 13 s in \u03c4 is needed to exceed normal test-to-test variations. In conclusion, O2 recovery kinetics are more reproducible for clinical purposes than O2 onset kinetics in moderately impaired patients with CHF. It should be recognized that this observation cannot be assumed to be generalizable to more severely impaired CHF patients.\nIntroduction\nOxygen (O2) kinetics describe the rate change of oxygen uptake \nduring onset or recovery of exercise and reflect changes in cardiac output and tissue oxygen extraction. Compared to healthy individuals, patients with chronic heart failure (CHF) have slower O2 onset and recovery kinetics, resulting in early fatigue and slow recovery after exertion due to a greater reliance on anaerobic metabolism (Wasserman et\u00a0al. 1996; Koike et\u00a0al. 1995). Although peak \nis widely accepted as a reliable indicator of maximal aerobic capacity in CHF patients (Weber et\u00a0al. 1982; Janicki et\u00a0al. 1990), O2 kinetics provide additional objective information on the ability to adapt to and recover from exercise that is indicative of daily life (Riley et\u00a0al. 1994; Koike et\u00a0al. 1995). Furthermore, O2 kinetics are potentially useful for risk stratification of CHF patients (Brunner-La Rocca et\u00a0al. 1999; Schalcher et\u00a0al. 2003) and for measuring the effects of exercise training, which has already been demonstrated in healthy individuals (Carter et\u00a0al. 2000) and patients with chronic obstructive pulmonary disease (COPD) (Puente-Maestu et\u00a0al. 2000).\nIn order to use O2 kinetics for these clinical purposes it is necessary to know more about the applicability and reproducibility of these exercise parameters in this specific patient group. Until now there has been no uniformity in the assessment of O2 kinetics in patients with CHF (Arena et\u00a0al. 2001). In addition, the reproducibility of O2 kinetics at submaximal exercise in CHF patients has not been studied extensively. Two studies that assessed O2 onset kinetics by different modeling techniques, suggest an acceptable reproducibility of nonlinear regression and an algebraic method (Belardinelli et\u00a0al. 1998; Sietsema et\u00a0al. 1994). In both studies, however, intra-class correlations and limits of agreement were not mentioned.\nThe purpose of this study is to evaluate the goodness of fit and reproducibility of previously described clinically applicable methods to characterize O2 onset and recovery kinetics in moderately impaired patients with CHF. Furthermore, we aim to define interventional changes that are required to distinguish from the normal test-to-test variations.\nMethods\nSubjects\nNineteen patients (15 men, 4 women) with stable CHF (New York Heart Association class II\u2013III and echocardiographical ejection fraction \u226440%) attributed to idiopathic dilated cardiomyopathy (n\u00a0=\u00a04) or ischemic heart disease due to myocardial infarction (n\u00a0=\u00a015) were selected at the cardiology outdoor clinic of the M\u00e1xima Medical Centre (Veldhoven, The Netherlands). Fifteen patients were in NYHA functional class II and four in class III. Subject characteristics are listed in Table\u00a01. Patients with recent myocardial infarction (<3\u00a0months), angina pectoris at rest, atrial fibrillation, or atrial flutter were not included. All patients performed a pulmonary function test using a spirometer (Masterlab, Jaeger, W\u00fcrzburg, Germany) including measurement of forced expiratory volume in 1\u00a0s (FEV1) and forced vital capacity (FVC) during a maximal forced expiratory effort. Patients with chronic airways obstruction, defined as FEV1\/FVC\u00a0<\u00a060% were excluded.\nTable\u00a01Characteristics of included patients with CHF (N\u00a0=\u00a019)VariablesMean\u00a0\u00b1\u00a0SDRangeAge (years)62\u00a0\u00b1\u00a0843\u201378Height (cm)172\u00a0\u00b1\u00a08155\u2013184Weight (kg)85\u00a0\u00b1\u00a01054\u201397Body mass index (kg\/m2)29\u00a0\u00b1\u00a0422\u201337Fat mass (%)a30\u00a0\u00b1\u00a0720\u201343Time since diagnosis (months)20\u00a0\u00b1\u00a0246\u201396Left ventricular ejection fraction (%)33\u00a0\u00b1\u00a0719\u201340a\u00a0Fat mass was assessed by skinfold measurements (biceps, triceps, subscapular, and suprailiacal) according to standard procedures (WHO 1995)\nFifteen patients used beta-blockers and angiotensin-converting enzyme inhibitors, three patients used an angiotensin-converting enzyme inhibitor only, and one patient used a beta-blocker only. Sixteen patients used diuretics. The average duration that patients were using beta-blockers was 34\u00a0\u00b1\u00a033\u00a0months (range 7\u2013112\u00a0months) and 32\u00a0\u00b1\u00a029\u00a0months for ACE inhibitors (range 7\u2013118\u00a0months). Patients who did not use beta-blockers were not different from the other patients with respect to age, peak-\nor left ventricular ejection fraction.\nThe research protocol was approved by the local Research Ethics Committee of the M\u00e1xima Medical Centre, and all patients provided written informed consent.\nExercise testing\nSubjects performed a symptom-limited, incremental exercise test, and on a separate day (at least 3\u00a0days later), a constant-load test at 50% of the maximum workload achieved at the first test. This test was repeated at the same time on another day within 2\u00a0weeks (mean difference between tests 6.7\u00a0\u00b1\u00a03.9\u00a0days). All subjects took their medication at the usual time and were instructed not to perform any extra physical activity on testing days. During the testing period all patients were on sinus rhythm. Furthermore, none of the patients reported changes in symptoms, functional status or medication use. Therefore, they could be considered to be in a stable physical condition during the study period.\nAll exercise tests were performed in an upright seated position on an electromagnetically braked cycle ergometer (Corival, Lode, Groningen, The Netherlands). Measurements of \nand respired fractions of \nand \nwere obtained breath by breath (Oxycon \u03b1, Jaeger, Germany). Volumes and gas analysers were calibrated before each test.\nThe incremental exercise test was performed using an individualized ramp protocol with a total test duration of 8\u201312\u00a0min (Working Group on Cardiac Rehabilitation & Excercise Physiology and Working Group on Heart Failure of the European Society of Cardiology 2001). During the tests all patients were instructed to maintain a pedaling frequency of 70 per minute. A 12-lead electrocardiogram was registered continuously, and blood pressure was measured every 2\u00a0min (Korotkoff). The test was ended when the patient was not able to maintain the required pedaling frequency. Maximal workload was defined as the final registered workload, peak-\nas the average \nof the last 30\u00a0s of the test. Predicted value of peak-\nwas calculated with use of the Wasserman equation, normalizing peak-\nfor age, gender, weight and height (Hansen et\u00a0al. 1984).\nThe ventilatory threshold was determined by two independent observers using the V-slope method as described by Beaver et\u00a0al. (1986). The constant-load tests included 2\u00a0min of rest, 2\u00a0min of unloaded pedaling, 6\u00a0min at 50% of the maximum workload and 5\u00a0min of rest.\nData analysis\nMono-exponential model\nTime constants (\u03c4) were calculated by fitting the \ndata of the constant-load tests to a first-order (mono-exponential) model using the non-linear least squares method (Whipp and Wasserman 1972). Calculations were performed with breath-by-breath data averaged into four different sampling intervals that were used in previous studies: 5\u00a0s (5\u00a0s) (Matsumoto et\u00a0al. 1999), 10\u00a0s (10\u00a0s) (Arena et\u00a0al. 2002), 5 breaths (5b) (Koike et\u00a0al. 1995) and 8 breaths (8b) (Pavia et\u00a0al. 1999). The following formulas were used: A=B=Td=time delay\u03c4=time constant (s)Baseline-\nwas defined as the average \nof the last minute of the unloaded-cycling stage and steady-state-\nas the average \nof the last minute of exercise. The time delay (Td) is a parameter allowed to vary in order to optimize the fit, representing the time between onset of exercise and the start of the mono-exponential increase of \nOne of the determinants of this time delay is the lag time between the computer signal to deliver the work rate and the actual response of the ergometer, which amounted to 2.2\u00a0\u00b1\u00a00.6\u00a0s in this study. Occasional errant breaths (e.g., due to coughing, swallowing or talking) were deleted from the data set when \nexceeded three standard deviations of the mean, defined as the average of two following and two preceding sampling intervals (Lamarra et\u00a0al. 1987). In total, about 1% of the breaths had to be deleted.\nAlgebraic method\nO2 onset kinetics were also evaluated by an algebraic method calculating mean response time (MRT) (Sietsema et\u00a0al. 1994), using the following formulas:MRTO2 deficitThe expected amount of O2-uptake \nwas calculated by multiplying the O2-amplitude \nwith exercise duration (6\u00a0min). Oxygen deficit was calculated by subtracting the summed \nabove the baseline-value \nfrom the expected \nStatistical analysis\nAll data (presented as mean\u00a0\u00b1\u00a0SD) were analyzed using a statistical software program (SPSS 11.0). The \u2018goodness of fit\u2019 for mono-exponential modeling was evaluated by the coefficient of determination (R2). The fitting procedure was considered acceptable when R2\u00a0\u2265\u00a00.85, as previously described by de Groote et\u00a0al. (1996). Differences between calculation methods were evaluated by one-way ANOVA with repeated measures and Bonferroni post hoc analyses. In order to assess differences between kinetic parameters of the two tests the paired Student\u2019s t test was used. Linear regression was used to define correlations between variables. Agreement between the kinetic parameters was assessed by intra-class correlation coefficients, limits of agreement (mean difference \u00b11.96\u00a0\u00d7\u00a0SD) (Bland and Altman 1986) and coefficients of variation (SD of difference as a percentage of the mean value). Probability values <0.05 were considered statistically significant.\nResults\nSymptom-limited exercise tests\nAll subjects completed the exercise tests. The maximum workload was 109\u00a0\u00b1\u00a032\u00a0W, peak-\nwas 20.0\u00a0\u00b1\u00a04.0\u00a0ml\u00a0min\u22121\u00a0kg\u22121 (73\u00a0\u00b1\u00a09% of predicted peak-\n) and the maximal respiratory exchange ratio was 1.13\u00a0\u00b1\u00a00.13. The ventilatory threshold could not be determined in three patients (16%) because of excessive ventilatory oscillations. In the remaining 16 patients the independent observers agreed on the determination of the ventilatory threshold (mean \n:16.4\u00a0\u00b1\u00a03.2\u00a0ml\u00a0min\u22121\u00a0kg\u22121, 60\u00a0\u00b1\u00a011% of predicted peak-\n).\nConstant-load exercise tests\nThe mean value of \nduring the second minute of unloaded pedaling was 655\u00a0\u00b1\u00a078\u00a0ml\u00a0min\u22121 (30\u00a0\u00b1\u00a05% of predicted peak-\n), and the steady-state value at 50% of the maximal work load was 1,185\u00a0\u00b1\u00a0228\u00a0ml\u00a0min\u22121 (53\u00a0\u00b1\u00a07% of predicted peak-\n). Figure\u00a01 shows changes in \nduring a constant-load test in a representative subject.\nFig.\u00a01-response to steady-state exercise at 50% of the maximal workload (50\u00a0W) in a representative subject. The solid line represents 10\u00a0s averages of \nThe curved dashed line is the computer-derived representation of the best fit of the mono-exponential model to the \n-response. The first dashed vertical line indicates onset of exercise and the second vertical line the end of exercise\nIn 16 subjects, in whom the ventilatory threshold could be determined reliably, steady-state-\nwas below the ventilatory threshold. None of the other three subjects demonstrated a significant rise of \ndefined as in increase from the third to the sixth minute of exercise of more than two times the SD of the mean \nin the fourth minute. This indicates that these three patients also exercised below the ventilatory threshold (Whipp 1994).\nComparison of calculation methods\nConcerning the onset phase, there were no significant differences between mono-exponential modeling and the algebraic method (differences between \u03c4 and MRT \u22126.6\u20131\u00a0s, SD 11\u201317\u00a0s). In the recovery phase, the use of different sampling intervals yielded significantly different time constants (P\u00a0<\u00a00.001). Paired comparisons showed that in two cases the time constants were not significantly different (5\u00a0s vs. 5 breaths, P\u00a0=\u00a00.14 and 10\u00a0s vs. 8 breaths, P\u00a0=\u00a00.31).\nWhen comparing the applicability of the mono-exponential model to the \n-response during onset and recovery of exercise, the results indicate a better \u2018goodness of fit\u2019 during recovery (P\u00a0<\u00a00.001) (Table\u00a02). Parameters of O2 onset kinetics showed significant correlations with peak-\nwhen calculating \u03c4 with sampling intervals of 10\u00a0s, 5 breaths and 8 breaths (Table\u00a03). However, mainly due to the limited number of patients, the differences between these correlation coefficients were not statistically significant. During recovery of exercise \u03c4 was only correlated with peak-\nwhen 5 breath sampling intervals were used.\nTable\u00a02O2 Kinetics using an algebraic method and a mono-exponential model with different sampling intervals (n\u00a0=\u00a019)Kinetic parametersMean\u00a0\u00b1\u00a0SD (s)Goodness of fit (R2) Mean\u00a0\u00b1\u00a0SDUseful measurements (%)aO2 Onset kinetics\u00a0MRT71\u00a0\u00b1\u00a019\u2013\u2013\u00a0\u03c4-5\u00a0s67\u00a0\u00b1\u00a0200.69\u00a0\u00b1\u00a00.1526\u00a0\u03c4-10\u00a0s74\u00a0\u00b1\u00a022 0.81\u00a0\u00b1\u00a00.1242\u00a0\u03c4-5b75\u00a0\u00b1\u00a0300.87\u00a0\u00b1\u00a00.0879\u00a0\u03c4-8b74\u00a0\u00b1\u00a0220.91\u00a0\u00b1\u00a00.0784O2 Recovery kinetics\u00a0\u03c4-5\u00a0s60\u00a0\u00b1\u00a0130.89\u00a0\u00b1\u00a00.0684\u00a0\u03c4-10\u00a0s66\u00a0\u00b1\u00a0140.94\u00a0\u00b1\u00a00.04100\u00a0\u03c4-5b61\u00a0\u00b1\u00a0130.96\u00a0\u00b1\u00a00.03100\u00a0\u03c4-8b64\u00a0\u00b1\u00a0130.98\u00a0\u00b1\u00a00.02100Average duration of 5 breath sampling interval 15.9\u00a0\u00b1\u00a03.6\u00a0s at baseline, 13.0\u00a0\u00b1\u00a03.0\u00a0s at steady-state. Average duration of 8 breath sampling interval 25.0\u00a0\u00b1\u00a05.2\u00a0s at baseline, 20.9\u00a0\u00b1\u00a04.7\u00a0s at steady stateMRT Mean response time, \u03c4 time constant calculated with a mono-exponential model, 5s 5\u00a0s, 10\u00a0s 10\u00a0s, 5b 5 breaths, 8b 8 breathsa The assessment of \u03c4 was considered useful when R2\u00a0\u2265\u00a00.85Table\u00a03Correlation coefficients between averaged kinetic parameters and peak \nKinetic parametersCorrelation with peak \n95% Confidence intervalO2 Onset kineticsMRT\u22120.33\u22120.680.15\u03c4\u22125\u00a0s\u2013\u03c4-10\u00a0s\u22120.88*\u22120.99\u22120.24\u03c4-5b\u22120.67*\u22120.90\u22120.16\u03c4-8b\u22120.57 *\u22120.83\u22120.10O2 Recovery kinetics\u03c4-5\u00a0s\u22120.43\u22120.760.08\u03c4-10\u00a0s\u22120.45\u22120.750.01\u03c4-5b\u22120.47*\u22120.76\u22120.02\u03c4-8b\u22120.39\u22120.720.08Only data with R2\u00a0>\u00a00.85 when using a mono-exponential model were included; differences between correlation coefficients were not statistically significant* P\u00a0<\u00a00.05MRT Mean response time, \u03c4\u00a0=\u00a0time constant calculated with a mono-exponential model, 5s 5\u00a0s, 10s 10\u00a0s, 5b 5 breaths, 8b 8 breaths \nReproducibility\nIn the two constant-load tests there were no statistically significant differences between \nduring unloaded pedaling (655\u00a0\u00b1\u00a078 vs. 633\u00a0\u00b1\u00a098\u00a0ml\u00a0min\u22121), \nat steady-state exercise (1,185\u00a0\u00b1\u00a0228 vs. 1,202\u00a0\u00b1\u00a0235\u00a0ml\u00a0min\u22121), and respiratory exchange ratios at steady-state exercise (0.94\u00a0\u00b1\u00a00.04 vs. 0.94\u00a0\u00b1\u00a00.06). In addition, there were no statistically significant differences between the kinetic parameters of both tests. Considering limits of agreement, coefficients of variation and intra-class correlation coefficients, recovery kinetics show better reproducibility than onset kinetics (Table\u00a04).\nTable\u00a04Comparison of kinetic parameters in two consecutive constant-load testsKinetic parameterNDifference mean\u00a0\u00b1\u00a0SDLimits of agreementCoefficient of variation (%)Intraclass correlation coefficientO2 Onset kinetics\u00a0\n(ml\u00a0min\u22121\u00a0kg\u22121)190.1\u00a0\u00b1\u00a01.2\u22122.32.58.50.97\u00a0MRT (s)192.8\u00a0\u00b1\u00a012.9\u2212232918.40.86\u03c4\u22125\u00a0s (s)0\u2013\u2013\u2013\u2013\u03c4-10\u00a0s (s)69.3\u00a0\u00b1\u00a011.8\u2212143317.10.79\u03c4-5b (s)125.9\u00a0\u00b1\u00a019.8\u2212344628.40.63\u03c4-8b (s)165.6\u00a0\u00b1\u00a016.7\u2212283923.00.77O2 Recovery kinetics\u03c4-5\u00a0s (s)16\u22120.4\u00a0\u00b1\u00a07.2\u2212151412.20.91\u03c4-10\u00a0s (s)19\u22120.9\u00a0\u00b1\u00a010.7\u2212222116.30.86\u03c4-5\u00a0b (s)19\u22120.9\u00a0\u00b1\u00a06.4\u2212141210.40.94\u03c4-8\u00a0b (s)19\u22121.4\u00a0\u00b1\u00a08.6\u2212191613.40.91Only data with R2\u00a0>\u00a00.85 when using a mono-exponential model were included Oxygen uptake at steady-state exercise, MRT mean response time; \u03c4 time constant calculated with a mono-exponential model, 5\u00a0s 5\u00a0s; 10\u00a0s 10\u00a0s; 5b 5 breaths, 8b 8 breaths\nFigure\u00a02 shows Bland Altman plots of the kinetic parameters during onset and recovery of exercise with the highest intra-class correlation coefficients.\nFig.\u00a02Bland Altman plots showing the difference of O2 kinetics between two constant-load tests during onset (upper graph) and recovery of exercise (lower graph), using an algebraic method and mono-exponential modeling with 5 breath sampling intervals, respectively. The solid lines represent the mean difference between the two tests, the dashed lines indicate the 95% confidence intervals of the difference. MRT mean response time. \u03c4 -Rec\u00a0=\u00a0time constant of \nduring recovery with 5-breath sampling intervals\nDiscussion\nThe principal finding of this study is that, using the applied exercise protocol, O2 recovery kinetics were more reproducible than O2 onset kinetics in moderately impaired patients with CHF. It should be recognized, however, that this observation cannot be assumed to be generalizable to more severely impaired CHF patients.\nO2 onset kinetics\nBecause of the lack of standardized protocols, previous authors used both time constants (\u03c4, non-linear regression) (Belardinelli et\u00a0al. 1998; Koike et\u00a0al. 1995; Matsumoto et\u00a0al. 1999) and mean response times (MRT, algebraic method) (Sietsema et\u00a0al. 1994) to assess O2 onset kinetics in CHF patients. Considering non-linear regression, the use of 10\u00a0s sampling intervals yielded the best results in terms of reproducibility in this study. However, the goodness of fit, which was comparable with the study of Arena et\u00a0al. (2002) (0.81 vs. 0.78, respectively), was insufficient in 58% of the patients. Moreover, the wide limits of agreement restrict its use for clinical applications. While the calculation of \u03c4 with larger sampling intervals resulted in a better goodness of fit, their reproducibility was even lower. To our knowledge, only one study previously addressed reproducibility of O2 onset kinetics using mono-exponential modeling in CHF patients. In that study five patients with a peak-\nthat was comparable to our study performed three constant-load tests at a workload of 50\u00a0W, starting from unloaded cycling. Although the low mean difference between the tests (2\u00a0s) suggests good reproducibility, the actual variability between the tests was not mentioned, making it impossible to compare these results to our study (Belardinelli et\u00a0al. 1998). In spite of the fact that almost all of the onset procedures showed a significant correlation with peak-\n(Table\u00a03), differences between these correlations were not statistically significant. Therefore, one could not suggest a preferential use of one of the procedures.\nAlthough showing a slightly higher intra-class correlation coefficient (Table\u00a04), the wide limits of agreement of the MRT also indicate low reproducibility of this parameter. In addition, this parameter did not correlate significantly with peak \nSietsema et\u00a0al. (1994) previously addressed reproducibility of the MRT in 18 CHF patients, reporting small mean differences between two tests. Again these results cannot be compared to our study, because intra-class correlations and limits of agreement were not mentioned.\nWhen comparing reproducibility of O2 onset kinetics in CHF patients with healthy subjects (Kilding et\u00a0al. 2005) and patients with COPD (Puente-Maestu et\u00a0al. 2001) we found a lower reproducibility of \u03c4 (coefficients of variation 17.1, 6.2 and 8.7%, respectively). There are several physiological and methodological factors that may explain this discrepancy.\nOne significant physiological factor that may cause the large variability of O2 onset kinetics in CHF patients is the influence of typical ventilatory oscillations, especially when small sampling intervals are used (Francis et\u00a0al. 2002). Although we did not quantify the effect of oscillations, we did observe ventilatory oscillations more clearly in patients with low coefficients of determination. Furthermore, it is postulated that ventilatory oscillations in patients with CHF increase in the transition from rest to exercise (Kremser et\u00a0al. 1987) and diminish with increasing exercise (Francis et\u00a0al. 2002). This suggests that these oscillations have a greater influence on O2 onset kinetics than O2 recovery kinetics. Based on our study, variations in \u03c4 during onset of at least 24\u00a0s are needed to exceed the limits of the 95% confidence interval, whereas a change of only 13\u00a0s in \u03c4 during recovery is sufficient to exceed the normal test-to-test variations. This latter variability is also observed during the on-transient response in patients with COPD (Puente-Maestu et\u00a0al. 2001), which supports our view that the relatively large variability of the on-kinetic parameters was mainly caused by ventilatory oscillations.\nAnother explanation for the limited reproducibility of O2 onset kinetics in CHF patients is their low exercise capacity, which reduces the amplitude of O2 uptake, and consequently the reliability of the determination of the time constant (Lamarra et\u00a0al. 1987). Furthermore, the period between the tests (6.7\u00a0\u00b1\u00a03.9\u00a0days) could be a long enough period of time to induce changes in the cardio circulatory condition of the patients. However, all patients were in a stable clinical or functional condition during the study period.\nConsidering methodological factors, the fact that all patients started the constant-load test with unloaded pedaling in stead of rest resulted in a relatively low \n-amplitude. This could be a major factor contributing to the relatively low reproducibility of the on-kinetic parameters found in this study. The reasons for the authors, however, to apply this exercise protocol were threefold. First, starting exercise from unloaded pedaling results in a reduction of the duration of the early rapid increase of oxygen uptake, representing an initial increase of pulmonary blood flow (cardiodynamic phase). Since in this phase the change of \nis functionally distinct from the subsequent mono-exponential increase of \n(phase II), reducing the relative contribution of this phase might result in a better fit of the mono-exponential model to the data. Second, starting exercise from unloaded cycling might reduce ventilatory oscillations at the onset of exercise because of a reduction in variation of pedaling frequency and upper limb muscle activity. Third, in daily life exercise will be frequently started from an active state. Given the results of this study, however, the authors feel that additional research is required to re-assess the reproducibility of O2 onset kinetics using an exercise protocol starting from rest in stead of unloaded cycling. Starting exercise from rest might result in a better reproducibility of O2 onset kinetics than observed in this study, which may in particular be of importance for more severely impaired CHF patients than used in this study.\nAnother factor that may have influenced reproducibility of O2 onset kinetics is the choice of the workload (i.e. 50% of the maximum workload). The approach to maximize \n-amplitude by relating the workload to the ventilatory threshold (e.g. 90% of ventilatory threshold) was not used in this study, because it was postulated that such a fixed threshold cannot be determined reliably in a substantial number of CHF patients (Meyer et\u00a0al. 1996). When looking at the patients of this study in whom determination of this threshold was successful, \nat steady state amounted to 86% of \nat the ventilatory threshold, with none of the patients exercising above this threshold. This suggests that the applied constant-load exercise protocol was adequate, because the exercise intensity remained below the ventilatory threshold.\nO2 recovery kinetics\nO2 recovery kinetics were more reproducible in moderately impaired patients with CHF than O2 onset kinetics. This difference in reproducibility is at least partly explained by the larger \n-amplitude in recovery due to the fact that subjects were not cycling in the recovery phase. From the results of this study it is difficult to conclude to what extent this difference between onset and recovery kinetics is caused by a smaller influence of the cardiodynamic phase during recovery or a more stable breathing pattern during recovery causing less ventilatory oscillations. Nevertheless, these observations are in line with previous studies in healthy individuals, which also reported a better reproducibility of O2 recovery kinetics than O2 onset kinetics (Kilding et\u00a0al. 2005; Ozyener et\u00a0al. 2001).\nIn terms of reproducibility, the most optimal method to characterize O2 recovery kinetics was mono-exponential modeling with sampling intervals of 5 breaths (intra-class correlation 0.94). Using this method a change of at least 13\u00a0s in \u03c4 is needed to exceed the normal test-to-test variations. In addition, this method yielded an excellent goodness of fit (R2 0.96\u00a0\u00b1\u00a00.03). To our knowledge, other data on the reproducibility of the recovery kinetics after submaximal exercise in CHF patients are not available. Cohen-Solal et\u00a0al. (1995) studied reproducibility of O2 recovery kinetics after maximal exercise in 10 patients with CHF (NYHA II-III) using a mono-exponential model with sampling intervals of 7 breaths. They found a coefficient of variation comparable to our study with 5 breath sampling intervals (12.3 vs. 10.4%, respectively).\nConclusion\nThis study shows that the reproducibility of O2 onset kinetics assessed by mono-exponential modeling is too low to warrant their use for measuring effects of therapeutic interventions in moderately impaired patients with CHF. This might be mainly attributable to physiological factors like ventilatory oscillations and the applied exercise protocol. Future studies should address the effect of different exercise protocols.\nThe determination of time constants of O2 recovery kinetics has been shown to be feasible and reproducible when using mono-exponential modeling with 5 breath sampling intervals. Since this variable represents recovery from exercise that is indicative of daily life, it is potentially useful for clinical purposes like grading of functional impairment in patients with CHF and measuring effects of therapeutic interventions.","keyphrases":["oxygen uptake kinetics","time constant","mean response time","exercise testing","cardiac disease"],"prmu":["P","P","P","P","R"]}
{"id":"Planta-3-1-2039816","title":"Relationship between petal abscission and programmed cell death in Prunus yedoensis and Delphinium belladonna\n","text":"Depending on the species, the end of flower life span is characterized by petal wilting or by abscission of petals that are still fully turgid. Wilting at the end of petal life is due to programmed cell death (PCD). It is not known whether the abscission of turgid petals is preceded by PCD. We studied some parameters that indicate PCD: chromatin condensation, a decrease in nuclear diameter, DNA fragmentation, and DNA content per nucleus, using Prunus yedoensis and Delphiniumbelladonna which both show abscission of turgid petals at the end of floral life. No DNA degradation, no chromatin condensation, and no change in nuclear volume was observed in P. yedoensis petals, prior to abscission. In abscising D.belladonna petals, in contrast, considerable DNA degradation was found, chromatin was condensed and the nuclear volume considerably reduced. Following abscission, the nuclear area in both species drastically increased, and the chromatin became unevenly distributed. Similar chromatin changes were observed after dehydration (24 h at 60\u00b0C) of petals severed at the time of flower opening, and in dehydrated petals of Ipomoea nil and Petunia hybrida, severed at the time of flower opening. In these flowers the petal life span is terminated by wilting rather than abscission. It is concluded that the abscission of turgid petals in D. belladonna was preceded by a number of PCD indicators, whereas no such evidence for PCD was found at the time of P. yedoensis petal abscission. Dehydration of the petal cells, after abscission, was associated with a remarkable nuclear morphology which was also found in younger petals subjected to dehydration. This nuclear morphology has apparently not been described previously, for any organism.\nIntroduction\nFloral life span depends on the fate of the petals. In several species the end of floral life span is due to petal withering or wilting. The petals in most of these species desiccate, and finally abscise or fall due to growth of the fruit. Only in a few species the desiccated petals remain attached to the fruit. In another large group of species the end of floral life span is determined by abscission of petals that are mostly or fully turgid.\nWilting in petals, at the end of their lifespan, is due to a series of highly regulated processes that lead to cell death, thus is a form of programmed cell death (PCD; van Doorn et al. 2003). Prior to visible PCD, many macromolecules in the petal cells are degraded to molecules that are suitable for phloem transport. These mobile molecules are transported to other parts of the plant (Winkenbach et al. 1970a, b). Typical for PCD in wilting petals is the disappearance of most of the organelles and accumulation of degrading enzymes in the vacuole. This is followed by rupture of the vacuolar membrane, whereby several hydrolases are released. Tonoplast rupture in numerous cells results in their collapse, which is visible as wilting (Winkenbach 1970a, b; van Doorn et al. 2003). PCD in several plant parts (Gunawardena et al. 2001; Wojciechowska and Olszewska 2003), including petals, is also usually accompanied by DNA degradation (van der Kop et al. 2003; Wagstaff et al. 2003; Yamada et al. 2006a, b) and chromatin condensation (Yamada et al. 2006a, b).\nIt is not known if the abscission of turgid petals is preceded by cellular changes that are indicative of PCD. We therefore investigated the time line of some of these changes, in Prunus yedoensis and Delphiniumbelladonna. In both species the petals abscise without any visible sign of turgor loss. We used DNA degradation (measured both on agarose gels and by using flow cytometry), chromatin condensation, and a decrease in nuclear diameter as parameters that indicate PCD. According to our hypothesis these parameters would not detectably change, prior to petal abscission.\nMaterials and methods\nPlant material\nPlants were grown in the garden of National Institute of Floricultural Science of Japan (P. yedoensis cv. Someiyoshino) or the greenhouse of the institute (D. belladonna cv. Bellamosum, Ipomoea nil cv. Violet, and Petunia hybrida cv. Double Duo Pink). Just before the onset of flower opening, cut branches (P. yedoensis) or potted plants (the other species) were transferred into a growth chamber at 24\u00a0\u00b1\u00a01\u00b0C, about 70% relative humidity (RH), and 12\u00a0h of daily illumination from cool-white fluorescent lights (100\u00a0\u03bcmol\u00a0m\u22122\u00a0s\u22121). The time to petal abscission and petal desiccation was observed daily, on plant material in the growth chamber. The timing of these symptoms was noted in six flowers from two plants. All determinations were made using petals that were collected from plant material in the chamber.\nDNA degradation\nDNA breakdown was detected by agarose gel analysis. Total DNA was extracted according to Yamada et al. (2003). The amount of DNA was estimated by molecular absorption spectrophotometry. Equal amounts of total DNA (3\u00a0\u03bcg) were immediately electrophoresed in a gel containing 3% agarose and stained with SYBR Gold nucleic acid gel stain (Molecular Probes, Eugene, OR, USA). The gel pattern was photographed, using an electronic UV transilluminator system (model FAS-III mini\u00a0+\u00a0DS-30; Toyobo, Tokyo, Japan). The DNA degradation index was calculated according to Yamada et al. (2006b).\nTwo biological replications, each containing several petals, were used for each stage of development. Depending on the species, two to several petals were used in each sample.\nFlow cytometry\nThe petal was chopped in nuclear extraction buffer, part of the kit (High Resolution kit) for plant DNA analysis (Partec, M\u00fcnster, Germany), a reagent set provided by the manufacturer of the flow cytometer used (Ploidy Analyzer, Partec). The extraction buffer in this kit was a low pH solution containing Triton X-100. The extract was filtered through 50\u00a0\u03bcm nylon mesh. The medium with the isolated DNA masses (nuclei) was collected, the buffer with the fluorescent DNA stain 4, 6-diamidino-2-phenylindole (DAPI) from a standard reagent set (Partec) was added, and the solution was vortex mixed. The DNA content of the isolated nuclei was analysed using the flow cytometer. The flow cytometer apparatus can be set to count all nuclei in a sample, and can be set to analyse a fixed number of nuclei. We here set it to detect 5,000 nuclei. DNA levels were obtained in a total of 5,000 nuclei. It should be noted that the large histogram peak was adjusted to 10 on the fluorescence scale. This means that nuclear condensation was not taken into account in the flow cytometry data.\nTwo biological replications, each containing several petals, were used for each stage of development. Depending on the species, three to several petals were used in each sample. In each species, the number of petals was the same at all stages of development.\nNuclear morphology; chromatin condensation\nNuclei were isolated from petals in the same way as for flow cytometry. Isolated nuclei were stained with DAPI, which stains DNA. The nuclei were observed under a fluorescence microscope (model PROVIS AX70, Olympus, Tokyo, Japan) using U-excitation (330\u2013385\u00a0nm). Digital photography of nuclei was obtained for each stain using low light cool CCD camera (model DP30BW, Olympus).\nDehydration and ethylene treatment\nFlowers were excised when the petals were fully open. Cut flowers were placed in air in the growth chamber (24\u00b0C, about 70% RH) or in a desiccator (at 60\u00b0C). For ethylene treatments, stems of cut flowers were placed in sterile distilled water, in a closed 70\u00a0l clear plastic box containing 2\u00a0\u03bcl\/l ethylene. Petal abscission was checked at hourly intervals and abscised petals were collected immediately. The petals were then used instantly for the measurement of the various parameters mentioned.\nStatistics\nThe number of replications is mentioned in the text and\/or in the figure legends. Statistical analysis used one way analysis of variance, at P\u00a0<\u00a00.05. All experiments were repeated at least once.\nResults\nSymptoms of petal life span cessation\nP. yedoensis flowers have five pale pink petals. After flower opening the petals remained attached to the flower for about 4\u00a0days, and then fell without visible wilting (Fig.\u00a01a, P2). During the first day following abscission, petals that were held at 24\u00b0C and 70% RH changed colour from pale pink to darker pink, and subsequently became infiltrated with liquid (results not shown). By the end of the first day following abscission, the petals started to shrivel at their margins (Fig.\u00a01a, P3). By the end of the second day after abscission the petals had much more decreased in size (Fig.\u00a01a, P4). The stages 2\u20134 (pictures P2\u2013P4) in Fig.\u00a01a use the same scale thus show the decrease in petal size.\nFig.\u00a01Petal abscission and petal dehydration in P. yedoensis, indicated by a P (a) and D.belladonna, indicated D (b). Cut branches of (P. yedoensis) and potted plants (D.belladonna) stood in a growth chamber (24\u00b0C, about 70% RH). The time to abscission and to dehydration symptoms (in days from full flower opening) is shown in the left lower corner of the pictures (n\u00a0=\u00a06). Stages P1 and D1: full flower opening; P2 and D2, petal abscission; P3 and D3, petals visibly dehydrated; P4 and D4, petals desiccated. All pictures have the same scale (bars\u00a0=\u00a010\u00a0mm)\nFlowers of Delphinium species contain five petals one of which has a large spur. The petals of D. belladonna fell about 7\u00a0days after flower opening (Fig.\u00a01b, D2). They did not show any visible wilting symptom by the time of abscission. Two days after abscission the petals had slightly shrivelled (Fig.\u00a01b, D3) but by day-3 after abscission the shrivelling had become very clear (Fig.\u00a01b, D4). The stages 2\u20134 (pictures D2\u2013D4) in Fig.\u00a01b have the same scale.\nDNA degradation\nFigure\u00a02 shows the petal DNA agarose gels of one of two repeat experiments. The pixel ratios under the lanes are the means of two experiments. In both species, the agarose gels showed no DNA fragments at the time of flower opening (Fig.\u00a02, lanes P1 and D1). By the time of abscission, the petals of P. yedoensis did not show a statistically significant (P\u00a0<\u00a00.05) increase in DNA fragmentation, compared with the time of floral opening (Fig.\u00a02, lane P2). In contrast, the petals of D. belladonna showed a considerable increase in DNA fragmentation by the time of abscission (Fig.\u00a02b, lane D2, pixel intensity had increased from 4.3 to 17.4). Following petal abscission the degree of DNA fragmentation, as observed on agarose gels, increased in P. yedoensis (Fig.\u00a02a, compare lane P3 with lane P2) and further increased in D. belladonna (Fig.\u00a02b, compare lane D3 with lane D2). By the time of petal desiccation (P4 and D4) the amount of fragmented DNA had become considerably lower than at the previous stage investigated, when the abscised petal showed visible wilting and dehydration (Fig.\u00a02).\nFig.\u00a02Agarose gel analysis of total DNA isolated from the petals of P. yedoensis, annotated as P (a) and D. belladonna, annotated as D (b). 3\u00a0\u03bcg of total DNA was extracted from the petals and electrophoresed in a 3% agarose gel. Lanes P1\u2013P4 and SD1\u2013SD4 refer to DNA isolated from petals at stages P1\u2013P4 and D1\u2013D4 as shown in Fig.\u00a01. Stages P1 and D1: full flower opening; P2 and D2, petal abscission; P3 and D3, petals visibly dehydrated; P4 and D4, petals desiccated. Pixel ratios below the lanes are means of two repeat experiments\nNuclear morphology\nIn P. yedoensis petals the nuclear morphology had not clearly changed by the time of petal abscission (Fig.\u00a03a). This is in contrast with D. belladonna where visual inspection showed that the nucleus was much smaller by the time of abscission, compared with the size observed at flower opening. The small nuclei of D. belladonna showed brighter DAPI fluorescence than the nuclei at flower opening. This indicates a high concentration of DNA per unit volume, thus chromatin condensation (Fig.\u00a03b, D2). Nuclear morphology was studied using light microscopy, giving a two-dimensional impression only. For this reason nuclear size will be referred to as nuclear area.\nFig.\u00a03Morphology of DAPI-stained nuclei in the petals of P. yedoensis, indicated by a P (a) and D.belladonna, indicated D (b). Two representative nuclei (-1 and -2) are shown at each stage of development. Nuclei were isolated from the petals and observed using a fluorescence microscope under U-excitation. Bars: 5\u00a0\u03bcm (a) or 15\u00a0\u03bcm (b). Stages as in Fig\u00a01. P1 and D1 1: full flower opening; P2 and D2, petal abscission; P3 and D3, petals visibly dehydrated; P4 and D4, petals desiccated\nAfter abscission, during the period of dehydration, the nuclei of both species showed an increase in area (Fig.\u00a03) In P. yedoensis the nuclear area at stage P4 seemed even considerably larger than that at floral opening (Fig.\u00a03a). In D. belladonna the nuclear area was apparently similar at D4 compared to the stage of floral opening (D1), but it was ostensibly much larger compared with the area at the stage of petal abscission (P2; Fig.\u00a03b). In both species the nuclear periphery was no longer smooth at stage P4 or D4 (Fig.\u00a03). In both species investigated, numerous nuclei showed the morphology shown in Fig.\u00a03, stages P4 and D4. The area of the remaining nuclei did not increase as much, whilst a few nuclei had not changed in nuclear area with respect to controls at flower opening.\nFlow cytometry of nuclei at various stages prior to and after abscission\nFlow cytometry of nuclei stained for DNA (with DAPI) shows the distribution of nuclei in fluorescence classes. The X-axis in Fig.\u00a04 is divided in numerous classes of increasing fluorescence, and the Y-axis shows the number of nuclei per fluorescence class. Flow cytometry of 5.000 petal nuclei at the time of P. yedoensis flower opening showed a peak of nuclei at phase G0\/G1 (which was set at 101 on the arbitrary logarithmic scale of the X-axis), and no mitotic nuclei (G2\/M), which have a fluorescence about two times that of normal G0\/G1 nuclei. By the time of petal abscission, no change had occurred in the DNA fluorescence histogram (Fig.\u00a04a, P2). Two days after abscission, the height of the G0\/G1 peak had diminished but the peak had not shifted. This is shown by mixing nuclei from day-0 to -6 (Fig.\u00a04a, P1\u00a0+\u00a0P4). The data suggest that a small amount of DNA had become degraded by day-2 after abscission.\nFig.\u00a04Fluorescence of DAPI-stained nuclei. Histograms were obtained by flow cytometry of 5,000 nuclei isolated from the petals of P. yedoensis (indicated by a P) as shown in a, and petals of D.belladonna (D) shown in b. Petals were severed from cut twigs placed in water (Prunus) or potted plants (Delphinium), which stood both in a growth chamber (24\u00b0C, about 70% RH). Stages as in Fig.\u00a01. P1 and D1: full flower opening; P2 and D2, petal abscission; P3 and D3, petals visibly dehydrated; P4 and D4, petals desiccated\nBy the time of petal abscission in D. belladonna, flow cytometry revealed a decrease in the height of the G0\/G1 phase nuclei, and several nuclei with a fluorescence less than that of G0\/G1 phase nuclei (Fig.\u00a04b, D2). During the 3\u00a0days following abscission the G0\/G1 peak height further decreased and the number of nuclei with fluorescence lower than G0\/G1 further increased. No clear shift in the G0\/G1 peak was observed: a mix of nuclei at day-0 and -10 did not result in a bimodal curve (Fig.\u00a04b, D1\u00a0+\u00a0D4). The results indicate extensive DNA degradation, before, during, and after abscission.\nEffects of ethylene\nEthylene treatment greatly hastened the time to petal abscission, in both species (Fig.\u00a05a, compared with Fig.\u00a01a). In both species, the rate of petal dehydration after abscission was not much affected by the ethylene treatment (Fig.\u00a05a) compared with the control without ethylene (Fig.\u00a01a). For example, 3\u00a0days after petal fall in D. belladonna the petals had extensively shrivelled, independent of the time of petal fall (day-7 in controls and day-2 in ethylene-treated flowers).\nFig.\u00a05Effect of ethylene on petal abscission and DNA fluorescence of nuclei in the petals of P. yedoensis and D.belladonna. Time to petal abscission and petal dehydration symptoms (a) and flow cytometric determination of 5,000 DAPI-stained nuclei (b) of ethylene-treated branches of P. yedoensis and potted plants of D.belladonna. Ethylene treatment is annotated by E. Compare stages of development with untreated controls shown in Fig.\u00a01, and with fluorescence data of controls in Fig.4. The left lower corner of the pictures series a show the times to abscission (EP2 and ED2) and the time to dehydration symptoms (both expressed in days from full flower opening; n\u00a0=\u00a06). Stages P1 and D1: full flower opening; EP2 and ED2, petal abscission; EP3 and ED3, onset of petal dehydration; EP4 and ED4, petals desiccated. All pictures have the same scale (bars\u00a0=\u00a010\u00a0mm)\nIn both species, flow cytometry revealed no effect of ethylene treatment on DNA fluorescence at the time of petal fall, compared to the fluorescence at the time of flower opening (Fig.\u00a05b). After petal fall, a clear shift to lower DNA fluorescence of the G0\/G1 peak of fluorescence was observed in P. yedoensis (Fig.\u00a05b, upper sequence), which was not found in controls without ethylene treatment (Fig.\u00a04, upper sequence). A small shift to lower DNA fluorescence (Fig.\u00a05b, lower sequence), also not found in controls (Fig.\u00a04 lower sequence), was also observed after ethylene treatment of D. belladonna. Superimposition of the stages P1 and EP4 (control and ethylene-treated stage 4 of P. yedoensis) showed two peaks of DNA fluorescence (Fig.\u00a05b, upper sequence). A similar tendency was observed by superimposing the stages D1 and ED3 (Fig.\u00a05b, lower sequence).\nDehydration of petals cut at flower opening\nFlowers were harvested just after opening and subjected to a dehydration treatment at 60\u00b0C for 24\u00a0h. The treatment resulted in a petal FW of about 20% of that of controls. Preliminary experiments showed that such a treatment resulted in a decrease of FW, and in changes of other parameters such as DNA degradation and nuclear morphology, that were very similar to dehydration on the laboratory bench (about 20\u00b0C and 70% RH) for about 96\u00a0h.\nTreatment of P. yedoensis flowers at 60\u00b0C for 24\u00a0h (Fig.\u00a06a, DP) resulted in a slight increase in petal DNA degradation, as shown on agarose gel (Fig.\u00a06b, DP). In contrast, this treatment induced a large increase in DNA degradation in D. belladonna petals (Fig.\u00a06b, DD).\nFig.\u00a06Effect of dehydration on DNA degradation, DNA fluorescence of nuclei, and nuclear morphology in the petals of P. yedoensis (P) and D.belladonna (D). Dehydration in the two species is indicated as DP and DD, respectively. a Flower morphology before (P1 and D1) and after (DP and DD) dehydration at 60\u00b0C for 24\u00a0h. Photographs show small branches with fully opened flowers and desiccated flowers of P. yedoensis (P1 and DP) and the same for individual flowers of D.belladonna (D1 and DD). All pictures have the same scale (bars\u00a0=\u00a010\u00a0mm). b Agarose gel analysis of total DNA isolated from the petals. c Flow cytometric determination of nuclei isolated from the petals. d Morphology of nuclei in the petals. Two representative nuclei (-1 and -2) are shown for each species, after the dehydration treatment (DP and DD). Bars\u00a0=\u00a05\u00a0\u03bcm (P1 and DP) or 15\u00a0\u03bcm (D1 and DD)\nThe dehydration treatment also had a small effect on DNA fluorescence of nuclei from P. yedoensis petals (Fig.\u00a06c). Superimposition of the nuclei prior to and after treatment showed no shift to lower DNA fluorescence (Fig.\u00a06c, P1\u00a0+\u00a0DP). In D. belladonna, the dehydration treatment resulted in lower G0\/G1 DNA fluorescence, and many nuclei with a lower than G0\/G1 fluorescence (Fig.\u00a06c). Superposition of the treatment and the control showed no shift of the G0\/G1 peak of DNA fluorescence (Fig.\u00a06c, D1\u00a0+\u00a0DD).\nFollowing the dehydration treatment, the nuclei of P. yedoensis had apparently somewhat increased in area, but no clear increase in nuclear area was discernable in D. belladonna (Fig.\u00a06d). The chromatin distribution in the nuclei, after the dehydration treatment (Fig.\u00a06d) was similar to that after dehydration due to abscission (Fig.\u00a03). Visual inspection indicated that about 80% of all nuclei had the morphology shown in Fig.\u00a06c, d. The area of the remaining nuclei had not as much increased.\nDehydration of petals in species that show petal wilting (Ipomoea and Petunia)\nSimilar dehydration treatments (60\u00b0C for 24\u00a0h) were carried out with I. nil and Petunia hybrida, species in which the petals wilt by the end of flower life span. Wilting is shown as inrolling in I. nil, which occurs at about 12\u00a0h after flower opening, in plants held at 24\u00b0C (Fig.\u00a07a). Wilting is observed after 7\u00a0days at 24\u00b0C in flowers attached to P. hybrida plants (Fig.\u00a07a).\nFig.\u00a07Effect of dehydration on fresh weight, DNA degradation, and DNA fluorescence of nuclei in the petals of I. nil and P. hybrida. Potted plants were grown in a greenhouse and then placed in a climate-controlled chamber (24\u00b0C, about 70% RH). Petals were detached from flowers that had just opened, and were placed at 60\u00b0C for 24\u00a0h to dehydrate. In a the time until about half of each petal had wilted (in days from full flower opening; n\u00a0=\u00a06) is shown in the left lower corner of the pictures. a Morphology of the flowers, at opening (left) and at petal wilting (right). All pictures have the same scale (bars\u00a0=\u00a010\u00a0mm). b Agarose gel analysis of total DNA isolated from the petals. c, d Flow cytometric determination of 5,000 DAPI-stained nuclei isolated from the petals of I. nil (c) and P. hybrida (d). Ip, Ipomoea; Pe, Petunia, D, dehydration\nFlowers were detached from potted plants, just after full opening. Fig.\u00a07b shows considerable DNA fragmentation on agarose gels, after the dehydration treatment (60\u00b0C for 24\u00a0h), in both species tested. Flow cytometry of Ipomoea petals held at 60\u00b0C for 24\u00a0h showed a large decrease in DNA fluorescence, compared to controls of Ipomoea (Ip) before desiccation (Fig.\u00a07c, compare DIp with Ip, whereby D means dehydrated). Fig.\u00a07d shows that DNA degradation was also found in Petunia (Pe). Superimposition of the fluorescence in controls and the treated samples showed two peaks (Fig.\u00a07c, d).\nThe morphology of DAPI-stained nuclei of I. nil and P. hybrida, after petal desiccation at 60\u00b0C for 24\u00a0h, is shown in Fig.\u00a08. Compared to nuclei of petals in flowers that had just fully opened (two panels upper left), relatively large parts not stained with DAPI were observed (two panels upper right). In I. nil the nuclear area had not become clearly increased, compared to those in petals that had not been desiccated (Fig.\u00a08a), but inflated nuclei were observed in P. hybrida (Fig.\u00a08b). Visual inspection indicated that about 80% of all nuclei showed the morphology shown in Fig.\u00a08b. The area of the remaining nuclei had not as much increased.\nFig.\u00a08Effect of dehydration on nuclear morphology in the petals of I. nil and P. hybrida. Nuclei were isolated from the petals of I. nil (a) and P. hybrida (b) after the indicated treatments, then observed using a fluorescence microscope under U-excitation. For comparison, the nuclei are shown during PCD of petals that remained attached to the plant (for data of Ipomoea and Petunia see also Yamada et al. 2006a, b). Stage S1: full flower opening; S2: onset of petal wilting; S3: full petal wilting; and S4: petals desiccated but still attached to the flower. Bars\u00a0=\u00a010\u00a0(\u03bcm\nBoth in I. nil (Fig.\u00a08a, S1\u2013S4) and in P. hybrida (Fig.\u00a08b, S1\u2013S4) the nuclear morphology after the dehydration treatment was quite different from the one observed during normal PCD. During PCD of petals that remained attached to the plant the area of the nuclei became increasingly smaller, whereas the brightness of DNA fluorescence remained high at least until the nuclei had become quite small.\nDiscussion\nIn reports on animal cells the suggestion that PCD occurs is usually backed up by showing changes such as phosphatidylserine externalization to the outer leaflet of the plasma membrane, caspase activation, and DNA degradation. Only the addition of a number of such parameters seems indicative of PCD. In the present study we took massive DNA degradation (measured in two ways), chromatin condensation, and a decrease in nuclear diameter as indicators of PCD. In other studies on petal PCD, it was shown that these parameters were each correlated with other indicators of PCD and also correlated with visible PCD symptoms such as petal wilting (Yamada et al. 2003, 2006a, b). We therefore suggest that these parameters, which separately represent only circumstantial evidence for PCD, are, when occurring together, adequate to indicate PCD.\nWe hypothesized that PCD parameters will not show in petal cells, prior to the abscission of turgid petals. The results of D. belladonna petals contradict the hypothesis, while those of P. yedoensis are in agreement with it. The data suggest that the degree of PCD, prior to abscission of turgid petals, depends on the species. The present data indicate that abscission of fully turgid petals may coincide with an advanced stage of PCD in many petal cells. The absence of visible wilting in abscising petals, therefore, does not seem to show absence of PCD. This is so because the wilting that is often associated with PCD is quite a late stage in the PCD process.\nIn petals the timing of PCD and that of abscission may be regulated independently. Independent regulation would explain the large variation of their relative timing, among the Angiosperms. In many species PCD comes much earlier than abscission (species showing petal wilting at the end of floral life), whereas in other species abscission comes first. In most species in which floral life span is ended by petal wilting, the petals eventually abscise, even when most of the petal surface has become desiccated. In these species the petal base, where abscission takes place, remains alive. Only in a few species fully desiccated petals are torn-off by the growing fruit, or (in still other species) remain attached to the fruit (van Doorn and Stead 1997; van Doorn 2001).\nThe agarose gel data (Fig.\u00a02) showed substantial DNA degradation prior to and during abscission in D. belladonna and less DNA degradation prior to and during petal fall in P. yedoensis. Flow cytometry data (Fig.\u00a04) confirmed the agarose gel data. DNA degradation seems a relatively late PCD process. Mass protein degradation, a late process, may serve as a yardstick. In Ipomoea petals, bulk degradation of proteins and DNA occurred at about the same time (Winkenbach 1970a, b). In Iris petals, 40% of total DNA had become degraded (van der Kop et al. 2003) by the time of the onset of mass protein degradation (Pak and van Doorn 2005). In Alstroemeria petals DNA breakdown (Wagstaff et al. 2003) started slightly before the decrease in protein content (Wagstaff et al. 2002).\nWhen using agarose gels to show DNA breakdown during PCD, a ladder of DNA fragments is often observed, at least in association with apoptosis in animal cells. DNA laddering on gels is the result of oligonucleosomal cleavage, resulting in DNA sequences of multiples of about 180\u00a0bp. In contrast to apoptosis in animal cells, the DNA breakdown pattern during plant PCD is quite variable and depends on the system. For example, during petal PCD clear DNA laddering without a background of DNA fragments of various lengths is rarely found. Most reports show DNA laddering against a background of fragments of various lengths (Orz\u00e1ez and Granell 1997; Wagstaff et al. 2003; Yamada et al. 2003) or only fragments of various lengths (van der Kop et al. 2003). The present results fall in the last category.\nNuclear morphology at the time of petal abscission also suggested advanced PCD in the petal cells of D. belladonna and little PCD in those of P. yedoensis. The nuclei of D. belladonna were much smaller and showed bright DAPI fluorescence, indicating chromatin condensation. No chromatin condensation was observed in P. yedoensis petals, but here it might possibly have occurred between the stages P2 and P3 and therefore may have been missed. We found that most but not all nuclei underwent the changes shown at the stages 2\u20134 of Fig.\u00a03. The difference might relate to differences in PCD progress, within the petal. It is known that cells associated with the vascular bundle undergo PCD at a later stage than the other petal cells. In some species the mesophyll cells also show earlier PCD than the epidermis cells (van Doorn et al. 2003).\nLittle is known about the physiology of petal cells, after abscission. The cells of abscised petals, as long as they remain alive, will be subject to a considerable decrease in water potential. Using young detached corollas (petals) of Ipomoea flowers, we observed that FW was about 65% of initial after 24\u00a0h on the laboratory bench (at about 20\u00b0C and 70% RH) and 50% of initial after 48\u00a0h (unpublished data). This indicated that some physiological processes would occur at least during day-1 and -2, but that these processes would be influenced by lower water potential.\nBoth agarose gel (Fig.\u00a02, P3 and D3) and flow cytometry analysis (Fig.\u00a04) showed DNA degradation after the petals had abscised. The data indicate that the PCD process, once started, goes on even under conditions of increasing water stress. However, by the time of total desiccation of the petals, the amount of degraded DNA on gels was smaller than at the time of visible petal wilting of the abscised petals (Fig.\u00a02, P4 and D4). This indicated that the DNA breakdown became halted by that time. This could be due to the fact that there was no more DNA, or might relate to the very low water potential in the tissue.\nAfter petal abscission, the petal nuclei unexpectedly increased in size. In both species the chromatin became unevenly distributed. The chromatin became also much less condensed, as indicated by decreasing DAPI brightness. The increase in nuclear size and uneven and reduced DAPI brightness were also observed after dehydration of young D. belladonna and P. yedoensis petals. In these experiments flowers were cut from the plant just after floral opening and then subjected to a desiccation treatment (24\u00a0h at 60\u00b0C). This treatment, apart from inducing dehydration, also involved an increase in temperature. The effects did not seem to be due to high temperature though, as preliminary experiments had shown that 72\u201396\u00a0h desiccation at room temperature had the same effects as 24\u00a0h treatment at 60\u00b0C. This indicates that the effect was due to dehydration rather than high temperature, and that it was not only taking place in cells that were relatively old, but also in much younger cells.\nI. nil and P. hybrida flowers both exhibit petal wilting rather than abscission at the end of floral life span. An increase in nuclear volume and a decrease in DAPI brightness were also found in the nuclei, if the flowers of these species were severed when just fully open and then subjection to a 24\u00a0h 60\u00b0C desiccation treatment. Preliminary experiments with these species had also shown that 72\u201396\u00a0h desiccation at room temperature had the same effects as 24\u00a0h treatment at 60\u00b0C. These results indicate that the particular nuclear morphology observed after dehydration in P. yedoensis and D. belladonna is also found in the petals of other species, which show petal wilting rather than petal abscission at the end of flower life. The particular nuclear morphology, therefore, seemed related to the low water potential as a result of the dehydration treatment. After the dehydration treatment in I. nil or P. hybrida the nuclear morphology was quite different from the one observed during wilting as the final phase of PCD. At the time of the normal senescence-related petal wilting in Ipomoea (Yamada et al. 2006b) and Petunia (Yamada et al. 2006a) flowers, the nuclei had become much smaller than at previous phases of development, and had a rather bright DAPI fluorescence. The desiccation treatment, therefore, induced changes quite different from those observed during PCD: a large nucleus with faint DAPI fluorescence rather than a small one with bright fluorescence.\nIt is concluded that the tested parameters indicated advanced stages of PCD prior to petal abscission in D. belladonna whereas PCD was much less obvious by the time of petal abscission in P. yedoensis. Dehydration of the petal cells, after abscission, was associated with a remarkable change in nuclear morphology and chromatin distribution. Similar changes in the nucleus were found after dehydration of younger petal cells, and after dehydration of the petal cells in two other species. This nuclear morphology has apparently not been described previously.","keyphrases":["abscission","programmed cell death","wilting","dna degradation","flow cytometry","petal senescence"],"prmu":["P","P","P","P","P","M"]}
{"id":"J_Abnorm_Child_Psychol-3-1-1915605","title":"Children\u2019s Feedback Preferences in Response to an Experimentally Manipulated Peer Evaluation Outcome: The Role of Depressive Symptoms\n","text":"The present study examined the linkage between pre-adolescent children\u2019s depressive symptoms and their preferences for receiving positive vs. negative feedback subsequent to being faced with an experimentally manipulated peer evaluation outcome in real time. Participants (n = 142) ages 10 to 13, played a computer contest based on the television show Survivor and were randomized to either a peer rejection (i.e., receiving the lowest total \u2018likeability\u2019 score from a group of peer-judges), a peer success (i.e., receiving the highest score), or a control peer evaluation condition. Children\u2019s self-reported feedback preferences were then assessed. Results revealed that participants assigned to the negative evaluation outcome, relative to either the success or the control outcome, showed a significantly higher subsequent preference for negatively tuned feedback. Contrary to previous work and predictions derived from self-verification theory, children higher in depressive symptoms were only more likely to prefer negative feedback in response to the negative peer evaluation outcome. These effects for depression were not accounted for by either state mood at baseline or mood change in response to the feedback manipulation.\n\u00a0\nDepression is a leading cause of disability across the life span (Murray & Lopez, 1996). Whereas the point prevalence of major depressive disorder (MDD) in school-age children is relatively modest (i.e., between 1 and 3 percent; Cohen et\u00a0al., 1993), epidemiological community surveys of childhood disorders suggest that 10 to 20 percent of school-aged children in the general population experience periods of elevated depressive symptoms, including depressed mood (e.g., Compas, 1997; Hammen & Rudolph, 1996). Several studies have shown that moderate levels of depression are associated with significant impairment in school and peer functioning (e.g., Nolen-Hoeksema, Girgus, & Seligman, 1992; Petersen, Sarigiani, & Kennedy, 1991), and may persist for years in some children (Nolen-Hoeksema et\u00a0al., 1992). Moreover, children displaying moderate levels of depressive symptoms are at high risk to suffer from severe, recurrent major depression later in life (e.g., Costello, Angold, & Keeler, 1999; Lewinsohn, Rohde, Klein, & Seeley, 1999). Taken together, although moderate levels of depressive symptoms may not meet DSM-IV-TR (APA, 2000) criteria for MDD, they are of significant concern.\nSeveral theorists have argued that feedback seeking may be implicated in the maintenance and\/or exacerbation of dysphoric states, including the development of full-blown depression. For instance, Coyne (1976) has asserted that people high in depression systematically alienate their relationship partners through excessive reassurance seeking. This behavior is particularly likely to elicit negative reactions in others\u2014including rejection, raising the risk of becoming even more depressed\u2014when people\u2019s feedback seeking reflects their suspicion that they will not receive such reassurance (e.g., constantly asking their partners if they still love them).\nWhereas Coyne suggested that depressed people merely inadvertently create the social conditions that make them suffer, the more recently advanced self-verification theory (e.g., Swann, 1990; Swann, Rentfrow, & Guinn, 2002) posits that people high in depression seek out negative social feedback more actively, as a means of verifying their negative self-conceptions. Specifically, according to self-verification theory (e.g., Swann, 1983; Swann, Pelham, & Krull, 1989; Swann, Rentfrow, & Guinn, 2002) individuals are drawn to feedback that confirms their self-conceptions because it tends to buttress their feelings of existential security (\u201cepistemic\u201d concerns) and\/or because they want their social interactions to unfold smoothly, and thus work to ensure that others do not form appraisals of them that are overly positive or overly negative (\u201cpragmatic\u201d concerns).\nNegative self-views including worthlessness, repulsiveness, and excessive self-criticism figure prominently in depression (e.g., Beck, 1967; Ellis, 1977). Consistent with self-verification theory, several studies among adults have provided evidence to suggest that people higher in depression are more inclined to actively seek out (or prefer) negative social feedback, as well as interaction partners likely to provide them with such unfavorable feedback. For instance, Swann and colleagues observed that depressed college students, relative to controls, preferred friends who evaluated them negatively, were more inclined to seek negative feedback from their roommates, and preferentially solicited unfavorable feedback from their dating partners (Swann, Wenzlaff, Krull, & Pelham, 1992). Moreover, when presented with self-discrepant (i.e., positive) feedback, adults high on depressive symptoms appeared to attempt to restore their sense of worthlessness by subsequently seeking feedback about their weaknesses from a different evaluator; whereas adults low on depressive symptoms were more likely to solicit feedback about their strengths (Swann, Wenzlaff, & Tafarodi, 1992). Finally, participants higher on depressive symptoms, relative to controls, were more likely to choose interacting with someone who appraised them negatively over the opportunity to avoid the encounter (Swann et\u00a0al., 1992).\nContrary to research with adults, the investigation of how depression in children is associated with feedback seeking preferences is still in its infancy. Joiner and colleagues (Joiner, Katz, & Lew, 1997) examined the linkage between depression and feedback seeking in a sample of inpatient youths. They found that children who were more depressed showed a greater preference for receiving feedback of a negative nature, based on responses to a questionnaire assessing feedback preferences in four distinct self-relevant domains (i.e., social, intellectual, athletic, and physical attractiveness). As noted by the authors, an important limitation of this study was the unknown predictive validity of their feedback preference questionnaire. Hence, it is unclear to what extent scores on this measure converge with participants\u2019 actual preference for negative feedback in the face of actually occurring events (see Joiner et\u00a0al., 1997).\nIn the only study to examine feedback seeking in children using a community sample, Cassidy and colleagues used a bogus feedback paradigm to examine children\u2019s actual feedback preferences (Cassidy, Ziv, Mehta, & Feeney, 2003). Specifically, participants provided personal information about themselves by completing a series of questionnaires. They were led to believe that their responses would be shown to peers from a different school who would use this information to answer specific questions of an evaluative nature about each participant. Next, participants were provided the opportunity to select some of the alleged answers to these questions, knowing that some answers would include negative feedback and others would include positive feedback. In line with findings for adults and children diagnosed with depression, children displaying higher depressive symptoms were more likely to display a relative preference for negative information about themselves. A similar pattern of findings consistent with self-verification theory was observed for the linkage between feedback preferences and global self-representations, as indexed by level of general self-esteem.\nAn important contribution of the Cassidy et\u00a0al. study is that it provides evidence to suggest that the tendency for depressed individuals to prefer rather negative feedback extends to moderately depressed children and adolescents, as indexed by level of self-reported depressive symptoms. However, the study is limited by its relatively narrow scope of inquiry with regard to the role of depressive symptoms. First, the potential role of state mood was not examined. Whereas self-verification theory claims that interest in negative feedback is motivated by negative self-views, an alternative explanation for the linkage between elevated depressive symptoms and more negative feedback preferences is that due to high levels of depressed state mood, people displaying elevated depressive symptoms are prone to be drawn to all that is negative, including unfavorable appraisals from others (e.g., Beck, 1967; Beck, Rush, Shaw, & Emery, 1979). Moreover, enhancing state mood with adults by providing them with experimentally manipulated favorable evaluations has been shown to increase levels of subsequent positive feedback seeking (Mischel, Ebbesen, & Zeiss, 1973).\nA second limitation of the Casssidy et\u00a0al. study is that the valence of the evaluative feedback was not manipulated. Beck has asserted that depressed people seek unfavourable feedback only insofar as their negative self-views have been activated by a stressful negative event. Work conducted by Swann and colleagues (Swann et\u00a0al., 1992) has shown that adults high in depressive symptoms respond quite differently with respect to their feedback preferences in the face of positive, negative, or neutral evaluations. Specifically, these individuals responded to positive\u2014i.e., self-discrepant\u2014evaluations with a more pronounced preference for negatively tuned feedback, relative to a control condition; whereas they were not more inclined to seek negative feedback subsequent to receiving unfavorable\u2014i.e., self-congruent\u2014evaluations.\nThe present study examined the linkage in pre-adolescent children between depressive symptoms and feedback preferences, in response to a salient and ego-involving emotion-eliciting event. Specifically, we examined these linkages in response to both a positive and a negative peer evaluation outcome. We chose peer evaluation as the emotion provocation stimulus because peer praise and peer rejection are common emotion-eliciting events in childhood (Coie, 1990). Moreover, ample evidence suggests that rejection ranks among the most aversive of human experiences and is associated with marked negative affect (e.g., Baumeister & Tice, 1990; Leary, Tambor, Terdal, & Downs, 1995). Finally, peer rejection figures prominently in the development and\/or maintenance of several forms of psychopathology, including depression (e.g., Nolan, Flynn, & Garber, 2003). The reason for our focus on pre-adolescent children is twofold. First, the most salient content of self-representations among pre-adolescents is one\u2019s social appeal and social skills\/attributes that influence interactions with others (Harter, 1998). Second, in this age group approximtely 50% of children\u2019s social activities involve peers (Grusec & Lytton, 1988; Rubin, Bukowski, & Parker, 1998), and peers as a reference group are of central importance in these children\u2019s lives (e.g., Hay, Payne, & Chadwick, 2004; Hartup, 1996).\nToward this aim, we devised an experimental peer evaluation manipulation based on the television show Survivor. In brief, participants were led to believe that they were playing an Internet version of Survivor against four same-sex contestants of comparable age (all of them were computerized bogus contestants) from four different schools in the same area. They were informed that a team of 16 same-age peer judges consisting of eight boys and eight girls would evaluate all participants. Specifically, each juror would give them a score between 0 and 100, with higher scores reflecting higher levels of perceived likeability. Moreover, the jurors would indicate what they liked most and what they disliked most about each contestant. Participants were randomized to one of the following three peer evaluation feedback conditions: (a) success feedback (i.e., having obtained the highest total score), (b) failure feedback (i.e., having obtained the lowest total score), or (c) control feedback (i.e., having obtained neither the highest nor the lowest score). These feedback conditions were designed to induce mood improvement, mood worsening, and no mood change, respectively. Subsequent to receiving the bogus peer evaluation feedback outcome, children\u2019s feedback preferences were assessed. The assessment of state mood at pre- and post-feedback afforded the possibility to examine the linkages between participants\u2019 level of depressive symptoms, feedback valence, (changes in) state mood, and subsequent feedback preferences. To increase the specificity of our findings for depressive symptoms, we also examined the potential role of children\u2019s peer-nominated social standing in affecting their feedback preferences.\nIn sum, the present study aimed to address the following specific research questions: (a) Does the type (valence) of peer feedback influence children\u2019s subsequent feedback preferences? (b) Are the effects of feedback valence moderated by children\u2019s level of depressive symptoms? (c) Do differences in state mood at baseline account for these potential effects of depression? and (d) Are the effects of feedback valence and depressive symptoms on subsequent feedback seeking mediated by changes in mood from pre to post-feedback?\nWe hypothesized that children assigned to receive a positive peer evaluation outcome would be more likely to prefer subsequent favorable feedback than those assigned to the neutral peer feedback condition; whereas children assigned to receive a negative peer evaluation outcome would not be more likely to prefer subsequent negative feedback. This set of predictions was based on findings with adults suggesting that positively valenced feedback leads to a subsequent preference for positively tuned feedback (Mischel et\u00a0al., 1973), whereas Swann and colleagues (Swann et\u00a0al., 1992) found that negative feedback exerted no effect on adults\u2019 subsequent preference for negative feedback. We also hypothesized that level of depressive symptoms would qualify the relationship between our peer feedback manipulation and children\u2019s subsequent feedback preferences, such that children higher on depressive symptoms would be more likely to respond to positive\u2014i.e., self-discrepant\u2014evaluations with a preference for negatively tuned feedback, relative to the control condition. Finally, we hypothesized that the effects of the peer feedback manipulation on subsequent feedback preferences would be mediated by changes in state mood from pre- to post-feedback.\nMethod\nParticipants\nParticipants were 142 children (73 boys, 69 girls) enrolled in 5th and 6th grade classes from two public elementary schools in the Netherlands. They were predominantly Caucasian (92.9%), ranged in age from 10 to 13 years (M\u00a0=\u00a011.2, SD\u00a0=\u00a0.66) and were predominantly from a middle-class SES background. For the initial sample of 185 children, classroom teachers sent parent permission letters home with children. Of the 165 letters returned, 142 parents (86.1%) gave their consent for their children to participate in the study, and 23 (13.9%) declined. We also obtained verbal permission to perform the study from the principal of the school and each child\u2019s teacher. Children were informed that they could discontinue their participation at any time.\nProcedure\nIn the first of two sessions, approximately one week apart, participants were administered the Children Depression Inventory (CDI, Kovacs, 1981) in their regular classrooms during school hours. Moreover, participants\u2019 social standing in their peer group was assessed via a commonly used procedure, which asks children to indicate whom among their classmates they like most and whom they like least (e.g., Newcomb, Bukowski, & Pattee, 1993). During administration of the measures the classroom teacher remained in the room. A research assistant read the directions aloud and children were encouraged to ask for help if they had questions or encountered problems completing the questionnaires. At the end of the first session, which lasted approximately 30\u00a0min, children were informed that later that week they would participate in a computer-contest. The second session was carried out in a quiet room on the school grounds. Participants were told that their class was selected to take part in an Internet computer-contest called \u2018Survivor.\u2019 In reality, the contest was a computer program written in Visual Basic designed to present the illusion of playing on-line with four other children.\nSurvivor contest\nUpon arrival, the participant was seated in front of a laptop computer equipped with a web-cam to have their photo taken. Participants were told that their picture would allow all the contestants to see what each of the other children looked like. Prior to beginning (Time 1), participants completed a computer-administered baseline mood measure, i.e., a one-item mood scale ranging from 1 (extremely negative) to 10 (extremely positive). In an attempt to add both to the credibility and the attractiveness of the contest, the opening bars of the hit \u2018Survivor\u2019 (produced by the band \u201cDestiny\u2019s Child\u201d) were played at the start of the game. In addition, an eye-catching logo of the American TV-show appeared on the computer display.\nThe objective and rules of the contest were presented on screen. Participants were encouraged to read the information, which was pre-tested on comprehensibility for children in this age-range, carefully in their own pace and click \u201ccontinue\u201d to progress to the next screen. Participants were informed that they would be playing against four same-sex contestants of comparable age (all of them were computerized fictitious co-players) from four different schools in the same area, and that all participants would be evaluated by a jury consisting of 16 members, eight boys and eight girls. Specifically, participants were explained that each judge would give them a score between 0 and 100, with higher scores reflecting higher levels of perceived likeability.\nAfter receiving this information, the computer displayed a screen announcing that in a moment pictures and names of all 16 judges would be presented one-at-a-time. The children whose pictures appeared were child actors from two different modeling agencies in the Netherlands. In a pilot study, 31 adults rated the attractiveness of 43 children\u2019s faces, (20\u00a0boys, 23 girls) on a scale ranging from 1 (extremely unattractive) to 10 (extremely attractive). For the purposes of the present study, we selected the eight children rated highest (four boys, M\u00a0=\u00a07.9, and four girls, M\u00a0=\u00a08.1) and the eight children rated lowest (four boys, M\u00a0=\u00a05.2, and four girls, M\u00a0=\u00a05.1). Following the viewing of these children, participants were directed through a series of screens in which they were asked to answer a series of questions that would give the members of the jury and the other contestants information about them. Participants responded to questions about their favorite musical group, hobbies, future occupation, things they liked and disliked about themselves, a number of character traits (e.g., sense of humor, agreeableness, intelligence, trustworthiness), how they got along with other children, and their academic performance. Most of the questions were in a multiple-choice format but some (e.g., \u201cwhat is your favorite musical group?\u201d) required an open-ended response. In view of the potential risk that children would respond to personal questions in a socially desirable fashion, instructions emphasized the importance of responding to questions honestly. To further minimize response bias, most questions were worded such that the \u201cbest\u201d or \u201coptimal\u201d answer was unclear (e.g., \u201cwhat is your favorite leisure activity\u201d?). Participants were informed on screen that their picture (previously taken by a web camera) along with the biographical information from their answers to the personal questions would be transmitted over the Internet and viewed by the judges who would then give them a \u2018likeability\u2019 score ranging from 0 to 100. Moreover, children were informed that the judges would also indicate what they liked most and what they disliked most of each participant (e.g., \u201c(s)he doesn\u2019t seem to be a nice person,\u201d \u201c(s)he is witty,\u201d \u201c(s)he is unattractive,\u201d \u201c(s)he seems fun to hang out with\u201d).\nSubsequent to answering all the biographical questions, participants were informed that pictures and descriptions of each of the other contestants would be presented one-at-a-time for review. Upon clicking \u201ccontinue,\u201d the picture of the first bogus co-player was displayed together with his or her self-description. The latter consisted of the alleged answers to the same questions that the participant had answered earlier. To enhance credibility of the bogus co-players, actual self-descriptions were taken from those of same-age children participating in another study. These participants gave their explicit consent to have this information viewed by other children, provided that the alleged self-description profiles would contain randomly combined personal information from at least three different children. Participants progressed through the game examining each of the profiles at their own pace.\nFollowing the participant scrutinizing the last profile, a message appeared on the screen indicating that the computer would now for every player add the scores from the judges to determine which player had received the highest total score and which player had received the lowest total score. After a 5\u00a0s waiting period, the names of the players with the highest and the lowest score appeared in capital letters on the screen. In the success condition, the name of the participant was displayed as having obtained the highest total score; one randomly chosen alleged co-player\u2019s name appeared as having obtained the lowest total score. Conversely, in the failure condition the name of the participant was displayed as having obtained the lowest total score, while one alleged co-player\u2019s name appeared as having obtained the highest total score. In the control condition, the participant received neither the highest nor the lowest score.\nFive seconds after receiving feedback (Time 2), participants were re-administered the mood measure via computer. Instructions emphasized the importance of rating how they felt \u2018right now.\u2019 Subsequent to completing this measure, participants were administered the feedback measure which assessed preferences for obtaining feedback from jurors that had evaluated them favorably versus jurors that had evaluated them unfavorably. Subsequently, a computer screen appeared announcing that children would now participate in another study. The participant was then accompanied to an adjacent room where a female research assistant debriefed him or her thoroughly.\nDebriefing\nEach child was thoroughly debriefed with the aim of removing any lingering effects of the false rejection feedback while participating in the Survivor contest. During the debriefing, the child was informed that the judges, the co-players and the received feedback were entirely fictitious and that this deception was a necessary part of the procedure. At this point it should be noted that in our previous work, more than 100 participants were assigned to receive Survivor-administered peer rejection feedback (Reijntjes et\u00a0al., 2006), including children with elevated depressive symptoms. Interviews with all participants, both immediately post-feedback as well as at one-week follow-up, indicated that the peer failure experience was not too emotionally upsetting. For instance, when asked, none of the participants made mention of any feelings of regret with regard to participation and none reported any objections to the procedure. On the contrary, several children\u2014spontaneously\u2014reported to consider the rejection experience an unnerving but useful experience, for themselves and\/or their peers. Still, during the investigation a registered clinical psychologist was available when needed. Moreover, before being dismissed children were interviewed at length about a recent positive social experience in which they felt positive and\/or were successful. In so doing, we aimed to maximize the remedy for children showing a strong emotional response to the peer feedback.\nTowards the end of the debriefing, participants were encouraged to ask questions or voice their concerns. All children reported that they understood the purposes of the research, as well as the necessity of having been deceived. The credibility of the deception manipulation was also assessed during the debriefing by asking each participant whether they had believed that they were playing against other children. With no exception, participants indicated that they believed that the game was genuine. Finally, all participants reported that prior to participating they had not talked with classmates about Survivor. At the conclusion of the debriefing, participants were urged to observe complete secrecy by not talking with their classmates about Survivor until all the other children had participated. To increase adherence to this instruction, children were asked to sign a non-disclosure agreement and were then provided a choice of one of several possible small gifts for playing the game (e.g., a small tape recorder, a gift certificate worth about 3 dollars).\nMeasures\nState mood\nSimilar to previous work among children (Reijntjes, Stegge, Meerum Terwogt, Kamphuis, & Telch, 2006) and adults (e.g., Gross, 1998), children rated their current feeling state using a one-item global mood index, ranging from 1 (extremely negative) to 10 (extremely positive). This Likert-scale was administered to assess participants\u2019 state mood at baseline (pre-feedback), and their changes in state mood from pre- to post-feedback. The mean score at baseline was 7.96 (SD\u00a0=\u00a01.36). Scores did not differ as a function of age, gender, or their interaction.\nChildren Depression inventory (CDI; Kovacs, 1981)\nThe CDI is a 27-item self-report measure designed to assess the social, behavioral, and affective symptoms of depression in children. Each item consists of three sentences that describe a symptom of depression in increasing degrees of severity. The respondent chooses the sentence that best describes him or her during the past week. Each item set is scored from 0 (symptom absent) to 2 (symptom is present always or most of the time). The CDI has adequate discriminant and convergent validity, test-retest reliability, and internal consistency (Saylor, Finch, Spirito, & Bennett, 1984). Coefficient alpha in the present sample, using the Dutch version of the instrument (Braet & Timbremont, 2002), was .80. Total scores ranged from 0 to 23; the median is 6. The top quartile (i.e., 24.6% of the participants) obtained a score of 12 or higher. Eleven participants (7.7%) obtained a score of 20 or higher, which is recommended as a cut-off point for general screening (Kovacs, 1992). Mean scores of this sample (M\u00a0=\u00a08.03, SD\u00a0=\u00a06.59) were similar to those previously reported (Reijntjes et\u00a0al., 2006; Smucker, Craighead, Craighead, & Green, 1986) and did not differ as a function of age, gender, or their interaction.\nTable 1Means and standard deviations of baseline measures by conditionFeedback conditionSuccess (n\u00a0=\u00a047)Neutral (n\u00a0=\u00a047)Failure (n\u00a0=\u00a045)MeasureMSDMSDMSDDepressive symptoms\u20077.837.18\u20078.346.78\u20078.116.00Social acceptance\u20073.7227.47\u20073.9027.63\u20073.0226.75Age (months)134.628.22134.178.11132.387.52\nSocial standing in the peer group\nParticipants completed a widely used nomination-based sociometric questionnaire, in which they identified the three classroom peers whom they liked most and the three classroom peers they disliked most (Newcomb et\u00a0al., 1993). Children who declined participation in the present study were included in the classroom lists, however the data collected on these children were not used. From these nomination data, two continuous scores for each participating child were computed. These scores included: (a) a measure of preference, by dividing the number of times each participant was nominated for the \u2018like most\u2019 question by the total number of participating children in the class, and (b) a measure of rejection, by dividing the number of times each participant was nominated for the \u2018like least\u2019 question by the total number of participating children in the class. Our measure of social acceptance (i.e., social standing in the peer group) was computed by subtracting the measure of rejection from the measure of preference. This figure was then multiplied by 100, yielding scores ranging from\u201484.6 to 61.5 (M\u00a0=\u00a03.56; SD\u00a0=\u00a027.10). These scores did not differ as a function of gender, age, or their interaction. For the majority of participants (58%), a positive social acceptance score was observed. Based on the widely used taxonomy for sociometric classification (Newcomb et\u00a0al., 1993), the number of children that were classified as \u201cpopular,\u201d \u201crejected,\u201d \u201cneglected,\u201d \u201ccontroversial,\u201d and \u201caverage\u201d amounted to 26, 26, 24, 8, and 58, respectively.\nSelf- reported feedback preference\nSubsequent to receiving the bogus peer evaluation outcome, participants responded to the measure assessing the valence of feedback preferences (i.e., positive versus negative). The specific dichotomous probe included: \u2018Which judges would you prefer to get feedback from, those that hold a positive view on you or those that hold a negative view on you\u2019?\nResults\nPreliminary analyses\nExploratory analyses revealed a departure from symmetry for the distribution of the CDI, as evidenced by a skewness value of 5.84. We therefore performed a square root transformation on the scores, which was successful in producing a distribution that was no longer skewed (skewness value is .73). This transformed variable (CDIt) was used in all analyses reported below. Participants reporting higher CDIt scores displayed lower levels of state mood at baseline (r\u00a0=\u00a0\u2212.26, p\u00a0<\u00a0.01). Moreover, higher CDIt scores were negatively associated with peer nominated social acceptance ratings (r\u00a0=\u00a0\u2212.21, p\u00a0<\u00a0.02).\nEquivalence of the experimental groups\nChildren were matched on age and gender, and then randomly assigned to one of the three experimental conditions. Means and standard deviations for Time 1 measures are presented in Table 1. To confirm that the randomization procedure resulted in comparable groups, baseline differences on continuous measures were examined using one-way ANOVAs. Results revealed no significant between-group differences.\nChange in state mood: Effects of the feedback manipulation\nMeans and standard deviations for state mood scores at Time 1 (baseline) and Time 2 (immediately post-feedback) are presented in Table 2. Two a priori repeated measures ANOVA\u2019s were performed to examine the magnitude and direction of emotional reactivity elicited by success feedback and failure feedback, each relative to the control (neutral) feedback condition. In the first planned contrast, state mood scores at Time 1 and Time 2 served as the dependent variables. Condition (Success vs. Neutral feedback) served as the between subjects factor, and Assessment phase (Pre-feedback vs. Post-feedback) served as the within subject repeated measures factor. Interaction effects were followed up with simple main effects analyses, comparing Time 1 vs. Time 2 scores for each feedback condition separately. In the second analysis, the planned contrast comparing Failure vs. Neutral feedback was tested using the same analytic approach as that outlined above for the Success vs. Neutral feedback contrast.\nTable 2Means and standard deviations for state mood at baseline (Time 1) and immediately post feedback (Time 2) by conditionFeedback conditionSuccess (n\u00a0=\u00a046)Neutral (n\u00a0=\u00a045)Failure (n\u00a0=\u00a042)MSDMSDMSDTime 17.831.528.031.088.021.47Time 28.831.258.191.196.842.05Note. Due to technical failures, data were not collected for 6 participants (1 randomized to the success condition, 2 to the neutral condition, and 3 to the failure condition).\nEffects for success feedback\nThe analysis comparing Time 1 vs. Time 2 scores yielded a significant effect for Time, F(1,\u00a089)\u00a0=\u00a034.70, p\u00a0<\u00a0.001, which was qualified by a significant Time by Condition interaction, F(1,\u00a089)\u00a0=\u00a018.53, p\u00a0<\u00a0.001, \u03b72\u00a0=\u00a0.17. Subsequent simple effect analyses for both conditions separately revealed a significant increase in state mood in the success condition, F(1,\u00a045)\u00a0=\u00a061.79, p\u00a0<\u00a0.001, \u03b72\u00a0=\u00a0.58, but no significant change in state mood in the neutral condition, p\u00a0>\u00a0.30. Taken together, these findings show that the success feedback condition elicited change in state mood in the expected positive direction, whereas the control feedback condition elicited no emotional reactivity.\nEffects for failure feedback\nThe analysis comparing Time 1 vs. Time 2 scores yielded a main effect for Time, F(1,\u00a085)\u00a0=\u00a012.84, p\u00a0<\u00a0.001, which was qualified by a Time by Condition interaction, F(1,\u00a085)\u00a0=\u00a021.08, p\u00a0<\u00a0.001, \u03b72\u00a0=\u00a0.20. Subsequent simple effect analyses for both conditions separately revealed a significant decrease in state mood in the failure condition, F(1,\u00a042)\u00a0=\u00a020.83, p\u00a0<\u00a0.001, \u03b72\u00a0=\u00a0.34, but no significant change in state mood in the neutral condition, p\u00a0>\u00a0.30 (see above). These findings indicate that the failure feedback condition elicited change in state mood in the expected negative direction.\nDo depressive symptoms, social acceptance, or gender moderate change in state mood?\nThe potential moderating effects of depressive symptoms, social acceptance, and gender on affective change were examined using the regression approach outlined by Aiken and West (1991). Separate hierarchical regression analyses were performed for the success feedback condition and the failure feedback condition, each relative to the control (neutral) feedback condition. In Step 1 of the first analysis, state mood score at Time 2 was regressed on state mood score at Time 1. In Step 2, feedback condition, gender (both dummy coded as 0 or 1), social acceptance score (centered), and CDIt score (centered) were entered. In Step 3, all three two-way interactions were entered, including the CDIt by Condition interaction term. Finally, the three-way interactions (e.g., Condition by Gender by CDIt) were entered in Step 4.\nModerator effects for success feedback\nChange in mood from pre to post-feedback was significantly predicted by feedback condition (see above). However, neither depressive symptoms, social acceptance score, nor gender moderated the relationship between feedback condition and mood change, as evidenced by non-significant interaction effects (p\u2019s\u00a0>\u00a0.25).\nModerator effects for failure feedback\nA similar analytic strategy as reported above for success vs. neutral feedback was employed. Results revealed that change in mood from pre to post-feedback was significantly predicted by feedback condition (see above). Again, none of the three within-person variables examined moderated the relationship between feedback condition and mood change, as evidenced by non-significant interaction effects (p\u2019s\u00a0>\u00a0.25).\nSelf-reported feedback preferences: Effects of the feedback manipulation\nSelf-reported feedback preferences for each of the three conditions are presented in Table 3. Results are reported for the total sample and for the top and bottom third as indexed by children\u2019s score on the CDI. Similar to the analyses reported above, we examined feedback preferences in response to success feedback and failure feedback, each relative to the control (neutral) feedback condition.Table 3Feedback preferences across conditions broken down by CDI statusFeedback conditionSuccess (n\u00a0=\u00a047)Neutral (n\u00a0=\u00a047)Failure (n\u00a0=\u00a045)PreferredPreferredPreferredPreferredPreferredPreferredCDI statuspositive (%)negative (%)positive (%)negative (%)positive (%)negative (%)Low (Bottom third) CDI<594.55.582.417.683.316.7High (Top third) CDI>1088.211.893.76.335.764.3Total sample91.58.591.58.566.733.3\nEffects of success feedback\nAs displayed in Table 3, there was no significant effect for Condition. Binominal tests showed that in both the success and the neutral condition children preferred significantly more positive feedback than expected by chance (p\u2019s\u00a0<\u00a0.001).\nEffects of failure feedback\nA Fisher-Exact test revealed a significant effect for condition, p\u00a0<\u00a0.004. This finding indicates that children randomized to receive a negative peer evaluation outcome, relative to controls, were significantly more inclined to subsequently prefer negatively tuned feedback. However, a binominal test revealed that children in the failure condition still preferred significantly more positive feedback than expected by chance, p\u00a0<\u00a0.04.\nDo depressive symptoms, social acceptance, or gender moderate feedback preferences?\nThe potential moderating effects of depressive symptoms, social acceptance, and gender on feedback preferences were examined using logistic regression analyses. In these analyses feedback preference (positive vs. negative) served as the binary outcome measure. In Step 1 of the first analysis, state mood at baseline (centered) was entered. In Step 2, feedback condition (success vs. control), gender (both dummy coded as 0 or 1), social acceptance score (centered), and CDIt score (centered) were entered. In Step 3, the two-way interaction terms were entered, including the CDIt by Condition interaction term. Finally, the three-way interaction terms (e.g., Condition by Gender by CDIt) were entered in Step 4. An identical analytic strategy as reported above was used for the failure vs. neutral contrast.\nModerator effects for success feedback\nFeedback preferences were not predicted by either state mood at baseline, gender, condition (see above), CDIt, or social acceptance score. Moreover, all interaction terms, including the interaction between CDIt and condition were not significant (p\u2019s\u00a0>\u00a0.20).\nModerator effects for failure feedback\nFeedback preferences were not predicted by state mood at baseline, gender, or social acceptance score. However, we observed a significant effect for condition (see above). Moreover, our findings yielded a significant effect for the interaction between CDIt and Condition (p\u00a0<\u00a0.05). Subsequent analyses revealed that children higher in depressive symptoms were significantly more likely to prefer negative feedback subsequent to the negative peer evaluation outcome (relative odds\u00a0=\u00a02.44, 95% CI\u00a0=\u00a01.14 to 5.22, p\u00a0<\u00a0.02). In contrast, no significant linkage between depressive symptoms and feedback preferences emerged in the control condition (p\u00a0>\u00a0.30).1\nAre the effects of feedback valence on feedback preferences mediated by changes in state mood from pre- to post-feedback?\nThe proposed mediation model depicted below was examined following Baron and Kenny (1986), who asserted that a variable functions as a mediator when it meets the following three conditions: (a) variations in the level of the independent variable account for variations in the presumed mediator (i.e., change in mood; path A); (b) variation in the level of the presumed mediator account for variations in the dependent variable (i.e., feedback preferences, path B); and (c) after controlling for paths A and B, a previously significant relation between the independent and dependent variables (i.e., path C) is no longer significant, with the strongest demonstration of mediation when path C becomes zero (Fig. 1).Fig. 1Mediational model of feedback preferences\nOur analyses revealed that the second condition for mediation was not met, as evidenced by a non-significant relation between changes in state mood and feedback preferences. Taken together, our findings show that neither state mood at baseline (see above), nor changes in affect from pre- to post-feedback were significantly associated with children\u2019s feedback preferences.\nDiscussion\nThe present study sought to advance our knowledge on how sub-clinical depression in pre-adolescent children is associated with self-reported feedback preferences. To our knowledge, this is the first study to investigate this linkage in response to an experimentally manipulated, ecologically relevant emotion-eliciting event in real time. In so doing, we included both a positive and a negative peer feedback manipulation, along with the assessment of the roles of state mood at baseline, changes in affect from pre- to post-feedback, and actual standing in the peer group. Data on participants\u2019 immediate changes in state mood in response to the feedback manipulation revealed that the Survivor contest was successful in eliciting differential affective reactions in the expected direction as a function of feedback valence. Moreover, our debriefing interviews revealed that children were involved in the activity and that none of the participants reported being aware that the feedback they received was bogus. Taken together, these data suggest that the Survivor contest was successful in achieving its major objective of providing a credible and ecologically relevant emotion-eliciting event.\nSeveral interesting findings emerged with respect to the examination of feedback preferences subsequent to receiving a positive (success) and a negative (failure) peer evaluation outcome, each relative to the control feedback condition. For instance, contrary to findings reported by Mischel and colleagues (1973), feedback preferences did not differ as a function of receiving a favorable versus a neutral control peer evaluation outcome. In contrast, relative to the control condition, the negative peer evaluation outcome lead to a significant increase in negatively tuned feedback preferences. However, it should be noted that the negative evaluation outcome merely induced a relative preference for negative feedback, as evidenced by the fact that in all three conditions children sought significantly more positive feedback than expected by chance.\nWhat might account for the finding that the effects of manipulated feedback on subsequent feedback preferences in the present study are at odds with those observed in prior work? One possible explanation may involve the (single and joint) effects of differences in the domain of feedback seeking, the age of the participants, and the specific measures that were employed. For instance, Mischel et\u00a0al. (1973) provided adult participants with feedback on an intellectual task, and observed that a favorable feedback outcome increased subsequent positive feedback seeking; whereas in the present study pre-adolescent children received feedback on a social task. Another explanation may be that our manipulated peer evaluation outcome differs from the types of manipulations used in other studies examining feedback preferences. It may be that the manipulation employed in the present study may have been more personally relevant (i.e., ego-involving), and hence elicited a different pattern of feedback preferences relative to exposure to less personally relevant feedback (e.g., impressions evaluators had allegedly formed on the basis of participants\u2019 displayed nonverbal behavior while delivering a speech; see Swann et\u00a0al., 1992). Specifically, it seems likely that participants in the present study were more concerned and\/or distressed after being rejected based on their personal and physical attributes, relative to participants receiving a negative evaluation based on their non-verbal behavior. Consequently, they may have displayed an increased interest in (preference for) negative feedback, which might be considered more diagnostic and informative on learning why the judges viewed them negatively.\nOur findings on the linkages between the valence of the manipulated feedback outcome and subsequent feedback preferences were not consistent with previous work among adults. In a similar vein, our results on the relationship between depressive symptoms and feedback preferences qualify the findings reported by Cassidy et\u00a0al. (2003). Specifically, in the present study the predictive role of depressive symptoms on subsequent feedback preferences emerged only for children in the negative feedback condition, such that children with higher levels of depressive symptoms displayed a significantly stronger preference for negative feedback. This depression effect was not observed in either the success or control conditions. Interestingly, across conditions we found that children\u2019s actual standing in the peer group was not associated with feedback preferences. In this context, it should be noted that in the Joiner et\u00a0al. (1997) study it was also observed that interest in negative feedback evidenced symptom specificity to depression.\nWhat might account for the observed effects of depressive symptoms on children\u2019s feedback preferences in response to the negative peer evaluation outcome? We considered the possibility that the observed effects were due to the higher levels of depressed mood observed at baseline. However, our findings indicated that this was not the case. Moreover, the observed linkage between depressive symptoms and feedback preferences cannot be explained by change in affect, given that change in affect from pre- to post-feedback did not mediate the single or joint effects of feedback valence and depressive symptoms on feedback preferences. These findings converge with the Joiner et\u00a0al. (1997) study showing that interest in negative feedback among depressed inpatient children was not associated with the emotional features of depression. We also considered the possibility that self-verification processes might account for the observed linkage between depressive symptoms and feedback preferences. However, our finding that the linkage was present in the failure condition but not in the success or neutral conditions is not in line with self-verification theory, which would predict depression effects across all three conditions. Rather, our findings are more consistent with the formulation put forth by Beck (1967), asserting that depressed (dysphoric) people seek negative feedback only insofar as their negative self-views have been activated by a potent stressful negative event.\nPossible implications of our findings deserve mention. The greater tendency for children high in depressive symptoms to seek out negative peer feedback in response to negative peer experiences might contribute to further negative evaluations from peers, and thus serve to maintain or exacerbate depressive symptoms in a cycle similar to that described by Coyne (1976). In line with this reasoning, considerable work has shown both concurrent and prospective linkages between heightened depressive symptoms (depression) in children and peer rejection (Panak & Garber, 1992), decreased peer interactions (Axelson et\u00a0al., 2003), and less secure peer attachment (Armsden & Greenberg, 1987; Armsden, McCauley, Greenberg, Burke, & Mitchell, 1990). Moreover, in a recent longitudinal study Borelli and Prinstein (2006) observed that a more negative feedback seeking style at baseline was positively associated with more negative perceptions of best friendship criticism at one-year follow-up, and positively associated with increases in depressive symptoms over time. However, this linkage was only found for girls.\nSeveral features of the present study deserve further comment. First, only self-reported feedback preferences were assessed, with no attention given to feedback seeking behavior. Future work should examine children\u2019s feedback seeking when they are free to choose how to divide their time between different sources of feedback information. Second, because all significant results are based on children\u2019s self-report, it is possible that our findings are partly due to shared method variance. Third, our findings are based on a community sample of children, rather than a clinical sample with a diagnosed mood disorder. Children\u2019s mean scores on the CDI suggest that depressive symptoms were relatively modest in magnitude. It is therefore an empirical question to what extent our findings can be generalized to children who meet criteria for major depressive disorder. Finally, this investigation focused only on children\u2019s feedback preferences in response to peer evaluation. While peer evaluation has the benefit of being both a potent elicitor of positive and negative affect as well as being ecologically sound, we should not assume that our findings are generalizeable to other relevant domains such as academic failure, interpersonal conflict, and loss or separation.\nNotwithstanding these limitations, the results of the present study extend our limited knowledge base on children\u2019s feedback preferences in several ways. First, our findings suggest that children respond quite differently with respect to their feedback preferences subsequent to positive or negative evaluations. Second, in line with the Joiner et\u00a0al. (1997) study, our findings contradict the view that the baseline feeling state of children high in depressive symptoms or manipulations of affect may be involved in governing their preference for more unfavorable feedback. Third, our findings provide evidence to suggest that children displaying elevated depressive symptoms may prefer favorable feedback so long as their negative self-views are not accessed by a potent negative stressful event.","keyphrases":["feedback preferences","peers","depressive symptoms","pre-adolescent children","sociometric status"],"prmu":["P","P","P","P","M"]}
{"id":"Eur_Radiol-2-2-1766022","title":"MR imaging of osteochondral grafts and autologous chondrocyte implantation\n","text":"Surgical articular cartilage repair therapies for cartilage defects such as osteochondral autograft transfer, autologous chondrocyte implantation (ACI) or matrix associated autologous chondrocyte transplantation (MACT) are becoming more common. MRI has become the method of choice for non-invasive follow-up of patients after cartilage repair surgery. It should be performed with cartilage sensitive sequences, including fat-suppressed proton density-weighted T2 fast spin-echo (PD\/T2-FSE) and three-dimensional gradient-echo (3D GRE) sequences, which provide good signal-to-noise and contrast-to-noise ratios. A thorough magnetic resonance (MR)-based assessment of cartilage repair tissue includes evaluations of defect filling, the surface and structure of repair tissue, the signal intensity of repair tissue and the subchondral bone status. Furthermore, in osteochondral autografts surface congruity, osseous incorporation and the donor site should be assessed. High spatial resolution is mandatory and can be achieved either by using a surface coil with a 1.5-T scanner or with a knee coil at 3 T; it is particularly important for assessing graft morphology and integration. Moreover, MR imaging facilitates assessment of complications including periosteal hypertrophy, delamination, adhesions, surface incongruence and reactive changes such as effusions and synovitis. Ongoing developments include isotropic 3D sequences, for improved morphological analysis, and in vivo biochemical imaging such as dGEMRIC, T2 mapping and diffusion-weighted imaging, which make functional analysis of cartilage possible.\nIntroduction\nArticular cartilage injuries are one of the most common types of injuries seen in orthopaedic practice. In a retrospective review of 31,510 knee arthroscopies, the incidence of chondral lesions was 63% [1]. Full-thickness articular cartilage lesions with exposed bone were found in 20% of patients, with 5% of these occurring in patients less than 40\u00a0years old [1].\nThe treatment of articular cartilage damage remains a challenge because cartilage has a limited capacity for spontaneous repair after a traumatic insult or degenerative joint disease [2]. Joint surface defects that exceed a critical size heal poorly and usually lead to osteoarthritis. As a result, several therapeutic strategies have been developed to restore articular cartilage and produce a durable repair. These methods may be arthroscopic or open surgical techniques and include marrow-stimulation techniques, osteochondral grafting, and chondrocyte implantation technique [3, 4].\nAs the use of articular cartilage repair techniques has become more widespread, techniques for imaging articular cartilage have become increasingly important. Arthroscopy is unsuitable for routine follow-up due to its invasive nature and associated risks. Conventional radiography does not allow direct visualisation of cartilage and arthrography combined with conventional radiography or computer tomography only provides information related to the cartilage surface [5]. By contrast, through the use of appropriate magnetic resonance imaging (MRI) techniques it is possible to evaluate the biochemical and biomechanical status of cartilage in addition to cartilage morphology. These benefits make MRI a powerful tool for the initial diagnosis and subsequent post-operative monitoring of cartilage lesions and cartilage repair tissue.\nArticular cartilage repair techniques\nMarrow stimulation\nThere are several established marrow stimulation techniques, including abrasion arthroplasty, subchondral drilling and microfracture. The principle of marrow stimulation techniques is to abrade (abrasion arthroplasty) or pierce (drilling and microfracture) the subchondral bone at the base of the cartilage defect, causing controlled bleeding and clot formation in the cartilage defect, which leads to the subsequent formation of fibrocartilage. The newly formed fibrocartilage may fill the defect and give relief of symptoms, but it lacks the structural, biomechanical and biochemical properties to sustain normal joint function in the long term [6]. Therefore, there is an increasing interest in osteochondral autografts and autologous chondrocyte implantation (ACI) therapies.\nOsteochondral grafting\nOsteochondral autograft transplantation is currently considered as the only surgical technique that provides and retrains proper hyaline articular cartilage [7]. Cylinders of autologous bone with their associated overlying hyaline cartilage are harvested from relatively non-weight-bearing areas of the knee and transferred into similarly sized holes created within the articular defect to be treated [8]. This technique is used most frequently at the knee and ankle joints [9, 10]. The main clinical indications for autologous osteochondral transplantation are focal cartilage defects, osteochondritis dissecans and osteonecroses. Autologous osteochondral transplantation is recommended for cartilage defects up to 3\u20134\u00a0cm2 [8].\nAutologous chondrocyte implantation (ACI)\nACI is currently considered the treatment of choice in young patients with a symptomatic full-thickness cartilage defect between 2 and 12\u00a0cm2 on the femoral condyle [11]. The classical ACI was first described in the mid-1990s and is a two-stage procedure. In the first stage, chondrocytes are harvested and cultured. In the second stage, a periosteal patch, usually harvested from the tibia, is sewn over the defect and the proliferated and differentiated cells are injected beneath the patch in order to fill the cartilage defect [12\u201314]. ACI provides significant and long-term benefits for patients in terms of diminished pain and improved function [15].\nMost complications are directly related to the periosteal graft and a revision arthroscopy rate between 4.8% and 60% has been described due to the problems with the periosteal flap [15, 16]. Early problems (<6\u00a0months) include periosteal graft detachment and delamination, and the most common late complication is periosteal hypertrophy. Delamination has been reported in 14% of patients undergoing ACI [17, 18]. Periosteal hypertrophy, has been reported to cause symptoms in 10\u201326% of the cases [11, 15, 19, 20].\nMatrix-associated ACT (MACT)\nThe necessity of a periosteal flap with classical ACI and the complications associated with this periosteal flap have led to the development of biomaterials as carrier for chondrocyte cells. The use of three-dimensional (3D) scaffolds has been shown to favour the maintenance of a chondrocyte-differentiated phenotype [21]. Thus efforts are now focused toward a tissue-engineering approach, which combines laboratory-grown cells with appropriate 3D biocompatible scaffolds.\nSecond generation ACI\nThe second generation of ACI technique use a bioengineered bi-layer collagen membrane rather than a periosteal flap [22, 23]. The use of a collagen membrane simplifies the surgical procedure and reduces overall surgical morbidity. Furthermore, the problem of periosteal hypertrophy can be avoided.\nThird generation of ACI\nFurther technological advances have led to the third generation of ACI, which use biomaterials seeded with chondrocytes as carriers and scaffolds for cell growth. These \u201call-in-one\u201d grafts do not need a periosteal cover or fixing stitches and can be trimmed to exactly fit the cartilage defect. The advantages of these new techniques are their technical simplicity, shorter operating time and the possibility to perform the surgery via a mini-arthrotomy or arthroscopy [23\u201326].\nMR techniques for cartilage and cartilage repair imaging\nAppropriate combinations of MRI techniques allow objective non-invasive measures of the properties of the grafted regions after biological cartilage repair, facilitating the longitudinal follow-up and evaluation of the repair tissue. MRI is currently the standard method for cartilage evaluation as it allows morphological assessment of the cartilage surface, thickness, volume and subchondral bone [27\u201333]. Additionally MRI techniques can be used to evaluate the biochemical and biomechanical status of articular cartilage. MRI is therefore ideal for the evaluation of the morphologic status of cartilage defects and the repair tissue throughout the post-operative period [34\u201337].\nMR evaluation of cartilage repair can be performed using the same acquisition techniques as used for native cartilage, as recommended by the International Cartilage Repair Society [29, 35]. The most commonly used MRI techniques are intermediate-weighted fast spin-echo (FSE), and 3D fat-suppressed gradient-echo (GRE) acquisition [27\u201329, 31\u201335]. Fast spin-echo (FSE) imaging combines heavy T2 weighting, magnetization transfer effects and relative preservation of high signal intensity in the marrow fat, so that the subchondral bone exhibits high signal intensity. In FSE imaging, cartilage appears dark against bright synovial fluid and there is consecutive high contrast between joint fluid and cartilage, and cartilage and bone marrow [40]. Collagen fibres with a highly regular structure, in particular near the bone-cartilage interface, tend to immobilize water molecules and promote dipolar interactions between their protons, thus accelerating T2 relaxation. T2-weighted FSE sequences are therefore useful for both the detection of surface and matrix damage assessed by intrachondral signal abnormalities. Proton density-weighted T2 FSE is of intermediate signal adjacent to the low signal subcortical bony plate, which allows to better visualise intrachondral abnormalities and lesions near to the cortical bone; whereas with T2-weighted FSE cartilage has a low signal intensity, which gives a high contrast to bright synovial fluid, resulting in better delineation of cartilage surface defects. The T2-weighted FSE sequence is relatively insensitive to magnetic susceptibility artefacts, which is advantageous in patients who have undergone previous surgery of the joint. FSE sequences are advocated because of the possibility of acquiring high-resolution images in a relatively short scan time and are normally included in the standard MRI protocol for the knee joint for detection of meniscal and ligamentous lesions [28, 31]. An exciting new development is a 3D FSE sequence [sampling perfection with application optimised contrasts using different flip angle evolutions (SPACE)] with isotropic voxels, which allows multi-planar reformatting in any plane without loss of resolution.\nThe advantage of fat-suppressed 3D spoiled gradient echo sequences is the relatively high signal intensity of articular cartilage in contrast to low signal intensity from the adjacent fat-suppressed tissue. Three-dimensional acquisitions yield images with higher resolution and contrast-to-noise ratio than 2D acquisitions. Fat-suppressed, 3D spoiled gradient echo imaging is easy to perform, widely available and, contrary to other cartilage imaging techniques, such as magnetization transfer imaging, it requires no post-processing of data and avoids misregistration artefacts [27, 31\u201333, 35, 38]. The 3D data set can be reformatted in any plane, allowing 3D visualisation and volume measurements [27, 30, 39]. New isotropic 3D gradient echo sequences such as DESS (double-echo steady-state), true FISP (fast imaging in steady state precession), balanced FFE (fast field echo), VIBE (volume interpolated breath-hold examination) and MEDIC (multi-echo data image combination), with a voxel size down to 0.5\u00a0mm3, at 3 T, and also at 1.5 T with a high gradient strength, have been developed and seem to be very promising for cartilage imaging. However, the usefulness of these techniques is yet to be validated in clinical studies. Caution should be exercised as a flare phenomenon in the double-echo steady-state sequence has been shown to sometimes cause a spurious wavy appearance of a normal graft or cartilage surface [41, 42]. Furthermore, there are limitations in the sensitivity of the fast low angle shot (FLASH) and double-echo steady-state sequences for depicting cartilage abnormalities and opinions vary regarding which sequence is superior for cartilage imaging [42\u201344].\nIndirect MR arthrography has been advocated in the evaluation of ACI [20, 29]. The indirect technique is performed by intravenous administration of contrast agent followed by a period of joint exercise, which leads to uptake of contrast agent by the synovial tissue and subsequent diffusion into the joint cavity. Indirect MR arthrography can be particularly helpful in differentiating delamination of the base of the graft from normal high signal intensity repair tissue in the immediate postoperative period [20, 29]. Thus, indirect MR arthrography may play a role in the evaluation of cartilage repair techniques in the early post-operative period.\nQuantitative MRI of articular cartilage\nQuantitative geometric measurements of cartilage parameters, such as cartilage thickness and volume, have been suggested as sensitive image based biomarkers for detecting and monitoring cartilage degeneration in osteoarthritis [45]. This has been possible due to the development of higher field magnets and stronger gradients systems as well as a parallel improvement in image analysis and processing techniques. New techniques allow rapid fully automated generation of accurate 3D reconstructions of articular cartilage layers [46]. Three-dimensional reconstructions can be generated from pre- and post-operative MR examinations and spatially registered to facilitate longitudinal comparisons of the cartilage repair site. This area holds a lot of promise for cartilage repair surgery planning; with on-going work to co-register biochemical and biomechanical sequences, such as dGEMRIC, with high-resolution 3D sequences.\nHigh-resolution MRI\nMost MRI studies on articular cartilage have tried to optimise pulse sequences that accentuate the contrast-to-noise ratio for cartilage, but less attention has been paid to image resolution.\nRubenstein et al. [47] demonstrated that the image resolution of standard MR sequences is insufficient to detect fraying of the articular surface of cartilage. It has also been reported that the smooth articular surface of healthy cartilage is indistinguishable from early superficial degenerative changes [27, 31\u201333]. Therefore, MRI of cartilage repair tissue must be performed at a sufficiently high resolution to detect early surface changes.\nIn order to obtain a sufficient resolution for cartilage repair imaging a 1.5-T MR scanner with a high performance gradient system and a dedicated extremity coil (quadrature\/phased array coil) is a minimum requirement. Three-Tesla clinical MR systems are becoming more widespread and can generate images with both a high signal-to-noise ratio and a high resolution.\nIn our experience with cartilage repair patients, the signal-to-noise ratio issue arising with standard field MR units can be partially resolved by using surface coils and optimising the pulse sequence to increase resolution for a given imaging time. Surface coils provide a high signal-to-noise ratio allowing a small field of view and high matrix size to be used resulting in an in-plane resolution of 0.234\u00a0mm2 acquired in a clinically acceptable time. However, the use of a surface coil is limited to the evaluation of one knee compartment; since cartilage repair is usually restricted to one compartment, this rarely poses a problem.\nUsing this approach an appropriate assessment of the cartilage repair tissue can be made. Moreover, this approach formed the basis for the definition of pertinent variables for describing articular cartilage repair tissue following biological cartilage repair [48].\nMR evaluation of the biochemical and biomechanical status of cartilage\nVarious tissue parameters which may be evaluated by MRI reflect the biomechanical properties of articular cartilage. The T1 and T2 relaxation times and apparent diffusion constants change during the cultivation of cartilage implants [49], indicating that the biomechanical properties of cartilage implants change as the graft matures. Furthermore, the spatial distribution of the T2 relaxation time can be used for in vivo monitoring of the biomechanical properties, pathological changes or aging of various cartilage layers [50, 51].\nGlycosaminoglycans (GAG) are the main source of fixed charge density (FCD) in cartilage and changes in GAG concentration are one of the features of cartilage graft maturation. Intravenously administered gadolinium diethylenetriamine pentaacetate anion (Gd-DTPA2\u2212), equilibrates in inverse relation to the FCD, which is in turn directly related to the GAG concentration. Therefore, T1 which is determined by the Gd-DTPA2\u2212 concentration becomes a specific measure of tissue GAG concentration. The delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) technique can be considered the method of choice for visualisation and quantitative evaluation of proteoglycan in articular cartilage [52, 53]. The dGEMRIC technique is feasible at 3 T [54] and is useful for evaluating cartilage repair [55].\nIn vitro studies show that the dGEMRIC index has a good correlation with cartilage biochemical properties [56]. However, in vivo studies of the dGEMRIC index in ACI grafted tissue are not so clearly correlated with stiffness of the repair tissue. The differences are presumably due to differences in the collagen content and architecture of the repair tissue. Therefore, the both GAG (dGEMRIC) and collagen (T2 mapping) imaging techniques are required when evaluating the biomechanical status of cartilage repair tissue.\nMR classification systems of cartilage implants\nFor the long-term follow-up, evaluation and classification of cartilage repair tissue clinical scores in addition to morphological and biochemical evaluation of biopsies taken during control arthroscopies have previously been used [37, 57].\nFor optimal use of MRI in the evaluation of cartilage repair tissue, a simple evaluation and a point scoring system that allows efficient statistical data analysis is necessary. A few different classification systems for the description of articular cartilage repair tissue have been proposed [37, 48] (personal correspondence with Dr C. Winalski, Brigham and Womens Hospital, Boston, Mass., USA); however, early systems have certain limitations and deficiencies that can potentially lead to confusion.\nRoberts et al. [37] used four parameters to assess cartilage repair on MR images: surface integrity and contour, cartilage signal in graft region, cartilage thickness and changes in underlying bone. A score is obtained by summing the values of the four parameters; scores range from 0, no repair, to a maximum of 4, complete repair. Unfortunately the system only assesses each parameter as normal or abnormal and provides no assessment of the graft integration, degree of defect fill or the presence of adhesions.\nThe magnetic resonance observation of cartilage repair tissue (MOCART) scoring system, defined by our working group and shown in Table\u00a01 [48], was designed to systematically record only those observations that can be most accurately and reproducibly determined and to avoid the use of ambiguous terms [48]. The MOCART system has been shown to be reliable and has excellent inter-observer reproducibility for the defined variables [58]. The system is very helpful for the longitudinal follow-up of cartilage repair patients [58], and facilitates prospective multi-centre studies comparing different cartilage repair techniques. \nTable\u00a01The MOCART scoring systemVariableClassesDegree of defect repair and defect fillingComplete (on a level with adjacent cartilage)Hypertrophy (over the level of the adjacent cartilage)Incomplete (under the level of the adjacent cartilage; underfilling)\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0>50% of the adjacent cartilage\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0<50% of the adjacent cartilageSubchondral bone exposed (complete delamination or dislocation and\/or loose body)Integration to border zoneComplete (complete integration with adjacent cartilage)Incomplete (incomplete integration with adjacent cartilage)demarcating border visible (split-like)Defect visible\u00a0\u00a0<50% of the length of the repair tissue\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0>50% of the length of the repair tissueSurface of the repair tissueSurface intact (lamina splendens intact)Surface damaged (fibrillations, fissures and ulcerations)\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0<50% of repair tissue depth\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0>50% of repair tissue depth or total degenerationStructure of the repair tissueHomogeneousInhomogeneous or cleft formationSignal intensity of the repair tissueDual T2-FSE\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Isointense\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Moderately hyperintense\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Markedly hyperintense3D GE-FS\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Isointense\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Moderately hypointense\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Markedly hypointenseSubchondral laminaIntactNot intactSubchondral boneIntactOedema, granulation tissue, cysts, sclerosisAdhesionsNoYesEffusionNo effusionEffusion\nMR findings following osteochondral autografting\nDue to the invasive nature of arthroscopy, MRI has become the most important tool for the follow-up of patients with osteochondral autografts. There have been a limited number of studies focusing on the morphologic assessment of osteochondral grafts during clinical follow up. With the exception of the study by Link et al. [59], these studies have evaluated only relatively small numbers of patients [60, 61].\nAn MRI evaluation of osteochondral grafts should include: the number and size of the grafts; bone and cartilage integration, the cartilage surface contour; the contour of the cartilage bone interface; an assessment of the signal in the graft, the adjacent bone marrow and at the donor site; details of any soft tissue abnormalities and an assessment of the contrast enhancement patterns.\nCartilage and bone integration and congruity\nWhen assessing integration and surface congruity following osteochondral grafting cartilage and bone should be considered separately. With respct to the cartilage, Link et al. [59] found only 15% of patients had an incongruity of the cartilage-cartilage interface. Furthermore, no substantial defects or irregularities of the cartilage overlying the bony cylinders were seen on MRI (Fig.\u00a01). Lastly, gaps between cartilage plugs and between cartilage plugs and adjacent native cartilage were rarely visualised in this study [59] (Fig.\u00a02).\nFig.\u00a01Normal cartilage integration of osteochondral autografts in the weight bearing region of the femoral condyle in a patient 2\u00a0years after osteochondral autograftsFig.\u00a02Cystic cavities in the osteochondral graft area with fissure-like gap (arrow) between cartilage caps of the grafts in a patient 2\u00a0years after osteochondral autografts. Both signs are associated with a poor prognosis\nSanders et al. [61] examined 21 patients between 1 and 22\u00a0months following osteochondral transplantation using a series of five dynamic contrast-enhanced sequences at 1-min intervals post contrast. Their findings were very similar to Link et al. [59] in terms of cartilage-cartilage interface incongruity.\nIn a study to evaluate the use of indirect MR arthrography in examinations of osteochondral graft patients Herber et al. [60] found that indirect MR arthrography helped to identify a persistent fissure gap between the implanted cartilage and native cartilage. They showed that the grafts were well seated and the cartilage-cartilage interface demonstrated a smooth contour in the majority of cases. Moreover, the authors felt that indirect MR arthrography was superior to unenhanced imaging in the assessment of the cartilage surface. Their results highlight a potential role for indirect MR arthrography in challenging cases.\nRegarding the bony integration, Link et al. [59] reported that poor bony integration of the osteochondral cylinder may be suggested by the presence of cystic cavities with fluid-like signal intensity and\/or a persistent oedema-like signal within the subchondral bone.\nCartilage and bone signal intensity\nFrom 105 osteochondral cylinders in 55 patients (51%) Link et al. [59] reported bone marrow signal intensities consistent with oedema (hypointensity on T1-weighted images and hyperintensity on the fat-suppressed T2-weighted or PD-weighted images), during the first 12\u00a0months post-operatively (Fig.\u00a03). During the 12\u201324\u00a0month period this dropped to 17%. They found cystic changes in the osseous component of the graft in four of 99 cylinders. Furthermore, eight cylinders showed no or partial enhancement after contrast agent was administered and was reported as necrosis. The T2 signal intensities varied depending on whether the osteonecrosis caused a fibro\/sclerotic or cystic degeneration. This limits the usefulness of T2-weighted images for diagnosing graft osteonecrosis. Interestingly, only two of the six patients who showed signs of osteonecrosis of one or more cylinders had associated clinical abnormalities [59]. These osteonecroses of the graft cylinders did not lead to collapse of the bone or pathological changes of the cartilage that could be visualised by the MRI. Since cartilage derives its nutrition almost exclusively from the synovial membrane, this may provide an explanation. By comparison, Sanders et al. [61] and Hangody and Fules [9] reported no cases of osteochondral necrosis in their patient groups.\nFig.\u00a03Coronal STIR images 12\u00a0weeks (a), 52\u00a0weeks (b) and 2\u00a0years (c) after osteochondral transplantation show gradual resolution of severe bone marrow oedema in and around the grafts as bony incorporation occurs\nWhen the cartilage layer of the osteochondral cylinder was examined, the cartilage signal intensity of the graft was similar to the surrounding cartilage in the vast majority of cases, 86% [59]. These findings are supported by Sanders et al. [61], who found that the graft cartilage signal intensity was similar to adjacent cartilage in most cases. Additionally they found similar rates of graft and perifocal graft oedema.\nGraft and adjacent bone\nSubchondral bone marrow oedema is often present in the early post-operative phase but usually resolves as the graft incorporates into the subchondral bone. A normal fatty marrow signal is seen within and around the plugs when solid bony incorporation occurs (Fig.\u00a04). Herber et al. [60] examined ten patients at 3, 6 and 12\u00a0months using indirect MR arthrography and noted a high rate of early post-operative subchondral marrow oedema, which settled in most cases by 12\u00a0months.\nFig.\u00a04a, bSagittal T1-weighted SE images of the normal development of autologous osteochondral transplants. a Marked oedema in and around the osteochondral plugs at the recipient site 12\u00a0weeks (arrows) after surgery and b bony incorporation of the grafts with fatty bone marrow in and around the grafts (arrows) and filling of the donor site with cancellous bone after 2\u00a0years\nNormal findings\nThe results of these earlier follow-up studies have helped to define normal and abnormal MR findings following osteochondral autografting and help in the identification of possible complications.\n\u201cNormal\u201d MR findings associated with after osteochondral autografting include bone marrow oedema in and around the grafts in approximately 50% of the subjects during the first 12\u00a0months, with a gradual reduction thereafter. However persistent oedema may be seen in a small number of cases for up to 3\u00a0years post-operatively. Joint effusion and synovitis appear to follow a similar trend. Incongruities at the bone-bone interface occur frequently, while incongruities at the cartilage-cartilage interface occur in approximately 15% of grafts. The sometimes substantial incongruities of the bone-bone interface should not be interpreted as an abnormal finding or complication. Differences occur because the cartilage thickness of the donor site commonly differs in thickness from the implant site, but the cylinder is inserted to a depth to ensure a smooth cartilage surface.\nAbnormal findings\nComplications which can be determined by MRI include graft loosening or migration, incongruencies of the cartilage-cartilage interface, significant gaps between osteochondral plugs and adjacent native cartilage and partial or complete necroses of the grafts (Figs.\u00a05, 6). However, it should be noted that over time fibrocartilagenous tissue fills the gaps between osteochondral plugs and adjacent native cartilage thereby improving surface congruity.\nFig.\u00a05Incongruity at the cartilage-cartilage interface on a medial femoral condyle 12\u00a0months after osteochondral autograftingFig.\u00a06Osteonecrosis of the osteochondral autograft 2\u00a0years after surgery shown by marked hypointense signal alteration of the graft on T1-weighted SE image in the sagittal plane (arrows)\nFuture studies in this field should provide better understanding of the pathophysiology of transplanted hyaline cartilage and its function, which is important for the long-term prognosis of these patients.\nMR findings following ACI and MACT\nAssessment and interpretation of MR examinations for ACI and MACT patients should be performed in a systematic fashion. Careful attention should be paid to the degree of defect filling, the integration of the graft to adjacent cartilage and underlying bone, the graft\u2019s internal structure and surface, its signal intensity and any changes in the subchondral bone. Last but not least, the presence of adhesions to the graft or joint effusion should be evaluated.\nDefect filling\nOne of the major goals of ACI and MACT is to ensure the graft cartilage has the same thickness as the adjacent native cartilage in order to restore the smooth contour of the articular cartilage surface (Fig.\u00a07). When evaluating defect fill MRI has been shown to be effective in detecting cases of incomplete filling, either focally or globally [20, 29, 34, 36].\nFig.\u00a07A sagittal FSE image of a stable cartilage implant at 2\u00a0years after MACT surgery shows complete filling of the defect (arrows mark the borders of the implant)\nLongitudinal MRI follow-up of ACI patients has shown that a consistent volume of repair tissue can be visualised after 3\u00a0months, which remains stable for at least 2\u00a0years post-operatively [62]. Tins et al. [63] demonstrated that 63% of patients in their series had normal cartilage thickness compared with native articular cartilage. Roberts et al. [37] also found a similar percentage of grafts to have normal cartilage thickness compared with adjacent native cartilage. In a different study with a 2-year MR follow-up [64], complete filling of the defect by repair tissue was seen in 65.2% of patients.\nGraft hypertrophy is often asymptomatic, but may produce pain and catching. It usually occurs between 3 and 7\u00a0months and has been reported to complicate between 10% and 39% of cases [15, 18, 65]. Graft hypertrophy is seen on MRI as the ACI graft protruding above the level of the native articular cartilage and may involve part or the full width of the graft (Fig.\u00a08). It is important to note that hypertrophy of grafts close to the intercondylar notch may cause impingement on the anterior cruciate ligament. Treatment consists of arthroscopic debridement of the hypertrophied tissue.\nFig.\u00a08Severe hypertrophy of cartilage implant 104\u00a0weeks after MACT surgery on a sagittal T2-weighted FSE image\nThe reported incidence of graft hypertrophy following MACT is lower than with the classical ACI with periosteal flap technique [22, 66]. We found an incidence of 20% graft hypertrophy following MACT surgery [64], which is similar to the hypertrophy rate following classical ACI surgery [18, 67]. Interestingly, however, in three out of four cases the graft hypertrophy resolved, with the cartilage thickness returning to the level of adjacent cartilage within one year. This was hypothesised to be due to increasing weight bearing during rehabilitation and subsequent remodelling of the repair site.\nHenderson et al. [19] analysed 81 lesions in 58 knees 12\u00a0months after ACI and reported that MRI showed 81.6% of the lesions had normal or nearly normal cartilage at the site of repair. These investigators analysed four categories, including the filling and the signal intensity of the repair site, as well as the presence and severity of bone-marrow oedema and effusion. A complete fill of the repair site was found in 79%, 13.6% had >50% filling, and 2.5% had <50% filling. By comparison, we found filling to the level of adjacent cartilage in 65.2% and underfilling in 17.4% in a 2-year follow-up of MACT patients using Hyalograft C [64].\nIntegration\nThe interface between ACI and native cartilage should be indiscernible, a fluid-like split, in particular a broad split or one that extends beneath the base of the ACI has been described as pathological [20, 34, 36] (Fig.\u00a09). Poor graft integration can be identified on high-resolution sequences by fluid signal clefts or ill-defined high signal intensity at the interface between the graft tissue and native cartilage [15, 18, 20, 34, 36, 67].\nFig.\u00a09A split-like integration defect, seen on a sagittal FSE image, 52\u00a0weeks after surgery. Arrows mark the borders of the implant, the right-hand arrow points to the defect\nThe clinical importance of a full-thickness fissure or a broader gap, suggesting incomplete integration, is still unclear. Potentially, a gap between the native cartilage and the edge of the graft may act as a focal point for cartilage wear [36]. We found integration to be complete in 18 out of 23 knees after 2\u00a0years [64]. One patient had incomplete integration with a split-like fissure between the graft and adjacent cartilage. Four patients showed incomplete integration with a slightly broader gap between the graft and adjacent cartilage (17.4%).\nPoor integration of the ACI repair tissue to the bone or to the adjacent native cartilage may result in delamination of the graft from the underlying bone (Fig.\u00a010). On MRI, a delaminated graft may appear as a loose body in the joint if it has dislocated, or if still in situ at the repair site, a thin rim of fluid between the base of the graft and the subchondral bone plate, resembling a cartilage flap, may be seen [20, 34]. Clinically, patients may complain of pain, swelling or locking. This complication may occur in between 5% and 14% of cases [15, 18].\nFig.\u00a010A sagittal FSE image of incomplete delamination of the repair tissue 24\u00a0weeks after MACT surgery. Fluid partially demarcates the bone interface\nStructure and surface\nThe articular surface of the ACI or MACT site should appear smooth and be continuous with the adjacent native cartilage. Irregularities of the graft surface on MRI have been described previously and seem to be relatively common [19, 20, 34, 68, 69]. This is in keeping with findings during follow-up arthroscopy [70].\nIn some patients, we have seen a transition from an initially irregular surface to a regular smooth surface over time (Fig.\u00a011). We believe this may represent continuous organisation of the graft as it matures. In contrast, the development of surface defects over time should be considered as abnormal [62].\nFig.\u00a011Gradual integration and normalisation of the graft surface and internal structure after MACT surgery from (a) 12\u00a0weeks to (b) 24\u00a0weeks on sagittal T2-FSE image\nThe internal structure of normal hyaline cartilage usually has a trilaminar appearance on most sequences [71]. Although the widths of these signal bands seen on MRI did not correlate with the thickness of histological layers and were affected by chemical shift and the magic angle effect, they represent a homogeneous layering present in normal collagen architecture. Regarding the internal structure of the graft we found only eight out of 23 patients had homogeneous repair tissue at their first follow-up scan. In a further eight patients the repair became homogeneous with signal layering during the follow-up period [62]. This most likely represents a normal maturation process, whereas the reverse process with repair tissue going from a homogeneous to inhomogeneous appearance over time may be considered abnormal.\nSignal intensity\nIn addition to a smooth surface and homogeneous signal layering, the signal intensity of repair tissue should resemble normal hyaline cartilage. The post-operative signal characteristics seen after ACI surgery are clearly affected by the choice of sequence used. In the uncomplicated case, there is a gradual change in the signal characteristics of the repair tissue over time to resemble those of normal articular cartilage [62, 72] (Fig.\u00a012). Typically, when a FSE sequence is used the signal intensity of the ACI\/MACT repair tissue steadily decreases with time. By contrast the signal intensity steadily increases over time with fat-suppressed T1-weighted GRE sequences [19, 62, 65].\nFig.\u00a012a\u2013eChanges of implant signal intensity, on PD and T2-FSE images following MACT. a, b Fluid-like signal after 4\u00a0weeks; c, d hypointensity at 24\u00a0weeks and e isointensity with native hyaline cartilage after 52\u00a0weeks\nIn the early post-operative period (4\u00a0weeks) the graft may have a fluid-like appearance, which may be misinterpreted as complete delamination. However, on high-resolution imaging the surface of the implant is seen as a thin dark line, this feature is more commonly seen with classical ACI [34]. Several report show that at approximately 12\u00a0months post-operatively the signal intensity of the ACI graft on both FSE and fat-suppressed GRE sequences resembles native hyaline cartilage and can no longer be differentiated from it [19, 20, 34, 68, 69]. By contrast, Brown et al. [17] found that a significant proportion of grafts (69%) remained hyperintense on FSE sequences more than 18\u00a0months after surgery.\nIn clinical follow-up studies, Henderson et al. [19] reported signals identical to the adjacent articular cartilage in 63% of patients following classical ACI grafting and nearly normal signals in 29.6% at 12\u00a0months. In our group of MACT patients, we found all grafts evaluated at 24\u00a0months were isointense [64]. These differences may be attributable to the different evaluation times and the further maturation of the graft over the additional 12-month period in our study.\nSubchondral lamina and bone\nThe subchondral lamina is normally not violated during the ACI or MACT procedure and therefore should be intact. However, cartilage repair for cases of osteochondritis dissecans or involving partial removal of subchondral sclerosis may be associated with defects of the cortical endplate. An intact cortical endplate which becomes damaged during follow-up may be the result of over-use or recurrent trauma [62]. The bone contour underneath the graft may show the presence of central osteophytes, defects, and irregularities. When central osteophyte formation occurs beneath a graft the surface of the overlying cartilage is usually smooth and level with adjacent cartilage. The importance of central osteophytes has still to be determined [63].\nSubchondral oedema commonly occurs at the repair site in the early postoperative period [17, 19, 29, 34, 63, 68], and has been described as part of the normal healing process during the first 3\u00a0months following surgery [20, 34]. Abnormal loads transmitted to the bone have also been suggested to account for the marrow oedema [73]. The presence of oedema-like marrow signal beyond 12\u00a0months or an increase in intensity of oedema should be considered abnormal and requires close clinical follow-up [20, 34]. Possible reasons for persistent or reappearing subchondral bone marrow oedema include: over-use, an abnormality of the leg axis and new trauma. Conversely, it has been reported that the persistence of oedema-like signal intensity is a sign of yet undetermined importance [34]. For the diagnosis of bone marrow oedema, a fat-suppressed sequence such as STIR or fat-suppressed FSE is required. Cystic changes in the subchondral bone underneath the cartilage implant have been described and indicate a problem with the graft which requires close clinical follow-up [19]. Cyst formation has been associated with oedema-like signal intensity in 10% of cases with these cases also demonstrating a fibrocartilage appearance rather than hyaline-like articular cartilage [63]. In our series of 23 patients who underwent MACT surgery, we found 13% had an oedema-like signal in the subchondral bone at 2\u00a0years [64].\nA comparison of ACI and MACT patient groups [19, 64] showed the rate of bone marrow oedema and effusion was similar, approximately 40%, with both techniques.\nAdhesions\nAdhesions are demonstrated on MRI as bands of intermediate to low signal intensity tissue traversing the joint and demonstrating contact to the repair tissue (Fig.\u00a013). Adhesions most commonly connect to the infra-patellar fat pad, suprapatellar pouch and parapatellar recesses [15, 18, 67]. Knee stiffness from intraarticular adhesions requiring arthroscopic release has been reported in up to 10% of ACI patients [15, 67, 73]. Patients who have undergone extensive cartilage repair or have multiple grafts appear to be more at risk [18]. Adhesion to the graft surface may lead to graft tearing or dislocation as the patients activity level increases.\nFig.\u00a013An adhesion seen as a thin band-like structure, running from the tibial articular surface to the cartilage implant on a sagittal T2-FSE image. Arrows mark the borders of the implant\nEffusion\nPost-operative reactive synovitis should be considered as a possible cause of pain in this patient group. For the evaluation of synovitis intravenous contrast media application is usually required. Synovitis is generally accompanied by effusion and clinical signs of swelling of the affected joint. The majority of cases with synovitis in the early postoperative period resolved during the follow-up, which can be explained by its reactive nature [64]. In addition to the cartilage repair surgery itself other causes of synovitis have to be considered, such as native cartilage defects or meniscal lesions.\nMaturation of cartilage repair tissue\nSerial follow-up MRI scans of MACT patients show that the cartilage repair is a dynamic process that can be non-invasively monitored [62]. High-resolution MRI examinations at 4, 12, 24, 52 and 104\u00a0weeks post-operatively revealed characteristic changes in the repair tissue over time, which we believe represent the normal maturation process of the repair tissue. The most significant features were: first, early filling defects showed progressive filling by 6\u201312\u00a0months (Fig.\u00a014); second, initial graft hypertrophy seen at 3\u00a0months resolved by 6\u00a0months; third, small surface defects became smooth over time. Finally, signal intensity gradually changes over time from fluid-like appearance in the early post-operative stage to iso-intensity with surrounding native hyaline cartilage by 6\u201312\u00a0months. One may conclude that inverse developments are associated with a poor prognosis.\nFig.\u00a014a, bProgressive defect filling at the repair site. After matrix-based autologous chondrocyte implantation, significant improvement of filling of the defect at the repair site from (a) 4\u00a0weeks to (b) 24\u00a0weeks post-operatively, depicted on sagittal FSE images\nHistology\nIn a histological analysis of follow-up biopsies after ACI surgery, 57% of patients after ACI demonstrated articular cartilage [74]. However, it is generally hypothesised that continuous remodelling of the graft occurs with the transplant becoming more like hyaline cartilage.\nTo our knowledge only one study has attempted to correlate MRI findings with ACI graft histological findings [63]. Tins et al. [63] studied 41 patients at 1\u00a0year following ACI grafting on the femoral condyle. The histological appearance of the graft was classified into one of the four morphological categories recommended by the ICRS. MRI findings were correlated to the histology findings. The authors found no relationship between any of the MRI features assessed and the histological appearance of the cartilage repair tissue; however, this is unsurprising given the sequences used. Studies of the correlation between histology and the dGEMRIC index and\/or T2 maps are a very interesting area for future studies.\nFunctional outcome\nMarlovits et al. [58] recently described the statistical correlation of clinical outcome scores with the radiological variables of the MOCART scoring system. For the variable \u201cfilling of the defect\u201d a statistically significant correlation with all KOOS (knee injury and osteoarthritis outcome score) variables and also with the VAS (visual analogue score) was observed. For the variable \u201cintegration to border zone\u201d and \u201csurface of the repair tissue\u201d no statistically significant correlation was found. The variable \u201cstructure of the repair tissue\u201d showed a statistically significant correlation with the VAS and nearly all KOOS group except for the variable \u201csymptoms\u201d. For the variables \u201cadhesion\u201d, \u201csubchondral lamina\u201d, and \u201ceffusion\u201d no statistically significant correlation with the clinical scores was found. In contrast the variable \u201csubchondral bone\u201d showed a statistically significant correlation with the VAS and nearly all KOOS group except for the variable \u201csymptoms\u201d. For the signal intensities a statistically significant correlation was found with the KOOS variables \u201csymptoms\u201d, \u201csport\u201d, and Activities of daily Living (ADL) function.\nRecommendations\nFrom reviewing our studies and those of other centres we recommend that follow-up MR studies should be performed at 3\u00a0months and 1\u00a0year. The initial imaging at 3\u00a0months allows the volume and adherence of repair tissue to be assessed. Imaging at 1\u00a0year demonstrates the maturation of the graft and allows complications to be identified both non-invasively and at a sufficiently early stage.\nConclusion\nIn the near future as the use of clinical high-field (3 Tesla) systems with modern multi-element coil configurations becomes more widespread and new high-resolution isotropic 3D sequences are utilised a further improvement in the morphological analysis of cartilage implants can be expected. Moreover, advanced cartilage imaging techniques which allow the biochemical composition of cartilage to be studied will be possible in vivo (Fig.\u00a015). This is particularly promising for evaluating the maturation of the graft and whether or not hyaline cartilage has developed. The ability to non-invasively assess graft maturity will help to define the optimal postoperative rehabilitation and to detect the early stages of graft failure.\nFig.\u00a015a, bA dGEMRIC image of a matrix-associated ACT 2\u00a0years after surgery. a The cartilage layer of the graft shows different T1 values, representing proteoglycan concentration, compared with hyaline cartilage. b a 3D-GRE image of the same patient, which shows morphology of cartilage implant with hypointense signal alteration of the cartilage implant in comparison with normal hyaline cartilage","keyphrases":["autologous chondrocyte implantation","articular cartilage","cartilage repair","mri","autologous osteochondral transplantation"],"prmu":["P","P","P","P","P"]}
{"id":"Anal_Bioanal_Chem-4-1-2226005","title":"Selected isotope ratio measurements of light metallic elements (Li, Mg, Ca, and Cu) by multiple collector ICP-MS\n","text":"The unique capabilities of multiple collector inductively coupled mass spectrometry (MC-ICP-MS) for high precision isotope ratio measurements in light elements as Li, Mg, Ca, and Cu are reviewed in this paper. These elements have been intensively studied at the Geological Survey of Israel (GSI) and other laboratories over the past few years, and the methods used to obtain high precision isotope analyses are discussed in detail. The scientific study of isotopic fractionation of these elements is significant for achieving a better understanding of geochemical and biochemical processes in nature and the environment.\nIntroduction\nThe development of multiple collector inductively coupled mass spectrometry (MC-ICP-MS) about 15\u00a0years ago immediately indicated the advantages of this technique, at least in ratio measurements from mass 80 and above. In general, the acquired data showed precision equal or better to the well-established thermal ionization mass spectrometry (TIMS) [1, 2]. The ease of data acquisition was remarkable, the ionization efficiency was significantly larger, and consequently the sensitivity was improved. When MC-ICP-MS was introduced it had to compete with TIMS, which at that time was fully developed and provided the best isotope ratio data for metallic elements in terms of precision and accuracy. Even in the stages of application tests MC-ICP-MS exhibited several advantages: high precision, high reproducibility, high analytical throughput, simple sample preparation, and, as mentioned above, improved sensitivity [3].\nMC-ICP-MS requires a more complicated instrument compared to TIMS. It uses an ion source at atmospheric pressure and therefore requires a specially designed ion beam introduction inlet system equipped with efficient pumping systems to reduce the argon gas pressure to 10\u22127\u221210\u22128\u00a0mbar. Further pumping systems along the ion flight tube maintain the vacuum at 2\u2009\u00d7\u200910\u22129\u00a0mbar. The ion beam emerging from the interface has a circular profile which requires a tunable quadrupole lens, efficiently changing it to a rectangular shape. This rectangular ion beam possesses ions with an energy spread of up to 30\u00a0eV. An electrostatic energy filter is used to separate an almost monoenergetic ion beam acceptable for the magnetic sector mass separator. A further inherent problem in an ICP-MS is the relatively high instability of the plasma, observed as fluctuations and drifts of the ion beam. A single collector system, whether a Faraday cup or an electron multiplier, is too slow to follow this instability; therefore an array of Faraday cups (and also a Daly electron multiplier) is mounted at the end of the instrument, allowing the simultaneous monitoring of two or more ion beam intensities. This technique had already been applied in the multiple collector TIMS instruments.\nPlasma 54 (P54), the first MC-ICP-MS instrument manufactured by VG Elemental in the UK [1], contained an ion collector equipped with seven Faraday cups and an analog Daly detector. A unique feature of all double focusing instruments is the wide flat-topped ion peak shape, which is of primary importance for high precision data acquisition. Two further modifications were applied to this instrument: a wide-end flight tube to allow simultaneous measurement of ions with large mass differences, such as U\u2013Pb or 6Li\u20137Li, and the addition of an electrostatic energy filter before the Daly detector to enhance abundance sensitivity measurements. Isotope ratio measurements using laser ablation have also been demonstrated [4]. Several years later VG Elemental-Thermo Elemental introduced the Axiom, a smaller double focusing instrument of vertical configuration, providing high mass resolution capability. This mass spectrometer was aimed for high precision, fast analytical work [5].\nSeveral other MC-ICP-MS instruments were manufactured by other companies applying different approaches for efficient reduction of ionic spectral interferences formed by argon and atmospheric gases, e.g., thermalizing the ions produced by the plasma, introducing a detection system comprising a fixed array of Faraday cups and ion counters, and including an adjustable ion beam dispersion device.\nMicromass in the UK introduced the IsoProbe MC-ICP-MS where an off-axis hexapole collision cell is mounted in a jacket between the second interface cone and a beam focusing system [5]. The cell is flushed with a low pressure inert gas which reduces the ion energy to less than 1\u00a0eV. Also spectral interferences such as ArC+, ArN+, ArO+, ArCl+, and  dimers produced in the atmospheric plasma, which interfere with 52Cr+, 54Fe+, 56Fe+, 75As+, and 80Se+, respectively, are removed by collision-induced cleavage of the Ar\u2013X bonds, allowing measurement isotope ratios at masses below 80. The remainder of the instrument comprises a magnetic mass separator and a multiple collector detection system.\nNu Instruments in the UK introduced the Nu Plasma MC-ICP-MS. This machine, as with the Plasma 54, was developed by P.A. Freedman who maintained the same basic concepts, but introduced a different design for almost all the modules. Essentially it is a forward geometry (electrostatic sector followed by magnetic sector), double focusing mass spectrometer with a C-shape configuration compared to the S-shape of the P54. The ion beam profile conversion system is of different construction and the electrostatic energy filter and the magnetic separator were changed in shape and size. The profound advance in this design is the multiple collector detection system. The detection module comprises an array of 15 fixed detectors, 12 Faraday cups, and three electron multipliers, in front of which a zoom lens system deflects each separated ion beam into a chosen detector. Also, one of the multipliers is equipped with a filter for cutting off the tail from highly abundant isotopes. A later model from Nu Instruments, the Nu1700, is a large geometry, high resolution MC-ICP-MS [6] that allows one to overcome most of the interferences.\nFinnigan MAT in Germany introduced the Neptune MC-ICP-MS, a double focusing C-shape instrument with a large magnet providing 16% mass dispersion, movable collector array, and high resolution capability. Flat-topped peaks are achievable at R\u2009>\u20094,000 [7].\nWhen discussing isotope ratio measurements utilizing plasma ionization, several further phenomena should be mentioned. Isotopic fractionation is of utmost importance. It is caused by repulsive forces in the intensive positive ion beam emerging from the plasma and supersonic ion expansion through the sample cone. Both effects yield radial repulsion of the lighter isotope from the beam center, i.e., increasing the heavy mass over light mass ratio. The fractionation effect is inversely mass dependent from several per mils in uranium to more than 10% in lithium. It is constant in time, since fresh sample solution is continuously aspirated into the ion source. This is in contrast to TIMS ionization, where a fixed sample is used, permanently changing in composition as the lighter isotope is preferentially vaporized. Fractionation may be corrected in one of three techniques: (1) internal and (2) external normalization, including double spike method; and (3) \u201cstandard-sample-standard bracketing\u201d; consecutive measurements of the same ratio in a sample and standard. Further details will be given when describing the ratio measurements in the elements discussed in this review.\nICP mass spectrometry is subject to other interferences originating from various sources. Spectral interferences are products of interactions between the carrier gas and atmospheric gases or the solvent molecules. As mentioned above they obscure the lower mass range. Sodium ions in the sample solution in the case of copper analysis may interfere as ArNa+ at mass 63. Further cases are molecular interferences such as oxides, nitrides, and hydrides of trace elements in the solution and isobaric ion interferences. These cases can be removed by using expensive high resolution MS. Other ways to reduce interferences include using of desolvation nebulizer and chromatographic separation of the analyte from the matrix prior to introduction into the MS. This separation is also necessary to prevent the additional effects of mass fractionation connected with the matrix effect. In the case of the heavy isotope ratio measurements, this fractionation is corrected by internal or external normalization, but in the case of standard-sample-standard bracketing, generally used for light masses correction, the analyte must be carefully and precisely separated from the matrix.\nAs with the stable isotopes of the light elements, the isotopic composition of a sample is given in \u03b4 units (\u2030) relative to standard (I is isotope, x and y are mass numbers).\nGenerally, the isotope variations are derived from bracketing the measured sample ratio with the mean ratios of a standard measured before (std1) and after the sample run (std2), and are presented as deviations in parts per 1,000 of the measured ratio from that of the standard (normalizing) ratio:\nResults and discussion\nLithium\nLithium isotopes are of significant importance in a number of fields, such as geochemistry [8\u201311], astrophysics [12\u201313], nuclear technology [14], and biomedicine [15]. MC-ICP-MS opened new frontiers in Li isotope measurements by providing high precision data (<0.3\u2030, 2\u03c3). Lithium isotopes fractionate during hydrothermal processes, and significant variations may be observed in 7Li\/6Li ratios in water derived from marine sedimentary rocks and from hydrothermal altered igneous rocks, thereby providing valuable information regarding regional ground-water flow paths. Lithium isotopes are a powerful tracer of recycling processes in the Earth. This is particularly the case for understanding the geochemical evolution of the Earth\u2019s mantle, because (a) Li is a moderately incompatible constituent of minerals in peridotite, (b) Li isotope ratios show large variations in the terrestrial system, caused by low-temperature fractionation and mixing, (c) Li is a fluid-mobile element. Therefore, combined with existing geochemical information, its isotopes could be powerful geochemical tracers, especially for fluid-related (metasomatic) mantle processes. In recent years, almost all lithium isotope studies dealing with geological and related systems have applied the MC-ICP-MS measurement technique.\nLithium isotope ratio measurements require careful separation of Li from the matrix in natural samples. For solid samples, digestion and ion exchange chromatography separation are used in general; liquid samples are processed by chromatographic separation [16\u201321]. Acid leaching [22, 23] and direct resin techniques [24] are also applied. The most rapid method of separation was published by Hall et al. [21] for TIMS and modified at the Geological Survey of Israel for MC-ICP-MS. Any traces of Na, K, Mg, Sr, and Rb were detected using this modified method, so the samples were sufficiently pure for ratio measurements by Nu Plasma MC-ICP-MS.\nThe values of the ratio in standard L-SVEC NBS (NIST, Li2CO3) are  [25], and in seawater  [26]. A typical analysis sequence consists of blank, standard, blank, sample 1, blank, standard, blank, sample 2, and so on. Lithium ratio measurements exhibit time-dependent drifts and random shifts. Tomascak et al. [16] reported a typical 7Li\/6Li ratio drift for the L-SVEC standard from 12.7 to 12.3 during an 8-h period. Nishio and Nakai [17] rejected data when two successive measurements of the bracketing standard ratio shifted by more than 2\u2030. These authors also reported a stability of  (2\u03c3) for an in-house isotope standard measured over an 8-month period. It is good practice to carry out the sample and standard measurements under identical instrumental and solution conditions. The analyte concentrations in both solutions should be as close as possible and the impurity levels identical and as low as possible.\nIt should be noted that sample purity in TIMS analysis relative to MC-ICP-MS is of major importance, since the large isotopic fractionation in Li is highly sensitive to impurities in the loaded sample, especially when Na and K are interfering elements [21]. It was reported that a Na\/Li ratio greater than approximately 5 may cause unstable instrumental fractionation [16]. Moriguti and Nakamura [27] developed a four-step chromatographic separation for Li purification, allowing precise TIMS Li ratio analyses.\nBouman et al. [28] studied a wide range of Li samples originating from islands in the Pacific, Atlantic, and Indian Oceans. Thirty six samples and three standard materials were analyzed. The precision of these measurement are 0.01\u20130.5(\u2030), 0.6\u20131.0(\u2030), and 1.2\u20131.9(\u2030) for 22, ten, and four samples, respectively. The data for standard materials are presented in Table\u00a01, which also summarizes selected MC-ICP-MS Li isotope ratio measurements in geological samples.\nTable\u00a01Li isotopic composition in standards and selected natural samples relative to NIST NBS L-SVEC (\u2030)Sample\u03b47Li\u00b12\u03c3ReferenceJR-2 rock standard3.840.18[24]Magmatic arc lavas (Kurile)[18]8322\/3 Onekotan4.2<1.0K33 Keli-Mutu5.1<1.0VB30 granite\u22121.41.0[19]MG20 granite0.81.0Inorganic calcite CM019\u22127.60.6[20]Coral Acropora21.00.4Indian Ocean water33.01.2[21]Atlantic Ocean water32.11.2Foraminifera Orbulina universa28.41.6Inorganically grown carbonates[23]Aragonite (salinity 10\u00a0psu)\u221210.90.8Calcite (salinity 50\u00a0psu)\u22121.90.8Seawater29.70.4[28]BHVO-1 rock standard5.01.5Seawater 680 W30.70.4[29]Mediterranean Sea30.590.26[30]Red Sea30.490.12Dead Sea28.780.11Yarkon spring15.140.21SC -1 olivine3.41.0[31]Ia\/211 clinopyroxene\u22122.41.0M 1 saprolite\u221211.61[32]10 saprolite0.21SH 65 Ocean Island basalt7.0<1[35]KRS9806 xenolith, Japan\u22127.70.83[36]9708 xenolith, Australia6.00.83Zagami meteorite4.40.5[38]15495 lunar low-Ti mare basalt5.60.214-1 amphibolite0.91[39]12-2 amphibolite\u221214.21\nBryant et al. [29] studied Li isotope ratio measurements by MC-ICP-MS under \u201ccold plasma\u201d conditions. The results are characterized by fewer baseline interferences and improved reproducibility as compared with conventional hot plasma techniques. The 2\u03c3 precisions for 1,200-W, 800-W, and 680-W plasma energy are conservatively estimated as 1.1\u2030, 0.7\u2030, and 0.5\u2030, respectively. The effects of analyte, acid, and matrix concentrations were discussed.\nSeitz et al. [31] determined the Li isotopic composition of coexisting olivine, clinopyroxene, and orthopyroxene from spinel- and garnet-bearing peridotite xenoliths. The degree of intramineral fractionation correlated negatively with equilibration temperature.\nThe lithium isotopic composition of saprolites developed on a granite and diabase dike from South Carolina was measured to document their behavior during continental weathering. A general trend of decreasing \u03b47Li with increasing weathering intensity was observed [32, 33]. A similar study was carried out by K\u0131sak\u00fcrek et al. [34]. The internal precision on 7Li\/6Li measurements was usually less than 0.20\u2030, and the external precision of Nu Plasma MC-ICP-MS, was 0.8\u2030.\nRyan et al. [35] defined lithium isotope variations in mantle sources (volcanic rocks from the Antarctica oceanic islands), and Nishio et al. defined lithium isotope variations in mantle-derived xenoliths [36]. These isotopic measurements have provided much information on the mantle source region. Similarly, Elliott et al. [37] showed that Li isotopes promise to provide significant new constraints on the distribution of recycled material in the mantle and its implications for mantle convection.\nLithium isotope compositions and concentrations of lunar samples, including basalts, breccias, and glass, and martian meteorites were measured using Thermo Finnigan MC-ICP-MS [38].\nTeng et al. [39] measured lithium isotopic compositions in the country-rock amphibolites and schists using Nu Plasma MC-ICP-MS. \u03b47Li decreased dramatically with contact distance (along a \u221210\u00a0m traverse from the pegmatite into amphibolite) from +7.6 to \u221219.9.\nPogge von Strandmann et al. [40] investigated the behavior of Li isotopes in glaciated basaltic terrain. The \u03b47Li value of the suspended load was always lower than that of the bedload due to preferential retention of 6Li in secondary minerals during weathering. In turn, the \u03b47Li value of the dissolved load was always greater than that of the bedload. The authors suggested that \u03b47Li decreases with increasing chemical weathering. Similarly, Hathorne and James [41] used Li isotopes in seawater as a tracer for silicate weathering and showed that between 16 and approximately 8\u00a0Ma silicate weathering rate has increased, while weathering intensity has decreased.\nNishio et al. [42] studied Li isotope ratios in North Atlantic and Indian Ocean waters and its relation to those of Sr and Nd. The Li isotope results support the recent proposal that significant amounts of recycled lower continental crust might produce the radiogenic isotope signatures of the Indian Ocean basalts. Rudnick and Ionov [43] examined Li isotopic disequilibrium in olivine and clinopyroxenes from far-east Russia. Jeffcoate et al. [44] studied Li isotope fractionation in peridotites and mafic melts. Their results highlight the potential of Li isotopes as a high resolution geospeedometer of the final phases of magmatic activity and cooling. Wunder et al. [45] studied Li isotope fractionation between Li-bearing staurolite, Li-mica, and aqueous fluids.\nMagnesium\nMagnesium has three naturally occurring isotopes at mass numbers 24, 25, and 26 with relative abundance of 78.99, 10.00, and 11.01%, respectively. Galy et al. [46] briefly summarized the various fields where natural variations in the isotopic composition of this element may arise: (1) stellar nucleosynthesis and incorporation of presolar grains into meteorites, (2) the decay of 26Al to 26Mg, (3) isotopic fractionation in volatilization\/condensation reactions, (4) isotopic fractionation during low temperature fluid\/rock interactions, and (5) kinetic and thermodynamic isotope effects in biological processes.\nGaly et al. [46] performed high precision magnesium isotope ratio measurements using MC-ICP-MS for three groups of materials: commercial Mg materials including the NIST isotopic standard SRM 980, natural magnesite, and commercial chlorophyll in spinach. The observed ratio 26Mg\/24Mg was 0.139828\u2009\u00b1\u20090.000037 (2\u03c3), compared to the NIST value of 0.13932\u2009\u00b1\u20090.000 6 (2\u03c3) [47]. The variations in the Mg materials reported in \u03b425Mg and \u03b426Mg units are given in Table\u00a02. A desolvation nebulizer was used to minimize the introduction of H2O, CO2, O2, and N2, therefore reducing the presence in the plasma of molecular interferences such as , C2H+, , CN+, and NaH+. Other possible interferences are doubly charged ions such as 48Ca2+, 48Ti2+, 50Ti2+, 50V2+, 50Cr2+, and 52Cr2+. These elements, if present, should be removed by purification.\nTable\u00a02Mg isotopic composition in standards and selected natural samples relative to DSM-3 (\u2030)Sample\u03b426Mg\u00b12\u03c3\u03b425Mg\u00b12\u03c3ReferenceAldrich Mg solution2.600.171.330.08[46]aDead Sea Mg metal3.960.152.030.08AG 177 magnesia2.030.041.030.01OUM 10988 magnesite, Italy1.220.040.600.03AG 27 chlorophill b, spinach1.060.070.540.03North Atlantic Sea water2.590.041.330.08[51]aMixed foraminifera\u22121.880.16\u22120.990.10Dolomite1.640.040.880.01Seawater[52]aEPR1 surface1.950.140.980.06WP45N 5800\u00a0m2.010.161.000.04Med-T1 surface2.020.071.050.09Hydrothermal 2,650 m\u22121.010.16\u22120.510.06River water\u00a0MB160.760.460.380.22MB60.090.220.040.11PH60.220.060.110.06PH5\u22121.010.16\u22120.510.14Ganges\u22121.390.06\u22120.70.09[53]Amazon\u22121.030.07\u22120.530.04Lena\u22121.280.08\u22120.660.02Seawater\u22120.840.130.430.15[54]MT 66 solute\u22121.740.07\u22120.900.07Ett113 bulk silicate rock\u22120.420.03\u22120.210.02M 201 biotite\u22120.070.09\u22120.010.00MO33 100\u2013110 soil0.020.010.010.01Ace78 travertine\u22124.010.11\u22122.060.07Dead Sea water\u22120.600.08\u22120.330.10[55]Seawater\u22120.740.05\u22120.360.12Sataf spring\u22122.500.10\u22121.300.10Carbonaceous chondrites[56]144A, Al-Ti diopside0.110.06\u22120.090.12144A, Al-Ti diopside\u22122.740.11\u22121.740.11Olivines[57]DR9894 Australia\u22121.430.2\u22120.660.13ZS56-2 Siberia\u22121.050.2\u22120.540.13aRelative to NIST isotopic standard SRM 980\nVariations of the Mg isotope ratio in the metallic chips of the NIST SRM 980 magnesium isotopic standard were shown using five different MC-ICP-MS instruments of two types [48]. The chips were 1\u201350\u00a0mg in size. The differences in \u03b425Mg and \u03b426Mg of the SRM 980 were up to 4.2 and 8.19\u2030, respectively, while the long-term repeatability of the \u03b4 values was up to 0.09 and 0.16\u2030 respectively. Because of the heterogeneity of the NIST reference material, two homogeneous isotope standards, DSM-3 (Dead Sea magnesium) and Cambridge1 were prepared and characterized. The heterogeneity of Mg isotopes was also reported by Zhu et al. [49]. Carignan et al. [50] discussed the isotopic homogeneity of existing reference materials and suggested the acceptance of DMS-3 as a new Mg isotope reference standard material.\nChang et al. [51] developed a Mg separation technique for low-Mg biogenic carbonates with yields close to 100%. The technique was applied to the determination of Mg isotopes in three natural samples: seawater, foraminifera, and dolomite. De Villiers et al. [52] established the magnesium isotopic composition of seawater and evaluated its constancy as a function of depth and geographic location. The authors demonstrated that the magnesium isotopic composition of ancient oceans can be used to make important inferences about the relative contribution of different lithologies to the global continental weathering flux.\nTipper et al. [53, 54] analyzed river water, rock, travertine, and soil and demonstrated that both Ca and Mg isotope ratios are fractionated during weathering. The Mg isotope composition of the rivers is intermediate between limestone and silicate rock. Silicate soil has a \u03b426Mg of \u22120.03\u2030, heavier than that of silicate rock by 0.5\u2030. This fractionation in the soil creates a complementary groundwater reservoir of light Mg. Seasonal variations in Mg isotope ratios in the dissolved load are small, but define an array which can be modeled as a mixture between a fractionated groundwater reservoir and surface runoff.\nMg isotope fractionation during brine evolution in the Dead Sea is presently being studied using the MC-ICP-MS technique [55]. Samples from the solar system were used for 26Al\u221226Mg dating, by measuring the variations in the Mg ratios using laser ablation combined with MC-ICP-MS [56].\nPearson et al. [57] investigated Mg isotopic variations in the lithospheric mantle by analyzing olivine in mantle-derived peridotite xenoliths and megacrysts using a laser-ablation microprobe and MC-ICP-MS. \u03b426Mg ranges from \u22123.01 to +1.03\u2030 and \u03b425Mg from \u22121.59 to +0.51\u2030, relative to the magnesium isotopic standard DSM-3, were found. The in situ measurement of Mg isotopes thus provides a powerful new method for investigating processes in the mantle.\nCalcium\nCalcium has six stable isotopes at mass numbers 40, 42, 43, 44, 46, and 48 with relative abundance of 96.941, 0.647, 0.135, 2.086, 0.004, and 0.187%, respectively. Halicz et al. [58] studied in detail the MC-ICP-MS isotope ratio measurements of calcium. The more important points are discussed here. Under normal instrumental operation conditions it is not possible to use the isotope 40Ca because it is masked by the intense 40Ar+ ion beam. Furthermore, the ion dispersion of Ca isotopes is too large to allow the 48Ca\u221242Ca mass range to be accommodated on the multicollector array; therefore only 42Ca, 43Ca, and 44Ca are monitored. Ar-scattered interferences were monitored at half masses for elevated background correction. Corrections of doubly charged Sr were made by measuring doubly charged 87Sr2+. A desolvation nebulizer was used to reduce molecular interferences such as ,  , and . The results for the Ca ratios in the studied sample are given in \u03b4 units in Table\u00a03 and were derived using the bracketing technique relative to the NIST SRM 915a Ca standard.\nTable\u00a03Ca isotopic composition in standards and selected natural samples relative to NIST SRM 915a (\u2030)Sample\u03b444Ca\u00b12\u03c3ReferenceCaCO3 Merck0.610.28[58]2-8-E3 speleothem0.250.32Acropora coral0.580.16Shell from marine organism[59]USGS EN-10.53a0.07CaCO3 JM 9912\u22120.630.07CaCO3 JM 4064\u22126.620.08Inorganic calcite[61]CM0400.090.2CM039\u22120.070.2Growth solution0.310.2Foraminifera[62]OMEX-12b0.210.10WIND-10b0.280.11Mediterranean Sea water0.980.14Seawater1.090.09[54]MT 66 solute0.540.06Ett113 bulk silicate rock0.310.05MO33 0-10 soil0.510.05MO33 100-110 soil0.540.13Ace78 travertine0.140.05Carbonate fluorapatite[63]OG-50.260.11D4\/010.420.08S30\/030.230.0132a\/010.520.1232\/01s0.130.03D49\/01\u22120.230.06aRelative to IAPSO seawater\nWieser et al. [59] developed a high precision Ca isotope ratio measurement technique for a Finnigan Neptune magnetic sector ICP-MS. Delta values including \u03b444Ca\/43Ca, \u03b444Ca\/42Ca, and \u03b448Ca\/42Ca were measured with an external reproducibility better than 0.2\u2030 in seawater and biogenic and non-biogenic marine carbonates. Fietzke et al. [60] developed a new technique for direct measurements of 44Ca\/40Ca ratios on an MC-ICP-MS using \u201ccool plasma\u201d. Reducing the plasma energy from the usually applied approximately 1,300\u00a0W to about 400\u00a0W significantly reduced the 40Ar+ isobaric effect, allowing simultaneous and precise measurements of the two Ca isotopes. Repeated measurements of the 44Ca\/40Ca ratios in various Ca standard materials were in good agreement with data reported in the literature.\nMarriott et al. [61] investigated temperature dependence of Ca isotopes in solution, inorganic calcite, and foraminifera and concluded that they are lighter than in the growth solution, and only weakly dependent on temperature. Sime et al. [62] also found negligible temperature dependence of calcium isotope fractionation in planktonic foraminifera.\nRiver water, rock, travertine, and soil were studied by Tipper et al. [54] who demonstrated that Ca, as well as Mg isotope ratios, are fractionated during weathering. Fractionation of Ca during continental weathering is of importance to the global cycle of Ca. The riverine input of Ca to the oceans is controlled not only by the composition of the primary continental crust, but also by the size and composition of the fractionated reservoir on the continents. The impact on the oceanic cycle of Ca depends on the relative residence times of dissolved Ca in the ocean and the storage time of fractionated Ca.\nSoudry et al. [63] explained fluctuations of Tethyan phosphogenesis through time, and whether or not they reflect long-term changes in ocean circulation or in continental weathering. A \u03b444Ca increase during the Late Cretaceous\u2013Eocene also reflects a decrease in weathering Ca2+ fluxes together with increased biological removal of isotopically light Ca2+, fostered by increased continental flooding and concomitant carbonate (chalk) sedimentation on shelves. The overall concordant trends between the measured \u03b444Ca, the eustatic sea level curve, and the sizes of the flooded continental area throughout the Early Cretaceous\u2013Eocene, point to a linkage between the Ca isotopic composition of paleoseawater and long-term paleogeographic and oceanographic changes.\nSteuber and Buhl [64] analyzed the calcium isotope composition in modern and ancient marine carbonates. No significant temperature dependence of Ca isotope fractionation was found in Cretaceous shells. \u03b444Ca of Cretaceous seawater was 0.3\u20130.4\u2030 lower than that of the modern ocean.\nChu et al. [65] developed a procedure for the precise determination of Ca isotope ratios in natural and organic samples, such as bones, milk, and other biological materials. The data demonstrated that geological\/environmental conditions do not cause large variability and it was suggested that diet is the major cause for variations in bones; so Ca isotope ratios may serve as a paleodiet tracer.\nSkeletal carbonates from the Tethyan realm were analyzed by Farka\u0161 et al. [66]. The observed late Mesozoic \u03b444Casw was simulated using a Ca isotope mass balance model, and the results indicated that the variation in \u03b444Casw can be explained by changes in oceanic input fluxes of Ca that were independent of the carbonate ion fluxes.\nSime et al. [67] interpreted Ca isotope behavior in marine biogenic carbonates. The 18 million year record of planktonic foraminifera in the Atlantic averages  and is a good match to Neogene Ca isotope record based on foraminifera, but is not similar to those in bulk carbonates. There are also publications on Ca using seawater as a standard for normalization in the bracketing method.\nCopper\nCopper isotope ratio measurements have been used to determine natural isotope variations in ore geology, geochronology, and archaeometry [68, 69]. Three different types of ICP-MS instrument, a quadrupole, a single collector magnetic sector, and a multicollector magnetic sector instrument, were compared by Diemer et al. [70]. Although precision of results significantly differed, excellent agreement was observed between results obtained using all the instruments. The results for the Cu-isotopic composition are given in Table\u00a04.\nTable\u00a04Cu isotopic composition in standards and selected natural samples relative to SRM NIST 976 (\u2030)Sample\u03b465Cu\u00b12\u03c3ReferenceNative copper[71]aOUM15126 Michigan, USA0.450.06OUM00061 Yekaterinburg, Russia\u22120.330.06OUM15127 Cornwall, England0.410.06Minerals\u00a0OUM23585 malachite, England\u22120.260.06OUM1616 azurite, USA1.590.06OUM25139 chalcopyrite, England0.070.06Acc. Ser. 25300 chalcopyrite, Canada0.400.06G-4 chalcopyrite, sulfide deposits\u22120.440.06Ultra-pure Cu standards[75]JMC Cu Axiom0.6190.058JMC Cu IsoProbe0.6410.019IMP Cu IsoProbe0.200.10IMP Cu IsoProbe0.2070.049Copper sulfide precipitates[78]A-Cu- 0 Cu(I)S bulk0.700.06A-Cu- 0 Cu(I)S precipitated\u22122.520.06A-Cu-48 Cu(I)S bulk0.620.06A-Cu-48 Cu(I)S precipitated\u22122.320.06C-Cu-0 Cu(OH)2 bulk\u22120.260.06C-Cu-0 Cu(OH)2 precipitated\u22120.540.06Chalcopyrite from the Grasberg deposit region[73]aF1-001 Grasberg skarn0.1070.031XC05-001 Ertsberg diorite0.6330.035ES-005 Ertsberg skarn0.1930.054XC22001 pyrite shell0.2180.023XC25-002 bornite-2.69 Dalum\u22120.2690.031Alexandrinka VHMS sulfides[76]SW-1(a) chalcopyrite0.184<0.07SW-1(b) quartz0.318<0.07HV-1(a) sphalerite0.330<0.07CA-1 pyrite0.054<0.07CB-1(b) covellite\u22120.300<0.07CB-2(a) silicate\u22120.058<0.07Supergene copper sulfides[81]Tt-1, El Teniente bornite0.370.16Tt-6, El Teniente chalcopyrite\u22120.150.16El-1, El Salvador chalcopyrite0.810.16M-1, Mocha chalcopyrite\u22120.060.16aOriginal results were given in \u025b units (parts per 10,000) and have been converted to \u03b4 units\nMC-ICP-MS ratio determinations of copper were comprehensively studied by Zhu et al. [71]. A sample purification procedure was described, noting that possible isotope fractionation must be avoided. Potential interferences in 65Cu\/63Cu ratio measurements are (23Na40Ar)+ and  at mass 63 and (25Mg40Ar)+ at mass 65. It has been shown that for samples where Na\/Cu and Mg\/Cu ratios are below 10\u22124 and 10\u22123, respectively, and under instrumental working conditions applied in this study, the abundances of the polyatomic ions relative to Na+ and Mg+ were approximately 10\u22124, i.e., isobaric interferences were negligible.\nTwo Cu isotope measurement procedures were used: the standard-sample-standard bracketing technique where variations are calculated relative to Cu isotope standard (SRM NIST 976) as in Eq.\u00a02, and the \u201cdoping\u201d technique [2] where the sample is doped with Zn at a concentration Cu\/Zn ratio close to 1. Early results were given in \u201c\u025b\u201d units, but in the last papers \u201c\u03b4\u201d is accepted. A constant value of the 68Zn\/66Zn ratio was used as a normalization factor to correct the measured 65Cu\/63Cu ratios. External precision (2\u03c3) better than 0.03\u2030 and 0.06\u2030 was achieved by applying the doping and bracketing techniques, respectively [71]. Borrok et al. [72] presented a new method for efficient separation of Cu, Fe, and Zn from the greater concentrations of matrix elements using a single anion-exchange column with hydrochloric acid media.\nThe possibility of laser ablation combined with a MC-ICP-MS for Cu isotope ratio determinations was presented by Graham et al. [73] About 160 solid samples were analyzed with a 2\u03c3 uncertainty of approximately 0.04\u2030. Jackson and G\u00fcnter [74] examined the influence of various processes of laser ablation on fractionation of the Cu isotope ratio. The data suggest that the dominant source of isotopic fractionation at high laser fluence was the preferential volatilisation of 63Cu during incomplete vaporization and ionization in the ICP of particles greater than approximately 0.5\u00a0\u03bcm in diameter.\nMason et al. [5, 75] performed a detailed study on spectral interferences across the mass range 63Cu to 70Zn and the mass discrimination corrections using two different MC-ICP-MS instruments: the double focusing VG Axiom and the single focusing Micromass IsoProbe. It was observed that the ion types and their intensities depend on the different instrumental configuration. The importance of removing problematic matrix components prior to the Cu and Zn measurements was also emphasized. Based on their method Mason et al. [76] studied Zn and Cu isotopic variability in the Alexandrinka sulfide ore deposit from the Urals, Russia. A further study on mass discrimination correction shows the importance of matrix removal, particularly Fe and Ti, the dependence of mass discrimination on Cu\/Zn ratio in the solution and that sample introduction system with a desolvating membrane causes variable behavior of the Cu standard, probably due to variations in Cu oxidation state in the solution [77].\nEhrlich et al. [78] studied copper isotope fractionation between aqueous Cu(II) and CuS, the latter being precipitated from CuSO4 solution with Na2S under anoxic conditions. Using Ni as a doping agent and the standard-sample-standard bracketing technique a 2\u03c3 error of 0.06\u2030 was achieved. The mean fractionation factor at 20\u00a0\u00b0C was derived: . Additional experiments over the temperature range 2\u201340\u00a0\u00b0C showed an inverse dependence of the fractionation factor on temperature. Markl et al. [79] used copper isotopes as monitors of redox processes in hydrothermal mineralization. The authors concluded that copper isotope analyses cannot be easily used as a reliable fingerprint for the source of copper in archaeology and geology because the variation caused by redox processes within a single deposit is usually much larger than between deposits. However, Asael et al. [80] showed that systematically large Cu isotope fractionation occurred during redox processes in sedimentary copper ore deposits. Cu isotope fractionation was also observed in bacterial oxidizing environments [81].\nThe roles of Cu and Zn isotopes in chondrites and iron meteorites were studied by Luck at al. [82, 83]. A typical error (external precision) for \u03b465Cu\u2009\u00b1\u20090.4\u20130.5\u2030 was reported. Moynier et al. [84] investigated isotopic composition of zinc, copper, and iron in lunar samples.\nRecently isotopic ratio studies of Cu and Zn were performed in seawater [85]. The very low concentration of these elements and high TDS matrix demanded development of preconcentration and purification, followed by separation processes. In this work accurate data and their uncertainties are given only for Zn.\nConclusions\nWe have reviewed a group of more than 60 papers that were published in the period from 2000 up to present. We also quote results from a few unpublished works made at the GSI. We have chosen to focus on the isotope ratio determinations of only four elements, namely lithium, magnesium, calcium, and copper, where high precision ratio measurements were for various reasons impossible to achieve with the early MC-ICP-MS instruments. The second-generation machines have detection systems with sufficient dispersion to accommodate Li ions and the capability to be tuned to partial mass numbers. The high resolution capability allows one in some cases to resolve interferences. New types of desolvation nebulizers allow significant reduction of solvent and atmospheric interferences and the \u201ccold plasma\u201d technique reduces nebulizing gas ion intensities. Consequently, precision (\u00b12\u03c3) for \u03b47Li, \u03b425, 26Mg, \u03b444Ca, and \u03b465Cu down to the range of (0.1\u20130.2)\u2030, (0.01\u20130.1)\u2030, (0.05\u20130.1)\u2030 and (0.06\u20130.15)\u2030, respectively, can now be achieved.","keyphrases":["isotope ratio","mc-icp-ms","light elements"],"prmu":["P","P","P"]}
{"id":"Pediatr_Radiol-4-1-2292491","title":"MRI evaluation of tissue iron burden in patients with \u03b2-thalassaemia major\n","text":"\u03b2-Thalassaemia major is a hereditary haemolytic anaemia that is treated with multiple blood transfusions. A major complication of this treatment is iron overload, which leads to cell death and organ dysfunction. Chelation therapy, used for iron elimination, requires effective monitoring of the body burden of iron, for which serum ferritin levels and liver iron content measured in liver biopsies are used as markers, but are not reliable. MRI based on iron-induced T2 relaxation enhancement can be used for the evaluation of tissue siderosis. Various MR protocols using signal intensity ratio and mainstream relaxometry methods have been used, sometimes with discrepant results. Relaxometry methods using multiple echoes achieve better sampling of the time domain in which relaxation mechanisms take place and lead to more precise results. In several studies the MRI parameters of liver siderosis have failed to correlate with those of other affected organs, underlining the necessity for MRI iron evaluation in individual organs. Most studies have included children in the evaluated population, but MRI data on very young children are lacking. Wider application of relaxometry methods is indicated, with the establishment of universally accepted MRI protocols, and further studies, including young children, are needed.\nIntroduction\n\u03b2-Thalassaemia major is a hereditary anaemia characterized by ineffective erythropoiesis and haemolysis [1]. The underlying mechanism is defective production of haemoglobin \u03b2-chains, resulting in excess of \u03b1-chains, which are unstable and precipitate to form intracellular inclusion bodies [2, 3]. This excessive intracellular deposition of \u03b1-chain material is responsible for accelerated apoptosis of the erythroid precursors and for peripheral haemolysis of the erythrocytes [3]. By the age of 3\u00a0months, severe anaemia develops leading to increased intestinal iron absorption. To maintain haemoglobin at a level of 10\u201312\u00a0g\/dl, patients suffering from \u03b2-thalassaemia major need to be given repeated blood transfusions [1]. A major drawback of this treatment is transfusion siderosis, which, in association with the increased intestinal iron absorption, apoptosis of the erythroid precursors and peripheral haemolysis, leads to iron overload [1].\nIron is ubiquitous in eukaryotic organisms. It is essential for cellular survival and proliferation and for haemoglobin synthesis [4, 5]. Human demands for iron are covered partly by intestinal absorption, but mainly from the recycling of iron from old or abnormal erythrocytes phagocytized by macrophages [6]. Iron released from macrophages binds mainly to transferrin, but also to citrate and albumin to form non-transferrin-bound iron (NTBI) which is a toxic form [7\u20139]. Extracellular iron bound to transferrin enters the cell via transferrin receptors by a process of endocytosis. Once iron is released into the cytoplasm, it enters a poorly defined cellular compartment termed the labile iron pool (LIP). Iron in this compartment is loosely bound and therefore is highly toxic [10]. Intracellular iron that is not needed for immediate use is stored in the form of ferritin which consists of an apoprotein storing up to 4,500 atoms of iron (loading factor 4,500) [5, 11], and is not cytotoxic. When ferritin storage capacity is exceeded the LIP increases and haemosiderin is generated from ferritin denaturation [10]. Iron in the form of haemosiderin is thought to be more cytotoxic [10]. The iron in the LIP is in both the ferrous (Fe2+) and ferric (Fe3+) forms [12]. Fe2+ reacts with hydrogen and lipid peroxides and generates highly toxic hydroxyl and lipid radicals (Fenton reaction) that damage cellular membranes, proteins and nucleic acids [4]. Iron overload, just as lack of cellular iron, may lead to cell death and organ dysfunction.\nIron overload is a major cause of morbidity and mortality in \u03b2-thalassaemia major [1]. Iron accumulates initially in the reticuloendothelial system (bone marrow, spleen, liver) and then in the hepatocytes, the heart (myocytes) and the endocrine glands [1, 9, 13]. In contrast to the reticuloendothelial cells, the turnover of iron in the hepatocytes, myocytes and endocrine glands is very low [9]. Chelation therapy has been used to eliminate excess iron [1, 7]. Chelatable iron is derived from the catabolism of haemoglobin in macrophages [14]. Chelators remove NTBI from the plasma, but they do not interact with ferritin and haemosiderin at clinically relevant rates [10, 15]. Desferrioxamine (DFO), which is the chelating agent most widely used over the last 30\u00a0years, requires parenteral administration. It removes mainly extracellular iron and only a fraction of the intracellular LIP iron [14, 15]. Deferiprone is an oral iron chelator that penetrates the cellular membrane and chelates intracellular species of toxic iron [16]. Cellular uptake of chelators takes place at different rates in different cells [15]. DFO is of low toxicity, mainly affecting the optic, auditory and skeletal systems [7]. Skeletal changes develop mainly due to the toxic effects of DFO on the growth cartilage and are manifested as disproportionate truncal shortening with loss of seated height [7]. Adverse effects of deferiprone are arthralgia, gastrointestinal symptoms, elevated liver enzymes and rarely agranulocytosis [7, 16].\nThe effective management of patients, and especially of children, with thalassaemia requires optimal monitoring of the toxic effects of both iron overload and excessive chelation therapy. Serum ferritin has been widely used as a surrogate marker and a target ferritin level of 1,000\u00a0\u03bcg\/l is generally recommended [7, 17]. However, serum ferritin represents only 1% of the total iron pool, and as an acute-phase protein it is not specific because the levels can be raised in inflammation (e.g. hepatitis) and liver damage [7, 18, 19]. Chronic liver inflammation is not rare in patients with thalassaemia, since over 40% of them have positive anti-hepatitis C virus (HCV) antibodies and more than 50% have chronic (persistent or active) hepatitis [20, 21]. Liver iron concentration (LIC) measured on needle biopsy is currently considered the gold standard for the evaluation of siderosis [7, 18, 19]. However, needle biopsy is an invasive technique, it is not easily repeatable and the accuracy of the resulting LIC measurement is greatly affected by hepatic inflammation-fibrosis and uneven iron distribution [22]. Furthermore, it appears that cardiac iron overload, which is the leading cause of death in thalassaemia, cannot be predicted from the degree of liver siderosis, probably because of differences in iron kinetics between liver and heart cells [9]. The use of non-invasive techniques for monitoring iron overload in each of the affected organs would be preferable, and to this end MRI has been used increasingly over the last two decades [19, 22\u201344]. Other non-invasive methodologies are too complex, too costly and not readily available (i.e. magnetic susceptometry), or lack imaging capability (i.e. magnetic resonance spectroscopy) [45, 46].\nMRI assessment of tissue iron\nRelaxation theories\nMRI evaluation of tissue iron overload is based on T2 relaxation enhancement induced from the interactions between high-molecular-weight iron complexes such as ferritin, haemosiderin and ferrioxamine with water molecules [5, 47, 48]. Ferrioxamine is produced after cellular uptake of DFO, and in extrahepatic tissues, where there is no active excretion and it may remain for days [7]. Ferrioxamine-based agents have been used in the past as contrast media because of their T2* effect [49]. \u201cInner sphere\u201d and \u201couter sphere\u201d relaxation theories have been proposed to explain the T2 relaxation enhancement induced in siderotic tissues [50\u201355]. According to the inner sphere theory, iron electron spins enhance the relaxation of protons of water molecules buried in iron-containing proteins. Enhanced relaxation of the bound water protons is then transferred to the free water protons by water or proton exchange [48, 56, 57]. According to the outer sphere theory, magnetic field gradients induced at the periphery of iron-containing proteins create loss of phase and relaxation enhancement of free water protons diffusing in the neighbourhood [53, 54].\nMR methodologies\nMR methods for assessing tissue iron can be separated into two groups: signal intensity ratio (SIR) methods and relaxometry methods. Various techniques have been described, including: (a) methods measuring SIR based on T2-weighted (spin-echo) or T2*-weighted (gradient-echo) sequences [19, 23\u201326, 30, 58\u201362], (b) relaxometry methods measuring absolute T2, (c) relaxometry methods measuring absolute T2*, and (d) hybrid relaxometry methods [30\u201332, 34\u201337, 39\u201343, 58, 63].\nSIR methods\nThese have been used for the study not only of the liver but also of other organs such as the spleen, pancreas, pituitary gland, bone marrow and abdominal lymph nodes [19, 23, 25, 27, 30, 58\u201362]. For SIR assessment the signal intensity of the target organ is divided by the signal intensity of a reference tissue (e.g. fat, muscle) or noise. Signal intensity measurements are performed in the same slice by using the region-of-interest (ROI) method. For large organs such the liver, spleen and pancreas more than one ROI is used, positioned in areas lacking vascular structures and movement artefacts [19, 23, 25, 27, 30, 58, 61, 62]. The mean signal intensity from the different ROIs is then divided by the signal intensity of the reference tissue. One ROI is used for signal intensity measurement of the pituitary gland, bone marrow, abdominal lymph nodes and the reference tissue [58\u201360]. For SIR evaluation of the liver various reference tissues have been used, of which paraspinal muscle appears to be the best choice because it combines good sensitivity with minimum intersite variability [19, 64]. A disadvantage of the SIR methods is that in most cases they use only one echo time (TE) and thus lose their detection sensitivity in tissues with heavy siderosis, where transverse relaxation is much faster than the TE. This occurs particularly in the liver at the upper range of LIC values where signal intensities are widely dispersed [39]. Gandon et al. [19], by using an algorithm that combined signal intensity ratios from multiple sequences with different TEs, achieved extension of the detection range up to about 21\u00a0mg Fe per gram dry liver tissue, with sensitivity and specificity similar to those of biochemical analysis.\nT2 relaxometry methods\nThese assess T2 relaxation time or R2 (1\/T2) by using the Carr-Purcell-Meiboom-Gill (CPMG) spin-echo sequence, which employs multiple (2\u201332) equidistant refocusing 180\u00b0 pulses, each followed by an echo [65, 66]. Most scanners, by using a pixel-by-pixel, log-linear fitting model, automatically derive the corresponding T2 maps. Signal intensity measurements in the T2 maps correspond to the mean T2 relaxation time of the included voxels [67]. To avoid motion artefacts from respiration, studies of abdominal organs should be performed with respiratory triggering. To this end a pressure-sensitive pad is attached to the abdomen and depending on the patient\u2019s respiratory cycle a delay time is chosen. Data acquisition takes place at the end-expiration phase [39, 40]. During cardiac MRI, in addition to respiratory triggering, cardiac triggering is necessary. To obtain a minimum repetition time (TR) of 2,000\u00a0ms two to three R\u2013R intervals should be allowed between successive excitations [39, 40]. The mid-systole phase and a cardiac trigger delay of around 250\u2013300\u00a0ms are usually chosen to image the myocardium with sufficient thickness for the best signal intensity measurements [39, 40]. A pseudo four-chamber or short-axis view of the heart is generally used. A T2 relaxometry method that received FDA approval for clinical liver iron estimation has recently been developed by St. Pierre et al. [44]. This method uses multiple T2-weighted single spin-echo sequences with different TEs acquired in half-Fourier mode to reduce acquisition time. The calculated mean R2 values combined with LIC values, obtained from liver biopsies, are used to create calibration curves.\nT2* relaxometry methods\nThese evaluate T2* or R2* (1\/T2*) by using multiple gradient-echo sequences with different TEs. These methods have been developed to further accelerate acquisition, in order to increase sensitivity and eliminate artefacts related to respiration or cardiac motion. To obtain R2* (1\/T2*) values, the signal decay curve is usually fitted with an exponential model: S=S0e\u2212TE\/T2*, where S is the net image signal intensity, TE is the echo time and S0 is a constant [68]. T2* relaxometry methods have been used mainly for myocardial iron assessment and cardiac gating is always applied [35, 42, 68\u201371]. Breath-hold sequences have greatly eliminated motion artefacts [35, 42, 68].\nHybrid relaxometry methods\nHybrid approaches have been applied in high fields and measure both R2 and R2* to calculate the inhomogeneity factor R2\u2032=R2*\u2212R2 [34, 72, 73]. These approaches assume that R2\u2032 is more specific to mechanisms of relaxation related to iron than R2 [34, 73].\nComparison of the MRI methodologies\nSIR versus relaxometry\nSIR methods require shorter acquisition times but lack a wide range of iron assessment [63]. Relaxometry methods, mainly the T2* method, by using multiple echoes create in- and out-of-phase effects between water and fat transverse magnetization (Fig.\u00a01) [35]. Relaxometry methods, although taking longer, are preferable because they achieve a better sampling of the time domain in which relaxation mechanisms take place and lead to more precise results [63].\nFig.\u00a01A 15-year-old male with \u03b2-thalassaemia major. Axial scan with a multiecho gradient-echo sequence (TR\/TE 150\/1.07, 2.14, 3.21, 4.28, 5.35, 6.42, 7.49, 8.56, 9.63, 10.7, 11.77, 12.84, 13.91, 14.98, 16.05, 17.12, 18.19, 19.26, 20.33, 21.4\u00a0ms; flip angle 35\u00b0). a Second echo shows pixel annulation (arrows) due to out-of-phase phenomena, at the interfaces of abdominal organs and muscles with fat. b Fourth echo: no pixel annulation is observed because water and fat protons are in phase (courtesy of Dr. M. Douskou)\nT2, T2* and hybrid methods\nThere is no general consensus on which relaxometry method (spin-echo or gradient-echo) or index (R2, R2*, R2\u2032) is best for tissue iron quantification. Theoretically, R2* and R2\u2032 are more sensitive than R2 to iron-induced field inhomogeneities. Gradient-echo sequences evaluating R2* are preferred for cardiac relaxometry, because of their short acquisition time, but gradient-echo sequences are more prone to artefacts and R2* is more dependent on factors unrelated to iron, such as susceptibility artefacts from the lungs and the blood oxygenation level-dependent (BOLD) effect [35, 74]. According to the BOLD effect, the T2* decreases as the concentration of intracapillary deoxyhaemoglobin increases [74]. A pronounced BOLD effect may be observed in early diastole and results in signal loss [75, 76]. Data collection during mid-systole helps to overcome BOLD effect interference with accurate T2* measurements of the myocardium. In liver and pituitary studies both T2 and T2* have been assessed. T2* is more sensitive to low iron content, but studies in the liver show that these methods suffer from inaccuracies at high iron concentrations [77]. Furthermore, in high fields susceptibility artefacts from the sphenoid sinus are more pronounced with gradient-echo sequences and may lead to inaccurate measurements of pituitary siderosis [43].\nMRI studies of individual iron overloaded organs\nThe degree of siderosis, the crystalline structure of ferritin, the rate of iron elimination under chelation therapy and the degree of ferrioxamine formation are all organ-specific [7, 15, 18, 78]. All these parameters may be responsible for differences in the T2 relaxation enhancement induced in the various organs. Individual organs should be considered separately, and the effect of age on iron overload should be taken into account. Higher survival probabilities have been reported in patients with thalassaemia born in the last 30\u00a0years [79]. Patient compliance with treatment regimens and effective chelation therapy are thought to be the main factors associated with improved survival [79]. The combination of DFO, deferiprone and the new oral chelators is considered very promising, but will require effective monitoring by non-invasive methods [80]. An increasing number of studies have evaluated iron in the various affected organs by MRI [19, 22\u201344]. Many of these studies have included paediatric patients, but very few have investigated children younger than 10\u00a0years and none has included children younger than 5\u00a0years [17, 18, 28, 39, 43, 58, 60\u201362, 77, 81\u201384]. In addition, very few studies have evaluated the effect of age on individual organ siderosis and only one has evaluated and compared MRI data from younger and older patients with thalassaemia [28, 81, 82, 84]. Studies have tended to focus on specific organs.\nLiver\nFor the MRI evaluation of liver siderosis both SIR and relaxometry techniques have been used (Fig.\u00a02) [18, 23, 28, 32, 44, 68, 85]. R2 of the liver demonstrates a significant positive correlation with serum ferritin and LIC determined from liver biopsy material [18, 23, 32, 39, 40, 44, 68]. Comparative evaluation of hepatic R2 and R2* in iron-overloaded patients demonstrates that both parameters correlate closely with LIC [85]. The relationship of SIR with LIC and serum ferritin varies among studies [18, 30, 58]. In most studies R2 and SIR show a better correlation with LIC than with serum ferritin [18, 23, 28, 32, 44, 68, 85]. This can be explained in part because the HCV-positive thalassaemia patients in the studies had higher serum ferritin levels than those who were HCV-negative [77, 86]. Liver R2 shows no association with the hepatic inflammation histological activity index or the type of hepatitis (chronic persistent or chronic active), but is affected by hepatic fibrosis [18, 39, 77].\nFig.\u00a02A 17-year-old male with \u03b2-thalassaemia major. Axial scan, fourth echo of a multiecho spin-echo sequence (TR\/TE: 2000\/20, 40, 60, 80, 100, 120, 140, 160\u00a0ms), shows low-signal intensity in the liver, bone marrow of the spinal body and the pancreas suggesting iron overload\nIn iron overload states, over 70% of body iron is found in the liver and LIC has been considered to be the best marker of total body iron burden [7, 18, 19]. Based on the good correlation between hepatic R2 or SIR and LIC a number of recent studies have tested the relationship between siderosis of the liver and other organs [28, 39, 40, 58\u201361, 87]. No correlation has been found between liver and pituitary siderosis [28, 58]. With regard to the heart, a correlation with liver siderosis has been found only in cases of heavy myocardial iron deposition [39, 40]. This lack of correlation can probably be explained by differences in transferrin receptor concentration, iron kinetics, the crystalline structure of ferritin and the degree of organ inflammation or fibrosis [7, 78, 88\u201392]. Furthermore, under chelation therapy with DFO intracellular paramagnetic ferrioxamine is formed, which exits slowly from cells unless there is an active excretion pathway as is present in the hepatocytes [88].\nYoung patients with thalassaemia studied longitudinally have shown absence of substantial improvement in the MR parameters of liver siderosis under different chelation therapy regimens [82]. This may be explained by the fact that liver siderosis progresses very fast in thalassaemia patients, and iron overload develops after only 2\u00a0years of transfusion therapy [93]. Therapy with the most widely used chelating agent is started at the age of about 3\u00a0years, and until growth is completed DFO should not exceed a dose of 40\u00a0mg\/kg per day [7]. An early start to monitoring the progress of tissue iron deposition with MRI might be useful in deciding whether to begin chelation therapy at a younger age and when to introduce new chelating agents.\nHeart\nCardiac failure is the leading cause of death from iron overload in patients with thalassaemia [94, 95]. Thalassaemia patients on a regimen of regular blood transfusions who are not receiving chelation therapy, develop heart enlargement by the age of 10\u00a0years and heart failure by the age of 16\u00a0years [22]. Cardiomyopathy secondary to iron overload is potentially reversible with vigorous chelation therapy [7]. Myocardial iron has been evaluated by MRI using SIR and relaxometry techniques (Fig.\u00a03) [22, 28, 39, 40, 68, 82, 96\u201398]. In the prechelation era, post-mortem examination of patients with thalassaemia showed a close correlation between cardiac and hepatic iron concentrations. MRI studies have demonstrated discordant results regarding the relationship of myocardial iron with hepatic iron and serum ferritin [28, 39, 40, 68, 97]. Differences in iron kinetics and variations in chelation schemes may be responsible for the lack of correlation of the MRI-determined myocardial iron with that of the liver [88]. More active elimination of iron from the hepatocytes than from the myocytes may play a role in the absence of correlation in patients receiving chelation therapy [28, 39, 68].\nFig.\u00a03A 16-year-old male with \u03b2-thalassaemia major. a T2 map of the short axis of the heart shows low values of the left ventricle (arrow) and the septum (arrowhead). b R2* (=1\/T2*) map of the short axis of the heart shows high signal of the septum (asterisk). Note the reduction of motion artefacts and better delineation of the heart boundary in the R2* image (courtesy of Dr. M. Douskou)\nAnother important issue is whether myocardial iron evaluated by MRI could predict which patient will develop cardiac failure and arrhythmia. Thalassaemia patients with significantly reduced left ventricular ejection fraction (LVEF) have myocardial T2* values about 20\u00a0ms [22, 37, 38, 68], although most patients with T2* values less than 20\u00a0ms have normal LVEF [68]. Furthermore, the relationship between myocardial R2 and LVEF is either non-existent or weak [22, 38\u201340]. Differences in cardiac functional indices among patients with similar MRI measurement of iron might also be explained by genetic factors that predispose to the more rapid development of cardiac failure when a critical level of myocardial siderosis is reached [99, 100].\nHeterogeneous iron distribution in the myocardium has been demonstrated in histological studies, and this would suggest the necessity for global MRI estimation of myocardial iron [42, 101]. Pepe et al. [42], using T2* multiecho cardiac MRI, demonstrated that global T2* shows a close correlation with midseptal T2*, suggesting that the latter may be used for a quick assessment of myocardial siderosis.\nMR studies have shown that cardiac siderosis increases with age, but that siderosis progresses more slowly in the heart than in the liver [28, 82]. Systematic MRI studies in children are necessary in order to evaluate the age at the start of cardiac iron overload.\nPituitary gland and brain\nIncreased iron deposition in the anterior pituitary gland is the cause of hypogonadotropic hypogonadism and growth hormone deficiency [102, 103]. Iron accumulates in all five cell types of the adenohypophysis, but preferentially in the gonadotropin-secreting cells [102, 103]. Gonadotropin cell death due to iron toxicity is probably the cause of the decreased pituitary gland height observed in thalassaemia patients with hypogonadotropic hypogonadism (Fig.\u00a04) [104]. MRI has been used to evaluate pituitary siderosis with SIR and relaxometry techniques [28, 43, 83, 105]. SIR and R2 findings show significant correlation with serum ferritin [83]. The very few studies evaluating simultaneously the pituitary and other solid organs have demonstrated no correlation between their MRI parameters of siderosis [28, 43, 81]. Pituitary siderosis appears to increase with age, but MR data are lacking for children under the age of 7\u00a0years [28, 81] [84].\nFig.\u00a04The pituitary. a A 12-year-old male with \u03b2-thalassaemia major. Midsagittal T1-weighted (TR\/TE 500\/20\u00a0ms) scan shows low-signal intensity of the anterior pituitary lobe (arrow) and the bone marrow (asterisks) suggesting iron overload. The pituitary gland is small measuring 3.5\u00a0mm (normal for age 5.3\u00b10.8\u00a0mm) [112]. This patient developed hypogonadotropic hypogonadism. b An 11-year-old male with \u03b2-thalassaemia major. Mid-sagittal T1-weighted (TR\/TE 500\/20\u00a0ms) scan shows normal signal intensity of the anterior pituitary lobe (arrow) along with normal pituitary gland height (6\u00a0mm). The bone marrow (asterisk) shows low-signal intensity suggesting iron overload\nThe single study assessing iron in the brain of thalassaemia patients by MRI [106] showed significantly higher R2 values in the cortex, the putamen and the caudate nucleus in patients than in controls. A lack of correlation between R2 and serum ferritin was found, possibly because serum ferritin, which does not cross the blood\u2013brain barrier, is not a good marker of brain siderosis [106].\nPancreas\nIron overload leads to impairment of the endocrine and exocrine functions of the pancreas [107, 108]. Pancreatic siderosis has been evaluated in thalassaemia patients using SIR techniques and relaxometry techniques [61, 84, 109]. Midiri et al. [109], using fat as the reference tissue, found a significant negative correlation of SIR with serum ferritin and trypsin. Papakonstantinou et al. [61], using muscle as the reference tissue, found no relationship of the pancreatic SIR with the hepatic SIR or serum ferritin. The findings of Argyropoulou et al. [84], using T2 relaxometry techniques, were similar. In addition, relaxometry techniques demonstrated that the T2 relaxation time of the pancreas of patients with thalassaemia is significantly lower in children than in adults [84]. Fatty degeneration of the pancreas in adult patients may explain these differences (Fig.\u00a05).\nFig.\u00a05Siderosis of the spleen, pancreas and adrenals. a A 14-year-old male with \u03b2-thalassaemia major. Axial scan, fourth echo of a multiecho sequence (TR\/TE 2,000\/20, 40, 60, 80, 100, 120, 140, 160\u00a0ms) shows low-signal intensity of the liver, pancreas (arrow) and spleen suggesting increased iron deposition. b A 27-year-old-male with \u03b2-thalassaemia major and diabetes. Axial T2-W (TR\/TE 1,800\/80\u00a0ms) scan shows low-signal intensity of the liver and adrenals suggesting siderosis. High signal intensity of the pancreas (arrow) suggests tissue damage and fatty degeneration\nAdrenals\nAbnormalities in adrenal function have been reported in patients with thalassaemia [103]. There is only one study evaluating adrenals for iron overload with MRI (Fig.\u00a05), which showed a significant correlation between adrenal and liver siderosis [87].\nSpleen, lymph nodes and bone marrow\nIn spite of the fact that the spleen, lymph nodes and bone marrow, which all contain reticuloendothelial cells, are among the first organs to be affected by iron overload [1, 13], there have been very few studies evaluating their iron overload in thalassaemia by MRI, and these used mainly SIR techniques [59, 60, 62, 84]. SIR of the spleen shows a significant correlation with serum ferritin but not with SIR of the liver (Fig.\u00a05) [62]. The absence of correlation between liver and spleen siderosis could be explained by differences in iron kinetics, by differences in the cluster size of iron proteins, by haemochromatosis gene mutations in \u03b2-thalassaemia major carriers, and by the presence of extramedullary haemopoietic tissue in the spleen [78, 110, 111]. Intraabdominal lymph nodes in \u03b2-thalassaemia have been related to chronic hepatitis C [60]. Lymph node siderosis correlates with liver, but not with spleen siderosis [60].\nIn the few studies that have been reported, SIR and relaxometry methods have shown discordant results for MR parameters of bone marrow siderosis and serum ferritin [59, 84]. Normal bone marrow signal associated with liver siderosis has been reported in a few patients with thalassaemia and this may be due to differences in genotype or differences in chelation therapy regimens [59].\nConclusion\nIt is evident that MR relaxometry has the potential to become the method of choice for non-invasive, safe and accurate assessment of iron load. Further theoretical research, along with studies monitoring wider age groups of patients are needed before a generally accepted protocol can be established. Until then, extreme caution is needed in its clinical application and interpretation. Experimental artefacts, non-optimized protocols, poor data analysis and unawareness of the inherent limitations of current methodologies in assessing a heavy body iron burden can result in misleading diagnosis and inappropriate management of thalassaemia patients with iron overload. Future experimental developments in relaxometry based on a better theoretical understanding of the contribution of the iron-containing proteins to the MR signal are expected to further strengthen its clinical role in the monitoring of patients with \u03b2-thalassaemia major.","keyphrases":["mri","iron","\u03b2-thalassaemia","relaxometry"],"prmu":["P","P","P","P"]}
{"id":"Placenta-1-5-1895600","title":"Oxidative Stress and the Induction of Cyclooxygenase Enzymes and Apoptosis in the Murine Placenta\n","text":"Placental oxidative stress has been implicated in many complications of human pregnancy, including preterm delivery and preeclampsia. It is now appreciated that reactive oxygen species can induce a spectrum of changes, ranging from homeostatic induction of enzymes to apoptotic cell death. Little is known regarding the occurrence of placental oxidative stress in other species. We investigated markers of oxidative stress in the labyrinthine (LZ) and junctional (JZ) zones of the murine placenta across gestational age, and correlated these with expression of the cyclooxygenase enzymes COX-1 and COX-2, and apoptosis. We tested a causal link between the two by subjecting placental explants to hypoxia-reoxygenation (H\/R) in vitro, a known stimulus for generation of oxidative stress. Western blotting demonstrated significant increases in the concentrations of hydroxynonenal (HNE), COX-1 and COX-2 with gestational age. Dual-labelling demonstrated co-localisation of HNE, and COX-1 and COX-2 within the trophoblast of the LZ, and glycogen cells of the JZ. An apoptotic index based on TUNEL-positivity demonstrated an increase with gestational age, and dual-labelling showed co-localisation of TUNEL labelling with HNE and active caspase-3 within the trophoblast of the LZ. H\/R significantly increased oxidative stress, induction of COX-1 and COX-2, and the apoptotic index. Co-localisation demonstrated the increases in COX to be within the trophoblast of the LZ, and in particular the glycogen cells of the JZ. Apoptosis was restricted to the LZ. We speculate that the induction of COX enzymes is a physiological response to oxidative stress, and may play a role in initiating or augmenting parturition. Generation of oxidative stress may also play a role in influencing the growth trajectory of the placenta, and its component cell types. The mouse may provide an experimental genetic model in which to investigate these phenomena.\n1\nIntroduction\nOxidative stress of the placenta has been implicated in the pathogenesis of many complications of human pregnancy, including miscarriage, preeclampsia, and preterm labour [1\u20133]. These complications all share the common predisposing feature of reduced trophoblast invasion and incomplete conversion of the uterine spiral arteries [4,5]. Therefore, it is generally held that malperfusion of the placenta leads to increased oxidative stress, resulting in placental dysfunction. In the case of preeclampsia it is thought that the stress induces the release of a cocktail of factors, including pro-inflammatory cytokines, anti-angiogenic factors and apoptotic debris [6], into the maternal circulation that causes activation of the peripheral endothelial cells. Placental inflammatory lesions have also been associated with premature onset of labour [7].\nBy contrast, there are few data relating to placental oxidative stress available for other species. The mouse is potentially a powerful model in which to study the mechanisms of placental and obstetrical pathologies, and is the best-studied mammalian experimental genetic model system. The definitive murine placenta is a discoid haemochorial organ as in the human, and there are three anatomically and physiologically distinct regions: the labyrinth zone (LZ), junctional zone (JZ) and decidua basalis (DB) [8]. The labyrinth zone is the principal area of exchange, and consists of a meshwork of maternal blood spaces lined by trophoblast. The trophoblast comprises three layers; an outer layer (I) composed of cytotrophoblast cells, and two layers (II & III) of syncytiotrophoblast [9]. Deep to the syncytiotrophoblast are the fetal capillaries, embedded in a small amount of connective tissue. The junctional zone, interposed between the labyrinth and the decidua basalis, is of unknown function, but contains spongiotrophoblast cells and trophoblastic glycogen cells (GC) [8].\nThe decidua basalis is composed principally of maternal uterine tissues, and contains maternal arteries and veins that are continuous with the arterial and venous channels traversing the JZ. Glycogen cells migrate into the DB and surround the maternal arteries that supply the placenta late in gestation. The mechanisms of myometrial stimulation and activation appear to parallel those of the human [10]. For example, prostaglandins (PGs) are central components of labour in both humans and mice [11]. There are fundamental similarities in the changes occurring prior to labour, including an increase in myometrial contractile activity, increased coupling of myometrial cells through the formation of gap junctions, an increase in PG receptors in the myometrium, and enhanced sensitivity to oxytocin at term. There is also a rise in intracellular calcium resulting in an increase in myosin light chain phosphorylation.\nIn this study we determined the degree of oxidative stress present in the murine placenta at different gestational ages in normal pregnancies. We also tested whether there was a temporal and spatial association between oxidative stress and two markers of placental function, one physiological and one pathological. These were the induction of cyclooxygenase (COX) enzymes and apoptosis respectively.\nEvidence from other systems points to potential links between oxidative stress and COX induction, resulting in the production of PGs [12\u201316]. PGs are derived from arachidonic acid (AA) through the actions of the COX enzymes, which are rate-limiting. There are two known isoforms of COX, COX-1 and COX-2, which share similar structures but differ in their function. COX-1 is believed to play a constitutive \u201chousekeeping\u201d role, whereas COX-2 is considered to be an inducible isoform. However, these contrasting roles have not been proven in the mouse [17]. In mice induction of COX-2 by administration of lipopolysaccharide leads to preterm labour, which can be blocked using the specific COX-2 inhibitor SC-236 [18].\nApoptosis has been described within the LZ of the normal mouse placenta, and is most frequent towards term and in post-mature placentas [19]. The stimulus for this is not known, but oxidative stress is a potent inducer of trophoblast apoptosis in the human placenta [20].\n2\nMaterials and methods\n2.1\nTissue collection\nPlacentas were collected from C57BL\/6J inbred mice, and all experiments were carried out in accordance with the UK Government Home Office licensing procedures. Stages E14, E16, E18, and E19 were studied, where E1 of gestation was the morning when a copulation plug was found.\n2.2\nWestern blotting\nPlacentas from three different animals at each gestational age were homogenized in ice-cold sample buffer, and centrifuged at 15,000\u00a0rpm. Protein concentration within the supernatant was determined using a colorimetric assay (Bio-Rad Bradford protein assay). An equal amount of protein from each sample (30\u00a0\u03bcg) was subjected to electrophoresis in a 10% SDS-polyacrylamide gel under reducing conditions and transferred onto nitrocellulose membrane by a semidry transfer machine. Membranes were incubated in a blocking solution (TBS containing 5% powdered non-fat milk and 0.01% Tween-20) at room temperature for 1\u00a0h, and then in the same solution containing primary antibody (Table 1) at 4\u00a0\u00b0C overnight. After washes in TBS-T, membranes were incubated with the horseradish peroxidase secondary antibody at room temperature for 1\u00a0h. The immunoreactive bands were visualized by enhanced chemiluminescence with the ECL system (Amersham Biosciences) according to the manufacturer instructions. The levels of protein expression were quantified densitometrically and normalised against their respective expressions of \u03b2-actin. Prestained protein markers were used as molecular weight standards for each analysis.\n2.3\nImmunohistochemistry\nAt each gestational age three placentas were fixed by immersion in 4% paraformaldehyde overnight, dehydrated and embedded in paraffin wax. Sections (6\u00a0\u03bcm thick) were dewaxed and immunostained according to our standard protocol [21]. When necessary, antigen retrieval was performed in a pressure cooker for 2\u00a0min in 0.01\u00a0M Citrate buffer at pH 6. Primary antibodies (Table 1), diluted in goat serum, were applied overnight in a humidified chamber at 4\u00a0\u00b0C. Binding was detected using Vectastain Elite ABC kits (Vector Laboratories) and SigmaFast DAB (Sigma), according to the manufacturers\u2019 instructions. Sections were then lightly counterstained with haematoxylin. Negative controls were performed by omitting incubation with the primary antibody. For each antibody slides from all the gestational ages were prepared and immunostained in the same batch, ensuring identical conditions for comparisons.\nIn order to localise expression of the antigens to specific cell types staining intensity was graded visually from 0\u20133, where 0 represented the negative control. Three people scored the sections independently, blinded to the gestational age or experimental protocol, and the mean value was taken.\n2.4\nImmunofluorescent dual-labelling\nDual immunofluorescent labelling was performed in order to test for co-localisation of markers of oxidative stress and COX expression in individual cells. Sections were dewaxed, permeabilised in TBS containing Triton X-100 (0.1%) and Tween 20 (0.1%) (TBS-TT) for 30\u201360\u00a0min, and blocked in 5% bovine serum albumin for 20\u00a0min at room temperature. Primary antibodies diluted in TBS-TT were applied overnight at 4\u00a0\u00b0C. Negative control sections had primary antibodies omitted. After three 10-min washes in TBS-TT, sections were incubated for 1\u00a0h at room temperature with a mixture of fluorescent secondary antibodies, containing anti-goat Alexa 488 and anti-rabbit Alexa 568 (both used 1\/100; from Molecular Probes) in TBS-TT. Sections were washed in TBS-TT as before and then twice in distilled water for 5\u00a0min and subsequently mounted in Vectashield mounting medium containing DAPI (Vector, UK). Images were captured using a Leica confocal microscope (LeicaTCS-NT, Leica Instruments GmbH).\nCo-localisation was identified in overlay images by a yellow signal caused by the combination of the green signal (COX-1\/COX-2) and the red signal (HNE).\n2.5\nTUNEL assay\nStaining was performed using In-Situ Cell Death Detection kit-Texas Red (Roche Applied Science) according to the manufacturer's instructions. Conditions were adjusted so that the staining pattern matched morphological assessments of apoptosis based on examination of resin-embedded samples. Once the protocol had been optimised an apoptotic index was calculated as follows:\n2.6\nDual-fluorescence TUNEL assay & Active Caspase-3, and HNE co-localisation\nStaining was performed using In-Situ Cell Death Detection kit-Texas Red (Roche Applied Science) according to the manufacturer's instructions, and using an Anti-Active Caspase-3 antibody (Promega) at 1:50, and Anti-HNE as previously at 1:200. Samples were also DAPI stained to intercalate the DNA and fluoresce cell nuclei. For negative controls, omission of primary antibody and TdT enzyme were performed separately. For positive controls, post-lactational mouse mammary glands were used. For confocal microscopy, all samples were analysed during one session to avoid bias, and multiple (usually 5) fields of view per labyrinth were saved for further analysis.\n2.7\nEx-vivo culture experiment\nIn order to induce oxidative stress placental samples were subjected to hypoxia-reoxygenation (H\/R) in vitro [22]. Placentas were taken from 3 animals at gestational age E18, halved on an ice block and immersed in ice-cold PBS prior to culture. Three samples, each 5\u201310\u00a0mm3, from each placenta were incubated in culture medium (TCS large vessel endothelial cell basal medium (TCS CellWorks, Milton Keynes, UK)), which had been maintained for several hours in the specified gas composition at 37\u00a0\u00b0C. Data for intraplacental oxygen concentrations are not yet available for the mouse, and so estimates were made on the basis of results for another small mammal, the guinea-pig. The umbilical venous pO2 towards term is 29.5\u00a0mmHg, which is close to that for fetal primates and ungulates [23]. Therefore, for normoxia a gas mix of 5% O2, 90% N2, 5% CO2 was used, whereas for hypoxia it was 1% O2, 94% N2, 5% CO2. Gas composition was monitored and controlled throughout using Biospherix (Redfield, NY) Pro-Ox and Pro-CO2 probes. Control samples were cultured for 4\u00a0h in normoxia, whereas samples subjected to H\/R experienced 1\u00a0h of hypoxia followed by 3\u00a0h of normoxia. Following culture the tissues were either frozen or fixed for Western blotting and immunohistochemistry respectively.\n2.8\nStatistical analyses\nStatistical significance was calculated by using one-way analysis of variance (ANOVA). Differences between groups were assessed using Fisher's Least Significant Difference Test. Statistical significance was assumed at a P-value <0.05.\n3\nResults\n3.1\nMarkers of oxidative stress\nHydroxynonenal (HNE) is a marker of lipid peroxidation. Western blotting revealed significant changes across gestational age (P\u00a0=\u00a00.003), with low concentrations of HNE at E14, a significant increase at E16, and a subsequent decline by E18 (Fig.\u00a01a,b).\nScoring based on IHC revealed different intensities of immunoreactivity in the different cell populations, and different temporal patterns. At E14 immunoreactivity was low in all cell types (Fig.\u00a01c,d). At E16 there was moderate staining (scored 2) in the syncytiotrophoblast of the LZ, which peaked (3) at E18 and declined to 1 by E19 (Fig.\u00a01e). The cytotrophoblast cells of the LZ stained uniformly at level 1 throughout, and no immunoreactivity was observed in the fetal endothelial cells. In the JZ strong staining was observed in the GCs at E16 (2.5) and E18 (3) (Fig.\u00a01f), and this was maintained at 2.5 at E19. The spongiotrophoblast cells were less immunoreactive, scoring only 0.5 throughout gestation, and no immunoreactivity was observed in the giant cells or the decidua.\nNitrotyrosine (NT) staining indicates the formation of the prooxidant peroxynitrite, and of an imbalance in the production of the superoxide ions and nitric oxide. Immunostaining revealed a similar pattern to HNE, with almost undetectable staining in early samples and significant increases from day 16 onwards (data not shown).\nTherefore, oxidative stress increases during gestation in the murine placenta, specifically in syncytiotrophoblast and glycogen cells.\n3.2\nCOX-1\nWestern blotting revealed significant changes in concentrations of COX-1 across gestational age (P\u00a0=\u00a00.013). There was an increase from E14 to E16, followed by a significant decrease by E18 (Fig.\u00a01a,b).\nAt E14 COX-1 immunoreactivity was not observed in any cell type, except for low intensity (level 1) staining in the GCs. At E16 strong reactivity (3) was visible in the GCs, with lower intensity staining (1) being present in the spongiotrophoblast and cytotrophoblast cells (data not shown). By E18 there was a general increase in staining, with many of the cytotrophoblast cells (2) reacting positively, along with\u00a0the syncytiotrophoblast (2), and all GCs (3) (Fig.\u00a02a,b). This pattern persisted at E19, when the decidua (2) was also\u00a0immunoreactive. By contrast, the spongiotrophoblast cells\u00a0displayed\u00a0low (0.5) immunoreactivity throughout gestation.\nTowards term COX-1 and HNE immunoreactivity were present within the same cells in both the LZ and JZ, as evidenced by the dual-labelling experiments (Fig.\u00a02c,d).\n3.3\nCOX-2\nWestern blotting revealed a significant change in concentrations of COX-2 across gestation (P\u00a0=\u00a00.002), peaking at E19 (Fig.\u00a01).\nIHC demonstrated that COX-2 expression is low (level 1) within the syncytiotrophoblast, spongiotrophoblast and GCs at E14. With advancing gestational age immunoreactivity increased, principally in the cytotrophoblast cells (2), GCs (2.5) and spongiotrophoblast (1) at E18 and E19 (Fig.\u00a03a,b). The syncytiotrophoblast displayed low immunoreactivity (0.5) throughout gestation.\nAgain there was strong co-localisation between immunoreactivity for COX-2 and HNE (Fig.\u00a03c).\n3.4\nDetection of apoptotic changes\nDuring apoptosis cytokeratins within trophoblast cells are cleaved by active-caspase 3 to yield a specific product detected by the M30 antibody [24]. The labyrinth showed low immunoreactivity for active-caspase 3 until E18 (Fig.\u00a04b), but thereafter it increased in intensity until E19. The staining was localised to both the syncytiotrophoblast and individual cytotrophoblast cells.\nImmunoreactivity for M30 also increased with gestational age, and was most abundant at E18, declining slightly at E19 (data not shown).\nIn order to estimate the number of apoptotic nuclear profiles TUNEL staining was performed and an apoptotic index calculated. TUNEL-positive nuclei were first observed at E16 when the apoptotic index was 2.7%, and steadily increased in frequency until E19, when the index reached 20.0% (P\u00a0<\u00a00.001). There was a strong temporal and spatial association between TUNEL staining and immunoreactivity for HNE (Fig.\u00a04a) and active caspase-3 (Fig.\u00a04b). At E16 a number of cells were caspase-positive but TUNEL-negative. Later, almost all TUNEL-positive nuclei were surrounded by cytoplasm immunoreactive for active caspase 3. TUNEL-positive nuclei were rarely observed in the absence of caspase-positivity, when they were considered to be necrotic.\nTo further confirm trophoblast cells were undergoing apoptosis, morphological evidence was sought by examining resin embedded samples. Cells displaying peripheral nuclear chromatin condensation, some in the classical crescent shape, were seen with increasing frequency towards E19 (Fig.\u00a04c). There was occasional evidence of apoptotic body formation, but other classical characteristics, such as plasma membrane blebbing, could not be resolved with light microscopy. The position of the nuclei suggested that the majority was within the syncytiotrophoblast, but the involvement of cytotrophoblast and endothelial cells could not be excluded at the light microscopic level.\n3.5\nEx-vivo culture experiments\nTo test the relationships between oxidative stress and the induction of COX enzymes and apoptosis experimentally, placental explants were subjected to hypoxia\/reoxygenation (H\/R), a known stimulus for the generation of reactive oxygen species [22].\nWestern blotting confirmed increased concentrations of HNE and COX-1 in the explants following H\/R compared to control samples frozen at the start of the experiment (time zero) or cultured under normoxic conditions. For COX-2 a significant increase was only observed compared to the time zero controls (Fig.\u00a05a,b).\nIHC localised the HNE to individual cytotrophoblast cells and to the syncytiotrophoblast layers of the labyrinth. In the JZ individual spongiotrophoblast cells were immunopositive, but the strongest staining was seen in the GCs within the JZ and decidua. Dual-labelling revealed strong co-localisation for COX-1 and COX-2 in the spongiotrophoblast and GCs in the JZ, and within the syncytiotrophoblast and cytotrophoblast cells in the labyrinth (Fig.\u00a05c).\nIHC revealed increased immunoreactivity for M30 within the trophoblast following H\/R. The apoptotic index based on TUNEL labelling increased from 2.8% and 1.6% in the time zero and normoxic controls respectively, to 20.6% following H\/R (P\u00a0=\u00a00.006). The majority (78%) of TUNEL positive cells were also immunoreactive for HNE on dual-labelling (Fig.\u00a05d). From resin-embedded material it appeared that most of the cells with condensed peripheral chromatin following H\/R were within the syncytiotrophoblast layers (data not shown).\nFrom these data it can be concluded that there is a significant increase in oxidative stress following H\/R, and that this is associated with increased expression of COX-1 and COX-2 expression in the LZ and JZ, and with increased apoptosis in the labyrinth.\n4\nDiscussion\nThese results indicate that oxidative stress increases with gestational age in the murine placenta during normal pregnancies, and that it may play a significant physiological role by inducing higher concentrations of the COX-1 and COX-2 enzymes, and hence increasing prostaglandin synthesis. Our data also indicate a close temporal and spatial association between oxidative stress and trophoblast apoptosis within the labyrinth. Trophoblast apoptosis has been linked to the pathophysiology of preeclampsia, and the mouse might provide a useful genetic model in which to elucidate the mechanisms underlying the shedding of apoptotic debris.\nWe have recently proposed that placental oxidative stress arises through fluctuations in oxygenation [3,25], and its development in the murine placenta towards term is consistent with this view. As fetal and placental oxygen extraction reach their peaks so any transient mismatch between maternal oxygen supply and feto-placental demand will lead to a dip in the intraplacental oxygen concentration. The fact that H\/R in vitro is a powerful inducer of oxidative stress, and that the stress localises to the same tissues as in late gestation supports this hypothesis. It is now accepted that oxidative stress induces a spectrum of cellular changes, ranging from the physiologically homeostatic to the frankly pathologic. Induction of enzymes is towards the more physiological end of that spectrum, whereas apoptosis is towards the opposite extreme.\nInduction of the COX enzymes has been linked to oxidative stress through activation of the p38MAPK and the NF-\u03baB family of transcription factors in other cell types [26\u201330]. In our study there appeared to be strong co-localisation between the formation of HNE and expression of COX-1 and -2 to individual cytotrophoblast, spongiotrophoblast and glycogen cells, both in vivo and in vitro. However, there was an apparent discrepancy between the Western blot results and our IHC findings, for the former indicated a peak in oxidative stress and COX-1 at E16 while the staining intensity for HNE and COX-1 increased until E19. This difference can be accounted for by the fact that the number of GCs declines by about half between E16 and E18 [31], so reducing their contribution to the overall tissue homogenate.\nExpression of the COX enzymes appears to be mainly altered in placental rather than decidual tissues. Changes with gestational age were observed in the trophoblast populations within the LZ, but the greatest changes in immunoreactivity were seen within the GCs and the spongiotrophoblast cells in the JZ. Expression of COX-1 and COX-2 has recently been reported in the rat placenta, and that of COX-2 similarly increases with gestational age, particularly in the JZ [32]. The placental cells in this zone have direct access to maternal blood as they line the maternal venous sinuses. Therefore, it is possible that PGs synthesized in this zone in response to oxidative stress are released into the maternal circulation. It is also notable that the GCs migrating into the DB are strongly immunoreactive for COX, and so PGs synthesised by these cells could exert local paracrine effects in the decidua. As PGs have been linked with the induction of parturition [10,11,33] this raises the possibility that placental oxidative stress could play a role in initiating or augmenting uterine contractions should the mismatch in maternal blood supply and feto-placental demands become extreme.\nTrophoblast apoptosis increased late in gestation as assessed by IHC for M30 and TUNEL, and cell morphology. There was a close temporal and spatial co-localisation with oxidative stress, although on Western blotting evidence of oxidative stress appeared to decline in the last days of gestation. This may again reflect changes in the cell populations within the placenta and associated decidua. The apoptotic index based on TUNEL-positivity showed a continual increase until term, although M30 immunoreactivity suggested a decline after E19. There is strong evidence that following initial cleavage, exposing the M30 epitope, cytokeratin 18 becomes further cleaved to smaller fragments [24]. In these late stages of apoptosis the M30 epitope may be lost, and so the antibody no longer detects all apoptotic trophoblast [34]. A causal link between oxidative stress and apoptosis was strengthened by the fact that apoptosis could be induced by H\/R in vitro. As in the human placenta this was associated with increased concentrations of active caspase-3 [20]. The mitochondrial pathway was strongly implicated in the apoptotic process in the human, but further work is required to determine whether the same mechanism operates in the mouse.\nThe increase in oxidative stress and apoptosis in the LZ with gestational age may explain some of the growth dynamics of the murine placenta. Overall placental volume increases with gestational age until E16.5 and then plateaus, coinciding with the rise in oxidative stress. It is notable that within the LZ the volume and surface area of the trophoblast plateaus at E16.5 whereas those of the fetal capillaries continue to increase until E18.5 [35]. The fetal endothelial cells did not display immunoreactivity for HNE or nitrotyrosine, and did not appear to label with TUNEL, suggesting that oxidative stress and its consequences are lower in this cell type.\nThe links between oxidative stress and apoptosis suggest that the mouse may provide a useful genetic model in which to investigate the relative roles of antioxidant enzymes and signalling pathways in regulating trophoblast apoptosis. Apoptosis increases towards term in normal human pregnancies [36], but increased rates have been reported in preeclamptic placentas and in placentas from cases of intrauterine growth restriction [37\u201339]. It is thought that microparticles of apoptotic, or aponecrotic, debris released from the apical surface of the syncytiotrophoblast into the maternal circulation result in an enhanced maternal inflammatory response in preeclampsia [6]. Whether apoptotic debris is shed from the murine labyrinth as in the human placenta towards the end of gestation remains to be determined.","keyphrases":["oxidative stress","cyclooxygenases","apoptosis","murine placenta"],"prmu":["P","P","P","P"]}
{"id":"Int_J_Colorectal_Dis-3-1-2039795","title":"The sentinel node procedure in colon carcinoma: a multi-centre study in The Netherlands\n","text":"Background Lymph node status is the most important predictive factor in colorectal carcinoma. Recurrences occur in 20% of the patients without lymph node metastases. The sentinel lymph node (SLN) biopsy is a tool to facilitate identification of micrometastatic disease and aberrant lymphatic drainage. We studied the feasibility of in vivo SLN detection in a multi-centre setting and evaluated nodal micro-staging using immunohistochemistry (IHC).\nIntroduction\nSurvival in patients with colon carcinoma is strongly correlated with lymph node status: the 5-year disease-free survival rate is 70\u201380% for patients with lymph node-negative disease (stage I\/II) but only 45\u201350% for those with node-positive disease (stage III) [1]. The presence of lymph node metastases indicates the use of adjuvant chemotherapy in these patients, which increases the 5-year survival rate with about 10% [2]. Despite the favourable prognosis of patients with localized colon carcinoma without regional lymph node metastasis, 20\u201330% of these patients will develop recurrent disease after apparently curative resection [3]. It is possible that in this group of patients, small lymph node metastases have been missed, resulting in understaging. This may be due to an inadequate surgical lymphadenectomy or insufficient pathological examination [4]. According to international guidelines, meticulous pathological examination of at least 12 lymph nodes is warranted for adequate staging of patients with colon carcinoma [5]. However, several studies showed that the minimal number of lymph nodes necessary for correct staging varied considerably from 6 to 18 to as many as possible in the study of Goldstein et al. [4, 6\u20139]. In addition, in-depth pathological examination of lymph nodes by immunohistochemical staining for cytokeratin or reverse transcriptase polymerase chain reaction (RT-PCR) may reveal micrometastases that could have been missed by routine haematoxylin and eosin (H&E) examination. Several authors have reported a decreased survival rate when micrometastases are detected in colon carcinoma [10\u201313]. The possible benefit of adjuvant therapy in this group of patients is not clear yet.\nThese (ultra-)staging techniques are time consuming, labour intensive and costly. For optimal staging, in depth examination of only the sentinel lymph node (SLN) could be helpful. In colon carcinoma, the SLNs are defined as the first one to four blue-stained nodes with the most direct lymph drainage from the primary tumour. They have the greatest potential to harbour metastatic disease when present, enabling focussed examination with multi-level micro-sectioning of the SLNs to provide a more efficient and cost-effective detection of micrometastases. In addition, patterns of aberrant lymphatic drainage can be visualized with SLN mapping, which may lead to a more extended resection. Several studies have reported varying results of the SLN procedure in colon carcinoma [14\u201320]. This study presents the results of the SLN procedure in six Dutch hospitals. The primary aim of this study is to test the accuracy and sensitivity of the SLN procedure in a multi-centre setting. Furthermore, we looked at upstaging and possible aberrant lymphatic drainage.\nMaterials and methods\nThis study was performed between May 2002 and May 2005 in five teaching hospitals and one university hospital. All procedures were supervised by one of the coordinating surgeons (Plukker, Braat). Only patients with histologically proven primary colon carcinoma were included in the study. Patients with distant metastases or gross lymph node involvement as shown by pre-operative examinations or palpation during surgery were excluded. The procedure was only performed when one of the study coordinators was available for supervision (Plukker, Braat, Kelder). The study was approved by the local scientific ethics committee, and all patients had given informed consent. Patients with rectal cancer were excluded from the study.\nSLN mapping was carried out through an open procedure by injection of 1\u20133\u00a0ml Patent Blue with a tuberculin syringe and 29-gauge needle sub-serosally in four quadrants around the tumour. The sub-serosal injection was carried out before vascular ligation. Within 5 to 10\u00a0min after the blue dye injection, the SLN could be identified by following the blue-stained lymphatic vessels leading to the blue-stained SLN. These lymph nodes were tagged with a long suture. SLNs were defined as the first one to four blue-stained lymph nodes seen within the regional basin. After marking of the SLNs, routine resection was performed. If the SLN was found outside the normal lymphatic basin, we performed an extended resection. The tumour and all lymph nodes were examined according to standard guidelines [5]. If the SLNs were negative after routine H&E staining, they were sectioned at 150-\u03bcm intervals and examined at three levels with H&E as well as immunohistochemistry on cytokeratins (CK7\/8 or 18). Metastases between 0.2 and 2\u00a0mm were referred to as micrometastases. Metastases smaller than 0.2\u00a0mm were referred to as isolated tumour cells [5]. Upstaging was defined as the presence of micrometastases or isolated tumour cells after immunohistochemistry (IHC) in patients with a negative lymph node status after H&E.\nDefinitions\nSee Fig.\u00a01. Identification rate is the number of patients with one or more SLNs identified (b)\/the total number of procedures (a)\u2009\u00d7\u2009100%. Negative SLNs were false negative if one of the other regional lymph nodes (non-SLNs) were tumour positive (d). The accuracy of the SLN procedure suggests a conformity of the SLN status and the regional nodal status, i.e. the total number of patients with a positive SLN (c)\u2009+\u2009the number of patients with a true-negative SLN (e)\/the number of patients with an identified SLN (b)\u2009\u00d7\u2009100%. Sensitivity is the number of patients with a positive SLN (c)\/The total number of node positive patients (c\u2009+\u2009d)\u2009\u00d7\u2009100%. Upstaging is the number of patients with positive SLNs by IHC (g)\/the number of patients who were node negative by H&E examination (e\u2009+\u2009g)\u2009\u00d7\u2009100%.\nFig.\u00a01Flowchart. SLN Sentinel lymph node, pts patients\nResults\nThe SLN procedure was performed in 69 patients. Tumour characteristics are shown in Table\u00a01. Figure 1 shows the total number of patients and SLN results. At pathological examination, a mean of 11 nodes per specimen was found; per hospital, this varied between 9 and 17 (9, 10, 12, 14, 17, 17, respectively). The mean number of SLNs was 2.3 per patient. The SLN was identified in 67 out of 69 patients (97%). One of the two failed procedures was in a patient with a carcinoma in the sigmoid colon surrounded by a concurrent diverticulitis. The other patient had extended lymph node metastases with angio-invasion at pathological examination. In 28 patients, lymph node metastases were identified at the pathological examination; this includes the one case where the SLN procedure failed because of extensive lymph node metastases (28 of 69, 41%). This one case with lymph node metastases and a failed procedure was excluded from further statistical analysis on the SLN procedure, leaving 27 node-positive patients in the final analysis. In 24 patients, the SLN was positive, either with H&E staining or with IHC, resulting in a sensitivity of 89% (24 of 27) in the group of 67 patients with a successful SLN procedure. If we leave out the patients who had a positive sentinel node only after IHC, the sensitivity is 15 of 18 (83%). In 15 of 24 SLN-positive patients, the SLN was the only involved lymph node (63%). In 9 of 27 lymph node-positive patients, metastases were found only after IHC. In four patients, these were micrometastases, whereas in five cases, isolated tumour cells were found. Therefore, without IHC, the number of node-positive cases would have been 27 minus 9, which is 18. This corresponds to a total of 49 node-negative cases by H&E in the group with a successful SLN procedure. With IHC, the upstaging is 9 of 49 or 18%. The SLN was negative in 43 patients. In 40 patients, the non-SLNs were also negative. This results in a negative predictive value of 93% (40 of 43). One of the three patients with a false-negative SLN had lymph node metastases with extra-nodal growth in the non-SLN. In another patient, a small tumour deposit was found in the mesocolon right next to the primary tumour. This was classified as N1 according to the American Joint Committee on Cancer classification, although it is unclear whether this is a true lymph node metastasis or some kind of \u2018in transit\u2019 metastasis. The last patient with a positive non-SLN showed micrometastases at H&E examination in a small peritumoural lymph node. Aberrant lymphatic drainage was seen in three patients (4%). In two cases, the SLN was found on the left side of the middle colic artery in patients with a tumour in the ascending colon. In both cases, an extended right hemicolectomy was performed. The third patient had a tumour near the rectosigmoid junction with a high para-aortal SLN. Therefore, we performed an extended left sided resection en-bloc with a partial para-aortal dissection. None of these lymph nodes contained metastases. All other SLNs were found in the mesocolon in close proximity to the tumour. In these cases, the central lymph node as identified by the pathologist was always a non-SLN. The accuracy of the SLN procedure in this study was 96%, as the pathological status of the SLN corresponds with the definitive lymph node status in 64 of the 67 patients.\nTable\u00a01Tumour characteristicsCharacteristicsValuesTumour location\u00a0Right colon35\u00a0Left colon2\u00a0Sigmoid colon32T-stage\u00a011\u00a0214\u00a0348\u00a046Mean number of lymph nodes11Mean number of SLN2.3\nDiscussion\nWith an identification rate of 97%, accuracy of 96%, sensitivity of 89% and negative predictive value of 93%, this study shows that it is possible to perform the SLN procedure properly in patients with localized colon carcinoma in a multi-centre setting. Other multi-centre studies showed varying results of this technique (Table\u00a02) [14\u201321]. Our results correlate with those from other larger studies, which show accuracy and sensitivity rates of 95\u201398 and 89\u201393%, respectively [15, 18\u201320]. Most smaller studies show worse results with low accuracy and success rates and corresponding low sensitivity rates and negative predictive values [14, 17, 21]. In one study, the time between injection of the blue dye and identifying the SLN was too long, leading to a larger number of SLNs [17]. It is very likely that not all of these blue nodes were true SLNs. In the study by Bertagnolli et al. [14], 79 patients were operated on by 25 different surgeons in 13 different hospitals. A mean of three procedures per surgeon seems insufficient to adequately learn this technique. It is known that the learning curve of the SLN in colon carcinoma stabilizes after about five procedures [20]. To minimize technical failures, the procedure in our study was performed by a few surgeons under direct supervision of one of the two surgeons coordinating this study (Braat, Plukker). Apart from too few procedures, the worse results in some studies might be explained by inclusion of patients with advanced disease. Some studies included patients with clinically apparent stage III or stage IV disease [21]. Widespread lymph node metastases could result in obstruction of lymphatic channels, and lymphatic drainage is bypassed to other (non-sentinel) lymph nodes. This phenomenon is called skip-metastasis. It was noted in one of the patients with a false-negative SLN in our study who had advanced lymphatic metastases with extra-nodal growth. Patient selection is therefore important for a reliable SLN procedure in colon carcinoma. In fact, the SLN procedure is not useful in patients with clinically apparent stage III or stage IV disease as false-negativity rates will be higher. Moreover, in these patients, metastases will be easily found at routine pathological examination, and the SLN procedure will not have any additional value. The SLN procedure could be useful in those patients with (micro)metastases that would not be identified with routine pathological examination. Furthermore, the failed procedure in one of our patients with concurrent diverticulitis also suggests the importance of an undisturbed lymphatic drainage for a successful SLN procedure.\nTable\u00a02 Results of multi-centre studies of the SLN procedure in colon cancerStudyNumber of patientsNumber of centresIdentification rate (%)Accuracy (%)Sensitivity (%)Upstaging (%)Bilchik et al. [15]40310010010010Saha et al. [18]131399979216Bertagnolli et al. [14]72139281420Read et al. [21]3827976253Kelder and Braat69697968913 or 18\nWe saw aberrant lymphatic drainage in three patients (4%). This percentage correlates with the literature [15, 18, 20]. In this study, none of these aberrant SLNs showed metastases. However, potentially, these aberrant SLNs are the only lymph nodes containing metastasis, as shown in a previous study [22]. In an experimental situation, it seems justified to perform an extended resection in these cases. Further study should be performed to justify an extended resection in the daily practice.\nLiterature not clearly indicates how many nodes should be examined to accurately predict lymph node status [6\u20138, 23, 24]. One study showed that a colon specimen usually contains about 50 lymph nodes and that more than 70% of the lymph nodes containing metastases are smaller than 5\u00a0mm [9]. It is also known that the prognosis in node-negative patients with colon carcinoma is better when more lymph nodes have been examined [24]. Taking this into account, the pathologist takes only a sample of the lymphatic basin of a resected colon specimen, even when international guidelines are followed, which state that at least 12 lymph nodes are needed for adequate staging [5]. The mean number of 11 lymph nodes in our study is not enough to predict lymph node status according to the international guideline. This fact could theoretically lower the chance to detect metastases in non-SLN and thus could lower the false-negative rate. However, we did not find any differences in false negative rates between the two hospitals with a mean number of nine and ten examined nodes (40 cases) and the hospitals with more than 12 examined nodes (29 cases). With regards to upstaging, most studies show an upstaging of 10\u201316% [15, 18\u201320]. However, they calculated upstaging by dividing the number of IHC-positive patients by the total number of patients (Fig.\u00a01g\/a, 9 of 69, 13% in our group). We think it is better to consider upstaging solely in the H&E node-negative group, as this is the group to be upstaged by IHC. Using this method, we find 18% upstaging in our series. In addition to this true upstaging, patients with a SLN as the only site of metastases could have been \u2018possibly upstaged,\u2019 as conventional pathological dissection of the mesentery might have missed this lymph node. The SLN procedure with patent blue might be able to improve adequacy of the lymph node examination by selecting the right lymph nodes, even small nodes less than 5mm, to be examined in depth by the pathologist. We found the SLN to be the single lymph node with metastasis in 15 (21%) of the patients (Fig.\u00a01 f). \u2018Possible upstaging\u2019 might play a role here, but we cannot prove this.\nAs we believe that even isolated tumour cells are important for staging, we assigned patients with micrometastases or isolated tumour cells to the group of node-positive patients. It must be remarked, however, that these cases were also used for the calculation of upstaging. Our idea of the biological importance of micrometastases and isolated tumour cells is based on a recent meta-analysis that showed that micrometastases detected retrospectively by RT-PCR correlated better with overall survival than IHC and carried significant prognostic value [12].\nRegarding the detection of micrometastases, two studies showed a high reliability of the SLN concept to predict micrometastases and\/or isolated tumour cells also in non-SLNs. Therefore, it seems sufficient to perform IHC only on the SLN, while examining the non-SLNs with H&E [25, 26]. Prospective studies are needed to evaluate the potential benefit of systemic chemotherapy in patients with these micrometastases. A reliable SLN procedure might facilitate this intensive pathological examination by allowing focussed examination of only the SLN and thereby aid in a better patient selection for adjuvant therapy in the future.","keyphrases":["colon carcinoma","sentinel lymph node","micrometastasis","minimal residual disease"],"prmu":["P","P","U","M"]}
{"id":"Acta_Neuropathol_(Berl)-3-1-1824787","title":"Intraneuronal A\u03b2 immunoreactivity is not a predictor of brain amyloidosis-\u03b2 or neurofibrillary degeneration\n","text":"Amyloid \u03b2 (A\u03b2) immunoreactivity in neurons was examined in brains of 32 control subjects, 31 people with Down syndrome, and 36 patients with sporadic Alzheimer\u2019s disease to determine if intraneuronal A\u03b2 immunoreactivity is an early manifestation of Alzheimer-type pathology leading to fibrillar plaque formation and\/or neurofibrillary degeneration. The appearance of A\u03b2 immunoreactivity in neurons in infants and stable neuron-type specific A\u03b2 immunoreactivity in a majority of brain structures during late childhood, adulthood, and normal aging does not support this hypothesis. The absence or detection of only traces of reaction with antibodies against 4\u201313 aa and 8\u201317 aa of A\u03b2 in neurons indicated that intraneuronal A\u03b2 was mainly a product of \u03b1- and \u03b3-secretases (A\u03b217\u201340\/42). The presence of N-terminally truncated A\u03b217\u201340 and A\u03b217\u201342 in the control brains was confirmed by Western blotting and the identity of A\u03b217\u201340 was confirmed by mass spectrometry. The prevalence of products of \u03b1- and \u03b3 -secretases in neurons and \u03b2- and \u03b3-secretases in plaques argues against major contribution of A\u03b2-immunopositive material detected in neuronal soma to amyloid deposit in plaques. The strongest intraneuronal A\u03b217\u201342 immunoreactivity was observed in structures with low susceptibility to fibrillar A\u03b2 deposition, neurofibrillary degeneration, and neuronal loss compared to areas more vulnerable to Alzheimer-type pathology. These observations indicate that the intraneuronal A\u03b2 immunoreactivity detected in this study is not a predictor of brain amyloidosis or neurofibrillary degeneration. The constant level of A\u03b2 immunoreactivity in structures free from neuronal pathology during essentially the entire life span suggests that intraneuronal amino-terminally truncated A\u03b2 represents a product of normal neuronal metabolism.\nIntroduction\nNeurofibrillary degeneration and brain amyloidosis with deposition of fibrillar amyloid \u03b2 (A\u03b2) in plaques are diagnostic features of Alzheimer\u2019s disease (AD). Intracellular processing of amyloid precursor protein (APP) with \u03b2- and \u03b3-secretases generates A\u03b21\u201340 and A\u03b21\u201342 in the endoplasmic reticulum, trans-Golgi network, and endosomal\u2013lysosomal system [10, 19, 25, 62, 66]. An amino-terminally truncated 3-kd peptide (A\u03b217\u201340\/42) is the product of APP cleavage with \u03b1- and \u03b3-secretase. Cells may produce and secrete several species of A\u03b2, including A\u03b21\u201340, A\u03b21\u201342\/3 and A\u03b217\u201340\/42 [8, 23, 24].\nHuman neurons are A\u03b2-immunoreactive [11, 17, 22, 38, 39, 57]. The nature, distribution, and role of intraneuronal A\u03b2 are the subject of controversy (see review by Takahashi [58]). Intraneuronal A\u03b2 immunoreactivity has been localized in lipofuscin deposits [4, 65], cathepsin D-positive vesicles of lysosomal origin [11], multivesicular bodies within presynaptic and postsynaptic compartments [57], and intracellular and extracellular neurofibrillary tangles (NFTs) [1, 21, 26, 27, 34, 42]. Several studies of cytoplasmic A\u03b2 immunoreactivity in neurons and glial cells in the human brain have been conducted to determine the properties of A\u03b2 in human neurons and its role in fibrillar plaque formation [11, 17, 22, 39, 57]. The key observation has been the absence of [64] or only minimal intraneuronal A\u03b2 immunoreactivity [22] in normal brain. Therefore, the appearance of or an increase in A\u03b2 immunoreactivity has been suggested as a sign of neuronal pathology [22] leading to fibrillar plaque formation in the brain of people with AD [11, 15, 22, 39, 63].\nOverexpression of APP, overproduction of A\u03b2, and early intracellular accumulation of A\u03b2 [33] have been considered the foundation for early onset of AD pathology and functional deterioration in adults with Down syndrome (DS) in their 40\u2019s [22, 39, 63]. The loss of A\u03b2 immunoreactivity in areas of plaque formation has led to the conclusion that neurons release intracellular A\u03b2, which initiates a seeding process leading to plaque formation [39]. According to Bahr et al. [2], the death of neurons, a prominent feature of AD, is associated with the release of oligomerized intracellular A\u03b242 into the surrounding milieu, which may stimulate the production of amyloidogenic fragments of APP, amplify the levels of intracellular A\u03b2 in neighboring cells, and act as a nidus for the deposition of secreted A\u03b2.\nThe association of intracellular A\u03b242 with intraneuronal tangles has been considered an indication that the growing concentration of A\u03b242 may contribute to NFT formation [40]. The presence of altered aspartyl residues in intracellular NFTs has been interpreted as indicating that racemized A\u03b2 peptides are involved in neurofibrillary degeneration [41, 52].\nThe pattern of intraneuronal A\u03b2 immunoreactivity observed in the control cohort in our previous study [61] was in conflict with the hypothesis that A\u03b2 immunoreactivity in neurons is the key and an early event in the cascade of pathology leading to AD. Therefore, the aim of this study was to reexamine the hypothesis that intraneuronal A\u03b2 immunoreactivity is an early manifestation of Alzheimer-type pathology leading to the formation of fibrillar plaque and\/or neurofibrillary degeneration. To test this hypothesis, we examined patterns of A\u03b2 accumulation in the human brain during development, adulthood, and aging, and patterns of changes in the amount and distribution of A\u03b2 in neurons in people with DS and DS\/AD, and sporadic AD.\nMaterials and methods\nHuman tissue\nThe brains of 99 individuals were examined. Control brains included 32 cases, from 3\u00a0months to 102\u00a0years of age (3-, 6-, 11-, and 13-month-old infants; 4-, 8-, 14-year-old children; and adults from 23 to 102\u00a0years of age; 19 males and 13 females). DS brains included 31 cases from 3\u00a0weeks to 72\u00a0years old (infants that were 3\u00a0weeks, and 3, 6, and 9\u00a0months old and adults from 28 to 72\u00a0years of age; 15 males and 16 females). In the AD cohort, the brains of 36 subjects from 65 to 97\u00a0years of age (17 males and 19 females) were studied. Diagnostically, three subjects had a mild cognitive impairment (MCI) corresponding to global deterioration scale (GDS) stage 3 [45, 46], a frequent clinical precursor of AD. GDS stage 4 (mild AD) was reported for three subjects; stage 5 (moderate AD) for three subjects; stage 6 (moderately severe AD) for 11 subjects, and stage 7 (severe AD) for 16 subjects.\nOne brain hemisphere was fixed in 10% buffered formalin for 1.5 to several months and then dissected into 1\u00a0cm thick slabs. The tissue blocks were dehydrated for 5\u00a0days in 70% ethanol, then 2\u00a0days in 80% ethanol, and finally 1\u00a0week in 96% ethanol. The samples were infiltrated with polyethylene glycol (PEG) 400 (Merck #807,485) for 6\u00a0days (two changes of 3\u00a0days each, at room temperature) and with PEG 1000 for another 6\u00a0days (two changes of 3\u00a0days each, at 42\u00b0C). The slabs were embedded in fresh PEG 1000 [28]. The tissue blocks were cut serially at 50\u00a0\u03bcm thick sections. The immunoproperties of intracellular A\u03b2 in tissues fixed in formalin for 1.5 to several months were also compared with immunostainings of sections from five brains (two DS and three AD) prefixed in 4% paraformaldehyde for 2\u00a0h, dissected into 1\u00a0cm thick slabs, fixed in 4% paraformaldehyde for the next 24\u00a0h, cryoprotected with 15 and 30% solutions of sucrose (4\u00a0days), frozen, and cut serially into 50\u00a0\u03bcm thick sections.\nThe methods selected for this study were approved by the New York State Institute for Basic Research (IBR) Institutional Review Board. The tissue samples were provided by the Silberstein Institute for Aging and Dementia at New York University, the Brain Bank at IBR, and the University of Miami Brain and Tissue Bank. In postmortem examination, all samples were identified only by an anonymous case number, and tissue was examined blind to clinical and demographic information.\nImmunostaining\nSeveral antibodies were applied to serial sections to evaluate A\u03b2 immunoreactivity in cortical and subcortical structures, the cerebellum, and brainstem. Monoclonal antibodies (mAbs) 6E10 and 6F\/3D were used for characterization of the amino-terminal portion of A\u03b2 (Table\u00a01). mAb 6E10 recognizes an epitope in residues 4\u201313 of A\u03b2 (Signet Laboratories, 1:10,000) [30, 36, 59]. mAb 6F\/3D recognizes an epitope in residues 8\u201317 of A\u03b2 (Novocastra Laboratories Ltd; NCL-\u03b2-amyloid). The central portion of A\u03b2 was detected with mAb 4G8, which recognizes an epitope in residues 17\u201324 of A\u03b2 [29]. The carboxyl terminus of A\u03b2 was characterized with pAbs purified from rabbit serum by epitope-specific affinity chromatography. These antibodies react with A\u03b2 residues 32\u201340 (Catalog nr. 44\u2013348) and 32\u201342 (Catalog nr. 44\u2013344; BioSource International, Inc., CA, USA). The reactivity towards other species of A\u03b2 peptides was eliminated through a series of preabsorption steps. Purified rabbit polyclonal antibodies, R164 and 165, specific for the carboxyl terminus of A\u03b21\u201342 (residues 35\u201342) were also used [37]. Fibrillar A\u03b2 was detected with rabbit polyclonal antibody R262 produced by immunization of rabbits with fibrillar A\u03b21\u201342. Rabbit antibodies were purified according to the protocol described by Miller et al. [36].\nTable\u00a01Mouse monoclonal and rabbit polyclonal antibodies used for immunocytochemistryTypeNameEpitopeDilutionSourcemAb6E104\u201313 aa A\u03b21:10,000Signet Laboratories (developed at IBR)mAb6F\/3D8\u201317 aa A\u03b21:50NovocastramAb4G817\u201324 aa A\u03b21:8,000IBR (Dr. R. Kascak)pAb44\u201334832\u201340 aa A\u03b21:500BiosourcepAb44\u201334432\u201342 aa A\u03b21:500BiosourcepAbR16435\u201342 aa A\u03b21:500IBR (Drs. D.L. Miller and P.D. Mehta)pAbR16535\u201342 aa A\u03b21:500IBR (Drs. D.L. Miller and P.D. Mehta)pAbR262Fibrillar A\u03b2 1\u201342aa1:200IBR (Drs. D.L. Miller and P.D. Mehta)mAbTau1189\u2013207 aa of tau1:100,000IBR (Dr. R. Kascsak)\nMonoclonal antibody 6E10 reacts with A\u03b2 both on western blots and in formalin fixed material [13, 14]. mAb 6E10 binds APP on western blots, as a native protein, but does not immunoreact with APP after fixation with formalin and dehydration. We have also shown that in cultures of cells overexpressing APP and formalin fixed and dehydrated both antibodies, 6E10 and 4G8 do not detect APP but they detect A\u03b2 [13]. Also, the study of cultured cells with elevated levels of APP and C-terminal fragments of APP revealed that mAb 4G8 does not detect APP and C-terminal fragments of APP by immunocytochemistry [12] (Table\u00a02).\nTable\u00a02A\u03b2n\u221240 and A\u03b2n\u221242 concentration (pmol\/g) in temporal cortexControl case #A\u03b2n\u221240A\u03b2n\u221242p3p4p3p42472210.325621211,1690.30.30.2a0.2aBrain samples were homogenized in formic acid as described in Materials and methods. Aliquots of the extracts were neutralized and subjected to PAGE and immunobloting. p4 bands migrated above the 3\u00a0kDa standards and bound mAb 6E10. p3 bands migrated near the 3\u00a0kDa standard and bound R162 or R226 but did not bind mAb 6E10aThere was only one band in this extract. Its level was too low to be revealed by mAb 6E10 and so could not be identified as p3 or p4\nThe endogenous peroxidase in the sections was blocked with 0.2% hydrogen peroxide in methanol. To enhance immunoreactivity of A\u03b2, sections were treated with 90% formic acid for 30\u00a0min [31]. The sections were then treated with 10% fetal bovine serum in phosphate buffer solution (PBS) for 30\u00a0min to block nonspecific binding. The antibodies were diluted in 10% fetal bovine serum in PBS and were incubated with sections overnight at 4\u00b0C. The sections were washed and treated for 30\u00a0min with either biotinylated sheep anti-mouse IgG antibody or biotinylated donkey anti-rabbit IgG antibody diluted 1:200. The sections were treated with an extravidin peroxidase conjugate (1:200) for 1\u00a0h and the product of reaction was visualized with diaminobenzidine (0.5\u00a0mg\/ml with 1.5% hydrogen peroxide in PBS). After immunostaining, the sections were lightly counterstained with cresyl violet.\nPhosphorylated tau protein of neurofibrillary tangles was detected with mAb Tau-1 (1:100,000). Tau-1 recognizes an epitope in residues 189\u2013207 of the human tau sequence [16]. To obtain optimum staining with Tau-1, sections were treated with alkaline phosphatase (Sigma, Type VII-L, 400 \u03bcg\/ml in PBS, pH 7.4, 0.01% H2O2) [20].\nA three-point classification was used to estimate semi-quantitatively the difference of immunoreactivity with mAb 4G8 in four brain structures of 25 control adult subjects. The amygdala and cornu Ammonis were selected as structures susceptible to amyloidosis \u03b2 and neurofibrillary degeneration. The lateral geniculate body and dentate nucleus were selected as structures resistant to AD pathology and almost free of plaques and NFTs even in severe AD. Grade 1 corresponded to weak immunoreactivity present in about 50% of neurons; grade 2 corresponded to moderate immunoreactivity in almost all neurons; and grade 3 corresponded to strong immunoreactivity present in almost all neurons in a given brain structure.\nSections from brain of DS subjects were used to determine whether intraneuronal A\u03b2 immunoreactivity differs in people with an extra copy of the gene encoding APP. The difference between intraneuronal A\u03b2 and amyloid in plaques was determined with antibodies detecting the amino- and carboxyl-terminus and the middle portion of A\u03b2 peptide. The relationship of neurofibrillary changes to A\u03b2 immunoreactivity in neurons was evaluated in brains of people with DS\/AD and sporadic AD.\nPartial purification and identification of A\u03b2 peptides\nSamples of the brain cortex (250\u00a0mg) from control males that were 31, 32 and 59\u00a0years-old were dispersed in 2.5\u00a0ml of 99% formic acid by brief sonification and centrifuged for 30\u00a0min at 100,000\u00a0g. Supernatants were dried by centrifugal evaporation, the residues were resuspended in 1\u00a0ml of 70% (v\/v) formic acid and centrifuged as above. The solutions were subjected to size-fractionation on a 1\u00a0\u00d7\u00a030\u00a0cm Pharmacia HR-12 column equilibrated with 70% formic acid as previously described [36]. The A\u03b2-containing fractions were identified by Western blotting and their contents were quantified by photodensitometry, as previously described [44]. The A\u03b2-containing fractions were dried under vacuum and were re-dried out of ammoniacal methanol to neutralize traces of formic acid. To solubilize A\u03b2 peptides, each residue was treated with 250\u00a0\u03bcl of 50\u00a0mM ammonia and centrifuged as above, and the supernatant liquid was adjusted to pH 7.4 with KH2PO4.\nAntibody R287, raised to A\u03b227\u201337 using the previously described methods [40] was purified on a peptide affinity column and was coupled to an Aminolink matrix (Pierce-Endogen) at a concentration of 360\u00a0pmol per 200\u00a0\u03bcl of settled matrix. The A\u03b2 isoforms were immuno-adsorbed to 8\u00a0\u03bcl of R287-agarose during a 3\u00a0h incubation. Following washes with PBS and water the A\u03b2 isoforms were eluted with 2\u00a0\u00d7\u00a0100\u00a0\u03bcl portions of 2.5% trifluoroacetic acid in 50% acetonitrile. About 50% of total A\u03b2 peptides present in the cortex samples were recovered by this method, as evaluated by Western blotting. The shape and size of the A\u03b2 band in Western blotting of the size-fractionated material and the immuno-purified peptide were the same, which suggested that the process did not selectively enrich any of the A\u03b2 peptides.\nMass spectrometry and Western blotting\nThe peptide preparation isolated by immunoadsorption on R287-agarose was dissolved in 30\u00a0\u03bcl of 40% (v\/v) formic acid and approximately 82% was loaded on a Symmetry\u00ae C18 nanoAcquity column (180\u00a0\u03bcm\u00a0\u00d7\u00a020\u00a0mm) as six sequential 4.1\u00a0\u03bcl injections. Following the sixth injection, the peptides were resolved on an Atlantis dC18 nanoAcquity column (100\u00a0\u03bcm\u00a0\u00d7\u00a0100\u00a0mm) with a 20\u00a0min gradient of 10 to 50% acetonitrile in water containing 0.1% formic acid at flow rate of 0.4\u00a0\u03bcl\/min. The eluate from the column was analyzed directly on a Qtof Micro (Waters Corp.) mass spectrometer equipped with a nanoflow electrospayer and scanned for m\/z 50\u20131,500 at 1.1\u00a0s intervals. An external lock mass standard (leucine enkephalin; m\/z\u00a0=\u00a0556.2771) was analyzed at 11\u00a0s intervals through a separate orthogonal electrosprayer. The data were processed using MassLynx 4.0 (Waters Corp.) software, including Accurate Mass Measure and MaxEnt3 algorithms. Briefly, chromatograms were created from the data set by plotting a 1\u00a0Da window around the m\/z\u2019s of interest. The peaks were confirmed to have the component of interest by summing the mass spectra across the peak, determining the centroid m\/z values, followed by deconvolution and transformation to the accurate mass value of the 1+ ion. The resulting ion envelope encompassing mass isomers containing zero to four 13C atoms was plotted to create the chromatogram of the specific peptide. For A\u03b217\u201340, the 2+ ion was by far the most prevalent and its ion envelope spanned m\/z values from 1196.3 through 1198.7. For A\u03b21\u201340, the 4+ and 5+ ions were the most prevalent, spanning m\/z values from 1082.8 through 1084.8 and 866.4 through 868.0, respectively.\nWestern blotting was performed by a previously described method, which allows the detection of sub-femtomol quantities of A\u03b240 or A\u03b242 [44]. Samples and appropriate standards were subjected to PAGE in tris-tricine 16% gels. Rabbit polyclonal antibodies R162 (raised to A\u03b231\u201340) and R226 (raised to A\u03b232\u201342) were used to detect the C-terminal sequences of A\u03b2 isoforms. These antibodies are highly selective for their targets. R162 showed no cross-reactivity with a 100-fold excess of A\u03b242 [44], and R226 is 2500-fold more reactive with A\u03b242 than with A\u03b240 [37]. Monoclonal antibody 6E10 [30] was used to detect the A\u03b2 sequence 4\u201313 [59]. The blots were developed wth NBT and BCIP and the bands were quantified by photodensitometry [44].\nResults\nAge-associated changes of A\u03b2 immunoreactivity in neurons in control and DS brains\nInfants and children. In the majority of the brain structures examined, including the entorhinal cortex and neocortex, amygdala, and hippocampus, A\u03b2 immunoreactivity was present in neurons of the 11 and 13 month-old normal infant brains and the 9\u00a0month-old infant diagnosed with DS (Fig.\u00a01). At this age, A\u03b2-immunoreactivity was observed in only about 5\u201310% of the neurons. However, fine and randomly dispersed A\u03b2-immunoreactive granules were present in the cytoplasm in almost all neurons in the dentate gyrus. In infants, strong A\u03b2-immunoreactivity was found in clusters of cytoplasmic granules in almost all neurons in the magnocellular portion of the lateral geniculate body, but reaction in small neurons was rare and weak. In the brains of 4 and 8\u00a0year-old children, about half of neurons in the cortex and basal ganglia were A\u03b2-immunopositive, whereas in the brain of the 14\u00a0year-old child, the percentage of immunoreactive neurons was more than 50%.\nFig.\u00a01Intraneuronal A\u03b2 immunoreactivity with mAb 4G8 (17\u201324aa) in the CA1 sector, dentate gyrus (DG), amygdala (AMY), lateral geniculate body (LGB), and cortex (COR; temporal superior gyrus) in 9\u00a0month-old infant with DS, 11\u00a0month-old control infant, and 33 and 83\u00a0year-old control subjects (C) reveals age-associated and structure-specific differences. A few immunopositive neurons are present in CA1 sector, amygdala, and cortex in infants; however, immunopositivity is detectable in the majority of neurons in infant dentate gyrus and lateral geniculate body. In young adult (33\u00a0year-old) and aged subject (83\u00a0year-old) immunopositivity is present in the majority of neurons and is much stronger than in infants. Dense deposits are present in almost all neurons in the LGB and amygdala, and dispersed fine granular staining is present in the majority of neurons in the dentate gyrus. Loose granular immunopositive material appears in about 80% of neurons in CA1 sector and cortical neurons\nAdults. In adults, A\u03b2 immunoreactivity was present in the majority of the neurons, but the amount and pattern of distribution of immunopositive material showed a broad range of cell-type- and brain structure-specific differences. Strong A\u03b2 immunoreactivity characterized all nuclei in the amygdala in adults, with the strongest reaction in the lateral and ventral subdivisions (mean grade, 2.3; SD\u00a0\u00b1\u00a00.4). In the amygdala, numerous A\u03b2-positive cytoplasmic granules were concentrated at one pole of the cell and formed a perinuclear cap. In the cornu Ammonis (CA), A\u03b2 immunoreactivity showed marked sector-specific differences. Strong and uniform reaction characterized neurons in the CA4 sector, and moderate reaction was observed in CA2 and 3 sectors, but A\u03b2 immunoreactivity in neurons in the CA1 sector was much weaker and less uniform (mean grade in the CA, 1.8; SD\u00a0\u00b1\u00a00.7). Apical dendrites in pyramidal neurons in the CA and subiculum proper were often marked with rows of A\u03b2-immunoreactive granules. Granule cells in the dentate gyrus contained numerous randomly dispersed A\u03b2-positive granules. Strong immunoreactivity appeared in large neurons, and moderate staining appeared in small neurons of the caudate nucleus and putamen. Reactions in neurons in the thalamus, globus pallidus, and n. accumbens were less uniform and weaker. A\u03b2 immunoreactivity in cortical pyramidal neurons was stronger and more common than in cortical granule cells. In the cerebellum, a moderate amount of fine, granular A\u03b2- immunoreactive material was found in the majority of Purkinje cells, but in granule cells, the reaction was weak and was present only in a minority of cells. Very strong A\u03b2 immunoreactivity was found in the cytoplasm in all neurons in the LGB and dentate nucleus (mean grade 2.8\u00a0\u00b1\u00a00.4), as well as in the nucleus olivaris.\nAged subjects. In general, in control people older than 65\u00a0years of age, A\u03b2 immunoreactivity was reduced in about 20% of neurons of the amygdala, nucleus basalis of Meynert (NBM), cornu Ammonis, large neurons in the caudate-putamen, and cortex, as compared to control adults. However, the amount and pattern of A\u03b2 immunoreactivity in the dentate gyrus, LGB, dentate nucleus, and nucleus olivaris inferior were comparable in aged subjects and normal adults.\nIn addition to the structure- and age-associated differences in neuronal A\u03b2 immunoreactivity, there were interindividual differences between people of the same age and gender. Strong and uniform A\u03b2 immunoreactivity was found in 44% of the control subjects; moderate and uniform in 40%, and weak and nonuniform in 16%. No difference between control and DS infants (Fig.\u00a01) and adult with DS who has incipient neurofibrillary degeneration (28\u00a0years-old) was detected.\nIntraneuronal A\u03b2 immunoreactivity in control brains with neurofibrillary degeneration and in DS\/AD or sporadic AD\nIn the control group, mAb Tau-1 revealed neurofibrillary degeneration in the brains of all persons older than 43\u00a0years of age. In the entorhinal cortex, subiculum, and amygdala, the number of neurons with NFTs increases with age. The increase in Tau-1-positive material was paralleled by the reduction and loss of cytoplasmic A\u03b2 immunoreactivity in affected neurons in the older subjects.\nAll subjects with DS older than 28\u00a0years-old were affected with neurofibrillary degeneration, and all people with DS older than 38\u00a0years of age had developed \u03b2 amyloidosis. The increase in neurofibrillary degeneration, neuronal death, and \u03b2 amyloidosis was associated with marked reductions of intraneuronal A\u03b2 immunoreactivity in the entorhinal cortex (Fig.\u00a02a, b), hippocampus, amygdala, NBM, and neocortex.\nFig.\u00a02Development of neurofibrillary tangles (arrowheads mAb Tau-1, a) in neurons in the second layer of the entorhinal cortex of a 43\u00a0year-old subject with DS\/AD is associated with loss of cytoplasmic A\u03b2 immunoreactivity (arrowheads mAb 4G8, b) without plaque formation in islands of stellate neurons. Amyloid plaque in the third layer (asterisk) is mAb 4G8-positive. The insula of a subject with mild AD (GDS 4) is affected by early and severe amyloidosis-\u03b2. Plaques are marked with arrowheads (c). mAb 4G8-positive material is present in both plaques (asterisk) and neurons (arrowheads, d). The strongest A\u03b2 immunoreactivity is present in almost all neurons (arrowheads) in the lateral geniculate body (low and high magnification; AD, GDS 6; e), dentate nucleus (DS\/AD, GDS 7; 72\u00a0year-old; f), and nucleus olivaris inferior (DS\/AD, GDS 7; 72\u00a0years-old; g); however, amyloid plaques do not develop in these structures, even in severe AD\nA similar pattern of reduction of A\u03b2 immunoreactivity associated with neurofibrillary degeneration, amyloidosis \u03b2, and neuronal loss was found in people with MCI and subjects with sporadic AD (GDS stage 4\u20137). In severe AD (GDS stage 7), almost all neurons in the second layer of the entorhinal cortex were affected by neurofibrillary degeneration detected with mAb Tau-1. These neurons contained only traces of A\u03b2 immunoreactivity or were free of cytoplasmic A\u03b2. However, in neocortex, the reduction of intracellular A\u03b2 immunoreactivity was less pronounced (Fig.\u00a02c, d).\nBoth in persons with DS\/AD and persons with sporadic AD, the percentage of A\u03b2- immunopositive Purkinje cells was reduced by about 25% compared to age-matched control subjects. The majority of granule cells failed to manifest A\u03b2 immunoreactivity. In the cerebellum, the decrease in neuronal A\u03b2 immunoreactivity was observed in the almost total absence of intraneuronal NFTs.\nIn contrast to these patterns of reduction in A\u03b2 immunoreactivity, a constant level of strong reaction was present in almost all neurons in LGB, dentate nucleus, and nucleus olivaris inferior in people with DS\/AD or sporadic AD. These structures were almost free of NFTs and amyloid plaques in all AD subjects, including those with severe AD (Fig.\u00a02e\u2013g).\nImmunoproperties of intraneuronal A\u03b2\nThe lack of A\u03b2 immunoreactivity or presence of very few cytoplasmic grains immunoreactive with antibodies detecting an epitope in residues 4\u201313 (mAb 6E10) and 8\u201317 (mAb 6F\/3D) and the presence of cytoplasmic immunoreactivity with antibodies detecting 17\u201324 (mAb 4G8), 32\u201342 (pAb 44\u2013344), and 35\u201342 (pAbs R164 and 165), indicates that neurons harbor mainly amino-terminally truncated A\u03b2 (Fig.\u00a03a, b, c, d, e, f). Stronger staining of intracellular A\u03b2 with pAb 44\u2013344 (residues 32\u201342) and pAb R164 (35\u201342) than with pAb 44\u2013348 (32\u201340), indicates that the major component of cytoplasmic A\u03b2 in neurons is A\u03b217\u201342. Intraneuronal A\u03b2 was not labeled with pAb R262 that detects fibrillar amyloid in cored plaques and in vessel wall (not shown). The pattern of immunostaining of A\u03b2 in fibrillar plaques confirmed that parenchymal plaques contain mainly A\u03b21\u201342.\nFig.\u00a03 a\u2013f shows immunoreactivity of intraneuronal A\u03b2 in the CA4 sector of a 32\u00a0year-old control subject. No reaction in neurons stained with mAb 6E10 (4\u201313 aa) (a) and mAb 6F\/3D (8\u201317 aa) (b) indicates that in the majority of neurons, the amino-terminal portion of A\u03b2 is not detectable. Immunoreactivity of intraneuronal A\u03b2 is shown in sections stained with mAb 4G8 (17\u201324 aa) (c), pAb R164 (35\u201342 aa) (d), pAb 44\u2013348 (32\u201340 aa) (e) and pAb 44\u2013344 (32\u201342 aa) (f). In the brain of subjects with AD some extracellular ghost tangles contain full length A\u03b2 peptides immunoreactive with antibodies 6E10 (g), 4G8 (h), and pAb 44\u2013344 (i)\nA\u03b2 immunoreactivity in ghost tangles\nWhile neuronal neurofibrillary changes were associated with loss of cytoplasmic A\u03b2 immunoreactivity, some ghost tangles (extracellular NFTs) were A\u03b2-positive (Fig.\u00a03g, h, i). In the sporadic AD group, among 31 examined subjects, A\u03b2-positive ghost tangles were found in 24 brains (77%). The proportion of subjects with A\u03b2-reactive ghost tangles increased from 50% in people with MCI to 70% in people with moderately severe AD and to 100% in people with severe AD. A\u03b2-positive ghost tangles were found in 100% of these subjects in the entorhinal cortex, in 87% in the CA1, 29% in the amygdala, 21% in the CA2 and CA4, 8% in the subiculum proper, and 4% in the CA3 and temporal cortex. Of AD subjects with A\u03b2-positive ghost tangles, they were numerous in 41%, moderate in number in 17%, and were rare in 21% of the subjects.\nIn 13 of 24 brains of persons with DS (54%), A\u03b2-immunoreactive ghost tangles were found in the entorhinal cortex, amygdala, and CA1 and CA2 sectors. The youngest subject with positive staining was 43\u00a0years of age at demise. A\u03b2-positive ghost tangles were found in the brains of all DS subjects from 55 to 72\u00a0years old. Of DS subjects in whom A\u03b2-immunoreactive ghost tangles were present, they were numerous in 46%, moderate in number in 39%, and rare in 15% of subjects.\nIn the control cases, no A\u03b2-immunoreactive ghost tangles were observed in spite of the presence of neurofibrillary changes in all subjects older than 43\u00a0years of age.\nA\u03b2-positive ghost tangles were strongly immunoreactive with mAbs 6E10 (residues 4\u201313), 6F\/3D (8\u201317), and 4G8 (17\u201324), and pAbs 44\u2013344 (32\u201342) and R165 (35\u201342). The reaction with pAb 44\u2013348 (32\u201340) was weak.\nA\u03b2 isoforms could be detected on Western blots of crude control brain extracts\nAntibody R162 revealed 2 bands containing the A\u03b232\u201340 sequence (Fig.\u00a04, lane 3), whereas antibody R226 revealed 2 bands containing the A\u03b233\u201342 C-terminal sequence (Fig.\u00a04, lane 4). Immunoreactivity with mAb 6E10 (Fig.\u00a04, lane 5) unequivocally showed that the upper band contained A\u03b2 peptide sequences possessing amino acid residues 4\u201313. This antibody does not bind to A\u03b217\u201340 even at a 15-fold higher concentration (Fig.\u00a04, lane 6). We found that the large amounts of other proteins in the extracts affected the electrophoretic migration rates and band shapes of the peptides, so that their migration rates did not unambiguously distinguish A\u03b21\u201340 from A\u03b217\u201340. Using synthetic A\u03b2 standards we showed that substances in a crude brain extract retarded the migration of A\u03b217\u201340 so that it migrated at the rate of A\u03b21\u201340 (in the absence of brain extract).\nFig.\u00a04A\u03b2 isoforms in extracts from control brain 247 revealed by immunoblotting. A formic acid extract of cerebral cortex from brain C247 was prepared as described in the methods section. A portion of the dried extract was dissolved in PAGE sample buffer and a 15 \u03bcl aliquot containing 30\u00a0\u03bcg of protein (equivalent to 300\u00a0\u03bcg of tissue) was applied to the lanes developed with R162 and R226. Because mAb 6E10 is much less sensitive than R162 or R226, the formic acid extract was fractionated by size-exclusion as described in the methods section. The fraction containing peptides in the mass range 2,000\u201312,000\u00a0Da was concentrated, and an aliquot containing peptides from 12\u00a0mg of tissue was applied to the lane developed with 6E10. The extracts were subjected to PAGE and blotting along with synthetic A\u03b2 standards and molecular mass markers as described in the text. The blots were developed with antibody R162 (to the C-terminus of A\u03b232\u201340), R226 (to the C-terminus of Abeta33\u201342) or mAb 6E10 (to A\u03b24\u201313). Lane 1 2\u00a0fmol A\u03b21\u201340, lane 2 2\u00a0fmol A\u03b217\u201340, lane 6 150\u00a0fmol A\u03b217\u201340, lane 7 10\u00a0fmol A\u03b21\u201340. Molecular mass markers denoted on the right-hand margin are insulin single chains and aprotinin\nPeptides with a defined C-terminus may have distinct N-termini, hence, we operationally define the p4 peptides as those that bind mAb 6E10 and migrate between the 3 and 6\u00a0kDa standards. Monoclonal antibody 6E10 binds to A\u03b2 residues 4\u201313; therefore, p4 may contain peptides whose N-terminal residues are Asp1\u2013Arg5. We define the p3 peptides as those that migrate near the 3\u00a0kDa standard and do not bind 6E10. The levels of A\u03b240 peptides in three normal brain cortex samples were between 0.3 and 2\u00a0pmol\/g of p3 and between 0.3 and 3\u00a0pmol\/g, and the levels of A\u03b242 peptides were between 0.2 and 2\u00a0pmol\/g of p3 and between 0.2 and 1\u00a0pmol\/g. Although these amounts are readily detectable, they are 3\u20134 orders of magnitude below the A\u03b2 levels in an AD brain sample that we analyzed (not shown).\nDetection of A\u03b217\u201340 by mass spectrometry\nWe immunoprecipitated 650\u00a0fmol of A\u03b2 peptides from brain C247 and characterized both the chromatographic behavior and the masses of the peptides by mass spectrometry using the coupled capillary HPLC-MS system. As shown in Fig.\u00a05, the identity of the A\u03b2 peptide A\u03b217\u201340 from the brain was confirmed by comparison to synthetic A\u03b217\u201340, which eluted at the same time and displayed the same m\/z profile. Deconvolution and transformation of the A\u03b217\u201340 2+ ion spectrum to the 1+ monoisotopic mass gave mass values of 2392.2852\u00a0Da (brain sample) and 2392.2813\u00a0Da (synthetic) compared to the theoretical value of 2392.2950\u00a0Da (4.1\u00a0ppm and 5.7\u00a0ppm errors, respectively). We also observed ion profiles consistent with A\u03b21\u201340 4+ and 5+ ions in the mass spectra. The presence of detergent in the sample and the complexity of the chromatogram and spectra complicated the identification of other A\u03b2 species.\nFig.\u00a05Chromatographic elution profile of A\u03b217\u201340. Samples from (a) control brain C247 and (b) synthetic A\u03b217\u201340 were prepared and analyzed by LC-MS as described in \u201cMaterials and methods\u201d. The chromatograms show only the MS ion counts for the M+2H+ ions of A\u03b217\u201340 mass isomers containing zero to four 13C atoms (m\/z\u00a0=\u00a01196.3\u20131198.7). The ion intensities determined at 1.1\u00a0s intervals were summed, background subtracted and smoothed. The y-axis scale is 400 ions per second full scale (100%). The identification of A\u03b217\u201340 in the peaks at 24.4 (a) and 24.5 (b) minutes was demonstrated by the characteristic ion envelope obtained from the centroid accurate mass spectra for the A\u03b217\u201340 M+2H+ ions from each peak (24.2 to 24.7\u00a0min) shown in the insets\nDiscussion\nA\u03b2 immunoreactivity in neurons in normal brain\nPrevious studies either did not find intracellular A\u03b2 immunoreactivity in brains of control subjects [64] or found only punctate A\u03b2 immunoreactivity in a minority (15%) of examined adults [22]. Therefore, it was proposed that the accumulation of intraneuronal A\u03b2 is involved in early AD pathology [17]. However, this study of control cases, from several months to 102\u00a0years of age demonstrates that intraneuronal A\u03b2 immunoreactivity appears in the first year of life, increases in childhood, stabilizes in the second decade of life, and remains high throughout adulthood. Detection of stable intracellular A\u03b2 immunoreactivity in neurons in control cases throughout the entire life may indicate that A\u03b2-immunoreactive material in the cell body reflects normal neuronal metabolism and is not neuronal pathology.\nThe observation that intraneuronal A\u03b2 in control, DS\/AD, and sporadic AD cases was almost exclusively amino-terminally truncated confirmed and extended the findings of the Mori et al. [39] study of people with DS and the Sergeant et al. [51] study showing that amino-truncated A\u03b2 peptides represent more than 60% of all A\u03b2 species in subjects with preclinical AD. A\u03b2 peptides with N-terminal deletions exhibit enhanced peptide aggregation relative to full-length species [43] and retain the neurotoxicity and \u03b2-sheet structure. It was hypothesized that A\u03b217\u201342 peptides may initiate and\/or accelerate plaque formation, perhaps by acting as nucleating centers that seed the subsequent deposition of relatively less amyloidogenic but apparently more abundant full-length A\u03b2 [18, 43, 48]. Gouras et al. [17] considered intracellular A\u03b242 accumulation to be an early event leading to neuronal dysfunction. However, the appearance of intraneuronal A\u03b2 immunoreactivity in the first year of life, and the stable and strong immunoreactivity throughout adulthood, in the absence of morphological signs of cell injury or degeneration, suggests that A\u03b2 detected in neurons with applied antibodies does not adversely affect cell structure. This lack of fibrillization and toxicity may indicate that these A\u03b2 species remain in inert form. Possibly this inert state is maintained by binding with blockers of fibrillization and toxicity. The transport of A\u03b2 within cytoplasmic vesicles or vacuoles [57] might be another factor preventing the expression of their cytotoxic activity. The detected intraneuronal A\u03b2 appears to be the physiological metabolite with unknown function. Higher levels of secreted APP and nonamyloidogenic secreted APP and lower levels of A\u03b2 40 in children with severely autistic behavior and aggression compared with controls [54] may indicate that modifications of APP processing and potentially A\u03b2 trafficking are clinically significant in absence of neurodegeneration or neuronal loss.\nThe very strong A\u03b2 immunoreactivity in the LGB, nucleus olivaris inferior, and dentate nucleus during the entire course of human adulthood and the absence of or minimal amyloid load in very severe AD oppose the hypothesis that strong intraneuronal A\u03b2 immunoreactivity is a predictor of fibrillar plaque formation. Convergent findings in PS1 tg mice have been reported by Chui et al. [9].\nThis study suggests also that the intensity of intraneuronal A\u03b2 immunoreactivity is not a predictor of neurofibrillary degeneration. The strongest A\u03b2 immunoreactivity was observed in neurons in the LGB, nucleus olivaris inferior, and dentate nucleus, which remain free of neurofibrillary degeneration in very severe AD. The moderate A\u03b2 immunoreactivity in the second layer of the entorhinal cortex and pyramidal neurons in the CA1, observed in this study, and the early onset of NFTs observed in these neuronal populations [5\u20137] also question the link between A\u03b2 immunoreactivity and susceptibility to neurofibrillary degeneration.\nThe lack of pathological changes in brain structures with the strongest intraneuronal A\u03b2 immunoreactivity during the entire human lifespan may indicate that amino-terminally truncated A\u03b2 is a product of cell metabolism that does not (1) limit cell survival, (2) predispose to fibrillar A\u03b2 deposition in plaques, nor (3) cause neurofibrillary degeneration.\nReduction of intraneuronal A\u03b2 immunoreactivity in DS\/AD and sporadic AD\nIn this study, the strongest intraneuronal A\u03b2 immunoreactivity was found in control cases, compared to those with AD and in DS\/AD. Significant weaker A\u03b2 immunoreactivity in neurons in people with DS\/AD and sporadic AD appear to be the result of AD pathology, including neurofibrillary degeneration and neuronal loss.\nIn the memory systems of persons with DS\/AD or sporadic AD, neurofibrillary degeneration is the major cause of neuronal loss and, therefore, the major factor contributing to the reduction of intraneuronal A\u03b2. Over the course of 22\u00a0years, an AD patient loses 87% of the neurons in the CA1 and 63% in the CA4 sector, and 77% in the subiculum [5]. People with DS\/AD older than 50\u00a0years of age are affected by severe loss of neurons including 98% loss in the second layer of the entorhinal cortex, 74% in the CA1 sector, 57% in the subiculum proper, and 71% in the amygdala [32, 60]. Large portions of surviving neurons in the brains of people with AD and DS\/AD are affected with neurofibrillary changes, yet they show little or no A\u03b2 immunoreactivity. These changes explain, in part, the loss of A\u03b2 immunoreactivity in neurons in the plaque perimeter, as noted in persons with DS\/AD [39]. The lower intraneuronal A\u03b2 immunoreactivity observed in people with sporadic AD or DS\/AD may also reflect a shift in APP processing. This shift would be from amino-terminally truncated A\u03b2 accumulation in cell cytoplasm in normal brain to APP processing with enhanced generation and secretion of A\u03b21\u201340\/42 in people with AD.\nAccumulation of A\u03b21\u201340\/42 in ghost tangles\nIntracellular and extracellular NFTs have been shown to be A\u03b2 immunoreactive [1, 21, 27, 34, 42, 49, 56]. However, studies of purified PHF revealed the absence of beta-pleated sheet conformation or any other characteristic of an amyloid [50]. It was proposed that the staining of intraneuronal PHF with antibodies to amyloid is due to the proximity of A\u03b2 molecules to the PHF in the cytoplasm of neurons [47]. Deposition of A\u03b2 within extracellular NFTs has been considered a secondary event [67].\nStrong immunoreactivity of extracellular NFTs with antibodies 6E10 (residues 4\u201313), 6F\/3D (8\u201317), 4G8 (17\u201324), 44\u2013344 (32\u201342) and R165 (35\u201342) could be an indicator of the presence of full length of A\u03b2 in extracellular space. The weak reaction of ghost tangles with pAb 44\u2013348 (32\u201340) may suggest a lower concentration of this form of A\u03b2 in the extracellular space. An increase in the percentage of A\u03b2-positive ghost tangles in people with progressing AD appears to reflect the increase in the percentage of neurons dying due to neurofibrillary degeneration and an increasing concentration of full length A\u03b2 in the extracellular space. Spatial and temporal separation of plaques and A\u03b2-positive ghost tangles indicates that ghost tangles may bind A\u03b2, but do not initiate fibrillar plaque formation.\nInterindividual differences in intraneuronal A\u03b2 immunoreactivity\nIntraneuronal A\u03b2 immunoreactivity revealed some interindividual differences. Gouras et al. [17] observed increased apo-E immunoreactivity in A\u03b242-immunoreactive neurons and suggested that apo E might be a powerful modifier of intraneuronal A\u03b2 accumulation. Neuron-generated apo-E may bind to neuronal A\u03b2 and affect both its secretory pathway and its storage in the cell cytoplasm. The lack of apo-E in the offspring of \u03b2APP tg mice crossed with apo-E knockouts reduces significantly the amyloid load [3]. Chronic inflammation is another factor that may influence amyloid formation [53].\nIn the human brain, there appear to be several pools of A\u03b2 in equilibrium [35]. In this study, we demonstrate by the immunocytochemical method abundant intraneuronal amino-terminally truncated A\u03b217\u201340\/42 with minor presence of the intraneuronal A\u03b21\u201340\/42. The presence of the A\u03b217\u201340 peptide in control cortex samples was confirmed by mass spectrometry. However, in extracts from cerebral cortex we identified by Western blotting the A\u03b240 peptides that were amino-terminally truncated (A\u03b217\u201340) and full length A\u03b21\u201340 in similar quantities. We also detected both the A\u03b217\u201342 peptide and the full length A\u03b21\u201342 peptide, although the levels of the latter were 2 or 3 times lower than those of the amino-terminally truncated one. The differences between the A\u03b2 peptides detected in the brain by the immunocytochemical and biochemical methods suggest that the A\u03b2 peptides accumulated intraneuronally are mainly those amino-terminally truncated, while the full length peptides are mainly dispersed in extracellular space, and hence are not detected by the immunocytochemical method as intracellular deposits. Deposition of full length A\u03b2 in ghost tangles and the increase of A\u03b2-positive ghost tangles could be considered an indicator of the presence of A\u03b21\u201342 in extracellular space and an increase of this extracellular form of A\u03b2 in advanced stages of AD.\nIt appears that the immunocytochemical methods applied in this and other studies [11\u201314, 17, 22, 39] do not monitor the neuronal secretory pathway of APP processing leading to normal secretion of A\u03b21\u201340 and A\u03b21\u201342. Stern et al. [55] have shown that full length APP is extremely sensitive to fixation methods and its immunogenicity could be easily destroyed. The possibly enhanced secretion of these peptides in AD that may result in fibrillar A\u03b2 deposition in plaques also is not detected. The lack of detection or poor detectability of the product of \u03b2- and \u03b3-secretases in autopsy material might be the result of tissue preservation method, the minute amount of A\u03b21\u201340\/42 that might be secreted without long-term storage, the prevalence of amino-terminally truncated A\u03b2 masking the detection of A\u03b21\u201340\/42, or the masking of the amino-terminal epitopes by interactions with other proteins. One may speculate that deposition of amino-terminally truncated A\u03b2 might be up- or down-regulated by physiological and pathological factors independently of any changes in the secretory pathway.\nThis study suggests that neuron-type- and brain-structure-specific patterns of intraneuronal A\u03b2 immunoreactivity, established in teenagers and maintained at a constant level during adulthood and aging, reflect normal cell metabolism rather than pathological changes. Reduction and loss of immunoreactivity in neurons with neurofibrillary changes appears to be a response to the progression of pathological changes in the neuron. The absence of a link between the age at onset and the progression of accumulation of intraneuronal A\u03b2-positive material in control subjects with the fibrillar plaque formation or neurofibrillary degeneration in people with AD indicates that the form of intraneuronal A\u03b2, detected by applied methods, does not predict plaque formation or neurofibrillary degeneration.","keyphrases":["down syndrome","alzheimer\u2019s disease","plaques","tangles","intraneuronal amyloid-\u03b2"],"prmu":["P","P","P","P","M"]}
{"id":"Pediatr_Nephrol-2-2-1764601","title":"Focal segmental glomerulosclerosis \u2013 epidemiology aspects in children and adults\n","text":"The histologic features of idiopathic forms of focal segmental glomerulosclerosis (FSGS) were first described by Theodor Fahr in the Handbuch der speziellen pathologischen Anatomie und Histologie in 1925 [1]. Over the subsequent eight decades much has been written about the histologic features and clinical characteristics of patients with FSGS, but it is only in recent years that attention has been directed to the incidence and prevalence of the disorder in various populations. This article will provide an overview of the epidemiology of FSGS by reviewing published surveys of renal biopsies, experiences from clinical registries of children with renal insufficiency, and data from the U.S. Renal Data Systems (USRDS).\nDiagnostic considerations\nPrimary FSGS can present at any age, and it always causes proteinuria. It is most commonly diagnosed in patients with overt nephrotic-range proteinuria, but any proteinuria that is fixed and persistent over several months (i.e., not orthostatic or transient proteinuria) may signal underlying FSGS [2, 3]. Since the diagnosis of FSGS depends on obtaining renal histologic material, the observed incidence and demographics of FSGS in both children and adults depend on the population examined and on the indications used for renal biopsy. The typical findings on renal biopsy in idiopathic FSGS are described elsewhere in this series of articles.\nIncidence and prevalence of FSGS in children\nAn estimation of the incidence and prevalence of FSGS in children is hampered by the fact that most children with nephrotic syndrome (NS), unlike the majority of adults, are not routinely subjected to renal biopsy. Therefore, prevalence and incidence figures are extrapolated from clinical reports, registries, and renal biopsy materials. Clinical reports often make the presumptive diagnosis of minimal change nephrotic syndrome (MCNS) based on steroid responsiveness [4, 5]. This usually leads to an under-diagnosis of FSGS because up to 15\u201320% of FSGS patients initially respond to steroids [6]. The largest pediatric registry is that of the North American Pediatric Renal Transplant Cooperative Study (NAPRTCS), which collects data not only on transplant patients but also on dialysis and chronic kidney disease (CKD) patients [6]. Based on results from nearly 6000 children with CKD, in whom the estimated glomerular filtration rate (GFR) was less than 75\u00a0ml\/min per 1.73\u00a0m2, it has been shown that FSGS carries the highest likelihood of progressing to end-stage renal disease (ESRD), exceeding the risk of hypoplasia, obstructive uropathy, and reflux nephropathy. According to NAPRTCS data, nearly 60% of such children with a diagnosis of FSGS progress to dialysis or transplantation within 24\u00a0months of entry into the registry. However, because reporting is entirely voluntary and involves mainly pediatric centers, there is significant underreporting, and selection bias may exist.\nClinical surveys from North America and the United Kingdom have reported the incidence of NS to be between two and four new cases per 100,000 children per year, with biopsy-confirmed FSGS comprising 15\u201320% of the total [5, 7\u201310]. However, as noted above, many children with NS do not undergo a diagnostic renal biopsy unless they are shown to be steroid-resistant. A study from Canada reported an incidence of FSGS of 0.37 to 0.94 new cases per 100,000 children per year [5], and similar results were obtained in the U.S. Midwest [9]. In the NAPRTCS database, patients with FSGS account for 14.2% of dialysis patients and for 11.5% of transplant patients; the differences may reflect a reluctance by some centers to transplant FSGS patients for fear of disease recurrence. Members of the Southwest Pediatric Nephrology Study Group (SPNSG) reported that FSGS was diagnosed in 75 of 1053 (7.1%) patients with NS who underwent a renal biopsy between 1972 and 1981 [11].\nWhile there is a male preponderance among children with MCNS, most reports have described no clear gender difference in patients with FSGS [4, 11, 12], although in the SPNSG series, all 18 children with FSGS below the age of 3\u00a0years were boys [11].\nEthnicity and age appear to play important roles in the prevalence of FSGS. The NAPRTCS and SPNSG reports include approximately equal numbers of Caucasian and African American children with FSGS despite the large difference in the racial distribution of the underlying populations [6, 11]. According to the NAPRTCS report, FSGS is the most common cause of ESRD in African-American children in that cohort, accounting for 23% of all pediatric patients with ESRD. In Caucasian children, FSGS is only the third most common primary disease and is responsible for approximately 10% of all pediatric patients with ESRD [5]. In contrast, African-American children with NS appear to be more likely to have FSGS than MCNS [4, 10, 13]. Whether the frequency of FSGS in Hispanic children differs from that in Caucasians is unclear [14, 15].\nThe prevalence of FSGS among all children with NS who undergo a renal biopsy increases with age. However, it is not clear if the true population-based incidence of FSGS is age-dependent. Two thirds of children with NS present before the age of 6\u00a0years, but only a minority of the younger children exhibit FSGS, whereas FSGS is the most common histology in the older group. The frequency of FSGS in NS patients presenting before 6\u00a0years of age is less than 10%, but increases to 20\u201350% or more in patients presenting in adolescence [4, 9\u201311, 16]. The combined effect of age and ethnicity is responsible for the reported high frequency of FSGS in kidney biopsies from adolescent African-American patients with NS [4, 10, 13]; both age and ethnicity are probably independent risk factors for FSGS histology.\nAn intriguing recent development has been the apparent increase in the incidence of FSGS in children [4, 5, 10, 17]. Differences in the ages and ethnicity of the subjects do not fully explain these findings. Filler et al., for instance, studied mostly Caucasian children in Canada and found that the calculated incidence of FSGS increased from 0.37 to 0.94 per 100,000 children per year from the first to the second half of a 17-year period between 1985 and 2002 [5]. The increase appears to be present mostly in older children [4]. Similar increases in incidence have not been seen in other types of NS in children [4, 5]. The cause of the increased incidence is not known but is unlikely to be due to genetic factors. The role of environmental pollution has been proposed but remains hypothetical [18]. Morbid obesity-associated FSGS has increased in renal biopsy material [19], but because it remains uncommon, the current adult and pediatric obesity epidemic probably does not explain the increase in FSGS.\nIncidence and prevalence of FSGS in adults\nSeveral publications suggest that the prevalence of idiopathic FSGS in adults may also be increasing [20, 21]. Observations from two metropolitan centers suggest that FSGS was found in 2.5\u20134% of native renal biopsies in the 1970s, but in 12.2\u201318.7% in this decade, making FSGS the most common diagnosis based on native kidney biopsies. FSGS becomes less common with advancing age, but it is still a significant problem in the elderly. In a series of 1368 renal biopsies from patients over 60\u00a0years of age, FSGS was present in 5.4% of those patients with nephrotic syndrome [20]. These trends in increased numbers of FSGS cases are observed in both African Americans and Caucasians and occurred despite stable or falling rates of other glomerular diseases, such as minimal change disease and membranous nephropathy [21].\nTwo large series in adults support the fact that primary FSGS is a risk factor for developing ESRD. In one report of outcomes from immunosuppressive therapy in 59 adult patients with FSGS, 30% of patients developed some level of renal insufficiency within 5\u00a0years [22, 23]. Wehrmann and colleagues reported results from 250 patients with idiopathic FSGS (average age: 32\u00a0years) in which they found an overall 10-year renal survival of 67% [23]. The risk of developing ESRD in idiopathic FSGS can be predicted at the time of diagnosis. Heavy proteinuria, either at the time of biopsy but particularly following treatment, portends a poor long-term outcome. Other features that increase the risk for progressive renal disease include elevated serum creatinine at the time of diagnosis, hypertension, age at time of diagnosis, and male gender [22, 24, 25]. The most compelling predictor for progression of FSGS, however, appears to be the response of proteinuria after treatment is initiated. One series suggests that if a patient with primary FSGS fails to respond to initial therapy (most often steroid-based), the likelihood of renal death is as great as if the patient received no treatment at all [25]. However, a full assessment of relative risk will require larger cohorts of patients with FSGS who received consistent interventions.\nThe risk of FSGS-related ESRD in adults is also supported by data published by the USRDS [26]. Documented FSGS accounts for more than 7000 patients currently receiving ESRD therapy in the United States, and it is likely that primary FSGS accounts for at least some of the 25,000 patients with unspecified forms of glomerulonephritis, the 100,000 patients with ESRD attributed to hypertension, and the 20,000 patients with an unknown cause of ESRD [26]. The USRDS also demonstrates a dramatic increase in the incidence of ESRD due to FSGS, particularly over the last decade in the African-American population. In the Caucasian U.S. population, new-onset ESRD due to FSGS has slowly increased to a rate of approximately five cases\/million population in 2003. In contrast, the incidence has increased nearly five-fold in the African-American population since the early 1980s to 30\u201340 cases per million population [26].\nFamilial FSGS\nOccasional cases of familial FSGS have long been recognized. In 1973 Habib suggested that 12% of all pediatric cases of FSGS had an affected sibling [2]. Although most cases of FSGS are sporadic with unknown pathophysiology, it is now clear that mutations in structural podocyte-associated proteins account for a portion of familial cases and for at least some of the sporadic cases. A mutation in the NPHS2 gene, which encodes the protein podocin, has been linked to autosomal-recessive steroid-resistant nephrotic syndrome in children [27]. However, there are large differences between different ethnic populations. For example, approximately one third of Israeli-Arab children but none of Israeli-Jewish or Japanese children with sporadic FSGS were reported to have NPHS2 mutations [27, 28]. The frequency of NPHS2 mutations in North American children is not known. Mutations in the a-actinin 4 gene (ACTN4) have been associated with autosomal-dominant FSGS in adults, a disease characterized by a variable risk of progression to ESRD. Most recently, autosomal-dominant FSGS has been linked to the gene that encodes the transient receptor potential cation channel, subfamily C, member 6 (TRPC6) [29]. Although more data are needed, it is likely that mutations in the known FSGS causative genes account for only a minority of cases of FSGS in children and adults. A more complete review of the genetic aspects of FSGS is provided elsewhere in this series of articles.\nConclusion\nIt is clear that there is still much to learn about the epidemiology of idiopathic FSGS. However, there are encouraging signs that the body of knowledge will be enhanced in coming years by the various registries and population-based studies currently underway.\nQuestions\n(Answers appear following the reference list) \nWhich of the following statements is correct? \nPatients with idiopathic FSGS often present with microscopic hematuria without proteinuria.All patients with FSGS have nephrotic range proteinuria.Patients with FSGS have proteinuria of varying severity.None of the above.Which of the following carries the highest risk for a patient with FSGS to progress to ESRD? \nMale gender.Persistent proteinuria after therapy.Hyperlipidemia.Caucasian race.Age.Which of the following is incorrect? \nAccurate data concerning the outcome of patients with FSGS is lacking since the disease was first described only 30\u00a0years ago.The incidence of the disease appears to be decreasing in the last 20\u00a0years.Most cases of FSGS can now be linked to specific gene mutations that disturb podocyte function.All of the above.None of the above.Which of the following is correct? \nFSGS is a more frequent cause of ESRD in Caucasian children than in African-American children.FSGS is more commonly associated with nephrotic syndrome in Hispanic children than in white children.Children with FSGS account for less than 10% of African-American children who progress to ESRD.All of the above.None of the above.","keyphrases":["focal segmental glomerulosclerosis","epidemiology","nephrotic syndrome"],"prmu":["P","P","P"]}
{"id":"Purinergic_Signal-3-4-2072912","title":"[3H]Adenine is a suitable radioligand for the labeling of G protein-coupled adenine receptors but shows high affinity to bacterial contaminations in buffer solutions\n","text":"[3H]Adenine has previously been used to label the newly discovered G protein-coupled murine adenine receptors. Recent reports have questioned the suitability of [3H]adenine for adenine receptor binding studies because of curious results, e.g. high specific binding even in the absence of mammalian protein. In this study, we showed that specific [3H]adenine binding to various mammalian membrane preparations increased linearly with protein concentration. Furthermore, we found that Tris-buffer solutions typically used for radioligand binding studies (50 mM, pH 7.4) that have not been freshly prepared but stored at 4\u00b0C for some time may contain bacterial contaminations that exhibit high affinity binding for [3H]adenine. Specific binding is abolished by heating the contaminated buffer or filtering it through 0.2-\u03bcm filters. Three different, aerobic, gram-negative bacteria were isolated from a contaminated buffer solution and identified as Achromobacter xylosoxidans, A. denitrificans, and Acinetobacter lwoffii. A. xylosoxidans, a common bacterium that can cause nosocomial infections, showed a particularly high affinity for [3H]adenine in the low nanomolar range. Structure\u2013activity relationships revealed that hypoxanthine also bound with high affinity to A. xylosoxidans, whereas other nucleobases (uracil, xanthine) and nucleosides (adenosine, uridine) did not. The nature of the labeled site in bacteria is not known, but preliminary results indicate that it may be a high-affinity purine transporter. We conclude that [3H]adenine is a well-suitable radioligand for adenine receptor binding studies but that bacterial contamination of the employed buffer solutions must be avoided.\nIntroduction\nPurinergic receptors play an important role in transmembrane signaling [1]. Currently, two distinct families are officially recognized by the International Union of Pharmacology (IUPHAR), i.e. receptors for the purine nucleoside adenosine (P1 or adenosine receptors) and receptors for purine and\/or pyrimidine nucleotides (P2 receptors) [2\u20134]. Whereas the P1 receptor family comprises four subtypes, A1, A2A, A2B, and A3, all of which are G protein-coupled receptors (GPCRs), the P2 family is further subdivided into two subfamilies, P2Y (GPCRs) and P2X (ligand-gated ion channels) [2\u20134]. In addition to the nucleoside (adenosine) and nucleotide receptors, a receptor for the nucleobase adenine has recently been discovered by a reverse pharmacological approach identifying adenine as the natural ligand for a rat orphan GPCR [5]. A mouse orthologue (mMrgA10) of the rat adenine receptor was subsequently identified by sequence comparison [5]. Very recently, a new adenine receptor has been cloned from mice showing 82% identity in its amino acid sequence to mMrgA10 and 76% to the rat adenine receptor, indicating that the new receptor is a distinct adenine receptor subtype (Genbank nucleotide sequence accession numbers: new mouse adenine receptor, DQ386867; mMrgA10, XM_195647; rat adenine receptor, AJ311952) [6, submitted]. Both adenine receptors that have been pharmacologically characterized are coupled to the inhibition of adenylate cyclase by Gi protein [5, 6]. So far, no human receptor for adenine has been identified, although initial clues for the possible existence of human adenine receptors have been found [7]. Adenine receptors are structurally unrelated to P1 and P2 receptors and therefore constitute a new family of purinergic receptors for which we proposed the designation P0 (\u201cP zero\u201d) receptors [8] based on the structural relationships of the physiological agonists, adenine (P0) representing a partial structure of adenosine (P1) and adenosine again being a partial structure of adenine nucleotides (P2), such as adenosine triphosphate (ATP) or adenosine diphosphate (ADP).\nRadioligand binding studies are widely used to characterize GPCRs on the protein level [9]. Adenine, the natural ligand of adenine receptors, is commercially available in tritium-labeled form ([3H]adenine) and has been used to characterize recombinant rat adenine receptors expressed in Chinese hamster ovary (CHO) cells [5] as well as natively expressed rat adenine receptors in rat brain [7, 10] using membrane preparations. The radioligand has been shown to be stable under the incubation conditions [10]. Furthermore, mouse adenine receptors natively expressed in NG108-15 (neuroblastoma \u00d7 glioma hybrid) cell membranes were labeled by [3H]adenine [7]. Recently, we successfully applied [3H]adenine binding to detect the mouse adenine receptor protein recombinantly expressed in Sf21 insect cell membranes, which constitute a null background because they do not endogenously express any high affinity binding site for adenine [6].\nTwo recent poster presentations reported on problems with [3H]adenine binding to adenine receptors. In one study using whole rat brain membrane preparations, a high-affinity binding site was detected (apparent Ki 57.5\u00a0nM) [11]. However, a very high Bmax value was found (281\u00a0pmol\/mg protein), and the binding was almost completely blocked by 10\u00a0\u03bcM of hypoxanthine and abolished in the absence of Mg2+, indicating that the detected binding site was not identical with the G protein-coupled rat adenine receptor [11]. In another study, high affinity binding of [3H]adenine was detected even in the absence of added protein, and the authors suggested that [3H]adenine bound in a highly specific manner to the glass fiber filters used in the filtration assays [12]. IC50 values were determined for five adenine derivatives and three compounds [adenine (18\u00a0nM [12]; rat: 29.9\u00a0nM [7], 18\u00a0nM [5]), 7-ethyladenine (30\u00a0\u03bcM [12]; rat: 47.3\u00a0\u03bcM [7]), 8-bromoadenine (14\u00a0\u03bcM [12]; rat: 17.3\u00a0\u03bcM [7]] showed similar IC50 values as those previously determined for the rat adenine receptor, but two compounds [5\u2032-deoxyadenosine (725\u00a0\u03bcM [12]; rat: 0.823\u00a0\u03bcM [10]), 2-fluoroadenosine (19\u00a0\u03bcM [12]; rat: 0.62\u00a0\u03bcM [7]] had much lower affinities for the unknown binding site labeled in the absence of added protein [12] than for the rat adenine receptor as previously determined [7, 10], indicating that the unknown binding sites labeled in the absence of added rat tissue were very different. Based on their results, the IJzerman group had suggested to avoid the use of [3H]adenine as a radiolabeled probe for the adenine receptor due to its putative specific, high-affinity binding to glass fiber filters [12].\nThat report prompted us to carefully reanalyze radioligand binding data obtained with [3H]adenine in our laboratory with the goal to find out the reason for the problems encountered in other laboratories. As a matter of fact, we had occasionally observed unusually high counts in a few experiments, which could, however, be avoided by repeating the experiments under carefully controlled experimental conditions, including the use of freshly prepared buffer solutions. We have now performed a systematic study clearly showing that [3H]adenine is a suitable radioligand for the labeling of adenine receptors in various cells and tissues if bacterial contaminations are excluded. Furthermore, we identified three common gram-negative aerobic bacteria that grow in cold Tris buffer and express high-affinity binding sites for [3H]adenine.\nMaterials and methods\nChemicals\n[8-3H]Adenine (27\u00a0Ci\/mmol) was obtained from Amersham Biosciences (Munich, Germany). Tris was obtained from Acros Organics (Leverkusen, Germany) and dimethyl sulfoxide (DMSO) was from Fluka (Switzerland). All other chemicals and reagents were obtained from Sigma unless otherwise noted.\nCell culture\nHuman embryonic kidney (HEK) 293 and CHO-K1 cells were grown as monolayers at 37\u00b0C (5% CO2) in Dulbecco\u2019s modified Eagle medium (DMEM) containing 10% fetal calf serum, 100 U\/ml penicillin, 100\u00a0\u03bcg\/ml streptomycin, and 2\u00a0mM L-glutamine.\nMembrane preparations\nFrozen rat brains were obtained from Pel Freez (Rogers, AR, USA) and thawed at 4\u00b0C. Cortex and striata were dissected, and membrane fractions were prepared as previously described [7]. Membrane preparations from CHO-K1 cells, and HEK293 cells were prepared as described [13, 14]. Membrane preparations from bacteria were obtained after growing them on agar plates and subsequent amplification of single strains in Lennox broth (LB) medium over night. Membranes were then prepared in analogy to the procedures used for mammalian-cell membranes [7, 13]. Protein concentrations were determined according to the method of Lowry [15].\n[3H]Adenine binding assays\nAdenine binding assays were carried out as previously described [7]; however, in the absence of Mg2+ and ethyleneglycoltetraacetic acid (EGTA), unless otherwise noted, as Mg2+ and EGTA were found to have no effect on determined Ki values (data not shown). In brief, membrane preparations (50\u00a0\u03bcg of protein, unless otherwise indicated) were incubated with 10\u00a0nM [3H]adenine in 50\u00a0mM Tris-HCl, pH 7.4 in a total volume of 200\u00a0\u03bcl. Inhibition curves were determined using six to nine different concentrations of adenine, spanning three orders of magnitude. Three separate experiments were performed, each in triplicate, unless otherwise noted. Nonspecific binding was determined in the presence of 100\u00a0\u03bcM unlabeled adenine. Incubations were carried out for 1\u00a0h at room temperature and terminated by rapid filtration through GF\/B glass fiber filters (Whatman, Dassel, Germany). Filters were washed three times, 2\u00a0ml each, with freshly prepared ice-cold 50\u00a0mM Tris-HCl buffer, pH 7.4 and immediately transferred to mini vials. Scintillation cocktail (Ultima Gold, Canberra Packard, 2.5\u00a0ml) was added and after an incubation of 9\u00a0h filter-bound radioactivity was measured by liquid scintillation counting at an efficiency of 54%. In some experiments, the addition of mammalian protein was omitted and replaced by 100\u00a0\u03bcl of different buffer samples. For competition experiments with bacteria, either 100\u00a0\u03bcl of 1:100 or 1:1000 dilution of an overnight culture of the bacteria in Tris buffer, 50\u00a0mM, pH 7.4, or a membrane preparation of bacteria (containing 0.4\u201310\u00a0\u03bcg of protein) was used.\nAfter isolation and classification of the bacteria from contaminated buffer solutions, experiments were performed with intact bacteria (approximately 6\u2009\u00d7\u2009104 bacteria\/sample, which roughly equals 0.5\u00a0\u03bcg of total protein\/sample), unless otherwise indicated, using the standard procedure (see above). The cell number was estimated from the optical density (OD) of the overnight culture suspension, and the assumption that  [16].\nData analysis\nData were analyzed using Prism 4.03 (Graph Pad, San Diego, CA, USA). IC50 values were determined by fitting data to sigmoidal concentration-inhibition curves. Results are presented as means \u00b1 standard error of the mean (SEM) from the number of observations.\nIsolation of microorganisms from contaminated buffer solutions and classification\nMicroorganisms were isolated from Tris-HCl buffer, pH 7.4, by plating 300\u00a0\u03bcl of the buffer on LB agar plates. Single colonies were isolated and incubated overnight in LB medium at 37\u00b0C with constant shaking at 230\u00a0rpm. Further separation was achieved by plating bacteria from overnight cultures on plate count (Merck, Darmstadt) and blood agar (Oxoid, Wesel). Morphology of colonies was visually examined. Conventional physiological and biochemical characterization assays, including gram staining, catalase and oxidase activity, motility, and oxidation\/fermentation (O\/F) test were carried out and analyzed according to  Bergey\u2019s Manual of Systematic Bacteriology using reagents from Merck (Darmstadt) [17]. In addition, the following kits and appliances were used: BBL OXI\/FERM Tube II (Schwarz Pharma GmbH, Germany), API 20 NE strips and apiweb software (Biomerieux, N\u00fcrtingen, Germany), and VITEK 2 fully automated system (Biomerieux, N\u00fcrtingen, Germany).\nResults\nProtein dependence of [3H]adenine binding\nAs a first step, we reevaluated (unpublished) data obtained in initial studies that had been performed to investigate the suitability of [3H]adenine as a radioligand for labeling adenine receptors. Figure\u00a01 shows [3H]adenine binding to different membrane preparations from (a) rat brain striatum, (b) rat brain cortex, and (c) HEK293 cells and CHO-K1 cells. For each membrane preparation, different amounts of protein were investigated (25, 50, 100, and 200\u00a0\u03bcg for striatum; 0, 25, 50, 100, and 200\u00a0\u03bcg for cortex; 50 and 100\u00a0\u03bcg for HEK and CHO cells). In all cases, we found a large, approximately linear, increase in specific binding with increasing protein concentration, whereas the increase in nonspecific binding was small. No specific binding was detected in the absence of protein (see Fig.\u00a01b).\nFig.\u00a01Protein dependence of [3H]adenine binding to adenine receptors in rat brain striatal membranes (a), in rat brain cortical membranes (b), and in human embryonic kidney (HEK293) cell membranes and in Chinese hamster ovary cell (CHO) membranes (c). Different amounts of protein were incubated for 60\u00a0min with 10\u00a0nM of [3H]adenine in Tris-HCl buffer, pH 7.4 (n\u2009=\u20093). Nonspecific binding was determined in the presence of 100\u00a0\u03bcM adenine\nMicrobial contaminations in buffer solutions\nAs a next step, we investigated whether microbial contaminations present in incubation buffers that were not freshly prepared might be responsible for high counts occasionally observed in [3H]adenine binding studies in our laboratory. We performed a systematic analysis of buffer solutions (50\u00a0mM Tris-HCl, pH 7.4) stored at different conditions (different periods of time and temperatures), in different containers (plastic, glass, different sizes) used in our laboratory. Samples of buffer solutions were taken, and radioligand binding studies were performed with [3H]adenine (10\u00a0nM) using the same procedure as for the labeling of adenine receptors, except that no tissue or cell membrane preparation was added. Most investigated buffer solutions did not show any specific [3H]adenine binding. However, one sample of Tris buffer that had been taken from a 5-l plastic container with a small orifice, stored at 4\u00b0C, exhibited high affinity binding of [3H]adenine (data not shown). In contrast, radioligands used for the labeling of adenosine receptors ([3H]2-Chloro-N6-[3H]cyclopentyladenosine (CCPA) (A1) [18], [3H][3H]3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine (MSX)-2 (A2A) [19], [3H]8-Ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1 ]purin-5-one (PSB)-11 (A3) [20]), or P2Y12 receptors ([3H]2-propylthioadenosine-5\u2032-adenylic acid (1,1-dichloro-1-phosphonomethyl-1-phosphonyl) anhydride (PSB)-0413) [21] did not show any specific binding to that buffer solution.\nFigure\u00a02 shows [3H]adenine binding determined in differently treated Tris-buffer solutions (in the absence of added protein). Freshly prepared buffer did not show any specific [3H]adenine binding. After storing the buffer for 1 day at 4\u00b0C, a small degree of [3H]adenine binding could be observed, and after 2 weeks, [3H]adenine binding was significant (562\u2009\u00b1\u200919\u00a0cpm specific binding). There was a large, exponential increase in specific [3H]adenine binding with time, and after 6\u00a0weeks at 4\u00b0C, approximately 7,000\u00a0cpm (specific binding) were measured. After the 6-week-old buffer was filtered through 0.2\u00a0\u03bcm filters, [3H]adenine binding was completely abolished. Heating of the buffer for 1\u00a0min at 80\u00b0C dramatically reduced [3H]adenine binding, whereas heating for 3\u00a0min at 80\u00b0C or heating at 121\u00b0C for 20\u00a0min in an autoclave led to complete loss of specific [3H]adenine binding.\nFig.\u00a02[3H]Adenine binding in the absence of added protein (cells or cell membranes). Incubation buffer (Tris-HCl, pH 7.4) was kept in a 5-l plastic container at 4\u00b0C for up to 6\u00a0weeks. Differently treated buffer samples (100\u00a0\u03bcl) were tested for [3H]adenine binding. The results shown represent means of three independent experiments \u00b1 standard error of the mean\nIsolation of microbial buffer contaminants\nAs our results indicated that adenine binding was due to microbial contaminations growing in the incubation buffer, we decided to isolate the contaminants in order to characterize and eventually identify them. The microorganisms were isolated by plating contaminated incubation buffer on agar plates. This led to identification of three bacterial strains differing in the morphology of the formed colonies. The three strains were then separately amplified in medium over night, and membrane preparations were obtained to perform homologous competition binding assays using [3H]adenine (Fig.\u00a03a).\nFig.\u00a03Competition curves for adenine versus 10\u00a0nM [3H]adenine obtained with membrane preparations from rat brain cortex and isolated microorganisms (contaminants 1\u20133) (a) and intact bacteria (b). IC50 values (a): ; ; ; contaminant 3\u2009=\u20092,350\u00a0nM, (n\u2009=\u20091). IC50 values (b): ; ; \nMembrane preparations of contaminant 1 showed a very high affinity for adenine (, n\u2009=\u20093), whereas membrane preparations of the other two contaminants appeared to have considerably lower affinities (contaminant 2: , n\u2009=\u20092; contaminant 3: IC50\u2009=\u20092351\u00a0nM, n\u2009=\u20091). For comparison, the binding curve for adenine at rat brain cortical adenine receptors () is shown (Fig.\u00a03a). Using amounts of membrane preparations of contaminant 1 that contained more than 1 \u03bcg protein\/assay tube led to depletion of the radioligand (more than 50% of the added radioligand was bound to the protein) (data not shown).\nIdentification of microbial contaminants\nIn order to identify the contaminants, standard procedures and classification kits were used (see Table\u00a01). Standard tests, including catalase, oxidase, fermentation, motility test, and gram staining indicated that contaminant 1 is Pseudomonas  spp. or a strain of Achromobacter spp., contaminant 2 is Achromobacter  spp., and contaminant 3 Acinetobacter spp. [17]. All three bacteria are strictly aerobic gram-negative rods. For further classification, a standardized system (Api 20 NE) for classification of bacteria was used, which combines conventional and assimilation tests for identification of gram-negative rods not belonging to the Enterobacteriaceae. It was found that contaminant 1 was positive for oxidase, nitrate reduction, glucose degradation, gluconate, caprate, adipate, maltose, citrate, and phenylacetate, whereas contaminant 2 was negative for glucose, and caprate and contaminant 3 only showed positive results for caprate, maltose, and phenylacetate. Those results confirmed contaminant 3, already presumed to belong to the species Acinetobacter, as A. lwoffii, with a probability of 98.1%, and led to the identification of contaminant 1 as A. xylosoxidans (94.5% probability) and of contaminant 2 as A. denitrificans (82.2% probability) (see Table\u00a01). Identification of the two Achromobacter strains with the VITEK fully automated system for rapid bacterial identification and antibiotic susceptibility confirmed both as members of the Achromobacter spp. with a probability of 90% each; however, differences were found in the results for phosphate, citrate, and proline assimilation (for details, see Table\u00a01). Table\u00a01 summarizes selected test results, which were positive for at least one of the strains.\nTable\u00a01Summary of classification test results using standard procedures and kitsTestContaminant 1Contaminant 2Contaminant 3Morphology of colonies\u00a0\u00a0FormRoundRoundRound\u00a0\u00a0Diameter1\u20132\u00a0mm1\u00a0mm1\u00a0mm\u00a0\u00a0ColorGreyBeigeWhite\u00a0\u00a0BrimSmoothSmoothSmooth\u00a0\u00a0SurfaceShinyShinyShiny\u00a0\u00a0Cross sectionRaisedRaisedRaised\u00a0\u00a0Gram stainNegativeNegativeNegativeBiochemistry\u00a0\u00a0Catalase+++\u00a0\u00a0Oxidase++\u2013\u00a0\u00a0O\/F test\u2013\/\u2013\u2013\/\u2013\u2013\/\u2013\u00a0\u00a0Motility++\u2013\u00a0\u00a0Lysine\/BBL Ox\/Ferm Tube II\u2013+n.d.\u00a0\u00a0ResultAchromobacter spp. or Pseudomonas spp.Achromobacter spp.Acinetobacter spp.Api 20 NE\u00a0\u00a0NO3, nitrate reduction++\u2013\u00a0\u00a0GLU, glucose+\u2013\u2013\u00a0\u00a0GNT, gluconate++\u2013\u00a0\u00a0CAP, caprate+\u2013+\u00a0\u00a0ADI, adipate++\u2013\u00a0\u00a0MLT, maltose+++\u00a0\u00a0CIT, citrate++\u2013\u00a0\u00a0PAC, phenylacetate+++\u00a0\u00a0OX, oxidase++\u2013\u00a0\u00a0ResultA. xylosoxidans (94.5%)A. denitrificans (82.2%)A. lwoffii (98.1%)VITEK 2\u00a0\u00a0N-LGGH, \u03b3-L-glutamyl (nutrient assimilation, NA)++n.d.\u00a0\u00a0P-BPHO, bis(p-nitrophenyl)phosphate+\u2013n.d.\u00a0\u00a0C-CIT, citrate\u2013+n.d.\u00a0\u00a0C-KGA, \u03b1-acetoglutaryc acid++n.d.\u00a0\u00a0N-LPROT, L-proline-NA+\u2013n.d.\u00a0\u00a0ResultAchromobacter spp. (90%)Achromobacter spp. (90%)n.d. n.d. not determined\n[3H]Adenine binding assays with isolated, intact bacteria\nAdenine competition binding studies were performed using isolated, intact bacteria (Fig.\u00a03b). The IC50 value obtained with whole bacterial cells of A. xylosoxidans () was in the same concentration range as that obtained with membrane preparations of the same bacteria, previously designated contaminant 1 (). For the other two bacteria, A. denitrificans and A. lwoffii, IC50 values were 5- to 10-fold lower when determined in whole bacterial cells compared with membrane preparations. From the homologous competition experiments, KD and Bmax values were estimated for A. xylosoxidans (membranes and intact cells). For bacterial membrane preparations, a KD value of 5.84\u2009\u00b1\u20091.12\u00a0nM and a Bmax value of 266\u2009\u00b1\u200965\u00a0pmol\/mg protein was calculated (n\u2009=\u20093). For the intact bacteria, a KD value of 11.0\u2009\u00b1\u20091.2\u00a0nM and a Bmax value of 780,000\u2009\u00b1\u2009120,000 sites\/cell (n\u2009=\u20098) was obtained.\nStructure\u2013activity relationships\nAffinities for selected compounds at adenine binding sites of A. xylosoxidans were determined in competition assays using whole bacterial cells and compared with data obtained in binding studies at the rat brain adenine receptor [7]. Figure\u00a04 shows competition curves for selected compounds, which exhibited high affinity, i.e., adenine (), hypoxanthine (), and 2-fluoroadenine (). The results for all compounds tested are summarized in Table\u00a02. Whereas the affinity of adenine for the binding sites of A. xylosoxidans was in the same range as for the rat adenine receptor, the affinities for hypoxanthine and 2-fluoroadenine differed substantially from those determined for the rat adenine receptor. Hypoxanthine showed very low affinity for the rat adenine receptor (Ki 45,000\u2009\u00b1\u200919,400\u00a0nM), but high affinity for the bacterial [3H]adenine binding sites, with an IC50 value in the low nanomolar range (59.1\u2009\u00b1\u20092.0\u00a0nM). For 2-fluoroadenine, the opposite was true: the Ki value for rat brain cortical adenine receptor was 620\u2009\u00b1\u2009140\u00a0nM [7], whereas the IC50 value for the A. xylosoxidans binding site was in the micromolar range (32,100\u2009\u00b1\u20093,000\u00a0nM).\nFig.\u00a04Competition curves for adenine, hypoxanthine and 2-fluoroadenine versus 10\u00a0nM [3H]adenine obtained with Achromobacter xylosoxidans (intact bacteria) (; ; Table\u00a02Comparison of affinities of adenine and selected compounds for the rat adenine receptor and bacterial binding sites determined in radioligand binding studiesCompoundAchromobacter xylosoxidans (intact bacteria)Rat brain cortical membranesIC50 \u00b1 SEM [\u03bcM]a (% inhibition \u00b1 SEM at 10\u00a0\u03bcM)Ki \u00b1 SEM [\u03bcM]a (% inhibition \u00b1 SEM at 100\u00a0\u03bcM)Adenine0.0138\u2009\u00b1\u20090.0027b0.0299\u2009\u00b1\u20090.0034c2-Fluoroadenine32.1\u2009\u00b1\u20093.00.62\u2009\u00b1\u20090.14c2-Hydroxyadenine\u226b10 (7\u2009\u00b1\u200915)d29.4\u2009\u00b1\u20096.42,6-Diaminopurine\u226b10 (3\u2009\u00b1\u20098)d4.95\u2009\u00b1\u20090.751,N6-Ethenoadenineca. 10 (53\u2009\u00b1\u20096)d34.4\u2009\u00b1\u20098.1Hypoxanthine0.0591\u2009\u00b1\u20090.019645.0\u2009\u00b1\u200919.4Uracil\u226b10 (6\u2009\u00b1\u20091)\u226b100 (\u22126\u2009\u00b1\u20092)Xanthine>10 (17\u2009\u00b1\u20096)>100 (23\u2009\u00b1\u20097)Uridine\u226b10 (5 \u00b1 5)d\u226b100 (\u22129 \u00b1 3)Adenosine\u226b10 (12 \u00b1 9)d19.4\u2009\u00b1\u20096.3caResults are from three independent experiments performed in triplicate, unless otherwise notedbResults are from eight independent experiments performed in triplicatecKi values and % inhibition values are from [7]dResults from two independent experiments performed in triplicate\nDiscussion\n[3H]Adenine has been successfully used by us [6, 7] and other laboratories [5, 10] to label the recently discovered rat and mouse adenine receptors. However, problems with [3H]adenine binding assays have been reported by two laboratories [11, 12]. These have led to the suggestion that [3H]adenine was not a suitable radioligand for the labeling of G protein-coupled adenine receptors [11, 12]. IJzerman and coworkers [12] reported that [3H]adenine binding was not protein dependent and that it bound with nanomolar affinity to glass fiber filters in the absence of added protein. These results, which were contradictory to our own data, prompted us to reexamine the [3H]adenine binding results that we had obtained during the past years, trying to find an explanation for the discrepancies.\nIn contrast to the results reported by IJzerman and coworkers [12], binding of [3H]adenine to various membrane preparations was strictly protein dependent in our hands, as expected (Fig.\u00a01). Specific binding linearly increased with increased amounts of protein and thus increased numbers of adenine receptors. Nonspecific binding was generally low for [3H]adenine, and there was only a minor increase in nonspecific binding with increasing protein concentrations. In the absence of protein, no specific binding was observed (Fig.\u00a01b). These results indicated that [3H]adenine was a suitable radioligand for labeling adenine receptors. In fact, we could recently perform [3H]adenine binding assays on a null background for the first time, namely, at the mouse adenine receptor heterologously expressed in Sf21 insect cells. Whereas membrane preparations of the nontransfected Sf21 cells did not exhibit any specific [3H]adenine binding, cell membranes prepared from cells infected with recombinant baculoviruses bound [3H]adenine with high affinity [6].\nHowever, when we looked carefully at all of our previous [3H]adenine binding data, we found a few [3H]adenine binding experiments that could not be evaluated due to unusually high radioactivity counts. These occasional problems had been solved by carefully controlling the experimental conditions, e.g., by using freshly prepared buffer solutions. Stimulated by the experiences reported by the IJzerman group [12], we decided to perform a systematic study to find out the reasons for those problems, which might also be causative for erroneous [3H]adenine binding results in other laboratories [11, 12].\nHigh affinity binding of [3H]adenine to filter paper, as suggested by IJzerman and coworkers [12], could be excluded by our experiments, as buffer solutions that were freshly prepared did not show any specific [3H]adenine binding in filtration assays using glass fiber GF\/B filters, the same filters that had been used by the IJzerman group [12]. On the contrary, we discovered that bacterial contaminations, which can be present in buffer solutions, express high-affinity binding sites for [3H]adenine and therefore impede adenine receptor binding assays. When we examined different buffer solutions stored in our laboratory, we discovered high [3H]adenine binding in Tris-HCl buffer solution (pH 7.4) stored at 4\u00b0C in a 5-l plastic container. That container had only a small orifice and was therefore difficult to purify. Thus, microbial contamination in this container was carried over when new buffer solution was prepared. When the contaminated buffer was filtered through a bacteria-tight filter (0.2\u00a0\u03bcm) or heated in order to denature proteins, specific [3H]adenine binding was abolished, strongly indicating that microbial proteins were responsible for the high affinity binding of adenine. A further indication that a living organism was involved was the fact that adenine binding increased exponentially with time. Interestingly, the microorganisms grew better at 4\u00b0C than at room temperature.\nOur first presumption, that the contaminants might belong to yeast, could not be proven. Saccharomyces cerevisiae was used as a control organism for binding studies but showed no [3H]adenine binding (data not shown). Three microorganisms were isolated from the contaminated Tris-buffer solution and identified using standard procedures and kits. Two of these contaminants were assigned to the genera Achromobacter and one to Acinetobacter. Both bacteria species are gram-negative rods and are strictly aerobic. They are commonly found in soil and water [17]. For healthy humans or animals, they are not pathogenic, but especially A. xylosoxidans and A. lwoffii have gained increasing importance due to their ability to cause nosocomial infections [22\u201332]. These bacteria are able to grow under nonoptimal conditions, such as low temperature and restricted nutrient supply [17, 24\u201326]. They are able to metabolize a wide variety of organic substances, such as chemical pollutants in the environment, and can therefore be used as bioreporters and for the degradation of pollutants [33\u201336]. This is consistent with the fact that these bacteria are able to grow in simple Tris-HCl buffer at low temperature.\nBoth A. lwoffii and Achromobacter spp., exhibit specific binding sites for adenine, with IC50 values as low as 13\u00a0nM for A. xylosoxidans. Thus, the detected adenine binding site in A. xylosoxidans exhibits an even higher affinity than the rat (29.9\u00a0nM [7], 18\u00a0nM [5]) or mouse (54.9\u00a0nM [6]) adenine receptor. The binding affinities for A. lwoffii and A. denitrificans were more that 70-fold lower, with affinities in the low micromolar range (1\u00a0\u03bcM and 2.4\u00a0\u03bcM, respectively) when membrane preparations were analyzed and about 20-fold lower when intact cells were investigated for binding (299\u00a0nM and 253\u00a0nM, respectively). For A. xylosoxidans, only a threefold difference was found when binding affinities for membranes were compared with those with intact cells (Fig.\u00a03). The specific, high-affinity [3H]adenine binding site in A. xylosoxidans appeared to be expressed in extraordinarily high density, as amounts of membrane preparations that contained more than 1\u00a0\u03bcg of protein\/assay tube led to depletion of the radioligand (i.e., more than 50% of the added radioligand was bound to the protein). Rough estimations of receptor densities by homologous competition assays confirmed the high expression levels.\nSo far, the nature of these high-affinity adenine binding sites in bacteria is not known. However, bacteria express a large number of transporters, including nucleobase transporters, in order to secure their nutrient supply (for review see [37]). Nucleobase transport in bacteria has been mainly studied in Escherichia coli and Bacillus spp., as well as in the fungi Aspergillus nidulans and Neurospora crassa [37\u201339]. Usually, these transporters fulfill two main functions. Firstly, purines can serve as preformed bases for nucleotide biosynthesis, and secondly, they serve as nitrogen sources [40, 41]. Distinct adenine uptake systems have been identified, e.g., in E. coli [42]. The high density of the detected [3H]adenine binding sites in A. xylosoxidans would be consistent with its function as a purine transporter and points to an important role of this protein, which appears to be upregulated when the bacteria are transferred to Tris buffer (unpublished observation), a medium poor in nutrients. Such an effect has been described for protozoa [43]: purine salvage enzymes and transporters can be dramatically up- or down-regulated according to growth stage and availability of purine sources [44]. Examples are the high-affinity hypoxanthine transporter in Trypanosoma brucei brucei, which shows a 450% increased transport rate after 24\u00a0h of purine deprivation, and the adenine transporter in Crithidia luciliae, which shows a >100-fold increase of adenine uptake after purine starvation [44, 45].\nFor E. coli as well as for B. subtilis, two adenine transport systems have been described: a low- and a high-affinity transport system [42, 46, 47]. The latter system is important when the concentration of adenine is low [46, 47]. Differences in adenine-binding affinity observed in our studies when intact cells were compared with membrane preparations (Fig.\u00a03) might be explained by the existence of different transporters in the bacteria (Achromobacter and Acinetobacter). Whereas bacterial membrane preparations were obtained directly from an overnight culture grown in complete medium, binding studies at whole bacteria were performed after growing them in Tris-HCl buffer, a nutrient-poor medium, in which they may have upregulated the high-affinity transporters [42, 47].\nIn order to investigate the structure\u2013activity relationships of the high-affinity adenine binding site in A. xylosoxidans, a series of compounds, including adenine derivatives, other purines, and pyrimidines (uracil, xanthine, hypoxanthine), and nucleosides (uridine, adenosine) were investigated in binding studies, and the results were compared with those obtained at the rat adenine receptor. As expected, structure\u2013activity relationships at the rat adenine receptor were very different from those at the bacterial adenine binding site. Hypoxanthine, for example, which exhibits a low affinity for the rat adenine receptor (45,000\u00a0nM), bound to the bacterial site with 760-fold higher affinity (59.1\u00a0nM), and 2-fluoroadenine, which showed a high affinity for murine adenine receptors (620\u00a0nM), bound with a 50-fold lower affinity (32,100\u00a0nM) to the bacterial site. Several compounds that had shown affinity in the micromolar range at rat adenine receptors (adenosine, 2-hydroxyadenine, 2,6-diaminopurine) were completely inactive at Achromobacter binding sites.\nAlexander had previously reported that hypoxanthine at a concentration of 10\u00a0\u03bcM completely blocked [3H]adenine binding to rat brain membranes in his experiments, the results of which were not consistent with the labeling of a G protein-coupled adenine receptor [11]. It might be speculated that he actually labeled a bacterial adenine binding site rather than the rat adenine receptor, which would explain the discrepant results. The limited number of compounds (five) investigated by IJzerman and coworkers [12] do not allow a full comparison of the structure\u2013activity relationships, but large differences were observed for two compounds\u20145\u2032-deoxyadenosine (725,000\u00a0nM [12] vs. 823\u00a0nM (rat adenine receptor) [10]) and 2-fluoroadenine (19,000\u00a0nM [12], 620\u00a0nM (rat adenine receptor) [7])\u2014indicating the labeling of a very different, presumably a bacterial, binding site by the authors [12]. The fact that hypoxanthine exhibits high affinity for the [3H]adenine binding site in A. xylosoxidans in the same concentration range as adenine itself is another indication that the labeled protein may be a purine transporter.\nNucleobase transporters have been identified in bacteria, fungi, protozoa, algae, plants, and mammals, but only few have been cloned and analyzed in detail [37, 48]. Five basic families of nucleobase transporters have been described: the nucleobase-ascorbate transporters (NAT), which include members from archaea, eubacteria, fungi, plants, and metazoa; the purine-related transporters (PRP), which are restricted to procaryotes and fungi; the purine permeases (PUP), which are purine transporters exclusively found in plants; and the equilibrative (ENT) and concentrative (CNT) nucleoside transporters, which not only transport nucleosides but may also transport nucleobases [37, 48\u201352]. Bacteria have developed different transport systems for related compounds, which allow them to independently absorb those compounds. This is an advantage when growing under nutritional deprivation [46, 53]. For C. luciliae, a nucleobase transporter that recognizes adenine and hypoxanthine equally well has been described [45]. In E. coli, adenine and uracil have different, specific transport systems, as do xanthine and guanine, whereas hypoxanthine might utilize the guanine transporter [53]. For B. subtilis, specific transport systems for guanine and hypoxanthine, for guanosine and inosine, as well as three independent uptake systems for adenine, adenosine and uracil, have been identified [46].\nFrom the described observations, we conclude that the high-affinity binding sites found in bacteria isolated from Tris-buffer solutions have not much in common with the adenine receptors found in mammals. The genomes of the bacteria A. baumannii and of several closely related bacteria, such as Pseudomonas spp. are known. Therefore, we performed a search to identify potential sequences with homology to the rat and mouse adenine receptors, which, however, yielded no hits. It appears likely that [3H]adenine labels a high-affinity nucleobase transporter for adenine and hypoxanthine in Achromobacter spp., a bacterium isolated as a contaminant from Tris-HCl buffer. The high affinity in the low nanomolar range is in fact unusual; therefore, it may be speculated that the labeled protein might belong to a new type of high-affinity bacterial nucleobase transporter.\nConclusions\nIn conclusion, we have demonstrated that [3H]adenine is a well-suited radioligand for the labeling of G protein-coupled adenine receptors, but precaution is advised for preparing and storing buffers used for the assays to avoid bacterial contaminations. After systematically analyzing occasionally encountered irregularities in [3H]adenine binding assays, we were able to isolate three bacteria, commonly found in soil and water, from Tris-HCl buffer. They were identified as A. lwoffii, A. xylosoxidans, and A. denitrificans and revealed high-affinity binding sites for [3H]adenine.","keyphrases":["[3h]adenine","adenine receptor","radioligand binding studies","achromobacter","acinetobacter","nosocomial infection","purine transporter","purinergic receptor","g protein-coupled receptor","bacterial buffer contamination"],"prmu":["P","P","P","P","P","P","P","P","P","R"]}
{"id":"Surg_Endosc-3-1-2169271","title":"Expert and construct validity of the Simbionix GI Mentor II endoscopy simulator for colonoscopy\n","text":"Objectives The main objectives of this study were to establish expert validity (a convincing realistic representation of colonoscopy according to experts) and construct validity (the ability to discriminate between different levels of expertise) of the Simbionix GI Mentor II virtual reality (VR) simulator for colonoscopy tasks, and to assess the didactic value of the simulator, as judged by experts.\nTraining skills in endoscopy for diagnostic and therapeutic procedures is essential and requires a great deal of hands-on training [1]. Virtual reality (VR) simulators offer a promising option to train these skills extensively prior to training in real-life colonoscopy, without jeopardizing patients or causing them unnecessary discomfort [2]. The use of VR training prior to performing real flexible endoscopy on patients enables novice endoscopists to go through part of their proficiency curve before submitting patients to their relatively insufficient endoscopy skills. This might not only be advantageous for the patients undergoing endoscopy, but might also prevent complications and potential consequences resulting in medicolegal litigation. One of the simulators in the field of flexible endoscopy is the GI Mentor II (see Figure\u00a01). VR simulators have been used extensively in different fields of expertise before applying these procedures to patients. In the United States of America simulator training is mandated by the Accreditation Council for Graduate Medical Education (ACGME) in laparoscopic procedures for surgical residents [3]. The first step is to validate the simulator construct properly and verify its didactic value, before implementing simulators in teaching programmes or developing a new curriculum for flexible endoscopy around them.\nFigure\u00a01.The GI Mentor II virtual reality simulator, the setup for training in lower endoscopy.\nSome studies have already been published on this subject [4\u20136], but the presented outcomes lacked power due to their relatively small sample sizes. In addition, some cases did not study the validity of endoscopy, but for example only the EndoBubble module, a computer simulation skills test measuring how long it takes a person to pop 20 balloons in a virtual tunnel.\nObjective\nThe main objectives of this study were: (1) to establish the degree of representation of real-life colonoscopy on the Simbionix GI Mentor II VR colonoscopy simulation, as judged by experts (expert validity), (2) to determine whether the GI Mentor II simulator can distinguish between various degrees of expertise in endoscopy, judged by novice, intermediate experienced, experienced and expert endoscopists performing VR colonoscopy (construct validity), and (3) to assess the didactic value of the simulator, as judged by experts.\nMaterial and Methods\nSimulator\nThe simulator used in this study was the Simbionix GI Mentor II (Simbionix Ltd. Israel, software version 2.7.3.0) (Figure\u00a01). The GI Mentor II can simulate upper GI tract endoscopies such as esophagogastroduodenoscopy, endoscopic retrograde cholangiopancreatographies, and endoscopic ultrasound. The lower GI tract endoscopies it simulates are sigmoidoscopy and colonoscopy. The simulator records a range of parameters upon each exercise, which can be used to assess performance objectively. The endoscope used is a customized Pentax ECS-3840F endoscope.\nParticipants\nParticipants were allocated to four groups to assess the validity and didactic value of the GI Mentor II simulator. The first group, the novices, was defined as participants without any flexible endoscopy experience; they were all medical interns or residents. The second group was intermediate experienced, with fewer than 200 colonoscopies performed before. In the third group experienced participants all performed more than 200 colonoscopies but fewer than 1,000. The fourth group consisted of experts, all of whom had performed more than 1,000 colonoscopies. These categories were chosen based upon several other studies, the demands for Dutch accreditation for colonoscopy, and the accreditation demands of the British Society of Gastroenterology, which advocates 200 colonoscopies under supervision during training [4, 6\u20138]. All persons were either invited to participate within our hospital, or participated during a national congress of the Dutch Society of Gastroenterology in spring 2006.\nThe groups consisted of at least 28 persons to ensure sufficient statistical power [9]. A post hoc sample size calculation based on the results for time to finish the EndoBubble task showed a minimal sample of 26 participants in the novices group to achieve a power of 0.95. Originally, the intermediate experienced and experienced participants formed one group, but as the expertise level and performance within this group varied considerably, this groups was split. A schematic setup of the study design is presented in Figure\u00a02.\nFigure\u00a02.The study design.\nQuestionnaire\nAll participants were asked to fill out a questionnaire on demographics and their general medical and endoscopy experience. It also included the number of endoscopies performed annually and number of years registered as a skilled professional endoscopist.\nAfter the simulator run the participants were asked to answer questions about their appreciation of the realism of the colonoscopy exercises performed. Appreciation was expressed on a four-point Likert scale [10] varying from very unrealistic (1) to very realistic (4). Questions were asked about the realism of imaging, simulator setup, endoscope control and both haptic and visual feedback. Experts were asked whether the GI Mentor II could be used as a teaching device for novice endoscopists and whether experience on the simulator could be useful in practice.\nSimulation modules\nAll participants first performed the hand\u2013eye coordination task (EndoBubble level 1) of popping all 20 balloons in the test as quickly as possible, without touching the walls. Next, the participants performed VR case numbers 1 and 3, both from colonoscopy module 1. These cases were carefully selected for their discriminative value; both cases are straightforward colonoscopies, without any abnormalities such as polyps, tumours, or inflammation. Case number 1 is a relatively easy colonoscopy to perform, whereas case number 3 is more difficult, requiring the endoscopist to apply techniques such as straightening the endoscope during loop formation and applying torque to the endoscope shaft. The assignment given for the VR colonoscopies was to reach the cecum as quickly as possible with as little patient discomfort as possible. Patient discomfort was defined as the estimated percentage of time the virtual patient was in excessive pain and the number of times excessive local pressure was caused. Other relevant test parameters were the percentage of time spent with clear view and the number of times view of the lumen was lost. The task was considered accomplished when the cecum was reached.\nData analysis\nSPSS 13.0 software was used to perform descriptive statistics and Kruskal\u2013Wallis tests for statistic analysis of the data. A separate analysis between groups was performed using a two-tailed Mann\u2013Whitney exact U test. A p-value of less than 0.05 was considered significant. The data showed a nonparametric distribution, therefore the median and range of performance parameters are presented as primary values.\nResults\nParticipants\nThirty-five novices, 15 intermediates, 20 experienced, and 35 expert endoscopists participated in the study. The average number of colonoscopies performed annually by experts was 445, and their mean number of years registered as a gastroenterologist was 7.7 (range 0\u201335 years).\nConstruct validity\nData output by the simulator are presented in Tables\u00a01 and 2. The EndoBubble task was completed faster by the experts and experienced endoscopists than by novices, with fewer wall collisions. These differences were statistically significant (Kruskal\u2013Wallis test) (Table\u00a01). Also the colonoscopy tasks were completed faster (p\u00a0<\u00a00,001, Kruskal\u2013Wallis test), with less patient comfort and better visibility by experts and experienced endoscopists (Table\u00a03). Novice endoscopists (N\u00a0=\u00a035) reached the cecum in a mean time of 29:57 (min:sec) in colonoscopy case 3, intermediate experienced (N\u00a0=\u00a015) in 5:45, experienced (N\u00a0=\u00a020) in 4:19, and experts (N\u00a0=\u00a035) in 4:56. Novices lost view of the lumen significantly more often than the other groups.\nTable\u00a01.EndoBubble hand\u2013eye coordination taskExperienceTime to finish (min:sec)Number of times wall touchedNovice N\u00a0=\u00a035Mean6:561.9Median5:581.0Range1:24\u201320:250\u201320Intermediate N\u00a0=\u00a015Mean1:561.1Median1:410.0Range0:54\u20134:020\u20135Experienced N\u00a0=\u00a020Mean1:370.9Median1:210.0Range0:43\u20135:330\u20139Expert N\u00a0=\u00a035Mean1:240.3Median1:130.0Range0:49\u20133:250\u20132Kruskal-WallisChi- square63.1519.374Asymp. sign.0000.025Table\u00a02.Colonoscopy module 1, cases 1 and 3ExperienceTime to reach cecum (hour:min:sec)% of time spent with clear viewLost view of lumenExcessive local pressure% of time patient was in painExcessive loop formedCase 1Novice N\u00a0=\u00a035Mean6:47960.40.513.30.83Median6:169700110Range 1:53\u201315:08 82\u201399 0\u201330\u201330\u201344 0\u20136Intermediate N\u00a0=\u00a015Mean1:36970080.6Median1:40980050Range0:55\u20132:5291\u2013100000\u2013300\u20133Experienced N\u00a0=\u00a020Mean1:239800.29.20.7Median1:21980081Range0:48\u20132:4389\u201310000\u201310\u2013270\u20133Expert N\u00a0=\u00a035Mean1:23980014.51.49Median1:179800121Range0:42\u20133:1694\u20131000\u2013100\u2013570\u201310Case 3Novice N\u00a0=\u00a035Mean29:57863.23.892.24.77Median23:42853301Range4:48\u20131:28:1972\u2013960\u2013121\u2013140\u2013240\u201334Intermediate N\u00a0=\u00a015Mean5:45891.12.10.91.13Median4:21921200Range2:28\u201313:4178\u2013970\u201340\u201360\u201340\u20138Experienced N\u00a0=\u00a020Mean4:19910.61.91.01.6Median3:50910101Range2:27\u20137:0273\u2013990\u201330\u201380\u201340\u20139Expert N\u00a0=\u00a035Mean4:56890.91.622.51Median4:0390111Range1:38\u201315:3968\u2013990\u201340\u201360\u2013100\u201312Table\u00a03.Statistics colonoscopy module 1, cases 1 and 3Time to reach cecum% of time spent with clear viewLost view of lumenExcessive local pressure% of time patient was in painExcessive loop formedCase 1Chi square69.04313.88918.41519.7837.10110.691Asymp. sig.0.0000.0030.0000.0000.0690.014Case 3Chi Square65.5596.97841.93628.7944.2844.856Asymp. sig.0.0000.0730.0000.0000.2320.183Kruskal-Wallis test\nA separate analysis between groups using a Mann\u2013Whitney exact U test demonstrated no significant difference between the intermediate, experienced and expert groups on all parameters. They all completed the task faster than the novices (see Table\u00a04).\nTable\u00a04.Differences between groups module 1, cases 1 and 3Time to reach cecum% of time spent with clear viewEost view of lumenExcessive local pressure% of time patient was in painExcessive loop formedCase 1Novice vs. intermediate0.0000.1770.0390.0130.0700.743Intermediate vs. experienced0.1660.6171.0000.2440.3850.547Experienced vs. expert0.9620.6211.0000.0430.0770.020Intermediate vs. expert0.1410.2591.0001.0000.0180.009Case 3Novice vs. intermediate0.0000.1040.0000.0040.5840.040Intermediate vs. experienced0.2570.3940.2850.5030.7710.184Experienced vs. expert0.9690.2970.1530.9420.1540.726Intermediate vs. expert0.3260.7570.8700.4160.1110.090Mann\u2013Whitney two-tailed test, exact significance\nExpert validity\nThe group of expert endoscopists rated the colonoscopy simulation 2.95 on a four-point Likert scale for overall realism. Anatomical representation was rated 2.58, and the simulator setup 3.14. Endoscope control scored 3.21. Haptic feedback was rated 2.57.\nDidactic value\nExpert opinion was that the GI Mentor II simulator should be included in the training of novice endoscopists (3.51 on a four-point Likert scale) and that expertise gained on the simulator was considered applicable in a clinical curriculum (rated 3.29 out of 4). The simulator was not considered suitable for certification of trained endoscopists (rated 2.29 out of 4).\nDiscussion\nThis study represents the largest and most detailed study on the validity of this type of colonoscopy simulator so far. The data show that the simulator can discriminate clearly between endoscopists of different expertise levels performing different colonoscopy tasks. Differences were statistically significant using relatively large sample sizes in all three exercises, the EndoBubble task as well as cases number 1 and 3. The difference between our study and previous studies by others is that we focused on the basic aspects of navigation for colonoscopy itself, rather than on the hand\u2013eye coordination task alone, used for example in the study by Ritter et al. [4], and that we included more participants in four separate groups with different levels of expertise [4\u20136, 11, 12]. in this way we were able to demonstrate that the GI Mentor II can distinguish between expertise levels up to the level of an intermediate experienced endoscopist, who has performed around 200 colonoscopies. In a similar study Sedlack et al. [5] describe a limited construct for a different simulator (AccuTouch, Immersion Medical). Felsher et al. [11] demonstrated differences between novices and experts in large sample sizes but did not compare novices to intermediate levels of expertise. In this study we have demonstrated convincing expert validity for colonoscopy on the GI Mentor II virtual simulator. This in contrast to other studies focusing on the EndoBubble task as a validation study [4] and not dealing with the subject of expert validity [4, 6, 7, 11, 12].\nThe colonoscopy tasks were considered as accomplished once the participants reached the cecum. Asking the participants to inspect the mucosa on the way back through the colon does not, in our opinion, provide a proper representation of the endoscopist\u2019s skills in manoeuvring through the colon, as other aspects besides the basic navigation skills of the endoscopist could influence the performance parameters provided by the simulator considerably in this case. This might lead to very different end times depending, for example, on the carefulness of the endoscopist.\nThis study demonstrates that the GI Mentor II simulator offers a convincing, realistic representation of colonoscopy according to experts. The overall assessment was good. Expert opinion was that the simulator can be used as a teaching tool for novice endoscopists. The simulator\u2019s haptic feedback is doubtful. Inexperienced residents can be trained in the skills necessary in flexible endoscopy such as steering control, straightening the endoscope during loop formation and applying torque up to a certain level.\nConclusion\nThe current study demonstrates that the GI Mentor II simulator offers a convincing, realistic representation of colonoscopy according to experts (expert validity) and that the simulator can discriminate up to the level of intermediate experienced endoscopists (construct validity) in colonoscopy. In the cases used the simulator could not discriminate between intermediate, experienced and expert endoscopists. The next step will be a study to determine whether novice endoscopists can develop a learning curve that will actually improve their endoscopic skills applied to real patients.","keyphrases":["validation","endoscopy","simulator","colonoscopy","training"],"prmu":["P","P","P","P","P"]}
{"id":"Virchows_Arch-3-1-1888715","title":"Discrepancies in the diagnosis of intraductal proliferative lesions of the breast and its management implications: results of a multinational survey\n","text":"To measure discrepancies in diagnoses and recommendations impacting management of proliferative lesions of the breast, a questionnaire of five problem scenarios was distributed among over 300 practicing pathologists. Of the 230 respondents, 56.5% considered a partial cribriform proliferation within a duct adjacent to unequivocal ductal carcinoma in situ (DCIS) as atypical ductal hyperplasia (ADH), 37.7% of whom recommended reexcision if it were at a resection margin. Of the 43.5% who diagnosed the partially involved duct as DCIS, 28.0% would not recommend reexcision if the lesion were at a margin. When only five ducts had a partial cribriform proliferation, 35.7% considered it as DCIS, while if \u226520 ducts were so involved, this figure rose to 60.4%. When one duct with a complete cribriform pattern measured 0.5, 1.5, or 4 mm, a diagnosis of DCIS was made by 22.6, 31.3, and 94.8%, respectively. When multiple ducts with flat epithelial atypia were at a margin, 20.9% recommended reexcision. Much of these discrepancies arise from the artificial separation of ADH and low-grade DCIS and emphasize the need for combining these two under the umbrella designation of ductal intraepithelial neoplasia grade 1 (DIN 1) to diminish the impact of different terminologies applied to biologically similar lesions.\nIntroduction\nThe concept of atypical hyperplasia was introduced decades ago within the continuum of intraductal breast proliferations, a continuum which encompasses benign proliferations of usual ductal hyperplasia to high-grade ductal carcinoma in situ (DCIS) [3\u20135, 7, 15, 32]. The term atypical ductal hyperplasia (ADH) was used initially for a vaguely defined group lesions that had \u201csome but not all of the requisite features of ductal carcinoma in situ\u201d [21]. Subsequently, the qualitative similarity to low-grade DCIS (LG-DCIS) was stressed, and quantitative measures were introduced to separate ADH from DCIS [21, 31].\nTotally arbitrary boundaries were drawn to separate ADH from LG-DCIS, a separation that resulted in drastically different management approaches in the 1980s \u2014i.e., mastectomy for those diagnosed as DCIS and follow-up for those with a diagnosis of ADH. Now, even with widespread use of conservative surgery, patient management differs significantly depending on whether a lesion is diagnosed as ADH or DCIS. The two most notable approaches for this separation have been based on: (a) the definition by Page et al. [21] who defined the minimum requirement for LG-DCIS as complete involvement of at least two spaces by a proliferation that cytologically and architecturally resembles DCIS and (b) the definition of Tavassoli and Norris [31] who required complete involvement of one or more ducts by a cribriform or micropapillary proliferation of uniform cells with low-grade nuclei exceeding 2\u00a0mm in aggregate diameter for a diagnosis of LG-DCIS. According to either of these two definitions, any intraductal proliferative lesion that demonstrated the qualitative cytologic and architectural features of LG-DCIS but failed to pass the defined quantitative threshold was to be designated as ADH.\nSince then, no objective molecular, ultrastructural, immunohistochemical, or morphometric feature has been identified to reliably distinguish ADH from LG-DCIS [1, 11, 15, 16, 18, 25]. Therefore, the distinction of the two continues to be based solely on application of the arbitrary criteria mentioned above. Frequently cited studies in the literature, which have attempted to calculate the relative risk of various proliferative lesions, may have claimed to adhere to the criteria of Page et al. have actually deviated from the two-space rule and have accepted single-space involvement as DCIS, thereby blurring the distinction between levels of hyperplasia and atypia and diminishing the practical utility of such a distinction [8].\nWe conducted a survey 15 and 20\u00a0years postintroduction of these two sets of criteria to determine how much uniformity or discrepancy exists in the interpretation of these lesions and how it would impact current patient management.\nMaterials and methods\nTo investigate pathologists\u2019 approach to selected problems in the diagnosis and management of intraductal proliferative lesions of the breast, a questionnaire was prepared with diagrammatic representations of five potentially problematic scenarios in breast pathology dealing with intraductal proliferative lesions (Figs.\u00a01, 2, 3, 4, 5). It was decided to use diagrammatic representations rather than glass slides to facilitate distribution of the survey among a greater number of pathologists and to ensure that the participants focused on the same diagnostic issue, given the presence of certain lesions in the background. The questionnaire also inquired whether the respondents considered breast pathology their area of expertise, the average number of breast biopsies and lumpectomies or mastectomies they signed out each week, the practice setting in which they worked in (academic, community, or private), and how long they had been in practice.\nFig.\u00a01The first scenario assessed how pathologists would diagnose a partially involved duct adjacent to unequivocal cribriform DCIS, and whether they would recommend reexcision if it were less than 0.1\u00a0mm from a resection marginFig.\u00a02In question 2, participants were asked whether the number of partially involved ducts affects their decision to make a diagnosis of ADHFig.\u00a03Responses to question 3 demonstrated what pathologists thought was the lowest size threshold required to make a diagnosis of DCISFig.\u00a04Question 4 evaluated how participants would manage flat epithelial atypia close to a resection marginFig.\u00a05Question 5 surveyed how pathologists measured invasive carcinoma in the presence of multifocal microinvasion\nThe questionnaire was distributed among over 300 pathologists who were either known for their expertise and specialization in breast pathology in various countries or who were interested in the field as evidenced by their participation in educational courses dedicated to breast pathology. If a participant left a response in the questionnaire blank, that response was considered null and excluded from calculations. Also, for all questions regarding diagnosis and management of lesions illustrated in the questionnaire, the respondents were given the option of choosing one of the two answers (i.e., ADH vs DCIS, Yes vs No). The responses of the small minority who chose both were similarly excluded from analyses. Also, if a participant responded to the questions regarding average number of biopsies or lumpectomies\/mastectomies a week by providing a numeric range, the average value of the range was considered for calculations. The collected responses were anonymized and tabulated in a custom Microsoft Access database (Microsoft Corporation, Redmond, WA, USA), and custom queries were written for data analysis. Statistical significance of the results was evaluated using chi-square and t tests.\nResults\nA total of 230 completed questionnaires were received and included in the study. Of the 230 respondents, 93 (40.4%) considered breast pathology their area of expertise, while 130 (56.5%) did not consider themselves experts but had special interest or responsibility for signing out breast pathology. The respondents signed out an average and standard deviation of 10.1\u00b19.5 biopsies and 7.5\u00b17.8 lumpectomies or mastectomies a week. These figures were 14.4\u00b111.4 biopsies and 10.3\u00b19.0 lumpectomies or mastectomies a week for the responding pathologists who considered breast pathology their area of expertise and 6.9\u00b16.0 biopsies and 5.3\u00b16.0 lumpectomies or mastectomies a week for those who did not. T test showed the differences between the means in these two groups to be statistically significant (p<0.0001 for both biopsies and lumpectomies\/mastectomies).\nAcademic pathologists constituted 85 (37.0%) of the respondents, while 113 (49.1%) of the respondents worked in community hospitals and 20 (8.7%) in private laboratories. Nine pathologists (3.9%) worked in more than one practice setting. Sixty-five pathologists (28.3%) had been practicing pathology for less than 5\u00a0years, 37 (16.1%) for 6 to 10\u00a0years, and 125 (54.3%) had over 10\u00a0years of practice experience.\nAs shown in Fig.\u00a01, the first question addressed whether respondents considered a partially involved duct adjacent to unequivocal cribriform DCIS as ADH or DCIS and, depending on their response, whether they would recommend reexcision if this partially involved duct was less than 0.1\u00a0mm from the excision margin. Of the 230 respondents, 130 (56.5%) considered this partial cribriform proliferation as ADH and, among these respondents, 49 (37.7%) recommended reexcision of the ADH if it were close to the excision margin. On the other hand, of the 100 (43.5%) who diagnosed this partially involved duct as DCIS, 28 (28.0%) would not recommend reexcision if the lesion were close to the excision margin. Although chi-square analysis showed that pathologists who made a diagnosis of DCIS were significantly more likely to recommend reexcision compared to those who made a diagnosis of ADH (p<0.001), the final impact on patient management was that, regardless of the diagnosis, 116 (50.4%) would recommend to have a reexcision while 108 (47.0%) would not.\nQuestion 2 documented how respondents categorized a partial cribriform proliferation involving a few (five) or numerous (20 or more) ducts (Fig.\u00a02). When only five ducts had a partial cribriform proliferation, 82 (35.7%) of the respondents considered it as DCIS. With \u226520 ducts so involved, 139 (60.4%) of the respondents designated the changes as DCIS. Chi-square testing showed this difference to be statistically significant (p<0.001).\nIn Question 3, as shown in Fig.\u00a03, the minimum size requirement for diagnosis of LG-DCIS in a single duct was addressed. When a single duct with a complete cribriform pattern measured 0.5, 1.5, or 4\u00a0mm, a diagnosis of DCIS was made by 52 (22.6%), 72 (31.3%), and 218 (94.8%) of the respondents, respectively. Chi-square testing confirmed that pathologists were significantly more likely to diagnose DCIS if the single duct measured >2\u00a0mm (p<0.001).\nQuestion 4 addressed the approach to management of flat epithelial atypia when it was present in multiple ducts located within less than 0.1\u00a0mm from the excision margin (Fig.\u00a04). In this scenario, when multiple ducts with flat epithelial atypia were close to an excision margin, 48 respondents (20.9%) recommended reexcision.\nFinally, question 5 evaluated how respondents measured invasive carcinoma when two foci of stromal microinvasion (measuring less than 1\u00a0mm each) emanated from two opposite poles of a 1-cm duct with high-grade comedo-type DCIS (Fig.\u00a05). Of the 230 respondents, 185 (80.4%) measured these as two separate foci of microinvasion (less than 1\u00a0mm each), while 37 (16.1%) considered the total size of invasive carcinoma as the aggregate diameter of the high-grade DCIS plus the two microinvasive foci, namely, 1.19\u00a0cm.\nChi-square testing was performed to compare the responses of expert breast pathologists and nonexperts to all five questions. No statistically significant difference was found between the two groups in the proportion of responses to any of the questions.\nDiscussions\nThese results indicate that 15 and 20\u00a0years postintroduction of criteria for separation of ADH from DCIS [21, 31], interobserver variability in the diagnosis of intraductal proliferative lesions of the breast has not diminished. Given the significance of margin involvement or proximity in current management of DCIS, this survey demonstrates an even wider variability that exists in the management of such lesions and ultimately patient care.\nIn a survey done by Rosai in 1991, 17 ductal and lobular proliferative breast lesions were distributed among five experts in breast pathology [24]. The participants were asked to categorize such lesions, which had already been circled on glass slides, as either hyperplasia, atypical hyperplasia, carcinoma in situ, or \u201cother\u201d (to be specified) based on the diagnostic criteria they used in their daily practice. In that study, there was no a single case in which all five pathologists agreed on the diagnosis, and there were only three cases (18%) in which four of the five pathologists agreed. Also, some pathologists tended to make more malignant diagnoses than others. In his report, Rosai considered this interobserver variability to be unacceptably high and suggested the adoption of a terminology such as mammary intraepithelial neoplasia with a grading system similar to that which was being used for the uterine cervix.\nA possible explanation for such a high degree of interobserver variability set forth in that report was that the pathologists were not using a standard set of criteria. Therefore, a year later, Schnitt et al. [27] tried a different approach to assess interobserver variability in the diagnosis of intraductal proliferative lesions of the breast. In their 1992 survey, they asked six experts in breast pathology to evaluate 24 proliferative ductal lesions. In this survey, the participating pathologists agreed to use the criteria of Page et al., and 15 teaching slides representing classic examples of usual ductal hyperplasia, atypical ductal hyperplasia, and noncomedo DCIS were circulated among the six pathologists to foster concordance before initiation of the study. The specific area of interest on each study slide was indicated by masking all the surrounding tissue so that all the participants focused on the same lesion and to prevent any bias that may result from assessment of changes in the surrounding breast tissue. The participants in this survey were asked to adhere to the provided criteria rather than the ones they used in their daily practice. Despite all these efforts, there was complete agreement among all six pathologists in only 14 (58%) of the 24 cases and among five or more pathologists in 17 cases (71%). The most common diagnostic problem was the distinction of atypical hyperplasia from DCIS in six cases. Although this study showed a significant improvement in interobserver agreement compared to Rosai\u2019s survey, the persistence of significant differences among expert breast pathologists even under optimal and highly artificial conditions conveyed a more widespread problem in the pathology community with potential impact on patient management.\nResponses to the current survey also demonstrate that although criteria to distinguish ADH from DCIS have been introduced over two decades ago, there is still no agreement on how to categorize these two types of mammary intraductal proliferation. While the sole partially involved duct described in question 1 would not qualify as DCIS according to the criteria set forth by either Page et al. or Tavassoli and Norris, 43.5% of practicing pathologists, nevertheless, diagnosed it as DCIS. This might seem logical because the partially involved duct probably reflects an extension of the same process present in the nearby unequivocal cribriform DCIS. However, the fact remains that there is no consensus on what to designate a partially involved duct adjacent to unequivocal LG-DCIS and how to manage such a lesion when it is near or on the surgical excision margin [10]. Even among the respondents who considered the partially involved duct in question 1 as ADH, more than a third recommended reexcision if it were close to the margin of resection, and of those who considered this partially involved duct as DCIS, close to 30% would not recommend reexcision even if the in situ carcinoma were within 0.1\u00a0mm of the resection margin. This interobserver variability in diagnosis and management was so high that, in the end, patients would have an almost 50:50 chance of having a reexcision or not, regardless of the diagnosis of the partially involved duct as ADH or DCIS. This was due to a combination of the high proportion diagnosing it as DCIS and the high proportion of those who recommended reexcision even though they interpreted it as ADH. Obviously, the impact of this issue goes beyond simply margin involvement; it is also crucial in the assessment of lesion size, resulting in widely varied assessment of size\/extent of LG-DCIS.\nQuestion 2 addressed the concept of extensive ADH. Cognizant of the fact that available criteria require complete involvement of duct cross sections for a diagnosis of low-grade (cribriform or micropapillary) variants of DCIS, it was surprising to find that even when only five ducts were involved by a partial cribriform proliferation, over 35% of the respondents considered it as DCIS. Furthermore, when greater than 20 ducts were partially involved by such a cribriform proliferation, the proportion of respondents who treated it as DCIS rose significantly to over 60%. Chi-square testing showed that as the number of partially involved ducts increased, the number of patients diagnosed with cancer significantly increased. Nevertheless, even with drastic differences in the number of involved ducts (5 vs \u226520), there was no unanimity in the diagnosis of carcinoma in situ vs atypical hyperplasia.\nThis scenario has become increasingly more frequent since the introduction of screening mammography. It was not addressed in the criteria introduced by either Page et al. or Tavassoli and Norris because both those retrospective studies were based on predominantly premammographic era biopsies. With the increasing number of such \u201cextensive ADH\u201d cases (\u2265\u00a020 partially involved ducts) seen in her consultation practice, Tavassoli suggested that even when these are designated as extensive ADH due to strict adherence to previously defined criteria, they should be managed as DCIS and reexcised if close to or at the margin of resection [29].\nQuestion 3 demonstrated that the majority of respondents used the 2-mm size criterion rather than the two spaces in diagnosing DCIS. When the single duct described in the question measured greater than 2\u00a0mm (4\u00a0mm), close to 95% of the respondents considered it DCIS. However, if this single duct measured less than 2\u00a0mm, i.e., 0.5 or 1.5\u00a0mm, 22.6 and 31.3% of the respondents considered it DCIS, respectively. Although this difference in the probability of diagnosing DCIS when the lesion measured less than or greater than 2\u00a0mm was statistically significant, it is interesting to note that over 30% of the respondents considered a single duct measuring 1.5\u00a0mm as DCIS and over 20% designated a single duct measuring only 0.5\u00a0mm as carcinoma in situ despite the fact that these two scenarios meet neither the criteria of Page et al., which require two completely involved spaces not otherwise specified as to size [21], nor the criteria of Tavassoli and Norris, which require one or more spaces greater than 2\u00a0mm in maximal cross-sectional diameter [31]. Even with a substantial 4\u00a0mm duct, 5.2% did not designate the lesion as DCIS presumably because of the two-space requirement of Page et al. These responses demonstrate that in the current day to day practice of pathology, some women with a 0.5-mm lesion would be diagnosed as having DCIS, with a reexcision if it were close to or at the resection margin, and most probably radiation therapy, whereas some women with 4\u00a0mm lesions interpreted as ADH would get no reexcision, would be simply followed up, or at most would enter some form of prevention trial with hormone therapy [2, 9].\nQuestion 4 in our survey documented the confusion that exists regarding the management of flat epithelial atypia, with over 20% of respondents recommending reexcision if the lesion were close to an excision margin. As a result, over a fifth of the patients would have reexcision, while the remaining 80% would not.\nThe last question showed the various approaches pathologists take in measuring invasive carcinoma when multifocal early invasion emanating from a single duct is present. Although over 80% of respondents would consider foci of invasion less than 1\u00a0mm emanating from opposite poles of a duct with DCIS as microinvasion, slightly over 16% of respondents would measure DCIS with its associated microinvasion from opposite poles as one continuous invasive carcinoma, which would entail a drastically different treatment approach.\nThe results of this survey raise numerous questions about studies performed in different countries and even different institutions within the same country regarding risk factors, treatment, prognosis, and outcome of intraductal proliferative lesions of the breast, which include LG-DCIS and\/or ADH\u2014a significant proportion of mammographically detected noninvasive lesions. Even when the criteria used are explicitly stated, application of criteria varies remarkably among pathologists and from one study to the next. Certainly, the issues raised in this study are not uncommon but have not been specifically addressed in any of the major single-, multiinstitutional, or multinational studies on DCIS cases that include LG-DCIS. Most if not all such studies lack a central review of the diagnosis and even many rely on multiple pathologists at sometimes multiple institutions. This study also illustrates that 15 to 20\u00a0years of education of pathologists at local, national, and international courses has not helped much in increasing the level of agreement and uniformity in the diagnosis and interpretation of these common lesions using the criteria available for separating ADH from LG-DCIS. Chi-square testing failed to reveal any statistically significant difference in the response behavior of expert breast pathologists and pathologists who did not consider themselves experts.\nSince the institution of widespread screening mammography, the number of early, LG-DCIS lesions that are diagnosed has dramatically increased [13, 14, 19]. How partially involved ducts, often spread around completely involved ducts, are interpreted could change the size\/extent of the lesion by several centimeters. This potentially undermines the internal consistency and comparability of epidemiological studies and clinical trials regarding such lesions.\nIn his 1991 survey, Rosai lamented the fact that there were no known morphometric, ultrastructural, immunohistochemical, or molecular features to distinguish ADH from LG-DCIS [24]. Unfortunately, this issue continues to be the case [1, 11, 15, 16, 18, 25]. Pathologists must assign lesions within the ADH\u2013DCIS continuum to one end of the spectrum or the other based on the morphologic features present on an H&E-stained slide and an arbitrary set of quantitative criteria that cannot be applied to every lesion encountered. Not only the subjective nature of interpreting the morphologic findings but also the existence of different and in some cases conflicting diagnostic criteria create considerable interobserver variability in distinguishing ADH from LG-DCIS.\nThis diagnostic variability in turn leads to confusion regarding the optimal management approach to such intraductal proliferative lesions because a diagnosis of LG-DCIS is automatically associated with a significantly worse prognosis and usually requires a more drastic surgical approach compared to ADH. Furthermore, the negative impact of the diagnosis of \u201ccarcinoma\u201d, albeit an in situ one, on patients\u2019 psychological well-being (depression and anxiety) has been well documented [12, 23] and should be a consideration in the choice of optimal terminology.\nMolecular, immunohistochemical, and morphologic similarities indicate that the only difference between ADH and LG-DCIS is a quantitative one [1, 11, 15, 16, 18, 25]. The claim that separation of ADH from LG-DCIS is justified based on the development of carcinomas post-ADH in either breast while those that develop after LG-DCIS occur in the same breast is questionable [20, 22, 26]. Over a period of 23\u00a0years, three papers have appeared on this topic based on a group of 28 women with a median follow-up of 31\u00a0years, noting that the invasive carcinomas that occur after LG-DCIS treated by biopsy alone develop in the same breast and in the same quadrant from which the original biopsy with DCIS was taken. Seven of the 11 women who developed subsequent invasive carcinoma were diagnosed within 10\u00a0years of the DCIS biopsy, 1 was diagnosed within 12\u00a0years, and the remaining 3 developed infiltrating carcinomas over 23\u201342\u00a0years. The authors conclude that these results \u201cindicate a striking dividing point biologically and histopathologically between LG-DCIS lesions and the cytologically similar but lesser lesions of ADH\u201d [26]. When 7 of the 28 women developed invasive carcinoma within 10 years, one wonders why the remaining patients were not contacted to receive appropriate therapy for their disease rather than continuing the study for another 20 years.\nJudging from the microscopic description and the sampling documented for these 28 cases, a substantial number of these lesions were probably not low grade because they had some atypia and\/or necrosis, albeit not diffusely, suggesting that those who died within 10\u00a0years most likely had substantial amount of residual disease and\/or higher grade DCIS lesions [6]. The gross and pathologic features of these 28 cases are described in detail in the initial paper published in 1982, which did not refer to them as low grade [20]. Therefore, it is more likely that this study reflects the natural history of a wide variety of intraductal carcinomas, only some of which were LG-DCIS. A more simple explanation for any differences that might exist between ADH and LG-DCIS in the frequency of subsequent progression would be that a minuscule lesion, once totally removed, results in near equalization of the risk for subsequent development of carcinoma in the two breasts of that individual patient. A more extensive process, however, is less likely to be completely eliminated by conservative surgical excision alone, leading to the development of recurrences at the same site due to residual disease [6].\nA recent epidemiological study has shown a nearly equal incidence rate (5.4 vs 4.5\/1,000 person-years) for development of subsequent invasive carcinoma in either breast after a diagnosis of DCIS among close to 37,700 patients diagnosed between January 1988 and December 2002, many of whom were treated by surgery and radiation therapy [17]. This confirms the fact that once a DCIS is adequately treated, it results in equalization of risk for either breast. Furthermore, it indicates that complete excision is an adequate therapy for small lesions. Therefore, complete excision with adequate margins should be explored further as the only therapy for small DIN 1 (DCIS grade 1) lesions that do not exceed 3\u20134\u00a0mm in maximum extent, particularly when there is no evidence of even any flat epithelial atypia around the DCIS.\nIt seems only logical to adopt a terminology in which two morphologically identical lesions are not given diagnostic designations as different as \u201chyperplasia\u201d and \u201ccarcinoma\u201d. The use of the DIN terminology may help solve this problem [28, 30]. Although the DIN terminology does not claim to reduce interobserver variability, it diminishes the effect of drastically different designations for similar lesions by including the spectrum of atypical ductal hyperplasia and LG-DCIS under the umbrella designation of grade 1 DIN.","keyphrases":["breast","ductal carcinoma in situ","hyperplasia","ductal intraepithelial neoplasia","interobserver variability"],"prmu":["P","P","P","P","P"]}
{"id":"Environ_Health_Perspect-114-9-1570082","title":"GIS Approaches for the Estimation of Residential-Level Ambient PM Concentrations\n","text":"Spatial estimations are increasingly used to estimate geocoded ambient particulate matter (PM) concentrations in epidemiologic studies because measures of daily PM concentrations are unavailable in most U.S. locations. This study was conducted to a) assess the feasibility of large-scale kriging estimations of daily residential-level ambient PM concentrations, b) perform and compare cross-validations of different kriging models, c) contrast three popular kriging approaches, and d ) calculate SE of the kriging estimations. We used PM data for PM with aerodynamic diameter \u226410 \u03bcm (PM10) and aerodynamic diameter \u2264 2.5 \u03bcm (PM2.5) from the U.S. Environmental Protection Agency for the year 2000. Kriging estimations were performed at 94,135 geocoded addresses of Women\u2019s Health Initiative study participants using the ArcView geographic information system. We developed a semiautomated program to enable large-scale daily kriging estimation and assessed validity of semivariogram models using prediction error (PE), standardized prediction error (SPE), root mean square standardized (RMSS), and SE of the estimated PM. National- and regional-scale kriging performed satisfactorily, with the former slightly better. The average PE, SPE, and RMSS of daily PM10 semivariograms using regular ordinary kriging with a spherical model were 0.0629, \u22120.0011, and 1.255 \u03bcg\/m3, respectively; the average SE of the estimated residential-level PM10 was 27.36 \u03bcg\/m3. The values for PM2.5 were 0.049, 0.0085, 1.389, and 4.13 \u03bcg\/m3, respectively. Lognormal ordinary kriging yielded a smaller average SE and effectively eliminated out-of-range predicted values compared to regular ordinary kriging. Semiautomated daily kriging estimations and semivariogram cross-validations are feasible on a national scale. Lognormal ordinary kriging with a spherical model is valid for estimating daily ambient PM at geocoded residential addresses.\nLarge-scale, population-based epidemiologic investigations of the health effects of ambient air pollution often rely on measurements from a network of air quality monitors maintained by the U.S. Environmental Protection Agency (U.S. EPA 1995a, 1995b, 2005). The Air Quality System (AQS) is the only national ambient air pollution database currently available for public use in the United States. The availability of individual-level health outcome and covariable data from national-scale studies that often characterize participants over the course of several years enables researchers to study the acute effects of ambient air pollution using individual-level data (Liao et al. 2004, 2005a; Sullivan et al. 2005; Wellenius et al. 2005; Whitsel et al. 2004). This approach requires measures of daily particulate matter (PM) exposures, ideally assessed as close to the individual level as possible, such as at participant residences or in immediate proximity to participants themselves. Because daily measures of ambient PM concentrations from the AQS are unavailable in the large majority of locations, spatial estimation methods using geographic information systems (GIS) are increasingly being considered to estimate geocoded location-specific ambient PM concentrations, such as kriging methods. Important methodologic and practical issues still need to be resolved, however. This study was designed to a) assess the feasibility of large-scale kriging estimation of daily residential-level ambient PM concentrations, b) perform and compare cross-validations of different kriging models, c) determine and contrast the most appropriate kriging approaches, and d) calculate the SEs of the kriging estimations.\nMaterials and Methods\nWe obtained from AQS the PM10 and PM2.5 (PM with aerodynamic diameter \u2264 10 and 2.5 \u03bcm, respectively) data from 1993\u20132004 (U.S. EPA 2005). The data from 2000 were used for this study after eliminating duplicate records and converting all measures to the same units and denominator. We calculated \u201cmonitor-specific\u201d daily averages based on \u2265 18 hourly measures. Monitor-specific daily averages were set to missing for monitors reporting < 18 hourly measures on any given day. If more than one monitor was operating at the same location on a given day, we then computed \u201csite-specific\u201d daily PM10 and PM2.5 averages by taking the mean of the monitors\u2019 measures. We also obtained the longitude and latitude for each site from the AQS database. These data served as pollutant- and site-specific daily source data for our study (Liao et al. 2005b).\nWe geocoded 94,135 addresses of Women\u2019s Health Initiative (WHI) Clinical Trial (CT) participant residences and examination sites in the contiguous 48 United States and District of Columbia, after assessing geocoding vendor error (Whitsel et al. 2004, 2005). Daily PM10 and PM2.5 concentrations and the associated estimation errors (SEs) are estimated at these geographic locations by the Environmental Epidemiology of Arrhythmogenesis in WHI study (Whitsel 2006).\nWe used ArcView GIS (version 8.3) and its Geostatistical Analyst Extension (ESRI Inc., Redlands, CA) for semivariogram determination and cross-validation and for subsequent spatial estimation of daily location-specific PM concentrations. Three frequently referenced spatial models (spherical, exponential, Gaussian) (Cressie 1993a; Davis 2002) were considered using the weighted least-squares method (Gribov et al. 2004; Jian et al. 1996) to obtain the \u201coptimal\u201d daily semivariogram parameters (range, partial sill, and nugget). Based on the daily semivariograms, we performed ordinary kriging to estimate the daily mean PM concentration and its SE at each of the 94,135 geocoded addresses. Next, we performed the standard cross-validation\u2014an iterative procedure that omits site-specific PM data points one at a time and refits the model using the remaining data to estimate the PM concentration at the site of the omitted observation. We assessed the validity (also termed \u201cgoodness of fit\u201d) of each semivariogram using three cross-validation parameters readily available from the ArcView software package: a) the average of prediction error (PE), where PE is the average of the difference between the predicted and measured daily PM values at each monitoring site; b) the average of standardized prediction error (SPE), where SPE is the PE divided by the SE of estimation across all sites; and c) root mean square standardized (RMSS), the standard deviation (SD) of all SPEs across all sites. Additionally, we assessed the goodness of fit of each semivariogram by the average of the SEs of the estimations, generated by the kriging procedure, across all 94,135 geocoded addresses. The expectations for a good-fitting semivariogram and kriging model are an average PE and SPE near 0, an RMSS near 1, and a small SE. If RMSS < 1, there is a tendency toward overestimation of the variance; if > 1, there is a tendency toward underestimation (ESRI Inc. 2001). These criteria were consistently used to guide our model selection processes throughout this study (Liao et al. 2005c).\nAs an alternative to using the automatically calculated semivariogram (calculated using the weighted least-squares method (Gribov et al. 2004; Jian et al. 1996), one can also manually specify the semivariogram parameters to improve the cross-validation parameters in ArcView. We selected six least satisfactory daily semivariograms throughout year 2000 and manually adjusted the semivariogram parameters to obtain the best achievable average RMSS and SPE (RMSS as close to 1 and average SPE as close to 0 as possible). The cross-validation parameters from the weighted least-squares method\u2013calculated semivariograms were then compared to those of the manually adjusted semivariograms.\nWe performed daily ordinary krigings on both the original scale (regular ordinary krig-ing) and the lognormal scale (lognormal ordinary kriging) (Cressie 1993b; Johnston 2001) for all WHI CT addresses for the year 2000 and compared the cross-validation parameters between the two kriging procedures. Log-normal ordinary kriging was used because it has the ability to eliminate the negative predicted values, which is a problem in ordinary kriging, especially when the source data contain extreme values.\nResults\nCharacteristics of the site-specific daily average PM10 and PM2.5 concentrations\nDuring 1994\u20132003, the number of monitoring sites that provided GIS-usable daily PM10 data varied widely (range, 120\u20131,340). On 17% of days, GIS-usable data were provided by \u2265 400 monitoring sites; on 39% of days, by 200\u2013400 sites; and on 44% of days, by 120\u2013200 sites. The corresponding values for PM2.5 during 1999\u20132003 were 33% of days by \u2265 400 sites and 67% of days by 148\u2013400 sites. Specific to the year 2000, there were averages of 325 PM10 and 456 PM2.5 monitoring sites operating per day across the contiguous United States, with minima and maxima of 148 and 1,061 sites for PM10 and 178 and 1,019 sites for PM2.5. As a result, there were 118,791 site-days during 2000 for which we can retrieve measured PM10 data and 166,796 site-days for PM2.5 data. The mean (\u00b1 SD) of PM10 and PM2.5 from these retrievable site-days were 26.29 \u00b1 58.13 and 13.14 \u00b1 8.59 \u03bcg\/m3, respectively, with medians of 21.33 and 11.20 \u03bcg\/m3, respectively. A right-skewed distribution of both PM10 and PM2.5 are evident, especially for PM10. Figure 1 illustrates the spatial relationships between the geocoded addresses and the PM monitoring sites on an optimal day and a typical day. The mean distance between each address and its nearest PM monitor was 12.35 km, with an SD of 13.98 km, a median of 7.81 km, an interquartile range of 10.53 km, and 99th percentile of 68.36 km.\nComparisons of three widely used spatial models\nTables 1 and 2 present summary statistics of the cross-validation parameters (PE, SPE, and RMSS) comparing three widely used spatial models (spherical, exponential, Gaussian) for PM10 and PM2.5, respectively. In general, both average PE and average SPE are very close to 0, with a very narrow range of variation from the 366 daily cross-validations. More specifically, > 95% of average PEs were within \u00b1 2 \u03bcg\/m3 of measured PM10, and \u00b1 0.5 \u03bcg\/m3 of measured PM2.5, an average measurement error that we considered acceptable. In terms of RMSS, we considered > 95% of cross-validations as acceptable, but there were days when RMSS indicated a slight over-or underestimation of the prediction variability. These data support the overall validity of using kriging-based estimation approaches to estimate location-specific PM concentrations across the contiguous United States.\nComparisons of default and manually adjusted semivariograms\nTable 3 presents the cross-validations and actual kriging estimations from the weighted least-squares mean method calculated semivariogram and manually adjusted semivariogram. For the 6 days when the PE, SPE, or RMSS indicated a less satisfactory default-calculated semivariogram, these three cross-validation parameters could be improved satisfactorily through adjustment of the semivariogram parameters by an operator. However, the application of such \u201cimproved\u201d semivariograms to the estimation of PM10 concentrations at geocoded locations across the United States did not necessarily provide better estimation of location-specific PM (i.e., smaller SEs). To the contrary, the average SEs from the default semivariograms were smaller than those from manually adjusted semivariograms. Because each average SPE of the default-calculated daily semivariograms was close to 0, and each default-calculated daily semivariogram produced a smaller estimation error, we recommend using the default-calculated semivariogram, even though the RMSS from the default-calculated semivariogram was not fully satisfactory.\nComparisons of regular versus lognormal ordinary krigings\nWe applied regular ordinary kriging (spherical model, default-calculated daily semivariograms) to estimate daily PM10 concentrations at geocoded addresses (n = 94,135) of WHI CT participants and examination sites in the contiguous United States. We examined the estimated PM10 concentrations and identified 22 days during 2000 when estimated values exceeded the range of observed values. In some cases, the estimated values were negative. The number of addresses affected by this problem ranged from a few on most days to 3.5% of all addresses. This problem was related to skewed PM10 distributions and to small numbers of extreme outlying values or operating sites on some days. We therefore compared regular ordinary kriging and log-normal ordinary kriging anticipating that log-normal kriging would attenuate this problem.\nTable 4 lists the 22 days on which regular ordinary kriging yielded estimated PM10 values that were outside the range of measured values. For comparison, the minima and maxima of the measured and estimated PM10 concentrations from both regular and log-normal ordinary krigings are also listed in Table 4. In summary, during 2000, lognor-mal ordinary kriging effectively reduced the number of problematic days from 22 to 1. Even on this one day, lognormal ordinary kriging yielded a minimum value that was closer to the range of measured data than that from regular ordinary kriging.\nTable 5 shows the mean values of cross-validation parameters of daily PM10 semivariograms for both regular ordinary kriging and lognormal ordinary kriging. Cross-validation parameters were within the acceptable range from both regular and lognormal ordinary krigings, except for the 22 \u201cout-of-range\u201d days as defined above. On these out-of-range days, the SPE was well within the acceptable range for both regular and lognormal krigings, but the RMSS was > 1 from both approaches. Even so, for these out-of-range days RMSS from lognormal ordinary kriging was closer to 1 than that from regular ordinary kriging.\nWe then performed regular and lognormal ordinary kriging to estimate PM10 concentrations at geocoded addresses of WHI CT participants and examination sites, based on year 2000 PM10 data (94,135 locations and 366 days). The mean, SD, median, and maximum of the daily mean SE of the estimated PM10 from the regular ordinary kriging were 27.36, 83.35, 13.93, and 1160.20 \u03bcg\/m3, respectively. In contrast, those from the lognormal ordinary kriging were 16.29, 6.65, 15.05, and 67.46 \u03bcg\/m3. Clearly, the distribution of the estimation errors from lognormal ordinary kriging was considerably less skewed and had fewer outlying values than that from regular ordinary kriging. Alternative methods (winsorizing extreme PM10 values; using ArcView\u2019s \u201cno-sector\u201d option to search for measured data points from a circle centered around a location that needs of an estimation\u2014i.e., disabling the default \u201csector\u201d search for measured data points in the four sectors of a circle, reducing the range or nugget) were less effective in estimating predicted values within the range of measured values (data not shown).\nSimilar to the situation observed in PM10 estimations, lognormal ordinary kriging also effectively eliminated the negative or out-of-range problem that occurred in about 5% of PM2.5 data when using regular ordinary kriging. Other cross-validation parameters were comparable between the lognormal and regular ordinary krigings (data not shown).\nComparisons between national and regional krigings\nFrom the 61 days when 900 or more monitoring sites were operating in the year 2000 in the 48 contiguous states, the first of such days from each month was selected for comparisons between ordinary kriging models on a national versus regional scale. National krigings and cross-validations were performed on these 12 selected days using daily site-specific PM10 data. Regional krigings and cross-validations were performed on the same data using the regional map (Figure 1) that divides the U.S. continent into five regions (northwest, southwest, middle north, southeast, and northeast). These five regions were created based on the assumption that different semivariogram parameters would be needed for different geographic areas. In general, for both regional and national krigings, the average SPE and RMSS from cross-validations of semivariograms calculated for the 12 selected days were very close to 0 and 1, respectively (Table 6)\nDiscussion\nClassical methods often assume that measures are uniformly or randomly distributed. The assumptions are often inappropriate for analysis of environmental measures because values at neighboring locations are rarely independent, particularly over short distances. This form of dependence (spatial autocorrelation) nonetheless makes it possible to interpolate values at unmonitored locations from known values at monitored locations. Kriging is one such interpolation method originally developed by mining engineers (Krige 1966). It is especially attractive in this setting because it takes the spatial autocorrelation structure function (variogram) into account by considering known values from monitored locations, weighting them with values read from the variogram at corresponding distances, and splitting weights among adjacent locations. The method thereby ensures that interpolations do not depend on monitor density (Legendre and Fortin 1989). By doing so, kriging yields best linear unbiased estimates, in this setting, of location-specific daily mean ambient PM concentrations and their SEs.\nLarge-scale population-based epidemiologic investigations of the health effects of ambient air pollution often rely on data collected from a network of air quality monitors maintained by the U.S. EPA\u2014the AQS data (U.S. EPA 1995a, 1995b, 2005). It is revealing to compare kriging with interpolation methods used in the well-known time-series and cohort studies of PM effects on mortality and cardiovascular disease (Abbey et al. 1991, 1999; Dockery et al. 1993; Katsouyanni et al. 1996, 2001; Miller et al. 2004, 2005; Pope et al. 2004; Samet et al. 2000a, 2000b). These studies uniformly estimated PM exposures using area-based arithmetic averaging or nearest-neighbor imputation\u2014alternative methods that have important limitations (Moore and Carpenter 1999). Such limitations include the assumption of homogeneous exposures within study areas and the inability (or failure) to estimate exposures or associated PEs. For example, when daily exposure was of interest and there were no operating PM monitors with a study area, data pairs (daily PM concentrations, death counts) were unavailable in these studies. In addition, when longer-term (monthly to yearly) exposure was of interest, area aggregated exposures were based on available measurements within a given time frame. If there were five 24-hr measures in a month, for example, the monthly average exposure was calculated as the mean of the five readings. In contrast, our kriging-based approach estimated daily mean exposures and SEs at geocoded addresses of participants and their examination sites across the contiguous United States that can be readily integrated over time with little influence of missing data. Studies in the geosciences have also found that kriging provides consistently improved interpolation accuracy over traditional inverse-distance weighting and other, simpler spatial interpolation methods (Zimmerman 1999). Another important advantage of GIS-based estimation over the traditional area-average approach is the availability of both the location-specific estimated pollutant concentrations and their SEs.\nOur goal in this study was to contribute methodologic and practical insights toward standardized, semiautomated GIS approaches to estimation of daily air pollution concentrations and their associated estimation errors. The air pollution data estimated using these approaches will support the Environmental Epidemiology of Arrhythmogenesis in WHI study (Whitsel 2006) examining the cardiac effects of air pollution in 68,133 post-menopausal women 50\u201379 years of age at baseline in the WHI CT (WHI Study Group 1998). Here we describe our experience resolving several important methodologic and practical issues in adopting a systematic, standardized, and semiautomated kriging approach to estimate daily air pollution concentrations and the associated estimation errors at geocoded addresses across the contiguous United States over 10 years.\nWe successfully downloaded from AQS the PM10 and PM2.5 raw data from 1993\u20132004. We then cleaned, calculated, and reconstructed site-specific daily PM concentration data ready for GIS applications. It is well known that the monitoring sites in AQS are not randomly distributed, which is one of the assumptions in kriging estimation, and the density of the monitoring sites is relatively low given the size of the contiguous United States. However, the AQS is the only currently available nationwide database. Our cross-validation studies suggest that the AQS data can be used as source data for kriging estimation of ambient pollution concentrations at various locations across the 48 contiguous states.\nIn this study, we performed cross-validation to assess the goodness of fit of various semivariogram and spatial models using four major parameters: the average PE, SPE, RMSS, and SE of estimation. Details can be found elsewhere (Webster and Oliver 2001), but it is worth noting that in addition to using the SE as a measure of the goodness of fit of a kriging model, one could improve the health effects models by incorporating SE in the models to account for the error in the estimation of location-specific PM concentrations. We consider this an important advantage of GIS-based estimation over the traditional area-average approach and are performing studies of using SE in health effects models.\nWe compared the performance of three widely spatial models (spherical, exponential, Gaussian) for PM10 and PM2.5 estimations using regular ordinary kriging on a national scale (Tables 1 and 2). In general, the cross-validation parameters suggest that all three models performed fairly well. Overall, the spherical model seemed to perform slightly better, consistent with the observation that the spatial distribution pattern of ambient air pollutants is closest to the assumption of the spherical model. The spherical model has been used most often in modeling spatially distributed data, providing a further rationale for its use in our large-scale population-based study of the health effects of PM. Furthermore, from the perspective of the cross-validation results, both average PE and average SPE are very close to 0, with a very narrow range of variation from the 366 daily cross-validations. These data support the overall validity of using kriging-based estimation approaches to estimate location-specific PM concentrations across the contiguous United States.\nWe completed an empirical analysis to investigate whether manually adjusting semivariogram parameters improves a) cross-validation parameters and b) estimated PM10 concentrations and their SEs (Table 3). From these data, we conclude that manually adjusting semivariogram parameters improves cross-validation parameters. However, the application of such \u201cimproved\u201d semivariograms to the estimation of PM10 concentrations at geocoded locations across the United States did not necessarily provide better estimation of location-specific PM. Therefore, we recommend using the default-calculated semivariogram.\nSemivariograms are sensitive to strong positive skewness. As a result, regular ordinary kriging can yield negative predicted values or values exceeding the range of the source data. Kriging works best if the input data have a normal distribution. One solution is to log-transform the input data\u2014using \u201clognormal kriging.\u201d In the ArcView software package, performing lognormal kriging is a standard option. This option log-transforms the input data to normalize its distribution and attenuate the impact of very large values. It also back-transforms the estimated values and the \u201cunbiased\u201d SE of the estimation to the original scale (Cressie 1993b; Johnston 2001). Our results comparing lognormal ordinary kriging versus regular-scale ordinary kriging suggest that lognormal ordinary kriging not only effectively estimated location-specific PM concentrations within the range of the measured data for the days regular ordinary kriging yielded negative or \u201cout of range\u201d PM estimations, but also yielded a smaller average SE than did regular ordinary kriging and estimations. Therefore, our results support the use of lognormal ordinary kriging as an acceptable solution to the problem commonly posed by positively skewed distributions of environmental data.\nOur comparisons of national- versus regional-scale kriging indicate that, in terms of cross-validation results, both performed similarly. However, such comparisons are based on krigings using the source data from optimal days (when > 900 sites across the country were reporting data), which account for only 17% of all days in a year. Therefore, there is additional justification for using national-scale kriging: Usually, there were very few operating sites within a region. On typical days\u2014when only about 200 monitoring sites were operating\u2014ability to derive stable and meaningful semivariograms was greatly impaired. Regional kriging also poses problems for estimation at locations near regional borders. For example, at locations within Washington State but near the Washington\u2013Idaho border, regional kriging is based solely on PM10 concentrations in the \u201cWashington\/Oregon, Northern California\u201d region. It is not based on PM10 concentrations measured immediately across the border in Idaho, despite the real possibility that they would have the largest weights in national-scale kriging estimation. For all these reasons, we recommend national-scale kriging.\nConsidering the number of study participants and the length of study period (1994\u20132003) for the Environmental Epidemiology of Arrhythmogenesis in WHI study, development of an automated procedure enabling large-scale daily krigings and semivariogram cross-validations was critical. In this study, we decided to use ArcView for predicting individuals\u2019 PM exposure concentrations because of the flexibility it offers for automation. Because ArcView GIS relies on either the weighted least-squares method or visual adjustment to create semivariograms, we did not compare the relative performance of semivariograms generated using alternative methods such as maximum likelihood and restricted maximum likelihood. For generating semivariograms, we compared only three popular spatial models (spherical, exponential, and Gaussian). Our results, however, do not invalidate alternative spatial models (e.g., power). In the end, we selected the spherical model for our study because it is the most studied model, and its assumption pertaining to the spatial correlation of data is probably closest to our pollutant data. Furthermore, the spherical model seemed to perform as well as or slightly better than the remaining models in terms of cross-validation parameters.\nWe chose ordinary kriging instead of universal or simple kriging for several reasons. First, the assumption for simple kriging of a known mean concentration on any given day across space is not practical for our data. Although it may seem more appropriate because of the \u201cvarying mean\u201d concentration across the contiguous U.S. assumption, universal kriging requires a predetermined set of \u201cexploratory variables\u201d to explain the varying means. The candidates, many of which are spatial variables, include emissions, land use, population, road network distribution, altitude, rainfall, latitude, climatology, and other quality data. Denby et al. (2005) recently recommended a method that uses measured concentration data in combination with some \u201cexploratory variables\u201d as suggested above. However, their approach may not be feasible for a national-scale study such as ours, because little guiding information is available as to how to identify a set of widely acceptable variables that can be applied to the entire nation. Moreover, even if we could identify a set of exploratory variables, we do not know the forms or shapes of their independent and joint relations to the air pollution measures. Further studies that involve large-scale national data using universal kriging are still needed. In this study, we empirically tested whether the non-constant mean assumption for universal kriging was needed; we performed five regional ordinary krigings so that different parts of the country would assume a different mean PM concentration. Our data suggested that regional and national ordinary kriging performed similarly. Therefore, our data indirectly validated and supported the use of national ordinary kriging.\nAlthough the primary objective of our study is to assess the short-term relationship between PM and cardiac responses, the proposed kriging method also enables us to calculate the long-term cumulative exposure of an individual by taking into account the change of his or her residences over time, because the WHI study recorded the residential location history over 10 years. Nevertheless, from the environmental perspective, an inherited limitation of the kriging-based approach is that the estimations of the PM concentrations will provide only surrogates, or the best guesses, of the true exposure levels at the locations of interest. Thus, the accuracy of the estimations depends highly on the quality of the measured data and their spatial correlation. Even if the estimations were made with a high level of confidence, they cannot be directly interpreted as the true individual-level exposures. However, to correlate individual level cardiac responses with a surrogate of location-specific exposure, our approach represents one of the best available methods for a large-scale population-based study.\nIn summary, our investigation of GIS approaches for estimating daily mean geocoded location-specific air pollutant concentrations and their SEs supports the use of a spherical model to perform lognormal ordinary kriging on a national scale. Our findings also support the use of default-generated semivariograms (estimated using the weighted least-squares method) without visual adjustment. We developed a semiautomated program to access and execute ArcView to implement these approaches for large-scale daily kriging estimations and semivariogram cross-validations. Detailed information about this program can be obtained on request.","keyphrases":["kriging","cross-validation","geographic information systems","population-based studies","particulate air pollution"],"prmu":["P","P","P","P","R"]}
{"id":"Rev_Endocr_Metab_Disord-3-1-1894829","title":"Monogenic diabetes in children and young adults: Challenges for researcher, clinician and patient\n","text":"Monogenic diabetes results from one or more mutations in a single gene which might hence be rare but has great impact leading to diabetes at a very young age. It has resulted in great challenges for researchers elucidating the aetiology of diabetes and related features in other organ systems, for clinicians specifying a diagnosis that leads to improved genetic counselling, predicting of clinical course and changes in treatment, and for patients to altered treatment that has lead to coming off insulin and injections with no alternative (Glucokinase mutations), insulin injections being replaced by tablets (e.g. low dose in HNF\u03b1 or high dose in potassium channel defects -Kir6.2 and SUR1) or with tablets in addition to insulin (e.g. metformin in insulin resistant syndromes). Genetic testing requires guidance to test for what gene especially given limited resources. Monogenic diabetes should be considered in any diabetic patient who has features inconsistent with their current diagnosis (unspecified neonatal diabetes, type 1 or type 2 diabetes) and clinical features of a specific subtype of monogenic diabetes (neonatal diabetes, familial diabetes, mild hyperglycaemia, syndromes). Guidance is given by clinical and physiological features in patient and family and the likelihood of the proposed mutation altering clinical care. In this article, I aimed to provide insight in the genes and mutations involved in insulin synthesis, secretion, and resistance, and to provide guidance for genetic testing by showing the clinical and physiological features and tests for each specified diagnosis as well as the opportunities for treatment.\nIntroduction\nMonogenic diabetes is diabetes that results from one or more mutations in a single gene. The mutation might have arisen de novo and hence be a spontaneous case or might be dominantly or recessively inherited. Molecular genetic testing specifies a diagnosis in 1\u20132% [1]. The mechanism can be easily understood from the underlying pathophysiology. In children, almost all monogenic diabetes results from mutations in genes that regulate \u03b2-cell function and infrequently from mutations resulting in very severe insulin resistance [2]. This article discusses the pathophysiology and clinical manifestations used to select patients eligible to genetic testing, and demonstrates the importance in the treatment of monogenic diabetes.\nWhy diagnose monogenic diabetes?\nMany patients with genetically proven monogenic diabetes were initially incorrectly diagnosed. They received a diagnosis of neonatal diabetes or diabetes diagnosed before the age of 6\u00a0months that was not further specified [3] or diagnosed as type 1 or type 2 diabetes [3\u20135]. It is important to correctly diagnose monogenic diabetes because not only does it help to elucidate the aetiology of the patient\u2019s diabetes and explain other associated clinical features, it can also predict the clinical course of the patient and guide the most appropriate treatment. For instance, patients might not need any treatment or might be able to switch from insulin injections to tablets such as sulfonylurea [6, 7]. Finally a diagnosis has implications for other family members often correcting their diagnosis, prognosis and treatment as well as allowing appropriate genetic counselling.\nNormal insulin release and normal insulin sensitivity\nThe \u03b2-cells form the core of the islets of Langerhans and are responsible for the synthesis and secretion of insulin and C-peptide (Fig.\u00a01, upper part). Insulin\u2019s main function, stimulating glucose uptake in peripheral tissue, is regulated by insulin binding to insulin receptors on the cell membrane of peripheral cells such as muscle cells. This binding initiates an intracellular signalling cascade that leads to an increase in the glucose influx (Fig.\u00a02).\nFig.\u00a01Pancreatic \u03b2-cell and the genes involved in monogenic diabetes. Mutations in different genes result in different phenotypes (Tables\u00a01, 2, 3, and 4). Also, different mutations in the same gene might lead to different phenotypes as shown in the spectrum of phenotypes in for instance Kir6.2.The upper part shows the physiological situation from insulin synthesis to packaging and from glucose sensing to insulin secretion. From centre to right: Insulin synthesis and packaging: Insulin (Ins) is synthesized in the nucleus regulated by transcription factors and after translation in the endoplasmatic reticulum (ER) and Golgi apparatus (Golgi) stored in granules. From left to right down: Glucose sensing and insulin secretion: Glucose enters the \u03b2-cell by passive diffusion facilitated by the glucose-transporter-2 (GLUT2). It is phosphorylated by the enzyme glucokinase (GCK) to glucose-6-phosphate (G6P) and metabolised to ATP via glycolysis or even further via the Krebs cycle in the mitochondria (Mito). ATP closes the KATP channel, preventing K+ efflux, depolarising the cell membrane. Depolarisation opens voltage dependent calcium channels (VDCC) allowing calcium influx. The rise in intracellular calcium (Ca2+) helps the insulin granules to fuse with the cell membrane resulting in insulin secretion.The lower part shows the pathological situation due to mutations in the genes involved in monogenic diabetes. The proteins encoded by the genes involved are given in bold type followed by the clinical presentation if mutated (in brackets and italics). From centre to right: Insulin synthesis is mainly influenced by nuclear transcription factors that may also be involved in pancreatic development and hence mutations may result in pancreatic atrophy (PTF1\u03b1, HNF1\u03b2) or agenesis (IPF1) rather than reduced insulin synthesis per se as in TNDM (ZAC) or MODY (HNF1\u03b1, HNF4\u03b1, NEUROD1). Mutations in genes that are involved in packaging of the insulin into granules in the ER and Golgi apparatus result in TNDM (HYMAI), \u03b2-cell destruction and PNDM (EIF2AK3 in Wolcott Rallison Syndrome\u2014WCRS), diabetes at a mean age of six as part of the Diabetes Insipidus, Diabetes Mellitus, Optic Atrophy and Deafness syndrome (DIDMOAD) alternatively called Wolfram syndrome (WRS) (WSF1), or in diabetes as part of the Thiamine Responsive Megaloblastic Anaemia Syndrome (TRMAS) alternatively called Roger\u2019s syndrome. Mutations in T-lymphocytes may also lead to \u03b2-cell destruction and PNDM as seen with mutations in FOXP3 as part of the Immunodysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX) syndrome. From left to right down: Glucose sensing is reduced by mutations in GLUT2- leading to TNDM as part of the Fanconi-Bickel Syndrome, GCK- resulting in MODY2 and mitochondrial DNA-interfering with oxidative phosphorylation. These latter three mutations reduce ATP and increase ADP leading to a decreased ATP\/Mg-adenosine diphosphate (ADP) ratio that activates the KATP channel to remain open. Activating mutations in the KATP channel itself (Kir6.2\/SUR1) reduce sensitivity to ATP and hence also favour the open state of the channel. The subsequent efflux of potassium prevents depolarisation of the cell-membrane and hence prevents insulin secretionFig.\u00a02Peripheral cell (e.g. muscle cell) showing the impact of diverse mutations that result in insulin resistance. In the physiological situation, insulin binds to the insulin receptor resulting in phosphorylation (P) of a tyrosine (Y) residue of the insulin receptor substrate-1 (IRS1). This phosphorylation activates phosphatidylinositol 3-kinase (PI3K) resulting in glucose transporter 4 (GLUT4) being translocated to the cell membrane which in turn leads to glucose influx. In the pathological situation, mutations in the insulin receptor interfere with insulin receptor synthesis, posttranslational processing and intracellular-transport of the receptor to the cell membrane or lead to reduced binding of insulin, reduced activation or increased degradation of the receptor. The result is no phosphorylation of Y and hence eventually no glucose uptake. This situation occurs in Type A severe insulin resistance, Rabson-Mendenhall and Leprechaunism. In the presence of high levels of triglycerides (TG) and hence free fatty acids (FFA) a serine (S) residue is phosphorylated preventing phosphorylation of Y and hence eventual glucose uptake. This situation occurs in congenital generalised lipoatrophy and familial partial lipodystrophy. In all these pathological situations, higher levels of insulin are needed for glucose uptake and hence lead to reduced insulin sensitivity and insulin resistance\nAbnormal insulin release and abnormal insulin sensitivity\nMost monogenic diabetes in children results from mutations in genes causing \u03b2-cell loss or \u03b2-cell dysfunction and hence affect insulin synthesis, packaging, glucose sensing or insulin secretion (Fig.\u00a01, lower part) [3, 8]. Mutations of pancreatic transcription factors may result in reduced pancreatic growth, (sub)total pancreatic agenesis or reduced insulin synthesis per se and may present as neonatal diabetes (ND), maturity onset diabetes of the young (MODY), or diabetes as part of a syndrome (Fig.\u00a01, lower part centre). Other mutations that affect insulin synthesis are not expressed in the pancreatic \u03b2 cell but in the CD4+ CD25+ regulatory T lymphocytes, where overactivation results in auto-immunity against \u03b2-cells typically causing diabetes in the first 3\u00a0months of life. Mutations affecting translation, cleavage and packaging of insulin may result in reduced \u03b2-cell number or reduced number of granules and lower concentrations of insulin in these granules (Fig.\u00a01 lower part right).\nMutations that affect insulin secretion involve genes that regulate glucose sensing and affect beta-cell function rather than its development or destruction. They include functional mutations in mitochondrial DNA (Fig.\u00a01, lower part left). All these mutations reduce sensitivity to glucose and its metabolism favouring the open state of the potassium channels prevent depolarisation and hence insulin secretion.\nIn children, monogenic diabetes infrequently occurs from mutations resulting in very severe insulin resistance (Fig.\u00a02) [2]. They are mainly caused by mutations in the insulin receptor gene, that affect its synthesis and posttranslational processing, increase receptor degradation, reduce binding of insulin or receptor activation [9]. These give rise to type A severe insulin resistance, Rabson-Mendenhall syndrome or Leprechaunism [2, 9\u201311]. Alternatively, insulin resistance may be the result of hypertriglyceridaemia associated with congenital generalised lipoatrophy or familial partial lipodystrophia [12, 13].\nWhen to consider a diagnosis of monogenic diabetes?\nGiven the limited resources available it is vital that genetic tests are used in situations where they are likely to be positive and will alter clinical care. This will involve careful clinical selection and physiological tests like C peptide and autoantibody measurement as well as examination of other family members before doing molecular genetic tests. Monogenic diabetes should be considered in any diabetic patient who has features inconsistent with their current diagnosis and clinical features of specific subtypes of monogenic diabetes.\nNo specified diagnosis or features inconsistent with current diagnosis\nA diagnosis of diabetes might have been made without further specifying the cause as in neonatal diabetes or diabetes diagnosed below the age of 6\u00a0months. Also, the majority of patients with genetically proven monogenic diabetes are initially incorrectly diagnosed as Type 1 or Type 2 diabetes [5].\nNo specified diagnosis of neonatal diabetes or of diabetes diagnosed below the age of 6\u00a0months\nNeonatal diabetes was defined as insulin requiring diabetes diagnosed within the first 3\u00a0months of life. It is an area that has rapidly transitioned from a clinical to a molecular genetic classification [3, 14]. There is good evidence that diabetes diagnosed in the first 6\u00a0months of life is not type 1 diabetes because neither autoantibodies typical for diabetes nor an excess of high type 1 HLA susceptibility are found in these patients [15]. Diabetes diagnosed before the age of 6\u00a0months irrespective of current age should always lead to further molecular genetic testing. Also because many years after diagnosis, patient and doctor will find difficulty in recalling the exact age of diagnosis and only be aware that it was \u2018at very young age\u2019. Features that help differentiate between the different subtypes and hence guide genetic testing are further described in the section below and in Table\u00a01.\nTable\u00a01Features of diabetes diagnosed before 6\u00a0months of age in addition to undetectable to low C-peptideProtein (Chromosome\/gene; Syndrome)Clinical pictureNumber of cases describedMedian birth weight ingrams SDS (standard deviation score)Median age at diagnosis in weeks (range)Family history reflected by inheritanceOther clinical featuresOther testsTreatment(% in consanguineous or isolated populations)Pancreatic appearance (present\/size)\u2022 ZAC\/HYMAI (6q24 imprinting defect)TNDM\u00b1150 (rare)2,100 (\u22122.94)0.5 (0\u20134)- Macroglossia (23%)NormalInsulin\/pump\u2009>\u2009relapse: diet\u2009>\u2009insulin\u2022 Kir6.2 (KCNJ11)TNDM10% PNDM90%\u00b1100 (rare)2,580 (\u22121.73)6 (0\u2013260)- Spontaneous- DKA (30%)NormalHigh dose sulfonylurea- Dominant(10%)- Developmentaldelay 20%)- Epilepsy (6%)\u2022 PTF1A(10p13-12)PNDM3 (100%)1,390 (\u22123.8)Recessive- Severeneurological dysfunctionAtrophyInsulin\/pump- Cerebellar hypoplasia\u2022 IPF1 (13q12.1)PNDM2 (50%)2,140 (\u22122.97)- RecessiveNo pancreasInsulin\/pump- Parents may have early onset diabetes as heterozygotes\u2022 HNF1\u03b2 (179)TNDMRare1,900 (\u22123.21)- Dominant (60%)- Renal development disordersAtrophyInsulin\/pump- Spotaneous\u2022 EIF2AK3(2p;Wolcott-Rallison Syndrome)PNDM30 (90%)13 (6\u201365)- Recessive- Epiphyseal dysplasia (90%)Exocrine dysfunctionInsulin\/pump- Developmental delay (80%)- Acute liver failure (75%)- Osteopenia (50%)- Hypothyroidism (25%)\u2022 FOXP3 (Xp11.23; IPEX Syndrome)PNDM14 (rare)2,860 (\u22121.2)6 (0\u201330)X-linked Hence only boys affected- Chronic diarrhoea with villous atrophy (95%)Insulin\/pump- Pancreatic and thyroid autoantibodies (75%)- Eczena (50%)- Anaemia (30%)- Thyroiditis (20%)- Often die in first year\u2022 GLUT2 (3q; Fanconi Bickel Syndrome)TNDMRecessive- Impaired utilisation of glucose and galactoseInsulin\/pump- Hepatorenal glycogen accumulation- Proximal renal tubular dysfunction\u2009>\u2009glucosuria\u2022 Glucokinase (GCK11 homozygote)PNDM6 (85%)1,720 (\u22122.75)- RecessiveNormalInsulin\/pump- Parents have fasting hyperglycaemia as heterozygotes\nClinical features that are unusual for type 1 diabetes\nType 1 diabetes shares clinical features with certain types of monogenic diabetes, such as young age of diagnosis, normal body weight and insulinopaenia [5] so that a monogenic diagnosis can be overlooked. Features in children initially thought to have type 1 diabetes but that should suggest a possible diagnosis of monogenic diabetes are shown below. None of these are absolute and should be considered collectively rather than in isolation [4]. The approximate percentage of patients with type 1 diabetes is given in brackets. \nAge of diagnosis below 6\u00a0months after birth (<1%) [15]Family history with a parent affected (2\u20134%) [16]Endogenous insulin production after 3\u00a0years of diabetes (the honeymoon phase), indicated by detectable C-peptide (>200\u00a0nmol\/l) in response to raised glucose (>8\u00a0mmol\/l) (1\u20135%).Absence of islet autoantibodies, especially when measured at diagnosis (3\u201330%) [17, 18]. The great variation in antibody prevalence in series probably represents differences in assays and means it is hard to apply published series directly into clinical practice. Absent antibodies should lead to other investigation\/consideration rather than leading directly to genetic tests.\nClinical features that are unusual for type 2 diabetes\nType 2 diabetes in children and young adolescents might share features which meet former classification criteria for MODY such as diagnosed <25\u00a0years, autosomal dominant inheritance and non-insulin dependency [19\u201322]. Patients might have a rather low body mass index and have other features that are unusual for type 2 diabetes. These should raise suspicion for the current diagnosis to be correct as chances for these features occurring in type 2 diabetic children or adolescents are low as shown here (percentage shown in brackets). \nNo evidence of insulin resistance with fasting C-peptide within the normal range (0\u201320%) [19\u201322].Ethnic background from a low prevalence type 2 diabetes race e.g. European Caucasian (0\u201345%) [19\u201322]Acanthosis nigricans not detected (10%) [19]Not markedly obese or diabetic family members who are normal weight (20%) [22].\nClinical features of specific subtypes of monogenic diabetes and their treatment\nTypical clinical presentations in children when a diagnosis of monogenic diabetes should be considered can mainly be classified into four categories that are further discussed below: \nNeonatal diabetes and diabetes diagnosed within the first 6\u00a0months of life (Table\u00a01)Familial diabetes with an affected parent (autosomal dominant) (Table\u00a02 upperpart)Mild (5.5\u20138.5\u00a0mmol\/l) fasting hyperglycaemia especially if young or familial (Table\u00a02 lower part).Features in other tissues as part of genetic syndromes associated with diabetes (Tables\u00a03 and 4)\nNeonatal diabetes and diabetes diagnosed before the age of 6\u00a0months irrespective of current age\nDiabetes diagnosed before the age of 6\u00a0months is most likely to have a genetic cause, and further clinical aspects should be investigated to guide genetic testing. Clinically two subgroups were recognised: transient neonatal diabetes mellitus (TNDM) that resolved at a median of 12\u00a0weeks and then did not require any treatment although as many as 50% of cases relapsed during the paediatric age range [23, 24]. In contrast permanent neonatal diabetes mellitus (PNDM) required lifelong insulin injections from diagnosis onwards. The majority of patients with TNDM have an imprinting abnormality of the transcription factor ZAC gene and HYMAI gene encoding an untranslated RNA chromosome 6q (Table\u00a01) [14, 23]. Apart from macroglossia seen in 23% there are no non-pancreatic features [23]. The second commonest cause of mutations in patients with diabetes diagnosed before the age of 6\u00a0months of life are mutations in the KCNJ11 gene encoding the Kir6.2 subunit of the KATP channel and that can result in either TNDM (10%) or PNDM (90%) [3, 25\u201327]. Despite being a heterozygous mutation most have no family history because 90% of cases are spontaneous mutations. Most patients have isolated diabetes although neurological features are seen in 20% of patients. The most severe is the Developmental delay early onset generalised Epilepsy and Neonatal Diabetes (DEND) syndrome [25]. More common is the intermediate DEND syndrome where patients have less severe developmental delay and do not have early onset generalised epilepsy and that is often associated with the V59M mutation [3, 26]. Recently, activating mutations in the ABCC8 gene encoding the SUR1 subunit of the KATP channel were found to be a similar cause of neonatal diabetes [28]. While the majority of cases in PNDM are the result of mutations in the KCNJ11 gene, in a small minority of patients, many different genetic mutations have been described [3]. If both parents are glucose intolerant, homozygous or compound heterozygous mutations in glucokinase are most frequent [29, 30]. Features that help differentiate between TNDM, PNDM and the different subtypes, guide which gene to test for and are described in Table\u00a01.\nConcerning treatment, imprinting abnormalities of the 6q24 locus show initially very high glucose values (range 12\u201357\u00a0mmol\/l) and so insulin is used initially although the dose can rapidly be reduced. Once patients have relapsed patients should remain under annual follow up due to the risk of diabetes relapsing. Relapse patients are not insulin dependent and can be treated with diet initially although subsequently often need insulin [14]. The long-term response to oral treatment such as sulfonylurea or metformin is uncertain.\nPatients with Kir6.2 mutations have all the clinical features of insulin dependency as 30% present with ketoacidosis and they usually do not have detectable C peptide and so were treated with insulin [26]. It has recently been shown that these patients cannot only be successfully treated with oral sulfonylureas but can also get better glycaemic control without an increase in hypoglycaemia and with neurological features improving as well [70]. The doses needed are high when calculated on a per kg body weight basis compared to adults, with patients typically needing 0.5\u00a0mg glibenclamide\/kg\/day although some may need as much as 1\u00a0mg\/kg\/day [31\u201336, 70]. With time many patients have been able to reduce their doses of sulfonylurea while maintaining excellent glycaemic control [7, 70]. Similar results have recently been found for patients with activating SUR1 mutations [37]. All other causes need to be treated with insulin. Some paediatricians find these patients are best managed on subcutaneous insulin pumps due to the fluctuations in glucose levels. In patients with pancreatic aplasia exocrine pancreatic supplements will additionally be required.\nFamilial diabetes with an affected parent\nChildren and young adults with a strong family history of diabetes\nGenetic causes of a family history of diabetes are autosomal dominant and non-insulin dependent. A diagnosis of Maturity Onset Diabetes of the Young (MODY) should be considered whenever a parent has diabetes even if they are thought to have type 1 or type 2 diabetes (Table\u00a02). MODY comes in two major subgroups: (1) the result of transcription factor mutations (IPF1-MODY4, HNF1\u03b2-MODY5, HNF1\u03b1-MODY3, HNF4\u03b1-MODY1, NeuroD1-MODY6 or CEL-MODY7) described in this section and (2) mutations reducing glucose sensing (glucokinase mutations-MODY2) in which a family history might be less evident because it only presents as mild hyperglycaemia as described in the following section. MODY due to transcription factor mutations often present in adolescence\/young adulthood, show progressive hyperglycaemia and frequently lead to complications. Of the transcription factor mutations, MODY3\u2014due to HNF1\u03b1 mutations\u2014is the commonest form [38]. The clinical characteristics of patients with HNF1\u03b1 mutations are: \nYoung onset diabetes that shows characteristics of not-being insulin dependent e.g., do not develop keto-acidosis in the absence of insulin, achieve good glycaemic control on a small dose of insulin. Detectable C-peptide is measured when on insulin with glucose >8\u00a0mmol\/l after 3\u00a0years of diabetes (the honeymoon period).Family history of diabetes. This might be insulin treated and considered to be type 1 diabetes. This would typically be diagnosed at their 20s, 30s or 40s. There may also be an affected grandparent although often these are diagnosed after 45\u00a0years.Glucosuria at relatively normal blood glucose levels are often seen as these patients have a low renal threshold [39].Oral glucose tolerance tests in early stages tend to show a very large glucose increment usually >5\u00a0mmol\/l [39]. Some patients might have a normal fasting value while still rise into the diabetic range at 2\u00a0h.Marked sensitivity to sulfonylurea resulting in hypoglycaemia despite poor glycaemic control before starting sulfonylurea [40,41].Table\u00a02Familial diabetes diagnosed, or undiagnosed due to mild hyperglycaemiaGene\/proteinClinical pictureNumber of cases describedMedian age at diagnosis in weeks (range)Family history reflected by inheritanceOther clinical featuresOther testsTreatmentGlucose at presentation in mmol\/l Median (range)OGTTFamilial diabetes diagnosedHNF-1\u03b1MODY319714 (4\u201318)DominantHyperglycaemia is rapidly progressive with age17 (11\u201326)Large increment (0\u00a0h\u20132\u00a0h usually >5\u00a0mmol\/l)Diet\u2009>\u2009low dose of sulfonylureaLow renal threshold\u2009>\u2009glucosuriaSensitive to sulfonylureaHNF-4\u03b1MODY12217 (5\u201318)DominantHyperglycaemia is rapidly progressive with age15 (9\u201320)Large increment (0\u00a0h\u20132\u00a0h usually >5\u00a0mmol\/l)Low dose of sulfonylureaNormal renal thresholdSensitive to sulfonylureaReduced levels of apoAIII, apoCIII, and triglyceridesOther unusual causes: IPF1 (MODY4), NeuroD1 (MODY6), CEL (MODY7)Familial diabetes undiagnosed due to mild fasting hyperglycaemiaGlucokinase (GCK, heterozygous)MODY215210 (0\u201318)DominantHyperglycaemia is mild (fasting 5.5\u20138\u00a0mmol\/l)11 (5.5\u201316)Small increment (0\u00a0h\u20132\u00a0h usually <3.5\u00a0mmol\/l)No treatment(The mild hyperglycaemia might not have been diagnosed in relatives\/parents)Hyperglycaemia is only slowly progressive with age> usually diagnosis is by incidental findingNormal renal threshold\nPatients with HNF1\u03b1 gene mutations can initially be treated through diet although they will have marked postprandial hyperglycaemia after high carbohydrate food as the \u03b2-cell defect results in insufficient increase in insulin secretion with hyperglycaemia [42]. Most patients will need pharmacological treatment as they show progressive deterioration in glycaemic control throughout life and are at risk of considerable micro-vascular and macro-vascular complications [43]. The first treatment to be used in children who are not controlled on insulin should be low dose sulfonylureas which results in a 4-fold greater lowering of glucose than metformin [41]. These patients are extremely sensitive to sulfonylurea and as long as they do not have problems with hypoglycaemia can be maintained on these for many decades [40]. Glycaemic control in sulfonylureas is often better than that achieved on insulin especially in children and young adults [44]. The dose of sulfonylureas should initially be low (1\/4 of the normal starting dose in adults) to avoid hypoglycaemia. If there is hypoglycaemia despite dose titration of a once or twice daily sulfonylurea preparation such as Gliclazide, a slow release preparation or mealtime doses with short-acting agents like nateglinidine may be considered [45].\nMutations in the hepatocyte nuclear factor 4\u03b1 (HNF4\u03b1)-gene resulting in MODY1 are considerably less common, have similar characteristics but might be diagnosed later and patients have no low renal threshold [46]. They also lead to reduced levels of triglycerides and the apolipoproteins apoAIII and apoCIII [47]. Diagnosis should be considered when HNF1\u03b1 tests negative while the clinical features are strongly suggestive for HNF1\u03b1 [46]. Patients with HNF4 \u03b1 mutations are usually sensitive to sulfonylurea [48].\nThe handful of families with autosomal dominant non-insulin dependent diabetes that have further been described include mutations in IPF1 (MODY4) [49], NeuroD1 (MODY6) [50,51], and recently the carboxyl ester lipase (CEL) gene (MODY7) [52], but these are so unusual they do not need to be tested for in children with diabetes except in a research setting or when there are additional phenotypes such as pancreatic exocrine dysfunction [52]. IPF has been described under neonatal diabetes, NeuroD1 encodes a transcription factor that binds to a critical Ebox motif on the insulin promoter and plays a role in both pancreatic and neuron development.\nMild (5.5\u20138.5\u00a0mmol\/l) fasting hyperglycaemia especially if young or familial\nRaised fasting blood glucose in the range of 5.5 to 8.5\u00a0mmol\/l is unusual in children and young adults. This always raises concern that they may be about to develop type 1 diabetes or have type 2 diabetes. However a considerable proportion of these patients with persistent mild fasting hyperglycaemia will have a heterozygous mutation in the glucokinase gene. The phenotype associated with glucokinase mutations is remarkably similar for all mutations with the following features suggesting a diagnosis. \nThe fasting hyperglycaemia is persistent and stable over a period of months or years [39]HbA1C is typically just below or just above the upper limit of normal (5.5 to 5.7%)In an oral glucose tolerance test the increment (2\u00a0h glucose-fasting glucose) is small (typically <3.5\u00a0mmol\/l) although because of the variability of the oral glucose tolerance test this should not be considered an absolute criteria [39].Parents may have \u2018type 2 diabetes\u2019 or may not be diabetic. On testing one parent will have a mildly raised fasting blood glucose, in the range of 5.5\u20138.5\u00a0mmol\/l, as this is an autosomal dominant condition [39]. Testing of fasting glucose in apparently unaffected parents is important when considering a diagnosis of a glucokinase mutation.\nThe fasting hyperglycaemia does not deteriorate significantly and the glucose is regulated at the higher set point [39]. As this is rarely associated with any microvascular or macrovascular complications even when no treatment is given throughout life and as there is very little if any response to either oral hypoglycaemic agents or insulin because the set point remains the same, these patients do not need treating in the paediatric age range [53].\nGenetic syndromes associated with diabetes\nWhen diabetes in a child is associated with other multi-system disease the possibility of a monogenic syndrome that explains all features should be considered. The online Mendelian inheritance in Man (OMIM) website (access through the NCBI website http:\/\/www.ncbi.nlm.nih.gov\/entrez\/query.fcgi) can help with clinical features and to know if the gene has been defined and hence molecular genetic testing is available. For described and previously undescribed syndromes see the ISPAD rare diabetes collection (contact through link on the ISPAD website or through http:\/\/www.diabetesgenes.org). The most common genetic syndromes that include diabetes are described below and in Tables\u00a03 (insulin synthesis\/secretion) and 4 (insulin resistance). \nTable\u00a03Syndromic features in addition to the diabetes: insulin synthesis\/secretionGene\/proteinClinical pictureNumber of cases describedMedian age at diagnosis in weeks (range)Family history reflected by inheritanceOther clinical featuresTreatmentHNF1\u03b2Rarely isolated PNDM or MODY 5- Renal developmental disorders, especially renal cysts and dysplasiaInsulin (+possibly treat exocrine deficiency?)HNF1\u03b2Renal cysts and diabetes syndrome (RCAD)- Uterine and genitalia developmental anomalies- Hyperuricaemia, gout- Abnormal liver function testsWSF1Diabetes insipidus, diabetes mellitus, optic atrophy, deafness (DIDMOAD) syndrome\/Wolfram syndrome (90% have mutations)Especially where consanguineous marriages are frequent6\u00a0years(Most <16\u00a0years)Dominant- Diabetes insipidus- Optic atrophy - Bilateral sensorineural deafness- Dilated renal tracts- Truncal ataxia- Protean neurological signs 75% has the complete phenotype, increasing with increasing ageInsulinSLC19A2(Thiaminetransporter protein)Thiamine responsiveMegaloblastic anaemia (TRMA) syndrome Roger\u2019s syndromeRareRecessive- Thiamine responsive megaloblastic anaemia- Sensorineural deafnessThiamine\u2009>\u2009insulin\u00a0\u00a0\u00a0tRNA(leu(UUR)) gene (3243 A to G; tRNA)- Maternally inherited diabetes (MID)- Mitochondrial myopathy, encephalopathy, lactic acidosis, stroke-like syndrome (MELAS)- Sensorineural deafness- Short stature- Subclinical exocrine deficiency- HeteroplasmyInsulinTable\u00a04Syndromic features in addition to the diabetes: insulin resistanceProteinClinical pictureMedian age at diagnosis in weeks (range)Family history reflected by inheritanceOther clinical featuresOther features\/testsTreatmentAcanthosis nigricansInsulin levelsAndrogen excess and hypertrichosisInsulin receptorType AAdolescenceRecessive (usually)Insulin resistance in absence of obesityYes\u2014marked\u2191\u2191\u2191\u2191\u2191\u2191\/PCO(Metformin\/glitazones)\u2009>\u2009insulin\/pumpInsulin receptorRabson-MendenhallCongenitalRecessive (usually)- Abnormal dentitionYes\u2014marked\u2191\u2191\u2191\u2191\u2191\/PCO(Metformin\/glitazones)\u2009>\u2009insulin\/pump- Extreme growth retardationInsulin receptorLeprechaunism (Donahue syndrome)CongenitalRecessive (usually)- Abnormal faciesYes\u2014marked\u2191\u2191\u2191\u2191\u2191\u2191\/PCO(Metformin\/glitazones)\u2009>\u2009insulin\/pump- SGA and growth retardation- Large genitalia- Rarely survive infancySeipin&AGPAT2Total lipodystrophyAdolescence or congenitalRecessive- Total loss of subcutaneous fatYes\u2014may be marked\u2191\u2191\u2191\u2191\u2191\/PCO+\/\u2212Recombinant \/insulinLamin AC&PPAR\u03b3Partial lipodystrophyDominant- Partial loss of subcutaneous fatMetformin\u2009>\u2009insulin\nMODY-5 due to an HNF-1\u03b2 mutation (renal cysts and diabetes syndrome\nThe transcription factor hepatocyte nuclear factor 1 \u03b2 is expressed in primitive pancreatic duct cells involved in early endocrine cell differentiation incidentally leading to PNDM, but mainly leading to MODY5 with pancreatic atrophy as a consistent finding when assessed by abdominal computed tomography [54, 55]. Patients with HNF-1\u03b2 mutations rarely present with isolated diabetes [56]. Renal developmental disorders especially renal cysts and renal dysplasia are present in almost all patients with mutations or gene deletions [57]. These may be diagnosed in utero and precede the diagnosis of diabetes. A diagnosis of HNF-1\u03b2 should be considered in any child with diabetes who also has non-diabetic renal disease. Other features are presented in Table\u00a03. Patients with HNF1-\u03b2 mutations, unlike patients with HNF-1\u03b1 mutations, are not sensitive to sulfonylurea and hence usually require insulin treatment [58]. Pancreatic size is reduced reflecting a reduction in both the endocrine and exocrine pancreas and sub-clinical exocrine deficiency is present in most patients [57] but it is uncertain if this should be treated if it is asymptomatic.\nWolfram or DIDMOAD syndrome (diabetes insipidus, diabetes mellitus, optic atrophy and deafness)\nThis autosomal recessive syndrome shows mutations in the gene for Wolfram syndrome (WFS1) in at least 90% of patients [59\u201361], and the association of diabetes with progressive optic atrophy below 16\u00a0years of age is diagnostic [62]. Other features are described in Table\u00a03 with the complete phenotype seen in 75% of patients with increasing prevalence with age. The order of appearance of the neurological features may vary, even within families. Patients with Wolfram syndrome die at a median age of 30\u00a0years [62]. The diabetes is non-autoimmune and insulin deficient and presents at a mean age of 6\u00a0years [62]. Patients require insulin treatment from the time of diagnosis but autoantibodies are not present [62].\nRoger\u2019s or TRMA syndrome (thiamine responsive megaloblastic anaemia)\nThe diabetes in this rare recessive genetic syndrome, is insulin deficient in nature and both the diabetes and the anaemia are responsive to thiamine. However, all seem to develop an insulin requirement in the long term [63]. Further details are described in Table\u00a03.\nMitochondrial diabetes\nMaternal transmission of mutated or deleted mitochondrial DNA (mtDNA) can result in maternally inherited diabetes [64] although they do not usually occur in the paediatric age range. Patients suffer from sensorineural deafness and short stature. In family members there might be some overlap between MIDD and MELAS syndrome [65, 66]. The heteroplasmy in proportions of mitochondrial DNA affected lead to a variety of phenotypes. The diabetes is characterised by progressive non-autoimmune \u03b2-cell failure, usually manifests outside the paediatric age range, and may progress to needing insulin treatment rapidly. Other features are described in Table\u00a03.\nInsulin resistance syndromes\nThe key features of all insulin resistance syndromes are acanthosis nigricans, massively raised insulin concentrations, and androgen excess while obesity is absent (Table\u00a04) [2]. The more severe the insulin resistance and the earlier the onset, the more likely is diabetes. The mechanism of the main insulin resistance syndromes, Type A, Rabson-Mendenhall, Leprechaunism and Lipodystrophy, is shown in Fig.\u00a02 while the key clinical features of the main insulin syndromes, are presented in Table\u00a04 [2]. Treatment of severe insulin resistance is very difficult and most patients with diabetes have poor glycaemic control and frequently develop long-term complications [2]. Treatment approaches include the use of the insulin sensitisers metformin and glitazones, but effectiveness is limited when the insulin resistance is very severe. Insulin is the main stay of treatment and U500 insulin and insulin pumps are usually required [2]. In partial lipodystrophy metformin may have benefit and insulin is not required in the early stages [67]. In total lipodystrophy the response of diabetes to recombinant lipodystrophy [68] can be dramatic but is only available on a research basis.\nTesting for a molecular monogenic diagnosis\nHow to test for monogenic diabetes?\nWhile in type 1 and type 2 diabetes there is no single diagnostic test, this is not the case in monogenic diabetes where in >80% of cases a molecular genetic diagnosis can be made by DNA testing. These are offered by many laboratories but should preferably be performed in a laboratory that is experienced and specialised in such diagnosis irrespective whether this requires sending samples abroad (http:\/\/www.diabetesgenes.org). Some recently described monogenic genes, such as Kir6.2 in patients in whom diabetes was diagnosed before the age of 6\u00a0months, can be screened for with no charge (http:\/\/www.diabetesgenes.org). Other tests are more expensive (up to 500 Euro or 635 USD) but make future patient care cheaper and hence testing would be cost-effective. For example, in a Swiss centre yearly costs for type 1 diabetes was approximately 2050 Euro (2603 USD) per type 1 patient compared to 410 Euro (521 USD) per MODY2 patient [69]. Approval from the patient\u2019s insurance company should be sought prior to sending DNA when applicable. Future analyses will show cost-effectiveness for the different subtypes and prove earlier diagnosis to be more beneficial.\nWhat if a monogenic diagnosis cannot be made?\nOccasionally molecular genetic test results are negative, despite unusual clinical features or typical features for a certain monogenic subtype. The certainty of such a negative result increases if a specialised centre performed testing. Even then, some cases remain unsolved. These can be referred to the ISPAD rare cases registry (see website or contact a.t.hattersley@exeter.ac.uk) to allow pattern finding and closer investigation by experts if a novel idea evolves, hence increasing chances for new and future insights, novel diagnoses and improved patient care.\nSummary\nMolecular genetic testing can define a diagnosis in 1\u20132% of all diabetic patients with monogenic diabetes. Advances in this field have led to the identification of the genes associated with many clinically identified subgroups of diabetes and explained clinical heterogeneity in conditions defined by age of diagnosis e.g. neonatal diabetes and MODY. Molecular genetic tests are now available to help define the diagnosis, and importantly alter prognosis and optimise treatment of children, young adults and their families with diabetes. As these tests are expensive genetic testing should be limited to those who on clinical grounds are likely to be positive. Considering testing for monogenic diabetes is hence a challenge and should be guided by unusual features of the current diagnosis, specific features concordant with monogenic subtypes and by the possibility of a change in treatment. This article discussed the pathophysiology and clinical manifestations used to select eligible patients and guide genetic testing, and demonstrates its importance in the treatment of monogenic diabetes.","keyphrases":["monogenic diabetes","kir6.2","insulin resistance syndrome","neonatal diabetes","gck","mody"],"prmu":["P","P","P","P","P","P"]}
{"id":"Arch_Sex_Behav-3-1-2042031","title":"Characteristics of Sexual Abuse in Childhood and Adolescence Influence Sexual Risk Behavior in Adulthood\n","text":"Childhood and adolescent sexual abuse has been associated with subsequent (adult) sexual risk behavior, but the effects of force and type of sexual abuse on sexual behavior outcomes have been less well-studied. The present study investigated the associations between sexual abuse characteristics and later sexual risk behavior, and explored whether gender of the child\/adolescent moderated these relations. Patients attending an STD clinic completed a computerized survey that assessed history of sexual abuse as well as lifetime and current sexual behavior. Participants were considered sexually abused if they reported a sexual experience (1) before age 13 with someone 5 or more years older, (2) between the ages of 13 and 16 with someone 10 or more years older, or (3) before the age of 17 involving force or coercion. Participants who were sexually abused were further categorized based on two abuse characteristics, namely, use of penetration and force. Analyses included 1177 participants (n=534 women; n=643 men). Those who reported sexual abuse involving penetration and\/or force reported more adult sexual risk behavior, including the number of lifetime partners and number of previous STD diagnoses, than those who were not sexually abused and those who were abused without force or penetration. There were no significant differences in sexual risk behavior between nonabused participants and those who reported sexual abuse without force and without penetration. Gender of the child\/adolescent moderated the association between sexual abuse characteristics and adult sexual risk behavior; for men, sexual abuse with force and penetration was associated with the greatest number of episodes of sex trading, whereas for women, those who were abused with penetration, regardless of whether the abuse involved force, reported the most episodes of sex trading. These findings indicate that more severe sexual abuse is associated with riskier adult sexual behavior.\nIntroduction\nChildhood and adolescent sexual abuse has been associated with a wide variety of adverse mental and physical health outcomes. Research also suggests that the greater the severity of the sexual abuse, the worse the health outcomes. Thus, more severe sexual abuse (e.g., sexual abuse involving force, more intimate sexual acts, a close relative, or repeated sexual abuse) has been associated with poorer social adjustment, less life satisfaction, and more severe psychological symptoms (Callahan, Price, & Hilsenroth, 2003; Carlson, McNutt, & Choi, 2003; Fassler, Amodeo, Griffin, Clay, & Ellis, 2005; Feinauer, Mitchell, Harper, & Dane, 1996). In a meta-analysis on the effects of child sexual abuse, Rind, Tromovitch, and Bauserman (1998) found that force was associated with more negative reactions but not with later psychological symptoms, whereas penetration was unrelated to these outcomes.\nSexual abuse severity also has been associated with subsequent sexual risk behavior, including more sexual partners (Merrill, Guimond, Thomsen, & Milner, 2003) and greater likelihood of sex with someone just met, earlier age at first intercourse, and a higher frequency of STD diagnoses (Walser & Kern, 1996). Although these studies suggest that more severe sexual abuse is associated with more sexual risk behavior, they provide only limited information regarding whether specific aspects of the abuse predict such outcomes. Needed is more fine-grained research to determine whether characteristics of the abuse experience (e.g., whether physical force was used during the abuse and the type of sexual act that occurred during the abuse) are associated with sexual health outcomes.\nTwo studies have examined the association between sexual abuse with force and later sexual risk behavior. Cinq-Mars, Wright, Cyr, and McDuff (2003) found that adolescent girls who experienced childhood or adolescent sexual abuse with force were more likely than girls who were sexually abused without force to have engaged in subsequent consensual sex during adolescence, to have more than one partner per year, and to have been pregnant. In a sample of men who have sex with men (MSM), Jinich et\u00a0al. (1998) reported that sexual abuse perceived as moderately or strongly coerced was associated with higher frequency of unprotected anal sex and higher HIV seroprevalence rates, relative to MSM who perceived the sexual abuse as voluntary or mildly coerced. Thus, in these two studies, sexual abuse involving force has been associated with greater sexual risk behavior.\nInvestigations of the effects of penetrative (vs. non-penetrative) sexual abuse have yielded mixed results. Cinq-Mars et\u00a0al. (2003) found that adolescent girls who experienced sexual abuse involving penetration were more likely to have engaged in consensual sex and experienced an unplanned pregnancy, compared to girls who experienced non-penetrative sexual abuse. Fergusson, Horwood, and Lynskey (1997) found that, compared to participants reporting no sexual abuse, those who experienced non-penetrative sexual abuse reported higher rates of unprotected sex. However, those who experienced penetrative sexual abuse reported the worst outcomes; compared to participants who were not sexually abused, those who reported penetrative sexual abuse were more likely to have been pregnant, to report more than five sexual partners by age 18, to have had unprotected sex, and to have had an STD.\nIn contrast, in a meta-analysis, Arriola, Louden, Doldren, and Fortenberry (2005) found that the effect size for the relation between sexual abuse and later sexual behavior (i.e., unprotected sex, sex with multiple partners, and sex work) did not differ for studies including non-contact abuse, studies including contact abuse only, and studies including penetration abuse only; their findings suggest that type of sexual act during sexual abuse was not associated with later sexual behavior. However, some of these categories included very few studies (e.g., there were only three studies that included penetration abuse only). Furthermore, effects may have been obscured if studies with less restrictive definitions of sexual abuse included a large number of participants who had exper- ienced more severe (i.e., contact or penetrative) sexual abuse.\nIn sum, evidence from a small number of studies suggests that force and penetration may be associated with adult sexual risk behavior. An important limitation of previous research is that few studies have investigated the differential effects of sexual abuse for men and women. Because women in heterosexual relationships often have less control or power over sexual encounters compared to men (see the Theory of Gender and Power, Connell, 1987, for an explanation of the power imbalance between men and women), it is important to study gender in relation to sexual health behaviors. Indeed, the limited research on this topic suggests that the association between sexual abuse and adult sexual behavior differs by gender (e.g., Futterman, Hein, Reuben, Dell, & Shaffer, 1993; Mason, Zimmerman, & Evans, 1998; Zierler et\u00a0al., 1991). It is possible that gender interacts with abuse characteristics to lead to different outcomes for males and females, an idea which is supported by research on the psychological sequelae of sexual abuse. For example, in a meta-analysis, Rind et\u00a0al. (1998) found that whether or not the sexual abuse experience was consensual was associated with later psychological adjustment for men, but not for women. Few studies have investigated the effects of the interaction of gender and abuse characteristic on later sexual behavior. Overall, previous studies investigating the association between abuse characteristics and later sexual behavior have tended to use small samples, or included only males or only females, thus precluding gender comparisons.\nThe primary purpose of this study was to determine whether use of force and type of sexual act was associated with sexual risk behavior in a group of patients receiving outpatient care from a sexually transmitted disease (STD) clinic. Based on previous research, we hypothesized that: (1) the use of force; and (2) sexual abuse involving penetration would be associated with greater sexual risk behavior. The secondary purpose of this study was to determine whether the effects of abuse characteristics on adult sexual behavior differed by gender.\nMethod\nParticipants\nParticipants were men and women attending a public STD clinic in upstate New York. All had been screened for possible inclusion in a randomized controlled trial (RCT) evaluating several different sexual risk reduction programs. Screening criteria for the RCT included: age 18 or older; not HIV positive; and sexual behavior (e.g., unprotected sex, multiple partners) that put them at risk for contracting an STD in the past 3 months. This study used baseline data from the RCT, prior to the receipt of the interventions. Baseline data were available from 1265 eligible participants. Data from participants who refused to answer sexual abuse (n=12) or demographic (n=1) questions, were inconsistent in reporting their sexual behavior (n=5), were outliers on sexual behavior data (n=30; defined as having a studentized deleted residual >4), or were recruited in error (n=1) were eliminated. Outliers on sexual behavior data were excluded because these individuals likely were members of an extremely high-risk population that merits separate investigation (Wegener & Fabrigar, 2000).\nOverall, the sample was 46% female (n=557), 65% African American (n=785), and 24% Caucasian (n=294). The majority of participants were unemployed (n=620; 51%), had a high school education or less (n=762; 63%), and had a household income of less than $15,000 per year (n=686; 57%). Most participants were single (never married; n=958; 79%); 75 participants (6%) were married, and 183 (15%) were divorced, separated, or widowed. Participants were, on average, 29.2 years of age (SD=9.7). Among women, 504 (90%) reported having sex with only men in the past 3 months; 53 (10%) reported having sex with both men and women. Among men, 612 (93%) reported having sex with only women in the past 3 months; 31 (5%) reported having sex with only men; and 15 (2%) reported having sex with both men and women in the past 3 months.\nProcedure\nPatients who registered for a clinic visit were invited to a private exam room by a trained research assistant (RA), and were asked to answer a series of brief screening questions. The RA explained the study to patients who met eligibility criteria and obtained informed consent. Participants then completed a 45-minute, Audio Computer-Assisted Self-Interview (ACASI) that included measures of demographic characteristics, health behaviors and beliefs, and psychosocial functioning, as well as questions about childhood sexual experiences and current sexual behavior. ACASI was used because it optimizes participant privacy (improving data quality) while allowing low-literacy persons to participate (Schroder, Carey, & Vanable, 2003). For the present study, we used data from measures of childhood\/adolescent sexual abuse and sexual behavior. After their clinic exam and counseling, participants were paid $20 to compensate them for their time. All procedures were approved by the IRBs of the participating institutions.\nMeasures\nChildhood\/adolescent sexual abuse\nThree items, adapted from Finkelhor\u2019s (1979) longer survey of childhood sexual experiences, were used to assess sexual abuse (see Appendix A).1 Participants who reported any contact sexual experiences (including kissing, fondling, giving oral sex, receiving oral sex, vaginal sex, or anal sex) (1) before age 13 with someone 5 or more years older or (2) between ages 13 and 16 with someone 10 or more years older, and those who reported (3) any contact sexual experience before age 17 involving force or coercion, were classified as sexually abused; all other participants were classified as not sexually abused. Those who were sexually abused were further categorized according to whether the abuse involved force and\/or penetration. Participants who reported a sexual experience before age 17 involving force or coercion were considered to have experienced sexual abuse with force. Sexually abused participants who reported any oral, vaginal, or anal sex were considered to have experienced sexual abuse with penetration. A single, four level categorical variable was created to examine the impact of different abuse characteristics on sexual risk behavior: (1) no sexual abuse; (2) sexual abuse without force or penetration; (3) sexual abuse with penetration but without force; and (4) sexual abuse with both force and penetration. Too few participants reported sexual abuse with force and without penetration (n=39, 5%) to include a sexual abuse with force only category.\nCurrent sexual behavior\nThe sexual risk behavior items were developed and tested in previous studies (Carey et\u00a0al., 1997, 2000, 2004). Participants were asked to report: the number of male and female sexual partners they had in their lifetime and in the past 3 months; the number of times they exchanged sex for money or drugs (lifetime); and the number of times they had been treated for an STD (lifetime).\nThe frequency of unprotected sex was also investigated. Participants were asked to report the number of times in the past 3 months that they had vaginal and anal sex with and without a condom with their: (1) steady partner; (2) other male partners; and (3) other female partners. Responses to these items were used to calculate the absolute number and the proportion (number of unprotected sex episodes\/number of protected and unprotected sex episodes) of unprotected sex episodes in the past 3 months.\nTable 1Sexual abuse characteristics by genderMen (n=643)Women (n=534)Total (n=1177)n% of menn% of womenn% of totalNo sexual abuse227351823440935Sexual abuse without force and without penetration10016591115914Sexual abuse with penetration only (no force)208321052031327Sexual abuse with both force and penetration108171883529625Table 2Demographic characteristics of sexual abuse groups formed by force and penetrationNo Sexual Abusea (n=409)Sexual Abuse (no force or penetration)b (n=159)Sexual Abuse (penetration)c (n=313)Sexual Abuse (force and penetration)d (n=296)n%n%n%n%Sex (male)227c,d56100d63208a,d66108a,b,c36Race (minority)269b,c,d66120a,c75279a,b,d89228a,c77Education (high school or less)212b,c,d52100a,c63244a,b,d78191a,c65MSDMSDMSDMSDAge (in years)28.4d9.628.79.529.29.830.5a9.7ap\u2009<\u2009.05, compared to No Sexual Abuse.bp\u2009<\u2009.05, compared to Sexual Abuse, No Force, No Penetration.cp\u2009<\u2009.05, compared to Sexual Abuse, No Force, Penetration.dp\u2009<\u2009.05, compared to Sexual Abuse, Force, Penetration.\nStatistical analyses\nAnalyses of variance (ANOVAs) were used to determine whether the four categories of sexual abuse (no sexual abuse; sexual abuse without force or penetration; sexual abuse with penetration; and sexual abuse with both force and penetration) were associated with later risky sexual behavior. If there was a significant overall effect of sexual abuse, Tukey tests were conducted to determine specifically which groups differed. Demographic variables that differed between groups were controlled for in these analyses. Thus, the ANOVAs included: (1) demographic covariates and (2) a main effect of sexual abuse. Continuous outcome variables that were not normally distributed (i.e., the number of lifetime partners, the number of partners in the past 3 months, the number of episodes of unprotected sex in the past 3 months, the number of times participants exchanged sex for money or drugs, and the number of previous STD diagnoses) were transformed using a log10 of (x+1) transformation (Tabachnick & Fidell, 2001). Unless otherwise stated, analyses associated with these variables used the log transformation.\nExploratory analyses were conducted to investigate whether gender moderated the relations between the sexual abuse characteristics and later sexual behavior. ANOVAs were conducted including demographic covariates, a main effect of abuse, and the interaction of abuse and gender.\nResults\nOf the 1216 patients who completed the survey, 66% reported childhood\/adolescent sexual abuse (n=807). Of these 807 participants who met criteria for sexual abuse, 159 (20%) reported sexual abuse without force and without penetration, 313 (39%) reported sexual abuse with penetration, 39 (5%) reported sexual abuse with force and without penetration, and 296 (37%) reported sexual abuse with both force and penetration. Because few participants reported sexual abuse with force but without penetration, those participants were excluded from the analyses, leaving a final sample size of N=1177. Sexual abuse characteristics by gender are reported in Table 1.\nTable 3Sexual risk behaviors of participants who reported sexual abuse with force, sexual abuse without force, and no sexual abuse (raw data)No Sexual Abusea (n=409)Sexual Abuse (no force or penetration)b (n=159)Sexual Abuse (penetration)c (n=313)Sexual Abuse (force and penetration)d (n=296)MSDMSDMSDMSDSexual partners (number, lifetime)31.7c,d80.927.6c,d28.260.1a,b211.564.2a,b172.4Sexual partners (number, past 3 months)2.5c,d2.12.72.23.2a2.73.5a4.0Unprotected sex (number of events, past 3 months)15.3c,d24.317.026.820.7a30.122.8a38.2Unprotected sex (proportion, past 3 months)0.680.320.640.330.700.300.670.33Exchanged sex for money or drugs (number, lifetime)4.9c,d55.95.6d42.06.5a,d42.417.6a,b,c89.6STD diagnoses (number, lifetime)2.4c,d3.02.6c,d3.23.4a,b3.64.0a,b4.1ap\u2009<\u2009.05, compared to No Sexual Abuse.bp\u2009<\u2009.05, compared to Sexual Abuse, No Force, No Penetration.cp\u2009<\u2009.05, compared to Sexual Abuse, No Force, Penetration.dp\u2009<\u2009.05, compared to Sexual Abuse, Force, Penetration.\nDemographic differences\nPreliminary analyses examined whether any demographic variables were associated with the sexual abuse characteristics (see Table 2). Sexual abuse was significantly associated with sex, race, education, and current age. Thus, for example, participants reporting a history of sexual abuse were more likely to be less well-educated and more likely to report a minority racial\/ethnic identity than nonabused participants. Importantly, sexual abuse involving force and penetration was more likely to be reported by women than by men. All pairwise comparisons for the demographic characteristics are presented in Table 2. Because of these associations, sex, race, education, and current age were used as covariates in subsequent analyses.\nRelation between sexual abuse characteristics and sexual behavior\nAfter controlling for relevant demographic covariates, sexual abuse was significantly associated with the number of lifetime partners, F(3, 1160)=21.08, p\u2009<\u2009.0001, the number of episodes of unprotected sex in the past 3 months, F(3, 1169)=3.97, p\u2009<\u2009.01, the number of partners in the past 3\u00a0months, F(3, 1169)=7.28, p\u2009<\u2009.0001, the number of times sex was traded, F(3, 1153)=14.23, p\u2009<\u2009.0001, and the number of previous STD diagnoses, F(3, 1169)=8.01, p\u2009<\u2009.0001 (see Table 3). Sexual abuse was not significantly associated with the proportion of unprotected sex episodes in the past 3\u00a0months.\nFollow-up Tukey tests showed that, compared to those who were sexually abused with penetration, those who were not sexually abused had significantly fewer: (1) lifetime sexual partners (Cohen\u2019s d=.40); (2) partners in the past 3\u00a0months (d=.23); (3) episodes of unprotected sex in the past 3 months (d=.19); (4) episodes of sex trading (d=.31); and (5) previous STD diagnoses (d=.22; all ps\u2009<\u2009.05). Similarly, compared to those who were sexually abused with both force and penetration, those who were not sexually abused had significantly fewer: (1) lifetime sexual partners (d=.49); (2) partners in the past 3 months (d=.29); (3) episodes of unprotected sex in the past 3 months (d=.21); (4) episodes of sex trading (d=.46); and (5) previous STD diagnoses (d=.34; all ps\u2009<\u2009.05).\nIn addition, compared to those who were sexually abused with penetration, those who experienced sexual abuse without force and without penetration had significantly fewer lifetime sexual partners (d=.32) and fewer previous STD diagnoses (d=.17; both ps\u2009<\u2009.05). Similarly, compared to those who were sexually abused with both force and penetration, those who experienced sexual abuse without force and without penetration had significantly fewer: (1) lifetime sexual partners (d=.34); (2) episodes of sex trading (d=.29); and (3) previous STD diagnoses (d=.30; all ps\u2009<\u2009.05). Finally, those who were sexually abused with both force and penetration reported significantly more episodes of sex trading than those who were sexually abused with penetration (d=.29; p\u2009<\u2009.05).\nBecause sex trading likely leads to a greater number of sexual partners, episodes of unprotected sex, and STD diagnoses, follow-up analyses were conducted to determine whether penetration was still associated with the sexual behavior outcomes after controlling for sex trading. All effects remained significant after controlling for sex trading (all ps\u2009<\u2009.05).\nGender as a moderator of the relation between sexual abuse characteristics and sexual behavior\nTo determine whether gender moderated the relation between sexual abuse characteristics and sexual behavior, gender-by-sexual abuse interactions were included in the ANOVAs. Relevant demographic covariates were included.\nThe gender-by-sexual abuse interaction was significantly associated with the number of episodes of sex trading, F(3, 1150)=3.56, p\u2009<\u2009.05. Analyses of simple main effects revealed that, for both women and men, those who were sexually abused with force and penetration reported significantly more episodes of sex trading than those who were not abused, or than those who were abused without force and without penetration. However, for women only, those who were sexually abused with penetration reported significantly more episodes of sex trading than those who were not abused (all ps\u2009<\u2009.05; see Fig. 1).Fig. 1The effect of the interaction of gender and sexual abuse status on the number of episodes of sex trading\nDiscussion\nThis study investigated whether characteristics of childhood and adolescent sexual abuse (i.e., force and type of sexual activity) were related to adult sexual risk behavior, and whether these associations differed by gender. This research benefited from several methodologic strengths. For example, we sampled a large group of both men and women who reported sexual abuse; this large and diverse sample allowed exploration of two sexual abuse characteristics and gender differences. We also used psychometrically sound measures and a computer-administered survey, known to result in higher, and presumably more candid, rates of socially stigmatized and sensitive behaviors (Schroder et\u00a0al., 2003). These strengths increase confidence in the validity and generalizability of the results.\nA key set of findings was that (1) sexual abuse with penetration as well as (2) sexual abuse with force and penetration were both related to higher rates of adult sexual behavior compared to (3) sexual abuse without force and without penetration and (4) no sexual abuse. This pattern of findings corroborates results from research investigating the mental health sequelae of sexual abuse, which indicate that force (e.g., Bulik, Prescott, & Kendler, 2001; Rind et\u00a0al., 1998; Rodriguez, Ryan, Kemp, & Foy, 1997) and penetration (e.g., Briere & Elliott, 2003; Bulik et\u00a0al., 2001) are associated with worse psychological outcomes; the current research also adds to the limited body of research suggesting a relation between force and penetration, and later sexual behavior (e.g., Cinq-Mars et\u00a0al., 2003; Fergusson et\u00a0al., 1997). The effect sizes for the association between sexual abuse and later sexual behavior were small to medium, indicating that other variables besides sexual abuse account for a large portion of the variance in adult sexual behavior. The latter finding is consistent with the idea that adult sexual behavior is influenced by multiple environmental as well as individual factors (Smith & Subramanian, 2006).\nPenetration by itself (i.e., without force) and penetration in combination with force were associated with increased sexual risk behavior relative to those who were abused without force and without penetration, and those who were not abused. The sole difference between the penetration only and the penetration plus force groups involved sex trading, where those who experienced sexual abuse with force and penetration reported engaging in a greater frequency of sex trading, relative to those who experienced sexual abuse with penetration and no force. However, this finding was qualified by a significant gender-by-abuse interaction. Because only a very small number of participants reported sexual abuse with force but without penetration (i.e., forced kissing or fondling), we were unable to investigate the impact of force only.\nA somewhat unexpected finding was that the group that reported sexual abuse without force and without penetration did not differ significantly from the nonabused group on any of the sexual behavior outcomes. Future investigation of the relation between sexual abuse and adult sexual behavior might find it fruitful to conduct more fine-grained assessments of the sexual experiences that involve only large age differentials to determine how these experiences are perceived by both men and women, and whether such experiences influence subsequent sexual behavior.\nIt may seem counter-intuitive that individuals who experienced more severe sexual abuse (i.e., sexual abuse with force or penetration) would engage in more sexual experiences than those who experienced less severe sexual abuse; that is, one might expect individuals who experienced severe sexual abuse to avoid sex because of the negative consequences. However, relative to individuals who experienced less severe sexual abuse, individuals who experienced more severe sexual abuse may use different strategies to cope with their sexual abuse experience(s). Thus, for both men and women, those who experienced more severe forms of sexual abuse may use alcohol or drugs to cope with the sexual abuse, which, in turn, may lead to the exchange of sex for money or drugs, and\/or to a greater number of sexual partners and episodes of unprotected sex. In addition, alcohol and other drug use may lead to a greater number of sexual partners and episodes of unprotected sex due to decreased ability to attend to distal concerns, such as acquiring an STD when intoxicated or high (cf. alcohol myopia; Steele & Josephs, 1990). Indeed, we have reported previously that substance use is an important mediator of the relation between sexual abuse and risky sexual behavior (Senn, Carey, Vanable, Coury-Doniger, & Urban, 2006); future research should explore whether substance use and other potential mediators operate differently for those who experienced different severity levels of sexual abuse.\nAn alternative explanation for the association between more severe sexual abuse and greater adult sexual risk behavior is Finkelhor and Browne\u2019s (1985) traumagenic dynamics model. This model proposes that one consequence of sexual abuse is traumatic sexualization, in which a child develops maladaptive scripts for sexual behavior, when rewarded for sexual behavior by affection. More severe sexual abuse, such as sexual abuse involving force or penetration, may lead to greater traumatic sexualization. As adults, those who experienced traumatic sexualization may believe sex is necessary to obtain affection from others. Thus, traumatic sexualization may lead to, for example, earlier consensual sex or a greater number of sexual partners (e.g., Cinq-Mars et\u00a0al., 2003; Fergusson et\u00a0al., 1997).\nAnother consequence of sexual abuse, according to Finkelhor and Browne (1985), is powerlessness, in which a child learns that his or her needs or requests are ignored by others; the child thus fails to develop self-efficacy to stop unwanted sexual advances. More severe sexual abuse, particularly sexual abuse involving force or penetration, may lead to greater feelings of powerlessness. Perhaps because they lack the interpersonal skills or the self-efficacy to stop unwanted sexual advances, these individuals may be less likely to refuse intercourse with aggressive partners, resulting in more sexual partners. Powerlessness could help explain findings linking more severe sexual abuse to more adult sexual risk behavior (e.g., Cinq-Mars et\u00a0al., 2003; Fergussion et\u00a0al., 1997). In this regard, Kallstrom-Fuqua, Weston, and Marshall (2004) found that sexual abuse severity had an indirect effect on maladaptive relationships, mediated through powerlessness; thus, having many sexual partners could be a consequence of difficulty forming close relationships. Further research is needed to examine whether the sexual abuse characteristics investigated in this study are associated with Finkelhor and Browne\u2019s (1985) traumagenic dynamics.\nAnother finding yielded by this study is that abuse characteristics were associated with different outcomes for men and women. For men, only abuse with both force and penetration was associated with a greater frequency of sex trading, whereas for women, abuse with penetration, regardless of whether or not force was involved, was associated with more sex trading. In the current cultural context, young males may view sex with an older woman as masculine and mature, rather than abusive. Males, therefore, may tend to view only experiences involving force or coercion as abusive. Women, on the other hand, may be more likely to view intercourse with an older individual as abusive, regardless of whether or not force was involved. This idea is supported by meta-analytic findings that boys\u2019 reactions to sexual abuse were less negative than were girls\u2019 reactions (Rind et\u00a0al., 1998). Different perceptions of whether or not the experience was abusive may lead to the use of different coping strategies.\nThese results should be interpreted mindful of the limitations of the study. One limitation involved the brevity of the sexual abuse assessment. Use of a brief survey allowed us to obtain a large and diverse sample, but limited the richness of the data collected. The survey did not assess other aspects of sexual abuse, such as duration, frequency, and relationship to the perpetrator, which may be important correlates of later outcomes (e.g., Banyard & Williams, 1996; Briere & Elliott, 2003). In addition, these brief questions did not allow for assessment of reactions to the sexual experience; many participants, especially those who did not report force or coercion, may not have considered themselves sexually abused, but may have viewed these sexual experiences as inconsequential or even consensual. Future research, involving mixed qualitative and quantitative methods, might help to elucidate the empirical relations observed in the current sample.\nA second limitation involves the correlational nature of the data. Clearly, such data limit causal inferences, although given the temporal sequence of childhood\/adolescent sexual abuse and adult sexual behavior, the limits may be less concerning in this context. Nonetheless, we acknowledge that unexplored variables that are related to both sexual abuse and greater sexual risk behavior (e.g., more adverse childhood experiences; Dong, Anda, Dube, Giles, & Felitti, 2003) should be included in future investigations of the sexual abuse\u2013risky sex relation.\nIt is important to recognize that participants in this study were recruited from a sexually transmitted disease clinic, and were included because they were currently engaging in sexual behavior that conferred risk for contracting an STD. The rates of sexual abuse reported in this sample were considerably higher than rates (i.e., 15% for men and 30% for women) reported in national samples (Briere & Elliott, 2003; Finkelhor, Hotaling, Lewis, & Smith, 1990; Vogeltanz et\u00a0al., 1999). In addition to engaging in sexual risk behavior, patients attending STD clinics may differ from the general population in other important ways as well; for example, patients attending STD clinics often report extremely high rates of alcohol and drug use (Cook et\u00a0al., 2006). Due to the nature of the sample, these results of the present study may not generalize to other populations.\nThese results have implications for both practice and research. Regarding public health and clinical practice, they suggest that a thorough sexual health assessment should include inquiry about the nature of the sexual abuse, particularly whether force was involved and what type of sexual act occurred. Given the likely impact of sexual abuse on sexual risk behavior (as well as other health outcomes), we recommend a more comprehensive approach to sexual health assessment, education, counseling, and\/or therapy. Indeed, these findings highlight the need to develop interventions tailored to the unique needs of persons with a history of sexual abuse to promote (and restore) sexual health and reduce sexual risk. With respect to research, these findings raise many questions about the conditions under which sexual abuse impairs healthy sexual development and expression, and about the mechanisms by which sexual abuse influences sexual development, behavior, and adjustment. This work will require sophisticated methods and analyses to overcome the limitations of what is inherently retrospective and correlational research.","keyphrases":["sexual behavior","hiv","sexually transmitted disease","child\/adolescent sexual abuse"],"prmu":["P","P","P","R"]}
{"id":"Eur_J_Nutr-2-2-1705489","title":"Digestibility of resistant starch containing preparations using two in vitro models\n","text":"Background Resistant starch (RS) is known for potential health benefits in the human colon. To investigate these positive effects it is important to be able to predict the amount, and the structure of starch reaching the large intestine.\nIntroduction\nResistant Starch (RS) is defined as starch that is not absorbed in the small intestine of humans. Three different types of RS were defined by Englyst et al. [1]. Type 1 is defined as physically inaccessible starch, type 2 (RS2) as native starch granules and type 3 (RS3) as retrograded starch. More recently, a fourth type of RS has been classified, comprising chemically modified starches [2, 3]. RS is claimed to be a good substrate for colonic fermentation and to be beneficial because of its high ratio of butyrate production, which may play a major role in the prevention of colon cancer, as shown in several studies on animal models [4, 5]. The fermentation products from RS are known to lower the pH in the colon which leads to less production, and\/or accumulation of potentially harmful by-products of protein fermentation, for example, ammonia or phenols which may promote tumorigenesis [6]. To estimate potential health benefits of RS it is important to be able to predict its behavior in the human gastrointestinal tract, in particular the amount and the structure of starch reaching the large intestine. The digestion of food and absorption of nutrients are spatiotemporal and dynamic processes involving complex enzymatic systems and transport reactions. Thus, the simulation of all these biochemical and physiological events in a single model is illusive. Nevertheless, several in vitro digestion methods, from basic batch systems to sophisticated dynamic models have been developed in order to quantify digestibility of food [7\u20139]. A realistic approach implies a well-defined system that takes into account the specific contributions of oral, gastric and intestinal digestion. Advantages of using in vitro instead of in vivo models are low costs, relatively easy performance, no limitations by ethical constraints, and the possibility to compare different substrates when applying standardized conditions. Therefore, the aim of this study was to compare a rather simple and a more sophisticated in vitro model as well as the Megazyme RS method to determine the indigestible fractions of two RS preparations containing RS2 and RS3. The digestion residues obtained by the two models were compared and characterized. Furthermore, the RS3 fractions were compared with ileostomy effluents obtained in an in vivo study [10].\nMaterials and methods\nSubstrates\nThe RS3 containing carbohydrate source, C\u22c6Actistar\u00ae, is a retrograded long chain maltodextrin product obtained after partial enzymatic hydrolysis, and subsequent retrogradation of tapioca starch (RTmd), produced according to United States Patent 6 043 229 [11] and obtained from Cerestar-Cargill (Vilvoorde, Belgium). Physico-chemical characteristics of RTmd were described recently by Pohu [12]. The RS2 containing carbohydrate source is native high amylose maize (HAM) starch and was obtained from Cerestar International (Neuilly-sur-Seine, France).\nBatch in vitro digestion model\nThe batch model used is a three step incubation at 37\u00b0C simulating the digestion in mouth, stomach and small intestine. The method was carried out as described by Lebet et al. [7] and modified by J\u00f6rger [13]. A sample of 30\u00a0g substrate was suspended in 500\u00a0ml phosphate buffer (20\u00a0mM, pH 6.9, Na2HPO4 1.42\u00a0g\/l, KH2PO4 1.36\u00a0g\/l, NaCl 0.58\u00a0g\/l), and incubated stepwise with (a) 0.5\u00a0ml human salivary \u03b1-amylase solution (Sigma A1031, Buchs, CH, 10\u00a0mg\/ml in CaCl2 1\u00a0mM) at pH 6.9 for 15\u00a0min, (b) 1.25\u00a0ml porcine pepsin suspension (Sigma P7012, 1\u00a0mg\/ml in NaCl 9\u00a0g\/l) at pH 2.0 for 30\u00a0min and (c) 10\u00a0ml porcine pancreatin (Sigma P7545, 0.5\u00a0mg\/ml in CaCl2 25\u00a0mM) at pH 6.9 for 3\u00a0h in the presence of 12\u00a0g bovine bile (Sigma B8381). Degradation products were removed by dialysis (Servapor 44146, Serva Feinbiochemica GmbH & Co., Heidelberg, Germany; cut-off 12\u201314\u00a0kDa) overnight under continuous movements against running deionized water <20\u00b0C. The retentate was freeze-dried. Based on total starch (TS) determinations in the starting materials and digestion residues, the amount of indigestible starch was calculated.\nDynamic in vitro digestion model (TIM-1)\nThe dynamic model used in this study has been described by Minekus et al. [8]. The model comprises four serial compartments simulating stomach, duodenum, jejunum and ileum. The in vitro digestion was performed for 6\u00a0h at 37\u00b0C. A mixture of 60\u00a0g substrate, 180\u00a0g electrolyte solution (NaCl 5\u00a0g\/l, KCl 0.6\u00a0g\/l, CaCl2\u00b72H2O 0.3\u00a0g\/l, NaHCO3 0.6\u00a0g\/l), 60\u00a0g water, 5\u00a0g pepsin solution (Sigma P7012, 0.28\u00a0g\/l in solution A: NaCl 3.1\u00a0g\/l, KCl 1.1\u00a0g\/l, CaCl2\u00b72H2O 0.15\u00a0g\/l, NaHCO3 7.1\u00a0g\/l) and 5\u00a0g lipase solution (Rhizopus lipase, Amano Pharmaceutical Co. F-AP 15, Ltd. Japan, 0.25\u00a0g\/l in solution A) were introduced into the gastric compartment. Computer controlled peristaltic valve pumps controlled meal transit through the individual compartments. The pH value was computer monitored by adding HCl (1\u00a0M) or NaHCO3 (1\u00a0M), respectively. In the stomach the values were preset to pH 4.5, 2.8, 1.8, 1.7 and 1.5 at 0, 20, 40, 60 and 90\u00a0min, respectively. In the small intestine, the pH was maintained at 6.5, 6.8 and 7.2 in the duodenum, jejunum and ileum, respectively. About 1\u00a0ml trypsin solution (Sigma T4665, 2\u00a0g\/l in solution A) was added to the duodenum at the beginning of the experiment. Secretions of porcine bile 4\u00a0g\/100\u00a0g in water (Sigma B8631) and pancreatic solution 7\u00a0g\/100\u00a0g in water (Pancrex-V powder, Paines & Byrne, Greenford, UK) entered the duodenal compartment at 0.5 and 0.25\u00a0ml\/min, respectively. The absorption of water and digestive products from the jejunal and ileal compartments was simulated using hollow-fiber devices (cut-off 5\u201310\u00a0kDa). Ileal effluents (indigestible fraction) were collected after 2, 4 and 6\u00a0h, pooled and freeze-dried. Substrate remaining in the jejunum and ileum at the end of the experiment was considered to be indigestible as well and was therefore mixed with the ileal effluent pool. TS determinations of the starting materials, the digestion residues and the remaining substrate in the model after the experiment were carried out. Starch degradation products (up to DP 7) were quantified in dialysates by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) in a BioLC System (Dionex Corp., Sunnyvale CA, USA) using a Carbo-Pac PA1 column and applying a sodium acetate gradient (unpublished, internal method). TS and oligosaccharide quantifications were recalculated as glucose content. The recovered glucose after the experiment (in the digestible and indigestible residues) was assumed as 100%, of which the indigestible fraction is expressed as a fraction.\nCharacterization\nBoth RS preparations were characterized before and after digestion. Additionally, freeze dried RTmd fractions from an ileostomy study feeding RTmd were pooled (7 subjects) and used for characterization analyses [10].\nTS was determined using the Megazyme TS assay kit (Megazyme, Co. Wicklow, Ireland) [14]. In brief, starch was hydrolyzed in two phases. In phase 1, starch was pre-treated with dimethyl-sulfoxide (DMSO), totally solubilized and partially hydrolyzed with \u03b1-amylase. In phase 2, dextrins were quantitatively hydrolyzed to glucose with amyloglucosidase (AMG). Glucose was subsequently determined enzymatically using the Hexokinase\/Glucose-6-phosphate-dehydrogenase assay according to Boehringer [15].\nThe RS fraction was determined using the Megazyme RS assay kit (Megazyme, Co. Wicklow, Ireland) [16]. The samples were incubated in a shaking water bath with pancreatic \u03b1-amylase and AMG at 37\u00b0C for 16\u00a0h, during which time non-resistant starch was solubilized and hydrolyzed to glucose. RS was recovered as a pellet after centrifugation, which was dissolved by stirring in KOH 2\u00a0M and quantitatively hydrolyzed to glucose with AMG [16]. Glucose was determined as described above.\nProtein was determined as sum of amino acids after acid hydrolysis and ion-exchange chromatography with post-column ninhydrin detection [17].\nWide-angle X-Ray powder diffraction (X-Ray) measurements were performed using a Scintag PADX Diffractometer (PANAlytical, Almelo, NL) operating at 45\u00a0mA and 40\u00a0kV at an angular range of 2\u03b8 from 1 to 40\u00b0 with a step size of 0.03\u00b0. Counting time was 5\u00a0s on each step.\nDifferential scanning calorimetry (DSC) measurements were performed using the Thermal Analyst system 2000 (DSC 2910, TA Instument Ltd, Newcastle, UK). Samples at a starch:water ratio of 1:4 were prepared and heated from 4\u00b0C to 200\u00b0C at 10\u00b0C\/min, thereafter the samples were cooled at 20\u00b0C\/min to 4\u00b0C and heated again at the same heating rate. All the DSC results were evaluated from the mean of three separate determinations for each sample, unless stated otherwise.\nStatistical analysis\nStatistical analyses were carried out using SPSS, version 12.0.1 for Windows (one-way ANOVA and Post-Hoc Tukey). Differences were considered to be significant at P\u00a0<\u00a00.05.\nResults\nComposition and digestibility of RS containing preparations\nIn Table\u00a01 the average TS contents and indigestible starch fractions of RTmd and HAM are shown. All data are mean values of at least five repetitions. Data resulting from the dynamic and the batch model are based on the TS content before and after digestion. Both preparations contain identical amounts of TS. The two in vitro digestion models as well as the Megazyme RS method which is accepted as the official AOAC method for RS determination, led to similar amounts of indigestible RS3 fractions. Digestibility of RS2 was similar using the dynamic model, and the Megazyme RS method, whereas the batch model led to a lower digestibility. Expressing the RS results obtained by the Megayzme RS method based on substrate fresh weight (instead of g\/100\u00a0g TS as shown in Table\u00a01) yielded to 50.2\u00a0\u00b1\u00a02.6, and 45.3\u00a0\u00b1\u00a04.1\u00a0g\/100\u00a0g for RTmd and HAM, respectively. This is very well in accordance with respectively, 48.3\u00a0\u00b1\u00a02.8 and 46.3\u00a0\u00b1\u00a03.9\u00a0g\/100\u00a0g found in a collaborative study published earlier by McCleary et al. [16]. Standard deviations after HAM digestion were found to be higher compared to RTmd digestion. The batch system showed a better reproducibility than the dynamic model and Megazyme RS method for both substrates.\nTable\u00a01Average total starch (TS) content of retrograded tapioca maltodextrins (RTmd) and high amylose maize starch (HAM) and their indigestible starch fractions obtained by dynamic digestion, batch digestion and the Megazyme resistant starch (RS) method (mean values\u00a0\u00b1\u00a0SD)SampleTS (g\/100\u00a0g\u00a0dm)Digestion methodIndigestible starch fraction (g\/100\u00a0g TS dm)RTmd94.3\u00a0\u00b1\u00a01.8 (n\u00a0=\u00a017)Dynamic59.6\u00a0\u00b1\u00a02.1 (n\u00a0=\u00a06)a,bBatch60.6\u00a0\u00b1\u00a00.9 (n\u00a0=\u00a05)aMegazyme57.7\u00a0\u00b1\u00a02.9 (n\u00a0=\u00a012)bHAM94.3\u00a0\u00b1\u00a01.1 (n\u00a0=\u00a07)Dynamic57.5\u00a0\u00b1\u00a04.7 (n\u00a0=\u00a05)dBatch65.8\u00a0\u00b1\u00a03.5 (n\u00a0=\u00a010)cMegazyme54.8\u00a0\u00b1\u00a05.0 (n\u00a0=\u00a017)dMeans with the same letters are not significantly different (Tukey P\u00a0<\u00a00.05)\nComposition of digestion residues\nIn Table\u00a02 the average TS and RS contents of RTmd and HAM digestion residues are shown. TS quantifications of in vitro digestion residues were carried out after each single experiment in duplicate. In contrast, RS contents as well as TS in ileostomy effluents were measured in the pooled fractions only. TS contents in digestion residues turned out to be lower than in corresponding starting materials (Table\u00a01), which are due to additional enzymes and bile added during digestion. Moreover, residues obtained from the batch digestion contained more starch than those from the dynamic model. The digestion residues of RTmd and HAM obtained from the dynamic model contained 1.5\u00a0g protein\/100\u00a0g\u00a0dm and 1.8\u00a0g protein\/100\u00a0g\u00a0dm, respectively, whereas in the batch digestion residues only 0.6\u00a0g\u00a0protein\/100\u00a0g\u00a0dm and 1.2\u00a0g protein\/100\u00a0g\u00a0dm were determined. Bile contains large amounts of glycine that was correspondingly found in higher amounts in the dynamically digested residues (0.2 and 0.3\u00a0g\/100\u00a0g\u00a0dm for RTmd and HAM, respectively) compared to the batch digested residues (0.3 and 0.4\u00a0g\/100\u00a0g\u00a0dm for RTmd and HAM, respectively). Also other amino acids like asparagine, glutamine, alanine, proline, and leucine were found in higher amounts in dynamically digested preparations. The lower amount of TS found in the ileal effluent pool compared to the in vitro digested samples is due to the dilution by additional endogenous and exogenous material such as diet derived protein, dietary fiber and fat.\nTable\u00a02Total starch (TS) and resistant starch (RS) contents of in vitro and in vivo digestion residues of retrograded tapioca maltodextrins (RTmd) and high amylose maize starch (HAM) (mean values\u00a0\u00b1\u00a0SD)SampleTS (g\/100\u00a0g dm)RS (g\/100\u00a0g dm)RTmd dynamic72.4\u00a0\u00b1\u00a03.7 (n\u00a0=\u00a012)36.4\u00a0\u00b1\u00a01.0 (n\u00a0=\u00a02)RTmd batch88.6\u00a0\u00b1\u00a00.6 (n\u00a0=\u00a010)61.4\u00a0\u00b1\u00a01.2 (n\u00a0=\u00a03)RTmd in vivo48.4\u00a0\u00b1\u00a00.0 (n\u00a0=\u00a02)31.3\u00a0\u00b1\u00a01.1 (n\u00a0=\u00a02)HAM dynamic74.9\u00a0\u00b1\u00a01.7 (n\u00a0=\u00a010)27.6\u00a0\u00b1\u00a02.0 (n\u00a0=\u00a02)HAM batch80.9\u00a0\u00b1\u00a02.6 (n\u00a0=\u00a012)35.7\u00a0\u00b1\u00a00.7 (n\u00a0=\u00a03)\nIt is expected that RS values close to 100% are found in the in vivo as well as the in vitro digestion residues. As shown in Table\u00a02 lower amounts of RS (28\u201362%) were found in the digestion residues. This can be explained by the fact that the digestion method carried out prior to the analytical RS determination has an influence on the starch structures leading to less resistant starch. Therefore, analysing RS in digestion residues may be interpreted as characterization method, indicating the highly resistant fraction. Different amounts of RS were found because of additional non-starch material present in the samples as described above. Calculating RS recoveries based on TS led to 50\u00a0g\/100\u00a0g TS for the dynamically digested RTmd fractions, whereas after batch and in vivo digestion similar amounts (69 and 65\u00a0g\/100\u00a0g TS, respectively) were found. In contrast, clearly lower RS amounts were found in both HAM digestion residues (37 and 44\u00a0g\/100\u00a0g) after dynamic and batch digestion, respectively.\nIn Fig.\u00a01 the absorption of digestion products from the jejunum and ileum compartments during the dynamic experiment can be followed. The main digestion products found were maltose and maltotriose despite the fact that larger molecules are able to pass the hollow fiber devices as well. Starch digestion products up to DP3 were found when RTmd was being digested, whereas after HAM digestion minor amounts of maltotetraose (less than 0.1% of total digestible glucose) were detected as well. The kinetics of RTmd and HAM digestion differed considerably. The digestible fraction of RTmd was degraded completely after 4\u00a0h, whereas degradation of HAM still continued after 4\u00a0h. The lower amounts of degradation products collected during the first 4\u00a0h from HAM digestion, and the higher total amounts separated from the ileum compartment show that HAM under these experimental conditions is being digested more slowly compared to RTmd. The enzyme used for starch digestion in the dynamic model is a \u03b1-amylase, therefore maltose is theoretically expected to be the smallest degradation product. As can be seen in Fig.\u00a01, glucose was found as well. Glucose could originate from added compounds during the dynamic digestion experiment, such as bile and enzymes. Moreover, a small contamination of \u03b1-amylase by AMG or \u03b1-amylase containing small amounts of side-activity cannot be excluded. The total amount of starch degradation products absorbed during the dynamic in vitro digestion experiment is slightly higher for HAM compared to RTmd. This is in accordance with a somewhat lower non-significant indigestible residue of HAM compared to RTmd as shown in Table\u00a01.\nFig.\u00a01Digestible starch degradation products (up to DP3) of retrograded tapioca maltodextrins (RTmd), and high amylose maize starch (HAM) recovered from the jejunum (a) and the ileum (b) compartments\nCharacterization of digestion residues\nX-ray diffraction\nX-ray diffraction patterns of RTmd and its dynamic, batch and in vivo digestion residues are presented in Fig.\u00a02. RTmd and indigestible RTmd fractions all led to a typical A-type pattern. However, the diffraction patterns, with exception of RTmd dynamically digested, exhibited an additional peak at \u03b82\u00a0=\u00a020\u00b0 which can be interpreted as V-form due to the presence of amylose\u2013lipid complexes. RTmd and RTmd batch digested residues were very similar concerning their crystallinity. The only differences were the peaks at 2\u03b8\u00a0=\u00a020\u00b0, and 2\u03b8\u00a0=\u00a023\u00b0, which were slightly lower and higher, respectively in the batch digested sample. In contrast to RTmd and its batch digested fraction, dynamically digested RTmd showed no peak at 2\u03b8\u00a0=\u00a027\u00b0C and no pronounced double peak at 2\u03b8\u00a0=\u00a017\u00b0 and 2\u03b8\u00a0=\u00a018\u00b0 (both typical for A-type structure), which indicate a loss in crystallinity due to digestion. The in vivo digestion residue showed an additional peak at 2\u03b8\u00a0=\u00a029\u00b0; this peak could be due to additional structures of proteins or lipids present in this sample.\nFig.\u00a02X-ray diffraction patterns of retrograded tapioca maltodextrins (RTmd) and their digestion residues\nHAM containing samples led to lower crystallinity compared to RTmd (Fig.\u00a03), which correspond to Gerard et al. [18], showing that high amylose maize starches have low crystallinity. HAM and its digestion residues led to B-type pattern, although the peak at 2\u03b8\u00a0=\u00a020\u00b0 observed in HAM and batch digested HAM indicated a mixture between B- and V-type which was described by Shi et al. [19] as well. Due to in vitro digestion of HAM and dynamically digestion of RTmd the peak \u03b82\u00a0=\u00a026\u00b0 disappeared which points to a loss in crystallinity. Similar as for RTmd (Fig.\u00a02) the dynamic digestion led to higher changes in crystallinity compared to batch digestion.\nFig.\u00a03X-ray diffraction patterns of high amylose maize starch (HAM) and their digestion residues\nDSC\nThe thermal behavior of the substrate melting was studied using DSC measurements. The results from the first and second heating are shown in Table\u00a03. During the first heating, endothermic peak temperatures (Tp) turned out to be slightly lower for RS2 compared to RS3. The peak ranges within the RTmd and HAM containing preparations were found to be similar with exception of a slightly narrower peak of in vivo digested RTmd. Enthalpies were found to be lower for HAM containing preparations compared to RTmd containing preparations. The melting transition of RTmd was separated by an exothermic effect (not shown). Such a melting characteristic can be interpreted as partial melting (endothermic), followed by recrystallization (exothermic) and final melting (endothermic) [20, 21]. This exothermic peak was not visible in the digestion residues, which indicates that due to digestion the fractions responsible for these rearrangements were removed. Within the RS3 fractions, in vivo digestion led to significantly lower enthalpies. Dynamic digestion of both preparations led to an exothermic peak when the sample was reheated (Table\u00a03). This indicates that the reorganization of the crystalline structures takes place during the second heating. RTmd and its batch and in vivo digested fractions did not show any heat flow during the second heating (therefore not shown in Table\u00a03), it can be interpreted that recrystallization was completed during cooling in these samples already. HAM containing samples, excluding the dynamically digested fraction, all led to an endothermic peak during reheating which indicates a reversible reconstruction of the amylose\u2013lipid complexes [20].\nTable\u00a03Average results (n\u00a0=\u00a03) from differential scanning calorimetry (DSC) for retrograded tapioca maltodextrins (RTmd) and high amylose maize starch (HAM) and their digestion residues. T0: onset temperatures (\u00b0C), Tp: peak temperatures (\u00b0C), Tc: completion temperatures (\u00b0C), \u0394H: transition enthalpy\u00a0\u00b1\u00a0SD (J\/g) (endothermic if nothing else stated)SampleT0(\u00b0C)Tp (\u00b0C)Tc (\u00b0C)\u0394H (J\/g)First heatingRTmd (n\u00a0=\u00a02)60.7112.8140.49.0\u00a0\u00b1\u00a01.1aRTmd dynamic58.4108.6132.49.1\u00a0\u00b1\u00a00.2aRTmd batch61.8109.1136.410.8\u00a0\u00b1\u00a02.0aRTmd in vivo58.4105.0123.55.2\u00a0\u00b1\u00a01.1bHAM61.896.9110.02.6\u00a0\u00b1\u00a00.3cHAM dynamic55.598.7127.04.2\u00a0\u00b1\u00a02.2cHAM batch (n\u00a0=\u00a02)59.991.5110.53.6\u00a0\u00b1\u00a00.7cSecond heatingRTmd dynamic79.787.9144.0*2.4\u00a0\u00b1\u00a00.4HAM60.088.2109.62.7\u00a0\u00b1\u00a00.9dHAM dynamic84.994.2146.1*2.8\u00a0\u00b1\u00a00.2dHAM batch59.883.2101.90.9\u00a0\u00b1\u00a00.0e*Exothermic, means with the same letters are not significantly different (Tukey P\u00a0<\u00a00.05)\nDiscussion\nDigestibility of RS preparations\nIn vitro digestion models use a strictly standardized procedure based on an average human digestibility. Therefore standard deviations of in vitro experiments are expected to be low. In vitro obtained HAM fractions showed higher standard deviations compared to in vitro digested RTmd fractions. Possibly the molecular structures of HAM have been more sensitive to small differences in experimental conditions such as mechanical stress, temperature or pH between experiments.\nRTmd in vitro digestion residues result in quantitatively similar RS fractions (Table\u00a01) which are very well in accordance with 59\u00a0g\/100\u00a0g found in vivo after feeding RTmd to ileostomy patients [10]. In contrast, in vitro digestion using HAM as a substrate led to different amounts of RS. These differences may be explained by methodological variations in the in vitro models used, suggesting that HAM was more affected by the experimental conditions. During the dynamic digestion experiment the substrate is exposed to the movements of the flexible walls [8], in the batch model the slurry is stirred [7], and in the Megazyme RS method the sample is shaken [16]. An effect of mechanical treatment on starch degradation of HAM but not of RTmd was shown by McCleary and Monaghan [22] before. They performed the Megazyme RS method comparing shaking and stirring. Stirring led to a higher digestibility of HAM, whereas RTmd did not show any differences. In the present study however, stirring applied in the batch model led to a lower digestibility compared to shaking as used in the Megazyme RS method. This shows that apart from mechanical forces, also other factors may have a significant impact on starch degradation. For example, different amounts and types of enzymes, their accessibilities to the substrate as well as the formation of the enzyme\u2013substrate complexes could have a larger influence on starch degradation of HAM compared to RTmd using in vitro digestion models. Furthermore, the incubation time during the dynamic digestion is nearly twice as long compared to the batch digestion and over four times as long in the Megazyme RS method. As pointed out in Fig.\u00a01, 99% of in vitro digestible RTmd fractions were degraded after 4\u00a0h, whereas only 86% of those of HAM were digested at this stage. If the dynamic digestion experiment would have been stopped after 4\u00a0h (the duration of the batch digestion experiment is 3.75\u00a0h), the remaining indigestible fraction of HAM would be identical to that obtained by the batch in vitro digestion. Whether a prolongation of the batch incubation would lead to HAM fractions similar to those obtained with the dynamic model, remains to be elucidated.\nThe analytical RS contents found in the dynamically digested residues was lower in both preparations when compared to the batch digested fractions (Table\u00a02). This leads to the assumption that in the dynamic approach starch structures are attacked to a higher extent compared to the batch model. Nevertheless, the amount of TS recovered after digestion of RTmd was not affected, whereas the TS recovery of HAM possibly was influenced by the different amount of analytical RS present in the sample; the results strongly indicate that structural features play an important role in digestibility.\nStructural aspects of the RS preparations\nX-Ray diffraction\nA mixture between A- and V-type structures for RTmd, and its in vivo digestion residues (Fig.\u00a02) was found by Pohu [12] as well. The tightly packed A-type structure is known to be very heat stable. Shamai et al. [23] investigated RS3 fractions from high amylose maize, maize flour and wheat starch, respectively. They found that RS3 produced at low retrogradation temperatures of 40\u00b0C lead to B-type patterns, whereas incubation at 95\u00b0C produced a mixture of A- and V-type polymorphs. This is in contrast to the present study, where RS3 produced from long chain tapioca maltodextrins was found to consist of a mixture between A- and V-type crystal structures, even though rather low retrogradation temperatures of 52\u201354\u00b0C were applied [11]. This shows that the structural behavior of RS3 cannot be predicted without knowing the exact process parameters and these parameters are rarely disclosed, particularly in patents.\nHAM led to a mixture of B- and V-type polymorphs, which is characteristic for native high amylose maize starch, as shown by several research groups [23\u201325]. The dynamically digested fraction did not show the V-type characteristic peak at 2\u03b8\u00a0=\u00a020\u00b0. This peak is known to represent amylose\u2013lipid complexes. It is possible that the extra-addition of lipase in the dynamic digestion model was able to degrade lipids present in HAM and RTmd to an extent where no complexation was possible anymore. During batch digestion no additional lipase was used since this enzyme is present in the pancreatin preparation. In earlier experiments it has been shown that the pH-optimum of the pancreatic lipase is approximatively 9 [26]. Therefore, the incubation at pH 6.9 in the batch model is expected to lead to a small lipid degradation only. This could explain that the peak at 2\u03b8\u00a0=\u00a020\u00b0 was only reduced during batch procedure, but disappeared using the dynamic digestion model. In vivo digested RTmd showed amylose\u2013lipid complexes (Fig.\u00a02) as well. However, it can not be stated whether the lipid degradation in the dynamic model was more efficient compared to in vivo digestion because diet-derived lipids led to a much higher lipid content before in vivo digestion (results not shown). The X-ray patterns of RTmd and HAM samples indicate that the molecules within the crystals are not packed in the same way which leads to different functional properties of HAM, and RTmd. Batch digestion of both preparations did not lead to a distinct change in crystallinity. However, in vivo digestion caused slightly broader, and dynamic digestion distinctly broader diffraction peaks, which indicate the presence of either imperfect or relatively small crystallites [27]. The latter could have occurred due to the mechanical stress which underlines that the dynamic model attacks the starch structures more strongly.\nDSC\nEndothermic transitions of the investigated samples took place at temperatures which are known to destroy the amylose\u2013lipid complexes. At lower temperatures (45\u201360\u00b0C) no peak was detected indicating that amylopectin is neither present in RTmd nor in HAM [20]. RTmd in vivo digested showed the lowest peak maximum and enthalpy of all the digestion residues. This may be due to the higher amount of non-starch material in the sample (Table\u00a01) which may compete for water binding and thus reduce the starch swelling.\nDynamically digested HAM showed a slightly broader endothermic peak compared to the other samples which indicates a broader polydispersity [28]. This observation coincides well with the statement that starch is degraded differently, and probably more strongly in the dynamic model. In vitro digestion of HAM led to slightly but not significantly higher enthalpies, especially when dynamically digested. Cooke et al. [27] suggested that the enthalpy of gelatinization primarily reflects the loss of the double helical order which could occur to a higher extent during dynamic digestion.\nConclusions\nThe results of this study show that both, a simple and a more sophisticated in vitro digestion model and the ileostomy study led to similar amounts of starch escaping digestion when using a RS3 containing carbohydrate source as substrate. In contrast, when the two in vitro digestion models were compared with a RS2 containing preparation, different amounts of indigestible starch were found. It is not possible to predict which model simulated the in vivo RS2 fraction more precisely. The more sophisticated in vitro digestion model attacked starch probably more strongly compared to the batch in vitro digestion model using an RS2 and an RS3 containing substrate. This was confirmed by differences in crystalline fractions and structural rearrangements as measured by X-ray and DSC measurements. These structural differences, caused by different experimental parameters influenced starch digestibility of HAM but not of RTmd. The behavior of other RS preparations in the two in vitro models described here can not be deduced from the obtained results and has to be examined case by case.","keyphrases":["in vitro digestion","x-ray","dsc","resistant starch type 2","resistant starch type 3"],"prmu":["P","P","P","R","R"]}
{"id":"J_Med_Internet_Res-1-1-1761705","title":"Can Examination of WWW Usage Statistics and other Indirect Quality Indicators Help to Distinguish the Relative Quality of Medical websites?\n","text":"Background The Internet offers a great amount of health related websites, but concern has been raised about their reliability. Several subjective evaluation criteria and websites rating systems have been proposed as a help for the Internet users to distinguish among web resources with different quality, but their efficacy has not been proven.\nIntroduction\nAfter the early enthusiasm generated by the potential use of the Internet in Medicine [1,2,3], concern has been raised about the quality of the resources available on the Internet compared to more academic media. It is technically very easy to publish on the Internet [4]. The lack of a review process of the documents on the Net, and the power of this media in transmitting the data has the risk of misinforming both lay people [5,6,7] and health care professionals [8]. However, only a few studies have tried to measure this risk of misinformation [9,10,11]. Nothing yet is known about the users' ability to discriminate between low and high quality resources.\nSeveral initiatives have been proposed which could be applied at different levels to improve the average quality of medical websites. For instance, we could apply certain basic methods for the websites to be correctly designed. In this sense, some academic organizations have proposed a set of basic information that every medical web site should provide about the author and sources of the web site contents, their potential conflicts of interest and funding, and the currency of the information [12]. But many of the available medical websites have been created without any quality control by a third party. How can Internet health care visitors distinguish between such different resources?\nInternet users can find health and medical related websites in several ways. World Wide Web search engines (e.g., AltaVista, Excite, Infoseek and many others) provide the users with a list of websites that match a given topic, with the results ordered by syntactic similarity with the query [13]. Unfortunately, the quality of contents is not guaranteed.\nOn the other hand, certain websites indexes and review services, such as Medical Matrix (http:\/\/www.medmatrix.org\/) and HealthAtoZ (http:\/\/www.HealthAtoZ.com\/), offer systematic evaluations of medical resources on the Web [14], as a post publication editorial process. These rating systems could be an useful tool for guiding the visitors of medical websites [12]. However, authors who have reviewed these Internet resources, point out the variability of their evaluation criteria and their doubtful efficacy [14].\nThe quality of a given medical article on the Internet could be measured by the users opinion about it, for example by counting the number of times it is retrieved [15]. However, this idea has been criticized because it would replace the scientific peer review process with the opinion of the Internet users, whatever their qualification [6].\nDespite the differences between the printed medical information and the Internet, several evaluation tools from the former could be useful if applied on the \"Net.\" Similarly to printed medical journals, medical documents on the Internet could be ranked by their citation analysis [15,16], but no methods have been proven for use with medical websites. When an article is quoted in a paper, certain agreement among the authors may be supposed. Similarly, when a webmaster makes a link from his web site to another, certain credibility is given to the latter. In fact, the International Committee of Medical Journals Editors recommend caution when a link is made from a peer reviewed journal site to other sites [17]. If linking on the web can be equivalent to quoting in printed medical articles, a citation analysis on the web could be performed by the quantification of the links to a given medical web site.\nThe ideal method for assessing the quality of medical websites should provide a means of rating great amounts of medical web resources while respecting the World Wide Web peculiarities, such as its multimedia capabilities and changing contents. At the same time, it should at least be as reliable as systematic reviews of those resources by editorial boards. In summary, it should be a method born in the Internet but with the efficacy of those used in the printed media.\nIn this study, we evaluated the reliability of four websites characteristics as medical websites quality indicators. The four characteristis used: their authors' impact factor, their grade of updating, their daily visits and inbound links. The evaluations of a sample of pediatric websites by a number of Internet rating systems was the gold standard with which these websites characteristics were compared.\nMethods\nDuring March 1998 a subset of websites rating systems were compiled. From these, we selected a sample of websites that were studied during the first week of April 1998.\nEight web rating systems, whose evaluations were offered as figures, were compiled from previous studies [13,14] (Table 1). One half of the selected rating systems gave the results of their evaluations by means of graphic analog scales, and the other half by numeric scales. Every web site evaluated by these rating systems that provided information about child health, whether for lay people or health professionals, was included in the study. Some of these rating systems (e.g., Lycos Top 5%) provides the visitors with a search tool by keyword. In these cases, the websites were selected using the keywords \"Pediatrics\", \"Infancy\", \"Child health\", and \"Child Care.\" For the remaining rating systems, the pediatric websites were compiled manually. Those websites not accessible twice during the study period were excluded.\nOnly three rating systems (Medical Matrix, Physician Choice, and Six Senses) gave information about their editorial boards. Most of their members were physicians. Two of the web rating systems only gave a global result of their websites evaluation (Medical Matrix and Magellan), while the rest (HealthAtoZ, Argus Clearinghouse, Lycos Top 5%, Sympatico Health, Physician Choice, and Six Senses) gave a result for each considered criterion. Content was a common criterion to all the eight ranking systems. Therefore, the results of the evaluation of each web site were divided in two categories, content and non-content (design) aspects. In order to make comparisons, the results of the evaluations of the websites supplied by each rating system were transformed to a one hundred scale.\nTable 1\nCompiled web sites ranking systems. The results of evaluations are showed as two possible types of scales, graphic analog (A) or numeric (N)\nRating systems (Included\/excluded web sites)\nUniform Resource Locator\nType of scale\nArgus Clearinghouse Seal of Approval (16\/1)\nhttp:\/\/www.clearinghouse.net\/cgi-bin\/chadmin\/viewcat\/Health___Medicine?kywd++\nA\nHealthAtoZ (241\/66)\nhttp:\/\/www.healthatoz.com\n*\nLycos Top 5% (8\/3)\nhttp:\/\/point.lycos.com\/topics\/Health_Overall.html\nN\nMagellan Internet Guide (40\/11)\nhttp:\/\/www.mckinley.com\/magellan\/Reviews\/Health_and_Medicine\/index.magellan.html\nA\nMedical Matrix (75\/11)\nhttp:\/\/www.medmatrix.org\/SPages\/Pediatrics.asp\nA\nPhysician's choice (4\/0)\nhttp:\/\/www.mdchoice.com\/pcsites.htm\nN\nSix Senses Seal of Approval (4\/0)\nhttp:\/\/www.sixsenses.com\/winners.html\nN\nSympatico Health (8\/1)\nhttp:\/\/www1.sympatico.ca\/Contents\/Health\/LISTS\/D3-C03_all1.html\nA\n* Graphic analog scale developed in numeric\nWhen provided, the daily visits registered by the websites visits counters were recorded. In some websites the date from which the counter was started was not available. Thus, their webmasters were asked for this information by electronic mail, and it was included in the statistical study if provided before the end of the observation period, 15th April 1998.\nThe websites authors and editors' names were searched in 1997 MEDLINE [18], and their articles were registered. Their impact factors of the journals wherein they were published were obtained by using the 1996 Science Citation Index (Institute for Scientific Information, Philadelphia, PA). The impact factor of a given web site author was the sum of the impact factors of his or her articles. For institutional websites only the name of the web editor was considered.\nWhen provided, the time since the last update was also recorded.\nFinally, by means of the Web search engine Infoseek [19], we calculated how many websites on the Internet linked to each web site of our sample. The searching strategy by syntax of this engine allows to know the websites that are linked to a given web site [20]. As a web site may be linked not only from external websites but also from websites of its own organization, we only considered external links. Although other search engines such as AltaVista, Excite or HotBot offer similar searching options, we chose Infoseek because it provided the results of the queries grouped by web site, which makes the exclusion of the internal links easier.\nComparison of means was performed by Mann-Whitney U test, and correlation analysis by means of Spearman's correlation coefficient ( rS). P values equal or less than .05 were considered significant. All computations were made with SPSS for Windows 7.0 (SPSS Inc., Chicago, IL) statistical package.\nResults\nAfter excluding 93 non-accessible websites, a total of 363 pediatric websites were compiled.\nTable 2\nCorrelations among the number of daily visits to the web sites, the impact factor of their authors or editors, the grade of update, and the number of links that receive. NS means not significant\nNumber of inbound links rS p\nVisits\/day rS p\nAuthor's impact factor rS p\nVisits\/day\n.46 .005\nAuthor impact factor\nNS\nNS\nWeeks since the last update\n-.36 <.001\nNS\nNS\nTable 3\nCorrelation among the number of links and visits to the web sites, the impact factor of their authors, and the time since the last update, and the results of their evaluation by HealthAtoZ and Medical Matrix. No significant correlations were demonstrated with the other systems. Medical Matrix only provides total results, does not specify results by contents and non-contents aspects\nNumber of Inbound Links\nVisits\/day\nAuthor impact factor\nWeeks since the last update\nrS\np\nrS\np\nrS\np\nrS\np\nHealthAtoZ\nTotal Contents Non contents\n.29 <.001 .30 <.001 .24 <.001\nNS\nNS\n-.19 .04 -.23 .00 NS\nMedical Matrix\nTotal\nNS\n.79 .03\nNS\nNS\nOn average, the websites of our sample received links from 470 other sites on the Internet (range, 0 to 3574). In 48% of the websites, information on their last update was given. On average, they had been updated 47.5 weeks before (range, 0 to 395). Only 10% of the websites had a visit counter, and the average daily visits were 470 (range, 1.2 to 3145). Seven visit counters did not distinguish among different visitors, that is, they registered any visit to their websites. In 137 websites (38%) the editor\/author's name was given, but only 60 of them had published at least one article since January 1997 in the journals included in MEDLINE database. Their average impact factor was 2.14.\nFigure 1\nWeeks since the last update for the total of the sample, n=363, and for the websites evaluated at least by two rating systems, n=25 (median, 25th and 75th percentiles)\nFigure 2\nNumber of inbound links to websites for the total of the sample, n=363, and for the websites evaluated at least by two rating systems, n=25 (median, 25th and 75th percentiles)\nTable 4\nTop 50 pediatric web sites of the sample (N= 363) by the number of their inbound links. The weeks since the last update, the number of daily visits to the web sites and their editor\/author's impact factor are also provided. In parenthesis, the place that each web site would obtain if ranked by the two latter criteria. In italics, those web sites indexed at least by two rating systems. Missing values are due to the lack of visits counter, editor's name, or information about the last update, for many web sites\nUniform Resource Locator\nNumber of inbound links\nDaily visits to web sites\nWeb site editor\/author's impact factor\nWeeks since the last update\n1\nhttp:\/\/www.merck.com\n3574\n-\n-\n13\n2\nhttp:\/\/www.ucalgary.ca\/~dkbrown\/index.html\n2355\n1620 (3\u00ba)\n0 ( 360\u00ba)\n-\n3\nhttp:\/\/KidsHealth.org\n1109\n-\n-\n-\n4\nhttp:\/\/www.psych.med.umich.edu\/web\/aacap\n927\n-\n-\n3\n5\nhttp:\/\/www.aap.org\n896\n-\n-\n1\n6\nhttp:\/\/www.chadd.org\n785\n-\n-\n4\n7\nhttp:\/\/www.castleweb.com\/diabetes\n767\n-\n-\n-\n8\nhttp:\/\/www.medconnect.com\n714\n-\n-\n-\n9\nhttp:\/\/www.aaaai.org\n677\n-\n-\n-\n10\nhttp:\/\/www.aacap.org\/web\/aacap\n612\n-\n-\n4\n11\nhttp:\/\/www.nas.com\/downsyn\n572\n-\n0 ( 360\u00ba)\n1\n12\nhttp:\/\/www.childbirth.org\n534\n-\n-\n-\n13\nhttp:\/\/web.syr.edu\/~jmwobus\/autism\n502\n-\n0 ( 360\u00ba)\n-\n14\nhttp:\/\/oncolink.upenn.edu\/disease\n487\n-\n10.1 (8\u00ba)\n9\n15\nhttp:\/\/www.jdfcure.com\/index.html\n428\n-\n-\n-\n16\nhttp:\/\/www.mic.ki.se\/Diseases\/index.html\n423\n1412 (5\u00ba)\n-\n-\n17\nhttp:\/\/www.asf.org\n365\n940 (6\u00ba)\n-\n1\n18\nhttp:\/\/www.mdcc.com\n365\n-\n-\n1\n19\nhttp:\/\/www.mc.vanderbilt.edu\/peds\n357\n-\n-\n-\n20\nhttp:\/\/www.ama-assn.org\/journals\/standing\/jama\/jamahome.htm\n330\n-\n-\n-\n21\nhttp:\/\/www.med.jhu.edu\/peds\/neonatology\/poi.html\n322\n253 (10\u00ba)\n9.3 (13\u00ba)\n2\n22\nhttp:\/\/www.wish.org\n317\n-\n-\n-\n23\nhttp:\/\/education.indiana.edu\/cas\/adol\/adol.html\n312\n-\n0 ( 360\u00ba)\n52\n24\nhttp:\/\/www.kidsdoctor.com\n297\n-\n0 ( 360\u00ba)\n-\n25\nhttp:\/\/www.xmission.com\/~gastown\/safe\n297\n94 (20\u00ba)\n-\n-\n26\nhttp:\/\/www.childquest.org\n287\n-\n-\n6\n27\nhttp:\/\/www.uab.edu\/pedinfo\n284\n-\n-\n-\n28\nhttp:\/\/www.childsecure.com\n255\n-\n-\n-\n29\nhttp:\/\/www.mc.vanderbilt.edu\/peds\/pidl\n254\n-\n0 ( 360\u00ba)\n1\n30\nhttp:\/\/www.stjude.org\n251\n-\n-\n-\n31\nhttp:\/\/www.nccf.org\n249\n70 (25\u00ba)\n-\n8\n32\nhttp:\/\/www.mda.org.au\n238\n-\n0 ( 360\u00ba)\n12\n33\nhttp:\/\/www.peds.umn.edu\n235\n-\n0 ( 360\u00ba)\n2\n34\nhttp:\/\/www.csmc.edu\/neonatology\n232\n117 (15\u00ba)\n-\n1\n35\nhttp:\/\/med-aapos.bu.edu\n225\n-\n0.4 (51\u00ba)\n3\n36\nhttp:\/\/www.jhbmc.jhu.edu\n220\n-\n-\n3\n37\nhttp:\/\/sids-network.org\n214\n3145 (1\u00ba)\n0.3 (54\u00ba)\n1\n38\nhttp:\/\/www.diabetes.com\n212\n-\n-\n-\n39\nhttp:\/\/sids-network.org\/index.htm\n208\n3145 (1\u00ba)\n0.3 (55\u00ba)\n1\n40\nhttp:\/\/www.oneworld.org\/scf\n205\n-\n-\n-\n41\nhttp:\/\/www.childmmc.edu\n204\n-\n-\n13\n42\nhttp:\/\/www.os.dhhs.gov\/hrsa\/mchb\n197\n-\n-\n7\n43\nhttp:\/\/www.wp.com\/pedsrheum\n197\n81 (23\u00ba)\n11.7 (6\u00ba)\n-\n44\nhttp:\/\/dem0nmac.mgh.harvard.edu\/neurowebforum\/neurowebforum.html\n188\n2441 (2\u00ba)\n0 ( 360\u00ba)\n-\n45\nhttp:\/\/pedsccm.wustl.edu\n179\n145 (13\u00ba)\n1.0 (39\u00ba)\n2\n46\nhttp:\/\/www.drgreene.com\n179\n-\n-\n-\n47\nhttp:\/\/www.medsch.wisc.edu\n162\n-\n-\n-\n48\nhttp:\/\/www.blindcntr.org\/bcc\n150\n-\n-\n-\n49\nhttp:\/\/home.coqui.net\/titolugo\n144\n68 (26\u00ba)\n0.2 (56\u00ba)\n1\n50\nhttp:\/\/www.chmcc.org\n141\n-\n-\n1\nOnly 25 websites of the sample were indexed and evaluated at least by two rating systems, and none by the eight. This subset of websites showed significantly better results of the evaluation of their contents and design by HealthAtoZ, and higher grade of updating (Figure 1) and higher number of inbound links (Figure 2). When the evaluations of these 25 websites by the different rating systems were compared, no significant correlations were found. Changes regarding the average impact factor of the authors of the websites or the number of daily visits could not be demonstrated in this subset of websites.\nSome interesting correlations between the results of the evaluations of the websites and the rest of study variables were found. The number of links received by the websites significantly correlated with their daily visits and with the time since the last update (Table 2). The number of inbound links also correlated with the results of the websites evaluation by HealthAtoZ (Table 3).\nThe number of daily visits significantly correlated with the results of the websites evaluation made by Medical Matrix, and the grade of updating significantly correlated with the results of the contents and designs evaluation made by HealthAtoZ (Table 3).\nFinally, no correlation was demonstrated between the average impact factor of the websites authors and the other variables.\nThe top fifty pediatric websites of the sample are shown in Table 4, ordered by the number of their inbound links according to the Infoseek indexing engine. More than a half of the 25 websites indexed by at least two rating systems may be found among these top fifty websites.\nDiscussion\nIn this study, certain websites characteristics that depend on the users' preferences have been compared with evaluations of pediatric resources on the Web by third parties. Although rating systems have been previously criticized because their editorial boards frequently do not employ uniform criteria [13], we have considered them as the standard method because it somewhat represents a post-publication review process.\nSome aspects of our method are open to discussion. Firstly, the reliability of the data regarding the daily visits and the updating frequency depends on the accuracy of the information that the websites editors offer in their sites. In this sense, we considered the grade of updating of the websites by the dates of their last changes. Clearly these changes could involve very different aspects and in different grades, and not necessarily provide more current contents. However, we believe that it could demonstrate the editor's efforts in maintaining or increasing the interest of his web site for the visitors.\nThe results regarding the number of daily visits to the websites must be considered with caution when comparing one web site to another, because some visit counters were set to register every visit, instead of every distinct visitor. Nevertheless, both can be considered usage indexes of a given web site.\nOn the other hand, quantification of links to the websites clearly depends on the power of the search engine we employ. By no means our results show the total number of links to the websites in our sample. In fact, a previous article states that it would be necessary to combine the databases from at least five large search engines to cover the most of the web [21].\nAlthough all bibliometric indexes have limitations [22,23], we employed the impact factor as a measure of the webmasters' publishing capacity because it is a classical indicator of the quality of biomedical articles. Recently, it has been suggested that every medical web site should be evaluated following some basic criteria [24]. One of the more accepted criteria is that the authorship must be clearly stated, as a basic means for assessing the reliability of the web site contents. However, we could not demonstrate that the more highly evaluated, the most updated, or the most linked or visited pediatric websites, had the authors with the highest publishing capacity measured by their impact factor. In other words, some web quality standards do not correlate with classical quality standards from the printed media such as the impact factor of a given author's articles.\nWe could not find statically significant correlations among the evaluations of the websites by the different rating systems. This is probably due to the small size of the subset of websites indexed and evaluated by all the systems, and their different evaluation criteria. However, some interesting data were found when we considered the correlations among the four websites characteristics and the evaluations. We found that the best websites for HealthAtoZ, the largest analyzed rating system, were the most updated and the most linked ones. On the other hand, the most valuable websites for Medical Matrix, the second rating system by size, were the most visited ones. In any case, both the number of daily visits and the time since the last update highly correlated with the number of inbound links. The lack of correlation among the four variables and the evaluations by the other rating systems could be due to their little contribution to our sample.\nMany efforts to establish quality criteria will have limited efficacy due to the dynamic behaviour of the Internet as a publishing medium. In fact, a recent article demonstrates the lack of consensus among the editorial boards of a large sample of evaluation and rating systems regarding the evaluation criteria they employ. The same authors pointed out that \"... it may be difficult or even inappropriate to develop a static tool or system for assessing health related websites.\" [25] Therefore, the question could be to provide context to this issue. That is, to know how good a given medical web site is in comparison with the rest of medical websites. A democratic and feasible method for reaching this objective could be let the Internet community say which medical websites are the best ones, that is, which they usually visit or which they usually recommend by linking to them. Moreover, we believe that the fact that these usage indexes correlate with the evaluations by third parties, qualifies them as quality markers.\nEysenbach and Diepgen [16] have recently proposed that an ideal quality control system for medical resources on the Internet should take in account the users opinion, and not only their evaluation by a third party, that is, a \"downstream filtering\" and not only an \"upstream filtering\" approach. More interestingly, our study demonstrates certain agreement among both approaches in identifying high quality resources.\nLaPorte et al [15] proposed an electronic publishing system in which the impact of a given resource on the Internet could be measured by counting how many times the document was retrieved or quoted. The introduction of the citation analysis of the medical resources on the web as a method to assess their quality has been recently proposed [16]. On the other hand, a very promising software system is being developed by Kleinberg [26,27]. This system would provide the users with a way of knowing the very best of the web on a given topic in a faster and more complete way than commercial human compiled directories. This system is based in the identification of two subsets of websites when a query on a given topic is made, those websites containing a lot of information about the topic (authoritative websites) and those which contain large amounts of links to the former (hub sites). Our work demonstrates that those authoritative websites, that is the more linked ones, are indeed the best ones regarding the evaluation of its contents and design by the editorial boards of some large web rating systems.\nThe citation analysis of biomedical journals has been a classic tool in assessing their relative quality [28]. Similarly, medical web resources could be ranked by a \"webcite index\" [16], which is not yet defined. Linking in the World Wide Web could be equivalent to quoting in printed publications, and its quantification could be useful for measuring the relative quality of medical websites. Some indexes could be created to make more rational comparisons among websites with different sizes. For example, in the same way that the calculation of the impact factor of a given medical journal takes into account the number of articles published by that journal yearly, the size of a given domain could be considered to obtain some indexes that would express more accurately the grade of linkage of a medical web site. Moreover, Platform for Internet Content Selection (PICS) [29], an infrastructure that could be applied as a filtering system of the medical information on the Net [16], could incorporate these indexes as one of the meta- data assigned to every medical document as electronic labels. Then, these electronic labels could be checked automatically by an user's browser, bypassing those documents with a \"webcite index\" not high enough. A problem could be how to avoid false \"self-labelling\" by dishonest webmasters. In any case, more work is needed to give answers to these and other technical questions on the emerging field of Webometrics [30].\nAn evaluation system based on this quantification would bring advantages and risks. Rankings could be generated very quickly and in an objective way, because the Internet community by itself would evaluate great amounts of medical websites. However, this evaluation process would be made a posteriori, and the potential harmful effects of the diffusion of documents without enough quality could not be avoided. Therefore, this method could not replace previous editorial effort that warrants a minimal quality for each resource.\nOur work demonstrates that the visitors of pediatric websites and the editors of websites on the \"Net,\" so called webmasters, show certain maturity when they have to identify the pediatric resources with high quality. We believe that the key point is how to augment the proportion of these resources. An important issue could be to establish a citation style not only for articles from peer reviewed electronic journals [31], but also for any medical document on the Net. The prestige that citation in a printed journal represents will stimulate high quality publishing on the Internet, and web site editors will employ enough review processes to obtain the necessary quality. A web site's ranking system based on the citation analysis on the web by the quantification of links would be an additional incentive. The more valuable resources will attract the Internet users' visits and the webmasters' links, and very likely the best funding and financial supports.\nIn summary, although the Internet provides a very different publishing medium, traditional means borrowed from printed journals could also be used with this electronic media for achieving minimal levels of quality. These include certain peer review processes, that enhance the rigor of the documents submitted for publication taking in account the peculiarities of this media, and linking analysis as a measure of the citation on the World Wide Web.","keyphrases":["internet","bibliometrics","webometrics","health education","information systems","computer communication networks","cybermetrics","web metrics"],"prmu":["P","P","P","M","R","M","U","M"]}
{"id":"Mol_Hum_Reprod-1-1-2408935","title":"Regulation of spindle and chromatin dynamics during early and late stages of oocyte maturation by aurora kinases\n","text":"Examination of factors regulating oocyte chromatin remodeling is crucial to circumvent embryonic aneuploidy and resulting defects. Aurora kinases (AURK) are involved in regulation of chromatin remodeling, however, little attention has been paid to AURKs in regard to oocyte maturation. Meiotically incompetent mouse oocytes contain transcripts for all three Aurk isoforms: A, B and C. Upon achieving meiotic competence, oocytes showed significant increases in transcript levels of all three Aurk isoforms and transcript levels remained unchanged as oocytes progressed through meiosis, with AurkA being the predominant isoform. Inhibition of oocyte AURKs during the prophase\u2013metaphase I (MI) transition via inhibitor ZM447439 (ZM) had no effect on germinal vesicle breakdown. However, meiotic spindles were malformed, and microtubule organizing centers and chromatin were scattered. Chromosomal spreads of MI oocytes indicated AURK inhibition resulted in abnormal chromosome condensation. Furthermore, inhibition of AURK during prophase I\u2013MII prevented completion of MII and extrusion of the polar body. Inhibition of AURKs during the MI\u2013MII transition resulted in significantly fewer cells progressing to MII and induced aberrant chromatin remodeling. Further analysis indicated that inhibition of AURKs resulted in absence of histone-H3 phosphorylation at serine 10 and 28. These data suggest a ZM-sensitive AURK may be an oocyte histone-H3 kinase capable of regulating chromatin remodeling throughout oocyte meiosis, both pre- and post-MI.\nIntroduction\nIn vitro oocyte maturation offers immense potential for treatment of infertility, however, current systems are relatively inefficient (Tan et al., 2007). Additionally, mammalian oocytes are notorious for high rates of chromosomal abnormalities (Hassold and Hunt, 2001), resulting in subsequent embryonic aneuploidy, infertility and congenital defects. Unfortunately, components of successful oocyte maturation and regulation of these events remains enigmatic. Therefore, understanding regulatory mechanisms involved in oocyte meiotic maturation, especially those controlling chromatin remodeling, is imperative to establish therapies to improve current assisted reproductive technologies and circumvent oocyte-derived infertility and aneuploidy-induced congenital defects.\nRemodeling of chromosomes during oocyte meiosis begins when homologues initially pair and condense via actions of the synaptonemal complex during initiation of prophase I, accompanied by homologous recombination and crossing-over events (Vallente et al., 2006). Chromatin subsequently decondenses as oocytes enter a phase of quiescence prior to completing prophase I. In response to the pre-ovulatory gonadotropin surge, follicle-enclosed oocytes resume meiosis and homologues condense in preparation for a reductional division (Mehlmann, 2005). A bipolar meiotic spindle forms, consisting of polymerized microtubules, and attaches to homologues at their centromeres. Subsequently, physical contact between homologous pairs at chiasmata counteract forces pulling apart homologues, resulting in alignment of chromosomes along the metaphase plate, signaling completion of metaphase I (MI). The meiotic spindle then facilitates separation and segregation as homologues are pulled toward opposite spindle poles at the beginning of anaphase. Oocytes progress through telophase, resulting in disproportionate cytokinesis and extrusion of the first polar body signaling completion of meiosis I (Wang and Sun, 2006). Subsequently, oocytes forego DNA replication and re-arrest at MII.\nReversible phosphorylation, controlled via actions of protein kinases and phosphatases, is an extremely important regulator of oocyte meiosis, and well-suited to the rapid changes required during the cell cycle (see reviews, Swain, 2007; Swain and Smith, 2007). Extensive research has been conducted on the role of kinases such as cyclin-dependant kinase (CDK1) and mitogen-activated protein kinase (MAPK) in mammalian oocytes (Motlik et al., 1998; Abrieu et al., 2001). Additionally, roles for protein phosphatases (PPP) in control of oocyte germinal vesicle breakdown (GVBD) (Alexandre et al., 1991; Gavin et al., 1991, 1994; Swain et al., 2003), spindle dynamics (Alexandre et al., 1991; Lu et al., 2002) and chromatin remodeling have been identified (Mailhes et al., 2003; Swain et al., 2007). However, relatively little attention has been paid to aurora kinases (AURK) in regard to mammalian oocyte maturation.\nAURKs are a family of serine\/threonine protein kinases responsible for regulating several mitotic cell cycle events important to maintaining proper cellular ploidy, including chromosome condensation, spindle dynamics and cytokinesis (see review, Carmena and Earnshaw, 2003). Thus, considering high rates of aneuploidy associated with oocyte meiosis, AURKs may play an instrumental role in the female gamete was well. Based primarily on research from lower eukaryotes and mitotically dividing mammalian cells, three AURKs exist categorized by sequence differences and subcellular localization; AURKA, AURKB and AURKC (Nigg, 2001). Aurora-A has been identified in mammalian oocytes localized to nuclei in GV-intact oocytes; to the spindle, spindle poles and condensing chromatin during MI; and to spindle poles at MII (Yao and Sun, 2005). Neutralization of oocyte AURKA activity delays GVBD and distorts MI spindle organization (Yao et al., 2004). Aurora-B kinase is known as the equatorial kinase and localizes to condensed chromatin during mitosis and meiosis in lower eukaryotes, where it is thought to regulate chromosome condensation (Hsu et al., 2000; Murnion et al., 2001) and homologue separation (Kaitna et al., 2002; Rogers et al., 2002). Aurora-C kinase is the least studied AURK isoform, previously reported to be testes-specific with localization to centrosomes (Tseng et al., 1998; Kimura et al., 1999; Hu et al., 2000); though transcript for AurkC has now been identified in human cumulus\u2013oocyte complexes (Assou et al., 2006) and reports indicate that it binds to chromosomal passenger proteins during mitosis (Li et al., 2004; Sasai et al., 2004; Yan et al., 2005).\nObjectives of this study were to determine which Aurk isoform transcripts are present in oocytes; examine functional roles of AURK activity during various meiotic stages of mouse oocyte maturation, and identify AURK phosphoprotein substrates and targets of actions, focusing primarily on regulation of chromatin remodeling during the first meiosis.\nMaterials and Methods\nAll procedures described within were reviewed and approved by The University Committee on Use and Care of Animals at the University of Michigan and were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.\nMouse stimulation and oocyte collection\nMeiotically incompetent GV-intact oocytes were collected from 11-day-old female CF1 mice (Harlan, Indianapolis, IN). Meiotically competent GV-intact oocytes were collected from 20\u201323-day-old CF1 female mice, 42\u201344 h following injection with 10 IU eCG (Sigma, St Louis, MO). Oocytes were isolated by manual rupturing of antral ovarian follicles in Hepes-buffered human tubal fluid medium (HTFH; Irvine Scientific, Santa Ana, CA) supplemented with 0.3% w\/v polyvinylpyrrolidone (Sigma).\nRNA isolation, reverse transcription and real-time PCR\nOocyte total RNA was extracted from 50 oocytes at each development stage using Picopure RNA isolation kit (Arcturus Bioscience, Mountain View, CA) following manufacturer\u2019s instructions. Oocyte cDNA was synthesized using 125 pmol random hexamer, 500 \u00b5M dNTP, 20 U RNase inhibitor and 62.5 U MultiScribeTM reverse transcriptase (ABI systems) in a final volume 50 \u00b5l. Primers for mouse AurkA, AurkB and AurkC were designed with no sequence overlap between isoforms (AurkA\u2014forward primer: 5\u2032 cactagcaaagagccaacca 3\u2032, reverse primer: 5\u2032 ggtggcttcaatagggtgtt 3\u2032; AurkB\u2014forward primer: 5\u2032 cctgacctactgccacaaga 3\u2032, reverse primer: 5\u2032 gccaaagtctgcaatcttca 3\u2032; AurkC\u2014forward primer: 5\u2032 ctgccatgagaagaaggtga 3\u2032, reverse primer: 5\u2032 gtccagagtcccacacattg 3\u2032). Real-time PCR was performed on Applied Biosystems 7300 Real-Time PCR system. Each PCR was performed with 1.5 oocyte equivalents of cDNA added to SYBR Green PCR Master Mix (Applied BioSystems, Foster City, CA). In addition, control reactions were conducted consisting of no template with primers and master mix. Real-time PCR reactions were carried out for 40 cycles (95\u00b0C for 15 s, 60\u00b0C for 1 min) after initial 10 min incubation at 95\u00b0C. Following PCR, products were isolated and run on a 2% agarose gel for 60 min at 100 V to verify size of the amplified product. Additionally, DNA was isolated from gels using QIAquick Gel Extraction Kit (Qiagen, Chatsworth, CA) and subjected to DNA sequencing to verify identity of the product. Standard curve method was used to compare relative abundance of a single Aurk isoform between oocyte meiotic stages using normalization of \u03b2-actin levels. Data were collected over three replicates, with triplicate samples for each isoform and fold increases were based on meiotically incompetent GV-intact oocytes levels, which were normalized to 1. Statistical significance was determined using unpaired Student\u2019s t-test, P < 0.05). To examine relative abundance of all three isoforms within a single time point, we ensured primer efficiency of all samples were within a 5% range of an internal \u03b2-actin control. We then analyzed data using comparative Ct method.\nOocyte culture and AURK inhibition\nAurora kinase inhibitor ZM447439 (ZM, Astra Zeneca, Wilmington, DE) was dissolved in dimethylsulphoxide (DMSO) to obtain a 10 mM stock. Stock solution was dissolved in HTF to obtain final concentrations of 0.625, 1.25, 2.5, 5, 10 and 20 \u00b5M. Control treatments contained DMSO.\nTo assess effects of AURK inhibition on oocyte maturation, meiotically competent GV-intact oocytes (prophase I) were placed into culture in presence or absence of varying doses of ZM. Oocytes were assessed for GVBD and MII development at 2 and 16 h, respectively. Experiments were performed in triplicate and statistical differences in development were assessed using chi-square analysis with differences considered significant if P < 0.05.\nTo determine effects of AURK on chromosome condensation and spindle formation during the prophase I to MI transition, prophase I oocytes were matured in vitro to MI (7 h) in the presence or absence of ZM (10 \u00b5M) then subjected to immunocytochemistry (ICC) or processed for chromosome spreading. Prophase-I oocytes were also cultured to a time point consistent with MII to assess spindle and chromatin characteristics following extended AURK inhibition.\nTo assess effects of AURK inhibition on oocyte meiosis during the MI\u2013MII transition, oocytes were matured for 7 or 9 h in the absence of any chemical manipulation to allow normal spindle formation and chromatin remodeling. Oocytes were then cultured to MII (an additional 9 or 7 h) in presence or absence of 10 \u00b5M ZM, followed by assessment of chromatin positioning and spindle configuration. All experiments were performed in triplicate and nonparametric parameters were analyzed for significant differences by Chi-square.\nFinally, to begin to determine substrates for oocyte AURK, histone-H3 phosphorylation at ser10 and ser28 was assessed following AURK inhibition at various time points utilizing ICC and western blot analysis.\nImmunocytochemistry\nTo examine effects of AURK inhibition on spindle formation and metaphase chromosome positioning, MI and MII oocytes were attached to poly-lysine coated coverslips, and fixed in 2% (w\/v) paraformaldehyde with 0.05% (v\/v) Triton X-100 in phosphate-buffered saline (PBS) (pH = 7.3) for 30 min. Oocytes were then blocked overnight with 2% (w\/v) bovine serum albumin, 0.1 M glycine and 5% (w\/v) dry milk in PBS at 4\u00b0C. Oocytes were incubated with \u03b2-tubulin antibody (Sigma, 1:200) and pericentrin antibody (Abcam, 1:1000). Negative controls included non-immune mouse serum in place of primary antibody. After three 5 min washes with blocking solution, samples were reacted with the appropriate Alexa 568 and 488 conjugated secondary antibodies (Molecular Probes) at a 1:750 dilution for 1 h at 37\u00b0C. Following washing, slides were incubated with Hoescht 33 342 (1 \u00b5g\/ml) in PBS for 20 min at 37\u00b0C. Coverslips were then mounted on glass slides with 90% glycerol in PBS for fluorescence microscopic visualization under \u00d71000 on a confocal microscope.\nChromosomal spreading and analysis\nFollowing culture to MI in ZM (10 \u00b5M), oocytes were collected and prepared for chromosomal spreading (Hodges and Hunt, 2002). Briefly, zona pellucida were removed by exposure to 1% pronase in HTFH. Zona-free oocytes were then washed and fixed by carefully placing them onto a microscope slide dipped in a solution of 1% paraformaldehyde in distilled water (pH 9.2) containing 0.15% Triton X and 3 mM dithiothreitol. Slides were then placed into a humidified chamber overnight before being subjected to triplicate 5 min washes in PBS and air-dried at room temperature. To analyze chromosomal condensation, slides were placed into a 1% solution of Hoescht 33 342 in PBS for 10 min and subjected to three more washes in PBS. Glycerol mounting solution and a coverslip were added and slides were sealed. Chromosomal spreads were analyzed blind to treatment at \u00d71000 on a Leica DMR microscope. Statistical differences between treatment groups were analyzed using chi-square analysis.\nElectrophoresis and western blot analysis\nTo assess effects of AURK inhibition on oocyte histone-H3 phosphorylation, groups of oocytes (n = 100) were prepared for western blot analysis. Oocytes were placed in 2\u00d7 sodium dodecyl sulphate (SDS)\u2013polyacrylamide gel electrophoresis (PAGE) sample loading buffer [80 mM Tris\u2013HCl (pH = 6.8), 20% glycerol, 4% SDS, 4% \u03b2-mercaptoethanol, 0.04% bromophenol blue], vortexed and placed on ice for 15 min. Following sonication on ice for 10 s, samples were denatured at 90\u00b0C for 10 min and loaded for electrophoresis. Total protein from equal numbers of mouse oocytes was loaded in each lane and separated by one-dimensional SDS\u2013PAGE. Resolving gels were cast using 12% acrylamide; stacking gels contained 5% acrylamide. HeLa cell histone lysate was used as a positive control for recognizing phospho-ser10. Gels were equilibrated and transferred to Hybond-P PVDF transfer membrane (Amersham Life Science, Little Chalfont Buckinghamshire, UK) by Semi-Dry Electrophoretic Transfer Cell (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer\u2019s instructions. Blots were blocked in 5% nonfat milk in Tris-buffered saline (TBS)+0.5% Tween (TBST) at room temperature for 1 h and incubated with the appropriate primary antibody diluted in TBST + 5% nonfat milk overnight at 4\u00b0C with agitation. Antibodies included anti-phospho-ser10-histone H3 (1:1000, Upstate) and anti-phospho-ser28-histone-H3 antibody (1:500, Upstate). After complete washing in TBST, blots were incubated with the appropriate horse-radish peroxidase-conjugated IgG secondary antibody (diluted 1:2000) at room temperature for 1 h, washed in TBST and developed with ECL Plus reagents (Amersham Life Sciences) according to the manufacturer\u2019s instructions. To verify equal protein loading of lanes to allow densitometric analysis blots were stripped for 30 min in a 50\u00b0C water bath with agitation in a stripping buffer (62.5 mM Tris\u2013HCl, pH 6.7, 100 mM \u03b2-mercaptoethanol and 2% SDS). Completely stripped blots were blocked in 5% nonfat milk in TBST for 1 h at room temperature, then incubated with histone-H3 antibody (diluted 1:1000, Chemicon) overnight at 4\u00b0C with agitation and processed further as described above. Band densities were assessed using NIH imaging software, Image J.\nResults\nAURK isoforms in mouse oocytes\nTo determine specific AURK isoforms present in mouse oocytes, isoform-specific primers were designed for AurkA, AurkB and AurkC. Transcripts were amplified utilizing real-time PCR for all three isoforms in meiotically incompetent GV-intact oocytes. Significant increases in transcript levels for all three Aurk isoforms were identified in meiotically competent GV-intact and meiotically maturing oocytes, compared with incompetent oocytes (Fig.\u00a01). No differences in transcript levels were identified for any isoform between meiotically competent GV-intact, GVBD, MI or MII oocytes. Additionally, AurkA appeared to be the predominant isoform transcript, displaying an approximate 12-fold increase compared with AurkB and AurkC, which displayed comparable levels, in meiotically competent GV-intact oocytes (data not shown). Single bands were present on agarose gels for each isoform, corresponding to predicted sizes (AurkA\u201490 bp, AurkB\u2014100 bp, AurkC\u2014145 bp). Sequencing verified that amplified gene products shared 100% homology with mouse Aurk isoforms (data not shown).\nFigure\u00a01:\nGraphical representation demonstrating fold-increases of aurora kinase (Aurk) isoform levels between various oocyte types obtained from real-time PCR.\nDay 11 germinal vesicle intact (GVI) oocytes were used at controls and normalized to 1. (A) AurkA, (B) AurkB and (C) AurkC. Presence of an asterick represents statistical significance compared with groups without an asterick, P < 0.05.\nAURKs and oocyte meiotic progression\nTo determine the influence of AURK on oocyte meiotic progression, meiotically competent GV-intact oocytes (prophase I) were matured in varying doses of the AURK inhibitor ZM and development was checked at 2 and 16 h. Though a significant reduction was observed with 2.5 \u03bcM, Inhibition of AURK had no effect on oocyte GVBD at 2 h at any other dose examined compared with control treatments (Table\u00a0I). Furthermore, no differences were visually apparent at the light microscope level between ZM-treated and control oocytes at a time point consistent with MI development (7 h; Fig.\u00a02, inset). However, concentrations of ZM at 2.5, 5, 10 and 20 \u00b5M prevented all oocytes from progressing to MII at 16 h (Fig.\u00a03). Concentrations of 1.25 and 0.675 \u00b5M allowed a small portion of oocytes to extrude the first polar body at 16 h, which was significantly less than the percentage of MII oocytes obtained from control treatments, P < 0.01 (Table\u00a0I). Additionally, culturing GV-intact oocytes for 7 h to MI in presence of 5 or 10 \u00b5M ZM, followed by thorough washing and 9 h of culture in the absence of the AURK inhibitor, indicated that, although ZM may be washed out from blocking the ATP binding pockets, defects caused by ZM treatment were not reversible, as oocytes were unable to complete meiosis and extrude the first polar body (data not shown). Based on these data, a dose of 10 \u00b5M ZM was selected for future experiments to ensure all AURK isoforms were inhibited. This dose has been used in other studies on mammalian oocytes (Jelinkova and Kulbeka, 2006), and is lower than the 20 \u00b5M used in other studies (Gadea and Ruderman, 2005).\nFigure\u00a02:\nRepresentative micrographs demonstrating effect of aurora kinase (AURK) inhibition on oocyte meiotic spindle formation.\nInhibition of mouse oocyte AURKs with 10 \u00b5M ZM447439 (ZM) for 7 h had no observable effect on oocyte development at the light microscope level (inset images). However, immunocytochemical examination demonstrated that morphology of the meiotic spindle of ZM-treated oocytes appeared abnormal, with polymerized microtubules (\u03b2-tubulin: green), microtubule organizing centers MTOCS (pericentrin: red\/orange) and chromatin (blue) was scattered around the metaphase plate, compared with vehicle treated controls. Furthermore, 16 h treatment of oocytes with ZM resulted in arrest of oocytes at a MI-like stage (inset). Immunocytochemical examination demonstrated aberrant polymerized microtubules, MTOCs and condensed chromatin (blue) and MTOCs (red\/orange). Polar body of control MII oocytes is indicated by PB.\nFigure\u00a03:\nRepresentative micrographs of oocyte chromosome spreads demonstrating negative impact of AURK inhibition on oocyte chromatin remodeling.\nTreatment of oocytes with ZM447439 (ZM-10 \u00b5M) for 7 h resulted in metaphase I oocytes (MI) with significantly greater defects in bivalent formation, compared with controls (P < 0.0001).\nTable I.\nDevelopment of mouse oocytes following treatment in varying doses of Aurora kinase inhibitor ZM447439 (ZM).\nZM concentration (\u00b5M)\n2 h GVBD\n16 h MII\n0\n115\/121 (95%)a\n38\/52 (73%)a\n0.625\n29\/30 (97%)\n8\/30 (27%)b\n1.25\n30\/35 (86%)\n8\/26 (31%)b\n2.5\n84\/99 (85%)b\n0\/54 (0%)c\n5\n59\/66 (89%)\n0\/28 (0%)c\n10\n55\/64 (86%)\n0\/28 (0%)c\n20\n55\/64 (85%)\n0\/28 (0%)c\nSignificant differences in development between treatments within a time point are indicated by different superscripts, P < 0.01.\nAURKs and oocyte spindle morphology and chromatin remodeling\nProphase\u2013MI transition\nTo begin to determine a temporal window when AURK inhibition may be conveying observed phenotypes, experiments examined the effects of AURK inhibition during the prophase I\u2013MI transition. Aberrant spindle morphology and improper positioning of chromatin were observed in oocytes cultured for 7 h in presence of 10 \u00b5M ZM (19% normal, n = 32) compared with controls (91% normal, n = 35; P < 0.0001; Fig.\u00a02). Similar patterns were also obtained from treatments containing 2.5, 5 and 20 \u00b5M ZM (data not shown).\nTo examine effects of AURK inhibition on oocyte chromosome condensation in greater detail, chromosomal spreads of MI oocytes were examined following 7 h of ZM (10 \u00b5M) treatment from prophase I. Inhibition of AURKs resulted in oocytes with a significant reduction in normal chromosome condensation (0%, n = 21) compared with control treatments (95%, n = 22), as evidenced by the inability to resolve bivalents, P < 0.001 (Fig.\u00a03).\nProphase I\u2013MII transition\nCulture of oocytes for 16 h in 10 \u00b5M ZM during the prophase I\u2013MII transition indicated that arrest of oocytes was not due to inability of microtubule polymerization as \u03b2-tubulin staining indicated microtubules polymerized around chromatin (Fig.\u00a02). Additionally, pericentrin staining indicated apparent microtubule organizing centers (MTOC) assembly. However, spindle formation and MTOC localization were disrupted compared with untreated controls. Furthermore, chromatin was scattered throughout the meiotic spindle, with the pattern of normal chromatin remodeling significantly reduced following ZM treatment (0%, n = 44) compared with controls (81%, n = 32), P < 0.001.\nMI\u2013MII transition\nTo determine effect of AURK inhibition following normal chromatin remodeling and spindle formation, oocytes were cultured for 7 or 9 h in the absence of ZM (cells typically at MI or AI, respectively). Subsequently, these oocytes were cultured to a time point consistent with MII (an additional 9 or 7 h) in presence or absence of ZM. Treatment of oocytes for 9 h with ZM resulted in significantly less cells progressing to MII (53%, n = 117) compared with 80% of control oocytes (n = 121), P < 0.05. Treatments of oocytes for 7 h with ZM also resulted in significantly fewer cells progressing to MII (58%, n = 88), compared with controls (73%, n = 78; P < 0.05). All MII oocytes obtained following ZM treatment displayed scattered chromatin around the metaphase plate and slightly irregular shaped spindles (Fig.\u00a04).\nFigure\u00a04:\nRepresentative micrographs of oocytes cultured to a time point consistent with MII in presence or absence of 10 \u00b5M AURK inhibitor ZM447439 during the MI-MII transition.\nControl MI oocytes developed normally to MII, extruding the first polar body and displaying condensed chromatin on the metaphase plate (blue) within the meiotic spindle with normally condensed \u03b2-tubulin (red) (A). However, following AURK inhibition, a portion of oocytes arrested prior to MII, displaying scattered chromatin within a malformed meiotic spindle (B). Those oocytes that did complete MII under AURK inhibition displayed disorganized chromatin within the meiotic spindle (C). Similar patterns of chromatin disorganization and inability to complete cytokinesis and progress to MII were also observed following culture of oocytes to MII (7 h) following AURK inhibition from a time point where cells are beginning to enter anaphase (E and F) compared with controls (D).\nAURKs and oocyte histone phosphorylation\nBecause of reported roles for histone-H3 phosphorylation in chromatin remodeling, phosphorylation of histone-H3 following AURK inhibition was examined as a possible cause of aberrant condensation during the prophase I to MI transition. Western blot analysis and ICC utilizing phospho-ser10 histone-H3 antibody was performed. Inhibition of AURK for 2 or 7 h with 10 \u00b5M ZM resulted in a total lack of histone-H3 ser10 phosphorylation (Fig.\u00a05A and B). Treatment of oocytes with ZM also completely inhibited phosphorylation of histone-H3 at ser28, as evidenced by ICC and western blot (Fig.\u00a06A and B). These data raise the possibility that a ZM-sensitive AURK is an oocyte histone-H3 kinase and that histone-H3 phosphorylation may be influencing normality of oocyte metaphase chromatin condensation and subsequent separation and segregation of homologues.\nFigure\u00a05:\nRepresentative micrographs and western blot demonstrating inhibition of oocyte aurora kinases (AURK) results in ser10-histone-H3 hypophosphorylation.\n(A) Culture of GV-intact oocytes for 2 h to allow germinal vesicle breakdown(GVBD) in presence of 10 \u00b5M ZM447439 resulted in a total lack of ser10-Histone-H3 phosphorylation (d), compared with controls (a). A similar reduction in ser10 phosphorylation was observed following 7 h (MI) of culture in the presence of ZM (j) compared with controls (g). Chromatin was stained with Hoescht and is pictured in blue (b,e,h,k). Overlays are also indicated (c,f,i,l). (B) Western blot analysis confirmed that ZM treatment inhibits ser10 phosphorylation (n = 100 oocytes\/lane).\nFigure\u00a06:\nRepresentative micrographs and western blot demonstrating inhibition of oocyte aurora kinases (AURK) results in ser28-histone-H3 hypophosphorylation.\nChromatin is stained in blue, while phospho-ser28-histone-H3 is stained in red. (A) Culture of GV-intact oocytes for 2 h to allow germinal vesicle breakdown(GVBD) in presence of 10 \u00b5M ZM447439 (ZM) resulted in a total lack of ser28 phosphorylation (b), compared with controls (a). A similar reduction in ser28 phosphorylation was observed following 7 h (MI) of culture in the presence of ZM (d) compared with controls (c). (B) Western blot analysis confirmed that ZM treatment inhibits ser28 phosphorylation (n = 100 oocytes\/lane).\nDiscussion\nMaintaining integrity of chromatin remodeling is especially important in the oocyte considering its extreme susceptibility to aneuploidy, primarily during the first meiosis (Hassold and Hunt, 2001). AURKs are a family of serine\/threonine kinases that regulate various structural elements and mechanistic events associated with the dynamic process of chromatin remodeling. Therefore, examination of AURKs during oocyte maturation is of interest when attempting to discern causative factors and molecular signaling pathways involved in aberrant oocyte chromosome modifications. We have determined that mouse oocytes contain transcripts for all three Aurk isoforms: AurkA, AurkB and AurkC and that levels of these transcripts increase significantly as oocytes gain meiotic competence, but do not change as oocytes progress through meiosis to MII. The predominant Aurk isoform transcript in meitocally competent and maturing oocytes appears to be AurkA. In agreement with these findings, during preparation of this manuscript, transcripts for AurkA, AurkB and AurkC were also reported in fully-grown immature bovine oocytes, with AurkA as the predominant isoform (Uzbekova et al., 2007).\nTo determine effects of AURK on oocyte maturation, we utilized the highly selective pharmacological AURK inhibitor, ZM. Inhibition of AURKs via ZM occurs through blockage of the ATP binding site at an adjacent cleft not present in other kinases (Ditchfield et al., 2003). Although ZM is a selective inhibitor of AURKs, it does inhibit other kinases, including CDK1, MAPK and CDC25. However, concentrations of ZM much higher than those utilized in the majority of our studies (20 \u00b5M) had no effect on CDK1, CDC25 or MAPK activities in Xenopus egg extracts, indicating ZM did not directly affect these kinases, or affect any upstream regulatory kinases involved in their activation (Gadea and Ruderman, 2005). Furthermore, cellular characteristics and phosphorylation patterns following inhibition of CDK1 (Marchal et al., 2001; Kubelka et al., 2002; Swain et al., 2003; Bui et al., 2004) and MAPK (Tong et al., 2003; Yu et al., 2007) in mammalian oocytes are dramatically different than those observed following ZM treatment in this study; suggesting observed effects are indeed the result of AURK inhibition. It should be mentioned that ZM displays differential inhibitory action toward different AURK isoforms, demonstrating inhibition of AURKB \u223c20 times more potently than AURKA (Girdler et al., 2006). Thus, differential phenotypes observed in our study using lower concentrations of ZM may indicate AURKB-specific functions. Future experiments will attempt to determine if lower doses of ZM, as well as utilization of other AURK inhibitors with differential inhibitory actions, can verify this and determine isoform-specific functions within the mammalian oocyte.\nAURK do not appear to play a role in oocyte meiotic resumption or regulation of oocyte nuclear envelope (NE) integrity during GVBD. Although ZM appeared to cause a slight delay in NE disassembly, no significant differences were apparent at any dose of ZM examined other than 2.5 \u03bcM. This is in agreement with findings that inhibition of bovine oocyte AURKs with VX680 has no effect on GVBD (Uzbekova et al., 2007). However, these findings are in contradiction to a study utilizing pig oocytes, which demonstrated lack of GVBD following exposure to elevated doses of ZM (7\u201310 \u00b5M; Jelinkova and Kubelka, 2006). Difference may be the result of varying experimental conditions or species-specific differences, as Jelinkova and Kulbelka (2006) utilized porcine cumulus\u2013oocyte complexes from abattoir ovaries, whereas the current study utilized denuded oocytes obtained from gonadotropin stimulated mice.\nIn our study, inhibition of AURKs during 16 h of culture inhibited progression to MII and polar body extrusion, similar to developmental observations in clam oocytes following treatment with another AURK inhibitor, Hesperadin (George et al., 2006), and bovine oocytes treated with VX680 (Uzbekova et al., 2007). Failure to complete cytokinesis has also been observed following AURK inhibition during mitosis (Ditchfield et al., 2003). Evidence exists suggesting AURKB may be the specific isoform responsible for failure of oocytes to extrude the first polar body, as specific inhibition of AURKB in Drosophila cells prevented cytokinesis (Adams et al., 2001; Giet and Glover, 2001). Interestingly, AURKB was localized to the region of the contractile ring in bovine MII oocytes; (Uzbekova et al., 2007). However, AURKA and AURKC were also found in the vicinity of the contractile ring in bovine oocytes, confounding interpretation. Whether failure to complete cytokinesis and extrude the polar body in our study is a primary effect of AURK inhibition on cytokinesis, or a secondary effect due to chromosome remodeling or spindle defects remains to be elucidated.\nTo begin to determine possible causes for oocyte arrest prior to MII, we examined effects of AURK inhibition on chromatin remodeling and spindle formation during the prophase I\u2013MI transition. Inhibition of AURKs during this time point resulted in improper positioning of chromatin, as evidenced by scattering throughout the meiotic spindle. This is in agreement with recent studies of AURK inhibition in mouse (Wang et al., 2006), pig (Jelinkova and Kubelka, 2006) and bovine oocytes (Uzbekova et al., 2007) reporting abnormal chromosome positioning at MI. Additionally, chromosomal spreading in our study indicates AURK inhibition negatively affects ability of MI oocyte chromosomes to condense properly and resolve bivalents. This may be due to premature decondensation, similar to that observed following AURK inhibition in Xenopus egg extracts (Gadea and Ruderman, 2005). It has been reported that AURKB inhibition via RNAi is responsible for chromosome misalignment in Drosophila cultured cells and results in amorphous chromatin (Adams et al., 2001) and only partial condensation (Giet and Glover, 2001). Thus, AURKB may be the AURK isoform responsible for chromatin defects observed in mammalian oocytes in this study. Indeed, a recent report demonstrates AURKB localized to condensed chromatin in MI and MII bovine oocytes (Uzbekova et al., 2007).\nFailure of ZM-treated oocytes to progress to MII does not appear to be the result of inability to polymerize microtubules or form MTOCs, as indicated by \u03b2-tubulin and pericentrin staining. This is in contrast to AURK inhibition studies in Xenopus egg extracts, where ZM treatment resulted in failure to form the mitotic spindle (Gadea and Ruderman, 2005). However, fidelity of spindle function in our study remains in question, as spindle morphology and MTOC localization was disrupted following AURK inhibition. AURKs, such as AURKA, regulate several components of the spindle apparatus and spindle poles (see review, Ducat and Zheng, 2004). Indeed, AURKA localized to spindle poles in mouse (Yao et al., 2004) and pig oocytes (Yao and Sun, 2005), but not bovine oocytes (Uzbekova et al., 2007) and specific neutralization of the kinase resulted in disorganization of the meiotic spindle. This disorganization is in agreement with initial reports of ZM influences on intact somatic cells (Ditchfield et al., 2003). Thus ZM inhibition of oocyte AURKA may account for observed defective spindle phenotypes. Alternatively, condensed chromosomes direct formation of the spindle apparatus via nucleation\/stabilization of microtubules (Merdes and Cleveland, 1997; Khodjakov et al., 2000). Therefore, defects in meiotic spindle morphology and scattering of chromatin observed in these experiments may be the result of aberrant chromatin condensation, possibly controlled by AURKB (see review, Shannon and Salmon, 2002). Reports in Xenopus mitotic cell-free extracts indicate AURK inhibition does interfere with chromatin driven microtubules assembly (Gadea and Ruderman, 2005).\nTo begin to determine if the inability of ZM-treated oocytes to reach MII following AURK inhibition was due only to defects incurred during the prophase\u2013MI transition, or if AURKs had roles at other meiotic transition time points, we matured oocytes in vitro to time points consistent with MI and AI oocytes, thus allowing normal spindle formation and chromatin remodeling to occur. Subsequently, we then cultured oocytes in presence of ZM to a time point consistent with MII. Inhibition of AURKs during the MI-MII transition resulted in a portion of oocytes failing to segregate chromosomes and extrude the first polar body, while those that did complete MII displayed severely scattered chromatin. Thus, it appears as if AURKs may not only control spindle formation and chromatin condensation during early meiotic events, but also regulate separation and\/or segregation of oocyte chromosomes during later meiosis. Effects may be directly on meiotic spindle components, possibly regulated by AURKA. Alternatively, defective chromosome remodeling could also be due to ZM inhibition of AURKB. Aurora B regulates kinetochores and their interactions with microtubules (Kaitna et al., 2002; Cimini et al., 2006). Another possible explanation for aberrant separation\/segregation following AURK inhibition during late oocyte meiosis may be interferences with regulation of cohesion. Aurora B regulates release of chromosome cohesion during meiosis in Caenorhabditis elegans, apparently via phosphorylation of REC-8 (Rogers et al., 2002). Future studies will attempt to determine if defects include premature separation of sister chromatids due to premature release of cohesion, or if aberrant phenotypes are the result of failure to separate homologous chromosomes.\nTo begin to determine possible targets of oocyte AURKs responsible for observed defects in chromosomal remodeling, we examined the phoshorylation state of histone-H3 at ser10 and ser28. It is thought that phosphorylation of histone-H3 may cause the histone to act as a receptor or recruitment factor for condensation factors (Hirano, 2000), or possibly reduce the affinity of histone-H3 for DNA and make the relatively compact chromatin fiber more readily accessible to remodeling factors (Hirano, 2000), such as the condensin complex. Condensin is a multi-subunit protein complex that play a central role in chromosome compaction and condensation and is reported to co-localize and bind with phosphorylated histone-H3 (Schmiesing et al., 2000; Ball et al., 2002). Interestingly, AURKB controls association of condensin with chromosomes during mitosis (Lipp et al., 2007; Takemoto et al., 2007), and may be functioning in a similar manner during mammalian oocyte meiosis, though this remains unknown. It should be mentioned that reports for the requirements of histone-H3 phosphorylation in chromatin condensation varies greatly (Van Hooser et al., 1998; Wei et al., 1998, 1999; Goto et al., 1999; Kaszas and Cande, 2000; Schmitt et al., 2002). These contradictions also appear to hold true in regard to oocyte meiosis, with differing reports on temporal and spatial localization and correlation with condensation (Jelinkova and Kubelka, 2006; Wang et al., 2006; Swain et al., 2007). However, contradictions may be explained by differences in experimental conditions or perhaps species-specific differences. Regardless, contradictions in the literature make it increasingly evident that differences in the role of histone phosphorylation exist depending on the organism and\/or type of cellular division examined (Fuchs et al., 2006), and that the specific histone-H3 AURK within mammalian oocytes remains unknown.\nIn summary, these studies indicate AURK plays a significant role in mouse oocyte maturation involving progression to MII, acting in both early (prophase\u2013MI transition) and late meiotic events (MI\u2013MII transition). When AURK is inhibited in early meiosis, microtubules polymerize and MTOCs form, but spindle morphology and MTOC localization is disrupted. Furthermore, metaphase chromatin does not condense or position normally. Inhibition of oocyte AURKs during late meiosis (MI\u2013MII transition), following chromatin condensation and spindle formation, negatively affects separation and segregation of chromosomes. This aberrant chromatin remodeling following AURK inhibition in oocytes appears to be due, in part, to hypophosphorylation of histone-H3 at both ser10 and ser28. We report amplification and sequencing of all three Aurk isoforms transcripts: AurkA, AurkB and AurkC. Levels of these transcripts increase as oocytes achieve meiotic competence, with AurkA being the predominant isoform. Future studies will focus on determining protein expression levels of AURK isoforms, as well as specific functional roles and intracellular targets.\nFunding\nFunding for research by Gary D. Smith provided by NIH RO1 grant #HD046768-01A2. Support for Jason E. Swain provided by an NIH T32 grant. Partial support for Jingwen Wu provided from the Lyle C. Roll Research Fund to Professor James O. Woolliscroft, Dean of Medical School, University of Michigan.","keyphrases":["oocyte","kinase","meiosis","chromosomal disorders","signal transduction"],"prmu":["P","P","P","M","M"]}
{"id":"Eur_Spine_J-2-2-1602205","title":"Epithelioid sarcoma in the thoracic spine\n","text":"Epithelioid sarcoma is a rare and highly malignant soft tissue tumor that is commonly found in the extremities and rarely in the trunk area. This malignant tumor often mimics granuloma or nodular fasciitis, which causes a delay in establishing the diagnosis. This type of cancer has a high recurrence rate. Surgical treatment requires wide radical resection. The objective of this case report is to highlight the unique location of a rare neoplasm and to illustrate the relentless course of epithelioid sarcoma despite initial radical resection. A 14-year-old boy was admitted to our facility with a soft tissue mass on the right lower thoracic spine. The large tumor mass had deeply penetrated into the muscles, infiltrated the neuroforamen of T9\u2013T10 level, and compressed the dural sac. Immunohistological study of the biopsy was highly consistent with an epithelioid sarcoma. Wide excision of the mass, laminectomy and spine fusion with instrumentation was performed. The patient received chemotherapy and irradiation. The first recurrence of the neoplasm was seen as a contralateral metastasis 21 months after the resection. On the last follow-up, 3 years postoperatively, the patient was in a good general condition. However, further progression of the sarcoma had to be recognized. Our case encompasses multiple features that represent negative prognostic factors. Initial wide excision of the neoplasm and adjuvant therapy including chemotherapy and irradiation seem to slow down the relentless course of epithelioid sarcoma in the trunk.\nIntroduction\nEpithelioid sarcoma is a rare type of a fibrohistiocytic tumor, which is not extensively documented in the literature. It is common at the extremities, and rare at the trunk area. The incidence of epithelioid sarcoma in the trunk is 4% with a range from 0 to 44% [2, 12, 14]. It affects mainly adolescent males [1\u20133, 11, 13, 14, 17]. To our knowledge there are only two cases reported of localization in the spine (lumbosacral junction and sacrum) [7, 14], and there are no reports on epithelioid sarcoma at the thoracic spinal level. Laskowski [9] has first described the unique histological appearance in 1961. It resembles a chronic inflammatory process, necrotizing granuloma, squamous cell carcinoma or other fibrohistiocytic tumors. Electron microscopic appearance suggests origin from synovial structures [6, 10]. There is 85% local recurrence rate and 30\u201350% likelihood for metastases predominately lymphatic or in the lungs [2, 4, 11]. The 5-year survival rate varies from 65 to 100% [1, 12, 13]. Factors adversely affecting the survival rate are [2, 12]:Size of the tumor;Infiltration of the tumor into muscular, neural or vascular structures;Primary location in the trunk;Pulmonary metastasis.Wide surgical resection is indicated, since epithelioid sarcoma frequently follows a relentless course with multiple recurrences [4, 17]. Chemotherapy and irradiation are recommended to decrease recurrence rate [2, 8].\nCase report\nA 14-year-old overweight boy presented at an orthopaedist\u2019s private practice with a painless, bulging mass on his back that was incidentally discovered by his grandmother. He was 183\u00a0cm tall and weighted 107\u00a0kg. Besides obesity no other medical problems were reported. There was no family history of a soft tissue mass. On the physical examination the tumor had a soft consistence and could not be shifted from the underlying tissue. Neurological examination did not reveal any deficit. Magnetic resonance imaging (MRI) with gadolinium of his thoracolumbar spine revealed a right paravertebral cystic mass. The soft tissue tumor extended from T7 to T11 level and measured 9.5\u00d73\u00d73\u00a0cm. The mass infiltrated via the neuroforamen at T9\u2013T10 levels compressing one quarter of the spinal cord (Fig.\u00a01). There was no osseous involvement. Open biopsy of the mass showed necrobiotic granuloma including epithelioid and fusiform cells. Immunohistochemical studies revealed a co-expression of vimentin and cytokeratin, with pathological diagnosis of epithelioid sarcoma grade III. During the tumor staging a craniolateral metastasis was depicted on the PET scan (Fig.\u00a02). Consecutive MRI confirmed a soft tissue mass of 1\u00a0cm diameter, located about 10\u00a0cm distant from the main mass. Wide mass resection, including the locoregional metastasis, led to a sacrifice of large parts of the paravertebral thoracic muscles. In addition a hemilaminectomy of T9\u2013T10 vertebra with resection of the adjacent pedicles and careful dissection from the dural sac was performed. There was no sparing of right ninth nerve root. The tumor resection was intralesional. Posterior transpedicular instrumentation from T6 to L1 was carried out. After surgery no new motor or sensory deficits were noted. Chemotherapy was initiated on the 6th postoperative week and irradiation on 10th postoperative week. The patient received nine courses of chemotherapy within 25\u00a0weeks (Table\u00a01) and a total dose of 3-Gy irradiation over 4\u00a0weeks. He developed a wound dehiscence 2\u00a0weeks after initiation of the irradiation therapy. Under daily dressing no infection was observed. The wound was finally closed with a pedicle musculocutaneous flap using the Latissimus dorsi muscle 4\u00a0weeks after the completing the course of irradiation. Routine follow-up consisted of a physical examination, plain radiograms of the thoracic spine plus MRI Scans every 3\u00a0month (Fig.\u00a03).\nFig.\u00a01Preoperative antero-posterior (a) and lateral (b) plain X-rays of the middle thoracic spine. On the ap view no irregular formation of the pedicles can be depicted\u2014the lateral view of the thoracic spine show apophyseal endplate changes as a feature of Scheuermans disease. No signs of osteolytic lesion can be detected. Preoperative transverse (c) and sagittal (d) MRI scan (T2 sequences: TR=3,070, TE=120) demonstrating the tumor mass in the region of the paravertebral muscles, reaching and compressing the dural sac via the right neuroforamen Th9\/10Fig.\u00a02On the PET scan a craniolateral metastasis was detected (a) and the paravertebral tumor mass was confirmed (b). No further satellite lesions were foundTable\u00a01Agents and dosage of the quadruple chemotherapyAgent (trade name)Total dose (mg)Ifosfamid (Holoxan\u00ae)100,440Vincristin (Onkocristin\u00ae)20Actinomycin D (Lyovac\u00ae)12Doxorubicin (Ribodoxo\u00ae)494Fig.\u00a03Postoperative antero-posterior (a) and lateral (b) plain radiograph of the thoracic spine at final follow-up. Postoperative transverse (c) and sagittal (d) MRI scan (T2 sequences: TR=3,682, TE=120) at 1.5\u00a0year showing no sign of recurrence\nA metastasis at the fifth intercostal space of the chest contralateral to the initial tumor location was observed at 21\u00a0months follow-up. At this stage the patient denied further intervention. Two years postoperatively pulmonary metastases were detected on CT scans in addition to a local recurrence on the right thoracic spine. There was no significant progression of the tumor growth at the last follow-up 37\u00a0month after surgical treatment. The young male was in good general condition fully participating in social life.\nDiscussion\nEpithelioid sarcoma is a rare soft tissue neoplasm with a high local recurrence rate. Epithelioid sarcoma presents histologic characteristics similar to inflammatory processes and other benign soft tissue tumors, which frequently delays the diagnosis and adequate treatment. Typical for this soft tissue sarcoma is an immunohistochemical co-expression of cytokeratin and\/or epithelial membrane antigen (EMA) and vimentin, and in half of the cases an immunoreactivity for CD34, an occasional reactivity for smooth muscle actin (SMA), desmin, neuron specific enolase and S100 protein [2, 5]. In our specimen a strong positive immunoreaction with antibodies to cytokeratin, vimentin and EMA was registered (Fig.\u00a04). Histomorphology and immunohistochemical pattern are typical and separates this tumor entity from other sarcomas. To differentiate from osteogenic sarcoma, which may also co-express cytokeratin and vimentin, and may demonstrate a immunoreactivity with SMA and CD99, epithelioid sarcoma, however, does not produce tumor osteoid, which is the key finding in osteogenic sarcoma.\nFig.\u00a04a Histomorphologic aspect of epithelioid sarcoma with solid and nodular growth pattern, H&E (10\u00d7). b The tumor cells are roundish, admixed with spindle shaped cells and are featuring an eosinophilic cytoplasm with vesicular, irregular nuclei (20\u00d7). c Immunohistochemical investigation with antibodies against cytokeratin shows a strong positive immunoreaction (20\u00d7). d The immunoreaction against epithelial membrane antigen (EMA) demonstrates a comparable picture (20\u00d7). e The immunoreactivity against smooth muscle actin (SMA) features a weak staining (20\u00d7). f The immunoreaction against CD 99 is negative (20\u00d7)\nWide local resection is the recommended treatment of epithelioid sarcoma [1, 8, 11, 14]. In the spine however, total eradication of the sarcoma by an enbloc excision is compromised by the presence of neural structures [16]. Thus the concept of intralesional resection was followed both for the main tumor mass and the metastasis. Nerve sheaths may serve as a pathway for the spread of the sarcoma [1, 2, 8, 11, 17]. Enzinger [4] reported six cases with a mass infiltration along nerve sheaths or vascular structures. In our case there was involvement of the right ninth nerve root. The mass expansion into the right T9\u2013T10 neuroforamen with compression of the dural sac necessitated a wide hemilaminectomy with resection of the facet joints and the adjacent pedicles. The wide paraspinal muscle resection required six levels segmental instrumentation and fusion to avoid scoliotic deformity due to muscular imbalance [15]. Pulmonary metastasis and local recurrence of the sarcoma were first diagnosed 24\u00a0month after the resection. In comparison, a survival period of 22\u00a0month after the initial surgery was described in a case report of epithelioid sarcoma in the lumbosacral spine [14]. Moreover, on first diagnosis, our case embodied many factors that promote recurrence of this neoplasm: large size, localization in the trunk and infiltration in both muscle and neural structures. Recurrence as the typical feature of epithelioid sarcoma cannot be prevented even in extremities, where a radical surgical regimen can be pursued [8]. In conclusion, epithelioid sarcoma of the spine is an extremely rare entity, which differs from other sarcoma subtypes in propensity for local recurrence and spread along the lymphatic pathways. Initial radical resection followed by adjuvant chemotherapy and irradiation can retard the relentless course of this malignancy.","keyphrases":["epithelioid sarcoma","thoracic spine","survival"],"prmu":["P","P","P"]}
{"id":"Eur_Radiol-3-1-2077913","title":"Dual-source computed tomography in patients with acute chest pain: feasibility and image quality\n","text":"The aim of this study was to determine the feasibility and image quality of dual-source computed tomography angiography (DSCTA) in patients with acute chest pain for the assessment of the lung, thoracic aorta, and for pulmonary and coronary arteries. Sixty consecutive patients (32 female, 28 male, mean age 58.1\u00b116.3 years) with acute chest pain underwent contrast-enhanced electrocardiography-gated DSCTA without prior beta-blocker administration. Vessel attenuation of different thoracic vascular territories was measured, and image quality was semi-quantitatively analyzed by two independent readers. Image quality of the thoracic aorta was diagnostic in all 60 patients, image quality of pulmonary arteries was diagnostic in 59, and image quality of coronary arteries was diagnostic in 58 patients. Pairwise intraindividual comparisons of attenuation values were small and ranged between 1\u00b16 HU comparing right and left coronary artery and 56\u00b19 HU comparing the pulmonary trunk and left ventricle. Mean attenuation was 291\u00b165 HU in the ascending aorta, 334\u00b193 HU in the pulmonary trunk, and 285\u00b166 HU and 268\u00b167 HU in the right and left coronary artery, respectively. DSCTA is feasible and provides diagnostic image quality of the thoracic aorta, pulmonary and coronary arteries in patients with acute chest pain.\nIntroduction\nAcute chest pain represents one of the most difficult diagnostic challenges in emergency medicine. Chest pain history alone often cannot identify a group of patients who could be treated without further diagnostic testing [1], and triage decisions based on initial cardiac enzyme levels [2] and electrocardiography (ECG) [3] are often insufficient. Imaging may improve patient triage by decreasing delay in diagnosis and treatment and thus morbidity and mortality [4, 5]. Most studies [6\u20138] have focused on patients being suspected of having acute coronary syndrome, but did not include additional differential diagnoses such as aortic dissection or pulmonary embolism, conditions that may clinically mimic coronary syndromes [9].\nMulti-detector row computed tomography (CT) angiography is widely accepted and routinely used as a primary tool in the emergency assessment of pulmonary embolism [10] and aortic dissection [11]. In addition, 64-slice CT coronary angiography has demonstrated the capability to diagnose and to rule-out coronary artery disease (CAD) [12, 13]. Some recent studies have shown that ECG-gated multi-detector row CT is logistically feasible [14] and yields promising results as a modality for evaluating chest pain patients with cardiac and non-cardiac disease in the emergency setting [15, 16]. Even though using 64-slice CT scanner technology, however, patients with elevated heart rates require medical heart rate control prior to CT coronary angiography by administering beta-blockers and\/or benzodiazepines [8, 13, 17].\nDual-source CT (DSCT) represents the most recent scanner technology and is characterized by two tubes and two detectors that are mounted in orthogonal orientation onto the gantry. As compared to 64-slice CT, this scanner configuration maintains a consistently high spatial resolution while enabling ECG-gated imaging with an increased temporal resolution of 83\u00a0ms [18]. First studies have shown robust results of DSCT angiography (DSCTA) regarding image quality of cardiac structures even at high heart rates [19\u201321].\nThe purpose of this study was to investigate the feasibility and image quality of ECG-gated DSCTA of the chest as a tool to evaluate cardiac and non-cardiac causes of acute chest pain in patients presenting to the emergency department.\nMaterials and methods\nPatient population\nSixty consecutive patients (32 females, 28 males, mean age 58.1\u00b116.3\u00a0years, age range 26\u201384\u00a0years) were prospectively included in this study. Intake was performed on weekdays from 7\u00a0am to 7\u00a0pm from August to October 2006. All patients suffered from acute chest pain and were referred to our department to diagnose or to rule out pulmonary embolism (n=56) or aortic dissection (n=4). Inclusion criteria were acute chest pain >5\u00a0min within the previous 24\u00a0h and\/or elevated serum D-dimer levels. Dyspnea and hemodynamic instability were not considered exclusion criteria. Similarly, all patients irrespective of their mean or regularity of heart rate and irrespective of their ability to perform breath-hold were included. Exclusion criteria included pregnancy, previous adverse reaction to iodinated contrast agent, nephropathy (serum creatinine >1.3\u00a0mg\/dl), elevated troponine-I or creatine kinase-MB level in the initial blood sample, initial diagnostic ECG changes indicating an acute coronary syndrome (i.e., ST elevation or depression >1\u00a0mm, T-wave inversion >4\u00a0mm in >2 anatomically contagious leads), and interference with standard clinical care of patients. The study was approved by the local ethics committee; informed consent was obtained.\nScan protocol and data reconstruction\nAll CT examinations were performed on a DSCT scanner (Somatom Definition, Siemens Medical Solutions, Forchheim, Germany). First, a single non-enhanced low-dose scan at the level of the aortic root was obtained. In this slice, a region of interest (ROI) was set in the lumen of the aorta for monitoring intraluminal contrast enhancement. The delay from start of contrast material injection to start of scanning was planned using the bolus-tracking technique. A total of 110\u00a0ml iodinated contrast material (iodixanol, Visipaque 320; 320\u00a0mg\/ml, GE Healthcare, Buckinghamshire, UK) was administered at a flow rate of 4\u00a0ml\/s via an 18-gauge needle placed into a superficial vein in the left antecubital fossa, followed by 30\u00a0ml saline solution at the same flow rate (4\u00a0ml\/s). After reaching the preset contrast enhancement level of 80 Hounsfield Units (HU) in the ROI, a breath-hold signal was given and the scan was initiated automatically after a delay of 6 s. A topogram was used for planning the examination and determining the scan range. Data acquisition was performed using a dedicated biphasic chest pain protocol. As illustrated in Fig.\u00a01, the lower chest including the heart was scanned with a tube current time of 320\u00a0mAs while the upper chest was scanned with a tube current time product of 160\u00a0mAs. The borderline between upper and lower scan range was set at approximately 2\u00a0cm below the carina. Data acquisition was performed in a cranio-caudal direction. Acquisition parameters were as follows: detector collimation 2\u00d732\u00d70.6\u00a0mm by using a z-flying focal spot for the simultaneous acquisition of 2\u00d764 overlapping 0.6-mm slices, gantry rotation time 330\u00a0ms, and tube potential 120\u00a0kV. The pitch varied according to the patient's heart rate and ranged from 0.2\u20130.43, with higher pitch at higher heart rates. ECG-pulsing for radiation dose reduction [22] was applied in all patients. At mean heart rates below 60\u00a0bpm, full tube current was applied from 60 to 70%, at 61\u201370\u00a0bpm from 50 to 80%, and at heart rates above 70 from 30 to 80% of the R-R interval.\nFig.\u00a01Scan topogram illustrating planning of the chest pain protocol. The scan range covered the entire chest (red box). Premonitoring for bolus tracking was performed at the level of the aortic root (white line). The border for full tube current for the heart and half tube current for the upper lung is set approximately 2\u00a0cm below the tracheal bifurcation. It is delimited by a virtual horizontal line connecting the upper ends of the blue boxes on both sides\nRetrospective ECG-gating for phase synchronization was used. For the heart, CT data sets were reconstructed at 70% of the R-R interval with a slice thickness of 0.75\u00a0mm (increment 0.5\u00a0mm) by using a medium soft-tissue convolution kernel (B26f) (mean field of view, FoV: 151\u00b117\u00a0mm, image matrix 512\u00d7512). If considered necessary, additional images were reconstructed in 5% steps using the same parameters within the time window of full tube current. Images of the mediastinum (mean FoV: 293\u00b143\u00a0mm) including the aorta and pulmonary arteries were reconstructed with a slice thickness of 1\u00a0mm (increment 0.8\u00a0mm) by using a medium soft-tissue convolution kernel (B30f), and images of the lung were reconstructed with a slice thickness of 2\u00a0mm (increment 1.5\u00a0mm) by using a sharp convolution kernel (B60f, same FoV as for the mediastinum). All images were transferred to a second Wizard (Siemens) equipped with cardiac post-processing software (Syngo Circulation, Siemens).\nData analysis\nAll data were qualitatively evaluated regarding image quality and artifacts of different thoracic structures by two independent readers who are both experienced in cardiovascular radiology. This evaluation was performed on transverse source images, multi-planar reformations (MPR), curved MPR, and thin-slab maximum intensity projections.\nImage quality and artifacts-lung parenchyma\nImage quality of lung parenchyma was independently rated using a two-point scale, adapted from a previous publication [23]. Lung parenchyma allowing diagnostic assessment due to distinct anatomic details of bronchial and parenchymal structures without significant artifacts and noise was rated with a score of 1 (diagnostic). Lung parenchyma with artifacts or noise causing reduction of image quality and diagnostic value was rated with a score of 2 (non-diagnostic).\nArtifacts were rated to quantify the cranio-caudal distribution of artifacts within the lung parenchyma for the right and left lung separately in coronal MPR. Because of the different tube current at upper and lower parts along the z-axis (see Fig.\u00a01), the following scores were separately applied for the apex and the basis of the lung, respectively: 1=\u2009no artifacts, 2=\u2009breathing artifacts (stair step artifacts), 3=\u2009ECG-gating (i.e., synchronization or interpolation artifacts), and 4=\u2009noise artifacts. If breathing and ECG-gating artifacts appeared at the same time in one patient, the artifact with the worst impact on image quality was noted.\nImage quality and artifacts-vascular structures\nImage quality of thoracic vascular structures was independently rated using the same two-point scale [23] as used for the rating of the lung parenchyma. Regarding the thoracic aorta, a score of 1 (diagnostic) indicated confident evaluation of the ascending aorta, the aortic arch, and descending aorta. Regarding pulmonary arteries, a score of 1 indicated confident evaluation of central, lobar, segmental, or subsegmental pulmonary arteries. Regarding coronary arteries, a score of 1 represented confident depiction (homogenous attenuation; no artifacts decreasing coronary analysis) of the right coronary artery (RCA), left main artery (LMA), left anterior descending artery (LAD), left circumflex artery (LCX), and their side branches. A score of 2 indicated decreased image quality of thoracic vascular structures with severe impairment of diagnostic value (non-diagnostic) due to breathing, motion, or ECG-gating artifacts.\nImage noise and attenuation-vascular structures\nImage noise was determined as the standard deviation of attenuation in a ROI placed in the ascending aorta [24]. Contrast attenuation was measured in each patient in the ascending aorta, pulmonary trunk, LMA, proximal segment of the RCA, and right and left ventricle. Image noise and contrast attenuation were assessed using a circular ROI positioned exactly within the vessel or ventricular lumen while avoiding superimposition or partial volume effects from the vessel wall or myocardium. Measurements of the ascending aorta and pulmonary trunk as well as of the right and left ventricle were performed on the same transverse image, while the ROI in the coronary arteries was individually placed on separate transverse images.\nImaging findings\nImaging findings indicating the possible underlying cause of acute chest pain were documented in each patient by both readers in consensus.\nStatistical analysis\nStatistical analysis was performed using commercially available software (SPSS 11.5, SPSS Inc., Il). Quantitative variables are expressed as mean \u00b1 standard deviation (SD) including 95% confidence intervals (CI) or range when appropriate. Categorical data were expressed as frequencies or percentages. Inter-observer agreement (kappa statistics) for image quality ratings was calculated. Two-tailed Student's t test for paired samples was used to explore significant differences in vessel attenuation among the ascending aorta, RCA, LMA, pulmonary trunk, and right and left ventricle. Intra-individual differences regarding vessel attenuation among the different vascular territories were performed using pairwise comparisons. Bonferroni correction for multiple comparisons was made, and a P-value <0.003 was considered statistically significant.\nResults\nAll CT scans were well tolerated and were successfully performed in all 60 patients without complications. All patients had a sinus rhythm, and the average heart rate during data acquisition was 74.9\u00b119.0 beats per minute (bpm) (range 45-130\u00a0bpm). No beta receptor antagonists or benzodiazepines were administered prior to CT; 13 patients (22%) took oral beta blockers as part of their baseline medication at the time of the scan. Mean scan time was 12.2\u00b12.3\u00a0s (range 9.7\u201317.5\u00a0s) and mean scan length was 24.1\u00b12.1\u00a0cm (range 19.3\u201329.7\u00a0cm). The reconstruction interval used for image reading was 70% of the R-R interval. In seven patients it was considered necessary to reconstruct additional data sets in 5% intervals within the window of full tube current to obtain images with diagnostic quality.\nImage quality and artifacts-lung parenchyma\nImage quality of lung parenchyma was rated as being diagnostic (score 1) by both readers in all 60 patients (100%; excellent inter-observer agreement, kappa\u2009=1.0), and pathology both at upper and lower lung parts could be diagnosed or excluded in all 60 patients.\nLung parenchyma was rated by both readers as being artifact-free in 42 patients (70%). Breathing artifacts causing stair-step artifacts were encountered in nine patients (15%) by one reader and eight (13%) of these nine patients by the other reader (excellent inter-observer agreement, kappa\u2009=0.93). Breathing artifacts were exclusively found at the lung base, i.e., at the end of the breath-hold period. ECG-gating-related artifacts were found in ten patients (17%) by one reader, and in nine (15%) of these ten patients by the other reader (excellent inter-observer agreement, kappa\u2009=0.84). The two types of artifacts were not encountered in the same patient, and image quality was diagnostic despite the artifacts.\nImage quality and artifacts-vascular structures\nImage quality of the thoracic aorta was rated by both readers as being diagnostic (score 1) in all 60 patients (100%; excellent inter-observer agreement, kappa\u2009=1.0), and pathologies of the thoracic aorta could be diagnosed or excluded by both readers in all 60 patients. Both readers rated image quality of the pulmonary arteries in the one (i.e., the same) patient (2%) as being non-diagnostic (score 2) due to insufficient attenuation of segmental and subsegmental pulmonary arteries. In this patient, attenuation did not allow excluding segmental or subsegmental pulmonary embolism, while attenuation in the pulmonary trunk and right and left lobar artery was considered sufficient for diagnosis or exclusion of central and lobar pulmonary embolism. In this patient, image quality of the coronary arteries was considered as being diagnostic by both readers. Image quality of coronary arteries was rated by both readers as being diagnostic (score 1) in the same 58 patients (97%; excellent inter-observer agreement, kappa\u2009=1.0). In the remaining two patients (3%), image quality of the coronary arteries was considered by both readers as being non-diagnostic (score 2) due to ECG-gating-related artifacts (mean heart rate 71\u00a0bpm, ranging from 50 to 84\u00a0bpm during scanning) in one and severe image noise due to obesity (body mass index 40.4\u00a0kg\/m2) in the other. In both patients, both readers rated image quality of the pulmonary arteries as being diagnostic.\nImage noise and attenuation-vascular structures\nMean image noise in the ascending aorta (mean ROI size 3.6\u00b10.2\u00a0cm2) was 29.8\u00b15.6.\nMean attenuation in the ascending aorta (same ROI and same ROI size as for measurements of image noise) was 291\u00b165\u00a0HU (95% CI: 274\u2013308\u00a0HU), mean attenuation in the pulmonary trunk (mean ROI size 2.4\u00b10.2\u00a0cm2) was 334\u00b193\u00a0HU (95% CI: 310\u2013358\u00a0HU), mean attenuation in the proximal RCA (mean ROI size 0.14\u00b10.02\u00a0cm2) was 285\u00b166\u00a0HU (95% CI: 268\u2013302\u00a0HU), and mean attenuation in the LMA (mean ROI size 0.12\u00a0cm2\u00b10.01) was 286\u00b167\u00a0HU (95% CI: 269\u2013304\u00a0HU). Mean attenuation in the right ventricle (mean ROI size 2.6\u00b10.2\u00a0cm2) was 313\u00b189\u00a0HU (95% CI: 290\u2013336\u00a0HU) and mean attenuation in the left ventricle (mean ROI size 2.6\u00b10.2\u00a0cm2) was 277\u00b166\u00a0HU (95% CI: 260\u2013294\u00a0HU) (Fig.\u00a02).\nFig.\u00a02Mean attenuation values (HU) within different vessels indicating a relatively homogenous contrast distribution between the different thoracic vascular territories. RCA\u2009=\u2009right coronary artery; LMA\u2009=\u2009left main coronary artery\nNo significant differences regarding attenuation were found between the RCA and LMA (P=n.s.), and between the ascending aorta and both coronary arteries (P=n.s.). No significant differences in attenuation were present between the right and left ventricle (P=n.s.), between the right ventricle and ascending aorta (P=n.s.), between the right ventricle and pulmonary trunk (P=n.s.), and between the right ventricle and the RCA (P=n.s.) and LMA (P=n.s.). Similarly, there were no significant differences in attenuation between the left ventricle and ascending aorta (P=n.s.), and between left ventricle and LMA (P=n.s.) and RCA (P=n.s.). Significant differences were only found between the pulmonary trunk and ascending aorta (P\u2009<0.003), between the pulmonary trunk and the RCA and LMA (P<0.003), respectively, and between the left ventricle and pulmonary trunk (P<0.003).\nPairwise comparisons of mean intra-individual differences regarding vessel attenuation were small and ranged between 1.18\u00b15.86\u00a0HU comparing the right and left coronary artery and 56.45\u00b18.96\u00a0HU comparing the pulmonary trunk and left ventricle (Table\u00a01). \nTable\u00a01Pairwise intra-individual comparisons between different thoracic vascular territories\u00a0Mean differenceStandard deviation95% confidence intervalLower boundaryUpper boundaryAortaPulmonary trunk-4311-75-11AortaRCA65-1022AortaLCA54-817AortaRight ventricle-2313-6318AortaLeft ventricle145-330Pulmonary trunkRCA49111682Pulmonary trunkLCA47121283Pulmonary trunkRight ventricle2012-1657Pulmonary trunkLeft ventricle5792984RCALCA-16-1917RCARight ventricle-2813-6913RCALeft ventricle85-824LCARight ventricle-2714-7015LCALeft ventricle96-1028RCA\u2009=\u2009right coronary artery; LCA\u2009=\u2009left coronary artery\nImaging findings\nNo pathologic findings were found in 33 patients (55%), whereas various pathologies of the aorta, pulmonary and coronary arteries, lung, and mediastinum were present in 27 patients (45%) (Table\u00a02, Figs.\u00a03, 4, 5). \nTable\u00a02Imaging findings in the study populationDiagnosisNumber of patients (n=60)No pathology33Pulmonary embolism11Aortic pathology, total5\u00a0Dissection type B3\u00a0Pseudoaneurysm of the aortic arch1\u00a0Plaque rupture1Coronary pathology, total3\u00a0Significant stenosis of the LAD2\u00a0Occlusion of the RCA1Pulmonary consolidation3Pericarditis\/pericardial effusion2Seropneumothorax1Non-small cell lung cancer1Synovial cell carcinoma1LAD, left anterior descending artery; RCA, right coronary arteryFig.\u00a03A 63-year-old female patient admitted to the emergency department with acute chest pain. (a) Curved multiplanar reformations along the centerline of the right coronary (RCA), left anterior descending (LAD), and the left circumflex artery (RCX) allow excluding significant coronary stenosis in this patient. Mean heart rate during DSCTA was 71\u00a0bpm. (b) Thin-slab transverse maximum intensity projection shows no evidence of pulmonary embolism. (c) Transverse image at the level of the pulmonary trunk demonstrates acute aortic dissection type B (arrow) with mild left-sided pleural effusionFig.\u00a04A 58-year-old female patient admitted to the emergency department with acute chest pain. (a) Curved multiplanar reformations along the centerline of the right coronary (RCA), left anterior descending (LAD), and the left circumflex artery (LCX) demonstrate normal coronary arteries and no evidence of stenosis. Mean heart rate during DSCTA was 63\u00a0bpm. (b) Thin-slab transverse maximum intensity projection show bilateral pulmonary embolism (arrows). (c) Oblique-sagittal thin-slab maximum intensity projection demonstrates the thoracic aorta without evidence of diseaseFig.\u00a05A 71-year-old male patient admitted to the emergency department with acute chest pain. (a) Curved multiplanar reformations along the centerline of the right coronary (RCA), left anterior descending (LAD), and the left circumflex artery (LCX) show occlusion of the proximal RCA (long arrow) and vessel wall calcifications without significant stenosis in the proximal and middle segment of the LAD and LCX (short arrows). Mean heart rate during DSCTA was 73\u00a0bpm. (b) Thin-slab transverse maximum intensity projection demonstrates normal opacification of pulmonary arteries with no evidence of embolism. (c) Oblique-sagittal thin-slab maximum intensity projection demonstrates the thoracic aorta with minimal atherosclerotic wall changes, but with no evidence of potential causes for acute chest pain\nDiscussion\nOur study demonstrates that DSCT allows performing an ECG-gated chest examination for visualizing the different thoracic vascular territories at the same time-by employing an adjusted contrast media protocol-within a reasonable breath-hold period, and thus provides a diagnostic image quality in almost all patients. One of the most important findings of this study is that diagnostic data of coronary arteries could be obtained without foregoing heart rate control.\nContrast media protocol and scan time\nWith increasing gantry rotation times and faster volume coverage of newer CT scanners, scan times successively shorten and higher injection rates are required to achieve sufficient contrast in the vascular territory of interest. Similar to a previous 64-slice CT study [15], we have set the ROI for bolus tracking in the ascending aorta. With a relatively low threshold of 80\u00a0HU above which the scan was initiated, a homogenous attenuation in both aortic\/coronary and pulmonary arteries could be achieved. By doing so, we aimed at the time interval where the contrast enhancement curve of the pulmonary circulation overlaps with enhancement of the aorta [25]. This has also resulted in comparable attenuation in left and right cardiac cavities. Recommended mean attenuation values for diagnostic image quality are approximately 300\u00a0HU for the pulmonary arteries [26] and 250\u2013350\u00a0HU in the ascending aorta and coronary arteries [27, 28]. We obtained a mean attenuation of 291\u00a0HU in the ascending aorta, 334\u00a0HU in the pulmonary trunk, 285\u00a0HU in the RCA, and 286\u00a0HU in the LMA with only small intra- and inter-individual variations. These attenuation values were considered diagnostic in 98% of our patients and are comparable to previously published data [15, 29]. We encountered only one patient with non-diagnostic attenuation of segmental and subsegmental pulmonary arteries. However, this may have been also caused by other reasons negatively affecting contrast attenuation in pulmonary arteries such as Valsalva maneuver [30].\nThe other major issue for CT in acute chest pain represents the issue of scan duration. As ECG-gated CT requires low pitch [31], scan time is increased as compared to non-gated chest CT examinations. This, however, negatively affects image quality by making examinations more prone to breathing artifacts. This holds particularly true for patients with acute chest pain who often suffer from dyspnea. In our study, mean scan time was approximately 12 s, which represents a further improvement as compared to a mean scan time of 21 s with 64-slice CT [15].\nRole of CT in the diagnostic pathway of acute chest pain\nFigure\u00a06 represents a simplified flow-chart of a generally accepted clinical pathway for patients with cardiac and non-cardiac causes of acute chest pain [9, 32, 33]. While CT is the accepted reference modality for the diagnosis and exclusion of pulmonary embolism and aortic dissection [10, 11], the role of CT in patients with a suspected cardiac cause for chest pain is currently under investigation. Gallagher and colleagues [6] have shown that 64-slice CT has accuracy that is comparable to that of stress nuclear imaging for the detection of acute coronary syndrome in low-risk patients with negative serial ECG and biomarker results. Hoffmann and coworkers [8] have demonstrated that 64-slice CT has good performance characteristics for ruling out acute coronary syndromes in patients presenting to the emergency department with acute chest pain. The authors demonstrated in patients in whom initial triage was inconclusive that the absence of coronary artery plaque or significant stenosis on CT angiography had an excellent negative predictive value for the subsequent diagnosis of acute coronary syndrome. Furthermore, in those patients with CAD on CT, the extent of coronary atherosclerotic plaque provided incremental information to standard baseline patient variables and clinical risk assessment. Goldstein and colleagues [7] have shown that 64-slice CT coronary angiography is able to definitely establish or exclude CAD as the cause of acute chest pain. The high negative predictive value of the studies suggests that CT coronary angiography may be useful for facilitating and optimizing triage of patients with acute chest pain and\/or inconclusive initial emergency department evaluation [7, 8].\nFig.\u00a06Flow chart of a generally accepted clinical pathway for patients with cardiac and non-cardiac causes of acute chest pain. CT represents the standard of reference in patients with suspicion of acute aortic syndromes or pulmonary embolism (light gray boxes). The added value of CT in the evaluation of acute coronary syndrome has been already demonstrated [8] (moderate gray box). The potential future role of CT in patients with acute chest pain might be at an even earlier point of diagnostic work-up (dark gray boxes) to rule out life-threatening coronary, pulmonary, and aortic disease and to guide adequate therapeutic interventions\nStudy limitations\nWe included only a relatively small number of patients and thus a small number of pathologies. Second, our study lacks a standard end-point to assess the final diagnosis and misses comparison with the reference standard invasive coronary angiography for investigating CAD for which diagnostic accuracy could be calculated. However, only a minority of our patients underwent invasive coronary angiography because in our emergency department standard management in patients with suspected acute coronary syndrome directly undergoing invasive coronary angiography without prior CT. That is in line with most studies involving patients referred to the emergency department [34]. Third, we did not investigate the optimal contrast media technique for obtaining homogenous contrast attenuation of different thoracic vascular territories. It remains to be determined if the use of the test bolus technique would yield better results with regard to vessel opacification. Finally, it needs to be assessed whether caudo-cranial or caudo-cranial scan direction will result in better image quality.\nConclusion\nFirst experience indicates that DSCTA is feasible in patients with acute chest pain and provides diagnostic image quality of the aorta, pulmonary arteries, and coronary artery system as well as of the lung parenchyma and mediastinum in a patient population without foregoing heart rate control. A dedicated contrast media protocol allows for homogenous attenuation of the different thoracic vascular territories.","keyphrases":["acute chest pain","coronary angiography","dual-source ct"],"prmu":["P","P","P"]}
{"id":"Breast_Cancer_Res_Treat-3-1-2001217","title":"The patient experience\n","text":"The impact of improved treatments for the management of hormone-sensitive breast cancer extends beyond clinical responses. Thanks to appropriate literature and access to the internet, patient awareness of treatment options has grown and patients are now, in many cases, able to engage their oncologists in informed conversations regarding treatment and what to expect in terms of efficacy and safety. Indeed, patients realize that although there is no cure for metastatic disease, treatment can greatly reduce the risk of progression and in the adjuvant setting, where treatment is administered with a curative intent, current treatment options reduce the risk of relapse. The approval of letrozole throughout the breast cancer continuum has provided patients with many reassuring options. The improvement in outcome with letrozole is achieved without a detrimental effect on overall quality of life. Adverse events such as hot flushes, arthralgia, vaginal dryness, and potential osteoporosis are most significant from the patient\u2019s perspective, and it is important that caregivers pay attention to patients experiencing these events, as they can impact compliance unless effectively explained and managed. The major benefits of letrozole are to improve prospects for long-term survivorship in the adjuvant setting and to delay progression and the need for chemotherapy in the metastatic setting.\nIntroduction\nThe diagnosis of breast cancer is a devastating blow for women and is associated with depression, anxiety, and a range of other psychological problems, such as self-blame and negative perception of body-image [1\u20135]. Newly diagnosed metastatic breast cancer (MBC) is also associated with high levels of psychological morbidity, particularly for younger women [6]. Prevention of relapse is a long-term therapeutic imperative, but the impact of therapy on quality of life (QOL) also needs to be taken into consideration when planning treatment strategies.\nAll therapeutic modalities for early breast cancer (mastectomy or lumpectomy, radiation, chemotherapy, antibody therapy and endocrine therapy) can have a significant impact on patients\u2019 QOL both in the short-term and in the transition period from primary treatment to long-term survivorship [7\u20139]. Receiving good quality information about prognosis, treatment options, side effects, and risks of breast cancer recurrence is, therefore, important for patients diagnosed with breast cancer [10\u201313].\nThe third-generation aromatase inhibitor (AI) letrozole was introduced a decade ago, but at that time, a limited amount of information on breast cancer and its treatment was available to patients. Since then, the international oncology community has made major advances in its knowledge about breast cancer biology, the individualization of treatment options, and communication with patients. Greater understanding of genetics and breast cancer risk has improved approaches for counseling individuals about their susceptibility [14]. In women who develop breast cancer, the individualization of treatment is becoming more sophisticated through the use of biomarkers as prognostic and predictive factors. These include human epidermal growth factor receptor (HER) 2 [15, 16], urokinase-type plasminogen activator and plasminogen activator inhibitor type 1 [17, 18], mRNA expression patterns [19, 20], and, more recently, genetic profiling techniques [21, 22]. Individualizing treatment is also made easier by the ability to more accurately determine the patient\u2019s risk factors and prognosis; for example, by using the Adjuvant! Online program [23\u201325]. Recognizing breast cancer overtreatment has also contributed to tailoring treatment according to individual risk [26\u201330]. Oncology teams have facilitated patient involvement in selecting the most appropriate therapy [24, 31, 32] and have improved the provision of patient information, support, and counseling [33, 34].\nAt the same time, patients began to organize effectively and expand their access to knowledge of new drugs and treatment paradigms [35\u201337]. Patients have learned to be informed so that they can have constructive dialogues with their doctors and feel they understand and contribute to treatment decisions [38\u201342]. Patients who take an active role in the decision-making process perceive that they have a treatment choice, in contrast to those who prefer a shared or passive role [43]. They know they cannot be told at any time during the course of their disease that they are or are not cured; they recognize that breast cancer is a chronic illness. Patients are now empowered with hope and the knowledge that breast cancer can be managed. Moreover, increased survival rates have prompted greater interest in the QOL of breast cancer survivors [44\u201346].\nBetter education and improved access to screening has resulted in fewer patients being diagnosed with advanced breast cancer at their first presentation, and consequently, mortality has decreased [29, 47\u201349]. In addition, more effective adjuvant therapy with third-generation AIs has significantly decreased the risk of recurrence as compared with the previous standard of care based on tamoxifen [50\u201353]. However, there remains a long-term risk of breast cancer recurrence over time [54, 55]. Hormone receptor-positive (HR+) breast cancer is particularly challenging because of the persistent risk of recurrence with this chronic \u201csmoldering\u201d disease. The Early Breast Cancer Trialists Collaborative Group meta-analysis showed that in untreated women with the same nodal status, the breast cancer death rate is greater for patients with estrogen receptor-negative (ER\u2212) versus ER+ tumors in the first 5\u20136\u00a0years, but substantially lower for ER\u2212 versus ER+ tumors over the next 10\u00a0years [56]. Similarly, Saphner reported that beyond 5\u00a0years, the risk of recurrence was higher for patients with HR+ tumors than for those with HR\u2212 tumors (P\u00a0=\u00a00.00002) [54].\nPatients may experience stress after medical therapy is ended because they feel they have lost a safety net [57, 58]. This fear is understandable and justifiable in view of the persistent risk of recurrence for HR+ breast cancer. An analysis of the MA.17 trial of extended adjuvant therapy recently showed an increasing risk of disease recurrence over time in patients treated with placebo after discontinuing tamoxifen [59]. Not surprisingly, therefore, patients may be willing to receive long-term therapy (extended adjuvant) to prevent recurrence provided that the benefits outweigh the risks and QOL is maintained [60]. Patients will try to tolerate treatment-related adverse events if there is the prospect of achieving a \u201ccure,\u201d i.e. remaining free of relapse during their lifetime.\nTamoxifen was the mainstay of breast cancer therapy, but its time-dependent efficacy and serious adverse events created a need for new therapies [61\u201363]. The suppression of estrogen was shown to be greater and more selective with third-generation AIs than with first- and second-generation compounds [64], and this has resulted in better clinical outcomes and improved tolerability [65]. This review examines the clinical use of the third-generation AI letrozole from the patient\u2019s perspective and assesses how it has improved treatment outcomes across the breast cancer continuum, including advanced or MBC, extended and initial adjuvant therapy, and neoadjuvant therapy.\nMetastatic setting\nEndocrine therapy is the first-choice treatment for women with HR+ breast cancer without acute life-threatening symptoms and should be administered for as long as possible before switching to cytotoxic regimens. Endocrine therapy is preferred to cytotoxic chemotherapy because of its more favorable safety profile [66]. Thus, extending the time to the initiation of chemotherapy is important with endocrine therapy, because a considerable proportion of patients who progress to chemotherapy will experience toxic side effects without gaining benefit [67]. The most common acute side effects of chemotherapy, such as leukopenia, alopecia, and nausea and vomiting, are significantly increased in women receiving combination regimens compared with single agents [68]. Chemotherapy can also adversely affect certain aspects of QOL, notably increasing fatigue [69], and some regimens may be associated with severe or life-threatening complications such as cardiac failure [70]. A meta-analysis to review the evidence and determine whether chemotherapy or endocrine therapy has the most beneficial effect on treatment outcomes (survival, response rate, toxicity, and QOL) concluded that in women with HR+\u00a0MBC, a policy of treating first with endocrine therapy rather than chemotherapy is recommended except in the presence of rapidly progressive disease [71].\nPrior to the introduction of third-generation AIs, postmenopausal patients with hormone-responsive MBC had few endocrine therapy options other than tamoxifen [62]. Second-line endocrine agents were limited by safety concerns, including weight gain or the risk of thromboembolism with megestrol acetate [72, 73] and cardiovascular toxicity with aminoglutethimide [65]. Randomized controlled trials demonstrated that letrozole is better tolerated and more effective than these second-line endocrine agents [65, 72, 73]. Furthermore, letrozole has also demonstrated superior early survival compared with tamoxifen as first-line therapy in postmenopausal patients with MBC [74]. Importantly, longer time to disease progression with letrozole versus tamoxifen was achieved without increased time with adverse events and resulted in more quality-adjusted survival for patients on letrozole [75]. Maintaining functional ability is an important goal for patients treated in this setting. A subanalysis by different sites of metastatic lesion and Karnofsky Performance Scores also showed the superiority of letrozole compared with tamoxifen in patients with nonvisceral metastases, with visceral metastases without liver involvement, and with liver metastases [76]. From the patient\u2019s perspective, it is important to receive the most effective therapy first-line; therefore, letrozole represents a more attractive option than tamoxifen for postmenopausal women.\nPharmacologic and clinical differences exist between third-generation inhibitors and should be considered in the selection of the most appropriate endocrine therapy [64, 77]. Data from a small randomized crossover trial in 72 postmenopausal women with HR+\u00a0MBC showed that overall QOL was significantly better with letrozole than with anastrozole (P\u00a0=\u00a00.002 for mean total Functional Assessment of Cancer Treatment-endocrine symptoms) [78]. Furthermore, letrozole was significantly better tolerated overall than anastrozole. Less nausea, fewer hot flashes, and less abdominal discomfort resulted in almost twice as many patients preferring letrozole to anastrozole [78].\nWhile letrozole is an appropriate first-line therapy for the majority of patients with hormone-responsive MBC, a small subset of patients with HR+ HER2+ tumors have high-risk disease and are candidates for early treatment with chemotherapy plus the anti-HER2 monoclonal antibody trastuzumab [79]. A meta-analysis of 12 studies involving 2,379 patients with MBC demonstrated that HER2+ tumors are less responsive than HER2\u2212 tumors to endocrine treatment (overall relative risk 1.42; 95% confidence interval, 1.32\u20131.52; P\u00a0<\u00a00.00001) [80]. Evidence from preclinical models suggests that trastuzumab may overcome relative resistance to endocrine therapy [81], providing the rationale for combining anti-HER2 and endocrine therapies in the clinical setting. Recently, the first published results of an AI in combination with an anti-HER2 antibody (i.e. trastuzumab) show that the combination demonstrated durable responses for at least 1\u00a0year in 25% of patients [82]. Preliminary results from a phase 3 trial (TrAstuzumab in Dual HER2 ER-positive Metastatic breast cancer) demonstrated that the combination of trastuzumab with an AI (anastrozole) was more effective than anastrozole alone in postmenopausal patients with HR+ HER2+ MBC [83]. Another phase 3 trial, which is enrolling more than 1,200 patients in this setting and has just finished recruitment, is investigating letrozole in combination with lapatinib, a dual inhibitor of HER2 and HER1 tyrosine kinases [84]. The strategy of combining letrozole with trastuzumab (as investigated in the evaluation of Letrozole combined with Trastuzumab trial), or another HER2-directed therapy, may allow patients with HR+, HER2+ tumors to safely delay the initiation of cytotoxic chemotherapy.\nAdjuvant therapy\nPatients receiving adjuvant therapy expect their treatment to prevent breast cancer recurrence and offer the prospect of cure. However, even modest gains in survival are sufficient to make adjuvant endocrine treatment worthwhile for premenopausal women with early-stage breast cancer [85]. This benefit threshold also appears to apply to women with a higher risk of recurrence for whom adjuvant chemotherapy is indicated [86]. Adjuvant therapy should be tailored to suit the needs of individual patients based on their clinical risk factors, attitudes, and personal life circumstances [25]. However, it is important to recognize that individual preferences cannot always be fully explained on the basis of treatment-related determinants and patient or clinical characteristics [87].\nThe Anastrozole versus Letrozole: Investigation into Quality of Life study compared the effects of anastrozole and letrozole on estrogen levels, QOL, lipids, and bone health [88]. A total of 185 postmenopausal women with invasive breast cancer were randomized to receive adjuvant therapy with either 12\u00a0weeks of letrozole followed by 12\u00a0weeks of anastrozole or vice versa. Of the patients who have completed the 12\u00a0weeks of treatment (n\u00a0=\u00a0146), 50 (34%) had a preference for neither drug, 50 (34%) preferred anastrozole, and 46 (32%) preferred letrozole. Both estradiol (E2) and estrone sulfate levels (E1S) were significantly lower on letrozole than on anastrozole (P\u00a0<\u00a00.000001). Thus, 2.5\u00a0mg of letrozole reduces circulating E2 and E1S levels to a significantly greater degree than 1\u00a0mg of anastrozole, with no significant difference in patient preference. The greater suppression of estradiol levels might translate into improved clinical efficacy, although further studies, such as the ongoing Femara Anastrozole Clinical Evaluation trial, are required to confirm these findings.\nPatients with HR+ tumors need to consider the prospect of life-long adjuvant therapy in view of the persistent risk of disease recurrence [54, 56, 89]. Following the introduction of third-generation AIs, there is much greater choice in endocrine therapy. The challenge for physicians is to select the most appropriate strategy to suit individual patient circumstances. Letrozole first entered the adjuvant setting as a treatment option for patients completing 5\u00a0years of tamoxifen [90] and has demonstrated clinical superiority over tamoxifen as initial adjuvant therapy [51] and neoadjuvant therapy [91].\nNeoadjuvant therapy \nNeoadjuvant endocrine therapy with letrozole is an attractive option for some postmenopausal patients with HR+ early breast cancer [92], including elderly patients who are unable or unwilling to undergo chemotherapy or surgery, and patients with locally advanced HR+ tumors who wish to have breast-conserving surgery but are not suited for preoperative chemotherapy [91, 93]. In a randomized controlled trial, letrozole demonstrated a superior overall objective response rate and rate of breast-conserving surgery compared with tamoxifen as neoadjuvant therapy [91]. Currently, letrozole is the only AI approved in the neoadjuvant setting (in the United Kingdom and 16 other countries worldwide). It provides a reasonable therapeutic alternative to preoperative chemotherapy in postmenopausal women with HR+ disease in clinical situations where the low toxicity of the regimen is considered an advantage, e.g. in women older than 70\u00a0years [91]. However, patients need to be aware that a longer course of therapy may be required to achieve an objective response than is the case with neoadjuvant chemotherapy [94]. In one study, neoadjuvant letrozole was safely given over 12\u00a0months to postmenopausal women with large operable or locally advanced HR+\u00a0breast cancers [94]. A longer treatment course may suit some patients and give them more time to consider their options for surgery, radiotherapy, and chemotherapy.\nNeoadjuvant therapy with letrozole could be considered a sensitivity test of endocrine therapy that might be incorporated into strategies to individualize adjuvant treatment according to response [95]. This would provide reassurance to patients that they will be receiving an adjuvant endocrine therapy that has shown activity against their tumor. Objective assessment of the antitumor activity of neoadjuvant letrozole can be made on the basis of response rate and by analysis of predictive biomarkers. In one study, neoadjuvant letrozole was found to inhibit tumor proliferation (determined by the biomarker Ki67) more effectively than tamoxifen [95]. Letrozole was effective independently of HER2 expression status, although the greatest difference between letrozole and tamoxifen was seen in tumors that were HR+ and HER1\/2+. Recent evidence has suggested that HER2+ tumors can continue to proliferate despite neoadjuvant letrozole or tamoxifen treatment, which could imply therapeutic resistance that may manifest later in the clinical course of the disease [96].\nBiomarker studies may also reveal differences between AIs. In a randomized comparative trial, letrozole and anastrozole significantly reduced proliferation in HR+, HER2+ or HER2\u2212 tumors [97]. Decreased proliferation was seen at all Allred ER expression levels with both agents, but only letrozole showed a significant effect in the lower ER cases. In addition, more cases showed a reduction in progesterone receptor (PgR) expression following letrozole than anastrozole [97]. Another study showed evidence of a decrease in HER2 expression after neoadjuvant treatment with letrozole [98].\nAlthough complex, biomarkers may become increasingly important from a patient\u2019s perspective, because they can help to improve the individualization of treatment. Furthermore, biomarkers may be useful to predict the risk of resistance to endocrine therapy and the need to consider alternative approaches in the future, such as combination therapies or cytotoxic chemotherapy. Knowing what to expect next is important for patients. However, further work is necessary to validate the use of clinical markers and biomarkers in the neoadjuvant setting as surrogate end points for long-term outcomes [96].\nEarly adjuvant therapy\nTamoxifen was the gold standard endocrine therapy for all women with HR+ breast cancer until recent results from large randomized trials challenged this paradigm for postmenopausal women [50, 51, 99]. Oncologists and patients now face an important choice when selecting adjuvant endocrine treatment [66, 100], whether to start with the most potent endocrine therapy (an AI) upfront or to start with tamoxifen and switch to an AI inhibitor after 2\u20135\u00a0years. The ongoing Breast International Group (BIG) 1-98 randomized trial is expected to provide more information on the benefits of switching to letrozole after 2\u20133\u00a0years on tamoxifen and will clarify which is the optimal strategy. The final results are expected to be released in 2008 [51], but until then, the most appropriate endocrine therapy will need to be selected on the basis of currently available evidence. The MA.17 trial has already demonstrated that letrozole treatment is beneficial after 5\u00a0years of adjuvant tamoxifen [90, 101, 102].\nPatients will ideally wish to receive treatment that provides the greatest long-term efficacy with the lowest risk of adverse events. BIG 1-98 showed that letrozole was significantly more effective than tamoxifen as initial adjuvant endocrine therapy [51]. Moreover, tamoxifen is perceived to be more toxic than AIs, largely because of well-publicized \u201cscares\u201d about increased risk of uterine cancer and thrombosis [61, 103]. These side effects and vaginal bleeding were reported for tamoxifen in BIG 1-98, whereas letrozole was associated with more skeletal events. The overall rate of cardiovascular events was not significantly different between the groups. Letrozole was also shown to be extremely well-tolerated in comparison with placebo in the MA.17 trial [90, 101]. With the exception of adverse events related to suppression of estrogen, there was no difference in adverse events (Table\u00a01).\nTable\u00a01Safety profile of letrozole in comparison with placebo (A) and tamoxifen (B) reported in postmenopausal women with early breast cancerPlacebo Acute toxicities reportedTotal number (%), any grade101Letrozole (n\u00a0=\u00a02,572)Placebo (n\u00a0=\u00a02,577)Edema571 (22)542 (21)Hypertension130 (5)129 (5)Hot flushes1,486 (58)a1383 (54)Fatigue999 (39)998 (39)Sweating782 (30)760 (29)Anorexia142 (6)a110 (4)Constipation363 (14)382 (15)Diarrhea168 (7)176 (7)Nausea308 (12)314 (12)Vaginal bleeding145 (6)196 (8)aInfection without neutropenia124 (5)112 (4)Arthritis167 (6)137 (5)Hypercholesterolemia418 (16)411 (16)Dizziness458 (18)441 (17)Insomnia166 (6)135 (5)Depression143 (6)131 (5)Headache706 (27)685 (27)Arthralgia651 (25)a532 (21)Myalgia380 (15)a310 (12)Bone pain141 (5)149 (6)Dyspnea161 (6)163 (6)Alopecia126 (5)a89 (3)Vaginal dryness147 (6)129 (5)TamoxifenWorst grade adverse events recorded within first 28\u00a0daysTotal number (%), any grade51Letrozole (n\u00a0=\u00a03,975)Tamoxifen (n\u00a0=\u00a03,988)CVA or TIA39 (1.0)41 (1.0)Thromboembolic event61 (1.5)140 (3.5)aCardiac event (IHD, CF)162 (4.1)153 (3.8)Other CV event19 (0.5)a8 (0.2)Vaginal bleeding132 (3.3)263 (6.6)Hot flushes1332 (33.5)1516 (38.0)aNight sweats554 (13.9)647 (16.2)aFracture225 (5.7)a159 (4.0)Arthralgia806 (20.3)a491 (12.3)Myalgia254 (6.4)243 (6.1)CV cardiovascular, CVA cerebrovascular accident, TIA transient ischemic attack, IHD ischemic heart disease, CF cardiac failureaSignificant difference\nBased on the results from the BIG 1-98 and the Anastrozole and Tamoxifen Alone or in Combination (ATAC) trial, which compared upfront AIs with tamoxifen, it is clear that all postmenopausal women with HR+ breast cancer should be given the opportunity to receive adjuvant use of an AI, and this recommendation is now reflected in internationally recognized treatment guidelines [66, 100, 104, 105]. Patients with HR+ breast cancer considered eligible for adjuvant chemotherapy because of their increased risk for relapse should also be candidates for the most effective adjuvant endocrine strategy [100]. Of note, the BIG 1-98 trial showed that adjuvant letrozole provides significant disease-free survival (DFS) benefits for patients at increased risk of recurrence, specifically patients with node-positive tumors, large primary tumors (>2\u00a0cm), and recipients of chemotherapy and also demonstrated a significant reduction in the risk of distant recurrence, a well-known predictor of breast cancer death [51]. Additional analyses of data from BIG 1-98 to determine the predictive value of centrally tested ER, PgR, and HER2 status on the response to letrozole and tamoxifen indicate that there is no difference in subgroups by ER\/PR or HER2 status regarding the superiority of upfront letrozole versus tamoxifen [106]. Low-risk patients with HR+ breast cancer who do not receive adjuvant chemotherapy are also candidates for AIs [104] because of the persistent risk of relapse [54, 56].\nWhile it is known that tamoxifen loses effectiveness after 5\u00a0years [62, 63], the optimal duration of initial therapy with an AI remains to be determined [107]. The MA.17 randomized controlled trial of extended adjuvant endocrine therapy has already shown that letrozole treatment is beneficial after 5\u00a0years of tamoxifen in postmenopausal patients [90, 101], but there are no equivalent data on the use of tamoxifen after initial AI therapy in the adjuvant setting [100]. Furthermore, the efficacy of switching to another AI in patients who discontinue treatment with letrozole is not known. Therefore, patients starting upfront therapy with letrozole will need reassurance that according to present knowledge, treatment can be continued for up to 5\u00a0years, and other options will be available down the line. In addition, patients will require information about the potential long-term impact of AIs on bone and the cardiovascular system [107].\nAccording to the current treatment guidelines [66, 100, 104, 105], patients already taking a course of adjuvant tamoxifen may wish to switch to anastrozole or exemestane, as these AIs have demonstrated efficacy in this setting [52, 108, 109]. Although recent data have shown that this sequential adjuvant strategy is associated with a survival advantage [53, 110, 111], it is important to note that these data were obtained from a selected population comprising patients at randomization who were disease-free after 2\u20133\u00a0years of tamoxifen. It is therefore not valid to make a direct comparison with an unselected population treated in trials of upfront AI therapy. The decision to switch endocrine therapy after 2\u20133\u00a0years, i.e. for a sequential tamoxifen\u2013AI strategy, should be based on an individual\u2019s risk for recurrence, risk for osteoporosis, and ability to tolerate tamoxifen. Importantly, BIG 1-98 is expected to provide more information on the benefits of switching to letrozole after 2\u20133\u00a0years of tamoxifen, and will clarify whether upfront or sequential AI use is the optimal strategy. The final results will be released in 2008 [51].\nExtended adjuvant (including late extended adjuvant) therapy\nMany patients fear breast cancer recurrence [112] and are reassured by the \u201csafety net\u201d of continuing medical treatment and monitoring [9]. The MA.17\u00a0trial showed that extended adjuvant therapy with letrozole gives patients the opportunity of retaining the safety net for at least 5\u00a0more years after adjuvant tamoxifen [101], and these results led to its approval in this indication. Currently, letrozole is the only AI approved as an extended adjuvant therapy.\nThe final analysis of MA.17 after a median follow-up of 30\u00a0months showed letrozole significantly improved DFS (42% reduction in risk vs. placebo), distant DFS (40% reduction in risk vs. placebo) and, in node-positive patients, overall survival (39% reduction in risk vs. placebo) [101]. A recent cohort analysis of the MA.17 trial data suggested that the longer patients are exposed to letrozole, the greater the benefit, at least out to 48\u00a0months [59]. In addition, an extension to the MA.17 trial (MA.17 Re-randomization) is being conducted to determine the benefits of continuing letrozole for a further 5\u00a0years [102].\nWhen a planned interim analysis of MA.17 revealed a significant advantage for letrozole, the trial was unblinded and patients on placebo were given the option to switch to letrozole [90]. In this non-randomized comparison, women from the placebo arm who elected to switch to letrozole also experienced an improvement in outcome when compared with those who elected to have no treatment [102].\nRecurrence risk persists beyond the completion of adjuvant therapy, even in patients at low risk of recurrence. One study shows that with systemic adjuvant therapy, patients with node-negative breast cancer have a \u226525% 10-year risk of relapse and a corresponding 10-year breast cancer death rate as high as \u226510%, depending on tumor grade and size [113]. Thus, the proven efficacy of letrozole given after a tamoxifen-free period means that physicians need to discuss the option of restarting endocrine therapy with almost all patients. Physicians have to consider how best to address this topic with patients who are up to 2\u20133\u00a0years out beyond their initial 5\u00a0years of tamoxifen, i.e. about 5\u20138\u00a0years after their initial diagnosis. This will be a major communication challenge and create a dilemma for patients who may feel well and have put their breast cancer behind them.\nSafety and compliance issues\nThe clinical benefits of AIs and tamoxifen are generally achieved without a major detrimental effect on overall QOL [114]. Data from the MA.17 trial, which is the only large adjuvant trial comparing AI therapy not with tamoxifen but with placebo, showed that overall QOL was maintained during extended therapy with letrozole, and only a minority of patients experienced substantial changes in QOL, which were compatible with a reduction in estrogen synthesis [115]. It is important to recognize that patients may attribute such changes to their treatment, whereas they could in fact be symptoms of menopause [116]. However, side effects do occur, which not only affect patient adherence to endocrine therapy [117] but can also lead to additional morbidity and even serious or life-threatening complications in a small minority of patients [51, 52, 90, 99, 108, 109].\nThe main safety concerns with long-term estrogen deprivation include potential effects on bone health, cardiac health, lipid profile, cognitive functioning, and sexual health [100]. The MA.17 trial showed that letrozole is well-tolerated in comparison with placebo [101], and BIG 1-98 showed that letrozole was better tolerated than tamoxifen [51] (Table\u00a01). A recent patient-reported outcomes study in 104 tamoxifen-intolerant women found that switching to letrozole was associated with a significant reduction in hot flushes (P\u00a0=\u00a00.001) and significant improvements in QOL (P\u00a0=\u00a00.001) and mood (P\u00a0=\u00a00.04). Furthermore, when given the choice of continuing therapy, 66% of women indicated that they preferred to remain on letrozole, while only 24% preferred to go back to tamoxifen [118].\nDespite an increase in newly diagnosed osteoporosis with AIs, no significant difference in clinical fracture rate was seen between letrozole and placebo [101]. A companion study to MA.17 showed that there was only a modest increase in bone resorption and reduction in bone mineral density (BMD) in the spine and hip with letrozole compared with placebo [119]. Consequently, patients should be made aware of the risk of osteoporosis and given advice on lifestyle measures (e.g. exercise, diet, vitamins) to reduce risk. A baseline BMD measurement should be obtained for all patients before starting therapy with an AI. BMD should then be measured annually, and patients at high risk of osteoporosis should be considered for prophylactic use of a bisphosphonate [120, 121]. However, recent data from ATAC may be reassuring for patients facing upfront AI therapy, since they indicate that women with normal initial BMD did not develop osteoporosis during a 5-year AI treatment [122].\nLetrozole has been associated with an increase in arthralgia compared with placebo or tamoxifen, and myalgia compared with placebo [51, 101]. Arthralgia and joint pain can be bothersome and may lead to impaired mobility. Patients need to be advised that constant exercising of the joints will alleviate this adverse event and that use of nonsteroidal anti-inflammatory drugs over a certain period of time may be helpful. Moreover, these complaints seem to be most frequent in the beginning of AI therapy [123].\nAnother potential consequence of endocrine therapy is a detrimental effect on sexual health [115, 116]. Vaginal dryness is a consequence of menopause and treatments that cause menopausal symptoms, and has an important bearing on sexual health and well-being of breast cancer survivors [124]. Vaginal dryness can be a significant problem that can interfere with the stability of relationships [125], and patients need to receive counseling with regard to this issue [126]. It has been shown that clinical assessment and an active intervention program for menopausal symptom management in breast cancer survivors can lead to an improvement in sexual functioning [127]. A recent report demonstrated that the use of local estradiol therapy may cause elevation of serum estradiol and may therefore be contraindicated in postmenopausal women on AI therapy [128]. So far, such data do not exist for estradiol-containing compounds; thus, local estradiol treatment may be indicated in individual cases. Androgen treatment may also improve sexual well-being in postmenopausal women [129], yet the oncological safety of this approach has not been validated in patients receiving AI therapy. Of note, the incidence of vaginal dryness in the MA.17 trial was similar in the letrozole and placebo groups (Table\u00a01) [101].\nDifferential effects on lipid profiles and cardiac risk have been reported between tamoxifen and AIs [51, 130, 131], but data from the Letrozole, Exemestane, and Anastrozole Pharmacodynamics trial, directly comparing the effects of anastrozole, letrozole, and exemestane on lipid profiles in healthy postmenopausal women, suggest that the steroidal and nonsteroidal inhibitors have a similar impact on lipid profiles [132]. On the basis of evidence from large randomized trials, when comparing letrozole with tamoxifen or placebo, it appears that letrozole does not have clinically relevant adverse effects on lipids or cardiac risk during long-term adjuvant therapy, although further assessment is warranted [51, 101, 133].\nWhile letrozole is well-tolerated, and any side effects that do occur can be managed, it is essential that patients are motivated to stay on therapy. This can be a particular challenge in the extended adjuvant setting. Thus, as patients now face the prospect of receiving endocrine therapy for 10\u00a0years or more, it is important to consider the overall life-long benefits and risks from the individual\u2019s perspective. A low burden of adverse events and maintenance of QOL is important in maintaining adherence to long-term treatment [60]. In the MA.17 trial, the rate of patients choosing to discontinue therapy during the first year was similar for letrozole (n\u00a0=\u00a0256\/2,575) and placebo (n\u00a0=\u00a0254\/2,582) [90]. Letrozole is ingested orally and can be safely and conveniently taken at home. Physician contact with oral endocrine therapy can be much less frequent than, for example, with orally active chemotherapy where regular blood tests, side-effect monitoring, and resulting dose modifications make frequent physician contact mandatory. Patients with breast cancer prefer oral cancer therapy providing that it does not compromise treatment efficacy [134].\nConclusions\nPatients with breast cancer face bewildering choices at a time when they are experiencing highly stressful circumstances. Moreover, many of these women are not well-informed and thus are not able to take part in treatment decisions. The Gathering Information on Adjuvant Endocrine therapy initiative decided to delineate women\u2019s knowledge and experience of adjuvant endocrine therapy. Results thus far indicate that only 22% of patients were fully or highly involved in the decision to start adjuvant endocrine therapy [135, 136]. Many of the women who took part in the survey were not satisfied with the degree to which they were involved in treatment decision-making, and women 60\u00a0years or older had the lowest levels of involvement [135, 136]. The results from this survey show that information provided to patients about adjuvant endocrine therapy is suboptimal and indicate the need for programs to raise patient awareness. The medical community has a responsibility to help patients understand their prospects for survival and make the right choices about treatment [24]. Doctor\u2013patient discussions and programs to raise patient awareness will increase patients\u2019 knowledge about the individualization of treatment and may increase the number of women who take an active role in treatment decisions.\nPatients with hormone-responsive breast cancer should be offered the opportunity of receiving the most effective endocrine therapy. Clinical evidence suggests that postmenopausal women should receive an AI rather than tamoxifen as their first option if tumor characteristics and individual side-effect profiles support this choice. Letrozole has consistently demonstrated superiority over tamoxifen in the metastatic and adjuvant treatment settings [51, 74, 91, 101]. As a result of its innovative clinical trial program [51, 74, 101], letrozole is approved for use in postmenopausal women throughout the breast cancer care continuum [137]. From the patient\u2019s perspective, the major benefits of letrozole are improving prospects for long-term survivorship (\u201ccure\u201d) in the adjuvant setting and delaying progression and the need for chemotherapy in the metastatic setting.\nEndocrine therapy is very effective, has a generally favorable safety profile, and adds to the efficacy of chemotherapy. However, a major challenge for both physicians and patients is ensuring compliance with long-term daily therapy. This may be a particular problem in the extended adjuvant setting, where the patient may face the prospect of life-long therapy. As patients no longer feel sick, it is understandable that they may forget to take a dose and gradually lose interest in continuing with the treatment. Letrozole is well-tolerated and, as with all AIs, the majority of adverse events are secondary to the suppression of estrogen. The most important adverse events from the patient\u2019s perspective are the \u201cvisible\u201d ones, such as hot flushes, vaginal dryness, and arthralgias. To achieve optimal compliance, patients need to feel that physicians are taking their adverse events seriously and taking appropriate steps to alleviate any problems. Physicians who treat very few breast cancer patients may not have sufficient experience with AIs to satisfactorily manage individuals who are experiencing these adverse events.\nLetrozole is likely to continue to play a major role in the management of breast cancer in all settings (see the paper in this supplement by Drs. Ellis and Ma, on Femara\u00ae and the future). Considering its efficacy and favorable side-effect profile, it is the logical choice for inclusion in new regimens, including combinations with novel agents. As the future unfolds, the management of breast cancer is set for further change, and it is essential that patients are informed and educated so that they can actively participate in treatment decisions and thus derive the most benefit from treatment advances.","keyphrases":["patient","breast cancer","letrozole","aromatase inhibitors","adjuvant therapy","neoadjuvant therapy"],"prmu":["P","P","P","P","P","P"]}
{"id":"J_Hum_Genet-4-1-2413114","title":"Variations in the FTO gene are associated with severe obesity in the Japanese\n","text":"Variations in the fat-mass and obesity-associated gene (FTO) are associated with the obesity phenotype in many Caucasian populations. This association with the obesity phenotype is not clear in the Japanese. To investigate the relationship between the FTO gene and obesity in the Japanese, we genotyped single nucleotide polymorphisms (SNPs) in the FTO genes from severely obese subjects [n = 927, body mass index (BMI) \u2265 30 kg\/m2] and normal-weight control subjects (n = 1,527, BMI < 25 kg\/m2). A case-control association analysis revealed that 15 SNPs, including rs9939609 and rs1121980, in a linkage disequilibrium (LD) block of approximately 50 kb demonstrated significant associations with obesity; rs1558902 was most significantly associated with obesity. P value in additive mode was 0.0000041, and odds ratio (OR) adjusted for age and gender was 1.41 [95% confidential interval (CI) = 1.22\u20131.62]. Obesity-associated phenotypes, which include the level of plasma glucose, hemoglobin A1c, total cholesterol, triglycerides, high-density lipoprotein (HDL) cholesterol, and blood pressure were not associated with the rs1558902 genotype. Thus, the SNPs in the FTO gene were found to be associated with obesity, i.e., severe obesity, in the Japanese.\nIntroduction\nObesity is the most common nutritional disorder in developed countries, and it is a major risk factor for hypertension, cardiovascular disease, and type 2 diabetes (Kopelman 2000; Wilson et al. 2003). Genetic and environmental factors contribute to obesity development (Maes et al. 1997; Barsh et al. 2000; Rankinen et al. 2006). Recent progress in single nucleotide polymorphism (SNP) genotyping techniques has enabled genome-wide association studies on common diseases (Herbert et al. 2006; Frayling et al. 2007; Scuteri et al. 2007; The Wellcome Trust Case Control Consortium 2007; Hinney et al. 2007). Using a large-scale case-control association study, we found that secretogranin III (SCG3) (Tanabe et al. 2007) and myotubularin-related protein 9 (MTMR9) (Yanagiya et al. 2007) are involved in susceptibility to the obesity phenotype. Genome-wide association studies have shown that the fat-mass and obesity-associated gene (FTO) is also associated with the obesity phenotype (Frayling et al. 2007; Scuteri et al. 2007; Hinney et al. 2007). This association was also found in many Caucasian and Hispanic American populations (Frayling et al. 2007; Scuteri et al. 2007; Dina et al. 2007; Field et al. 2007; Andreasen et al. 2008; W\u00e5hl\u00e9n et al. 2008; Peeters et al. 2008), whereas it was not found in the Chinese Han population (Li et al. 2008). Among Japanese, body mass index (BMI) was higher in subjects who had the A allele of rs9939609, similar to that observed in Caucasians; however, this finding was not significant (Horikoshi et al. 2007). Another group reported that rs9939609 was associated with BMI in the Japanese (Omori et al. 2008). Thus, the association of SNPs in the FTO gene with obesity in the Japanese remains controversial.\nTo investigate the relationship between the FTO gene and obesity in the Japanese, we performed a case-control association study using patients with severe adult obesity (BMI\u00a0\u2265\u00a030\u00a0kg\/m2) and normal-weight controls (BMI\u00a0<\u00a025\u00a0kg\/m2); we found that SNPs in intron 1 of the FTO gene were associated with severe adult obesity.\nMaterials and methods\nStudy subjects\nThe sample size for severely obese Japanese subjects (BMI\u00a0\u2265\u00a030\u00a0kg\/m2) was 927 (male:female ratio 419:508, age 48.7\u00a0\u00b1\u00a014.2\u00a0years, BMI 34.2\u00a0\u00b1\u00a05.4\u00a0kg\/m2), whereas that for Japanese normal weight controls (BMI\u00a0<\u00a025\u00a0kg\/m2) was 1,527 (male:female ratio 685:842, age 48.1\u00a0\u00b1\u00a016.5\u00a0years, BMI 21.7\u00a0\u00b1\u00a02.1\u00a0kg\/m2). The severely obese subjects were recruited from among outpatients of medical institutes. Patients with secondary obesity and obesity-related hereditary disorders were not included, and neither were patients with medication-induced obesity. The normal-weight controls were recruited from among subjects who had undergone a medical examination for screening of common diseases. Clinical features of the subjects are illustrated in Table\u00a01. Additionally, 1,604 subjects were recruited (male:female ratio 803:801, age 48.7\u00a0\u00b1\u00a016.9\u00a0years, BMI 22.66\u00a0\u00b1\u00a03.16\u00a0kg\/m2) from the Japanese general population. Each subject provided written informed consent, and the protocol was approved by the ethics committee of each institution and that of RIKEN.\nTable\u00a01Clinical characterization of obese and control subjectsObeseControlP valueGender (M\/F)419\/508658\/842Age (year)49.1\u00a0\u00b1\u00a014.248.2\u00a0\u00b1\u00a016.50.049Body mass index (kg\/m2)34.50\u00a0\u00b1\u00a05.3921.65\u00a0\u00b1\u00a02.08 <0.000001Glucose (mg\/dl)129.2\u00a0\u00b1\u00a049.697.7\u00a0\u00b1\u00a023.9<0.000001HbA1c (%)6.5\u00a0\u00b1\u00a01.85.1\u00a0\u00b1\u00a00.6<0.000001Total cholesterol (mg\/dl)209.9\u00a0\u00b1\u00a037.9201.2\u00a0\u00b1\u00a036.4<0.000001Triglycerides (mg\/dl)153.2\u00a0\u00b1\u00a099.5104.0\u00a0\u00b1\u00a073.2<0.000001High-density lipoprotein cholesterol (mg\/dl)53.1\u00a0\u00b1\u00a018.965.1\u00a0\u00b1\u00a015.7<0.000001Systolic blood pressure (mmHg)136.4\u00a0\u00b1\u00a018.1123.4\u00a0\u00b1\u00a017.8<0.000001Diastolic blood pressure (mmHg)83.8\u00a0\u00b1\u00a012.076.0\u00a0\u00b1\u00a011.1<0.000001P values were analyzed using Mann\u2013Whitney U test. Data are mean\u00a0\u00b1\u00a0standard deviation\nDNA preparation and SNP genotyping\nGenomic DNA was prepared from the blood sample of each subject by using the Genomix (Talent Srl, Trieste, Italy). We searched for dbSNPs with minor allele frequencies (MAF)\u00a0>\u00a00.10 in the FTO gene of Japanese people. We selected 90 SNPs and were able to construct Invader probes (Third Wave Technologies, Madison, WI) for them (Supplementary Table 1). SNPs were genotyped using Invader assays as described previously (Ohnishi et al. 2001; Takei et al. 2002). Nine SNPs (rs9937053, rs9939973, rs9940128, rs7193144, rs8043757, rs9923233, rs9926289, rs9939609, and rs9930506) reported in a previous genome-wide association study (Scuteri et al. 2007) were genotyped using TaqMan probes (C__29910458_10, C__11776771_10, C__29621384_10, C__29387650_10, C__29387665_10, C__29693738_10, C__30270568_10, C__30090620_10, and C__29819994_10; Applied Biosystems, Foster City, CA, USA).\nStatistical analysis\nGenotype or allele frequencies were compared between cases and controls in three different modes. In the first mode, i.e., the additive mode, \u03c72 test was performed according to Sladek et al. (Sladek et al. 2007). In the second mode, i.e., the minor allele recessive mode, frequencies of the homozygous genotype for the minor allele were compared using a 2\u00a0\u00d7\u00a02 contingency table. In the third mode, i.e., the minor allele dominant mode, frequencies of the homozygous genotype for the major allele were compared using a 2\u00a0\u00d7\u00a02 contingency table. A test of independence was performed using Pearson\u2019s \u03c72 method. P values were corrected by Bonferroni adjustment and P\u00a0<\u00a00.00017 [0.05\/99 (total SNP number)\/3 (number of modes)] was considered significant. The odds ratio (OR) and 95% confidence interval (CI) were calculated by Woolf\u2019s method. We coded genotypes as 0, 1, and 2, depending on the number of copies of the risk alleles. OR adjusted for age and gender was calculated using multiple logistic regression with genotypes, age, and gender as independent variables. Hardy\u2013Weinberg equilibrium was assessed using the \u03c72 test (Nielsen et al. 1998). Haplotype blocks were determined using Haploview (Barrett et al. 2005). Simple comparison of the clinical data among the different genotypes was performed using one-way analysis of variance (ANOVA). Simple comparison of the clinical data between case and control groups was analyzed using Mann\u2013Whitney U test. Difference in BMI between genotypes was analyzed using a multiple linear regression, with BMI as the dependent variable and genotype as the independent variable, and with gender and age as covariates for BMI. Statistical analyses were performed using StatView 5.0 (SAS Institute, Cary, NC, USA). Power was calculated by the Monte Carlo method.\nResults\nCase-control association studies\nWe searched for dbSNPs with MAF\u00a0>\u00a00.10 in the FTO gene. By using Invader and TaqMan assay, we successfully genotyped 99 SNPs spanning the FTO gene (Supplementary Table 1). Using these SNPs, we performed tests of independence between the phenotype and genotypes of obesity at each SNP by using severely obese subjects (BMI\u00a0\u2265\u00a030\u00a0kg\/m2) and normal weight controls (BMI\u00a0<\u00a025\u00a0kg\/m2). For each SNP, the lowest P value among the three different modes was selected as the minimum P value. All SNPs, including rs1421084, were in Hardy\u2013Weinberg equilibrium (P\u00a0>\u00a00.01) (Supplementary Table 1).\nThe power of the test was calculated by Monte Carlo method with different MAFs and different effect sizes. Effect of the risk allele on penetrance was assumed to be multiplicative; i.e., the penetrances for three genotypes were assumed to be a, ar, and ar2, respectively, where a and r denote the lowest penetrance and genotype relative risk, respectively. Supplementary Table 2 shows the calculated values of the power of the test with different MAFs and different genotype relative risks (r). The lowest penetrance (a) was calculated for each gender by assuming the affection rates of 2.3% for men and 3.4% for women (Yoshiike et al. 2002). Genotype relative risk (r) was assumed to be the same for both genders. Supplementary Table 2 shows that the test has significant power at relative high risk allele frequency when genotype relative risk is >1.7.\nAs shown in Fig.\u00a01 and Supplementary Table 1, 15 SNPs demonstrated significant associations with the obesity phenotype; the threshold of significance using Bonfferoni correction was P\u00a0<\u00a00.00017. These SNPs included rs9939609 (Frayling et al. 2007) and rs1121980 (Hinney et al. 2007) that were reported to be significantly associated with the obesity phenotype in the Caucasian population, as determined by genome-wide association studies; rs9930506 (Scuteri et al. 2007) showed marginal association with obesity in the Japanese. Linkage disequilibrium (LD) analysis revealed that these 15 SNPs were in almost complete LD (D'\u00a0>\u00a00.98, r2\u00a0>\u00a00.80) and were located within the same LD block of approximately 50\u00a0kb (Fig.\u00a01). The most significant association was observed for rs1558902 [additive mode, P\u00a0=\u00a00.0000041 and allele-specific OR (95% CI) adjusted for age and gender was 1.41 (1.22\u20131.62)]. The minor alleles of rs9939609 (MAF\u00a0=\u00a00.24) and rs1121980 (MAF\u00a0=\u00a00.26) were significantly more frequent in the obese group than in the normal-weight control group (additive mode, P\u00a0=\u00a00.000012 and P\u00a0=\u00a00.000051, respectively), and ORs were 1.38 (95% CI\u00a0=\u00a01.20\u20131.59) and 1.33 (95% CI\u00a0=\u00a01.16\u20131.52), respectively (Table\u00a02, Supplementary Table 1). The MAF of both SNPs in the control group was 0.18; this was consistent with data obtained from the haplotype map of the human genome (HapMap) (Supplementary Table 1). Our data indicated that the SNPs in the FTO gene were associated with severe obesity in the Japanese.\nFig.\u00a01Linkage disequilibrium (LD) mapping, polymorphisms, and P values obtained in the test of independence between the phenotype and genotypes of obesity at various single nucleotide polymorphisms (SNPs) in the fat-mass and obesity-associated gene (FTO) gene. P values are expressed as negative logarithm of the minimum P values obtained in the three models (additive, minor allele dominant, and minor allele recessive modes). LD coefficients (D') between each pair of SNPs were calculated and are displayed as a strand in the LD blocks. Minor allele frequencies of all SNPs used in this analysis are \u226510%. The genomic structure is shown in the upper. The gray bar marks the LD block associated with obesityTable\u00a02Associations of single nucleotide polymorphisms (SNPs) in the fat-mass and obesity-associated gene (FTO) gene with obesity existing in the 50-kb linkage disequilibrium (LD) blockdbSNP IDAlleleGenotypeAdditive modeRecessive modeDominant modeCaseControl1\/2111222Sum111222SumOR (95% CI)\u03c72P value\u03c72P valueOR (95% CI)\u03c72P valueOR (95% CI)rs9937053A\/G593604949136341477312501.31 (1.13\u20131.51)12.30.00047 2.00.161.30 (0.90\u20131.88)13.00.000311.37 (1.16\u20131.63)rs9939973A\/G613674969247550494115201.32 (1.15\u20131.51)15.70.000077a3.00.0811.36 (0.96\u20131.93)16.10.000061a1.40 (1.19\u20131.66)rs9940128A\/G603664989247550094115161.31 (1.15\u20131.50)15.20.00010a2.60.111.33 (0.94\u20131.89)15.90.000068a1.40 (1.19\u20131.65)rs1421085C\/T4933853792457443101915191.38 (1.20\u20131.59)19.60.000011a3.30.0681.44 (0.97\u20132.12)20.00.0000078a1.47 (1.24\u20131.74)rs1558902A\/T4834153692552449102115221.41 (1.22 -1.62)21.20.0000041a4.60.0321.55 (1.04\u20132.31)20.80.0000052a1.48 (1.25\u20131.75)rs1121980A\/G613674999277350494715241.33 (1.16\u20131.52)16.50.000051a3.60.0591.40 (0.99\u20131.99)16.50.000050a1.41 (1.19\u20131.66)rs7193144C\/T4933953292055447101415161.39 (1.21\u20131.61)20.40.0000067a4.00.0441.49 (1.01\u20132.22)20.30.0000067a1.47 (1.24\u20131.74)rs8043757T\/A4831954190854436102715171.36 (1.18\u20131.57)17.40.000037a4.20.0401.51 (1.02\u20132.25)16.40.000052a1.42 (1.20\u20131.69)rs8050136A\/C5133653892556450101815241.38 (1.20\u20131.59)19.40.000012a4.70.0311.53 (1.04\u20132.26)18.50.000017a1.45 (1.22\u20131.71)rs3751812T\/G5134053492555458101315261.38 (1.20\u20131.59)19.60.0000098a5.10.0241.56 (1.06\u20132.31)18.50.000017a1.45 (1.22\u20131.71)rs9923233C\/G5133553391955449101015141.38 (1.20\u20131.60)19.80.0000093a5.00.0251.56 (1.06\u20132.30)18.70.000015a1.45 (1.23\u20131.72)rs9926289A\/G503235319045642599314741.37 (1.19 -1.58)18.70.000020a3.90.0471.48 (1.00\u20132.19)18.10.000021a1.45 (1.22\u20131.72)rs9939609A\/T5133453491956443100515041.38 (1.20\u20131.59)19.50.000012a4.50.0341.52 (1.03\u20132.24)18.70.000015a1.45 (1.23\u20131.72)rs7185735G\/A5134053692755455101415241.38 (1.20\u20131.59)19.90.0000089a5.00.0251.55 (1.05\u20132.30)18.80.000014a1.45 (1.23\u20131.72)rs9931494G\/C643634949217150494215171.35 (1.18\u20131.55)18.40.000018a5.60.0181.52 (1.07\u20132.15)16.90.000039a1.42 (1.20\u20131.67)rs17817964T\/C623615009236852493015221.30 (1.14\u20131.49)13.50.00022 5.80.0161.54 (1.08\u20132.19)11.40.000751.33 (1.13\u20131.57)rs9930506G\/A673654889208252191315161.28 (1.12\u20131.46)12.80.00038 3.50.0611.37 (0.98\u20131.92)12.10.000511.34 (1.14\u20131.58)rs9932754C\/T663684919257852591915221.29 (1.13\u20131.48)13.60.00023 4.20.0401.42 (1.01\u20132.00)12.60.000401.35 (1.14\u20131.59)rs9922619T\/G663684899237852991915261.29 (1.13\u20131.48)13.50.00024 4.30.0381.43 (1.02\u20132.01)12.30.000441.34 (1.14\u20131.58)rs7204609C\/T13441837392527371752915190.83 (0.73\u20130.93)9.680.0022 5.00.0250.77 (0.62\u20130.97)7.50.00630.79 (0.67\u20130.94)rs12149832A\/G533495259276248098215241.33 (1.15\u20131.53)15.20.000098a3.50.0611.43 (0.98\u20132.08)14.80.00012a1.39 (1.17\u20131.64)The odds ratio (OR) for each SNP was adjusted simultaneously for age and gender using additive modelCI confidence interval, \u03c72 chi-squareaSignificant P value (P\u00a0<\u00a00.00017)\nAnalysis of various quantitative phenotypes with rs1558902\nTo investigate whether the genotypes of SNP rs1558902 are associated with the phenotypes of metabolic disorders, we compared the following among the different genotypes in the cases, controls, and combined groups: ANOVA results, BMI, levels of fasting plasma glucose, hemoglobin A1c (HbA1c), total cholesterol, triglycerides, HDL cholesterol, and blood pressure. As rs1558902 showed the most significant association with obesity and its call rate was the highest, we analyzed various quantitative phenotypes by using this SNP. The quantitative phenotypes regarding BMI and the levels of fasting plasma glucose, HbA1c, total cholesterol, triglycerides, HDL cholesterol, and blood pressure were not found to be significantly associated with the genotypes at rs1558902 in either the case or control group (Table\u00a03). Although there was no significant difference in BMI values among genotypes in either the control or case group, the direction of the difference (AA\u00a0>\u00a0AT\u00a0>\u00a0TT) was in accordance with the association between the qualitative obesity phenotype and the genotype shown.\nTable\u00a03Comparison of various quantitative phenotypes among different genotypes at single nucleotide polymorphism (SNP) rs1558902 in obese and control subjectsObeseControlAA (n\u00a0=\u00a048)AT (n\u00a0=\u00a0341)TT (n\u00a0=\u00a0536)AA (n\u00a0=\u00a052)AT (n\u00a0=\u00a0448)TT (n\u00a0=\u00a01022)Age (year)49.8\u00a0\u00b1\u00a015.349.6\u00a0\u00b1\u00a014.348.8\u00a0\u00b1\u00a014.146.9\u00a0\u00b1\u00a015.446.9\u00a0\u00b1\u00a016.748.8\u00a0\u00b1\u00a016.5P value0.640.098BMI (kg\/m2)35.16\u00a0\u00b1\u00a05.7034.61\u00a0\u00b1\u00a05.4334.39\u00a0\u00b1\u00a05.3321.94\u00a0\u00b1\u00a02.23 21.62\u00a0\u00b1\u00a02.1021.65\u00a0\u00b1\u00a02.06P value0.580.56Glucose (mg\/dl)142.8\u00a0\u00b1\u00a054.8125.4\u00a0\u00b1\u00a043.2130.8\u00a0\u00b1\u00a053.3101.7\u00a0\u00b1\u00a044.196.3\u00a0\u00b1\u00a018.198.2\u00a0\u00b1\u00a024.7P value0.0540.34HbA1c (%)6.9\u00a0\u00b1\u00a02.16.4\u00a0\u00b1\u00a01.76.5\u00a0\u00b1\u00a01.85.1\u00a0\u00b1\u00a01.25.0\u00a0\u00b1\u00a00.55.1\u00a0\u00b1\u00a00.7P value0.190.15Total cholesterol (mg\/dl)215.1\u00a0\u00b1\u00a046.7211.3\u00a0\u00b1\u00a038.8208.6\u00a0\u00b1\u00a036.6195.6\u00a0\u00b1\u00a038.8201.4\u00a0\u00b1\u00a037.8201.4\u00a0\u00b1\u00a035.6P value0.370.53Triglycerides (mg\/dl)171.7\u00a0\u00b1\u00a0119.5151.3\u00a0\u00b1\u00a0102.1153.2\u00a0\u00b1\u00a096.0111.7\u00a0\u00b1\u00a070.6102.0\u00a0\u00b1\u00a071.4104.4\u00a0\u00b1\u00a074.2P value0.420.63HDL cholesterol (mg\/dl)53.2\u00a0\u00b1\u00a013.854.8\u00a0\u00b1\u00a024.052.0\u00a0\u00b1\u00a015.462.1\u00a0\u00b1\u00a014.265.1\u00a0\u00b1\u00a015.965.3\u00a0\u00b1\u00a015.6P value0.140.53SBP (mmHg)134.2\u00a0\u00b1\u00a020.4137.0\u00a0\u00b1\u00a017.8136.2\u00a0\u00b1\u00a018.2122.7\u00a0\u00b1\u00a017.3123.2\u00a0\u00b1\u00a018.8123.5\u00a0\u00b1\u00a017.5P value0.610.91DBP (mmHg)80.3\u00a0\u00b1\u00a011.784.1\u00a0\u00b1\u00a012.083.9\u00a0\u00b1\u00a012.075.5\u00a0\u00b1\u00a011.175.2\u00a0\u00b1\u00a011.776.3\u00a0\u00b1\u00a010.9P value0.140.22Data of each quantitative phenotype were compared among different genotypes at the rs1558902 in obese and control subjects. P values were analyzed using analysis of variance in each group of obese and control subjects. Data are mean\u00a0\u00b1\u00a0standard deviation.HDL high-density lipoprotein, SBP systolic blood pressure, DBP diastolic blood pressure.\nFinally, we examined the BMI distribution of rs1558902 in the Japanese general population and found that rs1558902 genotype was significantly associated with BMI (Table\u00a04). This result would confirm the association of rs1558902 with obesity.\nTable\u00a04Association of body mass index (BMI) with rs1558902 genotypes in the Japanese general populationAAATTTP value (additive model)aBMI (kg\/m2) (n)23.17\u00a0\u00b1\u00a03.20 (59)22.79\u00a0\u00b1\u00a03.26 (482)22.57\u00a0\u00b1\u00a03.11 (1063)0.041aThe difference in BMI according to genotypes was analyzed using a multiple linear regression, with BMI as the dependent variable and genotype as the independent variable and with gender and age as covariates for BMI. Data are represented as mean\u00a0\u00b1\u00a0standard deviation\nDiscussion\nRecent genome-wide association studies have shown that the FTO gene is associated with obesity (Frayling et al. 2007; Scuteri et al. 2007; Hinney et al. 2007). The associations between variations in the FTO gene and the obesity phenotype have been observed in many Caucasian subjects (Frayling et al. 2007; Scuteri et al. 2007; Dina et al. 2007; Field et al. 2007; Andreasen et al. 2008; W\u00e5hl\u00e9n et al. 2008; Peeters et al. 2008). However, these associations were controversial with regard to Asian subjects (Horikoshi et al. 2007; Li et al. 2008; Omori et al. 2008). BMI values did not significantly differ among the genotypes in the general population of Chinese and Japanese (Horikoshi et al. 2007; Li et al. 2008). We performed a case-control association study with regard to severe obesity and found that the SNPs in the FTO gene were significantly associated with severe obesity. Although the SNPs demonstrated the most significant association in the Japanese, which was different from that in Caucasians, the significantly associated SNPs existed in a similar block as that in Caucasians. Therefore, the FTO gene could also contribute to the development of severe obesity in the Japanese.\nBMI was modestly different among rs1558902 genotypes in the general population in this study; rs9939609 was not significantly associated with BMI in the general population (AA 23.22\u00a0\u00b1\u00a03.14 vs AT 22.79\u00a0\u00b1\u00a03.25 vs TT 22.58\u00a0\u00b1\u00a03.13, P\u00a0=\u00a00.063). In the Japanese population, rs1558902 may be more tightly associated with BMI than rs9939609. The National Nutrition Survey of Japan reported that the prevalence of subjects with a BMI of \u226530\u00a0kg\/m2 is only 2.3% in men and 3.4% in women aged 20\u00a0years and older (Yoshiike et al. 2002), and the mean BMI was approximately 23\u00a0kg\/m2 for ages 15\u201384\u00a0years (Yoshiike et al. 1998). Inconsistency in the results of effects of variations in the FTO gene on BMI between Japanese and Europians may be due to the relatively small mean and variance of BMI in the former than the latter.\nThe significant SNPs were located in intron 1 of the FTO gene. The rs1558902 and other significant SNPs, for example, rs9939609 and rs1121980, would affect transcriptional activity of the FTO gene, although further investigation is necessary. The precise mechanism by which the FTO gene leads to obesity development is unclear (Gerken et al. 2007; Sanchez-Pulido et al. 2007). However, the FTO gene is expressed in the hypothalamus and regulated by fasting and leptin (Frayling et al. 2007; Gerken et al. 2007). Using large-scale case-control association studies, we determined that the SCG3 (Tanabe et al. 2007) and MTMR9 (Yanagiya et al. 2007) genes are involved in susceptibility to the obesity phenotype. These two genes are expressed in the hypothalamus. Genetic studies in mice have suggested that mutations in several genes, such as those encoding leptin, proopiomelanocortin, and melanocortin-4 receptor, are implicated in a monogenic form of inherited obesity (Barsh et al. 2000; Rankinen et al. 2006). Such mutations have also been reported in obese humans. As most such genes are expressed in the hypothalamus and have been indicated to play important roles in the regulation of food intake, genes expressed in the hypothalamus are likely to be good candidates for susceptibility to obesity.\nIn summary, we have identified the genetic variations in the FTO gene that may influence the risk of severe obesity in the Japanese.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\nSummary of the association of SNPs between cases and controls (DOC 315 kb)","keyphrases":["association","obesity","fat-mass and obesity-associated gene","snp","japanese population"],"prmu":["P","P","P","P","P"]}
{"id":"Cancer_Chemother_Pharmacol-4-1-2270367","title":"Cisplatin-DNA adduct formation in patients treated with cisplatin-based chemoradiation: lack of correlation between normal tissues and primary tumor\n","text":"Purpose In this study, the formation of cisplatin-DNA adducts after concurrent cisplatin-radiation and the relationship between adduct-formation in primary tumor tissue and normal tissue were investigated.\nIntroduction\nConcurrent chemoradiotherapy is more effective than radiotherapy (RT) alone, both in in vitro studies [1, 2] as well as in clinical studies in many different tumor types, including advanced head and neck squamous cell carcinoma (HNSCC) and cervical cancer, leading to improvements in locoregional control and\/or survival [11, 13, 21, 28]. In a metaanalysis on concurrent chemoradiation in HNSCC, the addition of concurrent single agent cisplatin to RT was the most effective treatment regime with the largest improvement on overall survival [5]. Concurrent cisplatin-based chemoradiation is now considered standard care in advanced-stage HNSCC and cervical cancer.\nIn addition to the increased efficacy of the combined treatment, it was shown that the concurrent regimens are accompanied by higher acute toxicity rates compared to radiation alone [11, 21], with more severe mucositis and gastrointestinal toxicity.\nSince a substantial number of patients treated with concurrent chemoradiation still fail to respond to this toxic treatment, there is a need for an accurate predictive assay, based on which patients that are likely to respond to the therapy can be selected. This strategy may also provide a tool to individualize and tailor treatment, based on evaluation of the predictive assay, early during therapy.\nOne potential predictive marker is the formation of cisplatin-DNA adducts, which are formed when cisplatin reacts with the cellular DNA by binding to nucleotides. The majority of adducts are either intrastrand adducts with cisplatin bound between two guanosine (GG) nucleotides or adenosine\u2013guanosine (AG) nucleotides [8]. Cisplatin-DNA adducts can be measured in tumor and normal tissue. The level of adducts has been shown to correlate with cytotoxicity in vitro [34], and with response to therapy in patients [3, 17, 32, 35]. In most of these studies [3, 32, 35], adduct measurements were performed in the normal tissue, with the assumption that normal tissue can be used as surrogate marker for tumor.\nIn our institute, study protocols with cisplatin-DNA adduct measurements are ongoing in patients with HNSCC and cervical cancer, who are all treated with concurrent cisplatin-based chemoradiation. The objectives of the current study are (1) to investigate the two major forms of cisplatin-DNA adducts (GG and AG adducts) after different schedules of cisplatin given concurrently with radiation and (2) to explore relationships between adducts in primary tumor and normal tissue. We specifically wanted to investigate whether the level of adducts in tumors are reflected by those in normal tissues. In studies focused on the predictive value of cisplatin-DNA adduct levels, this would then justify the use of more easily obtained normal tissues as a surrogate for tumor samples.\nPatients and methods\nConcurrent chemoradiation protocols\nThis study on adduct formation was approved by the medical ethical committee of the participating hospitals. The main eligibility criteria were: patients scheduled for cisplatin chemoradiation, no previous treatment with cisplatin, and informed consent. Eligible patients were informed about the nature of the protocol and after written informed consent they were entered in the study. Patients were recruited from one of the following regimens.\nIn advanced-stage HNSCC patients, two different cisplatin-based concurrent chemoradiation protocols (RADPLAT) were used. The RADPLAT 100 schedule, which is the most commonly administered schedule in HNSCC [11], consisted of cisplatin given intravenously (IV) at a dose of 100\u00a0mg\/m2, as a 30\u00a0min infusion, 1\u20132\u00a0h before RT at days 1, 22, and 43 of treatment. This treatment was part of a randomized trial on IV vs. intra-arterial chemoradiation. In RADPLAT daily LD, low-dose (LD) cisplatin was given as a 1\u20132\u00a0min IV infusion at a dose of 6\u00a0mg\/m2 daily, for a total number of 20 doses, 1\u20132\u00a0h prior to RT. This treatment was shown to be an effective alternative in HNSCC [6, 19]. Patients ineligible for or refusing the randomized trial on intra-arterial chemoradiation were treated with RADPLAT daily LD, since this treatment could be given on an outpatient basis. The RT target volumes for all schedules included the primary tumor and the bilateral neck at a dose of 46\u00a0Gy in 23 fractions. A boost was given to the macroscopic tumor extensions at the primary tumor site and lymph node metastases at a dose of 24\u00a0Gy in 12 fractions, resulting in a total dose of 70\u00a0Gy in 35 fractions.\nIn patients with advanced-stage squamous cell cervical cancer, concurrent chemoradiation (CERVIX 40) consisted of weekly administration of cisplatin IV as a 4-h infusion at a dose of 40\u00a0mg\/m2, followed by RT within 1\u20132\u00a0h. The total number of doses was 5\u20136, depending on external beam RT schedule and the number of intracavitary brachytherapy applications. The total radiation dose was usually 46\u00a0Gy to the cervical tumor, uterus, and pelvic lymph nodes, with a boost to the cervix tumor and other involved regions, to a total dose of 60\u201374\u00a0Gy, depending on treated volume and whether or not intracavitary brachytherapy was given.\nCisplatin-DNA adducts\nBefore and after chemotherapy, normal tissue samples [white blood cells (WBC) and buccal cells] were collected. In patients with an accessible primary tumor, a biopsy of the tumor was also taken. To avoid harvesting necrotic tissue, the biopsy was taken at the viable peripheral rim of the tumor. Samples were obtained at different times, due to logistic reasons: for the patients in the RADPLAT 100 study, this was done 23\u00a0h after the end of administration of the first cisplatin infusion (given on day 1 of treatment). In the patients in the CERVIX 40 study, samples were taken 20\u00a0h after the end of administration of the first weekly cisplatin infusion (given on day 1 of treatment). For the patients in the RADPLAT daily LD group, samples were taken 1\u00a0h after the 5th dose on day 5 of treatment. WBC were isolated from whole blood samples according to a previously published protocol [24]. Buccal cells were collected in phosphate-buffered saline by scraping the bilateral buccal mucosa using a cotton swab. In HNSCC patients, the buccal mucosa could be located within the RT treatment fields, depending on the tumor site. The harvested cells were centrifuged (5\u00a0min at 4\u00b0C, 1,000\u00a0rpm) and resuspended in a Tris\u2013EDTA buffer and stored at \u221280\u00b0C until analysis. Tumor biopsies were taken and immediately frozen at \u221280\u00b0C until analysis. Quantification of GG- and AG-intrastrand adducts was performed by a 32P-postlabeling technique as previously described [29]. Internal standardization was incorporated in the present analysis method, by adding 300\u00a0fmol of TT nucleotides to each sample. From previous work, the reproducibility of the assay is known by analysis of duplicate specimens within the same experiment (within-run reproducibility) and by analysis of duplicate specimens in separate experiments (between-run reproducibility) [29]. The reproducibility was described for WBC and tumor samples and was within 10% for the within-run precision and between 2\u201320% for the between-run precision. It was also determined for buccal cells in the same way and similar reproducibility was obtained. The concentration of DNA present in the samples was measured spectrophotometrically at 260\u00a0nm with the Nanodrop ND-1000 (Nanodrop Technologies Inc, Wilmington, DE, USA). The cisplatin-DNA adduct levels were expressed as fmol\/\u03bcg DNA. The lower limit of quantification for the Pt-GG and Pt-AG adducts was 0.087 and 0.053\u00a0fmol\/\u03bcg DNA, respectively.\nStatistical analysis\nAnalysis of data was performed in SPSS software (version 11.5, SPSS, Inc.). For quantitative comparison of numerical data between groups, the Student\u2019s t-test was applied. The Pearson correlation coefficient and Spearman\u2019s rank correlation coefficient were calculated for analysis of correlations between different samples (WBC, buccal cells, and primary tumor) on an intra-patient level.\nResults\nSamples for cisplatin-DNA adduct determination were obtained from 63 patients: 27 from RADPLAT daily LD, 15 from CERVIX 40, and 21 from RADPLAT 100. WBC samples were taken from 61 patients, buccal cells from 25, and tumor biopsies from 23 of these patients. The reasons for the missing data for the normal tissue samples were: no collection of samples due to logistics or (in minority of cases) not sufficient volume for analysis. The reason for missing primary tumor biopsy data were: tumor not accessible for direct outpatient-based biopsy (in HNSCC patients) or refusal (in cervix cancer patients).\nIn WBC, all but three of the 60 available baseline samples were below the LLQ for the GG adducts and all but two below the LLQ for the AG adducts. This was probably due to some background signal inherent in the postlabeling method, since all patients had not been treated before with platinum chemotherapy. The yield of DNA, obtained from the buccal cell samples was rather low, ranging from 1\u201310\u00a0\u03bcg. Baseline samples of buccal cells were available from 16 of 25 patients, of whom posttreatment samples were also available. The baseline values of GG adducts in buccal cells ranged from 0.067 to 0.745\u00a0fmol\/\u03bcg DNA (mean 0.282, SD 0.19) and baseline values of AG adducts in buccal the cells ranged from 0.087 to 1.538\u00a0fmol\/\u03bcg DNA (mean 0.398, SD 0.38). All but two of the baseline GG-adduct values were above the LLQ and all the baseline AG-adduct values were above the LLQ. This was probably due to the low DNA quantities obtained from the buccal cell samples. The difference in adduct levels from baseline to post-infusion values was significant for the GG adducts, but not for the AG adducts. This implies that for measuring low quantities of AG adducts in the low amounts of buccal cell DNA available, we reached the limits of quantification with this postlabeling method.\nIn Table\u00a01, the results are presented for the GG- and AG-adduct levels in normal tissue and primary tumor for the three different cisplatin-chemoradiation regimes. Adduct levels in primary tumor were two to five times higher than those in WBC for all three treatment regimes for both GG- and AG-adduct formation (Student t-test, P\u00a0<\u00a00.001 for both adduct types). For the comparison of the adduct levels from the three different treatment protocols, the data from the RADPLAT daily LD were omitted, since the daily administration schedule and sampling time (1\u00a0h after the infusion) were different from the other two regimens. The adduct levels in the RADPLAT 100 schedule were statistically significantly higher than those after the CERVIX 40 schedule for both tumors (Student t-test, P\u00a0=\u00a00.01) and normal tissues (Student t-test, P\u00a0<\u00a00.01).\nTable\u00a01Cisplatin-DNA adducts (in fmol\/\u03bcg DNA) in normal tissue and primary tumor after different schedules of cisplatin-based chemoradiationTreatment scheduleWBCBuccal cellsTumorGGAGGGAGGGAGRADPLAT daily LDN2626111166 Mean0.340.050.840.210.660.10 SD0.100.190.390.140.370.05CERVIX 40N1414771010 Mean0.440.060.870.221.940.26 SD0.170.030.260.061.470.26RADPLAT 100N21217777 Mean1.0470.1241.5630.3403.8660.413 SD0.3770.0490.4340.0901.1010.089See text for explanation of treatment schedulesN number of patients, SD standard deviation, WBC white blood cells, GG GG-adducts, AG AG adducts\nA highly significant linear correlation (Pearson correlation, r\u00a0=\u00a00.93, P\u00a0<0.001, n\u00a0=\u00a061) was observed between the level of GG and AG adducts in WBC (see Fig.\u00a01a), with a mean ratio of GG\/AG adducts of 7.6\u00a0\u00b1\u00a02.1 SD. Similar linear relationships and ratios were found for GG and AG adducts in primary tumor (r\u00a0=\u00a00.89, P\u00a0<\u00a00.001, n\u00a0=\u00a023, and ratio 8.5\u00a0\u00b1\u00a02.8; Fig.\u00a01b) and buccal cells (r\u00a0=\u00a00.85, P\u00a0<\u00a00.001, n\u00a0=\u00a023, and ratio 3.9\u00a0\u00b1\u00a01.3; Fig.\u00a01c).\nFig.\u00a01Correlation-plots of GG- and AG-adduct levels in white blood cells (WBC) (panel a), primary tumor biopsy (panel b), buccal cells (panel c). In each panel, the three different treatment groups are depicted: RADPLAT daily LD 5\u00a0\u00d7\u00a06\u00a0mg (red squares), CERVIX 40\u00a0mg (green circles), and RADPLAT 100\u00a0mg (blue triangles)\nA trend was observed between GG-adduct levels in WBC and buccal cells, although not significant (r\u00a0=\u00a00.38, P\u00a0=\u00a00.07, n\u00a0=\u00a024). No significant correlations were found between tumor and normal tissue: tumor vs. WBC (r\u00a0=\u00a00.35, P\u00a0=\u00a00.13, n\u00a0=\u00a021) and tumor vs. buccal cells (r\u00a0=\u00a0\u22120.003, P\u00a0=\u00a00.99, n\u00a0=\u00a09). See Fig.\u00a02 for scatter plots. Similar results were found for the AG adducts: no significant correlations were found between adducts in tumor vs. WBC (r\u00a0=\u00a00.14, P\u00a0=\u00a00.55, n\u00a0=\u00a021), tumor vs. buccal cells (r\u00a0=\u00a00.25, P\u00a0=\u00a00.58, n\u00a0=\u00a07) or WBC vs. buccal cells (r\u00a0=\u00a00.26, P\u00a0=\u00a00.24, n\u00a0=\u00a022).\nFig.\u00a02Correlation plots of GG adducts in white blood cells (WBC) vs. buccal cells (panel a), and normal tissue vs. tumor (panels b and c). In each panel, the three different treatment groups are depicted: RADPLAT daily LD 5\u00a0\u00d7\u00a06\u00a0mg (red squares), CERVIX 40\u00a0mg (green circles), and RADPLAT 100\u00a0mg (blue triangles)\nDiscussion\nThere are two main conclusions from the 63 patients included in these analyses. First, intra-tumoral adduct levels were substantially higher than those in normal tissue (WBC) at all cisplatin-dose levels examined. Second, no positive correlations were evident between adducts in tumors and normal tissues. It should be noted that the various schedules, the cisplatin doses, and the duration of infusions differed, as well as the sampling times. However, all analyses on adducts were performed on paired samples, within the same patient. This eliminates variance of these factors since the normal tissue and tumor samples all were obtained at similar time points after the cisplatin infusion. In the RADPLAT daily LD, some accumulation from the previous four daily 6\u00a0mg cisplatin infusions would have occurred and affected the day 5 measurement after the 5th infusion. In the RADPLAT 100 and CERVIX 40 patients, no such accumulation would have occurred, since the sampling was done 20\u201323\u00a0h after the first infusion of cisplatin.\nRelatively little information is available regarding the in vivo formation of intra-tumoral cisplatin-DNA adducts in clinical series [17, 23]. Most studies focused on intra-tumoral platinum concentrations, both in HNSCC [12, 33] and cervical cancer patients [15, 22]. These studies are mostly characterized by relatively low numbers of patients, probably due to the invasive nature of the procedure. The data on correlations between adducts and platinum content are contradictory: In an experimental study [37], no relationship could be established between the intra-tumoral adduct levels and platinum content, although in one clinical study, a significant correlation was found [23].\nAdduct levels in primary tumors were consistently two- to fivefold higher than in WBC. This was true for both GG and AG adducts. This finding was previously described in anecdotal clinical cases [9, 30]. Similar observations were made in platinum content studies in an experimental tumor model [18] and in HNSCC patients [33]. Adducts in tumor were also higher than in buccal cells in the CERVIX 40 and RADPLAT 100 group, but not in the RADPLAT daily LD group. We observed a linear relationship between the two major adduct forms (AG and GG) for both normal tissues and tumor (Fig.\u00a01a\u2013c), although GG-adduct formation was 5\u201312 times increased relative to the AG adducts, as reported earlier [8, 36]. The linear relationships between GG and AG adducts serve as a validation of the assay. Apparently within a sample, both types of adducts are present in an equal proportion, although the absolute amounts differ greatly. In previous studies, the type of adduct responsible for the cytotoxic effect of platinum compounds has been investigated. From two of these studies [7, 36] it was concluded that the AG adduct was responsible for the platinum cytotoxicity. From our study, such a conclusion cannot be made, since both types of adducts were present in equal proportions.\nAdduct formation in different tissue samples showed a lack of correlation between tumor and normal tissue. One might have expected that higher adduct levels in normal tissue would be accompanied by more adducts in tumor, although this was not the case. Similar results were found in an animal study [25] and in a clinical series of uterine cervix cancer patients [22], both demonstrating lack of correlation between tumor platinum and serum platinum concentrations. If adduct formation were merely a matter of cisplatin exposure, then a positive correlation would be expected. The reasons for the higher levels of adducts in tumor vs. normal tissue and the lack of correlation between them may be explained by differences between tumor and normal tissue in one of the following factors: Tumors are heterogeneous in terms of blood supply and perfusion, resulting in differences in cisplatin uptake and diffusion, drug-pumps may diminish intra-cellular cisplatin concentrations by active transmembrane transport of cisplatin, prohibiting adducts to be formed, and tumor cells may have less effective capacities to repair damage from cytotoxic agents.\nAdducts are formed rapidly and in a dose-dependent fashion within 1\u20132\u00a0h after cisplatin exposure, with a gradual decrease (repair) within the next 20\u201324\u00a0h [20, 32, 36]. The persistence of cisplatin-DNA adducts may therefore be regarded as a measure of repair and possibly be used as predictive assay. We therefore chose to measure adducts 20\u201323\u00a0h after the end of chemotherapy infusion. Ideally, more frequent measurements would have generated more information on the rate of adduct formation, repair, and total exposure to adducts (like an AUC analysis) [24]. However, obtaining repeated biopsies is not feasible in clinical practice.\nThe results from the buccal cell samples need to be interpreted with caution, especially the AG-adduct levels, since uncertainties remain. With the low quantities of AG-adducts in the low amounts of buccal cell DNA we could extract, we reached the limits of quantification of the postlabeling method.\nThe rationale for measuring cisplatin-DNA adducts is that it could be used as a predictive assay: higher levels of adducts would predict favorable treatment outcome. Many studies have been performed for this purpose, investigating adducts in normal tissue (WBC and buccal cells) [3, 4, 10, 14, 26, 31, 32, 35]. These study designs are based on the assumption that normal tissue can be used as a surrogate marker for tumor tissue with respect to cisplatin-DNA adduct formation. In our present study, however, we did not find such a correlation. This might explain why the results on the role of adduct formation to predictive outcome are heterogeneous and contradictory, as illustrated below.\nSeveral studies on adduct formation in WBC showed that the level of adducts was positively correlated with response to chemotherapy in patients with advanced disease in a variety of tumor sites [31, 32], while others showed no correlation [4, 26]. One study showed a positive correlation in one tumor site (ovarian cancer), but not in the other (breast cancer) [14], and in another study the level of adduct formation showed a negative association with survival for day-5 adducts, while there was no difference for day-1 adducts [10]. In studies on adduct levels in buccal cells, a positive correlation was found between adducts and either disease response [3] or better survival in non-small cell lung cancer (NSCLC) [35]. We recently showed that adduct formation in primary tumor appeared to be associated with better progression-free survival in HNSCC [17].\nDifferences were observed between the levels of intra-tumoral adducts for the three chemoradiation schedules, with lower adducts after lower dosages of cisplatin. Based on this, however, one cannot predict that the schedules with less adducts will result in less cytotoxicity, since not only the cisplatin dose, but also timing and schedule of cisplatin administration are crucial determinants of efficacy [1]. These factors may contribute to differences in the formation and rate of repair of adducts, resulting in different exposures to cisplatin-DNA adducts. These differences make it difficult to extrapolate from the observed adduct values in tumor and normal tissues to a prediction of superiority of one schedule over another.\nIn future studies, immunohistochemistry on repair proteins like ERCC1 [27] or gene expression profiling studies on platinum resistance [16] could be used to improve the prediction of cisplatin sensitivity and prediction of therapy response.\nIn conclusion, we have demonstrated that in concurrent chemoradiotherapy schedules, cisplatin adduct levels in tumors were significantly higher than in normal tissues (WBC). No evidence of a correlation was found between adduct levels in normal tissues and primary tumor biopsies. This lack of correlation may, to some extent, explain the inconsistencies in the literature regarding whether or not cisplatin-DNA adducts can be used as predictive test in anticancer therapy.\nConflict of interest: none declared.","keyphrases":["cisplatin","chemoradiation","cervical cancer","dna adducts","head and neck cancer"],"prmu":["P","P","P","R","R"]}
{"id":"Int_Urol_Nephrol-4-1-2268719","title":"Magnesium carbonate for phosphate control in patients on hemodialysis. A randomized controlled trial\n","text":"Background Magnesium salts bind dietary phosphorus, but their use in renal patients is limited due to their potential for causing side effects. The aim of this study was to evaluate the efficacy and safety of magnesium carbonate (MgCO3) as a phosphate-binder in hemodialysis patients.\nIntroduction \nNormalization of the serum phosphate level is the cornerstone of medical protocols aimed at preventing and treating secondary hyperparathyroidism. Moreover, it has been shown that elevated serum levels of phosphate and the calcium\u2013phosphate product (Ca\u00a0\u00d7\u00a0P) play an important role in the development of extraosseous calcifications and are associated with increased mortality in hemodialysis patients [1, 2]. The ingestion of phosphate-binding agents in conjunction with dietary phosphate restriction and its adequate removal by dialysis are the cornerstones of serum phosphate control in end-stage renal disease (ESRD) patients. However, four decades after the introduction of chronic hemodialysis in early 1960s, we have not yet found the ideal phosphate binder(s) in terms of combined efficacy, safety and low cost. Aluminum and calcium salts and non-aluminum and non-calcium agents, such as sevelamer and lanthanum carbonate, all have advantages and disadvantages [3]. Magnesium-containing agents are aluminum- and calcium-free and are inexpensive phosphate-binders and, to date, their potential has not been explored extensively. In studies involving small series of both hemodialysis and peritoneal dialysis patients, magnesium carbonate (MgCO3) and magnesium hydroxide [Mg(OH)2] have been administered either alone or in combination with calcium salts with good results [4\u201310]. However, these compounds are not widely used in ESRD patients because nephrologists have an inordinate fear of hypermagnesemia and the belief that Mg administration frequently is accompanied by gastrointestinal disorders.\nWe carried out this study in hemodialysis patients to evaluate the efficacy and safety of MgCO3 as a phosphate-binder when given with a concurrent low dialysate magnesium solution. The control of serum phosphate in the two groups of patients was the primary outcome, while secondary outcomes were changes in serum calcium, magnesium, Ca\u00a0\u00d7\u00a0P and PTH levels and changes in bowel movements.\nSubjects and methods\nPatients and study design\nStable ESRD patients on maintenance hemodialysis in our renal unit participated in the study. The protocol of this project was approved by the Ethics Committee of the hospital. Exclusion criteria were: age <18\u00a0years, hemodialysis for less than 6\u00a0months, psychiatric or other disorders leading to non-compliance, unlikeliness to continue hemodialysis for more than 6\u00a0months in the same facility, critical illness at the time of recruitment, previous parathyroidectomy, severe hyperparathyroidism [serum intact parathyroid hormone (iPTH) > 500\u00a0pg\/ml], normal serum phosphate (<5.5\u00a0mg\/dl) without phosphate-binders, diseases resulting in diarrhea and the lack of informed consent. A total of 54 patients met the criteria and were approached for enrolment. The inclusion period lasted 2\u00a0months, between March 2 and April 29, 2006. Enrolled patients signed an informed consent and thereafter entered a 4-week washout period during which we withdrew all phosphate-binders as well as vitamin D medications. After the washout phase, three patients were removed because they had serum phosphate levels lower than 5.8\u00a0mg\/dl; the remaining 51 patients were allocated to receive either MgCO3 tablets (MgCO3 group) or calcium carbonate tablets (CaCO3 group) for 6\u00a0months. We chose CaCO3 instead of calcium acetate because the latter is not commercially available in Greece. Four patients did not agree to consume MgCO3 but did agree to participate by taking their standard binder of CaCO3; we allocated these patients to the CaCO3 group, whereas the remainder of the patients were randomly allocated with a ratio 1:1 to either the CaCO3 (21 randomly assigned patients plus 4 = 25 patients) or to the MgCO3 (26) group. Each MgCO3 tablet contained 250\u00a0mg MgCO3, which is equivalent to 71\u00a0mg of elemental magnesium, and each CaCO3 tablet contained 420\u00a0mg of  CaCO3 equivalent to 168\u00a0mg of elemental calcium. All patients were on the same standard dialysis schedule (three times weekly\u00a0\u00d7\u00a04 h), and in all of the patients the delivered dose of hemodialysis, as calculated by applying the single pool Kt\/Vurea index using the second-generation formula of Daugirdas [11],, was \u22651.35. In both groups, calcium concentration in the dialysate bath was 1.50\u00a0mmol\/l, whereas magnesium concentration was 0.48\u00a0mmol\/l in the CaCO3 group and 0.30\u00a0mmol\/l in the MgCO3 group. The National Kidney Foundation (NKF) suggests a bath calcium dialysate concentration of 1.25\u00a0mmol\/l (based on opinion, not on evidence). We used a bath concentration 1.50\u00a0mmol\/l because we wanted to avoid episodes of hypocalcemia, particularly in the MgCO3 group. The patients should be more susceptible to manifest hypocalcemia since they did not receive vitamin D. The starting dose of both phosphate-binders were three tablets of MgCO3 or CaCO3 daily; the dose was adjusted thereafter according to the serum phosphate values: weekly for the first month and then monthly. The dosage of the study drug was increased to one or two tablets per meal as required to achieve the target of serum phosphate level \u22645.5\u00a0mg\/dl. Based on our clinical experience, to achieve adequate control of serum phosphate we set the maximum daily dose of CaCO3 to 3780\u00a0mg (nine tablets), which is equivalent to 1512\u00a0mg of elemental calcium, and the maximum daily dose of MgCO3 to 2250\u00a0mg (nine tablets), which is equivalent to 639\u00a0mg of elemental magnesium. Each patient\u2019s full biochemical profile was obtained at baseline, then weekly during the first month and monthly thereafter. Parathyroid hormone was measured at baseline and then at monthly intervals. If a patient developed hypercalcemia (serum Ca\u00a0>\u00a010.5\u00a0mg\/dl), the daily dose of CaCO3 was reduced by one or two tablets. The same was done in patients with severe hypermagnesemia, which is defined as a serum magnesium level >3.5\u00a0mg\/dl. If severe hypermagnesemia persisted for more than 3 weeks, the administration of MgCO3 was stopped, and the patient was dropped from the study. No patient received vitamin D or a calcimimetic agent during the study. Serum total calcium values were corrected to the serum albumin values.\nStatistical analysis\nThe laboratory values were expressed as mean\u00a0[\u00b1 one standard deviation (SD)].\nStudent\u2019s two-tailed unpaired t test was used to compare values between the two groups of patients at baseline and at 6\u00a0months. We used repeated measures of analysis of variance (repeated ANOVA method) to test for differences between the two treatment groups in average values of calcium, phosphate, Ca\u00a0\u00d7\u00a0P, magnesium, alkaline phosphatase (ALP) and PTH over time (time-averaged mean value differences). The chi-square test was applied to determine differences in the prevalence of laboratory data. Significance was set at a P level of <0.05.\nResults\nOf the 26 patients enrolled in the MgCO3 group, one dropped out due to non-compliance. Of the remaining 25 patients, two (8%) discontinued ingestion of MgCO3 and dropped out: one (4%) because of persistent diarrhea, and one (4%) because of recurrent hypermagnesemia. Of the 25 patients in the CaCO3 group; five were removed from the study (two received a kidney transplant, one died (pneumonia), one suffered a stroke and was unable to swallow tablets and one moved to another hospital). The use of phosphate-binders and vitamin D by the patients before the washout period is given in Table\u00a01. Patients\u2019 mean serum values at baseline and at the end of the follow-up period are shown in Tables\u00a02 and 3. The monthly follow-up of the mean biochemical parameters are shown in Figs.\u00a01\u20134. The mean Kt\/Vurea was 1.379\u00a0\u00b1\u00a00.026 in the MgCO3 and 1.381\u00a0\u00b1\u00a00.027 in the CaCO3 group.\nTable\u00a01Patients\u2019 data in terms of the use of phosphate-binders and vitamin D before the washout periodMgCO3 group (n = 25)CaCO3 group (n = 21)Vitamin D11\/258\/21Phosphate binders25\/2521\/21\u00a0\u00a0\u00a0\u00a0CaCO31916\u00a0\u00a0\u00a0\u00a0Sevelamer24\u00a0\u00a0\u00a0\u00a0CaCO3\u00a0+\u00a0sevelamer41Table\u00a02Mean serum values at baselineMgCO3 groupCaCO3 groupP value (t test)Age (years)63.2365.32P\u00a0=\u00a0nsSD12.1911.68Calcium (mg\/dl)a9.429.14P\u00a0=\u00a0nsSD0.540.43Phosphorous (mg\/dl)6.636.58P\u00a0=\u00a0nsSD0.860.88Ca\u00a0\u00d7\u00a0P product (mg\/dl)262.6260.08P\u00a0=\u00a0nsSD10.368.35Magnesium (mg\/dl)2.382.36P\u00a0=\u00a0nsSD0.280.29ALP (IU\/l)7686P\u00a0=\u00a0nsSD3735Intact PTH (pg\/ml)316296P\u00a0=\u00a0nsSD182157SD,\u00a0Standard deviation; ALP, alkaline phosphatase; PTH, parathyroid hormone; ns, not significantaSerum total calcium level corrected to serum albuminTable\u00a03Mean values at 6 monthsMgCO3 groupCaCO3 groupP value (t test)Calcium (mg\/dl)a8.979.72t\u00a0=\u00a02.16SD0.570.42P\u00a0<\u00a00.05Phosphorous (mg\/dl)5.125.28t\u00a0=\u00a00.49SD0.700.74P\u00a0=\u00a0NSCa\u00a0\u00d7\u00a0P product (mg\/dl)246.0451.38t\u00a0=\u00a00.30SD7.657.75P\u00a0=\u00a0NSMagnesium (mg\/dl)2.592.40t\u00a0=\u00a01.59SD0.430.41P\u00a0=\u00a0NSALP (IU\/l)8984t\u00a0=\u00a00.46SD2826P\u00a0=\u00a0NSIntact PTH (pg\/ml)251212t\u00a0=\u00a00.42SD118198P\u00a0=\u00a0NSaSerum total calcium corrected to serum albuminFig.\u00a01Monthly follow-up of serum phosphate (sP)Fig.\u00a02Monthly follow-up of serum calcium (sCa)Fig.\u00a03Monthly follow-up of serum Ca\u00a0\u00d7\u00a0P product (SCa \u00d7\u00a0P)Fig.\u00a04Monthly follow-up of serum PTH (siPTH)\nThe mean daily dose of CaCO3 was 6.76 tablets (range 3\u20139) containing a total of 2839\u00a0mg (range 1260\u20133780) of CaCO3, which is equivalent to 1136\u00a0mg of elemental calcium (range 504\u20131512\u00a0mg) The mean daily dose of MgCO3 was 6.21 tablets (range 3\u20139) containing a total of 1552\u00a0mg (range 750\u20132,250) of MgCO3, which is equivalent to 441\u00a0mg of elemental magnesium (range 213\u2013639\u00a0mg). A Shapiro\u2013Wilk test did not indicate any evidence against the normality assumption for the distribution of calcium, phosphate, Ca\u00a0\u00d7\u00a0P, magnesium, ALP and PTH levels within the two groups (CaCO3 and MgCO3) at any time period during the follow-up. The significance level for the normality tests was set to a\u00a0=\u00a00.001.\nAverage values of the biochemical data during months 1\u20136 are shown in Table\u00a04. The initial mean serum phosphate levels were 6.63\u00a0mg\/dl in the MgCO3 group and 6.58\u00a0mg\/dl in the CaCO3 group (P\u00a0=\u00a0ns), while at the end of the study serum phosphate values were 5.13 and 5.26\u00a0mg\/dl in the MgCO3 and CaCO3 groups respectively (P\u00a0=\u00a0ns; Tables\u00a02, 3). Time-averaged mean serum phosphate values were 5.47\u00a0mg\/dl in the MgCO3 group and 5.29\u00a0mg\/dl in the CaCO3 group (P\u00a0=\u00a0ns; Table\u00a04). Serum phosphate levels decreased by 23% in the MgCO3 group and by 19% in the CaCO3 group (P\u00a0=\u00a0ns). At the end of the study 17 of 23 (74%) patients in the MgCO3 and 13 of 20 (65%) of the CaCO3 group, (\u03c72\u00a0=\u00a00.10, P\u00a0=\u00a0ns) had serum phosphate values within the range recommended by the Kidney Disease Outcomes Quality Initiative (K\/DOQI) guidelines (upper threshold of 5.5\u00a0mg\/dl) (Table\u00a05).\nTable\u00a04Average serum values during months 1\u20136 of the follow-up periodMgCO3 groupCaCO3 groupP value (t test)Calcium (mg\/dl)a9.139.60P\u00a0<\u00a00.001SD0.530.45Phosphorous (mg\/dl)5.475.29P\u00a0=\u00a0nsSD0.810.93Ca\u00a0\u00d7\u00a0P product (mg\/dl)250.3550.70P\u00a0=\u00a0nsSD7.758.07Magnesium (mg\/dl)22.572.41P\u00a0=\u00a0nsSD0.410.32ALP (IU\/l)8880P\u00a0=\u00a0nsSD3227Intact PTH (pg\/ml)285231P\u00a0<\u00a00.01SD161177aSerum total calcium corrected to serum albuminTable\u00a05Patients with laboratory values within the Kidney Disease Outcomes Quality Initiative (K\/DOQI) guideline range at 6 monthsMgCO3 group (n, %)CaCO3 group (n, %)\u03c72, P valueCalcium 17\/23 (73.91)5\/20 (25)\u03c72\u00a0=\u00a08.42, P\u00a0<\u00a00.01Phosphorous17\/23 (73.91)13\/20 (65)\u03c72\u00a0=\u00a00.10, P\u00a0=\u00a0nsCa\u00a0\u00d7\u00a0P product20\/23 (86.95)14\/20 (70)\u03c72\u00a0=\u00a00.99, P\u00a0=\u00a0nsPTH11\/23 (47.82)8\/20 (40)\u03c72\u00a0=\u00a00.79, P\u00a0=\u00a0ns\nMean serum Ca values at baseline were 9.42\u00a0mg\/dl in the MgCO3 group and 9.14\u00a0mg\/dl in the CaCO3 group (P\u00a0=\u00a0ns) (Table\u00a02). Time-averaged mean serum values were 9.13\u00a0mg\/dl in the MgCO3 group and 9.60 in the CaCO3 group (P < 0.001) (Table\u00a04). At 6\u00a0months, these values were 8.97 and 9.72\u00a0mg\/dl, respectively (P\u00a0<\u00a00.05) (Table\u00a02). Seventeen patients (17\/23; 74%) who received MgCO3 and only 5\/20 (25%) of those on CaCO3 had serum Ca values within the K\/DQOI guidelines ( \u03c72\u00a0=\u00a08.42, P\u00a0<\u00a00.01) (Table\u00a05). We encountered six episodes of hypercalcemia, defined as a serum calcium level >10.5\u00a0mg\/dl, in the CaCO3 group in comparison to one episode in the MgCO3 group.\nThe two groups of patients had nearly equal values of serum Ca\u00a0\u00d7\u00a0P product at the beginning, at the end as well as during the period study (Tables\u00a02\u20134), with the MgCO3 group having 62.6\u00a0mg2\/dl2 and the CaCO3 group having 60.1\u00a0mg2\/dl2 (P\u00a0=\u00a0ns) (Table\u00a01). Mean levels of Ca\u00a0\u00d7\u00a0P during months 1\u20136 of the follow-up were similar \u2013 50.35\u00a0mg2\/dl2 in the MgCo3 group and 50.70\u00a0mg2\/dl2 in the CaCo3 group (P\u00a0=\u00a0ns) (Table\u00a04); at the end of the study, the corresponding values were 46.0 and 51.4\u00a0mg2\/dl2 (P\u00a0=\u00a0ns) (Table\u00a03), whereas 20\/23 (87%) and 14\/20 (70%) patients, respectively, had Ca\u00a0\u00d7\u00a0P product below the upper limits of K\/DQOI recommendations (P\u00a0=\u00a0ns) (Table\u00a05).\nStarting mean levels of serum iPTH were similar in both groups \u2013 316\u00a0\u00b1\u00a0182\u00a0pg\/ml in the MgCO3 group and 296\u00a0\u00b1\u00a0157\u00a0pg\/ml in the CaCO3 group (P\u00a0=\u00a0ns) (normal range 10\u201365\u00a0pg\/ml) (Table\u00a02). During the 6-month follow-up period, average iPTH levels were significantly higher in the MgCO3-treated patients (285\u00a0pg\/ml) than in those treated with CaCO3 (231\u00a0pg\/ml) (P\u00a0<\u00a00.01) (Table\u00a04). However, at the end of the study, those levels did not differ significantly \u2013 251\u00a0\u00b1\u00a0118 and 212\u00a0\u00b1\u00a0198\u00a0pg\/ml, respectively (P\u00a0=\u00a0ns) (Table\u00a03). In terms of PTH levels, there was no statistically significant difference between the CaCO3 group and the MgCO3 group \u2013 29 versus 21%, respectively  (\u03c72\u00a0=\u00a00.11, P\u00a0=\u00a0ns). Moreover, ten of 23 (43%) patients in the MgCO3 group and nine of 20 (45%) in the CaCO3 group had initial levels of iPTH above 300\u00a0pg\/ml; in three of these ten patients (30%) of the MgCO3 group and five of the nine (56%) of the CaCO3 group, the iPTH decreased below 300\u00a0pg\/ml [\u03c72\u00a0=\u00a01.38, P\u00a0=\u00a0ns; 11\/23 (48%)]. Eight of 20 patients (40%) in the CaCO3 group and six of 23 patients (26%) in the MgCO3 group (P\u00a0=\u00a0ns) had final values of iPTH below 150\u00a0pg\/ml. The number of patients of both groups, patients who received MgCO3, and eight of 20 (40%) patients who received CaCO3 who had serum phosphorus, calcium, Ca\u00a0\u00d7\u00a0P product and iPTH values at 6\u00a0months that fell within the range recommended by the K\/DOQI guidelines are shown in Table\u00a05 (\u03c72\u00a0=\u00a00.79, P\u00a0=\u00a0ns).\nDuring the follow-up period the average serum Mg levels were slightly \u2013 but not significantly \u2013 higher in the MgCO3 group than in the CaCO3 group: 2.57 vs. 2.41\u00a0mg\/dl (P\u00a0=\u00a0ns) (Table\u00a04). Similarly, at 6\u00a0months, these values were 2.59 and 2.40\u00a0mg\/dl (P\u00a0=\u00a0ns) (Table\u00a03). One patient of the 25 (4%) stopped taking MgCO3 because of recurrent high levels of serum magnesium (>3.5\u00a0mg\/dl, the upper threshold according to our protocol). Two more patients manifested a transient elevation of serum magnesium \u2013 from 3.18 to 3.36\u00a0mg\/dl. No patient in the CaCO3 group had a serum magnesium >3\u00a0mg\/dl.\nDiscussion\nThis is the first study that compares MgCO3 with only one other phosphate-binder in hemodialysis patients. Our results show that MgCO3 administered for a period of 6\u00a0months has a good phosphate-binding ability, is well tolerated by most patients and is accompanied by a low incidence of side effects.\nO\u2019Donovan et\u00a0al. [4] described 28 patients on hemodialysis who were given magnesium chloride in place of oral aluminum hydroxide. These patients were also switched from a dialysate containing 0.85\u00a0mmol\/l magnesium to one not containing any magnesium at all. After 24\u00a0months of treatment on this regimen, serum phosphate was effectively controlled in these patients. The researchers saw no evidence of increased secondary hyperparathyroidism. Delmez et\u00a0al. [5] conducted a 10-week, prospective, randomized crossover study of 15 hemodialysis patients, who were on MgCO3 with a dialysate magnesium concentration of 0.25\u00a0mmol\/l; with this regimen, these investigators were able to reduce the CaCO3 dose and use a higher dose of calcitriol. Mean serum phosphate levels in the patients were similar with the MgCO3\/CaCO3 combination as with the CaCO3 treatment alone (5.7\u00a0\u00b1\u00a00.2 vs. 5.2\u00a0\u00b1\u00a00.2\u00a0mg\/dl, respectively), and there were no adverse gastrointestinal effects. Parsons et\u00a0al. [6] described 32 continuous cycling peritoneal dialysis (CAPD) in patients who were dialyzed with magnesium-free dialysate and used a mixture of CaCO3 and MgCO3 as a phosphate-binder for over 1\u00a0year. These patients achieved satisfactory control of hyperparathyroidism, with normal serum calcium, phosphorous and magnesium concentrations. Other investigators have also shown that the use of magnesium-containing agents as phosphate-binders can provide effective control of serum phosphate and hyperparathyroidism and that serum magnesium levels remain within acceptable ranges [7\u201310]. Spiegel et\u00a0al. [12] recently compared a regimen of MgCO3 and CaCO3 versus one with calcium acetate and found that the two were equally effective as phosphate-binders.\nIn our study, MgCO3 was well tolerated by most of our patients \u2013 only two of the 25 patients (8%) of this group were removed from the study, one for diarrhea and the other for hypermagnesemia. A third patient complained initially of mild diarrhea and abdominal discomfort, but this resolved after the first week. In comparison, five patients in the CaCO3 arm complained of constipation, but none dropped out of the study.\nSerum phosphate levels after the 4-week wash-out period were 6.63\u00a0mg\/dl in the MgCO3 group and 6.58\u00a0mg\/dl in the CaCO3 group. These values are lower than those reported by other investigators: 7.7\u00a0mg\/dl by Qunibi et\u00a0al. [13] in the CARE study, 7.4\u20137.7\u00a0mg\/dl by Chertow et\u00a0al. [14] in the Treat to Goal study and 6.6\u20137.2\u00a0mg\/dl by Asmus et\u00a0al. [15]. However, in the recently presented preliminary results of the CARE 2 study [16], patients\u2019 mean serum phosphate values were 6.5\u20136.6\u00a0mg\/dl. Our values may have been lower because our patients adhered more closely to their diet, they were receiving an adequate dose of hemodialysis or they had neither marked hyperparathyroidism (siPTH <500\u00a0pg\/ml) nor did they receive vitamin D. The Mediterranean type of diet consumed by our population on the island of Crete is less likely to cause hyperphosphatemia.\nThe serum phosphate level was reduced equally in both groups, and after the second month of treatment the mean values of serum phosphate in the two groups were comparable. (Table\u00a03; Fig.\u00a01).\nIn terms of the biochemical findings, the main difference between the two groups was the levels of serum calcium. Hypercalcemia is the price one has to pay for adequate phosphate-binding control with calcium-containing binders; the absence of hypercalcemia is the major advantage of magnesium-based phosphate-binders.\nWe observed that patients receiving MgCO3 were more likely to have serum calcium levels within the K\/DQOI guidelines [17] than those on CaCO3. Both groups of patients achieved a mean serum Ca\u00a0\u00d7\u00a0P product of <55\u00a0mg2\/dl2 (Tables\u00a03, 4). Consequently, a significant proportion of patients had a Ca\u00a0\u00d7\u00a0P product within the K\/DQOI recommendations (Table\u00a05).\nThe risk of severe hypermagnesemia is a major concern when magnesium salts are administered to hemodialysis patients. In such patients serum magnesium levels depend chiefly on the dialysate concentration of this ion, which standardly ranges between 0.45 and 0.50\u00a0mmol\/l. In the MgCO3 group we used a low magnesium dialysate, 0.30\u00a0mmol\/l, and this enabled us to avoid extreme hypermagnesemia and keep serum magnesium levels within acceptable ranges.\nClinical symptoms and ECG disorders due to hypermagnesemia appear only when the level of serum magnesium >4\u00a0mg\/dl [18]. The ionized fraction that represents approximately 60% of the total serum magnesium is the biologically active form of this element. Truttmann et\u00a0al. [19] and Saha et\u00a0al. [20] recently demonstrated that the ionized fraction of magnesium lower in HD patients than in individuals with normal renal function; therefore, the incidence of \u2018real hypermagnesemia\u2019 may be overestimated in these patients. Of note is that serum magnesium accounts for approximately 1% of the total body magnesium since it is mainly an intracellular cation. Estimation of total body magnesium is based on the determination of intracellular magnesium (IcMg) levels in skeletal muscles or in peripheral lymphocytes [21]. Calculations on the levels of magnesium in these tissues in uremic patients have produced conflicting results, with positive or neutral or even a negative correlation with serum magnesium being found [22, 23].\nAlthough we did not examine the consequences of hypomagnesemia and high serum magnesium levels, various studies have shown that hypomagnesemia plays a significant role in the pathogenesis of cardiovascular diseases and that a high serum magnesium level may retard the development and\/or acceleration of arterial atherosclerosis [24, 25]. Meema et\u00a0al. [26] first showed that \u2018hypermagnesemia might be associated with retardation or improvement of arterial calcifications in peritoneal dialysis patients\u2019. Izawa et\u00a0al. [27] described a hemodialysis patient in whom soft-tissue calcifications resolved after treatment with a dialysate with a  high concentration of magnesium. Two studies by our team [28, 29] clearly demonstrated that the absence of mitral annular calcifications and a thinner carotid intima media thickness in hemodialysis patients both correlated with higher levels of serum and intracellular magnesium. In a previous study, we showed that both intracellular and serum magnesium concentrations were directly associated with 5-year survival in 94 hemodialysis patients of our unit [30].\nOne of the secondary objectives of the study was to investigate if MgCO3 effectively controls serum iPTH. Calcium carbonate was more effective than MgCO3 in decreasing serum iPTH. In the MgCO3-treated patients time-average mean values of iPTH during months 1\u20136 were significantly higher than in those treated with CaCO3 (Table\u00a04). However, the final levels of serum iPTH at 6\u00a0months did not differ significantly between the two groups. Furthermore, at the end of 6\u00a0months, a slightly higher number of patients (48%) in the MgCO3 group than in the CaCO3 (40%) group achieved serum iPTH values in the range of 150\u2013300\u00a0pg\/ml.\nIn the presence of almost equal serum P values in the two groups, we hypothesize that the relatively higher PTH reduction in the CaCO3 group was due to the significant higher concentration of serum calcium in this group. On the other hand, high levels of serum magnesium have a similar, but weaker, action in suppressing PTH secretion [31]. Figure\u00a04, which shows the monthly follow-up of serum iPTH levels, shows that the curve of the change in mean iPTH values is different in the MgCO3-treated patients in that the mean values increase during the first month of the treatment, subsequently falling (but slower than in the CaCO3 group) and finally, in the sixth month, they approach the mean iPTH values of the CaCO3 group. We speculate that the shape of this curve indicates that suppressive action of serum magnesium on the parathyroid glands is weaker but delayed compared to that of serum calcium. The number of patient who had final serum iPTH values of <150\u00a0pg\/ml, which could be associated with adynamic bone disease, did not differ in the two groups. However, the impact of the two regimens on the patients\u2019 bone turn over was not included in the objectives of the study, so we did not obtain data associated with bone metabolism markers.\nOur experience suggests that a significant proportion of hemodialysis patients need more than one agent to achieve a satisfactory phosphate-binding. In such cases, MgCO3 could be if not the primary at least the second constituent of the phosphate-binding regimen, combined ideally with a calcium-containing salt. An additional advantage is that MgCO3 is much less expensive than the newest sevelamer HCL and lanthanum carbonate.\nThe limitations of our study are: (1) it is a single-center study, although this has also a number of advantages; (2) the number of the participant patients was not large, although it is one of the largest groups reported to date; (3) the allocation of the patients to the two regimens was only partially random; (4) the relatively low starting serum phosphate level in conjunction with the no vitamin D use means that the patients were not typical of most HD patients; (5) we did not obtain data on the patients\u2019 bone metabolism markers.\nIn conclusion, our study showed that MgCO3 administered for a period of 6\u00a0months is an effective and inexpensive agent to control serum P levels in hemodialysis patients. Gastrointestinal disorders due to its use were minor, while its administration in combination with a low dialysate magnesium concentration reduces the risk of severe hypermagnesemia. Patients treated with MgCO3 had a mild suppression of PTH, an optimum regulation of Ca\u00a0\u00d7\u00a0P product, relatively low serum calcium and no episodes of hypercalcemia. We believe that this demonstration of the effectiveness of MgCO3 to bind phosphate warrants further investigation in a larger group of patients.","keyphrases":["magnesium carbonate","hemodialysis","phosphate-binders","end-stage renal disease"],"prmu":["P","P","P","P"]}
{"id":"Behav_Genet-4-1-2226022","title":"Heritability of Self-reported Phobic Fear\n","text":"Twin studies on fear and phobia suggest moderate genetic effects. However, results are inconclusive regarding the presence of dominant genetic effects and sex differences. Using an extended twin design, including male and female twins (n = 5,465) and their siblings (n = 1,624), we examined the genetic and environmental influences on blood-injury, social, and agoraphobic fear and investigated their interaction with sex and age. Data of spouses (n = 708) of twins were used to evaluate assortative mating for the three fear dimensions. Results showed that there was no assortative mating for blood-injury, social and agoraphobic fear. Resemblance between biological relatives could be explained by additive and non-additive genetic effects for blood-injury and agoraphobic fear in all participants, and social fear in participants aged 14\u201325 years. For social fear in participants aged 26\u201365 only additive genetic effects were detected. Broad-sense heritability estimates ranged from 36 to 51% and were similar for men and women.\nIntroduction\nFears and phobias are extremely disruptive and distressing disorders that have a great impact on an individual\u2019s life (Mendlowicz and Stein 2000; Saarni et\u00a0al. 2007). The prevalence of anxiety disorders is high and anxiety disorders represent a serious public health concern (Kessler et\u00a0al. 2005). However, the etiology of phobic fear is still poorly understood.\nFamily studies of clinically defined phobias have consistently found evidence for the familial aggregation (Fyer et\u00a0al. 1995; Stein et\u00a0al. 2001). Twin family studies of clinically defined fears and phobias have also indicated familiarity (Torgersen 1983; Skre et\u00a0al. 1993; Skre et\u00a0al. 2000). Due to their relatively small sample sizes, clinical studies often are unable to establish whether this familiarity results from genetic or environmental similarities. Through community based twin and twin family studies, large samples can be assessed, resulting in sufficient power to disentangle genetic and environmental influences in the etiology of fears and phobias. Such studies have suggested an important role for genetic and unique environmental factors (e.g., aversive experiences) in the origin of fears and phobias, while shared environmental influences appeared to be of little etiological significance (Hettema et\u00a0al. 2001; Sundet et\u00a0al. 2003).\nThe Virginia Twin Registry investigated the familial aggregation of phobia diagnoses in a large adult sample. In women, heritability estimates of 39% for agoraphobia and 30% for social phobia were found (Kendler et\u00a0al. 1992). In men, these estimates were 37 and 20% (Kendler et\u00a0al. 2001). Familial aggregation of blood-, needles-, hospital-, or illness-phobia was studied in female twins only and appeared to be due to shared environmental factors, although the statistical power to differentiate between additive genetic and common environmental influences was low (Neale et\u00a0al. 1994a). An Australian study of self-reported fear of blood-injury-injection in female twins also found significant familial aggregation of blood-injury-injection fears. It was not possible to differentiate between genetic and shared environmental influences as the explanation for familial aggregation. Multivariate genetic analysis of fainting and blood-injury-injection fear with and without fainting indicated that the genetic variance in blood-injury-injection fear was attributable to additive genetic factors shared with fainting (55%) (Page and Martin 1998).\nBased on the correlation structure several studies suggested the presence of non-additive genetic effects in phobic fear, but due to insufficient statistical power, these effects could not convincingly be demonstrated (Skre et\u00a0al. 2000; Neale et\u00a0al. 1994a; Rose and Ditto 1983; Kendler et\u00a0al. 1999; Page and Martin 1998).\nThe heritability estimates reported in the Kendler et\u00a0al. studies of female (1992) and male twins (2001) seem to indicate lower heritability estimates for fear in men than in women. Together with the fact that women display generally more phobias than men (Fredrikson et\u00a0al. 1996; Furmark 2002; Bekker 1996; Curtis et\u00a0al. 1998; Bijl et\u00a0al. 2002; Middeldorp et\u00a0al. 2005) this suggests that men and women differ in the extent to which genetic and environmental risk factors affect phobic fear. Sex differences in the genetic contribution to the etiology of phobic fear have only been formally tested in the Virginia Twin Registry, in the Netherlands Twin Register and in the Australian Twin Registry. In the Virginia Twin Registry, Kendler et\u00a0al. (2002) found equal heritability in men and women for agoraphobia and blood-injury phobia. For social phobia, on the other hand, resemblance in men was explained by genetic factors while in women resemblance was explained by shared environment. Because earlier studies relying on a female sample (Kendler et\u00a0al. 1992, 1999) indicated that familial resemblance was entirely due to genetic factors, the authors attributed this sex difference to stochastic factors. However, results from two multivariate studies using data from the Virginia Twin Registry (Hettema et\u00a0al. 2005, 2006), indicated that around 10% of the variance in social phobia was explained by shared environmental factors.\nQualitative sex differences on the liability for agoraphobia, social phobia and blood-injury phobia were suggested by Kendler et\u00a0al. (2002) based on the dizygotic opposite sex (DOS) twin correlation, which was lower than the dizygotic (DZ) same sex correlations. A second study of the Virginia Twin Registry (Hettema et\u00a0al. 2005) found no sex differences in the genetic and environmental risk factors for agoraphobia or social phobia. In a combined Dutch and Australian sample, Middeldorp et\u00a0al. (2005) failed to find quantitative sex differences in the familial influences on agoraphobia and social phobia. For agoraphobia a low correlation in DOS twins suggested qualitative sex differences. To summarize, most studies report no sex differences in the heritability of fear and phobia while some do suggest qualitative sex differences based on the DOS and DZ correlation structure.\nSpousal resemblance is often found for psychiatric disorders and related phenotypic traits. The most cited explanation for spousal similarity is assortative mating, meaning that spouses are more similar for a trait or disorder than expected under random mating (Merikangas 1982; Garrison et\u00a0al. 1968), although significant spousal correlation could also indicate, for example, marital interaction or social stratification. Spousal resemblance is an important issue to consider because increased phenotypic correlation between spouses increases genetic variance in the offspring generation. When non-random mating for a heritable trait is present but this is not specifically included in the twin model, heritability estimates may be downwardly biased. For depressive disorders, a meta-analysis reported marital resemblance for depression in 12 of 17 studies (Mathews and Reus 2001). An extensive study on spousal similarity for psychiatric disorders in a population-based sample was carried out by Maes et\u00a0al. (1998). Several psychiatric diagnoses were examined, including generalized anxiety disorder, major depressive disorder, panic disorder and phobias. Moderate spousal correlations were seen for most psychiatric diagnoses, but for phobias spousal correlations were not significant.\nSo far, age effects on the heritability of phobic fears have not received much attention in the literature. This is an omission because it has well been established that some specific fears decline from adolescence to later life (e.g., fear of blood, strangers or personal death) (Bienvenu and Eaton 1998; Hall 1897; Rose and Ditto 1983; Kessler et\u00a0al. 1994; Henderson et\u00a0al. 1998), while others increase or are not affected by age at all (e.g., fear of negative social interactions or small organisms) (Rose and Ditto 1983; Lapouse and Monk 1959). Rose and Ditto (1983) found evidence that with age there is a change in the genetic and environmental influences on fear of death. However, a more recent study reported that there were hardly any longitudinal changes in the genetic and environmental determinants of anxiety (Gillespie et\u00a0al. 2004).\nIn order to replicate and extend previous research on fears and phobias, we examined the heritability of three classes of phobic fears (i.e., blood-injury, social, and agoraphobic fear) using longitudinal data from Dutch twins and their siblings registered with the Netherlands Twin Register (Boomsma et\u00a0al. 2006). The inclusion of siblings and repeated observations increases statistical power to discriminate between additive genetic and non-additive genetic effects and to detect sex specific genetic effects. Adding siblings to the sample also provides the opportunity to examine twin-specific effects on phobic fears (Posthuma and Boomsma 1999, 2000). Data were also available for a subsample of spouses of twins which were used to estimate the correlation between spouses to examine whether there is evidence for assortative mating. Since the determinants of phobic fear in adolescence and young adulthood may differ from those in later life, we divided the dataset into two age cohorts so as to test for differences in heritability between the two cohorts. In addition, we tested for qualitative and quantitative sex differences in the etiology of phobic fear.\nTo summarize, in this report, we address the following questions. First, is there spousal similarity for blood-injury, social and agoraphobic fear? Second, what are the heritability estimates for these three subtypes of fear? Third, are there sex and age differences in the heritability estimates of these subtypes?\nMethods\nParticipants\nThis study is part of an ongoing study on health and lifestyle in twin families registered with the Netherlands Twin Register (Boomsma et\u00a0al. 2006). Initially, adolescent and adult twins and their family members were recruited through City Councils in 1990\u201391 and in 1992\u201393. After 1993, an effort was made to recruit older twins through a variety of approaches. Siblings and spouses of twins were recruited in the study since 1995 and 2000, respectively. Parents did not participate in 1997 and 2000. All registered individuals gave their informed consent before they filled out questionnaires. Further details on response rates and demographic characteristics of the sample can be found elsewhere (Boomsma et\u00a0al. 2002; Vink et\u00a0al. 2004; Boomsma et\u00a0al. 2006). The data on phobic fear were derived from surveys sent in 1997 and 2000. Table\u00a01 shows the complete sample configuration of two age cohorts in 1997 and 2000. In total, there were 7,089 subjects (5,465 twins and 1,624 siblings) from 3,471 families, with longitudinal data available for 2,537 individuals. Based on the age of the twins, we subdivided the participating families into two age groups: a 14\u201325\u00a0year group (n\u00a0=\u00a04,275 individuals) and a 26\u201365\u00a0year group (n\u00a0=\u00a02,814 individuals). Families with siblings but without twins were assigned to one of the two age groups according to the age of the youngest sibling. There was a small overlap between the two age cohorts in age of the non-twin siblings. In the 14\u201325\u00a0year age group, there were 1,987 families of which 1,004 included one or more family members who participated at both time points. The 26\u201365\u00a0year age group involved 1,484 families of which 435 contained one or more family members who participated at both time points. In the youngest age group, mean age was 21.2\u00a0years (SD\u00a0=\u00a03.5) in 1997 and 23.2\u00a0years (SD\u00a0=\u00a03.6) in 2000. In the oldest age group, mean age was 36.6\u00a0years (SD\u00a0=\u00a010.5) in 1997 and 38.0\u00a0years (SD\u00a0=\u00a010.4) in 2000. For the 2000 survey, we asked spouses of all twins aged 25\u201330\u00a0years to participate. In total 708 spouses completed a survey.\nTable\u00a01Family configuration in the sample according to zygosity, cohort and number of additional non-twin siblings of the two time points (separated by the slash; 1997\/2000)Zero siblingsBrotherSisterBrother and SisterTotalAge 14\u201325MZMTwin pair70\/10441\/2935\/279\/4155\/164Single twin21\/574\/610\/111\/336\/77DZMTwin pair44\/4533\/1526\/143\/7106\/81Single twin18\/444\/910\/101\/233\/65MZFTwin pair145\/23049\/3562\/5612\/9268\/330Single twin49\/10610\/1211\/173\/273\/137DZFTwin pair84\/10834\/1937\/277\/7162\/161Single twin33\/8710\/912\/232\/257\/121DOSTwin pair101\/10564\/3768\/4110\/3243\/186Single twin69\/18311\/2625\/326\/2111\/243No twins23\/4338\/722\/463\/119Total age 14\u201325634\/1069283\/240334\/33056\/451307\/1684Age 26\u201365MZMTwin pair16\/608\/67\/115\/836\/85Single twin20\/686\/34\/76\/136\/79DZMTwin pair11\/313\/63\/77\/324\/47Single twin20\/544\/27\/55\/536\/66MZFTwin pair64\/22314\/2223\/539\/12110\/310Single twin30\/1226\/1010\/162\/448\/152DZFTwin pair29\/958\/1411\/153\/651\/130Single twin39\/994\/914\/73\/060\/115DOSTwin pair27\/869\/710\/154\/750\/115Single twin36\/1206\/810\/226\/558\/155No twins21\/4446\/2512\/879\/77Total age 26\u201365292\/95889\/131145\/18362\/59588\/1331Total number of families926\/2027372\/371479\/513118\/1041895\/3015MZM\u00a0=\u00a0monozygotic males; DZM\u00a0=\u00a0dizygotic males; MZF\u00a0=\u00a0monozygotic females; DZF\u00a0=\u00a0dizygotic females; DOS\u00a0=\u00a0dizygotic opposite sex twins\nZygosity\nA total of 1,322 twins were classified as DZ because they were of opposite sex. Zygosity of same sex twins was determined from DNA polymorphisms (n\u00a0=\u00a01,205), or from survey questions about physical twin resemblance and confusion of the twins (n\u00a0=\u00a02,938). Based on the answers from all available surveys, zygosity was determined. When there was an inconsistency over time or persons, the majority of the judgements determined the final outcome. The agreement between zygosity based on survey data and on DNA typing was 97% (Willemsen et\u00a0al. 2005).\nMeasures\nParticipants completed the Dutch version of the Fear Questionnaire (FQ; Marks and Mathews 1979). The FQ is a widely used instrument that possesses good psychometric qualities (Van Zuuren 1988). The FQ contains 15 items that tap phobic avoidance in three domains: blood-injury fear (e.g., avoidance of hospitals), social fear (e.g., avoiding talking to people in authority), and agoraphobic fear (e.g., avoiding large, open spaces). Each domain is represented with five items. Subjects were instructed to indicate on a nine point scale (0 \u2018would not avoid it\u2019 to 8 \u2018always avoid it\u2019) how much they would avoid certain situations because of fear or other unpleasant feelings. Scores were summed across subscales to obtain scores for blood-injury fear, social fear, and agoraphobic fear. A score was only calculated if at least four out of five items of a subscale were answered. Missing answers and double entries were substituted by the mean item score. We checked the factor structure of the FQ in our sample. All items of the FQ were analyzed with PCA with promax rotation in SPSS. Analyses were done separately for the two time points with random selection of one person per family. At both time points we found the original three factor solution in our data which accounted for around 47% of the variance.\nTwin-family Studies\nTwin studies make use of the genetic relatedness of twins and their family members to address questions about the etiology of population variation. Monozygotic (MZ) twins are genetically (nearly) identical. Dizygotic (DZ) twins and siblings share on average 50% of their segregating genes. Additive genetic effects (A) are suggested if the correlation for a phenotype in MZ twins is larger than the correlation in DZ twins and siblings. When the DZ correlation is more than half the MZ correlation, this indicates environmental effects shared by members from the same family (C). When the DZ correlation is less than half the MZ correlation, this can be taken as evidence for non-additive genetic effects (D). Non-additive genetic effects can consist of interactions between alleles within a locus (dominance) or across different loci (epistasis). Differences in fear scores within MZ twin pairs are due to unique environmental influences (E), which also include measurement error. The observed variance in phobic fear can thus be decomposed in four possible sources of variance; A, C, D, and E. However, the observed variances and covariances only provide enough information to test either an ACE model or an ADE model. Based on the pattern of twin correlations (see results section), A, D, and E were modelled in this study.\nStatistical Analysis\nSince phobic fear is not normally distributed in the population we used a multiple threshold model (see Fig.\u00a01) to estimate the genetic and environmental contributions to the liability of phobic fear. Liability to phobic fear is assumed to be continuous and normally distributed in the population. Derks et\u00a0al. (2004) showed that this is the optimal approach when analyzing L-shaped distributed phenotypic data. We divided the liability to phobic fear in three categories separated by two thresholds so that all categories contained approximately equal numbers of subjects (see Fig.\u00a02). The validity of the bivariate threshold model to the fear data divided in three categories, was assessed in PRELIS (J\u00f6reskog and S\u00f6rbom 1996). The fit of the bivariate threshold model to the blood-injury, social and agoraphobic fear data was good; almost all RMSEA\u2019s were <0.05 and all P values exceeded the 0.002 significance level (Bonferroni correction for multiple testing).\nFig.\u00a01Threshold model of the liability for phobic fear. Latent factors are symbolized in circles, observed phenotype as squares. E, unique environmental factor; D, non-additive genetic factor; A, additive genetic factor; L, liability for phobic fear. Latent factors can be correlated within families and influence the liability to phobic fearFig.\u00a02Distribution of fear scores in 1997 for men and women age 14\u201325 and age 26\u201365\u00a0years. The Y-axis gives the percentage of males and females with each score. The dashed lines indicate the thresholds\nGenetic structural equation modelling was carried out in Mx (Neale et\u00a0al. 2003). Testing of submodels was done by means of likelihood-ratio tests, by subtracting the negative log likelihood (\u22122LL) for the more restricted model from the \u22122LL for the more general model. This yields a statistic that is distributed as \u03c72 with degrees of freedom (df) equal to the difference in the number of parameters in the two models. If the \u03c72-test yields a P-value higher than 0.05, the constrained model is deemed not significantly worse than the previous model and is kept as the most parsimonious model to which the next model will be compared.\nWe started with fitting saturated models to describe the correlational structure between twin pairs and between siblings in each zygosity group. We used repeated measures from two time points (Rebollo and Boomsma 2006). Twin and sibling correlations were constrained to be equal at the two time points. For each fear subtype (blood-injury, social, and agoraphobic fear) and within each age group (14\u201325 and 26\u201365\u00a0years), we estimated five twin correlations (one for each sex by zygosity group) and three sibling correlations (male\u2013male, female\u2013female, and male\u2013female sib pairs). Two cross-time within person correlations (one for each sex) and eight cross-twin cross-time correlations (one for each of the five sex by zygosity and the three sib pair groups) were estimated for each trait (not reported in the results section). Thresholds were estimated separately for men and women at each time point. The spouse correlations (Spearman\u2019s correlation coefficients) were calculated using SPSS 12.0 for windows.\nFirst, we tested whether the resemblance in sibling pairs was the same as in dizygotic twin pairs. Next, quantitative sex differences were investigated by constraining the correlations for same-sex male twin pairs and same-sex female twin pairs to be equal. Qualitative sex differences were investigated by constraining the correlations for DZ same-sex twin pairs and DZ opposite-sex twin pairs to be equal.\nIn a genetic model, the influence of latent factors A, D and E on the phobic fear score in 1997 and 2000 was estimated by the parameters (factor loadings) a, d, and e. The estimates for a, d and e were constrained to be equal in 1997 and 2000 for all three fear subtypes because we assume that the same etiological mechanism influences phobic fear at both time points. Correlations between the latent A and D factors were fixed at 1 for MZ twins. For DZ twins and siblings, the correlations between the latent A factors were fixed at 0.5, because they share on average half of their segregating genes. Dominant genetic effects result from the interaction or combination of alleles at a particular locus. Offspring receive only one allele from each parent and not a combination of two alleles. Parents and offspring share on average half of their genes so the chance that two siblings receive the same allele is 0.5\u00a0\u00d7\u00a00.5 resulting in a correlation of 0.25 between the latent D factor for DZ twins and sibling pairs.\nWe first tested whether a, d and e in the two age groups could be constrained to be equal. Next, different models (ADE, AE and E) were fitted to the data to test which sources of variance contribute to individual differences in phobic fear.\nResults\nFigure\u00a02 shows the distribution of fear scores in 1997 for males and females aged 14\u201325 and aged 26\u201365. The 2000 scores (not presented here) show a similar pattern. Women had significantly more often high scores on each fear dimension than men in each age group, except for blood-injury fear in the oldest age group for which there was no difference between men and women. There was no effect of age on the distribution of fear scores, except for women\u2019s scores on blood-injury fear, which were significantly higher in the youngest age group.\nThere was no significant correlation between twins and their spouses for blood-injury fear (rs\u00a0=\u00a00.05, P\u00a0=\u00a00.162), social fear (rs\u00a0=\u00a00.04, P\u00a0=\u00a00.324) or agoraphobic fear (rs\u00a0=\u00a00.04, P\u00a0=\u00a00.341).\nTable\u00a02 displays the twin correlations and confidence intervals for blood-injury, social, and agoraphobic fear for both age groups. Correlations for same sex DZ and DOS twin pairs, and for sibling pairs could be constrained to be equal, suggesting that there is no evidence for a special twin environment. MZ male\u2013male and female\u2013female correlations were the same and DZ same-sex correlations were also the same, indicating that there are no quantitative sex differences in the influence of genetic effects on the three fear types. The DZ opposite-sex twin correlation was equal to the same-sex DZ correlation indicating that the same genes affect fear in men and women. After these constraints, MZ correlations were more than twice as large as the correlations for DZ twins and siblings suggesting that genetic effects contribute to individual differences in phobic fear and that these effects act in a partly non-additive manner.\nTable\u00a02Twin and sibling correlations for blood-injury, social and agoraphobic fear for age group 14\u201325\u00a0years and age group 26\u201365\u00a0years (95% confidence intervals added in parentheses)Blood-injury fear Social fearAgoraphobic fearAge 14\u201325Age 26\u201365Age 14\u201325Age 26\u201365Age 14\u201325Age 26\u201365MZ males0.30 (0.17\u20130.42)0.36 (0.14\u20130.54)0.53 (0.41\u20130.63)0.29 (0.03\u20130.50)0.47 (0.34\u20130.56)0.49 (0.27\u20130.65)DZ males0.04 (\u22120.16\u20130.22)\u22120.04 (\u22120.32\u20130.25)0.15 (\u22120.05\u20130.34)0.49 (0.20\u20130.68)0.27 (0.09\u20130.43)0.22 (\u22120.10\u20130.48)MZ females0.34 (0.25\u20130.43)0.41 (0.32\u20130.50)0.48 (0.39\u20130.56)0.42 (0.31\u20130.52)0.40 (0.30\u20130.48)0.37 (0.26\u20130.47)DZ females0.18 (0.05\u20130.30)0.11 (\u22120.06\u20130.28)0.20 (0.06\u20130.33)0.37 (0.20\u20130.53)0.13 (\u22120.00\u20130.26)0.27 (0.08\u20130.44)DZ opposite sex0.23 (0.10\u20130.34)0.16 (\u22120.04\u20130.34)0.22 (0.10\u20130.34)0.25 (0.03\u20130.44)0.17 (0.04\u20130.29)0.22 (0.01\u20130.41)Brother\u2013brother0.16 (0.03\u20130.28)0.28 (0.10\u20130.44)0.12 (\u22120.02\u20130.25)0.19 (\u22120.03\u20130.38)0.09 (\u22120.04\u20130.22)\u22120.01 (\u22120.24\u20130.22)Sister\u2013sister0.10 (\u22120.01\u20130.20)\u22120.02 (\u22120.16\u20130.13)0.09 (\u22120.03\u20130.20)0.14 (\u22120.01\u20130.28)0.10 (\u22120.01\u20130.19)0.12 (\u22120.03\u20130.26)Brother\u2013sister0.10 (0.02\u20130.18)0.06 (\u22120.05\u20130.17)0.15 (0.06\u20130.23)0.11 (\u22120.02\u20130.23)0.08 (\u22120.00\u20130.02)0.07 (\u22120.05\u20130.18)All MZa0.33 (0.26\u20130.40)0.39 (0.30\u20130.48)0.50 (0.43\u20130.56)0.40 (0.30\u20130.49)0.42 (0.35\u20130.49)0.39 (0.30\u20130.48)All DZ\/siblingsa0.13 (0.08\u20130.18)0.09 (0.02\u20130.16)0.15 (0.10\u20130.20)0.20 (0.13\u20130.27)0.11 (0.07\u20130.16)0.12 (0.05\u20130.19)MZ\u00a0=\u00a0monozygotic; DZ\u00a0=\u00a0dizygoticaAfter constraining these correlations to be equal\nModel fitting results for blood-injury, social, and agoraphobic fear are shown in Table\u00a03. The data from two age groups were simultaneously analysed in one model with different parameter estimates for the two age groups. For each fear subtype, a general ADE model (model 1) was fitted. Next, we tested if the A, D, and E components were the same for the two age cohorts (model 2). Finally, in model 3, and in the case of social fear in models 4 and 5, the significance of D and A in the two age groups was tested by constraining the relevant parameters at zero. For blood-injury and agoraphobic fear, this resulted in a model in which variance components did not differ between the two age groups. Non-additive genetic effects could not be left out of the model for blood-injury and agoraphobic fear. A, D and E explained respectively 9.9, 25.6, and 64.5% of the variance in blood-injury fear, and 10.7, 29.9, and 59.5% of the variance in agoraphobic fear. For social fear in the youngest age group, A, D and E contributed to the variance, and explained 7.8, 42.9, and 49.3% of the variance, respectively, while in the oldest age group D did not contribute to the variance. In fact, 40.3% of the variance in social fear in the oldest age group could be explained by additive genetic effects and 59.7% by unique environmental effects (Table\u00a04).\nTable\u00a03Model fitting results for blood-injury, social, and agoraphobic fear; comparisons of models are shownModelsvs\u22122LLdf\u0394df\u0394\u03c72PBlood-injury fear1. ADE20254.5896082. ADE no age diff.120257.23961792.650.983. AE no age diff.220415.2796203158.030.00Social fear1. ADE19439.7296082. ADE no age diff.119468.909617929.180.003. AE youngADE old119454.059611314.330.004. ADE youngAE old119439.72961130.001.005. ADE youngE old419521.589614381.870.00Agoraphobic fear1. ADE 19967.6196142. ADE no age diff.119973.66962396.050.743. AE no age diff.219984.909626311.240.01vs\u00a0=\u00a0versus and indicates which model the sub model is compared to; \u22122LL\u00a0=\u00a0\u22122 log likelihood; df\u00a0=\u00a0degrees of freedomMost parsimonious solution per fear subtype is shown in boldTable\u00a04Parameter estimates of the best-fitting models in age group 14\u201325 and age group 26\u201365 for blood-injury fear, social fear and agoraphobic feara2 (%)d2 (%)e2 (%)Blood-injury fearAge 14\u2013659.925.664.5Social fearAge 14\u2013257.842.949.3Age 26\u20136540.3\u201359.7Agoraphobic fearAge 14\u20136510.729.959.5a2\u00a0=\u00a0additive genetic variance component; d2\u00a0=\u00a0dominant genetic variance component\u00a0; e2\u00a0=\u00a0unique environmental variance component\nComments\nThis is the first study that explored the genetic and environmental contributions to individual differences in blood-injury, social, and agoraphobic fear using an extended twin design. We tested whether there was evidence for assortative mating, for a specific twin environment, and if the impact of the genetic and environmental factors differed between men and women and between two age cohorts.\nWe observed that more women than men scored high on all fear dimensions, except for blood-injury fear in the oldest age group. This pattern is consistent with the large majority of studies that looked at sex differences in fear levels: overall, women tend to have higher scores on self-report measures tapping various fear categories, the exception being blood-injury phobia, where the sex distribution is somewhat more balanced (Bijl et\u00a0al. 1998; Kessler et\u00a0al. 1994; Middeldorp et\u00a0al. 2005).\nPrior studies showed that many fear types display a distinct developmental course, with, in general, a decline of fear levels from adolescence to later life. Our data suggest no large effect of age in fear scores: a pattern of declining fear scores was only seen for blood-injury fear, with younger participants having higher fear levels than older participants. This is in accordance with the literature (Bijl et\u00a0al. 2002; Page 1994). However, many developmental changes in fear levels occur before the adolescent stage (Marks 1987), an underrepresented age group in our sample. Future research will have to determine whether changes in blood-injury, social and agoraphobic fear emerge before adolescence.\nSpouse similarity is an important issue to consider when studying genetic influences on population variation. In accordance with what was reported previously for phobias (Maes et\u00a0al. 1998) and anxiety disorders (Low et\u00a0al. 2007), we did not find a significant association between spouses, indicating that there is random mating for blood-injury, social and agoraphobic fears.\nBroad-sense heritability estimates for blood-injury, social, and agoraphobic fear were moderate ranging from 35.5% (blood-injury fear) to 50.7% (social fear in the youngest age group). These heritability estimates for blood-injury fear and agoraphobic fear are comparable to the estimates reported by Kendler et\u00a0al. (1992, 2001). The heritability estimate for social fear (50.7% in the youngest age group and 40.3% in the oldest age group) was somewhat higher than that reported by Kendler et\u00a0al. (30% for women and 20% for men) but comparable to the heritability estimate found by Middeldorp et\u00a0al. (2005) (50%). Contrary to what was found by Hettema et\u00a0al. (2005, 2006) shared environmental factors did not significantly contribute to the variance in social fear. All in all, these results confirm the conclusion of Kendler et\u00a0al. (1992, p. 279) that \u201cthe estimated heritability of liability for phobias indicates that genetic factors play a significant, but by no means overwhelming role in the etiology of phobias.\u201d\nIn accordance with previous findings (Hettema et\u00a0al. 2005; Middeldorp et\u00a0al. 2005; Kendler et\u00a0al. 2002), no differences in heritability between men and women were observed. Regarding qualitative sex differences, the results are somewhat different from previous studies. Whereas results of Kendler et\u00a0al. (2002) and Middeldorp et\u00a0al. (2005) suggested the presence of qualitative sex differences for agoraphobic fear, we did not find any evidence that different genes influence the liability of phobic fear in men and women. Since the sample in our study was enriched with siblings of twins, a large number of opposite sex first degree relatives was added to the study. These data do not indicate a lower familial correlation in opposite-sex relatives.\nFor all three fear subtypes, MZ correlations were more than twice as high as the DZ\/sibling correlations, indicating that non-additive genetic effects may contribute to the variance in phobic fears. Several previous studies suggested the influence of non-additive genetic effects on fears and phobia, which makes sense because fears are likely to be exposed to selection during evolution. Skre et\u00a0al. (2000) investigated the genetic and environmental contributions to common phobic fears in a treatment sample of 61 twin pairs and found the twin correlations for social fear to fit best to a model including non-additive genetic effects. For blood-injection-injury fear, the correlational structure found by Skre et\u00a0al. suggested the influence of non-additive genetic effects but model fitting showed that due to violation of equal variances between MZ and DZ twins all variance was accounted for by unique environmental effects. Neale et\u00a0al. (1994b) also reported evidence for non-additive genetic effects in reanalysing the Rose and Ditto data (1983) of common social fears. In addition, Kendler et\u00a0al. (1999) found evidence for non-additive genetic effects in agoraphobia, but not in social phobia and blood-injury phobia, while Page and Martin (1998) suggested the possibility of non-additive genetic effects on the variance in blood-injury phobia. In 2002, Kendler et\u00a0al. (2002) did not model non-additive genetic effects explicitly, yet the correlation structure did suggest the presence of non-additive genetic effects for agoraphobia and blood-injury phobia. To summarize, many studies indicated the presence of non-additive genetic effects on the liability for fear and phobias but they often did not have sufficient statistical power to detect non-additive genetic effects. The large sample size of this study together with repeated measures enabled us to detect the influence of non-additive genetic effects on blood-injury fear, social fear (in the youngest age group), and agoraphobic fear, confirming the speculations discussed in previous studies. For social fear in the oldest age group, non-additive genetic effects were not detected. Thus, the mixture of genetic and environmental influences on social fear seems to differ between the two age cohorts, though the differences in correlation structure were small. These results could be relevant to gene mapping of anxiety and phobia. If many of the genes underlying anxiety and phobia have non-additive effects, as suggested by our results, then quantitative trait loci with little additive genetic effects may have been missed in linkage analysis (Purcell and Sham 2004). Explicitly modelling non-additive genetic effects may therefore be important in the linkage analysis of anxiety, fear and phobia.\nIn general, our data are well in line with the multifactorial model of fears and phobias (Muris and Merckelbach 2001). This model assumes that a genetic vulnerability predisposes individuals to develop maladaptive fears and that discrete learning experiences (i.e., unique environmental factors) in combination with genetic vulnerabilities produce persistent fears.\nResults of this study should be interpreted in context of the following limitations. First, the present study relied on non-clinical groups, and, although there is no reason to assume that heritabilities increase at the extreme end of the fear continuum (Stevenson et\u00a0al. 1992), it is important to replicate the findings in clinical samples. Second, data of our study were obtained with an instrument (i.e., the FQ) that primarily focuses on self-reported avoidance behavior related to a limited number of fear categories and our results may not be generalized to other specific fears such as fear of small animals.\nIn summary, we found random mating for blood-injury, social and agoraphobic fear. Individual differences in blood-injury fear and agoraphobic fear in both age groups and social fear in the youngest age group (14\u201325\u00a0years) could be explained by additive and non-additive genetic factors and unique environmental factors, while individual differences in social fear in the oldest age group (26\u201365\u00a0years) could be explained by additive genetic and unique environmental factors. The heritability of the three fear subtypes was similar for men and women and no effects of sex-specific genes were detected.","keyphrases":["fear","genetics","dominance","assortative mating","twin-family study"],"prmu":["P","P","P","P","P"]}
{"id":"Plant_Mol_Biol-3-1-2039854","title":"Local coexpression domains in the genome of rice show no microsynteny with Arabidopsis domains\n","text":"Chromosomal coexpression domains are found in a number of different genomes under various developmental conditions. The size of these domains and the number of genes they contain vary. Here, we define local coexpression domains as adjacent genes where all possible pair-wise correlations of expression data are higher than 0.7. In rice, such local coexpression domains range from predominantly two genes, up to 4, and make up \u223c5% of the genomic neighboring genes, when examining different expression platforms from the public domain. The genes in local coexpression domains do not fall in the same ontology category significantly more than neighboring genes that are not coexpressed. Duplication, orientation or the distance between the genes does not solely explain coexpression. The regulation of coexpression is therefore thought to be regulated at the level of chromatin structure. The characteristics of the local coexpression domains in rice are strikingly similar to such domains in the Arabidopsis genome. Yet, no microsynteny between local coexpression domains in Arabidopsis and rice could be identified. Although the rice genome is not yet as extensively annotated as the Arabidopsis genome, the lack of conservation of local coexpression domains may indicate that such domains have not played a major role in the evolution of genome structure or in genome conservation.\nIntroduction\nThe fast-growing data sets on genome annotation and genome-wide gene expression facilitate the study and comparison of gene activity between and among genomes. The genomic context of genes is supposed to play an important role in the regulation of gene expression (van Drunen et\u00a0al. 1997). Non-random clusters of similarly expressed (co-regulated, coexpressed, highly expressed and\/or broadly expressed) genes have been described in almost all organisms, ranging from prokaryotes to eukaryotes. In eukaryotes, from yeast to Arabidopsis to human, both short-range co-regulated\/coexpressed clusters of two to five genes (Cohen et\u00a0al. 2000; Ren et\u00a0al. 2005; Zhan et\u00a0al. 2006) and longer-range coexpression domains of up to 30 genes spanning up to 100\u00a0kb and more (Lercher et\u00a0al. 2003; Ma et\u00a0al. 2005; Spellman and Rubin 2002; Zhan et\u00a0al. 2006) have been described. Duplicated genes (Lercher et\u00a0al. 2003), shared promoter regions (Kruglyak and Tang 2000), shorter gene distance (Cohen et\u00a0al. 2000; Roy et\u00a0al. 2002; Semon and Duret 2006; Williams and Bowles 2004) and\/or functional relatedness (Cohen et\u00a0al. 2000; Lee et\u00a0al. 2004; Spellman and Rubin 2002; Williams and Bowles 2004) were found to account for only part of the coexpression between genes. Therefore, most studies postulate that the occurrence of coexpression domains, small or large, is regulated on the level of higher-order chromatin structure (Cohen et\u00a0al. 2000; Hershberg et\u00a0al. 2005; Ren et\u00a0al. 2005; Spellman and Rubin 2002; Williams and Bowles 2004), although alternative views exist (Semon and Duret 2006).\nPreviously we have defined and demonstrated the existence of local coexpression domains in the genome of Arabidopsis (Ren et\u00a0al. 2005). A local coexpression domain was defined as any set of physically adjacent genes that are highly coexpressed with a pair-wise Pearson\u2019s correlation coefficient larger than 0.7. It was shown that a small (5\u201310%) yet significant fraction of genes in the Arabidopsis genome is organized in such local coexpression domains. Genes in such local domains were for the major part not categorized in the same functional category (GOslim). Neither tandemly duplicated genes nor shared promoter sequence, nor gene distance, explained the occurrence of coexpression of genes in such chromosomal domains. This indicates that other parameters in genes or gene positions are important to establish coexpression in local domains of Arabidopsis chromosomes. Here it is analyzed whether a similar situation exists in the genome of the monocotyledonous model plant rice (Oryza sativa), for which an earlier study had shown the existence of longer-range domains (Ma et\u00a0al. 2005). We combined the whole genome rice annotation data (TIGR version 3; www.tigr.com) with Massively Parallel Signature Sequencing (MPSS; mpss.udel.edu\/rice) expression data as well as Affymetrix array expression data (GEO, GSE4438; www.ncbi.nlm.nih.gov\/geo) for the rice cultivar japonica in a way similar to the analysis performed for the Arabidopsis genome (Ren et\u00a0al. 2005). The results show that the characteristics of the two genomes with respect to the occurrence and configuration of local expression domains are remarkably similar.\nAlso in the rice genome, a small yet significant fraction of genes is organized in local coexpression domains that predominantly consist of two, up to 4, genes that are not categorized in the same functional category, irrespective of the expression platform used for analyses. The presence of tandemly duplicated genes, shared promoter sequence or gene distance is not fully explaining the occurrence of coexpression of genes in such chromosomal domains. Therefore, the regulation of local coexpression domains is postulated to be at the level of higher-order of chromatin structure. Given the similarities in the characteristics and occurrence of coexpression domains between Arabidopsis and rice, we investigated whether the genes involved showed microsynteny between the two genomes. These analyses did not identify the presence of syntenic local coexpression domains between Arabidopsis and rice.\nMaterial and methods\nGenome data\nThe rice (Oryza sativa) genome was obtained from the website of The Institute of Genomic Research (TIGR; www.tigr.org). The rice TIGR version 3 [Jan. 2005] annotation has 57,915 gene loci. In case of alternative splicing, the longest variant of the gene was used. The genes along each chromosome were sorted based on ascending start coordinates and were numbered consecutively. These rank numbers (rank ID) helped to eliminate any discontinuity in the unique Os gene identifiers of the annotated genes and facilitated analyzing physically adjacent genes. In case of overlapping gene loci, the smaller one of the overlapping genes was removed from the data set. This way the order of both gene and rank ID numbers was maintained.\nExpression data\nThe MPSS expression data for rice (cv. Japonica) were obtained from the rice MPSS database (http:\/\/mpss.udel.edu\/rice). Only the unique MPSS tags (mapping to the genome only once) and those mapping to unique gene identifiers in TIGR v3 were used in the analyses. The MA expression data were obtained from the Gene Expression Omnibus repository (GEO; www.ncbi.nlm.nih.gov\/geo). The data with accession number GSE4438 were chosen for analysis, because they represent the only Affymetrix chip-based data set in the public domain with a reasonable coverage of the Oryza sativa cv. japonica transcriptome under four different experimental conditions (Walia et\u00a0al. 2007). The earlier analysis in Arabidopsis also concerned Affymetrix chip data (Ren et\u00a0al. 2005). The MA expression data were mapped to the TIGR v3 annotation based on gene accession numbers.\nThe expression values in libraries representing biological replicates, i.e., the same tissues under the same experimental conditions, were averaged. This way, for the MPSS data 18 different libraries were generated, that cover expression in 9 tissues (callus, panicle, leaves, root, germinating seed and seedling meristem, ovary and stigma, pollen, stem) under different experimental treatments or in different developmental stages. From the microarray (MA) data, only the expression data from cv. japonica were used to allow comparison with the MPSS data. Here, 4 different libraries were generated: a sensitive japonica genotype under control conditions and under salt stress; and a tolerant japonica genotype under control conditions and under salt stress (Walia et\u00a0al. 2007). The expression data is from crown and growing point tissue.\nIdentification of local coexpression domains\nPearson\u2019s correlation coefficient (R) was calculated between all adjacent pairs (duplets) of genes using the expression data from all 18 libraries. If R was higher than 0.7, the gene pair concerned was considered to be coexpressed. The value of R\u00a0>\u00a00.7 is generally considered a rule-of-thumb threshold (see for example bbc.botany.utoronto.ca\/affydb\/BAR_instructions) and is used in various analyses (Cohen et\u00a0al. 2000; Lee et\u00a0al. 2004; Ren et\u00a0al. 2005). The number of coexpressed adjacent pairs was counted. To evaluate the statistical significance of these numbers, they were compared with the number of coexpressed pairs from 100 randomizations of the population of expressed genes using the cumulative binomial distribution (Cohen et\u00a0al. 2000). Previous analyses indicated that more than 100 randomizations did not result in significant changes in the numbers obtained (Ren et\u00a0al. 2005). In each round of randomization, non-adjacent pairs of genes were randomly selected with replacement from the list of expressed genes that have expressed neighbors till the same total number of pairs was obtained. Similarly, coexpressed adjacent triplets, quadruplets and pentaplets were identified as series of genes with consecutive IDs in which all possible (that is, (n!\/(n\u22122)!)*2; where n is the number of genes involved) pair-wise R\u2019s should be above the cut-off of 0.7. The significance of results was evaluated with randomizations equivalent to the procedure used in case of duplets.\nDuplicated genes\nDuplicated genes were identified by local pair-wise protein BLAST (BLASTP 2.2.6 [Apr-09-2003]; (Altschul et\u00a0al. 1997)), on all gene pairs in the rice genome. A gene pair was considered to be duplicated (dup) if BLASTP yielded an E-value\u00a0<\u00a00.2 (Fukuoka et\u00a0al. 2004; Lercher et\u00a0al. 2003; Williams and Bowles 2004). To determine duplicated triplets, quadruplets and pentaplets, it was required that any pair of the genes concerned had a BLASTP E-value <0.2.\nAnalyses of gene orientation and gene distance\nAdjacent gene pairs were separated into tandemly, divergently and convergently transcribed pairs according to their relative direction of transcription. The number of coexpressed pairs in each orientation group was expressed as percentage relative to the total number of adjacent pairs in that group. Random pairs were made by randomly picking two non-adjacent genes from the list of expressed genes represented in pairs, analyzed for their orientation and compared with the real genome using a variant of the two-sample t test for proportions for determining the significance of a difference between two population proportions (Ott and Longnecker 2001). The test statistic is based on the z statistic from the normal distribution and is given by (p1\u2212p2)\/ \u221a (p1*(1\u2212p1)\/n1\u00a0+\u00a0p2*(1\u2212p2)\/n2), with p1 and p2 the two sample proportions, n1 and n2 the two sample sizes, under the condition that n1*p1, n1*(1\u2212p1), n2*p2 and n2*(1\u2212p2) are all larger than 5. The z value is converted to a p value using standard normal tables.\nTo determine the gene distance, the intergenic distance is used. This distance is defined as the length in nucleotides from the annotated end of one gene to the annotated start of the next gene, including the UTRs when known, otherwise the translation start and stop sites were taken. The data sets excluding the duplicated gene pairs were analyzed. For each data set, gene pairs were sorted based on gene distance from short to long and bins of 1,000 pairs were taken and analyzed, excluding the last bin with less than 1,000 pairs. The advantage of using equal pair bin is that it avoids unequal number of gene pairs in different distance categories. Per 1,000-pair bin, gene distance was calculated as the average over all 1,000 pairs. For each 1,000-pair bin, the fraction of coexpressed pairs relative to the total number of pairs in each orientation group in each bin was calculated and plotted.\nFunctional categorization of genes \nTAIR\u2019s GOslim, the Gene Ontology (GO) developed for plants (Berardini et\u00a0al. 2004) was used to classify the genes present in local coexpression domains. The three aspects of GOslim, molecular function, biological process and cellular component, were analyzed in parallel. With Python scripts, the number of pairs of which both members could be classified in GOslim was determined, and the number of pairs of which both members fall into the same well-defined GOslim category was also determined. The GOslim categories of \u2018unknown\u2019 and \u2018other\u2019 were not included into well-defined categories, because they give less (or no) information about functional categorization. The percentage of coexpressed pairs falling into the same well-defined category was compared with that of non-coexpressed pairs to determine whether coexpressed genes are more enriched in the same functional category than non-coexpressed genes.\nAssessing synteny between Arabidopsis and rice\nThe Inparanoid Eukaryotic Orthologous groups database (inparanoid.cgb.ki.se; O\u2019Brien et\u00a0al. 2005) was used to download all known orthologous and inparalogous clusters between Arabidopsis and rice. Inparanoid defines inparalogs as paralogs that arose through gene duplication after speciation. Inparalogs can form a group of genes that together are orthologous to a gene in another species. There are 9,044 orthologous clusters between Arabidopsis (from Ensemble) and rice (from the Model Organism database) and all of them were taken into account. These clusters were downloaded on Dec. 12, 2005. In the orthologous clusters, 15,544 sequences (proteins) from Arabidopsis are inparalogs and 14,807 sequences (proteins) from rice are inparalogs. More than half of Arabidopsis and rice inparalogs are many-to-many or many to-one orthology cases. Less than half of the cases are one-to-one orthology cases.\nThe Ensembl protein IDs (for Arabidopsis) and the Model organism database protein IDs (for rice) in Inparanoid were first translated to their unique gene identifiers in the respective TIGR annotation by BLASTP using an E-value\u00a0<\u00a0e\u221220. This yielded 14,753 unique Arabidopsis genes and 12,428 unique rice genes as inparalogs. The pairs of genes in local coexpression domains were analyzed to determine which genes in a rice local coexpressed pair have orthologs in an Arabidopsis local coexpressed pair, and vice versa. Because of the larger coverage of genes and tissues, only the MPSS expression data were used for this analysis. As coexpressed triplets and quadruplets are always combinations of coexpressed pairs, they were not further analyzed. For comparison, the pairs of genes that are not coexpressed were analyzed to determine how many non-coexpressed pairs, or one of their member genes, have orthologs in the other plant species. The numbers were then compared between coexpressed pairs and non-coexpressed pairs to determine the significance of occurrence of syntenic local coexpression domains.\nResults\nLocal coexpression domains consist of two to four neighboring genes\nThe TIGR version 3 of the rice genome has 57,915 predicted genes. This is about twice the number of genes predicted for Arabidopsis (28,952 genes; TIGR5 annotation). The coverage of the MPSS expression data for the rice genome is 40%. The expression coverage in the Affymetrix array data set we used is 26% (Table\u00a01). This is about half or less of the expression coverage for the Arabidopsis genome (72% in the TIGR5 update; Ren et\u00a0al. 2005). This difference can reflect the more advanced annotation of the Arabidopsis genome at this time, and\/or the more complex (duplicated) organization of the rice genome. The rice genome annotation has more genes that are physically overlapping than the Arabidopsis genome. Excluding the smaller overlapping genes from the analyses, we were able to identify 12,920 gene pairs with MPSS expression data and 6,032 pairs with MA expression data in rice (Table\u00a01; see also Materials and methods). Of these, 584 (4.5%) in MPSS and 320 (5.3%) in MA were identified to represent a local coexpression domain as defined as being coexpressed with a pair-wise Pearson\u2019s correlation coefficient larger than 0.7 (Table\u00a01). This percentage is similar to what we have found previously for Arabidopsis (Ren et\u00a0al. 2005) and agrees well with other findings that \u223c3\u20135% of a genome is tightly coexpressed (Semon and Duret 2006).\nTable\u00a01Description of rice expression data used for whole-genome local coexpression analysisMPSSMAGenes with expressionExcluding overlapping genes23,14614,789Without expressed neighbor(s)5,0815,438represented in pairs18,0659,351Adjacent pairsTotal12,9206,032Tandemly duplicated pairs (td) 1,663 (12.9%)a573 (9.5%)aCoexpressed584 (4.5%)b320 (5.3%)bTotal excluding td11,2575,459 Coexpressed excluding td 438 (3.9%)c288 (5.3%)cCoexpressed adjacent pairsTotal584320Tandemly duplicated pairs146 (25%)d32 (10%)dTandemly duplicated pairsTotal1,663573Coexpressed146 (8.8%)e32 (5.6%) eaPercentage of tandemly duplicated pairs relative to the total number of adjacent pairsbPercentage of coexpressed adjacent pairs relative to the total number of adjacent pairscPercentage of coexpressed adjacent pairs excluding td relative to the total number of adjacent pairs excluding tandemly duplicated pairsdPercentage of coexpressed tandemly duplicated pairs relative to the total number of coexpressed adjacent pairsePercentage of coexpressed tandemly duplicated pairs relative to the total number of tandem duplicated pairs\nNotably duplicated genes are supposed to influence coexpression statistics due to their common origin (Lercher et\u00a0al. 2003), although a surprising finding for the Arabidopsis coexpression domains was that only a minor fraction of duplicated genes were actually coexpressed (Ren et\u00a0al. 2005). The occurrence of duplicated pairs in the rice set was determined with pair-wise protein BLAST using a cut-off of E\u00a0<\u00a00.2 (Fukuoka et\u00a0al. 2004; Lercher et\u00a0al. 2003; Williams and Bowles 2004). This identified 1,663 (12.9%) duplicated gene pairs in the MPSS gene pair data set and 573 (9.5%) duplicated gene pairs in the MA gene pair data set.\nOf these, only 146 (8.8%) in the MPSS data and 32 (5.6%) in the MA data were coexpressed (Table\u00a01). Although this percentage is somewhat higher than the percentage of coexpression in non-duplicated pairs (3.9%), the majority of all duplicated pairs (91.2% in MPSS data and 94.4% in MA data) are not coexpressed. This shows that also in rice gene duplication does not correlate well with coexpression and suggests that expression divergence is a common phenomenon after duplication (Williams and Bowles 2004). Excluding the duplicated pairs from the coexpressed sets, there are 438 gene pairs in the MPSS data and 288 gene pairs in the MA data coexpressed in rice. This accounts for 75% (=438\/584) in MPSS and 90% (=288\/320) in MA of all coexpressed pairs. Therefore, also in rice the occurrence of duplicated genes cannot explain the occurrence of local coexpression domains. Extending the size of the local coexpression domain to triplets, quadruplets, pentaplets and on, requiring that all pair-wise combinations of genes have a tightly correlated expression, shows that few larger local coexpression domains exist (Table\u00a02). No quadruplet domains could be identified when tandemly duplicated genes were excluded (Table\u00a02). To assess the significance of the occurrence of the various local coexpression domains, we compared the number of coexpressed pairs, triplets and quadruplets with the average of such domains in 100 randomly generated genomes using the cumulative binomial distribution (Cohen et\u00a0al. 2000). Such comparisons revealed that local coexpression pairs occur in the rice genome significantly more often than expected by chance alone (Table\u00a02). However, when excluding the duplicated genes, triplets and quadruplets do not occur significantly more often than by chance (at P\u00a0<\u00a00.05) in both expression datasets. Local coexpression domains therefore consist of at most 2 genes when duplicated genes are not taken into consideration. This number appears smaller than in the Arabidopsis genome (Ren et\u00a0al. 2005), but this may reflect the lesser coverage of the annotation of the rice genome.\nTable\u00a02Local coexpression domains in the rice genomeRice genomeRandom genome (100\u00d7)TotalaCoexpressedbAveragecP-valuedPairsMPSS\u00a0+\u00a0tde12,920584 (4.52%)408\u00a0\u00b1\u00a0171.46\u00a0\u00d7\u00a010\u221217MPSS-tdf11,257438 (3.89%)356\u00a0\u00b1\u00a0212.17\u00a0\u00d7\u00a010\u22126MA\u00a0+\u00a0tdg6,032320 (5.30%)301\u00a0\u00b1\u00a0170.012MA-tdh5,459288 (5.28%)271\u00a0\u00b1\u00a0160.014TripletsMPSS\u00a0+\u00a0td7,77523 (0.30%)8.78\u00a0\u00b1\u00a02.92.95\u00a0\u00d7\u00a010\u22125MPSS-td6,83113 (0.19%)7.74\u00a0\u00b1\u00a03.00.025MA\u00a0+\u00a0td2,4615 (0.20%)6.54\u00a0\u00b1\u00a02.7n.s.MA-td2,1493 (0.14%)5.10\u00a0\u00b1\u00a02.4n.s.QuadrupletsMPSS\u00a0+\u00a0td4,8873 (0.06%)0.24\u00a0\u00b1\u00a00.471.81\u00a0\u00d7\u00a010\u22123MPSS-td4,3180 (0%)0.18\u00a0\u00b1\u00a00.39n.s.MA\u00a0+\u00a0td1,0790 (0%)0.14\u00a0\u00b1\u00a00.37n.s.MA-tdndindndndaTotal number of pairs, triplets, quadruplets in each data setbCoexpressed pairs, triplets, quadruplets in each data set. Percentages in brackets are coexpressed relative to the totalcAverage plus\/minus standard deviation from 100 randomizationsdP-value according to the cumulative binomial distribution (Cohen et al. 2000) for obtaining such a result by chance. P\u00a0<\u00a00.05 is considered significant; n.s.: not significanteMPSS data set including tandemly duplicated genesfMPSS data set excluding tandemly duplicated genesgMA data set including tandemly duplicated geneshMA data set excluding tandemly duplicated genesiNot determined\nLocal coexpression domains seem randomly distributed over the genome (Fig.\u00a01; MPSS data only). Only 10 coexpressed pairs are common between the MPSS and MA coexpressed sets, out of a total of 4,643 common pairs (excluding tandemly duplicated pairs). Detailed information about locations, orientations, expressions and gene distances (only for the pairs) for all pairs, triplets and quadruplets in both the MPSS and MA data sets are given in the supplementary data. All subsequent analyses were focused on domains consisting of non-duplicated gene pairs, unless stated differently.\nFig.\u00a01Distribution of local coexpression domains over all 12 rice chromosomes. Rectangles are schematic representation of chromosomes 1\u201312 from top to bottom. The numbers on the top show the scale in million bases along the chromosomes. Each gene in a local coexpression domain is depicted with a black bar. Only MPSS datasets excluding tandemly duplicated genes are shown. The orders of the drawings in each rectangle are: first lane, coexpressed pairs; second lane, coexpressed triplets; third lane, coexpressed quadruplets, fourth lane, partially syntenic coexpression domains (PSCDs) between Arabidopsis and rice\nOrientation and distance do not solely explain the occurrence of local coexpression\nIn yeast, there are several examples that divergently transcribed promoter regions are the cause of co-regulated neighboring genes (Korbel et\u00a0al. 2004; Kruglyak and Tang 2000). If promoter sharing is an important mechanism for coexpression in the rice genome, divergently transcribed gene pairs should be over-represented in the sub-population of coexpressed pairs, compared to coexpressed pairs that are tandemly or convergently transcribed. For all three-orientation groups, the number of pairs and the number of coexpressed pairs in the rice genome were determined (Table\u00a03). For each orientation group, the fraction of coexpressed pairs relative to the total number of pairs in that group was calculated (Table\u00a03). None of the fractions are significantly different from each other using a statistical test for comparing population proportions (Ott and Longnecker 2001). The fraction of coexpressed divergent pairs in the MPSS data is the lowest of the three groups (Table\u00a03). Therefore, shared promoter regions cannot solely explain the coexpression of adjacent genes.\nTable\u00a03Orientation of coexpressed gene pairsOrientation groupsaTotalbCoexpressedcMPSStan-td5,621239 (4.25%)div-td2,41882 (3.39%)con-td3,218117 (3.64%)MAtan-td2,707143 (5.28%)div-td1,22472 (5.88%)con-td1,52873 (4.78%)atan-td, div-td, con-td, respectively are the sub-groups of tandemly, divergently, convergently transcribed pairs excluding tandem duplicatesbTotal number of pairs in each direction groupcNumber of coexpressed pairs in each direction group. Percentages in the brackets are number of coexpressed pairs relative to the total number of pairs. None of the proportions are significantly different from each other according to the z test for comparing population proportions\nThe physically closer two genes are, the higher the likelihood is that they are coexpressed due to either cis or trans-activation (Hershberg et\u00a0al. 2005). Therefore, we determined the intergenic distance, defined as the sequence length in nucleotides from the annotated end of one gene to the annotated start of the neighboring gene, including UTRs when known, otherwise taking the start and stop site for translation. This distance was used to investigate whether it would explain the characteristics of local coexpression domains. In Fig.\u00a02, the fraction of coexpressed pairs is plotted for each orientation and for each 1000-pair bin after sorting based on intergenic distance. The results show that the fraction of coexpressed pairs, irrespective of gene orientation, does not decrease with larger gene distance. When gene distance is defined as the sequence length from the start of one gene till the start of the next gene (Ren et\u00a0al. 2005), the result is similar (data not shown). As a consequence, increasing intergenic distances do not seem to be a barrier for the occurrence of local coexpression and short intergenic distances do not favor coexpression. Therefore, intergenic distance does not solely explain local coexpression in the rice genome, as it did not in the Arabidopsis genome (Ren et\u00a0al. 2005).\nFig.\u00a02Gene distance does not solely explain the occurrence of coexpression. Gene distance, defined as the length in nucleotides from the annotated end of one gene to the annotated start of the next gene relative to the strand the genome that is given, with annotated start always smaller than the annotated end. X-axis is the averaged gene distance (in base pair) in each 1,000-pair bin. The Y-axis depicts the number of pairs (A, D), number of coexpressed pairs (B, E) and the fraction of coexpressed pairs (C, F), relative to the total number of pairs in each orientation (tan: tandem pairs; div: divergent pairs; con: convergent pairs) in each 1,000-pair bin\nFunctional categorization of coexpressed genes\nTo characterize the kind of genes that are present in the rice coexpression domains, the gene ontology (GO) developed for plants (GOslim; Berardini et\u00a0al. 2004) was used. The GOslim ontology provides a controlled vocabulary to describe gene and gene product attributes in plants, focusing on three aspects of annotation: molecular function, biological process and cellular component. Each aspect has 15\u201316 categories with 4\u20135 categories having terms like \u201cunknown\u201d or \u201cother\u201d. To all pairs of genes, each aspect of the GOslim annotation was assigned. For each aspect, the number of pairs was determined for which both member genes were covered by a GOslim assignment. In addition, the number of pairs for which both member genes fall into the same well-defined categories (excluding the \u201cunknown\u201d and \u201cother\u201d subcategories) was determined. The fraction of the latter was compared between coexpressed pairs and non-coexpressed pairs to determine whether coexpressed pairs were enriched in the same categories (Table\u00a04). In the MPSS data, the GOslim annotation coverage for both member genes in a pair is 22% for molecular function, 12% for biological process and only 3.4% for cellular component. In the MA data, these 3 percentages are: 25%, 13% and 3.7%, respectively. Comparing these figures with the GOslim coverage of the Arabidopsis genes (data from Ren et\u00a0al. 2005, but updated to the TIGR5 annotation), which is \u223c94% for all 3 aspects, shows that currently the rice genome is considerably less well annotated than the Arabidopsis genome. When comparing the coexpressed and non-coexpressed pairs in rice for the fraction of gene pairs falling into the same well-defined GOslim category, there is no significant difference (Table\u00a04). Therefore, also in the rice genome coexpressed gene pairs are not enriched for the same functional category.\nTable\u00a04Distribution of gene pairs over GOslim categories (Non-duplicated pairs)AllaCoexpressedbNon-coexpressedcP-valuedMPSS\u00a0\u00a0\u00a0\u00a0GO_func\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0coverede250210024020.42\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0sameKnCatf365 (14.6%)12 (12.0%)353 (14.7%)\u00a0\u00a0\u00a0\u00a0GO_proc\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Covered13665013160.47\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0sameKnCat144 (10.5%)7 (14%)137 (10.4%)\u00a0\u00a0\u00a0\u00a0GO_comp\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Covered383173660.60\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0sameKnCat113 (29.5%)6 (35.3%)107 (29.2%)MA\u00a0\u00a0\u00a0\u00a0GO_func\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0coverede13658312820.13\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0sameKnCatf177 (13.0%)7 (8.43%)170 (13.3%)\u00a0\u00a0\u00a0\u00a0GO_proc\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Covered707436640.13\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0sameKnCat67 (9.48%)2 (4.65%)65 (9.79%)\u00a0\u00a0\u00a0\u00a0GO_comp\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Covered202101920.24\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0sameKnCat45 (22.3%)4 (40%)41 (21.4%)aNumber of neighboring pairs excluding td. All other pairs are all duplicate-free, unless stated otherwisebNumber of coexpressed pairscNumber of non-coexpressed pairsdP value from the standard normal tables of the z statistic for the difference of the population proportion between coexpressed pairs and non-coexpressed pairs in the rice genome; *, significant (two-tailed; P\u00a0<\u00a00.05). The P value is the probability under the null hypothesis that the two population proportions are the sameeNumber of pairs of which both members are assigned (covered) with GOslim categoriesfNumber of pairs of which both members fall into the same \u201cknown\u201d GOslim category (excluding the categories with the indications \u2018unknown\u2019 and \u2018other\u2019). Percentage is the number of pairs relative to the number of pairs covered\nMicrosynteny of local coexpression domains between rice and Arabidopsis\nThe structural characteristics of local coexpression domains in rice and in Arabidopsis (Ren et\u00a0al. 2005) are remarkably similar. This prompts the question whether such domains also share functional characteristics and possibly consist of the same or related genes. Microsynteny in local expression domains of these two genomes would reflect conservation of such domains. The Inparanoid Eukaryotic Orthologous database (O\u2019Brien et\u00a0al. 2005) was used to retrieve the current list of genes that are supposed to be orthologous between Arabidopsis (14,753 genes) and rice (12,428 genes), including all many-to-many relationships. The genes establishing coexpressed pairs based on MPSS expression data in either Arabidopsis (944 pairs including 116 duplicated pairs; data from Ren et\u00a0al. 2005, but updated to the TIGR 5 annotation) or rice (584 pairs, including 146 duplicated pairs) were searched against these lists. This way, we aimed to identify the pairs of which both genes in the pair have an ortholog in the other plant and these orthologs are also coexpressed The analyses showed that there was not a single coexpressed pair in either Arabidopsis or rice of which both genes are orthologous to a gene of a coexpressed pair in the other species. Therefore, given the current annotation of the two genomes, there are no syntenic local coexpression domains between Arabidopsis and rice.\nPartially syntenic local coexpression domains can occur by chance\nIn 34 cases though, one gene of a coexpressed pair in one plant species was orthologous to at least one gene of a coexpressed pair in the other plant. That is 3.6% of all (944) coexpressed pairs in Arabidopsis and 5.8% of all (584) coexpressed pairs in rice. We will refer to such a case as a partially syntenic coexpression domain (PSCD). To assess the significance of such partially syntenic domains, we evaluated all the genes in non-coexpressed pairs, comparing Arabidopsis (15,629 pairs including 617 duplicated pairs) and rice (12,336 pairs including 1,517 duplicated pairs) to establish whether PSCDs are more enriched in the genome than partially syntenic non-coexpressed domains (PSND). We identified 4,488 PSNDs (72 due to duplicated pairs) between all non-coexpressed pairs of genes in both plant genomes. This is 28.7% of all Arabidopsis non-coexpressed pairs and 36.4% of all rice non-coexpressed pairs. The percentage of PSNDs among non-coexpressed pairs is 6\u20138 times higher than that of PSCDs from coexpressed pairs. Therefore, PSCDs do not seem to occur more often than expected by chance alone.\nA complicating issue in the analysis of synteny is the occurrence of many-to-many orthologs. The Inparanoid database defines so-called inparalogs as paralogs arising through gene duplication after speciation. These can form a group of genes that together are orthologous to a gene in the other species. As a result, there can be many to many, many to one and one to one relationships. Individual member genes in many-to-many or many-to-one relationships may not be the main orthologs. Interestingly, there is one many-to-one case in which four Arabidopsis genes are all orthologs of the same single rice gene (Os07g43560.1). These 4 Arabidopsis genes are: At4g23140.2, At4g23150.1, At4g23230.1 and At4g23270.1. The first two, At4g23140.2, At4g23150.1, form a local coexpressed pair. The other two genes, At4g23230.1 and At4g23270.1, are not more than ten genes away from the previous two genes on the same chromosomal region. The latter two genes are separated from each other by a few genes. Further analysis shows that gene At4g23270.1 has a duplicated neighbor, At4g23280.1, but is not coexpressed with it. It is, however, coexpressed with its other neighbor At4g23260.1, but is not duplicated with it. Orthology is established between At4g23270.1 and the rice gene Os07g43560.1, but not between any of the neighbors of the Arabidopsis genes. The rice gene Os07g43560.1 is also coexpressed with one of its neighboring genes Os07g43540.1, but it is not a duplicate of it, while this rice gene itself is a duplicate of another neighboring gene (Os07g43570), but it is also not coexpressed with it. A schematic representation of the resulting gene configuration is given in Fig.\u00a03. Such detailed analyses may reveal local microsynteny in the twilight zone of statistical significance and evolutionary relevance.\nFig.\u00a03Schematic representation of the chromosomal regions covering genes involved in a four-to-one orthology between Arabidopsis and rice. Top part of the figure is the chromosomal region from rice (from gene locus Os07g43540.1 to gene locus Os07g43570.1). Bottom part of the figure is the chromosomal region from Arabidopsis, representing 23 genes (from gene locus At4g23120 to At4g23340; the numbers in the picture do not carry \u201cAt4g\u201d). Black arrows represent the four Arabidopsis and the one rice gene involved in this orthology, and dashed curved connecting lines show the orthology relationships. Black bracket-like lines depict duplication and genes connected and included within by black bracket line are duplicated to each other. Dotted lines depict coexpression relationship and genes connected and included by dotted line are coexpressed with each other\nDiscussion\nLocal coexpression domains represent only a small part of the genome\nSetting stringent criteria for coexpression using MPSS and microarray expression data, the rice genome was found to contain a small but significant number of local coexpression domains that range from predominantly two, up till 4, genes, irrespective of the expression platform used for analysis. This is similar to the situation in Arabidopsis (Ren et\u00a0al. 2005). It shows that a genome is essentially not a random entity with respect to the occurrence of local coexpression domains. Our results agree with other coexpression studies where strong coexpression was shown to occur only within close proximity of several genes (Cohen et\u00a0al. 2000; Hershberg et\u00a0al. 2005; Lercher et\u00a0al. 2003; Semon and Duret 2006). Although coexpression was shown to extend to regions covering up to 30 genes and more (Spellman and Rubin 2002) and to cover chromosomal regions up to 100\u00a0kb (Ma et\u00a0al. 2005; Spellman and Rubin 2002; Williams and Bowles 2004) and more, there appears to exist a decrease in the strength of coexpression with increasing distances. The local coexpression domains described here represent \u223c4\u20135% of the potential coexpression fraction in the whole genome as found in other studies (Semon and Duret 2006). Larger but looser coexpression domains might cover up to \u223c10% (Cohen et\u00a0al. 2000; Williams and Bowles 2004) till 20% (Spellman and Rubin 2002) of the genome. The difference in occurrence between local and longer-range weaker but still statistically significant coexpression domains is highly dependent on the method used (Semon and Duret 2006). Moreover, the expression platforms used focus on snapshots of gene expression at the RNA level. Such data ignore various post-transcriptional regulatory mechanisms that can for example result in poor correlations between RNA and protein levels. Although we have shown before that in a transgenic set-up the creation of an artificial local coexpression domain results in markedly improved RNA\/protein correlations (Mlynarova et\u00a0al. 2002), this does not need to be the case for all endogenous local coexpression domains now identified.\nThe terms cluster or chromosomal domain and associated terms such as neighboring are generally based on a (much) more loose definition compared to the definition used here to identify local coexpression domains. Local coexpression domains require a pair-wise correlation between the expressions of ALL adjacent genes above 0.7. The larger domains are defined on the basis of the use of a sliding window of either a given sequence length (number of nucleotides) or of a given number of genes (Spellman and Rubin 2002; Williams and Bowles 2004). In such a window, the average correlation is calculated and compared with simulated sets. This allows for the presence of genes within a domain that are not strongly (co)expressed but are \u201ccarried along for a ride\u201d in the open chromatin domain (Spellman and Rubin 2002).\nThe two expression data sets here analyzed show very little overlap in coexpressed pairs (only 10 out of 4,643 all common pairs). The low number of shared pairs is thought to reflect the biological background of the data sets. The MPSS data concern a broad range of tissues and experimental conditions, while the MA data only cover crown and growing point tissue under control and salt stress condition (Walia et\u00a0al et\u00a0al. 2007). The number of libraries taken into account, as well as their biological background, obviously influences the possibility of identifying significant numbers of coexpression domains above the number of domains expected by chance. Two genes evaluated in four comparably similar conditions (MA data) are predicted to have a higher likelihood of exhibiting a similar expression pattern, than when examined in 18 different (MPSS) conditions. When considering genome-wide local coexpression of genes, a wide diversity of tissues and\/or conditions should be taken for analysis. Highly tissue-specific coexpression may be masked in this approach and should be analyzed by other means.\nParameters shaping local coexpression domains\nThe existence of local coexpression domains in rice could not be explained solely by gene orientation, such as tandemly, divergently or convergently oriented gene pairs. No relative enrichment of the proportion of coexpressed pairs was seen. The fraction of coexpressed genes in the divergent orientation was even lower than for the other two orientations (Table\u00a03). So shared promoter regions (for divergent pairs) and transcriptional read-through (for tandem pairs) do not explain the local coexpression domains in rice, similar to what we have concluded for Arabidopsis (Ren et\u00a0al. 2005). This is in contrast to some other studies in which shared promoter region (for divergent pairs) and transcriptional read-through established coexpression domains (Semon and Duret 2006). Whereas we do not detect any preferred orientation to result in coexpression in rice, other studies show a higher degree of coexpression in divergent and tandemly oriented gene pairs (Williams and Bowles 2004; Zhan et\u00a0al. 2006). The differences in conclusions are most likely due to the different methods used, such as the definition of coexpression of neighboring genes as well as the dataset and\/or expression platform used.\nGene distance is not the explanatory factor for the occurrence of local coexpression domains in rice. No significant decrease in the fraction of coexpressed genes was observed with increasing intergenic distance (Fig.\u00a02). The fraction of coexpressed pairs does not decrease even with gene distances up to 12\u00a0kb. It shows that at a relatively large distance, neighboring genes can still be coexpressed. Another study reported that when genes >12\u00a0kb apart were taken into account, the negative correlation between coexpression and gene distance was gone (Williams and Bowles 2004). In a comparative study of 6 eukaryotic genomes, coexpression was shown to vary at chromosomal distances above 100\u00a0kb (Fukuoka et\u00a0al. 2004). This suggests that considerable coexpression of neighboring genes can occur even at large gene distance, although the coexpression may not be related to the physical distance anymore. While gene distance itself is not predictive for coexpression (Cohen et\u00a0al. 2000; Kruglyak and Tang 2000), the likelihood of coexpression would favor short gene distances (Hershberg et\u00a0al. 2005; Hurst et\u00a0al. 2002; Lercher et\u00a0al. 2003; Semon and Duret 2006). It should be kept in mind that the rice data set now analyzed is far from complete in terms of its annotation, so what are now far-apart neighboring genes may be no longer directly neighboring the moment the annotation is improved. Because the characteristics of the local coexpression domains in Arabidopsis and rice are remarkably alike, the actual numbers and possibly even genes involved in local coexpression domains may be subject to change, but not the occurrence of such domains in these genomes.\nWith the gene ontology developed for plants (GOslim), there is no evidence that coexpressed genes are more enriched in the same functional category in comparison to non-coexpressed genes (Table\u00a04). Previous studies suggested that clustering of functionally related genes would occur in all metazoans (Cohen et\u00a0al. 2000; Lercher et\u00a0al. 2003). Recent studies demonstrated a significant enrichment for coexpressed genes in the same metabolic pathway (Williams and Bowles 2004) or the same biological processes (Zhan et\u00a0al. 2006), although this appeared not to be the explanation for the coexpression of neighboring genes (Williams and Bowles 2004). In worm, clusters of similarly expressed genes cover similar biological functions (Roy et\u00a0al. 2002). In human, coexpression over the whole genome was shown to correlate with functional relationships between the genes (Lee et\u00a0al. 2004). Our study found no enrichment of coexpressed gene pairs in the same functional category than non-coexpressed pairs, suggesting that it is not necessarily true that the natural selection maintained regional coexpression by keeping genes with similar functions in adjacent positions (Cohen et\u00a0al. 2000; Semon and Duret 2006).\nThe genomic context of genes is supposed to play an important role in the regulation of gene expression (van Drunen et\u00a0al. 1997). In a number of coexpression studies in various organisms, the occurrence of coexpression domains, whether small (local) or larger (global), sometimes independent of gene orientation and gene distance, were all supposed to be regulated at the level of higher-order chromosomal structure (Cohen et\u00a0al. 2000; Hershberg et\u00a0al. 2005; Ren et\u00a0al. 2005; Spellman and Rubin 2002; Williams and Bowles 2004; Zhan et\u00a0al. 2006). It will be difficult to formally exclude the possibility of transcriptional regulation as a cause of local coexpression. Co-regulated transcription could occur through shared promoter elements between the neighboring genes in coexpression domains. However, previous experience with transgene expression data indicated that the particular position of neighboring genes in a genome affects the expression of that gene considerably (Mlynarova et\u00a0al. 1994, 1995). Two physically neighboring transgenes could only be made to into an artificial local expression domain, that is, show correlated expression, when chromatin-organizing elements were placed around the genes (Mlynarova et\u00a0al. 2002). Although this experimental result indicates the importance of chromatin organization in establishing local coexpression in a plant genome, this result does not need to be the case for all coexpression domains identified. With this caveat, this study of local coexpression domains in rice that are independent of duplication, gene orientation, or gene distance strengthen the notion that the regulation of genes in such domains resides at the level of higher-order chromatin structures. Future studies using for example advanced fluorescence in\u00a0situ hybridization (FISH) technology on interphase chromosomes (Walter et\u00a0al. 2006), or the transgenic approach outlined above (Mlynarova et\u00a0al. 2002), will generate further experimental support for the location and characteristics of local coexpression domains. In addition, the new approaches in and insights from epigenetics and epigenomics (Henderson and Jacobsen 2007), such as the genome-wide mapping and analysis of DNA methylation (Zhang et\u00a0al. 2006; Zilberman et\u00a0al. 2007) and future histone modification maps (Esteller 2007) of plants, possibly in combination with genetics (Jansen and Nap 2001), will be useful to get more insight into the occurrence and function of local coexpression domains in plant genomes.\nLack of microsyntenic coexpression \nFrom an evolutionary point of view, syntenic regions between species reveal genes for conserved and important traits. Macrosynteny is generally not easily detectable after a long evolutionary time, as colinearity erodes by various mechanisms, such as transposon activity, intra or inter-chromosomal rearrangements, duplications, translocations, inversions and\/or individual divergence after speciation (Salse et\u00a0al. 2002). While macrosynteny may not be detectable any more for genomes that diverged more than 100\u00a0million years ago (mya), microsynteny, i.e., conservation of local gene order and orientation, may still exist and be informative (Devos et\u00a0al. 1999; Salse et\u00a0al. 2002). Arabidopsis and rice are thought to have diverged about 120\u2013200\u00a0mya (Salse et\u00a0al. 2002). Microsyntenic local coexpression domains between Arabidopsis and rice would indicate the importance of the evolutionary conservation of regulatory systems beyond sequence similarity after the divergence of dicotyledonous and monocotyledonous plants. Analyses show that there is not a single coexpressed pair in either Arabidopsis or rice of which both genes are orthologous to a gene in a coexpressed pair in the other species. Therefore, there are no syntenic local coexpression domains between Arabidopsis and rice. Although the analyses were performed for only one monocot and one dicot and should be extended to many more genomes, the results could be taken to suggest that maintenance of coexpression has not been an important driving force in genome conservation during or after the divergence of dicotyledonous and monocotyledonous plants. Although individual genes in local coexpression domains in either rice or Arabidopsis may have an ortholog in the other species, establishing so-called partially syntenic coexpression domains (PSCDs), this does not seem to occur above chance in the context of whole-genome configurations. Without statistical significance, the occurrence of such PSCDs is unlikely to have any evolutionary relevance on a genome-wide scale. Detailed analyses of individual cases and gene locations may suggest the occurrence of local microsynteny and point to chains of evolutionary events in which the conservation of coexpression could be involved. However, more detailed studies are required to assess the functional relevance, if any, of such genomic constitutions.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material.\n(XLS 231 KB)","keyphrases":["coexpression domain","rice","microsynteny","arabidopsis","higher-order chromatin structures"],"prmu":["P","P","P","P","P"]}
{"id":"Eur_Radiol-2-2-1705545","title":"Accuracy of dual-source CT coronary angiography: first experience in a high pre-test probability population without heart rate control\n","text":"The aim of this study was to assess the diagnostic accuracy of dual-source computed tomography (DSCT) for evaluation of coronary artery disease (CAD) in a population with extensive coronary calcifications without heart rate control. Thirty patients (24 male, 6 female, mean age 63.1\u00b111.3 years) with a high pre-test probability of CAD underwent DSCT coronary angiography and invasive coronary angiography (ICA) within 14\u00b19 days. No beta-blockers were administered prior to the scan. Two readers independently assessed image quality of all coronary segments with a diameter \u22651.5 mm using a four-point score (1: excellent to 4: not assessable) and qualitatively assessed significant stenoses as narrowing of the luminal diameter >50%. Causes of false-positive (FP) and false-negative (FN) ratings were assigned to calcifications or motion artifacts. ICA was considered the standard of reference. Mean body mass index was 28.3\u00b13.9 kg\/m2 (range 22.4\u201336.3 kg\/m2), mean heart rate during CT was 70.3\u00b114.2 bpm (range 47\u2013102 bpm), and mean Agatston score was 821\u00b1904 (range 0\u20133,110). Image quality was diagnostic (scores 1\u20133) in 98.6% (414\/420) of segments (mean image quality score 1.68\u00b10.75); six segments in three patients were considered not assessable (1.4%). DSCT correctly identified 54 of 56 significant coronary stenoses. Severe calcifications accounted for false ratings in nine segments (eight FP\/one FN) and motion artifacts in two segments (one FP\/one FN). Overall sensitivity, specificity, positive and negative predictive value for evaluating CAD were 96.4, 97.5, 85.7, and 99.4%, respectively. First experience indicates that DSCT coronary angiography provides high diagnostic accuracy for assessment of CAD in a high pre-test probability population with extensive coronary calcifications and without heart rate control.\nIntroduction\nRecent advances in multi-detector-row computed tomography (CT) technology have continuously improved the quality of non-invasive coronary artery imaging. As a result, various studies have demonstrated a high accuracy of coronary angiography with 64-slice CT for the diagnosis of coronary artery disease (CAD) [1\u20138]. In particular, the high negative predictive value has made non-invasive coronary angiography using 64-slice CT a modality that allows significant coronary stenoses to be reliably excluded. Consequently, the Task Force on the Management of Stable Angina Pectoris of the European Society of Cardiology has recently recommended in their guidelines that CT coronary angiography be performed in patients with stable angina who have a low pre-test probability of CAD, and an inconclusive exercise electrocardiogram (ECG) or stress imaging test [9].\nTo date, many studies have been published assessing the diagnostic performance of CT coronary angiography with different generations of scanners. Four-slice CT coronary angiography showed limited resolution and required long breath-hold times of nearly 40\u00a0s, resulting in a high number of vessel segments that could not be assessed [10]. Sixteen-slice CT coronary angiography showed improved diagnostic accuracy because of reduced breath-hold times and an increased temporal and spatial resolution [11, 12]. Sixty-four-slice CT scanners were the first to allow highly accurate identification of significant coronary artery stenoses [1\u20138]. However, data analysis in several of these studies could not be performed in all coronary artery segments, with a percentage of non-assessable segments of up to 12% [2]. The main reasons were severe coronary artery wall calcifications and motion artifacts, the latter occurring especially with higher heart rates. Calcifications are known to complicate the assessment of coronary arteries [10], however, some authors have suggested that substantial coronary calcification should not be used as a reason to defer CT coronary angiography [13]. \nIn order to reduce motion artifacts, the administration of either intravenous or oral beta-blocker medication for heart rate control has been proposed even for 64-slice CT systems [2\u20136, 8, 11, 14]. In most studies, target heart rates for scanning were below 60\u201365 beats per minute (bpm). Another concept to comply with higher coronary velocities at higher heart rates is the use of multi-segment reconstruction algorithms that combine data from two or more consecutive cardiac cycles to improve temporal resolution at certain heart rates [15]. However, the latter approach relies on a complete periodicity of heart motion, limiting its use in patients with variable heart rates; does not take into account the inter-heartbeat variability of coronary artery position; and requires low pitch factors, prolonging data acquisition times [16]. In line with this, a recent 64-slice CT study demonstrated no significant overall benefit of multi-segment reconstruction with regard to image quality of coronary arteries [17].\nThe recently introduced dual-source CT (DSCT) scanner is characterized by two x-ray tubes and two corresponding detectors mounted onto the rotating gantry with an angular offset of 90\u00b0 [18]. Regarding cardiac imaging capabilities, the new scanner system offers a high temporal resolution of 83\u00a0ms in a mono-segment reconstruction mode. Temporal resolution is independent of the heart rate, which is a major difference from single-source CT systems that rely on multi-segment reconstruction techniques. The first feasibility studies have shown promising results with DSCT coronary angiography regarding image quality of coronary arteries, cardiac valves, and left ventricular myocardium independent of the actual heart rate of the patient [19, 20]. However, to date no study has investigated the diagnostic accuracy of DSCT coronary angiography for the diagnosis of CAD.\nThe purpose of our study was to determine the performance of DSCT coronary angiography in diagnosing significant stenoses in comparison to invasive coronary angiography (ICA) in a high pre-test probability patient population without heart rate control.\nMaterials and methods\nPatients\nIn 30 patients (6 women, 24 men; mean age 63.1\u00b111.3\u00a0years; age range 35\u201386\u00a0years) who had undergone ICA for evaluation of suspected CAD, CT coronary angiography was performed within 30\u00a0days of catheterization (mean 14\u00b19\u00a0days). Exclusion criteria for DSCT were allergy to iodine-containing contrast medium, renal insufficiency (creatinine level>120\u00a0\u03bcmol\/L), pregnancy, hemodynamic instability, and previous stent graft placement or bypass surgery. Symptoms and cardiovascular risk factors of all patients are reported in Table\u00a01. Based on a previously published clinical pre-test score [21], each patient in this population had a high pre-test probability of CAD. Patients with elevated or irregular heart rates were not excluded from the study. The study protocol was approved by our local ethics committee and all participating patients gave written informed consent. \nTable\u00a01Synopsis of cardiovascular risk factors and symptomsFrequency (%)Risk factorsFamily history16 (53%)Obesity23 (77%)Dyslipidemia18 (60%)Diabetes19 (63%)Smoking25 (83%)Hypertension23 (77%)SymptomsAngina pectoris21 (70%)Probable angina pectoris7 (23%)Atypical chest pain2 (7%)\nDual-source CT scan protocol\nAll CT examinations were performed on a DSCT scanner (Somatom Definition, Siemens Medical Solutions, Forchheim, Germany). The patients were centrally placed in the scanner to ensure that the entire heart was covered with the smaller field-of-view of the second tube detector array. Irrespective of the individual heart rate and the heart rate variability, no beta-blockers were given prior to the scan. Three patients took oral beta-blockers as part of their baseline medication.\nNonenhanced DSCT for calcium scoring was performed from 1\u00a0cm below the level of the tracheal bifurcation to the diaphragm in a cranio-caudal direction using the following scanning parameters: detector collimation 32\u00d70.6\u00a0mm, slice acquisition 64\u00d70.6\u00a0mm by means of a z-flying focal spot, gantry rotation time 330\u00a0ms, pitch of 0.2\u20130.39 adapted to the heart rate, tube current 80\u00a0mAs per rotation, and tube potential 120\u00a0kV.\nThereafter, all patients received a single dose of 2.5\u00a0mg isosorbiddinitrate s. l. (Isoket, Schwarz Pharma, Monheim, Germany) [22]. After 2\u00a0min, the coronary angiography scan was started by continuously injecting a bolus of 80\u00a0ml of iodixanol (Visipaque 320, 320\u00a0mg\/mL, GE Healthcare, Buckinghamshire, UK) followed by 30\u00a0ml saline solution into an antecubital vein via an 18-gauge catheter (injection rate 5\u00a0mL\/s). Contrast agent application was controlled by bolus tracking. A region of interest (ROI; mean diameter 10.1\u00b15.6\u00a0mm, range 7.5\u201317.0\u00a0mm) was placed into the aortic root, and image acquisition started 5\u00a0s after the signal attenuation reached the predefined threshold of 100 Hounsfield units (HU). Data acquisition was performed from 1\u00a0cm below the level of the tracheal bifurcation to the diaphragm in a cranio-caudal direction with a detector collimation of 32\u00d70.6\u00a0mm, slice acquisition 64\u00d70.6\u00a0mm by means of a z-flying focal spot, gantry rotation time 330\u00a0ms, pitch of 0.2\u20130.43 adapted to the heart rate, tube current 400\u00a0mAs per rotation, and tube potential 120\u00a0kV. ECG-pulsing for radiation dose reduction was applied in all patients. At mean heart rates below 60\u00a0bpm, full tube current was applied from 60 to 70%, at 61\u201370\u00a0bpm from 50 to 80%, and at heart rates above 70 from 30 to 80% of the RR-interval. The electrocardiogram (ECG) was digitally recorded during data acquisition and was stored with the unprocessed CT dataset.\nDual-source CT image reconstruction\nA retrospective gating technique was used to synchronize the data reconstruction with the ECG signal. A mono-segment reconstruction algorithm that uses the data from a quarter rotation of both detectors was used for image reconstruction [18]. In each patient, images were first reconstructed at 60 and 70% of the RR-interval. If considered necessary, additional images were reconstructed in 5% steps of the RR interval within the full tube current window. In case of irregular heart rates, the temporal variability in the reconstruction phase was compensated by manual ECG editing. In case of premature heart beats, the temporal window past the premature heart beat was deleted, and the next diastolic window was filled with one to three temporal windows to avoid data gaps. All ECG editing was performed by one experienced cardiovascular radiologist not involved in data read-out. For calcium scoring, non-overlapping images with a slice width of 3\u00a0mm were reconstructed using a medium-sharp convolution kernel (B35f). For DSCT coronary angiography, images were reconstructed from the contrast-enhanced DSCT scan with a slice thickness of 0.75\u00a0mm, a reconstruction increment of 0.5\u00a0mm, and using a medium soft-tissue convolution kernel (B26f). Depending on the individual anatomy, the reconstructed field-of-view (FoV) was adjusted to encompass the heart exactly (mean FoV 167\u00b119\u00a0mm; range 131\u2013187\u00a0mm; image matrix 512\u00d7512 pixels). After removing patient and ECG information, all reconstructed images were transferred to a dedicated workstation (Wizard, Siemens Medical Solutions) equipped with dedicated cardiac post-processing software (Syngo Circulation, Siemens Medical Solutions).\nDual-source CT data analysis\nThe mean Agatston score was calculated for each patient from the non-enhanced DSCT data with a detection threshold of 130\u00a0HU by using semi-automated software (Syngo Calcium Scoring, Siemens Medical Solutions).\nFor analysis of DSCT coronary angiography data, coronary arteries were segmented according to the guidelines of the American Heart Association [23]. The right coronary artery (RCA) was defined to include segments 1\u20134, the left main and left anterior descending artery (LM-LAD) to include segments 5\u201310, and the left circumflex artery (LCX) to include segments 11\u201315. The intermedial artery was designated as segment 16, if present. All segments with a diameter of at least 1.5\u00a0mm at their origin were included. Diameter measurements were performed with an electronic caliper tool. Segments distal to an occluded vessel were excluded from analysis.\nAll reconstructed images were evaluated and classified by two independent readers using axial source images, multi-planar reformations (MPR), and thin-slab maximum intensity projections (MIP) on a per-segment basis. Both readers semi-quantitatively assessed the image quality of each coronary segment on a four-point ranking scale as previously published [1]: 1, excellent (no artifacts, unrestricted evaluation); 2, good (minor artifacts, good diagnostic quality); 3, adequate (moderate artifacts, still acceptable and diagnostic), and 4, not assessable (severe artifacts impairing accurate evaluation). Causes of image degradation were noted by both observers as arterial wall calcifications, motion artifacts, or others. Motion artifacts were defined as any impairment of image quality caused by vessel movement resulting in blurred or doubled vessel contours. Other reasons included low vessel opacification, low signal-to-noise ratio, and disturbing adjacent structures such as contrast-enhanced cardiac veins.\nIn addition, both readers assessed all coronary artery segments for the presence of hemodynamically significant stenoses. Significant stenosis was defined as narrowing of the coronary luminal diameter exceeding 50%. The vessel diameters were measured on reconstructions perpendicularly oriented to the vessel course.\nFor any disagreement in data analysis, consensus agreement was achieved.\nInvasive coronary angiography\nICA was performed according to standard techniques, and multiple views were stored on a CD-ROM. The angiograms were evaluated by one experienced observer who was blinded to the results of the DSCT coronary angiography. Coronary artery segments were defined according to the same guidelines mentioned above [23]. Each vessel segment was scored as being significantly stenosed, defined as a diameter reduction of >50% or not. Coronary artery analysis was performed in all vessels with a luminal diameter of at least 1.5\u00a0mm, excluding those vessels distal to complete occlusions.\nStatistical analysis\nStatistical analysis was performed using commercially available software (SPSS 12.0, SPSS, Chicago, IL, USA). Quantitative variables were expressed as mean\u00b1SD and categorical variables as frequencies or percentages. In a subanalysis, patients were subdivided into mean heart rates of <70\u00a0bpm and \u226570\u00a0bpm and into Agatston scores of <400 and \u2265400. We took into account the clustered nature of the data (i.e., the fact that there were not 420 independent vessel segments but instead clusters of segments in 30 patients). Inter-observer agreements for image quality read-out and assessment of significant coronary artery stenoses were interpreted according to the guidelines of Landis and Koch [24] using the clustered data. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated from chi-squared tests of contingency, and the 95% confidence intervals (CI) were calculated from binomial expression on a per-segment basis. Because of the interdependencies between different segments, the statistics were also calculated on a per-patient basis (presence of at least one significant coronary artery stenosis or absence of any significant stenosis in each patient). ICA was considered the standard of reference.\nResults\nDSCT and ICA were successfully performed in all patients without complications. The DSCT protocol was well tolerated by all patients, and all were able to hold their breath during data acquisition. The average HR during scanning was 70.3\u00b114.2\u00a0bpm (range 47\u2013102\u00a0bpm). Seventeen patients (56.7%) had a heart rate below 70\u00a0bpm (mean 59.7\u00b15.9\u00a0bpm, range 47\u201366\u00a0bpm), while 13 patients (43.3%) had a heart rate of \u226570\u00a0bpm (mean 84.2\u00b18.4\u00a0bpm, range 72\u2013102\u00a0bpm).\nPrevalance of coronary artery stenosis and calcium score\nA total of 56 coronary artery stenoses with a luminal narrowing of more than 50% in diameter were identified in 15 patients (50.0%) using ICA. Single-vessel disease was present in 13.3% (4\/30), two-vessel disease in 10.0% (3\/30), and three-vessel disease in 26.7% (8\/30). Significant coronary artery stenoses could be excluded in 15 patients (50.0%).\nCalcified vessel wall deposits were present in 24 patients (80%). Fourteen of these patients (58.3%) had significant coronary artery stenoses, while 10 patients (41.7%) had calcifications without significant stenoses. The mean Agatston score was 821\u00b1904 (range 0\u20133,110). Agatston score was <400 in 15 patients (50%, mean score 85\u00b1118) and \u2265400 in the other 15 patients (50%, mean score 1,483\u00b1893).\nImage quality and image reconstruction intervals with DSCT\nA total of 420 coronary artery segments with a diameter \u22651.5\u00a0mm were evaluated (11 segments were missing because of anatomical variants, 26 segments had a diameter less than 1.5\u00a0mm at their origin, and 9 segments were distal to an occluding stenosis). An intermedial artery was present in 16 patients (53.3%). Inter-observer agreement for image quality rating using clustered data was moderate (kappa=0.68). The initially used reconstruction time-points of 60 and 70% provided excellent image quality (score 1) in 21.9% (92\/420) and 60.2% (253\/420), respectively, while additional reconstructions were necessary in 17.9% (75\/420) to improve image quality. Whereas in the subgroup of patients with heart rates \u226570\u00a0bpm additional reconstructions at 30 to 50% were considered necessary in 27.4% (48\/175), in patients with heart rates <70\u00a0bpm, the 60 and 70% interval provided excellent image quality (score 1) in 90.2% of the segments [30.2% (74\/245) and 60.0% (147\/245), respectively].\nUsing the individual reconstruction interval with best image quality for each segment, image quality was excellent (score 1) in 47.4% of the coronary segments (199\/420), good (score 2) in 37.9% (159\/420), adequate (score 3) in 13.3% (56\/420), and poor\/not assessable in 1.4% (6\/420). Non-assessable (score 4) coronary artery segments (segment 4, n=2; segment 8, n=1; segment 10, n=2; and segment 14, n=1) were present in three patients with heart rates of 72, 77, and 86\u00a0bpm, and Agatston scores of 30, 616, and 0, respectively.\nIn the 221 segments with image quality rated other than excellent (score 1), reasons for impaired image quality were arterial wall calcifications in 48.9% (108\/221), motion artifacts in 46.6% (103\/221), or other in 4.5% of segments (10\/221; low signal-to-noise ratio, n=5; low vessel opacification, n=4; overlaying adjacent structures, n=1). At heart rates \u226570\u00a0bpm, minor to moderate motion artifacts accounted for 68.2% (73\/107) of image quality impairment, while in patients with an Agatston score \u2265400, severe arterial wall calcifications were mainly responsible for decreased image quality [76.4% (94\/123)].\nDiagnostic accuracy of DSCT in comparison to ICA\nThe kappa value for coronary artery stenosis detection with DSCT was 0.83 indicating a good inter-observer agreement. DSCT coronary angiography correctly recognized 54 of the 56 significant stenoses (96.4%) detected with ICA. For both readers, nine false-positive (FP) and two false-negative (FN) ratings occurred with DSCT coronary angiography. Causes of FP and FN ratings were massive calcifications in nine segments (eight FP, one FN) and motion artifacts in two segments (one FP, one FN). The two FN ratings occurred in segments 8 and 9. Examples of three patients with suspicion of CAD who underwent DSCT coronary angiography are provided in Figs.\u00a01, 2, and 3.\nFig.\u00a01a, bDual-source CT coronary angiography in a 62-year-old woman with suspected coronary artery disease (mean heart rate during scanning 76\u00a0bpm, Agatston score 0). a Curved thin-slab maximum-intensity projections through the centerline of the right coronary (RCA), left anterior descending (LAD), and left circumflex artery (LCX). Slight blurring of the mid-RCA and mid-LAD rendered image quality as good (score 2) in these segments, while image quality was rated excellent (score 1) in all other segments. Coronary artery disease could be reliably excluded in this patient. b Volume-rendered image of the left coronary arteries and of the proximal RCA (insert) demonstrates accurate depiction of the coronary artery treeFig.\u00a02a, bDual-source CT coronary angiography in a 69-year-old man with suspected coronary artery disease (mean heart rate during scanning 77\u00a0bpm, Agatston score 1,316). a Curved-planar reconstruction of the right coronary artery demonstrates high-grade coronary artery stenosis of the proximal segment (arrow) and non-significant stenoses of the mid and distal segments (arrowheads). b Invasive coronary angiography in a left anterior oblique projection (45\u00b0) confirms significant stenosis of the proximal segment of the right coronary artery (arrow) and non-significant stenoses of the mid and distal segments (arrowheads)Fig.\u00a03a, bDual-source CT scan in a 73-year-old man with suspected coronary artery disease (mean heart rate during scanning 86\u00a0bpm, Agatston score 259). a Thin-slab maximum-intensity projection shows stenosis of the distal right coronary artery near the origin of the posterior descending artery. This stenosis was rated significant (i.e., >50% luminal diameter narrowing) by both readers. b Invasive coronary angiography in a right anterior oblique projection shows irregular luminal narrowing in the distal right coronary artery that was qualitatively graded as less than 50% luminal diameter narrowing, resulting in a false positive diagnosis in DSCT coronary angiography\nOn a per-segment analysis, overall sensitivity was 96.4% (54\/56; 95% CI: 87.7\u201399.6), specificity was 97.5% (355\/364; 95% CI: 95.4\u201398.9), positive predictive value was 85.7% (54\/63; 95% CI: 74.6\u201393.3), and negative predictive value was 99.4% (355\/357; 95% CI: 98.0\u201399.9). Table\u00a02 summarizes demographic data, overall image quality, and diagnostic accuracy of DSCT. \nTable\u00a02Demographic data, overall image quality, and diagnostic accuracy\u00a0\u00a0Mean heart rateAgatston score\u00a0Total<70\u00a0bpm\u226570\u00a0bpm<400\u2265400No. of patients3017131515Age (years)63.1\u00b111.363.2\u00b110.162.9\u00b113.362.8\u00b113.763.4\u00b18.9Male\/female24\/615\/29\/410\/514\/1BMI (kg\/m2)28.3\u00b13.928.9\u00b14.327.6\u00b13.528.1\u00b13.528.5\u00b14.4Mean heart rate (bpm)70.3\u00b114.259.7\u00b15.984.2\u00b18.470.6\u00b113.770.0\u00b115.1Agatston score821\u00b1904901\u00b1991674\u00b178085\u00b11181,483\u00b1893Overall image qualitya1.68\u00b10.751.60\u00b10.731.81\u00b10.771.59\u00b10.751.79\u00b10.75\u00a0Score 147.4% (199\/420)51.8% (131\/245)38.9% (68\/175)54.2% (116\/214)40.3% (83\/206)\u00a0Score 237.9% (159\/420)32.4% (82\/245)44.0% (77\/175)35.5% (76\/214)40.3% (83\/206)\u00a0Score 313.3% (56\/420)11.9% (30\/245)14.9% (26\/175)7.5% (16\/214)19.4% (40\/206)\u00a0Score 41.4% (6\/420)0.8% (2\/245)2.2% (4\/175)2.8% (6\/214)\u2013Sensitivity96.4% (54\/56)97.2% (35\/36)95.0% (19\/20)100% (5\/5)96.1% (49\/51)\u00a0\u00a095% CI87.7\u201399.685.5\u201399.975.1\u201399.947.8\u201310086.5\u201399.5Specificity97.5% (355\/364)97.1% (203\/209)98.0% (152\/155)99.5% (208\/209)94.8% (147\/155)\u00a095% CI95.4\u201398.994.2\u201398.894.5\u201399.697.4\u201310090.1\u201397.8PPV85.7% (54\/63)85.4% (35\/41)86.4% (19\/22)83.3% (5\/6)86.0% (49\/57)\u00a095% CI74.6\u201393.370.8\u201394.465.1\u201397.135.9\u201399.674.2\u201393.7NPV99.4% (355\/357)99.5% (203\/204)98.8% (152\/153)100% (208\/208)98.7% (147\/149)\u00a095% CI98.0\u201399.997.3\u201310096.4\u201310098.2\u201310095.2\u201399.8aApplied scores were 1 excellent (no artifacts), 2 good (minor artifacts, good image quality), 3 adequate (moderate artifacts, acceptable image quality), and 4 not assessable (severe artifacts impairing image evaluation). A score of 1\u20133 was considered acceptable for diagnosis.BMI Body mass index, CI confidence interval, PPV positive predictive value, NPV negative predictive value\nOn a per-patient analysis, sensitivity was 93.3% (14\/15; 95% CI: 68.1\u201399.8), specificity was 100% (15\/15; 95% CI: 78.2\u2013100), positive predictive value was 100% (14\/14; 95% CI: 76.8\u2013100), and negative predictive value was 93.8% (15\/16; 95% CI: 69.8\u201399.8).\nIn both heart-rate subgroups, diagnostic accuracy for the assessment of coronary artery stenosis was similar and the rate of false ratings was comparable (one FN and six FP at heart rates <70\u00a0bpm; one FN and three FP at heart rates \u226570\u00a0bpm). In contrast, the subgroup of patients with an Agatston score \u2265400 included more false ratings (two FN and eight FP) than the subgroup of patients with an Agatston score <400 (zero FN and one FP). Consequently, sensitivity and specificity were worse in severely calcified vessels (see Table\u00a02).\nDiscussion\nFour main conclusions can be drawn from this study. First, DSCT coronary angiography provides a high diagnostic accuracy for the evaluation of CAD. Second, this high diagnostic performance of DSCT could be achieved in a patient population with extensive calcifications and in whom no heart rate control using beta blocker medication prior to CT was performed. Third, taking into account these circumstances, only six (1.4%) segments had to be excluded from data analysis, all in the distal part of the coronary artery tree with small vessel diameters. Fourth, false ratings were primarily due to severe vessel wall calcifications rather than motion artifacts.\nTemporal resolution better than 100\u00a0ms in combination with submillimeter isotropic spatial resolution and examination times no longer than 10\u00a0s are considered prerequisites for successful implementation of cardiac CT into routine clinical algorithms [18]. DSCT scanners with 0.33-s gantry rotation time and 32\u00d70.6 mm collimation in combination with double z-sampling (i.e., simultaneous acquisition of 64 overlapping 0.6-mm slices) fulfill these requirements. \nEarly feasibility studies confirmed the technical capacity of DSCT to provide diagnostic image quality of coronary arteries in patients with high heart rates [19, 20]. This study is, to the best of our knowledge, the first to demonstrate a high diagnostic accuracy of DSCT coronary angiography for the diagnosis of CAD in comparison to ICA. By including a patient population with a high prevalence of coronary calcification and without heart rate control during scanning, we found an overall sensitivity of 96.4% and specificity of 97.5% for the detection of significant coronary stenoses. These results are comparable to those previously reported with 64-slice CT; however, beta-blocker administration for strict heart rate control was needed in those studies to compensate for motion artifacts. By including patients with heart rates up to 102\u00a0bpm, we found minor to moderate vessel wall blurring due to motion artifacts to be present at high heart rates; however, only 1.4% of all segments had to be excluded from analysis because they were non-diagnostic. \nFurther confirmation of our preliminary results in larger patient populations may broaden the clinical indications for CT coronary angiography from a modality that is recommended for ruling-out CAD in patients with a low pre-test probability [9] to use in populations with intermediate- or even high pre-test probabilities of the disease. This might also include the implementation of CT coronary angiography as a tool for preoperative planning before cardiac bypass surgery [25] and as a gatekeeper for invasive angiography in patients referred for cardiac valve surgery [26].\nOur patient population had a high prevalence of coronary wall calcification, with a mean Agatston score of 821\u2014being higher than the 75% percentile in an age- and gender-matched control population [27]. With 64-slice CT, Raff et al. [2] reported a considerable decline in diagnostic accuracy in patients with Agatston scores >400 with a sensitivity of 93%, a specificity of 67%, and positive and negative predictive values of 93 and 67%, respectively. Using the same cut-off Agatston score, we found a sensitivity of 96%, a specificity of 95%, and positive and negative predictive values of 86 and 99%, respectively. Considering that the spatial resolution of DSCT is the same as that of the single-source 64-slice CT scanner, this apparent difference in calcification-dependency could indicate that the blooming artifact of severely calcified vessel walls may be sometimes superimposed by additional motion artifacts.\nWe acknowledge the following study limitations. First, we included a relatively small group of only 30 patients. Certainly, future studies with larger patient populations are needed to confirm our first experience. Second, our study patients were a high pre-test probability population, which may have resulted in an overestimation of the ability of DSCT to detect and to rule-out stenoses. In particular the predictive value of positive and negative diagnostic tests is known to be strongly influenced by disease prevalence and pre-test probability. Third, we did not apply the multi-segment reconstruction mode, which possibly may further improve the image quality at elevated heart rates. Fourth, CT coronary angiography is associated with substantial irradiation to the patient. However, using the DSCT system allowed the variable use of ECG-pulsing for dose-saving purposes [28] in all of our patients. In the protocol applied in this study, the ECG-pulsing window width was chosen according to the mean heart rate during scanning, i.e., a relatively narrow window width at low and a larger window width at higher heart rates. As previously performed for 64-slice CT [17, 29], studies analyzing optimum reconstruction intervals for DSCT coronary angiography are mandatory to reduce the width of the pulsing window and the applied radiation dose further. Finally, the ability to use the two x-ray tubes simultaneously with different voltages to improve plaque composition characterization [30] and thus potentially to improve the accuracy of stenosis detection has not been investigated.\nConclusion\nFirst experience indicates that DSCT coronary angiography provides high diagnostic accuracy for assessment of CAD in a high pre-test probability population with extensive coronary calcifications and without heart rate control. Further studies are needed to confirm our results in appropriate clinical settings with larger patient populations.","keyphrases":["coronary angiography","dual-source computed tomography","coronary stenosis"],"prmu":["P","P","R"]}
{"id":"J_Antimicrob_Chemother-1-1-2386081","title":"Microbiological evaluation of a new growth-based approach for rapid detection of methicillin-resistant Staphylococcus aureus\n","text":"Objectives Recently, a rapid screening tool for methicillin-resistant Staphylococcus aureus (MRSA) has been introduced that applies a novel detection technology allowing the rapid presence or absence of MRSA to be determined from an enrichment broth after only a few hours of incubation. To evaluate the reliability of this new assay to successfully detect MRSA strains of different origin and clonality, well-characterized S. aureus strains were tested in this study.\nIntroduction\nThe increasing numbers of multidrug-resistant Gram-positive pathogens have generated worldwide concern in the medical community. In particular, methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of disease and healthcare expenditures in almost every continent. The emergence and spread of MRSA has been shown to be associated with both hospital- and community-acquired infections. Effective treatment options for these infections are limited and the situation may become more severe soon. For these reasons, a proactive management of MRSA in healthcare facilities is needed.1,2\nActive screening and compliance to appropriate infection control activities have been shown to play an important role in the control of MRSA.1 Rapid diagnostic tests have the potential to make efforts even more effective. Thus, infection prevention has taken a step forward with the introduction of various tests for rapid identification of MRSA carriers.1,2\nIn 2006, a new rapid method based on a novel bioluminescence detection technology for the rapid detection of MRSA directly from specimens was published.3 An improved version of this assay, the 3M\u2122 BacLite\u2122 Rapid MRSA Test (3M Company, Maplewood, MN, USA), was recently introduced, which allows the presence or absence of MRSA to be determined within 5 h. Although the clinical performance has been previously analysed,4,5 so far no data are available on the detection of MRSA strains with highly diverse genetic backgrounds.\nThe aim of this study was to assess the reliability of this assay to successfully detect MRSA strains circulating currently in Germany and other parts of Europe on the basis of several well-characterized S. aureus strain collections. For this purpose, S. aureus strains of different origin and clonality comprising 724 methicillin-susceptible and methicillin-resistant strains comprising >90% of all registered European MRSA spa types within the SeqNet network (www.SeqNet.org) were tested in this study.\nMaterials and methods\nAll staphylococcal strains were freshly isolated from clinical material at the University of M\u00fcnster or during the course of various multicenter studies. One hundred and sixty-four strains isolated from patients with S. aureus bacteraemia (including 12 MRSA strains) as well as 50 isolates from the anterior nares of patients who did not subsequently develop S. aureus bacteraemia in a subsequent observation period (4 MRSA strains), were included into this study. Apart from 28 isolates from 14 patients (S. aureus first recovered from the anterior nares and subsequently from blood, one infected with MRSA), only one isolate per patient was tested. In addition, 96 MRSA strains, each presenting a unique spa type, were selected from our institutional collection (Table\u00a01). Furthermore, four hundred MRSA isolates were collected during the course of a recent multicenter study, also including community-acquired MRSA. In total, the 724 S. aureus strains tested comprised 211 methicillin-susceptible S. aureus (MSSA) and 513 MRSA strains.\nTable\u00a01\nGrouping of spa types tested in this study into spa clonal complexes (spa-CCs)\nspa-CCa\nspa typesb\nPutative MLST typesc\n001\nt001, t002, t003, t010, t035, t039, t041, t045, t055, t057, t066, t105, t106, t109, t110, t143, t149, t151, t264, t265, t422, t820, t892, t1018\nST-5, ST-45, ST-46, ST-222, ST-225, ST-228, ST-231, ST-111, ST-228\n004\nt004, t028, t029, t033, t040, t043, t061, t065, t141, t142, t266, t911\nST-45, ST-45, ST-46\n015\nt015, t031, t069, t073, t102, t116, t133\nST-45\n024\nt008, t009, t024, t036, t051, t052, t068, t113, t115, t139, t146, t243, t305\nST-8, ST-235, ST-247, ST-250, ST-254, ST-247, ST-250\n032\nt005, t022, t032, t107, t290, t379, t432, t794\nST-22, ST-23, ST-60\n037\nt011, t030, t037, t047, t108, t135, t137, t138\nST-30, ST-239, ST-240, ST-241, ST-246\n038\nt038, t161, t247\nST-45\n044\nt042, t044, t131\nST-80\nSingletons\nt012, t091, t101, t104, t163, t321, t372, t417, t431, t907\nST-7, ST-30, ST-22\naNaming of spa-CC was derived from the group founder.\nbt026, t103, t111, t132, t145, t282, t322 and t1007 were excluded from determination of the spa-CCs due to low number of repeats.\ncPutative MLST types were taken from the public SeqNet.org spa server database.\nIf the biochemical identification of staphylococcal isolates using the ATB 32 Staph gallery (bioM\u00e9rieux, Marcy l\u2019\u00c9toile, France) was ambiguous or categorized as unacceptable, partial 16S rRNA gene and RNA polymerase B (rpoB) gene sequencing was performed as described previously.6 Isolates were confirmed to be methicillin-resistant by detection of the mecA gene.\nTo determine the clonal lineages of MRSA strains, the x region of the spa gene was amplified by PCR with primers 1095F (5\u2032-AGACGATCCTTCGGTGAGC-3\u2032) and 1517R (5\u2032-GCTTTTGCAATGTCATTTACTG-3\u2032). DNA sequences were obtained with an ABI 377 sequencer (Applied Biosystems, Foster City, CA, USA). spa types were determined using the Ridom StaphType software version 1.3 (Ridom GmbH, W\u00fcrzburg, Germany), and spa clonal complexes (spa-CCs) were assigned by using the BURP algorithm.7\nThe 3M\u2122 BacLite\u2122 Rapid MRSA Test is a novel culture-based test for the detection of MRSA performed on a semi-automated system comprising a sample processor and a luminometer. The assay consists of three successive selectivity steps: selective enrichment in a proprietary broth containing cefoxitin (2 mg\/L) and colistin (50 mg\/L), immuno-magnetic extraction using a highly specific anti-S. aureus monoclonal antibody and selective lysis using lysostaphin. The selectivity steps are followed by a detection step using a highly sensitive cell marker, adenylate kinase (AK). AK is an essential enzyme found in all living cells, which regulates energy provision by catalysing the equilibrium reaction ATP + AMP = 2 ADP. By supplying a continual source of purified ADP, this assay drives the AK reaction to generate up to 40 000 ATP molecules per min. These amplified levels of ATP are measured using the luminometer supplied with the system.\nFor the present study, test isolates were streaked on sheep blood agar plates and grown at 37\u00b0C to confirm purity. For each isolate, one to three colonies were picked and suspended in sterile saline (0.9%) to achieve a turbidity equivalent to that of a 0.5 McFarland standard. Usually, 43 isolates were processed in each run. The 3M\u2122 BacLite\u2122 Rapid MRSA Test was performed according to the instructions of the manufacturer. In brief, 10 \u00b5L of each prepared bacterial suspension was transferred into a vial containing 1 mL of selective enrichment broth. Vials were incubated at 37\u00b0C for 2 h before 150 \u00b5L aliquots of each test sample were transferred into two adjacent wells of a 96-well assay plate, each containing 20 \u00b5L of capture reagent. MRSA cells bound to the monoclonal antibody on the capture reagent were then extracted from the sample matrix using the sample processor and concentrated in 100 \u00b5L of broth. One well for each sample was processed automatically for a baseline signal (T0) in the BacLite luminometer. After a further incubation period of 2 h at 37\u00b0C, the second well for each sample was processed in the same way (T2 reading). Results are expressed in relative light units and interpreted as either a positive or negative screen result by a software embedded algorithm. Positive and negative controls as well as reagent and broth controls were included in each test run. A run took \u223c5 h with a total hands-on time of 45\u201350 min (<1.5 min per tested isolate).\nResults and discussion\nAnalysing a total of 724 S. aureus strains, all 513 MRSA strains tested were recognized as MRSA, whereas none of the 211 MSSA strains was detected positive by the 3M\u2122 BacLite\u2122 Rapid MRSA Test. These results are particularly impressive as the institutional MRSA strain collection used in this study represents more than 90 different spa types covering >90% of all registered European MRSA spa types within the SeqNet network (Table 1). Beside several singletons, the MRSA enrolled in this study were grouped into eight spa clonal complexes (Table 1). Thus, a very large number of MRSA strains with different genetic backgrounds were recognized as MRSA using this novel method.\nHospitals and other healthcare facilities across the world are faced with alarming rates of infections caused by MRSA. Continuous spread of these pathogens requires efficient strategies for infection control. Early identification of MRSA carriers among hospitalized patients is crucial to prevent its spread.1 Therefore, rapid availability of laboratory results is of utmost importance. However, conventional screening methods require prolonged incubation and confirmatory testing. During this time, MRSA-negative patients may be held in unnecessary isolation, whereas unidentified MRSA-positive individuals remain a hidden reservoir for cross-infection. A rapid negative result should allow more effective use of hospital isolation resources, whereas a rapid positive result should help reduce the spread of the infection and MRSA infection rates.2\nIn the past few years, several in-house and commercial rapid MRSA assays based on molecular techniques have been introduced for the detection of this pathogen directly from the specimen, mostly from the anterior nares. The first molecular assays developed were based on the detection of an S. aureus-specific sequence and the mecA gene, which encodes methicillin resistance.8 These tests are difficult to use for the direct detection of MRSA from non-sterile specimens, such as nasal samples, because of the likely co-presence of MSSA and methicillin-resistant coagulase-negative staphylococci.9 In a setting of low prevalence of MRSA, a molecular test targeting the mecA and an S. aureus-specific gene in parallel applied directly to clinical specimens would result in a high number of false positives and unacceptable performance.9\nThis technical limitation has been overcome in some assays, by linking detection of the presence of the mecA gene with detection of the neighbouring chromosome-borne orfX gene.10,11 In that approach, regions near the integration site of SCCmec were targeted as surrogate markers instead of the mecA gene itself. However, these flanking regions are known to be more heterogeneous than assumed so far.10 Thus, false-negative results due to variations within the primer binding sites may occur. Moreover, false-positive results due to the detection of DNA from non-viable MRSA, deletions or replacement of the mec region in vivo or \u2018ghost sequences\u2019 such as partial SCCmec sequences in MSSA can occur, albeit their incidence in the routine clinical setting is as yet unclear.12 Despite the technical improvements in recent molecular-based assays, their high costs and relatively high operator skill requirement remain obstacles to their widespread routine use.\nThe ability of a test to detect a broad range of MRSA clones is particularly important for an assay that may be used across a wide geographic region as tests with gaps in detection could potentially \u2018select out\u2019 strains whose spread would be uncontrolled.\nBeside a variety of method-inherent limitations, rapid DNA-based methods amplify the nucleic acid and not the organism, which means the MRSA strain is unavailable for further characterization, such as determination of the resistance profile and strain typing. In contrast, applying the rapid growth-based assay tested here, any further examinations to characterize the respective strains will be possible from the enrichment broth.\nFor the 3M\u2122 BacLite\u2122 Rapid MRSA Test, a diagnostic sensitivity of 94.6% and diagnostic specificity of 96.9% for nasal screening swabs and 95.9% sensitivity and 88.8% specificity for groin screening swabs, respectively, have been reported.4,5 The analytical limit of detection of the assay was shown to be \u226494 cfu (3M\u2122 BacLite\u2122 Rapid MRSA Test, Instructions for Use).4,5\nHere, the new growth-based rapid MRSA assay was shown to detect without exception all MRSA strains of large collections of strains comprising highly diverse genetic backgrounds. Such a phenotypic test might be potentially more likely to cope with new strains. Further studies are warranted to evaluate this method using clinical specimens.\nFunding\nThis work was supported in part by a grant from the Bundesministerium f\u00fcr Bildung und Forschung (BMBF), Germany (Pathogenomic Plus Network PTJ-BIO\/0313801B). The device and the respective assay consumables were given to us by 3M for this study.\nTransparency declarations\nTwo of the authors (A. W. F. and K. B.) delivered independent scientific presentations at 3M-supported symposia. C. v. E. is a member of 3M\u2019s Scientific Advisory Board. All other authors: none to declare.","keyphrases":["mrsa","infection control","spa typing","staphylococci"],"prmu":["P","P","P","U"]}
{"id":"Bioprocess_Biosyst_Eng-2-2-1705473","title":"On-line optimization of glutamate production based on balanced metabolic control by RQ\n","text":"In glutamate fermentations by Corynebacterium glutamicum, higher glutamate concentration could be achieved by constantly controlling dissolved oxygen concentration (DO) at a lower level; however, by-product lactate also severely accumulated. The results of analyzing activities changes of the two key enzymes, glutamate and lactate dehydrogenases involved with the fermentation, and the entire metabolic network flux analysis showed that the lactate overproduction was because the metabolic flux in TCA cycle was too low to balance the glucose glycolysis rate. As a result, the respiratory quotient (RQ) adaptive control based \u201cbalanced metabolic control\u201d (BMC) strategy was proposed and used to regulate the TCA metabolic flux rate at an appropriate level to achieve the metabolic balance among glycolysis, glutamate synthesis, and TCA metabolic flux. Compared with the best results of various DO constant controls, the BMC strategy increased the maximal glutamate concentration by about 15% and almost completely repressed the lactate accumulation with competitively high glutamate productivity.\nIntroduction\nFor more than a few decades, the Corynebacterium spp. bacteria have been used for amino acids productions, including the commercially important products of glutamate, glutamine, and lysine. Among the amino acids mentioned earlier, L-glutamate is the largest fermentative product, which occupies about 53% of the world\u2019s amino acids market [1]. It is also particularly important in food industries and widely used as an important starting substance for the synthesis of various and useful pharmaceutical and healthy products. The previous research works [2\u20134] have revealed that primary by-products, such as lactate, accumulated during L-glutamate fermentation if the operating condition was improperly controlled, which in turn deteriorated the fermentation performance in terms of both glutamate productivity and yield.\nTo cope with the problem, the metabolic reaction network (flux) model (MR model) or technique, as its appearance in the early 1990s, has been recognized as an useful system analysis tool and thus widely used in those areas such as metabolic flux distribution analysis [2, 5], determination of the bottleneck controlling the targeted metabolic product formation [6, 7], recognition of fermentation phases [8], and calculation of theoretical or maximum yields [9, 10], etc. However, the research reports of using MR model for on-line physiological state prediction or process control are very limited [3, 11, 12], and the study stayed on on-line recognition of different fermentation phases or physiological states so as to provide information for the subsequent process control, such as whether substrate should be added or whether the fermentation should be terminated [11], determination of glucose feeding rate to avoid the acetate or ethanol overproduction in Escherichia coli or Saccharomyces cerevisiae fed-batch cultivation [12], etc. Optimization of fermentation processes by MR model was mostly limited on using the feed-forward type\u2019s off-line control strategy with the MR model as the constraint conditions [13\u201314].\nOn the other hand, analysis of key enzymes in metabolic network of cells was also very important, as those key enzyme generally play important roles in regulating and overproducing the targeted metabolic products. As a result, extensive research works with regard to the enzymes activities under different operating conditions have been carried out. Among them, the enzymes activities in fermentation by Corynebacterium glutamicum such as the influence of NH4+ concentration on C. glutamicum growth and glutamate dehydrogenase (GDH) [15], activity of phosphoenolpyruvate carboxylase responsible for the anaplerotic reactions in lysine continuous production by C. glutamicum under different dilution rates [16, 17], as well as lactate dehydrogenase (LDH) for pyruvate overproduction under different dilution rates with lactate as the sole carbon source [18], were investigated. The metabolic network models were also built for C. glutamicum under different operating conditions, aiming at achieving metabolic flux distribution information concerning the overproduction of targeted metabolites or substrate utilization [17\u201319], as well as on-line prediction of the targeted products formation and analysis of the entire metabolic fluxes [3].\nIn our previous study [3], we found that dissolved oxygen (DO) largely affected the metabolic flux distribution and the glutamate fermentation performance. The comprehensive analysis or evaluation of a fermentation process by the MR model-based metabolic flux analysis integrated with the intracellular enzymes activities monitoring, might gain a deeper insight into the entire fermentation process and the metabolic regulation mechanisms, so as to supply a more comprehensive and accurate information base for the subsequent process control and optimization of the fermentation process.\nIn this study, combining our previous study on on-line metabolic flux analysis of the glutamate fermentation by C. glutamicum, the two major enzymes (GDH and LDH), which possibly dominated the overall glutamate metabolism, were carefully investigated. Then, based on the comprehensive evaluation results of both the metabolic flux analysis and intracellular enzymes activities monitoring, a new optimization strategy of \u201cbalanced metabolic control (BMC)\u201d was proposed and verified experimentally, aiming to increase the glutamate production yield, while completely repressing the by-product overproduction simultaneously.\nMaterials and methods\nMicroorganism and fermentation conditions\nCorynebacterium glutamicum S9114, kept by the laboratory was used throughout this study. The same medium and seed culture conditions described in our previous study [3] were used.\nCorynebacterium glutamicum S9114 was cultured for glutamate production at 32\u00b0C in a 5\u00a0L fermentor (BIOTECH-5BG, Baoxing Co., China) containing about 3.4\u00a0L medium. Concentrated glucose was fed based on requirement to ensure the substrate concentration above a suitable level (15\u00a0g\/L) throughout the fermentation period. pH was controlled at 7.1\u00a0\u00b1\u00a00.1 by automatic addition of 25% (w\/w) ammonia water which also supplied the nitrogen source required for glutamate synthesis. DO was controlled at various levels by automatically or manually controlling the agitation speed based on particular requirements. The air aeration rate and fermentor pressure were kept constantly at 1.60\u00a0vvm and 0.07\u00a0MPa, respectively.\nAnalytical methods\nThe concentrations of cells, glucose, glutamate, and lactate were measured with the same methods as reported in our previous study [3]. The CO2 and O2 concentrations (partial pressure) in the inlet and exhaust gas were on-line measured by a gas analyzer (LKM2000A, Lokas Co. Ltd, Korea). The collected on-line data were smoothly filtered, and then oxygen uptake rate (OUR) and CO2 evolution rate (CER) were on-line calculated based on the literature reported method [20]. Respiratory quotient (RQ) was determined by its definition (RQ\u00a0=\u00a0CER\/OUR) using the on-line measured OUR and CER data.\nExtraction and assay of the GDH and LDH\nThe two key enzymes dominating glutamate fermentation and metabolism, the NADPH and NADH dependent GDH and LDH were detected by spectrophotometric method, and the relevant stoichiometric reactions could be written as follows:\nThe enzymes in the reverse direction of glutamate and lactate consumption were not considered and assayed in this case. Cells were collected by centrifugation and then suspended in 25\u00a0mmol\/L Tris\u2013HCl buffer, pH\u00a07.5. The suspension was sonicated by a sonifier (JY92-II, Scientz Biotechnology Co., China) at 0\u00b0C for a total period of 10\u00a0min (pulse on, 1\u00a0s; pulse off, 3\u00a0s). Cell debris was removed by centrifugation at 9,400g, 4\u00b0C, for 20\u00a0min, the supernatant was then used as the cell-free crude enzyme. GDH was assayed with a spectrophotometer (UV-2100, Unico, Shanghai, China) at 340\u00a0nm by measuring the optical variation within 1\u00a0min, with 0.05\u00a0mL supernatant and 2.95\u00a0ml GDH reaction mixture consisting of 13.3\u00a0mmol\/L \u03b1-ketoglutaric acid, 15\u00a0mmol\/L NH4Cl, and 1.67\u00a0mmol\/L NADPH (Sigma Chemical Co., St Louis, MO, USA) in phosphate buffer (pH\u00a07.5) at 37\u00b0C. LDH was assayed in the same condition but with the LDH reaction mixture containing 0.757\u00a0mmol\/L pyruvic acid and 1.67\u00a0mmol\/L NADH (Sigma Chemical Co., St Louis, MO, USA). Protein was measured by Bradford method with bovine serum albumin as the standard. Enzyme-specific activity (GDH or LDH) was expressed as units\/mg-protein, where 1\u00a0U was defined as the quantity of enzyme that converted 1\u00a0\u03bcmol of NAD(P)H per minute.\nOn-line control system\nThe on-line measured RQ data were sent to a PC in which a control program written with Visual-Basic (Microsoft Inc., USA) was embedded. Based on the RQ set-points and the measured RQ, the PC on-line regulated the agitation rate of the fermentor (AGT) with a discrete PI control manner described by Eq.\u00a03 , which renewed the agitation rate at an interval of 5\u00a0min.\nIn Eq.\u00a03, k represented the current control instant; RQset was the RQ set-point which might be subject to changes during the control; KC and \u03c4I were proportional and integral constants of the feedback controller, respectively. KC and \u03c4I were determined by Coon\u2013Cohen method by observing the RQ response to a step change in the input (agitation rate) during a certain period of the glutamate production phase.\nResults and discussion\nThe changing patterns of glutamate and lactate production, as well as the GDH and LDH activities at different DO control levels\nConstant control of DO is generally considered as the simplest optimization control method for the aerobic fermentations with aeration. Fig.\u00a01a, c, d showed the time courses of glutamate, lactate, cells, and residual glucose concentrations when DO was constantly controlled at 10 and 50% (saturation level), respectively. The results showed that, glutamate and lactate formation pattern strongly depended on the DO control level. A higher glutamate production rate could be achieved when the DO was controlled at a lower level of 10% and the final glutamate concentration reached about 91.5\u00a0g\/L at 34\u00a0h, while the final glutamate concentration stopped at a lower level of 72.7\u00a0g\/L (30\u00a0h) when controlling DO at 50%. On the other hand, lactate severely accumulated up to 28\u00a0g\/L when DO was controlled at a lower level of 10%, while almost no lactate accumulation occurred when controlling DO at 50%. These results suggested that the enzymatic activities of GDH and LDH under lower and higher DO level might be quite different.Fig.\u00a01Time courses of glutamate, lactate, cells and glucose concentrations, as well as the key enzymes activities at different DO control levels. a Glutamate concentration: (filled circle) DO 10%, (filled triangle) DO 50%; lactate concentration: (open circle) DO 10%, (open triangle) DO 50%. b GDH activity: (filled circle) DO 10%, (open circle) DO 50%; LDH activity: (filled triangle) DO 10%, (open triangle) DO 50%. c Cells concentration: (filled circle) DO 10%, (filled triangle) DO 50%. d Glucose concentration: (filled circle) DO 10%, (filled triangle) DO 50%\nFigure\u00a01b indicated the changing pattern of the GDH and LDH activities under the same DO control levels. The GDH activity for the lower DO case (10%) was much higher and declined slower than that of the higher DO case (50%) after experiencing the enzymatic activity peak of about 1.8\u00a0U\/mg-protein around 12\u201313\u00a0h. This result could at least explain or account for the fact that, higher glutamate production rate and accumulation occurred at lower DO control level in the initial and middle production phases of 8\u201320\u00a0h. The slow down of glutamate production rate in the late production stage (20\u201330\u00a0h) and the final stoppage of glutamate production were due to the reduction of the co-enzyme NADPH regeneration rate, which will be further discussed in the following section. As for the LDH activities, from the result of severe lactate\u2019s accumulation at lower DO level, it was speculated that the LDH activity under lower DO level (10%) should also be much higher than that of the higher DO case. However, the result did not agree with the expectation. Although the LDH activity under lower DO level was slightly higher than that of the higher DO for the majority of the fermentation period, this result could not stand for the large difference in the lactate accumulation under different DO levels.\nIt should be noted that the cell concentration could reach a constant and stabilized level of about 20\u00a0g\/L prior to production phase (Fig. 1c) for all of the constant DO control cases, therefore using the unit of \u201cU\/mg-protein\u201d could actually reflect the total enzyme activity.\nThe changing patterns of glutamate and lactate production, as well as the GDH and LDH activities at anaerobic fermentation condition\nGenerally, it is considered that the anaerobic condition is extremely harmful to glutamate production. To verify the above speculation, an experiment under extremely low DO level was conducted. In the fermentation, DO was initially controlled at 30%, and the agitation rate was manually reduced to bring DO down to 0% instantly at 12\u00a0h. Then, the same agitation rate was kept for the next 6\u00a0h. During this period, the fermentation could be considered as implemented under anaerobic condition, glutamate production stopped and lactate overflowed as shown in Fig.\u00a02a. At 18\u00a0h, the automatic control of DO was resumed to quickly bring DO back to 30%, a partial recovery of glutamate production was observed. However, the final glutamate concentration ended at a very low level of about 45\u00a0g\/L. As shown in Fig.\u00a02b, during the anaerobic period, GDH activity sharply dropped to a level of 0.87\u00a0U\/mg from the original 1.86\u00a0U\/mg, and could not be recovered again even when DO was resumed back to 30% level. The results indicated that the occurrence of anaerobic condition even for a short period would be both fatal and irretrievable to glutamate fermentation.Fig.\u00a02The changing pattern of the products formation and the key enzymes activities when DO control level was subject to a sudden change during 12\u201318\u00a0h, and maintained at extremely low level during the period. a The changing patterns of glutamate and lactate concentrations, as well as the DO control level, filled circle: glutamate concentration, open triangle: lactate concentration, solid line: DO. b The changing patterns of the key enzyme activities, filled circle: GDH activity, open triangle: LDH activity\nThe glucose consumption rates (glycolysis rates) at different DO control levels\nFigure\u00a03 showed the average glucose consumption rates under different DO control levels during the production phase. Apparently, under regular (aerobic) fermentation, the glycolysis rate did not depend on the DO control level, and the changing patterns of glycolysis rate at different DO levels were basically the same.Fig.\u00a03The time courses of glucose consumption rate at different DO levels. Open circle: DO\u00a0=\u00a010%, batch no. 050331; open triangle: DO\u00a0=\u00a050%, batch no. 050407\nThe mechanism analysis of lactate overflow and the control strategy of BMC\nFor the purpose of easily interpreting the experimental results, the metabolic flow chart of glutamate fermentation by C. glutamicum as shown in our previous study [3] was simplified and re-depicted in Fig.\u00a04. The metabolic fluxes changes at 10, 20, and 30\u00a0h of the fermentations with the DO constant controls (10, 50%) and BMC were also depicted. As shown in the map, glucose was converted into pyruvate through EMP pathway, and supplemented with PP pathway to produce NADPH for structuring glutamate synthesis precursors by cells. The pyruvate formed either entered TCA cycle via pyruvate dehydrogenase, generating the energy as well as co-enzyme substances such as ATP, NADH, etc. for the entire metabolic network operations, or was converted into lactate with LDH as the enzymatic catalyst (r5, Eq.\u00a02). At \u03b1-KG (\u03b1-ketoglutaric acid) node of TCA cycle, the glutamate synthesis reaction (r6) with GDH as the enzymatic catalyst (Eq.\u00a01) occurred. As shown in Fig.\u00a04, with the constant DO control strategy, the decrease of the DO control level enhanced the metabolic fluxes of both glutamate and lactate formation; while the metabolic flux of TCA after the \u03b1-KG node (r4) sharply decreased, and the TCA cycle was almost completely shut down when controlling DO at 10% level (also refer to Fig.\u00a05).Fig.\u00a04Simplified metabolic flow chart and the relevant metabolic fluxes changes during the fermentations. Broken line: very low metabolic flux or channels shut down; solid line: moderate metabolic flux; bold solid line: enhanced metabolic flux. a: at 10\u00a0h; b: at 20\u00a0h; c: at 30\u00a0hFig.\u00a05Comparison of RQ and calculated TCA metabolic flux rate at different DO control levels\nA higher enzyme activity in vivo reflects a higher potential in reaction rate catalyzed by the enzyme. However, it never means that the relevant reaction rate must be high, as the reaction rate also depends on other factors, such as the coenzyme (NADPH, NADH) activity as well as the reactant concentration. As shown in Fig.\u00a01a, b, at the end of the fermentation, the glutamate synthesis completely stopped even though GDH activity still remained at a higher level. The calculation results in our previous report [3] showed that at the end of the fermentation, the coenzyme NADPH regeneration rate had decreased to 5\u201320% of its peak level (figure not presented). Therefore, it could be concluded that, the glutamate synthesis stoppage was due to the significant reduction of NADPH regeneration rate instead of the reduction in GDH activity.\nIn general, the enzymes catalyzing interaction assures an intracellular carbon fluxes balance. This means that in glutamate fermentation, glycolysis rate (r1) should balance with glutamate synthesis (r6), lactate formation (r5), as well as the TCA metabolic flux (r4) that was involved with CO2 release. As a result, from Figs.\u00a01, 4, and 5, we could speculate that the severe lactate accumulation at lower DO control (DO\u00a0=\u00a010%) was due to the carbon metabolic balance rather than the higher LDH activity by the following facts. First, the changing patterns of glycolysis rate (r1) at different DO levels were almost the same; second, no significant differences in LDH activities were shown under different DO level; Third, the metabolic flux of TCA significantly decreased with the decrease in DO control level. Under the lower DO level, even though GDH activity was higher, the higher glutamate synthesis rate (r6) still could not completely balance with the glycolysis rate (r1), as TCA cycle was almost closed completely and the TCA metabolic flux (r4) was very low. Under this circumstance, lactate had to be overflowed or excreted (r5) into the broth to achieve the entire intracellular carbon balance. On the other hand, under the higher DO level, GDH activity was relatively low, but the TCA cycle was nearly open for a complete oxidation. Under this condition, lactate was not necessarily overflowed, as the glutamate synthesis rate (r6) plus the higher TCA metabolic flux (r4) were big enough to balance with the glycolysis rate (r1), even though LDH exhibited almost equivalent activity as compared with that of the lower DO case.\nIs there an effective way to solve the problem of simultaneous accumulation of the main product (glutamate) and by-product (lactate)? Two ways of using genetic engineering technique could be considered as the solution: (1) to use genetic engineering technique to knock-out the genes coding for LDH; (2) to use certain LDH inhibitors to eliminate the LDH action without affecting GDH and entire fermentation activities at the same time. These are direct and straightforward solutions, but might be very complicated and difficult to realize. Based on the metabolic analysis mentioned earlier, an alternative solution, the \u201cBMC\u201d strategy as shown in Fig.\u00a04, which is adaptively regulating of the TCA metabolic fluxes at an appropriate level to achieve the metabolic balance among glycolysis, glutamate synthesis, and TCA metabolic flux for enhancement of the glutamate synthesis and repression of lactate overflow, seemed to be easier to implement and more realistic.\nPossibility of using and realizing the BMC strategy\nThe necessary conditions for realization of BMC existed in the following two aspects: (1) the rates of the glutamate synthesis (r6) plus the TCA metabolic flux (r4) should just balance with the glycolysis rate (r1); (2) DO should be controlled at a reasonably lower level to assure a higher GDH activity. To achieve the target, an on-line measurable or predictable state variable that could actually reflect the TCA metabolic flux changes, must be available. Based on our experimental results and the on-line metabolic flux calculation in the previous study, we found RQ are related with the TCA metabolic flux (r4) closely, and could be considered as the on-line feedback index for realization of BMC.\nFigure\u00a05 depicted the time course of RQ and the TCA metabolic flux (r4) for the cases of constantly controlling DO at 10 and 50%, respectively. For both cases, RQ almost followed the same changing patterns during the growth phase. However, during the glutamate production phase, the changing patterns of RQ and TCA metabolic flux under different DO levels were apparently different. RQ almost stayed constantly around 0.80 in the case of controlling DO at 50%, while RQ gradually declined into the range of 0.5\u20130.6 for the case of controlling DO at 10%. Theoretically, RQ\u00a0=\u00a01.0 means that TCA cycle is fully opened and glucose is completely converted into CO2 and H2O via TCA cycle. In glutamate fermentation, a lower RQ (for example, RQ\u00a0=\u00a00.5\u20130.6) reflected the fact that, TCA cycle was almost completely shut down in order to direct the carbon flux to the glutamate synthesis at \u03b1-KG node. On the other hand, a higher RQ meant that the TCA metabolic flux was too high that a large portion of carbon was oxidased into CO2 and H2O accompanyied with large quantities of NADH and ATP formation, which actually caused the \u201cinefficient carbon cycle\u2018\u2019. Based on Fig.\u00a05, If we could carefully and adaptively control RQ at a suitable level, maintaining the TCA metabolic flux at a moderate level in between the fluxes of lower and higher DO levels, while still directing major carbon flux to the glutamate synthesis, then the BMC might be realized and lactate overflow be relieved.\nThe performance and experimental result of the BMC strategy\nFigure\u00a06 showed one complete data set of the BMC control results. The BMC was activated at 11\u00a0h after the fermentation entered into production phase, where the two controller parameters, KC and \u03c4I were set at 20 and 3, respectively. As shown in Fig.\u00a06, the set-point of RQ was changed three times during the fermentation (RQset: 0.80, 11\u201315\u00a0h, initial stage of production phase; 0.70, 15\u201328\u00a0h, middle stage; 0.60, 28\u201335\u00a0h, final stage). The principle of changing RQset was to assure that DO does not subject to either a higher (\u226540%) or a very low level (\u22645%) for a relatively longer time (1\u00a0h) in order to keep a higher GDH activity: if DO stayed at higher level for relatively long time, then it initiatively reduced the RQset down; if DO stayed at very low level for relatively long time, then it initiatively brings the RQset up. By using DO as the complementary indicator in this way, RQ could be controlled around its set-point, with the agitation rate stably staying in the range of 450\u2013750\u00a0rpm without reaching its upper limit (950\u00a0rpm) or low limit (450\u00a0rpm) even for a short time. With this control strategy, DO fluctuated at a lower range of 5\u201340% so that a higher GDH activity could be maintained. The BMC control led to an excellent fermentation performance as compared with those of the DO constant controls: the maximal glutamate concentration reached 101.6\u00a0g\/L at 34\u00a0h which was about an 11% increase over the best result of the DO constant controls (91.5\u00a0g\/L, DO\u00a0=\u00a010%); the lactate concentration at the corresponding time was only 0.11\u00a0g\/L which was only 14% of the best result of the DO constant controls (0.8\u00a0g\/L, DO\u00a0=\u00a050%). To verify the repeatability of BMC control\u2019s performance, the same experiment was repeated once. Again, a maximal glutamate concentration of 98.8\u00a0g\/L was obtained at 38\u00a0h, with the lactate accumulation of only 1.04\u00a0g\/L at the same time.Fig.\u00a06The experimental results and control performance using the BMC optimization strategy. Filled circle: glutamate concentration; open triangle: lactate concentration; open circle: cells concentration; filled square: glucose concentration\nIt should be noted that constant RQ control strategy could not lead to a good fermentation performance. We did three constant RQ control experiments setting RQ at 0.6, 0.7, and 0.8, respectively. The glutamate production stopped at very low level (40\u201360\u00a0g\/L) for all of the three cases. In the first two cases, constantly controlling RQ at 0.6 and 0.7 caused DO to drop to zero level, or the anaerobic environmental condition in other word, in the initial production stage. The cells metabolic and GDH activities were largely damaged as suggested by Fig.\u00a02 and relevant discussion. In the third case, the high RQ set-point (RQ\u00a0=\u00a00.8) caused a continuous rise-up and high level of DO (from 20 to 70%), which reduced the GDH activity and deteriorated glutamate production in turn. As a result, the adaptive RQ control (step-wise changing RQ set-points) in simultaneous consideration of DO change has to be adopted to satisfy the two conditions of obtaining an appropriate r4 flux and a reasonable lower DO level for realizing the BMC, as described in the previous section.\nFigure\u00a07 showed the changing patterns of the TCA metabolic flux (r4) when using the BMC control and the DO constant controls. Apparently, the BMC strategy controlled the TCA metabolic flux at an appropriate level just in between those of higher and lower DO levels. This realized the target of \u201cbalanced metabolism\u201d, which led to a higher glutamate production and almost completely repressed the lactate accumulation.Fig.\u00a07The calculated TCA metabolic fluxes of the BMC strategy and the DO constant controls\nTable\u00a01 indicated that CO2 yield (total CO2 released quantity versus total glucose consumed quantity) of the BMC was also just in between those of higher and lower DO constant control cases, which supported the BMC idea from the other side. However, it should be noted that, besides channel r4, the CO2 release also occurred at the other routes in the entire metabolic network, the shutdown of TCA cycle never meant the total CO2 production would cease. The effectiveness of the BMC strategy was also supported by the metabolic fluxes changes results shown in Fig.\u00a04. The moderate TCA metabolic flux (r4) simultaneously ensured a higher glutamate synthetic flux and an almost nil lactate formation flux.Table\u00a01The materials and carbon balances of the balanced metabolic control (BMC) and dissolved oxygen (DO) constant controlsBatch no.Glucose consumed (g)Glutamate produced (g)Lactate produced (g)Cellsa (g)CO2 released (g)Carbon balanceb (%)CO2\/Glucose (\u2212) (%)DO\u00a0=\u00a010%839.2384.0102.090.8352.9C-content335.6156.740.846.996.2101.528.6DO\u00a0=\u00a050%530.2265.23.277.1262.9C-content212.1108.21.339.871.7104.233.9RQ-BMC716.7406.40.460.7335.9C-content286.7165.90.1831.491.6100.831.9aThe dry cell weight, using the composition of strain C. glutamicum AJ-3462 (C4.71H8.02O1.92N, Ref.#11) as the calculation referencebRatio of the total production (glutamate, lactate, cell, and CO2) to total consumption (glucose)\nTable\u00a02 summarized the results and performance of the BMC and the DO constant controls. In consideration of the repeatability of the different control strategies, the experiments of constantly controlling DO at 10 and 50%, as well as BMC were repeated at least twice and then the averaged values were calculated for performance evaluation. The table evaluated the two different control strategies in terms of the four major fermentation performance index: maximal glutamate concentration, lactate accumulation quantity, glutamate productivity, and conversion rate from glucose to glutamate. The BMC showed an equivalent performance in the terms of productivity and conversion rate as compared with the best results of the DO constant controls. However, BMC strategy greatly improved the other two performance index: maximal glutamate concentration was increased by about 15% (with respect to DO\u00a0=\u00a010% case) and lactate accumulation was decreased by about 36% (with respect to DO\u00a0=\u00a050% case).Table\u00a02The summarized results of the BMC and DO constant controlBatch no.Conversion rate (%)Glutamate concentration (g\/L)Lactate concentration (g\/L)Productivity (g\/L\/h)Constant DO controls\u00a0050331 (DO\u00a0=\u00a010%)49.3883.0027.902.86\u00a0050526 (DO\u00a0=\u00a010%)43.6291.5025.502.69\u00a0DO\u00a0=\u00a010%, average46.5087.2526.452.77\u00a0050407 (DO\u00a0=\u00a050%)44.5174.201.002.56\u00a0050512 (DO\u00a0=\u00a050%)53.4072.800.802.43\u00a0DO\u00a0=\u00a050%, average48.9673.500.902.49\u00a0050427 (DO\u00a0=\u00a030%)55.5583.2018.602.19Balanced metabolic control by RQ (RQ-BMC)\u00a0050509 (RQ-BMC)56.71101.600.112.99\u00a0050516 (RQ-BMC)49.8098.801.042.60\u00a0RQ-BMC, average53.26100.200.582.80\nSummary\nA novel fermentation optimization method\u2014the \u201cbalanced metabolic control\u201d (BMC) strategy was proposed and successfully used by feedback controlling RQ to regulate the TCA metabolic flux rate at an appropriate level to achieve the metabolic balance among glycolysis, glutamate synthesis, and TCA metabolic flux for glutamate fermentation. The proposed BMC strategy greatly improved the fermentation performance in the two terms of maximal glutamate concentration and the lactate overflow repression. The maximal glutamate concentration increased by about 15% compared with the best results of the DO constant controls, and furthermore, the lactate overproduction occurred in the DO constant control cases could be completely relieved when using the BMC strategy. As a result, the proposed BMC strategy is expected to be applicable for optimization of other aerobic amino acids fermentations in potential.","keyphrases":["on-line optimization","glutamate fermentation","metabolic balancing","metabolic flux analysis","intracellular enzymes activities"],"prmu":["P","P","P","P","P"]}
{"id":"J_Mol_Med-4-1-2374882","title":"Involvement of (pro)renin receptor in the glomerular filtration barrier\n","text":"(Pro)renin receptor-bound prorenin not only causes the generation of angiotensin II via the nonproteolytic activation of prorenin, it also activates the receptor\u2019s own intracellular signaling pathways independent of the generated angiotensin II. Within the kidneys, the (pro)renin receptor is not only present in the glomerular mesangium, it is also abundant in podocytes, which play an important role in the maintenance of the glomerular filtration barrier. Recent in vivo studies have demonstrated that the overexpression of the (pro)renin receptor to a degree similar to that observed in hypertensive rat kidneys leads to slowly progressive nephropathy with proteinuria. In addition, the handle region peptide, which acts as a decoy peptide and competitively inhibits the binding of prorenin to the receptor, is more beneficial than an angiotensin-converting enzyme inhibitor with regard to alleviating proteinuria and glomerulosclerosis in experimental animal models of diabetes and essential hypertension. Thus, the (pro)renin receptor may be upregulated in podocytes under hypertensive conditions and may contribute to the breakdown of the glomerular filtration barrier.\nIntroduction\nWhen the (pro)renin receptor binds to the \u201chandle\u201d region of inactive prorenin, the receptor-bound prorenin gains its enzyme activity (ability to generate angiotensin I) without the proteolytic cleavage of the prosegment of prorenin in COS-7 cells [1, 2], presumably as a result of a conformational change. On the other hand, receptor-bound prorenin also triggers its own intracellular signaling pathways independent of the generated angiotensin II. Studies have shown that the stimulation of the (pro)renin receptor by renin\/prorenin activates tyrosine phosphorylation leading to the activation of extracellular signal-related protein kinases (ERK) [3] and upregulates transforming growth factor-\u03b21 (TGF-\u03b21) and matrix proteins without involving angiotensin II generation in human and rat mesangial cells [4]. In this review, we focus on the latest progress in elucidating the localization, regulation, and pathophysiological roles of the (pro)renin receptor in the kidneys.\nLocalization in the kidney\nStudies have demonstrated that the (pro)renin receptor protein and messenger ribonucleic acid (mRNA) are expressed in the mesangium cells of human kidneys [3, 4]. However, double immunohistochemical analyses using a polyclonal antirat (pro)renin receptor antibody demonstrated that the (pro)renin receptor was colocalized with a podocyte marker, podocalyxin, but not with a mesangium marker, Thy1.1, in rat kidneys [5] and electron microscopic analyses appeared to indicate the predominant presence of the rat (pro)renin receptor in podocytes, excluding the foot processes, and its absence in mesangial cells [5]. In addition, we recently detected (pro)renin receptor mRNA in cultured human podocytes (unpublished data). Within the glomerulus, podocytes play an important role in the maintenance of the glomerular filtration barrier and podocyte injury leads to proteinuria and initiates glomerulosclerosis resulting in the progressive loss of renal function [6]. Therefore, (pro)renin receptor in the podocytes may contribute to proteinuria and renal injury through an angiotensin-II-dependent pathway, an angiotensin-II-independent pathway, or both pathways in chronic kidney diseases.\nPossible upregulation of (pro)renin receptor expression under hypertensive conditions\nIn the kidneys of young hypertensive stroke-prone spontaneously hypertensive rats (SHRsp), an increase by two- or threefold in the mRNA expression of the (pro)renin receptor has been observed [7]. However, the kidneys of older hypertensive SHRsp did not show an elevation in (pro)renin receptor mRNA expression. Because the binding of renin to the (pro)renin receptor lowers the (pro)renin receptor mRNA level through a promyelocytic zinc-finger-protein-mediated negative feedback mechanism [8], a further increase in the plasma renin levels in old hypertensive SHRsp might inhibit the increase in (pro)renin receptor mRNA levels through a negative feedback loop. More recently, enhanced mRNA expression of the (pro)renin receptor was also observed in the clipped kidneys of Goldblatt hypertensive rats [9]. These results seem to provide evidence of the importance of hypertensive conditions in the regulation of (pro)renin receptor expression in the kidneys.\nEffects of (pro)renin receptor overexpression on the kidney\nRecent studies in transgenic rats overexpressing the human (pro)renin receptor gene nonspecifically by three to seven times demonstrated that glomerulosclerosis with proteinuria developed at 5\u20136months of age even in the absence of an elevation in blood pressure [10]. In the kidneys of 5- to 6-month-old transgenic rats, mitogen-activated protein kinase(s) (MAPK(s)) were activated without recognizable tyrosine phosphorylation of the epidermal growth factor receptor and the expression of TGF-\u03b21 was enhanced. The in vivo administration of angiotensin-converting enzyme (ACE) inhibitor did not inhibit the development of glomerulosclerosis, proteinuria, MAPK activation, or TGF-\u03b21 expression in the kidneys despite a significant decrease in the renal angiotensin II level. As shown in Fig.\u00a01, recombinant rat prorenin stimulated MAPK activation in human-receptor-expressed cultured cells but human receptor was unable to evoke the enzyme activity of rat prorenin. Thus, the overexpression of the human (pro)renin receptor elicits slowly progressive nephropathy via angiotensin-II-independent MAPK activation but not through the stimulation of angiotensin II generation. However, hypertension developed at 7months of age in transgenic rats overexpressing the human (pro)renin receptor gene exclusively in smooth muscle cells [11]. Because young transgenic rats of this strain show an enhanced expression of macula densa cyclooxygenase-2 that suppresses the tubuloglomerular feedback system and contributes to the inhibition of hypertension development [12], hypertension may not yet have developed at 6months of age or earlier. Therefore, we can interpret these observations as indicating that the hypertension may have occurred as a result of the nephropathy. However, further studies are needed to elucidate the involvement of the (pro)renin receptor in the development of hypertension.\nFig.\u00a01Both rat and human prorenin stimulate human (pro)renin receptor (h(P)RR)-dependent intracellular signals, but the h(P)RR activates human prorenin but does not activate rat prorenin\nReceptor-bound prorenin in diabetic kidneys\nRecombinant prorenin binds to recombinant (pro)renin receptor expressed compulsorily on the cell surfaces of COS-7 cells and prorenin bound to the receptor on the cell surfaces exerts renin activity without undergoing any change in its molecular weight. However, synthetic peptides containing the amino acid sequence corresponding to the \u201chandle\u201d region of the prorenin prosegment competitively inhibit prorenin from binding to cell membrane receptors [13]. In the absence of experimental data showing the blockade of renin signaling by the handle region peptide, we believe that the possible inhibition of receptor signals and angiotensin II generation by the handle region peptide should be interpreted as indicating the blockade of prorenin binding.\nDiabetic animals or patients have lower renin levels and higher prorenin levels than normal healthy controls [14, 15]. The handle region peptide was administered to rats with streptozotosin-induced type I diabetes and the tissue angiotensin I and II levels in their kidneys, and the development of nephropathy were followed [16]. The administration of the handle region peptide for 6months significantly inhibited the increase in renal angiotensin II levels and the development of proteinuria and glomerulosclerosis, suggesting that the nonproteolytic activation of prorenin bound to the (pro)renin receptor plays an important role in the development of nephropathy. Although renal mRNA expression of the (pro)renin receptor was similar in the control and diabetic rats [17], nonproteolytically activated prorenin increased in the kidneys of the diabetic rats, and this increase was inhibited by the handle region peptide. In addition, the renal total renin content remained unchanged during the treatment with the handle region peptide. These results suggested that the handle region peptide inhibits the conformational change in prorenin that leads to prorenin\u2019s enzymatic activation but does not alter the prorenin levels. Because the mRNA expression of cathepsin B, which processes the conversion of prorenin to renin, simultaneously decreased in the kidneys of the diabetic rats, prorenin may have accumulated in their kidneys. Also, the amount of prorenin released to the circulatory system may be elevated in diabetes leading to an elevation in receptor-bound prorenin as a result of the increased prorenin levels with no change in (pro)renin receptor expression.\nTo investigate the contribution of angiotensin-II-independent, (pro)renin-receptor-dependent signal pathways to diabetic end-organ damage, the therapeutic effects of infusion were compared with the effect of the deletion of angiotensin II type 1a receptor (ATKO) and ACE inhibition on streptozotosin-induced type I diabetes [18]. Deletion of the angiotensin II type 1 gene attenuated the development of glomerulosclerosis and proteinuria but did not prevent these developments. ACE inhibitor also failed to prevent diabetic nephropathy despite the normalization of renal angiotensin II. On the other hand, the handle region peptide almost completely prevented the development of these conditions despite the absence of an increase in renal angiotensin levels. Of note, MAPKs were markedly activated in diabetic ATKO animals and, while the handle region peptide almost completely inhibited the activation, ACE inhibition failed to even ameliorate it. These results indicate that inhibition of the angiotensin-II-mediated pathway had a little therapeutic effect on nephropathy in the diabetic rats, whereas the blockade of receptor-bound prorenin seemed to suppress the two major pathways for diabetic nephropathy: excessive production of angiotensin II in the kidneys and renal MAPK activation. In this experiment, the renal angiotensin II levels of diabetic mice treated with the handle region peptide were similar to those of untreated wild-type mice. Because the handle region peptide did not seem to inhibit the binding of renin to the (pro)renin receptor, the physiological production of renal angiotensin II induced by renin or receptor-bound renin was probably unaffected by the handle-region-peptide-induced inhibition of receptor-bound prorenin. In addition, the handle region peptide did not influence the plasma levels of renin and prorenin (receptor-unbound free renin and receptor-unbound free prorenin) or blood pressure. Thus, the handle region peptide also had no effect on the physiology of the circulating renin\u2013angiotensin system.\nWe recently found that the handle region peptide also leads to the regression of nephropathy in rats with streptozotosin-induced type I diabetes [17]. Diabetes was induced by streptozotosin injection in 4-week-old rats. Increased urinary protein excretion and glomerulosclerosis were observed 12weeks later and the administration of the handle region peptide or vehicle was started. While urinary protein excretion and glomerulosclerosis had progressed in vehicle-treated 28-week-old diabetic rats, treatment with the handle region peptide significantly reduced the proteinuria and glomerulosclerosis from the levels observed at the start of treatment. This reversal of the glomerulosclerosis that had already developed in the diabetic rats suggested that receptor-bound prorenin contributes not only to the onset of nephropathy but also to its progression.\nThe increased plasma prorenin level might be supplied by the kidneys in which high glucose levels might inhibit the processing of prorenin to renin as described above. In addition, extrarenal tissues might also be sources of the increased prorenin level under high glucose conditions because plasma prorenin is detectable even after a bilateral nephrectomy [15]. Thus, high glucose levels may contribute to an increase in the plasma prorenin level via both renal and extrarenal mechanisms, and the elevated plasma prorenin level might trigger an increase in receptor-bound prorenin leading to nephropathy. These mechanisms may explain why the handle region peptide inhibited the development and progression of nephropathy in animal models of diabetes without affecting the high blood glucose level. Further study is needed to clarify these points.\nReceptor-bound prorenin in hypertensive kidneys\nSHRsp rats have high plasma levels of prorenin and renin and are used as a model of primary hypertension [19]. In addition to the increased mRNA expression of the (pro)renin receptor as described above, elevated angiotensin I and II levels and glomerulosclerosis with proteinuria have been observed in the kidneys of young hypertensive SHRsp; administration of the handle region peptide significantly suppressed these changes without affecting the development of hypertension [7]. In addition, profibrotic changes in morphology were observed in the clipped kidneys of Goldblatt hypertensive rats with enhanced mRNA expression of the (pro)renin receptor [9]. Therefore, the increased expression of the (pro)renin receptor may play an important role in the development of renal tissues showing characteristic signs of hypertensive damage. Because recent studies have demonstrated the presence of angiotensin-II-independent, (pro)renin-receptor-dependent MAPK pathways in mesangial cells [20] and vascular smooth muscle cells [21], the beneficial effects of the handle region peptide may be mediated by both of these two major pathways that are stimulated by receptor-bound prorenin. Thus, receptor-bound prorenin also contributes to the development of hypertensive end-organ damage. This was recently supported, in part, by Susic et al., who reported that the handle region peptide significantly reduced the left ventricular mass even though the myocardial collagen content remained unchanged [22].\nPossible inhibition of receptor-bound prorenin by high levels of renin\nFigure 2 shows the binding of renin and prorenin to the (pro)renin receptor. Because both renin and prorenin bind to the (pro)renin receptor [3], increased renin levels may increase the amount of renin and decrease the amount of prorenin that binds to the receptor. A recent review reported that the handle region peptide offered no benefit to renin-transgenic models or to a model with renovascular hypertension [23]. Because the ratio of renin to prorenin must be higher in these models than in control models, renin\u2014but not prorenin\u2014that has bound to the receptor must contribute to the pathogenesis that occurs in these models. Because no data showed the blockade of renin signaling by the handle region peptide, the handle region peptide did not benefit the renin-transgenic models or the model with renovascular hypertension [23]. In contrast, the handle region peptide greatly benefited diabetic animals [16\u201318]. Because diabetic animals are known to have a low ratio of renin to prorenin [15], they have increased levels of prorenin bound to the receptor and decreased levels of renin bound to the receptor. Thus, the handle region peptide was thought to benefit in vivo diabetic animals by reducing the amount of prorenin that bound to the receptor. In addition, the handle region peptide had a partial benefit on SHRsp [7, 24] and SHR [22] animals fed a high-salt diet. This benefit was reduced by changing from a high-salt diet to a low-salt diet (unpublished preliminary data), suggesting that dietary salt might alter the levels of prorenin bound to the receptor by regulating the renin levels. Thus, the efficiency of the handle region peptide may depend on the ratio of renin to prorenin.\nFig.\u00a02Renin and prorenin competitively bind to the (pro)renin receptor in vivo and to the (pro)renin receptor expressed on the cell surfaces of COS-7 cells\n(Pro)renin receptors in cultured cells\nUsing vascular smooth muscle cells (VSMC) harvested from human-(pro)renin-receptor-transgenic rats, Batenburg et al. recently demonstrated that prorenin binds to the (pro)renin receptor and that receptor-bound prorenin exerts a catalytic activity but that renin does not bind to the receptor or increase its own catalytic activity [25], consistent with the results of a previous study using human VSMC [21]. The structural difference between renin and prorenin arises from 43 amino acids in the prorenin prosegment and prorenin acquires its catalytic activity without changing its molecular weight when a specific protein binds to the \u201chandle\u201d region of the prorenin prosegment [1]. Nevertheless, the handle region decoy peptide, which inhibited the binding of prorenin to the receptor in a test tube and to receptors expressed on the cell surfaces of COS-7 cells [13], failed to inhibit prorenin\u2019s acquisition of catalytic activity by binding to the receptor [25]. A recent study using a human cultured cell line clearly demonstrated that the 39-kDa full-length (pro)renin receptor was predominantly present in the intracellular endoplasmic reticulum [8]. Because both renin and prorenin can permeate the cell membrane [8] but the handle region peptide cannot, the handle region peptide might be incapable of inhibiting intracellular binding between the (pro)renin receptor and its ligands (Fig.\u00a03).\nFig.\u00a03Possible fragmentation of (pro)renin receptor in cultured cells\nAlthough (pro)renin receptors of VSMC harvested from human-(pro)renin-receptor-transgenic rats were not bound to renin [25], the same authors also showed that both renin and prorenin bind to the (pro)renin receptor and stimulate receptor-dependent ERK pathways in U937 monocytes [26]. In addition, a renin-stimulated, (pro)renin-receptor-dependent ERK pathway was observed in rat and human mesangial cells [4, 20], whereas the stimulation of the receptor by prorenin in cultured cardiomyocytes resulted in the activation of p-38 MAPK, but not ERK [27], and renin stimulation caused the interaction between the receptor and the transcription factor promyelocytic zinc finger protein in HeLa-S3 cells and HEK293 cells [8]. These results suggest that the ability to bind renin and prorenin and the intracellular pathways involving these proteins may be influenced by the species and condition of the cultured cells. In addition, the presence of (pro)renin receptors with molecular weights smaller than the 39kDa of the full-length receptor in cultured cells has been reported in abstracts presented at several meetings. Based on the results of all these studies regarding the (pro)renin receptor in cultured cells, we propose the possible fragmentation of the (pro)renin receptor (Fig.\u00a03). Namely, when the 39-kDa full-length (pro)renin receptor in the endoplasmic reticulum is released to the cytoplasm, it may divide into a renin-binding part and a remnant part. In the cytoplasm of VSMC and cardiomyocytes, the remaining smaller (pro)renin receptor without the renin-binding part would be capable of binding prorenin but would be incapable of binding renin. However, in the cytoplasm of the mesangium and monocytes, the smaller receptor might readhere to the renin-binding part and resume its ability to bind renin. When the smaller receptor in the cytoplasm reaches the cell membrane, it might further divide into the prorenin-binding part, recognizing the \u201chandle\u201d region, and a remnant part. The prorenin-binding part would be released to the extracellular area and the smallest remnant receptor would remain at the cell membrane. Because the smallest receptor does not contain either the renin-binding part or the prorenin-binding part, it would no longer be capable of binding renin or prorenin. When the prorenin bound to the smaller receptor also reaches the cell membrane, it would divide into three parts: the prorenin prosegment adhered to the prorenin-binding part, renin, and the remaining smallest receptor. Because the former two proteins are released to the extracellular area, angiotensin I generation from external angiotensinogen would occur there. Thus, under cultured cell conditions, the (pro)renin receptor may be fragmented depending on the cell species. Further studies are needed to verify the possible fragmentation of the (pro)renin receptor.\nConclusion\nIn the glomerulus of the kidneys, the (pro)renin receptor is not only present in the mesangium but is also abundant in the podocytes and its expression was increased by two- to threefold in the kidneys of some animal models of hypertension. A transgene-induced overexpression of human (pro)renin receptor in rats resulted in the development of slowly progressive nephropathy with proteinuria. In addition, the handle-region-peptide-induced inhibition of receptor-bound prorenin significantly inhibited the development and progression of proteinuria in experimental diabetic and hypertensive animal models. Thus, the glomerular (pro)renin receptor appears to play an important role in the regulation of the glomerular filtration barrier.","keyphrases":["prorenin","angiotensin","nonproteolytic activation","podocytes","mitogen-activated protein kinases"],"prmu":["P","P","P","P","R"]}
{"id":"Graefes_Arch_Clin_Exp_Ophthalmol-3-1-2082068","title":"Ocular blood flow in patients with obstructive sleep apnea syndrome (OSAS)\n","text":"Background Sleep-related disorders are among the important risk factors for neurovascular diseases. Obstructive sleep apnea syndrome (OSAS) is characterized by snoring, excessive daytime sleepiness, and insomnia. Our aim was to investigate the presence of glaucoma in patients with OSAS and to reveal vascular pathology related to the pathogenesis of glaucoma in those patients.\nObstructive sleep apnea syndrome (OSAS) is characterized by snoring, excessive daytime sleepiness, and insomnia. Epidemiological studies revealed a prevalence between 2 and 20%. Among the risk factors, obesity, male gender, upper respiratory tract abnormality, consumption of alcohol, snoring, and thick neck are worth mentioning [5, 11, 12].\nIn previous studies, retinal vascular tortuosity and congestion [7], floppy eyelid syndrome [3, 18, 22, 27], keratoconus [22], papilledema, and optic neuropathy [2, 14, 20] have been described in patients with OSAS. It has been stressed that OSAS may be a predisposing condition for anterior optic neuropathy [14]. In a study, Mojon et al., investigating 69 patients with OSAS, found the incidence of glaucoma to be 7.2% [21]. Onen et al. found a high incidence (14.6\u201347.6%) of primary open-angle glaucoma (POAG) in patients with sleep-disordered breathing [24]. Similarly, in another study by Mojon et al., the prevalence of low-tension glaucoma (LTG) in patients with OSAS was found to be 50% [23]. Recently, in a study from Turkey, it was stated that OSAS is correlated with a proportional decrease in retinal nerve fiber layer (NFL) [15].\nThe study\u2019s aim was to investigate the ocular blood flow in patients with OSAS since pathological ocular blood flow has been suggested as a major mechanism in the etiology of glaucoma and also to detect the glaucoma prevalence in the study group.\nPatients and methods\nPatients were recruited from those who were referred for suspected OSAS to the \u201cSleep Unit\u201d of the Neurology Clinic (Erciyes University Faculty of Medicine, Kayseri, Turkey) between December 2003 and July 2004. These were consecutively all patients referred to the Sleep Unit during the 7-month period. A total of 42 patients were evaluated during this period. Among these, 31 patients fulfilled the inclusion criteria. The remaining 11 patients were excluded from the study because they had at least one of the exclusion criteria (such as those who had previous eye surgery or laser treatment, who had any anterior or posterior segment disease or a history of ocular trauma, those with secondary glaucoma, history of chronic steroid use, history of shock, and diabetes mellitus).\nThe study included 31 patients with OSAS and 25 healthy controls. Diagnosis of OSAS was made by the Sleep Unit of the Neurology Clinic.\nAtherosclerotic risk factors likely to affect ocular blood flow such as hypertension, cigarette smoking, and high blood cholesterol level were investigated in all subjects; since it was impossible to completely eliminate these factors in both groups, the distribution of these risk factors was investigated and it was found to be similar between the groups (chi-square: 1.97; p\u2009=\u20090.16).\nCriteria for the diagnosis of POAG were as follows (all four of the criteria listed had to be present for inclusion in the study):\nA cup to disc ratio (c\/d) over 0.5 or difference of c\/d between two eyes >0.2 with thinning of the neuroretinal rim; a careful search was made to detect the presence of any disc hemorrhages to strongly support the diagnosis. The c\/d ratio and the optic disc evaluation were derived with a +90 diopter aspherical Volk lens by two independent examiners who were masked to the condition of the patient.Any of the detected visual field defects such as localized defects, paracentral scotoma, Bjerrum scotoma, nasal step, temporal sector defect, and diffuse defect which cannot be explained by any neurologic or fundus lesion.Open iridocorneal angle.An intraocular pressure (IOP) >21\u00a0mmHg without treatment.\nDiagnosis of normotensive glaucoma was made with criteria similar to those stated for POAG except for the fact that IOP was <21\u00a0mmHg without treatment.\nThe control group consisted of 25 age- and sex-matched healthy subjects who were recruited from those attending the eye clinic for conditions such as refractive errors, etc. Control subjects had to meet all of the following ophthalmological criteria for inclusion in the study:\nNo IOP elevation over 21\u00a0mmHgNo evidence of glaucomatous optic nerve appearance (no disc hemorrhages) and a c\/d ratio less than 0.5Normal anterior chamber angle on slit lamp and gonioscopic examinationNormal visual field test resultsNo previous history of antiglaucomatous drug usage, ocular trauma, ocular surgery, or laser therapy\nPatients underwent orbital color Doppler ultrasonography (Toshiba Power Vision 6000, Osaka, Japan) at the Radiology Unit of the University. An 11-MHz linear surface probe was used and the measurements were performed between 12:00 a.m. and 13:00 p.m. The patient was supine; eyes were closed and aimed at the ceiling under the closed eyelids. Systolic blood velocity (PSV) and end-diastolic blood velocity (EDV) were calculated in the ophthalmic artery and central retinal artery; resistivity index (RI) was calculated for the central retinal artery (CRARI) and ophthalmic artery (OARI; RI=PSV-EDV\/PSV).\nPolysomnography was performed at the Sleep Research Unit of the University; it was constituted by two EEG channels, two EOG channels, one EMG channel recording from the submental muscle, one nasal current channel, one thoracic motion channel, one abdominal motion channel, one oximeter channel, a microphone, two leg movement channels from the right and left anterior tibialis muscles, and video recordings made between 2300 and 0700 hours. The parameter used was the apnea-hypopnea index (AHI). Diagnosis of OSAS was made when the AHI was over 5. When the AHI was between 5 and 15, OSAS was regarded as \u201cmild,\u201d between 16 and 30 as \u201cmoderate,\u201d and when it was over 30, it was regarded as \u201csevere.\u201d\nAll the subjects in the study underwent a complete ophthalmological examination including visual acuity, slit lamp biomicroscopy, IOP measurement, gonioscopy, and fundus examination. Perimetric examination was made with the Octopus 500 EZ perimeter (Interzeag AG, Schlieren, Switzerland); stimulus size: white, III, full threshold strategy was used. Only phases 1 and phase 3 of the G1 program were used. The Octopus perimeter has a prior education (preparation) program for possibly naive patients, which was used for all patients who were naive to the examination.\nA factor based on the number of false-positive and false-negative replies to the stimuli, and their ratio (<13%), was used to determine reliability. Unreliable results were retested on another day.\nDisc analysis was made with the Heidelberg Retinal Tomograph II (Heidelberg Engineering GmbH 2001, software version 3.0, Heidelberg, Germany). Mean nerve fiber layer thickness, rim area, rim volume, and linear c\/d ratio were noted.\nAll statistical analyses were carried out using statistical software (SPSS, version 10.0 for Windows, SPSS Inc., Chicago, IL, USA). Differences were considered significant at p\u2009<\u20090.05. Student\u2019s t-test was used for intergroup analysis; chi-square, and Kruskal-Wallis, and Mann-Whitney U tests for nonparametric tests; analysis of variance (ANOVA) was used for the correlation analysis.\nThe study was conducted in accordance with the Declaration of Helsinki and was approved by an Institutional Ethics Committee.\nResults\nThis study included 31 patients (7 female, 24 male) and 25 controls (7 female, 18 male). There was no significant difference among the groups in terms of sex distribution (Yates corrected chi-square\u2009=\u20090.02; p\u2009=\u20090.88). While the mean age of the patient group was 52.07\u2009\u00b1\u20099.35\u00a0years, it was 50.44\u2009\u00b1\u20096.38\u00a0years in the control group; there was no statistically significant difference between age distributions among the groups (p\u2009=\u20090.13, unpaired Student\u2019s t-test). There was no statistically significant difference between the two groups with respect to the distribution of arteriosclerotic risk factors and hypertension (Yates corrected chi-square\u2009=\u20091.97; p\u2009=\u20090.16).\nThe prevalence of glaucoma was 4 of 31 OSAS patients (12.9%). In two of these four patients, both eyes were affected. One of these patients had normotensive glaucoma and one had POAG. The other two patients had normotensive glaucoma only in one eye. The other eyes had large cupping without any visual field defect. All of these four patients belonged to the severe OSAS group (AHI\u2009>\u200930).\nThere was no significant difference between mild, moderate, or severe OSAS patients in terms of OARI or CRARI values (Kruskal-Wallis test p\u2009=\u20090.961, p\u2009=\u20090.774).\nThe patient and control groups did not differ significantly in terms of CRARI and OARI or IOP (p\u2009>\u20090.05; Table\u00a01).\nTable\u00a01Comparison of patients and controls with respect to intraocular pressure (IOP), ophthalmic artery resistivity index (OARI), and central retinal artery resistivity index (CRARI)ParametersPatient eyes (mean \u00b1 SD) (n\u2009=\u200962)Control eyes (mean \u00b1 SD) (n\u2009=\u200950)tpIOP (mmHg)13.37\u2009\u00b1\u20094.3714.00\u2009\u00b1\u20094.31\u22121.000.32OARI0.72\u2009\u00b1\u20090.060.71\u2009\u00b1\u20090.060.7690.44CRARI0.69\u2009\u00b1\u20090.060.70\u2009\u00b1\u20090.05\u22120.8030.42\nThere was a statistically significant difference between median IOP values of mild (10.5\u00a0mmHg; range: 9\u201318\u00a0mmHg) and severe (14.5\u00a0mm Hg; range: 9\u201327\u00a0mmHg) OSAS patients (p\u2009=\u20090.031; Mann-Whitney U test).\nWhen correlation was analyzed between variables, a statistically significant correlation was found between OARI and MD values (r\u2009=\u20090.339, p\u2009=\u20090.007, Fig.\u00a01), CRARI and MD values (r\u2009=\u20090.246, p\u2009=\u20090.05, Fig.\u00a02), CRARI and LV values (r\u2009=\u20090.253, p\u2009=\u20090.048, Fig.\u00a03), and AHI and IOP values (r\u2009=\u20090.426, p\u2009=\u20090.001, Fig.\u00a04).\nFig.\u00a01The positive correlation between ophthalmic artery resistivity index (OARI) and mean defect (MD) in the patient group is statistically significant (r\u2009=\u20090.339, p\u2009=\u20090.007)Fig.\u00a02The positive correlation between central retinal artery resistivity index (CRARI) and mean defect (MD) in the patient group is statistically significant (r\u2009=\u20090.246, p\u2009=\u20090.05)Fig.\u00a03The positive correlation between central retinal artery resistivity index (CRARI) and loss variance (LV) in the patient group is statistically significant (r\u2009=\u20090.253, p\u2009=\u20090.048)Fig.\u00a04The positive correlation between apnea-hypopnea index (AHI) and intraocular pressure (IOP) in the patient group is statistically significant (r\u2009=\u20090.426, p\u2009=\u20090.001)\nDiscussion\nThis study aimed to investigate the prevalence of glaucoma in patients with OSAS and also to investigate the association of ocular blood flow with OSAS and glaucoma. In previous studies, while the prevalence of glaucoma was found to be 2% [8], no correlation was obtained between respiratory disturbance index (RDI) and IOP. However, many other studies found higher incidences and a positive correlation between IOP and RDI. For instance, Mojon et al. stated that there was a positive correlation between IOP and RDI in 114 glaucoma suspects [21]. In our study, similar to Mojon et al.\u2019s study, there was a significantly positive correlation between IOP and AHI (r\u2009=\u20090.426, p\u2009=\u20090.001). In another study by Robert et al., which was carried out with 69 patients with OSAS, prevalence was found to be 7.2% [27]. At the same time, a strong positive correlation was found between OSAS and lid hyperlaxity [27]. In that study, incidence was 8.7% and six patients were referred for glaucoma treatment [27]. In the study of McNab et al. in which 8 patients with floppy eyelid syndrome were investigated for OSAS and 20 other patients with known OSAS were examined for floppy eyelid syndrome and other possibly associated ocular features, it was found that 1 of 8 patients with floppy eyelids had normotensive glaucoma [18]. Onen et al. found a high prevalence of sleep-disordered breathing (SDB) in POAG patients [24]. When only snoring was taken into consideration, the incidence of \u201conly snoring\u201d was 47.6%, \u201csnoring and excessive daytime sleeping\u201d was 27.3%, and \u201csnoring, daytime sleepiness, and insomnia\u201d was 14.6% [24]. Marcus et al. also stressed that SDB may be a risk factor for low-tension glaucoma (LTG) [17]. Mojon et al. found that the prevalence of OSAS was 50% in LTG patients aged between 45 and 64\u00a0years and 63% in those over 64\u00a0years of age [23]. However, Girkin et al. [9] reported an odds ratio of 2.02 for OSAS patients to develop glaucoma.\nSimilar to most of the above-mentioned studies, the authors of the present study also found a high prevalence of glaucoma (12.9%) in the study group of 31 patients suggesting that OSAS in conjunction with a vessel disease is an important risk factor for glaucoma.\nThe correlation between RDI and IOP was also investigated by Geyer et al. [8]; the authors stated that there was no statistically significant correlation [8]. On the contrary, Mojon et al. found a positive correlation between two variables [21]. We found a statistically significant difference between AHI and IOP in mild and severe OSAS patients. In our study, all four glaucomatous patients were in the severe OSAS group with an AHI\u2009>\u200930. In the patient group, there was a positive correlation between OARI and MD as well as CRARI and MD, and LV. Tsang et al. also found that moderate to severe OSAS patients were associated with a higher incidence of visual field defects [29]. These results suggest that visual field defects may be caused by optic nerve perfusion insufficiency and that when RI increases, field defects also increase. However, there was no significant difference between OARI and CRARI values of patients and healthy subjects. This may suggest that the effects of OSAS on body systems are seen not only during sleep but also during daytime; pathological changes may be more clearly documented if recordings and ultrasonography can be made during the sleeping period with a suitable technique to be developed in the future. Hypoxia in OSAS is of intermittent character and the thinning may occur in the progression of the disease in such patients. Long-term follow-up of these patients in terms of NFL thickness changes over time may clarify this issue. Purvin et al. suggested that intracranial pressure increases as a result of episodic nocturnal hypoxemia and hypercapnia, thereby leading to papilledema and increasing the risk of visual loss [25]. Since intracranial pressure increase in these patients is of intermittent character, cerebrospinal fluid pressure may be normal. In our group of patients, visual field defects were detected in ten patients despite normal ophthalmological examination. This may be due to optic nerve damage caused by cerebral ischemia and intermittent intracranial pressure increase in such patients.\nLow diastolic pressure and systemic hypertension are reported to be strongly associated with glaucoma [4, 6, 25, 26]. Some authors suggested that physiological nocturnal hypotension, in the presence of other vascular risk factors, can bring the optic nerve head circulation under the critical levels and thus play a role in the pathogenesis of anterior ischemic neuropathy and glaucoma [1, 10, 16, 28].\nThe pathogenesis of LTG is not clear; however, optic nerve head hypoperfusion has been suggested as the cause [30]. Hayreh stated that many systemic and ocular paralyses occur at nighttime and may be realized when the patients awaken early in the morning [13]. Furthermore, Hayreh [13] and Meyer [19] suggested that nocturnal hypotension might play a role in the pathogenesis of LTG.\nIn conclusion, in our group of patients with OSAS, a high prevalence was found (12.9%\u2009=\u20094 glaucoma patients of 31 OSAS patients) and it is interesting to note that all four of the glaucoma patients were in the severe OSAS group. The positive correlation detected between OARI and MD, and also between CRARI and MD as well as LV, suggests that visual field defects may be due to optic nerve perfusion defects and that these field defects also increase as the RI increases. The presence and progression of glaucoma should be investigated particularly in patients with severe OSAS in the long-term follow-up, and changes in retinal nerve fiber layer thickness and ocular Doppler ultrasonographic findings should also be monitored in such patients.","keyphrases":["ocular blood flow","obstructive sleep apnea syndrome","glaucoma","polysomnography","apnea-hypopnea index"],"prmu":["P","P","P","P","P"]}
{"id":"Antonie_Van_Leeuwenhoek-3-1-2140096","title":"Comparative genomics of Streptomyces avermitilis, Streptomyces cattleya, Streptomyces maritimus and Kitasatospora aureofaciens using a Streptomyces coelicolor microarray system\n","text":"DNA\/DNA microarray hybridization was used to compare the genome content of Streptomyces avermitilis, Streptomyces cattleya, Streptomyces maritimus and Kitasatospora aureofaciens with that of Streptomyces coelicolor A3(2). The array data showed an about 93% agreement with the genome sequence data available for S. avermitilis and also showed a number of trends in the genome structure for Streptomyces and closely related Kitasatospora. A core central region was well conserved, which might be predicted from previous research and this was linked to a low degree of gene conservation in the terminal regions of the linear chromosome across all four species. Between these regions there are two areas of intermediate gene conservation by microarray analysis where gene synteny is still detectable in S. avermitilis. Nonetheless, a range of conserved genes could be identified within the terminal regions. Variation in the genes involved in differentiation, transcription, DNA replication, etc. provides interesting insights into which genes in these categories are generally conserved and which are not. The results also provide target priorities for possible gene knockouts in a group of bacteria with a very large numbers of genes with unknown functions compared to most bacterial species.\nIntroduction\nStreptomyces are a group of aerobic high %G+C Gram positive bacteria that undergo complex differentiation to form filamentous mycelium, aerial hyphae and spores. In addition, they produce a broad range of secondary metabolites including antibiotics, antiparasitic agents, herbicides, anti-cancer drugs and various enzymes of industrial importance. Two Streptomyces species have had their complete genome sequences published, namely the model organism Streptomyces coelicolor (%G+C\u00a0=\u00a072.1) and avermictin producer Streptomyces avermitilis (%G+C\u00a0=\u00a070.7) (Bentley et\u00a0al. 2002; Ikeda et\u00a0al. 2003). Two important aspects of the genomes structures of Streptomyces were supported by sequence data. Firstly, that the genome size of Streptomyces is large compared to other bacteria; 8,667,507 basepairs for S. coelicolor (7,825 protein coding genes) and 9,025,608\u00a0bp (7,577 protein coding genes) for S. avermitilis. Secondly, that the genomes of these two species are linear and both ends contain unique terminal inverted repeats that probably covalently bind a terminal protein. Terminal inverted repeats and covalently bound terminal proteins are not found in the limited number of other bacteria that have linear chromosomes such as Borrelia burgdorferi and Agrobacterium tumefaciens and, up to the present, seem to be unique to the Streptomyces and perhaps other Actinobacteria (Lin et\u00a0al. 1993; Chen et\u00a0al. 2002; Goodner et\u00a0al. 1999; Huang et\u00a0al. 2004). Over 2,500 Streptomyces strains are present in the Ribosomal Database Project (http:\/\/www.rdp.cme.msu.edu), over 1,500 are available at the American Type Culture Collection (http:\/\/www.atcc.org\/) and many more are held in both public and private culture collections throughout the world. Analysis of the small subunit ribosomal RNA gene sequences of Streptomyces confirms that they form a monophyletic clade, but one with considerable diversity. In addition, there is significant gene diversity at the interspecies level across the genomes of both completely sequenced Streptomyces with 2,291 gene unique to S. avermitilis and 2,307 genes unique to S. coelicolor.. This makes them particularly interesting targets for comparative genomic studies. In this study we chose four species to begin an analysis of the genomic diversity of the Streptomyces. S. avermitilis was chosen because of the availability of the complete genome sequence of this species, while Streptomyces maritimus was chosen because of its intermediate position in terms of phylogeny within the Streptomyces. Streptomyces cattleya was chosen because, based on small subunit ribosomal RNA sequence, this species is phylogenetically quite divergent from S. coelicolor and branches near the root of the Streptomyces clade. Streptomyces cattleya is a \u03b2-lactam producing species. Finally, Kitasatospora aureofaciens was chosen as this genus is very closely related to the Streptomyces.\nThe availability of two microarrays for S. coelicolor (Lum et\u00a0al. 2004; Huang et\u00a0al. 2001; Vinciotti et\u00a0al. 2005; http:\/\/www.surrey.ac.uk\/SBMS\/Fgenomics\/Microarrays\/index.html) makes possible a comparative genomic analysis of Streptomyces species. The genes that make up the genome of S. coelicolor have been classified based on scheme of Riley and colleagues for E. coli and modified for S. coelicolor (http:\/\/www.sanger.ac.uk\/Projects\/S_coelicolor\/scheme.shtml). A microarray analysis of the genomes of these Streptomyces using the S. coelicolor microarray is able to provide a wide ranging comparative analysis of the conserved genome content of these Streptomyces. This type of approach, where a heterologous microarray is used to analyze the genome content of a range of strains or species, has been successfully used in a wide range of organisms (Akman and Aksoy 2001; Akman et\u00a0al. 2001; Behr et\u00a0al. 1999; Chan et\u00a0al. 2003; Cho and Tiedje 2001; Dorrell et\u00a0al. 2001; Dziejman et\u00a0al. 2002; Fitzgerald et\u00a0al. 2001; Gill et\u00a0al. 2002; Leonard et\u00a0al. 2003; Murray et\u00a0al. 2001; Porwollik et\u00a0al. 2002; Salama et\u00a0al. 2000; Israel et\u00a0al. 2001; Rajashekara et\u00a0al. 2004). The strains analyzed using this approach range from intraspecies comparisons such as Campylobacter jejuni, Vibrio cholerae and Staphylococcus aureus (Dorrell et\u00a0al. 2001; Dziejman et\u00a0al. 2002; Fitzgerald et\u00a0al. 2001) to interspecies comparisons such as Sodalis glossinidiusversus an Escherichia coli array, Salmonella bongori versus a Salmonella enterica array, Shewanella species versus Shewanella oneidensis and E. coli arrays and Brucella species versus a Brucella melitensis array (Akman et\u00a0al. 2001; Chan et\u00a0al. 2003; Murray et\u00a0al. 2001; Rajashekara et\u00a0al. 2004).\nIn this study, we used both versions of the S. coelicolor genome microarrays to compare the gene complements of the three Streptomyces species and one Kitasatospora species. The genus Kitasatospora is closely related to the genus Streptomyces in terms of morphology, chemical taxonomy and small subunit ribosomal RNA sequence analysis. Thus, the choice of a species from this genus acts as potential outgroup in terms of overall genome structure. In terms of genes that are conserved, the types of genes of particular interest include genes involved in secondary metabolism, genes involved in chromosome replication, genes in the terminal regions of the chromosome, sigma factors, genes involved in differentiation and hypothetical genes. In terms of gene absence, the distribution of such genes along the chromosome and the apparent absence of any major housekeeping genes in a specific species are of interest. This information provides insights into genes that make up the core complement for a member of the Streptomyces and into which genes are central to defining a Streptomyces species.\nMaterials and methods\n16S phylogeny\nThis was carried out on selected small subunit 16S ribosomal RNA gene sequences obtained from Ribosomal Database Project-II Release 9 (http:\/\/www.rdp.cme.msu.edu\/index.jsp) and aligned using CLUSTALX (Thompson et\u00a0al. 1997). The analysis was carried out using Neighbor-Joining algorithm from the same program. In the case of S. maritimus, the taxonomy of the strain was confirmed by DNA sequencing of the 16S ribosomal RNA gene.\nArrays\nTwo series of arrays that cover about 97% of the complete genome of Streptomyces coelicolor A3(2) (Lum et\u00a0al. 2004; http:\/\/www.surrey.ac.uk\/SBMS\/Fgenomics\/Microarrays\/index.html) were used in this study. Both arrays are PCR arrays, but from different sources, namely Stanford University, USA and the University of Surrey, UK and made up of different PCR products. The Stanford array as used in this study contained sequences covering 7603 open reading frames. The Surrey microarray is made up of 7,758 unique PCR amplified sequences, 7,563 from the chromosome and 195 from SCP1. There are an additional 376 non-unique, alternative and cross-hybridizing sequences that are also spotted on to the array together with no probe spots and control spots. The two types of arrays were used to improve validation with a system using heterologous hybridization; however, only the University of Surrey array was hybridized and analyzed in duplicate. The major difference between the two arrays was that the Surrey array did not include a number of transposition element related genes, although there were other overlap differences. The sequences of the PCR products are not available for either array due to intellectual property protection requirements.\nStrains and growth conditions\nS. coelicolor A3(2) (SCP1+) 104, S. avermitilis ATCC 31267, S. cattleya ATCC 35852, S. maritimus Yang-Ming and K. aureofaciens ATCC 10762 were used in these studies. Fresh spores were collected and mycelium cultured in TSB liquid medium with 0.5% glycine at 30\u00b0C overnight.\nPreparation of labeled DNA\nGenomic DNA from a stationary phase culture was purified by the salting out procedure (Pospiech and Neumann, 1995) and had been sonicated to <\u00a02\u00a0Kb. Four to six micrograms of sonicated genomic DNA were used as template and this was denatured in the presence of 12\u00a0\u03bcg of 72%-GC-content random hexamers in a total volume of 25\u00a0\u03bcl at 100\u00b0C for 10\u00a0min. The mixture was then snap-cooled on ice before adding the remaining reaction components: 1.5\u00a0\u03bcl of Cy3-dCTP or Cy5-dCTP (Amersham Pharmacia Biotech), 4\u03bcl Klenow fragment (NEB #212), 5\u03bcl Klenow buffer, 0.5\u00a0\u03bcl dNTP (4\u00a0mM dATP, 4\u00a0mM dTTP, 10\u00a0mM dGTP, and 0.2\u00a0mM dCTP), and 14\u00a0\u03bcl ddH2O. The random primed labeling reaction was carried out for 2\u20133\u00a0h at 37\u00b0C. Buffer exchange, purification and concentration of the DNA products was accomplished by three cycles of diluting the reaction mixture in 0.5\u00a0ml TE buffer (10\u00a0mM Tris and 1\u00a0mM EDTA pH\u00a08.0) and filtering though a Microcon-30 microconcentrators (Millipore).\nMicroarray hybridization and data analysis\nThe two DNA pools to be compared were mixed and applied to an array in a hybridization mixture that contained 3.68\u00a0\u00d7 SSC, 0.18% SDS, and 1\u00a0\u03bcg yeast tRNA (total 16.3\u00a0\u03bcl), which had been heated at 100\u00b0C for 5\u00a0min before being applied to array. Hybridization took place under a glass coverslip sealed by glue in a humidified Omnislide (Thermo Hybaid) at 60\u00b0C for 12\u201314\u00a0h. The slides were washed, dried and scanned for fluorescence using a GenePix TM 4000B scanner (Axon instruments). Average signal intensity and local background measurements were obtained for each spot on each array using GenePixPro software. The dataset was screened for aberrant spots and these were eliminated from the analysis after manual checking. Most genes are present in duplicate on the two arrays and the signal from each pair of spots was inputted into the computer program available from ScanAlyze (Eisen et\u00a0al. 1998; Gollub et\u00a0al. 2003). The data was then processed into a mean log2 Cy3\/Cy5 ratio format. The dataset was normalized for each array separately and outputted to Excel where after checking the alignment of the datasets from each array, a mean signal for each common gene was calculated. Genes that were absent from either array, mostly transposon related genes in the University of Surrey array, were not included in the analysis. Based on Bentley et\u00a0al. 2002, the mean signal and standard deviation for the core region of genes from SCO2050 to SCO5800 was calculated. The standard deviation was used to set a cut-off for gene absence at 2SD below the core mean. The microarray data is presented relative to the S. coelicolor standard in two ways. This is either as a color plot of the genes where green presents a negative hybridization signal, black represents an equal hybridization signal and red indicates a positive hybridization signal using the program Treeview (Eisen et\u00a0al. 1998) or as numeric values for the signal from each gene. The microarray data for the four species described here and additional unpublished species can be accessed via rkirby@ym.edu.tw.\nComparison of the microarray dataset for S. avermitilis with the complete genome sequence\nThe nucleotide sequences for all the identified open reading frame from the S. avermitilis genome sequence (Ikeda et\u00a0al. 2003) were compared with the genome sequence of S. coelicolor using blastn limiting the output to the best match. This E value dataset for the genes was then aligned with the S. avermitilis microarray dataset and a comparison plotted as a scatterplot. Genes showing disagreement between the two datasets were identified based on a 2 Standard Deviation (SD) cutoff for the microarray dataset and a E-10 cutoff for the blast value.\nAnalysis of gene presence across the chromosome\nA graphical display was created by counting the number of gene detected as present from the signal based on the 2SD cutoff from each normalized microarray dataset using a moving window of 10 genes in steps of one.\nResults and discussion\nComparison of S. avermitilis, S. cattleya, S. maritimus and K. aureofaciens with the S. coelicolor genome\nIn total, after spot and data validation, a total of 7,083 open reading frames were included in this analysis as presence on both types of array and giving analyzable signal on all three arrays. Validity in this study was initially obtained by using microarrays from two sources that presumably use different PCR products to create the arrays. In addition, the University of Surrey array was hybridized and analyzed in duplicate. In terms of gene absence based on two standard deviations as described in the \u201cMaterials and methods\" section, the agreement between the Stanford array and the duplicated University of Surrey array was about 95%, while the agreement between the two University of Surrey arrays was about 98%. In order to minimize the effect of divergent individual array spots, the signal mean for each gene from the three arrays was used throughout this study.\nIn this study, the genomic content of three Streptomyces species and one Kitasatospora species with divergent taxonomy, antibiotic production and SSU rRNA sequence are compared using two different S. coelicolor microarrays. It is clear that there are inherent limitations to this approach. Firstly, only gene absence or divergence rather than the presence of new genes can be identified. Secondly, it is not possible to clearly separate the absence of a gene from the presence of a divergent homologue of the same gene. Finally, although the order of the genes in S. coelicolor and S. avermitilis are known from their complete genome sequences and are well conserved, this does not mean that the synteny of most of them is conserved in other Streptomyces species. However, the detection of synteny across Actinobacteria including Mycobacterium tuberculosis, Corynebacteriun glutamicum and other species (Bentley et\u00a0al. 2002 and unpublished data) supports a conserved central core structure to the genomes of the Actinomycetes and a priori most Streptomyces. Thus, although major chromosomal reorganizations in the central core region cannot be detected by microarray data, a basic chromosomal structure can be assumed as a first approximation; namely, a linear chromosome with variable terminal regions and a relatively well conserved core region.\nWhen the pooled data from the two arrays for the four species was analyzed using Cy-3 labeled S. coelicolor A(3)2 chromosomal DNA compared to heterologous Cy-5 labeled chromosomal DNA, a wide range of signal variation could be noted and this is shown in Supplementary Fig.\u00a01. The SSU rRNA tree places the divergence of these four strains from S. coelicolor as S. cattleya\u00a0>\u00a0K. aureofaciens\u00a0>\u00a0S. maritimus\u00a0>\u00a0S.avermitilis (Fig.\u00a01). Gene differences were present in the order S. cattleya\u00a0>\u00a0K. aureofaciens\u00a0>\u00a0S. avermitilis\u00a0>\u00a0S. maritimus based on \u22122SD cutoff below the mean signal for the core region genes. The microarray data thus shows general agreement with S. cattleya and K aureofaciens being more divergent and the other two species being relatively closer. It is interesting to note that the Kitasatospora species used in this study, K. aureofaciens, shows the same general structure as the Streptomyces species. This is not unexpected and confirms the close relationship between Kitasatospora and Streptomyces and agrees with the SSU rRNA tree data.Fig.\u00a01SSU rRNA phylogenetic tree of selected Streptomyces species and other Actinomycetes that have known complete genome sequences. The species analyzed by microarray are indicated in bold\nFurther support of the reliability of the data comes from a comparison of the blastn E values for all genes and the microarray data as shown in the Fig.\u00a02 scatterplot. This indicated 232 out of 6,832 genes show gene absence by microarray when they seem to be present by blastn and 268 out of 6,832 gene show gene presence by microarray when they seem to be absent by blastn; these results are both based on cutoffs of \u22122SD for the microarray data and \u221210 for the E value. This gives an overall reliability for S. coelicolor compared to S. avermitilis of 93%. Potential errors factors include in the case of the former type of error, poor spotting of the array at that point and choice of the PCR product sequence (the comparison is with the whole gene, as the PCR products are not available) and in the latter case cross-hybridization between multiple gene copies or a unreliable hybridization signal due to poor washing in that area. However, the results for S. avermitilis clearly support the reliability of the genome comparisons produced by this study.Fig.\u00a02Scatterplot comparing gene presence\/absence based on the microarray data and gene presence\/absence based in blastn between Streptomyces coelicolor and Streptomyces avermitilis. See \u201cMaterial and methods\" for details. Box A and Box C includes genes identified as absent in S. avermitilis by the microarray dataset but present using blastn and genes present in S. avermitilis using blastn, but identified as absent by the microarray dataset. Box B includes genes that are correctly identified as absent by the microarray dataset\nDistribution of gene differences across the complete chromosome of S. coelicolor for all four other Streptomyces species\nThe whole chromosome microarray dataset supports the following structure for the Streptomyces chromosome. Based on Fig.\u00a03 and Supplementary Fig.\u00a01, there is a central core of conserved probably syntenous genes that can be found across many Actinomycetes and in the S. coelicolor genome this reach from about SCO2050 to SCO5800 (Bentley et\u00a0al. 2002). The regions between SCO1100 and SCO2050 and between SCO5800 and SCO7600 are also quite well conserved between the Streptomyces studied here as well as being syntenous between the S. coelicolor and S. avermitilis genome sequences. However they are not present when the genomes of these two species are compared bioinformatically to other divergent Actinomycetes. These two regions seem to be two genus specific areas. Figure\u00a03 also clearly shows that gene conservation drops off dramatically in the terminal region. The regions from the left terminus to SCO1100 and from SCO7600 to the right terminus show much higher gene divergence that the rest of the chromosome. This agrees with the results for the S. ambofaciens sequencing studies of that species\u2019 terminal regions (Choulet et\u00a0al. 2006a, b). The gene conservation levels averaged across the four species are as follows: left terminal region (SCO0001\u2013SCO1100) 40.9%; left genus specific region (SCO1101\u2013SCO2050) 84.8%; core region (SCO2050\u2013SCO5800) 79.4%; right genus specific region (SCO5801\u2013SCO7600) 69.6% and right terminal region (SCO7601\u2013SCO7845) 50.3%. It is noticeable that neither the size nor the distribution of conserved genes is symmetrical between the two terminal regions or the two genus specific regions. Notably, the genus specific region actually has a higher frequency of gene conservation than the core regions as a whole and that the left terminal region is much larger than the right terminal region. This possibly represents horizontal exchange of terminal regions by recombination between strains\/species that involves only one terminal region. Such an event would give rise to asymmetric gene conservation similar to that detected here.Fig.\u00a03Analysis of \u201cgene presence\u201d across the four species. Created using a moving window of 10 genes and counting the number of genes with a microarray signal >2SD below the mean for the core region genes. The Y axis is the count for \u201cgene presence\u201d\nIn the Karoonuthaisiri et\u00a0al. (2005) study of regional gene expression in S. coelicolor, the boundaries for higher transcript levels during vegetative growth were placed at 1.5\u00a0Mb for the left arm and 2.3\u00a0Mb for the right arm. The former is midway across the left genus specific region and the latter approximately agrees with the boundary between the core and the right genus specific region. As the core region boundaries are also defined in terms of synteny with the Mycobacterium and Corynebacterium genomes as well as the data presented here, this supports the idea that the S. coelicolor chromosome structure is asymmetrical with respect to both gene conservation and gene function. It should be noted that because we are using only S. coelicolor as the source of the array data, the results do not imply that the genomes of S. cattleya, S. maritimus and K. aurefaciens are asymmetric. However, it should be noted that the S. avermitilis genome is also asymmetric (Ikeda et\u00a0al. 2003).\nNotably, there are 22 identifiable regions where all four species show a significant degree of concurrent gene absence outside of the terminal regions (Table\u00a01). The regions of high gene divergence are shown in Supplementary Fig.\u00a02 in detail. Previously, Bentley et\u00a0al. identified 14 regions in the S. coelicolor chromosome that were potentially laterally acquired regions. This analysis pinpoints all of these regions and quite accurately, usually to within one or two open reading frames. This suggests that other eight regions are probably quite robust when designated as potential lateral transfer regions. It also supports the usefulness of the microarray approach. All 22 regions were analyzed using Frame Plot (Artemis v7.1) and except for region B, they show abnormalities for at least some of the open reading frames compared to the G+C bias expected for the 1st, 2nd and 3rd codon positions of Streptomyces genes. Eight regions, A, B, F, I, M, O, Q and T contain transposon related genes near to or within the region. Four regions, H, N, P and R are flanked by highly conserved genes such as a ribosomal protein or sigma factor genes, which could encourage interspecific recombination. Finally, five regions consist largely of hypothetical proteins with no known similarity to any known protein as yet; these regions are G, J, L, S and W. Region L is particularly interesting as there is a central core of conserved gene flanked by two subregions that are highly not conserved. One of these genes is a putative spore septum determining protein, while the rest have unknown functions. Taken as a whole, the results suggests that S. coelicolor may have recently acquired all these regions either by transposition or by interspecific\/intraspecific recombination (Wolf et\u00a0al. 2002; Zhang et\u00a0al. 2002). It is also unlikely that they were acquired from any of the four species studied here. There are other regions that could potentially be identified as lateral transfer positions using less stringent criteria and a wider screening of genomes might help to support these additional regions as being involved in hotizontal transfer. In addition, such a wider screen might allow the identification of possible origins of these regions in other species.Table\u00a01Areas of the Streptomyces coelicolor genome identified as potentially horizontally transferred regions based on microarray parallel gene absence in all four speciesRegionArea of chromosomeGenes missinga%aSignificant featuresRegion ASCO0996\u2013SCO101017\/2959Integrase, insertion sequenceRegion BSCO2860\u2013SCO287953\/7669Rifampin ribosyl transferaseRegion CSCO3249\u2013SCO328894\/15660Integrase, excisionaseRegion DSCO3471\u2013SCO3538198\/26873AgaraseRegion ESCO3584\u2013SCO359930\/6050Region FSCO3929\u2013SCO393722\/3268Integrase\/recombinase, fstK-likeRegion GSCO3980\u2013SCO400156\/6488Hypothetical proteinsRegion HSCO4052\u2013SCO4066132\/14492Boundary dnaZ geneRegion ISCO4210\u2013SCO422337\/5469Region JSCO4247\u2013SCO425721\/3658Hypothetical proteinsRegion KSCO4340\u2013SCO435434\/4085Integrase, DNA invertaseRegion LSCO4509\u2013SCO4547106\/14474Hypothetical proteinsRegion MSCO4613\u2013SCO463140\/6859Integrase, excisionaseRegion NSCO4686\u2013SCO470024\/4455Boundary ribosomal proteins operonRegion OSCO5323\u2013SCO535157\/8071Integrase, excisionaseRegion PSCO5605\u2013SCO562046\/6472Boundary sigma factor whiGRegion QSCO5632\u2013SCO564440\/4491Integrase, korSARegion RSCO5715\u2013SCO573557\/7279Boundary ribosomal protein, bldBRegion SSCO5906\u2013SCO592428\/5650Hypothetical proteins, xylanaseRegion TSCO6372\u2013SCO640682\/10082RecombinaseRegion VSCO6607\u2013SCO664862\/12052HelicaseRegion WSCO6806\u2013SCO695373\/13355Hypothetical proteinsaThis is calculated from the available normalized gene dataset from the two microarrays\nGene conservation in the terminal regions of the four Streptomyces species\nAs has been mentioned earlier, the two regions at either terminus are much less well conserved than the central core region; these extend from SCO0001 to about SCO1100 on the left arm of the chromosome and from about SC7600 to SCO7845 on the right arm. The boundaries of these regions are not absolutely clear-cut, but what is clear is that as one moves towards the centre of the genome, gene conservation increases beyond these points. This can be clearly seen in Fig.\u00a03 where the gene conservation is plotted using a moving window for the four species, but it is also clear that the lack of conservation is not uniform across the terminal regions and that areas of higher gene conservation can be identified.\nThe significant interest in the terminal regions arises because the genomes of all Streptomyces that have been examined are linear and the problem of how the termini of such a molecule replicate is of particularly importance. Recent studies have indicated that two genes in particular, tpgA (SCO7734) and tapA (SCO7733), are involved in this process (Yang et\u00a0al. 2002; Bao and Cohen 2001). tpgA encoding the terminal protein that covalently binds to the termini of many linear Streptomyces replicons is conserved across all four species. In S. avermitilis this is also true based on sequence data and, further more, there are multiple copies of tpgA unlike S. coelicolor. The signal level of the S. avermitilis gene at +1.2 supports the presence of these multiple copies. The signal levels for the other three species are between about \u22120.3 and \u22120.1, which supports a single slightly diverging copy of this gene in these species. However, if two copies are present then the sequence divergence may be higher. Furthermore, tapA is also conserved except for S. maritimus, which seems to be more divergent at \u22120.8. It should be noted that the presence of these two genes is not a criteria for defining a genome with a linear topology, but the presence of one or both is certainly suggestive (Dary et\u00a0al. 2000; Wang et\u00a0al. 1999; Huang et\u00a0al. 1998; Lin and Chen 1997). Finally, ttrA is known to be involved in chromosomal transfer and is found very close to the telomere of S. coelicolor and S. avermitilis. This is also conserved in all four species suggesting the genetic exchange is highly important in Streptomyces and related species.\nThe two terminal regions encompass the major areas that are prone to deletion in many Streptomyces species and are therefore not essential except for linear terminal replication and genetic exchange. Given the relatively high lack of conservation of genes in this region, genes that are present in all four species represent an interesting class. A full list of all genes conserved in all four species in the terminal regions is provided in Tables\u00a02a and 2b. There are 36 hypothetical genes that show high similarity in the two terminal regions. Analysis of these groups of conserved genes using Artemis v7 (The Sanger Institute) identifies a total of five groups of genes that may make up possible single transcriptional units. These are SCO0551\u2013SCO0552, SCO0705\u2013SCO0710, SCO1021\u2013SCO1024, SCO7677\u2013SCO7680 and SCO7682\u2013SCO7688. In addition to TpgA and TapA, it is possible that there are other genes involved in terminal replication and these may be among the conserved genes present in the terminal regions. Although possible candidates can be deduced from a direct comparison of the two known Streptomyces genome sequences, they are many in number. Using the microarray analysis of the Actinomycetes in this study, the candidates can be reduced significantly. From candidates in Tables\u00a02a and 2b, two possible transcriptional units seem to be potential candidates for involvement in terminal replication; these are SCO1021\u2013SCO1024 (hypothetical proteins), and SCO7677\u2013SCO7689 (including hypothetical proteins, an AMP-binding ligase and membrane proteins). Gene knockout studies may be able to identify possible functions for these and other gene candidates, especially the other hypothetical proteins that are conserved in these four species.Table\u00a02Genes from the (a) left terminal, (b) right terminal region of Streptomyces coelicolor showing microarray conservation in all four species(a)SCO0002 ttrASCO0800 putative TetR-family transcriptional regulatory proteinSCO0142 hypothetical proteinSCO0802 hypothetical proteinSCO0150 hypothetical proteinSCO0810 putative ABC transporter permeaseSCO0201 putative integral membrane proteinSCO0830 putative penicillin-binding proteinSCO0232 hypothetical proteinSCO0839 putative transmembrane transport proteinSCO0415 hypothetical proteinSCO0840 putative marR-family transcriptional regulatorSCO0443 hypothetical proteinSCO0854 hypothetical proteinSCO0452 putative SIR2-like regulatory proteinSCO0883 polypeptide deformylaseSCO0466 araC family transcriptional regulatorSCO0887 putative TetR-family transcriptional regulatorSCO0471 putative araC family transcriptional regulatorSCO0894 putative membrane proteinSCO0496 putative iron-siderophore permease transmembrane proteinSCO0895 RNA polymerase principal sigma factor HrdCSCO0536 hypothetical proteinSCO0900 putative transmembrane efflux proteinSCO0538 probable sugar transporter sugar binding lipoproteinSCO0905 putative membrane proteinSCO0544 hypothetical secreted proteinSCO0907 putative dehydrogenaseSCO0546 pyruvate carboxylaseSCO0925 putative lysR-family transcriptional regulatorSCO0551 putative histidine kinase proteinSCO0926 hypothetical proteinSCO0552 putative response regulatorSCO0931 putative secreted proline-rich proteinSCO0565 putative polyprenyl synthetaseSCO0942 putative RNA polymerase sigma factorSCO0584 putative cytochromeSCO0943 hypothetical proteinSCO0591 putative lysozyme precursorSCO0947 putative integral membrane proteinSCO0592 hypothetical proteinSCO0949 hypothetical proteinSCO0614 hypothetical proteinSCO1011 conserved hypothetical proteinSCO0619 putative membrane proteinSCO1015 hypothetical proteinSCO0637 hypothetical proteinSCO1018 putative isomeraseSCO0690 possible oxidoreductaseSCO1021 hypothetical proteinSCO0695 hypothetical proteinSCO1022 hypothetical proteinSCO0701 hypothetical proteinSCO1024 hypothetical proteinSCO0707 putative branched-chain amino acid ABC transport permeaseSCO1034 putative tetR-family regulatory proteinSCO0708 putative branched-chain amino acid ABC transport proteinSCO1036 putative phosphotriesterase-family proteinSCO0709 putative branched-chain amino acid transport ATP-binding proteinSCO1040 putative DNA repair proteinSCO0710 putative branched-chain amino acid transport ATP-binding proteinSCO1041 hypothetical proteinSCO0765 secreted endoglucanaseSCO1043 putative transcriptional regulatory proteinSCO0779 conserved hypothetical proteinSCO1044 putative secreted proteinSCO0788 hypothetical proteinSCO1046 putative metal transporter ATPaseSCO0790 putative hydrolase(b)SCO7649 putative two-component system sensor kinaseSCO7677 putative secreted solute-binding proteinSCO7678 putative metal transport integral membrane proteinSCO7679 putative transport system integral membrane proteinSCO7680 putative ABC transporter ATP-binding proteinSCO7681 putative AMP-binding ligaseSCO7682 putative non-ribosomal peptide synthaseSCO7684 conserved hypothetical proteinSCO7685 conserved hypothetical proteinSCO7687 putative thioesteraseSCO7688 hypothetical proteinSCO7689 putative ABC transporter ATP-binding proteinSCO7718 hypothetical proteinSCO7720 hypothetical proteinSCO7724 hypothetical proteinSCO7734 Tpg proteinBold indicates groups of consecutive genes that may form a single transcriptional unit\nConservation of functional groups of genes across the four Streptomyces species\nOne approach to analyzing genetic variation across these four Streptomyces species is to look at the functional groupings of genes. Such an approach should allow the identification of strain versus genus specific genes especially when there are large numbers of genes with related functions such as sigma factors or where there are two copies of a gene, such as ftsK. However, because microarray data paints a broad picture across a whole genome, it is essential that once a gene or genes has been targeted based on microarray data, that experimental verification by other means is carried out. However, it is hoped that this dataset will be able to help researchers prioritize their gene targets better. The genes of the S. coelicolor chromosome have been grouped based on the scheme of M. Riley and colleagues for E. coli (ecocyc.org) modified for S. coelicolor (http:\/\/www.sanger.ac.uk\/Projects\/S_coelicolor\/scheme.shtml) and we used this classification. The genes involved in ribosomal proteins synthesis and modification should be highly conserved and the results indicate that almost all of them are present in all four species (Table\u00a03; Supplementary Fig.\u00a04). The only exceptions are SCO0436, SCO0509 SCO3430 and SCO3909 in S. avermitilis and SCO4716 and SCO5514 in K. aureofaciens. Of these genes, SCO0436, SCO0509 and SCO5514 represent duplicate genes in the S. coelicolor genome and therefore the choice of the microarray sequence will have had a significant effect on the heterologous hybridization. There is no obvious explanation for the failure to hybridize of the other two genes, but as a whole, this dataset supports the integrity of the array system for analysis of genome content as these genes are scattered across the whole Streptomyces genome.Table\u00a03Microarray data for ribosomal proteins from the four species\u00a0S. avermitilisS. cattleyaS. maritimusK. aureofaciensSCO0436 probable 50S ribosomal protein\u22120.35\u22120.130.44\u22120.29SCO0569 putative 50S ribsomomal protein fragment\u22120.750.63\u22120.420.27SCO1150 50S ribosomal protein L310.56\u22120.47\u22120.240.14SCO1505 30S ribosomal protein S40.36\u22120.350.76\u22120.31SCO1598 50S ribosomal protein L201.500.630.890.34SCO1599 50S ribosomal protein L350.230.410.49\u22120.51SCO1998 30S ribosomal protein S11.390.770.990.91SCO2563 30s ribosomal protein S20\u22120.34\u22120.340.770.33SCO2596 50S ribosomal protein L271.010.590.080.82SCO2597 ribosomal protein L210.270.080.64\u22120.18SCO3124 ribosomal L25p family protein0.390.31\u22120.23\u22120.89SCO3427 putative 50S ribosomal protein L310.240.370.220.60SCO3428 putative 50S ribosomal protein L330.150.280.540.09SCO3429 putative 50S ribosomal protein L280.680.160.550.45SCO3430 putative 30S ribosomal protein S14\u22120.800.10\u22120.17\u22120.19SCO3880 putative 50S ribosomal protein L341.020.130.240.71SCO3906 putative 30S ribosomal protein S60.700.941.11\u22120.18SCO3909 putative 50S ribosomal protein L91.300.010.87\u22121.27SCO4648 50S ribosomal protein L111.670.430.900.47SCO4649 50S ribosomal protein L10.620.53\u22120.250.92SCO4652 50S ribosomal protein L100.42\u22120.430.64\u22120.35SCO4653 50S ribosomal protein L7\/L121.221.030.790.45SCO4659 30S ribosomal protein S120.740.650.70\u22120.53SCO4660 30S ribosomal protein S70.57\u22120.230.680.12SCO4701 30S ribosomal protein S101.191.171.16\u22120.21SCO4702 50S ribosomal protein L30.920.020.840.49SCO4703 50S ribosomal protein L41.160.910.590.23SCO4704 50S ribosomal protein L230.851.441.240.36SCO4705 50S ribosomal protein L20.85\u22120.120.840.22SCO4706 30S ribosomal protein S190.060.240.32\u22120.26SCO4707 50S ribosomal protein L220.960.690.640.15SCO4708 30S ribosomal protein S31.150.380.781.07SCO4709 50S ribosomal protein L160.520.671.091.26SCO4710 50S ribosomal protein L290.33\u22120.06\u22120.190.41SCO4711 30S ribosomal protein S170.590.920.51\u22120.13SCO4712 50S ribosomal protein L141.050.350.480.82SCO4713 50S ribosomal protein L241.090.880.630.64SCO4714 50S ribosomal protein L51.240.780.771.03SCO4715 30S ribosomal protein S140.390.030.190.12SCO4716 30S ribosomal protein S81.18\u22120.110.64\u22120.76SCO4717 50S ribosomal protein L60.960.820.79\u22120.02SCO4718 50S ribosomal protein L180.090.300.570.74SCO4719 30S ribosomal protein S51.560.661.02\u22120.09SCO4720 50S ribosomal protein L300.130.390.640.26SCO4721 50S ribosomal protein L151.790.420.800.84SCO4726 50S ribosomal protein L360.46\u22120.130.35\u22120.10SCO4727 30S ribosomal protein S130.63\u22120.230.62\u22120.17SCO4728 30S ribosomal protein S111.120.550.870.27SCO4730 50S ribosomal protein L170.690.270.860.51SCO4734 50S ribosomal protein L13\u22120.270.450.500.40SCO4735 30S ribosomal protein S90.36\u22120.020.00\u22120.01SCO5359 50S ribosomal protein L310.860.221.400.46SCO5564 putative 50S ribosomal protein L280.600.280.210.51SCO5591 30S ribosomal protein S160.440.030.57\u22120.60SCO5595 50S ribosomal protein L190.770.781.54\u22120.03SCO5624 30S ribosomal protein S21.160.481.520.30SCO5736 30S ribosomal protein S150.700.410.79\u22120.46Mean hybridization score for ribosomal protein genes0.670.350.610.18Bold values indicate that the signal for that gene is more than 2SD below the mean core signal for that species and such a value is suggestive of either gene absence or very low similarity\nTable\u00a04 shows genes identified as possible sigma factors, anti-sigma factors and ant-sigma factor antagonists. The genes found in the central core region are more conserved. As would be expected, the major sigma factors such as hrdA, hrdB, hrdC and hrdD are conserved as well as many of the other studied sigma factors of S. coelicolor such as are sigA, sigE, sigF, sigG, sigR, sigT and whiG. Overall, fewer regulation genes from this group (anti-sigma factors and anti-anti-sigma factors) are conserved than sigma factors themselves. This analysis allows the identification of new candidate sigma factors for further study outside of the well studied ones, but within S. coelicolor and in other species. Overall, the results support the hypothesis that there is a core of sigma factors essential to keeping protein synthesis in Streptomyces running smoothly. The functionality of the rest may vary and include complete silence of some gene fragments, duplication of function, involvement in specific secondary metabolic activities and species\/genus specific functions.Table\u00a04Conservation across the four species of genes annotated as sigma factors or related proteins in Streptomyces coelicolor\u00a0S.avermitilisS.cattleyaS. maritimusK. aureofaciens\u00a0SCO0037 putative sigma factor\u22121.04\u22120.90\u22120.88\u22121.49SCO0159 putative ECF sigma factor\u22121.26\u22120.652.05\u22120.60SCO0194 putative sigma factor\u22120.87\u22120.35\u22120.61\u22120.52SCO0255 putative transcriptional regulator\u22120.64\u22120.37\u22120.99\u22120.46SCO0414 putative RNA polymerase sigma factor\u22120.05\u22120.28\u22120.15\u22120.22ConservedSCO0598 putative anti anti sigma factor0.110.50\u22120.080.57ConservedSCO0599 putative regulator of sig8\u22121.41\u22121.07\u22120.84\u22121.08SCO0632 putative RNA polymerase sigma factor\u22120.140.19\u22120.820.11SCO0672 putative anti-sigma factor antagonist\u22120.10\u22120.40\u22120.190.12SCO0781 putative anti sigma factor antagonist\u22120.79\u22120.86\u22121.06\u22120.83SCO0803 putative RNA polymerase sigma factor\u22120.25\u22120.09\u22120.51\u22120.01SCO0864 probable ECF-family sigma factor\u22120.74\u22120.86\u22121.04\u22120.50SCO0866 probable ECF-family sigma factor\u22120.130.19\u22120.280.23ConservedSCO0869 putative anti-sigma factor antagonist\u22120.59\u22120.90\u22121.23\u22120.90SCO0895 RNA polymerase principal sigma factor HrdC0.520.431.120.34ConservedSCO0942 putative RNA polymerase sigma factor0.340.810.610.45ConservedSCO1263 putative ECF-sigma factor\u22120.17\u22120.260.080.35ConservedSCO1276 RNA polymerase ECF sigma factor\u22121.22\u22120.550.60\u22120.88SCO1564 putative RNA polymerase sigma factor1.04\u22120.331.34\u22120.47SCO1723 putative RNA polymerase sigma factor0.19\u22120.39\u22120.150.52ConservedSCO1876 putative RNA polymerase sigma factor\u22121.01\u22120.93\u22120.77\u22120.50SCO2465 RNA polymerase principal sigma factor0.760.740.970.64ConservedSCO2639 putative RNA polymerase sigma factor0.740.220.010.27ConservedSCO2954 putative RNA polymerase sigma factor1.12\u22120.510.94\u22120.46SCO3066 putative regulator of Sig150.530.290.880.31ConservedSCO3067 putative anti anti sigma factor\u22120.74\u22121.250.89\u22120.42SCO3068 putative RNA polymerase sigma factor0.33\u22120.061.07\u22120.41ConservedSCO3202 RNA polymerase principal sigma factor0.980.291.400.19ConservedSCO3323 putative RNA polymerase sigma factor0.760.491.080.27ConservedSCO3356 ECF sigma factor 37\u22120.050.110.730.46ConservedSCO3450 putative RNA polymerase sigma factor (ECF subfamily)0.16\u22120.09\u22120.79\u22120.23SCO3548 putative anti-sigma factor\u22120.570.500.49\u22120.06SCO3549 bldG putative anti-sigma factor antagonist\u22120.03\u22120.160.21\u22120.20ConservedSCO3613 putative RNA polymerase sigma factor0.570.150.080.46ConservedSCO3692 putative anti-sigma factor antagonist0.140.64\u22120.270.13ConservedSCO3709 putative ECF sigma factor0.020.06\u22120.210.61ConservedSCO3715 putative ECF sigma factor0.450.92\u22120.28\u22120.27ConservedSCO3736 putative RNA polymerase ECF sigma factor\u22120.06\u22120.290.28\u22120.09ConservedSCO3892 putative RNA polymerase sigma factor0.680.210.80\u22120.27ConservedSCO4027 putative anti sigma factor antagonist\u22120.041.11\u22120.590.58SCO4034 putative RNA polymerase sigma factor0.981.271.260.22ConservedSCO4035 RNA polymerase sigma factor (fragment)1.041.270.880.45ConservedSCO4146 putative ECF subfamily sigma factor\u22120.230.530.580.16ConservedSCO4409 putative RNA polymerase sigma factor\u22120.100.120.100.64ConservedSCO4410 putative anti anti sigma factor\u22120.890.070.16\u22120.81SCO4452 putative sigma factor\u22120.17\u22120.21\u22120.080.27ConservedSCO4769 ECF sigma factor0.09\u22120.380.680.61SCO4864 putative ECF sigma factor0.02\u22120.34\u22120.12\u22120.73SCO4866 putative ECF sigma factor0.120.190.090.38ConservedSCO4895 putative ECF sigma factor\u22120.32\u22121.15\u22120.11\u22120.56SCO4938 putative ECF-sigma factor0.170.430.240.64ConservedSCO4960 possible sigma factor\u22120.040.05\u22120.660.60SCO4996 putative RNA polymerase ECF sigma factor\u22120.54\u22120.040.580.48SCO5147 putative ECF-subfamily sigma factor\u22120.390.350.790.59SCO5217 anti-sigma factor\u22120.47\u22120.050.28\u22120.80SCO5244 anti-sigma factor\u22120.32\u22120.54\u22120.37\u22120.29SCO5386 putative anti-sigma factor antagonist0.150.370.00-0.07ConservedSCO5621 RNA polymerase sigma factor WhiG0.790.920.64\u22120.27ConservedSCO5820 hrdB, major vegetative sigma factor1.361.061.531.09ConservedSCO5934 putative sigma factor0.070.25-0.580.17SCO6239 putative sigma factor\u22120.92\u22121.27\u22121.84\u22120.74SCO6996 putative RNA polymerase sigma factor-0.340.290.00\u22120.04ConservedSCO7099 putative RNA polymerase sigma factor\u22120.20\u22120.380.380.29SCO7104 putative RNA polymerase sigma factor\u22120.70\u22120.02\u22120.790.56SCO7112 putative ECF-family RNA polymerase sigma factor\u22120.35\u22120.25\u22121.65\u22120.38SCO7144 putative ECF sigma factor\u22120.620.13\u22120.910.34SCO7314 probable RNA polymerase sigma factor\u22120.25\u22120.110.230.46ConservedSCO7323 anti-sigma factor antagonist0.30\u22120.010.230.31ConservedSCO7325 anti-sigma factor antagonist-0.37-0.68\u22120.23\u22121.15SCO7341 putative RNA polymerase secondary sigma factor\u22120.090.540.290.44ConservedSCO7573 putative anti-sigma factor antagonist\u22120.180.03\u22121.690.21SCO7619 putative anti sigma factor antagonist\u22120.280.37\u22120.97\u22120.64SCO7754 putative anti-sigma factor antagonist\u22121.20\u22120.51\u22121.890.02Mean hybridization score for ribosomal protein genes0.010.060.100.05NABold values indicate that the signal for that gene is more than 2SD below the mean core signal for that species and such a value is suggestive of either gene absence or very low similarity. A conserved gene is one that seems to be present in all four species. NA, Not applicable\nAll four species studied here undergo differentiation and spore formation and as such would be expected to retain most genes involved in cell division\/sporulation\/differentiation. This is supported by Table\u00a05. K. aureofaciens shows greater gene divergence for certain genes when compared to the three Streptomyces species and these are specifically ftsI (SCO2090) and a putative cell division protein (SCO2968). However, in general, the same genes in all four species show a higher divergence, for example sapA, which is a protein associated with the spore surface hydrophobicity. As spore morphology varies a lot in the Streptomyces, high variability\/gene loss in such a gene is not unexpected. Other genes that show higher divergence are those involved in partitioning and cell division. This suggests that the genes and thus the proteins involved in these functions may differ from species to species in order to create the variation seen in aerial mycelium and spore structure across Streptomyces species. Specifically, SCO3934, an ftsK family protein gene is less well conserved than its homologue. This suggests that SCO5750 may produce the major ftsK protein. Other Fts proteins show a similar pattern with at least one homologue being well conserved. This may well help an understanding of the relationships between the genes involved in cell division and will allow better identification of specific targets for further study. One anomaly that stands out is bldB. This gene consistently shows a low level of hybridization. A comparison of the bldB gene sequence between S. coelicolor and S. avermitilis shows a nucleotide identity of about 87%, which ought to give a signal in the region of 0.0 or better. As two different arrays are used in this study, mechanical problems with this spot can probably be eliminated as the source of the anomaly. We suggest that because this is a relatively small gene, the PCR product chosen for both arrays may be the reason for this result. This emphasizes that array data should be used with a degree of caution and needs to be backed up by other experimental evidence when specific genes are being investigated.Table\u00a05Conservation across the four species of genes in Streptomyces coelicolor annotated as involved in cell division, sporulation and differentiation\u00a0S. avermitilisS. cattleyaS. maritimus K. aureofaciensSCO0409 sapA spore-associated protein precursor\u22121.99\u22121.39\u22120.59\u22120.67SCO1454 putative amino oxidase1.000.451.18\u22120.17SCO1489 bldD putative DNA binding protein0.990.761.020.58SCO1772 putative partitioning or sporulation protein0.690.350.390.54SCO2082 ftsZ cell division protein1.440.890.970.95SCO2083 ftsQ sporulation protein0.320.740.060.26SCO2084 murG0.86\u22120.010.490.32SCO2085 fts W putative cell division protein0.820.810.590.50SCO2086 murD0.580.230.350.45SCO2087 murX0.410.050.51\u22120.30SCO2088 murF1.180.730.670.01SCO2089 murE0.730.470.360.31SCO2090 ftsl cell division protein0.800.010.45\u22120.50SCO2607 Sfr protein0.730.730.91\u22120.01SCO2608 penicillin binding protein\u22120.04\u22120.250.45\u22120.92SCO2609 mreD rod shape-determining protein0.090.070.660.43SCO2610 mreC rod shape-determining protein\u22120.42\u22120.160.17\u22120.52SCO2611 mreB rod shape-determining protein0.980.800.680.19SCO2620 putative cell division trigger factor0.81\u22120.120.500.25SCO2968 putative cell division protein0.35\u22120.170.34\u22120.39SCO2969 ftsE cell division ATP-binding protein0.37\u22120.430.00\u22120.51SCO3034 whiB sporulation regulatory protein0.230.500.810.26SCO3323 bldB putative RNA polymerase sigma factor0.760.491.080.27SCO3404 ftsH2 cell division protein ftsH homolog1.110.511.070.15SCO3549 bldG putative anti-sigma factor antagonist\u22120.03\u22120.160.21\u22120.20SCO3557 putative septum site determining protein0.310.45\u22120.190.92SCO3558 putative morphological differentiation-associated protein0.69\u22120.261.62\u22120.20SCO3846 putative FtsW\/RodA\/SpoVE family cell cycle protein1.110.311.070.61SCO3886 putative partitioning or sporulation protein0.00\u22120.83\u22120.43\u22120.98SCO3887 putative partitioning or sporulation protein\u22120.19\u22120.190.24\u22121.04SCO3934 ftsK\/spoIIIE family protein\u22120.560.39\u22121.18\u22120.53SCO4014 sporulation associated protein\u22120.87\u22120.93\u22120.81\u22121.17SCO4184 mfC aerial mycelium formation0.120.170.01\u22120.05SCO4508 putative cell division-related protein\u22120.62\u22120.270.070.59SCO4531 putative septum determining protein\u22120.56\u22120.880.12\u22120.09SCO4620 traB1 putative sporulation-related protein\u22120.49\u22120.680.19\u22120.34SCO4621 traA1 putative sporulation-related protein\u22120.03\u22120.511.33\u22120.38SCO4767 putative regulatory protein0.140.001.67\u22120.01SCO4768 bldM putative two-component regulator1.061.000.920.61SCO5006 minD1 putative septum site-determining protein\u22120.31\u22120.040.58\u22120.28SCO5008 minD3 putative septum site-determining protein0.04\u22120.21\u22120.11\u22120.07SCO5112 BldKA\u22120.420.92\u22120.750.51SCO5114 BldKC\u22120.39\u22120.08\u22121.23\u22120.23SCO5115 BldKD0.01\u22120.18\u22120.89\u22120.15SCO5116 bldKE putative peptide transport system ATP-binding protein\u22120.04\u22120.22\u22120.70\u22120.03SCO5314 whiE protein VII\u22121.24\u22120.230.03\u22120.67SCO5315 polyketide cyclase\u22120.39\u22120.82\u22120.31\u22120.25SCO5316 acyl carrier protein\u22120.42\u22120.290.750.03SCO5318 polyketide beta-ketoacyl synthase alpha\u22120.03\u22120.130.860.43SCO5321 polyketide hydroxylase0.12\u22120.091.500.27SCO5587 ftsH cell division protein FtsH homolog\u22120.050.310.230.21SCO5621 whiG RNA polymerase sigma factor WhiG0.790.920.64\u22120.27SCO5723 bldB putative regulator, BldB\u22121.47\u22120.76\u22121.27\u22121.05SCO5750 ftsK homolog0.670.160.652.52SCO5819 whiH, sporulation transcription factor0.680.120.160.13SCO6029 whiI two-component regulator0.14\u22120.260.770.92Mean hybridization score for ribosomal protein genes0.05\u22120.050.27\u22120.02Bold values indicate that the signal for that gene is more than 2SD below the mean core signal for that species and such a value is suggestive of either gene absence or very low similarity\nThe genes involved in DNA replication, repair, restriction\/modification are shown in Table\u00a06 and only about 20% of these genes are not conserved relatively well across all four species. This is to be expected as DNA replication and repair are core functions. Most of the genes that show higher levels of gene divergence are found in the terminal regions of the linear chromosome and probably are genes that perform functions that are not essential to cell survival because the terminal regions of Streptomyces chromosomes are unstable and liable to deletion without lethality. Of particular interest are SCO0183 and SCO0842 (deoxiribopyrmidine photolyases); these repair system would seem to be absent in S. lividans and S. maritimus, but a homologue is present in S. avermitilis (confirmed by the genome sequence) and in S. cattleya. This confirms the high variability found for this repair function across the Streptomyces (Kobayashi et\u00a0al. 1989). A similar situation of high variability is found for the mutT homologues, potential 8 hydroxy-dGTP hydrolases. Knockout of this gene has been shown to increase the A:T to G:T mutation rate and thus it has a possible repair function (Kamiya et\u00a0al. 2004). The genes for recA (SCO5769), recF (SCO3876) and recR (SCO3618) are present in all four species; however, the recX (SCO5770), is more divergent and gives a low signal for S. cattleya and S. maritimus. SCO6405, a putative DNA recombinase, is scored as absent in all four species suggesting that there is redundancy in the Streptomyces genes concerned with recombination or that this gene is transposon related. The latter is supported by low homology to S. avermitilis putative integrases\/recombinases. There are four genes encoding DNA gyrases on the microarray, namely, gyrA DNA gyrase subunit A (SCO3873) and gyrB DNA gyrase subunit B (SCO3874) together with SCO5836 and SCO5822 and these may be TopIV homologues involved in resolving chromosome concatenates. All are conserved although the conservation of SCO5822 gyrB2 is lower. Thus both sets of gyrase genes would seem to be important. As expected, SCO1518, a ruvB Holliday junction protein gene and SCO1520, a ruvC crossover junction endonuclease are conserved across all the species. Unexpectedly, although probably present in all species, SCO1519 ruvA is much more divergent that the other two gene in this Holliday junction complex. This diversity is unexpected and not easily explicable except by the fact that recombination in Streptomyces may occur via a more variable mechanism than in other groups of bacteria and this is then reflected in the greater divergence of SCO1519 ruvA. All three genes annotated as a DNA polymerase 1 homologue are conserved as are four out of the five DNA polymerase III homologues, suggesting that there are roles for all of these conserved genes in Streptomyces. Two other unclassified DNA polymerase type genes, SCO4495 and SCO6084 are also conserved and thus may have important functions. There is, however, more diversity among the helicases and methylases\/methyltransferases. With the helicases, three out of 14 show significant divergence and therefore most of the helicases probably have important cellular roles. Four out of nine methylases\/methyltransferases show divergence. As some of these genes may be involved in the DNA modification part of restriction\/modification, such diversity across strains in not unexpected. Finally, four out of six ligases show divergence, perhaps reflecting the fact that the origin of a number of these ligases might be from bacteriophages.Table\u00a06Conservation across the four species of genes in Streptomyces coelicolor annotated as involved in DNA replication, repair, restriction and modification\u00a0S. avermitilisS. cattleyaS. maritimusK. aureofaciensSCO0183 putative deoxyribodipyrimidine photolyase\u22121.38331\u22120.76404\u22121.15786\u22120.78973SCO0760 putative methyltransferase\u22120.23598\u22120.176030.079613\u22120.16712SCO0842 putative deoxyribodipyrimidine photolyase0.0024750.167429\u22120.051980.35453SCO0918 putative excinuclease ABC subunit A\u22120.28707\u22120.348130.010852\u22120.46861SCO0945 putative formamidopyrimidine-DNA glycosylase\u22120.34857\u22120.60698\u22120.4371\u22120.38083SCO1040 putative DNA repair protein0.0473150.5687230.4561680.367071SCO1050 putative DNA protection protein\u22120.474790.0932990.451487\u22120.92697SCO1114 uracil DNA glycosylase\u22120.345730.3571820.4339020.950894SCO1167 putative helicase (fragment)0.6048130.072648\u22120.40455\u22120.25074SCO1180 putative DNA polymerase III beta chain\u22120.347\u22120.21511\u22120.42838\u22120.20922SCO1202 putative DNA ligase0.3084970.202188\u22120.155240.096586SCO1203 putative MutT-like protein\u22120.17791\u22120.37050.324233\u22120.28871SCO1255 G\/U mismatch-specific DNA glycosylase0.5216790.419040.3212080.429148SCO1343 uracil-DNA glycosylase0.612940.003390.3114780.036821SCO1380 putative DNA damage inducible protein0.7630640.3139480.2149310.677262SCO1395 mutT-like protein0.0682150.5725270.170710.414092SCO1475 putative primosomal protein0.0488920.6892320.535450.457655SCO1518 ruvB holliday junction DNA helicase1.1364890.6388030.9300671.045593SCO1519 ruvA holliday junction DNA helicase\u22120.57721\u22120.23275\u22120.153740.008274SCO1520 ruvC crossover junction endodeoxyribonuclease1.0797860.7081310.8863630.979821SCO1534 putative DNA polymerase III0.32960.371242\u22120.100250.090739SCO1739 putative DNA polymerase III1.1280490.4231581.1119640.326701SCO1780 putative DNA repair protein\u22120.14578\u22120.091750.258713\u22120.32746SCO1792 putative 3-methyladenine DNA glycosylase\u22120.20485\u22120.077550.083499\u22120.2824SCO1827 putative DNA polymerase III0.5597150.7112650.5128730.343849SCO1966 ABC excision nuclease subunit B0.0473820.068961.085903\u22120.05934SCO1969 putative DNA-methyltransferase\u22120.19025\u22120.121840.1433670.56545SCO2003 DNA polymerase I1.1721760.1884930.4981260.201411SCO2468 DNA primase0.827150.5205521.256130.69797SCO2626 putative DNA repair hydrolase (fragment)0.2899160.3373650.5677830.326821SCO2863 putative helicase\u22120.47935\u22120.68124\u22121.92881\u22120.81052SCO2952 putative helicase protein0.5139520.388640.7505170.379556SCO3109 putative transcriptional-repair coupling factor\u22120.87255\u22120.296110.759684\u22120.33637SCO3351 putative DNA repair protein\u22120.95043\u22120.685210.24637\u22120.48466SCO3352 putative DNA-binding protein0.090569\u22120.096020.536392\u22120.2275SCO3434 putative DNA polymerase I0.85541.6517680.8742461.328528SCO3510 putative DNA methylase0.402433\u22121.11393\u22122.15274\u22122.07607SCO3541 putative DNA polymerase0.003597\u22120.402890.297076\u22120.75003SCO3543 probable DNA topoisomerase I0.7987051.334770.878841.226661SCO3550 putative helicase0.2636440.139350.5598720.216127SCO3618 putative recomination protein0.5334040.0526230.511469\u22120.36893SCO3873 DNA gyrase subunit A1.293458\u22120.208510.985546\u22121.2494SCO3874 DNA gyrase subunit B0.993330.9582471.2506690.37466SCO3878 DNA polymerase III0.053750.0260530.328502\u22120.62343SCO3879 chromosomal replication initiator protein (fragment)1.259831.5725310.731065\u22120.12482SCO4092 ATP-dependent helicase\u22120.007520.0551940.978098\u22120.21188SCO4143 putative mutT-like protein0.0210350.4354710.479161\u22120.28423SCO4272 putative mutT-like protein\u22120.13520.016206\u22120.329680.527274SCO4351 putative DNA invertase\u22121.26688\u22120.78144\u22121.29284\u22120.68489SCO4495 putative DNA polymerase related protein\u22120.09624\u22120.759370.5824190.316259SCO4577 putative helicase0.7269620.0531961.044155\u22120.09189SCO4797 putative ATP-dependent DNA helicase II0.3233660.2864210.7498680.026912SCO5064 putative bifunctional protein\u22120.20262\u22120.49259\u22121.90763\u22120.29433SCO5143 DNA-3-methyladenine glycosylase I\u22120.865940.3424890.2766110.981316SCO5183 putative ATP-dependent DNA helicase0.2356330.1207650.7675040.591935SCO5184 putative ATP-dependent DNA helicase0.180638\u22120.175870.2088390.666073SCO5188 putative ATP-dependent DNA helicase0.204048\u22120.360050.808168\u22120.04087SCO5331 putative DNA methylase\u22121.52864\u22122.37483\u22123.76436\u22122.18209SCO5494 putative DNA ligase0.1247810.2884290.215946\u22120.0149SCO5566 putative ATP-dependent DNA helicase0.4089951.1867140.4281830.825946SCO5567 putative methylase0.338198\u22120.822070.596196\u22120.83241SCO5573 formamidopyrimidine-DNA glycosylase0.387552\u22120.083530.6015850.435209SCO5760 DNA glycosylase0.8469850.022350.6758760.46121SCO5770 RecX protein\u22120.1193\u22120.72104\u22120.580750.079675SCO5802 putative ATP-dependent helicase0.823429\u22120.104330.5115950.058287SCO5803 SOS regulatory protein LexA0.143322\u22120.78751\u22120.0905\u22120.16946SCO5805 ribonucleotide reductase0.2351820.2715140.98906\u22120.06823SCO5815 probable ATP-dependent DNA helicase\u22120.52023\u22120.31138\u22120.75254\u22120.71096SCO5822 gyrB2, probable DNA gyrase0.1672750.2234730.4268490.393883SCO5836 DNA gyrase-like protein0.7257080.5074980.9375310.073608SCO6084 putative DNA polymerase\u22120.053810.1656340.084564\u22120.16407SCO6151 putative methylated-DNA-protein-cysteine methyltransferase\u22120.88705\u22120.081120.3752140.207725SCO6262 putative helicase 6884138:6887071 forward MW:1039120.260624\u22120.288360.753508\u22120.08897SCO6405 putative DNA recombinase\u22120.16958\u22120.65562\u22121.15936\u22120.30768SCO6462 putative methylated-DNA-protein-cysteine methyltransferase\u22120.09961\u22120.002850.2318360.026866SCO6640 putative ATP-dependent helicase\u22120.56659\u22120.52367\u22121.18266\u22120.38994SCO6707 putative DNA ligase\u22120.254090.618121\u22120.0252\u22120.3333SCO6844 putative DNA methylase.0.4878060.475711\u22120.593870.230037SCO6907 putative DNA ligase.\u22120.714910.181384\u22120.67564\u22120.69734SCO7345 probable ATP-dependent DNA ligase0.1766920.2076060.3402550.085159SCO7522 putative DNA ligase\u22120.31874\u22120.0533\u22120.637010.470458Mean hybridization score0.1136330.0074410.153752\u22120.04491Bold values indicate that the signal for that gene is more than 2SD below the mean core signal for that species and such a value is suggestive of either gene absence or very low similarity\nTable\u00a07 shows the genes involved in peptidoglycan and teichoic acid synthesis. In this area of metabolism, there is also a relatively high level of conservation of genes, particularly the murA, murA2, murB, murD, murE, murF,murG and murX genes. Also conserved are the shape-determining genes SCO2609, SCO2610 and SCO2611, which may form an operon. This probably represent a core of genes together with the genes involved in biosynthesis of the cell wall that are needed to give a basic structure to the cells of any Streptomyces species. The penicillin binding proteins show a higher degree of variability, except for SCO2897, SCO4013 and SCO5301. The peptidases SCO3580, SCO3596, SCO3011 and SCO4439 and the D-alanine:D-lactate ligase SCO3595 all show a low level of gene conservation, perhaps because they are involved in relatively broad cellular functions and not under a great deal of selective pressure.Table\u00a07Conservation across the four species of genes in Streptomyces coelicolor annotated as involved in peptidoglycan biosynthesis\u00a0S. avermitilisS.cattleyaS. maritimusK. aureofaciensSCO0237 putative oxidoreductase\u22120.07526\u22121.14854\u22120.09821\u22120.57347SCO0286 putative peptidoglycan binding protein\u22120.9459\u22120.93949\u22121.83967\u22121.02755SCO0830 putative penicillin-binding protein0.2434580.0813410.5560960.08585SCO0936 putative oligosaccharide deacetylase\u22120.78759\u22121.38608\u22120.58892\u22121.00821SCO1018 putative isomerase0.3905340.5192040.3926050.600019SCO1875 putative secreted penicillin binding protein\u22120.448310.0395980.3203560.256976SCO2084 murG0.85602\u22120.006770.4859890.319639SCO2085 putative cell division protein0.8166240.807420.5922140.502565SCO2086 murD0.5805060.2253480.3476740.449973SCO2087 murX0.4057310.0496450.509959\u22120.3047SCO2088 murF1.1750780.7309250.6673830.006107SCO2089 murE0.7340680.4708690.3634580.308295SCO2345 putative peptidodoglycan-binding membrane protein\u22120.03290.0163380.1333550.069404SCO2451 putative rod shape-determining protein0.6043270.1800280.6122430.205981SCO2589 putative glycosyl transferase0.093285\u22120.412170.478809\u22120.09528SCO2590 putative glycosyltransferase0.069933\u22120.52938\u22121.18523\u22120.89326SCO2608 penicillin binding protein\u22120.0413\u22120.246810.445143\u22120.92429SCO2609 rod shape-determining protein0.0858790.067210.6636090.427758SCO2610 rod shape-determining protein\u22120.41695\u22120.157980.168038\u22120.52083SCO2611 rod shape-determining protein0.9798840.7964030.6752040.186331SCO2706 putative transferase0.2334230.505652\u22120.555440.407847SCO2707 putative transferase0.014126\u22120.372250.088105\u22120.12772SCO2897 probable penicillin-binding protein0.5766460.4093880.6932370.120551SCO2949 murA0.4183630.2090240.3351590.062475SCO3580 putative transpeptidase\u22120.09884\u22120.76666\u22121.133470.212152SCO3595 putative D-alanine:D-lactate ligase\u22120.77707\u22121.21402\u22121.61266\u22121.31715SCO3596 putative D-alanine:D-alanine dipeptidase\u22121.47294\u22120.65926\u22121.4814\u22120.85496SCO3811 putative D-alanyl-D-alanine carboxypeptidase\u22120.50714\u22120.586170.155077\u22120.78413SCO3847 putative penicillin-binding protein0.128622\u22120.436430.640849\u22120.18456SCO3901 putative penicillin-binding protein\u22120.59684\u22120.78306\u22120.26692\u22120.39905SCO4013 putative penicillin binding protein\u22120.09095\u22120.01844\u22120.047970.164779SCO4132 putative secreted transglycosylase0.237226\u22120.17562\u22120.03522\u22120.2261SCO4439 putative D-alanyl-D-alanine carboxypeptidase\u22120.74865\u22120.930490.177585\u22121.21329SCO4643 murB\u22120.01505\u22120.206590.197283\u22120.35551SCO5039 putative penicillin-binding protein0.590106\u22120.709260.8918190.313533SCO5301 putative penicillin-binding protein\u22120.19347\u22120.131310.547233\u22120.22028SCO5365 putative transferase1.11236\u22120.273340.569152\u22121.16844SCO5467 muramoyl-pentapeptide carboxypeptidase\u22120.154480.387477\u22120.49044\u22120.02243SCO5560 D-alanine-D-alanine ligase0.7281120.2096020.1677690.330255SCO5998 murA20.8007260.7929751.2913680.051102SCO6060 putative UDP-N-acetylmuramoyl-L-alanine ligase0.199410.3770040.8173290.527531SCO7050 putative D-alanyl-D-alanine carboxypeptidase0.6059490.702030.3438270.524521Mean hybridization score0.125637\u22120.107440.118866\u22120.14494Bold values indicate that the signal for that gene is more than 2SD below the mean core signal for that species and such a value is suggestive of either gene absence or very low similarity\nConserved genes with no known function\nGenes with no known function and no homologue outside of S. avermitilis that are conserved across the other three Streptomyces species should represent genes important to specifically being a myceliate Actinobacteria and the phenotype of gene knockout strains for these genes will be particularly interesting in terms of Streptomyces biology. Based on the dataset here, 936 genes can be identified as annotated as either conserved hypothetical genes or non-conserved hypothetical genes and these are shown in Supplementary Table\u00a01. The proportion of these genes that are conserved across all four species are 9%, 20%, 13%, 16% and 12% for the left terminal region, left Streptomyces specific region, core region, right Streptomyces specific region and right terminal region, respectively. There is also a low frequency of conserved hypothetical genes in the left terminal region and right Streptomyces specific region, 0.78% and 0.96%, respectively compared to 3.4% for the left Streptomyces specific region, 1.80% for the core region and 2.11% for the right terminal region. It is clear that there is a need to further screen these genes by increasing the range of Streptomyces species analyzed by microarray hybridization. This will reduce the number to a manageable number and will allow prioritization of genes for knockout and detailed phenotypic analysis.\nAnother approach to the problem of identifying functionally important genes is by the pinpointing of functional groups of such genes that may form a transcriptional unit. Blocks of three or more hypothetical genes that are conserved across all species were identified and are shown in Table\u00a08. It is possible that these groups represent conserved functional groups of genes essential to core functions that make Streptomyces different from other bacteria. They are found mostly in the area between the Streptomyces terminal regions and the central core region. There are seven groups of conserved hypothetical genes larger than five genes (SCO1407\u2013SCO1413, SCO2362\u2013SCO2370, SCO2911\u2013SCO2919, SCO3846\u2013SCO3854, SCO5536\u2013SCO5543, SCO5762\u2013SCO5767 and SCO6522\u20136528). It is likely, due to the proximity of various genes around SCO3846\u2013SCO3854, that this complex is involved in cell division, development and DNA partitioning. The function of the others groups is unknown. Interestingly, none of these gene groups are upregulated shifting from exponential phase to stationary phase or under stress shift as indicated by Karoonuthaisiri et\u00a0al. (2005).Table\u00a08Hypothetical genes in S. coelicolor conserved as a group in the four species analyzedGenes (SCO)Operon structureaLinked function if anyb0614, 0616, 0617, 0618None\u20131317, 1318, 1319, 1320None\u20131521, 1522, 1523, 1524Possible operonRecombination1634, 1635, 1636Possible operon\u20131650, 1651, 1652, 1653Possible operonProteosome1788, 1789, 1790, 1791, 1794, 1795, 1796Possible operonBoth flanks of rRNA gene homologues2030, 2031, 2032Possible operon\u20132124, 2125, 2127, 2129, 2130Possible operonGlucose kinase2268, 2269, 2270Possible operonClose to heme oxygenase2913, 2915, 2916, 2917None\u20133115, 3117, 3118, 3119None\u20133150, 3151, 3152, 3153None\u20133406, 3407, 3408Possible operonPenicillin binding protein3950, 3951, 3952Possible operonOxidoreductase4028, 4029, 4030None\u20134801, 4803, 4804, 4805None\u20135307, 5308, 5309, 5310, 5312None\u20135600, 5601, 5602, 5603, 5604Possible operonHomology to Mycobacterium tuberculosis5762, 5763, 5764, 5765Possible operonDNA helicase6413, 6415, 6416, 6417, 6419, 6420, 6421, 6422None\u20136574, 6575, 6576, 6577, 6578, 6579, 6580Possible operonPossible DNA binding protein6671, 6672, 6674, 6675, 6676Possible operon\u20137070, 7071, 7072None\u2013a\u00a0Gene structure from Artemis v7 is compatible with an operon type structure with possible appropriate ribosome binding sitesb\u00a0Inside or linked to the conserved genes is a gene(s) of known function\nConservation of genes involved in secondary metabolism and similar functions\nGenes that are involved in secondary metabolism and antibiotic production are widely distributed in the Streptomyces and many if not most may have been involved in horizontal transfer. However, there is significant similarity between genes involved in similar pathways and thus significant cross-hybridization may occur between similar metabolic pathways. A large number of genes are also involved in secondary metabolism (165) and polyketide synthesis (102) in the S. coelicolor genome. These are grouped together in 23 pathway groups and are displayed in Supplementary Fig.\u00a03. Genes identified as secondary metabolic genes but existing on their own and not in a group of secondary metabolic genes have been eliminated to simplify the analysis leaving only genes involved in these functions with two or more genes together in a group. These include specific pathways producing secondary metabolic products such as melanin, actinorhodin, CDA and Red pathway. Many of the other potential pathways have not been studied in detail and the functions of these genes are unknown. Because of evolutionary similarity, the presence of genes hybridizing to a particular pathway does not mean that the specific pathway is present, but possibly that a related one is may be present. Similarly, a high level of hybridization can mean either a very close relationship between the pathways in the two species or the presence of multiple copies of related pathways. In general terms S. maritimus shows the greatest absence of secondary metabolic pathways that are present in S. coelicolor. Interesting, S. cattleya and K. aureofaciens seems to have pathways related to many of the S. coelicolor secondary metabolic pathways present in their genomes, although they are phylogenetically more distant than S. maritimus. The actinorhodin pathway would seem to be absent from S. avermitilis (as expected from the genome data), S. cattleya and S. maritimus although some related genes do seem to be present in K. aureofaciens. The WhiE pathway is conserved in all species, but with some genes showing a very low level of hybridization in certain cases and these include whiE protein VII and the acyl carrier protein. Genes from the Red pathway show varying levels of hybridization suggesting that distantly related pathways may be present in these species. The CDA pathway is conserved in all four species and in certain cases the genes seem to be over represented suggesting multiple examples of the same type of pathway in S. cattleya and S. maritimus. The presence of similar pathways at a level of about 50% for K. aureofaciens supports the well established idea that horizontal gene transfer of secondary metabolic pathways may have played a significant role in the evolution of the Streptomyces and any related genus.\nBecause the natural environment of Streptomyces is the soil, they are thought to play an important role in the recycling of lignocellulose material. However, there is relatively little information on what genes are involved in this process. Interestingly, melC1 and melC2, which encode tyrosinase (monophenol monooxygenase, SCO2700) and its cofactor (SCO2701) (Leu et\u00a0al. 1992) are conserved across the three Streptomyces species and probably also Kitasatospora (SCO2700 \u22120.76, SCO2701 0.08). On the other hand, the duplicate MelD1 (SCO2701) and MelD2 (SCO2700) genes found in S. coelicolor are not conserved and are phylogenetically distinct from MelC1 and MelC2 found in other Streptomyces (unpublished results). This perhaps represents a divergence of function between this two gene pairs. S. coelicolor does not produce a detectable amount of black melanin pigment and these results suggest that these enzymes may be involved in the metabolic conversion of lignocellulose byproducts rather than pigment formation. Evolutionary conservation of these genes to serve this function under particular conditions of induction would make more sense than retention of inducible black pigment formation. Other enzymes with a possible role in the lignocellulose cycle that are conserved across the species are shown in Table\u00a09. These include many oxygenases that may have a role in producing oxygen radicals capable of attacking lignin, genes involved in the sensing and breaking down hydrogen peroxide, cellulose metabolism genes, cellobiose metabolism genes, etc. Those found in the terminal regions may represent gene groups that are not conserved in a syntenous manner and subject to horizontal gene transfer, while those within the core and intermediate regions may be part of the basic group of genes essential to Streptomyces in the soil environment. Lignocellulose degradation is a difficult topic to study in the Actinomycetales and therefore these candidate genes may help to solve some of the problems associated with this.Table\u00a09Genes conserved in the four Streptomyces species that are potentially involved in lignocellulose cyclingSCO0333DioxygenaseSCO0560Catalase\/PeroxidaseSCO0765EndoglucanaseSCO1187CellulaseSCO1188Cellulose binding proteinSCO1338MonooxygenaseSCO1451EndoglucanaseSCO1923DioxygenaseSCO2016MonooxygenaseSCO2267Heme oxygenaseSCO2700Tyrosinase (monophenol monooxygenase)SCO2701Tyrosinase cofactorSCO2783MonooxygenaseSCO2798Cellobiose hydrolaseSCO2838EndoglucanaseSCO3172MonooxygenaseSCO3236OxygenaseSCO4416MonooxygenaseSCO4870MonooxygenaseSCO5033Hydrogen peroxide sensing regulatorSCO5293OxygenaseSCO5390Alkanal monoxygenaseSCO5773MonooxygenaseSCO6545CellulaseSCO7223MonooxygenaseSCO7637EndoglucanaseNote that the oxygeneases included as possible enzymes that make be able to attack lignin are all unclassified yet as to their real function. The core region is in bold\nConclusions\nThis study confirms that within the Streptomyces analyzed here there is conservation of a core set of genes in the middle of the linear S. coelicolor\/S. avermitilis chromosome structure. This is associated with a much higher diversity of gene in the terminal regions of the linear chromosome. Linking these regions are two intermediate regions where there seems to be conservation of genus specific genes and gene clusters. This study also identifies candidate genes that may be possibly involved in terminal replication and other myceliate growth related functions based on a classification of genes into conserved and none conserved groups. This study also provides insights into which genes in Streptomyces play a more significant role in the biochemical network of S. coelicolor, Streptomyces and myceliate Actinobacteria in general. Finally, the degree of gene conserved detected between the four species implies that that genome model of S. coelicolor may extent well beyond the borders of the Streptomyces. It includes at least one Kitasatospora species; furthermore, a similar structure by microarray analysis has been found for Saccharomonospora viridis and Streptosporangium roseum, but not Streptomyces rimosus ATCC10970 (unpublished data). Thus, the microarray approach to genome content analysis and exploration of genome evolution may be fairly widely applicable in the various Actinomycete genus close to Streptomyces that undergo complex morphogenesis.\nElectronic supplementary material\nBelow is the link to the electronic supplementary material\nESM (PDF 938 kb)","keyphrases":["actinomycetales","genomotyping","linear genome"],"prmu":["P","U","R"]}
{"id":"Int_J_Colorectal_Dis-4-1-2225995","title":"Certolizumab pegol, a monthly subcutaneously administered Fc-free anti-TNF\u03b1, improves health-related quality of life in patients with moderate to severe Crohn\u2019s disease\n","text":"Background and aims Certolizumab pegol, a polyethylene glycolated Fc-free Fab\u2019 was efficacious and well tolerated in patients with moderate-to-severe Crohn\u2019s disease in a previously reported randomized, placebo-controlled study. In this paper, we report the effect of certolizumab pegol on health-related quality of life (HRQoL).\nIntroduction\nCrohn\u2019s disease is a chronic relapsing and remitting, inflammatory bowel disease (IBD) that has a significant impact on health-related quality of life (HRQoL) [1, 2]. HRQoL has been recognized as an important health outcome and has been defined in the medical setting as \u201ca concept encompassing a broad range of physical and psychological characteristics and limitations which describe an individual\u2019s ability to function and derive satisfaction from doing so\u201d [3]. Crohn\u2019s disease frequently begins in early adulthood, causing a heavy disease burden in a relatively young patient population. Consequently, many patients will be affected for most of their adult life.\nIn Crohn\u2019s disease, health status is affected as much by psychosocial factors and functional status as by disease activity [4]. For example, the need to wear an ostomy bag may adversely affect self-image, and this has been reported to be a common worry for patients with Crohn\u2019s disease [5]. Moreover, previous studies have demonstrated a high correlation between disease severity, as measured by the Crohn\u2019s Disease Activity Index (CDAI) [6], and HRQoL, as assessed using the Inflammatory Bowel Disease Questionnaire (IBDQ) [7, 8]. A large longitudinal study prospectively assessed the HRQoL of patients with Crohn\u2019s disease over 1\u00a0year and determined influencing factors [9]. The following factors were associated with a negative impact on overall HRQoL: female gender, tobacco use, active Crohn\u2019s disease, involvement of the colon, hospitalization, corticosteroid treatment, and surgery in the past 3\u00a0months (order does not indicate priority). These findings were reflected in a survey of patients with a long-term diagnosis of Crohn\u2019s disease [10]. It is therefore important to include improvement in HRQoL as a key therapeutic goal in the treatment of patients with Crohn\u2019s disease.\nConventional treatment options for Crohn\u2019s disease include aminosalicylates, antibiotics, corticosteroids, immunomodulators, and surgery. However, in some patients, the disease can be refractory or unresponsive to certain pharmacologic treatments. Furthermore, corticosteroids are associated with a risk of dependency and potentially serious side effects (e.g., diabetes, osteoporosis, moon face, and acne), which are detrimental to a patient\u2019s HRQoL and may negatively affect self-esteem [11\u201313]. Such treatments therefore require careful management. Additionally, it is rarely possible to achieve long-term relief from Crohn\u2019s disease with a single surgical procedure, and as a result, patients with Crohn\u2019s disease fear surgery so much that their HRQoL is reduced [14].\nIt is now well accepted that tumor necrosis factor \u03b1 (TNF\u03b1) plays a pivotal role in the underlying inflammatory pathophysiology of Crohn\u2019s disease [15]. Furthermore, neutralization of TNF\u03b1 has been shown to improve the symptoms of this debilitating illness [13, 16]. Infliximab has been shown to improve HRQoL, as measured by the IBDQ and the Short-Form Health Survey (SF-36) [8, 17], but the occurrence of infusion reactions along with the risk of developing anti-infliximab antibodies, which can reduce long-term efficacy [18], could negatively impact HRQoL over time. Patients may feel that subcutaneous treatment has advantages over treatments requiring infusion; a long stay at the clinic may negatively impact a patient\u2019s daily life. This increased freedom in patient treatment management may lead to improved treatment compliance with a consequent impact on therapeutic outcomes. Thus, there is a need for alternative long-term therapies for moderate-to-severe Crohn\u2019s disease that have minimal negative impact on patients\u2019 everyday life while also reducing the impact of the disease on patients\u2019 HRQoL [19].\nCertolizumab pegol is the first polyethylene glycolated (PEGylated) anti-TNF\u03b1 antibody fragment (Fab\u2019) to be studied in Crohn\u2019s disease. Unlike conventional anti-TNF agents, certolizumab pegol lacks the crystallizable fragment (Fc) region, thus potentially avoiding unwanted effects [20]. PEGylation increases the plasma half-life and reduces the frequency of dosing required. Certolizumab pegol is administered by subcutaneous injection once every 4\u00a0weeks (more frequent dosing may be used during an induction phase). Certolizumab pegol 400\u00a0mg administered subcutaneously demonstrated efficacy and was well tolerated in patients with moderate-to-severe active Crohn\u2019s disease in phases II [21] and III (PRECiSE 1 and PRECiSE 2 [22, 23]) studies. Certolizumab pegol has also demonstrated clinical efficacy and good tolerability in patients with rheumatoid arthritis [24, 25].\nThis is the first paper to report the effect of certolizumab pegol, a monthly subcutaneous treatment, on the HRQoL of patients with moderate-to-severe active Crohn\u2019s disease in a phase II placebo-controlled study [18]. HRQoL was assessed using the IBDQ.\nMaterials and methods\nEthical conduct\nThis study was approved by the Independent Ethics Committee\/Institutional Review Board for each center before initiation. It was conducted in accordance with the International Conference for Harmonization Guidelines for Good Clinical Practice and the declaration of Helsinki (revised 1996).\nWritten informed consent was obtained from each patient at screening before any study procedures were performed.\nPatients\nAdult patients (age \u226518\u00a0years) with moderate-to-severe active Crohn\u2019s disease, as defined by a CDAI score of 220\u2013450 points during the week before the administration of the first dose of study drug, were eligible for the study. Exclusion criteria included suspected or diagnosed abscess, a bowel perforation or evidence of non-inflammatory obstruction in 6\u00a0months before screening, extensive bowel resection, a functional colostomy or ileostomy, or a known history of tuberculosis. Concomitant medication was allowed, provided that doses were stable and could be continued for the 12-week double-blind period and the 8-week follow-up.\nStudy design\nThis was a multicenter, randomized, double-blind, placebo-controlled, 12-week study conducted at 58 international centers between February 2001 and March 2002. An 8-week follow-up period allowed collection of additional safety data. At screening, patients with moderate-to-severe active Crohn\u2019s disease were stratified according to concomitant steroids, immunosuppressants, or long-term anti-infectives. Eligible patients were randomized (1:1:1:1) to receive certolizumab pegol (100, 200, or 400\u00a0mg) or placebo (0.9% w\/v saline) by subcutaneous injection at weeks 0, 4, and 8 (Fig.\u00a01). Before dosing, patients\u2019 demography, medical history, and concomitant diseases were recorded.\nFig.\u00a01Study design (showing the total numbers of patients recruited into each treatment group)\nOutcomes\nThe primary endpoint of the study was the proportion of patients achieving a clinical response [\u2265100-point decrease in CDAI score or CDAI score \u2264150 points (remission)] at week 12. HRQoL was a secondary endpoint evaluated at baseline (week\u00a00, pre-injection) and at weeks\u00a02, 4, 6, 8, 10, and 12 using the 32-item self-administered IBDQ. The IBDQ is a disease-specific HRQoL measure. The questionnaire assesses the four aspects of a patient\u2019s life that are affected by IBD: symptoms directly related to the primary bowel symptoms, systemic symptoms, emotional function, and social function [26]. The reliability, validity, and responsiveness of the IBDQ have been demonstrated by Guyatt et al. [26, 27] and Irvine et al. [7, 28]. The IBDQ is completed using a 7-point Likert response for each question. Total IBDQ scores range from 32 to 224, with a higher score indicating better HRQoL. Remission is generally indicated by a score of at least 170 points, and a change of 16 points is regarded as clinically meaningful [7]. In addition to a total score, domain scores can be calculated for the Bowel Symptoms, Systemic Symptoms, Emotional Function, and Social Function domains. IBDQ measurements were performed in parallel with determination of CDAI score and serum concentrations of C-reactive protein (CRP). Official IBDQ versions translated into Danish, French, German, and Swedish were used. Our own translations were used for Russian, Serbian, and Afrikaans. To assess whether our own translated versions of the IBDQ provided a valid measure of HRQoL, we compared scores of the translated versions with those of the official translated versions and evaluated those scores in relation to the patient\u2019s CDAI scores. A sensitivity analysis was also carried out to check for influence of specific item scores on the resulting total scores. The results of these analyses demonstrated that our own translated versions of the IBDQ provided a valid measure of HRQoL, comparable to the official, translated versions.\nStatistical analysis\nA sample size of 260 patients was calculated to give approximately 83% power to detect a true difference between treatment groups of 23% for the primary endpoint of the study, based on a placebo response rate of 12%. After screening, patients were to be randomized in a 1:1:1:1 ratio (65 patients to each of the four treatment groups). Therefore, 195 patients were to receive certolizumab pegol and 65 were to receive placebo.\nEfficacy was assessed for the intent-to-treat (ITT) population, which included all patients who received at least one injection and had at least one efficacy measurement after the first injection. Patients who terminated the trial prematurely were advanced to the end-of-study visit. The last observation carried forward method was used in cases where data were missing.\nPost hoc analyses of IBDQ total score and individual domain scores were performed. These were purely exploratory in nature. Relationships between CDAI and IBDQ global scores at week\u00a012 were assessed for the ITT population using Kendall\u2019s tau coefficient, a measure of the strength of dependence showing to what extent these two variables move in the same direction. Changes from baseline in IBDQ total score and individual domain scores for each treatment group were formally compared with the placebo group. Least-squares means, adjusted for stratum, pooled country, and baseline IBDQ score, were compared across treatment groups. Fisher\u2019s exact test was used to compare the proportion of patients who achieved remission (defined as an IBDQ total score of \u2265170 points) in each active treatment group with that for the placebo group at each week. The analyses were performed based on both the overall ITT population and in the subgroup of patients with elevated baseline CRP (\u226510\u00a0mg\/l). P values \u22640.05 were considered to be statistically significant.\nResults\nThe clinical efficacy and safety assessments of this phase II dose-finding study have been reported in detail elsewhere [21]. Because the 400\u00a0mg dose was identified as being the most appropriate dose, this paper focuses on data for patients receiving certolizumab pegol 400\u00a0mg.\nPatients\nIn total, 372 patients were screened of whom 292 patients were enrolled (CDAI score <220 points was the most common reason for screening failure). Of these 292 patients, 291 were included in the ITT population used to measure the efficacy endpoints according to CDAI scores [1 patient (400\u00a0mg group) was excluded because of missing efficacy data]. Baseline IBDQ scores were not obtained for one patient in the certolizumab pegol 100\u00a0mg treatment group. The ITT analyses for the HRQoL evaluations therefore included data for 290 patients. Seventy-five patients (25.7%) withdrew from the study by week\u00a012. The majority of withdrawals were a consequence of lack of improvement\/disease progression.\nBaseline CRP measurements were obtained for 290 of the 291 patients in the ITT population. Elevated baseline CRP levels (\u226510\u00a0mg\/l) were recorded for 119 patients (41.0%). One of these patients was the patient in the certolizumab pegol 100\u00a0mg treatment group who was excluded from analyses as a result of baseline IBDQ scores not being obtained. The subgroup analyses of IBDQ by baseline CRP status were therefore performed on data from 118 patients with an elevated baseline CRP concentration.\nThe four treatment groups were generally well matched in terms of demographic and clinical characteristics (including baseline IBDQ total score and concomitant medication profile; Table\u00a01). In the ITT population, 21.6% of patients had received previous anti-TNF\u03b1 therapy, and 15.5% had concomitant medication with both immunomodulators and steroids.\nTable\u00a01Baseline demographic and clinical characteristics (intent to treat population used to measure efficacy endpoints according to Crohn\u2019s Disease Activity Index scores)\u00a0Placebo(n\u2009=\u200973)Certolizumab pegol100\u00a0mg (n\u2009=\u200974)200\u00a0mg (n\u2009=\u200972)400\u00a0mg (n\u2009=\u200972)Mean CDAI score (range)291.5 (206\u2013448)299.2 (194\u2013520)310.7 (184\u2013446)304.5 (204\u2013461)Mean IBDQ score (range)a122.9 (61\u2013190)132.2 (66\u2013189)b122.9 (74\u2013189)126.5 (71\u2013177)Geometric mean plasma CRP, mg\/l (range)7.268 (0.27\u201386.10)6.231 (0.15\u2013141.00)b6.475 (0.17\u2013127.00)7.738 (0.35\u2013128.28)CRP\u2009\u2265\u200910\u00a0mg\/l, n (%)28 (38.4)31 (42.5)b28 (38.9)32 (44.4)Concomitant medications, n (%)\u00a0\u00a0Aminosalicylates29 (39.7)37 (50.0)32 (44.4)28 (38.9)\u00a0\u00a0Anti-infectives7 (9.6)6 (8.1)7 (9.7)6 (8.3)\u00a0\u00a0Antidiarrheals10 (13.7)19 (25.7)16 (22.2)12 (16.7)\u00a0\u00a0Steroids29 (39.7)24 (32.4)29 (40.3)22 (30.6)\u00a0\u00a0Codeine and derivatives6 (8.2)5 (6.8)5 (6.9)2 (2.8)Immunomodulators\u00a0\u00a0Azathioprine17 (23.3)13 (17.6)23 (31.9)22 (30.6)\u00a0\u00a06-Mercaptopurine4 (5.5)9 (12.2)2 (2.8)2 (2.8)\u00a0\u00a0Methotrexate5 (6.8)4 (5.4)4 (5.6)3 (4.2)CDAI Crohn\u2019s Disease Activity Index; CRP C-reactive protein; IBDQ Inflammatory Bowel Disease Questionnaire; n number of patientsaMaximum score is 224 points.bData missing for one patient.\nClinical response\nThe onset of effect (clinical response) of certolizumab pegol was evident at week\u00a02, when certolizumab pegol treatment produced a statistically significant improvement in CDAI score compared with placebo (certolizumab pegol 400\u00a0mg 33.3% vs placebo 15.1%; P\u2009=\u20090.010). Clinical response rates were highest in the certolizumab pegol 400\u00a0mg group at all time points, with maximal response at week\u00a010 (certolizumab pegol 400\u00a0mg 52.8% vs placebo 30.1%; P\u2009=\u20090.006) [21]. Lower CDAI score (representing lower disease severity) was associated with a higher IBDQ global score (representing better HRQoL), as reflected by a high negative Kendall\u2019s tau value (\u22120.48; P\u2009<\u20090.0001) Clinical response was observed irrespective of baseline CRP level.\nImprovement in HRQoL\nPatients demonstrated HRQoL improvements from baseline, as measured by change in IBDQ total score, by the first assessment point, week\u00a02 [certolizumab pegol 100\u00a0mg, 16.6 points; certolizumab pegol 200\u00a0mg, 21.8 points (P\u2009\u2264\u20090.05 vs placebo); certolizumab pegol 400\u00a0mg, 22.8 points (P\u2009\u2264\u20090.05 vs placebo); placebo, 10.6 points] (Fig.\u00a02). Patients receiving certolizumab pegol 400\u00a0mg experienced statistically significantly greater improvements from baseline in IBDQ total score at all time points (P\u2009\u2264\u20090.05) compared with those receiving placebo. The greatest increase in IBDQ total score was recorded at week\u00a010 (certolizumab pegol 400\u00a0mg 32.2 points vs 18.6 points for placebo; P\u2009\u2264\u20090.05). Approximately half of the patients treated with certolizumab pegol 400\u00a0mg demonstrated clinically meaningful improvements in IBDQ total score (\u226516-point increase) at week\u00a02 (52.8%) and at week\u00a012 (66.7%).\nFig.\u00a02Mean change from baseline in Inflammatory Bowel Disease Questionnaire (IBDQ) total score during 12\u00a0weeks\u2019 subcutaneous treatment with either certolizumab pegol or placebo in patients with moderate-to-severe Crohn\u2019s disease (intent-to-treat population); least-squares means (adjusted for stratum, pooled country, and baseline score)\nAs early as week\u00a02, patients receiving certolizumab pegol 400\u00a0mg demonstrated statistically significant improvements in the Bowel Symptoms and Emotional Function domains of the IBDQ relative to placebo (Fig.\u00a03). Improvement in the Systemic Symptoms domain score approached statistical significance at week\u00a02 (P\u2009=\u20090.054). These improvements were generally dose-related (Figs.\u00a03a\u2212d). Interestingly, patients in the certolizumab pegol 200\u00a0mg group achieved statistically significantly greater improvements than those receiving placebo in IBDQ total score and all four IBDQ domains at week\u00a02 (Figs.\u00a02 and 3a\u2013d).\nFig.\u00a03Mean change from baseline in Inflammatory Bowel Disease Questionnaire (IBDQ) domain scores during 12\u00a0weeks subcutaneous treatment with either certolizumab pegol or placebo in patients with moderate-to-severe Crohn\u2019s disease (intent-to-treat population); least-squares means (adjusted for stratum, pooled country, and baseline score): a Bowel Symptoms domain, b Systemic Symptoms domain, c Emotional Function domain, and d Social Function domain\nEmotional Function and Systemic Symptoms were the domains that patients reported as being most improved by certolizumab pegol treatment. At every assessment, patients receiving certolizumab pegol 400\u00a0mg showed statistically significantly (P\u2009\u2264\u20090.05) greater improvements in Emotional Function domain scores than those receiving placebo (Fig.\u00a03c). They also demonstrated statistically significantly greater improvements in Systemic Symptoms domain scores than those receiving placebo at weeks\u00a04, 8, 10, and 12 (Fig.\u00a03b). The improvements patients experienced in Social Function domain scores approached statistical significance (P\u2009=\u20090.054) at week\u00a04 (Fig.\u00a03d).\nThe remission rate in the certolizumab pegol 400\u00a0mg group was highest at week\u00a010. This was statistically significantly greater than in the placebo group (Table\u00a02). The proportion of patients achieving remission in the certolizumab pegol 400\u00a0mg group was also statistically significantly greater than in the placebo group at weeks\u00a06, 8, and 12. The subgroup of patients with elevated CRP showed similar improvements in IBDQ scores to those observed in the ITT population.\nTable\u00a02Proportion of patients achieving remission defined by Inflammatory Bowel Disease Questionnaire total score (\u2265170 points)Patients (%), intent-to-treat population (n\u2009=\u2009290)WeekPlacebo (n\u2009=\u200973)Certolizumab pegol100\u00a0mg (n\u2009=\u200973)200\u00a0mg (n\u2009=\u200972)400\u00a0mg (n\u2009=\u200972)217.832.919.427.8419.237.0a*26.433.3617.835.6*30.638.9*821.935.629.238.9*1027.443.836.147.2*1223.338.423.638.9*aThe higher remission rate at week\u00a02 in the certolizumab pegol 100\u00a0mg group compared with higher-dose groups may reflect the slightly higher mean Inflammatory Bowel Disease Questionnaire (IBDQ) score at baseline in this group. However, certolizumab pegol 400\u00a0mg resulted in the greatest mean change in IBDQ at all time points.*p\u2009\u2264\u20090.05 vs placebo\nDiscussion\nPatients with Crohn\u2019s disease often have poor HRQoL [29], with disease-related concerns, such as worry about needing surgery, being the main causal factor [10]. It therefore follows that treatments relieving the symptoms of Crohn\u2019s disease and\/or reducing the risk of surgery should improve HRQoL in patients with Crohn\u2019s disease.\nCertolizumab pegol administered subcutaneously every 4\u00a0weeks is a rapid and clinically effective treatment of moderate-to-severe Crohn\u2019s disease with the 400\u00a0mg dose (highest dose tested) inducing the highest response rates [21, 22, 23]. This analysis indicates that such clinical findings are accompanied by improvements in HRQoL.\nCertolizumab pegol 400\u00a0mg appears to exert a rapid, consistently positive effect on HRQoL in patients with moderate-to-severe Crohn\u2019s disease. Patients who received certolizumab pegol 400\u00a0mg demonstrated statistically significantly greater improvements in IBDQ total score than those in the placebo group as early as week 2 and at all subsequent time points. As a reflection of the lower baseline IBDQ scores in the 400\u00a0mg group, patients in this group experienced the greatest improvement, despite the remission rate (which is the proportion of patients reaching a pre-defined score) being higher in the 100\u00a0mg group. Improvements in HRQoL were not dependent on baseline CRP level. Higher baseline CRP concentration was associated with greater improvement in clinical variables in this phase II study [21], but such a relationship has not been evident in phase III studies [30]. In addition, patients receiving certolizumab pegol 200\u00a0mg showed a statistically significantly greater change from baseline in IBDQ total score than those receiving placebo at weeks\u00a02 and 4. Collectively, these findings suggest that certolizumab pegol has an early treatment effect in patients with moderate-to-severe Crohn\u2019s disease. The benefits of certolizumab pegol on HRQoL were also apparent in the phase III studies [22, 23, 31, 32].\nImprovements in emotional well-being (e.g. depression, anxiety, irritability, and anger resulting from bowel problems) and systemic symptom domains were particularly marked with certolizumab pegol treatment. It appears that the improvements in these two domains of the IBDQ are largely responsible for the reported beneficial effect of certolizumab pegol on HRQoL. Overall, these findings suggest that certolizumab pegol alleviates the psychological symptoms associated with Crohn\u2019s disease and improves a patient\u2019s general well-being, in addition to providing clinical benefits in terms of CDAI response.\nHRQoL provides an integral picture of the interactions between a patient\u2019s disease, their treatment, and quality of life\u2014positive effects resulting from the efficacy of a treatment and negative effects caused by adverse events are evaluated. Few trials have examined the effects of conventional agents upon HRQoL in patients with Crohn\u2019s disease. While corticosteroids are effective in controlling acute Crohn\u2019s disease and are reported to improve IBDQ scores [33], the potentially serious adverse events associated with corticosteroids may negate any positive effects on HRQoL gained through control of symptoms. Additionally, corticosteroids are not suitable for maintenance treatment. Improvements in HRQoL have been observed with methotrexate treatment in a large, randomized, double-blind, placebo-controlled study [34]. However, 17% of patients (16\/94) receiving methotrexate were withdrawn from the study because of adverse events compared with 2% of placebo-treated patients (1\/47).\nThe improvements in HRQoL observed with certolizumab pegol in this study appear to be similar to those reported in patients with Crohn\u2019s disease after treatment with infliximab [8, 17, 35]. Similarly, natalizumab has been shown to improve HRQoL in patients with Crohn\u2019s disease [36].\nGiven the detrimental effects of Crohn\u2019s disease on physical and psychosocial functioning, additional research is needed to assess the impact of currently available and newer therapies on HRQoL. Although HRQoL was evaluated using the self-administered IBDQ, which has proved to be a valid and reliable disease-specific instrument for determining health status in patients with Crohn\u2019s disease [7], the analyses presented in this paper were purely exploratory in nature, and firm conclusions cannot be drawn from the results described. However, clinical benefit was also accompanied by improved HRQoL in the phase III studies [22, 23, 31, 32]. Further benefits may be gained through the good tolerability of certolizumab pegol [37] and convenience of subcutaneous dosing with certolizumab pegol once every 4\u00a0weeks.\nConclusions\nThis study demonstrates that the clinical efficacy of certolizumab pegol in patients with moderate-to-severe Crohn\u2019s disease, which is evident within 2\u00a0weeks of injection, is paralleled by improvements in HRQoL. The benefits of certolizumab pegol were most pronounced at the highest dose tested (400\u00a0g). Improvements in emotional well-being and systemic symptoms appear to be largely responsible for the observed beneficial effect of certolizumab pegol on HRQoL.","keyphrases":["quality of life","crohn\u2019s disease","inflammatory bowel disease","immunotherapy"],"prmu":["P","P","P","U"]}
{"id":"Virchows_Arch-3-1-1888722","title":"Search for residual prostate cancer on pT0 radical prostatectomy after positive biopsy\n","text":"Reported incidence of no residual prostate cancer (i.e. pathological stage pT0) on radical prostatectomy ranges from 0.07 to 4.2%. The incidence is higher after neoadjuvant endocrine treatment. The aim of this study was to search for residual cancer on radical prostatectomy (RP) specimens when an initial sampling failed to find the cancer in patients with positive biopsy. Our database of 1,328 consecutive patients whose biopsies and RP specimen were both examined at the Polytechnic University-United Hospitals of the Marche Region between March 1995 and June 2006 was reviewed. The radical prostatectomies were grossly completely sampled and examined with the whole mount technique. We identified eight patients (i.e. 0.6%; three untreated and five hormonally treated preoperatively, i.e. 0.3 and 0.8%, respectively, of the total number of RPs included in the study) with positive biopsy and with no residual cancer in the initial routine histological examination of the RP. The RP of this group of eight was subjected to additional sectioning and evaluation of the paraffin blocks of the prostatectomy, also after block-flipping, immunostaining with an antibody against CAM 5.2, p63, PSA, and alpha-methylacyl-CoA racemase, and DNA specimen identity analysis. There were no cases with a false positive biopsy diagnosis, and cancer was not overlooked or missed in the initial routine histological examination of any of the 8 pT0 RPs. A minute focus of cancer (the diameter was always below 2.0 mm) was found on the additional sections in five. In particular, cancer was found after block-flipping in one of them. In an additional case, cancer was eventually discovered after immunostaining tissue sections for cytokeratin CAM 5.2, for p63 and PSA. In the remaining two cases (one untreated and the other hormonally treated), cancer was not found (0.15% of the 1,328 RPs included in the study); the review of the description of the macroscopic appearance of the RP and of its slides revealed that part of the peripheral zone corresponding to the site of the positive biopsy was missing, i.e. not removed from the patient at the time of the operation at least in one of the two. DNA specimen analysis confirmed the identity of the biopsy and prostatectomy in both. An extensive search for residual cancer reduces the number of pT0 RPs after a positive biopsy from 0.6 to 0.15%. It is recommended to have the needle biopsy reviewed, carefully look again at the radical prostatectomy, do deeper sections and then flip certain paraffin blocks. In addition, atypical foci should be stained for basal cell markers and often AMACR, especially in hormone-treated cases. If a block is missing part of the peripheral zone (capsular incision), this should be commented on. DNA analysis for tissue identity should be performed when the other steps have been taken without finding cancer.\nIntroduction\nIn 1995, Goldstein et al. [9] introduced the term vanishing cancer phenomenon referring to cases with minute or no cancer on radical prostatectomy after a positive biopsy. They reported 13 patients with minimal (11 patients) or no (two patients) cancer in prostatectomy specimens. In two cases with no residual cancer on RP, they confirmed the identity of the biopsy and the prostatectomy tissue by DNA matching.\nThe Johns Hopkins Hospital group documented a fivefold increase in the incidence of no residual cancer on prostatectomy in patients in whom both biopsies and prostatectomies were performed at that institution between 1997 and 2005 (0.07% in 1997, 0.13% in 2004 and 0.34% in 2005) [5, 7, 30]. In their first study, they also documented two cases, one in which the biopsy review revealed only high-grade PIN and in which a diagnosis of cancer had been established in another institution, and a second case in which the possibility of specimen switching could not be ruled out due to DNA mismatch between the biopsy and the prostatectomy. Most recently, they have reported a series of 46 patients, 11 with no residual cancer on prostatectomy and 35 with minute cancer, and in 40 cases, they documented specimen identity [5]. In five of the six remaining cases, the results could not be interpreted due to technical problems, and in one case, the tissue from the biopsy with cancer did not match the tissue from the radical prostatectomy.\nBostwick and Bostwick [4] found that 38 patients with no cancer on prostatectomy, identified among 6,843 radical prostatectomies performed at Mayo Clinic during a 30-year period, showed no disease recurrence or progression after a mean follow-up of 10\u00a0years. In their experience, the incidence of vanishing cancer declined tenfold, comparing prostatectomies performed before 1980 (2.1% incidence) to a more recent time interval from 1993 to 1995 (0.2% incidence). They have estimated the current incidence of vanishing cancer at 2 per 1,000 radical prostatectomies [4].\nRecently, Trpkov et al. [28] found an incidence of no residual cancer on prostatectomy of 0.67% after ten-core positive biopsy. They concluded that in most cases, finding no residual cancer on prostatectomy after additional sectioning and evaluation may indicate minimal patient disease. Very recently Zynger et al. [32] reported that their frequency of finding no residual cancer in RP specimens has increased from 0% in 2002 to 4.2% in 2006. In Canada, Srigley [25] has estimated the incidence of no residual cancer after positive biopsy in his practice at less than 0.5%.\nIn Germany, the incidence of pT0 in patients with prostate cancer without neoadjuvant treatment was analysed by Herkommer et al. [13]. Based on a database of 3,609 patients, no residual prostate cancer was found in 0.8% after RP. Most men in this pT0 series had previous TURP or open prostatectomy, which may have eliminated prostate cancer, whereas in two patients, cytology was used for primary diagnosis. Only in 0.3% of men with positive prostate needle biopsies was no residual tumour detected in the prostatectomy specimen [13]. The incidence of pT0 patients in a very recent French study was reported to be 0.5% [6].\nNo residual cancer may also result from preoperative endocrine therapy [11, 12, 15, 16, 19, 24, 26]. The incidence of pT0 after neoadjuvant endocrine therapy for prostate cancer is reported to be higher than in untreated patients [11]. Kollermann et al. [16] analysed a group of 174 men who underwent prolonged androgen deprivation therapy and observed a pT0 in 36 of 174 men (21%).\nSeveral questions are addressed to explain why cancer is not present in a small proportions of prostatectomies. Cancer is missed during the examination of the RP, all prostatectomy tissue is not sampled, and the cancer is overdiagnosed in the biopsy. Pathologists face additional challenges in eliminating the possibility of laboratory error resulting in specimen (blocks, slides) mislabelling or switching and excluding an information system error resulting in specimen mix-up [28].\nThe aim of this paper was to report the results of a search for residual cancer on RP after an initial stage pT0 evaluation in eight patients with positive biopsy.\nMaterials and methods\nWe reviewed our database of 1,328 consecutive patients whose biopsies and RP specimen were examined between March 1995 and June 2006 at the five Pathological Anatomy Services associated with the Polytechnic University-United Hospitals of the Marche Region, Ancona, Italy. Most of the biopsies and all the RPs of this series of 1,328 patients were reported by the same pathologist (RM). Seven hundred patients were untreated before the operation, whereas 628 had received neoadjuvant endocrine treatment for approximately 3 to 6\u00a0months.\nThe biopsies and RPs were routinely processed as follows. The core biopsies from each site were submitted in separate containers. In brief, during biopsy processing, no more than two biopsy cores were embedded in paraffin blocks, and all blocks were sectioned at 3\u00a0\u03bcm and were stained with haematoxylin\u2013eosin (HE). All cores were sectioned in two separate levels represented on separate slides, with each slide containing three additional sections [28].\nAll radical prostatectomy specimens (tissue was not harvested for research) were fixed en bloc in 10% buffered formalin for at least 24\u00a0h and were grossly completely sampled using a standard protocol [17, 18, 28]. In particular, each specimen was painted over the surface with India ink. The seminal vesicles were amputated at the prostate junction and were grossly completely sampled. The remainder of the specimen was serially cut with a domestic electric food slicer calibrated and set to deliver slices which are 3\u20134\u00a0mm in thickness, representing transverse planes parallel to the initial apical and basal sections. Each prostate slide was processed into complete whole mount section, and one HE slide was routinely sectioned per block. The apical and basal transverse 2- to 3-mm margins were sectioned perpendicularly to assess the prostatic apical and basal margins. Prostatectomies were grossly sectioned in 8.5 slices (mean; range, 6\u201316), and 15.3 slides (mean; range, 10 to 20) were generated per prostatectomy. Slides from pelvic lymph nodes were not included in the reported number of examined slides.\nWe identified eight patients with positive 6- to 12-core biopsy and with no residual cancer (pathological stage pT0) in the initial routine histological examination of the RP and successively subjected to additional sectioning and evaluation.\nSearch for residual cancer\nSince 1995, a special cancer searching protocol has been applied to those RPs in which an initial routine-based examination does not show residual cancer [18]. As mentioned earlier, this is basically done in those cases whose biopsies and RP specimen are both examined at the Ancona institution.\nThe following successive steps were undertaken by two pathologists (RM and RMa). The procedure was usually stopped at the step where cancer is found. \nThe diagnostic needle biopsies were reviewed to exclude the possibility of a false positive biopsy diagnosis and to assess the approximate location of the biopsy with tumour, such as apex, mid-zone and base, both left and right.The slides of the surgical specimens were reviewed for residual cancer that was initially overlooked or missed.If the prostate was not totally embedded, the remaining prostate tissue was processed in toto. If the prostate was completely sampled, then this step was skipped. This type of information was usually contained in the pathology form where the all the steps of the processing procedure were recorded. The information was further confirmed by searching the specimen\u2019s container for residual pieces. As we routinely sample completely all prostatectomies, this step was skipped.Additional deeper sections (i.e. three to five sections) of the prostatectomy area (paraffin block) corresponding to the location of the core with cancer were re-cut. Further sections were also obtained from all the other paraffin blocks.Additional deeper sections (i.e. three to five sections) of the area corresponding to the location of the positive core as well as of all the remaining blocks were re-cut after block-flipping.Immunostains for p63 and alpha-methylacyl-CoA racemase (AMACR) were performed to evaluate suspicious foci (When these two were not yet available, we used 34betaE12 immunostaining).Immunostain for cytokeratin CAM 5.2, for p63 and for PSA (prostate specific antigen) was performed to identify the so-called \u201cminimal residual cancer\u201d especially in patients receiving neoadjuvant hormonal therapy [17].Review the description of the macroscopic appearance of external and cut surfaces of the surgical specimen as well as inspect the contour of the tissue sections on the slides for hint or clues that might indicate that part of the tissue was missing either due to the surgical procedure or for technical reasons.DNA specimen analysis was performed on formalin-fixed tissue to confirm the identity of the biopsies and prostatectomies whenever necessary.\nDNA specimen identity analysis\nTested samples in an individual case included the biopsy core with cancer and a random block for the corresponding RP. The tissue was obtained as a direct section from formalin-fixed and paraffin-embedded blocks. DNA was extracted from paraffin-embedded tissue using the QIAmp DNA mini kit (Qiagen) according to manufacturer\u2019s protocol (Promega, Madison, WI, USA). Fifteen microsatellites and amelogenin locus were co-amplified by the AmpFlSTR identifiler kit (Applied Biosystems, Foster City, CA), and the amplified fragments were electrophorized on an ABIPrism3130 genetic analyzer (Applied Biosystems). Fragment sizing and allele designation were established by GeneMapperID v3.2 software (Applied Biosystems), and genetic profiles from both biopsy and prostatectomy samples were compared.\nResults\nResidual cancer was not found in eight RPs after an initial routine examination. They represent 0.6% of the 1,328 consecutive patients. Three (0.2%) of them were from the group of untreated patients, whereas five (0.4%) were from those who had received neoadjuvant treatment. The latter figure represents 0.8% of the treated patients.\nPatients\u2019 clinical data are summarised in Table\u00a01. The mean age of the patients was 65\u00a0years (range, 58\u201371\u00a0years). Mean serum PSA pre-biopsy was 5.1\u00a0ng\/ml (range, 1.25\u20139.0\u00a0ng\/ml). Mean gland volume was 43.9\u00a0ml (range, 28.1\u201393.5\u00a0ml). Digital rectal examination was abnormal in 37% of patients, and 25% of patients had abnormal transrectal ultrasound. \nTable\u00a01Patients\u2019 clinical dataPatient no.Age (years)PSA Prebiopsy (ng\/ml)Digital rectal examinationTRUSGland volume (cc)1689.0AbnormalNormal47.92715.4NormalAbnormal31.43646.1NormalNormal28.14671.25AbnormalNormal93.55605.9NormalNormal36.66663.0AbnormalNormal47.97583.9NormalAbnormal29.38666.5NormalNormal36.6Mean (range)65 (58\u201371)5.1 (1.25\u20139.0)43.9 (28.1\u201393.5)PSA Prostate-specific antigen; TRUS transrectal ultrasound\nPatients\u2019 biopsy findings are shown in Table\u00a02. In all patients, carcinoma was found in one core. Seven patients demonstrated biopsy Gleason score of 6 (3\u2009+\u20093), whereas in one, it was 7 (3\u2009+\u20094). Cancer occupied approximately 5% of the core length (Fig.\u00a01) in 6 and 10% of the core in two. The positive biopsy location was variable, and there was no side predilection. \nTable\u00a02Biopsy findingsPatient no.No. of positive coresGleason scoreCancer length (% of involvement)Positive core location11\/123\u2009+\u20093\u2009=\u200965Right apex21\/103\u2009+\u20094\u2009=\u2009710Right apex31\/63\u2009+\u20093\u2009=\u200965Left mid-zone41\/63\u2009+\u20093\u2009=\u200965Right mid-zone51\/63\u2009+\u20093\u2009=\u200965Right mid-zone61\/123\u2009+\u20093\u2009=\u2009610Left mid-zone71\/103\u2009+\u20093\u2009=\u200965Right mid-zone81\/123\u2009+\u20093\u2009=\u200965Left baseFig.\u00a01Biopsy finding of a small groups of atypical acini (a) devoid of basal cells (b). Section immunostained for p63 (case no. 5). The diagnosis is acinar adenocarcinoma, Gleason score 3\u2009+\u20093\u2009=\u20096. (The prostatectomy findings are those seen in Fig.\u00a02)\nSearch for residual cancer\nThere were no cases with a false positive biopsy diagnosis (step 1). Cancer was not overlooked or missed in any of the eight prostatectomies (step 2). Each prostate had been totally embedded (step 3).\nCancer was found on deeper sections (steps 4 and 5) in five cases. Cancer was in the right and left apex, respectively, in two of them. Cancer was in the mid-zone in another two, one in the left and the other in the right. The fifth was in the left base. In particular, the cancer found in the right apex was discovered after block-flipping (i.e. step 5; Fig.\u00a02). The location corresponded to site of the positive biopsy in four cases. Two out of five cases were from the untreated group and showed a Gleason score of 3\u2009+\u20093\u2009=\u20096. Three cases had the morphologic appearance of cancer with evident regressive changes due to endocrine therapy. The diameter of the tumour, measured on the slides, was always below 2.0\u00a0mm. In one of the three treated cases, an additional focus of cancer was seen at a distance from to that of the positive biopsy.\nFig.\u00a02Paraffin blocks, original sections and additional sections before and after block-flipping in case no. 1. a Is the right apex. b Is the left apex. c Includes the whole mount sections of the body of the prostate. d Is the right and left base. e Includes the seminal vesicles and deferens. A1 is the paraffin block. A2 is the original H-E-stained section. A3 includes the additional sections before block-flipping. A4 refers to the additional sections after block-flipping (Block-flipping was done only for a and b). The dotted area (see also the red arrow) on the A4 slides is that of the cancer. It corresponds to the lesion of the biopsy seen in Fig.\u00a01. The same identification procedure applies to b. Concerning c and d the paraffin blocks, the original sections and the additional sections (block-flipping was not done) are shown. For e the paraffin blocks and the original sections are shown. (Other slides contain some annotations and abbreviations to indicate additional findings, slide orientation, and section order, etc.)\nThe definitive diagnosis was established on steps 6 and 7 in an additional case (the patient was hormonally treated before RP) where cancer was not seen in the additional sections even after block-flipping. Immunohistochemistry for p63 and AMACR was applied to a small suspicious focus of crowded acini. There were a few scattered p63 positive (basal) cells, whereas AMACR was negative. The focus was considered to be benign (i.e. atrophy). Cancer was discovered after immunostaining for cytokeratin CAM 5.2, for p63 and PSA. It was represented by scattered isolated cells positively immunostained with the antibody against CAM 5.2 (Fig.\u00a03) and with PSA and negative for p63 (Fig.\u00a03). The diameter of the focus was 1.0\u00a0mm. The block selected for immunohistochemistry corresponded to an H&E-stained slide with hypercellular stroma. There was no exact correspondence with the site of the positive biopsy.\nFig.\u00a03Cancer is discovered after immunostaining for cytokeratin CAM 5.2 (case no. 6). It is represented by scattered isolated cells. The same cells are negative for the basal cell marker p63 and positive for PSA. Part of an atrophic duct is also present\nIn the remaining two cases (one untreated and the other treated preoperatively), cancer was not found on steps 4 through 7. The review of the description of the macroscopic appearance and the inspection of the contour of the tissue sections on the slides revealed that part of the peripheral zone of the prostate was missing (Fig.\u00a04). This corresponded to the location of the positive biopsy (step 8). DNA specimen analysis confirmed the identity of the biopsy and prostatectomy in both cases (step 9; Table\u00a03). Incidentally, prostate tissue was documented clinically in one of the two patients (patient no. 7, hormonally treated before operation; the same patient whose prostate is shown in Fig.\u00a04). In particular, a biopsy of the residual fragment showed normal prostate tissue and adenocarcinoma with features identical to those seen in the preoperative biopsy.\nFig.\u00a04Whole mount section (case no 7). Part of the peripheral zone, posteriorly, is missingTable\u00a03Results of the search for residual cancerPatient no.Neoadjuvant treatmentCancer foundID test done1NoIn recutNo2NoIn recutNo3YesIn recutNo4YesIn recutNo5YesIn recut after block-flippingNo6YesAfter cytokeratin stainNo7YesNot in RPaYes, identical8NoNoYes, identicalaA post-operative biopsy of the residual prostate fragment in the patient showed normal prostate tissue and adenocarcinoma with features identical to those seen in the preoperative biopsy.\nDiscussion\nReported incidence of no residual cancer on RPs ranges from 0.07 to 4.2% (1\u201311, 20). It is higher after neoadjuvant hormonal treatment [16].\nThere are multiple reasons why cancer may not be found on RP after a positive biopsy. In some cases, the cancer may be minute and completely removed by the initial procedure, either needle biopsy or transurethral resection of prostate. Small cancers may be removed from the RP during the technical preparation of the specimen, such as leveling of the paraffin blocks. Small cancer foci can also be completely obscured if the patient has undergone antiandrogen therapy or the RP shows extensive inflammation or granulomatous inflammation. Other reasons include false-positive diagnosis on the initial biopsy, resulting in cancer overdiagnosis, or false-negative diagnosis rendered on the RP. Review of the material by a second pathologist or a specialist in urological pathology can often resolve the latter scenario. Lastly, errors may occur either before a specimen is submitted for pathological assessment or in the pathology laboratory. These errors include switching of patients\u2019 requisitions, mislabelling or switching of specimen containers, mislabelling or switching slides or blocks and information system errors (e.g. incorrect case entry in the information system or mixed accession numbers) [28].\nIn our study, the incidence of RPs with no residual cancer after an initial routine examination of the 1,328 specimens was 0.6%. This figure includes both untreated and hormonally treated patients. The incidence was 0.2 and 0.4%, respectively, of the total number of RPs included in the study, or 0.4% (3 out 700 untreated patients) and 0.8% (5 out 628 treated patients) when the two groups were considered separately. The former is well in the range of values reported in the literature for untreated patients and very close to the figure published by Herkommer et al. [11, 13]. The latter value seen in our treated patients is much lower than that observed by Kollermann et al. [16].\nAdditional sectioning and evaluation of the cases can reduce the number of pT0 RPs after a positive biopsy. In particular, the current study showed that the final incidence was 0.15% and included only two RPs with missing parts, probably due to incomplete removal of the prostate. One of these two patients was hormonally treated preoperatively.\nOur findings on the role of immunohistochemistry to detect residual PCa cells are in agreement with previous studies. Gleave et al. [8] found that 50% of the cases that exhibited no residual cancer on routine pathologic assessment had remaining foci of cancer discovered by immunostaining. Without the aid of additional step sections and immunostaining for cytokeratin, these cases would have been reported as being stage pT0 [2].\nDNA identity on formalin-fixed tissue from the paraffin blocks is a useful test to establish specimen identity and to exclude the possibility of laboratory error when no residual cancer is found on RP after a positive biopsy. In particular, DNA analysis is a useful test that eliminates the possibility of specimen mishandling or switching by establishing the identity of the tissue from the positive biopsy and the RP. It can be performed in the formalin-fixed tissue from the paraffin blocks. Different methods are used to investigate tissue specimen identity. These include immunolabeling of blood group antigens [22], sex chromosome targeting using fluorescence in situ hybridization [21] and microsatellite analysis. According to some authors, microsatellite analysis is the gold standard for investigating tissue identity [1, 10, 23, 28, 31].\nIn our study, the clinical and the biopsy data in patients with no initial residual cancer on radical prostatectomy after positive biopsy are similar to the findings from a recent study in patients with single-core positive biopsies and minimal cancer on biopsy [28, 29]. The majority of patients with no residual cancer on prostatectomy demonstrated minute cancer foci in one or two biopsy cores with Gleason score of 6, which, in many cases, may reflect minimal disease [28]. Rare small-volume cancers of higher grade may also be encountered [28]. Follow-up investigations have, in general, shown that no pT0 patient has clinical or biologic evidence of prostate cancer recurrence or progression [6, 14, 20, 27].\nThere are four aspects that we have not explored in our study. One is that the carcinoma could be lost in facing off of the paraffin blocks. We are fully aware of this potential problem. Our technicians are instructed so that they have to collect tissue sections and not waste material when the paraffin blocks are levelled off. The second is whether the time, effort and expense of the sampling described in this paper are warranted on a routine basis for all pT0 cases. We have not done any analysis in these respects due to the fact that the number of cases is very small and that time and expense do not represent an issue of concern in our institution. The third is what degree of sampling would be necessary to serially section through the entire prostate. Probably thousands of sections would be required, and we were prepared to cut as many sections as needed to find cancer. The fourth is whether there is an outcome difference when the initial pT0 carcinomas are detected after more thorough sampling vs pT0 cases without additional sampling. This was not addressed in our study because the basic aim was to avoid that a pT0 report is rendered to the clinician and to the patient, thus triggering a potential legal issue with all the problems related to it.\nThere are few observations in the literature on pT0 cases and preoperative diagnosis of cancer made in TURP specimens. The real incidence of no residual cancer after cancer detected by TURP material is not known and is reported to be seen in 6 to 39% of cases [9], presumably due to tumour ablation during the initial resection. However, studies of this phenomenon are limited by variations in the number of tissue sections submitted for histological evaluation and by the small number of cases [3].\nConclusions and recommendations\nThe current study showed that an extensive search for residual cancer reduces the number of pT0 RPs after a positive biopsy. To achieve this, it is recommended to have the needle biopsy reviewed, carefully look again at the radical prostatectomy, do deeper sections and then flip certain paraffin blocks. In addition, atypical foci should be stained for basal cell markers and often AMACR, especially in hormone-treated cases. If a block is missing part of the peripheral zone (capsular incision), this should be commented on. DNA analysis for tissue identity should be performed when the other steps have been taken without finding cancer.","keyphrases":["residual prostate cancer","prostate cancer","vanishing cancer"],"prmu":["P","P","P"]}
{"id":"Breast_Cancer_Res_Treat-3-1-2001222","title":"Safety of aromatase inhibitors in the adjuvant setting\n","text":"The third-generation aromatase inhibitors (AIs) letrozole, anastrozole, and exemestane are replacing tamoxifen as adjuvant therapy in most postmenopausal women with early breast cancer. Although AIs have demonstrated superior efficacy and better overall safety compared with tamoxifen in randomized controlled trials, they may not provide the cardioprotective effects of tamoxifen, and bone loss may be a concern with their long-term adjuvant use. Patients require regular bone mineral density monitoring, and prophylactic bisphosphonates are being evaluated to determine whether they may protect long-term bone health. AIs decrease the risks of thromboembolic and cerebrovascular events compared with tamoxifen, and the overall rate of cardiovascular events in patients treated with AIs is within the range seen in age-matched, non-breast-cancer populations. AIs are also associated with a lower incidence of endometrial cancer and fewer vaginal bleeding\/discharge events than tamoxifen. Compared with tamoxifen, the incidence of hot flashes is lower with anastrozole and letrozole but may be higher with exemestane. Generally, adverse events with AIs are predictable and manageable, whereas tamoxifen may be associated with life-threatening events in a minority of patients. Overall, the benefits of AIs over tamoxifen are achieved without compromising overall quality of life.\nIntroduction\nTamoxifen became the standard adjuvant therapy for women with early breast cancer following the first demonstration of efficacy more than 20\u00a0years ago [1]. Administration of tamoxifen for 5\u00a0years has been shown to reduce breast cancer recurrence by 41% and mortality by 34% in women with hormone-responsive tumors [2]. Nevertheless, many limitations of tamoxifen have emerged with widespread use. In the landmark National Surgical Adjuvant Breast and Bowel Project B-14 trial, 66% of tamoxifen-treated patients experienced side effects compared with 58% of patients given placebo [3]. Severe, potentially life-threatening events such as thrombosis were more likely to occur in patients aged >60\u00a0years [3]. Long-term adverse effects associated with 5\u00a0years\u2019 adjuvant tamoxifen include venous thromboembolic events, vaginal bleeding, vaginal discharge, ischemic cerebrovascular events, endometrial and uterine cancer, and hysterectomy [3, 4]. Experiencing side effects significantly increases the likelihood of patients discontinuing tamoxifen therapy (odds ratio 4.0; 95% confidence interval [CI] 1.1, 13.9 in women aged\u00a0\u2265\u00a055\u00a0years) [5]. Over time, resistance to tamoxifen may develop [6], and therapy beyond 5\u00a0years is not recommended because neither further disease-free survival nor survival benefit is gained [7].\nThe third-generation aromatase inhibitors (AIs) letrozole, anastrozole, and exemestane are rapidly replacing tamoxifen as initial adjuvant therapy [8, 9] or sequential adjuvant therapy after 2\u20135\u00a0years of tamoxifen [10\u201313]. By potently inhibiting the aromatase enzyme, which converts androgens to estrogen [14, 15], AIs achieve almost total suppression of total body aromatization and dramatic reductions in estrogen concentrations in postmenopausal women [16\u201318]. AIs are now recommended in international guidelines for the management of breast cancer [19\u201321]. In addition, guidance is being developed for the management of common co-morbidities such as osteoporosis in postmenopausal women with hormone-sensitive breast cancer receiving AIs [20, 22]. This review examines the safety of AIs and assesses their advantages and disadvantages compared with tamoxifen. It also considers the impact of treatment on co-morbidities commonly encountered in this population.\nPossible impact of treatment on common co-morbidities\nAdjuvant therapy should be individualized on the basis of clinical and biologic risk factors [21], including the presence of co-morbidities [23\u201326]. The most prevalent co-morbidities in the postmenopausal patient population are hypertension, arthritis, heart disease, diabetes, chronic obstructive pulmonary disease, eye problems, anemia, depression, fractures, hearing problems, osteoporosis, Parkinson\u2019s disease, renal failure, and urinary tract problems [25]. Understanding the long-term effects of aromatase inhibition on bone and cardiovascular health are particularly important to consider because of the potential effects of altering estrogen concentrations.\nBone disease\nBone health typically may deteriorate as women age, particularly after reaching menopause [27, 28]. A decline in estrogen concentrations accelerates postmenopausal bone loss [29\u201331] while vitamin D deficiency also increases bone turnover and the risk of fracture [32, 33]. It is important to note that bone health is compromised in women with breast cancer compared with the general population [34]. In the Women\u2019s Health Initiative Observational Study, breast cancer survivors had significantly lower total body bone mineral density (BMD) and total hip BMD [34] and a significantly higher risk of clinical fractures [35]. Of concern, osteoporosis was undiagnosed in more than three quarters of breast cancer survivors and the reference population [34]. Multiple factors contribute to the increased risk of osteoporosis and fractures in postmenopausal women with breast cancer [34]. Furthermore, tumor cells can have a direct effect on bone remodeling [36], and breast cancer therapy can lead to cancer treatment-induced bone loss (CTIBL) [37\u201339]. In a large cohort study, patients with early breast cancer who received anticancer therapy had a 30% higher risk for osteoporosis\/osteopenia (odds ratio 1.29; 95% CI 1.13, 1.46) [38]. The study also showed that other factors such as poor health status, history of smoking, and alcohol abuse can contribute to CTIBL. The most serious consequence of CTIBL is an increased risk of fractures (Fig.\u00a01) [35], which increase morbidity and healthcare costs [40]. The presence of bone metastases can contribute to CTIBL and lead to serious complications, including fractures, spinal compression, bone pain, and hypercalcemia of malignancy [41].\nFig.\u00a01Age-standardized fracture incident rates by survivor status. Standardized rates were calculated using the age distribution of the entire Women\u2019s Health Initiative Observational Study cohort. Excess numbers of fractures per 10,000 person-years are above each set of bars [35]. \u00a92005 American Medical Association. Reproduced with permission\nAromatase inhibitors and bone disease\nIn a recent study, the bone health of 1,354 patients with breast cancer receiving an AI (anastrozole, exemestane, or letrozole) was compared with 11,014 controls [39]. Treatment with an AI increased the risk of bone loss (relative risk 1.3; 95% CI 1.1, 1.6; P\u00a0=\u00a00.01) and bone fracture (relative risk 1.4; 95% CI 1.2, 1.6; P\u00a0=\u00a00.001). The risks remained significantly higher for AI therapy after adjustment for age and co-morbidities [39]. An increase in the incidence of arthralgia is noted with all three AIs, when compared with tamoxifen.\nAnastrozole\nHowell and colleagues reported fracture rates after a median follow-up of 68\u00a0months in the Arimidex, Tamoxifen, Alone or in Combination (ATAC) trial [42]. Fractures were reported in 577 (9.3%) of the 6,186 patients and were more common with anastrozole than with tamoxifen (11 vs. 8%, respectively; P\u00a0<\u00a00.0001). The incidence of hip fractures was 1% in both groups. The rate of fractures was low at approximately 2% per year and decreased to baseline levels after completion of 5\u00a0years of treatment. The effects of anastrozole and tamoxifen on BMD were assessed in a sub-analysis of 167 patients from the ATAC trial [43]. Anastrozole-treated patients had significant decreases in lumbar spine BMD (\u22128.1%; 95% CI \u221210.1, \u22126.1; P\u00a0<\u00a00.0001) and total hip BMD (\u22127.4%; 95% CI \u22129.6, \u22125.3; P\u00a0<\u00a00.0001) relative to tamoxifen-treated patients, in whom small increases were observed. Bone loss was greatest in the first 2\u00a0years of anastrozole treatment, as reported previously [44], but the rate of loss appeared to slow down from years 2 to 5. In the updated analysis after a median follow-up of 68\u00a0months, osteopenia or osteoporosis was reported in 11% of patients receiving anastrozole compared with 7% receiving tamoxifen (P\u00a0<\u00a00.0001) [42, 45]. Another sub-analysis of the ATAC trial showed that the majority of joint symptoms occur within 24\u00a0months of initiating treatment [46]. After 68\u00a0months\u2019 median follow-up, joint symptoms were reported in 35.6 and 29.4% of patients in the anastrozole and tamoxifen arms, respectively. Most symptoms were mild in intensity, and 46% were reported as an exacerbation of a pre-existing condition. The incidence of serious joint symptoms was similar for anastrozole and tamoxifen (10.6 vs. 10.4%, respectively) and only 2.1 and 0.9%, respectively, discontinued treatment because of joint symptoms. After a median follow-up of 68\u00a0months, muscle cramps were less common with anastrozole than tamoxifen (4 vs. 8%, respectively; P\u00a0<\u00a00.0001), whereas carpal-tunnel syndrome was more common with anastrozole (3 vs. 1%, respectively; P\u00a0<\u00a00.0001) [42].\nThese updated results from the ATAC trial confirm that AIs are a well-tolerated initial treatment option in terms of bone health [43, 45, 46]. Although anastrozole is associated with BMD loss, no patient with normal bone at baseline became osteoporotic after 5\u00a0years of treatment, and the rate of bone loss in the lumbar spine region slowed down in years 2\u20135.\nThe ARNO\/ABCSG8 trials investigated the efficacy and safety of switching to anastrozole after 2\u00a0years of tamoxifen [12]. Although there were significantly more fractures in patients switching to anastrozole (2.1%) than in those continuing on tamoxifen (1.0%) [12], the rate was lower than that seen at a similar point in the ATAC trial [12]. In the Italian Tamoxifen Anastrozole (ITA) trial, switching to anastrozole after 2\u20133\u00a0years of tamoxifen was not associated with an increase in fracture rate, although differences may emerge with longer follow-up [13].\nLetrozole\nIn the Breast International Group (BIG) 1\u201398 trial of initial adjuvant therapy, there was a slight yet significant difference in the incidence of fractures (5.7% with letrozole vs. 4.0% with tamoxifen; P\u00a0<\u00a00.001) [8]. The MA.17 trial of extended adjuvant therapy showed that when compared with placebo, letrozole had no significant impact on fractures [10]. There was a small but significant difference in patient-reported diagnoses of new-onset osteoporosis (8% letrozole vs. 6% placebo, P\u00a0=\u00a00.003), and arthralgia and myalgia were significantly more common with letrozole than placebo [10]. A companion study to MA.17 demonstrated a significant decrease in lumbar spine BMD (\u22125.35 vs. \u22120.70%; P\u00a0=\u00a00.008) and total hip BMD (\u22123.6 vs. \u22120.71%; P\u00a0=\u00a00.044) over 2\u00a0years in patients treated with letrozole compared with placebo, although no patient went below the threshold for osteoporosis in total hip BMD [47]. Data from this companion study suggest that women with a BMD score of \u22121.0 or greater when starting letrozole after tamoxifen are less vulnerable to enhanced bone resorption and may not require prophylactic bisphosphonate therapy.\nExemestane\nIn a model of ovariectomized rats, the steroidal AI exemestane was shown to prevent bone loss, presumably via its androgenic properties (both exemestane and its metabolite 17-hydro-exemestane demonstrate affinity for the androgen receptor) [48]. However, a randomized study to compare the effects of progestins and AIs on bone remodeling markers in patients with metastatic breast cancer found that exemestane increased osteoclast activity [49]. In the adjuvant treatment setting, a randomized trial involving 147 patients with early breast cancer demonstrated a non-significant effect of exemestane compared with placebo on the annual rate of BMD loss in the lumbar spine (2.17 vs. 1.84%; P\u00a0=\u00a00.568) and a small but significant effect in the femoral neck (2.72 vs. 1.48%; P\u00a0=\u00a00.024) [50]. Of note was the finding that BMD may rapidly improve following AI discontinuation: this trial showed that bone resorption markers returned to or below baseline values, and bone formation markers remained moderately increased within 6\u00a0months of stopping exemestane [51].\nIn the Intergroup Exemestane Study (IES) of exemestane following 2\u20133\u00a0years of tamoxifen, fractures were reported more frequently with exemestane than with tamoxifen after a median follow-up of 30.6\u00a0months, although this difference was not statistically significant (3.1 vs. 2.3%; P\u00a0=\u00a00.08) [52]. However, the difference in incidence of fractures was statistically significant (7.0% with exemestane vs. 4.9% with tamoxifen; P\u00a0=\u00a00.003) after a median follow-up of 55.7\u00a0months [11]. The incidence of osteoporosis was also significantly higher with exemestane than with tamoxifen (9.2 vs. 7.2%, respectively; P\u00a0=\u00a00.01). Recent results from a 1-year sub-study revealed that patients on exemestane experienced a significant decrease in hip BMD, while patients on tamoxifen did not [53]. These results were confirmed by another recent study, which evaluated the effects of exemestane on bone turnover markers and BMD in 70 postmenopausal women (62.0\u00a0\u00b1\u00a08.9\u00a0years) with early breast cancer who were switched to exemestane after 2\u20133\u00a0years on tamoxifen [54]. Patients in the exemestane group had a significant decrease in BMD and early parathyroid hormone (at month 6) and an increase in bone alkaline phosphatase (B-ALP) and the carboxy-terminal telopeptide of type I collagen after 24\u00a0months. These studies suggest that switching postmenopausal women from tamoxifen to exemestane causes a marked increase in bone turnover markers with a consequent reduction in BMD.\nArthralgia was also significantly more common with exemestane than with tamoxifen (5.4 vs. 3.6%, P\u00a0=\u00a00.01) in the IES [52]. A study by L\u00f8nning et\u00a0al. discovered a high prevalence of vitamin D deficiency in postmenopausal women treated with exemestane (52 of 59 patients) or placebo (56 of 62 patients), and this could be the most important factor causing bone loss in both groups [55]. Vitamin D substitution is therefore recommended for postmenopausal women, particularly those with breast cancer receiving an AI. The incidence of carpal-tunnel syndrome in the IES was higher in the exemestane arm (2.8%) than in the tamoxifen arm (0.4%; P\u00a0<\u00a00.001) [11].\nComparative studies of aromatase inhibitors\nA randomized trial (Letrozole, Exemestane, and Anastrozole Pharmacodynamics [LEAP]) of healthy volunteers demonstrated that letrozole, exemestane, and anastrozole have similar effects on bone biochemical measurements and all result in increases in bone turnover [56]. There were no statistically significant differences between the AIs in changes from baseline to 24\u00a0weeks for B-ALP, serum C-telopeptide crosslinks, and propeptide of type I procollagen. The only difference in the bone remodeling markers was a greater decrease in parathyroid hormone with exemestane than with anastrozole (P\u00a0=\u00a00.04).\nThus, all AIs seem to have similar effects on bone health. The ATAC bone sub-study results are reassuring for the entire AI class, and women with breast cancer who have normal BMD measurements at the onset of AI treatment may be able to undergo 5\u00a0years of therapy without the risk of developing osteoporosis. Patients at risk of clinically relevant BMD loss during treatment should be identified and managed according to evolving clinical guidelines [20, 57].\nBisphosphonates\nIn the American Society of Clinical Oncology (ASCO) guidelines postmenopausal patients with breast cancer who receive AIs are identified as being at high risk for osteoporosis, and it is recommended that they have baseline BMD evaluation and regular monitoring to guide subsequent therapeutic interventions such as bisphosphonates [20, 58]. Preliminary results have been reported from a small number of clinical trials of bisphosphonates in women receiving adjuvant AI therapy. In one trial, premenopausal breast cancer patients receiving goserelin plus anastrozole or goserelin plus tamoxifen were randomly assigned to the bisphosphonate zoledronic acid (ZA) (4\u00a0mg IV every 6\u00a0months) or placebo. After 36\u00a0months, it was shown that ZA given every 6\u00a0months helped prevent bone loss in these premenopausal patients in both the lumbar spine and hip regardless of endocrine therapy [59]. Two randomized trials have shown that bisphosphonates may be beneficial in postmenopausal patients at a higher risk of osteoporosis [60, 61]. In the Zometa-Femara Adjuvant Synergy Trial (Z-FAST) (North American) trial, 602 postmenopausal women with hormone-responsive breast cancer starting adjuvant therapy with letrozole were randomized to receive upfront ZA (4\u00a0mg IV infusion every 6\u00a0months) or delayed ZA when indicated (either post-baseline T-score decreases\u00a0<\u00a0\u22122\u00a0SD or occurrence of fracture) [60, 62]. Preliminary results after 12\u00a0months\u2019 follow-up indicate that initial treatment with ZA may be used to prevent CTIBL, and results at 24\u00a0months confirm these initial findings [62, 63] although the rate of clinical fractures was not changed. In addition, the small proportion of patients (8%) requiring ZA in the first year highlights the short-term bone tolerability of letrozole [62]. Results from the similarly designed ZO-FAST (European; N\u00a0=\u00a01,065) trial also support the use of ZA to potentially manage CTIBL in postmenopausal women with early breast cancer receiving adjuvant letrozole [61].\nLipid metabolism: A cohort study demonstrated that total and low-density lipoprotein (LDL) cholesterol concentrations are positively correlated with years since diagnosis of breast cancer [64]. In addition, during menopause, women experience adverse changes in cardiovascular risk factors, including declines in concentrations of high-density lipoprotein (HDL) cholesterol and increases in concentrations of total cholesterol, LDL cholesterol, HDL3 cholesterol, and triglycerides [65, 66]. These changes are independent of age and body mass index.\nAssessing the impact of AIs on lipid profiles is difficult in trials where tamoxifen is the comparator. The selective estrogen-receptor modulators (SERMs) such as tamoxifen are known to have lipid-lowering properties [67, 68]. What is clear is that the studies comparing AIs with tamoxifen indicate only that the AIs lack the lipid-lowering effects of tamoxifen.\nAromatase inhibitors and lipid metabolism\nAnastrozole\nIn the ATAC trial, the incidence of hypercholesterolemia was higher in patients receiving anastrozole than tamoxifen (9 vs. 3%, respectively; P\u00a0<\u00a00.0001) [42]. In the ITA trial, lipid metabolism disorders were reported in 9.3% of patients treated with anastrozole and 4.0% receiving tamoxifen (P\u00a0=\u00a00.04) [13].\nA recent multicenter study in patients with estrogen-receptor positive breast cancer investigated the effects of adjuvant anastrozole and toremifene, a SERM, on serum lipids [68]. Results showed that only toremifene had a beneficial effect on lipid profile, indicated by a decrease in total cholesterol, LDL cholesterol, triglycerides, and apolipoprotein B, and an increase in HDL cholesterol and apolipoprotein A1. Changes in total cholesterol, HDL, LDL, and apolipoproteins were significantly different between toremifene and anastrozole at 6 and 12\u00a0months (P\u00a0<\u00a00.05).\nLetrozole\nIn the BIG 1\u201398 trial, according to the protocol, cholesterol concentrations (fasting or non-fasting) were collected systematically in the case-report forms every 6\u00a0months and even patients with only a single measurement above the upper limit of normal were defined as hypercholesterolemic [8]. Hypercholesterolemia was reported in 5.4% of the letrozole arm compared with 1.2% of the tamoxifen arm in patients with baseline values within normal limits, who then had an increase of 1.5\u00a0times the upper limit of normal [69]. Hypercholesterolemia was typically a single event and in the majority of these patients (80%) occurred at only grade 1 intensity (meaning a slight numerical increase above normal, not requiring medications). Moreover, the majority of cases were single measurements collected in non-fasting patients. Furthermore, when looking at total serum cholesterol levels, there was a 12% median decrease from baseline in total cholesterol in the tamoxifen arm after 6\u00a0months, consistent with previous reports demonstrating the lipid-lowering effect of tamoxifen [67], while in the letrozole group total cholesterol values remained stable [8]. Hypercholesterolemia was not predefined as an adverse event in the ATAC trial, and lipid concentrations were not routinely assessed [42].\nExemestane\nHypercholesterolemia was not reported in the IES trial of sequential exemestane after tamoxifen [11, 52].\nAnother study examined the longitudinal changes in body composition and lipid profiles in 55 postmenopausal women with early breast cancer switched to exemestane after at least 2\u00a0years of tamoxifen treatment [70]. Fat mass significantly decreased (P\u00a0<\u00a00.01) while the fat-free mass to fat mass ratio significantly increased (P\u00a0<\u00a00.05) by month 12 in the exemestane but not in the tamoxifen group. In addition, triglycerides and HDL cholesterol significantly decreased (P\u00a0<\u00a00.01 and P\u00a0<\u00a00.05, respectively) in the exemestane group, while LDL cholesterol significantly increased (P\u00a0<\u00a00.01) at the end of the 1-year study period.\nAromatase inhibitors versus placebo\nWhen compared with placebo (the most accurate way to assess the true impact of AIs on serum lipids), the final analysis of the MA.17 trial demonstrated the incidence of hypercholesterolemia was 16% in the letrozole and the placebo arms [10]. Results from an MA.17 lipid sub-study showed that in 347 postmenopausal women with primary breast cancer treated for up to 36\u00a0months, letrozole (n\u00a0=\u00a0183) does not significantly alter lipid profile (samples drawn under fasting conditions) compared with placebo (n\u00a0=\u00a0164) [71]. In a placebo-controlled study involving 147 postmenopausal women with early breast cancer, exemestane had no major effect on lipid profile except for a modest but significant decrease from baseline in HDL cholesterol (P\u00a0<\u00a00.001) and apolipoprotein A1 (P\u00a0=\u00a00.004) [50]. On the basis of these results, it is clear that when compared with placebo, AIs do not have a detrimental effect on lipid profile. However, it should be noted that there have been no placebo-controlled trials of adjuvant anastrozole in women with breast cancer.\nComparative studies of aromatase inhibitors\nThe LEAP trial directly compared safety parameters between the steroidal AI exemestane and the non-steroidal AIs anastrozole and letrozole in 90 healthy postmenopausal women (Table\u00a01) [72]. Initial results from the trial showed that there were no significant differences between anastrozole and letrozole in effects on LDL:HDL ratios, triglyceride concentrations, and non-HDL concentrations. Exemestane was associated with an increase in LDL:HDL ratio (+17) (P\u00a0=\u00a00.047) compared with anastrozole. There was no median change from baseline in total serum cholesterol for letrozole, a slight increase for anastrozole (+0.4), and a non-significant decrease for exemestane (\u22123.9) (P\u00a0=\u00a00.164 vs. anastrozole) [72].\nTable\u00a01Comparative effects of third-generation aromatase inhibitors on lipids [72]Percentage change from baselineAnastrozole (n\u00a0=\u00a029)Letrozole (n\u00a0=\u00a029)P value vs. anastrozoleExemestane (n\u00a0=\u00a032)P value vs. anastrozoleTotal cholesterol\u00a0Week 12\u22122.3\u22123.80.617\u22125.50.262\u00a0Week 24+0.4\u22120.00.900\u22123.90.164\u00a0Triglycerides\u00a0Week 12\u22122.9+9.60.037\u22127.70.417\u00a0Week 24+0.3+5.40.550+2.10.827Ratio of LDL-C:HDL-C\u00a0Week 12\u22120.0\u22123.10.486+8.80.048\u00a0Week 24+4.6+3.40.847+17.00.047\u00a0Non-HDL-C\u00a0Week 12\u22122.7\u22124.20.667\u22123.50.820\u00a0Week 24+1.3+1.20.975\u22120.60.630Ratio of apo B:apo A1\u00a0Week 12\u22121.0\u22123.30.452+4.40.069\u00a0Week 24+0.0\u22120.80.842+9.00.023LDL-C low-density lipoprotein cholesterol, HDL-C high-density lipoprotein, apo B apolipoprotein B, apo A1 apolipoprotein A1\nCardiovascular disease\nCardiovascular risk increases substantially and progressively in women aged \u226565\u00a0years [73\u201377]. Isolated systolic hypertension, associated with arterial stiffening, is predominant in middle- and older-aged hypertensives [75] and predisposes individuals to coronary heart disease, heart failure, stroke, vascular dementia, and chronic kidney disease [73]. The risk of cardiac disease is also influenced by ethnicity, smoking, obesity, physical inactivity, alcohol abuse, and the presence of co-morbid diseases such as diabetes.\nIn patients with breast cancer the presence of co-morbidities, including cardiovascular disease and diabetes, is associated with a poorer prognosis than when co-morbid disease is absent [78] and may explain disparities in outcome between different ethnic groups [79]. There is also evidence that breast cancer is associated with a higher prevalence of hypertension compared with other tumor types [80] and a significantly increased risk of stroke compared with the general population (relative risk 1.12; 95% CI 1.07, 1.17) [81]. Many breast cancer therapies increase the risk of cardiovascular events [82\u201388]; tamoxifen, however, may have some cardio-protective effects [89, 90].\nTamoxifen and cardiovascular disease\nSeveral studies have demonstrated the potential cardioprotective properties of tamoxifen, including a reduction in hospital admissions due to cardiac disease [89\u201391] and decreased mortality from cardiac disease [92]. In a meta-analysis, tamoxifen was associated with a significantly decreased incidence of myocardial infarction (relative risk 0.90) and death from myocardial infarction (relative risk 0.62) [93]. This finding is consistent with results from an earlier cohort study [94] and the Early Breast Cancer Trialists\u2019 Collaborative Group (EBCTCG) meta-analysis, which demonstrated decreases in the risk of cardiac death and overall mortality from vascular disease in patients receiving tamoxifen compared with those receiving placebo [2].\nAromatase inhibitors and cardiovascular disease\nAssessing the impact of different AIs on cardiovascular disease in postmenopausal women with breast cancer is difficult and inter-trial comparisons are confounded by differences in data collection and end points; for example, in the BIG 1\u201398 trial all potential adverse events were predefined in the case-report forms whereas the ATAC trial used non-specific case-report forms to report adverse events [8, 95]. Furthermore, comparisons with tamoxifen are complicated by its cardioprotective properties. Placebo-controlled trials thus provide the best source of data to delineate the effects of AIs in a patient population with an inherently elevated risk of cardiac events.\nAnastrozole\nThe ATAC trial provided data on the cardiovascular effects of anastrozole as initial adjuvant therapy compared with tamoxifen. The incidence of ischemic cardiovascular disease was higher (but not significantly) with anastrozole than placebo (127\/3092, 4.1% vs. 104\/3094, 3.4%; P\u00a0=\u00a00.1). The incidence of angina was also higher with anastrozole (71\/3092, 2.3% vs. 51\/3094, 1.6%; P\u00a0=\u00a00.07), while myocardial infarction occurred with similar frequency (37\/3092, 1.2% vs. 34\/3094, 1.1%; P\u00a0=\u00a00.7 [42]. Hypertension was statistically significantly more common with anastrozole than with tamoxifen (13 vs. 11%, respectively; P\u00a0=\u00a00.04) [42]. In the ARNO95 trial vascular events, including hot flashes, ischemic cardiovascular events, deep vein thrombosis, and ischemic cerebrovascular events, occurred in 9.2% of the anastrozole arm compared with 8.8% of the tamoxifen arm [96].\nLetrozole\nThe BIG 1\u201398 trial demonstrated a similar incidence of cardiac events in the letrozole and tamoxifen groups (4.1 vs. 3.8%, respectively; not significant). However, more women in the letrozole group had grade 3, 4, or 5 cardiac events (2.1 vs. 1.1%, respectively; P\u00a0<\u00a00.001), but these events remain rare [8]. Of note, a recent update of the monotherapy arms of BIG 1\u201398 after a longer median follow-up of 51\u00a0months showed that the overall incidence of cardiac events was comparable in the two groups (134 events [5.5%] in the letrozole group vs. 122 [5.0%] in the tamoxifen arm), thus confirming the safe cardiac profile of letrozole reported at 26\u00a0months [97].\nExemestane\nIn the IES, there was no significant difference between exemestane and tamoxifen in the incidence of combined cardiovascular disease\/thromboembolic events (22.1 vs. 20.9%, respectively; P\u00a0=\u00a00.34) after a median follow-up of 55.7\u00a0months [11]. The incidence of myocardial infarction was higher with exemestane than with tamoxifen, although the difference between treatment groups was not significant (1.3 vs. 0.8%, respectively; P\u00a0=\u00a00.08) [11].\nOverall, the rate of cardiovascular events in patients treated with AIs is well within the range seen in age-matched, non-breast-cancer populations; for example, for women 57\u201365\u00a0years of age, the rates of fatal myocardial infarction and other fatal coronary artery disease are 1.1 and 0.81 per 1,000\u00a0patient-years, respectively [98]. Similar rates were recorded in the UK General Practice Research Database and Swedish MI register [99]. Currently, there is insufficient information to fully determine the effect of AIs on cardiovascular disease, especially coronary heart disease.\nAromatase inhibitors versus placebo\nCardiovascular events occurred with similar frequency in the letrozole and placebo arms in the MA.17 trial (5.8 vs. 5.6%, respectively; P\u00a0=\u00a00.76) [10]. Similar incidences were reported in the letrozole and placebo arms for stroke\/transient ischemic attack (0.7 vs. 0.6%, respectively), myocardial infarction (0.3 vs. 0.4%, respectively), new or worsening angina (1.2 vs. 0.9%), angina requiring coronary artery bypass graft (0.2 vs. 0.5%), and thromboembolic events (0.4 vs. 0.2%, respectively) [10]. These results clearly indicate that when compared with placebo, AIs do not have a detrimental effect on cardiovascular safety.\nGynecologic health\nThe onset of menopause is characterized by numerous adverse events associated with a decline in estrogen concentrations [100\u2013102]. Early symptoms include abnormal vaginal bleeding, hot flashes, and mood changes, while vaginal dryness and irritation, osteoporosis, and heart disease are late symptoms [29, 103, 104]. Vasomotor symptoms, particularly hot flashes, are common during transition to menopause [105\u2013109] and may lead to disturbed sleep, depressive symptoms, and significant reductions in quality of life [110\u2013115]. Cigarette smoking may be associated with increased risk of hot flashes in menopausal women [116]. Sexual dysfunction is also prevalent in menopausal women and is associated with vaginal atrophy, vaginal\/genital dryness, dyspareunia (pain during sexual intercourse), vaginitis, cystitis, and urinary tract infections [117].\nAromatase inhibitors and gynecologic health\nAnastrozole\nIn the ATAC trial, the incidence of hot flashes was significantly lower with anastrozole than with tamoxifen (36 vs. 41%; P\u00a0<\u00a00.0001) [9]. In the latest analysis, anastrozole was associated with a significantly lower incidence of gynecologic events (endometrial hyperplasia, endometrial neoplasia, cervical neoplasm, and enlarged uterine fibroids: 3 vs. 10% with tamoxifen; P\u00a0<\u00a00.0001) [42]. A quality-of-life (QOL) analysis confirmed that vaginal discharge, vaginal itching\/irritation, and vaginal bleeding were less common with anastrozole but found that vaginal dryness, pain during intercourse, and loss of interest in sex were more common [118]. After 2\u00a0years of treatment there was a non-significant trend towards a lower incidence of endometrial abnormalities with anastrozole than tamoxifen (odds ratio 0.44; 95% CI 0.146, 1.314; P\u00a0=\u00a00.14) [119]. The latest update of the ATAC trial revealed reduced libido in significantly more patients receiving anastrozole (1%) than tamoxifen (<1%; P\u00a0=\u00a00.0001) [42]. Patients receiving anastrozole also experienced a significantly higher incidence of dyspareunia than those receiving tamoxifen (1 vs.\u00a0<\u00a01%, respectively; P\u00a0=\u00a00.002), whereas urinary incontinence and urinary tract infection were significantly less common among patients receiving anastrozole (urinary incontinence: 2 vs. 4%, respectively, P\u00a0<\u00a00.0001; urinary tract infection: 8 vs. 10%, respectively, P\u00a0=\u00a00.002).\nIn a randomized study of postmenopausal women in whom abnormal vaginal bleeding and\/or asymptomatic endometrial thickening occurred during treatment with tamoxifen, switching to anastrozole was associated with a significant reduction in mean endometrial thickness compared with continuation of tamoxifen (P\u00a0<\u00a00.0001) [120]. Significantly fewer anastrozole patients required a repeat hysteroscopy and dilation and curettage compared with those taking tamoxifen (4.8 vs. 33.0%, respectively; P\u00a0<\u00a00.0001).\nLetrozole\nIn the BIG 1\u201398 trial [8], endometrial biopsies were significantly less common in patients receiving letrozole than tamoxifen (2.3 vs. 9.1%, respectively; P\u00a0<\u00a00.001), and there was a trend towards fewer invasive endometrial cancers (0.1 vs. 0.3%, respectively; not significant). There was a significantly lower incidence of vaginal bleeding with letrozole than with tamoxifen (3.3 vs. 6.6%, respectively; P\u00a0<\u00a00.001), and the incidence of hot flashes was also significantly lower (33.5 vs. 38.0%, respectively; P\u00a0<\u00a00.001). In another study in patients intolerant of tamoxifen, switching to letrozole for 6\u00a0weeks was associated with a 53.7% decrease in hot flashes (hot-flash score 97.0\u201352.1; P\u00a0=\u00a00.001) [121]. In the MA.17 trial, letrozole was associated with less vaginal bleeding than placebo (6 vs. 8%, respectively; P\u00a0=\u00a00.005) but a greater incidence of hot flashes (58 vs. 54%, respectively; P\u00a0=\u00a00.003) [10]. There was no significant difference in the incidence of vaginal dryness between letrozole and placebo.\nExemestane\nIn the IES, there were no significant differences between the exemestane and tamoxifen treatment arms in the incidence of endometrial cancer (0.4 vs. 0.7%, respectively; P\u00a0=\u00a00.17) [11], or the incidence of hot flashes (42 vs. 40%, respectively; P\u00a0=\u00a00.28) [52]. Overall, gynecologic symptoms were lower with exemestane than with tamoxifen (6 vs. 9%; P\u00a0<\u00a00.001) [52]; however, vaginal dryness was significantly more common among women taking exemestane than those taking tamoxifen, while vaginal discharge was significantly more common with tamoxifen [122]. Vaginal bleeding was significantly more common in the tamoxifen arm (7.1%) than in the exemestane group (4.8%; P\u00a0=\u00a00.001) [11].\nOther adverse events\nSecondary cancer\nThe association between tamoxifen and endometrial and uterine cancers is well-established [4] and is not observed with AIs. However, a safety analysis of the ATAC trial [42] showed a surprisingly higher incidence of head and neck cancer with anastrozole compared with tamoxifen (10\/3092 vs. 3\/3094, respectively). Similarly, there was an excess of lung cancer (25\/3092 vs. 16\/3094) and lung cancer deaths with anastrozole; however, further analyses are required to confirm these findings. Of note, a higher incidence of secondary cancer was not noted in the IES (72 events exemestane vs. 107 tamoxifen) or in the BIG 1\u201398 trial (69 letrozole vs. 82 tamoxifen) [8, 11].\nA meta-analysis showed that tamoxifen is associated with a modest but statistically significant increase in the risk of developing gastrointestinal cancer (relative risk 1.31; 95% CI 1.01, 1.69), particularly for postmenopausal women (relative risk 1.77) [93].\nGastrointestinal health\nDiarrhea was significantly more common among patients receiving the steroidal AI exemestane than in those taking tamoxifen (4.2 vs. 2.2%, respectively) [123] but is not a typical side effect of the non-steroidal AIs letrozole and anastrozole. However, an updated safety analysis of the ATAC trial showed that anastrozole was associated with an increased incidence of diarrhea compared with tamoxifen (9 vs. 7%; P\u00a0=\u00a00.02) [42].\nNeurologic effects and visual disturbance\nIt has been suggested that endocrine therapy may affect cognitive function in patients with breast cancer [124]. In a study comparing patients from the ATAC trial with healthy controls, anastrozole was associated with significant impairments in a processing speed task and on a measure of immediate verbal memory [125]. Another study conducted in healthy, estrogen-treated postmenopausal women treated with testosterone did not reveal any effects of aromatase inhibition on cognition [126].\nThe impact of adjuvant AI therapy on cognition and other neurologic processes is clearly an important issue that will require further studies in the future. Neurologic effects reported with exemestane, including dizziness and vertigo [127] and significantly more visual disturbances compared with tamoxifen [52], are not characteristic of non-steroidal AIs.\nDry mouth\nThe latest analysis of the ATAC trial demonstrated a significantly greater incidence of dry mouth in patients receiving anastrozole (4%) compared with tamoxifen (2%; P\u00a0=\u00a00.003) [42].\nCosmetic effects\nWeight gain is common after breast cancer therapy and increases the risk of recurrence, cardiovascular disease, and diabetes [64]. A study of Japanese patients showed that more women reported weight gain in the anastrozole group than in the tamoxifen group (35.8 vs. 12.5%, respectively; P\u00a0\u2264\u00a00.0036) [128], but no difference was seen among patients from the ATAC trial included in a QOL sub-analysis [118].\nThe androgen structure of exemestane may lead to androgenic side effects. Hypertrichosis, hair loss, hoarseness, and acne were reported in about 10% of patients treated with daily exemestane doses of 200\u00a0mg or more in dose-finding studies [129, 130], but have not emerged as a significant issue in phase II or phase III trials with this agent.\nAnastrozole treatment was associated with a lower incidence of nail disorders (2 vs. 3%; P\u00a0=\u00a00.002) and fungal infection (1 vs. 1%; p\u00a0=\u00a00.01) compared with tamoxifen [42].\nQuality of life and patient preference\nAnastrozole\nThe QOL of patients treated in the ATAC trial was studied during a 5-year follow-up period [118, 131]. Anastrozole and tamoxifen had similar overall effects on QOL (Functional Assessment of Cancer Therapy-Breast [FACT-B] trial outcome index plus endocrine sub-scale) in the first 2\u00a0years of treatment [118], and an initial worsening of endocrine symptoms gradually improved over time [131]. The authors concluded that the benefits of anastrozole are achieved without detrimental effects on QOL. However, another study conducted in Japanese patients demonstrated that FACT-G, FACT-B, and FACT-ES scores were significantly better with tamoxifen than with anastrozole (P\u00a0=\u00a00.012, P\u00a0=\u00a00.010, and P\u00a0=\u00a00.015, respectively) [132].\nLetrozole\nThe MA.17 and BIG 1\u201398 trials have demonstrated that adjuvant letrozole is well-tolerated compared with placebo [10] and better tolerated than tamoxifen [8]. In another study of postmenopausal women who were experiencing distressing side effects while taking adjuvant tamoxifen and were switched to letrozole, after 6\u00a0weeks 66% of patients preferred to remain on letrozole, 24% preferred to go back to tamoxifen, and 10% stopped all therapy [121].\nIn the placebo-controlled MA.17 trial, letrozole significantly improved outcomes and did not impair overall QOL [133] (Fig.\u00a02). Minor differences seen in some domains (physical functioning, bodily pain, vitality, vasomotor, and sexual) were consistent with a minority of patients experiencing changes in QOL compatible with a reduction in estrogen synthesis. A sub-analysis of US subjects in MA.17 demonstrated no significant differences between letrozole and placebo in overall QOL summary scores (mental and physical) and five of eight sub-domains of SF-36 [134]. There were no differences in SF-36 mental and physical QOL scores and MENQOL (menopause symptom scale) psychosocial and physical domains [134].\nFig.\u00a02Mean change score in Short Form 36-item Health Survey. A positive score indicates a favorable change in quality of life. (A) Physical component summary; P\u00a0=\u00a0not significant for all time points. (B) Mental component summary; P\u00a0=\u00a0not significant for all time points. [133]. \u00a92005 American Society of Clinical Oncology. Reproduced with permission\nExemestane\nResults from the IES QOL sub-protocol indicate that switching to exemestane from tamoxifen improves outcome without a significant detrimental impact upon QOL [135]. At entry, there was a high prevalence of severe endocrine symptoms (vasomotor complaints and sexual problems), and these persisted with exemestane and tamoxifen during the study. No significant differences between groups were seen for any endocrine symptoms apart from vaginal discharge, which was more pronounced with tamoxifen (P\u00a0<\u00a00.001).\nConclusions\nClinical trials show that the third-generation AIs lack the serious risks of thromboembolism and endometrial cancers associated with tamoxifen and are generally well tolerated, with the majority of adverse events occurring at mild to moderate intensity [8\u201311].\nAIs are associated with a mild to modest increased risk of osteoporosis compared with tamoxifen, and it is therefore essential that patients have regular BMD assessments and be monitored proactively to minimize the risk of clinical fractures [20, 57]. The increased risk of fractures with an AI compared with tamoxifen needs to be balanced against the increased risk of endometrial and cerebrovascular\/thromboembolic morbidity with tamoxifen [136]. Of note, the updated ATAC analysis shows that the majority of excess adverse events associated with tamoxifen occurred during the first 2.5\u00a0years of treatment; there were 142 (8%) fewer predefined adverse events in the anastrozole arm [137]. Thus, it appears that many excess gynecologic, thromboembolic, and cerebrovascular adverse effects occurring in tamoxifen-treated patients could be avoided if patients were treated initially with an AI [136].\nAlthough AIs do not have the cholesterol-lowering and potential cardioprotective properties of tamoxifen, they do not significantly worsen total cholesterol concentrations and do not appear to increase cardiovascular risk when compared with placebo. Nevertheless, it is prudent to recommend that all patients at risk of cardiovascular effects are properly monitored and managed, and all breast cancer patients should be routinely monitored for cardiovascular disease. It is difficult to draw meaningful conclusions from comparisons of randomized trials of tamoxifen versus anastrozole, letrozole, or exemestane because of differences in assessing and reporting risk of cardiovascular disease [8, 52, 95, 138].\nCurrent information is insufficient to determine the effects of AIs on cardiovascular disease and coronary heart disease risk [20]. Similarly, further follow-up is required to determine the late consequences of AI therapy [20]. Despite these provisos, ASCO now recommends that optimal adjuvant hormonal therapy for a postmenopausal woman with receptor-positive breast cancer includes an AI as initial therapy or after treatment with tamoxifen. Results from several ongoing trials, including the Femara versus Anastrozole Clinical Evaluation, MA.27, the National Surgical Adjuvant Breast and Bowel Project, LATER, and MILER, should provide more information on the long-term tolerance and the optimal duration of adjuvant AI therapy and help determine which strategy has the best ratio of efficacy to tolerance.\nIn conclusion, the efficacy benefits of AIs outweigh the risks when AIs are used as adjuvant therapy in postmenopausal women with early breast cancer. Safety, QOL, and patient preference must all be considered in the determination of the optimal strategy for long-term endocrine therapy, bearing in mind that patients may require treatment for 10\u00a0years or more. Every patient is unique, and endocrine therapy must be individualized according to clinical, biologic, and patient factors such as lifestyle, the presence of significant co-morbidities, and use of concomitant medications. Tolerability should no longer be an obstacle to effective, long-term endocrine therapy.","keyphrases":["safety","aromatase inhibitors","letrozole","adjuvant therapy","early breast cancer"],"prmu":["P","P","P","P","P"]}
{"id":"J_Gastrointest_Surg-3-1-1852390","title":"The Extended Learning Curve for Laparoscopic Fundoplication: A Cohort Analysis Of 400 Consecutive Cases\n","text":"Many studies have looked at the learning curve associated with laparoscopic Nissen fundoplication (LNF) in a given institution. This study looks at the learning curve of a single surgeon with a large cohort of patients over a 10-year period. Prospective data were collected on 400 patients undergoing laparoscopic fundoplication for over 10 years. The patients were grouped consecutively into cohorts of 50 patients. The operating time, the length of postoperative hospital stay, the conversion rate to open operation, the postoperative dilatation rate, and the reoperation rate were analyzed. Results showed that the mean length of operative time decreased from 143 min in the first 50 patients to 86 min in the last 50 patients. The mean postoperative length of hospital stay decreased from 3.7 days initially to 1.2 days latterly. There was a 14% conversion to open operation rate in the first cohort compared with a 2% rate in the last cohort. Fourteen percent of patients required reoperation in the first cohort and 6% in the last cohort. Sixteen percent required postoperative dilatation in the first cohort. None of the last 150 patients required dilatation. In conclusion, laparoscopic fundoplication is a safe and effective operation for patients with gastroesophageal reflux disease. New techniques and better instrumentation were introduced in the early era of LNF. The learning curve, however, continues well beyond the first 20 patients.\nIntroduction\nIt is well known that there is a learning curve associated with laparoscopic Nissen fundoplication (LNF) for gastroesophageal reflux disease (GERD).1,2 A number of studies have shown a decrease in the number of complications with surgical experience and with modifications to the surgical technique of fundoplication over time. Watson et al.2 showed that the individual surgeon\u2019s complication rate and conversion rate were highest in the first five procedures and stabilized after the first 20 operations. There are, however, few studies with large patient numbers showing the learning curve of a single surgeon and his\/her trainees. We have evaluated this learning curve in our series of 400 consecutive patients undergoing laparoscopic fundoplication.\nMaterials and Methods\nBetween January 1993 and August 2002, 400 patients (262 males, 138 females) [mean age: 42.9\u00a0years (range 9\u201386)] underwent laparoscopic fundoplication in a District General Hospital. All procedures were performed or supervised by a dedicated upper gastrointestinal surgeon. Several trainees became the primary surgeons later in the series under direct supervision once they were deemed to have the appropriate laparoscopic skills. The indications for operation were: symptomatic GERD despite prolonged medical therapy; intolerance of medical therapy due to side effects; and volume regurgitation or patient preference for surgery.\nData were collected prospectively on a handheld computer database (Psion, Psion Ltd., England). All patients underwent preoperative endoscopy and 24\u00a0h ambulatory esophageal pH monitoring. After the first 75 cases, all patients also underwent stationary esophageal manometry using a standardized technique.\nThe operative technique was modified during the course of the study as new equipment became available. Initially, five 10-mm abdominal ports were used: toward the latter half of the study, two 5-mm and three 10-mm ports were used. For the first 35 patients, a 0\u00b0 laparoscope was used: all subsequent operations were carried out using a 30\u00b0 laparoscope. The lower esophagus was mobilized from the crural arch. All patients underwent division of the short gastric vessels initially using individually applied ligaclips: after case 215, a harmonic scalpel (Ethicon, Endosurgery, UK) was used. In the first 40 patients, the crura were repaired (using 2\/0 silk) only if a hiatal defect and a hernia were present. After this, all patients underwent crural approximation. A loose wrap of 1\u20132\u00a0cm length was constructed over a 56 French gauge bougie using nonabsorbable sutures (initially silk; later \u201c0\u201d Ethibond) incorporating the anterior esophagus. During the period of the study, 63 patients, who were included in the fourth to seventh cohorts, underwent a laparoscopic partial posterior fundoplication as part of a randomized trial.3 These patients were included in this study, as there was no difference in symptomatic outcome, complication rate, or operative time between this group and those undergoing a 360\u00b0 fundoplication. Fourteen pediatric patients underwent an LNF throughout the series. The surgical technique used was the same in adult and pediatric populations. Patients were encouraged to mobilize immediately and commenced on oral fluids, followed by a light diet, as soon as tolerated.\nThe overall patient group was divided into eight cohorts of 50 consecutive patients. These cohorts were analyzed separately to compare the following: (1) patient demographics, (2) preoperative symptom length, (3) operative time, (4) length of postoperative hospital stay, (5) conversion to open operation, (6) reoperation rate, (7) postoperative dilatation rate, and (8) perioperative mortality or other early (within 6\u00a0months) postoperative complications.\nResults\nThe mean age, weight, and length of preoperative symptoms for each group was similar (see Table\u00a01). This table also shows an overall decrease in the amount of time to accrue each cohort throughout the study period. There was a steady decrease in the mean operative time throughout the study period from 143\u00a0min in the first cohort to 86\u00a0min in the last cohort (Fig.\u00a01). The mean postoperative hospital stay was reduced from 3.7\u00a0days (range 2\u201325) to 1.2 \u00a0days (range 1\u20135) from the first to the last cohort. There were no perioperative deaths. \nTable\u00a01Demographics and Length of Preoperative Symptoms in Patients Undergoing Fundoplication for GERD\u00a0Patient Numbers1\u20135051\u2013100101\u2013150151\u2013200201\u2013250251\u2013300301\u2013350351\u2013400Time period to accrue cohort (months)2921121111121110Mean age (years) (range)36.3 (13\u201370)41.6 (9\u201382)43.9 (13\u201364)44.5 (12\u201386)44.3 (15\u201366)43.9 (17\u201366)45.4 (18\u201374)45.1 (15\u201381)Sex (M:F)34:1638:1228:2232:1829:2129:2134:1635:15Mean weight (kg) (range)71.1 (44\u2013102)75.7 (29\u201398)76.1 (49\u2013104)74.3 (30\u2013102)79.5 (51\u2013120)79.3 (44\u2013103)78 (48\u2013103)80.4 (53\u2013100)Mean preoperative symptomatic period (months) (range)91 (8\u2013420)85 (6\u2013540)106 (3\u2013480)92 (4\u2013516)106 (4\u2013430)96 (12\u2013360)141 (6\u20131,152)140 (4\u20131,152)Figure\u00a01Showing operative conversions to open procedure, rates of reoperation, and rates of dilatation in patients undergoing laparoscopic fundoplication for GERD.\nFigure\u00a01 also shows the rate of conversion from laparoscopic to open fundoplication, the reoperation rate, and the postoperative dilatation rate.\nConversions to Open Operation\nThe conversion rate in the first cohort of 50 patients was 14%. Compared to this, only one conversion was required in the last 250 patients in the series, and this was necessitated by equipment failure rather than surgical difficulties. Other conversions were undertaken for hemorrhage from short gastric vessels (seven patients), port-site bleeding (one patient), splenic bleeding (one patient), difficult access (two patient), instrumental esophageal perforation (one patient), and adhesions from previous surgery (two patients).\nPatients Needing Postoperative Dilatations\nIn the first 50 patients, 8 of them (16%) needed endoscopic balloon dilatation for persistent dysphagia or gas bloat syndrome between 10\u00a0days and 3\u00a0months postoperatively. They were dilated between one and three times. Nine patients (18%) were dilated in the second cohort between 9\u00a0days and 10\u00a0months postoperatively on one to four occasions. In the third cohort, six patients (12%) underwent dilatation between 1\u00a0week and 7\u00a0months, whereas in the fourth cohort, five patients (10%) were dilated between 3\u00a0weeks and 2\u00a0months postoperatively. They were all dilated once or twice. Two patients (4%) had two dilatations each between 2 and 9\u00a0months in the fifth cohort. No dilatations were needed by the last 150 patients to undergo laparoscopic fundoplication.\nPatients Needing Reoperation\nFigure\u00a01 illustrates a decline in the number of patients requiring reoperation from seven patients (14%) in the first 50 to three patients (6%) in the last 50 patients. Table\u00a02 shows the number of reoperations that took place for any given reason in our overall patient group. It also highlights the number of reoperations that occurred within 3\u00a0months of the original fundoplication. \nTable\u00a02Total Number and Timing of Patients Undergoing Reoperation after Laparoscopic FundoplicationCause of ReoperationTotal Number of patientsEarly (within 3\u00a0months)Late (after 3\u00a0months)Mediastinal wrap herniation16115Persistent reflux11Dysphagia despite dilatation211Gas bloat33Perforation of wrap11Port-site hernia11\nIn the first cohort, five patients underwent reoperation for mediastinal \u201cwrap\u201d herniation between 9 and 80\u00a0months postoperatively. Two patients required revisional surgery; one underwent a Watson fundoplication, whereas the other undertook a redo Nissen fundoplication at 2 and 6\u00a0months, respectively, for persistent dysphagia failing to respond to endoscopic dilatations. One reoperation for \u201cwrap\u201d herniation was attempted laparoscopically but was converted to an open procedure. All other reoperations were carried out as open procedures.\nIn the second cohort, three patients were reoperated on \"for mediastinal \u201cwrap\u201d herniation and wrap disruption at 2, 30, and 47\u00a0months postoperatively: one by open surgery and two laparoscopically. One patient underwent laparoscopic conversion of a 360\u00b0 to 270\u00b0 \u201cwrap\u201d for \u201cgas bloat\u201d at 11\u00a0months despite two endoscopic \u201cwrap\u201d dilatations.\nThere was one reoperation in the third cohort of patients for gas bloat 92\u00a0months later. The wrap was found to be mildly attenuated and was taken down laparoscopically. In the fourth cohort, one patient underwent laparoscopic conversion to a 270\u00b0 \u201cwrap\u201d for \u201cgas bloat\u201d syndrome 12\u00a0months later, and one patient was converted from a 270\u00b0 to a 360\u00b0 wrap for a persistent reflux. Two patients underwent open reoperations in the fifth cohort: one for a perforation of the \u201cwrap\u201d at 4\u00a0days, the other for a port-site hernia repair at 9\u00a0months.\nIn the sixth cohort, two patients underwent a redo LNF for wrap herniation and disruption at 23 and 36\u00a0months postoperatively. In the seventh cohort, two patients were found to have a wrap herniation, and one patient was found to have a large crural defect with wrap herniation at 18, 19, and 23\u00a0months, respectively. All underwent redo LNF; the patient with the large crural defect had a hiatal mesh placed. In the last cohort, three patients underwent redo LNF (two with hiatal mesh placement) for wrap herniation at 20, 27, and 36\u00a0months postoperatively.\nDiscussion\nThe postoperative complications most commonly associated with open fundoplication are dysphagia and gas bloat syndrome. The advent of the laparoscopic approach to fundoplication, first described in 1991,4 has introduced a number of procedure-specific complications, including pneumothorax, pneumomediastinum, major-vessel injury, mesenteric thrombosis, and gastrointestinal perforation.5\nThe first prospective randomized study comparing laparoscopic and open Nissen fundoplication6 showed similar complication rates and a better symptom outcome in those who had undergone laparoscopic surgery. There has, however, been a concern as to the severity of the reported complications in the laparoscopic approach.7\nBefore the commencement of this study, the surgeon had a 6-year experience with open fundoplications. In the early 1990s, formal courses were not available to learn laparoscopic fundoplication: consequently, the surgeons pioneering this procedure were mentored for the first few cases. After this, the surgeon would operate independently.\nOur study shows that as the surgeon\u2019s experience of laparoscopic fundoplication increases, the mean operating time becomes comparable to that of an open operation. The mean postoperative length of stay in hospital was 1.2\u00a0days in the last 50 patients compared with an average stay of 7\u00a0days in those having an open fundoplication.8 The decrease in postoperative length of stay in hospital, which was seen throughout this series, can be partly attributed to increased knowledge of recovery from laparoscopic procedures and from patient feedback of their postoperative recovery.\nThe high conversion rate to an open operation in our first 50 patients (14%) can be attributed to the surgical learning curve and poorer quality equipment leading to reduced quality of vision and the reduced ability to secure bleeding. Similarly, high conversion rates were seen in other early laparoscopic series.9,2 Only 1 of the last 150 patients needed conversion to an open procedure, and this was due to equipment failure. One patient underwent a splenectomy (0.3% of all patients) due to splenic bleeding, which is comparable to other studies.10 This compares with a splenectomy rate of 3.6% in open Nissen fundoplications in one study.6 Of the 15 patients requiring conversion throughout the series, 14 were in the preharmonic scalpel era. Nine of these were converted due to bleeding. The harmonic scalpel has greatly enhanced the ease of fundal mobilization in comparison to the application of individual ligaclips to the short gastric arteries. A decreasing trend in conversion rate can, however, be seen within the first four cohorts before the introduction of the harmonic scalpel.\nThe number of patients undergoing endoscopic dilatation decreased significantly from 17% in the first 100 patients to none in the last 150 patients. This was probably due to a number of factors. First, none of the last 250 patients had symptomatic \u201cwrap\u201d disruptions\/slippages causing dysphagia. The patients who were found to have wrap herniation presented with heartburn and not dysphagia. Secondly, it is now recognized that early dysphagia (less than 2\u00a0months postoperatively) is present in a significant proportion of patients but settles with time without the need for intervention.11 The exception to this is in children who are less tolerant of dysphagia after laparoscopic fundoplication and hence are more likely to require early endoscopic dilatation.12 Two studies12,13 have shown dilatation success rates of 56 and 67%, respectively, in resolving postfundoplication dysphagia. The study by Malhi-Chowla et al.13 also found that the only symptom that responded to dilatation was dysphagia.\nThroughout the series, two patients had been reoperated on for persistent dysphagia beyond 2\u00a0months. Both were in the first 25 cases, and both were found to have bowstringing of the wrap due to lack of division of the posterior gastric bands. One was converted to a Watson fundoplication, whereas the other underwent a redo Nissen fundoplication. Our low incidence of dysphagia may be in part due to the laparoscopic operation used. Hunter et al.14 showed that the incidence of early and late persistent dysphagia is significantly lower in both LFNs and Toupet fundoplications than in Rosetti\u2013Nissen fundoplications.\nTwo patients had undergone reoperation for gas bloat syndrome: both were converted to a 270\u00b0 posterior wrap and are now either asymptomatic or mildly symptomatic.\nAll patients in this study had been followed up for a minimum of 4\u00a0years. Overall, 6% of our patients required reoperation because principally of wrap herniation. After the first 40 operations, a routine posterior crural approximation was carried out with nonabsorbable sutures to reduce the incidence of thoracic \u201cwrap\u201d migration. Two studies15,16 have emphasized the importance of a crural repair in reducing the incidence of postoperative paraesophageal hiatus hernia. Basso et al.17 have proposed a mesh repair of the hiatus to prevent \u201cwrap\u201d migration after finding that in several reoperations, the sutures approximating the crura had cut out with consequent wrap herniation.\nParaesophageal wrap herniation is more common in laparoscopic than in open fundoplication.16 Several reasons have been proposed for this: (1) the tendency to extend esophageal dissection further into the thorax,18 (2) the increased risk of breaching the left pleural membrane during dissection,19 and (3) the reduced postoperative pain allowing increased abdominal pressure when vomiting\/coughing in the laparoscopic procedure.16 Wu et al.20 found that routine division of the short gastric arteries and posterior closure of the crura during LNF significantly reduced wrap slippage\/migration. This is the procedure that we have undertaken since the 40th patient. Despite of this, 5% of the last 150 patients underwent reoperation (all for heartburn due to wrap herniation). Smith et al.21 have also concluded that wrap herniation is now the most common mechanism of failure requiring a redo fundoplication.\nOf the eight patients reoperated on in the first cohort, only one procedure was attempted laparoscopically. All reoperations are now attempted laparoscopically where possible. This change in approach has occurred with increasing laparoscopic experience. Several studies22,23 have shown that laparoscopic reoperations are not only possible and safe but also produce good results.\nSeveral studies24,25,26 have now shown a 90% satisfaction rate at 5-year follow-up after LNF. Our own study25 on patient satisfaction 2\u20138\u00a0years postlaparoscopic fundoplication revealed that once over the initial problem of early postoperative dysphagia, the satisfaction rate was 91%. Furthermore, 90% remained free of significant reflux symptoms, and only 14% were subsequently taking regular antireflux medication. This has changed little throughout the course of the series.\nOur results show a decreasing trend in operative time, postoperative hospital stay, conversion rate, postoperative dilatation rate, and reoperation rate with increasing surgical experience and improved technology. Another factor in this improvement may be due to the increased frequency with which this procedure was performed with time.\nConclusion\nDysphagia is the Achilles\u2019 heel of laparoscopic antireflux surgery. To avoid this, the authors have routinely divided the short gastric vessels. This has led to an increased rate of conversion owing to hemorrhage especially during the period when individual ligaclips were used. Short gastric vessel division may, in addition, increase the rate of wrap herniation and clip or thermal injury to the gastric fundus leading to perforation.\nThe high rate of reintervention in the first two cohorts would not be acceptable a decade later. It must be recognized that at the start of this series, the visual acuity of the optical systems and the quality of the instrumentation were both substantially inferior to those of today. Furthermore, there were no formal training courses available. The pioneers of advanced laparoscopic surgery had to suffer high conversion and complication rates in laparoscopic cholecystectomy,27 antireflux,2 and groin hernia surgery.28,29\nWhen introducing complex techniques, surgeons tend to underestimate the learning curve: both of themselves and of their institution. Only by maintaining prospective data can these problems be identified and recognized.","keyphrases":["learning curve","laparoscopic nissen fundoplication"],"prmu":["P","P"]}
{"id":"Neuroimage-2-1-2387200","title":"Abnormal brain connectivity in first-episode psychosis: A diffusion MRI tractography study of the corpus callosum\n","text":"A model of disconnectivity involving abnormalities in the cortex and connecting white matter pathways may explain the clinical manifestations of schizophrenia. Recently, diffusion imaging tractography has made it possible to study white matter pathways in detail and we present here a study of patients with first-episode psychosis using this technique. We selected the corpus callosum for this study because there is evidence that it is abnormal in schizophrenia. In addition, the topographical organization of its fibers makes it possible to relate focal abnormalities to specific cortical regions. Eighteen patients with first-episode psychosis and 21 healthy subjects took part in the study. A probabilistic tractography algorithm (PICo) was used to study fractional anisotropy (FA). Seed regions were placed in the genu and splenium to track fiber tracts traversing these regions, and a multi-threshold approach to study the probability of connection was used. Multiple linear regressions were used to explore group differences. FA, a measure of tract coherence, was reduced in tracts crossing the genu, and to a lesser degree the splenium, in patients compared with controls. FA was also lower in the genu in females across both groups, but there was no gender-by-group interaction. The FA reduction in patients may be due to aberrant myelination or axonal abnormalities, but the similar tract volumes in the two groups suggest that severe axonal loss is unlikely at this stage of the illness.\nIntroduction\nAltered connectivity is likely to account for the symptoms and cognitive changes of schizophrenia (Friston and Frith, 1995). A disconnectivity model involving both loss of specialized cortical function and damage to connecting pathways is likely to apply to schizophrenia (Catani and ffytche, 2005). Imaging studies have repeatedly confirmed the presence of cortical abnormalities, especially in prefrontal and temporal heteromodal cortex (Shenton et al., 2001; Bagary et al., 2003; Foong et al., 2001), and synaptic pathology leading to aberrant cortical circuitry has been well documented in neuropathological studies (Harrison and Weinberger, 2005). By comparison, the study of abnormalities in intra- and interhemispheric pathways has received much less attention, although the occurrence of schizophrenia-like symptoms has been documented in diseases involving the white matter (Walterfang et al., 2006).\nAmong these connecting pathways the corpus callosum is of special interest in schizophrenia. The corpus callosum develops into the third decade of life (Pujol et al., 1993) and the topographical organization of its fibers makes it possible to relate its abnormalities to specific cortical regions. Fibers of small diameter from heteromodal cortex traverse the genu (connecting prefrontal cortex) or the splenium (connecting superior temporal cortex), and larger diameter fibers connecting unimodal motor and sensory cortex traverse the body. A model by Crow (1998), based on the evolutionary aspects of language and the emergence of psychosis in man, postulated that abnormalities in callosal pathways connecting areas of the cortex asymmetrically distributed between the two hemispheres were central to schizophrenia. Since then, conventional MRI studies (Shenton et al., 2001), including those in drug-naive patients with their first episode of schizophrenia (Keshavan et al., 2002), have reported reduction in the size of the corpus callosum, more marked anteriorly, but shape differences in the rostral and mid area of the body containing motor and sensory fibers have also been reported (Goghari et al., 2005). Similar findings have been reported by Bachmann et al. (2003) in a first-episode cohort that included patients with schizophreniform and schizoaffective psychoses. In addition, a longitudinal study of patients with childhood-onset schizophrenia (Keller et al., 2003) has suggested that a failure of normal callosal growth may result in area reductions, particularly in the splenium, by early adulthood. Other imaging studies using voxel-based analysis (Hulshoff Pol et al., 2004) or magnetization transfer imaging (Foong et al., 2001) have also reported abnormalities, predominantly in the genu. In addition Highley et al. (1999a), in a detailed neuropathological study, reported a decrease in axon density in all, but the rostral region of the corpus callosum in female schizophrenics compared to controls. The functional significance of the corpus callosum in schizophrenia has been well documented by studies of interhemispheric transfer of information (Innocenti et al., 2003).\nDiffusion tensor imaging (DTI) (Basser et al., 1994) has made it possible to study in vivo the integrity and orientation of neural tissue by measuring water diffusion in the brain. DTI studies have used a region-of-interest (ROI) methodology and have measured the apparent diffusion coefficient (ADC) and\/or fractional anisotropy (FA), indices of white matter integrity. An early study from our group (Foong et al., 2000) reported reduced FA in the splenium of the corpus callosum in patients with chronic schizophrenia and similar findings have been reported by others in the splenium (Agartz et al., 2001), or in the structure as a whole (Brambilla et al., 2005; Ardekani et al., 2003; Kubicki et al., 2005). Kumra et al. (2004), on the other hand, failed to find such abnormalities in a small group of patients with early-onset psychosis and the same was the case in the only available study in first-episode patients, also from our group (Price et al., 2005). The clinical correlations of these corpus callosum abnormalities remain uncertain. Brambilla et al. (2005) reported a correlation between DTI changes in the anterior part of the corpus callosum and severity of positive symptoms, but these findings have not been confirmed in other studies.\nThe information about the direction of diffusion encoded by the eigenvalues and eigenvectors of the diffusion tensor has been used in DTI tractography (Mori et al., 1999) to investigate the continuity of axonal orientation between voxels and thus to infer the paths of fiber tracts in 3 dimensions. DTI tractography has been used in studies of normal subjects (Toosy et al., 2004; Parker and Alexander, 2005; Parker et al., 2005), in patients with multiple sclerosis (Wilson et al., 2003), callosal dysgenesis (Lee et al., 2004) and stroke (Huang et al., 2005). To date only one study (Kanaan et al., 2006) has used tractography to study the corpus callosum in patients with chronic schizophrenia, reporting reduced FA in the genu and it also demonstrated the superiority of this method, over ROI studies, in detecting subtle abnormalities across the whole of a white matter tract.\nHere we present, to our knowledge, the first study of the corpus callosum (or indeed any white matter pathway) in patients with first-episode schizophrenia spectrum disorders using diffusion imaging tractography. The main aim of the study was to determine whether subtle abnormalities of interhemispheric connections could be detected using this new technique in a group of patients during the first-episode of psychosis, in whom chronicity-related factors could be excluded. We hypothesized that measures of tract coherence (FA), in white matter traversing the genu and splenium of the corpus callosum, would be significantly reduced in the patient group compared to controls.\nMaterials and methods\nThe patients were recruited as part of a prospective, longitudinal study of first-episode psychosis in West London. Patients were eligible to be recruited into the study if they fulfilled the following criteria; age between 16 and 50\u00a0years presenting with a psychotic illness for the first time and less than 12\u00a0weeks on antipsychotic medication. Patients eligible for the study were screened using the WHO Psychosis Screen (Jablensky et al., 1992). The diagnosis was established using a structured interview, the diagnostic module of the Diagnostic Interview for Psychosis (DIP, Jablensky et al., 2000), which includes items from the Operational Criteria Checklist for Psychosis (OPCRIT, McGuffin et al., 1991) and the World Health Organization Schedules for Clinical Assessment in Neuropsychiatry (SCAN, Wing et al., 1990). Using these data, a computerized algorithm generates diagnoses under several classification systems including DSM-IIIR and ICD-10. DSM-IIIR diagnoses were subsequently checked against DSM-IV criteria by separately entering OPCRIT items into OPCRIT for Windows (http:\/\/sgdp.iop.kcl.ac.uk\/opcrit\/). Exclusion criteria were the presence of a medical or neurological illness that might impair cognitive function including head injury and alcohol or drug dependency.\nEighteen patients with an initial diagnosis of schizophrenia, schizophreniform or schizoaffective disorder took part in this study. Fifteen of these patients were interviewed again a year later to review the diagnosis, and for the remaining three, who could not be interviewed, a final diagnosis was established also a year later by compiling information from clinicians looking after the patients and by reviewing the clinical notes. Thirteen patients received a final diagnosis of schizophrenia and the remaining five received a diagnosis of schizoaffective disorder (one bipolar, two manic and two depressed subtype). The patients had been ill for a mean of 12.6\u00a0months (SD\u00a0=\u00a019.3\u00a0months, range 0\u201372\u00a0months) at entry into the study.\nThe range and severity of symptoms were assessed at each time point with the Scales for the Assessment of Positive Symptoms (Andreasen, 1983) and Negative Symptoms (Andreasen, 1981), The Young Mania Scale (Young et al., 1978) and the Hamilton Rating Scale for Depression (Hamilton, 1960). The onset of psychosis was established using the Symptom Onset in Schizophrenia inventory (Perkins et al., 2000). Alcohol and drug use was assessed as part of the Diagnostic Interview for Psychosis (see above) and criteria for abuse and dependence were established using the Alcohol Use Scale and the Drug Use Scale (Drake et al., 1990).\nThe mean age of the patient group was 23.6\u00a0years (SD\u00a0=\u00a06.3; range\u00a0=\u00a017\u201338\u00a0years), composed of 8 males and 10 females. All patients were receiving antipsychotic medication at the time of scanning. Twenty-one healthy subjects with a mean age of 29.4\u00a0years (SD\u00a0=\u00a07.1; range\u00a0=\u00a016\u201342\u00a0years) with a gender composition of 6 males and 15 females served as controls. Handedness for all subjects was assessed using the Annett scale (Annett, 1970) as it may be associated with DTI findings in the corpus callosum (Westerhausen et al., 2003). Exclusion criteria were the same as in the patient group as well as history of psychiatric illness in themselves or their first-degree relatives. See Table 1 for a description of study subjects. Permission to conduct the study was obtained from Merton, Sutton and Wandsworth, Riverside and Ealing Research Ethics Committees. All participants gave written informed consent and were paid an honorarium for their time.\nMRI data acquisition\nMRI for all subjects was obtained using a GE Signa 1.5 T scanner (General Electric, Milwaukee, WI, USA), which underwent regular quality-control checks, using a standard quadrature head coil. Each subject had a dual-echo fast spin echo scan (TR\u00a0=\u00a02000\u00a0ms, TE1\/TE2\u00a0=\u00a019\/95\u00a0ms, matrix\u00a0=\u00a0256\u00a0\u00d7\u00a0192, field of view [FoV]\u00a0=\u00a024\u00a0\u00d7\u00a018\u00a0cm2), which provides both proton density and T2-weighted images. Twenty-eight 5-mm slices were collected, in an oblique-axial plane (parallel to the AC\/PC line). An axial plane, IR-SPGR sequence was also performed which obtained a series of 156 contiguous axial slices with the following parameters TE\u00a0=\u00a05.1\u00a0ms, matrix\u00a0=\u00a0256\u00a0\u00d7\u00a0128, field of view\u00a0=\u00a031\u00a0\u00d7\u00a016\u00a0cm2, slice thickness\u00a0=\u00a01.2\u00a0mm, TR\u00a0=\u00a014.3\u00a0ms, flip angle 20\u00b0, TI\u00a0=\u00a0450\u00a0ms.\nDiffusion-weighted single-shot echo planar images (DW-EPI) were acquired in the axial plane (TE\u00a0=\u00a096\u00a0ms, FoV\u00a0=\u00a022\u00a0\u00d7\u00a022\u00a0cm2, matrix\u00a0=\u00a096\u00a0\u00d7\u00a096, slice thickness\u00a0=\u00a02.3\u00a0mm, \u0394\u00a0=\u00a040\u00a0ms, \u03b4\u00a0=\u00a034\u00a0ms, resulting in a maximum b factor of 1000\u00a0s\/mm2). The acquisition of diffusion-weighted images was gated to the cardiac cycle using a pulse oximeter with a gating scheme optimized for diffusion imaging. Gradients for diffusion sensitization were applied in 54 non-collinear directions. Six images with no diffusion weighting (b\u00a0\u2248\u00a00\u00a0s\/mm2) were also collected for each slice, giving a total of 60 images per slice. Images were interpolated to a 128\u00a0\u00d7\u00a0128 matrix during reconstruction, yielding a final in-plane resolution of 1.72\u00a0mm.\nDiffusion processing and analysis\nThe diffusion tensor was estimated for each voxel according to the method of Basser et al. (1994) and used to compute FA. We also used a model-selection algorithm based on the fit of spherical harmonic series to the diffusion profile (Alexander et al., 2002) to detect the most parsimonious description of diffusion in every voxel. Diffusion within each voxel was classified as isotropic, anisotropic with a single principal direction of diffusion, or anisotropic with more than one direction of diffusion (as in the white matter of the centrum semiovale). The single tensor model was subsequently used for voxels characterized by isotropic diffusion or a single direction of diffusion population. The two-tensor model of diffusion, as described by Parker and Alexander (2003), was used for the remaining voxels.\nWe used a probabilistic tractography algorithm (PICo or \u2018probabilistic index of connectivity\u2019) which considers multiple pathways emanating from a seed point or region (i.e. a group of voxels in a region of interest) (Parker and Alexander, 2003, 2005). Due to the presence of noise in the data, there is some uncertainty associated with the determination of the principal direction of diffusion. The algorithm accounts for this uncertainty by generating a probability density function (PDF) of fiber alignment from the diffusion model of each voxel (which in this case is either the single or the two-tensor model). This provides voxel-wise estimates of confidence in fiber tract alignment, which are then used in the probabilistic tract-tracing procedure. Using the PDFs from a chosen seed point, streamline-based tracking is performed and repeated 10,000 times in a Monte Carlo fashion (sampling each PDF at random on each repeat) to produce tract maps that estimate the probability of connection of every voxel in the brain to a given seed point or region (Parker and Alexander, 2005). Streamlines were propagated using trilinear interpolation of PDFs, as suggested by Behrens et al. (2003), and were terminated if curvature over the scale of a single voxel exceeded 180\u00b0 or if the path left the brain.\nSeed regions were placed in the genu and splenium of the corpus callosum. The genu was defined as the most anterior point of the corpus callosum before it bends downwards and backwards in front of the septum pellucidum, and the splenium as the posterior end of the corpus callosum at its thickest part. Each seed region consisted of a six voxel rectangular shape on a single axial slice. This region size ensured that partial volume effects were completely avoided. Seed regions were placed by displaying the FA maps in three mutually orthogonal orientations using MRIcro (http:\/\/www.sph.sc.edu\/comd\/rorden\/) and identifying the sagittal and coronal planes in which the volumes of the genu and splenium were largest. The seed regions were placed on the axial view corresponding to the intersection of these sagittal and coronal planes.\nThe seed region placement was the only operator-dependent step in the diffusion processing. To assess the inter-rater reliability of the method, a second, independent rater performed the seed region placement in 8 cases, using the same procedure.\nThe voxel values of the PICo output maps for the corpus callosum range from 0 (no probability of connection) to 1 (certainty of connection) with an intensity resolution step of 0.001 representing the ratio of the number of times that voxel was reached by a streamline originating from the seed point to the total number of iterations in the Monte Carlo process. Each PICo map was thresholded at five probability values ranging from 0.001 (the lowest recorded value of probability of connection) to 0.03, at logarithmically spaced intervals, to generate five objective binary masks (thresholded at 0.001, 0.002, 0.006, 0.013 and 0.03). This multi-threshold approach, previously used in the study of visual pathways (Toosy et al., 2004), allows with increasing degrees of certainty the reconstruction of the core of the tract, where fiber alignment should be greatest, and the exploration of the probability of connection of distant voxels to the seed point. For each threshold the mean tract volume and the mean FA were calculated for each subject.\nThe spatial variability of the tract across each subject group was characterized as previously described (Toosy et al., 2004; Parker et al., 2005). Firstly the non-diffusion-weighted (b\u00a0=\u00a00) images (inherently co-registered with PICo output images) were normalized to a stereotactic space (Montr\u00e9al Neurological Institute, MNI) using the standard echo planar image template in SPM2 (Wellcome Department of Cognitive Neurology, Institute of Neurology, London, UK) running in MATLAB (MathWorks, MA). The normalization parameters thus obtained were then applied to the binary images of the tract obtained by thresholding the probability images at the level that maximized the difference in FA between patients and controls (see below). After normalization, these images were averaged on a voxel-by-voxel basis, producing a map used to display the degree of tract overlap between subjects within each group.\nIn order to detect the presence of ventricular enlargement in the patient group, which might affect tractography, we performed a voxel-based comparison of CSF maps between the two groups using SPM2 (Wellcome Department of Cognitive Neurology, Institute of Neurology, London, UK). The IR-SPGR images were segmented and normalized using the iterative procedure described in Good et al. (2001), thus performing first a segmentation in native space, next normalizing gray matter images using the standard a priori gray matter template available in SPM2 and applying the same normalization parameters to the whole volume. The normalized volume was then segmented again yielding white matter, gray matter and CSF probability images. Voxel values in segmented images were multiplied by the Jacobian determinants derived from spatial normalization (modulation) to provide intensity correction for induced regional volumetric changes, thus preserving within-voxel volumes that may have been altered during non-linear normalization (Ashburner and Friston, 2000). CSF images were also smoothed to 6\u00a0mm (Full-Width-Half-Maximum) Gaussian filter. Smoothing is required to accommodate anatomical variation between subjects and therefore results in more normally distributed data. Global CSF volumes in cm3 were also obtained by integrating the CSF image signal intensity over the whole volume for each subject.\nStatistical analysis\nAge, gender and handedness distributions in the two groups were compared using t-tests and \u03c72 tests.\nThe inter-rater reproducibility of the method was tested by comparing the tract volumes obtained by the two raters. Inter-rater reliability was assessed using the coefficient of variation (CoV), CoV\u00a0=\u00a0(\u03c3\u00a0\/\u00a0\u03bc)\u00a0\u00d7\u00a0100%, where \u03c3 is the standard deviation and \u03bc is the mean, with lower values indicating better reproducibility.\nMultiple linear regressions of mean FA were carried out to compare tract coherence in patients and controls, with gender, age and tract volume as covariates, to control for between-group differences in these covariates. To avoid spurious positive results due to multiple comparisons, the multiple regressions were carried out simultaneously for each of the five thresholds in the genu and the splenium (ten regressions) using Zellner\u2019s seemingly unrelated regression method (Zellner, 1962), which yields a single significance value (from the F-statistic) of the difference between patients and controls across all ten regressions, in addition to individual values for each regression. The F-statistic is obtained by comparing the ten regressions obtained including or excluding group membership and taking into account the correlations between the residuals from all ten equations. This method also allows a test of whether region (genu or splenium) or probability thresholds modify the differences in FA between patients and controls. Similar multiple linear regressions were performed to explore gender differences in FA (when adjusting for group membership, age and tract volume) as well as age differences in FA (adjusting for group membership, gender and tract volume).\nA voxel-based statistical comparison between CSF maps from patients and controls was performed in SPM2 based on the General Linear Model and Gaussian random field theory incorporating subject age and gender into this model.\nResults\nPatients were significantly younger than controls. The mean age for patients was 23.6\u00a0years and 29.4\u00a0years for controls (t\u00a0=\u00a0\u2212\u00a02.7, df\u00a0=\u00a037, P\u00a0=\u00a00.011). However, there was an age overlap between the two groups, allowing the results to be adjusted for age. There were no significant differences in gender distribution (\u03c72\u00a0=\u00a00.62, df\u00a0=\u00a01, P\u00a0=\u00a00.43) or handedness (\u03c72\u00a0=\u00a01.81, df\u00a0=\u00a01, P\u00a0=\u00a00.18) between the groups. All patients were receiving antipsychotic medication and two were on mood stabilizers at the time of the study (see Table 1). The average duration of treatment prior to scanning was 61\u00a0days (range 8\u2013153\u00a0days).\nThe coefficient of variation for the tract volume for the 8 repeated measurements performed by two raters was 1.36%, indicating a very high inter-rater reproducibility.\nThe tract overlap maps for the genu and splenium in patients and controls (Figs. 1 and 2) were compared with reference maps of the corpus callosum to ascertain their anatomical validity.\nThe estimated tract volumes in patients and controls decreased at higher connection probability thresholds as the core of the tract was progressively isolated. Conversely, FA increased with higher thresholds (Figs. 3A\u2013D).\nThe results of the multiple regression analysis comparing FA in patients and controls are shown in Table 2.\nThe single test of significance considering both regions of the corpus callosum at all thresholds was P\u00a0\u2264\u00a00.02, indicating a significant difference in FA between the two groups in either region, over all thresholds. Inspection of the differences in FA at the individual thresholds indicated that the differences in FA between the groups are due to lower FA in the patient group in the genu at probability thresholds 0.006 and 0.013, and to a lesser extent in the splenium at thresholds 0.013 and 0.03 (see Table 2).\nTo examine subgroup differences in FA, we performed a regression analysis to compare the 13 schizophrenic and 5 schizoaffective patients. Both subgroups had a lower FA than controls. Results from the regression analysis revealed that in the genu, the schizoaffective group had a lower FA than the schizophrenia group (P\u00a0=\u00a00.0806), whereas in the splenium, the schizoaffective group had higher FA values than the schizophrenia group (P\u00a0=\u00a00.0295). This suggests that, although there may be subtle differences between subgroups, they differed from controls in the same direction and amalgamating the subgroups does not obscure the differences between patients and controls.\nWe performed a separate analysis looking for gender and age differences in FA by considering patients and controls together. In the analysis of gender, a single test of significance considering all thresholds and both regions of the corpus callosum gave a P\u00a0=\u00a00.0001. This result indicates that there are gender differences in FA irrespective of subject status. Thus FA, adjusted for group membership, was lower in females than males in the genu at thresholds 0.001 and 0.002, but not in the splenium. There was no significant group membership-by-gender interaction (P\u00a0=\u00a00.2).\nWith respect to the effects of age, there was no evidence that the difference between patients and controls in FA varied with age (P\u00a0=\u00a00.791) for the global test over both regions (i.e. the ten equations), after adjusting for gender and tract volume.\nFinally, the SPM analysis using a voxel-based comparison of CSF maps between the two groups revealed no differences between patients and controls using a family wise error correction for multiple comparisons, with f\u00a0=\u00a034.42 and no voxels as an extent threshold. This indicates that no ventricular enlargement was present in the patient group relative to controls. There was no significant difference between the global CSF volumes of patients (287 cm3, SD\u00a0=\u00a033.6) and controls (283 cm3, SD\u00a0=\u00a051.6) (t\u00a0=\u00a0\u2212\u00a00.85, P\u00a0=\u00a00.40, CI\u00a0=\u00a0[\u2212\u00a041.7\u201317.2]).\nDiscussion\nOur findings provide evidence of altered interhemispheric connectivity in patients with first-episode schizophrenia spectrum disorders. In the patient group, abnormal tract coherence, as measured by reduction in FA, was present in tracts traversing the genu and, to a lesser extent, in those traversing the splenium. Tract coherence, as measured by FA, was lower in females, both in patients and controls, but we failed to find a gender-by-group interaction.\nThe genu and the anterior part of the body of the corpus callosum contain mainly connections from prefrontal cortex, cingulate and insula, and pathology in these cortical regions could result in the abnormalities of the corpus callosum described here. Similarly, a decreased thalamic input to the frontal and temporal cortex, perhaps due to excessive synaptic pruning, could also result in decreased cortical connections and hence in abnormalities in the corpus callosum (Innocenti et al., 2003), but it is also possible that primary white matter abnormalities could contribute to our findings (Walterfang et al., 2006). The corpus callosum develops alongside other midline structures, namely the fornix, hippocampus, septum and cingulate cortex, and developmental or maturational abnormalities involving the corpus callosum are also likely to affect these structures known to play a role in schizophrenia.\nOur diffusion-MRI-based tractography (PICo) used a probabilistic approach that takes into account branching, crossing and merging of tract fibers and this represents an advantage over previous deterministic approaches that do not take these anatomical features into consideration (Kanaan et al., 2006). In addition the use of different probability thresholds and simultaneous multiple regression analysis (Zellner, 1962) has allowed us to demonstrate evidence, from a single statistical test, that there is some difference in tract coherence between patients and controls and to explore the characteristics of the core of the tract in the genu and the splenium. The group differences in FA become more apparent at intermediate probability thresholds in the genu when fiber alignment in the core of the corpus callosum is greatest and aberrant branching that could have artificially reduced FA (Jones et al., 2005) less likely. The high inter-rater reliability in the placement of the seed points also suggests that the differences in FA between patients and controls are likely to be illness-related rather than artifactual. The same applies to the effects of gender and age, known to affect FA (Price et al., 2005; Jones et al., 2005), that were statistically controlled for in our study as our groups were not closely age matched. Antipsychotic medication may have played a role in reducing FA, although it seems unlikely that medication effects could fully account for the differences between the groups in first-episode patients who had been exposed to medication for short periods. Moreover, others (Flynn et al., 2003) have found no correlation between the presence of white matter abnormalities in first-episode psychosis and exposure to antipsychotic medication. The possible effect of mood stabilizers in explaining our results is likely to be negligible as they were used in only 2 patients.\nThe gender difference in tract coherence that we found in our subjects, particularly in the genu, supports our previous results in a different sample of first-episode patients and controls using a region-of-interest methodology (Price et al., 2005) and is in keeping with the lower anisotropy in normal females than males reported by Westerhausen et al. (2003). Gender dimorphism and maturational differences are likely to be relevant in explaining this finding. Dubb et al. (2003), in an MRI study of normal subjects, found a larger genu and smaller splenium in men compared with women and attributed the differences to different hormonal influences, to enhanced motor coordination in men (interhemispheric connections crossing the genu) and to greater bihemispheric representation of language in women (tracts crossing the splenium). Adult maturational changes in the corpus callosum, with a peak in the third decade of life, would further accentuate these differences. The differences in age between the two groups are a limitation of the study, although we tried to circumvent the problem using age as a covariate.\nThe precise neuropathological correlates of FA abnormalities remain to be fully elucidated. Axonal membranes are considered to be the main determinant of anisotropy in neural tissue, and pathological or experimental models of axonal degeneration have lead to reductions in FA. Myelin abnormalities are also responsible for changes in FA, although their contribution may be less important than those of axons or axonal membranes (Beaulieu, 2002). In our study the lower FA in the white matter tracts traversing the corpus callosum may be due, in addition to myelin abnormalities, to differences in axonal membranes, alterations in axonal packing density, mean axonal diameter (for example due to a bias towards axons of greater or lesser diameter in one or other group) or a less coherent fiber alignment in the patient group. The similar tract volumes we found in patients and controls may suggest that severe axonal loss is unlikely at this early stage of the illness. On the other hand, the greater variance in genu tract volumes in the patient group suggests that there may be more branching (i.e. less coherence or alignment) in the core of the tract. We have also excluded the possibility that our findings could be related to ventricular enlargement in the patient group.\nThe study of patients with first-episode psychosis has the advantage of minimizing the effects of chronicity and lengthy exposure to medication, on the other hand, such patients are diagnostically heterogeneous and this is one of the shortcomings of our study and it remains uncertain whether the changes in corpus callosum described here are a core neuropathological abnormality common to all psychosis or are only present in a subgroup of patients. Support for the former accrues from the study of Bachmann et al. (2003) who reported similar findings in a heterogeneous group of patients with first-episode psychosis. In addition, there is evidence to suggest that corpus callosum abnormalities are also present in patients with bipolar disorder (Brambilla et al., 2003, 2004) and other DTI studies looking at ROIs in frontal white matter, although not specifically at the corpus callosum, have reported low FA in a mixed group of patients with first-episode psychosis (Szeszko et al., 2005) and in those with first-episode mania (Adler et al., 2006). There is also evidence that oligodendrocyte and myelination genes may be downregulated both in schizophrenic and affective psychosis (Tkachev et al., 2003; Iwamoto et al., 2005). It is also likely that the abnormalities described here may also be present in other connecting tracts, as suggested by the neuropathological findings of Highley et al. (1999b) in the anterior commissure, and the results of DTI imaging studies (Kubicki et al., 2005). Our study suggests that the schizophrenia and schizoaffective subgroups may be heterogeneous based on FA, but both these groups differ from controls in the same direction of FA change. An intriguing possibility, in need of further study, is that genetic variability within schizophrenia may be associated with specific patterns of brain morphology including variations in the size and structure of the corpus callosum (Agartz et al., 2006).\nOur findings also demonstrate that tractography is capable of detecting subtle pathological abnormalities in patients with psychosis early in the disease that may go undetected using other DTI methods (Price et al., 2005; Kanaan et al., 2006). Tractography is likely to have a role in future psychosis research, in elucidating connections between relevant cortical regions and in giving specific information about white matter abnormalities and their evolution over time.","keyphrases":["first-episode psychosis","tractography","corpus callosum","diffusion tensor imaging","dti, diffusion tensor imaging","fa, fractional anisotropy","mri, magnetic resonance imaging","mti, magnetization transfer imaging","pdf, probability density function","pico, probabilistic index of connectivity","roi, region of interest"],"prmu":["P","P","P","P","R","R","M","M","R","R","R"]}
{"id":"Eur_Radiol-3-1-2077908","title":"Bone marrow edema-like lesions change in volume in the majority of patients with osteoarthritis; associations with clinical features\n","text":"It has been suggested that bone marrow edema-like (BME) lesions in the knee are associated with progression of osteoarthritis (OA). The purpose of our study in patients with OA was to evaluate prospectively changes of BME lesions over 2 years and their relationship with clinical features. Magnetic resonance (MR) images of the knee were obtained from 182 patients (20% male; aged 43\u201376 years; mean age 59 years) who had been diagnosed with familial symptomatic OA at multiple joint sites. MR images were made at baseline and at 2 years follow-up. BME lesions in 2 years were associated with clinical features assessed by Western Ontario and McMaster Universities Osteoarthritis (WOMAC) scores. A total of 327 BME lesions were recorded. Total size of BME lesions changed in 90 patients (66%). Size of individual lesions changed in 147 foci (45%): new lesions appeared in 69 (21%), existing lesions disappeared in 32 (10%), increased in size in 26 (8%) and decreased in size in 20 (6%) lesions. Increase or decrease of BME lesions, over a 2-year time period, was not associated with severity of WOMAC scores. BME lesions fluctuated in the majority of patients with OA over a 2-year time period. These changes were not associated with severity of WOMAC scores at the study end point.\nIntroduction\nKnee osteoarthritis (OA) is a chronic, progressive joint disease, leading to pain and loss of function in a considerable proportion of patients, with great impact and consequences in the ageing population of the industrialized world. Disease markers need to be identified in order to predict and quantify progression. One possible marker in OA is the so-called bone marrow edema-like (BME) lesions [1].\nUnfortunately, the role of BME in OA remains controversial, as contradictory results have been reported. BME detected with magnetic resonance (MR) imaging has been associated with clinical symptoms in patients with OA [2, 3]. However, other studies reported no association between BME lesions and clinical symptoms [4\u20136]. Further, the role of BME as a marker for progression of OA is open to discussion. In a study by Felson et al. [7], BME was associated with progression of OA as assessed by joint space narrowing on conventional radiographs. On the other hand, in a study by Phan et al. [5], changes in BME did not significantly change with progression of disease assessed by Western Ontario and McMaster Universities Osteoarthritis (WOMAC) scores.\nSince these contradictory results regarding the association between BME and clinical features have been reported, the purpose of our study was to evaluate changes in BME lesions over a 2-year period, and associate them with clinical features.\nPatients and methods\nPatients\nThe present prospective study is part of the ongoing GARP (Genetics, Osteoarthritis and Progression) study [8]. The primary goal of the GARP study is the identification of genetic susceptibility determinants to OA and disease progression in middle-aged sibling pairs with OA at multiple joint sites. MR image sets of the knee were obtained in 182 patients at study entry and after 2\u00a0years [6]. Only one knee, the most symptomatic, was imaged per patient. In the present, study 39% (71\/182) of the patients had symptomatic knee OA in their imaged knee, defined as pain or stiffness on most days in the month prior to study entry, and osteophytes on radiographs. As the purpose of the MR study was to assess progression of OA, no images were made of a knee that already had a maximum Kellgren and Lawrence score of grade 4 [9].\nClinical assessment\nClinical data were assessed by WOMAC to assess pain, stiffness and functional impairment of the imaged knee at the 2-year time point, not at baseline [10].\nMR acquisition\nKnees were imaged using a transmit-receive four-channel knee coil in a 1.5-T superconducting magnet (Philips Medical Systems, Best, the Netherlands). Each examination consisted of: coronal proton density and T2-weighted dual spin echo (SE) images (with repetition time (TR) of 2,200\u00a0ms; echo time (TE) of 20\/80\u00a0ms; number of excitations per data line (NEX) 2; 5\u00a0mm slice thickness; 0.5\u00a0mm intersection gap; 160\u00a0mm field of view (FOV); 256\u2009\u00d7\u2009205 acquisition matrix, 18 slices); sagittal proton density and T2-weighted dual SE images (TR 2,200\u00a0ms; TE 20\/80\u00a0ms; NEX 2; 4\u00a0mm slice thickness; 0.4\u00a0mm intersection gap; 160\u00a0mm (FOV); 256\u2009\u00d7\u2009205 acquisition matrix, 20 slices); sagittal 3D T1-weighted spoiled gradient echo (GE) frequency selective fat suppressed images (TR 46\u00a0ms; TE 2.5\u00a0ms; NEX 1; flip angle 40\u00b0; 3.0\u00a0mm slice thickness; slice overlap 1.5\u00a0mm; no gap; 180\u00a0mm (FOV); 256 acquisition matrix, 80 slices); and axial proton density and T2-weighted turbo spin echo (TSE) fat suppressed images (TR 2,500\u00a0ms; TE 7.1\/40\u00a0ms; NEX 2; 2\u00a0mm slice thickness; no gap; 180\u00a0mm (FOV); 256 acquisition matrix, 62 slices). Total acquisition time (including the initial survey sequence) was 30 min.\nMR interpretation\nAll MR images were analyzed in known chronological order by means of consensus between three experienced readers, using a comprehensive score form [11]. During the assessment, the readers were blinded to radiographic results, patient symptoms and patient age. In cases of disagreement between the readers the more conservative, less severe score was recorded. BME or cysic lesions was assigned to any one or more of the following anatomic locations: the crista patellae, medial or lateral patellar facets, the medial or lateral trochlear articular facets, the medial or lateral femoral condyles, the medial or lateral tibial plateaux.\nBME lesions were defined as an ill-defined area of increased signal intensity on T2-weighted images in the subchondral bone, extending away from the articular surface over a variable distance [12]. The lesions were graded as follows: grade 0, absent; grade 1, minimal (diameter <5\u00a0mm); grade 2, moderate (diameter 5\u00a0mm\u201320\u00a0mm); grade 3, severe (diameter >20\u00a0mm). The maximum two-dimensional diameter was measured to grade the lesion. A total BME score of the knee was calculated by adding all grades of each BME lesion in the knee. Maximum possible knee score was 27 (grade 3 times nine anatomic locations). In the total study a maximum of 1,638 BME lesions (182 patients times nine anatomic locations) could be scored.\nSubchondral cysts were defined as well-defined foci of high signal intensity, with low signal intensity margins, on T2-weighted images, in the bone underlying the joint cartilage. Their greatest dimension was measured and they were graded as follows: grade 0, absent; grade 1, minimal (<3\u00a0mm); grade 2, moderate (3\u20135\u00a0mm); grade 3, severe (>5\u00a0mm). A total score of the knee was calculated by adding all grades of each cystic lesion in the knee. Maximum possible knee score was 27 (grade 3 times nine anatomic locations). In the total study a maximum of 1638 cystic lesions (182 patients times nine anatomic locations) could be scored.\nStatistical analysis\nOdds ratios (ORs) with 99% confidence intervals (CIs) were used to show any association between BME size changes with cystic size changes.\nThe difference in WOMAC scores between patients without BME lesions (group A) and patients with BME lesions (group B: unchanged BME lesions over 2\u00a0years; group C: increasing size of BME lesions over 2\u00a0years; group D: decreasing size of BME lesions over 2\u00a0years) was calculated by linear mixed models in SPSS for Windows, version 12.0 (SPSS, Chicago, Ill.) with a random intercept to adjust for the familial effect within sib pairs. Adjustments were made for age, sex and body mass index (BMI). Estimates of fixed effects were reported with 95% CIs.\nResults\nIn total 182 patients were monitored over a period of 2\u00a0years (Table\u00a01). Forty-six (25%) patients did not have BME lesions; thus, 136 patients (75%) had one or more BME lesions at any time point. In 46 (34%) of patients, BME scores did not fluctuate, whereas they changed in the other 90 (66%) patients. The total BME score per individual patient increased in 54 (40%) patients. It decreased in 27 patients (20%), and total BME score remained unchanged in nine (7%) patients. Individual BME scores did change in this last group without resulting in a change of the total BME score. A total of 327 BME lesions were detected from a possible total of 1,638 lesions (Table\u00a02). One hundred and forty-seven (45%) BME lesions changed: 69 new lesions appeared on the second MR (21%), 26 (8%) increased, 20 (6%) decreased in size, and 32 (10%) were no longer visible on the second MR scan (Fig.\u00a01). It was noted that more lesions appeared or increased than decreased or disappeared. \nTable\u00a01Patient characteristics (n\u2009=\u2009182)\u00a0At baselineAge years, median (range)59 (43\u201376)Female sex, (%)157 (80%)Body mass index (kg\/m2), median (range)25.7 (20.2\u201340.0)Symptomatic knee OA, n (%)a71 (39%)Kellgren & Lawrence Score 0\/1\/2\/3\/4, No.59\/53\/60\/10\/0Over 2\u00a0yearsBone marrow edema-like lesions, n (%)b128 (70%)Grade 0\/1\/2\/3c54\/56\/62\/10Cysts, n (%)b100 (55%)Grade 0\/1\/2\/3c82\/60\/37\/3At 2\u00a0yearsWOMAC Pain scores, median (range)d13 (0\u201399)WOMAC Stiffness scores, median (range)d18 (0\u201399)WOMAC Function scores, median (range)d14 (0\u201398)aDefined as pain or stiffness on most days of the month prior to study entry, in combination with osteophytes on radiographsbDuring 2\u00a0yearscMaximal grade per patientdn\u2009=\u2009157Table\u00a02Cystic and BME lesions per patient changing over 2\u00a0years in 182 patients (n\u2009=\u20091,638)\u00a0BME lesionsNo BMENo changeIncreaseDecreaseTotalCystsNo cyst1,2807738211,416No change24872417152Increase21130346Decrease5531124Totals1,31118095521,638Fig.\u00a01a\u2013d Axial T2-weighted turbo spin echo fat suppressed images. Increase (a at baseline, b after two years) in size of bone marrow edema-like lesions over 2\u00a0years at the crista patella (arrow) and at the medial femoral condyle (arrowhead). Decrease (c at baseline, d after two years) in size of bone marrow edema-like lesions over 2\u00a0years at the crista and medial part of the patella (open arrowhead)\nOne hundred (55%) patients had one or more cystic lesions at any time. In 44 patients (44%), total cystic score did not fluctuate over time, whereas in the other 56 patients (56%) the total cystic score changed in size. In 36 of these 56 patients (64%) the cystic score increased, it decreased in 18 patients (32%), and the total cystic score remained unchanged irrespective of changes on the level of individual cysts in two patients (4%). A total of 222 cystic lesions were detected from a possible total of 1,638 sites (Table\u00a02). Seventy cystic lesions (32%) changed: 32 new cysts appeared on the second MR (46%), 14 (20%) increased, six (8%) decreased in size, and 18 (26%) were no longer visible on the second MR (Fig.\u00a02).\nFig.\u00a02a, bAxial T2-weighted turbo spin echo fat suppressed images. Disappearing cyst at the lateral femoral condyle (arrow). a At baseline, b after 2\u00a0years\nCystic and BME lesions were both present in the same anatomic location (associated lesions) in 191 cases. A change of BME lesions or cystic lesions was associated with a change in size of an adjacent cystic or BME lesion (OR: 6.2; 95% CI: 3.2\u201312.3). In 47 cases both BME lesions and cystic lesions changed. When cystic and BME lesions were both present in the same anatomic location, size changes (increase or decrease) were in same direction (OR: 37; 95% CI: 6\u2013210) (Table\u00a02).\nOne hundred and fifty-seven (86%) patients completed a WOMAC questionnaire of the imaged knee at 2\u00a0years. The WOMAC pain and function scores for patients without and with BME lesions over 2\u00a0years are shown in Fig.\u00a03a and b, respectively. The mean WOMAC scores did not differ between the different patient groups; even when BME lesions completely disappeared, lower WOMAC scores were not recorded. The mean difference in WOMAC pain scores between patients with unchanged BME lesions, with increasing BME lesions and with decreasing BME lesions compared with patients without BME lesions were 2 (95% CI-8 to 12), 2 (95% CI-8 to 11) and 1 (95% CI-11 to 12) respectively (Fig.\u00a03a). The mean difference in WOMAC function scores between patients with unchanged BME lesions, with increasing BME lesions and with decreasing BME lesions compared to patients without BME lesions were \u22122 (95CI-12 to 8), \u22124 (95CI-13 to 6) and \u22124 (95CI-15 to 8) respectively (Fig.\u00a03b).\nFig.\u00a03Association between WOMAC pain (a) or function (b) scores and bone marrow edema-like lesions. Group A: patients with no bone marrow edema-like lesions; Group B: patients in whom bone marrow edema-like lesions did not change over 2\u00a0years; Group C: patients in whom bone marrow edema-like lesions increased in size over 2\u00a0years; Group D: patients in whom bone marrow edema-like lesions decreased in size over 2\u00a0years. Box plots show the median, interquartile range, minimum and maximum values\nDiscussion\nThe majority (75%) of patients with familial OA at multiple sites have BME lesions visualized when two sequential MR scans are made with a 2-year time interval. In the majority (66%) of these patients with BME lesions, the total size of BME changed over this 2-year time period. Our study also demonstrates that cysts and BME fluctuations are associated. However, no association existed between changes in BME lesions and severity of WOMAC scores after 2\u00a0years.\nThe finding, that BME lesions fluctuate in 66% of the patients, indicates that BME is part of a dynamic process in OA. BME is not a constant finding, as opposed to hyaline cartilage loss for example. Thus, it is important to realize that the finding of BME lesions in patients with OA represents only a single snapshot in time. The variability of BME lesions has been noted before [5]. It is also interesting and important to note that 10% of BME lesions disappear completely. This is particularly noteworthy if BME is to be used as an inclusion criterion, outcome parameter or surrogate endpoint in drug trials or clinical outcome studies.\nThe second finding of the present study is that when cystic lesions and BME lesions are in close proximity; the direction in which they change is identical. This is an interesting finding as the role of cysts in OA is unclear. Their exact pathophysiology is uncertain, as is their prognostic significance. A recent study by Carrino et al. [13] also showed a change in cyst size was accompanied by a change in oedema-like signal size. That study also showed subchondral cysts developing in pre-existing regions of subchondral bone marrow oedema-like signal.\nThe third finding is that changes in BME lesions did not correlate with severity of WOMAC scores. Patients in whom BME increased did not have a higher WOMAC score than patients with a decrease in BME size. Even when BME completely disappeared, lower WOMAC scores were not recorded. Previous work has demonstrated that pain, as assessed by WOMAC scores, was not related significantly to changes in BME lesions [5]. However, in the studies by Felson et al. [2, 7], BME was associated with progressive radiographic knee OA and pain. Cross sectional associations between BME lesions and clinical findings remain controversial [2\u20136]. Hence, a lack of clarity about the relationship between changes in BME lesions and WOMAC scores is not surprising. Phan et al. [5] have suggested that the complexity of pain physiology and the difficulty of pain evaluation may explain these findings, as well as the fact that patients experience pain differently. Another important factor might be the stage of OA in the patients being studied. For instance, pain might be associated with BME lesions in a more developed stage of the disease and less so earlier on. Knee OA studied in the population by Felson [2] was more advanced than the GARP population [8]. Also Felson\u2019s population consisted of patients with knee OA only, whereas the GARP population is focused on patients with familial OA at multiple sites. A considerable proportion of patients in the present study did not have symptomatic radiographic knee OA in the imaged knee, and consequently average WOMAC scores in the present population were low (Table\u00a01). Nevertheless, associations between BME lesions and clinical findings are controversial and BME lesions may be excluded ultimately as a factor in pain sensation.\nThe present study has a number of limitations. Firstly, not all patients with a complete MRI follow-up completed the WOMAC signal knee. Secondly, the inclusion of patients who were first-degree relatives may have introduced an artefact. However, linear regression analysis, with robust standard errors that clustered on pairs, excluded this possibility. Thirdly, although referred pain from the hip may have been a confounder, hip OA occurred in only 7% of the patients and was not thought a contributory factor. Fourthly, 2\u00a0years of follow-up may be too short for an early OA population. However, we did find a considerable change in size of both cystic and BME lesions. Finally, the term BME used in this article probably should be described more properly as \u201cill defined signal intensity abnormalities\u201d, as the so-called BME pattern in OA knees represents on histological examination nonspecific abnormalities such as bone marrow necrosis, bone marrow fibrosis, trabecular abnormalities and only a small amount of bone marrow oedema [1]. Nevertheless, the term BME is a commonly accepted identity and widely used in the OA literature [2].\nIn conclusion, BME lesions are shown to be a variable parameter when followed over time in patients with knee OA and are not predictive of pain.","keyphrases":["oa","bme","knee","womac","mri"],"prmu":["P","P","P","P","P"]}
{"id":"Ann_Surg_Oncol-4-1-2277451","title":"Does Practice Make Perfect?\n","text":"Extensive literature supports the correlation between surgical volume and improved clinical outcome in the management of various cancers. It is this evidence that has catalysed the creation of centres of excellence. However, on closer inspection, many of these studies are poor quality, low weight and use vastly heterogenous end points in assessment of both volume and outcome. We critically appraise the English language literature published over the last ten years pertaining to the volume outcome relationship in the context of cancer care. Future balanced unbiased studies may enable equipoise in planning international cancer management strategies.\nNo longer is there room for eminence-based complacency or misguided arrogance in healthcare delivery. The day of the autonomous clinician is gone with a vogue towards standardised, evidence-based clinical excellence. Cynics would erroneously attribute this to a parallel increase in litigation but increasing patient knowledge and expectations with a move toward subspecialisation are the main catalysts driving change. When offered operative intervention, the question frequently asked by the patient is \u201cHow many of these have you done before?\u201d This article aims to critically analyse recent literature and explore the correlation between volume and clinical outcome in the context of cancer care.\nBackground\nMortality rates are reported to be influenced by the number of particular operations performed in a given hospital or by a specific surgeon (i.e. outcomes are better in high-volume centres). In 1999, the US National Cancer Policy Board recommended that patients requiring complex procedures be transferred from low- to high-volume hospitals in its report entitled \u201cEnsuring Quality Cancer Care\u201d.1 The following year, the Institute of Medicine held a workshop to discuss cancer care, publishing a document (\u201cInterpreting the Volume-Outcome Relationship in the Context of Cancer Care\u201d2) which concluded that existing evidence was strong enough to recommend the regionalisation of high-risk operations. Hence, the impact of volume on outcome has been assessed in several tumour types but the majority of data relates to gastrointestinal, hepatobiliary, urological, and breast cancers.\nThe literature\nPatients undergoing pancreatic and oesophageal procedures have lower operative mortality and shorter hospital stay in the hands of experienced surgeons in high-volume units.3 With regard to gastric cancer, the findings are less consistent. Some suggest that high-volume centres have lower in-house mortality4 but no change in long-term survival.5 Various end points have been examined in colorectal cancer (Table\u00a01). Schrang and Billingsley both showed that it was surgeon-specific experience combined with multidisciplinary support rather than centre experience that afforded significant survival advantage.6,7Table\u00a01.Colorectal cancer outcome: high- and low-volume unitsHigh volumeLow volumeRef.APR versus LAR\u2193 APR\u2191 APRMeyerhardt et\u00a0al. 19\u2191 LAR\u2193 LARSphincter preservation\u2191 \u2191\u2193Purves et\u00a0al.2030-day postoperative mortality\u2194\u2194Schrag et\u00a0al.21Survival (overall and cancer specific)\u2191\u2193Schrag et\u00a0al.21Permanent stoma formation\u2193\u2191McGrath et\u00a0al.22Colonic pouch formation\u2191\u2193McGrath et\u00a0al.22APR, abdominoperineal resection; LAR, low anterior resection.\u2191 increased, \u2193 decreased, \u2194 no variation.\nComprehensive albeit retrospective studies unequivocally state that patients who undergo radical prostatectomy at lower-volume institutions are at significant risk of requiring adjuvant therapy due to adverse surgical factors, prolonged hospital admission, increased hospital charges and postoperative complications.8 The hospital structure of high-volume units, including easy availability of consultative, diagnostic and ancillary services, were cited as likely contributors to the association between procedure volume and short-term cystectomy outcomes.9 A minimum case load of only 11 radical cystectomies per year was cited to be associated with the lowest mortality rate.10\nLess certainty exists in relation to breast cancer but a variety of end points have been examined, including number of visits required to obtain a nonoperative diagnosis, mastectomy rates for <15\u00a0mm tumours and rate of referral for adjuvant radiotherapy.11 Skinner et\u00a0al. were adamant that volume alone could not be used as a surrogate for expertise but, like most studies, conceded that patients with breast cancer operated on in high-volume units compared to very low-volume units had better survival. However there is ambiguity surrounding the exact causal relationship: volume effect alone versus surgical skill versus appropriate use of adjuvant therapy.12\nThe Flaws\nThe majority of studies are poor quality, heterogenous and potentially flawed. Even the best literature stems from retrospective review of large databases which are up to 40% inaccurate13 and pertain to selected patient groups. Most papers are published from a few very high-volume US centres, thus introducing immediate potential for bias. No consistent end point is used and, incredibly, few studies explore cancer-specific outcomes. Mortality rates, when not corrected for comorbidities or stage at diagnosis, are poor surrogates for more robust comparators of volume\u2013outcome analysis such as cancer-specific survival, patient satisfaction and quality of life.\nAmerican data cannot be used as a basis for the formation of European cancer strategies because so many inherent differences exist. These include earlier disease stage at time of operation (a function of screening),14 wide variance in population15 and socioeconomic status16 as well as insured versus noninsured outcomes.17 Comparison of very high-volume centres and very low-volume centres is as redundant as measuring revenue from a supermarket versus a corner store. Is it not intuitive that analysis of such extremes will yield vastly different results? Looking at the structure\u2013process\u2013outcome model, structure is relatively fixed (health service, institution) but process is entirely variable and volume is but one component of it. Therefore, in the absence of multivariable data, subgroup analysis is entirely inappropriate, rendering the aforementioned studies at best biased, at worst invalid.\nThe Surgeon\nTo identify procedure volume in a single institution is easy but not so for quantifying specific operations done by a single surgeon, many of whom practise in several centres. Without asking individuals to record caseload prospectively, obtaining accurate figures may be very difficult. Surely significant bias could occur in that those willing to share details of operations may be confident of their own justification in performing such procedures. Furthermore, who counts as more experienced: a mature surgeon who has performed two colonic resections a week for 20\u00a0years but now does only two a month or a younger, specialist trained surgeon who performs three a week? How do you weight lifetime experience against current volume? Should a centralised database of surgeons\u2019 logbooks exist, and should permission to operate be granted or denied based on this? What governing body should be afforded such a task? Should operating surgeons be ranked in their ability or would this border on defamation for the less fortunate ones? Regarding referral pattern, does volume attract quantity or do excellent clinicians attract patients? Many studies have compared the significance of surgeon experience to unit volume with varied conclusions as to which has more impact. Do excellent surgeons naturally aggregate in excellent high-volume units, thus giving a self-propagating explanation for improved outcome?\nThe Institution\nMany papers allude to the importance of the multidisciplinary approach in cancer care. Higher-volume units are far more likely to have subspecialised radiologists, radiation and medical oncologists, high dependency and intensive care units, cancer specialist nurses, dedicated psychologists and palliative care support. Anecdotally, the involvement of such services translates into better patient outcome regardless of unit volume, thus confounding results. The resounding evidence in favour of the volume\u2013outcome relationship pertains to those cancers requiring adjuvant therapy: oesophagus, pancreas and advanced colorectal. It seems, therefore, that good surgical technique or individual surgeon experience do not exclusively guarantee positive outcomes and that much depends on availability of radiation and medical oncology.\nMore often than not, within a short period of the introduction of a new service, its resources are saturated. At what point does a dedicated unit declare that available services can no longer provide for patient throughput? The worry is that potential exists for patients to receive suboptimal care just before this saturation point is reached (Fig.\u00a01). Surely stretched resources in a high-volume centre are just as dangerous as absence of resources in smaller institution. A study on the difference in time lapse between diagnosis and intervention between institutions with different volumes would be welcome. Would prognosis be improved if a patient with aggressive disease underwent early intervention at a lower-volume centre rather than late intervention at a higher-volume centre? As cancer management is a dynamic process, the question of safety in transferring postoperative follow-up of a patient operated on in a high-volume unit to a less experienced lower-volume local centre is a pertinent one. On the other hand, should surgery be performed in low-volume units and adjuvant therapies in specialist centres? Without a doubt, the idea of hospital units functioning as a syncitium rather than single buildings must be engendered and skill-appropriate tasks assigned to each component, as recommended in breast cancer management by the Clinical Oncology Information Network (COIN) group.18Fig.\u00a01.Volume\u2013outcome relationship.\nThe Patient\nQuality of life and patient satisfaction, apart from in colorectal and prostate cancers, are largely ignored when assessing impact of volume on clinical outcome. Is it preferential for elderly patients to travel long distances for management of a low-grade tumour or would they be better served by treatment in a lower-volume local centre where follow-up will be on their doorstep? Decisions like this are made on a daily basis in the context of primary care and so this begs the question of higher-volume specialist centres receiving selected patient cohorts, thus falsely improving outcome. This is confirmed by Morris et\u00a0al. who showed that patients treated in the Australian private sector were likely to be younger, male and to have an earlier disease stage.17\nThe Future\nThe literature supports a correlation between surgical volume and improved clinical outcome in cancer care. However, a rather simplistic approach is evident in many studies and much potential remains for unbiased, prospective, statistically sound investigations with the aim of numerically stratifying appropriate volume and its impact on disease specific cancer outcomes.\nWhile traditionally graph 1 in Fig.\u00a01 has been used to represent the volume\u2013outcome relationship, it seems probable that it is more accurately represented by graph 2 in Fig.\u00a01, and can be interpreted as an area under a curve rather than a strictly linear relationship.","keyphrases":["volume","outcome","cancer"],"prmu":["P","P","P"]}
{"id":"Pflugers_Arch-3-1-1915585","title":"The endothelial glycocalyx: composition, functions, and visualization\n","text":"This review aims at presenting state-of-the-art knowledge on the composition and functions of the endothelial glycocalyx. The endothelial glycocalyx is a network of membrane-bound proteoglycans and glycoproteins, covering the endothelium luminally. Both endothelium- and plasma-derived soluble molecules integrate into this mesh. Over the past decade, insight has been gained into the role of the glycocalyx in vascular physiology and pathology, including mechanotransduction, hemostasis, signaling, and blood cell\u2013vessel wall interactions. The contribution of the glycocalyx to diabetes, ischemia\/reperfusion, and atherosclerosis is also reviewed. Experimental data from the micro- and macrocirculation alludes at a vasculoprotective role for the glycocalyx. Assessing this possible role of the endothelial glycocalyx requires reliable visualization of this delicate layer, which is a great challenge. An overview is given of the various ways in which the endothelial glycocalyx has been visualized up to now, including first data from two-photon microscopic imaging.\nIntroduction\nThe endothelial glycocalyx was already visualized some 40\u00a0years ago by Luft using electron microscopy [66]. Still, relatively little is known of the composition and function of this layer. Over the past decades, it has been increasingly appreciated as an important factor in vascular physiology and pathology, as described in 2000 in a review by Pries et al. [86] and in other, more recent reviews [4, 74]. The interest in the (patho)physiological role of the glycocalyx started with the observation of low and variable capillary tube hematocrit, which depended on the level of metabolic and pharmacological activation of the vascular system [51, 52, 63, 83, 101]. The relation between metabolic and agonist-induced increases in red blood cell velocity on the one hand and tube hematocrit on the other could partly be explained by plasma skimming as direct continuation of the F\u00e5hraeus effect [83]. However, this relation dissociated upon local treatment of the microvessels with heparinase, an enzyme which breaks down heparan sulfates in the glycocalyx [17]. This finding was in agreement with theoretical estimates predicting a 1.2-\u03bcm thick slow-moving plasma layer over the endothelium [51]. In vivo studies have revealed the glycocalyx in muscle capillaries to be a layer of about 0.5\u00a0\u03bcm thick, covering endothelial cells and determining luminal domains for macromolecules, red and white blood cells [129]. More recent studies indicate that glycocalyx thickness increases with vascular diameter, at least in the arterial system, ranging from 2 to 3\u00a0\u03bcm in small arteries [125] to 4.5\u00a0\u03bcm in carotid arteries [67].\nTo date, many studies indicate a variety of (patho)physiological roles for the endothelial glycocalyx; in addition to modulating capillary red blood cell filling, the glycocalyx may affect many other (dys)functions of the vascular system. Whereas the vascular endothelium is currently believed to be actively involved \u201cin every pathology presenting vascular projections\u201d [28], the same saying might well prove true for the glycocalyx. Assessing this possible involvement of the endothelial glycocalyx requires reliable visualization of this delicate layer, which is a great challenge. This review provides basic insight into the present knowledge of composition and functions of the endothelial glycocalyx and gives an overview of the various ways in which it has been visualized up to now.\nComposition\nThe endothelial glycocalyx is a carbohydrate-rich layer lining the vascular endothelium. It is considered to be connected to the endothelium through several \u201cbackbone\u201d molecules, mainly proteoglycans and also glycoproteins. These form a network in which soluble molecules, either plasma- or endothelium-derived, are incorporated. More luminally, the glycocalyx is formed by soluble plasma components, linked to each other in a direct way or via soluble proteoglycans and\/or glycosaminoglycans (which will be discussed below). A dynamic equilibrium exists between this layer of soluble components and the flowing blood, continuously affecting composition and thickness of the glycocalyx. Furthermore, the glycocalyx suffers from enzymatic or shear-induced shedding. The dynamic balance between biosynthesis and shedding makes it hard to define the glycocalyx geometrically [62]. The composition of the membrane-bound mesh of proteoglycans, glycoproteins, and glycosaminoglycans and the composition of associated plasma proteins and soluble glycosaminoglycans cannot be viewed as a static picture. Instead, the layer as a whole\u2014also known as endothelial surface layer (ESL) [86]\u2014is very dynamic, with membrane-bound molecules being constantly replaced and no distinct boundary between locally synthesized and associated elements; membrane-bound hyaluronan may reach lengths of >1\u00a0\u03bcm. Direct visualization techniques (see section on Visualization techniques) fail to demonstrate clear compositional differences within the glycocalyx, from endothelial membrane towards vascular lumen, but rather indicate that the endothelial glycocalyx resembles an intricate, self-assembling 3D mesh of various polysaccharides. Enzymatic removal of any of its constituents dramatically affects glycocalyx properties, which exemplifies the importance of considering the synergetic interaction of all glycocalyx constituents as a whole. In this review we will, therefore, use the term (endothelial) glycocalyx for the total layer (Fig.\u00a01) and be as specific as possible when addressing its various elements.\nFig.\u00a01Schematic representation of the endothelial glycocalyx, showing its main components. Left: The endothelial glycocalyx can be observed in vivo as a red blood cell exclusion zone, located on the luminal side of the vascular endothelium. It consists of membrane-bound and soluble molecules. Right: Components of the endothelial glycocalyx. Bound to the endothelial membrane are proteoglycans, with long unbranched glycosaminoglycan side-chains (GAG-chain) and glycoproteins, with short branched carbohydrate side-chains. Incorporated in and on top of this grid are plasma and endothelium-derived soluble components, including hyaluronic acid and other soluble proteoglycans (e.g., thrombomodulin) and various proteins, such as extracellular superoxide dismutase (ec-SOD) and antithrombin III (AT III). Together, these components form the endothelial glycocalyx that functions as a barrier between blood plasma and the endothelium and exerts various roles in plasma and vessel wall homeostasis. Note that this figure is not drawn to scale; its purpose is to illustrate glycocalyx composition\nBelow, state-of-the-art knowledge on the various components of the endothelial glycocalyx will be provided. Although many molecules have been identified as being part of the glycocalyx, information on their distribution is still scarce; if present, such knowledge was mostly obtained indirectly and nonquantitatively.\nProteoglycans\nProteoglycans are generally considered to function as the most important \u201cbackbone\u201d molecules of the glycocalyx. They consist of a core protein to which one or more glycosaminoglycan chains are linked. There is a notable variation among the proteoglycan core proteins with regard to their size, number of attached glycosaminoglycan chains, and whether or not they are bound to the cell membrane (Table\u00a01). The core protein groups of syndecans (n\u2009=\u20094) and glypicans (n\u2009=\u20096) have a firm connection to the cell membrane via a membrane-spanning domain (syndecans) or a glycosylphosphatidylinositol anchor (glypicans) [12, 25]. The other proteoglycans, such as mimecan, perlecan, and biglycan, are secreted after their assembly and glycosaminoglycan chain modification [44, 50]. This leads to production of soluble proteoglycans, which reside in the glycocalyx or diffuse into the blood stream. \nTable\u00a01Characteristics of proteoglycan core proteins in the vascular endothelial glycocalyxCore protein groupCore protein size (kDa)Number of subtypesNumber of GAG-chains linkedType of GAG-chains linkedStructural relation to cell membraneSyndecan19\u20133545HS\/CSMembrane-spanningGlypican57\u20136963HS\/CSGPI-anchorPerlecan40013HS\/CSSecretedVersican370110\u201330CS\/DSSecretedDecorin4011CS\/DSSecretedBiglycan4012CS\/DSSecretedMimecan3512\u20133KSSecretedGAG Glycosaminoglycan, HS heparan sulfate, CS chondroitin sulfate, DS dermatan sulfate, KS keratan sulfate, GPI glycosylphosphatidylinositol\nProteoglycans are promiscuous in their binding of glycosaminoglycan chains, meaning that one core protein can contain different types of glycosaminoglycan chains. The proportion of the various chains may change under different circumstances and stimuli [89]. Therefore, naming of proteoglycans after one type of glycosaminoglycan is somewhat deluding. For example, syndecan-1 proteoglycan is often addressed as a heparan sulfate proteoglycan, while in fact, it usually contains similar numbers of heparan sulfate and chondroitin sulfate chains [70].\nThere are five types of glycosaminoglycan chains: heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, and hyaluronan (or hyaluronic acid). They are linear polymers of disaccharides with variable lengths that are modified by sulfation and\/or (de)acetylation to a variable extent. The disaccharides are each composed of a uronic acid and a hexosamine; classification of the glycosaminoglycans depends on which uronic acid or hexosamine is incorporated and on the pattern of sulfation (Table\u00a02). Each of the five glycosaminoglycans has been investigated and reviewed extensively [22, 27, 57, 58, 114]. Dermatan sulfate is often regarded as a separate class of glycosaminoglycans, although it actually is type B chondroitin sulfate. The difference between the two is possible epimerization of the glucuronic acids into iduronic acids in dermatan sulfate, which has important consequences for functionality. Whenever possible, we will try to separate the two as precisely as possible; elsewhere, they will be referred to as chondroitin sulfate\/dermatan sulfate. \nTable\u00a02Composition of the disaccharides of various glycosaminoglycan chains\u00a0Heparan sulfateChondroitin sulfateDermatan sulfateaHyaluronanKeratan sulfateUronic acidGlcA(2S) IdoA(2S)GlcAGlcA IdoA(2S)GlcAGal(6S)Disaccharide link1\u03b241\u03b231\u03b231\u03b231\u03b24HexosamineGlcNAc(NS)(3S)(6S)GalNAc4Sa GalNAc6SaGalNAc(4S)(6S)GlcNAcGlcNAc(6S)Polymerization link1\u03b241\u03b241\u03b241\u03b241\u03b23Note the various possibilities of sulfation in heparan sulfate. These may coincide (e.g., in heparan sulfate the hexosamine GlcNS3S). A rare but possible hexosamine in heparan sulfate is the N-unsubstituted glucosamine  which has been left out of the table for convenient reading. Also note the presence of IdoA in dermatan sulfate, in contrast to the other chondroitin sulfates, making it more alike to heparan sulfate.GlcA Glucuronic acid, IdoA iduronic acid, Gal galacturonic acid, GlcNAc N-acetyl-glucosamine, GalNAc N-acetyl-galactosamine, 2S 2-O-sulfated, 3S 3-O-sulfated, 4S 4-O-sulfated, 6S 6-O-sulfated, NS N-sulfatedaThere are three types of chondroitin sulfate. Type A only has 4-O-sulfated N-acetyl-galactosamines, type B is known as dermatan sulfate and type C only has 6-O-sulfated N-acetyl-galactosamines.\nIn vasculature, heparan sulfate proteoglycans represent roughly 50\u201390% of the total amount of proteoglycans present in the glycocalyx [43, 86]. However, this figure is variable, as the expression of proteoglycans by endothelial cells depends on various stimuli. Syndecans, for example, have a tightly regulated expression pattern which varies with endothelial cell activation or stimulation with different chemokines [119]. The second most common glycosaminoglycan in the endothelial cell glycocalyx is chondroitin sulfate\/dermatan sulfate. The presence of heparan sulfate and chondroitin sulfate is reported to have a typical ratio of 4:1 for the vascular endothelium [70, 90]. Expression of keratan sulfate glycosaminoglycans in vasculature and its importance in (patho)physiology is less well understood. Another important glycosaminoglycan in the glycocalyx is hyaluronan. This long polymeric molecule (up to 104\u00a0kDa) differs from other glycosaminoglycans in that it is not linked to a core protein. Its exact link to the cell membrane is unknown, but it can be bound to the receptor CD44 [72]. Alternatively, hyaluronan may be attached to its assembly proteins, the hyaluronan synthases [135], which are located at the cytosolic side of the cell membrane. Another possibility is that hyaluronan is (in part) not directly bound to the membrane at all. It is capable of forming strikingly viscous solutions [57]. More recently, intracellular hyaluronan binding proteins such as cdc37 [31] and P32 [15] have been identified, suggesting a role for this glycosaminoglycan within the cell [23, 58].\nHeparan sulfate and chondroitin sulfate\/dermatan sulfate containing proteoglycans are produced in the endoplasmic reticulum and Golgi apparatus of the endothelial cell. After the ribosomal translation of the core protein, a xylosyltransferase will transfer xylose (Xyl) from uracildiphosphate xylose to specific serine residues (Ser) in the core protein. The xylose-enriched core protein is transported to the cis-Golgi, and galactosyltransferases types I and II will add two galactose-groups (Gal) to the xylose, after which glucuronosyltransferase type I adds glucuronic acid, thus, completing the primary linker for glycosaminoglycan chains: \u2013GlcA\u2013\u03b23\u2013Gal\u2013\u03b23\u2013Gal\u2013\u03b24\u2013Xyl\u03b23\u2013[Ser].\nAfter formation of the primary linker, the following step determines the type of glycosaminoglycan chain that will be formed. In the case of heparan sulfate, an \u03b14-glucosamine is added; in the case of chondroitin sulfate and dermatan sulfate, a \u03b24-galactosamine is added, and both galactose-residues from the linker may be sulfated. From this point onward, glucuronic acids and glucosamines are linked to the core protein. After chain polymerization, the growing glycosaminoglycan chain will undergo modifications including N-sulfation, O-sulfation, and epimerization. The latter changes glucuronic acid residues into iduronic acid residues and will change any chondroitin sulfate chain into its type B dermatan sulfate. These chain modifications take place in both the cis- and the trans-Golgi, determining the final type and functionality of the proteoglycan and each of its side chains. In contrast, hyaluronan is assembled at the cytosolic side of the cell membrane, and it is not modified afterwards. As a consequence, it has no sulfate groups or modification pattern.\nAs glycosaminoglycan chains contain numerous specific binding sites for plasma-derived proteins, small chain modifications can have great functional consequences. Sequential enzymatic modifications of the individual saccharide units within glycosaminoglycan chains endow proteoglycans with unique functions. Typically 16\u201348 different sulfation patterns may exist per disaccharide and as functional domains are assumed to be usually penta- to deca-saccharide long, at least 163\u2009=\u20094,096 different sulfation patterns on a hexasaccharide backbone are theoretically possible, and such a structural diversity corresponds to diversified biological functions of glycosaminoglycans. Indeed, it has been shown that modification patterns vary in time and under different (patho)physiological stimuli [4, 131]. The diversity of glycosaminoglycan sulfation patterns and its effect on specific protein binding and modulation of protein function suggest that conditions that diminish glycocalyx thickness or modulate protein specific glycosaminoglycan sulfation patterns and charge are likely to modulate vascular permeability and alter specific protein binding and activity.\nGlycoproteins\nBesides the proteoglycans with their long linear side chains, certain glycoproteins are also regarded as \u201cbackbone\u201d molecules, connecting the glycocalyx to the endothelial cell membrane. This group of endothelial glycoproteins, characterized by relatively small (2\u201315 sugar residues) and branched carbohydrate side chains, comprises a number of molecules that all have been studied intensively; major classes that will be discussed in more detail below are the endothelial cell adhesion molecules and components of the coagulation and fibrinolysis system. It is beyond the scope of this review to categorically discuss all glycoproteins that can be expressed by endothelial cells. Furthermore, one should realize that the level of glycoprotein expression on the endothelial cell membrane varies considerably with cell activation or stimulation.\nThe endothelial cell adhesion molecules are well-defined glycoproteins that play a major role in cell recruitment from the bloodstream and in cell signaling. The three families of cell adhesion molecules present in the endothelial glycocalyx are the selectin family, the integrin family, and the immunoglobulin superfamily.\nGlycoproteins from the selectin family contain a cytoplasmic tail, a transmembrane domain, several consensus repeats, an epidermal growth factor-like domain, and a terminal lectin domain, which is primarily responsible for binding of carbohydrate groups on glycosylated proteins or lipids. However, the epidermal growth factor-like domain is involved in selectin-ligand recognition as well [29, 48]. Selectins found on the vascular endothelium are E-selectin and P-selectin, both involved in leukocyte\u2013endothelial cell interactions [108]. P-selectin is constitutively produced and subsequently stored in the Weibel\u2013Palade bodies of the endothelial cells. Exocytosis of Weibel\u2013Palade bodies as induced by stimuli such as thrombin and histamine allows a rapid translocation of P-selectin to the cell surface [19, 53]. However, this expression is short-lived due to P-selectin internalization and redirection to lysosomal granules or the Golgi apparatus, where it is restored in newly formed Weibel\u2013Palade bodies [109]. E-selectin is not stored in granules, but requires de novo mRNA and protein synthesis to be expressed on the cell surface. Stimulation of endothelial cells by cytokines such as interleukin-1, tumor necrosis factor \u03b1, and lipopolysaccharide upregulates E-selectin expression; this typically requires 2\u20136\u00a0h [47]. In some tissues like the skin, P- and E-selectin appear to be constitutively expressed on endothelial cells [78, 139].\nIntegrins are heterodimeric molecules, composed of non-covalently bound \u03b1 and \u03b2 subunits. Both subunits have a cytoplasmic tail and a transmembrane domain, and together, they constitute an integral membrane protein. To date, 18 different \u03b1-subunits and 8\u00a0\u03b2-subunits have been identified, which means that each integrin is characterized by the specific combination of its subunits [142]. Integrins are found on many cell types, including endothelial cells, leukocytes, and platelets. In their luminal membrane, endothelial cells express integrin \u03b1V\u03b23, which is an important mediator of platelet\u2013endothelial cell interactions [11]. Most other endothelial cell integrins are involved in binding to the basement membrane. These integrins, such as \u03b12\u03b21, \u03b15\u03b21, and \u03b16\u03b21, bind to multiple extracellular matrix ligands, and are as such, responsible for interactions with laminin, fibronectin, and collagen. Many studies have focused and still focus on the interactions between these integrins and the subendothelial matrix during angiogenesis [98].\nThe immunoglobulin superfamily of glycoproteins is characterized by a cytoplasmic tail, a transmembrane domain, and a variable number of immunoglobulin-like domains that protrude luminally. Best known examples are intercellular adhesion molecule 1 and 2 (ICAM-1 and -2), vascular cell adhesion molecule 1 (VCAM-1), and platelet\/endothelial cell adhesion molecule 1 (PECAM-1), which act as ligand for integrins on leukocytes and platelets and are crucial mediators of leukocyte homing to the endothelium and subsequent diapedesis. ICAM-1 and -2 and PECAM-1 have a baseline expression, whereas VCAM-1 is only present after endothelial cell stimulation by cytokines, which also upregulates ICAM-1 expression [71]. Paradoxically, the constitutive expression of PECAM-1 is decreased after cytokine treatment [113]. The role of ICAM-2 in inflammation is still unclear, as it is also downregulated by inflammatory stimuli. Recently, it was shown that ICAM-2 is involved in regulation of angiogenesis [41].\nBesides the cell adhesion molecules, the endothelial glycocalyx harbors glycoproteins with functionality in coagulation, fibrinolysis, and hemostasis. A good example is the glycoprotein Ib-IX\u2013V complex, which is expressed on endothelial cells and also on platelets. It consists of four glycoproteins: Ib\u03b1, Ib\u03b2, IX, and V, that are each membrane-spanning polypeptides. Glycoprotein Ib\u03b1 and Ib\u03b2 are covalently linked via a disulfide group, whereas IX and V are non-covalently attached to the Ib heterodimer. The Ib-IX\u2013V complex binds von Willebrand factor (vWf) and is primarily known as the platelet vWf-receptor [65, 99]. Furthermore, the complex also binds P-selectin, mediating the interaction of platelets with activated endothelial cells [7]. Like platelets, endothelial cells express all components of the Ib-IX\u2013V complex [115, 141], which on one hand, allows binding to the vWf substrate of the subendothelium, and on the other, binds Weibel\u2013Palade body derived vWf, secreted luminally by activated endothelial cells.\nSoluble components\nEmbedded within and layered on top of the mesh of proteoglycans and glycoproteins are soluble components of various types such as proteins and soluble proteoglycans. These components are either derived from the endothelium or from the bloodstream, such as albumin and orosomucoid, which are pivotal in preserving the (charge-)selectivity of the permeability barrier [42]. The soluble components of the glycocalyx contribute greatly to the functional importance of the glycocalyx, as will be described below. The structural attribution of these soluble components is less well established. At least some of the soluble components may contribute to the structural organization of the luminal glycocalyx, although this is hard to prove. There are indications that proteoglycans bind to each other and to proteins [54, 88]. Protein\u2013glycosaminoglycan\u2013protein complexes have been identified, although not in the glycocalyx in particular [145]. Still, it is presumable that interactions between membrane-bound proteoglycans, soluble proteins, and soluble proteoglycans create a cross-linked mesh and provide some stability to the luminal glycocalyx. Hyaluronan might play an important role in this respect, being a very large linear molecule and possibly unbound to the endothelial cell membrane (Fig.\u00a01). It has been shown to interact with itself, forming stable hyaluronan\u2013hyaluronan complexes [103, 104]. Still, the glycocalyx is a delicate layer, and removal of one specific component may result in loss of function of the total [117].\nFunctional importance\nThe endothelial glycocalyx as the endothelial gatekeeper\nLocated between the blood stream and the endothelium, the endothelial glycocalyx is an important determinant of vascular permeability [35, 130]. It is able to limit access of certain molecules to the endothelial cell membrane, as has been demonstrated in small rat mesenteric arteries with the use of fluorescently labeled dextrans of various molecular weights, showing increasing permeability for smaller molecules [125]. Enzymatic (partial) removal and subsequent loss of permeability barrier function of the glycocalyx in rat myocardial capillaries leads to myocardial edema [124]. Not only size and steric hindrance play a role in glycocalyx dependent permeability, but also, electrostatic charges of the glycocalyx and the permeating substance. With many of the glycosaminoglycan chains being highly sulfated, the glycocalyx presents a net negatively charged surface to the bloodstream. Accordingly, neutralization of the glycocalyx induces an increase in albumin uptake by cultured endothelial cells [121] and an increased permeability for fluorescently labeled dextrans in rat mesenteric arteries [126].\nThe classical model used to describe microvascular fluid exchange is that of Starling [112], stating that fluid filtration rate across capillary endothelium is determined by the hydraulic and colloid osmotic pressures in the vascular lumen and in the surrounding tissue. This balance has been applied across the entire transendothelial barrier, with the different pressures being assessed globally. The discovery of a relatively thick endothelial glycocalyx and its influence on, e.g., edema formation has led to a major revision of the Starling principle by Weinbaum [137] and Michel [68], who proposed to apply the pressure gradients to the endothelial glycocalyx only. Hu and Weinbaum used this idea to generate a 3-D model of permeability over different regions of the endothelial layer, such as the glycocalyx, the endothelial clefts, and the tight junctions [40]. Recently, this model was simplified into a 1-D description of the varying tissue concentration gradients and subsequent permeability of the endothelium [144]. Another model, known as the glycocalyx-junction-break model, applies the Starling mechanism over the glycocalyx and describes its effects on solute and water transport over the endothelium, based on theoretical \u201cpores\u201d in the endothelium [79]. Recently, Curry has written a review on this model and the influences of phenotypical changes on microvascular permeability [14]. The importance of the endothelial glycocalyx in controlling extravasation of colloids and fluids is also stressed by studies of Rehm et al. [91] and Jacob et al. [45], who show impaired endothelial barrier function after glycocalyx degradation in an isolated, perfused heart model. Infusion of 5% albumin or 6% hydroxethyl starch, a natural and an artificial colloid, led to decreased fluid extravasation. However, after 20\u00a0mins of warm ischemia, only albumin infusion prevented vascular leakage. This underscores the importance of an intact glycocalyx and the role of plasma-derived proteins for competent glycocalyx functioning. The revised Starling principle has provided more detailed insight into vascular permeability and stresses the importance of the endothelial glycocalyx as a major determinant.\nBesides its capacity to restrict molecules from reaching the endothelium, the glycocalyx also influences blood cell\u2013vessel wall interactions. It repulses red blood cells from the endothelium. In the microcirculation, a red blood cell exclusion zone flanking the endothelium can be observed in vivo, which is decreased upon light dye-induced breakdown of the glycocalyx [129]. Similarly, platelets are not often observed interacting with the endothelium in control conditions, whereas partial glycocalyx removal by infusion with oxidized low-density lipoprotein (ox-LDL) is accompanied by an increase in platelet\u2013vessel wall interactions [128]. The role of the glycocalyx in leukocyte\u2013vessel wall interactions seems dual: on the one hand, it harbors the endothelial cell adhesion molecules, such as P-selectin, ICAM-1, and VCAM-1; on the other hand, it attenuates adhesion of leukocytes to these molecules.\nIn healthy mouse cremaster muscle venules, breakdown of heparan sulfate side chains through heparitinase leads to an increase in leukocyte adhesion to the endothelium in a dose-dependent manner [13]. Administration of ox-LDL or TNF\u03b1 also induces leukocyte rolling and adhesion [13, 36]. Steric hindrance seems to play a role in this process. The endothelial glycocalyx is much thicker (ranging from 0.2\u20130.5\u00a0\u03bcm in capillaries [124] to 2\u20133\u00a0\u03bcm in small arteries [125] and 4.5\u00a0\u03bcm in carotid arteries [67]) than the length of the various cell adhesion molecules. P-selectin, for example, the molecule initiating leukocyte rolling, only extends about 38\u00a0nm from the endothelial surface [111]. On cultured cells transfected with P-selectin constructs with two to six consensus repeats that are consequently shorter than normal P-selectin (nine consensus repeats), the number of attaching neutrophils decreased with decreasing P-selectin length. Cells defective of glycosylation, having a thinner glycocalyx, showed increased attachment of neutrophils [81]. Hence, in normal conditions, the glycosaminoglycan chains and soluble components of the glycocalyx seem to shield adhesion molecules, thereby, preventing interaction. Stimuli which degrade the glycocalyx or induce a more open mesh, such as enzymes, cytokines, or ischemia and reperfusion, appear to uncover the adhesion molecules, which in turn, allows blood cells to interact with the endothelium [13, 36, 70, 128]. One should realize that ligands for endothelial adhesion molecules are not uniformly distributed over the cell membrane of leukocytes, but show association with microvilli and membrane ruffles [102]. As the glycocalyx is estimated to have a low stiffness [33, 106, 138], it is likely that the ligand bearing leukocyte membrane extensions protrude relatively easily into the glycocalyx to reach their receptor and enable leukocyte\u2013vessel wall interaction. In contrast to other tissues, leukocyte\u2013vessel wall interactions occur spontaneously in venules in the skin, even in normal conditions without preceding trauma or inflammation [46, 78]. Whether this is associated with a deviating glycocalyx structure and\/or composition in skin microvessels remains to be elucidated.\nThe presence of a relatively thick endothelial glycocalyx in vivo has great consequences for rheology, especially in the microvasculature [62, 84]. In this part of the circulation, local blood viscosity and hematocrit appear to be modulated by the glycocalyx. Using a physical model, based on hemodynamic and hematocrit measurements in microvascular networks in vivo, Pries and Secomb recently demonstrated that incorporation of realistic estimates of glycocalyx dimensions in reconstructed mesenteric microvascular networks introduces about a twofold increase in the apparent viscosity of blood. These changes are sufficient to minimize discrepancies between experimentally determined and theoretically predicted microvascular network resistances in previous studies, which were based on the apparent viscosity of blood in glycocalyx-devoid glass capillaries [85].\nThe endothelial glycocalyx as mechanotransducer\nThe endothelium is exposed to mechanical forces induced by blood flow. It has long been recognized that these forces, in particular, shear stress, determine endothelial cell morphology and function [16, 18]. Endothelial cells exposed to shear stress produce nitric oxide (NO) [96], which is an important determinant of vascular tone. However, the molecule(s) responsible for the translation of biomechanical forces into biochemical signals (mechanotransduction) have not been identified as yet. Recently, the glycocalyx has been added to the list of possible candidates. In studies with cultured endothelial cells, Florian and colleagues [24] showed that treatment with heparitinase to specifically break down heparan sulfate glycosaminoglycans results in inadequate responses to shear variations and impaired NO production. Similarly, ex vivo experiments on canine femoral arteries conducted by Mochizuki and coworkers exhibited reduced shear-induced NO production after infusion with hyaluronidase, which degrades the hyaluronan glycosaminoglycans in the glycocalyx [69]. Thus, both heparan sulfate and hyaluronan appear to play a role in detecting and amplifying flow-induced shear forces [69].\nInterestingly, exposure of human umbilical vein endothelial cells to shear stress was found to increase the amount of hyaluronan in the glycocalyx approximately twofold, which may represent a positive feedback for shear stress sensing by endothelial cells [30]. Another recent study showed a correlation between shear stress profile and glycocalyx dimensions in the mouse carotid artery; the laminar flow profiles in the common carotid were found to coincide with a glycocalyx thickness of 399\u2009\u00b1\u2009174\u00a0nm, whereas disturbed laminar flow in the sinus region of the carotid bifurcation coincided with a thinner glycocalyx of 73\u2009\u00b1\u200936\u00a0nm [123]. Moreover, the flow divider region of the carotid bifurcation, supposedly having an undisturbed high laminar flow profile, was covered with a glycocalyx of 308\u2009\u00b1\u2009185\u00a0nm, comparable to the common carotid glycocalyx thickness.\nFrom these data, it seems likely that the glycocalyx plays an important role in mechanotransduction and that its composition is, in turn (at least partly), shear-dependent. The different components of the glycocalyx probably operate together, which means that the glycocalyx, as a whole, is responsible for its role as mechanotransducer. This idea is confirmed by theoretical models based on a regular hexagonal distribution of core proteins over the endothelial cell membrane [138]. Recently, Tarbell and Pahakis reviewed the current concepts on mechanotransduction by the (membrane-bound) glycocalyx [116]. They conclude that the glycocalyx core proteins are responsible for transmission of shear stress signals into specific cell signaling processes, e.g., NO production and cytoskeletal reorganization. At the same time, shear stress is transmitted to other regions of the endothelial cell as well, such as intercellular junctions and basal adhesion plaques, which are responsible for additional shear sensing even in the absence of a glycocalyx.\nThe endothelial glycocalyx as control center for the microenvironment\nThe proteoglycans in the glycocalyx contribute greatly to its functional importance. The glycosaminoglycan chain variety arising from chain epimerization, elongation, and most notably, chain sulfation, gives rise to a heterogeneous surface to which a lot of plasma-derived molecules can dock. Table\u00a03 lists a number of molecules, which depend on interaction with the glycocalyx for their functionality. \nTable\u00a03Molecules dependent on interaction with the endothelial glycocalyx for proper functioningInteracting moleculePrimary function in vasculatureReference numberAntithrombin IIIPotent inactivator of pro-coagulant proteases such as thrombin, factor Xa and factor IXa; activity enhanced by heparin or heparan sulfate107Heparin cofactor IIInactivator of the procoagulant protease thrombin; activated by dermatan sulfate in the endothelial glycocalyx120TFPIAnticoagulant protein blocking activated factor VII and X38, 49LPLEnzyme involved in breakdown of low density lipoproteins133LDLTransports cholesterol and triglycerides through the circulation140VEGFPotent stimulator of angiogenesis, production of which is triggered by hypoxia92TGF\u03b21\/2Growth factor known to mediate in a lot of signaling pathways, including smooth muscle cell differentiation and vascular tone and reactivity64FGF(r)Growth factor (receptor) involved in endothelial cell proliferation and angiogenesis3, 26Ec-SODExtracellular quencher of reactive oxygen species11IL 2, 3, 4, 5, 7, 8, 12, RANTESChemotaxis of leukocytes to the subendothelium; involved in arrest and diapedesis2, 39, 100, 110, 143TFPI Tissue factor pathway inhibitor, LPL lipoprotein lipase, LDL low density lipoprotein, VEGF vascular endothelial growth factor, TGF\u03b21\/2 transforming growth factor \u03b21 or \u03b22, FGF(r) fibroblast growth factor (receptor), ec-SOD extracellular superoxide dismutase, IL interleukin, RANTES Regulated on Activation, Normal T Expressed and Secreted\u2014also known as chemokine CCL5\nDocking of plasma-derived molecules can influence the local environment in several ways: (1) Binding of receptors or enzymes and their ligands to the endothelial glycocalyx causes a localized rise in concentration of these substances, which enables proper signaling or enzymatic modification. Fibroblast growth factor (FGF) signaling is mediated in this way and is known to depend completely on the interaction of both ligand and receptor with the glycocalyx [3, 26]. Similarly, the glycocalyx is involved in the lipolytic system, binding both lipoprotein lipase and its ligand low-density lipoprotein (LDL) [133, 140]. (2) Binding of plasma-derived molecules to the glycocalyx can lead to a local concentration gradient, which is often seen in growth factor-regulated gene transcription and developmental processes [61, 82]. (3) Docking of enzymes and their agonists or inhibitors to the glycocalyx adds a vasculoprotective role to glycocalyx functionality. Several important anticoagulant mediators can bind to the glycocalyx, such as antithrombin III, heparin cofactor II, thrombomodulin, and tissue factor pathway inhibitor (TFPI). Antithrombin III is a strong inhibitor of procoagulant enzymes like thrombin and activated factors X and IX (FXa and FIXa) [87]. It is known to bind to specific regions in heparan sulfate, which enhances its anticoagulant activity [107]. Heparin cofactor II is a thrombin-specific protease inhibitor [80], which is activated by dermatan sulfate in the glycocalyx [120]. Thrombomodulin is a chondroitin sulfate containing protein expressed by endothelial cells and is able to convert thrombin from a procoagulant enzyme to an activator of the protein C pathway, thus, becoming anticoagulant [136]. TFPI is a potent inhibitor of FVIIa and FXa. TFPI is believed to bind to the glycocalyx via heparan sulfates, but other proteins could be involved as well [49]. Furthermore, uptake and degradation of TFPI\u2013FXa complexes depends on heparan sulfates in the glycocalyx [38]. All these anticoagulant molecules present in the glycocalyx contribute to the thromboresistant nature of healthy endothelium [21]. The endothelial glycocalyx also modulates inflammatory responses by binding cytokines and attenuating binding of cytokines to cell surface receptors. Shedding of heparan sulfate from the glycocalyx results in increased endothelial cell sensitivity to activation by cytokines [9, 10]. Another aspect of the vasculoprotective role of the endothelial glycocalyx is its capacity to bind quenchers of oxygen radicals, such as extracellular superoxide dismutase (SOD) [59]. These enzymes help to reduce the oxidative stress and keep up NO bioavailability, thus, preventing the endothelium from becoming dysfunctional.\nThe endothelial glycocalyx in pathophysiology\nIn healthy vessels, the endothelial glycocalyx determines vascular permeability, attenuates blood cell\u2013vessel wall interactions, mediates shear stress sensing, enables balanced signaling, and fulfills a vasculoprotective role. But when it is disrupted or modified, these properties are lost, as has been shown through direct targeting of the glycocalyx in experimental settings. In the last few years, evidence is emerging that (damage to) the glycocalyx plays a pivotal role in several vascular pathologies. Here, we will discuss its suspected roles in diabetes, ischemia\/reperfusion, and atherosclerosis.\nDiabetes\nDiabetes is a clinically well-known disease with far-reaching complications, such as retino- and nephropathy, and elevated risks for atherothrombotic cardiovascular events. One of the hallmarks of diabetes is insulin absence or resistance and subsequent hyperglycemia, impairing the protective capacity of the vessel wall [73], and resulting in enhanced endothelial permeability [1] and impaired NO synthase function [20]. However, a common pathway leading to these vascular dysfunctions has not been identified. Recently, it was shown that the systemic glycocalyx volume of healthy volunteers, as assessed by comparing the intravascular distribution volume of a glycocalyx-permeable and a glycocalyx-impermeable tracer, was halved within 6\u00a0h after induction of acute hyperglycemia [76]. Using the same methodology, the systemic glycocalyx volume in type 1 diabetics was found to be about half of that of healthy controls; it was further reduced in diabetics with microalbuminuria [75]. In the same study, plasma levels of hyaluronan and hyaluronidase were found to be elevated in patients with diabetes, reflecting increased synthesis and shedding of hyaluronan under hyperglycemic conditions. Both studies show that acute and long-standing hyperglycemia is associated with profound reduction of glycocalyx dimensions. It is tempting to speculate that this damage to the glycocalyx contributes to endothelial dysfunction in hyperglycemic conditions, which can be measured in nondiabetic subjects as well [118]. Further studies are needed to investigate whether glycocalyx perturbation is responsible for the (micro)vascular complications in diabetes.\nIschemia\/reperfusion\nDamage to tissues during a period of absent or decreased flow (total or partial ischemia) can paradoxically be exaggerated by restoration of blood flow (reperfusion). Although the severity of damage resulting from ischemia\/reperfusion varies between tissues, a common component of this pathologic process for all organs is microvascular dysfunction [32, 105]. Endothelial cells play a central role and exhibit swelling and detachment from the basement membrane after ischemia\/reperfusion [77]. Especially in postcapillary venules, endothelial cells suffer from increased oxidative stress [56], leukocytes adhere and transmigrate [8, 132], and vascular permeability increases [55]. These endothelial consequences of ischemia\/reperfusion allude at involvement of the endothelial glycocalyx. Indeed, it was recently shown by Mulivor and Lipowsky [70] that intestinal ischemia\/reperfusion led to significant reduction of glycocalyx thickness in rat mesenteric venules, most likely due to shedding of glycosaminoglycan chains. The effects of ischemia\/reperfusion on the glycocalyx could be attenuated by blockade of xanthine-oxidoreductase, which is an endogenous reactive oxygen species (ROS) producing enzyme bound to heparan sulfate domains in the glycocalyx. This way, a central role for ROS in disruption and shedding of the glycocalyx in ischemia\/reperfusion was established [97]. Infusion of exogenous hyaluronan to restore the glycocalyx or administration of pertussis toxin, which inhibits G-protein-mediated shedding of glycosaminoglycan chains, also reduced ischemia-\/reperfusion-induced damage [70]. Together, these data hint at a role for the endothelial glycocalyx in the pathophysiology of ischemia-\/reperfusion-induced tissue damage. However, its relative contribution to this process and the impact of possible therapeutical interventions are yet to be established [6, 122, 134].\nAtherosclerosis\nAtherosclerosis is a large artery disease and typically requires high plasma levels of LDL to develop at predilection sites [34] that are marked by disturbed flow profiles. Subendothelial retention of atherogenic lipoproteins and subsequent inflammatory responses lead to the formation of subendothelial plaques [60, 93]. The role of the endothelial glycocalyx in atherogenesis is, as yet, not well-established, but there are some interesting observations which point at its involvement.\nIn 2000, Vink and colleagues [128] showed that administration of clinically relevant doses of ox-LDL leads to a disruption of the glycocalyx in hamster cremaster muscle microcirculation and evokes local platelet adhesion. Co-infusion with SOD and catalase, enzymes catalyzing the dismutation of superoxide anion and the decomposition of hydrogen peroxide, abolished this effect of ox-LDL, implicating a role for oxygen-derived free radicals. Indeed, loss of glycocalyx results in shedding of endogenous protective enzymes, such as extracellular SOD, and increases the oxidative stress on endothelial cells. This was further illustrated by a recent study by van den Berg et al. [123], showing a reduction in glycocalyx dimensions due to a high-fat, high-cholesterol diet. Furthermore, an inverse relation between glycocalyx thickness and intima\u2013media ratio was found, reflecting a reduction of vasculoprotective capacity of the endothelial glycocalyx at sites with higher atherogenic risk. In healthy mice, regional variations were found showing a thinner glycocalyx in the internal carotid sinus region compared to the common carotid artery. Together, these data suggest that the endothelial glycocalyx is involved in the initiation and progression of the atherosclerotic process [74].\nIn summary, the endothelial glycocalyx appears to be perturbed in several vascular diseases. It remains to be elucidated whether glycocalyx perturbation is causally involved in the pathophysiology of these diseases. If so, restoration of the glycocalyx may be a therapeutic target of interest. Furthermore, identification of specific glycosaminoglycan domains involved in these diseases, as a platform for other substances or signaling pathways, might also prove to be of therapeutic value [4].\nVisualization techniques\nBecause of the functional importance of the endothelial glycocalyx, development of direct visualization techniques is crucial to establish its exact role. The glycocalyx can be labeled by administration of specific markers that attach to one or more of its components, making them fluorescent or detectable. Preparation of (parts of) the vessel would then allow specific microscopic imaging of the endothelial glycocalyx. Regretfully, the glycocalyx is very vulnerable and easily disturbed or dehydrated during vessel handling and preparation protocols. As a result, glycocalyx dimensions are easily underestimated, which is illustrated by the first images of the glycocalyx, made by transmission electron microscopy (TEM) in 1966 with the use of the probe ruthenium red; glycocalyx thickness measured this way approximated 20\u00a0nm in capillaries [66]. Since then, many other attempts were made to image the glycocalyx using TEM. On bovine aorta endothelial cells under shear stress conditions of 3.0\u00a0Pa, the glycocalyx was reported to be 40\u00a0nm thick [121]. These dimensions did not comply with theoretical estimates predicting the glycocalyx to be up to 1\u00a0\u03bcm thick [51]. Using a new staining protocol with Alcian blue 8GX, van den Berg et al. [124] recently applied TEM to measure endothelial glycocalyx dimensions in rat myocardial capillaries, which revealed that endothelial cells are covered by a 200- to 500-nm thick glycocalyx (Fig.\u00a02a). Hyaluronidase treatment before fixation and staining resulted in significant reduction of this layer to 100\u2013200\u00a0nm. The groups of Haraldsson [37] and of Rostgaard and Qvortrup [94, 95] improved the TEM staining protocol using fluorocarbon-based oxygen carrying fixatives, revealing glycocalyces as thick as 60\u2013200\u00a0nm in glomerular capillaries, and 50\u2013100\u00a0nm in intestinal fenestrated capillaries. Apparently, the new staining and preparation protocols improved glycocalyx conservation in TEM experiments. However, TEM cannot be used in the in vivo situation.\nFig.\u00a02Visualization of the endothelial glycocalyx with different microscopic techniques. a Endothelial glycocalyx of a rat left ventricular myocardial capillary stained with Alcian blue 8GX and visualized using electron microscopy. Bar represents 1\u00a0\u03bcm. Reproduced with permission from reference number [124]. b Intravital microscopic recording of the endothelial glycocalyx of a hamster cremaster muscle capillary. The anatomical diameter of 5.4\u00a0\u03bcm is larger than the red blood cell column width (left pane) or the plasma column width (right pane) labeled with fluorescent dextran (70\u00a0kD). This difference is caused by the presence of the endothelial glycocalyx. The bar in the left pane represents 5\u00a0\u03bcm. Reproduced with permission from reference number [129]. c Endothelial glycocalyx of a mouse common carotid artery. 3D-reconstruction of a series of optical slices obtained with two-photon laser scanning microscopy showing part of the vessel wall. The intact vessel was perfused with FITC-labeled lectin (WGA) to stain the endothelial glycocalyx (green) and SYTO 41 to label cell nuclei (blue). The arrows indicate the direction of the X, Y, and Z axis. The scanned volume approximates 200\u2009\u00d7\u2009200\u2009\u00d7\u200960\u00a0\u03bcm3. For details on methodology see also reference number [67]\nSome 30\u00a0years after the first TEM images were made, Vink et al. [129] used intravital microscopy to visualize the endothelial glycocalyx in hamster cremaster muscle capillaries in vivo using indirect approaches. The glycocalyx was recognized as a red blood cell \u201cexclusion zone\u201d or \u201cgap\u201d between the flowing red blood cells and the endothelium. In addition, the plasma was labeled by a fluorescent dextran, and the glycocalyx then appeared as a plasma exclusion zone (Fig.\u00a02b). Interestingly, no exclusion zone was found for rolling white blood cells, suggesting that they have the ability to compress the glycocalyx in these vessels, which complies with the estimated low stiffness of the glycocalyx [33, 106, 138]. Subtraction of the diameter of the plasma column from the anatomical internal diameter revealed the dimensions of the glycocalyx, which appeared to be 0.4\u20130.5\u00a0\u03bcm thick [129]. This method has been used in many studies since, primarily in the cremaster muscle microcirculation of hamsters [35, 36, 130] or mice [13, 97]. This tissue is suited for intravital microscopy because it is thin and translucent, allowing clear visualization of microvascular endothelial cells and flowing blood cells, with low or absent vessel wall motion (Fig.\u00a02b). In addition, local flow velocities can be measured. However, the estimation of the glycocalyx thickness using intravital microscopy-based methods is indirect. Furthermore, intravital microscopy cannot be applied to image the endothelial glycocalyx in larger vessels.\nDirect visualization of the glycocalyx has been performed via several approaches, mostly using lectins which are proteins that bind specific disaccharide moieties of glycosaminoglycan chains [5, 24, 70]. Other labels include antibodies for heparan sulfate, syndecan-1 or hyaluronan [24, 70]. When attaching these markers to a fluorescent probe, advanced microscopic techniques can be applied to visualize the glycocalyx. Confocal laser scanning microscopy (CLSM) enables optical sectioning with good optical resolution, allowing 3D reconstructions of the specimen. Lectin labeling of the glycocalyx of cultured human umbilical vein endothelial cells and subsequent CLSM imaging revealed a surface layer as thick as 2.5\u2009\u00b1\u20090.5\u00a0\u03bcm [5]. CLSM has also been used to detect concentration changes of fluorescently labeled lectins in the glycocalyx of fixated rat mesentery postcapillary venules in case of ischemia\/reperfusion and inflammation [70]. Because larger vessels have thicker walls, which results in lower light penetration depths with significant loss of resolution at higher depths (>40\u00a0\u03bcm) [127] due to increased scattering of signal, CLSM is less suitable for imaging of the glycocalyx in arteries.\nA promising technique to directly visualize the glycocalyx in larger vessels, both ex vivo and in vivo, is two-photon laser scanning microscopy (TPLSM). TPLSM depends on excitation of a fluorophore by simultaneous uptake (i.e., within 10\u221218\u00a0s) of two red photons, instead of one blue photon as in conventional fluorescence excitation. The use of long wavelength red photons reduces scattering, and hence, increases penetration depth into tissue. Excitation of the fluorophore and consequent fluorescence only occurs at the focal point of the illumination cone, as the probability of two-photon excitation depends on the squared intensity of the excitatory photons. Any light received by the photomultipliers has to originate from the focal position, so scattering of the emitted photons does not influence resolution and no pinholes are required. As a consequence, TPLSM offers good resolution and optical sectioning at reasonable acquisition speed, while bleaching and phototoxicity of the dyes is limited to the focal position. The combination of enhanced penetration depth, good resolution, optical sectioning, and low phototoxicity makes TPLSM a suitable technique to visualize the delicate endothelial glycocalyx in larger vessels. This idea was confirmed by a recent study by Megens and colleagues [67] in which the endothelial glycocalyx was imaged with TPLSM in intact mouse carotid arteries (Fig.\u00a02c); the glycocalyx thickness was found to be 4.5\u2009\u00b1\u20091.0\u00a0\u03bcm. As TPLSM is also applicable in vivo in rodents [127], it might prove to be a good approach of in vivo visualization of the glycocalyx in the macrocirculation of these animals.\nConclusions\nOverlying the vascular endothelium, the glycocalyx is a membrane-bound mesh in which plasma-derived molecules integrate. It exerts a variety of functions, important in normal vascular physiology and also in vascular disease. Although data from experiments in microcirculation, and more recently, in macrocirculation strongly suggests a vasculoprotective role for the glycocalyx, research on this subject is hampered by lack of a good visualization technique. Two-photon laser scanning microscopy may prove to be a successful tool in achieving direct visualization of the glycocalyx in larger arteries in rodents, both ex vivo and in vivo, with the possibility to analyze focal variations in the composition or integrity of this layer.","keyphrases":["endothelial glycocalyx","heparan sulfate","endothelial surface layer","hyaluronic acid","vascular disease","optical imaging","two-photon microscopy"],"prmu":["P","P","P","P","P","R","R"]}
{"id":"Pediatr_Nephrol-3-1-1784542","title":"Therapeutic approach to FSGS in children\n","text":"Therapy of primary focal segmental glomerulosclerosis (FSGS) in children incorporates conservative management and immunosuppression regimens to control proteinuria and preserve kidney function. In long-term cohort studies in adults and children with primary FSGS, renal survival has been directly associated with degree of proteinuria control. This educational article reviews the current therapeutic approach toward children with primary FSGS.\nIntroduction\nFocal segmental glomerulosclerosis (FSGS) is a histologic finding that may result from a variety of insults to the kidney. FSGS typically presents with proteinuria and has a high risk of progressive loss of renal function [1]. Treatment of secondary forms of FSGS targets control of the underlying condition. Therapy of primary FSGS incorporates conservative management and immunosuppression regimens to control proteinuria and preserve kidney function. In long-term cohort studies in adults and children with primary FSGS, renal survival has been directly associated with degree of proteinuria control (Fig.\u00a01) [2, 3]. Patients who are resistant to therapies have a significant likelihood of progressing to end-stage renal disease (ESRD) and are a group in need of novel therapies to delay or prevent this outcome [4].\nFig.\u00a01a Kaplan\u2013Meyer analysis of the risk of end-stage renal disease (ESRD) by proteinuria remission status in children with primary focal segmental glomerulosclerosis (FSGS). b Kidney survival by proteinuria remission status in adults with primary FSGS. CR complete remission, PR partial remission, NR no remission\nGenetic considerations\nAn undefined proportion of patients with classically defined primary FSGS harbor genetic mutations in podocyte-specific genes such as nephrin, podocin, \u03b1-actinin-4, and CD2AP [5, 6]. There are conflicting reports about the effectiveness of immune-based therapy in the setting of these mutations, but the likelihood of a positive response may be low [7]. Although mutation-linked cases of FSGS may have a lower response rate to conventional immunomodulatory treatment, these patients still manifest the progressive fibrosis that is observed in nongenetic FSGS.\nNonimmunosuppressive therapy\nDiuretic therapy\nControl of edema in nephrotic syndrome allows not only cosmetic improvement but is expected to decrease pulmonary effusions, decrease ascites, and lower the risk of peritonitis and skin-related problems from edema. Overaggressive diuresis in patients with intravascular depletion may be a risk factor, however, in developing thrombotic complications and acute renal insufficiency.\nLoop diuretics are often required for control of edema in patients with proteinuria in the nephrotic range. Delivery of the diuretic to the site of action (lumen of the tubule) is often impaired in nephrotic syndrome due to decreased glomerular filtration rate (GFR), increased binding of the diuretic to intraluminal albumin, and\/or decreased delivery of sodium to sites of diuretic activity. An increase of sodium reabsorption in the distal tubule in response to loop diuretic activity may add to resistance to loop diuretics. This distal compensatory mechanism may be diminished by the use of a combination of loop and distal diuretics (thiazides) [8]. Though the addition of aldosterone inhibitors (spironolactone) is theoretically attractive under the theory that edema is in part driven by aldosterone, it is unclear whether spironolactone or other similar medications are clinically helpful to control edema [9\u201311] An additional advantage to the use of aldosterone inhibitors such as spironolactone is suggested by the antifibrotic properties of these agents, which will be discussed below [12].\nCombined albumin and furosemide therapy for anasarca has been studied, as well. Na et al. showed evidence for a mild increase in water diuresis but little evidence that the concomitant use of albumin adds to the natruretic effect of furosemide [13, 14]. Fliser et al. [15] showed a moderate (20%) increase in water and salt excretion when comparing albumin and furosemide to furosemide alone. Haws et al. [16] also showed a mild but transient benefit of albumin and furosemide therapy but commented on the potential serious complications of hypertension, respiratory distress, congestive heart failure, and electrolyte disturbances. Thus, the combination of albumin and furosemide infusions, whether in combination or sequential, may provide a small transient benefit in the therapy of children with severe edema [17].\nTreatment of hyperlipidemia\nFor patients who become nephrotic from the progression of FSGS, hyperlipidemia is an almost universal finding. Whether the hyperlipidemia associated with nephrotic syndrome should be specifically targeted for treatment in children separately from nephrotic syndrome treatment itself has been a question for more than 20\u00a0years. The childhood origin of atherosclerotic disease and increased risk for cardiovascular disease secondary to chronic kidney disease supports an interventional approach.\nThe report of the expert panel on blood cholesterol levels in children and adolescents [18] from the National Cholesterol Education Program (NCEP) defined categories of hypercholesterolemia in children for total cholesterol and low-density lipoprotein (LDL) cholesterol levels. High levels for total cholesterol were defined as \u2265200\u00a0mg\/dl and for LDL cholesterol as \u2265130\u00a0mg\/dl. Dietary treatment of hyperlipidemia is the first-line intervention. In adults with nephrotic syndrome, soy-based vegetarian diets and supplemented low protein diets have been shown to have potential benefits, decreasing both proteinuria and cholesterol, but have not been shown to slow the decline in GFR [19, 20]. Dietary therapy for dyslipidemia has been effective in reducing lipid levels in children with primary lipid disorders [21].\nBased on the report from the NCEP, pharmacologic therapy for children ages 10\u00a0years and older should be considered after an adequate trial of diet therapy if LDL cholesterol remains \u2265160\u00a0mg\/dl in children with significant risk for cardiovascular disease, as is seen in children with FSGS. Recommendations for pharmacologic therapy for hyperlipidemia in children from the report suggested that bile acid sequestrants cholestyramine and colestipol should be the first-line agents for treating children with lipid disorders [18]. This was due mainly to concerns about the safety of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (statins) in children. While they are effective in lowering cholesterol and are relatively safe, bile acid sequestrants pose particular problems in children. They are not very palatable, and they may affect absorption of other medications being used, including thiazide diuretics, propranolol, corticosteroids, thyroid hormones, and loop diuretics. A new medication in this category, colesevelam, apparently does not have these problems but is not approved for use in children.\nSince the NCEP report in 1992, several studies have been published suggesting that statins are safe in children as young as 4\u00a0years of age with familial hypercholesterolemia and do not adversely affect growth, hormone levels, or sexual development [21\u201323]. Statins are effective in treating the hypercholesterolemia of nephrotic syndrome, with decreases in total cholesterol levels up to 45% [24]. The long-term benefit of statins on renal function may be positive. Down stream from glomerular injury, high levels of urinary protein pass to the renal tubule and are reabsorbed. Protein reabsorption may injure the renal tubule. Statins may inhibit this tubular protein reabsorption and thereby protect from additional renal injury. Whether statins provide renoprotective effects in children has not been well studied, but there are several studies in adults with nondiabetic proteinuria that indicate that statins may slow GFR decline [25]. In these studies, the greatest benefit seemed to accrue in patients with the greatest amount of proteinuria and renal insufficiency. The side effects of statins have mainly been limited to myopathy and hepatotoxicity [26, 27].\nDrugs in the fibrate class have also been used alone and in combination to treat hypercholesterolemia in children with nephrotic syndrome [28]. The use of gemfibrozil with a statin may increase the incidence of myopathy. Fenofibrate, approved for adult use in January 2006, may have less interaction due different hepatic metabolism [29]. The long-term safety of fibrates in children has not been well established.\nAlteration of the renin-angiotensin-aldosterone axis\nBlood pressure control for children with FSGS targets values less than or equal to the 90th percentile for age, gender, and height and is consistent with recommendations for all children with kidney disease. Evidence from many trials using angiotensin-converting enzyme inhibitor (ACE-I) and\/or angiotensin receptor blocker (ARB) therapy in patients with proteinuria indicate that, beyond their antihypertensive effect, both are effective in reducing proteinuria in a wide variety of renal diseases. Few studies have had significant numbers of patients with FSGS specifically, and fewer still have included children with FSGS. In 1988, Trachtman and Gauthier reported a 50\u201370% reduction in proteinuria in children with steroid-resistant nephrotic syndrome (SRNS) using ACE-I therapy [30]. Bagga et al. [31], in a randomized, crossover trial of low-dose (0.2\u00a0mg\/kg) vs. high-dose (0.6\u00a0mg\/kg) enalapril in 25 patients with SRNS showed dose related responses, with average urine albumin\/creatinine ratio reductions of 33% and 52%, respectively. Blood pressure control was similar between the two groups. Several other studies of enalapril and ramipril in children with a variety of proteinuric renal diseases have confirmed the efficacy of these drugs in reducing proteinuria in many, but not all, treated children [32\u201335]. In the Ramipril Efficacy in Nephropathy (REIN) study, a double-blind study in adults with nondiabetic nephropathy, treatment with ramipril seemed to reduce both proteinuria and the rate of GFR decline more than could be attributed to blood pressure control alone [36]. W\u00fchl et al., in a similar trial in almost 400 children with hypoplasia\/dysplasia (70%) and glomerulopathies (13%), had similar findings [37].\nThere have been many studies in adults with renal disease comparing combined ACE-I\/angiotensin receptor blocker (ARB) therapy with monotherapy alone [38, 39]. Though differing in design and findings, overall, the studies seem to indicate greater reduction in proteinuria without a greater frequency of side effects [39\u201341]. Yang et al. [41] reported greater reduction in proteinuria with combined therapy in a small group of five children with immunoglobulin (Ig)A nephropathy and heavy proteinuria, with no significant side effects noted. The most concerning side effect of ACE-I and ARB therapy is in females of childbearing years, with significant risk of fetal abnormalities reported with in utero exposure [42]. Other side effects of ACE-I therapy were noted in 2.4% of children in one large series with ramipril in children with chronic renal failure [37]. These side effects included decreases in GFR and hemoglobin and increases in serum potassium levels. Acute renal failure, often associated with hypovolemia, has been noted and seems to respond to discontinuation of the medicine until the acute illness resolves. Angioedema and nonproductive cough have been encountered with the use of ACE-I therapy in adults and are less frequently reported in children. ARBs seem to have a lower incidence of angioedema and cough [43, 44]. The incidence of recurrent angioedema in those who experience ACE-I-associated angioedema and are switched to ARB therapy appears to be low [45].\nAldosterone inhibitors have shown potential for alteration of the fibrotic mechanisms in animal models of kidney fibrosis, in the reduction of proteinuria in diabetic nephropathy, and in proteinuric chronic kidney diseases [12, 46, 47]. In the later study of 40 patients with a variety of proteinuric kidney diseases, the combination of ACE-I and aldosterone inhibitor therapy led to a decrease in mean urinary protein excretion in the ten patients assigned to this study arm [48]. Studies in humans with FSGS have not been published.\nAntioxidants\nThe potential for antioxidant therapy in FSGS stems from experimental data that supports a role for excessive free radicals in multiple disease states, including chronic kidney disease. Based on antioxidant properties, vitamin E has been evaluated as a potential therapy for FSGS in one small study by Tahzib et al. In this open-label study, 11 children with FSGS were treated with vitamin E for approximately 3\u00a0months. A reduction in protein excretion was noted [49]. The antioxidant properties of vitamin E and the relatively low risk for adverse effects of this agent make this an interesting, if unproven, therapeutic option. The combined conservative management approach may control morbidity but leave the majority of patients with uncontrolled urinary protein excretion and risk for progression of disease. The typical approach to FSGS therapy in children is to add immunosuppression at the onset of therapy.\nImmunosuppression\nCorticosteroids\nCorticosteroids have long been the mainstay of treatment for childhood nephrotic syndrome, regardless of its etiology. The huge role that these agents play is evident in the way this disease is classified: steroid responsive, steroid dependent, and steroid resistant. The International Study of Kidney Disease in Children (ISKDC) standard dosing has been, and generally continues to be, applied. That is, an 8-week course of oral prednisone at 60\u00a0mg\/m2\/day for 4\u00a0weeks, followed by 40\u00a0mg\/m2 on alternate days for 4\u00a0weeks [50]. Initial ISKDC data showed a corticosteroid response rate of approximately 30% of the 37 children with FSGS studied, and subsequent studies have been consistent in showing a response to oral corticosteroids in a minority of FSGS patients [51]. However, a response to corticosteroids is generally consistent with a more favorable prognosis, even when an initial response is followed by resistance after a subsequent relapse. There are also reported population differences in the response to steroid treatment, such as decreased response in African Americans and Hispanics [52]. Some literature is emerging that shows a lower response rate for patients with the nephrosis 2 homolog, podocin (human) (NPHS2) mutation [53]. The more recent debate has been about what steroid, how much steroid, and for how long, before a patient can be definitely declared unresponsive. Certainly, data in adult patients advocate prolonged oral corticosteroid administration, in some cases >6\u00a0months [54]. There have been no pediatric trials of prolonged oral corticosteroid use in patients with FSGS. The regimens used vary tremendously, with the most dramatic differences occurring between pediatric nephrologists and internist nephrologists. Due to the generally poor response to the standard oral dosing, some pediatric protocols have advocated high doses of intravenous methylprednisolone, with varying degrees of success [54, 55]. Corticosteroids remain a key component of many therapeutic regimens for FSGS, usually in combination with the various other drugs used to treat this disease, such as alkylating agents or calcineurin inhibitors. Of course, corticosteroid therapy is not without side effects. These include hypertension, growth impairment with prolonged therapy, susceptibility to infection, diabetes mellitus, and osteoporosis [55]. These side effects have led to the tendency toward lower corticosteroid doses and shorter, rather than prolonged, courses.\nCalcineurin inhibitors\nCyclosporine A\nThe rationale behind the initial use of cyclosporine A (CsA) in FSGS and other forms of nephrotic syndrome is the evidence in animal models that the disease may be mediated by lymphokines that mediate glomerular basement membrane damage although it is unclear that this is actually the case. CsA acts on T-helper cells to inhibit interleukin-2 (IL-2) production, cytotoxic T-cell proliferation, and activation of B-cell responses by helper T-cells. However, CsA likely induces remission in proteinuria by two other mechanisms: induction of vasoconstriction of the glomerular afferent arteriole and interference with glomerular basement membrane permselectivity to proteins [56].\nIt has been two decades since CsA was first reported to show some benefit for patients with idiopathic nephrotic syndrome, especially for patients with steroid-responsive disease who had frequent relapses. One randomized trial of 49 steroid-resistant patients assigned to either CsA or placebo for 6\u00a0months showed a benefit in the CsA arm, with a response rate of 70% (complete or partial remission). This is the only medication with documented efficacy for steroid-resistant FSGS in controlled clinical trials in both adults and children [57, 58]. A major concern of long-term CsA treatment is the well-documented potential for nephrotoxicity. Another is the high relapse rate after drug withdrawal. In the Cattran study [57], 60% of the patients who responded to treatment had relapsed by week 78. There is also now concern being raised about secondary resistance developing in patients treated with CsA: an initial induction of remission, relapse when the drug is withdrawn, and resistance on reinstitution of the drug [59]. In this study of 32 children, the diagnosis of FSGS and the presence of C4 or C1q during immunofluorescence staining of kidney tissue appear to correlate with an increased risk of secondary resistance. There are still no guidelines for standardized dosing or duration of treatment of children with steroid-resistant nephrotic syndrome, which probably accounts for the variability in reported response to treatment in the literature.\nSide effects of CsA treatment include hypertension, hirsutism, and gingival hyperplasia. As a result of these and the risk for nephrotoxicity, treatment with cyclosporine has not been without controversy in terms of perceived optimal daily dosing, blood level to be maintained, and duration of treatment. A recent Egyptian study in 117 children with nephrotic syndrome, which included 79 patients with FSGS, used low-dose, long-term CsA (more than 2\u00a0years of treatment) [60]. The starting dose of 4\u20135\u00a0mg\/kg per day was adjusted to maintain a whole-blood trough level of 100\u2013150\u00a0ng\/ml during the first 2\u00a0months and 50\u2013100\u00a0ng\/ml thereafter. In these subjects with steroid-resistant FSGS, the investigators were able to achieve an almost 70% complete remission rate during the 6\u00a0months of therapy. Unfortunately, the relapse rate was substantial upon withdrawal of CsA. Overall, it appears that, as with corticosteroids, a positive response to CsA, even if followed by a relapse, is a good prognostic indicator with regard to the risk for progression to ESRD [57].\nTacrolimus\nThis newer and more potent calcineurin inhibitor has not undergone a controlled clinical trial for the treatment of FSGS, but there are anecdotal reports of responses in patients with nephrotic syndrome, some of whom had FSGS [61]. One retrospective study of 16 children with treatment-resistant nephrotic syndrome, including 13 with FSGS, documented reduction in urinary protein excretion in 13 while on therapy and subsequent relapse in three of the 13 [62]. There are also two small prospective studies in adults that showed a positive response [63, 64]. In these small studies, tacrolimus appeared to present a problem similar to that of CsA, with a majority of patients relapsing on drug withdrawal [63].\nAlkylating agents\nDNA alkylating agents such as cyclophosphamide and chlorambucil have been in use since the 1980s for several glomerular diseases, including FSGS. The use of these agents has been limited due to potential side effects, including bone marrow suppression, infertility, hemorrhagic cystitis, and possible future malignancy risk. A retrospective cohort of 29 patients suggested that cyclophosphamide may have some survival benefit in those with at least a partial response measured by impact on proteinuria and progression of chronic kidney disease [65]. A randomized trial in 1996 from the ISKDC evaluated 60 children with FSGS and their response to daily oral cyclophosphamide and alternate day prednisone vs. alternate day prednisone alone. There was no difference in renal survival or proteinuria between the two groups [66]. Due to unfavorable toxic side effects and variable reported efficacy in the literature, alkylating agents are falling out of favor for primary therapy in FSGS.\nMycophenolate mofetil\nMycophenolate mofetil (MMF) was introduced in the mid-1990s as an immunosuppressive agent for organ transplantation. Due to its steroid-sparing effect, efforts have been made to expand its clinical application to several glomerular diseases, including FSGS. MMF blocks de novo synthesis of T- and B-cell lymphocytes through noncompetitive, reversible inhibition of inosine monophosphate dehydrogenase. Data on the use of MMF in FSGS has been limited to a few uncontrolled trials with small numbers of patients, but it shows early promise. Choi et al. reported 46 patients with primary glomerulopathies, including 18 patients with FSGS. They found a statistically significant decrease in proteinuria in patients receiving MMF as adjunctive therapy [67]. Cattran et al. reported an open-label, 6-month trial of MMF in 18 patients with steroid-resistant FSGS, 12 of whom were also resistant to alkylating agents and\/or calcineurin inhibitors. Although patients did not achieve complete remission, four of 18 had a reduction in proteinuria during therapy [68]. A similar decrease in proteinuria was documented in a series of nine children and young adults with steroid-resistant FSGS who were treated with pulse steroids and MMF [69]. Overall, MMF is showing early promise as a steroid-sparing therapy in FSGS, but questions remain about length of therapy, escalation of dosing, and long-term malignancy risks.\nSirolimus\nThe utility of sirolimus in the treatment of FSGS has been entertained in patients with intolerance or resistance to corticosteroid therapy. One prospective nonrandomized study documented a reduction of proteinuria in 12 of 21 patients treated with 6\u00a0months of sirolimus [70]. Conversely, a study of six FSGS patients treated with sirolimus documented a decline in kidney function in five patients. None had a complete remission [71]. In transplant recipients, the use of sirolimus in conjunction with calcineurin inhibitors has also been associated with acute renal failure [72]. Consequently, sirolimus is not recommended for the treatment of FSGS due to the associated renal toxicity.\nPlasmapheresis\nIn multiple-drug-resistant primary FSGS, the use of plasmapheresis has been considered a rescue option. The generally accepted rationale is for the removal of a circulating factor from the plasma that alters glomerular barrier function [73]. In primary FSGS in the native kidneys, two small studies encompassing 19 patients reported a response rate between 12% and 55% [74, 75]. The best response was seen using a protocol of plasmapheresis, corticosteroids, and cyclophosphamide, making it difficult to attribute the full response to plasmapheresis alone [75]. A single case report of a child with resistant disease demonstrated improved proteinuria and serum creatinine [76]. At present, plasmapheresis is considered a rescue therapy and is an invasive procedure with significant risks of infection, hypocalcemia, and bleeding. Plasmapheresis is considered an option for prevention or treatment of recurrent FSGS in the transplant recipient based on uncontrolled studies [77, 78].\nAntifibrotic therapy\nThere are a large number of patients with multiple-drug-resistant FSGS who are at substantial risk of progression to ESRD and for whom there are no proven therapeutic options. The past decade has witnessed striking advances in understanding the cellular and molecular basis of renal fibrosis and its contribution to progressive kidney failure. Several therapeutic targets have been identified in animal models of fibrosis in the kidney, including molecules involved in the recruitment and activation of mononuclear cells (e.g., chemokines, lymphokines, adhesion molecules), recruitment and activation of interstitial myofibroblasts, fibrogenic molecules [e.g., transforming growth factor (TGF)-\u03b2, endothelin-1, angiotensin II, tumor necrosis factor (TNF)-\u03b1, and platelet-derived growth factor (PDGF)-\u03b2], angiogenic factors [e.g., vascular endothelial growth factor (VEGF)], antiapoptotic molecules, inhibitors of matrix synthesis, and molecules that enhance matrix degradation (matrix-degrading proteases, blockers of protease inhibitors) [79\u201383]. Crossing many of these mechanisms, peroxisome proliferator activator receptor-\u03b3 (PPAR\u03b3) agonists alter regulation of renal cell differentiation and proliferation [84\u201387], extracellular matrix production, macrophage accumulation, tissue inflammation, and apoptosis [88]. The most effective treatment to prevent progression of fibrosis and kidney failure in FSGS is likely to entail a combination of drugs that modulate mediators of fibrosis. The progression of kidney fibrosis is interrupted in part by the use of ACE-I and ARB agents in FSGS therapy. Future therapeutic options are likely to emanate from this area of research.\nConclusions\nCurrent strategies for control of FSGS use a stepwise approach with a goal of normalization of urinary protein excretion and the prevention of kidney failure. Progress in this field remains a priority in order to prevent the trajectory toward renal failure for patients proven to be resistant to treatment and to identify therapeutic regimens with minimal toxicity.\nCME questions\n(Answers appear following the reference list) \nA 14-year-old boy presents with nephrotic syndrome, normal serum creatinine, and normal blood pressure. He is diagnosed with FSGS by kidney biopsy and treated with corticosteroids. What is the most likely response to corticosteroids in this setting? \nComplete remission with corticosteroid therapyDependence on corticosteroidsFailure to respond to corticosteroids but improves with cyclosporineFailure to control proteinuria and progression to kidney failureFactors that seem to confer an unfavorable prognosis in children with nephrotic syndrome are: \nPrimary resistance to corticosteroidsResistance to cyclosporinePresence of NPHS2 podocin mutationAfrican American or Hispanic ethnicityAll of the aboveIn the management of FSGS, progressive kidney fibrosis may be slowed by: \nFurosemideAngiotensin receptor blockadeCholestyraminePrednisoneBoth b and dFSGS patients with NPHS2 podocin mutation are more likely to respond to corticosteroids than patients without a podocin mutation (T\/F).Cyclophosphamide is considered a mainstay in therapy for FSGS to prevent progression to end-stage renal failure (T\/F).","keyphrases":["fsgs","children","proteinuria","esrd"],"prmu":["P","P","P","P"]}
{"id":"Pharm_World_Sci-3-1-2082655","title":"Understanding the meaning of medications for patients: The medication experience\n","text":"Objective: To understand and describe the meaning of medications for patients. Methods: A metasynthesis of three different, yet complementary qualitative research studies, was conducted by two researchers. The first study was a phenomenological study of patients\u2019 medication experiences that used unstructured interviews. The second study was an ethnographic study of pharmaceutical care practice, which included participant observation, in-depth interviews and focus groups with patients of pharmaceutical care. The third was a phenomenological study of the chronic illness experience of medically uninsured individuals in the United States and included an explicit aim to understand the medication experience within that context. The two researchers who conducted these three qualitative studies that examined the medication experience performed the meta-synthesis. The process began with the researchers reviewing the themes of the medication experience for each study. The researchers then aggregated the themes to identify the overlapping and similar themes of the medication experience and which themes are sub-themes within another theme versus a unique theme of the medication experience. The researchers then used the analytic technique, \u201cfree imaginative variation\u201d to determine the essential, structural themes of the medication experience. Results: The meaning of medications for patients was captured as four themes of the medication experience: a meaningful encounter; bodily effects; unremitting nature; and exerting control. The medication experience is an individual\u2019s subjective experience of taking a medication in his daily life. It begins as an encounter with a medication. It is an encounter that is given meaning before it occurs. The experience may include positive or negative bodily effects. The unremitting nature of a chronic medication often causes an individual to question the need for the medication. Subsequently, the individual may exert control by altering the way he takes the medication and often in part because of the gained expertise with the medication in his own body. Conclusion: The medication experience is a practice concept that serves to understand patients\u2019 experiences and to understand an individual patient\u2019s medication experience and medication-taking behaviors in order to meet his or her medication-related needs.\nIntroduction\nMedications are one of the main options in the cure, treatment, and prevention of numerous medical conditions. However, there is still a gap in society regarding who must be held accountable for the outcomes of pharmacotherapy. Pharmacists have recently recognized the societal need to take responsibility for drug therapy outcomes [1]. However, if a health care practitioner strives to meet a patient\u2019s drug-related needs then that practitioner must understand the meaning of medications for that patient [1\u20133]. However, the medication experience as a practice concept has not been comprehensively researched in pharmacy.\nMedical and social scientists have examined the subjective experience of medications for patients, typically within the illness experience. Studies have been conducted on the meaning of medications for patients with specific medical conditions including: asthma [4]; hypertension [5]; and schizophrenia [6]. Moreover, studies have explored the meaning of therapeutic classes including: selective serotonin reuptake inhibitors [7]; hormone-replacement therapy [8, 9]; and antipsychotic medications [10]. Studies have also focused on patients\u2019 medication practices or medication-taking behaviors [10, 11]; decision-making [8]; and cultural ideas of drug use [12]. Conrad [13] introduced the concept of medication practices or how patients manage their medications. He found that patients with epilepsy interpret the prescribed regimen and create medication practices that may vary from the prescribed one. Though many studies have examined the meaning of medications, most have focused on specific diseases or classes of drugs. The authors surmise that there could be a common experience of taking medications chronically that transcends the specificity of diseases and medications. Additionally, most studies on the meaning of medications have focused on patients\u2019 decision-making, medication-taking behaviors, or compliance. Synthesizing the findings from three studies that investigate the meaning of taking medications for patients with many chronic conditions could provide a deeper level of understanding about a concept of importance to pharmacy practitioners. These three studies focused on understanding how patients experience taking medications on a daily basis; meaning how they feel, react and think about medications. This understanding may help pharmacists to better comprehend how and why patients make the decisions and take the actions they do. It is the goal of this article to present an understanding of the meaning of medications for patients, introduced here as the medication experience.\nMethods\nThis paper is the result of a meta-synthesis of three different yet complementary qualitative studies that included aims to understand the medication experience. These three studies were conducted by the two authors. Table\u00a01 describes each study\u2019s method and participants\u2019 demographics.\nTable\u00a01Studies\u2019 methods and participant attributesStudyMedication experiencePharmaceutical care practiceIllness experience of medically uninsuredMethodologyPhenomenologyEthnographyInterpretive phenomenologyMethods InterviewsInterviews, focus groups, participant observationInterviewsSetting of interviewsUniversity conference rooms and officesParticipants\u2019 homes and clinic exam roomsParticipants\u2019 homes, public library rooms, & university roomsParticipantsStaff and faculty at public universityPatients of pharmaceutical care (PC)Medically uninsured individualsSelection criteria6+ months taking a chronic medication (actual 3\u201315 years)2+ years experience with PC1+ year uninsured, 2+ medical conditions, prescribed medsNumber of participants52511Age range24\u20136036\u20137427\u201364Gender of participants4 women, 1 man17 women, 8 men8 women, 3 menAverage number of medical conditions per participant2.844.5\nThe first study was a phenomenological study of individuals\u2019 experiences taking at least two prescription medications. The methods were guided by Van Manen\u2019s [14] insights using in-depth interviews, which were audio-taped and transcribed. The analysis involved reading and coding each interview transcript for the meaning units of each participant\u2019s experience. Meaning units are phrases or text sections that illustrate a segment of the meaning distinct from the adjacent text. Then the codes were reviewed to identify the common themes of the medication experience for all participants.\nThe second study was an ethnography that used a triangulation of methods to reach a comprehensive understanding of pharmaceutical care practice [3]. The study revealed patients\u2019 medication experiences as a significant part of patients\u2019 experiences with pharmaceutical care. The study was conducted over a period of eight months in six clinics and one community pharmacy. The sampling was theoretical sampling, which involved selecting participants who would yield information that was relevant to the understanding of experiences of those in the pharmaceutical care practices. The data analysis was conducted following the techniques of Wolcott [15].\nThe third study was an interpretive phenomenological study that used in-depth interviews to understand medically uninsured individuals\u2019 (in the United States) chronic illness experiences, including the medication experience [16]. The interviews were audiotaped and transcribed. The interview transcripts and field notes were segmented and labeled for one of the three experiential units - the illness, medication, and uninsured experience. A thematic analysis of the lived experience of each segment of text was conducted coding each segment with a descriptive code that captured the meaning conveyed. The codes were iteratively refined, and removed as appropriate. This was in part conducted using an analytic method called free imaginative variation. Free imaginative variation involves removing a theme, then asking if the essence of the phenomenon withstands [14]. The resultant essential codes became the themes of the illness and medication experience of medically uninsured individuals.\nThe researchers used meta-synthesis of the three studies to reach a greater breadth of understanding about individuals\u2019 medication experiences. Meta-synthesis is an analytic method that facilitates a fuller understanding of a phenomenon by providing an interpretive integration of qualitative findings. The meta-synthesis of qualitative findings is increasingly seen as essential to enhance the generalizability of qualitative research [17]. The two researchers who conducted these three qualitative studies performed the meta-synthesis. The process began with the researchers reviewing the themes of the medication experience for each study. The researchers then aggregated the themes to identify the overlapping and similar themes of the medication experience. The researchers used free imaginative variation to determine the essential themes of the medication experience and from these the prevailing themes were identified. The findings from the meta-synthesis of the three studies informed the meaning of medications (i.e., the medication experience) that is presented in this paper.\nResults\nThe meaning of medications for participants was revealed as the following themes: a meaningful encounter, bodily effects, unremitting nature, and exerting control.\nA meaningful encounter\nThe medication experience is first revealed as an encounter with a medication. It is an encounter that is embedded with meaning long before it occurs. The meaningful encounter can be revealed as a sense of losing control, a sign of getting older, cause questioning, and a meeting with stigma. Each of these sub-themes of the meaningful encounter is described below.\nThe encounter with a chronic medication for the first time can be experienced as a sense of losing control, as one participant states: I used to think I was immune to disease. This body is so tremendous that it will never let me down, but here I was\u2014I had to start taking medication.\nOne participant explicitly characterized her medical need for chronic medications as a loss of control:I lost control of my health. I wish I could handle it myself.\nAnother participant describes a similar desire to treat her condition without medication:I felt like if I took lorazepam that I\u2019m failing. If I could fight it myself, get over it myself without the drug then I\u2019m getting better.\nParticipants commonly ascribed a negative meaning to medications when they first encountered them or when they began taking an increasing number of medications chronically. Another study found loss of control as part of the meaning of medications for patients [18].\nMany participants described medications as signifying aging. One participant characterized starting a medication as depressing because it indicated he was \u201cgetting older.\u201d However, for a participant in her mid-twenties using several medications, the significance was even greater:I feel like a 90-year-old woman. Like my grandpa, he\u2019s on all these pills. If you\u2019re an old person that\u2019s okay, but if you\u2019re young it\u2019s really odd.\nMedications distinguish the healthy from the sick and the young from the old. Taking chronic medications often made participants feel that they were now part of that group of people who take medications because they are getting older.\nAs part of the initial encounter with a chronic medication, many participants question the actual need for the medication they are prescribed.I\u2019m not sure what I\u2019ll get from these medications.\nUncertainty arises when participants realize they are supposed to take a medication chronically.I thought about what would happen to me if I just decided not to take it.\nQuestioning the need for a medication when it is first prescribed can be interpreted as resistance by health care professionals, whereas for participants it is a way to reclaim a degree of control. Participants sensed that their individual autonomy was undermined when taking chronic medications until the point they questioned the taken-for-granted notion that medications are the right option.\nThe first reactions to initiating a medication can also be shaped by the social views of the medical condition. For example, the reaction of individuals to initiating psychotropic medications can be a response to the stigma of mental illness. As expressed by participants in these studies, taking an antidepressant carries the shame associated with mental illness.Maybe I\u2019m just embarrassed, even if nobody would know. I think people would see me as weak.\nWhen initiating a chronic medication, participants encountered different meanings that shaped their initial experience of the medication. Participants experienced the meaningful encounter with a chronic medication as a sense of losing control, a sign of getting older, causing them to question, and a meeting with stigma.\nBodily effects\nMedications have expected pharmacological benefits as well as anticipated side effects and unanticipated adverse events, all of which are bodily experiences. When a patient\u2019s experience of their body in illness is debilitating, a medication can provide relief and allow the patient to regain their \u201chealthy\u201d body. However, medications also can cause negative bodily sequelae that are part of a patient\u2019s medication experience. The bodily effects of medications theme was revealed as the experience of a magic elixir and trade-offs.\nThe medication can \u201cnormalize\u201d the patient or bring her body as a whole together: At first I was very grateful that insulin existed\u2014it saved my life. I am grateful that I\u2019m alive. It is scary that you\u2019re reliant on this magic elixir to be alive.\nParticipants who characterized their medications as \u201cmagic elixirs\u201d often had medical conditions that incapacitated them.I\u2019ve struggled with depression all my life. My psychiatrist started me on antidepressants. I\u2019m very grateful for these drugs. I wouldn\u2019t be able to work if it wasn\u2019t for them.\nSometimes the body, disrupted by a disease, can be brought back into balance with medications. Conrad [13] found that epileptic patients perceived taking their anticonvulsants as a \u2018ticket\u2019 to normality.\nAn important component of the bodily effects stemmed from what the medications did to improve their lives. In contrast, other participants experienced the negative sequelae of a medication. However, if the benefit experienced was sufficiently good they would be willing to accept the side effects as a trade-off. One participant indicates:You learn to decide if you\u2019re gonna take the side effect over how well it helps with your depression. Citalopram worked so well for me that I didn\u2019t care that I was peeing my pants.\nAnother participant describes:I\u2019m not worried about taking them, mostly because if they\u2019re going to kill me earlier I don\u2019t care because I just want the problem to go away.\nAnother study identified that patients are aware of the trade-offs that must be made while trying to maximize their well-being [10]. Participants also expressed concern about taking a medication until they experienced the benefits.Even though I was hesitant at the beginning, after seeing the benefits and realizing how life can be with \u201cjust a medication\u201d and feel good\u2026\nThis theme illustrates how patients may rationally approach the decision to take or continue a medication and that they are willing to take a medication once they believe that the medication\u2019s benefits outweigh its risks.\nUnremitting nature\nThe unremitting nature of a chronic medication, like a chronic condition, is a burden.The first time somebody told me I would have to take that for the rest of my life I got mad.\nAnother participant states: I feel that part of my life now is going to be me taking more and more medicine and relying on medications to continue living.\nThe expectation of taking a medication regularly positions the patient as a passive agent, and the medication then becomes a symbol of dependence.I think, what if I went a couple months without taking anything? But I don\u2019t feel I have that choice. Those pills keep you prisoner. If you go on a trip, that\u2019s one of the major factors - it\u2019s more important than packing enough underclothes.\nThis participant has lost autonomy and in her words she is held captive by her medications. Additionally, due to the chronicity of medications participants have to take responsibility.It\u2019s this constant thing you wish you could have a break from, but you can\u2019t.\nIn order to achieve normal function some participants have to take medications indefinitely, yet they still wish that wasn\u2019t the case.You feel sorry for yourself because you\u2019re still on these pills. I don\u2019t want to think - it\u2019s been this long and I\u2019m still on them.\nExerting control\nThe last theme of the medication experience was revealed as participants exerting control over their medications. After encountering the meaning of a medication, questioning it, realizing the bodily effects and the continuous nature of medications, participants experimented with becoming the managers of their treatment regimens. After taking chronic medications for a period of time participants became familiar with the effects of medications on their bodies. They discovered creative ways to manage their medications and exert control over them; in part because they were now knowledgeable.I don\u2019t think people understand how much you have to think about it. I call it thinking like a pancreas. You have to do all the work. You have to determine how much you take [insulin] and that changes from day to day and from hour to hour...\nParticipants have their own method of learning pharmacology\u2013through experience. They know their bodies and perceive the changes produced by medications. As a result, exerting control is a common practice in individuals taking medications chronically.I know how to go off of it, decrease it very slowly or you get sick.\nHealth care professionals rarely acknowledge this practice as a form of exerting control; instead it is labeled as noncompliance. Another participant offers a description of being attuned to her body and taking control: As the doctor upped the dose I felt like I was high \u2026 I started getting a really weird feeling in my head, I even had pains in my chest. I thought to myself, I\u2019m just gonna quit taking this. So I quit, and then as the week went on I was slowly getting better.\nParticipants also self manage their medications by reducing the dose particularly when they are uncertain about the effects or concerned about becoming \u201cdependent.\u201d I like being able to adjust my sertraline as I find necessary. I like to have that control.\nAs the participants in these studies convey and Carrick et\u00a0al. [10] also observed, patients are the ultimate managers of their treatments and create their own medication practices. From the patient\u2019s perspective, such behaviors are rational and help them preserve control. Ramalho de Oliveira and Shoemaker [2] argued that pharmacists typically take a paternalistic perspective, viewing patients\u2019 non-compliant behavior as an irrational act. The understanding of patients exerting control over their medications provides a context in which to challenge the notion that patients\u2019 medication practices are illogical.\nDiscussion\nThe authors define the medication experience as an individual\u2019s subjective experience of taking a medication in his daily life. It begins as an encounter with a chronic medication. It is an encounter that is given meaning before it happens and is often a reaction to the symbol that medication holds. The experience may include positive or negative bodily effects. The unremitting nature of a chronic medication often causes an individual to question the need for the medication. Subsequently, the individual may exert control by altering the way he takes the medication and often in part because of the gained expertise with the medication in his own body.\nIn this paper, the themes of the medication experience are described as being essentially independent of one another; however there was a temporal location in which the meanings seemed to arise in the participants\u2019 stories. It appeared that the four themes are essentially stages of the medication experience or the experience of taking a chronic medication has a natural progression from a meaningful encounter to participants exerting control. Other scholars have recognized a pattern or stages that patients go through with specific medications. A study of young women using selective-serotonin reuptake inhibitors (SSRIs) for depression revealed that SSRI users passed through stages corresponding to how they felt about themselves [7]. Further research is needed to explore the validity of patients\u2019 medication experiences as a progression through stages or a journey.\nSince medications are among the most common options in the treatment and mitigation of diseases, it is essential for health care practitioners to acknowledge an individual\u2019s medication experience in order to positively influence patients\u2019 medication-taking behaviors. Patients\u2019 decisions, which at first appear irrational, might be seen as intelligent when a practitioner understands a patient\u2019s unique medication experience. Cipolle et\u00a0al. [1] state \u201ca practitioner cannot make sound clinical decisions without a good understanding of the patient\u2019s medication experience\u201d and urge practitioners to take responsibility for improving each patient\u2019s medication experience. The findings of this study provide pharmacists insights on the meanings that medications can have for patients, which may explain and certainly impact their medication-taking behavior. For example, understanding the potential meaning of initiating a chronic medication (e.g., losing control) may help explain a patient\u2019s reticence to taking the medication.\nA limitation of this study is that the synthesis of findings was from studies that explored the medication experience across multiple chronic conditions, which makes it difficult to discern the aspects of participants\u2019 experiences related to the unique illness experience of a specific disease. Additionally, the participants were all in the U.S. (though not all native born) and the medication experiences may vary in different cultures. Even though meta-synthesis addresses the issue of small sample sizes in qualitative research, this study still has a relatively small sample size (n\u00a0=\u00a041), which limits the applicability of these findings to others\u2019 experiences. Lastly, the authors re-analyzed their own work, which could have led to bias and reinforcement of the original findings.\nConclusions\nThe medication experience is a practice concept that serves to understand patients\u2019 experiences and to understand an individual patient\u2019s medication experience in order to meet his or her medication-related needs. The understanding of the medication experience presented in this paper can serve as a guide for health care practitioners who are interested in meeting a patient\u2019s medication-related needs and provide a foundation from which to interpret each individual\u2019s unique medication experience. Additional research is needed to further understand the medication experience and examine how it can be used to optimize patients\u2019 medication-taking behaviors as well as if there are stages of the medication experience that patients pass through.","keyphrases":["patients","medication experience","qualitative research","pharmaceutical care","united states","pharmacy services","medication management","patient-centered care"],"prmu":["P","P","P","P","P","M","R","M"]}
{"id":"Childs_Nerv_Syst-2-2-1705512","title":"Genome-wide microRNA profiling in human fetal nervous tissues by oligonucleotide microarray\n","text":"Objects Our objective was to develop an oligonucleotide DNA microarray (OMA) for genome-wide microRNA profiling and use this method to find miRNAs, which control organic development especially for nervous system.\nIntroduction\nTo date, 326 microRNAs (miRNAs), a ubiquitous family of about 22-nt noncoding regulatory RNAs, have been identified in the human genome and published in a database (http:\/\/www.sanger.ac.uk\/Software\/Rfam\/mirna\/index.shtml) [1]. Although more than 200 distinct miRNAs are predicted in the human genome, little is known about their biological functions, especially during embryonic development. MiRNAs regulate target genes at the post-transcriptional level and play important roles in development and cell lineage decisions. However, in vertebrates, neither the targets of miRNAs nor their expression profiles during development are well understood. Some miRNAs are specifically expressed in individual tissues and at particular developmental stages. The developmental or tissue-specific patterns of miRNA expression observed may suggest analogous roles in regulating human development or cellular differentiation [2\u20134].\nA major obstacle in the study of miRNA function is the lack of methods for quantitative expression profiling. The miRNA microarray method, a powerful tool for global analysis of miRNA expression first reported by Krichevsky et al. [5], is based on a membrane array spotted with specific antisense mature miRNA oligonucleotides. Since then, other types of oligonucleotide array methods have been published, including arrays on glass slides and on beads [6\u20139].\nIn this study, a robust microarray-based technique was established and used to identify the expression of 158 miRNAs in human fetal organs. The oligonucleotide microarray (OMA) was designed according to the method of Liu [7], with modifications. We used miRNA microarrays to study the profiles of miRNA expression in nervous tissue samples and other organic samples from two fetuses. Some clusters of miRNA families were co-expressed, providing clues about the maturation processes of miRNAs. At the same time, we found a high concordance between our array results and those from Northern blots. The microarray described here offers more comprehensive coverage and higher throughput than other methods, represents a powerful tool to better understand miRNA expression profiles in human tissues, and provides clues to the mechanisms for regulating protein translation.\nMaterials and methods\nSamples and RNA extraction\nTwo fetal abortuses [12\u00a0weeks (G12w) and 24\u00a0weeks (G24w) gestational age] tissues were obtained from the National Infrastructure Program of the Chinese Genetic Resources after obtaining informed consent. The G12w tissues were liver, kidney, cerebrum, lung, and heart, and the G24w tissues were the same, plus ovary, spleen, hypothalamus, pancreas, and cervical, thoracic, lumbar, and sacral spinal cord. Total RNA was isolated using TRIZOL reagent (Invitrogen) according to the manufacturer\u2019s standard protocol, and mRNA was purified by oligotex (Qiagen).\nMicroarray design\nThe miRNA oligonucleotide microarray design was based on that of spotted OMAs by Liu et al. [7], with significant modifications. The mature miRNA sequences, which ranged from 19 to 24 nt, were too short, so two probes were designed for several miRNAs, one containing active sites that detected both the mature and the precursor forms (probe a), while the other did not contain the active sites located in the precursor (probe b). An oligo probe was designed that was not homologous to any human sequence and was used as normalization control (control oligo); the sequence was 5\u2032 ATGTCATCTTGCGCGGCAGCTCGTCGACCGTGGCGAGAGT GTCTCGTCGATCATC 3\u2032. All oligos were 40 nt long. In theory, the mature miRNA expression level can be determined by subtracting the (a) signal from the (b) signal. Nine probes for nine tRNA genes were used as positive controls and six probes for six types of miRNA genes from Arabidopsis thaliana were used as negative controls.\nMicroarray fabrication\nAll probes were dissolved in 150\u00a0mM phosphate acid buffer (pH\u00a07.5\u20138.0) to a final concentration of 25\u00a0pmol\/\u03bcl. Then the control oligo was added to each miRNA probe at 2\u00a0pmol\/\u03bcl. The OMAs were spotted by a GeneMachine OmniGrid 100 Microarrayer in a 1\u00d74 pin and 9\u00d78 spot configuration for each subarray with triplicates. The spotting conditions were 75% humidity and 20\u00b0C. After spotting, slides were hydrated overnight in saturated salt solution and then crosslinked with UV light at 600\u00a0mJ\/cm2 (UVP CL1000).\nSample labeling\nThe cDNA was labeled during first strand synthesis by using a fluorescence-tagged (Cy5) random octameric primer. Briefly, 10\u00a0\u03bcg total RNA and 1\u00a0\u03bcg random primer were mixed and incubated at 70\u00b0C for 10\u00a0min, then 5 \u00d7 first-strand buffer, 0.1\u00a0M DTT, 5\u00a0mM unlabeled dNTP mix, Cy5-dCTP, RNA inhibitor, and Superscript II (200\u00a0U\/\u03bcl) were added and incubated at 42\u00b0C for 2\u00a0h, then denatured at 70\u00b0C for 10\u00a0min. NaOH was added to hydrolyze RNA to stop the reaction of reverse transcription, and then HEPES was added to neutralize it. The labeled cDNA was purified by using the QIAquick Nucleotide Removal Kit (Qiagene).\nMicroarray hybridization\nThe labeled cDNAs and Cy3-tagged oligonucleotides complementary to the control probes were dissolved in 6 \u00d7 SSPE\/5 \u00d7 Denhardt hybridization buffer and were hybridized with the miRNA oligonucleotide microarray for 16\u00a0h at 42\u00b0C. Then the slides were washed with buffer I (2 \u00d7 SSC\/0.5% SDS) for 15\u00a0min at 42\u00b0C, buffer II (1 \u00d7 SSC\/0.1% SDS) for 10\u00a0min at 42\u00b0C, buffer III (0.1 \u00d7 SSC) for 5\u00a0min at room temperature, dipped in double-distilled water for 1\u00a0min at room temperature, and then dried. The slides were scanned by an Agilent scanner (G2565AA) at 535 and 635\u00a0nm.\nStatistical analysis\nThe images were split into two, Cy3 and Cy5 channels, and each channel was imported into the Imagene Software 7.0 to read the signal value. The Cy3 signal was used as reference for the spot size of each miRNA oligo on the slides. The expression level of each miRNA in the sample labeled by Cy5 was normalized by a median method according to the Cy3 signal between two microarrays. So, the Cy5 signal, after normalization, gave the expression level of each miRNA. Clustering was carried out by Genespring Software 8.0 according to Cy5 intensity.\nNorthern blot analysis\nForty micrograms of total RNA from each sample was separated on 15% acrylamide denaturing gels (8\u00a0M urea) and then transferred to Hybond N+ membranes (Amersham) by electrophoresis for Northern blots. The filters were crosslinked with 150\u00a0mJ of UV (Bio-Rad) and baked at 80\u00b0C for 1\u00a0h. The specific oligo probes complementary to the corresponding miRNAs were labeled at the 5\u2032 end by using T4 polynucleotide kinase with 32P-\u03b3-ATP (Amersham). The sequence list of probes for Northern blots were: miR-92-2: 5\u2032- CAGGCCGGGACAAGTGCAATA-3\u2032; miR-9: 5\u2032- TCATACAGCTAGATAACCAAAGA-3\u2032; miR-9*: 5\u2032-ACTTTCGGTTATCTAGCTTTA-3\u2032; miR-15a: 5\u2032- CACAAACCATTATGTGCTGCTA-3\u2032; miR-17: 5\u2032- ACAAGTGCCTTCACTGCAGT-3\u2032; miR-20: 5\u2032-CTACCTGCACTATAAGCACTTTA-3\u2032; miR-106a: 5\u2032- GCTACCTGCACTGTAAGCACTTTT-3\u2032, and U6: 5\u2032-CGTTCCAATTTTAGTATATGTGCTGCCGAAGCGA-3\u2032 [10]. U6 on the membrane served as loading control. Prehybridization and hybridization were carried out using ExpressHyb Hybridization Solution (Clontech) according to the manufacturer\u2019s instructions. Membranes were washed at room temperature, twice with 2 \u00d7 SSC, 0.1% SDS and twice with 0.5 \u00d7 SSC, 0.1% SDS. The blots were exposed on Molecular Dynamics Phosphorimager screens and signals were quantified using ImageQuant (Molecular Dynamics). For reuse, blots were stripped by boiling in 0.5% SDS for 20\u00a0min and scanned on Phosphorimager screens. Blots without radioactive signals were re-hybridized and re-used up to six times without influencing the quality of radioactive signals.\nResults\nMiRNA oligo nucleotide microarray construction\nWe designed 243 oligonucleotide probes and covered 158 human miRNAs from http:\/\/microrna.sanger.ac.uk\/cgi-bin\/sequences\/browse.pl and from publications. Nine tRNAs served as positive controls. The oligonucleotide probes were 40\u00a0nt in length. For 67 miRNAs, two different probes were designed, one containing the active site and the other specific for the precursor. Using this strategy, we were able to analyze the miRNA and pre-miRNA in the same experiment. A control oligo probe, which was not homologous to any human gene sequence, was designed for spotting and quality control. All miRNA probes were dissolved in phosphate buffer at 25\u00a0\u03bcmol\/ml and the control probe was mixed with each miRNA probe at 2\u00a0\u03bcmol\/ml. The microarray slides were printed on an Ominigride 100 machine (Gene Machine). Each oligo probe was spotted in triplicate.\nTo develop the optimal conditions for the OMA, we compared mRNA and total RNA as the starting sample. Two micrograms of mRNA and 10\u00a0\u03bcg of total RNA were separately labeled during the reverse transcription reaction and hybridized to the OMA. The results indicated that mRNA had a relatively low signal compared to the total RNA, suggesting that mRNA had little influence on the results (Fig.\u00a01d), so total RNA could be used for the microarray experiment. The microarray hybridization time was tested at 4, 6, and 16\u00a0h. The hybridization signals at 6\u00a0h were considerably higher than those at 4\u00a0h, while the signals at 16\u00a0h were only slightly higher than those at 6\u00a0h (Fig.\u00a01a). To test the OMA sensitivity, quantities of total RNA ranging from 5 to 20\u00a0\u03bcg were labeled and then hybridized to the OMA (Fig.\u00a01c). The strongest signal was obtained at 10\u00a0\u03bcg total RNA. To find the optimal temperature, we tested hybridization at 42, 50, and 55\u00b0C. The signal was very low at 50 and 55\u00b0C (Fig.\u00a01b). From the above results, we chose 10\u00a0\u03bcg total RNA and 42\u00b0C hybridization for 16\u00a0h in all OMA experiments.\nFig.\u00a01miRNA oligonucleotiede microarrary construction. a The signal at hybridization times of 6 and 16\u00a0h was much higher than that at 4\u00a0h. b A hybridization temperature of 42\u00b0C was optimal for the microarray. c On testing quantities of total RNA from 5 to 20\u00a0\u03bcg, the signal was highest at 10\u00a0\u03bcg. d Comparison of total RNA and mRNA as the starting sample. The 100 highest signal pairs were chosen, and signal values greater than 3,000 were shown as 3,000. The results indicated that mRNA had little influence on the signal, so total RNA could be used as the starting material\nDistinct miRNA profiling in different organs during human development\nTotal RNAs of samples from human fetal organs at G12w and G24w were labeled during reverse transcription. The labeled cDNAs hybridized to the OMA. After data processing, we found that 72\u201383% of the miRNAs were expressed in human fetal organs, in which the most miRNAs were expressed in G24w cerebrum. Distinct miRNA expression patterns were found in different organs at each developmental stage (Fig.\u00a02a). Some miRNAs were seen in all samples from both embryonic stages (e.g., let-7d, miR-193, miR-7-3, miR-185, and miR-328, among others); they represent a type of restricted spatial and temporal expression pattern. These miRNAs were uniformly expressed over time, which suggests more general roles in gene regulation.\nFig.\u00a02Profiles of miRNome expression in human G12w and G24w fetal organs. a Distinct patterns of miRNA expression in different human fetal organs (abbreviation listed below). All of the data represent the average of at least three replicates from the same organ. b The same G24w heart sample triplicate chip results showed good reliability. Each result was from three replicates of the array. c Three stage-specific miRNAs, miR-92-2, miR-25, and miR-321, were expressed in G24w and G12w liver and lung. The bar represents the microarray signal intensity from 1,000 to 10,000. The blue line on the left indicates that the four miRNAs were clustered by cluster software analysis. C cerebrum, Hr heart, K kidney, Li liver, Lu lung, O ovary, P pancreas, S spleen, Vc cervical spinal cord, Vl lumbar spinal cord, Vs sacral spinal cord, Vt thoracic spinal cord\nTo test the replicability of the microarray, the G24w heart sample was labeled and hybridized in triplicate. The results from different detection batches were highly reproducible (Fig.\u00a02b). Similar miRNA expression patterns were seen in samples from developmentally proximal organs, indicating that a miRNA expression profile can be a marker of developmental stage in organs originating from the same endodermal layer. Three different miRNAs, miR-92-2, miR-25, and mirR-321, were predominantly expressed in liver and lung at G24w compared with the same organs at G12w (Fig.\u00a02c). As an indicator of miRNA expression level, the color of samples from G24w organs was brighter than those from G12w organs. So, the miRNA expression profile can be used as a biomarker of developmental stage.\nCNS specifically and higher expressed microRNAs\nIn our study, we found four CNS specifically expressed microRNAs, miR-9, miR-9*, miR-124a, and miR-125b, in which miR-124a and miR-125b are expressed higher in G24w cerebrum than in G12w. At the same time, miR-15a, miR-106a, and miR-17\u201392 cluster members were found expressed relatively higher in CNS tissues than other tissues. To validate the reliability of the microarray data, we selected seven CNS specifically and higher expressed miRNAs and designed specific probes for Northern blot analysis. The miRNAs were: miR-9, miR-9*, miR-15a, miR-106a, and miR-17\u201392 cluster members including miR-17, miR-20 and miR-92-1, which are located in the genomic site of both terminals and in the middle of the cluster. We found good consistency between the Northern blot and microarray data, as shown by the results from the selected miR-15a and miR-106a (Fig.\u00a03).\nFig.\u00a03Expression of miR-15a and miR-106a, detected by Northern blot. were in accord with the signals detected by microarray. The abbreviations are as in the legend of Fig.\u00a02. The miR-15a and miR-106a are shown on the left, indicating the corresponding expression detected by Northern blot. The microarray results are presented numerically, indicated by Oligo. U6 snRNA detected by Northern blot was used as detection control\nMiR-9, miR-9*, miR-124a, and miR-125b known to be specifically expressed in rat nervous system, are also only expressed in the human fetal nervous system found by microarray and Northern blot at the same time (Fig.\u00a04). Analysis of the array results indicated that miR-9 and miR-9* probably originated from the expressed pre-mi-RNA of chromosome 5 and may play key roles in human nervous system development. So it can be concluded that miRNA microarrays can identify different pre-miRNA origins by using different oligo probes.\nFig.\u00a04miR-9 and miR-9* were expressed specifically in the human nervous system. a miR-9 and miR-9* can be expressed from human chromosomes 1, 5, and 15 and form three types of pre-miRNA. The secondary structures of pre-miRNAs from the Sanger miRNA register website were predicted with mfold software. The pre-miRNAs can be excised by Dicer into the same mature miRNAs\u2014miR-9 and miR-9*. b Northern blot results probed with miR-9 and miR-9*. The abbreviations are as in the legend of Fig.\u00a02. The microarray results are presented numerically, indicated by Oligo. U6 snRNA detected by Northern blot was used as detection control\nMembers of the miR-17\u201392 cluster, which contains six pre-miRNAs within about 1\u00a0kb on human chromosome 13, share the same expression profile [11]. The microarray and Northern blot results showed that these six miRNAs had the same expression pattern (Fig.\u00a05), suggesting that clustered miRNAs have the same transcript and share the same promoter element.\nFig.\u00a05Co-expressed miR-17-92 cluster miRNAs. a The three known human miRNA clusters. The miR-17\u201392 cluster contains six pre-miRNAs within about 1\u00a0kb on chromosome 13. b The miR-17, miR-20, and miR-92-1 expression profiles detected by Northern blot (the name of the miRNA and pre-miRNA are given on the left and the abbreviations are as in the legend of Fig.\u00a02.) and microarray (results given numerically and marked as miR-17-oligo, miR-20-oligo, and miR-92-1-oligo). U6 snRNA detected by Northern blot was used as detection control\nDiscussion\nThe small size of miRNAs requires more sensitive tools for quantitative analysis. Although the optimized method of RT-Q-PCR can indirectly detect mature miRNAs [12], the efficiency of detection is relatively low. Currently, the most reliable method for the study of miRNA expression is Northern blot analysis with polyacrylamide gels. This method can distinguish pre-miRNA and miRNA at the same time, although the technique is relatively insensitive owing to the large total RNA volume needed, and it is labor-intensive.\nThe miRNA oligonucleotide microarray provides a novel method to carry out genome-wide microRNA profiling in human samples. We used total RNA as the sample for the microarray test, not just labeling filtered low molecular weight RNA, which could change the ratio of pre-miRNA to miRNA. So the profile we generated was that of pre-miRNAs and miRNAs. Owing to its high throughput and small sample requirement, the miRNA OMA can be used as screening method in miRNA research.\nSome microRNAs are within the introns of host genes. Intronic miRNAs are usually expressed in coordination with their host gene mRNA, suggesting that they are generally derived from a common transcript [13]. Some human microRNAs are even processed from capped, polyadenylated transcripts and can function as mRNAs [14]. Therefore, recognition of these miRNA gene families should help in the identification of putative mRNA targets and in understanding the pathways of miRNA biogenesis. Through GeneCluster software analysis, we found that miR-17, miR-18, miR-19a, miR-19b, miR-20, and miR-92-1 form a cluster.\nBased on the bioinformatics study and previous work, we attempted to verify the hypothesis that the miR-17\u201392 cluster may share the same expression unit. We searched for the genome location and possible co-expressed mRNA and found the six pre-miRNAs within about 1\u00a0kb on human chromosome 13; the possible co-expressed mRNA is human chromosome 13 open reading frame 25 (C13 orf25), transcript variant 2 mRNA, indicating that the miR-17\u201392 cluster members are intronic miRNAs. Analysis of clustered miRNA expression profiles suggested that the six clustered miRNAs may have the same promoter element.\nIn our study, we found that four miRNAs were only expressed in the human fetal nervous system, indicating that they may play important roles in human nervous system development. Before our study, Krichevsky et al. found miR-9 and miR-9* specifically expressed in mouse brain, and they were de-regulated in presenilin-1 null mice, which exhibited severe developmental defects of the brain [5]. MiR-9 and miR-9* may modulate critical development processes in human brain development and changes in stage and level of expression may induce major developmental errors. Giraldez et al. showed that miR-430 regulates brain morphogenesis in zebrafish and MZdicer mutants undergo axis formation and differentiate multiple cell types but display abnormal morphogenesis during gastrulation, brain formation, somitogenesis, and heart development. Injection of miR-430 rescues the brain defects in MZdicer mutants, revealing essential roles for miRNAs during morphogenesis [15].\nNo human miR-430 is registered online at present, but miR-17, miR-20, and miR-106a have the same sequence at nucleotides 2\u20138. Giraldez thought this is most important for recognition. In our study, members of the miR-17\u201392 cluster were highly expressed in the nervous system, but no human miRNA expression homologous to miR-430 was detected by Northern blot with a pre-miR-430 probe (data not shown). In humans, it is not miR-430 that plays a critical role in nervous system morphogenesis. So these results indicated that there are differences in the mechanisms of brain morphogenesis between human and zebra fish.\nThis raises the question of whether the specifically expressed miR-9, miR-9*, and higher expressed miR-17\u201392 cluster are functionally linked, or perhaps this just reflects higher nonspecific Dicer activity. He et al. compared B-cell lymphoma samples and cell lines to normal tissues and found that the levels of the primary or mature miRNAs derived from the miR-17\u201392 locus are often substantially increased in these cancers, implicating the miR-17\u201392 cluster as a potential human oncogene [16]. O\u2019Donnell et al. found that c-Myc-regulated miR-17 and miR-20 modulate E2F1 expression [17]. These findings indicate that the miR-17\u201392 cluster may be a common channel to regulate cell differentiation.","keyphrases":["microrna","oligonucleotide microarray","expression profile","nervous system development"],"prmu":["P","P","P","P"]}
{"id":"J_Chem_Ecol-4-1-2239250","title":"Performance of Generalist and Specialist Herbivores and their Endoparasitoids Differs on Cultivated and Wild Brassica Populations\n","text":"Through artificial selection, domesticated plants often contain modified levels of primary and secondary metabolites compared to their wild progenitors. It is hypothesized that the changed chemistry of cultivated plants will affect the performance of insects associated with these plants. In this paper, the development of several specialist and generalist herbivores and their endoparasitoids were compared when reared on a wild and cultivated population of cabbage, Brassica oleracea, and a recently established feral Brassica species. Irrespective of insect species or the degree of dietary specialization, herbivores and parasitoids developed most poorly on the wild population. For the specialists, plant population influenced only development time and adult body mass, whereas for the generalists, plant populations also affected egg-to-adult survival. Two parasitoid species, a generalist (Diadegma fenestrale) and a specialist (D. semiclausum), were reared from the same host (Plutella xylostella). Performance of D. semiclausum was closely linked to that of its host, whereas the correlation between survival of D. fenestrale and host performance was less clear. Plants in the Brassicaceae characteristically produce defense-related glucosinolates (GS). Levels of GS in leaves of undamaged plants were significantly higher in plants from the wild population than from the domesticated populations. Moreover, total GS concentrations increased significantly in wild plants after herbivory, but not in domesticated or feral plants. The results of this study reveal that a cabbage cultivar and plants from a wild cabbage population exhibit significant differences in quality in terms of their effects on the growth and development of insect herbivores and their natural enemies. Although cultivated plants have proved to be model systems in agroecology, we argue that some caution should be applied to evolutionary explanations derived from studies on domesticated plants, unless some knowledge exists on the history of the system under investigation.\nIntroduction\nThe occurrence of pests in agroecosystems has long promoted the study of insect\u2013plant interactions in crop plants, such as cabbage, lima bean, maize, cotton, and tomato (Takabayashi et al. 1994; Turlings et al. 1995; De Moraes et al. 1998; Geervliet et al. 2000; Thaler et al. 2001). Studies with crop plants have generated a wealth of data, highlighting a number of important mechanisms that influence the structure and function of multitrophic interactions and communities (Root 1973; Sheehan 1986; Khan et al. 1997; Gols et al. 2005). However, critics have argued that the biology and ecology of crop plants is often dramatically different from wild populations, thus bringing into some question the evolutionary relevance of the conclusions generated from data that rely on crop plants (Benrey et al. 1998; van der Meijden and Klinkhamer 2000). For instance, plant breeding programs have been reported to disrupt the original plant defense strategies that were present in the wild progenitors of cultivated species (Evans 1993). Artificial selection of some crop plants, aimed at accentuating a specific plant trait or group of traits (e.g., the production of edible structures), has been shown to reduce the level of undesired constituents, such as defense compounds, while enhancing others (such as primary metabolites including proteins and sugars). Many of the undesired secondary plant compounds (SPC) are known to have evolved and function as putative defenses against herbivores, whereas the desired primary plant compounds act as nutrients and thus may actually enhance the performance of herbivores (Schoonhoven et al. 2005).\nLevels of SPC are dynamic and vary with such factors as season, soil conditions, and leaf age (reviewed by Schoonhoven et al. 2005). Moreover, plants may increase their levels of defenses in response to feeding damage (Karban and Baldwin 1997; Agrawal 1999a), which may reduce the costs of defenses by avoiding the allocation of resources to defense when the attacker is absent. Secondary plant compounds have also been shown to affect negatively the development of higher trophic levels that attack these herbivores, such as predators, parasitoids, and even hyperparasitoids (Barbosa et al. 1986, 1991; Francis et al. 2001; Harvey et al. 2003, 2005, 2007; Ode et al. 2004). Consequently, changes in plant chemistry, mediated by artificial selection, may influence the behavior and development of consumers over several trophic levels, and this may ultimately lead to broader effects on the communities associated with these plants (Harvey et al. 2003; Ode 2006). Most importantly, in wild plants, defense mechanisms have not been constrained by the \u201cdirectional selection\u201d that characterizes many crop plants. Therefore, to understand the evolution of plant defenses against insect herbivores, multitrophic interactions should also be studied in wild conspecifics of the cultivated plant species where plant defenses are the likely result of a range of biotic and abiotic selection pressures.\nOne appropriate plant family for studying the effects of artificial versus natural selection on multitrophic interactions is the Brassicaceae, which contains such important crops as cabbages and various types of mustard (G\u00f3mez-Campo and Prakash 1999). Of all plants that have been domesticated, few have been manipulated to produce so many different cultivars as Brassica oleracea L. (e.g., cabbage, broccoli, cauliflower, and Brussels sprout; G\u00f3mez-Campo and Prakash 1999). Wild types of B. oleracea grow naturally along rocky coastlines of Britain and France (Mitchell and Richards 1979). It has been speculated that the wild populations in the UK are derived from plants that were cultivated by the Greeks and Romans in the Mediterranean region between 1,000 and 2,000 BC (Mitchell 1976). These early cultivated forms were introduced to Britain, but have been naturalized for centuries (Mitchell 1976). However, more recent evidence points also at an Atlantic origin of domestication (Song et al. 1990).\nPlants in the Brassicaceae characteristically produce secondary metabolites called glucosinolates (hereafter GS) (Fahey et al. 2002). After tissue damage, myrosinases catalyze the hydrolysis of GS into (iso)thiocyanates and nitriles (Mithen 2001; Fahey et al. 2002), which play a role in defense against insect herbivores (Rask et al. 2000). Generalist herbivores produce enzymes that can detoxify a wide range of substrates (Krieger et al. 1971), whereas specialists have evolved enzyme systems that can detoxify specific plant compounds that are associated with herbivore diet (e.g., Johnson 1999; Ratzka et al. 2002). Thus, generalist herbivores are usually more sensitive to high levels of specific allelochemicals compared to specialists. (see e.g., Blau et al. 1978; Giamoustaris and Mithen 1995).\nSpecialist feeding on brassicaceous plants are adapted to plants containing GS, and they detoxify, excrete, or even sequester these harmful metabolites (M\u00fcller et al. 2001; Ratzka et al. 2002; Wittstock et al. 2004). Moreover, some insects use these compounds as indicators of food plant suitability (Nayar and Thorsteinson 1963; Renwick and Lopez 1999). Not all GS are equally effective as stimulants, and high levels of GS can reduce the performance of herbivores that are specialized on brassicaceous species (Stowe 1998; Li et al. 2000; Traw and Dawson 2002; Agrawal and Kurashige 2003). In addition, GS concentrations can increase in response to herbivore feeding damage (Bodnaryk 1992; Agrawal 1999b) and negatively affect subsequent herbivory by both generalists and specialists (Agrawal 1999b; Bartlet et al. 1999; Traw and Dawson 2002).\nThis study compares the development of several species of herbivores and endoparasitoids when reared on three Brassica populations that differ in their degree of domestication. Insects were reared on a cultivated and a wild population of B. oleracea, and a recently escaped (feral) Brassica species. Levels of GS were measured as indicators of direct plant defense. Initially, the development of two specialists on Brassicaceae, Plutella xylostella L. (Lepidoptera: Plutellidae) and Pieris rapae L. (Lepidoptera: Pieridae), and a generalist herbivore, Mamestra brassicae L. (Lepidoptera: Noctuidae), were examined when reared on the three populations. Finally, the development of a specialist and generalist parasitoid reared on the same host, P. xylostella, were compared. Separate cohorts of P. xylostella were parasitized by two species of endoparasitoids, Diadegma semiclausum Hell\u00e9n (Hymenoptera: Ichneumonidae) and a related species, D. fenestrale Holmgren (Hymenoptera: Ichneumonidae). These two parasitoids differ in host specialization, with D. semiclausum restricted to P. xylostella and D. fenestrale attacking several other hosts that feed on non-brassicaceous species (Legaspi 1986; Azidah et al. 2000).\nThe following hypothesis was tested: specialist herbivores and parasitoids are less affected than generalists by differences in host plant chemistry between various Brassica populations that differ in their degree of domestication. It is proposed that changes in plant biology via domestication have significant effects on community level interactions and processes.\nMethods and Materials\nPlants The B. oleracea variety gemmifera (Brussels sprout) cv. Cyrus was used. Compared to other vegetable crops of B. oleracea, Brussels sprout cultivars contain relatively high levels of GS (Kushad et al. 1999; Rosa 1999), but considerably lower levels than the wild B. oleracea populations in Dorset, Great Britain (Moyes et al. 2000; Gols et al. 2008). Seeds from several plants (>10) were collected from a wild population of B. oleracea growing on chalk cliffs along the south coast of Great Britain, near Swanage, Dorset (\u201cOld Harry,\u201d 50\u00b038\u2032N, 1\u00b055\u2032E). This population contains intermediate levels of GS compared to other Dorset wild populations (Moyes et al. 2000). A feral Brassica population, which was found in a roadside hollow about 15\u00a0km east of Wageningen (51\u00b095\u2032N, 5\u00b078\u2032E, The Netherlands), was also included.\nIn addition to comparing the development of different herbivores, we also compared the development of a specialist and generalist parasitoid reared on the same host (see section on Plutella xylostella and Diadegma species). To discriminate between food-plant quality mediated through the host and host quality itself, a second closely related wild brassicaceous plant species, black mustard, Brassica nigra L., was included in one of the experiments. Seeds of B. nigra were collected from a natural population growing in a small patch along the River Rhine, near Wageningen, The Netherlands (51\u00b094\u2032N, 5\u00b062\u2032E).\nSeeds from the different populations were germinated in the first week of March 2005. Seedlings were transferred to 1.1 l pots filled with potting soil (\u201cLentse potgrond\u201d no. 4, Lent, The Netherlands). Plants were grown in a greenhouse at 20\u201330\u00b0C, 40\u201380% r.h, with a photoperiod of at least 16\u00a0hr. If the light dropped below 500\u00a0\u03bcmol photons\/m2\/sec during the 16-hr photoperiod, supplementary illumination was supplied by high-pressure mercury lamps. Plants were watered daily. After the plants were 4\u00a0wk old, they were fertilized once a week with Kristalon Blauw (N\u2013P\u2013K) 19\u20136\u201320\u20133 micro (2.5\u00a0mg\/l), which was applied to the soil. B. oleracea plants were 7\u00a0wk old when they were used in experiments and attained similar amounts of biomass (25\u201330\u00a0g per plant). Plants from all three populations were in the vegetative state and developed new leaves during the experiments. Fertilization and watering continued during the experiments. Brassica nigra plants were 5\u00a0wk old and were not fertilized because the soil still contained enough nutrients for optimal growth. B. nigra matures much faster than B. oleracea.\nInsects All insects used originated from cabbage fields in the vicinity of Wageningen. Cultures of all the herbivores have been maintained in the laboratory on Brussels sprouts cv. Cyrus for many years in climate rooms at 22\u2009\u00b1\u20092\u00b0C, 40\u201380% r.h, with a light regime of 16:8 L\/D. The two parasitoid species were collected in the summer of 2004 and were thereafter reared on plants heavily infested with P. xylostella larvae for several generations. After pupation on the walls of the rearing cage, parasitoid cocoons were carefully removed and transferred to a clean cage. Emerged adult wasps were provided with water and honey ad libitum. For parasitism, we used females that were 5 to 10\u00a0d old after adult emergence.\nGlucosinolate Analyses As an indicator of direct defense, GS concentrations in leaf tissues of B. oleracea were measured. Leaf samples were taken during the performance experiments (see below) from three treatment groups: plants that were undamaged, plants damaged by unparasitized P. xylostella, and plants damaged by larvae that had been parasitized by D. semiclausum. When leaf samples were taken, the damaged plant groups had been exposed to herbivore feeding for 7\u00a0d. Undamaged control plants were maintained in the same greenhouse, but were physically separated from the plants with caterpillars. Larvae, feces, and pupae were removed from leaves. All fully developed leaves were harvested with the exception of the oldest leaves, which had turned yellow and did not contain feeding damage. Leaves were removed with a razor blade, pooled per plant, and stored at \u221280\u00b0C. Samples were later freeze-dried and pulverized with a mortar and pestle. Fifty milligram aliquots of freeze-dried material were weighed in 2-ml centrifuge tubes. GS were extracted and purified as described in van Dam et al. (2004) and were separated on a reverse phase C-18 column (Alltima C-18, 3\u00a0\u03bcm,150\u2009\u00d7\u20094.6\u00a0mm, Alltech, Deerfield, IL, USA) on HPLC (Dionex, Sunnyvale, CA, USA) with an acetonitrile water gradient. Detection was performed with a DIONEX PDA-100 Photodiode array detector set to scan from 200 to 350\u00a0nm. For quantification, sinigrin (Sigma, St. Louis, MO, USA) was used as an external standard. Peaks were integrated at 229\u00a0nm, for which standard response factors have been defined (EC 1990). The different GS were identified based on their retention times, and UV spectra were compared to those of pure compounds (sinigrin, Sigma, St. Louis, MO, USA; glucotropaeolin and glucobrassicin were kindly provided by M. Reichelt, Max Planck Institute for Chemical Ecology, Jena, Germany), or compared to a certified oil seed reference (EC Community Bureau of Reference BCR-367R, Fluka, Buchs, Switzerland).\nInsect Performance To investigate the effect of domestication on plant direct defenses, the different herbivores and parasitoids (see below) were reared on the three plant populations. For all insects, egg-to-adult development time, adult dry mass, and survival (to adult) were determined when reared on the different populations. Adult dry mass was obtained by weighing adults on a Cahn C-33 microbalance (Cahn Instruments, Cerritos, CA, USA) that had been dried to constant weight at 80\u00b0C (3\u00a0d). Plants with insects were maintained in a greenhouse under the same conditions as described in the Plant section. Plants of the same population that received the same insect treatment were placed together, and caterpillars were allowed to develop and move around freely on plants until they reached the final instar. Different herbivore treatments were randomly positioned in a greenhouse, but were all placed in a similar position relative to the light sources to minimize microclimatic differences among plant populations and treatments.\nPlutella xylostella and Diadegma Species To obtain eggs of P. xylostella, more than 150 adult moths were released with a 50:50 sex ratio in a plastic cage (37\u2009\u00d7\u200940\u2009\u00d7\u200930\u00a0cm). Folded strips of Parafilm served as substrate for females to lay eggs on. Females were allowed to oviposit on the Parafilm overnight. Subsequently, the strips with eggs were incubated for 4\u00a0d at 22\u00b0C until the eggs hatched. Pieces of Parafilm with neonate larvae were placed on top of individual plants of each of the three plant populations. Larvae were allowed to feed on these plants until they reached the third instar (L3).\nFor each plant population, one cohort of 60 larvae was transferred to new plants and served as an unparasitized control. A second cohort of 130 larvae was parasitized by D. semiclausum, and a third cohort of 180 larvae was parasitized by D. fenestrale. For parasitism, individual female wasps of both species were presented with a L3 P. xylostella host. A host was considered as parasitized when the female wasp was observed to insert into and remove her ovipositor from the larva. Individual female wasps were allowed to oviposit in up to 10 separate hosts. After this, they were removed. Parasitized larvae were transferred to new plants of the same population on which the larvae had fed previously. Five plants were used for the unparasitized controls and nine for each of the parasitoid treatments. The number of plants provided ample food for all larvae to complete their development.\nWhen caterpillars molted into L4, strips of corrugated cardboard were placed on top of the plants, as P. xylostella prefers to pupate in secluded areas. After pupation, cocoons were collected and stored in labeled vials until adult emergence. When the moths or wasps emerged, the time of eclosion and sex were recorded. Individuals were killed by freezing at \u221220\u00b0C and stored for dry mass determination. Vials with cocoons ready to emerge were checked every 2\u00a0hr. Development time for P. xylostella was measured in full days, as the exact time of oviposition had not been recorded. In the case of the two Diadegma species, the median time point of the period needed to parasitize the hosts (3\u20134\u00a0hr) was used as the time of oviposition.\nTo further investigate whether food plant quality or host quality was a more important factor in the development of D. fenestrale, the experiments described above with P. xylostella were repeated on a second (and closely related) wild brassicaceous species, B. nigra. A separate study (Gols et al. unpublished) has shown that B. nigra is a qualitatively superior plant for the development of P. xylostella, compared with B. oleracea. We reared 33 unparasitized larvae on three B. nigra plants, and 180 larvae parasitized by D. fenestrale on 10 plants. We recorded egg-to-adult development time, adult biomass, and survival as before. The plants provided ample food for all the larvae to complete their development.\nPieris rapae Neonate larvae were obtained from the general culture and transferred to seven plants of each population with a distribution of six larvae per plant. When larvae had developed into L5, they were transferred to plastic containers that contained some leaf material from the plants they had fed on previously. After pupation, pupae were collected and placed in new plastic containers lined with filter paper. At adult emergence, the time of eclosion and sex were recorded, and the individuals were killed by freezing, followed by dry mass determination (as above). Containers with pupae ready to emerge were checked every 2\u00a0hr. Development time was measured in full days, as the exact time of oviposition had not been recorded.\nMamestra brassicae Like P. xylostella, adult M. brassicae are primarily nocturnal. Females do not need plants as an oviposition substrate and readily lay batches of eggs onto the surface of paper. From the general culture, we obtained paper sheets with M. brassicae eggs that were laid the previous night. These sheets were incubated at 22\u2009\u00b1\u20092\u00b0C (5\u00a0d) until the eggs hatched. Neonate larvae were transferred to 10 plants of each population, with a density of five larvae per plant. Once they had reached late L5, M. brassicae larvae were collected from the three populations, counted, and transferred to plastic containers (15\u2009\u00d7\u200912\u2009\u00d7\u20096\u00a0cm) that contained 2\u00a0cm of potting soil mixed with vermiculite (1:1) and some leaf material from the plant on which they had been feeding previously. After the larvae had pupated, they were collected and placed in new plastic containers filled with a layer of vermiculite. At moth emergence, the date of eclosion was recorded, and the individuals were killed by freezing, followed by dry mass determination. Containers with pupae ready to emerge were checked every 2\u00a0h. Development was measured in full days as the exact time of oviposition had not been recorded.\nStatistical Analysis Data on adult dry mass and development time were analyzed by using ANOVA with plant population and sex and their interaction as factors. All larvae within one plant population were considered as independent samples. The Tukey\u2013Kramer method was used for multiple comparisons of the means. For each insect species, a G test for heterogeneity was performed on survival rates of the three plant populations with H0: survival on each of the three plant populations is equal.\nConcentrations of individual GS compounds were log(x+1) transformed to meet assumptions of normality. To examine differences in GS content, a Mixed Model was used with plant population and plant treatment (intact, damaged by unparasitized Plutella, damaged by parasitized Plutella) as the fixed factors in the model. There was no random factor in the analysis, and the estimation of effects in the model was based on restricted likelihood maximization. When the main factors or their interactions were significant, specific linear contrasts were applied to separate further factor levels. When necessary, correction for unbalanced sample sizes was carried out by using the Satterthwaite correction. Analysis was carried out with SAS 8.02 (1999\u20132001 \u00a9SAS Institute, Inc).\nResults\nGlucosinolate Analyses GS analyses of leaf tissues revealed considerable quantitative and some qualitative variation among the different Brassica populations (Fig.\u00a01). Three pentyl-derived (C5) GS, glucoalyssin, gluconapoleiferin, and glucobrassicanapin were only detected in the feral population, whereas the other 10 compounds were present in all three populations (Fig.\u00a01). Total GS concentrations in undamaged plants were 3.2 and 1.4 times higher in plants of the wild population than in the cultivated and feral populations, respectively (Fig.\u00a01). Furthermore, concentrations of all individual compounds were significantly different among the three populations (statistics not shown, but all significance levels were lower than 0.05).\nFig.\u00a01Glucosinolate (GS) concentrations (mean \u00b1 SE) in leaf tissues of a cultivated (a), feral (b), and wild (c) Brassica population. Concentrations were measured in leaf tissue from plants that were undamaged (black bars), damaged by unparasitized P. xylostella larvae (white bars) and damaged by parasitized (D. semiclausum) larvae (gray bars). GS abbreviations and scientific names: TOT total GS concentration, GBC glucobrassicin (= indol-3-ylmethyl GS), IBE glucoiberin (= 3-methylsulfinyl propyl GS), GNA gluconapin (= 3-butenyl GS), NEO neoglucobrassicin (= 1-methoxyindol-3-ylmethyl GS), RAP glucoraphanin (= 4-methylsulfinyl butyl GS), SIN sinigrin (= 2-propenyl GS), PRO progoitrin (= 2(R)-2-hydroxy-3-butenyl GS), GBN glucobrassicanapin (= 4-pentenyl GS), GNL gluconapoleiferin (= 2-hydroxy-4-pentenyl GS), ALY glucoallyssin (= 5-methylsulfinyl pentyl GS), 4OH 4-hydroxyglucobrassicin (= 4-hydoxyindol-3-ylmethyl GS), 4MeOH, 4-methoxyglucobrassicin (= 4-methoxyindol-3-ylmethyl GS), and NAS gluconasturcin (= 2-phenylethyl GS). ND not detectable\nDifferences between the cultivated and feral populations on the one side, and the wild population on the other side, became even more pronounced after the plants were induced by larval P. xylostella feeding (Fig.\u00a01). In the cultivated and feral plants, total levels of GS remained at similar levels before and after induction by P. xylostella feeding (cultivar: t50\u2009=\u20090.82, P\u2009=\u20090.42; feral population: t50\u2009=\u20090.39, P\u2009=\u20090.70), whereas in the wild population, concentrations were 1.5\u20132 times higher after herbivore feeding (t50\u2009=\u20093.11, P\u2009<\u20090.001). Levels of individual GS changed differentially in response to herbivore feeding. The indole GS, glucobrassicin, was induced by P. xylostella feeding in all the plant populations (t50\u2009=\u20096.90, P\u2009<\u20090.001). Moreover, glucobrassicin accounted for almost 70% of the GS composition in the wild population after induction, and for only 53% and 35% in the cultivated and feral population, respectively. In contrast, the relative amount of glucobrassicin in undamaged plants was only 21%, 5%, and 25% in the cultivated, feral, and wild populations, respectively. A second indole GS, neoglucobrassicin, was also induced in response to herbivory in the feral (t50\u2009=\u20092.55, P\u2009=\u20090.01) and the wild population (t50\u2009=\u20094.76, P\u2009<\u20090.001), but not in the cultivated population (t50\u2009=\u20090.37, P\u2009=\u20090.71) in which levels of this compound were very low.\nNot all GS concentrations increased after herbivory. Sinigrin was reduced after P. xylostella larval feeding in the cultivated (t50\u2009=\u20092.4, P\u2009=\u20090.02) and the wild population (t50\u2009=\u20093.67, P\u2009<\u20090.001), but not in the feral population (t50\u2009=\u20090.07, P\u2009=\u20090.95). Similarly, levels of glucoiberin decreased in response to P. xylostella feeding in the cultivar (t50\u2009=\u20092.79, P\u2009=\u20090.007) and the wild population (t50\u2009=\u20094.22, P\u2009<\u20090.001), but not in the feral population (t50\u2009=\u20090.02, P\u2009=\u20090.99). In the feral population, both sinigrin and glucoiberin were present in much lower concentrations than in the other two populations (Fig.\u00a01). In plants damaged by parasitized and unparasitized larvae of P. xylostella, concentrations of individual GS were not significantly different (statistics not shown, but all significance levels were lower than 0.05).\nInsect Performance: Herbivores In P. xylostella, plant population and sex had an effect on egg-to-adult development time (plant population: F2, 131\u2009=\u20095.95, P\u2009=\u20090.003; sex: F1, 131\u2009=\u20095.52, P\u2009=\u20090.02; Fig.\u00a02a). Female P. xylostella developed faster than males; the fastest development time was observed for females reared on the cultivated and the feral population. For adult biomass, the interaction between plant population and sex was significant (F2, 132\u2009=\u200911.1, P\u2009<\u20090.001). Plant population had a strong effect on female but not on male biomass (Fig.\u00a02b). The heaviest females were recovered from the cultivar, and the lightest from the wild population. In contrast, males were significantly lighter than females (F1, 132\u2009=\u2009317, P\u2009<\u20090.001) and obtained similar biomasses on all three plant populations (Fig.\u00a02b). For P. xylostella, plant population did not affect larval survival to the adult stage (\u03c72\u2009=\u20090.96, df\u2009=\u20092, P\u2009=\u20090.62, Fig.\u00a03).\nFig.\u00a02Egg-to-adult development time (a) and adult dry mass (b) of P. xylostella males (white bars) and females (gray bars) reared on either a cultivated, a feral, or wild Brassica population. Bars (mean \u00b1 SE) with different letters are significantly different from each other (Tukey multiple comparisons, \u03b1\u2009=\u20090.05). Numbers of individuals (N) were on the cultivar, escape, and wild population, respectively: males, 21, 22, and 31; females 23, 22, 18Fig.\u00a03Larval to adult survival of two specialist herbivores, P. xylostella and P. rapae, and one generalist, Mamestra brassica, when reared on either a cultivated, feral or wild Brassica population. P. xylostella was also reared on a wild population of B. nigra\nDevelopment time also varied with the population on which the P. rapae larvae had been reared (F2, 105\u2009=\u20097.8, P\u2009<\u20090.001, Fig.\u00a04a). Egg-to-adult development time of males was shortest on the cultivar, longer on the feral, and longest on the wild population (Fig.\u00a04a). Males took longer to complete their development than females (F1, 105\u2009=\u20097.5, P\u2009=\u20090.007). Plant population also had a significant effect on adult biomass (F2, 101\u2009=\u200917.5, P\u2009<\u20090.001, Fig.\u00a04b). Whereas adult biomass in P. rapae did not differ between the cultivated and the feral line, biomass of butterflies reared on the wild population was lower (Fig.\u00a04b). On average, females were marginally heavier than males (F1, 101\u2009=\u20093.2, P\u2009=\u20090.08). Irrespective of plant population, more than 92% of all P. rapae larvae successfully developed into adults, and survival rates were not significantly different (\u03c72\u2009=\u20090.22, df\u2009=\u20092, P\u2009=\u20090.90, Fig.\u00a03).\nFig.\u00a04Egg-to-adult development time (a) and adult dry mass (b) of P. rapae males (white bars) and females (gray bars) reared on either a cultivated, feral, or wild Brassica population. Bars (mean \u00b1 SE) with different letters are significantly different from each other (Tukey multiple comparisons, \u03b1\u2009=\u20090.05). Numbers of individuals (N) were on the cultivated, feral, and wild population, respectively: males, 22, 10, and 19; females 16, 20, 20\nIn the case of the generalist herbivore, M. brassicae, the effect of host-plant population on adult biomass was more pronounced compared to the two specialists. It is difficult to determine the sex of adult moths, therefore, the data were pooled. Adult mass on the cultivated line was twice as high, compared to the wild population, and was also significantly higher on the feral population (F2, 49\u2009=\u20098.28, P\u2009<\u20090.001, Fig.\u00a05b). Plant population did not affect development time (F2, 49\u2009=\u20090.33, P\u2009=\u20090.72, Fig.\u00a05a). Unlike the two specialists, survival of M. brassicae was affected by plant population (\u03c72\u2009=\u200920.4, df\u2009=\u20092, P\u2009<\u20090.001, Fig.\u00a03). The percentage of M. brassicae larvae that developed successfully into adults was highest on the cultivated population (58%), slightly lower on the feral population (42%), and the smallest (4%) on the wild population.\nFig.\u00a05Egg-to-adult development time (a) and adult dry mass (b) of M. brassicae reared on either a cultivated, feral, or wild Brassica population. Bars (mean \u00b1 SE) with different letters are significantly different from each other (Tukey multiple comparisons, \u03b1\u2009=\u20090.05). Numbers of individuals (N) were on the cultivated, feral, and wild population, respectively: 21, 29, and 2\nInsect Performance: Parasitoids In the specialist parasitoid, D. semiclausum, plant population had an effect on egg-to-adult development time (F2, 275\u2009=\u20096.49, P\u2009=\u20090.002, Fig.\u00a06a). The parasitoid developed fastest on the feral population and developed more slowly on the cultivated and wild populations of B. oleracea (Fig.\u00a06a). Egg-to-adult development time was longer in females than in males (F1, 275\u2009=\u200913.0, P\u2009<\u20090.001). Furthermore, plant population had an effect on adult biomass in D. semiclausum, (F2, 277\u2009=\u200911.3, P\u2009<\u20090.001, Fig.\u00a06b). The heaviest D. semiclausum wasps emerged from hosts that were reared on the cultivated population, whereas the lightest emerged from host reared on the wild and feral populations. D. semiclausum females were heavier than males (F1, 277\u2009=\u200957.4, P\u2009<\u20090.001). Between 67 and 80% D. semiclausum successfully completed development to eclosion on the three Brassica populations (Fig.\u00a07). Survival rates were not significantly different on the three plant populations (\u03c72\u2009=\u20093.77, df\u2009=\u20092, P\u2009=\u20090.15).\nFig.\u00a06Egg-to-adult development time (a) and adult dry mass (b) of D. semiclausum males (white bars) and females (gray bars) reared on P. xylostella feeding on either a cultivated, feral, or wild Brassica population. Bars (mean \u00b1 SE) with different letters are significantly different from each other (Tukey multiple comparisons, \u03b1\u2009=\u20090.05). Numbers of individuals (N) were on the cultivated, feral, and wild population, respectively: 87, 69, and 81 for males and 11, 12, 10 for femalesFig.\u00a07Egg-to-adult survival of a specialist parasitoid, D. semiclausum, and a generalist parasitoid, D. fenestrale, when reared from the host, P. xylostella, on either a cultivated, feral, or wild Brassica population. D. fenestrale was also reared from P. xylostella on a wild population of B. nigra\nAs with D. semiclausum, egg-to-adult development time in D. fenestrale varied with plant population (F2, 69\u2009=\u20095.27, P\u2009=\u20090.007, Fig.\u00a08a). However, dry mass in male D. fenestrale wasps did not vary significantly with the population on which the hosts had been reared (F2, 69\u2009=\u20090.25, P\u2009=\u20090.78, Fig.\u00a08b). As only thirteen D. fenestrale females in total successfully developed, data on females were excluded from the analysis and are not presented in the figures. The most dramatic effect of plant population was on the survival of D. fenestrale (Fig.\u00a07), which was significantly different for the three plant populations (\u03c72\u2009=\u200912.2, df\u2009=\u20092, P\u2009=\u20090.002). Only 9% of the parasitized hosts reared on the wild population successfully produced D. fenestrale wasps. By contrast, 33% survived on the cultivar and 16% on the feral population. Moreover, the lowest number of surviving D. fenestrale parasitoids, eight out of 172 (or 4.6%), was obtained on B. nigra. Male wasps (N\u2009=\u20095) developing in hosts reared on B. nigra plants were lighter (0.432\u2009\u00b1\u20090.033\u00a0mg, mean \u00b1 SE) and developed slower (17.8\u2009\u00b1\u20090.37 d) than males reared from hosts on B. oleracea (see Fig.\u00a06). In contrast, healthy P. xylostella moths performed better on B. nigra than on B. oleracea, in terms of survival (97%, Fig.\u00a03) and adult mass, 1.985\u2009\u00b1\u20090.068\u00a0mg (N\u2009=\u200916) and 1.104\u2009\u00b1\u20090.068\u00a0mg (N\u2009=\u200916), for females and males, respectively (see also Fig.\u00a02b). However, development time, 17.8\u2009\u00b1\u20090.26 d for both female and male moths, was slightly longer (see also Fig.\u00a02a).\nFig.\u00a08Egg-to-adult development time (a) and adult dry mass (b) of D. fenestrale males reared on P. xylostella feeding on either a cultivated, feral, or wild Brassica population. Bars (mean \u00b1 SE) with different letters are significantly different from each other (Tukey multiple comparisons, \u03b1\u2009=\u20090.05). Numbers of individuals (N) were on the cultivar, escape, and wild population, respectively: 44, 18, and 14\nDiscussion\nThrough artificial selection via domestication, levels of primary and secondary compounds in domesticated plants are often altered compared with their progenitors. The results of this study revealed that GS concentrations in leaf tissue varied significantly among the three different plant populations. Much higher concentrations of GS were recorded in wild B. oleracea than in cultivated and feral population. GS levels in the wild population studied in this paper were similar to concentrations reported earlier in other wild populations of B. oleracea (Mithen et al. 1995; Moyes et al. 2000). The most striking differences in GS concentrations were observed after induction by herbivore feeding, especially in the wild population. In wild plants, total GS concentrations were 1.7 times higher in induced plants than in uninduced conspecific plants, and were 2.7 and 4.9 times higher after herbivory than in induced plants of the cultivated and feral population, respectively. By contrast, the total GS concentrations in induced plants remained at the same level as in undamaged plants in the cultivated and feral populations, although differences were found for levels of individual compounds.\nThe feral population differed from the other two populations with respect to pentyl-derived GS, which were absent in the cultivated and wild population. These C5 GS usually are not found in B. oleracea crops (Rosa 1999), indicating that this population may have crossed with a closely related species such as B. napus or B. rapa, both of which contain pentyl GS (Giamoustaris and Mithen 1995). Moreover, both B. napus and B. rapa are cultivated and grow naturally in the Netherlands. In all plant populations, concentrations of the indole GS, glucobrassicin, increased the most after herbivore feeding, but this compound was dominant only in the wild population, accounting for 70% of the GS content. Several studies have reported on herbivore-induced changes in GS concentrations in both cultivated and wild brassicaceous species. In line with our results, previous studies have shown that levels of indole GS increase in response to insect wounding (Bodnaryk 1992; Agrawal et al. 1999; Bartlet et al. 1999; Gols et al. 2008). We found that concentrations of some of the aliphatic GS were lower in plants that had been damaged by P. xylostella than in undamaged plants. Previous studies that used different herbivores reported that levels of aliphatic GS were either unaffected (Bodnaryk 1992; Bartlet et al. 1999; Traw and Dawson 2002; Gols et al. 2008) or even increased (Traw and Dawson 2002) in response to insect wounding. These results suggest differential induction of aliphatic GS by different herbivores (see also Traw and Dawson 2002).\nGlucosinolates also play a major role in determining a plant\u2019s nutritional quality, not only for humans and livestock but also for pathogens and insect herbivores (Chew 1988; Mithen 1992). This study shows that plants from a wild population of B. oleracea are less suitable for the development of several herbivores than plants from a cultivated and a feral population. However, the severity of these effects differed between the specialists P. rapae and P. xylostella on one hand, and the generalist M. brassicae on the other. In P. rapae, emerging adult butterflies were smaller and took longer to complete development when reared on wild plants than on the other two populations. These effects were less pronounced in P. xylostella; however, where adult body mass was more negatively affected than development time. Importantly, in both of the specialist herbivores, survival was high, irrespective of the plant population on which the larvae had been reared. By contrast, adult body mass and survival in the generalist herbivore, M. brassicae, were significantly lower when reared on the wild B. oleracea strain. This reveals that costs in terms of reduced fitness are higher for generalist herbivores than for specialists when they feed on the more toxic wild plants. Similarly, Giamoustaris and Mithen (1995) found a negative relationship between GS content in oilseed rape (B. napus) and the amount of leaf damage by generalist herbivores, but a positive relationship for specialists. Levels of GS, especially indole GS, were higher in the wild population of B. oleracea than levels of GS found in B. napus by Giamoustaris and Mithen (1995). These results suggest that indole GS, especially neoglucobrassicin, which is present in very low concentrations in the cultivar, may play a role in reducing the performance of insect herbivores.\nSpecialist insect herbivores, in contrast with generalists, may use GS as indicators of host plant suitability. For example, GS serve as feeding stimulants for insect herbivores specialized on plants belonging to the Brassicaceae (Nayar and Thorsteinson 1963; David and Gardiner 1966; Renwick and Lopez 1999). However, not all GS are equally active as feeding stimulants (e.g., Nayar and Thorsteinson 1963), and high levels of GS can even be toxic for specialists (Agrawal and Kurashige 2003). As such, high levels of specific GS may be responsible for the reduced performance of the specialists P. xylostella and P. rapae when reared on the wild population. Furthermore, levels of the enzyme myrosinase, which catalyzes hydrolysis of GS into the more toxic (iso)thiocyanates and nitriles (Rask et al. 2000; Mithen 2001), may also have differed among the populations. In addition, host plant quality is not determined only by the presence of allelochemicals. Nutrients, such as proteins and carbohydrates, as well as digestibility reducers, also play a role (Slansky 1993). It is possible that levels of limiting nutrients such as nitrogen and other defense-related compounds also vary across the three populations and thus amplify differences in performance caused by GS (Slansky and Feeny 1977).\nThe performance of the two parasitoid species reared on P. xylostella also varied with the plant population on which the host had been reared. However, there were also significant differences in performance between the specialist parasitoid, D. semiclausum, and the congeneric generalist parasitoid, D. fenestrale. The development of D. semiclausum was directly affected by the development of the host. Adult body mass was reduced when developing on wild B. oleracea plants, whereas development time and survival were unaffected, revealing that D. semiclausum ontogeny is affected by quantitative changes in host quality as mediated through the diet of their host. Alternatively, the development of D. fenestrale was characterized less by direct differences in host quality than by indirect population-related variations in plant quality. Although P. xylostella survival was high (>80%) on all B. oleracea populations, as well as on B. nigra plants, mortality in D. fenestrale was much higher on the wild Brassica populations.\nAs in most endoparasitoids, larvae of D. fenestrale primarily consume host hemolymph and fat body during early development. They only begin to indiscriminately attack other tissues later during development (the so-called \u201cdestructive feeding phase\u201d). In this way, they do not kill the host until the last possible moment. Larvae of P. xylostella are known to utilize enzymes that convert GS into desulfo-GS in their gut, which are then excreted with their feces (Ratzka et al. 2002). Because of their polarity, GS presumably do not permeate the host-gut membrane but effectively remain in the gut before they are excreted. Consequently, the larvae of D. fenestrale probably ingest little, if any, GS when feeding on hemolymph. However, during the destructive feeding phase, the parasitoid larvae undoubtedly consume the host gut and its contents, and this is when the toxic effects of plant allelochemicals on non-adapted parasitoids may be realized. In hosts parasitized by D. fenestrale, mortality mainly occurred just before the parasitoids would have been expected to pupate (personal observation) supporting this argument. Furthermore, the gut of endoparasitoid larvae is not externally connected until after emergence from the host. The excretion of wastes into internal host tissues would facilitate bacterial infection and precocious death of both the host and the developing parasitoid (Harvey et al. 2003). Thus, low concentrations of allelochemicals that are ingested by parasitoid larvae are stored and presumably accumulate in their tissues. This may account for the high mortality recorded here with D. fenestrale that developed in P. xylostella caterpillars reared on mustard and wild cabbage plants, which contain high levels of GS.\nAll insects used in this study have been reared on the Cyrus cultivar for many generations and may have adapted to this plant population. However, P. xylostella developed more successfully on B. nigra plants than on the cultivar, with moths enjoying higher survival and larger body mass. Furthermore, the performance of Mamestra brassicae and Pieris rapae was almost similar on the feral and the cultivated population. We cannot exclude that the observed differences are the result of rearing history rather than true plant effects, but the fact that the insects have no history with the recently cultivated feral population of B. oleracea and developed with equal success as on the cultivar suggests that plant quality is affected by domestication. Moreover, the development of the insects was more strongly negatively affected on the wild \u201cOld Harry\u201d population than on the feral population.\nIn summary, this study has shown that plant quality in terms of development of herbivores and their natural enemies differs significantly between wild and cultivated populations of B. oleracea. The identity of the food plant and the degree of specialization exhibited by the herbivores and their parasitoids influenced the degree to which plant population affected performance. Most importantly, these results demonstrate that artificial selection in which certain plant traits are accentuated at the expense of others can alter a significant part of a plant\u2019s evolved physiology. This may in turn have large impacts on insect communities that are associated with these plants. In wild plants, defense mechanisms have evolved under natural selection pressures from herbivores and pathogens and by the effects of natural enemies on herbivore populations. The reduction of the levels of direct defenses in cultivated plants could partly explain why these plants have often become more susceptible to attack from a wide range of herbivores and pathogens. To better understand the relative contribution of insect herbivory as a selective agent on the evolution of plant defenses, these traits should be studied in wild populations in which defense mechanisms have not been constrained by the \u201cdirectional selection\u201d that characterizes many species of crop plants. Future studies should examine insect communities associated with plant populations, including cultivars that differ in resistance against insect herbivores in plots in which the structural heterogeneity is also manipulated. This will facilitate a better understanding of the role that artificial selection has played in shaping the structure of communities associated with cropping systems.","keyphrases":["diadegma fenestrale","plutella xylostella","glucosinolates","pieris rapae","mamestra brassicae","diadegma semiclausum","direct defense","induction","crucifer"],"prmu":["P","P","P","P","P","P","P","P","U"]}
{"id":"Eur_J_Appl_Physiol-3-1-2039810","title":"The effect of ambient temperature on gross-efficiency in cycling\n","text":"Time-trial performance deteriorates in the heat. This might potentially be the result of a temperature-induced decrease in gross-efficiency (GE). The effect of high ambient temperature on GE during cycling will be studied, with the intent of determining if a heat-induced change in GE could account for the performance decrements in time trial exercise found in literature. Ten well-trained male cyclists performed 20-min cycle ergometer exercise at 60% (power output at which VO2max was attained) in a thermo-neutral climate (N) of 15.6 \u00b1 0.3\u00b0C, 20.0 \u00b1 10.3% RH and a hot climate (H) of 35.5 \u00b1 0.5\u00b0C, 15.5 \u00b1 3.2% RH. GE was calculated based on VO2 and RER. Skin temperature (Tsk), rectal temperature (Tre) and muscle temperature (Tm) (only in H) were measured. GE was 0.9% lower in H compared to N (19.6 \u00b1 1.1% vs. 20.5 \u00b1 1.4%) (P < 0.05). Tsk (33.4 \u00b1 0.6\u00b0C vs. 27.7 \u00b1 0.7\u00b0C) and Tre (37.4 \u00b1 0.6\u00b0C vs. 37.0 \u00b1 0.6\u00b0C) were significantly higher in H. Tm was 38.7 \u00b1 1.1\u00b0C in H. GE was lower in heat. Tm was not high enough to make mitochondrial leakage a likely explanation for the observed reduced GE. Neither was the increased Tre. Increased skin blood flow might have had a stealing effect on muscular blood flow, and thus impacted GE. Cycling model simulations showed, that the decrease in GE could account for half of the performance decrement. GE decreased in heat to a degree that could explain at least part of the well-established performance decrements in the heat.\nIntroduction\nPerformance decrements are widely observed during exercise in the heat compared to thermo-neutral circumstances (Febbraio et al. 1996; Gonzalez Alonso et al. 1999; Parkin et al. 1999; Romer et al. 2003; Tatterson et al. 2000; Tucker et al. 2004). It has been shown that fatigue at exhaustion is related to factors associated with thermoregulation and hyperthermia (Febbraio et al. 1996; Nielsen et al. 1990; Parkin et al. 1999). Time trial performance and fatigue (evidenced by decrements in power output over the race) and finish time have received much less attention in the literature. Accordingly, the present study will focus on the effect of a hot ambient temperature on thermal and cardio respiratory strain during the exercise that might contribute to the well-established decrease in power output.\nTatterson et al. (2000) measured time trial performance on a 30\u00a0min self-paced cycling time trial in 32\u00b0C, 60% RH versus 23\u00b0C, 60% RH. They observed that power output was reduced in the heat by 6.5% (345\u00a0\u00b1\u00a09\u00a0W vs. 323\u00a0\u00b1\u00a08\u00a0W). Tucker et al. (2004) compared 20\u00a0km time trials in 35 and 15\u00b0C and found a comparable reduction in average power output in the heat of about 6.3% (255\u00a0\u00b1\u00a047\u00a0W vs. 272\u00a0\u00b1\u00a045\u00a0W). This change in average power output led to a difference in final time on a 20\u00a0km time trial of about 48\u00a0s (29.6\u00a0\u00b1\u00a01.9\u00a0min vs. 28.8\u00a0\u00b1\u00a01.8\u00a0min), which equals about 2.8%. Possible causes for this deterioration in performance are associated with elevated body temperature (Tatterson et al. 2000) and anticipatory changes in pacing strategy to avoid hyperthermia (Tucker et al. 2004). Although most research has revolved around neuromuscular function, central drive and fatigue at the point of exhaustion, a plausible explanation for the deterioration in time trial performance in heat may also be found in a temperature-induced change in gross-efficiency (GE). GE is an important variable in cycling performance (Lucia et al. 2002; Moseley et al. 2004) and a linear relationship between body temperature and GE has been observed (Daanen et al. 2006).\nA possible explanation for the decreased GE in the heat could be mitochondrial leakage. A temperature induced metabolic disruption caused by non-specific proton-leakage across the inner mitochondrial membrane has been shown to occur at high muscle temperatures (Brooks et al. 1971; Willis and Jackman 1994), resulting in a decrease in ADP:O ratio. Further, a heat induced skin vasodilatation could occur in heat. To prevent a resulting decrease in blood flow to exercising and respiratory muscles (Nielsen et al. 1990; Romer et al. 2003; Rowell et al. 1966) a higher cardiac output must exist to continue supplying the muscles with the same blood flow, but still sending extra blood to the skin for cooling.\nIn the present study we sought to determine the effect of heat on GE. GE can be assessed accurately during sub-maximal exercise at intensities as high as 60\u221280% VO2max by calculating the ratio between mechanical and metabolic (mainly dependent on aerobic energy metabolism) power output, as has been done in Daanen et al. (2006), Foster et al. (2003), Hettinga et al. (2006), Lucia et al. (2002), Moseley et al. (2001, 2004). The presence of a possible effect of ambient temperature on GE can serve as input for an energy flow model (De Koning et al. 1999) to quantify the impact of this effect on time trial performance.\nMaterials and methods\nSubjects\nTen healthy, non-smoking, well-trained male subjects, familiar with cycling exercise at the club-level, participated in this study. They were informed of the nature of the experiment and provided written informed consent. Subject characteristics are presented in Table\u00a01. The study was approved by the Medical Ethical Committee of the University Medical Centre Utrecht (The Netherlands).Table\u00a01Subject characteristicsMean value\u00a0\u00b1\u00a0SDAge (years)23.5\u00a0\u00b1\u00a04.4Height (cm)179.7\u00a0\u00b1\u00a09.1Body mass (kg)72.5\u00a0\u00b1\u00a07.2VO2max (l\u00a0min\u22121)4.78\u00a0\u00b1\u00a00.41(W)354\u00a0\u00b1\u00a029Values are mean\u00a0\u00b1\u00a0SD\nExperimental design\nIncremental test\nThe subjects first performed an incremental bicycle test to determine at which power output VO2max was attained  The incremental test was performed in the heat, under similar conditions as the constant intensity bouts, to make sure that  would not be overestimated in the hot condition. This incremental test was used solely to determine the relative intensities for the constant intensity cycling bouts. The test began at a power output (PO) of 150\u00a0W, after which PO was increased with 15\u00a0W every minute. Exercise was performed on an electronically braked cycle ergometer (Lode Excalibur, Lode NV, Groningen, The Netherlands) until exhaustion or until pedal frequency dropped below 80\u00a0rpm. Oxygen consumption (VO2) was measured breath by breath, using open circuit spirometry (Oxycon Pro-Delta, Jaeger, Hoechberg, Germany). The gas analyzer was calibrated using a Jaeger 2\u00a0l syringe, room air and a calibration gas mixture. Heart rate (HR) was monitored using radiotelemetry (Polar Electro, Kempele, Finland).\nConstant intensity exercise bouts\nOn separate days, subjects performed a constant-intensity exercise bout at 60% of  in a thermo-neutral climate (N) and in a hot, dry climate (H). Temperature in the thermo-neutral climate was 15.6\u00a0\u00b1\u00a00.3\u00b0C, relative humidity (RH) was 20.0\u00a0\u00b1\u00a010.3%. In the hot, dry climate, the temperature was 35.5\u00a0\u00b1\u00a00.5\u00b0C and RH was 15.5\u00a0\u00b1\u00a03.2%. These temperatures were equivalent to the temperatures in the studies of Tatterson et al. (2000) and Tucker et al. (2004). All tests were performed in a climate-controlled room with a continuous airflow of 0.2\u00a0m\u00a0s\u22121. RH was set low to increase the evaporative capacity of the environment. Further, all trials in the H-climate were performed with a simulated wind velocity of 1.72\u00a0m\u00a0s\u22121 (6.2\u00a0km\/h) to further increase evaporative heat loss, which is the most important form of heat loss in cycling in hot conditions (Saunders et al. 2005). Prior to the experiments, the subjects were asked to refrain from vigorous exercise for at least 48\u00a0h. They were also asked not to consume coffee, alcohol or drugs after 10\u00a0p.m. the day before the exercise and not to eat for two hours prior to the experiments. Subjects drank water ad libitum before the experiment.\nBefore the constant intensity bout in the H-condition, the subject stabilized for 50\u00a0min in the 35\u00b0C, 20% RH room. For the comfort of the subjects and to prevent them from shivering, the stabilizing period was reduced to 35\u00a0min for the N-condition in the 15\u00b0C, 20% RH room. During the test, oxygen consumption (VO2), respiratory exchange ratio (RER) and ventilation (VE) were measured breath by breath. Power output (PO) and heart rate were registered continuously.\nAfter stabilization, a 5-min warm-up was performed at 100\u00a0W with a pedal frequency of \u223c100\u00a0rpm. After 1\u00a0min of rest, the constant intensity bout was started (\u223c100\u00a0rpm). The subjects cycled for 20\u00a0min at a constant power output of 60% of the power output at which maximal VO2 was attained at the incremental test  in the H condition. Directly after the end of exercise, blood lactate concentration (BLC) was measured (Lactate Pro, Arkray, Kyoto, Japan).\nRectal temperature (Tre) was monitored every 5\u00a0s during the entire test using a thermistor temperature probe (YSI 701, Yellow Springs Instrument, Dayton, USA) inserted about ten centimeters in the rectum. Skin temperature (Tsk) was measured every 5\u00a0s at 14 different skin loci, conform ISO 9886, using thermocouples (YSI 709B, Yellow Springs Instrument, Dayton, USA). Data were recorded with a Data Translation acquisition board (DT2821, Viewdac, Keithley Instruments, Cleveland, USA). Further, muscle temperature (Tm) was measured at a minimum depth of two centimeters and recorded every 5\u00a0s.\nSince this measurement was invasive, measurements were restricted to the constant intensity bout in H (n\u00a0=\u00a06). Tm was measured with a sterile thermal thermocouple-probe (type MAC08170A275SM, Ellab A\/S, Rodovre, Denmark) in the right vastus lateralis muscle, inserted by a physician. One hour before inserting the temperature probe, a lidocaine plaster was attached to the skin, as a local anaesthetic.\nCalculating gross-efficiency (GE)\nMetabolic power (Pmet) was calculated by multiplying oxygen consumption with the oxygen equivalent: Pmet (W)\u00a0=\u00a0VO2\u00a0\u00d7\u00a0[(4,940\u00a0\u00d7\u00a0RER\u00a0+\u00a016,040)\/60] according to Garby and Astrup (1987), assuming that respiratory exchange ratio (RER) equaled respiratory quotient (RQ) at sub-maximal intensities. Further, we assumed that respiratory exchange ratios in excess of 1.00 were attributable to buffering of lactate by bicarbonate. Ratios in excess of 1.00 were thus treated as if they equaled 1.00. The measured mechanical power output (PO) divided by the calculated Pmet defined GE. GE was calculated from 90\u00a0s after the start of exercise until the end of exercise.\nEnergy flow model\nThe energy flow model as described by De Koning et al. (1999) was used to calculate the effect of changes in GE on performance, by simulating a 20\u00a0km time trial as is studied in literature (Tatterson et al. 2000; Tucker et al. 2004). This model is based on power equations and has been reasonably successful in predicting performance in cyclic events as cycling (De Koning et al. 1999) and speed skating (De Koning et al. 2005). The energy flow model, also referred to as power balance model, relates to power production and power dissipation: Pprod\u00a0=\u00a0Plost\u00a0+\u00a0dE\/dt, where Pprod is total power that can be produced, Plost power that is used to overcome frictional losses, and dE\/dt rate of change of kinetic energy of the mass centre of the body. These terms can be calculated as described in De Koning et al. (1999), and the influence of changing one single variable, in this case GE, can be predicted.\nStatistics\nPaired Student t test\u2019s were performed to test if data were significantly different between the H-condition and N-condition, P values less than 0.05 were accepted as statistically significant.\nResults\nPower output at 60%  was 211.5\u00a0\u00b1\u00a018.6\u00a0W. All subjects were able to complete the trials in both H and N.\nVO2 was significantly higher in H than in N, resulting in GE being significantly lower in H compared to N. Further, HR and VE were higher in H compared to N. For RER and BLC, no main effect of temperature was found. Mean values are shown in Table\u00a02.Table\u00a02Gross-efficiency (GE), VO2, respiratory exchange ratio (RER), respiratory minute volume (VE), heart rate (HR) and blood lactate concentration (BLC) at 60%  in the thermo-neutral (N) and in the hot dry (H) climate60% NHGE (%)20.5\u00a0\u00b1\u00a01.419.6\u00a0\u00b1\u00a01.1*VO2 (ml\u00a0min\u22121)3,002.8\u00a0\u00b1\u00a0290.13,126.5\u00a0\u00b1\u00a0268.3*RER0.89\u00a0\u00b1\u00a00.030.90\u00a0\u00b1\u00a00.01VE (l\u00a0min\u22121)76.9\u00a0\u00b1\u00a07.782.0\u00a0\u00b1\u00a09.4*HR (bpm)145.1\u00a0\u00b1\u00a06.5155.4\u00a0\u00b1\u00a012.0*BLC (mmol\u00a0l\u22121)2.6\u00a0\u00b1\u00a01.03.9\u00a0\u00b1\u00a02.1Values are mean\u00a0\u00b1\u00a0SD*\u00a0Significant differences with N (P\u00a0<\u00a00.05)\nMean GE over time is plotted for both conditions in Fig.\u00a01. The difference in GE between conditions was 0.9% over the entire trial. From minute 5\u20138, the N\u00a0\u2212\u00a0H difference was 0.6%\u00a0\u00b1\u00a00.7%, from minute 15\u201318, the difference was 1.1\u00a0\u00b1\u00a01.3%, both significant. No significant change in GE within trials was observed comparing GE over the first half (min 5\u20138) with GE over the second half (min 15\u201318).Fig.\u00a01Gross-efficiency (GE) plotted over time at 60%  in the thermo-neutral (N) climate (dashed line) and in the hot dry (H) climate (solid line) \u00b1SD (dotted lines)\nTsk and Tre were significantly higher in H. Rectal and skin temperature changes combined, the difference in body heat content between N and H amounts to \u223c138.5\u00a0\u00b1\u00a035.1\u00a0kJ, calculated assuming that the specific heat of the body tissue was 3.4\u00a0kJ\u00a0kg\u22121\u00b0C\u22121. Maximal muscle temperature (Tmmax) in H was 38.7\u00a0\u00b1\u00a01.1\u00b0C. Mean temperature values are shown in Table\u00a03. Figure\u00a02 shows Tre, Tsk and Tm plotted over time for both conditions. Figure\u00a03 shows the correlation between Tre and GE for both conditions. R2 in N was 0.04, R2 in H was 0.36.Table\u00a03Rectal temperature (Tre), skin temperature (Tsk) and maximal muscle temperature (Tmmax) at 60%  in the thermo-neutral (N) and in the hot dry (H) climate60% NHTre (\u00b0C)37.03\u00a0\u00b1\u00a00.5837.35\u00a0\u00b1\u00a00.63*Tsk (\u00b0C)27.74\u00a0\u00b1\u00a00.7133.39\u00a0\u00b1\u00a00.57*Tmmax (\u00b0C)38.7\u00a0\u00b1\u00a01.1 (n\u00a0=\u00a06)Values are mean\u00a0\u00b1\u00a0SD*\u00a0Significant differences with N (P\u00a0<\u00a00.05)Fig.\u00a02Top panel Rectal temperature (Tre) plotted over time at 60%  in the thermo-neutral (N) climate (dashed line) and in the hot dry (H) climate (solid line) \u00b1SD (dotted lines). Center panel Skin temperature (Tsk) plotted over time at 60%  in the thermo-neutral (N) climate (dashed line) and in the hot dry (H) climate (solid line) \u00b1SD (dotted lines). Bottom panel Muscle temperature (Tm) plotted over time at 60%  in the hot dry (H) climate (solid line) \u00b1SD (dotted lines)Fig.\u00a03Top panel Correlation between gross-efficiency (GE) and rectal temperature (Tre) for the thermo-neutral (N) climate. Bottom panel Correlation between gross-efficiency (GE) and rectal temperature (Tre) for the hot, dry (H) climate\nTo quantify the effect of the decrease in GE of 0.9%, a simulation with an energy flow model (De Koning et al. 1999) was performed. This simulation showed that a change in GE of 0.9% would result in a difference in final time of 25.6\u00a0s over 20\u00a0km. Thus, about half of the decrement in performance reported in the literature (a difference in final time of 48\u00a0s over 20\u00a0km, Tucker et al. 2004) could be accounted for by the measured decrease in GE. To explain the entire deterioration in time trial performance in the heat, the mean decrease in GE had to be 2%.\nDiscussion\nGE during sub-maximal cycling exercise in the heat was significantly reduced compared to exercise in N by 0.9%. Values for GE corresponded to values found in literature, between 17 and 22% (Foster et al. 2003; Moseley et al. 2001). It has to be kept in mind that in calculating GE at 60%  anaerobic contribution was assumed to be negligible. But it might have been influenced by the different climates. Gonzalez-Alonso et al. (1999) reported that exercise in a hot environment results in a higher anaerobic contribution and found an increase in carbohydrate utilization and lactate accumulation during exercise. Though not significant, BLC in the present study was higher in H. According to di Prampero et al. (1999), an increase of BLC of 1\u00a0mmol\u00a0l\u22121 of blood is equivalent to an O2 consumption of 3\u00a0ml\u00a0kg\u22121 body weight. Differences in BLC in the present study between climates (2.6\u00a0mmol\u00a0l\u22121 in N vs. 3.9\u00a0mmol\u00a0l\u22121 in H) thus correspond with a difference in O2 consumption equivalent of \u223c14.1\u00a0ml\u00a0min\u22121. Accordingly, differences in GE between climates might be even larger then reported in the present study. If one adds this \u2018anaerobic O2 equivalent\u2019 to the VO2, then GE decreases about \u223c0.19% in N compared to \u223c0.27% in H, which means that the difference in GE between N and H increases with \u223c0.07%.\nMean Tre was about 0.3\u00b0C higher in H compared to N. Mean Tsk was about 5.7\u00b0C higher in H than in N. Comparable differences of 0.2\u20130.4\u00b0C in Tre (Tatterson et al. 2000; Tucker et al. 2004) and \u223c6.0\u00b0C in Tsk (Tatterson et al. 2000) were reported during cycling bouts of \u223c30\u00a0min in climates comparable to the present study. Although the difference in rectal temperature between H and N was significant, it was not a large difference compared to literature. The measured absolute values for Tre were somewhat low, presumably due to the relatively short period of exercise in our protocol and the use of a fan to optimize losing heat to the environment, as was done in the studies of Tatterson et al. (2000) and Tucker et al. (2004). The fan was used to simulate competitive circumstances, and although wind velocity in this study was not as high as the cycling speed, combined with the relatively low RH, the effect would have been considerable (Saunders et al. 2005).\nThe differences in ambient temperature between H and N represent a considerable difference in thermal stress. Thermal strain is mainly visible in the increased skin temperature, but although the difference of 0.3\u00b0C in core temperature is small, it is significant. Rectal and skin temperature changes combined, the difference in body heat content between N and H amounts to 138.5\u00a0\u00b1\u00a035.1\u00a0kJ, which is considerable. Further, the differences between climates have been shown to be large enough to evoke a difference in GE of 0.9%, the main purpose of the present study.\nA possible explanation for the lower GE in the heat may be the higher core temperature. Daanen et al. (2006) found a strong linear relationship between body temperature, mainly determined by Tre, and GE at 60%  in 30\u00b0C. Their study showed that our difference in Tre of 0.3\u00b0C could account for a reduction in GE of \u223c0.2%, and thus was not large enough to explain the entire reduction in GE of 0.9%. In the study of Daanen et al. (2006), the skin temperature however was almost unchanged, while in the present study skin temperature in the H condition was about 5.6\u00b0C higher, indicating an increased blood flow to the skin. The present study did not find a strong correlation in heat (r2\u00a0=\u00a0of 0.36) between Tre and GE. Further, remarkably, in the N-condition, no correlation between Tre and GE was found at all, even though Tre reached values which are also observed in the H-condition, only later in time (see Fig.\u00a03). Apparently, Tre does not seem to be the main cause of the reduced GE. This is supported by the observation that even though Tre rose significantly during the time trial with about 1\u00b0C, GE did not increase significantly comparing the first half with the second half within the trials.\nTwo other explanations for the reduced GE in heat can be given. Firstly, an explanation for the temperature-induced reduction in GE might be a metabolic disruption that is known to occur at elevated muscle temperatures (Brooks et al. 1971; Willis and Jackman 1994). Though Ferguson et al. (2006) found no effect of increasing muscle temperature on energy turnover in dynamic exercise in a range of 34.2\u201338.3\u00b0C, ADP:O ratio has been shown to decrease at higher muscle temperatures (Brooks et al. 1971; Willis and Jackman 1994). During heavy exercise in the heat, non-specific proton leakage across the inner mitochondrial membrane is increased, resulting in a decrease in the efficiency of oxidative phosphorylation and increasing the resting metabolic rate (Willis and Jackman 1994). This will result in a reduction in GE. Willis and Jackman (1994) found a decrease of 10\u201320% in ADP:O ratio at muscle temperatures of 40\u00b0C and higher compared to 37\u00b0C. They found that this decrease resulted in a 400\u2013800\u00a0ml\u00a0min\u22121 increase in VO2. In the present study, a smaller but significant increase in VO2 of 124\u00a0ml\u00a0min\u22121 was found. Mean Tmmax in heat was 38.7\u00b0C, lower than the 40\u00b0C as found in Willis and Jackman (1994), but higher than 34.2\u201338.3\u00b0C, the Tm range in which Ferguson et al. (2006) found no effect of increasing Tm on energy turnover in dynamic exercise. Since Tm was not measured in both climates, it cannot be confirmed if muscle temperature was significantly different. Tsk and Tre were significantly different but did not approach 40\u00b0C in either condition. It seems that, as suggested by Brooks (1971), the core of the body functions as a heat sink for the skeletal musculature helping to maintain Tm below the point where significant reductions in the ADP:O ratio occur.\nAnother potential cause for the decrease of GE in a hot ambient temperature was the larger vasodilatation of the skin to lose heat, as was shown by the significantly higher Tsk in H compared to N. The resulting decreased blood flow to exercising and respiratory muscles may be compensated by increasing cardiac output (Nielsen et al. 1990; Rowell et al. 1966), since a significant increase in heart rate was found in heat. The extra VO2 in the H condition is at least partially attributable to the extra myocardial VO2, since a higher cardiac output has to exist to continue supplying the muscles with the same blood flow, but have to send extra blood to the skin for cooling. Additionally, ventilation was increased, which may also lead to a reduction in GE. Assuming that the mechanical work per breath is 80\u2013125\u00a0J (Milic-Amili et al. 2001), it can be estimated that the higher VE in this study can account for maximally 10% of the increase in VO2.\nLastly, it has to be noticed, that GE decreases if the proportion of energy expenditure that is used to maintain homeostasis is increased. Thus, the lower GE in H could have been solely due to an increase in resting metabolic rate, while net efficiency remained unchanged. This would be consistent with the hypothesis that muscle temperature in the hot conditions was not high enough to make mitochondrial leakage a likely explanation for the observed reduced GE. Unfortunately, resting metabolic rate has not been measured.\nTo determine the potential importance of the measured decrease in GE on time trial performance, a 20-km time trial, which was also studied by Tucker et al. (2004), was modeled by the use of the energy flow model (De Koning et al. 1999). A time trial of this distance can be seen as a mainly aerobic exercise bout. Tucker et al. (2004) found a difference in final time of 48\u00a0s between exercise in 35\u00b0C compared to 15\u00b0C. Using the energy flow model of De Koning et al. (1999), it was calculated that the measured difference in GE of 0.9% would lead to a difference in final time between H and N of 25.6\u00a0s. This explains about half of the 48\u00a0s found by Tucker et al. (2004). For the entire reduction in final time of 48\u00a0s, a decrease in GE of 2% would be necessary, which was not found. It can be concluded that about half of the decrease in time trial performance can be accounted for by the reduction in GE. It has to be noted that in this simulation, it is assumed that the difference in GE between H and N at higher intensities will not change. Although it has been shown that GE increases with exercise intensity, since the relative share of resting metabolism diminishes at higher sub-maximal intensities (Mosely and Jeukendrup 2001), this effect seems to be equal for both conditions and will at most have only a minor effect on the difference between conditions.\nConclusion\nGE was lower in the heat. Tm was not high enough to make mitochondrial leakage a likely explanation for the observed reduced GE. Neither was the increased Tre. The extra VO2 in the H condition seems to be at least partially attributable to the extra myocardial VO2, since a higher cardiac output has to exist to continue supplying the muscles with the same blood flow, but have to send extra blood to the skin for cooling and thus impacted GE. Based on our findings under the current circumstances, it can be concluded that the temperature-induced change in GE could account for about half of the well-established performance decrements found during time trial exercise in the heat.","keyphrases":["performance","heat","muscle temperature"],"prmu":["P","P","P"]}
{"id":"Eur_Radiol-2-2-1766021","title":"Value of tissue harmonic imaging (THI) and contrast harmonic imaging (CHI) in detection and characterisation of breast tumours\n","text":"The purpose of this study was to investigate the extent to which tissue harmonic imaging (THI), speckle reduction imaging (SRI), spatial compounding (SC) and contrast can improve detection and differentiation of breast tumours. We examined 38 patients (14 benign, 24 malignant tumours) with different combinations of THI, SRI and SC. The effect on delineation, margin, tissue differentiation and posttumoral phenomena was evaluated with a three-point score. Additionally, 1oo not palpable tumours (diameters: 4\u201315 mm) were examined by contrast harmonic imaging (CHI) with power Doppler. After bolus injection (0.5 ml Optison), vascularisation and enhancement were observed for 20 min. The best combination for detection of margin, infiltration, echo pattern and posterior lesion boundary was the combination of SRI level 2 with SC low. THI was helpful for lesions OF more than 1 cm depth. In native Power Doppler, vessels were found in 54 of 100 lesions. Within 5 min after contrast medium (CM) injection, marginal and penetrating vessels increased in benign and malignant tumours and central vessels mostly in carcinomas (p<0.05). A diffuse CM accumulation was observed up to 20 min after injection in malignant tumours only (p<0.05). THI, SRI and SC improved delineation and tissue differentiation. Second-generation contrast agent allowed detection of tumour vascularisation with prolonged enhancement.\nIntroduction\nAs a technique complementary to mammography, high-resolution ultrasound (US) is well accepted for differentiating between cystic and noncystic putative tumour lesions [1\u201311]. New developments such as tissue harmonic imaging (THI) and contrast harmonic imaging (CHI) have enhanced its value, particularly for primary detecting tumours in dense inhomogeneous mammary gland tissue, identifying mammographically occult tumours and appraising abnormalities in tissue architecture [8, 12\u201322]. Lesions that are only a few millimetres in size become detectable, and tumours can be characterised more precisely. Ultimately, the exact localisation of small tumours is a prerequisite for ultrasound-guided needle biopsy, vacuum biopsy or preoperative wire placement [15, 23\u201325]. Harmonic imaging allows better visualisation of tissue architecture and facilitates recognition of parenchymal changes with tumour infiltration, making it easier to wire mark small lesions with THI [19, 26, 27].\nFor tumour characterisation, it is also essential to appraise vascularisation in a manner comparable to that achieved by dynamic contrast-medium-enhanced magnetic resonance imaging (MRI) [4, 12, 16, 17, 28\u201331]. Even if computed evaluation programmes with pixel analysis facilitate appraisal of tumour blood flow, native vascular US with power Doppler (PD) has proved to be relatively unreliable [4, 9, 13, 17, 18, 23, 24, 29]. Contrast medium enhancement already enables more distinct visualisation of intratumoral and peritumoral vessels. However, the brief diagnostic time window after bolus injection of US contrast medium (CM) is often a limiting factor. The combination of CHI with PD opens up promising vistas for appraisal of vascularisation. Preliminary investigations indicate that tumour detection, characterisation and US-guided intervention can be improved by using a CM of the second generation with modified CHI technique [18, 25, 32]. The aim of our study was to evaluate their potential benefits and limitations for detecting and differentiating breast tumours.\nMaterials and methods\nPatients and US\nA total of 138 patients (age: 25\u201379\u00a0years, median 54\u00a0years) were investigated in a multicentre trial with complementary mammary diagnostic procedures over a 36-month period (November 2002 to November 2005). All women gave informed written consent for their results to be used for programme research and evaluation. On the basis of US findings, each lesion was evaluated based on size, shape (ellipsoid, round, irregular), margins (smooth, macrolobulated, microlobulated, speculated, angulated, ill-defined), posterior sonographic artefacts (shadowing, through transmission, no posterior artefacts), echogenicity (hyperechogenic, isoechoic, hypoechoic), presence of calcifications and ductal extension. Investigations were supplemented by clinical examination and mammography in two planes. Ultrasound lesions were examined in three planes using PD and digital imaging with the GE Logiq 9 (GE Medical Systems, Milwaukee, WI, USA) US units. All investigations were carried out with a high-resolution multifrequency linear matrix array transducer (Linear Array M10L, 5\u201310\u00a0MHz) with the modalities of THI, spatial compounding (crossbeam), speckle reduction imaging (SRI) and contrast-medium-enhanced PD with CHI and additional three-dimensional (3D) imaging. Imaging documentation of digital raw data in single-image and short sequences was effected using a Picture and Communication System (PACS).\nStudy population\nThe patient population was divided into two groups. In group 1, we focused on the advantage of THI, SC and an adaptive algorithm for SRI. In group 2, we focused on CHI with PD.\nGroup 1\nThirty-eight patients (37\u201362\u00a0years) with 14 benign and 24 malignant tumours were examined in fundamental mode and with harmonic imaging with a high-frequency matrix-array transducer with the modalities of THI, SC and an adaptive algorithm for SRI. Prospectively, fundamental and tissue harmonic mode were used alone and in combination with all available levels of SC (cross beam: low, medium, high, maximum) and five levels of SRI. In patients without tumour lesions, criteria were differentiation of normal fibroglandular tissue from premammary and retromammary fat, including the Cooper\u2019s ligaments and ducts. Criteria that were evaluated for differential diagnosis in patients with lesions were margin, infiltration of malignant tumours, echo pattern with tissue components, calcifications, posterior lesion boundary and posterior acoustic features. After optimising system settings, a three-level score was applied to enable comparison of the quality of scans with fundamental imaging (0=equal, \u22121=inferior, +1=superior).\nGroup 2\nOne hundred patients (25\u201376\u00a0years, median 53\u00a0years) comprised 29 probably benign [Breast Imaging Reporting and Data System (BI-RADS III)] and 71 malignant (BI-RADS IV\u2013V) tumours confirmed by vacuum-assisted biopsy in 100\/100 lesions and 78\/100 cases additionally in the course of surgery. Tumour diameters varied between 4 and 15\u00a0mm (mean: 9\u00a0mm). Histological confirmation was obtained in all cases. All patients were comparatively investigated with fundamental B scan, THI and CHI with PD. After the B scan and evaluation of vascularisation in native PD, we used an echo signal amplifier of the second generation with low mechanical index (MI) technology. A bolus injection of Optison was administered (0.5\u00a0ml Optison diluted with 20\u00a0ml with NaCl solution and subsequent injection of another 20\u00a0ml NaCl). The bolus of CM was injected intravenously, and spreading of the contrast enhancement and washout in the tumours were followed for at least 20\u00a0min. A low MI (0.15\u20130.25) was chosen to avoid early destruction of the microbubbles.\nCM enhancement was appraised for up to 20\u00a0min. Moreover, additional 3D evaluations of the cine sequences were also possible retrospectively in consequence of the archiving of dynamic digital images of up to 180 single images and 10- loop. The images archived in B scan and THI could also be processed retrospectively, with various phases of SRI, which enabled marginal contours of the tumours to be highlighted by homogenising tissue structures. The patients were given detailed information before each injection of CM, particularly with respect to a possible predisposition to allergic reactions. Written consent was obtained. Studies were performed only in patients with normal renal function. Approval had been obtained from the hospital\u2019s ethics committee.\nHistopathological correlation\nHistopathological correlation was determined based on surgical and biopsy findings. Tumour size, grade, histological subtype and presence of invasion were documented. Pathological imaging correlation was performed in conjunction with both pathologist and breast imagers with regard to lesion location and size to ensure that the imaged lesions were evaluated histologically.\nBreast biopsy\nUS-guided biopsies were performed as vacuum-assisted biopsies, mostly with an 11-gauge needle. Up to 12 representative tissue samples were taken. When there were malignant findings, US-guided wire marking was mostly undertaken before surgical excision. Afterwards, follow-up examinations were carried out at 6-month intervals.\nStatistical analysis\nWe carried out statistical analyses: \nElementary statistics were computed.Fisher\u2019s two-tailed exact test was applied for statistical assessment of powers for discriminating different vascularisation and comparison of benign tumours versus carcinomas after the use of Optison.\nResults\nGroup1\nTHI has no effect on image quality in the near field. THI needs at least 1\u00a0cm of tissue penetration. The effect is best seen in the middle and far field, especially around the focus zone. Cutis and the first 10\u00a0mm of fat and Cooper\u2019s ligaments are already seen in fundamental mode, with better visibility and delineation with SC (cross beam: level medium) and SRI. The reflex of the anterior mammary fascia appears thinner and brighter with SC (cross beam: level medium to high) than in fundamental imaging. Ducts in deeper areas are best visualised with a combination of SC and THI. The main disadvantage is a significant reduction of the frame rate caused by SC, especially when THI is possible in addition. The first impression from image analysis of all combinations of SC and SRI was that higher levels of SC could be avoided by using, in addition, an SRI level of 1 or 2, giving the same advantage over the fundamental image with less reduction of frame rate. The best compromise for screening examinations of breast tissue is the combination of SC (level low) and SRI (level 1 or 2) with THI in small breasts. Fat appears slightly more echogenic, which potentially helps to define small tumours. The main limitation of THI was the reduced penetration (16% of our patients). In patients with large breasts and abundant fibrous tissue, fundamental imaging should be preferred.\nLesion detection and differentiation\nIn four out of five cysts, THI clears artifactual echoes caused by reverberations, noise and speckle so that even small intracystic lesions could be depicted. Best visibility was achieved with a combination of THI, SC (level low) and SRI 2, giving clear cyst borders and the intracystic lesion. Using SC in two out of five cysts, the typical dorsal enhancement caused by sound transmission is less intense, as expected from the fundamental mode. Fibroadenomas were best detected with a combination of THI with SC (level medium) and SRI (2), showing better margin delineation, even in the edges. Better differentiation of tissue components and tumour infiltration could be achieved by the same combination (Fig.\u00a01). Solid nodules generally appear more hypoechoic compared with surrounding breast tissue, which affords a better chance of detection [33].\nFig.\u00a01Effect on delineation of tumour margin and tissue differentiation. On the left side is fundamental mode without spatial averaging and tissue harmonic imaging (THI). On the right side THI+cross beam level low+speckle reduction imaging (SRI) 2. a Improvement of delineation of tumour margin, especially if the tumour border is perpendicular to the ultrasound beam (fibroadenoma). b Improvement of tissue differentiation (haematoma)\nCalcifications detected in mammography were seen in US in six out of ten patients. Visibility was increased, especially after using the combination of THI and SRI or SC and SRI (Fig.\u00a02a,b). Results for malignant tumours are summarised in Table\u00a01.\nFig.\u00a02a, b Effect on visibility of calcifications and delineation of tumour margin a Fundamental B mode. b Tissue harmonic imaging (THI)+cross beam low+speckle reduction imaging (SRI) level 1. Well-differentiated invasive ductal carcinoma with microcalcification and infiltration better seen in cross beam+SRI 2. There is less attenuation retrotumoral due to spatial compounding (SC)Table\u00a01Results of comparison of fundamental versus combination of tissue harmonic imaging (THI), spatial compounding (SC) (cross beam: level low) and speckle reduction imaging (SRI) level 1Criteria\u221210+1Margin11112Infiltration (existent in 19\/24 malignant cases)1315Echo pattern\/tissue components31011Calcifications123Posterior lesion boundary1716Posterior acoustic features996Score : \u22121=fundamental superior, 0=no difference, +1=combination superior\nGroup 2\nSeventy-one cases confirmed by biopsy were classified as malignant (BI-RADS IV\u2013V), and 29 cases were categorised as most likely benign (BI-RADS III). Histological confirmation by vacuum biopsy was also performed at patients\u2019 expressed request and in cases of malignancy or borderline lesions also by surgery (82\/100).\nHistological appraisal\nBenign lesions\nHistological finding detected four cases with a scar or intraductal inflammatory origin or ductal lesions, two cases of papillomas, five cases of fibrocystic mastopathy and 18 cases of fibroadenomas.\nMalignant lesions\nHistological findings revealed 11 ductal carcinomas in situ (DCIS), two lobular carcinomas in situ (LCIS) and 58 cases of ductal invasive carcinomas. Sonomorphology defined in particular the extent of the tumour, marginal contours, change in tissue architecture, possible infiltration of surrounding tissue, intraductal spreading, altered echogenicity of lesions with possible dorsal sound enhancement or attenuation and unchanged compressibility. In PD, CM enhancement with harmonic imaging additionally enabled detection of peritumoral or intratumoral and irregular vessels penetrating the tumour as well as increased tumour vascularisation in intermittent dynamic appraisal. In particular, irregular penetrating vessels and prolonged enhancement for more than 5\u00a0min were found in only malignancies (65\/71 cases: Fig.\u00a03a\u2013d, Fig.\u00a04a\u2013d and Fig.\u00a05a\u2013f). In six cases of DCIS with a maximum diameter of 5\u00a0mm and low grading (G I), increased vascularisation could not be demonstrated, even after CM enhancement. All were detected as small lesions using a combination of THI with SC. Five out of six showed tiny peritumoral vessels after contrast administration. Proliferating fibroadenomas and scars may show an increased peritumoral vascularisation but did not show prolonged enhancement in harmonic imaging with PD (Table\u00a02).\nFig.\u00a03a\u2013d Well-differentiated invasive ductal carcinoma (75-year-old woman, non-palpable mass) a Irregular tumour of 12 mm with hidden distal border in fundamental B scan and tissue harmonic imaging (THI). b, c In combination with cross beam and speckle reduction imaging (SRI), better delineation of the pectoral (distal) aspect , in particular, showing infiltration of the pectoral fascia. d Good visibility of the biopsy needle in THI and THI with crossbeam in three-dimensional (3D) techniqueFig.\u00a04a\u2013d Same patient as Fig.\u00a03 with invasive ductal carcinoma. a Unenhanced power Doppler (PD) shows few peripheral vessels. b\u2013c After administration of 0.5\u00a0ml Option, increasing enhancement within the lesion with prolonged persistence of enhancement up to 6\u00a0min is seen. d Beginning washout after 8\u00a0minFig.\u00a05a, b Ductal carcinoma in situ (DCIS) (35-year-old woman, no palpable mass). Cervical carcinoma, breast cancer in mother and sister. No clinical signs. Tumour proven by vacuum-assisted biopsy (DCIS) a Fundamental (B scan). b Tissue harmonic imaging (THI) showing a small, distinct, irregular mass of 8\u00a0mm. c\u2013f Same patient as a, b. c Power Doppler (PD) before administration of contrast. d Pronounced visibility of the feeding vessel in the early phase. e Late and persistent enhancement of the tumour typical for malignant breast tumours. f Better delineation of the tumour even after 14\u00a0min in contrast harmonic imaging (CHI) modeTable\u00a02Comparison of benign tumours versus carcinomas. Vascularisation after administration of CM [0.5\u00a0ml Optison]\u00a0Benign tumours (n=29)Carcinomas (n=71)Power DopplerPower Doppler +CMPower DopplerPower Doppler + CMEarly phase (1\u20135 min)Marginal vessels11\/29 (38%)24\/29 (83%)43\/71 (60%)67\/71 (94%)Penetrating vessels4\/29 (14%)12\/29 (41%)28\/71 (39%)65\/71 (91%) (p<0.05)Central vessels0\/29 (0%)6\/29 (20%)21\/71 (29%)48\/71 (68%) (p<0.05)Late phase (5\u201320\u00a0min)Diffuse enhancement0\/290\/290\/7165\/71 (91%) (p<0.01)Marginal vessels are seen in both groups in a high percentage. Most carcinomas show diffuse enhancement through the late phase (statistical evaluation with the Fisher\u2019s exact test, significant differences p<0.05)\nThe results of our own investigations indicate that the tumours can be identified better during biopsy in echo-inhomogeneous tissue using CHI in biopsies of smaller tumours under 10\u00a0mm in size. This enables false negative findings to be avoided in the histopathological investigations. After injection of CM, satellite foci and lymph nodes may also show increased vascularisation, which is manifested especially in harmonic imaging with PD.\nDiscussion\nAfter clinical examination, breast US is the preferred method in symptomatic patients. In cases without symptoms, breast US is ascribed a higher sensitivity for detecting breast cancer in women with dense breast tissue, women under the age of 50 and high-risk women. Mammographically occult cancers can be detected by US in 10\u201340% of cases, depending on patients\u2019 breast density and age. For women without symptoms, breast US should be mandatory and complementary to mammography in the case of breast density grade II [American College of Radiology (ACR)] or more [34]. US is now the more accurate imaging test in women 45\u00a0years or younger who present with breast symptoms and should be the initial imaging investigation [5, 7, 35]. In preoperative assessment of the local extent of breast cancer, US showed higher sensitivity for invasive cancer than for DCIS [8, 35, 41]. In nonfatty breasts, US and MRI were more sensitive than mammography for detecting invasive cancer, but both MRI and US involved the risk of overestimating tumour extent [4, 7\u20139, 12, 35]. Mammographic sensitivity decreased from 100% in fatty breasts to 45% in extremely dense breasts.\nThe respective diagnostic accuracy of the different breast imaging modalities, i.e. mammography (Mx), high-resolution breast US, and dynamic contrast-enhanced breast scanning (MRI) regarding early diagnosis of familial (hereditary) breast cancer were investigated. Breast cancer detection rates were: Mx: 42%, US 25% and MRI 83%, and positive predictive values were: Mx 29%, US 30% and MRI 43% [8].\nTHI in combination with digital encoding of the US beam is an increasingly common option for B-mode imaging. It reduces reverberations, sidelobe effects and speckle. Both image contrast and lateral resolution are improved in harmonic mode compared with conventional fundamental US [36]. First applications are reported for liver disease [37], lymph node enlargement in children [38] and patients with pancreatitis. Benefits of harmonic imaging are more apparent in obese patients, improving accuracy of diagnosis from 60% to 80% [39]. To determine the impact of THI on visualisation of focal breast lesions, Szopinski et al. [21] performed a prospective study on 219 female patients. THI improved the grey scale contrast between fatty tissue and breast lesions in 230 lesions (90.6%; p<0.001) compared with fundamental frequency images. Contrast improvement was greater in breasts with predominantly fatty or mixed (fatty\/glandular) composition than in predominantly glandular breasts. Overall conspicuousness, lesion border definition, lesion content definition and acoustic shadow conspicuousness were improved or equal in the harmonic mode for all lesions.\nAfter optimising the system settings, our results showed better delineation of tumour borders and infiltration with the combination of SC and SRI. Better differentiation of tissue structure was seen in the combination of THI and SRI; microcalcifications could be depicted best with a combination of harmonic mode, SRI and SC [19, 27, 40]. Retrotumoral tissue and the posterior lesion boundary of tumours are significantly better identified with high values of SC. One must be aware of the potential problem that the typical posterior acoustic enhancement is significantly reduced, with the result that a cyst can be misinterpreted as a solid tumour.\nTHI and CHI can be helpful for detecting tumours less than 10\u00a0mm in size in echo-inhomogeneous dense tissue, for characterising tumour morphology with dynamic enhancement studies and for performing a definitive core needle biopsy or vacuum-assisted biopsy in cases of tumour lesions less detectable in fundamental B scan or tumours located in the deep tissue near the intercostal muscles. Harmonic imaging in combination with SRI and cross beam can facilitate detection of intraductal tumours such as papillomas. With 3D imaging, the needle could be more easily placed in the middle of these small intraductal tumours during biopsies [15].\nVarious criteria of image morphology have proved useful in appraising whether a breast tumour is benign or malignant on the basis of US. Smooth marginal contour, dorsal US amplification, displacement margin, bilateral US extinction and absence of structural discontinuity in that order rather indicate a benign lesion. An echodense border, tumour outliers, jagged marginal contour, dorsal extinction and structural discontinuity point to malignant lesions. In consequence of the increasing improvements in tumour detection and morphological imaging, sensitivity <80% and specificities <95% can be achieved with US in tumour diagnostics [5, 7, 20, 21, 35]. An even higher diagnostic certainty is aspired to. This can already be attained with the combination of mammography and breast US, especially in dense and inhomogeneous glandular tissue [2, 7, 22, 35].\nTumour lesions of BI-RADS IV and V have to be clarified by biopsy, in particular, punch biopsy or vacuum biopsy. Consequently, US-guided biopsy is appropriate when it allows unequivocal lesion identification. US-guided interventions are required for further clarification in mammography occult findings and preoperative wire marking. In our own investigations, harmonic imaging was employed for definitive preoperative wire marking, to carry out a US-guided vacuum biopsy and also with CM enhancement for improved tumour detection.\nAdditional Doppler methods have been used to analyse breast tumour vascularity [42, 43]. Detection of penetrating or central vessels proved to be an accurate sign of malignancy [12, 13, 17, 22, 24, 25, 29, 31]. With the application of CM (Levovist), additional vessels were detected within the lesions, increasing diagnostic accuracy. Sensitivity of colour-coded US was improved from 64% up to 86% using the echo signal amplifier. Specificity was 86% without and 82% with echo signal amplifier. MRI was found to have a sensitivity of 100% and a specificity of up to 82% [12].\nOur study with a modified technique of CM-enhanced PD and THI showed better visualisation of tumour vascularisation after bolus application of Optison. Persistent enhancement after 5\u00a0min was characteristic for malignant tumours, enabling tumour characterisation as has hitherto only been known from CM-enhanced MRI [4, 12, 16, 18, 24, 29\u201331, 44].\nWhen using CM containing protein, special attention must be paid to an allergic predisposition and kidney function disorder. The use of second-generation CM necessitates a change in MI and imaging with harmonic imaging. However, the modified technique allows a differentiated diagnosis of tumour morphology and vascularisation [4, 12, 25, 29, 44, 45].\nTo improve the detection and assessment of intratumoral vessels in malignant breast tumours, techniques to enhance visualisation of vessels within focal lesions have been developed and automated. These techniques focus on the early perfusion phase following CM administration. Contrast-enhanced MRI allowed semiquantitative assessment of the kinetics of contrast media [4, 18, 31]. Artefacts caused by pulsation and patient movement should be avoided. More frequently, 3D postprocessing has become important in assessment of vasculature. CHI US is already used to distinguish scars from recurrent tumours as well as in the optimisation of US-guided biopsies [14, 15, 20, 32].\nConclusion\nIn conclusion, we feel confident that US using THI and CHI technologies is a valuable tool for evaluating focal breast lesions, in particular in mammography-dense breasts. If utilised extensively, it will be a cost-effective tool to facilitate detection and differentiation of malignant breast lesions in early, well-treatable stages and will thus help in reducing breast cancer mortality.","keyphrases":["tissue harmonic imaging","contrast harmonic imaging","spatial compounding","breast ultrasound"],"prmu":["P","P","P","R"]}